Oxidative DNA Damage In Rats Research Articles

Classical conditioning of oxidative DNA damage in rats

This study investigated whether the formation of 8-hydroxydeoxyguanosine (8-OH-dG), a known oxidative DNA modification relevant to carcinogenicity, can be classically conditioned to a novel taste in order to clarify the possible role of the central nervous system (CNS) or psychological stress on cancer initiation via a classical conditioning mechanism. Male Wistar rats underwent one or two conditioned taste aversion (CTA) experiments in which ferric nitrilotriacetate (Fe-NTA), which has renal toxicity and can induce renal cell carcinoma, served as a visceral unconditioned stimulus (US), and a saccharin solution (SAC) was used as a conditioned stimulus (CS). The 8-OH-dG levels in the group conditioned with the combination of SAC and Fe-NTA significantly increased as compared to those of the uncombined groups by two repeats of the conditioning procedure ( P=0.013). The rats that showed a painful response at the Fe-NTA administration had significantly higher values of 8-OH-dG than those without pain ( P=0.003). These results not only provide the first evidence regarding classical conditioning of oxidative DNA damage using the CTA procedure, but also suggest the involvement of the CNS and psychological stress in the pathogenesis of cancer via oxidative DNA damage.

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 stress and DNA damage in Fischer rats following acute exposure to trichloroethylene or perchloroethylene

Oxidative DNA damage is emerging as an biomarker of effect in studies assessing the health risks of occupational chemicals. Trichloroethylene (TCE) and perchloroethylene (PERC) are used in the dry cleaning industry and their metabolism can produce reactive oxygen compounds. The present study examined the potential for TCE and PERC to induce oxidative DNA damage in rats that was detectable as increased urinary excretion of 8-hydroxydeoxyguanosine (8OHdG). Thiobarbaturic acid reactive substances (TBARS) and 8-epi-prostaglandin F 2α (8epiPGF) were also measured as biomarkers of increased oxidative stress. Male Fischer rats were administered a single i.p. injection of 0, 100, 500, or 1000 mg/kg of PERC or TCE. Control rats received only vehicle (1:4 v/v of Alkamuls/water). A positive control group received 100 mg/kg 2-nitropropane (2NP). Rats were sacrificed 24 h after dosing. In rats receiving 2NP or TCE but not PERC, TBARS and the 8OHdG/dG ratios were significantly elevated in liver. Lymphocyte 8OHdG/dG was not affected significantly by 2NP, TCE or PERC. In rats receiving 2NP, urinary excretion of 8OHdG and 8epiPGF2 were significantly increased. In rats receiving TCE or PERC, significant increases in 8epiPGF2 or 8OHdG were not evident. Results indicate that a single high dose of TCE, but not PERC, can induce an increase in oxidative DNA damage in rat liver. However, the usefulness of 8OHdG as a biomarker of TCE-induced oxidative DNA damage is questionable.

Increased urinary excretion of 8-oxo-2′-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers

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.

P1C62 - Effects of wine complex polyphenols against liver oxidative DNA damage in rats treated with 2-Nitropropane

Vitis vinifera cell suspensions were used to isolate and characterize the flavonoids (anthocyanins, catechins) and non-flavonoids (stilbenes) found in red wine. Furthermore, we showed that astringin is produced although this stilbene has not previously been reported to be a constituent of V. vinifera or wine. The ability of these compounds to act as radical scavengers was investigated using 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical. Antioxidant activities were assessed by their capacity to prevent Fe2+-induced lipid peroxidation in microsomes and their action on Cu2+-induced lipid peroxidation in low-density lipoproteins. The results showed that astringin has an important antioxidant effect similar to that of rans-resveratrol, and a higher radical scavenger activity than the latter. Astringinin appeared to be more active. These data indicate that phenolic compounds (stilbenes, catechins, anthocyanins) exhibit interesting properties which may account in part for the so-called “French paradox,” i.e. that moderate drinking of red wine over a long period of time can protect against coronary heart disease.

