Review Multiplicity of Mammalian Reductases for Xenobiotic Carbonyl Compounds

Drug Metab. Pharmacokinet. 21 (1): 1–18 (2006).

Toshiyuki MATSUNAGA, Shinichi SHINTANI and Akira HARA* Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: A variety of carbonyl compounds are present in foods, environmental pollutants, and drugs. These xenobiotic carbonyl compounds are metabolized into the corresponding alcohols by many mammalian NAD(P)H-dependent reductases, which belong to the short-chain dehydrogenaseWreductase (SDR) and aldo-keto reductase superfamilies. Recent genomic analysis, cDNA isolation and characterization of the recombinant enzymes suggested that, in humans, the six members of each of the two superfamilies, i.e., total of 12 enzymes, are involved in the reductive metabolism of xenobiotic carbonyl compounds. They comprise three types of carbonyl reductase, dehydrogenaseWreductase (SDR family)

member 4, 11b-hydroxysteroid dehydrogenase type 1, L-xylulose reductase, two types of a‰atoxin B1 aldehyde reductase, 20a-hydroxysteroid dehydrogenase, and three types of 3a-hydroxysteroid dehydrogenase. Accumulating data on the human enzymes provide new insights into their roles in cellular and molecular reactions including xenobiotic metabolism. On the other hand, mice and rats lack the gene for a protein corresponding to human 3a-hydroxysteroid dehydrogenase type 3, but instead possess additional ˆve or six genes encoding proteins that are structurally related to human hydroxysteroid dehydrogenases. Characterization of the additional enzymes suggested their involvement in species-speciˆc biological events and species diŠerences in the metabolism of xenobiotic carbonyl compounds.

Key words: Carbonyl reduction; short-chain dehydrogenaseWreductase superfamily; aldo-keto reductase superfamily; carbonyl reductase; hydroxysteroid dehydrogenase; species diŠerence

alcohols by NADPH-dependent reductases with broad Introduction substrate speciˆcity. Some NADPH-dependent reduc- Aldehydes, ketones and quinones are present in a tases reduce quinones through two-electron transfer to diverse range of natural and synthetic compounds to the corresponding hydroqinones, which is also mediated which living organisms are exposed. In addition, car- by NA(D)PH:quinone reductase. This group of reduc- bonyl compounds are formed through biological tases with broad substrate speciˆcity for xenobiotic transformation of endogenous components and carbonyl compounds was originally called `aldo-keto xenobiotics that are ingested. Aldehydes are chemically reductases'1) andWor `carbonyl reductases'.2) Subse- reactive and interact with the nucleophilic centers of quently, according to the accumulated knowledge on nucleic acids and proteins. a-Dicarbonyl compounds, the functions and structures of the reductases, most such as methyl glyoxal and diacetyl, are more reactive. carbonyl-reducing enzymes have been grouped into two Ketones are less reactive, and many drugs contain keto distinct protein families, the short-chain dehydrogenase group(s). Quinones have a toxic eŠect, i.e., quinone- (SDR)3) and aldo-keto reductase (AKR)4) superfamilies. induced oxidative stress, when they are reduced through There had been three excellent reviews on the single-electron transfer to the corresponding semiqui- carbonyl-reducing enzymes by 2000.5–7) Over the recent nones. Organisms have evolved several enzyme systems ˆve years, several new enzymes that may reduce for detoxifying reactive carbonyl compounds. Such well xenobiotic carbonyl compounds have been found on established pathways include the oxidation of aldehydes genomic analysis, and thus the enzyme names have been to the corresponding carboxylic acids by aldehyde changed. For example, well-known carbonyl reductase dehydrogenases and aldehyde oxidases, and the reduc- (CBR), a member of the SDR superfamily, is now tion of aldehydes and ketones into the corresponding named CBR1 according to the Human Gene Nomencla-

Received; September 30, 2005, Accepted; October 24, 2005 *To whom correspondence should be addressed:AkiraHARA, Laboratory of Biochemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu, Gifu 502-8585, Japan. Tel. & Fax. ＋81-58-237-8586, E-mail: hara＠gifu-pu.ac.jp

1 2 Toshiyuki MATSUNAGA et al.

Table 1. Human enzymes involved in the reduction of xenobiotic carbonyl compounds.

Gene name Subcellular Accession Enzymes (location) Other names Endogenous substrates localization numbera)

SDR family CBR1 CBR1 Carbonyl reductase, PG 9-ketoreductase PG, isatin, (21q22.13) ketosteroids Cytoplasm P16152 CBR3 CBR3 Unknown Cytoplasm O75828 (21q22.2) 11bHSD 1 HSD11B1 Corticosteroid 11b-dehydrogenase isozyme 1 11-Ketoglucocorticoids Microsomes P28845 (1q32-q41) DHRS4 Dhrs4 Peroxisomal short-chain alcohol dehydrogenase, Retinal Peroxisomes Q9BTZ2 (14q11.2) NADPH-dependent retinol dehydrogenaseWreductase (NDRD) L-Xylulose DXCR DicarbonylWL-xylulose reductase, diacetyl L-Xylulose, diacetyl Cytoplasm Q7Z4W1 reductase (17q25.3) reductase

AKR family

AKR7A2 AKR7A2 A‰atoxin B1 aldehyde reductase member 2, Succinic semialdehyde Golgi O43488 (1p35.1-p36.23) AFAR2

AKR7A3 AKR7A3 A‰atoxin B1 aldehyde reductase member 3, Unknown Cytoplasm O95154 (1p35.1-p36.23) AFAR1 AKR1C1 AKR1C1 20a-HSD, 3(20)a-HSD, DD1 3- and 20-ketosteroids Cytoplasm Q04828 (10 q15-q14) AKR1C2 AKR1C2 3a-HSD type 3, DD2, bile acid-binding protein 3-Ketosteroids Cytoplasm P52895 (10 q15-q14) AKR1C3 AKR1C3 3a-HSD type 2, 17b-HSD type 5, PGF synthase, 3-, 17- and 20-keto- Cytoplasm P42330

(10 q15-q14) DDX steroids, PGD2 AKR1C4 AKR1C4 3a-HSD type 1, DD4, chlordecone reductase 3-ketosteroids Cytoplasm P17516 (10 q15-q14) a)The structural and functional information is available under the accession numbers of UniprotKBWSwiss-Prot (http:WWkr.expasy.orgWsprotW).

ture Committee (HGNC), because of the occurrence of reductase exhibit broad substrate speciˆcities for four types of CBR. Several mammalian enzymes that xenobiotic carbonyl compounds (Tables 1 and 2). CBRs were regarded as CBRs in the previous reviews are now are classiêd into four types, CBR1, CBR2, CBR3 and classiêd into CBR1-CBR3 or have been identiêd as CBR4 (Table 3). members of the AKR superfamily. The enzymes that 1. Carbonyl reductases (CBRs, EC 1.1.1.181) belong to the AKR superfamily have also been named CBR1: The cDNA for the human enzyme was ˆrst according to the nomenclature for this superfamily. In cloned by Wermuth et al.,8) who also characterized its addition, novel physiological roles of several enzymes properties using the enzyme puriêd from the brain, and have been reported. Therefore, we have reviewed the proposed its identity with xenobiotic ketone reductase literature on mammalian carbonyl-reducing enzymes and prostaglandin (PG) 9-ketoreductase.9) The enzyme using the new nomenclature in order to clarify their is ubiquitously distributed in human tissues.10) The gene relationship with the previously known enzymes. is mapped to chromosome 21q22.12, very close to the superoxide dismutase 1 locus at position 21q22.11.11) Carbonyl-reducing Enzymes in the SDR Superfamily The mRNA expression in MCF-7 cells is induced 3- or The SDR superfamily includes about 3,000 primary 4-fold in 24 hours by 2,(3)-t-butyl-4-hydroxyanisole, structures of functionally heterogeneous proteins, and b-naphtho‰avone, or Sudan 1.12) Human CBR1 is a becomes one of the largest protein families to date.3) 30 kDa-monomer comprising 277 amino acids, and The sequence identity of the members of this family is belongs to the SDR family. Recently, Tanaka et al.13) low, but the three-dimensional structures of many solved the crystal structure of the enzyme-NADP＋- members exhibit highly similar aWb folding patterns inhibitor complex, which has provided important in- with a central b-sheet, typical of the Rossmann-fold that sights as to the substrate binding site and the design of participates in cofactor binding. The catalytic tetrad of potent inhibitors of the enzyme, although its tertiary Asn-Ser-Tyr-Lys is conserved in the members of this structure is highly similar to that of porcine CBR1.14) superfamiy. Among the members, CBRs, 11b-hydro- Human CBR1 catalyzes the NADPH-dependent xysteroid dehydrogenase (HSD) type 1, dehydrogenaseW reduction of various carbonyl compounds, the best sub- reductase (SDR family) member 4 and L-xylulose strates being p-ando-quinones derived from polycyclic Mammalian Carbonyl-Reducing Enzymes 3

Table 2. Typical xenobiotic substrates of human reductases.

Enzyme Drugs Others CBR1 Daunorubicin, doxorubicin, haloperidol, Quinones, aromatic aldehydes, aromatic ketones, NNK bromperidol, metyrapone, loxoprofen, wortmannin, dolasetron CBR3 Menadione 11bHSD 1 Ketoprofen, metyrapone, insecticidal metyrapone NNK, menadione, aromatic aldehydes and ketones, 7-ketocholesterol analogues, oracin DHRS4 Dicarbonyl compounds with aromatic rings, alkyl phenyl ketones, some aromatic aldehydes and ketones L-Xylulose Dicarbonyl compounds, some aromatic aldehydes and ketones reductase

AKR7A2 Daunorubicin, ethacrynic acid A‰atoxin B1 dialdehyde, dicarbonyl compounds, aromatic aldehydes

AKR7A3 A‰atoxin B1 dialdehyde, 9,10-phenanthrenequinone,4-nitrobenzaldehyde AKR1C1 Dolasetron, naloxone, naltrexone, oxycodone, oracin, Aromatic aldehydes and ketones, quinones, dicarbonyl compounds, befunolol, ketotifen, 10-oxonortriptyline, haloperidol, NNK, trans-dihydrodiols of aromatic hydrocarbons, alicyclic alcohols loxoprofen, acetohexamide, daunorubicin, ethacrynic acid AKR1C2 Dolasetron, naloxone, naltrexone, oxycodone, oracin, Aromatic aldehydes and ketones, quinones, dicarbonyl compounds, befunolol, ketoprofen, ketotifen, 10-oxonortriptyline, NNK, trans-dihydrodiols of aromatic hydrocarbons, alicyclic alcohols haloperidol, loxoprofen, acetohexamide, daunorubicin AKR1C3 Naloxone, naltrexone 9,10-Phenanthrenequinone, 4-nitrobenzaldehyde, trans-dihydrodiols of aromatic hydrocarbons, alicyclic alcohols AKR1C4 Dolasetron, naloxone, naltrexone, oxycodone, oracin, Aromatic aldehydes and ketones, quinones, dicarbonyl compounds, ketoprofen, loxoprofen, acetohexamide, NNK, trans-dihydrodiols of aromatic hydrocarbons, alicyclic alcohols ethacrynic acid, metyrapone, chlordecone

Table 3. Homologs of human enzymes in other mammalian species.

