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RECEIVED J(JN 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) CMA. 052685 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. ii CMA. 052686 CONTRIBUTING INVESTIGATORS G.H. BARROWS, M.D., ASSISTANT PROFESSOR DEPARTMENT OF PATHOLOGY JOHN L. CREECH, OR., 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 iii CMA. 052687 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 DISEAS MOUNT SINAI SCHOOL OF MEDICINE NEW YORK, NEW YORK JOHN P. SAN002, M.S., INSTRUCTOR DEPARTMENT OF COMMUNITY HEALTH G.R. SCHRODT, M.D., PROFESSOR AND CHAIRMAN DEPARTMENT OF PATHOLOGY G. SONNENFELD, PH.O., 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 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 8. 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 v <3^ 52689 ANIMAL STUDIES: Bl. Characterization of Hepatic Enzyme Changes in Rats with Prolonged Vinyl ChioriGe Exposure: Decreased Glucose-6-Phospnatase 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 ..................... 33 B4. Oxidative and Detoxifying Ability of Liver Mesenchymal versus Parenchymal Cells in the Metabolism of Xenobiotics ................. 41 HUMAN STUOIES: 65. 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 vi CMA 052690 D. Histologic and Morphometric Analysis: A Means of Assessing Hepatic Injury in Chemical'Workers; Investigators - 6.H. Barrows, G.R. Schrodt, and C.H. Tamburro................................. 56 Dl. Morphometric Assessment of Histological lesions Character istic of Vinyl ChlorideInjury ........................................................... 56 D2. Computer-Assisted Morphometric Analysis as a Rapid Means of Determining CollagenContent ................................................................. 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 vi i CMA 052691 G2. Circulating Antigens and Autoantibodies in Vinyl ChlorideAssociated Liver Disease........................................................................... SC H. Use of Isolated Mammalian Liver Cells for the Study of Chemical Monomer Metabolism; Investigator - R.C.Feldhoff ................................ 67 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................................................................................................Q7 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 andtheirMetabolites .... 92 IV. Lists of Publications, Abstracts, Preprints, and Publications in Preparation......................................................................................................... 99 A. Publications...................................................'................................................iQO B. Abstracts............................................................................................................. 102 C. Preprints.......................................................................................................... .... D. Publications in Preparation Titles ......................................................... 105 V. Appendix (copies of IV., A, B, C) ................................................................. 106 viii CMA 052692 INTRODUCTION In 1974 Dr. John !_. 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 1 CMA 052693 2 medical surveillance program which were ongoing program arc re nu . rese3rch periods of this study. This has led to a concomitantly GU1T1!?*ture which has changed the medical approach to tftg large bcay ot _t1on ancj verification of occupationally-related chemical screening, iaentir-a injury. Although the contractual funding of this grant was only three years, each vear 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 puolished 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 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 inpossible 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. CMA. 052694 SUMMARIES OF RESEARCH PROGRAMS 3 CMA 052695 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 immuno competence 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 4 CMA. 052696 5 evidence suggested that the tumor antigens found were distinctive for eac^ 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 3H-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 Mulvihlll 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-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 052697 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 nonparenchyma1 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 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 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 giucose-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 CMA 052698 7 studies were conducted in sacuential experiments exposing Spraaue-Dawley 'ats to 10,000 ppm vinyl chloride for a period of up to 300 exposure hours. These studies demonstrate various adaptive chances in liver parenchymal cal's 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 Oisse. S3. 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 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 450, glutathione reductase, GEST, and GAST in liver hepatocytes and endothelial cells. CMA. 052699 8 The mesenchymal cells were shown to be capable of oxidizing xenobiotics which require -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), gaimia 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 9Q0 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 CMA 052700 9 ho 1 a promise 25 2 potential oetector of early liver injury, whether chemical cr not. Bile ados have the aGvantage of being natural biological substances whicn 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 ana R. Warick ANIMAL STUDIES: Cl. The Study of G1ycosami nog 1yean 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 052701 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 Dl. 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, CMA 052702 n 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 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 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 3. Vinyl Chloride Metabolite Detection--Chioroacetic 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 CMA. 052703 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. 052704 PROGRAM G THE STUDY OF LIVER TISSUE ANTIGENS AND ANTIBODIES IN THE DETECTION OF VINYL CHLORIDE INJURY; Investigator - E. Espinosa Gl. 