Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. Individual susceptibility to AA-induced disease likely reflects individual differences in enzymes that metabolize AA. Herein, we evaluated AAI metabolism by human cytochrome P450 (CYP) 1A1 and 1A2 in two CYP1A-humanized mouse lines that carry functional human CYP1A1 and CYP1A2 genes in the absence of the mouse Cyp1a1/1a2 orthologs. Human and mouse hepatic microsomes and human CYPs were also studied. Human CYP1A1 and 1A2 were found to be principally responsible for reductive activation of AAI to form AAI-DNA adducts and for oxidative detoxication to 8-hydroxyaristolochic acid (AAIa), both in the intact mouse and in microsomes. Overall, AAI-DNA adduct levels were higher in CYP1A-humanized mice relative to wild-type mice, indicating that expression of human CYP1A1 and 1A2 in mice leads to higher AAI bioactivation than in mice containing the mouse CYP1A1 and 1A2 orthologs. Furthermore, an exclusive role of human CYP1A1 and 1A2 in AAI oxidation to AAIa was observed in human liver microsomes under the aerobic (i.e., oxidative) conditions. Because CYP1A2 levels in human liver are at least 100-fold greater than those of CYP1A1 and there exists a > 60-fold genetic variation in CYP1A2 levels in human populations, the role of CYP1A2 in AAI metabolism is clinically relevant. The results suggest that, in addition to CYP1A1 and 1A2 expression levels, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.

Department of Biochemistry Faculty of Science Charles University 12840 Prague 2 Czech Republic

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Aoyama T, Gonzalez FJ, Gelboin HV. Human cDNA-expressed cytochrome P450 IA2: Mutagen activation and substrate specificity. Mol. Carcinog. 1989;2:192–198. PubMed

Arlt VM, Ferluga D, Stiborová M, Pfohl-Leszkowicz A, Vukelic M, Ceovic S, Schmeiser HH, Cosyns JP. Is aristolochic acid a risk factor for Balkan endemic nephropathy-associated urothelial cancer? Int. J. Cancer. 2002;101:500–502. PubMed

Arlt VM, Levová K, Bárta F, Shi Z, Frei E, Schmeiser HH, Nebert DW, Phillips DH, Stiborová M. Role of CYP1A1 and CYP1A2 in bioactivation versus detoxication of the renal carcinogen aristolochic acid I: Studies in Cyp1a(−/−) knockout mice. Chem. Res. Toxicol. 2011a;24:1710–1719. PubMed

Arlt VM, Stiborová M, vom Brocke J, Simoes ML, Lord GM, Nortier JL, Hollstein M, Phillips DH, Schmeiser HH. Aristolochic acid mutagenesis: Molecular clues to the aetiology of Balkan endemic nephropathy-associated urothelial cancer. Carcinogenesis. 2007;28:2253–2261. PubMed

Arlt VM, Zuo J, Trenz K, Roufosse CA, Lord GM, Nortier JL, Schmeiser HH, Hollstein M, Phillips DH. Gene expression changes induced by the human carcinogen aristolochic acid I in renal and hepatic tissue of mice. Int. J. Cancer. 2011b;128:21–32. PubMed

Atanasova S, von Ahsen N, Toncheva DI, Dimitrov TG, Oellerich M, Amstrong VM. Genetic polymorphism of cytochrome P450 among patients with Balkan endemic nephropathy (BEN) Clin. Biochem. 2005;38:223–228. PubMed

Chan W, Cu L, Xu G, Cai Z. Study of the phase I and phase II metabolism of nephrotoxin aristolochic acid by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2006;20:1755–1760. PubMed

Cheung C, Ma X, Krausz KW, Kimura S, Feigenbaum L, Dalton TP, Nebert DW, Idle JR, Gonzalez FJ. Differential metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice humanized for CYP1A1 and CYP1A2. Chem. Res. Toxicol. 2005;18:1471–1478. PubMed

Debelle FD, Vanherweghem JL, Nortier JL. Aristolochic acid nephropathy: A worldwide problem. Kidney Int. 2008;74:158–169. PubMed

Dragin N, Uno S, Wang B, Dalton TP, Nebert DW. Generation of ‘humanized' hCYP1A1_1A2_Cyp1a1/1a2(−/−) mouse line. Biochem. Biophys. Res. Commun. 2007;359:635–642. PubMed PMC

