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The metabolic elimination of vinyl chloride in Rhesus monkeys is a dose-dependent, satruable process, as in rats. Below 200-300 ppm of vinyl chloride (VC) in atmosphere, elimination obeys a first-order law; the clearance rate is much closer to that found in man than the values for rats, mice and gerbils. The maximal velocity of metabolic elimination of VC at high concentrations in Rhesus monkeys is only about half that of rats when related to kg body weight. It s suggested that Rhesus monkeys mimic the quantitative aspects of VC metabolism better than rats or mice.

Proteins of isolated rat hepatic sinusoidal cells incubated with 14C-vinyl chloride, rat liver microsomes and an NADPH-regenerating system were alkylated by vinyl chloride metabolites formed by microsomes. This suggests that reactive vinyl chloride metabolites can penetrate sinusoidal cells. Protein alkylation in isolated hepatic sinusoidal cells was higher when these were co-incubated with isolated hepatocytes, indicating that reactive vinyl chloride metabolites formed by hepatocytes are stable enough to diffuse out of hepatocytes into sinusoidal cells. Glutathione added to the incubation medium inhibited the covalent protein binding of vinyl chloride metabolites in sinusoidal cells as well as in hepatocytes incubated separately and deleted the increased protein binding in sinusoidal cells co-incubated with hepatocytes. The data indicate that glutathione present in the incubation medium traps reactive vinyl chloride metabolites formed by hepatocytes which otherwise would react with cell constituents of sinusoidal cells. If similar conditions exist in vivo, the alkylation of DNA of liver endothelial cells by vinyl chloride metabolites formed in hepatocytes is possible. This would explain the induction of hemangioendotheliomas of the liver by vinyl chloride.

On the basis of previous determinations of pharmacokinetic parameters for inhaled vinyl chloride in men, rhesus monkeys, and rats, and on improved pharmacokinetic models a pharmacokinetic treatment of the problem of "peak concentrations" of vinyl chloride, as occurring in industrial practice, became possible. For the calculations, metabolic elimination kinetics of vinyl chloride was assumed to be first order as experiments in different species including rhesus monkeys showed "linear" pharmacokinetics up to atmospheric exposures of 200-300 ppm. The distribution of vinyl chloride between atmosphere and organism under different conditions was evaluated using "'steady-state-kinetics". After treating the processes of "influx", "efflux", and "metabolism", the numerical values for the parameters derived from a human kinetic experiment were used to theoretically calculate the time courses of concentration of vinyl chloride in the organism and of the cumulative amount of vinyl chloride metabolized, under the conditions of (a) a 2h constant exposure to 5 ppm vinyl chloride and (b) two subsequent "peaks" of 50 ppm with a duration of 5 min each. This model calculation suggested that, regardless of the exposure profile, the amount of (reactive) metabolites formed from vinyl chloride would solely be a function of the mean atmospheric vinyl chloride concentration over time. The general validity of this suggested rule could subsequently be demonstrated. As the concentration of the reactive metabolite of vinyl chloride responsible for the carcinogenic effect at the target site must be a resultant of both formation and inactivation, an evaluation of the differential risk of different exposure profiles can reasonably be based on biochemical examinations of the "detoxifying" pathways. This points out the relevance of studies of the patterns of different metabolites of vinyl chloride in man under varying exposure profiles.

The formation of liver sinusoidal cell tumors after exposure to vinyl chloride led to a survey of hepatocytes and sinusoidal cells isolated from rat liver as to their ability to metabolically activate vinyl chloride and vinyl bromide. It is shown that metabolism of these vinyl halides to reactive metabolites is confined primarily to hepatocytes. This favours the theory that in the liver reactive metabolites of vinyl chloride leave the hepatocytes to act on hepatic sinusoidal cells.

