Salt shield: intracellular salts provide cellular protection against ionizing radiation in the halophilic archaeon, Halobacterium salinarum NRC-1.

The halophilic archaeon Halobacterium salinarum NRC-1 was used as a model system to investigate cellular damage induced by exposure to high doses of ionizing radiation (IR). Oxidative damages are the main lesions from IR and result from free radicals production via radiolysis of water. This is the first study to quantify DNA base modification in a prokaryote, revealing a direct relationship between yield of DNA lesions and IR dose. Most importantly, our data demonstrate the significance of DNA radiation damage other than strand breaks on cell survival. We also report the first in vivo evidence of reactive oxygen species scavenging by intracellular halides in H. salinarum NRC-1, resulting in increased protection against nucleotide modification and carbonylation of protein residues. Bromide ions, which are highly reactive with hydroxyl radicals, provided the greatest protection to cellular macromolecules. Modified DNA bases were repaired in 2 h post irradiation, indicating effective DNA repair systems. In addition, measurements of H. salinarum NRC-1 cell interior revealed a high Mn/Fe ratio similar to that of Deinococcus radiodurans and other radiation-resistant microorganisms, which has been shown to provide a measure of protection for proteins against oxidative damage. The work presented here supports previous studies showing that radiation resistance is the product of mechanisms for cellular protection and detoxification, as well as for the repair of oxidative damage to cellular macromolecules. The finding that not only Mn/Fe but also the presence of halides can decrease the oxidative damage to DNA and proteins emphasizes the significance of the intracellular milieu in determining microbial radiation resistance.

The role of CSA in the response to oxidative DNA damage in human cells.

Cockayne syndrome (CS) is a rare genetic disease characterized by severe growth, mental retardation and pronounced cachexia. CS is most frequently due to mutations in either of two genes, CSB and CSA. Evidence for a role of CSB protein in the repair of oxidative DNA damage has been provided recently. Here, we show that CSA is also involved in the response to oxidative stress. CS-A human primary fibroblasts and keratinocytes showed hypersensitivity to potassium bromate, a specific inducer of oxidative damage. This was associated with inefficient repair of oxidatively induced DNA lesions, namely 8-hydroxyguanine (8-OH-Gua) and (5'S)-8,5'-cyclo 2'-deoxyadenosine. Expression of the wild-type CSA in the CS-A cell line CS3BE significantly decreased the steady-state level of 8-OH-Gua and increased its repair rate following oxidant treatment. CS-A cell extracts showed normal 8-OH-Gua cleavage activity in an in vitro assay, whereas CS-B cell extracts were confirmed to be defective. Our data provide the first in vivo evidence that CSA protein contributes to prevent accumulation of various oxidized DNA bases and underline specific functions of CSB not shared with CSA. These findings support the hypothesis that defective repair of oxidative DNA damage is involved in the clinical features of CS patients.

Damage to the bases in DNA induced by stimulated human neutrophils.

Leukocyte-induced DNA damage may partially account for the known association between chronic inflammation and malignancy. Since elucidation of the chemical nature of leukocyte-induced DNA damage may enhance our understanding of the mechanisms underlying leukocyte-induced DNA damage and the carcinogenesis associated with inflammation, the present study was undertaken to characterize the chemical modifications that occur in DNA exposed to stimulated human neutrophils. Calf thymus DNA was exposed to phorbol myristate acetate (PMA)-stimulated neutrophils in the presence or absence of exogenously added iron ions. DNA samples were subsequently hydrolyzed, derivatized and analyzed by gas chromatography-mass spectrometry with selected-ion monitoring. A variety of base modifications including cytosine glycol, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine were identified. The yield of these various base products was increased by the addition of iron ions. Specifically, in the presence of physiologic quantities of iron ions, approximately 7 of every 1,000 DNA bases were modified. Addition of the superoxide anion scavenger, superoxide dismutase, the hydrogen peroxide scavenger, catalase, the hydroxyl scavenger, dimethylsulfoxide, or the iron chelator, deferoxamine, to DNA mixtures containing PMA, neutrophils, and iron ions, greatly decreased the yield of the damaged DNA base products. Our results indicate that stimulated human neutrophils can damage each of the four bases in DNA. It is likely that hydroxyl radical, generated via an iron catalyzed Haber-Weiss reaction, mediates neutrophil-induced DNA base damage, since: (a) the chemical structure of neutrophil-induced DNA base damage is consistent with a hydroxyl radical-mediated mechanism, (b) hydroxyl radical generated via ionizing radiation in aqueous solution produces DNA base modifications that are identical to neutrophil-induced DNA base modifications, (c) iron ions increase neutrophil-induced DNA base damage, and (d) iron chelators or scavengers of superoxide anion, hydrogen peroxide or hydroxyl radical decrease neutrophil-induced DNA base damage.

Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry.

Measurement of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10(6) DNA bases can be detected using amounts of DNA as low as 2 microgram. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10(6) DNA bases, or even lower, when more DNA is used. Up to 50 microgram of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.

Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 protein and the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations.

We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as alpha-hOgg1, for excision of damaged bases from DNA exposed to gamma-irradiation. Excision products were identified and quantified using gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The GST-alpha-hOgg1 protein used in this study is a fusion of alpha-hOgg1 to the C-terminus of the GST protein. The results show that GST-alpha-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to gamma-irradiation in a solution saturated with N(2)O or air. Fourteen other lesions, including oxidised purines and pyrimidines, were not excised from these substrates. Catalytic constants were measured for the excision of 8-OH-Gua and FapyGua from DNA gamma-irradiated under N(2)O. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 4.47 x 10(-5)and 8.97 x 10(-5)(min(-1)nM(-1)), respectively. The substrate specificity and the catalytic parameters of the wild-type GST-alpha-hOgg1 protein were compared to that of a polymorphic form of alpha-hOgg1 harbouring a Ser-->Cys mutation at codon 326. In the Japanese population, 47.6% of individuals possess both alleles coding for the wild-type alpha-hOgg1-Ser(326)and mutant alpha-hOgg1-Cys(326)proteins. The GST-alpha-hOgg1-Cys(326)protein was purified and its substrate specificity was determined by GC/IDMS analysis. The results show that the GST-alpha-hOgg1-Cys(326)protein efficiently excises 8-OH-Gua and FapyGua from gamma-irradiated DNA. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 2. 82 x 10(-5)and 4.43 x 10(-5)(min(-1)nM(-1)), respectively. Furthermore, we compared the capacity of these two forms of alpha-hOgg1 to act on substrates containing 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Me-FapyGua). The k (cat)/ K (m)values for excision of Me-FapyGua were 278 x 10(-5)and 319 x 10(-5)(min(-1)nM(-1)), respectively. Cleavage of 34mer oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apurinic/apyrimidinic site paired with a cytosine was also investigated. The results show that both GST-alpha-hOgg1-Ser(326)and GST-alpha-hOgg1-Cys(326)catalyse the various cleavage reactions at very similar rates. Furthermore, both proteins efficiently complement the mutator phenotype of the fpg mutY mutant of Escherichia coli.

Effect of single mutations in the OGG1 gene found in human tumors on the substrate specificity of the Ogg1 protein.

