Base damage, local sequence context and TP53 mutation hotspots: a molecular dynamics study of benzo[a]pyrene induced DNA distortion and mutability.

The mutational pattern for the TP53 tumour suppressor gene in lung tumours differs to other cancer types by having a higher frequency of G:C>T:A transversions. The aetiology of this differing mutation pattern is still unknown. Benzo[a]pyrene,diol epoxide (BPDE) is a potent cigarette smoke carcinogen that forms guanine adducts at TP53 CpG mutation hotspot sites including codons 157, 158, 245, 248 and 273. We performed molecular modelling of BPDE-adducted TP53 duplex sequences to determine the degree of local distortion caused by adducts which could influence the ability of nucleotide excision repair. We show that BPDE adducted codon 157 has greater structural distortion than other TP53 G:C>T:A hotspot sites and that sequence context more distal to adjacent bases must influence local distortion. Using TP53 trinucleotide mutation signatures for lung cancer in smokers and non-smokers we further show that codons 157 and 273 have the highest mutation probability in smokers. Combining this information with adduct structural data we predict that G:C>T:A mutations at codon 157 in lung tumours of smokers are predominantly caused by BPDE. Our results provide insight into how different DNA sequence contexts show variability in DNA distortion at mutagen adduct sites that could compromise DNA repair at well characterized cancer related mutation hotspots.

Quantitative analysis of 3-OHB[a]P and (+)-anti-BPDE as biomarkers of B[a]P exposure in rats.

The aim of this study was to develop an analytical method for the determination the levels of metabolites of benzo[a]pyrene (B[a]P), 3-hydroxybenzo(a)pyrene (3-OHB[a]P) and (+)-anti-benzo(a)pyrene diol-epoxide [(+)-anti-BPDE, combined with DNA to form adducts], in rat blood and tissues exposed to B[a]P exposure by high-performance liquid chromatography with fluorescence detection (HPLC/FD), and to investigate the usefulness of 3-OHB[a]P and (+)-anti-BPDE as markers of intragastrical exposure to B[a]P in rats. The levels of 3-OH-B[a]P and B[a]P-tetrol I-1 released after acid hydrolysis of (+)-anti-BPDE in the samples were measured by HPLC/FD. The calibration curves were linear (r(2) > 0.9904), and the lower limit of quantification ranged from 0.34 to 0.45 ng/mL for 3-OHB[a]P and from 0.43 to 0.58 ng/mL for (+)-anti-BPDE. The intra- and inter-day stability assay data suggested that the method is accurate and precise. The recoveries of 3-OHB[a]P and (+)-anti-BPDE were in the ranges of 73.6 ± 5.0 to 116.5 ± 6.3% and 73.3 ± 8.5 to 141.2 ± 13.8%, respectively. A positive correlation was found between the concentration of intragastrical B[a]P and the concentrations of 3-OH-B[a]P and (+)-anti-BPDE in the blood and in most of the tissues studied, except for the brain and kidney, which showed no correlation between B[a]P and 3-OHB[a]P and between B[a]P and (+)-anti-BPDE, respectively. A sensitive, reliable and rapid HPLC/FD was developed and validated for analysis of 3-OHB[a]P and (+)-anti-BPDE in rat blood and tissues. There was a positive correlation between the concentration of 3-OHB[a]P or (+)-anti-BPDE in the blood and the concentration of 3-OHB[a]P or (+)-anti-BPDE in the most other tissues examined. The concentration of 3-OHB[a]P or (+)-anti-BPDE in the blood could be used as an indicator of the concentration of 3-OHB[a]P or (+)-anti-BPDE in the other tissues in response to B[a]P exposure. These results demonstrate that 3-OHB[a]P and (+)-anti-BPDE are potential biomarkers of B[a]P exposure, which would also be useful to assess the carcinogenic risks from B[a]P exposure.

Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore.

The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2'-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5' or 3' directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

Association between cigarette smoking and the vaginal microbiota: a pilot study.

