DNA oxidative damage and strand breaks in young healthy individuals: a gender difference and the role of life style factors

The aim of this study was to analyze background levels of DNA damage in young (19-31 years) non-smoking individuals and to correlate damage to gender and life style. DNA single strand breaks (SSB) and alkali labile sites (ALS) were measured in 99 subjects living in Stockholm, Sweden. Further, oxidative DNA damage was analyzed using the DNA repair glycosylase FPG as well as HPLC-ECD for specific analysis of 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG). We found that males had higher (P < 0.001) levels of SSB + ALS than females, but no difference was seen for oxidative lesions. There was no correlation between FPG sites and 8-oxodG. For females, there was a positive correlation between FPG levels and body mass index and a negative correlation between SSB + ALS and fruit intake. We conclude that the background level of oxidative DNA damage, analyzed with improved methods, is low and that gender, fruit intake and BMI can affect DNA damage.     

Endonuclease-sensitive DNA modifications induced by acetone and acetophenone as photosensitizers.

Repair endonucleases, viz. endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, were used to analyse the modifications induced in bacteriophage PM2 DNA by 333 nm laser irradiation in the presence of acetone or acetophenone. In addition to pyrimidine dimers sensitive to UV endonuclease, 5,6-dihydropyrimidines (sensitive to endonuclease III) and base modifications sensitive to FPG protein were generated. The level of the last in the case of acetone was 50% and in the case of acetophenone 9% of the level of pyrimidine dimers. HPLC analysis of the bases excised by FPG protein revealed that least some of them were 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). In the damage induced by direct excitation of DNA at 254 nm, which was analysed for comparison, the number of FPG protein-sensitive base modifications was only 0.6% of that of the pyrimidine dimers. Mechanistic studies demonstrated that the formation of FPG protein-sensitive modifications did not involve singlet oxygen, as the damage was not increased in D2O as solvent. Hydroxyl radicals, superoxide and H2O2 were also not involved, since the relative number of single strand breaks and of sites of base loss (AP sites) was much lower than in the case of DNA damage induced by hydroxyl radicals and since the presence of SOD or catalase had no effect on the extent of the damage. However, the mechanism did involve an intermediate that was much more efficiently quenched by azide ions than the triplet excited carbonyl compounds and which was possibly a purine radical. Together, the data indicate that excited triplet carbonyl compounds react with DNA not only by triplet-triplet energy transfer yielding pyrimidine dimers, but also by electron transfer yielding preferentially base modifications sensitive to FPG protein, which include 8-hydroxyguanine.     

Repeated inhalations of diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase (OGG1) deficient mice

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 × 1.5 h inhalations of DEP (20 mg/m³) in Ogg1^- / -  and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in Ogg1^- / -  mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1^- / -  mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1^- / -  mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1^- / -  mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP.     

Photochemical DNA modifications induced by 1,2-dioxetanes.

1,2-Dioxetanes are efficient sources of triplet excited carbonyl compounds, into which they decompose on thermal or photochemical activation. In the presence of DNA, the decomposition of dioxetanes gives rise to DNA modifications, which have been studied by means of specific repair endonucleases. Cyclobutane pyrimidine dimers, which are generated by triplet-triplet energy transfer, were detected by a UV endonuclease; they made up between 2% and 30% of the total modifications recognized by a crude repair endonuclease preparation from Micrococcus luteus. For various 1,2-dioxetanes, the yield of pyrimidine dimers was proportional to their triplet excitation flux. DNA strand breaks, sites of base loss (AP sites; recognized by exonuclease III and endonuclease IV) and dihydropyrimidines (recognized by endonuclease III) were found to represent only a small fraction of the modifications. The majority of the modifications detected were recognized by formamidopyrimidine-DNA glycosylase (FPG protein) and represent 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) residues or other yet not defined base modifications which are recognized by this enzyme. The modifications were generated in similar relative yields by thermal and photo-induced decomposition of the 1,2-dioxetanes and therefore emanate under both conditions from the excited carbonyl compounds. The formation of the FPG protein-sensitive modifications was efficiently quenched by azide anions; the Stern-Volmer quenching of these modifications was 150-fold more effective than that of the pyrimidine dimers. The relative amounts of the two types of modifications were strongly dependent on the structure of the 1,2-dioxetanes and on the concentration of molecular oxygen. Singlet oxygen appears to be involved only to some extent in the generation of the FPG protein-sensitive base modifications as their yield was only moderately (approximately 2-fold) increased in D2O as solvent. A mechanism is suggested in which oxidized guanine is predominantly formed by a single-electron-transfer reaction of the triplet excited carbonyl product derived from the 1,2-dioxetane, followed by unknown secondary oxidations, which involve molecular oxygen and/or undecomposed 1,2-dioxetane.     

