Document dDaxbVrYGmxvzd1Ybm7dnebY5

RECEIVED JUN 2 8 1982 RESEARCH TECHNIQUES AND METHODS FOR THE DETECTION AND PREVENTION OF CARCINOGENESIS IN THE INDUSTRIAL WORKER GRANT REFERENCE NO. VC 7,0 UNIVERSITY OF LOUISVILLE HEALTH SCIENCES CENTER SCHOOL OF MEDICINE GRADUATE SCHOOL CANCER CENTER REPORT FOR THE CHEMICAL MANUFACTURERS ASSOCIATION (FORMERLY MANUFACTURING CHEMISTS ASSOCIATION) COffFIDSNTIAL Subject to Protect! vs Order in *33. v Ccnzco. Ins. ,L ;ro. SO - <-3o7 1^-tn C,'1 Juriciel ti . -- : '--vist:clot f'oir-t CMA 003421 C Q^FIDIjN? IAL Subject tc rrot3ct_v3 Zi'Jz r in. Ross v. _Cc-rc'.o. j>c . , :td. Z'Z - it 37 14+h T :-;: - -i ft-'" ' r-1 - - - ~ '- CMA 003422 INVESTIGATORS CARLO H. TAMBURRO, M.D., PRINCIPAL INVESTIGATOR CHARLES E. KUPCHELLA, PH.D., CO-PRINCIPAL INVESTIGATOR JOHN L. WONG, PH.D,, CO-PRINCIPAL INVESTIGATOR PROGRAM INVESTIGATORS G.H. BARROWS, M.D. J. T. DU, PH.D. E. ESPINOSA, M.D. R.C. FELDHOFF, PH.D. H.P. FORTWENGLER, M.S. G.R. SCHRODT, M.D. G. SONNENFELD, PH.D. U.N. STREIPS, PH.D. M.T. TSENG, PH.D. W.J. WADDELL, M.D. CONFIDENTIAL Subject to Protect Order in Ess_v. Conoco, Ino . . "3. 90-4837 I4th Judicial District r,~" t Calcasieu Parish, Louisian ii CMA 003423  me. i 14th Judicial Distr Calcasieu Parish, I CMA 003424 CONTRIBUTING INVESTIGATORS G.H. BARROWS, M.D., ASSISTANT PROFESSOR DEPARTMENT OF PATHOLOGY JOHN L. CREECH, JR., M.D., CLINICAL ASSISTANT PROFESSOR DEPARTMENTS OF SURGERY AND COMMUNITY HEALTH MEDICAL DEPARTMENT, B.F. GOODRICH COMPANY J. T. DU, PH.D., RESEARCH ASSOCIATE LIVER RESEARCH CENTER DEPARTMENT OF MEDICINE E. ESPINOSA, M.D., PROFESSOR DEPARTMENT OF PATHOLOGY R.C. FELDHOFF, PH.D., ASSISTANT PROFESSOR DEPARTMENT OF BIOCHEMISTRY H.P. FORTWENGLER, JR., M.S., RESEARCH ASSOCIATE LIVER RESEARCH CENTER DEPARTMENT OF MEDICINE RICHARD A. GREENBERG, PH.D., PROFESSOR AND ACTING CHAIRMAN DEPARTMENT OF COMMUNITY HEALTH CHARLES E. KUPCHELLA, PH.D., ASSOCIATE PROFESSOR ASSOCIATE DIRECTOR OF CANCER CENTER; *PROFESSOR AND CHAIRMAN DEPARTMENT OF BIOLOGICAL SCIENCES MURRAY STATE UNIVERSITY GARY LISS, M.D., M.P.H., CLINICAL INSTRUCTOR DEPARTMENT OF COMMUNITY HEALTH NIOSH/ROBERT A. TAFT LABORATORY; CINCINNATI, OHIO CAROLYN MARLOWE, B.A., RESEARCH ASSOCIATE DEPARTMENT OF PHARMACOLOGY & TOXICOLOGY *presently CCr?ID3IITIAL_ _ Object to Protective Order in v- .Conoco, Inc.._ No, 90 - 4537 14th Judicial District Calcasieu Parish, Lou-'-- CMA 003425 CONTRIBUTING INVESTIGATORS (Continued) LASLO MAKK, M.D., CLINICAL INSTRUCTOR DEPARTMENTS OF PATHOLOGY AND COMMUNITY HEALTH DIRECTOR, DEPARTMENT OF PATHOLOGY ST. ANTHONY HOSPITAL HANS POPPER, M.D., PROFESSOR STRATTON RESEARCH LABORATORY FOR THE STUDY OF LIVER DISEASE MOUNT SINAI SCHOOL OF MEDICINE NEW YORK, NEW YORK JOHN P. SANDOZ, M.S., INSTRUCTOR DEPARTMENT OF COMMUNITY HEALTH G.R. SCHRODT, M.D., PROFESSOR AND CHAIRMAN DEPARTMENT OF PATHOLOGY G. SONNENFELD, PH.D., ASSISTANT PROFESSOR DEPARTMENT OF MICROBIOLOGY & IMMUNOLOGY U.N, STREIPS, PH.D., ASSOCIATE PROFESSOR DEPARTMENT OF MICROBIOLOGY & IMMUNOLOGY C. H. TAMBURRO, M.D., PROFESSOR LIVER RESEARCH CENTER DEPARTMENTS OF MEDICINE AND COMMUNITY HEALTH M.T. TSENG, PH.D., ASSOCIATE PROFESSOR DEPARTMENT OF ANATOMY W.J. WADDELL, M.D., PROFESSOR AND CHAIRMAN DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY RAYA WARICK, PH.D., RESEARCH ASSOCIATE CANCER CENTER JOHN L. WONG, PH.D., PROFESSOR, DEPARTMENT OF CHEMISTRY ^O - Object TT~'. v. T4th jvTV /--I-; u-t iv CMA 003426 CONTRIBUTING INVESTIGATORS (Continued) LASLO MAKK, M.D., CLINICAL INSTRUCTOR DEPARTMENTS OF PATHOLOGY AND COMMUNITY HEALTH OIRECTOR, DEPARTMENT OF PATHOLOGY ST. ANTHONY HOSPITAL HANS POPPER, M.D., PROFESSOR STRATTON RESEARCH LABORATORY FOR THE STUDY OF LIVER DISEASE MOUNT SINAI SCHOOL OF MEDICINE NEW YORK, NEW YORK JOHN P. SANDOZ, M.S., INSTRUCTOR DEPARTMENT OF COMMUNITY HEALTH G.R. SCHRODT, M.D., PROFESSOR AND CHAIRMAN DEPARTMENT OF PATHOLOGY G. SONNENFELD, PH.D., ASSISTANT PROFESSOR DEPARTMENT OF MICROBIOLOGY L IMMUNOLOGY U.N. STREIPS, PH.D., ASSOCIATE PROFESSOR DEPARTMENT OF MICROBIOLOGY & IMMUNOLOGY C. H. TAMBURRO, M.D., PROFESSOR LIVER RESEARCH CENTER DEPARTMENTS OF MEDICINE AND COMMUNITY HEALTH M.T. TSENG, PH.D., ASSOCIATE PROFESSOR DEPARTMENT OF ANATOMY W.J. WADOELL, M.D., PROFESSOR AND CHAIRMAN DEPARTMENT OF PHARMACOLOGY ANO TOXICOLOGY RAYA WARICK, PH.D., RESEARCH ASSOCIATE CANCER CENTER JOHN L. WONG, PH.D., PROFESSOR, DEPARTMENT OF CHEMISTRY COTiFIDMTIAL Subject to Protective Order in Boss y. Conoco, Irc^ . To. 90-4337 14th JudirT'T^'ist'i-t Court Calces1'-u rt 003427 RESEARCH TECHNIQUES AND METHODS FOR THE DETECTION AND PREVENTION OF CARCINOGENESIS IN THE INDUSTRIAL WORKER Program Final Report Table of Contents Investigators ............................................................................................................... ii Contributors .................................................................................................... iii I. Introduction .................................................................................................................. 1 II. Summaries of Research Programs ............................................................................ 3 III. Research Programs and Results ............................................................................ 16 A. Studies of Human Immunological Systems in the Detection of Vinyl Chloride and Other Chemical Injury; Investigators - H.P. Fortwengler, Jr., and C.H. Tamburro.................................................................... 17 Al. Evaluation of Immunocompetence of Workers Chronically Exposed to Vinyl Chloride ................................................................... 17 A2. Assessment of the Leukocyte Adherence Inhibition Test for the Detection of Angiosarcoma of the Liver.......................................21 A3. Assessment of Vinyl Chloride-Induced Angiosarcoma Antigen for the Detection of Latent Angiosarcoma in Exposed Workers ........................................................................................ 22 A4. Study of Human Leukocyte Antigen (HLA) Frequencies in Chemically-Exposed Workers ................................................................... 25 A5. Identification of the Endothelial Cell as the Cell of Origin for Vinyl Chloride-Induced Angiosarcoma of the Liver..................................................................................................................30 B. Study of Hepatic Biochemical and Enzymatic Systems for the Identification of Vinyl Chloride Chemical Injury and Cancer Development: Animal and Human Studies; Investigators - J.T. Du, M.T. Tseng, and C.H. Tamburro................................................................... 34 C0NFID2ITTIAL Subject to Protective Order in 88. v. Conoco, Ino, , No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003428 ANIMAL STUOIES: 81. Characterization of Hepatic Enzyme Changes in Rats with Prolonged Vinyl Chloride Exposure: Decreased Glucose-6-Phosphatase Activity............................................................ 34 B2. Morphological Alterations in Livers of Rats Exposed to Vinyl Chloride.............................................................................................................. 34 B3. Alterations in the Oxidizing and Detoxifying Systems of Rat Liver After Prolonged Exposure to Vinyl Chloride ...................... 38 B4. Oxidative and Detoxifying Ability of Liver Mesenchymal versus Parenchymal Cells in the Metabolism of Xenobiotics ................. 41 HUMAN STUDIES: B5. The Effectiveness of Indocyanine Green (ICG) Clearances in the Detection of Liver Injury...................................................... 43 B6. The Assessment of Bile Acids versus Indocyanine Green (ICG) Clearances in the Detection of Chemical Liver Injury Chemical in Exposed Humans....................................................................................46 C. Study of Glycosaminoglycan Changes in the Detection of Hepatic Fibrotic Injury in Chemical Exposure and Hepatic Cancer Development; Investigator - C. E. Kupchella and R. Warick ... 51 ANIMAL STUDIES: Cl. The Study of Glycosaminoglycan Changes in Livers of Animals Bearing Metastasizing Hepatomas ...................................................... 51 C2. Significance of Tissue Glycosaminoglycan Elevations in Hepatic Necrosis and Regeneration .................................................. 51 HUMAN STUDIES: C3. Characterization of Glycosaminoglycan Patterns in Humans with Angiosarcoma.........................................................................................51 C4. Evaluation of Glycosaminoglycan Analysis of Urines of Chemically-Exposed Workers .................................................................. 51 C5. Characterization of Glycosaminoglycan Patterns in the Identification of Chemical and Nonchemical Injury of the Liver.............................................................. 51 Cz'2zo " 0rdsi* in vuuicial D : Calcasieu j0unt nrish, Louisiana vi CMA 003429 ANIMAL STUDIES: 31. Characterization of Hepatic Enzyme Charges in Rats with Prolonged Viny' Cn 1 oza E rosure: Dac-etsao Glucose-6-Pncsonatase Activity.......................................................... 32. Morphological Alterations in Livers of Rats Exposed to Vinyl Chloride........................................................................................................ 34 83. Alterations in the Oxidizing and Detoxifying Systems of Rat Liver After Prolonged Exposure to Vinyl Chloride ..................... 33 B4. Oxidative and Detoxifying Ability of Liver Mesenchymal versus Parenchymal Cells in the Metabolism of Xenobiotics ................. 4] HUMAN STUDIES: B5. The Effectiveness of Inoocyanine Green (ICG) Clearances in the Detection of Liver Injury......................................................43 86. The Assessment of Bile Acids versus Indocyanine Green (ICG) Clearances in the Detection of Chemical Liver Injury Chemical in Exposed Humans ................................................................................... AS C. Study of Glycosaminoglycan Changes in the Detection of Hepatic Fibrotic Injury in Chemical Exposure and Hepatic Cancer Development; Investigator - C. E. Kupchella and R. Warick ... 51 ANIMAL STUDIES: Cl. The Study of Glycosaminoglycan Changes in Livers of Animals Bearing Metastasizing Hepatomas ...................................................... 51 C2. Significance of Tissue Glycosaminoglycan Elevations in Hepatic Necrosis and Regeneration .................................................. 51 HUMAN STUDIES: C3. Characterization of Glycosaminoglycan Patterns in Humans with Angiosarcoma................................................................................... 51 C4. Evaluation of Glycosaminoglycan Analysis of Urines of Chemically-Exposed Workers ................................................................... 51 C5. Characterization of Glycosaminoglycan Patterns in the Identification of Chemical and Nonchemical Injury of the Liver............................................................................................................51 win' AL -_ v. jjonozo, ir_0. _ ;rQ. ,, 14th -JodieTTTT7... , _ J"Ct Co'- vi CMA 003430 D. Histologic and Morphometric Analysis: A Means of Assessing Hepatic Injury in Chemical Workers; Investigators - G.H. Barrows, G.R. Schrodt, and C.H. Tamburro ......................... 56 01. Morphometric Assessment of Histological Lesions Character istic of Vinyl Chloride Injury .................................................. 56 D2. Computer-Assisted Morphometric Analysis as a Rapid Means of Determining Collagen Content .............................................................. 56 D3. Development and Assessment of the Morphometric Method of Analysis of Collagen Content from Human Liver Biopsies Relationship to Age............................................................................... 56 04. Light Microscopic Assessment of Various Histological Lesions Found in Liver Biopsy Tissue Obtained from Vinyl Chloride Workers ........................................................................................ 56 E. Studies of Vinyl Monomer Chemicals and Their Metabolites Using Chemical Structure and Synthesis in the Determination of Toxicity of These Agents; Investigator - J.L. Wong ......................... 67 El. Synthesis, Purification, and Utilization of Vinyl Chloride Metabolites in Mutagenicity and Carcinogenicity Studies . . 68 E2. Detoxification Studies of Vinyl Chloride and Its Metabolites................................................................................................ 70 E3. Vinyl Chloride Metabolite Detection--Chloroacetic Acid ... 72 F. Assessment of Assays for the Carcinogenic Potential of Industrial Chemicals Using Prokaryotic and Eukaryotic Systems; Investigators - U.N. Streips and G. Sonnenfeld................................. 74 FI. Further Development of Bacterial Systems for Testing Carcinogenicity and Mutagenicity of Chemical Agents Used in the Manufacturing of Vinyl Chloride and Synthetic Rubber................................................................................................................. 74 F2. Preliminary Studies on the Use of Interferon Induction as an Indicator of Mutagenicity and Carcinogenicity of Chemicals......................................................................................................... 76 G. The Study of Liver Tissue Antigens and Antibodies in the Detection of Vinyl Chloride Injury; Investigator E. Espinosa............................................................................................................. 80 Gl. The Study of Tissue Antigens From Liver Tumors, Angiosar coma, and Hepatomas.................................................................................... 80 CONFIDENTIAL Subject to Proteotiv 3 Grder in Ross v. Conoco. Inc Ho. 90-4337 14th Judicial C-a ! "-I S ' "ii ti ~ r lot Court vi i CMA 003431 G2. Circulating Antigens and Autoantibodies in Vinyl ChlorideAssociated Liver Disease ..... .............................................. H. Use of Isolated Mammalian Liver Cells for the Study of Chemical Monomer Metabolism; Investigator - R.C. Feldhoff ......................... * HI. Isolation of Mammalian Liver Cells for the Study of the Metabolism of Chemical Monomers ..................................................... 87 H2. The In Vitro Study of Albumin Synthesis by Liver from Human Biopsies................................................................................................. 87 I. The Study of Tissue Disposition of Industrial Chemicals: The Vinyl Chloride Example; Investigator - W.J. Waddell and C. Marlowe..........................................................................................................92 II. The Use of Whole Body Autoradiography in the Specific Tissues. Localization of Accumulated and Retained Industrial Chemicals and their Metabolites .... 92 IV. Lists of Publications, Abstracts, Preprints, and Publications in Preparation............................................................................................................. 99 A. Publications....................................................... '.............................................. 100 8. Abstracts................................................................................................................. 102 C. Preprints............................................................................................................. 1 D. Publications in Preparation Titles ............................................................ 105 V. Appendix (copies of IV., A, B, C)....................................................................106 C0TIFID5TITIAL Subject to Protective Order in p.oss v. Conoco. Inc - . 90-4337. 14th Judicial Court rlr"asiBu Parish, Louisiana vi ii Ct4A 003432 G2. Circulating Antigens arc Au toant i cod ies in Vinyl ChloriGeAssociated Live'- Disease......................................................................... H. Use of Isolates Mammalian Ca' Is for the Study of Chemical Monomer Metabolism; invest: gator - rsidnoft............................ Hi. isolation of Mammalian Liver Ceils for the Study of the Metabolism of Chemical Moncmers .................................................. 87 H2. Tne In Vitro Study of Albumin Synthesis by Liver from Human Biopsies........................................................................................... S7 I. The Study of Tissue Disposition of Industrial Chemicals: The Vinyl Chloride Example; Investigator - W.J. Waddell ana C. Marlowe....................................................................................................92 II. The Use of Whole Body Autoradiography in the Specific Tissues. Localization of Accumulated and Retained Industrial Chemicals and their Metabolites .... 92 IV. Lists of Publications, Abstracts, Preprints, and Publications in Preparation............................................................................................................ 99 A. Publications...................................................... '..............................................100 B. Abstracts............................................................................................................... 102 C. Preprints............................................. 1Q4 D. Publications in Preparation Titles ........................................................... 105 V. Appendix (copies of IV., A, B, C) .................................................................. 106 CONFIDENTIAL Subject to Protective Order in Boss v. Conoco, In?. . ?;Q_ SO-4337 14th Judicial ;istrict Court ^^lossieu P-iriar.. ucuisinns viii CMA 003433 INTRODUCTION In 1974 Dr. John L. Creech, Jr., a surgeon and the plant physician for B.F. Goodrich's Chemical Plant on Bells Lane in Louisville, Kentucky, identi fied the increased occurrence of hepatic angiosarcoma in workers involved in polyvinyl chloride manufacturing. This discovery led to the implementation of an industrial cancer control, detection and prevention program at the Louis ville B.F. Goodrich Plant involving approximately 1,800 workers: 1,200 were active employees and 600 were previously employed individuals. The University of Louisville, in cooperation with the B.F. Goodrich Company, developed and implemented the program and has continued to follow this cohort of workers clinically and epidemiologically. A proposal submitted to the Chemical Manufacturers Association, formerly the Manufacturing Chemists Association, to foster scientific research in better technical methods for the detection and prevention of chemical injury and carcinogenesis in industrial workers was awarded in 1976. These investi gations covered nine general areas of interest and involved both animal and human studies. The studies included: (A) the use of the human immunological system for the detection of injury from vinyl chloride and other chemicals, (B) the assessment of hepatic biochemical enzymatic systems to detect and characterize vinyl chloride and other chemical injury, (C) the use of tissue and urinary glycosaminoglycan changes for early detection and diagnosis of chemical injury including cancer development, (D) the histological evaluation of liver tissue from chemical workers and the assessment of its collagen content as an indicator of chronic latent injury, (E) synthesis and analysis of vinyl chloride intermediate and end products to further understand its biological metabolism and removal, (F) the identification and assessment of cell assays to determine the carcinogenic potential of industrial chemicals, (G) the evaluation of tissue antigenic systems for detecting chemicallyinduced carcinogenesis, (H) exploratory studies for the use of isolated human liver cells to determine individual ability to detoxify chemicals, and (I) the further assessment of vinyl chloride metabolism by the use of whole body autoradiographic technique. Each of these areas of study were directed toward four major objectives: (1) to better characterize and understand the pathogenesis of chemicallyinduced injury, especially vinyl chloride injury, (2) to characterize the mechanisms of chemical injury and the body's biological defense to this injury using animal studies, (3) to evaluate and assess old and new biochemical parameters for the detection and verification of chemical injury in humans, and (4) to develop new techniques that could be applied to the problem of identifying occupationally-related chemical injury for purposes of preven tion. Many of the original objectives of the individual studies have been changed or modified because of developments and discoveries in both the animal CONFIDENTIAL Subject to Protective Order in Rcss v. Conoco, Inc,, No. 90-4837 14th -Judicial Pi strict, "crt fnl pi"-' - - r %7 ^ CMA 003434 2 program and the human medical surveillance program which were ongoing concomitantly during the research periods of this study. This has led to a large body of literature which has changed the medical approach to the screening, identification and verification of occupationally-related chemical injury. Although the contractual funding of this grant was only three years, each year was funded separately with varying time intervals between the funding from year to year so that almost six years have evolved between the initiation of these studies and this final report. This report, therefore, summarizes the work accomplished during this six-year interval and the related work which is still ongoing. The investigators' reports vary in the degree of detail depending upon whether their work is in the process of being published or has already been published. Work not yet published is described in greater detail; published work is accompanied by a copy of the actual article(s) attached in the Appendix. The investigators wish to express their appreciation to the B.F. Goodrich Company and especially to the entire management of its Bells Lane, Louisville plant. The continued cooperation and help of the plant managers, Philip Lawrence, Gabriel Le Febvre, Edward L. Beeler and especially Raymond Pruitt, Personnel Manager, along with Or. John Creech, Jr., and the medical department staff, requires special acknowledgement. The investigators also wish to thank their laboratory and research stl^ for their devoted work, and to thank Or. Harold Boyer, Vice President for Health Affairs, and Dr. Joseph X. Musacchia, Dean of the Graduate School, for their administrative efforts, and especially, Mrs. Vicky Strong for her patient secretarial support. A special thanks is given to Dr. Kathleen O'Connell without whose help and support completion of the periodic and final reports would have been much more difficult. Finally, and very importantly, the participation and cooperation of the entire work force of the B.F. Goodrich Bells Lane Plant, and their representative unions--the Distillery Workers Union, the International Brotherhood of Electrical Workers #369, the International Association of Machinist Workers #681, and Pipefitters Union Local #522--is acknowledged with appreciation. This work woula have been impossible without the active involvement and help of the workers. The objectives of this task were all directed toward improving and maintaining the best possible work environment. Again, a thank you to all who have contributed to this effort. J&Ut in 90~433Z cas:*u Pari.,.*, Vct Court -3"' ^aian* 003435 2 program zr.z the r^zr. razics! surveillance program which were c'goirQ concomitantly curing ore research periocs of this study. This has lec "to a large bcay of 11ce^ itu^e wnich has changed the medical approach to toe screening, identification and verification of occupationally-related chemical injury. Although the contractual funding of this grant was only three years, each year was funded separately with varying time intervals between the funding from year to year so that almost six years have evolved between the initiation of these studies and this final report. This report, therefore, summarizes the work accomplished during this six-year interval and the related work which is still ongoing. The investigators' reports vary in the degree of detail depending upon wnether their work is in the process of being published or has already been published. Work not yet published is described in greater detail; published work is accompanied by a copy of the actual article(s) attached in the Appendix. The investigators wish to express their appreciation to the B.F. Goodrich Company and especially to the entire management of its Sells Lane, Louisville plant. The continued cooperation and help of the plant managers, Philip Lawrence, Gabriel Le Febvre, Edward L. Beeler and especially Raymond Pruitt, Personnel Manager, along with Dr. John Creech, Jr., and the medical department staff, requires special acknowledgement. The investigators also wish to thank their laboratory and research staff for their devoted work, and to thank Dr. Harold Boyer, Vice President for Health Affairs, and Dr. Joseph X. Musacchia, Dean of the Graduate School, for their administrative efforts, and especially, Mrs. Vicky Strong for her patient secretarial support. A special thanks is given to Dr. Kathleen O'Connell without whose help and support completion of the periodic and final reports would have been much more difficult. Finally, and very importantly, the participation and cooperation of the entire work force of the B.F. Goodrich Bells Lane Plant, and their representative unions--the Distillery Workers Union, the International Brotherhood of Electrical Workers #369, the International Association of Machinist Workers #681, and Pipefitters Union Local #522--is acknowledged with appreciation. This work would have been impossible without the active involvement and help of the workers. The objectives of this task were all directed toward improving and maintaining the best possible work environment. Again, a thank you to all who have contributed to this effort. Subject Boss v. _pnoco l4th Cal C'Si = 1) 51'll }J T14 t Tn 6 in Uo 90 - 4337 *-oZTl"t CMA 003436 SUMMARIES OF RESEARCH PROGRAMS CONFIDE?!? IAL Suhjeiat to Protective 0?der in v. ^ncco^no^ No. 90-4837 14th Judicial District Cou->~t Calcasieu Parish, 3 CMA 003437 PROGRAM A STUDIES OF HUMAN IMMUNOLOGICAL SYSTEMS IN THE DETECTION OF VINYL CHLORIDE AND OTHER CHEMICAL INJURY; Investigators - H. P. Fortwengler, Jr., and C. H. Tamburro Al. Evaluation of Immunocompetence of Humans Chronically Exposed to Vinyl Chloride The scientific literature is replete with the demonstrations of immunodepression as a manifestation of medical disease, especially cancer. Lymphocytes (T cells) can be cytotoxic to human tumor cells and are often found decreased or poorly functioning in cancer patients resulting in various degrees of immunodepression. The immunocompetence of a cohort of chemical workers was studied to determine if there was any evidence that (a) varying degrees of prolonged exposure to vinyl monomers was associated with immu nological suppression, (b) the immune response was impaired in workers with exposure-related liver injury including angiosarcoma, and (c) exposure caused changes in immunological function which were identifiable in the pre-cancerous stages. The immunocompetence of 75 employees with demonstrated liver disease and 225 individuals without clinical or laboratory evidence of disease was studied. No significant clinical or laboratory evidence of impaired immunocompetence was found in the immunological parameters studied, nor were differences seen among the various subpopulations of workers. A2. Assessment of the Leukocyte Adherence Inhibition Test for the Detection of Angiosarcoma of the Liver Hal 1 id ay et al. (1974) and Thompson (1976) had reported the development of a leukocyte adherence inhibition test which had identified the clinical stages in colon cancer. Similar findings were reported in individuals with hepatic cancer. This study was undertaken to determine the feasibility of applying this newly developed technique to screen vinyl chloride-exposed workers for angiosarcoma . of the liver. It was assessed in a number of chemical and nonchemical workers who had cancer. This test system lacked sufficient reproducibility to be considered for clinical use. A3. Assessment of Vinyl Chloride-Induced Angiosarcoma Antigen for the Detection of Latent Angiosarcoma in Exposed Workers New antigens arise from tumors formed as a response to carcinogens. Their presence in methylcholanthrene-induced sarcomas was discovered by Foley in 1953. This discovery in mice was verified and extended by Prehn and Main (1957) to conclude that there were antigens particular to and specific for tumor tissue. Subsequent evidence of tumor antigen was found in humans by the Hellstroms, Vankey, Halliday, Maluish, Thompson, and others. The majority of CONFIDENTIAL Subject to Protective Order in o^s v. Icr.coo, Inc. , No. 90-4837 "th Juiicicl Dtstr^t ''nu'-t CMA 003438 evidence suggested that the tumor antigens found were distinctive for each histological type of tumor. Studies were undertaken to determine whether angiosarcoma tumor antigens would provide specific immune reactions that could be utilized as a specific test for vinyl chloride-induced tumor development. Lymphocytes from normal and liver diseased individuals were isolated and then grown in the presence of a liver reagent prepared from either normal indi viduals or individuals who had angiosarcoma. Reactivity of lymphocytes was assessed by determining the incorporation of ^-thymidine into stimulated cultures as compared to unstimulated cultures. A comparison study was per formed between vinyl chloride plant workers and nonchemical plant workers utilizing reactions of lymphocytes to normal and angiosarcoma liver reagents. The lymphocytic response was also studied in vinyl chloride-exposed and non vinyl chloride-exposed workers with and without evidence of liver disease. Initially, specific reactivity was seen only among vinyl chloride workers. Although these inital results were provocative, subsequent studies with large control populations demonstrated similar reactivities. The lack of further occurrences of angiosarcoma in the cohort worker population prevented further characterization of the tumor antigen and restudy of the earlier findings. A4. Study of Histocompatibility Leukocyte Antigen (HLA) Frequencies in Chemically-Exposed Workers This study involved the search for HLA tissue types which may identify individuals susceptible to chemically-induced injury. In 1976 Mulvihi^ suggested genotyping as a means of screening potential employees for anti^H subtypes which may predispose them to neoplastic development after occupa tional exposure. Itwas prompted by the suggested increased occurrence of HLA-B27 antigen in workers with occupational asbestosis. The HLA frequencies in polyvinyl chloride manufacturing workers were compiled to determine if any increased genotype occurrences were associated with angiosarcoma or other chemically-related liver disease. Nine hundred individuals were eligible for this study: 538 individuals were HLA tissue typed and 30 different HLA-A, HLA-B, and HLA-C antigens were assessed. Preliminary comparisons were made with the HLA frequencies in vinyl chloride workers with and without liver disease, and later in those with liver disease of chemical vs. nonchemical origin. HLA frequencies were also analyzed in those individuals with and without biochemical evidence of liver injury. HLA-B15 was found to occur with a greater frequency among chemical workers with liver disease than in "control" and "normal" populations. The HLA-B15 occurrence was greater among those with chemical liver injury, although it was not statistically significant. Two other HLA markers occurred with unusual lower frequency. A5. Identification of the Endothelial Cell as the Cell of Origin For Vinyl Chloride Angiosarcoma of the Liver There has been considerable disagreement as to the cell of origin for liver angiosarcoma, a nonparenchymal cell malignancy of the liver. This is of CMA 003439 3 evidence suggested that t.he tu'ncn en''igers found -vere distinctive fcr each histological type of tumor. Studies were undertaken to determine whether angiosarcoma tuner antigens would provide specific immune reactions that could be utilized as a specific test for vinyl chloride-induced tumor development. Lymphocytes from normal and liver diseased individuals were isolated and then grown in the presence of a liver reagent prepared from either normal indi viduals or individuals who had angiosarcoma. Reactivity of lymphocytes was assessed by determining the incorporation of -^H-thymidine into stimulated cultures as compared to unstimulated cultures. A comparison study was per formed between vinyl chloride plant workers and nonchemical plant workers utilizing reactions of lymphocytes to normal and angiosarcoma liver reagents. The lymphocytic response was also studied in vinyl chloride-exposed and nonvinyl chloride-exposed workers with and without evidence of liver disease. Initially, specific reactivity was seen only among vinyl chloride workers. Although these inital results were provocative, subsequent studies with large control populations demonstrated similar reactivities. The lack of further occurrences of angiosarcoma in the cohort worker population prevented further characterization of the tumor antigen and restudy of the earlier findings. A4. Study of Histocompatibility Leukocyte Antigen (HLA) Frequencies in Chemically-Exposed Workers This study involved the search for HLA tissue types which may identify individuals susceptible to chemically-induced injury. In 1976 Mulvihill suggested genotyping as a means of screening potential employees for antigen subtypes which may predispose them to neoplastic development after occupa tional exposure. It was prompted by the suggested increased occurrence of HLA-B27 antigen in workers with occupational asbestosis. The HLA frequencies in polyvinyl chloride manufacturing workers were compiled to determine if any increased genotype occurrences were associated with angiosarcoma or other chemically-related liver disease. Nine hundred individuals were eligible for this study: 538 individuals were HLA tissue typed and 30 different HLA-A, HLA-B, and HLA-C antigens were assessed. Preliminary comparisons were made with the HLA frequencies in vinyl chloride workers with and without liver disease, and later in those with liver disease of chemical vs. nonchemical origin. HLA frequencies were also analyzed in those individuals with and without biochemical evidence of liver injury. HLA-B15 was found to occur with a greater frequency among chemical workers with liver disease than in "control" and "normal" populations. The HLA-815 occurrence was greater among those with chemical liver injury, although it was not statistically significant. Two other HLA markers occurred with unusual lower frequency. A5. Identification of the Endothelial Cell as the Cell of Origin For Vinyl Chloride Angiosarcoma of the Liver There has been considerable disagreement as to the cell of origin for liver angiosarcoma, a nonparenchyma1 cell malignancy of the liver. This is of COliFIDFITTI 4.T, Ross y. Co-o-o -- -n 90-4337 14 h Judicial District Cou-t `3 CMA 003440 6 great importance since the identification of the type of cell responsible for malignant transformation could provide the basis for a better understanding of chemically-induced carcinogenesis. This was particularly relevant to vinyl chloride-induced liver cancer since vinyl chloride is predominantly metabo lized and detoxified by hepatocytes and produces a malignancy of the liver that is nonparenchymal in origin. Considerable controversy arose as to whether the cell of origin was a macrophage (Kupffer cell), a fibroblast, or an endothelial vascular lining cell. The recent identification of Factor VIII production by endothelial lining cells led us to determine whether liver angiosarcoma tumor contained increased amounts of coagulation Factor VIII. Factor VIII is a large protein, often called antihemophilic factor, found differentially in endothelial cells, platelets, and megakaryocytes. The studies described herein demonstrated Factor VIII presence in endothelial lining cells of arterial and venous vessels in normal liver, as well as in the cells of angiosarcoma. Histological verfication of the cell type was deter mined by light microscopy of the same tissue sections of the tumor. This verified the endothelial cell as the cell of origin in angiosarcoma. PROGRAM B STUDY OF HEPATIC BIOCHEMICAL ANO ENZYMATIC SYSTEMS FOR THE IDENTIFICATION OF VINYL CHLORIDE CHEMICAL INJURY AND CANCER DEVELOPMENT: ANIMAL AND HUMAN STUDIES; Investigators - J. T. Du, M. T. Tseng, and C. H. Tamburro Bl. Characterization of Hepatic Enzyme Changes in Rats With Prolonged Vinyl Chloride Exposure: Decreased Glucose-6-Phosphatase Activity Considerable concern was initially raised concerning the best technique for screening individuals with chemical exposure. At present, federal testing requirements recommend enzyme studies related to liver parenchymal cells. However, clinical evidence indicated that not only were the hepatocytes injured but so were Kupffer and endothelial cells, and that the endothelial cell or Kupffer cell was the malignant cell of origin. In addition, studies to determine the sequential enzymatic changes which occurred with prolonged continuous vinyl chloride exposure similar to that seen with the worker population were needed. Most studies at this time had been conducted with short term exposures. The early animal studies included assessment of the standard clinical, biochemical, and enzymatic studies and later explored the enzymatic changes occurring in various subcellular organelles. These included (a) markers for microsomal enzymes related to the metabolism of vinyl chlo ride, -450, NADPH-cytochrome reductase, and mixed function oxidase, (b) cyto solic enzymes related to glutathione metabolism and glutathione content, (c) mitochondrial marker cytochrome-C-oxidase, (d) cytosolic enzymes glu cose- 6-phosphatase, glucose-6-phosphate dehydrogenase related to pentose phosphate pathway, and (e) nucleic acid synthesis. In addition, protein synthesis was evaluated by in-vitro incorporation of ^H-leucine. These confidential Subject to Protective Order in Ross v Conoco, Inc.. . 90 - 4337 14th Judicial Pist7''"* ^t r,-TT"t- p-x T* CMA 003441 7 studies were conducted in sequential experiments exposing Sprague-Oawley rats to 10,000 ppm vinyl chloride for a period of up to 300 exposure hours. These studies demonstrate various adaptive changes in liver parenchymal cells that were not identifiable by conventional clinical laboratory test (CCLT). These data illustrate the limited usefulness of CCLT in identifying early liver cell changes even during high levels of exposure. B2. Morphological Alterations in Livers of Rats Exposed to Vinyl Chloride Sequential morphological assessments were performed in the rats from both chronic exposure experiments utilizing light and electron microscopy. Light microscopic findings included an increase in liver cell polyploidy, double nucleated cells, and areas of focal hepatocellular hyperplasia without evidence of cellular injury or increased fibrosis. Electron microscopic changes included proliferation of smooth endoplasmic reticulum without evidence of other subcellular organelles, or increased collagen deposit in the Space of Disse. B3. Alterations of Oxidizing and Detoxifying Systems of Rat Liver After Prolonged Exposure to Vinyl Chloride A second long-term study, based partly on the results from the B1 study, was conducted to determine the adaptive capability of the detoxifying syste^^ of liver cells. Glutathione reductase activity, glutathione content, glut^B thione epoxide-S-transferase (GEST; Glutathione transferase E), and glutathione aralkyl-S-transferase (CAST; glutathione transferases A&B) were measured in rats exposed to vinyl chloride and to chloroethanol, a vinyl chloride metabolite. A third set of studies was conducted to compare these detoxifying enzymes in rats exposed to vinyl chloride and excessive alcohol consumption. These studies illustrated the adaptability of liver cells to prolonged exposure to xenobiotics. Liver cells showed increases in reduced glutathione and glutathione reductase activity and subsequent increases in GEST and GAST, reflecting a progressive increase in the liver cells' capacity for detoxification of vinyl chloride's toxic intermediates. Concomitant alcohol consumption appears to interfere with this detoxifying capability and appears to place the hepatocyte at greater risk of malignant transformation. B4. Oxidative and Detoxifying Ability of the Liver Mesenchymal vs. Parenchymal Cells in the Metabolism of Xenobiotics Another aspect of the prolonged vinyl chloride exposure studies was to determine the oxidative and detoxifying capability of various liver cells. In collaboration with Dr. Feldhoff, the oxidative and detoxifying ability of the liver mesenchymal versus parenchymal cells was studied by determining P450, glutathione reductase, GEST, and GAST in liver hepatocytes and endothelial cells. COjiFIUTTKiiAL Sub j ect^rV^^TT--r;,, HOSS.V. Conosc 14th in '~4357 Ca7"tV" Court Parish, Louisiana CMA 003442 io 10,000 ppm vinyl chloriaa for a period of up to 300 exposure hours. These studies demonstrate various adaptive chances in ' 'ver parenchymal cel's c"a: were not identifiable oy conventional clinical laboratory test (CCLT). These data illustrate the limited usefulness of CCLT in identifying early liver cell changes even during high levels of exposure. B2. Morphological Alterations in Livers of Rats Exposed to Vinyl Chloride Sequential morphological assessments were performed in the rats from both chronic exposure experiments utilizing light and electron microscopy. Light microscopic findings included an increase in liver cell polyploidy, double nucleated cells, and areas of focal hepatocellular hyperplasia without evioence of cellular injury or increased fibrosis. Electron microscopic changes included proliferation of smooth endoplasmic reticulum without evidence of other subcellular organelles, or increased collagen deposit in the Space of Disse. 33. Alterations of Oxidizing and Detoxifying Systems of Rat Liver After Prolonged Exposure to Vinyl Chloride A second long-term study, based partly on the results from the 81 study, was conducted to determine the adaptive capability of the detoxifying systems of liver cells. Glutathione reductase activity, glutathione content, gluta thione epoxide-S-transferase (GEST; Glutathione transferase E), and glutathione aralkyl-S-transferase (GAST; glutathione transferases A&B) were measured in rats exposed to vinyl chloride and to chloroethanol, a vinyl chloride metabolite. A third set of studies was conducted to compare these detoxifying enzymes in rats exposed to vinyl chloride and excessive alcohol consumption. These studies illustrated the adaptability of liver cells to prolonged exposure to xenobiotics. Liver cells showed increases in reduced glutathione and glutathione reductase activity and subsequent increases in GEST and GAST, reflecting a progressive increase in the liver cells' capacity for detoxification of vinyl chloride's toxic intermediates. Concomitant alcohol consumption appears to interfere with this detoxifying capability and appears to place the hepatocyte at greater risk of malignant transformation. B4. Oxidative and Detoxifying Ability of the Liver Mesenchymal vs. Parenchymal Cells in the Metabolism of Xenobiotics Another aspect of- the prolonged vinyl chloride exposure studies was to determine the oxidative and detoxifying capability of various liver cells. In collaboration with Dr. Feldhoff, the oxidative and detoxifying ability of the liver mesenchymal versus parenchymal cells was studied by determining P450, glutathione reductase, GEST, and GAST in liver hepatocytes and endothelial cells CMA 003443 /_^ Calcasieu Pari^shx,siri'i'cOilti Court s is nq 8 The mesenchymal cells were shown to be capable of oxidizing xenobiotics which require P-450 and mixed function oxidases, although this capability was less than that of the parenchymal cells by a 70 to 1 ratio. GEST and GAST activity were also present but'in the ratio of 1 to 65, and 1 to 500, respec tively. These results demonstrate that the nonhepatocytic liver cells have the capability to activate vinyl chloride and other similar xenobiotics, al though to a lesser degree than hepatocytes. However, their ability to de toxify vinyl chloride metabolites is significantly less and may contribute to the reason for nonmesenchymal cell malignant transformation. B5. Effectiveness of Indocyanine Green (ICG) Clearances in the Detection of Liver Injury A chemically-exposed worker cohort of almost 1,000 individuals were medically screened to determine the effectiveness of ICG clearances versus standard biochemical studies of the liver to identify latent hepatic injury. Alanine aminotransferase (ALT/SGPT), aspartic aminotransferase (AST/SG0T), gamma glutamyl transpeptidase (GGPT), alkaline phosphatase (AP), and total bilirubin (TB) were assessed in contrast to 3 different dose levels of ICG. Positive predictive values as well as the specificity and sensitivity of each of the screening tests were determined in "normal" and "abnormal" worker populations as well as in those with hepatic disease and nonhepatic disease. Chemical and nonchemical liver injury were also determined in analysis of their relationship of work histories to the biochemical abnormalities. These studies demonstrated that ALT/SGPT was the screening test with the highest sensitivity and specificity among federally-required studies. ICG clearance, even at the low dose, clearly remained the best overall screening test for the detection of subclinical hepatic disease. B6. Assessment of Bile Acids vs. Indocyanine Green (ICG) Clearances in The Detection of Liver Injury Due to Chemical Exposure in The Human Population Fasting serum bile acid levels and ICG clearances were performed for 400 employees with varying degrees of chemical exposure as well as biochemical injury related to vinyl chloride exposure. Bile acid levels, ICG clearances and standard enzymatic biochemical tests were assessed regarding their ability to identify normal individuals, individuals with liver disease of nonchemical origin, and individuals with liver disease with chemical origin, correctly. These more sensitive studies were to identify early chemical injury; six other enzyme studies were also conducted regarding more specific enzymes for hepa tocellular injury, such as sorbitol dehydrogenase. One thousand seven hundred and sixty human seras taken from 900 exposed workers- were assessed and the results compared to the vinyl chloride exposure. Fasting serum bile acids demonstrated a high sensitivity separating normal versus abnormal individuals with early or latent liver dysfunction, particularly chemically-induced types of injury. Fasting serum bile acids ive Order in Wo. 90-4837 14th Judicial Distrilectt C' ourt 9 hold promise as a potential detector of early liver injury, whether chemical or not. Bile acias have the advantage of being natural biological substances which may be taken orally for screening clearance studies. PROGRAM C STUDY OF GLYCOSAMINOGLYCAN CHANGES IN THE EARLY DETECTION OF HEPATIC FIBROTIC INJURY IN CHEMICAL EXPOSURE AND HEPATIC CANCER DEVELOPMENT; Investigators - C. E. Kupchella and R. Warick ANIMAL STUDIES: Cl. The Study of Glycosaminoglycan Changes in Livers of Animals Bearing Metastasizing Hepatomas C2. Significance of Tissue Glycosaminoglycan Elevations in Hepatic Necrosis and Regeneration Animal studies to determine the sequence of GAG changes in experi mentally-induced fibrotic liver injury, and the relationship between GAG patterns in tumor tissue and in urine were studied in animals with fast vs. slow growing, metastasizing vs. nonmetastasizing, chemically-induced and transplantable liver cells tumors. The experimental animal studies identified heparan sulfate ( a type of GAG) elevation in hepatic tissue undergoing fibrotic changes and that the increased levels were reflected in the urine. Both heparan sulfate and hyaluronic acid levels were 3-4 times higher in experimentally transplanted liver tumors than in normal controls. Urinary excretion reflects both tumor GAG composition and size. Livers from animals bearing metastasizing hepatomas had a 10-fold great concentration of nonsulfated neutral uronic acid positive material than animals bearing nonmetastasizing hepatomas. Finally, hepatic necrosis was shown to be accompanied by significant tissue GAG elevation; hepatic regeneration was not. HUMAN STUDIES: C3. Characterization of Glycosaminoglycan Patterns in Humans with Angiosarcoma C4. Evaluation of Glycosaminoglycan Analysis of Urines of Chemically-Exposed Workers CMA 003445 G se as a pete ;etec:or of eay'1y liver injury, nether cnamical 11 i a acits z atvar.tace of ceir.q natural biological substances wnicn may be taxen oral y ror screening clearance studies. PROGRAM C STUDY OF GLYCOSAMINOGLYCAN CHANGES IN THE EARLY DETECTION OF HEPATIC FIBRQTIC INJURY IN CHEMICAL EXPOSURE AND HEPATIC CANCER DEVELOPMENT; Investigators - C. E. Kupcnella ana R. Warick ANIMAL STUDIES: Cl. The Study of Glycosaminoglycan Changes in Livers of Animals Bearing Metastasizing Hepatomas C2. Significance of Tissue Glycosaminoglycan Elevations in Hepatic Necrosis and Regeneration Animal studies to determine the sequence of GAG changes in experi mentally-induced fibrotic liver injury, and the relationship between GAG patterns in tumor tissue and in urine were studied in animals with fast vs. slow growing, metastasizing vs. nonmetastasizing, chemically-induced and transplantable liver cells tumors. M The experimental animal studies identified heparan sulfate ( a type of GAG) elevation in hepatic tissue undergoing fibrotic changes and that the increased levels were reflected in the urine. Both heparan sulfate and hyaluronic acid levels were 3-4 times higher in experimentally- transplanted liver tumors than in normal controls. Urinary excretion reflects both tumor GAG composition and size. Livers from animals bearing metastasizing hepatomas had a 10-fold great concentration of nonsulfated neutral uronic acid positive material than animals bearing nonmetastasizing hepatomas. Finally, hepatic necrosis was shown to be accompanied by significant tissue GAG elevation; hepatic regeneration was not. HUMAN STUDIES: C3. Characterization of Glycosaminoglycan Patterns in Humans with Angiosarcoma C4. Evaluation of Glycosaminoglycan Analysis of Urines of Chemically-Exposed Workers CONFIDENTIAL Subject to Protective Order in - -- v~ _Co-"CGO , Inc , , No . 90-4837 14th Judici-.i hi <3--int CMA 003446 10 C5. Characterization of Glycosaminoglycan Patterns in the Identification of Chemical and Nonchemical Injury of the Liver The study of glycosaminoglycans, glycoproteins involved in wound healing and scar formation, as potential indicators of chemical injury, fibrosis, and cancer development was conducted in a large cohort of exposed workers. Analy sis of GAG in tissue and urine was performed in individuals with angiosarcoma and heptocellular tumors, in individuals with chemical and nonchemical liver injury, and in individuals with chemical liver abnormalities and in normal controls. The studies indicated that human hepatic angiosarcoma and fibrotic liver disease are accompanied by elevated tissue GAGs, that the tumor tissue is different from the adjacent fibrotic areas of the liver, and that patients with angiosarcoma and primary hepatocellular carcinoma (hepatoma) have charac teristic urinary GAG patterns. These patterns are not found in "normal" indi viduals. Urinary GAG determinations give a better indication of liver disease than ultrasound or radioisotopic scanning, and although they are not as sensi tive as transaminases or ICG clearances, fractionated GAG analysis appears to differentiate between active and inactive liver disease. PROGRAM D HISTOLOGIC AND MORPHOMETRIC ANALYSIS: A MEANS OF ASSESSING HEPATIC INJURY IN CHEMICAL WORKERS; Investigators - G. H. Barrows, G. R. Schrodt, and C. H. Tamburro 01. Systematic Assessment of Histological Lesions Characteristic of Vinyl Chloride or Vinyl Monomer Injury D2. Computer-Assisted Morphometric Analysis as a Means of Determining Collagen Content D3, Development and Assessment of Morphometric Method of Analysis of Collagen Content from Liver Biopsies - Normal Occurrence with Age Vinyl monomer injury has been associated with various hepatic histo logical abnormalities. These have included subcapsular, portal, and perisinusoidal fibrosis, as well as hyperplasia of the hepatocytes and nonparenchymal (sinusoidal) cells. The ability to assess light microscopic determina tions of fibrosis (liver tissue collagen content) using computer-assisted morphometric analysis quantitatively, was delineated. The methodologies determined the normal presence of collagen and the effect of age. Prospective analysis to determine the ability of specific histological findings to cor rectly identify vinyl monomer exposure was also done. Both manual and computer-assisted morphometric assessment of normal collagen content identified the distribution between perisinusoidal, Subject to Protective Order in v. .Conoco, Inc.. ir0. SO-4837 14th Judicial District Court Calcasieu Icnis^-a CMA 003447 periportal, and pericentral areas in 4 different anatomical locations of the human liver of 4 different age groups. These studies demonstrated that the collagen content of the liver varies with age, increasing after the fourth decade. The collagen distribution shows an increased desposition in the perisinusoidal and midzonal regions with age. The computerized morphometric readout demonstrated that the collagen estimates vary from 2-6% of the total liver globules. (This does not include capsular fibrosis or collagen support tissue about major vessels.) D4. Light Microscopic Assessment of Liver Tissue Obtained from Vinyl Chloride Workers Characterizing Various Histological Lesions Light microscopic assessment of liver tissue obtained from vinyl chloride and nonvinyl chloride workers illustrated that focal hepatocellular hyper plasia and focal mixed hyperplasia, sinusoidal di1itation and focal areas of increased reticulum characterized chemical injury could be identified in a prospective blind study and that those individuals with these identified lesions had the highest correlation with their vinyl chloride accumulative exposure ranked months; this correlation was not seen with other chemicals within their exposure environment. PROGRAM E STUDIES OF VINYL MONOMER CHEMICALS AND THEIR METABOLITES USING CHEMICAL STRUCTURE AND SYNTHESIS IN THE DETERMINATION OF TOXICITY OF THESE AGENTS; Investigator - J. L. Wong El. Synthesis, Purification, and Utilization of Vinyl Chloride Metabolites in Mutagenicity and Carcinogenicity Studies E2. Detoxification Studies of Vinyl Chloride and Its Metabolites E3. Vinyl Chloride Metabolite Detection--Chloroacetic Acid In order to further elucidate the intermediate metabolism of vinyl chloride, especially with regard to the carcinogenic potential of its inter mediate metabolites--chlorooxirane and chloroacetaldehyde--laboratory syn thesis of these two putative metabolites was performed. The materials were utilized to further study the detoxification of these agents and their effect on bacterial systems. Additional studies were conducted to determine the ability to identify chloroacetic acid by mass spectrometry and gas chromatog raphy. These studies illustrated that although chlorooxirane and chloroacetaldehyde eventually give the same final product, the rate of reaction and the """ CONFIDENTIAL Subject to Protective Order in Eoss^ v. Cor.c-o, Ire. . J7o. 90-4337 14th - - CMA 003448 periportal, and pericentral areas in 4 different anatomical locations of the numan liver of 4 different age groups. These studies demonstrated that the collagen content of the liver varies with age, increasing after the fourth decade. The collagen distribution shows an increased desposition in the perisinusoidal and midzonal regions with age. The computerized morphometric readout demonstrated that the collagen estimates vary from 2-6% of the total liver globules. (This does not include capsular fibrosis or collagen support tissue about major vessels.) D4. Light Microscopic Assessment of Liver Tissue Obtained from Vinyl Chloride Workers Characterizing Various Histological Lesions Light microscopic assessment of liver tissue obtained from vinyl chloride and nonvinyl chloride workers illustrated that focal hepatocel1u1ar hyper plasia and focal mixed hyperplasia, sinusoidal dilitation and focal areas of increased reticulum characterized chemical injury could be identified in a prospective blind study and that those individuals with these identified lesions had the highest correlation with their vinyl chloride accumulative exposure ranked months; this correlation was not seen with other chemicals within their exposure environment. PROGRAM E STUOIES OF VINYL MONOMER CHEMICALS AND THEIR META80LITES USING CHEMICAL STRUCTURE AND SYNTHESIS IN THE DETERMINATION OF TOXICITY OF THESE AGENTS; Investigator - J. L. Wong El. Synthesis, Purification, and Utilization of Vinyl Chloride Metabolites in Mutagenicity and Carcinogenicity Studies E2. Detoxification Studies of Vinyl Chloride and Its Metabolites E3. Vinyl Chloride Metabolite Detection--Chloroacetic Acid In order to further elucidate the intermediate metabolism of vinyl chloride, especially with regard to the carcinogenic potential of its inter mediate metabolites--chlorooxirane and chloroacetaldehyde--laboratory syn thesis of these two putative metabolites was performed. The materials were utilized to further study the detoxification of these agents and their effect on bacterial systems. Additional studies were conducted to determine the ability to identify chloroacetic acid by mass spectrometry and gas chromatog raphy. These studies illustrated that although chlorooxirane and chloroacetalde hyde eventually give the same final product, the rate of reaction and the Subject to Protective Order v Conoco, Inc.,- }Jo. 90 - < 14th Judicial District Dour CMA 003449 12 intermediates in the two reactions are different, Chlorooxirane conjugates instantaneously with the sulfhydral compounds, while chloroacetaldehyde takes about 2 1/2 hours for a comparative reaction. Although both routes are con verged to yield the cysteine-S-acetaldehyde conjugate, the reaction rates are vastly different, one taking minutes, the other hours to complete. Chloroacetic acid is identifiable by mass spectrometry in liquid solutions, solu tions as small as 0.02M (2 mg/ml), while gas chromatography sensitivity was limited to concentrations of 0.2 to 2 mg/ml H2O (200 to 2,000 ppm). Use of a porous polymer solid support and formic acid is expected to improve sensi tivity by 100-fold. Combination of GC-MS techniques appear to be the most promising approaches to the detection of end metabolic products and biological tissue. PROGRAM F ASSESSMENT OF ASSAYS FOR THE CARCINOGENIC POTENTIAL OF INDUSTRIAL CHEMICALS USING PROKARYOTIC AND EUKARYOTIC SYSTEMS; Investigators - U.N. Streips and G. Sonnenfeld FI. Further Development of Bacterial Systems for Testing Carcinogenicity and Mutagenicity of Chemical Agents Used in the Manufacturing of Vinyl Chloride and Synthetic Rubber Application of mutagenic assays for the determination of mutagenic potential of a list of environmental chemicals was conducted using improved modifications of their bacterial system. In addition, a new screening technique for carcinogenesis was developed involving the inhibition of interferon induction. This was applied to several carcinogens and mutagens and compared to the effectiveness of the standard viral bacteriological assays. These studies have demonstrated improved techniques for rapid screening of large numbers of chemical formulations being utilized within an environmental region. F2. Preliminary Studies on the Use of Interferon Induction as an Indicator of Mutagenicity and Carcinogenicity of Chemicals The new method assessing the mutagenic/carcinogenic capabilities of chemicals using an interferon assay is reported as well as related research regarding the controlled process of cell division in bacteria and how these findings relate to mammalian cells. Interferon induction inhibition in contrast to the Ames bacterial assay demonstrated the ability to separate more accurately agents with more carcinogenic potential related to human occurrences. CMA 003450 PROGRAM G THE STUDY OF LIVER TISSUE ANTIGENS AND ANTIBODIES IN THE DETECTION OF VINYL CHLORIDE INJURY; Investigator - E. Espinosa GI. The Study of Tissue Antigens From Liver Tumors, Angiosarcoma, and Hepatomas G2. Circulating Antigens and Autoantibodies in Vinyl Chloride-Associated Liver Disease The approach to the use of tissue antigens produced by neoplasms and identified in serum, either as the antigens or autoantibodies, were studied in angiosarcoma and in chemically-induced liver cancers. Several normal tissue antigens, a tumor-associated protein antigen, and a glycoprotein liver antigen absent in individuals with angiosarcoma were identified and characterized. The finding of a missing antigen in vinyl chloride-related angiosarcoma stimulated studies of antigenic deletion in chemically-induced hepatoma and cultured human liver carcinoma cells. Two liver antigens were found to be absent. One of these antigens was shown to be normally present in other tissues (kidney and spleen) in addition to the liver. The second antigen was detected only in the liver and was found to be unrelated to the liver-specific F antigen. Liver-specific F antigen was also studied as a possible sensit^^ indicator of chemically-induced liver tumor. F antigen appeared to be abse^P in fast growing hepatoma 7777 and undetectable or very low in the slow growing hepatomas. The level of the F antigen did not appear to correlate with the rate of growth of these tumors; however, an increased concentration of F antigen appears related to those tumors with the highest metastatic characteristics. Studies of cultured human cancer carcinoma cells also noted a deficiency in liver-specific F antigen similar to that of the experimental hepatoma 7777. These data support the clinical observation that tissue antigens appear to be more useful in treatment and follow-up care, while antigenic deletions may prove more important for screening and early detection. PROGRAM H USE OF ISOLATED MAMMALIAN LIVER CELLS FOR THE STUDY OF CHEMICAL MONOMER METABOLISM; Investigator - R. C. Feldhoff HI. Isolation of Mammalian Liver Cells for the Study of the Metabolism of Chemical Monomers H2. The In Vitro Study of Albumin Synthesis by Liver from Human Biopsies CONFIDENTIAL Subject to Protective Orier in ?-os. v Cor.ooo, Ir.c., No. 90-4337 14th Ju^ieiol Court CMA 003451 THE STUDY OF LIVER TISSUE ANTIGENS AND ANTIBODIES IN THE DETECTION OF VINYL CHLORIDE INJURY; Investigator - . Espinosa Gl. The Study of Tissue Antigens From Liver Tutors, Angiosarcoma, and Hepatomas G2. Circulating Antigens and Autoantibodies in Vinyl Chloride-Associated Liver Disease The approach to the use of tissue antigens produced by neoplasms and identified in serum, either as the antigens or autoantibodies, were studied in angiosarcoma and in chemically-induced liver cancers. Several normal tissue antigens, a tumor-associated protein antigen, and a glycoprotein liver antigen absent in individuals with angiosarcoma were identified and characterized. The finding of a missing' antigen in vinyl chloride-related angiosarcoma stimulated studies of antigenic deletion in chemically-induced hepatoma and cultured human liver carcinoma cells. Two liver antigens were found to be absent. One of these antigens was shown to be normally present in other tissues (kidney and spleen) in addition to the liver. The second antigen was detected only in the liver and was found to be unrelated to the liver-specific F antigen. Liver-specific F antigen was also studied as a possible sensitive indicator of chemically-induced liver tumor. F antigen appeared to be absent in fast growing hepatoma 7777 and undetectable or very low in the slow growing hepatomas. The level of the F antigen did not appear to correlate with the rate of growth of these tumors; however, an increased concentration of F antigen appears related to those tumors with the highest metastatic characteristics. Studies of cultured human cancer carcinoma cells also noted a deficiency in liver-specific F antigen similar to that of the experimental hepatoma 7777. These data support the clinical observation that tissue antigens appear to be more useful in treatment and follow-up care, while antigenic deletions may prove more important for screening and early detection. PROGRAM H USE OF ISOLATED MAMMALIAN LIVER CELLS FOR THE STUDY OF CHEMICAL MONOMER METABOLISM; Investigator - R. C. Feldhoff HI. Isolation of Mammalian Liver Cells for the Study of the Metabolism of Chemical Monomers H2. The In Vitro Study of Albumin Synthesis by Liver from Human Biopsies CMA 003452 14 Presently, the rat has provided the most useful means of studying hepatic metabolism in mamnalian cells. The intact liver, however, consists of at least four cell types which differ in their metabolic and functional characteristics. These studies were directed toward the development of clinically utilizable techniques to study human liver biopsy material in relationship to its ability to synthesize, retain, and secrete protein. Improved techniques were also utilized for isolation of hepatic paren chymal and nonparenchymal cells so that nearly homogeneous populations of cells could be studied for their oxidizing and detoxifying capabilities. Preliminary results of this late addition to our research techniques have proven encouraging in the adaptability of highly sophisticated laboratory techniques to the study of human tissue in an in vitro system. It has demon strated that animal and human tissue obtained~5y a clinical biopsy technique are viable in an j_n vitro system, being able to perform normal functions relative to albumin synthesis. The technique for the study of this tissue in an in vitro system may be applicable to human tissue from individuals with varying degrees and types of cellular injury. This technique would provide the ability to determine the oxidizing and detoxifying capability of liver cells to various xenobiotics in a quantitative fashion. Although very futuristic in its approach, it appears accomplishable since the present devel oped methodology is clinically applicable. PROGRAM I THE STUDY OF TISSUE DISPOSITION OF INDUSTRIAL CHEMICALS: THE VINYL CHLORIDE EXAMPLE; Investigators - W. 0. Waddell and C. Marlowe II, The Use of Whole Body Autoradiography in Specific Tissues. Localization of Accumulated and Retained Industrial Chemicals and Their Metabolites The need for methods which more accurately identify the sites of locali zation of chemical agents and their metabolites has obvious importance. The use of whole-body autoradiography to identify the location of radioactively tagged chemicals was studied with regard to ^C-vinyl chloride. Whole-body saggital sections of mice exposed for three hours in '4C-viny1 chloride demonstrated that the highest levels of radioactivity in mice sacrificed at 20 minutes and one hour after removal from the vinyl chloride environment were observed in the liver, pancreas, kidney, intestinal contents, urine and bile. Concentrations of metabolites in the organs of excretion decreased continually over a 24-hour period. After nine hours removal from the vinyl chloride environment, Harder's gland, epithelium of the esophagus and intestine, and sublingual glands retained the highest levels of radioactivity, while moderate concentrations were seen in the liver, kidney, and intestinal contents. At 24 hours, the primary organs of retention were the thymus, Harder's gland, liver, and esophagus and intestinal epithelium. The high concentration of CMA 003453 nonvolatile metabolites of vinyl chloride retained in the thymus after 24 hours suggested that there was covalent binding of these metabolites to the molecules in the thymus. Possible interactions of the thymus may reflect a dual mechanism of carcinogenic action, one related to tissue damage in the liver, the other to a suppressed immune surveillance system. subje fioss v. L\,"' V, rde*- in i' 90-4337 '-strict Court n, LcuiSiana CMA 003454 oonvol at i 1 7 * p / ' '", ^1 c '~ i ^ ^ in a * - *-, ^ - - * v hours suggested that there was covalent binding of these metabolites to molecules in the thymus. ^ossicle interactions of the thymus may ref". = dual mechanism of carcinogenic action, one related to tissue damage in liver, the other to a suppressed immune surveillance system. the CONFIDENTIAL Subject to Protective Order in Ross v. Conoco, Inc., No. 90-4337 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003455 RESEARCH PROGRAMS AND RESULTS CONFIDENTIAL Subject to Protective Order in Ross v. Conoco, Inc., Ho. 90-4837 14th Judicial District Court Calcasieu Parish,, Louisiana cMA 00345C PROGRAM A STUDIES OF HUMAN IMMUMOLOGICAL SYSTEMS IN THE DETECTION OF VINYL CHLORIDE AND OTHER CHEMICAL INJURIES; Investigators - H.P, Fortwengler, Jr., and C. H. Tamburro. Al. Evaluation of Immunocompetence of Workers Chronically Exposed to Vinyl Chloride Background The study of the human immunological systems as a means of detecting chemical injury was initiated to determine whether or not the body's ability to recognize substances that were foreign would make specific responses that could be identified clinically. The immune response can be divided loosely into two types: antibody formation and cell-mediated responses. Our initial studies were conducted to determine whether the mediators of immunity, i.e., lymphocytes, could be identified as having any immune defects which could be related to chemical exposure and be an early signal of an impaired immune system which might interfere with the body's normal defenses. These antibody forming cells constitute the humoral part of the human immune response. The lymphocytes are divided into at least two classes or types called B cells and T cells. The B cell lymphocytes are responsible for the synthesis and secre tion of antibody molecules, while the T cells are responsible for helping B cells. In addition, the T cells are also able to carry out a whole series of reactions in their own right. Generally these reactions involve tissue destruction, and since antibodies are usually not involved, they are called cell-mediated reactions. There has been recent thinking that the body's defense against tumors is a result of a tumor's having a unique antigen which is therefore recognized as foreign. The immune system constantly responds against these new antigens, and in this manner, there is an ongoing immune surveillance against tumors. Failure to react with the new antigen results in cancers, according to this theory. Objective 1. To determine the immune competence of chemical workers with prolonged exposure to vinyl chloride monomers 2. To compare their immune response to those workers who had developed liver injury, including angiosarcoma. CONFIDENTIAL Subject to Protective Order In $083 y. Conoco, Inc,, No . 