Relationship between antioxidant potential and oxidative damage to lipids, proteins and DNA in aged rats

Oxidative stress has been implicated to play a major role in aging and age-related diseases. In the present study, we investigated the effects of aging on the total antioxidant capacity, uric acid, lipid peroxidation, total sulfhydryl group content and damage to DNA in adult (6 months), old (15 months) and senescent (26 months) male Wistar rats. The antioxidant capacity, determined by phycoerythrin-based TRAP method (total peroxyl radical-trapping potential) was significantly decreased in the plasma and myocardium of old and senescent rats, whereas plasma level of uric acid was elevated in 26-month-old rats. Age-related decline in plasma and heart antioxidant capacity was accompanied by a significant loss in total sulfhydryl group content, increased lipid peroxidation and higher DNA damage in lymphocytes. Correlations between TRAP and oxidative damage to lipids, proteins and DNA suggest that the decline in antioxidant status may play an important role in age-related accumulation of cell damage caused by reactive oxygen species.     

Losartan reduces oxidative damage to renal DNA and conserves plasma antioxidant capacity in diabetic rats

Increased reactive oxygen species (ROS) levels produced by hyperglycemia and angiotensin-II (AT-II) are considered among the pathogenic factors in the malignant transformation of diabetic renal cells. We aimed to investigate the potential role of AT-II in the increased cancer risk seen in diabetes; measuring oxidative damage to renal DNA and protective antioxidant defenses, including adiponectin (Adp) and plasma antioxidant capacity by the Ferric Reducing Ability of Plasma (FRAP) method. In the kidney of streptozotocin (STZ)-induced (55 mg/kg) diabetic rats either treated or not treated for 3 weeks with losartan, an AT-II type 1 receptor antagonist (20 mg/kg/day); we measured 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) levels, as an index of oxidative DNA damage, circulating Adp and FRAP. Diabetic rats showed significantly higher 8-oxodGuo levels in renal DNA (8.48 ± 0.98 × 10⁻⁶ dG, mean ± SEM n = 11) than normoglycemic ones (1.18 ± 0.04 × 10⁻⁶ dG, mean ± SEM, n=7) and lower plasma Adp and FRAP levels in comparison to normoglycemics. The treatment of diabetic rats with losartan significantly (P < 0.01) reduced 8-oxodGuo levels (5.4 ± 0.58 × 10⁻⁶ dG, mean ± SEM n=9) in renal DNA and conserved FRAP values. Moreover, an inverse correlation was found between 8-oxodGuo in kidney DNA and circulating Adp levels in normoglycemic and diabetic rats. Losartan treatment preserves FRAP levels, reduces DNA oxidative injury and thus the carcinogenesis risk. Furthermore, our results indicate that Adp plasma levels are a further marker of oxidative injury to the kidney and confirm that it is an important part of the plasma antioxidant defense.     

Different onsets of oxidative damage to DNA and lipids in bone marrow and liver in rats given total body irradiation.

We examined time-dependent changes in antioxidant vitamins and oxidative damage to DNA and lipids in the bone marrow, liver, and plasma of rats given total body irradiation (TBI) with X-rays at 3 Gy. The oxidative damage to DNA and lipids was evaluated by measuring increases of 8-hydroxydeoxyguanosine (8OHdG) in DNA and 4-hydroxy-2-nonenal (HNE), respectively. After the TBI, marked increases in 8OHdG and HNE were detected at 3 to 5 h in the bone marrow, while gradual increases in these parameters were detected after a few days in the liver. These changes in 8OHdG and HNE were well correlated within each tissue. In the bone marrow, levels of both vitamin C and vitamin E were decreased by the TBI; however, the changes in vitamin C were earlier and greater than those in vitamin E. In the liver, the level of vitamin C did not decrease, but that of vitamin E decreased due to the TBI. Changes in HNE, vitamin C, and vitamin E in the plasma were similar to those in the liver. Within each tissue, the time of decrease in antioxidants was almost the same as that of the increase in oxidative damage. An increase in total iron due to the TBI was also detected in these tissues. In particular, the total iron in the bone marrow was markedly increased at a few hours after the TBI, with a slight increase in transferrin and no increase in ferritin. Exposure studies performed on cells or isolated DNA showed that an increase in 8OHdG was detected immediately after irradiation at more than 100 Gy in bone marrow cells and at less than 10 Gy in isolated DNA, suggesting that an increase in 8OHdG is undetectable even in bone marrow immediately after the TBI at 3 Gy. These results indicate that the onset of oxidative damage to DNA and lipids was delayed after TBI at 3 Gy, that it was quite different in the bone marrow and the liver, and that an increase in iron and decrease in antioxidant vitamins were involved in the mechanism of oxidative damage.     

