Benzo[a]pyrene Diol Epoxide Research Articles

A sandwich enzyme-linked immunosorbent assay for adducts of polycyclic aromatic hydrocarbons with human serum albumin

Adducts of benzo[ a]pyrene-diolepoxide (BPDE) with blood nucleophiles have been used as biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs). The most popular such assay is a competitive enzyme-linked immunosorbent assay (ELISA) that employs monoclonal antibody 8E11 to detect benzo[ a]pyrene tetrols following hydrolysis of BPDE adducts from lymphocyte DNA or human serum albumin (HSA). Here we used 8E11 as the capture antibody in a sandwich ELISA to detect BPDE–HSA adducts directly in 1-mg samples of HSA or 20 μl of serum/plasma. The assay employs an anti-HSA antibody for detection, and this is amplified by an avidin/biotinylated horseradish peroxidase complex. The sandwich ELISA has advantages of specificity and simplicity and is approximately 10 times more sensitive than the competitive ELISA. To validate the assay, HSA samples were assayed from three populations with known high PAH exposures (coke oven workers), medium PAH exposures (steel factory control workers), and low PAH exposures (volunteer subjects) ( n = 30). The respective geometric mean levels of BPDE–HSA adducts—67.8, 14.7, and 1.93 ng/mg HSA (1010, 220, and 28.9 fmol BPDE equiv/mg HSA)—were significantly different ( P < 0.05). The sandwich ELISA will be useful for screening PAH exposures in large epidemiologic studies and can be extended to other adducts for which capture antibodies are available.

Abstract A6: Ethnic differences in risk of lung cancer associated with repair capacity for tobacco carcinogen-induced DNA adduct

Abstract Lung cancer is initiated by tobacco carcinogens-induced DNA damage, such as DNA adducts caused by benzo[a]pyrene diol epoxide (BPDE). These adducts can be removed by an efficient nucleotide excision repair. We have previously reported that DNA repair capacity (DRC) measured by the host-cell reactivation assay is associated with risk of lung cancer in non-Hispanic whites. In the recent analysis with larger sample sizes including minorities, we found that the suboptimal DRC remains associated with increased risk of lung cancer in 1730 cases and 1683 controls of non-Hispanic whites. The mean values of DRC were 8.37% ± 2.8% (SD) in the cases vs. 9.04% ± 3.1% in the controls (P &amp;lt; 0.0001). The odds ratio (OR) was 1.42 (95% confidence interval [CI], 1.24 – 1.64) after adjustment for age, sex, pack-years of smoking, family history of cancer, and other assay-related covariates. The similar results and risk trend have been also found in 127 Mexican-American patients with lung cancer and 176 cancer-free controls with a mean DRC of 8.38% ± 2.7% in the cases vs. 9.04% ± 3.3% in the controls, and an adjusted OR of 1.67 (95% CI, 1.00 – 2.78), although the difference in mean DRC values was borderline significant (P = 0.056) because of the relatively small sample size. However, this difference in DRC for tobacco carcinogen-induced DNA adducts was not significant in 327 African-American patients with lung cancer and 325 cancer-free controls. The mean values of DRC were 8.90% ± 3.1% in the cases vs. 8.66% ± 2.8% in the controls (P = 0.301). The adjusted OR was 0.99 (95% CI, 0.70 – 1.39). The DRC level in African-American controls was non-significantly lower than other ethnic controls (P = 0.144); however, African-American patients had significantly higher DRC than non-Hispanic white patients and Mexican-Americans patients (P = 0.053). The results further confirmed that low DRC is associated with increased risk of lung cancer in non-Hispanic whites and also provided the evidence of this association in Mexican Americans. These findings help us understand the ethnic differences in the development of lung cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A6.

