Les Hydrocarbures Aromatiques Polycycliques (HAP) représentent une famille de polluants atmosphériques issus de la combustion incomplète de la matière organique. Ils sont ainsi présents dans l'atmosphère polluée des villes, la fumée de cigarette et certaines industries. Une exposition aux HAP peut être à l'origine de cancers du poumon, de la peau et de la vessie. A ce titre, certains HAP sont suspectés ou reconnus cancérigènes pour l'Homme, comme le Benzo[a]pyrène (B[a]P), par leur capacité à induire des dommages à l'ADN après métabolisation. Malgré une émission systématique des HAPs en mélanges, la majorité des études s'est intéressée à l'effet génotoxique des HAP purs et principalement au B[a]P. Afin de fournir des données mécanistiques sur la génotoxicité et le mode d'action des mélanges de HAP, nous avons réalisé une étude in vitro dans des lignées cellulaires de poumons (A549), de vessie (T24) et de foie (HepG2). Les dommages à l'ADN ont été suivis par la mesure des adduits par HPLC-MS/MS et des dommages oxydatifs par la méthode des comètes, ainsi que le métabolisme par l'induction des gènes par RT-qPCR et les activités enzymatiques des CYP540 de phase I (EROD) et de phase II (GST). Dans un premier temps, l'utilisation du B[a]P, comme composé modèle a montré une absence quasi-totale de métabolisation et de génotoxicité pour T24. Par contre, la formation d'adduits et l'induction de la métabolisation a été mise en évidence pour A549, avec des effets notamment de dose-réponse « en cloche » similaires à ceux observés dans d'autres modèles de poumons. Nous avons ensuite étendu cette démarche à 12 HAP prioritaires et étudié leur métabolisation et la formation d'éventuels adduits en se focalisant sur la lignée pulmonaire A549. La combinaison de ces HAP au B[a]P dans des mélanges binaires ou dans des mélanges plus complexes mimant des mélanges environnementaux entraine une forte inhibition de la formation des adduits issus du B[a]P sans apparition d'adduits d'autres HAP. Par ailleurs, nous avons observé dans le cas des mélanges complexes une bonne corrélation entre l'activité EROD et la formation des adduits à l'ADN, alors que les gènes de phase sont eux surexprimés par l'exposition au mélange par rapport au B[a]P pur. Les mécanismes par lesquels s'exerce cette inhibition des adduits restent encore à élucider mais la métabolisation des HAPs constitue une étape clé dans la génotoxicité des mélanges à travers des phénomènes d'inhibition ou de compétition au niveau des CYP entrainant une inhibition de l'activité EROD. Il est donc clair que l'étude des HAP de façon individuelle n'est pas suffisante pour appréhender la génotoxicité des mélanges complexes. L'approche FET, couramment utilisée pour évaluer le risque lié à l'exposition aux mélanges de HAP, repose sur l'additivité des effets toxiques et néglige les interactions métaboliques entre les différents HAP. L'amélioration de cet outil de prédiction est nécessaire et passe obligatoirement par l'étude des mécanismes sous-jacents qui relient la composition des mélanges, leur métabolisation et leur génotoxicité. / Polycyclic Aromatic Hydrocarbons (PAH) represent a family of ubiquitous atmospheric pollutants produced upon incomplete combustion and thus present in polluted atmosphere of the cities, in cigarette smoke and in certain industries. Exposure to HAP can cause lung, skin and bladder cancers. For this reason, some PAHs are suspected or recognized carcinogenic to humans, especially Benzo[a]pyrene (B[a]P), through their ability at inducing the formation of DNA damage after metabolization. In spite of the systematic emission of PAHs in mixtures, the majority of the studies was interested in the genotoxic effect of pure PAH and mainly B[a]P. In order to provide mechanistic data on the genotoxicity and the mode of action of PAH mixtures, we designed an in vitro study using cell lines representative of lungs (A549), bladder (T24) and liver (HepG2). DNA damage was investigated through the quantification of adducts by HPLC-MS/MS and of oxidative damage by the Comet assay. In addition, the metabolism was studied by analyzing genes induction by RT-qPCR and enzymatic activities of phase I CYP540 (EROD) and phase II (GST). First, the use of B[a] P, as a reference compound showed a quasi-total absence of metabolization and genotoxicity for T24. In contrast, the formation of DNA adducts formation and the induction of metabolization was highlighted for A549, with a bell-shaped dose-response curve similar to those observed in other lungs models. Then we extended this approach to 12 priority PAH and analyzed their metabolization and the possible formation of adducts focusing on the pulmonary cell line A549. The combination of these HAP to B[a]P in binary mixtures or in complex mixtures representative to environmental exposures led to a strong inhibition in adducts formation induced by B[a]P without outbreak of adducts from other PAH. In addition, we observed, in the case of complex mixtures, a good correlation between the EROD activity and the formation of adducts in DNA, while phase I genes were always overexpressed after exposure to mixtures when compared to pure B[a]P. The mechanisms involved in the inhibition of DNA adducts remain to be elucidated but PAHs metabolization represents a key step in the mixtures genotoxicity through inhibition or competition of CYP resulting in an inhibition of EROD activity. It is thus clear that the study of the HAP in an individual way is not sufficient to understand the genotoxicity of complex mixtures. The TEF Approach, usually used to asses the risk related to PAH mixtures exposure, relies on toxic effects additivity and ignores metabolic interactions between the various PAH. The improvement of this prediction tool is essential and involves necessarily the study of the underlying mechanisms which connect mixtures composition, their metabolization and their genotoxicity.

