某自来水厂水源水有机提取物的雌激素样活性研究

[目的]了解某自来水厂水源水有机提取物的雌激素样活性。[方法]用固相萃取法富集水样,分低、中、高剂量染毒组（体外试验为每毫升染毒量分别相当于10、100、1000mL水源水中有机提取物的剂量;体内试验为每千克体重灌胃量相当于2、5、10、25L水源水中有机提取物的剂量）、阴性对照组和雌二醇阳性对照组,通过体外人乳腺癌（MCF-7）细胞增殖试验、报告基因试验和初断乳大鼠子宫增重试验对水源水有机提取物的雌激素样活性进行研究。[结果]体外细胞增殖试验中,高剂量组较阴性对照组出现细胞增殖效应（P〈0．05）,细胞增殖率为（130．85±13．68）％;而报告基因试验中,中剂量组检测到荧光强度增加（P〈0．05）,相对荧光强度为（207．67±12．40）％。子宫增重试验中,相当于低、中、高剂量染毒组大鼠子宫湿重和黏液重脏器系数与阴性对照组相比差异无统计学意义。[结论]体外试验表明水源水中含有雌激素样活性物质,但体内试验尚不能确定该水源水具有明确的雌激素样活性。     

松花江水源水中有机提取物诱导MCF-7细胞增殖的研究

目的了解松花江水源水环境内分泌干扰素的污染状况,从而为松花江水体污染的防治提供科学的依据。方法于2008年3月,采集以松花江为水源水的某市水厂原水水样,采用GDX-102大孔树脂对水样中的有机物进行提取浓缩,分别以相当于原水量125、500、2000、8000和16000ml/ml浓度与MCF-7细胞共培养,采用MTT法、细胞集落形成法、紫外分光光度计法观察其对人乳腺癌MCF-7细胞的增殖作用。结果随着有机提取物浓度的升高,MCF-7细胞增殖效应呈先升高后下降的趋势;当有机提取物浓度相当于原水量2000ml/ml时,MCF-7细胞的增殖活性达到最高。结论松花江水源水有机提取物能明显促进体外培养的人乳腺癌MCF-7细胞的生长,显示出雌激素活性。     

污水处理传统工艺与新工艺出水中有机提取物的雌激素样作用比较

目的评价污水处理厂传统处理工艺和新技术处理工艺出水中有机提取物的雌激素样活性,比较两种处理工艺对微量有机污染物的去除效果。方法采用树脂吸附固相萃取富集水样中的微量有机污染物,利用重组基因酵母试验和小鼠子宫增重实验检测水样中有机提取物的雌激素样活性大小。结果重组基因酵母检测中,在原水样中微量有机污染物浓缩1000倍条件下,新技术出水有机提取物处理的酵母表现出β-半乳糖苷酶活性,而三级出水、二级出水和进水都为500倍,相同条件下的β-半乳糖苷酶活性,新技术出水组三级出水组二级出水组进水组。小鼠子宫增重实验中,仅进水有机提取物油溶液原液组和阴性对照组之间具有显著性差异(P0.05),其他各水样组之间皆无显著性差异。结论郑州市城市污水经处理后雌激素样活性显著下降,但仍对环境具有潜在危害;新技术对污水中微量有机污染物的去除效果明显好于传统技术。     

城市回用水有机提取物的雌激素效应

目的检测郑州市某污水处理厂进水及出水中有机提取物的雌激素样效应。方法利用固相萃取技术提取进水及不同工艺出水中的有机物,应用环境雌激素基因重组酵母,通过测定酵母经类雌激素化合物质激活后产生的β-半乳糖苷酶活性来判断各水样有机污染物的雌激素样活性。结果进水、二级和三级出水的有机提取物在1/2、1/4、1/8三个稀释浓度及新工艺出水的有机提取物在1/2、1/4两个稀释浓度下,酵母菌表现出β-半乳糖苷酶活性;相同染毒浓度下,二级出水和进水的β-半乳糖苷酶活性明显升高。结论郑州市城市污水及出水中所含有机物在一定浓度下表现出雌激素样活性,具有潜在的环境与健康风险。     

