Document 676K9wdymGDzo9JQD0G3y6Jg

r MANUFACTURING CHEMISTS ASSOCIATION 182b CONNECTICUT AVENUE, N W. WASHINGTON, 0. C 20009 (202)483-6126 June 14, 1977 TO: . Vinyl Chloride Research Coordinators SUBJECT: Second Progress Report (Dated June 6, 1977) from the University of Louisville on "Research Techniques and Methods for Detection and Prevention of Carcinogenesis in Industrial Workers," Gentlemen: Your copy of the subject report is attached. The Chairman, Dr. T. R. Torkelson, requests that all Research Coordinators re view carefully this report and submit comments or opinions on it to him with carbons to the Project Manager. Your prompt action on this request will be appreciated as no copies of the subject report will be submitted to the Technical Panel until it has been approved by the Research Coordinators. Sincerely, JTS:prc Attachment J. T. Seawell Project Manager Vinyl Chloride Research RECEIVED JUN 1 7 1977 Environmental Affairs SL 080485 - UNIVERSITY OF LOUISVILLE LOUISVILLE, KENTUCKY 40202 SCHOOL OF MEDICINE DEPARTMENT OF MEDICINE DIGESTIVE DISEASES AND NUTRITION SECTION June 6, 1977 HEALTH SCIENCE CENTER WALNUT & PRESTON STREETS '""SIP Mr. George E. Best Vice President, Secretary-Treasurer Manufacturing Chemists Association, Inc. 1825 Connecticut Avenue, N.W. Washington, D.C. 20009 RE: Second Progress Report for the Manufacturing Chemists Association's Agreement with the University of Louisville Dear Mr. Best: The following updates the initial progress report made April 11 and describes what has been completed during the past two months of the Manufacturing Chemists Association's agreement with the University of Louisville entitled "Research Techniques and Methods for Detection and Prevention of Carcinogenesis in Industrial Workers." The technical pro posal is again composed of seven parts. Progress for each part will be reported separately. Technical Proposal A -- Immunological Systems for the Detection of Vinyl Chloride and Other Chemical Injury Part I ^ i' 1/ Fortwengler and group have been evaluating three independent aspects of the immune system of industrial workers exposed to vinyl chloride to determine whether these various functions of the immunological system can be used for the detection of future cancer development and whether alternative systems may be able to identify high-risk groups. A study of a subpopulation of lymphocytes (T cells) that are responsible for eliminating foreign growths and tumors from the body is underway. Their total number and ability to function have already been studied in an additional 15 individuals (total now 90) with various de grees of exposure to vinyl chloride. Part II The immunology research on HLA tissue typing as a genetic marker for possible identification with the development of vinyl chloride injury and angiosarcoma has been completed on approximately 130 individuals with and without vinyl chloride liver injury and with varying degrees of vinyl chloride exposure. un mmri, uu'iujimj SL 080486 I Mr. George E. Best June 6, 1977 Page Two d / Part III Lymphocyte adherence studies directed toward finding a more specific test for angiosarcoma by demonstrating activation of an individual's lymphocytes by a hypothesized angiosar coma tumor antigen have been completed in about 75 individuals thus far -- 3 of which have been shown elevated reactions. Two additional extracts prepared from stored angiosarcomatous livers have been added to the test panel to determine if those individuals giving positive tests arc in fact re acting with an antigen common to angiosarcomatous livers. Technical Proposal B -- Biochemical Enzymatic Systems for the Detection of Vinyl Chloride and Other Chemical Injury and Cancer Develop ment in Industrial Workers Part I - Human Studies Two organ specific liver function tests, serum sorbitol de hydrogenase (SDH) and bile acid clearance (BAC) studies are continuing to be done by Du's group. Approximately 250 SDH and 10 BAC studies have been completed thus far; in addition to those previously reported. `-P---a--r-t----I--I--------A---n--i-m---a--l---S---t-u--d---ie---s- t ,, -x _Chlorc(ethanol is presently felt to be a major metabolite of , ^vinyl chloride and is further oxidized to chloroacetaldehyde, I-' the toxic metabolite of vinyl chloride believed to be the ul y timate carcinogen. Studies have begun to determine the usa / bility of chloroethanol administered either intraperitoneally or in drinking water to produce comparable effects as vinyl chloride. The preliminary results showed that acute chloroethanol injec tion (daily injections of 30 mg/kg/injection for 10 days) caused a toxic effect (i.e., a 46% decrease of the liver non-protein sulfhydryl contentX whereas the slow feeding of chloroethanol (15 mg/kg/day for 20 days) resembled Du's results with vinyl chloride gas in rats; slow administration of chloroethanol caused the non-protein sulfhydryl concentration in liver to increase slightly (+14%). ^ ( / / In addition, Du's group has just established methods to study aralkyl glutathione-JL-transferase and expoxide glutathione-jStransferase in liver cytosol. The activities of these enzymes are used to indicate the capacity of the liver cytosol t-1 detoxify metabolites; they intend to utilize the system with rego'd to vinyl chloride metabolites. SL 080487 Mr. George E. Best June 6, 1977 Page Three Technical Proposal C -- Glycosaminoglycan Changes in the Early Detection of Fibrotic Injury and Hepatic Cancer During the period ending May 31, 1977 Kupchella et al. have accomplished the following: \. / \Y rf /" 1. Studies of various improvements in the procedure used to measure urinary glycosatninoglycans (GAG) have been completed. The raw data on duplicate sets of eight urine samples analyzed for each of three procedural modifications indicates that they now have a procedure that gives a greater yield, with less variance and in less time than the widely used method of DiFarrante. The data Is currently being evaluated statistically. They have also evaluated recovery of standard GAG added to urine and their modified method gives 90% recovery for all added fractions. 2. An animal experiment has been designed to assess the kine tics of early GAG changes in liver tissue and urine in response to chronic chemical injury to the liver. This work will begin on June 20, 1977. 3. Kupchella et al. have completed data analysis and prepared a manuscript on a project dealing with liver histochemical, biochemical and urinary GAG changes from the beginning of chronic CCI4 injury to the appearance of dense fibrosis in the rat. 4. They have also completed the design, selected the subjects and begun a study in which 24-hour urinary GAG excretion pat terns will be measured in A) workers with histories of high (average total) vinyl chloride exposure histories and some evi dence of liver disease; B) workers with histories of high expo sure without evident disease; C) workers with histories of liver exposure with evidence of liver disease; D) workers with histories of liver exposure without evidence of liver disease. 