Human Enzymes Involved in the Metabolic Activation of Carcinogenic

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Human Enzymes Involved in the Metabolic Activation of Carcinogenic Aristolochic Acids: Evidence for Reductive Activation by Cytochromes P450 1A1 and 1A2 Marie Stiborova´,*,† Eva Frei,‡ Manfred Wiessler,‡ and Heinz H. Schmeiser‡ Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, The Czech Republic, and Department of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Received March 16, 2001

Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcinogen, has recently been associated with the development of urothelial cancer in humans. Determining the capability of humans to metabolize AA and understanding, which human enzymes are involved in AA activation is important in the assessment of individual susceptibility. Using the nuclease P1-enhanced version of the 32P-postlabeling assay, we compared the ability of human, minipig and rat hepatic microsomal samples to activate AA to metabolites forming DNA adducts. Human microsomes generated AA-DNA adduct profiles reproducing those found in renal tissues from humans exposed to AA. Identical patterns of AA-DNA adducts were also observed when AA was activated by minipig and rat microsomes. Therefore, microsomes of both animals are suitable in vitro systems mimicking the enzymatic activation of AA in humans. To define the role of specific P450 enzymes and NADPH:P450 reductase in the activation of AA by human microsomes we investigated the modulation of AA-DNA adduct formation by specific inducers or selective inhibitors of P450s and cofactors or inhibitors of NADPH:P450 reductase. The inducer of P450 1A1/2, β-naphthoflavone, significantly stimulated the levels of AA-DNA adducts formed by rat microsomes, but inducers of P450 2B1/2 and 2E1 had no such effect. Furthermore, only inhibitors of the P450 1A subfamily (R-naphthoflavone, furafylline) significantly decreased the amount of adducts formed by microsomes from humans, minipigs and rats. R-Lipoic acid, an inhibitor of NADPH:P450 reductase, inhibited adduct formation too, but to a lower extent. On the basis of these results, we attribute most of the microsomal activation of AA to P450 1A1 and 1A2, although a role of NADPH:P450 reductase cannot be ruled out. With purified enzymes (recombinant P450 1A1/2 and NADPH:P450 reductase) and microsomes from baculovirus transfected insect cells expressing recombinant human P450 1A1/2 and NADPH: P450 reductase, the participation of these enzymes in the formation of AA-DNA adducts was confirmed. These results are the first report on the activation of AA by human enzymes and clearly demonstrate the role of P450 1A1, 1A2, and NADPH:P450 reductase in catalyzing the reductive activation of AA.

Introduction Some of the most potent carcinogens known are natural products (1). Among those which have been identified in plants, including safrole, cycasin, and pyrrolizidine alkaloids, aristolochic acids have recently attracted considerable attention. We reported that aristolochic acid (AA)1 is implicated in an endemic renal fibrosis, designated Chinese herbs nephropathy (2), in * To whom correspondence should be addressed. Phone: +420-221952333. Fax: +420-2-21952331. E-mail: [email protected]. † Charles University. ‡ German Cancer Research Center. 1 Abbreviations: AA, aristolochic acid; AAI, 8-methoxy-6-nitrophenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid; AAII, 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid; Ah receptor, aryl hydrocarbon receptor; R-NF, R-naphthoflavone; β-NF, β-naphthoflavone; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate; dAp, deoxyadenosine 3′-monophosphate; dGp, deoxyguanosine 3′-monophosphate; 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; IC50, 50% inhibitory dose; PEI, polyethylenimine; RAL, relative adduct labeling; XO, xanthine oxidase.

young Belgian women who had followed a slimming regimen including Chinese herbs (3-6). An inadvertent replacement of one of these herbs (Stephania tetrandra) by Aristolochia fangchi has been ascertained by the absence of tetrandrine and the detection of carcinogenic AA in the herbal powder used for the slimming pills. We detected and identified three AA-specific DNA adducts, 7-(deoxyguanosin-N2-yl) aristolactam I (dG-AAI), 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI) and 7-(deoxyadenosin-N6-yl)aristolactam II (dA-AAII), in renal tissues and thereby demonstrated that all Chinese herbs nephropathy patients had ingested the plant ingredient AA, the major alkaloid of aristolochia species. AA is a mixture of structurally related nitrophenanthrene carboxylic acids, with 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI) and 6-nitro-phenanthro-(3,4d)-1,3-dioxolo-5-carboxylic acid (AAII), being the major components (Figure 1). To date, over 100 patients suffering from Chinese herbs nephropathy have been identified (7). A third of the patients have already received a kidney transplant, another third are receiving dialysis,

10.1021/tx010059z CCC: $20.00 © 2001 American Chemical Society Published on Web 07/24/2001

Activation of Aristolochic Acid by P450 1A1/2

Chem. Res. Toxicol., Vol. 14, No. 8, 2001 1129

Figure 1. Metabolic activation and DNA adduct formation of AAI (RdOCH3) and AAII (RdH). (1) Aristolochic acids; (2) cyclic nitrenium ion of AAI or AAII; (3) aristolactams; (4) 7-(deoxyadenosin-N6-yl)aristolactam I or II (dA-AAI or dA-AAII); (5) 7-(deoxyguanosin-N2-yl) aristolactam I or II (dG-AAI or dG-AAII).