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.     

Modulation of restraint stress induced oxidative changes in rats by antioxidant vitamins

In the present study we examined immobilization stress-induced antioxidant defense changes in rat plasma and also observed the antioxidant effects of pre and post vitamins A, E and C administration (15 mg/Kg of body weight) individually and in combination (vit E + C) on these alterations.Following immobilization stress the circulating activities of superoxide dismutase, catalase and glutathione-S-transferase were decreased, while the level of thiobarbituric acid reactive substances (TBARS) was increased as compared to non-stressed control rats.Post treatment with individual vitamins A, E and C (after exposure to stress) resulted in a less marked alteration of plasma TBARS levels and activities of SOD, GST and catalase as compared to pre vitamin stress or stress alone treatments. Both pre and post vitamin treatments were effective in preventing stress induced derangement of free radical metabolism with a relative dominance by latter. The combined treatment with vitamin E and C did not show any additive antioxidant effect on restraint stress induced altered free radical metabolism, rather a predominant effect similar to vitamin E alone was observed. The prevention of oxidative stress generated in response to restraint stress by the vitamins can be summarized as: vitamin (E + C) i.e. vit E > vit C > vit A, thus combined vitamin (E + C) treatment though showed maximum preventive effect, but was similar to vitamin E treatment alone, in terms of the circulating activities of SOD, GST, catalase and TBARS levels.     

Nitroxides are more efficient inhibitors of oxidative damage to calf skin collagen than antioxidant vitamins

Reactive oxygen species generated upon UV-A exposure appear to play a major role in dermal connective tissue transformations including degradation of skin collagen. Here we investigate on oxidative damage to collagen achieved by exposure to (i) UV-A irradiation and to (ii) AAPH-derived radicals and on its possible prevention using synthetic and natural antioxidants. Oxidative damage was identified through SDS-PAGE, circular dichroism spectroscopy and quantification of protein carbonyl residues. Collagen (2 mg/ml) exposed to UV-A and to AAPH-derived radicals was degraded in a time- and dose-dependent manner. Upon UV-A exposure, maximum damage was observable at 730 kJ/m2 UV-A, found to be equivalent to roughly 2 h of sunshine, while exposure to 5 mM AAPH for 2 h at 50 degrees C lead to maximum collagen degradation. In both cases, dose-dependent protection was achieved by incubation with muM concentrations of nitroxide radicals, where the extent of protection was shown to be dictated by their structural differences whereas the vitamins E and C proved less efficient inhibitors of collagen damage. These results suggest that nitroxide radicals may be able to prevent oxidative injury to dermal tissues in vivo alternatively to commonly used natural antioxidants.     

Metal-mediated oxidative damage to cellular and isolated DNA by gallic acid, a metabolite of antioxidant propyl gallate

Propyl gallate (PG), widely used as an antioxidant in foods, is carcinogenic to mice and rats. PG increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, but not in HP100, which is hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. Although PG induced no or little damage to 32P-5'-end-labeled DNA fragments obtained from genes that are relevant to human cancer, DNA damage was observed with treatment of esterase. HPLC analysis of the products generated from PG incubated with esterase revealed that PG converted into gallic acid (GA). GA induced DNA damage in a dose-dependent manner in the presence of Fe(III)EDTA or Cu(II). In the presence of Fe(III) complex such as Fe(III)EDTA or Fe(III)ADP, GA caused DNA damage at every nucleotide. Fe(III) complex-mediated DNA damage by GA was inhibited by free hydroxy radical (*OH) scavengers, catalase and an iron chelating agent. These results suggested that the Fe(III) complex-mediated DNA damage caused by GA is mainly due to *OH generated via the Fenton reaction. In the presence of Cu(II), DNA damage induced by GA occurred at thymine and cytosine. Although *OH scavengers did not prevent the DNA damage, methional inhibited the DNA damage. Cu(II)-mediated DNA damage was inhibited by catalase and a Cu(I) chelator. These results indicated that reactive oxygen species formed by the interaction of Cu(I) and H2O2 participates in the DNA damage. GA increased 8-oxodG content in calf thymus DNA in the presence of Cu(II), Fe(III)EDTA or Fe(III)ADP. This study suggested that metal-mediated DNA damage caused by GA plays an important role in the carcinogenicity of PG.     

