Measurement of oxidatively generated base damage in cellular DNA

This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.     

Free radical-induced damage to DNA: mechanisms and measurement

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.     

Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement

Oxidation reactions that involve several oxygen and nitrogen reactive species together with nucleobase radical cations give rise among various classes of lesions to modified bases. About 70 of oxidized nucleosides that include diastereomeric forms have been characterized in mechanistic studies involving isolated DNA and related model compounds. However, only eight modified bases have been accurately measured within cellular DNA upon exposure to either gamma or UVA radiations. Emphasis is placed in this survey on recent developments of HPLC associated with tandem mass spectrometry (MS/MS) operating in the mild electrospray ionization mode. Interestingly, the HPLC-MS/MS assay in the multiple reaction monitoring mode appears to be the more sensitive and accurate method currently available for singling out several oxidized nucleosides including 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 5-formyl-2'-deoxyuridine, 5-(hydroxymethyl-2'-deoxyuridine, 5-hydroxy-2'-deoxyuridine, and the four diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine within isolated and cellular DNA. However, one limitation of the assay that also applied to all chromatographic methods is the slight side-oxidation of normal bases during DNA extraction and subsequent work-up. This explains why the combined use of DNA repair glycosylases with either the comet assay or the alkaline elution technique is a better alternative to monitor the formation of low levels of oxidized bases within cellular DNA.     

Measurement of oxidatively generated base damage to nucleic acids in cells: facts and artifacts

The search for DNA biomarkers of oxidative stress has been hampered for several decades by the lack of relevant information on base oxidation products and the challenging issue of measuring low amounts of lesions, typically a few modifications within the range 10⁶?C10⁸ normal bases. In addition and this was ignored for a long time, there is a risk of artifactual oxidation of overwhelming nucleobases during DNA extraction and subsequent workup that has led to overestimation of some base damage up to 2?C3 orders of magnitude. The main aim of the survey is to critically review the available methods that have been developed for measuring oxidatively generated base damage in nuclear and mitochondrial DNA. Among the chromatographic methods, high-performance liquid chromatography associated with tandem mass spectrometry (HPLC?CMS/MS) is the most accurate and versatile approach whereas HPLC?Celectrochemical detection (ECD) is restricted to electrochemically active modifications. These methods allow measuring several single oxidized pyrimidine and purine bases, tandem base lesions and interstrand DNA cross-links in nuclear DNA. As complementary analytical tools, enzymatic methods that associate DNA repair enzymes with either the alkaline comet assay or the alkaline elution technique are suitable for assessing low variations in the level of different classes of oxidatively generated DNA lesions. Most of the immunoassays suffer from a lack of specificity due to the occurrence of cross-reactivity with overwhelming normal bases. One major exception concerns the immunodetection of 5-hydroxymethylcytosine, produced in a relatively high yield as an epigenetic DNA modification. HPLC?CMS/MS is now recognized as the gold standard for measuring oxidized bases and nucleosides in human fluids such as urine, saliva, and plasma.     

Room temperature derivatization of 5-hydroxy-2'- deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine for analysis by GC/MS

Hydroxylated DNA basesare one type of oxygen free radical-induced damage to DNA. Such damage has been implicated in the process of carcinogenesis, and the levels of hydroxylated DNA bases may serve as a marker of cancer risk in humans. Measurement of oxidative DNA damage can be hampered by the ease with which artifactual oxidative DNA damage can be induced via sample processing. In this report we describe convenient room temperature derivatization and stability of 5-hydroxy-2'-deoxycytidine (5-OHdCyd) and 5-hydroxymethyl-2'-deoxyuridine (5-OHmdU) using GC/MS analysis. The derivatization reagent was N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchloro-silane:acetonitrile, 2:1. This method avoids use of acid and is much milder than previously reported derivatization conditions which typically involve heating above 100C for at least 20 min. Although heating has been reported to be problematic, the calculated levels of 5-OHdCyd and 5-OHmdU in enzymatically-hydrolysed calf thymus DNA were very similar in our hands with and without heating the sample for 20 min. As an example of the technique, comparison of 5-OHdCyd and 5-OHmdU levels in calf thymus DNA indicated relatively higher endogenous levels of 5-OHdCyd. In DNA treated with hydrogen peroxide and ferric chloride, however, the levels of 5-OHmdU increased much more than that of 5-OHdCyd. In addition to these hydroxylated derivatives of deoxycytidine and thymidine, the method also appears to work well with 8-oxoguanine, 4,6-diamino-5-(formylamino)pyrimidine, and 5-methyl-2'-deoxycytidine. This method may therefore be useful with a variety of modified DNA bases and nucleosides.     

