Vinyl Chloride Metabolites Research Articles

Long-term dechlorination of cis-DCE to ethene with co-immobilized Dehalococcoides mccartyi BAV1 and Clostridium butyricum in silica gel system

Chloroethenes are common groundwater pollutants, and have been classified as toxic and carcinogenic to humans. The metabolites of chloroethenes, cis-dichloroethene (cis-DCE) and vinyl chloride (VC) commonly accumulate in groundwater due to their recalcitrant reductive dechlorination under anaerobic conditions. Dehalococcoides mccartyi (Dhc) is the key anaerobic bacteria for complete dechlorination of chloroethene, and Clostridium butyricum (C. butyricum) can provide hydrogen for supporting the growth of Dhc. In this study, we co-immobilized Dhc strain BAV1 and C. butyricum in a silica gel to determine the ability of the complete dechlorination of cis-DCE. Our results showed that our immobilized system could protect BAV1 from a high concentration (8 mM) of cis-DCE to carry out complete dechlorination. After the long-term use of our immobilized system, the activity of complete dechlorination was maintained for more than 180 consecutive days. Furthermore, we applied the immobilized system to remediate contaminated groundwater and uncovered the complete dechlorination of cis-DCE into ethene, a non-toxic product, within 28 days. Therefore, this novel co-immobilized system could serve a solution for bioremediation at chloroethene-contaminated sites.

Mechanism of subchronic vinyl chloride exposure combined with a high‐fat diet on hepatic steatosis

Vinyl chloride (VC) is a common industrial organic chlorine and environmental pollutant. In recent years, the dietary structure of residents especially Chinese has gradually shifted to western dietary patterns. VC aggravates dietary fatty acid-induced hepatic steatosis, but its mechanism is still unclear. And if the risk factors for steatosis persist, more severe diseases such as fibrosis and cirrhosis will occur. Therefore, we studied the effects and mechanisms of VC (160 and 800 mg/m3 ) and its metabolite (chloroacetaldehyde, 2.25, 4.5, and 9μM) on hepatic steatosis of high-fat diet (HFD)-fed mice and palmitic acid (PA, 100 μM) treated HepG2 cells. Liver and serum biochemical indicators and pathological staining of the liver showed that the hepatic steatosis of VC combined with HFD groups was more severe than that of single-exposure groups (HFD group, low-dose VC group, and high-dose VC group). Moreover, VC enhanced HFD-induced oxidative stress (OS) and endoplasmic reticulum stress (ERS) and further upregulated the expression of sterol regulatory element-binding protein 1 (SREBP-1) and FAS. Besides, antioxidants and ERS inhibitors reduced the steatosis of HepG2 cells induced by VC metabolites and PA. These results suggest that VC exposure can enhance the degree of hepatic steatosis in HFD-fed mice. VC combined with HFD led to OS and ERS and upregulated the expression of de novo lipogenesis-related proteins, which may be related to the occurrence of hepatic steatosis. And the increased expression of CYP2E1 induced by VC combined with HFD may be the cause of OS.

Adipose tissue-liver crosstalk during pathologic changes caused by vinyl chloride metabolites in mice

Volatile organic compounds (VOCs), such as vinyl chloride (VC), can be directly toxic at high concentrations. However, we have shown that ‘nontoxic’ exposures to VC and its metabolite chloroethanol (CE) enhances experimental non-alcoholic fatty liver disease (NAFLD), suggesting an unpredicted interaction. Importantly, VOC exposure has been identified as a potential risk factor for the development of obesity and its sequelae in humans. As there is a known axis between adipose and hepatic tissue in NAFLD, the impact of CE on white adipose tissue (WAT) inflammation and lipolysis was investigated. Mice were administered CE (or vehicle) once, after 10 weeks of being fed high-fat or low-fat diet (LFD). CE significantly enhanced hepatic steatosis and inflammation caused by HFD. HFD significantly increased the size of epididymal fat pads, which was enhanced by CE. The relative size of adipocyte lipid droplets increased by HFD + CE, which was also correlated with increased expression of lipid-associated proteins (e.g., PLINs). CE also enhanced HFD-induced indices of WAT inflammation, and ER stress. Hepatic-derived circulating FGF21, a major modulator of WAT lipolysis, which is hypothesized to thereby regulate hepatic steatosis, was significantly increased by CE in animals fed HFD. Taken together these data support the hypothesis that environmental toxicant exposure can exacerbate the severity of NAFLD/NASH, involving the liver-adipose axis in this process. Specifically, CE enhances local inflammation and alters lipid metabolism and WAT-mediated hepatic steatosis due to changes in WAT lipolysis.

