Increase In 8-OHdG Levels Research Articles

Non-genotoxic mode of action and possible threshold for hepatocarcinogenicity of Kojic acid in F344 rats

Kojic acid (KA), a naturally occurring compound, is contained in traditional Japanese fermented foods and is used as a food additive, preservative and a dermatological skin-lightening agent. In the present experiment, initiation (experiment 1) and promotion (experiment 2) effects of KA-induced hepatocarcinogenesis were studied by rat medium-term bioassay for carcinogenicity. Male F344 rats were administered a diet containing 0–2% KA. Experiment 1 demonstrated that KA had no effect on induction of liver preneoplastic lesions or glutathione S-transferase placental form (GST-P) positive foci, in either number or area. In experiment 2, 2% KA treatment significantly increased the number and area of GST-P positive foci, but concentrations less than 0.5% did not. Moreover, 2% KA treatment significantly increased 8-OHdG levels and PCNA positive hepatocytes. The results indicated that low concentrations of KA do not have initiation effects on rat hepatocarcinogenesis, while higher concentrations of KA do promote hepatocarcinogenesis in rats. Thus, the results indicate that KA is a non-genotoxic hepatocarcinogen, showing the possible existence of a perfect threshold.

Oxidative DNA damage and reporter gene mutation in the livers of gpt delta rats given non-genotoxic hepatocarcinogens with cytochrome P450-inducible potency

Previous reports have proposed that reactive oxygen species resulting from induction of cytochrome P450 (CYP) isozymes might be involved in the modes of action of hepatocarcinogens with CYP-inducible potency. In the present study, we investigated 8-hydroxydeoxyguanosine (8-OHdG) levels, in vivo mutagenicity and glutathione S-transferase placental form (GST-P)-positive foci in the livers of gpt delta rats treated with piperonyl butoxide (PBO) or phenobarbital (PhB) for 4 and 13 weeks. Significant elevations in Cyp 1A1 and Cyp 1A2 mRNA levels after PBO treatment, and in Cyp 2B1 mRNA levels after PBO or PhB treatment, appeared together with remarkable hepatomegaly through the experimental period. Time-dependent and statistically significant increases in 8-OHdG levels were observed in the PBO treatment group along with significant increases in proliferating cell nuclear antigen (PCNA)-positive hepatocytes at 4 weeks, while no increase in 8-OHdG levels was found in PhB-treated rats. No changes in mutant frequencies of gpt and red/gam (Spi(-)) genes in liver DNA from PBO- or PhB-treated rats were observed at 4 or 13 weeks. A 13-week exposure to either PBO or PhB did not affect the number and area of GST-P-positive hepatocytes. CYP 1A1 and 1A2 induction may be responsible for elevated levels of 8-OHdG in PBO-treated rats. However, neither GC:TA transversions nor deletion mutations, typically regarded as 8-OHdG-related mutations, were observed in any of the treated rats. We conclude that reactive oxygen species, possibly produced through CYP catalytic pathways, likely induced genomic DNA damage but did not give rise to permanent gene mutation.

Oxidative DNA damage: the thyroid hormone-mediated effects of insulin on liver tissue

Thyroid hormone affects glucose homeostasis with its actions between the skeletal muscle and liver and the altered oxidative and non-oxidative glucose metabolism. In our study three chemicals are considered biomarkers associated with oxidative stress for protein modifications were measured; 8-hydroxy-2-deoxyyguanosine (8-OHdG), a major lesion that can be generated by reactive oxygen species for DNA damage, protein carbonyl content (PCO), products of protein oxidation and advanced oxidation protein products (AOPPs) a dithyrosine containing cross-linked protein products. The purpose of the recent study was to determine the effects of insulin and T4 or their combination in diabetic, thyroidectomized, or diabetic-thyroidectomized rats and possible relations with oxidative DNA and protein damages. For this purpose, rats were assigned to eight groups: Group 1; control, Group 2; diabetes, Group 3; diabetes+insulin, Group 4; surgically thyroidectomized control, Group 5; thyroidectomized+diabetes, Group 6; thyroidectomized+diabetes+insulin, Group 7; thyroidectomized+diabetes+insulin+thyroid hormone, levothyroxin sodium, 2.5 μg/kg and Group 8; thyroidectomized+diabetes+insulin+thyroid hormone, levothyroxin sodium, 5.0 μg/kg for 5 weeks. After the genomic DNA of liver tissues was extracted, the ratio of 8-OHdG to deoxyguanosine and liver tissue protein oxidation markers was determined. The main findings of our recent study were the increased 8-OHdG levels during the diabetes, hypothyroidism, and hypothyroidism with diabetes, which can be regulated in different percentages with the treatment of 2.5 and 5.0 μg/kg doses of thyroid hormone and the altered protein carbonyl and AOPP levels of liver tissue. Consequently, it was observed that the DNA and protein damage induced by oxidative stress in diabetes could be regulated by dose-dependent thyroid hormone-mediated effects to insulin treatment.