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 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 usefulin 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 052705 14 Presently, the rat has provided the most useful means of studying hepatic metabolism in manmalian 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 nonparenchyma1 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 cTinical biopsy technique are viable in an in 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-vinyl 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 052706 15 nonvolatile metabolites of vinyl chloride retained in the thymus aftsr Zi 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.. 052'?0'7 CM* RESEARCH PROGRAMS AND RESULTS 16 CMA 052708 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 iirmune response to those workers who had developed liver injury, including angiosarcoma. 17 CMA 052709 is Research Results An evaluation of irrmmocompetence 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 -- 2S0 _ X a TM200 -- S 1 mo - 50- I 1; 1! i : VINYL CHLORIDE EXPOSURE ..s;,ABOVE NEDIAN dt5*> `:::::: saow KEDiAR oi-2#> JT ^= Li; '2 j ; 1I * PHYT0HEM6Q.UTINIK CQKCAAAVALUN A POIIVEED AITOGEK 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). CMA. 052710 TABU 1 imuNE PARAMETERS OF VC WORKERS ACCORDING TO EXPOSURE TEST STREPTOCOCCAL ANTIGEN STIMULATION ABOVE MEDIAN EXPOSURE * * 191 +31 (n-52) BELOW MEDIAN EXPOSURE 186+39 (n-24) STREPTOLYSIN 0 STIMULATION 8+2 Cn-S3) 24+12 (h-24) PPD STIMULATION 29+8 (n-49) 18+6 (n-23) VARIOASE STIMULATION 29+5 (n*$4) 41+12 (n-23) CANDIDA STIMULATION 11+3 Cn-53) 6+2 (n-23) ABSOLUTE LYMPHOCYTE COUNT 2355+132 (n-52) 2316+148 (n24) T-CELL ROSETTES AT 4C (absolute) 1503+101 (h-50) 15241132 (n-24) T-CELL ROSETTES AT 33C (absolute) 1227+80 (n-50) 1260+123 (n-24) **TlmiLATION INDEX "SEN STATISTICAL SIGNIFICANCE NONE NONE NONE NONE NONE NONE NONE NONE Mitogen induced lymphocyte transformation in VC workers and unexposed individuals VC workers (n-78) Hi Unexposed individuals (ns2l) 19 CMA 052711 20 No statistical differences were seen in these same immune parameters between workers with or without liver injury (Tables 2 and 3). tabu z irwUNE PARAMETERS OF VC WORKERS WITH ADD WITHOUT LIVER DISEASE TEST RMYTOHtFUeSU/TININ STIMULATION CONCANAVALUN A STIMULATION *0*0*110 fttTOHN STIMULATION STREPTOCOCCAL ANTI SEN stimulation STREPTOLYSIN 0 STIMULATION PRO STIMULATION VAJtlOASE STIMULATION CANOida stimulation LIVER DISEASE 253*i55** 25In- ) 195*36 25In- ) 100+12 (n-ZS) 2081R2 2(n- R) 8+2 2<n- S> 18+6 25In- ) 36+9 <n-ZS> 1R+5 In-ZS) NO LIVER DISEASE 17n+;35 In-RSI 162+29 8In-r ) 80+13 3<*R ) 131-37 In*R9) 16i6 9lirR ) 30i9 5In-R ) 5Jli (m-50) 7+1 (n-R9) stimulation index TABU 3 IfMJNE PARAMETERS OF VC 'WORKERS WITH AND WITHOUT LIVER DISEASE TEST -HIT- SLWD MU, COUNT LYrPHOCrTE COUNT (PERCENT) LIVER DISEASE 69001500* 25Cn- ) 35+2 25In* ) NO LIVER DISEASE 66501380 50(n- ) 33+2 Ik-RS) AlSOLUTt LYNNHOCTTE COUNT T-CEU. ROSETTES AT RC (PERCENT) .2350+200 25In- ) 67+3 25(n- ) t-cell rosettes at Rc (AtSOLUTE) lEOO+lOO 25In- ) t-CELL ROSETTES AT 33C (PfRCENT) 55+2 25Ik- ) T+CELL ROSETTES AT 33C (assolute) 1300+100 25(n- ) 2303+200 8Ih-R ) 52+2 9I*R ) 1500+100 9<-R ) 52+2 (n-RD 1200*100 7t-R ) *SEH 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 (P<.01), percentage of lymphocytes (Pc.OOl), and lymphocyte reactivity to Streptolysin 0 (Pc.05) and PPO (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 inmunological abnormalities result from chronic exposure to various levels of vinyl chloride. CMA 052712 TABLE A IfTUNE PARAMETERS OF VC WORKERS OU) VERSES YOUNG TEST PHYTOHtMAGGLUTlNlN stimulation CONCANAVALLIN A stimulation POKEWEED MITQGfN stimulation streptococcal antigen stimulation STREPTOLYSIN 0 STIMULATION PPO STIMULATION VARIDASE STIMULATION CANDIDA STIMULATION 'STinumTIOH IHDtX "SEN OLD IRo'tZO** (h-37) 16025 (n-37) 9015 (n-37) 135*35 (h-53> 5*2 (n-38> 36+12 (h-39) 25*6 (h*38> 9t3 (n*37> YOUNG 210i32 (m--40) 188+25 43) 90+20 (h-90) 195+35 (h-33) 21+7 P (n*33) 1J+3 P f n--35) 39+9 (h-39) 9+3 (h-39) 21 TABLE 5 ItnUNE PARAMETERS OF VC WORKERS OLD VERSES YOUNG TEST WHITE BLOOD CELL COUNT OLD 7150+900* (h-38) LYMPHOCYTE COUNT (PERCENT) 33i2 (h-36) ABSOLUTE LYMPHOCYTE COUNT 2300+200 (h-36) T-CELL ROSETTES AT itc (percent) Wl2 (h-36) T-CELL ROSETTES AT 40c (absolute) 1900+150 (h-39) T-CELL ROSETTES AT 33c (percent) 5212 (h-36) T-CELL ROSETTES AT 33c (absolute) *SH 1100+100 (h-39) YOUNG -6150+300 9 '.01 (h-90) 91+2 9'.001 (h-90) 2900+100 (h-90) 6912 (h--90) 1600+100 (h-90) 53+2 (h-90) 13001100 (h-90) 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 Halliday 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. 052713 CMK 22 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 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, 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). 052714 TABLE 6 TISSUE EXTRACT PANEL extract ANGIO TUMOR NORMAL LIVER HEPATOMA LIVER ANGIO "NORMAL" LIVER* NORMAL KIDNEY** MHMgRR 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 ALONE 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 23 5 23 20 53 38 200 00 8 57 17 5 36 14 200 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 CMA 052715 24 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 "a above Indian <n-5a) :: BELOW MEDIAN (N-2R) I J: ANGIOSARCOMA ANTIGEN EXTRACT LIVER 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 i 0.3* 2.6 + 0.4 1.5 + 0.2 1.8 0.2 'STIMULATION INDEX + SEM CMA. 052716 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 SB IN vitro LYMPHOCYTE STIMULATION BY ANGIOSARCOMATOUS LIVER EXTRACT SUBJECTS HUMBER TESTED HUMBER OF SUBJECTS REACTIVE AGAINST: NORMAL LIVER ONLY NORMAL & ANGIO ANGIO ONLY NON-PALLET PLANT WORKERS 16 1 11 PALLET PLANT WORKERS TOTAL 18 54 *ONE 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 B and T Cells, genetic control of inmune 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, CMA. 052717 25 target antigens, immune response, turner virus susceptibility, liver cyclic acenosine monopnosphate levels, hybrid resistance, and T-cell: B-cell inter actions. HLA antigens are found on almost all ce^ls of the body except red blood cells. The major histocompatibility complexes in man are identified by letters (A, B, C, D) and