and the remainder suffers from slowly progressing renal failure (8). The persistence of the three AA-DNA adducts in human tissues (kidney and ureter), several months or even years after cessation of the regimen, is noteworthy (3-6). In human tissues, the dA-AAI was the most prominent adduct (3, 4, 6), consistent with our previous reports on rats given pure AAI or the natural mixture AA (9). Both longer persistence and higher initial levels of dA-AAI probably contributed to the relative abundance of this adduct (3, 9, 10). Moreover, H-ras protooncogenes are activated with high frequency by an AfT transversion mutation in codon 61 of DNA from AAI-induced carcinomas in rats (11). This suggests a relevant role of the dA-AAI adduct not only in the AA-induced renal fibrotic process, but also in AA-induced mutagenesis and carcinogenesis. Indeed, an increasing number of urothelial carcinomas has been identified in patients with Chinese herbs nephropathy, even after renal transplantation (1215). Therefore, patients with Chinese herbs nephropathy should undergo regular follow-ups for urothelial cancer. Interestingly, to date only 2-3% of the patients treated with the slimming regimen 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 differences in the individual activities of the enzymes catalyzing the biotransformation (detoxication and/or activation) of AA. Many genes of enzyme metabolizing carcinogens are known to exist in variant forms or show polymorphism resulting in differing activities of the gene products. These genetic variations appear to be important determinants of cancer risk (16). Therefore, screening Chinese herbs nephropathy patients as well as healthy women treated with the slimming regimen for genetic

variations in genes of the enzymes involved in AA metabolism should help to find possible relationships between genotypes and nephropathy, AA-DNA adduct levels and urothelial cancer risk. Thus, the identification of the enzymes principally involved in the activation of AA in humans and detailed knowledge of their catalytic specificities is of major importance. Recently we found that, in vitro, xanthine oxidase (XO), rat liver microsomal preparations, and even peroxidases were competent in activating both AAI and AAII to form the same DNA adducts found in vivo in rodents (9, 10, 17) and in humans (3, 4, 6). The present study was undertaken to examine the capability of human microsomal enzymes to activate aristolochic acids and to identify the human enzymes involved in AA-DNA adduct formation.

Experimental Procedures Cautions: Aristolochic acids are mutagenic and carcinogenic and should be handled with care. Exposure to 32P 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: R-naphthoflavone (R-NF), NADH, NADPH, troleandomycin, diethyldithiocarbamate, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS), ketoconazole, nuclease P1, deoxyadenosine 3′-monophosphate (dAp), deoxyguanosine 3′-monophosphate (dGp), dilauroyl phosphatidylcholine, dioleyl phosphatidylcholine, dilauroyl phosphatidylserine, and quinidine from Sigma Chemical Co. (St. Louis, MO); β-naphthoflavone (β-NF) from Aldrich Chemical Co., (Milwaukee, WI), 7-pentoxy- and 7-ethoxyresorufin from Fluka Chemie AG (Switzerland), calf thymus DNA from Roche Diagnostics Mannheim (Germany), and furafylline from New England

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Biolabs (Beverley, MA). The natural mixture 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 (18). Sulfaphenazole was kindly provided by P. Anzenbacher (Institute of Experimental Biopharmacy, Pro.Med. CS, Czech Academy of Sciences, Hradec Kra´love´). All other chemicals were of analytical purity or better. Enzymes and chemicals for the 32Ppostlabeling assay were obtained commercially from sources described previously (3, 17). Preparation of Microsomes and Assays. Rat microsomes from the livers of 10 male Wistar rats were prepared as described previously (19). Microsomes from one human liver (a 34 year old man, who died after a traffic accident) and from the liver of a male minipig were a gift from P. Anzenbacher and isolated as described (20). Microsomal preparations containing recombinant human P450 1A1, 1A2, and 3A4 were from GENTEST (Woburn, MA). Microsomes from the livers of 10 male Wistar rats pretreated with β-NF (19) were isolated as described (19, 21), those pretreated with phenobarbital (PB) as reported by Hodek et al. (22), and those pretreated with ethanol were isolated using a procedure described by Yang et al. (23). Protein concentrations in all microsomal fractions were assessed using the bicinchoninic acid protein assay (Pierce, Rockford, IL) with serum albumin as a standard (24). The concentration of P450 was estimated according to the method by Omura and Sato (25) based on the complex of reduced P450 with carbon monoxide. Specific contents of P450s were 0.60, 0.89, and 0.38 nmol/mg of protein for control (uninduced) rat, minipig and human microsomes, respectively, and 1.30, 2.74, and 1.80 for liver microsomes isolated from rats pretreated with β-NF, PB, and ethanol, respectively. Each microsomal preparation was analyzed for specific P450 activities. The assays used were ethoxyresorufin O-deethylation (P450 1A1/2), pentoxyresorufin O-depentylation (P450 2B1/2 in rat microsomes) (26) and benzyloxyresorufin-O-debenzylation (P450 2B6 in human microsomes), bufuralol 1′-hydroxylation (P450 2D), tolbutamide methyl hydroxylation (P450 2C), chlorzoxazone 6-hydroxylation (P450 2E1), and testosteron 6β-hydroxylation (P450 3A) (27; and references therein). The activity of NADPH:P450 reductase was measured according to Sottocasa et al. (28) using cytochrome c as substrate (i.e., as NADPH:cytochrome c reductase). The activities of P450s and NADPH:P450 reductase are shown in Table S1, which is present in Supporting Information (see below). The concentration of NADPH:P450 reductase was estimated as described earlier (29). Enzyme Preparations. Recombinant rat P450 1A1 protein was purified to homogeneity from membranes of Escherichia coli, in which a modified P450 1A1 cDNA had been expressed, at the laboratory of H. W. Strobel (University of Texas, Medical School of Houston, Texas) by P. Hodek (Charles University, Prague) by a procedure described previously (30). Recombinant human P450 1A2 was from Oxford Biomedical Research, Inc. (Oxford, U.K.). Rabbit liver NADPH:P450 reductase was purified as described (31). Incubations. The deaerated and argon-purged incubation mixtures contained in a final volume of 0.75 mL: 50 mM potassium phosphate buffer (pH 7.4), 1 mM NADPH, 3.5 mg of microsomal protein, 0.5 mM AAI or AAII as sodium salts dissolved in water and 1 mg of calf thymus DNA (4 mM). The reaction was initiated by adding NADPH. Incubations were carried out at 37 °C for 60 min. Control incubations were carried out either without activating system (microsomes) or with activating system and AAI and AAII, but without DNA or with activating system and DNA but without AAs. Incubation mixtures, in which microsomes containing human recombinant P450s (Supersomes) were used to activate AAI and AAII, were of the same composition except that 50 pmol of supersomal P450s were added instead of hepatic microsomes. Furthermore, purified P450 1A1 and 1A2 reconstituted with NADPH:P450 reductase were used as the additional activation system. Recombinant rat P450 1A1 and human P450 1A2 reconstituted

