title: Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement.
abstract: Oxidation reactions that involve several oxygen and nitrogen reactive species together with nucleobase radical cations give rise among various classes of lesions to modified bases. About 70 of oxidized nucleosides that include diastereomeric forms have been characterized in mechanistic studies involving isolated DNA and related model compounds. However, only eight modified bases have been accurately measured within cellular DNA upon exposure to either gamma or UVA radiations. Emphasis is placed in this survey on recent developments of HPLC associated with tandem mass spectrometry (MS/MS) operating in the mild electrospray ionization mode. Interestingly, the HPLC-MS/MS assay in the multiple reaction monitoring mode appears to be the more sensitive and accurate method currently available for singling out several oxidized nucleosides including 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 5-formyl-2'-deoxyuridine, 5-(hydroxymethyl-2'-deoxyuridine, 5-hydroxy-2'-deoxyuridine, and the four diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine within isolated and cellular DNA. However, one limitation of the assay that also applied to all chromatographic methods is the slight side-oxidation of normal bases during DNA extraction and subsequent work-up. This explains why the combined use of DNA repair glycosylases with either the comet assay or the alkaline elution technique is a better alternative to monitor the formation of low levels of oxidized bases within cellular DNA.
authors.affiliation: Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, Département de Recherche Fondamentale sur la Matière Condensée, Grenoble, France. jcadet@cea.fr
authors.forename: Jean
authors.initials: J
authors.lastname: Cadet
authors.forename: Thierry
authors.initials: T
authors.lastname: Douki
authors.forename: Sandrine
authors.initials: S
authors.lastname: Frelon
authors.forename: Sylvie
authors.initials: S
authors.lastname: Sauvaigo
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Pouget
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Ravanat
mesh_terms: D016477:Artifacts; D002851:Chromatography, High Pressure Liquid; D020552:Comet Assay; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D018736:DNA Adducts / Q000032:analysis; D004249:DNA Damage; D011088:DNA Ligases; D006863:Hydrogen-Ion Concentration; D009705:Nucleosides / Q000737:chemistry*; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization; D014462:Ultrafiltration
chemical_list: D018736:DNA Adducts; D009705:Nucleosides; D004247:DNA; D011088:DNA Ligases