Isolation and characterization of rabbit liver xenobiotic carbonyl reductases

Xenobiotic carbonyl reductases have been isolated from rabbit liver by ammonium sulfate fractionation and isoelectric focusing. Although these enzymes are very heterogeneous, the above procedures resolve the majority of the reductases in good yield. Most of the carbonyl reduction of oxisuran, 3,7-dimethyl-1-(5-oxyhexyl)-xanthine, metyrapone and daunorubicin (pH6.0) was accomplished by two distinct enzymes of pI 4.84 and 4.98. Other reductases with lesser activities toward these same substrates also occurred at higher pI values. Also resolved were several forms of enzymes that reduced daunorubicin (pH8.5) (previously identified as aldehyde reductase), naloxone and naltrexone (dihydrornorphinone reductases), and the model compounds, p-nitrobenzaldehyde and p-nitroacetophenone. The hydrogen stereospeciflcity of each of the rabbit liver carbonyl reductases, as well as rat liver aldehyde reductase, was determined by reducing the carbonyl substrates with A- and B-labeled [4-³H]NADPH and examining transfer of label to alcohol products and retention of label in the resulting oxidized cofactors. All of the oxisuran, metyrapone and daunorubicin (pH 6.0) reductases displayed B-hydrogen stereospecificity. Some enzymes that reduce 3,7-dimethyl-1-(5-oxyhexyl)-xanthine, p-nitroacetophenone and p-nitrobenzaldehyde were also B-stereospecific while other forms of these same enzymes were A-stereospecific. Only daunorubicin (pH8.5) (rabbit and rat), naloxone and naltrexone reductases were exclusively A-stereospecific. Apparent deuterium isotope effects of A- and B-labeled [4-²H]NADPH with daunorubicin (pH 6.0) reductases, daunorubicin (pH 8.5) reductase and naloxone reductases confirm the above hydrogen stereospecificity assignments. The results confirm the hydrogen specificity of aldehyde reductases as A-stereospecific and the majority of ketone reductases as B-stereospecific. In addition, several significant A-stereospecific ketone reductases appear to represent exceptions to the generalization that enzymes which catalyze the same reaction have the same stereospecificity. Finally, the binding of rat liver aldehyde reductase to NADPH produced a red shift in the cofactor 340 nm absorbance maximum which is opposite to that predicted on the basis of its hydrogen stereospecificity.     

Heterogeneity of anthracycline antibiotic carbonyl reductases in mammalian livers.

The anticancer antibiotics, daunorubicin and adriamycin, are reduced to their corresponding alcohol and glycol metabolites by cytoplasmic carbonyl reductases occurring in human, rabbit, rat and mouse tissues. Our data indicate that at least two different groups of pH-dependent daunorubicin reductases occur in rabbit and human liver: (1) the pH activity profile for daunorubicin reductases shows distinct optima or significant activity at both pH 6.0 and 8.5, and (2) pH 8.5-dependent daunorubicin reductases are resolved from pH 6.0-dependent daunorubicin reductases by ion exchange chromatography on DEAE-cellulose columns, gel filtration chromatography on BioGel P150, and isoelectric focusing. Ion exchange chromatography and isoelectric focusing also resolve multiple forms of each class of activities. A similar analysis suggests that a single type of pH-dependent daunorubicin reductase occurs in rat and mouse livers: (1) rat and mouse livers show a single pH optima for daunorubicin at pH 8.5, and (2) isoelectric focusing of rat and mouse preparations confirms the existence of a pH 8.5 daunorubicin and the absence of significant pH 6.0 daunorubicin activity. Although total adriamycin reduction is lower than daunorubicin reduction at any pH, significant adriamycin reduction also occurs in rabbit liver at pH 6.0 and 8.5; however, neither of these activities can be distinguished from pH 6.0 daunorubicin reductase activity by ion exchange and gel filtration chromatography and isoelectric focusing. In comparison, very low levels of pH 6.0 optimum adriamycin reductase activity are seen in human, rat and mouse livers. Thus, all species have pH 8.5 daunorubicin reductase and probably pH 8.5 adriamycin reductase, whereas rabbit and human also have pH 6.0 daunorubicin reductase(s) and rabbit has a pH 6.0 adriamycin reductase(s), which accounts for the bulk of the active anthracycline antibiotic metabolism in these species.     

