Steroid Synthesis-Dependent, Oxygen-Mediated Damage of Mitochondrial and Microsomal Cytochrome P-450 Enzymes in Rat Leydig Cell Cultures*

Abstract

Treatment of primary cultures of rat Leydig cells with 1 mM 8-bromo-cAMP for 2 days at ambient oxygen tension (19%) caused a 59% decrease in mitochondrial cholesterol side-chain cleavage (P-450scc) activity. This decrease was completely prevented when the oxygen tension was reduced to 1% O2 or when steroid synthesis was inhibited by aminoglutethimide. When the endogenous concentration of pregnenolone was increased by inhibiting its further metabolism, P-450scc activity was reduced by 80% in unstimulated cultures and was completely eliminated in cAMP-treated cultures. These losses were prevented when cells were maintained at 1% O2. The amount of immunoreactive P-450scc was also decreased by treatments that reduced P-450scc activity. Stimulation with cAMP also lowered microsomal C17-20 lyase activity by an oxygen-mediated, steroid synthesis-dependent mechanism. Treatment of cultures with testosterone caused a similar oxygen tension-sensitive decrease in C17-20 lyase activity. These results suggest that the enhanced loss of mitochondrial and microsomal cytochrome P-450 activities in cAMP-treated cultures is caused by the increased production of pregnenolone and testosterone, respectively, which generate reactive damaging species derived from reduced dioxygen. The increased catalytic turnover of these P-450 enzymes may also contribute to their damage. Although P-450 activities were preserved at 1% O2, the ability of cAMP-treated cells to synthesis testosterone in response to subsequent cAMP stimulation was still reduced. If, however, 25-hydroxycholesterol was supplied to these cells the decrease in testosterone-producing capacity was prevented, which demonstrates that the reduced steroidogenic capacity of cAMP-treated Leydig cells is caused, primarily, by the reduced availability of endogenous cholesterol.