Benzo[a]pyrene Diol Epoxide Research Articles

Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer.

Mutations in ras genes are commonly found in human cancers and in animal models. Although mutations at codons 12, 13, and 61 of H-, N- and K-ras genes can activate their oncogenic function, mutations at codon 12 of K-ras are the most common mutations found among the three ras genes in human cancers. To investigate whether codon 12 of human K-ras is especially susceptible to carcinogens and/or whether carcinogen-DNA adducts at this codon are repaired less efficiently, we examined tobacco smoke carcinogen-induced DNA damage in normal human bronchial epithelial and fibroblast cells. We used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction to map the distribution of DNA adducts induced by benzo[a]pyrene diol epoxide (BPDE) and other bulky carcinogens within exons 1 and 2 in H-ras, N-ras, and K-ras. We also analyzed BPDE-DNA adduct repair efficiency in these three genes using the same method. Codons 12 and 14 of the K-ras gene were hotspots for carcinogen-DNA adduct formation, with little and no adduct formation at codons 13 and 61, respectively. The BPDE-DNA adducts formed at codon 14 were repaired almost twice as quickly as those formed at codon 12. There was some BPDE-DNA adduct formation at codons 12 of H-ras and N-ras, but this codon was not a hotspot. Furthermore, no substantial difference in repair rates between codon 12 and the other codons analyzed (codons 3 and 18) was observed in either the H-ras or N-ras genes. These findings link the human cancer mutational hotspot at codon 12 of K-ras to preferential DNA damage and poor repair.

Smoking, lung cancers and their TP53 mutations.

The TP53 suppressor gene possesses several properties that have facilitated its use as a reporter of genotoxic exposure (Biggs et al., 1993). In particular, it is mutated in a high proportion of many types of human cancer and it sustains a broad spectrum of mutations that can vary in both their position and type. In lung tumours from smokers, the high frequency of G→T transversions (30% compared with 10% in cancers without tobacco aetiology) has been attributed to DNA adducts of polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene, that are present in tobacco smoke (Hainaut and Pfeifer, 2001). Benzo[a]pyrene is activated to form benzo[a]pyrene diol epoxide (BPDE), which reacts with DNA predominantly at the N2-position of guanine, and both in vitro and in vivo studies have demonstrated that BP and BPDE can, in common with other PAHs, induce G→T transversions (Wei et al., 1991; DeMarini et al., 1995; Schiltz et al., 1999; Wijnhoven et al., 2000). A single point mutation in the TP53 gene results in a base change in both DNA strands with, for example, a G→T mutation in one strand corresponding to a C→A change in the complementary strand (G:C→T:A). The question then arises whether such a change was induced by modification of G in one strand or of the C in the complementing strand. Recently, Rodin and Rodin (2000) examined the spectra of mutations that they believed were induced by damage of each of the two DNA strands of TP53 and concluded that when each strand was examined individually, there were no significant differences between the spectra in lung adenocarcinomas in smokers and non-smokers. The plot of mutations that represents the core of their argument and around which other arguments and conclusions in their paper are based is shown in Figure 1A, where all 12 possible base pair substitution mutations are grouped into six pairs, each consisting of two complementary substitutions. In this figure the mutations above the line represent the changes presumed to be induced by damage in the non-transcribed strand (NTS) of TP53 whereas those below the line represent the same changes induced in the transcribed strand (TS). For example, on the left-hand side, the G→T above the line represents a mutation induced in the NTS, whereas C→A below the line represents the G→T mutation induced in the TS. (Note that by convention all mutations are referred to by the mutation found in the NTS.) The data as presented show: (i) that above the central line the ratio of the mutation frequency in smokers to that of the same mutations in non-smokers remains approximately constant; (ii) that a similar relationship, albeit with a different value of the ratio,

Binding stoichiometry of DNA adducts with antibody studied by capillary electrophoresis and laser-induced fluorescence.

Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE-16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE-16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE- 16-mer and an unlabeled BPDE-90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR-BPDE-16-mer molecule, antibody with two TMR-BPDE- 16-mer molecules, and antibody with one TMR-BPDE-16-mer and one BPDE-90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange.

Retinal Pigment Epithelial Cell DNA is Damaged by Exposure to Benzo[ a ]pyrene, a Constituent of Cigarette Smoke

This study examined the effect of exogenous benzo[ a ]pyrene (BaP), an important constituent of cigarette smoke, on cultured bovine retinal pigment epithelial (RPE) cells. Evidence is presented for its metabolic conversion into benzo[ a ]pyrene diol epoxide (BPDE) and the consequent formation of potentially cytotoxic nucleobase adducts in DNA. Cultured RPE cells were treated with BaP at concentrations in the range of 0–100μM. The presence of BaP was found to cause inhibition of cell growth and replication. BaP induced the expression of a phase I drug metabolizing enzyme which was identified as cytochrome P450 1A1 (CYP 1A1) by RT–PCR and by Western blotting. Coincident with the increased expression of CYP 1A1, covalent adducts between the mutagenic metabolite BPDE and DNA could be detected within RPE cells by immunocytochemical staining. Additional support for their formation was afforded by nuclease P1 enhanced 32P-postlabelling assays on cellular DNA. Single-cell gel electrophoresis (comet) assays showed that exposure of RPE cells to BaP rendered them markedly more susceptible to DNA damage induced by broad band UVB or blue light laser irradiation. In the case of UVB, this is consistent with the photosensitization of DNA cleavage by nucleobase adducts of BPDE. Collectively, these findings imply that BaP has a significant impact on RPE cell pathophysiology and suggest mechanisms whereby exposure to cigarette smoke might cause RPE dysfunction and cell death, thus possibly contributing to degenerative disorders of the retina.

Different Effects on Human Topoisomerase I by Minor Groove and Intercalated Deoxyguanosine Adducts Derived from Two Polycyclic Aromatic Hydrocarbon Diol Epoxides at or Near a Normal Cleavage Site

Topoisomerase I (top1) relieves supercoiling in DNA by forming transient covalent cleavage complexes. These cleavage complexes can accumulate in the presence of damaged DNA or anticancer drugs that either intercalate or lie in the minor groove. Recently we reported that covalent diol epoxide (DE) adducts of benzo[a]pyrene (BaP) at the exocyclic amino group of G(+1) block cleavage at a preferred cleavage site ( approximately CTT-G(+1)G(+2)A approximately ) and cause accumulation of cleavage products at remote sites. In the present study, we have found that the 10S G(+2) adduct of BaP DE, which lies toward the scissile bond in the minor groove, blocks normal cleavage, whereas the 10R isomer, which orients away from this bond, allows normal cleavage but blocks religation. In contrast to BaP, the pair of benzo[c] phenanthrene (BcPh) DE adducts at G(+2), which intercalate from the minor groove either between G(+1)/G(+2) or between G(+2)/A, allow normal cleavage but block religation. Both intercalated BcPh DE adducts at G(+1) suppress normal cleavage, as do both groove bound BaP DE adducts at this position. These studies demonstrate that these DE adducts provide a novel set of tools to study DNA topoisomerases and emphasize the importance of contacts between the minor groove and top1's catalytic site.

Removal of benzo(a)pyrene diol epoxide (BPDE)-DNA adducts as a measure of DNA repair capacity in lymphoblastoid cell lines from sisters discordant for breast cancer.

