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 (-)-[3R,4R]-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).     

The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation

Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by human epoxide hydrolase (EH) and CYP1A1. Human dihydrodiol dehydrogenase isoforms (AKR1C1-AKR1C4), members of the aldo-keto reductase (AKR) superfamily, activate trans-dihydrodiols by converting them to reactive and redox-active o-quinones. We now show that the constitutively and widely expressed human AKR, aldehyde reductase (AKR1A1), will oxidize potent proximate carcinogen trans-dihydrodiols to their corresponding o-quinones. cDNA encoding AKR1A1 was isolated from HepG2 cells, overexpressed in Escherichia coli, purified to homogeneity, and characterized. AKR1A1 oxidized the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene with a higher utilization ratio (V(max)/K(m)) than any other human AKR. AKR1A1 also displayed a high V(max)/K(m) for the oxidation of 5-methylchrysene-7,8-diol, benz[a]anthracene-3,4-diol, 7-methylbenz[a]anthracene-3,4-diol, and 7,12-dimethylbenz[a]anthracene-3,4-diol. AKR1A1 displayed rigid regioselectivity by preferentially oxidizing non-K-region trans-dihydrodiols. The enzyme was stereoselective and oxidized 50% of each racemic PAH trans-dihydrodiol tested. The absolute stereochemistries of the reactions were assigned by circular dichroism spectrometry. AKR1A1 preferentially oxidized the metabolically relevant (-)-benzo[a]pyrene-7(R),8(R)-dihydrodiol. AKR1A1 also preferred (-)-benz[a]anthracene-3(R),4(R)-dihydrodiol, (+)-7-methylbenz[a]anthracene-3(S),4(S)-dihydrodiol, and (-)-7,12-dimethylbenz[a]anthracene-3(R),4(R)-dihydrodiol. The product of the AKR1A1-catalyzed oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was trapped with 2-mercaptoethanol and characterized as a thioether conjugate of benzo[a]pyrene-7,8-dione by LC/MS. Multiple human tissue expression array analysis showed coexpression of AKR1A1, CYP1A1, and EH, indicating that trans-dihydrodiol substrates are formed in the same tissues in which AKR1A1 is expressed. The ability of this general metabolic enzyme to divert trans-dihydrodiols to o-quinones suggests that this pathway of PAH activation may be widespread in human tissues.     

Cloning and developmental expression of mouse aldehyde reductase (AKR1A4)

The aldo-keto reductase superfamily catalyzes the reduction of a broad range of aldehydes and ketones to their corresponding alcohols. Here we report the cloning of the mouse aldehyde reductase cDNA and its embryonic pattern of expression. From stages E7.5 to E13.5 the gene encoding for this enzyme is expressed at high levels in several tissues, including the neural ectoderm, gut endoderm, somites, branchial arches, otic vesicles, limb buds, and tail bud. In adult mice aldehyde reductase was expressed in all tissues examined.     

Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals

Aldehyde reductase (AKR1A), a member of the aldo–keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.     

A CASE-SAR analysis of polycyclic aromatic hydrocarbon carcinogenicity

A CASE SAR analysis was performed on a selected database of PAHs to investigate the possible use of the CASE method as an aid for preliminary assessment of carcinogenic potential of untested environmental PAHs. A data set, denoted LEARN, consisting of 78 PAHs and their experimental carcinogenicities was used to 'train' the CASE method and derive the CASE fragments. 8 activating fragments and 4 inactivating fragments were identified. These fragments predicted the activities of 94% of the LEARN set correctly. The biological significance of several of these fragments are rationalized in light of the current theories of PAH carcinogenesis. Using these fragments, the potential activities of a database of 106, mostly untested PAHs, denoted TEST, were predicted. These were compared to 'expert judgement' predictions based on mechanistic considerations in order to evaluate the extent of concordance between these two methods and their respective strengths and weaknesses. Initial poor agreement (64%) was attributed to limitations of the LEARN database involving inadequate representation of 2- and 3-ring PAH subclasses. When these subclasses were excluded from the TEST database, the concordance improved to 90%. The CASE fragments were also used to predict the activities of a database of 24 PAHs, denoted VALIDATE (not included in the LEARN set) for which carcinogenicity data were available. The total prediction accuracy of 75% (89% of the actives correctly identified), despite the structural diversity of the VALIDATE set, provided independent evidence of the utility of the present CASE results. A close examination of the CASE incorrect predictions was conducted to delineate inadequancies of these CASE results in order to provide cautionary guidance for future application of the method. Finally, the present results were compared to the results of a previous CASE analysis based on a more limited PAH data set, and were found to be of greater general utility. It is concluded that the CASE fragments derived in the current study should provide a useful tool for assisting and complementing 'expert judgement' in the preliminary screening of PAHs for carcinogenic activity.     

