Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.     

Antigenotoxic Effect of Ascorbic Acid and Resveratrol in Erythrocytes of Ambystoma mexicanum,Oreochromis niloticus and Human Lymphocytes Exposed to Glyphosate

Glyphosate is a controversial herbicide. Its genotoxicity and presence in various ecosystems have been reported. The use of ascorbic acid and resveratrol could protect different organisms from glyphosate-induced genetic damage. In the present study, specific genetic damage induced by glyphosate was evaluated in erythrocytes of Oreochromis niloticus, Ambystoma mexicanum and human lymphocytes. Simultaneously, the antigenotoxic capacity of various concentrations of ascorbic acid and resveratrol was evaluated by means of pretreatment and simultaneous treatment protocols. The 0.03, 0.05 and 0.07 mM concentrations of glyphosate induced significant genotoxic activity (p < 0.05) in human lymphocytes and in erythrocytes of the species studied, and could cause genomic instability in these populations. The reduction in genetic damage observed in human lymphocytes exposed to high concentrations of glyphosate is only apparent: excessive genetic damage was associated with undetectable excessive tail migration length. A significant (p < 0.05) antigenotoxic effect of ascorbic acid and resveratrol was observed in all concentrations, organisms and protocols used. Both ascorbic acid and resveratrol play an important role in maintaining the integrity of DNA. Ascorbic acid in Oreochromis niloticus, Ambystoma mexicanum reduced glyphosate-induced genetic damage to a basal level. Therefore, our data indicate that these antioxidants could help preserve the integrity of the DNA of organisms exposed to glyphosate. The consumption of antioxidants is a useful tool against the genotoxicity of glyphosate.     

Involvement of ectonucleotidases and purinergic receptor expression during acute Chagas disease in the cortex of mice treated with resveratrol and benznidazole

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi. CD affects people worldwide, primarily in tropical areas. The central nervous system (CNS) is an essential site for T. cruzi persistence during infection. The protozoan may pass through the blood–brain barrier and may cause motor and cognitive neuronal damage. Once in the CNS, T. cruzi triggers immune responses that the purinergic system can regulate. Treatment for CD is based on benznidazole (BNZ); however, this agent has negative side-effects and is toxic to the host. For this reason, we investigated whether resveratrol (RSV), a potent antioxidant and neuroprotective molecule, would modulate purinergic signaling and RSV alone or in combination with BNZ would prevent changes in purinergic signaling and oxidative damage caused by T. cruzi. We infected mice with T. cruzi and treated them with RSV or BNZ for 8 days. Increases in ATP and ADP hydrolysis by NTPDase in the total cortex of infected animals were observed. The treatment with RSV in infected group diminished ATP, ADP, and AMP hydrolysis compared to infected group. The combination of RSV + BNZ decreased AMP hydrolysis in infected animals compared to the INF group, exerting an anti-inflammatory effect. RSV acted as a neuroprotector, decreasing adenosine levels. Infected animals presented an increase of P2X₇ and A_2A density of purine receptors. RSV reduced P2X₇ and A_2A and increased A1 density receptors in infected animals. In addition, infected animals showed higher TBARS and reactive oxygen species (ROS) levels than control. RSV diminished ROS levels in infected mice, possibly due to antioxidant properties. In short, we conclude that resveratrol could act as a neuroprotective molecule, probably preventing inflammatory changes caused by infection by T. cruzi, even though the mice experienced high levels of parasitemia.     