Mouse Rat Human enzyme Gene name Enzyme name Accession Gene name Enzyme name Accession (CH)a) (SI)b) (CH)a) (SI)b)

CBR1 Cbr1 (16) CBR1 P48758 (86z)Cbr1(11)CBR1 P47727 (85z) (Non-inducible CBR) *c) Cbr2 (11) CBR2 P08074 Unknown (Mouse lung CBR) CBR3 Cbr3 (16) CBR3 Q8K354(85z)Cbr3predicted CBR3 XP22164 (85z) (11) CBR4 BC009118 (8) CBR4 NP663570 (86z) Cbr4 (16) CBR4 Q7TS56 (84z) 11bHSD1 Hsd11b1 (1) 11bHSD1 P50172 (78z) Hsd11b1 (13) 11bHSD1 P16232 (76z) DHRS4 Dhrs4 (14) DHRS4 Q99LB2 (81z)Dhr4(15)DHRS4 Q8VID1 (81z) (NDRD) (NDRD) L-Xylulose Dcxr (11) L-Xylulose reductase Q91X52 (84z) Dcxr (Unknown) L-Xylulose reductase Q920P0 (84z) reductase AKR7A2 Akr7a5 (4) AKR7A5 Q8CG76 (88z) RGD:620311 (5) AKR7A4 Q8CG45 (87z) (AFAR2) (AFAR2) AKR7A3 Not exist Akr7a3 (5) AKR7A1 P38918 (81z) (AFAR1) AKR1C1 Akr1c18 (13) AKR1C18 Q8K023 (68z) LOC171516 (17) AKR1C8 P51652 (71z) (20a-HSD) (20a-HSD) AKR1C3 Akr1c6 (13) AKR1C6 P70694 (69z) LOC364773 (17) RAKh NP001014240 (17b-HSD type 5) (17b-HSD type 5) (73z) AKR1C4 Akr1c14 (13) AKR1C14 Q91WT7 (67z) LOC191574 (17) AKR1C9 P23457 (69z) (3a-HSD) (3a-HSD) a)Most rat genes are named tentatively. CH, chromosome. b)Accession: The number in UniprotKBWSwiss-Prot or amino acid sequence in NCBI database. SI: sequence identity with the relevant human enzyme. c)The CBR2 gene does not exist in the human genome. aromatic hydrocarbons.9,15) In human liver and placen- CBR1 seems to be involved in both the detoxiˆcation ta, CBR1 acts as a major quinone reductase, but the and intoxication of quinones. The endogenous sub- enzymatic reduction of several o-quinones results in strates of CBR1 are suggested to be PGs, some 3- redox cycling of the quinones, leading to the generation ketosteroids and isatin, of which isatin is the best one, of semiquinones and the superoxide anion.16) Thus, showing a high a‹nity and turnover number that are 4 Toshiyuki MATSUNAGA et al. compatible with those of xenobiotic o-quinones.17) In been isolated from rabbit liver32) and Chinese hamster addition, CBR1 catalyzes the reduction of the 4-keto, ovary cells.33) Thus, these animal species probably have aldehyde and C＝C of 4-oxonon-2-enal, a product of two genes for CBR1 isoforms, in contrast to the lipid peroxidation.18) CBR1 homozygous null mice are existence of only one gene for human CBR1. The nonviable,19) suggesting that the enzyme plays a non- substrate speciˆcities of the CBR1s of these animals and redundant role in cell signaling during embryogenesis pig34) are essentially identical to that of the human and development. A study on the biological function of enzyme, except that pig CBR1 exhibits 20b-HSD CBR1 in A549 adenocarcinoma cells suggested that the activity. Mouse and Chinese hamster CBR1s, similar to enzyme is involved in serum-free-induced apoptosis in the human enzyme, are distributed in many tissues,33,35) the cells.13) On the other hand, several studies involving whereas the rat enzyme is speciˆcally expressed in tumor tissue specimens have indicated that a decrease in reproductive tissues and the adrenal gland.31) CBR1 expression is correlated with the degree of CBR2: This enzyme is not present in human tissues, dediŠerentiation in hepatocellular carcinomas,20) poor as its gene has not been found in the human genome. survival and lymph node metastasis in epithelial ovarian However, CBR2 is highly expressed in the mitochondria cancer,21) and tumor progression and angiogenesis in of epithelial cells of mouse, guinea-pig and pig lung cancer.22) Therefore, elucidation of the mechan- lungs.36–38) In addition, mitochondrial CBR2, which was isms underlying the suggested roles of the enzyme in cell previously known as a sperm protein, P26h, is present in signaling, apoptosis and cancer progression is an im- testis and epididymis of hamster.39) As a gene for CBR2 portant future goal. is not found in the rat genome (Table 3), CBR2 protein Human CBR1 reduces several therapeutic agents and has not been detected in rat tissues. CBR2s are toxicologically important compounds (Table 2). The homotetramers composed of 26 kDa-subunits, and drug substrates include daunorubicin and doxorubicin,6) exhibit low sequence identify with monomeric CBR1. loxoprofen,23) metyrapone,23) haloperidol,24) brom- The enzymes that reduce various aliphatic, alicyclic and 25) 26) 27) peridol, timiperone, and wortmannin, which has aromatic carbonyl compounds with lower Km values are been proposed to be a potential antineoplastic agent. activated by fatty acids and inhibited by pyrazole, a Since daunorubicinol, the reduced product of anti- known inhibitor of alcohol dehydrogenase. The proliferative daunorubicin, is cardiotoxic, CBR1 is diŠerences between hamster CBR2 and the lung thought to be responsible for the severe cardiotoxicity enzymes of other species are in the cofactor speciˆcity associated with daunorubicin treatment.6) This is and the reversibility of the reaction. The hamster supported by the ˆnding that mice heterozygous for a enzyme shows a cofactor preference for NAD(H) and null allele of CBR1 show reduced sensitivity to e‹ciently catalyzes the oxidation of 5a-dihydrosteroste- anthracycline-induced cardiotoxicity.19) The enzyme rone, whereas the lung enzymes utilize NADP(H) as a also reduces 4-methylnitrosamino-1-(3-pyridyl)-1-buta- preferred cofactor and do not exhibit signiˆcant none (NNK), a tobacco-speciˆc potent carcinogen, into dehydrogenase activity. The tissue distribution and (S)-4-methylnitrosamino-1-(3-pyridyl)-1-butanol, which catalytic properties suggest that lung CBR2s function in is more tumorigenic. The signiˆcance of the NNK the detoxication of carbonyl compounds derived metabolism by CBR1 and other reductases was through lipid peroxidation and xenobiotic carbonyl described in a recent review by Maser and Breyer- compounds ingested from the airways and blood.36–38) PfaŠ.28) The hamster CBR2 is thought to act as a 3a-HSD to Other animal enzymes that have similar sequences to control the intracellular concentration of a potent human CBR1 are classiêd as CBR1s. The genes for androgen, 5a-dihydrosterosterone, during spermato- CBR1s of mouse and rat have been identiêd through genesis.39) A crystallographic study of mouse CBR2 has genomic analyses (Table 3). In the mouse genome, an provided insights into its unique tetrameric structure additional gene (accession no.: LOC435489) encoding a and cofactor binding mode.40) The key residues in the protein exhibiting 87z sequence identity with mouse cofactor speciˆcity were also identiêd by site-directed CBR1 is predicted. Similarly, two cDNAs for mutagenesis studies of mouse and hamster CBR2s.39,41) gonadotropin-inducible and non-inducible CBR1 CBR3: The gene for this enzyme was ˆrst identiêd isoforms (86z sequence identity) have been isolated 62 kilobases apart from the CBR1 gene on chromosome from rat ovary.29) The inducible rat CBR is encoded by 21.42) The encoded protein is composed of 277 amino the genomic CBR1 gene, and identical to a testicular acids, and its sequence identity with human CBR1 is CBR that was previously characterized.30) The non- 71z and that with animal CBR2s is less than 23z. inducible isoform might correspond to another form of Recently, a natural allelic variant of CBR3 (V244M) was the two rat testicular CBRs,31) although the gene for this found, and the kinetic constants for NADPH and isoform was not found on the present genomic sequenc- menadione of the recombinant CBR3 and V244M ing. Two cDNAs for such CBR1 isoforms have also variant were determined.43) The mRNA for CBR3 is Mammalian Carbonyl-Reducing Enzymes 5 suggested to be expressed in various tissues,33) although proposed as a novel therapeutic strategy for many of the expression level is much lower than that of CBR1 these conditions. Mammalian HSD11B1s exhibit high (personal communication from Dr. T. Terada). cDNA sequence identity, and the crystal structures of guinea- microarray analysis in patients with keloid showed that pig and mouse HSD11B1s were recently solved.48,49) the CBR3 gene, together with eight other genes, in Selective and potent HSD11B1 inhibitors will be keloid tissue is consistently upregulated.44) The CBR3 developed in the near future through structure-based gene has been identiêd or predicted in the mouse and drug design using the crystal structures. rat genomes (Table 3). In Chinese hamster, CBR3 (86z HSD11B1 also plays a role in the metabolism of sequence identity with human CBR3) exhibits high non-glucocorticoid carbonyl compounds, i.e., oracin, activity toward daunorubicin and isatin compared to ketoprofen, metyrapone, insecticidal metyrapone CBR1, but conversely lacks oxidoreductase activity analogues, NNK, menadione, and some aromatic toward PGs, which are substrates for CBR1.33) The aldehydes and ketones.50–52) On the reduction of NNK, mRNA for CBR3 is detected in the order of kid- the human enzyme forms both the (S)- and (R)-enantio- neyÀbrainÀliver in Chinese hamster, which diŠers mers of 4-methylnitrosamino-1-(3-pyridyl)-1-butanol. from the expression patterns of the mRNAs for CBR1 This enantioselectivity is in contrast to those of human and its isoform. Thus, reports on CBR3 have been few, CBR1 and other AKR1C isoforms, which form the and the concentrations of CBR3 proteins in human and (S)-enantiomer on the reduction.28) Recently, HSD11B1 animal tissues are unknown. Further studies on the was reported to e‹ciently catalyze the reduction of 7- enzymology and gene regulation of CBR3 are necessary ketocholesterol, the major dietary oxysterol, into 7- to elucidate its roles in the metabolism of endogenous hydroxycholesterol, species-speciˆc diŠerences in the and exogenous compounds, and in the pathogeneses of stereospeciˆc reduction being observed between the rat, diseases. human, and hamster enzymes.53) The human and rat CBR4: The gene for this enzyme was predicted on enzymes reduce 7-ketoxycholesterol into 7b-hydroxy- recent genomic analyses, and is located on human chro- cholesterol, whereas the hamster enzyme catalyzes the mosome 4 (4q32.3). The encoded protein (accession no. interconversion of 7-ketoxycholesterol into both 7a- Q8N4T8) is composed of 237 amino acids, and exhibits and 7b-hydroxycholesterol. Although mammalian low sequence identity (º25z)withothertypesofCBR. HSD11B1s show similar substrate speciˆcities for The CBR4 gene is present in laboratory animals xenobiotic compounds, the stereoselective reduction (Table 3) and dog (accession no.: XP534547), suggest- may diŠer depending with the species andWor the ing a role in the metabolism of endogenous compounds reduced compound. for CBR4. Although the enzyme is named carbonyl or 3. DehydrogenaseWreductase (SDR family) member carbonic reductase 4, its enzymatic properties and tissue 4 (DHRS4, EC 1.1.1.-) distribution remain unknown. The cDNA for human DHRS4 was ˆrst cloned as a 2. 