蛋白质加合物作为分子生物标志物的分析研究

为了探索研究用人的静脉血中蛋白质 -环氧苯乙烯加合物作为人接触苯乙烯的分子生物标志物的可行性 ,用改进的 Ra-Ni方法分析测定蛋白质 -环氧苯乙烯加合物 .收集大白鼠鲜血与环氧苯乙烯体外反应的加合物样品 ,用定量苯乙烯 ,环氧苯乙烯染毒的大白鼠血样品和 2个现场接触苯乙烯的工人静脉血 (石棉厂 ,钢琴厂 )样品 ,测所有样品里的蛋白质加合物 .结果表明体外反应样品 ,所测的蛋白质 -环氧苯乙烯加合物与环氧苯乙烯剂量有较好的相关性 ;动物实验样品所测的蛋白质加合物分别与注射环氧苯乙烯 ,苯乙烯剂量呈较好的相关性 ;现场人群的样品 ,所测石棉厂的人群血中蛋白质加合物与接触苯乙烯剂量有一定的相关性 ,而钢琴厂的人群血样中所测的蛋白质加合物与接触苯乙烯剂量相关性不明显 .根据蛋白质 -环氧苯乙烯加合物与剂量的关系 ,初步确认用血红蛋白中的半胱氨酸 -环氧苯乙烯加合物作为人接触苯乙烯的分子标志物有可行性 .同时为用蛋白质 -环氧苯乙烯加合物作为人接触低浓度苯乙烯的分子生物标志物提供可靠的现场实验数据 .     

血红蛋白-环氧苯乙烯加合物的分析

采用环氧苯乙烯和血红蛋白中羧酸基和巯基位的加合物同时测定的方法，对大鼠血红蛋白ＳＯ加的进行了体外实 腹腔注射环氧苯乙烯和苯乙烯的体内实验。结果表明在血红蛋白中所分析的３种加合物（ＳＧ，１－ＰＥ，２－ＰＥ）均随着ＳＯ剂量而增加。     

苯处理SD大鼠器官和血中DNA加合物的形成与分布

以Ｐ１核酸酶提高灵敏度的＾３２Ｐ后标记法系统研究活体条件下腹腔注射苯处理的ＳＤ大鼠脏器和血细胞中ＤＮＡ加合物的形成与分布。结果表明，苯诱导或Ａｒｏｃｌｏｒ诱导对苯处理的ＳＤ大鼠体内ＤＮＡ加合物的形成有明显的促进作用，苯诱导的促进作用大于Ａｒｏｃｌｏｒ诱导。采用共价加合物标记指数对＾３２Ｐ后标记法数据进行计算的结果表明，此指数能反映出同一处理不同器官及不同处理相同器官中的ＤＮＡ加合物的形成与分布情况     

甲醛致核酸损伤作用的实验研究

应用单细胞凝胶电泳技术(SCGE)和液相色谱-电化学检测法(HPLC-EC)研究了典型环境污染物甲醛对DNA分子的损伤效应．结果表明：甲醛不仅可诱导人外周血淋巴细胞DNA发生链断裂,还可引起DNA-DNA,DNA-蛋白质交联;甲醛与小牛胸腺DNA的体外作用较弱,但在铁离子介导下对DNA的氧化能力增强,可产生一定量的8-羟基脱氧鸟苷(8-OHdG)加合物：动物实验表明甲醛诱导大鼠肺组织DNA氧化损伤生成8-OHdG,提示甲醛较强的遗传毒性．     

农药混配剂对大鼠肠酯酶同工酶的抑制作用研究

比较了速灭杀丁、杀螟松和增效剂NIA_(16388)的单剂和混剂在离体与整体条件下对大鼠肠脂酶同工酶的抑制作用.结果表明,大鼠肠至少有10条酯酶带,主要由羧酸酯酶组成.杀螟松能强烈抑制羧酸酯酶活力,而速灭杀丁与NIA_(16388)在处理2h时呈现抑制作用,而随处理时间的增加酶活力有恢复的现象,羧酸酯酶的这种抑制作用同杀虫剂的剂量有关.     

