Combined Analysis of Gamma-H2AX/53BP1 Foci and Caspase Activation in Lymphocyte Subsets Detects Recent and More Remote Radiation Exposures

<p>Analysis of gamma-H2AX foci in blood lymphocytes is a promising approach for rapid dose estimation to support patient triage after a radiation accident but has one major drawback: the rapid decline of foci levels post-exposure cause major uncertainties in situations where the exact timing between exposure and blood sampling is unknown. To address this issue, radiation-induced apoptosis (RIA) in lymphocytes was investigated using fluorogenic inhibitors of caspases (FLICA) as an independent biomarker for radiation exposure, which may complement the gamma-H2AX assay. Ex vivo X-irradiated peripheral blood lymphocytes from 17 volunteers showed dose-and time-dependent increases in radiation-induced apoptosis over the first 3 days after exposure, albeit with considerable interindividual variation. Comparison with gamma-H2AX and 53BP1 foci counts suggested an inverse correlation between numbers of residual foci and radiation-induced apoptosis in lymphocytes at 24 h postirradiation (P = 0.007). In T-helper (CD4), T-cytotoxic (CD8) and B-cells (CD19), some significant differences in radiation induced DSBs or apoptosis were observed, however no correlation between foci and apoptosis in lymphocyte subsets was observed at 24 h postirradiation. While gamma-H2AX and 53BP1 foci were rapidly induced and then repaired after exposure, radiation-induced apoptosis did not become apparent until 24 h after exposure. Data from six volunteers with different ex vivo doses and post-exposure times were used to test the capability of the combined assay. Results show that simultaneous analysis of gamma-H2AX and radiation-induced apoptosis may provide a rapid and more accurate triage tool in situations where the delay between exposure and blood sampling is unknown compared to gamma-H2AX alone. This combined approach may improve the accuracy of dose estimations in cases where blood sampling is performed days after the radiation exposure.��</p>

Radioprotection of targeted and bystander cells by methylproamine

<p>INTRODUCTION: Radioprotective agents are of interest for application in radiotherapy for cancer and in public health medicine in the context of accidental radiation exposure. Methylproamine is the lead compound of a class of radioprotectors which act as DNA binding anti-oxidants, enabling the repair of transient radiation-induced oxidative DNA lesions. This study tested methylproamine for the radioprotection of both directly targeted and bystander cells.</p><p>METHODS: T98G glioma cells were treated with 15 ��M methylproamine and exposed to (137)Cs ��-ray/X-ray irradiation and He(2+) microbeam irradiation. Radioprotection of directly targeted cells and bystander cells was measured by clonogenic survival or ��H2AX assay.</p><p>RESULTS: Radioprotection of directly targeted T98G cells by methylproamine was observed for (137)Cs ��-rays and X-rays but not for He(2+) charged particle irradiation. The effect of methylproamine on the bystander cell population was tested for both X-ray irradiation and He(2+) ion microbeam irradiation. The X-ray bystander experiments were carried out by medium transfer from irradiated to non-irradiated cultures and three experimental designs were tested. Radioprotection was only observed when recipient cells were pretreated with the drug prior to exposure to the conditioned medium. In microbeam bystander experiments targeted and nontargeted cells were co-cultured with continuous methylproamine treatment during irradiation and postradiation incubation; radioprotection of bystander cells was observed.</p><p>DISCUSSION AND CONCLUSION: Methylproamine protected targeted cells from DNA damage caused by ��-ray or X-ray radiation but not He(2+) ion radiation. Protection of bystander cells was independent of the type of radiation which the donor population received.</p>

Two potential clinical applications of the alkaline single-cell gel electrophoresis assay .1. Human bladder washings and transitional cell carcinoma of the bladder .2. Human sperm and male infertility

