Quinone reduction and redox cycling catalysed by purified rat liver dihydrodiol/3 alpha-hydroxysteroid dehydrogenase.

A highly active preparation of rat liver dihydrodiol/3 alpha-hydroxysteroid dehydrogenase was obtained using a newly developed, rapid purification scheme involving affinity chromatography on Red Sepharose. Depending on the coenzyme present, the purified enzyme was found to catalyse the oxidation of dihydrodiols and steroids or the reduction of substrates with carbonyl or quinone moieties. Using a wide range of synthetic quinones derived from polycyclic aromatic hydrocarbons (PAHs), we observed a pronounced regioselectivity of the quinone reductase activity. Good substrates were the o-quinones of phenanthrene, benz(a)anthracene, chrysene and benzo(a)pyrene with the quinonoid moiety in the K-region which were reduced at rates of 1-10 mumol/min.mg enzyme. 1,4-Benzoquinone, naphthalene-1,2-quinone and benz(a)anthracene-8,9-quinone were also reduced at high rates. In contrast, alkyl-substituted quinones such as duroquinone and menadione were poor substrates for the enzyme. During the enzymatic reduction of several o-quinones, but not 1,4-benzoquinone, we observed the oxidation of large amounts of NADPH and the consumption of molecular oxygen which is indicative of a redox-cycling process. Thus, the reduction of quinones of PAHs may lead to a facilitated conjugation and excretion of these highly lipophilic compounds, but may also initiate toxic processes due to the formation of reactive oxygen species.     

Specificity of human aldo-keto reductases, NAD(P)H:quinone oxidoreductase, and carbonyl reductases to redox-cycle polycyclic aromatic hydrocarbon diones and 4-hydroxyequilenin-o-quinone

Polycyclic aromatic hydrocarbons (PAHs) are suspect human lung carcinogens and can be metabolically activated to remote quinones, for example, benzo[a]pyrene-1,6-dione (B[a]P-1,6-dione) and B[a]P-3,6-dione by the action of either P450 monooxygenase or peroxidases, and to non-K region o-quinones, for example B[a]P-7,8-dione, by the action of aldo keto reductases (AKRs). B[a]P-7,8-dione also structurally resembles 4-hydroxyequilenin o-quinone. These three classes of quinones can redox cycle, generate reactive oxygen species (ROS), and produce the mutagenic lesion 8-oxo-dGuo and may contribute to PAH- and estrogen-induced carcinogenesis. We compared the ability of a complete panel of human recombinant AKRs to catalyze the reduction of PAH o-quinones in the phenanthrene, chrysene, pyrene, and anthracene series. The specific activities for NADPH-dependent quinone reduction were often 100-1000 times greater than the ability of the same AKR isoform to oxidize the cognate PAH-trans-dihydrodiol. However, the AKR with the highest quinone reductase activity for a particular PAH o-quinone was not always identical to the AKR isoform with the highest dihydrodiol dehydrogenase activity for the respective PAH-trans-dihydrodiol. Discrete AKRs also catalyzed the reduction of B[a]P-1,6-dione, B[a]P-3,6-dione, and 4-hydroxyequilenin o-quinone. Concurrent measurements of oxygen consumption, superoxide anion, and hydrogen peroxide formation established that ROS were produced as a result of the redox cycling. When compared with human recombinant NAD(P)H:quinone oxidoreductase (NQO1) and carbonyl reductases (CBR1 and CBR3), NQO1 was a superior catalyst of these reactions followed by AKRs and last CBR1 and CBR3. In A549 cells, two-electron reduction of PAH o-quinones causes intracellular ROS formation. ROS formation was unaffected by the addition of dicumarol, suggesting that NQO1 is not responsible for the two-electron reduction observed and does not offer protection against ROS formation from PAH o-quinones.     