Repeated inhalations of diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase (OGG1) deficient mice

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 × 1.5 h inhalations of DEP (20 mg/m³) in Ogg1^{? / ?} and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in Ogg1^{? / ?} mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1^{? / ?} mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1^{? / ?} mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1^{? / ?} mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP.     

DNA containing a chemically reduced apurinic site is a high affinity ligand for the E. coli formamidopyrimidine-DNA glycosylase.

The E. coli Formamidopyrimidine-DNA Glycosylase (FPG protein), a monomeric DNA repair enzyme of 30.2 kDa, was purified to homogeneity in large quantities. The FPG protein excises imidazole ring-opened purines and 8-hydroxyguanine residues from DNA. Besides DNA glycosylase activity, the FPG protein is endowed with an EDTA-resistant activity which nicks DNA at apurinic/apyrimidic sites (AP sites). In contrast, DNAs containing chemically reduced AP sites are not incised by the FPG protein. However, the DNA glycosylase activity of the FPG protein is strongly inhibited in the presence of a purified synthetic 24 base-pair double-stranded oligonucleotide which contains a single apurinic site transformed chemically through borohydride reduction into a ring-opened deoxyribose derivative. The ability of the FPG protein to form a complex with this synthetically modified DNA was studied by electrophoresis in non-denaturing polyacrylamide gels. The FPG protein specifically binds the double-stranded oligonucleotide containing an apurinic site previously reduced in the presence of sodium borohydride. The complex was identified as a single retardation band on non-denaturing polyacrylamide gel electrophoresis. Complex formation is reversible and an apparent dissociation constant, KDapp, of 2.6 x 10(-10) M was determined. In contrast, no such retardation band was obtained between the FPG protein and double-stranded DNA containing an intact apurinic site or single-stranded DNA containing either an intact or a reduced apurinic site.     

Influence of DNA torsional rigidity on excision of 7,8-dihydro-8-oxo-2'-deoxyguanosine in the presence of opposing abasic sites by human OGG1 protein

The human protein OGG1 (hOGG1) targets the highly mutagenic base 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) and shows a high specificity for the opposite DNA base. Abasic sites can arise in DNA in close opposition to 8-oxodG either during repair of mismatched bases (i.e. 8-oxodG/A mismatches) or, more frequently, as a consequence of ionizing radiation exposure. Bistranded DNA lesions may remain unrepaired and lead to cell death via double-strand break formation. In order to explore the role of damaged-DNA dynamics in recognition/excision by the hOGG1 repair protein, specific oligonucleotides containing an 8-oxodG opposite an abasic site, at different relative distances on the complementary strand, were synthesized. Rotational dynamics were studied by means of fluorescence polarization anisotropy decay experiments and the torsional elastic constant as well as the hydrodynamic radius of the DNA fragments were evaluated. Efficiency of excision of 8-oxodG was tested using purified human glycosylase. A close relation between the twisting flexibility of the DNA fragment and the excision efficiency of the oxidative damage by hOGG1 protein within a cluster was found.     

Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans.

