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Divisions of Pharmaceutics and Biopharmaceutics (R.H.T., O.S.B., K.W.R.) and Biomolecular and Pharmaceutical Chemistry (R.E.R.), Faculty of Pharmaceutical Sciences; and Life Sciences Institute, Department of Zoology, Faculty of Science, University of British Columbia, Vancouver, British Columbia (T.A.P., T.A.G.)
(Received January 4, 2008; accepted February 27, 2008)
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Abstract |
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). Unfortunately, the widespread use of the anthracyclines is limited by the development of dose-dependent chronic cardiotoxicity in patients. This side effect manifests as an irreversible disorder following long-term treatment, with approximately 20% of cases leading to congestive heart failure (Singal and Iliskovic, 1998). Although the mechanism by which this side effect develops is not yet identified, evidence implicates metabolism as an important factor (Olson and Mushlin, 1990). Following DOX or DAUN administration, the C-13 alcohol metabolites, DOXol and DAUNol, are the major identified metabolites (Takanashi and Bachur, 1976), and these metabolites are speculated to be responsible for causing cardiac tissue damage (Del Tacca et al., 1985; Boucek et al., 1987; Cusack et al., 1993; Behnia and Boroujerdi, 1999; Olson et al., 2003; Sacco et al., 2003).
Formation of DOXol and DAUNol is mediated by cytosolic oxidoreductase enzymes of the AKR and short-chain dehydrogenase/reductase families, with the AKR1A1, AKR1B10, AKR1C2, and carbonyl reductase enzymes identified as major catalysts (Licata et al., 2000; Martin et al., 2006; Jin and Penning, 2007; Oppermann, 2007). Inhibition of the anthracycline-metabolizing enzymes is suggested as a strategy for avoiding treatment-related cardiotoxicity (Behnia and Boroujerdi, 1999), and chemical inhibitors for reductases and anthracycline derivatives that are metabolically less labile have been explored for their potential roles as safer alternatives to the currently used anthracycline drug treatments. In part, the success of these strategies relies on accurately identifying the enzymes responsible for anthracycline metabolism.
In this paper, we characterize the involvement of two recombinant aldo-keto reductase enzymes, AKR1A1 and AKR1C2, in mediating the in vitro production of DAUNol and DOXol from DAUN and DOX using a specific and sensitive high-performance liquid chromatography (HPLC)-fluorescence assay for direct measurement of the alcohol metabolites.
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Materials and Methods |
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Cloning of Human AKR1A1 and 1C2. The AKR1A1 and AKR1C2 genes were subcloned from lab vector constructs (p2ZOp2N-AKR1A1 and p2ZOp2N-AKR1C2) using standard molecular cloning techniques to the prokaryotic expression vector pET28a (Novagen, Madison, WI) as previously described (Bains et al., 2008). The expression constructs encoded an AKR with a six-histidine affinity tag separated by a 23-amino acid residue linker attached to the amino terminus of the expressed protein.
Expression of Recombinant Enzymes. The AKR proteins were expressed and purified from Escherichia coli cultures. Briefly, pET28a-AKR constructs were transformed by heat shock into BL21 (DE3) pLysS and grown in 500 ml of low-salt Luria broth media supplemented with 50 µg/ml kanamycin sulfate and 25 µg/ml chloramphenicol at 37°C. Expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside administration to a final concentration of 1 mM, and the cultures were grown for a further 3 h. Bacterial pellets were collected by centrifugation (4000 rpm for 20 min at 4°C), then resuspended at 5 ml/g wet weight with 300 mM NaCl and 50 mM NaH2PO4, pH 8.0. Lysis of the cell suspensions and purification of the AKR proteins using nickel-nitrilotriacetic acid agarose were carried out according to the manufacturer's recommendations (QIAGEN, Mississauga, Ontario). Glycerol was added to the tagged purified protein to a final concentration of 20%, and the samples were stored at –20°C.
The recombinant proteins were detected by Western blot analysis using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) with the primary monoclonal AKR1A1 and AKR1C2 antibodies (Abnova Corporation, Taipei City, Taiwan) diluted 1:5000 and 1:3000, respectively, and secondary IRDye 800CW goat anti-mouse IgG antibody (LI-COR Biosciences), diluted 1:5000. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard (Bio-Rad, Hercules, CA). Protein purity was assessed by SDS-PAGE stained with Sypro Ruby (Invitrogen Canada, Inc., Burlington, ON, Canada).
Measurement of AKR Enzyme Activities. AKR activity was measured by monitoring the initial rate of NADP(H) oxidation/reduction reactions using standard conditions (Penning, 2004). Fluorescence measurements of reduced cofactor were made with excitation and emission wavelengths of 355 and 460 nm, respectively, using a Fluoroskan Ascent FL (Thermo Fisher Scientific Co.). The assays were carried out with 200 to 275 ng of purified protein, cofactor (180 µM NADPH for AKR1A1, 2.3 mM NADP+ for AKR1C2), and 1 mM test substrate (p-nitrobenzaldehyde or DL-glyceraldehyde for AKR1A1, 1-acenaphthenol for AKR1C2) in a reaction mixture of 150 µl of 100 mM potassium phosphate, pH 7, at 25°C. Maximal rates (minutes–1) were calculated from the Ascent program (version 2.6; Thermo Scientific) using a 5-min interval. Enzymatic activities (micromoles of cofactor consumed per minute per milligram of purified protein) were calculated based on a standard curve constructed from fluorescence measurements for known NADPH concentrations. Organic solvents at a concentration below 4% (v/v) in the final mixture were used to solubilize some substrates and were not observed to affect enzyme function. All incubations were conducted in quadruplicate for each purified protein preparation.
