Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A emodina é uma antraquinona estruturalmente semelhante à aloe-emodina e ambas tem sido apontadas como capazes de causar lesões oxidativas pela produção de ERO. Sua presença em produtos dermocosméticos e de higiene pessoal, associada às informações de que a fotoativação de antraquinonas levaria ao aumento de lesões oxidativas causadas por ERO, torna relevante o estudo da associação da emodina com a radiação UVA. O objetivo desse trabalho foi avaliar a citotoxicidade induzida pela associação da emodina com doses subletais de radiação UVA, em células de Escherichia coli (selvagem e cepas deficientes em enzimas do BER), através de ensaios de sobrevivência bacteriana (taxa de dose de UVA igual a 20J/m/s, totalizando 108kJ/m ao final de 90min de experimento), e em células da linhagem A549 pela exclusão do corante azul de tripan e sobrevivência clonogênica(taxa de dose de UVA igual a 20J/m/s, totalizando 36kJ/m ao final de 30min de experimento). Além disso, a genotoxicidade desses agentes foi estudada por eletroforese em gel de agarose de DNA plasmidial (taxa de dose de UVA igual a 16J/m/s, totalizando 57,6kJ/m ao final de 60min de experimento). De acordo com os resultados: i) Concentrações iguais ou abaixo de 5,55mM de emodina não alteraram a sobrevivência em nenhuma das cepas estudas; ii) As proteínas Xth e Fpg parecem ter um papel importante no reparo das lesões causadas pela emodina, em altas concentrações, sugerindo a participação do reparo por excisão de bases (BER) nesse processo; iii) A associação da emodina com a radiação UVA se mostrou citotóxica em todas as cepas de E. coli; iv) O gene nfo foi o mais importante na resistência bacteriana às lesões induzidas pela associação dos dois agentes, reforçando o envolvimento do BER e indicando uma possível participação do reparo por incisão de nucleotídeos (NIR); v) A emodina parece ter interagido com o DNA plasmidial, alterando seu padrão de migração no gel de agarose; vi) Em células da linhagem A549, a emodina causa efeitos tóxicos imediatos que parecem ser reparados ao longo do tempo. Porém, quando a droga permaneceu por 24 horas em contato com as células, houve uma diminuição na sobrevivência celular que parece ser dosedependente; vii) As concentrações de 10μM e 25μM de emodina, quando associadas ao UVA, se mostraram responsáveis pela redução de mais de 50% na sobrevivência nas células A549, chegando a 100% de morte quando a concentração de emodina foi de 50μM; viii) A radiação UVA potencializou os efeitos citotóxicos da emodina, nos 2 modelos experimentais do presente estudo, indicando que a interação da emodina com a radiação UVA seja genotóxica e portanto prejudicial à saúde. / Emodin is an anthraquinone structurally similar to aloe-emodin and both have been identified as capable of causing oxidative damage by ROS production. Its presence in skin cosmetics and toiletries, associated to the information that the photoactivation of anthraquinones leads to increased oxidative damage caused by ROS, make studies about the association of emodin with UVA relevant. The aim of this study was to evaluate the cytotoxicity induced by the combination of emodin with sublethal doses of UVA radiation in Escherichia coli cells (wild strain and strains deficient in enzymes of BER) by bacterial survival assay (UVA dose rate equal to 20J/m/s, totaling 108kJ/m at the end of 90min of experiment); and in A549 cell line by trypan blue exclusion assay and clonogenic survival(dose rate of UVA equal to 20J/m/s, totaling 36kJ/m at the end of 30 minutes of experiment). Furthermore, the genotoxicity of these agents was studied by electrophoresis on agarose gel of plasmid DNA (dose rate of UVA equal to 16J/m/s, totaling 57,6kJ/m at the end of 60 minutes of experiment). According to the results: i) concentrations equal or below 5.55mM of emodin did not affect the survival in any of the studied strains; ii) proteins Xth and Fpg appear to have an important role in the repair of lesions induced by emodin, at high concentrations, suggesting the involvement of base excision repair (BER) in this process, iii) the association of emodin with UVA showed to be cytotoxic in all strains of E. coli iv) The nfo gene was the most important in bacterial resistance to damages induced by the association of the two agents, reinforcing the involvement of BER and indicating a possible role of the nucleotide incision repair (NIR), v) emodin appears to have interacted with plasmid DNA, altering its migration pattern in the agarose gel; vi) in A549 cell line, emodin caused immediate toxic effects that seemed to be repaired with time. However, when the drug remained for 24 hours in contact with the cells, there was a reduction in cell survival which seems to be in a dose dependent mode, vii) The concentrations of 10μM and 25μM of emodin, when associated with UVA, were responsible for a survival reduction of more than 50% survival in A549 cells, reaching 100% of death with the concentration of 50μM of emodin, viii) UVA radiation potentiates the cytotoxic effect of emodin in two experimental models in the present study, indicating that this interaction is genotoxic and therefore harmful to health.

