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Background The advances in colorectal cancer (CRC) treatment include the identification of deficiencies in Mismatch Repair (MMR) pathway to predict the benefit of adjuvant 5-fluorouracil (5-FU) and oxaliplatin for stage II CRC and immunotherapy. Defective MMR contributes to chemoresistance in CRC. A growing body of evidence supports the role of Poly-(ADP-ribose) polymerase (PARP) inhibitors, such as Olaparib, in the treatment of different subsets of cancer beyond the tumors with homologous recombination deficiencies. In this work we evaluated the effect of Olaparib on 5-FU cytotoxicity in MMR-deficient and proficient CRC cells and the mechanisms involved. Methods Human colon cancer cell lines, proficient (HT29) and deficient (HCT116) in MMR, were treated with 5-FU and Olaparib. Cytotoxicity was assessed by MTT and clonogenic assays, apoptosis induction and cell cycle progression by flow cytometry, DNA damage by comet assay. Adhesion and transwell migration assays were also performed. Results Our results showed enhancement of the 5-FU citotoxicity by Olaparib in MMR-deficient HCT116 colon cancer cells. Moreover, the combined treatment with Olaparib and 5-FU induced G2/M arrest, apoptosis and polyploidy in these cells. In MMR proficient HT29 cells, the Olaparib alone reduced clonogenic survival, induced DNA damage accumulation and decreased the adhesion and migration capacities. Conclusion Our results suggest benefits of Olaparib inclusion in CRC treatment, as combination with 5-FU for MMR deficient CRC and as monotherapy for MMR proficient CRC. Thus, combined therapy with Olaparib could be a strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC.

Tellurium is a rare element that has been regarded as a toxic, nonessential element, and its biological role is not clearly established. In addition, the biological effects of elemental tellurium and some of its organic and inorganic derivatives have been studied, leading to a set of interesting and promising applications. Diphenyl ditelluride (DPDT), an organic tellurium derivate, showed antioxidant, antigenotoxic, antimutagenic, and anticancer properties. The antioxidant and prooxidant properties of DPDT are complex and depend on experimental conditions, which may explain the contradictory reports of these properties. In addition, DPDT may exert its effects through different pathways, including distinct ones to those responsible for chemotherapy resistance phenotypes: transcription factors, membrane receptors, adhesion, structural molecules, cell cycle regulatory components, and apoptosis pathways. This review aims to present recent advances in our understanding of the biological effects, therapeutic potential, and safety of DPDT treatment. Moreover, original results demonstrating the cytotoxic effects of DPDT in different mammalian cell lines and systems biology analysis are included, and emerging approaches for possible future applications are inferred.

In a cross-sectional study of women in a nursing team at a university hospital in southern Brazil, we studied DNA damage, salivary cortisol levels, and cognition. DNA damage was measured in blood leukocytes with the comet assay and the micronucleus test. Salivary cortisol levels were determined upon waking, 30 min later, and at bedtime. Cognition was evaluated according to the Stroop, Digit span and Word span tests. Cortisol levels on waking up were associated negatively with the number of years the employee worked at the institution and positively with the DNA damage in comet assay. Cognitive scores were lower when the cortisol levels were low at awakening and high at bedtime; and were associated positively with educational level. Cortisol status may influence overall health as well as essential work skills, such as attention.

The sodium valproate has been largely used as an anti-epilepsy drug and, recently, as a putative drug in cancer therapy. However, the treatment with sodium valproate has some adverse effects. In this sense, more effective and secure complexes than sodium valproate should be explored in searching for new active drugs. This study aims to evaluate the cytotoxicity of sodium valproate, mixed ternary mononuclear Cu(II) complexes based on valproic acid (VA) with 1,10-phenanthroline (Phen) or 2,2’- bipyridine (Bipy) ligands - [Cu2(Valp)4], [Cu(Valp)2Phen] and [Cu(Valp)2Bipy] - in yeast Saccharomyces cerevisiae, proficient or deficient in different repair pathways, such as base excision repair (BER), nucleotide excision repair (NER), translesion synthesis (TLS), DNA postreplication repair (PRR), homologous recombination (HR) and non-homologous end-joining (NHEJ). The results indicated that the Cu(II) complexes have higher cytotoxicity than sodium valproate in the following order: [Cu(Valp)2Phen] > [Cu(Valp)2Bipy] > [Cu2(Valp)4] > sodium valproate. The treatment with Cu(II) complexes and sodium valproate induced mutations in S. cerevisiae. The data indicated that yeast strains deficient in BER (Ogg1p), NER (complex Rad1p-Rad10p) or TLS (Rev1p, Rev3p and Rad30p) proteins are associated with increased sensitivity to sodium valproate. The BER mutants (ogg1Δ, apn1Δ, rad27Δ, ntg1Δ and ntg2Δ) showed increased sensitivity to Cu(II) complexes. DNA damage induced by the complexes requires proteins from NER (Rad1p and Rad10p), TLS (Rev1p, Rev3p and Rad30p), PRR (Rad6 and Rad18p) and HR (Rad52p and Rad50p) for efficient repair. Therefore, Cu(II) complexes display enhanced cytotoxicity when compared to the sodium valproate and induce distinct DNA lesions, indicating a potential application as cytotoxic agents.

Grapes are one of the most commonly consumed fruit, in both fresh and processed forms; however, a significant amount is disposed of in the environment. Searching for a use of this waste, the antigenotoxic, antimutagenic, and antioxidant activities of aqueous extracts from organic and conventional Vitis labrusca leaves were determined using V79 cells as model. The antigenotoxic activity was analyzed by the alkaline comet assay using endonuclease III and formamidopyrimidine DNA glycosylase enzymes. The antimutagenic property was assessed through the micronucleus (MN) formation, and antioxidant activities were assessed using 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH(●)) radical scavenging, as well as with superoxide dismutase (SOD) and catalase (CAT) activity assays. In addition, phenolic content and ascorbic acid levels of both extracts were determined. Data showed that both organic and conventional grapevine leaves extracts possessed antigenotoxic and antimutagenic properties. The extract of organic leaves significantly reduced intracellular reactive oxygen species (ROS) levels in V79 cells, and displayed greater ability for DPPH(●) scavenging and higher SOD and CAT activities than extract from conventional leaves. Further, the extract from organic leaves contained higher phenolic and ascorbic acid concentrations. In summary, extracts from organic and conventional grape leaves induced important in vitro biological effects.