5. A report on preliminary studies comparing normal controls and angiosarcoma in patients has been accepted (April 4) for publication in CANCER. Technical Proposal D -- Histological Systems of Detection Schrodt and Tamburro are continuing their analysis of light microscopic and electron microscopic findings of liver biopsy tissue obtained from B. F. Goodrich workers with and without vinyl chloride exposure. Of the eighty (80) biopsies being studied for light microscopy and sixty (60) for electronic y SL 080488 Mr. George E. Best June 6, 1977 Page Four microscopy, first stage review has been completed on 10 light microscopic biopsies and 10 electronic microscopies. The ad ditional 10 first stage review will be done after the computer program morphometric analysis on the first 10 is complete. The computer program for the morphometric analysis of the specimens is complete and photographing of the random selected field for study is underway. Technical Proposal E -- Chemical Systems of Detection Wong et al. continue to develop the methodology for the assay of vinyl chloride metabolites. The strategy is to fully elu cidate the intermediary metabolism of vinyl chloride, i.e. the metabolic activation and inactivation steps. This is essential in being able to properly determine the significant end products of vinyl chloride in body tissues and fluids. Accordingly, one of the major concerns is the fate of chlorooxirane, the immediate P450 product of vinyl chloride. They have / used synthetic chl.acgo.xig.ane to react with non-protein sulfhydrylsoj and obtainedCchloroethanol). Since all literature methods for the^ t preparation^of chlorooxirane do not result in the pure compound and"contaminants such as chlorine, hydrogen chloride, ethylene oxide, etc. are often present, it was desirable to have double assurance that the unique chlorooxirane-chloroethanol conversion would proceed with "absolutely pure" chlorooxirane. Thus, they set out to develop a method to achieve this objective. They now have an apparatus that can prepare > 99% pure chlorooxirane in gram quantities safely. Reactions involving the pure material are in progress. Vx* . L>( The reaction of chloroacetaldehyde (CAA) with glutathione (GSH) is another target. Unlike the reaction of CAA with cysteine which gives a well-defined product, the CAA-GSH reaction is much more complicated. It is plausible that CAA has formed a bis-conjugate with GSH. Purification and isolation work for this reac tion is now in progress. Technical Proposal F -- Assays for the Carcinogenic Potential of Industrial Chemicals Utilizing Prokaryotic and Eukaryotic Systems Since the last communication Dr. Streips et al. further expanded \' \ \-j their mutagen/carcinogen screening capabilities by adding a bacteriophage induction assay in Bacillus subtilis. If most chemlcal carcinogens induce a faulty repair system (postreplication i* V\ or"SOJPrepair) then, by'assaying for the induction of this re- ,pair system they can estimate the carcinogenic potential of any SL 080489 Mr. George E. Best June 6, 1977 Page Five chemical. Induction of bacteriophage from lysogeny (carrier state) is a measure of SOS repair activity. This test has been reported for Escherichia coli (1,2). We utilize B.. subtilis and temperate bacteriophage SP02 and 0105. Briefly, the lysogenized culture of B^. subtilis is exposed to varying concentrations of the test chemicals. Simultaneously, a nonlysogenized, phage free culture is also exposed. Loss of viability due to bacteriophage induction and lysis, as well as the presence of phage in the supernatant provide us with an estimate of carcinogenic potential. This test has been able to correctly identify as carcinogens some chemicals which are not active in the Ames assays (2). They have also continued the chemical screening work and have assayed hexachlorocyclopentadlene (Hexa) and iso-hexa, pro vided by Dr. Wong. Both chemicals are quite reactive in our repair assays, with Hexa being active at far lower concentra tions (5X10~^M) than isohexa (1X10"2m). For the next quarter they will continue utilizing the expanded screening assays to determine the relative mutagenicity of the chemicals listed in the first progress report. In addition, collaboration with several investigators will provide synthe tic chemicals that are of interest in the study of molecular mechanisms for carcinogenic activity. References: 1. Meyn, M.S., Rossman, T., and W. Troll. 1977. Proc. Natl. Acad. Sci. (USA), 74: 1152. 2. Moreau, P., Bailone, A., and R. Devoret. 1976. Proc. Natl. Acad. Sci. (USA), 73: 3700. Technical Proposal G -- Tissue Antigens and Antibodies in the Detection of Vinyl Chloride Injury Dr. Espinosa et al. have, since the last report, characterized the previously identified liver-specific antigen (LSA-I) and differentiated another liver-specific antigen (LSA-II). The differences between these two antigens are as follows: 1. LSA-I elutes from DEAE-cellulose with 0.01 M Na2HP04 buffer at pH 7.6 whereas LSA-II binds to DEAE-cellulose equilibrated with 0.01 M phosphate buffer at pH 7.6 and it elutes with 0.4 M Na2HP04 at pH 6.8. SL 080490 Mr. George E. Best June 6, 1977 Page Six 2. Preliminary Immunoelectrophoresis results indicate that LSA-I's electrophoretic mobility is similar to a serum gamma globulin while LSA-II's is between that of a serum beta globu lin and an alpha globulin. 3. The molecular weights as determined by elution patterns in Sephadex G-200 are different. LSA-I was calculated to be be tween 82,000-93,000 daltons and LSA-II between 127,000-150,000 daltons. Purification of these antigens is being currently pursued in an effort to be able to apply Lhis new finding to the detection of early chemical injury. This completes the second Progress Report of the Manufacturing Chemists Association - University of Louisville Cancer Center agreement. If there is need for any further information or questions, please feel at liberty to contact me. Sincerely yours CHT:aw Carlo H. Tamburro, M.D. Associate Professor of Medicine Principal Investigator SL 080491 PART A TECHNICAL PROPOSAL .RESEARCH TECHNIQUES AND METHODS FOR DETECTION AND PREVENTION OF CARCINOGENESIS IN INDUSTRIAL WORKERS UNIVERSITY OF LOUISVILLE CANCER CENTER LOUISVILLE, KENTUCKY 40201 SL 080492 TABLE OF CONTENTS I. Technical Proposal Titles A. Immunological Systems for the Detection of Vinyl Chloride and Other Chemical Injury B. Biochemical Enzymatic Systems for the Detection of Vinyl Chloride and Other Chemical Injury as a Potential Means of Detection of Cancer in Humans C. Tissue and Urinary Glycosaminoglycan Changes Related to .Vinyl Chloride Injury: Use and Early Detection and Diagnosis D. Electron Microscopic Evaluation of Liver