Rapamycin attenuates liver injury caused by vinyl chloride metabolite chloroethanol and lipopolysaccharide in mice

Vinyl chloride (VC) is a prevalent environmental toxicant that is rapidly metabolized within the liver. Its metabolites have been shown to directly cause hepatic injury at high exposure levels. We have previously reported that VC metabolite, chloroethanol (CE), potentiates liver injury caused by lipopolysaccharide (LPS). Importantly, that study showed that CE alone, while not causing damage per se, was sufficient to alter hepatic metabolism and increase mTOR phosphorylation in mice, suggesting a possible role for the mTOR pathway. Here, we explored the effect of an mTOR inhibitor, rapamycin, in this model. C57BL/6 J mice were administered CE, followed by rapamycin 1 h and LPS 24 h later. As observed previously, the combination of CE and LPS significantly enhanced liver injury, inflammation, oxidative stress, and metabolic dysregulation. Rapamycin attenuated not only inflammation, but also restored the metabolic phenotype and protected against CE + LPS-induced oxidative stress. Importantly, rapamycin protected against mitochondrial damage and subsequent production of reactive oxygen species (ROS). The protective effect on mitochondrial function by rapamycin was mediated, by restoring the integrity of the electron transport chain at least in part, by blunting the deactivation of mitochondrial c-src, which is involved mitochondrial ROS production by electron transport chain leakage. Taken together, these results further demonstrate a significant role of mTOR-mediated pathways in VC-metabolite induced liver injury and provide further insight into VC-associated hepatic damage. As mTOR mediated pathways are very complex and rapamycin is a more global inhibitor, more specific mTOR (i.e. mTORC1) inhibitors should be considered in future studies.

Dynamics of Excretion of Thiodiacetic Acid into Urine in Polyvinyl Chloride Production Workers.

Background: Thiodiacetic acid (TDAA) is the main metabolite of vinyl chloride (VC) and 1,2-dichloroethane (EDC) and its urinary level is correlated with the level of exposure to these chemicals. Objective: To study dynamics of the excretion of TDAA into urine of polyvinyl chloride (PVC) production workers. Methods: The study sample consisted of 65 workers of VC and PVC divisions with various time intervals following exposure to the chemicals, 10 shift workers from PVC division, and 34 workers not exposed to the chemicals (control group). Analysis of urinary TDAA was carried out with gas chromatography with mass-selective detector. Results:The concentrations of TDAA in the urine of workers of the VC division and in group of primary occupations who had a high level of exposure to the chemicals, were significantly (p<0.05) higher than that of workers of the PVC production division and group of auxiliary professions. The highest levels of TDAA in the urine of workers were found at the beginning of the next shift and during a long break, 24–48 hours after the cessation of the exposure. Conclusion: When conducting biomonitoring studies in PVC production workers, the optimal time for collecting urine samples is at the beginning of the next shift or during a long rest, 24–48 hours after the exposure.

