Szeto Y T, Benzie I F F, Collins A R, Choi S W, Cheng C Y, Yow C M N, Tse M M Y
Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, China.
Mutat Res. 2005 Oct 15;578(1-2):371-81. doi: 10.1016/j.mrfmmm.2005.06.014. Epub 2005 Aug 8.
The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.
彗星试验是一种广泛应用于DNA损伤检测的生物监测工具。在人体研究中最常用的细胞是淋巴细胞。迫切需要找到一种替代的人体靶细胞，这种细胞能够以最小的侵入性从正常受试者身上采集。有一些关于颊细胞的报道，颊细胞易于从口腔内部采集，已被用于DNA损伤与修复的研究，这些报道引起了人们的兴趣。然而，我们按照已发表的方案进行的初步研究表明，颊细胞在所用的高pH值条件下（[O. 奥斯特林，K.J. 约翰森。单个哺乳动物细胞中辐射诱导的DNA损伤的微电泳研究。生物化学与生物物理研究通讯。123 (1984) 291 - 298]）会遭受大量损伤并解体，但在较低pH值下对裂解（彗星试验中的一个关键步骤）具有极强的抗性。因此，本研究的目的是开发一种能使颊细胞在彗星试验中裂解并进行DNA损伤检测的方案，并利用该模型评估颊细胞模型在人体生物监测和营养研究中的潜在用途。具体而言，我们旨在研究颊细胞DNA损伤（以链断裂形式）的个体内和个体间差异、体外暴露于标准氧化剂刺激和抗氧化剂处理的影响，以及原位暴露于富含抗氧化剂的饮料和使用富含类胡萝卜素的食物进行补充相关的影响。使用0.25%的胰蛋白酶处理30分钟，随后用蛋白酶K（1mg/ml）处理60分钟，成功实现了细胞裂解。当对预先包埋在载玻片上琼脂糖中的细胞进行此操作，然后进行电泳（在0.01M氢氧化钠、1mM乙二胺四乙酸、pH 9.1的条件下，12V电泳18分钟）时，尽管个体间差异较大，但仍获得了令人满意的彗星图像。细胞在裂解前暴露于标准氧化剂刺激（由过氧化氢诱导）会以剂量相关的方式增加DNA链断裂，而将细胞置于生育三烯酚（一种水溶性维生素E类似物）中孵育可对随后的氧化剂刺激提供显著的保护作用（P<0.05）。颊细胞原位（即在口腔中）暴露于富含抗氧化剂的绿茶会导致基础DNA链断裂急剧减少。在一项对照人体干预试验中，14名受试者在补充富含类胡萝卜素的浆果（枸杞）28天后，颊细胞的DNA链断裂有小幅但具有统计学意义（P<0.05）的减少。这些数据表明，这种颊细胞彗星试验在人体生物监测和营养研究中是一种可行且潜在有用的替代工具，可替代常用的淋巴细胞模型。
