Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2016-09-15T21:49:15Z No. of bitstreams: 1 Rodrigo Tese Final PDF.pdf: 7598532 bytes, checksum: 876426158d6c8f445be96952d8f7c27b (MD5) / Made available in DSpace on 2016-09-15T21:49:15Z (GMT). No. of bitstreams: 1 Rodrigo Tese Final PDF.pdf: 7598532 bytes, checksum: 876426158d6c8f445be96952d8f7c27b (MD5) Previous issue date: 2016-03-28 / The use of vegetables with medicinal purposes contributes significantly to the care of the primary needs of assistance to health due to difficult access of the population to medical and pharmaceutical assistance, the high cost of manufactured medications and a tendency for people to use natural origin products. However, technical and scientific information about most of them, particularly with respect to the genotoxic potential, are still insufficient so that the use can be considered safe. In this context, the aim of this study was to evaluate the genotoxic effects of boldo extracts (Plectranthus ornatus) and soursop (Annona muricata), through the comet assay and micronucleus test in human lymphocytes with cytokinesis blocking (MNCtB). The tests were performed on human lymphocyte cultures exposed to the extracts in three concentrations: 1.000?g/ml, 500?g/ml and 250?g/ml. Hydrogen peroxide (1mM) was used as a positive control in the comet assay and vincristine sulfate (1mM) in MNCtB. As a negative control, 40% ethanol was used in both tests. The processing of the material for the comet assay and MNCtB was done according respectively to the protocols Tice et al. (2000) and Fenech (1993). The comet assay was performed in five repetitions being computed, in each of them, 100 comets in a total of two blades. For MNCtB three repetitions were performed and 1.000 binucleate lymphocytes were computed per slide. The occurrence of DNA damage (comet assay) did not differ between the cell of the cultures exposed to different concentrations of boldo extracts from each other nor compared to the negative control. Higher occurrence of micronuclei (MNCtB) was observed in the cell of the cultures treated with the extract at concentrations of 1.000?g/ml and 500?g/ml when compared to the negative control (p<0.05) and cultures treated with the extract concentration of 250?g/ml. The soursop extracts at concentrations of 1.000?g/ml to 500?g/ml induced damage occurrence to the DNA significantly higher than that observed in the negative control and cultures treated with extract at a concentration of 250?g/ml (p<0.001). The conditions in which this study was performed, the obtained results allow concluding that: 1) boldo extracts are not effective in inducing DNA damage, but depending on the concentration, they present clastogenic and/or aneugenic effects; 2) soursop extracts induce DNA damage under certain concentrations. The results of this study strongly raise the achievement of other addressing the issue, so that the actual genotoxic potential of vegetables analyzed here can be established. / O uso de vegetais com fins medicinais contribui de forma significativa para o cuidado das necessidades prim?rias de assist?ncia ? sa?de devido ao dif?cil acesso da popula??o ? assist?ncia m?dica e farmac?utica, aos altos custos dos medicamentos industrializados e ? uma tend?ncia das pessoas a utilizarem produtos de origem natural. No entanto, informa??es t?cnico-cient?ficas acerca da maioria deles, particularmente no que tange ao potencial genot?xico, s?o ainda insuficientes para que o uso possa ser considerado seguro. Neste contexto, o objetivo deste estudo foi avaliar os efeitos genot?xicos de extratos do boldo (Plectranthus ornatus) e da graviola (Annona muricata), atrav?s do ensaio cometa e do teste de micron?cleo em linf?citos humanos com bloqueio da citocinese (MNCtB). Os testes foram feitos em culturas de linf?citos humanos expostas aos extratos em tr?s concentra??es: 1.000?g/ml, 500?g/ml e 250?g/ml. Per?xido de hidrog?nio (1mM) foi utilizado como controle positivo no ensaio cometa e sulfato de vincristina (1mM) no MNCtB. Como controle negativo foi usado o etanol 40% em ambos os testes. O processamento do material para o ensaio cometa e MNCtB foi feito de acordo, respectivamente, com os protocolos de Tice et al. (2000) e Fenech (1993). O ensaio cometa foi realizado em cinco repeti??es, sendo computados, em cada uma delas, 100 cometas em um total de duas l?minas. Para o MNCtB foram realizadas tr?s repeti??es e computados 1.000 linf?citos binucleados por l?mina. A ocorr?ncia de danos ao DNA (ensaio