Lack of quinone reductase activity suggests that amyloid-beta peptide/ERAB induced lipid peroxidation is not directly related to production of reactive oxygen species by redoxcycling

Mitochondrial type II hydroxyacyl-CoA dehydrogenase (ERAB) has recently been shown to mediate amyloid-beta peptide (Abeta) induced apoptosis and neurodegeneration. The precise mechanism of cell death induction is unknown, however, Abeta inhibits ERAB activities and as a result of ERAB-Abeta interactions, enhanced formation of lipid peroxidation products occur. The possibility that ERAB mediates quinone reduction is therefore investigated, thus giving the potential of redoxcycling and production of reactive oxygen species, leading to lipid peroxidation. Recombinant human ERAB was produced in a bacterial expression system and enzymological properties were evaluated. Using several orthoquinones as substrates, no ERAB mediated quinone reductase activity was found either in the presence or absence of Abeta, suggesting that the observed in vivo lipid peroxidation is a result of other mechanisms than redoxcycling by quinones.     

Characterization of an oligomeric carbonyl reductase of dog liver: its identity with peroxisomal tetrameric carbonyl reductase

Dog liver contains an oligomeric NADPH-dependent carbonyl reductase (CR) with substrate specificity for alkyl phenyl ketones, but its endogenous substrate and primary structure remain unknown. In this study, we examined the molecular weight and substrate specificity of the enzyme purified from dog liver. The enzyme is a ca. 100-kDa tetramer composing of 27-kDa subunit, and reduces all-trans-retinal and alpha-dicarbonyl compounds including isatin, which are substrates for pig peroxisomal tetrameric carbonyl reductase (PTCR). In addition, the dog enzyme resembles pig PTCR in inhibitor sensitivity to flavonoids, myristic acid, lithocholic acid, bromosulfophthalein and flufenamic acid. Furthermore, the amino acid sequence of dog CR determined by protein sequencing and cDNA cloning was 84% identical to that of pig PTCR and had a C-terminal peroxisomal targeting signal type 1, Ser-His-Leu. The immunoprecipitation using the anti-pig PTCR antibody shows that the dog enzyme is a major form of soluble NADPH-dependent all-trans-retinal reductase in dog liver. Thus, dog oligomeric CR is PTCR, and may play a role in retinoid metabolism as a retinal reductase.     

In vivo exposure of Dreissena polymorpha mussels to the quinones menadione and lawsone: menadione is more toxic to mussels than lawsone

The principal aim of this study was to assess whether the two quinones, menadione (2-methyl-1,4-naphthoquinone) and lawsone (2-hydroxy-1,4-naphthoquinone), elicit differential toxicity in mussels as has been reported for higher organisms. Therefore, the effects of short-term (48 h) and long-term (20 days) exposure of the two quinones at concentrations of 0.56 and 1 mg l(-1) to zebra mussels, Dreissena polymorpha, under laboratory conditions were studied. After the short-term exposure, the specific activities of the two-electron quinone oxidoreductase (DT-diaphorase) and the one-electron catalysing quinone reductases NADPH-cytochrome c reductase and NADH-cytochrome c reductase were determined in the gills and the rest of the soft tissues (soft mussel tissues minus the gills) of both treated and control mussels. At the higher concentrations of menadione and lawsone used, a significant reduction of the activity of NADPH-cytochrome c reductase in the gills and in the rest of the soft mussel tissues (by 33-34% and 31-43%, respectively) was observed. The activities of DT-diaphorase and NADH-cytochrome c reductase were not significantly affected. Interestingly, DT-diaphorase was observed in the gills, an organ requiring protection against antioxidants. Furthermore, a single-cell electrophoretic assay (comet assay) performed with gill cells to assess DNA damage by the quinones did not show any significant difference between the treated and the control organisms. This indicates that the formation of reactive species by the quinone metabolism in vivo in the mussels was possibly suppressed through the concerted action of DT-diaphorase and antioxidant enzymes. The results of in vitro experiments with gill extracts confirmed the protective role of DT-diaphorase. The rate of the two-electron quinone reduction was found to be five times that of the one-electron quinone reduction. The results of the long-term exposure unambiguously demonstrated that in mussels menadione, unlike in higher organisms, is more toxic than lawsone. The lack of detectability of xanthine oxidase in the mussel tissues could explain the comparatively lower toxicity of lawsone in the invertebtrate, lending support to a previous suggestion that xanthine oxidase might be responsible for the mechanism of toxicity of lawsone in higher organisms in vivo.     

