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

Abstract

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.

Fig. 1.

Scheme for the metabolic activation of 3MI by mouse OM and lung microsomal P450s. Pathway A represents dehydrogenation and pathway B represents epoxidation [The figure has been drawn on the basis of the findings in D'Agostino et al. (2009)]. Shaded areas show metabolites preferentially formed by CYP2A5 (blue) or CYP2F2 (green).

Fig. 2.

LC-MS/MS detection of metabolites of 3MI formed by mouse OM and lung microsomal P450s. Complete reaction mixtures contained 100 mM potassium phosphate buffer, pH 7.4, 50 μM 3MI, 0.5 mg/ml OM (A–C) or lung (D–F) microsomal protein, and 1.0 mM NADPH. GSH was added at 5 mM final concentration, where indicated. Reactions were performed for 0 (A and D) or 30 min (B, C, E, and F), as shown. The samples were analyzed using LC-MS/MS, as described under Materials and Methods. Typical results are shown.