<p>Part 1: The alkaline single-cell gel electrophoresis (comet) assay was used to analyse the integrity and DNA content of exfoliated cells extracted from bladder washing specimens from 9 transitional cell carcinoma patients and 15 control patients. DNA damage, as expressed by % tail DNA and tail moment values, was observed to occur in cells from both control and bladder cancer samples. The extent of the damage was, however, found to be significantly greater in the cancer group than in the control group. Comet optical density values were also recorded for each cell analysed in the comet assay and although differences observed between tumour grades were not found to be statistically significant, the mean comet optical density value was observed to be greater in the cancer group than in the control population studied, These preliminary results suggest that the comet assay may have potential as a diagnostic tool and as a prognostic indicator in transitional cell carcinoma, Part 2: Baseline DNA damage in sperm cells from 13 normozoospermic fertile males, 17 normozoospermic infertile males and 11 asthenozoospermic infertile males were compared using a modified alkaline comet assay technique. No significant difference in the level of baseline DNA damage was observed between the 3 categories of sperm studied; however the untreated sperm cells were observed to display approximately 20% tail DNA. This is notably higher than the background DNA damage observed in somatic cells where the % tail DNA is normally less than 5%. Sperm from the 3 groups of men studied were also compared for sensitivity to DNA breakage, using the modified alkaline comet assay, following X-ray irradiations (5, 10 and 30 Gy) and hydrogen peroxide treatments (40, 100 and 200 mu M). Significant levels of X-ray-induced damage were found relative to untreated control sperm in the two infertile groups following 30 Gy irradiation. Significant damage in hydrogen peroxide-treated sperm was observed in sperm from fertile samples, at 200 mu M and in infertile samples at 100- and 200-mu M doses relative to controls. These results therefore indicate that fertile sperm samples are more resistant to X-ray- and hydrogen peroxide-induced DNA breakage than infertile samples. Further studies involving greater numbers of individuals are currently in progress to confirm these findings.</p>

Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells

<p>The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34 + cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript andor exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicingprocessing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expressionsplicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34 + cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processespathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.</p>

BRCA1, a 'complex' protein involved in the maintenance of genomic stability

BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mRNA splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1-related protein complexes and their roles in maintaining genomic stability and tumour suppression.

Leukaemogenesis, gene interplay, and the role of the haemopoietic environment

<p>Malignant initiation, leukaemic transformation, and disease progression in haematological malignancies involves a series of mutational events in genes involved in normal housekeeping functions of the cell. These acquired genetic changes can lead to either increased proliferation or a decreased rate of apoptosis, thus allowing expansion of the malignant clone. Although leukaemia can arise as a de novo disease, it has become increasingly clear that therapies, including the use of irradiation and/or chemotherapy, can give rise to malignancy. Therapy-associated myelodysplasia (t-MDS) and therapy-associated acute myeloid leukaemia (t-AML) account for 10-20% of new cases of these diseases. Although these secondary malignancies have been recognised as a clinical entity for nearly 30 years, molecular studies are now pinpointing various regions of the genome that are susceptible to DNA damage by these chemotherapeutic/radiotherapeutic strategies. The detection of new malignancies (both solid tumours and haematological tumours) following allogeneic bone marrow transplantation (BMT) is also providing us with some clues to the nature of leukaemogenesis, particularly with the observation that leukaemia can occur in donor cells postallogeneic BMT.</p>

Imaging and radiation effects of gold nanoparticles in tumour cells

<p>Gold nanoparticle radiosensitization represents a novel technique in enhancement of ionising radiation dose and its effect on biological systems. Variation between theoretical predictions and experimental measurement is significant enough that the mechanism leading to an increase in cell killing and DNA damage is still not clear. We present the first experimental results that take into account both the measured biodistribution of gold nanoparticles at the cellular level and the range of the product electrons responsible for energy deposition. Combining synchrotron-generated monoenergetic X-rays, intracellular gold particle imaging and DNA damage assays, has enabled a DNA damage model to be generated that includes the production of intermediate electrons. We can therefore show for the first time good agreement between the prediction of biological outcomes from both the Local Effect Model and a DNA damage model with experimentally observed cell killing and DNA damage induction via the combination of X-rays and GNPs. However, the requirement of two distinct models as indicated by this mechanistic study, one for short-term DNA damage and another for cell survival, indicates that, at least for nanoparticle enhancement, it is not safe to equate the lethal lesions invoked in the local effect model with DNA damage events.</p>

