Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Tamaril is a phytomedicine constituded of 5 medicinal plants well known for their laxative proprieties: Cassia fistula (soft extract), Cassia angustifolia (Senna), Coriandrum sativum L. e Glycyrrhiza glabra L. (AlcaÃuz) and Tamarindus indicus L. (soft extract). Every medication to be launched on the market must succeed in a series of research steps, where clinical toxicology evaluation is an important one among them. Genotoxic assessment, which aims on the processes altering DNA integrity, is a relatively recent field in drug development and stands on the interface between toxicology and genetics. This study consisted on the evaluation of clinical safety and genotoxic potential of Tamaril capsules in healthy volunteers. The clinical evaluation consisted of an open study with 25 healthy volunteers of both sexes (13 males and 12 females) who received a daily oral dose of two capsules Tamaril for 28 consecutive days. The volunteers were selected for the study if considered in good health after criterious clinical, physical and laboratorial evaluations. At the end of the 28 study days, blood samples (5 mL) were collected from each volunteer for the genotoxic assessment of Tamaril on peripheral lymphocytes through the comet assay. The mean age of the volunteers was of 30.1 6.9 years and the body mass index was of 24.21Â3.00 Kg/cm2 on the pre-study evaluation and 24.26Â3.05 Kg/cm2 on the post-study. Hematological, hepatic, renal and metabolic functions, as well as sodium and potassium did not show signs of abnormality in any volunteer throughout the weeks of the study. Soften faces, abdominal pain and flatulence were the adverse events regularly observed. Through the comet assay, score 1 DNA damage was most frequently registered on peripheral lymphocytes of volunteers treated with Tamaril (p<0.05). Clinical and genotoxic evaluation of healthy volunteers receiving Tamaril for 28 uninterrupted days did not show signs of toxicity related to the treatment. / O Tamaril à um fitoterÃpico composto de cinco plantas medicinais: Cassia fistula (extrato mole), Cassia angustifolia (Sene), Coriandrum sativum L., Glycyrrhiza glabra L. (AlcaÃuz), e Tamarindus indicus L. (extrato mole); todas com conhecida aÃÃo laxativa. Todo medicamento que vai ser registrado pela AgÃncia Nacional de VigilÃncia SanitÃria (Anvisa), passa por diversas etapas de pesquisa sendo uma delas a toxicologia clÃnica. A genotoxicidade à uma especialidade relativamente recente, e se situa na interface entre a toxicologia e a genÃtica. Esta visa o estudo dos processos que alteram o DNA (Ãcido desoxirribonuclÃico). O objetivo desse estudo foi avaliar a seguranÃa e o potencial genotÃxico da formulaÃÃo de Tamaril cÃpsulas em voluntÃrios saudÃveis. O ensaio clÃnico consistiu de um estudo aberto com 25 voluntÃrios de ambos os sexos, (13 homens e 12 mulheres), que receberam diariamente duas cÃpsulas de Tamaril v.o. por 28 dias ininterruptos. Os voluntÃrios foram incluÃdos no estudo apÃs avaliaÃÃo clÃnica, exames fÃsicos e laboratoriais. Ao final de 28 dias, amostras de sangue (5mL) foram coletadas de cada voluntÃrio, para avaliar o efeito genotÃxico do Tamaril em linfÃcitos perifÃricos humanos atravÃs do teste do cometa. A idade mÃdia dos voluntÃrios foi de 30,1  6,9 anos e o Ãndice de massa corpÃrea foi de 24,21  3,00 Kg/ cm2 no prÃ-estudo e 24,26  3,05 Kg/ cm2 no pÃs-estudo. As funÃÃes hematolÃgica, hepÃtica, renal e metabÃlica, bem como os eletrÃlitos sÃdio e potÃssio foram analisados semanalmente atravÃs dos exames laboratoriais, os quais nÃo evidenciaram sinal de toxicidade, estando todos os resultados dentro da faixa de normalidade. Fezes pastosas, dor abdominal e flatulÃncia foram os eventos adversos mais observados. Pelo teste do cometa, foram observados danos tipo 1 (p<0,05) nos linfÃcitos perifÃricos dos voluntÃrios tratados com TamarilÂ. Os estudos de Toxicologia ClÃnica e genotoxicidade nÃo evidenciaram nenhuma toxicidade nos voluntÃrios tratados com Tamaril por 28 dias ininterruptos 2 cÃpsulas por dia v.o.