numbers (A10, B12, _C3). An increased incidence of certain HLA antigens had been shown toTie 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 indiviauals 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-827 antigen which was found witn 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 cnloriae workers. Objective 1. T termine the HLA frequency in vinyl chloride workers 2. T ampare 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, B-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-B15 (12 percent-normal population versus 28 percent-Pallet Plant Cohort). CMA 052718 HU-a ANTIGENS Al A2 A3 A9 A10 All A2 A39 A30 AW31 AW32 Blaafc TABLE 9 HLA-A FREQUENCIES HEALTHY CONTROLS 34 1 33 10 7 13 7 9 NO** NO NO NO WHO 1974 WORKSHOP 33 49 33 17 13 t 11 7 5 7 S HD PALLET PLANT(BFG) 4 37 14 11 IS 7 IS 0 4 4 7 14 If Totml frvquMCy HOT BMI 900 10T SO 3 1701 3S BOOT NON PALLET FLANT(8FG) 30 S3 21 33 U 13 S S 0 7 19 510 SOOT table 10 HLA-B FREQUENCIES KLA-8 ANTIGENS BS 17 U B12 813 114 US 1116 117 116 1WS1 H22 837 H3S 140 Blank HEALTHY CONTROLS 10 31 27 30 3 5 10 HD 8 ND 2 7 5 11 ND N Total frequency 903 tSST 1H0 1975 WORKSHOP 11 23 20 24 0 11 7 13 7 9 4 S 1 17 12 ND PALLET PLANT/BPO 11 IS >1 21 0 U 29 7 14 4 4 7 7 11 18 IS 503 1761 28 301T NON PALLET PLANT/BFO 9 28 20 31 S s 12 4 12 0 4 5 11 17 10 13 S19 3011 27 CMA. 052719 r 28 Table 11 illustrates the frequency of HLA-815 artcng the entire works'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-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 BIS 3 427 ' 15 Total CD 491 (13) IS (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 (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 (LD). CMA. 052720 TABLE 12 DISTRIBUTION OF HLA-B15 ANTIGEN AMONG WORKERS WITH AND WITHOUT CHEfllCAL RELATED LIVER DISEASE (PALLET PLANT COHORT) Clinical Diagnosis 29 TABLE 13 DISTRIBUTION OF HLA-B15 ANTIGEN AMONG WORKERS WITH AND WITHOUT CHEMICAL RELATED LIVER DISEASE (MAIN PLANT COHORT) Clinical Diagnosis ____ NORMA1 1 3 Total A (Z) (25) nt 4 9 13 (31) N-29 LD 3 9 12 (25) NORMAI 65 430 Total" 495 (Z) (15) CLI 0 2 E (0) N-526 LD 3 13 IT (13) TABLE 14 OCCURRENCE OF HLA-B15 AMONG CHEMICAL WORKERS WITH AND WITHOUT CHEMICAL LIVER INJURY Clinical Diagnosis ____ NORMAI . CLI 65 4 430 Total 495 (2) (13) 11 15 (27) N-538 LD BOTH 6 10 22 33 28 43 (21) (23) CM& 052721 30 A5. laentification of the Endothelial Ceil as the Cell of Origin for Vinyl Chloriae-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 & 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. CM* 052722 31 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 imnunodepression 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 B or T cells will not provide any useful information in the early stages of disease even with excessive exposure. CM* 052723 / 32 (2) The use of tumor antigens for* the detection of developing angio sarcoma, alchcugr. 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 iiimunological 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. Halliday, W.J., Hal 1iday, 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. CMA 052724 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, D., 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, 0. (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- 1151. ---------- "------------------------------------------------------------- " World Health Organization Report (1977) Histocompatibility testinq. Bodmer, W. (ed.), Munksgaard, Copenhagen. CMfc 052725 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. 34 CMA 052726 FIGURE 1 WEBER*S MOLECULAR CORRELATION CONCEPT OF NEOPLASIA 35 w-- -- aiucoNtooMfsi* 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 phosphorlbosylpyrophosphate (PRPP) synthetase and glutamine PRPP ami notransferase, the first two enzymes channeling rlbose-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. CMA 052727 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-Oawley 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-ephosphatase (microsomal), cytochrome oxidase (mitochondrial) and glucose-6phosphatase 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. _IJ1 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). CMA. 052728 CONVENTIONAL CUNICAl BIOCHEMICAL STUDIES IN RATS EXPOSED TO VINYL CHLORIDE ____ CONTROL I VC TREATED SGPT JU/t 37 SOOT IU/1 LDH 1U/I TOTAL PKOTCIN OM/dl FIGURE 2 HOURS Of VINYL CHIORIM EXPOSURE 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. CMA 052729 38 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. B3. 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 a!., 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) LIVER MFO Cl CH-Oi, V , (chlorooxirane) DETOXIFICATION WITH GLUTATHIONE Cl CH2CH2OH +GSH GS CHjCHjOH (CHLOROETHANOL) ^ +GSH Cl CH2CHO GSjCHjCHO (CHLOROACETALDEHYDE) I Cl CHjCOOH N-Ae-S-(2-HYDROXY ETHYL) CYSTEINE GS CHjCOOH THIODIGLYCOL1C ACID (CHLOROACETIC ACID) In these experiments, the enzymes studied related to vinyl chloride oxidation, included -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). CMA 052730 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). G1utathione-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 reauctase (GR), the enzyme to regenerate glutathione, was also elevated significantly following vinyl chloride exposure (Figure 4). 12 O FIGURE 3 2 (70) 4 (MO) 6 (210) TIME IN WEEKS (HRS) FIGURE 4 2 (70) A (140) 6 (210) TIME IN WEEKS (HRS) CMK 052731 40 The oxidizing capability as reflected by cytochrome -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 1977; Ivanetich et al.',' 1977) that vTnyl chloride metabolites destroyed P-450. No differences were 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 CMA 052732 41 orimary neoatocellular malignant lential 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 P-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, 7% 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 CMA 052733 42 experimental studies would be needed to eventually validate this hypothesis (Du et al., 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. CMA. 052734 43 HUMAN STUDIES: B5. 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 [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 CMA 052735 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. 