Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide MOLECULAR INSIGHTS INTO THE GENOME INSTABILITY OF PEROXIREDOXIN-NULL STRAINS

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1 Delta tsa2 Delta) are more resistant to hydrogen peroxide than wildtype (WT) cells and consume it faster under fermentative conditions. Also, tsa1 Delta tsa2 Delta cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1 Delta tsa2 Delta cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu, Zn-superoxide dismutase (Sod1), whose expression and activity increased similar to 5- and 2-fold, respectively, in tsa1 Delta tsa2 Delta compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1 Delta tsa2 Delta cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of (14)C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1 Delta tsa2 Delta cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.

Accumulation of (5 ` S)-8,5 `-cyclo-2 `-deoxyadenosine in organs of Cockayne syndrome complementation group B gene knockout mice

Cockayne syndrome (CS) is a human genetic disorder characterized by sensitivity to UV radiation, neurodegeneration, premature aging among other phenotypes. CS complementation group B (CS-B) gene (csb) encodes the CSB protein (CSB) that is involved in base excision repair of a number of oxidatively induced lesions in genomic DNA in vivo. We hypothesized that CSB may also play a role in cellular repair of the DNA helix-distorting tandem lesion (5`S)-8,5`-cyclo-2`-deoxyadenosine (S-cdA). Among many DNA lesions. S-cdA is unique in that it represents a concomitant damage to both the sugar and base moieties of the same nucleoside. Because of the presence of the C8-C5` covalent bond, S-cdA is repaired by nucleotide excision repair unlike most of other oxidatively induced lesions in DNA, which are subject to base excision repair. To test our hypothesis, we isolated genomic DNA from brain, kidney and liver of wild type and csb knockout (csb(-/-)) mice. Animals were not exposed to any exogenous oxidative stress before the experiment. DNA samples were analysed by liquid chromatography/mass spectrometry with isotope-dilution. Statistically greater background levels of S-cdA were observed in all three organs of csb(-/-) mice than in those of wild type mice. These results suggest the in vivo accumulation of S-cdA in genomic DNA due to lack of its repair in csb(-/-) mice. Thus, this study provides, for the first time, the evidence that CSB plays a role in the repair of the DNA helix-distorting tandem lesion S-cdA. Accumulation of unrepaired S-cdA in vivo may contribute to the pathology associated with CS. Published by Elsevier B.V.

Aminoacetone, a putative endogenous source of methylglyoxal, causes oxidative stress and death to insulin-producing RINm5f cells

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4+ ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O-2(center dot-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 mu M) and Fe3+ ion (100 mu M) addition to the cell cultures had no effect, whereas Cu2+ (5.0-100 mu M) significantly increased cell death. Supplementation of the AA- and Cu2+-containing culture medium with antioxidants, such as catalase (5.0 mu M), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu2+ ions (10-50 mu M) relative to control cultures. This may imply higher activity of antioxidant enzymes C, in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) Plus Cu2+ (100 mu M) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

The role of mitochondrial DNA damage in the citotoxicity of reactive oxygen species

Mitochondria contain their own genome, a small circular molecule of around 16.5 kbases. The mitochondrial DNA (mtDNA) encodes for only 13 polypeptides, but its integrity is essential for mitochondrial function, as all 13 proteins are regulatory subunits of the oxidative phosphorylation complexes. Nonetheless, the mtDNA is physically associated with the inner mitochondrial membrane, where the majority of the cellular reactive oxygen species are generated. In fact, the mitochondrial DNA accumulates high levels of oxidized lesions, which have been associated with several pathological and degenerative processes. The cellular responses to nuclear DNA damage have been extensively studied, but so far little is known about the functional outcome and cellular responses to mtDNA damage. In this review we will discuss the mechanisms that lead to damage accumulation and the in vitro models we are establishing to dissect the cellular responses to oxidative damage in the mtDNA and to sort out the differential cellular consequences of accumulation of damage in each cellular genome, the nuclear and the mitochondrial genome.

Increased SOD1 association with chromatin, DNA damage, p53 activation, and apoptosis in a cellular model of SOD1-linked ALS

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multifactorial and remain unclear. Here we examined DNA damage;p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93) --> Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis. (C) 2010 Elsevier B.V. All rights reserved.

Coordenação entre as respostas da catalase e da ascorbato peroxidade em folhas de feijão caupi submetidas a diferentes fontes de peróxido de hidrogênio

