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PURIFICATION AND CHARACTERIZATION OF AKR1B10 FROM HUMAN LIVER: ROLE IN CARBONYL REDUCTION OF XENOBIOTICS

Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Kiel, Germany (H.-J.M., S.B., E.M.); Department of Pharmacology and Toxicology, University of Tuebingen, Tuebingen, Germany (U.B.-P.); Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovskeho, Hradec Kralove, Czech Republic (V.W.); and Ernst-Moritz-Arndt-University Greifswald, Laboratory for Functional Genomics, Greifswald, Germany (S.V.)

(Received October 25, 2005; Accepted December 21, 2005)

	Abstract

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Members of the aldo-keto reductase (AKR) superfamily have a broad substrate specificity in catalyzing the reduction of carbonyl group-containing xenobiotics. In the present investigation, a member of the aldose reductase subfamily, AKR1B10, was purified from human liver cytosol. This is the first time AKR1B10 has been purified in its native form. AKR1B10 showed a molecular mass of 35 kDa upon gel filtration and SDS-polyacrylamide gel electrophoresis. Kinetic parameters for the NADPH-dependent reduction of the antiemetic 5-HT₃ receptor antagonist dolasetron, the antitumor drugs daunorubicin and oracin, and the carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) to the corresponding alcohols have been determined by HPLC. K_m values ranged between 0.06 mM for dolasetron and 1.1 mM for daunorubicin. Enzymatic efficiencies calculated as k_cat/K_m were more than 100 mM^–1 min^–1 for dolasetron and 1.3, 0.43, and 0.47 mM^–1 min^–1 for daunorubicin, oracin, and NNK, respectively. Thus, AKR1B10 is one of the most significant reductases in the activation of dolasetron. In addition to its reducing activity, AKR1B10 catalyzed the NADP⁺-dependent oxidation of the secondary alcohol (S)-1-indanol to 1-indanone with high enzymatic efficiency (k_cat/K_m = 112 mM^–1 min^–1). The gene encoding AKR1B10 was cloned from a human liver cDNA library and the recombinant enzyme was purified. Kinetic studies revealed lower activity of the recombinant compared with the native form. Immunoblot studies indicated large interindividual variations in the expression of AKR1B10 in human liver. Since carbonyl reduction of xenobiotics often leads to their inactivation, AKR1B10 may play a role in the occurrence of chemoresistance of tumors toward carbonyl group-bearing cytostatic drugs.

AKR1B10 belongs to the aldose reductases (AKR1B subfamily) and was discovered as an enzyme being overexpressed in human liver cancers and in the search for aldose reductase homologs (Jez et al., 1997; Cao et al., 1998; Hyndman and Flynn, 1998; Scuric et al., 1998). It shares 70% identity with human aldose reductase AKR1B1. Expression levels are high in human small intestine and adrenal gland and lower in all other tissues tested so far (Cao et al., 1998; Hyndman and Flynn, 1998). Recent observations suggest that its physiological substrates are retinal isomers (Crosas et al., 2003). In previous studies, we purified oxidoreductases from human liver cytosol catalyzing redox reactions of various substrates (Atalla et al., 2000; Breyer-Pfaff and Nill, 2000). Based on this procedure, we could now purify AKR1B10 in an active state and identify its nature by ESI-MS/MS analysis. To investigate the role of this comparatively new member of the AKR family in the carbonyl reduction of xenobiotics, we overexpressed AKR1B10 in Escherichia coli and used both the native and recombinant enzymes to characterize enzymatic properties and assess kinetic parameters. Because AKR1B10 is overexpressed in lung cancer (Hyndman and Flynn, 1998; Penning, 2005), we checked the purified enzyme for its ability to accept NNK as substrate. There is strong evidence that NNK plays a major role in the causation of lung cancer in smokers (Hecht, 1998). In addition, the two anticancer drugs daunorubicin, which is, among others, used in the treatment of lung cancer, and oracin, a potential cytostatic drug for oral use, which is, at present, in phase II of clinical trials, were tested for carbonyl reduction. All of the above-mentioned compounds were converted by AKR1B10 to the corresponding alcohols. We also present data for carbonyl reduction of the opioid receptor antagonist naltrexone and the antiemetic 5-HT₃ receptor antagonist dolasetron, which are efficiently reduced in vivo (Gonzalez and Brogden, 1988; Dow and Berg, 1995). Whereas the reduction of naltrexone proceeded slowly, AKR1B10 was among the most efficient reductases of dolasetron characterized so far (Breyer-Pfaff and Nill, 2004).

