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首页> 美国卫生研究院文献>Genetics and Molecular Biology >Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura Eleutherodactylidae)
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Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura Eleutherodactylidae)

机译：通过彗星测定法对直接发育的青蛙斑潜蝇（Eleutherodactylus johnstonei）（阿努拉斑潜蝇科）的DNA损伤进行估算

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The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.
机译：这项研究的目的是使用彗星试验来评估直接发育的青蛙斑节虾（Eleutherodactylus johnstonei）的遗传损伤。 DNA扩散测定法用于评估碱性，酶促和碱性/酶促治疗裂解约翰斯通氏血细胞的有效性，并确定与凋亡和坏死相关的DNA链断裂量。还使用Comet分析评估了对诱变博来霉素（BLM）和4-硝基喹啉-1-氧化物（4NQO）的细胞敏感性，以及分析的可重复性。碱性处理不能溶解约翰斯通氏大肠杆菌血细胞的细胞质和细胞核膜，而蛋白酶K（40μg/ mL）的酶消化产生裸核。细胞凋亡和坏死（通过DNA扩散测定评估）对DNA损伤的贡献估计为0％至8％。在不同浓度和暴露时间下，BLM和4NQO诱导约翰斯通氏大肠杆菌血细胞中的DNA损伤。两种诱变剂的剂量效应曲线均具有很高的重现性，并且始终显示出较低的变异系数（CV≤10％）。讨论了在生态毒理学研究中可能使用改良的彗星测定法评估约翰斯通氏大肠杆菌对除草剂的暴露的结果。

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