The plants are often exposed to variations in environmental conditions that may trigger metabolic disturbances leading to a consequent loss in productivity of crops. These stressful conditions usually induce an accumulation of reactive oxygen species (ROS) in the cell, a condition known how oxidative stress. Among these species, hydrogen peroxide (H2O2) is an important molecule involved in numerous signaling mechanisms. The present study aimed to understand the relationship between the different enzymatic mechanisms of elimination of H2O2 by catalase (CAT) and ascorbate peroxidase (APX) in leaf tissues of seedlings of the species Vigna unguiculata L. Walp, under conditions of oxidative stress induced by application of CAT inhibitor, 3-amino-1,2,4-triazole (3-AT), and H2O2 itself on the roots. Three experiments were conducted. The first experiment was performed applying the compound 3-AT (5 mM) during the time (hours). In the second experiment, seedlings were exposed to different concentrations of H2O2 (2.5, 5.0, 7.5, 10 mM) for 48 h. The third strategy included the pre-treatment with H2O2 (2.5 mM) for 24 h, followed by subsequent treatment with the inhibitor 3-AT and recovery control condition. Treatment with 3-AT causes a strong inhibition of CAT activity in leaf tissues accompanied by an increase of activity of APX. However a decrease in oxidative damage to lipids is not observed as indicated by TBARS. It was observed that activity of APX is directly linked to the content of peroxide. Inductions in the activities of CAT and APX were observed mainly in the seedlings treated with 2.5 mM H2O2. This can be associated with a decrease in oxidative damage to lipids. In contrast, one same tendency was not observed in treatments with higher concentrations of this ROS. These results suggest that the concentration of 2.5 mM H2O2 can induce responses antioxidants later in seedling cowpea. This concentration when applied as pre-treatment for 24 h promoted an induction systems removers CAT and APX, both in activity and in terms of gene expression. However this increment was not observed in the recovered plants and the plants subsequently subjected to 3-AT. Additionally, the pretreatment was not sufficient to attenuate the inhibition of CAT activity and oxidative damage to lipids caused by the subsequent application of this inhibitor. The results showed that the application of 3-AT and H2O2 in the root systems of seedlings of cowpea promote changes in the parameters analyzed in leaf tissues that indicate a direct response to the presence of these factors or systemic signaling mecanisms. H2O2 appears to activate the responses of two antioxidant systems in this study thar does not promote greater protection in case of additional treatment with 3-AT. This demonstrates the importance of the CAT system. In this work, complete results indicate that there is a difference between the signaling and the effects caused by exposure to H2O2 and by treatment with 3-AT

Análise da Expressão de APE1 após Estresse Oxidativo Induzido em Células Proficientes e Deficientes na Via de Reparo por Excisão de Nucleotídeos

Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment.

Proteômica comparativa de linhagens celulares humanas expostas a estresse oxidativo induzido por riboflavina fotossensibilizada

Reactive oxygen species (ROS) are continuously generated and can be derived from cellular metabolism or induced by exogenous factors, in addition, have the capacity to damage molecules like DNA and proteins. BER is considered the main route of DNA damage oxidative repair, however, several studies have demonstrated the importance of the proteins participation of other ways to correct these injuries. NER enzymes deficiency, such as CSB and XPC, acting in the damage recognition step in the two subways this system influences the effectiveness of oxidative damage repair. However, the mechanisms by which cells deficient in these enzymes respond to oxidative stress and its consequences still need to be better understood. Thus, the aim of this study was to perform a proteomic analysis of cell lines proficient and deficient in NER, exposed to oxidative stress, in order to identify proteins involved, directly or not, in response to oxidative stress and DNA repair. For this, three strains of human fibroblasts, MRC5-SV, CS1AN (CSBdeficient) and XP4PA (XPC-deficient) were treated with photosensitized riboflavin and then carried out the differentially expressed proteins identification by mass spectrometry. From the results, it was observed in MRC5-SV increase expression in most of the proteins involved in cellular defense, an expected response to a normal cell line subjected to stress. CS1AN showed a response disjointed, it is not possible to establish many interactions between the proteins identified, may be one explanation for their sensitivity to treatment with riboflavin and other oxidants and increased cell death probably by induction of pro-apoptotic pathways. Already XP4PA showed higher expression of apoptosis-blocking proteins, as there was inhibition or reduced expression of others involved with the activation of this process, suggesting the activation of an anti-apoptotic mechanism in this lineage, which may help explain the high susceptibility to develop cancers in XPC individuals. These results also contribute to elucidate action mechanisms of NER in oxidative damage and the understanding of important routes in the oxidative stress correlation, repair and malignant tumors formation

Estudo do efeito dos metabólitos do benzeno e tolueno sobre as mitocôndris cerebrais e hepáticas de ratos

Aim: The aim of this work was to investigate the hypothesis that catechol and 3MC inhibit FADH2-linked basal respiration in mitochondria isolated from rat liver and brain homogenates. Moreover, catechol ability to induce DNA damage in rat brain cells through the comet assay (alkaline single-cell gel electrophoresis assay) was also observed. Methods: Two different catechols were evaluated: pirocatechol (derived from benzene) and 3-methylcatechol (derived from toluene); rat liver and brain homogenates were incubated with 1mM catechol at pH 7.4 for up to 30 minutes. After that, mitochondrial fractions were isolated by differential centrifugation. Basal oxygen uptake was measured using a Clark-type electrode after the addition of 10 mM sodium succinate for a period of 12 minutes. In additional experiments, rat brain cells were treated with 1, 5 and 10mM pirocatechol for up to 20 minutes at 37º C, and submitted to electrophoresis. Results: Catechols (pirocatechol and 3methylcatechol) induced a time-dependent partial inhibition of FADH2-linked basal mitochondrial respiration. Indeed, pirocatechol was able to produce a dosedependent DNA oxidative damage in rat brain cells by 2 and 4 injury levels. These results suggest that reactive oxygen species generated by the oxidation of catechols, induced an impairment on mitochondrial respiration and a DNA damage, which might be related to their citotoxicity. Conclusion: Catechols produced an inhibition of basal respiration associated to FADH2 in isolated liver and brain mitochondria; 3-methylcatechol, at the same concentration, produced similar toxicity in the mitochondrial model. Indeed, pirocatechol induced a DNA damage in rat brain cells, mainly observed in comets formation and consequent DNA degradation