90t 1002 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 ^symptomatic 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 CM* 052736 45 specificipy 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 SHEEN CLEARANCE SENSITIVITY AND SPECIFICITY OF INDOCYANINE GREEN CLEARANCE o. wit:sirrs 1500 ICS 2.5 .a/KO Best Nidical AiStSlIENT ICS Q.5 a/ (*) (-) (*) 233 140 (-) 33 1923 TOTAL 516 ZOU 2439 (*) 5cIT Nedical <*) 45 Ashmmcxt (*> 14 (-> 25 396 MMtTIVITr: 55.3 iPECiriettr: 95.3 UMITWTY: 45/73 61.3 lAteinetTY! 596/410 96.3 Tot"a1l 73 410 483 SENSITIVITY AND SPECIFICITY OF INDOCYANINE GREEN CLEARANCE ICG 5,0 fn/na 3cit No:cal 111 () 78 U. 6 AmtIMCMT (-> a_ a ScMtTivm: 78/ * 92.31 S/CCiAtem; JSV42 - 90.3 IfllAL 84 52 125 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 CHLORIDE (VC) WORKERS figure 6 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 CMA 052-73T 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 and 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. CMA 052738 TABLE 2 CORRELATION OF LIVER 3ICPSY WITH BILE ACID LEVELS AND ICG CLEARANCE ICGU<s) (0.5 g/kg) CG (og/dl) CCA (ug/dl) CL I 4.2 0.6 95.2 28.3 89.7 29.3 KCLD 3.2 0.1 27.3 4.4 25.3 4.5 NBX 3.3 0.2 34.60 7.1 52.5 26,6 NON BIOPSIED N0RT1AL . 3.1 0.01 14,9 0.9 18.7 1.2 47 Analysis of variance (on Tog transform data) showed significant differences for ICG clearance for the three biopsied groups and for all four groups. Values for the CIO 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 (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 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 CMA 052739 48 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). Livermi crosome-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, jJ. 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., and 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). Elevated glutathione content, glutathione-S-transferase and glutathione reductase in liver of rats exposed to vinyl chloride. Fed. Proc. 37, 1545. Du, J.T., Sandoz, J.P., Tseng, M.T., and Tamburro, C.H. (1979). Biochemical alterations in livers of rats exposed to vinyl chloride. _J. Toxicol. Environ. 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 chloroacetaldehyoe monomer gydrate. Biochem. Biophys. Acta 442, 405-419. ------------ ----- ------------------------ Fortwengler, H.P., and Tamburro, C.H. (1975) Use of dye clearance in the detection of hepatocellular injury among vinyl chloride workers. Clin. Res. 23, 264A. Green, T., and Hathway, D.E. (1977). The chemistry and biogenesis of S-containing metabolites of vinyl chloride in rats. Chem. Biol. Interact. 17, 137-150. ------- ------- CMA 052740 49 Greenberg, R.A., and Tamburro, C.H. (1931). Exposure indices for ecidemiologicai surveillance of carcinogenic agents in an industrial chemical environment. _J. Occup. Med., 23. 353-358. Greim, H., Bonse, G., Radwan, Z., Reichert, 0., 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. 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, $.0. (1974a). Effect of 18 hr. fast and glutathione depletion on 1, 1-dichioroethylene-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 '4C-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. CMfc 052741 50 McCann, 0., 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. VanOuuren, 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. j>, 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. Appl. Pharmacol. ^6, 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. The Molecular Biology of Cancer, ed. H. Busch, pp. 487-521. New YorkiAcademic. Weber, G. and Convery, H.J.H. (1966). Insulin: Inducer of glucose-6phosphate dehydrogenase. Life Sci. j), 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. CMA 052742 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 51 CMA 052743 systematic evaluation of the usefulness of urinary SAG 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 conmon 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. 052744 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 prelminary evaluation of the use of an exclusively chondroitin SULFATE EXCRETION PATTERN AS A SCREENING TEST FOR VINYL CHLORIDE - EXPOSURE - ASSOCIATED LIVER INJURY * TEST* RESULT VINYL CHLORIDE EXPOEURE PLUS N0N-AH51OSARCOMA LIVER INJURY +. 73 - 2 29 SUN 9 32 SENSITIVITY * 77,71 (951 LIMITS WJ.CX - 97.21) SPECiPieiTY 90.61 (951 limits 751 - 9SI) urine FRACTIONATION in WHICH the chondroitin sulpate fraction is POSITIVE BUT THE HYALURONIC AGIO AND HEFARIN FRACTIONS ARE BOTH negative. 052745 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 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, Vol. 1, Marcel Dektcer, 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. Gastroentero1oqy 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. CMA 052746 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, 0. 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. 1., 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.STTlPA, 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. CMA. 05274 7 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 D3. 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 liver biopsy. 56 CMA. 052748 57 Industrial vinyl monomer exposure, especially vinyl chloride, has been associated icn various hepatic histological acnormaliiies. These nave included nyperplasia 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 liver 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 CMA 052749 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 Area Percent 2. 03 4. 30 6,10 14.70 rCTi u/^UllDKCC 7 c. lourio*: wrictm* cn- /**"-/* **! nia. -1 t1eultcu*iiuAm* i.m m* TABLE 1 REPRODUCIBILITY Digitizer Inter assay Intra Assay 0. 40 0.19 0.55 1.27 0.018 0. 052 0. 054 0. 6075 Square Counting 0. 27 0. 35 1. 10 2. 