Document LK2Jrd20pzg4ZjnYD0VayO6d5

^'/ MANUFACTURING CHEMISTS ASSOCIATION 1825 CONNECTICUT AVENUE. N W , WASHINGTON, O.C. 20009 TELEPHONE. (202) 328-4200 TELEX. 89617 (MCA WSH) August 17, 1978 TO: Vinyl Chloride Research Coordinators SUBJECT: University of Louisville, Research Proposal, Third Year, 1979-1980, Entitled, "Research Techniques And Methods For The Detection And Prevention Of Carcino genesis In Industrial workers" Gentlemen: The Chairman, Dr. T. R. Torkelson, has requested that the ' subject document be sent to you with the request that you give it your prompt consideration. A completed copy of the enclosed ballot should be returned to the undersigned as soon as possible, certainly no later than September 11, 1978. As may be recognized, your opinions on the 1979-1980 University of Louisville proposal must be secured prior to its release to the Technical Panel. Panel balloting must then be completed prior to a proposal and commitment being sent to the Management Contacts. If it is your wish to preserve the continuity of this program, prompt action should be taken on the 1979-1980 proposal to prevent a reoccurrence of the interruption of research effort which occurred as a result of delayed sponsorship of the 1978- 1979 research year. Sincerely RECEIVED AUG 211978 JTS:ep Enclosure J. T. Seawell Environmental Affairs Project Manager Vinyl Chloride Research SL 08033-7 Manufacturing Chemists Association Vinyl Chloride Research Coordinators University of Louisville, Research Proposal, Third Year, 1979-1S80 I approve of the research proposal as it has been presented by Dr. Tamburro and his associates and recommend that it be submitted to the Technical Panel. _y I do not approve of the research proposal as presented and recommend changes and/or modifications recorded in the attached letter. Name (Signed) Name (Typed) Zeb G. Bell, Jr. Company------------PPG Industries, Tnt Address________One Gateway Center ---------------------------- Pittsburgh, PA__ L5222. Telephone_______ (412) 434-2585_________ Telex Number Please return one copy of this ballot no later than September 11, 1978 to: Mr. Joseph T. Seawell Manufacturing Chemists Association 1825 Connecticut Avenue, N.W. Washington, D.c. 20009 SL 080338 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 080339 l3 A 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 Uldis N. Streips Enrique Espinosa II. Budget Proposal A. Summary Budget Sheet B. Individual Budget 5 9 14 17 20 36 39 41 SL 080340 III. Addendum -- Additional Technical Proposals for Consideration Proposal Title Investigator H. Whole-Body Auto Radiographic Study of Vinyl Chloride W. J. Waddell I. Vinyl Chloride Metabolism in Isolated Mammalian Liver Cells Richard C. Feldhoff Additional Proposal Budgets A. Individual Budget Page 46 51 56 SL 080341 Technical Proposal A Cellular Immunological Systems for the Detection of Vinyl Chloride Injury H. P. Fortwengler, M.S. 1 SL 080342 1 > . I 1 U L LWyUg-l rb. Introduction. The proposed cellular immunological research for year three funding consists of a continuation and advancement of work in progress. This ongoing research has been composed of three components which will be briefly introduced before giving more detailed information. The first component is directed toward the identification of those Individuals who may be predisposed to chemical injury. Individuals with diagnosed chemically-related injuries as well as those at risk of injury are being tissue-typed to determine If certain genetic profiles can be correlated to increased incidence of disease. The second component of the cellular immunological research is directed toward the identification of an immune defect in the lymphocytes, I.e. the protagonist of the immune system. Immune defects have frequently been reported in cancer patients and could possibly be an early signal that a cancerous lesion is imminent. Our third research component is directed toward a search for evidence of a vinyl chloride (VC) induced tumor antigen. Using the responses of lymphocytes to hypothesized new products found in or on VC induced tumor antigen. Using the responses of lymphocytes to hypothesized new products found in or on VC induced tumors, we are seeking early specific clues that an individuals immune system has at least tried to challenge the tumor. Cellular Immunological Studies. Part 1. HLA Frequencies in Vinyl Chloride Workers. An increased incidence of certain HLA types has been shown by several reports to be associated with susceptibility to various diseases. Furthermore, Mulvihill in the Journal of the National Cancer Institute in 1976 stated that a challenge to science is to develop a means of screening potential employees for abnormal genotypes that predispose them to neoplasia after occupational exposure which is harmless to normal genotypes. The first occupational disease-HLA correlation may have already been found. HLA-B27 antigen has been found more often in workers suspected of having the occupational disease asbestosis than among a control population. HLA tissue typing procedures including the microdroplet lymphocyte cytotoxicity test have been initiated In our laboratory and we have typed approximately 300 individuals from the Louisville vinyl chloride polymerization plant. Individuals do not change their genetic complement of HLS antigens, so the determinations need not be repeated periodically as in various clinical assays. Tissue typing for 11 HLA-A antigens and 16 HLA-B antigens and their possible Increased association with angiosarcoma or other chemically related diseases is about one-third completed. A preliminary comparison has been made between the HLA frequencies of VC workers found to have liver disease and those without liver disease. Although the number of individuals tested is not large enough to allow statistical significance to emerge', potential difference in frequencies may be seen at antigens A9, B15, and B17. As an additional procedural and population check, our compiled data is being compared with the frequencies obtained by two other large HLA studies. Frequencies of the healthy individuals studied by Scott et al, 1977, and the World Health Organization compare favorably to the frequencies found in our study here at the University of Louisville. , It has been clearly established that the larger the population studies the greater the chances of obtaining statistically significant data. It is anticipated that the accumulation of our HLA tissue typing data on the majority of the VC popu lation plant workers will take an additional two years (three years total). SL 080343 H.P. Fortwengler, M.S. 3 Cellular Immunological Studies. Part 2. Evaluation of Immunocompetence of Humans Chronically Exposed to Vinyl Chloride. It has been demonstrated that lymphoid cells (T cells) can be cytotoxic to human tumor cells and are often found decreased or porrly functioning in cancer patients resulting in various degrees of immunodepression. Viola, et al, reported others reported the increased incidence of human angiosarcoma in VC production workers* It is the purpose of this study to determine the immunocomptence of indi viduals that have undergone prolonged exposure to VC monomer and have developed liver lesions which, in some Instances, are thought to presage the development of angio sarcoma. The scientific literature is replete with demonstrations of immunodepression as shown by one or more immune parameters in cancer patients at various stages of disease. There is very little information in the scientific literature concerning lmmunoevaluation prior to diagnosis of frand malignancy. We are using immunological assays that have demonstrated usefullness in indication immunodepression in cancer patients. These tests include enumeration of lymphocytes and certain lymphocyte subpopulations as well as tests for lymphocyte function. (For details, see annual report, year 1). These tests have been used to evaluate the Immunocompetence of individuals with possible pre-mallgnant lesions or other liver disease as demonstrated by biopy. A comparison of these diseased individuals with normal plant workers demonstrated nothing so far to suggest that the groups were immunologically different. A com parison of those VC workers having high VC exposure (defined as those with lifetime exposure above the plant median) with individuals having low exposure (below the plant median VC exposure) demonstratd a slight pattern of immunodepression when lymphocytes were stimulated by PHA and ConA. Preliminary evaluation of this data indicated'there is no statistical significance between the high and low exposure groups. Even after additional immunological parameters were examined, no statistical differences could be found between the two groups. At this time, our preliminary interpretation of these results is that there is no residual immunological depression as a result of chronic exposure to increased levels of VC as determined by our standard battery of immunological tests. Further computer analysis of our data in addition Co cocomltant correlations with data being received from clinical testing will five us more definitive statistical results. Definitive plans for the third year of this second component of the study must await the completion of computer programs and statistical analysis of data gathered to date. A more complete delineation of third year plans for this research component will appear in second year progress reports. Cellular Immunological Studies. Part 3. A Search for Evidence of a VC Induced Tumor Antigen. New antigens arise on tumors formed as a response to carcinogens. Their presence on methylcholanthrene-induced sarcomas was discovered by Foley in 1953. This discovery in mice was verified and extneded by Prehn and Main in 1957 to conclude that there were antigens peculiar to and specific for tumor tissue. Sub sequently, evidence for tumor antigens was found in humans by the Hellstroms, Vankey, Halliday and Maluish, Thompson and others. The majority of the evidence suggested that the tumor antigens found were distinctive for each histological type of tumor. In an effort to capitalize on the well established fact that the body mounts SL 080344 H.P, Fortwengler, M.S. 4 an immune reaction to cancer, we are endeavoring to find evidence of these specific immune reactions and then utilize them to develop a relatively specific test for possible VC Induced tumor development. Lymphocytes (the cells responsible for immunity) from the individual tested are being isolated by centrifugation over Ficoll-Hypaque. These cells are then grown in the presence of a liver reagent prepared from either a normal individual or an individual who had angiosarcoma. A positive reaction is considered a three times increased incorporation of H^-thymidine into stimulated cultures as compared to unstimulated cultures. A comparison of the responses between vinyl chloride plant workers and normal non-chemical plant workers indicated that there were many people in the general population having reactivity to tissue antigens irrespective of whether it was from angio or normal liver. Non-tumor specific reactions of this type may be due to sensitization by "natural" means, injections of human or animal substances, transfusions, etc. The reactions of lymphocytes to normal and angio liver reagent were quantitated and a comparison of the responses of VC workers according to known VC exposure depending on whether they had exposure above or below the median plant exposure. No statistical differences were noted between the reactivities of the two groups. On the chance that the non-specific reactions may be obscuring the specific reactions, all those VC workers having reactions to both angio liver and normal liver were grouped. Those with reactions to normal liver only were grouped as were those with no reactions. That left only those with reactions to angio liver, the group in which we are most interested. They have what appears to be a specific reaction to angio tumor extract, i.e., negative reactions to normal liver with concomitant positive reactions to angio tumor. The composition of this group was striking--the only reactions obtained with possible specific tumor reactivity were in the group of VC workers. These results, although provocative, must be interpreted cautiously. In the third year of funding, the data will be extended by testing additional VC workers, but more importantly, by testing a comparison group of individuals having no known VC exposure. Our plans for the future, also include a slight reorganization of experimental design to ensure maximal statistical strenght from the data obtained. Computer time and analysis will be used in the assessment of data obtained. / SL 080345, l1 5 Technical Proposal B Biochemical Enzymatic Systems for the Detection of Vinyl Chloride and Other Chemical Injury as a Potential Means of Detection of Cancer in Humans Julie T. Du, Ph.D. SL 080346 Julie T. Du, Ph.D. 6 Characterization of