EVIDENCE SUPPORTING THE FORMATION OF 2,3-EPOXY-3-METHYLINDOLINE - A REACTIVE INTERMEDIATE OF THE PNEUMOTOXIN 3-METHYLINDOLE

The existence of a cytochrome P450-dependent 2,3-epoxide of the potent pneumotoxin 3-methylindole was indirectly confirmed using stable isot ope techniques and mass spectrometry. Determination of hydride shift a nd incorporation of labeled oxygen in 3-methyloxindole and 3-hydroxy-3 -methyloxindole, metabolites that may be in part dependent on the pres ence of the epoxide, were utilized as indicators of the epoxide's exis tence. One mechanism for the formation of 3-methyloxindole involves cy tochrome P450-mediated epoxidation followed by ring opening requiring a hydride shift from C-2 to C-3. Through incubations of goat lung micr osomes with [2-H-2]-3-methylindole, the retention of H-2 in 3-methylox indole was found to be 81%, indicating a majority of the oxindole was produced by the mechanism described above. 3-Hydroxy-3-methylindolenin e is an imine reactive intermediate that could be produced by ring ope ning of the 2,3-epoxide. The imine may be oxidized to 3-hydroxy-3-meth yloxindole by the cytosolic enzyme aldehyde oxidase. Activities of thi s putative detoxification enzyme were determined in both hepatic and p ulmonary tissues from goats, rats, mice, and rabbits, but the activiti es could not be correlated to the relative susceptibilities of the fou r species to 3-methylindole toxicity. The O-18 incorporation into eith er 3-methyloxindole or 3-hydroxy-3-methyloxindole from both O-18(2) an d (H2O)-O-18 was determined. The O-18 incorporation into 3-methyloxind ole from O-18(2) was 91%, strongly implicating a mechanism requiring c ytochrome P450-mediated oxygenation. Incorporation of O-18 into 3-hydr oxy-3-methyloxindole indicated that the alcohol oxygen originated from molecular oxygen, also implicating an epoxide precursor. These studie s demonstrate the existence of two new reactive intermediates of 3-met hylindole and describe the mechanisms of their formation and fate.