Polycyclic Aromatic Hydrocarbons Trans-dihydrodiols Research Articles

Human aldo-keto reductases and the metabolic activation of polycyclic aromatic hydrocarbons.

Aldo-keto reductases (AKRs) are promiscuous NAD(P)(H) dependent oxidoreductases implicated in the metabolic activation of polycyclic aromatic hydrocarbons (PAH). These enzymes catalyze the oxidation of non-K-region trans-dihydrodiols to the corresponding o-quinones with the concomitant production of reactive oxygen species (ROS). The PAH o-quinones are Michael acceptors and can form adducts but are also redox-active and enter into futile redox cycles to amplify ROS formation. Evidence exists to support this metabolic pathway in humans. The human recombinant AKR1A1 and AKR1C1–AKR1C4 enzymes all catalyze the oxidation of PAH trans-dihydrodiols to PAH o-quinones. Many human AKRs also catalyze the NADPH-dependent reduction of the o-quinone products to air-sensitive catechols, exacerbating ROS formation. Moreover, this pathway of PAH activation occurs in a panel of human lung cell lines, resulting in the production of ROS and oxidative DNA damage in the form of 8-oxo-2′-deoxyguanosine. Using stable-isotope dilution liquid chromatography tandem mass spectrometry, this pathway of benzo[a]pyrene (B[a]P) metabolism was found to contribute equally with the diol-epoxide pathway to the activation of this human carcinogen in human lung cells. Evaluation of the mutagenicity of anti-B[a]P-diol epoxide with B[a]P-7,8-dione on p53 showed that the o-quinone produced by AKRs was the more potent mutagen, provided that it was permitted to redox cycle, and that the mutations observed were G to T transversions, reminiscent of those observed in human lung cancer. It is concluded that there is sufficient evidence to support the role of human AKRs in the metabolic activation of PAH in human lung cell lines and that they may contribute to the causation of human lung cancer.

Interception of Benzo[a]pyrene-7,8-dione by UDP Glucuronosyltransferases (UGTs) in Human Lung Cells

Polycyclic aromatic hydrocarbons (PAHs) are environmental and tobacco carcinogens. Proximate carcinogenic PAH trans-dihydrodiols are activated by human aldo-keto reductases (AKRs) to yield electrophilic and redox-active o-quinones. Interconversion among benzo[a]pyrene (B[a]P)-7,8-dione, a representative PAH o-quinone, and its corresponding catechol generates a futile redox-cycle with the concomitant production of reactive oxygen species (ROS). We investigated whether glucuronidation of B[a]P-7,8-catechol by human UDP glucuronosyltransferases (UGTs) could intercept the catechol in three different human lung cells. RT-PCR showed that UGT1A1, 1A3, and 2B7 were only expressed in human lung adenocarcinoma A549 cells. The corresponding recombinant UGTs were examined for their kinetic constants and product profile using B[a]P-7,8-catechol as a substrate. B[a]P-7,8-dione was reduced to B[a]P-7,8-catechol by dithiothreitol under anaerobic conditions and then further glucuronidated by the UGTs in the presence of uridine-5′-diphosphoglucuronic acid as a glucuronic acid group donor. UGT1A1 catalyzed the glucuronidation of B[a]P-7,8-catechol and generated two isomeric O-monoglucuronsyl-B[a]P-7,8-catechol products that were identified by RP-HPLC and by LC-MS/MS. By contrast, UGT1A3 and 2B7 catalyzed the formation of only one monoglucuronide, which was identical to that formed in A549 cells. The kinetic profiles of three UGTs followed Michaelis–Menten kinetics. On the basis of the expression levels of UGT1A3 and UGT2B7 and the observation that a single monoglucuronide was produced in A549 cells, we suggest that the major UGT isoforms in A549 cells that can intercept B[a]P-7,8-catechol are UGT1A3 and 2B7.