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-20Bitstream added on 2014-06-13T20:21:26Z : No. of bitstreams: 1 leme_dm_dr_rcla_parcial.pdf: 198180 bytes, checksum: 4c280b4c68a010c361d6ef4e0da11146 (MD5) Bitstreams deleted on 2015-06-25T13:00:52Z: leme_dm_dr_rcla_parcial.pdf,. Added 1 bitstream(s) on 2015-06-25T13:03:16Z : No. of bitstreams: 1 000638858_20151201.pdf: 243435 bytes, checksum: d1e6915fdf00c6f43d70add5c4248acb (MD5) Bitstreams deleted on 2015-12-01T09:08:34Z: 000638858_20151201.pdf,. Added 1 bitstream(s) on 2015-12-01T09:09:26Z : No. of bitstreams: 1 000638858.pdf: 2789008 bytes, checksum: 13a14092763ec9a00dd9bfbff49af27a (MD5) / A questão energética é hoje um tema de preocupação mundial. A limitação das fontes de energia fóssil e seus efeitos indesejáveis ao meio ambiente têm estimulado a busca de novas fontes alternativas, desde que renováveis. Em diversos países, como no Brasil, têm sido destinados grandes incentivos ao desenvolvimento do setor de biocombustíveis, no qual se inclui o biodiesel. O futuro promissor do biodiesel está relacionado não apenas ao fato de ser um combustível renovável, mas também às suas contribuições na redução da emissão de poluentes atmosféricos e à sua maior degradabilidade, em relação aos combustíveis fósseis. No entanto, poucos são os estudos realizados com o biodiesel para avaliar os seus possíveis impactos, quando usados puro ou em misturas ao óleo diesel, sobre o ambiente (principalmente corpos d’águas e solos) e sobre os organismos vivos, em caso de uma possível contaminação ambiental. O presente estudo teve como objetivo avaliar a citotoxicidade, genotoxicidade e mutagenicidade do biodiesel e de suas misturas com diesel (B5, B20 e B50), por meio de simulações de vazamento em água e solo. As simulações foram realizadas no verão, para caracterizar um vazamento em condições tropicais, sendo aplicados diferentes ensaios biológicos [teste de Allium cepa, ensaio Salmonella/microssoma, ensaios de mutagenicidade com células CHO-K1 e HepG2 in vitro, ensaio para detecção de indução de morte celular (alterações do ΔΨm, externalização da fosfatidilserina), avaliação da citotoxicidade em tempo real (sistema xCELLigence™)] nas amostras de água e solo obtidas nos experimentos. Foi observada no presente estudo uma significativa citotoxicidade, que foi relacionada com constituintes do óleo diesel, mais especificamente, com os hidrocarbonetos policíclicos aromáticos (PAHs). Já os efeitos genotóxicos/mutagênicos, observados... / The energy-related issue is currently a subject that causes world concern. The limitations of fossil energy sources and their undesirable effects upon the environment have encouraged the search for new alternative sources, as long as they are renewable ones. In several countries, like Brazil, great incentives have been allocated to the development of the biofuel sector, and that includes biodiesel. The promising future of biodiesel is related not only the fact of being a renewable biofuel, but also to its contribution to reducing release of air pollutants and its higher degradability when compared to fossil fuels. Nevertheless, not enough studies have been conducted with biodiesel to evaluate their possible impacts – either when they are used crude or in biodiesel blends – upon both the environment (especially water bodies and soils) and living organisms, should there be environmental contamination. The aim of this study was to assess toxicity, genotoxicity and mutagenicity of biodiesel and its diesel blends (B5, B20 and B50), by simulating spills into water and soil. The simulations were carried out in the summer in order to characterize a spill under tropical conditions, and different assays [Allium cepa test, Salmonella mutagenicity assay, in vitro mutagenicity assays with CHO-K1 and HepG2 cells line, detection of cell death inductions (changes in ΔΨm, phosphatidylserine externalization), cytotoxicity assessment by xCELLigence™ system] were conducted using soil and water samples obtained in the experiment. In this study, significant citotoxicity was observed, which was found to be related to biodiesel contaminants, even more specifically, to its polycyclic aromatic hydrocarbons (PAHs). On the other hand, the genotoxic/mutagenic effects observed in the other assays done with diesel and biodiesel proved to result from the action of pollutants present in the raw material used... (Complete abstract click electronic access below)

Resumo: O MTA é um cimento amplamente utilizado na endodontia devido ao potencial indutor da mineralização, sendo empregado em procedimentos de proteção do complexo dentino/pulpar, retrobturação, perfurações radiculares e de furca e em dentes com rizogênese incompleta. Inicialmente esse material não foi preconizado como cimento obturador de canais radiculares. Entretanto, recentemente foi criado um cimento endodôntico resinoso contendo MTA (MTA Fillapex) com essa indicação, porém não há estudos sobre este novo material. Como o MTA é utilizado sobre os tecidos periodontal, ósseo e pulpar, é importante conhecer seus efeitos citotóxicos e genotóxicos, uma vez que, alguns cimentos endodônticos podem liberar componentes tóxicos, prejudicando o processo de regeneração e reparação dos tecidos no qual entram em contato. Assim, o objetivo deste trabalho foi avaliar a citotoxicidade e genotoxicidade do MTA obturador (Fillapex) comparando-o com o MTA reparador e com o AH Plus. Fibroblastos de hamster Chinês (V79) foram colocados em contato com diferentes diluições de meios de cultura previamente expostos a esses materiais. A citotoxicidade foi avaliada através do ensaio de MTT, em espectrofotômetro, para a verificação da taxa de viabilidade celular. A genotoxicidade foi avaliada pela formação de micronúcleos (MNT), para detecção de mutações cromossômicas. A taxa de sobrevivência celular e o número de micronúcleos antes e após a exposição aos cimentos foram estatisticamente analisados pelo teste de Kruskal-Wallis e teste de Dunn (p<0,05). Os resultados mostraram que o MTA reparador manteve a viabilidade celular acima de 50% em todas as diluições. Porém, tanto o MTA Fillapex quanto o AH Plus foram altamente citotóxicos e capazes de aumentar a formação de micronúcleos em 8 vezes em