Hydrogen Peroxide Supports Human and Rat Cytochrome P450 1A2-Catalyzed 2-Amino-3-methylimidazo[4,5-f]quinoline Bioactivation to Mutagenic Metabolites: Significance of Cytochrome P450 Peroxygenase M. Reza Anari, † P. David Josephy, ‡ Tracey Henry, ‡ and Peter J. O’Brien* ,† Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 2S2, Canada, and Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada Received August 13, 1996 X We show that the naturally occurring hydroperoxide hydrogen peroxide is highly effective in supporting the cytochrome P450 1A2 peroxygenase-catalyzed metabolic activation of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to genotoxic me- tabolites. Mutagenicity was assessed by the Ames assay with Salmonella typhimurium strain YG1012 and an activation system consisting of hydroperoxides plus either 3-methylcholan- threne-induced rat liver microsomes (rP4501A) or human P450 1A2-containing microsomes (hP4501A2). The mutagenic response was dependent on the concentration of microsomal protein, IQ, and hydroperoxides. The addition of hydrogen peroxide or tert-butyl hydroperoxide to rP4501A greatly enhanced the yield of histidine prototrophic (His + ) revertants. This increase was inhibited, in a concentration-dependent manner, by R-naphthoflavone, a P450 1A inhibitor. Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with hP4501A2 (K m ) 0.1 mM). The hydroperoxide-supported activation of IQ produced reactive intermediates which bound to 2′-deoxyguanosine; LC/MS analysis of the adducts revealed the same major (protonated) adduct at m/z ) 464.4 as previously reported for the DNA adduct formed (in vivo or in vitro) by the mixed function-catalyzed bioactivation system. None of the peroxidase- catalyzed IQ metabolites (nitro-, azo-, or azoxy-IQ) were detected. In conclusion, hydrogen peroxide in the physiological/pathological concentration range may be able to support the metabolic activation of arylamines to genotoxic products through the cytochrome P450 peroxygenase pathway. Introduction Heterocyclic aromatic amines (HAAs 1 ) are formed in proteinaceous foods as a result of pyrolysis during cooking (1). Seventeen HAAs have been identified so far in a variety of foods at parts per billion levels (1, 2). IQ and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are abundant HAAs, potent bacterial mutagens, and rodent carcinogens (2, 3); IQ is also a liver carcinogen in monkeys (4). HAAs, like many carcinogens, must be metabolically activated in order to exert their genotoxic effects. Metabolic activation occurs through N-oxidation of the exocyclic amine group catalyzed primarily by cytochrome P450 1A2 (5-8). Cytochrome P450 (P450) enzymes are extraordinarily versatile hemoproteins that catalyze the oxidation of physiological substrates and foreign chemicals, such as drugs, pesticides, polycyclic aromatic hydrocarbons, and HAAs (9, 10). The versatility also extends to the oxygen donor. Molecular oxygen serves as the natural donor when electrons are supplied to cytochrome P450 by NADPH via NADPH-cytochrome P450 reductase in the P450 monooxygenase pathway (eqs 1 and 2). In the overall reaction, equimolar amounts of substrate, O 2 , and NADPH are consumed and equimolar amounts of oxi- dized substrate, H 2 O, and NADP + are formed (11). Cytochrome P450 can also utilize reduced oxygen equivalents, e.g., hydroperoxides, to support the oxidation of various substrates, in a reaction that is independent of molecular oxygen, NADPH, and NADPH-cytochrome P450 reductase (12-18). Cytochrome P450 functions as a “peroxygenase” in this reaction and forms iron-oxygen species identical to the iron-oxene complex in the P450 monooxygenase pathway and analogous to peroxidase compound I, as shown in eqs 3 and 4 (12, 19, 20). Analysis of the microsomal hydroperoxide-supported oxidation reaction has provided valuable insights into the * Corresponding author. Phone: (416) 978-2716. Fax: (416) 978- 8511. E-mail: peter.obrien@utoronto.ca. † University of Toronto. ‡ University of Guelph. X Abstract published in Advance ACS Abstracts, April 15, 1997. 1 Abbreviations: Hepes, 4-(2-hydroxyethyl)-1-piperazineethane- sulfonic acid; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; P450, cy- tochrome P450; 3-MC, 3-methylcholanthrene; HAAs, heterocyclic aromatic amines; His + , histidine-prototrophic; MROD, methoxyresoru- fin O-demethylation; hP4501A2, human P450 1A2-containing mi- crosomes; rP4501A, 3-MC-induced rat liver microsomes; tBHP, tert- butyl hydroperoxide. P450-Fe III + O 2 + 2NADPH + 2e - f P450 •+ (Fe IV dO) + H 2 O + 2NADP + (1) P450 •+ (Fe IV dO) + X-H f P450-Fe III + X-OH (2) P450-Fe III + ROOH f P450 •+ (Fe IV dO) + ROH (3) P450 •+ (Fe IV dO) + X-H f P450-Fe III + X-OH (4) 582 Chem. Res. Toxicol. 1997, 10, 582-588 S0893-228x(96)00144-0 CCC: $14.00 © 1997 American Chemical Society