In vitro metabolism of benzo[a]pyrene-7,8-dihydrodiol and dibenzo[def,p]chrysene-11,12 diol in rodent and human hepatic microsomes.

Polycyclic aromatic hydrocarbons (PAHs) are contaminants that are ubiquitously found in the environment, produced through combustion of organic matter or petrochemicals, and many of which are procarcinogens. The prototypic PAH, benzo[a]pyrene (B[a]P) and the highly carcinogenic dibenzo[def,p]chrysene (DBC) are metabolically activated by isoforms of the P450 enzyme superfamily producing benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol), dibenzo[def,p]chrysene-11,12 diol (DBC diol). Each of these diols can be further metabolized by cytochrome P450 enzymes to highly reactive diol-epoxide metabolites that readily react with DNA or by phase II conjugation facilitating excretion. To complement prior in vitro metabolism studies with parent B[a]P and DBC, both phase I metabolism and phase II glucuronidation of B[a]P diol and DBC diol were measured in hepatic microsomes from female B6129SF1/J mice, male Sprague-Dawley rats, and female humans. Metabolic parameters, including intrinsic clearance and Michaelis-Menten kinetics were calculated from substrate depletion data. Mice and rats demonstrated similar B[a]P diol phase I metabolic rates. Compared to rodents, human phase I metabolism of B[a]P diol demonstrated lower overall metabolic capacity, lower intrinsic clearance at higher substrate concentrations (>0.14µM), and higher intrinsic clearance at lower substrate concentrations (<0.07µM). Rates of DBC diol metabolism did not saturate in mice or humans and were highest overall in mice. Higher affinity constants and lower capacities were observed for DBC diol glucuronidation compared to B[a]P diol glucuronidation; however, intrinsic clearance values for these compounds were consistent within each species. Kinetic parameters reported here will be used to extend physiologically based pharmacokinetic (PBPK) models to include the disposition of B[a]P and DBC metabolites in animal models and humans to support future human health risk assessments.

Regioselective Benzoylation of Diols and Carbohydrates by Catalytic Amounts of Organobase.

A novel metal-free organobase-catalyzed regioselective benzoylation of diols and carbohydrates has been developed. Treatment of diol and carbohydrate substrates with 1.1 equiv. of 1-benzoylimidazole and 0.2 equiv. of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in MeCN under mild conditions resulted in highly regioselective benzoylation for the primary hydroxyl group. Importantly, compared to most commonly used protecting bulky groups for primary hydroxyl groups, the benzoyl protective group offers a new protection strategy.

Nuclear respiratory factor 1 overexpression attenuates anti-benzopyrene­7,8-diol-9,10-epoxide-induced S-phase arrest of bronchial epithelial cells.

Nuclear respiratory factor 1 (NRF-1) has important roles in the regulation of several key metabolic genes required for cellular growth and respiration. A previous study by our group indicated that NRF­1 is involved in mitochondrial dysfunction induced by the environmental pollutant benzo[a]pyrene in the 16HBE human bronchial epithelial cell line. In the present study, it was observed that its genotoxic metabolite, anti­benzopyrene­7,8­diol­9,10­epoxide (BPDE), triggered cell cycle arrest in S­phase in 16HBE cells by activating ataxia-telangiectasia (ATM)/checkpoint kinase (Chk)2 and ATM and Rad3 related (ATR)/Chk1 signaling pathways. NRF­1 expression was suppressed by BPDE after treatment for 6 h. Flow cytometric analysis revealed that NRF­1 overexpression attenuated cell cycle arrest in S­phase induced by BPDE. In line with this result, DNA­damage checkpoints were activated following NRF­1 overexpression, as demonstrated by increased phosphorylation of ATM, Chk2 and γH2AX, but not ATR and Chk1, according to western blot analysis. It was therefore indicated that NRF­1 overexpression attenuated BPDE­induced S­phase arrest via the ATM/Chk2 signaling pathway.

New insights into BaP-induced toxicity: role of major metabolites in transcriptomics and contribution to hepatocarcinogenesis.

