Alleviating Chlorinated Alkane Inhibition on Dehalococcoides to Achieve Detoxification of Chlorinated Aliphatic Cocontaminants.

Cocontamination by multiple chlorinated solvents is a prevalent issue in groundwater, presenting a formidable challenge for effective remediation. Despite the recognition of this issue, a comprehensive assessment of microbial detoxification processes involving chloroethenes and associated cocontaminants, along with the underpinning microbiome, remains absent. Moreover, strategies to mitigate the inhibitory effects of cocontaminants have not been reported. Here, we revealed that chloroform exhibited the most potent inhibitory effects, followed by 1,1,1-trichloroethane and 1,1,2-trichloroethane, on dechlorination of dichloroethenes (DCEs) in Dehalococcoides-containing consortia. The observed inhibition could be attributed to suppression of biosynthesis and enzymatic activity of reductive dehalogenases and growth of Dehalococcoides. Notably, cocontaminants more profoundly inhibited Dehalococcoides populations harboring the vcrA gene than those possessing the tceA gene, thereby explaining the accumulation of vinyl chloride under cocontaminant stress. Nonetheless, we successfully ameliorated cocontaminant inhibition by augmentation with Desulfitobacterium sp. strain PR owing to its ability to attenuate cocontaminants, resulting in concurrent detoxification of DCEs, trichloroethanes, and chloroform. Microbial community analyses demonstrated obvious alterations in taxonomic composition, structure, and assembly of the dechlorinating microbiome in the presence of cocontaminants, and introduction of strain PR reshaped the dechlorinating microbiome to be similar to its original state in the absence of cocontaminants. Altogether, these findings contribute to developing bioremediation technologies to clean up challenging sites polluted with multiple chlorinated solvents.

Effects of toluene, acrolein and vinyl chloride on motor activity of Drosophila melanogaster.

The data generated by current high-throughput assays for chemical toxicity require information to link effects at molecular targets to adverse outcomes in whole animals. In addition, more efficient methods for testing volatile chemicals are needed. Here we begin to address these issues by determining the utility of measuring behavioral responses of Drosophila melanogaster to airborne volatile organic compounds (VOCs) as a potential model system for discovering adverse outcome pathways and as a method to test for toxicity. In these experiments, we measured motor activity in male and female flies to determine concentration-effect functions for three VOCs that differ in their mode of action: toluene, a narcotic; acrolein, an irritant; and vinyl chloride, a hepatocarcinogen. These experiments were conducted in Flyland, an outbred population of flies derived from 40 lines of the Drosophila Genetics Reference Panel (DGRP) (Mackay et al., 2012), in preparation for subsequent experiments with individual lines of the DGRP. Systematic, concentration-related changes in activity were observed with toluene, but not with acrolein; high concentrations of vinyl chloride reduced activity by a small amount. Despite higher activity levels in males than in females under control conditions, the sexes were equally sensitive to toluene. Transient increases in activity at the onset and offset of exposure to toluene and vinyl chloride suggested that the flies detected changes in air quality at concentrations that did not persistently suppress activity. The effects and potency of toluene are consistent with those observed in rodents. The lack of clear concentration-related changes in response to acrolein and vinyl chloride shows limitations of this method is for screening toxicity attributed to VOCs. This abstract does not reflect U.S. EPA policy.

The novel molecule 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate inhibits phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signalling through JNK activation in cancer cells.