Stiborova´ et al. with rabbit NADPH:P450 reductase were used in these experiments. Reconstitution of P450s with NADPH:P450 reductase was carried out essentially as described earlier (32). Briefly, P450 was reconstituted as follows: 0.5 µM P450s, 0.5 µM NADPH:P450 reductase, 0.5 µg of CHAPS/µL, 0.1 µg of liposomes/µL [dilauroyl phosphatidylcholine, dioleyl phosphatidylcholine, dilauroyl phosphatidylserine (1:1:1)], 3 mM reduced glutathione and 50 mM HEPES/KOH (pH 7.4) were mixed. An aliquot containing appropriate amounts of reconstituted P450 (0.125-1.0 nmol) was used for activation of AAs by its adding, instead of microsomes, to incubation mixtures of composition described above. In incubations testing the activity of pure NADPH:P450 reductase, the reaction mixtures were essentially the same as in the reconstitution experiments except that the P450 was omitted from the reconstitution mixture. After incubation of all reaction mixtures (37 °C, 60 min), the mixtures were extracted twice with ethyl acetate (2 × 2 mL). DNA was isolated from the residual water phase by the phenol/chloroform extraction method as described earlier (17, 19, 33). The content of DNA was determined spectrophotometrically (34). Inhibition Studies. The following chemicals were used to inhibit the activation of AAs in rat, minipig and human hepatic microsomes and in Supersomes (specific P450 enzymes known to be inhibited): R-NF (P450 1A1 and 1A2), furafylline (P450 1A2), diethyldithiocarbamate (P450 2A6 and 2E1), sulfaphenazole (P450 2C), quinidine (P450 2D), troleandomycin, and ketoconazole (P450 3A) (35). Inhibitors dissolved in 7.5 µL of methanol, to yield final concentrations 1-500 µM, depending on the chemical (except diethyldithiocarbamate which was dissolved in water to yield final concentrations 1-1500 µM), were added to the incubation mixtures. The effect of R-lipoic acid (a selective inhibitor of NADPH:P450 reductase) (36) was also tested (1.65.0 mM R-lipoic acid was used). An equal volume of methanol alone was added to the control incubations. The mixtures were then incubated at 37 °C for 10 min with NADPH prior to adding the AAI or AAII. The reaction mixtures were further incubated at 37 °C for 60 min. After incubation, the mixtures were extracted by ethyl acetate and DNA was isolated by procedures described above. 32P-Postlabeling Analysis. The nuclease P1 enrichment version (37) and the 1-butanol extraction-mediated enrichment procedure (38) were used. DNA samples (12.5 µg) were digested with micrococcal nuclease (750 milliunits) and spleen phosphodiesterase (12.5 milliunits) in digestion buffer (20 mM sodium succinate, 8 mM CaCl2, pH 6.0) for 3 h at 37 °C in a total volume of 12.5 µL. Digests (2.5 µL) were removed and diluted 1:1500 to determine the amount of normal nucleotides. In the nuclease P1 version digests (10 µL) were enriched for adducts by incubation with 5 µg (5 units) of nuclease P1 in 3 µL of a buffer containing 0.8 M sodium acetate (pH 5.0), 2 mM ZnCl2 for 30 min at 37 °C. The reaction was stopped by adding 3 µL of 427 mM tris base. The extraction with 1-butanol to enrich the adducts was carried out as described earlier (38). Labeling mix (4 µL) consisting of 400 mM bicine (pH 9.5), 300 mM dithiothreitol, 200 mM MgCl2, 10 mM spermidine, 100 µCi of [γ-32P]ATP (15 pmol), 0.5 µL of 90 µM ATP, and 10 units of T4 polynucleotide kinase were added. After incubation for 30 min at room temperature, 20 µL were applied to a polyethylenimine (PEI)-coated cellulose TLC plate (Macherey-Nagel, Du¨ren, Germany) and chromatographed as described (39) except that D3 and D4 were adjusted to pH 4.0 and 9.1 for better resolution. To determine the amount of normal nucleotides 5 µL of the 1:1500 dilution of digests were mixed with 2.5 µL of tris buffer (10 mM, pH 9.0) and 2.5 µL of labeling mix (see above) and incubated for 30 min at room temperature. The labeling mixture was diluted by mixing 4 µL with 750 µL of 10 mM tris buffer (pH 9.0). This solution (5 µL) was applied to a PEIcellulose TLC plate and run in 0.28 M (NH4)2SO4, 50 mM NaH2PO4 (pH 6.5). Adducts and normal nucleotides were detected and quantitated by an Instant imager (Packard). Count rates of adducted fractions were determined from triplicate maps after subtraction of count rates from adjacent blank areas. Excess