biochemical and enzymatic changes occurring in progressive exposure to vinyl chloride in animal experiments will be continued. These studies will enable us to further identify the sequential cellular enzymatic changes occurring with chemical monomer exposure. As already has been reported, the changes in gluconeogenesis and in glucose 6-phosphate dehydrogenase already parallel the alterations seen in primary hepatocellular tumors. Our first experiments in animals exposed to one to two percent of vinyl chloride for 14 to 137 hours over a one- to four-week period, has shown this decrease in glucose 6-phosphatase and the increase in glucose 6-phosphate dehydrogenase accompanying the changes in glutathione reductase. These enzymatic changes occurred before there were any signs of physical abnormalities or any changes in the con ventional liver biochemical tests performed during this period. These biochemical changes are similar to the alterations found in hepatomas, suggesting that the metabolic changes may predict impending physical changes. In our human studies of vinyl chloride exposed individuals, Popper and Tamburro have identified focal hepatocellular hyperplasia and hyper plastic nodules as one of the earliest histological manifestations of prolonged vinyl chloride exposure. Our present working hypothesis is that these early biochemical changes are indicative of a shift toward increased nuclei acid synthesis and hepatocellular replication, leading eventually to the hyperplastic areas seen in the human livers. We are next planning to complete our study of the light microscopic slides of these animals and to determine whether similar morphological changes are occurring and are compatible with the enzymatic results already determined. These tissues have already been prepared for analysis and the first set is now being completed. These include selective groups from 28, 71, and 103 hours of exposure within paired controls. Similar studies will be completed with those animals from our second experiment which have a more prolonged exposure. We feel strongly, from our human studies, that these animal light microscopic evaluations will greatly assist us in understanding whether these changes seen in the human studies parallel those in the animals and are a correct reflection of the bio chemical changes seen. Our second major study is related to our working hypothesis that adult hepatocytes have adequate capability of detoxifying the carcinogenic metabolites of vinyl chloride, chloroxirane and chloroacetaldehyde, and thereby prevent the development of primary hepatocellular cancers. The adjacent cells, sinusoidal lining cells, have a decreased ability for detoxification and a greater susceptibility to DNA injury due to prolonged exposure of active metabolites, thereby leading to the development of angiosarcoma. This hypothesis is supported Indirectly by the development of both primary hepatocellular carcinomas and angiosarcomas in newborn rats as reported by Maltoni, and by adult rats given significant, con tinuous alcohol ingestion with their vinyl chloride exposure, as reported by Radike and her group in Cincinnati. SL 080347 Julie T. Du, Ph.D, 7 Toward this end our second experiment in animals exposed to two to three percent vinyl chloride for up to 6 weeks has shown an elevated glutathione-reductase in the liver, followed by an elevation of concen tration of glutathione. This was later followed by an elevation of the detoxifying enzymes, glutathione epoxide-S-transferase (GEST) and later glutathione aralkyl-S-transferase (GAST). These biochemical changes were found before the conventional clinical tests showed abnormalities. Vinyl chloride is believed to be metabolized by the microsomal P-450 enzyme system (mixed function oxidase) and detoxified mainly by way of glutathione via transferases. This suggests that the primary route of vinyl chloride metabolism is initial oxidation to chloroethylene oxide and then detoxification by GEST directly. The delayed induction of GAST suggests that the longer exposure and probable saturation of the direct route leads to greater rearrangement of the chloroethylene oxide to chloroacetaldehyde and detoxification with glutathione. Fiala (Journal of the National Cancer Institute, ji7:591-598, 1976) has found that administration of hepatocarcinogens to rats led to an increase in the concentration of glutathione in the liver, and that the concentration remained high until the development of hyperplastic nodules. Our findings of a higher level of glutathione, and glutathionerelated enzymes after vinyl chloride exposure may be an early biochemical precancerous alteration. OUr purpose was and still is to continue the exposure.studies to identify the sequential cellular enzymatic changes occurring from early injury to tumor development. The present working hypothesis for the nondevelopment of primary cell tumors in the adult animal is the ability of the adult hepatocyte to adequately detoxify the carcinogenic metabolites of vinyl chloride. The adjacent cells, sinusoidal lining cells, as well as newborn liver cells, most likely have a decreased ability of detoxification. This would explain why angiosarcomas occur in adult rats and primary hepatomas occur in newborn rats, as has been recently shown by Maltoni. Our next set of experiments will be a prolonged exposure study of rats to vinyl chloride for 6 months or longer until the development of hyperplastic nodules and tumors. In addition to examining the enzymes and metabolites related to oxidation and detoxification of vinyl chloride and, the concentration of glutathione in the subcellular fractions of the total liver homogenate, we will also attempt to isolate the hepatocyte and endothelial cells from control and exposed animals. We intend to compare the cell's ability and capacity to handle the toxic agents, and to elucidate the mechanism of tumor formation in the endothelial lining cells. SL 080348 Julie T. Du, Ph.D. 8 This requires a clear-cut isolation of liver tissue into hepatocytes and mesenchymal cells (Kupffer and endotheial cells). In order to obtain meaningful results, the mesenchymal fraction cannot have any contamination from hepatocyte debris. Kupffer and endothelial cells' densities are very close. A clear separation was not possible until the use of centri fugal elutriatlon introduced by Knook's group in Netherland recently (Knook, et al., Exp. Cell Res. 109:317-329, 1977). The method gives a high yield of endothelial cells and high viability. It involves perfusion with pronase which breaks hepatocytes specifically. For the isolation of hepatocytes, the original method of Berry and Friend (J. Cell Biology, 43:506-520, 1969), modified by Exton's group (Huston e_t al., J. Biol. Chera., 251:5200-5208, 1976) will be adapted. This method yields hepatocytes by perfusion of liver with collagenase. Further, we are also working on developing methods to study the glutathione transferases and glutathione reductase in blood as possible means of detecting early changes. Liver tissue for light microscopy from these animals exposed to vinyl chloride will continue to be studied for correlations of these biochemical changes with morphological alterations. In our next stage of animal exposure experiments, we plan to add weanling rats to the vinyl chloride animal group. In these animals, enzymatic assays, to document these hypotheses, 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 immunological and tissue antigen test systems by Dr. Espinosa's group. Urine from these rats will be used to further verify the urinary glycosomino glycan studies of Dr. Kupchella and the biological end-product analysis by Wong and Taylor. The final portion of our studies will include the selective electron microscopic studies of these animals to further elucidate the morphological changes occurring at various stages of vinyl chloride exposure, and hopefully, to identify characteristic changes paralleling the enzymatic results already shown. These tissues have already been prepared for analysis and are awaiting our completion of our human electron microscopic studies so that we may have as complete a data base as possible for comparison. SL 080349 9 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 080350 C. E, Kupchella, Ph.D. 10 Project Rationale: Work performed to date in our laboratory confirms the validity of the rationale described in original application, namely: Since hepatic cancer has been shown to produce increases in liver glycosaminoglycans; since hepatic fibrosis has also been shown to produce such increases; since the types of GAG's associated with these two processes are not identical; and, if urinary GAG changes are found to reflect liver tissue GAG changes, urinary GAG analysis may serve as a means of early detection and perhaps a means of differential diagnosis. Further, if the GAG are found to play an important role in the pathogenesis of chemically-induced liver disease, perhaps GAG directed strategies can be developed for the control of these diseases. Approach It should be noted that our study continues to include both animal studies and studies of human clinical material. We continue to evaluate the urines and tissues of animals bearing transplantable liver tumors and the urines and tissues collected from other animal models, i.e., animals with necrotic livers, fibrotic livers, and livers undergoing regeneration. We are also evaluating human urine and liver tissue including: a) patients with hepatic angiosarcoma, b) patients with hepatocellular carcinoma, c) p'atients with fibrotic liver disease related to occupational exposure, i.e., the chemicals like vinyl chloride, d) patients with liver diseases (hepatitis, cirrhosis, etc., unrelated to occupational exposures, e) normal "controls". It should also be noted that while our studies were stimulated by the vinyl chloride/hepatic angiosarcoma problem, our results will have relevance to hepatic cancer, hepatic fibrosis and other chemically-induced liver diseases. Summary of Work Completed to Date We have already found that: 1) Human hepatic angiosarcoma is accompanied by elevated hepatic GAG's. (1) 2) The GAG's in the angiosarcomatous tumors are different from those in fibrotic tissue adjacent to the tumors. (1) 3) Angiosarcoma and hepatoma patients have characteristic urinary GAG patterns -- patterns not found in workers with non-angiosarcomatous liver injury. (2) 4) Heparan sulfate (a type of GAG) is elevated in hepatic tissue under going experimentally induced fibrosis and heparan sulfate is evaluated in the urine of such animals. (3) SL 080351 SL. .^upcnei-ia, i'rwu, 11 5) yet uncharacterized, chondroitin sulfates and hyaluronic acid levels -- but not heparin -- are 3-4 fold higher in experimentally transplanted hepatomas than in normal liver and urinary excretion reflects both the tumor GAG composition and the size of tumors. 6) Livers of animals bearing metastasizing hepatoma (5123tc) have 50-fold greater concentrations of non-sulfated, neutral, uronic acid positive material than is found in the livers of animals bearing two other, non-metastasizing hepatomas. Work to be Performed in Year-3 In our proposal "update" dated May 1977, we listed the following activities to be completed in year-3: 1) assess the contribution to urinary GAG patterns made by hepatic regeneration, 2) study the kinetics of GAG incorporation into connective tissue and GAG tumors, 3) study vinyl chloride injured animals, i.e., the urines and liver tissues of experimental animals with vinyl chloride induced fibrosis and angiosarcoma. 4) evaluate urinary GAG excretion in vinyl chloride workers with evidence of liver injury -- and "control" patients with non-occupationally related liver disease. Some of these studies have already begun. Some studies originally planned to be conducted in year-2 will be conducted in the third year. We will also persue a number of newly developed leads in the third year. Our current plan for the third year is to complete the following investigations: Studies Already Initiated ( 1) THE VALUE OF URINARY C-AG ANALYSES IN THE DIAGNOSIS OF LIVER DISEASE IN MAN FIELD TESTING As part of a double blind study intended to compare 1) urinary GAG analyses, 2) ultrasound diagnosis, and 3) capillary (nail bed) changes as rapid, noninvasiva methods of detection; we evaluated 120 urine samples (occasional samples only -- not 24-hour urines) for urinary GAG's and ;declared each to be either normal or abnormal. The test population included 33 individuals with abnormal liver function tests.and 36 abormal biopsies. While the GAG test provided the best screening for abnormal biochemical results, the statistical results were borderline, i.e., even with "occa sional" random urine samples measuring only the total GAG content and expressing this as a function of creatinine content, the sum of sensi tivity plus specificity was 1.20. When compared to an expectation of 1, this is of borderline significance (z =* 1.89). We will repeat this study in year-3 focusing on specific GAG fractions now known to be affected most severely by liver disease. SL 080352 C. E. Kupchella, Ph.D. 12 2) THE CONTRIBUTION OF NECROSIS TO THE INCREASED GAG LEVELS IN LIVER INJURY We have tentative evidence that indicates that necrosis is responsible for the increased GAG levels in injured and tumor-bearing livers. Using both histochemical and biochemical methods, we plan studies in year-3 to definitively determine the effects on hepatic and urinary GAG's induced by chemical and ischemic necrosis. 