对特辛基苯酚干扰雌激素受体作用的分子基础及其对ERβ亚型选择性结合的理论研究

对特辛基苯酚(4-tert-octylphenol,PTOP)是一种环境内分泌干扰物。已有研究发现虽然其能够直接与雌激素受体(estrogen receptor,ER)的两种亚型(ERα,ERβ)结合并产生干扰效应,但其结合能力却各不相同,PTOP对ERβ表现出更强的结合活性。为了探究PTOP与ER结合的分子机制及其对ER两种亚型的选择性机制,本文采用分子动力学模拟对PTOP-ER复合物进行了研究,并利用MM-GBSA方法计算了结合自由能。结果表明,范德华作用是维持PTOP与ER结合的主要驱动力;而极性相互作用的差异是导致PTOP对ERα和ERβ产生选择性结合的重要因素,PTOP与ERα之间的极性溶剂化作用阻碍了两者的结合。将PTOP与ER的天然底物雌二醇进行比较,发现PTOP与ER口袋之间缺乏氢键稳定二者结合,因此PTOP的结合活性较低。计算模拟亦指出了PTOP结合过程中发挥重要作用的关键氨基酸。以上计算结果将有助于我们进一步理解PTOP影响ER介导生理过程的干扰机制。     

自剪切及活性相关位点的组合突变提高头孢菌素C酰化酶水解活力

7-氨基头孢烷酸是合成头孢类抗生素的重要中间体,利用头孢菌素C酰化酶直接催化头孢菌素C获得7-氨基头孢烷酸的一步酶法与其它方法相比更加经济和环保,其关键是获得高活性的头孢菌素C酰化酶.本文以来源于Pseudomonas sp.KAC-1的Ⅰ类头孢菌素C酰化酶(CPCase-kac)蛋白序列为模版,对其基因进行全局优化设计,人工合成目的基因,并克隆至表达载体p ET28b,实现了CPCase-kac的高效表达.但是重组蛋白CPCase-kac表达活性较低,且自剪切不彻底,表达蛋白中存在前体形式的CPCase-kac,而头孢菌素C酰化酶的自剪切和活性往往相关.对CPCase-kac分子中影响活性的Y150、Q220、F347氨基酸位点进行饱和突变发现,活力提高的突变体Q220W、Q220G其第二次自剪切明显降低,而活力提高的突变体Y150R、Y150N、Y150H、Y150W及F347H、F347Y,自剪切却没有显著影响.将这些突变进行组合,最终得到了比活力提高了8.3倍的突变体Y150W/Q220G/F347Y.虽然Y150W/Q220G/F347Y突变体活性显著提高,但是其也具有Q220G较差的第二次剪切的特性.同时发现,外部条件改变可以影响CPCase前体或α’亚基的继续自剪切.这些发现为进一步提高CPCase-kac活性及应用打下了基础.     

BDE-28对斑马鱼核受体介导的内分泌干扰效应研究

2,4,4’-三溴联苯醚(BDE-28)在环境中普遍存在,且在长江流域多种水生生物中检出。目前,国内外对高溴代PBDEs(如BDE-47、BDE-99等)的水生脊椎动物内分泌干扰效应报道较多,而BDE-28的有关研究则相对较少。将斑马鱼胚胎暴露于2、20和200μg·L~(-1)的BDE-28后,借助q-RT-PCR方法对幼鱼8个重要受体包括雄激素受体(AR)、甲状腺激素受体(TR)、芳香烃受体(AhR)、雌激素受体(ER)、糖皮质激素受体(GR)、孕烷X受体(PXR)、盐皮质激素受体(MR)和过氧化物酶体增殖物激活受体(PPAR)相关基因的转录水平进行了研究。结果表明,BDE-28暴露可导致AR、TR和Ah R的基因下调,其中核心受体AR和TR在低中高3种浓度下的下调倍数分别为3.03、2.64、10.10和2.21、2.18、2.31,芳香烃受体基因2(ahr2)在暴露于2和20μg·L~(-1)的BDE-28后,下调倍数分别为12.65和9.23,而雌激素受体(er1)基因在低中高浓度显著上调,上调倍数分别为12.29、12.67和15.87,雌激素受体(er2a)基因在2和20μg·L~(-1)BDE-28下的上调倍数分别为10.83和17.19。进一步采用分子对接和分子动力学模拟的方法研究BDE-28与AR、TR、Ah R和ER之间的相互作用。结果显示,BDE-28与这些受体通过疏水和氢键等相互作用稳定结合,动力学模拟后骨架原子的均方根偏差(RMSD)在5 ns后较稳定。由此可知,BDE-28通过AR、TR、Ah R和ER受体介导产生内分泌干扰效应。     

