Detection of 1,N6-propanodeoxyadenosine in acrolein-modified polydeoxyadenylic acid and DNA by 32P postlabeling.
The interaction of acrolein, an alpha,beta-unsaturated aldehyde, with polydeoxyadenylic acid and DNA has been investigated using 32P-postlabeling analysis. In preliminary experiments, polydeoxyadenylic acid was incubated with excess acrolein and then digested to 3' monophosphates prior to transfer of 32P from [gamma-32P]ATP with T4 polynucleotide kinase. The 3',5'-bisphosphates were 3'-dephosphorylated prior to two-dimensional thin layer chromatography on polyethyleneimine-cellulose layers. Autoradiography provided evidence for the formation of one extra spot of radioactivity, compared to the control. To determine the adduct structure, deoxyadenosine-5'-monophosphate was incubated with a 3-fold excess of acrolein. This material was mixed with a 32P-labeled digest of acrolein-polydeoxyadenylic acid, and the sample was analyzed by ion-pair high performance liquid chromatography. The spot of 32P observed by thin layer chromatography co-eluted with the major product of the acrolein nucleotide reaction mixture, which was purified by ion-pair high performance liquid chromatography. Two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry showed the adduct to be 3-(2'-deoxyribosyl-5'-monophosphatyl)-7,8,9-trihydro-9-hydro xy- pyrimido[2,3-i]purine(1,N6-propanodeoxyadenosine-5'-monophosphate) . High performance liquid chromatography was used to fractionate digests of acrolein-modified DNA prior to detection of this exocyclic adduct by 32P-postlabeling.
Smith
R A
RA
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198.
Williamson
D S
DS
Cerny
R L
RL
Cohen
S M
SM
D000171:Acrolein / Q000633:toxicity*; D000447:Aldehydes / Q000633:toxicity*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004249:DNA Damage; D003839:Deoxyadenosines / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011061:Poly A
D000447:Aldehydes; D003839:Deoxyadenosines; C064049:1,N(6)-propanodeoxyadenosine; D011061:Poly A; C015465:poly(dA); D000171:Acrolein