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.     

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.     

Supplementation with antioxidant vitamins prevents oxidative modification of DNA in lymphocytes of HIV-infected patients

There is evidence suggesting that patients infected with human immunodeficiency virus (HIV) are under chronic oxidative stress. In the present study, the level of oxidatively modified bases in lymphocyte DNA and some other parameters of oxidative stress were measured in HIV-infected patients (n = 30), as well as in control groups (10 healthy volunteers and 15 HIV-seronegative injected drug users). Additional experiments were conducted using lymphocyte DNA samples from asymptomatic seropositive, HIV-infected patients who were supplemented with antioxidant vitamins A, C, and E or received placebo. Significant increases in the amount of the modified DNA bases were observed in HIV-infected patients when compared with the control group. The concentration of thiobarbituric acid reactive substances (TBARS) was higher and activities of antioxidant enzymes (superoxide dismutase and catalase) were lower in the group of HIV-infected patients in comparison to the control group. Vitamin supplementation resulted in the significant decrease in the levels of all modified DNA bases when compared to the patients who received placebo. The reduction of TBARS and the restoration of the activity of the enzymes were also observed. Our data suggest that people infected with HIV can benefit from treatment with antioxidant vitamins.     

Role of inducible nitrogen oxide synthase in benzene-induced oxidative DNA damage in the bone marrow of mice

We investigated the interaction of BZ and lipolysaccharide (LPS), a well-known inflammation-promoting agent, in wild-type and inducible nitrogen oxide synthase (iNOS) knockout mice. BZ generated DNA strand breaks (SB) in the liver of both wild-type and iNOS-deficient mice. In the bone marrow (BM) BZ and LPS generated SB only in wild-type mice. The effects were additive, suggesting that both a redox cycling and an iNOS-dependent pathway may be involved. Formamidopyrimidine DNA glycosylase sensitive sites were elevated by BZ in the BM in both types of mice, whereas endonuclease III sensitive sites were not affected by any treatment. Since BZ is associated with leukemia in humans, it suggests that oxidative DNA base damage rather than SB may be important in the development of leukemia.     

Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats

Acrylamide (ACR), used in many fields from industrial manufacturing to laboratory personnel work is also formed during the heating process through interactions of amino acids. Therefore ACR poses a significant risk to human health. This study aimed to elucidate whether resveratrol (RVT) treatment could modulate ACR-induced oxidative DNA damage and oxidative changes in rat brain, lung, liver, kidney and testes tissues. Rats were divided into four groups as control (C); RVT (30 mg/kg i.p. dissolved in 0.9% NaCl), ACR (40 mg/kg i.p.) and RVT + ACR groups. After 10 days rats were decapitated and tissues were excised. 8-hydroxydeoxyguanosine (8-OHdG) is a biomarker of oxidative DNA damage. 8-OHdG content in the extracted DNA solution was determined by enzyme-linked immunosorbent assay method. Malondialdehyde (MDA), glutathione (GSH) levels and myeloperoxidase activity (MPO) were determined in tissues, while oxidant-induced tissue fibrosis was determined by collagen contents. Serum enzyme activities, cytokine levels, leukocyte apoptosis were assayed in plasma. As an indicator of oxidative DNA damage, 8-OHdG levels significantly increased in ACR group and this was reversed significantly by RVT treatment. In ACR group, GSH levels decreased significantly while the MDA levels, MPO activity and collagen content increased in the tissues suggesting oxidative organ damage. In RVT-treated ACR group, oxidant responses reversed significantly. Serum enzyme activities, cytokine levels and leukocyte late apoptosis which increased following ACR administration, decreased with RVT treatment. Therefore supplementing with RVT can be useful in individuals at risk of ACR toxicity.     