Screening a compound library of compound 48/80 analogues, we identified 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate (E1) as a novel inhibitor of the phosphoinositide 3-kinase/Akt pathway. In order to determine the mechanism of action of E1, we analysed the effect of E1 on components of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. E1 demonstrated dose-dependent and time-dependent repression of Akt and mTOR activity in prostate and breast cancer cell lines, PC-3 and MCF-7, respectively. Inhibition of Akt and mTOR activity by E1 also coincided with increased c-Jun NH2-terminal kinase (JNK) phosphorylation. However, the mode of action of E1 is different from that of the mTOR inhibitor rapamycin. Proliferation and cell cycle analysis revealed that E1 induced cell cycle arrest and cell death in PC-3 and MCF-7 cells. Moreover, pretreatment of cancer cells with the JNK inhibitor SP600125 abolished the repression of Akt and mTOR activity by E1, indicating that the inhibition of Akt and mTOR by E1 is mediated through JNK activation. Consistently, E1 repressed Akt and mTOR activity in wild-type and p38-null mouse embryonic fibroblasts (MEFs), but not in MEFs lacking JNK1/2, and JNK-null MEFs were less sensitive to the antiproliferative effects of E1. We further showed that E1 can function cooperatively with suboptimal concentrations of paclitaxel to induce cell death in PC-3 and MCF-7 cells. Taken together, these data suggest that E1 induces cancer cell death through the JNK-dependent repression of Akt and mTOR activity and may provide a valuable compound for further development and research.

Long-term aerobic cometabolism of a chlorinated solvent mixture by vinyl chloride-, methane- and propane-utilizing biomasses.

The aerobic cometabolic biodegradation of a mixture of chlorinated aliphatic hydrocarbons (CAHs) including vinyl chloride (VC), cis- and trans-1,2-dichloroethylene (cis-DCE, trans-DCE), trichloroethylene (TCE), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA) was investigated at both 25 and 17 degrees C by means of bioaugmented and non-bioaugmented sediment-groundwater slurry microcosm tests. The goals of the study were (i) to study the long-term aerobic biodegradation of a CAH mixture including a high-chlorinated solvent (1,1,2,2-TeCA) generally considered non-biodegradable in aerobic conditions; (ii) to investigate the efficacy of bioaugmentation with two types of internal inocula obtained from the indigenous biomass of the studied site; (iii) to identify the CAH-degrading bacteria. VC, methane and propane were utilized as growth substrates. The non-bioaugmented microcosms were characterized, at 25 degrees C, by an average 18-day lag-time for the direct metabolism of VC (accompanied by the cometabolism of cis- and trans-DCE) and by long lag-times (36-264 days) for the onset of methane or propane utilization (associated with the cometabolism of the remaining CAHs). In the inoculated microcosms the lag-phases for the onset of growth substrate utilization and CAH cometabolism were significantly shorter (0-15 days at 25 degrees C). Biodegradation of the 6-CAH mixture was successfully continued for up to 410 days. The low-chlorinated solvents were characterized by higher depletion rates. The composition of the microbial consortium of a propane-utilizing microcosm was determined by 16s rDNA sequencing and phylotype analysis. To the best of our knowledge, this is the first study that documents the long-term aerobic biodegradation of 1,1,2,2-TeCA.

Sequence-specific p53 gene damage by chloroacetaldehyde and its repair kinetics in Escherichia coli.

Oxidative stress and certain environmental carcinogens, e.g. vinyl chloride and its metabolite chloroacetaldehyde (CAA), introduce promutagenic exocyclic adducts into DNA, among them 1,N(6)-ethenoadenine (epsilonA), 3,N(4)-ethenocytosine (epsilonC) and N(2),3-ethenoguanine (epsilonG). We studied sequence-specific interaction of the vinyl-chloride metabolite CAA with human p53 gene exons 5-8, using DNA Polymerase Fingerprint Analysis (DPFA), and identified sites of the highest sensitivity. CAA-induced DNA damage was more extensive in p53 regions which revealed secondary structure perturbations, and were localized in regions of mutation hot-spots. These perturbations inhibited DNA synthesis on undamaged template. We also studied the repair kinetics of CAA-induced DNA lesions in E. coli at nucleotide resolution level. A plasmid bearing full length cDNA of human p53 gene was modified in vitro with 360 mM CAA and transformed into E. coli DH5alpha strain, in which the adaptive response system had been induced by MMS treatment before the cells were made competent. Following transformation, plasmids were re-isolated from transformed cultures 35, 40, 50 min and 1-24 h after transformation, and further subjected to LM-PCR, using ANPG, MUG and Fpg glycosylases to identify the sites of DNA damage. In adaptive response-induced E. coli cells the majority of DNA lesions recognized by ANPG glycosylase were removed from plasmid DNA within 35 min, while MUG glycosylase excised base modifications only within 50 min, both in a sequence-dependent manner. In non-adapted cells resolution of plasmid topological forms was perturbed, suggesting inhibition of one or more bacterial topoisomerases by unrepaired epsilon-adducts. We also observed delayed consequences of DNA modification with CAA, manifesting as secondary DNA breaks, which appeared 3 h after transformation of damaged DNA into E. coli, and were repaired after 24 h.

