Respective roles of CYP2A5 and CYP2F2 in the bioactivation of 3-methylindole in mouse olfactory mucosa and lung: studies using Cyp2a5-null and Cyp2f2-null mouse models.

The aim of this study was to determine whether mouse CYP2A5 and CYP2F2 play critical roles in the bioactivation of 3-methylindole (3MI), a tissue-selective toxicant, in the target tissues, the nasal olfactory mucosa (OM) and lung. Five metabolites of 3MI were identified in NADPH- and GSH-fortified microsomal reactions, including 3-glutathionyl-S-methylindole (GS-A1), 3-methyl-2-glutathionyl-S-indole (GS-A2), 3-hydroxy-3-methyleneindolenine (HMI), indole-3-carbinol (I-3-C), and 3-methyloxindole (MOI). The metabolite profiles and enzyme kinetics of the reactions were compared between OM and lung, and among wild-type, Cyp2a5-null, and Cyp2f2-null mice. In lung reactions, GS-A1, GS-A2, and HMI were detected as major products, and I-3-C and MOI, as minor metabolites. In OM reactions, all five metabolites were detected in ample amounts. The loss of CYP2F2 affected formation of all 3MI metabolites in the lung and formation of HMI, GS-A1, and GS-A2 in the OM. In contrast, loss of CYP2A5 did not affect formation of 3MI metabolites in the lung but caused substantial decreases in I-3-C and MOI formation in the OM. Thus, whereas CYP2F2 plays a critical role in the 3MI metabolism in the lung, both CYP2A5 and CYP2F2 play important roles in 3MI metabolism in the OM. Furthermore, the fate of the reactive metabolites produced by the two enzymes through common dehydrogenation and epoxidation pathways seemed to differ with CYP2A5 supporting direct conversion to stable metabolites and CYP2F2 supporting further formation of reactive iminium ions. These results provide the basis for understanding the respective roles of CYP2A5 and CYP2F2 in 3MI's toxicity in the respiratory tract.

Mechanism-based inactivation of lung-selective cytochrome P450 CYP2F enzymes.

3-Methylindole (3MI) is a pneumotoxin that requires P450-catalyzed metabolic activation (dehydrogenation), to an electrophilic methylene imine to elicit toxicity. Previous studies have shown that the human pulmonary cytochrome P450 enzyme, CYP2F1, and its goat analog, CYP2F3, catalyzed the dehydrogenation of 3MI. However, it was not known whether the dehydrogenation product could bind to active site nucleophilic residues to inactivate these enzymes. Therefore, the purpose of this study was to determine whether 3MI is a mechanism-based inhibitor of CYP2F3 and CYP2F1. The results showed that both enzymes were highly susceptible to 3MI-mediated suicide inactivation. The k(inact) and the K(I) for CYP2F3 were 0.09/min and 160 microM, respectively, and the approximate partition ratio was 220. Although CYP2F3 lost approximately 80% of its activity in 30 min, a concurrent loss of its reduced carbon monoxide complex was not observed, suggesting that the heme was not destroyed/modified during the inactivation. The exogenous nucleophile, glutathione, did not protect CYP2F1 from 3MI-mediated inactivation, suggesting that the reactive intermediate did not diffuse from the active site before inactivation events. Dialysis of 3MI-inactivated CYP2F3 did not restore activity, and alternate substrates protected CYP2F3. In addition, 3MI inhibited the 7-ethoxycoumarin deethylase activity of human CYP2F1 in a time- and concentration-dependent manner; the k(inact) and K(I) were 0.025/min and 49 microM, respectively. In conclusion, this study presents evidence that 3MI is a mechanism-based inhibitor of both CYP2F3 and CYP2F1, which are important enzymes in the bioactivation of pneumotoxicants such as 3MI or 1,1-dichloroethylene or carcinogens such as naphthalene, benzene, and styrene.

Specific dehydrogenation of 3-methylindole and epoxidation of naphthalene by recombinant human CYP2F1 expressed in lymphoblastoid cells.

3-Methylindole (3MI) is a naturally occurring pulmonary toxin that requires metabolic activation. Previous studies have shown that 3MI-induced pneumotoxicity resulted from cytochrome P-450-catalyzed dehydrogenation of 3MI to an electrophilic methylene imine (3-methyleneindolenine), which covalently bound to cellular macromolecules. Multiple cytochrome P-450s are capable of metabolizing 3MI to several different metabolites, including oxygenated products. In the present study, the role of human CYP2F1 in the metabolism of 3MI was examined to determine whether it catalyzes dehydrogenation rather than hydroxylation or ring oxidation. Metabolism was examined using microsomal fractions from human lymphoblastoid cells that expressed the recombinant human CYP2F1 P-450 enzyme. Expression of CYP2F1 in the lymphoblastoid cells proved to be an appropriate expression system for this enzyme. Products were analyzed using HPLC and the mercapturate, 3-[(N-acetylcystein-S-yl)methyl]indole, of the reactive intermediate was identified and quantified. Product analysis showed that human CYP2F1 efficiently catalyzed the dehydrogenation of 3MI to the methylene imine without detectable formation of indole-3-carbinol or 3-methyloxindole. High substrate concentrations of 3MI strongly inhibited production of the dehydrogenated product, a result that may indicate the existence of mechanism-based inhibition of CYP2F1 by 3MI. Recombinant CYP2F1 demonstrated remarkable selectivity for the bioactivation of 3MI to the putative dehydrogenated reactive electrophile. Bioactivation of naphthalene to its pneumotoxic epoxide by CYP2F1 was also demonstrated.