再生水有机提取物雌激素效应研究

目的 研究天津市某再生水厂各处理单元水体的雌激素效应。方法 采用反相C-18固相萃取柱提取各处理单元水样中目标物质,采用人乳腺癌细胞(MCF-7细胞)增殖试验和基因重组酵母试验检测水样有机提取物的雌激素效应。结果 2项试验中各处理单元水样有机提取物均表现出雌激素效应,且在一定范围内,呈现出明显的剂量．效应关系。二级出水、混凝后出水、连续膜过滤后出水和O3处理后出水均在相当于原水1．00ml的浓度水平时表现出对MCF-7细胞最强的增殖效应,最大相对增殖效应依次为33．87％,19．49％,20．42％和16．24％。而且上述水样有机提取物均在相当于原水20ml的浓度水平时具有最高β-半乳糖苷酶活力(8．15,7．50,6．07和5．33)。结论 天津市某再生水厂现有处理单元对水体雌激素效应具有有限的去除作用,但最终出水仍具有一定的雌激素效应。     

武汉市饮用水中有机提取物对HepG2细胞DNA的损伤作用

目的研究武汉市生活饮用水中有机提取物对人肝肿瘤HepG2细胞DNA损伤作用。方法用XAD-2/8复合树脂(XAD-2和XAD-8树脂等体积混合)等比富集武汉市生活饮用水中有机污染物,应用慧星试验检测有机提取物的遗传毒性。结果该市不同水源、不同水文期下饮用水中有机提取物对HepG2细胞均产生不同程度的DNA损伤作用。同一水文期不同水源比较,在剂量10和30ml/ml下,汉江水源末梢水有机提取物致DNA损伤程度大于长江水源(P0.05);同一水源不同水文期时比较,在剂量10和30ml/ml下,枯水期有机提取物致DNA损伤程度大于丰水期(P0.05)。结论武汉市主要水源汉江和长江的氯化饮用水有机提取物对HepG2细胞DNA均有损伤作用,损伤作用的严重程度从大到小为汉江水长江水,枯水期丰水期。     

加氯消毒对水中有机提取物致人胎肝细胞DNA损伤的影响

目的探讨自来水加氯消毒对水中有机提取物致细胞DNA损伤的影响。方法于2007年7月(夏季)和2008年3月(春季)采集以长江为水源的某水厂的水源水、经加氯消毒处理后的出厂水和末梢水,以人胎肝细胞(L-02)为靶细胞,采用单细胞凝胶电泳技术,检测水样有机提取物诱导细胞DNA损伤的效应。水样有机提取物设4个染毒浓度(1.2、6、30、150ml/ml),阳性对照为苯并(a)芘(200μmol/L),溶剂对照为二甲基亚砜(DMSO,10μl/ml)。结果春季水源水、出厂水和末梢水以及夏季出厂水和末梢水水样有机提取物在低、中、高浓度(6、30、150ml/ml)引起的L-02细胞DNA损伤较溶剂对照组明显增加。春夏两季加氯消毒后的出厂水和末梢水有机提取物在低、中、高浓度(6、30、150ml/ml)引起的L-02细胞DNA损伤较水源水明显增强。春季的水样有机提取物对细胞DNA的损伤作用高于夏季。结论水源水、出厂水和末梢水有机提取物均能引起L-02细胞的DNA损伤,氯化消毒增加了水源水的遗传毒性,春季水样的遗传毒性大于夏季。     

地表水和自来水中环境雌激素活性物质的检测

目的了解我国地表水和自来水的环境雌激素污染情况。方法应用本实验室构建的基因重组酵母检测系统,对用H103大孔吸附树脂吸附的水样富集物进行环境雌激素活性检测。结果本次所检测的50％的地表水样和25％的自来水样检出环境雌激素活性物质,一些水源水活性较高,其水样富集物有显著的剂量-效应关系,仅100ml水样富集物即呈现阳性反应。结论我国的一些地表水中已受到环境雌激素类物质的污染,而现行的城市自来水处理系统不能有效地去除水源水中环境雌激素活性物质,致使城市自来水中也含有环境雌激素活性物质。     

