DDT暴露引起肝脏损伤和致癌作用的分子毒理研究
发布时间：2018-09-18 19:06
【摘要】：持续性有机污染物DDT虽已被禁止或限用多年,但由于其具有生物累积性、持久性、脂溶性、难降解和食物链富集性等特点,在环境和人体仍有很高的检出率。此外,一些发展中国家仍用DDT来控制疟疾,使得这些国家成为DDT高暴露区。肝脏是DDT进行代谢、解毒和积累的主要场所和器官。有报道揭示,DDT可以引起肝脏肿大、损伤以及肿瘤等疾病。然而目前关于DDT残留和肝脏健康风险的研究主要集中在流行病学和实验动物毒性评价方面,具体的分子机制还不是很清楚,且关于DDT暴露引起的肝脏毒性干预研究尚未见报道。本研究采用低剂量(人体血液DDT检出量)和高剂量(高暴露区环境DDT含量)DDT主要成分p,p'-DDT暴露人肝癌细胞或人正常肝脏细胞,围绕氧化应激靶点以及可能的调控信号通路,分析p,p'-DDT暴露导致肝癌恶化以及肝脏损伤的分子毒理机制。本研究内容含以下四个部分：第一部分：低剂量p,p'-DDT暴露对人肝细胞癌生长的影响及其机制研究。采用MTT方法分析低剂量p,p'-DDT暴露对人肝癌细胞HepG2增殖的影响。结果显示低浓度p,p'-DDT暴露HepG2细胞四天后促进了细胞增殖,细胞中ROS含量升高,抗氧化酶y-GCS和SOD活性降低。蛋白印迹法检测p,p'-DDT对Wnt/β-catenin信号通路的影响,发现GSK3pβ、β-catenin及其下游与增殖密切相关的靶蛋白c-Myc和CyclinD1表达均升高。加入ROS抑制剂NAC后,这些蛋白表达被逆转。此外,p,p'-DDT促进的HepG2细胞增殖可被NAC或β-catenin siRNA处理所降低。利用肝癌裸鼠成瘤模型,采用免疫组化和蛋白印迹法检测5 nM/Kg p,p'-DDT暴露对裸鼠肝细胞癌肿瘤组织氧化应激水平和Wnt/β-catenin通路的影响。发现p,p'-DDT增加了裸鼠瘤的生长,激活了肝癌肿瘤组织氧化应激和Wnt/β-catenin信号通路。研究表明,当低剂量p,p'-DDT暴露于肝癌细胞后,首先激活细胞中ROS生成和氧化应激反应；进而促进GSK3β在丝氨酸第九位点的磷酸化；激活的β-catenin进入细胞核与核转录因子TCF相结合,促进下游与增殖相关的靶蛋白(c-Myc和CyclinD1)表达,从而促进人肝癌细胞的增殖和肝细胞癌的生长。第二部分：低剂量p,p'-DDT暴露对人肝细胞癌粘附的影响及其机制研究。采用细胞粘附方法检测低剂量p,p'-DDT对肝癌细胞粘附的影响。结果表明：p,p'-DDT暴露HepG2细胞六天后,降低了细胞与细胞之间的粘附,提高了细胞与基质之间的粘附。p,p'-DDT增加了HepG2细胞中ROS含量,激活了JAK/STAT3信号通路。此外,加入ROS抑制剂NAC后,这种效应被显著逆转。而加入雌激素受体拮抗剂ICI抑制雌激素受体ER后,对p,p'-DDT诱导的现象没有影响。表明p,p'-DDT可能通过ROS而非ER介导其调控的细胞粘附。p,p'-DDT还影响HepG2细胞粘附分子的表达,它的暴露导致E-cadherin降低,N-cadherin和CD29升高。进一步研究揭示,p,p'-DDT影响的粘附分子表达能够被JAK抑制剂或STAT3抑制剂所逆转。同样地,p,p'-DDT激活了肝癌裸鼠肿瘤组织中JAK/STAT3信号通路,影响了E-cadherin、N-cadherin和CD29的基因表达。研究表明,p,p'-DDT通过提高细胞中ROS含量,介导JAK/STAT3信号通路激活,调控下游粘附分子表达；降低细胞与细胞间的粘附,增强细胞与基质间的粘附,促进肝癌细胞的恶化。第三部分：高剂量p,p'-DDT暴露对人正常肝脏细胞的毒性及维生素C和E的拮抗效果研究。采用流式细胞技术检测p,p'-DDT对人正常肝脏细胞HL-7702存活的影响,发现大于10 μM的p,p'-DDT暴露HL-7702细胞后,显著诱导细胞凋亡。进一步检测线粒体膜电位以及caspases家族蛋白的表达,结果显示p,p'-DDT暴露HL-7702细胞后,显著提高了细胞中ROS含量和促凋亡基因Bax的表达,降低了抗凋亡基因Bcl-2的表达,从而提高线粒体膜的去极化致使膜受到损伤。细胞色素c从线粒体释放到细胞质中,诱导caspase-9和3的活性形式产生,激活线粒体凋亡信号通路。与此同时,p,p'-DDT通过ROS介导下游NF-κB的激活,活化的NF-κB进入细胞核促进下游FasL的表达,随后结合到其死亡受体Fas上,激活死亡受体介导的凋亡信号通路,促进HL-7702细胞凋亡。然而,加入天然抗氧化物维生素C和E后,抑制了p,p'-DDT诱导的ROS生成及下游凋亡信号通路的激活,由此降低p,p'-DDT的细胞毒性,缓解细胞凋亡,且维生素C和E复合加入的效果大于单独加入的效果。本研究表明,维生素C和／或E可以拮抗p,p'-DDT引起的HL-7702细胞毒性。第四部分：高剂量p,p'-DDT暴露对人正常肝脏细胞的基因毒性和肝脏毒性及维生素C和E的拮抗效果研究。采用了DAPI染色、彗星实验、微核试验、DNA和蛋白质交联实验等方法,多方面评价了p,p'-DDT对人正常肝脏细胞HL-7702的基因毒性。结果显示：p,p'-DDT暴露后,剂量依赖性地提高了细胞中染色质浓缩、彗星实验参数、微核诱导率以及DPC系数。表明p,p'-DDT暴露可以诱导HL-7702细胞产生基因毒性,引起DNA损伤和染色质浓缩。采用qRT-PCR检测p,p'-DDT暴露对HL-7702肝细胞毒性的影响,发现P,P'-DDT暴露对HL-7702细胞具有明显肝脏毒性,具体表现在Ⅰ相代谢酶基因(CYP1A1、CYP2B6和CYP3A4)表达升高,Ⅱ相代谢酶基因(UGT、GST和GCS)表达下调。本研究还揭示维生素C和E可以拮抗p,p'-DDT引起的HL-7702细胞DNA损伤和代谢酶变化,即基因毒性和肝脏毒性,且二者复合的拮抗效果大于单独加入的效果。综上所述,本研究评价了p,p'-DDT暴露对肝脏的毒性作用,包括低剂量p,p'-DDT暴露对肝癌细胞恶化的影响以及高剂量p,p'-DDT暴露对正常肝脏细胞的损伤；围绕氧化应激靶点及可能的调控信号通路,阐明了p,p'-DDT暴露引起肝脏健康风险的相关分子机制；分析了天然抗氧化物维生素C和E对p,p'-DDT毒性的拮抗作用。因此,本研究有助于阐明持续性有机农药DDT残留引起的人体肝脏健康风险及分子机制,为干预DDT暴露引起的肝脏毒性提供了一定的防控依据。
[Abstract]:DDT, a persistent organic pollutant, has been banned or restricted for many years, but because of its bioaccumulation, persistence, fat solubility, refractory degradation and food chain enrichment, it still has a high detection rate in the environment and human body. In addition, some developing countries still use DDT to control malaria, making these countries a high exposure area of DDT. The main sites and organs where T is metabolized, detoxified, and accumulated. It has been reported that DDT can