The present study investigated the effect of a single bout of exhaustive exercise on the generation of DNA strand breaks and oxidative DNA damage under normal conditions and at high-altitude hypoxia (4559 meters for 3 days). Twelve healthy subjects performed a maximal bicycle exercise test; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage. Urinary excretion of 8-oxodG increased during the first day in altitude hypoxia, and there were more endonuclease III-sensitive sites on day 3 at high altitude. The subjects had more DNA strand breaks in altitude hypoxia than at sea level. The level of DNA strand breaks further increased immediately after exercise in altitude hypoxia. Exercise-induced generation of DNA strand breaks was not seen at sea level. In both environments, the level of FPG and endonuclease III-sensitive sites remained unchanged immediately after exercise. DNA strand breaks and oxidative DNA damage are probably produced by reactive oxygen species, generated by leakage of the mitochondrial respiration or during a hypoxia-induced inflammation. Furthermore, the presence of DNA strand breaks may play an important role in maintaining hypoxia-induced inflammation processes. Hypoxia seems to deplete the antioxidant system of its capacity to withstand oxidative stress produced by exhaustive exercise.     

Oxidatively damaged DNA and its repair after experimental exposure to wood smoke in healthy humans

Particulate matter from wood smoke may cause health effects through generation of oxidative stress with resulting damage to DNA. We investigated oxidatively damaged DNA and related repair capacity in peripheral blood mononuclear cells (PBMC) and measured the urinary excretion of repair products after controlled short-term exposure of human volunteers to wood smoke. Thirteen healthy adults were exposed first to clean air and then to wood smoke in a chamber during 4h sessions, 1 week apart. Blood samples were taken 3h after exposure and on the following morning, and urine was collected after exposure, from bedtime until the next morning. We measured the levels of DNA strand breaks (SB), oxidized purines as formamidopyrimidine-DNA-glycosylase (FPG) sites and activity of oxoguanine glycosylase 1 (hOGG1) in PBMC by the comet assay, whereas mRNA levels of hOGG1, nucleoside diphosphate linked moiety X-type motif 1 (hNUDT1) and heme oxygenase 1 (hHO1) were determined by real-time RT-PCR. The excretion of 8-oxo-7,8-dihydro-oxoguanine (8-oxoGua) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in urine was measured by high performance liquid chromatography purification followed by gas chromatography with mass spectrometry. The morning following exposure to wood smoke the PBMC levels of SB were significantly decreased and the mRNA levels of hOGG1 significantly increased. FPG sites, hOGG1 activity, expression of hNUDT1 and hHO1, urinary excretion of 8-oxodG and 8-oxoGua did not change significantly. Our findings support that exposure to wood smoke causes systemic effects, although we could not demonstrate genotoxic effects, possibly explained by enhanced repair and timing of sampling.     

Detection of extracellular 8-oxo-7,8-dihydro-2′-deoxyguanosine as a biomarker of oxidative damage in X-irradiated fibroblast cultures: optimization of analytical procedure

We have developed a simple methodology, based on single-step solid-phase extraction followed by isocratic high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD), to determine extracellular 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in culture supernatants of normal human dermal fibroblasts. A standard addition method, using externally added 8-oxodG (0.5 and 1 pmol) was employed to eliminate matrix effects arising from the chemically complex, protein-rich medium. Secondly, applying this procedure to X-ray irradiated fibroblasts, we report a significant twofold increase in the levels of 8-oxodG at the radiobiologically relevant dose of 6 Gy. This suggests that extracellular 8-oxodG might be a useful biomarker for oxidative stress following moderate doses of X-irradiation.     

Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro.

pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IR)-sensitive sites. Seven Fpg protein-sensitive sites per PBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.     

