Jean Cadet

Oxidatively Generated Damage to Cellular DNA: Mechanistic Aspects

In this chapter emphasis is placed on recent aspects of the oxidative formation of several classes of modified bases in cellular DNA that arise from the reaction of the hydroxyl radical (•OH), singlet oxygen and hypochlorous acid. Degradation compounds are detected quantitatively and specifically after suitable DNA hydrolysis into either nucleosides or bases by HPLC-tandem mass spectrometry. Thus, 6 oxidized nucleosides including: the four cis and trans diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine, 5-(hydroxymethyl)-2′-deoxyuridine and 5-formyl-2′-deoxyuridine are found to be formed as the result of •OH radical mediated oxidation of thymidine. In addition, γ-irradiation of cellular DNA was found to generate 8-oxo-7,8-dihydropurine derivatives and related formamidopyrimidine compounds resulting from •OH radical oxidation of the guanine and adenine bases. Furthermore, singlet oxygen oxidation of guanine was found to give rise exclusively to 8-oxo-7,8-dihydro-2′-deoxyguanosine while HOCl reaction with cytosine, adenine and guanine led to the formation of 5-chlorocytosine, 8-chloroadenine and 8-chloroguanine nucleosides respectively in the DNA and RNA of human white blood cells. Interestingly, formation of these various degradation products has been rationalized in terms of existing mechanisms that were proposed previously from model studies, mostly involving free nucleosides.

Chapter 20:Reactions of Singlet Oxygen with Nucleic Acids

The chapter is aimed at providing an updated overview of the main available data on the oxidation of nucleic acids by singlet oxygen (1O2). Mechanistic insights into the selective oxidative pathways of guanine, the predominant DNA and RNA target, were gained from detailed model studies. These have also allowed identification of spiroiminodihydantoin and minor 8-oxo-7,8-dihydroguanine as the main decomposition products of nucleosides and nucleotides. Evidence has been provided that the latter guanine oxidation product and thionucleobases are also highly reactive to singlet oxygen. The 1O2 oxidation of isolated and cellular DNA and RNA is much more specific giving rise almost exclusively to 8-oxo-7,8-dihydroguanine though the intermediacy of 4,8-endoperoxide that subsequently rearranges into easily reducible 8-hydroperoxyguanine. It was also shown that 1O2 is not able to induce significant levels of strand breaks and/or alkali-labile sites in cellular DNA upon exposure to a chemical source of singlet oxygen. UVA irradiation of cells and human skin is able to oxidatively damage nuclear DNA as inferred from the measurement of 8-oxo-7,8-dihydroguanine. This has been rationalized in terms of the predominant implication of 1O2 produced by type-II photosensitization mechanism over Fenton-type reactions.

Oxidatively Generated Damage to DNA and Biomarkers

The first part of this chapter is devoted to the description of degradation pathways for DNA bases and sugar moieties in the cell that are mediated by hydroxyl radical, one-electron oxidants and singlet oxygen. Thus, 11 single modified nucleosides and nucleobases that may be part of more complex radiation-induced DNA damage have been shown to be generated in nuclear DNA. In addition, four clustered DNA addition products and one tandem base-sugar lesion that arises from •OH-mediated hydrogen abstraction at C4 and C5 of the 2-deoxyribose moiety of DNA have also been identified in cells. Mechanisms of formation that are inferred from model studies are available for the 16 single and complex lesions thus detected in nuclear DNA. The DNA oxidation products whose radiation-induced formation in cellular was found to vary between 2 and 100 per 109 normal nucleosides per Gray may be used as biomarkers of oxidative stress. The accurate measurement of single, clustered or tandem lesions was performed, once DNA was extracted and subsequently enzymatically or chemically hydrolyzed, using accurate chromatographic methods. These involve, in most cases, the association of high performance liquid chromatography with the electrospray ionization tandem mass spectrometry (HPLC-ESI/MS–MS) detection techniques operating in the highly accurate and sensitive multiple reaction monitoring mode. Another approach that is more sensitive and less prone to artefactual oxidation consists of pre-incubating oxidized DNA with repair enzymes to reveal classes of modifications as strand breaks prior to either single-cell electrophoresis analysis or alkaline elution analysis.