The mutagen sensitivity assay is one of the approaches used to investigate individual DNA repair capacity. This method is based on the premise that after in vitro treatment with a test mutagen, DNA from subjects with defective repair will be more damaged than DNA from those with an efficient repair system. However, very little is known about unmeasured processes that occur between cell treatment and final assessment of DNA damage. To develop a more precise assay, we modified the traditional mutagen sensitivity assay to also include measurement of DNA damage after culturing cells in the absence of mutagen. First, we treated apparently normal and xeroderma pigmentosum lymphoblastoid cell lines with various doses of benzo(a)pyrene diol epoxide (BPDE) and harvested cells at different time points. A polyclonal antiserum against BPDE-DNA was used to quantitate levels of adducts by immunoslot-blot and immunohistochemistry. Selected conditions included treatment with 10 microM BPDE, a 4-hr culture in mutagen-free medium, and immunohistochemical measurement of BPDE-DNA adducts. The method was then applied in a pilot study to 50 lymphoblastoid lines from sisters discordant for breast cancer. There was no significant difference between cases and controls in the level of BPDE-DNA adducts in lymphoblasts harvested immediately after BPDE treatment. However, after a 4-hr culture in mutagen-free medium, the level of adducts was significantly higher (P = 0.006) among cases than in controls. There was a two-fold increase in mean adduct removal in lines from nonaffected as compared to affected sisters (44% and 22% decrease, respectively). DNA repair capacity was predictive of case status (P = 0.04) in logistic regression analysis. This method, which can be easily applied to large numbers of samples, should be useful in studies to investigate the role of DNA repair in cancer risk.

Effect of soluble and particulate nickel compounds on the formation and repair of stable benzo[a]pyrene DNA adducts in human lung cells.

Nickel compounds are well-known human carcinogens, but the underlying mechanisms are still not fully understood. Even though only weakly mutagenic, nickel chloride has been shown previously to impair the repair of UV-induced DNA damage as well as oxidative DNA damage. However, the carcinogenic potential depends largely on solubility, with poorly water-soluble nickel subsulfide and nickel oxide being strong carcinogens. Within the present study we investigated the effects of particulate black NiO and soluble NiCl(2) on the induction and removal of stable DNA adducts formed by benzo[a]pyrene (B[a]P) measured by a highly sensitive high performance liquid chromatography (HPLC)/fluorescence assay. With respect to adduct formation, NiO but not NiCl(2) reduced the generation of DNA lesions by approximately 30%. Regarding their repair, in the absence of nickel compounds, most lesions were removed within 24 h; nevertheless, between 20 and 35% of induced adducts remained even 48 h after treatment. NiCl(2) and NiO reduced the removal of adducts in a dose-dependent manner. Thus, 100 microM NiCl(2) led to approximately 80% residual repair capacity; after 500 microM the repair was reduced to approximately 36%. Also, even at the completely non-cytotoxic concentration of 0.5 microg/cm(2) black NiO, lesion removal was reduced to approximately 35% of control and to 15% at 2.0 microg/cm(2). Furthermore, both nickel compounds increased the benzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE)-induced cytotoxicity. Taken together, our results indicate that the nucleotide excision repair pathway is affected in general by water-soluble and particulate nickel compounds and provide further evidence that DNA repair inhibition may be one predominant mechanism in nickel-induced carcinogenicity.

Synthesis, characterization, and applications of a fluorescent probe of DNA damage.

We have designed and generated a 90-mer oligonucleotide that contains a single adduct of benzo[a]pyrene diol epoxide (BPDE) and that is fluorescently labeled. The known amount of BPDE adduct in a given length of DNA makes this probe a useful standard for DNA damage assay. The BPDE-90-mer was fluorescently labeled with tetramethylrhodamine to allow for high sensitivity detection with laser-induced fluorescence (LIF). The binding of both double-stranded and single-stranded BPDE-90-mer with three anti-BPDE antibodies was studied using affinity capillary electrophoresis (CE). Formation of antibody complex with BPDE-90-mer results in a shift in migration time from that of the unbound BPDE-90-mer. Affinity CE/LIF studies suggest that antibody 8E11 has high-affinity suitable for immunoassay of BPDE-DNA adducts. A competitive immunoassay using the fluorescent probe and CE/LIF is demonstrated for the analysis of BPDE-DNA adducts in A549 human lung carcinoma cells incubated with 2.5, 5, and 10 microM BPDE for 2 h. The design of the 90-mer probe is flexible to substitute different DNA damage types with relative ease. The fluorescent 90-mer is composed of six shorter oligonucleotides. The sequence of the two center oligonucleotides may be changed depending on the desired DNA lesion measurement. By inserting different damaged oligonucleotides, a variety of DNA damage systems can be investigated using the same CE/LIF approach.

Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects sperm from DNA damage and apoptosis caused by benzo( a)pyrene

Benzo(a)pyrene (BaP), an aryl hydrocarbon receptor (AhR) ligand present in cigarette smoke and car exhaust, is thought to have negative effects on male reproduction. We hypothesized that BaP damages sperm through AhR activation, phase 1 enzyme induction, DNA adduct formation, and increased germ cell apoptosis in the testis, and that resveratrol, a natural competitive inhibitor of the AhR found in some red wines, could prevent the adverse effects of BaP on sperm. Male Balb C mice were injected subcutaneously (s.c.) for 5 weeks with a range of BaP doses (0.5 mg/kg to 50 mg/kg). Live sperm were obtained from the vas deferens, counted, and stained to measure annexin-V positive (apoptotic) cells. In a subsequent study, mice were injected for 5 weeks with corn oil (control), BaP (5 mg/kg/week), or BaP plus resveratrol (50 mg/kg/week) ( n = 3 per group). Immunohistochemistry (IHC) was performed on testis sections for the determination of CYP1A1, BaP diol epoxide (BPDE) DNA adducts, and apoptosis and the results quantified by using the HSCORE, a semiquantitative scoring system. Our results demonstrated that sperm counts after 5 weeks were inversely correlated to BaP dosage. BaP (0.5 to 5 mg/week) positively correlated with sperm apoptosis while higher doses increased sperm necrosis. CYP1A1 protein was observed mainly in interstitial cells of some testis sections, but there was no significant induction by BaP. BPDE DNA adducts were induced in all components of the seminiferous tubules by BaP and suppressed by resveratrol: median HSCORE (interquartile range) control 61 (52—71.5); BaP 213 (192—248), P = 0.01 compared to control; BaP plus resveratrol 83 (70—90). BaP significantly increased apoptosis, mainly in spermatogonia: medain HSCORE (interquartile range) BaP 189 (161—223) versus control 83 (57—93), P < 0.01; and this effect was abrogated by resveratrol. Median HSCORE for BaP plus resveratrol was 112 (range 99–121). In summary, BaP caused increased sperm cell BPDE DNA adduct formation and apoptosis in the mouse. The natural AhR antagonist, resveratrol diminished BaP-induced DNA adducts and apoptosis in seminiferous tubules.

Patterns of resistance to exonuclease digestion of oligonucleotides containing polycyclic aromatic hydrocarbon diol epoxide adducts at N6 of deoxyadenosine.

The effect of adduct stereochemistry on the susceptibility to hydrolysis by snake venom (VPD) and bovine spleen (SPD) phosphodiesterases was investigated with short deoxyoligonucleotides containing defined adducts derived from alkylation of the exocyclic 6-amino group of dA by polycyclic aromatic hydrocarbon diol epoxides (DEs). In accordance with several earlier reports, we have found that adducts with R configuration at the site of attachment of dA to the DE moiety derived from either benzo[a]pyrene (BaP) or benzo[c]phenanthrene (BcPh) are generally more resistant to hydrolysis by VPD than are their (S)-diastereomers. The reaction with VPD initially yields a fragment containing the adducted dA residue at its 3'-end, which slowly hydrolyzes to a dimer (pXpA*) with an intact 5'-phosphodiester bond to the adducted dA. With several of the adducts studied, this dimer underwent cleavage to release eventually the monomeric adduct p(dA*). Adducts derived from cis opening of the epoxide ring of both BaP and BcPh DEs were considerably more resistant to VPD than the corresponding trans-opened adducts. Although several previous investigations had suggested that oligonucleotides containing adducts which have S configuration at the site of attachment of the hydrocarbon to adenine are more resistant to cleavage by SPD than are their (R)-diastereomers, the present results with a more extensive set of oligonucleotides indicate that SPD, in contrast to VPD, exhibits little discrimination between adducts with R and S configuration at the site of attachment to the base. Notably, for both enzymes, the most resistant internucleotide linkage (the bond 3'-sugar to phosphate for VPD and 5'-sugar to phosphate for SPD) is between the modified base and the base immediately 5' to it, regardless of the configuration of the adduct.