Figure 2. Autoradiographic profiles of AAI-DNA (A) and AAII-DNA (B) adducts obtained from calf thymus DNA after activation by hepatic microsomes from human (Aa, Ba), minipig (Ab, Bb), and rat (Ac, Bc). 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 room temperature for 1 h (AAI adducts) or 2 h (AAII adducts). 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 NaH2PO4, pH 6.0. Spot 1, dG-AAI; spot 2, dA-AAI; spot 3, dA-AAII; spot 4, dG-AAII. [γ-32P]ATP after the postlabeling reaction was confirmed. Adduct levels were calculated in units of relative adduct labeling (RAL) which is the ratio of counts per minute of adducted nucleotides to counts per minute of total nucleotides in the assay. Enzymatic synthesis of reference compounds, dAp-AAI, dGp-AAI, dAp-AAII, and dGp-AAII and their 32P-postlabeling were carried out as described earlier (9). Co-Chromatography on PEI-Cellulose. Adduct spots detected by the 32P-postlabeling assay were excised from the thin-layer plates, extracted, and co-chromatographed with reference 3′,5′ bisphosphate adducts as reported previously (9). HPLC Analysis of 32P-Labeled 3′,5′-Deoxyribonucleoside Bisphosphate Adducts. HPLC analysis was performed essentially as described previously (9, 17, 40). Individual spots detected by the 32P-postlabeling assay were excised from thin layer plates and extracted (41). The dried extracts were redissolved in 100 µL of methanol/phosphate buffer (pH 3.5) 1:1 (v/v). Aliquots (50 µL) were analyzed on a phenyl-modified reversed-phase column (250 × 4.6 mm, 5 µm Zorbax Phenyl; Sa¨ulentechnik 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 µL cell, dwell time 6 s). Statistical Analyses. Statistical association between total P450 levels, P450- and NADPH:P450 reductase-linked catalytic activities in hepatic microsomal samples and levels of individual AA-DNA adducts formed by the same microsomes were determined by the Spearman correlation coefficient using version 6.12 Statistical Analysis System sofware. Spearman correlation coefficients were based on a sample size of 6. All Ps are twotailed and considered significant at the 0.05 level.

Results Activation of Aristolochic Acids by Human, Minipig, and Rat Microsomes. We investigated the

formation of adducts by AAI and AAII in calf thymus DNA in the presence of microsomes isolated from rat, minipig, and human liver. All in vitro incubations were performed under standardized conditions of AAs (0.5 mM), dNp (4 mM) as calf thymus DNA, microsomal protein (3.5 mg) and NADPH (1 mM) to ensure adduct levels easily detectable by the 32P-postlabeling method (RAL range 1 adduct in 106-108 nucleotides for enzymatic reactions catalyzed by microsomes). Because enrichment of AA-DNA adducts by n-butanol extraction yielded a lower recovery for these adducts (80-90%) as compared to the nuclease P1 version of the 32P-postlabeling assay (9), all further experiments were carried out with enrichment by nuclease P1. All microsomes were capable of activating both AAI and AAII to form DNA adducts. It is evident from Figure 2 that AAI and AAII activated by microsomes from different species generated the same major DNA adduct spots as those obtained in vivo in rats and humans and as reported previously (3, 4, 6, 9, 39). Quantitative analysis (Table 1) revealed that the extent of DNA binding by AAI was always higher than by AAII irrespective of the type of microsomes used for activation. In the species comparison, microsomes from minipig were the most effective activation system for both AAs, followed by human and rat microsomes (Table 1). Control incubations carried out in parallel either without microsomes, or without DNA, or without AAs were free of adduct spots in the region of interest even after prolonged exposure times. Adduct spots 1, 2, 3, and 4 formed by AAs (Figure 2) cochromatographed on PEI-cellulose TLC plates (not shown) and by reversed-phase HPLC (Figure 3) with those of synthetic standards (9). Thus, spot 1 was assigned to 3′,5′-bisphospho-7-(deoxyguanosin-N2-yl)aristolactam I (dG-AAI), spot 2 to 3′,5′-bisphospho-7-