Abstract 4903: Oxidative DNA damage and its relationship with GSTP1 methylation in prostate cancer

Abstract Prostate cancer is the most common cancer diagnosed in men and is the second leading cause of cancer-related death in the United States. The risk factor for prostate cancer includes both genetic and epigenetic modifications. Growing evidence suggests that epigenetic mechanisms such as DNA methylation affect gene expression in tissue-specific manner. The most frequent epigenetic alteration observed in human prostate cancer is hypermethylation of gluthathione-S-transferase-pi class (GSTP1) in the promoter region which causes loss or its decrease expression. Inactivation of GSTP1 renders prostate epithelial cells vulnerable to oxidative DNA damage from both oxidants and electrophile carcinogens to acquire relevant genetic alteration and neoplastic transformation. Extensive observations suggest that reactive oxygen species may cause accumulated DNA damage which might contribute to increased incidence of prostate cancer. However, the role of GSTP1 and its correlation with DNA damage has not been studied. Based on the assumption that DNA damage is prevalent in cancer cells compared to nontumorous tissue, we took a panel of 24 human prostate specimens obtained after surgery which comprised of matched primary tumor and adjacent nontumorous (benign) prostate tissues. Oxidative DNA damage was measured by 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels through highly sensitive and quantitative ELISA assay. GSTP1 methylation was determined by methyl-specific PCR and 5-methylcytosine estimation was performed by HPLC analysis. The 8-OHdG levels were higher in cancer specimen compared to their benign counterparts. The 8-OHdG levels in DNA of prostate cancer tissues ranged from 0.151 to 0.616ng/μg DNA, whereas the levels in benign tissue range from 0.149 to 0.318ng/μg DNA, respectively. The increase in 8-OHdG levels correlate with GSTP1 methylation which was higher in cancer specimens compared to the benign counterparts. Furthermore, levels of 5-methylcytosine were higher in tumor specimens compared to benign tissue. Taken together, our studies suggest that there is a link between GSTP1 methylation and DNA damage which correlates to epigenetic modification in prostate cancer. Our results are in agreement with the prediction that positive relationship occur between GSTP1 inactivation and DNA damage in prostate cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4903.

Genomic instability in quartz dust exposed rat lungs: Is inflammation responsible?

Exposure to quartz dusts has been associated with lung cancer and fibrosis. Although the responsible mechanisms are not completely understood, progressive inflammation with associated induction of persistent oxidative stress has been discussed as a key event for these diseases. Previously we have evaluated the kinetics of pulmonary inflammation in the rat model following a single intratracheal instillation of 2mg DQ12 quartz, either in its native form or upon its surface modification with polyvinylpyridine-N-oxide or aluminium lactate. This model has been applied now to evaluate the role of inflammation in the kinetics of induction of DNA damage and response at 3, 7, 28, and 90 days after treatment. Bronchoalveolar lavage (BAL) cell counts and differentials as well as BAL fluid myeloperoxidase activity were used as markers of inflammation. Whole lung homogenate was investigated to determine the induction of the oxidative and pre-mutagenic DNA lesion 8-hydroxy-2-deoxy-guanosine (8-OHdG) by HPLC/ECD, while mRNA and protein expression of oxidative stress and DNA damage response genes including hemeoxygenase-1 (HO-1) and apurinic/apyrimidinic endonuclease (APE/Ref-1) were evaluated using Western blotting and real time PCR. Isolated lung epithelial cells from the treated rats were used for DNA strand breakage analysis using the alkaline comet assay as well as for micronucleus scoring in May-Gruenwald-Giemsa stained cytospin preparations. In the rats that were treated with quartz, no increased 8-OHdG levels were observed, despite the presence of a marked and persistent inflammation. However, DNA strand breakage in the lung epithelial cells of the quartz treated rats was significantly enhanced at 3 days, but not at 28 days. Moreover, significantly enhanced micronucleus frequencies were observed for all four time points investigated. In the animals that were treated with the PVNO modified quartz, micronuclei scores did not differ from controls, while in those treated with the aluminium coated quartz intermediate effects were found. These findings were in line with the kinetics of inflammation and epithelial proliferation in the rat lungs for the different treatments. Notably, a highly significant correlation was observed between neutrophil numbers and micronucleus frequencies, indicative for a role of inflammation in eliciting genomic instability in target cells of quartz-induced carcinogenesis. Our ongoing investigations focus on the evaluation of the causality between both in relation to quartz exposure.

Effect of a Hemodiafiltration Session with On-Line Regeneration of the Ultrafiltrate on Oxidative Stress

Background: The influence of a dialysis session using hemodiafiltration with on-line regeneration of the ultrafiltrate (HFR) is compared with that of a conventional hemodialysis session with polysulfone (HD-PS) and with a group of healthy individuals. Methods: Total antioxidant capacity (TAC), antioxidants, i.e., superoxide dismutase (SOD) glutathione peroxidase (GPX), reduced glutathione (GSH) and catalase, and biomarkers of oxidative stress were evaluated in plasma, whole blood and lymphocytes. Results: The study showed decreased plasma TAC, decreased activities of antioxidant enzymes and decreased GSH levels along with increased thiobarbituric-acid-reactive substances and 8–hydroxy-2-deoxyguanosine (8-OHdG) levels in lymphocytes. The antioxidants and biomarkers of lipid and protein damage after dialysis sessions with HFR were similar or better than following HD-PS. Thus, the blood GPX activity was comparable to that of healthy controls and significantly better than following HD-PS. An increase in 8-OHdG levels was observed in the HD-PS group after dialysis but not in the HFR group. Conclusions: These results show that HFR induces less oxidative stress than HD-PS.

Abrin induced oxidative stress mediated DNA damage in human leukemic cells and its reversal by N-acetylcysteine

Abrin is a plant glycoprotein toxin, classified as ribosome inactivating protein (RIP) due to its property of damaging ribosomes in an irreversible manner. Many RIPs have direct DNA damaging activity. The objective of the present study was to evaluate the oxidative stress and DNA damaging potential of abrin in U937 human myeloleukemic cells. Cells were treated with abrin at IC50 of 8 ng/ml for 24h. Abrin induced a time dependent increase in reactive oxygen species and levels of antioxidant enzymes. There was significant depletion of reduced glutathione levels. DNA damage was assessed by comet assay in terms of percent head DNA, tail DNA, tail length and Olive tail moment. DNA damage was more pronounced at 4 and 8h at IC50 concentration. Abrin at 4, 8, 16 and 32 ng/ml concentration induced significant DNA damage at 4h. There was time dependent increase in levels of 8-OHdG in abrin treated cells indicating the oxidative stress mediated DNA damage. N-Acetylcysteine pretreatment at 10nM for 1h, considerably reversed the abrin induced DNA damage at 16 and 32 ng/ml. Our results clearly show oxidative DNA damage potential of abrin at low concentration.

Oxidative DNA damage and its repair in rat spleen following subchronic exposure to aniline

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Splenotoxicity of aniline is associated with iron overload and generation of reactive oxygen species (ROS) which can cause oxidative damage to DNA, proteins and lipids (oxidative stress). 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the most abundant oxidative DNA lesions resulting from ROS, and 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase/lyase enzyme, plays a key role in the removal of 8-OHdG adducts. This study focused on examining DNA damage (8-OHdG) and repair (OGG1) in the spleen in an experimental condition preceding a tumorigenic response. To achieve that, male Sprague–Dawley rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. Aniline treatment led to a significant increase in splenic oxidative DNA damage, manifested as a 2.8-fold increase in 8-OHdG levels. DNA repair activity, measured as OGG1 base excision repair (BER) activity, increased by ∼ 1.3 fold in the nuclear protein extracts (NE) and ∼ 1.2 fold in the mitochondrial protein extracts (ME) of spleens from aniline-treated rats as compared to the controls. Real-time PCR analysis for OGG1 mRNA expression in the spleen revealed a 2-fold increase in expression in aniline-treated rats than the controls. Likewise, OGG1 protein expression in the NEs of spleens from aniline-treated rats was ∼ 1.5 fold higher, whereas in the MEs it was ∼ 1.3 fold higher than the controls. Aniline treatment also led to stronger immunostaining for both 8-OHdG and OGG1 in the spleens, confined to the red pulp areas. It is thus evident from our studies that aniline-induced oxidative stress is associated with increased oxidative DNA damage. The BER pathway was also activated, but not enough to prevent the accumulation of oxidative DNA damage (8-OHdG). Accumulation of mutagenic oxidative DNA lesions in the spleen following exposure to aniline could play a critical role in the tumorigenic process.

Effects of Angiotensin II Type 1 Receptor Blocker on Albumin-Induced Cell Damage in Human Renal Proximal Tubular Epithelial Cells

Background/Aims: Proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. We investigated the effect of angiotensin II type 1 receptor blockers (ARBs) on albumin-induced cell damage in human renal proximal tubular epithelial cells (RPTEC). Methods: The N-acetyl-β- D-glucosaminidase (NAG) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in the medium after albumin treatment with ARBs were determined by commercially available kits. The levels of p22^phox protein in RPTEC were measured using Western blotting after albumin treatment with ARBs. Angiotensin II concentrations in cell media and cell lysates were assayed with a commercially available kit. Results: Human albumin (0.1–10 mg/ml) dose-dependently increased NAG release and olmesartan or valsartan (10^–9–10^–7 mol/l) showed a significant reduction on albumin (1 mg/ml)-induced NAG release in RPTEC. Albumin treatment (1 mg/ml) showed significant increases in p22^phox protein levels in RPTEC and ARBs significantly decreased albumin-induced p22^phox protein levels. Significant increases in 8-OHdG levels were observed in the albumin (1 mg/ml)-treated group and ARBs markedly reduced albumin-induced 8-OHdG levels in RPTEC. Human albumin dose-dependently increased angiotensin II concentrations in both cell media and lysates. Conclusion: These observations suggest renal tubular cell-protective properties of ARBs related to decreased oxidative stress during proteinuria.

Combined Ascorbic Acid and Sodium Nitrite Treatment Induces Oxidative DNA Damage-Associated Mutagenicity In Vitro, but Lacks Initiation Activity in Rat Forestomach Epithelium

Combination treatment with sodium nitrite (NaNO(2)) and ascorbic acid (AsA) is well known to promote forestomach carcinogenesis in rats and weakly enhance esophageal carcinogenesis under acid reflux conditions. Nitric oxide generation and oxidative DNA damage are considered to be related to the enhancement of carcinogenesis. The purpose of the present study was to investigate whether oxidative DNA damage-associated genotoxicity and tumor initiating potential are involved in the carcinogenesis. In the bacterial reverse mutation assay using Escherichia coli deficient in the mutM gene encoding 8-hydroxydeoxyguanosine (8-OHdG) DNA glycosylase, the combination with NaNO(2) and AsA increased the mutation frequency dramatically, slight increase being evident in the parental strain. In vivo, a significant increase in 8-OHdG levels in the rat forestomach epithelium occurred at 24 h after combined treatment. Six-week-old F344 male rats were given drinking water containing 0.2% NaNO(2) and a diet supplemented with 1% AsA in combination, or the chemicals individually or basal diet alone for 12 weeks. After an interval of 2 weeks, they received 1% butylated hydroxyanisole in the diet for promotion until the end of weeks 52 and 78. Although one squamous cell carcinoma was observed in the combined group, there was no significant variation in tumor development among the groups. The study indicated that the combination of NaNO(2) with AsA induces genotoxicity due to oxidative DNA damage in vitro, and elevates 8-OHdG levels in the forestomach epithelium, but lacks initiating activity in the rat two-stage carcinogenesis model.

Sulfotransferase 1A1 and glutathione S-transferase P1 genetic polymorphisms modulate the levels of urinary 8-hydroxy-2′-deoxyguanosine in betel quid chewers

Betel quid chewing has been associated with several human cancers. However, the role of betel quid in carcinogenesis remains uncertain. Piper betel contains high concentrations of safrole (an inducer of DNA oxidative damage). Safrole may be metabolized by hepatic sulfotransferase 1A1 (SULT1A1), or glutathione S-transferases (GSTM1, GSTT1, and GSTP1). Thus, we investigated the association of genetic polymorphisms of SULT1A1, GSTM1, GSTT1, and GSTP1 with DNA oxidative damage among betel quid chewers. A biomarker for oxidative stress, urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) level, was analyzed using isotope-dilution LC-MS/MS in 64 betel quid chewers and 129 non-betel quid chewers. Data on demographics and habits (smoking, alcohol drinking, and betel quid chewing) were obtained from questionnaires. Our results revealed that urinary 8-OHdG level was higher in chewers with SULT1A1 Arg-His genotype than in chewers with SULT1A1 Arg-Arg genotype. Urinary 8-OHdG level was also higher in chewers with GSTP1 Ile-Ile genotype. Furthermore, the combined effect of SULT1A1 and GSTP1 genotypes on urinary 8-OHdG was evaluated. Non-chewers with both SULT1A1 Arg-Arg and GSTP1 Val-Val/Ile-Val (reference group) had the lowest mean level (3.6 ng/mg creatinine), whereas chewers with either SULT1A1 Arg-His or GSTP1 Ile-Ile had the highest 8-OHdG mean level (6.2 ng/mg creatinine; vs. reference group, P = 0.04). Chewers with both of SULT1A1 Arg-Arg and GSTP1 Val-Val/Ile-Val (4.6 ng/mg creatinine), and non-chewers with either SULT1A1 Arg-His or GSTP1 Ile-Ile (4.7 ng/mg creatinine) had a moderately increased 8-OHdG level. Thus, the susceptible SULT1A1 and GSTP1 genotypes may modulate increased DNA oxidative stress elicited by betel-quid chewing.

Ebselen attenuates oxidative DNA damage and enhances its repair activity in the thalamus after focal cortical infarction in hypertensive rats

Oxidative DNA damage has been proposed to be a major contributor to focal cerebral ischemic injury. However, little is known about the role of oxidative DNA damage in remote damage secondary to the primary infarction. In the present study, we investigated oxidative damage within the ventroposterior nucleus (VPN) after distal middle cerebral artery occlusion (MCAO) in hypertensive rats. We also examined the possible protective effect of ebselen, one glutathione peroxidase mimic, on delayed degeneration in the VPN after distal MCAO. Neuronal damage in the ipsilateral VPN was examined by Nissl staining. Oxidative DNA damage and base repair enzyme activity were assessed by analyzing immunoreactivity of 8-hydroxy-2'-deoxyguanosine (8-ohdG) and 8-oxoguanine DNA glycosylase (OGG1), respectively. The number of intact neurons in the ipsilateral VPN decreased by 52% compared to the contralateral side in ischemia group 2 weeks after distal cerebral cortical infarction. The immunoreactivity of 8-ohdG significantly increased while OGG1 immunoreactivity significantly decreased in the ipsilateral VPN 2 weeks after distal cortical infarction (all p<0.01). Compared with vehicle treatment, ebselen significantly attenuated the neuron loss, ameliorated ischemia-induced increase in 8-ohdG level as well as decrease in OGG1 level within the ipsilateral VPN (all p<0.01). OGG1 was further demonstrated to mainly express in neurons. These findings strongly suggest that oxidative DNA damage may be involved in the delayed neuronal death in the VPN region following distal MCAO. Furthermore, ebselen protects against the delayed damage in the VPN when given at 24 h following distal MCAO.

Detection of oxidative DNA damage, cell proliferation and in vivo mutagenicity induced by dicyclanil, a non-genotoxic carcinogen, using gpt delta mice

To ascertain whether measurement of possible contributing factors to carcinogenesis concurrently with the transgenic mutation assay is useful to understand the mode of action underlying tumorigenesis of non-genotoxic carcinogens, male and female gpt delta mice were given dicyclanil (DC), a mouse hepatocarcinogen showing all negative results in various genotoxicity tests, at a carcinogenic dose for 13 weeks. Together with gpt and Spi − mutations, thiobarbituric acid-reactive substances (TBARS), 8-hydroxydeoxyguanosine (8-OHdG) and bromodeoxyuridine labeling indices (BrdU-LIs) in the livers were examined. Whereas there were no changes in TBARS levels among the groups, significant increases in 8-OHdG levels and centrilobular hepatocyte hypertrophy were observed in the treated mice of both genders. In contrast, BrdU-LIs and liver weights for the treated females, but not the males were significantly higher than those for the controls. Likewise, the gpt mutant frequencies (MFs) in the treated females were significantly elevated, GC:TA transversion mutations being predominant. No significant alterations were found in the gpt MFs of the males and the Spi − MFs of both sexes. The results for the transgenic mutation assays were consistent with DC carcinogenicity in terms of the sex specificity for females. Considering that 8-OHdG induces GC:TA transversion mutations by mispairing with A bases, it is likely that cells with high proliferation rates and a large amounts of 8-OHdG come to harbor mutations at high incidence. This is the first report demonstrating DC-induced genotoxicity, the results implying that examination of carcinogenic parameters concomitantly with reporter gene mutation assays is able to provide crucial information to comprehend the underlying mechanisms of so-called non-genotoxic carcinogenicity.

Body mass index and oxidative DNA damage: A longitudinal study

Leanness has been shown to be related to an increased risk of some cancer forms, including lung cancer. However, biological evidence supporting a causal link between leanness and carcinogenesis is limited. The authors investigated longitudinally the association between body mass index (BMI) and levels of urinary 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, using data from 174 healthy employees who participated in a lifestyle intervention study. 8-OHdG levels were measured using automated high-performance liquid chromatography and adjusted for urinary creatinine levels. Analysis of repeated measurements using a random effects model detected a statistically significant inverse association between BMI and 8-OHdG levels (P = 0.003); one unit decrease in BMI was associated with a 2.7% (95% confidence interval 0.9-4.4) increase in 8-OHdG levels. The association was pronounced among men consuming less than 20 cigarettes per day (8.8% increase per unit decrease in BMI) and among non-smoking men (3.7% increase). The results based on a longitudinal observation suggest that weight loss is associated with increased oxidative DNA damage, a state presumably related to an increased risk of cancer.

Elevation of 8-Hydroxydeoxyguanosine in DNA from Isolated Mouse Lung Cells Following In Vivo Treatment with Aflatoxin B1

Aflatoxin B(1) (AFB(1)) is a mycotoxin produced by some strains of Aspergillus and is a recognized pulmonary and hepatic carcinogen. The most widely accepted mechanism of AFB(1) carcinogenicity involves bioactivation to AFB(1)-8,9-exo-epoxide and binding to DNA to form AFB(1)-N(7)-guanine. Another potential cause of DNA damage is AFB(1)-mediated stimulation of reactive oxygen species formation, leading to oxidation of DNA bases. The objective of this study was to determine the ability of AFB(1) to cause oxidative DNA damage in lung cell types of the A/J mouse. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and nonciliated bronchial epithelial (Clara) cells was assessed by high-performance liquid chromatography with electrochemical detection. An approximately 3-fold increase in 8-OHdG formation occurred in both alveolar macrophage and Clara cell preparations isolated from A/J mice 2 h following treatment with a single tumorigenic dose of 50 mg/kg AFB(1) ip (n = 3, p < 0.05). Prior treatment with 300 kU/kg polyethylene glycol-conjugated catalase prevented the AFB(1)-induced increase in 8-OHdG levels in all mouse lung cell preparations (n = 3, p < 0.05). These results support the possibility that oxidative DNA damage in mouse lung cells contributes to AFB(1) carcinogenicity.

Effect of repeated intravenous iron administration in haemodialysis patients on serum 8-hydroxy-2'-deoxyguanosine levels

Iron supplementation is a mainstay for management of renal anaemia in patients receiving haemodialysis (HD). Although it is well known that a single intravenous iron (IVIR) administration transiently enhances oxidative stress in HD patients, the consequence of repeated IVIR administration is still unknown. This study aims to clarify the time course of changes in serum 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of DNA oxidative injury, during a period of repeated IVIR administration in HD patients. Twenty-seven patients (62+/-14 years and 23 males) on long-term HD participated in this study. All patients had been on HD more than 6 months and none had received a blood transfusion or iron therapy in previous 6 months. The patients were divided into three groups according to the baseline haematocrit (Ht) and serum ferritin (FTN) levels as a marker of body iron stores: IVIR group (Ht<30% and FTN<100 ng/ml; n=7); High FTN group (Ht>or=30% and FTN>or=100 ng/ml; n=11); and low FTN group (Ht>or=30% and FTN<100 ng/ml; n=9). The IVIR group patients received 40 mg of ferric saccharate i.v. after each HD session until Ht increased by 5%. Serum 8-OHdG and other parameters were prospectively monitored for 10 weeks. At baseline, the serum ferritin level was independently associated with 8-OHdG in a multiple regression model (total adjusted R2=0.47, P<0.01). All patients in the IVIR group achieved the target Ht level during the study. IVIR administration resulted in significant increases in 8-OHdG levels (0.22+/-0.07-0.50+/-0.16 ng/ml: baseline to 10 week) as compared with both the high FTN group (0.52+/-0.20-0.58+/-0.28 ng/ml) and the low FTN group (0.39+/-0.11-0.36+/-0.11 ng/ml) (ANOVA for repeated measures P<0.01). Additionally, serum 8-OHdG and serum ferritin changed in the same manner. Repeated IVIR administration for HD patients was associated with signs of increased oxidative DNA injury, as reflected by increased serum levels of 8-OHdG. As these changes were accompanied by increased serum ferritin levels, excess body iron stores might play an important role in oxidative stress.

Increased 8-OHdG levels in the urine, serum, and substantia nigra of hemiparkinsonian rats

8-Hydroxy-2′-deoxyguanosine (8-OHdG), the predominant marker of oxidative DNA damage, may be a good biomarker for monitoring the progression of Parkinson's disease (PD). Unfortunately, there are no basic laboratory data examining 8-OHdG levels in animal models of PD. In this study, we demonstrate that rats lesioned with 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle display significantly elevated 8-OHdG levels in urine, serum, and substantia nigra, but not cerebrospinal fluid and striatum, compared to sham controls. These increments in 8-OHdG levels were detected at 2 days, but not at 7 days after the lesion suggesting that oxidative stress is restricted to the acute phase of 6-OHDA neurotoxicity. The present results support 8-OHdG as a biomarker that may aid both in the diagnosis and in the documentation of progression in PD.

The Pathogen Inactivation of Platelets: Studies of the Possible Mechanisms of the Platelet Dysfunction Seen Following Photochemical Treatment.

Background: The platelet mitochondrial genome has many similarities to bacterial and viral genomes. These include the lack of DNA redundancy, the lack of introns, no protective histone proteins, and inefficient DNA repair mechanisms; all of which are important characteristics that likely contribute to the increased rate of mutations occurring with mitochondrial DNA (mtDNA) compared to that seen with nuclear genomic DNA. Recent studies have shown that the use of pathogen inactivation involving UVA phototherapy using the combination of ultraviolet A (UVA) light and either the photoactive psoralen 4′-aminomethyltrioxsalen hydrocholoride (AMT/UVA, known to induce cross-linking of DNA) or the B-vitamin riboflavin (RF/UVA, known to produce the oxidized guanine moiety 8-OH-dG) are effective approaches to reduce and/or inactivate the bacterial and viral contaminants often present in human blood products. Such treatments however have been shown to be associated with impaired in vivo platelet recovery, survival and hemostatic function.Methods and Results: In the current study, rabbit and human platelets were treated with the two photochemical pathogen inactivation methods, (AMT/UVA or RF/UVA), using various UVA exposure, and the resultant platelets compared to control platelets with regard to their in vivo recovery, survival, and hemostatic function, as well as evaluating evidence of damage to platelet mtDNA. Rabbit platelets treated with UVA (1 to 5 joules/cm2) alone, RF alone, or AMT alone, showed minimal or no effect on in vivo platelet function; without evidence of damage to mtDNA. In contrast, the photochemical treatment of rabbit platelets with either AMT/UVA or RF/UVA showed a UVA-dose-dependent effect on in vivo platelet function as well as on mtDNA damage. Platelets treated with AMT/UVA(1 to 5 joules/cm2) revealed a 10–30% UVA-dose-dependent reduction in platelet recovery, survival, and hemostatic function, as well as a widespread but a non-specific effect upon platelet mtDNA structure. The treatment of rabbit platelets with RF/UVA(1 to 5 joules/cm2) showed considerably greater impact on platelet recovery, survival, and hemostatic function than that seen with AMT/UVA, at equivalent UVA doses. This was associated with a reproducible impairment of the amplification of a 3.8kb fragment within the platelet mtDNA genome. This mtDNA fragment is specifically associated with the coding for the NADH dehydrogenase subunit 1. A threshold dose of 1.0 joule/cm2 was noted for RF/UVA treatment, below which damage to mtDNA was found to be UVA-dose-dependent. Using HPLC to determine the concentration of 8-OH-dG within photochemically treated human platelets resulted in the demonstration of a UVA-dose-dependent increase of 8-OH-dG production for RF/UVA treated platelets. Control (no treatment), UVA alone, or AMT/UVA treated platelets produced no increase in 8-OHdG levels, even at UVA doses of 5 joules/cm2.Conclusions: These results indicate that the reproducible focal damage of platelet mtDNA by RF/UVA may have a considerably greater effect upon platelet in vivo circulatory and hemostatic functions than does the introduction of random mtDNA cross-linking produced by AMT/UVA treatment, possibly because of the discrete reproducible defect caused by the RF/UVA on the platelet mtDNA. These data nonetheless support the hypothesis that platelet mtDNA damage produced by pathogen inactivation methodology is associated with and may affect subsequent in vivo platelet function following platelet transfusion to a recipient.