Impact of GSTM1 on aromatic-DNA adducts and p53 accumulation in human skin and lymphocytes.

The cellular response to DNA damage is often a p53-mediated cell cycle arrest to provide time for DNA repair or to direct damaged cells into apoptosis. In this study, the impact of glutathione-S-transferase M1 (GSTM1) on DNA damage and subsequent p53-protein accumulation was examined in lymphocytes of healthy volunteers in vitro exposed to benzo[a]pyrene-diol-epoxide (BPDE) and in skin of atopic eczema patients topically treated with coal tar. DNA adducts were determined by immunocytochemical staining (ICC) and 32P-postlabelling, p53 accumulation was studied by ICC and the GSTM1 genotype was assessed by polymerase chain reaction. In cultured lymphocytes treated with 2.5 microM BPDE for 18 h, increased levels of p53 were found, which were positively related to BPDE-DNA adduct levels assessed by ICC (rs = 0.66, P < 0.001) and 32P-postlabelling (rs = 0.56, P < 0.001) and appeared to be higher in GSTM1(-/-) than in GSTM1(+) subjects (P = 0.003). In skin biopsies of coal tar treated eczema patients, p53 levels were elevated in 7/10 patients and a correlation was observed between p53 and DNA adduct levels (rs = 0.50, P = 0.029). GSTM1(-/-) subjects contained higher levels of p53 in the stratum basale than GSTM1(+) individuals (P = 0.026), but no influence of GSTM1 on DNA adduct levels was observed. Thus, p53 accumulates in human skin and lymphocytes as a protective mechanism against polycyclic aromatic hydrocarbon induced DNA damage, and this is more pronounced in GSTM1(-/-) compared to GSTM1(+) individuals.

Detection of DNA adducts of benzo[ a]pyrene using immunoelectrophoresis with laser-induced fluorescence : Analysis of A549 cells

Detection of benzo[ a]pyrene diol epoxide (BPDE)-damaged DNA in a human lung carcinoma cell line (A549) has been performed using free zone affinity capillary electrophoresis with laser-induced fluorescence (LIF). Using BPDE as a model carcinogenic compound, the speed, sensitivity and specificity of this technique was demonstrated. Under free zone conditions, an antibody bound adduct was baseline-resolved from an unbound adduct in less than 2 min. The efficiencies of separation were in excess of 6·10 5 and 1·10 6 plates per meter for the antibody-bound and unbound adducts, respectively. Separation using a low ionic strength buffer permitted the use of a high electric field (830 V/cm) without the loss of resolving power. Using LIF detection, a concentration detection limit of roughly 3·10 −10 M was achieved for a 90-mer oligonuleotide containing a single BDPE. The use of formamide in the incubation buffer to enhance denaturing of DNA did not affect the stability of the complex between the antibody and the adducts. Using a fluorescently labeled BPDE-modified DNA adduct probe, a competitive assay was established to determine the levels of BPDE–DNA adducts in A549 cells.

Myeloperoxidase--463A variant reduces benzo[a]pyrene diol epoxide DNA adducts in skin of coal tar treated patients.