Benzo[a]pyrene diol epoxide suppresses retinoic acid receptor-β2 expression by recruiting DNA (cytosine-5-)-methyltransferase 3A

Tobacco smoke is an important risk factor for various human cancers, including esophageal cancer. How benzo [a]pyrene diol epoxide (BPDE), a carcinogen present in tobacco smoke as well as in environmental pollution, induces esophageal carcinogenesis has yet to be defined. In this study, we investigated the molecular mechanism responsible for BPDE-suppressed expression of retinoic acid receptor-beta2 (RAR-β2) in esophageal cancer cells. We treated esophageal cancer cells with BPDE before performing methylation-specific polymerase chain reaction (MSP) to find that BPDE induced methylation of the RAR-β2 gene promoter. We then performed chromatin immunoprecipitation (ChIP) assays to find that BPDE recruited genes of the methylation machinery into the RAR-β2 gene promoter. We found that BPDE recruited DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A), but not beta (DNMT3B), in a time-dependent manner to methylate the RAR-β2 gene promoter, which we confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis of the reduced RAR-β2 expression in these BPDE-treated esophageal cancer cell lines. However, BPDE did not significantly change DNMT3A expression, but it slightly reduced DNMT3B expression. DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-Aza) and DNMT3A small hairpin RNA (shRNA) vector antagonized the effects of BPDE on RAR-β2 expressions. Transient transfection of the DNMT3A shRNA vector also antagonized BPDE's effects on expression of RAR-β2, c-Jun, phosphorylated extracellular signal-regulated protein kinases 1/2 (ERK1/2), and cyclooxygenase-2 (COX-2), suggesting a possible therapeutic effect. The results of this study form the link between the esophageal cancer risk factor BPDE and the reduced RAR-β2 expression.

Single Molecule Study on Incorporation Efficiency of DPO4 and Klenow Fragment in the Presence of BPDE Adduct

It's well known that the binding of DNA adducts such as benzo[a]pyrene-diol-epoxide (BPDE) to DNA template strand can impede or block DNA synthesis during the process of DNA replication. While DNA synthesis involving high fidelity replicative A-family polymerases such as Klenow fragment are blocked by DNA adducts, members of Y-family DNA polymerases such as Sulfolobus solfataricus P2 DNA polymerase IV (DPO4) can bypass the DNA adducts and resume the DNA synthesis. Understanding the functional differences between A-family and Y-family DNA polymerases in the process of DNA replication and the mechanism of bypassing DNA adducts is of great value to explain the cause of mutagenesis. We introduce an assay by anchoring DNA molecules to the modified surface to study the incorporation efficiency of DPO4 and Klenow fragment with the presence of BPDE adduct at single molecule level. Specifically, we anchor fluorescent labeled DNA template onto this surface with the adduct site open for nucleotide incorporation, photobleach the labels and flow the polymerases and labeled nucleotides into the hybridization cell. Using Total Internal Reflection Fluorescence Microscopy (TIRFM) we identify the time sequence incorporation of the nucleotides onto the anchored DNA template by identifying the location of the labeled nucleotide from TIRF images. We further quantify the signal densities of the images obtained from the two different polymerases, thus examining whether incorporation reactions have been executed and quantifying the incorporation efficiency of the polymerases.

An aphidicolin-block nucleotide excision repair assay measuring DNA incision and repair capacity

The objective of the present study was to develop a cellular phenotype assay for nucleotide excision repair (NER), using benzo[a]pyrene diol epoxide (BPDE) as model mutagen. Since in vitro exposure to BPDE may lead to DNA strand breaks resulting from both direct interaction with DNA and incisions introduced by the repair enzymes, we aimed to discriminate between both types of breaks using the comet assay and quantified the DNA strand breaks after in vitro challenge of peripheral blood mononucleated cells (PBMCs) with BPDE in the presence or absence of the DNA polymerase inhibitor aphidicolin (APC). The assay was performed with a low (0.5 microM) and a high (2.5 microM) BPDE concentration. The individual NER capacity was defined as the amount of DNA damage induced by BPDE in presence of APC, diminished with the damage induced by BPDE and APC alone. First, the assay was applied to a NER-deficient human fibroblast cell line (XPA-/-) to validate the methodology. Lower repair capacity and a higher amount of BPDE-induced DNA adducts were observed for the XPA-/- fibroblasts as compared to the wild-type fibroblasts. Repeated experiments on PBMCs from four donors showed low intra-individual, intra-experimental and inter-assay variation for both concentrations, indicating the reliability of the method. To assess the inter-individual variation, the assay was applied to PBMCs from 22 donors, comparing the repair capacity after exposure to 0.5 microM (N = 10) and 2.5 microM (N = 12) BPDE. The repair capacity showed a higher inter-individual variation as compared to the intra-individual variation. Moreover, this difference was more pronounced using the low concentration. All these results indicate the adequacy of the method using this low concentration. Further improvement, however, should be recommended by applying the study with low BPDE concentration in a larger population and taking into account the relevant genotypes for NER.

