Search

Balkan endemic nephropathy (BEN) is a human chronic tubulointerstitial renal disease that occurs in rural areas of some Balkan countries. The disease is insidious and fatal, and mostly affects persons in their sixties or seventies. BEN areas have unusually high rates of otherwise rare upper urinary tract tumors (UTT). Since extensive production of reactive oxygen species leading to oxidative stress has been implicated in tumor development, the aim of this study was to see whether oxidative stress is involved in the development of BEN and UTT. Urine samples were collected from a BEN village (N = 22) and a control village (N = 16) residents and analyzed for malondialdehyde (MDA) and 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG). The levels of both oxidative stress parameters were significantly higher in the BEN village residents than controls. However, there was no correlation between MDA and 8-oxodG results. Our results confirm that oxidative stress could be implicated in development of both, BEN and UTT.

Background: Oxidative stress may be important in carcinogenesis and a possible risk factor for breast cancer. The urinary excretion of oxidatively generated biomolecules, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), represents biomarkers of oxidative stress, reflecting the rate of global damage to DNA in steady state., Methods: In a nested case-control design, we examined associations between urinary excretion of 8-oxodG and risk of breast cancer in a population-based cohort of 24,697 postmenopausal women aged 50 to 64 years with 3 to 7 years follow-up. The accruing cases of breast cancer were matched to controls by age at diagnosis, baseline age, and hormone replacement therapy (HRT). Spot urine samples collected at entry was analyzed for 8-oxodG by high-performance liquid chromatography with electrochemical detection. Incidence rate ratio (IRR; 95% confidence intervals) based on 336 matched pairs with all information was estimated per unit increase in 8-oxodG divided by creatinine for all and estrogen receptor (ER) positive and negative breast cancers., Results: There was a borderline significant positive association between 8-oxodG and risk of all breast cancer (IRR: 1.08; 1.00-1.17 per unit increase in nmol/mmol creatinine). This association was significant with respect to the risk of ER-positive cancer (IRR: 1.11; 1.01-1.23) and among women not using HRT (IRR: 1.11; 0.97-1.26) or with low dietary iron intake (IRR: 1.10; 1.06-1.37 per unit increase) for all breast cancer., Conclusions: We observed positive association between 8-oxodG excretion and risk of especially ER-positive breast cancer., Impact: Our results suggest that oxidative stress with damage to DNA is important for the development of breast cancer.

Aims: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability., Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (rp 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples)., Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker., Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended.

Of the DNA-derived biomarkers of oxidative stress, urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is the most frequently measured. However, there is significant discrepancy between chromatographic and immunoassay approaches, and intratechnique agreement among all available chromatography-based assays and ELISAs is yet to be established. This is a significant obstacle to their use in large molecular epidemiological studies. To evaluate the accuracy of intra/intertechnique and interlaboratory measurements, samples of phosphate buffered saline and urine, spiked with different concentrations of 8-oxoG, together with a series of urine samples from healthy individuals were distributed to ESCULA members. All laboratories received identical samples, including 2 negative controls that contained no added 8-oxodG. Data were returned from 17 laboratories, representing 20 methods, broadly classified as mass spectrometric (MS), electrochemical detection (EC), or enzyme-linked immunosorbant assay (ELISA). Overall, there was good within-technique agreement, with the majority of laboratories' results lying within 1 sd of their consensus mean. However, ELISA showed more within-technique variation than did the chromatographic techniques and, for the urine samples, reported higher values. Bland-Altman plots revealed good agreement between MS and EC methods but concentration-dependent deviation for ELISA. All methods ranked urine samples according to concentration similarly. Creatinine levels are routinely used as a correction factor for urine concentration, and therefore we also conducted an interlaboratory comparison of methods for urinary creatinine determination, in which the vast majority of values lay within 1 sd of the consensus value, irrespective of the analysis procedure. This study reveals greater consensus than previously expected, although concern remains over ELISA.-ESCULA [European Standards Committee on Urinary (DNA) Lesion Analysis], Evans, M. D., Olinski, R., Loft, S., Cooke, M. S. Toward consensus in the analysis of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine as a noninvasive biomarker of oxidative stress.

The imbalance between the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA and the efficiency of cellular systems of DNA protection and repair is considered an important factor in the age-dependent development of cancer. This study investigated the relationship between oxidatively damaged DNA and the activity of the DNA repair system and 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxodGTPase) activity in liver and lung tissue from mice at 10-100 weeks of age. The level of 8-oxodG increased with age, whereas the level of formamidopyrimidine DNA glycosylase sites was unaltered. The enzyme activity toward single oxygen-induced DNA damage and mRNA expression levels of Ercc1, Neil1, and Ogg1 remained unaltered with age. However, the 8-oxodGTPase activity in the liver was 18% (95% CI: 0.2-37%) lower in mice at 25 and 50 weeks than in 10-week-old mice. The 10- and 100-week-old mice had similar 8-oxodGTPase activity. In contrast, the mRNA expression of Nudt1 was statistically unaltered that likely resulted from higher variation of measurements. The accumulation of 8-oxodG with age is not a direct consequence of decreased enzyme activity toward singlet oxygen-induced substrate DNA. An age-related higher level of 8-oxodG even occurs concomitantly with high 8-oxodGTPase activity.