Molecular characterization of polycyclic aromatic hydrocarbon (PAH)-degrading methanogenic communities

Previous research demonstrated that methanogenic cultures enriched from Baltimore Harbor (Baltimore, MD) sediments were able to degrade naphthalene and phenanthrene. In this report, the degradation activity was maintained through a sequential transfer without adding additional sediments and the established polycyclic aromatic hydrocarbon (PAH)-degrading methanogenic communities were characterized via comparative sequence analysis of clone libraries of 16S rRNA genes amplified using bacteria-specific and Archaea-specific primers. The phylogenetic analysis indicated that the addition of PAHs clearly shifted the structure of the methanogenic community and resulted in an increase in populations of species previously found in other hydrocarbon-degrading communities. Of particular interest is the fact that the dominant microbial population of the naphthalene cultures was different from that of the phenanthrene cultures, suggesting that different species are involved in the degradation. Finally, this information may lead to the identification and isolation of methanogenic populations that can degrade PAHs.     

Induction of aldehyde dehydrogenase by polycyclic aromatic hydrocarbons in rats

Short-term intragastric administration of selected polycyclic aromatic hydrocarbons (100 mg/kg daily for 4 days) to male Wistar rats resulted in marked changes in liver cytosolic aldehyde dehydrogenase activity. Non-carcinogenic anthracene, phenanthrene and chrysene produced a 2.5–3-fold increase in the activity assayed with propionaldehyde as substrate and NAD as coenzyme. Weakly carcinogenic 1,2-benzanthracene enhanced aldehyde dehydrogenase activity 9-fold and the potent carcinogens 3,4-benzpyrene and 3-methylcholanthrene 30-fold. With benzaldehyde as substrate and NADP as coenzyme the differences between the groups were even more pronounced. Somewhat similar but less manifest effects on aldehyde dehydrogenase activity were detected also in the liver microsomes and in the postmitochondrial fractions of the small intestinal mucosa. On the basis of their ability to induce aldehyde dehydrogenase activity the compounds could be divided into three groups. This classification was found to correlate well with the carcinogenic potency of the compounds. It appeared that the exposure to polycyclic aromatic hydrocarbons, especially the carcinogenic ones, was followed by synthesis of a new aldehyde dehydrogenase form. This new form was differentiated from the normally existing cytosolic aldehyde dehydrogenase by its ability to oxidize benzaldehyde in the presence of NADP.     

Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, environmental contaminants that pose a potential risk to fish populations. Both field and laboratory studies suggest that exposure of the early life stages of fish to PAH can mimic the embryotoxic effects of the planar halogenated hydrocarbons (PHHs), the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin. PHH toxicity is mediated by the aryl hydrocarbon receptor (AHR) and PHH potency is predicted by its AHR-binding affinity and CYP1A induction potency. However, the role of the AHR, if any, in mediating the developmental effects of PAH to fish remains unknown. In this study we looked at the AHR binding affinity of a test set of PAH that had been previously ranked for their potency for inducing teleost CYP1A. PAH that induced CYP1A inhibited [3H]TCDD binding to in vitro-expressed AHRs from rainbow trout and the AHR expressed in PLHC-1 fish hepatoma cells. Generally, the relative rank order for AHR binding affinity predicted the rank order of these same PAH for inducing CYP1A reported in other studies. There was a strong, positive relationship between binding to the PLHC-1 AHR (stimulus) and the EC50s for CYP1A induction (response) in whole juvenile trout and in RTL-W1 cells, but EC50s were much higher than expected for a 1:1 stimulus/response relationship. These data show that the ability of PAH to bind to teleost AHR predicts PAH potency for CYP1A induction. If PAH toxicity is receptor-mediated and predicted by induction potencies, we will have a powerful mechanistic-based tool for rapidly assessing the risk of toxicity to fish of PAH from any source.     