Injury from Chemical Workers E. Multivariant Analysis of Biological End-Products in Vinyl Chloride Metabolite Synthesis F. Assays for Identification of the Carcinogenic Potential of Industrial Chemicals G. Tissue Antigenic Systems for Detection of Chemical-Induced Carcinogenesis Investigator H. Philip Fortwengler Julie T. Du Charles E. Kupchella G. Randolph Schrodt Carlo H. Tamburro John L. Wong K. Grant Taylor J. T. Joseph Uldis N. Streips Enrique Espinosa II. Budget Proposal A. Summary Budget Sheet B, Individual Budget C. Justification Page 1 4 8 12 15 18 21 25 27 32 SL 080493 TECHNICAL PROPOSAL A IMMUNOLOGICAL SYSTEMS FOR THE DETECTION OF VINYL CHLORIDE AND OTHER CHEMICAL INJURY H. PHILIP FORTWENGLER, M.S. SL 080494 H. ?. Fortwengler, M.3. TECHNICAL PROPOSAL A IMMUNOLOGICAL SYSTEMS FOR THE DETECTION OF VINYL CHLORIDE AND OTHER CHEMICAL INJURY The continuation of testing of lymphocytes from individuals with varying degrees of vinyl chloride exposure is planned to determine if the defects in the Immunological system precede or accompany the initiation of vinyl chloride disease. Individuals with a history of vinyl chloride exposure, those with no history of vinyl chloride exposure but other chemical exposures, and those individuals with histologically diagnosed liver disease will have their lymphocytes tested against a panel of 10 Immunological evaluations. Previous investigations have demonstrated that the lymphoid cells of thymus origin (T cells) can kill human tumor cells. Our own preliminary studies have shown that lympho cytes from workers heavily exposed to vinyl chloride react to angiosarcoma extracts, as did one worker with angiosarcoma. One tantalizing explana tion to this observation is that the workers with high exposure to vinyl chloride in the past, but without the development of the tumor, have had some of the endothelial sinusoidal cells transform into cancer and have been destroyed by the Individual's own T cells, thereby leaving behind sensitized lymphocytes in an individual whose own Immunological systems have eradicated a potentially developing tumor. A second vinyl chloride-exposed employee who developed angiosarcoma and was tested very late in the disease process did not demonstrate reactivity by his lymphocytes to the angiosarcoma extract. This may represent an inability of his lymphocytic system to respond to the devel oping tumor, or this may be a reflection of the immunological system's being overwhelmed by the tumor. Lymphocytes in future studies will continue to be isolated from various groups of workers as noted above, and the T cells will be enumerated and tested for their functional activity and capability. Dr. Herberman, from the National Cancer Institute, has suggested that since cancer patients often have a decreased number or an impaired function of lymphocytes, assessment of the immunocompetence may be an early indicator of cancer development. The second immunological approach under Investigation will be the tissue-typing of individuals for histocompatibility antigens as a means of determining possible markers for genetic predisposition. This tissue-typing, one-third completed, includes individuals with and without vinyl chloride exposure and injury. Those individuals with vinyl chloride injury will be matched as closely as possible for age, sex, race, and degree of chemical exposure with individuals having no evidences of SL 080495 H. P. Forcvengler, M.S. hepatic or other organ injury. The frequency of each of the 25 separate HLA-A and HLA-B factors is being continually tabulated and will be analyzed for comparison when 250 have been completed. Chi square tests will be performed by Dr. Richard Greenberg, our biostatistician and epidemiologist, to determine the potential statistical intergroup vari ations. These statistical variations will be used to determine the increased risk of individuals who carry a particular genetically deter mined antigen. Results of these analyses, hopefully, will enable the prediction of those HLA types which are genetically predisposed to vinyl chloride disease or other chemical injuries. The third immunological component development is a specific immunological assay for the detection of angiosarcoma. Individuals from the vinyl chloride plant with negligible vinyl chloride exposure, indi viduals from outside the plant with no vinyl chloride exposure, those with high vinyl chloride exposure, and those with histologically proven liver and other parenchymal injury are continuing to be tested. These results are in continuous tabulation and analysis. Individuals whose lymphocytes have given or give elevated test values on initial screening will be studied in greater depth to determine the specificity of the reaction and the cause of reactivity. The specificities of the reactions will be tested, using a panel of tissue reagents prepared from three separate angiosarcomatous and three normal livers, as well as the matched respective non-involved kidney. This system will allow the separation of cross reactivity due to histocompatibility reactions versus those re actions due to specific angiosarcoma antigens. The rationale for the development of this test for angiosarcoma lies in the established fact that the body mounts an immune response (becomes "hypersensitive") to the cancer. Thus, the detection of the body's specific immune response to the angiosarcoma will, hopefully, prove to be useful in indicating the presence or development of angio sarcoma tissue antigens in specific individuals and may assist in determining at which levels or degrees of exposure these antigens begin to emerge. This might further add to data supportive of a threshold level for vinyl chloride exposure and subsequent development of angio sarcoma or other cancers. SL 080496 TECHNICAL PROPOSAL B BIOCHEMICAL ENZYMATIC SYSTEMS FOR DETECTION OF VINYL CHLORIDE AND OTHER CHEMICAL INJURY AS A POTENTIAL MEANS OF DETECTION OF CANCER IN HUMANS JULIE T. DU, PH.D. SL 08049 Julie T. Du, Ph.D. TECHNICAL PROPOSAL B BIOCHEMICAL ENZYMATIC SYSTEMS FOR DETECTION OF VINYL CHLORIDE AND OTHER CHEMICAL INJURY AS A POTENTIAL MEANS OF DETECTION OF CANCER IN HUMANS Characterization of the biochemical and enzymatic changes occurring in progressive exposure in animal experiments will be con tinued. This includes the following studies: (1) The determination of microsomal P-450 content, the cytochrome protein that is responsible for the oxidation of vinyl chloride; (2) The activity of mixed function oxidase; i.e., the capacity for the microsomes to metabolize drugs fN-demethylation); (3) The activity of glucose-6-Pase, a gluconeogenic enzyme and a marker for preneoplastic change; (4) The activity of glucose-6-P dehydrogenase, a pentose shunt enzyme (an increase of this can lead to more ribose and nucleic acid biosynthesis; (5) The level of reduced glutathione in liver for detoxifi cation