Cytospectrophotometric research on hemoglobin in human erythrocytes. I. The methemoglobin content in intact erythrocytes and its alteration under the influence of chromosmon, ascorbic acid, riboflavin and glutathione

The blood of healthy men and patients with methemoglobinemia of different genesis was incubated with chromosmon, ascorbic acid, riboflavin and glutathione, the percentage of erythrocytes with thorn-shaped protuberances-echinocytes being subsequently determined in the blood smears. The absorbtion spectra at the range 400-650 nm were investigated both in the smooth erythrocytes and in echinocytes. A correlation was found between the percentage of echinocytes and the methemoglobin content in the blood. The methemoglobin amount in the echinocytes was determined to be higher than in the smooth erythrocytes. It is discovered that effects of chromosmon, glutathione and riboflavin on production of methemoglobin depend on the dose, individual peculiarities of erythrocytes and on the illness that caused methemoglobinemia. The calculation of echinocyte percentage may be used as an express-diagnostics of methemoglobinemia and for purposes of studying the effect of methemoglobin-producing substances and drugs.     

Antioxidant effect of red wine polyphenols on red blood cells

The protective effect of red wine polyphenols against hydrogen peroxide (H(2)O(2))-induced oxidation was investigated in normal human erythrocytes (RBCs). RBCs, preincubated with micromolar amounts of wine extract and challenged with H(2)O(2), were analyzed for reactive oxygen species (ROS), hemolysis, methemoglobin production, and lipid peroxidation. All these oxidative modifications were prevented by incubating the RBCs with oak barrel aged red wine extract (SD95) containing 3.5 mM gallic acid equivalent (GAE) of phenolic compounds. The protective effect was less apparent when RBCs were incubated with wines containing lower levels of polyphenols. Furthermore, resveratrol and quercetin, well known red wine antioxidants, showed lower antioxidant properties compared with SD95, indicating that interaction between constituents may bring about effects that are not necessarily properties of the singular components. Our findings demonstrate that the nonalcoholic components of red wine, mainly polyphenols, have potent antioxidant properties, supporting the hypothesis of a beneficial effect of red wine in oxidative stress in human system.     

Resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses

Scavenging or quenching of the reactive oxygen species (ROS) involved in oxidative stress has been the subject of many recent studies. Resveratrol, found in various natural food products, has been linked to decreased coronary artery disease and preventing cancer development. The present study measured the effect of resveratrol on several different systems involving the hydroxyl, superoxide, metal/enzymatic-induced, and cellular generated radicals. The rate constant for reaction of resveratrol with the hydroxyl radical was determined, and resveratrol was found to be an effective scavenger of hydroxyl, superoxide, and metal-induced radicals as well as showing antioxidant abilities in cells producing ROS. Resveratrol exhibits a protective effect against lipid peroxidation in cell membranes and DNA damage caused by ROS. Resveratrol was also found to have a significant inhibitory effect on the NF-kappaB signaling pathway after cellular exposure to metal-induced radicals. It was concluded that resveratrol in foods plays an important antioxidant role.     

Effects of resveratrol and 4-hexylresorcinol on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

The protective effects of resveratrol and 4-hexylresorcinol against oxidative DNA damage in human lymphocytes induced by hydrogen peroxide were investigated. Resveratrol and 4-hexylresorcinol showed no cytotoxicity to human lymphocytes at the tested concentration (10-100 μM). In addition, DNA damage in human lymphocytes induced by H 2 O 2 was inhibited by resveratrol and 4-hexylresorcinol. Resveratrol and 4-hexylresorcinol at concentrations of 10-100 μM induced an increase in glutathione (GSH) levels in a concentration-dependent manner. Moreover, these two compounds also induced activity of glutathione peroxidase (GPX) and glutathione reductase (GR). The activity of glutathione-S-transferase (GST) in human lymphocytes was induced by resveratrol. Resveratrol and 4-hexylresorcinol inhibited the activity of catalase (CAT). These data indicate that the inhibition of resveratrol and 4-hexylresorcinol on oxidative DNA damage in human lymphocytes induced by H 2 O 2 might be attributed to increase levels of GSH and modulation of antioxidant enzymes (GPX, GR and GST).     

