The Chemical Toxicology of 2-Deoxyribose Oxidation in DNA

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The Chemical Toxicology of 2-Deoxyribose Oxidation in DNA Peter C. Dedon* Department of Biological Engineering and Center for EnVironmental Health Sciences, Massachusetts Institute of Technology, NE47-277, 77 Massachusetts AVenue, Cambridge, Massachusetts 02139 ReceiVed August 5, 2007

Damage to DNA and RNA caused by oxidative mechanisms has been well-studied for its potential role in the development of human disease. Only recently, though, have we begun to appreciate that oxidation of the 2-deoxyribose moiety in DNA is also a determinant of the genetic toxicology of oxidative stress and inflammation, with involvement in more than just “strand breaks”, such as complex DNA lesions, protein–DNA cross-links, and protein and DNA adducts. As an update to a 1992 review of 2′-deoxyribose oxidation by bleomycin and the enediynes published in Chemical Research in Toxicology [Dedon, P. C., and Goldberg, I. H. (1992) Chem. Res. Toxicol. 5, 311–332], this review focuses on recent developments in the chemical biology, bioanalytical chemistry, and genetic toxicology of 2-deoxyribose oxidation products in DNA under biologically relevant conditions. Contents 1. Introduction 2. The Importance of Normalizing 2-Deoxyribose Oxidation Data 3. 1′-Chemistry 3.1. Model Systems 3.2. Methods for Measurement 3.3. Biological Implications 4. 2′-Chemistry 4.1. Model Systems 4.2. Methods of Measurement 4.3. Biological Consequences 5. 3′-Chemistry 5.1. Model Systems 5.2. Methods of Measurement 5.3. Biological Consequences 6. 4′-Chemistry 6.1. Model Systems 6.2. Methods of Measurement 6.3. Biological Consequences 7. 5′-Chemistry 7.1. Model Systems 7.2. Methods of Measurement 7.3. Biological Consequences 8. Solvent Exposure and Other Models for 2-deoxyribose Oxidation in DNA 9. 2-Deoxyribose Oxidation Chemistry in Cells 10. The Metabolic Fate of 2-Deoxyribose Oxidation Products: Glutathione Conjugation and Glutathione S-Transferases

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1. Introduction DNA damage caused by a variety of oxidizing, alkylating, halogenating, and nitrosating species may play a significant role in the pathophysiology of inflammation, cancer, and degenerative diseases (6–8). While studies of the chemistry of nucleobase damage have dominated the literature, there is growing evidence that oxidation of deoxyribose in DNA plays a critical role in * To whom correspondence should be addressed. Tel: 617-253-8017. Fax: 617-324-7554. E-mail: [email protected].

the genetic toxicology of oxidative stress, both as individual lesions and as part of complex DNA lesions with closely opposed strand breaks and oxidized abasic sites (10, 11). Oxidation of each of the five positions in 2-deoxyribose in DNA occurs, under the most biologically relevant conditions, with an initial hydrogen atom abstraction to form a carbon-centered radical that adds molecular oxygen at diffusion-controlled rates (∼109 M–1 s–1; ref 12) to form a peroxyl radical, as shown in Figure 1 for 1′-oxidation. There are clearly different product spectra for 2-deoxyribose oxidation produced under aerobic and anaerobic conditions, for example, by γ-radiation (13), with the more biologically relevant spectrum of products formed under aerobic conditions shown in Figure 1. This perspective represents essentially an update of an earlier review published in Chemical Research in Toxicology in 1992 (14), and it focuses on recent developments in the chemical biology, bioanalytical chemistry, and genetic toxicology of 2-deoxyribose oxidation under biologically relevant (i.e., aerobic) conditions. Ample attention has been paid over the past three decades to the descriptive chemistry of 2-deoxyribose oxidation, as covered, for example, in Von Sonntag’s wellknown texts (13, 15) and in reviews such as that of Pogozelski and Tullius (16). Unfortunately, limited space obviates a thorough coverage of many new developments in the chemistry of 2′-oxidation of DNA, such as the cyclic base–sugar adducts observed by several groups (17–20). The perspective proceeds from brief surveys of product spectra and consequences of oxidation of each position of 2-deoxyribose to discussions about the biological implications of 2-deoxyribose oxidation products.

2. The Importance of Normalizing 2-Deoxyribose Oxidation Data Prior to discussing individual 2-deoxyribose oxidation chemistries, it is important to highlight the value of normalizing quantitative 2-deoxyribose oxidation data to the total number of oxidation events occurring in the DNA. The absolute values for the quantities of the individual products are important data for comparisons to other types of oxidatively induced damage in DNA and RNA bases, lipids, and proteins. However, as discussed in detail throughout this perspective, there is a great

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Figure 2. Elimination products of the 2-deoxyribonolactone.

in the section on 3′-chemistry. While R-particle irradiation of DNA resulted in 0.13 3′-phosphoglycolaldehyde residues per 106 nucleotides per Gy, γ-radiation produced 1.5 3′-phosphoglycolaldehyde residues per 106 nucleotides per Gy (31, 32). A direct comparison of these data would suggest a 10-fold greater efficiency of formation of this DNA lesion with γ-radiation. However, when the data are normalized to an estimate of the total number of 2-deoxyribose oxidation events, which amounts to 2 and 141 per 106 nucleotides per Gy for R- and γ-radiations, respectively, R-particles turn out to be seven-fold more efficient at producing the 3′-phosphoglycolaldehyde than γ-radiation, as a result of its higher LET properties (31, 32). Normalizing the strand break data thus provides a means to compare different DNA-damaging agents in a meaningful way.

3. 1′-Chemistry

Figure 1. 2-Deoxyribose oxidation in DNA.

deal of interest in defining the relative quantities of the various lesions to identify candidate biomarkers of oxidative stress, to test various models of 2-deoxyribose reactivity in DNA, and to define chemical mechanisms on the basis of shifts in 2-deoxyribose oxidation chemistry. To this end, the proportions of specific damage products relative to total 2-deoxyribose oxidation damage frequency can be calculated with knowledge of the latter. There are many approaches to quantifying or at least estimating total 2-deoxyribose oxidation events, including the plasmid nicking assay for in vitro studies and the comet assay for studies in cells. The former is a well-established technique that exploits the conversion of supercoiled plasmid DNA to nicked and linear forms following direct strand breaks and, after derivatization with agents such as putrescine, oxidized abasic sites (21–30). Alternatively, oxidized purine and pyrimidine bases can be converted to strand breaks using formamidopyrimidine DNA glycosylase and endonuclease III, respectively (27). Both enzymes have abasic site-cleaving activity that obviates the need for putrescine, and base damage is quantified as the difference between enzyme-induced and putescine-induced breaks. The importance of an assay for “total 2-deoxyribose oxidation events” is illustrated in studies comparing the yield of 3′phosphoglycolaldehyde residues arising from 3′-oxidation of DNA produced by γ- and R-radiations (31), as will be discussed