3) URINARY GAG PATTERNS IN HUMAN ANGIOSARCOMA, HEPATIC FIBROSIS AND OTHER LIVER DISEASES We will continue to accession patients with angiosarcoma, hepatoma, hepatitis, and cirrhosis to determine if the observation we reported in Cancer (40):2040 holds its specificity and sensitivity. 4) EXPERIMENTALLY INDUCED FIBROSIS AND REGENERATION We will complete early in year-3 the ongoing studies of tissue and urinary GAG changes in animals undergoing hepatic fibrosis and hepatic regeneration -- as described in our previous update. 5) GAG PATTERNS IN EXPERIMENTAL HEPATIC TUMORS We already have data showing that experimentally transplanted Morris Hepatomas have large amounts of GAG's and that this is reflected in urinary excretion. We are evaluating the relationship of these patterns to the behavior of these tumors. Results may lead to the development of GAG directed strategies useful in the control of hepatic cancer and perhaps cancer in general. STUDIES TO BE INITIATED AND COMPLETED IN YEAR-3 1. Vinyl Chloride Exposure We plan to study vinyl chloride exposed animals -- to compare observations with those obtained with CCl^ treated and with hepatoma-bearing animals -- with respect to GAG patterns. 2. Metabolic Studies Using radiolabelled precursors, we will study the synthesis of hepatic GAG's in response to fibrosis, necrosis, regeneration, and the pathogenesis of hepatic cancer. Methods We continue to use -- with only minor modifications -- the methods we described in our original grant application. SL 080353 C. E. Kupchella, Ph.D. 13 Epilog: Our`results to date indicate that GAG changes may be among the earliest demonstrable changes to occur with liver injury. Our third year studies should confirm the degree of usefulness of urinary and tissue GAG measure ments in early detection and differential diagnosis. These studies should help zero in on the role of GAG's in fibrogenesis and the pathogenesis of cancer and illuminate the way toward possible GAG directed strategies for the control of these diseases. REFERENCES 1. Kupchella, C. E. and Tamburro, C. H., Urinary and Tissue Glycosaminoglycan Patterns in Hepatic Angiosarcoma. In: Detection and Prevention of Cancer, H. E. Nieburgs, Eds., Part 1, Volume 1, Marcel Dekker, Inc., New York. 2. Curran, K. L., Kupchella, C. E., and Tamburro, C, H., Urinary Glycosaminoglycans Patterns in Angiosarcoma of the Liver. Cancer 40:3050-3053, 1977. 3. Kupchella, C. E., Jarvis, J. 0., Curran, K. L., Greenberg, R. A., and Tamburro, C. H., Changes in Tissue and Urinary Glycosaminoglycans in Chemically-Induced Hepatic Fibrosis in the Rat. (Submitted) SL 080354 TECHNICAL PROPOSAL D ELECTRON MICROSCOPIC EVALUATION OF LIVER INJURY FROM CHEMICAL WORKERS G. RANDOLPH SCHRODT, M.D. CARLO H. TAMBURRO, M.D. SL 080355 JLU4UUL. (luU IcliiiUliJTiTO 15 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 glucosamlnoglycans 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 080356 Schrodt and Tamburro 16 collagen deposits, such as those associated with alcohol, drugs and viral hepatitis. These other non-occupational diseases are associated with the hepatocellular injury and necrosis and subsequently have in creases 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 sig nificant 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 Hewlett-Packard 9815 desk-top calculator with surface digitizer with interface, has increased both the capability of studying larger numbers of individuals as well as increased the accuracy and rapid reproducibility of the manual methods. The calculator has been programmed to evaluate in a semi-quantitative manner the various ranges of collagen deposition seen in vinyl chloride and other chemically exposed workers. Three normal* individuals of various ages have had their hepatic collagen content studied. There appears to be a clear increased collogen deposition with increasing age. This "normal" data will play a significant role in the interpretation of our chemical worker data. Our chemical worker data will be analyzed using the work exposure histories of each individual for comparison of the collagen disposition to the other 19 chemicals to which they had potential exposure and in comparison to "normal" findings. individual historical, clinical, or laboratory evidence of liver disease, and died from sudden traumatic causes. SL 080357 Technical Proposal E Multivariant Analysis of Biological End-Products and Vinyl Chloride Metabolite Synthesis John L. Wong, Ph.D. SL 080358 J. L. Wong, Ph.D. 18 Our study has been concerned with the chemical systems of detection of vinyl chloride (VC) toxicity. Since the vinyl chloride epoxide (chlorooxirane, COR) is an obligatory intermediate in the normal metabolism of the vinyl monomer and because of the high reacti vity of the epoxide and its tendency to rearrange to chloroacetaldehyde (CAA) which is also reactive, we will continue to develop chemical metho dologies pertaining to the study of: (1) detoxification of the primary metabolites (COR and CAA) in a cytoplasmic environment, i.e. identifica tion of all plausible reaction products, so that the metabolic fate of the epoxide and the aldehyde can be determined as the host factors vary, (2) distribution of the metabolites COR, CAA, and their reduction product chloroethanol (CE) in whole animals and in cells, and (3) putative action of the primary metabolites COR and CAA on nuclear materials with the intention of relating the identity and quantity of the altered nucleic acid bases to pathological findings. Below illustrates our work plan for the coming year. (l) Detoxification Study The trapping of the epoxide and carbonyl compounds in the cyto plasmic environment by sulfhydryl compounds are generally the detoxifica tion reactions. The identification of these sulfur-conjugates as urinary metabolites is still incomplete. There is 35 percent of the vinyl chloride carbon-14 activity in the urine unaccounted for. We have studied the reactions of COR and CAA with sulfhydryl compounds 3,4-dichlorobenzenethiol and N-acety1cysteine. The benzenethiol was used by the Stockholm group in a preliminary study to detect the formation of CAA and COR from VC. The cysteine derivative is a cellular sulfhydryl component as well as a close analog of glutathione. In the case of COR and benzenethiol, the sulfurconjugation product S-acetaldehyde' Is formed. However, CAA and benzenethiol forms the hemithioacetal. These two reactions are distinctly different. The formation of hemithioacetal is reversible but that of the S-acetaldehyde is not. With N-acety1cyteine, both COR and CAA yield the same cyclic con densation product, a dihydrothiazenecarboxylic acid, at low pH. This thiazene is a multi-step reaction product. Its isolation may have solved the mystery of the unknown urinary metabolite as well as the origin of the identified urinary metabolites: the cysteine S-acetic acid and the Sethyl alcohol. Basing on these model studies, two types of experiments will be conducted in collaboration with Dr. Tamburro's laboratory. A. In Vitro Experiments: The cytosol and microsomal fractions will be isolated from hepatocytes and other nonhepatocyte cell lines. They will be used as such or fortified with glutathione to treat the primary metabolites COR and CAA, and the resulting mixtures will be analyzed for the known products described above as well as covalent binding to microsomes. Also, the thiazene compound which is a chemical equivalent to cysteine S-acetaldehyde, will be tested for its capability to generate the urinary metabolites S-acetic acid and S-ethyl alcohol in the presence of cytosol. SL 080359 J. L. Wong, Ph.D. 19 B. In Vivo Experiments: Rats will be exposed to the metabolites COR, CAA, and CE (feeding or ip). Urine analysis for the metabolites and polyamine patterns will be made. It is noted that the latter has been associated with various cell injuries. (2) Distribu tion Study 14 C-labeled COR, CAA, and CE will be synthesized according to known methods. Exposure of whole animals to these tracer compounds followed by the freeze and slice method and autoradiography (with Dr. Waddell) will show the preferential accumulation of these metabolites in various organs. When bacteria and cell cultures are used (with Drs. Streips and Feldhoff), the distribution of the tracers in the cell compartments can be quantitated by scintillation counting. These distri bution data will lend credence to the speculation concerning the patho genesis of vinyl chloride. (3) Putative Action Study The detection study of the putative action of vinyl chloride has been started on the hypothesis that it is due to 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 cyto sine and adenine to form the etheno derivatives, very little is known about the reaction of CAA on the most reactive base guanine. By using a battery of modern analytical tools such as HPLC, GC-MS and FT-NMR, we have found that the guanine base in various forms gives rise to two tricyclic ethenoguanines, the linear etheno compound and the angular etheno compound, and a third product which is still unidentified. These products can dis rupt nucleic acid structures hence their functions by means of steric and electronic perturbation. It is also worthy of note that their fluorescence properties may allow their direct detection in a cell nucleus. We will continue this line of structural and detection study involving CAA. The reaction of COR with the guanine base is more tricky due to the instability of COR in aqueous medium. A multitude of products are formed which we have found to be different from those of the CAA reaction. It is important to note that this is the first indication that COR and CAA show different molecular events in their putative action. Continuation work on the CORreaction will unravel the structures of the major products. Furthermore, we will apply these metabolites to the bacteria systems (with Dr. Streips) in an attempt to correlate the mutation events with the extent and pattern of alkylations. In summary, our strategy is to fully elucidiate the molecular events in the metabolism of vinyl chloride, i.e. detoxification and putative action, and use it as a model to screen the mutagenic and carcinogenic potential of other industrial vinyl monomers. Preliminary results have shown promise in evolving a protocol for early detection and prevention of certain industrial carcinogenesis. SL 080360 20 TECHNICAL PROPOSAL F ANALYSIS AND IDENTIFICATION OF CARCINOGENIC POTENTIAL OF INDUSTRIAL CHEMICALS ULDIS N. STREIPS, PH.D. SL 080361 U. N. Streips, Ph.D. 21 ASSAYS FOR THE CARCINOGENIC POTENTIAL OF INDUSTRIAL CHEMICALS UTILIZING PROKARYOTIC AMD EUKARYOTIC SYSTEMS INTRODUCTION Our laboratory in collaboration with Dr. J. Wong, Department of Chemistry, has studied extensively the mutagenic properties of vinyl chloride and the potential metabolic derivatives of vinyl chloride. These studies have led to the identification of chloroacetaldehyde monomer hydrate as the probable mutagenic metabolite, and also have suggested that this chemical elicits replication repair in bacteria to rectify damage (1). Since replication repair is an error-prone process, this then may describe the potential carcinogenic mode of action of chloroacetaldehyde. In addition, Dr. Wong has discovered that formation of chloroacetaldehyde is inhibited in the presence of reducing agents such as cysteine, glutathione, and others- formation of chloroethanol, a nonmutagenic metabolite is favored. This finding suggests that the carcinogenic action of vinyl chloride may be blocked by appropriate chemicals. This hypothesis will be tested in vitro in the several experiments described in this proposal. Although to date our work has centered on vinyl chloride and will continue to do so until this model system has been completely investigated, our laboratory has begun in collaboration with Dr. Wong, to examine other industrial monomers such as styrene and acrylyl nitrile and their metabolites by these same techniques. OBJECTIVE Our objective is to implement a fully comprehensive screening facility for potentially carcinogenic industrial chemicals and their metabolites. Once the carcinogenic compounds have been identified they will be examined in several types of assays by our research unit to define their mode of action and examine possible mechanisms for blocking their carcinogenic activity. The latter study will bear directly on potential