Reductases for carbonyl compounds in human liver

Two aldehyde reductases with mol. wt 78,000 and 32,000 and one carbonyl reductase with mol. wt 31,000 were purified to homogeneity from human liver cytosol. The high molecular weight aldehyde reductase exhibited properties similar to alcohol dehydrogenase; it had a single subunit of mol. wt 41,000 and a pI value of 10 to 10.5, and showed preference for NADH over NADPH as cofactor and sensitivity to SH-reagents, pyrazole, o-phenanthroline and isobutyramide. The enzyme reduced aliphatic and aromatic aldehydes, alicyclic ketones and alpha-diketones and an optimal pH of 6.0, and oxidized various alcohols with NAD as a cofactor at an optimal pH of 8.8. The identity of the enzyme with alcohol dehydrogenase was established by starch gel electrophoresis and co-purification of the two enzymes. The other enzymes were NADPH-dependent and monomeric reductases; the aldehyde reductase reduced aldehydes, hexonates and alpha-diketones and was sensitive to barbiturates, diphenylhydantoin and valproate, while the carbonyl reductase showed a broad substrate specificity for aldehydes, ketones and quinones and was inhibited by SH-reagent, quercitrin and benzoic acid. The latter enzyme appeared in three multiforms with different charges which occurred in differing ratios in liver specimens. Comparison of kinetic constants for aldehydes among the enzymes indicated that alcohol dehydrogenase is the best reductase with the highest affinity and Kcat values. The enzyme also catalyzed oxidation and reduction of aromatic aldehydes in the presence of NAD at physiological pH of 7.2. Tissue distribution of the three enzymes and variation of their specific activities in human livers were examined.     

The metabolism and toxicity of quinones,quinonimines, quinone methides,and quinone-thioethers

Quinones are ubiquitous in nature and constitute an important class of naturally occurring compounds found in plants, fungi and bacteria. Human exposure to quinones therefore occurs via the diet, but also clinically or via airborne pollutants. For example, the quinones of polycyclic aromatic hydrocarbons are prevalent as environmental contaminants and provide a major source of current human exposure to quinones. The inevitable human exposure to quinones, and the inherent reactivity of quinones, has stimulated substantial research on the chemistry and toxicology of these compounds. From a toxicological perspective, quinones possess two principal chemical properties that confer their reactivity in biological systems. Quinones are oxidants and electrophiles, and the relative contribution of these properties to quinone toxicity is influenced by chemical structure, in particular substituent effects. Modification to the quinone nucleus also influences quinone metabolism. This review will therefore focus on the differences in structure and metabolism of quinones, and how such differences influence quinone toxicology. Specific examples will be discussed to illustrate the diverse manner by which quinones interact with biological systems to initiate and propagate a toxic response.     

Bioreductive activation of quinones: A mixed blessing

Quinones can be metabolized by various routes: substitution or reductive addition with nucleophilic compounds (mainly glutathione and protein thiol groups), one-electron reduction (mainly by NADPH: cytochrome P-450 reductase) and two-electron reduction (by D,T-diaphorase). During reduction semiquinone radicals and hydroquinones are formed, which can transfer electrons to molecular oxygen, resulting in the formation of reactive oxygen intermediates and back-formation of the parent quinone (redox cycling). Reaction of semiquinones and reactive oxygen intermediates with DNA and other macromolecules can lead to acute cytotoxicity and/or to mutagenicity and carcinogenicity. The enhanced DNA-alkylating properties of certain hydroquinones are exploited in the bioreductive alkylating quinones. Acute cytotoxicity of quinones appears to be related to glutathione depletion and to interaction with mitochondria and subsequent disturbance of cellular energy homoeostasis and calcium homoeostasis. These effects can to a certain extent be predicted from the electron-withdrawing and electron-donating effects of the substituents on the quinone nucleus of the molecule. Prediction of cytostatic potential remains much more complicated, because reduction of the quinones and the reactivity of the reduction products with DNA are modulated by the prevailing oxygen tension and by the prevalence of reducing enzymes in tumour cells.This article is based on a lecture given at the 16th LOF Symposium, 27 October 1989, Utrecht, the Netherlands.     