of carcinogenic metabolites; and (6) The activity of glutathione reductase. This enzyme activity indicates the capacity to regenerate and maintain the level of reduced glutathione. An increase can be an early alarm of the animal for overexposure to the toxic agent, indicating possible early hepatocellular injury. These studies will enable us to identify the sequential cellular enzymatic changes occurring from early injury to tumor development. As has already been reported, the changes in gluconeogenesis and in the pentosephosphate shunt already parallel the alteration seen in primary hepatocellular tumors. The present working hypothesis for the non development of primary cell tumors in the adult animal is the ability of the adult hepatocyte to adequately detoxify the carcinogenic metabo lites of vinyl chloride, chlorooxirane and chloroacetaldehyde. The adjacent cells, sinusoidal lining cells, as well as newborn liver cells, most likely have a decreased ability of detoxification. In this or similar manner, these cells have a greater susceptibility to DNA injury SL 080498 Julie T. Du, Ph.D. due to the prolonged exposure of chloroacetaldehyde to the DNA molecule. This would explain why angiosarcomas occur in adult rats and primary hepatomas occur in newborn rats, as has been recently shown by Maltoni. In our next stage of animal exposure experiments, we will add weanling rats to the vinyl chloride exposure animal group. In these animals, enzymatic assays to document this hypothesis will be performed in a manner similar to the adult rats. Both weanling and adult rats will be utilized for verification and further elucidation of the immuno logical and tissue antigen test systems by Espinosa's group. Urines from these same rats will be utilized to further verify the urinary glycosamino glycan studies by Kupchello's group and the biological end-product analysi by Wong and Taylor. In addition, we plan to investigate two more enzymes related to glutathione and detoxification: (A) Gamma-glutamyl transpeptidase (GGTP) - This enzyme is responsible for the breakdown of glutathione. It has also been used as a circulating enzyme for the detection of early hepatocellular injury. It has, under recent years of clinical evaluation, lost some of its clinical importance due to the high frequency of an elevation of its activity in circulating blood in the absence of any documentable hepatocellular injury. The activity of this enzyme in the liver has been found to increase considerably during several cases of chemical carcino genesis. Higher levels of the enzyme have also been seen in patients with either primary or secondary neoplasms. The possibility that the high enzyme activity in blood might reflect more breakdown of glutathione rather than hepatocellular injury is under evaluation. It may well be that the "falsely positive" elevated GGTP levels in vinyl chloride-exposed individuals without evidence of hepatocellular injury may really reflect a disturbance in the glutathione levels and indicate an impairment in the detoxification of the intermediate metabolite, chloroacetaldehyde, which sub sequently leads to hepatocellular injury. (B) Aralkyl glutathione-S-transferase - The activity of this enzyme can be used to indicate the capacity for detoxifi cation of vinyl chloride metabolites in the rat. The possibility that this might be a better predictor of whether the animal is more or less or equally susceptible to vinyl chloride injury at various exposure levels is the goal of this investigation. These studies in parallel with circulating glutathione reductase will give us a better understanding of the detoxifying capability of the SL 080499 Julie T. Du, Ph.D, animal and, hopefully, we may be able to extrapolate the findings to man to predict better about the worker's detoxifying capacity. There has been some thought that manipulation of the diet may add to the increased ability to form these enzymes for better capacity of detoxification. This potentiality is, at present, most speculative; and, if the previously stated investigations warrant, it would be followed by subsequent studies. The final portion of this study will include the electron and light microscopic studies of these animals exposed to determine the graded morphological changes due to vinyl chloride exposure and to compare these to the enzymatic results already determined. These tissues have already been prepared for analysis, and the first set is now being studied. These include selected groups from 28, 71, and 103 hours of exposure with their paired controls. Similar studies will be performed with the next group of more prolonged exposures. We feel strongly from our human studies that these animal electron microscopic evaluations will greatly assist us in understanding the changes we see in our human studies being performed by Drs. Schrodt and Tamburro. SL 080500 TECHNICAL PROPOSAL C TISSUE AND URINARY GLYCOSAMINOGLYCAN CHANGES RELATED TO VINYL CHLORIDE INJURY: USE AND EARLY DETECTION AND DIAGNOSIS CHARLES E. KUPCHELLA, PH.D. SL 080501 0 E. Kupcheiia, Ph.D. TECHNICAL PROPOSAL C TISSUE AND URINARY GLYCOSAMINOGLYCAN CHANGES RELATED TO VINYL CHLORIDE INJURY: USE AND EARLY DETECTION AND DIAGNOSIS During the second year of this proposal, work will continue to complete studies undertaken during the first year. Vnese will include a completion of our evaluation of the effects of vinyl chloride exposure with and without liver injury on the urinary glycosaminoglycan excretion patterns. We have begun and, hopefully, will complete during the middle of the second year the evaluation of 24-hour urine sample collections from workers who fall into high, medium, and low exposure categories (exposure data coming from the Vinyl Chloride Medical Surveillance Data Bank). In addition, individuals already demonstrated to have liver injury, with both high vinyl chloride.exposures as well as low, will also be studied. There will be two control groups involved in this study. One will be those derived from individuals with other fibrogenic liver diseases, such as cirrhosis, hepatitis, and alcoholic liver injury. Since the preliminary data suggests the possibility that the urinary excretion patterns for vinyl chloride injury are distinctly different from these other frequently found non-occupational illnesses, this work will involve determination of the specific GAG excretion fractions and their complete biochemical characterization. This is needed in order to determine the usefulness of specific GAGs in the assessment of vinyl chloride injury as well as other chemical liver injury. Such data will have great value in future assessments of the effects of many types of hepatic toxic chemicals and exposure to them in the work place. These studies are expected to result in the development of specific tests for early liver damage and would, hopefully, become part of the routine