Molecular and structural aspects of xenobiotic carbonyl metabolizing enzymes. Role of reductases and dehydrogenases in xenobiotic phase I reactions

The major metabolic pathways involved in synthesis and disposition of carbonyl and hydroxyl group containing compounds are presented, and structural and functional characteristics of the enzyme families involved are discussed. Alcohol and aldehyde dehydrogenases (ADH, ALDH) participate in oxidative pathways, whereas reductive routes are accomplished by members of the aldo-keto reductase (AKR), short-chain dehydrogenases/reductases (SDR) and quinone reductase (QR) superfamilies. A wealth of biochemical, genetic and structural data now establishes these families to constitute important phase I enzymes.     

Properties of NADPH-dependent carbonyl reductases in rat liver cytosol

Rat liver cytosol was shown previously by us to contain multiple forms of 3 alpha-hydroxysteroid dehydrogenase. Two (F4-II and -III) of the seven forms were purified to homogeneity, and four (F3-II, -III, -IV and F4-I) of them partially purified. One of them (F4-III) has been shown previously to catalyze the reduction of long-chain aliphatic and aromatic aldehydes or aromatic ketones as well as 3-oxosteroids [M. Ikeda et al., Biochem. Pharmac. 30, 1931 (1981)]. The reducing activity of such compounds was examined with the other F4 enzymes, and it was revealed that they also reduce a number of carbonyl compounds described above. In addition, quinones were tested for the first time in this report as substrates for all the F4 enzymes, and among them 9,10-phenanthrenequinone was found to be the best substrate for them, followed by hydrindantin and 2,6-dichlorophenolindophenol, while menadione was a poor substrate. The F4 enzymes did not catalyze the reduction of the oxo group at the 9-position of the prostaglandins of the E and A class with NADPH or NADH. On the basis of this evidence, the identity of ketone reductases (F4-I-III) in the rat liver is proposed to be 3 alpha-hydroxysteroid dehydrogenase, rather than prostaglandin 9-ketoreductase, which was demonstrated to correspond to ketone reductase in human brain [B. Wermuth, J. biol. Chem. 256, 1206 (1981)].     

Aminoacetylenic carbonyl compounds

Conclusion 2-Substituted 2-propargylindan-1,3-diones were prepared by treating the salt of 2-substituted 1,3-indandione with propargyl bromide. Aminomethylation of the product with paraformaldehyde and secondary amines afforded a series of 2-substituted 2-(-aminobutynyl)indan-1,3-diones which exhibit depressant effects on the central nervous system.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 8, pp. 18–22, August, 1968.     

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.     

Determination of toxic carbonyl compounds in cigarette smoke

Toxic carbonyl compounds, including formaldehyde, malonaldehyde, and glyoxal, formed in mainstream cigarette smoke were quantified by derivatization-solid phase extraction-gas chromatography methods. Cigarette smoke from 14 commercial brands and one reference (2R1F) was drawn into a separatory funnel containing aqueous phosphate-buffered saline. Reactive carbonyl compounds trapped in the buffer solution were derivatized into stable nitrogen containing compounds (pyrazoles for beta-dicarbonyl and alpha,beta-unsaturated aldehyde; quinoxalines for alpha-dicarbonyls; and thiazolidines for alkanals). After derivatives were recovered using C(18) solid phase extraction cartridges, they were analyzed quantitatively by a gas chromatograph with a nitrogen phosphorus detector. The total carbonyl compounds recovered from regular size cigarettes ranged from 1.92 mg/cigarette(-1) to 3.14 mg/cigarette(-1). The total carbonyl compounds recovered from a reference cigarette and a king size cigarette were 3.23 mg/cigarette(-1) and 3.39 mg/cigarette(-1), respectively. The general decreasing order of the carbonyl compounds yielded was acetaldehyde (1110-2101 microg/cigarette(-1)) > diacetyl (301-433 microg/cigarette(-1)), acrolein (238-468 microg/cigarette(-1)) > formaldehyde (87.0-243 microg/cigarette(-1)), propanal (87.0-176 microg/cigarette(-1)) > malonaldehyde (18.9-36.0 microg/cigarette(-1)), methylglyoxal (13.4-59.6 microg/cigarette(-1)) > glyoxal (1.93-6.98 microg/cigarette(-1)).     