Addiction to Runx1 is partially attenuated by loss of p53 in the E��-Myc lymphoma model

<p>The Runx genes function as dominant oncogenes that collaborate potently with Myc or loss of p53 to induce lymphoma when over-expressed. Here we examined the requirement for basal Runx1 activity for tumor maintenance in the E��-Myc model of Burkitt's lymphoma. While normal Runx1fl/fl lymphoid cells permit mono-allelic deletion, primary E��-Myc lymphomas showed selection for retention of both alleles and attempts to enforce deletion in vivo led to compensatory expansion of p53null blasts retaining Runx1. Surprisingly, Runx1 could be excised completely from established E��-Myc lymphoma cell lines in vitro without obvious effects on cell phenotype. Established lines lacked functional p53, and were sensitive to death induced by introduction of a temperature-sensitive p53 (Val135) allele. Transcriptome analysis of Runx1-deleted cells revealed a gene signature associated with lymphoid proliferation, survival and differentiation, and included strong de-repression of recombination-activating (Rag) genes, an observation that was mirrored in a panel of human acute leukemias where RUNX1 and RAG1,2 mRNA expression were negatively correlated. Notably, despite their continued growth and tumorigenic potential, Runx1null lymphoma cells displayed impaired proliferation and markedly increased sensitivity to DNA damage and dexamethasone-induced apoptosis, validating Runx1 function as a potential therapeutic target in Myc-driven lymphomas regardless of their p53 status.</p>