酵母双杂交技术构建重组人雌激素受体基因酵母

应用酵母双杂交技术构建重组人雌激素受体基因酵母,用以检测类/抗雌激素化合物和环境样品的类/抗雌激素活性.采用多聚酶链反应(PCR)法扩增人雌激素受体(hER)基因,构建诱饵质粒pGBKT7-ER LBD;分别提取并纯化两种含有ER共激活因子基因的靶质粒pGAD424-GRIP1和pGAD424-SRC1;诱饵质粒和靶质粒同时转染酵母细胞Y187,于营养缺陷型培养基(SD/-Trp/-Leu)上筛选阳性菌落,分别构建两种ER双杂交酵母ER+GRIP1和ER+SRC1.考察双杂交酵母与不同激素:17β-雌二醇(E2)、二氢睾酮(DHT)、孕酮(PG)以及三碘甲状腺原氨酸(T3)的结合情况,并考察了雌激素受体拮抗剂4-羟基他莫昔芬(4-OHT)与酵母的相互作用.结果表明:ER+GRIP1和ER+SRC1酵母均能够专一性的和E2结合,并存在显著的剂量-效应关系,E2对ER+GRIP1和ER+SRC1酵母β-半乳糖苷酶活性诱导的EC50值分别为7.3×10-11mol·L-1和1.5×10-10mol·L-1,其中ER+GRIP1酵母细胞诱导产生的酶活性值明显高于ER+SRC1酵母细胞.4-OHT能够抑制E2诱导ER+GRIP1酵母细胞产生的酶活性,并存在显著的剂量-效应关系,IC50值为1.0×10-7mol·L-1.表明重组人雌激素受体基因酵母可以用于检测化合物和环境样品的类/抗雌激素活性.     

多点突变提高DhaA对芥子气的活性和热稳定性

为提高烷基卤脱卤酶Dha A对芥子气的活性和热稳定性,采用Autodock软件计算Dha A突变前后与芥子气的结合情况,利用重叠延伸PCR和DNA无缝拼接结合的方法,改变Dha A活性空腔进出口通道的大小,构建包含5个位点的Dha A突变体(Ile135Phe+Cys176Tyr+Val245Phe+Leu246Ile+Tyr273Phe);将Dha A及其突变体构建在p ET-28a载体上后,在Escherichia coli BL21(DE3)中进行表达,比较纯化后的野生型与突变体在酶学性质方面的变化情况.Autodock分子对接结果显示,突变后的Dha A与芥子气的结合能、结合效率和抑制常数均小于野生酶,突变体对芥子气的比活性提高了1.4倍,对10 mg/m L芥子气的降解率提高了近20%.热稳定性实验发现,Dha A突变体在50℃水浴1 h后残余酶活为76%,比野生型Dha A提高了19%.Dha A突变体的Tm值为56℃,比野生型Dha A提高了6℃.综上表明改变Dha A活性空腔内的进出口通道可以提高Dha A的热稳定性和对芥子气的催化活性.     