Oxidative damage to DNA is regarded as an important step in carcinogenesis. These lesions may arise as a consequence of exposure to xenobiotics, but are also generated as a consequence of endogenous generation of oxidizing compounds. Measurements of oxidative damage to guanines, such as 8-oxo-7, 8-dihydroguanine (8-oxodG) are increasingly being regarded as reliable biomarkers of oxidative stress and they may have a predictive value of cancer risk, although this needs to be established independently in several cohort studies. A survey of intervention studies of the ingestion of antioxidant-containing foods or tablets of antioxidants indicate that about one-third of the studies reported a protective effect in terms of lower levels of oxidative damage to DNA in white blood cells or decreased urinary excretion of 8-oxodG. Although firm conclusions cannot be reached, there appears to be links between ingestion of antioxidants, oxidative damage to DNA, and risk of cancer.

Background: Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. The noninvasive assessment of such damage, i.e., in urine, and application to large-scale human studies are vital to understanding this role and devising intervention strategies., Methods: We have reviewed the literature to establish the status quo with regard to the methods and meaning of measuring DNA oxidation products in urine., Results: Most of the literature focus upon 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and whereas a large number of these reports concern clinical conditions, there remains (a) lack of consensus between methods, (b) possible contribution from diet and/or cell death, (c) no definitive DNA repair source of urinary 2'-deoxyribonucleoside lesions, and (d) no reference ranges for healthy or diseased individuals., Conclusions: The origin of 8-oxodG is not identified; however, recent cell culture studies suggest that the action of Nudix hydrolase(s) on oxidative modification of the nucleotide pool is a likely candidate for the 8-oxodG found in urine and, potentially, of other oxidized 2'-deoxyribonucleoside lesions. Literature reports suggest that diet and cell death have minimal, if any, influence upon urinary levels of 8-oxodG and 8-oxo-7,8-dihydroguanine, although this should be assessed on a lesion-by-lesion basis. Broadly speaking, there is consensus between chromatographic techniques; however, ELISA approaches continue to overestimate 8-oxodG levels and is not sufficiently specific for accurate quantification. With increasing numbers of lesions being studied, it is vital that these fundamental issues are addressed. We report the formation of the European Standards Committee on Urinary (DNA) Lesion Analysis whose primary goal is to achieve consensus between methods and establish reference ranges in health and disease.

Oxidative damage to DNA may be important in carcinogenesis and a possible risk factor for lung cancer. The urinary excretion of products of damaged nucleotides in cellular pools or in DNA may be important biomarkers of exposure to relevant carcinogens reflecting the rate of damage in steady state and may predict cancer risk. Oxidation of guanine in DNA or the nucleotide pool may give rise to 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) for urinary excretion. Oxoguanine glycosylase (OGG1) is the base excision enzyme repairing 8-oxodG in DNA by release of 8-oxoguanine. In a nested case-cohort design we examined associations between urinary excretion of 8-oxodG and risk of lung cancer as well as potential interaction with the OGG1 Ser326Cys polymorphism in a population-based cohort of 25 717 men and 27 972 women aged 50-64 years with 3-7 years follow-up. We included 260 cases with lung cancer and a sub-cohort of 263 individuals matched on sex, age and smoking duration for comparison. Urine collected at entry was analysed for 8-oxodG by HPLC with electrochemical detection. The excretion of 8-oxodG was higher in current smokers, whereas OGG1 genotype had no effect. Overall the incidence rate ratio (IRR) (95% confidence interval) of lung cancer was 0.99 (0.80-1.22) per doubling of 8-oxodG excretion and there was no interaction with OGG1 genotype. However, among never-smokers (eight cases and eight sub-cohort members) the IRR was 11.8 (1.21-115) per doubling of 8-oxodG excretion. The association between 8-oxodG excretion and lung cancer risk among never-smokers suggests that oxidative damage to DNA nucleotides is important in this group.

Ambient particulate matter (PM) has been associated with increased risk of lung cancer. One proposed mechanism is that PM induces oxidative stress mediated by transition metals contained within this mixture. We examined the relationship between the personal exposure to water-soluble transition metals in PM(2.5) and oxidative DNA damage. In 49 students from central Copenhagen, we determined PM(2.5) exposure by personal sampling twice in 1 year, and measured in these PM(2.5) samples the concentration of the soluble transition metals vanadium, chromium, iron, nickel, copper, and platinum. Collected lymphocytes and 24-hour urine samples were analyzed for DNA damage in terms of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG). We found that the 8-oxodG concentration in lymphocytes was significantly associated with the vanadium and chromium concentrations with a 1.9% increase in 8-oxodG per 1 microg/L increase in the vanadium concentration and a 2.2% increase in 8-oxodG per 1 microg/L increase in the chromium concentration. We have previously reported that in this study population the personal exposure to PM(2.5) was associated with an increase in 8-oxodG in lymphocytes. However, vanadium and chromium were associated with the 8-oxodG concentration in lymphocytes independently of the PM(2.5) mass concentration. The four other transition metals were not associated with 8-oxodG in lymphocytes and none of the transition metals was significantly associated with 8-oxodG in urine. Our results could indicate that vanadium and chromium present in PM(2.5) have an effect on oxidative DNA damage that is independent of particle mass and/or other possible toxic compounds contained within this particulate mixture.