nÃo hà / Na prÃtica odontolÃgica vÃrios fÃrmacos sÃo utilizados com diversas finalidades incluindo-se, entre eles, os agentes capeadores das polpas de dentes decÃduos. O formocresol à o composto mais utilizado em pulpotomias de dente decÃduos na diluiÃÃo de 1:5 da formulaÃÃo de Buckley. A seguranÃa clÃnica do formocresol tem sido questionada em virtude de possuir na sua composiÃÃo o formaldeÃdo, composto sabidamente tÃxico e carcinogÃnico. O presente trabalho tem como objetivo, estimar o potencial genotÃxico do formocresol em diferentes diluiÃÃes a partir da amostra comercializada. Esse estudo foi avaliado pelos ensaios de curta duraÃÃo in vivo e in vitro. Os animais foram tratados com o formocresol nas diluiÃÃes de 1:50, 1:100, 1:500 e 1:1000, e apÃs 24h e 48h sacrificados, sendo posteriormente a medula Ãssea extraÃda, e o material submetido Ãs observaÃÃes de perdas cromossÃmicas (micronÃcleos) em eritrÃcitos policromÃticos (PCE), alÃm disso, os fÃgados dos animais tratados (grupo 24h) foram submetidos à anÃlise histolÃgica. No ensaio in vitro o formocresol nas diluiÃÃes de 1:750, 1:1000 e 1:2000 foram incubados em cultura de linfÃcitos humanos por 45 min., sendo em seguida submetido ao procedimento de anÃlise do teste cometa. Na avaliaÃÃo da incidÃncia de micronÃcleo, no grupo 24h, nÃo houve diferenÃa quando da comparaÃÃo entre as diluiÃÃes de 1:50, 1:100, 1:500 e controle negativo, apenas quando comparada à diluiÃÃo de 1:1000 em relaÃÃo Ãs demais houve diferenÃa significativa (p < 0,001). PorÃm, quando a diluiÃÃo de 1:1000 foi comparada à ciclofosfamida, nÃo existiu diferenÃa significativa (p > 0,50). Enquanto a diluiÃÃo 1:1000 e da ciclofosfamida diferiram estatisticamente do controle negativo, no grupo 24h (p < 0,01). Entretanto, na observaÃÃo do grupo 48h, apenas a ciclofosfamida apresentou incidÃncia de micronÃcleos, diferindo estatisticamente dos grupos tratados e controle (p< 0,001). Jà no estudo histolÃgico do fÃgado, foi observado hepatotoxicidade nos animais tratados do grupo 24h. Na anÃlise do cometa, foi observado que em todas as diluiÃÃes do formocresol utilizadas houve a formaÃÃo de crosslinks no DNA, fator esse determinante para inferir o potencial genotÃxico do formocresol. ConcluÃmos que o formocresol à tÃxico e, quando diluÃdo atà 1000 vezes, à potencialmente genotÃxico e mutagÃnico. / In odontology practice, many drugs used have different purposes, such as primary teeth pulp covers. The Formocresol is the medicine most used in pulpotomy of primary teeth in 1:5 dilution of Buckleyâs formulation. The clinical safety of Formocresol has been questioned since its formulation is composed by formaldehyde, which is known as a toxic and carcinogenic compound. The present study has the objective of estimate the genotoxic potential of Formocresol in different dilutions using the commercialized sample. This study was evaluated by short period trials in vivo and in vitro. The animals were treated with Formocresol in the dilutions of 1:50, 1:100, 1:500 and 1:1000, and after 24h and 48h, they were sacrificed afterwards with the medulla extraction. This material was submitted to chromosomal damage observations (micronuclei) in polychromatic erythrocytes (PCE). The liver of the animals treated (24h group) were also submitted to histological analysis. In in vitro trial, the 1:750, 1:1000 and 1:2000 dilutions of Formocresol were incubated in human lymphocyte culture for 45 min and to comet test analysis next. There was no difference in micronuclei incidence evaluation when compared to 1:50, 1:100 and 1:500 dilutions and to negative control, in the 24h group. The difference appeared only when compared the 1:1000 dilution to the others, with a significant difference of p<0.001. However, when the dilution of 1:1000 was compared to the cyclophosphamide there was no significant difference (p>0.50). Meanwhile, the dilution of 1:1000 and of cyclophosphamide was statistically different from the negative control, in the 24h group. However, in the 48h group observations, only the cyclophosphamide presented micronuclei incidence, statistically differing from the treated groups and control (p<0.001). In the liver histological study it was observed hepatotoxicity in the animal treated in the 24h group. In comet analysis it was observed that in all used dilutions there was DNA crosslinks formation, which was an important factor to give a genotoxic potential to Formocresol. It was concluded that Formocresol is toxic and when diluted to 1:1000 is potentially genotoxic and mutagenic