Benzo(a)pyrene (BaP) is a ubiquitous carcinogen resulting from incomplete combustion of organic compounds and also present at high levels in cigarette smoke. A wide range of biological effects has been attributed to BaP and its genotoxic metabolite BPDE, but the contribution to BaP toxicity of intermediary metabolites generated along the detoxification path remains unknown. Here, we report for the first time how 3-OH-BaP, 9,10-diol and BPDE, three major BaP metabolites, temporally relate to BaP-induced transcriptomic alterations in HepG2 cells. Since BaP is also known to induce AhR activation, we additionally evaluated TCDD to source the expression of non-genotoxic AhR-mediated patterns. 9,10-Diol was shown to activate several transcription factor networks related to BaP metabolism (AhR), oxidative stress (Nrf2) and cell proliferation (HIF-1α, AP-1) in particular at early time points, while BPDE influenced expression of genes involved in cell energetics, DNA repair and apoptotic pathways. Also, in order to grasp the role of BaP and its metabolites in chemical hepatocarcinogenesis, we compared expression patterns from BaP(-metabolites) and TCDD to a signature set of approximately nine thousand gene expressions derived from hepatocellular carcinoma (HCC) patients. While transcriptome modulation by TCDD appeared not significantly related to HCC, BaP and BPDE were shown to deregulate metastatic markers via non-genotoxic and genotoxic mechanisms and activate inflammatory pathways (NF-κß signaling, cytokine-cytokine receptor interaction). BaP also showed strong repression of genes involved in cholesterol and fatty acid biosynthesis. Altogether, this study provides new insights into BaP-induced toxicity and sheds new light onto its mechanism of action as a hepatocarcinogen.

Hypoxia diminishes the detoxification of the environmental mutagen benzo[a]pyrene.

Hypoxia promotes genetic instability and is therefore an important factor in carcinogenesis. We have previously shown that activation of the hypoxia responsive transcription factor HIFα can enhance the mutagenic phenotype induced by the environmental mutagen benzo[a]pyrene (BaP). To further elucidate the mechanism behind the ability of hypoxia to increase mutagenicity of carcinogens, we examined the activation and detoxification of BaP under hypoxic conditions. To this end, the human lung carcinoma cell line A549 was treated with BaP under 20%, 5% or 0.2% oxygen for 18h and alterations in BaP metabolism were assayed. First, BaP-induced expression of key metabolic enzymes was analysed; expression levels of the activating CYP1A1 and CYP1B1 were increased, while the detoxifying enzymes UGT1A6 and UGT2B7 were significantly reduced by hypoxia. To evaluate whether these changes had an effect on metabolism, levels of BaP and several of its metabolites were determined. Cells under hypoxia have a reduced capacity to metabolise BaP leaving more of the parent molecule intact. Additionally, BaP-7,8-dihydrodiol, the pre-cursor metabolite of the reactive metabolite BaP-7,8-dihydroxy-9,10-epoxide (BPDE), was formed in higher concentrations. Finally, under hypoxia, DNA adducts accumulated over a period of 168 h, whereas adducts were efficiently removed in 20% oxygen conditions. The delayed detoxification kinetics resulted in a 1.5-fold increase in DNA adducts. These data indicate that the metabolism under hypoxic conditions has shifted towards increased activation of BaP instead of detoxification and support the idea that modulation of carcinogen metabolism is an important additional mechanism for the observed HIF1 mediated genetic instability.

Inhibition of HIV-1 reverse transcriptase-catalyzed synthesis by intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide adducts.

To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT), this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6)-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

Polycyclic aromatic hydrocarbons stimulate human CYP3A4 promoter activity via PXR.

