Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Carcinogenesis vol.24 no.10 pp.1695±1703, 2003 DOI: 10.1093/carcin/bgg119 Human cytosolic enzymes involved in the metabolic activation of carcinogenic aristolochic acid: evidence for reductive activation by human NAD(P)H:quinone oxidoreductase Marie Stiborova1,3, Eva Frei2, Bruno Sopko1, Klara kova1, a1, Martina Lan Sopkova1, Vladimira Markov Tereza Kumstyrov a1, Manfred Wiessler2 and Heinz H.Schmeiser2 X-ray structures. The results demonstrate for the first time the potential of human NQO1 to activate AAI by nitroreduction. 1 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, The Czech Republic and 2Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Introduction Abbreviations: AA, aristolochic acid; AAN, aristolochic acid nephropathy; AAI, 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid; AAII, 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid; dA±AAI, 7-(deoxyadenosin-N6 -yl)aristolactam I; dA±AAII, 7-(deoxyadenosin-N6 yl)aristolactam II; dG±AAI, 7-(deoxyguanosin-N2 -yl) aristolactam I; dG± AAII, 7-(deoxyguanosin-N2 -yl) aristolactam II; NQO1, NAD(P)H:quinone oxidoreductase; polyethylenimine; XO, xanthine oxidase. The so-called Chinese herbs nephropathy (CHN), a unique type of rapidly progressive renal fibrosis associated with the prolonged intake of Chinese herbs during a slimming regimen, was observed for the first time in Belgium in 1991 (1,2). About 100 CHN cases have been identified so far in Belgium, half of which needed renal replacement therapy, mostly including renal transplantation (3±5). The observed nephrotoxicity has been traced to the ingestion of Aristolochia fangchi containing carcinogenic and nephrotoxic aristolochic acid (AA) inadvertently included in slimming pills (2). CHN has been described in patients in other European and in Asian countries and in the USA (about 170 cases) (6), who were exposed to Aristolochia species containing AA and had no relationship with the Belgian slimming clinic. Therefore, it has been proposed to designate the interstitial nephropathy in which the unequivocal role of AA has been fully documented as aristolochic acid nephropathy (AAN) (7,8). Recently, a high prevalence of urothelial cancer was found in a large cohort of AAN patients in Belgium (9,10) and a case with urothelial cancer has also been described in the UK (11). It is also noteworthy that AA consumption may be a potential cause of the development of a similar type of fibrosis of the kidneys with malignant transformation of the urothelium, the Balkan endemic nephropathy (BEN) (12±15), which is widely found in certain areas of Rumania, Croatia, Bosnia, Serbia and Bulgaria along the Danube river basin (12,15). This highlights the carcinogenic potential of AA in human beings. AA is a mixture of structurally related nitrophenanthrene carboxylic acids, with 8-methoxy6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI) and 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAII), being the major components (Figure 1). Since the demonstration that AA forms covalent DNA adducts in rodents (16±18) as well as in AAN patients (6,19±22), AA±DNA adducts have been used as biomarkers of exposure and to investigate the mutagenic and carcinogenic potential of AA. The predominant AA±DNA adduct in vivo, 7-(deoxyadenosinN6 -yl)aristolactam I (dA±AAI), which is the most persistent of the adducts in target tissue, is a mutagenic lesion leading to A ! T transversions in vitro (23,24). This transversion mutation is found at high frequency in codon 61 of the H-ras oncogene in tumors of rodents induced by AAI, suggesting that dA±AAI might be the critical lesion in the carcinogenic process in rodents. DNA binding studies confirmed that both AAs bind to the adenines of codon 61 in the H-ras mouse gene Carcinogenesis vol.24 no.10 # Oxford University Press; all rights reserved. 1695 3 To whom correspondence should be addressed Email: [email protected] or [email protected] Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA metabolism is important in the assessment of an individual's susceptibility to this carcinogen. Using the 32 P-postlabeling assay we examined the ability of enzymes of cytosolic samples from 10 different human livers and from one human kidney to activate the major component of the plant extract AA, 8-methoxy6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI), to metabolites forming adducts in DNA. Cytosolic fractions of both organs generated AAI±DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N6 -yl)aristolactam I, 7-(deoxyguanosin-N2 -yl)aristolactam I and 7(deoxyadenosin-N6 -yl)aristolactam II, indicating a possible demethoxylation reaction of AAI, were identified as AA± DNA adducts formed from AAI by all human hepatic and renal cytosols. To define the role of human cytosolic reductases in the activation of AAI, we investigated the modulation of AAI±DNA adduct formation by cofactors or selective inhibitors of the NAD(P)H:quinone oxidoreductase (NQO1), xanthine oxidase (XO) and aldehyde oxidase. We also determined whether the activities of NQO1 and XO in different human hepatic cytosolic samples correlated with the levels of AAI±DNA adducts formed by the same cytosolic samples. Based on these studies, we attribute most of the activation of AA in human cytosols to NQO1, although a role of cytosolic XO cannot be ruled out. With purified NQO1 from rat liver and kidney and XO from buttermilk, the major role of NQO1 in the formation of AAI±DNA adducts was confirmed. The orientation of AAI in the active site of human NQO1 was predicted from molecular modeling based on published M.Stiborova et al. Fig. 1. Metabolic activation and DNA adduct formation of AA (Umlagerung rearrangement). (23,24) and preferentially to purines in the human p53 gene (5,6,25). Interestingly, to date only 5% of the patients treated with the slimming regimen in Belgium have suffered from nephropathy. Taking into account that AA is toxic, should it not have affected more of the patients? One possible explanation for the different responses of patients may be individual differences in the activities of the enzymes catalyzing the biotransformation (detoxication and/or activation) of AA. Many genes of enzymes metabolizing carcinogens are known to exist in variant forms or show polymorphisms resulting in differing activities of the gene products. These genetic variations appear to be important determinants of cancer risk (26). Therefore, screening AAN patients, as well as healthy persons treated with the slimming regimen, for