A proteína APE1 (do inglêsApurinic/Apyrimidinicendonuclease 1/ Redox Factor-1 - APE1/REF-1) é uma enzima multifuncional, cuja expressão encontra-se frequentemente aumentada em gliomas. Além de apresentar atividade no reparo por excisão de base (BER), o gene APE1 também atua como fator de redução, mantendo fatores de transcrição (FTs) em um estado reduzido ativo. A via BER de reparo do DNA tem sido apontada como um possível fator de resistência a terapias baseadas no uso de agentes alquilantes, tais como temozolomida (TMZ). No presente trabalho, utilizou-se a estratégia de inibição da transcrição do gene APE1 pelo método de RNA interferente(siRNA) e tratamento com a droga TMZ nas células de glioblastoma (GBM), T98G (resistente à TMZ) e U87MG (sensível à TMZ), a fim de verificar a influência do silenciamento do gene APE1 sobre as respostas celulares à droga avaliadas por vários ensaios, bem como os efeitos sobre a expressão transcricional dos genes alvos dos FTs regulados por APE1. O silenciamento de APE1 e o tratamento das células T98G com a TMZ foram eficazes no sentido de reduzir a proliferação e a capacidade clonogênica, além de intervir na progressão do ciclo celular com bloqueio na fase S. Tais efeitos foram acompanhados pelo aumento da indução de danos no DNA e da expressão de H2AX fosforilada (H2AX), o que justifica a queda na sobrevivência celular. O mesmo efeito não foi observado nas células U87MG silenciadas para APE1 e tratadas com a TMZ, havendo o predomínio dos efeitos causados pela TMZ, exceto por uma leve indução de danos no DNA e de H2AX. Adicionalmente, nas células T98G silenciadas e tratadas, verificou-se uma moderada indução de apoptose, que foi observada ao longo dos tempos avaliados (1 a 10 dias), com uma leve indução de caspase-3 (5 dias); nessas células, observou-se também a indução (3,8 vezes) de morte celular autofágica (5 dias). Entretanto, nas células U87MG,a indução de apoptose foi baixa e não houve indução de morte por autofagia, sugerindo outros mecanismos de morte envolvidos na eliminação dessas células em resposta ao tratamento com a TMZ. O silenciamento de APE1 causou uma redução acentuada na invasão das células T98G, de forma similar à observada nas células somente tratadas com a TMZ, sendo que a combinação (silenciamento de APE1 e tratamento com a droga) resultou em um efeito aditivo, enquanto que nas células U87MG a combinação foi eficaz no sentido de reduzir a proporção de células invasivas, fato não observado nas condições isoladas. Os genes COX2 e VEGF, alvos dos FT NFB e HIF-1 (regulados por APE1) foram reprimidos nas células T98G enquanto que o gene VEGF foi induzido nas células U87MG, entretanto, tais alterações no padrão de expressão transcricional foram observadas somente em resposta ao tratamento com a TMZ, independentemente do silenciamento de APE1, indicando nenhuma mudança na atividade redox de APE1, possivelmente pela existência de proteínas APE1 remanescentes na célula. Além disso, a expressão proteica de NFBp65(ser563) foi aumentada em ambas as linhagens silenciadas e tratadas com a TMZ, provavelmente devido à inibição da proliferação celular. Em geral, os resultados do presente trabalho demonstraram que a estratégia de inibição do gene APE1 (participante da via BER) mostrou-se potencialmente viável, suportando a contribuição do BER na resistência à TMZ, visto que nas condições testadas, observou-se uma sensibilização das células de GBM, com efeito restrito às células de GBM resistentes (linhagem T98G), sendo pouco eficaz no sentido de sensibilizar as células sensíveis (linhagem U87MG) a esse agente. Assim, há que considerar as