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) is mediated mainly by cytochrome P450 monooxygenases (CYP) CYP1A1, 1A2 and 1B1. Several PAH are known to induce these CYP via aryl hydrocarbon receptor (AhR) signaling. Recently, it was shown that the PAH benzo[a]pyrene (BaP) can induce CYP3A4 as well. The induction was suggested to be mediated by the pregnane X receptor (PXR) rather than AhR. Metabolism by CYP3A4 is only known for dihydrodiol metabolites of PAH but not for their parent compounds. In the present study, a CYP3A4 reporter gene assay, requiring the overexpression of PXR, was used to investigate whether the PAH parent compounds BaP, benzo[c]phenanthrene (BcP) and dibenzo[a,l]pyrene (DBalP) as well as their corresponding phase I metabolites, the respective dihydrodiols and diol epoxides, can induce CYP3A4 promoter activity. BaP, BcP and their dihydrodiols were found to significantly activate the CYP3A4 promoter. Moreover, activation of PXR by all four compounds was detected by using a PXR transactivation assay, supporting that PXR mediates CYP3A4 induction by PAH. Taken together, these results show that both PAH parent compounds as well as their phase I metabolites induce CYP3A4 promoter via the transcription factor PXR.

Aldo-keto reductases protect lung adenocarcinoma cells from the acute toxicity of B[a]P-7,8-trans-dihydrodiol.

Tobacco smoke exposure stimulates the expression of genes that are likely to be involved in the metabolism of its combustion products such as polycyclic aromatic hydrocarbons (PAH). Four of the smoke induced genes are aldo-keto reductases (AKR), enzymes that metabolically activate PAH to PAH o-quinones. Alternatively, PAHs are metabolized to (±)-anti-diol epoxides, such as (±)-anti-benzo[a]pyrene diol epoxide ((±)-anti-BPDE)), by the combined action of P4501A1/1B1 and epoxide hydrolase. (±)-anti-BPDE forms DNA adducts directly, while PAH o-quinones cause DNA damage by oxidative stress through a futile redox cycle. To address the role of AKRs in PAH cytotoxicity, we compared the cytotoxicity of PAH metabolites and the effects of overexpressing AKR1A1 in lung cells. (±)-anti-BPDE and B[a]P-7,8-trans-dihydrodiol, an intermediate in (±)-anti-BPDE metabolism, are toxic to A549 cells at concentrations with an IC(50) of ∼2 µM. In contrast, the PAH o-quinone B[a]P-7,8-dione was about 10-fold less toxic to A549 cells with an IC(50) > 20 µM. Similar differences in cytoxicity were observed with two other PAH o-quinones (benz[a]anthracene-3,4-dione and 7,12-dimethylbenz[a]anthracene-3,4-dione) compared with their respective diol-epoxide counterparts (BA-3,4-diol-1,2-epoxide and DMBA-3,4-diol-1,2-epoxide). In addition, both anti-BPDE and B[a]P-7,8-trans-dihydrodiol induced p53 expression ∼6 h post-treatment at concentrations as low as 1 µM consistent with extensive DNA damage. B[a]P-7,8-dione treatment did not induce p53 but generated reactive oxygen species (ROS) in A549 cells and induced the expression of oxidative response genes in H358 cells. We also observed that overexpression of AKR1A1 in H358 cells, which otherwise have low levels of AKR expression, protected cells 2-10-fold from the toxic effects of B[a]P-7,8-trans-dihydrodiol. These data suggest that overexpression of AKRs may protect lung cancer cells from the acute toxic effects of PAH.

Fatty acid hydroperoxides support cytochrome P450 2S1-mediated bioactivation of benzo[a]pyrene-7,8-dihydrodiol.

