饮用水中碘乙酸和碘仿的检测识别、遗传毒性和潜在致癌性研究
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摘要
碘系消毒副产物是饮用水消毒过程中新发现的一类未受控消毒副产物。原水碘离子含量高和氯胺消毒是形成碘系消毒副产物的重要直接因素。上海市地处长江入海口,水源水质易受咸潮影响,而且多数水厂以常规水处理工工艺(预氧化、混凝、沉淀、沙滤和消毒)生产饮用水,特殊的环境条件和所采用饮用水加工工艺易于形成碘系消毒副产物。尽管饮用水中碘系消毒副产物含量低至纳克,但毒性极大。有研究表明碘系消毒副产物比受控的消毒副产物具有更强的细胞毒性和遗传毒性,因此,本研究以上海市饮用水加工过程中代表性的碘系消毒副产物(碘乙酸和碘仿)的生成水平和影响因素为切入点,通过检测饮用水中碘乙酸和碘仿含量,了解人群碘乙酸和碘仿的暴露水平；利用四噻唑蓝试验、CCK-8试验、鼠伤寒沙门氏菌回复突变试验、细胞内γ-H2AX含量测定和胞质分裂阻滞法微核试验等系列组合实验研究碘乙酸和碘仿的细胞毒性和遗传毒性特征及它们可能的作用机制；继而通过细胞恶性转化实验研究碘乙酸和碘仿的潜在致癌性及可能机制。研究结果将为今后上海市不同水源消毒副产物的过程控制,制水工艺的升级改进提供重要的科学依据,也为碘乙酸和碘仿健康风险评价和饮用水水质卫生标准的研制奠定基础。
第一部分上海市饮用水中碘乙酸和碘仿的检测识别
1饮用水中氯、溴和碘离子检测方法的建立
本研究在借鉴美国环保局推荐的300.1方法基础上,建立基于离子色谱法测定饮用水中氯和溴离子的检测方法；通过质谱对碘化衍生物进行精确定性,继而建立基于气相色谱电子捕获检测器测定水中碘离子的检测方法。离子色谱分析结果显示,氯离子和溴离子分离度良好,峰形对称,无拖尾,溶剂和杂质峰干扰少,整个色谱分析历时12 min。采用标准曲线法进行定量,氯离子与溴离子的方法线性范围分别为1～1000 mg/L和1～1000μg/L,确定系数均为0.999。平均加标回收率在89.7%～112.3%之间,精密度小于2.9%,氯离子和溴离子方法检出限分别为55.6和0.37μg/L。连续校准值为87.9%～113.2%,替代物响应值为93.2%～103.4%,平行双样测定的相对偏差在0.0%～8.0%。含碘物质经衍生化后,质谱鉴定发现碘离子衍生化后生成碘代丁酮存在2个同分异构体,即1-碘-2-丁酮和3-碘-2-丁酮。采用双毛细管柱定性分析显示,碘代丁酮分离度好,整个色谱分析历时19.33 min。选择响应值较高的3-碘-2-丁酮进行定量检测,方法线性范围为1～1001μg/L,确定系数r2=0.999；方法检出限为0.13μg/L；平均加标回收率为102.0%,相对标准偏差为5.1%,连续校准值在97.2%-108.8%,内标物响应值在92.00%-110.05%,平行双样测定的相对偏差在4.4%-11.6%。研究结果表明所建立的氯、溴和碘离子的检测方法灵敏度高,定性定量准确,质量控制符合美国EPA检测方法的要求,适用于水中微量氯离子、溴离子和碘离子的分析。
2气相色谱电子捕获检测器法测定饮用水中碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸
借鉴美国环保局推荐的检测方法551.1和552.3,经优化前处理和检测条件,建立基于双柱定性气相色谱电子捕获检测器法测定饮用水中碘乙酸、碘仿、4种三卤甲烷(氯仿、溴仿、二氯一溴甲烷、二溴一氯甲烷)和9种卤乙酸(氯乙酸、二氯乙酸、三氯乙酸、溴乙酸、二溴乙酸、三溴乙酸、二氯一溴乙酸、二溴一氯乙酸、溴氯乙酸)的检测方法。气相色谱电子捕获检测器法检测结果显示,碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸分离度好,峰形对称,无拖尾,溶剂和杂质峰干扰少,基线平稳。碘仿和4种三卤甲烷整个色谱分离时间为40.67 min,而碘乙酸和9种卤乙酸整个色谱分离时间为23.50 mmin。采用内标工作曲线法定量,碘乙酸和碘仿的方法线性范围分别为0.01-2.5μg/L和0.05-5μg/L,4种三卤甲烷和9种卤乙酸的方法线性范围均为1-100μg/L,确定系数均大于0.994。碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸的平均加标回收率在93.5%-111.1%之问,精密度小于7.0%。碘乙酸和碘仿的方法检测限分别为6.2和17.7ng/L,4种三卤甲烷和9种卤乙酸的方法检测限为191.4-502.2 ng/L。碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸的连续校准值在87.1%-114.0%,内标物平均响应值在85.2%-120.0%,替代物响应值在85.8%-116.5%,平行双样测定的相对偏差在0.1%-10.3%。改进后的碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸测定方法分离度好、分析时间短、灵敏度高、准确度高、重现性好。仪器条件简单,可自动化批量处理,适合于普通实验室分析。
3上海市饮用水中碘乙酸和碘仿的污染现状研究
为了解上海市以长江和黄浦江为水源水厂各工艺环节水中碘乙酸和碘仿的水平,评价水源水质、生产工艺、消毒剂种类对碘乙酸和碘仿形成的影响,本研究以上海市中心城区13家水厂(11家水厂采用常规生产工艺,2家采用深度处理工艺)为对象,分别于枯水期和丰水期采集水厂不同工艺过程水样,测定水样的pH值、氨氮、可溶性有机碳、UV254、特征紫外吸光度、氯离子、溴离子、碘离子、碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸的水平生成和变化情况。
研究结果显示,黄浦江和长江水质呈弱碱性(pH=7.34),氨氮含量小于0.5 mg/L,有机物含量未见偏高,DOC、UV254和SUVA的中位数分别为6.00 mg/L、0.231/cm、3.90L/(mg·m)。黄浦江pH值稍低于长江,丰水期高于枯水期；丰水期黄浦江和长江的氨氮浓度相近,枯水期黄浦江的氨氮含量高于长江。黄浦江可溶性有机碳和UV254含量均高于长江,其中两江可溶性有机碳含量丰水期略高于枯水期,而两江UV254含量则是丰水期低于枯水期；黄浦江的特征紫外吸光度水平在丰水期稍高于长江,枯水期则低于长江；黄浦江氯、溴和碘离子高于长江,且丰水期低于枯水期。各水厂出厂水碘乙酸和碘仿含量在0.03～1.66μg/L。9种卤乙酸和4种三卤甲烷含量范围在0.28～63.74μg/L。以黄浦江为原水的水厂出厂水中碘乙酸和碘仿含量高于以长江为原水的水厂,而丰水期又低于枯水期。多元线性回归分析结果显示,pH值与碘乙酸和碘仿的形成呈负相关,碘仿的形成还与UV254和碘离子水平呈正相关。
氯胺预氧化较臭氧产生更多的碘乙酸和碘仿,浓度范围在0.2～1.7μg/L,而与液氯预氧化的差异无统计学意义。各水厂生产工艺对水质pH值、氨氮、可溶性有机碳、氯离子、溴离子浓度影响不大,但UV254、特征紫外吸光度水平明显下降。常规生产工艺水厂的出厂水中碘离子浓度明显下降。未在原水中发现碘乙酸和碘仿,但各水厂出厂水均检出碘乙酸和碘仿。常规生产工艺可形成碘乙酸、碘仿、9种卤乙酸和4种三卤甲烷；深度处理工艺在活性炭工艺前均未检出碘乙酸和碘仿,但可检出微量9种卤乙酸和4种三卤甲烷。上述结果表明上海市主城区各水厂的出厂水中均检出碘乙酸和碘仿,含量处于纳克至微克水平。低pH值、高有机物、高碘离子及以氯胺预氧化有利于形成碘乙酸和碘仿。常规和深度处理工艺对水质pH值、氨氮、可溶性有机碳、氯离子和溴离子影响不大,不能有效去除饮用水加工过程生成的碘乙酸和碘仿。