Association of hepatitis virus infection, alcohol consumption and plasma vitamin A levels with urinary 8-hydroxydeoxyguanosine in chemical workers.

Urinary 8-hydroxydeoxyguanosine (8-OHdG) DNA adduct has been used as a biomarker in epidemiological studies. However, the determinants for urinary 8-OHdG have not been clearly identified. We tested urinary 8-OHdG levels in 205 male workers who had been exposed to vinyl chloride monomer (VCM). Epidemiological information was obtained by an interviewer-administered questionnaire. Hepatitis B surface antigen (HBsAg) and anti-hepatitis C antibody (anti-HCV) were also determined by immunoassay. Plasma antioxidants including Vitamins A and E, alpha- and beta-carotenes were assayed by high performance liquid chromatography. Median of urinary 8-OHdG level was 9.8 ng/mg creatinine (range, 1.4-60.1). Multiple linear regression analysis showed that alcohol drinkers had higher urinary 8-OHdG than those who did not, but there was no dose-response between the amount of alcohol consumption and urinary 8-OHdG. Workers with positive HBsAg, anti-HCV and elevated plasma Vitamin A level were independently associated with higher levels of urinary 8-OHdG, whereas age, smoking, body mass index, plasma alpha- and beta-carotenes, Vitamin E levels, or VCM exposure did not show such an association. The results suggest that active inflammation of hepatitis B and C, alcohol consumption and higher Vitamin A level can induce oxidative stress. Thus, we conclude that potential determinants need to be considered in epidemiological studies when urinary 8-OHdG is used as a biomarker.

Simultaneous quantitation of N(2),3-ethenoguanine and 1,N(2)-ethenoguanine with an immunoaffinity/gas chromatography/high-resolution mass spectrometry assay.

We have previously described an immunoaffinity/gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (IA/GC/ECNCI-HRMS) assay for quantitation of the promutagenic DNA adduct N(2),3-ethenoguanine (N(2),3-epsilonGua) in vivo. Here we present an expanded assay that allows simultaneous quantitation of its structural isomer, 1,N(2)-ethenoguanine (1,N(2)-epsilonGua), in the same DNA sample. 1,N(2)-epsilonGua and N(2),3-epsilonGua were purified together from hydrolyzed DNA using two immobilized polyclonal antibodies. GC/ECNCI-HRMS was used to quantitate the 3,5-bis(pentafluorobenzyl) (PFB) derivative of each adduct against an isotopically labeled analogue. Selected ion monitoring was used to detect the [M - 181](-) fragments of 3,5-(PFB)(2)-N(2),3-epsilonGua and 3,5-(PFB)(2)-[(13)C(4),(15)N(2)]-N(2),3-epsilonGua and the [M - 201](-) fragments of 3,5-(PFB)(2)-1,N(2)-epsilonGua and 3,5-(PFB)(2)-[(13)C(3)]-1,N(2)-epsilonGua. The demonstrated limits of quantitation in hydrolyzed DNA were 7.6 fmol of N(2),3-epsilonGua and 15 fmol of 1,N(2)-epsilonGua in approximately 250 microg of DNA, which corresponded to 5.0 N(2),3-epsilonGua and 8.7 1,N(2)-epsilonGua adducts/10(8) unmodified Gua bases, respectively. 1,N(2)-epsilonGua was found to be the predominant ethenoguanine adduct formed in reactions of lipid peroxidation products with DNA. The respective ratios of 1,N(2)-epsilonGua to N(2),3-epsilonGua were 5:1 and 38:1 when calf thymus DNA was treated with ethyl linoleate or 4-hydroxynonenal, respectively, under peroxidizing conditions. Only N(2),3-epsilonGua was detected in DNA treated with the vinyl chloride (VC) metabolite 2-chloroethylene oxide and in hepatocyte DNA from rats exposed to 1100 ppm VC for 4 weeks (6 h/day for 5 days/week). These data suggest that 1,N(2)-epsilonGua plays a minor role relative to N(2),3-epsilonGua in VC-induced carcinogenesis, but that 1,N(2)-epsilonGua may be formed to a larger extent from endogenous oxidative processes.