relação ao grupo controle na diluição 1:4, mostrando que são cimentos cito e genotóxicos / Abstract: The MTA is a sealer widely used in endodontics and for be considered a material that inducing mineralization, can be used in procedures for protection of dentin/pulp complex, retrofilling root and furcal perforations and in teeth with incomplete root formation. Initially this material was not recommended as root canal sealer. However, recently a resin sealer based on MTA (MTA Fillapex) was created for this indication, but there are no studies on this new material. As the MTA is used on the periodontal tissues, bone and pulp, it is important to know the cytotoxic and genotoxic effects, as some sealers can release toxic components, impairing the process of regeneration and repair of tissues in which they come into contact. Thus, the purpose of this study was to evaluate the cytotoxicity and genotoxicity of MTA obturator (Fillapex) comparing it with the MTA repairer and AH Plus. Chinese hamster fibroblasts (V79) were placed in contact with different dilutions of culture media previously exposed to such materials. Cytotoxicity was evaluated by MTT assay in spectrophotometer to check the viability rate and cell survival. The genotoxicity was evaluated by micronucleus (MNT) to detect chromosomal mutations. The cell survival rate and number of micronuclei before and after exposure to the cement were statistically analyzed by Kruskal-Wallis and Dunn test (p<0.05). The results showed that MTA reparative remained cell viability above 50% in all dilutions. However, both the MTA Fillapex and AH Plus were highly cytotoxic / Orientador: Marcia Carneiro Valera / Coorientador: Samira Esteves Afonso Camargo / Banca: Caio Cézar Randi Ferraz / Banca: Ana Paula Martins Gomes / Mestre

Les composés perfluorés (PFC) de formule chimique générale CF3-(CF2)n-SO3- ( sulfonates) ou CF3-(CF2)n-1-CO2- (acides) sont des polluants organiques émergents, dont la persistance, la bioaccumulation et la toxicité sont maintenant considérées préoccupantes au plan sanitaire et environnemental. L'objectif de notre recherche a été de mettre en évidence les effets cancérogènes in vitro et le mécanisme d'action impliqué lors de l'exposition pendant 7 jours de cellules embryonnaires de hamster Syrien (SHE) aux principaux représentants perfluorés, le sulfonate de perfluorooctane (PFOS), le perfluorooctanoate (PFOA), et à leur substitut, le sulfonate de perfluorobutane (PFBS). Le test de transformation cellulaire dans sa version standard ou selon un protocole de type initiation-promotion a permis de détecter les substances cancérogènes de profil initiateur ou promoteur de tumeur. La génotoxicité des PFCs a été explorée par le test Comet en conditions alcalines. PFOS a présenté un profil cancérogène non génotoxique de type initiateur aux concentrations de 0,37 et 3,7 µM (p<=0,01), coïncidant avec les concentrations sériques des travailleurs exposés au PFOS. L'activation des gènes PPARs a été observée après 7 jours d'exposition au PFOS, avec une induction plus importante et plus précoce (dès 24 heures d'exposition) du gène ppar-bêta/gamma aux concentrations transformantes (p<=0,05). PFOA appliqué seul n'induit pas la transformation néoplasique des cellules SHE. Par contre, il induit la transformation des cellules présensibilisées par un initiateur. Il agit selon un profil cancérogène non génotoxique de type promoteur de tumeur aux concentrations de 3,7 x 10-4 à 37 µM. Ces concentrations coïncident avec les concentrations sériques mesurées dans les populations professionnellement et non professionnellement exposées. PFBS ne s'est révélé ni initiateur, ni promoteur de tumeur. La mise en cause de ces PFCs dans l'augmentation des cancers de la vessie (pour le PFOS) et celui de la prostate (pour le PFOA) chez les travailleurs exposés ne peut être exclue / Perfluorinated compounds (PFCs) is a collective name for fluorinated surfactants and polymers with the general structure CF3-(CF2)n-SO3- (sulfonates) or CF3-(CF2)n-1-CO2- .(acids). This group is characterized by a high persistence, bioaccumulation and long term toxicity which are rising environmental and public health concerns. In the present work, we analyzed the in vitro carcinogenic potential of the two major PFCs, perfluorooctane sulfonate (PFOS), and perfluorooctanoic acid (PFOA), and their substitute, perfluorobutane sulfonate (PFBS). Cell transformation assays were carried out on Syrian hamster embryo (SHE) cells in a 7 day-treatment using the standard and the initiation-promotion protocols. Genotoxicity was tested using the comet assay. PFOS was not genotoxic on SHE cells, but it induced cell transformation at non cytotoxic concentrations 0,37 and 3,7 µM (p<=0,01). These concentrations coincided with serum PFOS concentrations measured in occupationally exposed workers. An increased expression of PPARs was registered after 7 days. The ppar-beta/gamma mRNA appeared to increase rapidly (24 hours after PFOS treatment) at concentrations closely related to cell transformation (p<=0,05). PFOA was inactive alone, but induced cell transformation of SHE cells pre-initiated with benzo(a)pyrene (BaP). Therefore PFOA was shown to act as a tumor promoter and a non genotoxic carcinogen at a large range of concentrations (3,7 x 10-4 à 37 µM). This range of concentrations covered seric concentrations in non-occupationally exposed and occupationally exposed populations. PFBS was negative alone and on BaP-pretreated SHE cells. For this reason, its use as a substitute for PFOS appears to be justified. To conclude, the cell transforming potenty of PFOS and PFOA denotes in vitro carcinogenic potential. Consequently, the hypothesis of their implication in human cancer recorded in occupationally exposed populations cannot be ruled out