Background: Oxidative stress and related DNA damage in human sperm may be important for fecundity and pregnancy outcome., Methods: We studied the level of oxidative DNA damage in terms of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in sperm DNA among 225 first-pregnancy planners. Over the six menstrual cycle follow-up time, after cessation of contraception, 135 pregnancies were conceived., Results: The likelihood of pregnancy occurring in a single menstrual cycle was inversely associated with the 8-oxodG level (P < 0.01). The odds ratio of pregnancy in each of the first three or all six follow-up menstrual cycles was 0.42 (0.23-0.78; 95% CI) and 0.61 (0.36-0.91) per unit increase in the log 8-oxodG/100 000 dG ratio after adjustment for potential confounders, (including sperm concentration) respectively. The intra-individual coefficient of variation of 8-oxodG in 2-6 monthly repeated sperm samples from 116 men was 19% for the 8-oxodG/dG ratio, whereas the inter-individual coefficient of variation was 49%. The 8-oxodG level was not significantly associated with smoking, consumption of alcohol or caffeine, exposure to welding fumes or the plasma levels of sex hormones., Conclusions: The data suggest that oxidative damage to sperm DNA influences fecundity and the level of damage is relatively constant within an individual and not influenced by smoking.

Ozone (O3) is a well-known oxidant pollutant present in photochemical smog. Although ozone is suspected to be a respiratory carcinogen it is not regulated as a carcinogen in most countries. The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to ozone for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from BALB/c mice was determined by the comet assay as a measure of DNA strand breaks. Within the first 200 min after exposure, the BAL cells from the mice exposed to 1 or 2 ppm ozone had 1.6- and 2.6-fold greater tail moments than unexposed mice. After 200 min there was no effect. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidised amino acids in the lung tissue. The mRNA level of the repair enzyme ERCC1 was not increased in the lung tissue. Inflammation was measured by the cytokine mRNA level in lung homogenates. An up to 150-fold induction of interleukin-6 (IL-6) mRNA was detected in the animals exposed to 2 ppm ozone compared to the air-exposed control mice. Also at 1 ppm ozone, the IL-6 mRNA was induced. The large induction of IL-6 mRNA in the lung took place after DNA strand breaks were induced in BAL. This does not support the notion that inflammatory reactions are the cause of DNA damage. To determine whether these exposures were mutagenic, Muta Mice were exposed to 2 ppm ozone, 90 min per day for 5 days. No treatment-related mutations could be detected in the cII transgene. These results indicate that a short episode of ozone exposure at five times the threshold limit value (TLV) in US induces lung inflammatory mediators and DNA damage in the cells in the lumen of the lung. This was not reflected by an induction of mutations in the lung of Muta Mice.

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.

Many epidemiologic studies have addressed the possible preventive effects of antioxidants in disease causation and progression. With the use of molecular techniques, it is feasible to investigate specific properties of antioxidants in intervention studies. The most widely used techniques to investigate oxidative DNA damage in white blood cells are the measurement of 7-hydroxy-8-oxo-2'-deoxyguanosine and the comet assay. The types of antioxidant intervention studies include those involving single or multiple supplementations of vitamin C, vitamin E, or carotenoids and those involving various natural food products (eg, carrot juice). In short-term intervention studies (usually weeks or a few months), results have been mixed. Single-dosing studies found that decreased oxidative DNA damage lasted only hours after antioxidant supplementation, suggesting that the preventive effect is relatively short. In addition, many of the positive studies were not placebo-controlled, thus leaving a possibility of false-positive results caused by period effects, eg, seasonal variation, changes in the lifestyles of the subjects, or variation in measurements over time. Because participation in an antioxidant intervention study may cause changes in dietary habits and because seasonal changes may have profound effects, it is recommended that future studies have a placebo-controlled, parallel design rather than a crossover design.