Commentary the Measurement of Oxidative Damage to DNA by HPLC and GC/MS Techniques

Oxidative damage to DNA has been measured by quantitating 8-hydroxy-2′-deoxyguanosine (8-OHdGuo) after enzymic digestion of DNA, followed by HPLC separation and electrochemical detection. Alternatively, 8-hydroxyguanine (and a wide range of other base-derived products of free radical attack) may be measured after acidic hydrolysis of DNA or chromatin, followed by derivatization and gas-chromatography/mass spectrometry. Both techniques have comparable sensitivity, but GC/MS enables determination of a wide variety of chemical changes to all four DNA bases and it can be applied to DNA-protein complexes. However, the two techniques do not always give similar results. Potential reasons for this are discussed. Greater attention to methodological questions is required before using measurement of 8-OHdGuo as a “routine” marker of oxidative DNA damage in vivo.     

Recent aspects of oxidative DNA damage: guanine lesions,measurement and substrate specificity of DNA repair glycosylases

This review discusses recent aspects of oxidation reactions of DNA and model compounds involving mostly OH radicals, one-electron transfer process and singlet oxygen (1O2). Emphasis is placed on the formation of double DNA lesions involving a purine base on one hand and either a pyrimidine base or a 2-deoxyribose moiety on the other hand. Structural and mechanistic information is also provided on secondary oxidation reactions of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a major DNA marker of oxidative stress. Another major topic which is addressed here deals with recent developments in the measurement of oxidative base damage to cellular DNA. This has been mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents, including UVA and ionizing radiations. In addition, the modified comet assay, which involves the use of bacterial DNA N-glycosylases to reveal two main classes of oxidative base damage, is applicable to isolated cells and is particularly suitable when only small amounts of biological material are available. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision pathways are briefly reviewed.     

Potential artifacts in the measurement of DNA deamination

Attack on DNA by some reactive nitrogen species results in deamination of adenine and guanine, leading to the formation of hypoxanthine and xanthine, respectively. Published levels of these products in cellular DNA have varied widely. Although these two deamination products are often measured by GC-MS analysis, the procedure of acid hydrolysis to release DNA bases for derivatization poses a risk of artifactual deamination of the DNA. In this study, we demonstrated the artifactual formation of these two deamination products during acid hydrolysis and hence developed a method for detecting and measuring 2'-deoxyinosine, the nucleoside of hypoxanthine. Our assay for 2'-deoxyinosine employs nuclease P1 and alkaline phosphatase to achieve release of the nucleosides from DNA, followed by HPLC prepurification with subsequent GC-MS analysis of the nucleosides. This assay detected an increase in the levels of 2'-deoxyinosine in DNA when commercial salmon testis DNA was treated with nitrous acid. We also used it to measure levels in various rat tissues of both normal and endotoxin-treated rats, but could not find increased 2'-deoxyinosine formation in tissues even though *NO production was substantially increased.     

Comparison of the levels of 8-hydroxyguanine in DNA as measured by gas chromatography mass spectrometry following hydrolysis of DNA by Escherichia coli Fpg protein or formic acid