第二部分碘乙酸和碘仿的细胞毒性研究
采用一组不同细胞毒性终点的试验研究碘乙酸和碘仿的细胞毒性及其可能的作用机制。分别以不同剂量的碘乙酸和碘仿对NIH/3T3细胞染毒72 h后,以四噻唑蓝试验测定碘乙酸对NIH/3T3细胞增殖和线粒体损伤的影响,以CCK-8试验测定碘仿对NIH/3T3细胞增殖和线粒体损伤的影响；并在显微镜下观察细胞形态学的变化；测定碘乙酸和碘仿对细胞乳酸脱氢酶、ATP、还原型谷胱甘肽含量的影响；以流式细胞仪分析细胞周期和凋亡的改变。研究结果显示,碘乙酸和碘仿染毒72 h后可导致NIH/3T3细胞存活率下降,呈剂量-反应关系；碘乙酸的细胞毒性分别比溴乙酸和氯乙酸强2、218倍,碘仿的细胞毒性比溴仿强81倍。碘乙酸和碘仿染毒72 h后,光镜下观察到细胞增殖受抑制,出现细胞凋亡或坏死现象；还可引起细胞外乳酸脱氢酶含量上升和细胞内ATP含量下降(P<0.05),并呈剂量-反应关系；对细胞内还原型谷胱肽无明显影响(P>0.05)。流式细胞仪分析结果显示,碘乙酸和碘仿可分别导致细胞凋亡和坏死比例增多,碘乙酸可使细胞出现S期阻滞,而碘仿则使细胞出现G2/M期阻滞。研究结果表明,碘乙酸和碘仿产生细胞毒性可能与线粒体损伤、ATP耗竭和胞膜损伤有关。线粒体途径可能是碘乙酸和碘仿致细胞死亡的机制之一。而碘乙酸和碘仿致细胞周期阻滞提示碘乙酸和碘仿可损伤细胞DNA。
第三部分碘乙酸和碘仿的遗传毒性研究
应用鼠伤寒沙门氏菌回复突变试验、细胞内γ-H2AX含量测定和胞质分裂阻滞法微核试验评价碘乙酸和碘仿的遗传毒性。分别以不同剂量的碘乙酸和碘仿对鼠伤寒沙门氏菌TA100和TA98菌株进行染毒,其中碘乙酸采用非预培养法和预培养法,碘仿采用非预培养法,均培养48 h后计数鼠伤寒沙门氏菌回变菌落数。先通过NIH/3T3细胞生长曲线确定碘乙酸和碘仿的染毒时间,再以CCK-8试验明确碘乙酸和碘仿的染毒剂量,最后采用胞质分裂阻滞法微核试验测定不同剂量碘乙酸和碘仿染毒40 h后诱导细胞微核形成数。先以CCK-8试验确定碘乙酸和碘仿染毒24 h后的染毒剂量,继而以流式细胞仪测定不同剂量碘乙酸和碘仿染毒24 h后NIH/3T3细胞γ-H2AX表达量。结果显示,碘乙酸在加或不加S9条件下均未能诱导鼠伤寒沙门氏菌TA100和TA98回复菌落数显著增加；碘仿在加或不加S9条件下均可诱导鼠伤寒沙门氏菌TA100和TA98回复菌落数显著增加。碘乙酸可引起NIH/3T3细胞γ-H2AX含量增加,并呈剂量-反应关系；碘仿能引起NIH/3T3细胞γ-H2AX含量增加,但未见剂量-反应关系。碘乙酸和碘仿均未能引起NIH/3T3细胞微核形成率增加。鼠伤寒沙门氏菌回复突变试验、微核试验和γ-H2AX含量测定试验表明碘乙酸和碘仿为可疑遗传毒物。
第四部分碘乙酸和碘仿的潜在致癌性研究
应用体外细胞恶性转化试验研究碘乙酸和碘仿的潜在致癌性及其可能的作用机制。先通过细胞克隆形成试验确定细胞恶性转化试验的染毒剂量,然后以不同剂量的碘乙酸和碘仿对NIH/3T3细胞染毒72 h后继续培养10天,观察细胞转化灶形成数。继而以伴刀豆蛋白A凝集试验、软琼脂试验和裸鼠成瘤试验验证转化细胞的恶变情况,并以流式细胞仪分析恶性转化后细胞的周期变化,以免疫细胞化学试验分析恶性转化后细胞p53蛋白表达情况。研究结果显示,碘乙酸可诱导NIH/3T3细胞发生恶性转化,转化细胞能引起伴刀豆蛋白A凝集,可在软琼脂中形成克隆,并能在裸鼠皮下形成肿瘤,肿瘤组织学检查表明所形成肿瘤为分化程度较低的纤维肉瘤。细胞周期分析显示转化细胞出现G0/1期阻滞,S期和G2/M期细胞比例下降。免疫细胞化学分析显示p53蛋白表达未见增强。而碘仿在体外NIH/3T3细胞恶性转化试验中呈阴性。体外细胞恶性转化试验表明碘乙酸具有潜在致癌性,而未观察到碘仿具有潜在致癌性。
Iodo-disinfection byproducts are a kind of new unregulated disinfection byproducts in drinking water treatment process in recent years. Iodo-disinfection byproducts are easy to form when high iodide in raw water and using chloramination. Salt water intrusion strongly influences drinking water source because Shanghai located in the intersection zone of river and sea. Most of works used conventional treatment process (preoxidation, coagulation, sedimentation, sand filtration, and post chloramination.) to produce drinking water. Special environmental conditions and conventional treatment process are in favor of iodo-disinfection byproducts formation. Otherwise, the concentrations of iodo-disinfection byproducts are very low in drinking water but they are toxic strongly. Previous studies indicated that iodo-disinfection byproducts were more cytotoxic and genotoxic than regulated disinfection byproducts. Therefore, it is necessary to detect the levels and influencing factors of iodoacetic acid and iodoform in drinking water in Shanghai in order to evaluate population exposure. MTT assay, CCK-8 assay, Ames test, cellular y-H2AX, and cytokinesis-block micronucleus assay were used to evaluate their cytotoxicity and genotoxicity and their mechanism. Then the potential carcinogenicity and its mechanism of iodoacetic acid and iodoform were assessed by cell transformation assay in vitro. The study would provide the important scientific evidences for disinfection byproducts control and treatment process improvement. Meanwhile, it is helpful to the health risk assessment of iodoacetic acid and iodoform and the establishment of standard for drinking water quality.
