Mutagenic effects of vinyl chloride on Drosophila melanogaster with and without pretreatment with sodium phenobarbiturate

Treatment of Drosophila males with 1–20% vinyl chloride in air gave an increased frequency of complete and mosaic recessive lethals. Pretreatment with phenobarbiturate for 24 h caused an increase in the mutagenic effect of vinyl chloride. This indicates that the mixed-function oxygenase system can be induced in Drosophila in the same way as in mammals.     

The effect of three triterpene acids and sporidesmin on the enzyme activities of rat liver plasma membranes.

Vinyl chloride and vinyl benzene (styrene) are mutagenic in microbial tests, in Drosophila, in yeast, and in mammalian cells. Reports from various countries have shown an excess of chromosomal aberrations in the lymphocytes of workers exposed to vinyl chloride monomer when the workers were compared with controls. Workers occupationally exposed to styrene also revealed a clear increase in the rate of chromosome aberrations in their lymphocytes. Both chloroethylene oxide and styrene oxide, the primary biotransformation products of vinyl chloirde and styrene respectively, bind covalently to cellular macromolecules. Vinyl chloride is a carcinogen in both animals and man. Styrene is currently being tested in animals. These findings, the demonstration of mutagenic response via microbial and other test systems and with observations of significant excesses of chromosomal aberrations among workers exposed to these agents, raise scientific and health oriented concern about the possible genetic risks of vinyl chloride and styrene to man.     

Studies on metabolic activation of vinyl chloride in Drosophila melanogaster after pretreatment with phenobarbital and polychlorinated biphenyls.

It is known that vinyl chloride is metabolized by the mixed function oxygenase system in the liver to reactive mutagenic and carcinogenic metabolites. This metabolic activation was studied in Drosophila melanogaster by measuring the uptake of 14C from labelled vinyl chloride in different strains and with different pretreatments with phenobarbital and polychlorinated biphenyl (PCB) Clophen A50), well known inducers of cytochrome P-450. In accordance with previously obtained data on vinyl chloride induced sex linked recessive lethals, it was shown that pretreatment with inducers increased the uptake of labelled compound up to ten times. There was, however, a marked difference in response between the five strains used. In particular, the strain Hikone, known to be resistant to insecticides, had a comparatively high initial radioactivity from vinyl chloride without any pretreatment, but it was not or insignificantly inducible with phenobarbital or PCB. Crosses between Hikone and an inducible strain indicated essentially a dominance for the Hikone genotype. Tests on inducible strains showed the same response to phenobarbital by 2 h old larvae and adult male and females. Dimethylsulphoxide (DMSO) used as a solvent decreased both the initial uptake of 14C and particularly the induction by PCB. The use of Tween 80 as an emulsifier did not have such an effect. It is emphasized that the interstrain variation in metabolic activation and inducability has to be taken into consideration in order to optimize the use of Drosophila for mutagenicity testing. This variation also opens up new possibilities of analyzing the mixed function oxygenase system biochemically and genetically.     

Mutagenicity of vinyl chloride in man: comparison of chromosome aberrations with micronucleus and sister-chromatid exchange frequencies

The mutagenic effects of vinyl chloride monomer in man were studied in the lymphocyte culture with 3 methods: the chromosome aberration assay, the micronucleus assay and the sister-chromatid exchange method. Compared with control, values obtained by these tests are increased in workers occupationally exposed to vinyl chloride. In relation to non-smokers, smokers exposed to vinyl chloride show significant increases in sister-chromatid exchange frequencies. The problem of correlating the results of the chromosome aberration assay with micronucleus and sister-chromatid exchange frequencies is discussed.     

Aerobic vinyl chloride metabolism in Mycobacterium aurum L1.

Mycobacterium aurum L1, capable of growth on vinyl chloride as a sole carbon and energy source, was previously isolated from soil contaminated with vinyl chloride (S. Hartmans et al., Biotechnol. Lett. 7:383-388, 1985). The initial step in vinyl chloride metabolism in strain L1 is catalyzed by alkene monooxygenase, transforming vinyl chloride into the reactive epoxide chlorooxirane. The enzyme responsible for chlorooxirane degradation appeared to be very unstable and thus hampered the characterization of the second step in vinyl chloride metabolism. Dichloroethenes are also oxidized by vinyl chloride-grown cells of strain L1, but they are not utilized as growth substrates. Three additional bacterial strains which utilize vinyl chloride as a sole carbon and energy source were isolated from environments with no known vinyl chloride contamination. The three new isolates were similar to strain L1 and were also identified as Mycobacterium aurum.     