Detoxication of Benzo[a]pyrene-7,8-dione by Sulfotransferases (SULTs) in Human Lung Cells

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Human aldo-keto reductases catalyze the metabolic activation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active o-quinones. Benzo[a]pyrene-7,8-dione a representative PAH o-quinone is reduced back to the corresponding catechol to generate a futile redox-cycle. We investigated whether sulfonation of PAH catechols by human sulfotransferases (SULT) could intercept the catechol in human lung cells. RT-PCR identified SULT1A1, -1A3, and -1E1 as the isozymes expressed in four human lung cell lines. The corresponding recombinant SULTs were examined for their substrate specificity. Benzo[a]pyrene-7,8-dione was reduced to benzo[a]pyrene-7,8-catechol by dithiothreitol under anaerobic conditions and then further sulfonated by the SULTs in the presence of 3'-[(35)S]phosphoadenosine 5'-phosphosulfate as the sulfonate group donor. The human SULTs catalyzed the sulfonation of benzo[a]pyrene-7,8-catechol and generated two isomeric benzo[a]pyrene-7,8-catechol O-monosulfate products that were identified by reversed phase HPLC and by LC-MS/MS. The various SULT isoforms produced the two isomers in different proportions. Two-dimensional (1)H and (13)C NMR assigned the two regioisomers of benzo[a]pyrene-7,8-catechol monosulfate as 8-hydroxy-benzo[a]pyrene-7-O-sulfate (M1) and 7-hydroxy-benzo[a]pyrene-8-O-sulfate (M2), respectively. The kinetic profiles of three SULTs were different. SULT1A1 gave the highest catalytic efficiency (k(cat)/K(m)) and yielded a single isomeric product corresponding to M1. By contrast, SULT1E1 showed distinct substrate inhibition and formed both M1 and M2. Based on expression levels, catalytic efficiency, and the fact that the lung cells only produce M1, it is concluded that the major isoform that can intercept benzo[a]pyrene-7,8-catechol is SULT1A1.

The Role of Human Aldo-Keto Reductases in the Metabolic Activation and Detoxication of Polycyclic Aromatic Hydrocarbons: Interconversion of PAH Catechols and PAH o-Quinones

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants. They are procarcinogens requiring metabolic activation to elicit their deleterious effects. Aldo-keto reductases (AKR) catalyze the oxidation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active PAH o-quinones. AKRs are also found to be capable of reducing PAH o-quinones to form PAH catechols. The interconversion of o-quinones and catechols results in the redox-cycling of PAH o-quinones to give rise to the generation of reactive oxygen species and subsequent oxidative DNA damage. On the other hand, PAH catechols can be intercepted through phase II metabolism by which PAH o-quinones could be detoxified and eliminated. The aim of the present review is to summarize the role of human AKRs in the metabolic activation/detoxication of PAH and the relevance of phase II conjugation reactions to human lung carcinogenesis.

Detoxication of Structurally Diverse Polycyclic Aromatic Hydrocarbon (PAH) o-Quinones by Human Recombinant Catechol-O-methyltransferase (COMT) via O-Methylation of PAH Catechols

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Metabolic activation of intermediate PAH trans-dihydrodiols by aldo-keto reductases (AKRs) leads to the formation of electrophilic and redox-active o-quinones. We investigated whether O-methylation by human recombinant soluble catechol-O-methyltransferase (S-COMT) is a feasible detoxication step for a panel of structurally diverse PAH-catechols produced during the redox-cycling process. Classes of PAH non-K-region o-quinones (bay region, methylated bay region, and fjord region o-quinones) produced by AKRs were employed in the studies. PAH o-quinones were reduced to the corresponding catechols by dithiothreitol under anaerobic conditions and then further O-methylated by human S-COMT in the presence of S-[³H]adenosyl-l-methionine as a methyl group donor. The formation of the O-methylated catechols was detected by HPLC-UV coupled with in-line radiometric detection, and unlabeled products were also characterized by LC-MS/MS. Human S-COMT was able to catalyze O-methylation of all of the PAH-catechols and generated two isomeric metabolites in different proportions. LC-MS/MS showed that each isomer was a mono-O-methylated metabolite. ¹H NMR was used to assign the predominant positional isomer of benzo[a]pyrene-7,8-catechol as the O-8-monomethylated catechol. The catalytic efficiency (k(cat)/K(m)) varied among different classes of PAH-catechols by 500-fold. The ability of S-COMT to produce two isomeric products from PAH-catechols was rationalized using the crystal structure of the enzyme. We provide evidence that O-8-monomethylated benzo[a]pyrene-7,8-catechol is formed in three different human lung cell lines. It is concluded that human S-COMT may play a critical role in the detoxication of PAH o-quinones generated by AKRs.

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.

