Identification and Quantification of a Guanine−Thymine Intrastrand Cross-Link Lesion Induced by Cu(II)/H₂O₂/Ascorbate

Abstract

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes and they can damage biological molecules including nucleic acids. It was shown that X-or γ-ray irradiation of aqueous solutions of DNA, during which · OH is one of the major ROS, can lead to the formation of intrastrand crosslink lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. Previous 32 P-postlabeling studies suggested that the intrastrand crosslink lesions may arise from Fenton reaction, which also induces the formation of · OH; the structures of the proposed intrastrand crosslink lesions, however, have not been determined. Here we showed for the first time that the treatment of calf thymus DNA with Cu(II)/H 2 O 2 /ascorbate could lead to the formation of an intrastrand crosslink lesion, i.e., G^T, where the C8 of guanine is covalently bonded to the neighboring 3′ thymine through its methyl carbon. LC-MS/MS quantification results showed dose-responsive formation of G^T. In addition, the yield of the intrastrand crosslink was approximately three orders of magnitude lower than those of commonly observed single-base lesions, that is, 8-oxo-7,8-dihydro-2′-deoxyguanosine, 5-(hydroxymethyl)-2′-deoxyuridine and 5-formyl-2′-deoxyuridine. The induction of intrastrand crosslink lesion in calf thymus DNA by Fenton reagents in vitro suggests that this type of lesion might be formed endogenously in mammalian cells.

Derivatization with Girard Reagent T Combined with LC−MS/MS for the Sensitive Detection of 5-Formyl-2‘-deoxyuridine in Cellular DNA

“…Hela-S3 cells were from the National Cell Culture Center (Minneapolis, MN). [1,[3][4][5][6][7][8][9][10][11][12][13][14][15] N 2 , 2′-D]-5-formyl-2′-deoxyuridine was prepared according to previously described procedures 8 .…”

Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

“…The chemical synthesis of the labeled 5-hmdU and 5fdU standards is described in Ref. 26. To improve the detection limit of fdU in positive-ion electrospray ionization (ESI)-MS, the digested DNA was then derivatized with Girard reagent T to form a hydrazone conjugate harboring a precharged quaternary ammonium moiety, as reported previously (27).…”

Uptake of estradiol from sediment by hornyhead turbot (Pleuronichthys verticalis) and effects on oxidative DNA damage in male gonads

“…Vitellogenin and E2 as were measured as described previously (Rempel et al, 2006). Analysis of 8-oxodG by LC/MS-MS followed those outlined in Hong et al (2006) using UV treatment in MCF-7 cells as a positive control. Data were analyzed for normality of distribution by Shapiro-Wilk's test, for equality of variance by Bartlett's test and for significance by Dunnett's and Bonferroni tests (p < 0.05).…”