ASSESSMENT OF THE METABOLITE OF CHLORORGANIC POLLUTANTS CONTENT IN URINE OF WORKERS OF POLYVINYCHLORIDE PRODUCTION

Introduction. The control and study of changes in the composition of biomedical samples under the influence of toxicants makes it possible to carry out early diagnostics, to reveal occupationally caused diseases. Vinyl chloride (VC) is a compound widely used in the chemical industry, the production of which is continuously growing all over the world, as well as the number of workers employed in it. Thiodiacetic acid (TDAA) is the main metabolite of vinyl chloride and its urinary excretion is associated with its level of exposure. Material and methods. The study was conducted in a linked sample of 19 workers in the dynamics of two-day 12-hour shifts after a three-day weekend. For 10 workers from this group, a more in-depth study of the dynamics of TDАА excretion before and after the shift was carried out: three 12-hour shifts. The analysis of TDAA in urine was carried out by chromatography-mass spectrometry using an Agilent 7890A gas chromatograph with an Agilent 5975C mass-selective detector. To find the optimal parameters for temperature, reaction time, catalyst type, mathematical planning of the 3-factor experiment was carried out. The identification of the TDAА derivative on mass chromatograms was carried out by the absolute retention time (10.36 min) and the intensity ratio of the peaks of the detected ions (146, 178). Results. Studies have shown average levels of TDAA in the urine of professional groups of workers (apparatchiks, cleaners) to be significantly higher before the next shift (12 hours after the end of the previous shift) than at the end of the previous shift and 1.99 times for operators (p = 0.004) and 2.61 times for cleaners (p = 0.002). Further, there was a significant decrease in the average level of TDAA in the urine relative to the beginning of the shift: 1.72 times for operators (p = 0.006) and 1.62 times for cleaners (p = 0.003). Conclusion. This analyte can be considered as an informative indicator of the presence of the production effect of VC from the point of view of evidence-based medicine, further confirmation of the need for studies of markers in persons in contact with this toxicant.

Integrated methodological approach reveals microbial diversity and functions in aerobic groundwater microcosms adapted to vinyl chloride.

Vinyl chloride (VC), a known human carcinogen, is often formed in groundwater (GW) by incomplete reductive dechlorination of chlorinated ethenes. An integrated microbial ecology approach involving bacterial enrichments and isolations, carbon stable-isotope probing (SIP) and metagenome and genome sequencing was applied to ethene-fed GW microcosms that rapidly transitioned to aerobic growth on VC. Actinobacteria, Proteobacteria and Bacteroidetes dominated the microbial communities in ethene- and VC-grown cultures. SIP with 13C2-VC demonstrated that Nocardioides spp. significantly participated in carbon uptake from VC (52.1%-75.7% enriched in heavy fractions). Sediminibacterium, Pedobacter and Pseudomonas spp. also incorporated 13C from VC into genomic DNA. Ethene- and VC-assimilating Nocardioides sp. strain XL1 was isolated. Sequencing revealed a large (∼300 kbp) plasmid harboring genes encoding alkene monooxygenase and epoxyalkane: coenzyme M transferase, enzymes known to participate in aerobic VC and ethene biodegradation. The plasmid was 100% identical to pNOCA01 found in VC-assimilating Nocardioides sp. strain JS614. Metagenomic analysis of enrichment cultures indicated other bacteria implicated in carbon uptake from VC possessed the genetic potential to detoxify epoxides via epoxide hydrolase or glutathione S-transferase (Pseudomonas) and/or metabolize VC epoxide breakdown products and downstream VC metabolites. This study provides new functional insights into aerobic VC metabolism within a GW microbial community.

Vinyl chloride dysregulates metabolic homeostasis and enhances diet-induced liver injury in mice.

