Short Communication SELECTIVE DEHYDROGENATION/OXYGENATION OF 3-METHYLINDOLE BY CYTOCHROME P450 ENZYMES

3-Methylindole (3 MI) is a selective pulmonary toxicant, and cytochrome P450 (P450) bioactivation of 3 MI, through hydroxylation, epoxidation, or dehydrogenation pathways, is a prerequisite for toxicity. CYP2F1 and CYP2F3 exclusively catalyze the dehydrogenation of 3 MI to 3-methyleneindolenine, without detectable formation of the hydroxylation or epoxidation products. It was not known whether 3 MI is simply an excellent dehydrogenation substrate for all P450 enzymes, or whether certain cytochrome P450s responsible for 3 MI bioactivation have unique active sites that only catalyze the dehydrogenation of the molecule, while other P450s would catalyze only the oxygenation of 3 MI. Therefore, the kinetics of product formation by the CYP2F1 and CYP2F3 enzymes were compared with other cytochrome P450 enzymes. The enzymes tested were CYP1A1, CYP1A2, CYP1B1, and CYP2E1. The CYP1A1 and CYP1A2 enzymes produced all three 3 MI metabolites: the dehydrogenation product, 3-methyleneindolenine (Vmax/Km 5 4 and 22, respectively); the hydroxylation product, indole-3-carbinol (Vmax/Km 5 42 and 100, respectively); and the epoxidation product, 3-methyloxindole (Vmax/Km 5 4 and 72, respectively). These CYP1A enzymes catalyzed oxygenation of 3 MI at much faster rates than dehydrogenation. CYP1B1 produced indole-3-carbinol (Vmax/Km 5 85) and 3-methyloxindole (Vmax/Km 5 7), and CYP2E1 only produced 3-methyloxindole (Vmax/Km 5 98), but neither enzyme catalyzed the formation of the dehydrogenated product. Six additional P450 enzymes that were tested formed none of the dehydrogenation product. The ability of the various CYP1 family enzymes to catalyze the formation of all three major 3 MI metabolites, along with the specific oxygenation by CYP2E1, illustrates that dehydrogenation of 3 MI is not a substrate-directed process, but that the members of the CYP2F family possess unique active sites that specifically catalyze only the dehydrogenation mechanism. 3-Methylindole (3 MI) is a potent pneumotoxicant, found in tobacco smoke and intestinal or ruminant contents (Hoffman and Rathkamp, 1970; Carlson and Breeze, 1983; Yost, 1997). It requires bioactivation by cytochrome P450 (P450) enzymes before it elicits any toxic effects (Yost, 1989, 1997). The selective expression of certain cytochrome P450 enzymes in pulmonary tissue is a likely mechanism for the organ-selective toxicity (Gram, 1997) because metabolic bioactivation by P450 enzymes forms toxic electrophilic metabolites (Skordos et al., 1998a). Incubation of 3 MI with several vaccinia virus-expressed cytochrome P450s demonstrated (Thornton-Manning et al., 1996) that human P450s appeared to metabolize 3 MI to several metabolites such as indole-3-carbinol, 3-methyloxindole, and 3-methyleneindolenine (Fig. 1). Recently, metabolic studies with human CYP2F1 and goat CYP2F3 have shown that these lung-expressed enzymes form the dehydrogenation product, 3-methyleneindolenine, without apparent formation of any other 3 MI metabolites (Wang et al., 1998; Lanza et al., 1999). Examples of dehydrogenation of other substrates by P450 enzymes has generally been limited to aromatic compounds such as acetaminophen (Dahlin et al., 1984), butylated hydroxytoluene (Bolton and Thompson, 1991), and tamoxifen (Fan et al., 2000). However, the efficient dehydrogenation of “unactivated” alkanes has been demonstrated for chemicals such as ethyl carbamate by CYP2E1 (Lee et al., 1998); lauric acid by CYP2E1, CYP4A5/7, and CYP4B1 (Guan et al., 1998); and valproic acid (Rettie et al., 1995) by CYP4B1. Dehydrogenation of an azaindole-containing drug by rats, monkeys, and humans (Zhang et al., 2000), and an indole-containing drug, zafirlukast (Kassahun et al., 2000), by CYP3A enzymes, has also been recently documented. These examples validate the importance of the current studies that are designed to evaluate the catalytic preference of human P450 enzymes for the dehydrogenation versus oxygenation of a prototypical indole, 3 MI. The objective of the current study was to determine whether the selective dehydrogenation of 3 MI is primarily a function of the substrate, or whether ring oxidation (3-methyloxindole) or methyl oxygenation (indole-3-carbinol) can be the preferred route of metabolism by certain P450 enzymes. To that end, four P450s from outside the 2F subfamily were selected for metabolic/kinetic study using 3 MI as the substrate. These enzymes were chosen because they are expressed in human lung tissues and have been shown to bioactivate a variety of lung toxicants (Macé et al., 1998; Pelkonen and Raunio, 1997; Yost, 1997). Six additional P450 enzymes were evaluated at single saturating substrate concentrations of 3 MI to determine the This project was supported by USHPS Grant HL13645 from the National Heart, Lung, and Blood Institute. 1 Abbreviations used are: 3 MI, 3-methylindole; NAC, N-acetylcysteine; P450, cytochrome P450; HPLC, high-performance liquid chromatography. Address correspondence to: Dr. Garold S. Yost, Department of Pharmacology and Toxicology, 30 South, 2000 East, Room 201, University of Utah, Salt Lake City, UT 84112-5820. E-mail: [email protected] 0090-9556/01/2907-950–953$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 7 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 380/914003 DMD 29:950–953, 2001 Printed in U.S.A. 950 at A PE T Jornals on A ril 5, 2017 dm d.aspurnals.org D ow nladed from