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

• Published in: Chemical research in toxicology 2012-12, Vol.25 (12), p.2788-2795
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Acrolein - immunology; Air Pollutants - immunology; Animals; Antibodies, Monoclonal - immunology; Biomarkers; Cells, Cultured; Chromatography, Liquid; DNA Adducts - immunology; Enzyme-Linked Immunosorbent Assay; HT29 Cells; Humans; Keratinocytes; Mice; Mice, Inbred BALB C; Mouth - cytology; Tandem Mass Spectrometry
• ISSN: 0893-228X; 1520-5010
• 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 μg 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.