routes of therapy to exposed populations. SCIENTIFIC BASIS FOR STUDY Although the mechanism for chemical carcinogenesis is still relatively obscure, it is fairly clear that many of the chemicals which elicit cancer (precarcinogens) do so as a result of metabolic conversion into more carcinogenic substances (proximate carcinogens) and finally to chemically reactive electrophilic compounds (ultimate carcinogens) which can react with nucleic acids, proteins, cellular components and are also mutagenic (2). Such seems to be the case of the potent carcinogens 4nitroquinoline-l-oxide (4NQ0) and its reduced form 4-hydroxylaminoquinoline-l-oxide (4HAQ0) (3). Although it is important to determine all the parameters of carcinogenic action by any suspected compounds, mutagenesis can serve as an initial screen for potential carcinogens. To screen for mutagenesis several systems using bacteria (4,5) and eukaroytic cells (6,7) as indicator strains have been developed. Of these systems probably the most comprehensive is the Salmonella reversion assay developed by Ames and coworkers (5,8). His tester strains can identify both base pair changes as well as frame shift mutations (5). Recently, these tests have come under attack as unreliable where human carcinogenesis is concerned (47). However, the accuracy of these tests in predicting carcinogens cannot be ignored. SL 080362-- U. N, Streips, Ph.D. 22 In addition, repair assays can be performed using recombination and repair deficient strains of B_. subtilis and UV and X-ray sensitive strains of S_. cerevisiae (9,10). These tests determine effects on DNA which may not result in mutation but instead result in a potentially lethal event due to structural damage to the DNA. Repair deficient strains cannot make the necessary corrections and as a result are preferentially killed (9). Our laboratory has utilized both the Ames and the repair screens to determine the mutagenic potential and probable mechanisms of action of chloroacetaldehyde, a metabolic derivative of vinyl chloride (1). In addition, tests for the induction of the SOS repair system (48) have recently been reported (49,50). These tests measure the induction of prophage in _E. coli. Ron Yasbin has recently initiated a similar screen with B_. subtilis utilizing the induction of PBSX in polA mutants. (Yasbin et. al., and Streips and Yasbin, in preparation). The investigators consider these screens to be more sensitive toward potential carcinogens than the reversion assays described by Ames. Since these experiments are directed at specifically monitoring the induction of error-prone repair pathways, they may in fact be more representative of carcinogenesis induction. We must, of course, keep in mind the limitations of a single cell model system. There are several conceivable reasons why a compound may be mutagenic in one system but not another. 1) There may be permeability differences between the bacterial and mammalian cell. This probably is not important, since the usual mutagens are reasonably soluble in organic solvents , and the membrane probably acts as an organis solvent for the first step in mutagen attack on the cell. There should not be significant differences between the bacterial and mammalian cell membranes, for the. solubilization of the mutagen, at least. In fact, solubility in both kinds of membranes may be very important step for mutational events, since both mammalian and bacterial DNA are connected to the membranes (1). 2) Mammalian repair systems may have a different efficiency than bacterial repair systems. This could be an important factor since very little is known about mammalian DNA repair machinery. Since bacterial assays are commonly done with repair-deficient strains to accentuate the mutational event, differ ences between bacterial and mammalian cell lines could be even oore pronounced. This is one reason why the studies must be extended to mammalian cell lines for corroborative results. 3) Chromosomes with histones could have a different susceptibility than the bacterial chromosome. This probably is not very likely, since bacteria do have polyamines associated with their DNA, and the mammalian chromosome is not always covered by histones. 4) The mutagens could be differentially detoxified or metabolized. This is extremely likely. It is unlikely that bacteria could detoxify and/or metabolize whereas mammalian cells couldn't, since mammalian cells have a far wider variety of metabolic and detoxification systems than bacteria. However, the opposite is very likely true, and the host activation of potential mutagens has been demonstrated (12). SL 08036J U. N. Streips, Ph.D. 23 To control for the possible differential effects between the two systems, especially in the activation of mutagens, the liver microsomal enzyme activated system was developed by Slater (12) and the host mediated assay by Legator and Mailing (13). In the Slater system the potential mutagen is incubated with mouse liver homogenate either in the presence or prior to exposure to the indicator cells (12). Ames has modified this approach to include lung microsomal enzyme preparations as well (14). The Legator system can detect whether the animal can activate or detoxify compounds which might be mutagenic. The animal is administered the mutagen, then injected with the indicator microorganism. Mutagen and bacterium are administered by different routes. This allows the host to modify the mutagen prior to the time it reaches the bacteria. After a sufficient period of time of exposure, the bacteria are withdrawn, and the induction of mutants is determined. A comparison then can be made between the mutagenic action of the compound in vitro and in vivo (13). Recently, Legator (7) has described an jin vivo test utilizing Drosophila. He suggests there is a strong correlation between mammalian and Drosophila activation systems, and also that Drosophila is the only in vivo eukaryotic system which is both comprehensive, and where effects on chromosome alterations can be studied in short periods of time. A combination of these several screens would probably allow in depth evaluation of the mutagenic potential of any chemical under investigation and alos provide strong clues conerning that chemical mode of action in causing the genetic lesion. Most of these tests utilize mutations which bring about the restoration of gene functions. These are called back mutation or reverse mutations and are restricted to only certain, highly specific sites within the gene. They must be near (as in the addition or deletion mutations) or at the precise site of the original mutations (as in the base substitution mutations). The frequency of reversions, as a consequence, is less than that of forward mutations. However, these can be screened rapidly and easily, simply by plating on a medium which normally would not support the growth of the original mutant organism. Reversion studies have been done in bacterial (5,12) and mammalian (15) cells. On the basis of these studies, the mutagenic properties of several compounds could be determined, and the type of original mutation identified (6). Most of the bacterial studies were done with repair deficient organisms to accentuate the occurrence of reverse mutations (5). Nevertheless, following these initial screens, additional tests need to be made on suspected compounds to determine their carcinogenic activity. For instance, AF2, a nitrofuran derivative is positive for DNA damage in repair tests and is mutagenic toward Escherichia coli, yet is negative in the Ames Salmonella screen (16). Furthermore, most mutagenesis studies are generally concerned with the mechanism of base sequence alteration due to a minority product or relatively rare event. In these studies products or structural changes occurring in high yield without regard for capacity to induce mutation are ignored (17). Thus, the action of carcinogens which result in structural changes of DNA, such as single-strand scissions, UV photoproducts, or alkylation of bases may involve many cell processes of repair and synthesis of which mutation may only represent a small fraction indicating successful completion of the task. The majority of events may result in non-mutagenic repair or cell death. SL 080364 1 U. N. Streips, Ph.D. 24 Considerable research has been done to study physically the mechanism of action of several carcinogens. In most of these studies transforming DNA in the Bacillus sub tills sytem has been utilized. For instance, Strauss has described that the alkylating agent, methylmethanesulfonate (MMS) destroys transforming activity of DNA by causing single stranded breaks and depurination of the DNA molecule. If cells are treated with MMS and the DNA is extracted following the treatment, single stranded scissions predominate. When the experiment is performed on DNA in vitro, the DNA is inactivated at 50C, too low a temperature for thermal denaturation. This DNA is primarily inactivated by the formation of apurinic sites (18), although alkylation of bases and single strand scissions also occur. Furthermore, a uvr~ strain of B_. subtilis which cannot repair UV damage, can partially repair MMS induced damage in transforming DNA. Presumably this strain is deficient in either excision, or repair replication but not in the last step, the ligation of the strands. Apparently, the MMS induced alterations require only this last step for restoration of activity. Intensive research has also been done with the potent carcinogens 4NQ0, and 4HAQ0. Sugimura has described that 4NQ0 is converted to 4HAQ0 by rat liver enzymes (19) . Both of these compounds are mutagenic (20), though 4HAQ0 is the more potent mutagen. The difference between these compounds can be resloved by investigating their effect on DNA; thus, 4HAQO effects greater damage on naked DNA than does 4NQ0. It appears that a bacterial enzyme converts 4NQO to 4HAQO in vivo, because 4NQ0 is equally efficient in inactivating bacteria by damaging bacterial DNA (3). Here again the value of a transformation assay becomes apparent for the treatment of DNA in vitro or in vivo can give differential results (21). The action of 4HAQO has also been defined. It is active only In the presence of 02, therefore the carcinogenic process must be the result of an interaction with a proximal compound formed by the oxidation process from 4HAQ0 (20). In animals, cancer can be induced by single injection, yet in vitro 4HAQ0 changes to an azoxy compound which becomes inactive within 10 minutes (20). Therefore, the time interval necessary for the determination of the fate of the cell is short. The interaction of carcinogenic amines and amides as well as polycyclic hydro carbons with transforming DNA in B_. subtilis has been documented (3,22,23). In these studies it has been shown that all of these compounds interact specifically with the DNA causing it to lose biological activity. Moreover, this DNA, when introduced into recipient cells, brought in a significantly higher proportion of forward mutations than did control DNA samples. Furthermore, the biological activity was markedly decreased when the treated DNA was introduced into her- strains which are deficient in enzymes required to repair UV light damage (23). UV damage of trans forming DNA may have relation to the G+C content of the DNA. The greater amount of damage occurrs in AT rich segments, primarily because of the significant proportion of thymine dimers (24). Chloroacetaldehyde, the probable carcinogen in vinyl chloride mediated neoplasia (1,25) has also been shown to interact with DNA specifically (26). The action of carcinogens may also inluence profoundly the physiology of test cells. This may be directly or indirectly attributable to the action of the carcinogen on the DNA. Mifuchi et al and Negai have described the induction of respiration deficient mutants of Saccharomyces cerevisiae by 4NQ0 (27,28). These cells also produced a taxohormone-like substance (27). In these properties they showed similarity to cancer cells. SL 080365 U. N. Streips, Ph.D. 25 In conclusion, by integrating the several different screening procedures with basic research concerning the molecular basis for the carcinogenic attack, concrete conclusions may be drawn about the carcinogenesis potential of any chemical. More over, once the mode of attack upon the host cells is determined, methods for blocking carcinogenic action may be developed. PLAN OF WORK This section will be divided into two parts. First of all we will outline the mutagen screening facility, then describe the research component of our carcino genic chemical analysis laboratory. A. Mutagenicity screening Established screening procedures: 1. Salmonella assay - We have obtained all the necessary Salmonella typhimurium assay strains from Bruce Ames (8) and this assay is fully implemented in vinyl chloride research performed by our laboratory (1). 