95 CMA 052750 59 {2) Laiar 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. (r* < iqiMre eorretponds to distil printout SB). CMA. 052751 60 FIGURE 3B Digitil densities of retlcuMn stained Hvcr 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 Iobe Deep peri-venous FIGURE 4 CMA 052752 61 Two hundred and fifty three light micrcgraphs derived from 30 tissue blocks from 8 normal individuals were studied. A Hewlett Packard SS64-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 ZONES AGE PERICENTRAL PERIPORTAL MIDZONAL MIDZONAL 15 11.3; 1 3.6: 1 23 2.8: 1 1: 1 40 2.8; 1 1: 1 50 1.8: 1 1: 1 51 1; 1 1: 1 PERICENTRAL PER I PORTAL 1.4: 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 20 4o AGE GROUPS so CMA 052T53 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 (483!) of the exposed workers had hepatic lesions consistent with exposure, 17 (353!) had only focal hepatocellular hyperplasia, and 6 (133!) had focal mixed hyperplasia or more advanced lesions. CLINICAL FINDINGS TABLE 3 PROBABLE PHASE DEVELOPMENT OF VINYL CHLORIDE PRE'CANCEROUS LESIONS NONE BIOCHEMICAL PORTAL SPLENO HEPATIC ABNORMAL HYPER MEGALY SCAN TENSION ABNORMAL HEPATIS PELIOSIS CANCER HISTOLOGICAL NORMAL FOCAL PERI- FOCAL PORTAL AND FINDING HEPATOCYTIC SINUSOIDAL MIXED CAPSULAR HYPERPLASIA FIBROSIS HYPERPLASIA FIBROSIS SINUSOIDAL CELL DYSPLASIA ANGIO SARCOMA FOCAL HEPAT0CYT1C HYPERPLASIA 1 0------------------------------------------------TIME--------------------------------------- C< CMK 052T5A 63 FIGURE 6 lfocal hyperplasia op hepatocytes with GREAT VARIATIONS IN THEIR SIZE AS WELL AS THEIR NUCLEI.>*THE LATTER ARE FRE QUENTLY DOUBLE, POLYCHROMATOPHILlC OR vacuolated. he, 150x. TPOCAL INCREASE OF RETICULIN FRAMEWORK. SILVER IMPREGNA TION, IOOx. V^g 7. V.;>:rfvw J FIGURE _______ HYPERPLASIA OF SINUSOIDAL CELL^7 (CURVED ARROWS) AND HEPATOCYTES (straight arrows)^(hSe, IflOx) HYPERPLASIA FIGURE FOCAL CAPSULAR AND SUBCAPSULAR FIBROSIS,* ANILINE BLUE, 60x. CHA. 052755 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 CL!-CHEMICAL LIVER INJURY LD- LIVER DISEASE So-stanoard *norm 60 o<N- a. AVERAGE VC EXPOSURE 12 3 4 RATINGS (..D-5) Z3 CLI FIGURE 10 LD GROUP RATINGS SO CMA 052756 65 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 "normal ity*' 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., Or. and Johnson, M.N. (1974) Angiosarcoma of Liver in the Manufacture of Polyvinyl Chloride. J. Occup. Med. J_6:150. 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 Worxers 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. CMA 052757 66 6. Creech, J.L., Jr., Makk, l., Whelan, J.6. 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 052758 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 Salmonella 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 P450 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-l,4-thiazine--may be the precursor of the urinary metabolites S-hydroxyethylcysteine and S-carboxymethylcysteine. 67 CMA. 052759 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. CMA. 052760 H C-OH OH 69 HH G -CKfC-O- C- CH.-C1 OH OH och, 0^0 aCHl^O-^CHjC! HH a CDrC-o-c-coi a OD CD' H a-CH^-OCHjCHj ochjCh;- Studies of their reactivities have necessitated: a- Synthesis of etheno-modified components of RNA and DNA-- angularetheno-guanina, 1inear-etheno-guanina, 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, and El-0-1inear-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-O-angular-etheno-guanine, 1-0- 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 CMA. 052761 -t- ft H Oeony-ribosy l 9,*i*eiiiooioEpt)4(e rlboayL CAA 3',S`-gycllC MMpnB*PAtA r IBsiyi 70 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 idenTTfication 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-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 CMA 052762 71 cns. ana 30 psrc=rii conversion a: om tries a reactions oy nigh pressure alcehyde inaiccaatteeaa. Cn the other hand, ch'.cro anotner adaition product, which was ioentifieG cy iviR ana IR ana 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 SH 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 p'H.A and 7, their reaction was extremely fast at room temperature yield ng .T-acatyicysta1'ne-S- acetaldehyde. It was identified by iH and NMR and characterized as the 2,4-dinitrophenylhydrazone derivative. This aiaehyce is probably the precursor of the urinary metabolites, S-2-hydroxyethylcysteine and thiodi-- glycolic acid. The reaction of chloroacetaldehyde with N-acetylcysteine under controlled pH conditions in aqueous medium at 0*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 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 H3CQC0CH(NHC0CH3)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- (NHC0CH3)CH2-S-CH2CH0, 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-acetyl-methyl ester): 052763 CMA X-NH-CK-COOR h2sh vrcl --- C1-CH2CH0 v X-NH-CH-COOR ------------- ^ ch2-s-ch-ch0ci OH ^ X=H, Ac R=H, Me X-NH-CH-COOR ch2-s-ch2cho A x-nh-ch-coor CH2S-CH-pH2 72 COOR 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 (^CICH and ^C1CH2+) and 50,52 (^Cl^T and 37cich2T) are distinctive because rela tive intensities within each doublet reflect the 76:24 isotopic ratio of 35ci;37ci. Thus, the 6C-MS combination technique should be most useful in the detection of the above. CMA 052764 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. Detect. Cancer (Proc. Int. Symp), 3rd 1^ 155. Laumbach, A. D., Streips, U. N. and Wong, J. L. (1978) Chloroacetaldehyde-induced damage to Bacillus subtil is. Abs. Ann. Mtq. ASM, p. 125. Laumbach, A. D., Streips, U. N. and Wong, 0. L. (1979) Chloroacetal dehyde- induced damage to Bacillus subtil is, Abs. Twelfth Internat. Congress Microbiol. Joseph, J. T. and Wong, 0. L. (1982) Model study of vinyl chloride metabolism sulfate reactions with chlorooxirane, Carcinoqenesis (in prep aration). CMA 052765 PROGRAM F ASSESSMENT OF ASSAYS FOR THE CARCINOGENIC POTENTIAL OF INDUSTRIAL CHEMICALS USING PROKARYTQIC AND 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 Salmonella reversion. Bacillus subtil is repair, and Bacillus subtil is forward mutations assays for screening the mutagenic potential of chemicals (1-3). For that reason we became an integral part, first, for B. 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). 74 CMA 052766 * screening i moratory (using microbial assays.1' or insso`-'*`ic''a1 s purposes is being maintained. We have proposed to the state of Xentuck V a regional chemical testing laboratory be funded to serve the ir.cuot; university needs in this part of the Commonwealth. That proposal is sti 11 pending. TABLE 1 sueuirr of rmmjrr substances tisns *o* wtaccticity 1. -Mewchlorobeuxoyl-cyelooucenecerboxyl R*r*iSe 2, lenity L petosvde 3. v*cc5mia 4. *-evcloexcenecerbeyloxy-1<-ehft v Uce ca44e 5. 9i***ycloeutaAft eixpoxyl peroxide 4. Aflecoxle 7. Chleroeeheaol 9. lfieo<a)pyren 9. ***nltrO qulneitfta-t-oxide 10, ChloexeeetildeHyde 11. Epi efelerohydein 11. Qilorooxirefte u. keciM dlNvide 14, Seyrtne oxide 15. 