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Kaurenoic acid (KA) is a diterpene presents in the oil-resin (copaiba oil) from plants belongs to Copaifera spp. As copaiba oil, KA also displayed a great variability of medicinal applications. In the present study, the genotoxic and mutagenic potential of KA from Copaifera langsdorffii on human lymphocytes, human leukemia cells (HL60) and bone marrow cells was evaluated. KA did not show selective action between lymphocytes and leukemia cells, has been induced apoptosis and DNA damage at same magnitude as valuated by bromide etidium/orange acridine and comet assay. Due to this observation, lymphocytes were selected for further experiments. According with comet assay results, more than 80% of lymphocytes DNA damage was repaired after 48 hours post-treatment. Lymphocytes treated with KA (30 and 60Âg/mL) showed increases on micronucleus frequencies in relation to negative control group. On the chromosome aberration test, lymphocytes treated at phse G1 and transition phase G1/S showed great sensibility (cytotoxicity and chromosomes aberrations) in comparison to cells treated at another phases of cell cycle. After treatment, any increase of polyploidy cells number was noted. Mices were treated with KA (25, 50 and 100mg/kg), and after 24 and 48 hours, they were sacrificed afterwards with the medulla extraction. This material was submitted to chromosomal damage observations (microniclei) in polychromatic erythrocytes (PCE). A great occurrence of micronucleated PCE was noted only at animals groups sacrificed 24 hours after treatment. The rate between PCE and NCE (normochromatic erythrocytes) was lower for animals sacrificed later. These observations indicating toxicity effects on the bone marrow cells. The mutagenic assay with yeast Saccharomyces cereviseae showed that the cytotoxic and mutagenic effects of KA were more pronounced during exponential growth phase, when the access to DNA is facilitated. KA induced locus and frameshift mutations. Frameshift mutations induced by DNA-intercalanting drugs have been correlated with DNA strand breaks induced by inhibition of DNA topoisomerases. On the DNA relaxation assay, KA inhibited the action of topoisomerase I. This inhibition effect seens to be related to the intercalanting ability of kaurenoic acid between DNA bases of pair. Thus, DNA strand breaks, the occurrence of micronucleated cells and frameshift mutations could be explained by the intercalanting action of kaurenoic acid. And the absence of polyploidy cells suggests that kaurenoic acid did not interfere on mitotic apparatus of cell. In conclusion, kaurenoic acid showed genotoxic and mutagenic effects on all the assays used / O Ãcido caurenÃico (AC) à um diterpeno presente no Ãleo resinoso de espÃcies de Copaifera. Assim como o Ãleo resinoso, o AC tambÃm apresenta uma ampla variabilidade de aplicaÃÃes medicinais. O presente trabalho teve como objetivo avaliar o potencial genotÃxico e mutagÃnico do AC isolado da planta Copaifera langsdorffii em linfÃcitos, cÃlulas leucÃmicas HL60 e em cÃlulas da medula Ãssea de camundongos. O AC nÃo mostrou seletividade entre linfÃcitos e HL60 tendo induzido apotose e danos ao DNA na mesma intensidade, avaliados pela coloraÃÃo diferencial por brometo de etÃdio/acridina laranja e pelo teste do cometa, respectivamente. De acordo com o teste do cometa, mais de 80% dos danos induzidos ao DNA de linfÃcitos foi reparada 48 horas apÃs o tratamento. LinfÃcitos tratados com AC apresentaram aumento, siginificativo, na freqÃÃncia de micronÃcleos e maior sensibilidade (citotoxicidade e aberraÃÃes cromossÃmicas) nas fases G1 e G1/S do ciclo celular, sem induzir aumento no nÃmero de cÃlulas poliplÃides. Camundongos foram tratados com AC nas doses de 25, 50 e 100mg/kg e apÃs 24 e 48 horas sacrificados, sendo, posteriormente, extraÃda a medula Ãssea, e o material submetido Ãs observaÃÃes de perdas cromossÃmicas (micronÃcleos) em eritrÃcitos policromÃticos. Uma maior incidÃncia de micronÃcleos ocorreu no grupo de animais sacrificados 24 horas apÃs o tratamento. A avaliaÃÃo da razÃo entre eritrÃcitos policromÃticos e normocromÃticos, foi menor para os animais sacrificados 48 horas apÃs o tratamento, indicando toxicidade em cÃlulas da medula. Nos ensaios de mutagÃnese com a levedura Saccharomyces cerevisea, o efeito citotÃxico e mutagÃnico do AC foi mais acentuado durante o crescimento exponencial da levedura, no qual o DNA està mais acessÃvel ao composto. O AC induziu mutaÃÃes lÃcus especÃficas e de deslocamento do quadro de leitura. MutaÃÃes do tipo deslocamento do quadro de leitura tendem a serem induzidas por agentes intercalantes de DNA e tÃm sido correlacionadas com as quebras de fitas de cadeia de DNA induzidas pela inibiÃÃo da aÃÃo de topoisomerase. No teste de relaxamento do DNA, o AC inibiu a aÃÃo da topoisomerase I. A inibiÃÃo da aÃÃo da topoisomerase I parece estar relacionada à intercalaÃÃo do AC no DNA. Assim, as quebras de fitas no DNA e induÃÃo de micronÃcleos e mutaÃÃes de deslocamento do quadro de leitura, podem estar relacionadas à aÃÃo intercalante do Ãcido caurenÃico. A ausÃncia de cÃlulas poliplÃides sugere que o Ãcido caurenÃico nÃo interfere no aparelho mitÃtico da cÃlula. Em conclusÃo, o Ãcido caurenÃico apresenta potencial genotÃxico e mutagÃnico nos modelos estudados.

