Cytotoxicity of 1,2-epoxynaphthalene is correlated with protein binding and in situ glutathione depletion in cytochrome P4501A1 expressing Sf-21 cells

Naphthalene is metabolized by several cytochrome P-450 (CYP) monooxygenases to 1,2-epoxynaphthalene. However, the subsequent interactions of the epoxi de with macromolecules in the cells, and the significance of these interact ions to cellular injury, are not well characterized. Additionally, CYP1A1, which can metabolize naphthalene to 1,2-epoxynaphthalene, may be induced by a number of xenobiotics. Yet, the in situ interaction between naphthalene and CYP1A1 alone, without the influence of other xenobiotic metabolizing en zymes, has not been examined. Using a model eukaryotic expression system ca pable of over-expressing recombinant CYP1A1, we found that naphthalene was toxic to cells expressing CYP1A1 in a dose- (LC50: 0.3 mn?) and time-depend ent (LT50: 12 h) manner. Naphthalene treatment of CYP1A1-expressing cells r esulted in a 47% decrease in cellular glutathione (GSH) levels. Pretreatmen t with ethyl ester GSH, a GSH analog, protected CYP1A1-expressing cells suc h that viability was 30%, greater than for cells treated with naphthalene a lone. Cytotoxicity was strongly correlated (r(2): 0.96) with covalent bindi ng of cellular proteins. Alkaline permethylation techniques showed that cys teinyl-SH groups of cellular proteins are a nucleophilic target of the epox ide metabolite. These results suggest that, in the absence of other pathway s, naphthalene is modified by CYP1A1 to 1,2-epoxynaphthalene, which subsequ ently binds cellular sulfhydryl groups on proteins and GSH.