Rat liver microsomes metabolise 14 C-vinyl chloride to intermediates which irreversibly bind to the microsomal protein and to soluble proteins and RNA, when these compounds are added to the incubation. A superoxide (O2) generating system comprised of phenazine methosulfate and NADH also converts 14 C-vinyl chloride to metabolites which irreversibly bind to albumin. These data are consistent with the assumption of chloroethylene oxide being the primary reactive metabolite of vinyl chloride. If rats are exposed to 14 C-vinyl chloride, about half of the radioactive metabolites in the liver microsomal fraction is bound irreversibly to microsomal protein, when assessed immediately after exposure. Large amounts of polar, extractable, metabolites are present in the cytosol fraction. The amount of radioactivity in tissues of the rats, irreversibly bound immediately after exposure, comprises 10 - 40% of the total radioactivity in tissues. This percentage rises up to 70% after 48 hrs. Som radioactivity derived from 14 C-vinyl chloride is also incorporated into DNA and RNA of liver. Whereas the peak of incorporation of 14 C into DNA is already reached immediately after exposure to 14 C-vinyl chloride, specific labelling of RNA increases after exposure until its maximum after 24 hours.

[1,2-14C] Vinyl chloride and [1,2-14C] trichloroethylene were incubated with rat liver microsomes, NADPH and RNA (from yeast). Whereas trichloroethylene metabolites were irreversibly bound to proteins in microsomal incubations to a higher extent than vinyl chloride metabolites, irreversible binding to RNA was lower for trichloroethylene metabolites. Hydrolysis of the RNA which was reisolated from microsomal incubations with 14C-vinyl chloride or 14C-trichloroethylene and separation of the nucleosides showed different alkylation products arising from vinyl chloride and from trichloroethylene, characteristic for vinyl chloride being formation of 1,N6-ethenoadenosine and 3,N4-enthenocytidine. The different reactivities of metabolites of vinyl chloride and of trichloroethylene prompted a comparison of the oncogenic effects of both compounds against the rat liver cell. Newborn rats were exposed for 10 weeks to 2000 ppm vinyl chloride or trichloroethylene (8 h/day; 5 days/week). After this period livers of the animals were stained for nucleoside-5-triphosphatase. Whereas the vinyl chloride exposed rats showed focal hepatocellular deficiencies in this enzyme, which are supposed to represent an early sign of malignancy, no such changes were induced by trichloroethylene exposure. The data therefore suggest differences between the hepatocarcinogenic activity of vinyl chloride and possible effects of trichloroethylene on the liver.

Previous experiments have shown that a number of xenobiotics such as halogenated ethylenes cause an experimental acetonemia. In addition, under exposure of rats to vinylidene fluoride (one of the agents producing this effect), the urinary excretion rates of acetoacetate and 3-hydroxybutyrate are enhanced. The enhanced formation of ketone bodies is theoretically explained by a covalent interaction of reactive metabolites of the applied xenobiotic with hepatic cytosolic coenzyme A. This theory is further corroborated by the following experiments: Microsomal incubations of [14C]vinyl chloride and [3H]coenzyme A lead to one metabolite containing 2 moles vinyl chloride/mole coenzyme A and two other with equimolar ratios of both components. Exposure of rats to vinyl chloride leads to a progressive depletion of hepatic cytosolic CoASH, but not of CoASH in mitochondria. In the cytosol acetyl-CoA is also diminished after vinyl chloride exposure. These changes may cause secondary effects in lipid metabolism which are regarded as responsible for the enhancement of ketone bodies.

In order to study the dose-dependence of the genotoxic effect of vinyl chloride (VC) hepatocellular ATPase-deficient foci were evaluated after subchronic exposure of newborn rats. Wistar rats were exposed from day 1 after birth over 10 weeks to 10, 40, 70, 150, 500 and 2000 p.p.m. VC (8 h/day; 5 days/week). One week after cessation of exposure hepatic ATPase-deficient foci were quantitated. For a subsequent investigation lower dose range groups of female and male Wistar and Sprague-Dawley rats were exposed (8 h/day; 5 days/week) to 2.5, 5, 10, 20, 40 and 80 p.p.m. VC. Exposure started at day 3 of life and lasted for 3 weeks. After cessation of exposure the animals were maintained for 10 weeks without further treatment until ATPase-deficient foci were quantitated. Both sets of experiments revealed a straight linear relationship between the dose of VC and the % foci area induced. Within the dose range investigated, no obvious threshold for the induction of pre-neoplastic foci by VC was observed.