cometa) n?o diferiu entre as c?lulas das culturas expostas ?s diferentes concentra??es dos extratos do boldo entre si e nem quando comparadas ao controle negativo. Maior ocorr?ncia de micron?cleos (MNCtB) foi observada nas c?lulas das culturas tratadas com os extratos nas concentra??es de 1.000?g/ml e de 500?g/ml quando comparadas ao controle negativo (p<0,05) e ?s culturas tratadas com o extrato na concentra??o de 250?g/ml. Os extratos da graviola nas concentra??es de 1.000?g/ml e 500?g/ml induziram ocorr?ncia de danos ao DNA significativamente maior do que o observado no controle negativo e nas culturas tratadas com extrato na concentra??o de 250?g/ml (p<0,001). Nas condi??es em que este estudo foi realizado, os resultados obtidos permitem concluir que: 1) extratos do boldo n?o s?o efetivos em induzir danos ao DNA, mas na depend?ncia da concentra??o apresentam efeitos clastog?nicos e/ou aneug?nicos; 2) extratos da graviola induzem danos ao DNA sob determinadas concentra??es. Os resultados deste estudo suscitam fortemente a realiza??o de outros abordando o tema, para que o real potencial genot?xico dos vegetais aqui analisados possa ser estabelecido.

Algumas áreas do canal de Piaçaguera, localizado na Baixada Santista, SP-Brasil, apresenta alto nível contaminação por hidrocarbonetos policíclicos aromáticos (HPA) entre outros contaminantes nos sedimentos. O objetivo do presente estudo foi verificar diferentes biomarcadores de exposição em bile e de efeitos genotóxicos em sangue de peixes, coletados in situ, que poderiam ser utilizados como ferramenta de monitoramento. O canal de Bertioga foi selecionado como região de referência. Embora não seja uma região sem nenhuma interferência de contaminantes, os sedimentos apresentam baixos valores para HPA, metais e atividade mutagênica. A espécie Mugil curema foi selecionada por ser freqüentemente encontrada em ambas as regiões de estudo. Observamos que os peixes coletados no canal de Piaçaguera apresentaram maior porcentagem de micronúcleos e danos ao DNA (ensaio cometa) no sangue quando comparados com a região de referência. Para o teste de micronúcleo, realizamos leituras de 1000 e 4000 células/indivíduo e não foram observadas diferenças estatísticas, sugerindo que o número de 1000 células poderia ser suficiente para gerar dados confiáveis para a espécie Mugil curema. A avaliação de mutagenicidade da bile foi realizada pelo teste Salmonella/microssoma combinado à extração de bile utilizando Blue rayon (BR) com as linhagens TA98, TA100 e YG1041 com e sem S9 (ativação metabólica). As maiores respostas mutagênicas foram observadas para os peixes coletados no canal de Piaçaguera. A mutagenicidade com a linhagem YG1041 na presença de S9 foi mais elevada quando comparado à mutagenicidade observada na ausência de S9 e também em comparação com a sua linhagem parental TA98, indicando a provável presença de compostos da classe das aminas aromáticas na bile como responsáveis pelos efeitos observados. A quantificação de metabólitos equivalentes de HPA foi realizada por HPLC/fluorescência na bile bruta e embora os resultados não se correlacionem com a mutagenicidade, altos níveis também foram encontrados para os peixes coletados no canal de Piaçaguera em comparação com Bertioga. O protocolo de amplificação do gene ras para a espécie Mugil curema foi estabelecido e os genes foram seqüenciados para verificação da presença de mutações. Não foram observadas mutações nos poucos peixes analisados e mais estudos são necessários para verificar a utilização deste biomarcador. O teste de micronúcleo, ensaio cometa e análise de mutagencidade em bile extraída por Blue rayon parecem ser ferramentas biológicas adequadas para monitoramento da qualidade ambiental de estuários contaminados. / Some areas of the Piaçaguera channel at Baixada Santista, SP, Brazil present high levels of polycyclic aromatic hydrocarbons (PAHs) among other contaminants in the sediment. The aim of the present study was to verify if biomarkers of exposure in bile and effect in blood of fishes collected in the field could be used as a monitoring tool. We selected Bertioga channel as a reference area. Although it is not a pristine area, the sediment presents low values of PAHs, metals and mutagenic activity. We selected the fish species Mugil curema, because they are frequently found in both areas during the entire year. We observed that the fish collected at the Piaçaguera channel showed a higher percentage of micronuclei and DNA damage (comet assay) in blood when compared