2. Repair assay - We have four strains of B_. subtilis which are deficient in various steps of DNA repair; polA, MC-1, her, and FB 13. These fall in the classical repair deficient classes described by Dubnau (29) , and lack specific components of the repair-mechanism. Their use in vinyl chloride related studies by our laboratory is also documented (1). We have extended this study to include several recombinationdeficient classes of JB. subtilis, using CAA as the reactive chemical. Results from these studies are shown in Table 1. 3. SOS repair assay - Neither the Salmonella nor the Bacillus reversion tests directly address the carcinogenicity of the compounds tested. We have recently instituted a screen developed by Ronald Yasbin, which may approach, as closely as is possible in a bacterial system, the assay for carcinogenicity. This test will be described in detail in a forthcoming publication (U.N. Streips and R.E. Yasbin. Microbial Testers for Chemical Carcinogenesis, I.C. Felkner (ed). Marcel Dekker, N.Y.). Briefly, most investigators now envision that the carcinogenesis mechanism may be started by error-prone repair, the so-called SOS pathway. This happens when the cell cannot repair by normal means and now induces a repair system which fixes DNA lesions with no consideration for the correct template. As a restilt, not only has the chemical damaged the DNA of the cell, but the cell in attempting repair has created a mutational site. Therefore, any chemical which induces SOS repair is more than likely a carcinogen. The induction of SOS can be measured in several ways, but the induction of bacterial viruses is the easiest. Therefore, our test measures the extent of virus induction in a cell population as a measure of SOS Inducing potential. When virus is induced, the cell dies. Therefore, in Table 2 100% survival is no SOS induction, less than 100% means SOS has been induced. At the present time several laboratories are assaying various chemicals as well as other materials by the SOS test and correlating this data to known carcinogenicity. At this time the initial preliminary evaluation is that this test may be a very accurate predictor of the carcinogenic potential of a chemical. SL 080366 U. N. Streips, Ph.D. 26 In Table 2 I present a comparison of the various systems. I have tested over 60 various substances in the last year by the Salmonella and Bacillus repair tests, but preliminarily only a few so far by SOS. I have chosen these four compounds for this presentation because they illustrate the variance of these microbial tests. All four compounds are mutagenic by the Salmonella test. All but styrene oxide are positive in the Bacillus repair assay, and seem to require recombination repair. Methyl methane sulfonate also is reactive toward the strain lacking polymerase I (polA). However, the preliminary indication from the SOS repair assay demonstrates significant differences between these compounds. Both CAA and MMS are quite reactive in this assay, but styrene oxide and EMS (ethyl methane sulfonate) are essentially non-reactive. This suggests that neither styrene oxide nor EMS can induce significant levels of SOS repair. It is interesting that recent reports show EMS not to be carcinogenic while MMS is a potent carcinogen (R. Yasbin, personal communication), It is our intention in the next funding year to finish our studies with CAA, and elucidate the several mechanisms of action of this compound. In addition we will extend our now established research techniques to thoroughly test other vinyl monomers, notably styrene oxide and aerylyInitrile. We will also extend our catalogue of compounds assayed by the SOS screen and draw more reliable conclusion on the applicability of this test for carcinogenesis screening. All of these screens will be applied to any additional samples of industrial chemicals which will be provided for our laboratory either by the various Investi gators in the Department of Chemistry or any other source. Any screened chemicals which prove to be positive in one or more of these assays will be examined more fully, both by repeated screening and also by the research component of our laboratory utilizing the experiments described below. In this regard, due to its differential activity in our screens, styrene oxide is of great interest. B. Examination of potential carcinogens 1. Assays using enucleated cells. This is research performed in collaboration with Dr. Mansfield, a member of the Department of Microbiology and Immunology. Cytochalasin B, a metabolic product of the fungus Helminthosporum dematioideum causes enucleation of cells (30). Following exposure to this antibiotic both enucleated cytoplasts and karyoplasts (nuclei) can be isolated. These techniques are currently being used routinely in our laboratory. These nuclei and cytoplasma can be treated with carcinogenic chemicals then fused, using Sendai virus (31), back to untreated cytoplasma and nuclei respectively. The fused products are then cultured in agarose and examined for one of several parameters of neoplastic transformation (32). These experiments will be implemented to examine potential carcinogenic chemicals isolated in the initial screen. This will add a mammalian system to our research program and also examine the effect of potential carcinogens on cytoplasmic components (i.e. membrane, mitochondria, etc) as well as nuclear material. 2. Transformation studies Initially we will examine the direct effect of any mutagenic chemicals on transforming DNA. High molecular weight transforming DNA can be isolated by extraction of cells with phenol (33). This DNA will be placed in dialysis tubing and the tubing is immersed in a solution of the mutagen. The concentration of SL 080367 U. N. Streips, Ph.D. 27 mutagen as well as pH can be regulated and varied. The mixture is incubated for various lengths of time. The dialysis tubing containing the DNA is removed and dialyzed against buffer to remove any unreacted mutagen. As a control, the dialyzing solution can be assayed for the presence of the mutagen on recombination deficient strains of B. sub tills. The resulting DNA samples will be analyzed spectrophotometrically to detect changes in concentration and absorbance spectrum. Radioactively labeled samples will be analyzed on neutral and alkaline sucrose density gradients to detect fragmentation and single stranded regions. Finally, the biological activity of these samples will be determined by transformation of both repair-positive and repair-negative strains of J3. subtilis. (Initial experiments are shown in Tables V and VI of the publication attached to this proposal). Secondly, repair-positive and repair-negative strains will be exposed to the mutagens. Following incubation with the mutagen the cells will be washed and the DNA will be isolated by phenol extraction. This DNA will be analyzed by the same techniques as were employed for the extracted DNA (above) . These experiments should identify the physical damage done to the DNA by the mutagenic fractions and whether the bacterial repair systems can repair the damage. By exposing whole cells to the mutagen, activation of the mutagen to a proximal form by the bacteria can be tested. In addition, all of these assays will be repeated in the presence of both liver and lung microsomal enzyme preparations according to Ames (14). Dr. Laumbach of our group has performed these types of experiments in testing the 4NQ0 mutagenesis system (23). a.) Effect of DNA synthesis Many mutagens act preferentially at the replication fork of DNA (34). There by, DNA synthesis is necessary for these mutagens to act. There are mutants of 3J. subtilis which are temperature sensitive for DNA synthesis (35). At the permissive temperature (37C) they grow normally, however, at the restrictive temperature (45C), even though proteins and RNA synthesis are normal. DNA synthesis is arrested. Mutagenic fractions will be incubated both with wild type cells and the temperature sensitive mutants. The effect on the viability of the two types of cells as well as on the DNA (assayed by the techniques described in the previous section) by the mutagenic chemicals will be determined. All experiments will be repeated in the presence of microsomal enzyme preparations. Bacterial spores, as they germinate, initiate DNA synthesis and shortly thereafter excrete DNA in linear fashion starting with the replication origin (36). Therefore the appearance of specific markers in the supernatant fluid is a direct result of DNA replication. We will assay the effect of the chemicals both on the appearance and the physical structure of the DNA. This DNA can be used by transformation and can be examined for mutational events or alterations by the techniques described above. b.) Specific bacterial sequences If the mutagenic chemicals exhibit activity towards DNA, we can begin to determine if there are discrete regions which are preferentially attacked. This could be monitored by use of the restrictiion endonuclease degradation system. Restriction endonucleases hit specific sequences in DNA (37). Such sequences occur at random in DNA, but if you have a discrete segment of DNA such as bacteriophage genome, then degradation by the endonuclease will always result in the production of the same fragments. These can be resolved by gel electrophoresis (38). We are SL 080368- U. N. Strelps, Ph.D. 28 presently assaying the genome of the 33. subtilis bacteriophage 029 in this manner. In addition, we have had success in isolating discrete segments of the 13. subtilis chromosome both by isolating segments of DNA following excretion from germinating spores, and by isolating membrane bound DNA and removing the membrane (U.N. Streips and R.J. Doyle, manuscript in preparation). Both the bacteriophage and the bacterial systems can be used in assaying the effect of the mutagenic fractions on degradation patterns of those DNA segments by endonucleases. This work has been ongoing in our laboratory for the last year. Untreated 029 DNA and bacterial DNA will serve as controls. In test samples both 029 and bacterial DNA will be exposed to the mutagen in dialysis tubing as described above. These tests will be done in the presence or absence of microsomal enzyme preparations. Following treatment, both control and test samples will be incubated with restriction endonucleases (we have in our laboratory EcoRI from Eh coli, Hin from H. influenza, and Sma from Serratia) in appropriate buffers. The digested samples will be compared. With the help of Dr. Wong's group these experiments have already been initiated.. We have several samples of phage DNA which has been methylated in vitro with CAA. c.) Membrane interactions. As mentioned In the previous section, we have routinely been isolating DNAmembrane complexes from 13. subtilis. These complexes are enriched for markers near the terminus and near the origin of the chromosome (11,39). If the mutagenic chemicals alter membrane configuration, they may also alter the DNA-membrane association. To examine this parameter we will isolate DNA-membrane complexes from B^. sub tills and an L-form of 13. subtilis we have in the laboratory (40) which have been treated with the mutagenic chemicals. In these experiments, the cell wall is removed from the bacteria by lysozyme, then the resulting spheroplasts, as well as the L-form sample, are ruptured physically by dilution into buffer. The samples are washed exhaustively (15X) by centrifugation at 29,500 rpm, 20 minutes, to remove all non-membrane bound DNA. The resulting samples also will retain enough nuclease to degrade all remaining DNA not covered by membrane, thus enriching the samples for the markers at the origin and the terminus (39). The membrane is removed from these samples by phenol extraction. The DNA can be assayed by transformation for biological activity. Treatment with the mutagenic chamicals may alter the regions protected by the membrane or could abolish the binding altogether. These possibilites could be evaluated in this system. 