Regulation of Cdc45 in the cell cycle and after DNA damage

The Cdc (cell division cycle) 45 protein has a central role in the regulation of the initiation and elongation stages of eukaryotic chromosomal DNA replication. In addition, it is the main target for a Chk1 (checkpoint kinase 1)-dependent Cdc25/CDK2 (cyclin-dependent kinase 2)-independent DNA damage checkpoint signal transduction pathway following low doses of BPDE (benzo[a]pyrene dihydrodiol epoxide) treatment, which causes DNA damage similar to UV-induced adducts. Cdc45 interacts physically and functionally with the putative eukaryotic replicative DNA helicase, the MCM (mini-chromosome maintenance) complex, and forms a helicase active 'supercomplex', the CMG [Cdc45-MCM2-7-GINS (go-ichi-ni-san)] complex. These known protein-protein interactions, as well as unknown interactions and post-translational modifications, may be important for the regulation of Cdc45 and the initiation of DNA replication following DNA damage. Future studies will help to elucidate the molecular basis of this newly identified S-phase checkpoint pathway which has Cdc45 as a target.

Transforming Growth Factor-alpha reduces carcinogen-induced DNA damage in mini-organ cultures from head-and-neck cancer patients

Epidermal Growth Factor Receptor (EGFR) signalling is particularly important in the biology of the vast majority of solid human malignancies. EGFR is over-expressed in up to 90–100% of head-and-neck squamous cell carcinomas (HNSCC), and increased expression of EGFR and its ligand Transforming Growth Factor- alpha (TGF-α) is not limited to malignant cells, but also detected in histologically normal mucosa of HNSCC patients, supporting the hypothesis of field carcinogenesis. Permanent EGFR activation via an autocrine stimulatory pathway is thought to be a major factor forcing pre-neoplastic tissue towards malignancy. Our study evaluates the impact of stimulation by TGF-α on carcinogen-induced and oxidative DNA damage in mucosa tissue cultures of macroscopically normal biopsies from tumour patients and controls. Effects of TGF-α on DNA-repair capacity were investigated. To assess DNA fragmentation, alkaline single-cell gel electrophoresis (comet assay) was used. Stimulation of cultures during 24 h with TGF-α decreased benzo(a)pyrene diolepoxide (BPDE)-induced DNA damage by 36% in the tumour group ( p < 0.001) and by 7% in controls ( n = 30). No statistically significant impact on oxidatively induced DNA fragmentation in both groups ( n = 15 and 20, respectively), or DNA repair could be shown n ( n = 6). The exact mechanism by which TGF-α stimulation reduces BPDE-induced DNA fragmentation remains unclear. It was shown in clinical studies, that EGFR targeting has synergistic effects with chemotherapy in HNSCC and reverses chemo-resistance of epithelial tumours, which was shown to be the consequence of altered expression of multidrug resistance (mdr) efflux pumps. EGFR downstream signalling regulates the transcription of MDR1 (p-glycoproteine) and glutathione homeostasis. BPDE is a substrate of mdr1 and is detoxified by glutathione conjugation. However, our results show a strong DNA-stabilizing effect of stimulation by TGF-α in mucosa tissue cultures of tumour patients and may therefore be seen as a physiological response to continued carcinogenic impact on the epithelium of the upper aerodigestive tract.

