Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

Clustered DNA damages—two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands—are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1–1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

Progression of Lipid Peroxidation Measured as Thiobarbituric Acid Reactive Substances, Damage to DNA and Histopathological Changes in the Liver of Rats Subjected to a Methionine-Choline-Deficient Diet

Methionine-choline-deficient diet represents a model for the study of the pathogenesis of steatohepatitis. Male rats were divided into three groups, the first group receiving a control diet and the other two groups receiving a methionine-choline-deficient diet for 1 month (MCD1) and for 2 months (MCD2), respectively. The livers of the animals were collected for the determination of vitamin E, thiobarbituric acid reactive substances (TBARS), GSH concentration, DNA damages, and for histopathological evaluation. The hepatic TBARS and GSH content was higher (P < 0.05) in the groups receiving the experimental diet (MCD1 and MCD2) compared to control diet, and hepatic vitamin E concentration differed (P < 0.05) between the MCD1 and MCD2 groups, with the MCD2 group presenting a lower concentration. Damage to hepatocyte DNA was greater (P < 0.05) in the MCD2 group (262.80 DNA injuries/100 hepatocytes) compared to MCD1 (136.4 DNA injuries/100 hepatocytes) and control diet (115.83 DNA injuries/100 hepatocytes). Liver histopathological evaluation showed that steatosis, present in experimental groups was micro- and macro-vesicular and concentrated around the centrolobular vein, zone 3, with preservation of the portal space. The inflammatory infiltrate was predominantly periductal and the steatosis and inflammatory infiltrate was similar in the MCD1 and MCD2 groups, although the presence of Mallory bodies was greater in the MCD2 group. The study describes the contribution of a methionine-choline-deficient diet to the progression of steatosis, lipid peroxidation and hepatic DNA damage in rats, serving as a point of reflection about the role of these nutrients in the western diet and the elevated non-alcoholic steatohepatitis rates in humans.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

Design of new tools to improve the efficacy of DNA-damaging drugs used in cancer therapy

Résumé Les tumeurs sont diverses et hétérogènes, mais toutes partagent la capacité de proliférer sans contrôle. Une prolifération dérégulée de cellules couplée à une insensibilité à une réponse apoptotique constitue une condition minimale pour que l'évolution d'une tumeur se produise. Un des traitements les plus utilisés pour traité le cancer à l'heure actuelle sont les chimiothérapies, qui sont fréquemment des composés chimiques qui induisent des dommages dans l'ADN. Les agents anticancéreux sont efficaces seulement quand les cellules tumorales sont plus aisément tuées que le tissu normal environnant. L'efficacité de ces agents est en partie déterminée par leur capacité à induire l'apoptose. Nous avons récemment démontré que la protéine RasGAP est un substrat non conventionnel des caspases parce elle peut induire à la fois des signaux anti et pro-apoptotiques, selon l'ampleur de son clivage par les caspases. A un faible niveau d'activité des caspases, RasGAP est clivé, générant deux fragments (le fragment N et le fragment C). Le fragment N semble être un inhibiteur général de l'apoptose en aval de l'activation des caspases. À des niveaux plus élevés d'activité des caspases, la capacité du fragment N de contrecarrer l'apoptose est supprimée quand il est clivé à nouveau par les caspases. Ce dernier clivage produit deux nouveaux fragments, N 1 et N2, qui contrairement au fragment N sensibilisent efficacement des cellules cancéreuses envers des agents chimiothérapeutiques. Dans cette étude nous avons prouvé qu'un peptide, appelé par la suite TAT-RasGAP317-326, qui est dérivé du fragment N2 de RasGAP et est rendu perméable aux cellules, sensibilise spécifiquement des cellules cancéreuses à trois génotoxines différentes utilisées couramment dans des traitements anticancéreux, et cela dans des modèles in vitro et in vivo. Il est important de noté que ce peptide semble ne pas avoir d'effet sur des cellules non cancéreuses. Nous avons également commencé à caractériser les mécanismes moléculaires expliquant les fonctions de sensibilisation de TAT-RasGAP317-326. Nous avons démontré que le facteur de transcription p53 et une protéine sous son activité transcriptionelle, nommée Puma, sont indispensables pour l'activité de TAT-RasGAP317-326. Nous avons également prouvé que TAT-RasGAP317-326 exige la présence d'une protéine appelée G3BP1, une protéine se liant a RasGAP, pour potentialisé les effets d'agents anticancéreux. Les données obtenues dans cette étude montrent qu'il pourrait être possible d'augmenter l'efficacité des chimiothérapies à l'aide d'un composé capable d'augmenter la sensibilité des tumeurs aux génotoxines et ainsi pourrait permettre de traiter de manière plus efficace des patients sous traitement chimiothérapeutiques. Summary Tumors are diverse and heterogeneous, but all share the ability to proliferate without control. Deregulated cell proliferation coupled with suppressed apoptotic sensitivity constitutes a minimal requirement upon which tumor evolution occurs. One of the most commonly used treatments is chemotherapy, which frequently uses chemical compounds that induce DNA damages. Anticancer agents are effective only when tumors cells are more readily killed than the surrounding normal tissue. The efficacy of these agents is partly determined by their ability to induce apoptosis. We have recently demonstrated that the protein RasGAP is an unconventional caspase substrate because it can induce both anti- and pro-apoptotic signals, depending on the extent of its cleavage by caspases. At low levels of caspase activity, RasGAP is cleaved, generating an N-terminal fragment (fragment N) and a C-terminal fragment (fragment C). Fragment N appears to be a general Mocker of apoptosis downstream of caspase activation. At higher levels of caspase activity, the ability of fragment N to counteract apoptosis is suppressed when it is further cleaved. This latter cleavage event generates two fragments, N1 and N2, which in contrast to fragment N potently sensitizes cancer cells toward DNA-damaging agents induced apoptosis. In the present study we show that a cell permeable peptide derived from the N2 fragment of RasGAP, thereafter called TAT-RasGAP317-326, specifically sensitizes cancer cells to three different genotoxins commonly used in chemotherapy in vitro and in vivo models. Importantly this peptide seems not to have any effect on non cancer cells. We have also started to characterize the molecular mechanisms underlying the sensitizing functions of TAT-RasGAP317-326. We have demonstrated that the p53 transcription factor and a protein under its transcriptional activity, called Puma, are required for the activity of TATRasGAP317-326. We have also showed that TAT-RasGAP317-326 requires the presence of a protein called G3BP1, which have been shown to interact with RasGAP, to increase the effect of the DNA-damaging drug cisplatin. The data obtained in this study showed that it is possible to increase the efficacy of current used chemotherapies with a compound able to increase the efficacy of genotoxins which could be beneficial for patients subjected to chemotherapy.

