Microsomal cytochrome P-450 enzyme damage in cultured Leydig cells: relation to steroidogenic desensitization.

It is well established that treatment of animals with a single high dose of LH or hCG results in steroidogenic desensitization of Leydig cells.'*2 Desensitized Leydig cells exhibit a decreased capacity to produce testosterone in response to subsequent stimulation with gonadotropins or CAMP analogues, and have decreased activities of the microsomal P-450 enzymes, 17a-hydroxylase, and C17-20 lyase. The present study provides evidence that the decreases in microsomal P450 activities are caused by oxygen-derived, free-radical damage of the enzymes, and, furthermore, that the decrease in microsomal P-450 activities are not the primary cause of the decrease in testosterone-producing capacity observed in desensitized Leydig cells. Primary cultures of purified, adult mouse Leydig cells were maintained in serum-free medium at 32"C, in a humidified atmosphere of 19% O2 (95% air/5% C02) or 1% 02, in the presence and absence of the antioxidant dimethyl sulfoxide. The culture medium was changed daily and the activities of microsomal enzymes were determined by quantifying the conversion of 'H-substrate to 'H-products during a I hr incubation at 37°C in an atmosphere of 1% 02. The maximal capacity to produce testosterone was determined by incubating replicate cultures with 1 mM 8-Br-CAMP for a 3 hr period and measuring testosterone in the medium by radioimmunoassay. 1 mM 8-Br-CAMP was added to half of the Leydig cells, during the initial 24 hr of culture only, to induce steroidogenic desensitization. The other half served as controls. Testosterone production by desensitized Leydig cells was 15-fold and threefold greater than that of controls on days one and two of culture, respectively. The addition of dimethyl sulfoxide to the culture medium and/or reduction of the oxygen tension had no effect on testosterone production by control or desensitized Leydig cells. The data presented in TABLE 1 demonstrate that, when control Leydig cell cultures are incubated in 19% 02, the microsomal P-450 activities, 17 a-hydroxylase and C17-20 lyase, are stable during the first 24 hr but decrease markedly by 48