to the Bertioga channel. The micronucleus readings were done in 1000 and 4000 cells/fish and no statistical difference was observed, suggesting that the number of 1000 cells would be sufficient to generate reliable data for the species Mugil curema , in the studied area. The mutagenicity of the bile was performed using the Salmonella/microsome assay combined with Blue rayon (BR) extraction with TA98, TA100 and YG1041 strains with and without rat liver S9. We observed that the mutagenic responses were higher in fish collected in Piaçaguera. The mutagenicity in YG1041 with S9 in fishes collected in the Piaçaguera channel was higher when compared to the mutagenicity observed in the absence of S9 and also higher than the response with the parental strain TA98. The results suggest that mutagenic polycyclic compounds, probably from the class of the aromatic amines are causing the observed effect. PAH metabolites-equivalent were also determined using HPLC/fluorescence in crude bile, and although the results did not correlate with the mutagenicity, higher levels were observed in fish collected in the Piaçaguera channel when compared to Bertioga. A protocol for amplification of the ras gene for the Mugil curema species was established and gene sequenced. No mutations were observed in the liver of the few fishes analyzed, and more studies are required to verify the utility of this biomarker in this fish. The micronuclei, comet assay and mutagenicity in bile extracted with blue rayon seem to be suitable biological tools to monitor the environmental quality of contaminated estuaries.

Algumas áreas do canal de Piaçaguera, localizado na Baixada Santista, SP-Brasil, apresenta alto nível contaminação por hidrocarbonetos policíclicos aromáticos (HPA) entre outros contaminantes nos sedimentos. O objetivo do presente estudo foi verificar diferentes biomarcadores de exposição em bile e de efeitos genotóxicos em sangue de peixes, coletados in situ, que poderiam ser utilizados como ferramenta de monitoramento. O canal de Bertioga foi selecionado como região de referência. Embora não seja uma região sem nenhuma interferência de contaminantes, os sedimentos apresentam baixos valores para HPA, metais e atividade mutagênica. A espécie Mugil curema foi selecionada por ser freqüentemente encontrada em ambas as regiões de estudo. Observamos que os peixes coletados no canal de Piaçaguera apresentaram maior porcentagem de micronúcleos e danos ao DNA (ensaio cometa) no sangue quando comparados com a região de referência. Para o teste de micronúcleo, realizamos leituras de 1000 e 4000 células/indivíduo e não foram observadas diferenças estatísticas, sugerindo que o número de 1000 células poderia ser suficiente para gerar dados confiáveis para a espécie Mugil curema. A avaliação de mutagenicidade da bile foi realizada pelo teste Salmonella/microssoma combinado à extração de bile utilizando Blue rayon (BR) com as linhagens TA98, TA100 e YG1041 com e sem S9 (ativação metabólica). As maiores respostas mutagênicas foram observadas para os peixes coletados no canal de Piaçaguera. A mutagenicidade com a linhagem YG1041 na presença de S9 foi mais elevada quando comparado à mutagenicidade observada na ausência de S9 e também em comparação com a sua linhagem parental TA98, indicando a provável presença de compostos da classe das aminas aromáticas na bile como responsáveis pelos efeitos observados. A quantificação de metabólitos equivalentes de HPA foi realizada por HPLC/fluorescência na bile bruta e embora os resultados não se correlacionem com a mutagenicidade, altos níveis também foram encontrados para os peixes coletados no canal de Piaçaguera em comparação com Bertioga. O protocolo de amplificação do gene ras para a espécie Mugil curema foi estabelecido e os genes foram seqüenciados para verificação da presença de mutações. Não foram observadas mutações nos poucos peixes analisados e mais estudos são necessários para verificar a utilização deste biomarcador. O teste de micronúcleo, ensaio cometa e análise de mutagencidade em bile extraída por Blue rayon parecem ser ferramentas biológicas adequadas para monitoramento da qualidade ambiental de estuários contaminados. / Some areas of the Piaçaguera channel at Baixada Santista, SP, Brazil present high levels of polycyclic aromatic hydrocarbons (PAHs) among other contaminants in the sediment. The aim of the present study was to verify if biomarkers of exposure in bile and effect in blood of fishes collected in the field could be used as a monitoring