The pattern of p53 mutations caused by PAH o-quinones is driven by 8-oxo-dGuo formation while the spectrum of mutations is determined by biological selection for dominance.

PAHs (polycyclic aromatic hydrocarbons) are suspect lung cancer carcinogens that must be metabolically converted into DNA-reactive metabolites. P4501A1/P4501B1 plus epoxide hydrolase activate PAH to (+/-)- anti-benzo[ a]pyrene diol epoxide ((+/-)- anti-BPDE), which causes bulky DNA adducts. Alternatively, aldo-keto reductases (AKRs) convert intermediate PAH trans-dihydrodiols to o-quinones, which cause DNA damage by generating reactive oxygen species (ROS). In lung cancer, the types or pattern of mutations in p53 are predominantly G to T transversions. The locations of these mutations form a distinct spectrum characterized by single point mutations in a number of hotspots located in the DNA binding domain. One route to the G to T transversions is via oxidative DNA damage. An RP-HPLC-ECD assay was used to detect the formation of 8-oxo-dGuo in p53 cDNA exposed to representative quinones, BP-7,8-dione, BA-3,4-dione, and DMBA-3,4-dione under redox cycling conditions. Concurrently, a yeast reporter system was used to detect mutations in the same cDNA samples. Nanomolar concentrations of PAH o-quinones generated 8-oxo-dGuo (detected by HPLC-ECD) in a concentration dependent manner that correlated in a linear fashion with mutagenic frequency. By contrast, micromolar concentrations of (+/-)- anti-BPDE generated (+)- trans- anti-BPDE-N (2)-dGuo adducts (detected by stable-isotope dilution LC/MS methodology) in p53 cDNA that correlated in a linear fashion with mutagenic frequency, but no 8-oxo-dGuo was detected. Previous studies found that mutations observed with PAH o-quinones were predominately G to T transversions and those observed with (+/-)- anti-BPDE were predominately G to C transversions. However, mutations at guanine bases observed with either PAH-treatment occurred randomly throughout the DNA-binding domain of p53. Here, we find that when the mutants were screened for dominance, the dominant mutations clustered at or near hotspots primarily at the protein-DNA interface, whereas the recessive mutations are scattered throughout the DNA binding domain without resembling the spectra observed in cancer. These observations, if extended to mammalian cells, suggest that mutagenesis can drive the pattern of mutations but that biological selection for dominant mutations drives the spectrum of mutations observed in p53 in lung cancer.

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.

Hepatocyte nuclear factor-4 alpha/gamma and hepatocyte nuclear factor-1 alpha as causal factors of interindividual difference in the expression of human dihydrodiol dehydrogenase 4 mRNA in human livers.

Human dihydrodiol dehydrogenase (DD) catalyses the oxidation of trans-dihydrodiols of polycyclic aromatic hydrocarbons and the reduction of several ketone-containing drugs. About 40-fold interindividual difference in DD activities has been noted. Recently, we found that transcriptional factors, hepatocyte nuclear factor (HNF)-1 alpha, HNF-4 alpha and HNF-4 gamma were essential for the expression of DD4 mRNA, which is a major form of DDs. Thus, to clarify a possible mechanism(s) underlying the interindividual difference in DD activities, we investigated the sequences of genes and the expression levels of mRNA for DD4 and HNFs in human livers. We found no clear relationship between the genotypes of DD4 and HNF genes and the expression levels of DD4 mRNA in the subjects. The expression level of DD4 mRNA significantly correlated with that of HNF-1 alpha, HNF-4 alpha or HNF-4 gamma. These results suggest that the expression level of DD4 mRNA is cooperatively regulated by the amounts of HNF-1 alpha, HNF-4 alpha and HNF-4 gamma.

Polycyclic aromatic hydrocarbon o-quinones inhibit the activity of the catalytic fragment of protein kinase C.