Rat liver microsomes were incubated with NADPH, 1,2-[(14)C] vinyl chloride and poly-adenosine. The latter was reisolated from the incubations and hydrolyzed. The radioactivity, originating from [(14)C] vinyl chloride, which was irreversibly attached to the poly-adenosine was confined to 1-N(6)-etheno-adenosine (3beta-ribofuranosyl-imidazo [2,1,i] purine). When rats were exposed to 1,2-[(14)C] vinyl chloride, part of the radioactivity was incorporated into RNA of liver. This radioactive labelling exhibited a first maximum, 14 h, and a second maximum, 72 h after ending the exposure. Analysis of hydrolysate of liver RNA showed that all natural nucleosides of RNA were labelled. Besides, small amounts of radioactivity could be detected which were confined to 1-N(6)-etheno-adenosine. The experiments support the theory that vinyl chloride metabolites react with adenosine moieties of nucleic acid under formation of 1-N(6)-etheno-adenosine. Furthermore, the results show that measurement of incorporation of radioactivity into nucleic acids after exposure of animals to radioactive vinyl chloride is not applicable as a means of determining the alkylating potency of vinyl chloride metabolites towards nucleic acids in vivo.

Vinyl chloride and vinyl bromide are metabolically activated by liver microsomal enzymes to intermediates that covalently bind to proteins and nucleic acids. Several lines of evidence suggest the involvement of the epoxides, i.e., chloroethylene oxide or bromoethylene oxide. Proven targets for alkylation are adenine, cytosine and guanine moieties in nucleic acids, and sulfhydryl groups of proteins. For all the halogenated ethylenes studied so far, including vinyl chloride and vinyl bromide, metabolism in vivo is a dose-dependent, saturable process. The metabolic capacity of rats is saturated at atmospheric concentrations of 250 ppm vinyl chloride and 55 ppm vinyl bromide. As recent reports describe a diminishment of hepatocellular glutathione in rats after exposure to vinyl chloride concentrations of 50 ppm and more, we carried out a series of experiments measuring covalent binding of vinyl chloride metabolites after exposure to different concentrations of 14C-vinyl chloride. In all of these experiments, including one of an exposure to only 2 ppm vinyl chloride, hepatic covalent protein binding was related to the dose of vinyl chloride which was actually metabolized, and the ratio between bound and metabolized material was constant. This strongly suggests that hepatic glutathione levels must have only a very limited impact on covalent protein binding of vinyl chloride metabolites, an assumption which is supported by a lacking effect of a pretreatment with diethylmaleate. A scheme of hepatocellular compartimentation of metabolic steps is proposed which serves to explain these findings.

Rats were exposed to [1,2-14C] vinyl chloride. Liver RNA was isolated, hydrolyzed, and the nucleosides separated on Aminex-A-6. Besides the physiological bases and 1,N6-ethenoadenosine, radioactivity was also incorporated into 3,N4-ethenocytidine. Radioactive 3,N4-ethenocytidine moieties were also formed on incubation of polycytidylic acid with rat liver microsomes, NADPH and [14C] vinyl chloride. These alkylation mechanisms are consistent with the mutagenic and cancerogenic properties of vinyl chloride.

