Product Of Oxidative DNA Damage Research Articles

Measurement of Oxidative DNA Damage and Repair in Specific DNA Sequences

We describe two methods that were developed in our laboratory to measure the production and repair of oxidative DNA damage in specific DNA sequences. Both of these methods rely on the use of monoclonal antibodies against modified nucleotides to separate DNA sequences that contain damage from those in which repair has occurred. In one case, the modified base is bromodeoxyuridine, which is inserted into the DNA during the repair synthesis step of excision repair. An antibody against this modified base is used to detect the production of the bromodeoxyuridine in the repair patch. This approach allows for the measurement of repair of any DNA lesion whose removal is accompanied by the production of a DNA repair patch. In the other case, the modified base is thymine glycol, an oxidized base that is produced by hydrogen peroxide and ionizing radiation. Using a monoclonal antibody that recognizes this altered base, detection of the production and repair of a specific base damage in a DNA sequence can be accomplished. These approaches are used in our laboratory to examine the transcription-coupled repair of oxidative DNA damage in both yeast and mammalian cells.

Haptoglobin reduces renal oxidative DNA and tissue damage during phenylhydrazine-induced hemolysis

Haptoglobin knockout (Hp-/-) mice are more sensitive to phenylhydrazine-induced hemolysis than Hp+/+ mice. Hemolysis was induced in Hp-/- and Hp+/+ mice using phenylhydrazine. Relative renal tissue damage and function were then assessed. Hp-/- mice had higher basal levels of renal lipid peroxidation, as evidenced by levels of malonaldehyde and 4-hydroxy-2(E)-nonenal (MDA/HNE). After the administration of phenylhydrazine, levels of 8-hydroxyguanine (but not other products of oxidative DNA damage) were significantly elevated in the renal DNA. There was also increased induction of heme oxygenase-1. The more severe renal damage in Hp-/- mice was also evident in the delayed erythropoietin gene expression and poorer renal clearance of 3H-inulin. This reduction in glomerular filtration function in Hp+/+ and Hp-/- mice could be restored to baseline by vasodilators (prazosin or diazoxide), implicating renal vasoconstriction as a major mechanism of acute renal failure during induced hemolysis. Precipitation of hemoglobin in the kidney was not increased in Hp-/- mice. Haptoglobin appears to play an important physiological role as an antioxidant, particularly during hemolysis.

Efficient synthesis of 8-Oxo-dGTP: A mutagenic nucleotide

An efficient synthesis of mutagenic and oxidative DNA damage product, 8-oxo-dGTP ( 4 ) has been achieved in high yield, along with a serendipitous generation of 8-dimsyl-dG ( 2 ). In combination with dPTP ( 5 ), 8-oxo-dGTP ( 4 ) can be formulated into a kit for investigating DNA random mutagenesis.

Reduced glutathione oxidation ratio and 8 ohdG accumulation by mild ischemic pretreatment

A critical role of oxidative stress has been implicated in ischemic brain damage. Mild ischemic pretreatment and/or synthesis of heat shock proteins (HSPs) has been suggested to protect against oxidative brain damage. However, experimental support of this suggestion have proven to be difficult partly because sensitive indices to assess oxidative consequences of ischemic brain damage were few. In this study, we have attempted to establish biochemical assay systems to quantitate oxidative brain damage following ischemia. We produced experimental brain ischemia in the Mongolian gerbil ( Meriones unguiculatus) and examined the hippocampus for ischemic brain damage. The results obtained from ischemic gerbil hippocampus demonstrated that oxidative brain damage can be quantitated by determining glutathione oxidation ratio together with the accumulation of the oxidative DNA damage product, 8-hydroxy-2′-deoxyguanosine (8 ohdG). Our results also demonstrated a role for mild ischemic pretreatment and synthesis of HSPs against oxidative brain damage. We showed that mild 2-min ischemic pretreatment reduced the degree of both glutathione oxidation ratio and 8 ohdG accumulation in gerbil hippocampus subsequent to 10 min ischemic challenge. We also showed that the accumulation of HSP70 was closely associated with the reduction of oxidative brain damage. To our knowledge, this is the first report to investigate glutathione redox states and oxidative DNA damage levels to evaluate a protective role of mild ischemic pretreatment and HSP synthesis following brain ischemia. Our data validate the previous suggestions and provide new additional data that argue for the protective role of mild ischemic pretreatment and HSP70 synthesis against oxidative brain damage.