8-hydroxyguanine (8-OH-Gua) is one of many lesions generated in DNA by oxidative processes including free radicals. It is the most extensively investigated lesion, due to its miscoding properties and its potential role in mutagenesis, carcinogenesis and aging, and also to the existence of analytical methods using HPLC and gas chromatography mass spectrometry (GC/MS). Some studies raised the possibility of artifacts generated during sample preparation. We investigated several experimental conditions in order to eliminate possible artifacts during the measurement of 8-OH-Gua by GC/MS. Derivatization has been reported to produce artifacts by oxidation of guanine to 8-OH-Gua in acid-hydrolysates of DNA, although the extent of artifacts seems to depend on experimental conditions. For removal of 8-OH-Gua from DNA, we used either formic acid hydrolysis or specific enzymatic hydrolysis with Escherichia coli Fpg protein. Derivatization of enzyme-hydrolysates should not generate additional 8-OH-Gua because of the absence of guanine, which is not released by the enzyme, whereas guanine released by acid may be oxidized to yield 8-OH-Gua. The measurement of 8-OH-Gua in calf thymus DNA by GC/isotope-dilution MS (GC/IDMS) using these two different hydrolyses yielded similar levels of 8-OH-Gua. This indicated that no artifacts occurred during derivatization of acid-hydrolysates of DNA. Pyridine instead of acetonitrile and room temperature were used during derivatization. Pyridine reduced the level of 8-OH-Gua, when compared with acetonitrile, indicating its potential to prevent oxidation. Two different stable-isotope labeled analogs of 8-OH-Gua used as internal standards for GC/IDMS analysis yielded similar results. A comparison of the present results with the results of recent trials by the European Standards Committee for Oxidative DNA Damage (ESCODD) is also presented.     

Determination of oxidative DNA base damage by gas chromatography-mass spectrometry. Effect of derivatization conditions on artifactual formation of certain base oxidation products

GC-MS is a widely used tool to measure oxidative DNA damage because of its ability to identify a wide range of base modification products. However, it has been suggested that the derivatization procedures required to form volatile products prior to GC-MS analysis can sometimes produce artifactual formation of certain base oxidation products, although these studies did not replicate previously-used reaction conditions, e.g. they failed to remove air from the derivatization vials. A systematic examination of this problem revealed that levels of 8-hydroxyguanine, 8-hydroxyadenine,5-hydroxycytosine and 5-(hydroxymethyluracil) in commercial calf thymus DNA determined by GC-MS are elevated by increasing the temperature at which derivatization is performed in our laboratory. In particular, 8-hydroxyguanine levels after silylation at 140°C were raised 8-fold compared to derivatization at 23°C. Experiments on the derivatization of each undamaged base revealed that the artifactual oxidation of guanine, adenine, cytosine and thymine respectively was responsible. Formation of the above products was potentiated by not purging with nitrogen prior to derivatization. Increasing the temperature to 140°C or allowing air to be present during derivatization did not significantly increase levels of the other oxidized bases measured.

This work suggests that artifactual oxidation during derivatization is restricted to certain products (8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-[hydroxymethyluracil]) and can be decreased by reducing the temperature of the derivatization reaction to 23°C and excluding as much air possible. Despite some recent reports, we were easily able to detect formamidopyrimidines in acid-hydrolyzed DNA. Artifacts of derivatization are less marked than has been claimed in some papers and may vary between laboratories, depending on the experimental procedures used, in particular the efficiency of exclusion of O₂ during the derivatization process.     

Oxidative damage to DNA: formation, measurement and biochemical features

Emphasis is placed in the first part of this survey on mechanistic aspects of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) as the result of exposure to z.rad;OH radical, one-electron oxidants and singlet oxygen (1O(2)) oxidation. It was found that 8-oxoGua, which is generated by either hydration of the guanine radical cation or .OH addition at C8 of the imidazole ring, is a preferential target for further reactions with 1O(2) and one-electron oxidants, including the highly oxidizing oxyl-type guanine radical. Interestingly, tandem base lesions that involve 8-oxoGua and a vicinal formylamine residue were found to be generated within DNA as the result of a single .OH radical hit. The likely mechanism of formation of the latter lesions involves the transient generation of 5-(6)-peroxy-6-(5)-hydroxy-5,6-dihydropyrimidyl radicals that may add to the C8 of a vicinal guanine base before undergoing rearrangement. Another major topic which is addressed deals with recent developments in the measurement of oxidative base damage to cellular DNA. This was mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents including UVA and ionizing radiations. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision and nucleotide excision pathways are briefly reviewed. For this purpose modified oligonucleotides in which cyclopurine, and cyclopyrimidine nucleosides were site-specifically inserted were synthesized.     