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Table 1. Quantitative Analysis of Adducts Formed in DNA by AAI and AAII Activated by Hepatic Microsomal Samplesa RALb (mean ( SD/107 nucleotides) control microsomes from AA-DNA adduct AAI-DNA adduct dG-AAI dA-AAI dA-AAII total AAII-DNA adduct dG-AAII dA-AAII total b

microsomes of rats induced with

human

minipig

rat

β-NF

PB

ethanol

1.36 ( 0.14 4.73 ( 0.50 0.27 ( 0.03 6.36 ( 0.70

1.85 ( 0.19 7.07 ( 0.81 1.75 ( 0.18 10.67 ( 1.1

1.96 ( 0.20 3.50 ( 0.36 0.28 ( 0.03 5.74 ( 0.60

6.23 ( 0.59 10.8 ( 1.5 1.24 ( 0.13 18.3 ( 1.8

1.82 ( 0.18 3.28 ( 0.33 0.42 ( 0.18 5.52 ( 0.56

1.73 ( 0.18 3.00 ( 0.30 0.21 ( 0.02 4.94 ( 0.51

0.14 ( 0.01 0.62 ( 0.06 0.76 ( 0.07

0.52 ( 0.05 1.56 ( 0.16 2.08 ( 0.21

0.14 ( 0.01 0.59 ( 0.06 0.73 ( 0.07

0.53 ( 0.05 1.72 ( 0.18 2.25 ( 0.21

0.20 ( 0.03 0.69 ( 0.10 0.89 ( 0.14

0.10 ( 0.01 0.48 ( 0.05 0.58 ( 0.06

a Numbers are averages ( SD (n ) 6) of triplicate in vitro incubations, each DNA sample was determined by two postlabeled analyses. Relative adduct labeling.

Figure 3. Separation of 32P-labeled nucleoside 3′,5′-bisphosphate adducts derived from AAI and AAII on a phenyl-modified reversedphase column. Chromatographic conditions are described in Experimental Procedures. Adduct spots were excised and extracted from PEI-plates as shown in Figure 2, dissolved and injected. Standards were obtained from in vitro incubations as described (9). For clarity, HPLC profiles are shown in arbitrary units.

(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), spot 3 to 3′,5′-bisphospho-7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII), and spot 4 to 3′,5′-bisphospho-7-(deoxyguanosin-N2-yl)-aristolactam II (dG-AAII). These adducts are known to be generated from AAs by nitro reduction (9, 42, 43). Therefore, the microsomes tested in this study contain enzymatic systems capable of catalyzing the reductive activation of AAs leading to formation of these

DNA adducts. NADPH:P450 reductase, NADH cytochrome b5 reductase and even P450s present in microsomes are candidates for the reductive activation of AAs. To investigate these possibilities, the influences of various cofactors upon AA-DNA adduct formation catalyzed by rat microsomes were examined. As shown in Table 2 the formation of AA-DNA adducts had an absolute requirement for NADPH, a known



Table 2. Effect of Enzyme Cofactors on the AA-DNA Adduct Formation in Rat Liver Microsomes RALa (mean ( SD/107 nucleotides) AAI

a

AAII

cofactor (1 mM)

dG-AAI

dA-AAI

dA-AAII

dG-AAII

dA-AAII

none NADPH NADPH + NADH NADH

0.01 ( 0.001 1.96 ( 0.20 3.21 ( 0.33 0.09 ( 0.01

0.04 ( 0.005 3.50 ( 0.36 5.60 ( 0.62 0.35 ( 0.05

0.003 ( 0.001 0.28 ( 0.03 0.46 (0.05 0.03 ( 0.01

0.001 ( 0.0005 0.14 ( 0.01 0.17 ( 0.02 0.02 ( 0.001

0.005 ( 0.001 0.59 ( 0.06 0.66 ( 0.07 0.08 ( 0.001


Table 3. Spearman Correlation Coefficients (r) among P450- and NADPH:P450 Reductase-Linked Activities, Total P450 Content, and Levels of AA-DNA Adducts Formed by Human, Minipig, and Rat Microsomes

adduct

ethoxyresorufin O-deethylationd (P450 1A1/2)

tolbutamide methyl hydroxylationd (P450 2C)

bufurarol 1′ hydroxylationd (P450 2D)

chlorzoxazone 6 hydroxylationd (P450 2E1)

testosteron 6β hydroxylationd (P450 3A)

P450 contente

P450 contentd

NADPH:P450 reductased

dG-AAI dA-AAI dA-AAII total dG-AAII dA-AAII total

0.97a 0.94b 0.60 0.97a 0.77 0.83b 0.81c

0.29 -0.22 -0.42 -0.08 -0.29 -0.28 -0.28

-0.49 -0.04 0.17 -0.18 0.03 0.01 0.01

-0.46 -0.27 -0.36 -0.37 -0.43 -0.40 -0.41

-0.41 -0.70 -0.94b -0.68 -0.88c -0.87c -0.87c

0.88 0.93c 0.74 0.96b 0.89 0.92c 0.91c

0.04 0.12 -0.13 -0.13 0.12 0.06 0.08

-0.17 -0.37 -0.24 -0.31 0.04 -0.01 -0.01

a P < 0.001. b P < 0.01. c P < 0.05. d Data from hepatic microsomes of all species (uninduced rat, minipig, human) as well as rat induced with β-NF, PB, ethanol were used for correlation analysis. e Data from hepatic human, minipig, rat, and β-NF-rat microsomes were used for correlation analysis.