Vinyl chloride (VC), a common industrial organochlorine and environmental pollutant, has been shown to directly cause hepatic angiosarcoma and toxicant‐associated steatohepatitis at high exposure levels. However, the impact of lower concentrations of VC on the progression of underlying liver diseases (e.g., nonalcoholic fatty liver disease [NAFLD]) is unclear. Given the high prevalence of NAFLD in the United States (and worldwide) population, this is an important concern. Recent studies by our group with VC metabolites suggest a potential interaction between VC exposure and underlying liver disease to cause enhanced damage. Here, a novel mouse model determined the effects of VC inhalation at levels below the current Occupational Safety and Health Administration limit (<1 ppm) in the context of NAFLD to better mimic human exposure and identify potential mechanisms of VC‐induced liver injury. VC exposure caused no overt liver injury in mice fed a low‐fat diet. However, in mice fed a high‐fat diet (HFD), VC significantly increased liver damage, steatosis, and increased neutrophil infiltration. Moreover, VC further enhanced HFD‐induced oxidative and endoplasmic reticulum stress. Importantly, VC exposure dysregulated energy homeostasis and impaired mitochondrial function, even in mice fed a low‐fat diet. In toto, the results indicate that VC exposure causes metabolic stress that sensitizes the liver to steatohepatitis caused by HFD. Conclusion: The hypothesis that low‐level (below the Occupational Safety and Health Administration limit) chronic exposure to VC by inhalation enhances liver injury caused by an HFD is supported. Importantly, our data raise concerns about the potential for overlap between fatty diets (i.e., Western diet) and exposure to VC and the health implications of this co‐exposure for humans. It also emphasizes that current safety restrictions may be insufficient to account for other factors that can influence hepatotoxicity. (Hepatology Communications 2018;2:270‐284)

Role of dietary fatty acids in liver injury caused by vinyl chloride metabolites in mice

BackgroundVinyl chloride (VC) causes toxicant-associated steatohepatitis at high exposure levels. Recent work by this group suggests that underlying liver disease may predispose the liver to VC hepatotoxicity at lower exposure levels. The most common form of underlying liver disease in the developed world is non-alcoholic fatty liver disease (NAFLD). It is well-known that the type of dietary fat can play an important role in the pathogenesis of NAFLD. However, whether the combination of dietary fat and VC/metabolites promotes liver injury has not been studied. MethodsMice were administered chloroethanol (CE - a VC metabolite) or vehicle once, 10weeks after being fed diets rich in saturated fatty acids (HSFA), rich in poly-unsaturated fatty acids (HPUFA), or the respective low-fat control diets (LSFA; LPUFA). ResultsIn control mice, chloroethanol caused no detectable liver injury, as determined by plasma transaminases and histologic indices of damage. In HSFA-fed mice, chloroethanol increased HSFA-induced liver damage, steatosis, infiltrating inflammatory cells, hepatic expression of proinflammatory cytokines, and markers of endoplasmic reticulum (ER) stress. Moreover, markers of inflammasome activation were increased, while markers of inflammasome inhibition were downregulated. In mice fed HPUFA all of these effects were significantly attenuated. ConclusionsChloroethanol promotes inflammatory liver injury caused by dietary fatty acids. This effect is far more exacerbated with saturated fat, versus poly-unsaturated fat; and strongly correlates with a robust activation of the NLRP3 inflammasome in the saturated fed animals only. Taken together these data support the hypothesis that environmental toxicant exposure can exacerbate the severity of NAFLD/NASH.

Vinyl Chloride Metabolites Potentiate Inflammatory Liver Injury Caused by LPS in Mice.

Vinyl chloride (VC) is a ubiquitous environmental contaminant for which human risk is incompletely understood. We have previously reported that high occupational exposure to VC directly caused liver damage in humans. However, whether VC may also potentiate liver injury from other causes is not known. C57Bl/6J mice were administered chloroethanol (CE), a major metabolite of VC, and lipopolysaccharide (LPS) 24 h after CE. Samples were harvested for determination of liver damage, inflammation, and changes in carbohydrate and lipid metabolism. In mice, CE exposure alone caused no detectable liver damage. LPS exposure caused inflammatory liver damage, oxidative stress, lipid accumulation, and glycogen depletion; the effect of all of these variables was potentiated by CE pre-exposure. In vitro experiments suggest that VC metabolite chloroacetaldehyde (CAA) directly damages mitochondria, which may explain the sensitization effect observed in vivo Moreover, co-exposure of cells to CAA and TNFα caused increased cell death, supporting the hypothesis of sensitization by VC metabolites. Taken together, these data demonstrate that exposure to VC/metabolites at levels that are not overtly hepatotoxic can potentiate liver injury caused by another hepatotoxicant. This serves as proof-of-concept that VC hepatotoxicity may be modified by an additional metabolic stress such as endotoxemia, which commonly occurs in acute (eg, sepsis) and chronic (eg, NAFLD) diseases.