We have investigated the effect of single amino acid substitutions of conserved arginines on the catalytic activities of the human Ogg1 protein (alpha-hOgg1-Ser(326)) (wild-type alpha-hOgg1). Mutant forms of hOgg1 with mutations Arg(46)-->Gln (alpha-hOgg1-Gln(46)) and Arg(154)-->His (alpha-hOgg1-His(154)) have previously been identified in human tumors. The mutant proteins alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these proteins and wild-type alpha-hOgg1 were investigated using gamma-irradiated DNA and the technique of gas chromatography/isotope-dilution mass spectrometry. All three enzymes excised 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) from gamma-irradiated DNA containing a multiplicity of base lesions. Michaelis-Menten kinetics of excision were measured. Significant differences between excision kinetics of these three enzymes were observed. Excision of FapyGua and 8-OH-Gua by wild-type alpha-hOgg1 was greater than that by alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154). The latter mutant protein was less active than the former. The diminished activity of the mutant proteins was more pronounced for 8-OH-Gua than for FapyGua. Cleavage assays were also performed using (32)P-labeled 34mer oligonucleotide duplexes containing a single 8-OH-Gua paired to each of the four DNA bases. The results obtained with the oligonucleotide containing the 8-OH-Gua/Cyt pair were in good agreement with those observed with gamma-irradiated DNA. Wild-type alpha-hOgg1 and its mutants repaired the three mismatches less efficiently than the 8-OH-Gua/Cyt pair. The substitution of Arg(154), in addition to diminishing the activity on 8-OH-Gua, relaxes the selectivity found in the wild-type alpha-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) found in human tumors are defective in their catalytic capacities.

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.

Repair of oxidative DNA damage in Drosophila melanogaster: identification and characterization of dOgg1, a second DNA glycosylase activity for 8-hydroxyguanine and formamidopyrimidines.

In Drosophila, the S3 ribosomal protein has been shown to act as a DNA glycosylase/AP lyase capable of releasing 8-hydroxyguanine (8-OH-Gua) in damaged DNA. Here we describe a second Drosophila protein (dOgg1) with 8-OH-Gua and abasic (AP) site DNA repair activities. The Drosophila OGG1 gene codes for a protein of 327 amino acids, which shows 33 and 37% identity with the yeast and human Ogg1 proteins, respectively. The DNA glycosylase activity of purified dOgg1 was investigated using gamma-irradiated DNA and gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The dOgg1 protein excises 8-OH-Gua and 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from gamma-irradiated DNA. with k(ca)(t)/K:(M) values of 21.0 x 10(-5) and 11.2 x 10(-5) (min(-1) nM(-1)), respectively. Enzymatic assays using oligodeoxyribonucleotides containing a single lesion show that dOgg1 displays a marked preference for DNA duplexes containing 8-OH-Gua, 8-OH-Ade or an AP site placed opposite a cytosine. The cleavage of the 8-OH-Gua-containing strand results from the excision of the damaged base followed by a ss-elimination reaction at the 3'-side of the resulting AP site. Cleavage of 8-OH-Gua.C duplex involves the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre. dOgg1 complements the mutator phenotype of fpg mutY mutants of Escherichia coli. Whole-mount in situ hybridizations on tissues at different stages of Drosophila development reveal that the dOGG1 messenger is expressed uniformly at a low level in cells in which mitotic division occurs. Therefore, Drosophila possesses two DNA glycosylase activities that can excise 8-OH-Gua and formamidopyrimidines from DNA, dOgg1 and the ribosomal protein S3.

Repair of oxidative DNA base lesions induced by fluorescent light is defective in xeroderma pigmentosum group A cells.

Fluorescent light (FL) has been shown to generate free radicals within cells, however, the specific chemical nature of DNA damage induced by FL has not previously been determined. Using gas chromatography/isotope dilution mass spectrometry, we have detected induction of the oxidative DNA lesions 5-hydroxycytosine (5-OH-Cyt), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) in cultured cells irradiated with FL. We followed the repair of these lesions in normal and xeroderma pigmentosum group A (XP-A) cells. 5-OH-Cyt and FapyGua were repaired efficiently in normal cells within 6 h following FL exposure. XP-A cells were unable to repair these oxidative DNA base lesions. Additionally, to compare the repair of oxidative lesions induced by various sources, in vitro repair studies were performed using plasmid DNA damaged by FL, gamma-irradiation or OsO(4)treatment. Whole cell extracts from normal cells repaired damaged substrates efficiently, whereas there was little repair in XP-A extracts. Our data demon-strate defective repair of oxidative DNA base lesions in XP-A cells in vivo and in vitro.