11b-HSD type 1 (HSD11B1, EC 1.1.1.146) peroxisomal 2,4-dienoyl-CoA reductase-related pro- 11b-HSD is a microsomal protein that catalyzes the tein.54) DHRS4 belongs to the SDR family, and has a interconversion of active cortisol into inactive cortisone. C-terminal peroxisomal targeting signal 1 (Ser-Arg- Two isoforms, NADP(H)-dependent HSD11B1 and Leu). It has also been called SDR-SRL, peroxisomal NAD(H)-dependent 11b-HSD type 2 (HSD11B2), in short-chain dehydrogenase and NADPH-dependent mammalian tissues have been cloned and characterized. retinol dehydrogenaseWreductase (NDRD), because the HSD11B1 is expressed in a wide range of tissues, acts rabbit and pig homologues of human DHRS4 exhibit predominantly as a reductase in intact cells and tissues NADPH-linked retinal reductase activity.55) In addition, by regenerating active cortisol from cortisone, and two alternative splicing forms of human DHRS4, SDR- regulates glucocorticoid access to the glucocorticoid SRL1 (lacking residues 118–204) and SDR-SRL-2 receptor. HSD11B2 is mainly expressed in mineralocor- (lacking 85–204), have been isolated.54) DHRS4, SDR- ticoid target tissues such as kidney and colon, acts only SRL1 and SDR-SRL-2 are now designated as DHRS4 as a dehydrogenase by producing inactive cortisone, and isoforms 1, 2 and 3, respectively. Since there has been protects the mineralocorticoid receptor from high levels no literature on the properties of human DHRS4, we of receptor-active cortisol. The roles and enzymology of characterized the recombinant DHRS4 isoform 1. It is the two isoforms were described in recent reviews.45,46) an NADPH-dependent tetrameric reductase for Increases in the activity and expression of HSD11B1 xenobiotic carbonyl compounds, but diŠers from the have been implicated in the pathogeneses of many enzymes of other species (described below) in its low common conditions including obesity, insulin activity toward retinoids and instability at low tempera- resistance, the metabolic syndrome, the polycystic ture. The human enzyme reduces all-trans-retinal with a 47) ovarian syndrome, osteoporosis and glaucoma. low Vmax WKm (0.8 unitsWmgWmM), and does not exhibit Therefore, selective HSD11B1 inhibition has been signiˆcant 9-cis-retinol reductase or all-trans-retinol 6 Toshiyuki MATSUNAGA et al. dehydrogenase activity. The enzyme is gradually CBR2 in inhibitor sensitivity and tissue distribution. inactivated at 49C, in contrast to the stable enzymes of L-Xylulose reductase is inhibited by n-butyric acid, but other species. The cold inactivation might have prevent- not by pyrazole. The enzyme is distributed in many ed detection of the enzyme activity in human tissues in tissues, of which the liver and kidney show high expres- previous studies on CBRs. The mRNA for DHRS4 sion of the enzyme. Furthermore, the L-xylulose reduc- isoform 1 is ubiquitously expressed in human tissues, in tases of mouse, rat, guinea pig and hamster are rapidly which expression of the mRNA for the isoform 2 is low. dissociated into their inactive dimeric forms at low No enzymatic activity is observed for recombinant temperature.61,63) isoform 2. Thus, the physiological role of human Carbonyl-reducing Enzymes in the AKR Superfamily DHRS4 is not clear, but the enzyme reduces alkyl phenyl ketones and might be involved in the metabolism The AKR superfamily is a rapidly growing group of of the butyrophenone type of drugs. NAD(P)(H)-dependent oxidoreductases that metabolize ThecDNAsforothermammalianDHRS4shavebeen carbohydrates, steroids, prostaglandins, and other en- cloned and their genes are shown in Table 3. The rabbit dogenous aldehydes and ketones, as well as xenobiotic and pig DHRS4s are peroxisomal homotetramers, being compounds.4) Currently there are more than a hundred composed of 27-kDa subunits.55) The enzymes reduce known members of this superfamily that is classiêd alkyl phenyl ketones, a-dicarbonyl compounds, retinals into 14 families. The nomenclature system is similar to and some aromatic aldehydes using NADPH as a that for the cytochrome P450 superfamily, but, unlike cofactor, and are identical to a tetrameric CBR that was that system, it involves amino acid sequence compari- previously puriêd from rabbit heart.56) Of these sons. Within a given family, subfamilies are deˆned xenobiotic substrates, 9,10-phenanthrenequinone is according to À60z identity in amino acid sequence e‹ciently reduced by the enzymes, and the reduction among subfamily members. The largest family, AKR1, results in the formation of reactive oxygen species is subdivided into ˆve subfamilies: AKR1A, (ROS).57) The enzymes show high catalytic e‹ciency for mammalian aldehyde reductases; AKR1B, mammalian all-trans-retinal and 9-cis-retinal (the Vmax WKm values of aldose reductases; AKR1C, HSDs and PGF synthases; the pig enzyme are 618 and 30 unitsWmgWmM, respec- AKR1D, D4-3-ketosteroid-5b-reductases; and AKR1E, tively, at 259C and pH 7.4), and oxidize all-trans-retinol mouse keto-reductase. More information on this family 55) with a low Vmax WKm value of 1.5 unitsWmgWmM. is available on the AKR superfamily homepage Mouse DHRS4 also exhibits high reactivity towards (www.med.upenn.eduWakr). Among the enzymes in this retinoids, and its content in the liver increases with superfamily, aldose reductases, aldehyde reductases, 58) cloˆbrate feeding. In the three animals, mRNA for HSDs, PGF synthases and a‰atoxin B1 reductases DHRS4 is expressed in many tissues, of which liver and (AFARs, belonging to the AKR7A subfamily) exhibit kidney exhibit the highest expression. We have cloned a broad substrate speciˆcities for xenobiotic carbonyl cDNA for DHRS4 from dog liver, and characterization compounds. In this section, we describe recent studies of the recombinant enzyme revealed that it is identical on the characteristics of HSDs, PGF synthases and to a high-molecular weight CBR that is highly expressed AFARs, focusing mainly on their roles in xenobiotic in the liver.59) metabolism. Aldehyde and aldose reductases are not 4. L-Xylulose reductase (EC 1.1.1.10) included in this section, because they are not involved in L-Xylulose reductase catalyzes the NADPH- the reduction of drug ketones, except for daunorubicin dependent reduction of L-xylulose to xylitol in the and acetohexamide.23) A recent review described the uronate cycle of glucose metabolism. The human, properties and functions of aldose reductase and closely mouse, rat, guinea pig and hamster enzymes have been related aldo-keto reductases,64) of which AKR1B10, i.e., cloned and shown to be identical with diacetyl reductase human aldose reductase-like protein, has been (EC 1.1.1.5) that reduces various a-dicarbonyl recognized as a new diagnostic marker of hepatocellular compounds including o-quinones, and aromatic alde- carcinomas65) and smokers' non-small cell lung carcino- hydes and ketones.60,61) Because of its broad substrate mas.66) Since the mouse and rat genomes contain many speciˆcity, L-xylulose reductase is thought to be genes for the enzymes belonging to the AKR1C subfa- involved in the metabolism of xenobiotic carbonyl mily compared with the human genome, we divide the compounds, although drugs containing carbonyl groups enzymes into human and rodent ones in order to avoid have not been examined as substrates. The enzyme is a more confusion. homotetramer, and exhibits high sequence identity of 1. Enzymes in the AKR7A subfamily 65z with mouse CBR2. The crystal structure of human AFAR catalyzes the NADPH-dependent reduction of 62) L-xylulose reductase is similar to that of mouse CBR2, a‰atoxin B1 dialdehyde, a toxic metabolite produced but there is a signiˆcant diŠerence in their substrate from a‰atoxin B1 by CYP3A, into unreactive mono- binding clefts. L-Xylulose reductase also diŠers from and di-alcohol derivatives.67) The human, mouse and rat Mammalian Carbonyl-Reducing Enzymes 7 enzymes have been cloned and characterized, and their hypothetical polypeptide of 129-amino acids (accession genes are shown in Tables 1 and 3. The enzymes are no.: Q6ZN81), and the AKR1CL2 gene encodes a dimers, and show similar broad substrate speciˆcities monomeric protein of 320-amino acids (accession no.: for various aromatic aldehydes and dicarbonyl com- Q9BU71) that exhibits low reductase activity only pounds. Of these substrates, 2-carboxybenzaldehyde is toward 9,10-phenanthrenequinone.81) AKR1C1- useful as a diagnostic model substrate for AFAR, AKR1C4 are NADP(H)-dependent monomeric de- because it is an inactive or poor substrate for other hydrogenases composed of 323 amino acids and exhibit reductases in the SDR and AKR families.68) In human high sequence identity (À83z), but their biochemical and rat tissues, AFAR is present in two isoforms, which properties are diŠerent. The enzymes are 3a-, 17b-andW have been classiêd into two classes, AFAR1 and or 20a-HSDs, and play roles in the pre-receptor AFAR2, with respect to sequence similarity, a‹nity for regulation of steroid receptors, nuclear orphan succinic semialdehyde, intracellular localization and receptors andWor membrane-bound ligand-gated ion inducibility by xenobiotics. channels. The enzymes are suggested to be targets for AFAR1: This class of AFAR includes human the treatment of prostate cancer, breast cancer, AKR7A369) and rat AKR7A1,70) the subunits of which endometriosis and endometrial cancer.82) The other are composed of 327 amino acids and exhibit 81z characteristic of the enzymes diŠering from those of the sequence identity. The rat enzyme reduces various members of the AKR superfamily is that they show high aldehydes, but its Km value for succinic semialdehyde, dihydrodiol dehydrogenase activity (DD, EC 1.3.1.20), an endogenous substrate, is high.71) Theenzymeis which oxidizes the trans-dihydrodiols of aromatic expressed in liver and several extrahepatic tissues, and is hydrocarbons into the corresponding catechols. induced by phenolic antioxidants, ethoxyquin, couma- Therefore, AKR1C1, AKR1C2, AKR1C3 and AKR1C4 rin, and dietary indoles and isothiocyanates.70,72,73) are called DD1, DD2, DDX and DD4, respectively. DD Modulation of the level of AKR7A1 is also suggested to was ˆrst shown to be a detoxiˆcation enzyme in the correlate with the carcinogenesis caused by a‰atoxin metabolism of carcinogenic polycyclic aromatic 70,72) B1. In rat liver, AKR7A1 exists as both a homodimer hydrocarbons, but has been regarded as a toxication and a heterodimer with a subunit of AKR7A4 belonging enzyme, because oxidation by the enzyme yields reactive to another class of AFAR. The crystal structure of and redox-active o-quinones and ROS.83) In addition, AKR7A1 revealed the details of the dimeric structure AKR1C1-AKR1C4 metabolize PGs and a number of and substrate recognition.74,75) Little is known about the xenobiotic compounds. In this section, we summarize catalytic activity and inducibility of human AKR7A3, the main characteristics of the individual enzymes and which is reported to reduce some substrates (Table 2).69) describe their roles in xenobiotic carbonyl metabolism. AFAR2: This class includes human AKR7A2,76) AKR1C1: This enzyme is known as 20a-HSD (EC mouse AKR7A5,77) and rat AKR7A4,78) the subunits of 1.1.1.149), and is distributed in many tissues.84–86) which are composed of 359–367 amino acids and exhibit AKR1C1 exhibits additional 3a-and3b-HSD activities high sequence identity (À87z)witheachother.The depending on the steroid structure: It reduces 3-keto-5b- enzymes are constitutively expressed in many tissues, in dihydrosteroids into the 3a-hydroxy derivatives, which they are thought to be associated with the Golgi whereas 3b-hydroxy products are formed on the apparatus.79) Their substrate speciˆcities are similar to reduction of 3-keto-5a-dihydrosteroids.87,88) The 5a- that of AFAR1, but AFAR2 shows a low Km value for androstane-3b,17b-diol and 5a,3b-tetrahydroxypr- succinic semialdehyde.68,76–79) Human AKR7A2 is indeed egnanes produced are ligands of estrogen receptor b89) identical to succinic semialdehyde reductase,80) indicat- and antagonists for the g-aminobutyric acid type A 90,91) ing its physiological role in g-hydroxybutyate synthesis. (GABAA) receptor, respectively. Thus, the enzyme Human AKR7A2 reduces various xenobiotic aromatic plays important roles in the steroid metabolism through aldehydes and a-dicarbonyl compounds, but is the formation of the above steroids as well as inactiva- essentially inactive towards aliphatic and aromatic tion of progesterone, neuroactive 5aWb-pregnan-3a-ol- 68,76) ketones. Although the human enzyme reduces 20-ones (positive modulators of the GABAA receptor), daunorubicin and ethacrynic acid at low rates,68) other and 5a-dihydrotestosterone.82) AKR1C1 diŠers from drugs have not been examined as substrates. AKR1C2 (3a-HSD type 3) by only seven amino acids, of 2. Human enzymes in the AKR1C subfamily which the residue at position 54 (Leu in AKR1C1 and Four human enzymes, AKR1C1-AKR1C4, have been Val in AKR1C2) has been demonstrated to be a cloned and characterized. The genes exist in a cluster on determinant of the steroid speciˆcity of the two enzymes chromosome 10 (Table 1). Although two genes, in site-directed mutagenesis and crystallographic stu- AKR1CL1 and AKR1CL2, for AKR1C-like proteins dies.88,92,93) have been found near the cluster, their gene functions AKR1C1 reduces a variety of non-steroid carbonyl are unknown: The AKR1CL1 gene codes for a compounds including PGs and NNK. The enzyme 8 Toshiyuki MATSUNAGA et al.