90-4337 14th Judicial District Court Calcasieu Parish, Louisiana 17 CMA 003457 18 Research Results An evaluation of immunocompetence was carried on in individuals with documented chemical hepatic injury and in those without biochemical or clinical evidence of liver injury, A second comparison was conducted between workers with high versus low exposure to vinyl chloride, utilizing our cumula tive exposure rank months (CERM) ranking. Tests for immunocompetence included enumeration of lymphocytes, determination of certain lymphocyte subpopula tions, and response of lymphocyte to nonspecific antigens as a test of lympho cytic function. Results of lymphocyte response to stimulation with nonspecific antigens Phytohemagglutinin (PHA), Concanavallin A (Con-A), and Pokeweed mitogen (PWM) are shown in Figure 1. LYMPHOCYTE RESPONSE OF VINYL CHLORIDE WORKERS (non-specific antigens) 300 250 - I 200 -- 50E 1 iso _ 1 iS) 100 - 50 - 1T 1; I V. VINYL CHLORIDE EXPOSURE ABOVE HED IAN (N-5A) :::::: belov nedian (N-2d> 1 11 1 T: k PHYTOHEHAGGLUTININ CONCANAVALLIN A POXEVEED HITOGEN Figure 1 Response of the other immune parameters in high and low vinyl chloride exposure are shown in Table 1. No statistically significant difference was found between workers with high and low vinyl chloride exposure, or between unexposed invidivduals and chemical workers (Figure 2). CONFIDENTIAL ^2222^52^ Bo. 90-4837 14oh Juciiciqi District Court Calcasieu Perish. Lcui siara CMA 003458 13 Research Results An evaluation of immunocompetence was carr'ed cn in individuals with documented chemical hepatic injury and in those without oiocnemical or clinical evidence of liver injury. A second comparison was conducted between workers with high versus low exposure to vinyl chloride, utilizing our cumula tive exposure rank months (CERM) ranking. Tests for immunocompetence included enumeration of lymphocytes, determination of certain lymphocyte subpcpulations, and response of lymphocyte to nonspecific antigens as a test of lympho cytic function. Results of lymphocyte response to stimulation with nonspecific antigens Phytohemagglutinin (PHA), Concanavallin A (Con-A), and Pokeweed mitogen (PWM) are shown in Figure 1. LYMPHOCYTE RESPONSE OF VINYL CHLORIDE WORKERS (NON-SPECIFIC ANTIC-Ertj) Figure 1 Response of the other inroune parameters in high and low vinyl chloride exposure are shown in Table 1. No statistically significant difference was found between workers with high and low vinyl chloride exposure, or between unexposed invidivduals and chemical workers (Figure 2). CONFIDENTIAL Subject to Protective Order In Ross v. Conoco, Inc,, No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003459 'ABIE I IMMUNE =A?A,"ETERS OF VC WORKERS ACCORDING CO EXPOSURE TEST STREPTOCOCCAL ANTIGEN stimulation ABOVE MEDIAN EXPOSURE 19l'i3l" (n-52) BELOW "ED I AH EXPOSURE 136i39 (n-29) STREPTOLYStN 0 STIMULATION 8t2 Cn-53) 2All2 Ch-24) PPO STIMULATION VARlQASE STIMULATION 29l3 (n-99) 2915 (n-59) 13*6 (n-23) 91*12 (n-23) CANDIDA STIMULATION 1113 (n-53) 6l2 (n-23) absolute lymphocyte COUNT t-cell rosettes at 4c (absolute) 2355i132 (n-52) 1503im (n-50) 23161198 (n-29) 15291132 !n-29) T-CELL ROSETTES AT 5JC (absolute) 'STIMULATION 1NDCX "SEN 1227130 (n-50) 12691123 (n-29) STATISTICAL SIGNIFICANCE NONE NONE NONE NONE NONE NONE NONE NONE Mitogen induced lymphocyte transformation in' VC workers and unexposed individuals CH VC workers (n = 78) 111 Unexposed individuals (n = 2l) 19 CONFIDENTIAL Subject to Protective Order In Ross v, Conoco. Inc. , Wo. 90-433? 14th Judicial District r Calcasieu Parish. CMA 003460 No statistical differences were seen in these same immune parameters between workers with or without liver injury (Tables 2 and 3). TABLE 2 TONE PARAMETERS 3F VC WORKERS WITH AND WITHOUT LIVER DISEASE TEST PMYTOHENAGGLUTtNfN stimulation LIVER DISEASE 253**35** (n-25) NO LIVER DISEASE 17&i35 (n-mS) Concanavallin a STIMULATION 195*36 (h-25) 162*29 (h-A8) POKEWEED MITOGEN STIMULATION 109*12 (h-25) 80+13 (h-A3) STREPTOCOCCAL ANTIGEN STIMULATION 208iA2 < H--24) 181*52 (h-A9) streptolysin o STIMULATION PPO STIMULATION VARIOASE STIMULATION CANDIDA STIMULATION 8i2 (h-25) 18*6 (h-25) 36*9 (h-25) 1A5 (h-25) 16i6 (n-R9) 50*9 (m-A5) 31*5 (h-50) hi (n-A9) 'STIMULATION INOCX **! TABLE 3 [TONE PARAMETERS OF VC WORKERS WITH AND WITHOUT LIVER DISEASE TEST WHITE BLOOD CELL COUNT LIVER DISEASE 6900*500* (h-25) NO LIVES DISEASE 6650*330 (n-50) LYMPHOCYTE COUNT (percent) 36*2 (h-25) 3812 (h-48) absolute lyhphoctte COUNT t-cell rosettes at 4c (pisceht) 2350*200 (h-25) 67*3 (h-25) 2300*200 (h-48) 6212 (n-N9) t-cell rosettes at Ac (absolute) 1600*100 (n-25) ISOOilOO (h-49) T-CELL ROSETTES AT 33C (percent) 55+2 (h-25) 52l2 (h-A7) T-CELL ROSETTES AT 33C (absolute) 1300+100 (n-25) 1200+100 (h-H7) Tables 4 and 5 show the results of these immunological parameters examined in old and young workers. Older individuals demonstrated significant differences in their total white count (Pc.Ol), percentage of lymphocytes (P<-001), and lymphocyte reactivity to Streptolysin 0 (P<.05) and PPD (tuberculin) (P<.05) stimulation. We believe these findings to be age related with no relationship to the presence of liver disease or exposure to vinyl chloride. No other statistically significant differences were found between the various groups. There is no evidence from this data that immunological abnormalities result from chronic exposure to various levels of vinyl chloride. CONFIDENTIAL Subject to Protec Hesq s v.. Conoco. I--n--' " Orion in -L. 'Jo ."SO*4337 14th Judicial District Court Calcasieu Parish, Louisiana cm* 003461 20 No statistical differences were seen in these sane immune parameters cet^eer workers with or without liver injury ("aoles 2 ana 3). TABLE 2 t.'TWE PARAMETERS DP vc WCSXE.-.S WITH AM) WITHOUT LIVES DISEASE TIST W^tOHEJUGGlUT r N J N STIMULATION LIVER DISEASE 253**35** (*-2S) NO LIVER DISEASE 12"!53 (-08) concanavallin a STIMULATION 195-36 Ck-25) 152129 (-*3) MITOGEN STIMULATION 100--12 (f 25) Will (n-03) Smr^OCQCCAL ANT(iJE* STIMULATION 258102 Ch-20) 131132 (*-43) 5TJE?*OLv$IN 0 stimulation 8l2 (n-2S) 15i5 In-OS) MMO STIMULATION !3i5 (*-25) 3Di9 (*-45) VAJM0A3E STIMULATION CANOIDA STIMULATION stimulation iMoex 36:9 (if25) 10*5 (if25) Jll5 (*-50) 7*1 (**49) TASLE J immune rarateters or vc workers with ANO WITHOUT LIVER DISEASE TEST "HITS 3LCO0 CEU COUNT LIVER DISEASE 6900:500 * C-25) NO LIVER DISUSE sesciiao (50) LT."?"CCTTE CCLNT (descent) 3o;2 (--25) 33i2 (ifwa) ABSOLUTE LTMNLOCYTE COUNT T-CELL aOSETTEJ AT 4C (EEACENT) 2I5D1295 (if25) S7-3 (if25) 233Q1250 (-08) 5212 (*-49) T-CELL AOSETTE! AT 4C (absolute) 1600:100 (*-25) 1500:100 (n-49) T-CELL AOSETTES AT 33C (newcent) 55*2 (if25) 52:2 (-07) T-CILL HOStTTES AT 33C (absolute) 1300*100 (if25) 1200:100 (n-07) Tables 4 and 5 show the results of these inrnunological parameters examined in old and young workers. Older individuals demonstrated significant differences in their total white count (Pc.Ol), percentage of lymphocytes (Pc.OOl), and lymphocyte reactivity to Streptolysin 0 (P<,05) and PPD (tuberculin) (P<.05) stimulation. We believe these findings to be age related with no relationship to the presence of liver disease or exposure to vinyl chloride. No other statistically significant differences were found between the various groups. There is no evidence from this data that immunological abnormalities result from chronic exposure to various levels of vinyl chloride. CONFIDENTIAL Subject to Protect!ve Order in Ross v. Conoco. Inc. , No. 90-483? 14th Judicial District Court Calcasieu Parish, Louisiana cma 003462 TASLE 4 IfTHJNE PAIWET63S OF VC W0RK6RS OLD VERSES VOUHG TEST phytchemagglut i n i n stimulation CCNCANAVALLIN A STIMULATION POKEWEED MITOGEN STIMULATION STREPTOCOCCAL ANTIGEN STIMULATION STREPTOLYSIN 0 STIMULATION PPQ STIMULATION VARIQASE STIMULATION CANDIDA STIMULATION *srimji_ATiow index "sen OLD 140**20** (-37) 160:25 (-371 90*15 (-37) 135:35 (h-3S) 5:2 (n-38) 36:12 (n-39) 25:6 (*58) 9:3 (-37) YOUNG 210:32 (n--40) 163:25 (*40) 90:20 (*40) 195:55 (-33) 21:7 P-.Q5 (h-33) 13:3 P..05 (-35) 39:9 (>39) 9:3 (-39) 21 TABLE 5 KWNE PARAMETERS OF VC WORKERS OLD VERSES YOUNG TEST WHITE SLOCO CELL COUNT LYMPHOCYTE COUNT (percent) OLD 7150:400* (-38) 33:2 (-56) YOUNG 6150:300 *.0I (-40) 41:2 *.001 (40) ABSOLUTE LYMPHOCYTE COUNT 2300:200 (-36) 2400:109 (n40) T'CELL ROSETTES AT 4c (percent) 64:2 (-36) 64:2 (n-40) T-CELL ROSETTES AT 4c (absolute) 1400:150 (34) T-CELL ROSETTES AT 33c (percent) 52:2 (-36) 1600:100 (-40) 53:2 (-49) T-CELL ROSETTES AT 33c (absolute) 1100:190 (-311) *3EM 1300:100 (n-4,1) A2. Assessment of the Leukocyte Adherence Inhibition Test for the Detection of Angiosarcoma of the Liver Background Hal 1 id ay et al. (1974) reported the development of a leukocyte adherence inhibition test which identified the preclinical stages of colon cancer. Similar findings were reported in patients with liver cancer--the primary hepatocellular type. The need for a more specific screening test, especially for those who might be developing angiosarcoma that was not clinically de tectable, led us to assess this test in our cohort population. Objective 1. To verify the studies of Hal 1 iday and Maluish regarding the use of the leukocyte adherence inhibition test in the identification of carcinomas of the colon and its possible adaptation to vinyl chloride-induced angiosarcoma. CMA 003463 Research Results Dr. Maluish came from Australia and spent a number of days helping us develop and further validate the methodology used. Despite multiple attempts we were not able to validate the reproducibi 1 ity of the leukocyte adherence inhibition test, neither in colon cancer nor in liver angiosarcoma patients. Unfortunately, we must conclude that the leukocyte inhibition adherence test is not sufficiently reproducible to be used as a reliable indicator of the presence of tumors. A3. Assessment of Vinyl Chloride-Induced Angiosarcoma Antigen for the Detection of Latent Angiosarcoma in Exposed Workers Background New antigens arise on tumors formed as a response to carcinogens. Their presence on induced sarcomas was discovered by Foley (1953). This discovery in mice was verified and extended by Prehn and Maine (1957) who concluded that there were antigens particular to and specific for tumor tissue. Subse quently, evidence for specific tumor antigens was found in humans by Hellstroms, Vankey, Halliday and Maulish, Thompson, and others. The majority of the evidence suggests that tumor antigens found were distinctive for each histological type of tumor. This concept was applied to our cohort population for primary liver cancer. Objective 1. To determine whether the human body mounts an immune reaction to developing cancer cells that can be identified by specific immune reactions with tumor-specific antigen. Research Results Lymphocytes (the cells responsible for immunity) were obtained from individual workers, isolated, and grown in the presence of liver reagents prepared from either normal individuals or individuals who had had angiosar coma. A positive reaction was identified by incorporation of ^H-thymidine into stimulated lymphocyte cultures as compared to unstimulated (control) cultures. Comparison of responses to a panel of tissue extracts (Table 6) by lymphocytes from vinyl chloride workers and normal non-chemical plant workers indicated that many unexposed individuals have reactivity to these tissue antigens. Lymphocyte reactivity against angiosarcoma tissue alone occurred more frequently among vinyl chloride workers (Table 7). ............. ' Subject ts Hogs v. C~ :__ -7 _L 14th Puli:::.! i.sji-st : Calcasieu Parish, Louisiana CMA 003464 Researcn Results Dr, Maluish cane from Australia and spent a nun,Per of days haloing us develop and further validate the methodology used. Despite multiple attempts we were not able to validate the reproducibility of the leukocyte adherence inhibition test, neither in colon cancer nor in liver angiosarcoma patients. Unfortunately, we must conclude that the leukocyte inhibition adherence test is not sufficiently reproducible to be used as a reliable indicator of the presence of tumors. A3. Assessment of Vinyl Chloride-Induced Angiosarcoma Antigen for the Detection of Latent Angiosarcoma in Exposed Workers Background New antigens arise on tumors formed as a response to carcinogens. Their presence on induced sarcomas was discovered by Foley (1953). This discovery in mice was verified and extended by Prehn and Maine (1957) who concluded that there were antigens particular to and specific for tumor tissue. Subse quently, evidence for specific tumor antigens was found in humans by Hellstroms, Vankey, Hal 1iday and Maulish, Thompson, and others. The majority of the evidence suggests that tumor antigens found were distinctive for each histological type of tumor. This concept was applied to our cohort population for primary liver cancer. Objective 1. To determine whether the human body mounts an immune reaction to developing cancer cells that can be identified by specific immune reactions with tumor-specific antigen. Research Results Lymphocytes (the cells responsible for immunity) were obtained from individual workers, isolated, and grown in the presence of liver reagents prepared from either normal individuals or individuals who had had angiosar coma. A positive reaction was identified by incorporation of ^H-thymidine into stimulated lymphocyte cultures as compared to unstimulated (control) cultures. Comparison of responses to a panel of tissue extracts (Table 6) by lymphocytes from vinyl chloride workers and normal non-chemical plant workers indicated that many unexposed individuals have reactivity to these tissue antigens. Lynphocyte reactivity against angiosarcoma tissue alone occurred more frequently among vinyl chloride workers (Table 7). CMA 003465 TABLE 6 TISSUE EXTRACT PANEL EXTRACT ANGIO TUMOR NORMAL LIVER HEPATOMA LIVER ANGIO "NORMAL" LIVER NORMAL KIDNEY** NUMBER IN PANEL 3 3 1 2 2 '"NORMAL" AREAS OF LIVER TISSUE FROM ANGIOSARCOMA LIVERS. ''OBTAINED FROM THE SAME DONORS AS NORMAL LIVERS, 23 TABLE 7 LYMPHOCYTE REACTIVITY TO TUMOR AND NORMAL TISSUE PANEL LYMPHOCYTES REACTIVE AGAINST ANGIO LIVER ALOAE NORMAL AND ANGIO LIVER NORMAL LIVER ALONE NONREACTIVE TOTAL VINYL CHLORIDE WORKERS N PERCENTAGE NON-VINYL CHLORIDE WORKERS N PERCENTAGE INTERPRETATION OF RESULTS 6 16 7 13 5 13 20 53 38 100 00 8 57 17 5 36 14 100 INDIVIDUALS WITH POSSIBLE SPECIFIC ANTI-TUMOR REACTIVITIES INDIVIDUALS WITH NONSPE CIFIC REACTIVITIES MASKING ANY POSSIBLE SPECIFIC REACTIVITIES INDIVIDUALS WITH NONSPECIFIC REACTIVITIES INDIVIDUALS WITH NO TISSUE REACTIVITIES an T IAL DSubJect~^m^T^T^der in -2j222^_l50ii !Io.*90-4837 14 h Judicial District Court CRlcRsi.u Parish, Louisiana CMA 003466 Lymphocytic reaction to normal and angiosarcoma liver reagents were studied in workers with high and low exposure (above or below the median exposures for the plant) to vinyl chloride (Figure 3). No statistical dif ference was noted between the reactivities of these two groups. LYMPHOCYTE RESPONSE OF VINYL CHLORIDE WORKERS (TISSUE ANTIGENS) VINYL CHLORIDE EXPOSURE 1 ABOVE MEDIAN (N-5<l> :: BELOW MEDIAN (N-2<0 ANGIOSARCOMA ANTIGEN EXTRACT LIVER ANTIGEN EXTRACT Figure 3 Similar studies in workers with and without liver disease also failed show significant differences in their lymphocytic reactivity to tis^B reagents (Table 8A). TABLE 8A LYMPHOCYTE REACTIVITY TO NORMAL AND ANGIOSARCOMA TUMOR TISSUES IN VINYL CHLORIDE WORKERS WITH AND WITHOUT LIVER DISEASE LIVER NORMAL TISSUE TUMOR TISSUE LIVER DISEASE NORMAL 2.4 0.3* 2.6 0.4 1.5 + 0.2 1.8 0.2 `STIMULATION INDEX + SEM CONFIDENTIAL Subject to Protective Order in Ross y. Conoco, Inc., No. 90-483? 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003467 Lymphocytic reaction to normal and arc: csarccma liver reagents studied in workers wish nigh and low exdosu'-e (aocve or below the : exposures for the plant) to vinyl chlorice (rigure 3). No statistical r'erence was noted between the reactivities of these two groups. LYMPHOCYTE RESPONSE OF VINYL CHLORIDE WORKERS (TISSUE ANTIGENS) VINYL CHICS.ICE EXPOSURE I ABOVE YECU.'I (N-5i) :: BELOW MEDIAN (N2<0 an AUGIOSAECOrA ANTIGEN EXTRACT LIVES ANTIGEN EXTRACT Figure 3 Similar studies in workers with and without liver disease also failed to show significant differences in their lymphocytic reactivity to tissue reagents (Table 8A). TABLE 8A LYMPHOCYTE REACTIVITY TO NORMAL AND ANGIOSARCOMA TUMOR TISSUES IN VINYL CHLORIDE WORKERS WITH AND WITHOUT LIVER DISEASE LIVER NORMAL TISSUE TUMOR TISSUE LIVER DISEASE NORMAL 2.4 + 0.3* 2.6 0.4 1.5 0.2 1.8 0.2 'STIMULATION INDEX SEM CONF iTiiitlAb Subject to Protective Order in Ross v. Conoco, Inc. , No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana ^ 003468 25 On the chance that non-specific reactions might be obscuring the specific reactions, workers with vinyl chloride-suspected disease (working in a non-vinyl chloride environment--Pallet Plant) were compared to workers without known liver disease (working at the Main Plant). TABLE 8B IN VITRO LYMPHOCYTE STIMULATION BY ANGIOSARCOMATOUS LIVER EXTRACT SUBJECTS HUMBER TESTED NUMBER OF SUBJECTS REACTIVE AGAINST: NORMAL LIVER ONLY NORMAL & ANGIO ANGIO ONLY NON-PALLET PLANT WORKERS 16 1 11 PALLET PLANT WORKERS 18 TOTAL 3R *0N INDIVIDUAL HAD ANGIOSARCOMA 1 2 0 2* 13 At first, this appeared to be a specific reaction to the angiosarcoma tumor extract, i.e., negative reactions to normal liver with positive reactions to tumor antigen. Initially, this type of reactivity occurred only among the vinyl chloride workers. Subsequent studies among additional nonchemical workers demonstrated similar reactivities to these liver reagents. Although these results were provocative, the lack of further occurrences of angio sarcoma in our worker population prevented us from further characterizing this (these) possible angiosarcoma antigen(s) and having the opporunity to verify these observations. A4, Study of Human Leukocyte Antigen (HLA) Frequencies in Chemically-Exposed Workers Background The major histocompatibility complex of the immune system, HLA, is com posed of multiallelic genes located on one region of chromosome 6 in the human and on chromosome 17 in the mouse. The genes in this region control a wide variety of surface antigens and lymphoid functions. This major histocompati bility complex is intimately related to the understanding of transplantations, graft versus host reactions, interactions of 8 and T Cells, genetic control of immune responses and several other phenomena in the immunological system. Certain genetic traits appear to be controlled in animals by the genes of this complex. These include transplantation antigens, cell-mediated lympholysis. COIf FI jpg ITT IAL Subject to Protective Order in Ross_v. Conoco, Inc., No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana q03469 target antigens, immune response, tumor virus susceptibility, liver cyclic adenosine monophosphate levels, hybrid resistance, and T-cell: B-cell inter actions. HLA antigens are found on almost all cells of the body except red blood cells. The major histocompatibility complexes in man are identified by letters (A, B, C, 0) and numbers (A10, B12, C3). An increased incidence of certain HLA antigens had been shown to"He associated with susceptibility to certain diseases. Mulvihill (1976) proposed that screening for these genetic markers to identify abnormal genotypes in potential employees should be done so that individuals who might be predisposed to neoplasia after occupational exposure would be separated from those from normal genotypes. The first occupational disease correlation reported was HLA-B27 antigen which was found with increased frequency in workers suspected of having occupational asbestosis. The possibility that the histocompatible complex could be used to identify individuals at increased risk of developing chemical injury led to its study in vinyl chloride workers. Objective 1. To determine the HLA frequency in vinyl chloride workers 2. To compare the HLA frequency in vinyl chloride workers with and without liver disease. In order to deal with the statistical problem of multiple comparisons due to the large number of antigens to be studied, potential marka^ for liver disease were identified for study: A-9, 8-15 and B-17. Research Results HLA determinations were done using the Terasaki microdroplet lymphocyte cytotoxicity test. HLA typing was performed on 538 of our chemical cohort population. Tissue typing included 11 HLA-A antigens, 15 HLA-B antigens and 4 HLA-C antigens. Frequency distributions were determined in our "standard" (healthy population) and compared with the "normal" groups studied by Scott et al. (1977) and the World Health Organization. The comparison of these fre quencies are found in Tables 9 and 10. A preliminary analysis of these HLA frequencies included the comparison of a subcohort population consisting of individuals identified through the medical screening program as having liver disease. These individuals had been transferred from the main chemical plant to an ancillary plant making wooden shipping pallets, referred to subsequently as "Pallet Plant Cohort". As seen in Table 10 this cohort had increased frequency of HLA-B 15 (12 percent-normal population versus 28 percent-Pallet Plant Cohort). CONFIDENTIAL Subject to protective Order in Hoss_v. Conoco, Inc. , No. 90-4837 14th Judicial District Court Calcasieu Parish, Loui si an a CMA 003470 target antigens, immune ^escc-'se. .'--s l ., ic-.-~ c ' I i tv. liver cyclic acenosine mcnopnospnate levels, r_.:r'c -es i s c srce, see T-ceii: 3-cell incer- accicns. HLA antigens are "'sere al~:sc e;' :e;:s cf t^e body except red looa cells. The major n i stocompatioi11ty complexes in man are identified by letters (A, 3, C, D) and numbers (A10, 312, C3). An increased incidence of certain HLA antigens had been shown to "He associated with susceptibility to certain diseases. Mulvihill (1576) proposed that screening for these genetic markers to identify abnormal genotypes in potential employees should be done so that individuals who might be predisposed to neoplasia after occupational exposure would be separated from those from normal genotypes. The first occupational disease correlation reported was HLA-B27 antigen which was found with increased frequency in workers suspected of having occupational asbes- tosis. The possibility that the histocompatible complex could be used to identify individuals at increased risk of developing chemical injury led to its stuoy in vinyl cnloriGe workers. Objective 1. To determine the HLA frequency in vinyl chloride workers 2. To compare the HLA frequency in vinyl chloride workers with and without liver disease. In order to deal with the statistical problem of multiple comparisons due to the large number of antigens to be studied, potential markers for liver disease were identified for study: A-9, 8-15 and B-17. Research Results HLA determinations were done using the Terasaki microdroplet lymphocyte cytotoxicity test. HLA typing was performed on 538 of our chemical cohort population. Tissue typing included 11 HLA-A antigens, 15 HLA-8 antigens and 4 HLA-C antigens. Frequency distributions were determined in our "standard" (healthy population) and compared with the "normal" groups studied by Scott et al. (1977) and the World Health Organization. The comparison of these fre quencies are found in Tables 9 and 10. A preliminary analysis of these HLA frequencies included the comparison of a subcohort population consisting of individuals identified through the medical screening program as having liver disease. These individuals had been transferred from the main chemical plant to an ancillary plant making wooden shipping pallets, referred to subsequently as "Pallet Plant Cohort". As seen in Table 10 this cohort had increased frequency of HLA-B15 (12 percent-normal population versus 28 percent-Pallet Plant Cohort). CMA 003471 27 HLA- 4 ANTIGENS AI A2 A3 A9 AlO All ASS A29 AW30 AW 31 AW32 Blank TA3LE 9 MU-A FSE'jl'EIICiES HEALTHY COST HOLS 34 51 23 16 7 13 7 9 NP* HP HO HD *H0 1974 WORKSHOP 33 49 23 17 13 9 11 7 5 7 9 HD Pallet PLAwTOFG) 46 37 14 11 19 7 18 0 4 4 7 14 N TdC a 1 Frequency wr oo.n 900 160" 503 177", 2S 2001 HOW PALLET PLAHT(aFG) 30 53 21 S3 6 11 13 S s a 7 19 519 200% TABLE 10 HLA-3 FREQUENCIES HLA-B ANTIGENS 93 97 BA B12 B13 814 B13 0116 817 818 BW21 BW22 827 0W35 B40 Blank HEALTHY CONTROLS 10 31 37 30 3 3 10 HD A 8 HD 3 7 3 11 HP H Total Tr*quACy 903 133% WHO 1973 WORKSHOP 11 23 20 24 a ll 7 12 7 9 4 5 S 17 12 HO 503 176% PALLET PLANT/BFO 11 IS 21 21 0 11 29 7 14 4 4 7 7 11 18 1* 28 201% HON PALLET PLANT/8FG 9 38 30 31 3 S 12 4 13 4 3 n 17 16 13 319 301% CONFIDENTIAL Subject to Protective Order in Ppss V. Conoco. Inc. , No. 90-4837. 14th Judicial District Court C-l^asieu Parish, Louisiana CMA 003472 Table 11 illustrates the frequency of HLA-B15 among the entire worker cohort at the main chemical plant. This cohort was subdivided into those with liver disease versus normal individuals based on medical screening studies and liver biopsy. HLA-B15 occurred in 13 percent of the normal group versus 17 percent of the liver diseased group. TABLE 11 DISTRIBUTION OP HLA-815 ANTIGEN AMONG CHEMICAL WORKERS WITH AND WITHOUT LIVER DISEASE (MAIN PLANT COHORT) Clinical Diagnosis NORMAL N=5Q9 LIVER DISEASE 64 B15 3 427 15 Total (I) 491 (13) 18 (17) To determine if this increased occurrence of HLA-B15 was due to chemi cally-related liver injury and not due to incidental (non-occupational) liver disease, both the pallet plant and the main plant cohorts with liver disease were subclassified into those with chemical liver injury (CLI), and non chemical liver disease (LD). The frequency of HLA-B15 in these cohorts is shown in Tables 12 and 13. Table 14 illustrates the occurrence of HLA-B15 in the entire plant cohort subclassified into those without liver disease (NORMAL), those with liver disease, all types (BOTH). Those with liver disease are further subclassified into those with chemical liver injury (CLI) and those with liver disease, non-chemical in origin (LO). CONFIL-ITTIAL Subject to Protective Order In Boss v. Conoco , Inc., Mo. 50-4337 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003473 23 Table 11 illustrates trie frecue''cy cT~ LiLa-3:5 artrc the er'ti,_e cohort at the main chemical plant. This cohort was subdivided into those with liver disease versus normal individuals based cn medical screening studies and liver biopsy. HLA-815 occurred in 13 percent of the normal group versus 17 percent of the liver diseased group. TABLE 11 DISTRIBUTION OF HLA-B15 ANTIGEN AMONG CHEMICAL WORKERS WITH AND WITHOUT LIVER DISEASE (MAIN PLANT COHORT) Clinical Diagnosis NORMAL N-509 LIVER DISEASE 64 B15 3 427 15 Total (I) 491 (13) 18 (17) To determine if this increased occurrence of HLA-815 was due to chemi cally-related liver injury and not due to incidental (non-occupational) liver disease, both the pallet plant and the main plant cohorts with liver disease were subclassified into those with chemical liver injury (CLI), and non chemical. liver disease (LO). The frequency of HLA-815 in these cohorts is shown in Tables 12 and 13. Table 14 illustrates the occurrence of HLA-815 in the entire plant cohort subclassified into those without liver disease (NORMAL), those with liver disease, all types (BOTH). Those with liver disease are further subclassified into those with chemical liver injury (CLI) and those with liver- disease, non-chemical in origin (LD), CONFIDENTIAL ^^Abject to Protective Order in ^oss v Conoco, Inc., No. 90-4837 14th Judicial District fiou^t Calca P- -i e-V, CMA 003474 TABLE 12 DISTRIBUTION OF HLA-315 ANTIGEN AMONG WORKERS WITH AND WITHOUT CHEMICAL RELATED LIVER DISEASE (PALLET PLANT COHORT) Clinical Diagnosis 29 TABLE 13 DISTRIBUTION OF HLA-315 ANTIGEN AMONG WORKERS WITH AND WITHOUT CHEMICAL RELATED LIVER DISEASE (MAIN PLANT COHORT) Clinical Diagnosis normai 1 3 Total 4 O (25) CL_L. 4 9 13 (31) N*29 I LD 3 9 12 (25) NORMAI 65 430 Total-'495 (X) (15) N=52S CL1 l LD 03 2 15""...... (0) 13 IB" (19) TABLE 14 OCCURRENCE OF HLA-315 AMONG CHEMICAL WORKERS WITH AND WITHOUT CHEMICAL LIVER INJURY Clinical Diagnosis NORMAI 65 430 Total 495 (2) (13) N-538 n i LD BOTH 46 10 11 22 15 28 (27) (21) 33 43 (23) CONFIDENTIAL Subject to Protective Order in.' Ross v. Conoco, Inc., Mo. 90-4837 14th Judicial District Court Calcasieu Parish, I.ouisiar>a. CMA Q03475 A5. Identification of the Endothelial Cell as the Cell of Origin for Vinyl Chloride-Induced Angiosarcoma of the Liver Background A search for evidence of vinyl chloride-induced tumor antigen has led to the finding that these tumors have an increased concentration of antigenic coagulation Factor VIII. Factor VIII is a known marker for the vascular lining cells of the endothelial type. Further experiments in animals indicated that the normal endothelial cell found lining the liver sinusoids has little if any Factor VIII fluorescence. Endothelial cells found lining larger vessels on the other hand demonstrated a striking fluorescence, as did experimentally transplanted mouse angiosarcomas. Conversely, mouse hepatic Kupffer cells, a second type of hepatic lining cell distinguished in histo logical cross section by engorgement with carbon particles, failed to demon strate positive fluorescence. An increase in antigenic Factor VIII in hepatic angiosarcoma endothelial cells as compared to the normal, led us to believe that these cells have increased production or storage capacity for antigenic Factor VIII. Objective 1. To study the Factor VIII fluorescence in human angiosarcomas, primary hepatocellular carcinomas, and normal livers. Research Results Examination of angiosarcoma liver demonstrated a significant fluorescence due to Factor VIII content in the malignant cells of the liver sinusoids. An intermittent pattern of fluorescence of the sinusoidal lining cells was seen. These cells were histologically identified as angiosarcomatous on hematoxylin and eosin ( H 4 E) staining of adjacent tissue. This type fluorescence was not seen in individuals with primary hepatocellular carinomas and a different pattern was seen in normal livers. These cells and their fluorescent pattern are illustrated in Figures 4 and 5. Figure 4 illustrates the H & E stained cells and Figure 5 their Factor VIII fluorescence. C0II?ID5!ITIAL Subject to Protective Order in Hoss_v. Conoco, Lnc. , No. 90-4337 14th Judicial District Court Calcasieu Parish, Louisiana rMA. 003^ A5. Identification or tne Endothelial Ceil es tne Ce1! cf 0 r 1 - - n fzr Vinyl Cnlorice-InGucea Angiosarcoma of the Liver Background A search for evidence of vinyl chloride-induced tumor antigen has led to the finding that these tumors have an increased concentration of antigenic coagulation Factor VIII. Factor VIII is a known marker for the vascular lining cells of the endothelial type. Further experiments in animals indicated that the normal endothelial cell found lining the liver sinusoids has little if any Factor VIII fluorescence. Endothelial cells found lining larger vessels on the other hand demonstrated a striking fluorescence, as did experimentally transplanted mouse angiosarcomas. Conversely, mouse hepatic Kupffer cells, a second type of hepatic lining cell distinguished in histo logical cross section by engorgement with carbon particles, failed to demon strate positive fluorescence. An increase in antigenic Factor VIII in hepatic angiosarcoma endothelial cells as compared to the normal, led us to believe that these cells have increased production or storage capacity for antigenic Factor VIII. Objective 1. To study the Factor VIII fluorescence in human angiosarcomas, primary hepatocellular carcinomas, and normal livers. Research Results Examination of angiosarcoma liver demonstrated a significant fluorescence due to Factor VIII content in the malignant cells of the liver sinusoids. An intermittent pattern of fluorescence of the sinusoidal lining cells was seen. These cells were histologically identified as angiosarcomatous on hematoxylin and eosin ( H & E) staining of adjacent tissue. This type fluorescence was not seen in individuals with primary hepatocellular carinomas and a different pattern was seen in normal livers. These cells and their fluorescent pattern are illustrated in Figures 4 and 5. Figure 4 illustrates the H & E stained cells and Figure 5 their Factor VIII fluorescence. CONFIDENTIAL SubjectTto Protective Order in -'os- .Conocot Inc, , ifa, 90-4837 I4th Judicial District Court Calcasieu Parish,, Louisiana cMA 003477 31 FIGURE <1 FIGURE 5 Relevance to Industry These rather extensive, detailed studies of a well-defined human population provide very valuable data regarding the use of immunological screening tests for the detection and identification of chemically-related injury. (1) There appears to be no significant evidence that the immunodepression or change in the antibody responding components or the cell-mediated responding components of the human immune system is affected by vinyl chloride exposure. Therefore, studying immunological parameters of type 8 or T cells will not provide any useful information in the early stages of disease even with excessive exposure. CONFIG Subject to Protective^Q.>-rg a0r_4i8n37 Pnss v. Conoco . Inc. ^th Judicial District ^ Calcasieu Parish, Louisia CMA 003478 (2) The use of tumor antigens for the detection of developing angio sarcoma, although a provocative possibility, is far too non-specific in its present stage of development to provide any reasonably useful means of detecting early cancer development. Until there is better evidence that tumor antigens provide sufficient specificity to prevent cross reactivity with other tissue materials, this approach will not appear to be a clinically useful one. (3) The present data suggest that the HLA-B15 antigen may reflect an increased susceptibility to liver injury, especially of chemically-induced origin. Confirmation of these findings and the determination of the effec tiveness of this marker in screening those with increased susceptibility to chemical liver injury must yet be determined by prospective study. (4) The leukocyte adherence inhibition test, unfortunately, was not adequate in its technical development nor sufficiently reproducible to be useful as a screening test for either colon cancer or liver angiosarcoma. All of the above immunological studies, with the possible exception of HLA marker, have provided little supportive evidence that chemical injury interferes with human immunocompetence as determined by our present methods of study. Therefore, studies of the immunological system for screening purposes appear to be of little, if any, value in the early identification of chemical injury or disease. (5) The identification of Factor VIII production by human and anin^^ angiosarcoma tumors provides further evidence for the need to develp screenii^r methods to detect endothelial rather than hepatocytic cell injury or dysfunction since these are the cells of origin that malignantly transform when exposed to vinyl chloride and other agents such as arsenic. References Foley, E.J. (1953) Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer Research 13:835-337. Halliday, W.O., Halliday, J.W., Campbell, C.B. et al. (1974) Specific immunodiagnosis of hepatocellular carcinoma by leukocyte adherence inhibition. British Medical Journal, 18,:349-352. Hellstrom, I., Hellstrom, K.E., Sjogren, H.O. and Warner, G.A. (1971) Demonstration of cell-mediated immunity to human neoplasms of various histological types. International Journal of Cancer, 7,:1--16. Mulvihill, J.J. (1976) Host factors in human lung tumors: An example of co-genetics in oncology. Journal National Cancer Institute, 57:3-6. C0II7IDENTIAL iuoject to Protective Order in Poss v_ Conoco, Inc, , Uo 90 - 4937 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003479 '.a us* :ecc::n or ceve , oc'rc ir-z'z- 5 ui - * i -- 11 'w j ^ 5 5 i ^ i * v ^ , is *ar coo non-scecific in its present s:ice c* cave'cp-re^t rco^ provide any reascnaoly useful means cf ceiacvir.g early cancer development. Unt.i.l t-h--e--r-e- is tetter evidence that tumor antigens provide sufficient specificity to prevent cross reactivity with other tissue materials, this approach will no' (3) The present data suggest that the HLA-315 antigen may reflect an increased susceptibility to liver injury, especially of chemically-induced origin. Confirmation of these findings and the determination of the effec tiveness of this marker in screening those with increased susceptibility to chemical liver injury must yet be determined by prospective study. (4) The leukocyte adherence inhibition test, unfortunately, was not adequate in its technical development nor sufficiently reproducible to be useful as a screening test for either colon cancer or liver angiosarcoma. All of the above immunological studies, with the possible exception of HLA marker, have provided little supportive evidence that chemical injury interferes with human irnmunocomoetence as determined by our present methods of study. Therefore, studies of the immunological system for screening purposes appear to be of little, if any, value in the early identification of chemical injury or disease, (5) The identification of Factor VIII production by human and animal angiosarcoma tumors provides further evidence for the need to develp screening methods to detect endothelial rather than hepatocytic cell injury or dysfunction since these are the cells of origin that malignantly transform when exposed to vinyl chloride and other agents such as arsenic. References Foley, E.J. (1953) Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer Research 13:835-837. Hal 1 id ay, W.O., Halliday, J.W., Campbell, C.B. et al. (1974) Specific immunodiagnosis of hepatocellular carcinoma by leukocyte adherence inhibition, British Medical Journal, 18,:349--352. Hellstrom, I., Hellstrom, K.E., Sjogren, H.O. and Warner, G.A, (1971) Demonstration of cell-mediated imnunity to human neoplasms of various histological types. International Journal of Cancer, 7,-.1-16, Mulvihill, J.J. (1976) Host factors in human lung tumors: An example of co-genetics in oncology. Journal National Cancer Institute, 57:3-6. CONFi: L, _t J - -r- i- Subject to Protective Or*or Boss v. Conoco, Inc. , Ho. 90-4337 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003480 33 Prehn, R.T. and Maine, J.M. (1957) Immunity to methylcholthrene-induced sarcomas. Journal of the National Cancer Institute, 16:No. 6, 769- 775. -- Scott, B.B., Rajah, S.M. and Losowsky, M.S. (1977) Histocompatibility antigens in chronic liver disease. Gastroenterology, 72:112-125. Terasaki, P.I., Bernoco, 0., Park, M.S., et al. (1978) Microdroplet testing for HLA-A, -B, -C, and -D antigens. American Journal of Clinical Pathology, 2^:103-119. Thompson, D.M.P., Gold, P., Freedman, S.O. and Shuster, J. (1976) The isolation and characterization of tumor-specific antigens of rodent and human tumors. Cancer Research, 36:3518-3525. Vankey, F., Stjernsward, J. and Nilsonne, U. (1971b) Cellular immunity to human sarcoma. Journal of the National Cancer Institute, 46:1145- U51. ~~ World Health Organization Report (1977) Histocompatibility testing. Bodmer, W. (ed.), Munksgaard, Copenhagen. CONFIDENT IAL Subject to Protective Order in Ross v. Conoco, Inc., No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003481 PROGRAM B STUDY OF HEPATIC BIOCHEMICAL AND ENZYMATIC SYSTEMS FOR THE IDENTIFICATION OF VINYL CHLORIDE AND OTHER CHEMICAL INJURY IN CANCER DEVELOPMENT: ANIMAL AND HUMAN STUDIES; Investigators - J.T. Du, C.H. Tamburro and M.T. Tseng ANIMAL STUDIES: Bl. Characterization Of Hepatic Enzyme Changes In Rats With Prolonged Vinyl Chloride Exposure: Decreased Glucose-6-Phosphatase Activity B2. Morphological Alterations In Livers Of Rats Exposed To Vinyl Chloride Background Vinyl chloride has been shown to induce an identical malignancy in both animals and man. Vinyl chloride-induced liver cancer in animals, therefore, provided an excellent model for the study of chemically-induced carcino genesis. The rat model provides the opportunity to further elucidate the mechanism and the pathogenesis of this particular chemical agent, whose major oxidation occurs in the hepatocytes (primary liver cells) while inducing a malignant transformation in an adjacent, different cell type (endothelial lining cell of the sinusoids). Vinyl chloride had been shown to cause angiosarcoma and primary hepatocellular carcinoma in laboratory animals (Maltoni, 1975, 1976), and angiosarcoma in man (Creech and Johnson, 1974). However, little was known regarding the biochemical changes that vinyl chloride mediated to induce hepatocellular injury and subsequent malignant transformation of the mesenchymal cells. Enzymatic alterations in primary liver parenchymal cell cancers (hepato cellular carcinoma, hepatoma) have been studied extensively. Weber and Convery (1966) and Weber and Lea (1967) proposed that there was a biochemical pattern which could be related to the biological behavior of neoplastic cells. Using a spectrum of hepatomas with different growth rates, a basis for underlying neoplastic cell transformation was proposed as being related to the cell's progressive alterations in the molecular pattern which led to progressive increases in the growth rate of the tumors. Figure 1 outlines the pathways. C cifT'ff T DSJ T X AIj .onriSTectivo 0rd4iB7 7. Conoco. court icasieu Parish., Louis, 34 CMA 003482 FIGURE 1 WEBEHfS MOLECULAR CORRELATION CONCEPT OF NEOPLASIA OLUCONCOOINISIS Key enzymes of glycolysis were shown to increase while key enzymes in gluconeogenesis decreased with tumor growth. In addition, the pentose phosphate biosynthetic pathway, glucose-6-phosphate dehydrogenase (Weber and Morris, 1963) and transaldolase (Heinrich et al., 1974) were shown to be increased in all hepatoma studies. Furthermore, the activity of phosphoribo- sylpyrophosphate (PRPP) synthetase and glutamine PRPP aminotransferase, the first two enzymes channeling ribose-5-phosphate into purine, DNA and RNA synthesis were also increased. The PRPP sythetase was increased in rapidly growing hepatomas and the transferase in all the hepatomas irrespective of growth rates (Weber et al., 1975). Decreased enzyme activity was observed at an early stage of carcinogenesis before the morphological signs of a tumor appeared in the liver, and the activity of enzymes dependent upon the rate of hepatoma proliferation correlated with progressive malignant transformation. These findings and the lack of any long-term sequential studies of animals exposed to vinyl chloride directed us to our first three objectives. Objectives 1. To determine the sequential hepatic enzymatic changes in animals chronically exposed to vinyl chloride and their correlation to con ventional clinical tests of liver function. C0N7IB5TITI AL Subject to Protective Order in Ross v. Conoco, Inc., No, 90-4837 14th Judicial District Court C*lc0-slau Rar-f <=h. CMA 003483 FIGURE 1 W3ER'S MOLECULAR CORRELATION CONCEPT OF NEOPLASIA 4------------------------------------------------------- aiucofftooiNtsi) Key enzymes of glycolysis were shown to increase while key enzymes in gluconeogenesis decreased with turner growth. In addition, the pentose phosphate biosynthetic pathway, glucose-6-phosphate dehydrogenase (Weber and Morris, 1963) and transaldolase (Heinrich et al., 1974) were shown to be increased in all hepatoma studies. Furthermore, the activity of phosphoribosylpyrophosphate (PRPP) synthetase and glutamine PRPP aminotransferase, the first two enzymes channeling ribose-5-phosphate into purine, DNA and RNA synthesis were also increased. The PRPP sythetase was increased in rapidly growing hepatomas and the transferase in all the hepatomas irrespective of growth rates (Weber et al.t 1975), Decreased enzyme activity was observed at an early stage of carcinogenesis before the morphological signs of a tumor appeared in the liver, and the activity of enzymes dependent upon the rate of hepatoma proliferation correlated with progressive malignant transformation. These findings and the lack of any long-term sequential studies of animals exposed to vinyl chloride directed us to our first three objectives. Objectives 1. To determine the sequential hepatic enzymatic changes in animals chronically exposed to vinyl chloride and their correlation to con ventional clinical tests of liver function. nOIIFlDaNTIAL Subjectlo ?rotectiveTorder in 3ss v. Conoco, Inc .Jo. 90 "Uth Judicial District Court Rdirssieu CMA 003484 36 2. To determine if these sequential enzymatic patterns, especially with regard to carbohydrate metabolism and nucleic acid synthesis, would correlate with vinyl chloride-induced liver injury and ultimate malignant transformation. 3. To correlate the sequential, morphological changes with these en zymatic findings. Research Results 1. Characterization of Hepatic Enzyme Changes Sprague-Dawley adult male rats were chronically exposed to vinyl chloride. The initial studies characterized the metabolic alteration in the liver by studying various organelle enzyme markers which included glucose-6phosphatase (microsomal), cytochrome oxidase (mitochondrial) and glucose-ephosphatase dehydrogenase (cytosol). In addition, standard serum biochemical clinical liver studies of the liver (liver "function" tests) were performed including aspartate aminotransferase (SGOT), alanine aminotransferase (SGPT), alkaline phosphatase, lactic acid dehydrogenase, total protein, and bili rubin. Additional studies of subcellular enzymes related to nucleic acid synthesis--glutamine PRPP aminotransferase, and glycolysis--phosphofructokinase were done. _l vivo protein synthesis was assessed by ^H-leucine incorporation. The results of these studies illustrated that rats exposed to high chronic doses of vinyl chloride had a 25% lower activity of glucose-6-phosphatase, a key enzyme for gluconeogenesis after about 71 hours of exposure and a 50-100% increase in glucose-6-phosphate dehydrogenase, a rate limiting enzyme in the pentose pathway, after 84 hours of exposure. These studies demonstrated that (a) metabolic alterations in the liver cell are induced by vinyl chloride exposure, (b) gluconeogenesis is altered as reflected by a lower glucose-6-phosphatase and a higher glucose-6-phosphatase dehydrogenase activity, and (c) that these changes occur in a fashion similar to the lower gluconeogenic and higher pentose shunt activity of hepatomas. Although this could result in an enhanced production of PRPP and ultimately more nucleic acid synthesis, our studies of PRPP did not document this as a subsequent finding. These enzymatic changes, although similar to the biochemical changes seen in rapidly growing primary hepatocellular carcinomas, most likely reflect liver cell adaptation to vinyl chloride exposure but do not appear to significantly increase DNA synthesis. In addition, these changes were sufficiently limited so as not to be reflected by circulating enzyme levels traditionally used for the detection of .clinical hepatocellular injury (Figure 2). CONFIDENTIAL Subject to Protective Order in Ross v. Conoco. Inc. No. SO-4337 14th Judicial District Cou-t T5a''-'t=Vl , 1 CMA 003485 CONVENTIONAL CLINICAL BIOCHEMICAL STUOIES IN RATS EXPOSED TO VINYL CHLORIDE SOFT IU/| n 7S CONTROL l VC TREATED T T TT i i* -t---- 1 SCOT tu/l 10M W/l TOTAL PROTEIN GM/dl FIGURE 2 14 hn 2*Iyi 42 In HOURS Of VINYL CHLORIDE EXPOSURE 7Uhi XU In 2. Morphological Alterations: Light and Electron Microscopic Assessment Sequential morphological assessment was performed in both of the chronic experiments utilizing light and electron microscopy. The enzymatic findings discussed in B1 and subsequently in B3 were associated with light microscopic findings which showed a marked increase in liver cell polyploidy, double nucleated cells and areas of focal hepato cellular hyperplasia. There was no evidence of hepatocellular injury, necrosis, or increased fibrosis. The morphological changes seen, however, reflect an increased liver cell regeneration which was not measurable by the techniques used to measure ONA synthesis in our experiments. The electron microscopic changes demonstrated a proliferation of the smooth endoplasmic reticulum without evidences of changes in the hepatocytic cell, cell membrane, nucleic acid or rough endoplasmic reticulum structures. n ONFIDENTIAL Subject Ross v. Protective Order in Inc- , ' 90-483? 14th Jutiioiul District Court Calcasieu Parish, Louisiana CMA 003486 CONVENTIONAL CLINICAL BIOCHEMICAL STUDIES IN RATS EXPOSED TO VINYL CHLORIDE CONTROL T i VC TREATED SGPT ITJ/I rI ------------ rI - IT ---- 1 SGOT [U'l jjj ns 71 l DH IU/1 N 1*4 T T * r i TOTAL PROTEIN GM/dl h VT ----------- 1________ MW. T i T i 4} Kn FIGURE 2 HOURS Of VINYL. CHLORIDE EXPOSURE TT L1 T I7 i 7 *T L 7 IN. MW, 2. Morphological Alterations: Light and Electron Microscopic Assessment Sequential morphological assessment was performed in both of the chronic experiments utilizing light and electron microscopy. The enzymatic findings discussed in B1 and subsequently in B3 were associated with light microscopic findings which showed a marked increase in liver cell polyploidy, double nucleated cells and areas of focal hepato cellular hyperplasia. There was no evidence of hepatocellular injury, necrosis, or increased fibrosis. The morphological changes seen, however, reflect an increased liver cell regeneration which .was not measurable by the techniques used to measure DNA synthesis in our experiments. The electron microscopic changes demonstrated a proliferation of the smooth endoplasmic reticulum without evidences of changes in the hepatocytic cell, cell membrane, nucleic acid or rough endoplasmic reticulum structures. CONFIDENTIAL Subject to Protective Order in Ra@3 v. Conoco, Inc. , Mo. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CtfA 00348-7 33 There was no evidence of increased collagen deposition in the space of Disse which has been reported by others during terminal or final stages of angio sarcoma development in animals. 83. Alterations in the Oxidizing and Detoxifying System of the Rat Liver After Prolonged Exposure to Vinyl Chloride. Background Vinyl chloride metabolism had been shown to be blocked at low dosage levels (50 ppm) by inhibiting the alcohol dehydrogenase system and at higher levels (200 ppm) by metabolism via the microsomal mixed function oxidase (MFO) system (Hefner et al., 1975, Bolt et al., 1975). Vinyl chloride's major metabolites, chlorooxirane and chloroacetaldehyde, have been identified as probable ultimate carcinogens (Barbin et al, 1975; Van Duuren, 1975; and Jaeger et al., 1974). Further studies (Elmore et al., 1976; Malaveille et al., 1975; McCann et al., 1975) demonstrated the mutagenic activity of chloro oxirane and chloroacetaldehyde on microorganisms and the nonmutagenicity of chloroethanol and chloroacetic acid, other metabolites of vinyl chloride. Detoxification of these metabolites was reflected by a reduction in non protein sulfhydryl content of the liver (Hefner, 1975) and tracer studies showing major urinary metabolites of vinyl chloride to be N-acetyl-S-hydroxyethylcysteine and thiodiglycolic acid (Watanabe et al., 1976). The proposed metabolic fate of vinyl chloride is illustrated: Cl CH - CHj (v c) 4 LIVER MFO Cl CH-CH, \/ 0 (chlorooxirane) DETOXIFICATION WITH GLUTATHIONE Cl CH2CH20H +GSH GS CHjCHjOH (chloroethanol) ^^ +GSH ci ch2cho gs2ch2cho (chloroacetaldehyde) 4 Cl CHjCOOH N-Ac-S-(2-HYDROXY ETHYL) CYSTEINE GS CHjCOOH 4 THIODIGLYCOLIC ACID (chloroacetic acid) In these experiments, the enzymes studied related to vinyl chloride oxidation, included P-450, NADPH-cytochrome-C-reductase and mixed function oxidase. Those enzymes related to detoxification include non-protein sulfhydryl content (NPSC), glutathione content (GSH), glutathione reductase (GR), glutathione-epoxide-S-transferase (GEST, glutathione-E-transferase), and glutathione-aralkyl-S-transferase (GAST, glutathione A&B transferases). COITFIDentiAI) Subject to Protective Ordr Ross. V. .Conoco, Inc.. Ho. 90-4837 14th Judicial District Court alcasxsu Parish, Louisiana CMA 003488 39 Objective 1. To determine the sequential changes of key hepatic oxidizing and detoxifying enzymes with prolonged vinyl chloride exposure. Research Results The oxidizing and detoxifying capability of rat liver cells during prolonged exposure to chronic high doses of vinyl chloride demonstrated a 25-50* higher activity of glutathione reductase, the enzyme which regenerates reduced glutathione to detoxify the metabolites of vinyl chloride. Although our acute and single vinyl chloride exposure studies demonstrated a decrease in the nonprotein sulfhydryl content (48%) similar to that reported by Hefner (1975), repeated or chronic exposure to higher levels showed a progressive increase which was statistically significant after 70 hours of exposure (Figure 3). Glutathione-epoxide-S-transferase (GEST) and glutathione-aralkyl-S-transferase (GAST), the enzymes capable of conjugating the toxic vinyl chloride metabolites, were significantly elevated following vinyl chloride exposure; glutathione reductase (GR), the enzyme to regenerate glutathione, was also elevated significantly following vinyl chloride exposure (Figure 4). O NORMAL a VC EXP. AIR EXP CONTROLS RANGE o a vc ex*. # ix? canraoLs UJ* CO <HO-- 4> o w& IP UccJ X CO o FIGURE 3 l4J`h -'i 0 FIGURE 4 i 2 (70) i___ - 4 (140) 6 (210) TIME IN WEEKS (HRS) Crdsr in . 90-4837 Conrt r.ouish,)nq CMA 003489 '0 Objective 1. To determine the sequential changes of key nepatic oxidizing and detoxifying enzymes with prolonged vinyl chloride exposure. Research Results The oxidizing and detoxifying capability of rat liver cells during prolonged exposure to chronic highdoses of vinyl chloride demonstrated a 25-504 higher activityof glutathione reductase, the enzyme which regenerates reduced glutathione to detoxify themetabolites of vinyl chloride. Although our acute and single vinyl chloride exposure studies demonstrated a decrease in the nonprotein sulfhydryl content (484) similar to that reported by Hefner (1975), repeated or chronic exposure to higher levels showed a progressive increase which was statistically significant after 70 hours of exposure (Figure 3). Glutathione-epoxide-S-transferase (GEST) and glutathione-aralkyl-S-transferase (GAST), the enzymes capable of conjugating the toxic vinyl chloride metabolites, were significantly elevated following vinyl chloride exposure; glutathione reductase (GR), the enzyme to regenerate glutathione, was 3lso elevated significantly following vinyl chloride exposure (Figure 4). o NORMAL A VC EAR AH EAR CONTROLS .RAN.CE. _ UP. io nor mal 4 vc air EAR. Civ'rVoU .Range Ui1 to < o CJ S. aCD uu <x a e -w. e E x to 3 o TIME IN WEEKS (HRS) FIGURE 3 CONFIDENTIAL FIGURE 4 Subject to Protective Order in Boss v. Conoco, Inc.. lio, 90-4837 14th Judicial District Court ^Icasisu Par-i-h, Louisiana CMA 003490 40 The oxidizing capability as reflected by cytochrome P-450, the major protein involved in vinyl chloride metabolism, however, was reduced signifi cantly only after prolonged vinyl chloride exposure (Figure 3), supporting reports in vivo (Reynolds et al., 1975) and in vitro (Guengerich and Strickland 19//; Ivanetich et al., 1977) that vinyl chloride metabolites destroyed P-450. No differences we^e found in the conventional clinical serum biochemical* tests or in other cellular enzyme studies (cytochrome-C oxidase, NADPH-cytochrome-C-reductase and mixed function oxidase). These changes seem to reflect early hepatic cell adaptation to chronic vinyl chloride exposure by increasing their capacity for detoxification and decreasing their capability to further metabolize vinyl chloride into its toxic intermediates--both provide a means of natural defense to a toxic substance. This entire detoxifying sequence is shown in Figure 5 as a composite of the detoxifying enzyme changes over a prolonged period of exposure. It is interesting to note that there is an earlier increase in the GEST trans ferase than the GAST transferases with prolonged exposure. This may reflect an earlier (in time) need for adaptation to excessive production of the epoxide and subsequently the accumulation of the aldehyde metabolite. Whether this reflects the maximum adaptability as indicated by a leveling off of enzyme after 200 hours, or whether these changes reflect an adaptation to CONFIDSffTIAL Subject to Protective Order in Soss_ v. _Conoco, Inc.,;ro. 90-4837 14th Judicial District Court Calcasieu Parish. Louis^a CMA 003491 prevent cellular injury as well as prevent primary hepatocellular malignant transformation would require even longer sequential studies for confirmation; these were beyond our time constraints. B4. Oxidizing and Detoxifying Ability of Liver Mesenchymal or Parenchymal Cells in the Metabolism of Xenobiotics. Background Even though the malignant transformation induced by vinyl chloride occurred in the mesenchymal cells of the liver (endothelial cells), all meta bolic studies up to this time had been directed at the liver as a whole or to the parenchymal cell--the hepatocyte. There had been no differentiation between the oxidizing capability of the hepatocytes that demonstrated injury and the endothelial cells which were malignantly transformed. Little was also known regarding the variable ability of these two cell types in the removal of the ultimate carcinogens or active metabolites. Therefore, our next set of studies was directed toward the ability of two different cell types to metabolize vinyl chloride. Objective 1. To study the comparative ability of the hepatocytes versus the mes chymal cells to metabolize vinyl chloride and determine the detoxi fying capability of the various liver cell types. Research Results Enzymatic studies of the detoxifying activities of the subcellular fraction of liver cells, both hepatocytic and mesenchymal included the study of GEST, GAST, GR, as well as -450 and mixed function oxidase capability of the various cell fractions. The effectiveness of the isolation of the hepatocytes and mesenchymal cells was confirmed by studying pyruvate kinase activity. Mesenchymal cells were shown to have about half the GEST activity, 1% of the GAST activity, less than half of the microsomal function oxidase activity, and 73% of the GR capability compared to the hepatocytes. The activity per million cells indicated that the nonhepatocytes had about 1/70 MFO activity, 1/65 GEST activity, 1/500 GAST activity, and 1/45 GR activity. These results demonstrate that the nonhepatocyte has significantly less capability in both the activation and detoxification of vinyl chloride and other xenobiotics. The data supports the hypothesis that the endothelial cells have a more limited capability to detoxify vinyl chloride metabolites and that this limited capability provides a greater possibility for DNA injury and subsequent malignant transformation. Again, unfortunately much longer go:t?xde3tial Subject to Protective Order in ftcss v- Cor.o:o, Inc., No. 90-4337 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003492 prevent ceiiuiar inj^y as wail as zr transformation *qu:c racj'.ra even long; these were beyona our time constraints. cri~,ary ^aoatoce11 u 1 ar malignant s e-tcen f a 1 studies for confirmation; B4. Oxidizing and Detoxifying Ability of Liver Mesenchymal or Parenchymal Cells in the Metabolism of Xenobiotics. Background Even though the malignant transformation induced by vinyl chloride occurred in the mesenchymal cells of the liver (endothelial cells), all meta bolic studies up to this time had been directed at the liver as a whole or to the parenchymal cell--the hepatocyte. There had been no differentiation between the oxidizing capability of the hepatocytes that demonstrated injury and the endothelial cells which were malignantly transformed. Little was also known regarding the variable ability of these two cell types in the removal of the ultimate carcinogens or active metabolites. Therefore, our next set of studies was directed toward the ability of two different cell types to metabolize vinyl chloride. Objective 1. To study the comparative ability of the hepatocytes versus the mesen chymal cells to metabolize vinyl chloride and determine the detoxi- fying capability of the various liver cell types. Research Results Enzymatic studies of the detoxifying activities of the subcellular fraction of liver cells, both hepatocytic and mesenchymal included the study of GEST, GAST, GR, as well as -450 and mixed function oxidase capability of the various cell fractions. The effectiveness of the isolation of the hepatocytes and mesenchymal cells was confirmed by studying pyruvate kinase activity. Mesenchymal cells were shown to have about half the GEST activity. It of the GAST activity, less than half of the microsomal function oxidase activity, and 731 of the GR capability compared to the hepatocytes. The activity per million cells indicated that the nonhepatocytes had about 1/70 MFO activity, 1/65 GEST activity, 1/500 GAST activity, and 1/45 GR activity. These results demonstrate that the nonhepatocyte has significantly less capability in both the activation and detoxification of vinyl chloride and other xenobiotics. The data supports the hypothesis that the, endothelial cells have a more limited capability to detoxify vinyl chloride metabolites and that this limited capability provides a greater possibility for DNA injury and subsequent malignant transformation. Again, unfortunately much longer COU?IDhHTIAL "object to protective Order in - v. Conoco, Inc. , Ho. 90-4337 Tudicial District Court *qu Parish, Louisiana cMA 003493 42 experimental studies would be needed to eventually validate this hypothesis (Du et a!., preprint attached in Appendix). Relevance to Industry (1) These chronic sequential studies are highly relevant to the chemical industry's environmental health problems. The sequential, biochemical changes that occur in the carbohydrate metabolism of the liver hepatocytes reflect adaptation of the liver cell to chemical exposure. This is further reflected by the histological changes which show polyploidy, double nucleated cells and areas of focal hyperplasia. As the clinical studies in this report will subsequently show, these changes are characteristic of chemical injury but do not necessarily reflect premalignancy. This would be consistent with the enzymatic studies failing to demonstrate any evidence of increased nucleic acid synthesis which is seen in the premalignant hepatoma studies of Weber and others. The important fact is that these changes cannot be detected by the standard federally-required biochemical studies, because these are directed solely at the hepatocyte. The hepatocyte may become injured but it does not usually malignantly transform. (2) The oxidative and detoxifying results of this study demonstrate that the hepatocyte progressively adapts its ability to remove and detoxify the active metabolites. This occurs without evidences of hepatocellular injury, at least up to what would be equivalent to 6-20% of the working years of a chemical worker. The studies also emphasize the fact that at very high exposures, the detoxifying system is adaptive and the oxidizing system's capacity is reduced, thereby providing a relative defense against continued formation of toxic metabolites. (3) The massive ability of the hepatocyte to oxidize as well as detoxify the active metabolites of vinyl chloride can well explain why there is no hepatocellular carcinoma development in humans with vinyl chloride exposure. A markedly reduced ability of the nonhepatocyte to detoxify the metabolites while still having the capacity to oxidize vinyl chloride into its active metabolites would support the higher probability of this cell becoming malignantly transformed. Since all clinical screening studies are directed toward the hepatocyte, these animal studies emphasize the insensitiveness of clinical biochemical studies to correctly identify early injury in the more susceptible cell, i.e., endothelial cell. More sensitive indicators of nonhepatocytic cell injury or dysfunction are needed for the detection of early chemical injury. These indicators must take into account the function of both types of liver cells. (4) The morphological findings described in the animal experiments con firm earlier observations regarding the earliest histological manifestations of vinyl chloride injury. They support our clinical observations that these lesions can be used to identify chemical injury. Program D, on the morphormetric and histological analysis details the clinical results found in humans. COffFIDZjTTIAL ^ Subject to Protective Order in ---j=L v- Conoco. Inc., fro. 90-4337 14th Judicial district Court 003494 HUMAN STUDIES: 85. Effectiveness of Indocyanine Green Clearance in the Detection of Liver Injury. Background The increasing concern over the potential health hazard of synthetic chemicals in the occupational environment has led the government to require, and many industries to establish, medical laboratory screening programs for their employees. The primary objectives of these screening programs is to identify potential work-related disease, so that intervention may reduce disability, morbidity, and mortality. Most screening studies are directed toward the detection of abnormalities in certain body systems. The specific tests used are most often selected on the basis of medical experience in symp tomatic or hospitalized populations. Prior experiences utilizing non specific, multi-phasic health screening have not proven to be effective in this area. The effectiveness of standard medical testing used predominately in asymptomatic individuals with subclinical low-grade disease--both work and non-work related--had not been determined. This study assessed the effec tiveness of using clearance studies of anionic dyes in the detection of liver injury in such individuals. Objective 1. To determine the effectiveness of ICG clearances in detecting hepatic injury. 2. To compare ICG sensitivity and specificity to standard clinical biochemical screening in the detection of liver injury. Research Results The study group (cohort) consisted of 969 male employees who were tested between June 1, 1976 and May 31, 1977 with standard biochemical studies of the liver which included alanine aminotransferase (ALT/SGPT), aspartic aminotrans ferase (AST/SGOT), gamma glutamyl transpeptidase (GGTP), alkaline phosphatase (AP), and total bilirubin (TB). In addition, these employees were also screened by ICG clearances at the 0.5, 2.5, and 5.0 mg/kg doses, and by two more specific liver enzyme tests (isocitric dehydrogenase [ICD] and sorbitol dehydrogenase [SDH]). This main cohort was divided into a "standard" and a "non-standard" population for purposes of analysis. The division was based upon the best medical opinion. The "standard" group demonstrated no clinical evidence of significant medical disease--occupational or non-occupational in origin, and "non-standard" included all the others. A subcohort of 120 individuals who had undergone liver biopsies for medical reasons, whether related to work or not, were also studied. This subcohort population was divided into those individuals with and without CONFIDENTIAL Subject tc ?rc :ective Order in Koss v. Conoco. 90-4837 14th Judicial District Court Calcasieu Pa: ish, Louisiana CMA. 003495 STUDIES:LM * J * - L-' . 35. Effectiveness of Indocyanine Green Clearance in the Detection of Liver Injury. Background The increasing concern over the potential health hazard of synthetic chemicals in the occupational environment has led the government to require, and many industries to establish, medical laboratory screening programs for their employees. The primary objectives of these screening programs is to identify potential work-related disease, so that intervention may reduce disability, morbidity, and mortality. Most screening studies are directed toward the detection of abnormalities in certain body systems. The specific tests used are most often selected on the basis of medical experience in symotomatic or hospitalized populations. Prior experiences utilizing non specific, multi-phasic health screening have not proven to be effective in this area. The effectiveness of standard medical testing used predominately in asymptomatic individuals with subclinical low-grade disea$e--both work and non-work related--had not been determined. This study assessed the effec tiveness of using clearance studies of anionic dyes in the detection of liver injury in such individuals. Objective 1. To determine the effectiveness of ICG clearances in detecting hepatic injury. 2. To compare ICG sensitivity and specificity to standard clinical biochemical screening in the detection of liver injury. Research Results The study group (cohort) consisted of 969 male employees who were tested between June 1, 1976 and May 31, 1977 with standard biochemical studies of the liver which included alanine aminotransferase (ALT/SGPT), aspartic aminotrans ferase (AST/SGOT), gamma glutamyl transpeptidase (GGTP), alkaline phosphatase (AP), and total bilirubin (TB). In addition, these employees were also screened by ICG clearances at the 0.5, 2.5, and 5.0 mg/kg doses, and by two more specific liver enzyme tests (isocitric dehydrogenase [ICO] and sorbitol dehydrogenase [SDH]). This main cohort was divided into a "standard" and a "non-standard" population for purposes of analysis. The division was based upon the best medical opinion. The "standard" group demonstrated no clinical evidence of significant medical disease--occupational or non-occupational in origin, and "non-standard" included all the others. A subcohort of 120 individuals who had undergone liver biopsies for medical reasons, whether related to work or not, were also studied. This subcohort population was divided into those individuals with and without CONFIDENTIAL Subject to Protective Order in Ross v. Conoco. Inc. , No. 90-4837 14th Judicial District Court 11"'1 si u Perish, Xouisi aua. 003496 CMA 44 histological evidence of liver injury, and the former further subdivided into those with and without histological characteristics of chemical injury (see Program D2). All employees had individual work histories and rank-ordered exposure indices for 22 different chemicals used within the work place. The GGTP provided the highest positive predictive value as a screening test for non-standard individuals (population with medical disorder). It also provided the highest sensitivity and specificity sum among the 5 most frequently abnormal tests (AST/SGOT, ALT/SGPT, AP, ICG); 2 other tests also had high predictive values--the indirect bilirubin, due to the high number of congenital indirect hyperbilirubinemic individuals, and the triglyceride levels, most likely reflecting differences in age, weight, and diabetic status in the non-standard population. The ICG clearance (0.5 mg/kg) espressed as mean t 1/2 in minutes (min.) demonstrated differences not only between standard and non-standard population but also differences between those with high and low mean exposure to vinyl chloride (Table 1). TABLE 1 INDOCYANINE CLEARANCE IN A COHORT INDUSTRIAL POPULATION MEAN SUBGROUP NO, MIN. 90Z loot STANDARD 662 2,9 2,0-3.8 1.8-4.8 LOW VC EXPOSURE 453 2.9 1.8-4.0 1.7-4.3 NON-STANDARD HIGH VC EXPOSURE 257 466 3.4 2.1-5,6 2.0-6,6 3.1 2.1-4.4 1.9-4.8 The biochemical tests which best correlated with the presence of hepatic disease in the chemical workers with clinical evidence of liver disease were ALT (SGPT), GGTP, alkaline phosphatase, and ICG clearance (i.e. these were the tests with the highest sensitivity in detecting latent liver disease). Sensi tivity alone, however, is not an adequate indicator of a test's screening value, especially when used in asymptomatic individuals. More appropriate evaluation of the screening ability of the tests requires that both sensi tivity and specificity be determined, i.e., identify the diseased individuals and correctly exclude the non-diseased individuals. The sensitivity and C0NFID5ITTIAL Subject to Protective Order in Boss v. Conoco , Inc., Bo. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana 003497 45 specificity of the 4 most sensitive biochemical screening tests and the ICG clearances (all doses) demonstrated that GGTP although the most sensitive is the least specific; AP has the highest specificity but the lowest sensitivi ty. ICG clearances, even at the low dose, clearly remain the best test, i.e., in combined sensitivity and specificity for screening and detecting individu als with subclinical liver disease (Table 2 a,b,c). TABLE 2 A Bc SENSITIVITY AND SPECIFICITY OF INDOCYANINE GREEN CLEARANCE SENSITIVITY AND SPECIFICITY OF INDOCYANINE GREEN CLEARANCE NO. PATIENTS 1500 ICG 3.5 hg/ks ICG 0,5 Jest Medical Assessment <+) (*) 223 (-) 38 (-) m 1923 total 516 2011 2939 --<*) Best (+) Medical Assessment (-) 95 19 (-) 28 396 SENSITIVITY: specificity: 55.31 95.51 sensitivitt: specificity: 45/73 61,SI 396/410 - 96.51 Total --1-- 73 910 983 SENSITIVITY AND SPECIFICITY OF INDOCYANINE SPEEM CLEARANCE ICG 5.0 j/<a aT ill C+> 78 H 5 Nedical Assessment (-} j_ % Sensitivitt: Specificity: 78/89 - 92,91 38/42 - 90.51 In SR rj 125 In addition ICG clearance demonstrates excellent correlation with the hist logical presence of liver disease and liver cancer (Figure 6). LIVER HISTOLOGY ANO DYE CLEARANCE AMONG VINYL CHLORIDE (VC) WORKERS FIGURE 6 aveaaoe VC EXPOMN mocx The subcohort biopsied population was additionally studied with regard to the sensitivity and specificity of the screening tests, the specific histo logical interpretation of the liver biopsy, and the relationship to vinyl chloride exposure. All biopsied individuals were subclassified into three groups: those with histological evidence consistent with chemical liver T\Ci *7 r T * * *-TT ITA.J4 subject to Protective Order in --ss- v .Conoco, Inc,. No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003498 45 of tins 4 most sensitive biochemical screening tests and zre l'A clearances (ail Gases} demonstrated that GGTP althougn the most sensitive is era 'east specific; AF has the highest specificity but the lowest sensitivi ty. ICG clearances, even at the low dose, clearly remain the best test, i.e., in comoined sensitivity and specificity for screening and detecting individu als with subclinical liver disease (Table 2 a,b,c). TABLE 2 A BC sensitivity wo specificity OF WBOCYMUNE 3SEEY CLEARANCE SEOIT1VITY WO SPECIFICITY OF i.NTOCYAfll.NE sasEM clearance mo, at: = HTJ i~i ICS 0.5 O/tj T <-> C-) "3 140 Asssss^OiT 1:-) S3 1923 TOTAL 515 :ou Z4J9 ilj Z.5 -o/G () (-) 3it (<) 45 "nicn. AllfSiMtHT (-) 14 23 595 IfWlTlVItf! 