發(fā)布時間：2018-05-09 04:01

  本文選題：碘乙酸 + 飲用水　； 參考：《復(fù)旦大學(xué)》2014年博士論文

【摘要】：工農(nóng)業(yè)的迅猛發(fā)展導(dǎo)致廢水排放量激增,水體污染日趨嚴(yán)重,原水水質(zhì)不斷惡化。現(xiàn)行的常規(guī)水處理工藝已難以完全清除水中眾多的污染物,且在飲用水消毒過程中,消毒劑還將與水中存在的有機(jī)和無機(jī)前體物反應(yīng)生成具有有害效應(yīng)的消毒副產(chǎn)物。早期毒理學(xué)研究表明,飲用水有機(jī)提取物具有致突變性、遺傳毒性、生殖毒性和發(fā)育毒性,可以引起氧化應(yīng)激、細(xì)胞凋亡和細(xì)胞惡性轉(zhuǎn)化等。近年的流行病學(xué)研究也表明,飲用氯消毒的飲用水可導(dǎo)致膀胱癌、直腸癌和多種不良生殖結(jié)局,包括低體重兒、小于胎齡兒和早產(chǎn)等。由于飲用水中的污染物既可來自原水也可來自加工過程,種類多樣,組份極為復(fù)雜,受分析技術(shù)和研究工作復(fù)雜性的制約,以往的研究并不了解究竟是何種污染物引起的效應(yīng)。近年來,隨著污染物譜解析技術(shù)的長足進(jìn)步和暴露組理念的發(fā)展,基于GC-MS和]LC-MS/MS導(dǎo)向的污染物譜分析,增進(jìn)了對飲用水中污染物暴露特征和污染物種類數(shù)量的認(rèn)識。而毒理學(xué)研究也由傳統(tǒng)的基于整體動物的高劑量染毒、從動物外推至人,發(fā)展到基于毒性通路機(jī)制和人源性細(xì)胞系的污染物毒效應(yīng)研究,并且這種理念在學(xué)術(shù)界和管理機(jī)構(gòu)產(chǎn)生了重大反響。為此,美國科學(xué)委員會于2007年出版發(fā)布了《21世紀(jì)的毒性測試》,高度概括了化學(xué)物質(zhì)毒性測試研究的核心思想和發(fā)展遠(yuǎn)景。鑒于此,本研究在對飲用水污染物特征分析的基礎(chǔ)上,選擇了可對健康產(chǎn)生潛在影響的重要污染物碘乙酸(iodoacetic acid, IAA),開展了基于毒性機(jī)制的毒理學(xué)研究。飲用水污染物組份錯綜復(fù)雜,含量范圍從數(shù)十納克至毫克每升不等,因此,對飲水中污染物混合效應(yīng)的評價界定極為困難。然而,飲用水中污染物的毒性效應(yīng)仍然有一定規(guī)律可循。例如：我們先前研究已表明,氧化應(yīng)激在飲用水整體混合污染物和某些污染物引起的細(xì)胞毒性和遺傳毒性中具有重要作用,提示水污染引起的氧化應(yīng)激通路值得關(guān)注。由于氧化應(yīng)激是機(jī)體在內(nèi)外環(huán)境刺激下的基本防御機(jī)制,氧化應(yīng)激可直接或間接氧化及損傷DNA、蛋白質(zhì)和脂質(zhì),進(jìn)而誘發(fā)基因突變、蛋白質(zhì)變性和脂質(zhì)過氧化,因而氧化應(yīng)激在毒性機(jī)制中具有重要作用。現(xiàn)有研究表明氧化應(yīng)激和適應(yīng)性反應(yīng)可能是某些污染物毒性效應(yīng)的共同機(jī)制,因此,以特異的氧化應(yīng)激信號通路為基礎(chǔ)的毒效應(yīng)分析研究有望增進(jìn)了解飲用水中低劑量混合暴露對人群健康的潛在影響。Nrf-2 (nuclear factor E2-related factor 2)/ ARE (antioxidant response element)信號通路在機(jī)體氧化應(yīng)激反應(yīng)中極為關(guān)鍵。Nrf-2作為重要的核轉(zhuǎn)錄因子參與調(diào)控細(xì)胞內(nèi)抗氧化酶和Ⅱ相解毒酶的表達(dá),維持細(xì)胞氧化還原穩(wěn)態(tài)和抑制氧化損傷及炎癥。Nrf2缺失或激活障礙,將引起細(xì)胞對應(yīng)激原的敏感性增高,與化學(xué)促癌的發(fā)生和細(xì)胞凋亡等病變過程相關(guān)。研究表明氧化應(yīng)激在具有動物致癌性的MX和與人群腫瘤相關(guān)的微囊藻毒素聯(lián)合暴露的毒效應(yīng)機(jī)制中具有重要作用。由于飲用水中的污染物是機(jī)體可能的應(yīng)激源,因此,利用氧化應(yīng)激效應(yīng)通路模型研究飲用水混合污染物毒理學(xué)效應(yīng)將為污染物的混合暴露評價方法提供新的思路。圍繞上述思路,本研究在飲用水污染物特征分析的基礎(chǔ)上,首先研究了飲用水中混合污染物對Nrf2/ARE效應(yīng)通路的影響,發(fā)現(xiàn)單獨作用并未引起ARE活性改變的污染物,在低劑量混合暴露時可引起Nrf2/ARE效應(yīng)通路顯著改變。結(jié)合飲用水污染物特征分析,我們選擇了飲用水中含量雖低,但前期初步研究已經(jīng)發(fā)現(xiàn)具有極強(qiáng)毒效應(yīng)的新型消毒副產(chǎn)物碘乙酸,圍繞氧化應(yīng)激效應(yīng)通路在遺傳毒性中的作用進(jìn)行了較為系統(tǒng)的研究,證實了Nrf2通路的激活在IAA引起的細(xì)胞毒性和遺傳毒性機(jī)制中具有重要作用。繼而,采用基因芯片技術(shù)研究了IAA對HepG2細(xì)胞基因表達(dá)和調(diào)控的影響,以期找到IAA毒性相關(guān)分子機(jī)制。研究結(jié)果將為認(rèn)識IAA的毒效應(yīng)機(jī)制和開展基于機(jī)制的健康風(fēng)險評估奠定重要基礎(chǔ),同時,也為未來研究制定國家生活飲用水水質(zhì)衛(wèi)生標(biāo)準(zhǔn)提供了重要的基礎(chǔ)數(shù)據(jù)和科學(xué)依據(jù)。第一部分飲用水污染物低劑量混合暴露對Nrf2/ARE信號通路的影響低劑量混合暴露是飲用水污染物暴露的現(xiàn)實特征。傳統(tǒng)毒理學(xué)評價模式從單一污染物、高劑量的動物實驗結(jié)果外推至人群,存在諸多不確定性,同時也不能確定含量低、但毒效應(yīng)強(qiáng)的組分引起的健康效應(yīng)及其累加效應(yīng)。由于氧化應(yīng)激是機(jī)體重要防御機(jī)制,其中Nrf2在維持細(xì)胞氧化還原穩(wěn)態(tài)、抑制氧化損傷,調(diào)控細(xì)胞內(nèi)抗氧化酶和Ⅱ相解毒酶反應(yīng)中發(fā)揮核心作用。因此,本研究首先通過基于GC-MS、GC-ECD和LC-MS/MS的污染譜特征分析技術(shù)揭示飲用水污染物整體暴露特征,繼而以Nrf2介導(dǎo)的抗氧化反應(yīng)研究飲用水中提取的有機(jī)污染物引起的Nrf2蛋白聚積、Nrf2相關(guān)調(diào)控基因的表達(dá)和ARE熒光素酶報告基因活性等效應(yīng)。