淮河水有机提取物对小鼠遗传毒性和脂质过氧化作用

目的探讨N市淮河原水及出厂水中有机提取物对小鼠遗传毒性和脂质过氧化作用。方法XAD-Ⅱ树脂吸附原水及出厂水中有机污染物,采用腹腔注射染毒小鼠,进行微核实验、精子畸形实验及血清、肝、脑组织中脂质过氧化物(LPO)含量及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性测定。结果在0.01ml/g(相当100L/kg原水、出厂水及末梢水连续染毒5d)的条件下,微核率和精子畸形率显著高于阴性对照组(P<0.05或P<0.01),同时相应组小鼠血清中LPO含量及SOD、GSH-Px活性均有不同程度的改变(P<0.01);N段上游水组小鼠血清SOD和脑组织中GSH-Px活性改变明显(P<0.01)。微核率及精子畸形率与LPO呈显著正相关(P<0.05)。结论淮河N段水有机提取物对小鼠有潜在的遗传毒性,并能影响小鼠的脂质过氧化作用。     

地表水中有机提取物对人B淋巴细胞DNA的损伤效应

目的探究地表水中有机提取物对人B淋巴细胞DNA的损伤效应。方法于2010年6—8月,采集某江流域18个采样点水样并提取有机物。将处于对数生长期的人B淋巴母细胞悬液分别暴露于0(对照)、250、500、1 000 ml/ml水样有机提取物培养24 h;采用CCK-8活细胞计数试剂盒和彗星试验分别研究水样有机提取物对细胞存活率的影响及其DNA损伤效应,并检测细胞内活性氧自由基(ROS)含量的变化,以评价水样有机提取物的氧化损伤效应。结果与对照组比较,除250 ml/ml采样点5、11、12水样外,各采样点水样有机提取物在各暴露剂量下对人B淋巴母细胞的存活率均较低,差异有统计学意义(P0.01);且随着各采样点水样有机提取物暴露剂量的升高,人B淋巴细胞的存活率呈下降趋势。与对照组相比,采样点4、5、6水样有机提取物在250、500、1 000 ml/ml剂量下对人B淋巴母细胞彗星尾部DNA含量无明显影响,采样点9、11、12和15水样有机提取物仅在1 000 ml/ml剂量下导致人B淋巴母细胞尾部DNA含量增加,差异有统计学意义(P0.05,P0.01);其余各采样点水样有机提取物在500、1 000 ml/ml剂量下均可导致人B淋巴母细胞彗星尾部DNA含量增加,差异有统计学意义(P0.05,P0.01)。与对照组相比,采样点16水样有机提取物在500 ml/ml剂量下对人B淋巴细胞内ROS含量无显著影响(P0.05),其余各采样点水样有机提取物在250~1 000 ml/ml剂量范围内均可显著诱导人B淋巴细胞内ROS的产生,差异有统计学意义(P0.01);除采样点15外,随着各采样点水样有机提取物暴露剂量的升高,人B淋巴细胞中ROS的含量均呈上升趋势。结论该流域江水中有机提取物对人B淋巴细胞DNA有显著的损伤效应,可能与细胞内ROS含量增加有关。     

Screening of estrogen-like activity of mineral water stored in PET bottles

Bottled mineral water consumption is steadily rising in the World. Italy is the largest natural mineral water consumer in Western Europe, about 200L per capita per annum. Recently, research has concentrated upon emerging toxicological problems such as the presence in drinking water of substances which interfere with the function of the endocrine system; defined as endocrine disruptors (EDs). The aim of this study was to assess the risk of exposure of the population to chemicals with estrogen-like activity through mineral water consumption by monitoring the presence of estrogenic compounds in mineral water bottled in polyethylene terephthalate (PET). A solid phase extraction (SPE) with C18 cartridges was carried out. The estrogenic activity of the extracts was assayed using a yeast assay expressing the human estrogen receptor alpha (hERalpha). This preliminary study shows that more than 90% of the water samples did not exhibit any appreciable estrogenic activity. The highest estrogenic activity detected in water extracts was equivalent to the activity induced by 23.1ng/L of the natural hormone 17beta-estradiol. Some mineral water samples showed toxicity on yeast cells.     

