Masonic Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street SE, Minneapolis, Minnesota 55455, USA.
Chem Res Toxicol. 2011 Jan 14;24(1):119-24. doi: 10.1021/tx100321y. Epub 2010 Nov 22.
Cigarette smoking is a major source of human exposure to acrolein, a widespread environmental pollutant and toxicant that is also formed endogenously through metabolism of amino acids and polyamines and lipid peroxidation. Acrolein reacts with DNA, producing two pairs of regioisomeric 1,N(2)-propanodeoxyguanosine adducts: (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo). Previous studies indicate that these adducts might be involved in producing mutations in the p53 tumor suppressor gene, as observed in lung tumors in smokers, but there are only limited published data comparing acrolein-DNA adducts in smokers and nonsmokers. In this study, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to analyze Acr-dGuo adducts in human leukocyte DNA. The potential for artifactual formation was found in two steps of the assay: DNA isolation and DNA hydrolysis. This was eliminated by employing a Ficoll-Hypaque double density gradient to obtain leukocytes free of erythrocyte contamination and by adding glutathione to scavenge acrolein present in H(2)O. The accuracy and precision of the method were confirmed. Acr-dGuo adducts were analyzed in leukocyte DNA from 25 smokers and 25 nonsmokers. γ-OH-Acr-dGuo was the predominant isomer in all samples, while α-OH-Acr-dGuo was detected in only three subjects. There was no significant difference between the total Acr-dGuo levels in smokers (7.4 ± 3.4 adducts/10(9) nucleotides) and nonsmokers (9.8 ± 5.5 adducts/10(9) nucleotides). Although our study is limited in size, these results, together with the results of previous analyses of acrolein-derived mercapturic acids in the urine of smokers and nonsmokers, suggest that glutathione conjugation effectively removes acrolein from external exposures such as cigarette smoking, protecting leukocyte DNA from damage.
吸烟是人类接触丙烯醛的主要来源，丙烯醛是一种广泛存在的环境污染物和毒物，也可以通过氨基酸和多胺的代谢以及脂质过氧化反应内源性形成。丙烯醛与 DNA 反应，产生两对区域异构体 1,N(2)-丙酰脱氧鸟苷加合物：(6R/S)-3-(2'-脱氧核糖基-1'-基）-5,6,7,8-四氢-6-羟基嘧啶并[1,2-a]嘌呤-10(3H)酮（α-OH-Acr-dGuo）和(8R/S)-3-(2'-脱氧核糖基-1'-基）-5,6,7,8-四氢-8-羟基嘧啶并[1,2-a]嘌呤-10(3H)酮（γ-OH-Acr-dGuo）。先前的研究表明，这些加合物可能参与了 p53 肿瘤抑制基因的突变，如吸烟者的肺癌中观察到的那样，但比较吸烟者和非吸烟者中丙烯醛-DNA 加合物的已发表数据有限。在这项研究中，我们开发了一种液相色谱-电喷雾电离-串联质谱（LC-ESI-MS/MS）方法来分析人白细胞 DNA 中的 Acr-dGuo 加合物。在测定的两个步骤中发现了人为形成的可能性：DNA 分离和 DNA 水解。通过使用 Ficoll-Hypaque 双密度梯度获得不含红细胞污染的白细胞，并添加谷胱甘肽清除 H(2)O 中存在的丙烯醛，从而消除了这种可能性。该方法的准确性和精密度得到了确认。在 25 名吸烟者和 25 名非吸烟者的白细胞 DNA 中分析了 Acr-dGuo 加合物。在所有样品中，γ-OH-Acr-dGuo 都是主要异构体，而 α-OH-Acr-dGuo 仅在三个样品中检测到。吸烟者（7.4±3.4 加合物/10(9)核苷酸）和非吸烟者（9.8±5.5 加合物/10(9)核苷酸）之间的总 Acr-dGuo 水平没有显著差异。尽管我们的研究规模有限，但这些结果与先前对吸烟者和非吸烟者尿液中丙烯醛衍生的硫尿酸的分析结果一起表明，谷胱甘肽缀合可有效清除吸烟等外部暴露源中的丙烯醛，从而保护白细胞 DNA 免受损伤。