1,4 epoxide bueeoo 14. bie(9ote-ehloroechyl)phonyi phoeehete 17. *et* chUroethyl pho*piece (hte e4cloftSYU*ift* *eii> IS. Lu*9 eeplrecee (rot leokan etch center 19, Ltaip eeoirerea frox eetktri without eencer Selaonelle * SR v* sa ` 4*4 mr *4 *4* *4* + 444 44 44 4 Ml SR 4 4 * "* rttetioi ktdtfnle *** iietml/ Mtamie * " **f4#pil** --fwmnicUy +* - ecrvnitlr Subtilli sr nr VR MR 44 NR 4 44 44 4 44 4 4 SR NR N n 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 manmalian 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. CMA. 052767 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 cancercausing 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 j_n 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 oF alpha-beta interferon (17). These results created an interest in our laboratory to further study this phenomenon. CMA 052768 77 } "n a :ar i w tO * inhibited interferon induction. rr * " ?lp`tr0filtm0r't 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., washea 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-B] 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- nel 1 a assay but negative in all Bacillus assays done to date (11), aTso inhibited interferon induction. ftHy1 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 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. CMA. 052769 78 References 1. Horowitz, s.. Doylo, R.J.. and U M. Streips. ^7,8^^ chromosome-membrane association i Proceedings of the Fourtfi subtilis. in Transfomat,on-1978. 0 ion> European Meeting on Bacterial Transformation pp. 245-251. 2. Horowitz, $., 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. <3. Bacteriol. 138:915-922. 3. Streips, U.N., Horowitz, S., and R.J. Doyle. (1980) Genetic analysis of DNA-surface interactions in Bacillus subtilis. 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. _In 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: Bacillus subti1 is assays for mutation and repair In Microbial Testers for Chemical Carcinogenesis, I.C. Felkner, eel., Marcel Dekker, New York, pp. 131-145. 7. Tamburro, C.H., Wong, O.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. CMA. 052770 79 ' i CeVaeyer, E., ar.d DeMaai/er-Gu':: iCfiG i afitnre.'e w" i Tj w tcrcfi "Ot'iiidcieft in r^ c.^crvo csi >s infected with Sindbis virus. J. iNatl. 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. Antimicrcb. Agents Chair,other. 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 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-naphthyl hydroxyl amine 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 in vitro by chemical carcinoaens. 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. CM&. 052T71 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. 80 052772 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 (ISA) (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 Of LITER ANGIOSARCOMA* ABLATED ANTIGEN TISSUE EXTRACT USED AS ABSORBBNT * NOtOSAftCOMA- RELATED ANT1CEH* Liver i|lai*t(Mia Liver li ideey Spleea Lhd| a Aatiia|ioaer tenia urviq we* absorbed nltb lyepbillied tloetie extract*. 100 "if/mt eod teaiad agelnot aagleea rcema extract. -Piece.*. at aot|f*a ledlcated by +. CMA 052773 sci.7.u"iataa further stadias of antigenic Gelation in cnemically-inaucea hepatomas and in cultured human liver carcinoma cells. In studies performed with the fast growing and undifferentiated chemically-induced Morris hsoatoma 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 tp liver. The second antigen (Antigen II) was detected only in liver. Aqtigen II was found unrelated to liver-specific F-antigen, differing in a number of properties and in immunologic reactivity. In studies of their suocellular distribution in normal liver, Antigen I appeared localized in cjtosoi (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 immunoelectroDhoresis close to that of serum gamna-globulins and Antigen II to that of serum alpha-globulins. The two antigens were completely inactivated with Pronase indicating that both antigens arq 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 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 differentiate^ 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 leatst 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-antigeh 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 CMA 052774 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 immunologically 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 Fnant'igen 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, a|re 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 tovinyl 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., 24fj:231. CMA 052775 04 n ! "* 5 7 ^^ ^ ^f after cessation of 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 ini the manufacture of polyvinyl chloride. Ann. N.Y. Acad. Scl., 24tj:80. 4. Nairn, R.C., Richmond, H.G., McEntegart, M.G., arid Fothergill, J.E. (1960) Immunological difference between normal and malignant cells. Br. Med. J., 2:1355,. 5. Ccrruthers, C., and Baumler, A. (1965) Immunochemical staining with fluorescein-labeled antibodies as an aid in the study of skin cancer formation, j. Natl. Cancer Inst., 34:101. 6. Burton, R.M., Hope, N.J., Beyerle, M.P. and Espinpsa, E. (1977) Tissue antigens in ovarian carcinoma. Oncology 34:146. 7. Burton, R.M., Hope, N.J., Beyerle, M.P. and fspinosa, E. (1978) Gewegsantigene bein ovarialkarzinom. Onkologie 1:75. 8. Kay, H.E.M. (1957) A and B antigens in normal and malignant cells, Br. 0. 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 mammaity tumors in mice. Internat. J, Cancer, ^: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 antigpn from carcinogeninduced sarcomas. J. Imnunol., 102:28. CMA 052776 85 15. Hughes, L.E., and Litton, B. (1964) Antigenic properties of human tumors: Delayed cutaneous hypersensitivity readtions. Br. Med. 0. 2=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, 235:370. 18. Burton, R.M., McGrew, T.L., Barrow, G.H., Beyerle, MkP., 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. Invent., 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 pnd 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. 2n 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., V_:ZS2. 