Prevention of Oxidative DNA Damage in Rats by Brussels Sprouts

The alleged cancer preventive effects of cruciferous vegetables could be related to protection from mutagenic oxidative DNA damage. We have studied the effects of Brussels sprouts, some non-cruciferous vegetables and isolated glucosinolates on spontaneous and induced oxidative DNA damage in terms of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in groups of 6–8 male Wistar rats. Excess oxidative DNA damage was induced by 2–nitropropane (2-NP 100mg/kg).Four days oral administration of 3g of cooked Brussels sprouts homogenate reduced the spontaneous urinary 8–oxodG excretion by 31% (p<0.05) whereas raw sprouts, beans and endive (1:l), isolated indolyl glucosinolates and breakdown products had no significant effect. An aqueous extract of cooked Brussels sprouts (corresponding to 6.7 g vegetable per day for 4 days) decreased the spontaneous 8-oxodG excretion from 92 ± 12 to 52 ± 15 pmol/24 h (p < 0.05). After 2–NP administration the 8–oxodG excretion was increased to 132±26 pmol/24h (p<0.05) whereas pretreatment with the sprouts extract reduced this to 102 ±30 pmol/24 h(p < 0.05).The spontaneous level of 8-oxodG in nuclear DNA from liver and bone marrow was not significantly affected by the sprouts extract whereas the level decreased by 27% in the kidney (p < 0.05). In the liver 2–NP increased the 8–oxodG levels in nuclear DNA 8.7 and 3.8 times (p<0.05) 6 and 24h after dose, respectively. The sprouts extract reduced this increase by 57% (p < 0.05) at 6 h whereas there was no significant effect at 24 h. In the kidneys 2–NP increased the 8–oxodG levels 2.2 and 1.2 times (p<0.05) 6 and 24 h after dose, respectively. Pretreatment with the sprouts extract abolished these increases (p<0.05). Similarly, in the bone marrow the extract protected completely (p < 0.05) against a 4.9–fold 2-NP induced increase (p < 0.05) in the 8-oxodG level.These findings demonstrate that cooked Brussels sprouts contain bioactive substance(s) with a potential for reducing the physiological as well as oxidative stress induced oxidative DNA damage in rats. This could explain the suggested cancer preventive effect of cruciferous vegetables. The correspondence between the urinary excretion and 8–oxodG levels in 2–NP target organs supports its being the main repair product that reflects the rate of guanine oxidation in DNA.

Cancer risk and oxidative DNA damage in man.

In living cells reactive oxygen species (ROS) are formed continuously as a consequence of metabolic and other biochemical reactions as well as external factors. Some ROS have important physiological functions. Thus, antioxidant defense systems cannot provide complete protection from noxious effects of ROS. These include oxidative damage to DNA, which experimental studies in animals and in vitro have suggested are an important factor in carcinogenesis. Despite extensive repair oxidatively modified DNA is abundant in human tissues, in particular in tumors, i.e., in terms of 1-200 modified nucleosides per 10(5) intact nucleosides. The damaged nucleosides accumulate with age in both nuclear and mitochondrial DNA. The products of repair of these lesions are excreted into the urine in amounts corresponding to a damage rate of up to 10(4) modifications in each cell every day. The most abundant of these lesions, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), is also the most mutagenic, resulting in GT transversions which are frequently found in tumor relevant genes. A series of other oxidative modifications of base and sugar residues occur frequently in DNA, but they are less well studied and their biological significance less apparent. The biomarkers for study of oxidative DNA damage in humans include urinary excretion of oxidized nucleosides and bases as repair products and modifications in DNA isolated from target tissue or surrogate cells, such as lymphocytes. These biomarkers reflect the rate of damage and the balance between the damage and repair rate, respectively. By means of biomarkers a number of important factors have been studied in humans. Ionizing radiation, a carcinogenic and pure source of ROS, induced both urinary and leukocyte biomarkers of oxidative DNA damage. Tobacco smoking, another carcinogenic source of ROS, increased the oxidative DNA damage rate by 35-50% estimated from the urinary excretion of 8-oxodG, and the level of 8-oxodG in leukocytes by 20-50%. The main endogenous source of ROS, the oxygen consumption, showed a close correlation with the 8-oxodG excretion rate although moderate exercise appeared to have no immediate effect. So far, cross-sectional study of diet composition and intervention studies, including energy restriction and antioxidant supplements, have generally failed to show an influence on the oxidative DNA modification. However, a diet rich of Brussels sprouts reduced the oxidative DNA damage rate, estimated by the urinary excretion of 8-oxodG, and the intake of vitamin C was a determinant for the level of 8-oxodG in sperm DNA. A low-fat diet reduced another marker of oxidative DNA damage in leukocytes. In patients with diseases associated with a mechanistically based increased risk of cancer, including Fanconi anemia, chronic hepatitis, cystic fibrosis, and various autoimmune diseases, the biomarker studies indicate an increased rate of oxidative DNA damage or in some instances deficient repair. Human studies support the experimentally based notion of oxidative DNA damage as an important mutagenic and apparently carcinogenic factor. However, the proof of a causal relationship in humans is still lacking. This could possibly be supported by demonstration of the rate of oxidative DNA damage as an independent risk factor for cancer in a prospective study of biobank material using a nested case control design. In addition, oxidative damage may be important for the aging process, particularly with respect to mitochondrial DNA and the pathogenesis of inflammatory diseases.