液相色谱-串联质谱法检测甲醛暴露产生的两种DNA加合物

针对两种甲醛-DNA加合物,N~6-羟甲基腺嘌呤(N~6-HOMe-d A)和N2-羟甲基鸟嘌呤(N_2-HOMe-d G),本实验室建立了基于液相色谱串联质谱的分析方法,以便从DNA分子水平上探讨甲醛暴露对DNA的损伤作用机理,其中[~(15)N_5]N~6-Me-d A和[~(13)C_(10)~(15)N_5]N_2-Me-d G为内标;液相分离所用色谱柱为Thermo Polar Advantage II C_(18);方法的精密度较好(RSD5%)、灵敏度较高(检出限分别为0.014和0.0064 ng·m L~(-1))、回收率良好(94.9%~111%).后用小牛胸腺DNA进行体外染毒实验,设置甲醛染毒的浓度梯度(0.001,0.01,0.05,0.1,0.5和1 mmol·L~(-1))和染毒时间组(0、2、4、8、12、16、24 h),利用建立的LC-MS/MS方法检测了DNA中N~6-HOMe-d A和N_2-HOMe-d G的含量.结果表明,两种甲醛-DNA加合物N~6-HOMe-d A和N2-HOMe-d G的量与甲醛暴露存在剂量-反应和时间-反应关系.     

用增强灵敏度的~32P后标记法测定DNA-环氧苯乙烯加合物

DNA加合物是具有亲电性化学致癌物与DNA形成共价相联的化合物,是化学致癌物损伤DNA的主要形式,能够灵敏准确地分析DNA加合物的方法是研究DNA损伤     

Controlling enzymatic activity by immobilization on graphene oxide

In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells’ viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.     

乙醛对脱氧核糖核酸分子的损伤作用

应用单细胞凝胶电泳技术和液相色谱-电化学检测法研究了典型环境污染物乙醛对脱氧核糖核酸(DNA)分子的损伤效应.结果表明:乙醛不仅可诱导人外周血淋巴细胞DNA发生链断裂,还可引起DNA-DNA,DNA-蛋白质交联;乙醛与小牛胸腺DNA的体外作用较弱,但在铁离子介导下对DNA的氧化能力增强,可产生一定量的8-羟基脱氧鸟苷(8-OHdG)加合物;动物实验表明,乙醛诱导大鼠肺组织DNA氧化损伤生成8-OHdG, 提示乙醛具有潜在遗传毒性,可能与引起DNA交联及氧化有关.     

黄浦江上游工业废水对程序外DNA合成的影响

用SD大鼠原代肝细胞作程序外DNA合成(UDS)试验,检测黄浦江上游水源保护区9家工厂的工业废水,结果表明,在不同时间采集的9家工厂的两批工业废水UDS试验均呈阳性,并且呈剂量—反应关系。这说明9家工厂的工业废水有潜在的致突变作用和致癌作用,对这些工业废水的排放应加以控制。     

生物大分子加合物的检测及其在环境风险性评价中应用

环境中化学物质对人体致癌的风险性评价对于预防人类疾病和控制环境污染具有非常重要的意义。而反映暴露水平与生物效应的生物标记物研究不仅是导向预防研究的核心内容，也是环境致癌物风险评价的重要手段。传统的有毒化学物监测方法在研究人群暴露和危险度评价时得到的仅为近似接触量，而对个体在吸收、代谢、排泄，生物利用上的差异不能提供任何信息，分子生物标记物可用于检测生物暴露于环境致癌物的剂量和效应，生物体的遗传性或诱发性的敏感性差异，以及由环境致癌物引发的各种疾病的早期诊断，不仅提供个体有效接触量，还能提供许多化学物质结构与毒性之间关系及毒物代谢动力学方面的信息。与其他手段相比，分子生物标志物作为指示污染物危害效应的生物信号，其检测是从以往测定环境中化学物的含量估计生物体的接触水平，发展到直接检测化学物与生物受体结合产物的一种方法。文章对分子生物标志物一生物大分子加合物(如DNA，RNA，蛋白质加合物等)的形成机理以及在环境风险评价中的应用进行了综述：介绍了几种生物大分子加合物的检测方法，并对各种方法的特点进行评述，讨论了目前分子生物标志物研究存在的问题，并展望了该领域的应用前景。     

