Identification of phase I metabolites of 3-methylindole produced by pig liver microsomes.

A study was conducted to investigate qualitative and quantitative aspects of the phase I metabolism of 3-methylindole (3MI) by porcine liver microsomes. Microsomal suspensions were prepared from the liver of 30 intact (uncastrated) male pigs. Metabolites produced in microsomal incubations were identified and quantitated with HPLC-UV, HPLC-fluorescence, and UV-spectral analysis; liquid chromatography-mass spectrometry (LC-MS) and NMR were used for the identification of a metabolite for which a reference compound was not available. The results showed that seven major metabolites of 3MI are produced by porcine microsomes, three of which had already been identified in pigs (3-OH-3-methyloxindole, 5-OH-3-methylindole, and 6-OH-3-methylindole). The other four major 3MI metabolites identified were 3-OH-3-methylindolenine, 3-methyloxindole, indole-3-carbinol, and 2-aminoacetophenone. On average, the metabolite that was produced in larger amounts was 3-OH-3-methylindolenine (45.1%), followed by the two oxindoles 3-methyloxindole (27.9%) and 3-OH-3-methyloxindole (18.5%). Average percentage of production of 6-OH-3-methylindole was 4.9%, whereas indole-3-carbinol accounted for 2.7% of all metabolites produced; 2-amino-acetophenone and 5-OH-3-methylindole were the metabolites produced in lesser amounts (0.5 and 0.3%, respectively). Large interindividual differences in the rate of production of all metabolites were observed. This variation could be attributed to differences in the activity and/or level of expression of phase I biotransformation enzymes and this issue should be further investigated.

Thioether adducts of a new imine reactive intermediate of the pneumotoxin 3-methylindole.

Cytochrome P450 enzymes can potentially oxygenate 3-methylindole to form 2,3-epoxy-3-methylindoline which could rearrange to the stable metabolite 3-methyloxindole or open to form 3-hydroxy-3-methylindolenine, a putative electrophilic imine. The purpose of the current work was to determine if the imine was formed, and to characterize it via its adducts with thiol nucleophiles. Thiols were added to incubations of goat lung microsomes with 3-methylindole and deuterated analogues of 3-methylindole to trap the imine intermediate as its thioether conjugates. The N-acetylcysteine conjugate of 3-hydroxy-3-methylindolenine was detectable by LC/MS, but a molecular ion was not observed because the adduct rapidly dehydrated to form the 2-substituted indole. However, the imine was S-alkylated, and the intermediate carbinol was intramolecularly trapped using thioglycolic acid as a trapping agent that induced cyclocondensation to a lactone. The retention of one atom of deuterium from [2-2H]-3-methylindole and three from 3-[2H3-methyl]indole substantiated the mechanism in which the lactone adduct was produced by sulfur addition to either 3-hydroxy-3-methylindolenine or the epoxide. Tandem mass spectrometry of the lactone adduct produced a daughter ion spectrum consistent with this adduct. These studies demonstrated the existence of a new reactive intermediate of 3-methylindole, 3-hydroxy-3-methylindolenine, which may play a role in the pneumotoxicity of this chemical.

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 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 H218O 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.