M. R. Nocerini

Effect of glutathione status on covalent binding and pneumotoxicity of 3-methylindole in goats☆

A study was conducted to investigate the relationship between glutathione (GSH) status, in-vivo metabolite covalent binding and 3-methylindole (3MI)-induced lung injury in goats. Cysteine or diethylmaleate pretreatments were given to sustain or deplete GSH, respectively, prior to intravenous 14C-3MI administration. Cysteine pretreatment prolonged survival times, decreased (P less than .05) covalent lung injury. Diethylmaleate pretreatment shortened survival times, increased (P less than .05) covalent binding and enhanced lung injury. Covalent binding was higher (P less than .05) in lung compared to liver and kidney. 3-Methylindole alone depleted GSH in 4 hours to 36, 66, and 75% of controls in these tissues, respectively. The relationship between tissue GSH, covalent binding and toxicity supports the hypothesis that 3MI-induced pneumotoxicity results from the formation of activated intermediates and that GSH plays a role in detoxication of these 3MI metabolites.

Adducts of 3-methylindole and glutathione: Species differences in organ-selective bioactivation

Lung and liver microsomes of several species were evaluated for potential to form activated metabolites of 3-methylindole (3MI). Microsomes were incubated with [14C]3MI and glutathione (GSH). Electrophilic 3MI metabolites were trapped and quantitated as GSH adducts by HPLC, and by determining the amounts of activated intermediates which became covalently bound to microsomal protein. The highest rates of 3MI-GSH adduct formation by the lung were detected in microsomes of the goat, followed in decreasing order by pulmonary microsomes from the horse, monkey, mouse, and rat, respectively. In contrast, hepatic 3MI-GSH adduct production was highest in microsomes from the rat, followed by mouse, monkey, goat, and horse microsomes, respectively. These results suggest that the species and organ-selective toxicity of 3MI are primarily caused by differences in rates of oxidative metabolism of 3MI to an electrophilic intermediate.

Glutathione adduct formation with microsomally activated metabolites of the pulmonary alkylating and cytotoxic agent, 3-methylindole☆

Incubations with goat lung and liver microsomes were conducted to trap with exogenous glutathione (GSH) the electrophilic intermediate produced via cytochrome P-450-dependent metabolic activation of 3-methylindole (3MI). Microsomal incubation mixtures with [14C]3MI, a NADPH-generating system, and [3H]GSH produced a dual-labeled adduct which was isolated by reverse-phase high-performance liquid chromatography. Reactive 3MI intermediates were also trapped with cysteine. Adduct formation increased in proportion to the concentration of either thiol. Covalent binding of activated 3MI metabolites to microsomal protein was inversely related to adduct production. There were both qualitative and quantitative differences in the formation of GSH adducts by lung and liver microsomes. In the presence of 2 mM GSH, the adduct was produced at a rate of 1.8 nmol/mg protein/min by lung microsomes but only at 0.1 nmol/mg protein/min by hepatic microsomes. The addition of cytosolic fractions containing glutathione S-transferase activity increased GSH adduct formation by approximately 30%. These results support the view that electrophilic 3MI intermediates are trapped by conjugation with GSH, and that organ-selective toxicity is primarily due to much faster rates of cytochrome P-450 oxidation of 3MI in the lung than in the liver.

In Vitro Covalent Binding of 3-[14C]Methylindole Metabolites in Goat Tissues

Abstract Covalent binding of 3-[14C]methylindole (3[14C]MI) in crude microsomal preparations of goat lung, liver, and kidney was measured to determine if a reactive intermediate was formed during the in vitro metabolism of 3-methylindole (3MI). The bound radioactivity was highest in lung compared to liver and kidney. The amount of bound radioactivity per nanomole of cytochrome P-450 was approximately 10 times higher in the lung compared to liver. No detectable bound radioactivity was found when 3-[3H]methyloxindole was used as the substrate. Cofactor requirements and the effects of inhibitors indicate that a mixed function oxidase (MFO) system is involved in formation of a reactive intermediate. Inhibitors and conjugating agents that are known to reduce the severity of 3MI-induced lung injury such as piperonyl butoxide (MFO inhibitor) and glutathione (conjugating agent) significantly decreased the in vitro binding of 3[14C]MI. The results indicate that a reactive intermediate is produced during the metabolism of 3MI by the MFO system. The organ specificity in binding suggests that covalent binding by lung microsomes may be related to the mechanism of 3MI-induced lung injury.

Electrophilic metabolites of 3-methylindole as toxic intermediates in pulmonary oedema

[methyl-14C]-3-Methylindole (3MI) was incubated with goat-lung microsomes, an NADPH-generating system and glutathione. An adduct between an oxidative metabolite of 3MI and glutathione was formed only when the complete system was employed. The adduct, which was detected by u.v. absorbance and scintillation counting of h.p.l.c. fractions, was purified to homogeneity by reverse-phase h.p.l.c. The ability of 3MI to bind to microsomal protein was reduced to 52% and 46% of controls when 2 mM and 4 mM glutathione, respectively, were included in the incubations. These results suggest the involvement of an electrophilic metabolite as the toxic intermediate in 3MI-mediated pulmonary oedema.

Alkylation of bronchiolar epithelial cells by 3-methylindole metabolites in the horse

Autoradiographs of horse-lung explants incubated with [3H]3-methylindole (3MI) showed 8 times greater labeling per area to bronchiolar epithelial cells than to the interalveolar septa. Incubations of horse-lung microsomes with [14C]3MI resulted in alkylation of microsomal proteins, which could be reduced by exogenous glutathione. An apparent covalent adduct of glutathione and 3MI was isolated from these incubations. These results suggest that the target cells of 3MI-induced injury in the horse, the bronchiolar epithelial cells, are alkylated by an electrophilic 3MI intermediate.

Recent Findings on the Chemical Nature and Biochemical Mechanisms of the Toxic Agent Involved in 3-Methylindole-Induced Acute Lung Injury

Abstract3-Methylindole (3MI) is a ruminal fermentation product of cryptophan that causes acute lung injury in ruminants, horses, and some other animals. Injury to the lung is cellular and species specific. Cytotoxic effects art mediated through mixed function oxidase metabolism of 3MI which causes the formantion of a reactive intermediate that covalently binds to cellular macromolecules. The reactive intermediate can be detoxified by conjugation with glutathione (GSH) to form a 3MI-GSH adduct. This adduct was isolated using HPLC and the structure was determined using W spectrometry, thermospray liquid chromotography/mass spectrometry, and nuclear agnetic resonance spectrometry. The structure of the 3MI-GSH-adduct was shown to be 3-(glutathion-S-yl)-methyi Indole. The most likely structure of the reactive intermediate responsible for acute lung injury is the imine methide of 3MI.

Structure of the adduct of glutathione and activated 3-methylindole.

3-Methylindole (3MI) is a pneumotoxic metabolite of tryptophan fermentation in ruminants. The toxicity of 3MI is organ, cell, and species specific. In ruminants and horses, the most susceptible species, the lung is the target organ. Type I alveolar epithelial and nonciliated bronchiolar epithelial (Clara) cells are the most susceptible targets within the lungs of ruminants, 2,3 while Clara cells only are primarily damaged by 3MI in horses.4 Exposure of man to 3MI is through intestinal absorption and by cigarette smoke although the toxic manifestations of this exposure have not yet been assessed.5–7