Quantitative determination of cyclobutane thymine dimers in DNA by stable isotope-dilution mass spectrometry.

Abstract

In order to understand the role of UV-induced DNA lesions in biological processes such as mutagenesis and carcinogenesis, it is essential to detect and quantify DNA damage in cells. In this paper we present a novel and both highly selective and sensitive assay using capillary gas chromatography (GC) combined with mass spectrometry (MS) for the detection and accurate quantitation of a major product of UV-induced DNA damage (cis-syn cyclobutadithymine). Quantitation of the cyclobutane thymine dimer was achieved by the use of an internal standard in the form of a stable 2H-labeled analogue. Both isotopically labeled and nonlabeled dimers were prepared directly from their corresponding monomers. Each was identified as their trimethylsilyl ether derivative by GC-MS. Calibration plots were obtained for known quantities of both nonlabeled analyte and internal standard. Quantitation of cis-syn cyclobutadithymine was demonstrated in DNA exposed to UVC radiation over a dose range of 0 to 3500 J m-2. Under the conditions used, the limit of detection was found to be 20-50 fmol on column (equivalent to 0.02-0.05 nmol dimer per mg DNA). The results of the present study indicate that capillary GC-MS is an ideally suited technique for selective and sensitive quantification of cis-syn cyclobutadithymine in DNA and hence UV-induced DNA damage.