Document x57256R0dpL1NV3x7j9MKKwOG

Manufacturing Chemists Association Record of Meeting VINYL CHLORIDE AUDIT TASK GROUP MCA Conference Room 407 Washington, D.C. September 6, 1978 Dr. Z. G. Bell, Chairman, presided and opened the meeting at 9:20 a.m. The following members and guests were present: MEMBERS PRESENT Z. G; Bell, Chairman R. K.' Hinderer C. D. Kary: T. R.. .Torkelson J. T. Seawell PPG Industries^ Inc.` The BFGoodrich Company, Chemica1 Division Shell Oil Company. -'! The Doww Chemica 1 >,Compa ny MCA staff PRESENT BY INVITATION J. C. Van Horn (part-time) MEMBERS ABSENT T. J. Benya G. K. Hatfield Ethyl Corporation Diamond Shamrock Corporation 1.0 Meeting Objectives The Vinyl Chloride Audit Task Group (ATG) met for the purpose of considering the following topics: 1.1 The report submitted by Experimental Pathology Lab oratories, Inc. (EPL) based on EPL's analyses of data evolving from a three phased tissue inventory conducted by Industrial BIO TEST Laboratories, Inc. (IBT), Decatur involving wet tissues, tissue blocks, and slides. UCC 003219 9^ aw<* 2- - 1.2 Whether or not histopathologic studies on the slides, currently available at IBT, Decatur should be completed, and 1.3 Whether or not it appears feasible to conduct audits of data on exposure chamber concentrations. Decisions on items 1.2 and 1.3 are dependent on preparation and acceptance of a final EPL report. 2.0 The EPL Report Partial evaluation of the EPL report revealed serious dis crepancies and voids which have their origin in the IBT inventory data summaries. Until these serious discrepancies can be recti-, fied, members of the ATG cannot make proper scientific inter pretations of the findings of either the results of the IBT three phased tissue inventory or the EPL report, neither can action be taken on the preparation of an ATG Interim Report. Various ramifications of the EPL study were considered by the members of the ATG and it was concluded that insufficient data are currently available to enable further planning at this stage. R^&Bgfiii^Lon was given to the point that the EPL report is the key,that can unlock the door to future ATG and/or Panel action on this entire program. The revised EPL report (when accepted) will largely determine the course of action whether it be (1) the conduct of additional audit work, or (2) an attempt to salvage additional histopathologic data, or (3) a request that steps be taken to terminate the IBT research agreement. 3.0 Meeting with Dr. Busev Members of the ATG requested that the Project Manager meet with Dr. Busey and coordinate with him all of the changes in the EPL report that eminate from ATG deliberations. JTS:ep Record Subject to Approval September 21, 1978 T. Seawell Project Manager Vinyl Chldride Research OCC _ 003220 ... MANUFACTURING CHEMISTS ASSOCIATION - V 7 1825 CONNECTICUT AVENUE, N.W., WASHINGTON, D.C. 20009 V T. ,, N ; TELEPHONE: (202) 328-4200 TELEX: 89617 (MCA W5H) July 31, 1978 / 5 ^ * ^1 >,f v / ^ 'm^v. ; TO:- Vinyl CHlprdde Technical Panel Vinylchp5rlde Research Coordinators vinyl-tChxoride Medical Subcommittee m*., i i^bie4^ v `^ ; v. t' *' , - * .V 1" *I *' * -y * " > V ` ' A*'.'*' ' | ^s^erortea^ih-Section 6.0 of the Record of ^the Univers . ^^i^6&j&gie|Meetlnq on June 14, 1978, Drs.:Tamburro and ...^ ' ^^^O^^rinelgP^r^gise^d;to s end the ..Project Manager" complete fab'st-ra^'e^^^' 'f' ^"*ofjfali^esearch^reviews presented on .all component studies^- *&pis snai Covering ^fciti-rDlscxt Report,}that ..they have exceeded" their promise>bra? ipieMterfCtra'snfsc<r^diip3tsK>'o-fof tihV^actuai.T^lear'ch^revi^SSS*!' : S*"/'."' ,, -V. . *'*r~~ , - T*"- ' *: . t ^h^tpjbhe^stu^i^fy*- -- Jrrajathe^TeporS ^omponent^res'earch j5 . - projects as well, included in the enclosed collection of papers' are copies, of ail 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 -r S0 J. T. Seawell Project Manager Vinyl Chloride Research THE WRITER'S DIRECT DIAL NUMBER IS (202) 328-4258 IJCC 003221 TM-.7*'AR.r 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 CHLORIDE 1 ucc 003222 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 A. COUNSELING & REHABILITATION 5. TREATMENT PROGRAM > DETECTION AND PREVENTION SYSTEMS BASIC RESEARCH PATHOGENESIS OF THE CARCINOGEN (s) MOLECULAR STRUCTURE VINYL CHLORIDE 2 ucc 003223 SLIDE 3. This illustrates the past clinical investigations done in the detection and prevention systems. They cover a variety of areas such as #1, 1 oking 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. Slide 3 CANCER CONTROL PROGRAM VINYL CHLORIDE ANGIOSARCOMA >> MEDICAL SURVEILLANCE SYSTEMS .1 * APPLIED CLINICAL INVESTIGATION DETECTION AND PREVENTION SYSTEMS 1, BILE ACID CLEARANCE 2, blood/tissue ENZYMES 3, LEUKOCYTE ADHERENCE INHIBITION A. HISTOCOMPATIBLE (HL-A) IDENTIFICATION 5. URINARY 6LYC0SAMINOGLYCANS 6. TISSUE ANTIGENIC SYSTEMS 7. HISTOLOGICAL/ELECTRON MICROSCOPIC CHARACTERIZATION 8. MULTIVARIANT ANALYSIS FOR END PRODUCTS 9. IDENTIFICATION OF POTENTIAL CHEMICAL CARCINOGENS BASIC RESEARCH PATHOGENESIS OF CARCINOGENS MOLECULAR STRUCTURE VINYL CHLORIDE 3 ucc 003224 vc- h . The fourth slide illustrates some basic science research being conducted 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 f 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. 4 ucc 003225 VINYL CHLORIDE ANGIOSARCOMA Slide A 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 4. 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 s ucc 003226 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. HONG, Ph.D. 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 metabollealiy 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 nonenzymatically. 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 detoxlficati n' 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 epoxtde 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 14 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. ucc 003227 Slide 1 Chemical Systems of Detection of Vinyl Toxicity VINYL . METABOLIC ^ f OXIDATION EPOXIDE + CARBONYL (3) I CYTOPLASM J DETOXIFICATION (1) NUCLEUS T PUTATIVE ACTION (2) 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 hew are they detoxified. Do they yield the same products or different ones? We have studied their reaction with sulfhydryl compounds 3^"dichlorobenzenethiol and N-acetylcysteine. The benzenethiol was used by the Stockholm group tn 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 glut'.athione. In the case of COR and benzenethiol, the sulfur-conjugation product S-acetaldehyde, compound 3f i$ formed. However, CAA and benzenethiol forms the hemithioacetal compound 4. These two reactions are distinctly different. The formation of hemithioacetal is reversible but that of the S-acetaldehyde is not. With N-acetylcyteine, both COR and CAA yield the same cyclic condensation product, a dlhydrothiazenecarboxyltc 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 add, Conpound 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 'OSt 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> end 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. 8 ucc 003229 Slide 2 Detoxification of Vinyl Chloride primary metabolites INTERMEDIARY METABOLITES COR1 CAA2 s-aceialdehVde8 JHIAZENES5 hehithioacetal4 THIAZENES5 URINARY METABOLITES. S-ACETIC ACID6 S-ETHYL ALCOHOL7 CHLOROACETIC ACID8 1 C-l2' H--CHjCl 3 RS-CH2CH0 4 RS-CH-CH2Cl &5 ^C02H rpsc---rch--2--c--o--2--h- 7r---s------c--h- --2ch2oh 8 Cl-CH2C02H (RS FROM 3,4--DICHLOROBENZENETHIOL, N-ACETYLCYSTEINE) Ucc 003230 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 CAA. 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 5, and a third product which is possibly an acetyl guanine, compound 5. These products can disrupt nucleic acid structures hence their functions by means of sterlc 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. 10 Slide 3 Putative Action of Vinyl Chloride. Reaction with Nucleic Acid Bases cm etheno-c', etheno-a2 C-ETHENO-G3, a-ETHENO-G4, ACETYL-G5 COR G--7--ACETALDEHYDE6 + ... 