Polycyclic aromatic hydrocarbons (PAHs) require metabolic activation to exert their carcinogenic effects. PAH trans-dihydrodiol proximate carcinogens are oxidized by aldo-keto reductases (AKRs) to their corresponding reactive and redox-active o-quinones which may have the properties of initiators and promoters. To determine whether these o-quinones target protein kinase C (PKC), their effects on human recombinant PKCalpha and PKCdelta and the catalytic fragment of rat brain PKC were determined. Naphthalene-1,2-dione (NP-1,2-dione), benzo[a]pyrene-7,8-dione (BP-7,8-dione), and 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBA-3,4-dione) potently inhibited (IC(50) values 3-5 microM) the basal and stimulated activity of the holoenzymes PKCalpha and PKCdelta in a dose-dependent manner. Inhibition of PKC by BP-7,8-dione was observed irrespective of whether PKCalpha activity was stimulated with phorbol 12-myristate 13-acetate (PMA), phosphatidylserine (PS), or Ca(2+) or whether PKCdelta was stimulated with phorbol 12-myristate 13-acetate (PMA) or phosphatidylserine (PS), suggesting that the inhibition was not cofactor-specific. All three quinones inhibited the catalytic fragment of PKC in vitro, yielding identical IC(50) values (3-5 microM), indicating that they interact with the catalytic domain of PKC rather than the cofactor/activator sites. In contrast, no effect on either the holoenzyme or the catalytic fragment was observed with the corresponding PAH trans-dihydrodiols, indicating that inhibition was o-quinone-specific. Irreversible inhibition of the catalytic fragment of PKC was observed since activity could not be restored by dialysis, suggesting that arylation of the fragment had occurred. NP-1,2-dione and BP-7,8-dione also suppressed PKC activity in human breast cancer MCF-7 cell lysates which express PKCalpha, -beta, -delta, -epsilon, -iota, and -lambda isozymes. These data suggest that PAH o-quinones, generated by AKRs, may affect cellular signaling through suppression of the activity of PKC isoforms.

Activation of Polycyclic Aromatic Hydrocarbontrans-Dihydrodiol Proximate Carcinogens by Human Aldo-keto Reductase (AKR1C) Enzymes and Their Functional Overexpression in Human Lung Carcinoma (A549) Cells

Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants and suspected human lung carcinogens. In patients with non-small cell lung carcinoma, differential display shows that aldo-keto reductase (AKR1C) transcripts are dramatically overexpressed. However, whether AKR1C isoforms contribute to the carcinogenic process and oxidize potent PAH trans-dihydrodiols (proximate carcinogens) to reactive and redox active o-quinones is unknown; nor is it known whether these reactions occur in human lungs. We now show that four homogeneous human recombinant aldo-keto reductases (AKR1C1-AKR1C4) are regioselective and oxidize only the relevant non-K region trans-dihydrodiols. However, these enzymes are not stereo-selective, since they oxidized 100% of these racemic substrates. The highest utilization ratios (V(max)/K(m)) were observed for some of the most potent proximate carcinogens known (e.g. 7,12-dimethylbenz[a]anthracene-3,4-diol (DMBA-3,4-diol) and benzo[g]chrysene-11,12-diol). In vitro, DMBA-3,4-diol was oxidized by AKR1C4 to the highly reactive 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBA-3,4-dione), which was trapped in situ as its mono- and bis-thioether conjugates, which arise from the sequential 1,6- and 1,4-Michael addition of thiol nucleophiles. Human multiple tissue expression array analysis showed that AKR1C isoform transcripts were highly expressed in the human lung carcinoma cell line A549. Isoform-specific reverse transcriptase-PCR showed that AKR1C1, AKR1C2, and AKR1C3 transcripts were all expressed. Western blot analysis and functional assays confirmed high expression of AKR1C protein and enzyme activity in these lung cells. A549 cell lysates were found to convert DMBA-3,4-diol to the corresponding o-quinone. In trapping experiments, LC/MS analysis identified peaks in the cell lysates that corresponded to the synthetically prepared mono- and bis-thioether conjugates of DMBA-3,4-dione. This quinone is one of the most electrophilic and redox-active o-quinones produced by AKRs. Its unique ability to form bis-thioether conjugates parallels the formation of bis- and tris-glutathionyl conjugates of hydroquinone, which display end organ toxicity. The ability to measure DMBA-3,4-dione formation in A549 cells implicates the AKR pathway in the metabolic activation of PAH in human lung.

Co-operative regulation of the transcription of human dihydrodiol dehydrogenase (DD)4/aldo-keto reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)-4alpha/gamma and HNF-1alpha.

