title: Cytotoxicity of 1,2-epoxynaphthalene is correlated with protein binding and in situ glutathione depletion in cytochrome P4501A1 expressing Sf-21 cells.
abstract: Naphthalene is metabolized by several cytochrome P-450 (CYP) monooxygenases to 1,2-epoxynaphthalene. However, the subsequent interactions of the epoxide with macromolecules in the cells, and the significance of these interactions 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 enzymes, has not been examined. Using a model eukaryotic expression system capable of over-expressing recombinant CYP1A1, we found that naphthalene was toxic to cells expressing CYP1A1 in a dose- (LC50: 0.3 mM) and time-dependent (LT50: 12 h) manner. Naphthalene treatment of CYP1A1-expressing cells resulted in a 47% decrease in cellular glutathione (GSH) levels. Pretreatment with ethyl ester GSH, a GSH analog, protected CYP1A1-expressing cells such that viability was 30% greater than for cells treated with naphthalene alone. Cytotoxicity was strongly correlated (r2: 0.96) with covalent binding of cellular proteins. Alkaline permethylation techniques showed that cysteinyl-SH groups of cellular proteins are a nucleophilic target of the epoxide metabolite. These results suggest that, in the absence of other pathways, naphthalene is modified by CYP1A1 to 1,2-epoxynaphthalene, which subsequently binds cellular sulfhydryl groups on proteins and GSH.
authors.affiliation: Department of Entomology, University of California at Davis, 95616, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Greene
authors.forename: J
authors.initials: J
authors.lastname: Zheng
authors.forename: D F
authors.initials: DF
authors.lastname: Grant
authors.forename: B D
authors.initials: BD
authors.lastname: Hammock
mesh_terms: D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D002460:Cell Line / Q000187:drug effects / Q000201:enzymology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D009281:Naphthalenes / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000633:toxicity*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins; D018411:Spodoptera / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D013997:Time Factors
chemical_list: D009281:Naphthalenes; D011994:Recombinant Proteins; C042431:S-ethyl glutathione; D019363:Cytochrome P-450 CYP1A1; D005978:Glutathione