11 ucc 003232 SLIDE 4 It is now known that vinyl chloride-mediated mutagenesis in the 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 line 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 is 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 VINYL1"hcpb? EPOXIDE -7* CARBONYL 25* (1) nmr quant (1) nmr quant (2) blol screen (2) blol screen Styrene., butadiene, acrylonitrile, crotononitrile, acrylate, ethyl vinyl ether, vinyl acetate, vinyl BROMIDE, VINYL FLUORIDE, VINYLIDENE CHLORIDE, VINYLIDENE BROMIDE, TRICHLOROETHYLENE, DICHLORODIFLUOROETHYLENE,1,1~DICHLOROPROPENE 12 UCC 003233 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 greac importance to assess the relative carcinogenic potential of each of these materials before they pre 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 80Z. 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 UCC 003234 .... -................. -................ -- TABLE 1 REACTIVITY OF CHEMICALS IN BACTERIAL ASSAYS i CHEMICAL SALMONELLA REVERSION REV/PLATE--CONTROL *CAA (0.004M) "" 265 STYRENE OXIDE (0.01M) 342 EMS (O.OOIM) 312 MMS (O.OOIM) 355 SOS REPAIR Z KONTREATED 15% 100Z 80Z 1Z SUBTILIS REPAIR ASSAY W+ REC" HCR- UVR- POL gnu INHIBITION 0 18 2 2 3 0200 1 1 16 111 4 21 6 6 10 * 14 Occ 03235 (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. Ve 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 whan the call 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 UCC 003236 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 recFIS recFIS recF16 TABLE 2 REPAIR ASSAYS WITH B. SU3TILIS* Average Inhibition In mm/8 experiments <* 13.5 7.5 13.5 9.5 16.5 16.5 11.3 16.8 NR 11.5 NR 6.7 8.8 Strains REC H recH342 Average Inhibition In inn/8 experiments NR REC rec-4 rec-13 MTC iatc-41 (cafr) UVR uvr-35 (cafr) her uvr jff wt wt wt 15.4 5.4 NR NR NR NR NR NR NR 6.1 .7.4 7.5 8.42 3.1 *100niM chloroacetaldehyde used In all these experiments 16 UCC 003237 induction in a cell population as a measure of SOS inducing potential. When virus ia induced, the cell dies. Therefore, in Table 1 100Z survival is no SOS induction* less than 100Z 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 styreneoxide are positive in the Bacillus repair assay, and seem to require recombination repair. Methyl methane sulfonate also is reactive toward the strain lacking polymerase I (pol A). However, the preliminary indication from the SOS repair assay demonstrates significant differences between these compounds. Both CAA and MM5 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 MIS 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 acryioinltrlle. We will 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 ucc 003238 THE CHARACTERIZATION OF BIOCHEMICAL ENZYMATIC CHANGES OCCURRING WITH PROLONGED VINYL CHLORIDE EXPOSURE IN ANIMAL AND MAN J.T. Du, Ph.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 tritlated 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. 18 ucc 003239 CONVENTIONAL CLINICAL BIOCHEMICAL STUDIES Slide 1 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 SUBCELUJUR ENZYME 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 NO CHANGE NO CHANGE 4. GLUTATHIONE CONTENT 5. GLUTATHIONE REDUCTASE ELEVATED INCREASED 6. GLUCOSE-6-P ASE DECREASED 7. GUJCOSE-6-P DEHYDROGENASE INCREASED RATS WEREEXPOSED TO 10,000-20,000 PPM VC FOR 14, 28, 42, 7L 84, 103 AND 137 HOURS. 19 - .... ..................... .......... ucc 003240 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 c rrela 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 df vinyl chloride exposure. These histological findings of f cal 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. 20 Ucc 003241 EXPOSURE TIME (hours) 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. 21 ucc 003242 lO ^xtp mobs NADPH oxldriotl/m in/m g protein) 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. 22 ucc 003243 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. 23 ucc ^ 003244 WEBER'S MOLECULAR CORRELATION CONCEPT OF NEOPLAMA GLYCOLYSIS 4 GLUCONEOGENESIS PK I PYRUVATE IQ* l*' P-cxylot* > 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 cholorlde 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 metab 11c fate schema presently under use. Vinyl chloride being metabolised at higher levels by mixed-function oxidase within the liver may be converted to ehlorooxl- rane or the epoxide. This unstable Intermediate may then spontaneously revert to form chloroethanol and the chloroethanol may further be metabolised to chloroacetaldehyde. Secondly, the chlorooxlrane may be directly detoxified by GSA via the glutathione epoxide S-transferase or further oxidized to ehloroacetal- dehyde. Chloroacetaldehyde In turn may be shown by glutathione aralykly-S- transferase or further oxidised to chloroacetlc acid. Studies by Wong and Streips have already shown that vinyl chloride, chloroethanol and chloroacetlc acid have no mutagenic properties In bacteria. However, the chlorooxlrane 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 Hated In Slide 2. They Include an elevation of non-protein sulfhydryl content, (Slide 3) glut athione reductase, (Slide 4) glutathione epoxide-S-transferase (GEST) (Slide 5) and glutathione aralykly-S-transferase (GAST) (Slide 6) concomitant with the increase in glucose-6-phosphate dehydrogenase and reduction In F450 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 chlorooxlrane accumulation Is to be ucc 25 003246 handled by direct detoxification by the glutathl ne but with prolonged exposure more is converted to the chloroacetaldehyde which then is further detoxified by the CAST. These data are compatible with other findings of Increased amounts of chloroacetic add in the urine of individuals with very high prolonged exposures. This would imply that at Initial doses the vinyl chloride is able to be metabollcally 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 inthe sinusoidal lining cells has lead us to our more recent studies. 26 ucc 003247 Slide 1 f (v c) Cl CH = CH2 Cl CH2CH20H (chloroethanol) 4 i (chlorooxirane) Cl CH-CHo * DETOXIFICATION WITH GLUTATHIONE 6S CH2CH20H t N-Ac-S-(2-hydroxy ETHYL) CYSTEINE Cl ch2cho . OAST- (chloroacetaldehyde) Xy Cl ch2cooh GS CH2CH0 (CHLOROACETIC ACID) VINYL CHLORIDE METABOLIC FATE GS CHoCOOH - V THIODIGLYCOLIC AC1 uco 003248 Sijtie z 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 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 28 ucc 003249 o'er top CD 8 7 6 & o E4 * 3 2 I 0 TIME IN WEEKS (HRS) ; Slide 3 10 T Slide 4 OrC AIR EXP. ucc 00325, NORMAL V.C. EXP. AIR EXP. Slide 6 ucc 003253 UJ CO < o: UJ u_ CO * z <o O' o. h- i' ? COj 6 ' QC UJ > >- XL 3 _J . <E or . < Q. UJ s 2o OE X^ f-- x <I-- s 3 _ CD I 1 I I i i 2 (70) 4 (140) TIME IN WEEKS (HRS) 6 (210) 30 28 26 24 22 cc Iu > 2e --* o 20 18 16 14 12 10 8 6 4 2 .0 2 Slide 7 ucc 003254 m lr"> o con 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 lead 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 immunopathologle 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 XgG C. Antigens deleted 35 ucc 003256 Firstly, four classes of protein antigens were detected by Immunodiffusion and lmmunofluorescent 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 UCC 003257 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 (79Z) 7 (77Z) 5 (62Z) 8 (28Z) 1 (20Z) 0 ( OZ) 2 (25Z) 2(9Z) 8 (20Z) 3 (13Z) 8(231) 0 ( OZ) 0 ( OZ) 37 ucc 003258 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-lnduced 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 Malulsh, 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 inmunlty) 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^-thymldlne 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 angio 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 angio 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 38 ucc 003259 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 anglo 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 t ensure maximal statistical strength from the data obtained. 