Changes in rodent-erythrocyte methemoglobin reductase system produced by two malaria parasites, viz. Plasmodium yoelii nigeriensis and Plasmodium berghei

The methemoglobin reductase system plays a vital role in maintaining the equilibrium between hemoglobin and methemoglobin in blood. Exposure of red blood cells to oxidative stress (pathological/physiological) may cause impairment to this equilibrium. We studied the status of erythrocytic methemoglobin and the related reductase system during Plasmodium yoelii nigeriensis infection in mice and P. berghei infection in mastomys. Malaria infection was induced by intraperitoneal inoculation with 10⁶ infected erythrocytes. The present investigation revealed a significant decrease in the activity of methemoglobin reductase, with a concomitant rise in methemoglobin content during P. yoelii nigeriensis infection in mice erythrocytes. This was accompanied with a significant increase in reduced glutathione and ascorbate levels. The activity of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and glutathione reductase increased with a progressive rise in parasitemia. However, no methemoglobin or associated reductase activity was detected in normal and P. berghei-infected mastomys. P. berghei infection in mastomys resulted in an increase in the level of reduced glutathione and ascorbate in erythrocytes, and also in the activity of lactate dehydrogenase, glucose 6-phosphate dehydrogenase and glutathione reductase. These results suggest that antioxidants/antioxidant enzymes may prevent or reduce the formation of methemoglobin in the host and thereby protect the host from methemoglobinemia.     

Diminution of Oxidative Damage to Human Erythrocytes and Lymphocytes by Creatine: Possible Role of Creatine in Blood

Creatine (Cr) is naturally produced in the body and stored in muscles where it is involved in energy generation. It is widely used, especially by athletes, as a staple supplement for improving physical performance. Recent reports have shown that Cr displays antioxidant activity which could explain its beneficial cellular effects. We have evaluated the ability of Cr to protect human erythrocytes and lymphocytes against oxidative damage. Erythrocytes were challenged with model oxidants, 2, 2''-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H₂O₂) in the presence and absence of Cr. Incubation of erythrocytes with oxidant alone increased hemolysis, methemoglobin levels, lipid peroxidation and protein carbonyl content. This was accompanied by decrease in glutathione levels. Antioxidant enzymes and antioxidant power of the cell were compromised while the activity of membrane bound enzyme was lowered. This suggests induction of oxidative stress in erythrocytes by AAPH and H₂O₂. However, Cr protected the erythrocytes by ameliorating the AAPH and H₂O₂ induced changes in these parameters. This protective effect was confirmed by electron microscopic analysis which showed that oxidant-induced cell damage was attenuated by Cr. No cellular alterations were induced by Cr alone even at 20 mM, the highest concentration used. Creatinine, a by-product of Cr metabolism, was also shown to exert protective effects, although it was slightly less effective than Cr. Human lymphocytes were similarly treated with H₂O₂ in absence and presence of different concentrations of Cr. Lymphocytes incubated with oxidant alone had alterations in various biochemical and antioxidant parameters including decrease in cell viability and induction of DNA damage. The presence of Cr attenuated all these H₂O₂-induced changes in lymphocytes. Thus, Cr can function as a blood antioxidant, protecting cells from oxidative damage, genotoxicity and can potentially increase their lifespan.     

Chemopreventive effect of bilberry (Vaccinium myrtillus) against cisplatin-induced oxidative stress and DNA damage as shown by the comet assay in peripheral blood of rats