tool. We selected Bertioga channel as a reference area. Although it is not a pristine area, the sediment presents low values of PAHs, metals and mutagenic activity. We selected the fish species Mugil curema, because they are frequently found in both areas during the entire year. We observed that the fish collected at the Piaçaguera channel showed a higher percentage of micronuclei and DNA damage (comet assay) in blood when compared to the Bertioga channel. The micronucleus readings were done in 1000 and 4000 cells/fish and no statistical difference was observed, suggesting that the number of 1000 cells would be sufficient to generate reliable data for the species Mugil curema , in the studied area. The mutagenicity of the bile was performed using the Salmonella/microsome assay combined with Blue rayon (BR) extraction with TA98, TA100 and YG1041 strains with and without rat liver S9. We observed that the mutagenic responses were higher in fish collected in Piaçaguera. The mutagenicity in YG1041 with S9 in fishes collected in the Piaçaguera channel was higher when compared to the mutagenicity observed in the absence of S9 and also higher than the response with the parental strain TA98. The results suggest that mutagenic polycyclic compounds, probably from the class of the aromatic amines are causing the observed effect. PAH metabolites-equivalent were also determined using HPLC/fluorescence in crude bile, and although the results did not correlate with the mutagenicity, higher levels were observed in fish collected in the Piaçaguera channel when compared to Bertioga. A protocol for amplification of the ras gene for the Mugil curema species was established and gene sequenced. No mutations were observed in the liver of the few fishes analyzed, and more studies are required to verify the utility of this biomarker in this fish. The micronuclei, comet assay and mutagenicity in bile extracted with blue rayon seem to be suitable biological tools to monitor the environmental quality of contaminated estuaries.

Made available in DSpace on 2014-12-17T15:18:13Z (GMT). No. of bitstreams: 1 ThiagoMC.pdf: 54368 bytes, checksum: 8e14c2b4d3fe392bf725d169831359dc (MD5) Previous issue date: 2007-02-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / A contamina??o de reservat?rios de ?gua ? um dos principais problemas da atualidade. A Barragem Engenheiro Armando Ribeiro Gon?alves (EARG), (06?08 S; 37?07 W) localizada no estado do Rio Grande do Norte, ? a segunda maior barragem do nordeste brasileiro, respons?vel pelo abastecimento dom?stico de aproximadamente 415 mil habitantes do semi-?rido brasileiro. A ?gua da EARG ? captada por um sistema de adutoras, onde ? tratada e distribu?da para a popula??o. O presente trabalho teve o objetivo de avaliar o potencial genot?xico da ?gua da Barragem EARG. Para isso, foram realizados os testes cometa e micron?cleo com eritr?citos de til?pias (Oreochromis niloticus) capturadas nesse reservat?rio. Os testes Allium cepa e muta??o reversa com Salmonela typhimurium foram realizados com ?gua do reservat?rio antes e ap?s o tratamento. Al?m disso, an?lises quantitativa e qualitativa de cianobact?rias, assim como a quantifica??o das microcistinas produzidas pelas cianobact?rias presentes nesse reservat?rio foram realizados, apenas com amostras coletadas na barragem. Os resultados obtidos indicaram aumento significativo na freq??ncia de micron?cleos em eritr?citos de O. niloticus (p<0,05). A m?dia obtida foi de 2,38 ? 3,02 micron?cleos em mil c?lulas analisadas, enquanto que o controle negativo apresentou m?dia de 0,20 ? 0,41 micron?cleos. O ensaio cometa realizado com peixes da EARG foi analisado em uma escala crescente de danos (0 - 4), e mostrou resultados classificados nos n?veis 0, 1, 2 e 3, enquanto que o controle negativo apresentou resultados nos n?veis 0 e 1. Nos par?metros macrosc?picos avaliados no teste A. cepa n?o foi verificado altera??es estatisticamente significativas. Os par?metros microsc?picos indicaram diminui??o significativa no ?ndice mit?tico nos dois pontos estudados. Al?m disso, tamb?m foi detectado aumento na freq??ncia de met?fases e an?fases aberrantes em ambos os pontos, por?m estatisticamente significativo apenas na amostra sem tratamento para an?fases aberrantes. A freq??ncia de micron?cleos no teste A. cepa n?o foi significativo em rela??o ao controle negativo. Para o teste de muta??o reversa com S. typhimurium realizado com ?gua sem extra??o, os resultados n?o demonstraram mutagenicidade para ambos os pontos. Os