39 ucc 003260 Figure 1 LVffHOCYTE RESPONSE OF VC WORKERS ACCORDING TO EXPOSURE ; iiil `ABOVE MEDIAN' BELOW.MEDIAN; ;(N-2A) ! i'i* ** ucc 003261 003262 ANGIO LIVER NORMAL AND ANGIO LIVER i O'fpl'e. f LYMPHOCYTE REACTIVITY TO TUMOR AMD NORMAL TISSUE PANEL VINYL CHLORIDE WORKERS . ll twrcentatc NON-VINYL CHLORIDE WORKERS a nercentace O 8 INTERPRETATION Of . RESULTS . * 6 16 ; 0 0 _ Individuals with possible specific anti-tumor reec-f tivities.' H 7 18 ' 8 57 Individuals with nonspecific reactivities masking any possible specific reac tivities. NORMAL LIVER S 13 17 Individuals with nonspecific reactivities. NONREACTIVE TOTAL 20 53 38 100 S 36 . Individuals with no tissue reactivities. 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 chi ride 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. 42 ucc 003263 Zt has been clearly established that the larger the population studied the greater the chances of obtaining statistically significant data. The accumu lation of HIA data on the majority of the VC population plant workers will take an additional two years as originally anticipated. This is necessary s that the strongest statistical analysis can be performed upon the completion f all HLA testing. 43 ucc 0032frt 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 ND**** 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 2! 243 ' 199% * ### ** CURRENTLY TYPINS_EOR 11 ANTIGENS SCOTT ET AL.,,------ ,,,,,, DATA COMPILED JUNE; 1978 NOT DONE 44 UCC 003265 HLA - B FREQUENCIES IN VINYL CHLORIDE WORKERS HLA " Bm antigens* HEALTHY 1975 who CONTROLS** WORKSHOP ul/pallet PLANT*** ul/non-pallet PLANT B5 10 B7 31 B8 27 B12 30 B13 3 m .5 B15 10 B18 8 B27 .V- 7 B37 ND**** B40 11 BW16 ND BW21 , . ; ND V BV/22 2 BW35 '5 : B17 6 BLANK ' ND \ N 900 TOTAL FREQ. .155* li . .8 23 22 20 16 . 24 .. 26 ... 6 .. 11 12 7 38 90 8 ' ... 0 5 .12 4,, 16 ; 12 . 0 4 4. 5 4. 17- 8 7 22 ND 12 7 28 ; 22 .25 ; 6 . . . .12 12 2 12 * l 17 , 2 [: : . 6. * 13 10 -- 14 k 6877 181% 24 192Z, .243 193% CURRENTLY TYPIN6F0R 16 ANTIGENS *# SCOTT ET AL>* 1977 _ #*# DATA COMPILED JUNE., 1978 *** NOT DONE . 45 UCC 003266 EVALUATION OF IMMUNOCOMPETENCE OF HUMANS CHRONICALLY EXPOSED TO VINYL CHLORIDE H.Pt Fortvengler, M.S., and C.H. Tamburro, M.D. It has been demonstrated that lymphoid cells (T cells) can be cytot xic 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 immuno- competence 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 immuno- depression 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 cerning immunoevaluation prior to diagnosis of frank malignance. We have used im munological assays (see Table 1) that have demonstrated usefulness In indicating immunodepresalon In cancer patients. These tests have been used to evaluate the luminocompetence of individuals with possible pre-mallgnant 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 lnounologically 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 Immmodepresslon (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 exp sure groups when examined with student's t test. Even after additional Immmologleal parameters were examined (see Table 2), no statistical differences could be found between the two groups. 46 ucc 003266.01 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. inmunoglobulin quantitation) will give us more definitive statistical results. 47 Ucc 003267 BATTERY OF IMMUNOCOMPETENCE TESTS IN USE TABLE 1 TEST REASON FOR USE Absolute Lymphocyte Count SRBC Rosettes, 4C SRBC Rosettes, 33C FHA Stimulation Con A Stimulation PWM Stimulation SPL Stimulation Recall Antigenic Stimulation SLO PPD _ Candida Varldase Gives a gross examination of the Immune system Quantitates the total number and percent of T cells. (T cells can reject tumors and transplants.) Quantitates the total number and percent f 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 48 nCC 3268 300< 250- Figure 1 * LWHOQTTE FESPONSE OF VC WORKERS ACCOFDIKG TO EXPOSURE; ABOVE ffiDIAN GW*D EHfEDWl' (N4D 050100 50 fe;$' VA- ; PHAj AW. m .w. m OOK-A pA {$$ 1|iv|\ 1I ' >%V* oi: m 49 ucc 003269 Table 2 ADDITIONAL IMMUNE PARAMETERS OF VC WORKERS ACCORDING 70 EXPOSURE TEST .Above Median ' . Exposure SPL Stimulation Absolute Lymphocyte Count 191 31 (n- 52) 2355 + 132 (n- 52) SRBC Rosettes, 4C < * SRBC Rosettes, 33C 1503 101 (n- 50) 1227 + 80 (n-50) Recall Stimulation SLO PPD Candida Varidase ' 8+2 (n- 53) 29 + 8 (n 49) 11 + 3 (n* 53) 29 + 5 (n-"54) Belov Median .Statistical Difference Esposure Between Croups 186 34 (n-24) 2316 + 148 (n- 24) Hone None 1524 + 132 (n-,24) 1260 + 123 (n-24) None ' ,:None 24 + 12 (n* 24) 18 +, 6 (a- 23) 6+2 (n- 23) 41 + 12 (n- 23) None None None, None ( 50 ucc 003270 - TISSUE AND URINARY GLYCOSAMINOCLYCANS IN HEPATIC FIBROSIS AND HEPATIC CANCER Charles E. Kupchella, Ph.D. Because characteristic glycosaminoglycan (GAG) changes hav 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 chang s 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 _witb 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 (5l23tc) 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 indicat s that different GAGs are involved in these processes.. Ve 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). 51 ucc 003271 references 1. Kupchella, C, E. and Tamburro, C. H., Urinary and Tissue Glycosaminoglycan Patterns in Hepatic Angiosarcoma. Tn Detection and Preventionof 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 ucc 003272 SLIDE 1 Kupchella, et al. LIST OF STUDIES/EXPERIMENTS COMPLETED TO DATE AMD 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 C&4-lndueed fibrosis (48 animals) at 3, 6, and 9 weeks (hlstochem/bioehem). 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 hepateetomy. 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). 53 ucc 003273 ucc 003274 NOTE: These are some preliminary data gathered In our transplantable hepatoma studies. These studies are designed to evaluate the relationship betveen tumor GAG composition and various tumor properties (growth rate, metastatic potential, etc.) and to evaluate urinary GAG excretion as a function of tumor size. TABLE: URONIC ACID LEVELS BY CAC FRACTION IN HORRIS 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 0,03 M NaCl NON-GAG CARBOHYDRATES 176 + 38 176 + 15 524 + 81 664 + 203 6770+1629 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 28 + 6 31 + 7 39 + 10 kb. .. HtC. J. D. b>iV"'kl"' ^ .,,t****<% , Printed m U.S.A. URINARY GLYCOSAMINOGLYCAN PATTERNS IN ANGIOSARCOMA OF THE LIVER Kevin L. Curran, BA, MS, Charles E. Kupchella, PhD, and Carlo H. Tamburro, MD Glycosaminoglycans extracted from 24-hour urine specimens from patient* with hepatic angiosarcoma and from normal/controls were separated as cet ylpyridinium complexes into "hyaluronic acid," "chondroitin sulfate,'* and "heparin" fractions, then further separated and characterised by anion-ex change chromatography and hyaluronidase susceptibility. The chromato graphic pattern of the urinary chondroitin sulfate fraction in patient* 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 hyaluronidasc-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 determination* of hyaluronidase susceptibility indicated that the resistant fraction was heparan sulfate and that the susceptible flection was chondroitin-4-sulfate and/or chondroitin-d-sulfate. Caattr 40:3050-5053, 1977. he emergence of angiosarcoma of the urinary GAG or collagen degradative or syn Tliver and its relationship to vinyl chloride thetic products. Preliminary studies in our labo exposure4*' has prompted a search for methodrsatory, however, demonstrated an increase in to detect this lesion. Although systematic both liver and urinary GAG in patients wirh screening programs are currently in opera angiosarcoma, chronic active hepatitis, and cir tion, there is still no single chemical in rhosis.1* Most of the increase in urinary CAG dicator which' is specific for angiosarcoma or for occurred in the chondroitin sulfate fraction and, changes which may precede this disease. in contrast to what was found