The role of nGPx4 in resisting DEHP-induced DNA damage and reducing caspase‐independent cell death in male germ cells

Objective: Di(2-ethyl-hexyl) phthalate (DEHP) is a widely used plasticizer that adversely affects sperm quality and function by inducing DNA damage and caspase-independent cell death (CICD). Nuclear glutathione peroxidase 4 (nGPx4) has been implicated in maintaining the structural integrity of sperm chromatin. However, it remains unclear whether nGPx4 can counteract the DNA damage caused by DEHP exposure.Methods: We employed a germ cell line (GC-1) spg mouse cell model engineered to overexpress nGPx4 (OE-nGPx4). The cells were subsequently exposed to DEHP and its metabolite mono-2-ethylhexyl phthalate (MEHP) to simulate the DNA-damaging effects of environmental factors on reproductive cells. Following treatment, we assessed the proportion of apoptotic cells and the extent of DNA damage using molecular biological analyses, in addition to evaluating the expression of proteins associated with the apoptotic pathway in germ cells.Results: nGPx4 overexpression protected against DEHP-induced DNA damage in germ cells, reducing the incidence of CICD and potentially preserving sperm quality. This protective effect was mediated by enhanced chromatin condensation in mouse sperm cells and downregulation of phosphorylated H2A histone variant (γ-H2A.X). The reduction in DNA degradation is attributed to a diminished formation of the complex between γ-H2A.X and apoptosis-inducing factor (AIF), resulting in decreased DNA fragmentation. Additionally, compared to MEHP-treated cells, OE-nGPx4 cells exhibited reduced expression of Bcl 2-associated X (Bax), thereby diminishing activation of the γ-H2A.X/AIF axis.Conclusion: Our findings suggest that nGPx4 is involved in chromatin condensation and may contribute to downregulating the AIF/γ-H2A.X axis in male germ cells, ultimately reducing DNA damage-induced CICD.