Li H, Cui S, Wang S, Jiang X, Zhang S, Zhang R, Fu P P, Sun X
Key Laboratory of Chemical Safety and Health, Chinese Center for Disease Control and Prevention , Beijing , P. R. China.
Free Radic Res. 2015;49(9):1049-54. doi: 10.3109/10715762.2015.1006213. Epub 2015 Jul 1.
Etheno-DNA adducts are generated by interaction of cellular DNA with exogenous environmental carcinogens and end products of lipid peroxidation. It has been determined that 1,N(6)-etheno-2'-deoxyadenosine (εdA) and 3,N(4)-etheno-2'-deoxycytidine (εdC) adducts formed in human white blood cells can be used to serve as biomarkers of genetic damage mediated by oxidative stress. In this study, we developed an ultrasensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method used to detect and quantify εdA and dC adducts in human white blood cells. The percent recoveries of εdA and dC adducts were found to be 88.9% ± 2.8 and 95.7% ± 3.7, respectively. The detection limits were ∼ 1.45 fmol for εdA and ∼ 1.27 fmol for εdC in 20 μg of human white blood cell DNA samples, both εdA and εdC adducts could be detected using only ∼ 5 μg of DNA per sample. For validation of the method, 34 human blood cell DNA samples were assayed and the results revealed a significant difference (P < 0.01) between levels (fmol/μg DNA) of 0.82 ± 0.83 (standard deviation [SD]) (range: 0.15-3.11) for εdA, 3.28 ± 3.15 (SD) (range: 0.05-9.6) for εdC in benzene-exposed workers; and 0.04 ± 0.08 (SD) (range: 0.0-0.27) for εdA and 0.77 ± 1.02 (SD) (range: 0.10-4.11) for εdC in non-benzene-exposed workers. Our method shows a high sensitivity and specificity when applied to small amounts of human white blood cell DNA samples; background levels of εdA and εdC could be reproducibly detected. The ultrasensitive and simple detection method is thus suitable for applications in human biomonitoring and molecular epidemiology studies.
乙烯基 - DNA加合物是由细胞DNA与外源性环境致癌物以及脂质过氧化终产物相互作用产生的。已确定在人类白细胞中形成的1,N(6)-乙烯基-2'-脱氧腺苷（εdA）和3,N(4)-乙烯基-2'-脱氧胞苷（εdC）加合物可作为氧化应激介导的遗传损伤的生物标志物。在本研究中，我们开发了一种超灵敏的超高效液相色谱 - 串联质谱（UPLC-MS/MS）方法，用于检测和定量人类白细胞中的εdA和εdC加合物。发现εdA和εdC加合物的回收率分别为88.9% ± 2.8和95.7% ± 3.7。在20μg人类白细胞DNA样品中，εdA的检测限约为1.45 fmol，εdC的检测限约为1.27 fmol，每个样品仅使用约5μg DNA即可检测到εdA和εdC加合物。为验证该方法，对34份人类血细胞DNA样品进行了检测，结果显示，苯暴露工人中εdA的水平（fmol/μg DNA）为0.82 ± 0.83（标准差[SD]）（范围：0.15 - 3.11），εdC为3.28 ± 3.15（SD）（范围：0.05 - 9.6）；非苯暴露工人中εdA为0.04 ± 0.08（SD）（范围：0.0 - 0.27），εdC为0.77 ± 1.02（SD）（范围：0.10 - 4.11），两者存在显著差异（P < 0.01）。当应用于少量人类白细胞DNA样品时，我们的方法显示出高灵敏度和特异性；可以重复检测到εdA和εdC的背景水平。因此，这种超灵敏且简单的检测方法适用于人类生物监测和分子流行病学研究。
