Document XzN6rpVY59we4oBpyB9M9n74G

MANUFACTURING CHEMISTS ASSOCIATION 1825 CONNECTICUT AVENUE, N.W., WASHINGTON, D C. 20009 TELEPHONE. (202) 328-4200 TELEX. 89617 (MCA WSH) July 31, 1978 TO: Vinyl Chloride Technical Panel Vinyl Chloride Research Coordinators Vinyl Chloride Medical Subcommittee Gentlemen: As reported in Section 6.0 of the Record of the University of Louisville Meeting on June 14, 1978, Drs. Tamburro and O'Connell promised to send the Project Manager complete abstracts of all research reviews presented on all component studies. We are pleased to report that they have exceeded their promise by sending us complete transcripts of the actual research reviews covering Dr. Tamburro's "Overview of the University of Louisville Multi-Disciplinary Approach to the Study of Chemical Carcinogenesis" and the reports on the component research projects as well. Included in the enclosed collection of papers are copies of all slides which were presented during the June 14th meeting. We trust you will find these research summaries helpful and, for the benefit of those of you who were unable to attend, they afford an excellent insight into the overall value of this research program. Sincerely JTS:ep Enclosure J. T. Seawell Project Manager Vinyl Chloride Research THE WRITER'S DIRECT DIAL NUMBER IS (202) 328-4258 CMA 002339 UNIVERSITY OF LOUISVILLE CHEMICAL CARCINOGENESIS MULTI-DISCIPLINARY RESEARCH GROUP I. OVERVIEW Slide 1. The University of Louisville has a multi-disciplinary approach to the study of chemical carcinogenesis, especially, chemical monomers. Vinyl Chloride was one of the first chemical monomers under study; we combined a cancer control program which designed and implemented a prospective medical surveillance system for identifying occupationally related chemical cancer (as shown on. the left of the slide) with a clinical research group which worked closely with the medical surveillance program in developing better techniques for detection and prevention. Basic research in the molecular structure and pathogenesis of carcinogens, especially chemical monomers (as shown on the right) made the third link in our multi-disciplinary approach to the study of chemical carcinogenesis. Slide 1 MULTIDISCIPLINARY APPROACH TO CHEMICAL CARCINOGENESIS CANCER CONTROL PROGRAM APPLIED CLINICAL INVESTIGATION BASIC RESEARCH INYL CHLORIDE NGIOSARCOMA MEDICAL SURVEILLANCE SYSTEMS DETECTION AND PREVENTION SYSTEMS PATHOGENESIS OF CARCINOGENS MOLECULAR STRUCTURE VINYL CHL0RI1 1 CMA 002340 SLIDE 2. This simply outlines in more detail the various components of the cancer control program which include a screening program, a data bank, an epidemio logical study system, educational programs, counseling and rehabilitation programs, and the treatment program. Slide 2 VINYL CHLORIDE ANGIOSARCOMA CANCER CONTROL PROGRAM APPLIED CLINICAL INVESTIGATION MEDICAL SURVEILLANCE SYSTEMS 1. SCREENING PROGRAM 2. DATA BANK 3. EDUCATIONAL PROGRAMS 4. COUNSELING & REHABILITATION 5. TREATMENT PROGRAM > DETECTION AND PREVENTION SYSTEMS " BASIC RESEARCH PATHOGENESIS OF THE CARCINOGEN (s) MOLECULAR STRUCTURE VINYL CHLORIDE CMA 002341 2 SLIDE 3. This illustrates the past clinical investigations done in the detection and prevention systems. They cover a variety of areas such as #1, looking at the appearance of normal human bile acids as a means of determining liver function to the Identification of potential chemical carcinogens and their metabolites by new bacterial systems covering the gamut from clinical to basic research in the area of detection and prevention. CANCER CONTROL PROGRAM Slide 3 VINYL CHLORIDE ANGIOSARCOMA MEDICAL SURVEILLANCE SYSTEMS APPLIED CLINICAL INVESTIGATION BASIC RESEARCH DETECTION AND PREVENTION SYSTEMS 1. BILE ACID CLEARANCE 2. BLOOD/TISSUE ENZYMES 3. LEUKOCYTE ADHERENCE INHIBITION A. HISTOCOMPATIBLE (hL"A) IDENTIFICATION 5. URINARY GLYCOSAMINOGLYCANS 6. TISSUE ANTIGENIC SYSTEMS 7. HISTOLOGICAL/ELECTRON MICROSCOPIC CHARACTERIZATION 8. MULTIVARIANT ANALYSIS FOR END PRODUCTS 9. IDENTIFICATION OF POTENTIAL CHEMICAL CARCINOGENS PATHOGENESIS OF CARCINOGENS MOLECULAR STRUCTURE VINYL CHLORIDE Cma 002342 3 SLIDE 4. The fourth slide illustrates some basic science research being conduct^ rr in the pathogenesis of carcinogens. In this case, they are numbered from 9 through 1 indicating those areas of research most related to the clinical area such as looking for tissue antigen and antibody detection as a means of screening to the most basic one; that is looking for industrial vinyl monomers by mass spectrometry studies and their metabolites in the carcinogenic'process. These studies are presented in more detail further on. These slides simply illustrate that the inter-disciplinary group is investigating chemical monomers from molecular structure aspects to clinical application. CMA 002343 4 VINYL CHLORIDE ANGIOSARCOMA Slide 4 CANCER CONTROL PROGRAM MEDICAL SURVEILLANCE SYSTEMS APPLIED CLINICAL INVESTIGATION BASIC RESEARCH DETECTION AND PREVENTION SYSTEMS "-- PATHOGENESIS OF CARCINOGENS 9. VINYL CHLORIDE TISSUE ANTIGEN/ ANTIBODY DETECTION 8. HEPATIC FIBROSIS IN CHEMICAL CARCINOGENESIS ` 7. VINYL CHLORIDE CARCINOGENESIS INDUCTION 6. GLYCOSAMINOGLYCAN IN HEPATIC ANGIOSARCOMA 5. VINYL CHLORIDE NEOPLASTIC TRANSFORMATION A. VINYL CHLORIDE INDUCED METABOLIC ALTERATION 3. CARCINOGENESIS AND SULFUR AMINO ACID METABOLISM 2. MUTAGENESIS OF VINYL CHLORIDE AND METABOLITIES 1, INDUSTRIAL VINYL MONOMER CARCINOGENESIS MOLECULAR STRUCTURE VINYL CHLORIDE 5 CMA 002344 ANALYSIS AND SYNTHESIS OF CHEMICALS AND THEIR USE IN BILE ASSAYS FOR THE IDENTIFICATION OF CARCINOGENIC POTENTIAL PART A. CHEMICAL SYSTEMS OF DETECTION OF VINYL MONOMER TOXICITY JOHN L. WONG, Ph.D. I SLIDE 1 Our study is concerned with the chemical systems of detection of the toxicity of vinyl monomers in general and vinyl chloride (VC) in particular. Most vinyl monomers are not mutagenic or carcinogenic per se, but become so after they are metabolical1y oxidized. They are converted to the epoxides which are highly reactive and can undergo spontaneous rearrangement to the carbonyl compounds. These primary metabolites, i.e. the epoxides and carbonyls, are electrophilic compounds and have a propensity to react with cellular nucleophiles nonenzymatlcally. Such reactions can be cate gorized Into two groups, those reacting in the cytoplasm and those in the nucleus. The trapping of the epoxide and carbonyl compounds in the cyto plasmic environment by sulfhydryl compounds are generally the detoxification* reactions. Whereas the primary metabolites reacting with the nucleic acid materials in the nucleus would constitute the putative action and is most likely involved in vinyl carcinogenesis. Basing on this concept, we have made advances in three areas of study: (1) The detoxification of the primary metabolites of VC in a cytoplasmic environment. Here, we have identified key intermediary metabolites and are going to identify all reaction products so that the metabolic fate of the epoxide and carbonyl can be determined as the host factors vary. (2) The putative action of the primary metabolites of VC which'involves alteration of the nucleic acid constituents. We have isolated some unusual modified bases and our intention is to relate quanti tatively these modifications of nucleic acids to bioassay findings. (3) The mutagenic and carcinogenic potential of industrial vinyl monomers other than vinyl chloride. We have applied controlled chemical oxidation mimicking the metabolic conditions to 1* vinyl monomers which are chosen on the basis of their Industrial usage. So far 12 epoxides have been generated for testing. We have seen that this chemical study is beginning to pay off in promoting team work in unraveling the mechanism of how a vinyl monomer is being handled In a cell, which is a critical issue in industrial safety consideration. CMA 002345 6 Slide 1 Chemical Systems of Detection of Vinyl Toxicity VINYL metabolic oxidation V EPOXIDE + CARBONYL * (3) cytoplasm nucleus detoxification (1) putative action (2) 7 CMA 002346 SLIDE 2 This slide shows the significant results which we have obtained pertaining to the detection of vinyl chloride detoxification products. The chlorooxirane, COR, compound 1, and its rearrangement product chloroacetaldehyde, CAA, compound 2, are the two most commonly hypothesized primary metabolites of vinyl chloride. The central question is how are they detoxified. Do they yield the same products or different ones? W have studied their reaction with sulfhydryl compounds 3,^-dichlorobenzenethiol and N-acetylcysteine. The benzenethiol was used by the Stockholm group in a priliminary 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 glutvathione. in the case of COR and benzenethiol, the sulfur-conjugation product S-acetaldehyde, compound 3> is formed. However, CAA and benzenethiol forms the hemithioacetal compound 4. These two reactions are distinctly different. The formation of hem!thioacetal is reversible but that of the S-acetaldehyde is not. With N-acetylcyteine, both COR and CAA yield the same cyclic condensation product, a dihydrothiazenecarboxylic acid, compound 5. This thiazene is a multi-step reaction product. Its isolation may have solved the mystery of an unknown urinary metabolite as well as the origin of the identified urinary metabolites: S-acetic acid. Compound 6, and S-ethylalcohol, Compound 7* In the Dow paper reporting the fate of ^C labeled vinyl chloride in rats, there is also a major metabolite accounting for *v35% of the ^C label in urine which has not been identified. We believe that this thiazene compound is a key intermediary metabolite, that it can undergo further metabolic processing to yield compounds 6 and 7 and may itself be present in the urine. These results as presented here will enable us to undertake a more comprehensive detection study of all the vinyl chloride detoxification products in biologi cal specimens. 00^3^ C^ 8 Slide 2 Detoxification of Vinyl Chloride PRIMARY METABOLITES INTERMEDIARY METABOLITES COR1 S-ACETALDEHVDE3 THIAZENES5 CAA2 HEMITHIOACETAL4 THIAZENES5 urinary metabolites. S--ACETIC ACID6 S--ETHYL ALCOHOL7 CHLOROACETIC ACID8 H-C"CH2Cl RS--CH2CH0 4 RS-CH-CH-Cl OH rs-ch2co2h RS-CH2CH20H 8 Cl-CH2C02H (RS FROM 3/^--DICHLOROBENZENETHIOL., N-ACETYLCYSTEINE) 9 CMA 002348 SLIDE 3 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 CM. Although CAA is long known to react with nucleic acid bases such as cytosine and adenine to form the etheno derivatives, compound 1 and 2, 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, as the nucleoside, nucleotide and polyguanylic acid, gives rise to two tricyclic ethenoguanines, the linear etheno compound 3 and the angular etheno compound kt and a third product which is possibly an acetylguanine, compound 5 These products can disrupt nucleic acid structures hence their functions by means of steric and electronic perturbation. It is also worthy of note that their fluorescence properties would allow their direct detection in a cell nucleus. 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. Using HPLC and NMR techniques, we have tenta tively identified one major product as compound 6. It is important to note that this is the first indication that COR and CAA show different molecular events In their putative action. The significance of this information in terms of vinyl carcinogenesis will become clearer when we can correlate quantitatively the alterations in nucleic acids brought about by COR and CAA and the bioassay results of these primary metabolites. CMA 02349 10 Slide 3 Putative Action of Vinyl Chloride. Reaction with Nucleic Acid Bases CAA ETHENO--C1, ETHENO-A2 L-ETHENO-G3, a-ETHENO-G4, ACETYL-G5 COR G-7-ACETAU3EHYDE6 + ... 11 CMA 002350 SLIDE 4 It Is now known that vinyl chloride-mediated mutagenesis in the m Salmonella test requires microsomal activation. In order to extend the vinyl chloride model to scrutinize the mutagenic and carcinogenic potential ^ of other industrial vinyl monomers, we have started a methodical screening system. The conventional Salmonella screening involves treating the vinyl monomers with the S~9 preparation which contains the mixed function,oxidase system and a whole variety of other soluble enzymes prior to bacteria screening. . This is sort of a black box approach, the outcome of which often is hinged on the history of the S-9 preparation. We have used mild chemical conditions, a controlled MCPBA oxidation to oxidize 14 vinyl compounds to the corresponding epoxides. They are chosen mainly on the basis of their industrial significance. These epoxides have been identified and quantitated by means of nuclear magnetic resonance technique so that the bioassay results can be related to the amounts of epoxides formed.. These epoxides upon heating will rearrange to the carbonyl derivatives which can be similarly quantitated and tested. We are now vigorously pursuing this 1ine of vinyl screening study in collaboration with Dr. Streips of the Microbiology Department. You'll be hearing from him shortly about the interesting assays which he has carried out on both the vinyl compounds and their epoxide derivatives. To summarize, our strategy has been to fully elucidate the molecular events In the metabolism of vinyl chloride, that is, the detoxification and putative action, and use it as a model to screen the mutagenic and carcinogenic potential of other industrial vinyl monomers. This work ?s part of a group effort. Some of the collaboration results will now be expounded by Dr. Streips. Slide 4 Mutagenic and Carcinogenic Potential , of Vinyl Monomers VINYL'-Mcref EPOXIDE CARBONYL 25 0) nmr quant (1) nmr quant. (2) biol screen (2) biol screen Styrene, butadiene, acrylonitrile, crotononitrile, acrylate, ethyl vinyl ether, vinyl acetate, vinyl BROMIDE, VINYL FLUORIDE, VINYLIDENE CHLORIDE, VINYLIDENE BROMIDE, TRICHLOROETHYLENE, DICHL0R0DIFLUOROETHYLENE,1,1"DICHLOROPROPENE 12 CMA 002351 PART B. REACTIVITY OF CHEMICALS AND THEIR METABOLITES IN VARIOUS BACTERIAL ASSAYS - DR. ULDIS N. STREIPS, Ph.D. With thousands of chemicals being routinely used in manufacturing processes, and new compounds being created daily for specific industrial needs, it is of great importance to assess the relative carcinogenic potential of each of these materials before they are released into the environment in quantity. When considering carcinogenesis assays, the most accurate are animal models. However, these suffer from two problems: first, they are time comsuming; second, they may not reflect the true state in human exposure. Nevertheless, to determine if a chemical is a carcinogen with our present state of knowledge requires animal testing. There are a variety of rapid screen techniques available for chemicals. All of you are probably familiar with the Ames Salmonella reversion test. This test determines"the mutagenicity of a compound. However, the correlation of mutagenesis and carcinogenesis is only accurate to about 80%. The Ames test in itself makes no prediction on the carcinogenicity of a chemical. Our laboratory also has used extensively the Bacillus repair test. This screening technique measures whether a chemical elicits enough damage to the cellular DNA to require repair for cell survival. Then by measuring the relative killing of a variety of repair-deficient strains, we can determine if the chemical is reactive to the DNA and which type of repair is required to fix this damage. A good example in Table 1 is chloroacetaldehyde (CAA), the active metabolite from vinyl chloride oxidation. This potent chemical at the concentration indicated causes no inhibition of the wild type, repair-proficient strain (w+), or the strains lacking UV repair (her-, uvr~) and DNA polymerase I 13 CMA 002352 TABLE 1 REACTIVITY OF CHEMICALS IN BACTERIAL ASSAYS CHEMICAL `JAA (0.004M) STYRENE OXIDE (0.01M) EMS (O.OOIM) MMS (O.OOIM) SALMONELLA REVERSION P REV/PLATE--CONTROL 265 342 312 355 SOS REPAIR % NONTREATED 15% 100% 80% 1% SUBTILIS REPAIR ASSAY W REC~ HCR~ UVR~ POL mm INHIBITION 0 18 2 2 3 0200 1 1 16 1 1 1 4 21 6 6 10 CMA 02353 14 (pol A~). However, the strain missing recombination repair (rec~) is tremendously inhibited. This suggests that CAA interacts with DNA and that recombination repair is necessary to rectify this damage. We have done further research with this compound (Table 2) and find that very specific kinds of rec~ strains are inhibited by CAA. This forms the basis for much of our research in the third year on CAA. We will compare strains which are all rec~ but some are killed by CAA (e.g. rec A or rec B) and some are not (rec C and rec H). We will isolate enzymes and structural components from these strains which vary in their response to CAA, then relate these to known mammalian counterparts. These experiments should help to elucidate the mechanism of action of this chemical. Similar studies will be initiated for other vinyl monomers, namely styrene oxide and acrylyl nitrile. 