Induction of NAD(P)H:quinone reductase by probucol: a possible mechanism for protection against chemical carcinogenesis and toxicity

Dietary antioxidants protect laboratory animals against induction of tumours by a variety of chemical carcinogens. Among possible mechanism, protection against chemical carcinogenesis could be mediated via antioxidant-dependent induction of detoxifying enzymes, including quinone reductase and glutathione S-transferase (GSH transferase). Probucol is used cholesterol-lowering drug used in the clinic, with pronounced antioxidant effect that protect against chemical carcinogenesis and toxicity. In the present study we therefore examined the ability of probucol to induce activities of quinone reductase in the cytosolic fractions of various tissues of mice. Quinone reductase activity was increased significantly in 6 of 8 tissues examined from probucol-fed mice. The greatest proportionate increase, to 1.8 times control levels, was observed in liver. Probucol also increased quinone reductase activities of forestomach, heart, kidney, lungs and spleen. Quinone reductase is a major enzyme of xenobiotic metabolism that carries out obligatory two-electron reductions and thereby protects cells against toxicity of quinones. It is induced in many tissues coordinately with other enzymes that protect against electrophilic toxicity. The protective effects of probucol appear to be due, at least in part, to the ability of this antioxidant to increase the activities in rodent tissues of several enzymes involved in the non-oxidative metabolism of a wide variety of xenobiotics. The induction of such enzyme, quinone reductase by probucol suggests the potential value of this compound as a protective agent against chemical carcinogenesis and other forms of electrophilic toxicity. The significance of these results can be implicated in relation to cancer chemopreventive effects of probucol in various target organs.     

Evidence for distinct binding sites in the cumene hydroperoxide-dependent metabolism of benzo[a]pyrene catalyzed by cytochrome P-450

A few constitutive cytochrome P-450 isozymes in male rat liver microsomes catalyzed the metabolism of benzo[a]pyrene (BP) in cumene hydroperoxide (CHP)-dependent reactions, which produced predominantly 3-hydroxyBP and BP quinones. This process varied with the concentration of CHP. At 0.05 mM CHP, 3-hydroxyBP was the major metabolite. An increase in CHP concentration reduced 3-hydroxyBP formation but increased the level of BP quinones. This change in metabolic profile was reversed by preincubation with pyrene. Pyrene selectively inhibited quinone formation and enhanced 3-hydroxyBP formation. Naphthalene, phenanthrene and benz[a]anthracene nonspecifically inhibited total metabolism. BP binding to microsomal protein correlated with quinone formation, suggesting a common precursor reactive intermediate. BP metabolism by female rat liver microsomes also depended on CHP concentration but was much less effective than that in the male. With females, quinones were the major metabolites at all CHP concentrations, and their formation was again modulated by pyrene. These data indicate that two distinct binding sites are responsible for the formation of 3-hydroxyBP and BP quinones.     

High levels of expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin.

NAD(P)H:quinone oxidoreductase (NQO1) is a flavoprotein which catalyzes the two-electron reduction of quinones and azo-dyes and thus prevents the formation of free radicals and toxic oxygen metabolites that may be generated by the one-electron reductions catalyzed by cytochrome P450 reductase. Analysis of RNA indicated 20- to 50-fold higher levels of NQO1 gene expression in the liver tumors and in the tissue surrounding the tumors of patients with hepatocarcinoma than in normal individuals. An approximately 50-fold higher level of NQO1 mRNA was also observed in human hepatoblastoma (Hep-G2) cells than in normal liver. By deletion mutagenesis in the human NQO1 gene promoter and subsequent transfection into hepatic and nonhepatic cell lines, a 1.42 kb DNA segment has been identified to contain cis-acting elements responsible for high levels of expression of the NQO1 gene in tumor cells.     