Effects of DNA repair gene polymorphisms on DNA damage in human lymphocytes induced by a vinyl chloride metabolite in vitro

Background: Epidemiologic studies suggest that variability in DNA damage from vinyl chloride monomer (VCM) may be partially mediated by genetic polymorphisms in DNA repair. This study aimed to corroborate these observations with controlled experiments in vitro using cell lines from individuals with differing DNA repair genotypes to determine damage following VCM metabolite exposure.Methods: Matched pairs of lymphoblast cell lines (homozygous wild-type versus homozygous variant for either XRCC1 399 or XPD 751 polymorphism) were exposed to chloroacetaldehyde and analyzed by the cytokinesis-block micronucleus assay.Results: All cell lines demonstrated a dose–response of increasing micronuclei with increasing exposure, but for both XRCC1 and XPD, the polymorphic cells peaked at higher micronucleus frequencies and declined at a slower rate to baseline than the wild-type cells.Conclusion: This supports the findings that XRCC1 and XPD polymorphisms may result in deficient DNA repair of VCM-induced genetic damage.

Simultaneous analysis of 28 urinary VOC metabolites using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI/MSMS)

Volatile organic compounds (VOCs) are ubiquitous in the environment, originating from many different natural and anthropogenic sources, including tobacco smoke. Long-term exposure to certain VOCs may increase the risk for cancer, birth defects, and neurocognitive impairment. Therefore, VOC exposure is an area of significant public health concern. Urinary VOC metabolites are useful biomarkers for assessing VOC exposure because of non-invasiveness of sampling and longer physiological half-lives of urinary metabolites compared with VOCs in blood and breath. We developed a method using reversed-phase ultra high performance liquid chromatography (UPLC) coupled with electrospray ionization tandem mass spectrometry (ESI/MSMS) to simultaneously quantify 28 urinary VOC metabolites as biomarkers of exposure. We describe a method that monitors metabolites of acrolein, acrylamide, acrylonitrile, benzene, 1-bromopropane, 1,3-butadiene, carbon-disulfide, crotonaldehyde, cyanide, N,N-dimethylformamide, ethylbenzene, ethylene oxide, propylene oxide, styrene, tetrachloroethylene, toluene, trichloroethylene, vinyl chloride and xylene. The method is accurate (mean accuracy for spiked matrix ranged from 84 to104%), sensitive (limit of detection ranged from 0.5 to 20ngmL−1) and precise (the relative standard deviations ranged from 2.5 to 11%). We applied this method to urine samples collected from 1203 non-smokers and 347 smokers and demonstrated that smokers have significantly elevated levels of tobacco-related biomarkers compared to non-smokers. We found significant (p<0.0001) correlations between serum cotinine and most of the tobacco-related biomarkers measured. These findings confirm that this method can effectively quantify urinary VOC metabolites in a population exposed to volatile organics.

