Evidence Supporting the Formation of 2,3-Epoxy-3-methylindoline:  A Reactive Intermediate of the Pneumotoxin 3-Methylindole

• Published in: Chemical research in toxicology 1998-07, Vol.11 (7), p.741-749
• Main Authors: Skordos, Konstantine W, Skiles, Gary L, Laycock, John D, Lanza, Diane L, Yost, Garold S
• Format: Article
• Language: English
• Subjects: Aldehyde Oxidase; Aldehyde Oxidoreductases - metabolism; Animals; Chromatography, Liquid; Cytochrome P-450 Enzyme System - metabolism; Epoxy Compounds - chemistry; Gas Chromatography-Mass Spectrometry; Goats; Indoles - chemistry; Male; Mice; Microsomes - chemistry; Microsomes - metabolism; Oxygen Radioisotopes; Rabbits; Rats; Skatole - chemistry; Skatole - toxicity
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx9702087

The existence of a cytochrome P450-dependent 2,3-epoxide of the potent pneumotoxin 3-methylindole was indirectly confirmed using stable isotope techniques and mass spectrometry. Determination of hydride shift and incorporation of labeled oxygen in 3-methyloxindole and 3-hydroxy-3-methyloxindole, metabolites that may be in part dependent on the presence of the epoxide, were utilized as indicators of the epoxide's existence. One mechanism for the formation of 3-methyloxindole involves cytochrome P450-mediated epoxidation followed by ring opening requiring a hydride shift from C-2 to C-3. Through incubations of goat lung microsomes with [2-2H]-3-methylindole, the retention of 2H in 3-methyloxindole was found to be 81%, indicating a majority of the oxindole was produced by the mechanism described above. 3-Hydroxy-3-methylindolenine is an imine reactive intermediate that could be produced by ring opening of the 2,3-epoxide. The imine may be oxidized to 3-hydroxy-3-methyloxindole by the cytosolic enzyme aldehyde oxidase. Activities of this putative detoxification enzyme were determined in both hepatic and pulmonary tissues from goats, rats, mice, and rabbits, but the activities could not be correlated to the relative susceptibilities of the four species to 3-methylindole toxicity. The 18O incorporation into either 3-methyloxindole or 3-hydroxy-3-methyloxindole from both 18O2 and H2 18O was determined. The 18O incorporation into 3-methyloxindole from 18O2 was 91%, strongly implicating a mechanism requiring cytochrome P450-mediated oxygenation. Incorporation of 18O into 3-hydroxy-3-methyloxindole indicated that the alcohol oxygen originated from molecular oxygen, also implicating an epoxide precursor. These studies demonstrate the existence of two new reactive intermediates of 3-methylindole and describe the mechanisms of their formation and fate.