BACKGROUND: Smoking has been identified in observational studies as a risk factor for bacterial vaginosis (BV), a condition defined in part by decimation of Lactobacillus spp. The anti-estrogenic effect of smoking and trace amounts of benzo[a]pyrene diol epoxide (BPDE) may predispose women to BV. BPDE increases bacteriophage induction in Lactobacillus spp. and is found in the vaginal secretions of smokers. We compared the vaginal microbiota between smokers and non-smokers and followed microbiota changes in a smoking cessation pilot study. METHODS: In 2010-2011, 20 smokers and 20 non-smokers were recruited to a cross-sectional study (Phase A) and 9 smokers were enrolled and followed for a 12-week smoking cessation program (Phase B). Phase B included weekly behavioral counseling and nicotine patches to encourage smoking cessation. In both phases, participants self-collected mid-vaginal swabs (daily, Phase B) and completed behavioral surveys. Vaginal bacterial composition was characterized by pyrosequencing of barcoded 16S rRNA genes (V1-V3 regions). Vaginal smears were assigned Nugent Gram stain scores. Smoking status was evaluated (weekly, Phase B) using the semi-quantitative NicAlert® saliva cotinine test and carbon monoxide (CO) exhalation. RESULTS: In phase A, there was a significant trend for increasing saliva cotinine and CO exhalation with elevated Nugent scores (P value <0.005). Vaginal microbiota clustered into three community state types (CSTs); two dominated by Lactobacillus (L. iners, L. crispatus), and one lacking significant numbers of Lactobacillus spp. and characterized by anaerobes (termed CST-IV). Women who were observed in the low-Lactobacillus CST-IV state were 25-fold more likely to be smokers than those dominated by L. crispatus (aOR: 25.61, 95 % CI: 1.03-636.61). Four women completed Phase B. One of three who entered smoking cessation with high Nugent scores demonstrated a switch from CST-IV to a L.iners-dominated profile with a concomitant drop in Nugent scores which coincided with completion of nicotine patches. The other two women fluctuated between CST-IV and L. iners-dominated CSTs. The fourth woman had low Nugent scores with L. crispatus-dominated CSTs throughout. CONCLUSION: Smokers had a lower proportion of vaginal Lactobacillus spp. compared to non-smokers. Smoking cessation should be investigated as an adjunct to reducing recurrent BV. Larger studies are needed to confirm these findings.

Hydrogen-bonded intermediates and transition states during spontaneous and acid-catalyzed hydrolysis of the carcinogen (+)-anti-BPDE.

Understanding mechanisms of (+)-anti-BPDE detoxification is crucial for combating its mutagenic and potent carcinogenic action. However, energetic-structural correlations of reaction intermediates and transition states during detoxification via hydrolysis are poorly understood. To gain mechanistic insight we have computationally characterized intermediate and transition species associated with spontaneous and general-acid catalyzed hydrolysis of (+)-anti-BPDE. We studied the role of cacodylic acid as a proton donor in the rate limiting step. The computed activation energy (ΔG‡) is in agreement with the experimental value for hydrolysis in a sodium cacodylate buffer. Both types of, spontaneous and acid catalyzed, BPDE hydrolysis can proceed through low-entropy hydrogen bonded intermediates prior to formation of transition states whose energies determine reaction activation barriers and rates.

Effect of carcinogenic acrolein on DNA repair and mutagenic susceptibility.

Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic α- and γ-hydroxy-1, N(2)-cyclic propano-2'-deoxyguanosine adducts (α-OH-Acr-dG and γ-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair and mismatch repair. Although Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2, and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity.

Electrochemical study on the effects of epigenetic cytosine methylation on anti-benzo[a]pyrene diol epoxide damage at TP53 oligomers.

Anti-benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (anti-BPDE) is a known carcinogen that damages DNA, and this damage is influenced by the DNA sequence and epigenetic factors. The influence of epigenetic cytosine methylation on the reaction with anti-BPDE at a known hotspot DNA damage site was studied electrochemically. Gold electrodes were modified with thiolated DNA oligomers spanning codons 270-276 of the TP53 gene. The oligomers exhibited 5-carbon cytosine methylation at the codon 273 location on the bound probe, the acquired complementary target, or both. Redox active diviologen compounds of the form C(12)H(25)V(2+)C(6)H(12)V(2+)C(12)H(25) (V(2+) = 4,4'-bipyridyl or viologen, C12-Viologen) were employed to detect anti-BPDE damage to DNA. DNA was exposed to racemic (±)- or enantiomerically pure (+)-anti-BPDE solutions followed by electrochemical interrogation in the presence of C12-Viologen. Background subtracted square wave voltammograms (SWV) showed the appearance of two peaks at approximately -0.38 V and -0.55 V vs Ag/AgCl upon anti-BPDE exposure. The acquired voltammetry is consistent with singly reduced C12-Viologen dimers bound at two different DNA environments, which arise from BPDE damage and are influenced by cytosine methylation and BPDE stereochemical considerations. UV spectroscopic and mass spectrometric methods employed to validate the electrochemical responses showed that (+)-anti-BPDE primarily adopts a minor groove bound orientation within the oligomers while selectively targeting the nontranscribed ssDNA sequence within the duplexes.