重组植酸酶appAM8的异源表达与性质比较

为比较在不同宿主中表达的大肠杆菌植酸酶突变体app AM8的性质,将app AM8基因分别在大肠杆菌BL21(DE3)和毕赤酵母GS115中进行表达,对纯化后的植酸酶进行酶学性质分析与比较.结果显示:(1)在大肠杆菌中表达的app AM8(E-app AM8)和酵母中表达的app AM8(P-app AM8)的最适p H值均为4.5,最适反应温度均为65℃,与野生型的app A的最适p H无明显区别,比app A的最适温度高了5℃;(2)经SDS-PAGE检测,E-app AM8分子量(Mr)大小为47×103,而P-app AM8为53×103左右,由于在酵母中的糖基化修饰电泳显示为两条带;(3)在60-80℃之间,E-app AM8的热稳定性性明显下降,而且在70℃处理15 min后,E-app AM8残余活性仅为4%,而P-app AM8仍保留50%的残余活性,表明酵母表达的植酸酶经过糖基化修饰后提高了酶的热稳定性.(4)E-app AM8的Km=0.245 mmol/L,Vmax=3196 U/mg,P-app AM8的Km=0.36 mmol/L,Vmax=3333 U/mg.本研究表明,糖基化修饰对植酸酶app AM8的热稳定性起着重要作用,8个位点的突变对该植酸酶的稳定性也起到了一定作用.     

石墨烯与抑癌基因p53 DNA片段相互作用的分子模拟与光谱学验证

石墨烯(graphene,G)及其衍生物由于具有独特的理化性质,被广泛应用于能源、生物医学等领域,但尚缺乏其对生物体和环境潜在危害的研究。采用分子动力学模拟并结合光谱学方法(紫外可见吸收光谱、紫外变温实验及荧光光谱),分析了石墨烯与抑癌基因p53启动子区DNA片段(p53-DNA)间的相互作用,并探讨了相关作用机制。石墨烯的部分芳香环与p53-DNA碱基的芳香环之间存在π-π堆积作用,两者可以通过嵌插作用进行结合,同时还通过沟槽作用进一步结合。光谱实验进一步证实,在石墨烯作用下,p53-DNA的熔点(Tm)值升高,EB-DNA体系发生静态荧光淬灭,说明石墨烯能与p53-DNA结合;同时,p53-DNA与石墨烯结合后在260 nm处的吸光度升高,说明石墨烯对p53-DNA的双螺旋结构具有一定的破坏作用。上述研究结果从分子水平上分析了石墨烯与p53-DNA间的相互作用机制,有助于进一步阐明石墨烯的毒性作用机理。     

酵母双杂交技术构建稀有鮈鲫不同雌激素受体亚型的重组荧光酵母

目前关于环境雌激素(environmental estrogens,EEs)的效应评估大多数是基于人的雌激素受体(estrogen receptor,ER)的离体检测,缺乏EEs对鱼类ER影响的研究。本研究利用酵母双杂交技术构建模式生物稀有鮈鲫不同ER亚型重组荧光双杂交酵母,用以快速检测EEs的雌激素活性并研究不同ER亚型对17β-雌二醇(E_2)敏感度的差异。采用反转录多聚酶链反应法获取稀有鮈鲫ERα、ERβ1和ERβ2的配体结合域(LBD)序列,构建并鉴别诱饵质粒pGBKT7-ERα-LBD、p GBKT7-ERβ1-LBD和pGBKT7-ERβ2-LBD。将诱饵质粒和猎物质粒pGAD424-GRIP1同时转化荧光酵母Y187-Luc,分别构建3株稀有鮈鲫ERα-GRIP1、ERβ1-GRIP1和ERβ2-GRIP1重组荧光双杂交酵母,并考察了E_2、双氢睾酮(DHT)、9-顺维甲酸(9-cis RA)、三碘甲状腺原氨酸(T_3)和孕酮(PG)对荧光素酶的诱导情况。结果显示:ERα-GRIP1、ERβ1-GRIP1和ERβ2-GRIP1重组荧光双杂交酵母能够专一性地被E_2诱导产生荧光素酶,并存在显著的剂量-效应关系,半数效应浓度(EC_(50))值分别为1.98×10~(-9)、1.77×10~(-10)、和3.52×10~(-10)mol·L~(-1),对E_2的敏感程度排序为:ERβ1-GRIP1>ERβ2-GRIP1>ERα-GRIP1。研究表明,稀有鮈鲫不同ER亚型对E_2的响应具有差异,3株重组荧光双杂交酵母不仅可以应用于快速识别内分泌干扰物中的类雌激素物质,分析稀有鮈鲫不同ER亚型对EEs的敏感性,解析雌激素污染物对稀有鮈鲫的作用机制,评估鱼类接触EEs的潜在风险,以期为水质保障和污染治理提供重要依据。     