Table 4. Comparison of kinetic constants for xenobiotic substrates among human enzymes.

CBR1 HSD11B1 AKR1C1 AKR1C2 AKR1C4 Substrate Km Vmax WKm Km Vmax WKm Km Vmax WKm Km Vmax WKm Km Vmax WKm NNK 7 400 12 20 0.2 90 0.3 57 8 10 Metyrapone 0.9 534 ND NS NS 19 35 R-Ketoifen NS NT 0.01 500 0.008 7250 NS S-Ketotifen NS NT 0.05 1840 0.004 25000 NS Dolasetron NS NT 0.06 3280 0.03 900 0.2 2570 Naloxone NS NT 0.4 41 0.02 570 0.006 12400 Naltrexone NS NT 0.4 59 0.1 210 0.01 6440 Oxycodone NS NT 1.8 17 NS 0.4 79

Km＝mM and Vmax WKm＝unitsWmgWM(1unit＝nmol of reduced product or NADPH Wmin). ND: the activity was detected, but the kinetic constants have not been determined. NS: no signiˆcant activity was detected. NT: not tested as a substrate. The values for naloxone, naltlexone and oxycodone were determined at pH 7.4 and 379C. reduces a variety of xenobiotic carbonyl compounds, roles of AKR1C2 in the development and progression of but diŠers from the AKR7A enzymes in its ability prostate cancer,82) recent literature suggested possible to reduce aromatic ketones.68) It exhibits low PGF involvement of the enzyme in the pathogenesis of synthase activity.85,94) On the reduction of NNK, the glaucoma and obesity in women. Signiˆcantly high enzyme shows the lowest Km value of all known NNK expression and activity of AKR1C2 are observed in reductases (Table 4).28) Drug substrates, which have human glaucomatous optic nerve head astrocytes,101) been identiêd using the puriêd hepatic and recom- and the expression and activities of AKR1C2 and binant enzymes,23,24,95,96) are listed in Table 2. Although AKR1C1 in omental adipose tissue are positive corre- these drugs are not speciˆc substrates for AKR1C1, lates with adiposity in women.102) catalytic e‹ciency comparison indicated that dolase- The substrate speciˆcity of AKR1C2 for non-steroid 85,103) tron, an antiemeric 5-HT3 receptor antagonist, is most carbonyl compounds is similar to that of AKR1C1 e‹ciently reduced by the enzyme.95) The Z-andE-10- (Table 2), but AKR1C2 is inactive towards ethacrynic ketonortriptylines are also good substrates for this acid and metyrapone.23) The substrate speciˆcity for enzyme.24) AKR1C1 is selectively and potently inhibited drugs also overlaps those of the other enzymes in the by benzbromarone,97) suggesting a possible drug-drug SDR and AKR superfamilies (Table 4), and AKR1C2 interaction between this drug and the above drug sub- may be the major enzyme in the reductive metabolism strates of the enzyme. of ketotifen because of its extremely high reactivity AKR1C1 is induced by ethacrynic acid, polycyclic towards both the R-andS-forms of the drug.24) There aromatic hydrocarbons, other polycyclic aromatic has been no report on the induction of AKR1C2 by compounds, electrophilic Michael acceptors, phenolic xenobiotics. The enzyme is highly inhibited by bile antioxidants, ROS, 4-hydroxynonenal, and dietary acids, and is identical to a human bile acid-binding indoles and isothiocyanates.73,98–100) The induction by the protein.103,104) polycyclic aromatic hydrocarbons supports the role of AKR1C3: This enzyme exhibits a broad tissue the enzyme in the activation of polycyclic aromatic distribution.84–86) Although its tissue expression can be hydrocarbons,83) whereas the enzyme provides an detected by means of immunochemical methods105) and inducible cytosolic barrier to 4-hydoxynonenal in ampliˆcation of the cDNA, AKR1C3 has not been oxidative stress by metabolizing the toxic aldehydes.100) puriêd from human tissues, in contrast to the puriˆca- The inducers include dietary indoles and isothio- tion of other AKR1C enzymes.23,24,28,103,106) The inability cyanates, which may in‰uence the metabolism of to purify the enzyme from the tissues may result from steroids and drugs catalyzed by AKR1C1. the high liability of the enzyme in tissue homogenates AKR1C2: This enzyme is the type 3 isozyme of and cell lysates.107) AKR1C3 was ˆrst named 3a-HSD human 3a-HSD (EC 1.1.1.213), and its mRNA is type 2,108) but exhibits high 17b-HSD activity toward detected in many tissues.84–86) Despite its structural 4-androstene-3,17-dione, so it is also classiêd as similarity with AKR1C1, this enzyme mainly exhibits 17b-HSD type 5 according to the nomenclature for 17b- 3a-HSD activity. The enzyme is believed to play im- HSD isozymes (EC 1.1.1.62).109) In addition, AKR1C3 portant roles in the inactivation of 5a-dihydrotestoste- is identical to PGF synthase (EC 1.1.1.188) that cata- rone and the synthesis of neuroactive 5aWb-pregnan-3a- lyzes both the formation of PGF2a from PGH2,andthe 82) 110,111) ol-20-ones from their precursors. In addition to the interconversion between PGD2 and 9a,11b-PGF2. Mammalian Carbonyl-Reducing Enzymes 9

Thus, the enzyme is involved in the metabolism of diŠerences in the amounts of HNF-1a,HNF-4a and androgens and PGs, and is suggested to be involved in HNF-4g.127) In addition, a variant of AKR1C4 (S145CW the development of prostate cancer by forming L311V) that decreases the enzyme activity has been testosterone and the diŠerentiation of leukemia cells identiˆed.128) Such studies on interindividual diŠerence 82,112) caused by metabolizing PGD2. AKR1C13 is sig- are needed for the other AKR1C enzymes, because they niˆcantly up-regulated during the diŠerentiation of are involved not only in the metabolism of other drugs, 113) HL-60 cells by all-trans-retinoic acid and vitamin D3, but also in several diseases. and its mRNA expression in human ˆbroblasts is also 3. Rodent enzymes in the AKR1C subfamily induced by thyroid hormone.114) Recently, three crystal NADP(H)-dependent 20a-, 17b-and3a-HSDs, which structures of the enzyme were determined to elucidate correspond to human AKR1C1, AKR1C3 and the mechanism underlying the multiple speciˆcity and to AKR1C4, respectively, have been cloned and character- develop speciˆc inhibitors.115–117) Such several inhibitors ized from mouse and rat tissues (Table 3). The rodent selective for AKR1C1, AKR1C2 and AKR1C3 have enzymes exhibit the following diŠerences in substrate been reported.118) speciˆcity and tissue distribution from the relevant Only 9,10-phenanthrenequinone and 4-nitrobenzal- human enzymes, although data on rat aldo-keto dehyde have been reported to be xenobiotic substrates reductase h (RAKh) have not been published. (1) Mouse of AKR1C3.85,111) We tested several drugs as substrates, AKR1C1888,129) and rat AKR1C8130) are 20a-HSDs, and found that naloxone and naltrexone are reduced by exhibiting additional 3aW3b-HSD activity that is the enzyme (unpublished results). The Km and Vmax WKm observed with human AKR1C1. In contrast to the high values for naloxone are 1.3 mM and 2.6 unitsWmgWM, DD activity of human AKR1C1, the rodent enzymes respectively, the respective values for naltrexone being show low DD activity, and signiˆcantly high reductase 0.6 mM and 54 unitsWmgWMat379C and pH 7.4. activity toward nonsteroidal substrates such as a-dicar- Comparison of these values with those for other bonyl compounds, aromatic aldehydes and ketones.88) enzymes (Table 4) suggests that AKR1C3 is not a major In addition, the tissue distributions of the rodent en- enzyme in drug metabolism. zymes diŠer from that of the ubiquitous human enzyme. AKR1C4: This enzyme is 3a-HSD type 1, and Mouse AKR1C18 is highly expressed in the ovary and exhibits higher a‹nity and velocities for most 3-keto kidney, and at lower levels in many other tissues.88,131) and 3a-hydroxysteroids than the other types of the AKR1C8 is ovary-speciˆc in female rats130) and its enzyme do. It also exhibits low 20a-HSD and 17b-HSD expression in male rat tissues is quite low. (2) Mouse activities,86) and is inhibited by phenolphthalein and AKR1C6132) and rat RAKh are 17b-HSDs that are steroidal anti-in‰ammatory agents.97) Crystallization of associated with low 20a-HSD activity, and high DD and AKR1C4 has not been achieved. This enzyme is reductase activities toward a variety of nonsteroidal expressed speciˆcally in the liver, and is involved in the carbonyl compounds. Thus, they appear to be type 5 catabolism of circulating steroid hormones and the isozymes of 17b-HSD that correspond to human metabolism of bile acids.82,119) The liver-speciˆc AKR1C3. However, the rodent enzymes do not exhibit expression of AKR1C4 mRNA is regulated by transcrip- 3a-HSD and PGF synthase activities, which are high for tion factors, hepatocyte nuclear factor (HNF)-4 aWg, AKR1C3. AKR1C6 is a liver-speciˆc enzyme,132) and HNF-1a and variant HNF-1.120,121) RAKh is expressed in rat liver and kidney. This also As AKR1C4 was ˆrst cloned as a cDNA for chlorde- diŠers from the ubiquitous tissue distribution of cone (an insecticide) reductase,122) it reduces various AKR1C3. (3) Mouse AKR1C14133) and rat AKR1C9134) xenobiotic carbonyl compounds and drugs (Table 2). are 3a-HSDs, exhibiting high reductase activities toward AKR1C4 e‹ciently reduces naloxone, naltrexone and various nonsteroidal carbonyl compounds. In this oxycodone, although it is almost inactive towards respect, the rodent enzymes are counterparts of human befunolol, daunorubicin, haroperidol and ketotifen AKR1C4, but they do not exhibit 20a-or17b-HSD (Table 4). The enzyme probably plays an important role activity that is detected at low levels for the human in the reduction of these three drugs and chlordecone. enzyme.86) The rodent enzymes exhibit a ubiquitous On the other hand, several drugs such as sul- tissue distribution, which is also diŠerent from the liver- fobromophthalein,123) cloˆbric acid derivatives124) and speciˆc expression of human AKR1C4. (4) There has anti-in‰ammatory 2-arylpropionic acids125) enhance the been no report on a mouse or rat NADP(H)-dependent enzyme activity of AKR1C4. While there has been no 3a-HSD that has similar properties to human AKR1C2. report on the induction of AKR1C4 by xenobiotics so Recent genomic analyses have shown that eight and far, an interindividual diŠerence in hepatic chlordecone nine genes for enzymes belonging to the AKR1C subfa- reductase activity has been reported.126) Alargeinterin- mily are present in the mouse and rat genomes, respec- dividual diŠerence has also been noted in the hepatic tively. The proteins include the above rodent HSDs and expression of AKR1C4 mRNA, which is related to the partially characterized mouse aldo-keto reductases 10 Toshiyuki MATSUNAGA et al.

Table 5. AKR1C enzymes speciˆcally expressed in mouse and rat tissues. The genes exist as a cluster on chromosomes 13 and 17 for the genomes of mouse and rat, respectively.

Mouse AKR1C enzymes Rat AKR1C enzymes Gene Gene (product)a) Characteristics (product)a) Characteristics

Akr1c12 NAD(H)-preferring 17b-HSD with moderate DD ac- LOC36773 Speciˆcity for cofactors and substrates is essentially (AKR1C12) tivity and low 3(20)a-HSD activity. (AKR1C24) the same as that of AKR1C12. Akr1c13 NAD(H)-preferring DD, oxidizing alicyclic alco- Akr1c12predicted Speciˆcity for cofactors and substrates is essentially (AKR1C13) hols, cis-and trans-dihydrodiols, and nerol. It (AKR1C16) the same as that of AKR1C13. It slowly oxidizes reduces dicarbonyl compounds and some aromatic some 3a-, 17b-and20a-hydroxysteroids. aldehydes. Akr1c19 NADH-preferring reductase speciˆc for dicarbonyl LOC307096 NADH-preferring reductase for dicarbonyl com- (AKR1C19) compounds. It slowly oxidizes 3-hydroxyhexobar- (RAKi) pounds. bital. Akr1c21 NADP(H)-preferring 3(17)a-HSD with DD activity Akr1c21predicted Speciˆcity for cofactors and substrates is essentially (AKR1C21) towards cis-dihydrodiols. It reduces dicarbonyl com- (RAKg) the same as that of AKR1C21. pounds, and some aromatic aldehydes and ketones. Akr1c20 NADPH-preferring reductase for dicarbonyl com- LOC498790 NAD(H)-preferring 3a-HSD for bile aids, 5b-pre- (AKR1C20) pounds with aromatic ring(s). It exhibits low (AKR1C17) gnanes, and 4-androstenes. It reduces dicarbonyl 3a(17b)-HSD activity. compounds and some aromatic aldehydes. Akr1c11predicted NADPH-preferring reductase with broad speciˆcity (AKR1C15) for various aliphatic and aromatic carbonyl compounds, quinones, and 17-ketoandrostanes. a)Products are named according to the nomenclature of the AKR superfamily. RAKi and RAKg are tentative names, because they are not assigned in this superfamily.

