Search

Acrolein (Acr) is a ubiquitous environmental pollutantfound incigarette smoke and automobile exhaust. It can also be produced endogenouslyby oxidation of polyunsaturated fatty acids. The Acr-derived 1,N2-propanodeoxyguanosine (Acr-dG) adducts inDNA are mutagenic lesions that are potentially involved in human cancers.In this study, monoclonal antibodies were raised against Acr-dG adductsand characterized using ELISA. They showed strong reactivity and specificitytoward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N2-propanodeoxyguanosines, and weak or no reactivity toward1,N6-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 fordetecting these adducts directly in cells; second, a highly sensitivedirect ELISA assay for measuring Acr-dG in cells and tissues usingonly 1 μg of DNA without DNA digestion and sample enrichment;and third, a competitive ELISA for better quantitative measurementof Acr-dG levels in DNA samples. The assays were validated using Acr-treatedHT29 cell DNA samples or calf thymus DNA, and the results were confirmedby LC–MS/MS-MRM. An immunohistochemical assay was also developedto detect and visualize Acr-dG in HT29 cells as well as in human oralcells. These antibody-based methods provide useful tools for the studiesof Acr-dG as a cancer biomarker and of the molecular mechanisms bywhich cells respond to Acr-dG as a ubiquitous DNA lesion. [ABSTRACT FROM AUTHOR]