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Effect of hepatic cytochrome P450 oxidoreductase deficiency on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adduct formation in P450 reductase conditional null mice.2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochrome P450s (CYPs). In order to evaluate the role of hepatic CYPs in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to CYPs, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic CYP function. RCN mice were treated orally with 50 mg/kg body weight PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e. N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts per 10(8) deoxynucleosides), while adduct levels in liver were ~3.5-fold lower. While no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average ~2-fold lower in extra-hepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was ~8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extra-hepatic tissues and further activation resulting in the formation of dG-C8-PhIP. Besides hepatic CYPs, PhIP may be metabolically activated mainly by a non-CYP pathway in liver.