Borthakur Gayatri, Butryee Chaniphun, Stacewicz-Sapuntzakis Maria, Bowen Phyllis E
Department of Kinesiology and Nutrition, University of Illinois at Chicago M/C 517, 1919 West Taylor Street, Chicago, IL 60612, USA.
Cancer Epidemiol Biomarkers Prev. 2008 Jan;17(1):212-9. doi: 10.1158/1055-9965.EPI-07-0706.
The extent of oxidative DNA damage is considered a biomarker of carcinogenic process and could be investigated in population studies using easily obtained cells. The oxidized DNA base adduct 8-hydroxy-2-deoxyguanosine (8-OHdG) released by enzymatic hydrolysis of DNA is commonly assayed by high performance liquid chromatography with electrochemical detection. It is expressed as a ratio of 8-OHdG to unoxidized deoxyguanosine. We modified and improved this method, determined the optimal time for harvesting buccal mucosa cells (BMC), assessed whether they mirror peripheral circulating blood cell DNA damage, and compared the anticoagulants, heparin, and EDTA for consistency in measurement of leukocyte 8-OHdG. Thirty-one healthy participants, randomized into two groups, donated BMC and blood samples. Samples were collected at baseline and either 3 or 7 days after baseline. Results showed no correlation between 8-OHdG/deoxyguanosine ratios in BMC and peripheral blood leukocytes at any time point regardless of harvest time. BMC had much higher oxidative DNA damage, but displayed a 25.6% reduction in the oxidized DNA adduct level (P < 0.04) at 3 days after baseline. Leukocytes collected in heparin and EDTA had similar 8OHdG/deoxyguanosine ratios; however, EDTA was preferred, as it produced a clean nuclear pellet without hemoglobin contamination, and the results were less variable. This improved assay shows within subject stability over time in both leukocyte and BMC DNA damage, increasing the probability that small intervention differences can be detected in healthy subjects. Buccal cells provide an accessible pool of epithelial cells that represents higher levels of DNA damage than circulating leukocytes.
氧化DNA损伤的程度被视为致癌过程的生物标志物，并且可以在人群研究中使用易于获取的细胞进行调查。通过DNA酶促水解释放的氧化DNA碱基加合物8-羟基-2'-脱氧鸟苷（8-OHdG）通常采用高效液相色谱-电化学检测法进行测定。它表示为8-OHdG与未氧化脱氧鸟苷的比率。我们改进了该方法，确定了采集颊黏膜细胞（BMC）的最佳时间，评估了它们是否反映外周循环血细胞DNA损伤，并比较了抗凝剂肝素和乙二胺四乙酸（EDTA）在测量白细胞8-OHdG时的一致性。31名健康参与者被随机分为两组，捐献了BMC和血液样本。在基线以及基线后3天或7天采集样本。结果显示，无论采集时间如何，在任何时间点BMC和外周血白细胞中的8-OHdG/脱氧鸟苷比率之间均无相关性。BMC具有更高的氧化DNA损伤，但在基线后3天时氧化DNA加合物水平降低了25.6%（P<0.04）。用肝素和EDTA采集的白细胞具有相似的8-OHdG/脱氧鸟苷比率；然而，EDTA更受青睐，因为它产生的细胞核沉淀纯净，无血红蛋白污染，且结果变异性较小。这种改进的检测方法显示，在白细胞和BMC DNA损伤方面，受试者内部随时间具有稳定性，增加了在健康受试者中检测到微小干预差异的可能性。颊细胞提供了一个易于获取的上皮细胞库，其代表的DNA损伤水平高于循环白细胞。