genetic variations in the genes of the enzymes involved in AA metabolism should lead to possible relationships between genotypes and nephropathy. Thus, the identification of the enzymes principally involved in the activation of AA in humans and a detailed knowledge of their catalytic specificities is of major importance. The detection of specific DNA adducts (16±22) by 32 Ppostlabeling analyses has allowed us to use AA±DNA binding 1696 as a probe for metabolic activation of AA in in vitro systems. Recently we found that in vitro, both microsomal and cytosolic enzymes activate AAI and AAII to form the same DNA adducts found in vivo in rodents (27±30) and in humans suffering from AAN (6,19±22). However, in these experiments, only enzymes from animals, and human recombinant enzymes, were tested for their capabilities to activate AA. Rat hepatic microsomal and recombinant human cytochromes P450 1A1 and 1A2, rabbit NADPH:cytochrome P450 reductase (29,30), rat liver cytosolic NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase) (31), buttermilk xanthine oxidase (XO) (27) and peroxidases such as ovine prostaglandin H synthase (28) and milk lactoperoxidase (27) were efficient in reductive activation of AA. However, animal enzymes or human recombinant systems are usually not the ideal models of the catalytic properties of enzymes in human organs. Moreover, no data are available on the participation of authentic human reductases in AA activation, but these are necessary to evaluate the susceptibility of humans to develop AAN or BEN. The present study was therefore undertaken to determine the capability of human organs to activate the major component of aristolochiacea extract, AAI and to identify whether human Activation of AA by NAD(P)H:quinone oxidoreductase hepatic and renal cytosolic enzymes (and which of them) are involved in adduct formation by AAI. Materials and methods Caution AAs are mutagenic and carcinogenic and should be handled with care. Exposure to 32 P should be avoided, by working in a confined laboratory area, with protective clothing, plexiglass shielding, Geiger counters and body dosimeters. Wastes must be discarded according to appropriate safety procedures. Chemicals Chemicals were obtained from the following sources: NADH, NADPH, nuclease P1, deoxyadenosine 30 -monophosphate (dAp), deoxyguanosine 30 -monophosphate (dGp), buttermilk XO, hypoxanthine, cytochrome c, sodium dodecyl sulfate (SDS), dicoumarol, allopurinol and 2-hydroxypyrimidine from Sigma Chemical Co. (St Louis, MO), bicinchoninic acid from Pierce, (Rockford, IL), Sephadex G-150 from Amersham-Pharmacia (Uppsala, Sweden), menadione from Merck (Darmstadt, Germany), Affi-Gel Blue (Cibacron Blue Agarose, Porcine Blue HB, C.T. 61211 Agarose) from Bio-Rad (Richmond, CA), Sudan I from The British Drug Houses (UK), N1 -methylnicotinamide from Aldrich Chemical Co. (Milwaukee, WI) and calf thymus DNA from Roche Diagnostics Mannheim (Germany). The natural mixture AA consisting of 65% AAI and 34% AAII was a gift from Madaus (Cologne, Germany). AAI and AAII were isolated from the mixture by preparative HPLC; their purity was 99.7% as estimated by HPLC (32). All other chemicals were of analytical purity or better. Enzymes and chemicals for the 32 P-postlabeling assay were obtained commercially from sources described previously (19,27). Animal experiments To induce NQO1, 10 male Wistar rats were injected i.p. with Sudan I in maize oil (20 mg Sudan I/kg body wt) once a day for 3 consecutive days. Rats were placed in cages in temperature- and humidity-controlled rooms. Standardized diet and water were provided ad libitum. Animals were killed 24 h after the last treatment by cervical dislocation (33). Liver and kidney of animals were excised immediately after death, quickly frozen in liquid nitrogen and stored at ÿ80 C until the isolation of cytosolic and microsomal fractions. Preparation of cytosolic fractions Liver and renal fractions (cytosol and microsomes) from 10 rats pre-treated with Sudan I (see above) and renal fractions from one human kidney (specimen from European transplantation Centre was a gift from Dr J.Nortier, Brussels, Belgium) were prepared by differential centrifugation as described previously (31,33). The age, drug and alcohol history of the donor of the kidney used in the experiment is not known. The 105 000 g supernatant was taken as cytosol and used for studies presented in the paper. All tissue fractions were stored at ÿ80 C. Ten human hepatic cytosolic fractions were from Gentest (Woburn, MA) (numbers of samples are shown in Table I) and stored at ÿ80 C. The donors ranged in age from 2 to 71 years and included two men and eight women. A drug and/or alcohol abuse history of the samples is described in Gentest protocols. Each cytosolic preparation was analyzed for specific NQO1 and XO activities. The assays used were as follows. NQO1, XO and aldehyde oxidase assays NQO1 activity was measured essentially as described by Ernster (34). The standard assay system contained 25 mM Tris±HCl (pH 7.4), 0.2% Tween 20, 0.07% bovine serum albumin, 400 mM NADH (or NADPH) and 100 mM menadione (2-methyl-1,4-naphthoquinone) dissolved in methanol. The enzyme activity was determined by following the oxidation of NADH (NADPH) spectrophotometrically at 340 nm on a Hewlett-Packard 8453 diode array spectrophotometer. One unit of activity is defined as the amount of enzyme catalyzing the oxidation of 1 mmol of NAD(P)H in 1 min. The activities of XO in cytosolic fractions were measured as described by Ichikawa et al. (35) using hypoxanthine as substrate. The aldehyde oxidase activity was assayed as described by Felsted et al. (36) using N1 -methylnicotinamide as substrate. Protein concentration was assessed using the bicinchoninic acid protein assay with serum albumin as a standard (37). Isolation of NQO1 NQO1 was isolated as described earlier (33). Hepatic and renal cytosolic fractions from Sudan I-treated rats were used. Briefly, proteins in 20 ml cytosol (24 and 17.4 mg/ml for hepatic and renal cytosolic fractions, respectively) were fractionated with ammonium sulfate and the fraction between 30 and 90% saturation containing most of the NQO1 activity was dialyzed against 2000 ml of 150 mM KCl in 50 mM Tris (pH 7.4). The dialyzed enzyme preparation was chromatographed on a Sephadex G-150 column and NQO1 was eluted with the same buffer. Pooled fractions containing the NQO1 activity were applied onto a column of Affi-Gel Blue and non-NQO1 proteins were eluted with the same buffer and subsequently with a gradient of