The singular product arising from the oxidation of the 1-carbon of 2-deoxyribose in DNA by a variety of oxidants is the 2-deoxyribonolactone abasic site shown in Figure 1 (33–38). Included among the oxidants capable of forming the 2-deoxyribonolactone abasic site are copper–phenanthroline complexes (39), cationic manganese porphyrins (40), oxoruthenium complexes (41), ultraviolet irradiation (42), enediyne antibiotics (23, 43), and γ-radiation (13). While more stable than a native abasic site (∼10–50-fold; ref 44), the ribonolactone undergoes a rate-limiting β-elimination reaction to form a butenolide species (Figure 2) with a half-life of 20 h in single-stranded DNA (32–54 h in duplex DNA), followed by a rapid δ-elimination to release 5-methylene-2(5H)-furanone (44). 3.1. Model Systems. A variety of model systems exist for studying the 2-deoxyribonolactone abasic site in vitro and in vivo (33–38, 45), each with different utility and limitations depending on the chemical or biological nature of the question asked. As will be discussed with 3′- and 4′-oxidation chemistry, significant effort has been devoted to developing site-specific radical-generating systems, based mainly on the pioneering work of Giese and co-workers (e.g., see refs 46 and 47). For 1′-radicals in DNA, several groups have developed chemically analogous approaches that involve stable, photosensitive 1′derivatives of 2′-deoxyribonucleotides and their phosphosphoramidite derivatives that can be incorporated into oligonucleotides (34, 37, 38, 45, 48). When subjected to photoinduced cleavage of the 1′-derivative, the resulting 1′-radical proceeds with reactions supposedly typical of those occurring in native DNA, such as addition of molecular oxygen to form a peroxyl radical (k ) 109 M–1 s–1; ref 49) that subsequently undergoes degradation along several pathways depending on the biochemical environment. The efficiency of formation of the 1′-radical varies among the different precursor chemistries, but with attention to the stability of the 2-deoxyribonolactone, postirradiation purification of the ribonolactone-containing oligonucleotide is possible. Greenberg and co-workers have similarly prepared a phenyl selenide precursor of the 5,6-dihydrothymidin-5-yl base oxidation product (50). This radical adds molecular oxygen to form the corresponding peroxyl radical that then abstracts the

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1′-hydrogen atom from a neighboring nucleotide (50). This type of stereoselective and spatially fortuitous chemistry has been observed in other systems, such as the model 3′-radical system developed by Bryant-Friedrich and co-workers (4, 51), and may represent a mechanism for propagating DNA oxidation and forming complex DNA lesions without the need for ionizing radiation tracks. Other model systems include sequence-selective production of the 1′-radical by DNA-cleaving antibiotics and other small oxidants that cause hydrogen atom abstraction from 2-deoxyribose, such as the enediyne antibiotics (14, 23, 24). These agents do not provide the site specificity of the photolabile precursors, often oxidize more than one site in DNA, and are complicated by the persistent binding of the agent to the site of 2-deoxyribose hydrogen atom abstraction. However, many of these agents, the enediynes in particular, can be used to cause DNA damage in cells (a few examples are found in refs 52–57). One novel approach to studying 1′- and other 2-deoxyribose oxidation chemistries in DNA shifted attention from the oxidizing agent to the 2-deoxyribose. Tullius and co-workers synthesized 2′-deoxyribonucleotides containing site-specific deuterium labels at each position in the sugar (58) and then used a sequencing gel mobility-based assay to quantify the primary isotope effects associated with 2-deoxyribose oxidation by the well-studied generator of hydroxyl radical-like species, the Fe2+-ETDA complex (59). The results were consistent with a model in which solvent exposure of the various hydrogen atoms in 2-deoxyribose in DNA governs their reactivity with hydroxyl radical, with reactivity occurring in the following order: 5′ > 4′ > 3′ ≈ 2′ ≈ 1′ (59). While this model for the determinants of hydroxyl radical reactivity with DNA has its weaknesses, such as a major focus on direct strand breaks without attention to abasic sites, the approach represents a major step forward in tackling the complexity of oxidation chemistry in the three-dimensional structure of DNA. 3.2. Methods for Measurement. The past decade has witnessed an emerging appreciation for studying 2-deoxyribose oxidation chemistry with quantitative rigor under biologically relevant conditions. We must distinguish here between descriptive analytical methods and those that provide quantitative information. For example, 2-deoxyribonolactone has been qualitatively characterized as an elimination product or derivatized adduct using various combinations of gas, liquid, and other types of chromatography with mass spectrometric or UV spectroscopic detection (33, 60, 61). These approaches provide semiquantitative data at best and are intended primarily to provide structural information and confirmation of the existence of the lesion. To be truly quantitative, a method must employ some kind of standard, with the most rigorous methods employing internal standards to correct for losses during DNA isolation, processing, and analysis. To this end, several groups have developed analytical methods for quantifying 2-deoxyribose oxidation products in isolated DNA and cells. On the basis of the observed chemical reactivity of the 2-deoxyribonolactone (35, 44), Greenberg and co-workers have approached the problem by developing a chemical probe that is proposed to be selective for reaction with the 2-deoxyribonolactone abasic site (62, 63). The probe consists of a fluorescently labeled biotinylated cysteine derivative that reacts with the butenolide β-elimination product of the 2-deoxyribonolactone (Figure 2) to form a heat-stable cyclic amide (62, 63). The method requires a heating step to distinguish the 2-deoxyribonolactone adducts from those formed with other 2-deoxyribose oxidation products (62, 63). With an apparent