Evaluation of oxidative DNA damage and antioxidant defense in patients with nasal polyps

Abstract

Objectives

The presence of inflammatory cells indicates the development of epithelial cell injury in nasal polyposis (NP) and the potential for production of high levels of reactive oxygen and nitrogen species. The aim of our study was to clarify the role of oxidative stress and antioxidant status in the deterioration accompanying NP.

Methods

Twenty patients (11 men) aged 47.2 ± 17.0 years with nasal polyps were included in the study. Twenty healthy subjects (7 men) aged 48.2 ± 15.3 years formed the control group. The erythrocyte activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and plasma nitric oxide (NO) concentrations were measured. An alkaline comet assay was used to determine the extent of blood lymphocyte DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites.

Results

A significant increase of NO (P < 0.05) and non-significant decreases of SOD (P > 0.05), CAT (P > 0.05), and GPx (P > 0.05) were seen in NP patients compared to healthy controls. The level of blood lymphocyte oxidative DNA damage in NP patients was significantly higher compared to the control group (P = 0.01).

Discussion

The blood lymphocyte DNA damage level increased in patients with NP. Elevated DNA damage may be related to overproduction of reactive oxygen and nitrogen species and/or decreased antioxidant protection.     

Intrauterine exposure to flavonoids modifies antioxidant status at adulthood and decreases oxidative stress-induced DNA damage

Maternal intake of flavonoids, known for their antioxidant properties, may affect the offspring's susceptibility to developing chronic diseases at adult age, especially those related to oxidative stress, via developmental programming. Therefore, we supplemented female mice with the flavonoids genistein and quercetin during gestation, to study their effect on the antioxidant capacity of lung and liver of adult offspring. Maternal intake of quercetin increased the expression of Nrf2 and Sod2 in fetal liver at gestational day 14.5. At adult age, in utero exposure to both flavonoids resulted in the increased expression of several enzymatic antioxidant genes, which was more pronounced in the liver than in the adult lung. Moreover, prenatal genistein exposure induced the nonenzymatic antioxidant capacity in the adult lung, partly by increasing glutathione levels. Prenatal exposure to both flavonoids resulted in significantly lower levels of oxidative stress-induced DNA damage in liver only. Our observations lead to the hypothesis that a preemptive trigger of the antioxidant defense system in utero had a persistent effect on antioxidant capacity and as a result decreased oxidative stress-induced DNA damage in the liver.     

Metformin does not prevent DNA damage in lymphocytes despite its antioxidant properties against cumene hydroperoxide-induced oxidative stress

Metformin (1-(diaminomethylidene)-3,3-dimethyl-guanidine), which is the most commonly prescribed oral antihyperglycaemic drug in the world, was reported to have several antioxidant properties such as the inhibition of advanced glycation end-products. In addition to its use in the treatment of diabetes, it has been suggested that metformin may be a promising anti-aging agent. The present work was aimed at assessing the possible protective effects of metformin against DNA-damage induction by oxidative stress in vitro. The effects of metformin were compared with those of N-acetylcysteine (NAC). For this purpose, peripheral blood lymphocytes from aged (n = 10) and young (n = 10) individuals were pre-incubated with various concentrations of metformin (10–50 μM), followed by incubation with 15 μM cumene hydroperoxide (CumOOH) for 48 h, under conditions of low oxidant level, which do not induce cell death. Protection against oxidative DNA damage was evaluated by use of the Comet assay and the cytokinesis-block micronucleus technique. Changes in the levels of malondialdehyde + 4-hydroxy-alkenals, an index of oxidative stress, were also measured in lymphocytes. At concentrations ranging from 10 μM to 50 μM, metformin did not protect the lymphocytes from DNA damage, while 50 μM NAC possessed an effective protective effect against CumOOH-induced DNA damage. Furthermore, NAC, but not metformin, inhibited DNA fragmentation induced by CumOOH. In contrast to the lack of protection against oxidative damage in lymphocyte cultures, metformin significantly protected the cells from lipid peroxidation in both age groups, although not as effective as NAC in preventing the peroxidative damage at the highest doses. Within the limitations of this study, the results indicate that pharmacological concentrations of metformin are unable to protect against DNA damage induced by a pro-oxidant stimulus in cultured human lymphocytes, despite its antioxidant properties.     