Dialkyl phthalate esters are used in the plastic industry and widely distributed in the environment. Previously, it has been shown that di-n-butyl phthalate (DBP) produces testicular atrophy and liver enlargement in rodents, and the mechanisms behind this could involve reactive oxygen species (ROS). In this study, oxidative DNA damage was measured in terms of the premutagenic modified nucleoside 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in nuclear DNA from liver, kidneys, and testes from rats exposed to DBP in the perinatal or preadult period. In one experiment, pregnant rats were administered 0 or 0.5 g DBP/kg/d by gavage from d 7 after conception to d 17 after delivery and organs from male offspring were analyzed. In a second experiment, 25-d-old rats were administered 0, 0.5, or 2 g DBP/kg/d by gavage for 10 d. After perinatal exposure, body and organ weights were unchanged. The 8-oxodG/10(6) dG ratio in liver DNA increased significantly in the exposed group. In contrast, the 8-oxodG/10(6) dG ratio was significantly decreased in kidney DNA, whereas it remained unchanged in the testis. After preadult exposure (postnatal d 25 to 34) the testes weight of the exposed animals were significantly decreased and severe atrophy of the seminiferous tubules was observed. The body weight of the animals in the high-dose group was significantly decreased compared to the control. The 8-oxodG levels in liver, kidney, and testis DNA remained unchanged. Although ROS has been suspected of being involved in the formation of testicular atrophy in phthalate-exposed rats, no apparent sign of oxidative DNA damage was found after phthalate exposure perinatally or during the preadult stage. With respect to phthalate-induced oxidative DNA damage in the liver, it appears that the developmental stage during exposure is important.

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.

Estrogenic chemicals are suspected of affecting cancer risk and male reproduction, possibly involving oxidative DNA damage. In this study, formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), was measured in testicular cells from rats after 17 alpha-ethinylestradiol (EE) exposure in vivo and in vitro after incubation with EE with or without an antiestrogen. In vivo, preadult (30-35 days) and adult (110-120 days) Wistar rats received 0, 2.8, or 56 mg EE/kg body weight as intraperitoneal injections (n=6). After 1 or 4 h, the 8-oxodG/10(6) dG ratio was measured in the liver, kidneys, and testes. Testes DNA analysis revealed an age-related effect (adult animals had a higher ratio than the young animals) and a concentration effect in preadult rats (increased EE-concentration caused increased ratio), but no time effect. No differences were found in the liver or kidneys. In vitro, testicular cells were isolated and incubated with EE concentrations ranging from 0.1 to 1000 nM. The results indicated an increase in 8-oxodG/10(6) dG from 0 to 10 nM estrogen. At 1000 nM, the level was close to control level. Coincubation of 10 nM EE (maximum damage) with an estrogen antagonist, ICI 182.780, abolished the effect at 10 nM, indicating that the damaging effect is estrogen receptor mediated.

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.

Reactive oxygen species have been implicated in the pathogenesis of acute pancreatitis. Few studies have focused on the loss of endogenous antioxidants and molecular oxidative damage. Two acute pancreatitis models in rats; taurocholate (3% intraductal infusion) and cerulein (10 microg/kg/h), were used to study markers of oxidative stress: Glutathione, ascorbic acid, and their oxidized forms (glutathione disulfide and dehydroascorbic acid), malondialdehyde, and 4-hydroxynoneal in plasma and pancreas, as well as 7-hydro-8-oxo-2'-deoxyguanosine in pancreas. In both models, pancreatic glutathione depleted by 36-46% and pancreatic ascorbic acid depleted by 36-40% (p <.05). In the taurocholate model, plasma glutathione was depleted by 34% (p <.05), but there were no significant changes in plasma ascorbic acid or in plasma and pancreas dehydroascorbic acid, malondialdehyde, and 4-hydroxynoneal, and no significant changes in the pancreas glutathione disulfide/glutathione ratio. While pancreas glutathione disulfide/glutathione ratio increased in the cerulein model, there were no significant changes in plasma glutathione, plasma, or pancreas ascorbic acid, dehydroascorbic acid, 4-hydroxynoneal, and malondialdehyde, or in pancreas 7-hydro-8-oxo-2'-deoxyguanosine. Reactive oxygen species have a minor role in the intermediate stages of pancreatitis models.

We studied the long-term effects of vitamins E and C and their combination on lipid peroxidation in vivo and in vitro. The Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) trial is a double-masked placebo-controlled randomized clinical trial to study the effects of vitamin C (500 mg of slow release ascorbate per day), vitamin E (182 mg of RRR-alpha-tocopherol acetate per day), and the combination of both antioxidants. Lipid peroxidation measurements were carried out for 48 male participants at entry and at 12 and 36 months. Compared with placebo, vitamin E and the vitamin combination increased plasma lipid-standardized alpha-tocopherol during the first 12 months by 68.2% and 65.2% (P:<0. 001 for both), respectively, and reduced serum 7beta-hydroxycholesterol by 50.4% (P:=0.013) and 44.0% (P:=0.041), respectively. The net change of lipid standardized alpha-tocopherol was 63.8% after 36 months of vitamin E supplementation and 43.3% for the combination. Vitamin C supplementation elevated plasma total ascorbate level by 30.1% (P:=0.043) in 12 months and by 91.1% (P:=0. 001) in 36 months. Neither vitamin E, vitamin C, nor the combination influenced the urinary excretion rate of 7-hydro-8-oxo-2'-deoxyguanosine or the antioxidative capacity of plasma. Vitamin E and the combination of vitamins E and C enhanced the oxidation resistance of isolated lipoproteins and total serum lipids. Our data indicate that long-term supplementation of nondepleted men with a reasonable dose of vitamin E alone or in combination with slow release vitamin C reduces lipid peroxidation in vitro and in vivo, whereas a relatively high dose of vitamin C alone does not.