Grollman AP, Shibutani S, Moriya M, Miller F, Wu L, Moll U, Suzuki N, Fernandes A, Rosenquist T, Medverec Z, et al. Aristolochic acid and the etiology of endemic (Balkan) nephropathy. Proc. Natl. Acad. Sci. U.S.A. 2007;104:12129–12134. PubMed PMC

Grosse Y, Baan R, Straif K, Secretan B, El Ghissassi F, Bouvard V, Benbrahim-Tallaa L, Guha N, Galichet L, Cogliano V. A review of human carcinogens-Part A: Pharmaceuticals. Lancet Oncol. 2009;10:13–14. PubMed

Harris AL. Hypoxia–a key regulatory factor in tumour growth. Nat. Rev. 2002;2:38–47. PubMed

Ikeya K, Jaiswal AK, Owens RA, Jones JE, Nebert DW, Kimura S. Human CYP1A2: Sequence, gene structure, comparison with the mouse and rat orthologous gene, and differences in liver 1A2 mRNA expression. Mol. Endocrinol. 1989;3:1399–1408. PubMed

Lemy A, Wissing KM, Rorive S, Zlotta A, Roumeguere T, Muniz Martinez MC, Decaestecker C, Salmon I, Abramowicz D, et al. Late onset of bladder urothelial carcinoma after kidney transplantation for end-stage aristolochic acid nephropathy: A case series with 15-year follow-up. Am. J. Kidney Dis. 2008;51:471–477. PubMed

Levová K, Moserova M, Kotrbova V, Sulc M, Henderson CJ, Wolf CR, Phillips DH, Frei E, Schmeiser HH, Mares J, et al. Role of cytochromes P450 1A1/2 in detoxication and activation of carcinogenic aristolochic acid I: Studies with the hepatic NADPH: Cytochrome P450 reductase null (HRN) mouse model. Toxicol. Sci. 2011;121:43–56. PubMed

Mimura I, Nangaku M. The suffocating kidney: Tubulointerstitial hypoxia in end-stage renal disease. Nat. Rev. Nephrol. 2010;6:667–678. PubMed

Nebert DW. Comparison of gene expression in cell culture to that in the intact animal: Relevance to drugs and environmental toxicants. Focus on “development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes.“. Am. J. Physiol. 2006;290:C37–C41. PubMed

Nortier JL, Martinez MC, Schmeiser HH, Arlt VM, Bieler CA, Petein M, Depierreux MF, De Pauw L, Abramowicz D, Vereerstraeten P, et al. Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi) N. Engl. J. Med. 2000;342:1686–1692. PubMed

Phillips DH, Arlt VM. The 32P-postlabeling assay for DNA adducts. Nat. Protoc. 2007;2:2772–2781. PubMed

Rendic S, DiCarlo FJ. Human cytochrome P450 enzymes: A status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab. Rev. 1997;29:413–480. PubMed

Rosenquist TA, Einolf HJ, Dickman KG, Wang L, Smith A, Grollman AP. Cytochrome P450 1A2 detoxicates aristolochic acid in the mouse. Drug Metab. Dispos. 2010;38:761–768. PubMed PMC

Schmeiser HH, Bieler CA, Wiessler M, van Ypersele de Strihou C, Cosyns JP. Detection of DNA adducts formed by aristolochic acid in renal tissue from patients with Chinese herbs nephropathy. Cancer Res. 1996;56:2025–2028. PubMed

Schmeiser HH, Stiborová M, Arlt VM. Chemical and molecular basis of the carcinogenicity of Aristolochia plants. Curr. Opin. Drug Discov. Devel. 2009;12:141–148. PubMed

Shi Z, Chen Y, Dong H, Amos-Kroohs RM, Nebert DW. Generation of a 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahrd mouse line harboring the poor-affinity aryl hydrocarbon receptor. Biochem. Biophys. Res. Commun. 2008;376:775–780. PubMed PMC

Shibutani S, Bonala RR, Rosenquist T, Rieger R, Suzuki N, Johnson F, Miller F, Grollman AP. Detoxification of aristolochic acid I by O-demethylation: Less nephrotoxicity and genotoxicity of aristolochic acid Ia in rodents. Int. J. Cancer. 2010;127:1021–1027. PubMed PMC