55.31 iFeciFieiTY: 95.61 itmtTtvrrr: 45/73 - SI.SI J96/10 * 96.51 Total " 73 410 463 SENSITIVITY WD spec: OF INDOCYWiNE 3FEEN :c: 5.o -c/<g a`ST .Toicju. iri (*> ;s 111 s Ajiswcat (.) tj Smiitivjty: 73/34 - 92.31 Smcimcitv: 33/42 90.31 TY LICE 34 32 125 In addition ICG clearance demonstrates excellent correlation with the histo logical presence of liver disease and liver cancer (Figure 6). LIVER HISTOLOGY AND DYE CLEARANCE AMONG VINYL CHLORICE (VC) WORKERS FIGURE 6 The subcohort biopsled population was additionally studied with regard to the sensitivity and specificity of the screening tests, the specific histo logical interpretation of the liver biopsy, and the relationship to vinyl chloride exposure. All biopsied individuals were subclassified into three groups: those with histological evidence consistent with chemical liver C OITFIDSNTIAL Subject to Protective Order in Ross v. Conoco, Inc . , No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003499 46 injury, those with histological evidence of liver disease without evidence of chemical injury, and those with normal liver biopsies. Each group was then evaluated regarding the sensitivity and specificity of the biochemical tests and their total exposure to vinyl chloride based on a rank ordered scale. Analysis of the subcohort biopsied group's work histories demonstrated that those individuals with chemical liver injury had the highest average rating for vinyl chloride exposure and were best identified by screening with ICG clearance tests. B6. The Assessment of Bile Acids Vs. Indocyanine Green (ICG) Clearances in the Detection of Liver Injury in a Chemically-Exposed Worker Population Background Since highly sensitive and specific screening tests for the detection of latent hepatic injury were needed, studies were conducted to determine whether bile acid clearances could be utilized to detect occupationally-related hepatic injury. Bile acids are naturally occurring steroids which are synthesized and removed solely by the liver. These substances, in contrast to synthetic anionic dyes such as indocyanine green, have been reported to provide equal if not better, sensitivity in the detection of liver disease. The development of radioimmunoassays for bile acids provided an economical means of determining whether these natural substances could be used as effective screening measures in subclinical liver disease. Studies were conducted to determine the relative effectiveness of bile acid levels vs. ICG clearances in the detection of subclinical liver disease. Objective 1. To determine the effectiveness of bile acid clearance in the detection of subclinical liver injury 2. To compare bile acids to ICG clearance in identifying chemical and non-chemical liver injury. Research Results A subcohort (64) of the worker population who had liver biopsies done for medical reasons ana had both ICG clearances and serum bile acids--cholylglycine (CG) and conjugates of cholic acid (CCA)--performed, were studied. These individuals were subdivided on the basis of the histological features of their biopsies into individuals with chemical liver injury (CLI), individuals with non-chemical liver disease (NCLD), and individuals with no histological abnormalities (NBX). The mean plus or minus S.E.M. for ICG, CG and CCA in the CLI, NCLD, NBX, and a non-biopsied normal group are shown in Table 2. CONFIDENTIAL Fos1?'JvSCt7^^e^^~o?der in ' CMA 003 500 District Court ch. r.rm-f TABLE 2 CORRELATION OF LIVER BIOPSY WITH BILE ACID LEVELS AND ICG CLEARANCE ICG(^) tO.5 3ig/kg) CG (ug/dl) CCA (ug/dl) CL I 4.2 t 0.6 95.2 28,3 89.7 29.3 NCi.n 3.2 + 0.1 27.3 + 4.4 25.3 4.5 NBX 3.3 + 0,2 34,60+ 7.1 52.6 t 26.6 NON BIOPSIED NORflAL 3.1 t 0.01 14.9 t 0.9 18.7 i 1.2 Analysis of variance (on log transform data) showed significant differences for ICG clearance for the three biopsied groups and for all four groups. Values for the CLD were significantly different from the ICGs for the normal, but showed no significant difference between the NCLD and the NBX group. Analysis of variance for ICG clearance was not as discriminating for the 3 biopsied groups. ICG clearance gave a better separation (sensitiviti^k of the normal vs. abnormal at the 95 percentile level for the norm^P population in the serum bile acids. This preliminary analysis, therefore, shows that fasting serum bile acids hold promise as a potential detector of early liver dysfunction, particularly chemically-induced, in those individuals who are asymptomatic. This test should be further investigated to determine how this natural substance might be more effectively utilized as a screening technique. Bile acids can be taken orally for clearance study and are not limited to intravenous route as is the case for synthetic anionic dyes such as ICG, Studies in this area continued to be pursued in similar populations. Relevance to Industry These clinical study results provide the best scientific basis upon which to recommend effective medical screening tests for the detection of occupationally-related injury to the liver. The results also provide sound scientific reasons for recommending modification of presently required federal testing in environments utilizing potentially hepatotoxic agents. Although synthetic anionic dye presently provides the best means for detecting early latent liver injury, it is limited by the need to be injected (an invasive procedure). Although reactions to the injection of this dye have been minimal, it does present barriers to voluntary worker compliance. Therefore, development of a screening test which can effectively determine _C 0 " ? 15 5 t- I a T, Ho*s^V ?rot_e"tive Order in --No. 90-4837 l-h Julicinl District Court r-1, 'S^tl P- T'l 7r) OUT CMA 003S01 7 BILE ACID LEVELS AND ICG CLEARANCE ICGCtis) <0.5 Tsg/ks) CG tug/d1) CCA Cut/dl) CL I 4.2 0.5 95.2 28.3 89.7 1 29.3 3.2 + 0.1 27.3 + 4.4 25.3 1 4.5 3.3 0.2 34.60 7,1 52.5 i 26.6 NON BIOPSIED NGBCAL 3.1 0.01 14,9 l 0.9 13.7+1.2 Analysis of variance (on log transform data) showed significant differences for ICG clearance for the three biopsied groups and for all four groups. Values for the CLO were significantly different from the ICGs for the normal, but showed no significant difference between the NCL0 and the NBX group. Analysis of variance for ICG clearance was not as discriminating for the 3 biopsied groups. ICG clearance gave a better separation (sensitivity) of the normal vs. abnormal at the 95 percentile level for the normal population in the serum bile acids. This preliminary analysis, therefore, shows that fasting serum bile acids hold promise as a potential detector of early liver dysfunction, particularly chemically-induced, in those individuals who are asymptomatic. This test should be further investigated to determine how this natural substance might be more effectively utilized as a screening technique. Bile acids can be taken orally for clearance study and are not limited to intravenous route as is the case for synthetic anionic dyes such as ICG. Studies in this area continued to be pursued in similar populations. Relevance to Industry These clinical study results provide the best scientific basis upon which to recommend effective medical screening tests for the detection of occupationally-related injury to the liver. The results also provide sound scientific reasons for recomnending modification of presently required federal testing in environments utilizing potentially hepatotoxic agents. Although synthetic anionic dye presently provides the best means for detecting early latent liver injury, it is limited by the need to be injected (an invasive procedure). Although reactions to the injection of this dye have been minimal, it does present barriers to voluntary worker compliance. Therefore, development of a screening test which can effectively determine COH?IDeatTIATi Subject to Protective Order in ----v' _Cp^Q"Q, Inc. , No. 90-4837 14th Judicial District Court CMA 003502 overall hepatic function, can be given orally, and is a nonsynthetic, normal biological constituent approaches the most ideal test for screening. Bile acid clearance could be the least invasive and therefore the most acceptable method to test asymptomatic individuals undergoing screening. The bile acid studies described above are the basis for recommending its use in future screening. The standardization of these bile acid test techniques, however, requires further careful validation so that specific recommendations regarding their use will provide the most accurate delineation between those with disease and without disease and those with occupationally-related disorders versus naturally-occurring ones. References Barbin, A., Bresh, H., Croisy, A., Jacquignon, P., Malaveille, C., Malaveille, C., Montesano, R., and Bartsch, H. (1975). Livermicrosome-mediated formation of alkylating agents from vinyl bromide and vinyl chloride. Biochem. Biophys. Res. Commun. 67, 596-603. Bolt, H.M., Kappus, H., Butcher, A., and Bolt, W. (1975). Metabolism of vinyl chloride. Lancet 1, 1425. Creech, J.L., and Johnson, M.N. (1974). Angiosarcoma of liver in the manufacture of polyvinyl chloride. ^3. Occup. Med. 16, 150-151. Creech, J.L., Makk, L., Whelan, J.G., Jr., and Tamburro, C.H. Hepatotoxicity among polyvinyl chloride production workers during first year of surveillance program. Gastroenterology 67, 786 (1974). Du, J.T., ana Tamburro, C.H. (1976). Decreased glucose-6-phosphatase activity in liver in vinyl chloride exposed rats. Fed. Proc. 35, 329. Du, J.T., ana Tamburro,C.H. (1978). 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C0NPIS5!TTIAL Subject to Protective Order in ff.?5S. v cr.occ , Inc. , No. 90- 4837 14th Judicial strict Court Calcasieu Louisiana CMA 003503 49, Greenberg, R.A., and Tamburro, C.H. (1981). Exposure indices for epi demiological surveillance of carcinogenic agents in an industrial chemical environment. Qccup. Med., 23. 353-358. Greim, H., Bonse, G., Radwan, Z., Reichert, D., and Henschler, 0. (1975). Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmacol. 24, 2013-2017. Guengerich, F.P. and Strickland, T.W. (1977). Metabolism of vinyl chloride: Destruction of the heme of highly purified liver microsomal cytochrome P-450 by a metabolite. Mol. Pharmacol. 13, 993-1004. ----- ---------------- -- Hefner, R.E., Watanabe, P.G., and Gehring, P.J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer in rats. Ann. N.Y. Acad. Sci. 246, 135-148. Heinrich, P.C., Morris, H.P., and Weber, G. (1974). Increased phosphoribosylpyrophosphate synthetase activity in rapidly growing hepatomas. FEBS Lett. 42, 145-148. Ivanetich, K.M., Aronson, I., and Katz, I.D. (1977). The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 in vitro. Biochem. Biophys. Res. Commun. 74, 1411-1418. Jaeger, R.J., Connolly, R.B., and Murphy, S.D. (1974a). Effect of 18 hr. fast and glutathione depletion on 1, 1-dichloroethylene-induced hepatotoxicity and lethality in rats. xjj. Mol. Pathol. 20, 187-198. Jaeger, R.J., Reynolds, E.S., Connolly, R.B., Moslen, M.T., Szabo, S., and Murphy, S.D. (1974b). Acute hepatic injury by vinyl chloride in rats pretreated with phenobarbital. Nature (London) 252, 724-726. Kappus, H., Bolt, H.M., Buchter, A., and Bolt, W. (1976). Liver microsomal uptake of ^C-vinyl chloride and transformation to protein alkylating metabolites in vitro. ToxicoL. Appl. Pharmacol. 37, .461-471. Malaveille, C., Bartsch, H., Barbin, A. Camus, A.M., and Montesano, R. (1975). Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol. Biochem. Biophys. Res. Commun. 63, 363-370. Maltoni, C., Lefemine, G., Chieco, P. and Carretti, D. (1974). Vinyl chloride carcinogenesis: current results and prospective. Medicine Del. Lavoro. 65, 421-444. Maltoni, C., and Lefemine, C. (1975). Carcinogenicity bioassays of vinyl chloride: Current results. Ann. N.Y. Acad. Sci. 246, 195-218. CONFIDENTIAL Subject to Protective Order in P.055 v. C:r.coo. Inc. , No. 90-4837 14th T^Iicial District Court Calcasieu Parish, Louisiana CMA 003504 -9 Greenberg, R.A., end Tamour-o, C.H. (1531). Exposure iodices for epi demiological surveillance of carcinogenic agents in an industrial chemical environment. J_, Cccup. Med., 33. 353-353. Greim, H., Bonse, G., Radwan, Z., Reichert, 0., and Henschler, 0. (1975). Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmacol. 24, 2013-2017. Guengerich, F.P. and Strickland, T.W. (1977). Metabolism of vinyl chloride: Destruction of the heme of highly purified liver microsomal cytochrome P-450 by a metabolite. Mol. Pharmacol. 13, 993-1004. -- Hefner, R.E., Watanabe, P.G., and Gehring, P.d. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer in rats. Ann. N.Y. Acad. Sci. 246, 135-148. Heinrich, P.C., Morris, H.P., and Weber, G. (1974). Increased phosphoribosylpyrophosphate synthetase activity in rapidly growing hepatomas. FE3S Lett. 42, 145-148. Ivanetich, K.M., Aronson, I., and Katz, I.D. (1977). The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 in vitro. Biochem. Biophys. Res. Commun. 74, 1411-1418. Jaeger, R.J., Connolly, R.B., and Murphy, S.D. (1974a). Effect of 18 hr. fast and glutathione depletion on 1, 1-dichloroethylene-induced hepatotoxicity and lethality in rats. Exp. Mol. Pathol. 20, 187-198. Jaeger, R.J., Reynolds, E.S., Connolly, R.B., Moslen, M.T., Szabo, S., and Murphy, S.D. (1974b). Acute hepatic injury by vinyl chloride in rats pretreated with phenobarbital. Nature (London) 252, 724-726. Kappus, H., Bolt, H.M., Buchter, A., and Bolt, W. (1976). Liver microsomal uptake of ^C-vinyl chloride and transformation to protein alkylating metabolites in vitro. ToxicoL. Appl. Pharmacol. 37, 461-471. " Malaveille, C., Bartsch, H., Barbin, A, Camus, A.M., and Montesano, R. (1975). Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol. Biochem. Biophys. Res.Commun. 63, 363-370. Maltoni, C., Lefemine, G., Chieco, P. and Carretti, D. (1974). Vinyl chloride carcinogenesis: current results and prospective. Medicine Del. Lavoro. 65, 421-444. Maltoni, C., and Lefemine, C. (1975). Carcinogenicity bioassays of vinyl chloride: Current results. Ann. N.Y. Acad. Sci. 246, 195-218. CONJIDZNTIAL Subject to Protective Order in Ross v. Conoco, Inc,, No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003505 50 McCann, J., Simmon, V., Streitwieser, D., and Ames, B.N. (1975). Muta genicity of chloroacetaldehyde, a possible metabolic product of 1,2dichloroethane, chloroethanol, vinyl chloride and cyclophosphamide. Proc. Nat. Acad. Sci. USA 72, 3190-3193. Reynolds, E.S., Moslen, M.T., Szabo, S., and Jaeger, R.J. (1975). Vinyl chloride-induced deactivation of cytochrome P-450 and other components of the liver mixed function oxidase system: An in vivo study. Res. Commun. Chem. Pathol. Pharmacol. 12, 685-694. Tamburro, C.H., Makk, L., and Popper, H. (1979). Early hepatic histological alterations among chemical (vinyl monomer) workers. Gastroenterology 77, A33. VanDuuren, B.L. (1975). Possible mechanism of carcinogenic action of vinyl chloride. Ann. N.Y. Acad. Sci. 246, 258-267. Watanabe, P.G., Hefner, R.E., Jr., and Gehring, P.J. (1976a). Vinyl chloride-induced depression of hepatic non-protein sulfhydryl content and effects of bromosulphalein (BSP) clearance in rats. Toxicol. 6, 1-8. ----------------- " Watanabe, P.G., McGowan, G.R., and Gehring, P.J. (1976b). Fate of 14C-vinyl chloride after single oral administration in rats, Toxicol. App1. Pharmacol. 36, 339-352, Watanabe, P.G., Zempel, J.A., Pegg, D.G., and Gehring, P.J. (1978). Hepatic macromolecular binding following exposure to vinyl chloride. Toxicol. Appl. Pharmacol. 44, 571-579. Weber, G. (1974). The molecular correlation concept. J_n The Molecular Biology of Cancer, ed. H. Busch, pp. 487-521. New York:Academic. Weber, G. and Convery, H.J.H. (1966). Insulin: Inducer of glucose-ephosphate dehydrogenase. Life Sci. Si, 1139-1146. Weber, G. and Lea, M.A. (1967). The molecular correlation concept. In Methods in Cancer Research, ed. H. Busch, vol. 2, pp. 523-578. New York:Academic. CONFIDENTIAL Subject to Protective Order in Ross v. Conoco , Inc. , Ifo . 90 - 4837 14th Judicial district Court C^l na s 1 ftii T *vii CMA 003506 : CONFIDENTIAL . Subject to Protective Order in Boss v- Conoco, Inc., No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003507 PROGRAM C STUDY OF GLYCOSAMINOGlYCAN CHANGES IN THE DETECTION OF HEPATIC FIBROTIC INJURY IN CHEMICAL EXPOSURE AND HEPATIC CANCER DEVELOPMENT; Investigators - C.E. Kupchella and R. Warick ANIMAL STUDIES: Cl. The Study of Glycosaminoglycan Changes in Livers of Animals Bearing Metastasizing Hepatomas C2. Significance of Tissue Glycosaminoglycan Elevations in Hepatic Fibrosis, Necrosis, and Regeneration HUMAN STUDIES: C3. Characterization of Glycosaminoglycan Patterns in Humans with Angiosarcoma C5. Characterization of Glycosaminoglycan Patterns in the Identification of Chemical and Nonchemical Injury of the Liver C4. Evaluation of Glycosaminoglycan Analysis of Urines of Chemically- Exposed Workers Background It is well established that glycosaminoglycans (GAGs) are involved in wound healing and scar formation. Although it is less certain what role they play, it is known that certain GAGs are elevated in malignant tumors including hepatic tumors, and it has been postulated that GAGs may be impor- tant determinants of tumor-cell properties. A number of laboratories, including ours, have established that urinary GAG excretions may serve as markers in the pathogenesis of chemical injury, fibrosis, and cancer. Despite the fact that urinary GAG analyses have long been used clinically to detect and diagnose genetically-determined metabolic disorders of GAG metabolism, a ive Order in --No 90-4837 al District Court Parish, Louisiana 51 CMA 003508  systematic evaluation of the usefulness of urinary GAG patterns in the detec tion and diagnosis of acute and/or chronic, necrotic and/or fibrotic liver injury--or cancer--has never been made. Studies were begun to determine the practical utility of tissue and urinary GAG analysis in the detection and diagnosis of chemically-induced liver injury and cancers. Objective In Animals: 1. To determine the relationship between GAG patterns in tumor tissue (and urine) in animals with fast versus slow growing and in metasta sizing versus non-metastasizing, chemically-induced, transplantable, hepatocellular tumors. (As an initial means of exploring the func tional role of GAGs known to be elevated in hepatic cancer). 2. To determine the sequence of GAG changes (in tissue, urine and blood) associated with the onset of experimentally-induced fibrotic injury with special attention to any change coinciding with the transition from reversible to irreversible fibrosis. 3. To determine the degree to which chemically-induced necrosis of the liver results in altered urinary GAG excretion and confirm changes seen in tissue in earlier studies. In Humans: 4. To compare the relative accuracy of the common methods of evaluating urinary GAGs and find the least expensive and least time-consuming method of urinary GAG analysis able to give good specificity and sen sitivity. 5. To repeat in a double-blind clinical trial the evaluation of the ability of urinary GAG analysis to correctly identify active liver disease. 6. To evaluate urinary GAG patterns in groups of alcoholics with active liver injury over a time period. 7. To compare liver tissue and urinary GAG patterns in patients (a) with alcohol-injured livers, (b) hepatic angiosarcoma, (c) primary hepatocellular tumors, (d) livers with metastatic cancer, and (e) chemically-induced hepatitis and cirrhosis. Research Results In Animals: Livers of animals bearing metastasizing hepatoma (5123tc) have 10-fold greater concentrations of a non-sulfated, neutral, uronic acid-positive ^j ./RoSsusbyje. c_tC__oto_n_o_cP__or_.o__te__c_t_9iv0e- Order 14'h Judicial District Cou: Calois-to,, to,,,-,),. CMA 003509 -J ~ -- v lal District Court Calcasieu Parish, Louisiana 52 s barnacle evaluation of the usefulness of urinary GAG patterns in the detec,on and diagnosis of acute and/or chronic, necrotic and/or fibrotic liver ijury--or cancer--has never been made. Studies were begun to determine the ractical utility of tissue and urinary GAG analysis in the detection and iagnosis of chemically-induced liver injury and cancers. Objective In Animals: 1. To determine the relationship between GAG patterns in tumor tissue (and urine) in animals with fast versus slow growing and in metasta sizing versus non-metastasizing, chemically-induced, transplantable, hepatocellular tumors. (As an initial means of exploring the func tional role of GAGs known to be elevated in hepatic cancer). 2. To determine the sequence of GAG changes (in tissue, urine and blood) associated with the onset of experimentally-induced fibrotic injury with special attention to any change coinciding with the transition from reversible to irreversible fibrosis. 3. To determine the degree to which chemically-induced necrosis of the liver results in altered urinary GAG excretion and confirm changes seen in tissue in earlier studies. In Humans: 4. To compare the relative accuracy of the common methods of evaluating urinary GAGs and find the least expensive and least time-consuming method of urinary GAG analysis able to give good specificity and sen sitivity. 5. To repeat in a double-blind clinical trial the evaluation of the ability of urinary GAG analysis to correctly identify active liver disease. 6. To evaluate urinary GAG patterns in groups of alcoholics with active liver injury over a time period. 7. To compare liver tissue and urinary GAG patterns in patients (a) with alcohol-injured livers, (b) hepatic angiosarcoma, (c) primary hepatocellular tumors, (d) livers with metastatic cancer, and (e) chemically-induced hepatitis and cirrhosis. Research Results In Animals: Livers of animals bearing metastasizing hepatoma (5123tc) have 10-fold greater concentrations of a non-sulfated, neutral, uronic acid-positive CMA 003510 53 material than is found in the livers of animals bearing two others, non-metastasizing hepatomas (4). Heparan sulfate and hyaluronic acid levels--but not heparin--are 3-4 times higher in experimentally transplanted hepatomas than in normal liver and urinary excretion reflects both the tumor GAG composition and the size of tumors (4, 8). Hepatic necrosis is accompanied by significant tissue GAG elevations but hepatic regeneration is not (5). Heparan sulfate (a type of GAG) is elevated in hepatic tissue undergoing experimentally induced fibrosis and heparan sulfate is elevated in the urine of experimental animals (3). In Humans: Human hepatic angiosarcoma and fibrotic liver disease are accompanied by elevated tissue GAGs (1). The GAGs in the angiosarcomatous tumor tissue are different from those in fibrotic tissue adjacent to the tumor (2). Angiosarcoma and hepatoma patients have characteristic urinary GAG pat terns--patterns not found in normal controls (2). Gross (non-fractionated) urinary GAG determinations give a better indica tion of liver disease than ultrasound analysis. Exacting urinary GAG analysis (fractionated) is able to differentiated active from inactive liver disease (7). Table 1 illustrates this for chrondrotin sulfate fraction. TABLE 1 PRELIMINARY EVALUATION CF THE USE OF AH EXCLUSIVELY CHONDROITIN SULFATE EXCRETION PATTERN AS A SCREENING TEST FOR VINYL CHLORIDE - EXPOSURE - ASSOCIATED LIVER INJURY * TEST* RESULT VINYl. CHLORIDE EXPOSURE PLUS NON-AHOIOSARCOHA LIVER INJURY + 73 - 2 29 Si* 9 32 sensitivity 77,71 (951 LlHtTS 40.01 * 97.2TEJ SPECtRietTY * 90.61 (951 LintTS 751 - 981) *UR1NE FRACTIONATION IN NHICH THE CNONOROITIN SULFATE FRACTION IS POSITIVE *UT THE HYALURONIC ACIO AND MePARIN FRACTIONS ARE BOTH NEGATIVE. COIIFIDTTTTIAL Subject" to Protective Order in Ross v. Conoco, Inc.. Ho 90-48c7 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003 511 54 Relevance to Industry These studies address the need for useful screening tests for liver injury--urine tests of the type evaluated obviously fit the ideal of being non-invasive, have no associated morbidity/mortality, and do not require "time oTT" from work to perform. They could provide useful screening tests for early injury and for dif ferentiating active versus inactive disease. GAG analysis may help assess methods of therapeutic intervention, i.e., the elucidation of the role of the GAGs in the pathogenesis of fibrotic liver disease and the identification of strategies by which fibrogenesis can be blocked and/or reversed. Finally, they could provide useful means of identifying individuals at risk of chemical injury--by further characterization of GAG changes, one may be able to identify specifc injury due to chronic alcohol, chemical or other exposure agents in individuals with liver impairment. References 1. Kupchella, C. D., and Tamburro, C. H., (1978). Urinary and Tissue Glycosaminoglycans patterns in angiosarcoma and other vinyl chloride exposure associated liver injury in: Detection aM Prevention of Cancer. H.E. Neiburgs, Ed., Part 1, Vol. 1, MariiB Dekker, Inc., New York. 2. Curran, K. L., Kupchella, C. E., and Tamburro, C.H., (1977). Urinary Glycosaminoglycan Patterns in Angiosarcoma of the Liver. Cancer 40:3050-3053. 3. Kupchella, C. E., Jarvis, J. 0., Curran, K. L. and Tamburro, C. H., (1977). Tissue and urinary glycosaminoglycans (GAG) changes in hepatic fibrosis. Presented at the meeting of the American Association of Study of Liver Disease. Chicago, Illinois, November 1, 1977. Gastroenterology 73(5):1229. 4. Kupchella, C. E., Drake, E., Curran, K. L., Kennedy, J. and Tamburro, C. H., (1979). Tissue and Urinary Glycosaminoglycans in Transplantable Hepatomas. Gastroenterology 75(5):972. 5. Kupchella, C. E., Seeskas, E., Kennedy, J. S. and Espinosa, E., (1979). Glycosaminoglycan Changes Associated with Hepatic Tumors: The Contributions of Regeneration and Necrosis. Clinical Research 27(2):389. 1 1 AJj Subject to Protective Order - 031. v Conoco. Inc. , Uo. 90-4 14th Court Calccp- t 0035^ 5 <1 Relevance to Inaustry These studies address the need for useful screening tests fc" liver injury--urine tests of the type evaluated obviously fit the iceal of oeing non-invasive, have no associated morbidity/mortality, and do not require "time off" from work to perform. They could provide useful screening tests for early injury and for dif ferentiating active versus inactive disease. GAG analysis may help assess methods of therapeutic intervention, i.e., the elucidation of the role of the GAGs in the pathogenesis of fibrotic liver disease and the identification of strategies by which fibrogenesis can be blocked and/or reversed. Finally, they could provide useful means of identifying individuals at risk of chemical injury--by further characterization of GAG changes, one may be able to identify specifc injury due to chronic alcohol, chemical or other exposure agents in individuals with liver impairment. References 1. Kupchella, C. 0., and Tamburro, C. H., (1978). Urinary and Tissue Glycosaminoglycans patterns in angiosarcoma and other vinyl chloride exposure associated liver injury in: Detection and Prevention of Cancer. H.E. Neiburgs, Ed., Part 1, Vo). 1, Marcel Dekker, Inc., New York. 2. Curran, K. L., Kupchella, C. E., and Tamburro, C.H., (1977). Urinary Glycosaminoglycan Patterns in Angiosarcoma of the Liver. Cancer 40:3050-3053. 3. Kupchella, C. E., Jarvis, J. 0., Curran, K. L. and Tamburro, C. H., (1977). Tissue ana urinary glycosaminoglycans (GAG) changes in hepatic fibrosis. Presented at the meeting of the American Association of Study of Liver Disease. Chicago, Illinois, November 1, 1977. Gastroenterology 73(5):1229. 4. Kupchella, C. E., Drake, E., Curran, K. L., Kennedy, J. and Tamburro, C. H., (1979). Tissue and Urinary Glycosaminoglycans in Transplantable Hepatomas. Gastroenterology 75(5):972. 5. Kupchella, C. E., Seeskas, E., Kennedy, J. S. and Espinosa, E., (1979). Glycosaminoglycan Changes Associated with Hepatic Tumors: The Contributions of Regeneration and Necrosis. Clinical Research 27(2):389. CONFIDENTIAL Subject to Protective Order in --s-- v- Conoco, Inc., q, 90-4837 l^th Judicial District Court r--i T,,,, CMA 003513 55 6. Greenberg, R, A., and Tamburro, C. H. (with C. E. Kupchella et al.). (1978). Early Detection of Disease in Individuals Exposed to Vinyl Chloride. Presented at the 1978 Annual Meeting of the American Public Health Association, San Diego, CA. 7. Curran, K. L., Kupchella, C. E., Sandoz, J. and Tamburro, C. H., (1979). Urinary Glycosaminoglycan Patterns in Human Hepatic Angiosarcoma, Hepatoma and in Workers at Risk with Angiosarcoma. Gastroenterology 75(5):959. 8. Kupchella, C. E., Drake, E., Curran, K. L., Kennedy, J., and Warick, R., (1981). Tissue and Urinary Glycosaminoglycan Patterns in Three Types of Morris Hepatomas. Cancer Research 4(2):419-424. 9. Tamburro, E. H., Kupchella, C. E., Taylor, K., Landau, E., Green berg, R., Maricq, H., Whelan, J., and Seifter, J., (1981). Screening for the Early Detection of Disease in Individuals Exposed to Vinyl Chloride. Environmental Protection Agency Technical Report 560/6-81-002, U.S. EPA, Washington, D.C. 10. Espinosa, E., Caple, S., Kupchella, C. E., Chia, S., (1979), Two liver antigens undetectable in a fast frowing line of trans planted hepatomas (Morris Hepatoma 7777). Federation of American Societies for Experimental Biology (Houston, Texas). Presented at Dallas, Texas, April 7, 1979; Fed. Proc. 38:1069. COffFIDSNTIAL Sutfject to Protective Order in Fos3 v. Conoco. Inc., lio. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003514 PROGRAM 0 HISTOLOGIC AND MORPHOMETRIC ANALYSIS: A MEANS OF ACCESSING HISTOLOGICAL HEPATIC INJURY IN CHEMICAL WORKERS; Investigators - G.H. Barrows, R. Schrodt, and C.H. Tamburro 01. Morphometric Assessment of Histological Lesions Characteristic of Vinyl Chloride Injury D2. Computer-Assisted Morphometric Analysis as a Rapid Means of Determining Collagen Content 03. Development and Assessment of Morphometric Method of Analysis of Collagen Content from Human Liver Biopsies - Relationship to Age D4. Light Microscopic Assessment of Various Histological Lesions Found in Liver Biopsy Tissue Obtained from Vinyl Chloride Workers Background Observations made on liver biopsy specimens obtained from vinyl monomer polymerization workers have identified the association of periportal, subcapsular, and mid-zonal fibrosis, and activated sinusoidal lining cells with vinyl chloride exposure. Many have believed these findings to be early indicators of potentially dangerous chemical exposure since these histological findings are characteristic patterns in the terminal or late stages of disease. The early stages of increased collagen deposition are often difficult to identify due to a lack of data concerning the normal collagen content and normal variation of collagen in the sinusoidal spaces. Other types of chemical workers' injuries, such as copper smelters' arsenic-induced injury, are also associated with increased collagen formation especially in the perisinusoidal areas (1-3, 6-7). In addition, there had been no systematic analysis of the collagen content of the liver's parenchyma based on age. Only subjective data was available regarding "normal" increases in the quantity of fibrous tissue within the agingliver. Therefore, objective criteria were needed to determine (a) whether the increased collagen deposition in indus trial workers could be used as an indicator of developing cancer, and (b) whether this increased collagen could be identified solely on tissue obtained by 1iver biopsy. CONFIDENTIAL Subject to Protective Order in Foss v. Conoco, Inc,, No . 90-4837 14th Judicial District Court Calcasieu Farish, Louisiana 55 CMA 003515 Industrial vinyl monomer exposure, especially vinyl chloride, has been associated with various hepatic histological abnormalities. These have included hyperplasia of both the hepatocytes and sinusoidal cells. Early histological studies, mainly on autopsy material, have shown focal mixed hyperplasia (hyperplasia of the hepatocytes or sinusoidal cells) to be an early histological alteration associated with vinyl chloride exposure (4,7,8). A double-blind histological assessment was conducted on the liver biopsies of some 120 exposed chemical workers to substantiate this observation and determine its potential use in medical surveillance. Assessment of the degree and duration of the vinyl chloride exposure was determined utilizing rank ordered exposure indices as reported by Greenburg and Tamburro (9). Objectives 1. Develop a clinically usable method for morphometric analysis of collagen content of liver biopsies. 2. Determine whether computer-assisted morphometric analysis could be used as the standard means of determining collagen content from human 1iver biopsies. 3. Morphometic assessment of normal collagen content and changes associ ated with increasing age. 4. Review and analyze light microscopic findings of liver tiss^^ obtained from vinyl chloride workers to determine whether specif^^ histological lesions were sufficiently characteristic to be identi fied and associated with vinyl chloride exposure. Research Results (1) Initially, a manual calculator-assisted approach was used for quanitation of trichrome stainable collagen in liver biopsies. Randomly selected areas of the liver biopsy were photographed. These were projected in an 16 x 16 cm area on a Hewlett-Packard 9864A digitizing plate and area cal culated using a Hewlett-Packard 9815 calculator. Measurements of collagen content and distribution of the perisinusoidal spaces were made from a ran domly selected number of complete grid sections in the various sublobular zones; they were prenumbered from left to right as shown in Figure 1A. The areas were measured in micrographs by superimposing an array of sampling points (Figure 18) and measuring the number of those points which overlaid specific areas. Two randomly selected grid areas from 3 histological, (peri portal, mid-zonal, central) and 4 anatomical locations by 2 methods were studied from 2 to 3 individuals in each bracket. Portal to central distance was quantitated and divided into 3 sections: pericentral, midzonal, and periportal. A trained observer then manually Subject to Protective Order in Ross v. Cor.o Irc 'To - 90 -4837 14th Judicial Dtstri'-t. Court Calcasieu Parish. Lou: -.ie.ne. CMA 003516 57 Industrial vinyl ~zr,z~,er exposure, especially vinyl c.n'onoe, res :een associated win '.arms r.spatic nistolccica' abnormalities. ~nese nave included nyjaerpi as i a of ootn the hepatocytes anc sinusoidal cells. la^ly histological studies, mainly on autopsy material, have shown focal mixed hyperplasia (hyperplasia of the hepatocytes or sinusoidal cells) to be an early histological alteration associated with vinyl chloride exposure (4,7,8). A double-blind histological assessment was conducted on the liver biopsies of some 120 exposed chemical workers to substantiate this observation and determine its potential use in medical surveillance. Assessment of the degree and duration of the vinyl chloride exposure was determined utilizing rank ordered exposure indices as reported by Greenburg and Tamburro (9). Objectives 1. Cevelop a clinically usable method for morphometric analysis of collagen content of liver Diopsies. 2. Determine whether computer-assisted morphometric analysis could be used as the standard means of determining collagen content from human 1iver biopsies. 3. Morphometic assessment of normal collagen content and changes associ ated with increasing age. 4. Review and analyze light microscopic findings of liver tissue obtained from vinyl chloride workers to determine whether specific histological lesions were sufficiently characteristic to be identi fied and associated with vinyl chloride exposure. Research Results (1) Initially,a manual calculator-assisted approach was used for quanitation of trichrome stainable collagen in liver biopsies. Randomly selected areas of the liver biopsy were photographed. These were projected in an 16 x 16 cm area on a Hewlett-Packard 9864A digitizing plate and area cal culated using a Hewlett-Packard 9815 calculator. Measurements of collagen content and distribution of the perisinusoidal spaces were made from a ran domly selected number of complete grid sections in the various sublobular zones; they were prenumbered from left to right as shown in Figure 1A. The areas were measured in micrographs by superimposing an array of sampling points (Figure IB) and measuring the number of those points which overlaid specific areas. Two randomly selected grid areas from 3 histological, (peri portal, mid-zonal, central) and 4 anatomical locations by 2 methods were studied from 2 to 3 individuals in each bracket. Portal to central distance was quantitated and divided into 3 sections: pericentral, midzonal, and periportal. A trained observer then manually rmiyiDENTI AL _ . j._ Protective Order in ibject to Pro go-4837 a v. Conocoj^InCj^L 10 l . [k'th Judicial District -our^ 1 ion Pr'Vari1is- h, Loui o - a CMA 003517 58 outlined all areas staining as collagen and the total sinusoidal area (Figure 2). This was repeated for a total of 3 areas in each biopsy sample. These randomly selected areas provided sufficient sampling to obtain a reproducible estimate of collagen content of various areas of liver lobule and were highly reproducible (Table 1). Manual collagen quantitation outline FIGURE 1 TABLE 1 Area Percent 2.03 4.30 6.10 14.70 REPRODUCIBILITY Digitizer Inter assay Intra Assay 0. 40 0. 19 0.55 1.27 0. 018 0. 052 0. 054 0. 6075 C0IT3TDENTIAI. Subject to Protective Order in Pose v- Conoco. Inc., No. 90-4837 14th Judicial District Court Ci|"3sini] Parish, Louieierq. Square Counting 0. 27 0. 35 1. 10 2.95 CMA 003518  (2) Later developments for computer quantitation promise to facilitate the execution of morphometric collagen determination. In this approach a video microscopic image is digitized to 4 bit precision, and held in digital video storage (Morphometrix-156 Image Analyzer). This digital image (Figure 3A, 38) contains all density information on a 256 x 512 matrix. The amount of staining of a particular density can be rapidly quantitated by counting the percentage of a particular density which occupies an image. This approach allows rapid quantitation of the total biopsy and enables study of a large number of samples. A second advantage is the adaptability of this technique for 3-dimensional reconstruction and quantitation of liver fibrosis volume. Figure 3 illustrates a computer printout of a histological area and a drawing of the area morphometrically analyzed. FIGURE 3a Density printout of Figure 2, with 10:1 reduction (tree In squere corresponds to digital printout JB). CONFIDENTIAL Subject to Protective Order in Poss v. Conoco, ino., lio. 90-4837 14th " O-.'t'irt Court ('ll........................ ' ' 7 -------- 1 CMA 003519 59 (2) Later cave Icpments for computer quantitation promise to facilitate the execution of morpncmetric collagen determination. In this approach a video microscopic image is digitized to 4 bit precision, and held in digital video storage (Morphometrix-156 Image Analyzer). This digital image (Figure 3A, 3B) contains all density information on a 256 x 512 matrix. The amount of staining of a particular density can be rapidly quantitated by counting the percentage of a particular density which occupies an image. This approach allows rapid quantitation of the total biopsy and enables study of a large number of samples. A second advantage is the adaptability of this technique for 3-dimensional reconstruction and quantitation of liver fibrosis volume. Figure 3 illustrates a computer printout of a histological area and a drawing of the area morphometrically analyzed. c 0JIFIDB]STIAL_ CMA 003520 60 3B ofFIGURE Digital densities retieulin stained liver biopsy. (3) Histological tissue was obtained for the study of liver collagen content at various ages from individuals dying of sudden death without historical, clinical or autopsy evidence of hepatobiliary disease, chronic congestive heart failure, excessive alcohol consumption, exposure to hepatoxins or infectious agents known to produce long term hepatic injury, drug abuse, or prolonged medical use of drugs known to be hepatoxic or potentially hepatoxic. The groups were divided into four age brackets: 16-30, 31-45, 46-60, 60 and older. Autopsy tissue was obtained from areas of the liver most frequently obtained in living patients by (A) percutaneous needle, {R, L) open wedge, and (D) transjugular biopsy. Needle and wedge samples were obtained from 4 areas of the liver as illustrated in Figure 4. wedge needle SITES Anterior axillary Right lobe Left lobe Oeep peri-venous -CONFIDENTIAL 4FIGURE Subject""to Protective Order in Foss v. Conoco, Inc., No. 90-4837 14th Juiioiil District Court CMA 003 521 Two hundred and fifty three light micrographs derived from 30 tissue blocks from 8 normal individuals were studied. A Hewlett Packard 9864-A digitizer microcomputer was used to quantitate areas of trichrome stainable collagen Collagen estimates varied from .2 to 6.1* (mean = 1.25) with considerable variation even in different biopsies from the same patient. Subcapsular biopsies have more collagen than deep biopsies; however, deep biopsies have more variation in collagen content. Collagen content appears to increase with age and more in midzonal regions than in portal areas (Table 2). TABLE 2 PERCENT COLLAGEN RATIO IN THE VARIOUS LOBULE AGE 15 PERICENTRAL MIDZONAL 11.8: 1 ZONES PERIPORTAL MIDZONAL 8.6: 1 PERICENTRAL PERIPORTAL 1.4: 1 CM 2.8: 1 AO 2.8: 1 50 1.3: 1 51 1: 1 1: 1 1: 1 1: 1 1: 1 2.4: 1 2.3: 1 1.9: 1 1: 1 This increase appears to become detectable in the 4th and 5th decades (Figure 5). HEPATIC COLLAGEN CONTENT WITH AGE figure 5 zo 40 AGE GROUPS 50 co:i?:sential Subject co Protective Order in Hess v. Conoco, Inc., No. 90-4837 14th Judicial District Court Calc=-L=u Parish, Louisiana CMA 003 G Two hundred and fifty three Tight ric'-ogriths derived from 50 t t S 5 J 0 1 G C S from 3 normal individuals e-e striae, A Hewlett -ackard 3864-A oigitizer microcomputer was used to o^ar.titata a-eas of tric.hrcme stainable coflacan. Collagen estimates varied from .2 to 6.if, (mean = 1.25) with considerable variation even in different biopsies from the same patient. Subcapsular biopsies have more collagen than deep biopsies; however, deep biopsies have more variation in collagen content. Collagen content appears to increase with age and more in inidzonal regions than in portal areas (Table 2). TABLE 2 PERCENT COLLAGEN RATIO THE VARIOUS LOBULE ZONES AG c PERICENT-AL NIOZONAL 15 11,3: 1 per:?drtal MICZCNAL 3.6: 1 23 2.3: 1 40 2.3: 1 50 1.3: 1 51 1: 1 1: 1 1: 1 1; 1 1: 1 PERICENTRAL PERIPORTAL 1.4: 1 2.4: 1 2.3: 1 l.S: 1 1: 1 increase appears to become detectable in the 4th and HEPATIC COLLAGEN CONTENT WITH AGE U<ocJ < --! < figure 5 20 u0 AGE GROUPS 50 r.OKFIDSffTIAL ,bject~"to Protective O'rder in s v. banooo. me . No. 90-43a7 4th Judicial District Court Calcasieu Parish. Louisiana CMA 003523 62 Differences in central, mid-zonal and portal collagen vary from 0.49 - 0.63 percent in the youngest age group and from 4 - 5.35 percent in the older age group. This histological data is consistent with the ICG clearances in normal adults which show a progressive decrease with age. The decreased functional capacity of normal aging liver may be due to an increased collagen deposition. (4) Light microscopic assessment of liver tissue obtained from vinyl chloride workers with varying histological lesions was done. In order to substantiate the ability to identify early histological alterations indicative of chemical exposure, 93 liver biopsies from 78 individuals were investigated duplicatively in a double blind manner. These histological lesions included focal hepatocellular hyperplasia (Figure 6), focal mixed hyperplasia, sinusoidal dilitation (Figure 7), focal areas of increased reticulum (Figure 8), and focal subcapsular fibrosis (Figure 9). The progressive development of vinyl chloride liver lesions are illustrated in Table 3. Thirty-five of these individuals were exposed chemical workers with hepatic screening test abnormalities, and 13 were exposed workers without hepatic screening test abnormalities who had had liver biopsies for non-liver related medical reasons. A group of 30 individuals who were not chemical workers, who had liver biopsies for nonhepatic-related illness during the same period of time at the same hospital, were compared. Twenty-three (48%) of the exposed workers had hepatic lesions consistent with exposure, 17 (35%) had only focal hepatocellular hyperplasia, and 6 (13%) had focal mixed hyperplas'a or more advanced lesions. CLINICAL FINDINGS TABLE 3 PROBABLE PHASE DEVELOPMENT OF VINYL CHLORIDE PRE-CANCEROUS LESIONS NONE BIOCHEMICAL PORTAL SPLENO- HEPATIC ABNORMAL HYPER- NEGALY SCAN TENSION ABNORMAL HEPATIJ PELIOSIS CANCER HISTOLOGICAL NORMAL FOCAL PERI- FOCAL PORTAL AND- FINDING HEPATOCYTIC SINUSOIDAL MIXED CAPSULAR HYPERPLASIA FIBROSIS HYPERPLASIA FIBROSIS SINUSOIOAL CELL DYSPLASIA ANGIO SARCOMA Subject to Protective Order in Poss v. Cgdoco. Inc. , No. 90-4837 14th Judicial District Court "''lcasieu Parish, Louisiana CMA 003524 kFOCAL HYPERPLASIA OF HEPATOCYTES WITH GREAT VARIATIONS IN THEIR SIZE AS WELL AS THEIR NUCLEI.X.THE LATTER ARE FRE QUENTLY D0U3LE, POLYCHROMATOPHILIC OR VACUOLATED. H&E, I50x. FIGURE 8 #FOCAL INCREASE OF RET1CULIN FRAMEWORK. SILVER IMPREGNA TION, lOOx. CMA 003525 Ct'c-=3 1 53 FIGURE 6 L.FOCAL hyperplasia of hepatocytes with GREAT VARIATIONS IN THEIR SIZE AS WELL AS THEIR NUCLEI .^THE LATTER ARE FRE QUENTLY DOUBLE, PQLYCHROP.ATOPHILIC OR VACUOLATED. HE, 150X. FIGURE 8 *FOCAL INCREASE OF RETLCULIN FRAMEWORK. SILVER IMPREGNA TION, 100 x. STRAIGHT ARR0WS)4(Hi, lOOx) HYPERPLASIA CMA 003526 64 In contrast only 5 of the comparison group had similar findings. Four (13%) had focal hepatocellular hyperplasia, and 1 (3%) had focal mixed hyperplasia and sinusoidal dilitation. On subsequent biopsy this individual was found to have angiosarcoma and a history of using hair spray containing vinyl chloride as a propellent. Ten individuals had 28 biopsies reviewed double blindly, and 10 individuals had 23 readings of the same biopsy; 21 of 23 (91%) duplicate readings and 27 of 28 (96%) multiple biopsy readings in the same individuals were identical. Only 18% of the individuals (3 of 17) had either duplicate and/or multiple biopsy readings which disagreed with their prior biopsy assessment. Focal hepatocytic hyperplasia, in addition to the previously described mixed hyperplasia, appeared to be the earliest identifiable change consistent with chemical exposure. These lesions can be consistently identified and are useful in screening chemical workers for evidence of chemical exposure. Chemical exposure histories to 20 different occupational chemicals were analyzed with regard to the histological findings in the liver biopsy. Portal tract fibrosis, disruption of the limiting plate, and changes in the limiting plate of the portal area had a significant correlation in individuals identi fied as having histological features of chemical liver injury in contrast to those who had nonchemical liver disease. A larger percentage of individuals with histological evidence of chemical injury had higher levels of exposure to vinyl chloride (Figure 10). Changes in the sinusoidal areas also demonstrated the highest correlation with vinyl chloride's cumulative exposure rank months, but not with the cumulative exposure rank months of acrylonitrile, a com parison chemical. INJURY - EXPOSURE CORRELATION Cll-CHEHICAL LIVER INJURY LO- LIVER DISEASE so-stanoaRd -norm 60 r < O o a. uj z> U AVERAGE VC EXPOSURE 12 3 RATINGS 4 (>3.5) o0 UJ 0. 2 CLI FIGURE 10 CONFIDENTIAL Subject to Protective Order in Ross v. Conoco, Inc,, IIo. 90-4337 14th Judicial District Court Calcasieu Parish, Louis'arq LO GROUP RATINGS Z5 SO CMA 003527 Relevance to Industry These studies demonstrate some highly important and critical information regarding documentation of chemical exposure-induced liver injury: (1) Rapid methods of quantitatively assessing total collagen content in human liver tissue have been applied. These can provide both retrospective as well as prospective analysis. (2) Morphometric analysis of collagen can be performed for clinical pur poses from human liver biopsies. This can be automated by using a computer for large volume work. (3) The identification of increased liver collagen content with age demon strates the need for better standards of "normality" before one can properly determine whether increased liver collagen is a reflection of chronic injury that may be work-related. (4) There are histologically identifiable lesions that are consistent with chemical injury; these lesions can be separated from those histological findings of nonchemical liver injury. These characteristic chemical liver injury lesions are correlated with vinyl monomers, most significantly vinyl chloride. Therefore, at the present state of knowledge, one can reasonably determine the presence or absence of significant chemical exposure from the examination of human liver tissue. The present studies have not demonstrate^ any spontaneous progression in the earliest lesions, i.e., focal hepatd^J cellular hyperplasia when found alone. . The present opinion is that lesion may simply reflect cellular adaptation to chronic exposure while mixed hyperplasia may indicate a higher risk of future cancer development. References 1. Creech, J.L., Jr. and Johnson, M.N. (1974) Angiosarcoma of Liver in the Manufacture of Polyvinyl Chloride. J, Occup. Med. 16:150. 2. Falk, J,, Creech, J.L., Jr., Heath, C.W., Jr., Johnson, M.N. and Key, M.M. (1974) Hepatic Disease Among Workers at a Vinyl Chloride Polymerization Plant. JAMA 230:59. 3. Makk, L., Creech, J.L., Jr., Whelan, J.G. and Johnson, M.N. (1974) Liver Damage and Angiosarcoma in Vinyl Chloride Workers. JAMA 230:64. 4. Popper, H. and Thomas, L.B. (1975) Alterations of Liver and Spleen Among Workers Exposed to Vinyl Chloride. Ann. N.Y. Acad. Sci. 246:172-193. 5. Makk, L., Delmore, F., Creech, J.L., Jr., et a2- (1975) Clinical and Morphological Features of Hepatic Angiosarcoma in Vinyl Chloride Workers. Cancer 37:149-163. CONFIDENTIAL ctiv? in - . LA . 90-4337 i3zrirt Court h , T Oi11 ^ ^ CMA 003528 'Sigvance to Incus try i'nese studies demonstrate some nignly 'mportant end critica1 :nf c1*-.; v regaraing documentation of chemical exposure-induced liver injury: (1) Rapid methods of quantitatively assessing total collagen content in human liver tissue have been applied. These can provide both retrospective as well as prospective analysis. (2) Morphometric analysis of collagen can be performed for clinical pur poses from human liver biopsies. This can be automated by using a computer for large volume work. (3) The identification of increased liver collagen content with age demon strates the need for better standards of "normality" before one can properly determine whether increased liver collagen is a reflection of chronic injury that may be work-related. (4) There are histologically identifiable lesions that are consistent with chemical injury; these lesions can be separated from those histological findings of nonchemical liver injury. These characteristic chemical liver injury lesions are correlated with vinyl monomers, most significantly vinyl chloride. Therefore, at the present state of knowledge, one can reasonably determine the presence or absence of significant chemical exposure from the examination of human liver tissue. The present studies have not demonstrated any spontaneous progression in the earliest lesions, i.e., focal hepato cellular hyperplasia when found alone. . The present opinion is that lesion may simply reflect cellular adaptation to chronic exposure while mixed hyperplasia may indicate a higher risk of future cancer development. References 1. Creech, J.L., Jr. and Johnson, M.N. (1974) Angiosarcoma of Liver in the Manufacture of Polyvinyl Chloride. J. Occup. Med. 16:150. 2. Falk, J., Creech, J.L., Jr,, Heath, C.W., Jr., Johnson, M.N. and Key, M.M. (1974) Hepatic Disease Among Workers at a Vinyl Chloride Polymerization Plant. JAMA 230:59. 3. Makk, L., Creech, J.L., Jr., Whelan, J.G. and Johnson, M.N. (1974) Liver Damage and Angiosarcoma in Vinyl Chloride Workers. JAMA 230:64. 4. Popper, H. and Thomas, L.8. (1975) Alterations of Liver and Spleen Among Workers Exposed to Vinyl Chloride. Ann. N.Y. Acad. Sci. 246:172-193. 5. Makk, L., Delmore, F., Creech, J.L., Jr., et al_. (1975) Clinical and Morphological Features of Hepatic Angiosarcoma in Vinyl Chloride Workers. Cancer 37:149-163. COITFIDSTTTIAL Subject to Protectiva Order i-n Bo5a v. Conoco. Inc., No. 90-4837 "\4th Judicial strict Court CMA 003529 66 6. Creech, J.L., Jr., Makk, !_., Whelan, J.G. and Tamburro, C.H. (1974) Hepatotoxicity Among Vinyl Chloride (PVC) Production Workers During First Year of Surveillance Program. Gastro. 64:786. 7. Thomas, l.B. and Popper, H. (1975) Pathology of Angiosarcoma of the Liver among Vinyl Chloride-Polyvinyl Chloride Workers. Ann. N.Y. Acad. Sci. 246:268-277. ------- 8. Gedigk, P., Muller, R., and Bechtelsheimer, H. (1975) Morphology of Liver Damage among Polyvinyl Chloride Worders. A Report of 51 Cases. Ann. N.Y. Acad. Sci. 246:278-285, 1975. 9. Greenberg, R. and Tamburro, C.H. (1981) Exposure Indices for Epidemiological Surveillance of Carcinogenic Agents in an Industrial Chemical Environment. J. Occup. Med. 23:353-358. CMA 003530 PROGRAM E STUDIES OF VINYL MONOMER CHEMICALS AND THEIR METABOLITES USING CHEMICAL STRUCTURE AND SYNTHESIS IN THE DETERMINATION OF TOXICITY OF THESE AGENTS; Investigator - J. L. Wong El. Synthesis, Purification, and Utilization of Vinyl Chloride Metabolites in Mutagenicity and Carcinogenicity Studies E2. Detoxification Studies of Vinyl Chloride and Its Metabolites E3. Vinyl Chloride Metabolite Detection--Chloroacetic Acid Background The mutagenicity of vinyl chloride and its potential metabolites was studied in detail by a combination of chemical synthetic and microbiological assay techniques. We prepared and characterized the potential putative metabolites (chlorooxirane and four forms of chloroacetaldehyde), and Dr. Uldis Streips of Microbiology conducted the testing of these compounds in the Bacillus and Salmonel1 a systems. Since all literature methods for the preparation of chlorooxirane result in mixtures with contaminants such as chlorine, hydrogen chloride, ethylene oxide, polymers, etc., it was necessary to develop a method to prepare pure chlorooxirane. In order to elucidate the intermediary metabolism of vinyl chloride, i.e., the metabolic activation and inactivation steps, to determine the significant end products of vinyl chloride in body tissues and fluids, the fate of chlorooxirane, the immediate P.450 product of vinyl chloride, was studied using synthetic chlorooxirane to react with non-protein sulfhydryls. Another prominent reactive property of chlorooxirane is its facile rearrangement to chloroacetaldehyde (CAA). In dimethylformamide at 25*C, it has a half-life of ~0.5 min in rearranging to CAA. Therefore, the mechanism of deactivation of CAA by cysteine was studied. The plausible cyclic pro duct--3L-carboxy-2,3-dihydro-1,4-thiazine--may be the precursor of the urinary metabolites S-hydroxyethylcysteine and S-carboxymethylcysteine. R01T?IDt.ITTlAL s Order in No. 90'483? -let Court Tn'iiR' 67 CMA 003531 68 As a putative metabolite, CAA is well-known to react with adenine and cytosine nucleotides* We will demonstrate that CAA can also react with guanine derivatives at body temperature and physiological pH to yield covalent products. These products will alter the N-H hydrogen-bonding sites in the nucleic acid bonds, hence their formation in a biological host may be consequential in inducing the onset of neoplasia. Since previous animal studies have not demonstrated chloroacetic acid in the urines of animals at low exposure levels but have been reported in humans at higher exposure levels, we intend to develop analytical methodologies which will help to verify this observation. Such studies may lead to a method of warning of environmental exposure to humans. The first target metabolite for analysis was chloroacetic acid. Objective 1. To study the chemistry of vinyl chloride toxicity by identifying the intermediates of vinyl chloride metabolism and their reactions with cytoplasmic chemicals. 2. To determine the putative actions of the primary metabolites with regard to: a) the chemical reactions of metabolites, chlorooxirane (COR) and chloroacetaldehyde (CAA) with sulfhydryls (detoxification). b) reactions on nucleic acid constituents (mutagenesis and carcinogenesis). 3. To develop analytical methods to detect a target metabolite--chloro acetic acid. El. Synthesis, Purification, and Utilization of Vinyl Chloride Metabolites in Mutagenicity and Carcinogenicity Studies The detection study of the putative action of vinyl chloride is based on the hypothesis that such action comes from the modification of the nucleic acid materials by the primary metabolites COR and CAA. Although CAA is long known to react with nucleic acid bases such as cytosine and adenine to form the etheno derivatives, little is known about the reaction of CAA on the most reactive base, guanine. Our study of CAA with guanine base using analytical tools such as HPLC, GC-MS and FT-NMR have shown that the guanosine reaction is enormously complicated. The hetero-bifunctional alkylating structures of 2-chloroacetaldehyde are shown below. CONFIDENTIAL Subject to Protective Order in Ross v. Conoco. Inc.. 'To. 90-4837 14th JirUcicl rif3tT'i"t CMA 003532 ci -ckv f? C-H H a-CH^-C-OH OK HH ci-CHi-c-o-c-cH^ci OH OH cich2 <A> CICH^O^CHjCI HH a CDj-C-O-- C-CD2 CI OD OD' H Cl -- CH2C-OCH2CH3 OCH,CH~' Studies of their reactivities have necessitated: a. Synthesis of etheno-modified components of RNA and DNA-- angularetheno-guanine, 1inear-etheno-guanine, 1inear-etheno-guanosine, 1inear-ethenodeoxy-guanosine, etheno-adenine, etheno-adenosine, etheno-deoxy-adenosine, etheno-cytosine, etheno-cytidine, and etheno-deoxy-cytidine. b. Synthesis of acidic hydrolytic products of etheno-modified bases-- 2-aminobiimidazole, 2-formamidobiimdazole, El-D-angular-etheno-guanine, ^ El-D-1inear-etheno-guanine. ^ c. Synthesis of alkaline decomposition products of etheno-modified nucleosides--2-amino-biimidazole nucleoside and 2-formamido-biimidazole nucle oside. d. Synthesis of the deuterated analogues for detailed structural study--8-D-etheno-adenine, El-D-etheno-cytosine, El-D-angular-etheno-guanine, El-D- etheno-adenine, and El-D-1inear-etheno-guanine. The kinetics of guanosine with CAA at wide range pH value have been studied with careful pH control. The pK vs. pH plot showed that there are two plateaus, pHl and pH8, and there is minimum at pH 4.5. From the product profiles, two different mechanisms can be suggested (Figure 1). The putative action of vinyl chloride in respect to the physicochemical properties of the etheno derivatives of cytidine, adenosine, and guanosine is further studied. The ^C-FTNMR spectroscopic properties are particularly revealing. By means of deuteration and proton undercoupling technique in acquiring the carbon-13 spectra, the various carbon chemical shifts are assigned and the ^C-^H coupling constants determined. From the latter data, the conformational preferences of the sugar moiety in the modified nucleosides are deduced as follows: (1) anti conformation for ethenocytidine CONFIDENTIAL Subject to Protective Order in jig53 V. _Cr.-:oo, Inc., 'Jo. 90-4337 i4tr. Iriioicl District Court Calcasieu Parish, Louisiana CMA 003533 o r-c -- :-oh OH HH O -CK ;~C~0 -- C- CH2--Cl OH OH CICH. A9 CICHf^O^CH.C: HH a C Di-CI -O --I C-.CDj C! OO OD H a-CHjC-OCHjCH, OCHjCh;- Studies of their reactivities have necessitated; a. Synthesis of etheno-modified components of RNA and DNA-- angularetheno-cuanire, 1 inear-etheno-guanir.e, i inear-etheno-guanosine, 1 inear-ethenodeoxy-guanosine, etheno-adenine, etheno-adenosine, etheno-deoxy-adenosine, etheno-cytosine, etheno-cytidine, and etheno-deoxy-cytidine. b. Synthesis of acidic hydrolytic products of etheno-modified bases-- 2-aminobiimidazole, 2-formamidobiimdazole, El-D-angular-etheno-guanine, and El--D--1inear-etheno-guanine. c. Synthesis of alkaline decomposition products of etheno-modified nucleosides--2-amino-biimidazole nucleoside and 2-formamido-biimidazole nucle oside. d. Synthesis of the deuterated analogues for detailed structural study--8-D-etheno-adenine, El-D-etheno-cytosine, El-D-angular-etheno-guanine, El-D- etheno-adenine, and El-D-1inear-etheno-guanine. The kinetics of guanosine with CAA at wide range pH value have been studied with careful pH control. The pK vs. pH plot showed that there are two plateaus, pHl and pH8, and there is minimum at pH 4.5. From the product profiles, two different mechanisms can be suggested (Figure 1). The putative action of vinyl chloride in respect to the physicochemical properties of the etheno derivatives of cytidine, adenosine, and guanosine is further studied. The ^C-FTNMR spectroscopic properties are particularly revealing. 8y means of deuteration and proton undercoupling technique in acquiring the carbon-13 spectra, the various carbon chemical shifts are assigned and the ^C-^H coupling constants determined. From the latter data, the conformational preferences of the sugar moiety in the modified nucleosides are deduced as follows; (1) anti conformation for ethenocytidine CONFIDENTIAL Subject to Protective Order in Raaa y. Conoco, Inc . . l,ro . 90-4337 14th Judicial District Court ch , T*oiT i f^T CMA 003534 70 R-H fU&otyl Oeoxy-ritMsyL C AA 3 '-tnonoi)to cc rt6oyl 3`,S--cyeltC iBonprto0ft4t9 rtoyl -H figure 1 and (2) syn for etheno-adenosine. Since anti conformation of nucleosides is necessary for the formation and stability of the helix, the influence of the etheno bridge on the adenine nucleus in altering the ribosyl group to syn will impose considerable stress on the DNA chain which might result in biological damages. E2. Detoxification Studies of Vinyl Chloride and Its Metabolites A previous attempt by Gothe et al. to trap the putative metabolites COR and CAA in vitro from vinyl chloride with 3,4-dichlorobenzenethiol has led to the identification of the product as 3,4-dichlorophenylthioacetaldehyde. It was interpreted to be indicative of the formation of either COR or CAA. We have defined this experiment further. Thus, chlorooxirane in organic medium reacts slowly with 3,4-dichlorobenzenethiol to form 2-(3,4-dichlorophenylthio) acetaldehyde as 80 percent^of the products. This reaction at room temperature takes about 7 days. At 60*C the reaction is completed in 21 hours giving' the same products. (In both cases some disulfide Cl2Ph-S-S-Ph-Cl2 and other unidentified polymeric materials are observed.) The identity of the product was established by comparison of gas chromatography retention time with an authentic example, by PMR and by mass spectrum. In aqueous acetone (2:1) or aqueous acetonitrile (3:1), the reaction is much faster. As pH = 4, 55 percent conversion is observed in 15 min. which is the lifetime of COR under CONFIDENTIAL Subject to Protective Order in Ross v. Conoco, Inc., No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003535 71 these conditions, and 80 percent conversion at pH = 7. The sole product identified from these reactions by high pressure liquid chromatography is the aldehyde indicated. On the other hand, chloroacetaldehyde in CHCI3 gives another addition product, which was identified by PMR and IR and its facile reversion to the starting materials as 3,4-Cl2Ph-S-CH(0H)CH2Cl. In aqueous acetone or aqueous acetonitrile, CAA did not react with the thiol within 1/2 hour, indicating its lower reactivity to the aromatic $H group compared to COR. These results, combined with the in vitro experiment by Gothe, have confirmed that chlorooxirane is an obligatory intermediate in the metabolism of vinyl chloride. The detoxification of chlorooxirane was also studied in aqueous media at different pH's. N-Acetylcysteine was used as a typical cellular sulfydryl compound involved in detoxification. In aqueous solutions at pH4 and 7, their reaction was extremely fast at room temperature yielding N-acetylcysteine-Sacetaldehyde. It was identified by *H and -^C NMR and characterized as the 2,4-dinitrophenylhydrazone derivative. This aldehyde is probably the precursor of the urinary metabolites, S-2-hydroxyethylcysteine and thiodiglycolic acid. The reaction of chloroacetaldehyde with N-acetylcysteine under controlled pH conditions in aqueous medium at O'C produced an intermediate compound. Upon warming up the reaction mixture to room temperature, our previously identified thiazene was isolated as the final product. In order to elucidate the stepwise formation of the thiazene, N-acetylcysteine methyl ester w^ allowed to react with chloroacetaldehyde in chloroform at 0*C. The initi product, plausibly the hemithioacetal H3CO-CO-CH-(NHCOCH3)CH2S-CHOH-CH2C1, eliminated HC1 upon neutralization with aqueous sodium hydroxide to produce the corresponding epoxide H3C0C0CH(NHCQCH3)CH2S-CH0-CH2. This structural assignment is supported by its PMR spectrum. Furthermore, when this epoxide was extracted into chloroform, it rearranged to the S-acetaldehyde H3COCOCH- (NHCOCH3)CH2-S-CH2CHO, identified by its pmr spectrum and by comparison with that formed from the reaction of N-acetylcysteine with chlorooxirane. Even though chlorooxirane and chloroacetaldehyde eventually give the same final product with N-acetylcysteine, the rate of reaction and the inter mediates in the two reactions are different. The chlorooxirane conjugates instantaneously with the sulfhydryl compound, while chloroacetaldehyde takes about 2 1/2 hours for a comparable reaction. The comparative study of the reaction of chlorooxirane and chloroacetal dehyde with sulfhydryl compounds can now be summarized in the following reaction pathways with cysteines (unmodified, N-acetyl, and N-acety1-methyl ester): -COITPIDEjTtIAL \to -retset:vs Coder in -ij. 90-4337 -.ci-il District Court radish, Louisin na CMA 003536 20 ' j -ft Z - ^ lr Z % Z ~ I ^ i ] r. t -r ` .* i r z ^ r, - r - - -. *' c r. ^ <*,, cj c ,, n n , c n w d s . ^ c n v,! r i n 'j j ,,. c ri u _ a ar.u i l s aci ,e reversion to the starting materials as 3,4-Cl^?h-S-CH(CH)CHgCl. In aqueous acetone or aqueous acetonitrile, CAA did not react with the thiol within 1/2 hour, indicating its lower reactivity to the aromatic SH grouD comoared to CCR. These results, combi red w'th the in vitro a'.p arimor.t Gothe, have confirmed that chlorooxirane is an obligatory intermediate in the metabolism of vinyl chloride. The detoxification of chlorooxirane was also studied in aaueous media at different pH's. N-Acetylcysteine was used as a typical cellular sulfydryl compound involved in detoxification. In aqueous solutions at oH4 and 7, the^r rsscticn -was extremely fast at room temperature y ielo'rg i-acetylcysta'r.e-Sacetaldenyae. It was identified by `H and -^C (WR and characterized as the 2,4-dinitrophenylhyorazone derivative. Tnis aicehyoe is prooaoly the precursor of the urinary metabolites, S-2-hydroxyethylcysteir.e and thiodiglycolic acid. The reaction of chloroacetaldehyde with N-acatylcysteine under controlled pH conditions in aqueous medium at 0C produced an intermediate compound. Upon warming up the reaction mixture to room temperature, our previously identified thiazene was isolated as the final product. In order to elucidate the stepwise formation of the thiazene, N-acetylcysteine methyl ester was allowed to react with chloroacetaldehyde in chloroform at 0*C. The initial product, plausibly the hemithioacetal H3C0-C0-CH-(NHC0CH3)CH2S-CH0H-CH2C1, eliminated HC1 upon neutralization with aqueous sodium hydroxide to produce the corresponding epoxide H3C0C0CH(NHCQCH3)CH2S-CH0-CH2. This structural assignment is supported by its PMR spectrum. Furthermore, when this epoxide was extracted into chloroform, it rearranged to the S-acetaldehyde H3COCQCH- (NHCOCH3)CH2~S-CH2CHO, identified by its pmr spectrum and by comparison with that formed from the reaction of N-acetylcysteine with chlorooxirane. Even though chlorooxirane and chloroacetaldehyde eventually give the same final product with N-acetylcysteine, the rate of reaction and the inter mediates in the two reactions are different. The chlorooxirane conjugates instantaneously with the sulfhydryl compound, while chloroacetaldehyde takes about 2 1/2 hours for a comparable reaction. The conparative study of the reaction of chlorooxirane and chloroacetal dehyde with sulfhydryl compounds can now be summarized in the following reaction pathways with cysteines (unmodified, N-acetyl, and N-acety1-methyl ester): r.OiT?IDTTTI AL Subject to -rotct'v': C`qq^4S37 Boss v. 14th Conoco, T~~r- ,1 JuTi-'.-'T- Inc, bo. 1u^; se-rt-T-i Cjt ^ Court rnl ' -h T o'1 CMA 003537 72 X-NH-CH-COOR h2sh Cl-CH2CHO X-NH-CH-COOR CH2-S-CK2CHO Xi COOP X-NH-CH-COOR -------------- > CH0-S-CH-CHtC1 2 6h 2 X=H, Ac R=H, Me X-NH-CH-COOR ^CH02 S-CH-CH-2, Although both routes are converged to yield the cysteine S-acetaldehyde con jugate, their reaction rates are vastly different. In general, chlorooxirane completes its reaction within an hour while chloroacetaldehyde requires over night. These chemical observations can be applied in (1) specific assays for the two putative metabolites in cellular studies of vinyl chloride, and (2) detoxification mechanism of cells exposed to the vinyl carcinogen. E3. Vinyl Chloride Metabolite Detection - Chloroacetic Acid The methodology includes gas chromatography and mass spectrometry. Gas chromatography results are as follows. Solid supports containing Carbowax and FFAP liquid phases have been used for direct analysis of carboxylic acids. Accordingly, 20 percent FFAP on chromosorb W passes chloroacetic acid at 200* with modest tailing of the peak. However, with this simple column, sensi tivity is limited to concentration ranges of 0.2 - 2 mg/ml H2O (200 - 2,000 ppm). Application of the improvements reported by Ackman (use of porous poly mer solid support and use of formic acid in helium carrier gas) is expected to improve sensitivity by 100 fold. Identification of chloroacetic acid by mass spectrometry was carried out. Chloroacetic acid can be identified by mass spectroscopy of water solutions as dilute as 0.02M (2mg/ml). The 2 doublets of peaks at m/e 49, 51 (33C1CH and 37C1CH2+) and 50,52 (35ClCH3t and 37C1CH3T) are distinctive because rela tive intensities within each doublet reflect the 76:24 isotopic ratio of 35C1:37C1. Thus, the GC-MS combination technique should be most useful in the detection of the above. COITFIDHSHTIAL Subject to Protective Order in P.oss v. Ccr.000. ir.c. , Ho. 90-4337 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003538 73 Relevance of Industry Broadly speaking, this is a study for early detection and prevention of industrial cancers. Our chemical methodologies (synthesis, structure, and analysis), aplied as an integral part of the multidisciplinary approach, contribute to elucidate specific molecular events in the effects of vinyl monomers on industrial workers. This information will form a rational basis for safer use of chemicals and design of preventive measures. Our molecular studies also provide the opportunity to develop useful marker(s) in the form of metabolites in the pathogenesis of chemical injury. References Elmore, J. D,, Wong, J. L., Laumbach, A. D. and Streips, U. N. (1976) Vinyl chloride mutagenicity via the metabolites chlorooxirane and chloroacetaldehyde monomer hydrate. Biochem. Biophys. Acta, 442, 405. Laumbach, A. D., Lee, S., Wong, J. L. and Streips, U. N. (1977) Studies on the mutagenicity of vinyl chloride metabolites and related chemicals. Prev. Oetect. Cancer (Proc. Int. Symp), 3rd 1^, 155. Laumbach, A. D., Streips, U. N. and Wong, J. L. (1978) Chloroacetal- dehyde-induced damage to Bacillus subtilis. Abs. Ann. Mtq. ASM, p. 125. ----------------------------------- Laumbach, A. D., Streips, U. N. and Wong, J. L. (1979) Chloroacetal dehyde- induced damage to Baci1lus subtilis, Abs. Twelfth Internat. Congress Microbiol. Joseph, J. T. and Wong, J. L. (1982) Model study of vinyl chloride metabolism sulfate reactions with chlorooxirane. Carcinogenesis (in preparation). CONFIDENTIAL Subject^to Protective Order in -ss.7'. _Concco, Inc,. ?7n 90-4837 l^th Judicial District Court Calcasieu Parish, Louiissiiaannaa CMA 003539 PROGRAM F ASSESSMENT OF ASSAYS FOR THE CARCINOGENIC POTENTIAL OF INDUSTRIAL CHEMICALS USING PROKARYTOIC ANO EUKARYOTIC SYSTEMS. Investigators; U.N. Streips and G. Sonnenfeld FI. Bacterial Assays for Testing Carcinogenicity and Mutagenicity of Indus trial Chemicals. Background Our laboratory had instituted Salmonel1 a reversion, Bacillus subtilis repair, and Bacillus subtilis forward mutations assays for screening the mutagenic potential of chemicals (1-3). For that reason we became an integral part, first, for 8. F. Goodrich, then for the Manufacturing Chemists Association sponsored projects on industrial chemical risk assessment. Objective 1. To screen a series of industrial and environmental - health-related chemicals for mutagenic potential. 