Benzene and lead exposure assessment among occupational bus drivers in Bangkok traffic

IntroductionAirpollutionproblemsasaresultoffastgrowthinthetotalnumberofvehiclesinurbancitiesareontheincrease .PreliminaryepidemiologicalexposureassessmentsuggestthatthedetrimentaleffectofairpollutiongeneratedbyvehiculartrafficindevelopingcitieslikeBangkok…     

DNA damage of germ cell of rat induced by nitrotoluene chemicals

2,4-dinitrotoluene(2,4-DNT), 2,6-dinitrotoluene(2,6-DNT) and 4-nitrotoluene(4-NT) are typical pollutants in the Songhua River of Northeast China. Sertoli/germ call cocultures and single cell gel electrophoresis(SCGE) are applied to investigate whether they have genotoxicity on DNA damage of germ cell of Kunming male rat. The results showed that all three nitrotoluene compounds tested could induce DNA single-strand breaks of the germ call. A significant relationship is found between logarithm dose and the degree of DNA damage, which implies that 2,4-DNT, 2,6-DNT and 4-NT have genotoxicity and can induce the germ cell DNA strand to break in vitro.     

Improved preparation and identification of aristolochic acid-DNA adducts by solid-phase extraction with liquid chromatography-tandem mass spectrometry

Aristolochic acid (AA) is a known nephrotoxin and potential carcinogen, which can form covalent DNA adducts after metabolic activation in vivo and in vitro. A simple method for preparation and characterization of aristolochic acid-DNA adducts was developed. Four AA-adducts were synthesized by a direct reaction of AAI/AAII with 2’-deoxynucleosides. The reaction mixture was first cleaned-up and pre-concentrated using solid phase extraction (SPE), and further purified by a reversed-phase high performance liquid chromatography (HPLC). By the application of developed SPE procedure, matrices and byproducts in reaction mixture could be greatly reduced and adducts of high purity (more than 94% as indicated by HPLC) were obtained. The purified AA-DNA adducts were identified and characterized with liquid-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS/MS) and LC-Diode array detector-fluorescence (LC-DAD-FL) analysis. This work provides a robust tool for possible large-scale preparation of AA-DNA adduct standards, which can promote the further studies on carcinogenic and mutagenic mechanism of aristolochic acids.     

Detection and isolation of fetal cells from maternal blood using the flourescence-activated cell sorter (FACS)

The presence of fetal cells in the maternal circulation during pregnancy has been suggested by repeated observations of small numbers of cells containing Y chromatin or a Y chromosome in the blood of pregnant women. With the fluorescence-activitated cell sorter (FACS), we have used antibodies to a paternal cell surface (HLA) antigen, not present in the mother, to select fetal cells from the lymphocyte fractions of a series of maternal blood samples, collected as early as 15 weeks of gestation. These sorted cells have been examined for a second paternal genetic marker, Y chromatin. Y chromatin-containing cells were found among the sorted cells from prenatal maternal blood specimens in 8 pregnancies subsequently producing male infants whose lymphocytes reacted with the same antibodies to paternal antigen used for sorting with the FACS. In each of 17 pregnancies resulting in male infants who failed to inherit the antigen detected by the antibodies used for cell sorting, Y chromatin-containing cells were not found prenatally. The use of two paternal genetic markers, a cell surface antigen and nuclear Y chromatin, to identify fetal cells in maternal blood permits us to conclude that these cells are present in the mother's circulation, as early as 15 weeks gestation. Further development of the techniques reported here could lead to widespread screening of maternal blood samples during pregnancy for detection of fetal genetic abnormalities.     

