The Molecular Toxicology About DDT Exposure On Liver Damage And Liver Cancer Progression

Though having being banned or restricted for three decades, DDT concentrations in the environment and human body are still very high, due to its bioaccumulation, long-range transport and persistence in the environment properties.In addition, DDT is still being used for the control of malaria in some developing countries, which becomes one major sources of occupational exposure to pesticides. Liver is an important detoxification organ and a special tissue, in which persistent organic pollutants metabolize and accumulate. Liver symptoms, associated with DDT poisoning, include hepatomegaly, liver damage and liver function disorder. However, the current studies of DDT and liver health mainly focus on epidemiologic and toxicological aspects, scarce information exists on the molecular mechanism and relevant effective inhibitors.In this study, we adopted low doses and high doses of p,p'-DDT, the main component of DDT, to expose human hepatocellular carcinoma cells and human normal liver cells. Besides, we focused on the target of oxidative stress and possible signal pathways to analyze the molecular mechanism of p,p'-DDT exposure on HCC's progression and liver damage. The research includes the following four parts:The first part:Effects and molecular mechanisms of low doses p,p'-DDT exposure on hepatocellular carcinoma cell proliferation. The data indicated that the proliferation of HepG2 cells was strikingly promoted upon p,p'-DDT exposure from 10-12 to 10-6 M for 4 days. In addition, reactive oxygen species (ROS) content was significantly elevated, accompanied with inhibitions of ?-glutamylcysteine synthetase (y-GCS) and superoxide dismutase (SOD) activities. Effects of p,p'-DDT on the Wnt/?-catenin pathway were determined by western blots analysis. Interestingly, the levels of P-catenin and its downstream target genes (c-Myc and CyclinD1) were significantly up-regulated, and co-treatment of NAC, the ROS inhibitor, inhibited these over-expressed proteins. Moreover, the p,p'-DDT-stimulated proliferation of HepG2 cells could be reversed after NAC or ?-catenin siRNA co-treatment. Likewise,5 nM/Kg p,p'-DDT treatment increased the growth of tumor in nude mice, stimulated oxidative stress and Wnt/?-catenin pathway using immuno-histochemical and western blots analysis. In summary, low doses p,p'-DDT exposed hepatocellular carcinoma, first activated ROS and stimulated the oxidative stress. It then promoted phosphorylation of GSK3? at Ser 9. Active ?-catenin was accumulated in the cytoplasm and entered the nucleus where it binded to the transcription factor TCF. Binding to TCF allowed the regulation of its downstream target genes (c-Myc and CyclinDl) expressions, which were associated with proliferation, thus promoting the progression of hepatocellular carcinoma.The second part:Effects and molecular mechanisms of low doses p,p'-DDT exposure on hepatocellular carcinoma cell adhesion. The data showed that p,p'-DDT, exposing HepG2 cells for 6 days, decreased the cell-cell adhesion and elevated the cell-matrix adhesion. Strikingly, p,p'-DDT increased the ROS content, accompanied by the activation of JAK/STAT3 pathway. Moreover, ROS inhibitor supplement reversed these effects significantly. However, the addition of ER inhibitor, ICI, had no effect on the p,p'-DDT-induced effects, indicating that the p,p'-DDT-disrupted cell adhesion was mediated by ROS not ER in HepG2 cells. p,p'-DDT altered the mRNA levels of related adhesion molecules, including inhibition of E-cadherin and promotion of N-cadherin along with CD29. Interestingly, the p,p'-DDT-altered adhesion molecules could be reversed by the JAK inhibitor or STAT3 inhibitor. Likewise, p,p'-DDT stimulated the JAK/STAT3 pathway in nude mice, as well as altered the mRNA levels of E-cadherin, N-cadherin, and CD29. Taken together, these results indicated that p,p'-DDT profoundly promoted the adhesion process by decreasing cell-cell adhesion and inducing cell-matrix adhesion via the ROS-mediated JAK/STAT3 pathway. All these events accounted for the carcinogenic potential of p,p'-DDT in liver.The third part:The cytotoxicity of high doses p,p'-DDT exposure on human normal liver cells and protective effects of vitamins C and E. The results demonstrated that p,p'-DDT exposure at over 10 ?M depleted the cell viability of HL-7702 cells and led to the cell apoptotic adopting the flow cytometric analysis. The molecular mechanisms of p,p'-DDT-induced cell apoptosis were investigated through membrane potential and the expression of caspases family.p,p'-DDT treatment elevated the ROS generation, induced mitochondrial membrane depolarization, and released cytochrome c into the cytosol, with subsequent elevations of Bax and p53, along with the suppression of Bcl-2. In addition, the activations of caspase-3 and -8 were triggered. Furthermore, p,p'-DDT promoted the expressions of NF-?B and FasL. When the cells were exposed to the NF-?B inhibitor (PDTC), the up-regulated expression of FasL was attenuated. However, these alterations caused by DDT treatment were prevented or reversed by the addition of VC or VE, and the protective effects of co-treatment with VC and VE were higher than the single supplement with p,p'-DDT. Taken together, these findings provided novel experimental evidences supporting that VC or/and VE could reduce p,p'-DDT-induced cytotoxicity of HL-7702 cells via the ROS-mediated mitochondrial pathway and NF-xB/FasL pathway.The fourth part:The genotoxicity and hepatotoxicity of high doses p,p'-DDT exposure on human normal liver cells and protective effects of vitamins C and E. DAPI staining of chromatin condensation, comet, micronucleus (MN) and DNA-protein crosslink (DPC) assays were used to evaluate the genotoxicity of p,p'-DDT exposure on HL-7702 cells. Treatment with p,p'-DDT alone increased levels of chromatin condensation, some comet parameters, micronucleus inductions and DPC coefficient in a dose-dependent manner. Based on these observations, it could be suggested that p,p'-DDT induced the genotoxicity of HL-7702 cells, caused the DNA damage and chromatin condensation. Meanwhile, RT-PCR assay was performed to evaluate the hepatotoxicity of p,p'-DDT on HL-7702 cells. The data showed that an increase in mRNA levels of CYP1A1 as well as CYP3A4, and a decrease in UGT along with GST mRNA levels were observed. Interestingly, supplementation with VC or/and VE prevented p,p'-DDT-induced alterations in DNA damage and phase metabolism gene expression, and the combination of VC+ VE had more protective effects than VC or VE alone. Collectively, these results illustrated that p,p'-DDT was toxic to human liver, which was partly ameliorated by the presence of VC or/and VE.In summary, this work evaluates the toxicity of p,p'-DDT on human liver, including low doses on the progression of HCC and high doses on the damage of human normal liver, classifies the relevant molecular mechanism focused on oxidative stress along with possible signal pathways, and investigates the natural effective antagonist of VC and VE. Accordingly, this project assists to understand the association of persistent pesticides pollution such as DDT and risks of HCC development as well as normal liver damage, and provides the theoretical basis about the intervention and control of food nutrients on p,p'-DDT-induced human liver health hazards.