Combined effect of tributyltin and benzo[a] pyrene on the levels of sex hormone and vitellogenin in female Sebastiscus marmoratus

Tributyltin（TBT）, an organometal used as an antifouling biocide, has been reported to induce masculinization of fish. Benzo [a]pyrene （BaP）, a widespread carcinogenic polycyclic aromatic hydrocarbon, has been reported that its microsomal metabolites can produce an estrogenic response when tested in vitro. This study was therefore designed to examine the potential in vivo influence of TBT, BaP and their mixture on sex hormone levels in serum of Sebastiscus marmoratus, which were given 2 separate intraperitoneally （ip） injections（a single injection every 7 d） of TBT（0.5, 1, 5 and 10 mg/kg）, BaP（0.5, 1, 5 and 10 mg/kg）, or both in combination（0.5, 1, 5 and 10 mg/kg）; control fish received olive oil vehicle only. Six days after the 2nd injection, serum samples were collected and analyzed for sex hormone levels and alkali labile protein phosphorus （ALPP）, which is related to the yolk precursor protein vitellogenin. The pollutants at all doses significantly reduced serum testosterone, estradiol and ALPP content after 2 injections compared with the corresponding controls. The reduction of the estradiol levels should be response for the decrease of the vitellogenin levels. The results in the present study suggested that aromatase seems not the major target acted by TBT and BaP in fish. This study demonstrated that TBT or BaP exposure both inhibit the reproductive potential in female Sebastiscus marmoratus. Combined effect of TBT and BaP on the serum testosterone, estradiol and ALPP was not antagonism from the anticipation.     

全氟辛烷磺酸(PFOS)对斑马鱼卵黄蛋白原mRNA水平的影响

为了研究环境低剂量全氟辛烷磺酸（perfluorooctane sulfonate,PFOS）对水生生物的内分泌干扰效应和可能的作用机制,测定了PFOS对斑马鱼（Brachydanio rerio）肝脏中卵黄蛋白原（vitellogenin,VTG）mRNA水平的影响.将斑马鱼暴露于4个PFOS的环境低剂量浓度组（0.1、1、10、100μg.L-1）中进行21d毒性试验,收集肝脏样品,提取RNA,采用荧光定量PCR（qRT-PCR）分别检测VTG1和VTG3的mRNA水平.结果表明：①PFOS暴露引起雄性斑马鱼肝脏VTG1和VTG3 mRNA水平升高,VTG1 mRNA水平升高与剂量呈正相关,在100μg.L-1暴露浓度处与对照组呈现显著性差异;VTG3的mRNA水平变化与剂量呈倒U型曲线,呈现典型的毒物刺激荷尔蒙效应,在10和100μg.L-1暴露浓度处与对照组呈现显著性差异;②PFOS暴露引起雌性斑马鱼肝脏中VTG1 mRNA水平升高,在10μg.L-1暴露浓度处与对照组呈现显著性差异,但在高浓度（10和100μg.L-1）处试验结果误差较大;VTG3 mRNA水平只在10μg.L-1暴露浓度处升高,但相比于对照组均没有显著性差异.试验结果表明PFOS暴露对斑马鱼的内分泌干扰作用明显,其毒性作用机制可能是类雌激素效应,而肝脏中VTG1和VTG3mRNA水平可能作为PFOS内分泌干扰效应评价的敏感生物标志物,但VTG1和VTG3 mRNA水平的响应曲线呈现基因亚型和性别差异.     

Determination of alkali-labile phosphoprotein phosphorus from fish plasma using the Tb^3＋-tiron complex as a fluorescence probe

A sensitive method based on the fluorescence quenching effect of the Tb^3＋-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus （ALP） released from fish plasma. The detection limit was 5.4 ng/ml （S/N=2）, and the relative standard deviation of the quenching effect （6 replicates） was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.     

Vitellogenin induction in Cyprinus carpio treated with 17beta-estradiol and 4-nonylphenol

The aim of the present study was to demonstrate induction ofvitellogenin in the common carp (Cyprinus carpio) as abiomarker for monitoring freshwater ecosystems. Sexually undifferentiated specimens of common carp were treated experimentally with 17-estradiol and increasing doses of 4-nonylphenol and levels of plasma Vtg were measured in orderto: 1) validate an ELISA assay for plasma Vtg in the common carpusing the polyclonal rabbit anti-salmon Vtg antibody AA1 (Biosense, Norway); 2) check the sensitivity of carp juvenilesin producing Vtg in response to estrogen stimulation. The grouptreated with 17-estradiol showed high induction (156%)with respect to controls, also groups treated with 4-nonylphenolshowed induction of Vtg. The group treated with 100 mg kg^-1b.wt showed an induction of 61%. A statistically significantcorrelation was found between dose and response. This preliminary study demonstrate a response to the ELISA assay for Vtg in the common carp using rabbit anti-salmon antibody AA1. With further evidence the tested biomarker might be proposed for large scale monitoring of estrogenic effects caused by pollution in urban and industrial effluents.     