Differences in the response to oxidative stress and mutant frequency in CD (Sprague-Dawley) and Fisher 344 rats due to an induced inflammatory response.

In this study, the rodent air pouch model was used to examine the production and processing of oxidative DNA damage in two strains of rats commonly used in toxicity testing. An inflammatory response was induced by injecting zymosan A (50 mg) into an air pouch on male CD (Sprague-Dawley [S-D]) and Fisher 344 (F-344) rats, and the animals were then sacrificed at 1, 3, 7, 14, and 28 days (n = 6 per time point per strain). Tissues from the lining of the air pouch were collected for 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis and for paraffin embedding. Significant (P < 0.01) increases in 8-OH-dG were observed after 1 day in the DNA from cells lining the air pouch of zymosan A-treated versus control S-D (101.5 +/- 27.1 vs. 23.1 +/- 2. 7 8-OH-dG/dG x 10(5)) and F-344 (51.4 +/- 5.3 vs. 14.4 +/- 0.6 8-OH-dG/dG x 10(5)) rats. By 28 days, 8-OH-dG levels had returned to background in S-D rats, but remained elevated in F-344 rats. The frequency of apoptosis was evaluated using the in situ end-labeling (TUNEL) assay, which revealed that zymosan A-treated S-D rats had a significantly (P < 0.05) higher frequency of apoptosis compared to zymosan A-treated F-344 rats. To examine the potential consequences of these differences in endogenously produced DNA damage and apoptosis, we measured mutations at the hprt locus in fibroblasts of the pouch lining and observed a significant (P < 0.05) increase in the mutant frequency at day 28 in F-344 rats (54.2 +/- 13.6 mutants per 10(6) cells) compared to controls (4.5 +/- 2.0 mutants per 10(6) cells). The mutant frequency was not increased in S-D rats. These data demonstrate that strain differences in the production and processing of oxidative DNA damage due to an inflammatory response may impact the long-term pathologic consequences of chronic inflammation. Environ. Mol. Mutagen. 35:336-342, 2000 Published 2000 Wiley-Liss, Inc.

Structure of an oligonucleotide containing a N-(2-deoxy-β-D- erythro-pentofuranosyl)formamide residue facing a guanine

Formamide residue is a major oxidative DNA damage product from ionizing radiation on thymine residues in DNA. We report NMR and molecular modeling studies on a DNA duplex structure which contains guanine opposite formamide residue. Formamide residue exists as either the cis and trans isomer. For the trans and the cis isomers, we find that guanine and formamide are stacked inside the helix and are hydrogen bonded. The oligonucleotide adopts globally a B form structure for the two isomers. Conformational changes are observed between the two isomers.

Sequence and Stacking Dependence of 8-Oxoguanine Oxidation: Comparison of One-Electron vs Singlet Oxygen Mechanisms

The oxidation of 7,8-dihydro-8-oxoguanine (8-oxoG)-containing oligodeoxynucleotides has been investigated using a variety of oxidants, including one-electron oxidants (Ir(IV), Fe(III), NiCR/KHSO5, and SO4-•) as well as singlet oxygen, generated both photochemically and thermally. The extents of oxidation in single-stranded and duplex oligodeoxynucleotides are compared, confirming theoretical ionization potentials of 8-oxoG in different sequence contexts in duplex DNA. As with guanine, 8-oxoG residues stacked in a duplex with a 3‘ neighboring G are more readily oxidized by one-electron oxidants than those stacked next to other bases, although the effect of stacking appears to be less pronounced for 8-oxoG than for G. Regardless of sequence, 8-oxoG is always more easily oxidized than the four natural nucleobases, even in the presence of multiple G sequences. Reactions with singlet molecular oxygen, thought to proceed through a cycloaddition mechanism, show little sequence selectivity and a 7-fold higher reactivity with single-stranded compared to duplex 8-oxoG residues. One-electron oxidants, such as Ir(IV) complexes, showed a more modest 3−4-fold higher reactivity with single-stranded DNA. In contrast, the Schiff base complex [NiCR]2+, used in conjunction with a strong oxidant, KHSO5, shows a 2-fold preference for oxidation of duplex vs single-stranded 8-oxoG, perhaps because of the high driving force and the possibility for competing G oxidation to equilibrate to 8-oxoG oxidation via hole transfer. Overall, these results point to subtle mechanistic differences in one-electron oxidation but a major distinction between one-electron and 1O2-mediated oxidation. Furthermore, they suggest an important role for 8-oxoG, not only as a product of oxidative DNA damage but also as a substrate for further oxidation.