Structure and mechanism of hydroxyl radical-induced formation of a DNA-protein cross-link involving thymine and lysine in nucleohistone.

Hydroxyl radical-induced formation of a DNA-protein cross-link involving thymine and lysine in calf thymus nucleohistone in vitro is reported. Basic amino acids such as lysine constitute a very high proportion of the amino acids of histones, and help histones to bind to DNA in chromatin. For this reason, basic amino acids are likely to participate in DNA-protein cross-linking. For identification of the thymine-lysine cross-link in nucleohistone, hydroxyl radical-induced cross-linking of thymine to lysine was investigated first using a model system, i.e., an aqueous mixture of thymine and lysine. Hydroxyl radicals were generated by exposure of this mixture to ionizing radiation after N2O saturation. The technique of gas chromatography-mass spectrometry was used to analyze the samples for possible cross-links. One thymine-lysine cross-link was found and its structure was elucidated. Using gas chromatography-mass spectrometry with selected-ion monitoring, this thymine-lysine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone gamma-irradiated in N2O-saturated aqueous solution. The yield of this DNA-protein cross-link was also measured and found to be a linear function of radiation dose between 15 and 200 Gy. This yield amounted to 0.0085 mumol/J. Possible mechanisms for the formation of this DNA-protein cross-link in nucleohistone were proposed.

Modification of DNA bases in chromatin of intact target human cells by activated human polymorphonuclear leukocytes.

We investigated whether phorbol-12-acetate-13-myristate (PMA)-activated human polymorphonuclear leukocytes (PMNs) induce base modifications in target cell DNA in vivo. Human PMNs produced 9.4 +/- 0.8 (SD) nmol of H2O2/10(6) cells during 50 min of exposure to 2 micrograms/ml PMA and 13.7 +/- 2.8 nmol/10(6) cells during exposure to PMA plus 5 mM NaN3. Neither nonstimulated PMNs, nor PMA alone, nor NaN3 alone induced base modifications in chromatin-associated DNA of human Ad293 cells above control levels, when assayed by gas chromatography/mass spectrometry with selected-ion monitoring. However, a 60-min exposure to 1.7 +/- 0.4 x 10(6) PMNs/ml in the presence of 2 micrograms/ml PMA induced a 2-3-fold increase in the level of all modified bases detected by gas chromatography/mass spectrometry with selected-ion monitoring. The guanine-derived products 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and the adenine-derived product 4,6-diamino-5-formamidopyrimidine were induced to the highest levels among those bases detected. These data demonstrate that exposure to activated PMNs causes DNA base modifications in target cells in vivo typical of those induced by hydroxyl radical attack. The induction of potentially promutagenic modified bases may contribute to the mutagenicity of activated PMNs.

Nickel(II)- and cobalt(II)-dependent damage by hydrogen peroxide to the DNA bases in isolated human chromatin.