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA.

Endogenous 5-methylcytosine ((Me)C) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational 'hotspots' for smoking induced lung cancer. (Me)C enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5'-CCCGGCACCC GC[(15)N(3),(13)C(1)-G]TCCGCG-3', + strand) were prepared containing [(15)N(3), (13)C(1)]-guanine opposite unsubstituted cytosine, (Me)C, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2'-deoxynucleosides, N(2)-BPDE-dG adducts formed at the [(15)N(3), (13)C(1)]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N(2)-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N(2) position of guanine.

Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons.

We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 µg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30-60%) than levels of bulky DNA adducts measured in the same samples by (32)P-postlabelling. The BPDE-DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE-DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between (32)P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.

Phenylalanine 171 is a molecular brake for translesion synthesis across benzo[a]pyrene-guanine adducts by human DNA polymerase kappa.

Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol κ, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol κ and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol κ to template/primer DNA containing (-)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N(2)-dG by Pol κ and that the F171A derivative of Pol κ bypasses these DNA lesions more actively than does the wild-type enzyme.

Fluorescently imaged particle counting immunoassay for sensitive detection of DNA modifications.

Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE-DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE-DNA adducts as low as 180 fM in the presence of 1 billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methylation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotoxical and epigenetic analysis.

Evaluation of DNA adducts, DNA and RNA oxidative lesions, and 3-hydroxybenzo(a)pyrene as biomarkers of DNA damage in lung following intravenous injection of the parent compound in rats.

Biomarkers of exposure and effect were assessed in 40 male Sprague-Dawley rats injected intravenously with 40 micromol/kg of benzo(a)pyrene (BaP) to determine which biomarkers are more representative of BaP-induced DNA damage in lung. Lung, liver, blood, and urine were collected at t = 2, 4, 8, 16, 24, 33, 48, 72, and 360 h postdosing. Specific BaP-diol epoxide (BPDE)-DNA adducts, 8-hydroxy-7,8-dihydro-2'-deoxyguanosine (8-OHdGuo), were measured in lung, liver, and mononucleated blood cells by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Urinary 8-OHdGuo and 8-hydroxy-7,8-dihydroguanosine (8-OHGuo) were also determined by HPLC-MS/MS, and urinary 3-hydroxybenzo(a)pyrene was measured by HPLC/fluorescence. Between 2 and 72 h postdosing, BPDE-DNA adducts were significantly increased in lung, liver, and mononucleated blood cells of BaP-treated rats as compared to controls, with the highest levels found in lung. 8-OHdGuo levels also increased in lung of BaP-treated rats with values reaching statistical significance at 2, 8, and 16 h postinjection. No influence of BaP treatment was found on 8-OHdGuo and 8-OHGuo urinary excretions. BPDE-DNA adducts in lung were strongly correlated to urinary 3-OHBaP (r = 0.936 and p < 0.001) and to a lesser extent to blood BPDE-DNA adducts (r = 0.636 and p < 0.001), the latter of which were correlated to each other (r = 0.573 and p = 0.002). Urinary 3-OHBaP and BPDE-DNA adducts in mononucleated blood cells appear as relevant biomarkers of BaP genotoxic exposure and are highly promising for health risk assessment in humans.

Particulate matter air pollution and the expression of microRNAs and pro-inflammatory genes: Association and mediation among children in Jinan, China.

Exposure to particulate matter (PM) has been associated with increased risk of various diseases, possibly through its effect on inflammatory response. MicroRNAs (miRNAs), an epigenetic mechanism regulating gene expression, can affect the expression of pro-inflammatory genes. However, few epidemiological studies have examined the impact of PM on inflammation-related miRNAs and their target mRNAs, especially among vulnerable population. We recruited 160 and 113 children from areas with different PM level in Jinan, China. We measured benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydotetrol-albumin (BPDE-Alb) adducts in serum and the expression of 5 candidate miRNAs involved in inflammation regulation and 7 pro-inflammatory genes predicted to be their targets in leukocytes. Generally, children in the polluted area had higher miRNAs and lower mRNAs expression than those in the control area. An interquartile increase of BPDE-Alb adducts was associated with 12.66 %, 14.13 %, and 12.76 % higher of let-7a, miR-146a-5p, and miR-155-5p, as well as 21.61 %, 20.16 %, and 12.49 % lower of IL-6, CXCL8, and TLR2 mRNAs at false discovery rate<0.05, respectively. Additionally, let-7a, miR-146a-5p, and miR-155-5p were found to mediate the associations of BPDE-Alb adducts with IL-6 and/or TLR2 expression. Our findings suggested that PM exposure might attenuate inflammatory response among children in China, which was partly mediated by miRNAs regulating pro-inflammatory genes.