3. Membrane transport studies These studies will be performed in collaboration with Dr. R.J. Doyle, a member of the department of Microbiology and Immunology. In the membrane studies, the following cell types will be used: B. subtilis 168 (a gram positive bacterium) , Eh coli (gram negative) and S_. cerevisiae (a eukaryotic organism). Cells will be grown in minimal media at 320 to logarithmic and stationary phases, centrifuged, then suspended in dilute buffer solutions. At this time, the mutagen will be introduced to the system. The mixture will be incubated for various periods of time,and the loss of protein or nucleic acid into the medium will be measured using previously developed procedures (41). This should establish whether gross membrane damage has occurred in these cells. Controls, without the mutagen will be run simultaneously. SL 080369 i 0. ft. Streips, Ph.D. 29 In addition, the transport of specific metabolites by mutagenic chamical treated cells will be examined. We will examine basically two types of membranes transport, energy-dependent or active transport; and energy-independent, or facili tated diffusion. For _S. cerevisiae, transport of the non-metabolizable sugar, sorbose, can be determined (42). The sorbose transport system is particularly useful because the sugar enters S_. cerevisiae via the facilitated diffusion systems and is not metabolically altered upon entering the cell (42). In IJ. subtilis, amino acid permease systems have been well characterized and will be utilized in our studies (43). This is done by use of isopr-pyl-thio-g-galactoside (IFTG) as described by Wilson et al (44). IPTG induced the lac operon system which controls both 8-galactoside permease and S-galactosidase (44). Upon induction of E_. coli by IPTG, lactose (or any B-galactoside) enters the cell by an active transport system. In studies done elsewhere (Argonne National Laboratory by R.J. Doyle) we have found that the best assay for galactoside transport in JS. coli is based on the use of ortho-nitro-phenyl-3-D-galactoside (ONPG), a synthetic substrate which can be cleaved by S-galactosidease to yield a visible chromaphore of high extinction. Other assays can be utilized where needed. We will use the established procedures in all these experiments. For example, cells are washed in buffered salt solutions, suspended to a pre-determined density, then subjected to the substance to be transported. The permeants (sorbose and amino acids) are labeled. At intervals, samples are withdrawn, quickly filtered, and washed. The amount of radioactivity in the residue is then determined. This is a facile and reliable procedure which can be used to determine whether membrane transport has been impaired in microbial cells. We will subject the cells to the chemicals and determine the effects of time, concentration, pH and temperature on both facili tated diffusion and active transport systems. Should these results appear promising we will then examine the effects of chemical mutagens on membrane transport of sugars by human red blood cells and by murine fibroblasts. We propose to perform these assays spectrally as described by Stein in his monograph (45). In the murine fibroblast system we will also assay for alterations of the specific surface histo- compatability antigens following exposure to the chemicals (46). The described experiments are straightforward and can be performed quickly, thus, a significant study on the effects of mutagenic chemicals on biological membranes can be completed within a relatively short time. To our knowledge, no research has been published describing the effects of industrial monomer metabolites on membrane transport. We recognize that if a chemical interferes significantly with membrane transport, obvious health implications become apparant. For example, cells could not carry out normal metabolism if membrane transport has been impaired, thereby causing the cells to metabolize endogenous reserves or expire. If only a few components cause alterations of membrane transport it may then be possible to eliminate such injurious substances. That would be one of the long term goals of our research. 4. Blocking of the carcinogenesis process This is potentially the most important section of the research component of this proposal, since this relates directly to possible therapeutic mechanism in vivo. Our collaborator, Dr. J. Wong, Department of Chemistry, has discovered that in the presence of reducing agents, such as cysteine, glutathione, etc., the potentially carcinogenic chlorooxirane is very quickly converted to non-mutagenic chloroethanol. In the absence of these agents, chlorooxirane forms the highly SL 080370- > U. N. Streips, Ph.D. 30 mutagenic chloroacetaldhyde. Utilizing this observation as a model system, we will examine the effect of chlorooxirane in all of the research studies outlined above in the presence and absence of reducing agents. In this way we will attempt to block the mutagenesis activity of this chemical. Pre-treatment of the various components in all of the assays will be approximately varied until optimal conditions are achieved. In these kinds of experiments we may discover not only mechamisms for blocking carcinogenesis, but also other blocking agents which may fit this role and be applicable to in vitro studies. In this section we have described several series of experiments which would yield valuable information concerning the mutagenic potential of various chemicals which have been shown to be active in a preliminary screen. These experiments would examine both the effects of the chemicals on DNA and on the transport systems across the cell membrane. The ultimate aim of these studies is to identify the mutagenic chemicals, characterize their mode of action and seek methods for blocking their carcinogenic potential SL 080371 'i U. N. Streips, Ph.D. 31 Strains REC A recAl recAl REC B recB6 recB3 recB19 recB2 recB2 recB2 REC C recC7 REC D recD27 recD27 REC E recE61 recE4 REC F recF7 recF18 recF15 recF15 recFIS TABLE 1 REPAIR ASSAYS WITH B. SUBTILIS* Average Inhibition in mm/8 experiments Strains Average Inhibition in mm/8 experiments 13.5 7.5 13.5 9.5 16.5 16.5 11.3 16.8 NR jL X * 5 NR 6.7 8.8 6.1 7.4 7.5 8.42 3.1 REC H recH342 REC rec-4 rec-13 MIC mtc-41 (cafr) UVR uvr-35 (cafr) her uvr WT wt wt wt DNA POLYMERASE polA NR 15.4 5.4 NR NR NR NR NR NR NR NR * lOOmM chloroacetaldehyde used in all these experiments SL 080372 CHEMICAL CAA (0.004M) STYRENE OXIDE .01M) EMS (0.001M) MMS (0.001M) TABLE 2 REACTIVITY OF CHEMICALS IN BACTERIAL ASSAYS SALMONELLA REVERSION if REV/PLATE--CONTROL 265 342 312 355 SOS REPAIR % NONTREATED 15% 100% 80% 1% SUBTILIS REPAIR ASSAY W REC- HCR~ UVR- POL ram INHIBITION 0 18 2 2 3 02001 1 16 1 1 1 4 21 6 6 10 G 2t S tre ip s , Ph.D. SL 080373 . Ni U. N. Streips, Ph.D. 33 F. References 1. Laumbach, A. D., Elmore, J., Wong, J., Yen, S. E., and U. N. Streips, (Manuscript submitted for publication). 2. Miller, J, A., 1970. Cancer Res., 30: 559. 3. Yamamoto, N., Fukuda, S,, and H. Takabe, 1970. Cancer Res., 30: 2532. 4. Scherr, G. H., Fishman, M., and R. H. Weaver, 1954. Genetics, 39: 141. 5. Ames, B. N., Lee, F. D., and W. E. Durstan, 1973. Proc. Natl. Acad. Sci., 70: 782. 6. Legator, M. S., 1972, In Mutagenic Effects of Environmental Contaminants, H. E. Sutton, and M. I. Harris, Eds., Academic Press, New York, p. 67. 7. Legator, M. S., and S. Zimmering, 1975. Mutat. Res., 29: 181. 8. Ames, B. N., McCann, J., and E. Yamasaki, 1975. Mutat. Res. (in press). 9. Nagao, M., and T. Suqimura, 1972. Cancer Res., 32: 2369. 10. Koske, R. E., and H. F. Stich, 1973. Mutat. Res., 19: 265. 11. Sueoka, N., and H. Yoshikawa, 1963. Cold Spring Harbor Symp. Quant. Biol., 28: 47. 12. .Slater, E. E., Anderson, M. D., and H. S. Rosenkranz, 1971. Cancer Res., 31: 970. 13. Legator, M. S., and H. V. Mailing, 1971. In Chemical Mutagens, Principles and Methods for their Detection, A. Holleander, Ed., Plenum Publishing Company, New York, p. 569. 14. Kier, L. D., Yamoski, E., and B. N, Ames, 1974. Proc. Natl. Acad. Sci., 71: 4159. 15. Chu, E. J. Y., and H. V. Mailing, 1968. Proc. Natl. Acad. Sci., 61: 1306. 16. Workshop International Agency for Research in Cancer, Brussels, Belgium, reported in Nature, 248: 381 (1974). 17. Flanders, H., 1968. Ann. Rev. Biochem., 37: 175. 18. Strauss, B., and R. Wahl, 1964. Biochim. Biophys. Acta, 80: 116. 19. Sugimura, T., Otake, H., Matsushima, T., 1968. Nature, 218: 392. 20. Ishizana, M., and H. Endo, 1968. Biochem. Pharm., 16: 637. 21. Ishii, Y., and S. Kondo, 1971. Mutat. Res., 13: 193. 22. Maher, V. M., Lesko, S. A., Straat, P. A., and P. 0. P. Tso, 1971. J. Bacter., 108: 202. SL 080374 * U. N. Streips, Ph.D. 34 23. Laumbach, A. D., and I. C. Felkner, 1972. Mutat. Res., 15: 233. 24. Munakater, N., and Y. Xkeda, 1969. Mutat. Res., 7: 133. 25. Malavielle, C., Bartsch, H., Barbin, A., Canurs, A. M., and R. Montesano, 1975. Biochem. Biophys. Res. Commun., 63: 363. 26. Kochetkor, N. K., Shibaeu, V. N., and A. A. Kost, 1971. Tetrahedron Letters, 22: 1993. 27. Mifuchi, I., Morita, T., Yanagihara, Y., Hosoi, M., and M. N. Shlda, 1963. Japan. J. Microbiol., 7: 69. 28. Nagai, S., 1969. Mutat. Res., 7: 333. 29. Dubnau, D., and C. Cirigliano, 1974. J. Bacter., 117: 488. 30. Croce, C. M., Tomassini, N., and H. Koprowski, 1974. Meth. Cell. Biol., 8: 145. 31. Sethi, K. K., and H. Brandis, 1974. Nature, 250: 225. 32. Rapin, A. M. C., and M. Burger, 1974. Adv. Cancer Res., 20:1, Academic Press, N. Y. 33. Streips, U, N,, and F. E. Young, 1974. Molec. Gen. Genetics, 133: 47. 34. Schwartz, M., and A. Worcel, 1971. J. Molec. Biol., 61: 329. 35. Young, F. E., and G. A. Wilson, 1972. In Spores V, p. 77. 36. Borenstein, S., and E. Ephrati-Elizur, 1969. J. Molec. Biol., 45: 137. 37. Boyer, H. W., 1971. Ann. Rev. Microbiol., 25: 153. 38. Danna, K. J., Sack, G. H,, and D. Nathans, 1973. J. Molec. Biol., 78: 363. 39. Synder, R. W., and F. E. Young, 1969. Biochem. Biophys. Res. Comm., 35: 354. 40. Bettinger, G. E., and F. E. Young, 1973. Biochem. Biophys. Res. Comm., 55: 1105. 41. Doyle, R. J., and E. Spoerl, 1968. Radiation Res., 34: 326. 42. Kalsow, C., and R. J. Doyle, 1972. J, Theoret. Biol., 34: 125. 43. Dehauwer, G., Lavalle, R., and J. M. Wiame, 1964. Biochem. Biophys. Acta, 81: 257. 44. Wilson, T. H., Kashket, E. R., and M. Kusch, 1972. In The Molecular Basis of Biological Transport, J. F. Waessuer and F. Huying, Eds., Academic Press, N. Y. SL 08037-5 U. N. Streips, Ph.D. 35 45. Stein, W. D., 1967. In The Movement of Molecules Across Cell Membranes, Academic Press, N.Y. 46. Reisfeld, R. A., and B. D. Kahan, 1970. In Adv. in Immunology, F. J. Dixon and H. G. Kunkel, Eds., Academic Press, N. Y., 12: 117. 47. Ashby, J., and J. A. Styles, 1978. Nature, 271: 452. 48. Witkin, E. M., 1976. Bacterlol. Rev., 40: 869. 49. Meyn, M. S., Rossman, T., and W. Troll, 1977. Proc. Natl. Acad. Sci., 74: 1152. 50. Moreau, P., Bailone, A., and R. Devoret, 1976. Proc. Natl. Acad. Sci., 73: 3700. SL 080376 36 Technical Proposal G Tissue Antigenic Systems for Detection of Chemical-Induced Carcinogenesis E. Espinosa, M.D. SL 080377 E. Espinosa, M.D. 37 Important alterations in liver antigens and antibodies in workers of the vinyl chloride industry have been described in previous work done in this laboratory. Of these changes, appearance in VC-associated liver angiosarcoma of an angiosarcoma associated antigen, an antigenic deletion and demonstration of tumor-bound immuno globulin G appeared worth investigating in further detail for development of a test for the early detection of VC liver disease. In the course of these investigations 2 important questions arose which we propose to answer during the third year of support of this proposal. The first question relates to the time required for the VC-associated anti genic 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. The second question pertains to the specificity of the antigenic changes as related to vinyl chloride. In order to answer the first question, Sprague Dawley rats will be exposed to vinyl chloride monomer for up to 52 weeks. 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 one 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 antitumor antibody by the immunofluorescence procedure using frozen sections of the tumor as substrata. Liver extracts 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. Qur second question "Vinyl chloride specificity of the antigenic changes in the liver" will be answered in experiments using both animal and human tissues. In the animal experiments we propose comparing the antigenic changes induced by vinyl chloride with changes induced by other hepatocarcinogenic agents. An adequate experimental model for this study is Morris hepatoma; lines 7777, 5123tc and 9618A are being maintained in our laboratories for this purpose. Some of these experiments have already been initiated with the collaboration of Dr. C. Kupchella. In addition, possible relationship of these changes to early fibrosis and/or necrosis known to occur in VC-related liver disease will be studied in experiments using carbon tetrachloride liver fibrosis in rats. The antigenic changes will be studied also in human liver cirrhosis and liver tumors unrelated to vinyl chloride exposure. Antigens from the tissues indicated above will be extracted by homogeinization in saline and analyzed by immunodiffusion procedures. Antiserum to be used in the human tissue studies is already available and kept frozen at -35 C for further use. Antiserum to tissues and tumors of rat origin will be prepared in rabbits. Human tissue extracts prepared from VC-associated liver angiosarcoma, hepatomas and liver cirrhosis will be compared for titer of angiosarcoma-associated antigen and for antigen deletions using Immunodiffusion procedures. In the ex periments using rat tissues, antigens appearing to be novel, absent, decreased or SL 080378 E. Espinosa, M.D. 38 increased in concentration within the vinyl chloride, Morris hepatoma and liver fibrosis groups will be compared in order to establish their specificity as re lated to vinyl chloride. The chemical nature of these antigens will be determined by studying their susceptibility to proteases, nucleases, blycosldases and periodate. Other properties such as pH sensitivity, alcohol solubility, and heat stability will be determined. 