características genéticas de cada linhagem de GBM, visto que estas são cruciais para as respostas apresentadas pelas células aos tratamentos empregados. / APE1 (Apurinic/Apyrimidinic endonuclease 1/ Redox Factor-1 - APE1/REF-1) protein is a multifunctional enzyme whose expression is often increased in gliomas. Besides presenting activity in base excision repair (BER), APE1 also acts as a reduction factor, maintaining transcription factors (TFs) in an active reduced state. The BER pathway has been implicated as a possible factor of resistance to therapies based on the use of alkylating agents such as temozolomide (TMZ). In the present study, we have been using a strategy of small interference RNA (siRNA) to down-regulate the APE1 gene under conditions of treatment with TMZ in T98G (resistant to TMZ) and U87MG (sensitive to TMZ), glioblastoma (GBM), in order to determine the effects of APE1 gene silencing on cellular responses to this drug, evaluated by several assays, as well as the effects on the transcriptional expression of target genes of TFs regulated by APE1. APE1 silencing and TMZ treatment was effective to reduce the cell proliferation and clonogenic capacity of T98G cells, in addition to interfering in the cell cycle progression (S-phase arrest). These effects were accompanied by induction of DNA damage and phosphorylation of H2AX (H2AX), which may explain the decrease in cell survival. The same effect was not observed in silenced U87MG and TMZ-treated cells, being observed a predominance of the effects caused by TMZ itself, except for a slight induction of DNA damage and H2AX. Additionally, in silenced T98G and TMZ-treated cells, there was a moderate induction of apoptosis, as observed over time (1 to 10 days), with a slight induction of caspase-3 (on day 5); for those cells, we also observed autophagic induction (3.8 fold) at day 5. However, the induction of apoptosis and autophagy in U87MG cells was very low, suggesting that other mechanisms of cell death might be involved in the elimination of GBM cells under TMZ treatment. APE1 silencing caused a marked reduction on the invasiveness of T98G cells, similarly to that observed in TMZ treated cells, while the combination (APE1 silencing and drug treatment) led to an additive effect. For U87MG, the treatment combination was effective in reducing the proportion of invasive cells, in spite of an absence of any effect produced by each isolated condition tested. Regarding to the expression profile of target genes of TFs regulated by the APE1 redox activity, it was observed that COX2 and VEGF genes, targets of FTs NFB and HIF-1, were down-regulated in T98G while VEGF gene showed induced in U87MG cells; however, such alterations in the transcriptional expression pattern were observed only in response to TMZ treatment, independently of APE1 gene silencing, indicating no change in the APE1 redox activity, possibly due to the presence of APE1 remaining proteins inside cells. In addition, NFBp65(ser563) protein expression was increased in both cell lines (silenced and treated with TMZ), probably due to the reduced cell proliferation rates. In general, the present results show that the strategy of APE1 gene knockdown was potentially viable, supporting the BER contribution of the mechanism of TMZ resistance, since under the conditions tested, there was a sensitization of GBM cells. However, this effect was restricted to the resistant cell line (T98G cells). Thus, it should be considered the genetic characteristics of each GBM cell line, since these are crucial to the cellular responses to the conditions tested in the present work.

