Vinyl Chloride Metabolites Research Articles

Covalent protein binding of vinyl chloride metabolites during co-incubation of freshly isolated hepatocytes and hepatic sinusoidal cells of rats.

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.

Calculated properties of some possible vinyl chloride metabolites

AbstractThere is considerable evidence indicating that the carcinogenic action of vinyl chloride involves metabolic conversion to the epoxide (chlorooxirane) as the initial step. In order to learn more about its subsequent behavior, we have computed structures, energies and other properties for two different protonated forms of the epoxide, and also for two possible rearrangement products, chloroacetaldehyde and acetyl chloride. An ab initio SCF‐MO procedure (GAUSSIAN 70) was used. Oxygen protonation is found to weaken both CO bonds, the effect being greater for the bond involving the carbon bearing the chlorine. Chlorine protonation leads to a marked weakening of the CCl bond; this suggests a possible loss of HCl, leaving behind a carbonium ion (and possible alkylating agent or rearrangement precursor). Thus, while CO bond breaking is doubtless an important reaction pathway for chlorooxirane, our results indicate that attention should also be focused upon the CCl bond; its rupture may conceivably be a key step in the biological action of vinyl chloride.

Chloroacetaldehyde-treated ribo- and deoxyribopolynucleotides. 1. Reaction products.

The in vitro reaction of the vinyl chloride metabolite chloroacetaldehyde (CAA) with cytosine and adenine residues in ribo- and deoxyribopolynucleotides leads to the formation of the relatively stable hydrated etheno derivatives 3,N4-(N4-alpha-hydroxyethene)adenine (epsilon C . H2O) and 1,N6-(N6-alpha-hydroxyethene)cytosine (epsilon A . H2O). Under physiological conditions the hydrates are slowly converted to 3,N4-ethenocytosine (epsilon C) and 1,N6-ethenoadenine (epsilon A). The half-life at pH 7.25 of epsilon C . H2O in poly(rC) is 4.9 h at 50 degrees C and of epsilon A . H2O in poly(rA) is 1.4 h at 37 degrees C. These dehydration rates in polymers are similar to those for hydrates in monomers. The reactivity of A and C residues is greatly suppressed in double-stranded polymers. Adenine residues are about 10 times less reactive in poly(rA) . poly(rU) than A in single-stranded polymers. Under similar reaction conditions no reaction of C residues in poly(rC) . poly(rG) was detected. In vinyl chloride exposed cells, where CAA is formed, the cyclic etheno derivatives of A and C are likely to occur preferentially in single-stranded regions of nucleic acids, with the hydrate forming a major proportion of the modification.

The effect of repeated vinyl chloride exposure on rat hepatic metabolizing enzymes

Sprague-Dawley rats were exposed to 2.8% vinyl chloride for 2 (70 hr), 4 (140 hr), and 6 (210 hr) weeks to determine the sequential biochemical changes related to the oxidation and detoxification ability of hepatic tissue. Glutathione- S-transferase(s) activity using 1,2-epoxy-( p-nitrophenoxy)propane and p-nitrobenzyl chloride as substrates was elevated 17 to 24, 28, and 35 to 42% after 2, 4, and 6 weeks of exposure, respectively, suggesting enzyme(s) induction. Reduced glutathione, the major substrate required to conjugate the toxic metabolites of vinyl chloride, was also consistently elevated. Similarly, the activity of glutathione reductase, the enzyme necessary for the regeneration of reduced glutathione from its oxidized form, was also increased following vinyl chloride exposure. Cytochromes P-450, the major protein involved with vinyl chloride metabolism, was reduced after vinyl chloride exposure, confirming reports of others that vinyl chloride metabolites destroy P-450. No abnormalities of standard clinical biochemical blood tests of liver function were found during 6 weeks of vinyl chloride exposure. The only consistent ultrastructural modification was the dilation of endoplasmic reticulum. The biochemical and ultrastructural alterations could reflect early hepatocellular adaptation to vinyl chloride exposure.

Reactions of vinyl chloride with RNA and DNA of various mouse tissues in vivo.

