Metabolism of 3-methylindole by vaccinia-expressed P450 enzymes: correlation of 3-methyleneindolenine formation and protein-binding.
The toxicity of 3-methylindole (3 MI), a selective pneumotoxin, is dependent upon cytochrome P450-mediated bioactivation 3. Using vaccinia-expressed P450 enzymes, the metabolites of radiolabeled 3 MI produced by 14 individual P450s were identified and quantified by high performance liquid chromatography. Indole-3-carbinol was produced from incubations of 3 MI with only four enzymes. Although 3-methyloxindole was a product of several P450s, human 1A2 was most efficient in producing this metabolite. The toxic intermediate of 3 MI is believed to be a reactive methylene imine, 3-methyleneindolenine. In this study, this intermediate was detected as its mercapturate adduct, when N-acetylcysteine was added to the incubations. 3-Methyleneindolenine was produced by CYP2A6 at a rate of 50.9 +/- 8.9 pmol/mg protein/hr and by CYP2F1 at a rate of 205.7 +/- 12.5 pmol/mg/hr. The mouse 1a-2 and rabbit 4B1 enzymes produced the reactive intermediate in amounts that exceeded that of the human 2F1 enzyme by 1.4-fold and 1.9-fold, respectively. The toxicity of 3 MI is believed to be due to covalent binding of a P450-generated intermediate to critical pulmonary proteins. Comparison of covalent binding studies to the formation of the metabolites revealed a strong correlation between the amount of the 3 MI adduct detected and covalent binding. This study showed that the methylene imine electrophile is produced by only a few P450 enzymes and is the metabolite responsible for the covalent binding and presumably, the toxicity of 3 MI. Remarkable product preferences between the desaturation pathway to form the methyleneindolenine by CYP2F1 and the ring epoxidation pathway to form the oxindole by CYP1A2, were observed.
Thornton-Manning
J
J
Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, USA.
Appleton
M L
ML
Gonzalez
F J
FJ
Yost
G S
GS
D000818:Animals; D001711:Biotransformation; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008214:Lymphocytes / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D000078183:Oxindoles; D011485:Protein Binding; D011817:Rabbits; D012862:Skatole / Q000378:metabolism* / Q000493:pharmacokinetics / Q000633:toxicity; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D007211:Indoles; D007527:Isoenzymes; D000078183:Oxindoles; C035500:3-methyloxindole; C097745:3-methyleneindolenine; D003577:Cytochrome P-450 Enzyme System; D012862:Skatole; C016517:indole-3-carbinol