resultados encontrados com os extratos das amostras coletadas em ambos os pontos apresentaram diferen?as estatisticamente significativa quando foi utilizada a fra??o S9 como ativador metab?lico. Para a ?gua n?o tratada, essas diferen?as foram encontradas apenas para a cepa TA98, enquanto que para a ?gua tratada, as duas linhagens apresentaram diferen?as significativas. A an?lise qualitativa de cianobact?rias demonstrou a exist?ncia de cianobact?rias potencialmente t?xicas tais como Planktothrix agardii, Cylindrospermopsis raciborskii e Microcystis panniformes. O ensaio de HPLC indicou a presen?a de 38,1 &#956;g/L de microcistinas na ?gua da EARG. O teste de muta??o reversa realizado com o extrato contendo microcistinas, n?o apresentou aumento na raz?o de mutagenicidade para TA100. O conjunto dos resultados obtidos no presente trabalho sugere que as ?guas da Barragem EARG podem conter agentes genot?xicos, capazes de alterar a informa??o gen?tica dos indiv?duos. Esses resultados indicam a necessidade de um programa de monitoramento e controle desses poluentes

La fumée du tabac est un aérosol extrêmement complexe constitué de milliers de composés répartis entre la phase particulaire et la phase vapeur. Il a été démontré que les effets toxicologiques de cette fumée sont associés aux composés appartenant aux deux phases. Plusieurs composés biologiquement actifs ont été identifiés dans la fumée du tabac; cependant, il n’y a pas d’études démontrant la relation entre les réponses biologiques obtenues via les tests in vitro ou in vivo et les composés présents dans la fumée entière du tabac. Le but de la présente recherche est de développer des méthodes fiables et robustes de fractionnement de la fumée à l’aide de techniques de séparation analytique et de techniques de détection combinés à des essais in vitro toxicologiques. Une étude antérieure réalisée par nos collaborateurs a démontré que, suite à l’étude des produits de combustion de douze principaux composés du tabac, l’acide chlorogénique s’est avéré être le composé le plus cytotoxique selon les test in vitro du micronoyau. Ainsi, dans cette étude, une méthode par chromatographie préparative en phase liquide a été développée dans le but de fractionner les produits de combustion de l’acide chlorogénique. Les fractions des produits de combustion de l’acide chlorogénique ont ensuite été testées et les composés responsables de la toxicité de l’acide chlorogénique ont été identifiés. Le composé de la sous-fraction responsable en majeure partie de la cytoxicité a été identifié comme étant le catéchol, lequel fut confirmé par chromatographie en phase liquide/ spectrométrie de masse à temps de vol. Des études récentes ont démontré les effets toxicologiques de la fumée entière du tabac et l’implication spécifique de la phase vapeur. C’est pourquoi notre travail a ensuite été focalisé principalement à l’analyse de la fumée entière. La machine à fumer Borgwaldt RM20S® utilisée avec les chambres d’exposition cellulaire de British American Tobacco permettent l’étude in vitro de l’exposition de cellules à différentes concentrations de fumée entière du tabac. Les essais biologiques in vitro ont un degré élevé de variabilité, ainsi, il faut prendre en compte toutes les autres sources de variabilité pour évaluer avec précision la finalité toxicologique de ces essais; toutefois, la fiabilité de la génération de la fumée de la machine n’a jamais été évaluée jusqu’à maintenant. Nous avons donc déterminé la fiabilité de la génération et de la dilution (RSD entre 0,7 et 12 %) de la fumée en quantifiant la présence de deux gaz de référence (le CH4 par détection à ionisation de flamme et le CO par absorption infrarouge) et d’un composé de la phase particulaire, le solanesol (par chromatographie en phase liquide à haute performance). Ensuite, la relation entre la dose et la dilution des composés de la phase vapeur retrouvée dans la chambre d’exposition cellulaire a été caractérisée en utilisant une nouvelle technique d’extraction dite par HSSE (Headspace Stir Bar Sorptive Extraction) couplée à la chromatographie en phase liquide/ spectrométrie de masse. La répétabilité de la méthode a donné une valeur de RSD se situant entre 10 et 13 % pour cinq des composés de référence identifiés dans la phase vapeur de la fumée de cigarette. La réponse offrant la surface maximale d’aire sous la courbe a été obtenue en utilisant les conditions expérimentales suivantes : intervalle de temps d’exposition/ désorption de 10 0.5 min, température de