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Chichester CH, Buckpitt AR, Chang A, Plopper CG: Metabolism and cytotoxicity of naphthalene and its metabolites in isolated murine Clara cells. Mol Pharmacol. 1994 Apr;45(4):664-72.
Nonciliated bronchiolar epithelial (Clara) cells of mice are highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type expresses high levels of cytochrome P450 monooxygenases. To establish the capability of these cells to metabolize an agent that causes Clara cell-selective toxicity in vivo, we evaluated the metabolism of naphthalene in isolated cells under two distinct conditions, i.e., in homogenized cell preparations supplemented with glutathione and glutathione S-transferases and in intact cells. In homogenized cell preparations naphthalene was metabolized to dihydrodiol (minor) and a single glutathione adduct (major) derived from the 1R,2S-epoxide. In intact cells the rate of formation of glutathione adduct was much lower and dihydrodiol predominated. Approximately 3-10% of racemic naphthalene oxide added to isolated homogenized cells was converted to glutathione adducts and dihydrodiol in 3-min incubations. At high concentrations of naphthalene oxide (0.25 and 0.5 mM), formation of the adduct derived from the 1R,2s-epoxide was favored. The intracellular glutathione concentration, measured by high performance liquid chromatography as the fluorescence of the monobromobimane-glutathione derivative, was 1.14 +/- 0.13 nmol/10 (6) cells. To determine whether Clara cell injury results from cytotoxic metabolites of naphthalene, we assessed viability of intact cells in response to different concentrations of naphthalene and naphthalene metabolites. At high naphthalene concentrations (0.5 and 1.0 mM) cell viability decreased to 63% or less of control, whereas lower concentrations (0.1 or 0.05 mM) did not alter viability significantly. Naphthalene-induced decreases in cell viability were blocked by preincubation of Clara cells with the cytochrome P450 monooxygenase inhibitor piperonyl butoxide. The cytotoxicity of naphthalene metabolites varied. Incubation of cells with 0.5 mM dihydrodiol, 1-naphthol, or 1,2-naphthoquinone decreased cell viability to an extent similar to that produced by 0.5 mM naphthalene. In contrast, 0.5 mM naphthalene oxide and 1,4-naphthoquinone significantly decreased viability more than the parent compound. Preincubation of Clara cells with piperonyl butoxide did not affect the loss in cell viability associated with naphthalene oxide. We conclude that isolated Clara cells 1) are capable of metabolizing naphthalene, a Clara cell-specific cytotoxicant, to two major metabolites, 2) have a detectable intracellular glutathione pool, and 3) are more susceptible to specific naphthalene metabolites than to the parent compound naphthalene. |
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