Difenoconazole causes cardiotoxicity in common carp (Cyprinus carpio): Involvement of oxidative stress, inflammation, apoptosis and autophagy.

Difenoconazole, a commonly used broad-spectrum triazole fungicide, is widely applied to fish culture in paddy fields. Due to its high chemical stability, low biodegradability, and easy transfer, difenoconazole persists in aquatic systems, raising public awareness of environmental threats. Difenoconazole causes cardiotoxicity in carp, however, the potential mechanisms of difenoconazole-induced cardiotoxicity remain unclear. Here, common carp were exposed to difenoconazole, and cardiotoxicity was evaluated by measuring the creatine kinase (CK) and the lactate dehydrogenase (LDH) in the serum. Cardiac pathological injury was determined by HE staining. The content and expression of oxidative stress indicators were detected using biochemical kits and qPCR analysis. Changes in inflammation-related cytokines were examined by qPCR. Apoptosis levels were assessed by TUNEL assay and qPCR. The occurrence of autophagy was measured by western blotting detection of autophagy flux LC3II/LC3I, and autophagy regulatory pathways were detected using qPCR. The results showed that difenoconazole exposure induced cardiotoxicity accompanied by obviously elevated LDH and CK levels and caused myocardial fibers to swell and inflammatory cells to increase. Elevated peroxide MDA and reduced transcriptional and activity levels of the antioxidant enzymes CAT, SOD and GSH-Px were dependent on the Nrf2/Keap-1 pathway. Moreover, the proinflammatory cytokines IL-1β, IL-6, and TNF-α were upregulated, iNOS activity was enhanced, whereas the anti-inflammatory cytokines TGF-β1 and IL-10 were downregulated after exposure to difenoconazole. Moreover, apoptosis was observed in the TUNEL assay and mediated through the p53/Bcl-2/Bax-Caspase-9 mitochondrial pathway. Furthermore, difenoconazole increased the autophagy markers LC3II, ATG5 and p62 and regulated them through the PI3K/AKT/mTOR pathway. Altogether, this study demonstrated that difenoconazole exposure caused common carp cardiotoxicity, which is regulated by oxidative stress, inflammation, apoptosis and autophagy, providing central data for toxicological risk assessment of difenoconazole in the ecological environment.