<p>Ends of linear chromosomes are maintained by specialized structures known as telomeres. These structures are protected by a number of essential protein complexes including the shelterin complex and CST (CTC1 – STN1 – TEN1) complex. CST is an RPA-like ssDNA binding protein that is vital for telomere length maintenance <i>via</i> inhibition of telomerase and stimulation of DNA polymerase α -primase during C-strand fill-in synthesis. CST is also known to possess additional genome-wide roles in regulating DNA replication and repair including helping facilitate replication re-start at stalled forks, activating checkpoint signaling at double-strand breaks, and promoting replication origin firing. Proper and efficient repair of DNA is critical in order to protect the integrity of the genome and prevent extreme mutagenesis. Telomeres have a strong predisposition to oxidative DNA damage in the form of 8-oxoguanine caused by exposure to reactive oxygen species and free radicals. These oxidative lesions are repaired by the base-excision repair (BER) pathway. Previous work has implicated telomeric proteins such as the shelterin complex in mediating BER. Here we show for the first time that the CST complex and individual subunits robustly stimulate a myriad of proteins involved in the BER pathway including Pol β, APE1, FEN1, and LIGI. CST’s ability to augment these BER-associated proteins could be instrumental in promoting efficient DNA repair. Additionally, we find that CTC1 and STN1 are able to significantly enhance the polymerase activity of Pol δ and Pol α on both random-sequence and telomeric-sequence DNA substrates <i>in vitro</i>. What is more, we establish the ability of CST to resolve G4 structure and promote Pol δ synthesis, which we predict is a key feature of CST’s involvement in DNA replication at telomeres, which are known to form replication-inhibiting G4’s. Our results define important mechanistic insight into CST’s role in DNA replication and repair, and provide a strong foundation for future studies relating defective telomere maintenance to aging disorders and cancers which impact human health.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / The DNA Base Excision Repair (BER) pathway repairs DNA damaged by endogenous and exogenous agents including chemotherapeutic agents. Removal of the damaged base by a DNA glycosylase creates an apurinic / apyrimidinic (AP) site. AP endonuclease1 (Ape1), a critical component in this pathway, hydrolyzes the phosphodiester backbone 5’ to the AP site to facilitate repair. Additionally, Ape1 also functions as a redox factor, known as Ref-1, to reduce and activate key transcription factors such as AP-1 (Fos/Jun), p53, HIF-1α and others. Elevated Ape1 levels in cancers are indicators of poor prognosis and chemotherapeutic resistance, and removal of Ape1 via methodology such as siRNA sensitizes cancer cell lines to chemotherapeutic agents. However, since Ape1 is a multifunctional protein, removing it from cells not only inhibits its DNA repair activity but also impairs its other functions. Our hypothesis is that a small molecule inhibitor of the DNA repair activity of Ape1 will help elucidate the importance (role) of its repair function in cancer progression as wells as tumor drug response and will also give us a pharmacological tool to enhance cancer cells’ sensitivity to chemotherapy. In order to discover an inhibitor of Ape1’s DNA repair function, a fluorescence-based high-throughput screening (HTS) assay was used to screen a library of drug-like compounds. Four distinct compounds (AR01, 02, 03 and 06) that inhibited Ape1’s DNA repair activity were identified. All four compounds inhibited the DNA repair activity of purified Ape1 protein and also inhibited Ape1’s activity in cellular extracts. Based on these and other in vitro studies, AR03 was utilized in cell culture-based assays to test our hypothesis that inhibition of the DNA repair activity of Ape1 would sensitize cancer cells to chemotherapeutic agents. The SF767 glioblastoma cell line was used in our assays as the chemotherapeutic agents used to treat gliobastomas induce lesions repaired by the BER pathway. AR03 is cytotoxic to SF767 glioblastoma cancer cells as a single agent and enhances the cytotoxicity of alkylating agents, which is consistent with Ape1’s inability to process the AP sites generated. I have identified a compound, which inhibits Ape1’s DNA repair activity and may have the potential in improving chemotherapeutic efficacy of selected chemotherapeutic agents as well as to help us understand better the role of Ape1’s repair function as opposed to its other functions in the cell.

No description available.