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 result, 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 15 CMA 002354 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 recFl6 TABLE 2 REPAIR ASSAYS WITH B. SU3TILIS* Average inhibition in mm/8 experiments Strains Average in mm/8 exp REC H 13.5 7.5 t , recH342 * . REC -m 13.5 9.5 16.5 16.5 11.3 16.8 rec-4 rec-13 MTC mtc-41 (cafr) 15.4 5.4 NR UVR NR __ uvr-35 (cafr) her uvr NR NR NR 11.5 NR 6.7 8*8 WT wt NR wt NR wt NR - 6.1 7.4 7.5 8.42 3.1 . *100mM chloroacetaldehyde used In all these experiments 16 CMA 002355 induction in a cell population as a measure of SOS inducing potential.--^NtieW virus is induced, the cell dies. Therefore, in Table 1 100% 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. In Table 1 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 X (pol A). 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 acryllnitrile. We will also extend our catalogue of compounds assayed by the SOS screen and draw a more reliable conclusion -on the applicability of this test for carcinogenesis screening. 17 CMA 002356 THE CHARACTERIZATION OF BIOCHEMICAL ENZYMATIC CHANGES OCCURRING WITH PROLONGED VINYL CHLORIDE EXPOSURE IN ANIMAL AND MAN J.T. Du, Fh.D., and C,H. Tamburro, M.D. Conventional clinical biochemical studies have not been shown to be an effective early indicator of chemical injury to the liver. Little is known about the earliest cellular enzymatic changes with prolonged exposure to chemical monomers. An initial vinyl chloride exposure experiment was conducted to examine sequentially the early enzymatic changes in cellular function of the liver. Animals were exposed from 10,000 - 20,000 ppm from 14 to 137 hours, of total exposure being exposed five days a week. Conventional clinical biochemical studies and subcellular enzyme markers were studied. These included: (1) microsomal enzymes related to vinyl chloride metabolism: P450, NADPH-cytochrome C reductase, and mixed function oxidase. Mitochondrial marker cytochrome C oxidase and protein metabolism was studied by looking at in vivo incorporation of tritiated leucine. In addition, glucose-6-phosphatase and glucose-6-phosphate dehydrogenase were studied as cytosol markers as were glutathione content and glutathione reductase in relationship to detoxification. Overall results are shown in Slide 1 for conventional and subcellular enzymes. 0,4 02357 18 I. CQWBVTICNAL CLINICAL BIOCHEMICAL STUDIES Slide i 1. SGOT 2. SGPT 3. LDH 4. ALK PHOS 5. BILIRUBIN 6. TOTAL PROTEIN 7. ALBUMIN 8. CHOLESTEROL 9. TRIGLYCERIDES NO CHANGE NO CHANGE NO CHANGE NO CHANGE NO CHANGE NO CHANGE NO CHANGE NO CHANGE NO CHANGE SUBCELUULAR ENZVME AND METABOLITE STUDIES IN LIVER 1. MICROSOMAL ENZYMES RELATED TO VC METABOLISM: A. P-450 B. NADPH-CYT C REDUCTASE NO CHANGE C. MIXED FUNCTION OXIDASE 2. MITOCHONDRIAL MARKER; CYT C OXIDASE 3. IN VIVO INCORPORATION OF VlEU 4. GLUTATHIONE CONTENT 5. GLUTATHIONE REDUCTASE NO CHANGE NO CHANGE ELEVATED INCREASED 6. GLUCOSE-6-P ASE 7. GLUCOSE-6-P DEHYDROGENASE DECREASED INCREASED RATS WERE EXPOSED TO 10,000-20,000 PPM VC FOR 14, 28, 42, 71, 84, 105 AND 137 HOURS. 19 CMA 002358 SLIDE 2, 3. 4, 5. This illustrates an early increase in glutathione reductase activity followed closely by a fall in glucose-6-phosphatase which becomes signifi cant after 71 hours of total exposure (Slide 3). At 100 hours, a marked increase in glucose-6-phosphate dehydrogenase (Slide 4) similar to that seen in metabolic studies by Weber and his group concerning hepatic primary cell neoplasms was found. As shown in Slide 5, Weber's molecular correla tion concept of neoplasia shows an impairment of the glucose-6-phosphatase producing a decrease in gluconeogenesis and an increase in the pentose phosphate shunt pathways which eventually lead to an increase in purine biosynthesis and nucleic acid synthesis. This, presumably, leads to increased liver cell replication. These biochemical changes in the early stages of vinyl chloride exposure may well explain the histological findings in humans exposed to vinyl chloride which appear to be character istic of vinyl chloride exposure. These histological findings of focal nodular hyperplasia of the hepatocytes may well be the histological reflec tion of a shift in gluconeogenesis and an increase in nucleic acid synthesis, induced by a mechanism not yet completely understood during the early phases of vinyl chloride exposure. If one were to carry this to a logical conclusion one would presume that the hepatocyte would eventually develop into a malignant cell with continuous regeneration and exposure to carcino gens. The fact that the primary hepatocyte does not become malignant in the adult rat, but does become malignant in the newborn rat with similar vinyl chloride exposure, led us to study the detoxifying capabilities of the hepatocyte versus the sinusoidal lining cells. During this time, there were no significant differences in the usual biochemical clinical studies used for screening early vinyl chloride injury. EXPOSURE TIME (hours} t SLIDE 2 SHOWS THE GROUP MEANS OF THE SPECIFIC ACTIVITY OF GLUCOSE-6PHOSPHATASE WITH RESPECT TO TIME OF EXPOSURE*. THE CONFIDENCE 'INTERVALS FOR GROUP MEAN DIFFERENCES ON THE ACTIVITIES- OF ENZYMES DISCUSSED ARE BASED ON THE ERROR MEAN SQUARE FROM A TWO FACTOR ANALYSIS OF VARIANCE WITH INTERACTION. THE TWO MAIN EFFECTS BEING TIME IN HOURS AND EXPOSURENONEXPOSURE. THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN THE EXPOSED AND CONTROL GROUPS FOR THE FIRST THREE TIME POINTS. HOWEVER, THE MEAN LEVEL OF THE EXPOSED GROUP IS SIGNIFICANTLY LESS THAN THAT OF THE CONTROL AFTER 71 HOURS OF EXPOSURE. CMA 002360 21 (uiojoid Bui/uiut/pozipixo HdQVN so|oui rfjx^Q l Slide 3 Exposure Time (hours} SLIDE 3 SHOWS THE GROUP MEANS OF THE SPECIFIC ACTIVITY OF GLUTATHIONE REDUCTASE WITH RESPECT TO TIME OF EXPOSURE. THERE IS A SIGNIFICANT INCREASE THROUGHOUT THE ENTIRE EXPERIMENT. CMA 002361 22 SLIDE 4 SHOWS THE GROUP MEANS OF THE SPECIFIC ACTIVITY OF GULCOSE-6PHOSPHATE DEHYDROGENASE WITH RESPECT TO TIME OF EXPOSURE. THERE IS A SIGNIFICANT INCREASE AFTER ABOUT 100 HOURS .OF EXPOSURE. CMA 002362 23 weber's molecular correlation concept of neoplasia lAifj .--. _ Em * 11M P -------- 1 | 5-PRA I PRPP Amidolramtarai* (PR PP 1 Slide 5 PURINE B IO S Y N T H E S IS PENTOSE PHOSPHATE PATHWAYS GLYCOLYSIS GLUCONEOGENESIS PK PEP CK^CT^P~CXyl0 o o ------------------------ ---------------------- ------------- _____-- ---------------------------------VINYL CHLORIDE OXIDATION AND DETOXIFICATION J.T. Du, Ph.D., and C.H. Tamburro, M.D. The second phase of study of the biochemical changes associated with vinyl choloride exposure were directed at oxidation and detoxification. Sprague-- Dawley rats were randomly exposed to controls and vinyl chloride exposure at 28,000 ppm, 7 hours per day, five days per week, for a total of 210 hours of exposure, for six weeks duration. Slide 1 shows the vinyl chloride metabolic fate scheme presently under use. Vinyl chloride being metabolized at higher levels by mixed-function oxidase within the liver may be converted to chlorooxirane or the epoxide. This unstable intermediate may then spontaneously revert to form chloroethanol and the chloroethanol may further be metabolized to chloroacetaldehyde. Secondly, the chlorooxirane may be directly detoxified by GSA via the glutathione epoxide S-transferase or further oxidized to chloroacetal dehyde. Chloroacetaldehyde in turn may be shown by glutathione aralykly-Stransferase or further oxidized to chloroacetic acid. Studies by Wong and Streips have already shown that vinyl chloride, chloroethanol and chloroacetic acid have no mutagenic properties in bacteria. However, the chlorooxirane and the chloroacetaldehyde are both mutagenic. Animal studies demonstrated significant differences in the liver of vinyl chloride exposed animals who showed no physical or clinical abnormalities. These findings are listed in Slide 2. They include an elevation of non-protein sulfhydryl content, (Slide 3) glut athione reductase, (Slide 4) glutathione epoxlde-S-transferase (GEST) (Slide 5) and glutathione aralykly-S-transferase (GAST) (Slide 6) concomitant with the increase in glucose-6-phosphate dehydregenase and reduction in P450 content (Slide 7). A composite of the studies from 14 to 350 hours is shown in Slide 8 which illustrates the earliest increases occurring in glutathione reductase followed later by an Increase in glutathione content. It is interesting to note that the Increase in GEST occurred before the Increase in GAST around 70 to 80 hours of the total exposure. This implies that the chlorooxirane accumulation is to be CMA 002364 25 handled by direct detoxification by the glutathione but with prolonged exposure more is converted to the chloroacetaldehyde which then is further detoxified by the GAST. These data are compatible with other findings of increased amounts of chloroacetic acid in the urine of individuals with very high prolonged exposures. This would imply that at initial doses the vinyl chloride is able to be metabolically handled and properly detoxified and that there may be a number of mechanisms by which the active metabolites are handled within the hepatocyte thus, preventing it from inducing DNA injury and subsequently angiosarcoma. Whether these metabolic capabilities are also present in the sinusoidal lining cells has lead us to our more recent studies. CMA 002365 26 Slide 1 I (v c) Cl CH = CHL9 % Cl ch2ch2oh /f (chloroethanol) (chlorooxirane) Cl CH-CHo Y' / DETOXIFICATION WITH GLUTATHIONE CH2CH2OH XS=^> N-Ac-S-(2-hydroxy ETHYL) CYSTEINE 4 Cl ch2cho (+gsh) GAST- (chloroacetaldehyde) GS CH2CH0 GS ch2cooh V THIODIGLYCOLIC AC 002366 Cl ch2cooh n 5 (CHLOROACETIC ACID) VINYL CHLORIDE METABOLIC FATE Si.jde 2 RESULTS In comparison with the.two control groups,, the following STATISTICALLY SIGNIFICANT DIFFERENCES (P < 0.05) WERE FOUND IN THE LIVER OF THE VINYL CHLORIDE EXPOSED BEFORE ANY SIGN OF PHYSICAL OR CLINICAL ABNORMALITIES WERE SEEN. 1, AN ELEVATION OF NON-PROTEIN SULFHYDRYL CONTENT. 2, AN ELEVATION OF GLUTATHIONE REDUCTASE. 3. AN ELEVATION OF GLUTATHIONE EPOXIDE -.S- TRANSFERASE (GEST) . 4. AN ELEVATION OF GLUTATHIONE ARALKYL "S" TRANSFERASE (GAST). 5. A REDUCTION OF P-450 CONTENT. 6, AN ELEVATION OF. GLUCOSE-6-PHOSPHATE DEHYDROGENASE CMA 02367 28 'NON-PROTEIN SULFHYDRYL GROUP CO NTENT IN LIVER Slide 3 10 =j NORMAL V.C. EXR AIR EXR 8 Ui l CO < o I-- 6 O--) - Qa lit cm a uii 6 I X5 I-- o < Z> M --* o CD o a: LU > X m 1 I | 2 (70) 4 (140) Slide 4 6 (210) CMA 0 0 2 3 6 9 I 14 = NORMAL 13 12 iLl CO 11 < CX LU -S u. 10 co 2 Z <C ( Q IX E 9 1-- k 'a CO| 5 C a: u i lu a s > x' -JO- 0- 4 IU 1 o 8 7 6 LU "* Z* 5 28 i l-- < I- 4 3_J 3 CD 2 -- 1 O 0 2 V.C. EXP. CMA .0 0 2 3 7 1 30 Slide 7 in 28 26 normal V.C. EXP AIR EXR 24 CMA 0 0 2 3 7 2 22 20 18 IS 14 12 10 8 6 4 2  2 3 73 IMMUNOPATHOLOGY OF VC-ASSOCIATED LIVER ANGIOSARCOMA AND THE EFFECTS OF INDUSTRIAL CHEMICALS ON ANTIGEN ANTIBODIES E. Espinosa, M.D. It has long been known that chemicals can modify antibodies in antigens of serums, and tissues. The measuring of these changes, hopefully, would 1 ad to an early detection of the chemical-associated diseases and provide an approach to study the pathogenesis of such disorders. Chemicals or their metabolites can modify antibodies or antigens due to their strong interaction with proteins affecting the quality and quantity of these proteins. Secondly, their measurement may lead to early detection and understanding of the patho genesis of this injury. The first slide summarized briefly our immunopathologic observations in VC-assoclated angiosarcoma. Slide 1 IMMUNOPATHOLOGY OF VC-ASSOCIATED LIVER ANGIOSARCOMA A. Antigens investigated in liver angiosarcoma: 1. Tumor-associated antigen 2. Antigens shared with tissues: CTA-2, STA-3 3. Antigens shared with bile: BT-1, BT-2, BT-3 4. Antigens with liver specificity B. Tumor bound IgG C. Antigens deleted 0023^ 35 Firstly, four classes of protein antigens were detected by immunodiffusion and immunofluorescent in the liver tumors: a tumor associated antigen; antigens shared with tissues; antigens shared with bile; and antigens with liver specificity. Studies of the physicochemical properties of these antigens and their presence in circulation have been completed and reported (on some of these antigens, while additional studies are being continued on the other antigens). We have also reported the presence of IgG binding to liver tumors, indicating that these tumors do produce antigens which stimulate B cell antibodies. Work is still in progress to identify the antigen which stimulates such antibody production. Our third major finding is related to the deletion of normal antigens in the angiosarcoma tumor. This deletion of normal antigens has recently stimulated similar analysis in chemically induced hepatomas in rats. Similarly to the angiosarcoma', two tissue antigens were found to be missing in these hepatomas. Studies are now being conducted concerning the properties of these antigens and the significance of their deletion. It may well be that the deletion of certain antigens may be a more sensitive indicator of tumor development. Table 1 demonstrates the frequency of circulating tissue antigens in various clinical disorders in patients with various liver and non-liver diseases. One finds that circulating tissue antigen-2 (CTA-2) has been found most frequently in acute hepatocellular injury such as in acute viral hepatitis and acute alcoholic hepatitis but to a lesser degree in chronic forms of disease such as cirrhosis and carcinoma and not at all in those with liver angiosarcoma. This data would lend further support to the concept that the presence of tissue bound antigens or their deletion are better markers of tumor development than those in circulation. 