Nitrite and microcystins co-exposure triggers PINK1-mediated mitophagy in spermatogonia

Nitrite and Microcystins (MCs) frequently co-occur in eutrophic water bodies, and both are well-documented to elicit reproductive toxicity via direct impairment of reproduction-associated cells. However, the molecular mechanisms underlying their combined cytotoxic effects remain poorly defined. In this study, the mouse germ cell line (GC-1) was exposed to 1/2 IC₅₀ concentrations of sodium nitrite (NaNO₂) or microcystin-leucine-arginine (MC-LR), individually or in combination, for 24 h in vitro. Co-exposure synergistically diminished cell viability, which was accompanied by mitochondrial swelling, cristae loss, and enhanced autophagosome formation. These changes were associated with elevated levels of mitochondrial reactive oxygen species (mtROS) and impaired mitochondrial membrane potential. Transcriptomic profiling revealed significant enrichment of pathways related to autophagy and cell death. Treatment with the mitochondria-targeted antioxidant mitoquidone mesylate (MitoQ) attenuated the loss of cell viability and mitochondrial damage induced by NaNO₂ and MC-LR, while suppressing the expression of the autophagy-related protein PINK1. Furthermore, siRNA-mediated knockdown of PINK1 could reduce the expression of mitochondrial autophagy markers, including phospho-Parkin, Atg12, and LC3-II. These findings identify PINK1 as a key regulator of mitochondrial autophagy triggered by NaNO₂ and MC-LR co-exposure and suggest that mitochondrial antioxidants such as MitoQ may mitigate the reproductive toxicity of these environmental contaminants.