Efeitos genot��xicos e bioqu��micos de xenobi��ticos em animais aqu��ticos

No presente trabalho, foi estudado um largo espectro de efeitos genot��xicos e bioqu��micos na tainha-garrento (Liza aurata). Nos Cap��tulos II e III s��o descritos os efeitos de exposi����o de curta dura����o ao fenantreno, um hidrocarboneto arom��tico polic��clico (HAP). A exposi����o de curta dura����o (16 horas) demonstrou a capacidade deste composto induzir a actividade da enzima de fase I da biotransforma����o, etoxiresorufina O-desetilase (EROD), provocar decr��scimos de integridade no ADN hep��tico e aumento de anomalias nucleares eritroc��ticas (ANE). Em termos de respostas de stresse, os n��veis plasm��ticos de cortisol e glucose aumentaram face �� exposi����o a este HAP. A exposi����o ao fenantreno induziu o decr��scimo da glutationa peroxidase (GPx) nas guelras, enquanto que no f��gado a actividade da GPx aumentou. No rim, a actividade da glutationa S-transferase (GST) foi inibida. Nas guelras, verificou-se um aumento da catalase. O fenantreno demonstrou igualmente a capacidade de induzir um aumento dos n��veis de glutationa nas guelras e f��gado. Estas respostas demonstraram a sensibilidade de L. aurata, a este HAP, real��ando a especificidade das respostas em termos de ��rg��os. Apesar dos aumentos das defesas antioxidantes, o potencial t��xico deste composto foi demonstrado pelo aumento da peroxida����o lip��dica nos tr��s ��rg��os. Nos cap��tulos seguintes, s��o descritas as respostas de L. aurata capturada na Ria de Aveiro, em locais com diferentes perfis de contamina����o, inicialmente no Outono de 2005 (Cap��tulos III a IX) e posteriormente analisando respostas sazonais (Cap��tulos X e XI). A an��lise de respostas de stresse (cortisol, glucose e lactato) revelou que L. aurata capturada em Vagos (local contaminado por HAPs) apresentava n��veis baixos de cortisol, enquanto que no Laranjo (local contaminado por merc��rio) apresentavam elevados n��veis de glucose e lactato. Relativamente ��s hormonas do eixo hipot��lamo ��� hip��fise ��� tir��ide (HHT), foram observados elevados n��veis plasm��ticos da hormona estimuladora da tir��ide (TSH) nos organismos capturados no Laranjo, baixos n��veis de tiroxina (T4) nos organismos da Barra (local sujeito a tr��fego naval) e baixos n��veis de triiodotironina (T3) no Rio Novo do Pr��ncipe (pr��ximo de um antigo efluente de pasta de papel), Laranjo e Vagos. A avalia����o das defesas antioxidantes, dano oxidativo e genot��xico nas guelras, rim e f��gado revelou diferen��as significativas nas respostas dos ��rg��os. L. aurata capturada na Barra apresentou dano oxidativo nas guelras (Cap��tulo V). No rim foi detectada uma diminui����o da integridade do ADN no Rio Novo do Pr��ncipe e Vagos (Cap��tulo VI), enquanto que no f��gado foi observado dano lip��dico na Gafanha e Vagos (Cap��tulo VIII). O dano n��o esteve sempre associado a um decr��scimo das defesas. As an��lises da ��gua e do sedimento da Ria de Aveiro (Outono de 2005) revelaram elevadas concentra����es de metais (Cd, Hg, Cu e Zn),principalmente, no Laranjo e Rio Novo do Pr��ncipe. L. aurata capturada nestes locais apresentou os n��veis mais elevados de metalotioninas hep��ticas (Cap��tulo VII) que parecem respons��veis pela inexist��ncia de danos no f��gado (Cap��tulo VIII). O dano oxidativo no ADN, avaliado atrav��s da quantifica����o dos n��veis plasm��ticos de 8-hidroxi-2-desoxiguanosina (8-OHdG) e o dano clastog��nico/aneug��nico, avaliado atrav��s da quantifica����o da frequ��ncia de ANE, foram estudados, no Outono de 2005, em duas esp��cies de peixes (L. aurata e Dicentrarchus labrax - robalo) (Cap��tulo IX). Os resultados revelaram grande sensibilidade de D. labrax em termos de dano oxidativo no ADN na Gafanha, Rio Novo do Pr��ncipe e Vagos, enquanto que L. aurata apresentou dano oxidativo apenas no Laranjo. O aumento da frequ��ncia de ANE apenas foi detectado em L. aurata, em Vagos, n��o se tendo detectado correla����o entre estes dois par��metros. O estudo sazonal (Maio de 2006 a Mar��o de 2007) do dano oxidativo no ADN e frequ��ncia de ANE em L. aurata (Cap��tulo X) demonstrou a varia����o destes par��metros com a esta����o do ano, apesar de n��o se ter verificado correla����o com os par��metros hidrol��gicos determinados. No entanto, no local de refer��ncia n��o se verificaram diferen��as sazonais, o que sugere que estes biomarcadores reflectem varia����es de biodisponibilidade de contaminantes. A an��lise global dos resultados das diferentes esta����es do ano revelou que L. aurata capturada no Rio Novo do Pr��ncipe e em Vagos apresentou maior susceptibilidade a dano oxidativo no ADN. No entanto, apenas L. aurata capturada em Vagos apresentou frequ��ncia de ANE superior �� do local de refer��ncia. Os dados do estudo sazonal revelaram uma correla����o entre dano oxidativo e ANE, sugerindo o stresse oxidativo como um poss��vel mecanismo envolvido na forma����o de anomalias. A integridade do ADN das guelras, rim, f��gado e sangue de L. aurata foi igualmente estudada ao longo de um ano (Cap��tulo XI), tendo-se verificado uma grande variabilidade ao longo deste per��odo. N��o foi demonstrada sensibilidade a um perfil de contamina����o espec��fico, tendo-se verificando variabilidade sazonal no local de refer��ncia. Globalmente, os resultados demonstraram a import��ncia da utiliza����o de uma bateria de biomarcadores na monitoriza����o ambiental e a especificidade da resposta dos diferentes ��rg��os de L. aurata.