子宫实验和E-SCREEN实验在检测雌激素活性中的相关性

目的　以 1 7β 雌二醇 (1 7β E2 )和四种植物提取物为受试物 ,研究子宫实验和E SCREEN实验在检测雌激素活性中的相关性。方法　断乳雌性小鼠 (1 0～ 1 2g)按体重分为 6组 ,分别给予小茴香、山豆根、补骨脂和川牛膝提取物 (1 0g kgBW ,ig)、1 7β E2 (0 5mg kgBW ,sc)和蒸馏水 (ig) ,持续 9天后处死动物剥离子宫称重。分别在人类乳腺癌MCF 7细胞培养液中加入四种植物提取物 (终浓度 1 0mg L)和 1 7β E2 (终浓度0 3μmol L) ,计算各组细胞在 1 2 0h内的平均群体倍增时间 ,分析小鼠子宫重量与细胞群体倍增时间的相关性。结果　 4种植物提取物均可使小鼠子宫重量增加 (P <0 0 5或P <0 0 1 ) ,细胞群体倍增时间缩短。子宫重量与群体倍增时间呈显著负相关 (r=- 0 96 7,P <0 0 1 )。结论　子宫实验和E SCREEN实验结果在检测雌激素活性物质时具有一致性     

Bioaccumulation of the synthetic hormone 17α-ethinylestradiol in the benthic invertebrates Chironomus tentans and Hyalella azteca

The present study investigated the bioaccumulation of the synthetic hormone 17α-ethinylestradiol (EE2) in the benthic invertebrates Chironomus tentans and Hyalella azteca, in water-only and spiked sediment assays. Water and sediment residue analysis was performed by LC/MS–MS, while biota extracts were analyzed using both LC/MS–MS and a recombinant yeast estrogen receptor assay. At the lowest exposure concentration, C. tentans accumulated less EE2 than H. azteca in the water-only assays (p=0.0004), but due to different slopes, this difference subsided with increasing concentrations; at the exposure concentration of 1 mg/L, C. tentans had a greater body burden than H. azteca (p=0.02). In spiked sediments, C. tentans had the greatest EE2 accumulation (1.2±0.14 vs. 0.5±0.05 μg/g dw, n=4). Measurements in H. azteca indicated a negligible contribution from the sediments to the uptake of EE2 in this species. These differences were likely due to differences in the behavior and life history of the two species (epibenthic vs. endobenthic). Water-only bioaccumulation factors (BAFs) calculated at the lowest exposure concentration were significantly smaller in C. tentans than in H. azteca (31 vs. 142, respectively; p<0.0001). In contrast, the sediment bioaccumulation factor (BSAF) of C. tentans was larger than that of H. azteca (0.8 vs. 0.3; p<0.0001). Extracts of the exposed animals caused a response in a recombinant yeast estrogen receptor assay, thus confirming the estrogenic activity of the samples, presumably from EE2 and its estrogenic metabolites. The results of the present study suggest that consumption of invertebrate food items could provide an additional source of exposure to estrogenic substances in vertebrate predators.     

基因重组酵母测评系统在筛选环境样品雌激素活性中的应用研究

目的 用重组酵母测评系统研究DDTs和汽车尾气提取物的拟雌激素活性.方法 将终浓度为10~(-9)～10~(-15) mol/L的171β-雌二醇、终浓度为10~(-4)～10~(-9) mol/L 的DDTs(p,p'-DDT、o,p'-DDT、p,p'-DDD、p,p'-DDE)和终浓度为0.25～8.00ml/ml的汽车尾气提取物加入酵母细胞培养液,作用4 h,通过定量测定β-半乳糖苷酶活力表达受试物的雌激素作用强度.结果 DDTs是一类可直接象配体一样与雌激素受体(ER)结合并发挥雌激素效应的环境雌激素,单独作用时o,p'-DDT雌激素活性较高,p,p'-DDE的雌激索活性较低;汽油燃料车尾气在终浓度为2.00～4.00 ml/ml时,其β-半乳糖苷酶活力与DMSO对照组相比,差异有统计学意义(P＜0.05),天然气尾气各剂量组的β-半乳糖苷酶活力与对照组(蒸馏水)相比,差异无统计学意义(P＞0.05).结论 DDTs是经ER介导的环境雌激素,其中o,p'-DDT的拟雌激素活性最高.汽车尾气提取物中汽油燃料车尾气具有弱雌激素活性,天然气燃料车尾气未检出雌激素活性.

