Testicular CYP3A triggers BDE47-induced spermatogenic cell ferritinophagy via ROS-ALKBH5-m⁶A-ATG12 pathway

Cytochrome P450 CYP3A (CYP3A) is the most abundant metabolic enzyme in the liver and also mediates the metabolism and toxicity of xenobiotics. Previous studies have reported that CYP3A is also expressed in the testis; however, its role and molecular mechanism in mediating male reproductive damage remain unclear. In this study, 2,2’,4,4’-tetrabromodiphenyl ether (BDE47) was employed to clarify the role of CYP3A in BDE47-induced reproductive toxicity. The results showed that BDE47 induced CYP3A expression in mouse testes, leading to oxidative stress and ferroptosis through excessive reactive oxygen species (ROS) and ferrous iron (Fe²⁺) overload, which was confirmed by CYP3A overexpression or knockdown experiments in GC-2 cells. Mechanistically, in addition to direct ROS generation during metabolic processing, a large portion of Fe²⁺ was produced through the ROS-initiated ferritinophagy pathway. Specifically, ROS induced HIF-1α expression, upregulated the m⁶A demethylase ALKBH5, and reduced m⁶A modification on Atg12 mRNA, subsequently elevating ATG12 protein levels and driving autophagic degradation of FTH1 with consequent Fe²⁺ overload. These results were further confirmed by experiments using hydrogen peroxide or antioxidants in GC-2 cells, as well as by Atg12 knockdown mice. Our findings demonstrate that CYP3A plays a critical role in male reproductive toxicity induced by environmental toxicants.