Comparative study of base damage induced by gamma radiation and Fenton reaction in isolated DNA

摘要

Degradation of DNA by oxygen radicals is an importantngenotoxic mechanism associated with ageing and carcinogenesis.n1 A wide range of oxidising species are produced byncellular metabolism as well as by environmental stresses such asnradiation and chemicals. Identification of DNA degradationnpathways associated with specific deleterious oxidative speciesnis required for a better understanding of pathological processesnrelated to oxidative stress. In cells, highly reactive species, suchnas hydroxyl radicals (u0001OH), may be produced through thenreduction of hydrogen peroxide, the dismutation product ofnsuperoxide anion,2 by ferrous ions. This process, known as thenFenton reaction, becomes cyclic in the presence of a reducingnspecies able to reduce the resulting Fe3u0002 into Fe2u0002. When thenreducing species is superoxide anion, the iron-catalysed conversionnof hydrogen peroxide is referred to as the “Haber–Weissncycle”. To prevent the deleterious effect of the latter processes,niron storage is strictly controlled within cells,3 for instancenthrough its sequestration by ferritin. However, the cytotoxicnpotential of iron may be expressed under some pathologicalnconditions such as inherited hemochromatosis and iron overload.nIron release has also been observed under oxidative stressnconditions, for instance UVA irradiation.4 Altogether, the ironcatalysednproduction of reactive oxygen species is likely tondamage key biomolecules including DNA, and thereby to benassociated with genotoxicity.5,6 However, the identity of thenoxidising species produced during the Fenton reaction, innparticular the actual formation of u0001OH, is still a matter ofndebate.7–9nThe extensively studied decomposition of water by ionizingnradiation provides another source of u0001OH radicals. The DNAnbases degradation reactions induced under these conditionsnhave been widely investigated, in terms of formation of primarynnucleobase radicals 10 and identification of the final products.11nThe comparison between the relatively well understoodnγ-radiolysis of DNA and the damage produced in DNA undernthe conditions of Fenton reaction may shed some light on thenactual oxidising species involved in the latter process. The fewnextensive studies on the degradation of free nucleosides undernFenton reaction conditions have revealed some differences withnrespect to γ-radiation.12,13 A reliable comparison between thenradical-induced damage to isolated DNA under the two latternconditions is still needed and is likely to provide relevantnmechanistic information. Indeed, the distribution of basendamage within DNA has been shown to be very sensitive tonthe reaction conditions, such as the redox conditions and thenpresence of oxygen.11 A relevant example is provided by thencompetitive pathways involved in the u0001OH-induced degradationnof 2u0001-deoxyguanosine.14–16 Indeed, hydroxyl radicals may addnto the aglycone of the latter nucleoside at either the C4 or thenC8 position.10 In the latter case, the resulting neutral reducingnradical may be either oxidised into 8-oxo-7,8-dihydro-2u0001-ndeoxyguanosine (8-oxodGuo) or reduced into 2,6-diamino-4-nhydroxy-5-formamidopyrimidine (FapyGua) nucleoside.nUsing a specific and sensitive high performance liquidnchromatography–tandem mass spectrometry assay, we undertookna comparative study of the formation of radical-inducednbase damage (Scheme 1) produced upon either gamma irradi-ation or exposure to Fenton reaction conditions. In the latternexperiments, complexes of ferrous ions were used, instead ofnferric salts in the presence of reducing agents. Indeed, the latterncompounds may modify the chemical conversion of DNA basenradicals. In addition, in order to determine a possible role ofnmetal ions in the u0001OH-induced degradation of DNA, gammanirradiations were also carried out in the presence of iron. It wasnthus possible to reliably compare the effects of the reactivenspecies produced upon radiolysis of water on one hand andnFenton reaction on the other hand. The level of exocyclicnadducts, including 3,N4-ethenocytosine, 1,N2-ethenoguanine,n1,N2-ethenoadenine and the pyrimidinopurine malonaldehydenguanine adduct, proposed to arise from the reaction of DNAnbases with 2-deoxyribose degradation products (Scheme 2),17–19nwas also measured.