Aerobic vinyl chloride metabolism in Mycobacterium aurum L1.

Mycobacterium aurum L1, capable of growth on vinyl chloride as a sole carbon and energy source, was previously isolated from soil contaminated with vinyl chloride (S. Hartmans et al., Biotechnol. Lett. 7:383-388, 1985). The initial step in vinyl chloride metabolism in strain L1 is catalyzed by alkene monooxygenase, transforming vinyl chloride into the reactive epoxide chlorooxirane. The enzyme responsible for chlorooxirane degradation appeared to be very unstable and thus hampered the characterization of the second step in vinyl chloride metabolism. Dichloroethenes are also oxidized by vinyl chloride-grown cells of strain L1, but they are not utilized as growth substrates. Three additional bacterial strains which utilize vinyl chloride as a sole carbon and energy source were isolated from environments with no known vinyl chloride contamination. The three new isolates were similar to strain L1 and were also identified as Mycobacterium aurum.     

The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 in vitro.

In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome $\text{b}$₅ or NADPH-cytochrome $\text{c}$ reductase $\text{in vitro}$.     

The biological fate in rats of vinyl chloride in relation to its oncogenicity.

The main eliminative route for [14C]vinyl chloride after oral, i.v. or i.p. administration to rats is pulmonary; both unchanged vinyl chloride and vinyl chloride-related CO2 are excreted by that route and the other [14C] metabolites via the kidneys. After intragastric administration, pulmonary output of unchanged vinyl chloride is proportional to the logarithm of reciprocal dose. Excretion patterns after i.v. and i.p. injections are predictable from the characteristics of excretion following oral administration. Pulmonary excretion of unchanged vinyl chloride after oral dosing is complete within 3-4 h, but pulmonary elimination of CO2 and renal excretion of metabolites occupies 3 days. In comparison, 99% of a small i.v. dose is excreted unchanged within 1 h of injection; 80% within 2 min. The rate of elimination of a single oral doses of [14C]vinyl chloride is uninfluenced by up to 60 days' chronic dosing with the unlabelled substance. The distribution volume of vinyl chloride as displayed by whole-animal autoradiography agrees with deductions from excretion data. Small localization of 14C in the para-auricular region of appropriate sections occurs in sectioned tubules, belonging possibly to the Zymbal glands. Biotransformation of vinyl chloride into S-(2-chloroethyl) cysteine and N-acetyl-S-(2-chloroethyl) cysteine occurs through addition of cysteine, and biotransformation into: (i) chloroacetic acid, thiodiglycollic acid and glutamic acid, and (ii) into formaldehyde (methionine, serine), CO2 and urea is explicable in terms of an associative reaction with molecular O2 involving a singlet oxygen bonded transition state in dynamic equilibrium with a cyclic peroxide ground state. There is no evidence for chloroethylene oxide formation.Thiodiglycollic acid is the major metabolite of chloroacetic acid in rats; more than 60% of the dose. The interaction of vinyl chloride and of its primary metabolites with the intermediates of mammalian metabolism is discussed in relation to the oncogenicity of that substance.     

Chromosomal analysis in vinyl chloride exposed workers: Comparison of the standard technique with the sister-chromatid exchange technique

A group of 21 workers occupationally exposed to vinyl chloride and 6 controls were examined for the presence of chromosomal aberrations or sisterchromatid exchanges in their peripheral lymphocytes. These people comprised a second sampling from a group of exposed workers and controls first examined 18 months earlier. The vinyl chloride exposed workers showed levels of chromosomal aberrations elevated above those of the controls, but there was only a slight increase in sister-chromatid exchanges (per cell or per chromosome) and this increase was not statistically significant. Sister-chromatid exchanges (SCEs) were also examined from in vitro cultures of lymphocytes exposed in G₀/early G₁ and late G₁/early S phase to vinyl chloride, both with and without metabolic activation. There was no increase in SCEs in vitro without metabolic activation but there was a marked increase with metabolic activation and this increase was shown to be independent of cell-cylce phase. It thus was apparent that the small increases of SCEs in workers were not due to the inability of vinyl chloride to induce SCEs in human lymphocytes but were probably because of low exposures and SCE levels could have returned to normal relatively quickly after exposure. The present study suggested that the analysis of longer-living conventional chromosomal aberrations appeared to be a more sensitive monitor of exposure to vinyl chloride in exposed workers than the estimation of SCEs; however, it should be noted that in a 3rd sampling taken 24 months later the exposed workers had chromosomal aberration levels similar to the controls.     