cofactor of NADPH:P450 reductase and/or P450-dependent enzyme systems. Adduct levels were negligible when NADPH was omitted from the incubation mixture. NADH, a cofactor of the microsomal NADH:cytochrome b5 reductase serving as a second electron donor for P450dependent systems (44), stimulated AA-DNA adduct formation when added together with NADPH. NADH alone was much less efficient as cofactor than NADPH. These results indicate a minor role of NADH:cytochrome b5 reductase in AA activation. To resolve which microsomal enzymes are mainly responsible for activation of AAs, five experimental approaches were employed: (i) induction of NADPH:P450 reductase and specific P450s, (ii) correlation of the AAactivation efficiencies under consideration with known marker activities of NADPH:P450 reductase and individual P450s, (iii) selective enzyme inhibition, (iv) purification of enzymes (NADPH:P450 reductase and recombinant P450s) and reconstitution, and (v) use of heterologous baculovirus expression systems of human P450s. Activation of Aristolochic Acids by Microsomes of Rats Pretreated with Inducers of P450 Enzymes and NADPH:P450 Reductase. The induction of individual enzymes was performed with rats. Microsomes isolated from livers of uninduced rats and those induced by β-NF (enriched with P450 1A1/2), PB (enriched with NADPH:P450 reductase and P450 2B1/2) and ethanol (enriched with P450 2E1) were used in the experiments. The induction of NADPH:P450 reductase (PB-microsomes) had no significant effect on AA-DNA adduct formation (Table 1). Consistent with these results, no correlation was found between NADPH:P450 reductase activities in microsomes and AA-DNA adduct levels (Table 3). However, an inhibitor of NADPH:P450 reductase, R-lipoic acid (36), inhibited AA-DNA adduct formation by microsomes in a dose dependent manner. Significant inhibition was found when a 10-fold molar excess of this inhibitor over AAs was used.

Incubations of AAs with DNA and β-NF microsomes led to a 3-fold increase in total DNA-binding of AAI and AAII (Table 1). Induction with PB or with ethanol had no significant effect on AA-DNA adduct formation (Table 1). These results were consistent with the correlation analysis. Total AA-DNA adduct formation was highly correlated with the levels of total P450 (nmol P450), when P450 levels measured in all species and those in β-NF microsomes were used for analyses (Table 3). However, no correlation was found when the microsomal samples induced with PB and ethanol were included into correlation analysis. Furthermore, a highly significant correlation was found between ethoxyresorufin O-deethylase activity, a marker for P450 1A, in all microsomes, and the formation of AA-DNA adducts (Table 3). No significant correlation was determined between any other P450 activities (P450 2C, 2D, 2E1, and 3A) and formation of AA-DNA adducts (Table 3). Effect of Inhibitors on Activation of Aristolochic Acids by Microsomes. Inhibition experiments further supported the role of P4501A enzymes in the activation of AAs. R-NF, a selective inhibitor of P450 1A1/2, and furafylline of P450 1A2, were highly effective in inhibiting AA-DNA adduct formation (Table 4) with the lowest IC50 values of 8 µM R-NF for AAII adduct formation in human and minipig microsomes. However, the inhibition of AADNA adduct formation by both inhibitors was not complete. A 5-10% residual microsomal activity was always seen resulting from other enzymes (i.e., NADPH:P450 reductase). Inhibitors of other P450 enzymes caused either weak (diethyldithiocarbamate) or no inhibition (sulfaphenazole, quinidine, troleandomycin, ketoconazole) of AA-DNA adduct formation. Collectively, these results indicate a major role of P450 1A1/2 and a minor, but detectable, role of NADPH:P450 reductase in the reductive activation pathway of AAs. Activation of Aristolochic Acids by Purified NADPH:P450 Reductase, P450 1A1, and 1A2. To

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Stiborova´ et al.

Table 4. Inhibition Constants for Inhibitors of AA-DNA Adduct Formation in Human, Minipig, and Rat Microsomesc IC50 (µM),b microsomes from inhibitora R-naphthoflavone (P450 1A1/2) AAI adducts AAII adducts furafylline (P450 1A2) AAI adducts AAII adducts sulfaphenazol (P450 2C) AAI adducts AAII adducts quinidine (P450 2D) AAI adducts AAII adducts diethyldithiocarbamate (P450 2A6, 2E1) AAI adducts AAII adducts troleandomycin (P450 3A) AAI adducts AAII adducts ketoconazole (P450 3A) AAI adducts AAII adducts

human

minipig

rat

β-NF rat

45 ( 5 8(1

52 ( 5 8(1

58.( 5 21 ( 2

37 ( 3 58 ( 6

16 ( 2 80 ( 8

20 ( 2 100 ( 11

52 ( 5 100 ( 13

31 ( 2 15 ( 2

n.i. n.i.

n.i. n.i.

n.i. n.i.

n.d. n.d.

n.i. n.i.

n.i. n.i.

n.i. n.i.

n.d. n.d.

n.i. n.i.

n.i. 900 ( 89

n.i. n.i.

n.i. n.i.

n.i. n.i.

n.i. n.i.

n.i. n.i.

n.i. n.i.

n.i. n.i.

n.i. n.i.

n.i. n.i.

n.i. n.i.

a Isoforms of P450 inhibited by selective inhibitors are shown in brackets. b Estimated from concentration-dependent inhibition of AA-DNA adduct formation (using 1-1500 µM concentrations of inhibitors depending on the chemical) by interpolation. 0.5 mM AAI or AAII was present in the incubation medium. c n.i., no inhibition, that is IC50 greater than 1000 µM. n.d., not determined. Numbers are averages ( SD (n ) 6) of triplicate in vitro incubations, each DNA sample was determined by two postlabeled analyses.