for normal and The association of elevated tissue glycosami- other diseases, the urinary chondroitin sulfate nogiycans (CAG) with tumors, including angio fraction was the only uronic acid positive fraction sarcoma, has been established. Glyco- found in the urine of seven of nine cases of vinyl- saminogiycans are also known to be involved in chloride-exposure-associated liver injury other normal connective tissue synthesis and collagen than angiosarcoma. This study was undertaken deposition and are elevated in connective tissue to characterize more completely the urinary disorders.11 Since angiosarcoma of the liver has "chondroitin sulfate" fraction in hepatic angio both neoplasia and fibrogenesi* in its etiology*' sarcoma. GAG changes could be expected to serve to sig nal the appearance of early lesions and may be useful in evaluating advanced lesions. Clinical Summaries Galambos* suggested that since the liver con Case 1--(Hepatic Angiosarcoma--advanced) tributes very little to the overall connective tissue of the body, hepatic fibrogenesis should not be expected to result in significant increases in A 45-year-old white male worked as a chemical helper in a vinyl chloride polymerization plant for thirteen years prior to the diagnosis of angiosarcoma. From the University of Louisville, Cancer Center, and the Price Institute for Surgical Research Health Sciences Cen ter. Louisville, Kentucky 40201. TM* work was supported in part by an American Cancer Society Institutional Cram, IN-ill, a grant from the (l. F. Goodrich Company, and contract NOl-CN-55212 with the National Cancer Institute. Twelve yean after initial employment, the patient exhibited a persistent elevation of lactic dehy drogenase and underwent angiographie studies which demonstrated multiple area* of scattered tumor stain throughout both lobes of the liver with areas of central transiucency consistent with the diagnosis of angio sarcoma of the liver. Address for reprints: C. E. Kupchella. Cancer Center, University of Louisville. Louisville, KY 40201. Accepted for publication April IS, 1977. Exploratory laparotomy and liver biopsy confirmed this diagnosis, and the patient was treated with adriamycin, cyclophosphamide, and methotrexate; an 3050 55 UCC 003275 MOLARITY NeCt No. 6 Glycosaminoclycans i.n Angiosarcoma Curran tt 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 previously 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). Case 2 (Hepatic Angiosarcoma--moderately advanced) A 54-year-old vinyl chloride polymerization worker was first employed as a polymerization vat cleaner 28 years prior to the diagnosis of angiosarcoma. Two years prior to diagnosis, the patient had persistent biochemical liver, abnormalities although he was oth erwise asymptomatic with a normal liver-spleen scan. Angiographic studies showed pciiosis 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 analytes 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 analysts. Cetylpyridinium chloride (Sigma Chemical Company, St. Louis) was added to the entire 24-hour volume to precipitate the GAGs Fie. 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 NaCI 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 are 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 tt al. '* Cetylpyridinium chloride was removed14 and the GAGs were sub jected to anion-exchange chromatography as de scribed by Schiller tt al.'* Glycosaminoglycan fractions were applied to 1.0 X 44 cm AG1-X2 (200-400 mesh, chloride form) columns Bio Rad Laboratories, Richmond, California) and eluted stepwise with 0.0, 0.5, 1.0, 1.25, 1.50, 2.0, and 3.0 M NaCl. At a flow rate of 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 ucc 003276 3052 Cancer Dumber 1977 Vol. 40 . Tabu 1. Source Normal Normal Angiosarcoma, case 2, pre-chemotherapy1 Angiosarcoma, case 2, post-chemotherapy* Angiosarcoma, case 1, advanced Ratio ofTotal Uronic Acid Eluted in 1.25 M/1.5 M NaCl 0.364 0l316 0.S43 0.971 5.000 Nora: The flytosaminoglycans (GAC) in a 24-hour urine apecimen were precipitated with cetylpyridinium chloride (CPC) and icparated a> 0.4 M NaCl aoluble ("hyaluronic add"), 1.2 M NaCl aoluble ("chondroitin sulfate"), and 2.1 M NaCl aoluble ("heparin") fraction!. The CPC waa removed from the 1.2 M NaCl-CPC- aolubiliaed fraction and the GAGa further purified by anion-cxchanfe chromatography. The total amount of GAG in the resulting 1.25 M and 1.50 M NaCl columneluted fraction! waa determined and the ratio of the two fraction! waa calculated. ('One day prior to begin ning of chemotherapy; Two daya 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 1.50 M NaCl-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 eqtyltrimethylammonium-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 Tailb 2. Hyalurenidaie Susceptibility Source Depolymcrization %' Heparin, standard Hyaluronic acid, standard Chondroitin sulfate, standard 1.25 M NaCl column-eluatt, pooled fractions from angtosarcomatoui patients 1.50 M NaCl column-eiuate, pooled fractions from angiosarcomatous patients 1.50 M Nad eolwnn-duate. normal 5.0 93.1 97.6 43.5 100.0 100.0 1 Glycosaminoglycani isolated (ram 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 NaC) column-eluted GAC and consequently do not ap pear in this table. The chromatographic pattern found here for controls conforms to urinary glycosaminoglycan distributions reported by others. **" 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 I-X2 chromatographic patterns reported by Kao and Leslie** and by others. *" 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*'1* and are susceptible to hyaluronidase1* 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, it., those of neoplastic growth, fibrogenesis, or cell death. In this re gard, it should be noted that 1) the ratio of heparan sulfate to chondroitin sulfate reported here for angiosarcomatous urine is similar to that reported for cirrhotic human liver firm* by Becker,* and 2) the shift from a hyaluronidase- 57 ucc 003271 No. 6 Gi.vc0SAMiMO0LV.CANs in Angiosarcoma Curran tt al. 3053 susceptible to a bynluronidase-rcsistant GAG is consistent with the suggestion by Huttercr and Rubin" that the stabilization of collagen de pends on a shift to a hyaluronidasc-resistant GAG envelope surrounding the collagen bundle. Although Huttcrer and Rubin attribute this to an augmentation of dermatan sulfate, Becker* 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-associated 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-induccd liver disease. REFERENCES 1. Anghileri, L J.: Metabolism of acid mucopolysaccha rides in hepatoma and normal liver. Qncatagj 30:304-317, 1974. 2. Becker, K.: Acid mucopolysaccharides in experimental and human cirrhosis. Jn Collagen Metabolism in (he Liver. H. Popper and K. Becker, Eds. New York, Stratton Inter continental Medical Book Corporation, 1973; pp. 43-52. 3. Bitter, T., and Muir, H.: A modified uronie acid carbazoic reaction. Anal. Boehm. 4:330-334, 1942. 4. Creech, J. L., and Johnson, M. N.: Angiosarcoma of the liver in the manufacture of polyvinyl chloride. J- Qeeapalioaal Mid. 16:150-151, 1974. 5. DiFcrrante, N.: The measurement of urinary muco polysaccharides. Awl. Boehm. 21:98-106, 1967. 6. DiFerrante. N.: Turbidimetric measurement of acid mucopolysaccharides and hyaluronidase activity. J. Bui. Chm. 220:303-306, 1956. 7. Falk, H., Creech, J. L-, Heath, O. W., Johnson, M. N., and Key, M. M.. Hepatic disease among workers at a vinyl chloride polymerization plant. JAMA 230:59-63, 1974. 8. Gaiambos, J. T.. Connective tissue metabolijm and cirrhosis, fa Collagen Metabolism in the Liver, H. Popper and K. Becker, Eds. New York, Stratton Intercontinental Medical Bo* Corporation, 1973; pp, 57-61. 9. Gasic, C., and Gasic, T.: Removal of sialic acid from the cell coat in tumor cells and vascular endothelium and its efTects in metastasis. Prac. Mad. Acad- Set. U.S.A. 48:1172-1177, 1962. 