典型天然雌激素MCF-7细胞增殖实验的条件优化研究

目的 研究不同试验条件对MCF-7增殖实验检测雌激素效应的影响.方法 选用两种来源(ATCC和某肿瘤医院)的MCF-7细胞,研究不同浓度、不同培养时间、不同溶剂、不同培养液条件下MCF-7细胞对17B-雌二醇(E2)、乙炔雌二醇(EE2)、雌三醇(E3)、雌酮(E1)的反应,以优化MCF-7增殖实验条件.结果 不同E2...     

二氯二苯二氯乙烯的类雌激素活性的实验研究

目的研究二氯二苯二氯乙烯(DDE)的类雌激素活性对人乳腺癌细胞株MCF-7增殖的影响。方法采用了体外MCF-7细胞增殖试验检测了DDE(3×10-5、3×10-6、3×10-7、3×10-8、3×10-9 mol/L)的类雌激素活性,并用生长曲线分析对其作用机制进行了初步探讨。结果3×10-6 mol/LDDE染毒组吸光度值高于溶剂对照组且MST-7细胞增殖的生长曲线与10-9 mol/L17β-雌二醇染毒组相似。结论DDE具有刺激MCF-7细胞增殖的类雌激素活性,其机制可能与17β-雌二醇相同。     

氯化汞雌激素样作用及其机制的实验研究

目的评价氯化汞(HgCl2)的雌激素样作用及其作用机制.方法从体内和体外两个水平,观察不同浓度的HgCl2对MCF-7人乳腺癌细胞增殖作用、去卵巢SD大鼠子宫的诱导增生作用、过氧化物活力的变化及对雌激素结合雌激素受体的影响.结果 10-6～10-9 mol/L的HgCl2均可引起MCF-7人乳腺癌细胞增殖,10-7 mol/L的HgCl2使MCF-7人乳腺癌细胞增殖达最大.每天0.04、0.20及1.00 mg/kg连续3 d腹腔注射HgCl2能刺激大鼠子宫增生和过氧化物酶活力增加;但HgCl2不影响雌二醇(E2)与雌激素受体结合.结论 HgCl2可能通过雌激素受体介导而表现出雌激素样作用,但不与E2竞争结合雌激素受体.     

镉通过类雌激素样作用促进人乳腺癌MCF-7细胞增殖

[目的]研究镉对人乳腺癌MCF-7细胞增殖及雌激素受体(estrogen receptor,ER)蛋白表达水平的影响及其可能机制。[方法]用1×10~(-6)、1×10~(-7)、1×10~(-8)、1×10~(-9)、1×10~(-10)、1×10~(-11) mol/L的17β-雌二醇(后称雌激素)培养不同来源的A、B两株MCF-7细胞4 d,应用CCK8法筛选敏感细胞株并确定雌激素促细胞增殖作用最强的浓度。用1×10~(-6)、1×10~(-7)、1×10~(-8)、1×10~(-9)、10~(-10)、1×10~(-11) mol/L的镉溶液染毒敏感细胞株,确定镉促进细胞增殖的最大浓度。设置阴性对照组(去雌激素培养液)、实验组(1×10~(-8) mol/L镉)、阳性对照组(1×10~(-9) mol/L雌激素),通过克隆形成实验检测镉对MCF-7细胞集落形成能力的影响。利用ER抑制剂(ICI182780)初步探讨镉致细胞增殖的可能机制,增设实验抑制剂组(1×10~(-8) mol/L镉+1×10~(-7) mol/L ER抑制剂)、阳性抑制剂组(1×10~(-9) mol/L雌激素+1×10~(-7) mol/L ER抑制剂),通过CCK8法、流式细胞术和Western blot分别检测各组的细胞增殖率、细胞周期S期比例和ER表达水平。[结果]实验所用B株MCF-7细胞为雌激素敏感细胞株,且当雌激素浓度为1×10~(-9) mol/L时,对MCF-7细胞的促进增殖作用[(203.55±36.65)%]最大(P0.001)。与阴性对照组(100%)相比,1×10~(-8)~1×10~(-10) mol/L镉溶液可促进MCF-7细胞增殖[(163.78±31.90)%~(176.88±10.06)%](P0.001)。1×10~(-8) mol/L镉可促进细胞集落形成(P0.05),增加细胞S期比例(P0.001),提高细胞ER蛋白表达水平(P0.001)。与实验组相比,加入ER抑制剂能够抑制镉对MCF-7细胞的促增殖作用[由(135.17±23.96)%降至(107.66±7.64)%](P0.01),抑制镉诱导的细胞S期比例增加[由(24.17±0.53)%降至(12.36±0.43)%](P0.001),拮抗镉诱导的ER表达增加[由(56.19±3.67)%降至(38.84±1.04)%](P0.05)。[结论]镉可以促进人乳腺癌细胞MCF-7增殖和ER表达,这种作用可被ER抑制剂所抑制,提示镉可能具有雌激素样作用。     