研究發(fā)現(xiàn)：飲用水污染譜中的單一污染物在1000至100萬倍環(huán)境濃度才使Nrf2/ARE響應(yīng),但8倍于環(huán)境濃度的整體污染物即可改變Nrf2/ARE活性。而且,以污染譜組分配置的模擬混合污染物也在8倍環(huán)境濃度時可以改變Nrf2/ARE活性,證實了Nrf2效應(yīng)通路是分析評價混合污染物效應(yīng)的敏感方法,經(jīng)模擬水樣和實際水樣的雙重驗證,揭示了低劑量混合污染物引起的氧化應(yīng)激反應(yīng)特征。這一基于毒性機(jī)制的混合污染物評價方法有望在環(huán)境污染物混合暴露的風(fēng)險評估中得到廣泛應(yīng)用。與此同時,根據(jù)飲用水中污染物的污染特征分析,明確了飲用水中主要污染物。通過查詢分析毒理學(xué)資料,選擇了上海市飲用水中檢出率高、毒作用強(qiáng)的污染物碘乙酸進(jìn)行進(jìn)一步深入研究。第二部分碘乙酸對十種人源性細(xì)胞系的細(xì)胞毒性、遺傳毒性比較研究碘乙酸是原水中存在的碘化物在飲用水消毒過程中被液氯或氯胺消毒劑氧化后生成的新型消毒副產(chǎn)物。盡管IAA在飲用水中的濃度處于數(shù)百納克至微克水平,相對于主要的消毒副產(chǎn)物THMs和HAAs含量較低,但由于IAA對鼠傷寒沙門氏菌具有強(qiáng)致突變性,對哺乳動物細(xì)胞具有較強(qiáng)的細(xì)胞毒性和遺傳毒性,經(jīng)IAA惡性轉(zhuǎn)化的細(xì)胞可引起裸鼠腫瘤,且人群在日常生活中經(jīng)飲水終生暴露,因此,碘乙酸對人群健康的潛在影響備受重視。2012年美國消毒副產(chǎn)物高登論壇(Gordon Research Conference on Disinfection by-products)將IAA和NDMA類消毒副產(chǎn)物列為亟需關(guān)注的消毒副產(chǎn)物。目前,鑒于人群流行病學(xué)資料和毒理學(xué)資料的充分性不足,國際癌癥研究機(jī)構(gòu)(IARC)尚未對IAA的致癌性類別進(jìn)行劃分,世界衛(wèi)生組織(WHO)的飲用水水質(zhì)指南和包括中國在內(nèi)的所有國家暫未對飲用水中的IAA提出限量要求。但是,美國環(huán)境保護(hù)局(EPA)已經(jīng)開始關(guān)注飲用水中IAA的潛在危害。重要的是,上海地處長江入海口,受咸潮影響,海水中的鹵化物會隨著咸潮逆流而上,使得原水水源中的鹵化物明顯增加,當(dāng)采用氯化消毒時即會形成消毒副產(chǎn)物IAA。因此,IAA的毒效應(yīng)及其機(jī)制研究對于健康風(fēng)險評估和飲用水衛(wèi)生標(biāo)準(zhǔn)的研制具有重要價值。有關(guān)碘乙酸具有極強(qiáng)細(xì)胞毒性和遺傳毒性的證據(jù)主要來自中國倉鼠卵巢細(xì)胞和人TK6、HepG2細(xì)胞,但人源性細(xì)胞的遺傳毒性結(jié)果并不一致。而且,由于IAA進(jìn)入機(jī)體后將隨血液分配至其他器官和組織,因而研究IAA對不同類型人源性細(xì)胞系的細(xì)胞毒性和遺傳毒性,對于了解IAA的敏感性靶器官具有重要意義。因此,本研究以10種人源細(xì)胞系為對象,研究比較了不同細(xì)胞系暴露于IAA引起的細(xì)胞毒性、遺傳毒性差異。研究結(jié)果顯示,不同細(xì)胞對IAA的敏感性差異較大,IAA對人原代細(xì)胞系HEK-a的細(xì)胞毒性是Eca-109細(xì)胞的14倍,細(xì)胞毒性大小依次為：HEK-a SW579 5637 MGC 80-3 HepG2 HaCAT CaCo-2≈PBL293Eca-109細(xì)胞：胞質(zhì)阻滯微核實驗結(jié)果顯示,IAA對于不同細(xì)胞的相對遺傳毒性潛能大小依次為：PBL SW579 HepG2 MGC 80-3 293 CaCo2 HaCAT HEK-a細(xì)胞,對Eca-109和5637細(xì)胞無遺傳毒性。此外,通過對微核進(jìn)行著絲粒蛋白A (CENP-A)免疫熒光染色,發(fā)現(xiàn)含著絲粒的微核在所有微核中占據(jù)較高比例,提示IAA可能通過干擾姐妹染色單體或染色體分離,誘導(dǎo)非整倍性染色體而致遺傳毒性。第三部分Nrf2/ARE氧化應(yīng)激信號通路在碘乙酸毒效應(yīng)機(jī)制中的作用研究氧化應(yīng)激與適應(yīng)性反應(yīng)是毒物毒作用和機(jī)體防御的基本機(jī)制。以往研究發(fā)現(xiàn)IAA可以導(dǎo)致活性氧自由基(ROS)增加,提示氧化應(yīng)激反應(yīng)在IAA的毒性作用中具有重要作用,然而,準(zhǔn)確的機(jī)制尚不清楚。由于外源性化學(xué)物主要通過肝臟代謝,選擇肝細(xì)胞系作為研究對象可較好地反映受試物的基本毒性特征,且HepG2細(xì)胞具有DNA修復(fù)系統(tǒng)和Ⅱ相解毒酶系,因此,本研究以人源性的HepG2細(xì)胞系為對象,研究Nrf2/ARE氧化應(yīng)激信號通路在IAA毒效應(yīng)機(jī)制中的作用。本研究通過檢測Nrf2介導(dǎo)的抗氧化反應(yīng)和細(xì)胞內(nèi)谷胱甘肽(GSH)發(fā)現(xiàn),IAA可顯著增強(qiáng)NRF2蛋白、ARE熒光素酶報告基因活性、NRF2基因及其下游抗氧化酶、Ⅱ相解毒酶基因GCLC, NQOl, HO-1的表達(dá)和GSH水平,且具有劑量-效應(yīng)關(guān)系。為研究Nrf2在IAA毒效應(yīng)中的特定作用,應(yīng)用敲除NRF2基因的HepG2細(xì)胞觀察IAA引起的細(xì)胞毒性和遺傳毒性。結(jié)果發(fā)現(xiàn),NRF2基因缺失可使細(xì)胞對IAA細(xì)胞毒性的敏感性增加,且IAA在敲除NRF2基因的HepG2細(xì)胞中的微核形成率顯著增加。為判定IAA誘導(dǎo)的抗氧化反應(yīng)是否能被天然抗氧化劑姜黃素阻斷,在暴露IAA前,我們先以10μM姜黃素預(yù)處理HepG2細(xì)胞1小時,結(jié)果表明姜黃素預(yù)處理可以有效降低IAA誘導(dǎo)的細(xì)胞毒性和遺傳毒性,提示激活Nrf2信號通路可降低IAA的毒性。為驗證結(jié)果的可靠性,以IAA染毒SD大鼠24小時,發(fā)現(xiàn)100倍環(huán)境濃度的IAA急性暴露使雄性大鼠肝臟的Nrf2和Gclc基因的表達(dá)顯著增加,而1000倍環(huán)境濃度的IAA染毒可使Nrf2, Keapl, Ho-1, Nqol和Gclc基因的表達(dá)均顯著高于對照組。