Department of Environmental and Occupational Health, The Winthrop P. Rockefeller Cancer Institute at The University of Arkansas for Medical Sciences, Little Rock, AR, United States.
Toxicology. 2013 Nov 16;313(2-3):145-150. doi: 10.1016/j.tox.2012.11.012. Epub 2012 Dec 3.
Humans are constantly exposed to mixtures, such as tobacco smoke, exhaust from diesel, gasoline or new bio-fuels, containing several 1000 compounds, including many known human carcinogens. Covalent binding of reactive compounds or their metabolites to DNA and formation of stable adducts is believed to be the causal link between exposure and carcinogenesis. DNA and protein adducts are well established biomarkers for the internal dose of reactive compounds or their metabolites and are an integral part of science-based risk assessment. However, technical limitations have prevented comprehensive detection of a broad spectrum of adducts simultaneously. Therefore, most studies have focused on measurement of abundant individual adducts. These studies have produced valuable insight into the metabolism of individual carcinogens, but they are insufficient for risk assessment of exposure to complex mixtures. To overcome this limitation, we present herein proof-of-principle for comprehensive exposure assessment, using N-terminal valine adduct profiles as a biomarker. The reported method is based on our previously established immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with modification to enrich all N-terminal valine alkylated peptides. The method was evaluated using alkylated peptide standards and globin reacted in vitro with alkylating agents (1,2-epoxy-3-butene, 1,2:3,4-diepoxybutane, propylene oxide, styrene oxide, N-ethyl-N-nitrosourea and methyl methanesulfonate), known to form N-terminal valine adducts. To demonstrate proof-of-principle, the method was successfully applied to globin from mice treated with four model compounds. The results suggest that this novel approach might be suitable for in vivo biomonitoring.
人类不断暴露于混合物中，例如烟草烟雾、柴油、汽油或新型生物燃料的废气，其中包含数千种化合物，包括许多已知的人类致癌物。人们认为，反应性化合物或其代谢物与 DNA 的共价结合以及稳定加合物的形成是暴露与致癌之间的因果关系。DNA 和蛋白质加合物是反应性化合物或其代谢物的内剂量的良好生物标志物，是基于科学的风险评估的组成部分。然而，技术限制阻止了对广泛的加合物进行全面检测。因此，大多数研究都集中在测量丰富的单个加合物上。这些研究为个别致癌物的代谢提供了有价值的见解，但不足以进行复杂混合物暴露的风险评估。为了克服这一限制，我们在此提出了使用 N 末端缬氨酸加合物谱作为生物标志物进行全面暴露评估的原理验证。该方法基于我们之前建立的免疫亲和液相色谱-串联质谱（LC-MS/MS）方法，并进行了修改以富集所有 N 末端缬氨酸烷基化肽。该方法使用烷基化肽标准品和体外与烷基化剂（1,2-环氧-3-丁烯、1,2:3,4-二环氧丁烷、环氧丙烷、氧化苯乙烯、N-乙基-N-亚硝基脲和甲基甲烷磺酸盐）反应的球蛋白进行了评估，已知这些物质会形成 N 末端缬氨酸加合物。为了证明原理验证，该方法成功应用于用四种模型化合物处理的小鼠球蛋白。结果表明，这种新方法可能适合体内生物监测。