medical screening of workers with potential exposure to hepatic toxic chemicals. Methods to determine the chemical characterization and identification of these specific GAGs are cited in the original grant application. Animal studies to be conducted during the second year will be the experiments, described in the original application, designed to determine the direct effects of vinyl chloride exposure on the experi mental animals on the GAG changes In tissue and urine. These studies will involve both animals exposed to relatively high (10 to 20 thousand parts per million) and relatively low (25 to 50 parts per million) levels of vinyl chloride. These studies will enable us to identify the specific testing parameters that would lend themselves best to routine screening Q30502 SL W i ii. Rupchiilla, Ph.D. 10 In the working population, as well as to assess the specific role of the GAG in fibrogenesis both in their reversible and irreversible stages. Depending on these observations, those specific GAG changes in the urine that appear to be valuable as indicators of liver injury will be further developed into a "final" procedural modification for the determination of these urinary GAG fractions by quick simply methodology--spot tests, perhaps--that can be used as a routine medical screening. In the third year, studies will be made with other animal models; and we will conclude our evaluation of the human population. The animal model studies will include: Cl) A study of the effects of partial hepatectomy on urinary and liver GAG levels - These studies will be done to assess the effect of cell division and cell regeneration on the liver and urinary GAG levels. This is of vital importance since many individuals working in the indus trial environment also have non-occupationally related liver injury--such as viral hepatitis and alcoholic injury--which stimulate liver cell division and liver regeneration. (2) Studies of the role of GAG in fibrogenesis, generally One set of studies will involve the effect on urinary and tissue GAG after stopping chemical liver injury (carbon tetrachloride) before and after the stage of irreversibility. (3) Studies of the incorporation of radio sulfur labeled GAG subunits into liver tissue during fibrogenesis induced by both carbon tetrachloride and vinyl chloride exposure. (4) Studies of prolonged exposure to determine the specific effects of the presence of developing cancer on the GAG levels - This will be conducted both in the animals with vinyl chloride-induced angiosarcoma and/or primary hapatoma, as well as in experimental animals carrying transplantable hepatomas (Novikoff). SUMMARY Preliminary results, both in the human material and in the prelimi nary animal experiment studies, indicate that the GAG changes in both urine and tissue may well be in the earliest demonstrable change to occur with liver injury. Work in the second and third years will help confirm the usefulness of these parameters in early detection, identify the significant leads for the design of future studies of the role of GAGs and, hopefully, give further information that may permit interference SL 080503 C* Ei ^up^r u with and/or modification of GAG metabolism's leading to fibroses of the liver. Finally, these studies will help produce the data concerning the relative specific effects of cancer development and how this may differ from non-occupational hepatocellular injury, such as that due to viruses or alcohol. r SL 080504 TECHNICAL PROPOSAL D ELECTRON MICROSCOPIC EVALUATION OF LIVER INJURY FROM CHEMICAL WORKERS G. RANDOLPH SCHRODT, M.D. CARLO H. TAMBURRO, M.D. SL 080505 Schrodt and Tamburro TECHNICAL PROPOSAL D ELECTRON MICROSCOPIC EVALUATION OF LIVER INJURY FROM CHEMICAL WORKERS Recent studies in this and other laboratories have suggested that in individuals exposed to vinyl chloride there is an increase in intralobular collagen (fibrous tissue) deposition. The more obvious increase in portal and subcapsular fibrosis is now well known. Our own preliminary data indicates that this collagen deposition occurs much earlier in the parenchyma of the liver rather than in the portal or sub capsular area. The collagen deposits apparent in light microscopic studies only with special staining are more readily visualized with the electron microscope. Most of this collagen appears confined to the space of Disse along the surface of the hepatocyte. This occurs more frequently between the sinusoidal lining cell and the hepatocyte surface rather than between hepatocyte to hepatocyte junctions; not infrequently deep invaginations of the space of Disse creates the impression that collagen bundles are found within the hepatocyte. This gives the impression that the fibrous strands are actually compressing the hepatocyte. As part of the total patient evaluation of individuals exposed to vinyl chloride, electron microscopic studies of the liver biopsies of a selected number of individuals have already been made. Following these initial observations, evaluation of the ultrastructural findings, with both the light microscope as well as the electron microscope, has been underway. Observations in individuals who have developed angiosarcoma following extensive exposure to vinyl chloride have demonstrated a marked increase in the collagen deposition in the hepatic perisinusoidal spaces. This appears to eventually lead to the destruction of the hepatic cell cords and is associated with the activation of the sinusoidal lining cells with focal sinusoidal widening and sinusoidal cell atypia. This fibrosis and widening of the sinusoids with the sinusoidal atypia appear to be a premalignant indicator of hepatic injury due to vinyl chloride exposure. These studies are supportive of those findings by Kupchella et al. in which there are increased glucosaminoglycans in both tissue and urine samples of vinyl chloride-exposed individuals. The importance of recognizing this early increase in collagen deposition in the perisinusoidal space and its lobular distribution may well characterize the vinyl chloride-exposed injury from other types of SL 080506 Schrodt and Tamburro collagen deposits, such as those associated with alcohol, drugs and viral hepatitis. These other non-occupational diseases are associ ated with the hepatocellular injury and necrosis and subsequently have increases in collagen deposition. In contrast, it appears that the vinyl chloride injury is associated with earlier deposition of collagen in an unusual pattern (parenchymal distribution) and occurs before any significant hepatocellular injury. Systematic comparison of the sinusoidal cell structures and the collagen content of the perisinusoidal spaces in the various zones of the hepatic lobule in individuals with vinyl chloride exposure, as well as exposure to other chemicals, will be continued during the second and third years of this grant proposal. The