	Materials and Methods

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The following columns and materials used for protein isolation were purchased from Pharmacia (Freiburg, Germany): Superdex 75 column, 1-ml HiTrap chelating column, Sephadex G-100, Q Sepharose, CM Sepharose, Polybuffer Exchanger PBE 94, and Polybuffer 74. Hydroxyapatite Bio-Gel HTP was obtained from Bio-Rad (Hercules, CA); a 50-ml ultrafiltration chamber and filters YM10 from Amicon (Millipore GmbH, Schwalbach, Germany); and (S)-(+)- and (R)-(–)-1-indanol, glyceraldehyde, NADPH, and glucose 6-phosphate from Sigma-Aldrich (St. Louis, MO). The molecular weight standards were obtained from Sigma-Aldrich and Fermentas GmbH (St. Leon-Rot, Germany). NADP-tetrasodium salt was purchased from AppliChem (Darmstadt, Germany), and glucose-6-phosphate dehydrogenase from yeast, grade I, ca. 350 U/mg, was obtained from Roche Diagnostics (Mannheim, Germany). NNK was supplied by Campro Scientific (Emmerich, Germany). Naltrexone hydrochloride was kindly provided by DuPont Pharma (Bad Homburg, Germany) and dolasetron mesylate hydrate by Hoechst Marion Roussel (now Sanofi-Aventis, Bad Soden, Germany). Reduced dolasetron was prepared according to the method of Dow and Berg (1995) as described previously (Breyer-Pfaff and Nill, 2004). Oracin and dihydrooracin were gifts from the Research Institute for Pharmacy and Biochemistry (Prague, Czech Republic). Daunorubicin was obtained from Rhône-Poulenc (Cologne, Germany), and reduced daunorubicin was obtained from Pharmacia-Farmitalia Carlo Erba (Milan, Italy). Pfu-Polymerase and T4 ligase were purchased from Fermentas, and restriction enzymes from New England Biolabs (Beverly, MA). Bacterial strains used in this study and the pQE-70 vector were obtained from QIAGEN (Hilden, Germany). The human liver cDNA library Uni-ZAp XR was obtained from Stratagene (Heidelberg, Germany). Primers were prepared by MWG (Ebersberg, Germany). Ampicillin and kanamycin were obtained from AGS (Heidelberg, Germany).

Enzyme Isolation and Purification. Human liver samples were provided by the Department of Surgery, University of Tuebingen. They were either excess normal tissue removed on partial hepatectomy for liver metastases or materials excluded from transplantation for medical reasons. The samples were stored at –80°C for 0.5 to 7 years. Homogenates were prepared with 4 volumes of homogenization buffer (250 mM sucrose, 20 mM Tris-HCl, 5 mM EDTA, adjusted to pH 7.4 at 37°C) and cytosol was obtained by differential centrifugation. The enzyme isolation was a modification (Breyer-Pfaff and Nill, 2000) of the method of Hara et al. (1990). Cytosol from 5 to 7 g of liver was concentrated to half its volume by ultrafiltration and fractionated on a 2.5 x 100 cm column with Sephadex G-100 using 25 mM Tris-HCl, pH 7.4. Fractions testing positive for oxidoreductase activity were concentrated with the medium being changed to buffer A (400 ml of 6.25 mM Tris + 0.625 mM EDTA adjusted to pH 8.0 with HCl and 100 ml of glycerol, with 2-mercaptoethanol added to 5 mM before use). In all further purification steps, 2-mercaptoethanol was added to buffers to a concentration of 5 mM before use. The elution rate was 1 ml/min except on chromatofocusing, and fractions of 5 ml or 2.5 ml were tested for enzymatic activity, then combined, concentrated, and stored in buffer A at –80°C.

Three enzymatically active peaks were obtained on elution of a 2.5 x 10 cm column of Q Sepharose with a NaCl gradient in buffer A: 0 M (0–100 min), linear gradient to 0.1 M (100–340 min), and linear gradient to 0.5 M (340–440 min). The second peak (Q2), collected from 200 to 320 ml, exhibited (S)-1-indanol oxidase activity. It was applied to a 1 x 13 cm column of CM Sepharose in 10 mM sodium phosphate + 0.5 mM EDTA adjusted to pH 6.5. After 20 min of isocratic elution, a linear gradient was started that attained 0.1 M NaCl within 60 min. Peaks with (S)-1-indanol oxidase activity were eluted at 35 to 80 ml (Q2-CM1) and 80 to 165 ml (Q2-CM2). From the latter peak, AKR1C2 and carbonyl reductase forms I and II were isolated (Breyer-Pfaff and Nill, 2000). The Q2-CM1 concentrate was purified on a 1.1 x 9.5 cm column of Bio-Gel HTP equilibrated with 10 mM Tris-HCl, pH 7.5. Linear gradients were run in which the Tris buffer was replaced by 0.2 M sodium phosphate, pH 6.5. The concentration of phosphate was 0.05 M after 60 min, 0.1 M after 90 min, held at 0.1 M for 30 min, and increased to 0.2 M within 60 min. AKR1B10 activity monitored by oxidation of (S)-1-indanol was eluted at 63 to 105 ml. Final purification was achieved by chromatofocusing on a 0.9 x 56 cm column of Polybuffer Exchanger PBE 94 equilibrated with 25 mM imidazole-HCl, pH 7.4 and 5 mM 2-mercaptoethanol. The column was run with Polybuffer 74/water (1:7 v/v) adjusted to pH 5.0 with HCl. Fractions of 4 ml/30 min were collected, the enzyme being eluted at pH 7.1 in fractions 28 to 32 (108–128 ml).