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. SL 080379 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 080380 39 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 3 31,523 21,371 23,922 14,373 9,476 8,690 8,508 16,747 11,500 11,845 17,657 14,719 7,590 SL 080381 VIII. FRINGE BENEFITS IX.TOTAL (Personnel and Expenses include fringe benefits) X. INDIRECT COSTS TOTAL 40 Year 3 (11,596) 197,921 62,813 260,734 SL 080382 BUDGET M.C.A. PROPOSAL I. CLINICAL IMMUNOLOGY H. Philip Fortwengler, Investigator A. Toxicity/Tumorgenecity Study 1. Personnel a. H. Philip Fortwengler, MS b. Michael Dever, BS Research Technician c. Linda Smith, BS Research Assistant 2. Supplies and Expenses a. 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 e. Miscellaneous reagents, glassware SUBTOTALS 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 41 SL 080383 c. HL-A Tissue-Typing 1. 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 a. Cell cultures b. Cell maintenance c. Miscellaneous reagents, glassware SUBTOTALS E. Xeroxing and publication costs YEARLY TOTALS 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 3 550 5,840 330 6,720 266 1,590 1,210 3,066 250 52,894 13,622 10,300 23,922 1,200 1,078 2,278 2. Animal Expenses a. Animals b. Animal care 1. Short term (200) 2, Long term (300) SUBTOTALS YEARLY TOTALS CLINICAL BIOCHEMISTRY Charles E. Kupchella, Ph.D., Investigator A. Personnel 1. Charles E. Kupchella, Ph.D. (No Cost) 2. Raya Warlck, Technician SUBTOTALS B. Supplies and Expenses 1. Lab/supplies/chemicals 2. Enzyme preparations 3. Glassware 4. Equipment maintenance 5. High pressure liquid chromatography columns SUBTOTALS C. Computer D. Travel YEARLY TOTALS 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 Year 3 1,485 2,112 8,498 12,095 38,295 43 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 080385 V. MICROBIOLOGY Oldis N. Streips, Ph.D., Investigator A. Personnel 1. U. N. Streips, Ph.D. 2. Research Technician 3. Research Assistant SUBTOTALS 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 VI. 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 Travel to meetings to consult (for 2) YEARLY TOTALS 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 44 9,000 2,845 11,845 3,600 3,700 3,605 3,000 752 14,657 3,000 29,502 SL 0803H6- VII. PATHOLOGY Enrique Espinosa, M.D., Investigator A. Personnel 1. Enrique Espinosa, M.D. (No cost) 2. Virginia Ford, B.A. Research Assistant 3. Janice Gettelfinger Part-time Clerk-Typist SUBTOTALS B. Expenses and Supplies 1. Supplies/expendable 2. Rabbits, purchase and maintenance 3. Equipment maintenance 4. Publication costs ,, SUBTOTALS C. Travel to other laboratories for consultation (2 trips) YEARLY TOTALS Year 3 45 9,980 4,739 14,719 3,490 2,300 500 650 6,940 650 22,309 SL 080387 ADDENDUM Additional Technical Proposals for Consideration SL 080388 46 Technical Proposal H Whole-Body Auto Radiographic Study of Vinyl Chloride W. J. Waddell, M.D. SL 080389_ f W. J. Waddell, M.D. 47 Introductory Summary Dr. Waddell proposes to obtain detailed data on the physical and chemical disposition of vinyl chloride in the mouse. The general procedure will be to administer the compound to the experimental animals by inhalation, orally and by parenteral routes and to sacrifice these animals after brief ether anethesia at geometrically increasing time intervals by immersion in a mixture of dry ice and hexane. Whole sagittal sections of the frozen animals then will be taken on a microtome in a cryostat. No thawing or contact with any solvents will be allowed in order to prevent translocation or removal of the substance. After freeze-drying of the sections, they will be placed against X-ray film to allow the production of a whole-body autoradiograph. The distribution of radioactivity in the specific tissues and fluids will be quantitated by a photometric densitometer which will compare the radioactivity in the autoradiograph with the specific area of the section. Using these quantitative data with the geometric time intervals- will allow the calculation of elimination rates from each and every tissue and fluid of the animal. Kinetic data can be obtained by this technique which can be obtained by no other method. Procedures which dissect out organs and tissues are limited by the skill of the dissector; few if any dissectors can obtain clean preparations of islets of Langerhans, bronchial epithelium, pigmented epithelium of the eye, or corpora lutea. Each of these anatomic areas has, within recent years, been shown to accumulate and retain environmental compounds; the accumulation in each of these areas, as well as all areas of the body, can be quantitatively assessed by wholebody autoradiography. In some instances tissues will be removed either from the sections or from animals dosed in parallel experiments; these tissues will be solubilized and quantitated by liquid scintillation counting. Selected areas of accumulation of radioactivity which are of interest will be studied further to identify chemically the nature of the radioactivity. These areas of the section will be removed by dissection, microscopically if necessary, and extracted with appropriate solvents. The extract will then be subjected to chromatography for identification of the chemical nature of the radioactivity. Studies in some animals may include removal of selected tissues or organs for autoradiography plus combustion or homogenization for extraction to quantitate the vinyl chloride and its metabolites. Whole-body autoradiography offers the unique ability of studying the complete disposition of a substance in a single animal. Collection of feces, urine and expired air allows calculation of total loss from the body. Sectioning one-half of the animal provides quantitative information on the disposition of the total substance in every tissue of the body; the remaining half of the frozen animal can be used later for extraction and further quantitation of metabolites. Each animal can be used for total assessment. Dr. Waddell and his colleagues have extensive experience in the study of drug and xenobiotic disposition by this approach and are in a position to offer this experience in correlating the biological disposition of vinyl chloride with its toxiclties. Twenty-five consecutive years of research have been devoted by Dr. Waddell to research on the disposition and metabolism of drugs and xenobiotics. Most of the various techniques available for these various studies have been used. SL 080390 W. J. Waddell, M.D. 48 The facilities available in this laboratory for the proposed study include liquid scintillation counters, chromatography apparatus and equipment, and almost all of the supporting smaller items of equipment for such studies. Whole-body autoradiography for mice is currently in operation in the laboratory so that all of this equipment and apparatus is available. All of the supporting facilities, such as animal quarters, radiation and chemical safety rooms and protection, etc. are available. Statement of Work; General Technical Approach The initial studies will be done in pigmented mice. Since several compounds have been shown to have a remarkably intense and persistent affinity for melanin (in the pigmented epithelium of the eye, the brain, etc.) the initial experiments should be on animals containing melanin. The albine human constitutes such a small percentage of the population that the animal counterpart ranks low on priority for study. The mouse is usually preferred to the rat for Initial study because of easier handling and lower cost for animals and isotopes without loss of any information on biological disposition. In fact the mouse has a gall bladder as does the human; however, the rat does not which makes examination of bile more difficult in this species. Metabolic differences in metabolism may be as wide among strains of rats or strains of mice as are such differences between mice and rats. Therefore, rats would be justified for the initial study only if other information had been obtained on a specific strain of rat which would enhance the informational yield of the study oq the disposition of vinyl chloride. 14 The C-labeled vinyl chloride would be given by inhalation, oral and intra venous routes of administration since these are most appropriate for obtaining valid pharmacokinetic data relating to its use in the environment. The intra venous route most often provides more precise data on the kinetics of distribution and elimination. One must be careful in the interpretation of data from intra venous administration of compounds with very limited water solubility even though this route frequently provides essential pharmacokinetic information. If the compound' is solubilized in a system which does not maintain its solubility after dilution with blood, then the bulk of the material may appear in the lungs and spleen after intravenous injection (e.g., Kennedy and Waddell, Toxicol. Appl. Pharmacol. 22: 252, 1972; Waddell, et al., Toxicol. Appl. Pharmacol. 39: 339, 1977). This is due to a filtration by these tissues of the precipitate resulting when the material is injected. The papers cited above demonstrated this situation with the psychoactive compound A"-THC and the plasticizer DEHP. Another important reason for including a parenteral route of administration is to clarify what may otherwise have been interpreted as another type of ad ministration artifact. For example, when nicotine is given by inhalation or parenteral administration, the highest accumulation in the entire body is in the bronchial epithelium (Waddell and Marlowe, Drug Metabol. Disposition 4: 530, 1976). Had this compound only been given by its usual route of administration, i.e., inhalation, one might have concluded that the accumulation in the bronchial epithelium was simple an artifact from absorption through these cells. Another example is the intense accumulation of arsenic in intestinal epithelium following its intravenous administration (Deak, Csaky and Waddell, .J. Toxicol. Environ. Health 1: 981, 1976). Had this element been given only by its usual route of exposure, i.e., oral, then one might not have suspected any specific interaction with enzymes in the intestinal epithelium. Instead, these studies lead to further investigation showing specific inhibition of glucose-6-phosphatase in these cells as a result of the distribution of arsenic. SL 080391 W. J. Waddell, M.D. 49 14 Following administration of C-labeled vinyl chloride to the animal, time intervals for survival will be included which provide complete disposition data. In this laboratory, geometric time intervals on each side of 1 hour have been used routinely: i.e., 2 min., 6 min., 20 min., 1 hr., 3 hr., 9 hr., 24 hr., 3 days, 9 days and 27 days. Rarely, however, are all of these intervals used for the first series of animals; half or less may be sufficient, if the intervals are appropriately spaced and other information .is already available on the kinetics of disposition of the substance. Conversely, further, longer intervals or even intervening intervals may be required as the dispositional data unfold. The selection of the 3-fold geometric progression of time relative to one hour has proved to be a particularly useful framework. Plotting the concentration of a substance in a specific tissue or fluid against this type of time scale will allow a simple, direct estimate of the half-life of the substance in that bio logical compartment. (For further details see Waddell and Marlowe, Autoradiography. In: Drug Fate and Metabolism: Methods and Techniques, Marcel Dekker, Inc. New York, Vol. 1, Chapter 1, 1977). The animals will be sacrificed by immersion in a bath of hexane cooled to the temperature of dry ice; the animals will have been lightly anesthetized with ether prior to freezing. After freezing, the animals are never allowed to thaw and the tissues never are in contact with any solvents. These precautions are necessary to prevent any translocation or removal of the substance being studied. The frozen animal is sectioned on a microtome in a cryostat at -20C. The whole, sagittal sections, 20p to lOOy thick, are taken onto Scotch tape and allowed to dry in the freezer. The Slee model 250 cryostat, already in operation in this laboratory, will be used for studies in mice. All of the facilities and equipment for studies in mice are already in operation here. After the sections have dried in the freezer, they can be removed to room temperature (if the substance being studied or its metabolites are not volatile). The sections are then placed against x-ray film in the darkroom, placed in light tight boxes, and returned to the freezer for