Assessing the anti-estrogenic activity of sodium pentachlorophenol in primary cultures of juvenile goldfish (Carassius auratus) hepatocytes using vitellogenin as a biomarker

Both pentachlorophenol and 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） had been studied widely because of their probable anti-estrogenic activity. Sodium pentachlorophenol （PCP-Na）, as a industrial product used in many fields, usually contains a trace of TCDD. The aim of this study was to assess the anti-estrogenic effect of PCP-Na in juvenile goldfish （Gurassius auratus） hepatocyte cultures using vitellogenin （VTG） as the biomarker. The ID50 of PCP-Na was investigated and then a series of concentrations （0.001 0.5 μg/ml） of PCP-Na were evaluated to estimate the anti-estrogenic activity. Results showed that PCP-Na was cytotoxic for hepatocytes even at very low concentration 〈1.21 μg/ml, and it could not induce VTG at any concentrations tested. Since it failed to stimulate VTG production, the possibility of its anti-estrogenic effect was tested, and a well-known anti-estrogenic compound-tamoxifen was used as positive control. PCP-Na caused a reduction in VTG synthesis in juvenile goldfish （Carassius auratus） hepatocytes at concentrations 〉0.1μg/ml when co-exposure with 1μg/ml 17β-estradiol （E2）, making its anti-estrogenic activity approximately as potent as tamoxifen. Our results indicate that PCP-Na can act as negative modulators of estrogenic function in juvenile goldfish （Carassius auratus） hepatocytes.     

双酚-A对Medaka的类雌激素效应研究

利用Medaka进行了双酚-A与17β-雌二醇的短期暴露研究．研究结果发现实验室建立的流水式暴露体系可为实验提供连续、恒定的污染物暴露浓度．利用肝肉指数可灵敏响应和指示污染物暴露．双酚-A可以剂量相关的方式诱导雄性Medaka肝脏产生卵黄蛋白原，从而有效证实了其类雌激素效应．     

Endocrine disruption of water and sediment extracts in a non-radioactive dot blot/RNAse protection-assay using isolated hepatocytes of rainbow trout

Goal, Scope and Background In order to evaluate the estrogenic activity of sediments and XAD water extracts of selected sites of the catchment area of the River Neckar, a river system in Southern Germany, an integrative assessment approach was used to assess the ecological hazard potential of endocrine-disrupting compounds in sediment and water. Methods The approach is based on estrogen receptor-mediated vitellogenin synthesis induced in isolated hepatocytes of rainbow trout and quantified in a non-radioactive dot blot/RNAse protection-assay in parallel to comprehensive chemical analyses of estrogenic substances. Results and Discussion Numerous investigated extracts revealed an estrogen activity comparable to that of the positive control (1 nM 17?-estradiol corresponding to 270 ng/L in the test medium). Based on a concentration factor of 30 in the extracts and a recovery of XAD resins of approximately 80 %, 17?-estradiol equivalent concentrations between 20 and 26.7 ng/L could be calculated downstream of a sewage treatment plant (< 0.1 ng/L for a reference site). A comparison of the bioassay-derived Bio-TEQs (toxicity equivalents) and the Chem-TEQs revealed a high correlation with a Pearson coefficient of 0.85, indicating that the same ranking of the samples could be obtained with respect to the endocrine disrupting potential with both chemical and bioanalytical analysis. However, the TEQ concentrations computed from chemical analyses were significantly lower than the bioassay-derived TEQ concentrations. In fact, in none of the samples, more than 14 % of the vitellogenin-inducing potency could be attributed to the substances (steroids, alkylphenols, bisphenol A, diethylstilbestrol) analyzed. A comparison of the endocrine disrupting potential of sediments extracted by the solvents acetone and methanol revealed lower biological effects for acetone-extracted samples. Possible reasons may be a masking of endocrine effects in acetone extracts by cytotoxicity, a low extraction efficiency of the solvent acetone, or anti-estrogen potencies of some extracted sediment compounds. Using a mass balance approach, the contribution of the compounds analyzed chemically (Chem-TEQs) to the total endocrine activity (Bio-TEQs) was calculated. Based on the very low detection limits, particularly of the steroids with their high TEF factors, results revealed that a calculation of the Chem-TEQs is associated with considerable scale inaccuracy: Whereas only 7-15 % of the biological effectiveness (Bio-TEQs) could be explained by endocrine substances identified above the detection limits, the assumption of concentrations slightly below the given detection limits would result in a significant over estimation (137-197 %) of the Bio-TEQs. Even the interassay variation of the dot blot assay with different fish donors for primary hepatocyte (factor 2 - 2.5) is relatively low, when compared to the large range of the Chem-TEQ concentrations (factor 20) obtained when applying different modes of calculation. Conclusions and Outlook Overall, only a minor portion of the endocrine activity detected by bioassays could be linked to compounds identified by chemical analysis. In vitro assays for assessment of endocrine activities are useful as sensitive integrating methods that provide quantitative estimates of the total activity of particular receptor-mediated responses. Although discrepancies may also result from different bioanalytical approaches, it is overall likely that bioanalytical and not chemical analytical approaches give the correct estimate of endocrine disrupting potencies in environmental samples. As a conclusion, assessment of endocrine disruption based on chemical analysis alone does not appear sufficient and further research into the spectrum of substances with potential endocrine activity as well as into additive or even synergistic effects in complex environmental samples is urgently needed.     