Replication ofN2,3-Ethenoguanine by DNA Polymerases

DNA is prone to attack by physical and chemical agents generated endogenously and exogenously, producing modified DNA bases (i.e. DNA adducts/lesions), abasic sites, and inter- and intrastrand DNA crosslinks. DNA adducts, if not properly repaired, can lead to blocked replication, misincorporation, and mutation, potentially causing gene deregulation and cancer. Etheno (e) DNA adducts are exocyclic adducts that, in addition to their use as fluorescent nucleotidfe derivatives,[1] were first recognized as reaction products of DNA with reactive metabolites of the occupational carcinogen vinyl chloride (VC).[2] Endogenous etheno-DNA adducts, arising from lipid peroxidation-derived DNA damage, were also detected in rats[3] and humans[4] without VC exposure. VC is a known carcinogen that induces hepatic angiosarcomas.[5] The major DNA adduct formed by VC, N7-(2-oxoethyl)guanine,[3, 6] is generally not considered to be mutagenic, because in vitro experiments showed that it did not cause detectable miscoding in an assay with modified poly(GC).[7] However, etheno adducts formed by VC (e.g. 1,N6-ethenoadenine, 3,N4-ethenocytosine, N2,3-ethenoguanine (N2,3-eG), and 1,N2-ethenoguanine (1,N2-eG)) have all been shown to be mutagenic in vitro and in bacteria (see N2,3-eG and 1,N2-eG structures in Figure 1a).[8] N2,3-eG is the most abundant endogenous etheno adduct, with levels estimated to be approximately 36 N2,3-eG lesions/cell in livers of untreated rats or humans.[9] A common assumption is that N2,3-eG is highly mutagenic; N2,3-eG is considered to contribute to the carcinogenesis of VC and inflammation-driven malignancies.[10] The dominance of a GC to AT transition in five of six K-ras (oncogene) tumors from VC workers[9] suggests the importance of a G adduct, but the misincorporation characteristics of 1,N2-eG are not consistent with this transition.[8a,f] Little repair of N2,3-eG occurs in VC-exposed rats, since the half-life of this lesion in rat liver and lung (150 days) and in rat kidney (75 days) is quite long.[11] The lability of the glycosidic bond of N2,3-e-deoxyguanosine (N2,3-e-dG) makes it difficult to adequately discern its mutagenic potential.[12] Both C and T were incorporated opposite N2,3-eG in a polyribo(G/N2,3-eG) template by avian myeloblastosis virus (AMV) reverse transcriptase.[13] N2,3-e-Deoxyguanosine triphosphate is inserted opposite T by several polymerases (pols).[8d]

The role of AlkB protein in repair of 1,N6-ethenoadenine in Escherichia coli cells

Etheno (ε) DNA adducts, including 1,N(6)-ethenoadenine (εA), are formed by various bifunctional agents of exogenous and endogenous origin. The AT→TA transversion, the most frequent mutation provoked by the presence of εA in DNA, is very common in critical codons of the TP53 and RAS genes in tumours induced by exposure to carcinogenic vinyl compounds. Here, using a method that allows examination of the mutagenic potency of a metabolite of vinyl chloride, chloroacetaldehyde (CAA), but eliminates its cytotoxicity, we studied the participation of alkA, alkB and mug gene products in the repair of εA in Escherichia coli cells. The test system used comprised the pIF105 plasmid bearing the lactose operon of CC105 origin, which allowed monitoring of Lac(+) revertants that arose by AT→TA substitutions due to the modification of adenine by CAA. The plasmid was CAA-modified in vitro and replicated in E.coli of various genetic backgrounds (wt, alkA, alkB, mug, alkAalkB, alkAmug and alkBmug). To modify the levels of the AlkA and AlkB proteins, mutagenesis was studied in E.coli cells induced or not in adaptive response to alkylating agents. Considering the levels of CAA-induced Lac(+) revertants in strains harbouring the CAA-modified pIF105 plasmid and induced or not in adaptive response, we conclude that the AlkB dioxygenase plays a major role in decreasing the level of AT→TA mutations, thus in the repair of εA in E.coli cells. The observed differences of mutation frequencies in the various mutant strains assayed indicate that Mug glycosylase is also engaged in the repair of εA, whereas the role the AlkA glycosylase in this repair is negligible.