The genotoxic effects of DNA lesions induced by artificial UV-radiation and sunlight

Solar radiation sustains and affects all life forms on Earth. The increase in solar UV-radiation at environmental levels, due to depletion of the stratospheric ozone layer, highlights serious issues of social concern. This becomes still more dramatic in tropical and subtropical regions where radiation-intensity is still higher. Thus, there is the need to evaluate the harmful effects of solar UV-radiation on the DNA molecule as a basis for assessing the risks involved for human health, biological productivity and ecosystems. In order to evaluate the profile of DNA damage induced by this form of radiation and its genotoxic effects, plasmid DNA samples were exposed to artificial-UV lamps and directly to sunlight. The induction of cyclobutane pyrimidine dimer photoproducts (CPDs) and oxidative DNA damage in these molecules were evaluated by means of specific DNA repair enzymes. On the other hand, the biological effects of such lesions were determined through the analysis of the DNA inactivation rate and mutation frequency, after replication of the damaged pCMUT vector in an Escherichia coli MBL50 strain. The results indicated the induction of a significant number of CPDs after exposure to increasing doses of UVC, UVB, UVA radiation and sunlight. Interestingly, these photoproducts are those lesions that better correlate with plasmid inactivation as well as mutagenesis, and the oxidative DNA damages induced present very low correlation with these effects. The results indicated that DNA photoproducts play the main role in the induction of genotoxic effects by artificial UV-radiation sources and sunlight. (C) 2010 Elsevier B.V. All rights reserved.

Análise do efeito de polimorfismos não-sinônimos em genes de reparo de DNA da via BER na resposta inflamatória da meningite

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

Levels of DNA damage in blood leukocyte samples from non-diabetic and diabetic female rats and their fetuses exposed to air or cigarette smoke

