The pneumotoxin 3-methylindole is a substrate and a mechanism-based inactivator of CYP2A13, a human cytochrome P450 enzyme preferentially expressed in the respiratory tract.

3-Methylindole (3MI), a respiratory tract toxicant, can be metabolized by a number of cytochromes P450 (P450), primarily through either dehydrogenation or epoxidation of the indole. In the present study, we assessed the bioactivation of 3MI by recombinant CYP2A13, a human P450 predominantly expressed in the respiratory tract. Four metabolites were detected, and the two principal ones were identified as indole-3-carbinol (I-3-C) and 3-methyloxindole (MOI). Bioactivation of 3MI by CYP2A13 was verified by the observation of three glutathione (GSH) adducts designated as GS-A1 (glutathione adduct 1), GS-A2 (glutathione adduct 2), and GS-A3 (glutathione adduct 3) in a NADPH- and GSH-fortified reaction system. GS-A1 and GS-A2 gave the same molecular ion at m/z 437, an increase of 305 Da over 3MI. Their structures are assigned to be 3-glutathionyl-S-methylindole and 3-methyl-2-glutathionyl-S-indole, respectively, on the basis of the mass fragmentation data obtained by high-resolution mass spectrometry. Kinetic parameters were determined for the formation of I-3-C (V(max) = 1.5 nmol/min/nmol of P450; K(m) = 14 muM), MOI (V(max) = 1.9 nmol/min/nmol of P450; K(m) = 15 muM) and 3-glutathionyl-S-methylindole (V(max) = 0.7 nmol/min/nmol of P450; K(m) = 13 muM). The structure of GS-A3, a minor adduct with a protonated molecular ion at m/z 453, is proposed to be 3-glutathionyl-S-3-methyloxindole. We also discovered that 3MI is a mechanism-based inactivator of CYP2A13, given that it produced a time-, cofactor-, and 3MI concentration-dependent loss of activity toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, with a relatively low K(I) value of approximately 10 muM and a k(inact) of 0.046 min(-1). Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.