Effects Of T-2 Toxin On Antioxidative Defence System Of Litopenaeus Vannamei

T-2 toxin is one of the most poisonous substances among several trichothecenes, which is widespread in aquatic feed. The important mechanism of cytotoxicity is oxidative stress, resulting in emerging a large number of free radicals. Antioxidant defence system of crustaceans is the first line of defense to respond to external stimulation. If shrimps were exposed to T-2 toxin for a long time, the antioxidant defence system will be severely damaged.In this study, the shrimp was taken as experimental subjects. The feed had been artificial contamination of T-2 toxin, which was divided into five T-2 toxin exposure dose groups(0.5, 1.2, 2.4, 4.8, 12.2 mg/kg?feed) and the control group. 20 days cumulative toxicity test was conducted to get muscle and hepatopancreas. Firstly, muscle was taken to conduct species identification using mitochondrial 16 S rRNA gene. Through comparing with 16 S rRNA gene of humans and animals on NCBI, primers design were successfully synthesized, including 16 SAR-L and 16 SAR-H. The result showed that the sample was Litopenaeus vannamei.In order to evaluate the effect of T-2 toxin on the total antioxidant capacity and antioxidant enzyme, the method of kit was adopted to determine T-AOC, MDA, as well as three important representative antioxidant enzymes CAT, SOD and GSH-PX. The results showed that hepatopancreas is the mainly organ caused by oxidative stress and oxidative damage, which is more sensitive to T-2 toxin. T-2 toxin can improve total antioxidant capacity of hepatopancreas, while MDA(oxidative injury) were positively correlated with the doses of T-2 toxin. Because the magnitude of oxidative damage was significantly higher than the total antioxidant capacity of the stimulation effects, it behaved damage effects. Fitting curve showed: T-AOC、SOD and GSH-PX have the same fitting model GaussAmp; the fitting model of CAT activity in hepatopancreas and muscle were parabolic and Gauss, respectively; the fitting model of MDA in hepatopancreas and muscles were one dollar and parabolic. While the exposure doses of T-2 toxin is 3.63, 1.78, 3.31 mg/kg, T-AOC, SOD and CAT in hepatopancreas appeared stimulating effect, respectively. When 0.55 mg/kg, MDA occurred NOAEL. When the exposure dose of T-2 toxin 4.57, 5.37, 2.51, 4.79 mg/kg, it appeared NOAEL.The expression levels of SOD and CAT were determined by using the method of real-time PCR. Firstly, by repeatedly primer design, we worked out a set of experiments methods to improve the efficiency, namely combination of manual and softwar e design method. During the process of real-time PCR, the best amount of samples is 20 mg. To ensure the effective combination of primer and template and avoid non-specific binding, the annealing temperatures of CAT and SOD were 56℃ and 58℃. After exposed to T-2 toxin, gene expressions of CAT and SOD were firstly increased and then decreased. This is hormesis. Muscle is more sensitive than the hepatopancreas in level of CAT and SOD gene expressions. And CAT gene expression is about 2 times than SOD.The HPLC method was adopted to determine three kinds of fat-soluble vitamins(VA, VD3, VE) content and the method of kit was used to determine the content of GSH. The fat-soluble vitamins, GSH in muscle and GSH in hepatopancreas can be significantly affected by T-2 toxin(p < 0.05). Optimum chromatographic conditions: ① Column: C18; ② mobile phase: using methanol-water(100 : 0; v / v), the flow rate was 1.0 mL / min; ③ extraction solvent: using methanol to dissolve the sample; ④ Detector: using UV detector. According to the degree of absorption components, the maximum absorption wavelength of VA, VD3 and VE were 280, 264 and 285 nm, respectively. Detection limits were 0.09, 0.06 and 0.13 μg/m L, respectively.In muscle, content of VE was the highest. VA followed. VD3 was the lowest. In high dose of T-2 toxin, the content of VA and VD3 were significantly decreased(p < 0.05). However, the content of VE showed volatility. In lower dose of T-2 toxin, the content of VD3 and VE were significantly increased(p < 0.05), which show stimulatory effect. With the dose of T-2 toxin rise, the content of GSH in hepatopancreas increased. Fitting Curve showed that after exposed to T-2 toxin, fitting model of VA and VD3 in muscle conforms Gauss, consistent to the model of CAT activity in muscle; GSH complied GaussAmp model, consistent with T-AOC, SOD and GSH- PX. GSH in hepatopancreas complied Boltzmann model. Above all indicates show that in order to resist oxidative stress caused by T-2 toxin, various substances works like synergistic effect. VD3 and GSH appeared the maximum inhibitory effect at 8.31 and 2.09 mg/kg, respectively. VA showed the minimum inhibitory effect at 0.96 mg/kg.According to comprehensive analysis of all indexes, we found T-2 toxin had a significant effect on antioxidant defense system. Hepatopancreas is the main target organ and repository. It is sensitive to T-2 toxin, and oxidative damage enlarged as the exposure dose increasing. After exposed to different dose of T-2 toxin, the content of all indexes in muscle and hepatopancreas worked differently, and they could influence each other. The mechanism was very complex. Once shrimps were exposed to T-2 toxin, CAT mainly showed damage effect, SOD and GSH-PX mainly showed stimulatory effect in hepatopancreas. In hepatopancreas, SOD mainly showed stimulatory effect in low dose, and SOD and GSH-PX in medium dose. In muscle, VA, GSH-PX, GSH and VD3 showed damage effect, as a whole, VA and GSH showed damage effect in the low dose of T-2, GSH-PX and GSH in medium dose, and VD3 in high dose; CAT and SOD mainly showed stimulatory effect, CAT in low dose, SOD in medium dose. And MDA was the key markers of damage effect in antioxidant defense system, CAT in muscle and SOD in hepatopancreas were the key markers in stimulatory effect. Also we obtained the dose-response curve of all indexes. Normally, they showed stimulatory effect at low dose, while inhibiting action at high dose.