Metabolism and mechanism of toxicity of 3-methylindole in human and goat tissues

3-Methylindole (3MI), a highly selective pneumotoxin in ruminants, horses, sheep and rodents, is an anaerobic bacterial fermentation product of tryptophan formed in the rumen of ruminants and in the large intestine of man. Toxicity in humans is not known, despite significant human exposures to 3MI from intestinal absorption after tryptophan fermentation and from inhalation of cigarette smoke. Bioactivation of 3MI was investigated in human lung and liver tissues in order to provide information about the susceptibility of humans to 3MI toxicity. Human lung microsomes were prepared from eight organ transplantation donors. Human liver microsomes were prepared from one of the organ transplantation donors and three cadavers. Human liver cytochrome P-450 (CYP) content from the organ donor was 0.59 ± 0.13 nmol/mg. The CYP content in the livers from the cadavers (0.29 ± 0.17 nmol/mg) was lower than that of the organ donor. Even though the human lung CYP content was non-detectable, the 3MI turnover rate and covalent binding of radioactive 3MI to human lung microsomal proteins was measurable. The 3MI turnover rate with human lung microsomes was 0.23 ± 0.06 nmol/mg/min which was lower than the rate with the human liver microsomes (7.40 nmol/mg/min) from the organ donor. The activities were NADPH-dependent and inhibited by l-aminobenzotriazole (ABT), a potent CYP suicide substrate inhibitor. 3MI covalent binding to human lung microsomes was 2.74 ± 2.57 pmol/mg/min. The magnitude of this binding was only four percent of human liver microsomal binding. The covalent binding in human lung microsomes was NADPH- and protein-dependent and also was inhibited by ABT. Therefore, the bioactivation of 3MI to covalent-binding intermediates is catalyzed by CYP in human pulmonary tissues. Microsomal proteins from human and goat lung and liver were incubated with radioactive 3MI, and the radioactive alkylated proteins were analyzed by sodium dodecyl sulfate-polyacylamide gel electrophoresis and high pressure liquid chromatography (HPLC) and visualized by autoradiography and radiochromatography, respectively. The results showed that a 57 kDa protein in goat lung microsomes was clearly the most prominently alkylated target associated with 3MI reactive intermediates but a 53 kDa protein in human liver microsomes was the prominent target. A cysteinyl adduct of the methylene imine electrophilic intermediate of 3MI was analyzed by HPLC and identified by mass spectrometry. The results demonstrate that 3MI is bioactivated in vitro to an electrophilic methylene imine intermediate which covalently binds to the thiol group of pulmonary microsomal proteins, presumably to produce lung damage. The data from human tissues suggest that humans may be susceptible to 3MI-mediated toxicity. The specificity of 3MI covalent binding and the extent of binding to target proteins may play important roles in organ- and species-selective susceptibilities to 3MI-induced pneumotoxicity.