Experimental study of oxidative DNA damage

Animal experiments allow the study of oxidative DNA damage in target organs and the elucidation of dose-response relationships of carcinogenic and other harmful chemicals and conditions as well as the study of interactions of several factors. So far the effects of more than 50 different chemical compounds have been studied in animal experiments mainly in rats and mice, and generally with measurement of 8-oxodG with HPLC-EC. A large number of well-known carcinogens induce 8-oxodG formation in liver and/or kidneys. Moreover several animal studies have shown a close relationship between induction of dative DNA damage and tumour formation.

In principle the level of oxidative DNA damage in an organ or cell may be studied by measurement of modified bases in extracted DNA by immunohistochemical visualisation, and from assays of strand breakage before and after treatment with repair enzymes. However, this level is a balance between the rates of damage and repair. Until the repair rates and capacity can be adequately assessed the rate of damage can only be estimated from the urinary excretion of repair products albeit only as an average of the entire body.

A number of model compounds have been used to induce oxidative DNA damage in experimental animals. The hepatocarcinogen 2-nitropropane induces up to 10-fold increases in 8-oxodG levels in rat liver DNA. The level of 8-oxodG is also increased in kidneys and bone marrow but not in the testis. By means of 2-nitropropane we have shown correspondence between the increases in 8-oxodG in target organs and the urinary excretion of 8-oxodG and between 8-oxodG formation and the comet assay in bone marrow as well potent preventive effects of extracts of Brussels sprouts. Others have shown similar effects of green tea extracts and its components. Drawbacks of the use of 2-nitropropane as a model for oxidative DNA damage relate particularly to formation of 8-aminoguanine derivatives that may interfere with HPLC-EC assays and have unknown consequences. Other model compounds for induction of oxidative DNA damage, such as ferric nitriloacetate, iron dextran, potassium bromate and paraquat, are less potent and/or more organ specific.

Inflammation and activation of an inflammatory response by phorbol esters or E. coli lipopolysaccharide (LPS) induce oxidative DNA damage in many target cells and enhance benzene-induced DNA damage in mouse bone marrow.

Experimental studies provide powerful tools to investigate agents inducing and preventing oxidative damage to DNA and its role in carcinogenesis. So far, most animal experiments have concerned 8-oxodG and determination of additional damaged bases should be employed. An ideal animal model for prevention of oxidative DNA damage has yet to he developed.     

Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods.The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10⁶ dG were respectively 7.85 and 5.87 (p = 0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.     

In isolated DNA, formamidopyrimidine-DNA glycosylase-sensitive sites determined by electrophoresis correspond to the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine by HPLC-ECD.

Several methods have been developed for determining the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. In the present study, we compared an electrophoretic method that uses formamidopyrimidine-DNA glycosylase (FPG protein) with a HPLC-ECD method. Firstly, we produced 8-oxodG in lambda DNA with methylene blue and visible light and cleaved it in one-half of the modified DNA enzymatically with FPG protein. Then, we determined the number of FPG protein-sensitive sites by electrophoresis (Y) and the number of 8-oxodGs by HPLC-ECD (X) per 10(5)dG of isolated DNA. Simple regression analysis of the data showed Y=1.07X+1.52 to be the most likely relationship. The correlation coefficient was 0.97. The values obtained by the two methods were very similar. This result is noteworthy because the number of FPG protein-sensitive sites determined by other methods have not yet come close to the number obtained by HPLC-ECD. Thus, this method might be more quantitative than other methods that measure FPG protein-sensitive sites. Another reason this electrophoresis method might be more useful than HPLC-ECD is that we can determine some other types of oxidative DNA damage well, by changing the DNA glycosylase.     

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.     

No significant paraquat-induced oxidative DNA damage in rats

The metabolism of paraquat generates oxygen radicals. Paraquat has thus been suggested as a model compound to induce oxidative damage to DNA, lipids and proteins in different cells and tissues, although experimental data are inconsistent. In order to explore the possibilities for an animal model of oxidative DNA damage in vivo, rats were treated with 20 mg/kg paraquat or vehicle i.p. One and five days later we measured DNA oxidation in terms of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in the liver and lung as well as the urinary excretion of 8-oxodG. No significant effects on the level of 8-oxodG in the liver, the lung or the urinary excretion, could be distinguished following paraquat treatment. We found, however, a significant correlation (r = 0.69; p<0.0002) between the 8-oxodG level in the lung and the urinary excretion, but no significant correlation between the level in the liver and the urinary excretion or between the levels in the liver and the lung. During the experiment the rats were clearly affected by the paraquat as they were very lethargic compared to the controls. Accordingly, even at toxic doses, paraquat did not cause detectable oxidative damage to DNA. The data do not support the use of paraquat as a model compound in experiments investigating effects or prevention of oxidative damage to DNA.     