Differential mechanisms of cell killing by redox cycling and arylating quinones

 The role of mitochondrial dysfunction in the mechanism of cell killing by quinones of differing chemical reactivities was investigated. Freshly isolated hepatocyte suspensions were exposed to 2,3-dimethoxy-1, 4-naphthoquinone, 2-methyl-1,4-naphthoquinone, 1,4-naphthoquinone or 1,4-benzoquinone in the presence or absence of cyclosporine A, ruthenium red, fructose or the combination of fructose plus oligomycin. All of the quinones caused concentration-dependent cell killing as assessed by the leakage of lactate dehydrogenase. However, only 2,3-dimethoxy- and 2-methyl-naphthoquinone caused a depolarization of mitochondrial membrane potential; cell killing by 1,4-naphthoquinone or 1,4-benzoquinone was not accompanied by mitochondrial depolarization. Neither cyclosporine A nor ruthenium red protected against cell killing or loss of mitochondrial membrane potential caused by any of the quinones examined. In contrast, fructose protected cells against all four quinones. For the redox cycling naphthoquinones, oligomycin reversed the protection afforded by fructose. However, the cytoprotective effect of fructose against the arylating quinones, 1,4-naphthoquinone and 1,4-benzoquinone, was not reversed by oligomycin. The results suggest that cell killing by redox cycling naphthoquinones is a manifestation of mitochondrial depolarization, not ATP depletion. In contrast, the fructose-mediated protection from arylating quinones is consistent with ATP depletion being a critical event leading to cell death. Accordingly, although a vast array of quinone compounds are known to be cytotoxic, the mechanism of cell killing by individual members of this chemical class differs and is determined primarily by the chemical reactivity of the individual quinone. Received: 26 June 1995 / Accepted: 17 October 1995     

K-region epoxides of polycyclic hydrocarbons: formation and further metabolism by rat-lung preparations

Epoxides of 7-methylbenz[a]anthracene and of benzo[a]pyrene that have been identified as the K-region epoxides, 7-methylbenz[a]anthracene 5,6-oxide and benzo[a]pyrene 4,5-oxide, have been detected as microsomal metabolites using preparations from the lungs of rats that had been pretreated with the microsomal mixed function oxidase inducer, 3-methylcholanthrene. It was also possible, using lung microsomal preparations from uninduced animals, to demonstrate the formation of an epoxide identified as the K-region derivative, benz[a]anthracene 5,6-oxide, as a microsomal metabolite of benz[a]anthracene. The K-region epoxides of 7-methylbenz[a]anthracene and of benzo[a]pyrene could not always be detected as metabolites when lung microsomal preparations from uninduced rats were used. The activities of two other enzymes present in pulmonary tissue fractions that are involved in the further metabolism of polycyclic hydrocarbon epoxides have also been measured and the values compared with those obtained with rat-liver. When benz[a]anthracene 5,6-oxide was used as substrate, much lower levels of microsomal epoxide hydrase activity were found in lung than in liver, but soluble-supernatant fractions of rat-lung appeared to possess higher levels of glutathione S-epoxide transferase activity than were present in rat-liver.The significance of these results in relation to the metabolic activation of polycyclic hydrocarbons by epoxide formation and to the induction of tumours of the respiratory tract by members of this class of chemical carcinogens is discussed.     

Natural and synthetic naphthoquinones active against Trypanosoma cruzi: an initial step towards new drugs for Chagas disease

Chagas disease is one of the most important endemic diseases in Latin America, caused by Trypanosoma cruzi. The drugs used for the treatment of this disease, nifurtimox and benznidazole, are toxic and present severe side effects. The need of effective drugs, without adverse effects, has stimulated the search for new compounds with potential clinical utility. An overview of a number of natural naphthoquinones tested against T. cruzi parasites is provided. Among natural naphthoquinones, lapachol, β-lapachone and its α-isomer have demonstrated useful trypanocidal activities. In the search for new trypanocidal agents, this review outlines different structural modifications of natural quinones, as well as synthetic quinones, which have been subjected to trypanocidal studies. This review summarizes the mechanism of action and structure-activity relationships of the quinone derivatives, including some theoretical calculations that discuss the correlation of stereo electronic properties with the trypanocidal activity. In this context, this review will be useful for the development of new antichagasic drugs based mainly on structural modification of natural quinones.     