The irreversible binding of 14C-vinyl chloride metabolites to RNA and DNA of mouse brain, lung, liver, kidney, spleen, pancreas, and testes after a single i.p. injection has been studied. Hydrolysates of nucleic acids from selected organs were separated on Aminex A6 for quantitation of alkylation products. Radioactivity in nucleic acids was registered in all of the studied organs with the exception of brain. RNA from spleen, pancreas and liver, and DNA from spleen and liver contained the highest amounts of radioactivity. In nucleic acids from spleen and pancreas, both organs of high metabolic activity, the entire radioactivity was found metabolically incorporated as C1-fragments. In RNA of kidney and liver, a large part of the radioactivity was also present as incorporated C1-fragments, but 3,N4-ethenocytidine (in kidney) as well as 1,N6-ethenoadenosine and 1,N6-ethenoadenine (in kidney and liver) were identified as alkylation products. In liver DNA, incorporation of C1-fragments was insignificant, indicating different interactions of vinyl chloride metabolites (C1-fragments and alkylating products) with RNA and DNA. The elution profiles of radioactivity in hydrolysates of liver DNA were dominated by an alkylation product of unknown structure (probably a derivative of deoxyguanosine). Possibly, 1,N6-ethenodeoxyadenosine and 1,N6-ethenoadenine were also present in liver DNA. The results are consistent with the ability of vinyl chloride to act as a multipotent carcinogen by alkylation of DNA in several tissues.

Inhalation pharmacokinetics based on gas uptake studies. III. A pharmacokinetic assessment in man of "peak concentrations" of 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.

DNA alkylation by vinyl chloride metabolites: Etheno derivatives or 7-alkylation of guanine?

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, and 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, N 6- ethenodeoxyadenosine, 3, N 4- 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.

Rat hepatic vinyl chloride metabolites induce gene conversion in the yeast strain D7RAD in vitro and in vivo

We have tested the genetic activity of gaseous vinyl chloride in vitro and in vivo using the gene-conversion system trp5-12/ trp5-27 → TRP +) in the yeast strain D7 RAD. To induce, in vitro, TRP + convertants with 2.5% gaseous vinyl chloride, a rat-liver microsomal system for metabolic activation of the vinyl chloride and dividing yeast cells are required. Neither a deficiency in excision repair ( rad3) nor in the error-prone repair pathway ( rad6) increased the vinyl-chloride-induced conversion frequencies compared with the repair-competent D7 RAD strain. When logarithmically growing cells of the D7 RAD strain were injected intravenously into male Wistar rats which inhaled 1% vinyl chloride in air for 24 h, a significant enhancement of the TRP + conversion frequencies was found compared with that in cells re-isolated from untreated rats. These results indicate that vinyl chloride metabolites from the metabolizing hepatocytes diffuse into yeast cells, which accumulate in the liver capillaries. This supports the hypothesis that the endothelial cells of the liver sinuses, which have hardly any metabolic activity, but give rise to vinyl-chloride-induced hemangiotheliomas (rate type of liver tumor), are transformed by diffusible metabolites of the procarcinogen vinyl chloride.

Studies on the miscoding properties of 1,N6-ethenoadenine and 3,N4-ethenocytosine, DNA reaction products of vinyl chloride metabolites, during in vitro DNA synthesis.

1,N6-Ethenoadenine (epsilon A) and 3,N4-ethenocytosine (epsilon C) are formed when electrophilic vinyl chloride (VC) metabolites, chloroethylene oxide (CEO) or chloroacetaldehyde (CAA) react with adenine and cytosine residues in DNA. They were assayed for their miscoding properties in an in vitro system using Escherichia coli DNA polymerase I and synthetic templates prepared by reaction of poly(dA) and poly(dC) with increasing concentrations of CEO or CAA. Following the introduction of etheno groups, an increasing inhibition of DNA synthesis was observed. dGMP was misincorporated on CAA- or CEO-treated poly(dA) templates and dTMP was misincorporated on CAA- or CEO-treated poly(dC) templates, suggesting that epsilon A and epsilon C may miscode. The error rates augmented with the extent of reaction of CEO or CAA with the templates. Base-pairing models are proposed for the epsilon A.G. and epsilon C.T pairs. The potentially miscoding properties of epsilon A and epsilon C may explain why metabolically-activated VC and its reactive metabolites specifically induce base-pair substitution mutations in Salmonella typhimurium. Promutagenic lesions may represent one of the initial steps in VC- or CEO-induced carcinogenesis.

Modification of deoxyguanosine by chloroethylene oxide.