Arsenite enhances the benzo[a]pyrene diol epoxide (BPDE)-induced mutagenesis with no marked effect on repair of BPDE-DNA adducts in human lung cells

Arsenite effects on the benzo[a]pyrene diol epoxide (BPDE)-DNA adduct-induced mutation were evaluated in three human lung cell-lines – A549 (wild-type p53), WI38-VA13 (p53 inhibited by SV40 large-T antigen), and H1299 (p53-null) – by using the pSP189 shuttle vector, which carries a mutation target supF gene. Arsenite alone had no significant effect on the spontaneous supF mutation. BPDE modification of pSP189 enhanced the mutation rates of supF 4.37-fold, 2.96-fold, and 1.95-fold for A549, WI38-VA13, and H1299, respectively. Arsenite potentiated the BPDE-induced mutation rates of supF 2.30-fold, 2.31-fold, and 2.35-fold in A549, WI38-VA13, and H1299, respectively. These results suggest that arsenite potentiates the BPDE-induced supF mutation via a p53-independent mechanism. By using the host cell reactivation assay, we evaluated arsenite effect on repair of BPDE-DNA adducts. We found that the arsenite treatments resulting in relative survival rates ⩾65% had no significant effect on repair of BPDE-DNA adducts, indicating that p53 status did not significantly affect the repair of BPDE-DNA adducts. This study reveals that arsenite enhances the BPDE-DNA adduct-induced mutagenesis with no marked effect on repair of BPDE-DNA adducts, suggesting that arsenic may act as a co-mutagen to promote the development of human lung cancer.

Induction of PUMA-α and down-regulation of PUMA-β expression is associated with benzo(a)pyrene-induced apoptosis in MCF-7 cells

Benzo(a)pyrene (BP) forms benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts in human breast adenocarcinoma MCF-7 cells, leading to p53 protein induction and phosphorylation. Although BP-induced apoptosis in rodent cells is known, it is still unclear in human cells. Here we have analyzed the effects of BP on p53 related apoptotic proteins, cell cycle and cell death in MCF-7 cells. PUMA-protein (p53 up-regulated modulator of apoptosis) levels were changed after BP exposure so that PUMA-alpha protein was statistically significantly increased whereas PUMA-beta protein was statistically significantly decreased. PUMA-protein levels were also investigated in ZR-75-1 cells, where PUMA-alpha protein was statistically significantly increased. Cytochrome c, which is released from mitochondria during apoptosis to form the apoptosome, was increased in cytoplasmic fraction after BP exposure in MCF-7 cells. Increased apoptosis was also seen after 48 and 72 h BP exposure (2.5 and 5 microM). In addition, BP decreased dose dependently cell viability (2.5 and 5 microM) and increased ROS formation (1 and 10 microM). Our results suggest that PUMA-alpha protein is involved in BP-induced cell death most likely through a p53 dependent apoptotic pathway.

Detection of benzo(a)pyrene diol epoxide‐DNA adducts in mononuclear white blood cells by CE immunoassay and its application to studying the effect of glutathione depletion

High levels of benzo(a)pyrene diol epoxide (BPDE)-DNA adducts in white blood cells have been indicated as a risk factor for lung cancer. Sensitive, specific, fast and cost-efficient techniques for the detection of BPDE-DNA adducts in white blood cells are required for routine human biomonitoring. In the present study, an immunoassay based on CE/LIF was developed for the detection of BPDE-DNA adducts in mononuclear white blood cells (MNCs). Although glutathione (GSH) conjugation catalyzed by glutathione-S-transferase (GST) is considered to be the major pathway for inactivating BPDE, the effect of GSH depletion on BPDE-DNA adduct formation in MNCs has not been assessed. Therefore, we applied the newly developed method to study the effect of GSH depletion by D,L-buthionine-[S,R]-sulfoximine (BSO) on the level of DNA adducts. We found that pretreatment of MNCs with 150 microM BSO for 2 h prior to BPDE exposure increased the level of BPDE-DNA adducts appreciably (by approximately 70%). Further investigations revealed that the 2-h BSO treatment neither decreased the GSH level instantly nor affected GST activity; rather, it prevented the induction of GSH in response to subsequent BPDE incubation. The blocked synthesis of GSH might be responsible for the elevated level of BPDE-DNA adducts in MNCs after BSO and BPDE treatment.