NaCl (0±3.5 M) in this buffer. NQO1 was eluted from Affi-Gel Blue with 20 mM Tris buffer (pH 10.0) containing 1 mM NADH. In order to remove residual protein impurities, the NQO1 sample was applied onto a Sephadex G-150 column and re-chromatographed. The eluate was concentrated by ultrafiltration and stored at ÿ80 C. All steps of the isolation procedure were performed at 4 C. Table I. XO-, NQO1-dependent catalytic activities and DNA adducts formed by AAI in human hepatic and renal cytosolic samples Human hepatic (H) and renal (R) cytosolic samples XOa H803 H806 H823 H830 H856 H866 H870 H889 H893 H8112 40.0 20.0 22.8 21.9 20.9 30.4 26.2 38.0 39.0 43.8 Average valuef R1 30.3 (8.70) 4.3 NQO1a AAI±DNA adductsb Without cofactors With 2-hydroxypyrimidinec With hypoxanthined With NADHe With NADPHe 70.0 1.0 12.0 4.0 4.0 19.0 5.0 3.0 3.0 10.0 2.7 7.2 1.0 3.2 5.2 5.4 1.4 5.2 5.4 1.4 1.2 4.4 2.9 3.3 5.0 0.3 0.7 0.8 5.9 4.7 3.5 10.8 1.7 3.8 9.7 2.1 5.5 3.5 7.7 3.8 13.2 12.0 4.0 12.2 10.1 10.1 11.9 14.3 8.9 10.5 23.0 14.6 16.2 15.0 14.8 18.2 15.3 15.2 15.4 16.0 13.1 (19.6) 0.2 3.8 (2.0) 0.8 0.04 0.10 0.35 0.005 0.15 0.25 0.25 0.05 0.25 0.25 0.05 0.05 0.20 0.10 0.15 0.25 0.02 0.03 0.04 0.30 0.25 2.9 (1.9) n.m. 0.15 0.55 0.10 0.10 0.45 0.10 0.25 0.15 0.35 0.20 5.2 (3.0) 0.4 0.04 0.55 0.60 0.20 0.80 0.50 0.50 0.60 0.70 0.45 0.50 10.7 (2.7) 1.4 0.07 1.00 0.55 0.65 0.75 0.65 0.85 0.75 0.70 0.65 0.80 15.9 (2.5) 3.2 0.4 Data for XO and NQO1 activities are presented as means of duplicate experiments; assays for their activities are described in Materials and methods. n.m., not measured. a XO and NQO1 activities in nmol/min/mg protein. b Relative adduct labeling (RAL), adducts/108 nucleotides/mg cytosolic protein. The values are arithmetic means SEM (n 4) of duplicate incubations, each DNA sample was determined by two postlabeled analyses. Cofactor of: c aldehyde oxidase, d XO and e NQO1. f Arithmetic means for 10 hepatic cytosolic samples (H803±H8112), values in parentheses are standard deviations, representing the inter-individual variability. 1697 M.Stiborova et al. Incubations The de-aerated and argon-purged incubation mixtures contained in a final volume of 0.75 ml: 50 mM Tris±HCl buffer (pH 7.4) containing 0.2% Tween 20, 1 mM cofactors for cytosolic enzymes (NADH or NADPH or hypoxanthine or 2-hydroxypyrimidine), 1 mg cytosolic protein, 0.5 mM AAI as sodium salt dissolved in water and 1 mg calf thymus DNA (4 mM dNp). The reaction was initiated by adding cofactor. Control incubations were carried out either without activating system (cytosol) or with activating system and AAI, but without DNA or with activating system and DNA but without AAI. Incubations with purified NQO1 enzymes contained in a final volume of 0.75 ml: 50 mM Tris±HCl buffer (pH 7.4) containing 0.2% Tween 20, 1 mM NADH or NADPH, 0.5 mM AAI as sodium salts dissolved in water, 1 mg calf thymus DNA (4 mM) and 0.05±1.0 U of hepatic or renal NQO1 instead of cytosolic fractions. Incubations with buttermilk XO contained in a final volume of 0.75 ml: 50 mM sodium phosphate (pH 7.4) containing 1 mM hypoxanthine, 0.5 mM AAI as sodium salts dissolved in water, 1 mg calf thymus DNA (4 mM) and 0.05±1.0 U XO instead of cytosolic fractions. All reaction mixtures were incubated at 37 C, 10±60 min, then extracted twice with ethyl acetate (2 2 ml). DNA was isolated from the water phase by the phenol±chloroform extraction as described earlier (19,27±31). The content of DNA was determined spectrophotometrically (38,39). Inhibition studies The following chemicals were used to inhibit the activation of AAI in human cytosolic fractions: dicoumarol and allopurinol to inhibit NQO1 and XO, respectively (40±42). Inhibitors dissolved in 7.5 ml of methanol, to yield final concentrations of 10 mM, were added to the incubation mixtures. An equal volume of methanol alone was added to the control incubations and proceeded as described above. 32 P-Postlabeling analysis The nuclease P1 enrichment version (43) of the assay, known to be the most suitable version of the 32 P-postlabeling technique for AA±DNA adduct detection and quantification was used and performed exactly as described (6,16, 19±22,27±31,44). Enzymatic synthesis of reference compounds, dAp-AAI, dGp-AAI and dAp-AAII and their 32 P-postlabeling were carried out as described earlier (18). Co-chromatography on polyethylenimine-cellulose Adduct spots detected by the 32 P-postlabeling assay were excised from the thin-layer plates, extracted and co-chromatographed with reference 30 ,50 bisphosphate adducts as reported previously (18). HPLC analysis of 32 P-labeled 30 ,50 -deoxyribonucleoside bisphosphate adducts HPLC analysis was performed essentially as described previously (18,27,45). Individual spots detected by the 32 P-post-labeling assay were excised from thin layer plates and extracted (46). The dried extracts were redissolved in 100 ml of methanol±phosphate buffer (pH 3.5) 1:1 (v/v). Aliquots (50 ml) were analysed on a phenyl-modified reversed-phase column (250 4.6 mm, 5 mm Zorbax Phenyl; Saulentechnik Dr Knauer, Berlin, Germany) with a linear gradient of methanol (from 40 to 80% in 45 min) in aqueous 0.5 M sodium phosphate and 0.5 M phosphoric acid (pH 3.5) at a flow rate of 0.9 ml/min. Radioactivity eluting from the column was measured by monitoring Cerenkov radiation with a Berthold LB 506 C-1 flow through radioactivity monitor (500 ml cell, dwell time 6 s). Molecular modeling Crystalographic coordinates for human NQO1 with bound FAD were obtained from the Protein Data Bank (47). For modeling purposes, the physiological dimer form was used (MacroMolecular file 1qrd_1.mmol). The coordinates were used without further refinement. The modeling of the binding of AAI to the active site was performed with the program Autodock 3.0.3. (48) and Sybyl 6.6.5 (Tripos GmbH, Germany) by the procedure as described (31,49). Statistical analyses Correlation coefficients between the catalytic activities of NQO1 and XO in human hepatic cytosolic samples and the levels of individual AAI±DNA adducts formed by the same cytosolic samples were determined by linear regression using Statistical Analysis System software version 6.12. Correlation coefficients were based on a sample size of 10. P is two-tailed and considered significant at the 0.05 level. 