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limit of quantification in the high fmol/low pmol range, the method is claimed to be specific for the ribonolactone lesion in samples of oxidized DNA. However, this conclusion was based on a comparison with only the native abasic site and the 2-deoxypentos-4-ulose abasic site derived from 4′-oxidation; yet, there are other R,β-unsaturated mono- and dicarbonyl species generated during DNA oxidation [e.g., 3′-keto-2′-deoxynucleoside, 5′-(2-phosphoryl-1,4-dioxobutane), and 5′-nucleoside-5′aldehyde; see Figure 1] that have the potential to react with the probe nonspecifically. Consistent with a potentially limited specificity is the observation by Greenberg and co-workers that the 2-deoxyribonolactone represents 75–80% of all aldehydeor ketone-containing 2-deoxyribose oxidation products arising with γ-radiation and Fe2+-EDTA (62, 63). This high proportion of an abasic site relative to direct strand breaks, and of a single type of abasic site, is at odds with published studies with these agents, including, for example, similar oxime derivatization studies and plasmid-nicking assays with γ-radiation (e.g., see refs 25, 30, and 64–66), and the primary isotope studies of Tullius and co-workers with Fe2+-EDTA that pointed to 1′oxidation as a minor portion of all Fe2+-EDTA-mediated 2-deoxyribose oxidation events (59). However, even with a heatinduced denaturation prior to gel analyses of isotope effect, the latter studies may have been biased toward direct strand breaks, so that the proportion of chemistries producing abasic sites may have been underestimated. Support for a high proportion of 1′-chemistry comes from the studies of Razskazovskiy and co-workers. They took a different approach with greater specificity for the 2-deoxyribonolactone abasic site that entailed elimination of the ribonolactone as the 5-methylene-2(5H)-furanone product (Figure 2) followed by HPLC resolution and quantification by UV spectroscopy (67). They exploited their observation of a catalytic effect of polyamines and certain metal ions for increasing the efficiency of heat-induced release of 5-methylene-2(5H)-furanone from irradiated DNA, with maximal release occurring with polylysine (67). While the method appears to be less sensitive than the cysteine probe approach of Greenberg and co-workers (which is compensated by using more DNA for the analysis), its application to γ-irradiated DNA revealed that the 2-deoxyribonolactone comprised about 30% of total 2-deoxyribose oxidation, a relatively high proportion similar to that observed by Greenberg and co-workers (62, 63). Indeed, the 30% value may underestimate the true proportion due to the tendency of the 5-methylene-2(5H)-furanone to dimerize to anemonin (68) but probably only by about 15%, the degree to which we have observed loss of 5-methylene-2(5H)-furanone using isotopically labeled internal standards (Chen and Dedon, unpublished observations). However, using Fe2+-EDTA as the oxidant, Razskazovskiy and co-workers did not observe detectable levels of the 2-deoxyribonolactone (Razskazovskiy et al., unpublished observations), which is inconsistent with Greenberg’s observations (62, 63) yet consistent with the low level of 1′-oxidation expected for Fe2+-EDTA (59). These divergent results await validation using a more sensitive and specific analytical method and a more complete survey of 2-deoxyribose oxidation products. 3.3. Biological Implications. Perhaps the most significant developments in the past decade of study of 2-deoxyribose oxidation in DNA have arisen in our understanding of the biological implications of these ubiquitous DNA lesions. In this regard, the 2-deoxyribonolactone has emerged as a potentially highly toxic DNA damage product. By itself, the 2-deoxyribonolactone abasic site has been shown to be mutagenic by

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Figure 3. Mechanism of cross-link formation between the DNA repair enzymes and the 2-deoxyribonolactone abasic site.

several groups. The first evidence for the mutagenicity of this sugar oxidation product arose in studies performed by Povirk and Goldberg, with the ribonolactone-generating enediyne, neocarzinostatin (10, 69). They observed high levels of G:C to A:T transitions at the C of AGC motifs, a site at which neocarzinostatin was known to cause 1′-oxidation and to produce the 2-deoxyribonolactone abasic site (10, 69). This observation was later confirmed by Greenberg’s group working with Myron Goodman and by Saparbaev and co-workers using site specifically labeled M13 shuttle vector constructs in Escherichia coli (70, 71), with the revelation that the ribonolactone lesion does not obey the classical “A-rule” for incorporation of dA opposite native abasic sites. Greenberg and co-workers observed higher rates of insertion of dG opposite the abasic site than dA using a restriction enzyme postlabeling assay (70), while Saparbaev and co-workers used an M13-based genetic reversion assay (GGX sequence context) and observed insertion of dT at rates three times higher than dG or dA (71). The story changes yet again in yeast. Kow and Greenberg employed an oligonucleotide transformation assay to determine that the deoxyribonolactone was bypassed at 10-fold higher frequencies than a native AP site, with dA and dC inserted at similar frequencies opposite the 2-deoxyribonolactone (72). The major conclusion from all of these studies is that deoxyribonolactone has different mutational properties than native abasic sites. There is one important caveat here that applies to all mutational studies involving labile DNA lesions. The results of such studies must be interpreted with a degree of caution since the labile lesions undergo an uncontrollable hydrolysis or degradation during the many steps involved in preparation of the shuttle vectors, transfection of the vectors into host cells, and during the processing and replication in the cells. Few groups have fully accounted for the purity of the mutational substrate. Another biological consequence of the 2-deoxyribonolactone abasic site involves the formation of toxic protein–DNA crosslinks (73, 74). The first demonstration of this phenomenon was made by Cunningham and Greenberg with the E. coli DNA repair enzyme, endonuclease III (73). This enzyme normally functions in base excision repair pathways with both an initial N-glycosylase activity against oxidized pyrimidines and a subsequent incision of the resulting abasic site by a lyase activity (75). Upon binding to the 2-deoxyribonolactone abasic site, however, the active site lysine 120, which normally forms a Schiff base with the 1′-aldehyde in the ring-opened form of the native abasic site, performs a nucleophilic attack on the carbonyl group of the ribonolactone (Figure 3). The resulting cross-link is irreversible, unlike a Schiff base, and complicates the DNA repair process (73). A chemically analogous cross-linking reaction was observed by Demple and Greenberg with human DNA polymerase β (74). During short-patch base excision repair (BER) in human cells, a DNA N-glycosylase removes a damaged base to create a native AP site that is initially processed by the 5′-phosphodiesterase


Figure 4. Retroaldol degradation of the erythrose abasic site arising from 2′-oxidation of DNA.

Figure 5. In situ preparation of the erythrose abasic site of 2′-oxidation. Modified from ref 9.

activity of the main human abasic endonuclease enzyme, Ape1. The remaining 5′-phosphorylated 5′-abasic site residue is then removed by a β-elimination reaction carried out by DNA polymerase β (pol β). While the 2-deoxyribonolactone is an efficient substrate for the Ape1 step, the subsequent β-elimination by pol β results in cross-link formation with the sugar lesion (74). Again, this involves an active site lysine (position 72) that normally forms a Schiff base with the C1-aldehyde during excision of an unmodified abasic site and instead performs a nucleophilic attack on the lactone to produce an amide linkage (74). In light of this behavior, it is not surprising that the 2-deoxyribonolactone is repaired by a long-patch mechanism that avoids the pol β-induced β-elimination reaction (76).

