Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human cytochrome P4501B1.
Cytochrome P4501B1 (CYP1B1) is the most recently identified member of the dioxin-inducible CYP1 family. CYP1B1 is constitutively expressed in most human tissues, including colon and breast, and can activate numerous chemically diverse carcinogens. We evaluated the metabolism of the dietary heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by microsomes from yeast expressing the human CYP1B1 protein. PhIP metabolites were analysed by HPLC with fluorescence and absorbance detection. We found that human CYP1B1 metabolizes PhIP to three products: N2-OH-PhIP, a mutagenic activation product; 4'-OH-PhIP, a detoxification product; and 2-OH-PhIP, the mutagenic potential of which is unknown. Metabolite identity was confirmed by co-elution with authentic standards and synchronous fluorescence spectroscopy. The identity of the 2-OH-PhIP standard was additionally confirmed by mass spectrometry. Kinetic studies of the formation of N2-OH-PhIP, 4'-OH-PhIP and 2-OH-PhIP by CYP1B1 indicated apparent Km values of 5.7 +/- 1.3, 2.2 +/- 0.5 and 1.3 +/- 0.2 microM, respectively. Apparent turnover rates were 0.40 +/- 0.03, 0.93 +/- 0.02 and 0.04 +/- 0.00 nmol product/min nmol P450, respectively. At saturating levels of substrate, CYP1B1-mediated formation of the non-mutagenic metabolite 4'-OH-PhIP was favored two-fold over that of the mutagenic metabolite, N2-OH-PhIP and >10-fold over that of 2-OH-PhIP. The formation of N2-OH-PhIP, a potent mutagen implicated in the etiology of human colon and breast cancer, indicates that CYP1B1 may play an important role in PhIP-mediated carcinogenesis.
Crofts
F G
FG
Department of Environmental Health Sciences, The Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205, USA.
Strickland
P T
PT
Hayes
C L
CL
Sutter
T R
TR
D001189:Aryl Hydrocarbon Hydroxylases; D002273:Carcinogens / Q000378:metabolism / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000378:metabolism / Q000493:pharmacokinetics*; D008861:Microsomes / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002273:Carcinogens; D007093:Imidazoles; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1