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A busca por novos derivados citotÃxicos da nor-&#946;-lapachona à parte de uma crescente e contÃnua busca por novos compostos ativos. O presente estudo teve como objetivo avaliar o potencial citotÃxico de quatro derivados arilaminos (1-4) da nor-&#946;-lapachona e os provÃveis mecanismos envolvidos na citotoxicidade da nor-&#946;-lapachona e de seus derivados. TrÃs derivados (1, 3 e 4) e seu precursor apresentaram elevado potencial citotÃxico sobre todas as linhagens celulares tumorais humanas utilizadas. Em estudos com cÃlulas nÃo tumorais (CMSP), a nor-&#946;-lapachona nÃo induziu citotoxicidade, porÃm seus derivados apresentaram efeitos antiproliferativos, cuja intensidade variou de moderado a fraco com valores de CI50 iguais a 5,02 ÂM (1), 12,02 ÂM (3) e 10,65 ÂM (4). AlÃm disso, os compostos nÃo apresentaram efeitos hemolÃticos (EC50 > 219,29 ÂM). Tanto a nor-&#946;-lapachona quanto seus derivados (1, 3 e 4) induziram apoptose (via mitocondrial) em cÃlulas HL-60, como observado atravÃs dos ensaios de anÃlises morfolÃgicas e de citometria de fluxo, e distÃrbios sobre a progressÃo do ciclo celular de cÃlulas HL-60, apenas na maior concentraÃÃo (4 ÂM). O tratamento com os compostos induziu a formaÃÃo de EROS, assim como quebras das fitas da molÃcula de DNA em cÃlulas HL-60 (NQO1&#8722;) e DU-145 (NQO1+). A co-administraÃÃo de GSH reduziu a sensibilidade celular aos efeitos tÃxicos dos compostos estudados, e o prÃ-tratamento com agentes antioxidantes (NAC e IK) reduziu drasticamente a produÃÃo de radicais livres, o mesmo nÃo ocorrendo com culturas prÃ-tratadas com BH (agente depletor de GSH). PorÃm, o dicumarol (inibidor de NQO1) preveniu a formaÃÃo de EROS e os efeitos genotÃxicos apenas na linhagem DU-145, indicando que esses compostos possam ser metabolizados por outras redutases que nÃo a NQO1. As lesÃes ao DNA de cÃlulas DU-145 promovidas pela nor-&#946;-lapachona e seus derivados (1, 3 e 4) foram, parcialmente, reparadas apÃs um perÃodo de tempo de 24 horas, o que corrobora com a sÃntese nÃo programada de DNA detectada apÃs a exposiÃÃo a esses compostos. AlÃm disso, todos os compostos ativos interferiram sobre os processos de reparo dos danos ao DNA induzidos pelo MMS, o que indica que a interferÃncia sobre esses processos contribui para seus efeitos citotÃxicos. A nor-&#946;-lapachona e seus derivados arilaminos (1, 3 e 4) inibiram a taxa de proliferaÃÃo de cepas de S. cerevisiae defectivas na expressÃo de topoisomerases (Top1&#916;, Top3&#916; e Top1&#916;Top3&#916;) de maneira mais pronunciada do que observado na cepa selvagem (BY4741), indicando que a interferÃncia sobre a atividade das topoisomerases influencia, de algum modo, a citotoxicidade desses compostos. Nos estudos de genotoxicidade e mutagenicidade com fibroblastos de pulmÃo de hamster chinÃs (linhagem V79), todos os compostos citotÃxicos promoveram quebras nas fitas de DNA, oxidaÃÃo de bases nitrogenadas e induÃÃo de micronÃcleos, em concentraÃÃo (10 ÂM) muito superior do que as necessÃrias para induzir os mesmos eventos em cÃlulas tumorais. ReforÃando a participaÃÃo de EROS sobre os eventos mutagÃnicos, o prÃ-tratamento com NAC preveniu a formaÃÃo de cÃlulas micronucleadas e reduziu a sensibilidade celular aos efeitos tÃxicos dos compostos estudados. Esses resultados ressaltam as propriedades antitumorais da nor-&#946;-lapachona e seus derivados arilaminos e que eles podem ser considerados como protÃtipos para o desenvolvimento de novos agentes anticÃncer. / The search for new cytotoxic derivatives of nor-&#946;-lapachone is part of a growing and continuous search for new active compounds. The present study aimed to evaluate the cytotoxic potential of four arylamino-nor-&#946;-lapachone derivatives (1-4) and the probable mechanism involved in the cytotoxicity of these compounds. Among the tested derivatives, only three of them (1, 3 and 4) and the precursor molecule elicited a significant antiproliferative effects in all human tumor cell lines used in the study. In experiments with peripheral blood mononuclear cells (PBMC), nor-&#946;-lapachone did not induce cytotoxicity, however its derivatives showed antiproliferative effects whose intensity ranged from moderate to weak, with IC50 values equal to 5.02 ÂM (1), 12.02 ÂM (3) and 10.65 ÂM (4). Moreover, all the compounds showed no hemolytic effects (EC50> 219.29 ÂM). Nor-&#946;-lapachone and its derivatives (1, 3 and 4) induced apoptosis (mitochondrial pathway) in HL-60 cells, based upon morphological and flow cytometric analysis, and interference on HL-60 cell cycle progression, only at the highest concentration (4 ÂM). Compounds exposure induced intracellular ROS generation, as well as DNA strand breaks in HL-60 (NQO1&#8722;) and DU-145 (NQO1+) cell lines. The co-administration of GSH reduced the cellular sensibility to the toxic effects of the studied compounds. Unlike cells pre-treated with BH (glutathione depletor agent), cells pre-exposed to antioxidants agents (NAC or KI) showed low production of free radicals. On the other hand, dicoumarol (NQO1 inhibitor) prevented the ROS formation and DNA strand breaks only in DU-145 cells, indicating that these compounds can be metabolized by other reductases than NQO1. The DNA damage induced by nor-&#946;-lapachone and its derivatives (1, 3 and 4) in DU-145 cells were partially repaired after 24 h, which agrees with the data of unscheduled DNA synthesis. In addition, all active compounds interfere with DNA repair processes of DNA damage induced by MMS, indicating that this action contributes to compounds cytotoxic effects. All active compounds inhibited the proliferation rate of S. cerevisiae strains defective in the expression of DNA topoisomerases (Top1&#916;, Top3&#916; and Top1&#916;Top3&#916;) more pronounced than that observed in wild type strain (BY4741), suggesting that the interference on topoisomerases activities may contributes to the cytotoxicity of active compounds. Genotoxicity and mutagenicity studies with Chinese hamster lung fibroblast cell line (V79), showed that all cytotoxic compounds promoted DNA strand breaks, DNA bases oxidation and micronucleus formation in a concentration (10 ÂM) far higher than needed to induce the same events in tumor cells. Strengthening the ROS contribution on the mutagenic events, the pre-treatment with NAC prevented the formation of micronucleated cells and reduced the V79 cell sensibility to the cytotoxic effects of the studied compounds. These findings underscore the antitumor properties of nor-&#946;-lapachone and its arylamino derivatives and they can be considered as prototypes for the development of new anticancer agents.