36 CMA 002375 CIRCULATING TISSUE ANTIGEN TABLE 1 Frequency of CTA-2 in patients with liver diseases, other diseases and healthy subjects Patients- No.tested Acute hepatitis: Infectious Serum Alcoholic Liver cirrhosis Obstructive jaundice Liver angiosarcoma Pancreatitis Pneumonia Congestive heart failure Myocardial infarction Carcinomas Newborns Healthy controls- 29 9 8 28 5 3 8 22 39 .23 35 12 48 No.with CTA-2 23 (79%) 7 (77%) 3 (62%) 8 (28%) 1 (20%) 0 ( 0%) 2 (25%) 2 ( 9%) 8 (20%) 3 (13%) 8 (23%) 0 ( 0%) 0 ( 0%) Cpift. 0023'76 37 A SEARCH FOR EVIDENCE OF A VINYL CHLORIDE-INDUCED TUMOR ANTIGEN H.P. Fortwengler, M.S., and C.H. Tamburro, M.D. 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 extended by Prehn and Main in 1957 to conclude that there were antigens peculiar to and specific for tumor tissue. Subsequently, 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 an immune reaction to cancer, Fortwengler's group is 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 density gradient centrifugation. 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 a three times increased incorporation of H^-thymidine into stimulated cultures. A comparison of the responses between vinyl chloride plant workers and normal non-vinyl chloride workers indicated that there were many people in the general population having reactivity to tissue antigens irrespective of whether it was from anglo or normal liver. Non-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 anglo liver were quantitated. A comparison of the responses to angio or normal liver by VC workers according to known VC exposure was made. The VC workers were grouped as either CMA OO2377 38 high exposure or low exposure depending on whether they had exposure above the median plant exposure. As can be seen in Figure 1, no statistical differences were noted between 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 (see Table 1). Those with reactions to normal liver only were grouped as were those with no reactions. That left only those with reactions to angio liver. As can be seen in Table 1, the only reactions obtained with possible specific tumor reactivity were found in the group of VC workers. These results, although provocative, must 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 include an expansion of the above data and a slight reorganization of experimental design to ensure maximal statistical strength from the data obtained. 002378 39 Figure 1 ANGIOSARCO* ANTIGEN EXTRACT' LIVER ANTIGEN''EXTRACT:' 00*2* 40 ANGIO LIVER NORMAL AND ANGIO LIVER ( oVe / LYMPHOCYTE REACTIVITY TO TUMOR AND NORMAL TISSUE PANEL 08^00 VWD VINYL CHLORIDE WORKERS ti percentage 6 16 : NON-VINYL CHLORIDE WORKERS ii percentage INTERPRETATION OP . RESULTS ' 00 Individuals with possible specific anti-tumor reac-*' tivities.' 7 18 8 57 Individuals with nonspecific reactivities masking any possible specific reac tivities. NORMAL LIVER 5 13 . 17 Individuals with nonspecific reactivities. NONREACTIVE 20 53 5 36 Individuals with no tissue reactivities. TOTAL 38 100 14 100 HLA FREQUENCIES IN VINYL CHLORIDE WORKERS H.P. Fortwengler, M.S., and C.H. Tamburro, M.D. 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 em ployees for abnormal genotypes that predispose them to neoplasia after occupa tional exposure which are harmless to normal genotypes. An increased incidence of certain HLA types has been shown to be as sociated with susceptibility to various diseases. HLA-A27 antigen has been found more often in workers suspected of having the occupational disease asbestosis than among a control population. HLA tissue typing procedures 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 HLA 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 re lated diseases is about one-third completed. A comparison between those employees found to have liver disease, labeled "Pallet Plant", and those without liver disease, labeled "Non-Pallet Plant", may be seen in Tables 1 and 2. Although the number of individuals tested is not as yet large enough to allow statistical significance to emerge, potential difference in frequencies may be seen at A9, B15, B27, 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 the University of Louisville study. *These are Individuals who because of their liver disease, were initially reassigned to a shop manufacturing wooden pallets. 002381 It has been clearly established that the larger the population studied the greater the chances of obtaining statistically significant data. The accumu lation of HLA data on the majority of the VC population plant workers will take an additional two years as originally anticipated. This is necessary so that the strongest statistical analysis can be performed upon the completion of all HLA testing. CMA 002382 43 HLA - A FREQUENCIES IN VINYL CHLORIDE WORKERS HLA - A ANTIGENS* HEALTHY CONTROLS** who 1975 WORKSHOP ul/pallet PLANT ul/non-pallet PLANT ^ A1 34 A2 51 A3 23 A9 16 A10 7 ' All . 13' A28 7 A29 9 AW19 AW30 ^ - -ND AW31 ND AW32 ND BLANK ND N TOTAL FREQ, 900 160% 32 49 22 17 13 8 11 7 28 5 7 8 ND ' 6877 207% 38 58 20 8 12 8 20 0 ND 8 4 8 16 24 200% 30 ' 53 18 23 7 10 13 7 ND 3 7 7 21 .243 ' 199% #* ##* #*# CURRENTLY TYPING FiOR 11 ANTIGENS SCOTT ET ALis 1977 DATA COMPILED JUNE/ 1978 NOT DONE cMA 002383 44 HLA - B FREQUENCIES IN VINYL CHLORIDE WORKERS HLA - B,, ANTIGENS* HEALTHY CONTROLS** 1975 who WORKSHOP ul^ UL/NON-PALLET PLANT B5 B7 B8 B12 B13 m B15 B18 B27 B37 B^O BW16 BW21 , . BW22 BW35 B17 * BLANK 10 31 27 30 3 5 10 8 7 ND**** 11 ND ; ND 7 2 .5 6 ND \ N TOTAL FREQ, 900 155% 11 23 20 24 6. 11 .7 9 87 5 .12 12 . 4 5 17- 7 ND 6877 181% 87 22 - 28 16 22 . 26 . . 0 .25 . 6 . 12 . . 12 ' 38 12 0 2 0 12 * 4, 716 - 0 1 17 2 44 7 64. :.; * 8 13 22 10 12 ' ": 14 24 192%. .243 193% * *# ** # 16CURRENTLY TYPING FOR ANTIGENS SCOTT ET AL.> 1977 ' DATA COMPILED JUNE,, 1978 ;NOT DON 45 CMA 002384 * EVALUATION OF IMMUNOCOMPETENCE OF HUMANS CHRONICALLY EXPOSED TO VINYL CHLORIDE H.P. Fortwengler, M.S., and C.H. Tamburro, M.D. . It has been demonstrated that lymphoid cells (T cells) can be cytotoxic to human tumor cells and are often found decreased in cancer patients. Viola, et al, reported the development of neoplasms in rats exposed to vinyl chloride (VC). Creech and others reported the Increased incidence of human angiosarcoma in VC production workers. It is the purpose of this study to determine the immunocompetence of individuals that have undergone prolonged exposure to VC monomer and have developed liver lesions which, in some instances, are thought to presage the development of angiosarcoma. The scientific literature is replete with demonstrations of immunodepresslon 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 con ceming immunoevaluation prior to diagnosis of frank malignance. We have used im munological assays (see Table 1) that have demonstrated usefulness in indicating immunodepression in cancer patients. These tests have been used to evaluate the immunocompetence of individuals with possible pre-malignant lesions or other liver disease as demonstrated by biopsy. A comparison of these diseased individuals with normal plant workers demon strated nothing to suggest that the groups were immunologlcally different. A com parison of those individuals having high VC exposure (defined as those with lifetime exposure above the median) with individuals having low exposure (below the plant median VC exposure) demonstrated a slight pattern of Immunodepression (see Fig. 1) when lymphocytes were stimulated by PHA and Con A. Preliminary evaluation of this data indicated there is no statistical significance between the high and low exposure groups when examined with student's t test. Even after additional Immunological parameters were examined (see Table 2), no statistical differences could be found between the two groups. W 002385 At this time, our preliminary interpretation of these results, which will soon undergo a more complete data analysis, 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 to concomitant cor relations with data being received from clinical testing (e.g. immunoglobulin quantitation) will give us more definitive statistical results. 47 CMA 002386 TEST BATTERY OF IMMUNOCOMPETENCE TESTS IN USE REASON FOR USE TABLE 1 Absolute Lymphocyte Count SRBC Rosettes, 4C SRBC Rosettes, 33C PHA Stimulation Con A Stimulation FWM Stimulation SPL Stimulation Recall Antigenic Stimulation SLO PPD Candida Varidase Gives a gross examination of the immune syst^^ Quantitates the total number and percent of T cells. (T cells can reject tumors and transplants.) Quantitates the total number and percent of an active subset of T cells Tests the ability of T cells to respond to a non-specific stimulant Tests the non-specific function of T cells; overlaps, but not identical to PHA Tests the non-specific function of T cells; has B cell participation Tests the non-specific function of T cells; has B and Null cell participation Determines the ability of sensitized lymphocytes to be activated by rechallenge; everyone should react with at least one of these common antigens CMA 002387 48 table 2 ADDITIONAL IMMUNE PARAMETERS OF VC WORKERS ACCORDING TO EXPOSURE TEST .Above Median Exposure SPL Stimulation 191 31 (n* 52) Absolute Lymphocyte Count 2355 + 132 (n 52) i SRBC Rosettes, 4C ** * t 1503" + 101 (n- 50) SRBC Rosettes, 33C 1227 + 80 . (n50) 1 Recall Stimulation SLO- , ' " 8+2 (n- 53) PPD Candida Varidase f 29 + 8 (n- 49) | 11 + 3 (n- 53) 29 + 5 (n- 54) Below Median Esposure . Statistical Difference Between Groups 186 * 34 Cn-24) 2316 + 148 (n 24) None None - 1524 + 132' Cn-24) None 1260 + 123 <n-24) .None * * 24 + 12 (n- 24) 18 +. 6 <n"23) 6+2 (n- 23) 41 + 12 (n-"23) None None None. None t ( MA 002389 50 TISSUE AND URINARY GLYCOSAMINOCLYCANS IN HEPATIC FIBROSIS AND HEPATIC CANCER Charles E. Kupchella, Ph.D. Because characteristic glycosaminoglycan (GAG) changes have been found in many kinds of tumors and in tissues undergoing fibrogenesis, and because they have been shown to be present in large amounts in blood vessels, and because tissue GAG changes have been shown to be reflected in urinary excretion in certain disease states, we asked the question: "Are there characteristic glycosaminoglycan changes associated with hepatic angiosarcoma and other chemically-induced diseases of the liver and are they reflected in urinary excretion such that they are exploitable in early detection and/or diagnosis?" To date we have found that: 1. Human hepatic angiosarcoma is accompanied by elevated hepatic GAGs. (1) 2. The GAGs 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 undergoing experimentally induced fibrosis and heparan sulfate is elevated in the urine of such animals.(3) 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 composi tion and the size of tumors. 6. Livers of animals bearing metastasizing hepatoma (5123tc) have 50-fold greater concentrations of a non-sulfated, neutral, uronic, acid-positive material than is found in the livers of animals bearing two other, non-metastasizing hepatomas. Glycosaminoglycans are important in the etiologies of both hepatic fibrosis and hepatic cancer. To date, our work indicates that different GAGs are involved in these processes.. We feel that the roles of the GAGs in the hepatic fibrogenesis and as partial determinants of cancer cell behavior are of such importance that GAG-directed strategies may be evolved for the control of these diseases as well as for their detection and diagnosis. (See slides 1 and 2). CMA 002390 51 REFERENCES 1. Kupchella, C. E. and Tamburro, C. H., Urinary and Tissue Glycosarainoglycan 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) 52 CMA 002391 SLIDE 1 Kupchella, et al. LIST OF STUDIES/EXPERIMENTS COMPLETED TO DATE AND THOSE IN PROGRESS 1. Evaluate urinary GAG excretion total in 50 human samples -- angiosarcomas, hepatic fibrosis, normal controls, hepatitis, etc. 2. Evaluated human hepatic angiosarcoma, hepatoma cirrhotic liver tissues GAGs (10 cases). 3. Evaluated tissue and urinary GAG patterns in animals undergoing CCl4-induced fibrosis (48 animals) at 3, 6, and 9 weeks (histochem/biochem). 4. Repeated # 3 evaluating at 1, 2, and 3 weeks (42 animals) histology/ biochemistry. 5. Evaluating urinary and tissue GAG patterns in animals bearing different behavioral types of transplantable hepatomas (42 animals). 6. Evaluating tissue and urinary GAG patterns In animals following partial hepatectomy. 7. Evaluating diurnal variation in urinary GAG excretion In two angiosarcoma cases (vs. normal). Compared urinary GAG patterns in workers with known normal and known diseased livers -- 24 workers (complete fractionations using Dowex 1X2 exchange). 8. Compared urinary GAG patterns with other non-invasive tests as to diagnostic value. One hundred-twenty (120) samples (used occasional samples with creatinine determinations). 9. Evaluating the contribution made to urinary GAG excretion by hepatic necrosis (experimentally induced Ischemic necrosis in rats). CMA 002392 53 NOTE: These are some preliminary d^^kathered in our transplantable hepatoma studies. These stud^s are designed to evaluate the relationship between tumor GAG composition and various tumor properties (growth rate, metastatic potential, etc.) and to evaluate urinary GAG excretion as a function of tumor size. CMA 0 0 2 3 9 3 TABLE: URONIC ACID LEVELS BY GAG FRACTION IN MORRIS HEPATOMA TISSUE; LIVER OF TUMOR BEARING ANIMALS; AND, NORMAL LIVER SLIDE 2 TUMOR 7777 TUMOR 5123 TUMOR 9618 LIVER 7777 LIVER 5123 LIVER 9618 NORMAL LIVER 1 0,03 M NaCl NON-GAG CARBOHYDRATES 176 + 38 176 + 15 524 + 81 664 + 203 6770+L629 455 + 146 950 + 340 0.4 M NaCl HYALURONIC ACID 90 + 10 91 + 12 111 + 9 40+8 41 5 49 + 10 25 + 3 1.2 M NaCl CHONDROITIN SULFATE 261 +29 217 + 51 208 + 22 68 + 23 58 + 5 41 + 10 47 + 7 2.1 M NaCl HEPARIN 31+7 17+4 67 + 11 17 + 3 I 28 + 6 31+7 39 + 10 I'rmlcd in Li.S.A. URINARY GLYCOSAMINOGLYCAN PATTERNS IN ANGIOSARCOMA OF THE LIVER Kevin L. Curran, BA, MS, Charles E. Kupchella, PhD, and Carlo H. Tamijurro, MD Glycosnminoglycans extracted from 24-hour urine specimens from patients with hepatic angiosarcoma and from normal/controls were separated as cetylpyridinium complexes into "hyaluronic acid," "chondroitin sulfate," and "heparin" fractions, then further separated and characterized by anion-ex change chromatography and hyaluronidase susceptibility. The chromato graphic pattern of the urinary chondroitin sulfate fraction in patients with angiosarcoma of the liver differed from those of controls in that there was a relative increase in the total amount of uronic acid in a hyaluronidase-resistant fraction and a decrease in a fraction susceptible to hyaluronidase digestion. These changes appeared to become more pronounced with advancing disease. Chromatographic patterns and determinations of hyaluronidase susceptibility indicated that the resistant fraction was heparan sulfate and that the susceptible fraction was chondroitin-4-sulfate and/or chondroitin-6-sulfate. Canetr 40:3050-3053, 1977. HE EMERGENCE OF ANCIOSARCOMA OF THE -2- liver and its relationship to vinyl chloride exposure4,7 has prompted a search for methods to detect this lesion. Although systematic screening programs are currently in opera tion,10-1* there is still no single chemical in dicator which' is specific for angiosarcoma or for changes which may precede this disease. The association of elevated tissue glycosaminoglycans (GAG) with tumors, including angio sarcoma, has been established.''