饮用水中碘乙酸毒作用机制研究

发布时间：2018-05-09 04:01

本文选题：碘乙酸 + 饮用水　；参考：《复旦大学》2014年博士论文

【摘要】：工农业的迅猛发展导致废水排放量激增,水体污染日趋严重,原水水质不断恶化。现行的常规水处理工艺已难以完全清除水中众多的污染物,且在饮用水消毒过程中,消毒剂还将与水中存在的有机和无机前体物反应生成具有有害效应的消毒副产物。早期毒理学研究表明,饮用水有机提取物具有致突变性、遗传毒性、生殖毒性和发育毒性,可以引起氧化应激、细胞凋亡和细胞恶性转化等。近年的流行病学研究也表明,饮用氯消毒的饮用水可导致膀胱癌、直肠癌和多种不良生殖结局,包括低体重儿、小于胎龄儿和早产等。由于饮用水中的污染物既可来自原水也可来自加工过程,种类多样,组份极为复杂,受分析技术和研究工作复杂性的制约,以往的研究并不了解究竟是何种污染物引起的效应。近年来,随着污染物谱解析技术的长足进步和暴露组理念的发展,基于GC-MS和]LC-MS/MS导向的污染物谱分析,增进了对饮用水中污染物暴露特征和污染物种类数量的认识。而毒理学研究也由传统的基于整体动物的高剂量染毒、从动物外推至人,发展到基于毒性通路机制和人源性细胞系的污染物毒效应研究,并且这种理念在学术界和管理机构产生了重大反响。为此,美国科学委员会于2007年出版发布了《21世纪的毒性测试》,高度概括了化学物质毒性测试研究的核心思想和发展远景。鉴于此,本研究在对饮用水污染物特征分析的基础上,选择了可对健康产生潜在影响的重要污染物碘乙酸(iodoacetic acid, IAA),开展了基于毒性机制的毒理学研究。饮用水污染物组份错综复杂,含量范围从数十纳克至毫克每升不等,因此,对饮水中污染物混合效应的评价界定极为困难。然而,饮用水中污染物的毒性效应仍然有一定规律可循。例如：我们先前研究已表明,氧化应激在饮用水整体混合污染物和某些污染物引起的细胞毒性和遗传毒性中具有重要作用,提示水污染引起的氧化应激通路值得关注。由于氧化应激是机体在内外环境刺激下的基本防御机制,氧化应激可直接或间接氧化及损伤DNA、蛋白质和脂质,进而诱发基因突变、蛋白质变性和脂质过氧化,因而氧化应激在毒性机制中具有重要作用。现有研究表明氧化应激和适应性反应可能是某些污染物毒性效应的共同机制,因此,以特异的氧化应激信号通路为基础的毒效应分析研究有望增进了解饮用水中低剂量混合暴露对人群健康的潜在影响。Nrf-2 (nuclear factor E2-related factor 2)/ ARE (antioxidant response element)信号通路在机体氧化应激反应中极为关键。Nrf-2作为重要的核转录因子参与调控细胞内抗氧化酶和Ⅱ相解毒酶的表达,维持细胞氧化还原稳态和抑制氧化损伤及炎症。Nrf2缺失或激活障碍,将引起细胞对应激原的敏感性增高,与化学促癌的发生和细胞凋亡等病变过程相关。研究表明氧化应激在具有动物致癌性的MX和与人群肿瘤相关的微囊藻毒素联合暴露的毒效应机制中具有重要作用。由于饮用水中的污染物是机体可能的应激源,因此,利用氧化应激效应通路模型研究饮用水混合污染物毒理学效应将为污染物的混合暴露评价方法提供新的思路。围绕上述思路,本研究在饮用水污染物特征分析的基础上,首先研究了饮用水中混合污染物对Nrf2/ARE效应通路的影响,发现单独作用并未引起ARE活性改变的污染物,在低剂量混合暴露时可引起Nrf2/ARE效应通路显著改变。结合饮用水污染物特征分析,我们选择了饮用水中含量虽低,但前期初步研究已经发现具有极强毒效应的新型消毒副产物碘乙酸,围绕氧化应激效应通路在遗传毒性中的作用进行了较为系统的研究,证实了Nrf2通路的激活在IAA引起的细胞毒性和遗传毒性机制中具有重要作用。继而,采用基因芯片技术研究了IAA对HepG2细胞基因表达和调控的影响,以期找到IAA毒性相关分子机制。