Detection of the acrolein-derived cyclic DNA adduct by a quantitative 32P-postlabeling/solid-phase extraction/HPLC method: blocking its artifact formation with glutathione
| Autor: | Amrita K. Cheema, Armaghan Emami, Raghu G. Nath, Marcin Dyba, Fung-Lung Chung, Jishen Pan |
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| Rok vydání: | 2007 |
| Předmět: |
musculoskeletal diseases
Spectrometry Mass Electrospray Ionization Biophysics Biochemistry Article Adduct chemistry.chemical_compound DNA Adducts immune system diseases DNA adduct Deoxyguanosine Animals Humans Solid phase extraction Acrolein skin and connective tissue diseases Molecular Biology Chromatography High Pressure Liquid Detection limit Chromatography Mutagenesis Solid Phase Extraction Cell Biology Glutathione chemistry Cattle Artifacts HT29 Cells Phosphorus Radioisotopes DNA |
| Zdroj: | Analytical biochemistry. 374(1) |
| ISSN: | 0003-2697 |
| Popis: | Acrolein (Acr), a hazardous air pollutant, reacts readily with deoxyguanosine (dG) in DNA to produce cyclic 1, N 2 -propanodeoxyguanosine adducts (Acr-dG). Studies demonstrate that these adducts are detected in vivo and may play a role in mutagenesis and carcinogenesis. In the study described here, a quantitative 32 P-postlabeling/solid-phase extraction/HPLC method was developed by optimizing the solid-phase extraction and the 32 P-postlabeling conditions for analysis of Acr-dG in DNA samples with a detection limit of 0.1 fmol. It was found that Acr-dG can form as an artifact during the assay. Evidence obtained from mass spectrometry indicates that the Acr in water used in the assay is a likely source of artifact formation of Acr-dG. The formation of Acr-dG as an artifact can be effectively blocked by adding glutathione (GSH) to the DNA sample to be analyzed. In addition, Acr-dG was detected as a contaminant in the commercial dG and dT 3′-monophosphate samples. Finally, this method was used to detect Acr-dG in calf thymus and human colon HT29 cell DNA with an excellent linear quantitative relationship. |
| Databáze: | OpenAIRE |
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