*-1M,,w Glycosaminoglycans are also known to be involved in normal connective tissue synthesis and collagen deposition and are elevated in connective tissue disorders.11 Since angiosarcoma of the liver has both neoplasia and fibrogenesis in its etiology17 GAG changes could be expected to serve to sig nal the appearance of early lesions and may be useful in evaluating advanced lesions. Galambos* suggested that since the liver con tributes very little to the overall connective tissue of the body, hepatic fibrogenesis should not be expected to result in significant increases in From the University of Louisville, Cancer Center, and the Price Institute for Surgical Research Health Sciences Cen ter. Louisville, Kentucky 40201. This work was supported in part by an American Cancer Society Institutional Gram, IN-111, a grant from the R. F. Goodrich Company, and contract NOI-CN-55212 with the National Cancer Institute. Address for reprints: C. E. Kupchella, Cancer Center, University of Louisville, Louisville, KY 40201. Accepted for publication April 15, 1477. urinary GAG or collagen degradative or syn thetic products. Preliminary studies in our labo ratory, however, demonstrated an increase in both liver and urinary GAG in patients with angiosarcoma, chronic active hepatitis, and cir rhosis. " Most of the increase in urinary GAG occurred in the chondroitin sulfate fraction and, in contrast to what was found for normal and other diseases, the urinary chondroitin sulfate fraction was the only uronic acid positive fraction found in the urine of seven of nine cases of vinylchloride-exposure-associated liver injury other than angiosarcoma. This study was undertaken to characterize more completely the urinary "chondroitin sulfate'' fraction in hepatic angio sarcoma. Clinical Summaries Case 1--(Hepatic Angiosarcoma--advanced) A 46-year-old white male worked as a chemical helper in a vinyl chloride polymerization plant for thirteen years prior to the diagnosis of angiosarcoma. Twelve years after initial employment, the patient exhibited a persistent elevation of lactic dehy drogenase and underwent angiographic studies which demonstrated multiple areas of scattered tumor stain throughout both lobes of the liver with areas of central translucency consistent with the diagnosis of angio sarcoma of the liver. Exploratory laparotomy and liver biopsy confirmed this diagnosis, and the patient was treated with adriamycin, cyclophosphamide, and methotrexate; an 3050 55 CMA 002394 MOLARITY NoCI No. 6 Glycosaminoglycans in Angiosarcoma Curran et al. 3051 initial response was associated with a decrease in the alkaline phosphatase activity, improvement in in docyanine green clearance and an increase in radio isotopic uptake in areas of picvtously defective up take. After completion of the chemotherapy course, hepatic function deteriorated and the patient under went partial hepatic lobe radiation (total dose of 5,000 rads) over a two-month period. Despite radia tion therapy, the clinical course continued to deterio rate with the development of ascites, peripheral edema, increasing jaundice, hypoalbuminemia, and marked elevations of transaminases and alkaline phosphatase activities. This was followed by progres sive hepatic failure, hepatorenal syndrome and he patic coma. Autopsy findings showed extensive in volvement of the liver with angiosarcomatous tissue extending into the diaphragm and metastasis to retro peritoneal and mediastinal lymph nodes, lungs, right adrenal gland and cerebellum. The right lobe of the liver demonstrated near elimination of the angiosar coma, presumably due to the radiation treatment. Urinary GAG assays reported here were made on 24hour urine specimens collected over the two-week period before death (Fig. 1). rjl- NORMAL URINE 10 3 _rfr - HFPATIC ANGIOSARCOMA--Advanced \ L.-- , -I" 1_ 1______________-B________ _1L____ Case 2 (Hepatic Angiosarcoma--moderately advanced) A 54-ycar-old vinyl chloride polymerization worker was first employed as a polymerization vat cleaner 28 years prior to the diagnosis of angiosarcoma. Two years prior to diagnosis, the patient had persistent biochemical liver, abnormalities although he was oth erwise asymptomatic with a normal liver-spleen scan. Angiographic studies showed pcliosis hepatis. A liver biopsy revealed focal sinusoidal dilatation, mild chronic inflammatory reaction with portal fibrosis, Kupffer cell hyperplasia and dysplasia. Subsequent biopsies demonstrated continued sinusoidal dilitation, atypical and dysplastic Kupffer cells with premalignant changes. The peliosis hepatis pattern be came more pronounced and multiple radioisotopic defects were evident on liver scan. A repeat biopsy one year after initial biochemical abnormality demon strated malignant sinusoidal cells. The patient was treated with a combination of adriamycin, cytoxan, and methotrexate with limited clinical and biochemi cal response. Death was preceded by peripheral edema, ascites, progressive hepatic failure, and coma. The GAG analyses reported here were made 10 and 6 months before death (Fig. 1, [middle]). Materials and Methods Twenty-four hour urine specimens were col lected from two patients with angiosarcoma of the liver, and from two normal controls. Urine specimens were stored at --70C until analysis. Cetylpyridinium chloride (Sigma Chemical Company, St. Louis) was added to the entire 24-hour volume to precipitate the GAGs Fic. 1. Elution Patterns of the Urinary Chondroitin Sul fate Fraction. The glycosaminoglycans (GAG) in a 24-hour urine specimen were precipitated with cetylpyridinium chlo ride (CPC) and separated as 0.4 M NaCl soluble ("hyalu ronic acid"), 1.2 M NaCl soluble ("chondroitin sulfate"), and 2.1 M NaCl soluble ("heparin") fractions. Each fraction was then subjected to anion-exchange chromatography. Shown here arc typical 1.2 M (chondroitin sulfate) fraction elution patterns (Advanced * case 1). according to the method of DiFerrante. * The hyaluronic acid, chondroitin sulfate, and hepa rin fractions were eluted individually according to the method of Schiller et al. '* Cetylpyridinium chloride was removed14 and the GAGs were sub jected to anion-exchange chromatography as de scribed by Schiller et al.1* Glycosaminoglycan fractions were applied to 1.0 X 44 cm AG1-X2 (200-400 mesh, chloride form) columns Bio Rad Laboratories, Richmond, California) and eluted stepwise with 0.0, 0.5, 1.0, 1.25, 1.50, 2.0, and 3.0 M NaCl. At a flow rate of 1.0 ml/min, approximately sixteen 10.3 ml fractions of each molar strength of NaCl were collected and a sample of each fraction was analyzed for uronic acid by the method of Bitter and Muir.' Stan dards of heparin (Nutritional Biochemical Com pany), chondroitin sulfate (Sigma Chemical Company), and hyaluronic acid (Nutritional Biochemical Company) were also evaluated by ion exchange chromatography. The uronic-acid-positive fractions within each individual salt fraction were pooled, dialyzed to remove salt, and concentrated. The fractions eluted by 1.25 or 1.50 M NaCl were tested for 56 Oii\ 239S 3052 Cancer December 1977 Vol. 40 Table I. Source Normal Normal Angiosarcoma, case 2, prc-chemothcrapy1 Angiosarcoma, case 2, post-chemotherapy* Angiosarcoma, case 1, advanced Ratio of Total Uronic Acid Eluted in 1.25 M/t.5 M NaCl 0.364 0.316 0.843 0.971 5.000 Note: The glycosaminoglycaru (GAG) in a 24-hour urine specimen were precipitated with cetylpyridinium chloride (CPC) and separated as 0.4 M NaCl soluble ("hyaluronic acid"), 1.2 M NaCl soluble ("chondroitin sulfate"), and 2.1 M NaCl soluble ("heparin") fractions. The CPC was removed from the 1.2 M NaCl-CPCsolubilizcd fraction and the GAGs further purified by anion-exchange chromatography. The total amount of GAG in the resulting 1.25 M and 1.50 M NaCl columneluted fractions was determined and the ratio of the two fractions was calculated. (`One day prior to begin ning of chemotherapy; 'Two days following chemotherapy initiation.) acid and heparin fractions revealed no qualita tive differences between controls and angiosar coma patients. The anion exchange column pat terns of the 1.2 M NaCl solubilized GAGs are shown in Fig. 1. Chromatography of the urinary "chondroitin sulfate" fractions of patients with angiosarcoma yielded a comparatively large, uronic-acid positive peak in 1.25 M NaCl. The ratios of the total amount of uronic acid-positive material eluted with 1.25 M NaCl to the total amount eluted with 1.50 M NaCl are given in Table 1. The susceptibility of the GAGs eluted with 1.25 or 1.50 M NaCl to hyaluronidase degrada tion is given in Table 2. The GAG eluted with 1.25 M NaCl was resistant to hyaluronidase, the enzyme producing only a 43% reduction in tur bidity. The t.50 M NaCI-eluted GAG fraction was 100% susceptible to hyaluronidase degrada tion. Discussion and Conclusion susceptibility to testicular hyaluronidase (Nutri tional Biochemical Company) using a modifica tion of the cetyltrimethylammonium-bromide, turbidimetric assay described by DiFerrante.' Results The major GAG fraction observed in all urines--both from normal controls or from pa tients with angiosarcoma--was the fraction so lubilized by 1.2 M NaCl/1% cetylpyridinium chloride (the "chondroitin sulfate" fraction). Anion exchange chromatography of hyaluronic Table 2. Hyaluronidase Susceptibility Source Depolymerization %' Heparin, standard Hvaluronic acid, standard Chondroitin sulfate, standard 1.25 M NaCl column-eluate, pooled fractions from angiosarcomatous patients 1 30 \1 NaCl column-eluate, pooled fractions from angiosarcomatous patients 1.5CM NaCl column-eluate. normal 5.0 93.1 97.6 43.5 100.0 100.0 ` Glycosaminoglycans isolated from urine were tested for hyaluronidase susceptibility by measuring changes in turbidity developed with the addition of cetyltrimethylammonium bromide following incubation with hyaluronidase Normal controls exhibited only minor amounts of 1.25 M NaCl column-eluted GAG and consequently do not ap pear in this table. The chromatographic pattern found here for controls conforms to urinary glycosaminoglycan distributions reported by others.1*'*1 These pat terns suggest that there was a relative increase in urinary heparan sulfate and a decrease in chondroitin-4- and/or -6-sulfate in patients with hepatic angiosarcoma. This interpretation agrees with the Dowex 1-X2 chromatographic patterns reported by Kao and Leslie1* and by others.*'1* Heparan sulfate is reported to be partially susceptible to hyaluronidase digestion,** and this correlates well with the observed 43% diges tion of the GAG in our 1.25 M NaCl fraction. Since heparan sulfate has been shown to be associated with blood vessels,** an increase in the urinary excretion of this GAG is not surpris ing in this vascular lesion. Also, chondroitin-4and -6 sulfates are reportedly eluted from Do wex 1-X2 columns with 1.50 M NaCl*11* and are susceptible to hyaluronidase" suggesting that our 1.5 M fraction is chordroitin-4- and/or chondroitin-6-sulfate. Assuming that urinary GAG patterns de scribed here are reflections of hepatic changes, it will be important to determine what processes these changes reflect, i.e., those of neoplastic growth, fibrogenesis, or cell death. In this re gard, it should be noted that 1) the ratio of heparan sulfate to chondroitin sulfate reported here for angiosarcoinatous urine is similar to that reported for cirrhotic human liver tissue by Becker,* and 2) the shift from a hyaluronidase- 57 CMA 002396 No (' Gi.VCOSAMINOOLV.CANS in Angiosarcoma Curran tt al. 3053 susceptible to a hynluronidase-rcsistant GAG is consistent with the suggestion by Ii'uttercr and Rubin" that the stabilization of collagen de pends on a shift to a hyaluronidase-resistant GAG envelope surrounding the collagen bundle. Although Huttcrcr and Rubin attribute this to an augmentation of dermatan sulfate, Becker3 reported that the GAG pattern in human cir rhosis was characterized by the augmentation of dermatan sulfate and heparan sulfate. If the ob served changes in urinary GAG are reflective of vinyl chloride-exposure-associatcd fibrosis, the fact that fibrosis is a precursor of angiosarcoma" indicates that the observations reported here constitute a promising lead in early detection of vinyl-chloride-induced liver disease. REFERENCES 1. Anghilcri, L. J.: Metabolism of add mucopolysaccha rides in hepatoma and normal liver. Oncology 30:304-317, 1974. 2. Becker, K.: Acid mucopolysaccharides in experimental and human cirrhosis. In Collagen Metabolism in the Liver, H. Popper and K. Becker, Eds. New York, Stratton Inter continental Medical Book Corporation, 1973; pp. 45-52. 3. Bitter. T., and Muir, H.: A modified uronic acid carbazole reaction. And. Bmficm. 4:330-334. 1962. 4. Creech,.]. L., and Johnson, M. N.: Angiosarcoma of the liver in the manufacture of polyvinyl chloride. J. Occupa tional Med. 16:150-151, 1974. 5. DiFerrante, N.: The measurement of urinary muco polysaccharides. Anal. Btochcm, 21:98-106, 1967. 6. DiFerrante, N.; Turbidimetric measurement of acid mucopolysaccharides and hyaluronidase activity. J. Bid. Chem. 220:303-306, 1956. 7. Falk, H., Creech, J. L,, Heath, D. W., Johnson, M. N., and Key, M. M.: Hepatic disease among workers at a vinyl chloride polymerization plant. JAMA 230:59-63, 1974. 8. Galambos, J. T.: Connective tissue metabolism and cirrhosis. In Collagen Metabolism in the Liver, H. Popper and K. Becker, Eds. New York, Stratton Intercontinental Medical Boojt Corporation, 1973; pp. 57-61. 9. Gasic, G., and Gasic, T.: Removal of sialic acid from the cell coat in tumor cells and vascular endothelium and its effects in metastasis. Proc. Malt. Acad. Sci. U.S.A. 48.1172-1177, 1962. 10. Greenberg, R. A., Tamburro, C, H., and Kupchella, C. E.: A prospective medical surveillance program for the detection and prevention of occupationally-related cancer. In Prevention and Detection of Cancer. H. E. Nieburgs, Editor, Part 1, Volume 2, Marcel Dekker, Inc., NY (In press). 11. Hutterer, F., and Rubin, .: Mucopolysaccharides in reversible and irreversible experimental hepatic fibrosis. In Collagen Metabolism in the Liver, H. Popper and K. Becker. Eds New York, Stratton Intercontinental Medical Book Corporation, 1973; pp. 53-56. 