1698 Results DNA adduct formation by AAI in human hepatic and renal cytosol We determined the formation of DNA adducts by AAI in calf thymus DNA in the presence of cytosolic samples from 10 different human livers and from one human kidney. Both human hepatic and renal cytosolic fractions were capable of activating AAI to form DNA adducts. AAI activated by cytosol from liver or kidney generated the same major DNA adducts as those obtained in vivo in rats and humans reported previously (6,10,11,14±22). A cluster of three major adducts (spots 1, 2 and 3) was formed by AAI (Figure 2). These adduct spots formed by AAI showed the same migration on polyethylenimine-cellulose TLC plates and on reversed-phase HPLC as those of synthetic standards (18) (see Supplementary material, Figure 1). Thus, spot 1 was assigned to 30 ,50 -bisphospho-7-(deoxyguanosin-N2 -yl)-aristolactam I (dG±AAI), spot 2 to 30 ,50 -bisphospho-7-(deoxyadenosin-N6 -yl)-aristolactam I (dA±AAI) and spot 3 to 30 ,50 -bisphospho-7-(deoxyadenosin-N6 -yl)-aristolactam II (dA±AAII). A minor dA±AAII adduct was formed from AAI also in previous in vitro studies utilizing other activating enzymatic systems (27±31) or in forestomach DNA of rats treated with AAI (18). An explanation may be the initial formation of dA±AAI in doublestranded DNA, with subsequent demethoxylation to dA±AAII (18). Control incubations carried out in parallel either without cytosol, without DNA or without AAI were free of adduct spots in the region of interest even after prolonged exposure times of the autoradiograms. The adducts formed by AAI activated with human cytosolic samples are known to be generated from AAI by nitro reduction (6,18,50,51). Therefore, similar to rat hepatic and renal cytosols (31), the human cytosolic fractions tested in this study contain enzymatic systems capable of catalyzing the reductive activation of AAI. XO, NQO1 and aldehyde oxidase are major candidates for the reductive activation of AAI in cytosol. To investigate these possibilities, the influences on AAI±DNA adduct formation by various structurally diverse compounds, which are electron donors for these enzymes, were examined. As shown in Table I the formation of AAI±DNA adducts was stimulated by the cofactors of NQO1, NADPH and NADH (52). Adduct levels were significantly higher, 4.2- and 2.8-fold on average, when NADPH or NADH were added into the Fig. 2. Autoradiographic profiles of AAI±DNA adducts obtained from calf thymus DNA after activation by human hepatic cytosol in the presence of NADPH (sample H803) (A), purified rat renal NQO1 in the presence of NADPH (B) and by buttermilk XO in the presence of hypoxanthin (C). The nuclease P1-enrichment procedure was used for analysis. Origins, in the bottom left-hand corner were cut off before exposure. Screen enhanced autoradiography was at ÿ80 C for 1 h. Chromatographic conditions: D1, 1 M sodium phosphate, pH 6.8; D3, 3.5 M lithium formate, 8.5 M urea, pH 4.0; D4, 0.8 M LiCl, 0.5 M Tris±HCl, 8.5 M urea, pH 9.1; D5, 1.7 M NaH2 PO4 , pH 6.0. Spot 1, dG±AAI; spot 2, dA±AAI; spot 3, dA±AAII. Activation of AA by NAD(P)H:quinone oxidoreductase incubation mixture. Hypoxanthine, a cofactor of XO (53), also stimulated AAI±DNA adduct formation, but only to a much lesser extent (Table I). 2-Hydroxypyrimidine, an electron donor of the cytosolic aldehyde oxidase (54), had practically no effect. These results suggest a minor, but detectable, role of XO in AAI activation, NQO1 seems more important for this activation. If the organs are compared, cytosolic samples from liver exhibited much higher efficiencies to activate AAI than the single kidney sample (Table I). The lower efficacy to activate AAI corresponds to lower activities of reductases in cytosol of this tissue (Table I). To further resolve which cytosolic enzymes are responsible for activation of AAI, different experimental approaches were employed: (i) analysis of the correlation of XO and NQO1 activities with AAI±DNA adduct formation by human cytosols, (ii) selective enzyme inhibition, (iii) utilization of purified XO and NQO1 and (iv) computer modeling of the NQO1±AAI complex. Correlation of NQO1- and XO-linked enzyme activities in human hepatic cytosolic samples with DNA adduct formation by AAI Catalytic activities of NQO1 and XO with menadione (55) and hypoxanthine (35) as substrates, respectively, were analyzed in human hepatic cytosolic preparations from 10 different donors. Large inter-individual variations in both catalytic activities were found (Table I). The activity of another reductase, aldehyde oxidase, measured with N1 -methylnicotinamide as a substrate (36), was not detectable in any human cytosolic samples. Quantitative 32 P-postlabeling analyses, as shown in Table I, also showed wide individual variations in DNA binding of AAI in the human cytosolic incubations. Correlations between the NQO1 or XO activities and AAI±DNA adduct formation were analyzed to examine the role of human cytosolic reductases in AAI activation. Highly significant correlations were found between the NQO1 activities measured with NADPH as cofactor and the levels of total AAI±DNA adducts determined in the presence of the same cofactor (NADPH) (0.985, P 5 0.001). Likewise, highly significant correlations were found between the activities of this enzyme and the levels of individual AAI±DNA adducts (0.941, P 5 0.001 for dG±AAI, 0.889, P 5 0.001 for dA±AAI and 0.712, P 5 0.05 for dA± AAII). The correlations were calculated with data taken from Supplementary material Table Ie. Catalytic activities of XO did not exhibit significant correlations with the levels of AAI± DNA adducts determined in the presence of hypoxanthine as cofactor (ÿ0.349, P 0.322). These results suggest that human cytosolic NQO1 is the predominant enzyme participating in AAI activation in human hepatic cytosol. Effect of inhibitors of NQO1 and XO on activation of AAI by human cytosolic samples Inhibition experiments further supported the role of NQO1 in the activation of AAI in human cytosolic samples. Two hepatic cytosolic samples with high NQO1 or XO activities were selected, and incubations were carried out in the absence and presence of dicoumarol and allopurinol, known inhibitors of NQO1 and XO, respectively. The hepatic cytosolic AAI activation with dicoumarol resulted in an 85% decrease in the levels of AAI±DNA adducts, while no effect of allopurinol was observed (Table II). Likewise, dicoumarol effectively inhibited AAI±DNA adduct formation by renal cytosol, by 75% (Table II). These results emphasize the major role of NQO1 in AAI activation by cytosolic samples of both human tissues. Activation of AAI by purified XO and NQO1 enzymes Even though XO has only a minor contribution to the AAI± DNA adduct formation in human hepatic cytosols, we tested in more detail its participation in AAI