Dietzia菌随机插入突变体文库的构建及功能基因筛选

Dietzia属菌具有很好的烃类降解和良好的高盐、高碱耐受能力.为研究Dietzia属降解烷烃和耐受盐碱的分子机制,以Dietzia sp. DQ12-45-1b为研究菌株,利用双质粒系统p Tip-istAB-sacB和pRTSK-sacB,成功构建库容为30 720的随机突变体文库.随机挑选的突变克隆及突变克隆传代20次后的Southern blot结果表明:转座序列插入的随机性较好,均为单插入突变,并且具有较好的遗传稳定性,可用于长期筛选功能基因.在此基础上,分别以高盐培养和正十六烷为单一碳源培养,利用高通量基因筛选,成功获得了一个耐盐突变体M9G和5个降解利用正十六烷相关突变株M6A、M6B、M7A、M9A、M81C.与野生型相比,5个降解利用正十六烷相关突变株的降解率下降范围为16.67%-84.35%.插入位点分析结果显示,M9G、M6A、M6B、M7A、M9A和M81C的插入基因分别预测为丝氨酸蛋白酶、铁氧化还原蛋白、假定蛋白、铁氧化还原蛋白还原酶、ABC转运蛋白和细胞色素P450类CYP153烷烃羟化酶.其中除了预测假定蛋白外,有些基因与传统上确定的功能存在差异,暗示着其可能具有新的功能.Dietzia菌随机插入突变体文库的构建及筛选可为研究Dietzia菌的抗逆和烷烃降解机制奠定基础.(图6表3参28)     

2-巯基噻唑啉与过氧化氢酶的相互作用机理研究

2-巯基噻唑啉是一种被广泛应用的抗腐蚀剂和光亮剂,然而它的一些残留物会对环境造成损坏,为探究2-巯基噻唑啉的毒性作用,采用光谱学技术和分子对接技术来考察2-巯基噻唑啉和过氧化氢酶的相互作用影响,其中酶活性实验表明2-巯基噻唑啉会抑制过氧化氢酶的活性,分子对接结果显示2-巯基噻唑啉会结合在过氧化氢酶的活性位点处,并最终导致过氧化氢酶空间结构和微环境的变化,根据荧光测试结果,表明2-巯基噻唑啉和过氧化氢酶之间的猝灭方式为静态猝灭。通过测量不同温度下的结合位点数、结合常数以及热力学常数,显示2-巯基噻唑啉与过氧化氢酶主要通过氢键和范德华力相结合。该研究从分子水平上考察了2-巯基噻唑啉对蛋白质的毒性作用,对于阐明污染物的毒性机理具有重要意义。     

Pesticide Residues in Water From River Kaveri,South India

The residue levels of presistent chlorinated pesticides such as HCH (hexachlorocyclo-hexane) isomers and DDT (dichlorodiphenyl trichloroethane) compounds were quantified in water samples collected from the River Kaveri and its distributor River Coleroon in Tamil Nadu, South India. HCH showed higher levels in River Kaveri during premonsoon (July to September) and monsoon (October to December) months, reflecting the HCH usage during that season for paddy crops. But in the case of DDT no clear trend in residue level was observed. the α-HCH was detected as the dominant isomer in all the three sampling sites. Among DDT compounds, p,p'-DDT and p,p'-DDE showed higher percentage of the total. International comparison of residue levels revealed that the present values are comparable to the waters from Asian and South East Asian nations, but lower than some samples from other parts of India. the value of DDT is well below the EEC's maximum acceptable concentration for surface waters and lower than the recommended limit of 2000ng 1^-1 in USA water for protection of aquatic life (Water Quality Criteria, 1972).     