Nickel compounds are known to be carcinogenic to humans and animals. Cobalt compounds produce tumors in animals and are probably carcinogenic to humans. The mechanisms of the carcinogenicity of these metal compounds, however, have remained elusive. In the present work, we have investigated the ability of Ni(II) and Co(II) ions in the presence of H2O2 to cause chemical changes in DNA bases in chromatin extracted from cultured cells of human origin. Eleven modified DNA bases in chromatin were identified and quantitated by the use of gas chromatography-mass spectrometry. 2-Hydroxyadenine (isoguanine), which has not previously been shown to occur DNA or chromatin, was also identified. Products identified were typical hydroxyl radical-induced products of DNA bases, suggesting that the hydroxyl radical was involved in their formation. This idea was supported by partial inhibition of product formation by typical scavengers of hydroxyl radical. Partial inhibition of product formation indicated a possible "site-specific" formation of hydroxyl radical by unchelated Ni(II) and Co(II) ions bound to chromatin. Although treatment of chromatin for 1 h with Co(II)/H2O2 caused formation of significant amounts of products, treatment with Ni(II)/H2O2 required incubation times of more than 5 h and an increase in Ni(II) concentration before increases in product amounts above background levels became detectable. In both cases, ascorbic acid did not increase product yields. Glutathione at a physiologically relevant concentration had little overall effect on DNA base modification. Superoxide dismutase increased the yields of most products. Chelation of Ni(II) and Co(II) ions with EDTA almost completely inhibited product formation. Ni(II) in the presence of H2O2 produced greater base damage to the DNA in chromatin than to isolated DNA, unlike other metal ions tested. DNA damage in chromatin caused by Ni(II) and Co(II) ions in the presence of H2O2 may contribute to the established genotoxicity and carcinogenicity of these metal ions.

Measurement of oxidatively induced DNA damage and its repair, by mass spectrometric techniques.

Oxidatively induced damage caused by free radicals and other DNA-damaging agents generate a plethora of products in the DNA of living organisms. There is mounting evidence for the involvement of this type of damage in the etiology of numerous diseases including carcinogenesis. For a thorough understanding of the mechanisms, cellular repair, and biological consequences of DNA damage, accurate measurement of resulting products must be achieved. There are various analytical techniques, with their own advantages and drawbacks, which can be used for this purpose. Mass spectrometric techniques with isotope dilution, which include gas chromatography (GC) and liquid chromatography (LC), provide structural elucidation of products and ascertain accurate quantification, which are absolutely necessary for reliable measurement. Both gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), in single or tandem versions, have been used for the measurement of numerous DNA products such as sugar and base lesions, 8,5'-cyclopurine-2'-deoxynucleosides, base-base tandem lesions, and DNA-protein crosslinks, in vitro and in vivo. This article reviews these techniques and their applications in the measurement of oxidatively induced DNA damage and its repair.

Quantitative determination of oxidative base damage in DNA by stable isotope-dilution mass spectrometry.

For understanding of the role of oxidative DNA damage in biological processes such as mutagenesis and carcinogenesis, it is essential to identify and quantify this type of DNA damage in cells. This can be achieved by gas chromatography/mass spectrometry. The present study describes the quantification of modified bases in DNA by isotope-dilution mass spectrometry with the use of stable isotope-labeled analogues as internal standards. A number of isotopically labeled DNA bases were synthesized. The mass spectra of their trimethylsilyl derivatives were recorded. Calibration plots were obtained for known quantities of modified bases and their isotope-labeled analogues. Quantification of various modified DNA bases by isotope-dilution mass spectrometry was demonstrated in isolated chromatin exposed to ionizing radiation. The results indicate that gas chromatography/stable isotope-dilution mass spectrometry is an ideally suited technique for selective and sensitive quantification of modified bases in DNA.

Chemical determination of free radical-induced damage to DNA.

Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.

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.

Effect of single mutations on the specificity of Escherichia coli FPG protein for excision of purine lesions from DNA damaged by free radicals.

The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA repair enzymes.

Identification and quantification of 8,5'-cyclo-2'-deoxy-adenosine in DNA by liquid chromatography/ mass spectrometry.