An Anisotropic Model of Damage and Unilateral Effect for Brittle Materials

This work deals with the initial applications and formulation of an aniscitropic plastic-damage constitutive model proposed for non-linear analysis of reinforced concrete structures submitted to a loading with change of the sign. The original constitutive model is based on the fundamental hypothesis of energy equivalence between real and continuous medium following the concepts of the Continuum Damage Mechanics. The concrete is assumed as an initial elastic isotropic medium presenting anisotropy, permanent strains and bimodularity (distinct elastic responses whether traction or compression stress states prevail) induced by damage evolution. In order to take into account the bimodularity, two damage tensors governing the rigidity in tension or compression regimes are introduced. Then, some conditions are introduced in the original version of the model in order to simulate the damage unilateral effect. The three-dimensional version of the proposed model is analyzed in order to validate its formulation when compared to micromechanical theory. The one-dimensional version of the model is applied in the analyses of a reinforced concrete beam submitted to a loading with change of the sign. Despite the parametric identification problems, the initial applications show the good performance of the model.

Could zinc prevent reproductive alterations caused by cigarette smoke in male rats?

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

DNA damage and nitric oxide production in mice following infection with L. chagasi

Leishmania chagasi, which causes visceral leishmaniasis in South America, is an obligate intracellular protozoan. Production of nitric oxide by macrophages during the inflammatory response is one of the main microbicidal mechanisms against this parasite. The goal of this study was to evaluate whether L. chagasi infection causes DNA damage in peripheral blood and spleen cells of Balb/c mice and whether such damage may be related to NO production. Balb/c mice were either infected with L chagasi or maintained as controls. The single-cell gel electrophoresis (comet) assay was used to measure DNA damage in peripheral blood and spleen cells, and the Griess reaction was used to measure NO production in the spleen. L chagasi infection induced DNA damage in peripheral blood and spleen cells of infected mice. Macrophages from the control group, challenged with L. chagasi, showed significantly (p < 0.05) greater NO production, compared to non-challenged cells. Treatment of spleen cells with N(G)-monomethyl-L-arginine (LNMMA) caused a significant reduction of NO production and DNA damage (p < 0.05). Our results indicate that L. chagasi induces DNA damage in the peripheral blood and spleen cells and that NO not only causes killing of the parasite but also induces DNA damage in adjacent cells. (C) 2011 Elsevier B.V. All rights reserved.

DNA damage and aberrant crypt foci as putative biomarkers to evaluate the chemopreventive effect of annatto (Bixa orellana L.) in rat colon carcinogenesis

Chemoprevention opens new perspectives in the prevention of cancer and other degenerative diseases. Use of target-organ biological models at the histological and genetic levels can markedly facilitate the identification of such potential chemopreventive agents. Colon cancer is one of the highest incidence rates throughout the world and some evidences have indicated carotenoids as possible agents that decrease the risk of colorectal cancer. In the present study, we evaluate the activity of annatto (Bixa orellaria L.), a natural food colorant rich in carotenoid, on the formation of aberrant crypt foci (ACF) induced by dimethy1hydrazine (DMH) in rat colon. Further, we investigate, the effect of annatto on DMH-induced DNA damage, by the comet assay. Male Wistar rats were given s.c. injections of DMH (40 mg/kg body wt.) twice a week for 2 weeks to induce ACE They also received experimental diets containing annatto at 20, 200 or 1000 ppm for five 5 weeks before (pre-treatment), or 10 weeks after (post-treatment) DMH treatment. In both protocols the rats were sacrificed on week 15th. For the comet assay, the animals were fed with the same experimental diets for 2 weeks. Four hours before the sacrifice, the animals received an s.c. injection of DMH (40 mg/kg body wt.). Under such conditions, dietary administration of 1000 ppm annatto neither induce DNA damage in blood and colon cells nor aberrant crypt foci in rat distal colon. Conversely, annatto was successful in inhibiting the number of crypts/colon (animal), but not in the incidence of DMH-induced ACF, mainly when administered after DMH. However, no antigenotoxic effect was observed in colon cells. These findings suggest possible chemopreventive effects of annatto through their modulation of the cryptal cell proliferation but not at the initiation stage of colon carcinogenesis. (c) 2005 Elsevier B.V. All rights reserved.

Protective effects of lemongrass (Cymbopogon citratus STAPF) essential oil on DNA damage and carcinogenesis in female Balb/C mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dietary intervention with narrow-leaved cattail rhizome flour (Typha angustifolia L.) prevents intestinal inflammation in the trinitrobenzenesulphonic acid model of rat colitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)