1 Determination of iodoacetic acid and iodoform in drinking water in Shanghai
1.1 Establishment of methods for determining chloride, bromide, and iodide in drinking water
The method for determining chloride and bromide in drinking water by ion chromatography was established on the base of U.S. Environmental Protection Agency 300.1 method, Iodobutanone derivative was identified by gas chromatography/mass spectrometry, and then gas chromatography coupled to electron capture detector was made to analyze iodide in water. The results of ion chromatography showed that chloride and bromide were separated completely. Their peaks were symmetrical, no tail and not disturbed by peaks of solvent or impurity. The total time of chromatogram separation was 12 minutes. External standard calibration curve was selected to quantitative analysis. The linear ranges of chloride and bromide were 1-1000 mg/L and 1-1000μg/L respectively, and their coefficients of determination were both 0.999. The mean recoveries were between 89.7% and 112.3%, and the relative standard deviations were less than 2.9%. Method detections limits of chloride and bromide were 55.6μg/L and 0.37μg/L respectively. Their continuing calibration checks were between 87.9% and 113.2%. Surrogate response was between 93.2% and 103.4%. Their relative percent difference for duplicates were between 0.0% and 8.0%. The results of mass spectrometry showed that iodide would form iodobutanone which could generate 1-iodo-2-butanone and 3-iodo-2-butanone isomers by derivatization. The data of qualitative analysis by two capillary columns revealed that iodobutanone were separated completely and the total time of chromatogram separation was 19.33 minutes.3-iodo-2-butanone with the high response value was selected to quantitative analysis. The linear range was 1-100μg/L, and the coefficient of determination (r2) was 0.999. The limit of detection was 13 ng/L. The mean recovery was 102.0%, and relative standard deviation was 5.1%. The continuing calibration checks were between 97.2% and 108.8%. Internal standard responses were between 92.00% and 110.05%. The relative percent difference for duplicates were between 4.4% and 11.6%. The improved method possesses higher degree of sensitivity and accuracy of qualitative and quantitative analysis. Its quality control measured up to methods of United States Environmental Protection Agency. And it is fit for trace analysis of chloride, bromide, and iodide in water.
1.2 Determination of iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids in drinking water by gas chromatography with electron capture detection
Base on U.S. Environmental Protection Agency 551.1 and 552.3 methods, the pretreatment and detection conditions of methods for identifying iodoacetic acid, iodoform, four trihalomethanes(chloroform,bromoform, bromodichloromethane, and dibromochloromethane) and nine haloacetic acids (chloroacetic acid, dichloroacetic acid, trichloroacetic acid, bromoaceic acid, dibromoacetic acid, tribromoacetic acid, bromodichloroacetic acid, chlorodibromoacetic acid, and bromochloroacetic acid) with two capillary columns coupled to gas chromatography/electron capture detector were improved and optimized. The results of gas chromatography showed that iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids were separated completely. Their peaks were symmetrical, no tail and not disturbed by peaks of solvent or impurity. The total time of iodoform and four trihalomethanes separation was 40.67 minutes. The total time of iodoacetic acid and nine haloacetic acids separation was 23.50 minutes. Internal standard calibration curve was selected to quantitative analysis. The linear ranges of iodoacetic acid and iodoform were 0.01-2.5μg/L and 0.05-5μg/L respectively. The linear ranges of four trihalomethanes and nine haloacetic acids were both 1-100μg/L. Their coefficients of determination were more than 0.994. The mean recoveries were between 93.5% and 111.1%, and the relative standard deviations were less than 7.0%. Method detections limits of iodoacetic acid and iodoform were 6.2 ng/L and 17.7 ng/L respectively. Method detections limits of four trihalomethanes and nine haloacetic acids were between 191.4 ng/L and 502.2 ng/L. The continuing calibration checks were between 87.1% and 114.0%. Internal standard responses were between 85.2% and 120.0%. Surrogate response was between 85.8% and 116.5%. Their relative percent difference for duplicates were between 0.1% and 10.3%. The modified methods possesses good separation, fast, high sensitivity, high accuracy, and good consistency. It is fit for analysis in normal laboratory because of its economical and automatic characteristic.
1.3 Pollution of iodoacetic acid and iodoform in drinking water in Shanghai
The object of this study is to detect the levels of iodoacetic acid and iodoform in water of each treatment processes in works that their source waters come from Yangtze River and Huangpu River in Shanghai and evaluate the relationship of iodoacetic acid and iodoform formation to water qulity, treatment processes, and disinfectants. Thirteen water treatment works that are at central regions of Shanghai were chosen for investigation in low water period and high water period. Eleven works used conventional water treatment process. Two works used advanced water treatment process. Water samples of different treatment processes were selected to analyze for pH value, ammonia nitrogen, dissoluble organic carbon, UV absorbance, specific UV absorbance, chloride, bromide, iodide, iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids.