Sistkova J, Hudecek J, Hodek P, Frei E, Schmeiser HH, Stiborová M. Human cytochromes P450 1A1 and 1A2 participate in detoxication of carcinogenic aristolochic acid. Neuro. Endocrinol. Lett. 2008;29:733–737. PubMed

Stiborová M, Frei E, Arlt VM, Schmeiser HH. Metabolic activation of carcinogenic aristolochic acid, a risk factor for Balkan endemic nephropathy. Mutat. Res. 2008;658:55–67. PubMed

Stiborová M, Frei E, Hodek P, Wiessler M, Schmeiser HH. Human hepatic and renal microsomes, cytochromes P450 1A1/2, NADPH:Cytochrome P450 reductase and prostaglandin H synthase mediate the formation of aristolochic acid-DNA adducts found in patients with urothelial cancer. Int. J. Cancer. 2005a;113:189–197. PubMed

Stiborová M, Frei E, Sopko B, Sopkova K, Markova V, Lankova M, Kumstyrova T, Wiessler M, Schmeiser HH. Human cytosolic enzymes involved in the metabolic activation of carcinogenic aristolochic acid: Evidence for reductive activation by human NAD(P)H:Quinone oxidoreductase. Carcinogenesis. 2003;24:1695–1703. PubMed

Stiborová M, Frei E, Wiessler M, Schmeiser HH. Human enzymes involved in the metabolic activation of carcinogenic aristolochic acids: Evidence for reductive activation by cytochromes P450 1A1 and 1A2. Chem. Res. Toxicol. 2001;14:1128–1137. PubMed

Stiborová M, Mares J, Frei E, Arlt VM, Martínek V, Schmeiser HH. The human carcinogen aristolochic acid I is activated to form DNA adducts by human NAD(P)H:quinone oxidoreductase without the contribution of acetyltransferases or sulfotransferases. Environ. Mol. Mutagen. 2011;52:448–459. PubMed

Stiborová M, Martínek V, Rýdlová H, Hodek P, Frei E. Sudan I is a potential carcinogen for humans: Evidence for its metabolic activation and detoxication by human recombinant cytochrome P450 1A1 and liver microsomes. Cancer Res. 2002;62:5678–5684. PubMed

Stiborová M, Martínek V, Rýdlová H, Koblas T, Hodek P. Expression of cytochrome P450 1A1 and its contribution to oxidation of a potential human carcinogen 1-phenylazo-2-naphthol (Sudan I) in human livers. Cancer Lett. 2005b;220:145–154. PubMed

Stiborová M, Sejbal J, Borek-Dohalska L, Aimova D, Poljakova J, Forsterova K, Rupertova M, Wiesner J, Hudecek J, Wiessler M, et al. The anticancer drug ellipticine forms covalent DNA adducts, mediated by human cytochromes P450, through metabolism to 13-hydroxyellipticine and ellipticine N2-oxide. Cancer Res. 2004;64:8374–8380. PubMed

Stiborová M, Sopko B, Hodek P, Frei E, Schmeiser HH, Hudecek J. The binding of aristolochic acid I to the active site of human cytochromes P450 1A1 and 1A2 explains their potential to reductively activate this human carcinogen. Cancer Lett. 2005c;229:193–204. PubMed

Toncheva DI, von Ahsen N, Atanasova SY, Dimitrov TG, Amstrong VM. Identification of NQO1 and GSTs genotype frequencies in Bulgarian patients with Balkan endemic nephropathy. J. Nephrol. 2004;17:384–389. PubMed

Turesky RJ. Interspecies metabolism of heterocyclic aromatic amines and the uncertainties in extrapolation of animal toxicity data for human risk assessment. Mol. Nutr. Food Res. 2005;49:101–117. PubMed

Vanherweghem JL, Depierreux M, Tielemans C, Abramowicz D, Dratwa M, Jadoul M, Richard C, Vandervelde D, Verbeelen D, Vanhaelen-Fastre R, et al. Rapidly progressive interstitial renal fibrosis in young women: Association with slimming regimen including Chinese herbs. Lancet. 1993;341:387–391. PubMed

Xue X, Xiao Y, Zhu H, Wang H, Liu Y, Xie T, Ren J. Induction of P450 1A by 3-methylcholanthrene protects mice from aristolochic acid-I-induced acute renal injury. Nephrol. Dial. Transplant. 2008;23:3074–3081. PubMed

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