The aim of this study was to evaluate the chemopreventive effect of bilberry on cisplatin induced oxidative stress and DNA damage in rat blood. Twenty-one female Wistar-Albino rats were divided into three groups: group I — untreated; group II — treated with cisplatin (single dose 7.5 mg/kg b.w.); and group III — treated with cisplatin (single dose 7.5 mg/kg b.w.) and bilberry (200 mg/kg b.w. for 10 days). Antioxidant enzyme systems including superoxide dismutase, catalase, glutathione peroxidase and the level of malondialdehyde (MDA) that might occur on erythrocytes have been determined and single cell gel electrophoresis (comet) was utilized to evaluate the DNA damage in lymphocytes. Treatment with cisplatin has increased the levels of MDA and decreased antioxidant enzymes in erythrocytes. Comet assay showed significantly higher values at dose of 7.5 mg/kg cisplatin as the result of oxidative DNA damage when compared to the control group. Cisplatin treatment with bilberry resulted in a highly significant (P < 0.05) decreased in the lymphocytes DNA when compared to the cisplatin group. Bilberry has been effective on antioxidant enzyme systems and MDA level and significantly decreased the comets. Our results indicate that bilberry is capable of preventing genotoxic and cytotoxic damage caused by cisplatin in peripheral blood cells in rats.     

Antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to Chattonella marina and hydrogen peroxide: Implications on the cause of fish kills

Chattonella marina, a red tide or harmful algal bloom species, has caused mass fish kills and serious economic loss worldwide, and yet its toxic actions remain highly controversial. Previous studies have shown that this species is able to produce reactive oxygen species (ROS), and therefore postulated that ROS are the causative agents of fish kills. The present study investigates antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to C. marina, compared with responses exposed to equivalent and higher levels of ROS exposure. Even though C. marina can produce a high level of ROS, gills and erythrocytes of sea bream exposed to C. marina for 1 to 6 h showed neither significant induction of antioxidant enzymes nor lipid peroxidation. Antioxidant responses and oxidative damage did not occur as fish mortality began to occur, yet could be induced upon exposure to artificially supplied ROS levels an order of magnitude higher. The result of this study implies that ROS produced by C. marina is not the principal cause of fish kills.     

Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.     

Resveratrol analogues like piceatannol are potent antioxidants as quantitatively demonstrated through the high scavenging ability against reactive oxygen species and methyl radical

Resveratrol (RSV) analogues have attracted much attention because of the expected health functions including antioxidant activities. We have carried out a quantitative determination of the scavenging abilities of six trans-RSV analogues against various reactive oxygen species and methyl radical (hydroxyl radical, superoxide, alkoxyl radical, peroxyl radical, methyl radical, and singlet oxygen). RSV analogues are in general more potent scavenger than the parent RSV. Furthermore, piceatannol (PIC) having two OH groups in the ortho position of resveratrol was found to show 11 times higher scavenging ability against peroxyl radical than parent resveratrol. With the aid of previous theoretical studies, the enhanced antioxidant ability was interpreted based on the effects of substituent that modifies the original resveratrol structure and function.     

Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage

Radiation-induced cellular injury is attributed primarily to the harmful effects of free radicals, which play a key role in irradiation-induced apoptosis. In this study, we investigated the radioprotective efficacy of edaravone, a licensed clinical drug and a powerful free radical scavenger that has been tested against γ-irradiation-induced cellular damage in cultured human peripheral blood lymphocytes in studies of various diseases. Edaravone was pre-incubated with lymphocytes for 2 h prior to γ-irradiation. It was found that pretreatment with edaravone increased cell viability and inhibited generation of γ-radiation-induced reactive oxygen species (ROS) in lymphocytes exposed to 3 Gy γ-radiation. In addition, γ-radiation decreased antioxidant enzymatic activity, such as superoxide dismutase and glutathione peroxidase, as well as the level of reduced glutathione. Conversely, treatment with 100 μM edaravone prior to irradiation improved antioxidant enzyme activity and increased reduced glutathione levels in irradiated lymphocytes. Importantly, we also report that edaravone reduced γ-irradiation-induced apoptosis through downregulation of Bax, upregulation of Bcl-2, and consequent reduction of the Bax:Bcl-2 ratio. The current study shows edaravone to be an effective radioprotector against γ-irradiation-induced cellular damage in lymphocytes in vitro. Finally, edaravone pretreatment significantly reduced DNA damage in γ-irradiated lymphocytes, as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment) (p < 0.05). Thus, the current study indicates that edaravone offers protection from radiation-induced cytogenetic alterations.     

