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首页> 外文期刊>The Journal of toxicological sciences >An RNA synthesis inhibition assay for detecting toxic substances using click chemistry
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An RNA synthesis inhibition assay for detecting toxic substances using click chemistry

机译：使用点击化学检测有毒物质的RNA合成抑制测定

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Biological risk assessment studies of chemical substances that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication, but also with transcription. There is no simple method for the detection of the DNA lesion-induced inhibition of transcription. Here, we report an assay for estimating the toxicity of chemical substances by visualizing transcription in mammalian cells using nucleotide analog 5-ethynyluridine (EU) and its click chemistry reaction. Ultraviolet light and representative chemical substances (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) of DNAdamaging agents show toxicity, as indicated by RNA synthesis inhibition in response to DNA damage in HeLa cells. Using titanium dioxide, we observed RNA synthesis inhibition in response to the rutile form, but not the anatase form, indicating that rutile titanium dioxide is a toxic substance. Because this method is based on the transcriptional response to DNA lesions, we can use terminally differentiated neuronlike PC12 cells, the differentiation of which can be induced by nerve growth factors, for evaluating chemical substances. Ultraviolet light and some chemicals (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) inhibited RNA synthesis in non-differentiated PC12 cells. Conversely, camptothecin and cisplatin did not inhibit RNA synthesis in differentiated PC12 cells, but 4-nitroquinoline- 1-oxide, mitomycin C, and etoposide did. And using titanium dioxide, we did not observed any RNA synthesis inhibition. These data suggest that this method might be used to estimate the potential risk of chemical substances in differentiated mammalian cells, which are the most common cell type found in the human body.
机译：诱导DNA损伤的化学物质的生物风险评估研究主要基于复制过程中DNA聚合酶的作用。但是，DNA损伤不仅干扰复制，而且干扰转录。没有检测DNA损伤诱导的转录抑制的简单方法。在这里，我们报告了一种通过使用核苷酸类似物5-乙炔基尿苷（EU）及其点击化学反应观察哺乳动物细胞中的转录来估算化学物质毒性的测定方法。 DNA损伤剂的紫外线和代表性化学物质（喜树碱，4-硝基喹啉-1-氧化物，丝裂霉素C和顺铂，但不是依托泊苷）显示毒性，如对HeLa细胞中DNA损伤的RNA合成抑制作用所表明。使用二氧化钛，我们观察到响应金红石形式而不是锐钛矿形式的RNA合成抑制，表明金红石型二氧化钛是有毒物质。因为此方法基于对DNA损伤的转录反应，所以我们可以使用终末分化的神经元样PC12细胞（可通过神经生长因子诱导其分化）来评估化学物质。紫外线和某些化学物质（喜树碱，4-硝基喹啉-1-氧化物，丝裂霉素C和顺铂，但不是依托泊苷）抑制未分化PC12细胞中的RNA合成。相反，喜树碱和顺铂不会抑制分化的PC12细胞中的RNA合成，但4-硝基喹啉-1-氧化物，丝裂霉素C和依托泊苷却能抑制RNA合成。使用二氧化钛时，我们没有观察到任何RNA合成抑制作用。这些数据表明，该方法可用于估算分化的哺乳动物细胞中化学物质的潜在风险，而哺乳动物细胞是人体中最常见的细胞类型。

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