Arthromyces ramosus peroxidase produces two chlorinating species

We previously reported that the hemes of horseradish peroxidase (HRP) and Arthromyces ramosus peroxidase (ARP) undergo vinyl and meso-carbon modifications when the enzymes oxidize chloride ion. Here we demonstrate for ARP that, although both modifications exhibit the same pH profile with an optimum at approximately pH 4.0, monochlorodimedone suppresses the vinyl but not meso-carbon modifications. Furthermore, meso-chlorination occurs when ARP reacts with exogenous HOCl, implicating an Fe(III)-O-Cl intermediate in the reaction. These results establish that (a) the chloro species involved in meso-modification differs from that which reacts with the vinyl groups, (b) equilibration of the vinyl modifying species (HOCl) into the medium occurs more rapidly than vinyl group modification, and (c) the oxidation of chloride by ARP produces two reactive species: HOCl, which adds to the heme vinyl but not meso-positions, and a distinct second species that adds to the meso-carbon.     

Bacterial degradation of vinyl chloride

Summary Mycobacterium L1 can grow on vinyl chloride as sole carbon and energy source. Application of this bacterium to remove vinyl chloride from waste gases is proposed. From air containing 1% vinyl chloride 93% of the vinyl chloride was removed by passing the air through a fermentor containing a growing population ofMycobacterium L1.     

Genetic toxicology of vinyl chloride—a review

Vinyl chloride (VC) is a colorless gas with a mild, sweet odor. It is extensively used in the production of vinyl chloride polymer, copolymer resin, packaging materials, wire and cable coatings as well as in industrial and laboratory intermediates. It is toxic and also carcinogenic in experimental animals. The wide human exposure to this compound in different industries throughout the world causes great concern for human health. In the present review an attempt has been made to evaluate and update the genotoxic effects of vinyl chloride based on the available literature.     

Liver-microsome-mediated formation of alkylating agents from vinyl bromide and vinyl chloride.

When a mixture of vinyl chloride/oxygen or vinyl bromide/air was passed through a mouse-liver microsomal system, volatile alkylating metabolites were trapped by reaction with excess 4-(4-nitrobenzyl)pyridine. The absorption spectra of the adducts, either from vinyl bromide or vinyl chloride, were identical with that obtained by reaction of chloroethylene oxide with 4-(4-nitrobenzyl) pyridine. Chloroethylene oxide decomposes in aqueous solution with a half-life of 1.6 minutes. After reaction of chloroethylene oxide and 2-chloroacetaldehyde with adenosine and Sephadex chromatography the binding products were compared with those formed in the presence of vinyl chloride, mouse-liver microsomes and adenosine. A common product of these reactions was tentatively characterized as 3-β-ribofuranosyl-imidazo-[2,1-i]purine.     

Polymorphisms in glutathione S-transferases in French vinyl chloride workers.

The authors have recently demonstrated a significant gene-environment interaction between vinyl chloride exposure and polymorphisms in the DNA repair protein XRCC1 on the occurrence of mutant p53 biomarkers of vinyl chloride-induced genetic damage. The aim of this study was to examine the polymorphisms in the glutathione S-transferases (GSTs) as potential modifiers of this relationship, since these enzymes may be involved in the phase II metabolism of the reactive intermediates of vinyl chloride. A cohort of 211 French vinyl chloride workers was genotyped for common polymorphisms in GSTM1, GSTT1 and GSTP1. Although no independent, statistically significant effect of these polymorphisms on the occurrence of the mutant p53 biomarker was found, the null GSTM1 and null GSTT1 polymorphisms were found to interact with the XRCC 1 polymorphism to increase the occurrence of the biomarker such that, for example, workers with at least one variant XRCC1 allele who were null for both GSTM1 and GSTT1 had a significant odds ratio for the biomarker (OR =8.4, 95% CI = 1.3 54.0) compared with workers who were wild-type for all alleles, controlling for potential confounders including cumulative vinyl chloride exposure.     