Arsenite and its Mono- and Dimethylated Trivalent Metabolites Enhance the Formation of Benzo[a]pyrene Diol Epoxide−DNA Adducts in Xeroderma Pigmentosum Complementation Group A Cells

Recently, inorganic arsenite (iAs(III)) and its mono- and dimethylated metabolites have been examined for their interference with the formation and repair of benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in human cells (Schwerdtle, ., Walter, I., and Hartwig, A. (2003) DNA Repair 2, 1449 - 1463). iAs(III) and monomethylarsonous acid (MMA(III)) were found to be able to enhance the formation of BPDE-DNA adducts, whereas dimethylarsinous acid (DMA(III)) had no enhancing effect at all. The anomaly manifested by DMA(III) prompted us to further investigate the effects of the three trivalent arsenic species on the formation of BPDE-DNA adducts. Use of a nucleotide excision repair (NER)-deficient Xeroderma pigmentosum complementation group A cell line (GM04312C) allowed us to dissect DNA damage induction from DNA repair and to examine the effects of arsenic on the formation of BPDE-DNA adducts only. At concentrations comparable to those used in the study by Schwerdtle et al., we found that each of the three trivalent arsenic species was able to enhance the formation of BPDE-DNA adducts with the potency in a descending order of MMA(III) > DMA(III) > iAs(III), which correlates well with their cytotoxicities. Similar to iAs(III), DMA(III) modulation of reduced glutathione (GSH) or total glutathione S-transferase (GST) activity could not account for its enhancing effect on DNA adduct formation. Additionally, the enhancing effects elicited by the trivalent arsenic species were demonstrated to be highly time-dependent. Thus, although our study made use of short-term assays with relatively high doses, our data may have meaningful implications for carcinogenesis induced by chronic exposure to arsenic at low doses encountered environmentally.

Cooperative activation of the ATR checkpoint kinase by TopBP1 and damaged DNA

TopBP1, acting in concert with DNA containing bulky base lesions, stimulates ATR kinase activity under physiologically relevant reaction conditions. Here, we analyze the roles of the three components in ATR activation: DNA, base damage and TopBP1. We show that base adducts caused by a potent carcinogen, benzo[a]pyrene diol epoxide (BPDE), constitute a strong signal for TopBP1-dependent ATR kinase activity on Chk1 and p53. We find that the C-terminus of TopBP1 binds preferentially to damaged DNA and is sufficient to mediate damaged DNA-dependent ATR activation in a manner similar to full-length TopBP1. Significantly, we find that stimulation of ATR by BPDE-damaged DNA exhibits strong dependence on the length of DNA, with essentially no stimulation with fragments of 0.2 kb and reaching maximum stimulation with 2 kb fragments. Moreover, TopBP1 shows preferential binding to longer DNA fragments and, in contrast to previous biochemical studies, TopBP1 binding is completely independent of DNA ends. We find that TopBP1 binds to circular and linear DNAs with comparable affinities and that these DNA forms elicit the same level of TopBP1-dependent ATR activation. Taken together, these findings suggest a cooperative activation mechanism for the ATR checkpoint kinase by TopBP1 and damaged DNA.

