Department of Medicinal Chemistry and the Masonic Cancer Center, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Chem Res Toxicol. 2013 Oct 21;26(10):1486-97. doi: 10.1021/tx400213m. Epub 2013 Sep 12.
1,3-Butadiene (BD) is a high volume industrial chemical commonly used in polymer and rubber production. It is also present in cigarette smoke, automobile exhaust, and urban air, leading to widespread exposure of human populations. Upon entering the body, BD is metabolized to electrophilic epoxides, 3,4-epoxy-1-butene (EB), diepoxybutane (DEB), and 3,4-epoxy-1,2-diol (EBD), which can alkylate DNA nucleobases. The most abundant BD epoxide, EBD, modifies the N7-guanine positions in DNA to form N7-(2, 3, 4-trihydroxybut-1-yl) guanine (N7-THBG) adducts, which can be useful as biomarkers of BD exposure and metabolic activation to DNA-reactive epoxides. In the present work, a capillary HPLC-high resolution ESI⁺-MS/MS (HPLC-ESI⁺-HRMS/MS) methodology was developed for accurate, sensitive, and reproducible quantification of N7-THBG in cell culture and in human white blood cells. In our approach, DNA is subjected to neutral thermal hydrolysis to release N7-guanine adducts from the DNA backbone, followed by ultrafiltration, solid-phase extraction, and isotope dilution HPLC-ESI⁺-HRMS/MS analysis on an Orbitrap Velos mass spectrometer. Following method validation, N7-THBG was quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of DEB and in DNA isolated from blood of smokers, nonsmokers, individuals participating in a smoking cessation program, and occupationally exposed workers. N7-THBG concentrations increased linearly from 31.4 ± 4.84 to 966.55 ± 128.05 adducts per 10⁹ nucleotides in HT1080 cells treated with 1-100 μM DEB. N7-THBG amounts in leukocyte DNA of nonsmokers, smokers, and occupationally exposed workers were 7.08 ± 5.29, 8.20 ± 5.12, and 9.72 ± 3.80 adducts per 10⁹ nucleotides, respectively, suggesting the presence of an endogenous or environmental source for this adduct. The availability of sensitive HPLC-ESI⁺-HRMS/MS methodology for BD-induced DNA adducts in humans will enable future population studies of interindividual and ethnic differences in BD bioactivation to DNA-reactive epoxides.
1,3-丁二烯（BD）是一种高用量的工业化学品，常用于聚合物和橡胶生产。它也存在于香烟烟雾、汽车尾气和城市空气中，导致人类群体广泛暴露。进入人体后，BD 被代谢为亲电环氧化物、3,4-环氧-1-丁烯（EB）、双环氧丁烷（DEB）和 3,4-环氧-1,2-二醇（EBD），这些物质可以烷基化 DNA 碱基。BD 最丰富的环氧化物，EBD，将 DNA 中的 N7-鸟嘌呤位置修饰为 N7-（2,3,4-三羟基丁基）鸟嘌呤（N7-THBG）加合物，这可以作为 BD 暴露和代谢激活为 DNA 反应性环氧化物的生物标志物。在本工作中，开发了一种毛细管 HPLC-高分辨率 ESI⁺-MS/MS（HPLC-ESI⁺-HRMS/MS）方法，用于细胞培养和人白细胞中 N7-THBG 的准确、灵敏和可重现定量。在我们的方法中，DNA 经过中性热水解，从 DNA 主链上释放 N7-鸟嘌呤加合物，然后进行超滤、固相萃取和同位素稀释 HPLC-ESI⁺-HRMS/MS 分析，在 Orbitrap Velos 质谱仪上进行。经过方法验证，DEB 处理的人纤维肉瘤（HT1080）细胞和吸烟者、不吸烟者、参加戒烟计划者以及职业暴露工人血液中分离的 DNA 中的 N7-THBG 进行了定量。HT1080 细胞用 1-100 μM DEB 处理时，N7-THBG 浓度从 31.4 ± 4.84 线性增加至 966.55 ± 128.05 加合物/10⁹ 核苷酸。不吸烟者、吸烟者和职业暴露工人白细胞 DNA 中的 N7-THBG 含量分别为 7.08 ± 5.29、8.20 ± 5.12 和 9.72 ± 3.80 加合物/10⁹ 核苷酸，表明该加合物存在于内源性或环境来源。BD 诱导的人类 DNA 加合物的灵敏 HPLC-ESI⁺-HRMS/MS 方法的可用性将使未来能够对 BD 生物活化为 DNA 反应性环氧化物的个体和种族差异进行人群研究。