4. 2′-Chemistry Of the five carbons of 2-deoxyribose in DNA, the 2′-position has received the least attention until recently. Oxidation of the 2′-position during γ-irradiation and by photoinduced generation of a 2′-radical in a 5-iodouracil-containing oligodeoxynucleotide results in the formation of a D-erythrose abasic site, as shown in Figure 1 (60, 77). One chemically interesting feature of this abasic site, one that is probably biologically irrelevant, is that heating under alkaline conditions causes a retroaldol reaction that results in a strand break with phosphoroglycoaldehyde termini on each end (Figure 4) (77). The 3′-residue is chemically identical to, but mechanistically unrelated to, the 3′-phosphoglycolaldehyde residue arising from 3′-oxidation (discussed shortly). An important point here is that the erythrose abasic site is substantially more stable to hydrolysis than the native and other oxidized abasic sites, with a half-life in 0.1 M NaOH at 37 °C of 3 h (9). 4.1. Model Systems. In addition to the 5-iodouracil photochemical system developed by Saito and co-workers, Greenberg’s group has developed a system for in situ generation of the lesion in oligonucleotides (9, 78, 79). The approach is based on a synthetic strategy developed by Johnson and co-workers for introducing latent aldehydes into the DNA backbone as vicinal diols and exposing the aldehyde by mild periodate oxidation (80). Greenberg prepared a phosphoramidite derivative of a protected ribitol that, following incorporation into oligonucleotides, can be deprotected, and the vicinal diol oxidized to form the erythrose abasic site, as shown in Figure 5 (9). While incorporation of the precursor into oligonucleotides is only 50% efficient, the conversion of the precursor to the erythrose abasic site in situ was found to be virtually quantitative (9).


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Figure 6. Intramolecular oxidation of the 4′- and 5′-positions of 2-deoxyribose in DNA bv the 3′-radical (4, 5). Modified from refs 4 and 5).

4.2. Methods of Measurement. At present, there are no published methods for quantifying the erythrose abasic site of 2′-oxidation. 4.3. Biological Consequences. While there have been no studies of the erythrose abasic site in cells, the results of several in vitro studies suggest possible biological consequences for this DNA lesion. Primer extension studies using the Klenow fragment have revealed that the erythrose abasic site generally obeys the “A-rule” for preferential insertion of dA opposite the abasic site, but the lesion is a strong block to polymerase extension (78). In terms of repair, the presence of a phosphate ester in a position R to the 2′-aldehyde (Figure 1) obviates β-elimination reactions performed by some DNA repair enzymes, such as the lyase activities of formamidopyrimidine DNA glycosylase and endonuclease III (78, 79). An interesting feature of the enzymology here is that the active site lysine of endonuclease III forms a Schiff base with the 2′-aldehyde of the erythrose abasic site (78, 79). However, the erythrose abasic site is a substrate for hydrolysis by abasic site endonuclease activities of enzymes such as endonuclease IV and exonuclease III, albeit a less efficient substrate than other oxidized abasic sites and the native abasic site (78, 81). Care must be taken in interpreting the biological meaning of these results since only a limited number of sequence contexts have been studied.

5. 3′-Chemistry Oxidation of the 3′-position in DNA presents a more complicated case than that of 2′-oxidation. The current picture of 3′-oxidation chemistry is shown in Figure 1, with chemical partitioning along two pathways to form a strand break with 3′-phosphoglycolaldehyde, 5′-phosphate, and base propenoic acid residues (31, 32, 82) or a 3′-oxo-nucleotide residue that undergoes β-/δ-eliminations to release 2-methylene-3(2H)furanone (4, 5). The identification of the base propenoic acid and 3′-phosphoglycolaldehyde residues was originally made by Barton and co-workers using 3′-selective oxidants, rhodium complexes with phenanthrenequinone diimine (82). The identification of the 3′-oxo-nucleotide residue was predicted earlier (83) and more recently established experimentally using a site specifically generated 3′-radical (4, 51). 5.1. Model Systems. As noted for all of the positions in 2-deoxyribose in DNA, the availability of chemical species capable of performing site-selective 2-deoxyribose oxidation chemistry has greatly facilitated research in this field. Such is the case with the rhodium complexes characterized by Barton and co-workers (82). However, Bryant-Friedrich and co-workers developed a more broadly useful photoactivated precursor to the 3′-radical (4, 51, 84), analogous to systems used for 1′- and

Figure 7. Elimination of 2-methylene-3(2H)-furanone from the 3′-oxonucleotide product of 3′-oxidation of DNA.

4′-chemistry. When incorporated into an oligonucleotide, photoactivation leads to a site-specific 3′-radical that, under biologically relevant conditions, adds molecular oxygen to begin the cascade of reactions that lead to the final product spectrum. One of the revealing results from studies by Bryant-Friedrich and co-workers was the observation of products of 5′- and 4′oxidation of 2-deoxyribose in DNA in addition to the logical products of 3′-oxidation (4, 5). This “migration” of the radical to other sites in DNA is analogous to the thymidinyl radical studies discussed earlier (50) and the site specifically generated radical at C8 of dG that abstracts either the 5′- or the 2′-hydrogen atom (85). As shown in Figure 6, an intranucleotide abstraction of the 4′-hydrogen atom by the 3′-peroxyl radical generates a 3′-phosphoglycolate residue, while abstraction of the 5′hydrogen atom from a neighboring nucleotide produces a nucleoside 5′-aldehyde, a species observed by Byrant-Friedrich and co-workers (4, 5). 5.2. Methods of Measurement. The two sets of products arising from 3′-oxidation of DNA provide different properties that can be exploited to develop analytical methods. While no formal method has been published, the 3′-oxo-nucleotide-residue undergoes β-/δ-eliminations to release 2-methylene-3(2H)furanone (Figure 7) that should be readily extracted into organic solvents and amenable to quantification by gas chromatography and mass spectrometry (GC/MS), in a manner similar to the methylenefuranone derived from 1′-oxidation. The aldehyde moiety of 3′-phosphoglycolaldehyde residue provides another opportunity for quantitation. Collins et al. developed a method that exploits the aldehyde moiety by derivatization as a stable oxime with pentafluorobenzylhydroxylamine, followed by solvent extraction and quantification of the oxime by isotope dilution gas chromatography/negative chemical ionization/mass spectrometry (31, 32). The limit of quantification in the presence of DNA was 30 fmol per sample, corresponding to two molecules of PGA in 106 nucleotides for 170 µg of DNA. The use of GC has been criticized for the potential for adventitious formation of oxidative damage during sample preparation, such as during the silylization step that requires the application of heat that can lead to oxidative DNA damage (86). However, the recent studies initiated by the European Standards Committee on Oxidative DNA Damage (ESCODD) demonstrate that such adventitious DNA damage is more

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Figure 8. Glyoxal adducts of dG formed by the 3′-phosphoglycolaldehyde residue.