Effects of environmental air pollution on endogenous oxidative DNA damage in humans

Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associated with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activities of organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,we wanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidative DNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxidative DNA damage we have determined 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and cyclic pyrimidopurinone N-1,N² malondialdehyde-2′-deoxyguanosine (M₁dG) by the immunoslot blot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantly increased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts) and Sofia (M₁dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposure to environmental air pollution in individuals from Prague (8-oxodG and M₁dG adducts) and Kosice (M₁dG adducts). The average level of M₁dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Prague and Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice compared to those from Prague. A significant increasing trend according to the interaction of c-PAHs exposure and smoking status was observed in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M₁dG and 8-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that can be made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humans but the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in metabolism and detoxification and also differences in the DNA repair capacity and antioxidant status of the individuals could be possible explanations for the variation observed in the level of endogenous oxidative DNA damage for the different populations.     

No significant paraquat-induced oxidative DNA damage in rats

The metabolism of paraquat generates oxygen radicals. Paraquat has thus been suggested as a model compound to induce oxidative damage to DNA, lipids and proteins in different cells and tissues, although experimental data are inconsistent. In order to explore the possibilities for an animal model of oxidative DNA damage in vivo, rats were treated with 20 mg/kg paraquat or vehicle i.p. One and five days later we measured DNA oxidation in terms of 7-hydro-8-oxo-2′-deoxyguanosine (8-oxodG) in the liver and lung as well as the urinary excretion of 8-oxodG. No significant effects on the level of 8-oxodG in the liver, the lung or the urinary excretion, could be distinguished following paraquat treatment. We found, however, a significant correlation (r=0.69; p<0.0002) between the 8-oxodG level in the lung and the urinary excretion, but no significant correlation between the level in the liver and the urinary excretion or between the levels in the liver and the lung. During the experiment the rats were clearly affected by the paraquat as they were very lethargic compared to the controls. Accordingly, even at toxic doses, paraquat did not cause detectable oxidative damage to DNA. The data do not support the use of paraquat as a model compound in experiments investigating effects or prevention of oxidative damage to DNA.     

Oxidative DNA damage in vivo: relationship to age, plasma antioxidants, drug metabolism, glutathione-S-transferase activity and urinary creatinine excretion

Oxidative DNA modification has been implicated in development of certain cancers and 8-oxodG, the most abundant and mutagenic DNA modification, has for some time been considered a biomarker of this activity. Urinary excretion of 8-oxodG over 24h has been used to estimate the rate of damage to DNA, and animal studies have supported this rationale. Reported determinants include tobacco smoking, heavy exercise, environmental pollution and individual oxygen consumption. Samples from three published studies were used to determine the association of urinary 8-oxodG excretion with age, plasma antioxidants, the glutathione-S-transferase phenotype and the activity of the xenobiotic metabolising enzyme CYP1A2. In the age range 35-65 years, age was not related to urinary 8-oxodG excretion, and there were no relations to either the glutathione-S-transferase phenotype or to the plasma antioxidants: vitamin C, alpha-tocopherol, beta-carotene, lycopene or coenzyme Q10. The activity of CYP1A2 showed a significant correlation in two of the three studies, as well as a significant correlation of 0.26 (p < 0.05) in the pooled data set. Regression analysis of CYP1A2 activity on 8-oxodG indicated that 33% increase in CYP1A2 activity would correspond to a doubling of 8-oxodG excretion. This finding needs to be confirmed in independent experiments. Spot morning urine samples can under certain circumstances be used to estimate 8-oxodG excretion rate provided that creatinine excretion is unchanged (in paired experiments) or comparable (in un-paired experiments), as evaluated from the correlation between 8-oxodG excretion in 24 h urine samples and in morning spot urine samples corrected for creatinine excretion (r = 0.50, p < 0.05). We conclude that 8-oxodG excretion is determined by factors like oxygen consumption and CYP1A2 activity rather than by factors like plasma antioxidant concentrations.     

Measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine in peripheral blood mononuclear cells: optimisation and application to samples from a case-control study on cancers of the oesophagus and cardia

8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker to evaluate the level of oxidative stress. This study describes in its first part the optimisation of our analytical procedure (HPLC/electrochemical detection). Particular care was exercised to avoid artefactual oxidation and in the precision of measurement, which was evaluated with blood bags from hemochromatosis patients. The best results were obtained with a DNA extraction step using the "chaotropic method" recommended by the European Standards Committee on Oxidative DNA Damage (ESCODD). Other approaches such as anion exchange columns gave ten times as much 8-oxodG as this method. Moreover, a complete DNA hydrolysis using five different enzymes allowed improved precision. The optimised protocol was applied to peripheral blood mononuclear cells (PBMC) sampled during a case-control study on cancers of the oesophagus and cardia. With 7.2 +/- 2.6 8-oxodG/10(6) 2'-deoxyguanosines (2'-dG) (mean +/- SD), patients (n = 17) showed higher levels of 8-oxodG than controls (4.9 +/- 1.9 8-oxodG/10(6) 2'-dG, n = 43, Student's t-test: p < 0.001). This difference remained significant after technical (storage, sampling period, 2'-dG levels) and individual (age, sex, smoking, alcohol) confounding factors were taken into account (p < 0.0001, Generalised Linear regression Model). To our knowledge, this is the first report to demonstrate an increase of 8-oxodG in PBMCs of patients suffering from a cancer of the upper digestive tract. This elevated level of DNA damage in patients can raise interesting issues: is oxidative stress the cause or the result of the pathology? Could this biomarker be used to evaluate chemoprevention trials concerning digestive tract cancers?     

The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M₁dG and 8-oxodG adducts as well as M₁dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.     

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.     

Age-associated change in mitochondrial DNA damage

There is an age-associated decline in the mitochondrial function of the Wistar rat heart. Previous reports from this lab have shown a decrease in mitochondrial cytochrome c oxidase (COX) activity associated with a reduction in COX gene and protein expression and a similar decrease in the rate of mitochondrial protein synthesis. Damage to mitochondrial DNA may contribute to this decline.

Using the HPLC-Coularray system (ESA, USA), we measured levels of nuclear and mitochondrial 8-oxo-2'-deoxyguanosine (8-oxodG) from 6-month (young) and 23-month-old (senescent) rat liver DNA. We measured the sensitivity of the technique by damaging calf thymus DNA with photoactivated methylene blue for 30s up to 2h. The levels of damage were linear over the entire time course including the shorter times which showed levels comparable to those expected in liver. For the liver data, 8-oxodG was reported as a fraction of 2-deoxyguanosine (2-dG). There was no change in the levels of 8-oxodG levels in the nuclear DNA from 6 to 23-months of age. However, the levels of 8-oxodG increased 2.5-fold in the mitochondrial DNA with age. At 6 months, the level of 8-oxodG in mtDNA was 5-fold higher than nuclear and increased to approximately 12-fold higher by 23 months of age. These findings agree with other reports showing an age-associated increase in levels of mtDNA damage; however, the degree to which it increases is smaller. Such damage to the mitochondrial DNA may contribute to the age-associated decline in mitochondrial function.     

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.     

Inter-laboratory validation of procedures for measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CVof less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10(6) Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.     