氯氰菊酯对人乳腺癌细胞MCF-7增殖的影响

目的通过观察氯氰菊酯(cypermethrin,CYP)对人乳腺癌细胞株MCF-7细胞增值的影响,探讨氯氰菊酯促进细胞增殖的生物学机制。方法 MCF-7细胞在加受试物前7 d改为无酚红完全培养基,以耗尽细胞内储存的雌激素。实验设为6组:无水乙醇(EtOH)溶剂对照组、雌二醇(E2)雌激素对照组、他莫西芬(TAM)抗雌激素对照组及氯氰菊酯3个浓度梯度(1.0×10-7mol/L、1.0×10-8mol/L、1.0×10-9mol/L)实验组。采用CCK8法检测各组细胞的增值情况,采用Hoechst 33342和Propidium Iodide双染荧光显微镜观察氯氰菊酯对雌激素耗尽诱导细胞凋亡作用影响。结果第4、5天细胞增殖实验,与EtOH溶剂对照组相比,除TAM抗雌激素组与EtOH溶剂对照组细胞增殖活力低外,其他4组细胞增殖活力低,依次为E2雌激素对照组、氯氰菊酯1.0×10-7mol/L组、氯氰菊酯1.0×10-8mol/L组、氯氰菊酯1.0×10-9mol/L组,P均<0.05;第3天与EtOH溶剂对照组比,E2雌激素对照组MCF-7细胞增殖活力高,P<0.05,第2天差异无统计学意义。荧光显微镜下见,EtOH溶剂对照组相比和TAM抗雌激素组细胞的凋亡作用明显,后者强于前者,而E2雌激素对照组、氯氰菊酯1.0×10-7mol/L组、氯氰菊酯1.0×10-8mol/L组、氯氰菊酯1.0×10-9mol/L组对细胞凋亡作用不明显。结论氯氰菊酯有可能是通过抑制乳腺癌细胞凋亡而发挥其促进细胞增殖作用的。     

17β-雌二醇对乳腺癌细胞的增殖作用及其对雌激素受体相关受体的调控作用

目的 研究17β-雌二醇(17β-E2)对乳腺癌细胞MCF-7增殖作用及其对雌激素受体相关受体(ERRα)的调控作用.方法 分别用10-12、10-11、10-10、10-9、10-8、10-7、10-6mol/L的17β-E2作用于MCF-7细胞24h,以DMSO作为溶剂对照,采用MTr法测定细胞增殖情况.分别用10-10、10-9、10-8、10-7mol/L 17β-E2作用于MCF-7细胞24 h后,采用RT-PCR法检测ERRa表达水平的变化.结果 10-12、10-11、10-10、10-9、10-8、10-7mol/L剂量17β-E2作用于MCF-7细胞24h后,对细胞的增殖率分别为112.09%,122.09%,123.42%,120.46%,124.60%,109.23%,均能促进细胞生长,并且随着受试物浓度的增加,细胞增殖效应增加,在10-6mol/L剂量时达到最大,而10-6 mol/L剂量的17β-E2作用于MCF-7细胞24 h后,其增殖率为67.97%,对细胞生长的产生抑制.10-10、10-9、10-8、10-7 mol/L 17β-E2作用于MCF-7细胞24 h后,ERRa的表达水平上调.结论 一定剂量范围内17β-雌二醇对MCF-7细胞有增殖作用,并且可以上调ERRα表达,提示ERRα在调节乳腺癌细胞生长中可能发挥着重要作用.     