Human dihydrodiol dehydrogenase (DD) 4/aldo-keto reductase (AKR) 1C4 is a major isoform of hepatic DD that oxidizes trans-dihydrodiols of polycyclic aromatic hydrocarbons to reactive and redox-active o-quinones and that reduces several ketone-containing drugs. To investigate the mechanism of transcriptional regulation of the human DD4 gene, the 5'-flanking region of the gene was fused to the luciferase gene. The results of luciferase assays using HepG2 cells and of 1,10-phenanthroline-copper footprinting indicated that two positive regulatory regions were located in regions from -701 to -684 and from -682 to -666. The former region contained a putative hepatocyte nuclear factor (HNF)-4 binding motif, and the latter region contained an HNF-1 consensus binding sequence. DNA fragments of the HNF-4 or HNF-1 motif gave a shifted band in a gel-shift assay with nuclear extracts from HepG2 cells. The formation of the DNA-protein complex was inhibited by the HNF-4 or HNF-1 motif of the alpha(1)-antitrypsin gene. A supershift assay using antibodies to human HNF-4alpha, HNF-4gamma and HNF-1alpha showed that HNF-4alpha and HNF-4gamma bound to the HNF-4 motif, and that HNF-1alpha interacted with the HNF-1 motif. Introduction of mutations into the HNF-4 or HNF-1 motif lowered the luciferase activity to 10 or 8% respectively of that seen with the intact human DD4 gene. These results indicate that HNF-4alpha, HNF-4gamma and HNF-1alpha regulate co-operatively the transcription of the human DD4 gene in HepG2 cells.

Co-operative regulation of the transcription of human dihydrodiol dehydrogenase (DD)4/aldo–keto reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)-4α/γ and HNF-1α

Human dihydrodiol dehydrogenase (DD) 4/aldo-keto reductase (AKR) 1C4 is a major isoform of hepatic DD that oxidizes trans-dihydrodiols of polycyclic aromatic hydrocarbons to reactive and redox-active o-quinones and that reduces several ketone-containing drugs. To investigate the mechanism of transcriptional regulation of the human DD4 gene, the 5′-flanking region of the gene was fused to the luciferase gene. The results of luciferase assays using HepG2 cells and of 1,10-phenanthroline-copper footprinting indicated that two positive regulatory regions were located in regions from -701 to -684 and from -682 to -666. The former region contained a putative hepatocyte nuclear factor (HNF)-4 binding motif, and the latter region contained an HNF-1 consensus binding sequence. DNA fragments of the HNF-4 or HNF-1 motif gave a shifted band in a gel-shift assay with nuclear extracts from HepG2 cells. The formation of the DNA-protein complex was inhibited by the HNF-4 or HNF-1 motif of the α1-antitrypsin gene. A supershift assay using antibodies to human HNF-4α, HNF-4γ and HNF-1α showed that HNF-4α and HNF-4γ bound to the HNF-4 motif, and that HNF-1α interacted with the HNF-1 motif. Introduction of mutations into the HNF-4 or HNF-1 motif lowered the luciferase activity to 10 or 8% respectively of that seen with the intact human DD4 gene. These results indicate that HNF-4α, HNF-4γ and HNF-1α regulate co-operatively the transcription of the human DD4 gene in HepG2 cells.

Metabolic activation of polycyclic aromatic hydrocarbon trans-dihydrodiols by ubiquitously expressed aldehyde reductase (AKR1A1)

Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by CYP1A1 and epoxide hydrolase (EH). CYP1A1 or aldo–keto reductases (AKRs) from the 1C subfamily can further activate the trans-dihydrodiols by forming either anti-diol-epoxides or reactive and redox active o-quinones, respectively. To determine whether other AKR superfamily members can divert trans-dihydrodiols to o-quinones, the cDNA encoding human aldehyde reductase (AKR1A1) was isolated from hepatoma HepG2 cells using RT-PCR, subcloned into a prokaryotic expression vector, overexpressed in E. coli and purified to homogeneity in milligram amounts. Studies revealed that AKR1A1 preferentially oxidized the metabolically relevant (−)-[3 R,4 R]-dihydroxy-3,4-dihydrobenz[ a]anthracene. AKR1A1 also displayed high utilization ratios ( V max/ K m) for the following PAH trans-dihydrodiols: (±) trans-3,4-dihydroxy-3,4-dihydro-7-methylbenz[ a]anthracene, (±) trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[ a]anthracene and (±) trans-7,8-dihydroxy-7,8-dihydro-5-methylchrysene. Multiple tissue expression (MTE) arrays were used to measure the co-expressed of CYP1A1, EH and AKR1A1. All the three enzymes co-expressed to sites of PAH activation. The high catalytic efficiency of AKR1A1 for potent proximate carcinogen trans-dihydrodiols and its presence in tissues that contain CYP1A1 and EH suggests that it plays an important role in this alternative pathway of PAH activation (supported by CA39504).