cause liver enlargement, injury, and tumors. However, current studies on DDT residue and liver health risk mainly focus on epidemiology and toxicity assessment in laboratory animals, and the specific molecular mechanism is not clear, and about DDT outbreaks. In this study, low-dose (human blood DDT detection) and high-dose (high environmental DDT content) DDT main components p, p'-DDT were used to expose human hepatocellular carcinoma cells or human normal liver cells, and the oxidative stress targets and possible regulatory signaling pathways were analyzed. Molecular toxicological mechanisms of cancer progression and liver injury. This study includes the following four parts: Part I: Effects of low-dose p, p'-DDT exposure on the growth of human hepatocellular carcinoma and its mechanism. MTT method was used to analyze the effects of low-dose p, p'-DDT exposure on the proliferation of human hepatocellular carcinoma HepG2 cells. Four days later, G2 cells proliferated, ROS content increased, antioxidant enzymes y-GCS and SOD activity decreased. The effects of p, p'-DDT on Wnt/beta-catenin signaling pathway were detected by Western blotting. GSK3pbeta, beta-catenin and their downstream target proteins c-Myc and Cyclin D1, which were closely related to proliferation, were all increased. After adding ROS inhibitor NAC, the expression of GSK3pbeta, beta-catenin and Cyclin D1 was detected. In addition, the proliferation of HepG2 cells stimulated by p, p'-DDT could be reduced by NAC or beta-catenin siRNA treatment. The effects of 5 nM/Kg p, p'-DDT exposure on oxidative stress and Wnt/beta-catenin pathway in nude mice with hepatocellular carcinoma were detected by immunohistochemistry and Western blotting. - DDT increased tumor growth in nude mice and activated oxidative stress and Wnt/beta-catenin signaling pathway in liver cancer tissues. Combination of nucleus and transcription factor TCF promotes the expression of c-Myc and Cyclin D1 in the downstream region, thereby promoting the proliferation of human hepatocellular carcinoma cells and the growth of hepatocellular carcinoma. Part II: Effect of low-dose p, p'-DDT exposure on the adhesion of human hepatocellular carcinoma and its mechanism. The results showed that p, p'-DDT decreased cell-to-cell adhesion and increased cell-to-matrix adhesion six days after exposure to HepG2 cells. p, p'-DDT increased ROS content and activated JAK/STAT3 signaling pathway in HepG2 cells. Inhibition of estrogen receptor antagonist ICI on estrogen receptor ER did not affect the phenomena induced by p, p'-DDT. It was suggested that p, p'-DDT might mediate cell adhesion through ROS rather than ER. p, p'-DDT also affected the expression of adhesion molecules in HepG2 cells. Exposure to ICI resulted in the decrease of E-cadherin, the increase of N-cadherin and CD29. Similarly, p, p'-DDT activates the JAK/STAT3 signaling pathway and affects the gene expression of E-cadherin, N-cadherin and CD29 in liver cancer nude mice. Studies have shown that p, p'-DDT mediates JAK/STAT3 signaling pathway activation by increasing ROS content in cells. Controlling the expression of downstream adhesion molecules, reducing cell-cell adhesion, enhancing cell-matrix adhesion and promoting the