Measurement of Anthracycline Reduction. Identification and quantification of DOXol and DAUNol were performed using HPLC (Waters Alliance 2695; Waters, Milford, MA) and fluorescence detection with excitation and emission wavelengths of 460 and 550 nm, respectively (Waters 2475 Multi 
Detector). Quantification was based on peak area ratio of DOXol to the internal standard. Linear calibration was determined over the concentration range 25 to 500 nM using a 1/x2 weighting scheme. Because an authentic chemical standard for DAUNol could not be obtained, DAUNol was quantified as DOXol equivalents using a response ratio of 1.0. The limit of quantification for DOXol was estimated as 25 nM. All data processes were performed with Waters Empower software (version 2.0).
Purified tagged AKR1A1 and AKR1C2 proteins were incubated in 150-µl reaction mixtures containing 25 mM KH2PO4, pH 7.4, and 1 µM DOX or DAUN at 37°C. Reaction conditions were selected to reflect a human physiological environment. Reactions were initiated with the addition of 1 mM NADPH. Incubations were conducted with up to 1 µg of purified protein for 120 and 240 min for DAUN and DOX, respectively, to maximize metabolite generation given the apparent low activity of these enzymes. At the end of the incubations, reductase activity was stopped with the addition of 150 µlof ice-cold acetonitrile that contained idarubicin as an internal standard. Protein was removed from the sample by vortex mixing and centrifugation at 10,000g for 10 min at 4°C, and the supernatant was removed for HPLC analysis. All incubations were conducted in triplicate for each purified protein preparation.
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Results and Discussion |
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41–42 kDa) (Fig. 1). Total protein staining of an SDS-PAGE gel demonstrated that the only detectable bands for the purified preparations corresponded to the AKR1A1 and AKR1C2 proteins identified by Western blotting (Fig. 1).
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Activities of the recombinant AKR1A1 using the test substrates p-nitrobenzaldehyde and DL-glyceraldehyde were 5.39 ± 0.35 and 1.56 ± 0.16 µmol/min/mg purified protein, respectively, whereas the activity of the recombinant AKR1C2 using 1-acenaphthenol was 2.23 ± 0.08 µmol/min/mg. These activities are in good agreement with data reported for purified recombinant and native proteins by other laboratories: AKR1A1, p-nitrobenzaldehyde, 6.0 µmol/min/mg (Palackal et al., 2001); DL-glyceraldehyde, 1.26 µmol/min/mg (O'Connor et al., 1999); and AKR1C2, 1-acenaphthenol, 2.5 µmol/min/mg (Burczynski et al., 1998), providing confidence that the recombinant proteins have retained full reductase function.
Formation of DAUNol and DOXol by AKR1A1 and absence of metabolite generation by AKR1C2 were measured by HPLC-fluorescence (Fig. 2). The C-13 metabolites were positively identified by identical chromatographic retention times to an authentic chemical standard of DOXol and to the metabolites generated in incubations with human liver cytosolic fractions. Mordente et al. (2003) measured the formation of DAUNol and DOXol by human cardiac cytosol reductases with average rates of 27.5 and 2.3 pmol/min/mg protein, respectively. The high substrate specificity of AKR1A1 was also demonstrated in our results from recombinant enzymes where DAUN was metabolized to a much greater extent than DOX. The levels of DOXol generated were too low for quantification using the HPLC assay, whereas specific reductase activities of 1.71 ± 0.09 nmol/min/mg were determined for DAUN reduction.
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Our findings of a lack of AKR1C2 involvement in DOX and DAUN metabolism differ from previously published results (Ohara et al., 1995). This discrepancy may be due to contaminating proteins in previous enzyme preparations or nonspecific measurement of reductase activities. SDS-PAGE gel staining for our enzyme preparations shows absence of any detectable contaminating proteins following affinity chromatography. Sypro Ruby total protein staining provides detection of proteins at 1 to 2 ng (Berggren et al., 2000); therefore, we estimate protein contamination in our preparations at less than 0.01%. Monitoring NADPH absorbance is the standard assay to characterize reductase enzyme function; however, this method cannot distinguish contaminating protein- or nonenzyme-catalyzed cofactor usage, potentially leading to erroneous identification of reductase function or substrate use. In our attempts to use the spectroscopic method to measure cofactor usage in AKR1C2-catalyzed DOX and DAUN metabolism, we could not distinguish enzyme- and nonenzyme-catalyzed signal decrease with substrate concentrations of 10, 50, and 250 µM (data not shown). To specifically measure AKR activities for the anthracycline drugs DOX and DAUN, we have used a direct and sensitive HPLC-fluorescence assay and do not identify any alcohol metabolites formed by AKR1C2 that exceed the limit of quantification of the assay (25 nM). In our in vitro system, the initial anthracycline concentrations are 1 µM, a physiologically relevant concentration for patient cardiac tissue (Stewart et al., 1993).
The failure of AKR1C2 to generate alcohol metabolites in vitro suggests that it is unimportant for the generation of DOX or DAUN metabolites in vivo. We recognize that in vivo studies are needed to clearly define the role of AKR1C2 in anthracycline metabolism; however, the conduct of definitive studies will be difficult. Several reductase enzymes are involved in the metabolism of these drugs, and distinguishing their individual contributions to the total metabolism will be a challenge using the traditional approaches of correlation studies and chemical or antibody inhibition. It is with these considerations that we present our in vitro findings to focus future work on anthracycline metabolism on the other AKR isoforms and carbonyl reductases.
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Acknowledgments |
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: DOX, doxorubicin; DAUN, daunorubicin; DOXol, doxorubicinol; DAUNol, daunorubicinol; AKR, aldo-keto reductase; HPLC, high-performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
Address correspondence to: Dr. K. Wayne Riggs, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3. E-mail: riggskw{at}interchange.ubc.ca
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