Abstract：

Objective To research the estrogen-like activity of the DDTs and the extract of vehicle exhaust by recombinant yeast system.Methods Being added into the yeast cell culture fluid,the objects reached the final concentration of 10~(-9)～10~(-15) mol/Lfor 17β-estradiol,10~(-4)～10~(-9) mol/L for DDTs(p,p'-DDT,o,p'-DDT,p,p'-DDD,p,p'-DDE),0.25～8.00 ml/ml for the extract of vehicle exhaust,then the cultivation was performed for 4 hours,the quantimtive assay of β-galactosidase activity accounts for the estrogenlike effect of the samples was carried out.Results DDTs were a group of environmental estrogens hormones,which could integrate estrogen receptors as the stimulators do and express the estrogen-like effect.Being tested separately.the DDTs showed the estrogen-like activity,which was stronger for o,p'-DDT and weaker for p,p'DDE.Theβ-galactosidase activity of gasoline-fueled vehicle exhaust at the final concentration of 2.00～4.00 ml/ml was significantly different from that of the DMSO(P＜0.05).while theβ-galactosidase activity of natural gas-fueled vehicle exhaust wasn't significantly difirerent from that of the contrast distilled water(P＜0.05).Conclusion DDTs are environmental estrogens mediated by ER,among which the strongest i8 o,p,-DDT.Gasoline-fueled vehicle exhaust shows weaker estrogen activity,natural gas-fueled vehicle exhaust shows no estrogen activity.     

Estrogenic Effects of Cimicifuga racemosa (Black Cohosh) in Mice and on Estrogen Receptors in MCF-7 Cells

The estrogenic effects of Cimicifuga racemosa or Actacea racemosa (black cohosh, CR) extracts were tested in mice, and their effects on estrogen receptor (ER) levels in human breast cancer MCF-7 cells were also investigated. Four groups of weanling female Kunming mice were given 0 (control), 75, 150, or 300 mg/kg body weight CR extracts orally for 14 days. The estrus cycle and the weights of the uterus and ovary of mice, as well as serum estradiol (E(2)) were measured. The proliferation patterns of MCF-7 cells exposed to CR extracts or 17beta-estradiol were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Subsequently, growth of MCF-7 cells in 0 (control) or 4.75 &mgr;g/L of CR extracts or 0.3 nmol/L of 17beta-estradiol groups were observed for 5 days. ER levels in MCF-7 cells were analyzed by indirect immunofluorescence assay using flow cytometry. The uterine weights of mice increased with the increase in dosage of CR extracts, and the estrus duration was significantly prolonged in the group receiving 300 mg/kg body weight (P <.05). However, CR extracts did not increase the serum E(2) concentration significantly. In the in vitro study, a dose-response relationship was demonstrated when cells were treated with low doses of CR extracts, and the optimal enhancement concentration of CR extracts was 4.75 &mgr;g/L on MCF-7 cells. The doubling times (T(D)) of cell growth in the CR extracts group and the 17beta-estradiol group were 32.1 and 31.7 hours, respectively, both shorter than that of the negative control group (T(D) = 35.3 hours). Additionally, 4.75 &mgr;g/L of CR extracts resulted in significantly increased ER levels compared with the control group (P <.01). In conclusion, CR extracts produced an estrogenic action. The effect of increasing ER levels by CR extracts may be one of the potential mechanisms of its phytotherapeutic effects for postmenopausal symptoms.     