荧光光谱法研究木栓酮与牛血清白蛋白的相互作用

为了解三萜类化合物在体内的结合转运机制,采用荧光光谱法结合紫外吸收光谱研究从中药爵床中分离的有效成分木栓酮(Friedelin,FDL)与牛血清白蛋白(Bovine serum albumin,BSA)的结合反应.通过研究木栓酮对牛血清白蛋白内源性荧光的淬灭作用,发现木栓酮可以与牛血清白蛋白相互结合,且其作用是一个静态猝灭的过程.通过计算木栓酮与牛血清白蛋白的结合常数和结合位点,发现在生理弱碱性溶液中二者具有较强的结合作用,且以1:1结合.根据热力学参数推测木栓酮与牛血清白蛋白的作用力类型为氢键和范德华力.根据F rster非辐射能量转移机理,计算出木栓酮与牛血清白蛋白结合距离为2.86 nm.研究表明木栓酮在体内可以通过血清白蛋白进行结合和转运.     

Einfluss organischer Substanz auf die Reproduktion, Biomasse und Akkumulation hydrophober Schadstoffe in Lumbriculus variegatus

Background, aim, and scope Sediment-Water Toxicity Tests are an important instrument for the environmental risk assessment of native, contaminated sediments within the European Water Framework Directive. Thus, it must be guaranteed to distinguish between natural and anthropogenic adverse effects. The content of particulate (POC) and dissolved (DOC) organic carbon can affect the mobility of sediment pollutants. Different studies demonstrated that the DOC-content can influence the bioavailability of contaminants and can directly affect aquatic organisms. The aim of this study was to investigate the influence of increasing sediment organic matter on reproduction and growth of the benthic oligochaete Lumbriculus variegatus. Furthermore, the accumulation factors of the hydrophobic substances 1,1-dichloro-2,2-bis(p-chlorphenyl)ethylene (p,p'-DDE), 2´, 3, 4, 4´, 5´-Hexachlorbiphenyl (PCB 138) und 2, 2´, 4, 4´, 5, 5´-Hexachlorbiphenyl (PCB 153) were calculated considering the TOC-content and the uptake route (water-only exposure or sediment ingestion). Materials and methods The test was performed according to the OECD Guideline 225 (2007) using artificial sediment with different peat concentrations. The effect of an increasing TOC-concentration was studied in three different testmodifications with and without spiked sediment. The nominal concentration of the contaminants in spiked sediment was 50?µg/kg. Results The results showed maximum accumulation factors of 13 for both, PCB 138 and 153 and 11 for p,p'-DDE in sediments with low organic carbon content. With increasing peat content accumulation factors decreased. Thereby the ingestion of sediment was the main exposure route. Moreover preponderantly negative correlations between the organic carbon content in unspiked sediments and reproduction as well as growth of the testorganisms were detected. In combination with the test substances reproduction and growth increased with increasing organic carbon content. Discussion Hence it can be assumed that organic matter, especially humic substances, directly effected the testorganisms in the tests without spiked sediment. Due to the decrease of accumulation factors in spiked sediment with increasing TOC-content it might be possible that the availability of humic substances was reduced by conglomeration with the tested chemicals. Conclusions Therefore the possible influence of dissolved organic matter on the testorganism should be considered when assessing the results of toxicity tests and bioaccumulation studies with Lumbriculus variegatus. Recommendations and perspectives To receive detailed information about a critical DOC-concentration influencing the testorganisms further investigations about the amount of organic matter in the sediment and the DOC-concentration in the overlaying water are recommended.     