DNA repair phenotype and cancer susceptibility—A mini review

DNA repair is a complicated biological process, consisting of several distinct pathways, that plays a fundamental role in the maintenance of genomic integrity. The very important field of DNA repair and cancer risk has developed rapidly in the past decades. In this review of selected published data from our laboratory, we describe mostly our work on the study of phenotypic markers of nucleotide excision repair (NER), as measured by the benzo(a)pyrene diol epoxide (BPDE)/ultraviolet (UV)-induced mutagen sensitivity assays, BPDE-induced adduct assay, host cell reactivation (HCR)-DNA repair capacity (DRC) assay, reverse transcription-polymerase chain reaction (RT-PCR) assay and reverse-phase protein lysate microarray (RPP) assay, by using peripheral blood lymphocytes in a series of molecular epidemiological studies. Results of our studies suggest that individuals with reduced DRC have an elevated cancer risk. This finding needs additional validation by other investigators, and we also discussed issues in conducting this kind of research in the future.

Involvement of Oxidative Stress and Activation of Aryl Hydrocarbon Receptor in Elevation of CYP1A1 Expression and Activity in Lung Cells and Tissues by Arsenic: An In Vitro and In Vivo Study

Epidemiological evidence indicated that there was a synergistic interaction between arsenic and cigarette smoke on enhancement of lung cancer risk. Benzo[a]pyrene (B[a]P), a component in cigarette smoke, is one of the most carcinogenic compounds known. Animal studies have demonstrated that there were increased benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) adduct formation and lung tumorigenesis in animals when they were coexposed to B[a]P and arsenic. Since BPDE adduct is a by-product of B[a]P metabolism, elevation of B[a]P metabolism by arsenic is suspected. However, the effects of arsenic on cytochrome P450 1A1 (CYP1A1) status (expression and activity), which is essential for B[a]P metabolism, either in lung cells or in lung tissues, are never demonstrated. We hypothesized that arsenic would enhance aryl hydrocarbon receptor (AhR) activation leading to CYP1A1 expression and activity in lung cells. Indeed, our present study successfully demonstrated the elevation of CYP1A1 messenger RNA expression in H1355 cells, a human lung adenocarcinoma cell line, as well as CYP1A1 expression and activity in lung tissues of arsenic-exposed mice. We further demonstrated that this elevation of CYP1A1 expression could be effectively blocked with AhR antagonist, 3',4'-dimethoxyflavone, indicating that the arsenic-induced CYP1A1 expression and activity were via AhR activation. Furthermore, we found that arsenic-induced AhR activation and -enhanced CYP1A1 expression can be further increased by a prooxidant, buthionine-(S,R)-sulfoximine, and suppressed by antioxidants, such as N-acetylcysteine and catalase. Our findings provided clear evidence that arsenic can enhance CYP1A1 expression and activity via AhR activation, and the arsenic-induced AhR activation is probably triggered by oxidative stress.

Endogenous cytosine methylation and the formation of carcinogen carcinogen-DNA adducts

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine ((Me)C, X = Me in Scheme 1). The same sites (e.g. p53 codons 157, 158, 245, 248, and 273) are mutational hotspots in smoking induced lung cancer, suggesting that methylated CG dinucleotides may be preferentially targeted by the reactive metabolites of tobacco carcinogens. We employed a stable isotope labeling HPLC-ESI-MS/MS approach to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diolepoxide metabolites of bay region polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene diol epoxide (BPDE). In contrast, cytosine methylation was protective against O(6)-guanine alkylation by tobacco tobacco-specific nitrosamines, e.g. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), To investigate the mechanisms behind these effects, a series of structural analogs of (Me)C were prepared, and their effects on reactivity of base the paired dG towards BPDE was examined. We found that the presence of the C-5 substituent on cytosine influences the reactivity of its partner guanine towards BPDE and modifies the stereoisomeric composition of the resulting N(2)-BPDE-dG adducts.