2. To initiate with Dr. G. Sonnenfeld a rapid mammalian assay to better predict human-related bacterial chemical activity and carcinogenic potential. Research Results In our previous progress reports we have documented the work which delineated the involvement of chloroacetaldehyde and chlorooxirane as the probable active components of vinyl chloride-mediated carcinogenesis. In addition, we screened a multitude of other chemicals generated by Dr. John Wong (our collaborator in this study) or by other investigators in the university or industry (1-3). This is documented in Table 1. Recently, in collaboration with Dr. Gerald Sonnenfeld, a new, exciting, potential screen for carcinogens was developed (4,5). This involved the inhibition of inter feron induction and was specific for several carcinogens (including chloro acetaldehyde) but not for non-carcinogenic analogues (including some muta gens). It should be illustrative of some of the molecular mechanisms of carcinogenesis attack on susceptible cells. We continue to develop this system even though our support from CMA has ended (6-8).. CONFIDENTIAL Subject"to Protective Order in - S- V- Jj.u22G- Tro., lb. 90-4837 14th Judicial District Court = parish, Louisiana 74 CMA 003540 75 A screening laboratory (using microbial assays) for institutional service purposes is being maintained. We have proposed to the state of Kentucky that a regional chemical testing laboratory be funded to serve the industry and university needs in this part of the Commonwealth. That proposal is still pending. TABLE 1 SUMMARY OP PERTINENT SUBSTANCES TESTED FOR IVTaCENICITY 1, Neca-chldrobenxoyl-cyclobucaneearbonyl peroxide 2. Benzoyl peroxide 2. N*4cecoxy-N-phenylacecamidfl 4. N-cyeiobucenecarboxyloicy*N-phenyiacet4nide 5. bts-cyelpbucane carboxyl peroxide 6, A/lacoxln 7. CHloroethanol 0. Benxofa)pyrtne 9- 4-nlcro quinoline*1-oxtde 10. Chloroacecaldehyde 11. Eplchlorohydrln 12. Oilorooxirane 13- Butane dlepoxlde 14. Styrene oxide 15. 3,4 epoxide butene 16. blx(Beco-ehloroechyl)phenyl phoephace 17. *ta chloroethyl phosphate (bla cyclohexyla*lne salt) 10- Lung ajplratee from smoker* with cancer 19, Lung aaptraces from smokera without cancer Salmonella + NR NR NR NR ++* NR +++ +4- +++ ++ + NR SR + a w reaction " tuciwntc +*+ - eitre*aly iuU|inU - " borderline #ta*enUUy ++ - etrongiy nttafenle Subtilt* NR NR NR NR NR ++ NR + -H*+ ++ f b NR NR NR NR NR To further elucidate molecular mechanisms of carcinogenesis, in collaboration with John Wong, we inititated a study of DNA-interaction with carcinogens, and with Ron Doyle, control of cell division processes in bacteria. Progress in this area has allowed us to secure a grant from the National Science Foundation to carry on with the work. Soon, we will be able to describe the cellular controls which maintain proper cell division in bacteria, then relate these findings to mamnalian cells. At that time, we will be able to examine carcinogen attack on these processes and determine which events lead to loss of cell division control and neoplastic growth. C0N7IDENTIAL Subject to Protective Order in Cor.ovo, Ir.c . , No. 90-4337 J-h' Judicial District Court ,isieu Parish, Louisiana CMA 003541 zzrzcsss is ce'ir,: -a:-,-, a d r - ' 0 " 5 "i C 1S ~ C i " d 5 jnr.ers'iiy neecs in pms part of tna Cortmonwea ' tn. pending. Tnat orcoosai is still TAB'- 1 ^OF ?SR?l!'EST j(,"35~v?CE5 TSSTT3 ^VTACr'lt3tTv -*Ci-C.hl3raftnjyl--jycioouCAneejrbanyi paraxld* 2. J*rtfoyi ?*cc<:d* Sj.^n* Lli a mr VR * * M-CveisouCaflcCar loTcy-s1--*- /13e< Pir-l A* 5' = -5-ty:*:m;; i r to < v L ;*r3x;^i 6. A/iaCaxXrt 7. CMocs*!\anoI 9. *-V.:rO ^wi-no, Li*-L-o*:d 10. CilorQ4iE4ldthya n. E?t ehiorahvdrln 12. CUorao*i,rn 13. Iuc4n dlteoxld* Scyr*n xld 15. J.4 <?xl4 SuCena 16. bl*(S4ca-*pilora*chl)9Sc>yl pnasohdCc 17. 8*ta cn Laroat.ty 1 ahoianaca (V.i x * Laming 13, Uuflf iplrC* (xam jiM n with Cinetr 19, Lwnp 4*Olraes fr ino6yi vlcnouC C4ner SR s* : * n - *o rttetlso * - *jC4fflU *** - *cr*aly me * * br4*rliM mtattnUlty +* - *crA||l7 Mjca^aAlc MR MR V* MR MR To further elucidate molecular mechanisms of carcinogenesis, in collaboration with John Wong, we inititated a study of DNA-interaction with carcinogens, and with Ron Doyle, control of cell division processes in bacteria. Progress in this area has allowed us to secure a grant from the National Science Foundation to carry on with the work. Soon, we will be able to describe the cellular controls which maintain proper cell division in bacteria, then relate these findings to mammalian cells. At that time, we will be able to examine carcinogen attack on these processes and determine which events lead to loss of cell division control and neoplastic growth. CON?IDINIIAL Subject to Protecting Order in P.css v. Conoco, Inc. , No. 90-4837 14th judicial""': strict Court Calcasieu Porish, Louisiana CtAA 003$42 76 Support from the CMA has been instrumental in generating and maintaining research activity in our laboratory relating to carcinogenesis and cell cycle events. We deeply appreciate this support. Relevance to Industry With the tremendous number of chemical formulations generated yearly, a battery of rapid screens are crucial for examining the environmental and health-related impact of these chemicals. A regional laboratory can do such screening effectively and economically as shown by the results previously described. In addition we have demonstrated that such research also leads to innovation in terms of new assays (the interferon assay), and related research (the studies on cell division). Industrial support also has led to other granting. Valuable mutagenesis information has been provided to industry regarding crucial chemicals. In turn, industry has provided us with the opportunity to expand and solidify our research efforts. The interaction has been optimal. F2. Preliminary Studies on the Use of Inhibition of Interferon Induction as an Indicator of Mutagenicity and Carcinogenicity of Chemicals Background Interferon, which was originally described as an antiviral agent, has since been shown to have several other activities, including an antitumor activity (9). Clinical trials are now in progress to determine the efficacy of interferon as an anti-cancer agent (10). We became interested in the interactions of carcinogens, or cancer- causing substances, and interferon. Early studies by DeMaeyer and others suggested that there was indeed an interaction between carcinogens and interferon induction (11-13). Pretreatment of rat embryo fibroblasts with the carcinogens triethyleneamine, 4-nitroquinoline-N-oxide, benzo-(a)-pyrene, and 3-methylcholanthrene all inhibited the induction of alpha-beta interferon by viruses, without affecting the gross viability of the cultures or the replication of the viruses (11-13). Several poorly or noncarcinogenic analogs, such as benzo-(e)-pyrene, had no effect on the production of interferon (11-13). The effect was observed after only one day of incubation of very small amounts of carcinogen with the tissue cultures. In addition, Hahon and co- workers have shown that incubation of cell cultures with various forms of aflatoxin also inhibited the induction of interferon in the cultures (14). In this case, the degree of inhibition correlated with the jm vivo carcinogenic potential of the aflatoxin form (14). Asbestos fibers and coal dust have also been reported to inhibit interferon induction in tissue culture (15,16). In vivo, injection of methylcholanthrene into mice inhibited the induction 6T alpha-beta interferon (17). These results created an interest in our laboratory to further study this phenomenon. CONFIDENTIAL Subject to Protective Order in Boss v. Conoco, Inc. , Ho. 90-4837 14th Judicial District Court P^r'sh, Louisiana CMA 77 Objective 1. To repeat the early studies to determine if carcinogen pretreatment inhibited interferon induction. 2. To begin studies to determine if effects on interferon induction could be used as a reliable indicator of carcinogenic potential of chemicals. 3. To study the mechanisms of the observed effects. Research Results Our first step was to establish the system in a mouse culture model, since this was more appropriate to current interferon technology. Mouse embryo tissue cultures were prepared, treated with carcinogen or analogue for 24 hr., washed with fetal bovine serum-containing medium and with serum-free medium to help remove carcinogen, challenged with an interferon inducer (either Newcastle disease virus or poly I:C), and then assayed for antiviral activity. Earlier data was able to be duplicated and those findings extended. Several known carcinogens including 7,12-dimethylbenz-(a)-anthracene, benzo-(a)-pyrene, 2-aminofluroene, aflatoxin-Bi and the #4 fraction of tobacco smoke condensate all inhibited interferon induction (5). Styrene oxide, an important industrial chemical which was positive in the Ames Salmo nella assay but negative in all Bacillus assays done to date (11), alf^ inhibited interferon induction. Ethyl methanesulfonate (EMS) and metf^| methanesulfonate, differ with respect to their carcinogenic potential as determined by tumor induction (18,19). MMS is a mutagen and highly carcino genic, while EMS is a potent mutagen but rarely carcinogenic. In our assay, MMS strongly inhibited alpha/beta interferon induction, while EMS had no effect. Additional studies in our laboratory extended these findings to the potent carcinogen chloroacetaldehyde and its noncarcinogenic analogues chloroethanol and chloroacetic acid (4). Only chloroacetaldehyde inhibited inter feron induction. An additional finding of interest was the apparent requirement for activation of carcinogens before they could inhibit interferon induction. In a preliminary study, we were able to show that addition of reduced glutathione to our cultures blocked the inhibition of interferon induction by benzo-(a)pyrene (4). Reduced glutathione can block the active metabolites of carcinogens (20), which suggested that these active metabolites were required for the effects of benzo-(a)-pyrene on interferon induction. Relevance to Industry We believe these studies to be highly relevant to the chemical industry because: 1) the studies yielded information on the effects of toxic chemicals on the interferon system, an important body defense against cancer, and 2) the studies may eventually lead to a new assay system for discriminating between carcinogenic and noncarcinogenic chemicals. CONFIDENT I AT. Subject to P CMA 003544 I .district Court T.O'l'i Si nnq ' - -- .3 - - ''-i. `TM in n i Z/ i z c 0 i n c 5 r" c r c r n li u c ^ i o n - i -- - - ~ ^ a n ^ rns *"* ^ 2. To begin studies to determine if effects on interferon induction could be used as a reliable indicator cf carcinogenic potential of " S ^rr\ i .* a 1 ^ 3. To study the mechanisms of the observed effects. Research Results Our first step was to establish the system in a mouse culture model, S'nc^ zn i s `*i is t'j r 9 c r 2 o r i ^ H9 zo t^cnr.olcQV. Mouss embryo tissue cultures were prepared, treated with carcinogen or analogue for 24 hr., wasnea witn fetal Devine serum-containing medium and with serum-free medium to help remove carcinogen, challenged with an interferon inducer (either Newcastle disease virus or poly I:C), and then assayed for antiviral activity. Earlier data was able to be duplicated and those findings extended. Several known carcinogens including 7,12-dimethylbenz-(a)-anthra- cene, benzo-(a)-pyrene, 2-aminofluroene, aflatoxin-3i and the #4 fraction of tobacco smoke condensate all inhibited interferon induction (5), Styrene oxide, an important industrial chemical which was positive in the Ames Salmo nella assaybut negative in all Bacillus assays done to date (11), also inhibited interferon induction. Ethyl methanesulfonate (EMS) and methyl methanesulfonate, differ with respect to their carcinogenic potential as determined by tumor induction (13,19). MMS is a mutagen and highly carcino genic, while EMS is a potent mutagen but rarely carcinogenic. In our assay, MMS strongly inhibited alpha/beta interferon induction, while EMS had no effect. Additional studies in our laboratory extended these findings to the potent carcinogen chloroacetaldehyde and its noncarcinogenic analogues chloro- ethanol and chloroacetic acid (4). Only chloroacetaldehyde inhibited inter feron induction. An additional finding of interest was the apparent requirement for activation of carcinogens before they could inhibit interferon induction. In a preliminary study, we were able to show that addition of reduced glutathione to our cultures blocked the inhibition of interferon induction by benzo-(a)pyrene (4). Reduced glutathione can block the active metabolites of carcinogens (20), which suggested that these active metabolites were required for the effects of benzo-(a)-pyrene on interferon induction. Relevance to Industry We believe these studies to be highly relevant to the chemical industry because: 1) the studies yielded information on the effects of toxic chemicals on the interferon system, an important body defense against cancer, and 2) the studies may eventually lead to a new assay system for discriminating between carcinogenic and noncarcinogenic chemicals. CONFIDENTIAL Subject to Protective Order in Eoss v. Conoco, Ino., Ho. 90-4337 CMA 003545 14th Judicial district Court Calcasieu Parish, Louisiana 78 References 1. Horowitz, S., Doyle, R.J., and U.N. Streips. (1978) Restricted chromosome-membrane association in a stable L-form of Bacillus subtil is. In Transformation-1978. Proceedings of the Fourth European Meeting on Bacterial Transformation and Transfection, pp. 245-251. .2 Horowitz, S., Doyle, R.J., Young, F.E., and U.N. Streips. (1979) Selective association of the chromosome with membrane in a stable L-form of Bacillus subtil is. J. Bacteriol. 138:915-922. 3. Streips, U.N., Horowitz, S., and R.J. Doyle. (1980) Genetic analysis of DNA-surface interactions in Bacillus subtil is. Microbiology 1980, ASM Press, pp. 284-287. In 4. Sonnenfeld, G., Barnes, M.C., Schooler, J., and U.N. Streips. (1980) Inhibition of interferon induction as a secreen for the carcino genic potential of chemicals. _I_n Interferon: Properties and Clinical Uses. Leland-Fikes Foundation Press, Dallas, pp. 589. 5. Barnes, M.D., Streips, U.N., and G. Sonnenfeld. (1981) Effect of carcinogens and analogs on interferon induction. Oncology, 38:98-101. ---------- .6 Streips, U.N., Laumbach, A.D., and R.E. Yasbin. (1981) Bacterial mutation monitors for active metabolites of clinical carcinogens: Baci1lus subtil is assays for mutation and repair In Microbial Testers for Chemical Carcinogenesis, I.C. Felkner, ecf., Marcel Dekker, New York, pp. 131-145. 7. Tamburro, C.H., Wong, J.L., and U.N. Streips. (1982) Approaches to occupational cancers. In Clinical Medicine for the Occupational Physician, Alderman, an<3"Hanley (eds.), Marcel Dekker, New York, pp. 255-295. .8 Clarke, J.S., Streips, U.N., Hoffman, J.L., Fok, F.F., and J.A. Yankeelove. (1980) J. Toxicology and Environmental Health (submitted). 9. Gresser, I. Cell. Immunol., (1977) Commentary on the varied biologic effects of interferon 34:406-416. 10. Merigan, T.C. (1982) Interferon Therapy in Human Viral Infections and Malignant Disease, pp. 88-90, In, Stiehm, E.R. Moderator, Ann. Intern. Med. 96:80-93. 79 11. DeMaeyer, E., and DeMaeyer-Guignard, 0. (1964) Inhibition by 3-methylcholanthrene of interferon formation in rat embryo cells infected with Sindbis virus. J. Natl. Cancer Inst. 32:1317. 12. DeMaeyer-Guignard, J., and DeMaeyer, E. (1965) Effects of carcinogenic and noncarcinogenic hydrocarbons on interferon synthesis and virus plaque development. J. Natl. Cancer Inst. 34:264. ------------------------------------- 13. DeMaeyer-Guignard, J., and DeMaeyer, E. (1967) Inhibition of interferon synthesis by triethylenemelamine and 4-nitroquinoline-N-oxide. Arch. Gesamte Virusforch. 22:61-68. 14. Hahon, N., et al. (1979) Aflatoxin inhibition of viral interferon induction. Antimicrob. Agents Chemother. 16:277-282. 15. Hahon, N. (1974) Depression of viral interferon induction in cell monolayers by coal dust. Br. J. Indus. Med. 31:201-208. 16. Hahon, N., and Eckert, H.L. (1976) Depression of viral interferon induction in cell monolayers by asbestos. Environ. Res. 11:52-65. 17. Waker, A., Feller, H., and Gericke, Da Hemmung. (1971) Der inter feron-induction durch 20-methylcholanthrene j_n vivo. Naturwissenschaften 58:274-275. 18. Roe, F.L.C., et al. (1963) Tests for carcinogenesis using newborn mice: 1,2-benzanthracene, 2-napthylamine, 2-naphthylhydroxylamine and ethyl methanesulfonate. Br. J. Cancer 17:255-260. 19. Casto, B.C., et. al. (1977) Development of a focus assay for trans formation of hamster cells jjn vitro by chemical carcinogens. Cancer Res. 37:3508-3515. 20. Chasseaud, L.F. (1979) Glutathione (reduced) and glutathione-Stransferase block the carcinogenic event by trapping the ultimate metabolites. Adv. Cancer Res. 29:176-255. CONFIDENTIAL ^^bjoct to rrotset i vs Ordor in ?oss_v. Conoco, Ir.c., !To. 90-4837 'th Judicial District Court '"sieu Parish, Louisiana CMA 003547 12. QeMaeyer-Guignard, J,, and DeMaeyer, E. (1965) Effects of carcinogenic and noncarcinogenic hydrocarbons on interferon synthesis and virus plaque development. J. Natl. Cancer Inst. 34:264. ------------------------------------- 13. DeMaeyer-Guignard, J., and DeMaeyer, E. (1967) Inhibition of interferon synthesis by triethylenemelamine and 4-nitroquinoline-N-oxide. Arch. Gesamte Virusforch. 22:61-68. 14. Hahon, N., et al. (1979) Aflatoxin inhibition of viral interferon induction. Antlmicrcb. Agents Che,mother. 16:277-232. 15. Hahon, N. (1974) Depression of viral interferon induction in cell monolayers by coal dust. Br. J. Indus. Med. 31:201-208. 16. Hahon, N., and Eckert, H.L. (1976) Depression of viral interferon induction in cell monolayers by asbestos. Environ. Res. 1 1:52-65. 17. Maker, A., Feller, H., and Gericke, Da Henunung. (1971) Der inter feron-induction durch 20-methylcholanthrene in vivo. Naturwissenschaften 58:274-275. 18. Roe, F.L.C., et al. (1963) Tests for carcinogenesis using newborn mice: 1,2-benzanthracene, 2-napthylamine, 2-naphthylhydroxylamine and ethyl methanesulfonate. Br. J. Cancer 17:255-260. 19. Casto, B.C., et. al. (1977) Development of a focus assay for trans formation of hamster cells jn vitro by chemical carcinogens. Cancer Res. 37:3508-3515. 20. Chasseaud, L.F. (1979) Glutathione (reduced) and glutathione-Stransferase block the carcinogenic event by trapping the ultimate metabolites. Adv. Cancer Res. 29:176-255. CONFIDENTIAL Subject to Protective Order in Boss v. Conoco, ino . ilo 90-4837 14th Jud.i e1 * or"i"t r-ourt Calcas1'1'" 55' CMA 003548 PROGRAM G THE STUDY OF TISSUE ANTIGENS AND ANTIBODIES IN THE DETECTION OF VINYL CHLORIDE INJURY; Enrique Espinosa, M.D. Gl. The Study of Tissue Antigens from Liver Tumors, Angiosarcoma, and Hepatomas. G2. Circulating Antigens and Autoantibodies in Vinyl Chloride-Associated Liver Disease Background In hepatic fibrosis and angiosarcoma associated with vinyl chloride exposure of industrial workers, manifestations of the disease could not be detected in most cases until the process was far advanced (1). Normal values of liver function tests were reported in a case with significant vinyl chloride hepatic fibrosis (2), and only a small percentage of workers of a plant unit where 7 cases of liver angiosarcoma were diagnosed had abnormal blood screening tests (3). Thus, conventional liver function tests do not appear to be sensitive indicators of vinyl chloride liver disease. Develop ment of more sensitive and specific methods for detecting the disease in early stages would be of great importance. An approach to this may be provided by antigenic studies in view of the tissue antigenic modifications which are known to occur in neoplasia. For example, in human carcinoma, loss of antigens have been reported in squamous cell carcinoma (4,5) and in ovarian carcinoma (6,7), loss of the ABH blood group isoantigens in some solid tumors (8,9), and of HL-A isoantigen in lymphoma (10). Also, tumor-specific trans plantation antigens have been demonstrated in a number of experimentally induced tumors (11-14) as well as tumor-associated antigen in spontaneous tumors in man (e.g. 15-18). In such an antigenic study of liver angiosarcoma the question arises whether the fibrotic and angiosarcomatous livers contain antigens that are quantitatively or qualitatively different from those present in normal tissue and whether such changes could stimulate an immunologic response. If such hypothetical changes are demonstrable and proved to be specific, they may be of use in diagnosis of vinyl chloride related liver disease. Objective 1. To test for liver antigenic changes in a) angiosarcomatous liver tissue, b) chemically-induced hepatomas, and c) cultured human liver carcinoma cells. CONFIDENTIAL Subject to Protective Order in Foss v. Cor.oco, Inc., No. 90-4837 14th Judicial District Court 1 si eu Perish, T oui siane CMA 003549 81 2. To test for abnormal serum antigens and autoantibodies in patients with histories of vinyl chloride exposure including individuals with liver angiosarcoma, liver dysfunction with fibrosis, and with normal liver function tests. Antigens studied included liver specific antigen (LSA) (19), and F-antigen (20,21), tissue antigens of wide organ distribution (22,23), and bile antigens (24). Autoantibodies were to nuclei, mitochondria and smooth muscle. Patients' sera were also tested by immunofluorescence for possible reactivity with rats exposed to vinyl chloride (25). Research Results 1. Liver antigenic changes a) angiosarcomatous liver tissue Liver angiosarcomatous tissue was analyzed in this work for presence of neoantigens, normal tissue antigens and tumor bound immunoglobulins. Several normal tissue antigens were found by immunodiffusion to be present in the tumor. These included the previously described liver-specific antigen, (LSA) (19) and antigens which are shared with several tissues (22,23). In addition, a tumor associated protein antigen and a glycoprotein liver antigen found to be absent in angiosarcoma were identified and characterized (25). Tissue distribution of liver angiosarcoma related antigen is shown in Table 1. Preparations containing tumor associated antigen were found to inhibit lymphocyte transformation tests in vinyl chloride workers suggesting that these workers have a decreased lymphocyte response to these preparations (26). Further, antigen coagulation Factor VIII was detected in the angio sarcoma cells, pointing to an endothelial cell origin of the tumor (25). Finally, immunoglobulin G bound to the angiosarcomatous tissue was demon strated by immunofluorescence and elution experiments suggesting antibody stimulation by the tumor (25). TABLE 1 TISSUE DISTRIBUTION HW LIVVR ANGIOSARCOMA* ABLATIO ANTIGEN TISSUE EXTRACT USED AS AOSOROBNT A NO 103 A ft COM A - RELATED A NTIGEN* Llr wj(taai f Coma tt*r Kidney $?( Luftf * 4 4 Aaili|ioire0tiii irvm ibiofbed lyopfellUed *strct, 100 atf/ml, 4*4 tested fgalAti <i|l4i rc<ni tunct. COHFIDSITTIAL f ia(|ffl Il4lealt< by . Subject to Protective Order in r03s V. Conocn. 9Q--S37 14rh Judical riser let Court Calcasieu Parish, Louisiana CMA 003550 82 b) chemically-induced hepatomas Our finding of an antigen missing in VC-related liver angiosarcoma stimulated further studies of antigenic deletion in chemically-induced hepatomas and in cultured human liver carcinoma cells. In studies performed with the fast growing and undifferentiated chemically-induced Morris hepatoma 7777, 2 liver antigens were found to be absent. These antigens were characterized and partially isolated. In studies of their occurrence in other tissues, one of these antigens (Antigen I) was shown to be present in kidney and spleen in addition to liver. The second antigen (Antigen II) was detected only in liver. Antigen II was found unrelated to liver-specific F-antigen, differing in a number of properties and in immunologic reactivity. In studies of their subcellular distribution in normal liver. Antigen I appeared localized in cytosol (54%) and mitochondrial (38%) fractions. Antigen II was about equally distributed in cytosol, mitochondria and nuclei fractions with little amounts in microsomes. Antigen I has a electrophoretic mobility in immunoelectrophoresis close to that of serum ganma-globulins and Antigen II to that of serum alpha-globulins. The two antigens were completely inactivated with Pronase indicating that both antigens are proteins or protein associated. Both antigens were relatively thermolabile; they were partially inactivated following incubation at 56C and completely inactivated at higher temperatures. Both antigens were completely inactivated when incubated irupH buffer lower than 3.5. In Sephadex-G200 gel filtration. Antigen I beMkd like a protein of approximately 51,000 Daltons, using as standards s^ran albumin, ovalbumin, chymotrypsinogen and ribonuclease. The molecular size of Antigen II (determined on a Bio-gel A5m column) was approximately 240,000 Daltons, using aldolase, catalase and ferritin as markers. The two antigens were found in the more differentiated and slower growing hepatomas 5123tc and 9618A at about the same concentration as normal liver. The fact that hepatoma 7777 is the fastest growing and least differentiated of the tumors studied suggests a possible functional relationship between the absent antigens and these properties (28). These antigenic deletions may be used as indicators in the early detection of liver tumors and in the evaluation of the rate of growth, histologic differentiation and metastatic properties of such hepatomas. Another liver constituent which may serve as a sensitive indicator of chemically-induced liver tumors, liver-specific F-antigen, was studied. In studies on the behavior of this antigen in Morris hepatomas, it was found that different types of these chemically-induced tumors have quite different levels of F-antigen. F-antigen appeared to be absent in the fast growing hepatoma 7777. In the slow growing hepatoma 9618A, the concentration was very low ranging from less than 2% to 10% of the normal liver concentration. The medium growing hepatoma, 5123tc, highly metastatic, had about twice the concentration as cor r 11 In 90-4337 t Court T -I --iana CMA 003551 in in6m\ c & * v in cultural r.LT.an liver carcinoma cells. In studies perrormea witn the fast growing and undifferentiated cheri c 2' ' 1 r z .tec 'jct's ^ ec it'ci "'7~7J ? 1' er sn'iij"; .'/ors 'c.rc c* b'5 absent. These antigens were cnaracterized and partially isolated. In studies of their occurrence in other tissues, one of these antigens (Antigen I) was snown to be present in kidney and spleen in addition to liver. The second antigen (Antigen II) was detected only in liver. Antigen II was found unrelated to liver-specific F-antigen, differing in a number of properties and in immunologic reactivity. In studies of cneir suocellular distribution in normal liver, Antigen I appeared local iced in c,,-:asui (;':) ano mi cocnonarial (38%) fractions. Antigen II was about eaually distributed in cytosol, mitochondria and nuclei fractions with little amounts in microsomes. Antigen I has a electrophoretic mobility in immunoel actrooho'-es is close to that of serum gamma-globulins and Antigen II to tnat of serum alpha-globulins. The two antigens were completely inactivated witn Pronase indicating that both antigens are proteins or protein associated. Both antigens were relativelythermo!abi1e; they were partially inactivated following incubation at 56C and completely inactivated at higher temperatures. Both antigens were completely inactivated when incubated in pH buffer lower than 3.5. In Sephadex-G20Q gel filtration, Antigen I behaved like a protein of approximately 51,000 Daltons, using as standards serum albumin, ovalbumin, chymotrypsinogen and ribonuclease. The molecular size of Antigen II (determined on a Bio-gel A5m column) was approximately 240,000 Daltons, using aldolase, catalase and ferritin as markers. The two antigens were found in the more differentiated and slower growing hepatomas 5123tc and 9618A at about the same concentration as normal liver. The fact that hepatoma 7777 is the fastest growing and least differentiated of the tumors studied suggests a possible functional relationship between the absent antigens and these properties (28). These atvtigenic deletions may be used as indicators in the early detection of liver tumors and in the evaluation of the rate of growth, histologic differentiation and metastatic properties of such hepatomas. Another liver constituent which may serve as a sensitive indicator of chemically-induced liver tumors, liver-specific F-antigen, was studied. In studies on the behavior of this antigen in Morris hepatomas, it was found that different types of these chemically-induced tumors have quite different levels of F-antigen. F-antigen appeared to be absent in the fast growing hepatoma 7777. In the slow growing hepatoma 9618A, the concentration was very low ranging from less than 2% to 10% of the normal liver concentration. The medium growing hepatoma, 5123tc, highly metastatic, had about twice the concentration as CON TIDE !TT IA.L Subject to Protective Order in Ross v. Conoco. Inc. , No. 90-4337 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003552 83 normal liver. F-antigen of hepatoma 5123tc and of normal liver were found localized in the cytosol subcellular fraction and were determined to be immunological ly identical and to have equivalent electrophoretic mobility and molecular weight (29). Since the antigen was undetectable in the fast growing hepatoma and undetectable or very low in the slow hepatoma, the level of F-antigen does not appear to correlate with the rate of growth of these tumors. A possible relationship between metastatic properties and F-antigen is now being considered because the hepatoma with the increased concentration of F-antigen was by far the most highly metastatic. This may prove useful in treatment of tumors. c) cultured human hepatoma cells In studies on cultured human liver carcinoma cells (after establishing optimal conditions required for the maintenance in serum free media of PLC/PRF/5 human liver carcinoma cells) it was determined that these hepatoma cells, similar to the experimental Morris hepatoma 7777, are deficient in liver-specific F-antigen. Nevertheless, these cells, like normal liver cells, produce serum albumin, fibrinogen, transferrin, alpha-1 antitrypsin and alpha-2 macroglobulin as shown by immunodiffusion (30,31) and immunofluorescence (32). These data add further support to the clinical observation that tissue antigens are more useful for treatment and follow-up care than screening and early detection, and that antigenic deletions may prove useful in early screening. 2. Abnormal Serum Antigens and Autoantibodies Serum autoantibodies to nuclei, mitochondria and smooth muscle were negative in all patients examined. In addition, serum from these patients did not show reactivity with liver from rats exposed to vinyl chloride. Liver-specific antigen LSA (19), bile antigens (24) and other tissue antigens (22, 23) associated with liver damage were not detected in these patients (25). Relevance to Industry These studies are relevant to chemical industry in view of the evidence provided showing important changes in the levels of angiosarcomatous liverantigenic components in association with vinyl chloride exposure. Deletion of liver antigens were also demonstrated in chemically - induced hepatoma. In addition, these observations may eventually prove useful in the development of new approaches for screening of chemical-associated tumors. References 1. Heath, C.W., Jr., Falk, H., and Creech, J.L. (1975) Characteristics of cases of angiosarcoma of the liver among vinyl chloride workers in the United States. Ann. N.Y. Sci., 246:231. CONFIDENTIAL Subject to Protective Order in Foss v. Ccr.oco, Inc. . No. 90-4837 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003553 2. Berk, P.D., Martin, J.F., and Waggoner, J.G. (1975) Persistence of vinyl chloride-indeuced liver injury after cessation of exposure. Ann. N.Y. Acad. Sci., 246:70. 3. Wyatt, R.H., Kotchen, J.M., Hochstrasser, D.L., Buchanan, J.W., Jr., Campbell, D.R., Slaughter, J.C., and Doll, A.H. (1975) An epidemiologic study of blood screening tests and illness histories among chemical workers involved in the manufacture of polyvinyl chloride. Ann. N.Y. Acad. Sci., 246:80. 4. Nairn, R.C., Richmond, H.G., McEntegart, M.G., and Fothergill, J.E. (1960) Immunological difference between normal and malignant cells. Br. Med. J., 2^: 1355. 5. Carruthers, C., and Baumler, A. (1965) Inmunochemical staining with fluorescein-labeled antibodies as an aid in the study of skin cancer formation. J. Natl. Cancer Inst., 34:191. 6. Burton, R.M., Hope, N.J., Beyerle, M.P. and Espinosa, E. (1977) Tissue antigens in ovarian carcinoma. Oncology 34:146. 7. Burton, R.M., Hope, N.J., Beyerle, M.P. and Espinosa, E. (1978) Gewegsantigene bein ovarialkarzinom. Onkologie 1:75. 8. Kay, H.E.M. (1957) A and B antigens in normal and malignant cells, Br. J. Cancer, 11:409. 9. Davidson, I., and Ni, L.Y. (1969) Loss of isoantigens A, B and H in carcinoma of the lung. Am. J. Pathol., 57:307. 10. Seigler, H.F., Kremer, W.B., Metzgar, R.S., Ward, F.E., Haung, A.T., and Amos, D.B. (1971) HL-A antigenic loss in malignant trans formation. J. Natl. Cancer Inst., 46:577. 11. Cryan, W.S., Hide, R.M., and Garb, S. (1966) Demonstration by gel diffusion of antigen in spontaneous mouse tumors. Cancer Res., 26:145 12. Heppner, G.H., and Pierce, G. (1969) In vitro demonstration of tumor-specific antigens in spontaneous mammary tumors in mice. Internat. J. Cancer, _4:212. 13. Isojima, S., Yagi, Y., and Pressman, D. (1969) Antigens conmon to rat hepatoma induced with 2-acetylaminofluorene. Cancer Res., 29:140. 14. Kahan, B.D., Holmes, E.C., Reisfeld, R.A., and Morton, D.L. (1969) Water soluble guinea pig transplantation antigen from carcinogeninduced sarcomas. J. Immunol., 102:28. Pqrish, - v?*er in -0. 90-4BZ7 ct Court Louisiana CMA 003554 _ i ' , '.w,; ,! 71 j1' '.il: ' ":f* ` 3 ^ * 'J Wyatt, R.H., Kotchan, J.M., Hcchstrasser, D.L., Buchanan, J.W., Jr., Campbell, D.R., Slaughter, O.C., and Doll, A.H. (1975) An epidemiologic study of blood screening tests and illness histories among che-ic = i wcrkers involves in the manufacture of polyvinyl chloride. Ann. N.Y. Acad. Sci., 246:80. 4. Nairn, R.C., Richmond, H.G., McEntegart, M.G., and Fothergill, J.E. (I960) Immunological difference between normal and malignant cells. 8r. Med. J., 2:1355. - fluorescein-laoeiea antiDodies as an aid in the study of skin cancer formation. J. Natl. Cancer Inst., 34:191. 6. Burton, R.M., Hope, N.J., Beyerle, M.P. and Espinosa, E. (1977) Tissue antigens in ovarian carcinoma. Oncolcqy 34:146. 7. Burton, R.M., Hope, N.J., Beyerle, M.P. and Espinosa, E. (1978) Gewegsantigene bein ovarialkarzinom. Onkoloqie 1:75. 8. Kay, H.E.M. (1957) A and B antigens in normal and malignant cells, Br. J. Cancer, 11:409. 9. Davidson, I., and Ni, l.Y. (1969) Loss of isoantigens A, B and H in carcinoma of the lung. Am. J. Pathol., 57:307. 10. Seigler,H.F., Kremer, W.B., Metzgar, R.S., Ward, F.E., Haung, A.T., and Amos, D.B. (1971) HL-A antigenic loss in malignant trans formation. J. Natl. Cancer Inst., 46:577. n. Cryan, W.S., Hide, R.M., and Garb, S. (1966) Demonstration by gel diffusion of antigen in spontaneous mouse tumors. Cancer Res., 26:145, . 12. Heppner, G.H., and Pierce, G. (1969) In vitro demonstration of tumor-specific antigens in spontaneous mammary tumors in mice. Internat. J. Cancer, 4:212. 13, Isojima, S., Yagi, Y., and Pressman, D. (1969) Antigens common to rat hepatoma induced with 2-acetyl aminofluorene. Cancer Res., 29:140. 14. Kahan, B.D., Holmes, E.C., Reisfeld, R.A., and Morton, D.L. (1969) Water soluble guinea pig transplantation antigen from carcinogeninduced sarcomas. J. Immunol., 102:28. CONFIDENTTM T. ^ PoSsUsbi9Ctr t0 ?7^^r^der ln, V1 v Conoco^^ ff0. 90.48 ' 14th Judicial District Court Calcasieu Parish. Louisian CMA 003555 85 15. Hughes, L.E., and Litton, B. (1964) Antigenic properties of human tumors: Delayed cutaneous hypersensitivity reactions. Br. Med. J. J_:209. ------------- 16. Itakura, K. (1963) Studies on human cancer antigens by gel diffusion methods. Gann, 54:93. 17. McKenna, J.M., Sanderson, R.P., and Blakemore, W.S. (1962) Extrac tion of distinct antigens from neoplastic tissue. Science, 135:370. ------------ 18. Burton, R.M., McGrew, T.L., Barrow, G.H., Beyerle, M.P., Fortwengler, P.H., Day, T.G., Kuhns, S.H., and Espinosa, E. (1979) Occurence of a thermostable antigen of ovarian carcinoma in normal tissues and secretions. Cancer, 43:2385. 19. Espinosa, E. (1973) Circulating tissue antigens. II. Studies on an organ-sepcific antigen of human liver. Lab. Invest., 29:556. 20. Espinsoa, E. (1977) F-antigen in liver injury. Gastroenterology, 72:985. 21. Espinosa, E. and Caple S. (1981) Extrahepatic immunofluorescent reactivity of antisera detecting liver F antigen. Fed. Proc,, 40:764. 22. Espinsoa, E. (1974) Circulating tissue antigens. I. Tissue antigens in serum of patients with diseases involving injury of the liver and of other organs. Clin. Exp. Immunol., 16:153. 23. Espinosa, E. (1976) On two tissue antigens detected in pathologic sera. Lab. Invest., 34:8. 24. Espinosa, E. (1976) Circulating tissue antigens. III. Identifica tion and characterization of antigens of limited and of wide body distribution in human gallbladder bile. Presence of serum of patients with acute hepatitis, Clin, Exp, Immunol., 25:410. 25. Espinosa, E. (1976) Immunopathologic observations in liver angio sarcoma. In Nieburgs, H.E. (ed.). Third international symposium on detection and prevention of cancer proceedings, New York, Marcel Dekker, Inc. 26. Fortwengler, H.P., Dever, M.E., Tamburro, C.H. and Espinosa, E. (1978) Lymphocyte transformation tests in vinyl chloride (VC) workers. Fed. Proc., 37:362. 27. Fortwengler, H.P., Jr., Jones, D., Espinosa, E. and Tamburro, C.H. (1981) Evidence for endothelial cell origin of vinyl chloride induced hepatic angiosarcoma. Gastroenterology, 80:1415. CONFIDENTIAL '^ Subject to Protective Order Inf ' Poss v. Cor.cco, Inc. , Ko. 90-4837i 14th Judicial District Court Calcasieu Parish, Louisiana 003536 86 28. Espinosa, E., Caple, S., Kuchella, C. and Chia, S. (1979) Two liver antigens undetectable in a fast growing line of transplanted hepatoma (Morris Hepatoma 7777). Fed. Proc., 38:1069. 29. Espinosa, E., Chia, S., Caple, S. and Kupchella, C. (1979) Liverspecific antigen in transplantable hepatomas having different growth rates. Fed. Proc., 38, 1069. 30. Johnston, P.B., Espinosa, E., Chia, S. and Caple S. (1979) Proper ties of 14 week matintenance cultures of PLC/PRF/5 cells. In vitro, J_5:227. -- 31. Espinosa, E., Johnston, P.B., El-Naggar, M.M. and Caple, S. (1980) Serum and liver antigens in cell sheets and culture fluid of PLC/PRF/5 human hepatoma cells. Lab. Invest., 42:18. 32. Espinosa, E., Johnston, P.B., and Caple, S. (1980) Immunofluorescent localization of plasma proteins in cultured human hepatoma cells. Fed. Proc., 39:777. cunr'i 1 v. S22!Jr^, 90.4S yV District Court alcasieu Parish, Louisiana CMA 003557 PROGRAM H THE USE OF ISOLATEO MAMMALIAN LIVER CELLS FOR THE STUDY OF CHEMICAL MONOMER METABOLISM; Investigator, R. C. Feldhoff HI. Isolation of Mammalian Liver Cells for the Study of the Metabolism of Chemical Monomers H2. The In Vitro Study of Albumin Synthesis by Liver from Human Biopsies Background Presently there is no simple or even complex method to assess the functional capability of the human liver to handle exposure to various chemicals and/or their metabolites. In general, the rat has proven to be the most useful experimental animal for studying hepatic metabolism. Perfused liver slices and isolated hepatocyte systems have been developed to better study the interrelationship and to control the myriad processes of liver metabolism. In the perfused liver the architecture of the organ is preserved but separated from other body influences. Newer in situ techniques allow the liver tissue to never be without a supply of oxygenated red cells. The same technique can also be used to prepare isolated liver cells enzymatically. The intact liver consists of at least four cell types, whereas isolated liver cells are nearly homogeneous in population. These cell suspensions provide technical advantages in that a number of conditions can be tested at one time and many uniform serial samples can be collected. Preliminary investigations were therefore done to determine whether the human liver biopsy material can function in a manner equivalent to liver slices and whether these tissues would demonstrate similar findings to those of isolated cell suspensions. If this technique were made functionally feasible it could then be used to study the metabolism of xenobiotics using viable human liver tissue in an in vitro manner. Objective 1. To determine if an in vitro adaptation could be developed which would utilize animal/human biopsy material for assessing the functional capability of human liver cells. Protein metabolism has been used as the standard indicator and criteria for comparison VCal<^ 87 CMA 003558 88 .2 To determine the usefulness of this technique in human biopi material obtained for routine medical purposes via percutaneous and transvenous approaches. Research Results Our first step was to verify the clinical and biochemical conditions needed for handling human liver biopsy material obtained during routine medi cal procedures. Appropriate assay conditions are being determined to assess the functional capability of the liver at the time of the biopsy to synthesize retained and secreted protein. At the same time, the biopsy's histopathology will be assessed and correlated with the biochemical data. The technique used to study both protein synthesis and secretion is shown in Figure 1. The his topathology is illustrated in Figure 2. IN VITRO TECHNIQUE FOR STUDY OF HUMAN LIVER TISSUE CUT' - RETAINED PROTEIN - SECRETORY PROTEIN LIGHT MICROSCOPY LIVER BIOPSY FIGURE 1 c*fmT*A*rtw*tA*t-*ltp*Utfm)mt #*tr DIVIDED LIVER BIOPSY Of IX (pmuTAM*S*4i.9m r* '*0t**f04* jvrnpe a**lv*i FIGURE 2 Or dor - 'io. 90-4331; T7r;ct Oourt -v, T'O'i AiTfi ftt^rtfi u/nfirt a rl_Ui Alfa<#***p4*i.*k*<4|nK 003559 2. To cetermire the usefulness of to is technique in ,T. - C- *- ' w ^ r J ^ ' m -r : ! * : i V r 1.7 J v C.7 C Li i -r',-'1 - - -r *1 " 3 * Research Results Our first step was to verify the clinical and biochemical conditions neeaed for handling human liver biopsy material obtained during routine medi cal procedures. Approoriate assay conditions are being determined to assess the functional capability of the liver at the time of the biopsy to synthesize retained and secreted protein. At the same time, the biopsy's histopathology will be assessed and correlated with the biochemical data. The technique used to study both protein synthesis and secretion is shown in Figure 1. The his topathology is illustrated in Figure 2. IV V'T-0 nr.-u'jmi'r FOR STUDY OF HUMAN UVES TISSUE CUT - RETAINED PROTEIN - IECPE7CRY PROTEIN LIGHT MICROSCOPY LIVER 3I0PSY FIGURE 1 *44* DIVIDED LIVER SICPSY o* 7** jW47ym *nm*v+us*4if*47S944cmnn/t. m44p04 H 7 4444C*Mp *-*4.**w*fW* CMA 003560 89 The initial experimental studies utilizing liver perfusion and the isola tion of hepatocytes techniques provided the hepatocytic and nonparenchyma 1 cells for Du and Tamburro's study of the oxidizing and detoxifying capabili ties of these cells. Subsequent experiments have been used to quantitate the intracellular albumin synthesis, the rate of albumin secretion by the use of specific antibody fractions available for human and rat albumin. Initial results are illustrated in the rocket immunoelectrophoresis patterns from rat liver biopsy study (Figure 3) and the amino acid analysis is seen in Figure 4. The technique for liver cell identification after cell separation by the perfusion technique is illustrated in Figure 5. FIGURE 3 cg:;?i32ittial Subject to Protective Crier ini Ecss v. Ccr.oeo. Ire., :,`o. 90-4S3'3f 14th Judicial Disc:1 Let '"curt j Calcasieu Parish, Louisiana , CMA 003561 90 FIGURE 4 MARKER CELL TYPE dna/rna ALL CELLS KC EC FB Relevance to Industry Although this study was a last year addition to our initial proposal, it has provided the preliminary exploration into the adaptation of highly sophisticated laboratory techniques to human tissue in an in vitro system. While the work of these studies is still developmental, it provides promise for future clinical use. Such use could include determination of an indi vidual liver's ability to oxidize and detoxify xenobiotics. The degree and nature of liver cellular injury or impairment could be determined in a quantitative biochemical fashion, and could be directly correlated with CO:TF INITIAL Subject to Protective Order in Ross v. Conoco, Inc., No. 90-48371, 14th Judicial District Court j Calcasieu Parish, Louisiana: VL CMA 003562 figure 4 MARKER CELL TYPE dna/rna ALL CELLS KC EC FIGURE 5 EB Relevance to Industry Although this study was a last year addition to our initial proposal, it has provided the preliminary exploration into the adaptation of highly sophisticated laboratory techniques to human tissue in an in vitro system. While the work of these studies is still developmental, it provides promise for future clinical use. Such use could include determination of an indi vidual liver's ability to oxidize and detoxify xenobiotics. The degree and nature of liver cellular injury or impairment could be determined in a quantitative biochemical fashion, and could be directly correlated with CONFIDENTIAL '' v'$ Subject to Protective Order in'' Poss v. Cor.oco , Ir.o . , I.o . 90-48371 Cioorict Court CMA 003563 91 morphological findings. These techniques could be also used to determine the degree of biochemical adaptations of the human tissue in individuals who have been exposed to xenobiotics. finally, these methods could, on sequential biopsy, provide clinical information regarding whether these alterations persist or revert back to a normal state. Although this is a very futuristic attempt to adapt presently developed methodology, it does accurately reflect the direction in which clinical research should be directed in order to better assess the human capability of sustaining environmental changes. References 1. Du, J. and Tamburro, C.H. (1980) Oxidative and detoxifying ability of liver mesenchymal vs. parenchymal cells in the metabolism of xenobiotics. Gastroenterology 79, 1013, 2. Exton, J.H. (1975) The perfusion rat liver. Methods in Enzymoloqy 37, 25-37. 3. Feldhoff, R.C., Taylor, J.M., and Jefferson, L.S. (1977) Synthesis and secretion of albumin in vivo, in perfused liver and isolated hepatocytes: Effects of~ hypophysectomy and growth hormone treatment. J. Biol. Chem. 252, 3611-16. 4. Feldhoff, R.C., Taylor, J.M., and Jefferson, L.S. (1977) Albumin synthesis by isolated rat hepatocytes. Effects of amino acids, bovine albumin, rat serum and hormones. Fed. Eur. Bioch, Soc. (Abstract). 5. Ledden, D.J., Feldhoff, R.C. and Gray, R.D. (1980) Purification and characterization of human albumin fragments. Fed. Proc. 39, 1676 (Abstract). 6. Leevy, C.M. (1963) In vitro studies of hepatic DNA synthesis in percutaneous liver biopsy specimens from man. J. Lab. Clin. Med. 61, 761-766. 7. Peters, T., Jr. (1975) Serum albumin. J_n The Plasma Proteins, 2nd ed., Putnam, F.W. ed., Vol. I, pp. 133-181, Academic Press, NY. 8. Peters, T., Jr. (1980) Albumin: An overview and bibliography. Miles Laboratories, Inc., Elkhart, IN. 9. Whelan, J.F., Jr., Creech, J., Jr. and Tamburro, C.H. (1981) The adequacy and safety of transvenous hepatic biopsy in complicated hepatic disease. Clinical Research 29 313A. Subject to Ftotecttve Order la.. POSJLV. CJS2224JSU."- court" j "14th Judicial ,j Cnlr'.asiRU P*rish' L CMA 003564 PROGRAM I THE STUDY OF TISSUE DISPOSITION OF INDUSTRIAL CHEMICALS: THE VINYL CHLORIDE EXAMPLE; Investigators - W.O. Waddell and C. Marlowe II. The Use of Whole Body Autoradiography in Specific Tissues. Localization of Accumulated and Retained Industrial Chemicals and Their Metabolites Background Vinyl chloride has been classified by regulatory agencies as a carcinogen. Chronic exposure of workers to this carcinogen has resulted in angiosarcoma of the liver, as well as other tumors (Creech and Johnson, 1974). Several types of tumor also are observed after vinyl chloride exposure to animals (Maltoni and Lefemine, 1974). How or why these various body sites developed tumors was unclear. The need for methods which more accurately identify the sites of localization of chemical agents and their metabolites has obvious importance. The use of whole body autoradiography to identify the ultimate location of radioactively tagged chemicals was studied utilizing 14C-vinyl chloride. Objective 1. To determine the sites of localization and retention of the metabolites of vinyl chloride in mice. 2. Identification of the tissues of localization should help elucidate the mechanism of carcinogenic action of vinyl chloride. Research Results The sites of localization of radioactivity in tissues of the mouse following exposure to ^C-vinyl chloride were studied by the technique of whole-body autoradiography (Waddell and Marlowe, 1977). Male CD-I mice purchased from Charles River, weighing 25--3Ig-, were exposed to `4C-vinyl chloride in room air in a sealed chamber for 3 hours. The radioactive vinyl chloride was synthesized from ^c_ethyiene dichloride (New England Nuclear, Lot all94--143, specific activity 3.2 mCi/mmole) immediately prior to exposure by the method of Wagner et al. (1975). The initial concentration of the vinyl chloride was 50 ppm with a specific activity of 1.84 mCi/mmole. At the end of the 3 hour exposure of the mice to ^C-vinyl chloride- the mice were removed to room air. Twenty minutes, 1, 3, 9 or 24 hours after removal of the mice to ^'ib.-ect to rrotect-ive Order in' Foss v, Gor.ooo, Ins., IFo. 90-48371' 14th Judicial District Court- -i Calcasieu Parish, Louisiana V| 92 CMA 003565 93 room air, a mouse was briefly anesthesized with ether and sacrificed by freezing in a dry ice/hexane bath at -75C. The preparation of the radioactive vinyl chloride, exposure of the mice to this isotopic gas, and the sacrifice of the mice were done in collaboration with Dr. William T. Stott at Dow Chemical in Midland, Michigan. All remaining procedures were performed at the University of Louisville. Whole-body sagittal sections of the mice, 20u- and 40y- thick were taken onto Scotch tape at -20*C. After freeze-drying, these sections were placed against Kodak AA X-ray film and allowed to expose in light-tight containers at -14*C for 6 to 39 weeks. The sites of localization of all nonvolatile metabo lites of vinyl chloride were visualized in the developed X-ray film (auto radiograph) and reveals the j_n vivo disposition of these metabolites at the time of sacrifice of the mouse"! These procedures for whole-body autoradi ography, first described by U11 berg in 1954 do not allow thawing or contact with any solvents. Thus, there is no translocation nor loss of radio activity. A complete description of these procedures have been published previously (Waddell and Marlowe, 1977). Representative autoradiographs from each mouse were used as negatives to produce the prints included with this report. Therefore, white areas in the prints represent the sites of radioactive accumulation of the nonvolatile metabolites of ^C-vinyl chloride. The highest levels of radioactivity in the mice sacrificed 20 minutes 1 hour after removal from the ^C-vinyl chloride environment were obserl^P in liver, pancreas, kidney, intestinal contents, urine and bile (Figures 1 and 2). Additional sites of localization of radioactivity in the 1-hour animal were thymus, thyroid and seromucous glands (Figure 2). The concentration of metabolites in the organs of excretion decreased continually over the 24 hour period (Figures 3-5). By 9 hours after removal from the vinyl chloride, thymus, Harder's gland, epithelium of the esophagus and intestine, urine and sublingual gland retain the highest levels of radioactivity; moderate concentrations of radioactivity were observed in liver, kidney and intestinal contents (Figure 4). After 24 hours the primary organs of retentions of radioactivity were thymus and Harder's gland; some radioactivity could be visualized in liver and in esophageal and intestinal epithelium (Figure 5). Figure 6 shows the relative concentration of nonvolatile metabolites in the cortex and medulla of the thymus at the earliest and latest time intervals studied. Twenty minutes after removal from the vinyl chloride, the concentration of radioactivity in the thymus was only slightly increased above that of blood (Figure 1 and 6); however, by 1 hour the thymus showed the highest uptake of radioactivity in the body (Figure 2) and remained the highest throughout the 24 hour period (Figures 2-6). COH-'--' A1 Crdsr Sub!set '-o T-.c., 'jo- 90 - 43313' PCSS V. -- Court -i Cgl("is',-0u pOT'l, "Vl , Louisiana. .'Ji CMA 003566 3' '; ' ' .v : " ifly ar-53:'3: ' z?d with ether = 33th ;;;r : = . c: :re rad1, oac: i ve vinyl chloride, exposure of the mice to this ;sot:oic gas, and the sacrif're of the mice were done in collaboration witn Or. William T. Stott at Dow Che 1 ica 1 in Midland, Michigan. All remaining procedures were performed at the University of Louisville. wnoie-oody sagittal sections of the mice, 20u- and 40u- thick were taken onto Scotch tape at -20'C. After freeze-drying, these sections were placed against Kodak AA X-ray film and allowed to expose in light-tight containers at -14C for 6 to 39 weeks. The sites of localization of all nonvolatile metabo lites of vinyl chloride were visualized in the developed X-ray film (auto- rarficgraDh) and revea s the in vivo disposition of these metabolites at the -a ~ - ' U 5 0 These procedures for ; `lOic--oooy auscrac`- w-F 1iy, first oescribed by Ul'berg in 1954 do not al low tnawing or contact '.rtnW any solvents. Thus, there is no translocation nor loss of radio- activity. A complete description of these procedures have been published previously (Waddell and Marlowe, 1977). Representative autoradiographs from each mouse were used as negatives to proGuce the prints included with this report. Therefore, white areas in the prints represent the sites of radioactive accumulation of the nonvolatile metabolites of ^C-vinyl chloride. The highest levels of radioactivity in the mice sacrificed 20 minutes and 1 hour after removal from the ^C-vinyl chloride environment were observed in liver, pancreas, kidney, intestinal contents, urine and bile (Figures 1 and 2). Additional sites of localization of radioactivity in the 1-hour animal were thymus, thyroid and seromucous glands (Figure 2). The concentration of metabolites in the organs of excretion decreased continually over the 24 hour period (Figures 3-5). By 9 hours after removal from the vinyl chloride, thymus, Harder's gland, epithelium of the esophagus and intestine, urine and sublingual gland retain the highest levels of radioactivity; moderate concentrations of radioactivity were observed in liver, kidney and intestinal contents (Figure 4). After 24 hours the primary organs of retentions of radioactivity were thymus and Harder's gland; some radioactivity could be visualized in liver and in esophageal and intestinal epithelium (Figure 5). Figure 6 shows the relative concentration of nonvolatile metabolites in the cortex and medulla of the thymus at the earliest and latest time intervals studied. Twenty minutes after removal from the vinyl chloride, the concentration of radioactivity in the thymus was only slightly increased above that of blood (Figure 1 and 6); however, by 1 hour the thymus showed the highest uptake of radioactivity in the body (Figure 2) and remained the highest throughout the 24 hour period (Figures 2-6). C0Ii?ID3UTIAL Subject to Protect Fos*_ v. ir,'Vnni'T} *. T /-l 1 `-W T I ^" g Order in No. 90 - 483 -'t- Court CMA 003567 co.v?iss:ttiat._ Subject tc Protec-rive Grier 'inf Ross v. Ccroo J- -"c. , Ho . 90 - 43371 14th Judicial District Court CaloaSip,j Parish, Louis's-, 14, C-VINYL CHLORIDE; 20 MIN AFTER REMOVAL 94 BRAIN THYMUS LUNG LIVER ADRENAL KIDNEY PANCREAS Figure 1: A print of a whole^-body autoradiograph from a male CD-I mouse which was exposed for 3 hours to ^C-vinyl chloride and then frozen 20 minutes after removal from the vinyl chloride environment. White areas correspond to radioactivity. I4C-VINYL CHLORIDE; I HR AFTER REMOVAL SEROMUCOUS GLAND THYROID BLOOD LUNG LIVER PANCREAS URINE MINOR AND MAJOR SALIVARY GLANDS THYMUS BILE CONTENTS OF INTESTINE Figure 2: A print of a whole-body autoradiograph from a male CD-I mouse which was exposed for 3 hours to ^C-vLnyl chloride and then frozen'1 hour after removal from the vinyl chloride environment. White areas correspond to radioactivity. CMA 003568 ^ ' r la1' J r337i . -- _ - - - . > I4c-vinyl chIorioe*; '^ hr :af^er 3-removal : f HARDER'S GLAND PARATHYROID BLOOD PANCREAS KIDNEY URINE 95 SEROMUCOUS GLAND THYMUS LIVER CONTENTS OF STOMACH AND INTESTINE Figure 3: A print of a whole-body autoradiograph from a male CD-I mouse which was c*\posed for 3 hours to ^HC-vinyI chloride and then frozen } hours after removal from Lhe vinyl chloride environment. White areas correspond to radioactivity. I4C-VINYL CHLORIDE; 9 HR AFTER REMOVAL HARDER'S GLAND SUBLINGUAL GLAND LIVER SPLEEN KIDNEY PANCREAS Figure 4: A print of a wiiolc-buUy nutoradi o.,rnpn .1Uc-v inyl chloride and then tro?ou ^ White areas correspond to r.id ioac t iv ll . ,*n a iTinlu CD-I nioui-e w:ri<_n wjs u^po^uj lor 3 ri^tirs to s a:ter removal rrum thu vinyl chloride euviron^oat, CMA 003569 14'C-ViN':'L 14th Juci:i' Cnl-" ? Order itl _ :;c. 90-433.71 T ? , - =; 1 l-'q. > f* * SEROMUCOUS GLAND THYMUS LIVER CONTENTS OF STOMACH AND INTESTINE Figure 3: A print of a *-hole-body autoradiograph from a m.ile CD-I mouse which was v.soosed fur 3 hour^ zo ^C-vinyl chLoride And then frozen 3 hours after removal from Lne vinyl chloride environment. White areas correspond co radioactivity. I4C-V!NYL CHLORIDE; 9 HR AFTER REMOVAL LINGUAL MUCOSA THYMUS BLOOD GASTRIC MUCOSA WALL OF INTESTINE URINE Figure A print of a whole-body .lutorad; frum a siale CD-I mounc v.riun vus opposed for 3 hours to lAc-vtnyL chloride and then frozen 9 Hours arter removal from the vinyl chloride envirur.Touc. White areas correspond zo rodiu.ici Lv wty . CMA 003570 *"m T^n , *0 . 90 -43371 ;T^ . l4C-VINYL CHLORIDE; 24 HR AFTER REMOVAL 96 Figure 5: k print of a *ho Le-imdy au to rad to craoh from a male CD-I mouse which was exposeJ for 3 hours to i-,C-vinvL cnlondo nod then frozen 2- hours after removal from the vinyl chloride environment, rtnite ureas correspond :o radioactivity. i4c-vinyl chloride 20 MIN AFTER REMOVAL 24 HR AFTER REMOVAL CORTEX AND MEDULLA OF THYMUS CORTEX AND MEDULLA OF THYMUS o: ? c i ' c s at tie thymus areas of '.ho ie-oou,. iuto rad logrnpiis from maLe CD-I mice w:,ich wore exposed to `'C-virvi ri.l-'rido for 3 uours mu then trozen 20 minutes or 2^ hours after rer.o'.al from :;,e . tu.i onlnri^e e v i r 'rv.unt. Vhitc areas correspond to r ad io.ic c t v 11 v . Note the m.m retention toe n ;n'v la t: "otnooLitcs in the coitcx of tne thymus after 24 hours. CMA 003571 97 The sites of localization of the nonvolatile metabolites of vinyl chloride in the mouse are similar to those reported for the rat (Duprat et al. 1977). The high concentration of nonvolatile metabolites of vinyl chloride retained in the thymus after 24 hours suggests that there is covalent binding of these metabolites to molecules in the thymus. It is possible that these molecular interactions in the thymus could have an effect on the immune system. The high concentration in the thymus may be involved in the stimulation of the immune system in mice seen by Sharma and Gehring ( 1979) and in the reduction in peripheral T-lymphocytes seen in man by Ward et al. (1976). Vinyl chloride may exert its carcinogenic action by a dual mechanism. It may suppress the immune surveillance system mediated by the thymus and concurrently damage several tissues including the liver. Relevance to Industry Whole-body autoradiographic studies on the biological disposition of industrial chemicals offers the most thorough approach to the study of the interaction of these chemicals with tissues in the bpdy. Knowledge of these tissue interactions allows predictions of potential toxicity or, conversely, to a lack of toxic effect if the chemical is rapidly and completely eliminated (Waddell et al., 1977). When chemicals are retained in specific tissues as in the case with vinyl chloride, the tissue of retention gives an indication of possible mechanisms of toxic action. Vinyl chloride appears to be having a toxic effect on the immune system; the direct evidence for an interaction the chemical with this system was not available until these studies were don^B References Creech,J.L. and Johnson, M.N. (1974) Angiosarcoma of liver in the manufacture of polyvinyl chloride. Journal of Occupational Medicine 16: 150. Duprat, P., Fabry, J.P., Gradiski, D. and Magadur, J.L. (1977) Metabolic approach to industrial poisoning: blood kinetics and distribution of ^C-vinyl chloride monomer (V.C.M.) Acta Pharmacoloqy and Toxicology 41:142. Maltoni, S. and Lefemine, G. (1974) Carcinogenicity bioassays of vinyl chloride. Research plan and early results. Environmental Research 7: 387. Sharma, R.P. and Gehring, P.J. (1979) Immunologic effects of vinyl chlo ride in mice. Annals New York Academy of Science, 320:551. Ullberg, S. (1954) Studies on the distribution and fate of S3^-label1ed benzylpenicillin in the body. Acta Radiology, Supplment 118:1. Subject to Protective Order lr* ;; Eoss_ v. Conoco, Ir.e.^ No. 90-4337!j 14th Judicial District Court jj. 'Icasisu Parish, Louisiana i CMA 003572 '3 J<l . ` ` 2 4 J C 1 -- J -- ' ~ -- V ' W ^ " 1 -4 * -- W ' 4 -4 . ( o ^ 1-4 ,7 / ' 7;;j ]:zn ccr.zzziri'zicn z~ :, 2 ' 22 ` e rre2i:;`'22s -;r v -vi *'*: "'i Z'Z z nyr^ij s dr" ^ f r 44 "C S^j^dSdS 2 " c 2 t rg rg 'is cc/dicnz c i pc i nc dr c ~g 5 5 metabol'tss to molecules in the thymus. It is possible that these molecular interactions in the thymus could have an effect on the immune system. The high concentration in the tnyinus may be involved in the stimulation of the i~mure svstem in mice seen by Charma and Ge^rinn 119791 arc i" th- ^^j^ in peripheral T-lymphocytes seen in man by Ward et al. (1976). Vinyl chloride may exert its carcinogenic action by a dual mechanism, it may suppress the immune surveillance system mediated by the thymus and concurrently damage several tissues including the liver. 'ihole-body autoradicgraphic studies on the biological disposition of industrial chemicals offers the most thorough approach to the study of the interaction of these chemicals with tissues in the body. Knowledge of these tissue interactions allows predictions of potential toxicity or, conversely, to a lack of toxic effect if the chemical is rapidly and completely eliminated (Waddell et al,, 1977). When chemicals are retained in specific tissues as in the case with vinyl chloride, the tissue of retention gives an indication of possible mechanisms of toxic action. Vinyl chloride appears to be having a toxic effect on the immune system; the direct evidence for an interaction of the chemical with this system was not available until these studies were done. References Creech,J.L. and Johnson, M.N. (1974) Ang iosarcoma of liver in the manufacture of polyvinyl chloride. Journal of Occupational Medicine 16: 150. Duprat, P., Fabry, J.P., Gradiski, D. and Magadur, J.L. (1977) Metabolic approach to industrial poisoning: blood kinetics and distribution of '^C-vinyl chloride monomer (V.C.M.) Acta Pharmacology and Toxicology Al * 1A9 Maltoni, S. and Lefemine, G. (1974) Carcinogenicity bioassays of vinyl chloride. Research plan and early results. Environmental Research 7^ 387. -------------------------------------- Sharma, R.P. and Gehring, P.J. (1979) Immunologic effects of vinyl chlo ride in mice. Annals New York Academy of Science, 320:551. Ullberg, S. (1954) Studies on the distribution and fate of S35Mabelled benzylpenici11 in in the body. Acta Radiology, Supplment 118:1. co^fjdfftial 3 Subject to jrotective Order in Ross v. --C--a-n--o Tnc- N- 90-4832 14th Judicial district Court ; alcasieu Parish, Louisia^-a V1 CMA 003573 98 Waddell, W.J. and Marlowe, C, (1977) Autoradiography. IN: Garrett, E.R. and Hirtz, J.L. (eds.), Drug Fate and Metabolism: Methods and Techniques. Vo 1. 1, pp. 1--25. New York: Marcel Dekker. Waddell, W.J., Marlowe, C., Miripol, J.E., and Gravin, P.J. (1977) The distribution in mice of intravenously administered plasma solutions of ]Di--2--Ethy1 hexyl Phthalate Determined by Whole-Body Autoradiography. Toxicology and Applied Pharmacology 39:339-353. Wagner, E.R., Muelder, W.M., Watanabek, P.G., Hefner, R.E., Jr., Braun, W.H. and Gehring, P.J. (1975) A gas chromatographic method for the preparation of i^c-labelled vinyl chloride. Journal of Labelled Compounds XI (4):535. Ward, A.M., Udnoon.S., Watkins, J., Walker, A.E. and Darke, C.S. (1976) Immunological mechanisms in the pathogenesis of vinyl chloride disease. British Medical Journal, 1:936. CQyJIBZn'TIAL Subject to Protective Order itt' ''i Bess v. Cor.cco , _Ir. No. 90 - 4833 14th Judicial District CourtCalcasieu Parish, Louisiana CMA 003574 LISTS OF PUBLICATIONS, ABSTRACTS, PREPRINTS AND PUBLICATIONS IN PREPARATION cc:~:- Subject to Pro F.css v, Conoco, 14th JullTc-l" Ca]nasiu P'" :Sre Criar ire' ' ^ So. 90-4S3'71'] t-rot Court \ 99 CMA 003575 TOO PUBLICATIONS 1. Fortwengler, Jr., H.P., Jones, 0., Espinsoa, E. and Tamburro, C.H. (1981) Evidence of Endothelial Cell Origin of Vinyl Chloride-Induced Hepatic Angiosarcoma. Gastroenterology, 80:1415-1419. 2. Ou, J.T., Sandoz, J.P., Tseng, M.T. and Tamburro, C.H. (1979) Biochemical Alterations in Livers of Rats Exposed to Vinyl Chloride. Journal of Toxicology and Environmental Health, ^5:1119-1132. 3. Tamburro, C.J. (1978) The Hepatic Role in Carcinogenesis and Its Early Detection--The Vinyl Chloride Model. The Vale Journal of Biology and Medicine, Inc., 51:67-80. 4. Du, J.T., Tseng, M.T. and Tamburro, C.H. (1982) The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes. Toxicology and Applied Pharmacology, 62:1-10. 5. Tamburro, C.H. and Greenberg, R.A. (1981) Effectiveness of Federally Required Medical Laboratory Screening in the Detection of Chemical Liver Injury. Environmental Health Perspectives, 41:117-122. .6 Curran, K.L., Kupchella, C.E. and Tamburro, C.H. (1977) Urinary Glyco- saminoglycan Patterns in Angiosarcoma of the Liver. Cancer, 40: 3050-3053. ---------- ~ 7. Kupchella, C.E. and Tamburro, C.H. (1978) Urinary and Tissue Glycosaminoglycan Patterns in Hepatic Angiosarcoma. Detection and Prevention of Cancer, H.E. Neiburgs, Ed., Part 1, Vol I, Marcel uekker, Fnc., New York. .8 Kupchella, C.E., Drake, E.E., Kennedy, J., Curran, K.L., Warick, R. and Morris, H.P. (1981) Tissue and Urinary Glycosaminoglycan Patterns Associated with a Fast, an Intermediate, and a Slow-growing Morris Hepatoma. Cancer Research, 41:419-424. 9. Tamburro, C.H,, Kupchella, C.E. and Greenberg, R.A., et al. (1981) Screening for the Early Detection of Disease in Individuals Exposed .to Vinyl Chloride. 002 -U-.S--. --E-n-v-ir-o-n-m-e-n-t-a-l --P-r-o-te-c-t-io-n---A-g-en-cy, -5-6-0-/6---8-1-- 10, Laumbach, A.D., Lee, S., Wong, J. and Streips, U.N. (1976) Studies on the Mutagenicity of Vinyl Chloride Metabolites and Related Chemidals. Prev., Detect. Cancer (Proc. Int. Symp.) 3rd, Vol. 1:155-170. 11. Elmore, J., Wong, H., Laumbach, A.D. and Streips, U.N. (1976) Vinyl Chloride Mutagenicity and Carcinogenicity via the Metabolites Chlorooxirane and Chloroacetaldehyde Monomer Hydrate. Biochem. Biophys. Acta 442:405. X '3 -j vi^'zive Order in ; 7. jCcno 'c Ire . . No . 90-4333' 14th Judi: i'.l" District Court 4, Calebs t-u ^ -- i sh. Louisiuni CMA 003575 12. Streips, U.N., Laumbach, A.D. and Yasbin, R.E. (1981) Bacterial Mutation Monitor for Active Metabolites of Chemical Carcinogens: B. Subtilis Assays for Mutation and INA Repair, In Microbial Testers. I. Cecil Felkner, Ed., Vol. 5, Marcel Dekke" Inc., New York. 13. Horowitz, $., Doyle, R.J., Young, F.E. and Streips, U.N. (1981) Selective Association of the Chromosome with Membrane in a Stable L-Form of Bacillus Subtilis. Journal of Bacteriology, 138:915-922. 14. Streips, U.N., Horowitz, S. and Doyle, R.J. (1980) Genetic Analysis of DNA-Surface Interactions in Bacillus Subtilis. Microbio logy:284-287. 15. Horowitz, S., Doyle, R.J. and Streips, U.N. (1978) Restricted ChromosomeMembrane Association in a Stable L-form of Bacillus Subtilis. In Transformation. 1978. Proceedings of the Fourth Intern ationTT Meeting on Bacterial Transformation. 16. Sonnenfeld, G., Barnes, M.C., Schooler, J. and Streips, U.N. (1980) Inhibition of Interferon Induction as a Screen for the Carcinogenic Potential of Chemicals, J_n Interferon: Properties and Clinical Uses, A. Khan, N.O. Hill and G.L. Dorn, Eds. pp. 589-598, Wadley Institutes of Molecular Medicine, Dallas, Texas. 17. Barnes, M.C., Streips, U.N. and Sonnenfeld, G. (1981) Effect of Carcinogens and Analogs on Interferon Induction. Oncology 38:98-101J^y 18. Espinosa, E. (1976) Immunopathologic observations in liver angiosarcoma. In Prevention and Detection of Cancer, Vol. 1, Neiburgs, H.E., Ed. pp. 927-36, Marcel Dekker, Inc., New York. 19. Espinosa, E. Circulating Tissue Antigens. (1976) Identification and Characterization of Antigens of Limited and of Wide Body Distribution in Human Gall Bladder Bile. Clin. Exp. Immunol., 25:410-417. object to . rotective Grder in "i Fo?g. v- C-r.JQO. T"-i, No. 90-4332'.' J14th Junici-il District Court. Calcasisu Parish, Louisiana CMA 003577 Mutation Monitor for Active Metacoiirss of Chemical C 3. Suoti 1 is Assays *sr `-`utatitr. a-,: i:,A f.etai-', Testers. I. Cecil felkner, Ed., Vol. 5, Marcel De'kke~r\ York. ;' :t : a` r.r,c., New 13. Horowitz, S., Ooyle, R.J., Young, F.E. and Streips, U.N. (1981) Selective Association cf tea Chromosome *itn Membrane in a Stable L-Porm of Saci 11 us Subti1 is. Journal of Bacteriology, 138:915-92?. 14. Streips, U.N., Horowitz, S. and Ooyle, R.J. (1980) Genetic Analysis of DNA-Surface Interactions in Bacillus Subtilis. Microbiology:2S4-287. 15. Horowitz, S., Doyle, R.J. and Streips, U.N. ( 1978) Restricted Membrane Association in a Stable L-form of BacillusSubtilis. in Transformation. 1978. Proceedings of the Fourth internaticrfiT Meeting on Bacterial Transformation. 16. Sonnenfeld, G., Barnes, M.C., Schooler, J. and Streips, U.N. (1980) Inhibition of Interferon Induction as a Screen for the Carcinogenic Potential of Chemicals, j_n Interferon: Properties and Clinical Uses. A. Khan, N.O. Hill and G.L. Dorn, Eds. pp. 589-598, Wadley Institutes of Molecular Medicine, Dallas, Texas. 