dThe objective of the present study was to evaluate DNA damage level in blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke, and to correlate the findings with levels of DNA damage detected in blood leukocyte samples from their fetuses. A total of 20 rats were distributed into four experimental groups: non-diabetic (control; G1) and diabetic exposed to filtered air (G2): non-diabetic (G3) and diabetic (G4) exposed to cigarette smoke. Rats placed into whole-body exposure chambers were exposed for 30 min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. Diabetes was induced by a pancreatic beta-cytotoxic agent, streptozotocin (40 mg/kg b.w.). At day 21 of pregnancy, each rat was anesthetized and humanely killed to obtain maternal and fetal blood samples for genotoxicity analysis using the alkaline comet assay. G2, G3 and G4 dams presented higher DNA damage values in tail moment and tail length as compared to G1 group. There was a significant positive correlation between DNA damage levels in blood leukocyte samples from G2 and G3 groups (tail moment); G3 and G4 groups (tail length) and G3 group (tail intensity) and their fetuses. Thus, this study showed the association of severe diabetes and tobacco cigarette smoke exposure did not exacerbate levels of maternal and fetal DNA damages related with only diabetes or cigarette smoke exposure. Based on the results obtained and taking into account other published data, maternal diabetes requires rigid clinical control and public health and education campaigns should be increased to encourage individuals, especially pregnant women, to stop smoking. (C) 2008 Elsevier B.V. All rights reserved.

Evaluation of the interactions of DNA with the textile dyes Disperse Orange 1 and Disperse Red 1 and their electrolysis products using an electrochemical biosensor

A disposable pencil graphite electrode modified with dsDNA was used in combination with square wave voltammetry in order to evaluate the interaction of DNA with the textile dyes Disperse Orange 1 (DO1) and Disperse Red 1 (DR1), and with the products of their electrolysis. Significant changes in the characteristic oxidation peaks of the guanine and adenine moieties of immobilized dsDNA were observed after incubation of the modified electrode for 180 s in solutions of the dyes in their original forms. The same was observed using the electrolysis products obtained by oxidation and reduction conversions. The oxidation peak currents of the guanine and adenine moieties decreased when the concentrations of DO1 and DR1 were increased up to 5.0 × 10 -6 and 1.0 × 10-6 mol L-1, respectively; the signal decreases were more pronounced after interaction with the oxidized dyes, compared to the reduced compounds. The interactions between DNA and DO1, DR1, and the electrolyzed dyes were further investigated by UV-vis spectrophotometry in solution, and different effects such as hypochromism and hyperchromism were observed in the resulting DNA spectra. The investigated interactions showed clear evidence of changes in the DNA structure, and suggested a predominant intercalation mode leading to damage in the biomolecule. © 2013 Elsevier B.V.

SNPs in DNA repair genes associated to meningitis and host immune response

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis. The patient genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. A higher frequency (P<0.05) of APE1 Glu allele in bacterial meningitis (BM) and aseptic meningitis (AM) patients was observed. The genotypes Asn/Asn in control group and Asn/Glu in BM group was also higher. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs is significantly higher in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 Glu allele or OGG1 Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1 Asn148Glu, OGG1 Ser326Cys or PARP-1 Val762Ala. Moreover, reduction in the levels of IL-6, IL-1Ra, MCP-1/CCL2 and IL-8/CXCL8 was observed in the presence of APE1 Glu allele in BM patients. In conclusion, we obtained indications of an effect of SNPs in DNA repair genes on the regulation of immune response in meningitis.

Abortive base-excision repair of radiation-induced clustered DNA lesions in Escherichia coli

It has been postulated that ionizing radiation produces a unique form of cellular DNA damage called “clustered damages” or “multiply damaged sites”. Here, we show that clustered DNA damages are indeed formed in Escherichia coli by ionizing radiation and are converted to lethal double-strand breaks during attempted base-excision repair. In wild-type cells possessing the oxidative DNA glycosylases that cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during postirradiation incubation and also in a dose-dependent fashion. E. coli mutants lacking these enzymes do not form double-strand breaks postirradiation and are substantially more radioresistant than wild-type cells. Furthermore, overproduction of one of the oxidative DNA glycosylases in mutant cells confers a radiosensitive phenotype and an increase in the number of double-strand breaks. Thus, the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.

Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus.

Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. Using confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number of XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using beta-galactosidase-XPG fusion constructs (beta-gal-XPG) transfected into HeLa cells, we have identified a single region of XPG that is evidently responsible both for foci formation and for the UV dynamic response. The fusion protein carrying the C terminus of XPG (amino acids 1146-1185) localized beta-gal specific antigenic signal to foci and to the nucleolus regions. After UV irradiation, antigenic beta-gal translocated reversibly from the subnuclear structures to the whole nucleus with kinetics very similar to the movements of XPG protein. These findings lead us to propose a model in which distribution of XPG protein may regulate the rate of DNA repair within transcriptionally active and inactive compartments of the cell nucleus.

Study of DNA damage with a new system for irradiation of samples in a nuclear reactor

FAPESP:97/5550

Análise do efeito de polimorfismos não-sinônimos em genes de reparo de DNA da via BER na resposta inflamatória da meningite

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.