The results showed that pH values of Yangtze River and Huangpu River were alkalescent (pH=7.34). The concentrations of ammonia nitrogen were less than 0.5 mg/L. The levels of organic matter were not high. Medians of dissoluble organic carbon, UV absorbance, and specific UV absorbance were 6.00 mg/L、0.23 l/cm、3.90 L/(mg·m) respectively. The pH value of Huangpu River was lower than that of Yangtze River. The pH values of two rivers in high water period were higher than low water period. The ammonia nitrogen level of Huangpu River was closed to Yangtze River in high water period. In low water period, the ammonia nitrogen level of Huangpu River was higher than Yangtze River. The dissoluble organic carbon and UV absorbance of Huangpu River was higher than that of Yangtze River. The dissoluble organic carbon levels of two rivers in high water period were higher than low water period, but the UV absorbance levels reversely. In high water period, specific UV absorbance level of Huangpu River was higher than Yangtze River, but reversely in high water period. Chloride, bromide, and iodide levels of Huangpu River were higher than that of Yangtze River. But chloride, bromide, and iodide levels of two rivers in high water period were lower than low water period. The concentrations of iodoacetic acid and iodoform were between 0.03μg/L and 1.66μg/L in finished water in works. The level of four trihalomethanes and nine haloacetic acids were between 0.28μg/L and 63.74μg/L in finished water in works.
The concentrations of iodoacetic acid and iodoform in finished water in works which based on Huangpu River as water source were higher than that of works which based on Yangtze River as water source. The concentrations of iodoacetic acid and iodoform in finished water in low water period were higher than high water period. The results of multiple liner regression analysis showed that the relationship of pH value and iodo-disinfecton byproducts formation were negative correlation. Otherwise, the relationship of UV254 and iodide levels and iodoform formation were positive correlation.
More iodo-disinfecton byproducts were formed when using chloramination compared with ozone. The level of iodo-disinfecton byproducts were between 0.2μg/L and 1.7μg/L when using chloramination. There were not sighnificant differences between chloramination and chlorination. The treatment processes of each work had no effect on the level of pH value, ammonia nitrogen, dissoluble organic carbon, chloride, and bromide. But the levels of UV254 and specific UV absorbance decreased. The level of iodide decreased in finished water in conventional treatment process works. Iodoacetic acid and iodoform were not detected in raw water. But iodoacetic acid and iodoform were detected in finished water in every work. Iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids were formed in conventional treatment process. Iodoacetic acid and iodoform were not detected before activated carbon in advanced treatment process. Only few four trihalomethanes and nine haloacetic acid were detected in finished water in advanced treatment process works. The results indicated that trace iodoacetic acid and iodoform were detected in finished water in every work at the central regions of Shanghai. Low pH value, high natural organic matter, high iodide, and chloramination were easy to form iodoacetic acid and iodoform. The conventional and advanced treatment processes had no effect on pH value, ammonia nitrogen, natural organic matter, chloride, bromide, iodoacetic acid and iodoform.
2 Cytotoxicity of iodoacetic acid and iodoform
The object of this research is to study the cytotoxicity and its mechanism of iodoacetic acid and iodoform by a group of different cytotoxic end points assay. NIH/3T3 cells were treated by different doses iodoacetic acid and iodoform for 72 hours respectively. Then the proliferation and mitochondria damage of NIH/3T3 cell induced by iodoacetic acid and iodoform were evaluated by methylthiazoletrazolium assay and CCK-8 assay. The morphology of cells was observed in the microscope. The level of lactate dehydrogenase, ATP, and reduced glutathione were measured respectively. Cell cycle and apoptosis of cells were analyzed by flow cytometer. The results showed that NIH/3T3 cells viability decreased after they were exposed to iodoacetic acid and iodoform for 72 hours. There were also dose-response relationships. Iodoacetic acid was 2×more cytotoxic than bromoacetic acid and 218×more cytotoxic than chloroacetic acid in NIH/3T3 cells. Iodoform was 81×more cytotoxic than bromoform. The prohibition of cellular proliferation, apoptosis, and necrosis also could be observed in the microscope. Iodoacetic acid and iodoform could cause extracellular lactate dehydrogenase increase and intracelluar ATP decrease after 72 hours incubation (P<0.05). But they had no effect on reducing glutathione (P>0.05). Iodoacetic acid and iodoform could induced an increase in the proportion of apoptosis and necrosis. Cells cycle were arrested in S and G2/M phase respectively after incubation with iodoacetic acid and iodoform. Mitochondria damage, ATP depletion, and membrane damage are possibly involved in the induction of cytotoxicity by iodoacetic acid and iodoform. Mitochondria pathway would be possible one of mechanisms of cell death caused by iodoacetic acid and iodoform. Cells cycle arrest indicated that iodoacetic acid and iodoform can damage cellular DNA.
3 Genotoxicity of iodoacetic acid and iodoform
The genotoxicity of iodoacetic acid and iodoform were evaluated by Ames test, cellularγ-H2AX, and cytokinesis-block micronucleus assay. Salmonella typhimurium TA100 and TA98 were treated by different doses iodoacetic acid and iodoform. Un-preincubation and preincubation methods were used in iodoacetic acid treatment process. Un-preincubation was just used in iodoform treatment process. Numbers of Salmonella typhimurium were counted after 48 hours. In cytokinesis-block micronucleus assay, the treatment time of iodoacetic acid and iodoform were detected by NIH/3T3 cell growth curve. Then doses of iodoacetic acid and iodoform were selected by CCK-8 assay. Micronuclei were counted after cells were treated by iodoacetic acid and iodoform for 40 hours using cytokinesis-block micronucleus assay. Doses of iodoacetic acid and iodoform were determined by CCK-8 assay. Then levels ofγ-H2AX were analyzed by flow cytometer after treatment for 24 hours. The results showed that iodoacetic acid could not induce Salmonella typhimurium TA100 and TA98 increase significantly with or without S9. Iodoform was mutagenic in Salmonella typhimurium TA100 and TA98 with or without S9. Iodoacetic acid could cause an increase in cellularγ-H2AX. And the results also showed a dose-response relationship. Iodoform could induceγ-H2AX formation in the cell, but not a dose-response relationship. Iodoacetic acid and iodoform both have no effect on micronucleus. The results of Ames test, concentration ofγ-H2AX in the cell, and cytokinesis-block micronucleus assay showed that iodoacetic acid and iodoform were possible genotoxicants.