Deficiency of N-methylpurine-DNA-glycosylase expression in nonparenchymal cells, the target cell for vinyl chloride and vinyl fluoride.

The ability to repair promutagenic damage resulting from exposure to carcinogens is a critical factor in determining quantitative relationships in carcinogenesis, including the target cell for neoplasia. One major pathway for the repair of alkylating agent-induced DNA damage involves removal of alkylated bases by N-methylpurine-DNA-glycosylase (MPG), the first enzyme in base excision repair. We have measured the expression level of MPG mRNA in liver, lung, and kidney of Sprague-Dawley rats as a function of age. A quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) method was used to measure cellular MPG mRNA. MPG mRNA was readily detectable in each tissue analyzed and the age-dependent and tissue specific expressions were not statistically different. The lowest amount of mRNA was measured in preweanling liver and the highest amounts were found in preweanling lung and kidney. Since MPG is reported to be responsible for excision of 1,N(6)-ethenoadenine and N(2),3-ethenoguanine, two promutagenic DNA adducts of vinyl chloride (VC) and vinyl fluoride (VF), we examined the regulation of this enzyme after carcinogen exposure. Expression of MPG was induced in rat liver by these carcinogens. In order to determine the repair capacity in different cell populations of liver, we measured MPG gene expression in isolated hepatocytes and nonparenchymal cells (NPC). The amount of MPG mRNA was 4.5-5 times higher in hepatocytes than in NPC of control rats. Induction of MPG expression was observed in hepatocytes of VF exposed-rats but not in NPC. The expression of MPG in NPC was only 15% of that of the hepatocytes from exposed rats. Western blots of MPG protein confirmed the cell type differences, but did not show increased protein in exposed vs. control liver and hepatocytes. Since metabolism of VC and VF requires CYP2E1, an enzyme exhibiting much greater activity in hepatocytes, formation of etheno adducts preferentially occurs in hepatocytes. These data suggest that cellular differences in the repair of N-alkylpurines may be a critical mechanism in the development of cell specificity in VC carcinogenesis.

Oral administration of dimethylvinyl chloride increases frequency of forestomach papillomas in Tg.AC mice.