deterioration of hepatocellular carcinoma cells. Part III: Toxicity of high-dose p, p'-DDT to human normal liver cells and antagonistic effect of vitamin C and E. Flow cytometry was used to detect the effects of p, p'-DDT on human normal liver cells. L-7702 cells were exposed to p, p'-DDT of more than 10 mu M, which significantly induced apoptosis. Further detection of mitochondrial membrane potential and caspases family protein expression showed that p, p'-DDT exposure to HL-7702 cells significantly increased ROS content and the expression of apoptotic gene Bax, and decreased the expression of anti-apoptotic gene. Cytochrome C releases from mitochondria to cytoplasm, induces the production of caspase-9 and 3, and activates the mitochondrial apoptosis signaling pathway. At the same time, p, p'-DDT mediates the activation of downstream NF-kappa B through ROS. Activated NF-kappa B enters the nucleus and promotes the downstream FasL. However, the addition of natural antioxidants vitamin C and E inhibited the production of ROS induced by p, p'-DDT and the activation of downstream apoptotic signaling pathways, thereby reducing the cytotoxicity of p, p'-DDT and alleviating apoptosis. This study showed that vitamin C and/or E could antagonize HL-7702 cytotoxicity induced by p, p'-DDT. Part IV: genotoxicity and hepatotoxicity of human normal liver cells exposed to high dose p, p'-DDT and antagonistic effect of vitamin C and E. DAPI staining was used. The genotoxicity of p,p'-DDT to human normal hepatocyte HL-7702 was evaluated by comet assay,micronucleus assay,DNA and protein cross-linking assay.The results showed that the concentration of chromatin,comet assay parameters,micronucleus induction rate and DPC coefficient were increased in a dose-dependent manner after p,p'-DDT exposure. HL-7702 cells were induced to produce genotoxicity, resulting in DNA damage and chromatin concentration. The effects of p, p'-DDT exposure on the hepatotoxicity of HL-7702 cells were detected by qRT-PCR. It was found that P, P'-DDT exposure had significant hepatotoxicity on HL-7702 cells. The expression of phase I metabolic enzyme genes (CYP1A1, CYP2B6 and CYP3A4) was increased, and phase II metabolic enzyme genes (UGT) This study also revealed that vitamin C and E could antagonize DNA damage and metabolic enzymes changes induced by p, p'-DDT in HL-7702 cells, i.e. genotoxicity and hepatotoxicity, and that the combined antagonistic effect of the two was greater than that of single addition. The effects of exposure to p, p'-DDT on the deterioration of hepatocellular carcinoma cells and the damage of high dose p, p'-DDT on normal hepatocytes were studied. Therefore, this study is helpful to elucidate the human liver health risks and molecular mechanisms caused by persistent organic pesticide DDT residues, and provide a basis for prevention and control of DDT exposure-induced liver toxicity.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R114
本文编号：2248824