Determination of oxidative DNA base damage by gas chromatography-mass spectrometry. Effect of derivatization conditions on artifactual formation of certain base oxidation products

GC-MS is a widely used tool to measure oxidative DNA damage because of its ability to identify a wide range of base modification products. However, it has been suggested that the derivatization procedures required to form volatile products prior to GC-MS analysis can sometimes produce artifactual formation of certain base oxidation products, although these studies did not replicate previously-used reaction conditions, e.g. they failed to remove air from the derivatization vials. A systematic examination of this problem revealed that levels of 8-hydroxyguanine, 8-hydroxyadenine,5-hydroxycytosine and 5-(hydroxymethyluracil) in commercial calf thymus DNA determined by GC-MS are elevated by increasing the temperature at which derivatization is performed in our laboratory. In particular, 8-hydroxyguanine levels after silylation at 140°C were raised 8-fold compared to derivatization at 23°C. Experiments on the derivatization of each undamaged base revealed that the artifactual oxidation of guanine, adenine, cytosine and thymine respectively was responsible. Formation of the above products was potentiated by not purging with nitrogen prior to derivatization. Increasing the temperature to 140°C or allowing air to be present during derivatization did not significantly increase levels of the other oxidized bases measured.

This work suggests that artifactual oxidation during derivatization is restricted to certain products (8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-[hydroxymethyluracil]) and can be decreased by reducing the temperature of the derivatization reaction to 23°C and excluding as much air possible. Despite some recent reports, we were easily able to detect formamidopyrimidines in acid-hydrolyzed DNA. Artifacts of derivatization are less marked than has been claimed in some papers and may vary between laboratories, depending on the experimental procedures used, in particular the efficiency of exclusion of O₂ during the derivatization process.     

The effect of experimental conditions on the levels of oxidatively modified bases in DNA as measured by gas chromatography-mass spectrometry: how many modified bases are involved? Prepurification or not?

Recently, an artifactual formation of a number of modified DNA bases has been alleged during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC-MS). These modified bases were 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra), and 5-formyluracil, which represent only a small percentage of more than 20 modified DNA bases that can be analyzed by GC-MS. However, relevant papers reporting the levels of these modified bases in DNA of various sources have not been cited, and differences in experimental procedures have not been discussed. We investigated the levels of modified bases in calf thymus DNA by GC-MS using derivatization at three different temperatures. The results obtained with GC/isotope-dilution MS showed that the levels of 5-OH-Cyt, 8-OH-Ade, 5-OH-Ura, and 5-OHMeUra were not affected by increasing the derivatization temperature from 23 degrees C to 120 degrees C. The level of 8-OH-Gua was found to be higher at 120 degrees C. However, this level was much lower than those reported previously. Formamidopyrimidines were readily analyzed in contrast to some recent claims. The addition of trifluoroacetic acid (TFA) adversely affected the levels of pyrimidine-derived lesions, suggesting that TFA is not suitable for simultaneous measurement of both pyrimidine- and purine-derived lesions. The data obtained were also compared with those previously published. Our data and this comparison indicate that no artifactual formation of 5-OH-Cyt, 8-OH-Ade, and 5-OHMeUra occurred under our experimental conditions in contrast to recent claims, and no prepurification of DNA hydrolysates by a tedious procedure is necessary for accurate quantification of these compounds. The artifactual formation of 8-OH-Gua can be eliminated by derivatization at room temperature for at least 2 h, without the use of TFA. The results in this article and their comparison with published data indicate that different results may be obtained in different laboratories using different experimental conditions. The data obtained in various laboratories should be compared by discussing all relevant published data and scientific facts, including differences between experimental conditions used in different laboratories.     

Oxidative base damage to DNA: specificity of base excision repair enzymes

Base excision repair (BER) is likely to be the main mechanism involved in the enzymatic restoration of oxidative base lesions within the DNA of both prokaryotic and eukaryotic cells. Emphasis was placed in early studies on the determination of the ability of several bacterial DNA N-glycosylases, including Escherichia coli endonuclease III (endo III) and formamidopyrimidine DNA N-glycosylase (Fpg), to recognize and excise several oxidized pyrimidine and purine bases. More recently, the availability of related DNA repair enzymes from yeast and human has provided new insights into the enzymatic removal of several.OH-mediated modified DNA bases. However, it should be noted that most of the earlier studies have involved globally modified DNA as the substrates. This explains, at least partly, why there is a paucity of accurate kinetic data on the excision rate of most of the modified bases. Interestingly, several oxidized pyrimidine and purine nucleosides have been recently inserted into defined sequence oligonucleotides. The use of the latter substrates, together with overexpressed DNA N-glycosylases, allows detailed studies on the efficiency of the enzymatic release of the modified bases. This was facilitated by the development of accurate chromatographic and mass spectrometric methods aimed at measuring oxidized bases and nucleosides. As one of the main conclusions, it appears that the specificity of both endo III and Fpg proteins is much broader than expected a few years ago.     