Nano-Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (Nano-LC-ESI-MS/MS). The purified protein was subjected to SDS-PAGE and stained with Colloidal Coomassie Brilliant Blue according to the manufacturer's instructions (GE Healthcare, Freiburg, Germany). For identification by mass spectrometry, the protein was excised from the gel stained with Colloidal Coomassie Brilliant Blue, washed (two times in 100 µl of 50 mM NH₄HCO₃/50% v/v MeOH, 30 min, 37°C), and dehydrated with 100% acetonitrile for 15 min. After removal of acetonitrile, trypsin solution (10 ng/µl trypsin in 20 mM ammonium bicarbonate) was added until the gel pieces stopped swelling, and digestion was allowed to proceed for 16 to 18 h at 37°C. For peptide extraction, gel pieces were subsequently covered with 15 µl of 1% (v/v) formic acid and incubated in an ultrasonic bath for 20 min. A second peptide extraction was done with 20 µl of 50% (v/v) acetonitrile. Both peptide-containing supernatants were transferred into micro-vials for mass spectrometric analysis. High pressure liquid chromatography separation was performed on an Ultimate system (LC Packings, Amsterdam, The Netherlands). The system was coupled via a nano-LC inlet (New Objective, Woburn, MA) to the Q-TOF mass spectrometer (Q-Star Pulsar i; Applied Biosystems, Foster City, CA) equipped with a nano-electrospray source (Protana, Odense, Denmark). Peptide chromatography and tandem mass spectrometry were performed according to the method of Eymann et al. (2004). The resulting MS/MS data were analyzed with the Bioanalyst Software (Applied Biosystems) and the integrated Mascot script. For database searches, the Mascot search engine (Matrix Science Ltd., London, UK) was used with a human sequence database. Proteins were regarded as positively identified when a molecular weight search score of 53, corresponding at least to a p value of 0.05, was obtained.

Gel Permeation Chromatography. Gel filtration was performed on an Amersham Pharmacia AKTA fast protein liquid chromatography system using a Pharmacia Superdex 75 column. The column was equilibrated with a buffer containing 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 0.2 mM 2-mercaptoethanol, and 10% glycerol. Elution of 12 µg of native AKR1B10 was detected by recording the absorption at 280 nm. The standard curve was generated by using the low molecular weight calibration kit from Amersham Pharmacia.

Cloning and Expression of AKR1B10 in E. coli. The coding sequence of AKR1B10 (National Center for Biotechnology Information Reference Sequence accession number NM_020299 [GenBank] ) was amplified from a human liver cDNA library (Stratagene) by PCR using primers of the following sequence: forward primer, ATGGCCACGTTTGTGGAG; reverse primer, CCTCAATATTCTGCATCGAAGG. PCR was performed with Pfu-Polymerase in 30 cycles with an annealing temperature of 52°C. In a second PCR, BgI II restriction sites were added using appropriate primers, and the resulting construct was ligated into the pQE-70 (QIAGEN) vector such that the protein was translated in frame from the vector's translation start site resulting in a protein containing a C-terminal His-tag. For expression of AKR1B10 protein, SG13009 cells (QIAGEN) were transformed with the expression plasmid, and a 200-ml culture containing ampicillin (100 µg/ml) and kanamycin (25 µg/ml) was grown to OD₆₀₀ = 0.6 at 37°C. Expression was induced by supplementing the culture medium with 1 mM isopropyl-1-thio-galactopyranoside. Cells were harvested after 3 h by centrifugation (6000g, 10 min) and resuspended in 20 ml of Tris buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM imidazole) before lysation in a French-pressure cell. The resulting suspension was centrifuged for 1 h at 100,000g at 4°C, and the supernatant was either stored at –80°C or directly used for Ni-affinity chromatography.

