Tannic acid antagonizes atrazine exposure-induced autophagy and DNA damage crosstalk in grass carp hepatocytes via NO/iNOS/NF-κB signaling pathway to maintain stable immune function

Abstract

Atrazine (ATR) is a herbicide widely used in grass crops. The pollution of the soil and water environment is extremely harmful to aquatic animals and their offspring. iNOS/NO upregulation, DNA damage and cellular autophagy affect the immune function of fish liver cells. The effects of ATR at exposure doses on grass carp hepatocytes in terms of autophagy and DNA damage effects in genotoxicity, as well as the antagonistic effects of TAN on the above phenotypes and the internal mechanisms are not known. Therefore, we constructed control (Con group), ATR exposure (ATR group), TAN exposure (TAN group) and mixed group (ATR + TAN group) models on grass carp hepatocytes. Validation was performed by comet assay, MDC staining, qRT-PCR and protein blotting assay as well as iNOS/NO indicator levels and expression of immune factors as these experimental methods. Our data indicate that iNOS/NO assay kit measured that ATR treatment resulted in a significant increase in iNOS/NO activity and levels in grass carp hepatocytes (p < 0.05). We also found that NO/iNOS/NF-κB pathway genes were significantly activated (p < 0.05) at the exposure dose of ATR (3 μg mL−1). In addition, the proportion of cells that died due to DNA damage, autophagy, and immunotoxic effects was significantly increased at the exposure dose of ATR. Comet assay protein blotting detected increased DNA damage in cells at the ATR exposure dose (p < 0.05). MDC staining and qRT-PCR and protein blotting to detect the proportion of autophagic cells and autophagy-related genes also appeared upregulated at the exposed dose of ATR (p < 0.05). In brief, this study showed that ATR exposure caused cellular DNA damage and autophagy via the NO/iNOS/NF-κB axis, which led to immunotoxic effects and eventual death of grass carp hepatocytes. The present study facilitates the demonstration of the molecular mechanism of TAN alleviation of ATR cytotoxicity from the perspective of NO-mediated iNOS/NF-κB axis. It provides insights into the protection of farmed fish from agricultural contaminants and opens up new horizons in the use of natural plant-derived monomers for the clinical treatment of antagonistic triazine pesticide poisoning.