4 Potential carcinogenicity of iodoacetic acid and iodoform
The potential carcinogenicity and its mechanism of iodoacetic acid and iodoform were assessed by cell transformation assay in vitro. Doses of iodoacetic acid and iodoform were determined by cell colony formation assay. Then NIH/3T3 cells were treated by different levels of iodoacetic acid and iodoform for 72 hours. Transformation colonies were counted after 10 days culture. Then transformed cells were identified by Concanavalin A agglutination assay, soft agar assay, and assay of tumorigenicity in nude mice. Cell cycle of transformed cells was analyzed by flow cytometer. The expression of p53 protein in transformed cells was detected by immunocytochemistry assay. The results showed that iodoacetic acid could induce NIH/3T3 cells transformation. Transformed cells could be agglutinated by Concanavalin A. Meanwhile, they could form colony in soft agar and tumor in nude mice. The results of histological examination showed that the tumor was low pathologic differentiated fibrosarcoma. Cell cycle of transformed cells was arrested in G0/G1 phase. The proportions of cells in S phase and G2/M phase decreased. The expression of p53 protein in transformed cells was not high. Iodoform could not cause NIH/3T3 cells transformation in vitro. Cell transformation assay in vitro indicated that iodoacetic acid has potential carcinogenicity. But iodoform has not yet.
第一部分上海市饮用水中碘乙酸和碘仿的检测识别
1饮用水中氯、溴和碘离子检测方法的建立
本研究在借鉴美国环保局推荐的300.1方法基础上,建立基于离子色谱法测定饮用水中氯和溴离子的检测方法；通过质谱对碘化衍生物进行精确定性,继而建立基于气相色谱电子捕获检测器测定水中碘离子的检测方法。离子色谱分析结果显示,氯离子和溴离子分离度良好,峰形对称,无拖尾,溶剂和杂质峰干扰少,整个色谱分析历时12 min。采用标准曲线法进行定量,氯离子与溴离子的方法线性范围分别为1～1000 mg/L和1～1000μg/L,确定系数均为0.999。平均加标回收率在89.7%～112.3%之间,精密度小于2.9%,氯离子和溴离子方法检出限分别为55.6和0.37μg/L。连续校准值为87.9%～113.2%,替代物响应值为93.2%～103.4%,平行双样测定的相对偏差在0.0%～8.0%。含碘物质经衍生化后,质谱鉴定发现碘离子衍生化后生成碘代丁酮存在2个同分异构体,即1-碘-2-丁酮和3-碘-2-丁酮。采用双毛细管柱定性分析显示,碘代丁酮分离度好,整个色谱分析历时19.33 min。选择响应值较高的3-碘-2-丁酮进行定量检测,方法线性范围为1～1001μg/L,确定系数r2=0.999；方法检出限为0.13μg/L；平均加标回收率为102.0%,相对标准偏差为5.1%,连续校准值在97.2%-108.8%,内标物响应值在92.00%-110.05%,平行双样测定的相对偏差在4.4%-11.6%。研究结果表明所建立的氯、溴和碘离子的检测方法灵敏度高,定性定量准确,质量控制符合美国EPA检测方法的要求,适用于水中微量氯离子、溴离子和碘离子的分析。
2气相色谱电子捕获检测器法测定饮用水中碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸
借鉴美国环保局推荐的检测方法551.1和552.3,经优化前处理和检测条件,建立基于双柱定性气相色谱电子捕获检测器法测定饮用水中碘乙酸、碘仿、4种三卤甲烷(氯仿、溴仿、二氯一溴甲烷、二溴一氯甲烷)和9种卤乙酸(氯乙酸、二氯乙酸、三氯乙酸、溴乙酸、二溴乙酸、三溴乙酸、二氯一溴乙酸、二溴一氯乙酸、溴氯乙酸)的检测方法。气相色谱电子捕获检测器法检测结果显示,碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸分离度好,峰形对称,无拖尾,溶剂和杂质峰干扰少,基线平稳。碘仿和4种三卤甲烷整个色谱分离时间为40.67 min,而碘乙酸和9种卤乙酸整个色谱分离时间为23.50 mmin。采用内标工作曲线法定量,碘乙酸和碘仿的方法线性范围分别为0.01-2.5μg/L和0.05-5μg/L,4种三卤甲烷和9种卤乙酸的方法线性范围均为1-100μg/L,确定系数均大于0.994。碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸的平均加标回收率在93.5%-111.1%之问,精密度小于7.0%。碘乙酸和碘仿的方法检测限分别为6.2和17.7ng/L,4种三卤甲烷和9种卤乙酸的方法检测限为191.4-502.2 ng/L。碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸的连续校准值在87.1%-114.0%,内标物平均响应值在85.2%-120.0%,替代物响应值在85.8%-116.5%,平行双样测定的相对偏差在0.1%-10.3%。改进后的碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸测定方法分离度好、分析时间短、灵敏度高、准确度高、重现性好。仪器条件简单,可自动化批量处理,适合于普通实验室分析。
3上海市饮用水中碘乙酸和碘仿的污染现状研究
为了解上海市以长江和黄浦江为水源水厂各工艺环节水中碘乙酸和碘仿的水平,评价水源水质、生产工艺、消毒剂种类对碘乙酸和碘仿形成的影响,本研究以上海市中心城区13家水厂(11家水厂采用常规生产工艺,2家采用深度处理工艺)为对象,分别于枯水期和丰水期采集水厂不同工艺过程水样,测定水样的pH值、氨氮、可溶性有机碳、UV254、特征紫外吸光度、氯离子、溴离子、碘离子、碘乙酸、碘仿、4种三卤甲烷和9种卤乙酸的水平生成和变化情况。
研究结果显示,黄浦江和长江水质呈弱碱性(pH=7.34),氨氮含量小于0.5 mg/L,有机物含量未见偏高,DOC、UV254和SUVA的中位数分别为6.00 mg/L、0.231/cm、3.90L/(mg·m)。黄浦江pH值稍低于长江,丰水期高于枯水期；丰水期黄浦江和长江的氨氮浓度相近,枯水期黄浦江的氨氮含量高于长江。黄浦江可溶性有机碳和UV254含量均高于长江,其中两江可溶性有机碳含量丰水期略高于枯水期,而两江UV254含量则是丰水期低于枯水期；黄浦江的特征紫外吸光度水平在丰水期稍高于长江,枯水期则低于长江；黄浦江氯、溴和碘离子高于长江,且丰水期低于枯水期。各水厂出厂水碘乙酸和碘仿含量在0.03～1.66μg/L。9种卤乙酸和4种三卤甲烷含量范围在0.28～63.74μg/L。以黄浦江为原水的水厂出厂水中碘乙酸和碘仿含量高于以长江为原水的水厂,而丰水期又低于枯水期。多元线性回归分析结果显示,pH值与碘乙酸和碘仿的形成呈负相关,碘仿的形成还与UV254和碘离子水平呈正相关。