activation in vitro and compared its efficacy with that of NQO1. For such studies we utilized buttermilk XO and NQO1 enzymes isolated from rat liver and kidney. Incubations of AAI with DNA in the presence of either purified NQO1 and its cofactor, NADPH, or XO and its cofactor, hypoxanthine, under the standardized conditions (0.5 mM AAI, 1 mM cofactors, 0.05±1.0 U of the enzymes, pH 7.4, 37 C, 60 min) resulted in the formation of AAI±DNA adducts (Table III). The same DNA adducts (dG±AAI, dA±AAI and dA±AAII, Figure 1) as those determined in either cytosol (present paper) or in vivo (6,10,11,14±22) were generated by both enzymes. The identity of adducts was confirmed by co-chromatography on TLC and HPLC. The NQO1- and XO-catalyzed binding of AAI was shown to be dependent on the time of incubation, being linear up to 60 min (not shown). The levels of individual AAI-adducts formed by XO differ from those produced by cytosol (present paper) or in vivo (6,10,11,14±22). In contrast to the adduct formation catalyzed by NQO1, which resembles that in vivo, where the levels of Table II. The effect of NQO1 and XO inhibitors on the AAI±DNA adduct formation in human cytosol Human cytosolic sample H803 NADPH H803 NADPH dicoumarol H893 hypoxanthine H893 hypoxanthine allopurinol R1 NADPH R1 NADPH dicoumarol R1 hypoxanthine R1 hypoxanthine allopurinol RALa (mean SEM/108 nucleotides) dG±AAI dA±AAI dA±AAII Total 10.2 1.5 3.4 3.4 1.3 0.26 0.15 0.15 13.7 2.1 4.0 4.7 1.5 0.41 0.21 0.21 1.5 0.1 0.3 0.3 0.4 0.14 0.04 0.05 23.0 3.7 7.7 8.4 3.2 0.81 0.40 0.41 0.50 0.05 0.30 0.25 0.05 0.04 0.005 0.005 0.60 0.10 0.35 0.35 0.05 0.03 0.035 0.030 0.05 0.005 0.02 0.05 0.05 0.005 0.005 0.005 1.10 0.20 0.40 0.45 0.20 0.70 0.035 0.040 Numbers are averages SEM (n 4) of duplicate in vitro incubations, each DNA sample was determined by two post-labeled analyses. Experimental conditions used for incubations with inhibitors are described in Materials and methods; concentrations of dicoumarol and allopurinol were 10 mM. a Relative adduct labeling. 1699 M.Stiborova et al. Table III. AAI±DNA adduct formation by buttermilk XO and rat hepatic and renal NQO1 Enzyme XOb Hepatic NQO1 Renal NQO1 RALa (mean SEM/108 nucleotides) dG±AAI dA±AAI dA±AAII Total 25.3 1.30 10.2 0.50 11.7 0.55 18.3 0.9 31.2 1.5 24.8 1.25 6.4 0.3 4.4 0.2 2.8 0.15 50.0 5.1 45.8 4.6 39.3 1.9 Numbers are averages SEM (n 4) of duplicate in vitro incubations, each DNA sample was determined by two post-labeled analyses. Relative adduct labeling. b The DNA binding was assayed as described in Materials and methods with 0.5 mM AAI, 1 mM hypoxanthine with 0.4 U of XO or 1 mM NADPH with 0.4 U of rat hepatic or renal NQO1. a dA±AAI adduct are always higher than those of dG±AAI, the dG±AAI adduct is prevalent in the XO-dependent system. Moreover, different dependencies of AAI±DNA adduct levels on the amount of NQO1 and XO were observed (Figure 3). Amounts of XO 40.4 U (400 nmol/min/mg) per incubation much more effectively activated AAI than the same amount of NQO1 while at lower more physiological enzyme concentrations NQO1 is more effective (Figure 3). These results might explain a lower contribution of XO to activate AAI in human cytosolic samples and confirm the major role of NQO1 in this human subcellular fraction; XO activities in all cytosolic samples were less than the critical XO activity needed for the pronounced AAI activation (compare Table I). Fig. 3. Dependence of DNA adduct formation from 0.5 mM AAI on the concentration of hepatic and renal NQO1 with NADPH as cofactors, and buttermilk XO in the presence of hypoxanthine. Experimental details are described in Materials and methods. aRelative adduct labeling. Molecular modeling In order to examine the molecular basis of the reductive activation of AAI by human NQO1, computer modeling was used in the present study. Molecular modeling clearly explains the efficiency of human NQO1 in activating AAI. The calculated model structure for the human NQO1±AAI complex is shown in Figure 4. It is evident from this figure that AAI fits well into the active site of human NQO1, being bound near the isoalloxazine ring of the flavin prosthetic group of the enzyme. This allows for an electron transfer during the reductive activation of AAI. The value of the apparent dissociation constant for the human NQO1±AAI complex is calculated to be 1.48 mM. Discussion Recently, specific AA±DNA adducts were found to be associated with a unique nephropathy, AAN, and urothelial cancer in a cohort of women who had followed a weight reducing treatment consisting of Chinese herbs containing AA in Belgium. This disease was observed also in other European and Asian countries and in the USA (5,6). Specific AA±DNA adducts were also detected in renal specimens of two patients suffering from malignant transformation of urothelium and living in BEN areas (14). Since the detection and quantification of specific DNA adducts by the 32 P-postlabeling procedure has proven a useful tool to monitor exposure to the plant carcinogen AA in vivo (6,10,11,14±22) and in vitro (27±31), they were utilized in the present study to identify human enzymes involved in AA activation. In mammalian tissues, both cytosol and microsomes contain enzymes catalyzing the reduction of nitro aromatic compounds 1700 Fig. 4. AAI is shown docked to the active site of human NQO1 where several key amino acid residues position the AAI substrate parallel to a flavin prosthetic group. (40±42,56,57). We have already identified rat hepatic cytosolic NQO1 to be capable of AA activation, generating AA± DNA adduct profiles identical to those found in renal tissue in humans (31). The present paper reports on the identification of human cytosolic enzymes, which participate in the reductive bioactivation of the major component of AA, AAI. Here, we clearly demonstrate that human cytosolic samples from livers of 10 different human donors and kidney of one donor are capable of activating AAI leading to the same DNA Activation of AA by NAD(P)H:quinone oxidoreductase adduct pattern as that formed in humans exposed to AA (6,10,11,19±22). Formation of AAI±DNA adducts was strongly correlated with NQO1 activities in different human hepatic cytosolic samples. Inhibition of DNA adduct formation by dicoumarol, the inhibitor of NQO1, provided additional evidence for the role of this enzyme in AAI activation in humans. Molecular modeling where the AAI molecule was docked to the active site of human NQO1 suggests that AAI binds where the other NQO1 substrates (i.e. duroquinone) (58) are found in the X-ray