This work was initiated to determine the potential for the Tg.AC mouse model to identify chemical carcinogens by an oral route of administration. Tg.AC v-Ha-ras transgenic mice were exposed to dimethyvinyl chloride (DMVC; 1-chloro-2-methylpropene), a structural analog of the human carcinogen vinyl chloride. In the National Toxicology Program 2-yr bioassay, DMVC induced tumors in the oral, nasal, and gastric epithelia of rats and mice. Initial studies were performed in female Tg.AC mice to determine an appropriate oral dose of DMVC to evaluate the potential for stratified gastric or oral epithelia of Tg.AC mice to serve as a target tissue for a transgene-dependent induced tumorigenic response. DMVC was administered to 13- to14-wk-old Tg.AC mice by gavage at doses of 0, 50, 100, and 200 mg/kg five times a week for 20 wk. The forestomachs of DMVC-treated Tg.AC mice had an increasing number of papillomas, which were associated with an increase in the dose of DMVC. The average numbers of papillomas per mouse per dose were 2.4, 7.6, 14.1, and 12.6 for the 0, 50, 100, and 200-mg/kg dose groups, respectively. The optimum papillomagenic dose of 100 mg/kg DMVC was established and administered for 5, 10, and 15/wk to investigate the kinetics of papilloma induction in Tg.AC mice. The average numbers of papillomas per animal were 1.8, 8.8, and 19.0 at 5, 10, and 15 wk, respectively. Reverse transcription-polymerase chain reaction assays determined that the v-Ha-ras transgene was transcriptionally active in all tumor tissues but not in nontumor tissues. In situ hybridization assays performed in conjunction with bromodeoxyuridine in vivo labeling localized the transgene-expressing cells of the forestomach papillomas to the proliferating cellular component of the tumors, as previously seen in skin papillomas of Tg.AC mice. The present results confirm that DMVC is tumorigenic and that oral routes of administration can be used to rapidly elicit a transgene-associated tumor response in the forestomach of Tg.AC mice.

Common conformational effects in the p53 protein of vinyl chloride-induced mutations.

The tumor suppressor gene p53 has been identified as the most frequent site of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen, has been associated with specific A --> T transversions at codons 179, 249, and 255 of the p53 gene. The mutations result in amino acid substitutions of His --> Leu at residue 179, Arg --> Trp at residue 249, and He --> Phe at residue 255 in highly conserved regions of the DNA-binding core domain of the p53 protein. We previously used molecular dynamics calculations to demonstrate that the latter two mutants contain certain common regions that differ substantially in conformation from the wild-type structure. In order to determine whether these conformational changes are consistent for other p53 mutants, we have now used molecular dynamics to determine the structure of the DNA-binding core domain of the Leu 179 p53 mutant. The results indicate that the Leu 179 mutant differs substantially from the wild-type structure in certain discrete regions that are similar to those noted previously in the other p53 mutants. One of these regions (residues 204-217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure, but accessible in the mutant structure, and another region (residues 94-110) contains the epitope for the monoclonal antibody PAb1620, which is accessible in the wild-type structure, but concealed in the mutant structure. Immunologic analyses of tumor tissue known to contain this mutation confirmed these predicted conformational shifts in the mutant p53 protein.

Kinetic characterization of CYP2E1 inhibition in vivo and in vitro by the chloroethylenes.

Trans- and cis-1,2-dichloroethylene (DCE) isomers inhibit their own metabolism in vivo by inactivation of the metabolizing enzyme, presumably the cytochrome P450 isoform, CYP2E1. In this study, we examined cytochrome P450 isoform-specific inhibition by three chloroethylenes, cis-DCE, trans-DCE, and trichloroethylene (TCE), and evaluated several kinetic mechanisms of enzyme inhibition with physiological models of inhibition. Trans-DCE was more potent than cis-DCE, and both were much more effective than TCE in inhibiting CYP2E1. The kinetics of in vitro loss of p-nitrophenol hydroxylase (pNP-OH) activity (a marker of CYP2E1) in microsomal incubations and of the in vivo gas uptake results were most consistent with a mechanism in which inhibition of the metabolizing enzyme (CYP2E1) was presumed to be related to interaction of a reactive DCE metabolite with remaining substrate-bound, active CYP2E1. The kinetics of inhibition by TCE, a weak inhibitor in vitro, were very different from that of the dichloroethylenes. With TCE, parent compound concentrations influenced enzyme loss. Trans-DCE was a more potent inhibitor of CYP2E1 than cis-DCE based on both in vivo and in vitro studies. Quantitative differences in the inhibitory properties of the 1,2-DCE isomers may be due to the different stability of epoxides formed from bioactivation by CYP2E1. Epoxide intermediates of DCE metabolism, reacting by water addition, would yield dialdehyde, a potent cross-linking reagent.