上述結(jié)果表明Nrf2介導(dǎo)的抗氧化反應(yīng)在IAA引起的毒效應(yīng)中具有重要作用,提示Nrf2可以作為IAA毒效應(yīng)的生物標(biāo)志物。第四部分基于基因芯片數(shù)據(jù)分析的碘乙酸毒作用分子機(jī)制研究從基因組或系統(tǒng)水平上闡明生命現(xiàn)象是人類后基因組時代的基本要務(wù)。盡管IAA已被證實具有極強(qiáng)的細(xì)胞毒性、遺傳毒性和致瘤性,其毒性機(jī)制研究較為有限,僅有的高通量試驗是基于DNA損傷信號通路和氧化應(yīng)激／抗氧化防御信號通路的PCR Array,尚無從整體層面剖析IAA毒作用分子機(jī)制的毒理基因組學(xué)研究。由于任何毒作用的發(fā)生都是細(xì)胞調(diào)節(jié)通路及其網(wǎng)絡(luò)整體紊亂的結(jié)果,從單個信號通路或某一功能性“模塊”來剖析細(xì)胞對環(huán)境刺激的應(yīng)答,頗具局限性。本研究以期應(yīng)用毒理基因組學(xué)的方法研究IAA對HepG2細(xì)胞基因表達(dá)和調(diào)控的影響,通過"cDNA芯片—miRNA芯片”數(shù)據(jù)聯(lián)合分析,高通量篩選出負(fù)相關(guān)差異表達(dá)基因和niRNA,大幅度縮小目的基因和miRNA的選擇范圍：進(jìn)而通過基因間相互作用關(guān)系的構(gòu)建,將基因間的調(diào)控關(guān)系以信號轉(zhuǎn)導(dǎo)網(wǎng)絡(luò)的形式整理出來,從而發(fā)現(xiàn)基因信號轉(zhuǎn)導(dǎo)的脈絡(luò),尤其是找到與IAA染毒關(guān)系密切,在網(wǎng)絡(luò)中連接度高且對網(wǎng)絡(luò)的穩(wěn)定性起到重要作用的核心調(diào)控因子。生物信息學(xué)分析結(jié)果顯示,IAA誘導(dǎo)的差異表達(dá)基因和miRNA主要與細(xì)胞增殖、凋亡相關(guān)；涉及的主要通路包括結(jié)直腸癌通路、前列腺癌通路、凋亡通路、TGF-beta信號通路、Wnt信號通路等。
[Abstract]:The rapid development of industry and agriculture leads to the increase of wastewater discharge, the pollution of water body is becoming more and more serious and the water quality of the original water is deteriorating. The current conventional water treatment process is difficult to completely remove many of the pollutants in the water. In the process of disinfection of drinking water, the disinfectant will also react with the existing organic and inorganic precursors in the water to produce harmful effects. Early toxicological studies have shown that the organic extracts of drinking water have mutagenicity, genetic toxicity, reproductive toxicity and developmental toxicity, which can cause oxidative stress, cell apoptosis and cell malignant transformation. Recent epidemiological studies also suggest that drinking water with chlorine disinfection can lead to bladder cancer, rectal cancer and a variety of adverse events. Reproductive outcomes, including low weight infants, less than gestational age and preterm birth. As pollutants from drinking water can come from both raw and processed, diverse and complex components, the previous studies do not understand the effects of what contaminants are caused by the complexity of analytical techniques and research. The rapid progress in pollutant spectrum analysis and the development of the concept of exposure group, based on the GC-MS and]LC-MS/MS oriented pollutant spectrum analysis, have enhanced the understanding of the exposure and number of pollutants in the drinking water, and the toxicological study is also contaminated by the traditional high dose of the whole animal, extrapolating from animals to human beings, and developing to Based on the mechanism of toxic pathway and the study of the toxic effects of human source cell lines, this concept has produced great repercussions in academia and management institutions. For this reason, the