Benzo(a)pyrene Diol Epoxide-Induced Chromosome 9p21 Aberrations Are Associated with Increased Risk of Bladder Cancer

Loss of chromosome 9p21 is one of the most frequent genomic alterations in bladder cancer. Alterations of 9p21 and p16 are also frequently seen in the epithelial cells of chronic smokers. We hypothesize that 9p21 is a molecular target of benzo(a)pyrene diol epoxide (BPDE), the metabolic product of tobacco carcinogen benzo(a)pyrene, and 9p21 BPDE sensitivity is a genetic susceptibility factor for bladder cancer. In this case-control study of 203 bladder cancer cases and 198 matched healthy controls, we compared the frequencies of BPDE-induced 9p21 aberrations in cultured peripheral blood lymphocytes using fluorescent in situ hybridization and evaluated the association between 9p21 BPDE sensitivity and bladder cancer risk. We found that BPDE-induced chromosome 9p21 aberrations were significantly higher in peripheral blood lymphocytes of bladder cancer cases (20.76 +/- 6.97 per 1,000) than those of controls (16.58 +/- 7.07 per 1,000; P < 0.0001). However, no difference was observed for CEP9, a control centromere locus on chromosome 9. Using the median aberration value in the controls as a cutoff point to dichotomize BPDE sensitivity and after adjustment by age, sex, ethnicity, and smoking status, 9p21 BPDE sensitivity was associated with a significantly increased risk of bladder cancer (odds ratio, 5.29; 95% confidence interval, 3.26-8.59), whereas the odds ratio for the CEP9 locus was 0.99 (95% confidence interval, 0.66-1.50). There was also a dose-response relationship between the 9p21 BPDE sensitivity and increased risk for bladder cancer. 9p21 may be a molecular target for BPDE damage in bladder cancer cases and 9p21 BPDE sensitivity may be a marker of bladder cancer susceptibility.

Particulate matter inhibits DNA repair and enhances mutagenesis

Exposure to ambient air pollution has been associated with adverse health effects including lung cancer. A recent epidemiology study has established that each 10 μg/m 3 elevation in long-term exposure to average PM 2.5 ambient concentration was associated with approximately 8% of lung cancer mortality. The underlying mechanisms of how PM contributes to lung carcinogenesis, however, remain to be elucidated. We have recently found that transition metals such as nickel and chromium and oxidative stress induced lipid peroxidation metabolites such as aldehydes can greatly inhibit nucleotide excision repair (NER) and enhance carcinogen-induced mutations. Because PM is rich in metal and aldehyde content and can induce oxidative stress, we tested the effect of PM on DNA repair capacity in cultured human lung cells using in vitro DNA repair synthesis and host cell reactivation assays. We found that PM greatly inhibits NER for ultraviolet (UV) light and benzo( a)pyrene diol epoxide (BPDE) induced DNA damage in human lung cells. We further demonstrated that PM exposure can significantly increase both spontaneous and UV-induced mutagenesis. These results together suggest that the carcinogenicity of PM may act through its combined effect on suppression of DNA repair and enhancement of DNA replication errors.

Development of human cell models for assessing the carcinogenic potential of chemicals

To develop human cell models for assessing the carcinogenic potential of chemicals, we established transgenic human cell lines and tested the sensitivity of known carcinogens using a cell transformation assay. A retroviral vector encoding an oncogenic allele of H-Ras (HBER) or c-Myc (HBEM) was introduced into human bronchial epithelial cells (HBE) immortalized by SV40 large T (LT) antigen, leading to increased cell proliferation but failing to confer a transformed phenotype characterized by anchorage-independent cell growth and tumor formation of immunodeficient mice. When these pre-transformed cells were treated with nickel sulfate (NiSO 4), we found that it shortened the latency of malignant transformation at least by 19 wk in HBER cells or 16 wk in HBEM cells compared to vector control cells. Similarly, the latency of cell transformation was shorter by 15 wk in HBER cells or 9 wk in HBEM cells when cells were treated with benzo(a)pyrenediol epoxide (BPDE). HBER cells appeared to be more sensitive to TPA, NiSO 4 or BPDE-induced cell transformation compared to human embryonic kidney cells expressing H-Ras (HEKR), implying that cell-type specificity is one of important factors determining the effectiveness of the assay. Using AFB 1 and BaP as the representative pro-carcinogens, we also compared the efficiency of three different metabolic conditions in mediating cell transformation. Low dose chemical induction seems to be a prospective system used for metabolic activation of pro-carcinogens. Our findings provided direct evidence that a genetically modified human cell transformation model can be applied to the assessment of potent carcinogens.