Preferential formation of deletions following in vivo exposure of postmeiotic Drosophila germ cells to the DNA etheno-adduct-forming carcinogen vinyl carbamate.

DNA sequence changes induced in the vermilion gene of Drosophila following in vivo treatment of postmeiotic male germ cells with vinyl carbamate (VCA), an etheno-adduct-forming carcinogen, are primarily deletions. With VCA, 65% (13/20) of the vermilion mutants isolated from crosses of NER+ (nucleotide excision repair) males with NER+ females and 40% (6/15) obtained from matings with NER- females were intra- or multi-locus deletions. Due to the insufficiently low mutagenic activity in NER+ genotypes of vinyl bromide (VB), another epsilon-adduct-forming carcinogen, vermilion mutants could only be isolated from crosses of VB-treated males with NER- females. Of 14 vermilion mutants induced by VB, three carried large deletions. Twenty-two of 23 base substitutions derived from either VCA or VB experiments fell into one of the four categories expected from epsilon-adducts: three vermilion mutants had GC-->AT transitions, five had AT-->GC transitions, 7 carried GC-->TA transversions, and 7 were AT-->TA transversions. In view of the similarities in the response of mouse and Drosophila germ lines to several classes of alkylating agents, a high incidence of deletions is predicted to occur as well in postmeiotic germ cells of mice exposed to these types of agents.

Protection from 1,1-dichloroethylene-induced Clara cell injury by diallyl sulfone, a derivative of garlic.

Bronchiolar Clara cell damage ensues after treatment of mice with 1,1-dichloroethylene (DCE). The cytotoxicity is mediated by CYP2E1, a cytochrome P450 isozyme that is highly localized in the Clara cells. Bioactivation of DCE produces the primary metabolites 2,2-dichloroacetaldehyde, which hydrolyzes to the acetal, and DCE epoxide, which reacts with glutathione to form the conjugates 2-(S-glutathionyl) acetyl glutathione [B] and 2-S-glutathionyl acetate [C]. In this study, we investigated the potential of diallyl sulfone (DASO2) to inhibit CYP2E1, to suppress the bioactivation of DCE to reactive intermediates and to abrogate DCE-induced Clara cell cytotoxicity. Our results showed that treatment of mice with DASO2 (100 mg/kg p.o.) produced decreases in CYP2E1-dependent p-nitrophenol hydroxylation that were apparent at 1 h. Enzyme activity plummeted to about 20% of the control by 2 h and remained at this low level from 3 to 8 h. Recovery of activity was evident at 16 h and returned to the control level by 24 h. Immunoreactivity of the CYP2E1 protein was decreased in immunoblots of lung microsomes from DASO2-treated mice. Treatment with DASO2 did not cause any structural alterations in lung tissue; in contrast, treatment with DCE (75 mg/kg) produced Clara cell damage. This lesion was not manifested in mice treated with DASO2 in conjunction with DCE. The lack of cytotoxicity observed in vivo correlated with a reduction of about 45% in the levels of both the acetal and the DCE epoxide-derived conjugates [B] and [C] in vitro. These results demonstrated that DASO2 significantly inhibited the CYP2E1 enzyme, decreased the production of DCE metabolites and protected Clara cells from DCE-induced cytotoxicity.