nÃo hà / A utilizaÃÃo de agrotÃxicos na agricultura elevou rapidamente seu consumo, especialmente de forma indiscriminada, sendo o Brasil um dos maiores mercados, representando 16% da venda mundial. No PiauÃ, a expansÃo agrÃcola na regiÃo dos cerrados contribuiu para o aumento do seu uso, expondo os agricultores a danos ao DNA. O objetivo desse estudo foi avaliar os efeitos tÃxicos e genotÃxicos nos agricultores piauienses expostos aos agrotÃxicos, com o uso de biomarcadores hematolÃgicos, bioquÃmicos e genotÃxicos. A populaÃÃo estudada consistiu de 60 trabalhadores expostos aos agrotÃxicos dos municÃpios de Barras e Josà de Freitas e 55 indivÃduos controle, sem histÃria de exposiÃÃo a agroquÃmicos. Para caracterizaÃÃo da populaÃÃo foi aplicado questionÃrio sÃcio epidemiolÃgico, de acordo com a International Commission for Protection Against Environmental Mutagens and Carcinogens-ICPEMC. Foram coletados 10 mL de sangue perifÃrico para realizaÃÃo das anÃlises hematolÃgicas, bioquÃmicas e ensaio cometa, que foram processadas pelo LACEN-PI. A mÃdia de idade foi de 34 anos, de etnia negra, na maioria, com tempo de trabalho, em mÃdia de 13,55 anos, carga horÃria de 41,5 horas semanais e 50% dos trabalhadores utilizavam pelo menos um tipo de EPI. Quanto aos hÃbitos de vida, 66,7% dos trabalhadores expostos informou nÃo consumir vegetais, 41,7 % eram fumantes e 73,3% consumiam bebidas alcoÃlicas. Do total do grupo exposto, 33,3% usava medicamentos prescritos e 66,7% usava medicamentos nÃo prescritos. No estudo foi evidenciado maior uso na agricultura de herbicidas (81,1%) e inseticidas (16,3%). No grupo dos trabalhadores expostos, 55% apresentaram leucopenia e 6,7% apresentaram diminuiÃÃo na contagem de cÃlulas vermelhas. Foram evidenciadas alteraÃÃes na creatinina plasmÃtica (p < 0,05); nas transaminases e fosfatase alcalina (p< 0,01) quando comparado o grupo exposto com o nÃo exposto. Nos resultados do ensaio cometa, o grupo exposto apresentou, em relaÃÃo ao grupo nÃo exposto, uma mÃdia de (32,13 vs 10,12) de Ãndice de dano, e frequÃncia do dano (21,82 vs 9,38), respectivamente. Na classe 1, a genotoxicidade observada foi de 17% para os expostos e 9% para os nÃo expostos. NÃo houve significÃncia entre os danos no DNA em relaÃÃo Ãs variÃveis: tempo de trabalho, nÃo uso de EPI, hÃbito de fumar, consumo de Ãlcool e nÃo consumo de vegetais. Conclui-se que os trabalhadores expostos a agrotÃxicos apresentaram alteraÃÃes enzimÃticas, hematolÃgicas (leucopenia) e instabilidade genÃtica, avaliados por parÃmetros bioquÃmicos e genotÃxicos, demonstrando assim a importÃncia do biomonitoramento dos trabalhadores como uma estratÃgia de vigilÃncia em saÃde do trabalhador no Estado do PiauÃ. / The use of pesticides in agriculture rapidly increased their consumption, especially indiscriminate consumption, being Brazil currently the largest market for pesticide in the world, representing 16% of worldwide sales. In the state of PiauÃ, the agricultural expansion in the region of Cerrado contributed to their increased use, exposing farm workers to damages to the DNA. The purpose of this study was to evaluate the toxic and genotoxic effects in farm workers exposed to pesticides in PiauÃ, with the use of hematologic, biochemical and genotoxic biomarkers. The population in analysis consisted of 60 farm workers from the municipalities of Barras and Josà de Freitas occupationally exposed to pesticides and 55 control individuals with no history of exposure to agrochemicals. To obtain the characteristics of the population, a social-epidemiological questionnaire was applied, recommended by International Commission for Protection Against Environmental Mutagens and Carcinogens-ICPEMC. 10 mL of peripheral blood were collected for haematological, biochemical and comet assay analyses, all of which were processed by LACEN-PI. The mean age was 34 years, of black ethnicity, mostly with an average of 13.55 years of work, workload of 41.5 weekly hours and 50% of workers used at least one type of PPE. In what concerns lifestyle, 66.7% of the exposed workers said they did not consume vegetables, 41.7% were smokers and 73.3% consumed alcohol. Of the total of the exposed group, 33.3% used prescribed medication and 66.7% used non-prescribed medication. In the study, it was evidenced a higher use of herbicides (81.1%) and insecticides (16.3%) in agriculture. In the group of exposed workers, 55% had leucopenia and 6.7% showed a decrease in the red blood cell count. It was found variation in plasmatic creatinine (p < 0.05); in liver enzymes and alkaline phosphatise (p < 0.01) when comparing the exposed and the non-exposed groups. In the results of the comet assay, the exposed group showed, in comparison with the non-exposed group, a mean of (32.13 vs. 10.12) of damage index and damage frequency of (21.82 vs. 9.38), respectively. In class 1, the genotoxicity observed was 17% for the exposed and 9% for the non-exposed. There was no significance between DNA damage and the following variables: workload, non-use of PPE, smoking, consumption of alcohol and non-consumption of vegetables. We concluded that workers exposed to pesticides presented toxic variations and genetic instability, which was evidenced by enzymatic variation and damages to the DNA, which thus demonstrates the importance of biomonitoring of workers as a strategy of occupational health surveillance in the state of PiauÃ.