Association between Common Genetic Variation in Cockayne Syndrome A and B Genes and Nucleotide Excision Repair Capacity among Smokers

Mutagen sensitivity in in vitro cultured lymphocytes challenged by benzo[a]pyrene diolepoxide (BPDE) has been validated as an intrinsic susceptibility factor for several cancers. Bulky BPDE-DNA adducts are repaired via either transcription-coupled repair or global genome nucleotide excision repair depending on the location of lesions. Cockayne syndrome A (CSA) and B (CSB) play essential roles in integrating the recognition of damage, chromatin remodeling, and the core nucleotide excision repair proteins. This study evaluated the hypothesis that common genetic variation in CSA and CSB is associated with mutagen sensitivity induced by BPDE in 276 cancer-free smokers. Tag single nucleotide polymorphisms (SNP; n = 37) selected across the entire coding and putative regulatory regions of CSA and CSB based on a high-density SNP database were genotyped by the Illumina Golden Gate assay. Major principal components of CSA and CSB that captured the linkage disequilibrium from multiple SNPs were globally associated with the number of breaks per cell at the threshold of 80% (P < or = 0.02 for both genes). Haplotype H125 in CSA and H97 in CSB as well as SNPs in high linkage disequilibrium with these two haplotypes were significantly associated with a 13% to 15% reduction in the mean number of chromatid breaks per cell (P < 0.05). A resampling-based omnibus test supported the significant association between SNPs and haplotypes in CSA and mutagen sensitivity induced by BPDE (P = 0.035). This study implicates transcription-coupled repair in protecting the cell from BPDE-induced DNA damage.

Genotypes and haplotypes of ERCC1 and ERCC2/XPD genes predict levels of benzo[a]pyrene diol epoxide-induced DNA adducts in cultured primary lymphocytes from healthy individuals: a genotype-phenotype correlation analysis

Benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts are a risk factor for tobacco-related cancers. Excision repair cross-complementing complementation group 1 (ERCC1) and excision repair cross-complementing complementation group 2/xeroderma pigmentosum D (ERCC2/XPD) participate in the nucleotide excision repair (NER) pathway that removes BPDE-DNA adducts; however, few studies have provided population-based evidence for this association. Therefore, we assayed for levels of in vitro BPDE-induced DNA adducts and genotypes of single-nucleotide polymorphisms (SNPs) of the NER genes ERCC1 (rs3212986 and rs11615) and ERCC2/XPD (rs13181, rs1799793 and rs238406) in 707 healthy non-Hispanic whites. The linear trend test of increased adduct values in never to former to current smokers was statistically significant (P(trend) = 0.0107). The median DNA adduct levels for the ERCC2 rs1799793 GG, GA and AA genotypes were 23, 29 and 30, respectively (P(trend) = 0.057), but this trend was not observed for other SNPs. After adjustment for covariates, adduct values larger than the median value were significantly associated with the genotypes ERCC1 rs3212986TT [odds ratio (OR) = 1.89, 95% confidence interval (CI) = 1.03-3.48] and ERCC2/XPD rs238406AA (OR = 0.64, 95% CI = 0.41-0.99) and rs238406CA (OR = 0.63, 95% CI = 0.45-0.89) compared with their corresponding wild-type homozygous genotypes. The results of haplotype analysis further suggested that haplotypes CAC and CGA of ERCC2/XPD, TC of ERCC1 and CACTC of ERCC2/XPD and ERCC1 were significantly associated with high levels of DNA adducts compared with their most common haplotypes. Our findings suggest that the genotypes and haplotypes of ERCC1 and ERCC2/XPD may have an effect on in vitro BPDE-induced DNA adduct levels.