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: The role of AlkB protein in repair of 3,N 4-ethenocytosine and 3,N 4-α-hydroxyethanocytosine

Etheno (ɛ) adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate ɛ-adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of ɛ-adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (Cupples and Miller (1989) [17]) which allowed to monitor Lac + revertants, the latter arose by GC → AT or GC → TA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in Escherichia coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of ɛC proceeds via a relatively stable intermediate, 3,N 4-α-hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and mug, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA +, alkB +, and mug + controls. Considering the levels of CAA-induced Lac + revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of ɛC and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs ɛC and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and ɛC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein.

AlkB Influences the Chloroacetaldehyde-Induced Mutation Spectra and Toxicity in the pSP189 supF Shuttle Vector

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogen vinyl chloride, reacts with DNA to form cyclic etheno ()-lesions. AlkB, an iron-/alpha-ketoglutarate-dependent dioxygenase, repairs 1, N (6)-ethenodeoxyadenosine (A) and 3, N (4)-ethenodeoxycytidine (C) in site-specifically modified single-stranded viral genomes in vivo and also protects the E. coli genome from the toxic effects of CAA. We examined the role of AlkB as a cellular defense against CAA by characterizing the frequencies, types, and distributions of mutations induced in the double-stranded supF gene of pSP189 damaged in vitro and replicated in AlkB-proficient (AlkB (+)) and AlkB-deficient (AlkB (-)) E. coli. AlkB reduced mutagenic potency and increased the survival of CAA-damaged plasmids. Toxicity and mutagenesis data were benchmarked to levels of -adducts and DNA strand breaks measured by LC-MS/MS and a plasmid nicking assay. CAA treatment caused dose-dependent increases in A, C, and 1, N (2)-ethenodeoxyguanosine (1, N (2)-G) and small increases in strand breaks and abasic sites. Mutation frequency increased in plasmids replicated in both AlkB (+) and AlkB (-) cells; however, at the maximum CAA dose, the mutation frequency was 5-fold lower in AlkB (+) than in AlkB (-) cells, indicating that AlkB protected the genome from CAA lesions. Most induced mutations in AlkB (-) cells were G:C to A:T transitions, with lesser numbers of G:C to T:A transversions and A:T to G:C transitions. G:C to A:T and A:T to G:C transitions were lower in AlkB (+) cells than in AlkB (-) cells. Mutational hotspots at G122, G123, and G160 were common to both cell types. Three additional hotspots were found in AlkB (-) cells (C133, T134, and G159), with a decrease in mutation frequency and change in mutational signature in AlkB (+) cells. These results suggest that the AlkB protein contributes to the elimination of exocyclic DNA base adducts, suppressing the toxic and mutagenic consequences induced by this damage and contributing to genetic stability.

Auxiliary Substrates for Elimination of Trichloroethene, Monochlorobenzene, and Benzene in a Sequential Anaerobic–Aerobic GAC Biobarrier

Sequential anaerobic-aerobic barrier is a novel concept for groundwater bioremediation. Trichloroethene (TCE), monochlorobenzene (MCB), and benzene (BZ) were used as model contaminants representing contaminant cocktails frequently found in the contaminated subsurface. The autochthonous microflora from a contaminated field was inoculated to eliminate model contaminants in a set of sequential anaerobic–aerobic granulated activated carbon (GAC) columns and batch studies. In the anaerobic column, the TCE was reductively dechlorinated through cis-dichloroethene (cis-DCE), vinyl chloride (VC), and ethene (ETH). Ethanol and sucrose as auxiliary substrates were added to donate electrons. In the second stage, MCB, BZ, and the lower chlorinated metabolites of TCE degradation, i.e. cis-Dichloroethene (cisDCE) and vinyl chloride (VC), were oxidatively degraded with addition of hydrogen peroxide and nitrate. This paper examines the influence of auxiliary substrates on the biological degradation of model pollutants. In the anaerobic barrier, the auxiliary substrates supply should be maintained low but stoichiometrically adequate for supporting reductive dechlorination. Supplying higher amount of auxiliary substrates provoked competitive reactions in anaerobic conditions, such as sulfate reduction and methanogenesis. If the auxiliary substrates are not utilized completely in the anaerobic phase, the remaining compounds flow into the aerobic phase. This led to unwanted conditions, i.e. oxidation of auxiliary substrates instead of pollutant elimination, and a higher consumption of electron acceptors. In the aerobic barrier, in particular, ethene proved to be a suitable auxiliary substrate for cometabolic degradation of cisDCE.