<p>Ageing is associated with a progressive decline in cognitive and physical function, as well as neurodegeneration. The DNA damage theory of ageing postulates that phenotypes associated with chronological ageing result from a time dependent accumulation of DNA damage caused by endogenously generated reactive oxygen species (ROS). In this work, we have used a host cell reactivation (HCR) technique to examine base excision repair (BER), the major pathway for removal of ROS generated damage, in fibroblasts from normal individuals and from patients with Cockayne syndrome (CS). The HCR assay utilizes an adenovirus encoded β-galactosidase (β-gal) reporter gene treated with methylene blue plus visible light (MB+VL) to measure BER of 7,8-dihydro-8-oxoguanine (8-oxoG). The results presented here demonstrate that host cell repair mechanisms remove MB+VL generated 8-oxoG from viral DNA and that reactivation of gene expression correlates with cellular repair capacity and requires CSA and CSB. Using the HCR assay, we demonstrate that culturing of primary human fibroblasts in media containing low levels of MB increases BER, suggesting increased DNA repair capacity may play a role in the therapeutic application of MB in Alzheimer’s disease treatment. We also demonstrate that BER decreases <em>in vitro </em>with increasing number of cell divisions, and that HCR of the damaged reporter gene is lower in fibroblasts from older donors. Using a second β-gal reporter gene assay, the enhanced expression assay, we were unable to show a relationship between the degree of decreased BER in CS and severity of clinical phenotype. However, we identified an interaction between CSB and the telomere protein TRF2. Overexpression of TRF2 leads to decreased nucleotide excision repair of UVC induced damage in a CSB dependent manner. We also demonstrate defective telomeres in the absence of functional CSB. The data presented in this work provide additional support for the DNA damage theory of ageing.</p> / Doctor of Philosophy (PhD)

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Pesticides are among the most pervasive environmental contaminants and they are an important potential risk for human health. Agricultural workers are constantly exposed to pesticide spray, drift and residues in the soil and foliage. Many agricultural pesticides are readily absorbed by the body, through contact with the skin, the respiratory track, the eyes, and the gastrointestinal system. Multiple studies have reported a strong association between pesticide exposure and various health outcomes including cancer. Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects and neurological diseases. The focus of the present translational study is to examine the relationship between human exposure to the organophosphate pesticide azinphos methyl (AZM) and oxidative stress by measuring biomarkers of oxidative stress in biological fluids (i.e., urine, serum) and peripheral blood lymphocytes (PBLs) of agricultural workers. The findings from these field studies will be validated in vitro by examining cultures of human lymphocytes treated with AZM for similar biomarkers of oxidative stress. Since the collection of PBLs from study participants is highly invasive and not suitable for studies involving younger subjects, we also examined buccal cells for biomarkers of oxidative stress (i.e., DNA damage) as a more universal source of human tissue to assess oxidative stress in pesticide exposed individuals. We demonstrated in this study that AZM induces oxidative stress and causes DNA damage in human tissues. Agricultural workers who had been exposed to AZM showed elevated serum levels of lipid peroxides, increased urinary levels of 8-OH-dG, and lymphocytes from these individuals showed increased DNA damage and associated changes in oxidative DNA repair enzymes. Biomarkers of oxidative stress were also elevated in human lymphocytes treated with physiologically relevant concentrations of AZM. In cultures of human lymphocytes, AZM caused a concentration-dependent loss of viability and associated increases in ROS and a reduction in intracellular GSH. We also demonstrated that viable leukocytes from the oral cavity can be readily obtained from humans and these buccal cells can be used to assess DNA damage following exposure to occupational and environmental genotoxicants. We also noted that oral leukocytes are especially sensitive to cryopreservation with DMSO and thus, these cells must be cryoprotected with 5% DMSO to preserve the viability of these cells for subsequent biochemical studies. In summary, these in vivo and in vitro studies demonstrated that AZM induces oxidative stress in a dose-dependent matter and that oral lymphocytes are a good source of human tissue for assessing DNA damage and possibly other biochemical changes. The possible health implications of the variations in these biomarkers of oxidative stress and DNA damage are undetermined. Yet the findings from these studies have provided a strong foundation for determining the mechanism by which pesticide induce oxidative stress, to explore the putative relationship between pesticide-induced oxidative stress and disease (e.g. cancer, neurodegenerative disorders) and determine whether tissue damage in humans is brought about by direct or by indirect action of organophosphate pesticides. / Graduation date: 2010

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Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.