Serial review: oxidative DNA damage and repairFree radical-induced damage to DNA: mechanisms and measurement1, 2
Introduction
Free radicals are produced in living cells by normal metabolism and by exogenous sources such as carcinogenic compounds and ionizing radiations (reviewed in [1]). Of the free radicals, the highly reactive hydroxyl radical (OH) causes damage to DNA and other biological molecules (reviewed in [2], [3]). This type of DNA damage is also called “oxidative damage to DNA” and is implicated in mutagenesis, carcinogenesis, and aging [1]. Oxidative damage to DNA is encountered by cellular repair systems and can be repaired (reviewed in [4]). DNA base damage is thought to be repaired mainly by base-excision repair (reviewed in [5]).
Oxidative damage to DNA can be measured by a variety of analytical techniques, which have their own advantages and drawbacks (reviewed in [6], [7]). Most of these techniques measure only a single product with no spectroscopic evidence for identification. Techniques that use mass spectrometry provide unequivocal identification and quantification of DNA damage (reviewed in [7]). For over a decade, gas chromatography/mass spectrometry (GC/MS) has been used for the measurement of DNA base and sugar lesions, and DNA-protein crosslinks in cells and in vitro [7]. Recently, liquid chromatography/tandem mass spectrometry (LC/MS/MS) and liquid chromatography/mass spectrometry (LC/MS) emerged as new techniques for the measurement of modified nucleosides in DNA. LC/MS/MS was first used for the measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) only, and then for that of several other modified nucleosides [8], [9], [10], [11], [12], [13], [14]. LC/MS was recently applied to the measurement of 8-OH-dGuo, (5′R)-8,5′-cyclo-2′-deoxyadenosine [(5′R)-8,5′-cdAdo], (5′S)-8,5′-cyclo-2′-deoxyadenosine [(5′S)-8,5′-cdAdo)], 8-hydroxy-2′-deoxyadenosine (8-OH-dAdo), (5′R)-8,5′-cyclo-2′-deoxyguanosine [(5′R)-8,5′-cdGuo], and (5′S)-8,5′-cyclo-2′-deoxyguanosine [(5′S)- 8,5′-cdGuo)] [15], [16], [17], [18]. The present article reviews mechanistic aspects of oxidative damage to DNA and its measurement by mass spectrometric techniques.
Section snippets
Mechanisms of oxidative damage to DNA
Hydroxyl radical adds to double bonds of heterocyclic DNA bases at second-order rate constants of 3–10 × 109 M−1 s−1 and abstracts an H-atom from the methyl group of thymine and each of the five carbon atoms of 2′-deoxyribose at rate constants of ∼2 × 109 M−1 s−1 (reviewed in [19]). Addition reactions yield OH-adduct radicals of DNA bases, whereas the allyl radical of thymine and carbon-centered sugar radicals are formed from abstraction reactions. If present, oxygen adds to OH-adduct radicals
Measurement of oxidative damage to DNA
In the past, various analytical techniques were used to measure oxidative damage to DNA (reviewed in [7]). These included immunochemical techniques, post-labeling assays, comet assay, alkaline elution with the use of DNA repair enzymes, high-performance liquid chromatography (HPLC) with electrochemical detection (ECD), GC/MS, LC/MS/MS, and LC/MS. In this article, we will briefly review some recent developments in this field and refer to previous studies whenever possible, because of space
Conclusions
Free radicals most notably OH generate a large number of modifications in DNA by a variety of mechanisms. These include sugar and base modifications, strand breaks and DNA-protein cross-links. The measurement of resulting products presents an enormous challenge. A number of analytical techniques with their own advantages and drawbacks are employed for this purpose. Unfortunately, no consensus exists between laboratories in terms of the measurement of DNA damage, most notably in the case of
Acknowledgements
Certain commercial equipment or materials are identified in this paper in order to specify adequately the experimental procedures. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
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This article is part of a series of reviews on “Oxidative DNA Damage and Repair.” The full list of papers may be found on the homepage of the journal.
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Guest Editor: Miral Dizdaroglu