Developmental retardation in neonates of aldehyde reductase (AKR1A)-deficient mice is associated with low ascorbic acid and high corticosterone levels

Aldehyde reductase encoded by the Akr1a gene catalyzes the NADPH-dependent reduction of a variety of aldehyde compounds, and it plays a role in the biosynthesis of ascorbic acid (AsA) by converting D-glucuronate to L-gulonate. Although supplementing drinking water with AsA (1.5 mg/mL) ameliorates the fertility of Akr1a ^−/− (KO) female mice, litter sizes in the KO mice are typically smaller than those for Akr1a ^+/+ (WT) mice, and about one-third of the neonates have a reduced stature. Half of the neonates in the smallest, developmentally retarded group died before weaning, and the remaining half (less than 6 g in weight) also barely grew to adulthood. While no difference was found in the number of fetuses between the KO and WT mice at 14.5-embryonic days, the sizes of the KO fetuses had already diverged. Among the organs of these retarded KO neonates at 30 d, the spleen and thymus were characteristically small. While an examination of spleen cells showed the normal proportion of immune cells, apoptotic cell death was increased in the thymus, which would lead to thymic atrophy in the retarded KO neonates. Plasma AsA levels were lower in the small neonates despite the fact that their mothers had received sufficient AsA supplementation, and the corticosterone levels were inversely higher compared to wild-type mice. Thus, insufficient AsA contents together with a defect in corticosterone metabolism might be the cause of the retarded growth of the AKR1A-deficient mice embryos and neonates.     

Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.     

Environmental polycyclic aromatic hydrocarbon (PAH) exposure and DNA damage in Mexican children

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants presenting a public health risk, particularly to children, a vulnerable population. PAHs have genotoxic and carcinogenic properties, which depend on their metabolism. Many enzymes involved in PAH metabolism, including CYP1A1, CYP1B1, GSTM and GSTT are polymorphic, which may modulate the activation/deactivation of these compounds. We evaluated PAH exposure and DNA damage in children living in the vicinity of the main petrochemical complex located in the Gulf of Mexico, and explored the modulation by genetic polymorphisms of PAH excretion and related DNA damage. The participants (n=82) were children aged 6-10y attending schools near the industrial area. Urinary 1-hydroxypyrene (1-OHP; a biomarker of PAH exposure) was determined by reverse-phase-HPLC; DNA damage by the comet assay (Olive Tail Moment (OTM) parameter); CYP1A1*2C and CYP1B1*3 polymorphisms by real time-PCR; and GSTM1*0 and GSTT1*0 by multiplex PCR. The median value of 1-OHP was 0.37μmol/mol creatinine; 59% of children had higher 1-OHP concentrations than those reported in environmentally exposed adults (0.24μmol/mol creatinine). A stratified analysis showed increased DNA damage in children with 1-OHP concentrations greater than the median value. We observed higher 1-OHP concentrations in children with CYP1A1*2C or GSTM1*0 polymorphisms, and a positive influence of CYP1A1*2C on OTM values in children with the highest PAH exposure. The data indicate that children living in the surroundings of petrochemical industrial areas are exposed to high PAH levels, contributing to DNA damage and suggesting an increased health risk; furthermore, data suggest that polymorphisms affecting activation enzymes may modulate PAH metabolism and toxicity.     

Inhibition of 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) by aldose reductase inhibitors

Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is a member of the aldo-keto reductase superfamily that catalyses the oxido-reduction of steroid hormones such as estrogens, androgens and neurosteroids. Inhibitors of aldose reductase (AR), a member of the same superfamily, were evaluated against AKR1C21. Models of the enzyme-inhibitor complexes suggest that Tyr118 and Phe311 are important residues for inhibitor recognition and orientation in the active site of AKR1C21.     

Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase

A high molecular model compound of polycyclic aromatic hydrocarbon was synthesised by coupling pyrene to PEG(5000). The pyrene-PEG was used for the study of a laccase-mediator-system. To prevent direct contact between the substrate and the enzyme the two were kept in their own compartments separated by a membrane. The low molecular mediators, 1-hydroxybenzotriazole and 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), which were oxidised by laccase to the corresponding radicals or cations permeated the membrane and reacted with the pyrene-PEG model compound. Oxidation of the model compound resulted in an alpha-oxidation of the alkyl-chain leading to two main oxidation products. The same oxidation products were obtained in the reaction system without a membrane.     