10. Grccnbetg, R. A., Tamburro, C H., and Kupchella, C. E.: A prospective medical surveillance program lor the detection and prevention of occupationally-related cancer. la Prevention and Detection of Cancer. H. E. Nieburgs, Editor, Part 1, Volume 2, Marcel Dekker, Inc., NY (In press). 11. Hutterer, F., and Rubin, E.: Mucopolysaccharides in reversible and irreversible experimental hepatic fibrosis. fit 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. C.: Micro fractionation and determination of urinary glycosaminoglycans. Button. MM. 9:317-326, 1974. 13. Koizumi, T., Nakamura. N., and Abe. H.: Changes in acid mucopolysaccharide in the liver in hepatic fibrosii. Biaehim. Btaphji. Ada. 148:749-756, 1967. 14. Kom, E. P,: Isolation of heparin from mouse mast cell tumor. J. But. Chm. 234.1325-1329, 1959. 15. Kupchella, C- E-, and Tamburro, C. H.: Urinary and tissue glycosaminoglycan patterns in hepatic angiosarcoma. la Prevention and Detection of Cancer, H- E. Nieburgs, Editor, Part 1, Volume I, Marcel Dekker, Inc., NY (In press). 16. Makk, L, Creech, J. L, Whelan, J. G-. and Johnson, M. N.: Liver damage and angiosarcoma in vinyl chloride workers: A systematic detection program. JAMA 230:64-68, 1974. 17. Popper, H., and Thomas, L. B.: Alterations of liver and spleen among workers exposed to vinyl chloride. Am. MY Arad. Sti. 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 Ota. Mid. 54:223-228,1959. 19. Schiller, S.. Slover, C. 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. Bui. Own. 236:983-987, 1961. 20. Sharon, N.: Complex Carbohydrates: Their Chem istry, Biosynthesis, and Functions. Reading, Massachusetts, Addison-Wcsley Publishing Company, 1975. 2t. Varadi, D, P., Cifonelli, J. A., and Dorfman, A.: The acid mucopolysaccharides in normal urine. Biathua. BtafhriAda. 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. Caam Rii. 33:2257-2264, 1973. 58 I ucc 003278 HEPATIC CELL IDENTIFICATION TECHNIQUE H.F. FORTWENGLER AND C.H. TAHBURRO, 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 microscopy, 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 trltlated thymidine. Iron particles, tritlated hydroxyproline or Factor VIII antibody, one can Identify cells as being active in incorporation of ^HT into DNA, having a phagocytic capability, having collagen forming ability or simply identles 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 ucc 003279 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. \ 60 Ucc 0032Q0 SLIDE I LIVER BIOPSY IN VITRO TECHNIQUE FOR HEPATIC CELL IDENTIFICATION A ELECTRON MICROSCOPY B CELL TYPE IDENTIFICATION C LIGHT MICROSCOPY DIVIDED LIVER BIOPSY MARKER CELL TYPE dna/rna ALL CELLS KC -\ EC FB 61 ucc 003281 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 surveill ->nce 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 is 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 ucc 003282 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 II 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 63 UCC 003283 ------------------------- ---------------------------------------------------- ------------------SLIDE 2----- OUTLINE OF EXISTING PROTOCOL FOR SCREENING ucc 003284 SLIDE 3 PROCEDURE FOR EVALUATION OF MEDICAL SCREENING DATA ON INDIVIDUAL BASIS 1. DETERMINATION OF SCREENING TESTS 2. DETERMINATION OF STANDARD RANGE (SR=90Z) 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 UCC 003285  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-oeeupatlonal disease which may simulate occupational injury such as hepatitis, diabetes, hyperlipidemia and obesity. Individuals found to have occupationally-related disease undergo thorough diagnostic 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 \Y.' ti code for the entire plant or industry, and estimated exposure (Slide 5). to various chemicals. The degree of exposure is ranked intheorder of 0 to 6. Slide 6 indicates the rating basis for the rank ordering of chemical exposure. These renk 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 code (3 digits), and the job code (an additional 3 digits) and 66 UCC 003286 SLIDE 4 REASONS FOR SCREENING PROGRAMS 1. PROTECTION FOR ECONOMIC BUSINESS 2. PROTECTION FOR OTHER INDIVIDUALS 3. ALTERNATIVE TO PERSONAL HEALTH SERVICES 4. ACQUISITION OF CLINICAL BASELINE INFORMATION 5. DETECTION OF TREATABLE CONDITIONS SLIDE 5 EXPOSURE SUMMARY A, JOB CLASSIFICATION CODE' 3. EMPLOYEE WORK HISTORY C. CHEMICAL EXPOSURES D. _ EXPOSURE INDICES 1, AVERAGE EXPOSURE 2, TOTAL EXPOSURE 3, INTERACTION EXPOSURE 67 ucc 003287  SLIDE 6 RATING BASIS 0 - ABSENT FROM ENVIRONMENT 1 - LOWEST EXPOSURE 2 - MINIMAL EXPOSURE TO LOW LEVELS 3 - MODERATE EXPOSURE 4 - WORKS IN AREA SUBJECT TO OCCASIONAL HIGH EXCURSIONS 5 - WORKS IN AREAS WHERE LEVEL IS HIGH 6 - INTIMATE CONTACT - SKIN OR HIGH INHALATION ucc 003288 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? 69 `JCC 003289 DETAILED WORK AND EXPOSURE HISTORY YEAR WORK HISTORY EXPOSURE RANK FOR EACH CHEMICAL BUILDING 1 JOB NO. MONTHS VINYL CHLORIDE 2* 3* 1944 000 1945 000 1945 111 1946 111 1947 111 1948 111 1948 121 1949 121 1949 112 aa 1957 a 121 t 576 576 194 194 194 194 192 192 253 a zis a 1972 sb 1972 000 574 1973 000 574 1974 000 574 1974 TERMINATE! 6 5 7 12 12 8 4 4 8 a a a 5 7 12 8 H* - CM - 2 11 2 11 5 41 6 41 6 43 5 43 4 31 4 31 2 G3 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 nurter 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 end 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 veil as retrospective data to document the safety of the environment or to determine if an occupational risk is truly present. 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 veil 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 UCC 003291 SLIDE 9 EMPLOYEE WORK AND EXPOSURE HISTORY YEAR 1945 1950 1957 1965 COMPANY WORK HISTORY MILLING; INC. 177* - 268** 177 - 269 178 - 438 PUSTIC INTERNATIONAL 013 - 291 018 - 548 013 - 620 J.K. MANWORK; INC, G.P, CHEMICAL CO. 100 - 001 100 - 020 001 - Ill # OF MONTHS 14 26 20 24 12 48 48 48 CURRENT AREA OR BUILDING CODE JOB CUSS I FI CATION CODE EXPOSURE DATA FOR EACH CHEMICAL FOR EACH JOB CUSSIFICATION IS KEPT BY THE COMPANY. t ucc 003292 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. 73 ucc 0032Q3 SLIDE 11 OCCUPATIONAL MEDICAL SURVEILLANCE PACKAGED COMPUTER PROGRAMS 1) WILL SAVE MONEY 2) WILL SAVE TIME 3) WILL GUARANTEE UNIFORM DATA COLLECTION A) 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 74 ucc 003294 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 hlgest exposure t 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 es a solvent for these catalysts, and e) 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. 75 Occ 00329$ 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. A. 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? 76 Ucc 003296 NUMBER AND TYPES OF MALIGNANCY IN CHEMICAL WORKER COHORT-1960-1978 l 1960-64 1965-68 1969-71 1972-74 LIVER ^ LUNG COLON BRAIN GU ucc 003297 RANK OF INDIVIDUAL S EXPOSURE VINYL CHLORIDE EXPOSURE RANK ANGIOSARCOMAS 'C AND MATCHED CONTROLS C SLIDE 14 `I ACRYLONITRILE EXPOSURE RANK OF INDIVIDUALS WITH ANGIOSARCOMA 1 SLIDE 15 gOwrsjcoOx CO CD ANGIOSARCOMAS AND MATCHED CONTROLS c "SLIDE 16 LIST OF SELECTED CHEMICALS FOR EXPOSURE INDICES CHEMICAL CODE 01 02 03 04 05 06 07 08 09 10 11 . 12 13 14 15 16 17 18 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 CHLORIDC VINYLIDENE CHLORIDE VINYL ACETATE PVC DUST CATALYSTS STYRENE HEXANE 80 ucc rirmnn VINYL CHLORIDE EXPOSURE RANK OF INDIVIDUALS WITH LUNG CANCER > LUNG CANCERS 3. AND MATCHED CONTROLS ucc 003301 SLIDE 18 ucc 003302 i ACRYLONITRILE EXPOSURE RANK OF INDIVIDUALS KITH1LUNG CANCER. UJ oc aCoO_ 2 CO =c tooo (=> o s I II III IV V ==> LUNG CANCERS 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 bio d 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 88Z 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 maxima dose 'capacity for the liver, one can correctly identify underlying hepatocellular Injury in almost 981 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 qhanggs 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 ox 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 83 ucc 003303 ; SLIDE 19 e Frequency of biochemical abnormalities being present and correctly Indicating the preaencej&lgnlfleant histological abnormalities (SCFT " alanine aminotransferase; GGT gamma glutamyl transpeptidase; AZJC PHOS Alkaline Phosphatase; SCOT * aspartic aminotransferase; ICD Isocetrlc dehydrogenase and ICG " indocyanine green clearance) 84 UCC 003304 FUNCTIONAL vs BIOLOGICAL SCREENING FOR HEPATOCELLULAR INJURY IN AN INDUSTRIAL POPULATION # TEST VALUES SLIDE 20 oC OO o 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, la 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 te 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 86 ucc 003306 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 87 Occ 0330? 