Zinc oxide nanoparticles (ZnO NPs) are important nanomaterials with myriad applications and in widespread use. The main aim of this study was to evaluate the direct effect of ZnO NPs on steroidogenesis by considering mouse testicular Leydig cells (TM3) as an in vitro model system. The uptake, intracellular behaviour, cytotoxicity and morphological changes induced by ZnO NPs (0-200 µg/ml) in a time-dependent manner in the TM3 were assessed. A significant ( p < 0.05) decrease in TM3 viability was observed at 2 µg/ml ZnO NP after a 1-h incubation time period. Increased antioxidant enzyme activity, namely, superoxide dismutase (SOD) and catalase, was regularly observed. Not surprisingly, apoptosis also increased significantly after a 4-h exposure period. Transmission electron micrographs illustrated that ZnO NPs were taken up by Leydig cells and resulted in the formation of autophagosomes, autolysosomes and autophagic vacuoles. Concomitant real-time data indicated that ZnO NPs significantly increased the expression of steroidogenesis-related genes (steroidogenic acute regulatory protein and cytochrome P450 side-chain cleavage enzyme) and significantly ( p < 0.05) decreased antioxidant enzyme gene (SOD) expression after a 4-h incubation period. Moreover, ZnO NPs exposure significantly increased testosterone production at 2 µg/ml concentration after a 12-h incubation period. Our findings confirm the adverse effects of ZnO NPs by being cytotoxic, enhancing apoptosis, causing steroidogenic effect in Leydig cells and increasing autophagic vacuole formation possibly via alteration of antioxidant enzyme activity in TM3 cells.
Keywords: TM3; ZnO nanoparticles; apoptosis; cytotoxicity; steroidogenesis; testosterone.