Ethanol inhibition of vinyl chloride metabolism in isolated rat hepatocytes.

Isolated rat liver cells convert [14C]vinyl chloride into non-volatile metabolites. The metabolism is not increased by in vivo pretreatment with phenobarbital. It is sensitive to inhibition by ethanol, which at a concentration of 4 mM inhibits vinyl chloride metabolism to 50% in hepatocyte suspensions. The metabolic activity is NADPH-dependent and is localized in the microsomal fraction of the liver. The enzyme is also strongly inhibited by tetrahydrofuran, indicating that it could be identical to an ethanol-inducible cytochrome P-450 described in the literature [1].     

Polymorphisms in glutathione S-transferases in French vinyl chloride workers

The authors have recently demonstrated a significant gene–environment interaction between vinyl chloride exposure and polymorphisms in the DNA repair protein XRCC1 on the occurrence of mutant p53 biomarkers of vinyl chloride-induced genetic damage. The aim of this study was to examine the polymorphisms in the glutathione S-transferases (GSTs) as potential modifiers of this relationship, since these enzymes may be involved in the phase II metabolism of the reactive intermediates of vinyl chloride. A cohort of 211 French vinyl chloride workers was genotyped for common polymorphisms in GSTM1, GSTT1 and GSTP1. Although no independent, statistically significant effect of these polymorphisms on the occurrence of the mutant p53 biomarker was found, the null GSTM1 and null GSTT1 polymorphisms were found to interact with the XRCC1 polymorphism to increase the occurrence of the biomarker such that, for example, workers with at least one variant XRCC1 allele who were null for both GSTM1 and GSTT1 had a significant odds ratio for the biomarker (OR=8.4, 95% CI=1.3–54.0) compared with workers who were wild-type for all alleles, controlling for potential confounders including cumulative vinyl chloride exposure.     

Aerobic biodegradation of vinyl chloride in groundwater samples

Studies were conducted to examine the biodegradation of 14C-labeled vinyl chloride in samples taken from a shallow aquifer. Under aerobic conditions, vinyl chloride was readily degraded, with greater than 99% of the labeled material being degraded after 108 days and approximately 65% being mineralized to 14CO2.     

Aerobic biodegradation of vinyl chloride in groundwater samples.

Studies were conducted to examine the biodegradation of 14C-labeled vinyl chloride in samples taken from a shallow aquifer. Under aerobic conditions, vinyl chloride was readily degraded, with greater than 99% of the labeled material being degraded after 108 days and approximately 65% being mineralized to 14CO2.     

Aerobic mineralization of vinyl chloride by a bacterium of the order Actinomycetales

A gram-positive branched bacterium isolated from a trichloroethylene-degrading consortium mineralized vinyl chloride in growing cultures and cell suspensions. Greater than 67% of the [1,2-14C]vinyl chloride was mineralized to carbon dioxide, with approximately 10% of the radioactivity appearing in cell biomass and another 10% appearing in 14C-aqueous-phase products.     

Vinyl chloride mutagenesis in Drosophila melanogaster.

In inhalation experiments, Drosophila males were exposed to vinyl chloride at concentrations of 200, 850, 10,000 30,000 or 50,000 ppm for 2 days, and to 30 or 850 ppm for 17 days. VCM was mutagenic in the recessive-lethal test both after short-term and long-term exposures. The lowest effective concentration (LEC) was 850 ppm after 2 day exposure, and this value could be lowered to 30 ppm by prolonging the exposure time to 17 days. With the concentration levels tested, the mutation frequency increased with concentrations and reached a plateau at 10,000 ppm. This indicates a substrate saturation effect. In contrast with the recessive lethal assay, negative results were obtained when tests on dominant lethals, translocations, entire and partial sex-chromosome loss were carried out with VCM at 30,000 ppm for 2 days. This finding of a false negative seems a logical consequence of the observed saturation effect, and strengthens the concept that there exist two effective concentrations for point mutations vs the induction of chromosome breakage events. Vinyl chloride monomer provides another example to support our view that chromosome breakage is not a reliable measure of mutagenic activity.