Resveratrol ameliorates ionizing irradiation-induced long-term hematopoietic stem cell injury in mice

Our recent studies showed that total body irradiation (TBI) induces long-term bone marrow (BM) suppression in part by induction of hematopoietic stem cell (HSC) senescence through NADPH oxidase 4 (NOX4)-derived reactive oxygen species (ROS). Therefore, in this study we examined whether resveratrol (3,5,4′-trihydroxy-trans-stilbene), a potent antioxidant and a putative activator of Sirtuin 1 (Sirt1), can ameliorate TBI-induced long-term BM injury by inhibiting radiation-induced chronic oxidative stress and senescence in HSCs. Our results showed that pretreatment with resveratrol not only protected mice from TBI-induced acute BM syndrome and lethality but also ameliorated TBI-induced long-term BM injury. The latter effect is probably attributable to resveratrol-mediated reduction of chronic oxidative stress in HSCs, because resveratrol treatment significantly inhibited TBI-induced increase in ROS production in HSCs and prevented mouse BM HSCs from TBI-induced senescence, leading to a significant improvement in HSC clonogenic function and long-term engraftment after transplantation. The inhibition of TBI-induced ROS production in HSCs is probably attributable to resveratrol-mediated downregulation of NOX4 expression and upregulation of Sirt1, superoxide dismutase 2 (SOD2), and glutathione peroxidase 1 expression. Furthermore, we showed that resveratrol increased Sirt1 deacetylase activity in BM hematopoietic cells; and Ex527, a potent Sirt1 inhibitor, can attenuate resveratrol-induced SOD2 expression and the radioprotective effect of resveratrol on HSCs. These findings demonstrate that resveratrol can protect HSCs from radiation at least in part via activation of Sirt1. Therefore, resveratrol has the potential to be used as an effective therapeutic agent to ameliorate TBI-induced long-term BM injury.     

Antioxidants reveal an inverted U‐shaped dose‐response relationship between reactive oxygen species levels and the rate of aging in Caenorhabditis elegans

Reactive oxygen species (ROS) are potentially toxic, but they are also signaling molecules that modulate aging. Recent observations that ROS can promote longevity have to be reconciled with the numerous claims about the benefits of antioxidants on lifespan. Here, three antioxidants [N‐acetylcysteine (NAC), vitamin C, and resveratrol (RSV)] were tested on Caenorhabditis elegans mutants that alter drug uptake, mitochondrial function, and ROS metabolism. We observed that like pro‐oxidants, antioxidants can both lengthen and shorten lifespan, dependent on concentration, genotypes, and conditions. The effects of antioxidants thus reveal an inverted U‐shaped dose–response relationship between ROS levels and lifespan. In addition, we observed that RSV can act additively to both NAC and paraquat, to dramatically increase lifespan. This suggests that the effect of compounds that modulate ROS levels can be additive when their loci of action or mechanisms of action are sufficiently distinct.     

Influence of novel naphthalimide-based organoselenium on genotoxicity induced by an alkylating agent: the role of reactive oxygen species and selenoenzymes

Abstract

Objective

The protection conferred by a series of synthetic organoselenium compounds against genotoxicity and oxidative stress induced by a reference mutagen cyclophosphamide (CP) was assessed.

Method

Genotoxicity was induced in mice by CP treatment (25 mg/kg b.w.) for 10 consecutive days. Organoselenium compounds (3 mg/kg b.w.) were administered orally in a concomitant and pretreatment schedule. DNA damage in peripheral blood lymphocytes and frequency of chromosomal aberration in the bone marrow cells were measured. Liver tissues were collected for analysis of the activity of antioxidant and detoxifying enzymes, lipid peroxidation (LPO) level, glutathione content, and histopathology.

