Abstract
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 kinact and the KI for CYP2F3 were 0.09/min and 160 μM, 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 kinact and KI were 0.025/min and 49 μM, 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.
Footnotes
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This work was supported by U.S. Public Health Service grants from the National Heart, Lung, and Blood Institute (HL13645 and HL60143) and the National Institutes of General Medical Sciences (GM074249).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.017897.
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ABBREVIATIONS: P450, cytochrome P450; 3MI, 3-methylindole; CHZX, chlorzoxazone; LB, Luria-Bertani; TB, Terrific Broth; EC, 7-ethoxycoumarin; GSH, glutathione; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; L-745,870, 3-{[4-(4-chlorophenyl)piperazin-1-yl]-methyl}-1H-pyrrolo-2,3-β-pyridine; MK-0524, (3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl acetic acid; SPD-304, 6,7-dimethyl-3-{[methyl-(2-{methyl-[1-(3-trifluoromethyl-phenyl)-1H-indol-3-ylmethyl]-amino}-ethyl)-amino]-methyl}-chromen-4-one.
- Received July 26, 2007.
- Accepted October 22, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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