To investigate the factors responsible for the high sensitivity of the livers of young rats to the carcinogenic stimulus of vinyl chloride (VC) adult and 11-day-old Wistar rats were exposed to [1,2-14C] VC. Adult rats received either a single 6-h exposure, or 2 single 6-h exposures separated by a treatment-free time interval of 15 h. Eleven-day-old rats received 2 single 6-h exposures, according to the same treatment schedule. The animals were sacrificed 1 h after the end of the corresponding exposure period; liver DNA was isolated, enzymatically hydrolyzed and analyzed by column chromatography. Incorporation of [14C]VC-derived radioactivity into the physiological deoxyribonucleosides (presumably reflecting the activity of DNA replication) was observed for all three sets of experiments. Virtually no difference in 14C-incorporation was observed between adult rats sacrificed immediately after one single 6-h exposure to [14C]VC and those which received a second exposure on the following day. In contrast, an about 8-fold increase in 14C-incorporation into the physiological purines of DNA of young versus adult rats was detected. This difference is indicative of a significantly elevated DNA synthesis/cell replication in the liver of young (11-d) rats. Radioactivity associated with 7-(2-oxoethyl)guanine was taken as an indicator of DNA alkylation by [14C]VC. Analysis of 7-(2-oxoethyl)guanine revealed that in adult animals the amount of this alkylation product formed is increased by a second exposure to VC. About 5-fold of the amount of 7-(2-oxoethyl)guanine present in adults could be determined in liver DNA of young (11-d) animals exposed under the same exposure conditions. Our results suggest that the high sensitivity of young rats to VC-induced hepatocarcinogenesis can reasonably be explained by enhanced DNA-alkylation and by increased cellular proliferation at an early age.

The age-dependence of the induction of pre-neoplastic enzyme-altered hepatic foci was investigated. Rats were exposed (8 h/day, 7 days/week) to 2000 p.p.m. vinyl chloride (VC) either 'transplacentally' (exposure of pregnant females), or immediately after birth for different time intervals (5, 11, 17, 47, 83 days) or from an age of 7 or 21 days onwards. The animals were then kept without further treatment; livers were evaluated for ATPase-deficient foci at the age of 4 months. 'Transplacental' exposure and exposure from day 1 through 5 caused no increase over controls in ATPase-deficient foci, probably due to the lack of hepatocellular proliferation and the low rate of VC metabolism at this developmental stage. However, foci area was steeply increased when newborn rats were exposed for 11 and 17 days; but this was not further enhanced by a 47- or 83-day exposure. Only a few ATPase-deficient foci occurred when exposure started 21 days after birth. Exposure of adult rats did not result in more ATPase-deficient foci than were seen in untreated controls; control values could not be increased by a preceding partial hepatectomy. The results indicate that the induction of pre-neoplastic hepatocellular lesions in rats by VC is restricted to a well defined period (approximately day 7-21) in the early lifetime of the animals. This period of highest sensitivity is characterized by the beginning of rapid liver growth.

Comparative experiments with vinyl chloride and 2,2'-dichlorodiethyl ether confirm that chloroacetaldehyde, which is formed during metabolism of both compounds, binds covalently to proteins, but not to nucleic acids. The putative nucleic-acid-binding agent, chloroethylene oxide, is formed from vinyl chloride, not from 2,2'-dichlorodiethyl ether. The degree of potential carcinogenicity of epoxides formed from substituted ethylenes is considered to be determined by factors of stability/reactivity and by the rate of breakdown of epoxide and the rate of its formation. Hence, pharmacokinetic data are of considerable importance in assessing the comparative carcinogenic potencies of ethylene derivatives.

When rats are exposed to [14C]vinyl chloride in a closed system, the vinyl chloride present in the atmosphere equilibrates with the animals' organism within 15 min. The course of equilibration could be determined using rats which had been given 6-nitro-1,2,3-benzothiadiazole. This compound completely blocks metabolism of vinyl chloride. The enzymes responsible for metabolism of vinyl chloride are saturated at an atmospheric concentration of vinyl chloride of 250 ppm. Pharmacokinetic analysis shows that no significant cumulation of vinyl chloride or its major metabolites is to be expected on repeated administration of vinyl chlorides. This may be consistent with the theory that a reactive, shortly living, metabolite which occurs in low concentration only, may be responsible for the toxic effects of vinyl chloride.