Genotoxicity and cytotoxicity of Cr(VI) and Pb��+ in Pisum sativum

A toxicidade dos metais �� uma problem��tica que envolve a sa��de humana e o ambiente, sendo necess��ria uma vigil��ncia constante e uma avalia����o dos danos precisa e robusta. As plantas, como principal fonte alimentar e de produtos, s��o de vital import��ncia �� sociedade humana. Devido a serem seres sesseis, este grupo �� um dos mais afectados por poluentes, tornandoos objectos de estudo extremamente interessantes. O objectivo desta tese foi avaliar os efeitos genot��xicos e citot��xicos do Cr(VI) e Pb2+ na esp��cie modelo Pisum sativum L. No capitulo I �� introduzida a problem��tica da toxicidade de ambos os metais, com especial relevo nas plantas, bem como as abordagens mais actuais no estudo da geno e citotoxicidade. No capitulo II s��o apresentados os resultados dos estudos da genotoxicidade do Pb2+ (II-1) e Cr(VI) (II-2 e II-3), tendo sido realizados analises de dano ao DNA a v��rios n��veis e altera����es do ciclo celular (II-1 e II-2), bem como a detec����o de instabilidade de microssatelites (II-1 e II-3), que �� um indicador do estado funcional do mecanismo de repara����o do DNA. O cap��tulo III aborda o efeito de stresses abi��ticos na capacidade fotossint��tica da esp��cie modelo. No cap��tulo III-1, realizou-se um estudo pioneiro de avalia����o da aplicabilidade da citometria de fluxo no estudo da fotoss��ntese, mais concretamente no estado funcional e estrutural dos cloroplastos, quando expostos a um inibidor da fotoss��ntese (Paraquat). Os dados obtidos neste estudo encorajaram a aplica����o da t��cnica nos cap��tulos III-2 e III-3, nos quais se analisaram os efeitos dos metais Pb2+ (III- 2) e Cr(VI) (III-3) na capacidade fotossint��tica de plantas expostas a este metal; em estudos que envolveram v��rios marcadores cl��ssicos, para alem dos da citometria de fluxo. Finalmente, no cap��tulo IV s��o apresentadas as conclus��es finais do trabalho, uma comparativa entre os efeitos e n��veis de toxicidade dos dois metais em estudo e s��o apontadas algumas perspectivas para futuros estudos, levantadas pelos dados obtidos.

Genotoxic risk of herbicides to Anguilla anguilla L.