Synergistic effects of concurrent challenge with bovine respiratory syncytial virus and 3-methylindole in calves.

OBJECTIVE: To evaluate the potential synergy between bovine respiratory syncytial virus (BRSV) and 3-methylindole (3MI) in inducing respiratory disease in cattle. ANIMALS: 20 mixed-breed beef calves. PROCEDURE: A 2 X 2 factorial design was used, with random assignment to the following 4 treatment groups: unchallenged control, BRSV challenge exposure (5 X 10(4) TCID50 by aerosolization and 5.5 X 10(5) TCID50 by intratracheal inoculation), 3MI challenge exposure (0.1 g/kg of body weight, PO), and combined BRSV-3MI challenge exposure. Clinical examinations were performed daily. Serum 3MI concentrations, WBC counts, PCV, total plasma protein, and fibrinogen concentrations were determined throughout the experiment. Surviving cattle were euthanatized 7 days after challenge exposure. Pulmonary lesions were evaluated at postmortem examination. RESULTS: Clinical respiratory disease was more acute and severe in cattle in the BRSV-3MI challenge-exposure group than in cattle in the other groups. All 5 cattle in this group and 3 of 5 cattle treated with 3MI alone died or were euthanatized prior to termination of the experiment. Mean lung displacement volume was greatest in the BRSV-3MI challenge-exposure group. Gross and histologic examination revealed that pulmonary lesions were also most severe for cattle in this group. CONCLUSIONS AND CLINICAL RELEVANCE: Feedlot cattle are commonly infected with BRSV, and 3MI is produced by microflora in the rumen of all cattle. Our results suggest that there is a synergy between BRSV and 3MI. Thus, controlling combined exposure may be important in preventing respiratory disease in feedlot cattle.

Cloning and expression of CYP2F3, a cytochrome P450 that bioactivates the selective pneumotoxins 3-methylindole and naphthalene.

Members of the CYP2F gene subfamily are selectively expressed in lung tissues and have been implicated as important catalysts in the formation of reactive intermediates from several pneumotoxic chemicals. Human CYP2F1 bioactivates 3-methylindole (3MI), while mouse CYP2F2 bioactivates naphthalene. Although 3MI is a potent pneumotoxin in ruminants and rodents, the participation of cytochrome P450s from the 2F subfamily in 3MI bioactivation has not been fully defined. To test the hypothesis that a goat lung 2F homologue uniquely catalyzes the dehydrogenation of 3MI to the putative electrophile 3-methylene-indolenine, the CYP2F3 cDNA was cloned from a goat lung cDNA library and expressed in Escherichia coli. The predicted amino acid sequence of CYP2F3 possessed 82% identity to both human CYP2F1 and mouse CYP2F2. CYP2F3 was mutated at the 5' end, expressed in E. coli, and shown to have a molecular mass of 50 kDa. The reconstituted enzyme uniquely catalyzed only the dehydrogenation of 3MI to form 3-methylene-indolenine, an electrophilic intermediate, without detectable formation of other products, thus demonstrating highly unusual selectivity for dehydrogenation rather than hydroxylation of a substrate. Immunoinhibition studies demonstrated that about 20% of the production of the intermediate in goat lung microsomal samples was produced by CYP2F3. The CYP2F3 enzyme had a specific activity that was similar to that of human cDNA-expressed CYP2F1. CYP2F3 also stereoselectively catalyzed the formation of the 1R,2S-oxide from naphthalene; this stereoisomer is the putative pneumotoxin. The enzyme, however, lacked catalytic activity with other common P450 substrates including 7-ethoxycoumarin, a substrate for CYP2F1, indicating that the substrate selectivity of CYP2F3 appears to be high.

Metabolism of 3-methylindole by vaccinia-expressed P450 enzymes: correlation of 3-methyleneindolenine formation and protein-binding.

