title: Further development of 32P-postlabeling for the detection of alkylphosphotriesters: evidence for the long-term nonrandom persistence of ethyl-phosphotriester adducts in vivo.
abstract: DNA phosphate oxygens are sites for alkylation leading to phosphotriester adducts (PTEs). PTEs are reported to be both abundant and persistent and so may serve as long-term markers of genotoxicity. Previously, we reported a 32P-postlabeling assay for the specific detection of PTEs plus identification of nucleosides located 5' to PTEs. Using this, we demonstrated the nonrandom nature of ethyl-PTEs (Et-PTEs) in vivo, these results being suggestive of either the nonrandom formation of Et-PTEs in vivo or sequence specific Et-PTE repair. Presently, we report the further development and validation of the 32P-postlabeling assay, to permit the more straightforward determination of nucleosides 5' to PTEs and, using this, have investigated the long-term persistence of PTEs in vivo. Analysis of liver DNA of mice treated in vivo with N-nitrosodiethylamine reveals an initial decline in the level of Et-PTEs (t1/2<24 h) as well as their nonrandom persistence for the duration of the time course, with approximately 37 and approximately 15% of the initial Et-PTEs remaining 4 and 56 days after treatment, respectively. From this, we conclude that Et-PTEs are suitable as long-term markers of genotoxic exposure and that putative PTE repair is not responsible for their nonrandom manifestation. However, the possibility of active repair contributing to the initial decline of Et-PTEs is considered.
authors.affiliation: Department of Cancer Studies & Molecular Medicine, Biocenter, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Le Pla
authors.forename: Yves
authors.initials: Y
authors.lastname: Guichard
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Bowman
authors.forename: Margaret
authors.initials: M
authors.lastname: Gaskell
authors.forename: Peter B
authors.initials: PB
authors.lastname: Farmer
authors.forename: George D D
authors.initials: GD
authors.lastname: Jones
mesh_terms: D000477:Alkylating Agents / Q000737:chemistry / Q000633:toxicity; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D004247:DNA / Q000187:drug effects; D018736:DNA Adducts / Q000032:analysis*; D004249:DNA Damage; D004052:Diethylnitrosamine / Q000737:chemistry / Q000633:toxicity; D015226:Dinucleoside Phosphates / Q000032:analysis*; D008099:Liver / Q000737:chemistry / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D010761:Phosphorus Radioisotopes; D013463:Sulfuric Acid Esters / Q000737:chemistry / Q000633:toxicity
chemical_list: D000477:Alkylating Agents; D015415:Biomarkers; D018736:DNA Adducts; D015226:Dinucleoside Phosphates; D010761:Phosphorus Radioisotopes; D013463:Sulfuric Acid Esters; D004052:Diethylnitrosamine; D004247:DNA; C011612:diethyl sulfate