The state of the literature led to a re-investigation of the alkylation products caused by vinyl chloride metabolites in DNA. When rat liver microsomes, an NADPH-regenerating system, DNA and [14C]vinyl chloride were incubated and, when the DNA was subsequently re-isolated and (enzymatically) hydrolyzed, chromatograms (on Aminex A-6) showed the presence of 1,N6-ethenodeoxyadenosine, 3,N4-ethenodeoxycytidine and 7-N-(2-oxoethyl)guanine (the product of hydrolysis of 7-N-(2-oxoethyl)-deoxyguanosine). By contrast, when rats were exposed to [1,2-14C]vinyl chloride and when the liver DNA of these rats was subjected to similar procedures, no radioactive 'etheno' derivatives could be detected, but a radioactive peak was eluted with 7-N-(2-oxoethyl)guanine. This peak could be transformed into 7-N-(2-hydroxyethyl)guanine; the chromatographic behaviour of which was identical to the reference compound used by Ostermann-Golkar et al. (Biochem. biophys. Res. Commun., 76 (1977) 259). Thus, it is concluded that the compound described by these authors, 7-N-(2-oxoethyl)guanine is in fact the major product of base alkylation in DNA after exposure to vinyl chloride.

The roles of chloroethylene oxide (CEO) and chloroacetaldehyde (CAA) in carcinogenicity of vinyl chloride (VC) have been studied by comparing biological effects of VC exposure with those of 2,2'-dichlorodiethylether (bis(chloroethyl)ether, BCEE) as a metabolic precursor of CAA. Biological end-points investigated were covalent protein binding, nucleic acid (RNA and DNA) alkylation and the potency of the two chemicals to induce preneoplastic ATPase-deficient foci in rat liver. After exposure of rats to [1-14C]BCEE, BCEE derived radioactivity was bound to liver proteins. Analysis of hydrolysates of liver RNA and DNA gave no indication for the formation of either 7-N-(2-oxoethyl)guanine, 1,N6-ethenoadenine or 3,N4-ethenocytosine residues within the nucleic acids. After application of VC, BCEE or chloroethanol [CE), also a precursor of CAA) to young rats, only animals exposed to VC developed preneoplastic hepatocellular ATPase-deficient foci. From these investigations it is concluded, that CEO (which is not formed during metabolism of BCEE and CE), not CAA, is the ultimate carcinogenic principle in VC carcinogenicity.

Rats have been exposed in a closed system to the chlorinated ethylenes vinyl chloride and trichloroethylene and to carbon tetrachloride as a reference compound. Data of uptake of the compounds, of urinary excretion of metabolites, and of exhalation after exposure show that the chlorinated ethylenes are metabolized much faster than carbon tetrachloride, probably due to their common ethylene structure. To eliminate differences in uptake, calculation of metabolites of the three compounds in tissues was based on the amount actually taken up by the animals. Vinyl chloride, trichloroethylene, and carbon tetrachloride showed irreversible binding of metabolites to tissue proteins, mainly of the liver. Irreversible protein binding of either of these compounds ranged within the same order of magnitude, if related to the amount of compound which had been taken up. Also, no differences in the relative portion of irreversibly bound metabolites were found after exposure to different atmospheric concentrations of the three compounds. As already shown for vinyl chloride, trichloroethylene is metabolized in vitro by rat liver microsomes in presence of NADPH-regenerating system to intermediates that irreversibly bind to proteins. Albumin (bovine and rabbit) was a preferred target for binding. In contrast to vinyl chloride, significant irreversible binding of trichloroethylene metabolites also occurred to non-SH-proteins (gamma-globulin, concanavalin A) and to polylysine. Hence it should be inferred that, unlike vinyl chloride, trichloroethylene metabolites not only bind to sulfhydryl groups but also, to a lesser extent, to free amino groups of proteins.