title: Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies.
abstract: 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.
authors.affiliation: Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, United States.
authors.forename: Jishen
authors.initials: J
authors.lastname: Pan
authors.forename: Bisola
authors.initials: B
authors.lastname: Awoyemi
authors.forename: Zhuoli
authors.initials: Z
authors.lastname: Xuan
authors.forename: Priya
authors.initials: P
authors.lastname: Vohra
authors.forename: Hsiang-Tsui
authors.initials: HT
authors.lastname: Wang
authors.forename: Marcin
authors.initials: M
authors.lastname: Dyba
authors.forename: Emily
authors.initials: E
authors.lastname: Greenspan
authors.forename: Ying
authors.initials: Y
authors.lastname: Fu
authors.forename: Karen
authors.initials: K
authors.lastname: Creswell
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Deborah
authors.initials: D
authors.lastname: Berry
authors.forename: Moon-Shong
authors.initials: MS
authors.lastname: Tang
authors.forename: Fung-Lung
authors.initials: FL
authors.lastname: Chung
mesh_terms: D000171:Acrolein / Q000276:immunology*; D000393:Air Pollutants / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015415:Biomarkers; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D018736:DNA Adducts / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D019073:HT29 Cells; D006801:Humans; D015603:Keratinocytes; D051379:Mice; D008807:Mice, Inbred BALB C; D009055:Mouth / Q000166:cytology; D053719:Tandem Mass Spectrometry
chemical_list: D000393:Air Pollutants; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D018736:DNA Adducts; D000171:Acrolein