Different mechanisms for the photoinduced production of oxidative DNA damage by fluoroquinolones differing in photostability.

Several fluoroquinolone antibacterial agents exhibit an adverse phototoxic effect in humans and are photo-cocarcinogenic in mice. The UV-induced production of reactive oxygen species plays a role in the toxicity and may be involved in carcinogenicity. Four fluoroquinolones were examined for the ability to photochemically produce oxidative damage in naked DNA. The major structural difference in the fluoroquinolones that would have an effect on their photostability is the functionality at the 8-position. At this position, 1-cyclopropyl-7-(2,8-diazbicyclo[4.3.0]non-8-yl)-6, 8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (BAY y3118) contains a chlorine atom, lomefloxacin a fluorine atom, ciprofloxacin a proton, and moxifloxacin a methoxy group. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in calf thymus DNA was assessed by HPLC with electrochemical detection, and strand breaks were measured in pBR322 with agarose gel electrophoresis. The relative photolability of the fluoroquinolones correlated to the extent of production of 8-oxodGuo and strand breaks, with both UVA and UVB irradiation, in the following order: BAY y3118 approximately lomefloxacin > ciprofloxacin > moxifloxacin. Experiments were performed to determine whether the mechanism of damage was due to a type I (radical) or type II (singlet oxygen) pathway. Nitrogen depletion of oxygen resulted in a decrease in the extent of formation of 8-oxodGuo, suggesting that oxygen was involved. The use of selective radical or singlet oxygen inhibitors was inconclusive with respect to which pathway was involved. The use of D(2)O as a solvent, which would extend the lifetime of singlet oxygen, suggested that this species is involved in the formation of 8-oxodGuo by moxifloxacin and ciprofloxacin, but not by lomefloxacin and BAY y3118. Similarly, it was found that singlet oxygen was not involved in strand break formation. Thus, the evidence suggests that fluoroquinolones can photochemically produce DNA damage by both type I and type II mechanisms.

A Serendipitous Synthesis of 8-Dimsyl-2′-deoxyguanosine

A serendipitous synthesis of 8-dimsyl-dG (2) has been achieved along with the known 8-benzyloxy-dG (3) in a nucleophilic substitution reaction of 8-bromo-dG (1) with in situ generated dimsyl and benzyloxy sodium. Compound 3 was directly converted into the mutagenic oxidative DNA damage product, 8-oxo-dGTP (4).

Indirect mutagenesis by oxidative DNA damage: formation of the pyrimidopurinone adduct of deoxyguanosine by base propenal.

Oxidation of endogenous macromolecules can generate electrophiles capable of forming mutagenic adducts in DNA. The lipid peroxidation product malondialdehyde, for example, reacts with DNA to form M1G, the mutagenic pyrimidopurinone adduct of deoxyguanosine. In addition to free radical attack of lipids, DNA is also continuously subjected to oxidative damage. Among the products of oxidative DNA damage are base propenals. We hypothesized that these structural analogs of malondialdehyde would react with DNA to form M1G. Consistent with this hypothesis, we detected a dose-dependent increase in M1G in DNA treated with calicheamicin and bleomycin, oxidizing agents known to produce base propenal. The hypothesis was proven when we determined that 9-(3-oxoprop-1-enyl)adenine gives rise to the M1G adduct with greater efficiency than malondialdehyde itself. The reactivity of base propenals to form M1G and their presence in the target DNA suggest that base propenals derived from oxidative DNA damage may contribute to the mutagenic burden of a cell.

