Abstract
Plastics (PS) and titanium (Ti) are utilized extensively in industrial, household, and medical applications due to their advantageous physicochemical properties. However, these materials have given rise to growing concerns regarding their biosafety. In this study, we investigated the impact of TiO2 nanoparticles (TiO2 NPs) and nanoplastics (nPS) on the reproductive function of male zebrafish (Danio rerio). Following a 30-day exposure period, the accumulation of both TiO2 NPs and nPS in testicular tissue was observed. Male zebrafish demonstrated significant decreases in secondary sexual characteristics, substantial reductions in sex hormone levels, and impaired sperm quality. While these effects were evident following individual exposures to either TiO2 NPs or nPS, co-exposure significantly intensified the extent of reproductive toxicity. Furthermore, the offspring of exposed males displayed pronounced developmental toxicity. Subsequent RNA sequencing analysis revealed significant upregulation of apoptosis-related genes in testicular tissue after exposure. Concomitant in vitro investigations employing GC-1 spermatogonial cells substantiated that co-exposure instigated apoptosis, plausibly through the activation of the PPARγ/RIPK3 signaling pathway. Collectively, our findings provide robust evidence that co-exposure to TiO2 NPs and nPS compromises male zebrafish reproductive function and induces transgenerational developmental toxicity, offering important theoretical insights for the prevention and risk assessment of emerging environmental co-contaminants.