氯胺预氧化较臭氧产生更多的碘乙酸和碘仿,浓度范围在0.2～1.7μg/L,而与液氯预氧化的差异无统计学意义。各水厂生产工艺对水质pH值、氨氮、可溶性有机碳、氯离子、溴离子浓度影响不大,但UV254、特征紫外吸光度水平明显下降。常规生产工艺水厂的出厂水中碘离子浓度明显下降。未在原水中发现碘乙酸和碘仿,但各水厂出厂水均检出碘乙酸和碘仿。常规生产工艺可形成碘乙酸、碘仿、9种卤乙酸和4种三卤甲烷；深度处理工艺在活性炭工艺前均未检出碘乙酸和碘仿,但可检出微量9种卤乙酸和4种三卤甲烷。上述结果表明上海市主城区各水厂的出厂水中均检出碘乙酸和碘仿,含量处于纳克至微克水平。低pH值、高有机物、高碘离子及以氯胺预氧化有利于形成碘乙酸和碘仿。常规和深度处理工艺对水质pH值、氨氮、可溶性有机碳、氯离子和溴离子影响不大,不能有效去除饮用水加工过程生成的碘乙酸和碘仿。
第二部分碘乙酸和碘仿的细胞毒性研究
采用一组不同细胞毒性终点的试验研究碘乙酸和碘仿的细胞毒性及其可能的作用机制。分别以不同剂量的碘乙酸和碘仿对NIH/3T3细胞染毒72 h后,以四噻唑蓝试验测定碘乙酸对NIH/3T3细胞增殖和线粒体损伤的影响,以CCK-8试验测定碘仿对NIH/3T3细胞增殖和线粒体损伤的影响；并在显微镜下观察细胞形态学的变化；测定碘乙酸和碘仿对细胞乳酸脱氢酶、ATP、还原型谷胱甘肽含量的影响；以流式细胞仪分析细胞周期和凋亡的改变。研究结果显示,碘乙酸和碘仿染毒72 h后可导致NIH/3T3细胞存活率下降,呈剂量-反应关系；碘乙酸的细胞毒性分别比溴乙酸和氯乙酸强2、218倍,碘仿的细胞毒性比溴仿强81倍。碘乙酸和碘仿染毒72 h后,光镜下观察到细胞增殖受抑制,出现细胞凋亡或坏死现象；还可引起细胞外乳酸脱氢酶含量上升和细胞内ATP含量下降(P<0.05),并呈剂量-反应关系；对细胞内还原型谷胱肽无明显影响(P>0.05)。流式细胞仪分析结果显示,碘乙酸和碘仿可分别导致细胞凋亡和坏死比例增多,碘乙酸可使细胞出现S期阻滞,而碘仿则使细胞出现G2/M期阻滞。研究结果表明,碘乙酸和碘仿产生细胞毒性可能与线粒体损伤、ATP耗竭和胞膜损伤有关。线粒体途径可能是碘乙酸和碘仿致细胞死亡的机制之一。而碘乙酸和碘仿致细胞周期阻滞提示碘乙酸和碘仿可损伤细胞DNA。
第三部分碘乙酸和碘仿的遗传毒性研究
应用鼠伤寒沙门氏菌回复突变试验、细胞内γ-H2AX含量测定和胞质分裂阻滞法微核试验评价碘乙酸和碘仿的遗传毒性。分别以不同剂量的碘乙酸和碘仿对鼠伤寒沙门氏菌TA100和TA98菌株进行染毒,其中碘乙酸采用非预培养法和预培养法,碘仿采用非预培养法,均培养48 h后计数鼠伤寒沙门氏菌回变菌落数。先通过NIH/3T3细胞生长曲线确定碘乙酸和碘仿的染毒时间,再以CCK-8试验明确碘乙酸和碘仿的染毒剂量,最后采用胞质分裂阻滞法微核试验测定不同剂量碘乙酸和碘仿染毒40 h后诱导细胞微核形成数。先以CCK-8试验确定碘乙酸和碘仿染毒24 h后的染毒剂量,继而以流式细胞仪测定不同剂量碘乙酸和碘仿染毒24 h后NIH/3T3细胞γ-H2AX表达量。结果显示,碘乙酸在加或不加S9条件下均未能诱导鼠伤寒沙门氏菌TA100和TA98回复菌落数显著增加；碘仿在加或不加S9条件下均可诱导鼠伤寒沙门氏菌TA100和TA98回复菌落数显著增加。碘乙酸可引起NIH/3T3细胞γ-H2AX含量增加,并呈剂量-反应关系；碘仿能引起NIH/3T3细胞γ-H2AX含量增加,但未见剂量-反应关系。碘乙酸和碘仿均未能引起NIH/3T3细胞微核形成率增加。鼠伤寒沙门氏菌回复突变试验、微核试验和γ-H2AX含量测定试验表明碘乙酸和碘仿为可疑遗传毒物。
第四部分碘乙酸和碘仿的潜在致癌性研究
应用体外细胞恶性转化试验研究碘乙酸和碘仿的潜在致癌性及其可能的作用机制。先通过细胞克隆形成试验确定细胞恶性转化试验的染毒剂量,然后以不同剂量的碘乙酸和碘仿对NIH/3T3细胞染毒72 h后继续培养10天,观察细胞转化灶形成数。继而以伴刀豆蛋白A凝集试验、软琼脂试验和裸鼠成瘤试验验证转化细胞的恶变情况,并以流式细胞仪分析恶性转化后细胞的周期变化,以免疫细胞化学试验分析恶性转化后细胞p53蛋白表达情况。研究结果显示,碘乙酸可诱导NIH/3T3细胞发生恶性转化,转化细胞能引起伴刀豆蛋白A凝集,可在软琼脂中形成克隆,并能在裸鼠皮下形成肿瘤,肿瘤组织学检查表明所形成肿瘤为分化程度较低的纤维肉瘤。细胞周期分析显示转化细胞出现G0/1期阻滞,S期和G2/M期细胞比例下降。免疫细胞化学分析显示p53蛋白表达未见增强。而碘仿在体外NIH/3T3细胞恶性转化试验中呈阴性。体外细胞恶性转化试验表明碘乙酸具有潜在致癌性,而未观察到碘仿具有潜在致癌性。
Iodo-disinfection byproducts are a kind of new unregulated disinfection byproducts in drinking water treatment process in recent years. Iodo-disinfection byproducts are easy to form when high iodide in raw water and using chloramination. Salt water intrusion strongly influences drinking water source because Shanghai located in the intersection zone of river and sea. Most of works used conventional treatment process (preoxidation, coagulation, sedimentation, sand filtration, and post chloramination.) to produce drinking water. Special environmental conditions and conventional treatment process are in favor of iodo-disinfection byproducts formation. Otherwise, the concentrations of iodo-disinfection byproducts are very low in drinking water but they are toxic strongly. Previous studies indicated that iodo-disinfection byproducts were more cytotoxic and genotoxic than regulated disinfection byproducts. Therefore, it is necessary to detect the levels and influencing factors of iodoacetic acid and iodoform in drinking water in Shanghai in order to evaluate population exposure. MTT assay, CCK-8 assay, Ames test, cellular y-H2AX, and cytokinesis-block micronucleus assay were used to evaluate their cytotoxicity and genotoxicity and their mechanism. Then the potential carcinogenicity and its mechanism of iodoacetic acid and iodoform were assessed by cell transformation assay in vitro. The study would provide the important scientific evidences for disinfection byproducts control and treatment process improvement. Meanwhile, it is helpful to the health risk assessment of iodoacetic acid and iodoform and the establishment of standard for drinking water quality.