Increased oxidative damage in nuclear and mitochondrial DNA in Alzheimer's disease

Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age-matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10-fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight-hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10-fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD.     

Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells?

The most commonly measured marker of oxidative DNA damage is 8-oxo-7,8-dihydroguanine (8-oxoGua) or its deoxyribonucleoside (8-oxodGuo). Published estimates of the concentration of 8-oxoGua/8-oxodGuo in DNA of normal human cells vary over a range of three orders of magnitude. Analysis by chromatographic methods (GC-MS, HPLC with electrochemical detection (ECD) or HPLC-MS/MS) is beset by the problem of adventitious oxidation of guanine during sample preparation. An alternative approach, based on the use of the DNA repair enzyme formamidopyrimidine DNA N-glycosylase (FPG) to make breaks in the DNA at sites of the oxidised base, gives much lower values. ESCODD, the European Standards Committee on Oxidative DNA Damage, has been testing the ability of different laboratories using a variety of methods to measure 8-oxoGua in standard samples of 8-oxodGuo, calf thymus DNA, pig liver, oligonucleotides, and HeLa cells, and in lymphocytes isolated from blood of volunteers. HPLC-ECD is capable of measuring 8-oxodGuo induced experimentally in calf thymus DNA or HeLa cells with high accuracy. However, there is no sign of consensus over the background level of this damage, suggesting that, even though standard extraction procedures were used, variable oxidation of Gua is still occurring. GC-MS failed to detect a dose response of induced 8-oxoGua and cannot be regarded as a reliable method for measuring low levels of damage. HPLC-MS/MS as yet has not proved capable of measuring low levels of oxidative DNA damage. FPG-based methods seem to be less prone to the artefact of additional oxidation. Although they can be used quantitatively, they require careful calibration and standardisation if they are to be used in human biomonitoring. The background level of DNA oxidation in normal human cells is likely to be around 0.3-4.2 8-oxoGua per 10(6) Gua. An effort should be made to develop alternative, validated methods for estimating oxidative DNA damage.     

Lipid peroxidation-induced DNA damage in cancer-prone inflammatory diseases: a review of published adduct types and levels in humans

Persistent oxidative stress and excess lipid peroxidation (LPO), induced by inflammatory processes, impaired metal storage, and/or dietary imbalance, cause accumulations and massive DNA damage. This massive DNA damage, along with deregulation of cell homeostasis, leads to malignant diseases. Reactive aldehydes produced by LPO, such as 4-hydroxy-2-nonenal, malondialdehyde, acrolein, and crotonaldehyde, react directly with DNA bases or generate bifunctional intermediates which form exocyclic DNA adducts. Modification of DNA bases by these electrophiles, yielding promutagenic exocyclic adducts, is thought to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. Ultrasensitive detection methods have facilitated studies of the concentrations of promutagenic DNA adducts in human tissues, white blood cells, and urine, where they are excreted as modified nucleosides and bases. Thus, immunoaffinity-(32)P-postlabeling, high-performance liquid chromatography-electrochemical detection, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, immunoslotblot assay, and immunohistochemistry have made it possible to detect background concentrations of adducts arising from endogenous LPO products in vivo and studies of their role in carcinogenesis. These background adduct levels in asymptomatic human tissues occur in the order of 1 adduct/10(8) and in organs affected by cancer-prone inflammatory diseases these can be 1 or 2 orders of magnitude higher. In this review, we critically discuss the accuracy of the available methods and their validation and summarize studies in which measurement of exocyclic adducts suggested new mechanisms of cancer causation, providing potential biomarkers for cancer risk assessment in humans with cancer-prone diseases.     