In cancer treatment, chemotherapy is one of the main strategies used. The knowledge of the cellular and molecular characteristics of tumors allows the use of more specific drugs, making the removal of tumors more efficient. Among the drugs of choice in these treatments, topoisomerase inhibitors are widely used against different types of tumors. Topoisomerases are enzymes responsible for maintaining the structure of DNA, altering its topological state temporarily during the processes of replication and transcription, in order to avoid supercoiling and entanglements at the double helix. The DNA damage formed as a result of topoisomerase inhibition can be repaired by DNA repair mechanisms. Thus, DNA repair pathways can modulate the effectiveness of chemotherapy. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the main pathways involved in the removal of double strand breaks (DSBs); while nucleotide excision repair (NER) is mainly characterized by the removal of lesions that lead to significant structural distortions in the DNA double helix. Evidence has shown that DSBs are the main type of damage resulting from the inhibition of the DNA topoisomerase II enzyme, and therefore the involvement of HR and NHEJ pathways in the repair process is well established. However, some topoisomerase II inhibitors induce other types of lesions, like DNA adducts, interstrand crosslinks and reactive oxygen species, and studies have shown that other DNA repair pathways might be participating in removing injury induced by these drugs. This review aims to correlate the involvement of proteins from different DNA repair pathways in response to these drugs, with an emphasis on NER.

Garcinia mangostana,popularly known as “mangosteen fruit,” originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 μg/mL] in established test assays (Comet assay, micronucleus test, andSalmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains ofSalmonella typhimurium(with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeastSaccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application.

The present study evaluates antigenotoxic and antimutagenic properties of diphenyl ditelluride (DPDT) against several known mutagens in Chinese hamster lung fibroblasts (V79 cells). DPDT was not cytotoxic and genotoxic at concentrations ranging from 0.01 to 0.1 μM. The pre-treatment for 2h with this organotellurium compound at non-cytotoxic dose range (0.01, 0.05 and 0.1 μM) increased cell survival after challenge with hydrogen peroxide (H2O2), t-butyl hydroperoxide (t-BOOH), methylmethanesulphonate (MMS) or ultraviolet (UV)C radiation. In addition, the pre-treatment with DPDT decreased the DNA damage and Formamidopyrimidine DNA-glycosylase (Fpg)- and Endonuclease III (Endo III) sensitive sites induction by the studied genotoxic agents, as verified by comet assay and modified comet assay, respectively. The pre-treatment also reduced micronucleus frequency, revealing the protector effect of DPDT against MMS and UVC-induced mutagenesis. Our results demonstrate that DPDT-treated cells at concentration range of 0.01-0.1 μM do not change thiobarbituric acid reactive species (TBARS) levels and ROS generation. Moreover, DPDT pre-treatment at this concentration range decreases the ROS induction by H2O2 and t-BOOH treatment indicating antioxidant potential. On the other hand, concentrations higher than 0.1 μM increase TBARS formation and inhibited superoxide dismutase (SOD) activity, suggesting pro-oxidative effect of this compound at high concentrations. Our results suggest that DPDT presents antigenotoxic and antimutagenic properties at concentration range of 0.01-0.1 μM. The protection effect could be attributed to antioxidant capacity of DPDT at this concentration range in V79 cells.

Baccharis trinervis leaves are used in the traditional medicine for the treatment of high fevers, edema, inflammation, sores and muscle cramps, snakebites and as antiseptics.To investigate the cytotoxic, genotoxic, and mutagenic effects of extracts and fractions of B. trinervis from Brazil and Colombia in Chinese Hamster Ovary (CHO) cells, as well as the mutagenic activity in Salmonella typhimurium.Aqueous extracts (AE) of aerial parts of B. trinervis from Brazil (B) and Colombia (C) were fractioned in ethyl acetate fraction (EAF), butanol extract (BF) and aqueous residue fraction (ARF). Qualitative chemical screening and determination of total flavonoid content were made. Identification of chemical constituents was performed by High Performance Liquid Chromatography (HPLC) and High Resolution Mass Spectrometry (HRMS). For the in vitro tests, CHO cells were treated for 3hours with extracts and fractions of B. trinervis. The cytotoxic activity was evaluated by clonal survival and 3-(4.5-dimethylthiazole-2-yl)−2.5-biphenyl tetrazolium bromide reduction assay (MTT). Also, genotoxic and mutagenic effects were evaluated by the alkaline comet assay and Cytokinesis-blockage micronucleus test (CBMN), respectively. Additionally, Salmonella/microsome assay was carried out to determinate the mutagenic effects in EAF from Brazil and Colombia.Phytochemical analyses indicated the presence of saponins and flavonoids. AE and EAF were the samples with the highest quantity of total flavonoids. HPLC showed the presence of luteolin only in AEC, and caffeic acid, ellagic acid, rosmarinic acid, and rutin were identified in AEB and AEC. However, these compounds had higher concentrations in AEC. The HRMS in positive mode of EAFB and EAFC showed presence of two carboxylic acids, coumarin, and two terpenoids. In addition, in negative mode were identified one terpenoid and two carboxylic acids in AE, BF and ARF of B. trinervis from both countries. Dose-dependent cytotoxic effects were observed in CHO cells treated with B. trinervis extracts and fractions by using clonal survival and MTT at concentrations higher than 0.05mg/mL. All the extracts and fractions induced DNA strand breaks in CHO cells with dose-dependent response, mostly EAFB and EAFC. The EAF from Brazil and Colombia showed mutagenic effect at 0.5mg/mL, while the other fractions did not show a significant difference. No mutagenic effects were found in EAF from both countries by the Salmonella/microsome assay.Phytochemical analyses showed that AE from Colombia presented higher concentrations of the identified compounds than AE from Brazil. Additionally, EAF from both countries showed higher content of the identified compounds than AE, BF an ARF. Furthermore, in vitro toxicological tests showed cytotoxic and genotoxic effects in all extracts and fractions used, although only EAF showed mutagenic effects by CBMN, but not by Salmonella/microsome assay. Our results suggest that flavonoids, phenylpropanoids, coumarins, and diterpenes may be responsible for the cytotoxic, genotoxic and mutagenic effects observed.