1 Determination of iodoacetic acid and iodoform in drinking water in Shanghai
1.1 Establishment of methods for determining chloride, bromide, and iodide in drinking water
The method for determining chloride and bromide in drinking water by ion chromatography was established on the base of U.S. Environmental Protection Agency 300.1 method, Iodobutanone derivative was identified by gas chromatography/mass spectrometry, and then gas chromatography coupled to electron capture detector was made to analyze iodide in water. The results of ion chromatography showed that chloride and bromide were separated completely. Their peaks were symmetrical, no tail and not disturbed by peaks of solvent or impurity. The total time of chromatogram separation was 12 minutes. External standard calibration curve was selected to quantitative analysis. The linear ranges of chloride and bromide were 1-1000 mg/L and 1-1000μg/L respectively, and their coefficients of determination were both 0.999. The mean recoveries were between 89.7% and 112.3%, and the relative standard deviations were less than 2.9%. Method detections limits of chloride and bromide were 55.6μg/L and 0.37μg/L respectively. Their continuing calibration checks were between 87.9% and 113.2%. Surrogate response was between 93.2% and 103.4%. Their relative percent difference for duplicates were between 0.0% and 8.0%. The results of mass spectrometry showed that iodide would form iodobutanone which could generate 1-iodo-2-butanone and 3-iodo-2-butanone isomers by derivatization. The data of qualitative analysis by two capillary columns revealed that iodobutanone were separated completely and the total time of chromatogram separation was 19.33 minutes.3-iodo-2-butanone with the high response value was selected to quantitative analysis. The linear range was 1-100μg/L, and the coefficient of determination (r2) was 0.999. The limit of detection was 13 ng/L. The mean recovery was 102.0%, and relative standard deviation was 5.1%. The continuing calibration checks were between 97.2% and 108.8%. Internal standard responses were between 92.00% and 110.05%. The relative percent difference for duplicates were between 4.4% and 11.6%. The improved method possesses higher degree of sensitivity and accuracy of qualitative and quantitative analysis. Its quality control measured up to methods of United States Environmental Protection Agency. And it is fit for trace analysis of chloride, bromide, and iodide in water.
1.2 Determination of iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids in drinking water by gas chromatography with electron capture detection
Base on U.S. Environmental Protection Agency 551.1 and 552.3 methods, the pretreatment and detection conditions of methods for identifying iodoacetic acid, iodoform, four trihalomethanes(chloroform,bromoform, bromodichloromethane, and dibromochloromethane) and nine haloacetic acids (chloroacetic acid, dichloroacetic acid, trichloroacetic acid, bromoaceic acid, dibromoacetic acid, tribromoacetic acid, bromodichloroacetic acid, chlorodibromoacetic acid, and bromochloroacetic acid) with two capillary columns coupled to gas chromatography/electron capture detector were improved and optimized. The results of gas chromatography showed that iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids were separated completely. Their peaks were symmetrical, no tail and not disturbed by peaks of solvent or impurity. The total time of iodoform and four trihalomethanes separation was 40.67 minutes. The total time of iodoacetic acid and nine haloacetic acids separation was 23.50 minutes. Internal standard calibration curve was selected to quantitative analysis. The linear ranges of iodoacetic acid and iodoform were 0.01-2.5μg/L and 0.05-5μg/L respectively. The linear ranges of four trihalomethanes and nine haloacetic acids were both 1-100μg/L. Their coefficients of determination were more than 0.994. The mean recoveries were between 93.5% and 111.1%, and the relative standard deviations were less than 7.0%. Method detections limits of iodoacetic acid and iodoform were 6.2 ng/L and 17.7 ng/L respectively. Method detections limits of four trihalomethanes and nine haloacetic acids were between 191.4 ng/L and 502.2 ng/L. The continuing calibration checks were between 87.1% and 114.0%. Internal standard responses were between 85.2% and 120.0%. Surrogate response was between 85.8% and 116.5%. Their relative percent difference for duplicates were between 0.1% and 10.3%. The modified methods possesses good separation, fast, high sensitivity, high accuracy, and good consistency. It is fit for analysis in normal laboratory because of its economical and automatic characteristic.
1.3 Pollution of iodoacetic acid and iodoform in drinking water in Shanghai
The object of this study is to detect the levels of iodoacetic acid and iodoform in water of each treatment processes in works that their source waters come from Yangtze River and Huangpu River in Shanghai and evaluate the relationship of iodoacetic acid and iodoform formation to water qulity, treatment processes, and disinfectants. Thirteen water treatment works that are at central regions of Shanghai were chosen for investigation in low water period and high water period. Eleven works used conventional water treatment process. Two works used advanced water treatment process. Water samples of different treatment processes were selected to analyze for pH value, ammonia nitrogen, dissoluble organic carbon, UV absorbance, specific UV absorbance, chloride, bromide, iodide, iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids.
The results showed that pH values of Yangtze River and Huangpu River were alkalescent (pH=7.34). The concentrations of ammonia nitrogen were less than 0.5 mg/L. The levels of organic matter were not high. Medians of dissoluble organic carbon, UV absorbance, and specific UV absorbance were 6.00 mg/L、0.23 l/cm、3.90 L/(mg·m) respectively. The pH value of Huangpu River was lower than that of Yangtze River. The pH values of two rivers in high water period were higher than low water period. The ammonia nitrogen level of Huangpu River was closed to Yangtze River in high water period. In low water period, the ammonia nitrogen level of Huangpu River was higher than Yangtze River. The dissoluble organic carbon and UV absorbance of Huangpu River was higher than that of Yangtze River. The dissoluble organic carbon levels of two rivers in high water period were higher than low water period, but the UV absorbance levels reversely. In high water period, specific UV absorbance level of Huangpu River was higher than Yangtze River, but reversely in high water period. Chloride, bromide, and iodide levels of Huangpu River were higher than that of Yangtze River. But chloride, bromide, and iodide levels of two rivers in high water period were lower than low water period. The concentrations of iodoacetic acid and iodoform were between 0.03μg/L and 1.66μg/L in finished water in works. The level of four trihalomethanes and nine haloacetic acids were between 0.28μg/L and 63.74μg/L in finished water in works.