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-25T20:56:40Z No. of bitstreams: 2 Dissertação - Laise Rodrigues de Andrade - 2012.pdf: 1986605 bytes, checksum: 71d7e662c6200f1a50a70ae9addb99b0 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luanna Matias (lua_matias@yahoo.com.br) on 2015-03-26T13:07:25Z (GMT) No. of bitstreams: 2 Dissertação - Laise Rodrigues de Andrade - 2012.pdf: 1986605 bytes, checksum: 71d7e662c6200f1a50a70ae9addb99b0 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-26T13:07:25Z (GMT). No. of bitstreams: 2 Dissertação - Laise Rodrigues de Andrade - 2012.pdf: 1986605 bytes, checksum: 71d7e662c6200f1a50a70ae9addb99b0 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The rapid advancement of nanotechnology has created a vast array of nanoparticles promising for industrial, energy and environmental sectors. Furthermore, many biological applications have been proposed for the nanoparticles, such as transport of genes and drugs for disease treatments, including cancer and infections. So, it is important to clarify if nanoparticles may represent a hazard to the environment and human health. In this study, we investigated the toxic potential and ability to induce DNA damage of different nanoparticles: four species of carbon nanotubes (NTC, NT, CS e MW), silver (AgNP) and nanoceria (CeO2-NP) nanoparticles. It was employed the somatic mutation and recombination test (SMART) in Drosophila melanogaster that detects the loss of heterozygosity of two genetic markers involved in the metabolic pathways of wing hairs formation - multiple wing hairs (mwh) and flare3 (flr3). In larvae, the proliferating of wings imaginal disc cells can produce hairs with mutant phenotypes expressed on the adult wings. Using the standard cross, third-stage larvae were treated with different concentrations of the nanoparticles until pupal stage. The wings of adult flies were examined microscopically for the identification of phenotypic abnormalities. In all concentrations the survival rates were higher than 90%, indicating the absence of chronic toxicity for nanoparticles evaluated. Using the conditional binomial test, the results of different treatments were compared with the respective negative control (distilled water), demonstrating no significantly increase in the NTC, NT and CS mutation and recombination frequencies (p>0.05) in mwh/flr3 genotype. In all carbon nanotubes tested, only the two higher concentrations of nanotubes MW (0.4 and 1 mg/mL) were able to induce genetic changes, mainly by mitotic recombination. The concentration of 10 mg/mL AgNP also promoted changes in the DNA and 61% of the phenotypic abnormalities were caused by recombination. The nanoceria was able to produce genotoxic effects at all concentrations tested (0.64-10 mg/mL). Overall, the mutational events were predominant, ranging from 46 to 72% of the total genotoxic effect induced by nanoceria, showing no dose-response relationship. In conclusion, our results demonstrated that carbon, silver and cerium dioxide nanoparticles have different genotoxic potential in D. melanogaster, so, another studies should be performed before any clinical and/or industrial application of nanoparticles. / O rápido avanço da nanotecnologia permitiu a criação de uma grande variedade de nanopartículas promissoras para os setores industrial, energético e ambiental. Além disso, muitas aplicações biológicas têm sido propostas para as nanopartículas, como o transporte de genes e fármacos para o tratamento de doenças, incluindo o câncer e infecções. Portanto, é importante esclarecer se as nanopartículas podem representar um perigo ao ambiente e à saúde humana. Neste estudo, nós investigamos a toxicidade potencial e a capacidade de induzir danos ao DNA de diferentes nanopartículas: quatro espécies de nanotubos de carbono (NTC, NT, CS e MW), nanopartículas de prata (AgNP) e nanocéria (CeO2-NP). Foi utilizado o teste para detecção de mutação e recombinação somática (SMART) em Drosophila melanogaster que detecta a perda de heterozigose de dois