Origin of the heterogeneous distribution of the yield of guanyl radical in UV laser photolyzed DNA

Oxidative guanine lesions were analyzed, at the nucleotide level, within DNA exposed to nanosecond ultraviolet (266 nm) laser pulses of variable intensity (0.002-0.1 J/cm(2)). Experiments were carried out, at room temperature, in TE buffer (20 mM Tris-HCl, pH 7.5; 1 mM EDTA) containing 35 mM NaCl, on 5'-end radioactively labeled double-stranded and single-stranded oligomer DNA at a size of 33-37 nucleobases. Lesions were analyzed on polyacrylamide gel electrophoresis by taking advantage of the specific removal of 8-oxodG from DNA by the formamidopyrimidine DNA glycosylase (Fpg protein) and of the differential sensitivity of 8-oxodG and oxazolone to piperidine. The quantum yields of lesions at individual sites, determined from the normalized intensities of bands, were plotted against the irradiation energy levels. Simplified model fitting of the experimental data enabled to evaluate the spectroscopic parameters characterizing excitation and photoionization processes. Results show that the distribution of guanine residues, excited to the lowest triplet state or photoionized, is heterogeneous and depends on the primary and secondary DNA structure. These findings are generalized in terms of excitation energy and charge-migration mediated biphotonic ionization. On the basis of the changes in the yield of the guanyl radical resulting from local helical perturbations in the DNA pi-stack, it can be assessed that the distance range of migration is <6-8 bp.     

Increased rOGG1 expression in regenerating rat liver tissue without a corresponding increase in incision activity

Rapidly proliferating tissue with synthesis of a large number of cellular macromolecules including DNA, may require enhanced DNA repair capacity in order to avoid fixation of promutagenic DNA lesions to mutations. This hypothesis was addressed by assessing the incision activity and the mRNA level of the DNA repair protein rat 8-oxodeoxyguanosine glycosylase (rOGG1) as well as the level of the oxidative stress biomarker 8-oxodeoxyguanosine (8-oxodG) in rat liver tissue before and after partial hepatectomy. A five-fold increase in rOGG1 expression was found at 24h after PHx relative to the control levels. At 48h the rOGG1 mRNA levels were reduced to three-times the control values. The corresponding incision activities of rOGG1 in the crude tissue extract as measured by the incision assay were slightly increased both at 24 and 48h after partial hepatectomy although the changes failed to be statistically significant (P=0.07 and 0.06, respectively). The levels of 8-oxodG were unaltered at 24h but increased to 1.8 times the control values at 48h after partial hepatectomy. The study showed that rapid proliferating liver tissue in vivo had an increased expression of the DNA repair protein rOGG1, without significantly increased incision activity on a 8-oxodG-containing substrate and with unchanged levels of 8-oxodG/10(6) dGuo after 24h of regeneration. At 48h the rOGG1 expression was decreased, and the levels of 8-oxodG/10(6) dGuo increased but still significant changes in the incision activity could not be detected. Thus, we can conclude that the rOGG1 expression is temporarily up-regulated by the proliferating events elicited by partial hepatectomy.     

Anti-8-oxo-2'-deoxyguanosine phage antibodies: isolation, characterization, and relationship to disease states

We have used human single chain Fv (scFv) phage display antibody libraries to isolate recombinant antibodies against the DNA adduct 8-oxo-2'-deoxyguanosine (8-oxodG). One of these scFvs (175G) bound to several 8-oxodG-containing oligonucleotides whilst demonstrating no cross-reactivity with G-containing control oligonucleotides, and bound to 8-oxodG lesions introduced into DNA by treatment with methylene blue and white light. In addition, 175G inhibited the cleavage of an 8-oxodG-containing oligonucleotide by the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg). The nucleotide sequence of the 175G V(H) gene segment was 98% homologous to the published V(H) sequence of a human hybridoma derived from a patient with systemic lupus erythematosus (SLE). Sera from two SLE patients bound to damaged DNA, and this binding could be inhibited by 175G. The use of human scFv phage display libraries has thus produced a unique reagent with specificity for 8-oxodG, which may have a role in damage detection and quantitation and in modifying DNA repair activity. 175G also offers support to the hypothesis that SLE might be associated with oxidative damage to DNA.     