use of a Hewlitt-Packard 9815 desk-top calculator with surface digitizer with interface, which has recently arrived, will increase both the capability of studying larger numbers of Individuals as well as in crease the accuracy and rapid reproducibility of the manual methods. The calculator is now being programmed to evaluate in a semi-quantitative manner the various ranges of collagen deposition seen in vinyl chloride and other chemically exposed workers. This data will later be analyzed using the work exposure histories of these individuals for comparison of the collagen disposition to the other 19 chemicals to which they had potential exposure. SL 080507 TECHNICAL PROPOSAL E MULTIVARIANT ANALYSIS OF BIOLOGICAL END-PRODUCTS AND VINYL CHLORIDE METABOLITE SYNTHESIS " JOHN L. WONG, PH.D, K. GRANT TAYLOR, PH.D. J. T. JOSEPH, PH.D. SL 080508 John L. Wong, Ph.D., et al. 16 TECHNICAL PROPOSAL E multivariant analysis of biological END-PRODUCTS AND VINYL CHLORIDE METABOLITE SYNTHESIS The methodology for the analysis of vinyl chloride end-products in body fluids and tissues will be developed by the end of the first year of this proposal. With the completion of the methodology, the quantitation of the metabolic end-products of vinyl chloride and the establishment of an exposure level-product concentration relationship will begin, first in animals' tissue and urine. Animal tissue and urines will be collected during the next phase of animal exposure studies and stored for future analysis. The first target metabolite for analysis is chloroacetic acid. The methodology will include gas chromotography and mass spectrometry (GC/MS), as well as high pressure liquid chromatography and Fourier Transform Nucleur Magnetic Resonance (HPLC/ FT-NMR). We have just received a $152,000 FT-NMR spectrometer and plan to couple a gas chromatograph to our present mass spectrometer to make the GC/MS system for use in the developed methodology. Dr. Grant Taylor is the co-principal investigator to this aspect of the project. Since previous animal studies have not demonstrated chloroacetic acid in the urines of animals at low exposure levels but have been re ported in humans at higher exposure levels, these methodologies will help to verify this observation and can lead to the determination of specific threshold exposure levels and could greatly aid in the deter mination of what constitutes a safe environmental exposure for humans, as well as being used as an indicator of excessive exposure for individual employees. The second part of this research proposal involves the meta bolic activation and inactivation of vinyl chloride. The identification of urinary metabolites is still incomplete. There is 35 percent of the vinyl chloride carbon-14 activity in the urine unaccounted for. We will continue to investigate the reactions of nonprotein sulfhydryls (e.g., cysteine and glutathione) with chloroacetaldehyde (CAA) to determine the sequence of conversions. In addition to our recent observations of the double reaction (bis-alkylation) of CAA with cysteine to form the cyclic product mentioned in our first progress report, we have made additional preliminary observations that glutathione (GSH) yielded GS-CHOH-CH2-SG with CAA. These intermediates or their subsequent oxidation/hydrolysis products may account for the elusive urinary metabolites. Furthermore, we have had some initial success with the isolation of nucleic acid derivatives from the putative metabolite reactions; viz., with chlorooxirane and chloroacetaldehyde. Our future studies will continue to elucidate their structures, properties, and reactivities. These metabolic activation and inactivation data will form part of the basic picture which will facilitate the interpretation of the cellular and clinical symptoms, SL 080509 John L. Wong, Ph.D., et al. as well as help us in the early determination of damage induced by vinyl chloride. This information is also essential in identifying these urinary metabolites and in developing the specificity of the methodology for detecting these end-products in biological fluids and tissues. The third portion of this proposal will deal with the prediction of vinyl fluoride as a noncarcinogen and vinyl bromide, iodide, and chloride as carcinogens. On the basis of the physicochemical properties of chlorooxirane in chloroacetaldehyde which we have found, we can make the following reasonable predictions: (1) Fluorooxirane will also be the obligatory intermediate in the metabolic process; and it will behave like ethylene oxide, which is not carcinogenic. It will not have diradical character; hence, the fluoroethanol ' will not be formed from vinyl fluoride. (2) The fluoroacetaldehyde can be formed from fluoro oxirane via the NIH shift. It will have similar activities as acetaldehyde, which is not known to be a carcinogen. (3) The major urinary metabolite would be fluoroacetic acid. (4) Vinyl bromide and vinyl iodide will be very much like vinyl chloride in the metabolic conversions, and they have carcinogenic potential. It would be very Important to confirm these predictions by actual studies of the chemical and biological behavior of the respective metabolic intermediates. If confirmed, the practical implications of the above predictions for the chemical industry would be far-reaching. A graduate student has already expressed an interest in working in this project. Thus, the support of a research assistant can rapidly get this project underway. SL 080510 TECHNICAL PROPOSAL F ANALYSIS AND IDENTIFICATION OF CARCINOGENIC POTENTIAL OF INDUSTRIAL CHEMICALS ULDIS N. STREIPS, PH.D. SL 080511 TECHNICAL PROPOSAL F ANALYSIS AND IDENTIFICATION OF CARCINOGENIC POTENTIAL OF INDUSTRIAL CHEMICALS During the next two successive years of this proposal, this group will continue its mutation screening, part of which has already been completed and detailed in the first progress report. These mutation screening capabilities will include both the salmonella rever sion assays (which measure the base substitutions and deletions) and the bacillus repair assays (which measure susceptibility of repair deficient strains to chemicals). Using these tests, we examined several samples for mutagenic activity. (See the attached table.) The forward mutation rate assay will be added; it measures the ability of a chemical to cause cell mutations directly. Similar assays can be performed in mammalian cells. In the forthcoming years of this program, we plan to implement a mammalian tissue culture assay for mutagenesis. The chemicals to be assayed and tested for mutagenicity include the 19 chemicals for which exposure indices are already available within the medical surveillance data bank and their metabolites, which are to be provided by Dr. Wong. The metabolites will be purified samples of industrially relevant chemicals. Dr. Yankeelov in the Department of Biochemistry and