The skin of atopic dermatitis patients provides an excellent model to study the role of inflammation in benzo[a]pyrene (BaP) activation, since these individuals are often topically treated with ointments containing high concentrations of BaP. In this study we have determined, by HPLC with fluorescence detection, the BaP diol epoxide (BPDE)-DNA adduct levels in human skin after topical treatment with coal tar and their modulation by the -463G-->A myeloperoxidase (MPO) polymorphism, which reduces MPO mRNA expression. BPDE-DNA adduct levels were 2.2 and 14.2 adducts/10(8) nt for MPO-463AA/AG and -463GG, respectively. The predominant BaP tetrol observed was tetrol I-1, which is derived after hydrolysis of the anti-BPDE-DNA adduct. The tetrol I-1/II-2 ratio, corresponding to the anti/syn ratio, was 6.7. The (32)P-post-labeling assay was also performed and thin layer chromatograms showed a major spot with a chromatographic location corresponding to BPDE-DNA. The mean values of the BPDE-DNA adduct spots were 3.8 +/- 2.4 per 10(8) nt for MPO-463AA/AG (n = 3) and 18.4 +/- 11.0 per 10(8) nt for MPO-463GG (n = 7), respectively (P = 0.03). One individual with the homozygous mutant genotype (-463AA) even had a 13-fold lower adduct level (1.4 per 10(8) nt) as compared to MPO-463GG subjects. In conclusion, these data show for the first time: (i) the in vivo formation of BPDE-DNA adducts in human skin treated with coal tar; (ii) that the MPO-463AA/AG genotype reduced BPDE-DNA adduct levels in human skin.

Studies of dose distribution, premutagenic events and mutation frequencies for benzo[a]pyrene aiming at low dose cancer risk estimation.

Cancer risk assessment of polycyclic aromatic hydrocarbons (PAH) is complicated by several of these compounds exerting a promoter action leading to high tumour incidences at high doses. Cancer risks at low doses corresponding to the uptake from air and food in the general environment would best be estimated on the basis of measurement of in vivo target doses of genotoxic (mutagenic) intermediates and a determination of mutation frequency per unit of dose. In experiments ultimately aiming at a risk assessment of environmental PAH from in vivo doses benzo[a]pyrene (BaP) was chosen as a model. gamma-Radiation has earlier been used as a reference standard in cancer risk estimation of genotoxic chemicals where dose equivalents (rad-equivalents) have been shown to give reliable risk estimates for several alkylating agents. Variation in dose of BaP diolepoxide between organs was studied by measurement of deoxyguanosine-N(2) adducts in DNA after administration of BaP by gavage to mice of a strain with reduced DNA repair (Xpa(-/-)). The adduct levels in spleen, forestomach, stomach and small intestine were approximately the same; with the adduct level in spleen as reference it was twice as high in liver and lung and about half as high in colon tissue. A chemical or radiation dose is proportional to the cumulative frequency of putatively premutagenic changes (premutagenic hits) in DNA. The mutation frequency per premutagenic hit (genotoxic chemicals) and per unit of dose (gamma-radiation) were calculated from acutely exposed V79 cells in order to determine the mutagenic effectiveness of each agent. Based on the mutagenic effectiveness determined in this study 10(-4) Gy can be regarded equally effective in causing phenotypically expressed HPRT mutations as the dose of BaP which causes the formation of one deoxyguanosine-N(2) adduct per cell.

Stereochemical, structural, and thermodynamic origins of stability differences between stereoisomeric benzo[a]pyrene diol epoxide deoxyadenosine adducts in a DNA mutational hot spot sequence.

Benzo[a]pyrene (BP), a prototype polycyclic aromatic hydrocarbon (PAH), can be metabolically activated to the enantiomeric benzo[a]pyrene diol epoxides (BPDEs), (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the (-)-(7S,8R,9R,10S) enantiomer. These can react with adenine residues in DNA, to produce the stereoisomeric 10S (+)- and 10R (-)-trans-anti-[BP]-N(6)-dA adducts. High-resolution NMR solution studies indicate that in DNA duplexes the 10R (-) adduct is intercalated on the 5'-side of the modified adenine, while the 10S (+) adduct is disordered, exhibits multiple adduct conformations, and is positioned on the 3'-side of the modified adenine. Duplexes containing the 10S (+) adduct positioned at A within codon 61 of the human N-ras sequence CAA are thermodynamically less stable and more easily excised by human DNA repair enzymes than those containing the 10R (-) adduct. However, the molecular origins of these differences are not understood and represent a fascinating opportunity for elucidating structure-function relationships. We have carried out a computational investigation to uncover the structural and thermodynamic origins of these effects in the 11-mer duplex sequence d(CGGACAAGAAG).d(CTTCTTGTCCG) by performing a 2-ns molecular dynamics simulation using NMR solution structures as the basis for the starting models. Then, we applied the MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) method to compute free energy differences between the stereoisomeric adducts. The 10R (-) isomer is more stable by approximately 13 kcal/mol, of which approximately 10 kcal/mol is enthalpic, which agrees quite well with their observed differences in thermodynamic stability. The lower stability of the 10S (+) adduct is due to diminished stacking by the BP moiety in the intercalation pocket, more helix unwinding, and a diminished quality of Watson-Crick base pairing. The latter stems from conformational heterogeneity involving a syn-anti equilibrium of the glycosidic bond in the modified adenine residue. The lower stability and conformational heterogeneity of the 10S (+) adduct may play a role in its enhanced susceptibility to nucleotide excision repair.

Microarray expression profiles in breast cancer MCF-7 cells following exposure to benzo[a]pyrene and its diol epoxide

Maintenance of genome integrity in mammalian cells may be compromised by the concomitant accumulation of DNA damage and loss of repair functions. Polycyclic aromatic hydrocarbons are classic DNA-damaging and tumor-promoting agents. Benzo[a]pyrene (B[a]P), a prototype PAH, induces several genotoxic responses, including cell cycle arrest, oxidative stress and DNA adduct formation. Exposure of MCF-7 cells to B[a]P (1−5 M) and the B[a]P diol epoxide (BPDE) (50−100 nM) induced transient S-phase arrest, which was followed by accumulation in G2 and M and 70% loss of cell viability. In cells treated with B[a]P and BPDE, we observed segregation of nucleolar material and a drastic reduction in the number of ribosomal fibrillar centers. To identify the expression profile in cells treated with B[a]P or BPDE, we analyzed the levels of expression of 1,152 selected genes using a GeneMAP CancerArray in control and treated cells. Changes in gene expression were evaluated twice in duplicate (n=4). Compared with the expression levels in untreated cells, transcripts that were upregulated by both B[a]P and BPDE included members of the cytochrome P450, AP-1 (Fra-1, Fra-2) and glutathione S-transferase families; PARP; and G1/S-specific cyclins. In contrast, transcript levels for BRCA-1, G2/M cyclins, bcl-2, junB and fosB were downregulated in cells treated with B[a]P or BPDE. Treatment with the metabolite BPDE tended to elicit a stronger response than treatment with B[a]P. Analysis of expression profiles by microarray may be useful to understand the interactions among overlapping pathways and assess exposure to environmental carcinogens.

Effect of Benzo[a]pyrene Diol Epoxide on Expression of Retinoic Acid Receptor-β in Immortalized Esophageal Epithelial Cells and Esophageal Cancer Cells

Expression of retinoic acid receptor-beta (RAR-β) is frequently lost in tobacco-related cancers. Benzo[a]pyrene diol epoxide (BPDE) is an active metabolite of tobacco procarcinogen benzo[a]pyrene and plays an important role in tobacco carcinogenesis. We therefore exposed SV-40 immortalized esophageal epithelial cells and esophageal cancer cells to BPDE to understand possible interactions between BPDE and RAR-β expression. Our data showed that BPDE decreased RAR-β mRNA and protein levels by suppression of transcription of RAR-β. Retinoic acid was able to partially block the inhibitory effect of BPDE on RAR-β expression and to increase G1 phase of cell cycles. Furthermore, induction of COX-2 expression by BPDE was associated with RAR-β inhibition. This study suggests that one way by which BPDE causes esophageal carcinogenesis may be through the inhibition of RAR-β.

Resveratrol Protects Sperm from DNA Damage and Apoptosis Caused by Exposure to Environmental Toxicants

Background: Benzo(a)pyrene (BaP) is a dioxin-like chemical present in cigarette smoke and car exhaust. It is an aryl hydrocarbon receptor (AhR) ligand with known negative effects on male reproduction. Men who smoke have an increased incidence of sperm BaP diol epoxide (BPDE) DNA adducts that may be transmitted to their offspring. We hypothesized that resveratrol, a natural inhibitor of the AhR, could suppress BaP induced CYP1A1 induction, prevent DNA adduct formation and decrease sperm apoptosis.

In vivo formation of (+)- anti-benzo[ a]pyrene diol-epoxide–plasma albumin adducts in fish

Benzo[ a]pyrene (BaP), a procarcinogenic polycyclic aromatic hydrocarbon (PAH), is bioactivated to BaP diol-epoxides (BPDEs) that can form adducts with DNA and blood proteins. We report here for the first time the in vivo formation of adducts between BPDE and plasma albumin (Alb) from two fish species experimentally exposed to BaP. Brook trout ( Salvelinus fontinalis) received either a single i.p. dose (10 mg/kg) or two separate i.p. doses (25 mg/kg; 7 days apart) of BaP, and blood was collected 2 (single exposure) or 3 (multiple exposure) days post-treatment. Arctic charr ( Salvelinus alpinus) received 10 i.p. doses (3 mg/kg; a single dose every 6 days), and blood was collected 2 days after the second, sixth, and 10th injections. BPDE–Alb adducts were measured by an improved HPLC/fluorescence method developed to detect and quantify BaP-tetrols released after acid hydrolysis of adducted Alb. HPLC/fluorescence chromatograms of Alb from BaP-treated fish revealed only BaP-tetrol I-1, thus indicating the formation of adducts exclusively via the (+)- anti-BPDE metabolite. Levels of (+)- anti-BPDE–Alb adduct ranged from 0.68 to 19.6 ng of tetrol I-1 per gram of Alb. Notably, adduct level was not related to BaP dose and there was no accumulation of adducts with repeated exposure, which may indicate a very short half-life (<2 days) of plasma Alb in fish. The data suggest that BPDE–Alb adducts in fish could be useful as a non-destructive biomarker of recent exposure to bioactivated BaP.

DNA Repair of Benzo[A]Pyrene Diol Epoxide-DNA Adducts in the DHFR Gene of a Human Embryonic Kidney Cell Line

The repair of polycyclic aromatic hydrocarbon (PAH)-DNA adducts by nucleotide excision repair is a critical factor in the human response to exposure to these environmental carcinogens. In this study, we utilized a laser cleavage technique and Southern Blotting to measure the repair of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) adducts in the transcribed and nontranscribed strands of a human cell line containing an amplified DHFR gene. Treatment of the cultures with 1.5 μM BPDE resulted in little adduct removal for 24 hours, whereas 1 μM BPDE-treated cells removed most of the adducts over a 24 hour period. Southern analyses of repair in individual strands of the human cell line revealed that adducts on the transcribed strand are repaired more rapidly than those on the non-transcribed strand during the first 8 hours after 1 μM BPDE treatment. These results suggest that human cells can repair BPDE-DNA adducts through a transcription-coupled repair process.