12. Kao, K. Y. T., and Leslie, J. G.: Micro fractionation and determination of urinary glycosaminoglycans. Biochem. Med. 9:317-326, 1974. 13. Koizumi, T., Nakamura, N., and Abe, H,: Changes in acid mucopolysaccharide in the liver in hepatic fibrosis. Biochim. lliophys. Ada, 148:749-756, 1967. 14. Korn, E. D,: Isolation of heparin from mouse mast cell tumor. J. Bid. Chan. 234:1325-1329, 1959. 15. Kupchella, C. E., and Tamburro, C. H.: Urinary and tissue glycosaminoglycan patterns in hepatic angiosarcoma. In Prevention and Detection of Cancer, H. E. Nieburgs, Editor, Part 1, Volume 1, Marcel Dekker, Inc., NY (In press). 16. Makk, I-, Creech, J. L-, Whelan, J. G., and Johnson, M. N.: Liver damage and angiosarcoma in vinyl chloride workers: A systematic detection program. JAMA 230:64-68, 1974. 17. Popper, H., and Thomas, L. B,: Alterations of liver and spleen among workers exposed to vinyl chloride. Ann. NT Acad. Sci. 246:172-194, 1975. 18. Rich, C,, and Myers, W, P, L.: Excretion of acid mucopolysaccharides in the urine of patients with malignant neoplastic diseases. J. Lab. and Clin. Med. 54:223-228, 1959. 19. Schiller, S., Slover, G. A., and Dorfman, A.: A method for the separation of acid mucopolysaccharides: Its application to the isolation of heparin from the skin of rats. J. Biol. Chem. 236:983-987, 1961. 20. Sharon, N.: Complex Carbohydrates: Their Chem istry, Biosynthesis, and Functions. Reading, Massachusetts, Addison-Wesley Publishing Company, 1975. 21. Varadi, D. P., Cifonelli, J. A., and Dorfman, A.: The acid mucopolysaccharides in normal urine. Biochim. Biophyi. Ada. 141:103-117, 1967. 22. Yamamoto, K., and Terayama, H.: Comparison of cell coat acid mucopolysaccharides of normal liver and vari ous ascites hepatoma cells. Coma Her. 33:2257-2264, 1973. 002397 58 HEPATIC CELL IDENTIFICATION TECHNIQUE H.P. FORTWENGLER AND C.H. TAMBURRO, M.D. It became very evident early in our vinyl chloride metabolism studies that in order to be able to determine various cell capabilities of oxidizing and detoxifying one needed both an in vivo and in vitro method for determining cell oxidizing and detoxifying capability as well as cell identification. Modification of the technique by Leevy, et al has recently been developed in which the In vitro technique utilizes biopsy tissue for the identification of the various hepatic cell types. This Is illustrated in Slide I. A liver biopsy, either human, or animal, is cut in a Y shape in which each of the components of the biopsy can be utilized for different studies; electron microscbpy, light electron microscopy and cell type identification. The tissue for cell type Identification is then incubated in a modified Eagles medium for two hours at 37. By incubating this tissue with various markers, such as tritiated thymidine, iron particles, tritiated hydroxyproline or Factor VIII antibody, one can identify cells as 3 being active in incorporation of HT into DNA, having a phagocytic capability, having collagen forming ability or simply identies of endothelial cells respect fully. This technique has already been put to use In verifying the cell of origin that malignantly transforms Into the angiosarcoma. This has been shown to be the endothelial lining cell and not, as some had thought, the Kupffer or macrophagic cell. This now allows us to direct our attention to the variation in metabolic capability between the primary hepatocyte and the endothelial lining cell. We now hope to be able, using this technique for cell identification, to identify early metabolic capability and morphological changes In cells as they are exposed to various chemicals for prolonged periods of time. This will allow us to determine whether or not these metabolic and morphological effects are progressive, at what stage the metabolic changes are morphologically manifested, 59 CMA 002398 whether these morphological changes are permanent and irreversible, and whether with discontinuance of exposure, these changes revert to normal or progress on to develop malignantcy, We hope that these new developing techniques may be applicable to human tissue via biopsies techniques and help to identify mor phological changes with greater specificity as to their etiological cause. 'v CMA 002399 60 SLIDE I CUT- IN VITRO TECHNIQUE FOR HEPATIC CELL IDENTIFICATION ELECTRON MICROSCOPY CELL TYPE IDENTIFICATION LIGHT MICROSCOPY livhr BIOPSY DIVIDED LIVER BIOPSY MARKER ^HT/^HU latex/ carbon/fe CELL TYPE dna/rna ALL CELLS FACTOR VIII FLUORESCENCE 3h proline/ VIT A FLUORESCENCE EC FB CMA 002400 61 MEDICAL SURVEILLANCE SYSTEM FOR INDUSTRIAL ENVIRONMENTS The University of Louisville has, through cooperative endeavors with St. Anthony's Hospital and the B. F. Goodrich Company, (Louisville Plant) developed a medical industrial surveillance system which is applicable for industry-wide use. The surveillance system Is composed of three levels of screening programs as shown in Slide 1. Level I applies to the industrial environment which does not contain any known or potentially toxic or carcinogenic chemicals. This is also the basis or foundation upon which the more advanced levels of surveillance are built. Level I data base is composed of an employee work history, a job exposure history and a medical illness history including illnesses of the past as well as the present and future. Level II surveillance is composed of Level I components plus a basic medical history and physical examination, rank ordering of exposure to any industrial chemical thought to be potentially carcinogenic or toxic, and medically and/or Federally required screening tests. Level III surveillance i3 designed for an industrial environment actually utilizing a known carcinogen or toxin. At this level a complete medical history and physical examination are conducted. Individual job or person monitoring is done where scientific capability is available; otherwise rank ordering of exposure to the various chemicals is used for each job classification. In addition to Federally required tests, specialized screen ing studies are performed. A triage protocol is shown in Slide 2, which has been utilized for a combined synthetic rubber and plastic production plant. Slide 3 illustrates the procedure used for medical evaluation of screening data on an individual employee basis. Regional standard range is determined for all laboratory screening tests. Individual tests results outside of the 62 0240l SLIDE 1 INDUSTRIAL MEDICAL SURVEILLANCE SYSTEM THE SCREENING PROGRAM LEVEL I A. EMPLOYEE WORK HISTORY B. JOB EXPOSURE HISTORY C. MEDICAL ILLNESS HISTORY (1) INITIAL (PAST) (2) MORBIDITY (PRESENT) (3) MORTALITY (FUTURE) ~ LEVEL 11 A. BASIC MEDICAL HISTORY B. BASIC PHYSICAL EXAMINATION C. RANK ORDER EXPOSURE - AREA MONITORING D. BASIC MEDICAL SCREENING LEVEL III A. COMPLETE MEDICAL HISTORY B. COMPLETE PHYSICAL EXAMINATION C. INDIVIDUAL MONITORING D. SPECIALIZED MEDICAL SCREENING CMA 002402 63 ------------------------------------ ----------------------------------------------------------------------------------------------------- SLIDE 2------- OUTLINE OF EXISTING PROTOCOL FOR SCREENING 64 CMA 002403 SLIDE 3 PROCEDURE FOR EVALUATION OF MEDICAL SCREENING DATA ON INDIVIDUAL BASIS 1. DETERMINATION OF SCREENING TESTS 2. DETERMINATION OF STANDARD RANGE (SR=90%) 3. INDIVIDUAL RESULTS OUTSIDE SR - REPEAT A. PERSISTENT ABNORMALITY - DIAGNOSTIC WORKUP 5, R - 0 NATURAL VARIATION - NON-DISEASE EX, (A) AGE - ALK PHOS (B) RACE - IGG <(C) GENETIC - INDIRECT BILIRUBIN 6, R - 0 NON-OCCUPATIONAL DISEASE E.G.(A) HEPATITIS (b) DIABETES (c) HYPERLIPIDEMIA (D) OBESITY 7, INVESTIGATIVE DIAGNOSTIC STUDIES E.G-. (a) SPECIAL PROCEDURES (B) HOSPITALIZATION (C) BIOPSY 8, TREATMENT (a) PRIMARY DISEASE (B) OCCUPATIONAL CHANGE 65 CMA 002404 standard range are repeated and individuals with persistent abnormalities undergo diagnostic work-up. There are two major variations in screening tests which may simulate occupationally related disease. These include natural variations, or non-diseases, as exemplified by an elevated alkaline phosphatase associated with age, or elevation of the IgG immunoglobulins associated with race and other areas such as a congenital indirect hyper bilirubinemia which are genetically related. The second category is the non-occupational disease which may simulate occupational injury such as hepatitis, diabetes, hyperlipidemia and obesity. Individuals found to have occupationally-related disease undergo thoroughdiagnostic studies to determine the etiological cause and are given the appropriate treatment as Indicated. Reasons for surveillance programs are listed in Slide 4. Although the medical surveillance system for industrial environments are designed mainly for the fifth reason, that is, the detection of treatable conditions, the design of this system acutally meets all five reasons for the program, (Slide 4). BASIC MEDICAL SURVEILLANCE SYSTEM The employee work history is composed of a uniform job classification code for the entire plant or industry, and estimated exposure (slide 5). to various chemicals. The degree of exposure is ranked in.theorder of 0 to 6. Slide 6 indicates the rating basis for the rank ordering of chemical exposure. These rank orders are used to develop exposure indices such as average, total, or interaction exposure indices. Slide 7 illustrates the selection criteria for the original nineteen chemicals used in our medical surveillance system at the B. F. Goodrich Plant. Similar criteria can be used for chemical toxins or potential carcinogens. Slide 8 is an illustration of a detailed work and exposure history; on the left are listed the years in which the Individual was employed, the building qs4#--.(3-4iglts), and the job code (an additional 3 digits) and 66 CMa 0024QS SLIDE 4 REASONS FOR SCREENING PROGRAMS PROTECTION FOR ECONOMIC BUSINESS PROTECTION FOR OTHER INDIVIDUALS ALTERNATIVE TO PERSONAL HEALTH SERVICES ACQUISITION OF CLINICAL BASELINE INFORMATION DETECTION OF TREATABLE CONDITIONS SLIDE 5 EXPOSURE SUMMARY A, J03 CLASSIFICATION CODE' 3. EMPLOYEE WORK HISTORY C. CHEMICAL EXPOSURES D. EXPOSURE INDICES 1, AVERAGE EXPOSURE 2, TOTAL EXPOSURE 3, INTERACTION EXPOSURE 67 002406 CMA- SLIDE 6 RATING BASIS 0 - ABSENT FROM ENVIRONMENT 1 - LOWEST EXPOSURE 2 " MINIMAL EXPOSURE TO LOW LEVELS 3 - MODERATE EXPOSURE A - WORKS IN AREA SUBJECT TO OCCASIONAL HIGH EXCURSIONS 5 - WORKS IN AREAS WHERE LEVEL IS HIGH 6 - INTIMATE CONTACT - SKIN OR HIGH INHALATION CMA 002407 68 SLIDE 7 SELECTION CRITERIA FOR ORIGINAL 19 CHEMICALS 1, IS IT A KNOWN HEPATOTOXIN? 2, IS IT A SUSPECTED CARCINOGEN? 3, HOW TOXIC IS IT? 4, DO EMPLOYEES FREQUENTLY COME INTO INTIMATE CONTACT WITH IT? 5, IS IT FOUND IN THE VINYL CHLORIDE POLYMERIZATION AREA? CMA 002408 69 SLIDE 8 CMA 0 0 2 4 0 9 DETAILED WORK AND EXPOSURE HISTORY WORK HISTORY EXPOSURE RANK FOR EACH CHEMICAL YEAR BUILDING JOB NO. MONTHS VINYL CHLORIDE 2* 3* 1944 000 576 6 1945 000 576 5 1945 111 194 7 1946 111 194 12 1947 111 194 12 1948 111 194 8 1948 121 192 4 1949 121 192 4 1949 112 it 253 8 i 2 11 2 11 5 41 6 41 6 43 5 43 4 31 4 31 2 63 1957 i 1972 1972 1973 1974 m 235 i 5^3 ii7 000 000 000 JTERM1NATEE 574 574 574 5 7 12 8 4 31 1 21 2 11 2 11 2 11 OTHER CHEMICAL 22* 4 4 1 1 1 1 1 1 2 1 1 4 4 4 number of months worked at that job in that building. This composes the work history for the employee. The exposure ranking of each chemical is shown on the right. Vinyl chloride exposure is illustrated first and the numbers 2 to 22 are used to indicate the other chemicals. A multi-employee continuous work and exposure history form for employees is illustrated in Slide 9. This demonstrates a simple means of keeping a work history using a six digit code system for both job classifications and building codes specific for each industry or company. This would provide useful prospective as well as retrospective data to document the safety of the environment or to determine if an occupational risk is truly oesent. DATA BANK The need for a data information resource to allow prospective investigative epidemiological studies have been well illustrated by the University of Louisville's medical surveillance Data Bank System. The material acquired during a three-four year period is illustrated in Slide 10. Packaged computer programs to analyze this occupational medical surveillance data are presently being shown to be cost effective, time saving, able to guarantee uniformity of data collection, as well as security to the workers as far as personal information is concerned (Slide 11). This data, collected on a prospective basis, provides immediately to industry and others, the best available human epidemiological information for validating preliminary results of other Industries or animal data. The use of this information for various types of evaluation are shown in Slide 12, Illustrations of the effectiveness of the program are demonstrated in Slides13, 14, and 15. Slide 13 simply lists the number and types of malignancies found in this cohort of chemical workers between 1960 and 1978. At a glance, one has the impression that there is an increasing number of tumors among liver. 71 CMA 002410 SLIDE 9 AEMPLOYEE WORK AND EXPOSURE HISTORY YEAR COMPANY WORK HISTORY 1945 MILLING, INC. 177*1 - 268** 177 - 269 178 - 438 1950 PLASTIC INTERNATIONAL 013 - 291 018 - 548 013 - 620 1957 ~ J.K. MANWORK, INC. 100 - 001 100 - 020 1965 G.P. CHEMICAL CO. 001 - Ill # OF MONTHS 14 26 ' 20 24 12 48 48 48 CURRENT # AREA OR BUILDING CODE * JOB CLASSIFICATION CODE EXPOSURE DATA FOR EACH CHEMICAL FOR EACH JOB CLASSIFICATION IS KEPT BY THE COMPANY. 061 ooatii 72 SLIDE 10 SOURCE AND USE OF DATA BANKS: SOURCE: UNIVERSITY OF LOUISVILLE DATA BANK CONTENTS AS OF MARCH 25, 1977 TYPE OF INFORMATION WORK HISTORIES "exposures INDICES BLOOD CHEMISTRIES LIVER SCANS SPLEEN SCANS CHEST X-RAYS HISTORY & PHYSICALS (1974) HISTORY & PHYSICALS (1975) HISTORY & PHYSICALS (1976) BIOPSIES NUMBER OF EMPLOYEES 1672 1672 1347 1222 1217 853 679 875 831 90 . "EXPOSURE INDICES ARE AVAILABLE FOR 21 CHEMICALS IN ADDITION TO VINYL CHLORIDE, CMA 002412 73 SLIDE 11 OCCUPATIONAL MEDICAL SURVEILLANCE PACKAGED COMPUTER PROGRAMS 1) WILL SAVE MONEY 2) WILL SAVE TIME 3) WILL GUARANTEE UNIFORM DATA COLLECTION 4) WILL GUARANTEE SECURITY TO THE WORKERS 5) WILL BE AVAILABLE IMMEDIATELY TO MEET THE NEEDS OF INDUSTRY 6) WILL BE AVAILABLE TO BOTH SMALL INDUSTRIES AS WELL AS LARGE INDUSTRIES CMA 002413 74 lung, and possibly, colon. Analysis of exposure work history helped in identifying correlations between chemicals and cancer. All the cases of angiosarcoma discovered in this cohort population since the start of the program in 1974 were compared with matched controls who had begun to work the same year as the angiosarcoma cases and had worked the same periods of time. Each control and case was then ranked as to their total exposure to vinyl chloride as shown in Slide 14. As one sees, the angiosarcoma cases are among those with the higest exposure to vinyl chloride based on their rank ordering. This was significant to a P <.05. In contrast, acrylonitrile (Slide 15) exposure ranking of individuals with angio sarcomas and their match controls shows no such correlation of that chemical to the cases of angiosarcoma. Of the additional 20 chemicals studied, only three others showed significant correlations, as did vinyl chloride, with angio sarcoma. They were a) the specific catalysts for the vinyl chloride polymerization process, b) hexane,used as a solvent for these catalysts, and c) diethyl maleate, a specialized catalyst used only for a specialized vinyl chloride poly merization process showed such relationships (Slide 16). This data validates the ability of rank ordering of exposure to identify causative relationships and/or the absence of such when used retrospectively. This will be even more effective as it is continued to be used prospectively. Additional studies have been conducted to determine if there exists any relationship between lung cancer and vinyl chloride exposure. As shown in Slide 17 and 18, no correlations were found between vinyl chloride, acrylonitrile, or any of the other 21 chemicals when the cases overall lung cancers were matched against their controls. This data would support the lack of a relationship between these chemical exposures and lung cancer development. CMA 002414 75 SLIDE 12 EVALUATION 1. ARE SCREENING PROGRAMS TO DETECT DISEASE LIKELY TO HAVE IMPORTANT EFFECTS ON HEALTH? 