Dr. Taylor in the Department of Chemistry will synthesize various derivatives of both biologically and environmentally occurring molecules; these will also be tested for mutagenicity. In addition, we will continue the carcinogenesis model study of the molecular mechanism for chloroacetaldehyde-mediated mutagenesis/ carcinogenesis. This, hopefully, will result in the isolation of specific cell products which may participate in the mutagenesis process, as well as the identification of other components which may block this mutagenesis. Agents which may shift a cell's predisposition to chloroacetaldehyde-caused mutagenesis and thus develop resistance to this chemical will also be examined. The carcinogenesis model would then be applicable for use in the study of other potential chemical carcinogens or chemicals whose intermediates are carcinogenic. These studies have great practical application since the under standing of the mechanism by which chemicals or their intermediates induce mutagenesis or carcinogenesis will be of enormous assistance In characterizing which chemicals have the practical potential for cancer development. It will also demonstrate those whose mutagenic capabilities are only of biological importance. In addition, these studies will identify those chemicals and/or their metabolites to which priority attention should be given. SL 080512 j TABLE SUMMARY OF PERTINENT SUBSTANCES TESTED FOR MUTAGENICITY Salmonella Bacillus 1. Meta-chlorobenzoyl-cyclobutanecarbonyl peroxide 2. Benzoyl peroxide 3. N-acetoxy-N-phenylacetamide 4. N-cyclobutanecarboxyloxy-N-pheny1acetamide 5. bls-cyclobutane carboxyl peroxide 6. Aflatoxin 7. Chloroethanol 8. Benzo(a)pyrene 9. 4-nltro qulnoline-1-oxide 10. Chloroacetaldehyde 11. Epichlorohydrin 12. Chlorooxirane 13. Butane diepoxide 14. Styrene oxide 15. 3,4 epoxide butene 16. bis(Beta-chloroethyl)phenyl phosphate 17. Beta chloroethyl phosphate (bis cyclohexylamine salt) 18. Lung aspirates from smokers with cancer 19. Lung aspirates from smokers without cancer + NR NR NR NR +++ NR ++ +++ +++ ++ +++ ++ ++ + NR NR + + NR NR NR NR NR ++ , NR + ++ ++ + ++ + + NR NR NR NR NR NR - no reaction + - mutagenic +++ - extremely mutagenic - borderline mutagenicity ++ - strongly mutagenic SL 080513 TECHNICAL PROPOSAL G TISSUE ANTIGENIC SYSTEMS FOR DETECTION OF CHEMICAL-INDUCED CARCINOGENESIS 'S( ENRIQUE ESPINOSA, M.D. SL 080514 Lc1.i^.ipinosa, M.D. 22 TECHNICAL PROPOSAL G TISSUE ANTIGENIC SYSTEMS FOR DETECTION OF CHEMICAL-INDUCED CARCINOGENESIS Tissue antigenic studies during the next two years of this proposal will be addressed to two aspects: (1) Those related to experi ments on human vinyl chloride-associated liver injury nnd (2) those to experiments on rats exposed to vinyl chloride and other related potential chemical carcinogens. A. Experiments on Human Vinyl Chloride-Associated Liver Injury (1) The antigen (S) involved in the stimulation of antibody bound to the angiosarcoma tumor will be further investigated. In parallel studies with Fortwengler et al. we will study the antigen (S) stimulating the cell-mediated immune response to angiosarcoma extract described by this latter investigator. We will study the role in these phenomena of the angiosarcomaassociated antigen described by us and of the other antigenic constituents of the anglosarcomatous liver tissue. These antigens will be prepared from liver angiosarcoma tumor which is now stored frozen. Isolation of the antigen will involve fractionation of the extract by ammonium sulfate, ion exchange chromotography, Sephadex filtration, gel clectrofocusing, and acrylamide electrophoresis. Antigen purification will be monitored by double diffusion tests measuring the highest dilution of test fractions giving precipitin reactions; or, if feasible, a single radial immunodiffusion procedure will be used. Immunoelectrophoresis and disc electrophoresis will be used to control homogeneity of the antigen preparations. Molecular parameters to be estimated will include molecular weight, sedimentation and diffusion coefficients, molecular radius and frictional ratios. These will be measured by sucrose gradient ultracentrifugation and Sephadex filtration. The antigens in these procedures will be monitored by double immunodiffusion tests. The specific antisera to be used are stored frozen. (2) A radioimmunoassay for the detection of the angiosarcomaassociated antigen will be developed. This will be used for the assay of this antigen in serum samples from the B. F. Goodrich Company workers with history of vinyl chloride exposure. The value of this method for the diagnosis of vinyl chlorideassociated liver disease will thus be determined. Following SL 080515 Lru'ique Espinosa, M,D. n isolation of the antigen by the procedures described above, this will be labeled with 1125 by a method described by McCatnmom and Yohn. The radioimmunoassay will be used for - the detection of the tumor-associated antigen in patients' sera and will be done according to the Egan et al. procedure. B. Experiments on Rats Exposed to Vinyl Chloride The changes in liver antigens and antibodies described in workers in the vinyl chloride industry will also be studied in rats chronically exposed to vinyl chloride in order to determine, under well-controlled experimental conditions, the time required for these changes to occur. This is of obvious great importance in order to determine the value of these alterations in the early detection of the disease. Sprague Dawley rats will be exposed to vinyl chloride monomer for up to 52 weeks and used for both the biochemical (Dr. Du) and the present studies. Some rats will be sacrificed on a weekly interval during the first six weeks of exposure and on a monthly interval thereafter. Non-exposed animals kept in similar conditions to the experimental ones will be used ' as controls. Abnormalities found at sacrifice will be recorded, and the livers will be examined histologically. Immunoglobulins bound to the angiosarcomas or the fibrotic livers identified at . sacrificing will be examined by indirect immunofluorescence using frozen tissue sections. Serum prepared from the blood obtained from these rats will be examined for presence of anti tumor antibody by the immunofluorescence procedure using frozen sections of the tumor as substrate. Liver abstracts will be prepared in saline solutions and examined for antigenic changes. These will be tested by immunodiffusion using both anti-human and anti-rat angiosarcoma sera prepared in rabbits. The practical value of these studies is now of even greater importance since Fortwengler et: al. have demonstrated by the lymphocyte transformation test cell-mediated reactivity against an angiosarcomanous liver extract in one of two individuals with liver angiosarcoma. This cell-mediated reactivity was also in two individuals of a group that had extended exposure to elevated quantities