confirm the role of P450 1A1, 1A2 and NADPH:P450 reductase in the activation of AAs, these enzymes were purified and NADPH:P450 reductase used alone or reconstituted with P450 1A1 or 1A2. Figure 4 shows that incubations of AAs with DNA, purified NADPH:P450 reductase and its cofactor, NADPH, resulted in the formation of the same pattern of DNA adducts as that determined in either microsomes (present paper) or in vivo (3, 4, 6, 9) and in the complete reconstitution system with P450 1A1. The identity of adducts were confirmed by TLC cochromatography and HPLC (not shown). AAIDNA adduct formation was dependent on the molar ratio of P450/NADPH:P450 reductase. Optimal ratios of P450 enzymes to reductase were close to a value of 1.0 for both P450 enzymes. Nitroreductive activation of AAI was more prominent with P450 1A1 than with P450 1A2. Under optimal ratios of P450s to reductase, a 5.5- and 2.3-fold increase of AAI-DNA adduct levels were found for P450 1A1 and 1A2, respectively. P450 1A1- and 1A2-mediated DNA-binding of AAI increased with increasing amounts of P450s reconstituted with reductase up to 500 pmol of both enzymes and then reached a plateau level at higher enzyme concentrations (Figure S1 in Supporting Information). In the case of AAII, higher concentrations of reconstituted enzymes were needed to reach saturation (Figure S2 in Supporting Information). Aristolochic Acids Are Activated by Recombinant Human P450 1A1 and 1A2, but Not by P450 3A4. Only purified P450 1A2 was available from man. P450 1A1 used in the experiments was the rat enzyme. It is known that the P450s of rats are sometimes not suitable models to mimic the catalytic properties of orthologous human enzymes. The human P450 1A1 enzyme however is available in microsomes from a baculovirus insect cells expression system (Supersomes). We compared Supersomes containing either human P450 1A1 or 1A2 and NADPH:P450 reductase. Moreover, Supersomes with P450 3A4 were also tested. Supersomes containing P450 1A1 and P450 1A2 were capable of catalyzing AA-adduct

formation in calf thymus DNA to nearly the same extent and both R-NF and furafylline inhibited DNA adduct formation (Table 5). The effect of these two inhibitors on NADPH:P450 reductase is not known. Both compounds were no inhibitors of NADPH:P450 reductase catalyzed AA reduction but on the contrary had a slight stimulating activity. Essentially no AA-adducts were detected in DNA using Supersomes containing human CYP3A4 as the activation system.

Discussion The detection and quantitation of specific DNA adducts by the 32P-postlabeling procedure has proven a useful tool to monitor exposure to the plant carcinogen AA in vivo (3, 4, 6, 9, 10, 45). Based on the structures of the adducts identified, we reported that nitro reduction of AAI and AAII to the corresponding aristolactams I and II is the main activating pathway in animals (9, 42, 43, 45) and humans (3, 4, 6). As shown in Figure 1 an intermediate cyclic nitrenium ion with a delocalized positive charge was postulated by us as the ultimate electrophilic species binding to DNA, via the C7, to the exocyclic amino groups of dG and dA. Recently, specific AA-DNA adducts were found to be associated with an unique nephropathy, Chinese herbs nephropathy, and urothelial cancer in women who had followed a weight reducing treatment consisting of Chinese herbs containing AA. Not all participants in the slimming procedure are affected by Chinese herbs nephropathy. Differences in carcinogen activation could be the reason for individual susceptibility. In mammalian tissues, both cytosol and microsomes contain enzymes catalyzing the reduction of nitro aromatic compounds (46-48). Nitroreduction by microsomal enzymes is catalyzed by P450s and NADPH:P450 reductase (47). The present paper reports on the identification of microsomal enzymes of different species including man, which participate in the reductive bioactivation of AA.



Table 5. Quantitative Analysis of Adducts Formed in DNA by AAI and AAII Activated by Supersomes Containing 50 pmol of Recombinant Human P450 1A1 or 1A2 in the Absence or Presence of 100 µM Selective P450 Inhibitors r-Naphthoflavone and Furafyllinea RALb (mean ( SD/107 nucleotides) supersomes with P450 1A1 AA-DNA adduct AAI-DNA adducts dG-AAI dA-AAI dA-AAII total AAII-DNA adducts dG-AAII dA-AAII total b

P450 1A2

without inhibitor

+R-NF

without inhibitor

+R-NF

+furafylline

5.2 ( 0.5 15.5 ( 1.3 1.2 ( 0.02 21.9 ( 2.1

1.7 ( 0.2 5.7 ( 0.4 0.4 ( 0.03 7.8 ( 0.8

5.2 ( 0.4 12.9 ( 1.2 0.9 ( 0.1 19.0 ( 1.8

0.4 ( 0.04 0.8 ( 0.06 0.1 ( 0.01 1.3 ( 0.1

0.3 ( 0.03 0.4 ( 0.05 0.1 ( 0.02 0.8 ( 0.09

0.5 ( 0.05 2.0 ( 0.2 2.5 ( 0.3

0.1 ( 0.03 0.6 ( 0.05 0.7 ( 0.08

0.1 ( 0.01 0.4 ( 0.05 0.5 ( 0.05

0.08 ( 0.01 0.2 ( 0.02 0.28 ( 0.03

0.4 ( 0.04 1.6 ( 0.2 2.0 ( 0.2


Figure 4. Autoradiographic profiles of AAI- (A) and AAII-DNA (B) adducts obtained from calf thymus DNA after activation by either NADPH:P450 reductase (Ab, Bb) or P450 1A1 reconstituted with NADPH:P450 reductase (Aa, Ba). The nuclease P1enrichment procedure was used for analysis. Origins, in the bottom left-hand corner were cut off before exposure. Screen enhanced autoradiography was at room temperature from 10 to 20 min. 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 NaH2PO4, pH 6.0.