American Science Committee published the toxicity test of the <21 century in 2007, and highly summarized the core ideas and development prospects of chemical substance toxicity test. On the basis of the analysis of the characteristics of drinking water pollutants, this study selected the iodoacetic acid (IAA), an important pollutant which could have a potential impact on health, and carried out a toxicological study based on the toxicity mechanism. The evaluation of mixed effects of pollutants in water is extremely difficult to define. However, the toxic effects of pollutants in drinking water still have some regularity. For example, our previous studies have shown that oxidative stress plays an important role in the cytotoxicity and genotoxicity of mixed pollutants and certain pollutants in drinking water, suggesting water pollution. The oxidative stress pathway is worthy of attention. Oxidative stress is the basic defense mechanism under the environment of internal and external environment. Oxidative stress can directly or indirectly oxidize and damage DNA, protein and lipid, and then induce gene mutation, protein denaturation and lipid peroxidation, and oxidative stress plays an important role in the toxicity mechanism. Studies have shown that oxidative stress and adaptive response may be a common mechanism for the toxic effects of certain pollutants. Therefore, the analysis of toxic effects based on specific oxidative stress signaling pathways is expected to enhance the potential impact of low dose exposure on drinking water on population health.Nrf-2 (nuclear factor E2-related factor 2). ARE (antioxidant response element) signaling pathway is very important in the oxidative stress response of the body. As an important nuclear transcription factor, it participates in the regulation of the expression of intracellular antioxidant enzymes and phase II detoxification enzymes, maintaining the redox homeostasis and inhibiting oxidative damage and the deletion or activation of.Nrf2, which will lead to the corresponding cell irritable cells. The increase in sensitivity is associated with the process of carcinogenesis and cell apoptosis. The study shows that oxidative stress plays an important role in the mechanism of the toxic effects of MX and the combined exposure to tumor associated microcystins in the population. The effect of oxidative stress effect pathway model on the toxicological effect of drinking water mixed pollutants will provide a new idea for the mixed exposure evaluation method of pollutants. Based on the analysis of the characteristics of drinking water pollutants, this study first studies the effect of mixed pollutants in drinking water on the Nrf2/ARE effect pathway. The pollutants that did not change the activity of ARE could cause a significant change in the Nrf2/ARE effect pathway at low dose of mixed exposure. The effect of the effect pathway in the genotoxicity