structure, with the planar aromatic AAI rings parallel to the flavin ring. This allows for an efficient electron transfer during the reductive activation. Recently, we showed that rat cytosolic NQO1 was also efficient to activate AAI (31). This indicates similarities of NQO1 enzymes from both species. Nevertheless, some differences were observable in catalytic activities of human and rat NQO1s. NQO1 in human hepatic cytosolic samples more efficiently activates AAI in the presence of NADPH than NADH (see Table I), while no difference was seen with rat hepatic NQO1 (31). This also corresponds to a ~2-fold lower activity of human NQO1 to reduce menadione in the presence of NADH than NADPH (data not shown). But, even though the NQO1 activities with menadione as substrate in human hepatic cytosols are lower than those in hepatic cytosol of rats, similar efficacies of subcellular systems of both species to activate AAI were observed (31 and the present paper). The higher binding affinity of AAI for human NQO1 than for the rat enzyme can explain the observed differences. AAI fits better into the active centre of human NQO1 than into that of the rat enzyme (compare Figure 4 in the present paper and figure 8 in 31). Likewise, the value of the calculated dissociation constant for the human NQO1±AAI complex (1.48 mM) is lower than for the rat enzyme (16.4 mM) (31). In comparison with NQO1, XO had only a minor impact on the capacity to activate AAI to form DNA adducts in human samples assayed. In contrast to this finding we observed that the isolated buttermilk XO was an effective activator of this compound (27 and the present paper), but the enzyme levels needed are not physiological. Another reason for the observed discrepancies might be the different substrate specificities of human cytosolic and buttermilk XO. The involvement of human NQO1 in the reductive activation of nitroaromatics like AAI is consistent with previous reports demonstrating that the enzyme functions efficiently as a nitroreductase of substrates like dinitropyrenes, nitrophenylaziridines and nitrobenzamides (59 and references cited herein). NQO1 activity is ubiquitously present in all tissue types (59± 62). Levels of expression and activities of NQO1 in humans differ considerably among individuals, because the enzyme is influenced by several factors like smoking, drugs, environmental chemicals and genetic polymorphisms (59,63,64). Biochemical studies have already demonstrated that NQO1 activity is induced by a wide range of chemicals (59,65±67). Two distinct regulatory elements in the 50 flanking region of the NQO1 gene, the antioxidant response element (ARE) and the xenobiotic response element (XRE), involving the liganded aromatic hydrocarbon (Ah) receptor, have been shown to regulate NQO1 expression in many cellular systems. Moreover, the anti-estrogens tamoxifen and hydroxytamoxifen stimulate the expression of NQO1 by activating the estrogen receptor, which is different from the Ah locus (68,69). AREmediated NQO1 gene expression is increased by a variety of phenolic antioxidants, tumor promoters and H2 O2 (59,65,67). The XRE of NQO1 shares significant homology with the XRE of CYP1A1 (70), the gene of the enzyme, which also activates AAs (6,29). Both NQO1 and CYP1A1 genes can be induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons, Sudan I and b-naphthoflavone (59). Because NQO1 activity is increased in rats treated with AA (33), it might also be induced in the Belgian AAN patients. So far two polymorphisms in the human NQO1 gene have been found in the general population. One of them the 609 C to T variant, designated NQO1 2, has profound phenotypic consequences and has been associated with an increased risk of urothelial tumors (71), therapy-related acute myeloid leukemia (72), cutaneous basal cell carcinomas (73) and pediatric leukemia (74). The frequency of homozygous NQO1 2 mutation varies across ethnic groups and was reported to be ~5% in Caucasians (59). Collectively, the data suggest that variations of this enzyme and regulatory proteins controlling its expression (Ah receptor, or its associated transcription factor, the Ah receptor nuclear translocator or Arnt protein) (26), might play a role in the risk of cancer by AAs. To confirm this hypothesis, the study screening Belgian AAN patients and other participants in the slimming regimen for polymorphisms of the genes encoding NQO1, cytochrome P450 1A1 and the Ah receptor is underway in our laboratories. Supplementary material Supplementary material can be found at http://www.carcin. oupjournals.org Acknowledgments Supported by Grant Agency of the Czech Republic (grant 203/04/0923) and the Ministry of Education of the Czech Republic (grant MSM 1131 00001). We are grateful to A.Breuer and K.Klokow (German Cancer Research Center, Heidelberg) for excellent technical assistance. References 1. Vanherweghen,J.L., Depierreux,M., Tielemans,C. et al. (1993) Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet, 341, 387±391. 2. Vanhaelen,M., Vanhaelen-Fastre,R., But,P. and Vanherweghen,J.L. (1994) Identification of aristolochic acid in Chinese herbs. Lancet, 343, 174. 3. Vanhaelen,M., Cuykens,J.J., Vandenberg,Ph., Bouman,K.P. and Hagens,Y. (1998) Valvular heart disease and Chinese-herbs nephropathy. Lancet, 351, 991. 4. Martinez,M.-C., Nortier,J., Vereestaeten,P. and Vanherweghem,J.-L. (2002) Progression rate of Chinese herb nephropathy: impact of Aristolochia fangchi ingested dose. Nephrol. Dialysis Transplant., 17, 408±412. 5. Cosyns,J.-P. (2003) Aristolochic acid and `Chinese Herbs Nephropathy'. A review of the evidence to date. Drug Safety, 26, 33±48 6. Arlt,V.M., Stiborova,M. and Schmeiser,H.H. (2002) Aristolochic acid as a probable cancer hazard in herbal remedies: a review. Mutagenesis, 17, 265±277. 7. Gillerot,G, Jadoul,M., Arlt,V.M., van Ypersele de Strihou,C., Schmeiser,H.H., But,P.P.H., Bieler,C.A. and Cosyns,J.-P. (2001) Aristolochic acid nephropathy in a Chinese patient: time to abandon the term `Chinese herbs nephropathy'? Am. J. Kidney Dis., 38, E26. 8. Olez,K., Daugirdes,J., Gregory,M.C., Frohnert,P.P., Blownik,D.M., Iha,V. and Cosyns,J.-P. (2001) Is `Chinese Herbs Nephropathy' a prejudical term? Am. J. Kidney Dis., 38, 1141±1142. 1701 M.Stiborova et al. 9. Cosyns,J.P., Jadoul,M., Squifflet,J.P., Wese,F.X. and van Ypersele de Strihou,C. (1999) Urothelial lesions in Chinese-herb nephropathy. Am. J. Kidney Dis., 33, 1011±1017. 10. Nortier,J.L., Muniz Martinez,M.C. et al. (2000) Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi). N. Engl. J. Med., 342, 1686±1692. 