Rabbit 3-hydroxyhexobarbital dehydrogenase is a NADPH-preferring reductase with broad substrate specificity for ketosteroids,prostaglandin D2, and other endogenous and xenobiotic carbonyl compounds

3-Hydroxyhexobarbital dehydrogenase (3HBD) catalyzes NAD(P)⁺-linked oxidation of 3-hydroxyhexobarbital into 3-oxohexobarbital. The enzyme has been thought to act as a dehydrogenase for xenobiotic alcohols and some hydroxysteroids, but its physiological function remains unknown. We have purified rabbit 3HBD, isolated its cDNA, and examined its specificity for coenzymes and substrates, reaction directionality and tissue distribution. 3HBD is a member (AKR1C29) of the aldo-keto reductase (AKR) superfamily, and exhibited high preference for NADP(H) over NAD(H) at a physiological pH of 7.4. In the NADPH-linked reduction, 3HBD showed broad substrate specificity for a variety of quinones, ketones and aldehydes, including 3-, 17- and 20-ketosteroids and prostaglandin D₂, which were converted to 3α-, 17β- and 20α-hydroxysteroids and 9α,11β-prostaglandin F₂, respectively. Especially, α-diketones (such as isatin and diacetyl) and lipid peroxidation-derived aldehydes (such as 4-oxo- and 4-hydroxy-2-nonenals) were excellent substrates showing low K_m values (0.1–5.9 μM). In 3HBD-overexpressed cells, 3-oxohexobarbital and 5β-androstan-3α-ol-17-one were metabolized into 3-hydroxyhexobarbital and 5β-androstane-3α,17β-diol, respectively, but the reverse reactions did not proceed. The overexpression of the enzyme in the cells decreased the cytotoxicity of 4-oxo-2-nonenal. The mRNA for 3HBD was ubiquitously expressed in rabbit tissues. The results suggest that 3HBD is an NADPH-preferring reductase, and plays roles in the metabolisms of steroids, prostaglandin D₂, carbohydrates and xenobiotics, as well as a defense system, protecting against reactive carbonyl compounds.     

The determination of volatile carbonyl compounds in diagnostic samples

Recently metaldehyde toxicity was reviewed. The review article made two references to analytical methodology of which one gave a personal communication as a method and the other was for a simple colorimetric test which suffered the disadvantage of being only semi-quantitative. Since the review a detailed gas-chromatographic technique specific for metaldehyde has been reported for plasma and urine. We now report on an extremely sensitive and rapid determination of general volatile carbonyl compounds, including acetaldehyde liberated from metaldehyde, as their 2,4-dinitrophenylhydrazone adducts whilst overcoming all of the difficulties and cumbersome manipulation previously needed with these adducts. The test has the advantage of being colorimetric and furthermore gas chromatography can be utilized to quantitate the acetaldehyde liberated by acid hydrolysis of metaldehyde and to separate it from the adducts formed by formaldehyde, acetone and similar compounds. Acetaldehyde and its polymers can be separately analyzed, when desired, by a simple additional step.     

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.     

Stereoselective acetonyl side chain reduction of warfarin and analogs. Partial characterization of two cytosolic carbonyl reductases.

The reductase activity mediating the ketone reduction of the acetonyl side-chain of warfarin and analogs has been partially purified from rabbit liver cytosol. The reductase activity is resolved in two different fractions (A and B) by DEAE-Sephacel chromatography. Both fractions reduce the acetonyl group of warfarin analogs with marked substrate (R-enantiomers), as well as product stereoselectivity (alcohols of the S-configuration). The reductases are NADPH-dependent, which is absolute for fraction B. The enzyme kinetics of the R-enantiomers of warfarin and three 4'-derivatives (4'-nitro-, 4'-chloro-, and 4'-methoxywarfarin) has been investigated. In contrast to fraction B, fraction A is sensitive in its KM for 4' substitution: the KM values of 4'-nitro- and chloro-analogs are approximately 6 times lower than the KM values of the 4'-methoxy analog or warfarin itself. On the other hand, the Vmax values of fraction A are all in the range of about 1 to 2 nmol/mg x min, whereas the Vmax values of fraction B vary from about 1(4'-methoxywarfarin) to 12 (the 4'-nitro analog). The intrinsic activities (Vmax/KM) of both enzymes show the same rank order: 4'-nitro greater than 4'-chloro greater than 4'-methoxy = warfarin. Warfarin reductase activity of both enzymes is not inhibited by pyrazole, sodium barbitone, or dicoumarol, but is strongly inhibited by quercetin, indomethacin, furosemide, and prostaglandin E2 (PGE2). In addition, fraction A is inhibited by menadione and androsterone; fraction B is inhibited by estrone. Various compounds were tested as substrates for these enzyme fractions.(ABSTRACT TRUNCATED AT 250 WORDS)