marcadores genéticos envolvidos nas vias metabólicas de formação de pelos da asa – pelos múltiplos (mwh) e flare3 (flr3). Nas larvas, a proliferação das células dos discos imaginais das asas pode produzir pelos com fenótipos mutantes expressos nas asas dos indivíduos adultos. Usando o cruzamento padrão, larvas de terceiro estágio foram tratadas com diferentes concentrações das nanopartículas até atingir o estágio de pupa. As asas das moscas adultas foram examinadas microscopicamente para identificação de alterações fenotípicas. Em todas as concentrações as taxas de sobrevivência foram superiores a 90%, indicando ausência de toxicidade crônica das nanopartículas avaliadas. Usando o teste binomial condicional, os resultados dos diferentes tratamentos com NTC, NT e CS foram comparados com o respectivo controle negativo (água destilada), demonstrando que não houve aumento estatisticamente significativo (p>0,05) nas frequências de mutação e recombinação no genótipo mwh/flr3. De todos os nanotubos de carbono avaliados, apenas as duas maiores concentrações do nanotubo MW (0,4 e 1 mg/mL) foram capazes de induzir alterações genéticas, principalmente via recombinação mitótica. A concentração de 10 mg/mL de AgNP também promoveu alterações no DNA e 61% das anormalidades fenotípicas foram causadas por recombinação. A nanocéria foi capaz de produzir efeitos genotóxicos em todas as concentrações testadas (0,64-10 mg/mL). Destes efeitos, os eventos mutacionais foram predominantes, variando de 46 a 72% do efeito genotóxico total induzido pela nanocéria, sem demonstrar efeito dose resposta. Em conclusão, nossos resultados demonstraram que nanopartículas constituídas de carbono, prata e dióxido de cério têm potenciais genotóxicos distintos em D. melanogaster, portanto, outros estudos devem ser realizados antes de qualquer aplicação clínica e/ou industrial das nanopartículas.

La toxicité cellulaire potentielle de la sulcotrione 2-(2-chloro-4-(methylsulfonyl)benzoyl)-1,3-cyclohexanedione, un herbicide sélectif tricétonique a été évaluée sur Vicia faba et Allium cepa. La génotoxicité a été étudiée sur une culture hydroponique pour des traitements à différentes concentrations de sulcotrione 10-5, 10-4 et 2.10-4 M pendant 45 h. Nos résultats ont montré que la sulcotrione provoque une augmentation dose-dépendante de la fréquence des micronoyaux dans les cellules méristèmatiques des racines. Elle induit des altérations chromosomiques à la concentration la plus faible (10-5M) mais aussi une baisse de l’indice mitotique, ce qui indique l'effet mutagène puissant de cette molécule. C’est le premier travail montrant une génotoxicité de la sulcotrione. Les signes d’intoxication se manifestent par les perturbations de la croissance foliaire et racinaires accompagnées d’un brunissement des racines traitées de Vicia faba. Sulcotrione, Mikado®, marc de raisin et des mélanges de sulcotrione ou Mikado® et de marc de raisin induisent la mort cellulaire. La présence des herbicides ainsi que celle des cocktails ont entrainé l’installation d’un état de stress oxydant caractérisé par une production accrue d’H2O2. La production de radicaux d’oxygène actif s’accompagne d’une augmentation de la production de MDA et un accroissement de la mort cellulaire. L’ajout de l’extrait de raisin aux herbicides, soit à la sulcotrione ou au Mikado®, modifie l'expression des gènes habituellement associés au stress cellulaire. Les cocktails et les herbicides modifient l’expression des gènes hsp70.1, cat, ubiquitin, APX, CuZnSOD cy et CuZnSOD ch. Des mécanismes de défense, d’induction de gènes associés au stress et de génotoxicité sont discutés. / Potential cell toxicity of sulcotrione 2-(2-Chloro-4-(methylsulfonyl)benzoyl)-1,3 cyclohexanedione), a selective triketonic herbicide was evaluated on Vicia faba and Allium cepa . Genotoxicity was studied in hydroponic culture conditions for treatment at different pesticide concentrations 10-5, 10-4 and 2.10-4 M for 45 h. Our results showed that sulcotrione treatments caused a dose dependent increase of micronucleus frequencies in root meristematic cells. Sulcotrione induced chromosomal alterations at the lowest concentration used (10-5M) when incubated for 42 h. We have shown a decrease in mitotic index, indicating a potent mutagenic effect of this element. This is the first report for the genotoxicity of such a sulcotrione herbicide. It induced a growth inhibition in both leaves and roots and a brownish color in treated roots. Sulcotrione, trade mark Mikado®, grape marc and mixtures of sulcotrione or Mikado® and grape marc induce cell death. The herbicides, cocktails of products with sulcotrione, such as adjuvant in commercial product, induced several changes for antioxidant cell state characterized by an overproduction of H2O2. Production of harmful radicals was accompanied by increased production of MDA and increase of the cell death rate. Addition of grape extracts to herbicides, either sulcotrione or Mikado®, had different effects and results in different expression of genes usually associated to cell stress. Mixture of grape marc and herbicides enhanced transcript accumulation for different effects and results in different expression of some stress-related genes like hsp70.1, cat, ubiquitin, APX, CuZnSOD cy et CuZnSOD ch. Mechanisms which could be associated to gene expression, cell defense and genotoxidity are discussed.