17. Barnes, M.C., Streips, U.N. and Sonnenfeld, G* (1981) Effect of Car cinogens and Analogs on Interferon Induction. Oncology 38:98-101. 18. Espinosa, E. (1976) Immunopathologic observations in liver angiosarcoma. In Prevention and Detection of Cancer, Vol. 1, Neiburgs, H.E., Ed. pp. 927-36, Marcel Dekker, Inc,, New York. 19. Espinosa, E. Circulating Tissue Antigens. (1976) Identification and Characterization of Antigens of Limited Distribution in Human Gall Bladder Bile. 25:410-417, and of Wide Body Clin. Exp. Immunol., ---------------------------------- ^ 1^r ifr Subject to '' .-0 . 2,0-48371 Ross, v- o, `-vE trict Court , (I "l4th Juaic ill 7is Calcasieu parisb Louis iani CMA 003578 102 ABSTRACTS 1. Fortwengler, H.P., Dever, M.E., Tamburro, C.H., and Espinosa, E. (1978). Lymphocyte transformation tests in vinyl chloride workers. Fed. Proc., 37: 362. ---------------- 2. Fortwengler, H.P., Jones, D., Tamburro, C.H. and Espinosa, E. (1979). Factor VIII content as evidence for endothelial origin of vinyl chloride associated liver angiosarcoma. Fed. Proc., 38:999. 3. Fortwengler, H.P. and Tamburro, C.H. (1976). Use of dye clearance in the detection of hepatocellular injury among vinyl chloride workers. Clin. Res., 23^264A. 4. Du, J.T. and Tamburro, C.H. (1976). Decreased glucose-6-phosphatase activity in liver in vinyl chloride exposed rats. Fed. Proc., 35:1422. 5. Du, J.T. and Tamburro, C.H. (1978). Elevated glutathione content, glutathione-S-transferase and glutathione reductase in liver of rats exposed to vinyl chloride. Fed. Proc., 37:1545. 6. Liss, G. and Tamburro, C.H. (1982). Serum bile acids in screening for chemical hepatoxicity. The Toxicologist, July 1982. 7. Kupchella, C.E., Jarvis, J.O., Curran, K.L., Greenberg, R. A. and Tamburro, C.H. (1977). Tissue and urinary glycosaminoglycan changes in hepatic fibrosis. Gastroenterology, 73:1299. 8. Kupchella, C.E. and Tamburro, C.H. (1977). Urinary chrondroitin sulfate fraction patterns in hepatic angiosarcoma. Clin. Res., 25:35A. 9. Kupchella, C.E. and Tamburro, C.H. (1977). Urinary glycosaminoglycan excretion patterns in chemically induced liver injury and cancer. Clin. Res., 25, 329. 10. Kupchella, C.E. and Curran, K.L., Drake, E., Kennedy, J. and Tamburro, C.H. (1978). Tissue and urinary glycosaminoglycans in transplantable hepatomas. Gastroenterology, 75:972. 11. Curran, K.L., Kupchella, C.E., Sandoz, J. and Tamburro, C.H. (1978). Urinary glycosaminoglycan patterns in human hepatic angiosarcoma, hepatoma, and in workers at risk for angiosarcoma. Gastroenterology, 75:959. 12. Kupchella, C.E., Secskas, E.M., Kenndy, J.S., and Espinosa, E. (1979). Glycosaminoglycan changes associated with hepatic tumors: The contributions of regerneration and necrosis. Clin. Res., 27:389. I al Subject to Protacti ve Order in: L No. 90-48571', Ross v. Conoco , 1r4-t1hoaJsuieduicPiaal riDshis, t)rLieout isCiaonurat- | -A CMA 0035*79 13. Greenberg, R.A. and Tamburro, C.H. (1978). Early detection of disease in individuals exposed to vinyl chloride. American Public Health Association Ann. Mtg. 14. Barrows, G.H., Joyce, M.J., Schrodt, G.R., Greenberg, R.A. and Tamburro, C.H. (1979). Computer-assisted morphologic quantitation of collagen in human liver biopsies. Laboratory Investigations, 40:3. 15. Tamburro, C.H., Mala, L. and Popper, h. (1979). Early hepatic histological alterations among chemical (vinyl monomer) workers. Gastroenterology, 77:A33. 16. Laumbach, A.C., Streips, U.N. and Wong, J.L. (1978). Chloroacetaldehyde-induced damage to Bacillus subtilis. Ann. Mtq. Amer. Soc. Microbiol., p. 125. 17. Streips, U.N. and Wong, J.L. (1978). Chloroacetaldehyde-induced damage to Bacillus subtilis. XII Intern. Congress Microbiol., p. 105. 18. Sonnenfeld, G., Barnes, M.C. and Streips, U.N. (1979). Inhibition of interferon induction as a screen for the carcinogenic potential of chemicals. J. Clin, Hematol. and Oncol., 9:291. 19. Johnston, P.B., Espinosa, E., Chia, S. and Caple, S. Properties of 14 week maintenance cultures of PLC/PRF/5 cells. In Vitro, 15:227. .20 Espinosa, E., Chia, S., Caple, S. and Kupchella, C.E. (1979). Liver-specific F antigen in transplantable hepatomas having different growth rates. Fed. Proc., 38:1069. .21 Espinosa, E., Caple, S., Kupchella, C.E. and Chia, S. (1979). Two liver antigens undetectable in a fast growing line of transplanted hepatoma (Morris Hepatoma 7777). Fed. Proc., 1069. .22 Du, J.T. and Tamburro, C.H. (1980). Oxidative and detoxifying ability of liver mesenchymal vs. parenchymal cells in the metabolism of xenobiotics. Gastroenterology, 79:1013. 23. Liss, G. and Tamburro, C.H. (1982). Toxic carcinogenic and safe exposure levels in vinyl chloride-induced hepatic angiosarcoma. Toxicologist, July 1982. Foss_ y. Cor. 14th JudicTr'_ Calcasieu pa ^ Order in -o. 50-433'Z "t rourt ouislana CMA 003S80 4 . , Joyce, ".J., Gc.irocfc, G.R., Greanoerg, R.A. and Tamourro, Computer-assisted morohologic cuantitatio.n or collagen in human liver biopsies, l. aboratory 1 n vest icat ions. bC:3. 15. Tamburro, C.H., Makx, L. and Popper, H. (1973). Early heoatic histological alterations among chemical (vinvl monomer) workers. Gastroenterology, _77:A33. 16. Laumbach, A.D., Streips, EI..N. and Wong, J.L. ( 1973). Cnlo-oacet aldehyde-i nauced damage to Sac 11 "us suttili's. ^n. Mtc. >?', Sc'. 17. Streips, u.N. ana *ong, J.L. ):97S). Chlcroacetaldehyde-induced damage to Saci1lus subtilis . XII Intern. Congress Microbiol., p. 105. 18. Sonnenfeld, G., ?arres, .C. interferon induction as a chemicals. J, Clin. Hematol. .`1. ) I 9 79) . Inhibition of carcinogenic potential of 19. Johnston, P.3., Espinosa, E., Chia, S. and Caple, S. Properties of 14 week maintenance cultures of PLC/PRF/5 cells. In Vitro, 15:227, 20. Espinosa, _ E., Chia, S., Caple, S. and Kupchella, C.E. ( 1979). Liver-specific F antigen in transplantable hepatomas having different growth rates. Fed. Proc., 38:1069. 21. Espinosa, E., Caple, S., Kupchella, C.E. and Chia, S. (1979). Two liver antigens undetectable in a fast growing line of transplanted hepatoma (Morris Hepatoma 7777). Fed. Proc., 1069. 22. Du, J.T. and Tamburro, C.H. (1980). Oxidative and detoxifying ability of liver mesenchymal vs. parenchymal cells in the metabolism of xenobiotics. Gastroenterology, 79:1013. 23. Liss, G. and Tamburro, C.H. (1982), Toxic carcinogenic and safe exposure levels in vinyl chloride*induced hepatic angiosarcoma. Toxicologist, July 1982. -------------- ------ .CONFID Subject-to"p^ Ross V. Cooanooccoo,, TT r - .* r' "-IAI $ 003^81 104 preprints 1. Tamburro, C.H., Makk, L. and Popper, H. Early Hepatic Histological Alterations among Chemical (Vinyl Monomer) Workers. 2. 0u, J.T., Eades, O.S., and Tamburro, C.H. Oxidative and GlutathioneRelated Detoxifying Enzyme Capabilities in Hepatocytes and Nonhepatocytes of Rat Liver. 3. Kupchella, C.E., Greenberg, R.A. , Warick, R.A. and Tamburro, C.H. Preliminary Assessment of the Usefulness of Urinary Total Glycosaminoglycan. "S-VriAL -i Subject Csotive Order in.' PpS3 v. { h1 ' + 7- "o. 90-4837! district Court C^.lc*3 s tea ?Irish Louis! CMA 003582 105 PUBLICATIONS IN PREPARATION TITLES 1. Tamburro, C.H., Miller, B. and Greenberg, R.A. Specificity of ICG Clearance TEst of Hepatotoxicity. Sensitivity and 2. Tamburro, C.H., Miller, B. and Chan, C. Indocyanine Green (ICG) Clearance Test Safety and Toxicity. 3. Fortwengler, P. and Tamburro, C.H. Use of HLA Tissue Typing in the Identification of Chemical Injury in Vinyl Monomer-Exposed Workers. 4. Fortwengler, P. and Tamburro, C.H. Immunocompetence of Humans Chemically Exposed to Finyl Monomer Chemical. 5. Barrow, G., Schrodt, G.R. and Tamburro, C.H. Collagen Changes in Normal Aging Liver. XiVG 141 r CMA 003583 T ambur'-c, B. : d G-a an: 3", Spec ificicy or iCu Clearance Test of nepatotoxicity. Ca c. ,: c . yd 2. Tamburro, C.H., Miller, 8. and Chan, C. Indocyanine Green (ICG) Clearance Test Safety and Toxicity. 3. Fortwengler, P. and Tamburro, C.H. Use of HLA Tissue Typing in the Identification of Chemical Injury in Vinyl Monomer-Exposed Workers. 4. Fortwengler, P. and Tamburro, C.H. Immunocompetence of Humans Chemically Exposed to Finyl Monomer Chemical. 5. Barrow, G., Scorect, G..T. me Aging Liver. C.H, Coi'aoen C^arc - ^ in c p 1 CONFIDENTIAL Subject to Protective Order in Roaa v. Conoco, Inc., Ho. 90-4837 14th Judin''1 ^'s:-i.''t CMA 003584 APPENDIX CONFIDENTIAL Subject to Protective Order in Foss v. Conoco, Inc., No. 90-4837. 14th Judicial District Court Calcasieu Parish, Louisiana CMA 003585 PUBLICATIONS COITFIPETfTIAL Subject to Protective Order In' Ross v. Conoco, Inc. . No. 90 -4837, 14th Judicial District Court Calcasieu Parish,, Louisiana CMA 003586 CASTROENTEROLOCY 19Sl;80:14t5-19 1 Evidence for Endothelial Cell Origin of Vinyl Chloride-Induced Hepatic Angiosarcoma H. PHILIP FORTWENGLER. JR.t DOUGLAS JONES, ENRIQUE ESPINOSA, and CARLO H. TAMBURRO Department of Medicine, Division of Digestive Diseases and Nutrition, Department of Pathology and Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky Previous reports of hepatic angiosarcoma have not clearly defined the cellular type front which this tu mor arises, as evidenced by the terminology of endo thelioma, Kupffer cell sarcoma, endothelial cell sar coma, and hemangioendothelial sarcoma, etc., which have been used interchangeably. In addition, there has been no consensus on the separate entity of Kupffer and sinusoidal endothelial cells. In the work presented here, evidence for the endothelial cell origin of this tumor is provided by the demon stration of factor Vlll, a known endothelial cell marker, in the tumor cells. Fluorescence due to the presence of factor VIII appeared intense in the tu mor sinusoidal ceils of all four vinyl chloride-associ ated angiosarcomas studied, whereas normal liver sinusoidal lining cells showed negligible fluores cence. Hepatic angiosarcoma has been associated with ex posure to thorotrast (1), arsenic (2), androgenic-ana bolic steroids (3), and vinyl chloride (4). However, most cases are of undetermined origin. Regardless of etiology, earlier studies (5-8) made no clear dis tinction between endothelial and Kupffer cells, and gave various histologic designations to this type of tumor, e.g,, hemangioblastoma, Kupffer cell sar- Received April 22. 1980- Accepted January 12, 1981. Address requests for reprints to: Carlo H. Tamburro, M.D., Di vision of Digestive Diseases and Nutrition, Department of Medi cine, Health Sciences and Cancer Center. University of Louisville. Louisville. Kentucky 40292. This investigation was supported by a Research Grant from the Manufacturing Chemists Association. This work was presented in part at the Federation of American Societies for Experimental Biology meeting in Dallas, Texas in April, 1979. The technical assistance of Mr. Larry Wilder is greatly ac knowledged. We would like to thank Dr. Hans Popper for his re view of the histology and his suggestions. 1981 by the American Gastroenterological Association 0016-5Q8V81/06141S-05SQ2.50 coma, angiosarcoma, and endothelioma (9), thus raising the question of its true cellular origin. This question is of particular current interest in view of recent findings by electron microscopy which clearly support a distinct origin for Kupffer and endothelial cells (10), as well as indicating the ab sence of endothelial cell to Kupffer cell transition (Ill- Factor VIII, a coagulation factor, was shown by Hoyer et al. (12), to be present in endothelial cells, megakaryocytes, and platelets. In the work pre sented here, using immunochemical evidence, the presence of abundant factor VIII in proliferating si nusoidal lining cells of angiosarcomatous liver tissue is demonstrated; it supports the endothelial cell ori gin of vinyl chloride-associated angiosarcoma. Materials and Methods Hepatic tissue studies were conducted in eight in dividuals--three with hepatic angiosarcoma related to heavy vinyl chloride exposure as production workers, one hepatic angiosarcoma associated with vinyl chloride used as hair spray propellant, and four normal subjects (trau matic death with no liver injury) who were used as con trols. Reagents. Purified human factor VIII was pro vided by the Louisville Red Cross Blood Center. Corre sponding antiserum prepared in rabbits was from Behring Diagnostics (American Hoechst Corp., Somerville, N.J.). A single precipitin line in immunoelectrophoresis was seen with this material when tested against human plasma and purified factor VIII. Fluorescein-conjugated goat anti serum to rabbit IgG (GARI) was obtained from Meloy Lab oratories, Inc. (Springfield. Va.}. Tissue specimens. Both normal and hepatic angio sarcoma tissue were obtained at postmortem examination within 30 min to 8 h of death. Portions of normal and tu mor tissues were processed for standard histologic and immunofluorescent staining. Immunofluorescence technique. Frozen sections (8 CMA 003587 1416 FORTWENGLER ET AL. /im) were rut on a cryostat, air dried, fixed for 5 min in cold acetone and rehydrated in phosphate-buffered saline (PBS). The sections were then treated for 30 min with anti serum to factor VIII. washed with PBS, and stained with fluorescein-conjugated GARI for 45 min. After three 5-min washes in PBS, the sections were examined by immuno- fluorescent microscopy utilizing a Vanox microscope (Olympus Corporation of America), and an HB0200 UV il luminator with a 3-mm BF 12 exciter filter and a G530 bar rier filter. Control sections were incubated with nonimmunized rabbit serum. Specificity of factor VIII staining was demonstrated by inhibition of the staining reaction after absorption of the anti-factor VIII rabbit serum with the purified factor VIII and by the negative staining reaction found using nonimmune rabbit serum. Results Factor VIII immunofluorescence of endo thelial cells in human umbilical cord shows a ho mogeneous continuous staining pattern (Figure 1). This preparation served as a positive control. Sec tions from normal liver did not display any specific factor VIII immunofluorescence in the sinusoidal GASTROENTEROLOGY Vol. so, No. 6 Figure 1. Umbilical cord, endothelial lining cells (arrows) show ing factor VIII related immunofluorescence in white (x 100). Figure 2. Fluorescent staining of normal liver showing minimal granular punctate factor VIM fluorescence in hepatic si nusoids (white arrows) (x 100). areas except for occasional spotty granular areas of minimal intensity along the sinusoidal borders (Fig ure 2). Only the linings of the hepatic arteries and veins consistently gave intense staining reactions. In contrast, vinyl chloride hepatic angiosarcoma tumor tissue demonstrated intense fluorescence occurring in the proliferating cells lining the enlarged sinu soids (Figure 3). These areas of specific immuno fluorescence were limited to the sinusoidal spaces, were multifocal and occasionally occupied up to 20% of the tissue specimen. There was no demonstrable difference among the four angiosarcoma specimens as to the frequency of stained cells or the pattern of specific fluorescence. Adjacent tissue stained with H & E documents angiosarcomatous cells within the enlarged sinusoids lining the hepatocytic cords (Fig ure 4). Fluorescence was present in the cytoplasm and cell surface but not in the nuclei of the sinusoidal cells as illustrated in Figure 5. This distinguishable pattern of factor VIII immunofluorescent staining was seen in all the angiosarcomas examined and in none of the normal hepatic tissue. CMA 003588 |un>- ISBt CELLULAR ORIGIN OF Ht.PA.TtC ANGIOSARCOMA H17 described it as composed of ceils closely rcsembli Kupffer cells and indicated that some tumor ce were phagocytic. Nevertheless, the tumor was desig nated "endothelial cell sarcoma of the liver." Later, in reporting a tumor, Baker et al. (16). made a dis tinction between the apparent maturation stages of sinusoidal lining cells, i.e. "Kupffer cells" and "nor mal resting sinusoidal endothelia." He noted that the tumor cells were phagocytic and therefore, de scribed the tumor as originating from Kupffer cells, as did Burston (17). Edmonson's classical description of this tumor (9) makes no distinction between endo thelial and Kupffer cells, but describes it as com posed of malignant endothelial cells. In the initial report of arsenic-associated "hemangioendothelial sarcoma" of the liver due to ingestion of Fowler's solution, Regelson et al. (18), indicated the presence of neoplastic endothelial cells but did not differentiate Kupffer from endothelial cells. On the other hand, Blackwell et al. (19), made no dis Figure 3. A section of liver angiosarcoma tissue exhibiting one of the multifocal areas displaying marked fluorescence in the proliferating sinusoidal Cells (white arrows), with some nonspecific background staining of hepatocytes (x 200). Discussion Descriptions and definitions of hepatic angio sarcoma have varied in the past and have relied pre dominantly on morphologic criteria for identifying the various sinusoidal cells. Some investigators have proposed that both Kupffer and sinusoidal endothelial cells are transitory in respect to each other and that Kupffer cells merely represent activated endothelial cells (5,13), In con trast, others support the view that the Kupffer cell is independent in nature and represents a hepatic macrophage (14,15). As early as 1939, Miller considered the Kupffer cell as a class of endothelial cells and distinguished them as "endothelial cells of Kupffer (13).'' He sug gested the term endothelioma or endothelioblastoma for the tumors arising from Kupffer cells and heman gioendotheliomas or hemangioendothelioblastoma for those derived from vascular endothelium. In the first communication of a thorotrast-associated ma lignant hepatic vascular tumor, McMahon et al. (1), figure 4. A frozen section of hepatic angiosarcoma tissue dem onstrating individual and small groupa of proliferating endothelial cells in dilated sinusoids (block arrows) (H & F. x 400). CAfA 03589 ` \ sinusoidal lining cells, i.e. "Kupffer cells" and "nor mal resting sinusoidal endotheha." He noted ihai the tumor cells were phagocytic and there:-.:?, de scribed the tumor as originating from Kuph'er cells, --J ^ * j. 2 vlUJjiLUt w Li * l of this tumor (9) makes no distinction between endo thelial and Kupffer cells, but describes it as com posed of malignant endothelial cells. In the initial report of arsenic-associated "heman- gioendothelial sarcoma" of the liver due to in?estion of Fowler's solution, Regelson et al. (13), indicated 1C,th? prcoiTiCO oi "cwuiuii.u c*"iuC2:.S ClQ not dmerentiace Kupffer rrom endotdeiial cells. On the other hand. Blackwell e: al. (19). made no dis- figure 3. A section of liver angiosarcoma tissue exhibiting one of the multifocal areas displaying marked fluorescence in the proliferating sinusoidal cells (white arrows), with some nonspecific background staining of hepatocytes (x 200). Discussion Descriptions and definitions of hepatic angio sarcoma have varied in the past and have relied pre dominantly on morphologic criteria for identifying the various sinusoidal cells. Some investigators have proposed that both Kupffcr and sinusoidal endothelial cells are transitory in respect to each other and that Kupffer cells merely represent activated endothelial cells (5,13). In con trast, others support the view that the Kupffer cell is independent in nature and represents a hepatic macrophage (14,15). As early as 1939, Miller considered the Kupffer cell as a class of endothelial cells and distinguished them as "endothelial cells of Kupffer (13)." He sug gested the term endothelioma or endothelioblastoma for the tumors arising from Kupffer cells and heman gioendotheliomas or hemangioendothelioblastoma for those derived from vascular endothelium. In the first communication of a thorotrast-associated ma lignant hepatic vascular tumor, McMahon ct al. Co Figure 4. A frozen section of hepatic angiosarcoma tissue dem onstrating individual and small groups of proliferating endothelial cells in dilated sinusoids (black arrows) (H 4 E x Q). CMA 003590 1418 FORTWENGLER et al GASTROENTEROLOGY Vol. 30. No. 6 Kupffer cells which arc peroxidase positive and phagocytic. In the present work we have shown by an immunofluorescent procedure that Factor VIII is present in the proliferating cells of vinyl chloride-associated angiosarcoma, thus confirming their endothelial cell origin. Our observation that normal hepatic tissue displays intense factor VIII fluorescence in cross sections of vessels, but limited fluorescence of the si nusoidal endothelial cells in comparison to angiosarcomatous tissue, suggests that the aberrant cells may have an increased production or storage capac ity for factor VIII or a decreased transport from the cell. In addition, staining for factor VIII may provide a means for identification of vascular cell in volvement in hepatic tumors and could be used to identify specific endothelial cell changes and their relationship to chemical biotransformation in both animals and humans. In view of these findings, we suggest that the term "endothelial cell angiosarcoma" be used as an ap propriate term for this tumor. References Figure 5. High-powered frozen section of angwsarcomatous tis sue showing factor VIII immunofluorescence of indi vidual proliferating cells lining hepatic cords (white ar rows). Fluorescent staining of cytoplasm but not cell nucleus is seen in one of the cells (X 1000). tinction between these cells but called the tumor Kupffer cell sarcoma. In more recent reports involving vinyl chloride-as sociated angiosarcoma, the question of cellular ori gin of the tumor persists. This malignancy has been variously described as pleomorphic malignant endo thelial cells (9), malignant spherical and spindle cells (20). "tumor cells" (21), sinusoidal cells (22), and as a tumor believed to be malignant Kupffer cells (23). Makk et al. (24) and Popper et al. (25) depicted this tumor as arising from sinusoidal endothelial cells. In comparing the pathology of liver angiosarcoma due to vinyl chloride, thorotrast, and arsenic. Popper et al. (25) concluded from light and electron micros copy that the tumors were probably endothelial and suggested confirmation by use of histologic markers. An appropriate marker which concentrates in the endothelial cells is provided by coagulation factor VIII which differentially appears in the endothelial cells, megakaryocytes and platelets (12) and not in 1. MacMahon HE. Murphy AS. Bates MI. Endothelial-cell sar coma of liver following thorotrast injections. Am j Pathol 1947:23:585. 2. Roth F. The sequelae of chronic arsenic poisoning in moselle vintners. Ger Med Mon 1957:2:172. 3. Falk H. Popper H. Thomas LB. et al. Hepatic angiosarcoma associated with androgenic-anabolic steroids. Lancet 1979:ii:1120. 4. Creech JL. Johnson MN. Angiosarcoma of the liver in the manufacture of polyvinyl chloride. [ Occup Med 1974:16.150. 5. Gibbs WN. Connor CR. Hutchison HE. Malignant hemangio endothelioma associated with thrombocytopenia. J Pathol Bact 1966:92:207. 6. MacSween RNM, Vetters JM, Ross SK. et al. Haemangiocndothelial sarcoma of the liver. J Pathol 1973:109:39. 7. Stout AP. Hemangio-endothelioma: a tumor of blood vessels featuring vascular endothelial cells. Ann Surg 1943:116:445. 8. Greenberg M. Kupffer cell sarcoma of the liver. Report of 2 cases in South African blacks. SA Med | 1977:52:244. 9. Edmonson HA. Tumors of the liver and intrahepatic bile ducts. Atlas of Tumor Pathology, Section 7, Fascile 25. Wash ington. D.C., Armed Forces Institute of Pathology. 1956:13945. 10. Van Furth R, et al. The bone marrow origin of Kupffep cells. In: Wisse E. Knook DL. eds. Kupffer cells and other liver si nusoidal cells. Amsterdam: Elsevier/North Holland. 1977:471. 11. Wisse , Knook DL The investigation of sinusoidal cells: a new approach to the study of liver function. Prog Liver Dis 1979:6:153. 12. Hoyer LW. de los Santos RP. Hoyer JR. Antihemophilic factor antigen. Localization in endothelial cells by immunofluorescent microscopy. J Clin Invest 1973:53:2737. 13. Miller JK. Primary sarcoma of the liver: endothelioblastoma. Am J Surg 1939:45:459. 14. Wisse E. Ultrastructure and function of Kupffer cells end other sinusoidal cells in the liver. In: Wisse E. Knook DL. eds. CMA 003591 fane 1961 CELLULAR 0R1CIN OF HEPATIC ANCIOSARCOMA 1417 described it as composed of cells closely resembling KupfTer cells and indicated that some tumor cell? were phagocytic. Nevertheless, the tumor was desig nated "endothelial cell sarcoma of the liver." Later, in reporting a tumor, Baker et al. (16), made a dis tinction between the apparent maturation stages of sinusoidal lining cells, i.e. "Kupffer cells" and "nor mal resting sinusoidal endothelia." He noted that the tumor cells were phagocytic and therefore, de scribed the tumor as originating from Kupffer cells, as did Burston (17). Edmonson's classical description of this tumor (9) makes no distinction between endo thelial and Kupffer cells, but describes it as com posed of malignant endothelial cells. In the initial report of arsenic-associated "hemangioendothelial sarcoma" of the liver due to ingestion of Fowler's solution, Regelson et al, (18), indicated the presence of neoplastic endothelial cells but did not differentiate Kupffer from endothelial cells. On the other hand, Blackwell et al. (19), made no dis Figure 3, A section of liver angiosarcoma tissue exhibiting one of the multifocal areas displaying marked fluorescence in the proliferating sinusoidal cells (white arrows), with some nonspecific background staining of hepatocytes (x 200). Discussion Descriptions and definitions of hepatic angio sarcoma have varied in the past and have relied pre dominantly on morphologic criteria for identifying the various sinusoidal cells. Some investigators have proposed that both Kupffer and sinusoidal endothelial cells are transitory in respect to each other and that KupfTer cells merely represent activated endothelial cells (5,13). In con trast, others support the view that the Kupffer cell is independent in nature and represents a hepatic macrophage (14,15). As early as 1939, Miller considered the Kupffer cell as a class of endothelial cells and distinguished them as "endothelial cells of Kupffer (13)," He sug gested the term endothelioma or endothelioblastoma for the tumors arising from Kupffer cells and heman gioendotheliomas or hemangioendothelioblastoma for those derived from vascular endothelium. In the first communication of a thorotrast-associated ma lignant hepatic vascular tumor, McMahon et al. (1), figure 4. A frozen section of hepatic angiosarcoma tissue dem onstrating individual and small groups of proliferating endothelial cells in dilated sinusoids (block arrows) (H 4 E X 400). CMA 003592 laR \ HF? a.-;c \s.*S ^ - i_.J J'-j .v COmpOSCG 0* J1. if i'< r i ? - ,, J u f; j `7 ` T\ d '1 ` h j t ,, ^^T':. m; - * r 1 n r ` z t1 **' i:' < i ' . **"; r ^ 1: * .1 ' " 1 ; tmction between the apparent maturai.on stales of sinusoidal lining ceils, i.e. ' Kupner ceils ' and nor mal resting sinusoidal endothelia." He noted that the tumor cells were phagocytic and therefore, de scribed the tumor as oncin atmr from Kucft-r ceils as did Burston (17). Edmonson's classical description of this tumor (9) makes no distinction between endo thelial and Kupffer cells, but describes it as com posed of malignant endothelial cells. In the initial report of arsenic-associated heman- gioendothelial sarcoma" of the liver due to incestion of Fowler's solution Rnoaienn =.( i'^i, ; the presence of neoplastic endothelial cells out did not differentiate Kupffer from endothelial ceils On the other hand, Blackwell et al. (19). made no dis Figure 3 A section of liver angiosarcoma tissue exhibiting one of the multifocal areas displaying marked fluorescence in the proliferating sinusoidal cells (white orruwsi. with some nonspecific background staining of hepatocytcs (X 200). Discussion Descriptions and definitions of hepatic angio sarcoma have varied in the past and have relied pre dominantly on morphologic criteria for identifying the various sinusoidal cells. Some investigators have proposed that both Kupffer and sinusoidal endothelial cells are transitory in respect to each other and that Kupffer cells merely represent activated endothelial cells (5,13). In con trast. others support the view that the Kupffer cell is independent in nature and represents a hepatic macrophage (14,15). As early as 1939, Miller considered the Kupffer cell as a class of endothelial cells and distinguished them as "endothelial cells of Kupffer (13)." He sug gested the term endothelioma or endothelioblastoma for the tumors arising from Kupffer cells and heman gioendotheliomas or hemangioendothelioblastoma for those derived from vascular endothelium. In the first communication of a thorotrast-associated ma lignant hepatic vascular tumor. McMahon et al. (1). figure 4. A frozen section of hepatic angiosarcoma tissue dem onstrating individual and smell groups of proliferating endothelial ceils in dilsted sinusoids (black arrows) (H 4 E X 400). CMA 003593 lunr 1*481 CELLULAR ORIGIN OF HEPATIC ANGIOSARCOMA 1419 Kupffer cells end other liver sinusoidal i ells, Amsterdam Elsevicr/North Holland. 1977 3d 15. Naito M, Wisse E. Observations on the fine structure and cytochemistry of sinusoidal cells in fetal and neonatal rat liver. In: Wisse E. Knook DL. eds. Kupffer cells and other liver sinusoidal cells. Amsterdam. Elsevier/North Holland, 1977.497. 18. Baker H de C. Paget GE. Davson J Hemangioendotheliomas (Kupffer-cell sarcoma) of the liver, | Pathol Bact 1956:72:173. 17. Burston J. Kupffer cell sarcoma. Cancer 1958:11,798. 18. Regelson W. Kim U, Ospino [. et al. Hemangioendothelial sar coma of liver from chronic arsenic intoxification by Fowler's solution. Cancer 1968:21:514. 19. Blackwell JB, |oske RA. Kupffer cell sarcoma. Dig Dis 1970:15:133. 20 Leu FI. Harry DS. Angiosarcoma of the liver in a vinyl chlo ride worker. Lancet 1974:1.1316. 21 Pollard SM, Mdlward-Sadler GH Malignant haemangioendothelioma involving the liver, [ Clin Pathol 1974:27 214. 22. Thomas LB. Popper H. Berk PD. et al. Vinyl chloride-induced liver disease N Engl | Med 1975:292.17 23 Smith PM. Williams M[. Evans DMD. Hepatic angiosarcoma in vinyl chloride workers. Bull NY Acad Med 1976.52,447, 24. Makk L. Delmore F, Creech |L |r. et al. Clinical and morpho logic features of hepatic angiosarcoma in vinyl chloride workers. Cancer 1976:37-149. 25 Popper H, Thomas LB. Telle3 N, et al. Development of hepatic angiosarcoma in man induced by vinyl chloride, thorotrast, and arsenic. Am | Pathol 1978:92:349. CMA 003594 2 BIOCHEMICAL ALTERATIONS IN LIVERS OF RATS EXPOSED TO VINYL CHLORIDE Julie T. Du, John P. Sindoi, Michael T. Tseng, Carlo H. Tamburro Division of Digestive Diseases and Nutrition, Department of Medicine, Department of Anatomy, and Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky Sprague-Oawley rats were exposed to vinyl chloride to determine the earliest sequential biochemical changes occurring with liver injury before angiosarcoma development. Activity of glucose-6-phosphatase, a key gluconeogenic enzyme in the liver microsomal fraction, decreased 25% with respect to controls after 70 h of exposure. GlucOse-6-phosphate dehydrogenase activity Increased twofold after more than 100 h of exposure. Nanprotein su/fhydryl levels (glutathione andfor cysteine) showed a slight but progressive elevation, whereas glutathione reductase activity increased 50-00% during exposure to vinyl chloride. NADPH-cytochrome c reductase and mixed function oxidase were unchanged in the same microsomal fraction. There were no changes in seven conventional clinical biochemical thee tests or in four other markers of liver mitochondrial, cytosol, and microsomal function. No significant histological changes were found on light microscopic examination during this exposure period. However, with electron microscopy, dilation of rough endoplasmic reticulum was seen in the animals exposed for more than 137 h. These enzymatic changes are considered to reflect- early hepatocellular adaptation to vinyl chloride exposure with very mild or limited hepatocellular Injury In Its earliest stage. INTRODUCTION Vinyl chloride has been shown to induce tumors, including angio sarcoma, in laboratory animals (Maltoni and Lefemine, 1975; Viola et al., 1971) and angiosarcoma in humans (Creech and Johnson, 1974). At high concentrations, vinyl chloride is believed to be metabolized by the microsomal mixed function oxidase (MFO) system of the liver to toxic metabolites (Bolt et al., 1975; Hefner et al., 1975; Johnson, 1967; Watanabe et al., 1976b, 1976c). Animals pretreated with phenobarbital, an inducer of We wish to express our sincere thanks to the people in the 8. F. Goodrich PUnt in `Louisvilfo.^*. for their cooperation in the exposure studies. Or. R. A-.Grecnberg for help with statistics, and'Ms. Ruth Shelton for technical assistance. This work was supported by a grant from the Manufacturing Chemists Association, Washing ton, DC. Requests for reprints should be sent to Carlo H. Tamburro. Division of Digestive Diseases and Nutrition, Health Sciences Center, MDR 33J, University of Louisville, Louisville, Kentucky 40232, 1119 leertul of Toxicology and Environmental Health, 511119-1132, 1979 Copyright O 1979 by Hemisphere Publishing Corporation 009S-4108/79/051119-l4 2-25 CMA 003595 1120 J.T. OU ETal. MFO, and exposed to 5% vinyl chloride (50,000 ppm) had increased serum alanine aminotransferase, a conventional biochemical indicator of liver injury, and increased serum sorbitol dehydrogenase, a liver specific enzyme (Jaeger et ah, 1974). However, there have been no sequential enzymatic studies to determine what progressive metabolic alterations of the hepatocyte occur during vinyl chloride exposure preceding the development of angiosarcoma. Although the nepatocyte is the major cell for oxidation and, probably detoxification of vinyl chloride and its metabolites, angiosaccoma develops not in the hepatocyte but in adjacent mesenchymal cells. During this time, the hepatocytes undergo changes (focal nodular hyperplasia) suggesting focal regeneration, probably from limited hepatocellular injury (Tamburro et ah, 1979). The purpose of our study was to characterize the earliest sequential enzymatic changes that occurred in vinyl chloride exposure to elucidate the hepatocytes' role in vinyl chloride-induced injury and angiosarcoma develop ment. Portions of this study have been presented elsewhere (Du and Tamburro, 1976). METHODS Animals and Experimental Design Sprague-Dawley male rats (300-500 g) were randomly assigned before each experiment to either a group to be exposed to vinyl chloride gas (15,000 ppm) or a nonexposed conirol group kept outside the chamber in the animal room. Animals were fed" standard laboratory chow pellets ad libitum. Four sequential experiments were done. In experiments A and B, rats were exposed 2-4 h/d, 4 d/wk, for 1-2 wk with total accumulated exposure times of 14, 2S, and 42 h. In experiment C, rats were exposed 4-8 h/d, 5 d/wk, for 2-3 wk with total accumulated exposure periods of 71 and 103 h. In experiment D, rats were exposed 6-8 h/d, 5 d/wk, for 3-4 wk with'total accumulated exposure periods of 84 and 137 h. The animals were exposed in a modified chamber consisting of an airtight vat that had been used for manufacturing polyvinyl chloride. An aliquot of vinyl chloride (185 g) was added to the vat per 4-d period to make an average concentration of 15,000 4000 ppm. The air was constantly circulated by a stirrer. The chamber volume was 4400 I (1100 gal), so the' respiration of ihe rats had a negligible effect on the composition of the chamber's atmosphere. Experiments with an added control group exposed only to air in an identical modified var showed no differences from the controls kept in the animaHroom (Table 1). All animals were anesthetized with ether, had blood drawn by cardiac puncture, and were sacrificed at the same time of day (within 3 h after final exposure) to avoid diurnal effects. CMA 003596 VINYL CHLORIDE EFFECTS ON RAT LIVER mi TABLE 1. Comparison of Hepatic Eniyme Acti\it;; in Animal Control Groups Band in Animal Quartan and In the Exposure Chamber Air exposed Glueose-fi-phosphate dehydrogenase Glutathione reductase 12.82 a 1.32f A.77 1 0.27^ Chamber 11.06 i 1.78c 5.28 t OAOa Kept in chamber 7 h/d, 5 d/wk, (or 6 wk; total, 210 h. *No significant. "Not significant. Sample Preparation Liver was excised rapidly and was immediately rinsed in ice-cold 0.15 M KCI with 0.02 M Tris buffer, pH 7.4, A portion of the tissue was homogenized with 9 volumes of the cold KCI-Tris buffer in a PotterElvehjem homogenizer. Each sample was prepared from a single organ and kept at 4C during preparation. Remaining liver was frozen rapidly and stored at --20C. For assays with frozen tissue, livers from control and experimental rats were frozen in an identical manner for the same length of time. Subcellular Fractionation and Biochemical Determination Homogenate was centrifuged at 600 and 8000 Xg for 10 min and at 100,000 Xg for 60 min to obtain the nuclear, mitochondrial, microsomal, and cytosol fractions, respectively, by the differential technique of Schneider and Hogcboom (1950). Centrifugation was at 4C in a Sorvall refrigerated RC5 supercentrifuge with a fixed angle rotor and a Beckman model L-5 ultracentrifuge with a swinging bucket rotor. Enzyme activities and cytochrome P-450 were determined in the isolated subcellular frac tions: cytochrome oxidase in the mitochondrial fraction; NADPHcytochome c reductase, mixed function oxidase, cytochrome P-450, and glucose-6-phosphatase in the microsomal fraction; and glutathione reductase and gluco$e-6*phosphate dehydrogenase in the 100,000 supernatant fraction. Cytochrome P-450, NADPH-cytochrome c reductase, and cytochrome oxidase were determined in fresh samples; all other determinations were in TM freshly isolated fractions from frozen tissue. '"Glucose-6-phosphatase was estimated by measuring release of inorganic phosphate (Harper, 1965). Cytochrome P-450 was estimated by the maximum absorption difference between the dithionite-reduced cytochrome and its CO complex (Omura and Sato, 1964). The P-450 concentration was calculated by using 91,000 CMA 003597 1- Pi.'.icn ~t .-1:21:1c c";y.Tie in Ammil Cuniroi Croups 3md in Anirnil Qumerj m3 in [he Expoiurs Chinhe.-17 Air exposed A .*n ii "5orrr rv --- w Cfuco$e-6-pfioipJuc dcttydrogenAse Gluutoion* rtduciue 12.5 Z 1 I.32c *.77 : 0,27d 11.05 r ].73c 5.23 i 0.AC* aViluej ire meeni t SEM (n *= 6). &Kept in chimber 7 h/d, 5 d/wfc, for 6 wk; tout, 310 h. (Not lijmfieinu ^Not significant. Sample Preparation Liver was excised rapidly and was immediately rinsed in ice-cold 0.15 M KCI with 0.02 M Tris buffer, pH 7.4. A portion of the tissue was homogenized with 9 volumes of the cold KCI-Tris buffer in a PotterElvehjem homogenizes Each sample was prepared from a single organ and kept at 4C during preparation. Remaining liver was frozen rapidly and stored at --20C. For assays with frozen tissue, livers from control and experimental rats were frozen in an identical manner for the same length of time, Subcellular Fractionation and Biochemical Determination Homogenate was centrifuged at 600 and 8000 Xg for 10 min and at 100,000 Xg for 60 min to obtain the nuclear, mitochondrial, microsomal, and cytosol fractions, respectively, by the differential technique of Schneider and Hogcboom (1950). Centrifugation was at 4C in a Sorvall refrigerated RC5 supercentrifugc with a fixed angle rotor and a Beckman model L-5 ultracentrifuge with a swinging bucket rotor. Enzyme activities and cytochrome P-450 were determined in the isolated subcellular frac tions: cytochrome oxidase in the mitochondrial fraction; NADPHcytochome c reductase, mixed function oxidase, cytochrome P-450, and glucose-6-phosphatase in the microsomal fraction; and glutathione reductase and glucose-6-phosphate dehydrogenase in the 100,000 Xg supernatant fraction. Cytochrome P-450, NADPH-cytochrome c reductase, and cytochrome oxidase were determined in fresh samples; all other determinations were in t> freshly isolated fractions from frozen tissue. .~Glucose-6-phosphatase was estimated by measuring release of inorganic phosphate (Harper, 1965). Cytochrome P-450 was estimated by the maximum absorption difference between the dithionite-reduced cytochrome and its CO complex (Omura and Sato, 1964). The P-45Q concentration was calculated by using 91,000 003598 CMA M'x cm'1 as the extinction coefficient for the increase in peak height between 490 and 450 nm. Cytochrome oxidase was measured as described by Wharton and Tzagoloff (1967), and NADPH-cytochrome c reductase according to Degroot and Dunn (1964). Mixed function oxidase was determined according to Holtzman et al. (1968) by measuring the hydroxylation of analine. Nonprotein sulfhydryl was estimated by the method of Sedlak and Lindsay (1968). Rate of oxidation of NADPH by oxidized glutathione was used as a measure of enzymatic activity of glutathione reductase (Carlberg and Mannervik, 1975). Glucose-ephosphate dehydrogenase was measured spcctrophotometrically, as the rate of NADPH formation (Lohr and Waller, 1965), Assays were conducted under conditions of linearity with respect to both rime and protein. Protein content was determined by the method of Lowry et al. (1951). Conventional clinical analyses of serum included aspartate aminotransferase (AST, SGOT), alanine aminotransferase (ALT, SGPT), alkaline phosphatase, bilirubin,albumin, cholesterol, and triglyceride, determined by rhe technicon sequential multiple analyzer computer (SMAC) system. i Materials The NADPH, cytochrome c, and oxidized glutathione were obtained from Sigma Chemical Co., St. Louis, Mo. Analine and other chemicals were reagent grade. Double-distilled water was used throughout. Light and Electron Microscopy Small strips of liver were removed under ether anesthesia and immersed immediately in ice-cold 3% glutaraldchyde (pH 7.4). Tissues were sliced into small cubes and fixed for 2 h at 4C. Subsequently, samples were washed overnight in phosphate buffer and postfixed in 1% osmium tetroxide for 1 h before being dehydrated in ascending alcohol and embedded in Epon. Tissue blocks were polymerized at 60C for 2 d. Thin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate before examination on a Philips 300 electron microscope. For ultrastructural analysis, control rats and rats exposed for 42 and 137 h to vinyl chloride were reviewed. For light microscopy, a block of tissue was fixed in buffered formalin and processed routinely for paraffin embedding. Sections 6 ftm thick were stained with hematoxylin and eosin before examination. RESULTS The protein contents (milligrams per gram of liver) of the subccllular fractions in control and vinyl chloride-exposed groups were the same throughout the exposure; therefore, the enzymatic results were expressed as micromoles converted per minute per milligram of subccllular protein. The statistical analysis (sec below) showed certain significant 003599 CMA VINYL CHLOKIDE EFFECTS ON RAT LIVER 1123 enzymatic differences between the exposed and control groups in glucose6-phosphatasc, glutathione reductase, and glucose-6-phosphatc dehydro genase. Glucose-6-phosphatase. No significant differences in glucose-6-phosphatase activity were detected between the two groups up to 42 h; however, after 71 h, the glucose-6-phosphatase activity was significantly less in the exposed than in the control animals. Figure 1 a shows the 25% decrease in mean activity of glucose-6-phosphatasc after 71 h; this activity remained significantly lower up to 137 h. Figure 1 b shows the 95% confidence intervals for D, the mean difference between the exposed group and the control groups, in each experiment. (*) FIGURE 1. |a) Compose carve at group mans of specific activity of glucosc-6-phosphatase with respect to exposure time; (i) 9556 confidence intervals tor D, the mean difference between the exposed end control groups. There is no significant difference between the exposed and control groups until after 42 h of exposure. After 71 h the mean level of the exposed group Is significantly less than that of the control group. CMA 003600 enzymatic differences between the exposed end control groups in glucose6-p.nosphacasc, g/Uiatnione reductase, and giucose-6-phosphatc dehydro genase. Glucose-S-phosphatase. No significant differences in giucose-6-ohn;p.'ctote act. .:;/ vrero detected between the two groups up to ~>2 n; however, after 71 h, the glucose-6-phosphatase activity was significantly less in the exposed than in the control animals. Figure 1<7 shows the 25% decrease in mean activity of glucose-6-phosphatase after 71 h; this activity remained significantly lower up to 137 h. Figure If? shows the 95% confidence intervals for D, the mean difference between the exposed group and the control groups, in each experiment. Ul FIGURE 1. (a) Compov'te curve of (roup mum of specific activity of (lueoie-6-phosphatase with respect to exposure time; (0) 95% confidence intervals for 0, the mean difference between the exposed and control (roups. There Is no sifniflcant difference between the exposed and control (roups until after 42 h of exposure. After 71 h the mean level of the exposed (roup is significantly less than that of the control group. .. CMA 003601 1124 I. T, DU ET AL. Glutathione Reductase. Figure 2, a and 6, illustrates the significant differences (p<0.05) between the exposed and control groups in all 4 experiments except at 71 h of exposure Co < 0.06). The glutathione reductase level was 25% greater in exposed than in nonexposed animals up to 71 h and approximately 50% greater after 84 and 137 h. Glucose-6-phosphate Dehydrogenase. There was no significant differ ence between exposed and control groups from 14 to 42 h and at 71 and 84 h. However, after 103 and 137 h of exposure there was a statistically significant difference as shown in Fig. 3, a and b. After about 100 h of exposure, the mean glucose-6-phosphate dehydrogenase level in exposed animals was about twice that in nonexposed animals. While these differences were occurring, none of ihe conventional biochemical liver function tests (e.g., serum aminotransferases, alkaline phosphatase) showed any significant change, nor were there any changes in cytochrome oxidase, mixed function oxidase, P-450, or NADPHcytochrome c reductase. C a < *4 *<3 *J lie Vl4 o Exposure Tm (hr*/ o >t 0 - - jf* 14,11,4} C O *s> If fIGURE 2. (o) Composite curve of group mean* of specific activity of glutathione reductase with respect to exposure time; (p) confidence interval, for D, the mean difference between the exposed and control (roup. The mean value for the exposed group is sipnifieantfy preater than that for the controls throughout the entire experiment (r> < 0.05) except at 71 H of exposure. CMA 003602 VINYL CHLORIDE EFFECTS ON RAT LIVER 1125 FIGURE 3. (o) Composite curve of (roup meins of specific ictivity of tlucote-6-phosohite dehydrogenise with respect to exposure time; (ft) 95% confidence Intervals for D, the mean difference between the exposed and control groups. The mean value for the exposed group Is not significantly different from that for the control group until after 84 h of exposure. The concentration of reduced glutathione in the livers of rats decreased to 52% of the control value after a single 2-h exposure to 15,000 ppm vinyl chloride (Table 2). However, the reduced glutathione concentration appeared to be the same or slightly elevated when the rats underwent multiple exposures. This elevation was not significant at the 5% level. The vinyl chloride-exposed rats suffered a weight loss of 4-13% and the control group gained 2-10% (Table 3). All rats survived to the termination of the experiment without noticeable ill effects. Statistical Analysis of Results The 95% confidence interval for group mean differences in each enzyme study was based on the error mean square from a two-factor analysis of variance with interaction,, the two main effects being exposure time in hours and cxposurc-noncxposurc. This analysis was performed for CMA 003603 Expo.ur* "'V' (h/.) "-'V> C o .' FIGURE 3. (p) Composite curv* of (roup meins of specific ictlvity of glucose-6-phosphite dehydrogenise with respect to exposure time; (ft) 9S34 confident* Intertill for D, the mein difference between the exposed and control groups. The mean value tor the exposed group is not significantly different from that for the control group until after 84 h of exposure. The concentration of reduced glutathione in the livers of rats decreased to 52% of the control value after a single 2-h exposure to 15,000 ppm vinyl chloride (Table 2). However, the reduced glutathione concentration appeared to be the same or slightly elevated when the rats underwent multiple exposures. This elevation was not significant at the 5% level. The vinyl chloride-exposed rats suffered a weight loss of 4-13% and the control group gained 2-10% (Table 3). All rats survived to the termination of the experiment without noticeable ill effects. -d*"1 Statistical Analysis of Results The 95% confidence interval for group mean differences in each enzyme study was based on the error mean square from a two-factor analysis of variance with interaction,, the two main effects being exposure time in hours and cxposurc-nonexposure. This analysis was performed for CMA 003604 1126 J.T. 0(J ETAL. TABLE 2. Effect of Vinyl Chloride Exposure on Concentration of Liver Nonprotein Sulfhydryl Compound in Rati Exposure tfm (h) 2 14 28 42 71 84 103 137 Concentration ratio (exposcd/control) 0.52(3} 1.02 (6) 0.96 (6) 0.82 (6) 1.21 (5) 1.07 (3) 1.49 16) 1.29 (3) ^Single exposure. Number of animals in control or experimental group Is shown in parentheses. cMuliiple exposures (4-8 h/d, 4-5 d/wk) for 14-137 h. TABLE 3. Body Weights of Rats before and after Vinyl Chloride Exposure0 Accumulated exposure N 14 28 42 71 84 103 137 Time Before After Before After Before After Before After Before After Before Afrter Before After Control 442 * 6.2 (3)e 454 9.5 (3) 436 9.2 (3) 445 12.1 (3) 433 5.2 (3) 455 t 6.4 (3) 437.6 12.2 (5) 447 19.4 (5) 412.7 7.9 (3) 455.fr 6,8 (3) 453 12.9 (6) 465 i 16.9 (6) 404.7 Jl 1.2 (3) 425,0 i 5.2 (3) Change <%) +3 2 5 +2 10 +3 S Vinyl chlorideexposed 453 t 1.5 (3) 421 t 4.1 (3) 448 6.0 (3} 408 3.0 (3) 450 4.3 {3) 410 2.8 (3) 422.7 9.2 (5) 385.0 * 4.2 (5) 425 * 17.4 (3) 399 72 (3) 441.5 6.8 (6) 397.0 * 5.7 (6} 415.0 15.7 (3) 398 i 28 (3) Multiple exposures to 1.5% vinyl chloride for 14-137 h, 4-8 h/d, 4-5 d/wk. 6Results are expressed as mean a SEM. ^Number of animals is given in parentheses. Change w -7 -9 -9 -13 -6 -10 --4 CMA 003605 VINYL CHLORIDE EFFECTS ON RAT LIVER 1127 each of the four experiments. Overall significant (p<0.05) differences between the exposed and control groups are found when the 95% confidence intervals (0 values) do not contain zero. In Fig. lb", there is no significant difference (p>0.05) between the 2 groups in experiments A and B because a D value of zero is within these two confidence intervals. However, for experiments C and D, the con fidence intervals do not contain a D value of zero, indicating a significant difference [p < 0.05) between the exposed and control groups. When the D value is less than zero (as in Fig. 1b), the experimental group is significantly lower than the control throughout the experiment; when the D value is greater than zero (as in Figs. 2 and 3), the experimental group is significantly higher than the control. Further, in experiments C and D of Fig. 1b, a single confidence interval is shown even though there are two time periods of exposure each. This results from the nonsignificance of the interaction term in the analysis of variance, meaning that the observed difference at 71 h of exposure is not statistically different from the difference at 103 h. Similar conclusions hold for experiment D in Fig. 1b. Figure 2b shows 2 confidence intervals for the set of experiments C at 71 and 103 h because the interaction of the time and exposure factors was significant; that is, the difference between the exposed and control groups after 103 h of exposure was significantly greater than the difference after 71 h. This is also reflected by the differences of the mean values at 71 and 103 h as shown in Fig. 2a. For the same reasons, there are also two confidence intervals for the C (71 and 103 h) and D (84 and 137 h) sets of experiments in Fig. 3b. Morphological Studies Morphological light microscopic studies were performed in a blind (coded) and randomized fashion and did not show any evidence of hepatocellular injury or changes usually seen in the latter stages of vinyl chloride exposure. Besides some variations in glycogen content, no difference in the fine structure of the hepatocytes was observed between controls (Fig. 4) and those exposed to vinyl chloride for 42 h. Dilation of rough endoplasmic reticulum was observed in a small number of hepatocytes after 137 h of vinyl chloride exposure (Fig. 5). Although no noticeable change in the amount of smooth endoplasmic reticulum was associated with this change, a concomitant increase in cytoplasmic density was evident in these cells. Another type of lesion found in other hepatocytes was characterized by the presence of small patches of clear spaces, which tended to aggregate near the cell periphery (Fig. 6). Such lesions usually affect the adjacent cell equally. No other cell type appeared to be affected by vinyl chloride exposure in this study. CMA 003606 VINYL CHLO'IOH ' 3 ON 3aT LI v; = i V27 each of the four experiments. Overall significant (p < 0.05) differences between the exposed and control groups are found when the 95% confidence intervals [D values) do not contain zero. in Fig. \cr, there is no significant difference (p>0.05) between the 2 groups in experiments A and B because a D value of zero is within these two confidence intervals. However, for experiments C and D, the con fidence intervals do not contain a D value of zero, indicating a significant difference {p <0.05) between the exposed and control groups. When the D value is less than zero (as in Fig. Id), the experimental group is significantly lower than the control throughout the experiment; when the D value is greater than zero (as in Figs. 2 and 3), the experimental group is significantly higher than the control. Further, in experiments C and D of Fig. lb, a single confidence interval is shown even though there are two time periods of exposure each. This results from the nonsignificance of the interaction term in the analysis of variance, meaning that the observed difference at 71 h of exposure is not statistically different from the difference at 103 h. Similar conclusions hold for experiment D in Fig. 16. Figure 26 shows 2 confidence intervals for the set of experiments C at 71 and 103 h because the interaction of the time and exposure factors was significant; that is, the difference between the exposed and control groups after 103 h of exposure was significantly greater than the difference after 71 h. This is also reflected by the differences of the mean values at 71 and 103 h as shown in Fig. 2a. For the same reasons, there are also two confidence intervals for the C (71 and 103 h) and D (S4 and 137 h) sets of experiments in Fig, 36. Morphological Studies Morphological light microscopic studies were performed in a blind (coded) and randomized fashion and did not show any evidence of hepatocellular injury or changes usually seen in the latter stages of vinyl chloride exposure. Besides some variations in glycogen content, no difference in the fine structure of the hepatocytes was observed between controls (Fig. 4) and those exposed to vinyl chloride for 42 h. Dilation of rough endoplasmic reticulum was observed in a small number of hepatocytes after 137 h of vinyl chloride exposure (Fig. 5). Although no noticeable change in the amount of smooth endoplasmic reticulum was associated with this change, a concomitant increase in cytoplasmic density was evident in these cells. Another type of lesion found in other hepatocytes was characterized by the presence of small patches of dear spaces, which tended to aggregate near the ceil periphery (Fig. 6). Such lesions usually affect the adjacent cell equally. No other cell type appeared to be affected by vinyl chloride exposure in this study. CMA 003607 ms ). T. DU ET At.. FIGURE 4. Portion of i hepatocyte from a control rat. A food complement of mitochortdria and rough endoplasmic reticulum is shown (XI2,000). FIGURE 5. Dilation of RER (arrows) shown in hepatoeyte 137 h after exposure to 15,000 ppm vinyl chloride during 2-3 wk (X35,000). FIGURE 6. Subplasmalemmal lesions (*) in hepatocytes 137 h after exposure to 15,000 ppm vinyl chloride during 2-3 wfc. The lesions are present in two adjacent ceils and the dear spaces seem to coalesce (XI 2,000). DISCUSSION Decreased glucose-6-phosphatase activity and increased glucosee-phosphate dehydrogenase and glutathione reductase activity in rat liver after exposure to vinyl chloride (as in our studies) may be the biochemical alterations indicative of early liver injury, adaptation to increased, detoxi fication activity, or preparation for increased nucleic acid synthesis. These differences were observed before any abnormalities were detectable from conventional liver function tests, such as scrum aminotransferases, or from changes in other subccllular organelle markers, such as mitochondrial cytochrome oxidase activity. In primary hepatocellular cancer (hepatomas), the activity of key enzymes for gluconeogcnesis (glucosc-6-phosphatase, etc.), decreased with increased rate of tumor growth (Weber, 1974; Weber and Convcry, 1966; Weber and Lea, 1967). In contrast, two enzymes for the pentose phosphate pathway, glucosc-6-phosphate dehydrogenase (Sclmcci and CMA 003608 VINYL CHLORIDE EFFECTS ON RAT LIVER 1119 Weber, 1976; Weber and Morris, 1963) and transaldolasc (Heinrich ct at., 1974) , increased in all hepatomas. Further, the activity of gluclosc-6phosphatase decreased before and during the development of hepatomas when carcinogens such as nitrosamine and dimethylaminonzobenzene were fed to rats (Isok and Teras, 1973; Weber and Cantero, 1955). Whether our similar findings in this study are indicators of eventual cancer development (angiosarcoma) is not yet known. We used a shorter exposure period in order to identify the biochemical changes that would best reflect the morphological and cellular changes anticipated on the basis of previous human and animal studies. No attempt was made to determine what effects these shorter exposure periods would induce with long-term observation. This is now in progress. However, Maltoni and Lefemine (1975) showed that of 69 Sprague-Dawley rats exposed to 10,000 ppm vinyl chloride, 16 (26%) developed Zymbal gland carcinomas after 50 wk, 5 (8%) developed nephroblastomas after 59 wk, and 9 (15%) developed angiosarcomas after 64 wk. The vinyl chloride-exposed groups lost weight (4-13%) whereas the control group gained weight (2-10%) during the entire experimental period. However, the differences in the three enzymes cannot be accounted for by lack of dietary food intake in the experimental group, because fasting increases glucose-6-phosphatase (Ashmore et al., 1954) and decreases glucose-6-phosphate dehydrogenase (Winberry and Holtcn, 1977), and we found decreased glutathione reductase and reduced glutathione content in fasted animals (unpublished data). Rats exposed to a high dose (5%) of vinyl chloride (Reynolds et al., 1975) had decreased mixed function oxidase activity. An in vitro study (Ivanetich et al., 1977) showed that the metabolites of vinyl chloride from the microsomal enzyme system decreased the levels of cytochrome P-450. In our studies; the cytochrome P-450 content and the activity of mixed function oxidase and NAOPH-cytochrome c reductase in rats repeatedly exposed to 1-2% vinyl chloride did not show any such changes. Sub sequent experiments in which rats were exposed to 28,000 ppm vinyl chloride for 70-210 h in 2-6 wk showed a significant decrease of cytochrome P-450 concentration (in preparation). Differences in dose level or experimental design may account for these variations. Drew et al. (1975) found that the activity of mixed function oxidase depended on the time interval between cessation of exposure to vinyl chloride and sacrifice of animals. The main detoxification route for vinyl chloride metabolism is thought to be conjugation with glutathione (johnson, 1967); sulfur-containing urinary metabolites of radioactively labeled vinyl chloride have been found in rats' urine (Green and Hath way, 1975, 1977; Watanabe et al., 1976b, 1976c). We found that under the present experimental conditions-the nonprotcin sulfhydryl content of liver (glutathione and/or cysteine) tended to 003609 V; N L " L v ^ j i r f i `^ ` Weber, 1975; Weber and Morris, 1953) era.-iezd'Jolese {He-nrkh et i!,, 1974), increased in all hepatomas. Further, the activity of glucIcsc-5- pnosphatase decreased before and during the development of hepatomas when carcinogens such as n'tresamme and dimethy!am:roaaobeuaene fed to rats (Isok and Teras, 1973; Weber and Cantero, 1955). Whether our similar findings in this study are indicators of eventual cancer development (angiosarcoma) is not yet known. We used a shorter exposure period in order to identify the biochemical changes that would best reflect the morphological and cellular changes anticipated on the basis of previous human and animal studies. No attempt was made to determine what <1w- 'c-v r-:2esrr ex'p'noesiu'-rse npse-ri co!s `.vou m .-i i < -* a I n n * a ---i oosorviticri, i"ti5 is new in progress. *xG'-i'ev5r( I'liuoni tincl rr.,r \t^- q ~' -\ :wcd that of 59 Scrague-^awla/ rats exposed to 13,200 per vinyl chloride, 16 (26%) developed Zymbal gland carcinomas after 50 wk, 5 (S%) developed nephroblastomas after 59 wk, and 9 (15%) developed angiosarcomas after 64 wk. The vinyl chloride-exposed groups lost weight (4-13%) whereas tne control group gained weight (2-10%) during the entire experimental period. However, the differences in the three enzymes cannot be accounted for by lack of dietary food intake in the experimental group, because fasting increases glucose-6-phosphatase (Ashmore et al., 1954) and decreases glucose-6-phosphate dehydrogenase (Winberry and Holtcn, 1977), and we found decreased glutathione reductase and reduced glutathione content in fasted animals (unpublished data). Rats exposed to a high dose (5%) of vinyl chloride (Reynolds et al., 1975) had decreased mixed function oxidase activity. An in vitro study (Ivanetich et al., 1977) showed that the metabolites of vinyl chloride from the microsomal enzyme system decreased the levels of cytochrome P-450. In our studies; the cytochrome P-450 content and the activity of mixed function oxidase and NADPH-cytochrome c reductase in rats repeatedly exposed to 1-2% vinyl chloride did not show any such changes. Sub sequent experiments in which rats were exposed to 28,000 ppm vinyl chloride for 70-210 h in 2-6 wk showed a significant decrease of cytochrome P-450 concentration (in preparation). Differences in dose level or experimental design may account for these variations. Drew et al. (1975) found that the activity of mixed function oxidase depended on the time interval between cessation of exposure to vinyl chloride and sacrifice of animals. The main detoxification route for vinyl chloride metabolism is thought to be conjugation with glutathione (Johnson, 1967); sulfur-containing urinary metabolites of radioactively labeled vinyl chloride have been found in rats' urine (Green and Hathway, 1975, 1977; Watanabe et al., 1976b, 1976c). We found that under the present experimental conditions-the non protein sulfhydryi content of liver (glutathione and/or cysteine) tended to CMA 003610 1130 1. T. DU ET AL. increase in rats repeatedly exposed to vinyl chloride (Tabic 2). Although the difference was not significant in this experiment, subsequent exposure of rats to 28,000 ppm for 70, HO, and 210 h in 2, 4, and 6 wk led to a significant elevation of the nonprotcin sulfhydryl content in liver (Du and Tamburro, 1978). Watanabe et al. (1976a) and Johnson (1967) found a depletion of nonprotein sulfhydryl content in rats after only a single exposure to vinyl chloride or chloroethanol. Hefner et al. (1975) found that a single vinyl chloride exposure reduced the glutathione content by one-half, but the decrease became smaller and even unnoticeable after repeated exposure to vinyl chloride. Fiala et al. (1976) showed that the reduced glutathione content increased after prolonged exposure of rats to various chemical carcinogens. These results are thought to reflect the exposed animals' attempt to make more glutathione to meet the unusually strong demand for detoxification. Glutathione reductase was elevated in our system after rats were exposed to vinyl chloride for 42 h. This enzyme generates reduced glutathione from its oxidized form as a compensatory mechanism to maintain the level of glutathione, and the simultaneous elevation of glucose-6-phosphate dehydrogenase regenerates NADPH, vvhich can be used as a cofactor for various synthetic pathways including nucleic acid synthesis. Elevated glutathione reductase activity was found in rats with primary hepatocellular cancer induced by diethylnitrosamine (Pinto and Bartley, 1973). This consistent increase in glutathione reductase activity after exposure to vinyl chloride suggests that it may be one of the earliest biochemical manifestations of exposure and injury. These metabolic changes could also play a role in the early changes observed by light and electron microscopy. Hepatic lesions such as dilation of smooth endoplasmic reticulum and loss of microvilli were reported in mice as early as 1 mo after vinyl chloride exposure (Schaffner ct al., 1976). In contrast, we observed dilation of rough endoplasmic reticulum and patchy, lesions near the plasmalemma. The dilated rough endoplasmic reticulum could be related to the increased enzyme synthesis (i.e, glucose6-phosphate dehydrogenase and glutathione reductase) induced by vinyl chloride. The absence of smooth endoplasmic reticulum proliferation in our animals suggests a comparatively milder effect rn our short-term study. In a subsequent study where long-term effects of vinyl chloride exposure was assessed, dilation of smooth endoplasmic reticulum in hepatocytcs was observed (in preparation). The nature of the patchy lesions found near the plasmalemma remains to be established. The location of the lesion, however, suggests that some toxic agents may be entering or exiting the hepatocytcs in these sites. We believe these early enzymatic changes reflect adaptation of the liver cell to early mild injury. The alterations in gluconeogcncsfs and in the pentose phosphate shunt appear to indicate the liver's adaptation while undergoing repetitive and prolonged exposure to the mildly toxic chemical CMA 0036X1 VINYL CHLORIOE EFFECTS ON RAT LIVER . 1131 vinyl chloride, which, when inadequately metabolized, produces inter mediates that lead to the formation of cancer. REFERENCES Ashmore, J., Hastings, A. B,, and Nesbctt, F. B. 1954. The effect of diabetes and fasting on liver gluco*e-6-phosphatasc. Proc. Natl. Acad. Sd. (J.S.A. 40:673-678. Bolt, H. M., Kappus, H., Buchter, A., and Bolt, W. 1975. Metabolism of vinyl chloride. Lancet 1:1425. Cariberg, I. and Mannervik, B. 1975. Purification and characterization of the ftavoenzyme glutathione reductase from rat liver. /. Biol. Chtm. 250:5475-5480. Creech, J. L. and Johnson, M. N, 1974. Angiosarcoma of liver In the manufacture of polyvinyl chloride./. Occup. Med. 16:150-151. Oegroot, L. ). and Dunn, A. D. 1964. Electron-transport enzymes of calf thyroid, Biochlm. Biophys. Acta 92:205-222. Drew, R. T., Harper, C,, Gupta, B. N., and Talley, F. A, 1975. Effects of vinyl chloride exposures to rats pretreated with phenobarbital. Environ. Health Perspcct. 11:235-24Z Du, J. T. and Tamburro, C. H. 1976. Decreased glucose-6-phosphatase activity in liver in vinyl chloride exposed rats. Fed. Proc. 35:329. Dtl, J. T. and Tamburro, C. H. 1978. Elevated glutathione content, glutathione-5-transferase and glutathione reductase in liver of rats exposed to vinyl chloride. Fed. Proc. 37:1545. Flala, S., Mohlndru, A., Kettering, W. G., Fiala. A. E., and Morris, H. P. 1976. Glutathione and gamma glutamyl transpeptidase in rat liver during chemical carcinogenesis. /. Natl. Cancer Inst. 57:591-598. Green, T. and Hathway, 0. E. 1975. The biological fate in rats of vinyl chloride in relation to Its oncogenicity. Chtm. Biol. Interact. 11:545-562. Green, T. and Hathway, D. E. 1977. The chemistry and biogenesis of S-containing metabolites of vinyl chloride in rats. Chtm. Biol. Interact. 1 7:137-150. Harper, A. E. 1965. Glucose-6-phosphatase. In Methods of Enzymatic Analysis, ed. H. U, Bergmeyer, pp. 788-792, New York: Academic. Hefner, R. E., Waunabe, P. G., and Gehring, P, |. 1975. Preliminary studies of the fate of inhaled vinyl chloride monomer In rats. Ann. N.Y. Acad. 5c/. 246:135-148. Heinrich, P. CL, Morris, H. P., and Weber, G. 1974. Increased phosphoribosylpyrophosphate synthetase activity In rapidly growing hepatomas. FE8S Lett. 42:145-148. Holtzman, J. 1_, Gram, T. ., Cion, P. L., and Gillette, J. R. 1968. The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells. Biochttn. J. 110:407-412. Isok, M. E. and Teras, L. E. 1973. Glucose-6-phosphatase activity In liver carcinogenesis and In transplantable hepatoma In mice. Vopr. Med. Khim. 19:568-570. Ivanetich, K. M., Aronson, I., and Katz, I. D. 1977. The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 In vitro. Blochem. Blophys. Res. Common. 74:1411-1418. Jaeger, R. J., Reynolds, E. 5., Connolly, R. 8., Moslen, M. T., Szabo, A., and Murphy, S. M. 1974. Acute hepatic injury by vinyl chloride in rats pretreated with phenobarbital. Nature (Land.) 252:724-726. Johnson, M. K, 1967, Metabolism of chloroethanol in the rat. Biochtm. Pharmacol, 16:185-199. Lohr, G. W. and Waller, H. D. 1965. Glueose-6-phosphate dehydrogenase. In Methods of Enzymatic Analysis, ed. H, U. Bergmeyer, pp. 744-751. New York: Academic. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folln phenol reagent. /. Biol. Chtm. 193:265-275. Maltoni, C. and Lcfemine, G. 1975. Carcinogenicity bioassays of vinyl chloride: Current results. Ann. N.Y. Acad. Sd. 246:195-218. NYL CMLO'J vinyl chloride, which, when inadequately metabolized, produces inter mediates that lead to the formation of cancer. REFERENCES Ashmore, ).. Hastings, A. 8., and Nesbett, F. B. 1954. The effect of diabetes and fasting on liver glueose-6-pho*phacas. Proc. Natl, Acad, S:i. J.S.A. 40-673-678. Bolt, H. M., Kappus, H., Suehter, A., and Bolt, IV. 1375. Metabolism of vinyl chloride. Lancet 1:1425. v. 9. 1975 'a*n fit liver. n Oeech, J. L. and Johnson, M. N. 1974. 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Glucosc-6-phosphatase activity fn liver carcinogenesis and In transplantable hepatoma In mice. Vopr. Med. Khim. 19:568-570. Ivanetlch, K. M, Aronson, I., and Katz, I. 0. 1977. The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 In Htro. Blocftem, Blophys. Res, Commute. 74:1411-1418. Jaeger, R. J., Reynolds, . 5., Connolly, R. 8., Moslen, M, T,, Szabo, A., and Murphy, S. M. 1974, Acute hepatic injury by vinyl chloride in rats precreated with phenobarbitaJ, Nature (Land.) 252:724-726. Johnson, M. K. 1967. Metabolism of chloroethanol In the rat. Biochem. Pharmacol. 16:185-199. Lohr, G. W. and Walter, H. 0. 1965. Glucose-6-phosphatt dehydrogenase. In Methods of Entymatic Analysis, ed. H, U. Bergmeyer, pp. 744-751, New York: Academic. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Falln phenol reagent. /. Biol. Chem. 193:265-275. Maltoni, C and Lefemine, G. 1975. Carcinogenicity bioassays of vinyl chloride: Current results. Ann. N.Y. Acad. 5c/. 246:195-218. CMA 003613 1132 J. T. DU ET AL. Omufi, T. and Sato, R, 1964. The carbon monoxide-binding pigment of liver microtomes, I. Evidence for Its hemoprotein nature. /. Biol. Chem, 239:2370-2378. Pinto, R. E. and Bartley, W. 1973. Glutathione reductase and glutathione peroxidase activities in hepatomom livers of rats treated with diethyl nitrosamine. FEBS Lett. 32:307-309. Reynolds, E. S., Moslen, M. T., Saabo, S-, and Jaeger, R. J. 1975. Vinyl chloride-induced deactivation of cytochrome P-450 and other components of the liver mixed function oxidase system: An in vivo study. Res. Common. Chcm. Pathol. Pharmacol. 