Detection of the acrolein-derived cyclic DNA adduct by a quantitative 32P-postlabeling/solid-phase extraction/HPLC method: blocking its artifact formation with glutathione.
Acrolein (Acr), a hazardous air pollutant, reacts readily with deoxyguanosine (dG) in DNA to produce cyclic 1, N2-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 32P-postlabeling/solid-phase extraction/HPLC method was developed by optimizing the solid-phase extraction and the 32P-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.
Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA.
Armaghan
A
Emami
Marcin
M
Dyba
Amrita K
AK
Cheema
Jishen
J
Pan
Raghu G
RG
Nath
Fung-Lung
FL
Chung
D000171:Acrolein / Q000737:chemistry*; D000818:Animals; D016477:Artifacts; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018736:DNA Adducts / Q000032:analysis*; D005978:Glutathione / Q000494:pharmacology*; D019073:HT29 Cells; D006801:Humans; D010761:Phosphorus Radioisotopes; D052616:Solid Phase Extraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D000171:Acrolein; D005978:Glutathione