| 【Background】Due to the geological pollution,the sewage of industrial and mining waste and the improper disposal of Ni-Cd battery, it has reported that there are more than 30 water and soil polluted by cadmium in most areas of our country, involving 16 provinces and autonomous regions nationwide. In these pollution areases, the cadmium content in rice, aquatic products and cadmium in drinking water content exceed the standard seriously. The population exposed to cadmium pollution is large,and cadmium exposure is reported to lead to abnormal bone metabolism, renal toxicity and reproductive toxicity,which is a serious threat to human health. However, there is no effective methods to solve the problem. The solution of the health problem caused by cadmium exposure has become an urgent medical problem. Studies had showed that the toxicity of cadmium in kidney, liver and reproductive system may be associated with oxidative stress.Previous studies had showed that the toxicity induced by cadmium exposure has been researched widely, and most of these researches were focused on the abnormal bone metabolism involoving in cadmium toxicity.Genotoxicity induced by cadmium exposure were also reported. However, the exact mechanisms involoved in the Cd genotoxicity and liver toxicity are still not clear. Some studies had suggested that the genetic toxicity induced by cadmium may be mainly related with testis damage, and oxidative stress plays a impotant role in the pathological process. Manystudies also confirmed that liver damage and kidney damage caused by Cd may be also due to oxidative stress. Selenium is an essential trace element for human beings,and selenium as a free radical scavenger has the function of antioxidant and anti-aging activity and is also closely related with the occurrence and prevention of many human diseases. Selenium is also the essential trace element in body and is the active ingredient in a variety of selenoprotein. Selenium not only has the antioxidant activity to inhibite oxidative damage, it may also act as a direct chemical to combinate with cadmium, which will play an important role of detoxification with Cd to alleviate cadmium toxicity. Therefore, to explore the antioxidant effects of selenium against cadmium and the dose effect relationship may be impotant to find an ideal and feasible way to help these populations exposed to cadmium, who are needed to reasonable supplied with proper selenium. 【Aims】The aims of this research is to observe the protective effects of different dose of selenium on liver and testicular toxicity in rats and cells exposed to cadmium and explore the related molecular mechanisms. 【Methods】1. The animal experiments: The main aim of the experiments is to clarify the toxicity and oxidative damage induced by cadmium and to explore the dose-effect relationship in antagonism between selenium and cadmium. SD rats were given cadmium chloride by intragastric administration to induce chronic oxidative damage with or without sodium selenite treatment. The effects of selenium on ROS levels in liver and testicular tissues in rats exposed to cadmium were investigated by immunofluorescence using DCFH-DA porbe. The effects of selenium on apoptosis in liver and testicular tissues in rats exposed to cadmium were investigated by Bcl-2 and Bax proteins detection by Western blot. The effects of selenium on oxidative stress in liver and testicular tissues in rats exposed to cadmium were investigated by MDA detection by kit. The effects of selenium on oxidative stress in liver and testicular tissues in rats exposed to cadmium were investigated by the determination of GSH levels and antioxidant enzymes(SOD, CAT and GPX) activities by corresponding kits and the protein expressions of Nrf-2 and relative antioxidant enzymes by Western blot.2. The cells experiments: The main aim of the experiments is to clarify the toxicity and oxidative damage induced by cadmium and to explore the protective effects of proper dose of selenium against cadmium toxicity. Brl-3A cells(rat liver cells) and Sertoli cells(the rat Sertoli cells) were incubated with sodium selenite and then exposed to cadmium chloride. The effects of selenium on cells viability in cells exposed to cadmium were investigated by MTT assay. The effects of selenium on apoptosis in cells exposed to cadmium were investigated by flow cytometry by Annexin V-FITC/PI apoptosis detection kit and Bcl-2/Bax proteins detection by Western blot. The effects of selenium on oxidative stress in cells exposed to cadmium were investigated by ROS level by DCFH-DA and DHE, and the MDA detection by kit. The effects of selenium on oxidative stress in cells exposed to cadmium were investigated by the protein expressions of Nrf-2 and relative antioxidant enzymes by Western blot. The effects of selenium on mitochondrial dysfunction in cells exposed to cadmium were investigated by the detection of mitochondrial ROS level by Mito SOX and MMP by rhodamine 123.3. Comprehensive analysis: we will compare with the animal experiment and cell experiments to find the common and different points, which will help to provide new insight on the antagonism between cadmium and selenium. 