A exposição dos peixes a poluentes provoca danos nos organismos que podem ser identificados precocemente através de respostas biológicas. O presente estudo visou avaliar os efeitos do naftaleno e benzo(a)pireno em pampos da espécie Trachinotus carolinus. Foram avaliados os efeitos citogenotóxicos, histopatológicos e bioquímicos após exposições às concentrações de 0,9 M; 2,7 M e 8,1 M de NAP e BAP por períodos de 12, 24, 48 e 96 horas. O NAP causa quebra no DNA de eritrócitos de pampos em concentrações de 8,1 M e a partir de 12 horas de exposição. O BAP revelou ser genotóxico a partir da menor concentração e de 24 horas. A mutagenicidade de ambos os poluentes, avaliada através da indução de formação de micronúcleos e anormalidades nucleares eritrocitárias, também ocorre a partir de curtos períodos de exposição e freqüências de MN e ANE estão relacionadas com a duração da exposição. O período de exposição aos HPAs foi determinante na intensidade e severidade das lesões observadas nos tecidos dos peixes. A especificidade de CYP1A, observada segundo análise imunohistoquímica, ocorreu de maneira dose-dependente e evidenciada principalmente nos maiores períodos experimentais. Os poluentes orgânicos, nas condições experimentais utilizadas, não provocaram alteração significativa na atividade das enzimas catalase e GST da espécie. Os biomarcadores, citogenotóxicos e histopatológicos utilizados neste estudo, demonstraram ser ferramentas eficientes para aferir a toxicidade, genotoxicidade e mutagenicidade de NAP e BAP como também sua relação dose-resposta na espécie T. carolinus. / Effects of exposure of fish to pollutants can be identified through stress responses. The present study aims to evaluate the effects of naphthalene and benzo(a)pyrene in Florida pompanos, Trachinotus carolinus. Evidences from citogenotoxical, histopathological and biochemical studies showed that alterations caused by exposures to 0.9 M, 2.7 M and 8.1 M of NAP and BAP occurred within 12 to 96 hours. NAP at 8.1 M induced erythrocyte DNA strand breaks in pompanos since early periods of exposure. Genotoxic effects of BAP at the lowest concentration were documented soon after 24 hours of exposure. Mutagenotoxicity of both pollutants, as seen by the induction of MN and ENA, was revealed since early periods and their frequencies are related to the duration of exposure. Exposures to these PAHs, for longer periods, resulted in increased frequency and severity of lesions observed in fish tissues. Specificity of CYP1A, observed through immunohistochemical analyses, was related to the dose of the pollutants and mainly at longer periods of exposure. These organic pollutants, under the experimental conditions, did not interfere with the activity of liver catalase and GST of the species. The citogenotoxic and histopathologic biomarkers used in this study proved to be efficient tools to ascertain the toxicity, genotoxicity and mutagenesis of NAP and BAP, as well as their dose related response, in the species T. carolinus.

O lodo de esgoto gerado em estações de tratamento de esgoto pode conter substancias tóxicas ainda não regulamentadas nas legislações nacionais e internacionais. Dentre elas as genotóxicas tem recebido especial atenção. Este trabalho teve como objetivos avaliar a genotoxicidade de amostras de lodo de esgoto tratado de diferentes Estações de Tratamento de Esgoto (ETE) do Estado de São Paulo com o teste de micronúcleo em Tradescantia e ampliar os conhecimentos sobre o potencial de utilização da Tradescantia pallida em substituição ao clone 4430. Todas as ETEs estudadas apresentaram pelo menos uma amostra positiva para o teste de micronúcleo em Tradescantia sendo necessários mais estudos para elucidar quais os compostos são responsáveis pelo efeito observado. Os resultados sugerem que a Tradescantia pallida pode substituir o clone 4430 no teste de micronúcleo, porém esse ensaio tem aplicabilidade limitada em programas de monitoramento. / The sludge produced in sewage treatment plants can contain toxic substances which are not yet regulated by national and international legislation. Among these, the genotoxic substances are of great concern. The present paper aimed at evaluating the genotoxicity of treated sludge samples collected in different Sewage Treatment Plants (STP) located in the State of São Paul, Brazil. The micronucleus assay in Tradescantia was the test chosen for this evaluation. Another objective of the study was to verify it Tradescantia pallida could replace the clone 4430 in the Trad-MN assay. All the STPs studied have presented at least one positive sample for the micronucleus assay in the Tradescantia. Further studies are required in order to determine which compounds are responsible for the observed effect. The results obtained suggest that T. pallida can replace clone 4430 in the micronucleus assay, however, this assay presents limited applicability in monitoring programs.