Polycyclic Aromatic Hydrocarbon Trans-Dihydrodiol Specificity of four Recombinant Human Dihydrodiol Dehydrogenase Isoforms

A major metabolic route of polycyclic aromatic hydrocarbon (PAH) activation proceeds through trans-dihydrodiol intermediates. We have previously shown that a member of the aldo-keto reductase (AKR) superfamily, rat liver dihydrodiol dehydrogenase (DD), catalyzes the NAD(P)+-dependent oxidation of PAH trans-dihydrodiols with the concomitant production of reactive oxygen species and o-semiquinone anion radicals on route to cyto- and geno-toxic o-quinones. The relevance of this pathway in humans, however, is unknown. In these studies, four homogeneous recombinant human homologs of rat liver DD (DD1, DD2, DD4 and DDX) were tested for their ability to oxidize a structural series of PAH trans-dihydrodiols of increasing ring size and methylation. The results indicate that human DDs preferred non-K-region trans-dihydrodiols and that methyl substitution enhanced oxidation rates by human DDs. Thus multiple human AKRs can contribute to the activation of structurally diverse procarcinogenic PAH by catalyzing the NAD(P)+-dependent oxidation of PAH trans-dihydrodiols to detrimental o-quinones.

Expression of mRNAs for dihydrodiol dehydrogenase isoforms in human tissues.

Dihydrodiol dehydrogenase (DD) [EC 1. 3. 1. 20] catalyzes the NADP+-linked oxidation of /trans-dihydrodiols of polycyclic aromatic hydrocarbons to corresponding catechols, and controls the formation of both their carcinogenic dihydrodiol epoxides (Oesch, et al.,, 1984) and cytotoxic o-quinones through autoxidation of the catechol metabolites (Flowers, et al.,., 1996). In addition, DD in mammalian liver is implicated in the metabolism of xenobiotic carbonyl compounds, steroids and prostaglandins because of its broad substrate specificity (Penning, et al.,, 1986; Hara, et al.,, 1986; Ohara, et al.,, 1994; 1995).KeywordsPolycyclic Aromatic HydrocarbonAbundant RabbitDihydrodiol DehydrogenaseCatechol MetaboliteMultiple cDNAsThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Expression and characterization of four recombinant human dihydrodiol dehydrogenase isoforms: oxidation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene to the activated o-quinone metabolite benzo[a]pyrene-7,8-dione.

The bioactivation of polycyclic aromatic hydrocarbons (PAHs) to their ultimate carcinogenic forms proceeds via the formation of proximate carcinogen trans-dihydrodiols. Previous studies demonstrated that rat liver 3 alpha-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3 alpha-HSD/DD), a member of the aldo-keto reductase (AKR) superfamily, oxidizes PAH trans-dihydrodiols to redox-cycling o-quinones. Multiple closely related AKRs exist in human liver; however, it is unclear which, if any, participate in PAH activation by catalyzing the NADP+ -dependent oxidation of PAH trans-dihydrodiols. In this study, cDNAs encoding four human DD isoforms were isolated from HepG2 cells using isoform-selective RT-PCR. The recombinant proteins were overexpressed in Escherichia coli, purified to homogeneity, and kinetically characterized. Calculation of KM and kcat values of each isoform for model substrates revealed that they possessed enzymatic activities assigned to native human liver DD1, DD2, DD4, and type 2 3alpha-HSD (DDX) proteins. The ability of human DDs to oxidize the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene (BP-diol) was then examined. A reverse phase HPLC radiochemical assay demonstrated that all four isoforms oxidize (+/-)-BP-diol in the following rank order: DD2 > DD1 > DD4 > DDX. Each DD consumed the entire racemic BP-diol mixture, indicating that both the minor (+)-S,S- and major (-)-R,R-stereoisomers formed in vivo are substrates. First-order decay plots showed that DD1 and DD2 displayed preferences for one of the stereoisomers, and circular dichroism spectroscopy indicated that this isomer was the (+)-7S, 8S-enantiomer. The products of these reactions were trapped as either glycine or thiol ether conjugates of benzo[a]pyrene-7,8-dione (BPQ), indicating that the initial oxidation product was the reactive BPQ. Thus, human liver possesses multiple AKRs which contribute to PAH activation by catalyzing the NADP+-dependent oxidation of PAH trans-dihydrodiols to redox-active o-quinones.

