Abstract
The formation of oxidative lesions arising from double stranded DNA damage is of major significance to chemical biology from the perspective of application to human health. The quantification of purine lesions arising from γ-radiation-induced hydroxyl radicals (HO•) has been the subject of numerous studies, with discrepancies on the measured 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) lesions reported by different groups. Here we applied an ameliorative protocol for the analysis of DNA damage with quantitative determination of these lesions via isotope dilution liquid chromatography coupled with tandem mass spectrometry. Tandem-type purine lesions were quantified along with 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) and 7,8-dihydro-8-oxo-2′-deoxyadenosine (8-oxo-dA) in single and double stranded DNA, generated during DNA exposure to diffusible HO• radicals in the absence or presence of physiological levels of oxygen. The cdA and cdG lesions in absence of oxygen were found ~2 times higher in single than double stranded DNA, with 5′R being ~6.5 and ~1.5 times more predominant than 5′S in cdG and cdA, respectively. Interestingly, in the presence of 5% molecular oxygen the R/S ratios are retained with substantially decreased yields for cdA and cdG, whereas 8-oxo-dA and 8-oxo-dG remain nearly constant. The overall lesion formation follows the order: 8-oxo-dG >> 8-oxo-dA > 5′R-cdG > 5′R-cdA > 5′S-cdA > 5′S-cdG. By this method, there was a conclusive evaluation of radiation-induced DNA purine lesions.
Highlights
Genetic information of all living organisms is stored in DNA, a polymer consisting of 2′deoxynucleosides
For this purpose 200 μL of N2O-saturated aqueous solutions containing calf thymus DNA (0.5 mg/mL) at natural pH were irradiated under steady-state conditions with a dose rate of 4.1 Gy min−1, followed by enzymatic DNA digestion optimized by us and LC-mass spectrometric (MS)/MS analysis
The use of purine 5′,8-cyclonucleosides as marker of DNA damage and reporter of DNA structures at the moment of the HO insult is increasingly appreciated in free radical research, together with the advantage that cyclopurine markers do not suffer the stability problems and artifactual oxidative process of the most known 8-oxo-dG
Summary
Genetic information of all living organisms is stored in DNA, a polymer consisting of 2′deoxynucleosides. The H5′ of sugars in DNA have been found quite vulnerable with a 55% probability of abstraction by HO, in respect to the rest of the sugar hydrogen atoms (Aydogan et al, 2002). This fact leads to the generation of peculiar lesions containing a carboncarbon bond between the sugar and the purine, rather than to abasic sites (Chatgilialoglu et al, 2011a). Oxidative artifacts during work-up of biological DNA samples are reported in the evaluation of 8-oxo-dG lesions, whereas cyclopurines are “pure” radical-derived products, and cannot derive from accidental oxidation of the material
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