【Results】1. Animal experiments show that cadmium exposure conditions, liver and testicular cells elevated ROS and MDA levels, lower levels of GSH, cells showed elevated levels of oxidation. Meanwhile, after cadmium exposure, rat liver, testis cells Nrf-2 and its regulation of antioxidant enzymes GPx-1, SOD-1, SOD-2 and CAT activity and protein expression levels lower, indicating that the liver, testis cells against oxidative capacity decreased, these results suggest that the liver of rats exposed to cadmium under conditions at a distinct testicular oxidative stress. The study of rats exposed to cadmium shows that the level of ROS and MDA in liver and testis increased, while the level of GSH and Nrf-2 decreased. The level of cell oxidation elevated. Meanwhile, both the activity and protein level of antioxidant enzymes GPx-1, SOD-1, SOD-2 and CAT decreased in rat liver and testis cells, indicating that the capacity of liver and testis cells against oxidation decreased. These results suggest that the cadmium exposure could induce distinct oxidative stress in rat liver and testis.2. When the rats exposed to cadmium, the protein level of pro-apoptotic factor Bax increased while the anti-apoptotic factor Bcl-2 decreased in liver and testis cells. ROS was involved in the apoptosis process of liver and testis cells. The process of cell oxidative stress and apoptosis resulted in down-regulation of ALT, AST and testosterone in serum, which contributed to the severe toxicity of liver and testicular in rats.3. On the level of cell experiments, the exposure of Brl-3A and Sertoli cells to cadmium resulted in increased cell apoptosis rate, decreased mitochondrial membrane potential, changes of cell proton concentration gradient and reduced cell viability, which was associated with ROS. These results indicate that mitochondria are the key intracellular target for cadmium-induced cell apoptosis.4. When the rats were administrated with selenium before exposed to cadmium, the levels of ROS, MDA and Nrf-2 in liver and testes were significantly decreased, and the GSH level increased. The protein levels of Nrf-2 regulating antioxidant enzymes were increased while the enzyme activity was reduced. The protein level of Bax decreased while the Bcl-2 increased in liver and testis cells. These results indicate that selenium can effectively antagonize toxicity of cadmium in rats with a dose-dependence manner as well as organ specificity.5. When the Blr-3A and Sertolie cells were pretreated with selenium before exposed to cadmium, the levels of ROS and the rate of cell apoptosis both decreased, while the mitochondrial membrane potential and the antioxidant protein level increased. Which suggests that pretreatment of cells with low dose selenium could effectively antagonize cell cadmium toxicity. 【Conclusions】1. Cadmium(cadmium chloride of 5 mg Cd/kg.bw)exposure by intragastric administration could induce apoptosis, necrosis and oxidative stress in rat liver and testis.2. The mitochondria damage is the key target of cadmium-induced apoptosis, necrosis process.The mitochondrial oxidative damage is crucial factor to generate the cell apoptosis and necrosis in rat liver and testis induced by cadmium.3. Selenium treatment could improve the damages of liver and testis, and oxidative stress in rat and cell induced by cadmium, but there were determinate dose range and dose selectivity of selenium that is safety and utility under 1.0mg Se/kg of dosage.4. Intervention by dose of 2.0mg Se/kg selenium merely intragastric administration is safe, but using this dosage to antagonize cadmium toxicity exists aggravating damage risks of liver and testis toxicity.5. The Nrf-2 of antioxidant core factor participates in inhibition of selenium on damages of liver and testis cells exposed to cadmium. |
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