dependent on the operator than on the technique, with LC/MSMS methods producing some of the most spurious results with 8-oxo-dG measurements (87). With proper precautions, DNA damage artifacts can be well-controlled if not eliminated. For example, purification of 2-deoxyribose oxidation products away from genomic DNA, DNA fragments, and the nucleotides generated by enzymatic digestion is critical for reducing adventitious 2-deoxyribose oxidation, in much the same way that prepurification limits the formation of 8-oxo-dG from dG during sample processing (88). A second precaution is to reduce temperatures for steps such as silylization or β|δ-elimination reactions and to add truly effective antioxidants such as deferoxamine (also known as desferrioxamine and desferal) during all steps of sample processing. GC/MS methods remain among the most sensitive analytical methods available and, with proper precautions, can have background levels of the analyte as low as or lower than LC/MS methods (89, 90). Finally, the most rigorous analytical methods are validated using synthetic or quantitatively well-defined substrates such as oligonucleotides, containing known quantities of the analyte. For example, Collins et al. synthesized an oligonucleotide possessing a 3′phosphoglycolaldehyde (32). As noted earlier in this perspective, application of a method for quantifying 3′-phosphoglycolaldehyde residues revealed different yields for γ- and R-radiations (31). While R-particle irradiation of DNA resulted in 0.13 3′-phosphoglycolaldehyde residues per 106 nucleotides per Gy, γ-radiation produced 1.5 3′-phosphoglycolaldehyde residues per 106 nucleotides per Gy (31, 32). Correction for the different yields of total 2-deoxyribose oxidation events for R- and γ-radiations reveals that the former is seven-fold more efficient at producing the 3′phosphoglycolaldehyde than the latter, probably as a result of its higher LET properties (31, 32). Whether this difference reflects a shift between several branches of 3′-chemistry or between the various sites in 2-deoxyribose remains to be determined. By either mechanism, there are clearly different product spectra for different oxidants, a phenomenon that may affect the cellular responses to oxidative stress. 5.3. Biological Consequences. While the obvious consequence of not repairing a 3′-phosphoglycolaldehyde is a persistent strand break, a less obvious consequence of this lesion involves the formation of DNA adducts. Awada et al. observed that 2-phosphoglycolaldehyde, a model for the 3′-phosphoglycolaldehyde in DNA, reacted with dG and DNA to form the diastereomeric 1,N2-glyoxal adducts of dG, 3-(2-deoxy-β-Derythro-pentofuransyl)-6,7-dihydro-6,7-dihydroxyimidazo-[1,2a]purine-9(3H)-one (Figure 8) (91). The reaction was slow relative to the reaction of glyoxal with dG in DNA (10-6 vs 0.08 M-1 s-1, respectively). The mechanistic basis for the generation of glyoxal from 2-phosphoglycolaldehyde was found to involve a nonradical, oxygen-independent reaction apparently


Figure 9. Phosphate-phosphonate rearrangement mechanism for the generation of glyoxal from the phosphoglycolaldehyde product of 3′oxidation of DNA.

with a phosphate-phosphonate rearrangement, as shown in Figure 9 (91). These results explain the observation by Kasai and co-workers that oxidation of DNA with the Fe2+-EDTA complex results in the formation of the glyoxal adducts of dG (92).

6. 4′-Chemistry As shown in Figure 1, the chemistry of 4′-oxidation pathway partitions along either of two pathways to form a variety of stable and electrophilic products. One pathway involves formation of an oxidized abasic site containing a 2-deoxypentos-4ulose residue. The other pathway has recently been shown to consist of two distinct reactions depending on the oxidizing agent (7, 93, 94). The invariant branch of this pathway results in a strand break with a 3′-phosphoglycolate residue, while the variable product consists of either a base propenal for peroxynitrite, bleomycin and enediyne antibiotics (7, 14), or malondialdehyde and a free nucleobase for γ-radiation and Fe2+EDTA (93, 94). 6.1. Model Systems. Among the 2-deoxyribose oxidation chemistries, oxidation of the 4′-position has been the most exhaustively studied due to the availability of many model 4′chemistry systems. The antitumor antibiotic, bleomycin, causes 4′-oxidation exclusively in single- and double-strand DNA lesions and has been thoroughly reviewed in the 1992 Chemical Research in Toxicology article (14). The pioneering work of Giese and co-workers with precursors of the 4′-radical species led to the development of the entire family of analogous site-specific radical-generating nucleotides discussed earlier (46, 47, 95–97). They developed a 4′-Cacylthymidine that could be incorporated as a phosphoramidite into oligonucleotides and photoactivated in situ to produce the 4′-radical (96, 97). Interestingly, the resulting peroxyl radical was stable enough to be observed by MALDI-TOF mass spectrometry (95). Following photoactivation, subsequent product formation was monitored by mass spectrometry and other approaches, with the observation of 3′-phosphoglycolate residues and 5′-phosphate groups as the products of 4′-oxidation (46, 47, 95, 96). The major weakness of the selenated dA and acylated dT model systems for 4′-radical generation was the apparent lack of formation of the oxidized abasic site (46, 47, 95, 96), that is characteristic of 4′-chemistry generated by bleomycin (83, 98) and γ-radiation (98–100), for example (Figure 1). This highlights a potential idiosyncrasy of the current set of model radical-forming systems: The homolytic bond scission to form the sugar radical is accompanied by a partner radical that has the potential to interfere with the subsequent chemistry of the sugar radical, thus altering the chemistry of 2-deoxyribose oxidation, for example, by radical recombination or secondary oxidation of the initial radical. A similar argument has been made for participation of bleomycin in subsequent steps of the 4′-chemistry that it initiates (101, 102). Furthermore,


Figure 10. Model systems for in situ generation of the 2-deoxypentos4-ulose abasic site (2, 3).