Substrate specificity of Schizosaccharomyces pombe Nth protein for products of oxidative DNA damage.

A gene from Schizosaccharomyces pombe, which encodes a protein with a strong sequence similarity to the Nth protein of Escherichia coli, has recently been identified [Roldán-Arjona, T., Anselmino, C., and Lindahl, T. (1996) Nucleic Acids Res. 24, 3307-3312]. The functional analysis of this eukaryotic enzyme indicated that it is a homologue of E. coli Nth protein. The gene has been subcloned and the protein (Nth-Spo) purified to apparent homogeneity. We investigated the substrate specificity of this eukaryotic enzyme for modified bases in oxidatively damaged DNA, using the technique of gas chromatography/isotope-dilution mass spectrometry (GC/IDMS). DNA substrates containing up to 17 types of modified bases were prepared by gamma-irradiation or by treatment with H2O2 in the presence of Fe(III)-EDTA or Cu(II). The results revealed an efficient excision of five pyrimidine-derived lesions, 5-hydroxycytosine, thymine glycol, 5-hydroxy-6-hydrothymine, 5,6-dihydroxycytosine, and 5-hydroxyuracil. None of the other pyrimidine or purine lesions was excised. Excision was measured as a function of enzyme concentration, time, substrate concentration, and temperature. Kinetic constants were determined. Although some DNA base lesions removed by Nth-Spo protein were similar to those previously described for E. coli Nth protein, differences between substrate specificities of these two enzymes were noted.

Analysis of a form of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis.

8-hydroxy-2'-deoxyguanosine (8-OH-dG) was first reported in 1984 as a major form of oxidative DNA damage product by heated sugar, Fenton-type reagents and X-irradiation in vitro. 8-OH-dG has been detected in cellular DNA using an HPLC-ECD method in many laboratories. Analyses of 8-OH-dG in animal organ DNA after the administration of oxygen radical-forming chemicals will be useful for assessments of their carcinogenic risk. Its analysis in human leucocyte DNA and in urine is a new approach to the assessment of an individual's cancer risk due to oxidative stress. The increase of the 8-OH-dG level in the cellular DNA, detected by HPLC-ECD method, was supported by its immunochemical detection and its enhanced repair activity. The validity of the general use of 8-OH-dG as a marker of cellular oxidative stress is discussed.

Types of mutations induced by glyoxal, a major oxidative DNA-damage product, in Salmonella typhimurium

We have analyzed the types of mutations induced by glyoxal, a major oxidative DNA-damage product, in Salmonella typhimurium. A set of six strains, TA7001 to TA7006, was used to detect base-pair substitutions, and the TA98 strain was employed to detect frameshift mutations. Glyoxal did not induce mutations at A:T base pairs. The majority of the mutations induced by glyoxal were base-pair substitutions at G:C base pairs, and a small level of frameshift mutations was detected in the TA98 strain.

No effect of supplementation with vitamin E, ascorbic acid, or coenzyme Q10 on oxidative DNA damage estimated by 8-oxo-7,8-dihydro-2'-deoxyguanosine excretion in smokers

The protective effect of fruit and vegetables against cancer has been related to their high antioxidant content. However, results from intervention trials have not been conclusive on the protective effect of antioxidant supplementation. In a randomized placebo-controlled trial we investigated the effect of dietary supplementation with antioxidants on a biomarker of oxidative DNA damage with mechanistic relation to carcinogenesis. One hundred forty-two smoking men aged 35-65 y were randomly assigned to one of the following seven treatments for 2 mo: 100 mg D-alpha-tocopheryl acetate plus 250 mg slow-release ascorbic acid twice a day (n = 20), 100 mg D-alpha-tocopheryl acetate twice a day (n = 20), 250 mg ascorbic acid twice a day (n = 21), 250 mg slow-release ascorbic acid twice a day (n = 21), 30 mg coenzyme Q10 in oil three times a day (n = 20), 30 mg coenzyme Q10 as granulate three times a day (n = 20), or placebo twice a day (n = 20). The trial outcome was the urinary excretion rate of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG)-a repair product of oxidative DNA damage. Two months of supplementation did not result in significant changes in the urinary excretion rate of 8-oxodG in any group. The lack of effect of antioxidant supplementation on the excretion rate of 8-oxodG, despite substantial increases in plasma antioxidant concentrations, agrees with the results from recent large intervention studies with cancer as an endpoint. The cancer-protective effect of fruit and vegetables seems to rely not on the effect of single antioxidants but rather on other anticarcinogenic compounds or on a concerted action of several micronutrients present in these foods.

