AN INVESTIGATION OF THE FORMATION OF CYTOTOXIC, GENOTOXIC, PROTEIN-REACTIVE AND STABLE METABOLITES FROM NAPHTHALENE BY HUMAN LIVER-MICROSOMES

Chemically reactive epoxide metabolites have been implicated in variou s forms of drug and chemical toxicity. Naphthalene, which is metaboliz ed to a 1,2-epoxide, has been used as a model compound in this study i n order to investigate the effects of perturbation of detoxication mec hanisms on the in vitro toxicity of epoxides in the presence of human liver microsomes. Naphthalene (100 muM) was metabolized to cytotoxic, protein-reactive and stable, but not genotoxic, metabolites by human l iver microsomes. The metabolism-dependent cytotoxicity and covalent bi nding to protein of naphthalene were significantly higher in the prese nce of phenobarbitone-induced mouse liver microsomes than with human l iver microsomes. The ratio of trans-1,2-dihydrodiol to 1-naphthol was 8.6 and 0.4 with the human and the induced mouse microsomes, respectiv ely. The metabolism-dependent toxicity of naphthalene toward human per ipheral mononuclear leucocytes was not affected by the glutathione tra nsferase mu status of the co-incubated cells. Trichloropropene oxide ( TCPO; 30 muM), an epoxide hydrolase inhibitor, increased the human liv er microsomal-dependent cytotoxicity (19.6 +/- 0.9% vs 28.7 +/- 1.0%; P = 0.02) and covalent binding to protein (1.4 +/- 0.3% vs 2.8 +/- 0.2 %; P = 0.03) of naphthalene (100 muM), and reversed the 1,2-dihydrodio l to 1-naphthol ratio from 6.6 (without TCPO) to 2.6, 0.6 and 0.1 at T CPO concentrations of 30, 100 and 500 muM, respectively. Increasing th e human liver microsomal protein concentration reduced the cytotoxicit y of naphthalene, while increasing its covalent binding to protein and the formation of the 1,2-dihydrodiol metabolite. Co-incubation with g lutathione (5 mM) reduced the cytotoxicity and covalent binding to pro tein of naphthalene by 68 and 64%, respectively. Covalent binding to p rotein was also inhibited by gestodene, while stable metabolite format ion was reduced by gestodene (250 muM) and enoxacin (250 muM). The stu dy demonstrates that human liver cytochrome P450 enzymes metabolize na phthalene to a cytotoxic and protein-reactive, but not genotoxic, meta bolite which is probably an epoxide. This is rapidly detoxified by mic rosomal epoxide hydrolase, the efficiency of which can be readily dete rmined by measurement of the ratio of the stable metabolites, naphthal ene 1,2-dihydrodiol and 1-naphthol.