(AKR1C12 and AKR1C13),135,136) but the functions of ty of the two enzymes. The rat counterpart (92z the other enzymes have not been studied. We have sequence identity) is AKR1C16, which is encoded in the isolated the cDNAs for AKR1C12, AKR1C13 and other cDNA for RAKb.138) The properties of the recombinant functionally unknown enzymes from mouse and rat AKR1C16 are essentially identical to those of mouse tissues, and have characterized the recombinant en- AKR1C13, except that the rat enzyme shows low zymes. Table 5 summarizes the genes for the enzymes dehydrogenase activity toward some 3a-, 17b-and20a- and their characteristics, which include our unpublished hydroxysteroids. The tissue expression patterns of the results. The tissue distributions of the enzymes, and mRNAs for AKR1C13131) and AKR1C16 are similar to their relationship with previously isolated enzymes and those of AKR1C12 and AKR1C24. In rat liver, the proteins are presented below. activity of AKR1C16 is lower than that of AKR1C24. (1) Mouse AKR1C12 has the same sequence as AKRa, Thus, the physiological roles of AKR1C13 and which is encoded by the interleukin-3-regulated gene.136) AKR1C16 remain unknown, although the enzymes may The rat counterpart exhibiting high sequence identity be involved in the oxidative metabolism of morphine. (90z) is AKR1C24, one form (TBER1) of the two (3) Mouse AKR1C19 shows high NADH-linked reductases for 6-tert-butyl-2,3-epoxy-5-cyclohexene- reductase activity only toward dicarbonyl compounds, 1,4-dione,137) andmaybeidenticaltoRAKf,whichwas but its NAD＋-linked dehydrogenase activity is low and previously isolated as a partial cDNA.138) The recom- directed toward some alcohol substrates.141) The enzyme binant AKR1C12 and AKR1C24 utilize NAD(H) as a is thought to act as a reductase based on the low preferred cofactor, and e‹ciently oxidize various 17b- inhibition of its reductase activity by NAD(P)＋.Therat hydroxysteroids. In addition, they exhibit moderate DD counterpart is RAKi (91z sequence identity), and the activity, and low 3a-and20a-HSD activities, as well as recombinant RAKi shows similar NADH-linked high reductase activity toward a-dicarbonyl compounds reductase activity and tissue distribution to and aromatic aldehydes. As AKR1C12 was found as a AKR1C19.131) (4) Mouse AKR1C21 is a NADP(H)- stomach aldo-keto reductase,135) both AKR1C12131) and dependent 3(17)a-HSD that is identical to DD puriêd AKR1C24 are highly expressed in the gastrointestinal from kidney.142–144) Although the enzyme is highly tract and liver, and at low levels in other tissues of the expressed in the kidney, its mRNA is detected in other two animals. (2) Mouse AKR1C13 is probably the same tissues. The rat counterpart of AKR1C21 is RAKg, enzyme as NAD＋-preferring DD, which is designated as the sequence identity being 86z. NADP＋-linked AKR1C22 in the AKR superfamily,139) because they 3(17)a-HSD activity was also observed for recombinant diŠer only by four amino acids. Its amino acid sequence RAKg, but the mRNA for RAKg is only expressed in is also highly similar to that of mouse morphine 6- liver and kidney, in which its alternative splicing form is dehydrogenase (EC 1.1.1.218),140) suggesting the identi- detected. (5) AKR1C17 and AKR1C15 are rat-speciˆc Mammalian Carbonyl-Reducing Enzymes 11 enzymes. AKR1C17 has been identiêd as a kidney- speciˆc 3a-HSD that diŠers from ubiquitous NADP(H)- dependent 3a-HSD (AKR1C9) with respect to its cofactor preference for NAD(H) compared to NADP(H), and substrate speciˆcity for bile acids, 5b-pregnanes and 5a-androstanes. AKR1C15 is a NADPH-dependent reductase, which is highly expressed in rat lung. It reduces a variety of carbonyl compounds with low Km values (0.3–20 mM). In particular, the ability of the enzyme to reduce aliphatic ketones and aldehydes is a unique characteristic that is not observed for the other human and rodent enzymes in the AKR7A and AKR1C subfamilies. The broad substrate speciˆcity of the enzyme resembles that of mouse lung CBR2, and the only diŠerence being in the steroid speciˆcity: AKR1C15 reduces the 17-keto groups of 5aW5b- androstanes, whereas mouse CBR2 reduces 3- ketosteroids. Since the CBR2 gene has not been identiêd in the rat genome and such a tetrameric CBR is indeed absent in rat lung, AKR1C15 may play an important role in the pulmonary metabolism of carbonyl compounds derived through lipid peroxidation, and androgens and xenobiotics. (6) Mouse AKR1C20 shows 89z sequence identity with mouse 17b-HSD type 5(AKR1C6),anditsmRNAisexpressedonlyinthe Fig. 1. Phylogenetic relationship among the human, rat and mouse liver.131) The recombinant AKR1C20 exhibits low 17b- members of the AKR1C subfamily. Cluster analysis of the human HSD and moderate 3a-HSD activities using NADP(H) (Hs), rat (Rn) and mouse (Mm) enzymes were performed using Clustal W145) and the tree was drawn with the TreeView program.146) as the preferred cofactors. However, the Vmax WKm values for 3a-hydroxy- and 3-keto-steroids of AKR1C20 (0.01–20 unitsWmgWmM) are much lower than those of mouse 3a-HSD, AKR1C14 (200–4860 unitsWmgWmM). and laboratory animals. Although the substrate speciˆc- AKR1C20 shows high reductase activity towards a- ities for ketone-containing drugs of all the enzymes have dicarbonyl compounds with aromatic ring(s), but does not been studied comparatively, such a diŠerence not reduce aromatic and aliphatic ketones. between the human and rodent enzymes may be respon- The genes for all the enzymes belonging to the sible for the species diŠerence in drug metabolism. In AKR1C subfamily exist as a cluster on chromosomes addition, the occurrence of many HSDs belonging to 10, 13 and 17 for the genomes of humans, mouse and the AKR1C subfamily in mouse and rat tissues suggests rat, respectively. This genomic organization suggests that the enzyme system to regulate cellular concentra- that these genes arose from ancient duplication events tions of active steroids in laboratory animals is more followed by divergence. As shown in a phylogenetic complex than that in humans. Human AKR1C1- analysis of the results of the multiple sequence align- AKR1C4 are potential targets for the treatment of ment (Fig. 1), the enzymes form a monophyletic gene several cancers, endometriosis and anxiety, and selective family comprised of three separate clades: the rodent inhibitors of the individual enzymes have been suggest- 3(17)a-HSD (AKR1C21 and RAKg) clade, the rodent ed to be new pharmaceutical agents for the treatment of 3a-HSDs and 20a-HSD clade, and the clade of other these diseases.86) The multiplicity of the mouse and rat enzymes. The phylogenetic tree reveals that initial enzymes should be considered when the eŠects of inhibi- duplication of the ancestral gene common to the tors are examined using laboratory animals. AKR1C enzymes shared among the these clades, and the Conclusion ancestral gene was subsequently duplicated also, giving rise to three clusters: the human enzymes, rodent Recent genomic and cDNA analyses suggested that enzymes (AKR1C15, RAKh, AKR1C6 and AKR1C20) the reductive metabolism of xenobiotic carbonyl com- and other rodent enzymes (AKR1C12–RAKi). pounds is mediated by many enzymes that diŠer in As described above, there are signiˆcant diŠerences in intracellular localization. The number of the human the multiplicity and tissue distribution of the enzymes enzymes is 12, which include uncharacterized CBR4. belonging to the AKR1C subfamily between humans Most enzymes function in the cellular metabolism of 12 Toshiyuki MATSUNAGA et al. their natural components, and convert toxic and lipid- (2000). soluble carbonyl compounds into less-reactive alcohols 8) Wermuth, B., Bohren, K. M., Heinemann, G., von because of their broad substrate speciˆcity. Thus, the Wartburg, J.-P. and Gabbay, K. H.: Human carbonyl omnipresent reductive potential must play an important reductase: nucleotide sequence analysis of a cDNA and part not only in normal cellular physiology, but also in amino acid sequence of the encoded protein. J. Biol. the metabolism of ketone-containing drugs and Chem., 263: 16185–16188 (1988). 9) Wermuth, B.: Puriˆcation and properties of an xenobiotic carbonyl compounds ingested. The literature NADPH-dependent carbonyl reductase from human indicates that human CBR1, HSD11B1, AKR1C1, brain: Relationship to prostaglandin 9-ketoreductase AKR1C2 and AKR1C4 are major reductases of ketone- and xenobiotic ketone reductase. J. Biol. Chem., 256: containing drugs, but suggests that their speciˆcity and 1206–1213 (1981). catalytic e‹ciency for several therapeutic drugs are 10) Wirth, H. and Wermuth, B.: Immunohistochemical signiˆcantly diŠerent. In future studies on the metabolic localization of carbonyl reductase in human tissues. J. fate of ketone-containing drugs, it will be necessary to Histochem. Cytochem., 40: 1857–1863 (1992). comparatively examine the substrate speciˆcities of all 11) Lemieux, N., Malfoy, B. and Forrest, G. L.: Human the enzymes, expecting roles of the newly-found carbonyl reductase (CBR) localized to band 21q22.1 by high-resolution ‰uorescence in situ hybridization dis- enzymes, CBR3, CBR4, DHRS4 and L-xylulose plays gene dosage eŠects in trisomy 21 cells. Genomics, reductase, in the metabolism. 15: 169–172 (1993). In rats and mice, additional ˆve or six reductases 12) Forrest,G.L.,Akman,S.,Krutzik,S.,Paxton,R.J., belonging to the AKR1C subfamily are suggested to be Sparkes,R.S.,Doroshow,J.,Felsted,R.L.,Glover, involved in the metabolism of xenobiotic carbonyl C. J., Mohandas, T. and Bachur, N. R.: Induction of a compounds. In addition, the tissue distributions of human carbonyl reductase gene located on chromo- rodent 20a-HSDs, 17b-HSD type 5 isozymes and 3a- some 21. Biochim. Biophys. Acta, 1048: 149–155 HSDs are signiˆcantly diŠerent from those of the corre- (1990). sponding human enzymes, AKR1C1, AKR1C3 and 13) Tanaka, M., Bateman, R., Rauh, D., Vaisberg, E., AKR1C4, which play important roles in pre-receptor Ramachandani, S., Zhang, C., Hansen, K. C., regulation of steroid actions. Such additional enzymes Burlingame, A. L., Trautman, J. K., Shokat, K. M. are perhaps involved in the animal-speciˆc cellular and Adams, C. L.: An unbiased cell morphology-based screen for new, biologically active small molecules. metabolism. This, together with the diŠerences in the PLoS Biol., 3: e128 (2005). tissue distributions of the other HSDs, may be responsi- 14) Ghosh, D., Sawicki, M., Pletnev, V., Erman, M., ble for the diŠerence in the metabolism of ketone-con- Ohno, S., Nakajin, S. and Duax, W. L.: Porcine taining drugs between humans and laboratory animals. carbonyl reductase. Structural basis for a functional References monomer in short chain dehydrogenasesWreductases. J. Biol. Chem., 276: 18457–18463 (2001). 1) Bachur, N. R. Cytoplasmic aldo-keto reductases: A 15) Wermuth, B., Platt, K. L., Seidel, A. and Oesch, F.: class of drug metabolizing enzymes. Science, 193: Carbonyl reductase provides the enzymatic basis of 595–597 (1976). quinine detoxication in man. Biochem. Pharmacol., 33: 2) Felsted, R. L. and Bachur, N. R.: Mammalian carbonyl 1277–1282 (1986). reductases. Drug Metab. Rev., 11: 1–60 (1980). 16) Jarabak, J. and Harvey, R. G.: Studies on three 3) Oppermann, U., Filling, C., Hult, M., Shafqat, N., reductases which have polycyclic aromatic hydrocarbon Wu,X.,Lindh,M.,Shafqat,J.,Nordling,E.Kallberg, quinines as substrates. Arch. Biochem. Biophys., 303: Y., Persson, B. and J äornvall, H.: Short-chain de- 394–401 (1993). hydrogenasesWreductases (SDR): the 2002 update. 17) Usami, N., Kitahara, K., Ishikura, S., Nagano, M., Chem. Biol. Interact., 143–144: 247–253 (2003). Sakai, S. and Hara, A.: Characterization of a major 4) Hyndman,D.,Bauman,D.R.,Heredia,V.V.,and form of human isatin reductase and the reduced Penning, T. M.: The aldo-keto reductase superfamily metabolite. Eur. J. Biochem., 268: 5755–5763 (2001). homepage. Chem. Biol. Interact., 143–144: 499–525 18) Doorn, J. A., Maser, E., Blum, A., ClaŠey, D. J. and (2003). Petersen, D. R.: Human carbonyl reductase catalyzes 5) Maser, E.: Xenobiotic carbonyl reduction and physio- reduction of 4-oxonon-2-enal. Biochemistry, 43: logical steroid oxidoreduction. The pluripotency of 13106–13114 (2004). several hydroxysteroid dehydrogenases. Biochem. 19) Olson,L.E.,Bedja,D.,Alvey,S.J.,Cardounel,A.J., Pharmacol., 49: 421–440 (1995). Gabrielson,K.L.andReeves,R.H.:Protectionfrom 6) Forrest, G. L. and Gonzalez, B.: Carbonyl reductase. doxorubicin-induced cardiac toxicity in mice with a null Chem. Biol. Interact. 129: 21–40 (2000). allele of carbonyl reductase 1. Cancer Res., 63: 7) Oppermann, U. C. and Maser, E.: Molecular and struc- 6602–6606 (2003). tural aspects of xenobiotic carbonyl metabolizing en- 20) Suto, K., Kajihara-Kano, H., Yokoyama, Y., Hayashi, zymes. Role of reductases and dehydrogenases in M., Kimura, J., Kumano, T., Takahata, T., Kudo, H. xenobiotic phase I reactions. Toxicology, 144: 71–81 and Tsuchida, S.: Decreased expression of the perox- Mammalian Carbonyl-Reducing Enzymes 13

