DDT暴露引起肝臟損傷和致癌作用的分子毒理研究
發(fā)布時(shí)間：2018-09-18 19:06
【摘要】：持續(xù)性有機(jī)污染物DDT雖已被禁止或限用多年,但由于其具有生物累積性、持久性、脂溶性、難降解和食物鏈富集性等特點(diǎn),在環(huán)境和人體仍有很高的檢出率。此外,一些發(fā)展中國(guó)家仍用DDT來(lái)控制瘧疾,使得這些國(guó)家成為DDT高暴露區(qū)。肝臟是DDT進(jìn)行代謝、解毒和積累的主要場(chǎng)所和器官。有報(bào)道揭示,DDT可以引起肝臟腫大、損傷以及腫瘤等疾病。然而目前關(guān)于DDT殘留和肝臟健康風(fēng)險(xiǎn)的研究主要集中在流行病學(xué)和實(shí)驗(yàn)動(dòng)物毒性評(píng)價(jià)方面,具體的分子機(jī)制還不是很清楚,且關(guān)于DDT暴露引起的肝臟毒性干預(yù)研究尚未見(jiàn)報(bào)道。本研究采用低劑量(人體血液DDT檢出量)和高劑量(高暴露區(qū)環(huán)境DDT含量)DDT主要成分p,p'-DDT暴露人肝癌細(xì)胞或人正常肝臟細(xì)胞,圍繞氧化應(yīng)激靶點(diǎn)以及可能的調(diào)控信號(hào)通路,分析p,p'-DDT暴露導(dǎo)致肝癌惡化以及肝臟損傷的分子毒理機(jī)制。本研究?jī)?nèi)容含以下四個(gè)部分：第一部分：低劑量p,p'-DDT暴露對(duì)人肝細(xì)胞癌生長(zhǎng)的影響及其機(jī)制研究。采用MTT方法分析低劑量p,p'-DDT暴露對(duì)人肝癌細(xì)胞HepG2增殖的影響。結(jié)果顯示低濃度p,p'-DDT暴露HepG2細(xì)胞四天后促進(jìn)了細(xì)胞增殖,細(xì)胞中ROS含量升高,抗氧化酶y-GCS和SOD活性降低。蛋白印跡法檢測(cè)p,p'-DDT對(duì)Wnt/β-catenin信號(hào)通路的影響,發(fā)現(xiàn)GSK3pβ、β-catenin及其下游與增殖密切相關(guān)的靶蛋白c-Myc和CyclinD1表達(dá)均升高。加入ROS抑制劑NAC后,這些蛋白表達(dá)被逆轉(zhuǎn)。此外,p,p'-DDT促進(jìn)的HepG2細(xì)胞增殖可被NAC或β-catenin siRNA處理所降低。利用肝癌裸鼠成瘤模型,采用免疫組化和蛋白印跡法檢測(cè)5 nM/Kg p,p'-DDT暴露對(duì)裸鼠肝細(xì)胞癌腫瘤組織氧化應(yīng)激水平和Wnt/β-catenin通路的影響。發(fā)現(xiàn)p,p'-DDT增加了裸鼠瘤的生長(zhǎng),激活了肝癌腫瘤組織氧化應(yīng)激和Wnt/β-catenin信號(hào)通路。研究表明,當(dāng)?shù)蛣┝縫,p'-DDT暴露于肝癌細(xì)胞后,首先激活細(xì)胞中ROS生成和氧化應(yīng)激反應(yīng)；進(jìn)而促進(jìn)GSK3β在絲氨酸第九位點(diǎn)的磷酸化；激活的β-catenin進(jìn)入細(xì)胞核與核轉(zhuǎn)錄因子TCF相結(jié)合,促進(jìn)下游與增殖相關(guān)的靶蛋白(c-Myc和CyclinD1)表達(dá),從而促進(jìn)人肝癌細(xì)胞的增殖和肝細(xì)胞癌的生長(zhǎng)。第二部分：低劑量p,p'-DDT暴露對(duì)人肝細(xì)胞癌粘附的影響及其機(jī)制研究。采用細(xì)胞粘附方法檢測(cè)低劑量p,p'-DDT對(duì)肝癌細(xì)胞粘附的影響。結(jié)果表明：p,p'-DDT暴露HepG2細(xì)胞六天后,降低了細(xì)胞與細(xì)胞之間的粘附,提高了細(xì)胞與基質(zhì)之間的粘附。p,p'-DDT增加了HepG2細(xì)胞中ROS含量,激活了JAK/STAT3信號(hào)通路。此外,加入ROS抑制劑NAC后,這種效應(yīng)被顯著逆轉(zhuǎn)。而加入雌激素受體拮抗劑ICI抑制雌激素受體ER后,對(duì)p,p'-DDT誘導(dǎo)的現(xiàn)象沒(méi)有影響。表明p,p'-DDT可能通過(guò)ROS而非ER介導(dǎo)其調(diào)控的細(xì)胞粘附。p,p'-DDT還影響HepG2細(xì)胞粘附分子的表達(dá),它的暴露導(dǎo)致E-cadherin降低,N-cadherin和CD29升高。進(jìn)一步研究揭示,p,p'-DDT影響的粘附分子表達(dá)能夠被JAK抑制劑或STAT3抑制劑所逆轉(zhuǎn)。同樣地,p,p'-DDT激活了肝癌裸鼠腫瘤組織中JAK/STAT3信號(hào)通路,影響了E-cadherin、N-cadherin和CD29的基因表達(dá)。