exposure of the film by the isotope in the tissue section. Currently the best film for and isotopes of similar energies is Kodak type AA. Until recently, it was not practical to use for whole-body autoradiography; however, a new film has been introduced in the last several months by Ceaverken A in Sweden designed specifically for this use. It reduces the exposure time for tritium essentially to that required for and has proved to be free of artifacts for whole-body autoradiography. Conversations with representatives from Eastman Kodak indicate that a similar film will be introduced by this company in the near future. After exposure of the autoradiograph, the section is removed in the darkroom and the x-ray film is developed. The sections can be stained with hematoxylin and eosin or any special stain if necessary to identify the sites of accumulation of the activity. Also, since the tissue has been subjected only to freeze-drying, the section can be used for histochemical studies to make further inferences correlating the disposition of the substance and its effect on the tissue (e.g., Deak, Csaky, and Waddell, J. Toxicol. Environ. Health 1:981, 1976). SL 080392. W. J. Waddell, M.D. 50 Quantitation of the activity in specific organs and areas will be done with the C.H.G.l densitometer manufactured by ADG instruments which is already in use in this laboratory. Photometric densities of the areas in the autoradiograph are compared on a scale relative to density of blood seen on the autoradiograph. The actual concentration in blood will then be determined by removal of a sample of blood from the section or frozen remains of the animal, solubilizing the blood, and counting it by liquid scintillation. Then the concentration in blood (or any other uniformly labeled reference tissue chosen) can be used with the relative photometric densities to give the concentration in any tissue or area. This technique provides information that cannot be obtained with any other method currently in use. For example, the concentration of radioactivity can be de termined in the islets of Langerhans, bronchial epithelium, etc. Dissection of these tissues to obtain such areas without surrounding tissue is very difficult or impossible without allowing loss by diffusion or contamination. But the con centrations in these areas can be readily and simply obtained by the technique offered. The major error which results when organs are removed and sampled either by homogenization or excision of a small portion is that areas within the tissue are not identified. For example, if the whole lung is removed for analysis, the very high accumulation of nicotine (Waddell and Marlowe, Drug Metabol. Disposition 4_: 530, 1976) or of certain PCB's in the bronchial epithelium is not observed (Brandt, Acta Pharmacol. Toxicol. 40 (Suppl. 11): 1977). The precision of quantitation by this technique is adequate to allow calcu lations that can be done by no other method currently available. This approach was utilized recently to evaluate the distribution of water-soluble substances in specific cellular layers of the developing teeth of young mice. These quanti tative data were sufficient to allow calculation of the pH gradients existing among these different layers of cells and structures (Lyman and Waddell, Am. J. Physiol. 232:F358, 1977; _232:F364, 1977). It is necessary or desirable to identify chemically the nature of the radioactivity in the area seen in the autoradiograph. This will be done by excising the areas of interest from the 100*i-thick sections which were contiguous to the 20*i-thick sections used for autoradiography. These tissues will then be extracted with appropriate solvents and chromatographed by thin-layer chroma tography. The thin-layer chromatographs will then be placed against x-ray film for localization for the different compounds. Quantitation can be further defined by removal of the material from the thin-layer plates and counting by liquid scintillation. For an example of the use of this method in the disposition of diphenylhydantoin see Waddell and Mirkin, Biochem. Pharmacol. 2_1:547, 1972, Further details of the technique with whole-body autoradiography may be found in: Waddell and Marlowe, Autoradiography, In: Drug Fate and Metabolism: Methods and Techniques. Marcel Dekker, New York, Vol. 1, Chapter 1, 1977). Examples of its usefulness may be found in Waddell, Autoradiography in Drug Distribution Studies. In: Fundamentals of Drug Metabolism and Drug Disposition. Williams and Wilkins. Baltimore, Chapter 24, pages 505-514, 1971; Waddell, Ann. Rev. Pharmacol. _L3:153, 1973 and in the chapter on Disposition of Drugs in the Fetus cited above. Reprints of any of these articles will be supplied on request. SL 080393 51 Technical Proposal I Vinyl Chloride Metabolism in Isolated Mammalian Liver Cells Richard C. Feldhoff, Ph.D. SL 080394 R t cha.rd C . Fe i ahc Vinyl Chloride Metabolism in Isolated Mammalian Liver Cells Richard C. Feldhoff, Ph.D., Assistant Professor, Department of Biochemistry, University of Louisville School of Medicine, Louisville, KY 40232. The Vinyl Chloride Study Group at the University of Louisville is in vestigating the metabolism of this important chemical monomer with a multi faceted yet interrelated attack. It Is clear from the work of Tamburro, Wong, Streips, Du and others that the reactive vinyl chloride metabolites chlorooxirane and chloroaceta1dehyde are produced in the liver and intimately related to vinyl chloride toxicity and carcinogenicity. The regulation of vinyl chloride metabolism at the cellular and molecular levels is however only poorly understood. An experi mental approach which should facilitate vinyl chloride investigation, is the use of isolated mammalian liver cells. The research program which is briefly described below would combine the knowledge and experience of the Vinyl Chloride Study Group at the University of Louisville with my own research training such that a model system can be developed where individual steps in the metabolism of vinyl chloride and other potential carcinogens can be routinely investigated following acute and chronic exposure. Initially, due to the lack of continuous human liver cell lines these studies would be performed with isolated rat liver cells and extended to human liver cells when they become more available. The effects of ethanol consumption on the metabolism of vinyl chloride will be one aspect of the proposed studies. The mammalian liver is the principal organ responsible for the metabolism and/or detoxifI cation of many drugs, hormones and other organic metabolites including potential carcinogens. The exposure of the liver to an hepatotoxin such as ethanol is likely to interfere with its ability to detoxify potential carcinogens. Many of the metabolic and hepatic effects of alcohol have been the'subject of extensive investigations, however the concept of the potentiation of carcinogenesis by ethanol is relatively new and has not been adequately investigated. Indeed, recent animal experiments suggest that alcohol intake greatly increases the incidence of cancer when the animals are also exposed to vinyl chloride. In order to better protect industrial workers and the general public from chemically-induced cancers, it appears desirable to define pathways for metabolism and detoxificat ion of vinyl chloride and other chemical monomers by the liver and to assess the role of alcohol consumption on these pathways. Studies of vinyl chloride metabolism can be facilitated by reducing the complexities associated with whole animal experiments to the level of Individual liver cells. Recently perfusion techniques have been developed which permit the bonds holding liver cells together to be cleaved when the enzyme collagenase is added to the perfusion medium. The perfusion procedure is depicted in Fig. 1. With the proper perfusion equipment, preparations of isolated liver cells can be readily obtained which exhibit very high viability and have been clearly shown by myself and others to retain normal liver specific functions. An example of some data which I obtained utilizing isolated liver cells is shown in Fig. 2. The synthesis and the secretion of albumin and many other plasma proteins is a specific function of the liver which the isolated cells retain. Additionally, as shown in Fig. 3, the intracellular morphology of the isolated cells when investigated by high resoltuion electron microscopy is identical to that of the intact tissue. SL 080395 Richard C. Feldhoff -53 There are also major technical advantages associated with the use of cell suspensions including 1) the ease of uniform sample collection (media or cells) as a function of time, 2) the ability to assay many different conditions or variabl simultaneously and 3) the ability to employ radioactive metabolites at a much higher specific activity than is feasible in other in vivo systems. With respect to vinyl chloride metabolism, isolated liver cells will be used to investigate: 1) the uptake, intracellular transport (binding proteins) and metab olism by different cell fractions of radioactive precursors (Feldhoff, Tamburro, Du, Wong, (Wittliff - cytosol receptors)). 2) identification and quantitation of metabolites released into the medium (Feldhoff, Wong). 3) effects of individual metabolites on normal parenchymal cell functions (Feldhoff, Tamburro); mutagenic capacities of newly identified compounds (S t reips). k) potentiation of carcinogenesis by concurrent exposure to hepatotoxins, such as ethanol (Feldhoff, Tamburro). SL 080396 Richard C. Feldhoff 54 FiO. ^ Diagram depicting cnnnulation and perfusion procedures. A, ligature tying portal vein cannula and placed above entry of .jdenie vein: B. inferior vena cava and abdominal aorta sectioned after insertion of portal rannula: C, infusion tubing and cannula inserted into rubber tubing insert in inflow line; D. inflow hue taped to aluminum block to hold portal cannula in place- E. ligature around crliac axis and superior mesenteric arteries, tied on after insertion of portal cannula; F, ligature around inferior vena cava above R renal vein, tied off after insertion of vena cava cannula; G. vena cava cannula inserted through right atrium into inferior vena cava, not tied: H. outflow line taped to aluminum block to hold vena cava cannula m place. HOURS OF IHCUS4TIOH Fig. 2 Total protein and albumin secretion by isolated hepatoeytes. Cells were prepared as described under "Experimental Proce dures" and preincubated for 30 nun in Krebs-Henseleit bicarbonate buffer containing 7.5 times the normal plasma levels of amino acids. Cells were transferred to 40 volumes of bicarbonate buffer containing normal plasma 1-ctels of amino aciJ- and pfllleucine at 10 ^Ci/utl. At each time point, aliquots of the suspension were centrifuged and assayed for incorporation into total secreted protein and albumin as described under "Experimental Procedures." ------ o. hepatoevtes from normal rats; ------, hepatocytes from hypophysectomized rats. SL 080397 Richard C. Feldhoff 55 Fig. 3. Isolated rat hepatocyte. Cell suspensions were prepared by collagenase perfusion and then incubated for 2 hours at 37 C in Krebs-Heseleit bicarbonate buffer. Utilizing standard procedures the cells were fixed for 1 hour at 23 C in 0.1 M cacodylate buffer, pH 1.k containing 2.7% g1utara1edhyde. Cell preparations were dried in graded ethanol solutions, transferred to propylene oxide and embedded in Epon. The sections were stained with lead citrate and uranyl acetate. Magni fication: 7700X. (Feldhoff, R.C. & Hillman, 8., Unpublished observation). SL 080398 Addendum Proposed to Third Year Budget Only Proposal H 56 Personnel W. Waddell, Principal Investigator Research Technician 8,500 TOTAL PERSONNEL 8,500 Supplies v.Chemical, BiochemicaL Glassware Isotopes and Materials Animals 500 1,000 2,500 1,QQQ TOTAL SUPPLIES 5,000 TOTAL $13,500 SL 080399 57 1 Proposal I Personnel Richard C. Feldhoff, Ph.D., Principal Investigator P. Patrick Hess, B.S., Research Assistant -05,500 Equipment One perfusion box 3,700 The apparatus consists of a Plexiglas box (120x40xA0 cm) on a Formica-covered instrumentation box. The instrumentation in cludes an air circulating system, a.motor for rotating the oxygenation chambers, a temperature control system and appropriate electronic controls. The air in the Plexiglas box is continuously circulated and kept at 38 C by a thermostat whose probe is placed near the livers. A large number of precision components are included in the perfusion chamber such as cylindrical Plexiglas oxygenation chambers with a non-rotating module of A stainless steel tubes for influent and effluent media and gases, sliding stainless steel trays for each animal, bubble traps, etc. Two perfusion pumps - Pharmacia ($850 each) Supplies Glassware Chemicals, Biochemicals, Media Animals and Animal Care Radioisotopic Supplies Disposable Sterile Tissue Culture Supplies 1,700 500 500 650 500 500 TOTAL $13,550 SL 080400