某鱼塘养殖污水有机提取物的遗传毒性

目的评价养殖污水有机提取物的遗传毒性.方法健康昆明种小鼠,按体重随机分组,每组8只,雌雄各半.以养殖污水有机提取物原液、1/2原液、1/4原液、1/8原液对小鼠染毒,分别相当于25、12.5、6.25、3.125L/kg,以DMSO为阴性对照,环磷酰胺(40 mg/kg)为阳性对照,连续染毒3天.应用小鼠骨髓嗜多染红细胞(PCE)微核试验、小鼠外周血淋巴细胞彗星试验对养殖污水有机提取物进行遗传毒性检测.结果养殖污水有机提取物原液、1/2原液组小鼠PCE微核率均高于阴性对照组,差别均有统计学意义(P＜0.01,P＜0.05);各剂量组小鼠外周血淋巴细胞拖尾率均高于阴性对照组(P＜0.01),原液、1/2原液组彗星平均尾长均高于阴性对照组,差别均有统计学意义(P＜0.01,P＜0.05).结论养殖污水中含有机致突变物质,可能对人体健康产生潜在的危害.     

Identification of estrogenic compounds in wastewater effluent

In order to identify the dominant contributors to estrogenic activity in environmental waters, a comprehensive fractionation method using silica gel column chromatography, combined with recombinant yeast assay for detecting estrogenic activity and with gas chromatography-mass spectrometry for quantifying endocrine disruptors and natural estrogens, was developed. The method was applied to the municipal sewage treatment plant (STP) secondary effluent discharged to the Tamagawa River in Tokyo, Japan, where endocrine disruption was observed in wild carp. The instrumental analysis demonstrated that averaged concentrations of nonylphenol, bisphenol A, estrone (E1), and 17beta-estradiol (E2) were 564 +/- 127, 27 +/- 19, 33 +/- 11, and 4.6 +/- 3.0 ng/L, respectively. Based on the concentration and relative potency of these compounds, the natural estrogens E1 and E2 represented more than 98% of the total estrogen equivalent concentration (EEQ) in the STP effluent, while the contribution of phenolic compounds to total EEQ was less than 2%. Estrogenic activities associated with the dissolved phase of the effluent samples were detected by a recombinant yeast assay. By using silica gel column chromatography, the dissolved phase was separated into several fractions that were subjected to the bioassay. The polar fractions exhibited estrogenic activity. The greatest estrogenic activity was found in a polar fraction containing E1 and E2 and represented 66 to 88% of the total estrogenic activities estimated from the bioassay data. These results lead to the conclusion that E1 and E2 were the dominant environmental estrogens in the STP effluent, but a significant contribution to estrogenic activities stems from unidentified components in the effluents.     

用Ames试验和小鼠原代细胞彗星试验检测A市生活饮用水遗传毒性

目的　用Ames试验和小鼠原代肝细胞彗星试验检测A市生活饮用水遗传毒性并比较两种方法的敏感性。方法　采用Ames试验和彗星试验分别对A市南郊水厂的水源水、出厂水、自来水的有机浓集物的诱变性进行检测。结果　出厂水和自来水的Ames试验于试样浓度为 3 0L/皿时才表现为阳性结果 ,而肝细胞的彗星试验在水样量为0 1L时就出现明显的阳性结果 (与阴性对照比P <0 0 1) ,后者的浓度比前者低 3 0倍。水源水的Ames试验于试样浓度为 6 0L/皿时仍表现为阴性结果 ,而原代肝细胞的彗星试验在水样量为 0 5L时就出现明确的阳性结果。结论　Ames试验只能检测出氯化消毒后饮用水的致突变阳性 ,而彗星试验能同时检测出水源水和氯化消毒后饮用水的致突变性 ,后者的敏感性远远高于前者。     

小鼠肝细胞彗星实验对饮用水氯化消毒副产物诱变性研究

目的 用小鼠肝细胞彗星实验检测生活饮用水氯化消毒前后的诱变性。方法 采用彗星实验分别对泸州市南部水厂的水源水、出厂水、自来水的有机浓集物的诱变性进行检测。结果 水源水的有机浓集物的彗星实验在高剂量组(相当于原水样量500ml)才出现阳性，而出厂水、自来水的望星实验在较低剂量组(相当于原水样量100m1)呈阳性反应。结论 水源水和氯化消毒饮用水都能诱导动物细胞DNA损伤，说明水源水和氯化消毒饮用水里存在致突变物质，2者对人类健康都存在潜在的威胁，但经氯化消毒过后的饮用水其消毒产生的氯化副产物比水源水具有更强的致突变性。