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首页> 外文期刊>Mutation Research. Reviews in Mutation Research >Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress
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Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress

机译：开发高通量报告基因检测试剂盒以检测遗传毒性和氧化应激

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The lack of toxicological information on many of the compounds that humans use or are exposed to, intentionally or unintentionally, poses a big problem in risk assessment. To fill this data gap, more emphasis is given to fast in vitro screening tools that can add toxicologically relevant information regarding the mode(s) of action via which compounds can elicit adverse effects, including genotoxic effects. By use of bioassays that can monitor the activation of specific cellular signalling pathways, many compounds can be screened in a high-throughput manner. We have developed two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. To exclude non-specific effects by compounds influencing the luciferase reporter-gene expression non-specifically, a third assay was developed to monitor changes in luciferase expression by compounds in general (Cytotox CALUX). To facilitate interpretation of the data and to avoid artefacts, all three reporter-gene assays used simple and defined reporter genes and a similar cellular basis, the human U2OS cell line. The three cell lines were validated with a range of reference compounds including genotoxic and non-genotoxic agents. The sensitivity (95%) and specificity (85%) of the p53 CALUX was high, showing that the assay is able to identify various types of genotoxic compound, while avoiding the detection of false positives. The Nrf2 CALUX showed specific responses to oxidants only, enabling the identification of compounds that elicit part of their genotoxicity via oxidative stress. All reporter-gene assays can be used in a high-throughput screening format and can be supplemented with other U2OS-based reporter-gene assays that can profile nuclear receptor activity, and several other signalling pathways.
机译：人类有意或无意使用或接触的许多化合物缺乏毒理学信息，这在风险评估中构成了一个大问题。为了填补这一数据空白，将更多的重点放在快速的体外筛选工具上，这些工具可以添加有关作用方式的毒理学相关信息，化合物可以通过作用方式引发不良反应，包括遗传毒性作用。通过使用可以监测特定细胞信号通路激活的生物测定，可以以高通量的方式筛选许多化合物。我们已经开发了两种新的特异性报告基因检测方法，可以检测化合物对两种感兴趣途径的影响：对基因毒性的p53途径（p53 CALUX）和对氧化应激的Nrf2途径（Nrf2 CALUX）。为了排除非特异性影响荧光素酶报道基因表达的化合物的非特异性作用，开发了第三种检测方法来监测一般化合物的荧光素酶表达的变化（Cytotox CALUX）。为了便于解释数据并避免人为因素，所有三种报告基因测定均使用简单且明确的报告基因以及相似的细胞基础（人类U2OS细胞系）。这三种细胞系已通过一系列参考化合物（包括遗传毒性和非遗传毒性剂）进行了验证。 p53 CALUX的灵敏度（95％）和特异性（85％）高，表明该测定法能够鉴定各种类型的遗传毒性化合物，同时避免了假阳性的检测。 Nrf2 CALUX仅显示出对氧化剂的特异性反应，从而能够鉴定出通过氧化应激引起部分遗传毒性的化合物。所有报告基因测定都可以以高通量筛选形式使用，并且可以补充其他基于U2OS的报告基因测定，这些分析可以描述核受体活性以及其他几种信号传导途径。

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