Received for publication August 2, 2004.
Revised October 20, 2004.
Accepted for publication October 26, 2004.

Bioactivation of the Pulmonary Toxicants Naphthalene and 1-Nitronaphthalene by Rat Cytochrome P4502F4

Abstract

Naphthalene, a ubiquitous environmental contaminant, produces cytotoxicity in nonciliated bronchiolar epithelial (Clara) cells in mice; rats are refractory to lung cytotoxicity from naphthalene. In contrast, 1 nitronaphthalene is a potent toxicant in both species. Naphthalene is metabolized by cytochrome P4502F to a 1,2 epoxide, the first and obligate step in events leading to cytotoxicity. 1-Nitronaphthalene is metabolized to both the 5,6- and the 7,8 epoxides with the 7,8-epoxide predominating in lung. Previous studies have demonstrated recombinant CYP2F2 (mouse) to efficiently metabolize both naphthalene and 1 nitronaphthalene. To better understand the mechanism for the unique toxicity profiles for both compounds, a CYP2F orthologue (CYP2F4) was isolated from rat lung and expressed using a baculovirus system. Recombinant CYP2F4 efficiently generates 1R,2S naphthalene oxide (K_m = 3 µM, V_max = 107 min^-1) and the 5,6- and 7,8-epoxides of 1 nitronaphthalene (K_m = 18 µM, V_max = 25 min^-1 based on total generated glutathione conjugates ). Kinetics and regio/stereoselectivity of rat CYP2F4 were indistinguishable from mouse CYP2F2. These results, combined with our recent immunomapping studies demonstrating minimal pulmonary CYP2F expression in rats, indicate that CYP2F expression is the factor most clearly associated with susceptibility to naphthalene-induced pneumotoxicity. CYP2F4 failed to display an enhanced ability to bioactivate 1 nitronaphthalene, an ability which could have potentially compensated for the lower CYP2F pulmonary expression levels in the rat, yet equal species susceptibilities. These results suggest the importance of other CYP enzymes in the epoxidation/bioactivation of 1 nitronaphthalene. Expression of recombinant CYP2F1 (human) yielded an immunoreactive protein with no detectable CO-difference spectrum suggesting inadequate heme incorporation.

Key words: 1-Nitronaphthalene, Baculovirus, Clara Cell, Cytochrome P450 2F, Naphthalene, Pulmonary Toxicity

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