isomal bifunctional enzyme and carbonyl reductase in H. and Maeda, M.: Further characterization of Chinese human hepatocellular carcinomas. J. Cancer Res. Clin. hamster carbonyl reductases (CHCRs). Chem. Biol. Oncol., 125: 83–88 (1999). Interact., 143–144: 373–381 (2003). 21) Umemoto, M., Yokoyama, Y., Sato, S., Tsuchida, S., 34) Nakjin, S., Tamura, F., Takase, N. and Toyoshima, Al-Mulla, F. and Saito, Y.: Carbonyl reductase as a S.: Carbonyl reductase activity exhibited by pig testicu- signiˆcant predictor of survival and lymph node lar 20b-hydroxysteroid dehydrogenase. Biol. Pharm. metastasis in epithelial ovarian cancer. Br. J. Cancer., Bull., 20: 1215–1218 (1997). 85: 1032–1036 (2001). 35) Ishikura, S., Yamamoto, Y., Matsuura, K., Wei, J., 22) Takenaka, K., Ogawa, E., Oyanagi, H., Wada, H. and Hodes, M. E. and Hara, A.: Properties and tissue Tanaka, F.: Carbonyl reductase expression and its distribution of mouse monomeric carbonyl reductase. clinical signiˆcance in non-small-cell lung cancer. Biol. Pharm. Bull., 21: 879–881 (1998). Cancer. Epidemiol. Biomarkers Prev., 14: 1972–1975 36) Nakayama, T., Yashiro, K., Inoue, Y., Matsuura, K., (2005). Ichikawa,H.,Hara,A.andSawada,H.:Characteriza- 23) Ohara, H., Miyabe, Y., Deyashiki, Y., Matsuura, K. tion of pulmonary carbonyl reductase of mouse and and Hara, A.: Reduction of drug ketones by guinea pig. Biochim. Biophys. Acta, 882: 220–227 dihydrodiol dehydrogenases, carbonyl reductase and (1986). aldehyde reductase of human liver. Biochem. Phar- 37) Oritani, H., Deyashiki, Y., Nakayama, T., Hara, A., macol., 50: 221–227 (1995). Sawada, H., Bunai, Y. and Ohya, I.: Puriˆcation and 24) Breyer-PfaŠ, U. and Nill, K.: High-a‹nity stereoselec- characterization of pig lung carbonyl reductase. Arch. tive reduction of the enantiomers of ketotifen and of Biochem. Biophys., 292: 539–547 (1992). ketonic nortriptyline metabolites by aldo-keto reduc- 38) Matsuura, K., Hara, A., Sawada, H., Bunai, Y. and tases from human liver. Biochem. Pharmacol., 59: Ohya, I.: Localization of pulmonary carbonyl 249–260 (2000). reductase in guinea-pig and mouse: Enzyme histochem- 25) Someya, T., Muratake, T., Hirokane, G., Shibasaki, ical and imuunochemical studies. J. Histochem. M., Shimoda, K. and Takahashi, S.: Interindividual Cytochem., 38: 217–223 (1990). variation in bromperidol metabolism and relationship 39) Ishikura, S., Usami, N., Kitahara, K., Isaji, T., Oda, to therapeutic eŠects. J. Clin. Psychopharmacol., 20: K.,Nakagawa,J.andHara,A.:Enzymaticcharacteri- 175–180 (2000). zation and subcellular distribution of a short-chain 26) Shimoda, K., Shibasaki, M., Inaba, T., Cheung, S. W., dehydrogenaseWreductase family protein, P26h, in Someya, T. and Takahashi, S.: Carbonyl reduction of hamster testis and epididymis. Biochemistry, 40: timiperone in human liver cytosol. Pharmacol. 214–224 (2001). Toxicol., 83: 164–168 (1998). 40) Tanaka, N., Nonaka, T., Nakanishi, M., Deyashiki, 27) Holleran, J. L., Fourcade, J., Egorin, M. J., Eiseman, Y., Hara, A. and Mitsui, Y.: Crystal structure of the J.L.,Parise,R.A.,Musser,S.M.,White,K.D., ternary complex of mouse lung carbonyl reductase at Covey, J. M., Forrest, G. L. and Pan, S. S.: In vitro 1.8 Å resolution: The structural origin of coenzyme metabolism of the phosphatidylinositol 3-kinase inhibi- speciˆcity in the short-chain dehydrogenaseWreductase tor, wortmannin, by carbonyl reductase. Drug Metab. family. Structure, 4: 33–45 (1996). Dispos., 32: 490–496 (2004). 41) Nakanishi, M., Matsuura, K., Kaibe, H., Tanaka, N., 28) Maser, E. and Breyer-PfaŠ, U.: Competing roles of Nonaka, T., Mitsui, Y. and Hara, A.: Switch of reductase in the detoxiˆcation of the tobacco-speciˆc coenzyme speciˆcity of mouse lung carbonyl reductase nitrosamine ketone NNK. In Penning T. M. and by substitution of threonine 38 with aspartic acid. J. Petrash, J. M. (ed.): Aldo-keto Reductases and Tox- Biol. Chem., 272: 2218–2222 (1997). icant Metabolism, Washington DC, American Chemi- 42) Watanabe, K., Sugawara, C., Ono, A., Fukuzumi, Y., cal Society, 2004, pp. 67–82. Itakura, S., Yamazaki, M., Tashiro, H., Osoegawa, 29) Aoki, H., Okada, T., Mizutani, T., Numata, Y., K., Soeda, E. and Nomura, T.: Mapping of a novel Minegishi, T. and Miyamoto, K.: Identiˆcation of two human carbonyl reductase, CBR3, and ribosomal closely related genes, inducible and noninducible pseudogenes to human chromosome 21q22.2. Genom- carbonyl reductases in the rat ovary. Biochem. ics, 52: 95–100 (1998). Biophys. Res. Commun., 230; 518–523 (1997). 43) Lakhman, S. S., Ghosh, D. and Blanco, J. G.: 30) Wermuth, B., Maeder-Heinemann, G. and Ernst, E.: Functional signiˆcance of a natural allelic variant of Cloning and expression of carbonyl reductase from rat human carbonyl reductase 3 (CBR3). Drug Metab. testis. Eur. J. Biochem., 228, 473–479 (1995). Dispos., 33: 254–257 (2005). 31) Iwata, N., Inazu, N. and Satoh, T.: The puriˆcation 44) Na,G.Y.,Seo,S.K.,Lee,S.J.,Kim,D.W.,Kim,M. and properties of NADPH-dependent reductases from K. and Kim, J. C.: Upregulation of the NNP-1 (novel rat ovary. J. Biochem.(Tokyo), 105: 556–564 (1989). nuclear protein-1, D21S2056E) gene in keloid tissue 32) Gonzalez,B.,Sapra,A.,Rivera,H.,Kaplan,W.D., determined by cDNA microarray and in situ hybridiza- Yam, B. and Forrest, G. L.: Cloning and expression of tion. Br.J.Dermatol., 151: 1143–1149 (2004). the cDNA encoding rabbit liver carbonyl reductase. 45)Tomlinson,J.W.,Walker,E.A.,Bujalska,I.J., Gene, 154: 297–298 (1995). Draper,N.,Lavery,G.G.,Cooper,M.S.,Hewison, 33) Terada, T., Sugihara, Y., Nakamura, K., Mizobuchi, M.andStewart,P.M.:11b-hydroxysteroid de- 14 Toshiyuki MATSUNAGA et al.

hydrogenase type 1: a tissue-speciˆc regulator of Res. Toxicol., 17: 1145–1150 (2004). glucocorticoid response. Endocr. Rev., 25: 831–866 58) Lei, Z., Chen, W., Zhang, M. and Napoli, J. L.: (2004). Reduction of all-trans-retinal in the mouse liver peroxi- 46) Blum, A. and Maser, E.: Enzymology and molecular some fraction by the short-chain dehydrogenaseWreduc- biology of glucocorticoid metabolism in humans. Prog. tase RRD: induction by the PPAR a ligand cloˆbrate. Nucleic Acid Res. Mol. Biol., 75: 173–216 (2003). Biochemistry, 42: 4190–4196 (2003). 47) Tomlinson, J. W.: 11b-Hydroxysteroid dehydrogenase 59) Hara, A., Nakayama, T., Deyashiki, Y., Kariya, K. type 1 in human disease: a novel therapeutic target. and Sawada, H.: Carbonyl reductase of dog liver: Minerva. Endocrinol., 30: 37–46 (2005). Puriˆcation, properties, and kinetic mechanism. Arch. 48) Ogg, D., Elleby, B., Norstrom, C., Stefansson, K., Biochem. Biophys., 244: 238–247 (1986). Abrahmsen, L., Oppermann, U. and Svensson, S.: The 60) Ishikura, S., Isaji, T., Usami, N., Kitahara, K., crystal structure of guinea pig 11b-hydroxysteroid Nakagawa, J. and Hara, A.: Molecular cloning of a dehydrogenase type 1 provides a model for enzyme- cDNA for hamster diacetyl reductase that is identical lipid bilayer interactions. J. Biol. Chem., 280: with L-xylulose reductase. Chem. Biol. Interact., 3789–3794 (2005). 130–132: 879–889 (2001). 49) Zhang,J.,Osslund,T.D.,Plant,M.H.,Clogston,C. 61) Nakagawa, J., Ishikura, S., Asami, J., Isaji, T., L.,Nybo,R.E.,Xiong,F.,Delaney,J.M.and Usami, N., Hara, A., Sakurai, T., Tsuritani, K., Oda, Jordan, S. R.: Crystal structure of murine 11b-hydro- K., Takahashi, M., Yoshimoto, M., Otsuka, N. and xysteroid dehydrogenase 1: an important therapeutic Kitamura, K.: Molecular characterization of target for diabetes. Biochemistry, 44: 6948–6957 (2005). mammalian dicarbonylWL-xylulose reductase and its 50) Maser, E. and Oppermann, U. C.: Role of type-1 11b- localization in kidney. J. Biol. Chem., 277: hydroxysteroid dehydrogenase in detoxiˆcation proc- 17883–17891 (2002). esses. Eur. J. Biochem., 249: 365–369 (1997). 62) El-Kabbani, O., Carbone, V., Darmanin, C., Ishikura, 51) Wsol, V., Szotakova, B., Skalova, L. and Maser, E.: S. and Hara, A.: Structure of the tetrameric form of The novel anticancer drug oracin: diŠerent human L-xylulose reductase: Probing the inhibitor- stereospeciˆcity and cooperativity for carbonyl reduc- binding site with molecular modeling and site-directed tion by puriˆed human liver 11b-hydroxysteroid mutagenesis. Proteins, 61: 688–690 (2005). dehydrogenase type 1. Toxicology, 197: 253–261 63) Ishikura, S., Usami, N., El-Kabbani, O. and Hara, A.: (2004). Structural determinant for cold inactivation of rodent 52) Hult, M., Nobel, C. S., Abrahmsen, L., Nicoll-Gri‹th, L-xylulose reductase. Biochem. Biophys. Res. Com- D. A., J äornvall, H. and Oppermann, U. C.: Novel mun., 308: 68–72 (2003). enzymological proˆles of human 11b-hydroxysteroid 64) Petrash, J. M.: All in the family: aldose reductase and dehydrogenase type 1. Chem. Biol. Interact., 130–132: closely related aldo-keto reductases. Cell Mol. Life Sci., 805–814 (2001). 61: 737–749 (2004). 53) Schweizer, R. A., Zurcher, M., Balazs, Z., Dick, B. 65) Zeindl-Eberhart, E., Haraida, S., Liebmann, S., and Odermatt, A.: Rapid hepatic metabolism of 7- Jungblut,P.R.,Lamer,S.,Mayer,D.,Jager,G., ketocholesterol by 11b-hydroxysteroid dehydrogenase Chung, S. and Rabes, H. M.: Detection and identiˆca- type 1: species-speciˆc diŠerences between the rat, tion of tumor-associated protein variants in human human, and hamster enzyme. J. Biol. Chem., 279: hepatocellular carcinomas. Hepatology, 39: 540–549 18415–18424 (2004). (2004). 54) Fransen, M., Van Veldhoven, P. P. and Subramani, S.: 66) Fukumoto, S., Yamauchi, N., Moriguchi, H., Hippo, Identiˆcation of peroxisomal proteins by using M13 Y., Watanabe, A., Shibahara, J., Taniguchi, H., phage protein VI phage display: molecular evidence Ishikawa, S., Ito, H., Yamamoto, S., Iwanari, H., that mammalian peroxisomes contain a 2,4-dienoyl- Hironaka, M., Ishikawa, Y., Niki, T., Sohara, Y., CoA reductase. Biochem. J., 340: 561–568 (1999). Kodama, T., Nishimura, M., Fukayama, M., Dosaka- 55)Usami,N.,Ishikura,S.,Abe,H.,Nagano,M., Akita, H. and Aburatani, H.: Overexpression of the Uebuchi, M., Kuniyasu, A., Otagiri, M., Nakayama, aldo-keto reductase family protein AKR1B10 is highly H., Imamura, Y. and Hara, A.: Cloning, expression correlated with smokers' non-small cell lung carcino- and tissue distribution of a tetrameric form of pig mas. Clin. Cancer Res., 11: 1776–1785, (2005). carbonyl reductase. Chem. Biol. Interact., 143–144: 67) Guengerich, F. P., Cai, H., McMahon, M., Hayes, J. 353–361 (2003). D., Sutter, T. R., Groopman, J. D., Deng, Z. and