The diphenyl ditelluride (DPDT) is a prototype for the development of new biologically active molecules. In previous studies, DPDT showed an elevated cytotoxicity in Chinese hamster fibroblast (V79) cells but the mechanisms for reduction of cell viability still remain unknown. DPDT showed mutagenic properties by induction of frameshift mutations in bacterium Salmonella typhimurium and yeast Saccharomyces cerevisiae. This organotelluride also induced DNA strand breaks in V79 cells. In this work, we investigated the mechanism of DPDT cytotoxicity by evaluating the effects of this compound on cell cycle progression, apoptosis induction and topoisomerase I inhibition. Significant decrease of V79 cell viability after DPDT treatment was revealed by MTT assay. Morphological analysis showed induction of apoptosis and necrosis by DPDT in V79 cells. An increase of caspase 3/7 activity confirmed apoptosis induction. The cell cycle analysis showed an increase in the percentage of V79 cells in S phase and sub-G1 phase. The yeast strain deficient in topoisomerase I (Topo I) showed higher tolerance to DPDT compared with the isogenic wild-type strain, suggesting that the interaction with this enzyme could be involved in DPDT toxicity. The sensitivity to DPDT found in top3∆ strain indicates that yeast topoisomerase 3 (Top3p) could participate in the repair of DNA lesions induced by the DPDT. We also demonstrated that DPDT inhibits human DNA topoisomerase I (Topo I) activity by DNA relaxation assay. Therefore, our results suggest that the DPDT-induced cell cycle arrest and reduction in cell viability could be attributed to interaction with topoisomerase I enzyme.

Background: We aimed to assess the level of DNA damage and susceptibility to exogenous mutagens in peripheral blood cells of Chronic Obstructive Pulmonary Disease (COPD) patients and healthy individuals by comet assay. Oxidative stress was also evaluated by means of thiobarbituric acid reactive species (TBARS) in blood plasma. Methods: Case-control study enrolling 51 COPD patients and 51 controls. Peripheral blood was used to perform the alkaline (pH>13) and neutral (pH=8.5) comet assay. For the assessment of susceptibility to exogenous DNA damage, the cells were treated with methylmethane sulfonate (MMS) for 1-hour or 3-hour at 37° C. The percentage of residual DNA damage after 3-h MMS treatment was calculated using the value of 1-h MMS treatment for each subject as 100%. Lipid peroxidation was evaluated by measuring TBARS in blood plasma. Results: DNA damage in patients was significantly higher than in controls as measured by the neutral and alkaline comet assay. Residual DNA damage detected after MMS treatment increased in patients, in contrast to controls, indicating higher susceptibility to alkylation damage and/or repair inhibition. High susceptibility to exogenous DNA damage in COPD patients correlates with high amount of TBARS and low forced vital capacity and expiratory volume. Conclusion: The positive correlation between increased susceptibility to exogenous DNA damage and TBARS levels in COPD patients suggests the possible involvement of oxidative stress in damage induction and/or repair inhibition.

Thiazacridine derivatives (ATZD) are a novel class of cytotoxic agents that combine an acridine and thiazolidine nucleus. In this study, the cytotoxic action of four ATZD were tested in human colon carcinoma HCT-8 cells: (5Z)-5-acridin-9-ylmethylene-3-(4-methylbenzyl)-thiazolidine-2,4-dione — AC-4; (5ZE)-5-acridin-9-ylmethylene-3-(4-bromo-benzyl)-thiazolidine-2,4-dione — AC-7; (5Z)-5-(acridin-9-ylmethylene)-3-(4-chloro-benzyl)-1,3-thiazolidine-2,4-dione — AC-10; and (5ZE)-5-(acridin-9-ylmethylene)-3-(4-fluoro-benzyl)-1,3-thiazolidine-2,4-dione — AC-23. All of the ATZD tested reduced the proliferation of HCT-8 cells in a concentration- and time-dependent manner. There were significant increases in internucleosomal DNA fragmentation without affecting membrane integrity. For morphological analyses, hematoxylin–eosin and acridine orange/ethidium bromide were used to stain HCT-8 cells treated with ATZD, which presented the typical hallmarks of apoptosis. ATZD also induced mitochondrial depolarisation and phosphatidylserine exposure and increased the activation of caspases 3/7 in HCT-8 cells, suggesting that this apoptotic cell death was caspase-dependent. In an assay using Saccharomyces cerevisiae mutants with defects in DNA topoisomerases 1 and 3, the ATZD showed enhanced activity, suggesting an interaction between ATZD and DNA topoisomerase enzyme activity. In addition, ATZD inhibited DNA topoisomerase I action in a cell-free system. Interestingly, these ATZD did not cause genotoxicity or inhibit the telomerase activity in human lymphocyte cultures at the experimental levels tested. In conclusion, the ATZD inhibited the DNA topoisomerase I activity and induced tumour cell death through apoptotic pathways.

The present study aimed to investigate thein vitromutagenic activity ofOriganum majoranaessential oil. The most abundant compounds identified by GC-MS wereγ-terpinene (25.73%),α-terpinene (17.35%), terpinen-4-ol (17.24%), and sabinene (10.8%). Mutagenicity was evaluated by theSalmonella/microsome test using the preincubation procedure on TA98, TA97a, TA100, TA102, and TA1535Salmonella typhimuriumstrains, in the absence or in the presence of metabolic activation. Cytotoxicity was detected at concentrations higher than 0.04 μL/plate in the absence of S9 mix and higher than 0.08 μL/plate in the presence of S9 mix and no gene mutation increase was observed. For thein vitromammalian cell micronucleus test, V79 Chinese hamster lung fibroblasts were used. Cytotoxicity was only observed at concentrations higher than or equal to 0.05 μg/mL. Moreover, when tested in noncytotoxic concentrations,O. majoranaessential oil was not able to induce chromosome mutation. The results from this study therefore suggest thatO. majoranaessential oil is not mutagenic at the concentrations tested in theSalmonella/microsome and micronucleus assays.

