RESUMO
Nodularin 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. Nodularin can cause oxidative stress-induced apoptosis on fish lymphocytes. The regulatory effects of epigallocatechin-3-gallate (EGCG) at 10, 100, and 1000 µg/L levels on the antioxidant defense system and apoptosis of Carassius auratus lymphocytes exposed to a high dose of nodularin (100 µg/L) were quantified in vitro. 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 with 10, 100, and 1000 µg/L EGCG for 12 h reached 27.9%, 19.1%, and 13.7%, 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-2 and the decreased expression of bax and caspase-3 in 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-2 and blocking the downstream of mitochondrial apoptosis pathway with increased intracellular antioxidant enzyme activity.
Assuntos
Antioxidantes/metabolismo , Catequina/análogos & derivados , Carpa Dourada/metabolismo , Linfócitos/efeitos dos fármacos , Peptídeos Cíclicos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Apoptose/efeitos dos fármacos , Western Blotting/veterinária , Catequina/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo/veterinária , Linfócitos/citologia , Linfócitos/metabolismo , Masculino , Toxinas Marinhas/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nodularia/química , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Nodularin, a metabolite of Nodularin spumigena, is widely detected in water blooms worldwide and causes serious negative effects on fish. The apoptosis-related cytotoxic effects and mechanisms of nodularin on Carassius auratus lymphocytes were investigated. 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-G1 DNA peaks. These cell characteristics confirmed the occurrence of apoptosis in lymphocytes. Flow cytometric results showed that the percentages of apoptotic cells incubated with 1, 5, 10, and 100 µg/L nodularin for 12 h reached 15.76%, 17.36%, 20.34% and 44.21%, respectively; controls showed low rates of apoptosis (2.4%). The mechanism of apoptosis induced by nodularin was determined, and results showed 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 Ca²âº, downregulation of Bcl-2 and upregulation of Bax expression at the mRNA and protein levels, and activation of caspase-3 and caspase-9 without 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.