Purification of Recombinant AKR1B10. Purification of AKR1B10 was achieved by fast protein liquid chromatography (AKTA-Purifier; Amersham Pharmacia) on a 1-ml Ni²⁺ HisTrap-FF column. A linear gradient of 10 mM to 500 mM imidazole in 20 mM NaH₂PO₄, 100 mM NaCl, 10% glycerol, pH 7.4 was used. AKR1B10 was eluted from the column at an imidazole concentration of approximately 200 mM. Protein-containing fractions were analyzed by SDS-PAGE. Samples containing pure AKR1B10 were pooled and 5x concentrated by ultrafiltration, thereby exchanging the elution buffer with 10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 20% glycerol, 5 mM 2-mercaptoethanol. Purified protein was stored in this buffer at –80°C until further use.

Immunoblotting. Antisera against full-length AKR1B10 were prepared by Eurogentec (Cologne, Germany) by using recombinant protein produced in our laboratory. Electroblotting was performed in a semidry blotting system. Proteins were transferred on a nitrocellulose membrane, and antigen-antibody complexes were visualized by chemoluminescence (ECL Plus detection kit; GE Healthcare, Munich, Germany). Primary antibodies were diluted 1/1000; the secondary antibody (peroxidase-conjugated swine anti-rabbit immunoglobulin; DakoCytomation, Glostrup, Denmark) was used in a 1/10,000 dilution.

Enzymatic Tests. Oxidoreductase activities in gel filtration eluates were monitored by HPLC measurement of (Z)-10-hydroxynortriptyline production from (Z)-10-oxonortriptyline (Breyer-Pfaff and Nill, 2000). Alternatively, naltrexone reduction by aliquots of the protein fractions was assayed spectrophotometrically. In further purification steps, NADPH generation with (S)-1-indanol as substrate served for activity testing. In a total volume of 0.6 ml, samples were incubated at 37°C with 100 mM Tris-HCl pH 7.4, 8 mM MgCl₂, 25 mM KCl, and either 0.1 mM NADPH or 0.25 mM NADP⁺. The decrease or increase of absorption at 339 nm in the presence of 0.1 mM naltrexone or 0.5 mM (S)-1-indanol, respectively, was registered for 5 to 10 min. Under the same conditions, (R)-1-indanol oxidation and DL-glyceraldehyde reduction were measured.

Enzyme Kinetics. Dolasetron reduction kinetics were determined by incubating 0.01 to 0.3 mM substrate with 1 µg of purified AKR1B10 in 0.36 ml of the above buffer containing 0.2 mM NADP⁺, 2 mM glucose 6-phosphate, and 1 U/ml glucose-6-phosphate dehydrogenase for NADPH generation. After 20 min at 37°C, the reaction was stopped by adding 40 µl of 2 M perchloric acid and cooling on ice. Reduced dolasetron was analyzed in the supernatant by HPLC (Breyer-Pfaff and Nill, 2004). In analogous fashion, naltrexone reduction was checked in a few samples. NNK (0.05–1.5 mM), oracin (0.02–0.75 mM), and daunorubicin (0.15–2 mM) reduction kinetics were assayed with 2.72, 1.36, and 0.68 µg of purified AKR1B10, respectively, in a total volume of 0.1 ml in 0.1 M sodium phosphate buffer, pH 7.4, and an NADPH-generating system (final concentrations: NADP⁺, 3 mM; glucose 6-phosphate, 20 mM; glucose-6-phosphate dehydrogenase, 1.8 units/0.1 ml; MgCl₂, 10 mM). Incubation times were 60, 30, and 10 min, respectively. Increase of product concentration was linear during the given periods of time. Reactions were stopped by the addition of 40 µl of 30% ammonia and cooling on ice. The mixtures were extracted twice with 300 µl of ethyl acetate (three times in the case of daunorubicin), and the combined organic phases were evaporated under vacuum. The residue was dissolved in the respective mobile phase and analyzed by HPLC as described previously (Wsol et al., 1996; Breyer-Pfaff et al., 2004). For daunorubicinol detection, a modified method of Fogli et al. (1999) was used (mobile phase, 50 mM sodium phosphate/acetonitrile (75:25), pH 4.0; flow rate, 1.5 ml/min). (S)-1-Indanol (0.25–4.0 mM) oxidation kinetics was assayed spectrophotometrically as described above. For the determination of kinetic constants, the results were analyzed by least-squares nonlinear fitting of a Michaelis-Menten hyperbola or the Hill equation (GraphPad Prism; GraphPad Software Inc., San Diego, CA).

	Results

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In the search for enzymes participating in the reduction of xenobiotic ketones, a purification scheme was developed which led to the simultaneous purification of the three aldo-keto reductases AKR1C1, -1C2, and -1C4, and the short-chain dehydrogenase carbonyl reductase (Breyer-Pfaff and Nill, 2000). Monitoring (S)-1-indanol oxidation activity led to the isolation of an additional enzyme which we could now unequivocally identify as AKR1B10 by ESI-MS/MS analysis (Table 1). The purified protein proved to be homogeneous on SDS-PAGE. The estimated molecular mass corresponded to that of the primary structure deduced from the cDNA sequence and was the same as that of AKR1C1 and AKR1C2 (34–36 kDa; Fig. 1A). A molecular mass of 35 kDa for AKR1B10 was also found by size-exclusion chromatography (Fig. 2).

View larger version (39K): [in this window] [in a new window]   FIG. 1. SDS-PAGE of purified aldo-keto reductases (Coomassie stain). A, the purification of aldo-keto reductases AKR1B10, AKR1C1, and AKR1C2 from human liver cytosol yielded single products with molecular masses of approximately 35 kDa. B, recombinant AKR1B10 was obtained by overexpression in E. coli. Cells were disrupted in a French-pressure cell and soluble proteins were subjected to Ni-affinity chromatography (see Materials and Methods). Lane assignments are as follows: M, marker proteins; 1, soluble protein fraction; 2, flowthrough fraction; 3, wash fraction; 4; proteins eluted with low (ca. 10–60 mM) imidazole concentrations; 5, AKR1B10 eluted with ca. 200 mM imidazole. The molecular mass of the recombinant protein (including His-tag) is 37.8 kDa in comparison to 36.0 kDa calculated from the primary structure of the native enzyme.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Gel filtration of 12 µg of native AKR1B10. The molecular mass (on the logarithmic scale) was plotted against the corresponding Kav value for each protein standard derived from its elution pattern (measured by the absorption at 280 nm). The standard proteins (ribonuclease A, 13.7 kDa; chymotrypsinogen A, 25 kDa; ovalbumin, 43 kDa; bovine serum albumin, 67 kDa) are represented by open circles. The Kav of AKR1B10 (black diamond) was located on the calibration curve and the molecular mass was calculated as 35 kDa.

The coding sequence of human AKR1B10 was obtained by PCR from a human liver cDNA library. After ligation into an expression vector, the protein was overexpressed in E. coli cells and purified in an active state by metal chelate chromatography (Fig. 1B). The purified recombinant enzyme was soluble and could be concentrated to at least 16 mg/ml without precipitation.

Western blot of human liver cytosol probed with antibodies raised against recombinant AKR1B10 showed the presence of the enzyme in only two of five livers (Fig. 3). This finding indicates interindividual variation in the expression of AKR1B10 in human tissues.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Western blot of human liver cytosol and E. coli cells overexpressing AKR1B10. An amount of 20 µg of cytosolic proteins, prepared from five different human liver samples (lanes 1–5), and soluble proteins from E. coli cells overexpressing AKR1B10 (lane 6) were loaded. Only in two of five cytosolic fractions is a signal at 36 kDa detectable (lanes 1 and 5).

View larger version (23K): [in this window] [in a new window]   FIG. 4. Kinetics of oracin, daunorubicin, and dolasetron carbonyl reduction and (S)-1-indanol oxidation by native AKR1B10. Reduced reaction products were quantified by HPLC or, in the case of (S)-1-indanol oxidation, the increase in absorbance at 339 nm was measured. Individual data points or means of four independent experiments ± S.D. are shown. A, reduction of oracin by AKR1B10 displays Michaelis-Menten kinetics with substrate inhibition. B, kinetic data for daunorubicin reduction by AKR1B10 are significantly (P = 0.0013) better fitted by the Hill equation than by the Michaelis-Menten hyperbola. The inset shows the deviation from linearity when the same data are graphed in a Hanes-Woolf plot. C, oxidation of (S)-1-indanol by native AKR1B10 (open circles) and recombinant AKR1B10 expressed in E. coli (filled squares). D, reduction of dolasetron by AKR1B10.

	Discussion

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A modification of the original purification scheme of Hara et al. (1990), consisting of several chromatographic steps, allowed for the simultaneous isolation of the three aldo-keto reductases AKR1C1, -1C2, and -1C4, and the short-chain dehydrogenase/reductase carbonyl reductase from human liver cytosol (Breyer-Pfaff and Nill, 2000). An additional fraction could be isolated irregularly and shown to contain a protein with (S)-1-indanol oxidizing activity. This protein was now identified by ESI-MS/MS as the aldo-keto reductase AKR1B10. This is the first time that this relatively new member of the aldo-keto reductase superfamily has been purified from human tissue in its native state. AKR1B10 mRNA levels were reported to be high in small intestine, colon, and adrenal gland, but only moderate amounts were found in liver (Cao et al., 1998; Hyndman and Flynn, 1998). This is in accordance with the occasional failure to detect a peak of enzymatic activity when cytosol of different livers was subjected to the purification procedure. Interindividual variability in AKR1B10 expression was also shown in the present study by Western blot analysis, where no AKR1B10 protein was detected in three of five human livers tested. To obtain a larger enzyme quantity, we cloned AKR1B10 in bacteria and isolated the recombinant protein by metal chelate chromatography. Native and recombinant enzyme forms were not identical regarding their kinetic parameters. Catalytic efficiencies of the native enzyme were 1.6 to 20 times higher compared with the recombinant form, depending on the substrate. At the moment, we cannot tell whether this loss of activity (Table 3) is due to adverse effects of the additional amino acids derived from the cloning procedure, suboptimal purification conditions, or a lack of post-translational modifications.

To assess the importance of AKR1B10 in the detoxification of xenobiotics, we determined the kinetic parameters of several carbonyl group-containing compounds. Recently, it was shown that AKR1B10 is a very efficient retinal reductase (Crosas et al., 2003). Published data indicate that the preferred substrates for this enzyme are aldehydes (Cao et al., 1998; Crosas et al., 2003), but both the native and the recombinant enzyme reduced the carbonyl group of various ketones (Table 3). In addition to its well documented function as a reductase, AKR1B10 oxidized indanol in the presence of NADP⁺ in vitro. The reaction rate with (S)-1-indanol was similar to that of AKR1C2 and AKR1C4, and about half that of AKR1C1 (Breyer-Pfaff and Nill, 2000). AKR1B10 exhibited an approximately 10-fold lower activity toward (R)-1-indanol (Table 2). The opiate receptor antagonist naltrexone was reduced by AKR1B10 very slowly, as was also found for AKR1C1 and AKR1C2 (Breyer-Pfaff and Nill, 2004).

In contrast, AKR1B10 is one of the most active reductases toward dolasetron, a 5-HT₃ receptor antagonist used as an antiemetic drug, which is efficiently reduced to its corresponding alcohol in vivo. The k_cat/K_m value exceeded 100 mM^–1 min^–1 in three independent kinetic experiments (Table 3). The enzymatic efficiency is at least as high as that of AKR1C1 and superior to that of AKR1C2 in dolasetron reduction (Breyer-Pfaff and Nill, 2004). Reduced dolasetron is the active metabolite with a half-life (t_1/2) of 7 to 8 h, whereas a t_1/2 of 0.13 to 0.24 h is observed for the parent carbonyl compound (Gan, 2005, and references cited therein). Carbonyl reductase was held responsible for this rapid elimination, because conversion to the alcohol also took place in whole blood (Dow and Berg, 1995). In view of its widespread tissue distribution (Wirth and Wermuth, 1992), carbonyl reductase may in fact have a role, but its k_cat/K_m value in dolasetron reduction is only about one-third that of AKR1B10 and AKR1C1 (Breyer-Pfaff and Nill, 2004). Participation of AKR1B10 in first-pass reduction of dolasetron may be of importance if the enzyme is highly expressed in human small intestine as reported by Cao et al. (1998) and as reflected by its original name, "human small intestine reductase" (Hyndman and Flynn, 1998). Although the reduction of dolasetron is the prerequisite for its binding to the receptor and the resulting antiemetic effect, the reduction of carbonyl groups in cytostatic drugs often leads to their inactivation. We found that the anthracycline derivative daunorubicin and the novel anticancer drug oracin, which is currently in phase II of clinical trial, both were substrates for AKR1B10, albeit both were reduced with much lower enzymatic efficiency than dolasetron (Table 3). Surprisingly, daunorubicin reduction kinetics showed no Michaelis-Menten hyperbola, but a sigmoidal pattern that was fitted by the Hill equation with a Hill coefficient of n = 1.5. Hill coefficients >1 indicate a cooperative mode of action that is typical for multimeric enzymes. Nonetheless, size-exclusion chromatography with the native enzyme revealed the expected molecular mass of the monomer (found, 35 kDa; calculated, 36 kDa; Fig. 2). Although some multimeric aldo-keto reductases are known, most members of this superfamily are monomers. Daunorubicin is mainly reduced in the cytosol by carbonyl reductase (k_cat/K_m = 64 mM^–1 min^–1) and, to a lesser extent, by AKR1C2 and AKR1A1 [previously designated as dihydrodiol dehydrogenase 2, DD2, and aldehyde reductase, ALR, respectively, in Ohara et al. (1995)]. In comparison, the catalytic efficiencies of AKR1B10 for daunorubicin (k_cat/K_m = 1.3 mM^–1 min^–1) and oracin (k_cat/K_m = 0.43 mM^–1 min^–1) are low. As detailed above, AKR1B10 expression is low or absent in normal liver. On the other hand, it was easily detectable in all five hepatocellular carcinoma tissues tested, whereas mRNA levels in nontumor samples were minimal (Scuric et al., 1998). Analogously, no or low AKR1B10 expression was found in human lung (Cao et al., 1998; Hyndman and Flynn, 1998); but, recently, Fukumoto et al. reported on the overexpression of AKR1B10 in smokers' non-small-cell lung carcinomas and suggested it as a diagnostic marker (Fukumoto et al., 2005; Penning, 2005). Because daunorubicin is used in the treatment of this disease, its carbonyl reduction (i.e., deactivation) by AKR1B10 may become a critical determinant in chemotherapy (Soldan et al., 1999; Ax et al., 2000; Lee et al., 2001).

The overexpression of AKR1B10 in squamous cell carcinoma of the lung raises the question whether inhibition of this enzyme might be beneficial for patients, inasmuch as the physiological role of AKR1B10 is not yet fully understood. Our results show that, on the one hand, AKR1B10 may contribute to drug resistance in cancer chemotherapy. On the other hand, one of the most potent lung carcinogens in tobacco smoke, NNK, is also reduced to the corresponding alcohol 4-methylnitrosamino-1-(3-pyridyl)-1-butanol by this enzyme. This reduction constitutes the first step in the major detoxification pathway of NNK in humans and can be followed by conjugation with glucuronic acid. In human lung and liver cytosol, mostly aldo-keto reductases of the AKR1C family and carbonyl reductase of the SDR superfamily contribute to the reduction of NNK (Atalla et al., 2000; Breyer-Pfaff et al., 2004). In cancer tissues, where AKR1B10 may obviously be overexpressed manyfold, the contribution of individual enzymes to NNK reduction has yet to be determined.

In conclusion, we purified AKR1B10 from human liver in an active state and cloned it from a cDNA library. The enzyme proved to be an efficient reductase of the antiemetic prodrug dolasetron to the corresponding alcohol, a high-affinity 5-HT₃ receptor blocker. Two anticancer drugs, daunorubicin and oracin, and the tobacco-specific carcinogen NNK were found to be substrates of the enzyme, although with low catalytic efficiency. In noncancerous human lung tissue, AKR1B10 mRNA is rarely found, and expression levels of AKR1B10 in human liver are low and show high interindividual variability, as revealed by Western blot analysis. Since AKR1B10 is overexpressed in human non-small-cell lung carcinoma and human hepatocellular carcinoma, it may become an important factor in drug resistance against carbonyl group-bearing antitumor drugs in these tissues.

	Footnotes

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (BR 478/14-1; MA 1704/3-2; MA 1704/3-3) and the Alexander von Humboldt Foundation.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.007971.

ABBREVIATIONS: AKR, aldo-keto reductase; EDTA, EDTA disodium salt; HT, hydroxytryptamine; PAGE, polyacrylamide gel electrophoresis; ESI-MS/MS, electrospray ionization-tandem mass spectrometry; NNK, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; SDR, short-chain dehydrogenase/reductase.

Address correspondence to: Edmund Maser, Institute of Toxicology and Pharmacology for Natural Scientists, D-24105 Kiel, Brunswikerstr. 10, Germany. E-mail: maser{at}toxi.uni-kiel.de

	References

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Atalla A, Breyer-Pfaff U, and Maser E (2000) Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol. Xenobiotica 30: 755–769.[CrossRef][Medline]

Ax W, Soldan M, Koch L, and Maser E (2000) Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction. Biochem Pharmacol 59: 293–300.[CrossRef][Medline]

Bohren KM, Bullock B, Wermuth B, and Gabbay KH (1989) The aldo-keto reductase superfamily. cDNAs and deduced amino acid sequences of human aldehyde and aldose reductases. J Biol Chem 264: 9547–9551.[Abstract/Free Full Text]

Breyer-Pfaff U and Nill K (2000) High-affinity stereoselective reduction of the enantiomers of ketotifen and of ketonic nortriptyline metabolites by aldo-keto reductases from human liver. Biochem Pharmacol 59: 249–260.[CrossRef][Medline]

Breyer-Pfaff U and Nill K (2004) Carbonyl reduction of naltrexone and dolasetron by oxidoreductases isolated from human liver cytosol. J Pharm Pharmacol 56: 1601–1606.[CrossRef][Medline]

Breyer-Pfaff U, Martin HJ, Ernst M, and Maser E (2004) Enantioselectivity of carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone by tissue fractions from human and rat and by enzymes isolated from human liver. Drug Metab Dispos 32: 915–922.[Abstract/Free Full Text]

Cao D, Fan ST, and Chung SS (1998) Identification and characterization of a novel human aldose reductase-like gene. J Biol Chem 273: 11429–11435.[Abstract/Free Full Text]

Crosas B, Hyndman DJ, Gallego O, Martras S, Pares X, Flynn TG, and Farres J (2003) Human aldose reductase and human small intestine aldose reductase are efficient retinal reductases: consequences for retinoid metabolism. Biochem J 373: 973–979.[CrossRef][Medline]

Dow J and Berg C (1995) Stereoselectivity of the carbonyl reduction of dolasetron in rats, dogs and humans. Chirality 7: 342–348.[Medline]

Eymann C, Dreisbach A, Albrecht D, Bernhardt J, Becher D, Gentner S, Tam le T, Buttner K, Buurman G, Scharf C, et al. (2004) A comprehensive proteome map of growing Bacillus subtilis cells. Proteomics 4: 2849–2876.[CrossRef][Medline]

Fogli S, Danesi R, Innocenti F, Di Paolo A, Bocci G, Barbara C, and Del Tacca M (1999) An improved HPLC method for therapeutic drug monitoring of daunorubicin, idarubicin, doxorubicin, epirubicin and their 13-dihydro metabolites in human plasma. Ther Drug Monit 1: 367–375.

Fukumoto S, Yamauchi N, Moriguchi H, Hippo Y, Watanabe A, Shibahara J, Taniguchi H, Ishikawa S, Ito H, Yamamoto S, et al. (2005) Overexpression of the aldo-keto reductase family protein AKR1B10 is highly correlated with smokers' non-small cell lung carcinomas. Clin Cancer Res 11: 1776–1785.[Abstract/Free Full Text]

Gan TJ (2005) Selective serotonin 5-HT3 receptor antagonists for postoperative nausea and vomiting: are they all the same? CNS Drugs 19: 225–238.[CrossRef][Medline]

Gonzalez JP and Brogden RN (1988) Naltrexone. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficacy in the management of opioid dependence. Drugs 35: 192–213.[Medline]

Hara A, Taniguchi H, Nakayama T, and Sawada H (1990) Purification and properties of multiple forms of dihydrodiol dehydrogenase from human liver. J Biochem (Tokyo) 108: 250–254.[Abstract/Free Full Text]

Hecht SS (1998) Biochemistry, biology and carcinogenicity of tobacco-specific N-nitrosamines. Chem Res Toxicol 11: 559–603.[CrossRef][Medline]

Hyndman D, Bauman DR, Heredia VV, and Penning TM (2003) The aldo-keto reductase superfamily homepage. Chem-Biol Interact 143–144: 621–631.

Hyndman DJ and Flynn TG (1998) Sequence and expression levels in human tissues of a new member of the aldo-keto reductase family. Biochim Biophys Acta 1399: 198–202.[Medline]

Jez JM, Flynn TG, and Penning TM (1997) A new nomenclature for the aldo-keto reductase superfamily. Biochem Pharmacol 54: 639–647.[CrossRef][Medline]

Lee KW, Ko BC, Jiang Z, Cao D, and Chung SS (2001) Overexpression of aldose reductase in liver cancers may contribute to drug resistance. Anticancer Drugs 12: 129–132.[CrossRef][Medline]

Ohara H, Miyabe Y, Deyashiki Y, Matsuura K, and Hara A (1995) Reduction of drug ketones by dihydrodiol dehydrogenases, carbonyl reductase and aldehyde reductase of human liver. Biochem Pharmacol 50: 221–227.[CrossRef][Medline]

Penning TM (2004) Introduction and overview of the aldo-keto reductase superfamily, in Aldo-Keto Reductases and Toxicant Metabolism (Penning TM and Petrash JM eds) pp 3–21, American Chemical Society, Washington DC.

Penning TM (2005) AKR1B10: a new diagnostic marker of non-small cell lung carcinoma in smokers. Clin Cancer Res 11: 1687–1690.[Free Full Text]

Scuric Z, Stain SC, Anderson WF, and Hwang JJ (1998) New member of aldose reductase family proteins overexpressed in human hepatocellular carcinoma. Hepatology 27: 943–950.[CrossRef][Medline]

Soldan M, Ax W, Plebuch M, Koch L, and Maser E (1999) Cytostatic drug resistance. Role of phase-I daunorubicin metabolism in cancer cells. Adv Exp Med Biol 463: 529–538.[Medline]

Wirth H and Wermuth B (1992) Immunohistochemical localization of carbonyl reductase in human tissues. J Histochem Cytochem 40: 1857–1863.[Abstract]

Wsol V, Kvasnickova E, Szotakova B, and Hais IM (1996) High-performance liquid chromatographic assay for the separation and characterization of metabolites of the potential cytostatic drug oracine. J Chromatogr B 681: 169–175.[Medline]

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