研究表明,p,p'-DDT通過(guò)提高細(xì)胞中ROS含量,介導(dǎo)JAK/STAT3信號(hào)通路激活,調(diào)控下游粘附分子表達(dá)；降低細(xì)胞與細(xì)胞間的粘附,增強(qiáng)細(xì)胞與基質(zhì)間的粘附,促進(jìn)肝癌細(xì)胞的惡化。第三部分：高劑量p,p'-DDT暴露對(duì)人正常肝臟細(xì)胞的毒性及維生素C和E的拮抗效果研究。采用流式細(xì)胞技術(shù)檢測(cè)p,p'-DDT對(duì)人正常肝臟細(xì)胞HL-7702存活的影響,發(fā)現(xiàn)大于10 μM的p,p'-DDT暴露HL-7702細(xì)胞后,顯著誘導(dǎo)細(xì)胞凋亡。進(jìn)一步檢測(cè)線粒體膜電位以及caspases家族蛋白的表達(dá),結(jié)果顯示p,p'-DDT暴露HL-7702細(xì)胞后,顯著提高了細(xì)胞中ROS含量和促凋亡基因Bax的表達(dá),降低了抗凋亡基因Bcl-2的表達(dá),從而提高線粒體膜的去極化致使膜受到損傷。細(xì)胞色素c從線粒體釋放到細(xì)胞質(zhì)中,誘導(dǎo)caspase-9和3的活性形式產(chǎn)生,激活線粒體凋亡信號(hào)通路。與此同時(shí),p,p'-DDT通過(guò)ROS介導(dǎo)下游NF-κB的激活,活化的NF-κB進(jìn)入細(xì)胞核促進(jìn)下游FasL的表達(dá),隨后結(jié)合到其死亡受體Fas上,激活死亡受體介導(dǎo)的凋亡信號(hào)通路,促進(jìn)HL-7702細(xì)胞凋亡。然而,加入天然抗氧化物維生素C和E后,抑制了p,p'-DDT誘導(dǎo)的ROS生成及下游凋亡信號(hào)通路的激活,由此降低p,p'-DDT的細(xì)胞毒性,緩解細(xì)胞凋亡,且維生素C和E復(fù)合加入的效果大于單獨(dú)加入的效果。本研究表明,維生素C和／或E可以拮抗p,p'-DDT引起的HL-7702細(xì)胞毒性。第四部分：高劑量p,p'-DDT暴露對(duì)人正常肝臟細(xì)胞的基因毒性和肝臟毒性及維生素C和E的拮抗效果研究。采用了DAPI染色、彗星實(shí)驗(yàn)、微核試驗(yàn)、DNA和蛋白質(zhì)交聯(lián)實(shí)驗(yàn)等方法,多方面評(píng)價(jià)了p,p'-DDT對(duì)人正常肝臟細(xì)胞HL-7702的基因毒性。結(jié)果顯示：p,p'-DDT暴露后,劑量依賴性地提高了細(xì)胞中染色質(zhì)濃縮、彗星實(shí)驗(yàn)參數(shù)、微核誘導(dǎo)率以及DPC系數(shù)。表明p,p'-DDT暴露可以誘導(dǎo)HL-7702細(xì)胞產(chǎn)生基因毒性,引起DNA損傷和染色質(zhì)濃縮。采用qRT-PCR檢測(cè)p,p'-DDT暴露對(duì)HL-7702肝細(xì)胞毒性的影響,發(fā)現(xiàn)P,P'-DDT暴露對(duì)HL-7702細(xì)胞具有明顯肝臟毒性,具體表現(xiàn)在Ⅰ相代謝酶基因(CYP1A1、CYP2B6和CYP3A4)表達(dá)升高,Ⅱ相代謝酶基因(UGT、GST和GCS)表達(dá)下調(diào)。本研究還揭示維生素C和E可以拮抗p,p'-DDT引起的HL-7702細(xì)胞DNA損傷和代謝酶變化,即基因毒性和肝臟毒性,且二者復(fù)合的拮抗效果大于單獨(dú)加入的效果。綜上所述,本研究評(píng)價(jià)了p,p'-DDT暴露對(duì)肝臟的毒性作用,包括低劑量p,p'-DDT暴露對(duì)肝癌細(xì)胞惡化的影響以及高劑量p,p'-DDT暴露對(duì)正常肝臟細(xì)胞的損傷；圍繞氧化應(yīng)激靶點(diǎn)及可能的調(diào)控信號(hào)通路,闡明了p,p'-DDT暴露引起肝臟健康風(fēng)險(xiǎn)的相關(guān)分子機(jī)制；分析了天然抗氧化物維生素C和E對(duì)p,p'-DDT毒性的拮抗作用。因此,本研究有助于闡明持續(xù)性有機(jī)農(nóng)藥DDT殘留引起的人體肝臟健康風(fēng)險(xiǎn)及分子機(jī)制,為干預(yù)DDT暴露引起的肝臟毒性提供了一定的防控依據(jù)。
[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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R114
本文編號(hào)：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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R114
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