was systematically studied, which confirmed that the activation of the Nrf2 pathway played an important role in the cytotoxicity and genotoxicity of IAA. Then, the gene chip technology was used to study the effect of IAA on the gene expression and regulation of HepG2 cells, in order to find the mechanism of IAA toxicity related molecular mechanism. The results will provide an important basis for understanding the toxic effect mechanism of IAA and the assessment of mechanism based health risk. At the same time, it also provides important basic data and scientific basis for the future research and formulation of the sanitary standards for drinking water quality of national living water. The first part is the shadow of Nrf2/ARE signaling pathway in the low dose exposure of drinking water pollutants. The low dose of mixed exposure is a realistic feature of the exposure of drinking water pollutants. The traditional toxicological evaluation model is extrapolated from the single pollutant and high dose of animal experiment to the population, and there are many uncertainties, but also the health effect and the cumulative effect caused by the toxic effects are also indeterminate, but the oxidative stress is the result of oxidative stress. It is an important defense mechanism in the body, in which Nrf2 plays a core role in maintaining the redox homeostasis, inhibiting oxidative damage and regulating the reaction of intracellular antioxidant enzymes and phase II detoxification enzymes. Therefore, this study first revealed the overall exposure characteristics of drinking water pollutants through the pollution spectrum characteristic analysis techniques based on GC-MS, GC-ECD and LC-MS/MS. The Nrf2 mediated antioxidant reaction studies the accumulation of Nrf2 protein caused by organic pollutants extracted from drinking water, the expression of Nrf2 related regulatory genes and the effect of ARE luciferase reporter gene activity. It is found that the single pollutant in the drinking water pollution spectrum is 1000 to 1 million times the environmental concentration to respond to Nrf2/ARE, but 8 times that of the ring. The overall pollutant concentration can change the Nrf2/ARE activity. Moreover, the simulated mixed pollutants configured by the pollution spectrum component can also change the Nrf2/ARE activity at 8 times the environmental concentration. It is proved that the Nrf2 effect pathway is a sensitive method for the analysis and evaluation of the mixed pollutant effect. The low dosage of the simulated water sample and the actual water sample is verified by the dual validation of the simulated water samples and the actual water samples. The characteristics of oxidative stress caused by mixed pollutants. This method of evaluation of mixed pollutants based on toxic mechanism is expected to be widely used in the risk assessment of mixed exposure of environmental pollutants. At the same