Serial review: oxidative DNA damage and repair
Free radical-induced damage to DNA: mechanisms and measurement1, 2

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Abstract

Free radicals are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells, including DNA. The resulting damage to DNA, which is also called oxidative damage to DNA, is implicated in mutagenesis, carcinogenesis, and aging. Mechanisms of damage involve abstractions and addition reactions by free radicals leading to carbon-centered sugar radicals and OH- or H-adduct radicals of heterocyclic bases. Further reactions of these radicals yield numerous products. Various analytical techniques exist for the measurement of oxidative damage to DNA. Techniques that employ gas chromatography (GC) or liquid chromatography (LC) with mass spectrometry (MS) simultaneously measure numerous products, and provide positive identification and accurate quantification. The measurement of multiple products avoids misleading conclusions that might be drawn from the measurement of a single product, because product levels vary depending on reaction conditions and the redox status of cells. In the past, GC/MS was used for the measurement of modified sugar and bases, and DNA-protein cross-links. Recently, methodologies using LC/tandem MS (LC/MS/MS) and LC/MS techniques were introduced for the measurement of modified nucleosides. Artifacts might occur with the use of any of the measurement techniques. The use of proper experimental conditions might avoid artifactual formation of products in DNA. This article reviews mechanistic aspects of oxidative damage to DNA and recent developments in the measurement of this type of damage using chromatographic and mass spectrometric techniques.

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 (radical dotOH) 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 radical dotOH 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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    1

    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.

    2

    Guest Editor: Miral Dizdaroglu

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