Homogeneous Escherichia coli FPG protein. A DNA glycosylase which excises imidazole ring-opened purines and nicks DNA at apurinic/apyrimidinic sites.

The repair of 2,6-diamino-4-hydroxy-5-N-methyl-formamidopyrimidine (Fapy) residues in DNA is performed by a Fapy-DNA glycosylase activity which is encoded for by the fpg gene in Escherichia coli. Besides DNA glycosylase activity, this protein, the FPG protein, is endowed with an EDTA-resistant activity nicking DNA at apurinic/apyrimidinic (AP) sites. To overproduce the FPG protein, the fpg gene was placed under the control of the tac promoter in the expression vector pKK223-3 yielding the pFPG230 plasmid. The production of the FPG protein in cells harboring the pFPG230 plasmid was 800-fold higher than that of the wild type strain after induction by isopropyl-beta-D-thio-galactopyranoside. From these cells, the FPG protein was purified to homogeneity in sufficient quantity to study its physical and catalytic properties. In its active form, the FPG protein is a globular monomer of 31 kDa and has an experimentally measured isoelectric point of 8.5. When the FPG protein is heat-denatured in the presence of EDTA the two activities are more rapidly inactivated than when heated in the absence of EDTA, suggesting that the FPG protein possesses a tightly bound metal ion. Atomic absorption spectrophotometric analysis shows that there is one zinc/FPG protein molecule. The FPG protein is different from previously described DNA glycosylases and AP-nicking enzymes in E. coli. The contribution of the AP-nicking activity associated with the FPG protein represents 10-20% of the total EDTA-resistant AP-nicking activities in E. coli.     

OGG1 mRNA expression and incision activity in rats are higher in foetal tissue than in adult liver tissue while 8-oxo-2'-deoxyguanosine levels are unchanged

This study was set up to investigate the relationships between the formation and removal of DNA damage in form of 8-oxodeoxyguanosine (8-oxodG) in neonatal (day 16 of gestation) as compared to adult rats. The hypothesis addressed was whether the rapidly dividing foetal tissue has an enhanced requirement of DNA repair providing protection against potentially mutagenic DNA damages such as 8-oxodG. The activity of the primary 8-oxodG-repair protein OGG1 was measured by a DNA incision assay and the expression of OGG1 mRNA was measured by Real-Time PCR normalised to 18S rRNA. The tissue level of 8-oxodG was measured by HPLC-ECD. We found a 2-3-fold increased incision activity in the foetal control tissue, together with a 3-15-fold increase in mRNA of OGG1 as compared to liver tissue from adult rats. The levels of 8-oxodG in the foetal tissue were unaltered as compared to the adult groups. To increase the levels of 8-oxodG, the rats received an injection (i.p.) of the hepatotoxin 2-nitropropane. The compound induced significant levels of 8-oxodG in male rat livers 5h after the injection and in the foetuses 24h after the injection, while the female rats showed no increase in 8-oxodG. The incision activity was slightly depressed in both male and female liver tissue and in the foetal tissue 5h after the injection, but significantly increased from 5 to 24h after the injection. However, it did not reach levels significantly above the control levels.In conclusion, this study confirms that foetal tissue has increased levels of OGG1 mRNA and correspondingly an enhanced incision activity on an 8-oxodG substrate in a crude tissue extract.     

DNA repair and aging in mouse liver: 8-oxodG glycosylase activity increase in mitochondrial but not in nuclear extracts