Age-related sensitivity to lung oxidative stress during ozone exposure

As immature and aged rats could be more sensitive to ozone (O(3))-linked lung oxidative stress we have attempted to shed more light on age-related susceptibility to O(3) with focusing our interest on lung mitochondrial respiration, reactive oxygen species (ROS) production and lung pro/antioxidant status. For this purpose, we exposed to fresh air or O(3) (500 ppb 12 h per day, for 7 days) 3 week- (immature), 6 month- (adult) and 20 month-old rats (aged). We determined, in lung, H(2)O(2) release by mitochondria, activities of major antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)], heat shock protein (HSP(72)) content and 8-oxodG and dG-HNE nDNA contents, as DNA oxidative damage markers. In adult rats we did not observe alteration of pro/antioxidant status. In contrast to adults, immature rats exposed to O(3) higher nDNA 8-oxodG content and HSP(72) and without antioxidant enzymes modification. Aged rats displayed mild uncoupled lung mitochondria, increased SOD and GPx activities, and higher 8-oxodG content after O(3) exposure. Thus, in contrast to adults, immature and aged rats displayed lung oxidative stress after O(3) exposure. Higher sensitivity of immature to O(3) was partly related to ventilatory parameters and to the absence of antioxidant enzyme response. In aged rats, the increase in cytosolic SOD and GPx activities during O(3) exposure was not sufficient to prevent the impairment in mitochondrial function and accumulation in lung 8- oxodG. Finally, we showed that mitochondria seem not to be a major source of ROS under O(3) exposure.     

Controlled oxidation of calf thymus DNA to produce standard samples for 8-Oxodeoxyguanosine analysis; Effects of freeze-drying,storage and hydrolysis conditions

Calf thymus DNA containing defined levels of 8-hydroxy-2′-deoxyguanosine (8-oxodG) was prepared by treatment with visible light in the presence of photosensitiser Ro 19-8022. The DNA was checked for stability; after freeze-drying, the amount of 8-oxodG did not increase during 6 weeks' storage at room temperature. However, freeze-drying itself can introduce additional oxidative damage. Two enzymic hydrolysis regimes (DNase I, phosphodiesterases I and II, and alkaline phosphatase; or P1 nuclease and alkaline phosphatase) give similar values for 8-oxodG.     

Controlled oxidation of calf thymus DNA to produce standard samples for 8-oxodeoxyguanosine analysis; effects of freeze-drying, storage and hydrolysis conditions

Calf thymus DNA containing defined levels of 8-hydroxy-2'-deoxyguanosine (8-oxodG) was prepared by treatment with visible light in the presence of photosensitiser Ro 19-8022. The DNA was checked for stability; after freeze-drying, the amount of 8-oxodG did not increase during 6 weeks' storage at room temperature. However, freeze-drying itself can introduce additional oxidative damage. Two enzymic hydrolysis regimes (DNase I, phosphodiesterases I and II, and alkaline phosphatase; or P1 nuclease and alkaline phosphatase) give similar values for 8-oxodG.     

Dexamethasone ameliorates oxidative DNA damage induced by benzene and LPS in mouse bone marrow

Mice were grouped to receive vehicle, dexamethasone (DEX), lipopolysaccharide (LPS), benzene (BZ, 200 mg/kg) and combinations: LPS + DEX, BZ + DEX, LPS + BZ, LPS + DEX + BZ. The DNA damage in bone marrow cells from BZ group was enhanced 2.8-fold measured by nuclear 8-hydroxy-2 '-deoxyguanosine (8-oxodG) and 1.4-fold measured by Comet score (index of DNA breaks) (p < 0.05). In the BZ + DEX group, 8-oxodG level and the Comet score were lowered to 65% and 76% respectively of that in the BZ group (p < 0.05). The BZ + LPS caused a 3.9-fold increase in 8-oxodG and a 1.6-fold increase in the Comet score (p < 0.05). The LPS + DEX + BZ lowered 8-oxodG level and the Comet score to 50% and 78% of the values in the LPS + BZ group, respectively (p < 0.05). Nitrate/nitrite levels in serum were higher after BZ + LPS treatment than after all other treatments. Both 8-oxodG level and the Comet scores were correlated to the serum nitrate/nitrite level across all the treatments (r = 0.55, p < 0.01 and r = 0.69, p < 0.01, respectively). In bone marrow cells the 8-oxodG correlated with the Comet scores (r = 0.80, p < 0.01). We conclude that DEX administration can reduce the DNA damage from BZ treatment and from the combination of BZ and LPS. The correlation of DNA damage with nitrate/nitrite indicates the possible involvement of reactive nitrogen species (RNS) in the interaction between BZ and the inflammatory reaction stimulated by LPS. The 8-oxodG determination is more sensitive than strand break analysis by the Comet assay in bone marrow in vivo in mice for measuring the BZ-induced DNA damage.     

Inter-laboratory Validation of Procedures for Measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2′-deoxyguanosine in DNA

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CV of less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10 6 Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.