11. Lord,G.M., Cook,T., Arlt,V.M., Schmeiser,H.H., Williams,G. and Pusey,C.D. (2001) Urothelial malignancy and Chinese herbal nephropathy. Lancet, 358, 1515±1516. 12. Ivic,M. (1970) The problem of etiology of endemic nephropathy. Acta Fac. Med. Naiss., 1, 29±37. 13. Tatu,C.A., Oren,W.H., Finkelman,R.B. and Feder,G.L. (1998) The etiology of Balkan endemic nephropathy: still more questions than answers. Environ. Health Perspect., 106, 689±700. 14. Arlt,V., Ferluga,D., Stiborova,M., Pfohl-Leskowicz,A., Vukelic,M., Ceovic,S., Schmeiser,H.H. and Cosyns,J.-P. (2002) Is aristolochic acid a risk for Balkan endemic nephropathy-associated urothelial cancer? Int. J. Cancer, 101, 500±502. 15. Pfohl-Leskowicz,A., Petkova-Bocharova,T., Chernozemsky,I.N. and Castegnaro,M. (2002) Balkan endemic nephropathy and associated urinary tract tumours: a review on aetiological causes and the potential role of mycotoxins. Food Additives Contamin., 19, 282±302. 16. Schmeiser,H.H., Schoepe,K.-B. and Wiessler,M. (1988) DNA adduct formation of aristolochic acid I and II in vitro and in vivo. Carcinogenesis 9, 297±303. 17. Pfau,W., Schmeiser,H.H. and Wiessler,M. (1990) 32 P-Postlabelling analysis of the DNA adducts formed by aristolochic acid I and II. Carcinogenesis, 11, 1627±1633. 18. Stiborova,M., Fernando,R.C, Schmeiser,H.H., Frei,E., Pfau,W. and Wiessler,M. (1994) Characterization of DNA adducts formed by aristolochic acids in the target organ (forestomach) of rats by 32 Ppostlabeling analysis using different chromatographic procedures. Carcinogenesis, 15, 1187±1192. 19. Schmeiser,H.H., Bieler,C.A., Wiessler,M., van Ypersele de Strihou,C. and Cosyns,J.-P. (1996) Detection of DNA adducts formed by aristolochic acid in renal tissue from patients with Chinese herbs nephropathy. Cancer Res., 56, 2025±2028. 20. Bieler,C.A., Stiborova,M., Wiessler,M., Cosyns,J.-P., van Ypersele de Strihou,C. and Schmeiser,H.H. (1997) 32 P-Post-labeling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy. Carcinogenesis, 18, 1063±1067. 21. Stiborova,M., Frei,E., Breuer,A., Bieler,C.A. and Schmeiser,H.H. (1999) Aristolactam I a metabolite of aristolochic acid I upon activation forms an adduct found in DNA of patients with Chinese herbs nephropathy. Exp. Toxic. Pathol., 51, 421±427. 22. Arlt,V.M., Pfohl-Leszkowicz,A., Cosyns,J.P. and Schmeiser,H.H. (2001) Analyses of DNA adducts formed by ochratoxin A and aristolochic acid in patients with Chinese herbs nephropathy. Mutat. Res., 494, 143±150. 23. Schmeiser,H.H., Janssen,J.W.G., Lyons,J., Scherf,H.R., Pfau,W., Buchmann,A., Bartram,C.R. and Wiessler,M. (1990) Aristolochic acid activates ras genes in rat tumors at deoxyadenosine residues. Cancer Res., 50, 5464±5469. 24. Arlt,V.M., Wiessler,M. and Schmeiser,H.H. (2000) Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene. Carcinogenesis, 21, 235±242. 25. Arlt,V.M., Schmeiser,H.H. and Pfeifer,G.P. (2001) Sequence-specific detection of aristolochic acid-DNA adducts in the human p53 gene by terminal transferase-dependent PCR. Carcinogenesis, 22, 133±140. 26. Smith,G., Stanley,L.A., Sim,E., Strange,R.C. and Wolf,C.R. (1995) Metabolic polymorphism and cancer susceptibility. Cancer Surv., 25, 27±65. 27. Schmeiser,H.H., Frei,E., Wiessler,M. and Stiborova,M. (1997) Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32 P-post-labeling: evidence for reductive activation by peroxidases. Carcinogenesis, 18, 1055±1062. 28. Stiborova,M., Frei,E., Breuer,A., Wiessler,M. and Schmeiser,H.H. (2001) Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthaseÐ32 P-postlabeling analysis of DNA adduct formation. Mutat. Res., 493, 153±164. 29. Stiborova,M, Frei,E., Wiessler,M. and Schmeiser,H.H. (2001) Human enzymes involved in the metabolic activation of carcinogenic aristolochic acids: evidence for reductive activation by cytochrome P450 1A1 and 1A2. Chem. Res. Toxicol., 14, 1128±1137. 30. Stiborova,M., Frei,E., Hajek,M. and Schmeiser,H.H. (2001) Carcinogenic and nephrotoxic aristolochic acids upon activation by NADPH:cytochrome P450 reductase form adducts found in DNA of 1702 patients with Chinese herbs nephropathy. Gen. Physiol. Biophys., 20, 375±392. 31. Stiborova,M., Frei,E., Sopko,B., Wiessler,M. and Schmeiser,H.H. (2002) Carcinogenic aristolochic acids upon activation by DT-diaphorase form adducts found in DNA of patients with Chinese herbs nephropathy. Carcinogenesis, 23, 617±625. 32. Schmeiser,H.H., Pool,B.L. and Wiessler,M. (1984) Mutagenicity of the two main components of commercially available carcinogenic aristolochic acids in Salmonella typhimurium. Cancer Lett., 23, 97±101. 33. Stiborova,M., Hajek,M., Vosmikova,H., Frei,E. and Schmeiser,H.H. (2001) Isolation of DT-diaphorase [NAD(P)H dehydrogenase (quinone)] from rat liver cytosol: identification of new enzyme substrates, carcinogenic aristolochic acids. Collect. Czech. Chem. Commun., 66, 959±972. 34. Ernster,L. (1967) DT-Diaphorase. Methods Enzymol., 10, 309±317. 35. Ichikawa,M., Nishino,T., Nishino,T. and Ichikawa,A. (1992) Subcellular localization of xanthine oxidase in rat hepatocytes: high-resolution immunoelectron microscopic study combined with biochemical analysis. J. Histochem. Cytochem., 40, 1097±1103. 36. Felsted,R.L., Chu,A.E.-Y. and Chaykin,S. (1973) Purification and properties of the aldehyde oxidase from hog and rabbit livers. J. Biol. Chem., 248, 2580±2587. 37. Wiechelman,K.J., Braun,R.D. and Fitzpatrick,J.D. (1988) Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal. Biochem., 175, 231±237. 38. King,R., Teitel,C.H. and Kadlubar,F.F. (2000) In vitro bioactivation of Nhydroxy-2-amino-a-carboline. Carcinogenesis, 21, 1347±1354. 39. Yamazoe,Y., Zenser,T.V., Miller,D.W. and Kadlubar,F.F. (1988) Mechanism of formation and structural characterization of DNA adducts derived from peroxidative activation of benzidine. Carcinogenesis, 9, 1635±1641. 40. Watanabe,T., Kaji,H., Takashima,M., Kasai,T., Lewtas,J. and Hirayama,T. (1997) Metabolic activation of 2- and 3-nitrodibenzopyranone isomers and related compounds by rat liver S9 and the effect of S9 on the mutational specificity of nitrodibenzopyranones. Mutat. Res., 388, 67±78. 41. Fu,P.P. (1990) Metabolism of nitro-polycyclic aromatic hydrocarbons. Drug Metab. Rev., 22, 209±268. 42. Ritter,C.L., Decker,R.W. and Malejka-Giganti,D. (2000) Reduction of nitro and 9-oxo groups o.d. environmental nitrofluorenes by the rat mammary gland in vitro. Chem Res. Toxicol., 13, 793±800. 43. Reddy,M.V. and Randerath,K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32 P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis, 7, 1543±1551. 