DA CASTANHA DE CAJU Apesar da importância social e econômica do beneficiamento da castanha de caju para o Rio Grande do Norte, a produção ainda é realizada de forma artesanal. Para a coleta da amêndoa da castanha é necessário assá-la. A fumaça gerada durante a queima da castanha possui altas concentrações de Material Particulado (MP) e esse MP produzido é inalado diariamente por grupos familiares por um período que pode exceder a 10 horas diárias. Em geral, os poluentes atmosféricos oriundos da queima de biomassa são potencialmente nocivos a saúde, relacionando-se com eventos de genotoxicidade, aumento no número de internações hospitalares e ambulatoriais, e mortalidade por doenças cardiovasculares e respiratórias. O presente trabalho teve três objetivos principais: 1° Realizar medições na concentração de O3, NO2, MP, Black Carbon (BC) e composição elementar do MP 2,5 provenientes da queima da castanha. 2° Identificar o potencial mutagênico associado ao beneficiamento artesanal da castanha de caju durante a estação seca, chuvosa e intermediária no RN (Brasil) com o auxílio do teste de micronúcleo (MN) em Tradescantia pallida. 3° Verificar o efeito mutagênico da atividade em MN de células esfoliadas de mucosa oral de trabalhadores envolvidos no beneficiamento. Para isso, três locais distintos foram definidos como pontos testes: Ponto 1. Comunidade do Amarelão, situada no perímetro rural do município de João Câmara-RN (local onde ocorre a queima da castanha de caju); Ponto 2. Fazenda Santa Luzia, situada próxima à região de queima da castanha de caju (local com as mesma condições ambientais do Ponto 1, porém sem a influência da atividade); Ponto 3. Universidade Federal do Rio Grande do Norte (UFRN), zona urbana de Natal-RN. Os resultados obtidos para o O3 e NO2 não excederam os limites estabelecidos pela legislação brasileira. No entanto, os resultados da medição de MP obtidos com o medidor portátil \"DUSTTRAK (TM)Aerossol Monitor \" indicou que entre as 8 medições de MP realizadas no Ponto 1, 7 excederam o nível de exposição definido como \"estado de emergência \"descrito na legislação brasileira (500µg/m 3 ), diferindo significativamente dos resultados obtidos nos Pontos 2 e 3 (6µg/m 3 ). As avaliações realizadas nos meses de Janeiro, Maio e Setembro de 2009, com o Mini-sampler confirmaram os resultados previamente obtidos com o \"DUSTTRAK (TM)Aerossol Monitor \".O valor médio de MP 2,5 (Jan - 548,412 µg/m 3 ; Mai - 1022,232 µg/m 3 ; Set - 1291,946 µg/m 3 ) e BC (Jan - 46,798 µg/m 3 ; Mai 70,068- µg/m 3 ; Set - 69,432 µg/m 3 ) obtido nas três campanhas para o Ponto 1 foram significativamente maiores que o Ponto 2 e 3. Para o Ponto 1 os elementos Si, S, Cl, K, Ni, Cu e Zn quando presentes estiveram em concentrações superiores aos Pontos 2 e 3. Os testes de genotoxicidade com T. pallida indicou aumento significativo no número de MN em todas as campanhas. Os resultados com células de mucosa oral humana corroboraram com o biomonitor vegetal, sendo verificado aumento significativo na freqüência de MN. Os resultados obtidos caracterizaram um dos piores níveis de exposição humana ao MP já relatado na literatura, excedendo amplamente os limites da legislação brasileira assim como os da OMS. Os resultados obtidos apresentaram um problema ocupacional grave, sendo necessária intervenção imediata dos gestores públicos na tentativa de minimizar os efeitos lesivos da atividade. / x Despite the social and economic importance of the processing of cashew nuts to Rio Grande do Norte, the production is still carried out artisanally. To collect the almond nut is necessary to roasting. The smoke generated during the burning of the nut has high concentrations of particulate matter (PM) and the PM produced is inhaled daily by families for a period which can exceed 10 hours a day. In general, air pollutants come from burning biomass are potentially harmful to health, relating to genotoxicity events, increase in the number of hospitalizations and outpatient, and mortality from cardiovascular and respiratory diseases. This project had three main objectives: 1 Make measurements in the concentration of O3, NO2, MP, Black Carbon (BC) and elemental composition of the PM 2.5 from the combustion of the cashew nut. 2 Identify the mutagenic potential associated with artisanally processing of cashew nut during the dry season, wet and intermediate in RN (Brazil) by using a micronucleus (MN) bioassay of T. pallida tetrads. 3 To investigate the effect of mutagenic activity in exfoliated cells of oral mucosa of workers involved in processing. For this, three test sites were chosen for this purpose: Site 1. the Amarelão community - where the roasting occurs, Site 2. the Santa Luzia farm - an area near the roasting site, though without direct influence on the process and Site 3. the Universidade Federal do Rio Grande do Norte (UFRN) - an urban area of Natal, Brazil. The results obtained for the O3 and NO2 do not exceed the limits established by Brazilian legislation. However, the results of the measurement of PM obtained with the portable meter \"DUSTTRAK (TM) Aerosol Monitor\" has indicated that between 8 PM measurements made at Site 1, 7 exceeded the level of exposure defined as \"emergency rule\" described in the Brazilian legislation (500 ? g/m 3 ), differing significantly from the results obtained in Sites 2 and 3 (6 ? g/m 3 ). Evaluations in January, May and September 2009, with the Mini-sampler confirmed the results previously obtained with the \"DUSTTRAK (TM)Aerosol Monitor.The average value of 2.5 MP (Jan - 548.412 mg/m 3 ; May - 1022.232 mg/m 3 ; Set - 1291.946 mg/m 3 ) and BC (Jan - 46.798 mg/m 3 ; May 70.068 - mg/m 3 ; Set - 69.432 mg/m 3 ) obtained in the three campaigns for Site 1 were significantly higher than the Site 2 and 3. To the Site 1 the elements Si, S, Cl, K, Ni, Cu and Zn when present were at concentrations higher than the Sites 2 and 3. Genotoxicity tests with T. pallida showed a significant increase in the number of MN in all campaigns. The results with cells of human oral mucosa have confirmed the biomonitoring, and found significant increase in the frequency of MN. The results marked one of the worst levels of human exposure to PM has been reported in the literature and greatly exceeded the limits of the Brazilian legislation as well as the WHO. The results showed a serious occupational problem, requiring immediate intervention of public officials in an attempt to minimize the harmful effects of the activity.