Simultaneous measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine by HPLC-MS/MS

An assay with high selectivity and sensitivity has been developed which, for the first time, allows quantitative, simultaneous measurement in DNA of both 8-oxo-2'-deoxyguanosine (8-oxodG) and 8-oxo-2'-deoxyadenosine (8-oxodA)-important biomarkers of oxidative DNA damage in vivo. Using reversed-phase HPLC coupled to electrospray tandem mass spectrometry (HPLC-MS/MS) in multiple reaction monitoring (MRM) mode it was possible to detect background levels of these lesions in commercially available calf thymus DNA (85 +/- 3 and 7.1 +/- 0.2 per 10(6) DNA bases for 8-oxodG and 8-oxodA respectively; n = 3). Levels of 8-oxodG determined by HPLC coupled to an electrochemical detection system (HPLC-EC) were found to be similar (75 +/- 6 per 10(6) DNA bases; n = 3) to those obtained using tandem mass spectrometry.     

Facts about the artifacts in the measurement of oxidative DNA base damage by gas chromatography-mass spectrometry

Recently, several papers reported an artifactual formation of a number of modified bases from intact DNA bases during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC/MS). These reports dealt with 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra) and 5-formyluracil that represent only a small percentage of the 20 or so modified DNA bases that can be analyzed by GC/MS. Removal of intact DNA bases by prepurification of calf thymus DNA hydrolysates using HPLC was shown to prevent artifactual formation of these modified bases during derivatization. It needs to be emphasized that the procedures for hydrolysis of DNA and derivatization of DNA hydrolysates used in these papers substantially differed from the established procedures previously described. Furthermore, a large number of relevant papers reporting the levels of these modified bases in DNA of various sources have been ignored. Interestingly, the levels of modified bases reported in the literature were not as high as those reported prior to prepurification. Most values for the level of 5-OH-Cyt were even lower than the level measured after prepurification. Levels of 8-OH-Ade were quite close to, or even the same as, or smaller than the level reported after prepurification. The same holds true for 5-OHMeUra and 8-OH-Gua. All these facts raise the question of the validity of the claims about the measurement of these modified DNA bases by GC/MS.

A recent paper reported a complete destruction of 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) by formic acid under the conditions of DNA hydrolysis prior to GC/MS. The complete destruction of FapyGua and FapyAde by formic acid is in disagreement with the data on these compounds in the literature. These two compounds were measured by GC/MS following formic acid hydrolysis for many years in our laboratory and by other researchers with no difficulties. These facts clearly raise the question of the validity of the claims made about the previous measurements of these compounds by GC/MS.     

Facts about the artifacts in the measurement of oxidative DNA base damage by gas chromatography-mass spectrometry

Recently, several papers reported an artifactual formation of a number of modified bases from intact DNA bases during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC/MS). These reports dealt with 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra) and 5-formyluracil that represent only a small percentage of the 20 or so modified DNA bases that can be analyzed by GC/MS. Removal of intact DNA bases by prepurification of calf thymus DNA hydrolysates using HPLC was shown to prevent artifactual formation of these modified bases during derivatization. It needs to be emphasized that the procedures for hydrolysis of DNA and derivatization of DNA hydrolysates used in these papers substantially differed from the established procedures previously described. Furthermore, a large number of relevant papers reporting the levels of these modified bases in DNA of various sources have been ignored. Interestingly, the levels of modified bases reported in the literature were not as high as those reported prior to prepurification. Most values for the level of 5-OH-Cyt were even lower than the level measured after prepurification. Levels of 8-OH-Ade were quite close to, or even the same as, or smaller than the level reported after prepurification. The same holds true for 5-OHMeUra and 8-OH-Gua. All these facts raise the question of the validity of the claims about the measurement of these modified DNA bases by GC/MS.

A recent paper reported a complete destruction of 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) by formic acid under the conditions of DNA hydrolysis prior to GC/MS. The complete destruction of FapyGua and FapyAde by formic acid is in disagreement with the data on these compounds in the literature. These two compounds were measured by GC/MS following formic acid hydrolysis for many years in our laboratory and by other researchers with no difficulties. These facts clearly raise the question of the validity of the claims made about the previous measurements of these compounds by GC/MS.