Detection of Acrolein-Derived Cyclic DNA Adducts in Human Cells by Monoclonal Antibodies

作者：Pan Jishen; Awoyemi Bisola; Xuan Zhuoli; Vohra Priya; Wang Hsiang Tsui; Dyba Marcin; Greenspan Emily; Fu Ying; Creswell Karen; Zhang Lihua; Berry Deborah; Tang Moon Shong; Chung Fung Lung^*

来源：Chemical Research in Toxicology, 2012, 25(12): 2788-2795.

DOI：10.1021/tx3004104

Acrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N-2-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity toward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N-2-propanodeoxyguanosines, and weak or no reactivity toward 1,N-6-ethenodeoxyadenosine and 8-oxo-deoxyguanosine. Using these antibodies, we developed assays to detect Acr-dG in vivo: first, a simple and quick FACS-based assay for detecting these adducts directly in cells; second, a highly sensitive direct ELISA assay for measuring Acr-dG in cells and tissues using only 1 jig of DNA without DNA digestion and sample enrichment; and third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA samples. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA, and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion.

• 出版日期2012-12