Exposição a Hidrocarbonetos policíclicos aromáticos (HPAs) está relacionada com o aumento de risco de câncer. Esses carcinógenos dependem da sua ativação para intermediários eletrofílicos para causar danos em biomoléculas. As vias de ativação melhor estudadas incluem (i) a formação de diol-epóxido nas regiões de baía ou fjord dos HPAs através de epoxidações catalisadas pela citocromo P450 (CYP450), com uma hidrólise intermediária pela epóxido hidrolase e (ii) oxidação (CYP450 ou peroxidases) levando à formação de um cátion radical reativo. Outras vias incluem a formação de derivados metilados, quinonas e metabólitos de anel aberto cujas contribuições para a carcinogênese ainda são pouco estudadas, assim como o papel do estresse oxidativo na toxicidade dos HPAs. Neste trabalho foi investigado a citotoxicidade e genotoxicidade do indeno[1,2,3-cd]pireno, trifenileno e coroneno e seus produtos de oxidação (quinonas e hidroquinonas). Linhagens de carcinoma hepatocelular humano (HepG2) e de hepatócitos humanos normais (THLE-2) foram incubadas com os HPAs e suas respectivas quinonas e hidroquinonas acetiladas para análise de viabilidade celular (MTT) em diferentes condições de cultivo (por 16 horas, de 20 a 200 uM). Células HepG2 foram incubadas por 16 horas com indeno[1,2,3-cd]pireno (50 &#181;M), coroneno (20 &#181;M), trifenileno (10 uM) ou seus produtos de oxidação (quinonas e hidroquinonas acetiladas) para análise de dano oxidativo em DNA e peroxidação lipídica. Nas concentrações descritas acima, esses HPAs estruturalmente diferentes e seus produtos de oxidação são citotóxicos e levam ao aumento dos níveis de 7,8- dihidro-8-oxo-2\'-desoxiguanosina e malonaldeído. Tais danos podem contribuir para o aumento do risco de desenvolvimento de doenças, como o câncer, na população exposta. / Exposure to Polycyclic Aromatic Hydrocarbons (PAH) is related to the increase in the risk of cancer. These carcinogens depend on their activation by electrophilic intermediates to cause damage on biomolecules. The best understood activation pathways include (i) the formation of diol-epoxide in the bay and fjord regions of the PAH through epoxidations catalyzed by cytochrome P450 (CYP450), with an intermediate hydrolysis by epoxide hydrolase and (ii) oxidation (CYP450 or peroxidases) leading to the formation of a reactive cationic radical. Other pathways include the formation of methylated derivatives, quinones and open-ring metabolites whose contributions to carcinogenesis, as well as the role of oxidative stress on PAH toxicity, have not been extensively studied yet. This work investigated the cytotoxicity and genotoxicity of indene[1,2,3-cd]pyrene, triphenylene and coronene and their oxidation products (quinones and hydroquinones). Strains of human hepatocellular carcinoma (HepG2) and of normal human hepatocytes (THLE-2) were incubated with PAH and their respective quinones and acetylated hydroquinones and afterwards analyzed for cell viability (MTT) under different culture conditions (for 16 hours, 20 to 200 BM). HepG2 cells were incubated for 16 hours with indene[1,2,3-cd]pyrene (50 BM), coronene (20 BM), triphenylene (10 BM) or their oxidation products (quinones and acetylated hydroquinones) and afterwards analyzed for oxidative damage on DNA and lipid peroxidation. In the mentioned concentrations, these structurally different PAHs and their oxidation products are cytotoxic and lead to an increase in the levels of 7,8-dihydro-8-oxo-2\'-deoxyguanosine and malonaldehyde. Such damages may contribute to increase the risk of diseases like cancer in the exposed population.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2015-09-10T00:55:01Z No. of bitstreams: 1 Nilton C N Santos Tese Geno Cito Endo 10 ago 20h.pdf: 2096544 bytes, checksum: fd3a63f3f5235dae758262d72d8a3bb2 (MD5) / Made available in DSpace on 2015-09-10T00:55:01Z (GMT). No. of bitstreams: 1 Nilton C N Santos Tese Geno Cito Endo 10 ago 20h.pdf: 2096544 bytes, checksum: fd3a63f3f5235dae758262d72d8a3bb2 (MD5) Previous issue date: 2015-06-29 / Pulp therapy for deciduous teeth is the last resort for preventing tooth loss, but among the products used for this, i.e. so-called filling pastes, none is considered to be ideal. The objective of this study, using the micronucleus test on the bone marrow of mice and the comet assay on human lymphocytes, was to evaluate the genotoxic and cytotoxic effects of four filling pastes that are used in this therapy: zinc oxide, calcium hydroxide P.A., mineral trioxide aggregate and an iodoform paste (Guedes-Pinto paste). To perform the micronucleus test, male Swiss mice (Mus musculus) were divided into groups of ten animals each: four groups were each exposed to one of the filling pastes, administered intraperitoneally at dilutions of 1/10, 1/50, 1/500 and 1/1000. Cyclophosphamide was used as the positive control. The negative controls used were the dilution vehicles: dimethylsulfoxide (DMSO) for the Guedes paste and zinc oxide; and phosphate-buffered saline solution (PBS) for the calcium hydroxide P.A. and mineral trioxide aggregate. The animals were sacrificed 24 h and 48 h after the treatment. The bone marrow was extracted, in order to calculate the micronucleus occurrence rate in 1000 polychromatic erythrocytes (PCE) in each of the animals, under an optical microscope (1000 X), in a blinded test. Cytotoxicity was evaluated by determining the PCE/NCE (normochromatic erythrocyte) ratio in 200 erythrocytes/animal. For