2. DOES TREATMENT OF RISK FACTORS INFLUENCE DEVELOPMENT OF DISEASE? 3. INDIVIDUAL COMPLIANCE IN SCREENING PROGRAMS, 4. DOES THE SCREENING PROGRAM REALLY ALTER OUTCOME OF TARGET DISEASE? 5. ARE THE PRESENT METHODS OF EVALUATIONS MISLEADING AS TO EFFECTIVENESS OF POSSIBLE SIDE EFFECTS OF SCREENING DISEASE, LABELLING OF INDIVIDUAL AND LONG-TERM THERAPY? CMA 002415 76 NUMBER AND TYPES OF MALIGNANCY IN CHEMICAL WORKER COHORT-1960 -1978 1960-64 1965-68 1969-71 1972-74 1975-78 LIVER " LUNG COLON C T OrTT'TO BRAIN O GU too H <Ti 00 o O VINYL CHLORIDE EXPOSURE RANK OF INDIVIDUALS WITH ANGIOSARCOMA 40'-, LJ 35 OC => to o 30 CL X 25 to n< 20 a > -4 15 Q 10 O X5 z <a: 0 II III IV ANGIOSARCOMAS 7 ' ZU AND MATCHED CONTROLS I. ACRYLONITRILE EXPOSURE RANK OF INDIVIDUALS WITH ANGIOSARCOMA ex^zoo I II III IV ANGIOSARCOMAS r AND MATCHED CONTROLS i ~i SLIDE 16 LIST OF SELECTED CHEMICALS FOR EXPOSURE INDICES CHEMICAL CODE 01 02 03 04 05 06 07 03 09 *7" 10 11 12 13 14 15 16 17 13 19 20 21 22 ACRYLIC ACID ACRYLAMIDES - ACRYLAMIDE, METHYL, N-OCTYL, nMA ACRYLONITRILE ACETYLENE ACRYLATES -- ETHYL, METHYL, METHYL-METH, 2 ETHYL HEXYL, N-BUTYL BISPHENOL A BUTADIENE CAPRYLYL CHLORIDE CHLORINATED SOLVENTS -- CARBON TETRACHLORIDE CHLOROFORM, TRICHLOROETHYLENE, EDC CHLORO ETHYL VINYL ETHER DIETHYL MALEATE MECURIC CHLORIDE METHANOL PHENOL TOLUENE VINYL CHLORIDE VINYLIDENE CHLORIDE VINYL ACETATE PVC DUST CATALYSTS STYRENE HEXANE MA 002419 80 i VINYL CHLORIDE EXPOSURE RANK OF INDIVIDUALS WITH LUNG CANCER r--* LUNG CANCERS ii AND MATCHED CONTROLS I I 002420 ACRYLONITRILE EXPOSURE RANK OF INDIVIDUALS WITH*LUNG CANCER LU oc oo oD_ oo <c oNo> Q O g- tyro o o K> 3 LUNG CANCERS Aj 3 AND HATCHED CONTROLS SCREENING TESTS The use of screening studies to determine the presence of hepatocellular injury have relied mainly on enzymatic studies determined in circulating blood or serum. Slide 19 illustrates the frequency with which the presently avail able clinical biochemical studies correctly indicated the presence of hepatic injury. The best enzymatic biochemical study was the SGPT which correctly correlated with hepatic injury in about 88% of the cases. The use of functional studies such as dye or bile acid clearances which is shown to the far right, demonstrated that using a maximum dose capacity for the liver, one can correctly identify underlying hepatocellular injury in almost 98% of the cases. The major limitation of clearance studies, however, is the lack of specificity as to the cause of the injury. In addition, our data indicates that clearance studies are able to demonstrate progressive changes much earlier in the developing of hepatocellular injury than enzymatic studies can. This is illustrated in Slide 20 by the demonstration of progressive, increasing changes in dye clearance in the absence of changes in alkaline phosphatase during progressive exposure to the hepatoxic chemicals over many years. More attention to development of more specific and sensitive single screening procedures are needed rather than a "shot gunapproach" of multiple tests. The medical surveillance system is a far more cost effective means of deter-, mining industrial environment safety than any presently useable animal or bacteriological screening procedure. Although bacteriological studies are relatively inexpensive, they only indicate which chemicals may be of potential danger. They do not provide any data as to the degree of exposure or duration of exposure needed to present a significant risk. Animal studies can provide further information, however, the correlation CMA 002422 83 . SLIDE 19 f ! Frequency of biochemical abnormalities being P"*"C correctly Indicating the presenceftignlfleant h^tological abnormalities (SOFT - Uanlne aminotransf erase i GGT - ICJ . ScSc^rC^nLe^i^'- Socyenin. green clearance) CMA 002423 84 FUNCTIONAL vs BIOLOGICAL SCREENING FOR HEPATOCELLULAR INJURY IN AN INDUSTRIAL POPULATION #' TEST VALUES SLIDE 20 8 O o to to TIME IN YEARS between developing cancers in animals is not necessarily nor consistently equal to the frequency and occurrence of chemically induced cancers in man. The cost of studying chemical carcinogens (Slide 21) in the four million chemical formulations now available, or the 600 new chemicals reportedly synthesized yearly, or even the 63,000 chemicals estimated to be in common use, are prohibitive in cost. The minimum estimate of cost to study a single chemical used at a single dose level, in a single animal specie for two hundred animals over a two year period, is approximately $40,000. This cost is a conservative one. If this were applied to all 600 new chemicals per year the cost would be $24,000,000. If one were to do these studies solely for the 63,000 chemicals in common use, this would be over $2,000,000,000. In contrast, a medical surveillance system institution in a single plant of 1,000 workers costs roughly $45,000. Once developed, its record maintenance would run about $5,000 per year, or approximately $5.00 per individual. Analyses of the data on a semi-annual, or annual basis, would cost approximately $20,000 for an initial plant and 5-10,000 for each additional plant. This is a relatively inexpensive and provides a much more relevent means of determining environmental safety in a prospective manner. This system can also be utilized to determine and demonstrate whether or not primary preventative methods are effective in reducing the occurrences of cancers in those environments where they are shown to be higher than expected. Such prospective surveillance systems, will provide the means of determining the need and effectiveness of costly preventive requirements which on theoretical grounds are thought to be needed but not proven to be useful. Slide 22 shows how Cancer Centers and Health Science Centers may play an external objective scientific role in assisting corporate medical directors in assessing industrial environmental safety. The corporate medical director responsible for multiple plants working with their part-time or full-time plant physicians can work CMA 002425 86 SLIDE 21 CHEMICAL CARCINOGENS 4,000,000 CHEMICAL FORMULATIONS 600 NEW chemicals/year 63,000 chemicals in common use 1 CHEMICAL, 1 DOSE, 1 SPECIES, 200 ANIMALS, 2 YEARS COST FOR CHEMICAL TESTING $2,520,000,000 in common use *24,000,000 FOR NEW CHEMICALS CMA 002426 87 collaboratively with scientific communities on these problems (Line one). This slide (22) outlines such a working relationship. Various plants dependent on size and need may have, as shown on Line two, no plant physician or full-time (FT) or part-time (PT) one. Line three illustrates various types of plants. A single circle indicates a plant manufacturing a single product (i.e., plastics); multiple circles - multiple products (i.e., plastic and rubbers). The letters below the circles identify the carcinogenic or toxic environment: X - no known carcinogen or toxin A = actual carcinogen or toxin P - possible carcinogen or toxin Line four identifies the type of medical surveillance program at each plant. Line five indicates the type of medical screening program being conducted at each plant and Line six indicates the working team of corporate medical director and his staff, Medical Center or Cancer Center medical epidemiologist and medical consultants for prospective ongoing design, review and analysis. The medical surveillance program's screening studies may be carried on either by local hospitals, or Industrial laboratories would supply the test results and medical information to the regional data banks where medical epidemiologists and specialists would analyze the data for the industrial plants in their geographic areas. This information would have the objective scientific status and would prevent claims of biased analysis. If the data were to show problems developing or existing, industry could then apply proper resources to correct them or determine their validity. If the information showed no evidence of developing problems, the data would be utilized to support the safety of the work environment. This system has been successfully used for four years now at the B. F. Goodrich Company, Louisville Plant, in cooperation with local medical facilities, the unions and management. Each component collaborated in the data collection, analysis and validation. Conclusions drawn here have academic, managerial and labor support of their validity and scientific accuracy. 88 CMA 02427 Line 1. CANCER CENTER CORPORATE MEDICAL DIRECTOR (CMP) SLIDE 22 SCHOOLS PUBLIC HEALTH MSP LEVEL MEDICAL 5. SCREENING III A LOCAL HOSPITAL LOCAL EXTERNAL HOSPITAL ` LABORATORY \ 6. 89 BIOASSAY OF CHEMICAL MONOMERS IN ISOLATED MAMMALIAN LIVER CELLS Richard C. Feldhoff, Ph.D. Assistant Professor, Department of Biochemistry, University of Louisville School of Medicine, Louisville, KY *10232 The mammalian liver is the principal- organ responsible for the metabolism and/or detoxification 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 detoxification of chemical monomers by the liver and to assess the role of alcohol consumption on these pathways. Studies of chemical monomer 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 htgh 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 ts 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 re solution electron microscopy is identical to that of the intact tissue. There are also major technical advantages associated with the use of ceil 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 variables simultaneously and 3) the ability to CMA 002429 ;o Dr. Richard C. Feldhoff employ radioactive metabolites at a much higher specific activity than is feasible in other in vitro or in vivo systems. Fm. 1 Diagram depicting cannulation and perfusion procedures. A, ligature tying portal vein cannula and placed above entry of splenic vein: B. inferior vena cava and abdominal aorta sectioned after insertion of portal cannula; C, infusion tubing And cannula inserted into rubber tubing insert in inflow line; D, inflowline taped ro aluminum block to hold portal cannula in [/lace- E, ligature around celiac axis and superior mesenteric arteries, tied off 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 in place. 91 002430 CMA Dr. Richard C. Feldhoff Fic. 2 Total protein and albumin secretion by isolated hepatocytes. Cells were prepared ns described under "Experimental Proce dures" and pivincubated for 30 min in Krebs-Henselrii bicarbonate buffer containing 7.5 times the normal plasma levels oramino acids. Cells were transferred to 40 volumes ofbicarbonate buffer containing normal plasma levels of amino acids and ['Hikw.ke at 13 /*Ci/ml. At each time point, aliquots of the suspension were centrifuged and assayed fir incorporation into total secreted protein and albumin as described under "Experimental Procedures." O------O, hcpatocytes from normal rats; O------ , hcpatocytes from hypophysectomized rats. With respect to chemical monomer metabolism, isolated liver cells can be used to investigate: 1) the uptake, intracellular transport (binding proteins) and metabolism by various cell fractions of radioactive pre cursors (Feldhoff, Tamburro, Du, Wong, (Wittliff - cytosol receptors)). 2) identification and quantitation of metabolites released into the medium (Feldhoff, Wong, (Hoffman - HPLC)). 3) effects of individual metabolites on normal parenchymal cell functions (Feidhoff, Tamburro); mutagenic capacities of newly Identified compounds (Streips). k) potentiation of carcinogenesis by concurrent exposure to hepatotoxins such as ethanol (Feldhoff, Tamburro). This proposed research program 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 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. CMA 002431 92 93 CMA 002432 SUMMARY OVERVIEW THE YA^ JOURNAL OF ulOLOGY AND, MEDiClMc Si (i Wa), u7^>0 rr^ --" , -r-iL - * A #*G a CJi<V/^V<4CSaS -Cs 2r]y Detector*--71:e Vinyl Cnori^e Model*1*2 CARLO H. TAM3URR0 University of Louisville School of Medicine, Louisville, Kentucky Received October 17, 1977 The liver's role in vinyl chloride toxicity and carcinogenicity is providing a better understanding of the chemical carcinogenesis mechanism. A variety of both malignant and benign hepatic tumors has been demonstrated with prolonged exposure to vinyl chloride. The multi-system involvmcnt ofthis carcinogen and toxin has provided a model for the study ofchemical carcinogenesis common to both man and animal. Clinical studies have shown the usefulness of biochemical, radioisotopic, and radiological studies in the detection of toxic and carcinogenic lesions. Animal studies have demonstrated the biochemical metab olism by the liver of vinyl chloride-produced intermediates which are mutagenic in bacterial systems and may be the ultimate carcinogens. Hepatic subccilular enzyme studies prove preliminary evidence of cellular adaptation and increased detoxification. Disruption of this oxidization and detoxification balance may be the key to the malignant transformation of ceils. A working hypothesis is presented which may explain the metabolism of vinyl chloride into mutagenic intermediates by the liver cell and the develop ment of malignant transformation by extra hepatic sinusoidal lining cells, lung cells, and brain tissue. INTRODUCTION Currently there is a growing concern that chemical compounds may be respon sible for most human cancer through environmental contact. Although 100,000 to 200,000 new chemicals are introduced into industry each year, little is known about their effects. These compounds are primarily synthetics and thus not natural to the environment. The view that industrial chemicals may be latent carcinogenic hazards has again been brought into sharp focus by the discovery ofvinyl chlorideinduced angiosarcoma. Vinyl chloride (CH2 =* CH-C1 monochloroethyene, a gas) is the basic molecule or monomer of polyvinyl chloride and its co-polymers and one of the most important organic intermediates in the plastics industry. The resulting plastic resin, polyvinyl chloride, is used in innumerable consumer and industrial products, such as containers, wrapping film, electrical insulation, pipelines, credit cards, etc. Until recently (early 1970's) vinyl chloride was regarded as being rela tively non-toxic [1,2]. Initially this opinion seemed to be supported by the facts that it was used transiently as an anaesthetic and had been used commercially in indus try for many years [3]. The direct information on the toxicity of vinyl chloride to man was obtained from experiments by research workers on themselves, from the evaluation of its suit- 67 1Portions ofthis work were supported by a grant from Manufacturing Chemists Association and by NationalCancer Institutes Contract #K0I-CN-552I2. 