Protective Effects And Mechanisms Of Natural Antioxidant Against Nodularin-ind Uced Cytotoxicity On Fish Lymphocytes In Vitro

Nodularin, a metabolite of Nodularin spumigena, is one of the most conspicuous and widespread pollutants that elicit water ecological hazards to fish, causing serious damage on the immune system and physiological functions. Thus, the objective of this study is to examine the apoptotic responses of immune systems of fish upon exposure to nodularin using isolated lymphocytes from Carassius auratus. The apoptosis-related cytotoxic effects and mechanisms of nodularin on Carassius auratus lymphocytes through the apoptosis rate detection, confocal laser microscope and transmission electron microscope observation, agarose gel electrophoresis of DNA-Ladder detection were investigated. In particular, the mechanism to induce apoptosis responses by nodularin is investigated.Confocal laser microscope results showed that control cyteblast present homogeneous fluorescence, the nodularin-induced group have granular fluorescent fragments. Transmission electron microscopy results showed that nodularin-treated lymphocytes display a series of morphological changes, including condensed cytoplasm, nuclear chromatin agglutination and marginalization. DNA fragmentation was verified by the DNA-ladder and formation of sub-G1DNA peaks. These cell characteristics confirmed the occurrence of apoptosis in lymphocytes. Flow cytometric results showed that the percentages of apoptotic cells incubated with1,5,10, and100ug/L nodularin for12h reached15.76%,17.36%,20.34%and44.21%, respectively; controls showed low rates of apoptosis (2.4%).The study also found that nodularin exposure caused a significant increase in intracellular reactive oxygen species (ROS), loss of mitochondrial transmembrane potential in a dose-dependent manner, upregulation of intracellular Ca2+, downregulation of Bcl-2and upregulation of Bax expression at the mRNA and protein levels, and activation of Caspase-3and Caspase-9without Caspase-8. In summary, all the results suggest that nodularin induces lymphocyte apoptosis via the mitochondrial apoptotic pathway and destroys the immune response of fish. Epigallocatechin-3-gallate (EGCG) is a kind of natural antioxidant, which has the ability to clear oxygen free radicals, inhibit cell apoptosis, enhance immunity. Nodularin can cause oxidative stress-induced cytotoxicity on fish lymphocytes.The regulatory effects of epigallocatechin-3-gallate (EGCG) at10,100, and1000μg/L levels on the antioxidant defense system and apoptosis of Carassius aura/us lymphocytes exposed to a high dose of nodularin (100μg/L) were quantified in vitro, which may be a detoxicant to nodularin.EGCG reduced nodularin-induced oxidative damage on fish immune cells. This compound significantly increased the activities of superoxide dismutase and catalase and the level of glutathione but decreased the levels of intracellular reactive oxygen species and malondialdehyde. Flow cytometry results showed that the percentages of apoptotic cells after treatment with10,100, and1000μg/L EGCG for12h reached27.9%,19.1%, and13.7%, respectively. By contrast, the nodularin alone-induced group showed a high percentage of apoptosis (44.2%). Western blot analysis showed the increased expression of Bcl-2and the decreased expression of bax and caspase-3in EGCG-treated fish lymphocytes. EGCG also inhibited the potential collapse of the mitochondrial membrane. Overall, EGCG can inhibit nodularin-induced apoptosis and protect the normal immunity of fish by regulating Bax/Bcl-2and blocking the downstream of mitochondrial apoptosis pathway with increased intracellular antioxidant enzyme activity.