Lipid peroxidation of polyunsaturated fatty acids (PUFAs) is an endogenous source of α,β-unsaturated aldehydes that react with DNA producing a variety of cyclic adducts. The mutagenic cyclic adducts, specifically those derived from oxidation of ω-6 PUFAs, may contribute to the cancer promoting activities associated with ω-6 PUFAs. ( E)-4-Hydroxy-2-nonenal (HNE) is a unique product of ω-6 PUFAs oxidation. HNE reacts with deoxyguanosine (dG) yielding mutagenic 1, N-propanodeoxyguanosine adducts (HNE-dG). Earlier studies showed HNE can also be oxidized to its epoxide (EH), and EH can react with deoxyadenosine (dA) forming the well-studied εdA and the substituted etheno adducts. Using a liquid chromatography-based tandem mass spectroscopic (LC-MS/MS) method, we previously reported the detection of EH-derived 7-(1',2'-dihydroxyheptyl)-1, N-ethenodeoxyadenosine (DHHεdA) as a novel endogenous background adduct in DNA from rodent and human tissues. The formation, repair, and mutagenicity of DHHεdA and its biological consequences in cells have not been investigated. To understand the roles of DHHεdA in carcinogenesis, it is important to develop an immuno-based assay to detect DHHεdA in cells and tissues. In this study we describe the development of monoclonal antibodies specifically against DHHεdA and its application to detect DHHεdA in human cells.
Dietary polyunsaturated fatty acids (ω-3 and ω-6 PUFAs) play a role in certain human cancers. The oxidation of PUFAs produces reactive α,β-unsaturated aldehydes (enals) that can modify DNA producing mutagenic lesions. Of particular interest, 7-(1′,2′-dihydroxyheptyl)-1,N6- ethenodeoxyadenosine (DHHϵdA) is a novel DNA adduct in vitro and in vivo derived from lipid peroxidation of ω-6 PUFAs. Despite its recent identification in vivo, the role of DHHϵdA in tumorigenesis is not yet known. Previously, the endogenously formed DHHϵdA was detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). Due to its low levels in vivo (1-30 adducts/109 DNA bases), a relatively high quantity of DNA is needed for its detection and quantification. In this study we developed a monoclonal antibody against DHHϵdA in order to detect and quantify this adduct in cells and tissues. Two antigens were synthesized by conjugating activated hapten: 5′-carboxy-7-(1′,2′-dihydroxyheptyl) adenosine (5′-carboxy DHHϵA) to BSA and KLH carrier proteins. KLH-conjugated hapten was used for mice immunization, whereas BSA-based hapten was used to test for an immune response. After immunization and several screenings, 6 monoclonal cell lines were chosen for further tests. Normal and competitive ELISA was performed to characterize the antibodies from all cell lines. Competitive ELISA revealed no cross-reactivity towards normal nucleosides and α- and γ-OHPdG, the cyclic 1,N2-propano adduct derived from acrolein, and 8-oxo-dG for all of the tested antibodies. However, we observed a weak (< 1:100) specificity for HNE-dG, an adduct derived from (E)-4-hydroxy-2-nonenal produced from oxidized ω-6 PUFAs. Because of the relatively low level of endogenous HNE-dG compared to DHHϵdA, this weak cross-reactivity should not affect the specificity of the developed antibody to detect DHHϵdA. Two cell lines were discarded after antibodies from them showed moderate competitive effects against 1,N6-etheno-2′-deoxyadenosine (ϵdA). From the remaining 4 cell lines the best one was chosen to produce purified antibody. We believe that this antibody will be a useful tool in the studies of the role of DHHϵdA as an endogenous DNA lesion in cancer development associated with ω-6 PUFAs. This work was supported by the NCI grant CA134892. Citation Format: Marcin Dyba, Brandon Da Silva, Jishen Pan, Fung-Lung Chung. Development of novel monoclonal antibodies for a cyclic DNA adduct derived from oxidation of ω-6 polyunsaturated fatty acids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2733. doi:10.1158/1538-7445.AM2015-2733
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