| ANTX-a, the representative of neurotoxins in the freshwater bodies, is detected in water blooms of many countries. ANTX-a is a kind of low-molecular-weight alkaloid, its neurotoxic effects and toxic mechanism studies are relatively clear, but other toxicity studies are relatively few. Therefore, this study utilize the immune cells isolated from common aquatic carp as the object and ANTX-a as the target pollutant, the concentration of ANTX-a is 0.01,0.1,1,10 mg/L and exposure time for 12 h by in vitro exposure method. To study the toxic effects of ANTX-a on immune cells in carp, flow cytometry was used to detect the apoptosis rate, transmission electron microscope was used to observe the structure changes and agarose gel electrophoresis was used to detect the breaks of DNA. And on this basis, studies on the molecular mechanism of ANTX-a on the antioxidant system of immune cells and the death pathway of cells were also carried out.Flow cytometry results showed that 0.01-10 mg/L ANTX-a could induce apoptosis of immune cells of carp, the apoptotic rates of treated cells reached 18.89%, 22.89%,39.23%, and 35.58%, respectively. Meanwhile, it indicate that ANTX-a can lead to the disorder of cell cycle, speed of division were slow down; Transmission electron microscopy results showed a condensed cytoplasm with hollow bubbles and a slight swelling of the mitochondria in immune cells exposed to 10 mg/L ANTX-a compared to untreated cells. DNA ladder gel electrophoresis results showed that ANTX-a can induce DNA breakage in immune cells of carp. Thus, it can be say that ANTX-a has an effect on the immune system of aquatic crucian carp.The relevant indicators of antioxidant system in immune cells were detected. Results showed that ANTX-a exposure induces a significant increase in reactive oxygen species and malonaldehyde in lymphocytes; a decrease in the antioxidant enzymes superoxide dismutase and catalase; a reduction in glutathione contents; and an inactivation of glutathione reductase, glutathione peroxidase, and glutathione-s-transferase. All these results suggest that ANTX-a destroyed the antioxidantsystem and induced oxidative stress of fish immune cells. The results of RT-PCR and Western Blot indicated that mRNA level of Bcl-2 in lymphocytes was decreased significantly by ANTX-a exposure, while Bax mRNA level was up-regulated in a dose-dependent manner. Meanwhile, the mitochondrial membrane potential and Cytochrome c oxidase subunit 6a were all decreased by ANTX-a in fish lymphocytes. These changes increased the cleaved caspase-3 and caspase-9 protein level, also shows that ANTX-a induced apoptosis of carp immune cells associated with the mitochondrial apoptosis pathway. Subsequently, receptor-interacting serine-threonine kinases RIP3, mixed lineage kinase domainlike protein MLKL and mitochondrial protein phosphatase PGAM5 were determined in our experiment, and results discovered that they were all increased in fish lymphocytes. However, tumor necrosis factor TNF-a and phosphor-IKXα/β were all decreased markedly, phosphor-NF-κB was increased at the first, but decreased in the concentration ANTX-a is 10 mg/L. These results suggested that necrosis may happen at 10mg/L ANTX-a. In summary,0.01-10 mg/L neurotoxin ANTX-a can damage the balance of antioxidant system in immune cells of crucian carp and induce apoptosis of carp immune cells. In addition, the apoptosis of immune cells were related to the mitochondrial pathway of apoptosis and TNF-a receptor pathway, and 10 mg/L ANTX-a can causes necrosis of immune cells, all these results stated that ANTX-a not only has obvious neurotoxicity, but also affected the immune system of aquatic organisms. |
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