8-oxo-deoxyguanosine (8-oxodG) is one of the major DNA lesions formed upon oxidative attack of DNA. It is a mutagenic adduct that has been associated with pathological states such as cancer and aging. Base excision repair (BER) is the main pathway for the repair of 8-oxodG. There is a great deal of interest in the question about age-associated accumulation of this DNA lesion and its intracellular distribution, particularly with respect to mitochondrial or nuclear localization. We have previously shown that 8-oxodG-incision activity increases with age in rat mitochondria obtained from both liver and heart. In this study, we have investigated the age-associated changes in DNA repair activities in both mitochondrial and nuclear extracts obtained from mouse liver. We observed that 8-oxodG incision activity of mitochondrial extracts increases significantly with age, from 13.4 + or - 2.2 fmoles of oligomer/100 microg of protein/16 h at 6 to 18.6 + or - 4.9 at 14 and 23.7 + or - 3.8 at 23 months of age. In contrast, the nuclear 8-oxodG incision activity showed no significant change with age, and in fact slightly decreased from 11.8 + or - 3 fmoles/50 microg of protein/2 h at 6 months to 9.7 + or - 0.8 at 14 months. Uracil DNA glycosylase and endonuclease G activities did not change with age in nucleus or mitochondria. Our results show that the repair of 8-oxodG is regulated differently in nucleus and mitochondria during the aging process. The specific increase in 8-oxodG-incision activity in mitochondria, rather than a general up-regulation of DNA metabolizing enzymes in those organelles, suggests that this pathway may be up regulated during aging in mice.     

No evidence of mitochondrial respiratory dysfunction in OGG1-null mice deficient in removal of 8-oxodeoxyguanine from mitochondrial DNA

Accumulation of high levels of mutagenic oxidative mitochondrial DNA (mtDNA) lesions like 8-oxodeoxyguanine (8-oxodG) is thought to be involved in the development of mitochondrial dysfunction in aging and in disorders associated with aging. Mice null for oxoguanine DNA glycosylase (OGG1) are deficient in 8-oxodG removal and accumulate 8-oxodG in mtDNA to levels 20-fold higher than in wild-type mice (N.C. Souza-Pinto et al., 2001, Cancer Res. 61, 5378-5381). We have used these animals to investigate the effects on mitochondrial function of accumulating this particular oxidative base modification. Despite the presence of high levels of 8-oxodG, mitochondria isolated from livers and hearts of Ogg1-/- mice were functionally normal. No differences were detected in maximal (chemically uncoupled) respiration rates, ADP phosphorylating respiration rates, or nonphosphorylating rates with glutamate/malate or with succinate/rotenone. Similarly, maximal activities of respiratory complexes I and IV from liver and heart were not different between wild-type and Ogg1-/- mice. In addition, there was no indication of increased oxidative stress in mitochondria from Ogg1-/- mice, as measured by mitochondrial protein carbonyl content. We conclude, therefore, that highly elevated levels of 8-oxodG in mtDNA do not cause mitochondrial respiratory dysfunction in mice.     

Modulation of hOGG1 DNA repair enzyme in human cultured cells in response to pro-oxidant and antioxidant challenge

The putative modulation of the base excision repair enzyme, human 8-oxoguanine glycosylase (hOGG1), important in the removal of the potentially mutagenic lesion 8-oxo-2'-deoxyguanosine (8-oxodG), was investigated in human cell culture models. The expression of specific mRNA and protein was measured following pro-oxidant and antioxidant treatments in one human lymphoblastoid and one keratinocyte line. The measurement of intracellular reactive oxygen species generation was monitored by a fluorogenic assay and potential genotoxic effects confirmed by the dose-dependent increase in formamidopyrimidine-DNA glycosylase (Fpg) sensitive sites by alkaline unwinding following sub-lethal doses of hydrogen peroxide. The generation of a potentially antioxidant environment was assessed by the intracellular increase and extracellular depletion in ascorbic acid, confirmed by capillary electrophoresis. Despite these pro-oxidant and antioxidant treatments no significant change in mRNA of hOGG1 was observed in either cell line. Western analysis revealed that relatively high, yet noncytotoxic, doses of hydrogen peroxide caused a consistent approximate 50% decrease in hOGG1 protein in lymphoblastoid cells. The lack of upregulation of hOGG1 suggests the gene is constitutively expressed, which is further supported by studies examining the sequence of its promoter region. However, hOGG1 protein turnover may be sensitive to intracellular redox changes.