PhD

dissertationRThe heterocyclic alkaloid 3-methylindole (3MI), a naturally occurring systemic toxin that is has a high degree of selectivity for pulmonary tissue. EMI is formed by gastrointestinal fermentation of dietary tryptophan and causes a naturally occurring respiratory disease in cattle. Pulmonary pathology can also be experimentally produced in other species but with varying of efficiency; among some of the species studied the order of susceptibility is: goats > mice = rats > rabbits. Bioactivation of the compound by Cytochrome P-450 is necessary for toxicity, and reactive metabolites covalently bind to cellular proteins in the lungs and other tissues, particularly the liver. The binding that occurs in goat lung and liver tissue directly correlates with toxicity; however, covalent binding in mouse or rat lung and liver tissue is inversely correlated with toxicity. Several limited studies on the in vivo metabolism of 3MI in goats have shown that 3-methylosindole ((3MOI), indole-3-carbinol (I3COH) and indole-3-carbosylic acid are metabolites, but little else about the metabolic fate of 3MI is known. A reactive intermediate of 3MI has been trapped with glutathione in goat-lung microsomal incubations of 3MI and identified as the methylene imine metabolite 3-methylene-indolenine, but the formation of this compound has not been demonstrated in vivo. Moreover, the exact chemical mechanisms of the methylene imine's formation have not been elucidated. In the present studies, the metabolic fate of 3MI was determined in mice and the in vivo formation of the methylene imine was confirmed. In addition, a novel unique 3MI metabolite, 3-hydroxy-3-methyoxindole (HMOI), was indicative of one or more possible metabolic pathways that might contribute to 3MI toxicity were identified. Other metabolites identified were phenyl-ring hydroxylated glucuronide conjugates of HMOI and 3MOI, as well and indole-3-carboxylic acid and its glucuronide. The mechanism of methylene imine formation was elucidated by inter- and intramolecular kinetic deuterium-isotope effect studies and quantitation of 18O incorporation from 18O2 and H218O into the closely related metabolite 13COH. Results showed that the methylene imine was formed by a P-450-catalyzed hydration of I3COH are not an important mechanism of methylene imine formation. Mechanistic studies on the in intro formation of 2MOI and HMOI in goat-lung and mouse-liver microsomes and cystosol were conducted to better understand the differences in 3MI-mediated toxicity between these two tissues. A previously unknown bioactivation pathway for 3MI was deducted by determining the relative incorporation of 18O from H2[18O] into 2MOI and HMOI, the requirement for aldehyde oxidase in the formation of HMOI and the effect of thiol trapping agents on the metabolic profile of 3MI. The data suggest that a reactive intermediate, other that the methylene imine, is formed and the goat-lung tissue lacks that ability to detoxify this metabolite whereas mouse-liver enzymes efficiently oxidize the compound to HMOI.esults showed that the methylene imine was formed by a P-450-catalyzed hydrogen abstraction followed by a one-electron oxidation, and that dehydration of I3COH is not an important mechanism of methylene imine formation. Mechanistic studies on the in vitro formation of 3MOI and HMOI were also conducted. A novel bioactivation pathway for 3MI was deduced by determining the source of $/sp[18]$O incorporated into 3MOI and HMOI, the requirement for aldehyde oxidase in the formation of HMOI and the effect of thiol trapping agents on the metabolic profile of 3MI. The data suggest that a 3-hydroxyindolenine is formed that is oxidized to HMOI in mouse liver but not goat lung