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 (l.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 direct r 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 shew 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 ucc 003308 Line 1. 2. PLANT PHYSICIANS 3, PLANTS MSP 4. LEVEL MEDICAL 5. SCREENING SLIDE 22 PJ III t LOCAL HOSPITAL II t LOCAL HOSPITAL II i EXTERNAL LABORATORY 6, ucc 003309 89 8I0ASSAY 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 A0232 The mamnalJan 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 hepatotoxln 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 cotlagenase is added to the perfusion medium. The perfusion procedure is depicted in Fig. 1. With the proper perfusion equipment, preparations of isolated liver cells can be readily obtained which exhibit very high viability and have been clearly shown by myself and others to retain normal liver specific functions. An example of some data which 1 obtained utilizing Isolated liver cells is shown in Fig. 2. The synthesis and the secretion of albumin and many other plasma proteins is a specific function of the liver which the isolated cells retain. Additionally, as shown in Fig. 3, the intracellular morphology of the Isolated cells when investigated by high re solution electron microscopy is Identical to that of the intact tissue. There are also major technical advantages associated with the use of cell suspensions including 1) the ease of uniform sample collection (media or cells) as a function of time, 2) the ability to assay many different conditions or variables simultaneously and 3) the ability to 90 UCC 003310 Or. Richard C. Feldhoff employ radioactive metabolites at a much higher specific activity than Is feasible in other -in vitro or in vivo systems. Fra. 1 Diagram depicting cumulation and perfuaion procedure*. A, ligature tying portal rein cannula and placed above entry of *|ita>ie vein: B, inferior vena cava and abdominal aorta tectioned after imenion of portal cannula; C. infution tubing and cannula inaerted into rubber tubing ioeert in inflow line; D, inflow line taped to aluminum block to hold portal rannula in place: , ligature around celiac axia and tuperior nicacntrric arterie*. tied off after insertion of porta) cannula: F. ligature around inferior vena cava above R renal rein, tied off after ineerttoa of vena cava cannula; G, vena cava cannula inaerted through right atrium into inferior vena cava, not tied; H. outflow lino taped to aluminum block to hold vena cava cannula in place. 91 ucc 003311 Dr. Richard C. Feldhoff Flo. 2 Total protein and albumin secretion by isolated hepatoeytej. Cells v.tr* prepared as described under 'Experimental Proce dures' and preineubated for 30 min in Krebs-Henselrii bicarbonate butfer eontuimns '.5 times the normal plasma levels of amino acids. Cells were transferred to 40 volumes ofbicarbonate buffer containing normal plasma level; of amino adds and rili'endne at 10 /.Cb'ml, At each time point, aliquots of the suspension were centrifuged and assayed for incorporation into total secreted protein and albumin as described under "Experimental Procedures." O------O, hcpatocyies from normal rats; e------ , hepatocytes from hypopbvsectomired 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, (Wlttllff - 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 (Feldhoff, Tamburro); mutagenic capacities of newly identified compounds (Streips). A) 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. 92 ucc 003312 Fig. 3 Isolated rat hepatocyte. Cell suspensions were perpared by cotlagenase perfusion and then Incubated for 2 hours at 37* C in Krebs-Henseleit bicarbonate buffer. Utilizing standard procedures the cells were fixed for 1 hour at 23* C In 0.1 H cacodylate buffer, pH 7.4 containing 2.1% glutaraldehyde. Cell preparations were dried in graded ethanol solutions, transferred to propylene oxide and embedded in Epon. The sections were stained with 93 1 SUMMARY OVERVIEW THE YALE JOUWiAL OF ItlOLOGY AND. MEDICINE 51 (lV7b), o7-S0 Tlie Sepsiic Roie in Cnrcir.ogsnesis and Its 2ariy Detection--Tne Vinyl Chloride Model1,2 CARLO H. TAM3URRO University of Louisville School ofMedicine, Louisville, Kentucky Received October 17. 1977 The liver't role in vinyl chloride toxicity and caicinocenicity ii providing a bettergndentandinf ofthe chemical catciooEeneaia oiechanitm. A variety of both malignant and benign hepatic tuition haa been demooitialnd with prolonged expoaurc to vinyl chloride. The tnuhi-tytlcm involvment ofthi> carcinogen and toxin haa provided a model for the ttudy ofchemical caicinogeneiii common to both man and animal. Clinical atudies have ahown the uiefulneaa of biochemical, radioiaotopic. and radiological atudiea in the detection of toxic and carcinogenic leiiona. Animal atudiea have demonatnted the biochemical maiab oliam by the liver of vinyl cMotide-produced intertnediatea which are mutagenic in bacterial eyatema and may be the diwa cardnogcna. Hepatic aubccilular engyme atudiea prove prehmiaary evidence of cellular adaptation xnd increaaed detoxillcatiort. Dunrption otthia nririiiation and delotdifation balance may be the key to the --itg---- transformation of cede, a working bypotheaia ie pieaanted which may explain the mataboliam of vinyl chloride into mutagenic intatmediatea by the liver cell and the davakpmool of malignant iranctotmattoo by extra hepatic ainuvotdal lining ceih, lung relli. and brain liaaoe. 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 (CHi -- CH--Cl monochloroethyene, agas) is the basic molecule ormonomerofpolyvinyl 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'$) 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 ofvinyl chloride to man was obtained from experiments by research workers on themselves, from the evaluation of its suit- 67 ^------- ppnrtinl hr gnnt fi--it Matniftrturiog rhimilll ft iinrininn mil by Nxiinuxl fim n Institute* Coronet ON01-CN-JJ2I2. Thil snick u th llurtOTotb in a series entitled. "Semioirs on Livvr Disease." that kv* bun presented M part of ih* Training Program in Liver Diieaas it tht Vuwnni AdminiMrarion Hoipitil. West Hma, Connecticut. Dr. Harold O. Conn. Profasor ofMedicine. Ytit University School ofMedicine, and Directoroflb*Training Prcpnm in Liver Diaaaac. ii guest editor. PlaaM address rapitat requests to; Cario H. Tamburto. M.D.. ProfessorofMedicmc, Chiaf. DigaMiva Diaeaeei and Nutrition Diviaioa. Diractor, Vinyl Cblorida Project. S11 South Floyd Stnet, P.O. Bon J5WQ, Louisvflle, KY 4001 0044-0036/78/5101-CQ67 $01.40 Copyright O 1*71 by Tba Yala Journal of Biology and Kedidae, lac. Ail righta of rapwdnation in any faroiraaatyad. 