Generation of reactive oxygen species during the enzymatic oxidation of polycyclic aromatic hydrocarbon trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) catalyzes the oxidation of polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols (proximate carcinogens) to catechols which rapidly autoxidize to yield o-quinones (Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) J. Biol. Chem 263, 1814-1820). Although this pathway suppresses the formation of the PAH anti- and syn-diol epoxides (ultimate carcinogens), the process of autoxidation is anticipated to yield reactive oxygen species (ROS). We now show that the NADP+ dependent oxidation of (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene (Npdiol) and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (Bpdiol) catalyzed by homogeneous DD is accompanied by the consumption of molecular oxygen and the production of H2O2. With both trans-dihydrodiol substrates, oxygen consumption was stoichiometric with H2O2 production consistent with the reaction: QH2 + O2 = H2O2 + Q, where QH2 is the catechol and Q is the o-quinone. Using Npdiol or Bpdiol as substrates, a burst of superoxide anion production is catalyzed by DD which can be detected as the rate of cyt c reduction that is inhibited by superoxide dismutase. Using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as spin-trapping agent, secondary spin adducts corresponding to DMPO-CH3 were formed during the enzymatic oxidation of Npdiol and Bpdiol. The formation of the CH3. radical arises from the OH. attack of DMSO, which was used as cosolvent. These spin adducts were attenuated by superoxide dismutase and catalase, implying that O2-. and H2O2 are obligatory for the formation of DMPO-CH3. It is proposed that O2-. is the radical that propagates autoxidation and that the resultant H2O2 undergoes Fenton chemistry to produce the OH. radical. Identical spin adducts were observed using a superoxide anion generating system (hypoxanthine/xanthine oxidase) and DMPO as spin-trapping agent in the presence of DMSO. The ability of DD to generate ROS during the oxidation of PAH trans-dihydrodiols (proximate carcinogens) may have important implications for tumor initiation and promotion.

Development of chiral stationary phases for the enantiomeric resolution of dihydrodiols of polycyclic aromatic hydrocarbons by π-donor-acceptor interactions

Chiral stationary phases (CSPs) derived from ( R)-(−)-2-(2,4,5,7-tetranitrofluoren-9-ylideneaminooxy) propionic acid (TAPA) covalently bound to silica gel have been developed by altering the alkyl group at the chiral centre or the number of aromatic rings and their degree of nitration. The chromatographic properties of the CSPs were characterized by use of a racemic model solute. Depending on the solvent strength of the mobile phase, the CSPs exhibit the quality of a normal or a reversed phase. The chromatographic behaviour of 30 racemic hydroxylated derivatives of polycyclic aromatic hydrocarbons (PAHs) on ( R)-(−)-TAPA CSP revealed the structural requirements for chiral recognition. The applicability of the CSPs for the enantiomeric separation of trans-dihydrodiols of PAHs on an analytical as well as preparative scale and for investigating the enantioselectivity of the biotransformation and genotoxicity of PAHs is demonstrated.

A novel regio- and stereocontrolled synthesis of diol epoxide and trans-dihydriol metabolites of polycyclic aromatic hydrocarbons. An application to the synthesis of the bay-region syn- and anti-diol epoxides of the carcinogen 1,4-dimethylphenanthrene

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) require metabolic activation in order to exert their tumorigenic activity, typically to the diol epoxides as predicted by the bay-region concept. While a number of synthetic methods toward these diol epoxides are described in the literature, it was felt that a new, entirely different approach may be needed that achieves regio- and stereochemically controlled synthesis of these metabolites, particularly the bay-region analogues. The authors delineate a generally applicable, efficient synthesis of PAH diol epoxides and trans-dihydrodiols and its application to the first synthesis of the putative active metabolites, the bay-region diol epoxides of the carcinogen 1,4-dimethylphenanthrene (1,4-DMPh).