Cytochrome P450 2A13 efficiently catalyzes the bioactivation of several tobacco-specific nitrosamines in vitro. This efficient bioactivation together with the selective expression of P450 2A13 in the human lung suggests that this P450 may play an important role in the initiation of lung cancer in smokers. Therefore, the identification of potent and selective inhibitors/inactivators of P450 2A13 could potentially help to lower the risk of lung cancer in smokers. In this study, we investigated the ability of 8-methoxypsoralen (8-MOP), a known inhibitor of P450 2A6, to inhibit and inactivate the activities of heterologously expressed P450 2A13 in reconstituted systems. We found that 8-MOP is a potent inhibitor of P450 2A13-mediated metabolism of several compounds, including testosterone, which had not been known to be a P450 2A13 substrate. The KI for the non-competitive inhibition of P450 2A13-mediated coumarin 7-hydroxylation by 8-MOP was 0.11 μM. The inhibition of P450 2A13 was accompanied by inactivation of the enzyme. Therefore, the observed decrease in activity is most likely due to the inactivation of the enzyme together with competitive or non-competitive inhibition of P450 2A13 by 8-MOP. The inactivation did not result in a loss of native heme, or a significant change in the reduced-CO spectrum of the P450, and did not generate any detectable heme adducts. Instead, the inactivation of P450 2A13 by 8-MOP occurred through the formation of an adduct to the apoprotein. LC/MS analysis of the adducted protein indicated an increase in the mass of 232 Da compared with the unadducted protein. This mass shift correlates with the addition of one molecule of 8-MOP plus one atom of oxygen atom to the P450 apoprotein.
Bibliographical noteFunding Information:
The research described in this article was supported in part by a postdoctoral fellowship to L.B.vW. from Philip Morris USA. The work was also supported in part by research grants CA 092596 (to X.D.) and CA 16954 (to P.F.H.) from the National Institutes of Health.