Effect of dihydrotestosterone on hydrogen peroxide-induced apoptosis of mouse embryonic stem cells

Steroid hormones have been reported to activate various signal transducers that trigger a variety of cellular responses. Among these hormones, testosterone has been identified as an antioxidant that protects against cellular damage. Therefore, using mouse embryonic stem (ES) cells as a model system, this study evaluated the effects of dihydrotestosterone (DHT), a biologically active testosterone metabolite, on H₂O₂-induced apoptosis. H ₂O₂ increased the release of lactate dehydrogenase (LDH) and DNA fragmentation but reduced the cell viability in a time-dependent manner (≥8 h). Moreover, H₂O₂ decreased the level of DNA synthesis and the levels of the cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4]. These effects of H ₂O₂ were inhibited by a pretreatment with DHT. However, a treatment with flutamide (androgen receptor inhibitor, 10^-3 M) abolished the protective effects of DHT. This result was supported by the presence of the androgen receptor in mouse ES cells. The activity of the antioxidant enzyme, catalase, was increased by the DHT treatment but not by a co-treatment with DHT and flutamide. Using CM-H₂DCFDA (DCF-DA) for the detection of intracellular H₂O₂, DHT decreased the intracellular H₂O₂ levels but flutamide blocked this effect. H₂O₂ also increased the level of p38 MAPK, JNK/SAPK, and NF-κB phosphorylation, which were inhibited by the DHT pretreatment. Catalase inhibited the effect of H₂O₂ on MAPKs and NF-κB. However, the flutamide treatment abolished the inhibitory effects of DHT on the H₂O₂-induced increase in the levels of p38 MAPK, JNK/SAPK, and NF-κB phosphorylation. DHT inhibited the H₂O₂-induced increase in caspase-3 expression and decreased the level of Bcl-2 and the cellular inhibitor of apoptosis protein (clAP)-2. These effects were abolished by the flutamide treatment. In conclusion, DHT prevents the H₂O₂-induced apoptotic cell death of mouse ES cells through the activation of catalase and the downregulation of p38 MAPK, JNK/SAPK, and NF-κB via the androgen receptor.