Oxidative DNA damage correlates with oxygen consumption in humans.

Generation of reactive oxygen species from mitochondrial respiration has been proposed as an important determinant of longevity and cumulative cancer risk. Interspecies correlations and animal calorie restriction studies of metabolic rate and oxidative DNA damage support this notion. In the present study we have demonstrated a close association between oxidative DNA damage as assessed by the urinary excretion of 8-oxo-7,8-dihdro-2'-deoxyguanosine (8-oxodG) and oxygen consumption in 33 healthy premenopausal women (r = 0.64; p = 0.00007). In the 12 women who smoked, 8-oxodG excretion was increased by 35%, although oxygen consumption increased only 10% compared with the 21 nonsmoking women. Apparently, the rate of oxidative DNA damage relates to mitochondrial respiration in humans and is aggravated by smoking.

2] Assays of oxidative DNA damage biomarkers 8-oxo-2′-deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by high-performance liquid chromatography with electrochemical detection

High-performance liquid chromatography with electrochemical detection is a highly sensitive and selective method for detecting oxo8dG and oxo8Gua, biomarkers of oxidative DNA damage. When employed together with the DNA isolation and monoclonal antibody-based immunoaffinity purification methods described, oxo8dG and oxo8Gua in DNA and urine can be readily detected and quantitated, offering a powerful approach for assessing oxidative DNA damage in vivo. Application of the technique to the detection of oxo8dG from DNA permits quantitation of the steady-state levels of this oxidatively modified deoxynucleoside and overcomes the detection problems associated with the extremely low levels present in DNA. In addition, the selectivity gained by this detection method eliminates the problem of separating the signal for oxo8dG from those of normal deoxynucleosides. The quantitation of oxo8dG and oxo8Gua in biological fluids is noninvasive and complements the measurement of oxo8dG in DNA by estimating the rate of oxidative DNA damage occurring within the body or in a population of cells. This analytical approach may allow one to estimate oxidative DNA damage in an animal or individual exposed to prooxidant conditions associated with lifestyle, genetic predisposition, degenerative diseases, or environmental toxins. Furthermore, these assays may allow one to assess the potentially beneficial effects of intervention strategies that protect DNA from such damage.

8-Hydroxydeoxyguanosine as a urinary biomarker of oxidative DNA damage.

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)

Formation of DNA adducts and oxidative DNA damage in rats treated with 1,6-dinitropyrene.

In vitro metabolism studies have indicated that the tumorigenic environmental pollutant 1,6-dinitropyrene has the potential to bind covalently to DNA and to induce oxidative DNA damage. We have determined if 1,6-dinitropyrene treatment will cause both types of DNA damage in vivo. Female Sprague-Dawley rats were given a single intraperitoneal injection of 1,6-dinitropyrene, and covalent DNA adduct formation, as indicated by the presence of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene, and oxidative DNA damage, as indicated by increases in 5-hydroxymethyl-2'-deoxyuridine and 8-hydroxy-2'-deoxyguanosine, were assessed at 3, 12, 24 and 48 h after dosing. 32P-postlabeling analyses of DNA isolated from liver, mammary gland, bladder and nucleated blood cells indicated the formation of N-(deoxy-guanosin-8-yl)-1-amino-6-nitropyrene, with the levels being highest in the bladder. 5-hydroxymethyl-2'-deoxyuridine was detected in DNA from each of these tissues, and the levels of this oxidized nucleoside were higher in the mammary glands and livers of 1,6-dinitropyrene-treated rats. 1,6-Dinitropyrene dosing did not affect the levels of 8-hydroxy-2'-deoxyguanosine in these two tissues. These results indicate that exposure to 1,6-dinitropyrene can result in increased levels of 5-hydroxymethyl-2'-deoxyuridine in addition to covalent DNA adduct formation.

The rate of DNA damage and aging.

