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
AUTOR(ES)
D'Agostino, Jaime
FONTE
The American Society for Pharmacology and Experimental Therapeutics
RESUMO
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 (Vmax = 1.5 nmol/min/nmol of P450; Km = 14 μM), MOI (Vmax = 1.9 nmol/min/nmol of P450; Km = 15 μM) and 3-glutathionyl-S-methylindole (Vmax = 0.7 nmol/min/nmol of P450; Km = 13 μM). 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 KI value of ∼10 μM and a kinact of 0.046 min−1. Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2769036Documentos Relacionados
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