[Abstract]:DDT, a persistent organic pollutant, has been banned or restricted for many years, but because of its bioaccumulation, persistence, fat solubility, refractory degradation and food chain enrichment, it still has a high detection rate in the environment and human body. In addition, some developing countries still use DDT to control malaria, making these countries a high exposure area of DDT. The main sites and organs where T is metabolized, detoxified, and accumulated. It has been reported that DDT can cause liver enlargement, injury, and tumors. However, current studies on DDT residue and liver health risk mainly focus on epidemiology and toxicity assessment in laboratory animals, and the specific molecular mechanism is not clear, and about DDT outbreaks. In this study, low-dose (human blood DDT detection) and high-dose (high environmental DDT content) DDT main components p, p'-DDT were used to expose human hepatocellular carcinoma cells or human normal liver cells, and the oxidative stress targets and possible regulatory signaling pathways were analyzed. Molecular toxicological mechanisms of cancer progression and liver injury. This study includes the following four parts: Part I: Effects of low-dose p, p'-DDT exposure on the growth of human hepatocellular carcinoma and its mechanism. MTT method was used to analyze the effects of low-dose p, p'-DDT exposure on the proliferation of human hepatocellular carcinoma HepG2 cells. Four days later, G2 cells proliferated, ROS content increased, antioxidant enzymes y-GCS and SOD activity decreased. The effects of p, p'-DDT on Wnt/beta-catenin signaling pathway were detected by Western blotting. GSK3pbeta, beta-catenin and their downstream target proteins c-Myc and Cyclin D1, which were closely related to proliferation, were all increased. After adding ROS inhibitor NAC, the expression of GSK3pbeta, beta-catenin and Cyclin D1 was detected. In addition, the proliferation of HepG2 cells stimulated by p, p'-DDT could be reduced by NAC or beta-catenin siRNA treatment. The effects of 5 nM/Kg p, p'-DDT exposure on oxidative stress and Wnt/beta-catenin pathway in nude mice with hepatocellular carcinoma were detected by immunohistochemistry and Western blotting. - DDT increased tumor growth in nude mice and activated oxidative stress and Wnt/beta-catenin signaling pathway in liver cancer tissues. Combination of nucleus and transcription factor TCF promotes the expression of c-Myc and Cyclin D1 in the downstream region, thereby promoting the proliferation of human hepatocellular carcinoma cells and the growth of hepatocellular carcinoma. Part II: Effect of low-dose p, p'-DDT exposure on the adhesion of human hepatocellular carcinoma and its mechanism. The results showed that p, p'-DDT decreased cell-to-cell adhesion and increased cell-to-matrix adhesion six days after exposure to HepG2 cells. p, p'-DDT increased ROS content and activated JAK/STAT3 signaling pathway in HepG2 cells. Inhibition of estrogen receptor antagonist ICI on estrogen receptor ER did not affect the phenomena induced by p, p'-DDT. It was suggested that p, p'-DDT might mediate cell adhesion through ROS rather than ER. p, p'-DDT also affected the expression of adhesion molecules in HepG2 cells. Exposure to ICI resulted in the decrease of E-cadherin, the increase of N-cadherin and CD29. Similarly, p, p'-DDT activates the JAK/STAT3 signaling pathway and affects the gene expression of E-cadherin, N-cadherin and CD29 in liver cancer nude mice. Studies have shown that