Metabolic activation of polycyclic aromatic hydrocarbons and aryl and heterocyclic amines by human cytochromes P450 2A13 and 2A6

Abstract

Human cytochrome P450 (P450) 2A13 was found to interact with several polycyclic aromatic hydrocarbons (PAHs) to produce Type I binding spectra, including acenaphthene, acenaphthylene, benzo[c]phenanthrene, fluoranthene, fluoranthene-2,3-diol, and 1-nitropyrene. P450 2A6 also interacted with acenaphthene and acenaphthylene, but not with fluoranthene, fluoranthene-2,3-diol, or 1-nitropyrene. P450 1B1 is well-known to oxidize many carcinogenic PAHs, and we found that several PAHs (i.e., 7,12-dimethylbenz[a]anthracene, 7,12-dimethylbenz[a]anthracene-5,6-diol, benzo[c]phenanthrene, fluoranthene, fluoranthene-2,3-diol, 5-methylchrysene, benz[a]pyrene-4,5-diol, benzo[a]pyrene-7,8-diol, 1-nitropyrene, 2-aminoanthracene, 2-aminofluorene, and 2-acetylaminofluorene) interacted with P450 1B1, producing Reverse Type I binding spectra. Metabolic activation of PAHs and aryl- and heterocyclic amines to genotoxic products was examined in Salmonella typhimurium NM2009, and we found that P450 2A13 and 2A6 (as well as P450 1B1) were able to activate several of these procarcinogens. The former two enzymes were particularly active in catalyzing 2-aminofluorene and 2-aminoanthracene activation, and molecular docking simulations supported the results with these procarcinogens, in terms of binding in the active sites of P450 2A13 and 2A6. These results suggest that P450 2A enzymes, as well as P450 Family 1 enzymes including P450 1B1, are major enzymes involved in activating PAHs and aryl- and heterocyclic amines, as well as tobacco-related nitrosamines.

Figure 1

Absolute (A, B, and C) and Reverse Type I difference spectra of P450 1B1 (D) and Type I difference spectra of P450 2A13 (E) and 2A6 (F) induced by different concentrations of FA (A, D, and a), FA-2,3-diol (B, E, and b), and acenaphthene (C, F, and c), respectively. Inserts a, b, and c show the difference spectra for the α and β bands of the P450 enzymes. P450 concentrations used were 1.0 µM in 100 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol (v/v). The concentration of chemicals added varied from 0.25–16 µM.

Figure 2

Reverse Type I binding spectra for P450 1B1 with different concentrations of (A) FA, (B) B[c]Phe, (C) 7,12-DMBA, (D) 7,12-DMBA-5,6-diol, (E) B[a]P-4,5-diol, (F) 1-NP, and (G) 2-AF. Experimental details are the same as in the legend to Figure 1.

Figure 3

Metabolic activation (A, B, and C) and cytotoxicity (D, E, and F) of procarcinogens by P450 2A13 (A and D), P450 2A6 (B and E), and P450 1B1 (C and F) in S. typhimurium NM2009. Procarcinogens used were B[c]Phe (○), FA-2,3-diol (●), 2-AA (△), 7,12-DMDA-3,4-diol (▲), B[a]P-7,8-diol (□), 2-AF (■), and MeIQ (◇) in Parts A, B, D, and E and B[a]P-7,8-diol (■) in Parts C and F. Metabolic activation of procarcinogens by P450 enzyme system was determined by induction of umu gene expression in S. typhimurium NM2009 and cytotoxicity was determined by measuring decreased bacterial OD₆₀₀. Data are means of duplicate determinations.

Figure 4

Docking simulations of interaction of 2-AF (A, C) and 2-AA (B, D) with P450 2A13 (A , B) and P450 2A6 (C, D). The distance (2.95 Å) between the atom in the H-10 moiety of 2-AF and the NH of Asn297 in P450 2A13 is shown in Part A. U values indicate the interaction energy.

Figure 5

Docking simulations of interaction of (A) B[c]Phe, (B) FA, and (C) FA-2,3-diol (D), 7,12-DMBA, (E) 5-MeCh, (F) B[a]P, and (G) B[a]P-7,8-diol with P450 1B1. The heme group of the P450 is shown in the lower part of each part. U values indicate the interaction energy.

Figure 6

Docking simulation of interaction of (A) B[c]Phe, (B) FA, and (C) FA-2,3-diol with P450 2A13 and (D) B[c]Phe, (E) FA, and (F) FA-2,3-diol with P450 2A6. U values indicate the interaction energy.