12:685-694, Sehaffner, F.. Popper, H., and Selikoff, I. J. 1976. Initial features of vinyl chloride hepatic Injury. Gastroenterology 71 :A35 (abstr.). Schneider, W. C. and Hogeboom, G. H. 1950. Intracellular distribution of eniymes. V. Further studies on the distribution of cytochrome e in rat liver homogenates. /. Biot. Chem. 183:1 23-1 2S. Sedlak, J. and Lindsay, R. H. 1968. Estimation of total protein bound and non-protein sulfhydryl groups in tissue with Ellman*s reagent. Anal. Biochem. 25:192-205. Selmeci, L, E. and Weber, G. 1976. Increased gluCOse-6-phosphate dehydrogenase concentration In hepatoma 3924A: Enzymic and immunological evidence. FEBS Lett. 61:63-67. Tamburro, C. H., Makk, L., and Popper, H. 1979. Early hepatic histological alterations among chemical (vinyl monomer) workers. Gastroenterology, in press. Viola, P, L., Bijotti, A,, and Caputo, A. 1971. Oncogenic response of rat skin, lungs-, and bones to vinyl chloride. Cancer Res. 31:516-522. Watanabe, P. Q., Hefner, R. E., Jr., and Gehring, P. J, 1976a. Vinyl chloride-induced depression of hepatic non-protein sulfhydryl content and effects on bromosulphaiein (BSP) clearance In rats. Toxicology 6:1-8, Watanabe, P. G., McGowan, G. R., and Gehring, P, M. 1976b. Fate of ,'1C-vfnyl chloride after single oral administration in rats. Toxicol. Appl. Pharmacol. 36:339-352, Watanabe, P. G., McGowan, G. R., Madrid, E. O., and Gehring, P. J. 1976c. Fate of ,4C-vfnyl chloride following inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37:49-50. Weber, G. 1974. The molecular correlation concept. In The Molecular Biology of Cancer, ed. H. Busch, pp. 487-521. New York: Academic. Weber, G. and Cantero, A. 1955. Glucose-6-phosphatase activity In normal, precancerous, and neoplastic tissues. Cancer Res, 15:105-108. Weber, G. and Con very, H. J. H. 1966. insulin: Inducer of glucosc-6-pbofphatc dehydrogenase. Life Sci. 5:1139-1146. Weber, G. and Lea, M. A. 1967. The molecular correlation concept. In Methods in Cancer Research, ed. H. Busch, vol. 2, pp, 523-578. New York: Academic. Weber, G, and Mortis, H. P. 1963. Comparative biochemistry of hepatomas. III. Carbohydrate enzymes In liver tumors of different growth rates. Cancer Res. 23:987-994. Wharton, D. C and Tzagoloff, A. 1967. Cytochrome oxidase from beef heart mitochondria. In Methods In Enrymology, eds. R. W. Estabrook and M: E. Pullman, vol. 10, pp. 245-250. New York: Academic. Wlnberry, L and Holten, D. 1977. Rat liver glucose-6-phosphate dehydrogenase. Oietary regulation of the rate of synthesis. /. 5/of, Chem. 252:7796-7801. Received March 20, 1979 Accepted fufy 23, 1979 CMA 003614 i i THE YALE JOURNAL OF BIOLOGY AND MEDICINE 51 (1978). 67-80 The Hepatic Role in Carcinogenesis and Its Early Detection--The Vinyl Chloride Model12 CARLO H. TAMBURRO University of Louisville School of Medicine, Louisville, Kentucky Received October 17, 1977 The liver's role in vinyl chloride toxicity and carcinogenicity is providing! better understanding of the chemical carcinogenesis mechanism. A variety of both malignant and benign hepatic tumors has been demonstrated with prolonged exposure to vinyl chloride. The multisystem involvment of this carcinogen and toxin has provided a mode! for the study of chemical carcinogenesis common to both man and animal. Clinical studies have shown the usefulness of biochemical, radioisotopic, and radiological studies in the detection of toxic and carcinogenic lesions. Animal studies have demonstrated the biochemical metal** olism by the liver of vinyl chloride-produced intermediate* which arc mutagenic in baefena/ system* amd may be the ultimate carcinogens. Hepatic subcellular enzyme studies prove preliminary evidence of cellular adaptation and increased detoxification. Disruption of this oxidization and detoxification balance may be the hey to the malignant transformation of ceils. A working hypothesis is presented which may explain the metabolism of vmyl chlondc into mutagenic intermediates by the liver cell and the develop ment of malignant transformation by extra hepatic sinusoidal lining ceils, lung cells, and brain tissue. INTRODUCTION Currently there is a growing concern that chemical compounds may be respon sible for most human cancer through environmental contact. Although 100,000 to 200,000new chemicals are introduced into industry each year, little is known about their effects. These compounds are primarily synthetics and thus not natural to the environment. The view that industrial chemicals may be latent carcinogenic hazards has again been brought into sharp focus by the discovery of vinyl chlorideinduced angiosarcoma. Vinyl chloride (CHi = CH--C1 monochloroethyene, a gas) is the basic molecule or monomer of polyvinyl chloride and its co-polymers and one of the most important organic intermediates in the plastics industry. The resulting plastic resin, polyvinyl chloride, is used in innumerable consumer and industrial products, such as containers, wrapping film, electrical insulation, pipelines, credit cards, etc. Until recently (early 1970's) vinyl chloride was regarded as being rela tively non-toxic (1,2]. Initially this opinion seemed to be supported by the facts that it was used transiently as an anaesthetic and had been used commercially in indus try for many years (3]. The direct information on the toxicity of vinyl chloride to man was obtained from experiments by research workers on themselves, from the evaluation of its suit- 67 * Portion* ofthis work were supported by a gram from Manufacturing Chemists Association and by National Cancer Institutes Contract #N0I-CN*53212. *77i>* article is the thirteenth in a series entitled. "Seminars on Liver Disease/" that have been presented as pan of the Training Program in Liver Disease at the Veterans Administration Hospital. West Haven. Connecticut. Dr. Harold O. Conn. Professor of Medicine. Yale University School of Medicine, and Director of the Training Program in Liver Disease, is guest editor. Please address reprint request* to: Carlo H. Tamburro, M-D . Frofc.vsor of Medicine. Chief. Digestive Diseases and Nutrition Division. Director, Vinyl Chloride Project. 511 South Floyd Street, P.O. Box 35260, Louisville, KY 40232 0044-0086/78/5101-0067 SOI.40 Copyright 1978 by The YaJe Journal of Biology and Medicine, Inc. All rights of reproduction in any form reserved. 3 CMA 003615 68 CARLO H. TAMBURRO ability as an anaesthetic [4.51, and from study of the cases of vinyl chloride poisoning contracted during industrial use (6, 7, 8, 9], Sporadic studies with vinyl chloride polymerization workers demonstrated vary ing degrees of hepatic biochemical derangements, hematological abnormalities, and skin changes [9], Acute short term exposures led to disturbances of the central nervous system, cardiac arrythmias, severe irritation of the mucosal membrane of the eyes and the respiratory tract, and in some cases--severe pulmonary edema with obstruction of the liver and kidneys. Chronic inhalation trials in animals clearly showed that vinyl chloride was toxic to the liver and kidneys as well as irrita ting to the mucosal membranes and the lungs. Microscopic examination of liver sections showed degeneration of the central lobules while the damage to the kidneys chiefly involved the tubule and the interstitial lining somewhat like that of carbon tetrachloride damage [10, 11, 12]. BACKGROUND The literature, however, contained virtually no information on damage to man after chronic exposure until Filatova et al. reported disturbances of the blood vessels and nerves in individuals exposed with 20-300 ppm of vinyl chloride on a continuous basis [13], Cordier et al. [ 14] and Wilson et al. [ 15] were the first to re port the hitherto unrecognized disorder termed occupational acroosteolysis (AOL) which included the symptoms of tenderness ofthe fingertips, gradual destruction of bony integrity of the fingers and a Raynaud's-like phenomena. Studies were initiated in animals to reproduce the acroosteolysis. In 1971, Violi et al. [ 16] while exposing animals to 30,000 ppm to induce acroosteolysis, accidentally discovered cancer, Maltoni et al. [17] while studying various levels of exposure demonstrated that angiosarcoma occured at 250 ppm; the Manufacturing Chemists Association's studies [18] demonstrated these liver cancers even at 50 ppm. At the same time. Dr. John Creech, at the B.F. Goodrich Chemical Company Plant in Louisville, Kentucky, discovered an hepatic angiosarcoma in one employee. Creech, recalling an earlier hepatic angiosarcoma at the plant, reviewed the medical histories of previous employees. Four additional angiosarcomas were found, further supporting the connection between vinyl chloride exposure and tumor development. Measures to control the levels of vinyl chloride exposure were then instituted following federal regulation. THE CHEMICAL Vinyl chloride's chemical structure is a double-bonded, 2-carbon halogenated hydrocarbon which has structural similarities to tri-chloroethylene, an inhalation anaesthetic. As previously noted, vinyl chloride was once considered as an anaesthetic but was discarded because it caused myocardial irritability. Some of its important physical properties include a low boiling point, a high specific gravity, a low solubility in water, and a half-life in air which ranges from 3-20 hours. Knowledge of these physical properties may be important in determining how this agent produces a cumulative effect in the environment which ultimately leads to cancer formation. Vinyl chloride is both toxic and carcinogenic, as recognized by the wide variety of associated disorders which have been found among vinyl chloride polymeri zation workers and vinyl chloride-exposed animals. A yet incomplete list of these CMA 003616 VINYL CHLORIDE--RELATED CARCINOGENESIS TABLE 1 Occupational Vinyl Chloride Exposure Associated Disorders 1. Thrombocytopenia 2. Reticulocytosis 3. Splenomegaly 4. Hepatic fibrosis 5. Scleroderma-like skin changes 6. Acro-osteolysis 7. Raynaud's phenomenon 8. Leukopenia 9. Hepatomegaly 10. Pulmonary functional impairment 11. Angiosarcoma 12. Cardiac arrhythmia 13. Nephroblastomas* 14. Zymbal gland carcinomas* 15. Large cell lung cancer 16. Brain cancer *!a rats only 69 associated disorders is shown in Table 1. Animal and epidemiological studies indicate the probability that cancer induction at other sites is also directly attributed to prolonged and excessive vinyl chloride exposure [19], In order to develop effective methods of prevention, accurate knowledge of the pathogenesis of this environmental chemical in ultimately producing its most destructive effect--cancer--is needed. EXPERIMENTAL ANIMAL STUDIES--TUMOR FORMATION The carcinogenicity of vinyl chloride has been demonstrated in a variety of animals as well as in humans at exposure levels that vary from 50-30,000 ppm. As illustrated in Table 2, a variety of tumor types have been found in rats, mice, and hamsters. An extensive list of benign tumors have also been reported in mice, rats, and hamsters exposed to vinyl chloride. Maltoni's group has now demonstrated primary liver cell cancers in exposed newborn rats. Direct hepatocellular injury as well as pulmonary, mucosal and skin injuries have been shown in directly exposed animals. Pretreatment with many agents increases the toxicity of vinyl chloride; they include phenobaibital, ethanol, polychlorinated biphenyls and pesticides such as hexachlorobenzene [20], This relationship to vinyl chloride's ability to induce cancer is under study, particularly in view of the industrial environment which allows exposure to many chemicals to occur con currently. HUMAN STUDIES Epidemiological studies in the human strongly suggest that exposure beyond 10 years is associated with increased cancer mortality, mainly digestive system cancers, primarily hepatic [21, 22]. There also appears to be a higher incidence of large cell carcinomas of the lung, brain glioblastoma multiforms, and lymphomas [23,24], although there is some disagreement as to the interpretation of this aspect of the epidemiological data. CMA 003617 . 'S'! L CHLORIDE--RELATED C 'i?.C;S0CENE~;3 T*RLE I Occupational Vrtn'.l C" j.-'de Exposure As^ci-urJ 1. Th'ooibocytopenta 2. Reuculocytosis 3. Splenomegaly 4. Hepatic fibrosis 5. Scleroderma-like skin changes 6. Acro-osteolyxis 7. RayfUu4`i phenomenon i.Leukopenia 9 Hepatomegaly 10, Pulmonary functional impairment 11, Angiosarcoma 12, Cardiac arrhythmia 13, Nephroblaatomaa* 4* -f t*icOj vor6tiiufn1 U. Large ce.l lung cancer 16. Brain cancer *In rata only 69 associated disorders is shown in Table 1. Animal and epidemiological studies indicate the probability that cancer induction at other sites is also directly attributed to prolonged and excessive vinyl chloride exposure f 19]. In order to develop effective methods of prevention, accurate knowledge of the pathogenesis of this environmental chemical in ultimately producing its most destructive effect--cancer--is needed. EXPERIMENTAL ANIMAL STUDIES--TUMOR FORMATION The carcinogenicity of vinyl chloride has been demonstrated in a variety of animals as well as in humans at exposure levels that vary from 50-30,000 ppm. As illustrated in Table 2, a variety of tumor types have been found in rats, mice, and hamsters. An extensive list of benign tumors have also been reported in mice, rats, and hamsters exposed to vinyl chloride. Maltooi's group has now demonstrated primary liver ceil cancers in exposed newborn rats. Direct hepatocellular injury as well as pulmonary, mucosal and skin injuries have been shown in directly exposed animals. Pretreatment with many agents increases the toxicity of vinyl chloride; they include phenobarbital, ethanol, polychlorinated biphenyls and pesticides such as hcxachlorobcnzene [20]. This relationship to vinyl chloride's ability to induce cancer is under study, particularly in view of the industrial environment which allows exposure to many chemicals to occur con currently. HUMAN STUDIES Epidemiological studies in the human strongly suggest that exposure beyond 10 years is associated with increased cancer mortality, mainly digestive system cancers, primarily hepatic [21,22). There also appears to be a higher incidence of large cell carcinomas of the lung, brain glioblastoma multifonns, and lymphomas [23,24], although there is some disagreement as to the interpretation of this aspea of the epidemiological data. CMA 003618 70 CARLO H. TAMBURRO TABLE 2 Carcinogenicity of Vinyl Chloride fExposure- 5Q"IQ.QQQ ppm) Tumor Type Species 1 Liver--angiosarcoma 2. Liver--hepatocellular carcinoma 3. Lung--aiienocaictnoma 4. Lung--'large cdl carcinoma 5. Mammary adenocarcinoma 6. ZymbaJ gland tumors 7. Nephroblastoma 8. Osteochondromas 9. Skin epitheliomas 10. Melanomas 11. Glioblastoma multiforme 12. Lymphoma Adult humans, rats, mice and hamsters Newborn rats Rats and mice Humans* Mice Rats Rais Rats Hamsters Hamsters Humans* Humans* 'Strongly suggested epidemiologicaily The multisystem involvement of this carcinogenic and toxic chemical is further illustrated in man. Early physical findings of vinyl chloride-injury include hepato megaly, portal hypertension, possible mild systemic pulmonary hypertension, bilateral midzonal pleural thickening of the lung, and splenomegaly with and without increased portal pressure [25]. These anatomical and physiological findings most frequently occur in the absence of the traditional clinical biochemical derangement of the liver. Screening studies of vinyl chloride workers during the past two and a half years have clearly illustrated the irregular and often delayed appearance ofabnormalities in aspartate and alanine aminotransferases (SGOT and SGPT), alkaline phosphatase as well as other hepatocellular enzymes. Gamma glutamic transpeptidase (GGTP), believed to be a more sensitive indicator of hepatocellular injury, has proven to have too high a false-positive rate to warrant its use in the screens for hepatocellular injury. Sorbitol dehydrogenase (SDH) studies indicate that this enzyme, which is liver tissue specific, is too insensitive for primary screening but is useful for confirmatory testing. Anionic dye clearance studies (Indocyanine Green, ICG) have demonstrated the highest specificity and sensitivity of all the primary screening procedures tested when performed at the 5 mg/kg dose level. At the traditional 0.5 mg/kg level, it has the same effectiveness as the aminotransferases and alkaline phosphatase com bined. The frequency of abnormal ICG dye clearances increases with prolonged exposure to vinyl chloride as illustrated in Fig. 1 and correlates well with the cumulative exposure to vinyl chloride as well as the histological evidence of hepatocellular injury. These functional studies for detecting hepatocellular injury do not, however, identify the cause. Clinical studies have illustrated the usefulness of radioisotopic liver-spleen scans as a primary screening procedure for anatomical lesions of the liver and spleen. This procedure has proven to be the single most reliable method oftumor detection. Sixteen of the 19 individuals with anatomical lesions were detected by liver-spleen scan. In contrast, only 32 of 950 normal individuals had scan abnormalities which further diagnostic studies proved incorrect. This method provides an 84% sensitiv ity and 97% specificity, with only a 3% false-positive rate. MA 003619 VINYL CHLORIDE--RELATED CARCINOGENESIS 71 Diagnostic angiographic studies of these radioisotopic abnormalities in 80 individuals have demonstrated 3 major lesions. The first is petiosis hepatis, illustrated in Fig. 2. These lesions are usually numerous involving the entire liver, and have a diffuse stain throughout the nodules which persists into the late venous phase without central hypovascularity [26J. A second, similar lesion has been discovered in individuals with splenomegaly, and named lienal peliosis (Fig. 3). These splenic lesions demonstrate a shortened celiac artery to portal vein circulation time, normal portal vein diameters, and increased spleen size. This has been found only in individuals with long-term vinyl chloride exposure. The final lesion is that of angiosarcoma (Fig. 4), This tumor has characteristic angiographic features of central hypovascularity, midarterial puddling, and a prolonged peripheral tumor stain which continues up to 30-36 seconds after injection. These characteristic findings have allowed differentiation from other primary hepatocellular cancers, benign tumors and benign vascular lesions [26]. These angiographic lesions have been pathologically confirmed with the additional histological finding including peliosis hepatis, sinusoidal dilatation, and activated sinusoidal cells with increased deposits of collagen in the sinusoidal space ofDisse, Exploratory wedge biopsies have in addition demonstrated increased subcapsular fibrosis with subcapsular bile duct proliferation plus the often described portal fibrosis [27], VINYL CHLORIDE METABOLISM AND CARCINOGENESIS Present biochemical knowledge indicates that vinyl chloride is most likely metabolized by the liver in a three step process [28]. At concentrations less than 50 ppm, vinyl chloride is metabolized by the alcoholic dehydrogenase system into chloroacetaldehyde and monochloroacetic acid.. Cl-CH=CHi-->C1--CHj-CHj-OH alcohol Cl-CHi-CHO--CICHi-COOH dehydrogenase An alternative pathway which appears to become operative at 220 ppm is oxidation by the peroxidase-catalase system. Cl--CHz-CHz-OH -- CICHa-CHiOOH catalase ClCHz-CHO TOTAL Ha. TOO % I | OA-a/H* IP j.Omg/Hi */. 7. FIG. 1. Frequency of abnormal indocyanine green dye clearance among vinyl chloride woricer* utili zing0.J mg/kg and J .0 mg/kgdoi. total vc evoaune a CMA 003620 Diagnostic ar.c'.czrzc.i.c 5tcu.es o:' mess rauioijvKCpic abnormally V. 22 individuals have demonstrated 3 major lesions. The first is peliosis nepu.U, illustrated in Fig. 2. These lesions are usually numerous involving the entire liver, and have a diffuse stain throughout the nodules which persists into the late venous phase without central hypovascularity [26]. A second, similar lesion has been discovered in individuals with splenomegaly, and named lienal peliosis (Fig. 3). These splenic lesions demonstrate a shortened celiac artery to portal vein circulation time, normal portal vein diameters, and increased spleen size. This has been found only in individuals with long-term vinyl chloride exposure. The final lesion is that of angiosarcoma (Fig. 4). This tumor has characteristic angiographic features of central hypovascularity, midanerial puddling, and a prolonged peripheral tumor stain which continues up to 30-36 seconds after injection. These characteristic findings have allowed differentiation from outer primary hepatocellular cancers, benign tumors and benign vascular lesions [26]. These angiographic lesions have been pathologically confirmed with the additional histological finding including peliosis hepaiis, sinusoidal dilatation, and activated sinusoidal cells with increased deposits of collagen in the sinusoidal space of Disse. Exploratory wedge biopsies have in addition demonstrated increased subcapsular fibrosis with subcapsular bile duct proliferation plus the often described portal fibrosis [27], VINYL CHLORIDE METABOLISM AND CARCINOGENESIS Present biochemical knowledge indicates that vinyl chloride is most likely metabolized by the liver in a three step process [28]. At concentrations less than 50 ppm, vinyl chloride is metabolized by the alcoholic dehydrogenase system into chloroacetaldehyde and monochloroacetic acid, q-CH*CHi--fCl-CHj-CHi-OH-----aJcoho1 ... .a-CHi-CHO-^ClCH^-COOH dehydrogenase An alternative pathway which appears to become operative at 220 ppm is oxidation by the peroxidase-catalase system. a-CHr-CHa-OH---301 .GCHi-CHiOOH---------OCHi-CHO catalase TOTAL a*. % I | aa --/W ESS .o*n/a v* % V. FTC. |. Frequeacy of abnormal indocyanine grata dye clearance umg vinyl chlorate norkars utili zing0.3 m*/V**nd5.0m*/lndo. CMA 003621 72 CARLO H. TAMBGRRO FIG. 2. Hepatic arteriogram: Ve nous phase. Changes of peliosis hepatis are present throughout the left lobe. The multiple nodular stains represent peliosis hepatis lesions (arrows) ranging from 2-3 mm to 2 cm in size. FIG. 3. Splenic arteriogram: Ve nous phase (15 seconds). There are 3--4 circular and oval stains (ar rows) in the superior and infenorlateral portions of the spleen. Nor mal pancreatic stain occurs jisl above the splenic vein. CMA 003622 VINYL CHLORIDE--RELATED CARCINOGENESIS 73 FIG. 4. Hepatic uieriogrom: Venom phue. At approximately 14-15 accondi the peripheral stain is identified (arrows), lasting through the entire phase. Scattered areas of puddling are also present in and around the area of central hypovascularity. In this case chloroacetaldehyde is again formed. At higher levels oxidation appears to be by the mixed function oxidase system, forming chloroethylene oxide which spontaneously rearranges to form chloroacetaldehyde which then can be further oxidized to form monochloroacetic acid. oxidase C1-CH=CHj- CL-CH CHi- V Cl-CHi-CHO CICHi-COOH As illustrated in Table 3, vinyl chloride oxidation intermediates chloroethanol and chloroacetaldehyde, at low doses, are most likely detoxified via the glutathi one-cysteine conjugation system. This system, however, is saturable and at higher levels vinyl chloride is excreted via the lungs [28]. It appears that at higher vinyl chloride levels increased amounts of chloroethanol and chloroacetaldehyde are further oxidized to chloroacetic acid which is excreted in the urine. This is further supported by the absence of chloroacetic acid in urines ofrats exposed to low, short term levels of vinyl chloride but found in urine of rats exposed to 5,000 ppm for an extended time and reported in workers exposed to levels greater than 250 ppm for a prolonged time [29, 30], Elmore et al., utilizing a modified Ames system and pure synthesized vinyl chloride intermediates, has demonstrated that vinyl chloride, chloroethanol, and CMA 003623 FIG. 4. Hepatic arteriogram: Venous phase. Ai approximately 14-13 secotub the peripheral $uin is identified (snowj). lasting through the entire phase. Scattered areas of puddling are also present in and around the area of central hypovascularity. In this case chloroacetaldehyde is again formed. At higher levels oxidation appears to be by the mixed function oxidase system, forming chloroethylene oxide which spontaneously rearranges to form chloroacetaldehyde which then can be further oxidized to form monochloroacetic acid. oxidase Cl-CH-CHa- CL-CH----CHj \/ hCI-CHj-CHO OCHi-COOH As illustrated in Table 3, vinyl chloride oxidation intermediates chloroethanol and chloroacetaldehyde, at low doses, are most likely detoxified via the glutathiooe-cysteine conjugation system. This system, however, is saturable and at higher levels vinyl chloride is excreted via the lungs [28], It appears that at higher vinyl chloride levels increased amounts of chloroethanol and chloroacetaldehyde are further oxidized to chloroacetic acid which is excreted in the urine. Thi is further supported by the absence of chloroacetic acid in urines ofrats exposed to low, short term levels of vinyl chloride but found in urine of rats exposed to 5,000 ppm for an extended time and reported in workers exposed to levels greater than 250 ppm for a prolonged time [29, 30]. Elmore et al., utilizing a modified Ames system and pure synthesized vinyl chloride intermediates, has demonstrated that vinyl chloride, chloroethanol, and CMA 003624 74 Cl CH - CHt (VC) Liver MFO Cl CH-CH; \l O (Chlorooxirane) CARLO H. TAMBURRO TABLE 3 Proposed Meubolic Fate of Vinyl Chloride Cl CHj CHj OH (Chloroethanol) +GSH Cl CHiCHO---------(Chloroacetaldehyde) GS CHi CHO Cl CHi COOH (Chloroacetic acid) GS CHi COOH Thiodiglycolic *cid chloroacetic acid arc not mutagenic in bacteriological systems [31]. This may indicate that the vinyl chloride monomer is neither hcpatotoxic nor carcinogenic until it has been metabolized to its intermediate forms by the liver and/or other tissues. Alternatively, chloroethanol--the most transportable of the vinyl chloride metabolites--may be transferred or diffused to adjacent cells, such as the sinusoi dal lining cells, where it could be converted to chloroacetaldehyde but less likely to be detoxified or further oxidized. Since vinyl chloride appears to bind the serum albumin, it may, itself be transported to and oxidized by extra-hepatic cells which are unable to fully oxidize or completely detoxify its metabolites, and thereby lead to molecular DNA injury and cancer formation at distant tissue sites. These quandaries led to further study of the hepatochemical changes in rats undergoing progressively increased exposure to vinyl chloride. Subcellular en zymes and metabolites were studied in animals exposed to from 10-20,000 ppm vinyl chloride, ranging from 14-137 hours. Microsomal enzymes, including PASO, NADPH cytochrome c reductase, and mixed function oxidases were studied. Determinations of cytochrome c oxidase as the mitochondria], tritiated-Ieucine incorporation as the protein synthesis and glucose-6-phosphatase as the carbo hydrate metabolism markers were also done. Glutathione and glutathione reduc tase as oxidative and detoxification markers were determined in addition to the conventional clinical biochemical studies which included the aspartate (SGOT) and alanine aminotransferase (SGPT), alkaline phosphatase, bilirubin, lactic acid dehydrogenase (LDH), total protein, albumin, cholesterol, and triglycerides. During the entire 137 hours of exposure there were no significant changes in the mitochondria] and the microsomal enzymes, the tritiated-Ieucine incorporation, or in the glutathione content. There was however, after 71 hours, a rise in the gluta thione reductase and a concomitant fall in glucose-6-phosphate. This occurred without any histologically discernible changes in the hepatocytes by light micro scopy nor any significant changes in the conventional clinical biochemical studies. CMA 003625 VINYL CHLORIDE--RELATED CARCINOGENESIS 75 The discovery of a decreased glucose-6-phosphatase after "simulated" chronic exposure led to the study of enzymes in the pentose phosphate shunt pathway. Weber and Lea [32] had found similar changes for primary hepatocellular neo plasms, demonstrating that in a rapidly developing primary hepatocellular tumor, there is decreased gluconeogenesis with a reduction in the glucose-6-phosphatase, followed by an increase in glucose-6-phosphate dehydrogenase and transaldolase. These biochemical changes were also followed by an increase in purine biosynthe sis (increased phosphoribosylpyrophosphate aminotransferase (PRPP) and in creases in the production of ATP and GTP leading to increased nucleic acid synthesis. Vinyl chloride-exposed animals showed no significant changes in the glucose-6phosphatase dehydrogenase activity during the initial 84 hours of exposure. However, after 103 hours, there was significant increase in glucose-6-phosphatase dehydrogenase. Studies of PRPP, at least up to 137 hours, have as yet shown no significant changes. Studies are now underway using animals exposed to 130 to 250 hours to determine if the biochemistry in vinyl chloride injury is similar to that in primary hepatocellular tumors. This, however, does not explain why the hepatocyte, which is the primary cell for oxidizing and detoxifying vinyl chloride, is not the primary target for cancer trans formation. How do the hepatocytic biochemical changes, seen in the early phase of high vinyl chloride exposure, relate to the later morphological changes that occur in the adjacent sinusoidal cells? MORPHOLOGICAL FINDINGS Electron microscopic examination of liver sections of mice exposed from 1 to 6 months to 2,500-6.000 ppm vinyl chloride, for 5 hours/day, 5 days/week--a level known to induce angiosarcoma [33]--have demonstrated hepatocellular changes as early as one month. These changes included hypertrophy of the smooth endo plasmic reticulum (believed to reflect vinyl chloride metabolism) and, plasma membrane loss of microvilli with invaginations--possibly reflecting the movement of injurious metabolites across the membrane and out of the cell, allowing the metabolites to be picked up by the sinusoidal cells [34], The sinusoidal cell reactions were multicellular. Increasing numbers and sizes of lipocytes were seen with little fibrosis. Macrophages were seen filled with phago somes, sometimes containing long needle-like crystals. Although there were many mononuclear cells present, the main abnormalities were seen in the endothelial lining cells. In the early stages they are larger and thicker--possibly swollen. Later, they became bulky and in places, multi-layered containing increased organelles, especially mitochondria and endoplasmic reticulum. Later disruptions in the sinu soidal walls seen were consistent with beginning peliosis hepatis. The lining cells, probably the precursors of angiosarcoma, often resembled fibroblasts. However, their endoplasmic reticulum did not contain any collagen components. These observations by Schaffneret al. [34] give support to the suggestion that metabolites of vinyl chloride produced in the hepatocytes may be transported through the plasma membrane and enter sinusoidal lining cells, eventually leading to angio sarcoma. Attempts at screening for vinyl chloride hepatic injury might be better aimed at the endothelial cells and the hepatic sinusoidal circulation rather than the hepatocytes. Our work in humans has identified similar findings. One major difference at CMA 003626 Wsbsr ar.d Lea [32] had found similar changes for primary hepatocellular neo plasms, demonstrating that in a rapidly developing primary hepatocellular tumor, there is decreased gluconeogenesis with a reduction in the giucose-6-phosphatase, followed by an increase in g!uccse-6-phcsphate dehydrogenase and transaldclase. These biocnerrucai cnanges were also followed oy an increase in purine oiosynthesis (increased phosphoribosyipyrophosphate aminotransferase (FRPP) and in creases in the production of ATP and GTP leading to increased nucleic acid synthesis. Vinyl chloride-exposed animals showed no significant changes in the gIucose-6phosphatase dehydrogenase activity during the initial 84 hours of exposure. However, after 103 hours, there was significant increase in gluccse-6-phosphatase cehycregenase. studies of P.T??. at least up to 137 hour:, have as yet shown no significant changes. Studies are now underway using animals exposed to 130 to 250 hours to determine if the biochemistry in vinyl chloride injury is similar to that in primary hepatocellular tumors. This, however, does not explain why the hepatocyte, which is the primary cell for oxidizing and detoxifying vinyl chloride, is not the primary target for cancer trans formation. How do the hepatocytic biochemical changes, seen in the early phase of high vinyl chloride exposure, relate to the later morphological changes that occur in the adjacent sinusoidal cells? MORPHOLOGICAL FINDINGS Electron microscopic examination of liver sections of mice exposed from 1 to 6 months to 2,500-6,000 ppm vinyl chloride, for 5 hours/day, 5 days/week--a level known to induce angiosarcoma [33]--have demonstrated hepatocellular changes as early as one month. These changes included hypertrophy of the smooth endo plasmic reticulum (believed to reflect vinyl chloride metabolism) and, plasma membrane loss of microvilli with invaginations--possibly reflecting the movement of injurious metabolites across the membrane and out of the cell, allowing the metabolites to be picked up by the sinusoidal cells [34], The sinusoidal cell reactions were multicellular. Increasing numbers and sizes of lipocytes were seen with little fibrosis. Macrophages were seen filled with phago somes, sometimes containing long needle-like crystals. Although there were many mononuclear cells present, the main abnormalities were seen in the endothelial lining cells. In the early stages they are larger and thicker--possibly swollen. Later, they became bulky and in places, multi-layered containing increased organelles, especially mitochondria and endoplasmic reticulum. Later disruptions in the sinu soidal walls seen were consistent with beginning peliosis hepatis. The lining cells, probably the precursors of angiosarcoma, often resembled fibroblasts. However, their endoplasmic reticulum did not contain any collagen components. These observations by Schafiner et al. [34] give support to the suggestion that metabolites of vinyl chloride produced in the hepatocytes may be transported through the plasma membrane and enter sinusoidal lining cells, eventually leading to angio sarcoma. Attempts at screening for vinyl chloride hepatic injury might be better aimed at the endothelial ceils and the hepatic sinusoidal circulation rather than the hepatocytes. Our work in humans has identified similar findings. One major difference at CMA 003627 76 CARLO H. TAMBURRO present is an increased collagen deposition, characteristic of human vinyl chloride injury and likely species specific. Light microscopic studies utilizing special stains on hepatic tissue from individuals with extensive exposure to vinyl chloride but without clinical biochemical hepatic abnormalities have shown distinctive midzonal increased deposition of collagen in the space of Dissc [35]. Routine light microscopic studies using hematoxylin and eosin failed to easily demonstrate this midzonal increased collagen. The increased deposition along the hepatic cell surface is associated with larger sinusoidal space and activation of the sinusoidal lining cells illustrated by increased nuclear size and cytoplasmic content. The in creased collagen deposition, when studied electron microscopically, demonstrates compression of the hepatocytes by the collagen bundles which initially give the ap pearance of /nrra-hepatocellular collagen bundles as illustrated in Fig. 5. The strands of collagen appear to compress the hepatocytes causing cords of hepato cytes to be broken and to coalesce with adjoining sinusoids eventually leading to peliosis hepatis-like lesions. These observations led us to the study of the proteroglycan role in collagen form ation in vinyl chloride-exposed workers. It had been suggested in the literature that glycosaminoglycans in blood and/or urine might be useful as means of early cancer detection since a number of studies had demonstrated the production of sulfated glycosaminoglycans with malignant states. Pathologists have often used this feature as a diagnostic aid in characterizing malignant vascular tumors of the skin ^---* - t "'i. v-w.'* FIG. 5. Electron microscopy showing collagen (CB) bundles (arrows) invaginatmg into the hep* atocyte. giving the appearance of inter-hepatocytic collagen. N nucleus; S - sinusoidal space: IM " invagination into the cell membrane (small arrows). t 'If /-J / > ' *. . r t - ; , r" - H.v- *. /.. -j? - * ft - .. .'STy 'J* * . .1- CMA 003628 VINYL CHLORIDE--RELATED CARCINOGENESIS 77 [36]. Others have noted a strong positive Alcian blue glycosaminoglycan staining reaction in human angiosarcoma tissue [37]. This suggested that quantitative and qualitative determinations of glycosaminoglycan production in individuals with neoplasm, either by serum or urine, might be used to identify those at high risk or as an early indicator of neoplastic formation. The feasibility of glycosaminoglycan "spot test" for vinyl chloride production workers made this an attractive possibil ity for mass screening. Urinary glycosaminoglycans, measured as uronic acid, were studied in individu als with alcoholic cirrhosis, viral hepatitis, secondary liver metastasis, hepatic angiosarcoma and normal controls. The percentage of total glycosaminoglycans that was dialyzable and the percent age ofunfractionated total that appeared in the hyaluronic acid, chondroitin sulfate, or in the heparin fractions was similar for all groups. However the distribution of positive fractions varied with the different groups studied. Seven of the nine vinyl chloride-exposed individuals, other than those with angiosarcoma, had glycosaminoglycan positive chondroitin sulfate fractions with negative hyaluronic acid and heparin fractions whereas this occurred in only 3 of the 32 urines from other hepatic diseases [38]. In addition, study of the total tissue glycosaminoglycan levels in angiosarcoma tumors and fibrotic tissue adjacent to the tumor demonstrated that tumor tissue itself had higher levels of hyaluronic acid and heparin fractions as compared to the non-tumor adjacent tissue which had higher levels of chondroitin sulfate fractions. A similar relationship was found in cirrhotic liver tissue and normal controls. This data conforms to reports by others that both hepatic connective tissue disorder [39,40,41,42,43] and hepatic cancer [44] result in increased hepatic glycosamino glycan levels. It may be significant that the angiosarcoma patient has half the urinary glycosaminoglycan excretion of patients with liver metastasis and that analysis ofangiosarcoma tumor tissue exhibits halfthe glycosaminoglycan content reported by Kojima et al. [44] for hepatocellular carcinoma. The increases in liver and urinary glycosaminoglycans may well reflect the importance of these substan ces in the process of fibrogenesis and tumor growth. Although no significant differences were found in total glycosaminoglycans of vinyl chloride-exposed indi viduals with associated liver injury, there was a significant difference in the excre tion patterns of these individuals. Seventy-eight percent of them had positive chondroitin sulfate fractions in contrast to only nine percent of the non-exposed liver injury cases. Thus the change in the glycosaminoglycan excretion pattern in individuals with pre-cancerous injuries may be of significant prognostic and diagnostic importance [45], The urinary glycosaminoglycan excretion patterns in an angiosarcoma patient 3 months to 2 weeks prior to death demonstrated an increase in the urinary chondroitin sulfate fraction with a change in its composition as the disease progressed. During this time, the chondroitin sulfate composition showed a continuous increase in the ratio of the 1.25 M NaCl to the 1.5 M NaCl fractions. This was due to an increase in the 1.25 M eluate and a decrease in the 1.5 M eluate fraction and was 2.3 times greater than the controls. In the most advanced stage ofthe angiosarcoma the ratio increased to 13.7 times greater [46]. These very preliminary studies would suggest that alterations in the ratios of these fractions' compositions may be useful in evaluating the severity and subsequent progression of disease. Early lesions may produce small changes in the ratio which would become more pronounced as the disease worsened. The CMA 003629 v;>'\ i. live r.c ,1, :irvo: :^n s:air:r,2 1 C*. * r.--ir.3rtp^ qualitative determinations of glycosaminoglycan production in individuals with neoplasm, either by serum or urine, might be used to identify those at high risk or as an early indicator of neoplastic formation. The feasibility of glycosaminoglycan "spot test" for vinyl chloride production workers made this an attractive possibil ity for mass screening. Urinary glycosaminoglycans, measured as uronic acid, were studied in individu als with alcoholic cirrhosis, viral hepatitis, secondary liver metastasis, hepatic angiosarcoma and normal controls. The percentage of total glycosaminoglycans that was dialyzable and the percent age of unfractionated total that appeared in the hyaluronic acid, chondroitin sulfate, or in the heparin fractions was similar for all groups. However the distribution of positive fractions varied with the different groups studied. Seven of the nine vinyl chloride-exposed individuals, other than those with angiosarcoma, had glycosaminoglycan positive chondroitin sulfate fractions with negative hyaluronic acid and heparin fractions whereas this occurred in only 3 of the 32 urines from other hepatic diseases [38]. In addition, study of the total tissue glycosaminoglycan levels in angiosarcoma tumors and fibrotic tissue adjacent to the tumor demonstrated that tumor tissue itself had higher levels of hyaluronic acid and heparin fractions as compared to the non-tumor adjacent tissue which had higher levels of chondroitin sulfate fractions, A similar relationship was found in cirrhotic liver tissue and normal controls. This data conforms to reports by others that both hepatic connective tissue disorder [39,40,41,42,43] and hepatic cancer [44] result in increased hepatic glycosamino glycan levels. It may be significant that the angiosarcoma patient has half the urinary glycosaminoglycan excretion of patients with liver metastasis and that analysis of angiosarcoma tumor tissue exhibits halfthe glycosaminoglycan content reported by Kojima et al. [44] for hepatocellular carcinoma. The increases in liver and urinary glycosaminoglycans may well reflect the importance of these substan ces in the process of fibrogenesis and tumor growth. Although no significant differences were found in total glycosaminoglycans of vinyl chloride-exposed indi viduals with associated liver injury, there was a significant difference in the excre tion patterns of these individuals. Seventy-eight percent of them had positive chondroitin sulfate fractions in contrast to only nine percent of the non-exposed liver injury cases. Thus the change in the glycosaminoglycan excretion pattern in individuals with pre-cancerous injuries may be of significant prognostic and diagnostic importance [45]. The urinary glycosaminoglycan excretion patterns in an angiosarcoma patient 3 months to 2 weeks prior to death demonstrated an increase in the urinary chondroi tin sulfate fraction with a change in its composition as the disease progressed. During this time, the chondroitin sulfate composition showed a continuous increase in the ratio of the 1.25 M NaCl to the 1.5 M NaG fractions. This was due to an increase in the 1.25 M eiuate and a decrease in the 1.5 M eluate fraction and was 2.3 times greater than the controls. In the most advanced stage ofthe angiosarcoma the ratio increased to 13.7 times greater [46]. These very preliminary studies would suggest that alterations in the ratios of these fractions' compositions may be useful in evaluating the severity and subsequent progression ofdisease. Early lesions may produce small changes in the ratio which would become more pronounced as the disease worsened. The CMA 003630 78 CARLO H. TAMBURRO determination of this ratio might also be useful in identifying those individuals with significant injury or continuing progression of disease despite changes in the environment. Finally therapeutic measures for intervention might be better evalu ated for their effectiveness in arresting cancer development as reflected by the glycosaminoglycan changes which in turn reflect changes in collagen formation. SUMMARY AND HYPOTHESIS Vinyl chloride appears to enter the body through the respiratory tract, the skin, or by swallowing; it is absorbed and transported to the liver by the systemic and portal circulatory systems. In the liver, the primary site of metabolism, it is oxidized by various enzyme systems: alcohol dehydrogenase at lower exposure levels; the peroxidase-catalase system at intermediate levels; and mixed function oxidases at higher levels. At the higher levels, the mixed function oxidase system transforms vinyl chloride to chJorooxiranes which are then spontaneously trans formed into choroethanol and chloroacetaldehyde. These two intermediate metabolites, chloroethanol and chloroacetaldehyde, are detoxified by conjugation with glutathione and cysteine-SH groups and are excreted in the urine. At even higher doses increasing amounts of the chloroacetal dehyde are further oxidized to chloroacetic acid and excreted as an end product in the urine. However, when chloroacetaldehyde and/or the chlorooxiranes exceed the detoxification threshold of the hepatocyte, this leads to hepatocellular toxicity and/or stimulation of the sinusoidal cells. This acute event in tum acts as a stimulat ing mechanism for increased collagen deposition in the space of Disse and sinusoidal areas as shown by electron and light microscopy studies. The increase in the collagen depostion in sinusoidal spaces then leads to disruption of hepatic cell surface function, disruption of the hepatic cords, coalition of the sinusoidal spaces and eventual peliosis hepatis. These lesions alternately lead to sufficient vascular dysfunction to add further to the biochemical manifestation of hepatic cellular injury. Since it is highly unlikely that the unstable chlorooxiranes are able to be trans ported to adjacent cells and that the chloroacetaldehyde would most likely be conjugated or detoxified within the hepatocyte, an intermediate form, such as chloroethanol, which is transportable from the hepatocyte, may then move on to the adjacent sinusoidal lining cells, or possibly even further to other extrahepatic tissue. At these extrahepatic sites, an intermediate, such as chloroethanol, may then be converted to chloroacetaldehyde. The extrahepatic tissue sites are most likely unable to further convert the chloroacetaldehyde to chloroacetic acid nor to detoxify it sufficiently, if at all, by their own detoxification systems. This would allow a longer contact period with the cell's DNA. In addition many of the extrahepatic cells normally are regenerating at faster rates than hepatocytes, thus increasing the possibility of DNA derangement and ultimate carcinogenesis. Alternatively, vinyl chloride itself may be taken up by extrahepatic tissue, oxidized but incompletely detoxified, allowing the cell itself to become susceptible to direct DNA injury. In this or similar manner, chemical metabolites may induce injury to the DNA in rapidly replicating cells at sites beyond the liver, thus accounting for other cancers developing with vinyl chloride. The recent work by Maltoni's group, showing that exposure of newborn rats to the same dose of vinyl chloride as adult rats, results in primary hepatocellular carcinoma (40-45%) rather than in angiosarcoma (8-12%), lends further support to VINYL CHLORIDE--RELATED CARCINOGENESIS 79 the concept that the hepatocyte's ability to resist cancer transformation is dependent upon its ability to detoxify the mutagenic/carcinogenic metabolite of vinyl chloride. This review of our present knowledge of vinyl chloride injury and cancer formation in man is, at best, a very rough hypothetical outline. With continued investigation and study it will allow us to more accurately and completely fill in the missing pieces ofthis fascinating puzzle, thus leading us to a better understanding of the pathogenesis of chemically induced cancer in the biologically complex human system. REFERENCES 1. Elkins HB: The Chemistry of Industrial Toxicology. Second Edition. New York. New York, John Wiley A Sons Inc. 1950 2. Oettei H: In Ullmann's Etizyklopadic derTechnichenChetnie. Third Edition, 3:489. Munehen-Berlin. Urban and Schwaraenberg, 1954. 3. Kunststoflkomission dcs Bundesgesundhcrtsautcs (Plastics Committee of the Federal German Ministry of Health), Bundesgenundherishlaa. 8:369, 1975 4. vonOrtingen WF:The Halogenated Aliphatic. Olefmic. Cyclic. Aromatic, and Aliphatic-Aromatic Hydrocarbons Including the Halogenated Insecticides, Their Toxicity and Potential Dangers. (PHS Publication No. 414). Wash ington. D.C.. Government Printing Office, 1955 J. Oster RH, Can- CT, Krantx JC. el a): Anesthesia XXVI). Narcosis with vinyl chloride. 8:359-61,1947 6. Schottek W: The toxicity of vinyl chloride. Chem Techn 21:708-711, 1969 7. Gauvatn S; Vinyl chloride. Proc Roy Soc Med. 69:275-310, 1975 8. Braun P, DruckmanE, Eds: Public-health rounds at the Harvard School of Public Health. Vinyl chloride: Can the worker be protected? New Eng J Med 294:653-657. 1976 9. Selikoff U. Hammond EC. Eds: Toxicity of vinyl chloride--polyvinyl chloride. Ann NY Acad Sci 246:1-337, 1975 10. Irish DD: In Aliphatic halogenated hydrocarbons. Industrial Hygiene and Toxicology. Second Edition. Edited by FA Patty. New York. Interscience Publishers. 1963. 2:1241-1332 11. Mastromatteo E, Fisher M, Christie H. et al: Acute inhalation toxicity of vinyl chloride to laboratory animals. Arner Ind Hyg Assoc J 21:394-398. 1960 12. Lester D. Greenberg LA, Adams WR; Effects of single and repeated exposures of humans and rats to viny| chloride. Amer Ind Hyg Assoc J 24:265-275. 1963 13. Filatova VS, Balakhonova I, Gronsberg ES. Hygienic characteristics of vinyl chloride production. GigTr Prof Zabol 2:6, 1958 14. Cotdier JM. Ftevez C, Lt Fiver, et al: Acroostcolyse et lesions cutan6cs auociies chez deux ouvriers. affectes au nettoyage d'autoclavcs. Med Trav 4:14-19. 1966 13. Wilson RH, McCormick WF. Tatum CF. et al: Occupational acroosteolysis: Report of 31 eases. JAMA 201:577- 581, 1967 16. Vioti PL. Bigotti A, Caputo A: Oncogenic response of rat skin, lungs, and bones to vmyl chloride. Cancer Res 31:516-522. 1971 17. MaltoniC, Lcfemine GL: Carcinogenicity bioassays of vinyl chloride 1. Research plan and early results. Environ Res 7:387-405, 1974 18. Kepiinger ML. Goode JW, Gordon DE. et al: Interim results of exposure of rats, hamsters, and mice to vinyl chloride. Ann NY Acad Sci 246:219-224. 1975 19. Nicholson WJ, Hammond EC. Scidman H, et al: Mortality experience of a cohort of vinyl chloride--polyvinyl chloride workers. Ann NY Acad Sci 246:225-230, 1975 20. Star Series: Scientific and technical assessment report on vinyl chloride and polyvinyl chloride. EPA 600/6-75- 004:48. 1975 21. Tabenhaw 1R, Gaffey WR; Mortality study of workers m the manufacture of vinyl chloride and polymers of vinyl chloride. J Occup Med 16:509-318, 1974 22. Tabershaw-Cooper Associates Inc: Supplementary epidemiological study of vinyl chloride workers 1. Manuf Chem Assoc 5:1-30, 1973 23. Duck BW, Taylor KJW, Williams DMJ- Mortality study of workers in a polyvinyl chloride production plant. Lancet ii: 1197-99. 1975 24. Wzxweiller RJ. Stringer W, Wagoner JK. et al: Neoplastic risk among workers exposed to vinyl chloride. Ann NY Acad Sci 271:40-8. 1976 03t VINYL CMLGFG A"D C the concept tr.jc 1.er_. . te > ;. i..i ..l.'l-.T.-tc.-t ii dependent upon :i3 or...:;- :o tr.e eore:nrren:c ~=:^roi,;e vinyl chloride. This review of our present Vtnowiecge of 'inyl chloride injury and cancer formation in man is, at best, a very rough hypothetical outline. With continued investigation and study it will allow us tc more accurately and completely fill in the missing pieces of this fascinating puzale. thus leading us to a better understanding of the pathogenesis of chemically induced cancer in the biologicaily complex human system. REFERENCES 1. Elkins HB: The Chemistry of Industrial Toxicology Second Edition, New York. New York. John Wdy A Sons Inc. 1950 2. Oettei H; In L'Umann's EnzykJopidie 2er Techr.tchen Chcmie Third Edition. 5:489. Munchen-Berhn. Urban and Scnwari^ncers, i>.?- 3. KunststOtTkomusion oes Bunaesgesunanerisautes t Plastics Committee of the Federal German Ministry of Health), Bunde*jenur.dhsr'it'i". J59, \7~: 4. vonOtUrtgen WF. The Halogenated Aliphatic. Olefmic. Cyclic. Aromatic, and Aliphatic-Aromatic Hydrocarbons Including the Halogenatec Lssecaoides, Their Toxicity and Potential Danger*. t?HS Publication No. 414). Wash ington. D C. Government Printing Office. 1955 5. Osier RH. CarrCT. Kraniz JC, et all Anesthesia XXVll. Narcosis with vinyl chloride. 8 359-^1. 1947 6. Schotiek W; The tonicity of vinyl chloride Chem Techn 21:703--711, 1969 7. Gauvain S: Vinyl chJonde. Proc Roy Soc Med. 2.75--310. 1975 S. Braun P. Druckman E. Eds: Public-health rounds at the Harvard School of Public Health. Vinyl chlonde. Can the worker be protected? New Eng J Med 294.633-657, 1976 9. SeiikoffU. Hanunond EC. Eds: Toxicity of vinyl chJonde--polyvinyl chlonde. Ann NY Acad Sci 246:1-337,1973 10. Irish DD: In Aliphatic halogenated hydrocarbons. Industrial Hygiene and Toxicology. Second Edition. Edited by FA Patty. New York. Interscience Publishers. 1963, 2:1241-1332 11. Mastromatteo E. Fisher M, Christie H. et ai: Acute inhalation toxicity of vinyl chloride to laboratory animals. Amer Ind Hyg Assoc J 21:394-398, I960 12. Lester D. Greenberg L4. Adams WR; Effects of single and repeated exposures of humans and rats to vinyl chlonde. Amer Ind Hyg Assoc J 24,263-275. 1963 13. FUaiova VS, Balakhonova I. Gronsbcrg S. Hygienic characteristics of vmyl chlonde production. GigTr Prof Zabol 2:6, 1958 M. Cordier JM. Fievez C. Li Fever, et ai: Acroostcolyse et lesions cutanees associees chez deux ouvrien, affect** an nettoyage d'autodaves. Med Trav 4:14-19, 1966 15, Wilson RH, McCormick WF. Tatum CF. et ai' Occupational acroosteolysis: Reponof 31 cases. JAMA 201:577- 581, 1967 16, Violi PL. Btgocti A. Capoto A: Oncogenic response of rat skin, lungs, and bones to vinyl chlonde. Cancer Res 31:516-522. 1971 17, Maitoni C, Lefemm* GL: Carcinogenicity bioassays of vinyl chloride 1. Research plan and early results. Environ Res 7:317-405. 1974 IS, Kepiinger ML, Goode JW, Gordon D, et al: Interim results of exposure of rats, hamsters, and mice to vinyl chloride. Ann NY Acad Sd 246:219-224. 1973 19. Nicholson WJ. Hammond EC. Seidman H. et ai: Mortality experience of a cohort of vinyl chloride--polyvinyl chloride workers. Am NY Acad Sci 246:225-230. 1975 20. Star Scries: Scientific and technical assessment report on vinyl chloride and polyvinyl chloride. EPA 600/6-75* 004:48, 1975 21. Taberihaw R. Gaffey WR: Mortality study ofworkers m the manufacture of vinyl driond* and polymers of vinyl chloride. J Occup Med 16:509-518. 1974 22. Tatwrshaw-Cooper Associates Inc: Supplementary epidemiological study of vinyl chloride workers I. Manuf Cbem Assoc 5:1-30. 1975 23. Duck BW. Taylor KJW, Williams DMJ: Mortality study of workers in a polyvinyl chloride production plant. Lancet ii; 1197-99. 1975 24. Waxwetller RJ. Stringer W, Wapxtcr JK. et ai: Neoplastic risk among workers exposed to vinyl chloride. Ann NY Acad Sci 271:40-8. 1976 MA 003633 80 CARLO H. TAMBURRO 25. Tamburro, CH: Unpublished results 26. Whelan JC, Creech JL, Tamburro CH: Angiographic and radionuclide characteristics of hepatic angiosarcoma found in vinyl chloride workers. Radiology US 549-J57, 1976 27. Popper H. Thomas LB: Alterations of liver and spleen among workers exposed to vinyl chloride. Ann NY Acad Sci 246:172-193, 1975 25. Hefner RE Jr., Watanabc PG. Gehring PJ. Preliminary studies of the fate of inhaled vinyl chloride monomer in ruts. Ann NY Acad Sci 246:135-148. 1975 29. Watanabe PG, McGowan GR. Gehring PJ: Fate of ,4C- vinyl chloride after single oral adminstration in rau. Toxicol Appl Pharmacol 36:339-352, 1976 30. McGowan GR. Watanabe PG, Gehring PJ: Vinyl chloride urinary metabolites: Isolation and identification. Personal Communication, 1977 31. Elmore JD, Wong JL, Laumbach AD, et al: Vinyl chloride mutagenicity via the metabolites chlorooxirane and chloracetaldehyde monomer hydrate. Biochim Biophys Acta 442:405--119, 1976 32. Weber G. Lea MA: The molecular correlation concept. In Methods in Cancer Research. Edited by NH Busch. NY, Academic Press Inc, 2:523-578, 1967 33. Malioni C. Lefemine G: Carcinogenicity bioassays of vinyl chloride: current results. Ann NY Acad Sci 246:195-- 219, 1975 34. SchafTner F, Popper H, SelikofT U, et at: Initial features of vinyl chloride hepatic injury. Gastroenterology ii:(No, 5) A35/928, 1976 35. Schrodt R, Tamburro CH: Unpublished data 36. Girard D, Johnston WC. Grahm JH: Cutaneous angiosarcoma. Cancer 25-868-83, 1970 37. Ban- R. Bower M: ``Letters'' JAMA 231 <9):9t4. 1975 38. Curran KL. Kupchella CE. Tamburro CH: Urinary glycosaminoglycan patterns in angiosarcoma of the liver. Cancer 40:3050-53, 1977 39. GaJambos JT, Shapira R: Natural history of hepatitis: IV glycosaminoglycuronans and collagen io the hepatic connective tissue. J Clin Inves 52 (ll);2952-62, 1973 40. Koizumi T, Nakamura N, Abe H: Changes in acid mucopolysaccharide in the liver in hepatic fibrosis. Biochim Biophys Acta 148:749^56* 1967 41. Kojima J: Studies on the metabolism of hepatic connective tissue in fibrosis of the liver. Med J Osaka UnW 16:419-29, 1964 42. Rubin E: Autoradiographic characterization of sulfated acid mucopolysaccharides in experimental cirrhosis. J Histochem Cytochem 14:688-89, 1966 43. Patrick R5. Kennedy JS: The synthesis of the sulfted mucopolysaccharides at sites of hepatic fibrosis is induced by carbon tetrachloride* amyloidosis, and the implantation of catgut. J Pathol Bartend 88 549-55. 1964 44. Kojima J, Kanatani M. Ohmori K: The glycosaminoglycan* in human hepatic cancer. Cancer Res 35 (3):542~57* 1975 45. Kupchella C* Tamburro CH: Urinary glycosaminoglycan excretion patterns in chemically induced liver injury and cancer. Clin Res 25:329, 1977 46. Kupchella CE, Tamburro CH: Urinary and tissue glycosaminoglycan patterns in hepatic angiosarcoma. In Pro ccedings ofthe III International Symposium on Detection and Prevention ofCancer. Edited by HE Niebergs. NY, Marcel Dekker Inc. 1977 Carlo H. Tamburro, M.D. Cancer Center and Digestive Diseases and Nutrition Division Department of Medicine University of Louisville School of Medicine Louisville, Kentucky 40201 CMA 003634 TOXICOLOGY AND APPLIED PHARMACOLOGY 62, 1 -10 ( 1982) 4 The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes' Du.Julie T. 3 Michael T. Tseng, and Carlo H. Tamburro3 Liver Research Center, Division of Digestive Diseases and Nutrition, Departments of Medicine and Anatomy, and Regional Cancer Center, University of Louisville School of Medicine, Louisville. Kentucky 40292 Received July 17, 1980: accepted September 12. 1981 The Effect of Repeated Vinyl Chloride Exposure on Rat Hepatic Metabolizing Enzymes, Du, J T , Tseng, M T . and Tamburro, C H. (1982). Toxtcoi Appi. Pharmacol. 62, I10. Sprague-Dawky rats were exposed to 2.8% vinyl chloride for 2 (70 hr). 4 (140 hr), and 6 (210 hr) weeks to determine the sequential biochemical changes related to the oxidation and detoxification ability of hepatic tissue. Glutathione-5-transferase(s) activity using 1,2* cpoxy-(p-nitrophenoxy)propane and /vmtrobenzyl chloride as substrates was elevated 17 to 24, 28. and 35 to 42% after 2. 4, and 6 weeks of exposure, respectively, suggesting enzyme(s) induction. Reduced glutathione, the major substrate required to conjugate the toxic metabolites of vinyl chloride, was also consistently elevated. Similarly, the activity of glutathione reductase, the enzyme necessary for the regeneration of reduced glutathione from its oxidized form, was also increased following vinyl chloride exposure. Cytochromes f-450, the major protein in volved with vinyl chloride metabolism, was reduced after vinyl chloride exposure, confirming reports of others that vinyl chloride metabolites destroy P-450. No abnormalities of standard clinical biochemical blood tests of liver function were found during 6 weeks of vinyl chloride exposure. The only consistent ultrastructural modification was the dilation of endoplasmic reticulum. The biochemical and ultrastructural alterations could reflect early hepatocellular adaptation to vinyl chloride exposure. Vinyl chloride, at high concentrations, has been shown to be carcinogenic in both lab oratory animals (Maltoni and Lefemine, 1975; Viola et at., 1971) and man (Creech and Johnson, 1974). Present data support the metabolism of vinyl chloride by hepatic microsomal mixed-function oxidase system into toxic intermediates, chloroethylene ox ide (Bolt et al., 1975; Hefner et al.. 1975; Kappus et al., 1976) and chloroacetaldehyde ' This work was supported by a grant from the Man ufacturing Chemists Association. Washington, DC. Portions of this study have been presented (Fed. Proc. 37, 1545, 1978). ; Present address: Clement Associates, Inc,. 1010 Wisconsin Avenue, N.W., Suite 660, Washington, D.C. 20007. 'Address requests for reprints to: Carlo H. Tam burro, University of Louisville. (Gross and Freiberg, 1969). These two in termediates are considered to be the ultimate carcinogens (Barbin et al., 1975; Jaeger et al., 1974b; Van Duuren, 1975), to be mu tagenic in bacterial systems (Elmore et at,, 1976; Greim et al,, 1975; Malaveille et al., 1975; McCann et al,, 1975), to act as an alkylating agent by reacting with adenosine (Barbin et al., 1975) and cytidine (Laib and Bolt, 1978), and to bind with protein (Bolt et al., 1976; Kappus et al., 1976; Watanabe et al., 1978). Detoxification of these metab olites occurs mainly by conjugation with glu tathione and is catalyzed by hepatic gluta thione transferases; the conjugates are excreted in the urine as substituted cysteine derivatives (Watanabe et al., 1976b,c; Green and Hathway, 1975, 1977). Chloroacetal dehyde can be further oxidized to chloro- I 0O4I-0O8X/82/0I0OOI- I0J02.00/0 Copyright C 1982 by Pro*. Inc All rights of reproduction to ny form rwrvid CMA 003635 i DU, TSENG, AND TaMBURRO acetic acid (Hefner et al., 1975). These data are compiled in a metabolic scheme in Fig. I as an updated hypothesized metabolic fate of vinyl chloride in the adult rat. The me tabolism of chloroethylene oxide via epoxide hydratase is not listed in the scheme because its product has not been identified. There have been a few in vivo studies con cerning the effects of vinyl chloride exposure on hepatic concentrations of glutathione (Hefner et al., 1975; Watanabe et al., 1976c; Du and Tamburro, 1978), cytochromes P450 (Reynolds et al., 1975) and on mixedfunction oxidase activity (Drew et al., 1975; Reynolds et al., 1975). However, informa tion about the sequential alterations in he patic oxidation and detoxification of vinyl chloride, during prolonged exposure, simu lating the occurrence in workers, is still lack ing. It was reported previously that the en zymatic changes in rat liver following prolonged exposure to vinyl chloride were similar to those found in rat hepatoma (Du and Tamburro, 1976; Du et al., 1979). The sequential biochemical changes related to the hepatic oxidation and detoxification of vinyl chloride following prolonged exposure are reported here. METHODS Animals and experimental design. Eight- to ten* week-old Sprague-Dawley male rats (-*'300g), sup plied by Laboratory Supply of Indianapolis. Indiana, were randomized prior to the experiment into three groups: a vinyl chloride-exposed group and the air-ex posed group housed in identical chambers and a second control group housed in the University's Central Animal Care Center. The exposure level was 28,000 ppm vinyl chloride, 7 hr/day, 5 days/week for 2, 4, and 6 weeks. The exposure chambers were 4400-liter airtight vats. Vinyl chloride (^300 to 340 g) was added to the vat to give a time-weighted average concentration of 28,000 1000 ppm. The chamber air was changed daily and the vinyl chloride concentration was determined by gas chromatography. The air was constantly circulated by a stirrer. The rats' respirations had negligible effect on the composition of the chamber's atmosphere because of the chamber's large volume. Animals were fed on standard laboratory chow pellets ad libitum. All animals were anesthetized with ether, blood was drawn from the inferior vena cava, and the animals were Fig. I. The proposed metabolic fate of vinyl chloride. (GSH, glutathione; MFO. mixed-function oxidase; VC, vinyl chloride; GEST, glutathione S-epoxide transferase; and GAST, glutathione 5-al dehyde transferase). CMA 003636 VINYL CHLORIDE EFFECT ON ENZYMES 3 killed approximately 20 hr after exposure at 1 00 pm each day. NADPH. glutathione, and glutathione disulfide were obtained from Sigma Chemical Company. St, Louis, Missouri, 1,2-epoxy-3-(p-mtrophenoxy)propanc was purchased from Eastman Kodak Company, Rochester. New York: p*mtrobenzyl chloride was obtained from Matheson. Coleman and Bell, East Rutherford, New Jersey: benzphetamine was donated by the Upjohn Company, Kalamazoo, Michigan. Double-distilled water was used throughout. Sample preparation and biochemical determination, Homogenates and subcellular fractions were prepared as described previously (Du et al., 1979). Each sample was prepared from a single organ and kept at 4C during preparation. The remaining liver was frozen rapidly in liquid nitrogen and stored at -- 70C. Cytochromes P450 concentrations were determined in the frozen mi crosomal fractions the day following sacrifice. The glu tathione (GSH) concentration, as well as glutathione5-transferase, glutathione reductase, and mixed-func tion oxidase activities were determined in the freshly fractionated frozen liver. For the assays using frozen tissue, the livers from control and experimental rats were frozen in an identical manner for the same length of time. Cytochromes P-450 concentration (Omura and Sato, 1964), nonprotein sulfhydryl content (Sedlak and Lindsay. 196$). and glutathione reductase activity (Carlberg and Mannervik. 1975) were determined in the microsomal or cytosol fractions by methods de scribed previously (Du et al.. 1979). Glutathione-5-transferase activity was determined using the 100,000 X g supernatant fraction. 1,2-Epoxy3-(p-nitrophenoxy)propane and p-nitrobenzyl chloride were the substrates for glutathione-5-epoxide transfer ase and glutathione-S-aralkyl transferase (GAST), re spectively. Enzyme activity was determined as described by others (Habig et al., 1974; Kaplowitz et al., 1975). All assays were linear functions of protein concentration and timed for at least 2 min. Solutions of l.2-epoxy-3(p-mtrophenoxy)propane and p-nitrobenzyl chloride were prepared in absolute ethanol; the final ethanol con centration in the incubation mixture was 0.5%. Mixedfunction oxidase activity was estimated in the micro somal fraction by measuring NADPH disappearance in the NADPH-dependent demethylation reaction of benzphetamine (Lu et al., 1972). The protein content was determined by the method of Lowry et al. (1951). The serum clinical liver tests including aspartate ami notransferase, alanine aminotransferase, alkaline phos phatase. bilirubin, cholesterol, and triglyceride were determined by Technicon sequential multiple analyzer computer (SMAC) system. Light and electron microscopy. Small strips of liver were removed under ether anesthesia, sliced into small cubes, placed immediately in ice-cold 1% osmium tetroxide (pH 7.4). and fixed for 2 hr at 4C. Subse quently. samples were washed overnight in phosphate buffer, dehydrated in ascending alcohol, and embedded in Epon. Tissue blocks were polymerized at 60C for 2 days. Thin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate before ex amination on a Philips 300 electron microscope. For ultrastructural analysis, three rats randomly selected from controls and groups exposed for 2. 4. and 6 weeks to v.nyl chloride were studied. For light microscopy, a block of tissue was fixed in buffered formalin and processed routinely for paraffin embedding. Sections 6 jim thick were stained with he matoxylin and eosin. Statistical analysis. Analysis of variance was per formed for the various groups at the different time pe riods and multiple comparisons were performed based on the results of the analysis of variance. RESULTS The protein content (mg protein/g liver) in the subcellular fractions in both control and vinyl chloride-exposed groups was the same throughout the exposure (data not shown); the enzymatic results, therefore, are expressed as micromoles of substrate con verted per minute per milligram of protein. There were no statistical differences be tween the normal and air-exposed groups in the glutathione and cytochromes R-450 con tents or in any of the enzyme activities. The nonprotein sulfhydryl content (Table 1) was significantly elevated from 26 to 54% at 2, 4, and 6 weeks in the vinyl chloride-exposed group compared to both control groups. Al though the nonprotein sulfhydryl content in creased with exposure, the increases were not statistically significant. Glutathione re ductase activity (Table 1) in the exposed group was increased by 53 to 77% at all three time periods. The increase in glutathione reductase was the same at 2 and 4 weeks of exposure but showed a further significant increase after 6 weeks of exposure. Glutathione-S-epoxide transferase (GEST, Table 1) and glutathione-5-aralkyl transferase (GAST, Table I) activities were signifi cantly higher than controls after 6 weeks of exposure, 37 and 45%, respectively. The cy tochromes /*-450 content, on the other hand. CMA 003637 ' :n'( L GHLORiDE EFFECT ON E.\Z\ mes .. ximalcly '0 i- ^ ^ \^PH, glutathione. inJ glut.:;'..one tlisul: ue *erc ` u. `;J :*:m Sigmj Ot.T. ;Cd! St Louij, Missouri. i ,2-eDOx>-3-fp-nitrophenoxv ipropane was pur.'hied :rom Eastman Kodak Company, Rochester. New York, ^*nttrobcnzyl chloride was obtained from Matneson, Coleman and Bell. East Rutherford. New Jersey; benzpnetamme was donated by the L'pJonn Company, Kalamazoo. Micmgan Douoie-distiiied water was used throughout. Sample preparation and biochemical determination. Homogenates and subcellular fractions were prepared as described previously t Du et at., 1979). Each sample was prepared from a single organ and kept at 4aC during preparation. The remaining liver was frozen rapidly in Ituuid nitrogen and itored at -7QaC, Cytochromes /*- were oetermir.cU in \zc .rjzen mic.-jjoma* fractions ihe day following sacririce. The glu tathione iGSH) concentration, as well as glutathione* 5-transferase, glutathione reductase, and mtxed-func* tion oxidase activities were determined in the freshly fractionated frozen liver. For the assays using frozen tissue, the livers from control and experimental rats were frozen in an identical manner for the same length of time. Cytochromes F*-4:0 concentration (Omura jnd Sato, 1964). nonprotein sulfhydryl content iSedlak and Lindsay, 1963). and glutathione reductase activity (Carlberg and Mannervik. 1975) were determined in the microeomal or cytosol fractions by methods de scribed previously (Du et ai. 1979). GluUthione-5-transferase activity was determined using the 100.000 X g supernatant fraction. 