analogous radical recombination chemistry is well-known for the DNA nitration chemistry performed by peroxynitrite and nitrosoperoxycarbonate, which undergo homolytic bond scission to form nitrogen dioxide radical and either hydroxyl radical or carbonate radical anion, with the former reacting with the base radical formed initially by one-electron oxidation by the latter (7). A second type of model system for studying 4′-chemistry involves site-specific generation of individual products, as illustrated earlier for the erythrose abasic site of 2′-oxidation in DNA. Two groups have succeeded in preparing the 2-deoxypentos-4-ulose abasic site in oligonucleotides, as illustrated in Figure 10 (2, 3). The approach used by Stubbe and co-workers involves incorporation of 4′-azido-2′-deoxyuridine-5′-triphosphate into duplex oligonucleotides by primer extension. Treatment with uracil-DNA glycosylase to release uracil and mild reduction of the azide with triphenylphosphine generates the 4′-abasic site (Figure 10) (2). The resulting abasic site was found to be more labile than a native abasic site (half-life of 26 vs 130 h at 37 °C, pH 7) (2). The approach taken by Greenberg and co-workers involves photolytic cleavage of 1′- and 4′-Onitroveratryl moieties to release the abasic site (3). They determined a half-life at 37 °C (pH 7.5) of 8 h (3), again less stable than a native abasic site. 6.2. Methods of Measurement. Several groups have developed methods to provide quantitative information about the 3′phosphoglycolate residue. For example, 3′-phosphoglycolate has been detected using a 32P-postlabeling method that involves nuclease-mediated release of 2′-deoxynucleotides with attached 3′-phosphoglycolate residues (103). This approach has proved to be quite sensitive, with fmol quantities detected in 0.5 µg of DNA (103). However, the absence of internal standards and the need to analyze four separate signals representing 3′phosphoglycolate residues attached to the four canonical nucleotides makes the method more semiquantitative (103). Attempts to develop a GC/MS method for quantifying glycolic acid derived from the 3′-phosphoglycolate residues in DNA (104, 105), again highly sensitive, have been hampered by high background contamination of glycolic acid from a variety of sources (105). Chen et al. recently developed GC/MS methods to quantify both the 3′-phosphoglycolate and the 2-deoxypentos-4-ulose abasic site (98). The abasic site was converted to a 3′-phosphoro3-pyridazinylmethylate derivative by treatment with hydrazine, and the derivative was released from DNA as 3-hydroxymethylpyridazine (HMP) by enzymatic hydrolysis. Similarly, 3′phosphoglycolate was released as 2-phosphoglycolic acid by enzymatic hydrolysis. Following HPLC prepurification, both 2-phosphoglycolate and 3-hydroxymethylpyridazine were silylated and quantified by GC/MS with limits of detection of 100 and 200 fmol and sensitivities of two and four lesions per 106 nucleotides in 250 µg of DNA, respectively. The abasic site method was validated using the model oligonucleotide developed by Stubbe and co-workers (2), while validation of the 3′phosphoglycolate method was achieved with a site specifically labeled oligonucleotide synthesized by mild oxidation of the 3′-phosphoglycolaldehyde-containing nucleotide from 3′-chemistry (91, 106). The method was then applied to DNA damaged

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by bleomycin and γ-radiation. As expected, bleomycin produced ∼eight phosphoglycolate residues and ∼three abasic sites per 10 total 2-deoxyribose oxidation events quantified as described for 3′-chemistry, with damage frequencies of 32 and 12 lesions per 106 nucleotides per µM, respectively. For γ-radiation, 4′oxidation was found to comprise only 13% of 2-deoxyribose oxidation events, with ∼three abasic sites (four per 106 nt per Gy) and ∼10 phosphoglycolates (13 per 106 nt per Gy) for every 100 2-deoxyribose oxidation events. 6.3. Biological Consequences. The biological consequences of the 4′-oxidation products are many. In terms of mutagenesis, Povirk and co-workers have thoroughly assessed the mutational consequences of bleomycin-induced DNA lesions, in addition to analogous damage produced by the enediynes (reviewed in ref 57). Both sets of agents produce double-strand breaks with defined chemistries, as well as abasic sites with closely opposed strand breaks. Bleomycin-induced mutagenesis in bacteria appears to result from replicative bypass of abasic sites, with repair blocked by the proximity of closely opposed strand breaks, while the bistranded lesions break down in mammalian cells to form double-strand breaks that induce deletions and rearrangements probably involving nonhomologous end joining (57). Greenberg and co-workers have studied the mutagenesis of the 2-deoxypentos-4-ulose abasic site (107). They observed a uniquely high level of three-nucleotide deletion products under SOS conditions in E. coli, deletions found to be dependent on DNA polymerases II and IV, and single nucleotide insertions that obeyed the “A-rule” (107). In terms of DNA repair, incision of the abasic site by exonuclease III and endonuclease IV was determined to be ∼six-fold less efficient than a native abasic site (107). Demple and co-workers observed that the major human abasic site endonuclease, Ape1, performed incision at the abasic site only a few-fold slower than a native abasic site, while polymerase β excised the 5′-terminal oxidized and native abasic site residues at similar rates (108). Further studies revealed that Ape1 hydrolyzed 3′-phosphoglycolate residues 25fold more slowly than the abasic site (108). There is also evidence for the formation of DNA adducts with one of the products of 4′-oxidation of DNA. The base propenal species accompanying the formation of 3′-phosphoglycolate residues by several oxidants (e.g., peroxynitrite, bleomycin, and enediynes) has been shown to be an important if not major source of the endogenous pyrimidopurinone adduct of dG, M1dG (93, 109–111). This adduct was originally observed by Marnett and co-workers as one of several species formed in in vitro reactions of dG and DNA with the lipid peroxidation product, malondialdehyde (112–114). Marnett and co-workers later discovered that M1dG is a ubiquitous endogenous DNA lesion presumably derived from ongoing lipid peroxidation reactions (115–118). However, the analogous chemical structures of base propenals and the reactive β-hydroxyacrolein form of malondialdehyde motivated studies that revealed a 100-fold greater reactivity of base propenals to form M1dG (109). Furthermore, base propenals were shown to be mutagenic, presumably by virtue of the formation of M1dG and other malondialdehyde-like adducts (110, 119). Recent studies in E. coli demonstrated an inverse correlation between the membrane content of polyunsaturated fatty acids, the major source of malondialdehyde, and the level of M1dG in cells subjected to oxidative stress and lipid peroxidation by peroxynitrite (93). This not only pointed to DNA oxidation and base propenals as a major source of M1dG but also raised the possibility of kinetic or thermodynamic selectivity in the

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Figure 11. Furfural as the elimination product of the 5′-nucleoside5′-aldehyde residue.

reactions of oxidants with cellular molecules, with polyunsaturated fatty acids representing a preferential target for oxidation. The question of the source of M1dG in human cells and tissues remains to be defined under different circumstances given the evidence for contributions from both base propenals and malondialdehyde (111). As mentioned earlier, γ-radiation leads to 4′-oxidation chemistry that results in the formation of malondialdehyde and free base rather than base propenal, which explains the absence of increased M1dG levels in irradiated E. coli cells in spite of evidence for lipid peroxidation (93). γ-Radiation thus represents a potentially useful tool to define the contribution of malondialdehyde to M1dG formation in human cells and tissues.