p, p'-DDT mediates JAK/STAT3 signaling pathway activation by increasing ROS content in cells. Controlling the expression of downstream adhesion molecules, reducing cell-cell adhesion, enhancing cell-matrix adhesion and promoting the deterioration of hepatocellular carcinoma cells. Part III: Toxicity of high-dose p, p'-DDT to human normal liver cells and antagonistic effect of vitamin C and E. Flow cytometry was used to detect the effects of p, p'-DDT on human normal liver cells. L-7702 cells were exposed to p, p'-DDT of more than 10 mu M, which significantly induced apoptosis. Further detection of mitochondrial membrane potential and caspases family protein expression showed that p, p'-DDT exposure to HL-7702 cells significantly increased ROS content and the expression of apoptotic gene Bax, and decreased the expression of anti-apoptotic gene. Cytochrome C releases from mitochondria to cytoplasm, induces the production of caspase-9 and 3, and activates the mitochondrial apoptosis signaling pathway. At the same time, p, p'-DDT mediates the activation of downstream NF-kappa B through ROS. Activated NF-kappa B enters the nucleus and promotes the downstream FasL. However, the addition of natural antioxidants vitamin C and E inhibited the production of ROS induced by p, p'-DDT and the activation of downstream apoptotic signaling pathways, thereby reducing the cytotoxicity of p, p'-DDT and alleviating apoptosis. This study showed that vitamin C and/or E could antagonize HL-7702 cytotoxicity induced by p, p'-DDT. Part IV: genotoxicity and hepatotoxicity of human normal liver cells exposed to high dose p, p'-DDT and antagonistic effect of vitamin C and E. DAPI staining was used. The genotoxicity of p,p'-DDT to human normal hepatocyte HL-7702 was evaluated by comet assay,micronucleus assay,DNA and protein cross-linking assay.The results showed that the concentration of chromatin,comet assay parameters,micronucleus induction rate and DPC coefficient were increased in a dose-dependent manner after p,p'-DDT exposure. HL-7702 cells were induced to produce genotoxicity, resulting in DNA damage and chromatin concentration. The effects of p, p'-DDT exposure on the hepatotoxicity of HL-7702 cells were detected by qRT-PCR. It was found that P, P'-DDT exposure had significant hepatotoxicity on HL-7702 cells. The expression of phase I metabolic enzyme genes (CYP1A1, CYP2B6 and CYP3A4) was increased, and phase II metabolic enzyme genes (UGT) This study also revealed that vitamin C and E could antagonize DNA damage and metabolic enzymes changes induced by p, p'-DDT in HL-7702 cells, i.e. genotoxicity and hepatotoxicity, and that the combined antagonistic effect of the two was greater than that of single addition. The effects of exposure to p, p'-DDT on the deterioration of hepatocellular carcinoma cells and the damage of high dose p, p'-DDT on normal hepatocytes were studied. Therefore, this study is helpful to elucidate the human liver health risks and molecular mechanisms caused by persistent organic pesticide DDT residues, and provide a basis for prevention and control of DDT exposure-induced liver toxicity.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R114
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