We have already identified microsomes from rat liver which generate AAI- and AAII-DNA adduct profiles very similar or identical to the profiles found in the target tissue, as the most appropriate activation system in vitro (17). The results of the present study clearly demonstrate that AAs are bioactivated also by human microsomes forming dG and dA adducts identical to those found in humans exposed to AA (3, 4). The reductive formation of AA-DNA adducts is strongly dependent on the catalytic activities of P450s present in microsomes. In microsomes of three different species (human, minipig, and rat), nitroreduction of AA leading to DNA adduct formation was predominantly catalyzed by P450 1A. This conclusion is supported by a strong correlation coefficient between the levels of AA-DNA adducts formed in various microsomes and rates of ethoxyresorufin O-deethylation, a

P450 1A-dependent reaction. A drastic inhibition of AADNA adduct formation by R-NF and furafylline, specific inhibitors of P450 1A1/2 and P450 1A2, respectively, provided additional evidence for the role of P450 1A1/2 in AA activation. Using pure recombinant enzymes (P450 1A1/2) reconstituted with NADPH:P450 reductase and microsomes containing human recombinantly expressed P450 1A1/2 confirmed these results. The identification of human P450s in reductive activation of the nitroaromatic substances AAI and AAII is consistent with results reported by Chae et al. (46). There the authors showed that human recombinant P450 1A2 reconstituted with NADPH:P450 reductase was able to reduce 4-nitropyrene and to a lower extent also 1- and 2-nitropyrene. P450 3A4 present in human hepatic microsomal samples as well as human recombinant P450 3A4 in the reconstitution system was also involved in the nitroreduction of 4-nitropyrene (46), but this enzyme was ineffective in reductive activation of AAI and AAII (present paper). Purified rabbit NADPH:P450 reductase alone also catalyzed AA-DNA adduct formation in vitro. If NADPH: P450 reductase is, however, present in the microsomal fractions of human and rats, it seems to have a rather less, but measurable, capacity to activate AA compared to P450s of the 1A subfamily. At present, we can only speculate on the discrepancy between the isolated enzyme and the intact system. A competition between the active sites of the two systems may arise in microsomes with a higher accessibility of the active center of P450 enzymes for AAI and AAII than that of the reductase. Soluble enzymes with reducing activity such as buttermilk XO are known to be effective nitroreductases activating several nitro-aromatics including AAs (17, 42, 43, 47, 48). Although the contributions of human cytosolic XO or other human soluble reductases (D,T-diaphorase, aldehyde oxidase) to the nitroreductive metabolism of AA have not been examined, the present study clearly indicates a significant role of microsomal P450 1A1/2 enzymes in the nitroreduction of AA. While the human P450 1A2 protein is constitutively expressed in livers and intestine, human P450 1A1 is an extrahepatic enzyme expressed in lungs, kidneys, gastrointestinal, and urinary tract upon induction. Levels of expression and activities of both P450s in humans are influenced by several factors (nutrition, smoking, drugs, environmental chemicals, and genetic polymorphisms) (49-53) and differ considerably among individuals. The polymorphic expression of P450 1A1 and P450 1A2 has been attributed to altered expres-

1136


sion of the transcription factor that modulates its regulation, that is, the aryl hydrocarbon (Ah) receptor, or its associated transcription factor, the Ah receptor nuclear translocator or Arnt protein (16). Moreover, the human P450 1A1 gene is genetically polymorphic (49-51). The variability of P450 1A1/2 levels and activities might play a major role in determining risk to the carcinogenicity of AA for participants in the slimming cure. To test this hypothesis, patients with Chinese herbs nephropathy and other participants in the slimming regimen will be screened for genetic polymorphisms of genes of enzymes involved in xenobiotic metabolism in the next phase of our work.

Acknowledgment. Supported by Grant Agency of the Czech Republic (Grant 303/99/0893) and the Ministry of Education of the Czech Republic (Grant MSM 1131 00001). We are grateful to Prof. Dr. H. W. Strobel (University of Texas, Houston, TX) for the possibility to isolate recombinantly expressed rat P450 1A1 in his laboratory, to M. Sˇ ulc (Charles University, Prague) for isolation of rabbit NADPH:P450 reductase, to V. BorˇekDohalsky´ for statistical expertise, and to A. Breuer and K. Klokow (German Cancer Research Center, Heidelberg) for excellent technical assistance. Supporting Information Available: Table of cytochrome P450- and NADPH:P450 reductase-dependent catalytic activities in hepatic microsomal samples, and figures of dependence of DNA adduct formation from 0.5 mM AAI on the concentrations of NADPH:P450 reductase alone and on those of P450 1A1 or P450 1A2 reconstituted with equimolar NADPH:P450 reductase. dG-AAI (A), dA-AAI (B), dA-AAII (C), and total adducts (D), and dependence of DNA adduct formation from 0.5 mM AAII on the concentrations of NADPH:P450 reductase alone and those of P450 1A1 or P450 1A2 reconstituted with equimolar NADPH:P450 reductase. dG-AAII (A), dA-AAII (B), and total adducts (C). This material is available free of charge via the Internet at http://pubs.acs.org.

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Human Enzymes Involved in the Metabolic Activation of Carcinogenic

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