五氯酚的类雌激素效应初探

目的 研究五氯酚(PCP)的类雌激素活力对MCF-7细胞增殖的影响.方法 采用离体MCF-7细胞增殖试验检测PCP的类雌激素活力,并用生长曲线对其作用机制进行初步探讨.结果 PCP在7.5×10-7～7.5×10-5 mol/L有明显刺激MCF-7细胞增殖的类雌激素活力,在7.5×10-6 mol/L增殖效应最大.细胞周期分析PCP组细胞增殖指教均明显高于溶剂对照组.结论 PCP在7.5×10-7 ～7.5×10-5 mol/L具有刺激MCF-7细胞增殖的类雌激素活力.     

金雀异黄素对DDT类农药诱导乳腺癌细胞增殖效应的抑制作用

目的 探讨金雀异黄素( genistein,Gen)与DDT类有机氯农药(o,p’-DDT,p,p’-DDT和p,p’-DDE)联合作用对乳腺癌(MCF-7)细胞增殖效应的抑制作用.方法 取处于对数生长期的MCF-7细胞,分别加入含100 μmol/Gen 和0.1 μmol/L o,p’-DDT、0.1μmol/Lp,p’-DDT、10 μmol/Lp,p’-DDE单独作用组以及100 μmol/L Gen +0.1 μmol/Lo,p’-DDT、100 μmol/L Gen +0.1 μmol/Lp,p’-DDT、100 μmol/L Gen+10 μmol/LP,p’-DDE的培养基,并设溶剂对照(0.1％无水乙醇)组和雌激素对照(10-3 μmol/L雌二醇)组.培养48 h后,采用噻唑蓝(MTT)实验检测MCF-7细胞的增殖情况.结果 与溶剂对照相比,100 μmol/L的Gen作用组MCF-7细胞的增殖率下降,0.1 μmo/L的o,p’-DDT作用组MCF-7细胞的增殖率升高,差异均有统计学意义(P＜0.05).与相应DDT类农药单独组相比,Gen+o,p’-DDT、Gen+p,p’-DDT联合作用组MCF-7细胞的增殖率明显下降,差异均有统计学意义(P＜0.05).结论 Gen能够抑制DDT类农药诱导的MCF-7细胞增殖效应.     

五溴联苯醚对MCF-7细胞c-myc和p53 mRNA及蛋白表达的影响

[目的]观察五溴联苯醚(5-BDEs)对乳腺癌细胞MCF-7c-myc和p53 mRNA及蛋白表达的影响,以探讨5-BDEs雌激素活性的分子生物学作用机制.[方法]将MCF-7细胞在DMEM培养液中进行常规传代培养.采用MTT比色法观察5-BDEs对MCF-7细胞增殖的影响,用逆转录聚合酶链反应(RT-PCR)技术和免疫组化方法检测5-BDEs对c-myc原癌基因和p53抑癌基因表达的影响.[结果]与溶剂对照组相比,在1×(10-4～10-8)mol/L浓度范围内,5-BDEs与1×10-8mol/L雌二醇作用类似可显著促进MCF-7细胞增殖(P<0.05),并表现出良好的剂量-效应和时间-效应关系.RT-PCR及免疫组化结果显示,1×(10-4～10-8)mol/L的5-BDEs可促进MCF-7 c-myc和抑制p53 mRNA及蛋白表达.[结论]5-BDEs具有雌激素样生物活性,可促进雌激素反应性肿瘤细胞MCF-7增殖,这一效应主要是通过影响c-myc mRNA和蛋白质的表达而实现的.     

