Chem. Res. Toxicol. 2008, 21, 2313–2323
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Interaction of Polycyclic Aromatic Hydrocarbons with Human Cytochrome P450 1B1 in Inhibiting Catalytic Activity Tsutomu Shimada,*,† Norie Murayama,‡ Katsuhiro Tanaka,† Shigeo Takenaka,† Yoshio Imai,† Nancy E. Hopkins,§ Maryam K. Foroozesh,| Willam L. Alworth,§ Hiroshi Yamazaki,‡ F. Peter Guengerich,⊥ and Masayuki Komori† Laboratory of Cellular and Molecular Biology, Department of Veterinary Science, Osaka Prefecture UniVersity, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan, Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical UniVersity, Machida, Tokyo 194-8543, Japan, Departments of Chemistry and Cell and Molecular Biology, Tulane UniVersity, New Orleans, Louisiana 70118, Chemistry Department, XaVier UniVersity, New Orleans, Louisiana 70125, and Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232-0146 ReceiVed August 8, 2008
Eleven polycyclic aromatic hydrocarbons (PAHs) and 14 acetylenic PAHs and biphenyls were used to analyze interactions with cytochrome P450 (P450) 1B1 in inhibiting catalytic activity, using 7-ethoxyresorufin O-deethylation (EROD) as a model reaction. Most of the chemicals examined were direct inhibitors of P450 1B1 except for 4-(1-propynyl)biphenyl, a mechanism-based inhibitor. In the case of direct inhibition of EROD activity {15 of 24 chemicals, e.g., benzo[a]pyrene, 1-(1-propynyl)pyrene, and 3-(1-propynyl)phenanthrene}, restoration of the EROD activity occurred with increasing incubation time, and kinetic analysis showed that EROD Km values were higher with these inhibitors at initial stages of incubation but became lower with increasing incubation time. With the other nine chemicals, the Km values for P450 1B1-mediated EROD increased during the incubations. Acetylenic inhibitors, but not the 11 PAHs, induced reverse type I spectral changes with P450 1B1, and the low dissociation constants (Ks) suggested a role for such interaction in the inhibition of catalytic activity. Studies of quenching of P450 1B1-derived fluorescence with inhibitors demonstrated that acetylenic inhibitors and PAHs interacted rapidly with P450 1B1, with Kd values