L'exposition humaine aux contaminants environnementaux est inévitable du fait de leur présence dans l'eau, l'air et l'alimentation. La plupart d'entre eux sont reconnus comme étant mutagènes et/ou carcinogènes chez l'animal mais ils sont souvent seulement suspectés de l'être chez l'Homme. Le manque de connaissance vis-à-vis des substances chimiques a conduit l'UE à lancer le programme REACH avec l'objectif d'évaluer la toxicité d'environ 30 000 molécules. Cette évaluation nécessiterait l'utilisation de plus de 4 millions d'animaux et la pertinence controversée de ces modèles pourrait aboutir à des conclusions discutables. Les méthodes in vitro sont considérées comme une alternative potentielle à l'expérimentation animale. Néanmoins, le choix du modèle cellulaire et des conditions expérimentales restent à préciser. Les hépatocytes humains en culture primaire représentent le modèle le plus pertinent en toxicologie malgré de nombreuses contraintes (variabilité inter-individuelle, changements phénotypique précoces, obtention aléatoire). La lignée HepaRG constitue une alternative intéressante puisque ces cellules peuvent proliférer de manière illimitée et expriment les EMXs à des niveaux proches des hépatocytes humains. L'expression de ces enzymes restant stable pendant plusieurs semaines, ce modèle permet l'évaluation du risque lié à une exposition chronique aux contaminents environnementaux, essentielle en génotoxicité. Il reste cependant nécessaire de caractériser plus amplement cette lignée vis-à-vis des EMXs et de l'adapter aux tests de toxicologie actuels. Dans ces travaux, nous avons développé un test haut débit utilisant la quantification in situ des histones phosphorylées γH2AX avec l'objectif de pouvoir évaluer le risque génotoxique d'une exposition unique ou répétée aux contaminants environnementaux. Ce test a été validé avec succès par l'évaluation de la génotoxicité associée à une exposition de 1, 7 et 14 jours pour 10 polluants. Nous avons ensuite généré des lignées recombinantes biosenseurs, dérivées du modèle HepaRG et permettant d'identifier les xénobiotiques altérant l'expression transcriptionnelle des EMXs. Par transfection transitoire, nous avons dans un premier temps validé à l'aide d'inducteurs prototypiques et de nos 10 contaminants nos constructions contenant le gène rapporteur de la luciférase sous le contrôle des promoteurs de plusieurs EMXs. Ensuite, nous avons généré des lignées stables exprimant la GFP comme gène rapporteur et permettant une détection rapide des xénobiotiques capables d'induire l'expression des EMXs. Parmi les EMXs, le CYP2E1 joue un rôle important en santé humaine. En effet, cette enzyme induite dans certaines conditions physiopathologiques comme le diabète et l'obésité est responsable de l'activation de nombreux procarcinogènes et est à l'origine d'une production d'EROs. Les cellules HepaRG pourraient constituer un modèle pertinent pour l'étude du CYP2E1. Cependant, l'expression et l'activité de cette enzyme au sein de ce modèle nécessitent d'être mieux caractérisées en regard des données discordantes de la littérature. A l'aide de la chlorzoxazone, un marqueur spécifique de l'activité du CYP2E1, nous avons démontré l'influence du métabolisme de phase II sur l'activité apparente du CYP2E1. Nous proposons ici quelques recommandations afin de mieux quantifier l'activité du CYP2E1 sur les hépatocytes humains et sur le modèle HepaRG à l'aide la chlorzoxazone. / Human exposure to toxic chemicals is virtually unavoidable due to contamination of air, water and food. A number of environmental contaminants are recognized as mutagenic and/or carcinogenic in animal but they are often only suspected to have similar effects in Humans. The lack of knowledge on the effects of most industrial-made chemicals has led the EU to launch the REACH program with the aim of evaluating the toxicity of more than 30.000 molecules. Such evaluation would require the use of at least 4 millions of animals for an estimated cost of 2.8 billions €. While the relevance of these in vivo models remains controversial.