甘草次酸和甘草甜素雌激素样作用研究

目的探讨甘草次酸和甘草甜素两种三萜类成分的雌激素样作用,为开发新的活性植物雌激素成分奠定基础。方法采用溴化噻唑蓝四氮唑(MTT)法观察两种三萜成分对雌激素受体(ER)阳性人乳腺癌细胞系MCF-7细胞增殖的影响,Western Blot法和半定量RT-PCR法测定两种三萜成分对MCF-7细胞ER亚型蛋白及mRNA表达的影响。实验数据(±s)采用SPSS 23.0进行统计分析,基于单因素方差分析的方法,组间比较用SNK法和LSD法。结果 100μmol/L和10μmol/L甘草甜素能够抑制MCF-7细胞的增殖;100μmol/L、1μmol/L和10-2μmol/L甘草次酸能够促进MCF-7细胞的增殖;10μmol/L的甘草次酸可诱导MCF-7细胞ERα、ERβ蛋白表达,mRNA表达均显著升高(P0.05);加入1×10~(-7)μmol/L的雌激素受体拮抗剂ICI182780均能抑制甘草次酸的以上效应。结论甘草次酸具有促进MCF-7细胞增殖作用,其作用机制可能是上调MCF-7细胞ERα、ERβ基因和蛋白的表达,显示有雌激素样作用,且其雌激素样作用可能是ER介导产生的。抗雌激素样作用的甘草甜素的作用机制还有待于深入研究。     

Predictability of Copper, Irgarol, and Diuron Combined Effects on Sea Urchin Paracentrotus lividus

The aim of this work was to investigate the mixture toxicity of Irgarol (2-methylthio-4-t-butylamino-6-cyclopropylamino-s-triazine), Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), and copper upon the sea urchin Paracentrotus lividus and to compare the observed data with the predictions derived from approaches of Concentration Addition (CA) and Independent Action (IA). Copper spermiotoxicity was more sensitive (EC50 = 0.018 mg/L) than embryotoxicity (EC50 = 0.046 mg/L). The offspring malformations were mainly P1 type (skeletal alterations) in both cases, probably because copper competes to fix Ca²⁺. Irgarol and Diuron toxicity has been previously investigated. EC50 mixture embryotoxicity showed an EC50 of 1.79 mg/L, whereas spermiotoxicity mixture effects were lower than 11%. Both CA and IA modeling approaches failed to predict accurately mixture toxicity. For embryotoxicity, the IA model overestimated the mixture toxicity at effect levels of <80%. CA does not represent the worst-case approach showing values lower than IA (embryotoxicity) or similar (spermiotoxicity).     

Two-Stage Prediction on Effects of Mixtures Containing Phenolic Compounds and Heavy Metals on <Emphasis Type="Italic">Vibrio qinghaiensis</Emphasis> sp. Q67

Two-stage prediction (TSP) model had been developed to predict toxicities of mixtures containing complex components, but its prediction power need to be further validated. Six phenolic compounds and six heavy metals were selected as mixture components. One mixture (M1) was built with equivalent-effect concentration ratio and four mixtures (M2–M5) were designed with fixed concentration ratio. In M1–M5, the toxicities were well predicted by TSP model, while CA overestimated and IA underestimated the toxicities. In M1–M5, compared with the actual mixture EC50 value, the prediction errors of TSP model (13.9%, 17.9%, 19.2%, and 17.3% and 15.8%, respectively) were significantly lower than those in the CA (higher than 30%) and IA models (20.9%, 33.0%, 20.6%, 21.8% and 12.5%, respectively). Thus, the TSP model performed better than the CA and IA model.     

Mixture toxicity of three toxicants with similar and dissimilar modes of action to Daphnia magna

Mixture toxicity of similar- and dissimilar-acting toxicants can be predicted by the models concentration addition (CA) and independent action (IA) using single substance toxicity data. Knowledge of the toxicants mode of action is thus required in order to use the models. In order to test the predictive capability of the models, we conducted Daphnia magna 48 h immobilization experiments with three toxicants with known modes of action (dimethoate, pirimicarb and linear alkyl benzene sulfonate) singly, and in binary and ternary mixtures. Our results indicate that CA and IA predict binary mixtures of similar- and dissimilar-acting toxicants equally well. CA and IA also equally predicted the ternary mixture consisting of both similar- and dissimilar-acting chemicals. The paper discusses the concept of mode of action and the implications the definition of mode of action has on the choice of reference model for mixture toxicity studies.     