Tin or stannous (Sn 2+ ) compounds are used as catalysts, stabilizers in plastic industries, wood preservatives, agricultural biocides and nuclear medicine. In order to verify the Sn 2+ up-take and toxicity in yeast cells we utilized a multi-elemental analysis known as particle-induced X-ray emission (PIXE) along with cell survival assays and quantitative real-time PCR. The detection of Sn 2+ by PIXE was possible only in yeast cells in stationary phase of growth (STAT cells) that survive at 25 mM Sn 2+ concentration. Yeast cells in exponential phase of growth (LOG cells) tolerate only micro-molar Sn 2+ concentrations that result in intracellular concentration below of the method detection limit. Our PIXE analysis showed that STAT XV185-14c yeast cells demonstrate a significant loss of intracellular elements such as Mg, Zn, S, Fe and an increase in P levels after 1 h exposure to SnCl 2 . The survival assay showed enhanced tolerance of LOG yeast cells lacking the low-affinity iron and zinc transporters to stannous treatment, suggesting the possible involvement in Sn 2+ uptake. Moreover, our qRT-PCR data showed that Sn 2+ treatment could generate reactive oxygen species as it induces activation of many stress-response genes, including SOD1 , YAP1 , and APN1 .

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA.

Plant extracts have a long history to be used in folk medicine.Cassia alataextracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract ofC. alataby reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity forPseudomonas aeruginosa,Staphylococcus epidermidis,S. aureus, andBacillus subtilis. CaRP also inhibited biofilm formation ofS. epidermidisandP. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity againstS. aureusandS. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In theSalmonella/microsome assay CaRP showed weak mutagenicity (MI<3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate thatC. alataleaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications.

The susceptibility of individuals to the genotoxic effect of pesticides can be modulated by genetic variations in the xenobiotic detoxification and DNA repair processes. This study evaluates if the two BER polymorphisms (XRCC1Arg194Trp and OGG1Ser326Cys) or the combined genotypes of these polymorphisms with PON1Gln192Arg could modify individual susceptibility to pesticide exposure in vineyard workers, as measured by micronucleus formation and DNA damage induction in peripheral leukocytes. The study population comprised 108 agricultural workers exposed to pesticides and 65 nonexposed. Our results demonstrate that individuals with the variant allele (OGG1Cys) showed higher DNA damage, detected by the comet assay, in relation to individuals carrying the wild-type OGG1Ser allele. Considering the combined influence of metabolizing PON1 and the DNA repair OGG1 genes, we observed significantly higher DNA damage in the comet assay in the exposed group when a less efficient OGG1Cys allele was acting independently of the PON1 genotype, reinforcing the importance of the OGG1 repair enzyme in the response to DNA damage by pesticide exposure. The association of the PONGln/Gln genotype with higher MN frequency suggests that the PON1 genotype is a major determinant of genotoxic risk in individuals exposed to pesticides. Analysis of the compared effect of XRCC1 and PON1 genotypes in the exposed group suggested that, among the poorly metabolizing PON1Gln/Gln individuals, the XRCC1Arg/Trp genotype has a protective effect with respect to MN formation. These results indicate that enhanced XRCC1 function may provide some protection from the enhanced genotoxic risk associated with inefficient xenobiotic detoxification in the studied population. Environ. Mol. Mutagen. 52:20–27, 2011. © 2010 Wiley-Liss, Inc.

This study presents the first application of an in vivo alkaline comet assay using haemocytes of Drosophila melanogaster larvae. These cells, which play a role similar to that of mammalian blood, can be easily obtained and represent an overall exposure of the treated larvae. To validate the assay, we evaluated the response of these cells to three well-known mutagenic agents: ethyl methanesulfonate (EMS), potassium dichromate (PD), and gamma radiation (γ-irradiation). Third-instar Drosophila larvae were exposed to different concentrations of EMS (1, 2, and 4 mM) and PD (0.5, 1, and 2.5 mM) and to different doses of γ-irradiation (2, 4, and 8 Gγ). Subsequently, haemolymph was extracted from the larvae, and haemocytes were isolated by centrifugation and used in the comet assay. Haemocytes exhibited a significant dose-related increase in DNA damage, indicating that these cells are clearly sensitive to the treatments. These results suggest that the proposed in vivo comet test, using larvae haemocytes of D. melanogaster, may be a useful in vivo assay for genotoxicity assessment.

Ultraviolet radiation is known to cause adverse effects to aquatic species and aquatic environments. The fish Colossoma macropomum (tambaqui) and Arapaima gigas (pirarucu) live in the Amazon basin, near the Equator, and thus receive high intensity of ultraviolet radiation. Deforestation further aggravates the situation by reducing shade at ground level. The aim of this study was to evaluate the genotoxic effects of UVA and UVB radiation on erythrocytes of tambaqui and pirarucu fish using Micronuclei test and Comet assay. Our study showed that UV radiation caused DNA damage in both species as detected by Comet assay. In addition, there were differences in response to genotoxicity between both species, which are possibly related to their evolutionary history. Tambaqui fish exposed to ultraviolet radiation for different periods presented clear dose–response in DNA damage profile. Significant damage repair was observed 24 h after cessation of ultraviolet radiation exposure. At the test conditions used, no significant increase in micronucleated cells was observed in tambaqui and pirarucu fish. Tambaqui proved to be more sensitive to ultraviolet radiation than Pirarucu, as detected by Comet assay, showing statistically higher baseline DNA damage. The present results demonstrated that alkaline Comet assay was very sensitive for detecting the UV-induced genotoxicity during the short exposure period in our study. In addition, the present study also suggests that tambaqui and pirarucu fish are useful sentinel organisms, as their UV sensitivity allows them to be effective monitors of biological hazards in the Amazon region.

Diphenyl ditelluride (DPDT) is a potential prototype for the development of novel biologically active molecules. Thus, it is important to evaluate the toxic effects of this compound. In the present study, we evaluated the cytotoxic, genotoxic and mutagenic properties of DPDT in Chinese hamster fibroblast (V79) cells, in strains of the yeast Saccharomyces cerevisiae both proficient and deficient in several DNA repair pathways and in Salmonella typhimurium. DPDT induced frameshift mutations in both S.typhimurium and a haploid wild-type strain of S.cerevisiae. Mutants of S.cerevisiae defective in base excision repair and recombinational repair were more sensitive to DPDT. The results of a lactate dehydrogenase leakage assay suggest that DPDT is cytotoxic to V79 cells. At cytotoxic concentrations, this compound increased thiobarbituric reactive species levels and decreased the glutathione:GSSH ratio in yeast and V79 cells. DPDT generated single- and double-strand DNA breaks in V79 cells, both with and without metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, an induction of oxidative DNA base damage was indicated by a modified comet assay using formamidopyrimidine DNA glycosylase and endonuclease III. Treatment with DPDT also induced micronucleus formation in V79 cells. Pre-incubation with N-acetylcysteine reduced DPDT's oxidative, genotoxic and mutagenic effects in yeast and V79 cells. Our results suggest that the toxic and mutagenic properties of DPDT may stem from its ability to disturb the redox balance of the cell, which leads to oxidative stress and the induction of DNA damage.