Results

Exposure to CP not only led to a significant increase in the percent of chromosomal aberration and DNA damage, but also enhanced generation of hepatic reactive oxygen species (ROS) and LPO level. The organoselenium compounds demonstrated marked functional protection against CP-induced genotoxicity. DNA damage and chromosomal aberration along with ROS generation were attenuated in the organoselenium-treated mice compared with the CP-treated control mice. CP caused marked depression in the activities of the selenoenzymes (glutathione peroxidase (GPx) and thioredoxin reductase (TRxR)) and other detoxifying and antioxidant enzymes, while treatment with organoselenium compounds restored all these activities towards normal.

Discussion

The protective effect of these compounds may be primarily associated with the improvement of the activity of antioxidant and detoxifying enzymes (including the selenoenzymes, GPx, and TRxR) that are known to protect the DNA and other cellular components from oxidative damage.     

The effect of antioxidants on in vivo and in vitro methemoglobin formation in erythrocytes of patients with Parkinson’s disease

Methemoglobin formation was examined in erythrocytes of 16 patients with Parkinson’s disease (PD) (stage 3–4 by the Hoehn and Yahr scale). The patients receiving levodopa-containing drugs (madopar, nakom) were also treated with intramuscular injections of mexidol (daily dose 100 mg/day) for 14 days. Control group included 12 clinically healthy persons. The erythrocyte methemoglobin content was determined by electronic paramagnetic resonance (EPR) using the EPR signal intensity with the g-factor 6.0. The methemoglobin content was significantly higher in erythrocytes of PD patients than in healthy donors. The complex therapy with mexidol normalized the methemoglobin content in erythrocytes of PD patients. Incubation in vitro of erythrocytes of donors and PD patients with acrolein increased the methemoglobin content, while incubation with carnosine normalized the methemoglobin content in erythrocytes of PD patients. Prophylactic (i.e. before acrolein addition) and therapeutic administration of carnosine to the incubation system with acrolein decreased the methemoglobin content to its initial level. Results of this study suggest that inclusion of the antioxidants mexidol and carnosine in the scheme of basic therapy of PD may reduce side effects associated with methemoglobinemia.     

MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis

DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation.     

Daily grape juice consumption reduces oxidative DNA damage and plasma free radical levels in healthy Koreans

Grape contains flavonoids with antioxidant properties which are believed to be protective against various types of cancer. This antioxidative protection is possibly provided by the effective scavenging of reactive oxygen species (ROS), thus defending cellular DNA from oxidative damage and potential mutations. This study of healthy adults tested whether a daily regimen of grape juice supplementation could reduce cellular DNA damage in peripheral lymphocytes and reduce the amount of free radicals released. Sixty-seven healthy volunteers (16 women and 51 men) aged 19-57 years were given 480 ml of grape juice daily for 8 weeks in addition to their normal diet, and blood samples were drawn before and after the intervention. The DNA damage was determined by using the single cell gel (comet) assay with alkaline electrophoresis and was quantified by measuring tail length (TL). Levels of free radicals were determined by reading the lucigenin-perborate ROS generating source, using the Ultra-Weak Chemiluminescence Analyzer System. Grape juice consumption resulted in a significant decrease in lymphocyte DNA damage expressed by TL (before supplementation: 88.75 +/- 1.55 microm versus after supplementation: 70.25 +/- 1.31 microm; P=0.000 by paired t-test). Additionally, grape juice consumption for 8 weeks reduced the ROS/photon count by 15%, compared to the beginning of the study. The preventive effect of grape juice against DNA damage was simultaneously shown in both sexes. These results indicate that the consumption of grape juice may increase plasma antioxidant capacity, resulting in reduced DNA damage in peripheral lymphocytes achieved at least partially by a reduced release of ROS. Our findings support the hypothesis that polyphenolic compounds contained in grape juice exert cancer-protective effects on lymphocytes, limiting oxidative DNA damage possibly via a decrease in free radical levels.