The toxicity of 3-methylindole (3 MI), a selective pneumotoxin, is dependent upon cytochrome P450-mediated bioactivation 3. Using vaccinia-expressed P450 enzymes, the metabolites of radiolabeled 3 MI produced by 14 individual P450s were identified and quantified by high performance liquid chromatography. Indole-3-carbinol was produced from incubations of 3 MI with only four enzymes. Although 3-methyloxindole was a product of several P450s, human 1A2 was most efficient in producing this metabolite. The toxic intermediate of 3 MI is believed to be a reactive methylene imine, 3-methyleneindolenine. In this study, this intermediate was detected as its mercapturate adduct, when N-acetylcysteine was added to the incubations. 3-Methyleneindolenine was produced by CYP2A6 at a rate of 50.9 +/- 8.9 pmol/mg protein/hr and by CYP2F1 at a rate of 205.7 +/- 12.5 pmol/mg/hr. The mouse 1a-2 and rabbit 4B1 enzymes produced the reactive intermediate in amounts that exceeded that of the human 2F1 enzyme by 1.4-fold and 1.9-fold, respectively. The toxicity of 3 MI is believed to be due to covalent binding of a P450-generated intermediate to critical pulmonary proteins. Comparison of covalent binding studies to the formation of the metabolites revealed a strong correlation between the amount of the 3 MI adduct detected and covalent binding. This study showed that the methylene imine electrophile is produced by only a few P450 enzymes and is the metabolite responsible for the covalent binding and presumably, the toxicity of 3 MI. Remarkable product preferences between the desaturation pathway to form the methyleneindolenine by CYP2F1 and the ring epoxidation pathway to form the oxindole by CYP1A2, were observed.

Isolation of a mercapturate adduct produced subsequent to glutathione conjugation of bioactivated 3-methylindole.

Bioactivation of the pneumotoxin 3-methylindole (3MI) to a methylene imine intermediate has been demonstrated previously by trapping the electrophile with glutathione in goat lung microsomal incubations. To determine whether the same bioactivation process occurs in whole animals, 3MI was administered to goats, mice, and rats, and the urinary metabolites from these three species were analyzed by HPLC for the presence of the mercapturate that would be expected as the processed and excreted form of the 3MI-glutathione adduct. The mercapturate, 3-[(N-acetylcysteine-S-yl)-methyl]indole (3MI-NAC), was identified in the urine from all three species and was isolated from rat urine for structural identification by uv, NMR, and mass spectrometry. Synthetic 3MI-NAC had uv, NMR, and chromatographic characteristics identical to the isolated metabolite. The presence of this mercapturate in the urine of treated animals unequivocally demonstrates that 3MI is bioactivated to the methylene imine in vivo and that the glutathione adduct is also formed, presumably to detoxify the methylene imine.

Isolation and identification of 3-hydroxy-3-methyloxindole, the major murine metabolite of 3-methylindole.

3-Methylindole (3MI) is pneumotoxic to ruminants and rodents subsequent to metabolic oxidative activation by cytochrome P-450 monooxygenases. Goats are much more susceptible than mice and rats to 3MI-mediated lung damage, and these differences in species susceptibility may be reflected by differences in the metabolic products of 3MI. Radioactive 3MI was administered ip to Swiss-Webster mice, and the major nonpolar urinary metabolites were fractionated and separated by HPLC. Although 3-methyloxindole has been shown to be the major urinary metabolite of 3MI in goats, it was not detected in mouse urine. Instead, the major metabolite, 3-hydroxy-3-methyloxindole, was isolated and purified and its structure elucidated by 1H and 13C NMR, mass spectrometry, and IR spectroscopy. This is the first identification of this highly oxidized indole from mammalian sources. The production of this metabolite may be indicative of the formation of an electrophilic methyleneoxindole intermediate, which could be responsible for pneumotoxicity in this species.

Inhibition of 3-methylindole bioactivation by the cytochrome P-450 suicide substrates 1-aminobenzotriazole and alpha-methylbenzylaminobenzotriazole.

The cytochrome P-450 suicide substrates 1-aminobenzotriazole (ABT) and alpha-methylbenzylaminobenzotriazole (alpha MB) were used as probes to examine the participation of cytochrome P-450 monooxygenases in the metabolism and covalent binding of 3-methylindole. ABT was a potent inactivator of 3-methylindole turnover and covalent binding of [methyl-14C]3-methylindole to protein in goat lung microsomal incubations. Both covalent binding and 3-methylindole turnover were decreased approximately 50% at 0.01 mM and 100% at 0.1 mM concentrations of ABT. The effects of ABT indicated that toxicity, as related to covalent binding, was directly dependent upon cytochrome P-450 catalysis. The inactivation of 3-methylindole turnover was greater with a 0.01 mM concentration of the isozyme-selective inhibitor alpha MB, 74% as compared with 47% for ABT. alpha MB (0.01 mM) decreased benzphetamine N-demethylase activity by 82% but decreased 7-ethoxyresorufin O-deethylase activity by only 28%. Thus, both 3-methylindole metabolism and benzphetamine oxidation were selectively inactivated by alpha MB. These findings suggest that 3-methylindole is metabolized to alkylating, electrophilic intermediates preferentially by the homologues of "phenobarbital-inducible" isozymes (presumably forms 2 and 5 in analogy to rabbit lung isozymes) to cytochrome P-450 in pulmonary microsomes, rather than by the polycyclic aromatic hydrocarbon-inducible isozymes.