27. Fortwengler, H.P., Jr., Jones, D., Espinosa, E. and Tflmburro, C.H. (1981) Evidence for endothelial cell origin of vinyl chloride induced hepatic angiosarcoma. Gastroenterology, 8:1415. 052'7'7'7 86 28. Espinosa, E., Caple, S., Kuchella, C. and Chia, S. (1979) Two liver antigens undetectable in a fast growing Tide of transplanted hepatoma (Morris Hepatoma 7777). Fed. Proc., 3^: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, _15,: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. CMA. 052778 PROGRAM H THE USE OF ISOLATED MAMMALIAN LIVER CELLS FOR THE STUDY OF CHEMICAL MONOMER METABOLISM; Investigator, R. C. Feldhoff HI. Isolation of Mamnalian 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 methpd to assess the functional capability of the human liver to handle exposure to various chemicals and/or their metabolites. In general, the rat hap 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 brgan is preserved but separated from other body influences. Newer _i 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 jji 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 metabolisijt has been used as the standard indicator and criteria for comparison 87 CMA 052779 2. To determine the usefulness of this technique in human bi-cosy Research Results Our first step was to verify the clinical and biochemical conditions neeoed 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 LIVES TISSUE CUT- A * RETAINED PROTEIN 3 - SECRETORY PROT{IN C LIGHT MICROSCOPY LIVER BIOPSY FIGURE 1 DIVIDED LIVER BIOPSY FIGURE 2 ri i CMA 052780 89 The initial experimental studies utilizing liver perfusion and the isola tion of hepatocytes techniques provided the hepatocytic apd nonparenchymal cells for Ou 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 plbumin. Initial results are illustrated in the rocket immunoelectrophoresis patterns from rat liver biopsy study (Figure 3) and the amino acid analysis is $een in Figure 4. The technique for liver cell identification after cell separation by the perfusion technique is illustrated in Figure 5. CMA 052781 I- i; V';; ' 70 - !:! [:, i -.j 90 FIGURE 4 TECHNIQUE FOR HEPATIC CELL TYPE IDENTIFICATION MARKER CELL TYPE DNA/R|NA ALL CELLS KC EC FIGURE 5 FB Relevance to Industry Although this study was a last year addition to our initial proposal it has provided the preliminary exploration into the adaptation of highly ,s,?*?,ist.cated. ab.oratory techniques' to human tissue in an in vitro system. While the work of these studies is still developmental, it provides promise for future clinical use. Such use could include determination of an indi vidual liver s ability to oxidize and detoxify xenobiotics, The degree and nature of liver cellular injury or impairment could be determined in a quantitative biochemical fashion, and could be directly correlated with CM* 052782 91 morphological findings. These techniques could be also used to determine the degree of biochemical adaptations of the human tissue in individuals who have been exposed to xenobiotics. Finally, these methods could* on sequential biopsy, provide clinical information regarding whether these alterations persist or revert back to a normal state. Although this is a very futuristic attempt to adapt pre$ently developed methodology, it does accurately reflect the direction in which clinical research should be directed in order to better assess the humin capability of sustaining environmental changes. References 1. Ou, J. and Tamburro, C.H. (1980) Oxidative and detoxifying ability of liver mesenchymal vs. parenchymal cells in th 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 oT~ 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. Bur. 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. 2jl The Plasma Proteins, 2nd ed., Putnam, F.W. ed., Vo 1. I, pp. 133-181, Academic Press, NY. 8. Peters, T., Jr. (1980) Albumin: An overview and bib!ipgraphy. Miles Laboratories, Inc.7flkhart, 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. CMA 052783 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 Theitr Metabolites Background Vinyl chloride has been classified by regulatory agencies as a carcinogen. Chronic exposure of workers to this carcinogeh has resulted in angiosarcoma of the liver, as well as other tumors (Crqech 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 l^C-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--31g., were exposed to '^C-vinyl chloride in room air in a sealed chamber for 3 hours. The radioactive vinyl chloride was synthesized from '^C-ethylene dichloride (New England Nuclear, Lot <*1194-143, 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 92 CMA 052784 93 v-. i ; i: t'-iafly anesir.esized with ether" and sacrificed zj ' ' : _v ;ca,',.=.<ar,e bath at -75C. 7 .e aracicn of che radioacaive vinyl chloride, exposure of the mice to this isotcoic gas, and the sacrifice of the mice were dpne in collaboration with Dr. William T. Stott at Oow Cheiical in Midland, Michigan. All remaining procedures were performed at the University of Louisville. Whole-oody sagittal sections of the mice, 20u- and 4Qv- 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 lightt-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 vivo disposition of these metabolites at the time of sacrifice cf the mouse. These procedures for whole-body autoradi ography, first described by Ullberg in 1954 do not allow tnawing 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). 4 Representative autoradiographs from each mouse were u$ed 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 14C-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 Herder'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). 052785 l4C-VINYL CHLORIDE; 20 MIN AFTER REMOVAL 94 Figure 1: A print of a whole-body autoradiograph from a male CD-I mouse which was exposed for 3 hours to l^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 SER0MUC0US GLAND THYROID BLOOD LUNG LIVER PANCREAS URINE Figure 2: 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 1 hour after removal from the vinyl (chloride environment. White areas correspond to radioactivity. CMA 052786 C-'/li'i . L unLur.---- , o ,nf > -- .i > ,_w.w ifatJAage ...________rw. fljAsilw .1 H " - -'-Lr.* ~?Er^i#^S~re5 -*J--- - '-- ----'.kSsT~-itjTM -- ---.iv^-cC'3t};%"V _____ - `*? <d*aaac " ' " wfl n----, -- - ; atrL"-^' SEROMUCOUS GLAND THYMUS LIVER ,,- - , --" ^ -%-u.-,-. .- - ..j^rSS -............ -'-J--" -.vwy - ;*?..- -._.- \-r-r-4. ---.-*$; '- --- - -*'--' ---1------------- --* -it -**< CONTENTS OF STOMACH AND INTESTINE Figure 3: A print of a whole-body aucoradiograoh frdm a male CD-I mouse which was imposed for 3 liours to ^C-vinyl chloride and clien frozen 3 hours after removal from the vinyl chloride environment. White areas correspond to radioactivity. 14,C-VINYL CHLORIDE; 9 HR AFTER REMOVAL HARDER'S GLAND SUBLINGUAL GLAND LIVER SPLEEN KIDNEJY PANCREAS ws UNGUAL MUCOSA THYMUS BLOOD GASTRIC MUCOSA WALL OF INTESTINE URINE Figure U*. A, print of a whole-body autoradiograph from a male CD-I mouse **':twexposed for 3 hours to ^C-vinyi chloride and then frozen 9 hours after removal from the vinyl chloride environment. White areas correspond to radioactivity. CMk 052787 14C'VINYL CHLORIDE;' 24 HR AFTER REMOVAL HARDER'S GLAND BLOOD KIDNEY PANCREAS 96 V1BRISSAL FOLLICLES THYMUS LIVER Figure 5: A print of a whole-body autoradiograph from a male CD-I mouse which was,exposed for 3 hours to 1"C~vinvl chloride and then frozen 24 hours after removal from the vinyl chloride environment. Unite areas correspond to radioactivity. l4C-VtNYL CHLORIDE 24 HR AFTER REMOVAL CORTEX AND MEDULLA OF THYMUS Figure 6; Prirts of the thymus areas of whole-body autoradiographs from male CD-I mice which were exposed to ^"C-vinvl chloride for 3 ;u>urs and then frozen 20 minutes or 24 hours after removal from the vinyl chloride environment. White areas correspond to radioactivity. INote the high retention of the nonvolatile metabolites in the cortex of tne thymus after 24 hours. CM* 052788 97 c j z ^ .* - i w Of tile nC'.T/G iatl ie TBZaZC :tgs or '/Try; c^crioe ,1,3 r,ir,Crj.ui ^j?: c',<*2 j i*.i i ,aj ;o t\lose reported for tr.d ra \UuPrfdt t Si* I d / / j * The nig.i concentration of nonvolatile reiabo'iitss cilleries in tne 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 Gehrina (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. ? e 1 av ar. c 3 to Indus try 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 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) Angiosarcoma of liver in the manufacture of polyvinyl chloride. Journal of Occupational Medicine 16: 150. -------------------------**------------------------ Duprat, P., Fabry, J.P., Gradiski, 0. 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 Maltoni, S. and Lefemine, G. (1974) Carcinogenicity bioas$ays of vinyl chloride. Research plan and early results. Environmental Research 7: 387. Sharma, R.P. and Gehring, P.0. (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 S^-labelled benzylpenicillin in the body. Acta Radiology, Supplftent 118:1. CMA. 052789 98 Waddell, W.J. and Marlowe, C. (1977) Autoradiography. IN: Garrett, E.R. and Hirtz, O.L. (eds.), Drug Fate and Metabolism: Methods and Techniques. Vol. 1, pp. l-2b. New Vork: 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 [l4C]0i-2-Ethylhexyl 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 Gehrinq. P.J. (1975) A gas chromatographic ipethod for the preparation of *4c-labelled vinyl chloride. Journal ^>f 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. CMA 052790 LISTS OF PUBLICATIONS, ABSTRACTS, PREPRINTS AND PUBLICATIONS IN PREPARATION 99 Ctih 052791 100 PUBLICATIONS 1. Fortwengler, Jr., H.P., Jones, D., Espinsoa, E. and Tamburro, C.H. (1981) Evidence of Endothelial Cell Origin of Vinyl Chloride-Induced Hepatic Angiosarcoma. Gastroenterology, 80:1415-1419. 2. Du, 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, :1119-1132. 3. Tamburro, C.J. (1978) The Hepatic Role in Carcinogenesis and Its Early Detection--The Vinyl Chloride Model. The Yale 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. Toxi cology and Applied Pharmacology, 62:1-10. 5. Tamourro, 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 Glycosamino- glycan Patterns in Hepatic Angiosarcoma. Detection and Prevention of Cancer, H.E. Neiburgs, Ed., Part 1, Vol i, Marcel uekker, Inc.", 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., Kupcnella, C.E. and Greenberg, R.A., et al. (1981) Screening for the Early Detection of Disease in Individuals Exposed 0to02.Vinyl Chloride. -U-.-S-.--E--n-v-i-ro-n--m-e--n-ta-l---P-r-o-t-e-c-t-i-o-n---Aqency, -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 Chemicals. 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. CMA 052792 101 .12 Streips, U.N., laumbach, A,D. and '''astir, .E Mutation Monitor for Active MetaDolit as of Chemical Carcinogens: B. Subti1 is Assays for Mutation and INA Repair, _I_n Microoial Testers. T. Cecil Felkner, Ed., Vol. 5, Marcel Dekker, Inc., New York. 13. Horowitz, S., Doyle, R.O., 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 ONA-Surface Interactions in Bacillus Subtilis. Microbiology-.284-287. 15. Horowitz, S., Doyle, R.J. and Streips, U.N. (1978) Restricted ChrnmoscmeMembrane 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, _l Interferon: Properties and Clinical Uses. A. Khan, N.O. Hill and G.t. 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 and of Wide Body Distribution in Human Gall Bladder Bile. Clin. Exp. Iimiunol., 25:410-417. --------------------------------- CMA. 052793 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, CM* 052794 Greenberg, A. A. anc 1 1 f.C i v i uij a . s e/.pC Sec Association Ann. Mtc. ^ .in;': ^7" \ " 5 " / - -i " 2/' h ="i r"^ * * r -- ' - - ' * c.'icrice. American Public -lea'th 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., Makk, L. and Popper, H. (1979). Early hepatic histological alterations among chemical (vinyl monomer) workers. Gastroenterology, 77:A33. 16. Laumbach, A.D., Streips, U.N. and Wong, J.L. (1978). Chloroacetaldehyde-induced damage to Baci11 us subtilis. Ann. Mtg. Amer. Soo. Microbiol., p. 125. 17. Streips, U.N. and Wong, 0.1. (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. ------------- ------ CMA 052795 104 PREPRINTS 1. Tamburro, C.H., Makk, L. and Popper, H. Early Hepatic Histological Alterations among Chemical (Vinyl Monomer) Workers. 2. Du, J.T., Eades, D.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. CMA 052796 'IONS 'Tt: 1 . Tamburro, C.'-.., >'111 s'-, 3. 2nd Greenberg, R Specificity of ICG Clearance TEst of Hepatotoxicity. wC . i 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. D 3arrow, G., Scnrcct, G.R. and Tamourro, C.H. Collagen Changes in Normal Aging Liver. CMAl 052797