壬基酚对鲫鱼(Carassius auratus)的雌激素效应研究

实验研究了环境雌激素4-壬基酚(NP)对鲫鱼(Carassiusauratus)的剂量-效应关系。雄性鲫鱼腹腔注射不同剂量的17β-雌二醇(E2)(质量分数分别为0 01,0 10,1 00mg kg(注射剂量 鱼体重,下同))和4-壬基酚(NP)(质量分数分别为1,10,50,100,200mg kg),用ELISA方法测定鲫鱼血浆中卵黄蛋白原含量。结果显示,与对照相比,在注射后14d内,0 01mg kgE2处理组和1 00mg kgNP处理组鲫鱼血浆卵黄蛋白原含量没有显著差异;其他各处理组在注射后8d,血浆卵黄蛋白原含量显著升高(P<0 05),且与暴露浓度呈依赖关系;注射后14d,10,50,100和200mg kgNP处理组的含量依次是对照组的10,59,340和435倍。研究表明,NP对鲫鱼具有明显的雌激素效应。      

实时定量RT-PCR方法检测雌二醇诱导原代培养青鳉鱼肝细胞的基因表达

利用原代培养的雄性青鳉鱼肝细胞进行了相同雌二醇(E2)浓度(100 nmol·L-1)下的不同时间(2、4、6、8d)和相同暴露时间(4d)下的不同浓度(1、10、100、1000、10000 nmol·L-1)的雌二醇(E2)实验,采用实时定量RT-PCR方法对青鳉鱼VTG-Ⅰ、VTG-Ⅱ、CHG-H、CHG-L和ERα的基因表达进行了研究,结果表明,VTG-Ⅰ、VTG-Ⅱ、CHG-H、CHG-L和ERα的基因表达与E2的暴露浓度呈现很好的剂量-效应关系,且1nmol·L-1E2暴露就能显著诱导肝细胞内VTG-Ⅰ、VTG-Ⅱ、CHG-H、CHG-L和ERα的基因表达.作为生物监测雌激素活性的in vitro方法,实时定量RT-PCR的灵敏度高于其它已经发表的方法.因此,实时定量RT-PCB方法与原代培养青鳉鱼肝细胞相结合的方法在环境雌激素效应的评价上具有很大地应用潜力.     

Effects of p,p′-DDE exposure on gonadal development and gene expression in Japanese medaka （Oryzias latipes）

Although 1,1-dichloro-2,2-bis（p-chlorophenyl）-ethylene （p,p′-DDE）, the major and most persistent metabolite of dichlorodiphenyltrichloroethane （DDT）, was continually detected in wild fishes that showed abnormal gonad development such as intersex, little is known about the impact of p,p′-DDE exposure on gonad development in fishes. To survey the effects of p,p′-DDE on gonadal development and gene expressions, male juvenile （20-d post hatch） Japanese medaka （Oryzias latipes） was exposed to 1, 5, 20, and 100 μg/L p,p′-DDE for two months. Increased hepatosomatic index （HSI） and decreased gonadosomatic index （GSI） were found in the p,p′-DDE-treated groups. Intersex was found in 100 μg/L p,p′-DDE exposure group, as well as 100 ng/L 17α-ethynylestradiol （EE2） group. By quantitative real-time RT-PCR, it was found that gene expressions of vitellogenins （VTG-1, VTG-2）, choriogenins （CHG-H, CHG-L）, and estrogen receptor α （ER-α） in the liver of the fish were significantly up-regulated by p,p′-DDE exposure. VTG-1 and VTG- 2 were recommended as the preferred biomarker for assessing anti-androgenic p,p′-DDE because they were the highest up-regulated among the genes and showed good dose-response relationship. The up-regulated ER-α suggested that a potential synergetic effect would occur when p,p′-DDE coexists with other ER-α-binding endocrine-disrupting chemicals （EDCs）.