Studies On The Mechanisms Of Cytotoxic Effect Induced By Environmental Endocrine Disrupters-dioxin

Endocrine disruptors (EDs) are widely dispersed environmental and food contaminants in our country that poses a great risk to the public health.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs) compounds are persistent organic pollutants (POPs) and the typical environmental endocrine disruptors (EEDs), which the present international society pays attention extremely. At present, they are one of the hot spots in the field of environmental toxicology because of their extensive disposition and coexistence in the environment, persistence, bioaccumulation, migration and high toxicity. It mainly comes from waste incineration, the use and manufacture of pesticide, fertilizer, herbicides and other production with chlorine compounds. The prototype chemical for toxicity study is TCDD, which is recognized as the world's strongest carcinogens and the most toxic congener of dioxins. It is a ubiquitous environmental pollutant. In recent years, several dioxin contamination incidents have taken place, so many countries in the world pay great concern about dioxin on human health and environmental safety of the potential threat. Thus, TCDD is one of the hot spots in the field of environmental toxicology. Although preliminary achievements of the mechanism have been gained on the research of TCDD, the cytotoxic mechanism of TCDD is still not entirely clear. In this study, the toxic mechanism of TCDD has been researched both in vivo and in vitro. In addition, in order to provide a theoretical and experimental basis,we have studied the protective effect of zinc to TCDD. This study can be divided into two parts.1.The effect of individual and combined exposure to TCDD and PCBs in peripheral blood lymphocyte of ratsThe Sprague-Dawley rats were exposured to TCDD and Aroclor 1254 individual and combined, and detect the integrity of DNA by single cell gel electrophoresis(SCGE). Factorial design analysis of variance was used for determining the joint action of toxicological endpoints in TCDD and Aroclor 1254. The goal of this experiment is to further study the combined effects of TCDD and PCBs and the possible modes and mechanisms of and combined effects were investigated. In the present study, Twenty Sprague-Dawley rats were subjected to 2×2 factorial experimental design and randomly divided into four groups, and were treated intragastrically with vehicle(olive oil), Aroclor 1254(Aroclor 1254 10 mg/kg), TCDD(TCDD 10μg/kg), and the combination (Aroclor1254 10 mg/kg + TCDD 10μg/kg)once a day for six consecutive days, respectively. After 6 d exposure, peripheral blood lymphocytes were obtained and detected for DNA damage by single cell gel electrophoresis(SCGE). Tail Length, Tail DNA% and Tail Moment were analyzed by SCGE image analysis software(CASP). The possible interactions between the two compounds and the involvement of different mechanisms are discussed. The consequence suggested that The Tail Length, Tail DNA% and Tail Moment in both TCDD group and combined group were higher than those in the control(p<0.01). However, no obvious DNA damage was observed in Aroclor 1254 group. The result of the two-way analysis of variance revealed that the combined effects of TCDD and PCBs were synergistic under the present experimental condition.2. The cytotoxic effect of TCDDThis part we studied the oxidative stress and the protective effect of zinc on cytotoxicity induced by TCDD. The ROS production, DNA damage, Content of antioxidants, activity of antioxidant enzymes, expression of protein and Apoptosis were measured to investigate the role of toxic effects of TCDD-induced oxidative damage in HepG2 cells. HepG2 cells were cultured and treated with TCDD 1 nM.10 nM,100 nM,TCDD 100 nM+ZnCl265μM,TCDD 100 nM+ZnCl2 100μM for 24 h. Cell viability was assayed by MTT method; DCFH-DA was used for detecting the production of ROS in cells; GSH and MDA content, activity of SOD in cells were measured by colorimetry method; Apoptosis was measured with Hoechst 33258; The integrity of DNA was detected by SCGE; The express levels of CYP1A1,SODⅠ,SODⅡand HSP70 were measured by western blot. The results of this partial study suggest that TCDD decreased the cell viability in a well dose-response way; Formation of intracellular ROS and MDA content induced by TCDD increased in a dose-response way, and achieved the highest level in TCDD 100 nM(p<0.01). The GSH content and activity of SOD in cells decreased exposed by TCDD for 24 h(p<0.05); Obvious DNA damage and apoptosis was induced by TCDD(p<0.05). Compared with the control group, the expression of CYP1A1 and HSP70 increased induced by TCDD(P<0.05); But the expression of SODⅠand SODⅡdecreased by TCDD-induced(p<0.01). TCDD-induced cytotoxicity could be inhibited by ZincBased on the results of this study, the following conclusions could be made under the present experimental condition:①TCDD can significantly induced DNA damage in peripheral blood lymphocytes, and the effect was significant in the combined group.②TCDD induced oxidative stresses in HepG2 cells significantly:Formation of intracellular ROS increased, and the antioxidant defense system reduced by TCDD-induced. The expression of CYP1A1 and HSP70 increased, but the expression of SODⅠand SODⅡdecreased by TCDD-induced. The cellular lipid, DNA oxidative damage and apoptosis occurred at last.③Zinc as a radical scavenger, play protective effects on cytotoxicity induced by TCDD. In another word, zinc could be the antioxidant for TCDD-induced oxidative stress. It is the first time that Zinc was used in the inhibition for TCDD-induced oxidative stress in this study.