Formation of methyl ester of 2-methylglyceric acid from thymine glycol residues: a convenient new method for determining radiation damage to DNA

Abstract

Thymine glycol residues in DNA or thymidine were converted to methyl 2-methylglycerate by reaction with alkaline borohydride followed by methanolic HCl. The product was labeled either from [3H]DNA or from [3H]borohydride and was followed by cochromatography with authentic 14C-labeled material. Following acid hydrolysis, the identity of 2-methylglyceric acid was confirmed by high-resolution mass spectrometry, NMR, IR, and elemental analysis. Treatment of DNA or thymidine with X-irradiation, with H2O2 and Fe2+, with H2O2, Cu2+, and ascorbate, and with H2O2 and ultraviolet light, permanganate, or sonication all produced methyl 2-methylglycerate in varying amounts after alkaline borohydride and methanolic HCl, whereas untreated DNA did not. The data indicate that certain oxidants including hydroxyl radicals generated by chemical means or from radiolysis of water convert thymine residues to thymine glycols in DNA, which can be determined as methyl 2-methylglycerate.

Gas chromatographic-mass spectrometric method for the assessment of oxidative damage to double-stranded dna by quantification of thymine glycol residues

“…Following optimization of derivatization and chromatography of methyl-2-methylglycerate-<TBDMS)z' we tested conditions for its generation from thymine glycol. The optimal conditions for hydrolysis and [3H]borohydride reduction of thymine glycol were described previously by Schellenberg and Shaeffer [23] and adapted for [2H]borohydride. The resulting product from crystalline thymine glycol contains a single deuterium atom, as shown in Figure 3b, exhibiting an [M -57] fragment ion shifted from m/z 305 to 306, compared with the product obtained when NaBH 4 is used (Figure 3a).…”

“…There have been several published assays for quantifying thymine glycol in DNA using radiochemical, immunoassay, chromatographic, and mass spectrometric procedures [10,[17][18][19][20][21][22][23]. These have been successful in measuring the oxidative damage in solutions of DNA exposed to various chemical or radiochemical agents but have generally lacked the sensitivity required to be applied to clinical measurements, in vivo animal studies, or even cultured cell experiments, except when cells have been prelabeled with radioactive thymine [24][25][26]; however, one of the analytical procedures seemed particularly well suited for adaptation for gas chromatography-mass spectrometry (GC-MS) analysis.…”

“…These have been successful in measuring the oxidative damage in solutions of DNA exposed to various chemical or radiochemical agents but have generally lacked the sensitivity required to be applied to clinical measurements, in vivo animal studies, or even cultured cell experiments, except when cells have been prelabeled with radioactive thymine [24][25][26]; however, one of the analytical procedures seemed particularly well suited for adaptation for gas chromatography-mass spectrometry (GC-MS) analysis. Schellenberg and Shaeffer [23] used a radiochemical procedure employing alkaline hydrolysis, fol- (Figure 2). This report summarizes a GC-MS (electron ionization) method for thymine glycol quantification and its application to preparations of DNA.…”