Synergistic reproductive toxicity of microcystin-LR and polystyrene micro/nano-plastics in male zebrafish

Microcystins (MCs) and micro- or nano-plastics (MPs or NPs) coexist in aquatic environment. The combined toxic effects and underlying mechanisms of MCs and MPs or NPs in male reproductive toxicology remain poorly studied. To investigate potential male reproductive interference, male zebrafish were exposed to microcystin-LR (MCLR; 0, 5, 25 μg/L), polystyrene micro-plastics (PSMPs; 5 μm, 1 mg/L), polystyrene nano-plastics (PSNPs; 80 nm, 1 mg/L), and MCLR + PSMPs or PSNPs for 45 days. Co-exposure to MCLR and PSMPs or PSNPs enhanced the bioavailability of MCLR and potentially exerted synergistic effects. Exposure to PSMPs, PSNPs, or MCLR individually resulted in the disruption of testicular architecture and increased oxidative stress in vitro and in vivo, while co-exposure enhanced these toxic effects, especially with PSNPs. Our findings indicated that simultaneous exposure to PSMPs or PSNPs in conjunction with MCLR significantly exacerbates DNA damage compared to exposure to MCLR, PSMPs, or PSNPs alone. Consequently, this caused aberrant expression of cell cycle-related proteins in zebrafish testes and cell cycle arrest in GC-2 cells. Moreover, exposure to MCLR and PSNPs disrupted mitochondrial morphology and inhibited mitochondrial biogenesis and dynamics, resulting in mitochondrial membrane potential loss, adenosine triphosphate (ATP) content reduction in GC-2 cells. This research underscores the potential risks associated with MCLR and PSMPs or PSNPs on reproductive processes, as evidenced by the induction of DNA and mitochondrial damage in spermatogenic cells.