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The acrolein derived cyclic 1,N 2 -propanodeoxyguanosine adduct (Acr-dG), formed primarily from ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) under oxidative conditions, while proven to be mutagenic, is potentially involved in DHA-induced apoptosis. The latter may contribute to the chemopreventive effects of DHA. Previous studies have shown that the levels of Acr-dG are correlated with apoptosis induction in HT29 cells treated with DHA. Because Acr-dG is shown to be repaired by the nucleotide excision repair (NER) pathway, to further investigate the role of Acr-dG in apoptosis, in this study, NER-deficient XPA and its isogenic NER-proficient XAN1 cells were treated with DHA. The Acr-dG levels and apoptosis were sharply increased in XPA cells, but not in XAN1 cells when treated with 125 μM of DHA. Because DHA can induce formation of various DNA damage, to specifically investigate the role of Acr-dG in apoptosis induction, we treated XPA knockdown HCT116 + ch3 cells with acrolein. The levels of both Acr-dG and apoptosis induction increased significantly in the XPA knockdown cells. These results clearly demonstrate that NER deficiency induces higher levels of Acr-dG in cells treated with DHA or acrolein and sensitizes cells to undergo apoptosis in a correlative manner. Collectively, these results support that Acr-dG, a ubiquitously formed mutagenic oxidative DNA adduct, plays a role in DHA-induced apoptosis and suggest that it could serve as a biomarker for the cancer preventive effects of DHA. [ABSTRACT FROM AUTHOR]

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]