1.2-Epoxy_Mp-niirophenoxy)Dropane and ^-nitrobenzyl chloride were the substrates for glutathione*5-epoxide transfer ase and glutathionc-5-aralkyl transferase (GaST), re spectively. Enzyme activity was determined as described by others (Habig et ai, 1974; Kaplowitz et ai, 1975). All assays were linear functions of protein concentration and timed for at least 2 min. Solutions of 1.2-epoxyG* (^mtrophenoxy)propane and p-nitrobenzyf chloride were prepared in absolute ethanol; the final ethanol con centration in the incubation mixture was 0.5%. Mixed* function oxidase activity was estimated in the micro somal fraction by measuring NADPH disappearance in the NADPH-dependent demethyiation reaction of beazpfeetamine (Lu et ai, 1972). The protein concent was determined by the method of Lowry et ai (1951). The scrum clinical liver test* including aspartate ami* notransferase, alanine aminotransferase, alkaline phos phatase, bilirubin, cholesterol, and triglyceride were determined by Technicon sequential multiple analyzer computer (SMAC) system. Light and electron microscopy. Small strip* of liver were removed under ether anesthesia, sliced into small cubes, placed immediately in ice-cold 1% osmium tetroxide (pH 7.4), and fixed for 2 hr at 4*C. Subse- bu'fer. Jinvcratcd in ascending aicohoi. jnd tnoeoced n Epon T'isue ?locks were polymerized ai o0C for 1 ^ am T":n .cctions were cut with a diamond o:fs and stained with uranyl acetate and lead curate before ex amination on a Philips 300 electron microscope For ulirasiructural analysis, three rats randomly selected from controls and groups exposed for 2. 4, and 6 weeks to vinyl chloride were studied. For hgnt microscopy, a block of tissue was fixed m buffered formalin and processed routinely for paraffin embedding. Sections 6 urn thick were stained with he matoxylin and eostn. Statistical analysts Analysis of variance was per formed for the various groups at the different time pe riods and multiple companions were performed based on the results of the analysis of variance. RESULTS The protein content (mg protein/g liver) in the subcellular fractions in both control and vinyl chloride-exposed groups was the same throughout the exposure (data not shown); the enzymatic results, therefore, are expressed as micromoles of substrate con verted per minute per milligram of protein. There were no statistical differences be tween the normal and air-exposed groups in the glutathione and cytochromes P-450 con tents or in any of the enzyme activities. The nonprotein sulfhydryl content (Table 1) was significantly elevated from 26 to 54% at 2, 4. and 6 weeks in the vinyl chloride-exposed group compared to both control groups. Al though the nonprotein sulfhydryl content in creased with exposure, the increases were not statistically significant. Glutathione re ductase activity (Table 1) in the exposed group was increased by 53 to 77% at all three time periods. The increase in glutathione reductase was the same at 2 and 4 weeks of exposure but showed a further significant increase after 6 weeks of exposure. Glutathione-5-epoxide transferase (GEST, Table 1) and glutathione-S-aralkyl transferase (GAST, Table 1) activities were signifi cantly higher than controls after 6 weeks of exposure, 37 and 45%, respectively. The cy tochromes P-450 content, on the other hand. CMA 003638 TABLE I Sequential Changes in Hepatic Nonprotein Sulfhydryl, Cytochromes P-450 Content and Activities of Glutathione Reductase and Glutathione-S-Transferases (Epoxide and Aralkyl) in Rats Exposed to Vinyl Chloride" Time (weeks) DU. TSENG, AND TAMBURRO Nonprotein sulfhydryl (urnol/g liver) Glutathione reductase {100 X pmol/min/mg protein) GEST {100 X nmol/ min/mg protein) GAST (10 X pmol/ min/mg protein) Cytochrome JM50 {nmol/g liver) Treatment Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control Normal control Vinyl chloride-exposed Air control 0 7.9 0 3 -- -- 5.0 0.2 -- -- 9.1 1 6 -- " 2.4 0.4 -- -- 17.0 3.7 -- -- 2 7.8 0.4* 9.4 0 2*' 7.1 0.3' 4.3 0 4* 6.7 + 0.6*' 4.5 0.2' 7.8 0.4 9.1 1.7 8.1 1.2 2.1 0.3 2.6 0.4 2.2 0.2 17.1 1.7 13.2 l.l 15.5 1.3 4 7.1 + 0.4* 10.2 + 0.6*' 6.9 0.4' 4 2 0.4* 6 J Y 0 5*' 3.7 0 3' 7.5 + 1 0 9.7 0.7' 6.3 + 0.7' 1.9 02 2.4 0 3 1.9 0 2 197 1.3* 15.3 + 1.3* 19.6 + 2.7 6 69 0 4* 11 4 + 0 6*' 7.9 o y 4.8 f 0.3* 8.9 i 0 7*' 5.3 Y 0 4' 7 7 1 0* 11 0 x t 3*' 8 4 0.9` 2.4 0.3* 3.2 0.1*' 2.1 0 2' 15.5 + 1 4* 106 + 1 0*' 14.3 1.8' Rats were exposed lo 28,000 ppm of vinyl chloride; normal controls and the air controls were exposed to air only. Each number represents the mean and the SEM from a group'of six rats, * Normal vs vinyl chloride-exposed, p < 0.0S. ' Air control vs vinyl chloride-exposed, p < 0.05. 4 Vinyl chloride (6 weeks) exposed vs vinyl chloride (2 and 4 weeks) exposed, p < 0.05. vinyl chloride effect on enzymes 5 was significantly lower than controls after 6 weeks of exposure to vinyl chloride (Table 1). No differences were found in the hepatic mixed-function oxidase activity or in the serum clinical liver tests. After 2 weeks of exposure, the two control groups had gained weight but the vinyl chloride-exposed group did not (Table 2). After 4 weeks of exposure, the normal control group housed at the an imal care center had gained significantly more weight than either the air-control or vinyl chloride-exposed group. After 6 weeks of exposure, however, the vinyl chloride-ex posed group failed to gain weight; the nor mal control group gained more than the aircontrol group (Table 2). Morphological examination revealed poly hedral hepatocytes arranged in irregular plates interposed by vascular sinusoids in the livers of the control rats. This general cytoarchitecture was maintained after vinyl chloride exposure. Hepatocytes in control rats contained a prominent spherical nu cleus, numerous ovoid mitochondria, stacks of rough endoplasmic reticulum (RER), some aggregates of smooth endoplasmic re ticulum (SER), and varying amounts of ly- sosomes and glycogen particles (Fig. 2a).Few interstitial cells were scattered among the hepatocytes. These cells contained few cy toplasmic organelles and could be readily discerned at the light microscopic level by their hyperchromatic nuclei. The sinusoids were linked by fenestrated endothelium and some of the lining cells displayed phagocytic activity. After 2 to 6 weeks of vinyl chloride exposure, the principal organelle affected appeared to be the endoplasmic reticulum. Cisternae of the RER became dilated in a relatively small population of the hepato cytes in the 2-week treatment group. Four weeks after exposure to vinyl chloride, patches of dilated endoplasmic reticulum were prominently displayed in some hepa tocytes (Fig. 2b). At this stage the SER was relatively unaffected. In the 6-week treat ment group, vesiculation of SER and dis tention of RER were easily discernible in a large number of hepatocytes (Fig. 2c). How ever, other cell organelles showed no de monstrable change. These changes, though, are still beyond the resolving limit of the light microscope. The nonhepatocyte com ponents showed minimum changes which TABLE 2 Body Weights of Rats before and after Vinyl Chloride Exposure* Duration (week) 2 4 6 Treatment Normal control VC-exposed Air control Normal control VC-*xposed Air control Normal control VC-exposed Air control Initial weight (8) 400 - 15 405 12 396 17 398 8 410 16 395 10 402 14 396 9 398 14 U O Ov It Final weight (8) 433 16 14 414 18 450 15 421 15 419 5 486 14 398 10 449 4 Percentage gain 8* 5' 13" 3* 6* 21" <!* 13" * Analysis of body weight was by regression analysis followed by an analysis of variance on the residuals from the regression equation. (Residual " observed final weight - predicted final weight from regression equation.) * Normal control vs vinyl chloride exposed, p < 0 05. r Air control vs vinyl chloride exposed, p < 0.05. * Air control vs normal control, p < 0.05. CMA 003640 i) No differences round :n the hepatic mixed-function oxidase activity or m the serum clinical iiver tests. After 2 weeks of exposure, the two comrm 'roups had gained weignt out ine ir.yi nivr:ue-c.veuscd gruup did not (Table 2). After 4 weeks of exposure, the normal control group housed at the an imal care center had gained significantly more weight than either the air-control or vinyl chloride-exoosed group. After 6 weeks of exoosure. however, the vinyl chloride-ex- mal control group guinea more man me aircontroi group iTao.e 2; Morphological examination revealed poly hedral hepatccytes arranged in irregular plates interoosed by vascular sinusoids in the livers of the control rats. This genera; cytoarchitecture was maintained after vinyl chloride exposure. Hepatccytes tn control rats contained a prominent spherical nu cleus, numerous ovoid mitochondria, stacks of rough endoplasmic reticulum (RER), some aggregates of smooth endoplasmic re ticulum (SER), and varying amounts of ly* hepatccytes. These cells contained few cy toplasmic organelles and could be readily discerned at the light microscopic level by their hyoerchromatic nuclei. The sinusoids were linked cy ienestratew encode..urn ,,.ca some of the lining cells displayed phagocytic activity. After 2 to 6 weeks of vinyl chloride exposure, the principal organelle affected appeared to be the endoplasmic reticulum. Cisternae of the RER became dilated in a relatively small population of the hepatocytes in the 2-wee,< treatment group. Fear weeks after exposure to vmyi chloride, patches of dilated endoplasmic reticulum were prominently displayed in some hepatocytes (Fig. 2b). At this stage the SER was relatively unaffected. In the 6-week treat ment group, vesiculatton of SER and dis tention of RER were easily discernible in a large number of hepatocytes (Fig. 2c). How ever, other cell organelles showed no de monstrable change. These changes, though, are still beyond the resolving limit of the light microscope. The nonhepatocyte com ponents showed minimum changes which TABLE i Body Weights of Rats before and after Vinyl Chloride Exposure" Duration (week) 2 4 6 Treatment Normal control VC-exposed Air control Normal control VC*capoaed Air control Normal control VC-exposed Air control Initial weight (g> 400 r 15 405 12 396 17 398 r 8 410 16 395 r 10 402 - 14 396 9 398 - 14 Final weight (g) 433 16 396 14 414 r 18 450 15 421 15 419 5 486 14 398 10 449 4 Percentage gain 8* -2" y 13" 3* e 21" <!*" 13" * Analysis of body weight was by regression analysis followed by an analysis of variance on the residuals from the regression equation. (Residual - observed final weight - predicted final weight from regression equation.) * Normal control vs vinyl chloride exposed, p < 0.05. Air control vs vinyl chloride exposed, p < 0.05. ` Air control vs normal control, p < 0.05. CMA 003641 6 DU. TStNG, AND TaMBURRO CMA 003642 VINYL CHLORIDE EFFECT ON ENZYMES 7 were characterized by an increased accu mulation of lysosomal-like substances in some of the sinusoidal lining cells as well as a greater tendency to accumulate lipids in the interstitial cells (Fig, 2d). DISCUSSION Glutathione conjugation is an important pathway for the metabolism of potentially harmful electrophilic metabolites of xenobiotics. Studies by Watanabe et al. (I976b,c) indicate this to be the major route for in activation of the vinyl chloride metabolites. Glutathione-S-transferases are a group of cytosol enzymes catalyzing the reaction of glutathione and electrophilic compounds to form less toxic and more water-soluble con jugates. Their activity during chronic ex posure to xenobiotics, like vinyl chloride, could be a key determinate in the ultimate outcome of such exposures as illustrated by the longer arrow in Fig. 1. The increase in hepatic GEST activity at 4 weeks and the later increase in GAST activity at 6 weeks suggested that in the earlier stages of ex posures most of the chlorooxirane interme diate is being adequately detoxified. During the later stages of chronic exposure, more chlorooxirane may become rearranged to yield more chloroacetaldehyde and, in turn, react with other available glutathione trans ferases or become further metabolized to chloroacetic acid. Alternatively, the exces sive chlorooxirane could rearrange sponta neously to form chloroethanol and be further oxidized to chloroacetaldehyde, which in turn may react with glutathione, or be oxi dized to monochloroacetic acid (Johnson, 1967). This would be consistent with the later increases in the aralkyl-transferases and the finding by Hefner et al. (1975) that monochloroacetic acid is found only in the urine of rats exposed for an extended time to higher levels (5000 ppm) of vinyl chloride. The increased use of alternate pathways, for chlorooxirane and chloroacetaldehyde de toxification, may reflect increased concen tration of these active metabolites allowing greater opportunity for DNA injury. A single exposure to vinyl chloride de creased hepatic nonprotein sulfhydryl com pounds in rats (Watanabe, 1976a): similar decreases of glutathione concentrations were produced in rats by other xenobiotics such as 1.1-dichloroethylene (Jaeger et al., 1974a; Reichert et al., 1978) and acetaminophen (Mitchell et al., 1973). In the presenf study, repeated exposure to vinyl chloride caused a significant increase of nonprotein sulfhy dryl concentrations (Table 1) analogous to the elevation of glutathione concentrations seen after the administration of carcinogens to rats (Fiala et al., 1976). In addition, the results showed that repeated exposure to vi nyl chloride also caused an increase in he patic glutathione-S'-transferase activity (Ta ble l) similar to that seen after the administration of phenobarbital and 3-methylcholanthrene to rats (Mukhtar and Bresnick, 1976). These data suggest a mecha nism for compensatory synthesis of hepatic glutathione and glutathione-5-transferases after repeated exposure to vinyl chloride. The decreased concentration of cyto chromes P-450 found in rats after repeated exposure to vinyl chloride (Table 1) is con- FlO. 2. (a) Portion of a hepatocyte from control. Stacks of rough endoplasmic reticulum (RER) are separated by many ovoid mitochondria (M). Chromatin is finely dispersed in the nucleus (N). 9300X. (b) Hepatocyte after 2 weeks of vinyl chloride exposure. Dilation of RER appeared widespread in these two cells. Bile (B) canaliculus appeared unaltered in these rats. 5300X, (c) Four weeks after vinyl chloride exposure. Golgi complex (G) appeared unaffected while cisternal dilation continued. Distinction between SER and RER is complicated by the detachment of ribosomes. Lipid droplets (L) and glycogen (GL) often accumulated. 9500X. (d) A fat-storing interstitial cell is surrounded by several hepatocytes in a vinyl chloride-treated animal. Unlike lipid stored in hepatocytes, the shape of lipids (L) appeared irregular in these cells. 8000X. CMA 003643 V L C H L 7,-r D b E F "1 7 v\-*. `vc were chjructcrizdd V i." .- rr'1j*ti^!*rof !' -"o^crioi'fiw m-;-)-1 o some of the sinusoidal lining ceiis as well as a greater tendency to accumulate lipids in the interstitial cells (Fig. 2d). DISCUSSION Glutathione conjugation is an important pathway for the metabolism of potentially harmful electrophilic metabolites of xeno- biotics. Studies by Watanabe et al. 1 1976b,c) I ^ i ' t ' a ''"i? f a - *- r --n> r * , .icio* ihc `.;n>l cmor'iCw Giutathione-5-transferases are a group of cytosol enzymes catalyzing the reaction of glutathione and electrophilic compounds to form less toxic and more water-soluble con jugates. Their activity during chronic ex posure to xenobiotics, like vinyl chioride, could be a key determinate in the ultimate outcome of such exposures as illustrated by the longer arrow in Fig. 1. The increase in hepatic GEST activity at 4 weeks and the later increase in GAST activity at 6 weeks suggested that in the earlier stages of ex posures most of the chlorooxirane interme diate is being adequately detoxified. During the later stages of chronic exposure, more chlorooxirane may become rearranged to yield more chloroacetaldehyde and, in turn, react with other available glutathione trans ferases or become further metabolized to chloroacetic acid. Alternatively, the exces sive chlorooxirane could rearrange sponta neously to form chloroethanol and be further oxidized to chloroacetaldehyde, which in turn may react with glutathione, or be oxi- m -- .'-'chler>' ic;*.' : . : l'-1-'This mid be . .. :h the later increases in the aruiky 1-iransierases and the tindmg by Hefner et ai. (1925) that monochloroacetic acid is found only in the urine of rats exposed for an extended time f n 1 `"ihcT \ * < '-t * 'kl ^-4* The increased use of alternate pathways, for chlorooxirane and chloroacetaldehyde de toxification, may reflect increased concen tration of these active metabolites allowing greater opportunity for DNA injury. A single exposure to vinyl chloride de- :r-r;ied b.rpzv.: ~:-protc:" :r I ;j-r.- pounds in ,ats \ atanabe, 19, od*: simitar decreases of glutathione cencer `.rations were produced in rats by other xenobiotics such as l.l-dichloroethylene (Jaeger et al., 1974a: Reichert et al., 1978) and acetaminophen (Mitchell et al., 1972). In the present study, repeated exposure to vinyl chloride caused a significant increase of nonprotein sulfhydryl concentrations (Table 1) analogous to the elevation of glutathione concentrations seen after the administration of carcinogens to rats (Fiala et al., 1976). In addition, the results showed that repeated exposure to vi nyl chloride also caused an increase in he patic glutathione-5-transferase activity (Ta ble 1) similar to that seen after the administration of phenobarbital and 3-mcth- ylcholanthrene to rats (Mukhtar and Bresnick, 1976). These data suggest a mecha nism for compensatory synthesis of hepatic glutathione and glutathione-5-transferases after repeated exposure to vinyl chloride. The decreased concentration of cyto chromes P-450 found in rats after repeated exposure to vinyl chloride (Table l) is con- FlC. 1 (*) Portion of a hepatocyte from control. Stacks of rough endoplasmic reticulum 1RER) are separated by many ovoid mitochondria (M). Chromatin is finely dispersed in the nucleus (N). 9300X. (b) Hepaiocyte after 2 weeks of vinyl chloride exposure. Dilation of RER appeared widespread in these two cells. Bite (B) canaliculus appeared unaltered in these rats. 5300x. (c) Four weeks after vinyl chloride exposure. Coigi complex (G) appeared unaffected while cisternal dilation continued. Distinction between SER and RER is complicated by the detachment of ribosomes. Lipid droplets 1L) and glycogen (GL) often accumulated. 9500X. (d) A fat-storing interstitial cell is surrounded by several hepaiocytes in a vinyl chloride-treated animal. Unlike lipid stored in hepatocytes. the shape of lipids (L) appeared irregular in these cells. SOOOX, CMA 003644 8 DU, TSENG. AND TAMBURRO sistent with work by Reynolds et al (1975). This decrease in cytochromes P~450 content was also shown in vitro (Guengerich and Strickland, 1977; Ivanetich et al1977) sug gesting that a metabolite of vinyl chloride destroys the cytochrome. Mixed-function oxidase activity, with benzphetamine as sub strate was unaltered. With regard to the structural alterations produced by vinyl chloride, the present find ings confirmed previous observations on the selective effect of this carcinogen in the en doplasmic reticulum (Du et al., 1979). A gradual increase in the number of hepatocytes affected and the involvement of both smooth and rough ER were shown in this study. The endoplasmic reticulum is the pri mary site of protein synthesis and its dilation suggested the presence of a vinyl chloriderelated effect. The lack of a concomitant Golgi hypertropy is noteworthy since this organelle serves as the site of glycosylation and packaging of many exportable proteins. It may be inferred that the vinyl chloride effect is mainly an intracellular phenome non. The relatively late involvement of SER could reflect a further attempt at detoxifi cation by the hepatocyte. The tendency for the interstitial cells to accumulate lipid and the heightened phagocytic activity correlate with increases in collagen formation and may be evidence of low-grade cellular injury. This may prove to be the initial histological response to vinyl chloride exposure. Ultrastructural responses to chronic in halation of vinyl chloride were reported by Feron et al. (1979). Unlike our findings, the principal effects observed were swollen mi tochondria and some proliferation of SER. The mitochondrial swelling presumably re flects the extensive vacuolization observed by light microscopy. The discrepancy prob ably resulted from differences in the dose and duration in vinyl chloride exposure since Feron et al. (1979) exposed rats to 5000 ppm vinyl chloride for 52 weeks. Such an exten sive mitochondrial lesion could result in bio chemical modifications such as a change in ATPase or cytochrome oxidase levels. The only enzyme activity measured, glucose-6phosphatase, was reduced. The authors believed that the altered glu tathione metabolism, as reflected by the in creased nonprotein sulfhydryl content, and the increased activities of glutathione-5'transferases and glutathione reductase, in rat liver after repeated exposure to high doses of vinyl chloride represent an early hepatocellular adaptation to vinyl chloride exposure. ACKNOWLEDGMENTS The authors wish to express their gratitude to the staff of the B. F. Goodrich Plant in Louisville, Kentucky, for their cooperation tn the exposure studies, to Dr. Richard A. Greenberg for his review, to Mr. John P. Sandoz for his help with the statistical analysis, and Mr. John Kretsie and Ms. Debra S. Eades for technical as sistance. REFERENCES Barbin, A., Bresil, H., Croisy, A.. Jacquionon. P,, Malaveille. C., Montesano, R., and Bartsch. H. (1975). Liver-mtcrosotne-mediated formation of alkylating agents from vinyl bromide and vinyl chlo ride. Biochem. Biophys. Res. Commun. 67, 596-603. Bolt. H. M., Kappus, H., Buchter. A., and Bolt, W (1975). Metabolism of vinyl chloride. Lancet 1, 1425. Bolt. H. M.. Kappus, H., (Caufmann. R.. Appel. K. E.. Buchter. A., and Bolt. W. (1976). Metabolism of "C-vinyl chloride in vitro and in vivo. Inserm Sym posia Ser. 52, 151-164,1ARC Scientific Publications No. 13. CarlberG. I., and MaNNERVIIC, B. (1975). Purifica tion and characterization of the flavoenzyme gluta thione reductase from rat liver. J. Biol. Chem. 250, 5475-5480, Creech, J. L.. and Johnson, M. N. (1974). Angios arcoma of liver in the manufacture of polyvinyl chlo ride. J. Occup. Med. 16, 150-151. Drew, R. T., Harper. C.. Gupta, B. N., and Talley. F. A. (1975). Effects of vinyl chloride exposures to rats pretreated with phenobarbital. Environ. Health Perspec. 11, 235-242. Du. J. T.. AND TambURRO, C. H. (1976). Decreased glucose-6-phosphatasc activity in liver in vinyl chlo ride exposed rats. Fed. Proc. 35, 329, CMA 003645 VINYL CHLORIDE EFFECT ON ENZYMES 9 Du. J. T., AND TambuRRO, C H. (1978) Elevated glutathione content. glutathione-S-transferase and glutathione reductase in liver of rats exposed to vinyl chloride. Fed. Proc 37. 1 545. Du. J T. Sandoz, J, P., Tseng. M T, and Tamburro. C. H. (1979). Biochemical alterations in liv ers of rats exposed to vinyl chloride, J Toxicol. En viron. Health 5. 1119-1132, Elmore. J. D.. Wong. J. L., Laumbach, A. D.. and Streips, U N (1976). Vinyl chloride mutagenicity via the metabolites chlorooxirane and chloroacetaldehyde monomer hydrate. Biochim. Biophys. Acta 442, 405-419. Feron. v J., Spit, B. J., Immel. H. R., and Kroes, R. (1979) One-year time sequence inhalation toxicity study of vinyl chloride in rats. Ill Morphological changes in the liver. Toxicology 13, 143-154. Fiala. S., Mohindru. A.. Kettering, W G.. Fiala. A E., and Morris. H. P. (1976), Glutathione and gamma glutamyl transpeptidase in rat liver during chemical carcinogenesis. J Nat Cancer Inst 57, 591 598, Green, T,, and Hathway, D. E. (1975). The biolog ical fate in rats of vinyl chloride in relation to its oncogenicity. Chem -Biol. Interact. 11, 545-562. Green, T.. and Hathway, D. E. (1977). The chem istry and biogenesis of S-containing metabolites of vinyl chloride in rats. Chem.-Biot. Interact. 17, 137150. Greim. H., Bonse, G.. Radwan, Z., Reichert. D,, and HenSCHlER. D. (1975). Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation. Biochem. Pharmacol 24, 2013-2017. Gross, H.. and Freiberg, J. (1969). Alpha-halo ethers. 41. Existence of chloroethylene oxide. J. Prakt. Chem. 311, 506-510. GuENGERiCH. F. P . and Strickland. T. W. (1977). Metabolism of vinyl chloride' Destruction of the heme of highly purified liver microsomal cytochrome P-450 by a metabolite. Mol. Pharmacol 13, 993-1004, HabiG. W. H,, Pabst. M. J,, and JakobY. W. B. (1974). Glutathione-S-transferases. The first enzy matic step in mercapturic acid formation. J. Biol. Chem. 249, 7130-7139. Hefner, R. E.. Watanabe, P. G., and Gehring. P. J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer in rats. Ann. /V. Y. Acad. Sci. 246, 135-148. Ivanetich. K. M., Aronson. L, and Katz, I. D. (1977). The interaction of vinyl chloride with rat he patic microsomal cytochrome P-450 in vitro. Biochem. Biophys. Res. Commun. 74, 1411-1418. Jaeger, R. J., Connolly, R. B.. and Murphy, S. D. (1974a). Effect of 18 hr. fast and glutathione deple tion on l.l-dichloroethylene-induced hepatotoxtcily and lethality in rats. Exp. Mol. Pathol. 20, 187-198. Jaeger, R. J., Reynolds. E. S., Connolly. R. B, Moslen. M. T.. Szabo, S.. and Murphy, S. D (1974b). Acute hepatic injury by vinyl chloride in rats pretreated with phenobarbital. ,\ature ILondonj 252, 724-726, Johnson, M. K. (1967), Metabolism of chloroethanol in the rat Biochem. Pharmacol. 16, 185-199. Kaplowitz, N.. Kuhlenkamp, J., and Clifton, G. (1975) Drug induction of hepatic glutathione-5transferases in male and female rats. Biochem. J. 146, 351-356. Kappus, H.. Bolt. H. M., Buchter. A., and Bolt. W (1976). Liver microsomal uptake of "C-vinyl chloride and transformation to protein alkylating metabolites in vitro. Toxicol. Appl. Pharmacol. 37, 461-471. LaIB. R. J., and Bolt. H. M. (1978), Formation of 3. N4-ethenocytidine moieties in RNA by vinyl chloride metabolites in vitro and in vivo. Arch. Toxicol 39, 235-240. Lowry, O. H., Rosebrough, N. J.. Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem. 193, 265275. Lu. A. Y. H., Kunztzman, R., West, S., Jacobson, M.. AND Conney. A. H. (1972). Reconstituted liver microsomal enzyme system (hat hydroxylates drugs, other foreign compounds and exogenous substrates. J Biol Chem. 247, 1727-1734. Malaveille. C,, Bartsch. H.. Barbin. A.. Camus. A. M.. and Montesano, R. (1975). Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol. Biochem. Biophys. Res. Commun. 63, 363-370. Maltoni, C., and Lefemine, C. (1975). Carcinoge nicity bioassays of vinyl chloride: Current results. Ann. /V. K, Acad. Sci. 246, 195-218. McCann. J., Simmon, V., Streitwieser, D., and Ames, B. N. (1975). mutagenicity of chloroacetaldehyde, a possible metabolic product of 1.2-dichlo- roethane, chloroethanol, vinyl chloride and cyclo phosphamide. Proc. Nat. Acad. Sci. USA 72, 31903193. Mitchell, J. R.. Jollow, D. J., Potter. W. a., Gil* lete. J. R.. and BrODIE, B. B. (1973). Acetaminophen-induced hepatic necrosis. [V. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211217. Mukhtar. H.. and Bresnick. E. (1976). Effects of phenobarbital and 3-methylcholanthrcne administra tion on glutathione-5-cpoxide transferase activity in rat liver. Biochem. Pharmacol- 25, 1081-1084, Omura. T,, and Sato, R. (1964). The carbon mon oxide-binding pigment of liver microsomes. I. Evi dence for its hemoprotein nature. J. Biol. Chem. 239, 2370-2378. Reichert, d., Werner, H. w,, and Henschler. D CMA 003646 j t. 4'u r. . I' c .txthione coiuer'. . .... v.,1.:-v giLiatmone reauc'-ase 3 i-cr ;i chloride Fid, P'oc 3", 1545 --T." '33 .: ...i;. Dl. J T . Sandqz, 1, P . Tseng. M T . ivj TaW3LRRQ. C H (1979) Biochemical al'erations m livers of rats exposed to vinvi chloride J Toxicol En viron Heahn 5. 11!9-132 Elmore. J D.'Aong, J L,, Lalmbalh. a. D . >sd Streips. Li N (19'6). Vmil chloride mutagenicity via the metabolites cnlorooxirnne and chloroace'.aidehyde monomer hydrate. Biocmm Biophit A,:a 442, 405-419, Feron. v j,. Spit. 8 J . 1mm el. H, R . oo Kaots. R ! 14791, One-vear time sequence inhalation tosicity study of vinyl chloride in rats III Morphological Fiala, S., MGHiNORL. 4 . i\L77cn.."j, -s G . I iac.a. 4, E. and Morris. H P i!4"ai C,\ctatn ,-d gamma glutamyl transpeondave in rat liver curing chemical carcinogenesis. J \n Cancer fnsi 57. 541- 598 Green. T . AND Hathway. D E, i 1475). The biolog ical fate m rats of mhvi chloride in reiat.on to us oncogcmcitv Chem -Biot Interact II. 545-:rG. Green, T . and H athva ay, D E M9""j The -hemistry and biogenesis of S-cantainmg metabolites of vinyl chloride in rats. Chem.-Biol. Interact 17, 137- 150. Greim. H., Bonse. G.. Radwan. Z,, Reichert. D.. and Henschler. D (1975). Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylenes as a function of metabolic oxirane formation Biochem. Pharmacol 14,2013-3017 Gross. H., and Freiberg. J (1969). Alpha-halo ethers. 41. Existence of chloroethylene oxide J, Prakt. Chtm. 311, 506-510. Guencerich, F. P.. and Strickland, T. W. (l *977) Metabolism of vinyl chloride; Destruction of the heme of highly purified liver microsomal cytochrome P-450 by a metabolite. Mol. Pharmacol 13, 993-1004. Habig. W H.. PaBST. M. J.. and JaKObY, W. B. (1974). Giuuthione-5-transferases. The first enzy matic step in mcrcaptunc add formation. J Biol. Chtm. 149, 7130-7139. Hefner, R. E. Watanabe, P. G.. and Gehring, P. J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer in rau. Ann .V. Y. Acad. Set. 146, 135-148. Ivanetich. K, M., Aronson. I., and Katz, I. D. (1977). The interaction of vinyl chloride with rat he patic microaomai cytochrome P-150 in vitro. Biochem. Biophvs. Res. Commun 74, 1411-1413. Jaeger. R. J., Connolly. R. B.. and Morphy. S. D. (1974a). Effect of 18 hr. fast and glutathione deple tion on l.l-dichloroethylenc-induced hepalotoxicity and lethality in rats. Exp, Mol. Pathol. 20. 187-198. . . . J _ -' , . -J . - , a . A - P . N ,J pretrejted with onenoourbuai Vj/urr Lonatm 252. ':4--;p Johnson. M K (196') Metabolism of on: iroethanoi in the rat Biochem Pharmacol 16, 155-199 SIPIOAITT N V.`, "LEV-A amp. J . AND C. C 19'5) Drug mductiun of hepatic giutatnicne-Jtransferases m male and femaie rats. Biochem J 146, 351 -356. Kappls. H. Bolt. H M. Blchter. a., and 3olt 'V (1976), Liver microsomal uptake of *C - -1 ny 1 cnloride and transformation to protein atkvtacmg metabolites in vitro Toxicol Appl Pharma, f 3T 46 1-4 [ s,a .5 . . A-.li rtu. r, h '-1 i 147j i ro--r-ut`:n 3 .NNetncnocytiOme moieties in R.Na ov vmyi cniorme metjhi,'"'.jA m ,nd .t wvo. Arch, Tox.-ii 59. 235-240 Lowry. O H,. RosebrolGh. N J, Farr, A, L., and Randall. R. J < 1951). Protein measurement with the Folm phenol reagent. J Biol Chem 193. 265- 275 Ll, A V H.. Klnztzm.an. R , West, S . J Acossos. M.. and Conney. A. H. 11972). Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and exogenous substrates. J. Biot. Chem. 247. 1727-1734. Malaveille, C. Bartsch. H., Barbin, A.. Camus. A. M.. and MONTESANO, R. ( 1975). Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehvde and chloroethanol. Biochem Biophvs Res. Common. 63, 363-370. MalTONI. C- and Lefemine. C. (1975). Carcinoge nicity bioassays of vinyl chloride' Current results. Ann. .VK .-lead. Sci. 246, 195-218. McCann. J.. Simmon, V., Streitwieser. D. and Ames, B. N. (1975). mutagenicity of chloroaceialdehyde. a possible metabolic product of 1.2-dtchlo- roethane, chloroethanol. vinyl chloride and cyclo phosphamide. friar Mat. Acad. Sci. USA 72. 31903193. Mitchell. J. R., Jollow. D. J., Potter. W a.. Gil- lete. J- R.. and Brodie. 8. B. (1973). Acetamtnophen-induced hepatic necrosis. [V. Protective role of glutathione. J. Pharmacol, Exp. Ther. 187, 211217. Mukhtar. H.. and Bresnicr, E. (1976). Effects of phcnobarbital and 3-methylcholanthrene administra tion on glutathione-5-epoxide transferase activity la rat liver. Biochem. Pharmacol. IS, 1081-1084. Omura, T., and Sato, R. (1964). The carbon mon oxide-binding pigment of liver microtomes. (. Evi dence for its hemoprotein nature. J. Biol. Chtm. 239, 2370-2378. Reichert. D,, Werner, H. W,, and Henschlejl D. CMA 003647 10 DU. TSENG, AND TaMBURRO (1978). Role of liver glutathione in 1.1 -dichloroethylene metabolism and hepatotoxicity in intact rats and isolated perfused rat liver. Arch. Toxicol. 41, 16917S Reynolds. E. S., Moslen, M. T. Szabo. S., and Jaeger. R J. (1975). Vinyl chlortde-induced deac tivation of cytochrome P-450 and other components of the liver mixed function oxidase system: An in vivo study Res. Commun. Chem. Pathol. Pharmacol. 12, 685-694. Sedlak. J., AND Lindsay, R. H. (1968). Estimation of total protein bound and non-protein sulfhydryl groups in tissue with Ellman's reagent. Artal. Biochem. 25, 192-205. VanDuuREN, B. L. (1975). Possible mechanism of car cinogenic action of vinyl chloride. Ann. V Y Acad. Set. 246, 258-267. Viola, P. L.. Bigotti. A.. AND CaputO, A. (1971). Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res. 31. 516-522. Watanabe. P. G., Hefner, R. E.. Jr., and Gehring, P J ( 1976a). Vinyl chloride-induced depression of hepatic non-protein sulfhydryl content and effects of bromosulphalein (BSP) clearance in rats. Toxicology 6, 1-8. Watanabe, P. G.. McGowan, G. R.. and Gehring, P J. (1976b). Fate of ''C-vinyl chloride after single oral administration in rats. Toxicol. Appl Pharma col. 36. 339-352. Watanabe, P G., McGowan, G. R,, Madrid. E. O.. and Gehring. P. J. (1976c). Fate of "C-vinyl chlo ride following inhalation exposure in rats. Toxicol. Appl Pharmacol. 37, 49-50. Watanabe. P. G.. Zempel, J. A.. Pegg. D. G., and Gehring, P. J. (1978). Hepatic macromolecular binding following exposure to vinyl chloride, Toxicol. Appl. Pharmacol. 44, 571-579. CMA 003648 5 EKi'tronrtmntal Health /Vrv^riv *'v Vnl Ut. pp. 117-UJ. IWI Effectiveness of Federally Required Medical Laboratory Screening in the Detection of Chemical Liver Injury by Carlo H. Tamburro* and Richard Greenberg* The increasing concern of industrialized societies over the potential health hazard of synthetic chemicals in the occupational environment has led to government requirements for medical laboratory screening of workers. The specific tests for such screening programs are most often selected on the basis of medical experience which utilized them in symptomatic or hospitalized populations. Required screening tests for hepatic injury including cancer in vinyl chloride workers has been systematically and prospectively studied in an industrial population working with synthetic rubber and plastics. Approximately 1.100 employees were studied over a five-year period. A cohort of 909 male employees, for the purposes of analysis, were divided into a "standard" and "nonstandard" population based upon the absence or presence of significant medical disease {including liver disease). \ suhcuhort of 110 individuals was further identified based on availahiliity of liver biopsy. Kvalualion of federally required studies included alkaline phosphatase (Al'). y-glutamyl transpeptidasc (GGTI'I, alanine aminntranserase (ALT. SGI*T), aspartic am ini (transferase (AST, SGI IT) and bilirubin (IIR). Also studied were indocyanine green clearance (ICG) and radioisotopic liver spleen scans l I.-S scans). The GGT1* provided the highest positive predicted value as a screening test for identifying "nonstandard" individuals (individuals with all types of medical disease) followed by ICG, AST, ALT, L-S scan. Al', and BR. In the identification of asymptomatic liver disease the GGTI* had the least specificity due to a high false positive rate, while the Al' provided the highest specificity. The ICG clearance however, provided the best combination of positive predictive value and sum of specificity and sensitivity. The AP provided additional increase in specificity as a follow-up study. There was no evidence that any of the other federally required tests added any additional benefit and did add significant increase in the false positive rate. These studies support the need for evaluating screening tests as to their sensitivity, specificity and positive predictive value, in asymptomatic individuals, before they are made established requirements. Introduction Industrialized societies throughout the world have become increasingly concerned over the poten tial health hazard of synthetic chemicals in the occupational environment. Governmental regula tions have increased the number and types of medical laboratory screening required for a largevariety of halogenated hydrocarbons as well as other potential environmental hazards. The pri mary objective of these screening programs is to `Liver Research Center. Division nf Digestive Diseases, Department of Medicine, and Department of Community Health and Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky 40201. October 1981 reduce disability, morbidity and mortality in work ers, especially as related to serious low-grade health hazards. In general, screening programs are instituted because of the presence in the work environment of a suspected or proven environmen tal toxin or carcinogen, which has the potential of producing low-grade injury over long periods of exposure. Most screening studies are directed toward the detection of abnormalities in certain body systems. The specific tests are frequently selected on the basis of medical experience which utilized them in symptomatic or hospitalized populations. Prior expe riences utilizing nonspecific multiphasic health sur veillance screening and maintenance have not proven to be cost effective except under certain limited 117 CMA 003649 conditions (i). The cost effectiveness of such tests, however, in the determination of medical screening requirements, has played a limited role due to the potential seriousness of these occupational agents. Little attention has been paid as to whether the effectiveness of federally required screening pro vides the best or, more importantly, a necessary benefit when applied to asymptomatic and other wise healthy worker populations. The discovery in 1974 of hepatic toxicity and cancer formation in vinyl chloride workers provided the opportunity to systematically and prospectively study the effectiveness of federally required and federally recommended medical screening proce dures for the detection of chemical liver injury, including cancer development (2). Table 1 lists the federally required medical screening procedures since 1974 for environments utilizing vinyl chloride or polyvinyl chloride. Table 2 lists the federally recommended studies for these same environments. This paper will present a preliminary assessment of the effectiveness of these federally required studies in the accurate detection and identification of chem ically induced liver injury due to halogenated hydro carbons, especially vinyl chloride. Materials and Methods The industrial population studied consisted of approximately 1200-1400 employees of a chemical plant whose two major products were synthetic rubber and plastics. The industrial plant had been in operation for over 35 years and had a predomi nance of male employees (96%), approximately 80-87% of the work force being white, 11-12% black, less than 1% of other racial origins. Turnover of the plant was approximately 10 to 15% per year with 65-70% of the work force having worked five years or more at the plant. Employee ages ranged from 18-65, with a mean of 52 years. A cohort consisting of 969 male employees who worked continually from June 1, 1976 to May 31, 1977 was, for purposes of this analysis, divided into a "standard" and a "nonstandard" population. These designations were given on the basis of a review of Table 1. Federally required studies for vinyl chloride workers. History and physical < 10 years as vinyl chloride worker--(annual) > 10 years as vinyl chloride worker--(semiannual) Biochemical studies SCOT (AST) SGPT (ALT) GGTP AP TB 118 Table 2. Federally recommi tided (not reuuired) studies. Hepatic studies LDH isoenzyme Total protein Protein electrophoresis HbsAp Radioisotopic scan Kidney dysfunction (urine examination) Albumin RBC Exfoliative abnormal cells Pulmonary system FVC FEV, Chest x-ray (PA and lateral) all present standard medical data on each employ ee, including the federally required studies. Other screening studies of the medical surveillance pro grams were not utilized in the classification of overall medical status because, at that time, their clinical usefulness was unknown or controversial. All studies were performed on an annual basis; those individuals with ten years or more of employ ment were examined and screened semiannually. Compliance with medical screening studies during the five-year study period showed a continuous participation in the history and physical examina tions by over 75% of the work force, laboratory tests and chest x-rays by 86%, and liver-spleen scans by 85%. Seventeen percent failed to undergo at least one history and physical examination, 9% did not have any of the radiological studies, and only 4% failed to have laboratory studies during this period. Approximately 40-50% of these individuals who did not undergo an examination claimed to have been examined by their private physician. A subcohort of 120 individuals was further identified based on the availability of a liver biopsy performed for medical reasons, both related and not related to their work. The term "standard" is used for those individuals who, based upon the best medical opinion, demon strated no evidence of any significant medical disease, occupational or nonoccupational in origin. The "nonstandard" population included all others not included in the standard population. The subcohort population was divided into those individuals with and without histological evidence of liver injury and further subdivided into those with and without histological features characteris tic of chemical injury. All employees had individual work histories. These consisted of a standardized job classification for all jobs within the plant since its opening and a Environmental Health Perspectives CMA 003650 rank ordering of exposure for 22 different sus pected or potentially hazardous heptatoxic chemi cals used within the work place (J-.5). The agents were rank ordered on the basis of the intensity of exposure for each of the job classifications for each of the years that the plant was in operation. From this detailed work history, a cumulative exposure ran month ration (CERM) was determined for each employee for each of the 22 chemicals. All histologi cal material was classified as to the presence or absence of liver disease, and to whether the abnor malities were consistent with chemical or non chemical injury. This classification was conducted double blindly by three experienced physicians, two pathologists, and a hepatologist (6), without knowledge of any medical data, exposure or work history. Results Although 50 or more biochemical screening tests were performed during this study period, this paper will limit itself to the evaluation of the federally required studies, the indocyanine green clearance (ICG) study at the 0.5 mg/kg dose (', 8) and radioisotopic liver and spleen scan (9). The 100 GOT? iCG SCOT SGPT _iv R SPIES n OS A S i" alt site TESTS Figure 1. Positive predictive values of screening tests in identification of medical disease in an asymptomatic working population (,V = 969). All those screening tests with positive predictive values of greater than 70 are shown except for indirect bilirubin (due to high number of congenital indirect hyperbilirubinemia) and triglyceride determination. Above each bar in the graph are shown the sum values for sensitivity and specificity of each test. They generally follow the same ranking. October 1981 T'v'v''vl I mW'aJ ? I 1 i.v,My'i 1 . . t:V.. .. I : SGPT GGT ALK SGOT ICO BILI- OSrnq 50mg PHOS RUBIN ICG Figure 2. Frequency with which clinical biochemical tests cor rectly reflex the presence of hepatic damage in chemical workers suspected of having liver disease1 (SGPT' alanine aminotransferase (ALT) (GGT) y-glutamvl transpeptidase, (SCOT) aspartic aminotransferase (AST). (Aik. Phos.) alka line phosphatase, (ICD) isocitric dehydrogenase (ICG) Indo cyanine Green clearances at 0.5 and 5.0 mg'kg dose. federally required biochemical studies include alka line phosphatase (AP), y-glutamyl transpeptidase (GGPT), alanine aminotransferase (ALT SGPT), bilirubin (ALT/SGPT), and the aspartic amino transferase (AST/SGOT). The positive predictive values of these screening tests in identifying medical disease (including liver disease) in this asymptomatic working populati^^k are shown in Figure 1. The GGTP provided t^^r highest positive predictive value as a screening test for "nonstandard" individuals. It also provided the highest sensitivity and specificity sum shown in brackets. The predictive value of the other tests, in decreasing positivity were ICG, AST, ALT, liver and spleen scan, AP, and bilirubin. Further evaluations were conducted on the subcohort population in whom we had both histolog ical and biochemical data concerning hepatocellular damage. If one looks at only those individuals who received liver biopsies for suspected liver disease then one would find the percent of positive tests as illustrated in Figure 2. The ALT (SGPT). GGTP, AP and AST (SCOT) demonstrate a very high degree of sensitivity in identifying individuals with hepatic disease. As shown on the right, ICG clearances at the 0.5 mg/kg level provide a similar degree of sensitivity to SGOT and AP. The higher dose ICG clearance (5 mg/kg) appears to provide the most sensitivity for latent hepatic disease. These findings are consistent with the general medical experience with hospitalized patients. Sensitivity alone however is not an adequate indicator of a test's screening value, especially when used in asymptomatic individuals. More appro priate evaluation of these tests' value as screening instruments are shown by their sensitivity, specificity CMA 003651 were rank ordered on the basis of the intensity of exposure for each of the job classifications for each of the years that the plant was in operation. From this detailed work history, a cumulative exposure ran month ration (CERM) was determined for each employee for each of the 22 chemicals. All histologi cal material was classified as to the presence or absence of liver disease, and to whether the abnor malities were consistent with chemical or non chemical injury. This classification was conducted double blindly by three experienced physicians, two pathologists, and a hepatologist (o'), without knowledge of any medical data, exposure or w irk history'. Results Although 50 or more biochemical screening tests were performed during this study period, this paper will limit itself to the evaluation of the federally required studies, the indocyanine green clearance (ICG) study at the 0.5 mg/kg dose (~, 8) and radioisotopic liver and spleen scan (9). The Figure l. Positive predictive values of screening testa in identification of medical disease in an asymptomatic working population (,V = 969). All those screening tests with positive predictive values of greater than 70 are shown except for indirect bilirubin (due to high number of congenital indirect hyperbilirubinemia) and triglyceride determination. Above each bar in the graph are shown the sum values for sensitivity and specificity of each test. They generally follow the same ranking. October 1981 0 L-1----- Li---LLi' i.-. --Li------ Li------- Li------1 SGPT GGT ALK SGOT ICO 3lt_t- 05mg50mg PHOS RUBIN ICG Floras 2. Frequency with which clinical biochemical te.-t.- cor rectly rertex the presence of nepatic damage ,r. chemical workers -usuected of havir.e liver disease SGPT' alanine am.njtramferrje ALT 'JUT -'-glutamyl trii.-.aDe'otria^e, 'sGuTl aspartic ammocransferase 'AST), iAik. Phos.) alka line ofosnr.acase. ` 1CD) isocitric dehydrogenase ' ICG1 Indo cyanine Green clearances at 0,5 and 5.0 mg kg dose. federally required biochemical studies include alka line phosphatase <APL y-glutamyl transpeptidase (GGPT), alanine aminotransferase (ALT SGPT), bilirubin (ALT SGPT). and the aspartic amino transferase (AST-SGOT). The positive predictive values of these screening tests in identifying medical disease (including liver disease) in this asymptomatic working population are shown in Figure 1. The GGTP provided the highest positive predictive value as a screening test for "nonstandard" individuals. It also provided the highest sensitivity and specificity sum shown in brackets. The predictive value of the other tests, in decreasing positivity were ICG. AST, ALT, liver and spleen scan, AP, and bilirubin. Further evaluations were conducted on the subcohort population in whom we had both histolog ical and biochemical data concerning hepatocellular damage. If one looks at only those individuals who received liver biopsies for suspected liver disease then one would find the percent of positive tests as illustrated in Figure 2. The ALT (SGPT), GGTP, AP and AST (SGOT) demonstrate a very high degree of sensitivity in identifying individuals with hepatic disease. As shown on the right, ICG clearances at the 0.5 mg/kg level provide a similar degree of sensitivity to SGOT and AP, The higher dose ICG clearance (5 mg/kg) appears to provide the most sensitivity for latent hepatic disease. These findings are consistent with the general medical experience with hospitalized patients. Sensitivity alone however is not an adequate indicator of a test's screening value, especially when used in asymptomatic individuals. More appro priate evaluation of these tests' value as screening instruments are shown by their sensitivity, specificity 119 CMA 003652 >0 TESTS Fiuuke 3. Sensitivity and specificity of various biochemical screening tests and their sensitivity and specificity sum values (S & S) based on 78 with biopsy documentation of their hepatic status and all of the biochemical screening studies listed. All screening tests with S & S sums less than 110 (e.g. bilirubin and isocitnc dehydrogenase, are not illustrated. Ficcre 4. Correlation between the histologic findings on liver biopsy and each individual's average total vinyl chloride exposure based on their average CERM (Cumulative Expo sure Rank Months) ratings. Rankings: 1 = lowest possible exposure: 2 = minimal exposure, low levels: 3 = moderate exposure; 4 = worked in areas subject to occasional high excursions, or frequently high and/or had intimate contact. and sum values shown in Figure 3 in the biopsied subpopulation. Here again, y-glutamyl transpeptidase and ICG clearance (0.5 mg dose) show the greatest sensitivity for identifying individuals with liver disease. However, GGPT had the least specificity, reflecting its high incidence of false positives. Specificity increased with the use of AST, ALT, and ICG clearance. The alkaline phosphatase pro vided the highest specificity, suggesting that mild or low grade chronic hepatic injury due to environ mental agents may be activating hepatic AP syn thesis in the absence of biliary tract obstruction or cholestasis. The ICG clearances, even at the low dose (0.5 mg/kg), clearly remains the test with the best combined sensitive and specific screening study for detection of individuals with subclinical hepatic disease. This subcohort biopsied group was further exam ined on the basis of the histological interpretation of their liver biopsies and their work exposure to vinyl chloride. All biopsied individuals were subdivided into three groups: 19 with histological evidence consistent with chemical liver injury; 30 with histo logical evidence of liver disease, nonchemical liver injury; and 29 with normal liver biopsies. Each of the histological subgroups were further subdivided based on their vinyl chloride exposure, on a scale of 1 to 4 (Fig. 4). The chemical liver injury group contained the highest percentage of individuals with the highest average rating (CERM) for vinyl chloride expo sure. In contrast, with those with liver disease, nonchemical, and those with normal livers have a 120 more even distribution of individuals relative to their degrees of vinyl chloride exposure. In our previous studies we noted that almost all individuals with histologically specific lesion of vinyl chloride injury or angiosarcoma had a total average CERM rating of 3.5 or greater. The asterisk in Figure 3 indicates the percentage of individuals in each of the three histological groups with exposure ratings of 3.5 or greater. Again, the chemical liver injury group have the highest per centage of individuals with the high exposure ratings. This further supports previous work (4,10) identifying focal hepatocellular hyperplasia as the earliest histological characteristics of chemical injury in liver disease. A study of the frequency with which these tests are positive among those individuals with liver disease, based on their histological findings (chem ical versus nonchemical), provides additional data supporting the clinical observation that an increased AP has a greater specificity for chronic liver injury. Figure 5 shows the ratio of the proportion of positive screening tests in those with histological chemical liver disease divided by the proportion of positive tests in those whose disease is not of chemical origin. All tests, independent of their sensitivity and specificity for liver injury, were more frequently abnormal in the presence of nonchemical, subclinical liver injury, except for AP. In contrast, AP was far more frequently abnormal in those individuals with chemical liver injury, which tended to be more chronic than acute and generally less severe. Environmental Health Perspectives CMA 0036S3 TESTS Ficire 5. Frequency with which biochemical test.-; were abnor mal in those with different type of hepatic injury expresses as a ratio: (CLI) chemical liver injury. (LI)) liver disease, nonchemical Discussion This preliminary systematic review of the posi tive predictive values and the sensitivity and specificity of federally and some non-federally required tests for chemical workers provides the first scientific and biological basis for the selection of medical screening tests for liver injury in occupa tional environments. Although these commonly used medical tests have been found by clinical experience to be effective as diagnostic tools in the symptomatically ill or hospitalized population, little clinical work has been done to determine their ability to accurately separate biological variations from early latent or underlying disease in asymp tomatic individuals. Tests which provide very high false-positive rates (decreased specificity) such as GGTP, interfere with the screening process identi fication of the high risk worker by the extra time and cost required for repeat testing, the decreased productivity for the employer, the employees' increased anxiety, and by the loss of confidence in the effectiveness of the testing program by both employees and employer. Determination of the sensitivity and specificity of screening studies for asymptomatic individuals is essential if effective recommendations are to be made a federal require ment. This evaluation process also provided the October 1981 best means of developing effective triage protocols for the screening program. For example, in this particular population of industrial workers, we have shown that the assessment of hepatic function is best accomplished by low dose ICG clearance (O.o mg'kg). The ICG clearance is somewhat a more complicated technique (i.e., injection of substance and repeated blood sampling) but requires only 10 min to perform, and needs only one needle stick. In exchange it provides the best singular screening test for latent hepatic injury. If adequate medical facilities are not easily accessible, then ALT should be substituted. If either ICG clearance and/or ALT studies are found to be abnormal, an AP should be done and a diagnostic work-up instituted to deter mine the etiology (ID. The rationale for these recommendations is based on the actual study of chronic subacute chemical injury in an asymptomatic population, not preselected because of signs or symptoms. Therefore the test's ability to correctly differentiate disease from nondisease or one type of injury from another is more accurately determined. Chemical and envi ronmental agents of low toxicity tend to produce repeated or persistent injury which accumulates over time. Tests which measure overall functional capacity quantitatively or semiquantitatively. rather than measuring acute low-grade injury over tim^^ are more likely to detect changes. For this reason^^ clearance or tolerance studies provide the best means for identifying latent hepatic disease, while enzyme studies like ALT, GGPT, and SGOT usu ally reflect acute cellular injury of higher grade or degree and cannot accurately reflex accumulative damage until very late in the disease process. Tests which provide information concerning the progres sion or nonprogression of injury will be far more helpful to the practicing occupational physician. They provide him/her with a better capability to discern between nonoccupational and occupational disease, and the best available reassurance for the worker of his or her safety while allowing the greatest possibility for continued productivity and employment. Finally, these data provide a sound scientific basis upon which to modify federal requirements. The removal of specific testing requirements which, with field experience, prove not to have any significant positive predictive value, or effective sensitivity and specificity will aid in reducing overall cost and help maintain continued compliance by industry and workers. There may be theoretical reasons to maintain or continue some of the present federallyrequired screening studies, but these reasons should be separately identified and not be confused with the purposes of the more effective test in the CMA 003654 ICO - ao 1- 60 ^ iOn CL 40 \ NON c-eTm.c-l 1 ! ii !: i ! ! 0 ...... , 1 -1 i_____ i_J_____ i. L...... -1 AST ALT GGTP AP ICG (SGOT) (SGPT) 05 TESTS 5.?-<, ?,y. Frequency-Aiih u ".icr,-nnur- mal tn :ho?e ^i:n ihfferent ;yu "cp^ii: injury f\|T*c--o'. as a ratio- (CLI> chermcai liver injury, (I.l>i tivr dt.-fiiMu nonchemicaJ, Discussion This preliminary systematic review of the posi tive predictive values and the sensitivity and specificity of federally and some non-federallv required tests for chemical workers provides the first scientific and biological basis for the selection of medical screening tests for liver injury in occupa tional environments. Although these commonly used medical tests have been found by clinical experience to be effective as diagnostic toots in the symptomatically ill or hospitalized population, little clinical work has been done to determine their ability to accurately separate biological variations from early latent or underlying disease in asymp tomatic individuals. Tests which provide very high false-positive rates (decreased specificity) such as GGTP, interfere with the screening process identi fication' of the high risk worker by the extra time and cost required for repeat testing, the decreased productivity for the employer, the employees' increased anxiety, and by the loss of confidence in the effectiveness of the testing program by both employees and employer. Determination of the sensitivity and specificity of screening studies for asymptomatic individuals is essential if effective recommendations are to be made a federal require ment. This evaluation process also provided the October 1981 nave shown :ha: the assessment of hepatic function is best accomplished by low dose ICG clearance . o. 3 mg kg). The ICG clearance is somewhat a more complicated technique (i.e.. injection of substance anti repeated blood -anvil big1 hut 'v'1 pi min to perform, and needs only one needle stick. In exchange it provides the best singular screening test for latent hepatic injury. If adequate medical facilities are not easily accessible, then ALT shoum be substituted. If either ICG clearance and or ALT studies are found to be abnormal, an AP should be done and a diagnostic work-up instituted to d-ter- The rationale lui" tnese recoinmcnuntiuns ij uus<wl on the actual study of chronic subacute them,'..:?; injury in an asymptomatic population, not preselected because of signs or symptoms. Therefore the test's ability to correctly differentiate disease from nondisease or one type of injurs from another is more accurately determined. Chemical and envi ronmental agents of low toxicity tend to produce repeated or persistent injury which accumulates over time. Tests which measure overall functional capacity quantitatively or semiquantitauvely. rather than measuring acute low-grade injury over time are more likely to detect changes. For this reason, clearance or tolerance studies provide the best means for identifying latent hepatic disease, while enzyme studies like ALT. GGPT, and SGOT usu ally reflect acute cellular injury of higher grade or degree and cannot accurately reflex accumulative damage until very late in the disease process. Tests which provide information concerning the progres sion or nonprogression of injury will be far more helpful to the practicing occupational physician. They provide him/her with a better capability to discern between nonoccupational and occupational disease, and the best available reassurance for the worker of his or her safety while allowing the greatest possibility for continued productivity and employment. Finally, these data provide a sound scientific basis upon which to modify federal requirements. The removal of specific testing requirements which, with field experience, prove not to have any significant positive predictive value, or effective sensitivity and specificity will aid in reducing overall cost and help maintain continued compliance by industry and workers. There may be theoretical reasons to maintain or continue some of the present federally required screening studies, but these reasons should be separately identified and not be confused with the purposes of the more effective test in the 121 003655 detection of occupationally related liver injury. The capabilities and limitations of any new tests or old tests for new screening purposes in detection of other potential occupational hazards, should be validated before making them a required screening procedure. This would lay the foundation for sys tematic determination of effectiveness of screening procedure against a proven standard. Unvalidated federal requirements provide the worker and employer with a false sense of security and safety by what is believed to be effective monitoring. More importantly, such a situation can lead to delay in effective correction of cause and disease preven tion. Conclusions Biochemical screening for hepatic injury in asymp tomatic chemical workers can be done most effec tively by the use of liver specific clearance studies. ICG clearance provides the best combination of positive predictive value and sensitivity and specificity for functional hepatic testing at the present time. None of the present federally required studies provide any significant degree of sensitivity with out marked reduction in specificity in asymptomatic individuals. The ALT (SGOT) is the most useful among those Federal tests presently required and the alkaline phosphatase may provide additional specificity as a follow-up study in those individuals with positive ICG or ALT screening studies. There is no evidence that any of the other federal studies add any benefit and strong evidence that they significantly increase the false-positive results in well individuals. All screening studies should undergo evaluation as to their positive predictive value and sensitivity and specificity in asymptomatic individuals before becoming permanent or established requirements. Portions of this work were supported by National Cancer Institute Contract No-l-CN'-.55Z12 and Manufacturing Chem ists Association Grant, The authors wish to acknowledge the help and cooperation of Hynson. WeStcott & Dunning and B. F. Goodrich Chemical Company REFERENCES 1. Collen, M, F. Cost effectiveness of multiphasic health testing services. In: Multiphasic Health Testing Services. M. F. Collen, Ed., Wiley, New York, 1978. Chapter 17. Section F. pp. 487-530. 2. Creech, J. L., Makk, L., Whelan, J. G., Jr., and Tamburro, C. H. Hepatotoxicity among polyvinyl chloride production workers during first year of surveillance program. Gastro enterology 67: 786 (1974). 3. Greenberg, R. A., Tamburro, C. H., and Kupchella. E. C. Prospective medical surveillance program for detection and prevention of industrial related cancer. In: Prevention and Detection of Cancer; Part 1, Vol. 2: H. Nieburgs, Ed.. Marcel Dekker, New York, 1978, pp. 1921-28. 4. Tamburro, C. H., Greenberg, R. A.. Newby. L. G.. and Turns, D. M. Implementation and assessment of a demon stration cancer control detection and prevention program in a cohort of industrial workers. Program Contract #N0lCN-55212 Final Report, Division of Cancer Control and Rehabilitation, National Cancer Institute. Bethesda, Mary land, 1978. 5. Greenberg, R. A., and Tamburro, C. H. Exposure indices for epidemiological surveillance of carcinogenic agents in an industrial chemical environment. J. Occup. Med., in press. 6. Tamburro, C. H., Makk, L., and Popper, H. Early hepatic histological alterations among chemical (vinyl monomer) workers. Gastroenterology 77: A33 (November 1979). 7. Fortwengler, P., and Tamburro, C. H. Use of dye clearance in the detection of hepatocellular injury among vinyl chlo ride workers. Clin. Res. 23: 264A (1975). 8. Tamburro, C. H., Creech. J. L, Davis, A., and Greenberg. R. A. Indocyanine green clearance as a prospective indica tor of hepatocellular chemical toxicity. Gastroenterology 75: 989 (1978). 9. Whelan, J. G., Jr., Greenberg, R., and Tamburro. C. H. Radioisotopic scans and gray scale ultrasonography in detection of liver damage. Gastroenterology 79- 1129 (1980). 10. Popper, H. and Thomas, L. B. Alterations of liver and spleen among workers exposed to vinyl chloride. Ann. N. Y. Acad. Sci. 246: 172 (1976). 11, Tamburro, C. H. Chemical hepatitis, pathogenesis, detec tion and management, Med. CUn. N. Amer. 63: 545 (1979). 122 Environmental Health Perspectives CMA 003656 Reprinted from CANCER. Vol. 40. No 6. Dcccm* ber [977 Copyright, () 1^77, by [he Nmeritjn Can cer Society, (nc, J, B. Lippincou Company Printed m L S A URINARY G LYCOS AM I NO G LYCAN PATTERNS IN ANGIOSARCOMA OF THE LIVER Kevin L. Curran, BA, MS, Charles E. Kupchella, PhD, and Carlo H. Tamburro, MD Glycosaminoglycans extracted from 24-hour urine specimens from patients with hepatic angiosarcoma and from normal/controls were separated as cetylpyridinium complexes into "hyaluronic acid," "chondroitin sulfate," and "heparin" fractions, then further separated and characterized by anion-ex change chromatography and hyaluronidase susceptibility. The chromato graphic pattern of the urinary chondroitin sulfate fraction in patients with angiosarcoma of the liver differed from those of controls in that there was a relative increase in the total amount of uronic acid in a hyaluronidase-resistant fraction and a decrease in a fraction susceptible to hyaluronidase digestion. These changes appeared to become more pronounced with advancing disease. Chromatographic patterns and determinations of hyaluronidase susceptibility indicated that the resistant fraction was heparan sulfate and that the susceptible fraction was chondroitin-4-sulfate and/or chondroitin-6-sulfate. Cancer 40:3050-3053, 1977. he emergence of angiosarcoma of tiif. Tliver and its relationship to vinvl chloride exposure*1-7* *h*a*s* prompted a search for methods to detect this lesion Although systematic screening programs are currently in opera tion,101' there is still no single chemical in dicator which is specific for angiosarcoma or for changes which may precede this disease. The association of elevated tissue glycosamirioglycans (ClACl) with tumors, including angio sarcoma, has been established.18 "1"" Glycosaminoglycans are also known to be involved in normal connective tissue synthesis and collagen deposition and are elevated in connective tissue disorders.13 Since angiosarcoma of the liver has both neoplasia and fibrogenesis in its etiology17 GAG changes could be expected to serve to sig nal the appearance of early lesions and may he useful in evaluating advanced lesions. Galambos" suggested that since the liver con tributes very little to the overall connective tissue of the body, hepatic fihrogenesis should not be expected to result in significant increases in From ihe University of Louisville. Cancer Center, and the Pnce Institute for Surgical Research Health Sciences Cen ter. Louisville. Kentucky 40201. I li a work vs,is Supported in pan liv .Ml \merii an ('ant rr Sot rets Insfifuttottal Crant, IN-lll. a grant from the H E Coodru h Cnmpajtv. and i onirai t N() 1.(.N-SS /12 ss it Is the Niiiiniiitl ('aiiMcr InsiHtiic AdHrrss for reprints (' l'. Kup< Ih-II.i, (l.mirr (Vnirr, l nisrrMiv '*! Lmiiwillr. Louisville. K\ 4U20) ,\i < rptcfi tor piililiu.iiion \pnl l s I1/"*'* urinary GAG or collagen degradative or syn thetic products. Preliminary studies in our labo ratory. however, demonstrated an increase in both liver and urinary GAG in patients with angiosarcoma, chronic active hepatitis, and cir rhosis 15 Most of the increase in urinary GAG occurred in the chondroitin sulfate fraction and, tn contrast to what was found for normal and other diseases, the urinary chondroitin sulfate Inn lion was the only uronic acid positive fraction found in the urine of seven of nine cases of vinylt hloride-exposure-associated liver injury other than angiosarcoma. This study was undertaken to characterize more completely the urinary "chondroitin sulfate" fraction in hepatic angio sarcoma. Clinical Summaries Case l--(Hepatic Angiosarcoma--advanced) A 46-ycar-old white male worked as a chemical helper in a vmvl chloride polymerization plant for ihirteen years prior to the diagnosis of angiosarcoma. I'welve years after initial employment, the patient exhibited a persistent elevation of lactic dehy drogenase and underwent angiographic studies which demonstrated multiple areas of scattered tumor stain throughout both lobes of the liver with areas of central translueenrv consistent with the diagnosis of angiosari oma of the liver Exploratory laparotomv and liver hiopsy confirmed this diagnosis, and the patient was treated with adnamyctn, cyclophosphamide, and methotrexate, an 3050 6 CMA 003657 M O LARlTf NaCl No 6 Glvcosaminoclscans in Am.iosakcoma Curran tt al. 3051 initMl response was assrx not'd wuh a decrease in ihe alkaline phosphatase activity, improsemem in in docyanine green clearance and an increase in radio isotopic uptake in areas of previously defective up take \fter completion of the chemotherapy course, hepatic function deteriorated and the patient under went partial hepatic lobe radiation (total dose of 5,000 rads) over a two-month period Despite radia tion therapy, the clinical course continued to deterio rate with the development of ascites, peripheral edema, increasing ]aundice. hvpoalbuminemia. and marked elevations of transaminases and alkaline phosphatase activities. This was followed by progres sive hepatic failure, hepatorenal svndrome and he patic coma Autopsy findings showed extensive in volvement of the liver with angiosarcomatous tissue extending into the diaphragm and metastasis to retro peritoneal and mediastinal Ivmph nodes, lungs, right adrenal gland and cerehellum. The right lobe of the liver demonstrated near elimination of the angiosar coma. presumably due to the radiation treatmeni Urinary GAG assays reported here were made on 24hour urine specimens collected over the two-week period before death (Fig D Case 2 (Hepatic Angiosarcoma--moderately advanced) A 54-year-old vinyl chloride polymerization worker was first employed as a polymerization vat cleaner 28 years prior to the diagnosis of angiosarcoma Two years prior to diagnosis, the patient had persistent biochemical liver, abnormalities although he was oth erwise asymptomatic with a normal liver-spleen scan Angiographic studies showed peliosis hepatis. A liver biopsv revealed focal sinusoidal dilatation, mild chronic inflammatory reaction with portal fibrosis. KupITer cell hyperplasia and dysplasia. Subsequent biopsies demonstrated tontinued sinusoidal dilitation, atypical and dvsplaslit Klipfler tells with [ire* malignant changes The peliosis hepatis pattern be came more pronounced and multiple radioisotopic defects were evident on liver scan. A repeat biopsv one year after initial biochemical abnormality demon strated malignant sinusoidal cells. The patient was treated with a combination of adriamycin, cytoxan. and methotrexate with limited clinical and biochemi cal response. Death was preceded by peripheral edema, ascites, progressive hepatic failure, and coma The GAG analyses reported here were made 10 and 6 months before death (Fig. I. |middle|) Materials and Methods Twenty-four hour urine specimens were col let ted from two p.uienls with angiosarcoma til the liver, and from two norni.il tontrols. I'l ine spot imcns were stored ;u until analysis. Gctylpvridinium t hloride (Sigma Chemieul Company, St. Louis) was added m the entire 24-hour volume to precipitate the (i.