44. Gupta,R.C. and Early,K. (1988) 32 P-Adduct assay comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures. Carcinogenesis, 8, 1687±1693. 45. Pfau,W. and Phillips,D.H. (1991) Improved reversed-phase highperformance liquid chromatographic separation of 32 P-labeled nucleoside 30 ,50 -bisphosphate adducts of polycyclic aromatic hydrocarbons. J. Chromatogr., 570, 65±76. 46. Schmeiser,H.H., Dipple,A., Schurdak,M.E., Randerath,E. and Randerath,K. (1988) Comparison of 32 P-postlabeling and high pressure liquid chromatographic analyses for 7,12-dimethylbenz[a]anthraceneDNA adducts. Carcinogenesis, 9, 633±638. 47. Berstein,F.C., Koetzle,T.F., Williams,G.J.B., Meyer,E.F.,Jr, Brice,M.D., Rodgers,J.R., Kenhard,O., Shimanouchi,T. and Tasumi,M. (1977) The Protein Data Bank: a computer-based archival file for macromolecular structures. J. Mol. Biol., 112, 535±542. 48. Morris,G.M., Goodsell,D.S., Halliday,R.S., Huey,R., Hart,W.E., Belew,R.K. and Olson,A.J. (1998) Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. J. Computat. Chem., 19, 1639±1662. 49. Dauber-Osguthorpe,P., Roberts,V.A., Osguthorpe,D.J., Wolff,J., Genest,M. and Hagier,A.T. (1988) Structure and energetics of ligand binding to protein: Escherichia coli dihydrofolate reductasetrimethoprim, a drug receptor system. Proteins, 4, 31±47. 50. Pfau,W., Schmeiser,H.H. and Wiessler,M. (1990) Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides in DNA. Carcinogenesis, 11, 313±319. 51. Pfau,W., Schmeiser,H.H. and Wiessler,M. (1991) N6 -Adenyl arylation of DNA by aristolochic acid II and a synthetic model for the putative proximate carcinogen. Chem. Res. Toxicol., 4, 581±586. 52. Hajos,A.K.D. and Winston,G.W. (1991) Dinitropyrene nitroreductase activity of purified NAD(P)H-quinone oxidoreductase: role in rat liver cytosol and induction by Aroclor 1254 pretreatment. Carcinogenesis, 12, 697±702. Activation of AA by NAD(P)H:quinone oxidoreductase 53. Swaminathan,S. and Hatcher,J.F. (1986) Xanthine oxidase-mediated mutagenicity of the bladder carcinogen 4-nitrobiphenyl. Mutat. Res., 172, 37±45. 54. Bauer,S.L. and Howard,P.C. (1991) Kinetics and cofactor requirements for the nitroreductive metabolism of 1-nitropyrene and 3-nitrofluoranthene by rabbit liver aldehyde oxidase. Carcinogenesis, 12, 1545±1549. 55. Pius,J., Xu,Y. and Jaiswal,A.K. (1996) Non-enzymatic and enzymatic activation of mitomycin c: identification of a unique cytosolic activity. Int. J. Cancer, 65, 263±271. 56. Chae,Y.-H., Thomas,T., Guengerich,F.P., Fu,P.P. and El-Bayoumy,K. (1999) Comparative metabolism of 1-, 2-, and 4-nitropyrene by human hepatic and pulmonary microsomes. Cancer Res., 59, 1473±1480. 57. Arlt,V.M., Stiborova,M., Hewer,A., Schmeiser,H.H. and Phillips,D.H. (2003) Human enzymes involved in the metabolic activation of the environmental contaminant 3-nitrobenzanthrone: evidence for reductive activation by human NADPH:cytochrome P450. Cancer Res., 63, 2752± 2761. 58. Chen,S., Wu,K., Zhang,D., Sherman,M., Knox,R. and Yang,C.S. (1999) Molecular characterization of binding of substrates and inhibitors to DTdiaphorase: combined approach involving site-directed mutagenesis, inhibitor-binding analysis, and computer modeling. Mol. Pharmacol., 56, 272±278. 59. Ross,D., Kepa,J.K., Winski,S.L., Beall,H.D., Anwar,A. and Siegel,D. (2000) NAD(P)H:quinone oxidoreductase (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms. Chem.-Biol. Interact., 129, 77±97. 60. Ernster,L. (1987) DT-diaphorase: a historical review. Chem. Scr., 27A, 1±13. 61. Riley,R.J. and Workman,P. (1992) DT-diaphorase and cancer chemotherapy. Biochem. Pharmacol., 43, 1657±1669. 62. Long,D.J.,II, and Jaiswal,A.K. (2000) NRH:Quinone oxidoreductase2 (NQO2). Chem.-Biol. Interact., 129, 99±112. 63. Schulz,W.A., Krummeck,A., Rosinger,I., Eickelman,P., Neuhaus,C., Ebert,T., Schmitz-Drager,B.J. and Sies,H. (1997) Increased frequency of a null-allele for NAD(P)HÐquinone oxidoreductase in patients with urological malignancies. Pharmacogenetics, 7, 235±239. 64. Chen,H.W., Luni,A., Seifried,A., Wilkens,L.R. and Le Marchand,L. (1999) Association of the NAD(P)H-quinone oxidoreductase C-609-T polymorphism with a decreased lung-cancer risk. Cancer Res., 59, 3045± 3048. 65. Dhakshinamoorthy,S., Long,D.J.,II and Jaiswal,A.K. (2000) Antioxidant regulation of genes enconding enzymes that detoxify xenobiotics and carcinogens. Curr. Top. Cellul. Regul., 36, 201±216. 66. Joseph,P., Jaiswal,A.K., Stobbe,C.C. and Chapman,J.D. (1994) The role of specific reductases in the intracellular activation and binding of 2nitroimidazoles. Int. J. Radiat. Oncol. Biol. Phys., 29, 351±355. 67. Talalay,P. and Prochaska,H.J. (1987) Mechanism of induction of NAD(P)H:quinone reductase. Chem. Scr., 27A, 61±66. 68. Montano,M.M. and Katzenellenbogen,B.S. (1997) The quinone reductase geneÐa unique receptor-regulated gene that is activated by antiestrogens. Proc. Natl Acad. Sci. USA, 94, 2581±2586. 69. Montano,M.M., Jaiswal,A.K. and Katzenellenbogen,B.S. (1998) Transcriptional regulation of the human quinine reductase gene by antiestrogen-liganded estrogen receptor-alpha and estrogen receptor-beta. J. Biol. Chem., 273, 25443±25449. 70. Nebert,D.W. and Jones,J.E. (1989) Regulation of the mammalian cytochrome P1-450 (CYP1A1) gene. Int. J. Biochem., 3, 243±252. 71. Schulz,W.A. Krummeck,A., Rosinger,I., Eickelman,C., Neuhas,C., Ebert,T., Schmitz-Drager,B. and Sies,H. (1997) Increased frequency of a null allele for NAD(P)H:quinone oxidoreductase in patients with urological malignancies. Pharmacogenetics, 7, 235±239. 72. Larson,R.A., Wang,Y., Banerjee,M., Wiemels,J., Hartford,C., Beau,M.M. and Smith,M.T. (1999) Prevalence of the inactivating 609C ! T polymorphism in the NAD(P)H:quinone oxidoreductase (NQO1) gene in patients with primary and therapy-related myeloid leukemia. Blood, 94, 803±807. 73. Clairmont,A., Sies,H., Ramachandran,S., Lear,J.T., Smith,A.G., Bowers,B., Jones,P.W., Fryer,A.A. and Strange,R.C. (1999) Association of NAD(P)H:quinone oxidoreductase (NQO1) null with numbers of basal cell carcinomas use of a multivariate model to rank the relative importance of this polymophism and those at other relevant loci. Carcinogenesis, 20, 1235±1240. 74. Wiemels,J., Pagnamenta,A., Taylor,G.M., Eden,O.B., Alexander,F.E. and Greaves,M.F. (1999) A lack of functional NAD(P)H:quinone oxidoreductase allele is selectively associated with pediatric leukemias that have MLL fusions. United Kingdom Childhood Cancer Study Investigators. Cancer Res., 59, 4095±4099. Received May 2, 2003; revised June 25, 2003; accepted June 30, 2003 1703