Mytilus galloprovincialis and Parapenaeus longirostris as bioindicators of heavy metal and organochlorine pollution

The worth of the data obtained from Mytilus galloprovincialis and Parapenaeus longirostris for the purpose of assessing the extent to which the areas they come from are polluted by heavy metals (Fe, Zn, Cu, Pb, Mn, Ni, Cd, Cr, Co) and chlorinated hydrocarbons (PCBs, p,p'DDE, p,p' DDT, p,p'DDD, BHCs, heptachlor epoxide, dieldrin, endrin), was thoroughly investigated. The work embraced the accuracy of the determinations, the spot and area variability, the overall coefficient of variation, the effect of the season and the size, together with, in the case of P. longirostris, the differences observed in four sections of the Saronikos Gulf, in Greece, the degree of pollution, which was estimated independently by measuring the concentration in the water of nutrients (phosphate, ammonia, nitrite, nitrate, silicate) and dissolved oxygen. The study proved the feasibility of employing the organisms as bioindicators of pollution. It also established that the individual variability of response, the season and the size do not cause as much assessment error as the commonly huge temporal fluctuations and the frequent inadequacies of the analytical procedures. A scheme is described for the collection of a truly representative sample.     

Organochlorine insecticide residues in Egyptian mothers’ milk

Two hundred ninety‐two mother's milk samples were collected during 1994 from ten Egyptian governorates representing rural, metropolitan, and newly reclaimed desert areas. The samples were analyzed for 14 organochlorine insecticides and metabolites using electron capture gas chromatography and confirmed by gas chromatography/mass spectrometry (GC/MS). The overall samples average showed that the most abundant organochlorine insecticide residues were p,p'‐DDE, α‐endosulfan, β‐HCH and, p,p’‐DDT. Residues in the range of not detectable to less than 14μg/l whole milk were recorded for α‐HCH, heptachlor, heptachlor epoxide, aldrin, dieldrin, endrin endrin aldehyde and p,p'‐DDD. The percentage of samples exceeding the acceptable daily intake for children (ADIs) set by the FAO/WHO ranged from 19% to 44% in all governorates included in the study except the governorate of Minia in which only 2.6% of the samples exceeded the ADI values. The insecticides exceeding ADI values are mostly heptachlor and heptaclor epoxide (ADI = 3.12 μg/1 whole milk).     

Organochlorine levels in human blood from residents in areas of limited pesticide use in Sudan

Ninety-nine human blood samples were collected from the riverine region of northern Sudan and the traditional and mechanized rain-fed areas of western and eastern Sudan, representing areas of limited pesticide use in Sudan. Blood samples were analyzed for organochlorine pesticides by gas liquid chromatography (GLC) followed by electron-capture detection (ECD). p,p′-DDE (a metabolite of DDT), heptachlor epoxide, β-hexachlorocyclohexane (β-HCH), and dieldrin were detected in all locations surveyed. The level of total organochlorine burden was highest in the traditional rain-fed area, followed by the mechanized rain-fed area, and the riverine area. A highly significant correlation was observed between total organochlorine blood burden and the age of the donors (r = 0.608**).     

The significance of male-male aggression for yearling dispersal in roe deer (Capreolus capreolus)

The significance of adult buck aggression as a proximate factor driving yearling male dispersal in roe deer, Capreolus Capreolus, was studied in Sweden during the dispersal season (April–June) in 1988–1991. In roe there is a well established positive correlation between yearling antler size and dispersal tendency. I tested the prediction that yearlings with large antlers will experience more agonism from adult bucks than those with smaller antlers. Additionally, I studied the relationship between antler size and level of sexual maturity, by recording date of velvet shedding, and sampling testes weights from culled yearlings during the breeding season. The amount of agonism experienced by yearlings was positively correlated with the size of their antlers during April and May, whereas in June no such relationship was found. Aggression towards all yearlings tended to increase as the season progressed. A positive correlation between antler size and testes weight was evident, and the larger the antlers of yearlings the earlier the velvet shedding occured. These results indicate: firstly, that adult buck agonism is an important proximate factor underlying yearling male dispersal; and secondly, that agonism is mainly directed towards the sexually most mature yearlings. This, in turn, suggests mate competition to be the ultimate cause of male-male agonism, and thus male dispersal in this species.     