désorption de 200°C pour 2 min et température de concentration cryogénique (cryofocussing) de -75°C. La précision de la dilution de la fumée est linéaire et est fonction de l’abondance des analytes ainsi que de la concentration (RSD de 6,2 à 17,2 %) avec des quantités de 6 à 450 ng pour les composés de référence. Ces résultats démontrent que la machine à fumer Borgwaldt RM20S® est un outil fiable pour générer et acheminer de façon répétitive et linéaire la fumée de cigarette aux cultures cellulaires in vitro. Notre approche consiste en l’élaboration d’une méthodologie permettant de travailler avec un composé unique du tabac, pouvant être appliqué à des échantillons plus complexes par la suite ; ex : la phase vapeur de la fumée de cigarette. La méthodologie ainsi développée peut potentiellement servir de méthode de standardisation pour l’évaluation d’instruments ou de l’identification de produits dans l’industrie de tabac. / Tobacco smoke is an extremely complex aerosol composed of thousands of constituents distributed amongst the particulate and vapor phases. Toxicological effects have been linked to compounds present in both of these phases. Many biologically active compounds have been identified within tobacco smoke; however, there is a lack of studies correlating specific in vitro or in vivo biological responses to components within whole tobacco smoke. The goal of this research was to develop reliable and robust smoke fractionation methods using analytical separation and detection techniques in combination with in vitro toxicological assays. In a previous study by our collaborators, toxicological assessment of the particulate phase combustion products of twelve individual tobacco components revealed that the combustion products of chlorogenic acid were the most cytotoxic using the in vitro micronucleus test. Therefore, a preparative liquid chromatography method was developed in this work to fractionate the combustion products of chlorogenic acid to assess the bioactivity of these fractions and to identify the compounds responsible for the toxicity observed. The sub-fraction responsible for the most cytotoxic response comprised catechol, which was identified by liquid chromatography/time-of-flight mass spectrometry. Emerging studies have highlighted the toxicological significance of whole tobacco smoke and specifically the vapor phase, which shifted our focus to whole smoke analyses. The Borgwaldt RM20S® smoking machine in combination with British American Tobacco’s in vitro cell exposure chamber allow for the generation of fresh cigarette smoke in various doses and delivery to cell cultures. In vitro biological assays have a high degree of variability, thus, all other sources of variability must be accounted for to accurately assess toxicological endpoints; however, the reliability of dose delivery of the instrument had not been assessed until now. We have determined the reliability (RSD from 0.7-12%) of smoke generation and dilution by quantifying two reference standard gases (CH4 by flame ionization detection and CO by infrared absorption) and the tobacco particulate phase marker, solanesol (by high performance liquid chromatography-ultraviolet absorption detection). The relationship between dose and diluted vapor phase components found within the exposure chamber was then characterized by developing a headspace stir-bar sorptive extraction-gas chromatography/mass spectrometry method. The method repeatability gave an RSD from 10-13% for five reference compounds identified in the vapor phase of cigarette smoke. The maximal peak area response was obtained using the following experimental conditions: exposure-to-desorption time interval of 10  0.5 min, desorption temperature of 200 °C for 2 min, and a cryofocussing temperature of -75 °C. The dilution precision was found to yield a linear response of analyte abundance and was observed to be a function of concentration (RSD from 6.2-17.2 %) with quantities of 6-450 ng for the reference compounds. The findings obtained suggest the Borgwaldt RM20S® is a reliable tool to generate and deliver repeatable and linear doses of cigarette smoke to in vitro cell cultures. Our approach began with designing the methodology to work with an individual tobacco component, which could then be applied to a more complex sample, e.g., the vapor phase of cigarette smoke. The methodology developed can potentially serve as standardized methods for the assessment of instrumentation or screening of products for the Tobacco Industry.