7. 5′-Chemistry As occurs with 3′- and 4′-oxidation, the chemistry of 5′-oxidation branches along either of two pathways (Figure 1). One path yields a strand break with 3′-formylphosphate- and 5′-(2-phosphoryl-1,4-dioxo-2-butane)-ended fragments (14, 120), while the other results in strand break with a 3′-phosphate and a 5′-nucleoside-5′-aldehyde residue (14). In spite of the apparently high proportions of 5′-chemistry occurring with agents such as Fe2+-EDTA (59), the chemistry of 5′-oxidation of 2-deoxyribose in DNA has not received the same attention as 1′- and 4′-positions in terms of both model 5′-radical-generating systems and biological studies. One reason may be the lack of an abasic site with 5′-chemistry and the associated interest in polymerase bypass and mutagenicity. However, the highly electrophilic products of 5′-oxidation have generated interest in terms of their biological consequences. 7.1. Model Systems. In addition to the enediyne antibiotics that produce mixtures of 5′-oxidation along with either 1′- or 4′-chemistry (14, 22, 23, 121, 122), Manetto et al. have developed a model system for site-specific generation of the 5′-radical. They synthesized 5′-tert-butyl ketone derivatives of thymidine and 2′-deoxyguanosine that, upon photoactivation, lead to the formation of the thymidin-5′-yl and 2′-deoxyguanosin-5′-yl radicals, respectively (123). Although these precursors have not been incorporated into oligonucleotides, photoactivation of the dG 5′-tert-butyl ketone was observed to lead to the formation of 5′,8-cyclo-2′-deoxyguanosine, presumably via intramolecular attack of the 5′-radical at the C8-N7 double bond of dG (123). This system could prove quite useful with the synthesis of a stable phosphoramidite version. [Note: The 5′-0-nitrophenyl system of Marx and co-workers does not produce a 5′-radical species mimicking biologically relevant 5′oxidation chemistry (124).] 7.2. Methods of Measurement. There have been few studies aimed at developing quantitative analytical methods for the 5′oxidation products. However, the chemical properties of the various products lend themselves to method development in several ways. First, the 5′-nucleoside-5′-aldehyde residue undergoes β,δ-elimination reactions to release furfural (Figure 11) (125–127), the physicochemical properties of which are similar to those of the methylfuranone species generated from β,δ-

elimination reactions with the 3′-oxonucleotide and 2-deoxyribonolactone products described earlier. A second feature of the products of 5′-oxidation is the presence of reactive carbonyls: the formyl ester of 3′formylphosphate and the aldehydes in the other products (Figure 1). Chen et al. exploited the dialdehydic structure and β-elimination properties of the 2-phosphoryl-1,4-dioxo-2-butane residue accompanying the 3′-formylphosphate species (Figure 1) to develop two analytical methods (120). The first approach involves reaction with O-benzylhydroxylamine to form a stable dioxime derivative (Figure 12), the β-elimination of which yielded the dioxime of trans-1,4-dioxo-2-butene that could be quantified by GC/MS (120). Using a benzylhydroxylamine dioxime derivative of [2H4]-cis-1,4-dioxo-2-butene as an internal standard, the dose response for the formation of 5′-(2-phosphoryl-1,4-dioxobutane) was determined to be linear for γ-radiation, with six lesions per 106 nt per Gy (120). This amounted to 0.04 lesions per 2-deoxyribose oxidation event when normalized for total 2-deoxyribose oxidation (120). The second approach involves treatment of the 5′-(2-phosphoryl-1,4-dioxobutane) species with hydrazine, which leads to a cyclization reaction and β-elimination to release pyridazine (Figure 12). This is analogous to the reaction of hydrazine with the 2-deoxypentose4-ulose abasic site, which arises from 4′-oxidation of 2-deoxyribose in DNA, to form a 3′-phosphopyridazine residue (98). The pyridazine is extracted into organic solvent and quantified by GC/MS (120). The limit of quantification of assay was determined to be ∼80 fmol in 500 µg of DNA. 7.3. Biological Consequences. The variety of electrophilic species generated by 5′-oxidation leads to a host of potentially important biological consequences. First among these is the reaction of the 5′-(2-phosphoryl-1,4-dioxobutane) residue to form bicyclic oxadiazabicyclo-(3.3.0)octaimine adducts of dC, dG, and dA (Figure 13) (128–132). Akin to the mutagenic cis1,4-dioxo-2-butene metabolite of furan (133, 134), the trans1,4-dioxo-2-butene elimination product of the 5′-(2-phosphoryl1,4-dioxobutane) residue was observed to react with dC in DNA to form the bicyclic adduct (131). Later studies revealed the formation of analogous adducts with dA and dG (129, 130, 132, 135) and, more importantly, in DNA subjected to oxidation by a variety of agents (128). These adducts may thus represent another form of endogenous DNA damage. A second biological consequence of 5′-oxidation of DNA involves the formation of protein adducts. More specifically, Jiang et al. observed transfer of isotopically labeled formyl groups from the 3′-formylphosphate residue to histone proteins in cells and, using an LC/MS approach, characterized the adduct as an N-formyl modification of the -amino group of lysine (Figure 14) (136). The adduct was detected only in histone proteins from a variety of sources, with controls for adventitious oxidation of DNA during protein isolation, which suggests that the adduct represents an endogenous secondary modification of histones (136). Its chemical analogy to the physiologically important lysine N-acetylation suggests that lysine N-formylation may interfere with signaling mediated by histone modifications.

8. Solvent Exposure and Other Models for 2-deoxyribose Oxidation in DNA One of the more controversial facets of 2-deoxyribose oxidation is the question of how DNA secondary structure affects the reactivity of the various hydrogen atoms toward oxidation. The most thorough and systematic approach to this problem was taken by Tullius and co-workers in their measure-


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Figure 12. Approaches to the quantification of the 5′-(2-phosphoryl-1,4-dioxobutane) residue of 5′-oxidation.

electrophorestic approach used by Tullius and co-workers to quantify the isotope effects. While the samples were heated at 90 °C for 3 min in an alkaline loading solution prior to electrophoresis (59), it is possible that a portion of the alkalinelabile abasic sites, such as the 2-deoxyribonolactone, survived, which would reduce the quantity of strand breaks and thus the apparent reactivity of a site such as the 1′-position. Clearly, a complete quantitative analysis of 2-deoxyribose oxidation products for different oxidants will resolve this issue.

9. 2-Deoxyribose Oxidation Chemistry in Cells Figure 13. DNA adducts derived from the 5′-(2-phosphoryl-1,4dioxobutane) residue of 5′-oxidation.