The DNA damage-sensing NER repair factor XPC-RAD23B does not recognize bulky DNA lesions with a missing nucleotide opposite the lesion.

The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were -2.5-3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 ß-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes.

DNA as an in vitro trapping agent for detection of bulky genotoxic metabolites.

The instability of electrophilic reactive metabolites in in vitro metabolism studies makes their accurate analysis challenging. To stabilise the reactive compounds prior to their analysis, different trapping agents, such as thiols, amines and cob(I)alamin, have earlier been tested depending on the metabolites to be analysed and the type of study. In the present work, DNA is introduced as a trapping agent for measuring the formation of bulky electrophilic metabolites. Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), was used as a model compound in a rat liver S9 metabolic system. Under physiological incubation conditions, B[a]P metabolises to diol epoxide (BPDE) metabolites which were trapped by DNA resulting in the formation of covalently bound DNA adducts. The methodology for analysis of these adducts included extraction of the DNA from the metabolic system, digestion of the DNA to yield nucleosides and analysis of the BPDE-adduct to deoxyguanosine (BPDE-dG) by liquid chromatography coupled to high resolution mass spectrometry (HRMS). The chromatographic conditions in combination with the high mass accuracy data (±3 ppm) was useful in resolving BPDE-dG in its protonated form from the complex set of ions present in the metabolic matrix. The method was validated in terms of sensitivity, specificity, accuracy, precision and recovery, and applied to provide a preliminary estimate of BPDE-dG levels from the metabolism of B[a]P in rat S9. The use of DNA as a trapping agent for in vitro metabolites has a potential to aid in cancer risk assessment procedure of PAHs, for instance, in inter-species comparison of metabolism to reactive metabolites and can be adapted for screening of genotoxic metabolites, e.g., from emerging environmental contaminants.

Synthesis and characterization of DNA fluorescent probes containing a single site-specific stereoisomer of anti-benzo[a]pyrene diol epoxide-N2-dG.

We present a comprehensive study of synthesis and characterization of DNA probes containing covalently bound benzo[a]pyrene diol epoxide (BPDE) isomers at a defined site. Short oligonucleotides of 16mers containing a single trans-(+)- or trans-(-)-anti-BPDE-N(2)-guanine adducts (BPDE-16mer) were first synthesized and then ligated with a fluorescently labeled single-stranded oligonucleotide. The ligation products (double-stranded or single-stranded 90mers) contained a single BPDE adduct of defined stereochemistry and a fluorescent label. The BPDE adduct could be recognized by a specific antibody, and the fluorescent label was useful for highly sensitive laser-induced fluorescence detection. The incorporation of single BPDE in the 16mers was validated by liquid chromatography, UV spectroscopy, and tandem mass spectrometry analysis. The stereochemistry of the single BPDE adducts in the 16mers was further identified by enzyme digestion-coupled stereoselective chromatography analysis. The ligation of BPDE-16mer with normal oligonucleotides for the synthesis of tetramethylrhodamine (TMR)-BPDE-90mers was evaluated. It was found that the modification of the 16mer by anti-BPDE could significantly reduce the ligation yield of ds90mer and lead to the formation of gapped DNA. The incorporation of a single anti-BPDE adduct in ligated ds90mers was confirmed using an antibody specific to the anti-BPDE-dG and affinity capillary electrophoresis. The detection limits of the TMR-BPDE-90mers by capillary electrophoresis coupled with laser-induced fluorescence are below 4 x 10(-19) mol.

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.

Detection of phosphodiester adducts formed by the reaction of benzo[a]pyrene diol epoxide with 2'-deoxynucleotides using collision-induced dissociation electrospray ionization tandem mass spectrometry.