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.

Exocyclic DNA Lesions Stimulate DNA Cleavage Mediated by Human Topoisomerase IIα in Vitro and in Cultured Cells

DNA adducts are mutagenic and clastogenic. Because of their harmful nature, lesions are recognized by many proteins involved in DNA repair. However, mounting evidence suggests that lesions also are recognized by proteins with no obvious role in repair processes. One such protein is topoisomerase II, an essential enzyme that removes knots and tangles from the DNA. Because topoisomerase II generates a protein-linked double-stranded DNA break during its catalytic cycle, it has the potential to fragment the genome. Previous studies indicate that abasic sites and other lesions that distort the double helix stimulate topoisomerase II-mediated DNA cleavage. Therefore, to further explore interactions between DNA lesions and the enzyme, the effects of exocyclic adducts on DNA cleavage mediated by human topoisomerase IIalpha were determined. When located within the four-base overhang of a topoisomerase II cleavage site (at the +2 or +3 position 3' relative to the scissile bond), 3,N(4)-ethenodeoxycytidine, 3,N(4)-etheno-2'-ribocytidine, 1,N(2)-ethenodeoxyguanosine, pyrimido[1,2-a]purin-10(3H)-one deoxyribose (M(1)dG), and 1,N(2)-propanodeoxyguanosine increased DNA scission approximately 5-17-fold. Enhanced cleavage did not result from an increased affinity of topoisomerase IIalpha for adducted DNA or a decreased rate of religation. Therefore, it is concluded that these exocyclic lesions act by accelerating the forward rate of enzyme-mediated DNA scission. Finally, treatment of cultured human cells with 2-chloroacetaldehyde, a reactive metabolite of vinyl chloride that generates etheno adducts, increased cellular levels of DNA cleavage by topoisomerase IIalpha. This finding suggests that type II topoisomerases interact with exocyclic DNA lesions in physiological systems.

DNA damage and mutations produced by chloroacetaldehyde in a CpG-methylated target gene

Chloroacetaldehyde (CAA) is a metabolite of the human carcinogen vinyl chloride. CAA produces several types of DNA adducts including the exocyclic base adducts 3, N 4-ethenocytosine, 1, N 6-ethenoadenine, N 2,3-ethenoguanine, and 1, N 2-ethenoguanine. Adducts of CAA with 5-methylcytosine have not yet been characterized. Here we have analyzed the mutational spectra produced by CAA in the supF gene of the pSP189 shuttle vector when present in either an unmethylated or CpG-methylated state. The vectors were replicated in human nucleotide excision repair-deficient XP-A fibroblasts. The mutational spectra obtained with the unmethylated and methylated supF target genes were generally similar with a preponderance of C/G to T/A transitions and C/G to A/T transversions. CAA-induced DNA adducts were mapped along the supF gene by using thermostable thymine DNA glycosylase (TDG) in conjunction with ligation-mediated PCR or by a Taq polymerase stop assay. Prominent CAA-induced TDG-sensitive sites were seen at several CpG positions but were independent of methylation. Methylated CpG sites were sites of CAA-induced mutations but were not the major mutational hotspots. Taq polymerase arrest sites were observed at numerous sequence positions in the supF gene and reflected the rather broad distributions of mutations along the sequence. We conclude that methylated CpG sites are not preferential targets for chloroacetaldehyde-induced mutagenesis.