Reaction of deoxyguanosine in glacial acetic acid with chloroethylene oxide, a proposed reactive metabolite of vinyl chloride, led to a single, strongly fluorescent product in nearly quantitative yield. The u.v. spectra indicated alkylation of N-7 of guanine, which was confirmed following reduction of the reaction product by sodium borohydride to 7-(2-hydroxyethyl)guanine, and the synthesis of the same modified guanine via a stereoselective 7-N hydroxy alkylation using 2,3-epoxy-1-propanol. In agreement with the expected structure 7-(2-oxoethyl)guanine reacted with the carbonyl specific reagent 2,4-dinitrophenylhydrazine (2,4-DNPH). However, its i.r. and proton n.m.r. spectra did not support the existence of a simple aldehyde group. Moreover, the 2,4-dinitrophenylhydrazone was labile, 7-(2-oxoethyl)guanine being produced when excess 2,4-DNPH was removed. This instability was interpreted as being due to the reversible formation of a hemiacetal ring between O6 of the guanine residue and the aldehyde carbon of the 2-oxoalkyl group resulting in O6,7-(1'-hydroxyethano)guanine. This conformation was supported by the occurrence in field desorption mass spectra of the ions of m/e = 175 and 292 which are interpreted as O6,7-ethenoguanine and O6,7-ethenodeoxyguanosine resulting from the elimination of H2O of the hydroxyethano residue. O6,7-(1'-hydroxyethano)guanine might be expected to cause faulty base pairing during replication of DNA, which may be the molecular basis of the carcinogenicity of vinyl chloride.

Immunotoxicologic studies with vinyl chloride in rabbits and mice

Our previous studies in mice indicated that the exposure to vinyl chloride (VC) produced a state of immunostimulation. The metabolism of VC was an important factor in this phenomenon. The present paper describes the effects of VC exposure on induced immunologic responses in rabbits. No consistent effect of VC exposure was noticed on skin reactivity to tuberculin or serum anti-tetanus titers in sensitized rabbits. Vinyl chloride produced no change in the number of antibody secreting cells in the lymph nodes of immunized rabbits. An increase in the spontaneous splenic lymphocyte transformation in immunized rabbits was observed when the animals were exposed to VC. Two known metabolites of VC, namely thiodiglycolic acid and N-acetyl- S-(hydroxyethyl)-cysteine produced little or no effect when added to mouse splenic lymphocyte cultures in vitro but in vivo administration of thiodiglycolic acid produced apparent immune stimulation in mice. The study indicated that although VC may cause an apparent enhancement of immune reactivity, it does not alter the immunologic response to simultaneously administered antigens.

Effects of inducers on the in vivo covalent binding of a vinyl chloride metabolite to liver fractions

Effects of inducers on the in vivo covalent binding of a vinyl chloride metabolite to liver fractions

Formation and inactivation of a chemically reactive metabolite of vinyl chloride

The mechanisms responsible for, and protecting against, metabolic activation of vinyl chloride were investigated in the rat. When [ 14C]vinyl chloride was incubated with hepatic microsomes, a 14C-labeled material became irreversibly bound to microsomal proteins; binding required NADPH, was decreased by CO, SKF 525-A, or glutathione, and was increased by 1,1,1-trichloropropene-2,3-oxide (TCPO). Inhalation of a 5% vinyl chloride atmosphere decreased hepatic cytochrome P-450 and glutathione. Pretreatment of the animals with DDT or phenobarbital (1) increased in vitro irreversible binding to microsomal proteins measured in the presence, but not in the absence, of TCPO and increased the in vivo loss of cytochrome P-450 during inhalation of vinyl chloride, but (2) decreased the in vitro irreversible binding to 10,000 g supernatant proteins and the in vivo loss of glutathione during inhalation of vinyl chloride. These results are consistent with the views that (1) vinyl chloride is activated by cytochrome P-450 into its chemically reactive epoxide, (2) the epoxide may be inactivated by epoxide hydrase or by binding to glutathione, (3) pretreatment with DDT or phenobarbital increases both the formation rate of the epoxide and its inactivation rate by epoxide hydrase, and (4) cytochrome P-450 destruction is related to the formation rate of the expoxide, whereas glutathione depletion seems related to only that fraction of the formed epoxide which has escaped inactivation by microsomal epoxide hydrase and has diffused in the cytosol.