time, the main pollutants in drinking water are clarified according to the pollution characteristics of drinking water. The second part of the cytotoxicity of iodiacetic acid to ten human derived cell lines, the comparison of the genotoxicity study of iodide acetic acid is a liquid chlorine or chloramine disinfectant in the process of drinking water disinfection. Although the concentration of IAA in drinking water is at the level of hundreds of NNG to microgram, the content of THMs and HAAs is low relative to the main disinfection by-products, but because IAA has strong mutagenicity to Salmonella typhimurium, it has strong cytotoxicity and genotoxicity to mammalian cells, and IAA is malignant. The transformed cells can cause tumor in nude mice, and the population is exposed to the drinking water for life in daily life. Therefore, the potential impact of iodiacetic acid on the health of the population has been paid much attention to the disinfection byproducts of the United States of America (Gordon Research Conference on Disinfection by-products) for the disinfection byproducts of IAA and NDMA class as an urgent need to pay attention to disinfection. At present, in view of the insufficiency of population epidemiological and toxicological data, the International Cancer Research Institute (IARC) has not yet divided the carcinogenicity of IAA, and the WHO (WHO) guidelines for drinking water quality and all countries including China have not required limited requirements for IAA in drinking water. However, the United States The Environmental Protection Agency (EPA) has begun to pay attention to the potential hazards of IAA in drinking water. It is important that Shanghai is located in the mouth of the Yangtze River, affected by the salt tide, and the halide in the sea water will go up with the salt tide, which makes the halide in the raw water obviously increased. When the chlorination is used, the disinfection by-product IAA. will be formed and the toxic effect of IAA The study of its mechanism is of great value to health risk assessment and the development of sanitary standards for drinking water. The evidence for the extremely strong cytotoxicity and genotoxicity of iodized acetic acid mainly comes from Chinese hamster ovary cells and human TK6, HepG2 cells, but the genetic toxicity of human derived cells is not consistent. Moreover, as IAA enters the body With the distribution of blood to other organs and tissues, the study of the cytotoxicity and genotoxicity of IAA on different types of human derived cell lines is of great significance for understanding the sensitive target organs of IAA. Therefore, 10 human source cell lines were used to compare the cytotoxicity and genetic toxicity of different cell lines exposed to IAA. The results showed that the sensitivity of different cells to IAA was different. The cytotoxicity of IAA to the human primary cell line HEK-a was 14 times