Szeto Y T, Collins A R, Benzie I F F
Department of Nursing and Health Sciences, The Hong Kong Polytechnic University, Kowloon, SAR, Hong Kong, China.
Mutat Res. 2002 Mar 20;500(1-2):31-8. doi: 10.1016/s0027-5107(01)00298-6.
A modified version of the comet assay was employed to investigate the effect in vitro of dietary antioxidants in the subcellular environment. Human lymphocytes were isolated, embedded in agarose gel, lysed in high ionic strength solution with Triton X-100, and then incubated for 30 min with antioxidants at different concentrations. Gels were washed, and the comet assay performed on cells stressed by 5 min incubation with 45 microM hydrogen peroxide and on unstressed cells in parallel. Results showed that alpha-tocopherol was protective against oxidant stress, whereas caffeic acid did not protect, and at high concentration (100 microM) caused increased DNA damage. Results for quercetin suggested a direct damaging effect, but this did not reach statistical significance. However, at low concentration (3.1 microM), quercetin appeared protective. Thus some dietary antioxidants that have been shown previously to have a protective effect in the 'standard', whole-cell, comet assay cause DNA damage in this lysed-cell version. The cell membrane may have an important role in limiting cellular access of these 'double-edged' antioxidants. Furthermore, the absolute concentration and the presence of complementary or synergistic intracellular antioxidants may delineate the type of action of a putative antioxidant. We suggest that, used in conjunction with the standard comet assay, this lysed-cell version is useful for assessing the effect of the cell membrane and intracellular systems on susceptibility of DNA to oxidative damage, and will help determine the mechanism of protection or damage by phytochemicals.
采用改良的彗星试验来研究膳食抗氧化剂在亚细胞环境中的体外作用。分离出人淋巴细胞，将其包埋在琼脂糖凝胶中，在含有Triton X-100的高离子强度溶液中裂解，然后与不同浓度的抗氧化剂孵育30分钟。洗涤凝胶，并对经45微摩尔过氧化氢孵育5分钟应激的细胞和未应激的平行细胞进行彗星试验。结果表明，α-生育酚对氧化应激具有保护作用，而咖啡酸没有保护作用，且在高浓度（100微摩尔）时会导致DNA损伤增加。槲皮素的结果表明有直接损伤作用，但未达到统计学显著性。然而，在低浓度（3.1微摩尔）时，槲皮素似乎具有保护作用。因此，一些先前在“标准”全细胞彗星试验中显示具有保护作用的膳食抗氧化剂，在这种裂解细胞版本中会导致DNA损伤。细胞膜可能在限制这些“双刃剑”抗氧化剂进入细胞方面起重要作用。此外，绝对浓度以及互补或协同的细胞内抗氧化剂的存在可能会描绘出一种假定抗氧化剂的作用类型。我们建议，结合标准彗星试验使用这种裂解细胞版本，对于评估细胞膜和细胞内系统对DNA氧化损伤易感性的影响是有用的，并且将有助于确定植物化学物质的保护或损伤机制。