IMMUNOLOGIC EFFECTS OF VINYL CHLORIDE IN MICE

Male CD-1 mice were exposed to 10, 100 or 1000 ppm vinyl chloride (VC) for 2--8 weeks at 6 hr/day, 5 days/week. A slight increase in the spleen weight of mice was noted at the highest exposure level. Spleens were obtained from these animals (4 mice/group after 2, 4, and 8 weeks of exposure) and their lymphocytes cultured in vitro with or without the presence of phytomitogens, phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Relative blast formation and the DNA synthesis was measured by the incorporation of 3H-thymidine in the cultured cells. The response of splenic lymphocytes to the phytomitogens was increased several-fold by VC exposure. The effects were apparent at 1000 ppm VC after 2 weeks of exposure and at all levels of VC exposure after 4--8 weeks. The effects were generally more pronounced at 100 ppm VC exposure than those at 1000 ppm. In vitro culture of splenic lymphocytes from control or VC-exposed mice in the VC atmosphere did not show an enhancement of blast formation. Alteration of VC metabolism during the VC exposure in vivo yielded results that indicated that metabolites of VC may be responsible for the stimulation of lymphocyte transformation observed in splenic cultures.

Metabolism of [ 14C]- and [ 36Cl]-labeled vinyl chloride in vivo and in vitro

Label from [ 14C]vinyl chloride was covalently bound to protein and nucleic acids in vivo and in vitro in the presence of rat liver microsomal fractions or highly purified cytochrome P-450 and NADPH-cytochrome P-450 reductase preparations. The ratio of bound to total non-volatile metabolites increased in going from the in vivo to the microsomal to the purified system. [ 36Cl]vinyl chloride was metabolized by microsomes and highly purified systems: no label was bound and most could be accounted for as chloride ion. Phenobarbital pretreatment of rats did not induce total metabolism of vinyl chloride in vivo at either 10 or 250 ppm exposure levels; however, binding to protein and RNA was enhanced at the 10 ppm but not the 250 ppm level. Phenobarbital pretreatment increased the in vitro microsomal conversion of vinyl chloride to both total and bound metabolites. A sizeable fraction of the label of [ 14C]vinyl chloride metabolized in vivo was recovered in the microsomal fraction of the liver, but sodium dodecyl sulfate polyacrylamide gel electrophoresis of in vitro incubations indicated that the metabolites were distributed among many microsomal proteins and not localized to cytochrome P-450. Evidence was obtained for the metabolism of the suspected vinyl chloride metabolite chloroethylene oxide by microsomal epoxide hydratase. However, the epoxide hydratase inhibitor 3,3,3-trichloropropylene oxide, which blocks the microsomal degradation of chloroethylene oxide, did not enhance the level of vinyl chloride bound to either protein or adenosine.

Vinyl chloride and trichloroethylene: Comparison of alkylating effects of metabolites and induction of preneoplastic enzyme deficiencies in rat liver

[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.

In vivo trapping of a vinyl chloride metabolite by means of 3,4-dichlorobenzenethiol.

The successful trapping of a vinyl chloride metabolite and a metabolite of 2,2′ dichrorodiethylether is reported. The reaction products are excreted as S-2 (3,4 dichlorothiophenyl) acetic acid in the urine of rats. This has been confirmed by GC-MS measurements.

Formation of 3,N4-ethenocytidine moieties in RNA by vinyl chloride metabolites in vitro and in vivo

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.

Comparative Mammalian Metabolism of Vinyl Chloride and Vinylidene Chloride in Relation to Oncogenic Potential

Elucidation of the role of vinyl chloride metabolites in the various reaction sequences which comprise the metabolic pathway, including the interaction of reactive metabolities with some purine and pyrimidine residues of target-organ DNA, provides some explanation for the (oncogenic) properties associated with the original substance. Comparative investigation of the biological fate of vinylidene chloride reveals an agent of low oncogenic potential which is likely to be damaging only under special circumstances, and species differences which suggest that the mouse is more susceptible than the rat towards vinylidene chloride oncogenicity.

Alkylation of RNA by vinyl chloride metabolites in vitro and in vivo: Formation of 1- N6-etheno-adenosine

Rat liver microsomes were incubated with NADPH, 1,2-[ 14C]vinyl chloride and poly-adenosine. The latter was reisolated from the incubations and hydrolyzed. The radioactivity, originating from [ 14C]vinyl chloride, which was irreversibly attached to the poly-adenosine was confined to 1- N 6-etheno-adenosine (3β-ribofuranosyl-imidazo[2,1,i] purine). When rats were exposed to 1,2-[ 14C]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.