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sup 32 P-Postlabeling measurement of adenine N-1-oxide in cellular DNA exposed to hydrogen peroxide

Journal Article · · Chemical Research in Toxicology; (USA)
OSTI ID:6411490
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  1. Centre d'Etudes Nucleaires de Grenoble (France)
A {sup 32}P-postlabeling assay has been developed for monitoring the formation within DNA of adenine N-1-oxide, the specific H{sub 2}O{sub 2}-mediated oxidation product under nonradical conditions. This has required the chemical synthesis of both 2{prime}-deoxyadenosine N-1-oxide 3{prime}-monophosphate and 2{prime}-deoxyadenosine N-1-oxide 5{prime}-monophosphate, the substrate and the product of polynucleotide kinase mediated phosphorylation. Isolation of the substrate from the other nucleotides was found to be necessary in order to improve the rate of phosphorylation and to prevent self-radiolysis processes. ({sup 32}P)-2{prime}-deoxyadenosine N-1-oxide 5{prime}-monophosphate was obtained after successive ion exchange and reverse-phase HPLC and was characterized by a microreaction. The sensitivity of the assay, which is close to 1 modified adenine N-1-oxide/10{sup 6} bases, allowed the determination of this lesion within the DNA of cells exposed to nonlethal levels of H{sub 2}O{sub 2}.
OSTI ID:
6411490
Journal Information:
Chemical Research in Toxicology; (USA), Journal Name: Chemical Research in Toxicology; (USA) Vol. 3:2; ISSN CRTOE; ISSN 0893-228X
Country of Publication:
United States
Language:
English