No contexto dos contaminantes aqu��ticos, os herbicidas s��o considerados como um dos grupos mais perigosos. Uma vez aplicados, estes s��o facilmente transportados para cursos de ��gua, quer devido a uma pulveriza����o pouco cuidada ou devido a fen��menos de escorr��ncia superficial e/ou subterr��nea. A presen��a destes agroqu��micos no ambiente tem vindo a ser associada a efeitos nefastos em organismos n��o-alvo, como �� o caso dos peixes. Contudo, existe ainda uma grande lacuna no que diz respeito �� informa����o cient��fica relacionada com o seu impacto genot��xico. Deste modo, a presente tese foi delineada com o intuito de avaliar o risco genot��xico em peixes de duas formula����es de herbicidas: o Roundup��, que tem como princ��pio activo o glifosato, e o Garlon��, que apresenta o triclopir na base da sua constitui����o, produtos estes largamente utilizados na limpeza de campos agr��colas, assim como em florestas. Foi ainda planeado desenvolver uma base de conhecimento no que diz respeito aos mecanismos de dano do ADN. Como ��ltimo objectivo, pretendeu-se contribuir para a mitiga����o dos efeitos dos agroqu��micos no biota aqu��tico, nomeadamente em peixes, fornecendo dados cient��ficos no sentido de melhorar as pr��ticas agr��colas e florestais. Este estudo foi realizado adoptando a enguia europeia (Anguilla anguilla L.) como organismo-teste, e submetendo-a a exposi����es de curta dura����o (1 e 3 dias) dos produtos comerciais mencionados, em concentra����es consideradas ambientalmente realistas. Para a avalia����o da genotoxicidade foram aplicadas duas metodologias: o ensaio do cometa e o teste das anomalias nucleares eritroc��ticas (ANE). Enquanto o ensaio do cometa detecta quebras na cadeia do ADN, um dano pass��vel de ser reparado, o aparecimento das ANE revela les��es cromossomais, sinalizando um tipo de dano de dif��cil repara����o. O ensaio do cometa foi ainda melhorado com uma nova etapa que incluiu a incuba����o com enzimas de repara����o (FPG e EndoIII), permitindo perceber a ocorr��ncia de dano oxidativo no ADN. No que diz respeito ao Roundup��, o envolvimento do sistema antioxidante como indicador de um estado pr��oxidante foi tamb��m alvo de estudo. Uma vez que as referidas formula����es se apresentam sob a forma de misturas, o potencial genot��xico dos seus princ��pios activos foi tamb��m avaliado individualmente. No caso particular do Roundup��, tamb��m foram estudados o seu surfactante (amina polietoxilada; POEA) e o principal metabolito ambiental (��cido aminometilfosf��rico; AMPA). Os resultados obtidos mostraram a capacidade do Roundup�� em induzir tanto dano no ADN (em c��lulas de sangue, guelras e f��gado) como dano cromoss��mico (em c��lulas de sangue). A investiga����o sobre o poss��vel envolvimento do stresse oxidativo demonstrou que o tipo de dano no ADN varia com as concentra����es testadas e com a dura����o da exposi����o. Deste modo, com o aumento do tempo de exposi����o, os processos relacionados com o envolvimento de esp��cies reactivas de oxig��nio (ERO) ganharam preponder��ncia como mecanismo de dano no ADN, facto que �� corroborado pela activa����o do sistema antioxidante observado nas guelras, assim como pelo aumento dos s��tios sens��veis a FPG em hepat��citos. O glifosato e o POEA foram tamb��m considerados genot��xicos. O POEA mostrou induzir uma maior extens��o de dano no ADN, tanto comparado com o glifosato como com a mistura comercial. Apesar de ambos os componentes contribuirem para a genotoxicidade da formula����o, a soma dos seus efeitos individuais nunca foi observada, apontando para um antagonismo entre eles e indicando que o POEA n��o aumenta o risco associado ao princ��pio activo. Deste modo, real��a-se a necessidade de regulamentar limiares de seguran��a para todos os componentes da formula����o, recomendando, em particular, a revis��o da classifica����o do risco do POEA (actualmente classificado com ���inerte���). Uma vez confirmada a capacidade do principal metabolito do glifosato ��� AMPA ��� em exercer dano no ADN assim como dano cromoss��mico, os produtos da degrada����o ambiental dos princ��pios activos assumem-se como um problema silencioso, real��ando assim a import��ncia de incluir o AMPA na avalia����o do risco relacionado com herbicidas com base no glifosato. A formula����o Garlon�� e o seu princ��pio activo triclopir mostraram um claro potencial genot��xico. Adicionalmente, o Garlon�� mostrou possuir um potencial genot��xico mais elevado do que o seu princ��pio activo. No entanto, a capacidade de infligir dano oxidativo no ADN n��o foi demonstrada para nenhum dos agentes. No que concerne �� avalia����o da progress��o do dano ap��s a remo����o da fonte de contamina����o, nem os peixes expostos a Roundup�� nem os expostos a Garlon�� conseguiram restaurar completamente a integridade do seu ADN ao fim de 14 dias. No que concerne ao Roundup��, o uso de enzimas de repara����o de les��es espec��ficas do ADN associado ao teste do cometa permitiu detectar um aparecimento tardio de dano oxidativo, indicando deste modo um decaimento progressivo da protec����o antioxidante e ainda uma incapacidade de reparar este tipo de dano. O per��odo de p��s-exposi����o correspondente ao Garlon�� revelou uma tend��ncia de diminui����o dos n��veis de dano, apesar de nunca se observar uma completa recupera����o. Ainda assim, foi evidente uma interven����o eficiente das enzimas de repara����o do ADN, mais concretamente as direccionadas ��s purinas oxidadas. A avalia����o das metodologias adoptadas tornou evidente que o procedimento base do ensaio do cometa, que detecta apenas o dano n��oespec��fico no ADN, possui algumas limita����es quando comparado com a metodologia que incluiu a incuba����o com as enzimas de repara����o, uma vez que a ��ltima mostrou reduzir a possibilidade de ocorr��ncia de resultados falsos negativos. Os dois par��metros adoptados (ensaio do cometa e teste das ANE) demonstraram possuir aptid��es complementares, sendo assim recomendado a sua utiliza����o conjunta com vista a efectuar uma avalia����o mais adequada do risco genot��xico. Globalmente, os resultados obtidos forneceram indica����es de grande utilidade para as entidades reguladoras, contribuindo ainda para a (re)formula����o de medidas de conserva����o do ambiente aqu��tico. Neste sentido, os dados obtidos apontam para a import��ncia da avalia����o de risco dos herbicidas incluir testes de genotoxicidade. A magnitude de risco detectada para ambas as formula����es adverte para a necessidade de adop����o de medidas restritivas em rela����o �� sua aplica����o na proximidade de cursos de ��gua. Como medidas mitigadoras de impactos ambientais, aponta-se o desenvolvimento de formula����es que incorporem adjuvantes selecionados com base na sua baixa toxicidade.

