Mechanism by which ethane dimethanesulfonate kills adult rat leydig cells: Involvement of intracellular glutathione

We have previously demonstrated that ethane-1,2-dimethane-sulfonate (EDS) kills adult, but not immature, rat Leydig cells in vivo and in vitro. The mechanism responsible for this selective toxicity is not known. Here we report that the cytotoxic effects of EDS on adult rat Leydig cells were not dependent upon new protein synthesis or cytochrome P450 enzyme activity. To determine whether inhibition of glutathione synthesis protects Leydig cells from the cytotoxic effects of EDS, adult rat Leydig cells were cultured in the presence or absence of 4 mM buthionine sulfoximine (BSO; 2 hr), a specific inhibitor of glutathione synthesis, and subsequently with increasing doses of EDS (3 hr). Following EDS addition, the ability of the cells to produce testosterone in response to LH stimulation, and to synthesize protein ([³⁵S]methionine incorporation) were evaluated. In both cases, Leydig cells cultured in the presence of BSO were far less sensitive than Leydig cells cultured in medium alone (control) to EDS effects on testosterone production (control: EC₅₀ 60 μg EDS/ml; BSO: EC₅₀ > 1500 μg EDS/ml) and [³⁵S]methionine incorporation (control: EC₅₀ = 95 μg EDS/ml; BSO: EC₅₀ = 1560 μg EDS/ml). This protective effect of BSO was abolished by restoring intracellular glutathione levels with glutathione ethyl ester (8 mM; GSHEE). Interestingly, none of these treatments altered the viability (i.e., [³⁵S]methionine incorporation) of immature rat Leydig cells (EC₅₀ = 420 μg EDS/ml for control, GSHEE, and BSO groups). Taken together, these data suggest that the mechanism by which EDS kills adult rat Leydig cells may involve Leydig cell glutathione.