The concentrations of iodoacetic acid and iodoform in finished water in works which based on Huangpu River as water source were higher than that of works which based on Yangtze River as water source. The concentrations of iodoacetic acid and iodoform in finished water in low water period were higher than high water period. The results of multiple liner regression analysis showed that the relationship of pH value and iodo-disinfecton byproducts formation were negative correlation. Otherwise, the relationship of UV254 and iodide levels and iodoform formation were positive correlation.
More iodo-disinfecton byproducts were formed when using chloramination compared with ozone. The level of iodo-disinfecton byproducts were between 0.2μg/L and 1.7μg/L when using chloramination. There were not sighnificant differences between chloramination and chlorination. The treatment processes of each work had no effect on the level of pH value, ammonia nitrogen, dissoluble organic carbon, chloride, and bromide. But the levels of UV254 and specific UV absorbance decreased. The level of iodide decreased in finished water in conventional treatment process works. Iodoacetic acid and iodoform were not detected in raw water. But iodoacetic acid and iodoform were detected in finished water in every work. Iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids were formed in conventional treatment process. Iodoacetic acid and iodoform were not detected before activated carbon in advanced treatment process. Only few four trihalomethanes and nine haloacetic acid were detected in finished water in advanced treatment process works. The results indicated that trace iodoacetic acid and iodoform were detected in finished water in every work at the central regions of Shanghai. Low pH value, high natural organic matter, high iodide, and chloramination were easy to form iodoacetic acid and iodoform. The conventional and advanced treatment processes had no effect on pH value, ammonia nitrogen, natural organic matter, chloride, bromide, iodoacetic acid and iodoform.
2 Cytotoxicity of iodoacetic acid and iodoform
The object of this research is to study the cytotoxicity and its mechanism of iodoacetic acid and iodoform by a group of different cytotoxic end points assay. NIH/3T3 cells were treated by different doses iodoacetic acid and iodoform for 72 hours respectively. Then the proliferation and mitochondria damage of NIH/3T3 cell induced by iodoacetic acid and iodoform were evaluated by methylthiazoletrazolium assay and CCK-8 assay. The morphology of cells was observed in the microscope. The level of lactate dehydrogenase, ATP, and reduced glutathione were measured respectively. Cell cycle and apoptosis of cells were analyzed by flow cytometer. The results showed that NIH/3T3 cells viability decreased after they were exposed to iodoacetic acid and iodoform for 72 hours. There were also dose-response relationships. Iodoacetic acid was 2×more cytotoxic than bromoacetic acid and 218×more cytotoxic than chloroacetic acid in NIH/3T3 cells. Iodoform was 81×more cytotoxic than bromoform. The prohibition of cellular proliferation, apoptosis, and necrosis also could be observed in the microscope. Iodoacetic acid and iodoform could cause extracellular lactate dehydrogenase increase and intracelluar ATP decrease after 72 hours incubation (P<0.05). But they had no effect on reducing glutathione (P>0.05). Iodoacetic acid and iodoform could induced an increase in the proportion of apoptosis and necrosis. Cells cycle were arrested in S and G2/M phase respectively after incubation with iodoacetic acid and iodoform. Mitochondria damage, ATP depletion, and membrane damage are possibly involved in the induction of cytotoxicity by iodoacetic acid and iodoform. Mitochondria pathway would be possible one of mechanisms of cell death caused by iodoacetic acid and iodoform. Cells cycle arrest indicated that iodoacetic acid and iodoform can damage cellular DNA.
3 Genotoxicity of iodoacetic acid and iodoform
The genotoxicity of iodoacetic acid and iodoform were evaluated by Ames test, cellularγ-H2AX, and cytokinesis-block micronucleus assay. Salmonella typhimurium TA100 and TA98 were treated by different doses iodoacetic acid and iodoform. Un-preincubation and preincubation methods were used in iodoacetic acid treatment process. Un-preincubation was just used in iodoform treatment process. Numbers of Salmonella typhimurium were counted after 48 hours. In cytokinesis-block micronucleus assay, the treatment time of iodoacetic acid and iodoform were detected by NIH/3T3 cell growth curve. Then doses of iodoacetic acid and iodoform were selected by CCK-8 assay. Micronuclei were counted after cells were treated by iodoacetic acid and iodoform for 40 hours using cytokinesis-block micronucleus assay. Doses of iodoacetic acid and iodoform were determined by CCK-8 assay. Then levels ofγ-H2AX were analyzed by flow cytometer after treatment for 24 hours. The results showed that iodoacetic acid could not induce Salmonella typhimurium TA100 and TA98 increase significantly with or without S9. Iodoform was mutagenic in Salmonella typhimurium TA100 and TA98 with or without S9. Iodoacetic acid could cause an increase in cellularγ-H2AX. And the results also showed a dose-response relationship. Iodoform could induceγ-H2AX formation in the cell, but not a dose-response relationship. Iodoacetic acid and iodoform both have no effect on micronucleus. The results of Ames test, concentration ofγ-H2AX in the cell, and cytokinesis-block micronucleus assay showed that iodoacetic acid and iodoform were possible genotoxicants.
4 Potential carcinogenicity of iodoacetic acid and iodoform
The potential carcinogenicity and its mechanism of iodoacetic acid and iodoform were assessed by cell transformation assay in vitro. Doses of iodoacetic acid and iodoform were determined by cell colony formation assay. Then NIH/3T3 cells were treated by different levels of iodoacetic acid and iodoform for 72 hours. Transformation colonies were counted after 10 days culture. Then transformed cells were identified by Concanavalin A agglutination assay, soft agar assay, and assay of tumorigenicity in nude mice. Cell cycle of transformed cells was analyzed by flow cytometer. The expression of p53 protein in transformed cells was detected by immunocytochemistry assay. The results showed that iodoacetic acid could induce NIH/3T3 cells transformation. Transformed cells could be agglutinated by Concanavalin A. Meanwhile, they could form colony in soft agar and tumor in nude mice. The results of histological examination showed that the tumor was low pathologic differentiated fibrosarcoma. Cell cycle of transformed cells was arrested in G0/G1 phase. The proportions of cells in S phase and G2/M phase decreased. The expression of p53 protein in transformed cells was not high. Iodoform could not cause NIH/3T3 cells transformation in vitro. Cell transformation assay in vitro indicated that iodoacetic acid has potential carcinogenicity. But iodoform has not yet.
引文
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