Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells
Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells
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DOI：
10.1007/s00204-014-1229-3
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发表时间：
2014-05-01
影响因子：
6.1
通讯作者：
Wiesmueller, Lisa
中科院分区：
文献类型：
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作者：
Ireno, Ivanildce C.;Baumann, Cindy;Wiesmueller, Lisa
众所周知，体外遗传毒性测试存在几个缺点，特别是基于哺乳动物细胞的分析，其特异性较低。遵循遗传毒性检测的新概念，我们在活的人类细胞中开发了一种基于荧光的方法。该方法对DNA双链断裂和损伤诱导的复制叉停滞引发的DNA重组事件进行了量化，预计可检测到广泛的遗传毒性作用模式。为了最大限度地提高灵敏度，我们设计了一种DNA底物，它包含了来自人类基因组的化学反应元件。使用这种底物，我们筛选了不同类型的人类肿瘤和未转化细胞的DNA损伤反应，这表明遗传毒性致癌物的检测不依赖于P53状态，但被细胞凋亡所消除。选择能够进行稳健和灵敏的遗传毒性检测的细胞类型，以产生具有染色体整合的DNA重组底物的报告克隆。对报道的细胞系进行了21种化合物的仔细研究，根据用于鉴定致癌化合物的既定类别将其分成五组：遗传毒性致癌物质(“真阳性”)、非遗传毒性致癌物质(“真阴性”)和非致癌化合物(“真阴性”)，据报道，这些化合物在基于哺乳动物细胞的分析中会导致染色体异常或突变(“假阳性”)。我们的结果记录了在独立细胞克隆中检测到遗传毒性致癌物，细胞毒性水平为85%，特异性=90%，检测到假阳性化合物<；17%。重要的是，通过环磷酰胺与原代肝细胞培养相结合的测试，我们还利用这一新的检测系统为识别需要代谢激活的致癌物提供了概念验证。
In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA doublestrand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities 85 %, specificity of >= 90 % and detection of false-positive compounds < 17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.