of vinyl chloride. The identification and purification of the angiosarcoma antigens will make the specificity and sensitivity of the angiosarcoma lymphocyte trans formation test and the development of immunological methods of early detection of greater clinical use. SL 080516 PART B BUDGET PROPOSAL RESEARCH TECHNIQUES AND METHODS FOR DETECTION AND PREVENTION OF CARCINOGENESIS IN INDUSTRIAL WORKERS UNIVERSITY OF LOUISVILLE CANCER CENTER LOUISVILLE, KENTUCKY 40201 SL 080517 BUDGET SUMMARY I. CLINICAL IMMUNOLOGY Personnel Supplies and Expenses II. CLINICAL BIOCHEMISTRY Personnel Supplies and Equipment III. CLINICAL BIOCHEMISTRY Personnel Supplies and Equipment IV. CLINICAL PATHOLOGY Personnel Supplies and Expenses V. MICROBIOLOGY Personnel Supplies and Expenses VI. CHEMISTRY Personnel Supplies and Expenses VII. PATHOLOGY Personnel Supplies and Expenses Year 2 Year 3 30,605 31,523 19,441 ' 21,371 23,225 13,075 23,922 14,373 8,900 "* 8,800 9,476 8,690 7,735 8,508 15,160 12,400 16,747 11,500 11,100 16,452 11,845 17,657 13,990 7,200 14,719 7,590 SL 080518 VIII. FRINGE BENEFITS IX.TOTAL (Personnel and Expenses include fringe benefits) X. INDIRECT COSTS TOTAL Year 2 (11,033) 188,083 Year 3 (11,596) 197,921 59,765 62,813 247,849 260,734 SL 080519 BUDGET M.C.A. PROPOSAL CLINICAL IMMUNOLOGY H. Philip Fortwengler, Investigator A. Toxicity/Tumorgenecity Study 1. Personnel d * H. Philip Fortwengler, MS b. Michael Dever, BS Research Technician c. Linda Smith, BS Research Assistant Supplies and Expenses & * Animals, 300 per year b. Animal Maintenance c. Cell Culture Reagents d. Sterile Culture Flasks e. Chemical Reagents f. Needles, Syringes g- Sterile Cell Culture, Disposable Plasticware h. Miscellaneous Expendables SUBTOTALS B. Immunocompetence Study 1. Personnel (No additional cost) 2. Supplies and Expenses a. Lymphocyte isolation reagents b. Cell culture media c. 3H isotopes d. Scintillation reagents 6 Miscellaneous reagents, glassware SUBTOTALS Year 2 15,500 9,305 5,800 30,605 1,100 1,195 1,300 1,000 500 300 900 300 6,595 1,500 500 450 1,100 250 3,800 Year 3 15,965 9,584 5,974 31,523 1,210 1,315 1,330 1,100 550 330 990 330 7,155 1,650 550 495 1,210 275 4,180 SL 080520 c HL-A Tissue-Typing X. Personnel (No additional cost) 2. Supplies/Expenses a. Hamilton syringes b. Tissue-typing antisera and plates c. Miscellaneous reagents, glassware SUBTOTALS D. LAI Test for Angiosarcoma 1. Personnel (No cost) 2. Supplies and Expenses' 3 Cell cultures b. Cell maintenance c. Miscellaneous reagents, glassware SUBTOTALS E. Xeroxing and publication costs YEARLY TOTALS II. CLINICAL BIOCHEMISTRY - ENZYME Julie T. Du, Ph.D., Investigator A. Personnel 1. Julie T. Du, Ph.D. 2. Ruth Shelton, MS SUBTOTALS B. Supplies and Expenses 1. Laboratory Supplies a. Enzymes b. Radioactive SUBTOTALS Year 2 Year 3 500 5,300 300 6,100 550 5,840 330 6,720 250 1,346 1.100 2,696 250 266 1,590 1,210 3,066 250 50,046 52,894 13.225 10,000 23.225 13,622 10,300 23,922 1,100 979 2,079 1,200 1,078 2,278 SL 080521 29 Animal Expenses a. Animals b. Animal care 1. Short term (200) 2. Long term (300) SUBTOTALS YEARLY TOTALS Year 2 1,350 1,920 7,726 10,996 36,300 III. CLINICAL BIOCHEMISTRY Charles E. Kupchella, Ph.D., Investigator A. Personnel 1. Charles E. Kupchella, Ph.D. (No cost) 2. Leslie Caine, Procurrent Technician 8,900 SUBTOTALS 8,900 B. Supplies and Expenses 1. Lab/supplies/chemicals 2. Enzyme preparations 3. Glassware 4. Equipment maintenance 5. High pressure liquid chromatography columns 2,900 600 1,550 350 1,500 ^ SUBTOTALS 6,900 C. Computer 1,000 D. Travel 900 YEARLY TOTALS 17,700 CLINICAL PATHOLOGY G. Randolph Schrodt, M.D., Investigator A. Personnel 1. G. Randolph Schrodt, M.D. (No cost) B. Supplies and Expenses 1. Lab materials 2. Chemicals 3. EM preparation and tissue storage material 4. Photography equipment and supplies YEARLY TOTALS 2,550 650 1,435 3,100 7,735 Year 3 1,485 2,112 8,498 12,095 38,295 9,476 9,476 3,590 600 1,200 350 1,050 6,790 1,000 900 18,166 2,700 800 2,508 2,500 8,508 - SL 080522 MICROBIOLOGY Uldis N. Streips, Ph.D., Investigator A. Personnel 1. U. N. Streips, Ph.D. 2. Research Technician 3. Research Assistant SUBTOTALS B. Supplies and Expenses 1. Media 2. Glassware 3. Isotopes 4. Equipment maintenance 5. Publishing costs SUBTOTALS C. Travel to national meetings to consult with experts in the field YEARLY TOTALS CHEMISTRY John L. Wong, Ph.D., Investigator A. Personnel 1. John L. Wong, Ph.D. (No cost) 2."-.Joseph P. Joseph, Ph.D. Post-doctoral 3. Research Assistant SUBTOTALS B. Supplies and Expenses 1. Chemicals, VPC, HPLC supplies 2. Glassware 3. _ Lab supplies 4. Publication costs 5. Equipment maintenance SUBTOTALS C. Travel to meetings to consult (for 2) YEARLY TOTALS Year 2 2,000 7,500 5,660 15,160 4,500 2,000 3,000 500 1,000 11,000 1,400 27,560 8,600 2,500 11,100 3,500 3,800 3,400 2,800 552 14,052 2,400 16,452 Year 3 2,000 8,087 6,660 16,747 5,000 1,000 2,100 600 1,200 9,900 1,600 28,247 9,000 2,845 11,845 3,600 3,700 3,605 3,000 752 14,657 3,000 17,657 30 SL 080523--- 4 ' VII. PATHOLOGY Enrique Espinosa, M.D., Investigator Personnel 1. Enrique Espinosa, M.D. (No cost) 2. Virginia Ford, B.A. Research Assistant 3. Janice Gettelfinger Part-time Clerk-Typist SUBTOTALS Expenses and Supplies 1. Supplies/expendable 2. Rabbits, purchase and maintenance 3. Equipment maintenance 4. Publication costs SUBTOTALS Travel to other laboratories for consultation (2 trips) YEARLY TOTALS Year 2 Year 3 9,330 4,660 13,990 3,300 2,200 500 600 6,600 600 21,190 9,980 4,739 14,719 3,490 2,300 500 650 6,940 650 22,309 SL 080524 ADDITIONAL BUDGETARY JUSTIFICATIONS In the individual budget summaries of each proposal, there will be noted an 18 percent increase in budgetary personnel costs and a 12 percent increase in the cost of supplies and materials. This con stitutes an over-all increase in the budget of approximately 16 percent. This increase in both personnel and supplies budgets is totally a reflection of inflationary costs and is not an increase in actual fund requests. The major increase in the budget is in indirect costs. The University overhead cost is based on a fixed value of 65 percent of wages and salaries. From these indirect costs comes the physical and structural support from the University to the investigative projects. In addition, this year this includes the availability of a Type 3 exposure facility and its safety and operational personnel. This facility is under construction and was funded by the University through general University funds, and its operation and maintenance will be supported by general University funds. The indirect costs reflect the depreciation costs for the availability of these facilities. In the absence of such indirect costs, the direct charges to various investi gators and projects for housing hereof and exposure of experimental animals would be substantially and significantly increased. SL 08052