Anthracyclines have been widely used as antitumor agents, playing a crucial role in the successful treatment of many types of cancer, despite some side effects related to cardiotoxicity. New anthracyclines have been designed and tested, but the first ones discovered, doxorubicin and daunorubicin, continue to be the drugs of choice. Despite their extensive use in chemotherapy, little is known about the DNA repair mechanisms involved in the removal of lesions caused by anthracyclines. The anthracycline cosmomycin D is the main product isolated from Streptomyces olindensis, characterized by a peculiar pattern of glycosylation with two trisaccharide rings attached to the A ring of the tetrahydrotetracene. We assessed the induction of apoptosis (Sub-G1) by cosmomycin D in nucleotide excision repair-deficient fibroblasts (XP-A and XP-C) as well as the levels of DNA damage (alkaline comet assay). Treatment of XP-A and XP-C cells with cosmomycin D resulted in apoptosis in a time-dependent manner, with highest apoptosis levels observed 96 h after treatment. The effects of cosmomycin D were equivalent to those obtained with doxorubicin. The broad caspase inhibitor Z-VAD-FMK strongly inhibited apoptosis in these cells, and DNA damage induced by cosmomycin D was confirmed by alkaline comet assay. Cosmomycin D induced time-dependent apoptosis in nucleotide excision repair-deficient fibroblasts. Despite similar apoptosis levels, cosmomycin D caused considerably lower levels of DNA damage compared to doxorubicin. This may be related to differences in structure between cosmomycin D and doxorubicin.

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organoselenium (OS) compounds. The aim of the present study was to investigate the mutagenic effects of three symmetrical derivatives from the prototype DPDS (p-chloride-phenyl-diselenide, p-methyl-phenyl-diselenide and p-methoxy-phenyl-diselenide) in yeast. In summary, the cellular effects of the three OS compounds studied in this work appear to be variable according to the substituent group in the aromatic ring and are concentration-dependent. The presence of methoxyl group in the aromatic ring of DPDS structure results in elevation of the cytotoxic and mutagenic potential while the introduction of a methyl group increases the cytotoxic effect.

The photoprotective effect of the methanolic extracts of three Antarctic plant species - Deschampsia antarctica Desv., Colobanthus quitensis (Kunth) Bartl., and Polytrichum juniperinum Hedw. against UV-induced DNA damage was investigated in hamster lung fibroblasts (V79 cells) and in a biomonitor organism Helix aspersas, using comet assay. The protective, mutagenic, and antimutagenic profiles of these extracts were also evaluated using haploid strains of the simple eukaryote Saccharomyces cerevisiae, and antioxidant activity were investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay, as well as the hypoxanthine/xanthine oxidase assay. At the concentration range employed, the extracts were not cytotoxic or mutagenic to S. cerevisiae. In addition, the treatment with these extracts enhanced survival, and decreased induced reverse, frameshift, and forward mutations in a dose-response manner in all UVC doses employed. The plants extracts did not generate DNA strand breaks in V79 cells, and the treatment significantly decreased DNA damage induced by UVC. Extracts significantly decreased UVC-induced lipid peroxidation in V79 cells, showing a clear antioxidant property. Moreover, results of comet assay in V79 cells, employing Fpg, Endo III, and Endo V enzymes, demonstrated significant reduction of UVC-induced DNA damage after pre-incubation with these extracts. The treatment with all tested extracts were much less efficient against UVC-induced cytotoxicity in the yeast strain defective in photolyase as compared to the wild type strain, suggesting that this DNA repair pathway is stimulated by substances present in the extracts. All extracts showed a significant inhibitory effect in the hypoxanthine/xanthine oxidase assay, and they had the ability to scavenge DPPH. In H. aspersas, the treatment was able to protect against UVC-induced damage. In conclusion, D. antarctica, C. quitensis, and P. juniperinum extracts present photoprotective properties, which can be attributed to molecules, such as flavonoids and carotenoids, which act as UV-absorbing molecules and as antioxidants, as well as stimulate DNA-repair processes.

In order to clarify the molecular mechanisms of Sn(2+) genotoxicity, we evaluated the induction of strand breaks, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (Endo III) sensitive sites, and the interference with the repair of methyl methane sulfonate (MMS)-caused DNA damage in V79 Chinese hamster lung fibroblasts exposed to stannous chloride by comet assay. A concentration-related increase in the DNA damage induced by 2 h SnCl(2) treatment at a concentration range of 50-1,000 microM was observed (r = 0.993; P0.01). Significantly elevated DNA migration in relation to the control level was detected at doses 100, 500 and 1,000 microM in normal alkaline and at doses 500 and 1,000 microM in modified (with Fpg and Endo III) comet assay. Although 50 microM SnCl(2) concentration did not increase significantly the DNA migration by itself in comet assay, it was capable to inhibit the repair of MMS-induced DNA damage during the post-treatment period of 24 h. Our results demonstrate the genotoxic and comutagenic effects of stannous chloride in V79 cells. The inhibitory effect of Sn(2+) on repair of MMS-induced DNA damage suggests that this metal can also interfere in DNA repair systems thus contributing to increased mutation by shifting the balance from error-free to error-prone repair processes.

Arrabidaea chica Verlot (Bignoniaceae) has been used as a medicinal herb to treat anemia, hemorrhage, inflammation, intestinal colic, hepatitis, and skin infections in the Brazilian Amazon region. Studies have demonstrated the healing properties of extracts obtained from A. chica leaves, which contain anthocyanins and flavonoids. However, few investigations have assessed the safe use of this plant species. In this study, mutagenic and genotoxic effects of a crude aqueous extract, a butanolic fraction, and aqueous waste from A. chica leaves were evaluated using the Salmonella/microsome assay in TA98, TA97a, TA100, TA102, and TA1535 strains and the alkaline comet assay in Chinese hamster ovary (CHO) cell culture with and without metabolic activation. The crude aqueous extract, butanolic fraction, and aqueous waste were not mutagenic in any of the Salmonella typhimurium strains tested, and showed negative responses for genotoxicity in CHO cells. High-performance liquid chromatography (HPLC) analysis indicated the presence of phenolic acids and flavonoids such as rutin and luteolin. The lack of mutagenic/genotoxic effects might be due to phytochemical composition with high concentrations of known anti-inflammatory compounds. Thus, the crude aqueous extract, butanolic fraction, and aqueous waste from A. chica leaves do not appear to pose short-term genotoxic risks.