Figure 14. Lysine N-formylation by the 3′-formylphosphate residue of 5′-oxidation.

ments of the 1° isotope effects for oxidation of each position in 2-deoxyribose in DNA sites selectively labeled with deuterium (59). Using Fe2+-EDTA as a putative hydroxyl radical generator, they observed reactivity in terms of 1° isotope effects in the order 5′ > 4′ > 3′ ≈ 2′ ≈ 1′, which parallels the solvent accessibility of the various positions, so they concluded that HO• reactivity reflects the solvent exposure of the hydrogen atoms (59). However, as discussed earlier, recent observations by Greenberg and co-workers and Razskazovskiy and coworkers challenge the generality of this model, with a predominance of 1′-oxidation for γ-radiation (62, 63, 67), and we have observed lower than expected proportions of 4′-oxidation chemistry (98). That the solvent exposure model might only be applicable to Fe2+-EDTA is perhaps not surprising given the net negative charge of this complex at pH 7 and the possibility of restricted access to the charge-shielded grooves of DNA. One possible contribution to the observed reactivity of 2-deoxyribose with γ-radiation may arise from the relative stability of the carbon-centered radicals formed in the first step of 2-deoxyribose oxidation. Sevilla and co-workers calculated the order of radical stability as 1′ > 4′ > 2′ > 3′ > 5′ (137). It is also possible that abasic sites were not accounted for in the gel

There are several important questions motivating studies of 2-deoxyribose oxidation in cells and tissues. With implications for developing biomarkers of inflammation and oxidative stress, which 2-deoxyribose lesions predominate in cells? What are the relative quantities of native abasic sites (from repair of damaged bases) and oxidized 2-deoxyribose? How does the cellular environment affect the chemistry of 2-deoxyribose oxidation? To address these questions, several groups have obtained indirect evidence for the formation of 3′-phosphoglycolate residues in cells (138, 139), while Ciriolo et al. qualitatively demonstrated the formation of base propenals in bleomycin-treated nuclei (140). The observation of copper- and radiation-induced DNA–protein cross-links by Oleinick and coworkers may be related to the formation of Schiff bases between protein amino groups and 2-deoxyribose aldehyde/ketone residues (141). Along similar lines, Okada and co-workers have proposed that DNA–protein cross-links in cells treated with arsenic compounds arise as a result of alkali-labile abasic sites (142). Collins et al. applied a GC/MS analytical method for 3′-phosphoglycolaldehyde residues to quantify the lesion in γ-irradiated human cells, with the observation of a 1000-fold protective effect of the cellular environment against radiationinduced 2-deoxyribose oxidation (31). This is similar to the 1000-fold protective effect of the cellular environment on γ-radiation-induced 8-oxo-dG formation observed by Cadet and co-workers (143, 144).

10. The Metabolic Fate of 2-Deoxyribose Oxidation Products: Glutathione Conjugation and Glutathione S-Transferases With the ultimate goal of identifying candidate biomarkers of oxidative stress and inflammation, there has been an effort to move studies of 2-deoxyribose oxidation along a logical pathway from formation to fate, by focusing on the metabolic or, more broadly, biotranformational fate of 2-deoxyribose oxidation products. Such fates include chemical reactions while attached to DNA, such as β-/δ-elimination reactions, enzymatic release from DNA by repair enzymes, and subsequent chemical and enzymatic reactions of the released products. An illustration of these issues is found in the work of Marnett and co-workers for the pyrimidopurinone adduct of dG, M1dG, which is released

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These observations suggest that GST activity may represent a form of DNA repair by reacting with base propenals and other DNA-bound or freely diffusible 2-deoxyribose oxidation products, as well as the more commonly recognized lipid peroxidation targets. For example, a nuclear or chromatin GST activity could mitigate the reaction of base propenals with dG to form M1dG (93, 111) and the reaction of 5′-(2-phosphoryl-1,4dioxobutane) elimination product to form stable adducts with dG, dA, and dC (128–131). Acknowledgment. This work was supported by NIH Grants CA103146, GM059790, CA26735, CA110261, and CA116318 and by NIH/NIEHS Center Grant ES002109. Figure 15. GSH conjugation of cytosine propenal by direct reaction and catalyzed by GST (1).

by nucleotide excision repair and enters the bloodstream with subsequent oxidative metabolism (145, 146). One potentially important metabolic fate for sugar oxidation products involves glutathione conjugation. Cells are protected against oxidative stress by a host of antioxidant molecules and enzymes, with prominent roles played by glutathione and glutathione S-transferases (GSTs), with the latter catalyzing the conjugation of glutathione to a variety of native and exogenous electrophilic compounds (147, 148). Indeed, loss of GST activity has been correlated with an increased risk for cancer and other diseases (149, 150). GSTs are comprised of at least three families of enzymes distinguished by amino acid sequence as well as location: mitochondrial, cytosolic, and microsomal; the latter have been renamed membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) (151). While there is a single class of mitochondrial GST, there are six microsomal GSTs in three classes (I, II, and IV; mainly associated with eicosanoid metabolism) and at least 17 cytosolic enzymes in seven classes (A, M, P, S, T, Z, and O) (151). However, GST activity has also been observed in nuclei (152–156) and in nonhistone chromatin proteins (157). The cytosolic version of GST T1-1 (GST5-5) has been detected biochemically and immunologically in nuclei (152, 156), and the nuclear activity of this enzyme appears to be enriched over that in the cytosol (153). Several pieces of evidence suggest that many 2-deoxyribose oxidation products will be susceptible to reactions with glutathione and will be substrates for GSTs. First, there are two general rules for identifying GST substrates: that the molecule contains an electrophilic atom (many 2-deoxyribose oxidation products have two highly electrophilic sites) and that it reacts nonenzymatically with glutathione (158). This is likely to be the case for the many R,β-unsaturated carbonyl species arising from 2-deoxyribose oxidation (Figure 1), and it has been shown to be true for base propenals derived from 4′-oxidation (1). Second, GSTs have been shown to react with R,β-unsaturated aldehydes arising from lipid peroxidation (e.g., malondialdehyde and hydroxynonenal; ref 1), many of which are chemical analogues of 2-deoxyribose oxidation products (109). As illustrated in Figure 15, it has been observed that base propenals derived from 4′-oxidation of 2-deoxyribose are among the best substrates for GSTs of the Pi family (1). This is not surprising given the chemical similarities between the R,β-unsaturated aldehydes derived from lipid peroxidation and the R,β-unsaturated aldehyde present in base propenals. GSH conjugates have also been observed with the cis-1,4-dioxo-2-butene metabolite of furan, which is analogous to the trans-1,4-dioxo-2-butene elimination product of the 5′-(2-phosphoryl-1,4-dioxobutane) residue from 5′-oxidation (128–131).

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The Chemical Toxicology of 2-Deoxyribose Oxidation in DNA

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