Mass Spectrometric Assays for the Tandem Lesion 8,5'- Cyclo-2' Deoxyguanosine in Mammalian DNA
Pawel Jaruga, M Birincioglu, H Rodriguez, M Miral Dizdar
8,5'-Cyclopurine-2'-deoxynucleosides are among major lesions in DNA that are formed by attack of hydroxyl radical. These compounds represent a concomitant damage to both sugar and base moieties of the same nucleoside and thus can be considered tandem lesions. Because of the presence of a covalent bond between the sugar and purine moieties, these tandem lesions are not repaired by base-excision repair, but by nucleotide-excision repair. Thus, they may play a role in diseases with defective nucleotide-excision repair. We recently reported the identification and quantification of 8,5'-cyclo-2'-deoxyadenosine (8,5'-cdAdo) in DNA by liquid chromatography/mass spectrometry with the isotope-dilution technique (LC/IDMS) [Dizdaroglu, M., Jaruga, P., and Rodriguez, H. (2001) Free Radical Biol. Med. 30, 774-784]. In the present work, we investigated the measurement of 8,5'-cyclo-2'-deoxyguanosine (8,5'-cdGuo) in DNA by LC/IDMS. A methodology was developed for the separation of both (5'R)- and (5'S)-diastereomers of this compound in enzymic hydrolysates of DNA. The mass spectra were recorded using atmospheric pressure ionization-electrospray process in the positive ionization mode. For quantification, stable isotope-labeled analogues of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo were prepared and isolated by semipreparative LC to be used as internal standards. The sensitivity level of LC/MS in the selected-ion monitoring mode (LC/MS-SIM) was determined to be approximately 15 fmol of these compounds on the LC column. The yield of 8,5'-cdGuo was measured in DNA exposed in aqueous oslution to ionizing radiation at doses from 2.5 to 40 Gy. For comparison, gas chromatography/mass spectrometry with the isotope-dilution technique (GC/IDMS) was also employed to measure both (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo in DNA. Both techniques yielded nearly identical results. The radiation chemical yield of 8,5'-cdGuo was similar to those of other major purine-derived lesions in DNA. The sensitivity level of GC/MS-SIM was determined to be significantly greater than that of LC/MS-SIM was determined to be significantly greater than that of LC/MS-SIM (1 vs. 15 fmol). The background levels of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo were measured in calf thymus DNA and in DNA samples isolated three different types of cultured human cells. The levels of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo were approximately 2 lesions/106 DNA nucleosides and 10 lesions/106 DNA nucleosides, respectively. No significant differences between tissues were observed in terms of these background levels. The results showed that both LC/IDMS and GC/IDMS are well suited for the sensitive detection and precise quantification of both (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo in DNA.
cclonucleosides, DNA repair, gas chromatography, liquid chromatography, mass spectrometry, oxidative DNA damage