Is mixture toxicity measured on a biomarker indicative of what happens on a population level? A study with Lemna minor

For plants, pigment content has shown to be a remarkably consistent biomarker across chemicals with different modes of action. In this study, we evaluated the use of pigment content as endpoint in binary mixture toxicity studies compared to three growth endpoints on the floating plant Lemna minor. Six binary combinations of six herbicides with different mode of action were used. Data were tested against both the concentration addition (CA) and independent action (IA) reference models. For CA, two statistical approaches were used. The study showed that for some herbicide combinations the mixture toxicity measured on pigment content did not reflect the results measured on plant population growth, emphasizing the importance of measuring growth in parallel with biomarkers. CA explained the data just as well as IA, and the two different statistical models used to test the data in relation to CA showed very similar results.     

Effects of Single and Mixed Estrogens on Single and Combined Cultures of D. subspicatus and P. subcapitata

This paper investigates the effect of estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) individually and mixed at equal proportions (1:1:1) on Desmodesmus subspicatus and Pseudokirchneriella subcapitata in single and combined cultures (S+) at different exposure times based on algal growth (in vivo chlorophyll fluorescence and cell counting) and coenobium formation. EE2 and E2 were more toxic to individual and combined (S+) cultures than was E1. The frequency of coenobium formation by D. subspicatus increased significantly for all estrogens and all concentrations. After 96 h, D. subspicatus prevailed in S+. The results of the exposure to E+ suggested a less-than-additive effect on D. subspicatus and S+ and additive effect on P. subcapitata. Toxic effects occurred for both species exposed to E+ with individual estrogen concentrations below the NOEC of each species. Assays must include changes in response due to the exposure of more than one species to more than one estrogen.     

Estimating the combined toxicity by two-step prediction model on the complicated chemical mixtures from wastewater treatment plant effluents

The toxicities of chemical mixtures containing 10 compounds, detected in wastewater treatment plant (WWTP) effluents, were investigated using Daphnia magna in a two-step prediction (TSP) model. The 10 chemicals determined by gas chromatography/ mass spectrometry in WWTP effluents included three groups: Three acetylcholinesterase inhibitors, six narcosis inhibitors, and one seedling root inhibitor. In the first step, a concentration addition (CA) model was used to predict the mixture toxicities for the three component groups with similar modes of action; in the second step, an independent action (IA) model was used for the newly developed concentration-response curves from the three CA predictions. The CA predictions did not show a statistically significant difference from the observed results with respect to the three groups of chemicals, whereas the IA model did not conform to the experimental results. Therefore, the concentration-response curves obtained from the mixture toxicity tests in each group was considered as a single curve and applied in the next step of the mixture toxicity prediction. However, the observed toxicity of the 10-chemical mixture showed large differences from the results of the IA and CA model predictions, whereas the TSP model predicted the toxicity well and with statistical significance (p = 0.0501, n = 17). This suggests that the TSP model would provide a valid prediction for a randomly selected chemical mixture having various modes of action if the concentration-response function for an individual component is obtained.     

Joint toxicity of chlorpyrifos and esfenvalerate to fathead minnows and midge larvae

The joint toxicity of esfenvalerate and chlorpyrifos to the fathead minnow (Pimephales promelas) and the aquatic midge larvae (Chironomus tentans) was determined using comparisons to independent action (IA) and concentration addition (CA) models. Equipotent mixtures of the two insecticides were used for initial testing of both species. A secondary study evaluating the effects of low-level chlorpyrifos on esfenvalerate toxicity also was performed. For fathead minnows, the equipotent mixture and the low-level chlorpyrifos exposure resulted in toxicity greater than that predicted by either model. In both studies, however, the observed concentrations causing 50% effect (EC50) were within a factor of two of the values predicted by the CA model. For midges, the observed EC50s were similar to the values predicted by the CA model, whereas the IA model slightly underpredicted toxicity. The observance of toxicity that was not predicted by either of the conceptual models tested likely results from a toxicokinetic interaction occurring between the toxicants.     

Different stages in the temporal course of estrogen treatment produce opposite effects on voluntary alcohol consumption in male rats.