Recent studies suggested that 8,5'-cyclo-2'-deoxyadenosine may play a role in diseases with defective nucleotide-excision repair. This compound is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the sugar moiety of 2'-deoxyadenosine. It is likely to be repaired by nucleotide-excision repair rather than by base-excision repair because of a covalent bond between the sugar and base moieties. We studied the measurement of 8,5'-cyclo-2'-deoxyadenosine in DNA by liquid chromatography/isotope-dilution mass spectrometry. A methodology was developed for the analysis of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography in DNA hydrolyzed to nucleosides by a combination of four enzymes, i.e., DNase I, phosphodiesterases I and II, and alkaline phosphatase. Detection by mass spectrometry was performed using atmospheric pressure ionization-electrospray process in the positive ionization mode. Results showed that liquid chromatography/isotope-dilution mass spectrometry is well suited for identification and quantification of 8,5'-cyclo-2'-deoxyadenosine in DNA. Both (5'R)- and (5'S)-diastereomers of 8,5'-cyclo-2'-deoxyadenosine were detected. The level of sensitivity of liquid chromatography/mass spectrometry with selected-ion monitoring amounted to 2 fmol of this compound on the column. The yield of 8,5'-cyclo-2'-deoxyadenosine was measured in DNA in aqueous solution exposed to ionizing radiation at doses from 2.5 to 80 Gray. Gas chromatography/mass spectrometry was also used to measure this compound in DNA. Both techniques yielded similar results. The yield of 8,5'-cyclo-2'-deoxyadenosine was comparable to the yields of some of the other major modified bases in DNA, which were measured using gas chromatography/mass spectrometry. The measurement of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography/mass spectrometry may contribute to the understanding of its biological properties and its role in diseases with defective nucleotide-excision repair.

Measurement of 8-hydroxy-2'-deoxyadenosine in DNA by liquid chromatography/mass spectrometry.

8-Hydroxyadenine (8-OH-Ade) is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the C-8 position of adenine followed by oxidation. We describe the measurement of the nucleoside form of this compound, 8-hydroxy-2'-deoxyadenosine (8-OH-dAdo) in DNA by liquid chromatography/mass spectrometry (LC/MS). The developed methodology enabled the separation by LC of 8-OH-dAdo from intact and modified nucleosides in enzymic hydrolysates of DNA. Measurements by MS were performed using atmospheric pressure ionization-electrospray process. Isotope-dilution MS was applied for quantification using a stable isotope-labeled analog of 8-OH-dAdo. The level of sensitivity of LC/MS with selected-ion monitoring (SIM) for 8-OH-dAdo amounted to approximately 10 femtomol of this compound on the LC column. This level of sensitivity is similar to that previously reported using LC-tandem MS (LC/MS/MS) with multiple-reaction monitoring mode (MRM) (7.5 femtomol). This compound was quantified in DNA at a level of approximately one molecule/10(6) DNA bases using amounts of DNA as low as 5 microg. The results suggested that this lesion may be quantified in DNA at even lower levels, when more DNA is used for analysis. In addition, gas chromatography/isotope-dilution mass spectrometry with SIM (GC/IDMS-SIM) was applied to measure 8-OH-Ade in DNA following its removal from DNA by acidic hydrolysis. The background levels of 8-OH-dAdo and 8-OH-Ade measured by LC/IDMS-SIM and GC/IDMS-SIM, respectively, were nearly identical. In addition, DNA samples, which were exposed to ionizing radiation at different radiation doses, were analyzed by these techniques. Nearly identical results were obtained, indicating that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results. The level of sensitivity of GC/MS-SIM for 8-OH-Ade was also measured and found to be significantly greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS-MRM for 8-OH-dAdo. The results show that the LC/MS technique is well suited for the measurement of 8-OH-dAdo in DNA.

A novel activity of E. coli uracil DNA N-glycosylase excision of isodialuric acid (5,6-dihydroxyuracil), a major product of oxidative DNA damage, from DNA.

We describe a novel activity of E. coli uracil DNA N-glycosylase (UNG) that excises isodialuric acid from DNA. Isodialuric acid is formed in DNA as a major oxidative product of cytosine. DNA substrates, which were prepared by gamma-irradiation, were incubated with UNG. Following precipitation of DNA, analyses of pellets and supernatant fractions by gas chromatography/mass spectrometry showed an efficient excision of isodialuric acid from DNA by UNG. None of the other 15 identified DNA base lesions was excised. The excision of isodialuric acid indicates that the non-aromaticity of a substrate may not be a limiting factor for UNG.