Using the comet assay and lysis conditions to characterize DNA lesions from the acrylamide metabolite glycidamide
Using the comet assay and lysis conditions to characterize DNA lesions from the acrylamide metabolite glycidamide
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DOI：
10.1093/mutage/gex036
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发表时间：
2018-01-01
期刊：
影响因子：
2.7
通讯作者：
Brunborg, Gunnar
中科院分区：
文献类型：
--
作者：
Hansen, Siri Helland;Pawlowicz, Agnieszka J.;Brunborg, Gunnar
用碱性彗星试验和无细胞系统研究了食品污染物丙烯酰胺的代谢物缩水甘草胺(GA)对DNA损伤的影响。在彗星试验中加入甲酰胺嘧啶-DNA-糖基酶(FPG)酶，可敏感地检测到GA诱导的DNA损伤。我们使用LC-MS来表征GA处理过的裸DNA的修饰碱基，以及随后的FPG处理和不处理。在上清液中检测到N7-GA-鸟嘌呤和N3-GA-腺嘌呤苷元，表明加合物碱基有一定程度的脱除；用FPG处理裸DNA，没有进一步增加加合物产量，也没有出现其他加合碱基。我们用GA处理人淋巴细胞，发现FPG检测到的DNA损伤水平有很大差异，这取决于裂解步骤的持续时间和pH。GA诱导的FPG敏感部位的这些裂解依赖的变化与用甲基甲烷磺酸(MMS)处理细胞后观察到的变化平行。另一方面，光活性化合物(RO12-9786)加光诱导的氧化损伤(8-oxo-guanine)和X射线诱导的DNA链断裂在很大程度上与裂解条件无关。结果表明，GA诱导的损伤主要是N7-GA-DG加合物，与标准裂解程序一样，N7-GA-DG加合物在pH为10时缓慢开环，这种结构是FPG导致链断裂的底物。这些数据表明，某些DNA损伤特有的碱溶解依赖性可用于研究特定类型的DNA修饰。彗星试验越来越多地用于化学品的监管测试；在这种情况下，依赖裂解的变异代表了一种新的方法，可以深入了解遗传毒性侮辱的分子性质。
The alkaline comet assay and a cell-free system were used to characterise DNA lesions induced by treatment with glycidamide (GA), a metabolite of the food contaminant acrylamide. DNA lesions induced by GA were sensitively detected when the formamidopyrimidine-DNA-glycosylase (Fpg) enzyme was included in the comet assay. We used LC-MS to characterise modified bases from GA-treated naked DNA with and without subsequent Fpg treatment. N7-GA-Guanine and N3-GA-Adenine aglycons were detected in the supernatant showing some depurination of adducted bases; treatment of naked DNA with Fpg revealed no further increase in the adduct yield nor occurrence of other adducted nucleobases. We treated human lymphocytes with GA and found large differences in DNA lesion levels detected with Fpg, depending on the duration and the pH of the lysis step. These lysis-dependent variations in GA-induced Fpg sensitive sites paralleled those observed after treatment of cells with methyl methane sulfonate (MMS). On the other hand, oxidative lesions (8-oxoGuanine) induced by a photoactive compound (Ro 12-9786) plus light, and also DNA strand breaks induced by X-rays, were detected largely independently of the lysis conditions. The results suggest that the GA-induced lesions are predominantly N7-GA-dG adducts slowly undergoing imidazole ring opening at pH 10 as in the standard lysis procedure; such structures are substrate for Fpg leading to strand breaks. The data suggest that the characteristic alkaline lysis dependence of some DNA lesions may be used to study specific types of DNA modifications. The comet assay is increasingly used in regulatory testing of chemicals; in this context, lysis-dependent variations represent a novel approach to obtain insight in the molecular nature of a genotoxic insult.