Re-evaluation of a reported increased micronucleus frequency in lymphocytes of workers occupationally exposed to formaldehyde

A replicate evaluation of increased micronucleus (MN) frequencies in peripheral lymphocytes of workers occupationally exposed to formaldehyde (FA) was undertaken to verify the observed effect and to determine scoring variability. May���Gr��nwald���Giemsa-stained slides were obtained from a previously performed cytokinesis-block micronucleus test (CBMNT) with 56 workers in anatomy and pathology laboratories and 85 controls. The first evaluation by one scorer (scorer 1) had led to a highly significant difference between workers and controls (3.96 vs 0.81 MN per 1000 cells). The slides were coded before re-evaluation and the code was broken after the complete re-evaluation of the study. A total of 1000 binucleated cells (BNC) were analysed per subject and the frequency of MN (in ���) was determined. Slides were distributed equally and randomly between two scorers, so that the scorers had no knowledge of the exposure status. Scorer 2 (32 exposed, 36 controls) measured increased MN frequencies in exposed workers (9.88 vs 6.81). Statistical analysis with the two-sample Wilcoxon test indicated that this difference was not significant (p = 0.17). Scorer 3 (20 exposed, 46 controls) obtained a similar result, but slightly higher values for the comparison of exposed and controls (19.0 vs 12.89; p = 0.089). Combining the results of the two scorers (13.38 vs 10.22), a significant difference between exposed and controls (p = 0.028) was obtained when the stratified Wilcoxon test with the scorers as strata was applied. Interestingly, the re-evaluation of the slides led to clearly higher MN frequencies for exposed and controls compared with the first evaluation. Bland���Altman plots indicated that the agreement between the measurements of the different scorers was very poor, as shown by mean differences of 5.9 between scorer 1 and scorer 2 and 13.0 between scorer 1 and scorer 3. Calculation of the intra-class correlation coefficient (ICC) revealed that all scorer comparisons in this study were far from acceptable for the reliability of this assay. Possible implications for the use of the CBMNT in human biomonitoring studies are discussed.