'This article is the thirteenth in a series entitled, "Seminars on Liver Disease," that have been presented as part of the Training Program in Liver Disease at the Veterans Administration Hospital, West Haven, Connecticut. Dr. Harold O. Cons. Professor of Medicine. Yale University School of Medicine, and Directorofthe Training Program in Liver Disease, is guest editor. Please address reprint requests to: Carlo H. Tamburro, M.D., Professor ofMedicine, Chief, Digestive Diseases and Nutrition Division, Director, Vinyl Chloride Project. 511 South Floyd Street, P.O. Box 35260, Louisville, KY 40232 0044-0086/78/5101-C067 $01.40 Copyright 1973 by The Yale Journal of Biology and Medicine, Inc. All rights of reproduction in any form reserved. 94 CMA 002433 68 CARLO H. TAM3LRRO ability as an anaesthetic [4, 5], and from study of the cases of vinyl chloride poison ing contracted during industrial use [6, 7, 8, 9]. Sporadic studies with vinyl chloride polymerization workers demonstrated vary ing degrees of hepatic biocnemical derangements, hematological abnormalities, and skin changes [9j. Acute short term exposures led to disturbances of the central nervous system, cardiac arrythmias, severe irritation of the mucosal membrane of the eyes and the respiratory tract, and in some cases--severe pulmonary edema with obstruction of the liver and kidneys. Chronic inhalation trials in animals clearly showed that vinyl chloride was toxic to the iiver and kidneys as well as irrita ting to the mucosal membranes and the lungs. Microscopic examination of liver sections showed degeneration of the central lobules while the damage to the kidneys chiefly involved the tubule and the interstitial lining somewhat like that of carbon tetrachloride damage [10, 11, 12]. BACKGROUND The literature, however, contained virtually no information on damage to man after chronic exposure until Filatova et al. reported disturbances of the blood vessels and nerves in individuals exposed with 20-3G0 ppm of vinyl chloride on a continuous basis [13]. Cordier et al. [14] and Wilson et al. [15] were the first to re port the hitherto unrecognized disorder termed occupational acroosteolysis (AOL) which included the symptoms of tenderness ofthe fingertips, gradual destruction of bony integrity of the fingers and a Raynaud's-like phenomena. Studies were initiated in animals to reproduce the acroosteolysis. In 1971, Violi et al. [ 16] while exposing animals to 30,000 ppm to induce acroosteolysis, accidentally discovered cancer. Maltoni et al. [17] while studying various levels of exposure demonstrated that angiosarcoma occured at 250 ppm; the Manufacturing Chemists Association's studies [18] demonstrated these liver cancers even at 50 ppm. At the -same time. Dr. John Creech, at the 3.F. Goodrich Chemical Company Plant in Louisville, Kentucky, discovered an hepatic angiosarcoma in one employee. Creech, recalling an earlier hepatic angiosarcoma at the plant, reviewed the medical histories of previous employees. Four additional angiosarcomas were found, further supporting the connection between vinyl chloride exposure and tumor development. Measures to control the levels of vinyl chloride exposure were then instituted following federal regulation. THE CHEMICAL Vinyl chloride's chemical structure is a double-bonded, 2-carbon halogenated hydrocarbon which has structural similarities to tri-chloroethylene, an inhalation anaesthetic. As previously noted, vinyl chloride was once considered as an anaesthetic but was discarded because it caused myocardial irritability. Some of its important physical properties include a low boiling point, a high specific gravity, a low solubility in water, and a half-life in air which ranges from 3-20 hours. Xnowledge of these physical properties may be important in determining how this agent produces a cumulative effect in the environment which ultimately leads to cancer formation. Vinyl chloride is both toxic and carcinogenic, as recognized by the wide variety of associated disorders which have been found among vinyl chloride polymeri zation workers and vinyl chloride-exposed animals. A yet incomplete list of these 95 002^4 CtAfc- VINYL 4 CARCINOGENESIS TABLE 1 Ocoopotional Vinyl Chloride Exposure Associated Disorders 1. Thronibocyiopenia 2. Rcliculocylosis 3. Splenomegaly 4. Hepatic fibrosis 5. Scleroilcrnia-like skin changes 6. Acro-osicolysis 7. Raynaud's phenomenon 8. Leukopenia 9. Hepatomegaly 10. Pulmonary functional impairment 11. Angiosarcoma 12. Cardiac arrhythmia 13. Nephroblastomas* 14. Zymbal gland carcinomas* 13. Large cell lung cancer 16. Brain cancer In rats only 69 associated disorders is shown in Table 1. Animal and epidemiological studies indicate the probability that cancer induction at other sites is also directly attributed to prolonged and excessive vinyl chloride exposure [19J. In order to develop effective methods of prevention, accurate knowledge of the pathogenesis of this environmental chemical in ultimately producing its most destructive effect--cancer--is needed. EXPERIMENTAL ANIMAL STUDIES--TUMOR FORMATION The carcinogenicity of vinyl chloride has been demonstrated in a variety of animals as well as in humans at exposure levels that vary from 50-30,OCO ppm. As illustrated in Table 2, a variety of tumor types have been found in rats, mice, and hamsters. An extensive list of benign tumors have also been reported in mice, rats, and hamsters exposed to vinyl chloride. Maltoni's group has now demonstrated primary liver cell cancers in exposed newborn rats. Direct hepatocellular injury as well as pulmonary, mucosal and skin injuries have been shown in directly exposed animals. Pretreatment with many agents increases the toxicity of vinyl chloride; they include phenobarbital, ethanol, polychlorinated biphenyls and pesticides such as hexachlorobenzene [20]. This relationship to vinyl chloride's ability to induce cancer is under study, particularly in view of the industrial environment which allows exposure to many chemicals to occur con currently. HUMAN STUDIES Epidemiological studies in the human strongly suggest that exposure beyond 10 years is associated with increased cancer mortality, mainly digestive system cancers, primarily hepatic [21,22]. There also appears to be a higher incidence of large cell carcinomas of the lung, brain glioblastoma multiforms, and lymphomas [23,24], although there is some disagreement as to the interpretation of this aspect of the epidemiological data. 96 70 CARLO H. TAN.uURRO TABLE 2 Carcinogenicity of Vinyl Chloride (Exposure: 50-10.000 ppm) lumorType Species 1. Liver--angiosarcoma 2. Liver--hepatocellular carcinoma 3. Lung--adenocarcinoma 4. Lung--large cell carcinoma 5. Mammary adenocarcinoma 6. Zymbul gland tumors 7. Nephroblastoma 8. Osteochondromas 9. Skin epitheliomas 10. Melanomas 11. Glioblastoma multiforme 12. Lymphoma Adult humans, rats, mice and hamsters Newborn rats Rats and mice Humans' Mice Rats Rats Rats Hamsters Hamsters Humans* Humans* 'Strongly suggested epidemiologically The multisystem involvement of this carcinogenic and toxic chemical is further illustrated in man. Early physical findings of vinyl chloride-injury include hepato megaly, portal hypertension, possible mild systemic pulmonary hypertension, bilateral midzonal pleural thickening of the lung, and splenomegaly with and without increased portal pressure [25]. These anatomical and physiological findings most frequently occur in the absence of the traditional clinical biochemical derangement of the liver. Screening studies of vinyl chloride workers during the past two and a half years have clearly illustrated the irregular and often delayed appearance ofabnormalities in aspartate and alanine aminotransferases (SGOT and SGPT), alkaline phosphatase as well as other hepatocellular enzymes. Gamma glutamic transpeptidase (GGTP), believed to be a more sensitive indicator of hepatocellular injury, has proven to have too high a false-positive rate to warrant its use in the screens for hepatocellular injury. Sorbitol dehydrogenase (SDH) studies indicate that this enzyme, which is liver tissue specific, is too insensitive for primary screening but is useful for confirmatory testing. Anionic dye clearance studies (Indocyanine Green, ICG) have demonstrated the highest specificity and sensitivity of all the primary screening procedures tested when performed at the 5 mg/kg dose level. At the traditional 0.5 mg/kg level, it has the same effectiveness as the aminotransferases and alkaline phosphatase com bined. The frequency of abnormal ICG dye clearances increases with prolonged exposure to vinyl chloride as illustrated in Fig. 1 and correlates well with the cumulative exposure to vinyl chloride as well as the histological evidence of hepatocellular injury. These functional studies for detecting hepatocellular injury do not, however, identify the cause. Clinical studies have illustrated the usefulness ofradioisotopic liver-spleen scans as a primary screening procedure for anatomical lesions of the liver and spleen. This procedure has proven to be the single most reliable method oftumor detection. Sixteen of the 19 individuals with anatomical lesions were detected by liver-spleen scan. In contrast, only 32 of 950 normal individuals had scan abnormalities which further diagnostic studies proved incorrect. This method provides an 84% sensitiv ity and 97% specificity, with only a 3% false-positive rate. 97 71 Diagnostic angiographic studies of these radioisotopic abnormalities in SO individuals have demonstrated 3 major lesions, "he first is peliosis hepatis, illustrated in Fig. 2. These lesions are usually numerous involving the entire liver, and have a diffuse stain throughout the nodules which persists into the late venous phase without central hypovascularity [26]. A second, similar lesion has been discovered ir, individuals with splenomegaly, and named lienal peiiosis (Fig. 3). These splenic lesions demonstrate a shortened ceiiac artery to portal vein circulation time, normal portal vein diameters, and increased spleen size. This has been found only in individuals with long-term vinyl chloride exposure. The final lesion is that of angiosarcoma (Fig. 4). This tumor has characteristic angiographic features of central hypovascularity, midarterial puddling, and a prolonged peripheral tumor stain which continues up to 30-36 seconds after injection. These characteristic findings have allowed differentiation from other primary hepatocellular cancers, benign tumors and benign vascular lesions [26]. These angiographic lesions have been pathologically confirmed with the additional histological finding including peliosis hepatis, sinusoidal dilatation, and activated sinusoidal cells with increased deposits ofcollagen in the sinusoidal space of Disse. Exploratory wedge biopsies have in addition demonstrated increased subcapsular fibrosis with subcapsular bile duct proliferation plus the often described portal fibrosis [27]. VINYL CHLORIDE METABOLISM AND CARCINOGENESIS Present biochemical knowledge indicates that vinyl chloride is most likely metabolized by the liver in a three step process [28]. At concentrations less than 50 ppm, vinyl chloride is metabolized by the alcoholic dehydrogenase system into chloroacetaldehyde and monochloroacetic acid. Cl-CH=CH:--CI--CH*-CKz--OH- alcohol Cl-CHj-CHO--CICHz-COOH dehydrogenase An alternative pathway which appears to become operative at 220 ppm is oxidation by the peroxidase-catalase system. Cl-CHz--CHi-OH HlQl iCICHi-CHiOOH catalase CICHz-CHO TOTAL Me. too r I I 60 - FIG. 1. Frequency of abnormal indocyanine green dye clearance among vinyl chloride workers utili zing O.S mg/kg and 1.0 mg/kg doses. 0 0 TOTAL VC EXPOSURE MDCX 98 CMA 002437 72 CARLO H. TAMRURRO A i i' ! FIG. 2. Hepatic arteriogram: Ve nous phase. Changes of peliosis hepatis are present throughout the left lobe. The multiple nodular stains represent peliosis hepatis lesions (arrows) ranging from 2-3 mm to 2 cm in size. V* FIG. 3. Splenic arteriogram: Ve nous phase (15 seconds). There are 3-4 circular and oval stains (ar rows) in the superior and inferiorlateral portions of the spleen. Nor mal pancreatic stain occurs just above the splenic vein. v--............. .... 99 <9 VNYI* 4 l.