Conformational effects in the p53 protein of mutations induced during chemical carcinogenesis: molecular dynamic and immunologic analyses.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen that induces the rare sentinel neoplasm angiosarcoma of the liver, has been associated with specific A-->T transversions at the first base of codons 249 and 255 of the p53 gene. These mutations result in an Arg-->Trp amino acid substitution at residue 249 and an Ile-->Phe amino acid substitution at residue 255 in a highly conserved region in the DNA-binding core domain of the p53 protein. To determine the effects of these substitutions on the three-dimensional structure of the p53 protein, we have performed molecular dynamics calculations on this core domain of the wild-type and the Trp-249 and Phe-255 mutants to compute the average structures of each of the three forms. Comparisons of the computed average structures show that both mutants differ substantially from the wild-type structure in certain common, discrete regions. One of these regions (residues 204-217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure but accessible in both mutant structures. In order to confirm this conformational shift, tumor tissue and serum from vinyl chloride-exposed individuals with angiosarcomas of the liver were examined by immunohistochemistry and enzyme-linked immunosorbent assay. Individuals with tumors that contained the p53 mutations were found to have detectable mutant p53 protein in their tumor tissue and serum, whereas individuals with tumors without mutations and normal controls did not.

The study of health effects of vinyl chloride air pollution on population.

A series of indicators, including serum lysozyme activity, G-banding chromosome aberration (G-banding CA) analysis, sister chromatid exchanges (SCEs), chromosome aberration (CA), T-lymphocyte transformation rate (TcTR), gamma-GT, GPT and AKP, were employed in the present survey among occupationally vinyl chloride (VC) exposed workers and inhabitants living in VC polluted area in a polyvinyl chloride (PVC) factory. The results showed that the serum lysozyme (S-LZM) activities in Group 3 (adult inhabitants exposed to 0.20 mg/m3 VC for at least 8 years), Group 2 (workers exposed to 4.1 mg/m3 for at least 7 years occupationally), Group 1 (workers exposed to 25.7 mg/m3 for at least 2 years) were significantly higher than control. G-banding CA analysis showed that the total chromosome breakage rates in both Groups 1 and 2 were higher, but no difference existed between Group 3 and control. Only Group 1 was observed having higher SCEs, CA level and lower TcTR than control. AKP levels in Groups 1 and 2 were higher than control, but no gamm-GT and GPT differences were found among groups. The study also suggests that G-banding CA analysis is more sensitive than CA and SCEs.

Mutagenic and genotoxic effects of three vinyl chloride-induced DNA lesions: 1,N6-ethenoadenine, 3,N4-ethenocytosine, and 4-amino-5-(imidazol-2-yl)imidazole.

The mutagenic and genotoxic properties of 1,N6-ethenoadenine (epsilon Ade), 3,N4-ethenocytosine (epsilon Cyt), and 4-amino-5-(imidazol-2-yl)imidazole (beta) were investigated in vivo. The former two modified bases are known DNA adducts formed by the human carcinogen vinyl chloride; beta is formed by pyrimidine ring-opening of epsilon Ade. Chemically synthesized deoxyhexanucleotides containing epsilon Ade and beta, d[GCT-(epsilon A)GC], and d[GCT(beta)GC], respectively, were described previously [Biochemistry (1987) 26, 5626-5635]. epsilon Cyt was inserted into an oligonucleotide, d[GCTAG(epsilon C)], by a mild enzymatic synthetic procedure, which avoided exposure of the base to alkaline conditions. 3,N4-Etheno-2'-deoxycytidine 3',5'-bisphosphate coupled with reasonable efficiency (30-40%) to the 3'-nucleoside of an acceptor pentamer, d(GCTAG), in a reaction catalyzed by T4 RNA ligase in the presence of ATP. Each of the three modified hexanucleotides and an unmodified control were inserted into a six-base gap positioned at a known site in the genome of bacteriophage M13-NheI. A nick was placed in the DNA strand opposite that containing the single DNA lesions, enabling the formation of singly adducted single-stranded genomes by denaturation. After transfection of the adducted phage DNAs into Escherichia coli, each of the adducts was found to be genotoxic. The most toxic lesion was beta, which reduced survival of the genome by 97%. epsilon Cyt and epsilon Ade reduced survival by 90% and 65%, respectively. An examination of the surviving phage populations revealed that each of the three adducts was mutagenic. The least mutagenic lesion was epsilon Ade (0.1% of the survivors were mutant), which showed primarily A-->G transitions.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunobiochemical profiles of workers differing in the degree of occupational exposure to vinyl chloride.