研究结果将为认识IAA的毒效应机制和开展基于机制的健康风险评估奠定重要基础,同时,也为未来研究制定国家生活饮用水水质卫生标准提供了重要的基础数据和科学依据。第一部分饮用水污染物低剂量混合暴露对Nrf2/ARE信号通路的影响低剂量混合暴露是饮用水污染物暴露的现实特征。传统毒理学评价模式从单一污染物、高剂量的动物实验结果外推至人群,存在诸多不确定性,同时也不能确定含量低、但毒效应强的组分引起的健康效应及其累加效应。由于氧化应激是机体重要防御机制,其中Nrf2在维持细胞氧化还原稳态、抑制氧化损伤,调控细胞内抗氧化酶和Ⅱ相解毒酶反应中发挥核心作用。因此,本研究首先通过基于GC-MS、GC-ECD和LC-MS/MS的污染谱特征分析技术揭示饮用水污染物整体暴露特征,继而以Nrf2介导的抗氧化反应研究饮用水中提取的有机污染物引起的Nrf2蛋白聚积、Nrf2相关调控基因的表达和ARE荧光素酶报告基因活性等效应。研究发现：饮用水污染谱中的单一污染物在1000至100万倍环境浓度才使Nrf2/ARE响应,但8倍于环境浓度的整体污染物即可改变Nrf2/ARE活性。而且,以污染谱组分配置的模拟混合污染物也在8倍环境浓度时可以改变Nrf2/ARE活性,证实了Nrf2效应通路是分析评价混合污染物效应的敏感方法,经模拟水样和实际水样的双重验证,揭示了低剂量混合污染物引起的氧化应激反应特征。这一基于毒性机制的混合污染物评价方法有望在环境污染物混合暴露的风险评估中得到广泛应用。与此同时,根据饮用水中污染物的污染特征分析,明确了饮用水中主要污染物。通过查询分析毒理学资料,选择了上海市饮用水中检出率高、毒作用强的污染物碘乙酸进行进一步深入研究。第二部分碘乙酸对十种人源性细胞系的细胞毒性、遗传毒性比较研究碘乙酸是原水中存在的碘化物在饮用水消毒过程中被液氯或氯胺消毒剂氧化后生成的新型消毒副产物。尽管IAA在饮用水中的浓度处于数百纳克至微克水平,相对于主要的消毒副产物THMs和HAAs含量较低,但由于IAA对鼠伤寒沙门氏菌具有强致突变性,对哺乳动物细胞具有较强的细胞毒性和遗传毒性,经IAA恶性转化的细胞可引起裸鼠肿瘤,且人群在日常生活中经饮水终生暴露,因此,碘乙酸对人群健康的潜在影响备受重视。2012年美国消毒副产物高登论坛(Gordon Research Conference on Disinfection by-products)将IAA和NDMA类消毒副产物列为亟需关注的消毒副产物。目前,鉴于人群流行病学资料和毒理学资料的充分性不足,国际癌症研究机构(IARC)尚未对IAA的致癌性类别进行划分,世界卫生组织(WHO)的饮用水水质指南和包括中国在内的所有国家暂未对饮用水中的IAA提出限量要求。但是,美国环境保护局(EPA)已经开始关注饮用水中IAA的潜在危害。重要的是,上海地处长江入海口,受咸潮影响,海水中的卤化物会随着咸潮逆流而上,使得原水水源中的卤化物明显增加,当采用氯化消毒时即会形成消毒副产物IAA。因此,IAA的毒效应及其机制研究对于健康风险评估和饮用水卫生标准的研制具有重要价值。有关碘乙酸具有极强细胞毒性和遗传毒性的证据主要来自中国仓鼠卵巢细胞和人TK6、HepG2细胞,但人源性细胞的遗传毒性结果并不一致。而且,由于IAA进入机体后将随血液分配至其他器官和组织,因而研究IAA对不同类型人源性细胞系的细胞毒性和遗传毒性,对于了解IAA的敏感性靶器官具有重要意义。因此,本研究以10种人源细胞系为对象,研究比较了不同细胞系暴露于IAA引起的细胞毒性、遗传毒性差异。研究结果显示,不同细胞对IAA的敏感性差异较大,IAA对人原代细胞系HEK-a的细胞毒性是Eca-109细胞的14倍,细胞毒性大小依次为：HEK-a SW579 5637 MGC 80-3 HepG2 HaCAT CaCo-2≈PBL293Eca-109细胞：胞质阻滞微核实验结果显示,IAA对于不同细胞的相对遗传毒性潜能大小依次为：PBL SW579 HepG2 MGC 80-3 293 CaCo2 HaCAT HEK-a细胞,对Eca-109和5637细胞无遗传毒性。