\J CAACINOCiENESiS 73 t ,1 11 \ i a t iV FIG. 4. Hepatic arteriogram: Venous phase. At approximately 14-13 seconds the peripheral stain is identified (arrows), lasting through the entire phase. Scattered areas of puddling are also present in and around the area of central hypovascularity. In this case chloroacetaldehyde is again formed. At higher levels oxidation appears to be by the mixed function oxidase system, forming chloroethylene oxide which spontaneously rearranges to form chloroacetaldehyde which then can be further oxidized to form monochloroacetic acid. Cl-CH=CHi oxidase CL-CH +CI-CH1-CHO CICHi-COOH As illustrated in Table 3, vinyl chloride oxidation intermediates chloroethanol and chloroacetaldehyde, at low doses, are most likely detoxified via the glutathi one-cysteine conjugation system. This system, however, is saturable and at higher levels vinyl chloride is excreted via the lungs [28]. It appears that at higher vinyl chloride levels increased amounts of chloroethanol and chloroacetaldehyde are further oxidized to chloroacetic acid which is excreted in the urine. This is further supported by the absence ofchloroacetic acid in urines ofrats exposed to low, short term levels of vinyl chloride but found in urine of rats exposed to 5,COO ppm for an extended time and reported in workers exposed to levels greater than 250 ppm for a prolonged time [29, 30]. Elmore et al., utilizing a modified Ames system and pure synthesized vinyl chloride intermediates, has demonstrated that vinyl chloride, chloroethanol, and 100 002439 74 Cl CH = CHj (VC) Liver MFO Cl CH-CHi \l O (Chlorooxirane) CARLO rL TAi^SUAIiO TABLE 3 Proposed Metabolic Fate ot' Vinyl Chloride Cl CHj CHi OH (Chloroethunol) | r-GSH Cl CHi CHO----------(Chloroacetaldehyde) GS CHj CKO Cl CHj COOH (Chloroacetic acid) OS CHj COOH Thiodiglycolic acid chloroacetic acid are not mutagenic in bacteriological systems [31]. This may indicate that the vinyl chloride monomer is neither hepatotoxic nor carcinogenic until it has been metabolized to its intermediate forms by the liver and/or other tissues. Alternatively, chloroethanol--the most transportable of the vinyl chloride metabolites--may be transferred or diffused to adjacent cells, such as the sinusoi dal lining cells, where it could be converted to chloroacetaldehyde but less likely to -- be detoxified or further oxidized. Since vinyl chloride appears to bind the serum albumin, it may, itself be transported to and oxidized by extra-hepatic cells which are unable to fully oxidize or completely detoxify its metabolites, and thereby lead to molecular DNA injury and cancer formation at distant tissue sites. These quandaries led to further study of the hepatochemical changes in rats undergoing progressively increased exposure to vinyl chloride. Subcellular en zymes and metabolites were studied in animals exposed to from 10-20,000 ppm vinyl chloride, ranging from 14-137 hours. Microsomal enzymes, including P-450, NADPH cytochrome c reductase, and mixed function oxidases were studied. Determinations of cytochrome c oxidase as the mitochondrial, tritiated-leucine incorporation as the protein synthesis and glucose-6-phosphatase as the carbo hydrate metabolism markers were also done. Glutathione and glutathione reduc tase as oxidative and detoxification markers were determined in addition to the conventional clinical biochemical studies which included the aspartate (SGOT) and alanine aminotransferase (SGPT), alkaline phosphatase, bilirubin, lactic acid dehydrogenase (LDH), total protein, albumin, cholesterol, and triglycerides. During the entire 137 hours of exposure there were no significant changes in the mitochondrial and the microsomal enzymes, the tritiated-leucine incorporation, or in the glutathione content. There was however, after 71 hours, a rise in the gluta thione reductase and a concomitant fall in glucose-6-phosphate. This occurred without any histologically discernible changes in the hepatocytes by light micro scopy nor any significant changes in the conventional clinical biochemical studies. 101 CMA 002440 VINYL CH^GIUDL--RELATED CARCINOGENESIS 75 The discovery of a decreased giecose-6-phosphaiase afler "simulated" chronic exposure led to the study of enzymes in the pentose phosphate shunt pathway. Weber and Lea [32] had found similar changes for primary hepatocellular neo plasms, demonstrating that in a rapidly developing primary hepatocellular tumor, there is decreased giuconeogenesis with a reduction in the gIucose-6-phosphatase, followed by an increase in glucose-d-phosphate dehydrogenase and transaldolase. These biochemical changes were also followed by an increase in purine biosynthe sis (increased phospheribosyipyrophosphate aminotransferase (PRPP) and in creases in the production of ATP and GTP leading to increased nucleic acid synthesis. Vinyl chloride-exposed animals showed no significant changes in the glucose-6phosphatase dehydrogenase activity during the initial 84 hours of exposure. However, after 103 hours, there was significant increase in gIucose-6-phosphatase dehydrogenase. Studies of PRPP, at least up to 137 hours, have as yet shown no significant changes. Studies are now underway using animals exposed to 130 to 250 hours to determine if the biochemistry in vinyl chloride injury is similar to that in primary hepatocellular tumors. This, however, does not explain why the hepatocyte, which is the primary cell for oxidizing and detoxifying vinyl chloride, is not the primary target for cancer trans formation. How do the hepatocytic biochemical changes, seen in the early phase of high vinyl chloride exposure, relate to the later morphological changes that occur in the adjacent sinusoidal cells? MORPHOLOGICAL FINDINGS Electron microscopic examination of liver sections of mice exposed from 1 to 6 months to 2,500-6,000 ppm vinyl chloride, for 5 hours/day, 5 days/week--a level known to induce angiosarcoma [33]--have demonstrated hepatocellular changes as early as one month. These changes included hypertrophy of the smooth endo plasmic reticulum (believed to reflect vinyl chloride metabolism) and, plasma membrane loss of microvilli with invaginations--possibly reflecting the movement of injurious metabolites across the membrane and out of the cell, allowing the metabolites to be picked up by the sinusoidal cells [34], The sinusoidal cell reactions were multicellular. Increasing numbers and sizes of lipocytes were seen with little fibrosis. Macrophages were seen filled with phago somes, sometimes containing long needle-like crystals. Although there were many mononuclear cells present, the main abnormalities were seen in the endothelial lining celis. In the early stages they are larger and thicker--possibly swollen. Later, they became bulky and in places, multi-layered containing increased organelles, especially mitochondria and endoplasmic reticulum. Later disruptions in the sinu soidal walls seen were consistent with beginning peiiosis hepatis. The lining cells, probably the precursors of angiosarcoma, often resembled fibroblasts. However, their endoplasmic reticulum did not contain any collagen components. These observations by SchafFner et al. [34] give support to the suggestion that metabolites of vinyl chloride produced in the hepatocytes may be transported through the plasma membrane and enter sinusoidal lining cells, eventually leading to angio sarcoma. Attempts at screening for vinyl chloride hepatic injury might be better aimed at the endothelial cells and the hepatic sinusoidal circulation rather than the hepatocytes. Our work in humans has identified similar findings. One mruor difference at 102 CMA 002441 76 CAaLU /i* t AkMiURRO present is an increased collagen deposition, characteristic of human vinyl chloride injury and likely species specific. Light microscopic studies utilizing special stains on hepatic tissue from individuals with extensive exposure to vinyl chloride but without clinical biochemical hepatic abnormalities have shown distinctive midzonal increased deposition of collagen in the space of Disse [35]. Routine light microscopic studies using hematoxylin and cosin failed to easily demonstrate this midzonal increased collagen. The increased deposition along the hepatic cell surface is associated with larger sinusoidal space and activation of the sinusoidal lining cells illustrated by increased nuclear size and cytoplasmic content. The in creased collagen deposition, when studied electron microscopically, demonstrates compression of the hepatocytes by the collagen bundles which initially give the ap pearance of m/ra-hepatocellular collagen bundles as illustrated in Fig. 5. The strands of collagen appear to compress the hepatocytes causing cords of hepato cytes to be broken and to coalesce with adjoining sinusoids eventually leading to peliosis hepatis-like lesions. These observations led us to the study of the proteroglycan role in collagervformation in vinyl chloride-exposed workers. It had been suggested in the literature that glycosaminoglycans in blood and/or urine might be useful as means of early cancer detection since a number of studies had demonstrated the production of sulfated glycosaminoglycans with malignant states. Pathologists have often used this feature as a diagnostic aid in characterizing malignant vascular tumors of the skin /i. rr o -r7 \ 1s * n 'ji; Vf v*; a r v. * V</ FIG. 5. Electron microscopy showing collagen (CB) bundles (iarrows) iavaginating into the hepatocyte, giving the appearance of inter-hepatocytic collagen. N _ nucleus; S " sinusoidal space: IM - invagination into the cell membrane (small arrows). 103 CMA 002442 ViNVi- C*iLOHiO.,--^A^CINOGENESiS 77 [36]. Others have noted a strong positive Aldan blue glycosaminogiycan staining reaction in human angiosarcoma tissue [37j. This suggested that quantitative and qualitative determinations of glycosaminogiycan production in individuals with neoplasm, either by serum or urine, might be used to identify those at high risk or as an early indicator of neoplastic formation. The feasibility of glycosaminogiycan ``spot test" for vinyl chloride production workers made this an attractive possibil ity for mass screening. Urinary giycosaminoglyeans, measured as uronic acid, were studied in individu als with alcoholic cirrhosis, viral hepatitis, secondary liver metastasis, hepatic angiosarcoma and normal controls. The percentage of total glycosaminoglycans that was dialyzable and the percent age of unfractionated total that appeared in the hyaluronic acid, chondroitin sulfate, or in the heparin fractions was similar for all groups. However the distribution of positive fractions varied with the different groups studied. Seven of the nine vinyl chloride-exposed individuals, other than those with angiosarcoma, had glycosaminogiycan positive chondroitin sulfate fractions with negative hyaluronic acid and heparin fractions whereas this occurred in only 3 of the 32 urines from other hepatic diseases [33]. In addition, study of the total tissue glycosaminogiycan levels in angiosarcoma tumors and fi'orotic tissue adjacent to the tumor demonstrated that tumor tissue itself had higher levels of hyaluronic acid and heparin fractions as compared to the non-tumor adjacent tissue which had higher levels of chondroitin sulfate fractions. A similar relationship was found in cirrhotic liver tissue and normal controls. This data conforms to reports by others that both hepatic connective tissue disorder [39,40,41,42,43] and hepatic cancer [44] result in increased hepatic glycosamino giycan levels. It may be significant that the angiosarcoma patient has half the urinary glycosaminogiycan excretion of patients with liver metastasis and that analysis of angiosarcoma tumor tissue exhibits halfthe glycosaminogiycan content reported by Kojima et al. [44] for hepatocellular carcinoma. The increases in liver and urinary glycosaminoglycans rnay well reflect the importance of these substan ces in the process of fibrogenesis and tumor growth. Although no significant differences were found in total glycosaminoglycans of vinyl chloride-exposed indi viduals with associated liver injury, there was a significant difference in the excre tion patterns of these individuals. Seventy-eight percent of them had positive chondroitin sulfate fractions in contrast to only nine percent of the non-exposed liver injury cases. Tnus the change in the glycosaminogiycan excretion pattern in individuals with pre-cancerous injuries may be of significant prognostic and diagnostic importance [45]. The urinary glycosaminogiycan excretion patterns in an angiosarcoma patient 3 months to 2 weeks prior to death demonstrated an increase in the urinary chondroi tin sulfate fraction with a change in its composition as the disease progressed. During this time, the chondroitin sulfate composition showed a continuous increase in the ratio or the 1.25 M NaCl to the 1.5 M NaCl fractions. This was due to an increase in the 1.25 M eluate and a decrease in the 1.5 M eluate fraction and was 2.3 times greater than the controls. In the most advanced stage of the angiosarcoma the ratio increased to 13.7 times greater [461. These very preliminary studies would suggest that alterations in the ratios of these fractions' compositions may be useful in evaluating the severity and subsequent progression of disease. Early lesions may produce small changes in the ratio which would become more pronounced as the disease worsened. The 104 002443 T6 CARLO H. 7AM5URRO determination of this ratio might also be useful in identifying those individuals with significant injury or continuing progression of disease despite changes in the environment. Finally therapeutic measures for intervention might be better evalu ated for their effectiveness in arresting cancer development as reflected by the glycosaminoglycan changes which in turn reflect changes in collagen formation. SUMMARY AND HYPOTHESIS Vinyl chloride appears to enter the body through the respiratory tract, the skin, or by swallowing; it is absorbed and transported to the liver by the systemic and portal circulatory systems. In the liver, the primary site of metabolism, it is oxidized by various enzyme systems: alcohol dehydrogenase at lower exposure levels; the peroxidase-catalase system at intermediate levels; and mixed function oxidases at higher levels. At the higher levels, the mixed function oxidase system transforms vinyl chloride to chlorooxiranes which are then spontaneously trans formed into choroethanol and chloroacetaldehyde. These two intermediate metabolites, chloroethanol and chloroacetaldehyde, are detoxified by conjugation with glutathione and cysteine-SH groups and are excreted in the urine. At even higher doses increasing amounts of the chloroacetal dehyde are further oxidized to chloroacetic acid and excreted as an end product in the urine. However, when chloroacetaldehyde and/or the chlorooxiranes exceed the detoxification threshold of the hepatocyte, this leads to hepatocellular toxicity and/or stimulation ofthe sinusoidal cells. This acute event in turn acts as a stimulat ing mechanism for increased collagen deposition in the space of Disse and sinusoidal areas as shown by electron and light microscopy studies. The increase in the collagen depostion in sinusoidal spaces then leads to disruption of hepatic cell surface function, disruption of the hepatic cords, coalition of the sinusoidal spaces and eventual peliosis hepatis. These lesions alternately lead to sufficient vascular dysfunction to add further to the biochemical manifestation of hepatic cellular injury. Since it is highly unlikely that the unstable chlorooxiranes are able to be trans ported to adjacent cells and that the chloroacetaldehyde would most likely be conjugated or detoxified within the hepatocyte, an intermediate form, such as chloroethanol, which is transportable from the hepatocyte, may then move on to the adjacent sinusoidal lining cells, or possibly even further to other extrahepatic tissue. At these extrahepatic sites, an intermediate, such as chloroethanol, may then be converted to chloroacetaldehyde. The extrahepatic tissue sites are most likely unable to further convert the chloroacetaldehyde to chloroacetic acid nor to detoxify it sufficiently, if at all, by their own detoxification systems. This would allow a longer contact period with the cell's DNA. In addition many of the extra hepatic cells normally are regenerating at faster rates than hepatocytes, thus increasing the possibility of DNA derangement and ultimate carcinogenesis. Alternatively, vinyl chloride itself may be taken up by extrahepatic tissue, oxidized but incompletely detoxified, allowing the cell itselfto become susceptible to direct DNA injury. In this or similar manner, chemical metabolites may induce injury to the DNA in rapidly replicating cells at sites beyond the liver, thus accounting for other cancers developing with vinyl chloride. 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