32P-postlabeling of acrolein-deoxyguanosine adducts in DNA after nuclease P1 digestion.
In order to study the relationship between the level of acrolein-DNA adducts and their biological effects, sensitive methods are needed to quantitate DNA adducts. 32P-postlabeling is one such method that has been widely used and we have adapted the technique to detect acrolein-deoxyguanosine adducts. Adducts formed by the reaction of acrolein and deoxyguanosine-3'-monophosphate were isolated by HPLC. Based on their UV spectra and cochromatography with standards after dephosphorylation with acid phosphatase, these adducts were identified as the nucleotide equivalents of cyclic 1,N2-propanodeoxyguanosine adducts formed by acrolein that have been described by Chung et al. [15]. As nucleotides, the adducts were good substrates for polynucleotide kinase-mediated transfer of phosphate from ATP and were able to be detected by 32P-postlabeling. These adducts were resistant to the activity of nuclease P1 and dinucleoside monophosphates in the form d(G*pN) where G* is the acrolein-guanine adduct also resisted digestion by nuclease P1. Digestion of DNA by nuclease P1 and acid phosphatase resulted in the conversion of normal nucleotides to nucleosides and selective enrichment of the adducts as dinucleoside monophosphates. Using nuclease P1/acid phosphatase digestion, followed by 32P-postlabeling and TLC separation, levels of the two adducts in acrolein-treated DNA were found to be about 6185 and 19,222 nmol/mol.
Maccubbin
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Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, NY 14263.
Lee
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Struck
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Gurtoo
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D000135:Acid Phosphatase / Q000378:metabolism; D000171:Acrolein / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003500:Cyclization; D004247:DNA / Q000378:metabolism*; D003849:Deoxyguanosine / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism; D007553:Isotope Labeling; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D015226:Dinucleoside Phosphates; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D000171:Acrolein; D000255:Adenosine Triphosphate; D004247:DNA; D000135:Acid Phosphatase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D003849:Deoxyguanosine