The purpose of this study was to examine alcohol consumption in different stages of the estrogen treatment. Three groups of castrated male Wistar rats were used. One group was treated with 5 microg of estradiol benzoate (E) per day per rat for 6 days and oil from days 7 to 12 (EO group). The second group was treated with oil for 6 days and E from days 7 to 12 (OE) and the third with E for 12 consecutive days (EE). The three groups were exposed to a choice of both water and ethanol (10%) before treatment (PreT), from days 7 to 12 of the oil or the E treatment (T2), and during 6 additional days in the post-treatment period (PosT). Alcohol was not available from days 1 to 6 of the oil or the E treatment (T1). Alcohol consumption in the EO group during T2 was higher than in PreT and PosT periods and all periods in the other two groups. In contrast, alcohol consumption during T2 was significantly lower than during the PreT of the OE group and T2 of the EE group. At the same time in the EE group, alcohol intake in the T2 was higher than in the PreT and the PosT periods. These results reveal the opposite effects of estrogen treatment on alcohol consumption, which apparently depended on the physiological conditions produced by the temporal course of hormone treatment.     

Development of a short-term reproductive endocrine bioassay using steroid hormone and vitellogenin end points in the estuarine mummichog (Fundulus heteroclitus)

We have developed a short-term gonadal recrudescence test with the estuarine mummichog (Fundulus heteroclitus) and determined endocrine end points sensitive to a strong estrogen agonist (ethynylestradiol; EE2) and an antiestrogen (ZM 189,154; ZM) at concentrations of 0 to 1,000 ng/L in three separate experiments. A protocol was developed to ensure a year-round supply of recrudescing fish. A protocol for determining steroid production (testosterone and 11-ketotestosterone [11-KT] in incubated testes tissue and testosterone and 17-estradiol [E2] in incubated prematurational follicles) was optimized. Recrudescing fish (males, gonadosomatic index = 2%; females = 10%) were exposed to graded doses of EE2 or ZM for 7 to 15 d using a static daily-renewal protocol. At high EE2 (>250 ng/L), the effect on males was depression of androgen steroidogenesis and plasma steroid levels. In females, high EE2 depressed gonadal production and circulating E2 levels; however, EE2 concentrations <100 ng/L caused increased gonadal production and plasma E2. Low ZM (<100 ng/L) had little effect on male and female fish, while higher concentrations (>250 ng/L) increased E2 and 11-KT production while decreasing plasma 11-KT and E2 (1,000 ng/L only). Male and female plasma vitellogenin responded in a concentration-dependent fashion to EE2 with no effect by ZM. The low observable effect concentrations for the endocrine parameters were 1 ng/L for EE2 and 250 ng/L for ZM. The bioassay and results encompassing the environmentally relevant exposure range (1-100 ng/L) will be useful for assessing effects of endocrine-active contaminants in estuarine environments.     

Chemical and biological analysis of endocrine-disrupting hormones and estrogenic activity in an advanced sewage treatment plant

The steroid hormones estrone (E(1)), 17beta-estradiol (E(2)), estriol (E(3)), 17alpha-ethinylestradiol (EE(2)), and their conjugated forms were surveyed throughout an advanced sewage treatment plant (STP). The estrogen concentrations in water and sludge samples, collected in October 2004 and April 2005, were determined by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Simultaneously, the estrogenic activity was quantified using estrogen-responsive reporter cell lines (MELN) to investigate the behavior of overall estrogenic compounds. The estrogen concentrations in the inlet ranged from 200 to 500 ng/L, with the contribution of conjugated forms being higher than 50%. The major estrogens in influent were E(1) and E(3). The estrogenic activity was between 25 and 130 ng/L of E(2) equivalents (EEQs). Estrogen concentrations and estrogenicity measured in the inlet and in primary treated sewage were similar, showing a weak impact of primary treatment on hormone removal. In contrast, both estrogen concentration and estrogenicity decreased during biological treatment, with high removal efficiencies (>90%). Estrone, E(2), and EE(2) persisted in the treated water below 10 ng/L, whereas the estrogenicity was lower than 5 ng/L of EEQs. Estrogen mass flux in the effluent and sludge represented less than 2 and 4%, respectively, of the inlet. Consequently, the fraction of estrogens sorbed into the sludge was very small, and biodegradation was the main vehicle for estrogen elimination. This dual approach, comparing chemical and biological analysis, allowed us to confirm that most of the estrogenic activity occurring in this STP, which receives mainly domestic sewage, resulted from sex hormones.