Polystyrene-microplastics and DEHP co-exposure induced DNA damage, cell cycle arrest and necroptosis of ovarian granulosa cells in mice by promoting ROS production

The joint pollution of microplastics (MPs) and di-(2-ethylhexyl) phthalic acid (DEHP) often occurs, and consequently poses a serious threat to human and animal health, which has attracted widespread attention. However, the damage to the female mammalian ovary caused by the single exposure and co-exposure of MPs and DEHP and its specific mechanisms are not clear. Here, we established mouse models of single and co-exposures to polystyrene-microplastics (PS-MPs) and DEHP. The results showed that exposed to 100 mg/L PS-MPs and 200 mg/kg DEHP for 35 days destroyed the ovarian granulosa cell layer of mice, leading to follicular fragmentation and atresia. We cultured ovary granulosa cells in vitro to perform further mechanism studies and found that PS-MPs and DEHP had synergistic effects. Both of them promoted the excessive production of ROS and induced oxidative stress by triggering the CNR1/CRBN/YY1/CYP2E1 signaling axis, which in turn caused DNA oxidative damage. Additionally, we provided compelling evidence that oxidative stress mediated-hippo signaling pathway played a critical role in PS-MPs and DEHP caused ovary damage, resulting in ovarian granulosa cell cycle arrest and necroptosis. Using oxidative stress inhibitor AM251 or DAS could reverse these changes markedly and alleviate the reproductive toxicity caused by PS-MPs and DEHP, effectively. Overall, these results demonstrated that co-exposure of PS-MPs and DEHP adversely affected the integrity of ovary granulosa cell layer, resulting in DNA oxidative damage, cell cycle arrest and increased necroptosis of mouse ovarian granulosa cells by inducing oxidative stress. Our study shed new light on the co-exposure toxicity of PS-MPs and DEHP, provided novel insights for the reproductive toxicity of PS-MPs combined exposure with DEHP in female animals from a new free radical generation pathway perspective.