In this study, we investigated the products formed following the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE) with 2'-deoxynucleoside 3'-monophosphates. The B[a]PDE plus 2'-deoxynucleotide reaction mixtures were purified using solid phase extraction (SPE) and subjected to HPLC with fluorescence detection. Fractions corresponding to reaction product peaks were collected and desalted using SPE prior to analysis for the presence of molecular ions corresponding to m/z 648, 632, 608 and 623 [M-H]- consistent with B[a]PDE adducted (either on the base or phosphate group) 2'-deoxynucleotides of guanine, adenine, cytosine and thymine, respectively, using LC-ESI-MS/MS collision-induced dissociation (CID). Reaction products were identified having CID product ion spectra containing product ions at m/z 452, 436 and 412 [(B[a]Ptriol+base)-H]-, resulting from cleavage of the glycosidic bond between the 2'-deoxyribose and base, corresponding to B[a]PDE adducts of guanine, adenine and cytosine, respectively. Further reaction products were identified having unique CID product ion spectra characteristic of B[a]PDE adduct formation with the phosphate group of the 2'-deoxynucleotide. The presence of product ions at m/z 399 and 497 were observed for all four 2'-deoxynucleotides, corresponding to [(B[a]Ptriol+phosphate)-H]- and [(2'-deoxyribose+phosphate+B[a]Ptriol)-H]-, respectively. In conclusion, this investigation provides the first direct evidence for the formation of phosphodiester adducts by B[a]PDE following reaction with 2'-deoxynucleotides.

Detection of BPDE-DNA adducts in human umbilical cord blood by LC-MS/MS analysis.

Benzo [a]pyrene (BaP) is a model compound for the study of polycyclic aromatic hydrocarbon (PAH) carcinogenesis. Upon metabolism, BaP is metabolized to the ultimate metabolite, BaP trans-7,8-diol-anti-9,10-epoxide (BPDE), that reacts with cellular DNA to form BPDE-dG adducts responsible for BaP-induced mutagenicity, carcinogenicity, and teratogenicity. In this study, we employed our developed LC-MS/MS method to detect and quantity BPDE-dG adducts present in 42 normal human umbilical cord blood samples and 42 birth defect cases. We determined that there is no significant difference in the level of BPDE-dG formation between the normal and birth defect groups. This represents the first time to use an LC-MS/MS method to quantify BPDE-dG in human umbilical blood samples. The results indicated that under experimental conditions, BPDE-dG adducts were detected in all the human umbilical cord blood samples from the normal and birth defect groups.

Poleta, Polzeta and Rev1 together are required for G to T transversion mutations induced by the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts in yeast cells.

Benzo[a]pyrene is an important environmental mutagen and carcinogen. Its metabolism in cells yields the mutagenic, key ultimate carcinogen 7R,8S,9S,10R-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, (+)-anti-BPDE, which reacts via its 10-position with N2-dG in DNA to form the adduct (+)-trans-anti-BPDE-N2-dG. To gain molecular insights into BPDE-induced mutagenesis, we examined in vivo translesion synthesis and mutagenesis in yeast cells of a site-specific 10S (+)-trans-anti-BPDE-N(2)-dG adduct and the stereoisomeric 10R (-)-trans-anti-BPDE-N2-dG adduct. In wild-type cells, bypass products consisted of 76% C, 14% A and 7% G insertions opposite (+)-trans-anti-BPDE-N2-dG; and 89% C, 4% A and 4% G insertions opposite (-)-trans-anti-BPDE-N2-dG. Translesion synthesis was reduced by approximately 26-37% in rad30 mutant cells lacking Poleta, but more deficient in rev1 and almost totally deficient in rev3 (lacking Polzeta) mutants. C insertion opposite the lesion was reduced by approximately 24-33% in rad30 mutant cells, further reduced in rev1 mutant, and mostly disappeared in the rev3 mutant strain. The insertion of A was largely abolished in cells lacking either Poleta, Polzeta or Rev1. The insertion of G was not detected in either rev1 or rev3 mutant cells. The rad30 rev3 double mutant exhibited a similar phenotype as the single rev3 mutant with respect to translesion synthesis and mutagenesis. These results show that while the Polzeta pathway is generally required for translesion synthesis and mutagenesis of the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts, Poleta, Polzeta and Rev1 together are required for G-->T transversion mutations, a major type of mutagenesis induced by these lesions. Based on biochemical and genetic results, we present mechanistic models of translesion synthesis of these two DNA adducts, involving both the one-polymerase one-step and two-polymerase two-step models.