as high as that of Eca-109 cells, and the cytotoxicity of the cells was: HEK-a SW579 5637 MGC 80-3 HepG2 HaCAT CaCo-2 PBL293Eca-109 cells: the cytoplasmic hysteresis micronucleus test showed that IAA was for different cells. The relative genotoxicity potential is: PBL SW579 HepG2 MGC 80-3293 CaCo2 HaCAT HEK-a cells, no hereditary toxicity to Eca-109 and 5637 cells. Furthermore, micronuclei containing centromeric micronucleus is found to occupy a high proportion in all micronucleus by micronucleus centromere protein A (CENP-A) immunofluorescence staining, suggesting that IAA may pass through the stem. Disturbing sister chromatid or chromosomal separation and inducing aneuploidy chromosomes to induce genotoxicity. Third the role of Nrf2/ARE oxidative stress signaling pathway in the mechanism of iodiacetic acid toxicity study oxidative stress and adaptive response are the basic mechanisms of toxicant and body defense. Previous studies found that IAA could lead to reactive oxygen species. The increase of base (ROS) suggests that oxidative stress plays an important role in the toxicity of IAA. However, the exact mechanism is not clear. Since exogenous chemicals mainly pass through the liver metabolism, the selection of hepatocyte lines as the research object can better reflect the basic toxic characteristics of the subjects, and the HepG2 cells have the DNA repair system and the phase II phase. Detoxification enzyme system, therefore, studies the role of Nrf2/ARE oxidative stress signaling pathway in the mechanism of IAA toxicity in human HepG2 cell lines. This study shows that IAA can significantly enhance the NRF2 protein, ARE luciferase reporter gene activity and NRF2 gene by detecting Nrf2 mediated antioxidant response and intracellular glutathione (GSH). And its downstream antioxidant enzymes, the expression of GCLC, NQOl, HO-1, and GSH levels, and the dose effect relationship. In order to study the specific role of Nrf2 in the IAA toxic effect, the cytotoxicity and genotoxicity of IAA induced by the HepG2 cells that knock off the NRF2 gene were observed. The results showed that the deletion of NRF2 gene could make cells to IAA cytotoxicity. The sensitivity of sex increased, and the micronucleus formation rate of IAA in the HepG2 cells that knocked out the NRF2 gene was significantly increased. To determine whether the antioxidant response of the IAA induced by the natural antioxidant curcumin was blocked. Before exposure to IAA, we pretreated HepG2 cells with 10 u M curcumin for 1 hours. The results showed that the curcumin pretreatment could effectively reduce IAA. The induction of cytotoxicity and genotoxicity indicated that activation of Nrf2 signaling pathway could reduce the toxicity of IAA. In order to verify the reliability of the results, IAA staining was used.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R123

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