56) Imamura, Y., Migita, T., Otagiri, M., Choshi, T. and Harris, T. M.: Reduction of a‰atoxin B1 dialdehyde by Hibino, S.: Puriˆcation and catalytic properties of a rat and human aldo-keto reductases. Chem. Res. Tox- tetrameric carbonyl reductase from rabbit heart, J. icol., 14: 727–737 (2001). Biochem.(Tokyo), 125: 41–47 (1999). 68) O'connor, T., Ireland, L. S., Harrison, D. J. and 57) Shimada, H., Oginuma, M., Hara, A. and Imamura Hayes, J. D.: Major diŠerences exist in the function

Y.: 9,10-Phenanthrenequinone, a component of diesel and tissue-speciˆc expression of human a‰atoxin B1 exhaust particles, inhibits the reduction of 4-benzoyl- aldehyde reductase and the principal human aldo-keto pyridine and all-trans-retinal and mediates superoxide reductase AKR1 family members. Biochem. J., 343: formation through its redox cycling in pig heart. Chem. 487–504 (1999). Mammalian Carbonyl-Reducing Enzymes 15

69) Knight, L. P., Primiano, T., Groopman, J. D., semialdehyde reductase from human brain. Identity

Kensler, T. W. and Sutter, T. R.: cDNA cloning, with a‰atoxin B1 aldehyde reductase. Eur. J. Biochem.,

expression and activity of a second human a‰atoxin B1- 265: 1056–1060 (1999). metabolizing member of the aldo-keto reductase super- 81) Nishinaka, T., Azuma, Y., Ushijima, S., Miki, T. and family, AKR7A3. Carcinogenesis, 20: 1215–1223 Yabe-Nishimura, C.: Human testis speciˆc protein: a (1999). new member of aldo-keto reductase superfamily. 70) Hayes, J. D., Judah, D. J. and Neal, G. E.: Resistance Chem. Biol. Interact., 143–144: 299–305 (2003).

to a‰atoxin B1 is associated with the expression of a 82) Bauman, D. R., Steckelbroeck, S., Penning, T. M.: novel aldo-keto reductase which has catalytic activity The roles of aldo-keto reductases in steroid hormone towards a cytotoxic aldehyde-containing metabolite of action. Drug News Perspect, 17: 563–578 (2004). the toxin. Cancer Res., 53: 3887–3894 (1993). 83) Penning, T. M.: Aldo-keto reductases and formation of 71) Ellis, E. M. and Hayes, J. D.: Substrate speciˆcity of polycyclic aromatic hydrocarbon o-quinones. Method an a‰atoxin-metabolizing aldehyde reductase. Enzymol., 378: 31–67 (2004). Biochem. J., 312 (Pt 2): 535–541 (1995). 84) Shiraishi, H., Matsuura, K., Kume, T. and Hara, A.: 72) Kelly,V.P.,Ellis,E.M.,Manson,M.M.,Chanas,S. Expression of mRNA for dihydrodiol dehydrogenase A.,MoŠat,G.J.,McLeod,R.,Judah,D.J.,Neal,G. isoforms in human tissues. Adv. Exp. Med. Biol., 463:

E. and Hayes, J. D.: Chemoprevention of a‰atoxin B1 539–544 (1999). hepatocarcinogenesis by coumarin, a natural benzopy- 85) Nishizawa, M., Nakajima, T., Yasuda, K., Kanzaki,

rone that is a potent inducer of a‰atoxin B1-aldehyde H.,Sasaguri,Y.,Watanabe,K.andIto,S.:Close reductase, the glutathione S-transferase A5 and P1 kinship of human 20a-hydroxysteroid dehydrogenase subunits, and NAD(P)H:quinone oxidoreductase in rat gene with three aldo-keto reductase genes. Genes Cells, liver. Cancer Res., 60: 957–969 (2000). 5: 111–125 (2000). 73)Bonnesen,C.,Eggleston,I.M.andHayes,J.D.: 86) Penning,T.M.,Burczynski,M.E.,Jez,J.M.,Hung, Dietary indoles and isothiocyanates that are generated C.F.,Lin,H.K.,Ma,H.,Moore,M.,Palackal,N. from cruciferous vegetables can both stimulate apopto- and Ratnam, K.: Human 3a-hydroxysteroid sis and confer protection against DNA damage in dehydrogenase isoforms (AKR1C1-AKR1C4) of the human colon cell lines. Cancer Res., 61: 6120–6130 aldo-keto reductase superfamily: functional plasticity (2001). and tissue distribution reveals roles in the inactivation 74) Kozma,E.,Brown,E.,Ellis,E.M.andLapthorn,A. and formation of male and female sex hormones. J.: The Crystal structure of rat liver Akr7A1: A dimeric Biochem. J., 351: 67–77 (2000). member of the aldo-keto reductase superfamily. J. 87) Steckelbroeck, S., Jin, Y., Gopishetty, S., Oyesanmi, Biol. Chem., 277: 16285–16293 (2002). B. and Penning, T. M.: Human cytosolic 3a-hydro- 75) Kozma,E.,Brown,E.,Ellis,E.M.andLapthorn,A. xysteroide dehydrogenases of the aldo-keto reductase J.: The high resolution crystal structure of rat liver superfamily display signiˆcant 3b-hydroxysteroid de- AKR7A1: understanding the substrate speciˆcities of hydrogenase activity. J. Biol. Chem., 279: 10784–10795 the AKR7 family. Chem. Biol. Interact., 143–144: (2004). 289–297 (2003). 88) Ishikura, S., Nakajima, S., Kaneko, T., Shinitani, S., 76) Ireland, L. S., Harrison, D. J., Neal, G. E. and Hayes, Usami, N., Yamamoto, I., Carbone, V., El-Kabbani, J. D.: Molecular cloning, expression and catalytic O. and Hara A.: Comparison of stereoselective reduc- activity of a human AKR7 member of the aldo-keto tion of 3- and 20-oxosteroids among mouse and primate reductase superfamily: evidence that the major 2-car- 20a-hydroxysteroid dehydrogenases. In Weiner H., boxybenzaldehyde reductase from human liver is a Plapp,B.,Lindahl,R.andMaser,E.(ed.):Enzymolo-

homologue of rat a‰atoxin B1-aldehyde reductase. gy and Molecular Biology of Carbonyl Metabolism 12, Biochem. J., 332: 21–34 (1998). West Lafayette, Purdue University Press, 2005, pp. 77) Hinshelwood, A., McGarvie, G. and Ellis, E.: Charac- 341–351. terization of a novel mouse liver aldo-keto reductase 89) Weihua, Z., Lathe, R., Warner, M. and Gustafsson, AKR7A5. FEBS Lett., 523: 213–218 (2002). J.-Å.: An endocrine pathway in the prostate, ERb,AR, 78) Kelly, V. P., Ireland, L. S., Ellis, E. M. and Hayes, J. 5a-androstane-3b,17b-diol, and CYP7B1, regulates D.: Puriˆcation from rat liver of a novel constitutively prostate growth. Proc.Natl.Acad.Sci.USA, 99: expressed member of the aldo-keto reductase 7 family 13589–13594 (2001). that is widely distributed in extrahepatic tissues. 90) Wang,M.,He,Y.,Eisenman,L.N.,Fields,C.,Zeng, Biochem. J., 348: 389–400 (2000). C.M.,Mathews,J.,Benz,A.andFu,T.:3b-Hydroxy-

79) Kelly, V. P., Sherratt, P. J., Crouch, D. H. and Hayes, pregnane steroids are pregnenolone sulfate-like GABAA J. D.: Novel homodimeric and heterodimeric rat g- receptor antagonists. J. Neurosci., 22: 3366–3375 hydroxybutyrate synthases that associate with the Golgi (2002). apparatus deˆne a distinct subclass of aldo-keto reduc- 91) Turkmen, S., Lundgren, P., Birzniece, V., Zingmark, tase7familyproteins.Biochem. J., 366: 847–861 E., Backstrom, T. and Johansson, I. M.: 3b,20b- (2002). Dihydroxy-5a-pregnane (UC1011) antagonism of the 80)Schaller,M.,SchaŠhauser,M.,Sans,N.and GABA potentiation and the learning impairment in- Wermuth, B.: Cloning and expression of succinic duced in rats by allopregnanolone. Eur. J. Neurosci., 16 Toshiyuki MATSUNAGA et al.

20: 1604–1612 (2004). tissue are positive correlates of adiposity in women. 92) Matsuura, K., Deyashiki, Y., Sato, K., Miwa. G., Am.J.Physiol., 288: E398-E404 (2005). Ishida, N. and Hara, A.: Identiˆcation of amino acid 103) Deyashiki, Y., Taniguchi, H., Amano, T., Nakayama, residues responsible for diŠerences in substrate speciˆc- T., Hara, A. and Sawada, H.: Structural and function- ity and inhibitor sensitivity between two human liver al comparison of two human liver dihydrodiol dihydrodiol dehydrogenase isoenzymes by site-directed dehydrogenases associated with 3a-hydroxysteroid mutagenesis. Biochem. J., 323: 61–64 (1997). dehydrogenase activity. Biochem. J., 282: 741–746 93) Couture, J.-F., Legrand, P., Cantin, L., Luu-The, V., (1992). Labrie, F. and Breton, R.: Human 20a-hydroxysteroid 104) Hara, A., Matsuura, K., Tamada, Y., Sato, K., dehydrogenase: Crystallographic and site-directed Miyabe, Y., Deyashiki, Y. and Ishida, N.: Relationship mutagenesis studies lead to the identiˆcation of an of human liver dihydrodiol dehydrogenases to hepatic alternative binding site for C21-steroids. J. Mol. Biol., bile-acid-binding protein and an oxidoreductase of 331: 593–604 (2003). human colon cells. Biochem. J., 313: 373–376 (1996). 94) Ohara, H., Nakayama, T., Deyashiki, Y., Hara, A., 105) Lin,H.K.,Steckelbroeck,S.,Fung,K.M.,Jones,A. Miyabe, Y. and Tsukada, F.: Reduction of prostaglan- N. and Penning, T. M.: Characterization of a

din D2 to 9a,11b-prostaglandin F2 by a human liver monoclonal antibody for human aldo-keto reductase 3a-hydroxysteroid dehydrogenase isoenzyme. Biochim. AKR1C3 (type 2 3a-hydroxysteroid dehydrogenaseW Biophys. Acta, 1215: 59–65 (1994). type 5 17b-hydroxysteroid dehydrogenase); immuno- 95) Breyer-PfaŠ, U. and Nill, K.: Carbonyl reduction of histochemical detection in breast and prostate. naltrexone and dolasetron by oxidoreductases isolated Steroids, 69: 795–801 (2004). from human liver cytosol. J. Pharm. Pharmacol., 56: 106) Hara, A., Taniguchi, H., Nakayama, T. and Sawada, 1601–1606 (2004). H.: Puriˆcation and properties of multiple forms of 96) Wsol, V., Szotakova, B., Skalova, L. and Maser, E.: dihydrodiol dehydrogenase from human liver. J. Contribution of aldo-keto reductases to the metabolism Biochem.(Tokyo), 108: 250–254 (1990). of the novel anticancer drugs oracin in man. In Weiner 107) Dufort,I.,Rheault,P.,Huang,X.F.,Soucy,P.and H.,Plapp,B.,Lindahl,R.andMaser,E.(ed.):En- Luu-The, V.: Characteristics of a highly labile human zymology and Molecular Biology of Carbonyl type 5 17b-hydroxysteroid dehydrogenase. Endocrinol- Metabolism 12, West Lafayette, Purdue University ogy, 140: 568–574 (1999). Press, 2005, pp. 249–255. 108) Khanna,M.,Qin,K.N.,Wang,R.W.andCheng,K. 97) Higaki, Y., Usami, N., Shintani, S., Ishikura, S., El- C.: Substrate speciˆcity, gene structure, and tissue- Kabbani, O. and Hara, A.: Selective and potent inhibi- speciˆc distribution of multiple human 3a-hydro- tors of human 20a-hydroxysteroid dehydrogenase xysteroid dehydrogenases. J. Biol. Chem., 270: (AKR1C1) that metabolizes neurosteroids derived from 20162–20168 (1995). progesterone. Chem. Biol. Interact., 143–144: 503–513 109) Peltoketo, H., Luu-The, V., Simard, J. and Adamski, (2003). J.: 17b-Hydroxysteroid dehydrogenase (HSD)W17- 98) Ciaccio, P. J., Jaiswal, A. K. and Tew, K. D.: Regula- ketosteroid reductase (KSR) family; nomenclature and tion of human dihydrodiol dehydrogenase by Michael main characteristics of the 17HSDWKSR enzymes. J. acceptor xenobiotics. J. Biol. Chem., 269: 15558–15562 Mol. Endocrinol., 23: 1–11 (1999). (1994). 110)Matsuura,K.,Shiraishi,H.,Hara,A.,Sato,K., 99) Burczynski, M. E., Lin, H. K. and Penning, T. M.: Deyashiki, Y., Ninomiya, M. and Sakai, S.: Identiˆca- Isoform-speciˆc induction of a human aldo-keto tion of a principal mRNA species for human 3a-hydro- reductase by polycyclic aromatic hydrocarbons xysteroid dehydrogenase isoform (AKR1C3) that ex-