Purpose : To evaluate DNA damage in lymphocytes and cognitive deficits in a nursing team occupationally exposed to ionizing radiation in a university hospital in southern Brazil. Method : Cross-sectional study, case-control design, included 79 women working in a nursing team in Hemodynamics, Radiology, Ambulatory and Midwifery Units. They were classified in two groups: 1_Exposed to ionizing radiation, n=38 (Hemodynamics and Radiology Units) and 2_Unexposed to ionizing radiation, n=41 (Ambulatory and Midwifery Unit). Blood was collected and DNA damage in lymphocytes was analyzed using the comet assay and micronucleus test (MN). The cells were classified according to the damage frequency (DF) and index (DI) based on the comet tail size. Cognition was also evaluated according to the scores obtained in Stroop test, the digit and word span. Results : No difference was found for the variables DI, DF, MN, Stroop_word, Stroop_color, Stroop_color/word between the groups. However, when the groups were analyzed independently of their exposure condition, the damage index in the Hemodynamics was lower than in all other Units. The damage frequency in Midwifery Unit was higher than in Ambulatory and Hemodynamics. The frequency of micronucleus was not different among the employees from the four Units. Conclusions : DNA damage in lymphocytes did not differ between nursing teams occupationally exposed or not exposed to ionizing radiation. However, higher damage in a non exposed group may indicate other risks to the healthcare team.

The hsp60 expression pattern and catalase activity in the freshwater planarian Dugesia schubarti exposed to copper under laboratory conditions were investigated. In the hsp60 induction experiments, planarians were exposed to a range of copper concentrations (0-960 microgCu/L) for 4 or 24h, to concentrations of 50 or 100 microgCu/L for 2, 4, 8, and 24h at 19 degrees C, and to heat shock at 27 degrees C for 24h. The concentrations of hsp60 in whole-body homogenates were determined immunochemically by Western blotting. Stress protein induction was detected only after 24h treatment at 27 degrees C. The tissue concentration of hsp60 remained unaltered in Cu-exposed planarians under the experimental conditions used. Catalase activity was significantly induced at concentrations of 40, 80, and 160 microgCu/L after 24h exposure. Our results suggest that catalase levels in planarians could represent biomarkers of interest for the estimation of copper effects in freshwater ecosystems.

The alkaline single-cell gel electrophoresis, or comet assay, was used to evaluate the genotoxic potential of copper sulphate in planarians. Concentration-related increase in DNA damage was induced after 2 h and 7 days exposure to CuSO 4 dissolved in culture water. To study the influence of copper ions on the persistence of mutagen-induced DNA lesions, planarians were treated with methyl methanesulphonate (MMS), and further incubated in the absence (post-incubation) or presence (post-treatment) of CuSO 4 . After 2 h of post-treatment enhanced persistence of DNA effects in relation to the corresponding post-incubation value was detected, which indicate inhibition of DNA repair by CuSO 4 . At 4 h an increase of DNA migration in relation to the 2 h value was observed, which is significant for the post-incubation group. After 24 h, DNA damage decreased but was still significantly elevated in relation to the control. From our results, we conclude that planarians are suitable organisms for in vivo detection of copper genotoxicity in the comet assay, and can be used to assess both acute and chronic exposure to this chemical in aquatic ecosystems. The inhibition effect of copper ions on repair of MMS-induced DNA damage suggests that copper could modulate the genotoxic effects associated with complex mixture exposure in the environment.

Orange juice (OJ) is among the most consumed fruit juices worldwide, and its chemopreventive action is fairly addressed in the literature. This review critically presents the available evidence linking OJ with cancer chemoprevention and on discussing the putative mechanisms and negative health effects. The chemopreventive action of OJ is related to its effect on metabolic enzymes and its antiinflammatory, cytoprotective/apoptotic, hormonal, cell signaling-modulating, antioxidant, and antigenotoxic effects. Most studies on OJ are in vitro, and few are conducted in vivo. Results from in vitro studies must be interpreted carefully because these findings do not consider in vivo bioavailability. However, such results are useful for studying the impact of different processing and storage methods on OJ's chemopreventive effect. Evidence of OJ's chemoprevention in humans is limited. OJ is antimutagenic in bacteria and antigenotoxic in humans and rodents. Studies using rodent cancer models showed that OJ is cancer chemopreventive, influencing either the induction stage or the promotion stage. The composition and, therefore, the chemopreventive action of OJ might be influenced by different cultivars, climates, extraction methods, packaging, storage temperatures, and shelf lives, among other factors. Epidemiological studies and randomized controlled intervention studies in humans evaluating the chemopreventive effect of OJ, taking into consideration variability in OJ composition, are needed.

The organoselenium compound, dicholesteroyl diselenide (DCDS) is a structural analogue of diphenyl diselenide (DPDS) and may be considered as a promising antioxidant drug in vivo. Nevertheless, little is known about the toxicological properties of DCDS. In the present study we evaluated the cytotoxic, genotoxic and mutagenic properties of DCDS in Chinese hamster lung fibroblasts (V79) and in strains of the yeast Saccharomyces cerevisiae, proficient and deficient in several DNA-repair pathways. The results with V79 cells show that DCDS induced cytotoxicity, GSH depletion and elevation of lipid peroxidation at lower concentrations than did DPDS. DCDS also generated single- and double-strand DNA breaks in V79 cells, both in the presence and in the absence of metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, the induction of oxidative DNA base-damage was demonstrated by means of a modified comet assay with formamidopyrimidine-DNA glycosylase and endonuclease III. Treatment with DCDS also induced micronucleus formation in V79 cells as well as point and frame-shift mutations in a haploid wild-type strain of S. cerevisiae. Yeast mutants defective in base excision-repair proteins were the most sensitive to DCDS. Pre-incubation with N-acetylcysteine reduced DCDS's oxidative, genotoxic and mutagenic effects in yeast and in V79 cells. Our findings indicate that the presence of cholesteroyl substituents in DCDS results in elevation of its cytotoxic and genotoxic potential compared with that of DPDS in yeast and in V79 cells. However, due to dose-dependent contrasting behaviour of organoselenium compounds and differences in their toxicity in in vitro and in vivo systems, further studies are needed in order to establish the non-toxic concentration range for treatment in mammals.

