PhD

dissertation3-Methylindole (3MI), a fermentation product of tryptophan, is a selective pneumotoxin whose toxicity is also highly species-selective. Among the species examined, goats are most susceptible to 3MI pneumotoxicity and rabbits are least susceptible. Although humans are exposed to 3MI from several sources, including cigarette smoke, the potential toxicity of this chemical to humans is not well defined. In the present studies, bioactivation and metabolism of 3MI were examined using HepG2 cell lysates containing vaccinia-expressed cytochrome P450 enzymes. The human P450s, CYP2C8, CYP3A4, CYP2F1 and CYP2A6, and rabbit CYP4B1 and mouse CYP1A2 metabolized 3MI to an intermediate that became covalently bound to cellular macromolecules. The putative reactive intermediate of 3MI metabolism, 3-methyleneindolenine, was identified in 3MI incubations with HepG2 lysates containing human CYP2F1 and CYP2A6 and rabbit CYP4B1 and mouse CYP1A2, as its mercapturate, 3- ((N-acetylcystein-S-yl)-methyl) indole, when N-acetylcysteine (NAC) was added to the incubations. Correlation analyses revealed that the production of 3-methyleneindolenine and the formation of a covalently bound intermediate are related, indicating that the metabolite that becomes covalently bound to cellular macromolecules is probably 3-methyleneindolenine. 3MI bioactivation and metabolism were also examined in isolated cells and subcellular fractions from rabbit lung tissue. In incubations of macrophages and Clara cells with 3MI and NAC, a polar NAC adduct tentatively identified as an adduct of the putative intermediate, 3-hydroxy-3-methylindolenine (3H3MIN), 3 ((N-glutathion-S-yl)-methyl) indole (3MI-GSH) and 3MOI were identified. In rabbit lung microsomal incubations with 3MI and glutathione, 3MI-GSH, 3MOI, indole-3-carbinol and a GSH adduct of 3H3MIN were identified. These metabolites have previously been shown to be formed in goat lung microsomal incubations with GSH. These studies indicate that the species-selectivity of 3MI toxicity is due to factors other than the presence of pulmonary cytochrome P450 enzymes capable of bioactivating 3MI. Only a few P450 enzymes were capable of bioactivating 3MI. However, the ability of human cytochrome P450 enzymes to bioactivate 3MI suggests that this compound is a potential human toxin