\(G is - HEPATIC ANGIOSARCOMA-Advanced 10 5- ] Fit, I Klunon Patterns of the Urinary Chondroitin SulFat Fruition The glvrnsaminoglycans (GAG) in a 24.hour urine specimen were precipitated with ceiylpvndmium chlo ride (CPC') and separated as 0,4 M NaC! soluble ("hyalu ronic acid"), 1 2 M NaCl soluble ("chondroitin sulfate"}, and 2.1 M NaCl soluble (,lhepann") fractions Each fraction was then subjected to anion-exchange chromatographv Shown here are rypical 1 2 M (chondroitin sulfate) fraction elution patterns (Advanced - case \ ) according to the method of DiFerrante.5 The hyaluronic acid, chondroitin sulfate, and hepa rin fractions were eluted individually according lo the method of Schiller el at. 14Cetylpvridinium chloride was removed14 and the GAGs were sub jected to anion-exchange chromatography as de scribed by Schiller el al.Glycosaminoglycan fractions were applied to 1.0 X 44 cm AG1-X2 (200-400 mesh, chloride form) columns Bio Rad Laboratories, Richmond, California) and eluted stepwise with 0 0, 0.5, 1.0, 1.25, 1.50, 2.0, and 3.0 M NaCl. At a flow rate of L0 ml/min, approximately sixteen 10.3 ml fractions of each molar strength of NaCl were collected and a sample of each fraction was analyzed for uronic acid by the method of Bitter and Muir.3 Stan dards of heparin (Nutritional Biochemical Com pany), chondroitin sulfate (Sigma Chemical Company), and hyaluronic acid (Nutritional Biochemical Company) were also evaluated by mo exchange chromatography I hc iironii -acid-positive fractions within each individual salt fraction were pooled, dialyzed io remove salt, and concentrated. The fractions cluicd by I 25 or I 50 M NaCl were tested for CMA 003658 3052 Cancer December 19 Vul - T VS1F 1 Source RcUiu of [ ntal L ronic \ctd Muted in I 25 M/1 5 M \aCl Norma! Normal \niziosarcorna. case 2, pre-chemotherapy1 \nmosarroma, c<i>e 2. pn*r-< hcmother.ipv * Am<io*.ir< om.i, ta'C I, advanced 0564 0 5 Hi 0 84 \ 007| 5OOO Non- J'hr v c osammoqlvcans (CAP) in a 24-hour urine specimen were precipitated with telylpvndimum < blonde (CPC) ,md separated .is 0 4 M soluble l``hvaiuronic acid I 2 \1 NaCl soluble ("chondroitin sulfate"), and 2 I M NaCl soluble ("heparin") fractions The CPC was removed from the l 2 M NaCl-CPC* solubilized fracnon and the GACs further purified by anion-exchange chromatography The total amount of GAG m the resulting 1 25 M and 1,50 M NaCl column- eluted fractions was determined and the ratio of the two fractions was calculated, (`One day prior to begin ning of chemotherapy. Two days following chemotherapy initiation ) acid and heparin fractions revealed no qualita tive differences between controls and angiosar coma patients. The anion exchange column pat terns of the 1 2 M NaCl solubilized C.AGs are shown in Fig. 1 Chromatography of the urinary "chondroitin sulfate" fractions of patients with angiosarcoma yielded a comparatively large, uronic-acid positive peak in 1.25 M NaCl. The ratios of the total amount of uronic acid-positive material eluted with 1.25 M NaCl to the total amount eluted with 1.50 M NaCl are given in Table 1. I he susceptibility of the GAGs eluted with I 25 or I 50 \1 NaCl to hyaluronidase degrada tion is given in Table 2. The GAG eluted with 125 M NaCl was resistant to hyaluronidase. the enzyme producing only a 43% reduction in tur bidity. The 1.50 M NaCi-eluted GAG fraction was 100% susceptible to hyaluronidase degrada tion. Discussion and Conclusion susceptibility to testicular hyaluronidase (Nutri tional Biochemical Company) using a modifica tion of the cetvltrimethylammoniurn-bromide, turbidimetric assay described by DiFerrante.' Kl-.Sft.TS The major GAG fraction observed in all urines--both from normal controls or from pa tients with angiosarcoma--was the fraction so lubilized by 1.2 M NaCl/t% cetylpyrtdinium chloride (the "chondroitin sulfate" fraction). Anion exchange chromatography of hyaluronic Table 2. Hyaluronidase Susceptibility Source Pepolvmerizaijon -V Heparin, standard Hvaiuronic acid, standard Chondroitin sulfate, standard 1 25 M NaCl column-rliiate. pooled fraction* from anqiosari nm.itons patients \ SO M N.i< l column-eluaie. pooled fractions from antpo>ar< omatous patients 1 5() M NaCl column-eluate, normal 50 i b*6 i\ s 100 o 100 0 1 (llycosaminoglvcans isolated from urine were tested lur hvaiurnmdase suscepubdits bv rneasunnR changes m turbtditv developed wtih the addition of t etvltrimrthslammomum bromide followmtr incubation with hv.iluromd.iw Normal controls exhibited nnk minor aniounis ,,! I M \'aC:l column-fluted (,.\(, and i nnsrquenilv do not ap pear in this table The chromatographic pattern found here for controls conforms to urinary glycosaminoglycan distributions reported by others.13-31 These pat terns suggest that there was a relative increase in urinary heparan sulfate and a decrease in rhondroitin-4- and/or -6-sulfate in patients with hepatic angiosarcoma. This interpretation agrees with the Dowex 1-X2 chromatographic patterns reported by Kao and Leslie13 and by others 3-" Heparan sulfate is reported to be partially susceptible to hyaluronidase digestion,30 and this correlates well with the observed 43% diges tion of the GAG in our 1.25 M NaCl fraction. Since heparan sulfate has been shown to be associated with blood vessels,30 an increase in the urinarv excretion of this GAG is not surpris ing in this vascular lesion. Also, chondroitin-4and -6 sulfates are reportedly eluted from Do wex 1-X2 columns with 1.50 M NaCl31*and are suscepiihle to hyaluronidase30 suggesting that our 1 5 \| frac tion is c hordroitin-4- and/or chondroitin-6-sulfate. Assuming that urinary GAG patterns de scribed here are reflections of hepatic changes, it will be important to determine what processes these changes reflect, i.e., those of neoplastic growth, fibrogenesis, or cell death. In this re gard, it should be noted that t) the ratio of heparan sulfate to chondroitin sulfate reported here for angiosareomatous urine is similar to that reported for cirrhotic human liver tissue by Becker.1 and 21 the shift from a hvaluronidnse- CMA 003659 \ r~,A ^or^tl \^4,o'5nrromal c^>e 2. prcM'hemoihf f\*pv1 V'lt'H iSGIsT''r'Gi \ ,', -r* Z imst nrmoi her inv * Vnti'us^rf tim.v uor l iCv.inc ed <) ') > 1 o ') \ 1 ' ,f' I ' w> V-n f hr u\,ifnini^!\t m* f \( > :n ,i 2i-h.>'ir urine ^penmen were pnxipumed ^ith t rMvipvridininm "noriuf 'C P(\ md irp.4r.ucd .u u x M Vt( I mlublr ' hyaluronic .tnd I 2 M Yi( I 'I'iunte 'chofuinnim iuiftitc' ^nd 1 ! M YtC I aoiub'e heparin fraction', T-* rpr .1, J '( \.i/Y i'( , -oiMOtiizcd trscvn ire *",e < rViJ r paired 3>, imon-exchange oromato^nohv The romi amount >f (t \<. :n 'he r-SLiuna : 2i M arid I '<> \[ \jf:i coiumn- eiuied fractions was determined and the ratio of the '.wo fractions was calculated `One dav prior to hf'stin- run* of chemotherapv *Two day* Jnllrmin* chemotherapy mutation ' tern; ot ;n.r 1 2 \f N'aC! solubilized OaOs are shown m Fig 1 Chromatography of the urinarv "chondroitin suifate" fractions of patients with angiosarcoma yielded a comparatively large. ironic-.*i id po'iiive Beak .r. i 2^ M N'aC! The ratios ot the total amount of uronic ucid-posune material eluted with I 25 \I NaC! 'o the total amount e'uted with I 50 \( N'aC! ure riser. r Table ! 1 he susceptibility of the GAGs eluted with 1 2w or I id M N'aCl to hvaluronidase degrada tion is given in Table 2. The GAG eluted with i M \`..r! loi-.iptroUw. -Ida:-' erzvme pend^.jm^ an;-, a -2" reduction in biditv The I 50 M NaC.i-eluted GAG fraevon was 100,Ja susceptible to hvaluronidase degrada tion Dtscrssr.v VND COXCLL'SIO.N susceptibility to testicular hvaluronidase (Nutri tional Biochemical Company) using a modifica tion of the cetvltrimethylammonium-bromide. turbidimetric assay described by DiFerrantc.* Rl-.-ll.-LIS The major GAG fraction observed in all urines--both from normal controls or from pa tients with angiosarcoma--was the fraction so lubilized bv 1 2 M N'aCl/1% cetylpvridinium chloride (the "chondroitin sulfate" fraction) Anion exchange chromatography of hyaluronic Tasle l Hvaluronidase Susceptibility Source Depolvmenzaimn V Heparin, standard Hyaluronic acid, standard Chondroitin sulfate, standard 1 25 M NaC! column-Huate. pooled fraction* fnm .ingiwuirrommoirt pm tents 1 M NaCl t-oluirm-eluaie. ponied fractions from .tn^tusarcomatnu^ patients 1 M NaCl column-eluate. normal ;o `>s i l>~ b -11 1 I IK) 0 100 0 1 (liycnuminoqlvcans isolated from urine were lested for hvaluronidase susceptibility bv measurinq changes in turbidity developed with the addition of retvhrimetnvlammonium bromide fnilowinq incubation -un hvaiunmuia.-c Normal controls exhibited onlv minor amounts of ! 2SI NaC'l cnlumn-eluied t.Atl and I'linseipientlv do noi ap pear in (his table The chromatographic pattern found here for controls conforms to urinary glvcosaminogivcan distributions reported by others l, 2i These pat terns suggest that there was a relative increase in urinary heparan sulfate and a decrease in chondroiiin-4- and/or -fi-sulfatc in patients with hepatic angiosarcoma. This interpretation agrees with the Dowex 1-X2 chromatographic patterns reported by Kao and Leslie12 and bv others 3 '* Heparan sulfate is reported to be partiallv susceptible to hvaluronidase digestion." and this correlates well with the observed 43% diges tion of the GAG in our 1.25 M NaCl fraction. Since heparan sulfate has been shown to be associated with blood vessels.3" an increase in the urinary excretion of this GAG is not surpris ing in this vascular lesion. Also, chondroitin-4and -A sulfates are reportedly eluted from Do wex 1-X2 columns with 1.30 M NaCl31*and are susceptible to hvaluronidase" suggesting that imr I 5 M fraction is < hordrmtin-4- and/or chnndmitin-A-sulfate. Assuming that urinarv GAG patterns de scribed here are reflections of hepatic changes, it will be important to determine what processes these changes reflect, t.e., those of neoplastic growth, fibrogenesis. or cell death. In this re gard. it should be noted that 1) the ratio of heparan, sulfate to chondroitin sulfate reported here for angiosarcomatous urine is similar to that reported for cirrhotic human liver tissue bv Becker,3 and 2' the shift from a hvalurontdase- CMA 003660 No 6 GlycosaminoglyCans in Angiosarcoma Curran tt at 3053 susceptible to a hvaluronidase-resistant GAG is consistent with the suggestion bv Hutterer and Rubin11 that the stabilization of collagen de pends on a shift to a hvaluromdase-resistam GAG ens elnpe surrounding the collagen bundle. Mihotigh Hutterer and Rubin attribute this in an augmentation of dermatan sulfate, Meeker1 reported that the GAG pattern in human cir rhosis was characterized bv the augmentation of dermatan sulfate and heparan sulfate If the ob served changes in urinary GAG are reflective of vinvl chloride-exposure-associated fibrosis, the fact that fibrosis is a precursor of angiosarcoma17 indicates that the observations reported here constitute ,i promising lead in early detection of vinyl-chloride-induced liver disease Kl.I 1 Id N< I'.N ! \nuhilrn l, j Metabolism ol <i< id nun opolvsac ( ha- ndes in hepatoma And normal liver Oncvin^y ^*1 |0M 2 Betker, K \cid mucopolvsaci handes in experimental Anri hum.in cirrhosis In Collagen Metabolism in the Liver H Popper and K Bet ker Kds New Mirk. Oration Inter continental Mcriual Book Corporation |0"V pp 4ds2 ^ Bitter 1 . and Muir. H A modified uromc at id (.trh.o /ole rent lion Anal Hvuntm 4 DH-M4 1002 -1 ( rerc h, | ( , and Johnson, M N \m<iosnr( um,i of die liver m the inamita< tore of polwiml c hloride 7 Dom/w* tmmi \l.r/ 16 IM |T4 s I )||ci i.iiiie \ the mr.isui nneni <>l m iiuiv nunn- | tub n.h i li.it i< lex \,<,,l ItuHkrm 21 0* ion |0td o Dil'cnante N I urbidimeirii mcnnit'emeui <4 at ul mm opolvs.u ( harides and hv nluronirinse atitviiv J ^l'd ( hmi 22(1 VD W, 10S6 Ktlk, 11 , ('rrec h, J L , I leatli, D W , Johnson M \ and Kev M M Hepatic disease nmony workers ai a wnvl c hloride polymerization pl.int JAMA 2VMO-6Y 19"'4 M dnlnmbos, J I' Connective tissue metabolism and cirrhosis In Collagen Metabolism in the I.off, H Popper and K Br< ker, Kds New York, station Intert ontmental Medical Book Corporation, 10"l, pp s7-6| o (>,ai( C and Cnsu I Kemoval of sialic acid from the c ell c o.*t in tumor c ells and vast that endoiht hum and iis elicits m meiastasis Pr<* \utl Uml. .S< t l V l 4* l I"2 ! rH |0n2 Id (Ireenberii K V. lamburro C H . and Ku[>< Bella, ( l A prospective medical surveillance program for ihc delet iion and prevention of occ upaiumallv-relaied i.uuer In Prevention .md Heteition of (amt er It K Nieburm I'.ditor, Part I Volume 2. Martel Dekker, In< , N\ (In press) 1! I loiterer V and Kubin, K : Mucopolysa<< handes m reversible and irreversible experimental hepatic librosis In Collagen Metabolism in the Liver, II Popper and K Becker Kds New Mirk, Siratton Intercontinental Medical Book ('orporatton !07K pp B-i6 12 Kao K T and Leslie, J 0 Micro fractionation and determination of unnarv ^Ivcosamino^lvcnns Biochem Med 'Mr MO, 10'4 H Koi/umi. I Nakamura N and Abe, H Changes in a< ul mut opolv sa< charide in the liver in hepatic fibrosis Itanium Alia 1 4X 740-7S6, 1067 M Korn I. I) Isolation of heparin from mouse mast i ell tumor ) HaU (hem 2^4 1^-1129, !0S0 I' Kupc liella, (' l; . md Tamhurro. C H, Urinary and fissile n K < osainmoylvc an patterns In hepatic angiosarcoma In Pnvetilion and Defection of dancer. II K Niebunjs, Lriitoi. Part I 'volume 1, Martel Dekker. Inc , NY (In pf css ) Ui M.ikk, I . Creech. | L , Whelan J (I , and Johnson, M N Liver damage .md nnmnsarroma in vmvl chloride wot ker s \ svsirmaiit derm ion program JAM A 210 64-6X, IT4 r Popper. H, and 1 homas. L B Alterations of liver and spleen amoni' workers exposed tn vinvl chloride dnn W A<ad S,i. 246 P2-I04. 19" lx Kith C and Mvers, \V P I. Kxcrenon of acid rniic opolvsac c handes in the urtne of patients with malignant neoplasm diseases J Lah and (dm Med 14 221-22H, |0S0 10 Schiller. S, Sloven d \. and Dnrfman. \ \ mctliod lor ihr separation of at id mm opolvsat (.handes Its .ipplu alum to the isolation of heparin from the skin of rats / Hmi ( hrtn 2V> OH V OH7, 1061 20 Sharon N domplex ( arbohvdrates I heir dhem- isirv BiosvmheMS and I'unc nuns, Heading. Massat husetis. Xdcliscm-Wc'sli V Pulllisimu; ('omp.uiv, |0"s 21 \ararii, D P Cilonelli, I \ , and Dorfman, \ 1 he at id mm opolv sac c handes in normal urine Bwchim Btophys htti Ml ID I I'. 1067 22 Yamamoto. K . and Teravama. H Comparison of t ell c oat at id mucopolv sat eharides of normal liver and van* oils ,isi ues hepatoma cells (.timer Rt\ M 22^-2264, [0^ A CMA 003661 Prevention and Detection of Cancer PARTI. PREVENTION Volume 1. Etiology Edited by Herbert E. Nieburgs Mount Sinai School of Medicine of The City University of New York New York, New York COPYRIGHT 1977 by MARCEL DEKKER, INC. MARCEL DEKKER. INC. New York and Basel CMA 003662 URINARY AND 1 ISSUE GIYCOSAMINOGLYCAN PATTERNS IN HEPATIC ANGIOSARCOMA Charles E. Kupchella and Carlo H. Tamburro Cancer Center and Department of Medicine University of Louisville School of Medicine Louisville, Kentucky 40201 I. INTRODUCTION The recent discovery of a relationship between vinyl chloride and angiosarcoma of the liver has received much attention (1-3). Although there are now systematic detection programs for vinyl chloride workers (3,4), there is as yet no specific chemical abnormality that serves as a good indicator of early, vinyl-chloride-induced liver injury and angio sarcoma. Alpha feto-protein has been a relatively valuable serological marker for hepatocellular carcinoma (5), but is has not as yet proven use ful in the detection of angiosarcoma (6). New leads are needed if more specific tests are to be developed for angiosarcoma. The literature suggests that the glycosaminoglycans in the urine and/or blood should be evaluated as a possible aid in early detection. The production of sulfated glycosaminoglycans is characteristic of malig nant vascular tumors of the skin and some pathologists use this feature as a diagnostic aid (7). Barr and Bonin (8) observed a strong positive alcian-blue, glycosaminoglycan staining reaction in human angiosarcoma tissue and suggested than an attempt be made to qualitate and quantitate the production of glycosaminoglycans in the neoplasms, serum, and urine of those at risk. They pointed out that the urinary glycosaminoglycans may have diagnostic significance in angiosarcoma and, if so, a glycosami noglycan spot test might easily be employed as a gross screening test of vinyl chloride production workers. A number of other observations place the glycosaminoglycans in a relevant position with regard to angiosarcoma. Angiosarcoma is accom panied by connective tissue abnormalities (2,9) and changes in tissue, urinary, and blood glycosaminoglycans have been found to occur in many connective-tissue disorders -- including connective tissue disorders of the liver (10-14) -- as well as in hepatic cancer (15-17), Supported in part by grants from the B. F, Goodrich Company and the American Cancer Society (IN-111) and a contract with the National Cancer Institute (N01-CN-55212). 9 IS CMA 003663 The purpose of this study was to make a preliminary determination of the glycosaminoglycan oatterns in tissue and urine associated with angiosarcoma of the liver and with vinyl-chloride-induced liver injury Other than angiosarcoma and to compare these patterns with those in normal controls and those associated with other liver disease. Our goal was to evaluate the use of glycosaminoglycan patterns in the early detec tion of vinyl-chloride-induced liver injury and angiosarcoma and to explore the role of the glycosaminoglycans in the etiology of vinyl chloride injury. II. PROCEDURES AND MATERIALS USED Urine specimens were collected as occasional samples from: 9 normal controls; 9 individuals with histories of occupational exposure to vinyl chloride and having abnormal, liver, biochemical studies; 6 with "other" cancers prior to surgery; 3 with angiosarcoma; 8 with active viral hepatitis; 6 with cirrhosis; 2 with lung-liver metastases; and 4 with metabolic disorders of the liver (congenital and indirect hyperbiliru binemia) . In one case of angiosarcoma, 24-hr urines were collected on alter nate days beginning 2 weeks prior to death. Urine samples were collected without preservative and frozen at -76 until analysis. Specimens were divided into two 25 ml samples and one 5 ml sample. Urinary creatinine was measured on the 5 ml sample using a Technicon Autoanalyzer. The degree of urinary glycosaminoglycan polymerization was estimated by dialyzing one 25 ml sample for 24 hours in tap water; the sample was then treated identically to an undialyzed sample by the method of DiFerrante (18) using cetylpyridinium chloride as a precipitant. After resolubilization of the glycosaminoglycans in water, duplicate samples were assayed for total uronic acid by the modi fied carbozole reaction of Bitter and Muir (19). The remaining glyco saminoglycans were reprecipitated with cetylpyridinium chloride and separated into the wash, hyaluronic acid, chondroitin sulfate, and heparin fractions as described by Schiller et. al.(20). Each of the fractions was assayed for uronic acid ( pg per mg of creatinine). Autopsy tissue was obtained in 2 cases of hepatic angiosarcoma (tumor tissue and non-tumor tissue adjacent to tumor), 2 cases of cirrhosis, and in 3 control cases (gun-shot wound victims without liver pathology) and analyzed for glycosaminoglycans by a previously reported modification (21) of the method of Schiller et. al. (20). Uronic acid was determined in each of the hyaluronic acid, chondroitin sulfate, and heparin fractions. Pieces of tissue were subjected to alcian-blue-periodie-acidSchiff staining with and without hyaluronidase and diastase pretreatment. These procedures were carried out according to the methods described by Howry (22), Ascitic fluid was also obtained at autopsy in one case of angio sarcoma and analyzed for glycosaminoglycans. The fluid was centrifuged 916 CMA 003664 The purpose o'" s study was to mass a preliminary determination of tne glyccsaminoglycan patterns in tissue and urine associated with angiosarcoma of ore -i-er and with vinyl-cnloriae-induced liver injury otner tnan angiosarcoma and to compare these patterns with those in ' C +"u. coot";,; -it t'csp associate' with otiier < . , c r disease, uu r gcai was to evaluate tne use of glycosammoglycan patterns in the early detec tion of vmyl-cn'oride-induced liver injury and angiosarcoma and to explc'e the role of the glycosaminoglycans in the etiology of vinyl Cnloride injury. I! PROCEDURES AND MATERIALS -JSEO urine specimens were collected as occasional samples from: 9 no--'al contrp't, ) 'rc dua!s wth nistories of occupational exposure to vmyi cnloride and saving aDnormal, liver, bioc.nemical studies; 6 with "other1' cancers prior to surgery, 3 with ang'osarcoma; 3 with active viral hepatitis; 5 with cirrnosis; 2 with lung-liver metastases; and A with metabolic disorders of the liver (congenital and indirect hyperbiliru binemia) . In one tase pf angiosarcoma, 24-hr urines were collected on alter nate days beginning 2 weeks prior to death. Urine samples were collected without preservative and frozen at -76 until analysis. Specimens were divided into two 25 ml samples and one 5 ml sample. Urinary creatinine was measured on the 5 ml sample using a Technicon Autoanalyzer. The degree of urinary glycosaminoglycan polymerization was estimated by dialyzing one 25 ml sample for 24 hours in tap water, toe sample was then treated identically to an undialyzed sample by tne method of DiFerrante (18) using cetylpyridmium chloride as a orecipitant. After resolubi1ization of the glycosaminoglycans in water, duplicate samples were assayed for total uronic acid by the modi fied carbozole reaction of Bitter and Muir (19). The remaining glyco saminoglycans were reprecipitated with cetylpyridinium chloride and separated into the wash, hyaluronic acid, chondroitin sulfate, and heparin fractions as described by Schiller et. al.(2Q). Each of the fractions was assayed for uronic acid ( ug per mg of creatinine). Autopsy tissue was obtained in 2 cases of hepatic angiosarcoma (tumor tissue and non-tumor tissue adjacent to tumor), 2 cases of cirrhosis, and in 3 control cases (gun-shot wound victims without liver pathology) and analyzed for glycosaminoglycans by a previously reported modification (21) of the method of Schiller et. al. (20). Uronic acid was determined in each of the hyaluronic acid, chondroitin sulfate, and heparin fractions. Pieces of tissue were subjected to alcian-blue-periodic-acidSchiff staining with and without hyaluronidase and diastase pretreatment. These procedures were carried out according to the methods described by Mowry (22). Ascitic fluid was also obtained at autopsy in one case of angio sarcoma and analyzed for glycosaminoglycans- The fluid was centrifuged 916 CMA 003665 and the sediment analyzed as tissue above. The supernatant was treated by the method for urine described above. III. RESULTS A summary of the urinary glycosaminoglycan measurement is given in Table I. Normal controls had the least urinary glycosaminoglycans (measured as uronic acid) of all groups. All other groups showed some elevation. The levels in angiosarcoma, hepatitis, cirrhosis, and liver metastases were significantly elevated (P < .05) over normal controls. The cirrhotic group exhibited the greatest variance in urinary glycosami noglycans. No significant differences were found in urinary creatinine levels between groups. There were no significant differences between groups in either the percentage of the total glycosaminoglycans that was dialyzable (Table I) or in the percentage of the unfractionated total that appeared in the hyaluronic acid, chondroitin sulfate, and heparin fractions. Seven of 9 vinyl-chloride-exposed individuals other than those with angiosarcoma had positive chondroitin sulfate fractions with nega tive hyaluronic acid and heparin fractions. This was true in only 3 of 32 other urines evaluated in this same manner. The pattern of daily glycosaminoglycan excretion prior to death due to angiosarcoma in one individual is given in Figure 1. Total tissue glycosaminoglycan levels for angiosarcoma tumors, fibrotic tissue adjacent to tumors, cirrhotic liver tissue and normal liver tissue are shown in Figure 2. Fractional hyaluronic acid, chon droitin sulfate, and heparin levels are given in Figure 3. Histochemically, angiosarcomatous tissue exhibited a strong alcianblue positive staining reaction.' Alcian-blue staining was only slightly less in "non-tumor" tissue adjacent to tumor masses. The staining reaction in tissue from normal liver was very weak and only slightly stronger in cirrhotic liver tissue. The strong alcian-blue reaction in angiosarcomatous tissue did not occur if sections were pretreated with hyaluronidase. Ascitic fluid sediment was uronic-acid-positive in only the hyaluronic acid fraction -- 112 ug uronic acid per gram of dry, defatted sediment; ascitic fluid supernatant contained 1.7, 1.2, and 0.2 /jg uronic acid per ml in the hyaluronic acid, chondroitin sulfate, and heparin fractions, respectively. IV. DISCUSSION The literature indicates that normal male creatinine excretion is 1.5 g per 24 hrs (23). Thus, our normal mean (Table I) of 3;I ; .4 ug cetylpyridinium chloride-precipitable uronic acid per mg creatinine falls in the middle of the normal ranges reported by Varma et. al. (24), 917 CMA 003666 2.6 - 4.7 ,,g/mg; DiFerrante and Rich (25), 2.9 - 4.8 pg/mg; and Kao and Leslie (26), 1.8 - 1.9 ug/mg. Although our study was not controlled for age, Goldberg and Cotlier (27) have shown that urinary glycosaminoglycan excretion is constant from ages 20-70. Manley et. al. (28) have shown that the proportion of urinary glycosaminoglycans in the chondroitin sulfate fraction is constant from ages 20-70. Manley et. al. also reported that the chondroitin sulfate fraction is highest at birth and tnat it gradually drops until age 20. suggesting that urinary chondroitin sulfate reflects tissue growth. The fact that we found no differences between groups in the creatinine concentration is significant in that it indicates that occa sional samples do reflect 24-hour excretion when normalized to creatinine. Precedent for expressing glycosaminoglycan measurements as a function of creatinine content in occasional urine samples has been established by DiFerrante and Rich (25) and Pennock (29). Manley et. al. (28) have shown that the creatinine/uronic acid ratio is steady from ages 20-70. Our results indicate that the liver diseases evaluated are accom panied by elevated urinary glycosaminoglycan excretion. Our tissue data suggests that this reflects liver-tissue glycosaminoglycan elevation and conforms to the reports by others that both hepatic connective tissue disorders (10-14) and hepatic cancer (15) result in increased hepatic glycosaminoglycan levels. It may be significant that the angiosarcoma patients had half the urinary glycosaminoglycan excretion of patients with liver metastases and that our analysis of angiosarcomatous tumor tissue exhibited half the glycosaminoglycan content reported by Kojima et. al. (15) for hepatocellular carcinoma. While our data suggest that liver disease results in a decrease in the proportion of highly polymerized glycosaminoglycans, variance was large within each group and none of the differences between groups were statistically significant. Although we have not completed the characterization of isolated glycosaminoglycan fractions, our data indicate: 1) that the chondroitin sulfates are the primary urinary glycosaminoglycans in both normal controls and in dise^e states; 2) that the chondroitin sulfates and heparin are the dominant glycosaminoglycans in normal and cirrhotic livers (Figure 3). Chondroitin sulfate is elevated in the fibrotic, non tumor, portions of angiosarcomatous livers while heparin is the predomi nant glycosaminoglycan in tumor tissue. Hyaluronic acid is also apparent ly elevated relative to chondroitin sulfate in angiosarcomatous tumors (Figure 3); and 3) that hyaluronic acid is the sole glycosaminoglycan in ascites fluid sediment. These qualitative data are in general agreement with those reported by others. Goldberg and Cotlier (27), Douglas et. al. (30), and Varma et. al. (24) have reported that the chondroitin sulfates are the predominant urinary glycosaminoglycans. Varma et. al. reported that 2/3 of urinary glycosaminoglycans are chondroitin-4-and chondroitin-6-sulfate and this agrees with our data on normal controls and on those with liver disease. Kojima et. al. (15) reported that in hepatocellular carcinoma y is CMA 003667 ;3J, Iceberg a-.d Cotlier . Z7 nave snown t'at jrrj'/ 3'/css smi -a z'.ycan excretion is constant from aces 20-70. '-'an 1 Jy a"., a .-3! n5: Z-Cwr :i]: Z:"e orzzort' zr z " .irirarv glycosammoglycarc n tie or zrcro::: r, i^-^te -Yjtt'bn is constant -rcm "''-action 15 nignest at airtn ana tnat .: gradually crops ur.ui age 20, suggesting that jcnaty znondroitin Sonata reflects tissue growth. The ''act that we -"o.,no -o oi "e-erces between grouts ;n tne creatinine concentration is significant ir. gnat it indicates tnat occa sional samples do '?~'?cz 24-'gur excretizr wren normalized to creatinine, Precedent `or expressing 17.1 -.5 ami nog 'yea" measurements as a funct'on of creatinine content m tctas'tral :a*:'e; nas oeen estao'isne: by cur -as.its 'ricr.; '.rat t- = 11 .y c-sease; evaijated are accom panied oy e'evated urinary gl/ccs am'nog' yea'1 excreren. Gur t'SS-e data suggests that this '-e'lects 1 ive^-ti ssue glycosaminoglycan elevation and conforms to tne reports oy others that both oeoati: connective tissue a'seders (12-14) irz `, = tat'i isnes'" 1S 'es.ji t ir - r;'ea se: -etatic g 1/Cosami nool yean 'eve's 1: -jv be s - or -'ican; that tre ang'osarcoma catients rad naif :re 1 ' yc 0 s im nog 1 <r an exfet'on of catierts witn liver metastases and tnat our i^ai/sis of ang;osarccmatons tumor tissue exmii ted naif tno gi ycosami nog i yca.n content reoorted by Kojima et. ai. (15) for nepatocei1j'ar carcinoma. While our data suggest that liver disease results in a decrease in the proportion of highly polymerized g1ycosaminoglyeans, variance was large witnin each group and none of the differences oetween groups were statistically significant. Altnough we have not completed the characterization of isolated glycosaminoglycan fractions, our data indicate: 1) tnat tne cnondroitin Sulfates are the primary urinary glycosaminoglycans in both normal controls and in disease states, 2) that the chondroitin sulfates and heparin are the dominant glycosaminoglycans in normal and cirrhotic livers (Figure 3). Chondroitin sulfate is elevated in the fibrotic, non tumor, portions of angiosarcomatGus livers while heparin is the predomi nant glycosaminoglycan in tumor tissue. Hyaluronic acid is also apparent ly elevated relative to chondroitin sulfate in angiosarcomatous tumors (Figure 3); and 3) that hyaluronic acid is the sole glycosaminoglycan in ascites fluid sediment. These qualitative data are in general agreement with those reported by others. Goldberg and Cotlier (27), Douglas et. al. (30), and Varma et. al. (24) have reported that the chondroitin sulfates are the predominant urinary glycosaminoglycans. Varma et. al. reported that 2/3 of urinary glycosaminoglycans are chondroitin-4-and chondroitin-6-sulfate and this agrees with our data on normal controls and on those with liver disease. Kojima et. al. (15) reported that in hepatocellular carcinoma j LS CMA 003668 tissue, chondroitin sulfates and hyaluronic acid were increased 33 and 10 times, respectively, over amounts found in healthy livers; the heparin and heparan sulfate proportions dropped. This contrasts with our data on angiosarcoma tissue, i.e. heparin and hyaluronic acid increased 5 and 10 times, respectively, over normal tissue, cnondroitin sulfate levels rose but fell in proportion to Other glycosaminoglycans, Galambos and Shapira (10) reported that the chondroitin sulfates are dominant in normal livers and in hepatic fibrosis, but Kojima et. al. (15) report that chondroitinase-resistant and hyaluronidase-resistant glycosaminoglycans are dominant. Kuroda et. al. (31) also reported that heparan sulfate is the dominant glycosaminoglycan in the normal liver. Our histochemica1 observation that nearly all of tne increased alcian-blue positive material in angiosarcomatous livers was susceptible to hya1uronidase digestion suggests that the observed chondroitin sulfate elevation is due to chondroitin-4- and/or chondroitin-6-sulfate. The increases in liver and urinary glycosaminoglycans may well reflect an important role of these substances in the process of fibrogenesis and in tumor growth. Galambos and Shapira (10) reported that hyaluronic acid was elevated during hepatic fibrogenesis. If a similar fibrotic process is operative in angiosarcoma, it may be that the ob served tumor-tissue heparin increase is reflective of tumor growth. We did observe a four- to six-fold greater heparin level in tumor tissue than in adjacent, non-tumor tissue. The observation that the chondroitin sulfates tend to be the exclusive uronic-acid-positive constituents in the urine of individuals is paradoxical in that those glycosaminoglycan fractions that are most elevated in angiosarcomatous tissue are those that are absent from the urine of individuals who may well have early, vinyl-chloride-induced liver injury. This pattern may be due to the selective action of lyso somal , glycolytic enzymes in the liver and/or may reflect the role of the Chondroitin sulfates in early fibrotic changes in the liver. Certainly the potential usefulness of this pattern in early detection warrants the more complete evaluation now ongoing in our laboratory. V. SUMMARY Glycosaminoglycans were measured in urine and tissue of patients with hepatic fibrosis and hepatic cancer including vinyl-chlorideexposure-associated liver injury and angiosarcoma. Angiosarcoma, hepa titis, cirrhosis, and liver metastatic patients exhibited significantly elevated glycosaminoglycan excretion. Angiosarcoma tissue exhibited elevated glycosaminoglycan levels with the greatest increases in the heparin fraction. Histochemically, angiosarcomatous tissue gave a strong alcian-blue staining reaction which could be prevented by pretreatment with hyaluronidase. Although vinyl-chloride-exposure-associated liver injury other than angiosarcoma was not accompanied by a significantly elevated glycosaminoglycan excretion, this condition tended to be associated with a urinary glycosaminoglycan excretion pattern in which the chondroitin sulfate fraction was the only uronic-acid-positive fraction. <J 19 CMA 003669 TABLE I. Urinary Glycosaminoglycan levels in yg Uronic Acid per mg Creatinine by Liver Diseases Category Patient qroup normal control vinyl chloride exposed Cases 9 9 ug uronic acid per mg creatinine (* 1 S.E.) 3.2 * .4 4.1 .4 % uronic acid not dialyzable ( 1 S.E.) 65 * 5 39 t 6 other cancer 6 4.5 1.5 39 6 other liver disease 4 5.1 t 0.8 37 i 13 angiosarcoma hepatitis 3 a 7.6 1.6 8.5 - 1.8 41 t 22 52 t 14 cirrhosis 6 12.7 - 3 53 i 9 1iver metastasis 2 13.8 i .9 42 920 CMA 003670 car La .el a m .g Jrzr.-c nC i j L*af* CreiCl n i ne oy Live1- Cise53S dti-C-y Patient qrouD hc-ciai ccnt'o! v'nyl chloride exposea Cases 9 } jg uranic acid per mg creatinine (; I S.E.) 3.3 * 4,1 - .4 % uronic acid not dialyzable ( 1 S.E.) 55 * 5 39 5 other cancer other liver disease 6 4 4.5 - 1.5 5.1 i 0.8 39 i 5 37 i 13 angiosarcoma 3 7.6 - 1.6 41 4 22 in hepatitis 8 6.5 * 1.8 cirrhosis 6 12.7 t 3 53 4 9 1 iver metastasis 2 13.3 i .9 42 920 CM^- 003671 oL DAYS PRIOR TO LIVER DEATH FIG. I. Urinary glycosaminglycan output in one angiosarcoma patient dming the 16-day period prior to death. 921 CMA 003672 1200 r u \o NMJ n o o OJ u> FIG. 2. Glycosaminoglycan concentration in angiosarcomatous, cirrhotic and normal human liver tissue. 1200 - u Z o a. *>s KJ n Tumor Cot* i N.on-lumof Ad|0ccnl Cqt* I Tumor Cat* 2 Non-tumor Adjacent Cot* 2 Cot* i t\>i* 4 Cot* *> Cote t> Flti. 2. Giycosaminog lycan concentration in angLusarcomatous, cirrhotic and noraal human liver tissue. Cat* ? 003674 GLYCOSAMINOGLYCAN c o n c e n t r a t io n IN MICROGRAMS URONIC ACID PER GRAM DRY DEFATTED LIVER TISSUE/ 600 r soo Hyaluronic Aod " Chondroilm Sulfate " n o o OJ cn Tumor Cos# 1 ANGIOSARCOMA------------------------- ------------------ CIRRHOSIS Non - lumor Adjoctnt T umor Non * lumof Adjocenl Com 1 Com 2 Com 2 Com 3 Cote 4 NORMAL Cos* 5 Cost 6 . 3. Fractional concentrations of glycosaninoglycans in angiosarcomatoust cirrhotic and normal human liver tissue. VI. REFERENCES 1. Creech, J. L. and Johnson, M, N. Angiosarcoma of the Liver in the Manufacture of Polyvinyl Chloride. J. Occup. Med. 16: 150-151, 1974. -- 2. Falk, H., Creech, J. L., Heath, D. W., Johnson. M. N., and Key, M. M. Hepatic Disease Among Workers at a Vinyl Chloride Polymerization Plant. JAMA 230: 59-63, 1974. 3. Makk, L., Creech, J. L., Whelan, J. G., and Johnson, M. N. liver Damage and Angiosarcoma in Vinyl Chloride Workers: A Systematic Detection Program. JAMA 230: 64-68, 1974. 4. Creech, J. L,, Makk, L,, Whelan, J. and Tamburro, C. H, Hepatotoxicity Among Polyvinyl Chloride Production Workers During First Year of Surveillance Program. Gastroenterology 67: 786, 1974. 5. Kohn, J. and Weaver, P. C. Serum Alpha Fetoprotein in Hepatocellular Carcinoma. Lancet 2; 334-336, 1974. 6. Tamburro, C. H., Makk, L. and Creech, J. L. Unpublished observation. 7. Girard, D., Johnston, W. C., and Grahm, J. H. Cutaneous Angiosar coma. Cancer 25: 868-883, 1970. 8. Barr, R. and Bonin, M. "Letters." JAMA 231(9): 914, 1975. 9. Popper, H., and Thomas, L. 8. Alterations of Liver and Spleen Among Workers Exposed to Vinyl Chloride. Ann. NY Acad. Sci. 246: 172-194, 1975. 10. Galambos, J. T., and Shapira, R. Natural History of Hepatitis: IV Glycosaminoglycuronans and Collagen in the Hepatic Connective Tissue. J. Clin. Invest. 52(11): 2952-2962, 1973. 11. Koizumi, T., Nakamura, N., and Abe, H. Changes in Acid Mucopoly saccharide in the Liver in Hepatic Fibrosis. Biochim. Biophys. Acta. U8: 749-756, 1967. 12. Kojima, J. Studies on the Metabolism of Hepatic Connective Tissue in Fibrosis of the Liver. Med, J. Osaka Univ. 1: 419-429, 1964. 13. Rubin, . Autoradiographic Characterization of Sulfated Acid Mucopolysaccharides in Experimental Cirrhosis. J. Histochem. Cytochem. 1: 688-689, 1966. 14. Patrick. R. S. and Kennedy, J. S. The Synthesis of Sulfated Mucopolysaccharide at Sites of Hepatic Fibrosis Induced by Carbon Tetrachloride, Amyloidosis, and the Implantation of Catgut. J. Pathol. Bacterid. 88; 549-555, 1964. 15. Kojima, J., Nakamura, N., Kanatani, M, and Ohmori, K. The Glycosaminoglycans in Human Hepatic Cancer. Cancer Res. 35(3): 542-547, 1975. 92a CMA 003676 1. Creech, J. L. and Jonnscn, M, N. Angiosarcoma of Che Liver fn Che Manufacture of 3o1yvin/l Ch'rr^de. J. Cocoa, "''ed '6- ,cC-!5i L974. -- 2. FaN, H., C<e=cn, J. 'eatn, D. Jonnson, M. is., anc Key, M. M. Hepatic Disease Among Workers at a Vinyl Chloride Polymerization Plant, JAMA 230: 53-53, 1971. 3. Makk, l., Creech, J, L., nhe'an, J, 5., and Jonnson, M. S. Liver Damage and Angiosarcoma in Vinyl Chloride Workers: A Systematic Detection Program, JAMA 230: 1-53, 1971. I. Creed, J, l., `'akk. L. , '..-e'an, i-g C. - -etstttsx'- cty tr.org Poiyvnyl Cr'cr-te 2rodction Wervars Dori-g rirst fear or" Surveillance 3rcgram. Gast'oenterology 5_7: ;'SS, 1971. 5. Konn, J. and Weaver, P. C. Serum Alpha Fetoprotein in Hepatocel1ular 'Carcinoma. Lancet 2: 331-336, 1971. 5. Tamburro, C. H., s'akk, L. and Creech, J. 1. Unpublished observation, 7. Girard, 3., Johnston, W. C., ard jra.nm, J. M. Cutaneous Angiosar coma. Cancer 2S_: 358-333, 1370. a, 3arr, R. and Sonin, M. "Letters." JAM 231(9): 914, 1975. 9. Popper, H., and Thomas, L. 3. Alterations of liver and Spleen Among Workers Exposed to Vinyl Chloride. Ann. NY Acad. Sci. 246: 172-194, 1975. 10. Gaiambos, J. T. , and Shapira, S. .Natural History of Hepatitis: IV Giycosamlnoglycuronans and Collagen in the Hepatic Connective Tissue. J. Clin. Invest. 52( 11): 2952-2962, 1973. II. Koizumi, T,, Nakamura, N., and Abe, H. Changes in Acid Mucopoly saccharide in the Liver in Hepatic Fibrosis. Siochim. Biophys. Acta. 148: 749-7SS, 1967. 12. Kojima, 0. Studies on the Metabolism of Hepatic Connective Tissue in Fibrosis of the liver. Med. J. Osaka Univ. 16_: 419-429, 1964. 13. Rubin, . Autoradiographic Characterization of Sulfated Acid Mucopolysaccharides in Experimental Cirrhosis, J. Histochem. Cytochem. 14: 688-689, 1966. 14. Patrick. R. S. and Kennedy, J. S. The Synthesis of Sulfated Mucopolysaccharide at Sites of Hepatic Fibrosis Induced by Carbon Tetrachloride, Amyloidosis, and the Implantation of Catgut. J. Pathol. Bactenol. 38: 549-555, 1964. 15. Kojima, J., Nakamura, N., Kanatani, M. and Ohmori, K. The Glycosaminoglycans in Human Hepatic Cancer. Cancer Res. 35(3): 542-547, 1975. 924 CMA 003677 16. Anghileri, L. J. Metabolism of Acid Mucopolysaccharides in Hepatoma and in Normal Liver. Oncology 30: 304-317, 1974. 17. Yamamoto, K.. and Teryama, H, Comparison of Cell Coat Acid Muco polysaccharides of Normal Liver and Various Ascites Hepatoma Cells. Cancer Res. 33: 2257-2264, 1973. 18. OiFerrante, N. M. The Measurement of Urinary Mucopolysaccharides. Anal. Biochem. 2k 98-106, 1967, 19. Bitter, T., and Muir, H. A Modified Uronic Acid Carbazole Reaction. Anal. Biochem. 4: 330-334, 1962. 20. Schiller, S., Slover, G. A., and Dorfman, A. A Method for the Separation of Acid Mucopolysaccharides: Its Application to the Isolation of Heparin from the Skin of Rats. J. Biol. Chem. 236(4): 983-987, 1961. 21. Kupchella, C., and Steggerda, F. The Distribution of Acid Muco polysaccharides in the Canine Gastrointestinal Mucosa. Trans. NY Acad. Sci. 34: 351-360, 1971. 22. Mowry, R. W. Alcian Blue Techniques for the Histochemical Study of Acidic Carbohydrates. J. Histochem. and Cytochem, : 407, 1956. 23. Sunderman, F, W, and Boerner, F. Normal Values in Clinical Medicine. W. B. Saunders. Philadelphia, p. 353, 1949. 24. Varma, R. S., Varma, R., Allen, W. S., and Wardi, A. H. Urinary Excretion of Acid Mucopolysaccharides in Schizophrenia. Biochem. Med. 11(4): 358-369, 1974. 25. DiFerrante, N, and Rich, C. The Determination of Acid Aminopolysaccharide in Urine. J. Lab. Clin. Med. 48: 491-494, 1956. 26. Kao, K. and Leslie J. Micro Fractionation and Determination of Urinary Glycosaminoglycans. Biochem. Med. 9(4): 317-326, 1974. 27. Goldberg, J. and Cotlier, E. Specific Isolation and Analysis of Mucopolysaccharides (Glycosaminoglycans) from Human Urine. Clin. Chim. Acta. 41: 19-27, 1972. 28. Manley, G., Severn, M. and Hawksworth, J. Excretion Patterns of Glycosaminoglycans and Glycoproteins in Normal Human Urine. J. Clin. Pathol. 21: 339-34S, 196B. 29. Pennock, C. A, A Modified Screening Test for Glycosaminoglycan Excretion. J. Clin. Path. 22: 310, 1969. 925 CMA 003678 30. Douglas, C., Nowak, J. and Danes, B. Mucopolysaccharides in Urine During Normal Human Development. Pediatr. Res. 7: 724-727, 1973, 31. Kuroda, J., Saito, S., Seno. N.. Nagase, S. , and Anno, K. Isolation and Chemical Characterization of Mucopolysaccharides from Rat Tumors. Cancer Res. 34(2): 308-312, 1974. 926 CMA 003679 C. E. Kupchella et al. Tabi* 1 Charectenstics of the tranaotantabfe hepatomas studied Tumor line Growth rate designation Time (days) until tumor reached 3cm4ong axis Histology Metastatic General metastatic char potential4 acteristic observed here Coltagan4 7777 Fast 3123tc Intermediate 96 ISA Slow 18 Poorly differen tiated 35 Moderately dif ferentiated 89-09 Well differen tiated Scattered lung micro- + metastases but no gross metastases ev ident at sacrifice + + Multiple large metasta ses to lungs grossly 0 evident in lungs of all animals at sacrifice 0 No lung metastases ev 0 ident by sampling at sacrifice 4 According to tha data of Hruban er at 0 4) Anion-Exchange Chromatography. Following uronic acid measurement, the uronic acid-positive material was pooled by tumor line with tumor and liver tissue material pooled sepa rately. The cetylpyridinium chloride was removed as described by Korn (17), and then each fraction was dialyzed and sub jected to Dowex 1-X2 (200 to 400 mesh) anion-exchange chromatography as described by Schiller et al. (28). The 0.0, 0.5, 1.0.1.25, 1.5, and 2.0 m NaCI eluate fractions were eluted stepwise in 15 to 30 fractions (10 ml) each. One-ml samples were assayed for uronic acid to produce an elution profile. Chromatographic fractions were pooled across all tissue groups, dialyzed, and concentrated, yielding 6 pooled fractions which were then subjected to enzymatic characterization. Enzymatic Characterization. Each of the fractions were subjected to digestion by Streptomyces hyaluronidase (digests only hyaluronic acid), bovine testicular hyaluronidase (digests hyaluronic acid, chondroitin, and chondroitin sulfate but not heparin, dermatan sulfate, or heparan sulfate), and chondroitinase ABC (digests hyaluronic acid, chondroitin, chondroitin sulfate, and derma sulfate but not heparin or heparan sulfate) as described by Kojima etal. (16). Histochemistry. Small pieces of liver and tumor as well as lung and intestine in selected animals were fixed in Zenker's fluid, embedded in paraffin, cut at 6 pm, and subjected to hematoxylin and eosin, Alcian blue-periodic acid-Schiff (21), and Masson's trichrome (21) staining. Alcian blue-periodic acid-Schiff staining was also carried out with and without prior digestion in chondroitinase ABC and bovine testicular hyalu ronidase (9). For the condroitinase ABC study, hydrated tissue sections were incubated with enzyme (16) for 2 hr at 37; control sections were incubated in buffer only. Statistical Analysis. Statistical analysis of tissue GAG data was carried out in a sequential format starting with an analysis of variance. Significant differences among the means were further evaluated using the Newman-Keuls procedure de scribed by Snedecor and Cochran (29). Uronic acid levels in the 0.04 m NaCI fraction isolated from the 3 tumors were similar and significantly higher (p < 0.05) than the levels found in normal liver. In the 1.2 m NaCI fraction, tumors exhibited 5- to 6-fold greater (p < 0.05) uronic acid levels than normal liver. In the 2.1 m NaCI fraction, uronic acid levels were similar for all tissues except that Tumor 9618A had significantly higher (p < 0.05) levels than did Tumor 5l23tc. There was a statistically significantly greater (p < 0.05) level of uronic acid in the 0.03 m NaCI fraction for the livers of animals bearing Tumor 5123tc compared to normal liver and the livers of animals bearing the other 2 lines. Except for this, differences among livers were unremarkable. 1000 RESULTS The amounts of GAG in Tumors 7777, 5123tc, and 9618A and in normal liver are presented by fractions in Chart 1. "Uronic acid" levels in the 0.03 m NaCI fraction were similar in Tumors 7777 and 5123tc but were significantly (p < 0.05) lower than the levels found in tumor 9618A and in normal liver. 420 0 03M Nad 04*4 NoCi t'ZMNaCi ZiMNaG Fraction in --Uteri cntytpynOinium efttood* comoN* n uiubta Chant 1. QAQ iavaia measured an uromc acid in laai-growtng (F) Tumor 7777. intarmadiata-rata </) Tumor 51231c. and atam-qmiiAnq (5) Tumor M18A and m tha Kvara of non-tumor-Ooartng (40 antmaia tor aach ot 4 laauannaity csdactad aatt tracttona. Sara, gaomatrtc maana. OMfarancaa Batwaan Fit and S/N in ma 0.03 m NaCI traction, baturnon tumor ttaaua and normal Avar ki both The 0.4 u and 1.2 u NaCI traction, and batwaan / and S ai tha 2.1 u NaCI fraction ara atatudcaay aiflnihcant (P < 0.05). CANCER RESEARCH VOL. 41 CMA 003681 Chromatographic data for the 0.4 m NaCI and 1.2 m NaCI GAG fractions, those fractions which were appreciably larger in tumor tissue versus normal liver, are presented in Chart 2 together with the patterns obtained for these same fractions isolated from normal liver and the liver of tumor-bearing ani mals These data indicate that, even though the uranic levels in the initial fractions were similar from tumor line to tumor line, there were some qualitative GAG differences in the fractions isolated from different tumor lines. This is even more apparent in composite Chart 3, which was derived by multiplying the mean of the individual uronic acid levels shown in Chart 1 by the percentage of distribution shown in Chart 2 and then summing within each chromatographic fraction. Enzymatic Characterization of Tumor GAG'S The results of the enzymatic characterization of GAG's iso lated from tumor tissue are summarized in Table 2. On the basis of the elution patterns reported by Kao and Leslie (15) and corroborated in our own study of authentic GAG's and on the enzyme susceptibilities for each fraction given in Table 2, we arrived at the identities of the predominant GAG in each chromatographic fraction given in Table 2. Column 7. Histochemical Observations 1000 too o0 p t0ov 100 o co 3 a 100 100 GAG .5 in Morns Hepatomas 0 Tumors. Each of the 3 tumor types exhibited significantly more intense Alcian blue staining than did normal liver or host liver; GAG's were generally distributed throughout the tumor tissue. Tumor sections subjected to hyaluronidase or to chon- I 0 4M NaCl*Olubl material 1.2M NaCtsoiuble material H20 0$M i 0M l 25M 1 5M 20M Amon exchange column NaCI friction* Chart 3. Anion-exchange chromatographic pattern# obtained lor the GAG* isolated initially #s 0.4 m NaGI-eotubie and 1.2 m NCl-#otuble cetytoyridinium Chloride complexes. A, Tumor 7777; 8, Tumor 5l23tc; C. Tumor 9618A; O. livers of tumor-bearing animals pooled across tumor types; , normal liivveqr. Tiihis chart Is a composite of Charts 1 and 2 and is baaed on the percentagjeessj^given in Chart 2) of the arithmetic mean of the individual uronic acid values { Chart 1) for each tissue type by fraction. II lil I 1.. B - II c 1 | 0 -ll _____________ .1. H20 0$M 1 0M H20 0.5M 1.0M 1 25M 1.5M 2QM Amon exchange column NaCI fraction* Chart 2. Antoft-axehanga chromatographic profiles for the GAG-cetyipyndm* ium chloride complex## solubilized initially in 0.4 m and 1.2 m NaCI. A, Tumor 7777; B, Tumor 51231C; C. Tumor 9618A; D, pooled liver tissue from tumor* bearing animals; E, normal liver. Recoveries ranged from 65 to 107%. droitinase ABC exhibited reduced levels of Alcian blue staining consistent with biochemical measurements and the enzymatic characterization of chemically isolated fractions. Host Livers. The livers of animals bearing Tumors 7777 and 9618A were histologically indistinguishable from normal liver. Liver tissue from animals bearing Tumor 5123tc consistently exhibited a slightly greater vacuolar appearance than did nor mal liver (Fig. 1B). Urinary GAG Excretion Over the entire study, mean total uronic acid excreted per pair of animals per 24 hr for control animals and animals bearing Tumors 7777, 5123tc. and 9618A were 118 7 (S.E.). 116 3, 203 12, and 201 7 >ig, respectively, GAG excretion by animals bearing Tumors 5123tc and 9618A were statistically significantly (p < 0.05) elevated over controls and animals bearing Tumor 7777. There were 22, 8, 8, and 27 twenty-four-hr collections assayed, respectively. The small number of 7777 and 5l23tc samples was due to the rapid growth of these tumors and time in transit after inoculation. Urinary excretion profiles for animals bearing Tumor 5123tc over time and in relation to tumor size are illustrated in Chart 4. A similar pattern over a longer time span was observed in animals bearing Tumor 9618A. In both 5123tc and 961 BAbearing animals, uronic acid excretion appeared to be greater after the tumors reached larger sizes, but no regression with tumor size was apparent in either case. FEBRUARY 1981 CMA 003682 421 Chromatpcrspnic data `cr ;he 0.- m SaC- arc : 2 m .\aC; G-G `'ac:cr3. '.hpzs !rac;.c-.s .>r;ch vers scp.-eccpiy arger in tumor tissue versus normal liver, are creseoteo in Cbarf 2 `oget'-er :.n :re pzcir.-s o2;amso -or r.ssa same mac:.cos so'aiT'; os'-r ,..ar sno :ne .:" :..rcr-:ear.rg a.-,`- ^3 = T-jzs ~2:a no cats that, eve- though ~ *"t " * 1 *'j. `` z~ z 5`iT> 1 nr "">? ,,c = .-y.s in$. there //ere some Qualitative GAG differences ;n the fractions isolated from different tumor lines. This is even more apparent in composite Chart 3, which was derived by multiplying the mean of the individual uronic acid levels shown in Chart 1 by the percentage of distribution shewn in Chart 2 and th9ri summing within each chromatographic fraction. Enzymatic Characterization of Tumor GAG's l A The results of the enzymatic characterization of GAG's iso lated from tumor tissue are summarized in Table 2. On the basis of the elution patterns reported by Kao and Leslie (15) and corroborated in our own study of authentic GAG's a^d on tne enzyme susceptibilities for each fraction given in Tab.'s 2, we arrived at the identities of the predominant GAG in each cnromatographic fraction given in Table 2. Column 7. Histochemieal Observations Tumors. Each of the 3 tumor types exhibited significantly more intense Alcian blue staining than did normal liver or host liver; GAG's were generally distributed throughout the tumor tissue. Tumor sections subjected to hyaluronidase or to chon- OAM NCI40iu0< 50 0 '.ZM NaCtsolg&to mat*n*l A 3 A 'O bB .Q 3 90 05 0 I. IB D E HjO OSW 1 OM HjO 0.3M 1.0M I ZSM ISM Z.QM Anion *xchng column MaCI fractiona Owl 7- AnlenwxstMnga enromatograoMc proflla* lor tf GAG-carylpynOwv aim cMonOa compiaaaa aoluoauoo vwtiaav IflOlw and 1 2 u NaCI. A. Tumor 7777: 8. Tumor SIZQtc: C. Timor MtSA: 0. ooolad livar ttaaua from tumor' baanog ammaM; E. normal Mvor. Racowma rangad earn SS to t07%. Chon 3 Anio/nxehanga cnfamatogratmlc oaitama otitainad h)r tha GAG i laolaiao mmauy n 0.x u NaCl-wluoio and > 2 u NaCt-aoiuOia catyioy/idifmun chionda comoiaiaa. A, Tumor 7777: S. Tumor Si23ic: C. Tumor SSiaA. 0. ilvara ot tumor-oaartne ammata poolad acroaa tumor rypaa; E. normal bvar. Thia chart la a comooaita of Charta 1 and 2 and ia baaad on tha parcantagaa (gtvan In Chart 2) of tha anthmaoc mean of tha ndtvidual uromc acid valuaa (given in Chart 1J for aach tiaaua typo Oy fraction. droitinase ABC exhibited reduced levels of Alcian blue staining consistent with biochemical measurements and the enzymatic characterization of chemically isolated fractions. Host Livers. The livers of animals bearing Tumors 7777 and 9618A were histologically indistinguishable from normal liver. Liver tissue from animals bearing Tumor 5123tc consistently exhibited a slightly greater vacuolar appearance than did nor mal liver (Fig. 10). Urinary GAG Excretion Over the entire study, mean total uronic acid excreted per pair of animats per 24 hr for control animals and animals bearing Tumors 7777, 5123tc, and 9618A were 118 7 (S.E.), 116 3, 203 12, and 201 7 /ig, respectively. GAG excretion by animals bearing Tumors 5123tc and 9618A were statistically significantly (p < 0.0S) elevated over controls and animals bearing Tumor 7777. There were 22. 8, 6, and 27 twenty-four-hr collections assayed, respectively. The small number of 7777 and 5l23tc samples was due to the rapid growth of these tumors and time in transit after inoculation. Urinary excretion profiles for animals bearing Tumor 5123tc over time and in relation to tumor size are illustrated in Chart 4. A similar pattern over a longer time span was observed in animals bearing Tumor 9616A. in both 5l23tc and 9618Abearing animals, uronic acid excretion appeared to be greater after the tumors reached larger sizes, but no regression with tumor size was apparent in either case. FEBRUARY 1981 CMA 003683 421 C. E KupcheHa et at. Table 2 identification Qt the prvdortunant GAG m each of Ouramon-exchange chrometograp/uc fractions The identification (Column 7) of the predominant GAG or GAG $ oresent m each of our anion-exchange chromatographic fractions (Column 1) was deduced from (a) solubilities of cetylpyridmium chloride complexes, (b) anion-exchange chromatographic patterns compared to those that we obtained for authentic GAG s and those reported by Kao and Leslie (15) (Columns 2 and 3). and <c) the susceptibility of each fraction to mucopoiysacchandases (Columns 4. 5, and 6> Dowex t -X2 traction (m NaCt) GAG s reported By Kao and Leslie {15) to Dp primarily eluted in this fraction Other GAG s reported to be partially eluted in this fraction digested by Str*ptomyce$ hyaiuronidase % digested by bovine tesnrular hyaluroni- dase ^digested by chondroiti- na$e ABC Predominant GAG m Our fraction 00 05 1.0 1,25 15 2,0 Hyaluronic acid (84)* Heparan sulfate (76) Chondroitin 4-sutfate ' (66) Chondroitin 6-sul<ate (63) Heparin (72) None Hyaluronic acid (12) Heparan sulfate < M) Heparin (18) Dermatan sulfate (19) Chondroitin 6-Sulfate 09) Dermatan sutfate (67) Chondroitan 4-sul fate 03) 70-100 35-61 0 0 30 0 80-100 61 0 <2 25 100 ? 0 0 Hyaluronic ac*d Hyaluronic acid Heparan sulfate Heparan sulfate 22 Heparan sulfate and/or heparin Heparin 0 * Numbers in parentheses, percentage eluted per fraction. A Chart A. Urinary GAG excretion in relation to tumor growth for antmaia bearing Tumor 5l23tc and in control animals. . tumor*beanng animals; O. control animals. We had sufficient urinary GAG'S for anion-exchange chro matography only in the case of control animals and animals bearing Tumor 9618A. A chromatographic comparison of these 2 profiles indicated that the elevation in urinary GAG's occurred across all column fractions to about the same degree. DISCUSSION The results depicted in Charts 1 to 3 conform to the gener alization (7, 16, 18) that tumors, including hepatic tumors, exhibit high levels of GAG's relative to the tissue of origin. Charts 2 and 3 suggest that differences between tumors may be related to the behavioral properties of the tumors. Chart 3 reveals a gradation from normal liver, through the liver of tumorbearing animals, well-differentiated, slowly growing tumor tis sue to faster-growing, metastatic tumor lines. The gradation shown in Chart 3 is even more striking if the patterns for tumor lines 7777 and 5123tc are inverted. Since tumor line 5123tc was more highly metastic than was line 7777 (Table 1), such an inversion would arrange the tissues according to metastatic potential and raises the possibility that the patterns, particularly Fraction 1.0 m. are related in some way to metastatic potential. It is postulated that the 1.0m NaCI chromatographic fraction contains an undersuifated form of heparan sulfate such as that described by Kuroda el a/. (18) in AH109 hepatomas. Both Kuroda et at. (18) and Saito (25) reported that heparan sulfate is the major GAG constitutent in AH109A hepatic tumors. Kuroda et at. also reported that most of this heparan sulfate is eluted in 1.0 m NaCI in anion-exchange chromatography and that heparan sulfate is also the predominant GAG in normal liver and suggested that this may mean that the tumor heparan sulfate comes from tumor cells and not from connective tissue elements within the tumors. The predominance of heparan sulfate in Morris hepatomas and in AH109A hepatomas do not conform with the fact that hyaluronic acid and chondroitin sulfate have generally been identified as the predominant GAG's in animal tumors (a, 7). These findings likewise do not conform with the report by Kojima et el. (16) that chondroitin sutfate and hyaluronic acid are the predominant GAG's in human hepatocellular cancer. The possibility that tumor heparan sulfate is related in some direct way to tumor behavior has been raised by others. A role for sulfated GAG's in cell recognition and adhesion has been proposed by Dietrich et at. (8), and Chiarugi and Vannucchi (3) have proposed that cell surface heparan sulfate regulates both cell division and transport. The histological appearance of the tumors studied here and the histological normalcy of the livers of tumor-bearing animals agree with the findings reported by Hruban et at. (14). Although the vacuolar appearance of the livers of animals bearing Tumor 5123tc is not abnormal in rodents, this characteristic was uniformly present in all Tumor 5123tc-bearing animals and was found in no others. The possibility exists that this structural feature is related to the high concentrations of the uncharac- 422 CMA 003684 CANCER RESEARCH VOL. 41 GAG s in Morns Hepatomas terized, 0.03 m NaCl-soluble, uronic acid-positive-matenal found m these livers. 8 Oietnch. C P . Samoaio, L O . Toledo, O M 5 , and Cassaro, C m F recognition and adhesiveness' a possible biological rote tor me mucopolysaccharides Biochem Biophys. Res. Commun . 75 329^^^B The fact that urinary GAG excretion was elevated in animals 1977. bearing Tumors 5123tc and 961SA but not in animals bearing Tumor 7777 suggests that urinary GAG excretion may reflect 9 Drury, R A 0,. and Wallington. Carleton s Histological Technique, pp 210-219 New York Oxford University Press. 1967 10 Friman C and Juvam, M Urinary excretion and glycosaminoglycans in properties of certain tumors and is not simply an indirect resull of the presence of tumor Overall levels of excretion of GAG s in our control and experimental animals agree with levels Scand l rj- l 22 1975malignant diseases of the fmemopoiotic and lympnatic tissues Acra Med i I Gordm, A , Edgren, J Fnman. C and Holmstrom, T A case of disseminated hemagiomatosis with cutaneous. hepatic and skeletal manifestations and reported for rats by Lehtonen et al. (19). It remains to be determined what the source(s) of the elevated urinary GAG is (are). There have been reports of striking increased urinary excretion of glycosaminoglycans, Acta Med Scand 196 525-530.1975 12 Hatae. Y . Yoda, Y . and Makita. A. Glycosaminoglycans in small cell carcinoma of human lung histologically characteristic pattern. Gann. 70' increases in GAG synthesis in tumor cells (see Ref. 20). but Kojima et al. (16) have suggested that decreased degradation may account for increased GAG in some tumors. Our investi 389*390. J 979 13 Hruban. Z-. Mochizuki, Y. Slesers. A., and Morns. H. P A comparative study of cellular organelles Of Morns hepatomas Cancer Res.. 32 853867. 1972. gations provide no evidence that the source of the abnormal GAG increases in tumor tissue and urine is anything other than the tumor cells themselves: however, a possibility that these results stem from an impaired ability of the liver to degrade circulating GAG's cannot be ruled out. 14 Hruban, Z . Morris. H P , Mochizuki. Y,, Meranze, D. R , and SJesers. A Light microscopic observations of Morns hepatomas. Cancer Res. 31 752762.1971. 15. Kao. K Y. T . and Leslie, J G. Microfractionation end determination of urinary glycosaminoglycans Biochem. Med., 9 317-326. 1974 16. Koiima, J., Nakamura, n,, Kanatani. M , and Ohmon. K. The giycosammoglycans in human hepatic cancer. Cancer Res., 35. 542-547. 1975, 17. Korn, E. 0, The isolation of hepann from mouse mast ceil tumor J. Biol CONCLUSION Chem.. 234: 1325-1329. 1959. 18. Kuroda, J.. Saito. S . Seno, N.. Nagase. 5., and Anno. K Isolation and chemical characterization of mucopolysaccharides from rat tumors Cancer Our data clearly show that hepatomas 7777, 5l23tc, and 9618A have GAG compositions that are appreciably different than that of normal liver. These data show that there are also qualitative differences in GAG between tumor lines and that heparan sulfate patterns parallel growth rate and possible Res.. 34 308-312. 1974. 19. Lehtonen. A., Nanto. V,, and Kasanen. A The effects of some antunftam- matory agents on the urinary excretion of mucopolysaccharides m the rat. Ann. Med exp : Fenn . 45. 32-34, 1967. 20. Manley. G . Bower. L.. and Anson, A Urinary excretion of gfycosaminogly* cans in disseminated neoplasm. J. Cltn. Pathol., 31 * 447-453. 1978. 21. McManus. J. F. A., and Mowry, R. w Staining Methods: Histological and degree of malignancy. Heparan sulfate is the predominant GAG in the hepatomas studied. The increased urinary excretion of GAG's for 2 of the 3 tumor lines examined suggests that urinary Histochemical. New York: Paul B Hoeber. Inc. (Harper and Brothers), 1960 22. Nakamura, N., Mun, Y., Tanigaki. Y., and Kottma, J. Changes m the cellular glycosaminoglycans of cultured mastocytoma celts induced by sodium bu tyrate Biochim. Biophys. Acta 627. 60-70, 1980. GAG analysis may prove useful in the detection and diagnosis 23 Ozzeiio. L . and Speer. F. 0. The mucopolysacchandes m the normal and t of some hepatic tumors. diseased breast: their distribution and significance. Am. J. Pathoi. 34 1005, 1956. 24. Rich. C . and Meyers. W. P. L. Excretion of acid mucopoiysacchan REFERENCES the urine of patients with malignant neoplastic diseases J Lab Cim. Med. 54 223-228. 1959. 25 Saito. S. Mucopolysaccharides of rat ascites hepatoma cells Gann. 64 1. Sitter, T., and Muir, H. A modified uronic acid carbazoie reaction. Anal 247-255. 1973, Biochem., 4` 330-334, 1962 2. Cameron, E.. and Pauling, l. Ascorbic acid and the glycosaminoglycans Oncology (Basel) 2 7. 161-192, 1973-. 3. Chiarugi. V. P.. and Vannucehi, S. Surface heparan sulfate as a control element in eukanotic cells: a working model, J. Theor. 8ol.. 6/ 459-475. 26 Sakaki, T., Tsurumi, N,. Maeda. J.. and Matsuda. H, Studies on the influ ences of acid mucopolysaccharides on the growth of Tawa sarcoma J. Osaka Dental Umv,. 4. 113-122, 1970. 27 Sanders, F. K,. and Smith. J. Effect of collagen and acid polysaccharides on the growth of BHK/21 cells in semi-soiid media. Nature (Lond ). 227 513- 1976, 4. Choi. H. V.. Meyer, K., and Swarm, R. Mucopolysaccharide and protein- polysaccharide Of a transplantable rat chondrosarcoma. Proc. Natl. Acad. Sci, U S. A.. 68: 677-879, 1971. 5. Cudkowicz, G. The potysacchandes of a human carcinoma. Br. J. Cancer. 10. 759-762, 1956. 6. Curran, K. L.. Kupchefla. C. .. and Tamburro, C, H. Urinary giycosamino- glycan patterns in angiosarcoma of the liver. Cancer (Phiia ), 40. 30503053. 1977. 7. Danishefsky, I., Oppenheimer. E. T.. Heritier-Watkins. O . and Wilhite, M Mucopolysaccharides in animat tumors. Cancer Res.. 26. 229-232. t966 515, 1970 28. Schiller. S.. Siover. G. A., and Dorfman. A. A method for the separation of acid mucopolysaccharides- its application to the isolation of heparm from the skm of rats. J. Biol. Chem.. 236` 983*967. 1961. 29. Snedecor. G. W,. and Cochran. W. G. Statistical Methods, Ed 6, p. 273. Ames. Iowa: Iowa State University Press, 1967. 30 Takeuchi, J. Growth promoting effect of acid mucopolysaccharides on Ehrlich ascites tumor. Cancer Res., 26 797-802. 1966, 31. Winterbourne. 0 J.. and Mora. P. T. Distribution of glycoconjugatos m mouse fibroblasts with varying degrees of tumoriQenicity. J Supramol Struct.. 7, 91-100. 1977. FEBRUARY 1981 CMA 003685 'acr t^-a; v.-;n?.'y GAG .ircr-.c / ;a = " _;,,r .u.'u., ara a"ii,.a.3 a^'ctr ',i*..a a<';a 'iddfCd `:r '.rs Sy Lid:c*sr.' if j/ (13) It remains to be determined wnat the source(s) of the elevated urinary GAG is (are). Tnere hare ceen reports or striKing moreases n GAG 3yn*he3'S n tumor oe'ls (see Pet 2C). Put Kc;ima er at (to) nave suggested tna: oecreasea degradation may account for increased GAG in some tumors Our .nvesti- gations provide no evidence that the source of the abnormal GAG increases ;n tumor tissue and unne is anything other than the tumor cells themselves; however, a possibility that these results stem from an impaired ability of the liver to degrade circulating GAG's cannot be ruled out. Cur data clearly snow that hepatomas 7777. 5`2utc, and 951 BA have GAG compositions that are appreciably different than that of normal liver. These data show that there are aiso Qualitative differences m GAG fcetv/een tumor lines and that heparan sulfate patterns parallel growth rate and possible degree at malignancy. Heparan sulfate .s the predcm.nant GAG in the hepatomas studied Tne increased urinary excretion of GAG s for 2 of the 3 tumor lines examined suggests that urinary GAG analysis may prove useful m the detection and diagnosis of some hepatic tumors. REFERENCES 1 Bitter. T , ana Muir. M A modified vromc acid caroazole reaction Anal Biochem , 4 330-334, *962 2 Cameron ,. and Pauling, L Ascorbic acid and the glycosammogtycans Oncology (Basel) 2 7 iai-192. 197G. 3. Chiarugi, v, P , and vannuccht. 5, Surface heoaran sulfate as a control element m eukanotic cells: a working model. J. Theor. SoJ.. 61 *59-475, 1976 4 Choi. H. V,, Meyer, K,. add Swarm. R Mucopolysaccharide and protempoiywccharide of a transplantable rat chondrosarcoma. Proc. Natl. Acad. Sci. u, S. A,. 6a. 877-379. 1371 5. CudkQwicz, G. The polysaccharides of a human carcinoma. Br. J. Cancer, f0` 759-752. 1956. 5. Curran. K. L.. Kuochaila. C. .. and TambuiTO, C- H Unnary glycosamino glycan patterns tn angiosarcoma of the kver, Cancer (Phifa ), 40 30503053, 1977 7 Danishefsky, I., Oppenheener, E. T., Henber-Watkm*. Q . and Wilhite, M MucooofyseccftencM* m ammaf tumors. Cancer Res., 26 229-232. (966, - * wim Cu crocus -eD.V'C a,"d ia 4!.Gr>5 ~ i-- J.S51- - `-t z` %';? =25-;3C 1975 ' 2 Harae y Veda > , and Makita A Glyccsamxngfycans r *n-ail cert laronoma of !urg his;aiogn:aUy cnaraciansnc pa*(err> Gann 70 369-190 1979 `in " *.! i ;iizu^i v A *!"-; m x<j p a ^ study of cellular organelles of Morris hepatomas Cancer Res , 32 353- 357. t 972 * 4 Hrudan, 2,. Morns, h P , Mocnizuki. V Meranze. 0 R , and Siesers, a u.grt micrcicootc ooservations of Morris r.epaiomas Cancer Rea 31 ,`52- 752. i97t 15 Kao. K Y. T,, and Leslie, J G Microfractionaticn and deterrnmatrcn of urinary giYcasammogiyCans a*och*m Med . 9 3(7-326.1374 16 Kciima. J . Nakamura, N., Kanatam, M,, and Ohmon. K The g*>oosarr,ino- 1 7 giycans m numan heoafic cancer Career Res , 35 543-547 13/5 Ken. E 0 The isoiarcn c* *?oann fr~m mpuse mast cel 'u--:r j 2.gi C"*m , 2J-t 1225-1529 1 i n^roda, j oawo. 5.. Seng. 'l, Naga^e. o . and Anno, K isolation and o,*emical Characterization of mucccciysacc.-ances from rat tumors Career Res 34 2C6-3I2, (974. 19 Lehtonen, A.. Nanto, V , and Kasanen. A The effects of some antiinflam matory agents On tne urinary excretion pf mucccoiysaccnar-des ,0 ;ne rat. Ann Med exp.; Penn . 45. 32-34. J967 20 Manley. G . Bower. L,. And Anson. A Urinary excretion of gfycosammogiy- :ans in diSiamirated necciasm j C',r Ra:hcl.3f 447-453 '973 2 J McManus. J f A., and Mowry. R W Staining Methods: H*src'ogfcaf and HistcchemcaJ. New York; Paul 8 hceoer. ;nc. harper and Brctrersj, 1950 22 Ndiiamira. N . Mun. Y , Tamgaki, Y , ano Koiima, j Charges tre caUuiar giycosammogtycans of cultured mestocyroma certs induced py sodium bu tyrate, Biochim BtOQhyS, Acta 62 7 50-73. 1980. 23 Ozzetlo, L . and Speer, F. 0. The mucopolysaccharides m the normal and diseased breast: mew distribution and significance. Am j Patnot, 34 993- 1005. 1956. 24 Rich, C . and Meyers. W P. L. Excretion of od mucooofYaacchandes m the unne of patients with maiignant neoofesttc dtseeses. J Lab Cun Med 54 223-228. (959. 25 Sarto S. Mucoooiy**Chandes of rat ascites hepatoma ccff$ Gann 64 247-255. 1973. 26 Sasaki, r , Tsurumi. N.. Maeda, J., and Matsuda. H. Studies on tne influ ences of acid mucooolysacchandea on me growm of Tawa sarcoma j, Osaka Gentai Unw., 4, U3-I22. 1970. 27. Sanders. F, K,. and Smith. J. Effect of cortagen and aod polysaccharides on the growth of 8HK/21 ceils m semi-soud media Nature (Land ). 22 7 513- SI 5. 1970. 23 Schitter. S.. Steeer. G. A., and Oortman. A. A method for the separation of acid mucopolysaccharides: its application to the isolation of hepann from the stun of rats. J. Stof. Chera.. 236. 963-967. 1961. 29. Snedecor, G. W. and Cochran, w. G. Sfatnhcal Methods. Ed 6. p, 273. Ames, tow: iow Slate University Press, 1967, 30 Takeuchi, J. Growth promoting effect of acid mucopolysaccharides on Ehrlich ascites tumor. Cancer Res.. 26 797*602. 1966. 31 Winterbourne. 0 3.. and Mora, P T Distribution of giycocontugates m mouse fibroblasts with varying degrees of tumongeruerty J. Suoramol. Struct., 7. 91-fOO. 1977. FEBRUARY 7 981 CMA 003686 C. E. Kupchella et al. Fig. 1. At typical lung metastases in animats bearing Tumor 7777. These were found more frequently end earlier and were larger in animals bearing Tumor 5i23tc. No lung metastases were found in any of the animals bearing Tumor 9616A XSO. B, liver of animals bearing Tumor 5123tc showing clear cytoplasm-vacuolar appearance character istic of such livers. x50. 424 CANCER RESEARCH VOL. 41 CMA 003687