Testicular oxidative damage and role of combined antioxidant supplementation in experimental diabetic rats

The present study was designated to assess oxidative damage and its effect on germ cell apoptosis in testes of streptozotocin (STZ)-induced diabetic rats. The role of antioxidant supplementation with a mixture of vitamins E and C and alpha lipoic acid for protection against such damage was also evaluated. Forty-five adult male rats were randomly divided into three groups: group I, control, non-diabetic rats; group II, STZ-induced, untreated diabetic rats; group III, STZ-induced diabetic rats supplemented with a mixture of vitamins E and C and alpha lipoic acid. Glycated hemoglobin (HbA_1C), glucose, and insulin levels were estimated in blood samples. Malondialdehyde (MDA), the activities of the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and caspase-3 in addition to testosterone (T) level were all determined in testicular tissues. Histopathological studies using H&E stain, as well as, immunohistochemical detection of apoptosis using (TUNEL) method were also performed. Blood glucose and HbA_1c were significantly increased while insulin was significantly decreased in STZ-induced diabetic rats as compared with controls. In rat testicular tissues, MDA, and caspase-3 activity were significantly elevated while SOD and GPx enzymatic activities as well as T level were significantly decreased in diabetic rats as compared with control group. Antioxidant supplementation to diabetic rats restored the testicular enzymatic activities of SOD and GPx to almost control levels, in addition, MDA and caspase-3 activity decrease while T increase significantly as compared with untreated diabetic group. Prominent reduction of germ cell apoptosis was found in diabetic rats supplemented with antioxidants. An important role of testicular oxidative damage and germ cell apoptosis in diabetes-induced infertility could be suggested, treatment with antioxidants has a protective effect by restoring SOD and GPx antioxidant enzymatic activity.     

Measuring oxidative damage to DNA and its repair with the comet assay

Background

Single cell gel electrophoresis, or the comet assay, was devised as a sensitive method for detecting DNA strand breaks, at the level of individual cells. A simple modification, incorporating a digestion of DNA with a lesion-specific endonuclease, makes it possible to measure oxidised bases.

Scope of review

With the inclusion of formamidopyrimidine DNA glycosylase to recognise oxidised purines, or Nth (endonuclease III) to detect oxidised pyrimidines, the comet assay has been used extensively in human biomonitoring to monitor oxidative stress, usually in peripheral blood mononuclear cells.

Major conclusions

There is evidence to suggest that the enzymic approach is more accurate than chromatographic methods, when applied to low background levels of base oxidation. However, there are potential problems of over-estimation (because the enzymes are not completely specific) or under-estimation (failure to detect lesions that are close together). Attempts have been made to improve the inter-laboratory reproducibility of the comet assay.

General significance

In addition to measuring DNA damage, the assay can be used to monitor the cellular or in vitro repair of strand breaks or oxidised bases. It also has applications in assessing the antioxidant status of cells. In its various forms, the comet assay is now an invaluable tool in human biomonitoring and genotoxicity testing. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Genotoxic risk and oxidative DNA damage in HepG2 cells exposed to perfluorooctanoic acid

Perfluorooctanoic acid (C8HF15O2, PFOA) is widely used in various industrial fields for decades and it is environmentally bioaccumulative. PFOA is known as a potent hepatocarcinogen in rodents. But it is not yet clear whether it is also carcinogenic in humans, and the genotoxic effects of PFOA on human cells have not yet been examined. In this study, the genotoxic potential of PFOA was investigated in human hepatoma HepG2 cells in culture using single cell gel electrophoresis (SCGE) assay and micronucleus (MN) assay. In order to clarify the underlying mechanism(s) we measured the intracellular generation of reactive oxygen species (ROS) using dichlorofluorescein diacetate as a fluorochrome. The level of oxidative DNA damage was evaluated by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG) in PFOA-treated HepG2 cells. PFOA at 50-400 microM caused DNA strand breaks and at 100-400 microM MN in HepG2 cells both in a dose-dependent manner. Significantly increased levels of ROS and 8-OHdG were observed in these cells. We conclude that PFOA exerts genotoxic effects on HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS.