Formation Of 8-hydroxydeoxyguanosine Research Articles

Mechanistic Insights into Acetamiprid-Induced Genotoxicity on the Myocardium and Potential Ameliorative Role of Resveratrol

The current study aimed to explore the genotoxic impacts of the insecticide acetamiprid (ACP) on the myocardium and assess the ameliorative role of resveratrol (RSV). Male rats (10/group) were treated via oral route for 90 days: control; ACP (25mg/kg); RSV (20mg/kg); ACP+RSV. Peripheral blood micronucleus test, oxidative stress analysis, comet assay, 8-hydroxydeoxyguanosine and gene expression assessment were performed. The findings revealed that ACP has myocardial genotoxic effects, as demonstrated by increased micronucleus and 8-hydroxydeoxyguanosine formation and increased all comet parameters. Oxidative stress analysis demonstrated that ACP elevated H2O2 and NO levels while decreasing catalase and GST activities. Acetamiprid dysregulated the expression of genes related to oxidative stress and DNA damage response. However, RSV co-treatment resulted in significant protection against these genotoxic impacts. Resveratrol reduced DNA damage and restored the oxidative balance in the myocardium. Moreover, RSV modulated the Nrf2/HO-1 and Atm/P53 pathways, potentiating antioxidant defense and DNA repair.

Accumulation of 8-hydroxydeoxyguanosine, L-arginine and Glucose Metabolites by Liver Tumor Cells Are the Important Characteristic Features of Metabolic Syndrome and Non-Alcoholic Steatohepatitis-Associated Hepatocarcinogenesis.

To uncover mechanisms and explore novel biomarkers of obesity, type 2 diabetes (T2DM) and nonalcoholic steatohepatitis (NASH)-associated hepatocarcinogenesis, cellular and molecular alterations in the liver, and hepatocellular carcinomas (HCCs) were investigated in NASH model 60-week-old Tsumura, Suzuki, Obese Diabetic (TSOD) mice and NASH HCC patients. Markedly elevated lipid deposition, inflammation, fibrosis, and peroxisome proliferation in the liver, preneoplastic lesions, and HCCs of TSOD mice were accompanied by accumulation of polysaccharides in the cellular cytoplasm and nuclei and increase of oxidative DNA damage marker, 8-hydroxydeoxyguanosine (8-OHdG) formation in the liver and altered foci. Metabolomics of TSOD mice HCCs demonstrated significant elevation of the concentration of amino acid L-arginine, phosphocreatine, S-adenosylmethionine/S-adenosylhomocysteine ratio, adenylate, and guanylate energy charges in coordination with tremendous rise of glucose metabolites, mostly fructose 1,6-diphosphate. L-arginine accumulation in HCCs was associated with significant under-expression of arginase 1 (ARG1), suppression of the urea cycle, methionine and putrescine degradation pathways, activation of Ser and Thr kinase Akt AKT, phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) kinases, β-catenin, mammalian target of rapamycin (mTOR), and cell proliferation. Furthermore, clinicopathological analysis in 20 metabolic syndrome/NASH and 80 HCV-positive HCC patients demonstrated significant correlation of negative ARG1 expression with poor tumor differentiation, higher pathological stage, and significant decrease of survival in metabolic syndrome/NASH-associated HCC patients, thus indicating that ARG1 could become a potential marker for NASH HCC. From these results, formation of oxidative stress and 8-OHdG in the DNA and elevation of glucose metabolites and L-arginine due to ARG1 suppression in mice liver cells are the important characteristics of T2DM/NASH-associated hepatocarcinogenesis, which may take part in activating oxidative stress resistance, synthesis of phosphocreatine, cell signaling, methylation, and proliferation.

Protective effects of oral glutathione on fasting-induced intestinal atrophy through oxidative stress

AIMTo determine whether oral glutathione (GSH) administration can alleviate the effects of fasting-induced intestinal atrophy in the small intestinal mucosa.METHODSRats were divided into eight groups. One group was fed ad libitum, another was fed ad libitum and received oral GSH, and six groups were administrated saline (SA) or GSH orally during fasting. Mucosal height, apoptosis, and cell proliferation in the jejunum were histologically evaluated. iNOS protein expression (by immunohistochemistry), nitrite levels (by high performance liquid chromatography, as a measure of NO production), 8-hydroxydeoxyguanosine formation (by ELISA, indicating ROS levels), glutathione/oxidized glutathione (GSH/GSSG) ratio (by enzymatic colorimetric detection), and γ-glutamyl transpeptidase (Ggt1) mRNA levels in the jejunum (by semi-quantitative RT-PCR) were also estimated.RESULTSOral GSH administration was demonstrated to drastically reduce fasting-induced intestinal atrophy in the jejunum. In particular, jejunal mucosal height was enhanced in GSH-treated animals compared to SA-treated animals [527.2 ± 6.9 for 50 mg/kg GSH, 567.6 ± 5.4 for 500 mg/kg GSH vs 483.1 ± 4.9 (μm), P < 0.01 at 72 h]. This effect was consistent with decreasing changes in GSH-treated animals compared to SA-treated animals for iNOS protein staining [0.337 ± 0.016 for 50 mg/kg GSH, 0.317 ± 0.017 for 500 mg/kg GSH vs 0.430 ± 0.023 (area of staining part/area of tissue), P < 0.01 at 72 h] and NO [2.99 ± 0.29 for 50 mg/kg GSH, 2.88 ± 0.19 for 500 mg/kg GSH vs 5.34 ± 0.35 (nmol/g tissue), P < 0.01 at 72 h] and ROS [3.92 ± 0.46 for 50 mg/kg GSH, 4.58 ± 0.29 for 500 mg/kg GSH vs 6.42 ± 0.52 (8-OHdG pg/μg DNA), P < 0.01, P < 0.05 at 72 h, respectively] levels as apoptosis mediators in the jejunum. Furthermore, oral GSH administration attenuated cell proliferation decreases in the fasting jejunum [182.5 ± 1.9 for 500 mg/kg GSH vs 155.8 ± 3.4 (5-BrdU positive cells/10 crypts), P < 0.01 at 72 h]. Notably, both GSH concentration and Ggt1 mRNA expression in the jejunum were also attenuated in rats following oral administration of GSH during fasting as compared with fasting alone [0.45 ± 0.12 vs 0.97 ± 0.06 (nmol/mg tissue), P < 0.01; 1.01 ± 0.11 vs 2.79 ± 0.39 (Ggt1 mRNA/Gapdh mRNA), P < 0.01 for 500 mg/kg GSH at 48 h, respectively].CONCLUSIONOral GSH administration during fasting enhances jejunal regenerative potential to minimize intestinal mucosal atrophy by diminishing fasting-mediated ROS generation and enterocyte apoptosis and enhancing cell proliferation.

Effect of scaling and root planing on levels of 8-hydroxydeoxyguanosine in gingival crevicular fluid of chronic periodontitis patients with and without Type II diabetes mellitus.

Context:Chronic periodontitis (CP) and diabetes mellitus are associated with increased oxidative damage to DNA with formation of 8-hydroxydeoxyguanosine (8-OHdG). The aim of this study was to evaluate the change in gingival crevicular fluid (GCF) levels of 8-OHdG and glycosylated hemoglobin (HbA1c) by 3 months after scaling and root planing (SRP), in CP patients with and without Type II diabetes mellitus.Settings and Design:Sixteen patients with CP, 16 patients with CP and Type II diabetes mellitus (CP-D), and 16 systemically healthy individuals with clinically healthy periodontium who served as controls were included in the study.Materials and Methods:The clinical parameters (plaque index [PI], probing depth [PD], clinical attachment level [CAL], and bleeding on probing [BOP%]), HbA1c levels, and GCF 8-OHdG levels were measured at baseline. All the patients except controls were treated with SRP followed by evaluation of the above-mentioned clinical and biochemical parameters after 3 months.Statistical Analysis Used:Statistical analysis was performed using paired t-test, independent t-test, and Mann–Whitney U-test.Results:After SRP, CP-D group showed a greater reduction in PI, PD, BOP%, and greater gain in CAL when compared to CP patients (P < 0.05). Levels of 8-OHdG and HbA1c in CP-D patients also showed a greater reduction, 3 months after SRP when compared to CP patients (P < 0.05).Conclusions:GCF 8-OHdG levels, HbA1c levels, and clinical parameters were reduced significantly in CP and CP-D patients, with maximum reduction achieved in CP-D patients 3 months after SRP.

Cocaine effects on generation of reactive oxygen species and DNA damage: formation of 8-hydroxydeoxyguanosine in active abusers

Cocaine abuse continues to be a major public health problem in the world. An upper numbers of individuals are initiating cocaine use with a stable rate of growth each year with an increasing number of people with cocaine related problems. Following cocaine oxidative pathways a ROS formation are generated. Oxidative stress has been demonstrated to play an important role in cocaine addiction and toxicity due to its oxidized metabolites produced by cytochrome P450 during cocaine biotransformation. The ROS induced genotoxicities include DNA damage, gene mutation, chromosome aberrations and micronuclei formation. 8-Hydroxy-2’-deoxyguanosine (8-OHdG) an oxidative modified DNA product, is the most representative product that may reflect oxidative damage induced by ROS. The present study was designed to investigate whether a systemic cocaine administration and its metabolism increase 8-OHdG production. Our findings clearly showed that cocaine promoted the ROS formation with significant increased of urinary 8-OHdG and MDA with a decreased of total scavenging capacity (TSC).

Lyophilized strawberries prevent 7,12-dimethylbenz[α]anthracene (DMBA)-induced oral squamous cell carcinogenesis in hamsters

This study evaluated the ability of lyophilized strawberries to prevent 7,12-dimethylbenz[α]anthracene (DMBA)-induced tumor development in hamster cheek pouch. The experimental diet containing 5% strawberries was given to hamsters during or after DMBA treatment, or throughout the entire bioassay. Our data indicate that tumor incidence, multiplicity, volume and histologic grade of oral precancerous lesions were reduced in animals fed the experimental diet compared to those fed the control diet. We chemically identified and quantified the main phytochemicals by high performance liquid chromatography/tandem mass spectrometry. Fourteen quantified compounds were classified to three categories: anthocyanins (48.8%, w/w of the phenolics), ellagitannin/ellagic acid/ellagic acid derivates (42.9%) and flavonols (8.3%). We further elucidated the molecular mechanisms underlying preventive activities of strawberries. Our mechanistic studies show that strawberries suppress cell proliferation, angiogenesis and oncogenic signaling. Moreover, feeding 5% strawberries also significantly decreased arachidonic acid metabolism and formation of 8-hydroxydeoxyguanosine. These observations suggest that strawberries inhibit DMBA-induced oral cancer in hamsters through suppression of multiple oncogenic events, and thus may serve as alterative agents for oral cancer prevention in humans.

Genotoxic and oxidative stress effects of 2-amino-9H-pyrido[2,3-b]indole in human hepatoma G2 (HepG2) and human lung alveolar epithelial (A549) cells

2-Amino-9H-pyrido[2,3-b]indole (AαC), which is present in high quantities in cigarette smoke and also in fried food, has been reported to be a probable human carcinogen. However, few studies have reported on the genotoxicity and oxidative stress induced by AαC. This study investigated the genotoxic effects of AαC in human hepatoma G2 (HepG2) and human lung alveolar epithelial (A549) cells using the comet assay. Significant increases in DNA fragment migration indicated that AαC causes serious DNA damage in HepG2 and A549 cells. The role of oxidative stress in the mechanism of AαC-induced genotoxicity was clarified by measuring the level of intracellular reactive oxygen species (ROS), the GSH/GSSG ratio and the formation of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage. The results showed that the levels of ROS and 8-OHdG increased, whereas the GSH/GSSG ratio decreased. The concentration of 8-OHdG was positively related to DNA damage. Taken together, these results indicate that AαC can induce genotoxicity and oxidative stress and that AαC likely exerts genotoxicity in HepG2 and A549 cells through ROS-induced oxidative DNA damage. This is the first report to describe AαC-induced genotoxic and oxidative stress in HepG2 and A549 cells.

Lactoferrin directly scavenges hydroxyl radicals and undergoes oxidative self-degradation: a possible role in protection against oxidative DNA damage.

In this study, we examined the protective effect of lactoferrin against DNA damage induced by various hydroxyl radical generation systems. Lactoferrin (LF) was examined with regard to its potential role as a scavenger against radical oxygen species using bovine milk LF. Native LF, iron-saturated LF (holo-LF), and apolactoferrin (apo-LF) effectively suppressed strand breaks in plasmid DNA due to hydroxyl radicals produced by the Fenton reaction. In addition, both native LF and holo-LF clearly protected calf thymus DNA from fragmentation due to ultraviolet irradiation in the presence of H2O2. We also demonstrated a protective effect of all three LF molecules against 8-hydroxydeoxyguanosine (8-OHdG) formation in calf thymus DNA following ultraviolet (UV) irradiation with H2O2. Our results clearly indicate that native LF has reactive oxygen species-scavenging ability, independent of its nature as a masking component for transient metals. We also demonstrated that the protective effect of LF against oxidative DNA damage is due to degradation of LF itself, which is more susceptible to degradation than other bovine milk proteins.

Reactive Oxygen Species Induce Epigenetic Instability through the Formation of 8-Hydroxydeoxyguanosine in Human Hepatocarcinogenesis

Chronic hepatitis C (CHC) triggers oxidative stress and contributes to the emergence of hepatocellular carcinoma (HCC). We previously reported that tumor suppressor gene (TSG) methylation is a critical factor during the early stages of hepatocarcinogenesis. In this study, we clarify the association between oxidative stress and epigenetic alterations during hepatocarcinogenesis. We examined DNA oxidation and methylation profiles in 128 liver biopsy samples from CHC patients. The DNA oxidation and methylated TSG numbers were quantified using immunohistochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG) and quantitative PCR for 11 TSGs, respectively. The quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) assay in HepG2 and fetal liver Hc cells treated with H₂O₂ was used to quantify trimethyl-H3K4, acetylated-H4K16 (an active chromatin marker), trimethyl-H3K27 (a repressive chromatin marker) and 8-OHdG. We analyzed 30 promoters of 25 different TSGs by qPCR. The high levels of 8-OHdG was the only variable that was significantly associated with the increased number of methylated TSGs in CHC (p < 0.0001). The ChIP-qPCR revealed that after H₂O₂ treatment of the cell lines, the 8-OHdG-bound promoters showed a modification from an active chromatin (trimethyl-H3K4 and acetylated-H4K16 dominant) to a repressive chromatin (trimethyl-H3K27 dominant) status. We conclude that oxidative stress alters the chromatin status, which leads to abnormal methylation of TSGs, and contributes to hepatocarcinogenesis in CHC patients.

Quercetin protects human-derived liver cells against mercury-induced DNA-damage and alterations of the redox status

Aim of this study was to investigate the cytotoxic and genotoxic properties of inorganic and organic mercury compounds, i.e., HgCl2 and methylmercury (MeHg). In addition, the DNA-protective and antioxidant effects of the flavonoid quercetin (QC) were studied. All experiments were conducted with human-derived liver cells (HepG2), which possess antioxidant and drug-metabolizing enzymes in an inducible form. 8-Hydroxydeoxyguanosine (8-OHdG) and comet formation were monitored as endpoints of DNA damage. The impact of the metal compounds on the redox status was also investigated, since it is assumed that their toxic effects are due to oxidative damage. A number of biochemical parameters related to oxidative stress, namely glutathione, malondialdehyde, protein carbonyl and formation of reactive oxygen species (ROS) were measured after treatment of the cells with the mercury compounds in the presence and absence of quercetin. To elucidate the mechanisms that underlie the effects of QC, three protocols (pre-, simultaneous and post-treatment) were used. Both mercury compounds (range 0.1–5.0μM) caused induction of DNA migration and formation of 8-OHdG. In combination with the flavonoid (range 0.1–5.0μM), DNA-protective effects of QC were observed after pre- and simultaneous treatment but not when the flavonoid was added after treatment with the metal compounds. Exposure to the metal compounds led also to substantial changes of all parameters of the redox status and co-treatment experiments with QC showed that these alterations are reversed by the flavonoid. Taken together, the results of our experiments indicate that these two mercury compounds cause DNA damage and oxidative stress in human-derived liver cells and that the flavonoid reduces these effects. Since the concentrations of the metals and of the flavonoids used in the present work reflect human exposure, our findings can be taken as an indication that QC may protect humans against the adverse effects caused by the metal.

Selenite induces oxidative DNA damage in primary rat hepatocyte cultures

The effects of selenite ions on oxidative DNA damage in primary rat hepatocyte cultures were examined. Selenite ions dose-dependently stimulated 8-hydroxydeoxyguanosine formation in DNA from the hepatocyte cultures at concentrations of 1, 5 and 10μmol/L (1.6-, 2.5- and 3.6-fold). Selenite ions also induced DNA fragmentation in nucleus from the hepatocyte cultures at concentrations of 5 and 10μmol/L. Selenite ions could not affect protein expression levels of 8-oxoguanine DNA glycosylase 1 which is responsible for excision of 8-hydroxydeoxyguanosine from DNA, but could increase 2′,7′-dichlorofluorescein-fluorescence intensity from 2′,7′-dichlorodihydrofluorescein, an indicator of reactive oxygen species generation, at concentrations of 5 and 10μmol/L. Furthermore, an increase in 8-hydroxydeoxyguanosine induced by selenite ions (10μmol/L) was inhibited by free radical scavengers (α-tocopherol, N-acetyl-l-cysteine, and melatonin) at concentrations more than 50μmol/L. These observations imply that selenite ions have the potential to induce oxidative DNA damage in the liver through an increase in reactive oxygen species formation, but not a decrease in 8-oxoguanine DNA glycosylase 1 protein expression levels.

Indicators of oxidative stress and apoptosis in mouse whole lung and Clara cells following exposure to styrene and its metabolites

In mice, styrene is hepatotoxic, pneumotoxic, and causes lung tumors. One explanation for the mechanism of toxicity is oxidative stress/damage. Previous studies have shown decreased glutathione levels, linked to increased apoptosis, in lung homogenates and isolated Clara cells 3 h following styrene or styrene oxide (SO) administration or in vitro exposure. The objective of the current studies was to determine what effects styrene and its active metabolites, primarily styrene oxide, had on indicators of oxidative stress and attendant apoptosis in order to understand better the mechanism of styrene-induced toxicity. Three hours following in vitro exposure of Clara cells to styrene or SO there were increases in reactive oxygen species (ROS). Following administration of styrene or styrene oxide ip, increases in ROS, superoxide dismutase (SOD), and 8-hydroxydeoxyguanosine (8-OHdG) formation were observed. Since increases in ROS have been linked to increases in apoptosis ratios of bax/bcl-2, mRNA and protein expression were determined 3–240 h following the administration of styrene and R-styrene oxide (RSO). The bax/bcl-2 mRNA ratio increased 12 and 24 h following R-SO and 120 h following styrene administration. However, the bax/bcl-2 protein ratio was not increased until 240 h following R-SO, and 24 and 240 h following styrene administration. However, only a slight increase in caspase 3 was observed. These results indicated that oxidative stress occurred 3 h following styrene or styrene oxide as evidenced by increased ROS and SOD. This increased ROS may be responsible for the increased 8-OHdG formation. Our findings of limited apoptosis in Clara cells following acute exposure to styrene or SO are in agreement with others and may reflect the minimal extent to which apoptosis plays a role in acute styrene toxicity. It is clear, however, that oxidative stress and oxidative effects on DNA are increased following exposure to styrene or styrene oxide, and these may play a role in the lung tumorigenesis in mice.

Olaquindox-induced genotoxicity and oxidative DNA damage in human hepatoma G2 (HepG2) cells

Olaquindox, a synthetic antimicrobial compound, is widely used in China as feed additive for growth promotion. However, it is a mutagen with its functional mechanism yet to be unclear. The purpose of this study was to investigate the genotoxic effects of olaquindox in human hepatoma G2 (HepG2) cells and to determine whether the oxidative DNA damage participated in the mechanism of olaquindox toxicity. The results of cell survival assay revealed that the HepG2 cells viabilities were significantly inhibited by olaquindox in a dose- and time-dependent manner. The cytokinesis-block micronucleus (CBMN) assay demonstrated a clear dose–response relationship between olaquindox treatments and micronucleus (MN) frequencies. Moreover, marked increases of DNA fragment migration were observed in the single cell gel electrophoresis (SCGE) assay. These data suggest that olaquindox treatment produced serious chromosome damage and DNA damage in HepG2 cells. To elucidate the possible oxidative DNA damage mechanism of olaquindox genotoxic activity, the levels of the intracellular reactive oxygen species (ROS) and the formation of 8-hydroxydeoxyguanosine (8-OHdG) were detected. The results showed that olaquindox induced the increased levels of ROS and 8-OHdG in HepG2 cells. Considering all the results, it is inferred that olaquindox exerts genotoxic effects in HepG2 cells probably through the ROS-induced oxidative DNA damage.

Cytoprotective effects of the lipoidic‐liquiform pro‐vitamin C tetra‐isopalmitoyl‐ascorbate (VC‐IP) against ultraviolet‐A ray‐induced injuries in human skin cells together with collagen retention, MMP inhibition and p53 gene repression

Irradiation with ultraviolet-A (UVA) ray at doses of 20-100 J/cm(2) diminished the cell viability of human keratinocytes HaCaT and human melanoma cells HMV-II, both of which were protected by pre-irradiational administration with the ascorbic acid (Asc) derivative, VC-IP (2,3,5,6-O-tetra-2'-hexyldecanoyl-L-ascorbic acid; vitamin C-isopalmityl tetraester), which is the first lipoidic-liquiform pro-vitamin C by itself that is materialized by esterization of all four intramolecular hydroxyl groups of an Asc molecule with branched chain fatty groups, resulting in molecular fluidity higher than that of the corresponding straight chains. Irradiation with UVA to HaCaT keratinocytes was shown to cause the formation of 8-hydroxydeoxyguanosine (8-OHdG), translocation of phosphatidylserine in the inner layer into the outer layer of cell membrane, and lowering of a mitochondrial membrane potential, all of which were repressed by pre-irradiational administration with VC-IP. Expression of p53 gene, another hallmark of UV-induced DNA damages, was promoted by UVA irradiation to the keratinocytes but also repressed by VC-IP. Administration with VC-IP of 10-50 microM to human fibroblasts NHDF achieved the enhancement of collagen synthesis, repression of matrix metalloprotease-2/9 activity, and increasing of intracellular Asc contents more markedly than that with Asc itself of the same concentrations. Thus UVA-induced diverse harmful effects could be prevented by VC-IP, which was suggested to ensue intrinsically from the persistent enrichment of intracellular Asc, through esterolytic conversion of VC-IP to a free-form Asc molecule, resulting in relief to UVA-caused oxidative stress.

Hydroquinone-induced genotoxicity and oxidative DNA damage in HepG2 cells

Hydroquinone (HQ) is used as an antioxidant in rubber industry and as a developing agent in photography. HQ is also an intermediate in the manufacture of rubber, food antioxidant and monomer inhibitor. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of HQ and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. DNA strand breaks and DNA–protein crosslinks (DPC) were measured by the proteinase K-modified alkaline single cell gel electrophoresis (SCGE) assays. Using the SCGE assay, a significant dose-dependent increment in DNA migration was detected at concentrations of HQ (6.25–25 μM); but at the higher tested concentrations (50 μM), a reduction in the migration compared to the maximum migration at 25 μM was observed. Post-incubation with proteinase K significantly increased DNA migration in cells exposed to higher concentrations of HQ (50 μM). A significant increase of the frequency of micronuclei was found in the range from 12.5 to 50 μM in the micronucleus test (MNT). The data suggested that HQ caused DNA strand breaks, DPC and chromosome breaks. To elucidate the oxidative DNA damage mechanism, the 2,7-dichlorofluorescein diacetate (DCFH-DA) and o-phthalaldehyde (OPT) were chosen to monitor the levels of reactive oxygen species (ROS) and glutathione (GSH), respectively. The present study showed that HQ induced the increased levels of ROS and depletion of GSH in HepG2 cells, the doses being 25–50 and 6.25–50 μM, respectively. Moreover, HQ significantly caused 8-hydroxydeoxyguanosine (8-OHdG) formation in HepG2 cells at concentrations from 12.5 to 50 μM. All these results demonstrate that HQ exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress. GSH, as a main intracellular antioxidant, is responsible for cellular defense against HQ-induced DNA damage.

Changes in the antioxidant defense and hepatic drug metabolizing enzyme and isoenzyme levels, 8-hydroxydeoxyguanosine formation and expressions of c-raf.1 and insulin-like growth factor II genes during the stages of development of hepatocellular carcinoma in rats

This is an extensive study in a defined initiation-promotion hepatocellular carcinoma model of hepatocarcinogenesis (in rats) in which many important marker enzymes and isoenzymes and 8-hydroxydeoxyguanosine formation have been studied together with two very important cellular proliferating genes, insulin-like growth factor II and c-raf.1, known for their role in hepatocellular cancer development. Experiments were carried out on hepatic tissues of male Sprague-Dawley rats. Variations in different enzyme/isoenzyme activities/contents/expression pattern and 8-hydroxydeoxyguanosine-positive cells were studied. Insulin-like growth factor II and c-raf.1 gene expressions were monitored. A direct shift with increase in size and numbers of lesions was found to occur in different experimental groups. In this study, glutathione peroxidase (1.14 and 1.46-fold) and reduced triphosphopyridine nucleotide (TPNH)-cytochrome-c-reductase (1.94 and 2.94-fold) activities, cytochrome b5 (1.57 and 3.28-fold) and P-450 contents (1.45 and 1.22-fold), glutathione content (1.27 and 1.45-fold) and superoxide dismutase and catalase (1.16 and 1.39-fold) activities in group A animals were found to be lower than those in initiation and promotion studies, respectively. 8-Hydroxydeoxyguanosine-positive nuclei count showed that oxidative damage of nuclear DNA enhanced with the progress of the disease. The insulin-like growth factor II expression was found to be predominant in hepatocellular carcinoma and in early preneoplastic lesions. Unlike insulin-like growth factor II, c-raf.1 expression was located in the late basophilic lesions associated with hepatocellular carcinoma. During the various stages of the development of hepatocellular carcinoma, the enzymes played a significant role in metabolizing carcinogens and thereby scavenging various toxic metabolites or free radicals produced. A sequence of cellular changes starting from the appearance of glycogen storage foci to basophilic foci leading to hepatocellular carcinoma via mixed cell foci varied the activity/content or expression pattern of the enzymes and isoenzymes and in 8-hydroxydeoxyguanosine formation. It has been established that c-raf.1-induced signaling pathways activated by insulin-like growth factor II is implicated in the late stage of development of cancer.

Estrogen-Induced Generation of Reactive Oxygen and Nitrogen Species, Gene Damage, and Estrogen-Dependent Cancers

In addition to the direct effect of estrogen on mitochondria and the redox cycling of catechol estrogen, estrogen-induced proinflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), also generate reactive oxygen and nitrogen species (RO/NS). Different cellular signaling pathways may operate in response to varying levels of estrogen-induced RO/NS, leading to genotoxic damage, cell apoptosis, or cell growth. At high levels of RO/NS, cells receiving genotoxic insults, if not repaired, may engage the apoptotic pathways. There is increasing evidence supporting that estrogen-induced alterations in the genome of cells is produced by oxidative attack. Furthermore, ROS generated by estrogen exposure and/or active metabolites of estrogen in combination with receptor-mediated proliferation of genetically damaged cells may be involved in tumor development. This view is supported by the findings of DNA modifications produced in vitro or in vivo by natural and synthetic estrogens in the target organs of cancer both in experimental models and in humans. Interaction of estrogen-induced oxidants and estrogen metabolites with DNA was shown to generate mutations in genes. Cotreatment with an inhibitor of IL-1β and TNF-α synthesis, pentoxifylline, decreased stilbene estrogen-induced levels of myeloperoxidase (MPO), 8-hydroxydeoxyguanosine formation, and gene mutations, and prevented stilbene estrogen-induced lesions. Stable MCF-7 clones overexpressing IL-1β resulted in a high level of IL-1β peptide secretion undergoing cell apoptosis, and an elevated level of p53 protein in response to high oxidative stress when compared to nontransfected cells, whereas MCF-7 clones overexpressing IL-1β that resulted in a moderate level of IL-1β secretion stimulated the clonal expansion of MCF-7 and TM3 cells. Estrogen-induced MCF-7 cell growth and cyclin D1 expression were suppressed by antioxidants and mitochondrial blockers. These studies support that in addition to ovarian estrogen-mediated ER signaling, mitogenic signals may also come from estrogen-induced RO/NS. Further validation of this concept that the concentration of the RO/NS within the cellular microenvironment determines its stimulatory or inhibitory growth signals as well as its genotoxic effects regulating the growth of estrogen-dependent tumors may result in novel preventive strategies.

Development of quantitative analysis of 8-nitroguanine concomitant with 8-hydroxydeoxyguanosine formation by liquid chromatography with mass spectrometry and glyoxal derivatization

Under inflammatory conditions, both 8-nitroguanine (NO 2Gua) and 8-hydroxydeoxyguanosine (8-OHdG) are found in tissues. Measurements of the two types of damaged bases on nucleotides are expected to provide information pointing to the possible correlation between inflammation and carcinogenesis. For the establishment of an in vivo model, in this study, a sensitive and precise method for the determination of NO 2Gua, which uses liquid chromatography with mass spectrometry (LC–MS) and 6-methoxy-2-naphthyl glyoxal (MTNG) derivatization, was developed in vitro. The procedure for DNA digestion in this method is identical to that widely used for 8-OHdG measurement, which enables us to detect the two damaged bases in the same DNA sample. In order to validate our method, we measured NO 2Gua levels in DNA sample using LC–MS. A mass spectrometer equipped with an electrospray atmospheric pressure ionization source and operated in the negative ion mode (ESI −) was set up with selective ion monitoring at m/ z 391 and 394 for NO 2Gua-MTNG and [ 13C, 15N 2]-NO 2Gua-MTNG as surrogate standard, respectively. The average recoveries from DNA samples spiked with 25, 50 and 250 nM NO 2Gua were 99.4, 99.8 and 99.1% with correction using the added surrogate standard, respectively. The limit of quantification was 3.0 nM for NO 2Gua. To ascertain the applicability of our method to DNA samples harboring the two damaged bases, we measured NO 2Gua and 8-OHdG levels in calf thymus DNA treated with ONOO −. As a result, both NO 2Gua and 8-OHdG levels were clearly increased with ONOO − dose dependency, the amount of NO 2Gua at the high dose ONOO − being almost the same as those of 8-OHdG. LC–MS was able to determine NO 2Gua in a small amount of DNA sample, and is therefore expected to be a very powerful tool for the evaluation of DNA damage induced by reactive nitrogen species.

Mutagenicity of UV-irradiated maltol in Salmonella typhimurium

We investigated the photomutagenicity of maltol (3-hydroxy-2-methyl-4H-pyran-4-one) in bacterial cells. Maltol has a caramel-butterscotch odour and is used as a food additive to impart flavour to bread and cakes. Unirradiated maltol was not mutagenic up to 5 mg/plate in the Ames test. When maltol was irradiated with either UVA (a black light, 320-400 nm, 230 microW/cm(2)) for 5-30 min or UVC (a germicidal lamp, 610 microW/cm(2)) for 3 min in sodium phosphate buffer (pH 7.4) prior to the exposure of bacterial cells, it was mutagenic to Salmonella typhimurium TA100, TA104 and TA97. Mutagenic activation of maltol by UVA-irradiation was more evident in neutral and alkaline conditions (pH 7.0-9.0) than in acidic conditions. On the other hand, photomutagenicity was not observed when maltol was irradiated with UVA in 100 mM NaCl solution or water. The mutagenic photoproduct was stable for at least 60 min after UVA-irradiation. However, addition of thiol compounds (cysteine or glutathione) to the UVA-irradiated maltol diminished the mutagenicity. Mutational spectrum analysis revealed that the predominant base-substitutions induced were G:C-->T:A transversions and G:C-->A:T transitions. An increase of 8-hydroxydeoxyguanosine formation in salmon sperm DNA exposed to maltol and UVA in vitro was detected by HPLC-ECD, but it was too small to explain the photomutagenicity. We are considering the formation of DNA adducts as the photomutagenic mechanism.

Microcystin-induced 8-hydroxydeoxyguanosine in DNA and its reduction by melatonin, vitamin C, and vitamin E in mice

Microcystin LR (MC-LR), a liver-specific toxin synthesized by Microcystis aeruginosa, was investigated. MC-LR initiated reactive oxygen species formation followed by damaging DNA and some other cellular components. We investigated the ability of MC-LR to induce oxidative DNA damage by examining the formation of 8-hydroxydeoxyguanosine (8-OH-dG) using HPLC with electrochemical detection. Melatonin, vitamin C (ascorbate), and vitamin E (as Trolox), all of which are free radical scavengers, markedly inhibited the formation of 8-OH-dG in a concentration-dependent manner. The concentration that reduced DNA damage by 50% (IC(50)) was 0.55, 31.4, and 36.8 microM for melatonin, ascorbate, and Trolox, respectively. The results show that melatonin is 60- and 70-fold more effective than vitamin C or vitamin E, respectively, in reducing oxidative DNA damage. These findings are consistent with the conclusion that melatonin's highly protective effect against microcystin toxicity relates, at least in part, to its direct hydroxyl radical scavenging ability.