A new theory of aging based on the rate of DNA damage is presented, and the relationship between the rate of oxidative DNA damage and maximum life span (MLS) of mammalian species is explored. In humans the level of oxidative DNA damage, as measured by urinary biomarkers, can be modulated by caloric restriction and dietary composition. Consequently, longevity may depend not only on the basal metabolic rate but also on dietary caloric intake and the type of diet. The theory may provide the basis for a practical approach for reduction of degenerative diseases in general, extension of life expectancy, and optimization of individual lifestyles.

Ascorbic acid protects against endogenous oxidative DNA damage in human sperm.

Damage to the DNA of germ cells can lead to mutation, which may result in birth defects, genetic diseases, and cancer. The very high endogenous rate of oxidative DNA damage and the importance of dietary ascorbic acid (AA) in preventing this damage has prompted an examination of these factors in human sperm DNA. The oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (8-oxo-7,8-dihydro-2'-deoxyguanosine; oxo8dG), 1 of approximately 20 major products of oxidative damage to DNA, was measured in DNA isolated from human sperm provided by healthy subjects and compared to the seminal fluid AA levels. This relationship was studied in two groups. In a group of 24 free-living individuals 20-50 years old high levels of oxo8dG were correlated with low seminal plasma AA. The endogenous level of oxo8dG in this group was 13 fmol per microgram of DNA or approximately 25,000 adducts per sperm cell. The second group of individuals was maintained on a controlled diet that varied only in AA content. When dietary AA was decreased from 250 to 5 mg/day, the seminal fluid AA decreased by half and the level of oxo8dG in sperm DNA increased 91%. Repletion of dietary AA for 28 days (from 5 mg/day to 250 or 60 mg/day) caused a doubling in seminal fluid AA and reduced oxo8dG by 36%. These results indicate that dietary AA protects human sperm from endogenous oxidative DNA damage that could affect sperm quality and increase risk of genetic defects, particularly in populations with low AA such as smokers.

Oxidative damage to DNA during aging: 8-hydroxy-2'-deoxyguanosine in rat organ DNA and urine.

Oxidative damage to DNA is shown to be extensive and could be a major cause of the physiological changes associated with aging and the degenerative diseases related to aging such as cancer. The oxidized nucleoside, 8-hydroxy-2'-deoxyguanosine (oh8dG), one of the approximately 20 known oxidative DNA damage products, has been measured in DNA isolated from various organs of Fischer 344 rats of different ages. oh8dG was present in the DNA isolated from all the organs studied: liver, brain, kidney, intestine, and testes. Steady-state levels of oh8dG ranged from 8 to 73 residues per 10(6) deoxyguanosine residues or 0.2-2.0 x 10(5) residues per cell. Levels of oh8dG in DNA increased with age in liver, kidney, and intestine but remained unchanged in brain and testes. The urinary excretion of oh8dG, which presumably reflects its repair from DNA by nuclease activity, decreased with age from 481 to 165 pmol per kg of body weight per day for urine obtained from 2-month- and 25-month-old rats, respectively. 8-Hydroxyguanine, the proposed repair product of a glycosylase activity, was also assayed in the urine. We estimate approximately 9 x 10(4) oxidative hits to DNA per cell per day in the rat. The results suggest that the age-dependent accumulation of oh8dG residues observed in DNA from liver, kidney, and intestine is principally due to the slow loss of DNA nuclease activity; however, an increase in the rate of oxidative DNA damage cannot be ruled out.

Mutagenesis and carcinogenesis: endogenous and exogenous factors.

The understanding of mutagenesis and its relation to carcinogenesis and aging is developing rapidly. A number of new findings are relevant to our understanding and are discussed: 1) The endogenous rate of oxidative DNA damage is estimated to be 10(4) hits/cell/day in humans and an order of magnitude higher in rodents. 2) The induction of cell proliferation may be a critical factor in both human cancer and the cancer caused by the maximum tolerated dose (MTD) of chemicals in rodents. 3) About half of all chemicals tested, whether synthetic or natural, are carcinogens, including the group of nature's pesticides, the main (greater than 99%) toxic chemicals in our diet. It is not clear how much, if any, of this high percentage of carcinogens is due to bias in selection of chemicals. A reasonable explanation for a very high percentage of all chemicals being carcinogenic at the MTD is that the MTD causes cell proliferation and inflammation, risk factors for cancer. 4) In the evolutionary war between plants and animals, animals have developed layers of general defenses, almost all inducible, against a world of natural toxic chemicals. This means we are well buffered against toxicity at low doses from both man-made and natural chemicals. Thus, low doses of carcinogens appear to be both much more common and less hazardous than is generally thought.