Using the HepaCometChip Assay for Broad-Spectrum DNA Damage Analysis.
Using the HepaCometChip Assay for Broad-Spectrum DNA Damage Analysis.
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DOI：
10.1002/cpz1.563
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发表时间：
2022-09
期刊：
影响因子：
--
通讯作者：
Engelward, Bevin P
中科院分区：
文献类型：
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作者：
Owiti, Norah A;Kaushal, Simran;Martin, Lincoln;Sly, Jamie;Swartz, Carol D;Fowler, Jasmine;Corrigan, Joshua J;Recio, Les;Engelward, Bevin P
接触 DNA 损伤剂可能会导致突变，从而导致癌症。肝脏特别脆弱，因为它含有高水平的细胞色素 P450 酶，可以将异生素转化为 DNA 反应性代谢物，从而形成潜在致癌的大体积 DNA 加合物。因此，目前临床前测试的要求包括肝脏 DNA 损伤的体内测试，这通常需要许多动物。鉴于许多国家正在努力减少或消除在研究中使用动物，因此迫切需要快速而可靠的体外测试来辨别潜在的药剂是否具有损害肝细胞基因组的能力。一种可能的方法是利用碱性彗星测定，其中可以通过电泳期间受损 DNA 自由迁移到阳极的能力来评估遗传毒性。然而，彗星测定有几个局限性：(i) 测定速度慢； (ii) 检测方法容易受到实验噪音的影响； (iii) 很难检测大体积的 DNA 加合物，因为它们不直接影响 DNA 迁移； (iv) 通常使用的细胞类型不具有强大的代谢能力。为了解决其中一些问题，我们开发了“HepaCometChip”（又名 HepaRG™ CometChip™），其中代谢活性细胞被整合到更高通量的 CometChip 平台中。为了提高对大块损伤的敏感性，使用了“修复捕获”，其中无法检测到的大块损伤被转化为可以通过测定检测到的修复中间体（特别是单链断裂）。在这里，我们描述了执行 HepaCometChip 测定的协议，包括 HepaRG 细胞的处理和剂量以及执行 CometChip 测定。凭借其更高的吞吐量、捕获代谢激活的能力以及对大块病变的敏感性，HepaCometChip 为使用动物进行遗传毒性测试提供了潜在的替代方案。 HepaRG 细胞培养和剂量彗星芯片测定。
Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because there it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that can form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether potential pharmaceutical agents have the ability to damage the genome of hepatocytes. One possible approach is to leverage the alkaline Comet assay, wherein genotoxicity can be assessed by the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The Comet assay, however has several limitations: (i) the assay is slow; (ii) the assay is vulnerable to experimental noise; (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration; and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the “HepaCometChip” (a.k.a. the HepaRG™ CometChip™) wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. To increase sensitivity for bulky lesions, “repair trapping” is used, wherein undetectable bulky lesions are converted into repair intermediates (specifically, single strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. HepaRG cell Culturing and Dosing CometChip Assay.