Received for publication May 30, 2007.
Revised January 21, 2008.
Accepted for publication January 23, 2008.

Metabolism of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by Human CYP1B1 Genetic Variants

Abstract

Human cytochrome P450 1B1 (CYP1B1) plays a critical role in the metabolic activation of a variety of pro-carcinogens, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The existence of human CYP1B1 missense genetic variants has been demonstrated but their activities in metabolizing PhIP are unknown. In this study, we expressed 15 naturally-occurring CYP1B1 variants (with either single or multiple amino acid substitutions) and determined their activity changes in metabolizing PhIP to its two major metabolites, 2-hydroxyamino-PhIP (N²-OH-PhIP) and 4'-hydroxy-PhIP (4'-OH-PhIP). While the PhIP-metabolizing activities of four variants (Ala¹¹⁹Ser, Pro³⁷⁹Leu, Ala⁴⁴³Gly, Arg⁴⁸Gly/Leu⁴³²Val) were comparable to that of the expressed wild-type CYP1B1, five variants (Trp⁵⁷Cys, Gly⁶¹Glu, Arg⁴⁸Gly/Ala¹¹⁹Ser, Arg⁴⁸Gly/Ala¹¹⁹Ser/ Leu⁴³²Val, Arg⁴⁸Gly/ Ala¹¹⁹Ser/Leu⁴³²Val/Ala⁴⁴³Gly) exhibited more than two fold decrease in activity and a reduction in the catalytic efficiency (V_max/K_m) for both N- and 4-hydroxylation of PhIP. Six variants (Gly³⁶⁵Trp, Glu³⁸⁷Lys, Arg³⁹⁰His, Pro⁴³⁷Leu, Asn⁴⁵³Ser, Arg⁴⁶⁹Trp) showed little activity in PhIP metabolism but the molecular mechanisms involved are apparently different. The microsomal CYP1B1 protein level was significantly decreased for the Trp³⁶⁵, Lys³⁸⁷, and His³⁹⁰ variants and was not detectable for the Ser⁴⁵³ variant. In contrast, there was no difference between the Trp⁴⁶⁹ variant and the wild-type in the microsomal CYP1B1 protein level and P450 content but the Trp⁴⁶⁹ variant totally lost its metabolic activity towards PhIP. The Leu⁴³⁷ variant also had a substantial amount of CYP1B1 protein in the microsomes but lacked detectable P450 peak and activity. Our results should be useful in selecting appropriate CYP1B1 variants as cancer susceptibility biomarkers for human population studies related to PhIP exposure.

Key words: CYP expression, CYP1B, cytochrome P450 isoforms, gene/environment interactions, HPLC, human CYP enzymes, kinetics, microsomes