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Development and application of human cell lines engineered to metabolically activate structurally diverse environmental mutagens

机译:经过工程改造以代谢激活结构多样的环境诱变剂的人类细胞系的开发和应用

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Abstract: Cytochromes P450 are responsible for themutagenic/carcinogenic activation of many environmentalpromutagens/procarcinogens. These enzymes are presentat highest concentrations in liver in vivo but aremarkedly absent in tester organisms for most in vitromutagenicity test systems. Two approaches have beenused to supply needed metabolic activation,incorporation of an extracellular activating system,usually derived from a rodent liver and introduction ofactivating enzymes into the target cell. The latterapproach appears to result in a more sensitive testingsystem because of the close proximity of the activatingenzymes and the target DNA. Human cell lines have beendeveloped which stably express human cytochromes P450and other cDNAs which have been introduced individuallyor in combination. The resulting cell lines areexquisitely sensitive to exposure to promutagens andprocarcinogens. Mutagenicity is measured at thehypoxanthine phosphoribosyl transferase (hprt) andthymidine kinase (tk) gene loci. The most versatilecell line, designated MCL-5, stably express five cDNAsencoding all of the human hepatic P450s known to beprincipally responsible for known human procarcinogenactivation. The induction of mutation is observed inMCL-5 cells upon exposure to ng/ml levels of modelcompounds such as nitrosamines, aflatoxin B$-1$/ andbenzo(a)pyrene. A lower volume mutagenicity assay hasbeen developed for use with samples available inlimited amounts. Human lymphoblast mutation assays havebeen used to screen for mutagenic activity sedimentsamples from a polluted watershed. Two sediment sampleswere found to have mutagenic activity to humanlymphoblasts.!17
机译:摘要:细胞色素P450负责许多环境promutagens /致癌物的致癌/致癌活化作用。这些酶在体内肝脏中的浓度最高,但对于大多数体外致突变性测试系统而言,在测试生物中却不存在。已经使用了两种方法来提供所需的代谢活化,掺入通常源自啮齿动物肝脏的细胞外活化系统以及将活化酶引入靶细胞中。由于活化酶和靶DNA的紧密接近,后一种方法似乎导致更灵敏的测试系统。已经开发出稳定表达人细胞色素P450和其他cDNA的人细胞系,所述细胞色素P450和其他cDNA已经被单独或组合引入。所得细胞系对暴露于链霉菌素和致癌物非常敏感。在次黄嘌呤磷酸核糖基转移酶(hprt)和胸苷激酶(tk)基因位点测量致突变性。最通用的细胞系,称为MCL-5,稳定表达五个cDNA,编码所有已知主要负责已知人类致癌物激活的人类肝P450。暴露于ng / ml ng / ml水平的模型化合物(如亚硝胺,黄曲霉毒素B $ -1 $ /和苯并(a)re)后,在MCL-5细胞中观察到突变的诱导。已经开发出了一种较低体积的致突变性测定法,可用于无限量的样品。人类淋巴母细胞突变检测已用于筛选受污染流域的诱变活性沉积物样品。发现有两个沉积物样品对人类成淋巴细胞具有诱变活性!17

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