Cruciferous vegetables have cancer preventive effects which may be due to reduction of oxidative DNA damage. We investigated the effect of an aqueous extract of cooked Brussels sprouts on formation of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) in calf thymus DNA in vitro. Damage was induced by a Fenton reaction, UVC (254 nm), UVA (365 nm), sunlamp light, and methylene blue with visible light. The extract inhibited 8-oxodG formation in all systems except visible light with methylene blue. The IC50 values were 6-20 microg/ml corresponding to the extract of 5-20 g of Brussels sprouts distributed in a volume of 50 L. The protective effect in the Fenton reaction was unaffected by addition of EDTA. After HPLC separation fractions were identified with similar DNA protective effects. Sinigrin, a glucosinolate abundant in Brussels sprouts, co-eluted with the most effective fraction and had DNA protective effects. In comparison with other antioxidants the patterns of effect of the extract in the five damage systems were more similar to that of sodium azide than to those of dimethylsulfoxide and vitamin C. Constituents of Brussels sprouts can protect DNA by direct scavenging, e.g. hydroxyl radical and other oxidants, without prooxidant effects at concentrations potentially achievable by modest intake of the vegetable.

We investigated whether acute iron intoxication causes oxidative DNA damage, measured in terms of 7-hydro-8-oxo-2'-deoxyguanosine, 8-oxodG, in nuclear DNA in testes and epididymal sperm cells in vivo and in vitro in rats. In addition, we investigated levels of the modified nucleoside in liver and kidney and measured its urinary excretion. Sperm cells were isolated from the epididymides and the testes cells were isolated after homogenisation. In vitro, the sperm and testes cells were incubated with increasing concentrations of FeCl2 ranging from 0 to 600 microM. The median (range) levels of 8-oxodG/10(5) dG in the epididymal sperm cells increased from 0.48 (0.42-0.90) to 15.1 (11.4-17.6) (p < 0.05), whereas the level rose from 0.63 (0.22-0.81) to 8.8 (4.5-11.6) (p < 0.05) at 0 and 600 microM, respectively, in the testicular cells. In vivo groups of 7-8 rats received 0, 200 or 400 mg iron/kg as dextran i.p. After 24 h, epididymal sperm cells, testes, kidneys and liver were collected for analysis. Kidney and sperm DNA showed a significant increase in 8-oxodG in the iron-treated animals. The median (range) values of the 8-oxodG/10(5) dG in the epididymal sperm cells rose from 0.66 (0.38-1.09) to 1.12 (0.84-5.88) (p < 0.05) at 0 and 400 mg iron/kg, respectively, whereas the values in the testes and liver showed no significant change. In the kidneys the 8-oxodG/10(5) dG median (range) values were 0.98 (0.73-1.24), 1.21 (1.13-1.69) and 1.34 (1.12-1.66) after 0, 200 and 400 mg iron/kg, respectively (p < 0.05). The 8-oxodG-excretion rate was measured in 24h urine before and after iron treatment. The rate of urinary 8-oxodG excretion increased from 129 (104-179) pmol/24 h before treatment to 147 (110-239) pmol/24 h after treatment in the group receiving 400 mg iron/kg (p < 0.05). The results indicate that acute iron intoxication may increase oxidative damage to sperm and kidney DNA.

Oxidative damage to DNA could be involved in the increased risk of cancer associated with exposure to polluted urban air, which contains a number of oxidants. CYP1A2 is induced by and metabolizes polyaromatic hydrocarbons (PAH) and aromatic amines and could modify effects of exposure to ambient air pollution. Similarly, DNA repair may be influenced by occupational and other exposures as well as modify the effect of DNA damaging agents. As part of a large investigation of the genotoxic burden to diesel exposed workers in transport sectors we studied oxidative DNA damage in 57 non-smoking bus drivers from the greater Copenhagen area. The drivers were studied on a workday and on a day off work. Comparisons were made between drivers from the central (n=30) and rural/suburban (n=27) areas of Copenhagen. The rate of oxidative DNA damage was estimated from 24 h urinary excretion of 8-oxo-2'-deoxyguanosine (8-oxodG), a repair product of the highly mutagenic oxidation of guanine in DNA or the cellular pool of GTP. CYP1A2 activity was estimated from the urinary excretion of metabolites of dietary caffeine. The DNA repair was estimated by unscheduled DNA synthesis (UDS) in mononuclear cells isolated on the workday. Repeated measures ANOVA and multifactorial ANCOVA with CYP1A2 activity, age and UDS as covariates were used for statistical evaluation. On the workday, the 8-oxodG excretion was 190+/-108 and 146+/-89 pmol/kg 24 h in the bus drivers from central and the suburban/rural areas Copenhagen, respectively (p<0.05). The 8-oxodG excretion was not significantly different between the workday and the day off. CYP1A2 activity was not affected by driving area but was correlated with the 8-oxodG excretion on the workday (r=0.53; p<0.05). UDS was not significantly affected by driving area or correlated with the 8-oxodG excretion. The increased excretion of 8-oxodG in bus drivers from central Copenhagen as compared with drivers from rural/suburban greater Copenhagen suggests that exposure to ambient air pollution causes oxidative damage to DNA. This effect may be modified by the activity of CYP1A2 or a coregulated enzyme., (Copyright 1999 Elsevier Science B. V.)

The role of dietary fats and energy in carcinogenesis has been partly related to oxidative damage to DNA. We have investigated the effect of dietary fat content and saturation on the urinary excretion of 8-oxo-7,8dihydro-2'-deoxyguanosine (8-oxodG) in male and female rats. Groups of Fischer F344 rats (n = 6-10) were fed control chow (3.4% fat) or diets containing 21.8% corn oil or 19.8% coconut oil + 2% corn oil for 12-15 weeks. At the end of the diet intervention period 24h urine was collected for determination of 8-oxodG by HPLC. In the male groups fed control, corn oil and coconut oil diet the excretion of 8-oxodG was 403+/-150, 932+/-198 and 954+/-367pmol/kg 24 h, respectively (p < 0.05). In the female groups fed control and corn oil diet the excretion of 8-oxodG was 752+/-80 and 2206+/-282 pmol/kg 24 h, respectively (p < 0.05). Calculated per whole animal the excretion was 137+/-51, 324+/-70 and 328+/-128 pmol/24 h in the control, corn and coconut oil male groups and 156+/-21 and 464+/-56 pmol/24 h in the control and corn oil female groups, respectively ( p < 0.05). Thus, per animal or per consumed energy there was much less difference in 8-oxodG excretion between the corresponding male and female groups and only significant difference between the high fat groups. There was a close correlation (r = 0.7; p < 0.05) between 8-oxodG excretion and the energy intake. The present study suggests that a high fat diet increases oxidative DNA modification substantially irrespective of the saturation level of the fat. Energy intake appears to be the major determinant of the rate of modification.

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.

Background: Reactive oxygen species from, e.g. tobacco smoke are suggested to be involved in carcinogenesis by oxidative modification of DNA. The urinary excretion rate of the oxidized nucleoside 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) has been validated as a biomarker of the rate of oxidative DNA modification with mechanistic relation to carcinogenesis. In cross-sectional studies, the urinary excretion rate of 8-oxodG has been shown to be elevated in smokers compared with non-smokers., Purpose: In this randomised, controlled smoking cessation study, we investigated whether cigarette smoking per se causes oxidative DNA modification., Methods: Of the 182 healthy smokers included, 100 were randomized to quit smoking after baseline samples had been taken, and 82 were randomized to continue usual smoking. Before the start of the study and after 4 weeks, the subjects collected 24-h urine samples that were analysed for 8-oxodG content by high-pressure liquid chromatography with electrochemical detection. The subjects randomized to smoking cessation were followed up after 26 weeks., Results: Four weeks of smoking cessation resulted in a 21% decrease in 8-oxodG excretion rate (from mean +/- SD, 30.5 +/- 13.9 to 24.1 +/- 10.5 nmol/24 h, P < 0.001) in 58 quitters included in per-protocol data analysis. Sixty-five continued smokers included in per-protocol analysis showed a 9% decrease in 8-oxodG excretion rate (from 31.6 +/- 13.2 to 28.7 +/- 12.6 nmol/24 h, P = 0.026). After 4 weeks, the 8-oxodG excretion rate was 16% (95% confidence interval 4 to 28%) higher in the continued smokers than in the quitters (P = 0.0085, ANCOVA), demonstrating the effect of smoking per se. A 23% (P < 0.005) decrease in 8-oxodG excretion rate was sustained for 26 weeks in 27 quitters who completed the study., Conclusion: Smoking cessation significantly reduces the urinary excretion rate of 8-oxodG, giving direct and controlled evidence that cigarette smoking causes an increased rate of oxidative DNA modification. This could represent a mechanism by which tobacco smoke is carcinogenic.

Dietary antioxidants may influence cancer risk and aging by modifying oxidative damage. The effect of graded dietary doses of the antioxidant vitamins C and E on oxidative DNA damage was studied in the liver of guinea-pigs under normal conditions. Like human beings, guinea-pigs cannot synthesize ascorbate and alpha-tocopherol. In one experiment, three groups of 6-8 guinea-pigs were fed diets containing 15 mg of vitamin E/kg chow and three different amounts of vitamin C (33,660 or 13,200 mg/kg) for 5 weeks. In a second experiment, three groups of seven guinea-pigs were fed diets containing 660 mg of vitamin C/kg and three different amounts of vitamin E (15, 150 or 1500 mg/kg) for 5 weeks. The three graded levels of each vitamin respectively represent marginal deficiency, an optimum supplementation and a megadose. Oxidative damage to liver DNA was estimated by measuring 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG) referred to deoxyguanosine (dG) by means of high-performance liquid chromatography with simultaneous electrochemical-coulometric and ultraviolet detection. The level of ascorbate in the liver was 0.034 +/- 0.051, 1.63 +/- 1.06 and 1.99 +/- 0.44 micromol/g in the low, medium and high dose ascorbate groups (59-fold variation). The liver concentration of alpha-tocopherol was 28 +/- 11, 63 +/- 18 and 187 +/- 34 nmol/g in the low, medium and high dose alpha-tocopherol groups (7-fold variation). The level of oxo8dG in the liver DNA was 1.89 +/- 0.32, 1.94 +/- 0.78 and 1.93 +/- 0.65 per 10(5) dG in the low, medium and high dose ascorbate groups (no effect: P > 0.05). In the low, medium and high dose alpha-tocopherol groups oxo8dG level in the liver DNA was 2.85 +/- 0.70, 2.74 +/- 0.66 and 2.61 +/- 0.92 per 10(5) dG (no effect: P > 0.05). It is concluded that even very large variations in the content of the antioxidant vitamins C and E in the diet and liver have no influence on the steady-state level of oxidative damage to guanine in the liver DNA of normal unstressed guinea-pigs.

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.

Menadione (2-methyl-1,4-naphthoquinone) induces oxidative stress in cells causing perturbations in the cytoplasm as well as nicking of DNA. The mechanisms by which DNA damage occurs are still unclear, but a widely discussed issue is whether menadione-generated reactive oxygen species (ROS) directly damage DNA. In the present study, we measured the effect of menadione on formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), an index of oxidative DNA base modifications, and on DNA fragmentation. Isolated hepatocytes from phenobarbital-pretreated rats were exposed to menadione, 25-400 microM, for 15, 90 or 180 min with or without prior depletion of reduced glutathione (GSH) by diethyl maleate. Menadione caused profound GSH depletion and internucleosomal DNA fragmentation, which was demonstrated by a prominent fragmentation ladder on agarose gel electrophoresis. We found no oxidative modification of DNA in terms of increased 8-oxodG formation. In contrast, the positive control of sunlamp light increased 8-oxodG 5-fold in rat hepatocytes. We conclude that oxidative modification of DNA bases is unlikely to be important in menadione-induced DNA damage.

Reactive oxygen species (ROS) induce 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, which has been proposed as a key biomarker relevant to carcinogenesis. 8-OHdG has been induced in a number of different ways, most often without knowledge of the specific type and amount of ROS generated. We have measured 8-OHdG formation in calf thymus DNA exposed to ionizing radiation under conditions generating either hydroxyl radicals (OH.), superoxide anions (O2-) or both. Additionally, we investigated the relationship between the scavenger effect of the drug 5-aminosalicylic acid (5-ASA) and increasing OH. exposure toward 8-OHdG formation. The effect of this drug was compared to those of the physiological scavengers ascorbate and reduced glutathione (GSH). We found that OH. generated 8-OHdG in a dose-dependent manner, whereas O2- did not cause 8-OHdG formation. 5-ASA, ascorbate and GSH all acted as hydroxyl radical scavengers, although with different concentration-effect curves, emphasizing the importance of using relevant pharmaco-/physiological concentrations in studies focusing on therapeutic applications of scavengers. The scavenger effect of 5-ASA at concentrations > or = 0.1 mM was similar at 30 and 100 Gy radiation, i.e. within a wide range of OH. exposure, which is useful information considering clinical applications where the exact amount of ROS formed is unknown. Both 5-ASA and ascorbate at low concentrations (< or = 0.1 mM) were less efficient in preventing 8-OHdG formation from X-ray generated OH. than was shown in a previous comparable study using light as the source of ROS. This differentiation probably reflects variations in both number and type of ROS formed in the two systems.

Living organisms are continuously exposed to reactive oxygen species as a consequence of biochemical reactions as well as external factors. Oxidative DNA damage has been implicated in aging, carcinogenesis and other degenerative diseases. The urinary excretion of the DNA repair product 8-hydroxydeoxyguanosine (8OHdG) has been proposed as a noninvasive biomarker of oxidative DNA damage in humans in vivo. We have developed a three-dimensional HPLC analysis with electrochemical detection for the analysis of 8OHdG in urine and studied factors affecting the excretion of this biomarker in 83 healthy humans and in various laboratory animals, including dog, pig, and rat. Previously, other groups have used comparable HPLC methods or gas chromatography-mass spectrometry with selective ion monitoring for measuring the excretion of 8OHdG in humans, rats, mice, and monkeys. In the 169 humans studied so far, the average 8OHdG excretion was 200-300 pmol/kg per 24 h with a sevenfold range, and the coefficient of variation was 30-40%. This excretion corresponds 140-200 oxidative modification of guanine bases per cell per day. Thirty-two smokers from our study population excreted 50% (31-69%; 95% confidence interval) more 8OHdG than 53 nonsmokers. This indicates a 50% increased rate of oxidative DNA damage from smoking, adding to the other well-known health hazards of smoking. The biochemical-physiological basis is unknown but may be related to smoke constituents including or generating reactive oxygen species and/or consuming antioxidants and/or the well-known enhancing effect of smoking on the metabolic rate. In our 83 healthy subjects the 8OHdG excretion correlated with body composition. Thus, lean and/or male subjects excreted more than obese and/or female subjects, possibly related to differences in metabolic rate. In accordance, the excretion of 8OHdG decreased after calorie restriction, which will cause a decline in the metabolic rate. Across the investigated species, humans, dogs, pigs, and rats, the excretion of 8OHdG correlated with the specific metabolic rate, confirming data from other groups on humans, monkeys, rats, and mice. The excretion of 8OHdG decreased with age in rats in parallel with the decline in metabolic rate with advancing age. The excretion of 8OHdG reflects the formation and repair of only one out of approximately 20 described oxidative DNA modifications. So far, methods are not available for the determination of the corresponding repair products, except 8OHdG and thymidine glycol, in urine. Moreover, the importance in terms of mutagenicity, particularly regarding tumour suppressor genes and oncogenes, is mainly documented for 8OHdG in DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxidative DNA damage may be implicated in ageing, carcinogenesis and other degenerative diseases. Oxidative DNA damage can be assessed in humans in vivo from the urinary excretion of the DNA-repair product 8-hydroxydeoxyguanosine (8OHdG). We investigated factors influencing the excretion of 8OHdG in 24 h urine from 83 randomly selected healthy subjects (52 women) aged 40-64 years. For 2 weeks prior to urine collection the subjects kept a weighed diet record. 8OHdG was quantified by an automatic three-dimensional HPLC analysis with electrochemical detection. The 8OHdG excretion was 252 +/- 103 (mean +/- SD) pmol kg body weight/24 h with a range from 78 to 527. Multiple regression analysis identified three factors, smoking, body mass index (BMI) and gender, as significant predictors of the 8OHdG excretion. In 30 smokers the 8OHdG excretion was 320 +/- 99 pmol/kg/24 h opposed to 213 +/- 84 pmol/kg/24 h in 53 non-smokers. According to multiple regression analysis smokers excreted 50% (31-69%; 95% confidence interval) more 8OHdG than non-smokers. In 52 women the 8OHdG excretion was 240 +/- 106 pmol/kg/24 h opposed to 271 +/- 96 pmol/kg/24 h in 31 men. According to the multiple regression analysis men excreted 29% (10-48%) more 8OHdG than women. According to multiple regression analysis the 8OHdG excretion decreased with 4% (2-6%) per increment in BMI measured in kg/m2. The dietary distribution of energy demonstrated no important predictive value with respect to 8OHdG excretion. The intake of the antioxidant vitamins C and E and of vitamin A equivalents, including beta-carotene, was not associated with 8OHdG excretion. The results suggest that smoking increases oxidative DNA damage by approximately 50%. This effect implies potential serious health effects adding to the other well-known health hazards of smoking. The higher 8OHdG excretion in men and lean subjects may be related to a higher rate of metabolism with increased availability of reactive oxygen species. The apparent 7-fold individual variation in oxidative DNA damage carries implications regarding the rate of ageing and the risk of cancer and other degenerative diseases. The excretion of 8OHdG into urine offers a valuable tool for testing such hypotheses in humans.

Oxidative DNA damage, as expressed by 8-hydroxydeoxyguanosine (8-OHdG), was investigated in calf thymus DNA exposed to either ultraviolet radiation or to FeCl2/H2O2 in a Fenton-like reaction. The influence of iron (absent in the UV system and present in the FeCl2/H2O2 system) and pH (7.4 and 4.0) on the effect of glutathione (GSH), ascorbate, and 5-aminosalicylic acid (5-ASA, a drug used in the treatment of chronic inflammatory bowel diseases) was examined in these systems. Without iron, all three compounds considerably reduced 8-OHdG formation (i.e., acted as scavengers), while in the presence of iron salts, 8-OHdG formation was accelerated (except for GSH at pH 7.4), i.e., the compounds acted as prooxidants. This effect was augmented at low pH. The prooxidant property of 5-ASA may have implications for its clinical use. Maximum scavenging effect for all the compounds investigated was obtained at much lower doses than the maximum enhancing effect. This demonstrates that to the end of oxy-radical scavenging, the concentration of the GSH, ascorbate, and 5-ASA, respectively, should be chosen to obtain maximum antioxidant effect and minimum prooxidant effects. The significance of this finding for the selection of antioxidant dose is important but remains to be investigated further.