Effects of quinones and flavonoids on the reduction of all-trans retinal to all-trans retinol in pig heart

We have recently purified a tetrameric carbonyl reductase from the cytosolic fraction of pig heart (pig heart carbonyl reductase). Since pig heart carbonyl reductase efficiently reduces all-trans retinal as the endogenous substrate, it probably plays an important role in retinoid metabolism in the heart. The purpose of the present study was to evaluate the inhibitory effects of quinones and flavonoids on the reduction of all-trans retinal to all-trans retinol catalyzed by pig heart carbonyl reductase, using pig heart cytosol. Of quinones tested, 9,10-phenanthrenequinone, a component of diesel exhaust particles, was the most potent inhibitor for the all-trans retinal reduction, and a significant inhibition was also observed for plumbagin and menadione. The order of the inhibitory potencies for flavonoids was kaempferol > quercetin > genistein > myricetin = apigenin = daidzein. However, the inhibitory potencies of flavonoids were much lower than that of 9,10-phenanthrenequinone. 9,10-Phenanthrenequinone competitively inhibited the all-trans retinal reduction, whereas kaempferol exhibited a mixed-type inhibition. It is likely that 9,10-phenanthrenequinone strongly inhibits the reduction of all-trans retinal to all-trans retinol by acting as the substrate inhibitor of pig heart carbonyl reductase present in pig heart cytosol.     

Metabolism of chrysene by brown bullhead liver microsomes.

We have investigated the regio- and stereoselective metabolism of chrysene, a four-ring symmetrical carcinogenic polycyclic aromatic hydrocarbon (PAH), by the liver microsomes of brown bullhead (Ameriurus nebulosus), a bottom-dwelling fish species. The liver microsomes from untreated and 3-methylcholanthrene (3-MC)-treated brown bullheads metabolized chrysene at the rate of 30.1 and 82.2 pmol/mg protein/min, respectively. Benzo-ring diols (1,2-diol and 3,4-diol) were the major chrysene metabolites formed by liver microsomes from control and 3-MC-treated fish. However, the control microsomes produced a considerably higher proportion of chrysene 1,2-diol (benzo-ring diol with a bay region double bond) plus 1-hydroxychrysene, than 3,4-diol plus 3-hydroxychrysene, indicating that these microsomes are selective in attacking the 1,2- position of the benzo-ring. On the other hand, 3-MC-induced microsomes did not show such a regioselectivity in the metabolism of chrysene. Control bullhead liver microsomes, compared to control rat liver microsomes, produced a considerably higher proportion of chrysene 1,2-diol, the putative proximate carcinogenic metabolite of chrysene. Like rat liver microsomes, bullhead liver microsomes produced only trace amounts of the K-region diol. Chrysene 1,2-diol and 3,4-diol formed by the liver microsomes from both control and 3-MC-treated bullheads consisted predominantly of their R,R-enantiomers. Chrysene is metabolized by bullhead liver microsomal enzymes to its benzo-ring diols with a relatively lower degree of stereoselectivity compared to benzo[a]pyrene (a five-ring PAH), but with a higher degree of stereoselectivity compared to phenanthrene (a three-ring PAH). The data of this study, together with those from our previous studies with phenanthrene, benzo[a]pyrene and dibenzo[a,l]pyrene (a six-ring PAH), indicate that the regioselectivity in the metabolism of PAHs by brown bullhead and rainbow trout liver microsomes does not vary greatly with the size and shape of the molecule, whereas the degree of stereoselectivity in the metabolism of PAHs to benzo-ring dihydrodiols does.     