This study assessed the chronic effects of physical exercise on the level of DNA damage and the susceptibility to exogenous mutagens in peripheral blood cells of chronic obstructive pulmonary disease (COPD) patients. The case-control study enrolled COPD patients separated into two groups (group of physical exercise (PE-COPD; n=15); group of nonphysical exercise (COPD; n=36)) and 51 controls. Peripheral blood was used to evaluate DNA damage by comet assay and lipid peroxidation by measurement of thiobarbituric acid reactive species (TBARS). The cytogenetic damage was evaluated by the buccal micronucleus cytome assay. The results showed that the TBARS values were significantly lower in PE-COPD than in COPD group. The residual DNA damage (induced by methyl methanesulphonate alkylating agent) in PE-COPD was similar to the controls group, in contrast to COPD group where it was significantly elevated. COPD group showed elevated frequency of nuclear buds (BUD) and condensed chromatin (CC) in relation to PE-COPD and control groups, which could indicate a deficiency in DNA repair and early apoptosis of the damaged cells. We concluded that the physical exercise for COPD patients leads to significant decrease of lipid peroxidation in blood plasma, decrease of susceptibility to exogenous mutagenic, and better efficiency in DNA repair.

By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal β-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells.

Eukaryotic cells have developed mechanisms to prevent genomic instability, such as DNA damage detection and repair, control of cell cycle progression and cell death induction. The bifunctional compound furocumarin 8-methoxy-psoralen (8-MOP) is widely used in the treatment of various inflammatory skin diseases. In this review, we summarize recent data about the role of chromatin remodeling in the repair of DNA damage induced by treatment with 8-methoxypsoralen plus UVA (8-MOP+UVA), focusing on repair proteins in budding yeast Saccharomyces cerevisiae, an established model system for studying DNA repair pathways. The interstrand crosslinks (ICL) formed by the 8-MOP+UVA treatment are detrimental lesions that can block transcription and replication, leading to cell death if not repaired. Current data show the involvement of different pathways in ICL processing, such as nucleotide excision repair (NER), base excision repair (BER), translesion repair (TLS) and double-strand break repair. 8-MOP+UVA treatment in yeast enhances the expression of genes involved in the DNA damage response, double strand break repair by homologous replication, as well as genes related to cell cycle regulation. Moreover, alterations in the expression of subtelomeric genes and genes related to chromatin remodeling are consistent with structural modifications of chromatin relevant to DNA repair. Taken together, these findings indicate a specific profile in 8-MOP+UVA responses related to chromatin remodeling and DNA repair.

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organoselenium compounds. Studies have shown its interesting pharmacodinamic properties, as antioxidant, antimutagenic and antitumoral effects. Here we report the antigenotoxic properties of DPDS against tamoxifen (TAM)-induced oxidative DNA damage in MCF-7 cultured cell line. We determined the cytotoxicity by lactate dehydrogenase (LDH) leakage assay and evaluated oxidative DNA damage by modified comet assay employing the enzymes formamidopyrimidine DNA-glycosylase (Fpg) and endonuclease III (Endo III). Our results demonstrate that the cellular effects of DPDS appear to be complex and concentration-dependent. The present findings show that DPDS is not genotoxic (at concentrations lower than 2.0μmol/L) in MCF-7 cells, as observed in the modified comet assay. Moreover, DPDS protects against TAM-induced oxidative DNA damage, probably by its antioxidant activity, without interfering with its cytotoxicity. In this manner, the treatment with low concentrations of DPDS, a synthetic organoselenium compound, could be used as a potent antigenotoxic agent to prevent the risk of cancer induction triggered by tamoxifen hormone therapy. Thereby, more studies concerning the toxicity of DPDS and its structural derivatives are still necessary for future safe therapeutic application and development of novel chemopreventive compounds for combined therapy in breast cancer.

Patulin, a known mycotoxin, is considered a significant contaminant in apples, apple-derived products and feeds. This study investigated the genotoxic effects of patulin in multiple organs (brain, kidney, liver and urinary bladder) of mice using an in vivo comet assay. We assessed the mechanism underlying this genotoxicity by measuring the GSH content and the thiobarbituric acid-reactive species (TBARS) level. Male CF-1 mice were given 1.0–3.75 mg/kg patulin intraperitoneally. The effect of patulin was dose-dependent and the highest patulin dose induced DNA strand breaks in the brain (damage index, DI, in hippocampus increased from 36.2 in control animals to 127.5), liver (44.3–138.4) and kidneys (31.5–99); decreased levels of GSH (hippocampus – from 46.9 to 18.4 nmol/mg protein); and an increase in lipid peroxidation (hippocampus – from 5.8 to 20.3 MDA equivalents/mg protein). This finding establishes an interrelationship between the pro-oxidant and genotoxic effects of patulin. Pre-treatment administration of N-acetyl-cysteine reduced patulin-induced DNA damage (hippocampus – DI from 127.5 to 39.8) and lipid peroxidation (hippocampus – 20.3 to 12.8 MDA equivalents/mg protein) by restoring cellular GSH levels, reinforcing the positive relationship between patulin-induced GSH depletion and DNA damage caused by systemic administration of this mycotoxin.

Biflorin is a natural quinone isolated from Capraria biflora L. Previous studies demonstrated that biflorin inhibits in vitro and in vivo tumor cell growth and presents potent antioxidant activity. In this paper, we report concentration-dependent cytotoxic, genotoxic, antimutagenic, and protective effects of biflorin on Salmonella tiphymurium, yeast Saccharomyces cerevisiae, and V79 mammalian cells, using different approaches. In the Salmonella/microsome assay, biflorin was not mutagenic to TA97a TA98, TA100, and TA102 strains. However, biflorin was able to induce cytotoxicity in haploid S. cerevisiae cells in stationary and exponential phase growth. In diploid yeast cells, biflorin did not induce significant mutagenic and recombinogenic effects at the employed concentration range. In addition, the pre-treatment with biflorin prevented the mutagenic and recombinogenic events induced by hydrogen peroxide (H(2)O(2)) in S. cerevisiae. In V79 mammalian cells, biflorin was cytotoxic at higher concentrations. Moreover, at low concentrations biflorin pre-treatment protected against H(2)O(2)-induced oxidative damage by reducing lipid peroxidation and DNA damage as evaluated by normal and modified comet assay using DNA glycosylases. Our results suggest that biflorin cellular effects are concentration dependent. At lower concentrations, biflorin has significant antioxidant and protective effects against the cytotoxicity, genotoxicity, mutagenicity, and intracellular lipid peroxidation induced by H(2)O(2) in yeast and mammalian cells, which can be attributed to its hydroxyl radical-scavenging property. However, at higher concentrations, biflorin is cytotoxic and genotoxic.