A semi-empirical SCF-MO study on the base-pairing properties of 8-oxopurines: significance for mutagenicity

C8-Oxidised purines like 7-hydro-8-oxoguanine (8OG) and 7-hydro-8-oxoadenine (8OA) are known as products of oxidative DNA damage. Semiempirical molecular orbital calculations at the PM3 SCF-MO level are used to investigate the base-pairing properties of these bases in an attempt to understand their mutagenic properties. A detailed analysis of the base-pairing properties of these bases leads to an identification of the most probable pairing schemes involved in mutagenic base-mispairing. It is suggested that both bases are capable of inducing transversional as well as transitional mutations via base-mispairing. The results presented are largely in consonance with available experimental reports.

Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage.

Uracil DNA N-glycosylase is a repair enzyme that releases uracil from DNA. A major function of this enzyme is presumably to protect the genome from pre-mutagenic uracil resulting from deamination of cytosine in DNA. Here, we report that human uracil DNA N-glycosylase also recognizes three uracil derivatives that are generated as major products of cytosine in DNA by hydroxyl radical attack or other oxidative processes. DNA substrates were prepared by gamma-irradiation of DNA in aerated aqueous solution and incubated with human uracil DNA N-glycosylase, heat-inactivated enzyme or buffer. Ethanol-precipitated DNA and supernatant fractions were then separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results demonstrated that human uracil DNA N-glycosylase excised isodialuric acid, 5-hydroxyuracil and alloxan from DNA with apparent K(m) values of approximately 530, 450 and 660 nM, respectively. The excision of these uracil analogues is consistent with the recently described mechanism for recognition of uracil by human uracil DNA N-glycosylase [Mol,C.D., Arval,A.S., Slupphaug,G., Kavil,B., Alseth,I., Krokan,H.E. and Tainer,J.A. (1995) Cell, 80, 869-878]. Nine other pyrimidine- and purine-derived products that were identified in DNA samples were not substrates for the enzyme. The results indicate that human uracil DNA N-glycosylase may have a function in the repair of oxidative DNA damage.

Superoxide and the production of oxidative DNA damage.

The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron.

Age-Associated Change of 8-Hydroxyguanine Repair Activity in Cultured Human Fibroblasts

8-hydroxyguanine (8-OH-Gua) is a major oxidative DNA damage product. Its repair activity was determined in the human fibroblast cell line, TIG-3, during the aging process. The repair activity increased in the first part of the aging process and decreased in the latter part. The highest repair activity was observed at the age of 30 PDL. These results suggest that the defense capacity for replication errors due to oxidative DNA damage increases greatly when the cells are rapidly dividing.

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 γ-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.

Coal tar residues produce both DNA adducts and oxidative DNA damage in human mammary epithelial cells.

In the present study we compare the metabolic activation of coal tar, as measured by the production of both DNA adducts and oxidative DNA damage, with that of a single carcinogen that is a constituent of this complex mixture in human mammary epithelial cells (HMEC). We find that a significant level of DNA adducts, detected by 32P-postlabeling, are formed in HMEC following exposure to coal tar residues. This treatment also results in the generation of high levels of oxidative DNA damage, as measured by the production of one type of oxidative base modification, thymine glycols. The amounts of both DNA adducts and thymine varied considerably between the various coal tar residues and did not correlate with either the total amount of polycyclic aromatic hydrocarbons (PAH) or the amount of benzo[a]pyrene (B[a]P) present in the residue. Fractionating the residue from one of the sites by sequential extraction with organic solvents indicated that while the ability to produce both types of DNA damage was contained mostly in a hexane-soluble fraction, a benzene-soluble fraction produced high levels of reactive oxygens relative to the number of total DNA adducts. We find that the total amount of PAH or B[a]P present in the coal tars from the various sites was not a predictor of the level of total DNA damage formed.