Characterization of the enzymes involved in the in vitro metabolism of amrubicin hydrochloride

The in vitro metabolism of amrubicin by rat and human liver microsomes and cytosol was examined. The main metabolic routes in both species were reductive deglycosylation and carbonyl group reduction in the side-chain. In vitro metabolism of amrubicinol by rat and human liver microsomes and cytosol was also examined and the main metabolic route of this active metabolite was reductive deglycosylation. Metabolism of amrubicin in human liver microsomes was inhibited by TlCl(3) and that in human liver cytosol was inhibited by dicumarol and quercetin. Generation of amrubicinol was inhibited only by quercetin. The results indicate that metabolism of amrubicin is mediated by NADPH-cytochrome P450 reductase, NADPH:quinone oxidoreductase and carbonyl reductase. In addition, generation of amrubicinol is mediated by carbonyl reductase. Metabolism of amrubicinol in human liver microsomes was inhibited by TlCl(3) and that in human liver cytosol was inhibited by dicumarol. The results indicate that metabolism of amrubicinol is mediated by NADPH-cytochrome P450 reductase and NADPH:quinone oxidoreductase. To investigate the influence of cisplatin on the metabolism of amrubicin and amrubicinol, human liver microsomes and cytosol were pre-incubated with cisplatin. This did not change the rates of amrubicin and amrubicinol metabolism in either human liver microsomes or cytosol.     

Stereoselectivity of cytochrome P-450 isozymes and epoxide hydrolase in the metabolism of polycyclic aromatic hydrocarbons

Enantiomeric compositions of epoxides formed in the metabolism of planar benz[a]anthracene (BA), benzo[a]pyrene (BaP), and chrysene (CR), and nonplanar benzo[c]phenanthrene (BcPh), 12-methylbenz[a]anthracene (12-MBA) and 7,12-dimethylbenz[a]anthracene (7,12-DMBA) by liver microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats are determined either by direct chiral stationary phase HPLC analysis or by the enantiomeric compositions of metabolically formed trans-dihydrodiols. Cytochrome P-450 isozymes contained in various liver microsomal preparations have varying degrees of stereoselectivity in catalyzing the epoxidation reactions at various formal double bonds of the polycyclic aromatic hydrocarbons studied. In general, cytochrome P-450c, the major cytochrome P-450 isozyme contained in liver microsomes from 3-methylcholanthrene-treated rats, has the highest degree of stereoselectivity. Regardless of absolute configuration, non-K-region epoxides are converted to trans-dihydrodiols by epoxide hydrolase-catalyzed water attack at the allylic carbon. The S-center of K-region S,R-epoxide enantiomers derived from planar BA, BaP and CR is the major site of epoxide hydrolase-catalyzed water attack. In contrast, the R-center of K-region S,R-epoxide enantiomers derived from nonplanar BcPh, 12-MBA and 7,12-DMBA is the major site of epoxide hydrolase-catalyzed water attack. However, the K-region R,S-epoxide enantiomers of the six polycyclic aromatic hydrocarbons studied are hydrated by microsomal epoxide hydrolase with varying degrees of regioselectivity. Thus the enantiomeric composition of a metabolically formed dihydrodiol is determined by (i) the stereoselective epoxidation at a formal double bond of a parent hydrocarbon by microsomal cytochrome P-450 isozymes and (ii) the enantioselective and regioselective hydration of the metabolically formed epoxide by microsomal epoxide hydrolase.     

Carbonyl Reductases and Pluripotent Hydroxysteroid Dehydrogenases of the Short-chain Dehydrogenase/reductase Superfamily

Carbonyl reduction of aldehydes, ketones, and quinones to their corresponding hydroxy derivatives plays an important role in the phase I metabolism of many endogenous (biogenic aldehydes, steroids, prostaglandins, reactive lipid peroxidation products) and xenobiotic (pharmacologic drugs, carcinogens, toxicants) compounds. Carbonyl-reducing enzymes are grouped into two large protein superfamilies: the aldo-keto reductases (AKR) and the short-chain dehydrogenases/reductases (SDR). Whereas aldehyde reductase and aldose reductase are AKRs, several forms of carbonyl reductase belong to the SDRs. In addition, there exist a variety of pluripotent hydroxysteroid dehydrogenases (HSDs) of both superfamilies that specifically catalyze the oxidoreduction at different positions of the steroid nucleus and also catalyze, rather nonspecifically, the reductive metabolism of a great number of nonsteroidal carbonyl compounds. The present review summarizes recent findings on carbonyl reductases and pluripotent HSDs of the SDR protein superfamily.