4-Nitroquinoline 1-Oxide Forms 8-Hydroxydeoxyguanosine in Human Fibroblasts through Reactive Oxygen Species

4-Nitroquinoline 1-oxide (4NQO) is thought to elicit its carcinogenicity by producing DNA adducts after being metabolized to 4-hydroxyaminoquinoline 1-oxide, which forms 8-hydroxydeoxyguanosine (8OHdG), oxidative damage. To determine whether reactive oxygen species (ROS) are involved in the generation of 8OHdG by 4NQO, we used high-performance liquid chromatography and immunohistochemistry to measure the levels of 8OHdG in normal human fibroblasts treated with 4NQO. The extent of ROS induced by 4NQO was determined by using fluorescent probes to detect ROS, electron paramagnetic resonance spectrometry using a cell-free system, and measurement of intracellular glutathione (GSH) levels. In fibroblasts, 4NQO dose dependently increased 8OHdG levels. Hydrogen peroxide (H2O2) and superoxide were detected in cells treated with 4NQO by using dichlorofluorescin diacetate and hydroethidine, respectively. The addition of catalase to culture medium reduced 8OHdG levels and the intensity of dichlorofluorescin fluorescence, while 4NQO generated hydroxyl radicals in the cell-free system. These findings suggest that 4NQO treatment leads to formation of superoxide, H2O2, and hydroxyl radicals, resulting in the production of a substantial amount of 8OHdG in DNA. Neither the level of 8OHdG nor that of GSH had returned to the basal level 24 h after removal of 4NQO even at a concentration as low as 1 microM. Our results suggest that generation of ROS and depletion of GSH in cells are also important factors for the generation of 8OHdG by 4NQO. This paper describes practical and sensitive ways to detect ROS and 8OHdG and discusses a new functional pathway to elicit genotoxicity.

Benzo[ a]pyrene and its metabolites combined with ultraviolet A synergistically induce 8-hydroxy-2′-deoxyguanosine via reactive oxygen species

We previously reported that benzo[ a]pyrene (BaP) and UVA radiation synergistically induced oxidative DNA damage via 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation in vitro. The present study shows that microsomal BaP metabolites and UVA radiation potently enhance 8-OHdG formation in calf thymus DNA about 3-fold over the parent compound BaP. Utilization of various reactive oxygen species scavengers revealed that singlet oxygen and superoxide radical anion were involved in the 8-OHdG formation induced by microsomal BaP metabolites and UVA. Two specific BaP metabolites, benzo[ a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (+/−) (anti) (BPDE) and BaP-7,8-dione, were further tested for synergism with UVA. BaP-7,8-dione showed an effect on 8-OHdG formation induced by UVA radiation that was similar to that of the parent BaP, whereas BPDE exhibited significantly higher induction of 8-OHdG than BaP. At as low as 0.5 μM, BPDE plus UVA radiation substantially increased 8-OHdG levels about 25-fold over the parent BaP. BPDE increased the formation of 8-OHdG levels in both BPDE concentration- and UVA dose-dependent manners. Additionally, singlet oxygen was found to play a major role in 8-OHdG induction by BPDE and UVA. These results suggest that BaP metabolites such as BPDE synergize with UVA radiation to produce ROS, which in turn induce DNA damage.