In the accompanying report (p. 1031), we showed that a novel dioxin-inducible cytochrome P450, CYP2S1, efficiently metabolizes benzo[a]pyrene-trans-7,8-dihydrodiol (BaP-7,8-diol) into the highly mutagenic and carcinogenic benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BaP-diol-t-epoxide), using cumene hydroperoxide in lieu of NADPH/O(2). Lipid hydroperoxide-supported P450 oxidation has been reported in several cases. However, it has not yet been described for the bioactivation of BaP-7,8-diol. In this report, we demonstrate that CYP2S1 can use various fatty acid hydroperoxides to support epoxidation of BaP-7,8-diol at a much higher rate than with cumene hydroperoxide. Kinetic analyses with several fatty acid hydroperoxides revealed that 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) was the most potent oxidant tested (K(m), 3.4 +/- 0.8 microM; turnover, 4.51 +/- 0.13 min(-1)), followed by 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K(m), 2.8 +/- 0.7 microM; turnover, 3.7 +/- 0.1 min(-1)), 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (K(m), 2.7 +/- 0.8 microM; turnover, 3.69 +/- 0.09 min(-1)), and 15S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K(m), 11.6 +/- 0.3 microM; turnover, 0.578 +/- 0.030 min(-1)). The antioxidant butylated hydroxyanisole inhibited CYP2S1-catalyzed epoxidation by 100%, suggesting that epoxidation proceeds by a free radical mechanism. Other cytochromes P450, including CYP1A1, CYP1B1, CYP1A2, and CYP3A4, were also able to epoxidize BaP-7,8-diol using various fatty acid hydroperoxides, although at slower rates than CYP2S1. The cytotoxicity of BaP-7,8-diol significantly increased in mammalian cells overexpressing CYP2S1, and BaP-diol-t-epoxide formation in these cells also increased in the presence of 13-HpODE. Together, these results suggest that fatty acid hydroperoxides can serve as physiological cofactors in supporting in vivo CYP2S1-catalyzed oxidation of BaP-7,8-diol, and that fatty acid hydroperoxides and CYP2S1 may play important roles in benzo[a]pyrene-induced carcinogenesis.

Differential protection by human glutathione S-transferase P1 against cytotoxicity of benzo[a]pyrene, dibenzo[a,l]pyrene, or their dihydrodiol metabolites, in bi-transgenic cell lines that co-express rat versus human cytochrome P4501A1.

Polycyclic aromatic hydrocarbons (PAHs) are activated by cytochrome P450 (CYP) isozymes, and a subset of the reactive metabolites generated is detoxified via conjugation with glutathione (GSH) by specific glutathione S-transferases (GSTs). We have used V79MZ cells stably transfected with either human or rat cytochrome P4501A1 (CYP1A1), alone or in combination with human GSTP1 (hGSTP1), to examine the dynamics of activation versus detoxification of benzo[a]pyrene (B[a]P), dibenzo[a,l]pyrene (DB[a,l]P), and their dihydrodiol metabolites. The cytotoxicity of B[a]P or DB[a,l]P was 9-11-fold greater in cells expressing human, as compared to rat CYP1A1, despite similar enzymatic activities. Co-expression of the hGSTP1 with the hCYP1A1 conferred 16-fold resistance to B[a]P cytotoxicity, compared to only 2.5-fold resistance when hGSTP1 was co-expressed with rat CYP1A1. The lower B[a]P cytotoxicity in the cells expressing rat CYP1A1, and weaker protection by hGSTP1 co-expression in these cells, were attributable to the much lower fraction of B[a]P metabolism via formation of the 7,8-dihydrodiol intermediate by the rat CYP1A1 compared to hCYP1A1. Resistance to the DB[a,l]P cytotoxicity conferred by hGSTP1 expression was also greater in cells co-expressing hCYP1A1 (7-fold) as compared to cells co-expressing rCYP1A1 (<2-fold). Resistance to B[a]P conferred by hGSTP1 was closely correlated with the activity level in two clonal transfectant lines with a 3-fold difference in hGSTP1-1 specific activity. Depletion of GSH to 20% of control levels via pretreatment with the de novo GSH biosynthesis inhibitor buthionine sulfoximine reduced the protection against B[a]P cytotoxicity by hGSTP1 from 16-fold to 5-fold, indicating that catalysis of conjugation with GSH, rather than binding or other effects, is responsible for the resistance. The cytotoxicity of the dihydrodiol intermediates of B[a]P or DB[a,l]P was much greater, and similar in cell lines expressing either human or rat CYP1A1. Again, however, the protection conferred by hGSTP1 co-expression was 2-5-fold greater in cells with hCYP1A1 than with rCYP1A1 expression. These results indicate that GST expression can effectively limit cytotoxicity following activation of B[a]P by human or rat CYP1A1, but is less effective as a defense against exposure of cells to the intermediate metabolite B[a]P-7,8-dihydrodiol.

Revised assignment of absolute configuration of the cis- and trans-N6-deoxyadenosine adducts at C14 of (+/-)-11beta,12alpha-dihydroxy-13alpha,14alpha-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene by stereoselective synthesis.

We have reassigned relative and absolute configurations by unambiguous stereoselective syntheses of the cis- (13s and 13R) and trans-N6-deoxyadenosine (dAdo) adduct diastereomers (14S and 14R) derived from (+/-)-11beta,12alpha-dihydroxy-13alpha,14alpha-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]P DE-2), previously reported by Li et al. [(1999) Chem. Res. Toxicol. 12, 758-767]. Two stereoselective methods, asymmetric aminohydroxylation of the (+/-)-trans-11,12-dihydrodiol (3) with 3',5'-di-O-(tert -butyldimethylsilyl)-2'-deoxyadenosine (4) and the highly stereoselective cis addition of 4 to (+/-)-DB[a,l]P DE-2 in hexafluoropropan-2-ol (HFP), were employed. Both afforded a 1:1 mixture of the cis-N6-dAdo adduct diastereomers, which were separated as triacetates (5S and 5R) in comparable yields (approximately 80%). The corresponding trans adduct diastereomers (10S and 10R) were obtained by coupling the aminotriol derived from trans opening of (+/-)-DB[a,l]P DE-2 with 6-fluoro-(2'-deoxy-3,5-di-tert-butyldimethylsilyloxy-beta-D-erythro-pentafuranosyl)purine (9) and subsequent acetylation in approximately 70% yield. The cis-5S and -5R and trans-10S and -10R were separately treated with 7% HF-pyridine followed by ammonolysis in NH3-saturated MeOH to give the dAdo adducts with all hydroxyl groups free (13S, 13R, 14S, and 14R). Comparison of the 1H NMR and CD spectra of these presently synthesized dAdo adducts with spectra of the previously reported compounds revealed that the interpretation of the 1H NMR and CD spectra and assignment of the relative stereochemistry (cis/trans) and absolute configuration made by Li et al. were at variance with our results. The above highly stereoselective syntheses of (+/-)-DB[a,l]P DE-2 adducted dAdo derivatives enabled efficient preparation of each of the four possible stereoisomeric 5'-dimethoxytrityl-3'-phosphoramidites for use in oligonucleotide synthesis.

Oxidation of PAH trans-dihydrodiols by human aldo-keto reductase AKR1B10.

AKR1B10 has been identified as a potential biomarker for human nonsmall cell lung carcinoma and as a tobacco exposure and response gene. AKR1B10 functions as an efficient retinal reductase in vitro and may regulate retinoic acid homeostasis. However, the possibility that this enzyme is able to activate polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols to form reactive and redox-active o-quinones has not been investigated to date. AKR1B10 was found to oxidize a wide range of PAH trans-dihydrodiol substrates in vitro to yield PAH o-quinones. Reactions of AKR1B10 proceeded with improper stereochemistry, since it was specific for the minor (+)-benzo[a]pyrene-7S,8S-dihydrodiol diastereomer formed in vivo. However, AKR1B10 displayed reasonable activity in the oxidation of both the (-)-R,R and (+)-S,S stereoisomers of benzo[g]chrysene-11,12-dihydrodiol and oxidized the potentially relevant, albeit minor, (+)-benz[a]anthracene-3S,4S-dihydrodiol metabolite. We find that AKR1B10 is therefore likely to play a contributing role in the activation of PAH trans-dihydrodiols in human lung. AKR1B10 retinal reductase activity was confirmed in vitro and found to be 5- to 150-fold greater than the oxidation of PAH trans-dihydrodiols examined. AKR1B10 was highly expressed at the mRNA and protein levels in human lung adenocarcinoma A549 cells, and robust retinal reductase activity was measured in lysates of these cells. The much greater catalytic efficiency of retinal reduction compared to PAH trans-dihydrodiol metabolism suggests AKR1B10 may play a greater role in lung carcinogenesis through dysregulation of retinoic acid homeostasis than through oxidation of PAH trans-dihydrodiols.

Evidence for the aldo-keto reductase pathway of polycyclic aromatic trans-dihydrodiol activation in human lung A549 cells.

Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (+/-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-trans-dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione), a redox-active o-quinone. We show that B[a]P-7,8-trans-dihydrodiol (AKR substrate) and B[a]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (+/-)-trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene or the diol-epoxide, (+/-)-anti-7,8-dihydroxy-9alpha,10beta-epoxy-7,8,9,10-tetrahydro-B[a]P. B[a]P-7,8-trans-dihydrodiol and B[a]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP(+)/NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([(15)N(5)]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[a]P-7,8-trans-dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O-methyl transferase (COMT) inhibitor, suggesting that COMT protects against o-quinone-mediated redox cycling. We conclude that activation of PAH-trans-dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.

Comparisons of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol activation by human cytochrome P450 and aldo-keto reductase enzymes: effect of redox state and expression levels.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are metabolically activated to proximate carcinogenic trans-dihydrodiols. PAH trans-dihydrodiols are further activated in humans by cytochrome P450 (P450) 1A1 and 1B1 to yield diol-epoxides or by aldo-keto reductases (AKR) 1A1 and 1C1-1C4 to yield reactive and redox-active o-quinones. Reconstituted in vitro systems were used to compare the steady-state kinetic constants for human P450 (P450 1A1 and 1B1) and AKR (AKR1A1, AKR1C1-1C4) mediated metabolism of (+/-)- trans-7,8-dihydroxy-7,8-dihydrobenzo[ a]pyrene ((+/-)-B[ a]P-7,8-diol) at physiological pH. It was found that P450 isoforms yielded much greater k cat/ K m values than AKR enzymes. Initial rates of (+/-)-B[ a]P-7,8-diol oxidation were measured for AKR1A1, AKR1C2, P450 1A1, and P450 1B1 as the ratio of NADPH/NAD (+) cofactors was varied to determine the redox state necessary for AKRs to successfully compete for trans-dihydrodiols. P450 and AKR enzymes equally competed for (+/-)-B[ a]P-7,8-diol substrate at an NADPH/NAD (+) ratio equal to 0.001. The resting NADPH/NAD (+) ratio was determined in A549 human lung adenocarcinoma cells to be 0.28. These data suggest that the P450 pathway would be favored over the AKR pathway if the enzymes were equally expressed. Basal mRNA transcript levels of AKR1C1-1C3 exceed those of both basal and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced P450 1A1 and 1B1 by up to 90-fold in A549 cells as measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods. AKR expression levels were comparable to TCDD-induced P450 1A1 and 1B1 in HBEC-KT immortalized normal human bronchial epithelial cells. Functional assays of both A549 and HBEC-KT cell lysates demonstrated a lack of TCDD-inducible P450 1A1/1B1 activity but robust basal expression of AKR1A1 and AKR1C activities, where the functional assay for P450 detection is 300-fold more sensitive than the functional assay for AKR isoforms. These data suggest that AKR enzymes may effectively compete with P450 1A1/1B1 for PAH trans-dihydrodiol activation in human lung cells.

Degradation of metabolites of benzo[a]pyrene by coupling Penicillium chrysogenum with KMnO4.

Several main metabolites of benzo[a]pyrene (BaP) formed by Penicillium chrysogenum, Benzo[a]pyrene-1,6-quinone (BP 1,6-quinone), trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP 7,8-diol), 3-hydroxybenzo[a]pyrene (3-OHBP), were identified by high-performance liquid chromatography (HPLC). The three metabolites were liable to be accumulated and were hardly further metabolized because of their toxicity to microorganisms. However, their further degradation was essential for the complete degradation of BaP. To enhance their degradation, two methods, degradation by coupling Penicillium chrysogenum with KMnO4 and degradation only by Penicillium chrysogenum, were compared; Meanwhile, the parameters of degradation in the superior method were optimized. The results showed that (1) the method of coupling Penicillium chrysogenum with KMnO4 was better and was the first method to be used in the degradation of BaP and its metabolites; (2) the metabolite, BP 1,6-quinone was the most liable to be accumulated in pure cultures; (3) the effect of degradation was the best when the concentration of KMnO4 in the cultures was 0.01% (w/v), concentration of the three compounds was 5 mg/L and pH was 6.2. Based on the experimental results, a novel concept with regard to the bioremediation of BaP-contaminated environment was discussed, considering the influence on environmental toxicity of the accumulated metabolites.

Protective efficacy of hGSTM1-1 against B[a]P and (+)- or (-)-B[a]P-7,8-dihydrodiol cytotoxicity, mutagenicity, and macromolecular adducts in V79 cells coexpressing hCYP1A1.

Transgenic cell lines were constructed to study the dynamics of competition between activation versus detoxification of benzo[a]pyrene (B[a]P) or B[a]P-7,8-dihydrodiol metabolites. Stably transfected V79MZ cells expressing human cytochrome P4501A1 (hCYP1A1) alone or in combination with human glutathione-S-transferase M1 (hGSTM1) were used to determine how effectively this GST isozyme protects against cytotoxic, genotoxic, and mutagenic effects of B[a]P or the enantiomeric dihydrodiol metabolites (+)-benzo[a]pyrene-7,8-dihydrodiol ((+)-B[a]P-7,8-diol) and (-)-benzo[a]pyrene-7,8-dihydrodiol ((-)-B[a]P-7,8-diol). Expression of hGSTM1 in the presence of hCYP1A1 conferred significant 8.5-fold protection against B[a]P-induced cytotoxicity, but protection against cytotoxicity of either B[a]P-7,8-diol enantiomer was not significant. Mutagenicity of B[a]P at the hprt locus was dose and time dependent in cells that expressed hCYP1A1. Mutagenicity of B[a]P was reduced by 21-32% and mutagenicity induced by the B[a]P-7,8-diols was reduced 20-58% in cells further modified to coexpress hGSTM1-1 compared to cells expressing hCYP1A1 alone. Expression of hGSTM1-1 reduced adducts in total cellular macromolecules by twofold, in good correlation with the reduction in B[a]P mutagenicity. These results indicate that while hGSTM1-1 effectively protects against hCYP1A1-mediated cytotoxicity of B[a]P, a significant fraction of the mutagenicity that results from activation of B[a]P and its 7,8-dihydrodiol metabolites by hCYP1A1 is derived from B[a]P metabolites that are not detoxified by hGSTM1.

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (+/-)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1.

We have used V79MZ hamster lung fibroblasts stably transfected with human cytochrome P450-1A1 (hCYP1A1; cell line designated V79MZh1A1) or P450-1B1 (hCYP1B1; cell line designated V79MZh1B1) alone, or in combination with human glutathione-S-transferase (GST) alpha-1 (hGSTA1), in order to examine GST protection against cytotoxicity and mutagenicity of dibenzo[a,l]pyrene (DBP) and the intermediate dihydrodiol metabolite (+/-)-DBP-11,12-dihydrodiol (DBPD). At comparable expression levels of hCYP1A1 and hCYP1B1, both DBP and DBPD were more cytotoxic in V79MZ1A1 (IC(50)=2.7 and 0.7nM, respectively) than in V79MZh1B1 (IC(50)=6.0 and 4.8nM, respectively). In contrast, both DBP and DBPD were two- to four-fold more mutagenic in V79MZh1B1 than in V79MZ1A1. Co-expression of hGSTA1 with hCYP1A1 decreased DBP cytotoxicity two-fold compared to V79MZh1A1 with hCYP1A1 alone, and provided a small, yet still statistically significant, 1.3-fold protection against DBPD. Protection against mutagenicity of these compounds was comparable to that for cytotoxicity in cells expressing hCYP1A1. In V79MZh1B1 cells, co-expression of hGSTA1 conferred up to five-fold protection against DBP cytotoxicity, and up to nine-fold protection against the (+/-)-DBP-dihydrodiol cytotoxicity relative to the cells expressing hCYP1B1 alone. Co-expression of hGSTA1 also reduced mutagenicity of DBP or its dihydrodiol to a lesser extent (1.3-1.8-fold) than the protection against cytotoxicity in cells expressing hCYP1B1. These findings demonstrate that the protective efficacy of hGSTA1 against DBP and DBPD toxicity is variable, depending on the compound or metabolite present, the specific cytochrome P450 isozyme expressed, and the specific cellular damage endpoint examined.

Benzo[a]pyrene-7,8-dihydrodiol promotes checkpoint activation and G2/M arrest in human bronchoalveolar carcinoma H358 cells.

Polycyclic aromatic hydrocarbons (PAHs) are potent carcinogens that require metabolic activation inside cells. The proximate carcinogens PAH-diols can be converted to o-quinones by aldo-keto reductases (AKRs) or to diol-epoxides by cytochrome P450 (P450) enzymes. We assessed the effect of benzo[a]pyrene-7,8-dihydrodiol (BPD) on proliferation in p53-null bronchoalveolar carcinoma H358 cells. BPD treatment led to a significant inhibition of proliferation and arrest in G2/M in H358 cells. The relative contribution of the AKR and P450 pathways to cell cycle arrest was assessed. Overexpression of AKR1A1 did not affect cell proliferation or cell cycle progression, and benzo[a]pyrene-7,8-dione did not cause any noticeable effect on cell growth, suggesting that AKR1A1 metabolic products were not involved in the antiproliferative effect of BPD. On the other hand, blockade of P450 induction or inhibition of P450 activity greatly impaired the effect of BPD. Moreover, P450 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly enhanced the antiproliferative effect of BPD. Mechanistic studies revealed that BPD caused a DNA damage response, Chk1 activation, and accumulation of phospho-Cdc2 (Tyr15) in H358 cells, effects that were impaired by an ataxia-telangectasia mutated (ATM)/ATM-related (ATR) inhibitor. Similar results were observed in human bronchoepithelial BEAS-2B cells, arguing for analogous mechanisms in tumorigenic and immortalized nontumorigenic cells lacking functional p53. Our data suggest that a p53-independent pathway operates in lung epithelial cells in response to BPD that involves P450 induction and subsequent activation of the ATR/ATM/Chk1 damage check-point pathway and cell cycle arrest in G2/M.

Effect of DNA modifications on DNA processing by HIV-1 integrase and inhibitor binding: role of DNA backbone flexibility and an open catalytic site.

Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.

Competing roles of aldo-keto reductase 1A1 and cytochrome P4501B1 in benzo[a]pyrene-7,8-diol activation in human bronchoalveolar H358 cells: role of AKRs in P4501B1 induction.

Benzo[a]pyrene (BP) requires metabolic activation to electrophiles to exert its deleterious effects. We compared the respective roles of aldo-keto reductase 1A1 (AKR1A1, aldehyde reductase) and P4501B1 in the formation of BP-7,8-dione and BP-tetrols, respectively, in intact bronchoalveolar cells manipulated to express either enzyme. Metabolite formation was confirmed by HPLC/MS and quantitatively measured by HPLC/UV/beta-RAM. In TCDD-treated H358 cells (P4501B1 expression), the anti-BPDE hydrolysis product BP-tetrol-1 increased over 3-12 h to a constant level. In H358 AKR1A1 transfectants, formation of BP-7,8-dione was elevated for 3-12 h but significantly decreased after 24 h. Interestingly, BP-tetrols were also detected in AKR1A1 transfectants even though they do not constitutively express P4501A1/P4501B1 enzymes. Northern and Western blotting confirmed the induction of P4501B1 by BP-7,8-dione in parental cells and the induction of P4501B1 by BP-7,8-diol in AKR1A1-transfected cells. P4501B1 induction was blocked in AKR1A1 transfectants by the AKR1A1 inhibitor (sulfonylnitromethane), the o-quinone scavenger (N-acetyl-l-cysteine), or the cytosolic AhR antagonist (diflubenzuron). Attenuation of P4501B1 induction in these cells was verified by measuring a decrease in BP-tetrol formation. Our studies show that the formation of BP-7,8-dione by AKR1A1 in human bronchoalveolar cells leads to an induction of P4501B1 and that a functional consequence of this induction is elevated anti-BPDE production as detected by increased BP-tetrol formation. Therefore, the role of AKR1A1 in the activation of BP-7,8-diol is bifunctional; that is, it directly activates BP-7,8-diol to the reactive and redox-active PAH o-quinone (BP-7,8-dione) and it indirectly trans-activates the P4501B1 gene by generating the aryl hydrocarbon receptor (AhR) ligand BP-7,8-dione.