(PAHs), electrophiles, and oxidative stress: implica- hibits high prostaglandin D2 11-ketoreductase activity. tions for the alternative pathway of PAH activation J. Biochem.(Tokyo), 124: 940–946 (1998). catalyzed by human dihydrodiol dehydrogenase. Can- 111) Suzuki-Yamamoto, T., Nishizawa, M., Fukui, M., cer Res., 59: 607–614 (1999). Okuda-Ashitaka, E. and Nakajima, T.: cDNA cloning, 100) Burczynski, M. E., Sridhar, G. R., Palackal, N. T. and expression and characterization of human prostaglan- Penning, T. M.: The reactive oxygen species and din F synthase. FEBS Lett., 462: 335–340 (1999). Michael acceptor-inducible human aldo-keto reductase 112) Desmond, J. C., Mountford, J. C., Drayson, M. T., AKR1C1 reduces the a,b-unsaturated aldehyde 4- Walker,E.A.,Hewison,M.,Ride,J.P.,Luong,Q. hydroxy-2-nonenal to 1,4-dihydroxy-2-nonene. J. Biol. T., Hayden, R. E., Vanin, E. F. and Bunce, C. M.: The Chem., 276: 2890–2897 (2001). aldo-keto reductase AKR1C3 is a novel suppressor of 101) Agapova,O.A.,Yang,P.,Wang,W.H.,Lane,D.A., cell diŠerentiation that provides a plausible target for Clark,A.F.,Weinstein,B.I.andHernandez,M.R.: the non-cyclooxygenase-dependent antineoplastic ac- Altered expression of 3a-hydroxysteroid de- tions of nonsteroidal anti-in‰ammatory drugs. Cancer hydrogenases in human glaucomatous optic nerve head Res., 63: 505–512 (2003). astrocytes. Neurobiol. Dis. 14: 63–73 (2003). 113) Mills,K.I.,Gilkes,A.F.,Sweeney,M.,Choudhry,M. 102) Blouin, K., Blanchette, S., Richard, C., Dupont, P., A.,Woodgate,L.J.,Bunce,C.M.,Brown,G.and Luu-The, V. and Tchernof, A.: Expression and activity Burnett, A. K.: Identiˆcation of a retinoic acid respon- of steroid aldoketoreductases 1C in omental adipose sive aldoketoreductase expressed in HL60 leukaemic Mammalian Carbonyl-Reducing Enzymes 17

cells. FEBS Lett., 440: 158–162 (1998). 1148–1153 (1998). 114) Moeller, L. C., Dumitrescu, A. M., Walker, R. L., 126) Molowa, D. T., Shayne, A. G. and Guzelian, P. S.: Meltzer, P. S. and RefetoŠ, S.: Thyroid hormone Puriˆcation and characterization of chlordecone responsive genes in cultured human ˆbroblasts. J. Clin. reductase from human liver. J. Biol. Chem., 261: Endocrinol. Metab., 90: 936–943 (2005). 12624–12627 (1986). 115) Lovering,A.L.,Ride,J.P.,Bunce,C.M.,Desmond, 127) Ozeki, T., Takahashi, Y., Nakayama, K., Funayama, J.C.,Cummings,S.M.andWhite,S.A.:Crystal M., Nagashima, K., Kodama, T. and Kamataki, T.:

structures of prostaglandin D2 11-ketoreductase Hepatocyte nuclear factor-4aWg and hepatocyte nuclear (AKR1C3) in complex with the nonsteroidal anti-in- factor-1a as causal factors of interindividual diŠerence ‰ammatory drugs ‰ufenamic acid and indomethacin. in the expression of human dihydrodiol dehydrogenase Cancer Res., 64: 1802–1810 (2004). 4 mRNA in human livers. Pharmacogenetics, 13: 49–53 116) Komoto, J., Yamada, T., Watanabe, K. and (2003). Takusagawa, F.: Crystal structure of human 128) Kume, T., Iwasa, H., Shiraishi, H., Yokoi, T., prostaglandin F synthase (AKR1C3). Biochemistry, 43: Nagashima, K., Otsuka, M., Terada, T., Takagi, T., 2188–2198 (2004). Hara,A.andKamataki,T.:Characterizationofa 117) Qiu,W.,Zhou,M.,Labrie,F.andLin,S.X.:Crystal novel variant (S145CWL311V) of 3a-hydroxysteroidW structures of the multispeciˆc 17b-hydroxysteroid dihydrodiol dehydrogenase in human liver. Phar- dehydrogenase type 5: critical androgen regulation in macogenetics, 9: 763–771 (1999). human peripheral tissues. Mol. Endocrinol., 18: 129) Ishida, M., Chang, K.-T., Hirabayashi, K., Nishihara, 1798–1807 (2004). M. and Takahashi, M.: Cloning of mouse 20a-hydro- 118) Bauman, D. R., Rudnick, S. I., Szewczuk, L. M., Jin, xysteroid dehydrogenase. J. Reprod. Dev., 45: 321–329 Y., Gopishetty, S., Penning, T. M.: Development of (1999). nonsteroidal anti-in‰ammatory drug analogs and 130) Ma, H. and Penning, T. M.: Characterization of steroid carboxylates selective for human aldo-keto homogeneous recombinant 20a-hydroxysteroid de- reductase isoforms: potential antineoplastic agents that hydrogenase: ‰uorescent properties and inhibition work independently of cyclooxygenase isozymes. Mol. proˆle. Biochem. J., 341: 853–859 (1999). Pharmacol., 67: 60–68 (2005). 131) Vergnes, L., Phan, J., Stolz, A. and Reue, K.: A cluster 119) Russell, D. W.: The enzymes, regulation, and genetics of eight hydroxysteroid dehydrogenase genes belonging of bile acid synthesis. Ann. Rev. Biochem., 72: 137–174 to the aldo-keto reductase supergene family on mouse (2003). chromosome 13. J. Lipid Res., 44: 503–511 (2003). 120) Ozeki, T., Takahashi, Y., Kume, T., Nakayama, K., 132) Deyashiki, Y., Ohshima, K., Nakanishi, M., Sato, K., Yokoi, T., Nunoya, K., Hara, A. and Kamataki, T.: Matsuura,K.andHara,A.:Molecularcloningand Co-operative regulation of the transcription of human characterization of mouse estradiol 17b-dehydrogenase dihydrodiol dehydrogenase (DD)4Waldo-keto reductase (A-speciˆc), a member of the aldoketo reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)4aW family., J. Biol. Chem., 270: 10461–10467 (1995). g and HNF-1a. Biochem. J., 355: 537–544 (2001). 133) Ishikura, S., Usami, N., Nakajima, S., Carbone, V., 121) Ozeki, T., Takahashi, Y., Nakayama, K. and El-Kabbani, O. and Hara, A.: Site-directed mutagene- Kamataki, T.: Hepatocyte nuclear factor (HNF)-4aWg, sis of the coenzyme-binding residues of mouse 3a-and HNF-1a, and vHNF-1 regulate the cell-speciˆc expres- 3(17)a-hydroxysteroid dehydrogenases. Their roles in sion of the human dihydrodiol dehydrogenase (DD)4W the speciˆcity for coenzymes and substrates. In Weiner AKR1C4 gene. Arch. Biochem. Biophys., 405: 185–190 H.,Plapp,B.,Lindahl,R.andMaser,E.(ed.):En- (2002). zymology and Molecular Biology of Carbonyl 122) Winters, C. J., Molowa, D. T. and Guzelian, P. S.: Metabolism 12, West Lafayette, Purdue University Isolation and characterization of cloned cDNAs encod- Press, 2005, pp. 324–332. ing human liver chlordecone reductase. Biochemistry, 134) Pawlowski, J. E. and Penning, T. M.: Overexpression 29: 1080–1087 (1990). and mutagenesis of the cDNA for rat liver 3a-hydro- 123) Matsuura, K., Tamada, Y., Deyashiki, Y., Miyabe, Y., xysteroid dehydrogenase. Role of cysteines and tyro- Nakanishi, M., Ohya, I. and Hara, A.: Activation of sines in catalysis. J. Biol. Chem., 269: 13502–13510 human liver 3a-hydroxysteroid dehydrogenase by sul- (1994). phobromophthalein. Biochem. J., 313: 179–184 (1996). 135) Ikeda, S., Okuda-Ashitaka, E., Masu, Y., Suzuki, T., 124) Matsuura, K., Hara, A., Kato, M., Deyashiki, Y., Watanabe, K., Nakano, M., Shingu, K. and Ito, S.: Miyabe, Y., Ishikura, S., Sugiyama, T. and Katagiri, Cloning and characterization of two novel aldo-keto Y.: Activation of human liver 3a-hydroxysteroid reductases (AKR1C12 and AKR1C13) from mouse dehydrogenase by cloˆbrate derivatives. J. Pharmacol. stomach. FEBS Lett., 459: 433–437 (1999). Exp. Ther., 285: 1096–1103 (1998). 136) Du, Y., Tsai, S., Keller, J. R. and Williams, S. C.: 125) Yamamoto, T., Matsuura, K., Shintani, S., Hara, A., Identiˆcation of an interleukin-3-regulated aldoketo Miyabe, Y., Sugiyama, T. and Katagiri, Y.: Dual reductase gene in myeloid cells which may function in eŠects of anti-in‰ammatory 2-arylpropionic acid autocrine regulation of myelopoiesis. J. Biol. Chem., derivatives on a major isoform of human liver 3a- 275: 6724–6732 (2000). hydroxysteroid dehydrogenase. Biol. Pharm. Bull., 21: 137) Tajima, K., Hashizaki, M., Yamamoto, K., 18 Toshiyuki MATSUNAGA et al.

Narimatsu, S. and Mizutani, T.: Puriˆcation and some Matsuura, K., Hara, A. and Sawada, H.: Identiˆcation properties of two enzymes from rat liver cytosol that of two dihydrodiol dehydrogenases associated with catalyze carbonyl reduction of 6-tert-butyl-2,3-epoxy- 3(17)a-hydroxysteroid dehydrogenase activity in mouse 5-cyclohexene-1,4-dione, a metabolite of 3-tert-butyl- kidney. J. Biochem.(Tokyo), 106: 633–638 (1989). 4-hydroxyanisole. Arch. Biochem. Biophys., 361: 143) Ishikura,S.,Usami,N.,Nakajima,S.,Kameyama,A., 207–214 (1999). Shiraichi,H.,Carbone,V.,El-Kabbani,O.andHara, 138) Qin, K.-N. and Cheng, K.-C.: Structure and tissue- A.: Characterization of two isoforms of mouse 3(17)a- speciˆc expression of the aldo-keto reductase superfa- hydroxysteroid dehydrogenases of the aldo-keto reduc- mily. Biochemistry, 33: 3223–3228 (1994). tase family. Biol. Pharm. Bull., 27: 1939–1945 (2004). 139) Deyashiki, Y., Takatsuji, T. and Hara, A.: Molecular 144) Bellemare, V., Faucher, F., Breton, R. and Luu-The, cloning and characterization of dihydrodiol de- V.: Characterization of 17a-hydroxysteroid dehydrogenase from the mouse. In Penning T. M. and hydrogenase activity (17a-HSD) and its involvement in Petrash, J. M. (ed.): Aldo-keto Reductases and the biosynthesis of epitestosterone. BMC-Biochem., 6: Toxicant Metabolism, Washington, DC, American 12 (2005). Chemical Society, 2004, pp. 101–114. 145)Thompson,J.D.,Higgins,D.G.,Gibson,T.J.: 140) Toki, S. and Yamano, S.: Production of morphinone CLUSTAL W: improving thesensitivity of progressive as a metabolite of morphine and its physiological role. multiple sequence alignment through sequenceweight- Yakugaku-Zasshi, 119: 249–267 (1999). ing, positions-speciˆc gap penalties and weight matrix 141) Ishikura,S.,Horie,K.,Sanai,M.,Matsumoto,K.and choice. Nucleic Acids Res., 22: 4673–4680 (1994). Hara, A.: Enzymatic properties of a member 146) Page, R. D. M.: TREEVIEW: An application to dis- (AKR1C19) of the aldo-keto reductase family. Biol. play phylogenetic trees on personal computers. Com- Pharm. Bull., 28: 1075–1078 (2005). put. Appl. Biosci., 12: 357–358 (1996). 142) Nakagawa, M., Tsukada, F., Nakayama, T.,