Characteristics of residual organochlorine pesticides in soils under different land-use types on a coastal plain of the Yellow River Delta

The residual levels of organochlorine pesticides (OCPs) were examined in soils covering five types of land use along a salinity gradient on the Yellow River Delta. The most prominent OCPs were dichlorodiphenyltrichloroethane (∑DDT, arithmetic mean = 5.11 μg kg^?1), hexachlorocyclohexane (∑HCH, 1.69 μg kg^?1) and ∑endosulfan (10.4 μg kg^?1). The spatial variability of OCPs composition shifted from γ-HCH and o,p′-DDT dominated pesticides in coastal soils to p,p′-DDE dominated pesticides in inland soils. In different land-use types, the percentages of β-HCH and p,p′-DDE are characterized by more recalcitrant components in decreasing order of vegetable fields, cereal fields, cotton fields, wetlands and tidal flats with increasing soil salinity. However, the less recalcitrant components, γ-HCH and o,p′-DDT, showed an opposite trend. Endosulfan sulfate predominated in all land-use types. Residual levels of β-HCH were affected by soil organic matter. The correlations between γ-HCH and clay content and between p,p′-DDE, o,p′-DDT and salinity might associate with the influence of sediment cotransport by the Yellow River and the density of anthropogenic activities in coastal region. Depth distribution of the OCPs in typical soil profiles also implied that local historical usage and sediment transport by the Yellow River both affected the OCPs residual in this region.     

Accumulation of persistent organic pollutants in the food chain of lake baikal

Lake Baikal is the deepest and oldest freshwater lake in the world containing about 20% of the world's fresh water. The present state of its contamination with organic pollutants was studied during 1997–1998 by collecting samples of sediments, phytoplankton, Zooplankton, sponges, aquatic plants, algae, muscles of various fish species, eggs of various birds and blubber of seals. Samples were prepared and analysed using GC‐MS for semivolatile priority pollutants on the US EPA list, for the presence of organochlorine pesticides and for PCBs. The results showed that there was pronounced bioaccumulation of persistent organochlorine compounds along the trophic chain, leading to substantial residues of organochlorine compounds in seals (e.g. 0.87–25.3 ppm of p,p'‐DDT in blubber).     

Assessment of estrogenic chemicals using an estrogen receptor α (ERα)- and ERβ-mediated reporter gene assay in fish

In vitro reporter gene assays using vertebrate cell lines or yeast have been used for assessment of the estrogenic chemicals. However, estrogen receptor α (ERα)- and ERβ-mediated reporter gene system in fish has yet to be developed. In the present study, we developed an ERα- and ERβ-mediated reporter gene assay in fish and estimated estrogenic activities of 17β-estradiol (E2), p-nonylphenol (NP), bisphenol A (BPA), p,p′-DDE, and genistein (Gen) using the in vitro reporter assay. The activity was intensely induced by transfection with either ERα or ERβ expression plasmid under E2 or Gen administration, whereas it was significantly induced by transfection with ERα expression plasmid, but not with ERβ expression plasmid under NP administration. On the other hand, the activity was induced more intensely by transfection with ERα expression plasmid than ERβ expression plasmid under BPA or p,p′-DDE administration. These results indicate that there are obvious differences in the activity through ERα and ERβ among the estrogenic chemicals examined in vitro. Thus, the in vitro reporter assay provides an excellent system for elucidating the action mechanism of estrogenic chemicals in fishes. Physical and Chemical Impacts on Marine Organisms, a Bilateral Seminar Italy–Japan held in November 2004