Vitamin A (Vit A) is widely suggested to be protective against oxidative stress. However, different studies have been demonstrated the pro-oxidant effects of retinoids in several experimental models. In this work, we used the yeast Saccharomyces cerevisiae as a model organism to study the Vit A effects on superoxide dismutase (SOD)-deficient yeast strains. We report here that Vit A (10, 20 and 40 mg/ml) decreases the survival of exponentially growing yeast cells, especially in strains deficient in CuZnSOD (sod1Delta) and CuZnSOD/MnSOD (sod1Deltasod2Delta). We also observed the protective effect of vitamin E against the Vit A-induced toxicity. Possible adaptation effects induced by sub-lethal oxidative stress were monitored by pre-, co- and post-treatment with the oxidative agent paraquat. The enzymatic activities of catalase (CAT) and glutathione peroxidase (GPx), and the total glutathione content were determined after Vit A treatment. Our results showed that CuZnSOD represents an important defence against Vit A-generated oxidative damage. In SOD-deficient strains, the main defence against Vit A-produced reactive oxygen species (ROS) is GPx. However, the induction of GPx activity is not sufficient to prevent the Vit A-induced cell death in these mutants in exponential phase growth.

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair (NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gammaH2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase IIalpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions.

The effect of dietary diphenyl diselenide (1 ppm) on N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis was examined in female Wistar rats. Beginning at 5 weeks of age, the animals were fed with either control or diphenyl-diselenide-supplied diets until the end of the study (210 days). At 50 days of age, mammary tumor was induced by the administration of three doses of NMU (50 mg/kg body wt, intraperitoneally) once a week for 3 weeks. In experimental trials, latency to tumor onset was extended in rats fed with diet supplemented with diphenyl diselenide (P < 0.05). The incidence and frequency of tumors were significantly small in animals supplemented with diphenyl diselenide. However, the multiplicity of tumors was not altered by dietary diphenyl diselenide. Diphenyl diselenide supplementation also restored superoxide dismutase (SOD) activity and vitamin C levels altered in the NMU group (P < 0.05). Our results suggest that diphenyl diselenide can be considered a chemopreventive agent, even when supplemented at a relatively low concentration.

Objectives: To evaluate the antioxidant status and repair capacity in breast cancer patients as well as the relationship between these parameters and expression of critical proteins in breast cancer tissue. Design and methods: Blood samples were obtained from 25 female breast cancer patients and 19 healthy women. The antioxidant status was determined by the concentration of thiobarbituric-reactive substances (TBARS) and activity of superoxide dismutase (SOD) and catalase (CAT). The basal DNA damage and repair capacity in lymphocytes were evaluated by comet assay. The expression of p53, c-erbB2, Ki-67, estrogen receptor (ER) and progesterone receptor (PR) in cancer tissue was detected by immunohistochemical staining. Results: The breast cancer patients presented significantly elevated endogenous DNA damage in lymphocytes and lower susceptibility to DNA damage induced by H2O2 when compared to the control group. There is a negative correlation between TBARS and sensitivity to peroxide induced DNA damage in patients. The percentage of residual damage after H2O2 treatment followed by 3h of post-incubation is significantly higher in patients and also correlates positively with SOD activity, ER and PR expression and negatively with the basal DNA damage. Conclusions: Our results demonstrate low repair capacity in lymphocytes of breast cancer patients and suggest that the regulation of DNA repair is sensitive to cellular redox state and can be modulated by ER/PR status.

Background We investigated a potential link between genetic polymorphisms in genes XRCC1 (Arg399Gln), OGG1 (Ser326Cys), XRCC3 (Thr241Met), and XRCC4 (Ile401Thr) with the level of DNA damage and repair, accessed by comet and micronucleus test, in 51 COPD patients and 51 controls. Methods Peripheral blood was used to perform the alkaline and neutral comet assay; and genetic polymorphisms by PCR/RFLP. To assess the susceptibility to exogenous DNA damage, the cells were treated with methyl methanesulphonate for 1-h or 3-h. After 3-h treatment the % residual damage was calculated assuming the value of 1-h treatment as 100%. The cytogenetic damage was evaluated by buccal micronucleus cytome assay (BMCyt). Results COPD patients with the risk allele XRCC1 (Arg399Gln) and XRCC3 (Thr241Met) showed higher DNA damage by comet assay. The residual damage was higher for COPD with risk allele in the four genes. In COPD patients was showed negative correlation between BMCyt (binucleated, nuclear bud, condensed chromatin and karyorrhexic cells) with pulmonary function and some variant genotypes. Conclusion Our results suggest a possible association between variant genotypes in XRCC1 (Arg399Gln), OGG1 (Ser326Cys), XRCC3 (Thr241Met), and XRCC4 (Ile401Thr), DNA damage and progression of COPD.

Pso2 protein, a member of the highly conserved metallo-β-lactamase (MBL) super family of nucleases, plays a central role in interstrand crosslink repair (ICL) in yeast. Pso2 protein is the founder member of a distinct group within the MBL superfamily, called β-CASP family. Three mammalian orthologs of this protein that act on DNA were identified: SNM1A, SNM1B/Apollo and SNM1C/Artemis. Yeast Pso2 and all three mammalian orthologs proteins have been shown to possess nuclease activity. Besides Pso2, ICL repair involves proteins of several DNA repair pathways. Over the last years, new homologs for human proteins have been identified in yeast. In this review, we will focus on studies clarifying the function of Pso2 protein during ICL repair in yeast, emphasizing the contribution of Brazilian research groups in this topic. New sub-pathways in the mechanisms of ICL repair, such as recently identified conserved Fanconi Anemia pathway in yeast as well as a contribution of non-homologous end joining are discussed.