94 68 CARLO H. TAM3URK.0 ability as an anaesthetic [4,5], and from study ofthe cases ofvinyl chloride poison ing contracted during industrial use [6, 7, 8, 9]. Sporadic studies with vinyl chloride polymerization workers demonstrated vary ing riogrws of hepatic biochemical derangements, hematological abnormalities, and skin [9], Acute short term exposures led to disturbances ofthe 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 iiverand kidneys as well as irrita ting to the mucosal membranes and the lungs. Microscopic examination of liver sections showed degeneration of the central lobules while the damage to the kidneys chiefly involved the tubule and the interstitial lining somewhat like that of carbon tetrachloride damage [10, 11, 12]. BACKGROUND The literature, however, contained virtually no information on damage to man after chronic exposure until Filatova et al. reported disturbances of the blood vessels and nerves in individuals exposed with 20-300 ppm of vinyl chloride on a continuous basis [13]. Cordieretal. [14] and Wilson et al. [15] were the first to re port the hitherto unrecognized disorder termed occupational acroosteolysis (AOL) which included the symptoms oftenderness 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.C30 ppm to induce acroosteolysis, accidentally discovered cancer. Maltoni et al. [17] while studying various levels of exposure demonstrated that angiosarcoma occured at 250 ppm; the Manufacturing Chemists Association's studies [18] demonstrated these liver cancers even at 50 ppm. At the .same time, Dr. John Creech, at the B.F. Goodrich Chemical Company Plant in Louisville, Kentucky, discovered an hepatic angiosarcoma in one employee. Creech, recalling an earlier hepatic angiosarcomaat 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 halogenaied 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 ofits important physical properties include a low boiling point, a high specific gravity, a low solubility in water, and a half-life in air which ranges from 3-20 hours. Knowledge ofthese 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 UCC 003315 VINYL CKLO.UOn--ItLL., ,*il> CARCINOGENESIS TABLE 1 Oecuj&iionaJ Vinyl Chloride Exposure Auociated Diionlen i. Thrombocyiopxnix 2- Keiiculocytom 3. Splenomegaly 4. Hepatic fibrosis 3. Sclcruderma'ltjte sltin channel 6. Aero-oiteolyiia 7. Raynaod t phenomenon I. Leukopenia 9. Hepatomegaly 10- Pulmonary functional impairment 11. AngkMircoina 12. Cardiac arrhythmia 13. Nephrobhutomaa* 14. Zymbel gland carctnomaa* 15. Large cell hint cancer IS. Brain cancer In rata only 69 associated disorders is shown in Table 1. Animal and epidemiological studies indicate the probability that cancer induction at othersites is alsodirectlyattributed to prolonged and excessive vinyl chloride exposure [19]. In order to develop effective methods ofprevention, accurate knowledge of the pathogenesis of this environmental chemical in ultimately producing its most destructive effect--cancer--is needed. EXPERIMENTAL ANIMAL STUDIES---TUMOR FORMATION The carcinogenicity of vinyl chloride has been demonstrated in a variety of animals as well as in humans at exposure levels that vary from 50-30,000 ppm. As illustrated in Table 2, a variety of tumor types have been found in rats, mice, and hamsters. An extensive list of benign tumors have also been reported in mice, rats, and hamsters exposed to vinyl chloride. Maltoni's group has now demonstrated primary liver cell cancers in exposed newborn rats. Direct hepatocellularinjury as well as pulmonary, mucosal and skin injuries have been shown in directly exposed animals. Pretreatment with many agents increases the toxicity ofvinyl chloride; they include phenobarbital, ethanol, polychlorinated biphenyls and pesticides such as hexachlorobcnzene [20]. This relationship to vinyl chloride's ability to induce cancer is under study, particularly in view of the industrial environment which allows exposure to many chemicals to occur con currently. HUMAN STUDIES Epidemiological studies in the human strongly suggest that exposure beyond 10 years is associated with increased cancer mortality, mainly digestive system cancers, primarily hepatic [21,22]. There also appears to be a higher incidence of large cell carcinomas of the lung, brain glioblastoma multifonns, and lymphomas [23,24], although there is some disagreement as to the interpretation ofthis aspect of the epidemiological data. 96 UCC 0033A6 70 CARLO H. 7AtaUfLRO TABLE 2 Cftrcinogtnicity of Vinyl Chloride (Exposure: 50-10,000 ppm) TumorType Specie* 1. Liver--angiosaitoma 2. Liver--hepatocellular carcinoma 3. Luns--adenocarcinoma 4. Lung--iaige cell carcinoma 3, Mammary adenocarcinoma & Zymbai gland tumor* 7. Nephrablaitoma 5. Osteochondroma* 9. Skin epithelioma, 10. Melanoma* 11. GlioNauooa multiform* 12. Lymphoma Adult human*, nts. mac* and hamster* Newborn rats Rat* and mice Humans* Mice Rats Rats Rat* Hamster, Hamster* Humitt* 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]. Theseanatomical 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 transpeptidasc (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 Tig. 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 oftumordetection. 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 UCC 003317 ViNYL CisLO^U^E-^aUELATaji.* C 71 Diagnostic angiographic studies of these radioisotopic abnormalities in 80 individuals have demonstrated 3 major lesions. The 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 in individuals with splenomegaly, and named lienal peliosis (Fig. 3). These splenic lesions demonstrate a shortened celiac artery to portal vein circulation time, normal portal vein diameters, and increased spleen size. This has been found only in individuals with long-term vinyl chloride exposure. The final lesion is that of angiosarcoma (rig. 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 ofDisse. Exploratory wedge biopsies have in addition demonstrated increased subcapsular fibrosis with subcapsular bile duct proliferation plus the often described portal fibrosis [27]. VINYL CHLORIDE METABOLISM AND CARCINOGENESIS Present biochemical knowledge indicates that vinyl chloride is most likely metabolized by the liver in a three step process [28]. At concentrations less than 50 ppm, vinyl chloride is metabolized by the alcoholic dehydrogenase system into chloroacetaldehyde and monochloroacetic acid. Cl-CH-CHi--a~CHi-CH*-OH--- -C-~--CI-CHj-CHO--CJCHj-COOH dehydrogenase An alternative pathway which appears to become operative at 220ppm is oxidation by the peroxidase-catalase system. Cl-CHr-CHi-OH--.ClCHi-CHiOOH------- CICHi-CHO catalase WIL M. % 1 I Mu*/* U M M/W */m % V, 98 ucc 003318 ViNYi, CKLOAiOE--CA.tCINQGENESiS j i 73 * $ Zi. FIG. 4. Hepatic arteriogram: Venou* phacc. At approximately 14-15 teconda the peripheral uaia U identified (emewe). laating through the entire phau- Scattered ateat pf puddling are alio present in and around the area of central hypovaacularity. 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 monochioroacetic acid. Cl-CH-CHx- oxidase -+CL-CH---CH o' -*C1--CHa-CHO--CICHa--COOH As illustrated in Table 3, vinyl chloride oxidation intermediates chloroethanol and chloroacetaldehyde, at low doses, are most likely detoxified via the glutathione-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 3,000 ppm for an extended time and reported in workers exposed to levels greater than 250 ppm fora 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 UCC 003320 72 CARLO H. TAMRURXO r" < '.K FIG. 2. Hepatic arteriogram: Vtoout ptaatc. Change! of peliotit hepatia are prueat throughout tho loft lobe. The multiple nodular ttnini reprttent peliotii hepatia Iciiona (arnwi) ranging from 2-3 mm to 2 cm in tire. ''$ I'M FIG. 3. Splenic arteriogram: Venoua phi** US aeconda). There an 3-4 circular and oval Maim (nrror) in tha wperior and inferior lateral portion* of the tpleen. Nor mal pancreatic Main oceuri prat above the (picnic vein. 99 V* Occ 00331Q 74 Cl CH - CHi (VO' . CARLO K. TAMSURKO TABLE} Proposed Metabolic Fate of Vinyl Chloride Deloxificatioti with gluiithiooe O CHi CHi OH (CMoraeihanoi) | +GSH OCHi CHO----------(Chioroocetnldnhyde) <CS CHi CHO ClCHiCOOH (Chloraocetic add) ICHiCOOH Thiodjglycolk 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, chlorocthanol--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, itselfbe 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 ofexposure 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-d-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 UCC 003321 ft VINYL CHLORiOii--RELATED CARCINOGENESIS 75 The discovery of a decreased glucose-S-phosphatase after "simulated" chronic exposure led to the study of enzymes in the pentose phosphate shunt pathway. Weber and Lea [32] had found similar changes for primary hepatocellular neo plasms, demonstrating that in a rapidly developing primary hepatocellular tumor, there is decreased giuconeogenesis with a reduction in the glucose-6-phosphatase, followed by an increase in gIucose-6-phosphate dehydrogenase and transaldolase. These biochemical changes were also followed by an increase in purine biosynthe sis (increased phosphoribosyipyrophosphate aminotransferase (PREP) 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 S4 hours of exposure. However, after 103 hours, there was significant increase in glucose-6-phosphatase dehydrogenase. Studies of PRPP, at least up to 137 hours, have as yet shown no significant changes. Studies are now underway using animals exposed to 130 to 250 hours to determine if the biochemistry in vinyl chloride injury is similar to that in primary hepatocellular tumors. This, however, does not explain why the hepatocyte, which is theprimary 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 occurin 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 liningcells. In the early stages they are largerand thicker--possibly swollen. Later, they became bulky and in places, multi-layered containing increased organelles, especially mitochondria and endoplasmic reticulum. Later disruptions in the sinu soidal walls seen were consistent with beginning peliosis hepatis. The lining cells, probably the precursors of angiosarcoma, often resembled fibroblasts. However, their endoplasmic reticulum did not contain any collagen components. These observations by 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 m^jor difference at 102 UCC 003322 76 CARLO H. 'i AMit'ORRO present is an increased collagen deposition, characteristic ofhuman 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 mid~zonal 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 ofthe hepatocytes by the collagen bundles which initially give the ap pearance of ih/rn-hepatoceiluiar 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 oftheproteroglycan role in collagen form ation in vinyl chloride-exposed workers. Ithad been suggested in the literature that glycosaminoglycans in blood and/or urine might be useful as means ofearly 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 ofthe skin FIG. 3. Electron microscopy showing collagen (CB) bundles (brows) invaginaiing into the hepatocyte, giving the appearance of tnter-hcpalocytic collagen. N nucleus; S -- sinusoidal space; IM - invagination into the cell membrane (smail brows). 103 UCC 003323 VINYL CHLORIDE'--ALcA'ILD CARCINOGENESIS 77 {36J, Others have noted a strong positive Aician blue glycosaminoglycan staining reaction in human angiosarcoma tissue [37J. This suggested that quantitative and quaiitative determinations of glycosaminoglycan production in individuals with neoplasm, either by serum or urine, might be used to identify those at high risk oras an early indicator of neoplastic foimatioa. The feasibility of glycosaminoglycan "spot test" for vinyl chloride production workers made this an attractive possibil ity for mass screening. Urinary glycosaminoglycans, measured as uronic acid, were studied in individu als with alcoholic cirrhosis, viral hepatitis, secondary liver metastasis, hepatic angiosarcoma and normal controls. The percentage oftotal glycosaminoglycans that was dialyzablc and the percent age ofunfractionated total that appeared in thehyaluronicacid, chondroitin sulfate, or in the heparin fractions was similar for all groups. However the distribution of positive fractions varied with the different groups studied. Seven of the nine vinyl chloride-exposed individuals, other than those with angiosarcoma, had glycosaminoglycan positive chondroitin sulfate fractions with negative hyaluronic acid and heparin fractions whereas this occurred in only 3 of the 32 urines from other hepatic diseases [38]. In addition, study of the total tissue glycosaminoglycan levels in angiosarcoma tumors and fibrotic tissue adjacent to the tumor demonstrated that tumor tissue itselfhad higher levels ofhyaluronic acid and heparin fractions as compared to the non-tumor adjacent tissue whichhad higher levels ofchondroitin 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, <0,41,42,43] and hepatic cancer [44] result in increased hepatic glycosamino glycan levels. It may be significant that the angiosarcoma patient has half the urinary glycosaminoglycan excretion of patients with liver metastasis and that analysis ofangiosarcoma tumor tissue exhibits halfthe glycosaminoglycan content reported by Kojima et al. [44] for hepatocellular carcinoma. The increases in liver and urinary glycosaminoglycans may well reflect the importance ofthese substan ces in the process of fibrogenesis and tumor growth. Although no significant differences were found in total glycosaminoglycans ofvinyl chloride-exposed indi- ^ viduals with associated liver injury, there was a significant difference In the excre tion patterns of these individuals. Seventy-eight percent of them had positive chondroitin sulfate fractions in contrast to only nine percent of the non-exposed liver injury cases. Thus the change in the glycosaminoglycan excretion pattern in individuals with pre-cancerous injuries may be of significant prognostic and diagnostic importance [43]. The urinary glycosaminoglycan excretion patterns in an angiosarcoma patient 3 months to 2 weeks prior to death demonstrated an increase in the urinary chondroi tin sulfate fraction with a change in its composition as the disease progressed. During this time, the chondroitin sulfate composition showed acontinuous increase in the ratio of the 1.25 M NaCl to the 1.5 M NaCl fractions. This was due to an increase in the 1.25 Meluate and a decrease in the 1.5 Meluate fraction and was 23 times greater than the controls. In the most advanced stage ofthe angiosarcoma the ratio increased to 13.7 times greater [46]. These very preliminary studies would suggest that alterations in the ratios of these fractions' compositions may be useful in evaluating the severity and subsequent progression ofdisease. Early lesions may produce small changes in the ratio which would become more pronounced as the disease worsened. The 104 UCC 003324 4 li CARLO H. 7AHEURRO determination ofthis 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 tum 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 ami cysteine-SH groups and are excreted in the urine. At even higherdoses increasing amounts ofthe chloroacetal dehyde are forther oxidized to chloroacetic add and excreted as an end product in the urine. However, when chloroacetaldehyde and/or the chlorooxiranes exceed the detoxification threshold ofthe hepatocyte, this leads to hepatocellular toxicity and/or stimulation ofthe sinusoidal cells. This acute event in turn acts as astimulat ing mechanism for increased collagen deposition in the space of Disse and sinusoidal areas as shown by electron and lightmicroscopy studies. The increase in the collagen depostion in sinusoidal spaces then leads to disruption ofhepatic cell surface function, disruption ofthe hepatic cords, coalition ofthe sinusoidal spaces and eventual pdiosis hepatis. These lesions alternately lead to sufficientvascular dysfunction to add further to the biochemical manifestation of hepatic cellular injury- _ V. Since it is highly unlikely that the unstable chlorooxiranes are able to betrans 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 chloroacedc add nor to detoxify it suffidently, if at all, by their own detoxification systems. This would allow a longer contact period with the cell's DNA. In addition many ofthe 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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