内分泌扰乱化学品活性筛选和测试方法

对近年来对内分泌扰乱化学品(EDCs)的筛选和测试方法进行了述评。目前倾向于采用包括与激素受体结合的亲和力测试、体外和活体测试在内的层式检测法对EDCs的活性进行筛选和测试,并评价人体和野生动物内分泌扰乱的性质、可能性和剂量-反应关系。目前也致力于EDCs的结构-效应关系特别是定量关系的研究,旨在提供一种简便低廉的筛选方法。总的说来,稳定可靠的标准化筛选和检测方法的建立仍待努力。      

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.     

镉致黑斑蛙肝脏中ROS生成及其蛋白质氧化损伤作用

在实验条件下,将健康性成熟黑斑蛙(Rana nigromaculata)暴露于0.005、0.010、0.050和0.100mg·L-1浓度的镉溶液中30d,采用2,4二硝基苯肼比色法测定肝组织蛋白质羰基含量,KCl-SDS沉淀法测定DNA-蛋白质交联含量,并测定了肝组织中活性氧自由基(ROS)的水平,以探讨镉对黑斑蛙肝组织蛋白质的氧化损伤作用及其作用机制.结果表明,随染镉浓度的增加,黑斑蛙肝线粒体中的ROS水平明显升高,各染毒组与对照组相比有显著性差异;肝蛋白质羰基(PCO)含量和DNA-蛋白质交联(DPC)也随镉暴露浓度的增加而升高,且均呈明显的浓度-效应关系,但这种升高仅在镉浓度为0.05、0.10mg.L-1时才具有显著意义.结果还显示,低浓度镉的长期暴露可引起黑斑蛙肝蛋白质氧化损伤和DNA损伤,诱导产生大量自由基可能是导致蛋白质和DNA产生损伤的主要机制之一.     

应用~(32)P-后标记技术对苯醌和氢醌处理人血淋巴细胞形成DNA加合物的研究

应用核酸酶Ｐ１促进的３２Ｐ－后标记技术对苯醌和氢醌处理人血淋巴细胞后形成的ＤＮＡ加合物进行了研究。结果表明，人血淋巴细胞被苯醌和氢醌处理２４ｈ之后，均形成了同一种ＤＮＡ加合物。两种化合物在不同浓度下处理人血淋巴细胞每１０７核苷酸中有０．０１～１０个核苷酸形成加合物。为了达到同等ＤＮＡ加合物水平，氢醌比苯醌需要更高的浓度。小牛胸腺ＤＮＡ与苯醌、氢醌反应可生成５种ＤＮＡ加合物。用苯醌或氢醌处理淋巴细胞所形成的加合物种类与上述纯ＤＮＡ与苯醌，氢醌形成的５种加合物在双向薄层层折图谱上完全不相符合。这些结果意味着苯醌、氢醌与ＤＮＡ的加合作用在细胞内存在着修饰机制。     

Zebrafish embryo toxicity of 15 chlorinated, brominated, and iodinated disinfection by-products

Disinfection to protect human health occurs at drinking water and wastewater facilities through application of non-selective oxidants including chlorine. Oxidants also transform organic material and form disinfection by-products (DBPs), many of which are halogenated and cyto- and genotoxic. Only a handful of assays have been used to compare DBP toxicity, and researchers are unsure which DBP(s) drive the increased cancer risk associated with drinking chlorinated water. The most extensive data set employs an in vitro model cell, Chinese hamster ovary cells. Traditionally, most DBP research focuses on the threat to human health, but the effects on aquatic species exposed to DBPs in wastewater effluents remain ill defined. We present the developmental toxicity for 15 DBPs and a chlorinated wastewater to a model aquatic vertebrate, zebrafish. Mono-halogenated DBPs followed the in vivo toxicity rank order: acetamides > acetic acids > acetonitriles ~ nitrosamines, which agrees well with previously published mammalian in vitro data. Di- and tri-halogenated acetonitriles were more toxic than their mono-halogenated analogues, and bromine- and iodine-substituted DBPs tended to be more toxic than chlorinated analogues. No zebrafish development effects were observed after exposure to undiluted or non-concentrated, chlorinated wastewater. We find zebrafish development to be a viable in vivo alternative or confirmatory assay to mammalian in vitro cell assays.