Efeitos da ingest��o de simbi��tico e indol-3-carbinol sobre o processo de carcinog��nese qu��mica de c��lon em ratos Wistar alimentados com dieta contendo heme

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Notch tumor suppressor function

Cancer development results from deregulated control of stem cell populations and alterations in their surrounding environment. Notch signaling is an important form of direct cell-cell communication involved in cell fate determination, stem cell potential and lineage commitment. The biological function of this pathway is critically context dependent. Here we review the pro-differentiation role and tumor suppressing function of this pathway, as revealed by loss-of-function in keratinocytes and skin, downstream of p53 and in cross-connection with other determinants of stem cell potential and/or tumor formation, such as p63 and Rho/CDC42 effectors. The possibility that Notch signaling elicits a duality of signals, involved in growth/differentiation control and cell survival will be discussed, in the context of novel approaches for cancer therapy

Deletion of the RNR4 gene causes hyperresistance to the carcinogen 4-NQO in the yeast model

La stabilit�� g��nomique, qui est essentielle �� la vie, est possible gr��ce �� la r��plication et la r��paration de l���ADN. Une des enzymes responsables de la r��plication et de la r��paration de l���ADN est la ribonucleotide reductase (RNR), qui est retrouv��e chez la levure et chez l���humain. Cette enzyme catalyse la formation de d��oxyribonucl��otides et maintien le pool de dNTP requis pour la r��paration et la r��plication de l���ADN. L���enzyme RNR est un t��tram��re ��2��2 constitu�� d���une grande (R1, ��2) et d���une petite (R2, ��2) sous-unit��. Chez S. cerevisiae, les g��nes RNR1 et RNR3 encodent la sous-unit�� ��2 (R1). L���activit�� catalytique de RNR d��pend d���une interaction avec le fer et de la formation d���un complexe entre R1 et R2. L���expression de toutes les sous-unit��s est inductible par les dommages caus��s �� l���ADN. Dans cette ��tude, nous d��montrons que des cellules qui n���expriment pas une des sous-unit��s, Rnr4, du complexe RNR sont sensibles �� divers agents endommageant l���ADN, tels que le m��thyl m��thane sulfonate, la bl��omycine, le p��roxyde d���hydrog��ne et les rayons ultraviolets (UVC 254 nm). Au contraire, le mutant est r��sistant au 4-nitroquinoline-1- oxide (4-NQO), un compos�� qui engendre des l��sions encombrantes. Par cons��quent, le mutant rnr4�� d��montre une r��duction marqu��e en mutations induites par le 4-NQO comparativement �� la souche parentale. Nous voulions identifier la voie de r��paration de l���ADN qui conf��rait cette r��sistance au 4-NQO ainsi que les prot��ines impliqu��es. Les voies BER, NER et MMR n���ont pas aboli la r��sistance au 4-NQO de la souche rnr4��. La prot��ine recombinante Rad51 ne joue pas un r��le critique dans la r��paration de l���ADN et dans la r��sistance au 4-NQO. La d��l��tion du g��ne REV3, qui encode une polym��rase de contournement, impliqu��e dans la r��paration post-r��plication, a partiellement aboli la r��sistance au 4-NQO dans rnr4��. Ces r��sultats sugg��rent que la polym��rase Rev3 et possiblement d���autres polym��rases transl��sion (Rev1, Rev7, Rad30) pourraient ��tre impliqu��es dans la r��paration de l��sions encombrantes dans l���ADN dans des conditions de carence en dNTP. La r��paration de l���ADN, un m��canisme complexe chez la levure, implique une vaste gamme de prot��ines, dont certaines encore inconnues. Nos r��sultats indiquent qu���il y aurait plus qu���une prot��ine impliqu��e dans la r��sistance au 4-NQO. Des investigations plus approfondies seront n��cessaires afin de comprendre la recombinaison et la r��paration post-r��plication.