Metabolism of the polycyclic aromatic hydrocarbon pyrene by Aspergillus niger SK 9317

The metabolism of pyrene, a polycyclic aromatic hydrocarbon consisting of four rings, by Aspergillus niger SK 9317 was investigated. The metabolites formed were isolated and identified as 1-hydroxypyrene, 1,6- and 1,8-pyrenequinone, 1,6- and 1,8-dihydroxypyrene, 1-pyrenyl sulphate and 1-hydroxy-8-pyrenyl sulphate. This is the first report of 1-hydroxy-8-pyrenyl as a metabolite in the microbial metabolism of pyrene. The results suggest that A. niger metabolizes pyrene by cytochrome P-450 monooxygenase enzyme systems.     

Induction of choline kinase by polycyclic aromatic hydrocarbon carcinogens in rat liver

The administration to rats of polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, 3,4-benzo(a) pyrene and β-naphthoflavone caused a significant elevation of hepatic choline kinase activity. On the other hand, phenobarbital-type inducers (phenobarbital, 1,1,1-trichloro 2,2-bis (ρ-chlorophenyl) ethane (DDT) and hexachlorobenzene) did not stimulate the activity at all. The administration of either cycloheximide or actinomycin D completely depressed the elevation of choline kinase activity induced by polycyclic aromatic hydrocarbons, indicating that the elevated activity by these chemicals could be due to the change in the enzyme level. These results strongly suggest that induction of choline kinase are involved in the sequence of events leading to the induction of hepatic drug metabolism by polycyclic aromatic hydrocarbons.     

Metabolic activation of selected aromatic amines and polycyclic hydrocarbons by isolated subcellular fractions of Drosophila melanogaster

The applicability of microsomal preparations from Drosophilamelanogaster as the metabolic factor in the Salmonella mutagenicity assay with strains TA98 and TA100 was evaluated. Isolated cellular fractions (S27) from PB-pretreated flies activated N-acetyl-2-aminofluorene (2-AAF), N-hydroxy-N-aceyl-2-aminofluorene (N-OH-AAF), benzo[a]pyrene (BP), 9,10-dimethylanthracene (DA) and 2 -naphythylamine (NA)_into mutagenic metabolites, 7,-12-Dimethylbenz[a]-anthracene (DMBA) was ineffective under the conditions of the test.This study was performed in an effort to determine optimal conditions for activating, by Drosophila enzymes, aromatic amines and polycyclic hydrocarbons, with 2-AAF and BP as model mutagens. The following alterations improved the sensitivity of this combined Salmonella/Drosophila assay. (1) Incubation of the plates at 25°C for 1 night instead of permanent exposure at 37°C. (2) Isolation of S27 fractions instead of the conventional S9, because 9000 × g was not sufficient tio spin down Drosphila mitochondria.     

Metabolic activation of polycyclic aromatic hydrocarbons to mutagens in the Ames test by various animal species including man

6 polycyclic aromatic hydrocarbons were assayed for mutagenicity in the Ames test, in the presence of hepatic post-mitochondrial preparations isolated from the mouse, rat, hamster, pig and man. Benzo[a]pyrene, dibenzo[a,i]pyrene and benz[a]anthracene gave a positive mutagenic response only in the presence of activation systems derived from the hamster. With the exception of the pig, activation systems derived from all animal species could convert 3-methylcholanthrene to mutagens, the hamster being the most efficient. With the exception of the rat and pig, all animal species activated 7,12-dimethylbenz[a]-anthracene to mutagens, the human preparation being the most effective followed by the hamster and mouse. Dibenz[a,h]anthracene was not activated by any of the hepatic preparations. It is concluded that, among the animal species studied the hamster is generally the most efficient in activating polycyclic aromatic hydrocarbons to mutagens in the Ames test.     

Human pancreas protein 2 (PAN2) has a retinal reductase activity and is ubiquitously expressed in human tissues

Human gene for pancreas protein 2 (PAN2) is a novel member of the short-chain dehydrogenase/reductase gene superfamily. The properties of PAN2 protein have not yet been characterized. We present the first evidence that human PAN2 is a ubiquitously expressed microsomal enzyme that recognizes retinoids but not steroids as substrates with the apparent K(m) values between 0.08 microM and 0.4 microM. PAN2 is approximately 4-fold more efficient in the reductive than in the oxidative direction. The apparent K(m) values for NADP(+) and NADPH are 0.65 microM and 0.32 microM versus 1200 microM and 1060 microM for NAD(+) and NADH, respectively. Kinetic constants and expression pattern of PAN2 suggest that it is likely to function as a reductase in vivo and might contribute to the reduction of retinaldehyde to retinol in most human tissues.