THE ANTIOXIDANT DEFENSE SYSTEM OF ISOLATED GUINEA-PIG LEYDIG-CELLS

Utilization of highly enriched preparations of steroidogenic Leydig ce lls have proven invaluable for studying the direct effects of various hormones and agents on Leydig cell function in vitro. However, recent work indicates that isolated Leydig cells are often subjected to oxyge n (O-2) toxicity when cultured at ambient (19%) oxygen concentrations. Because intracellular antioxidants play an important role in protecti ng cells against oxygen toxicity, we have investigated the intracellul ar antioxidant defense system of isolated Leydig cells. The cellular l evels of several antioxidants including catalase, glucose-6-phosphate dehydrogenase (G-6-PDH), superoxide dismutase (SOD) of the Cu/Zn & Mn variety, glutathione peroxidase, glutathione reductase and total gluta thione were quantitated using enriched populations of Leydig cells iso lated from adult male guinea pig testes. Compared to whole testicular homogenates, Leydig cells contained significantly (P < 0.01) less G-6- PDH, total SOD, glutathione reductase and total glutathione, but signi ficantly (P < 0.001) more glutathione peroxidase. Compared to hepatic values previously reported in the guinea pig, Leydig cells contain nea rly 400 times less catalase, about 14 times less glutathione peroxidas e and almost 11 times less glutathione reductase. Since G-6-PDH and gl utathione reductase are both necessary to regenerate reduced gluthathi one (GSH) which couples with glutathione peroxidase to breakdown hydro gen peroxide (H2O2) under normal conditions, it is plausible that the oxygen toxicity observed in isolated Leydig cells is due to the intrac ellular accumulation of H2O2. Using the dichlorofluorescin diacetate ( DCF-DA) assay, we found that Leydig cells incubated in the presence of 19% O-2 produced significantly (P < 0.001) higher levels of H2O2 with time in culture compared to Leydig cells maintained at 3% O-2. These results support the hypothesis that the increased susceptibility of is olated Leydig cells to oxygen toxicity may be due, in part, to decreas ed amounts of certain antioxidant defenses and an increased production of the reactive oxygen species H2O2.