BaP and 17β-estradiol affect retromer-VPS35, restricting pro-survival transcription and exacerbating neurotoxicity in a reclaimed water environment

Under the reclaimed water system, Endocrine-disrupting chemicals and persistent organic pollutants often co-occur in realistic mixed-pollution environments and may jointly enhance neurotoxicity. In this study, we investigated the combined effects of 17β-estradiol (E2) and benzo[a]pyrene (BaP) by integrating seasonal monitoring of secondary wastewater effluent with mechanistic assays in NGF-differentiated PC12 cells. Pollutant levels in the effluent were highest during the dry season, supporting the construction of a co-exposure scenario relevant to industrially impacted reclaimed-water systems. Compared with single exposure, combined exposure to BaP and E2 resulted in markedly greater oxidative stress and reduced cell viability, with ROS levels increasing to approximately 2.9-fold of the control level and cell viability declining to about 61 %. These data indicate a synergistic neurotoxic effect and suggest that, although upstream survival-related signaling pathways were activated in response to stress, this activation was insufficient to elicit effective downstream neuroprotective transcription. Molecular docking further indicated that both pollutants could stably interact with VPS35, a core component of the retromer complex, suggesting impaired endosome-to-Golgi retrieval and enhanced cargo misdirection toward lysosomal degradation. Collectively, these findings identify retromer-VPS35 dysfunction as a potential mechanistic link between BaP/E2 co-exposure and synergistic neurotoxicity, and emphasized the importance of considering intracellular transport interference in the mixed risk assessment of pollutants derived from reclaimed water.