Kei Ishii, Masamichi Ishiai, Hiroko Morimoto, Mito Kanatsu-Shinohara, Ohtsura Niwa, Minoru Takata, Takashi Shinohara, The Trp53-Trp53inp1-Tnfrsf10b Pathway Regulates the Radiation Response of Mouse Spermatogonial Stem Cells, Stem Cell Reports, Volume 3, Issue 4, 14 October 2014, Pages 676-689, ISSN 2213-6711 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18685号 / 医博第3957号 / 新制||医||1007(附属図書館) / 31618 / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 藤田 潤, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Martina Klaučová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Consultant: PharmDr. Ivona Pávková, Ph.D. Diploma thesis title: Ames test in the drug development Background: Thesis objective is the determination of potential genotoxicity of newly developed drugs within primary testing and the introduction of the Ames microfluctuation test which can be used in common laboratory conditions. Methods: I used commercially supplied kit based on the principles of Ames test which detects reverse mutation through colour changes of the samples using bacterial strains S. typhimurium. At first I had to study literary sources and then I could design the procedures of the Ames microfluctuation test, preparation of the chemicals and storage of the strains which are optimal for all laboratories. Results: The drug samples T6445 and T6447 with 30 µM concentration tested by metabolic activation S9 on bacterial strain ST TA 98 show genotoxicity. The sample UOCHB1 with 30 µM concentration tested without activation shows possible genotoxicity on both strains ST TA 98 and ST TA 100. Other samples do not show any toxicity. I used 3 different procedures during the designation of assay. The most suitable version of the...

Adverse outcome pathways (AOPs) provide a framework to organize and weigh the evidence linking a toxicant’s initial interactions with molecules in the cell to adverse outcomes of regulatory concern. AOPs are constructed in modules that include key events (KEs) and key event relationships (KERs). Quantitative understanding of the KERs is critical for the development of predictive toxicological models. The objective of this project was to investigate the ability to define the quantitative associations of the KERs upstream, and contained in, an existing AOP (#296): “Oxidative DNA Damage Leading to Chromosomal Aberrations and Mutations”. The data supporting quantitative associations between these KERs was gathered through literature review and experimental methods. I first used systematic literature review tools to develop and apply a pragmatic and transparent method to search the literature for AOP evidence. A broad search, covering all of the KERs of interest, was initially conducted. This search, which retrieved more than 230 thousand articles, demonstrates the data-rich nature of the AOP. An artificial intelligence informed prioritization of the top 100 articles were then examined in detail. This approach identified 39 articles containing qualitative empirical support for the AOP, but limited quantitative evidence of the KERs. A second search was conducted to address the need for quantitative evidence as well as the lack of evidence for the KER between and increase in reactive oxygen species (ROS) and oxidative DNA damage. The second search retrieved 12 articles that could be used to define a quantitative relationship between cellular ROS and oxidative DNA damage. To begin to address gaps in quantitative understanding, I then conducted experiments in the laboratory to measure oxidative DNA damage, DNA strand breaks, chromosomal aberrations, and mutations in TK6 cells after exposure to a range of concentrations of 4-Nitroquinoline 1-oxide (4NQO: a prototype ROS producing agent). An increase in both oxidative DNA damage and DNA strand breaks was observed after 2, 4, and 6 h exposures with the high throughput comet assay (CometChip). An increase in the incidence of micronuclei was observed after a 24 h exposure to a low concentration of 4NQO, as measured with the flow cytometry micronucleus assay, while high cytotoxicity was found at higher concentrations. Lastly an increase in mutation frequency was observed with Duplex Sequencing, an error-corrected sequencing technology. Additionally, an increase in the proportion of C>A transversions was observed, consistent with the expected mutations following oxidative DNA lesions. Overall, my work contributes to the quantitative understanding of AOP #296 and this project serves as a key example of AOP-informed study design, highlighting notable challenges in characterizing quantitative relationships.