the comet assay, human lymphocytes were cultured in different dilutions of each of the filling pastes (1:500, 1:750, 1:1000 and 1:2000), for 3 h at 37 ?C, under an atmosphere containing 5% CO2. Two positive controls were used: methyl-methanesulfonate (0.4 ?M) for the calcium hydroxide P.A. and mineral trioxide aggregate; and doxorubicin (0.6 ?M) for the Guedes paste and zinc oxide. Two negative controls were also used here: distilled water for the calcium hydroxide P.A. and mineral trioxide aggregate; and DMSO for the Guedes paste and zinc oxide. Comets were identified by means of fluorescence microscopy (400 X), and 100 of them were counted on each of the three slides that were analyzed for each drug test. The statistical analysis on the results from the micronucleus test was performed using the conditional test for comparing proportions in situations of rare events. Analysis of variance, followed by the Tukey test, was used to assess the PCE/NCE ratio obtained from the different treatments and also to compare the means from the DNA damage indices that were obtained through the comet test, using the Prisma software, version 4.0. The micronucleus occurrence rate was significantly higher among the animals treated with Guedes paste, at all the dilutions tested and at both sacrifice times and also among the animals treated with zinc oxide and sacrificed 48 h after treatment at the dilutions of 1:50, 1:500 and 1:1000. Cytotoxic effects from these pastes were detected in the animals sacrificed at both times. Calcium hydroxide P.A. and mineral trioxide aggregate did not present any cytotoxic or genotoxic effects. The results obtained from the comet assay also showed that zinc oxide and Guedes paste presented genotoxicity, whereas calcium hydroxide P.A. and mineral trioxide aggregate did not. These results show that there is a need to reassess the use of zinc oxide and Guedes paste and provide encouragement for conducting additional studies to evaluate the genotoxicity and cytotoxicity of these pastes. / A terapia pulpar de dentes dec?duos se constitui no ?ltimo recurso de preven??o da perda dent?ria, mas, dentre os produtos empregados para tal, as denominadas pastas obturadoras, nenhum ? considerado como ideal. O objetivo deste trabalho foi avaliar, com o uso do teste de micron?cleo em medula ?ssea de camundongo e do ensaio cometa em linf?citos humanos, os efeitos citot?xicos e genot?xicos de quatro pastas obturadoras utilizadas nesta terapia: ?xido de zinco, hidr?xido de c?lcio P.A., agregado tri?xido mineral e uma pasta iodoformada (pasta Guedes-Pinto). Para realiza??o do teste de micron?cleo, camundongos Swiss (Mus musculus), machos, foram divididos em grupos de dez animais, que foram expostos ?s pastas obturadoras, administradas via intraperitoneal nas dilui??es de 1/10, 1/50, 1/500 e 1/1000. Ciclofosfamida foi utilizada como controle positivo. Os controles negativos foram: dimetilsulf?xido (DMSO) para a pasta Guedes-Pinto e ?xido de zinco; e solu??o salina tamponada (PBS) para o hidr?xico de c?lcio P.A. e agregado trioxido mineral. Os animais foram sacrificados 24h e 48h ap?s tratamento, a medula ?ssea foi extra?da e foram analisados 1000 eritr?citos policrom?ticos (PCE) de cada um dos animais, sob microscopia ?ptica (1000X) e em teste cego. A citotoxicidade foi avaliada pela rela??o PCE (eritr?cito policrom?tico) / NCE (eritr?cito normocrom?tico) em 200 eritr?citos/animal. Para o ensaio cometa, linf?citos humanos foram cultivados nas dilui??es de 1:500, 1:750, 1:1000 e 1:2000 das pastas obturadoras, durante 3h, a 37?C, em atmosfera de 5% de CO2. Foram utilizados dois controles positivos: metil-metanosulfonato (0,4?M) para o hidr?xido de c?lcio P.A. e agregado tri?xido mineral, e doxorrubicina (0,6 ?M) para a pasta Guedes-Pinto e ?xido de zinco. Foram tamb?m dois os controles negativos utilizados: ?gua destilada para o hidr?xido de c?lcio P.A. e agregado tri?xido mineral, e DMSO para pasta Guedes-Pinto e ?xido de zinco. A identifica??o do cometa foi realizada sob microscopia de fluoresc?ncia (400X), sendo computados 100 deles em cada uma das tr?s l?minas analisadas para cada droga teste. A an?lise estat?stica dos resultados do teste de micron?cleo foi realizada com o uso do teste condicional para compara??o de propor??es em situa??o de eventos raros. An?lise de vari?ncia, seguida do teste de Tukey, foram utilizados para avalia??o da rela??o PCE/NCE obtida com os diferentes tratamentos e tamb?m para compara??o das m?dias dos ?ndices de danos ao DNA obtidos no ensaio cometa com o software Prisma vers?o 4.0. A ocorr?ncia de micron?cleos foi significativamente maior nos animais tratados com a pasta Guedes-Pinto em todas as dilui??es testadas, nos dois tempos de sacrif?cio e tamb?m para os animais tratados com ?xido de zinco e sacrificados 48h ap?s tratamento, nas dilui??es 1:50; 1:500 e 1:1000. Efeitos citot?xicos destas pastas foram detectados nos animais sacrificados nos dois tempos. O hidr?xido de c?lcio P.A. e o agregado tri?xido mineral n?o apresentaram efeitos citot?xicos nem genot?xicos. Os resultados obtidos com o ensaio cometa tamb?m apontaram para a genotoxicidade do ?xido de zinco e pasta Guedes-Pinto, e n?o do hidr?xido de c?lcio P.A. e agregado tri?xido mineral. Estes resultados mostram a necessidade de reavalia??o do uso do ?xido de zinco e pasta Guedes-Pinto e suscitam a realiza??o de estudos adicionais avaliando a genotoxicidade e citotoxicidade destas pastas.