The immunobiochemical studies were conducted in a group of 98 production workers engaged in polyvinyl chloride manufacture from ethylene (group A workers) and in a group of 59 vinyl chloride workers from a chemical plant employing classic production technology from acetylene (group B workers). Both groups of workers were matched by age (group A workers: 37.7 +/- 8.66 years; group B workers: 34.9 +/- 11.2 years) and average exposure length (group A workers: 8.6 +/- 3.0 years; group B workers: 10.7 +/- 8.4 years). All workers were examined for the serum concentrations of immunoglobulins IgG, IgA and IgM and acute reactants lysozyme (LYS), transferrin (TRF), ceruloplasmin (CPL), alpha-l-antitrypsin (AlAT), alpha-2-macroglobulin (A2M) and orosomucoid (ORO). The statistical analysis included calculations of means, standard deviations and 95% confidence intervals. Differences in means were evaluated by t-test, differences in the distribution pattern of values by F-test. Abnormality of values was assessed by comparisons to normal values valid in Czechoslovakia. Group A worked in conditions meeting the MAC 10 mg VC.m-3 comparing with group B workers had elevated levels of IgG (P less than 0.005), IgA and IgM (P less than 0.001 both). Group B workers differed from group A workers by exhibiting significantly elevated levels of AlAT, and CPL. (P less than 0.001). The differences in the frequency of abnormal values between group A and group B worked in substantially less favourable hygienic conditions were significant for immunoglobulins elevated in group A and for ORO (P less than 0.01) and CPL (P less than 0.001) elevated in group B. The possible relationship of these immunobiochemical findings with the degree of vinyl chloride exposure are critically analyzed.

Deoxyhexanucleotide containing a vinyl chloride induced DNA lesion, 1,N6-ethenoadenine: synthesis, physical characterization, and incorporation into a duplex bacteriophage M13 genome as part of an amber codon.

Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N6-ethenoadenine (epsilon Ade) DNA adduct at an amber codon in the genome of an M13mp19 phage derivative. The deoxyhexanucleotide d[GCT(epsilon A)GC] was chemically synthesized by the phosphotriester method. Mild nonaqueous conditions were employed for deprotection because of the unstable nature of the epsilon Ade adduct in aqueous basic milieu. Physical studies involving fluorescence, circular dichroism, and 1H NMR indicated epsilon Ade to be very efficiently stacked in the hexamer, especially with the 5'-thymine. Melting profile and circular dichroism studies provided evidence of the loss of base-pairing capabilities attendant with formation of the etheno ring. The modified hexanucleotide was incorporated into a six-base gap formed in the genome of an M13mp19 insertion mutant; the latter was constructed by blunt-end ligation of d(GCTAGC) in the center of the unique SmaI site of M13mp19. Phage of the insertion mutant, M13mp19-NheI, produced light blue plaques on SupE strains because of the introduced amber codon. Formation of a hybrid between the single-strand DNA (plus strand) of M13mp19-NheI with SmaI-linearized M13mp19 replicative form produced a heteroduplex with a six-base gap in the minus strand. The modified hexamer [5'-32P]d-[GCT(epsilon A)GC], after 5'-phosphorylation, was ligated into this gap by using bacteriophage T4 DNA ligase to generate a singly adducted genome with epsilon Ade at minus strand position 6274. Introduction of the radiolabel provided a useful marker for characterization of the singly adducted genome, and indeed the label appeared in the anticipated fragments when digested by several restriction endonucleases. Evidence that ligation occurred on both 5' and 3' sides of the oligonucleotide also was obtained. The adduct was introduced into a unique NheI site, and it was observed that this restriction endonuclease was able to cleave the adducted genome, albeit at a lower rate compared to unmodified DNA. The M13mp19-NheI genome containing epsilon Ade will be used as a probe for studying mutagenesis and repair of this DNA adduct in Escherichia coli.