此外,通过对微核进行着丝粒蛋白A (CENP-A)免疫荧光染色,发现含着丝粒的微核在所有微核中占据较高比例,提示IAA可能通过干扰姐妹染色单体或染色体分离,诱导非整倍性染色体而致遗传毒性。第三部分Nrf2/ARE氧化应激信号通路在碘乙酸毒效应机制中的作用研究氧化应激与适应性反应是毒物毒作用和机体防御的基本机制。以往研究发现IAA可以导致活性氧自由基(ROS)增加,提示氧化应激反应在IAA的毒性作用中具有重要作用,然而,准确的机制尚不清楚。由于外源性化学物主要通过肝脏代谢,选择肝细胞系作为研究对象可较好地反映受试物的基本毒性特征,且HepG2细胞具有DNA修复系统和Ⅱ相解毒酶系,因此,本研究以人源性的HepG2细胞系为对象,研究Nrf2/ARE氧化应激信号通路在IAA毒效应机制中的作用。本研究通过检测Nrf2介导的抗氧化反应和细胞内谷胱甘肽(GSH)发现,IAA可显著增强NRF2蛋白、ARE荧光素酶报告基因活性、NRF2基因及其下游抗氧化酶、Ⅱ相解毒酶基因GCLC, NQOl, HO-1的表达和GSH水平,且具有剂量-效应关系。为研究Nrf2在IAA毒效应中的特定作用,应用敲除NRF2基因的HepG2细胞观察IAA引起的细胞毒性和遗传毒性。结果发现,NRF2基因缺失可使细胞对IAA细胞毒性的敏感性增加,且IAA在敲除NRF2基因的HepG2细胞中的微核形成率显著增加。为判定IAA诱导的抗氧化反应是否能被天然抗氧化剂姜黄素阻断,在暴露IAA前,我们先以10μM姜黄素预处理HepG2细胞1小时,结果表明姜黄素预处理可以有效降低IAA诱导的细胞毒性和遗传毒性,提示激活Nrf2信号通路可降低IAA的毒性。为验证结果的可靠性,以IAA染毒SD大鼠24小时,发现100倍环境浓度的IAA急性暴露使雄性大鼠肝脏的Nrf2和Gclc基因的表达显著增加,而1000倍环境浓度的IAA染毒可使Nrf2, Keapl, Ho-1, Nqol和Gclc基因的表达均显著高于对照组。上述结果表明Nrf2介导的抗氧化反应在IAA引起的毒效应中具有重要作用,提示Nrf2可以作为IAA毒效应的生物标志物。第四部分基于基因芯片数据分析的碘乙酸毒作用分子机制研究从基因组或系统水平上阐明生命现象是人类后基因组时代的基本要务。尽管IAA已被证实具有极强的细胞毒性、遗传毒性和致瘤性,其毒性机制研究较为有限,仅有的高通量试验是基于DNA损伤信号通路和氧化应激／抗氧化防御信号通路的PCR Array,尚无从整体层面剖析IAA毒作用分子机制的毒理基因组学研究。由于任何毒作用的发生都是细胞调节通路及其网络整体紊乱的结果,从单个信号通路或某一功能性“模块”来剖析细胞对环境刺激的应答,颇具局限性。本研究以期应用毒理基因组学的方法研究IAA对HepG2细胞基因表达和调控的影响,通过"cDNA芯片—miRNA芯片”数据联合分析,高通量筛选出负相关差异表达基因和niRNA,大幅度缩小目的基因和miRNA的选择范围：进而通过基因间相互作用关系的构建,将基因间的调控关系以信号转导网络的形式整理出来,从而发现基因信号转导的脉络,尤其是找到与IAA染毒关系密切,在网络中连接度高且对网络的稳定性起到重要作用的核心调控因子。生物信息学分析结果显示,IAA诱导的差异表达基因和miRNA主要与细胞增殖、凋亡相关；涉及的主要通路包括结直肠癌通路、前列腺癌通路、凋亡通路、TGF-beta信号通路、Wnt信号通路等。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R123

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