No description available.

La fumée du tabac est un aérosol extrêmement complexe constitué de milliers de composés répartis entre la phase particulaire et la phase vapeur. Il a été démontré que les effets toxicologiques de cette fumée sont associés aux composés appartenant aux deux phases. Plusieurs composés biologiquement actifs ont été identifiés dans la fumée du tabac; cependant, il n’y a pas d’études démontrant la relation entre les réponses biologiques obtenues via les tests in vitro ou in vivo et les composés présents dans la fumée entière du tabac. Le but de la présente recherche est de développer des méthodes fiables et robustes de fractionnement de la fumée à l’aide de techniques de séparation analytique et de techniques de détection combinés à des essais in vitro toxicologiques. Une étude antérieure réalisée par nos collaborateurs a démontré que, suite à l’étude des produits de combustion de douze principaux composés du tabac, l’acide chlorogénique s’est avéré être le composé le plus cytotoxique selon les test in vitro du micronoyau. Ainsi, dans cette étude, une méthode par chromatographie préparative en phase liquide a été développée dans le but de fractionner les produits de combustion de l’acide chlorogénique. Les fractions des produits de combustion de l’acide chlorogénique ont ensuite été testées et les composés responsables de la toxicité de l’acide chlorogénique ont été identifiés. Le composé de la sous-fraction responsable en majeure partie de la cytoxicité a été identifié comme étant le catéchol, lequel fut confirmé par chromatographie en phase liquide/ spectrométrie de masse à temps de vol. Des études récentes ont démontré les effets toxicologiques de la fumée entière du tabac et l’implication spécifique de la phase vapeur. C’est pourquoi notre travail a ensuite été focalisé principalement à l’analyse de la fumée entière. La machine à fumer Borgwaldt RM20S® utilisée avec les chambres d’exposition cellulaire de British American Tobacco permettent l’étude in vitro de l’exposition de cellules à différentes concentrations de fumée entière du tabac. Les essais biologiques in vitro ont un degré élevé de variabilité, ainsi, il faut prendre en compte toutes les autres sources de variabilité pour évaluer avec précision la finalité toxicologique de ces essais; toutefois, la fiabilité de la génération de la fumée de la machine n’a jamais été évaluée jusqu’à maintenant. Nous avons donc déterminé la fiabilité de la génération et de la dilution (RSD entre 0,7 et 12 %) de la fumée en quantifiant la présence de deux gaz de référence (le CH4 par détection à ionisation de flamme et le CO par absorption infrarouge) et d’un composé de la phase particulaire, le solanesol (par chromatographie en phase liquide à haute performance). Ensuite, la relation entre la dose et la dilution des composés de la phase vapeur retrouvée dans la chambre d’exposition cellulaire a été caractérisée en utilisant une nouvelle technique d’extraction dite par HSSE (Headspace Stir Bar Sorptive Extraction) couplée à la chromatographie en phase liquide/ spectrométrie de masse. La répétabilité de la méthode a donné une valeur de RSD se situant entre 10 et 13 % pour cinq des composés de référence identifiés dans la phase vapeur de la fumée de cigarette. La réponse offrant la surface maximale d’aire sous la courbe a été obtenue en utilisant les conditions expérimentales suivantes : intervalle de temps d’exposition/ désorption de 10 0.5 min, température de désorption de 200°C pour 2 min et température de concentration cryogénique (cryofocussing) de -75°C. La précision de la dilution de la fumée est linéaire et est fonction de l’abondance des analytes ainsi que de la concentration (RSD de 6,2 à 17,2 %) avec des quantités de 6 à 450 ng pour les composés de référence. Ces résultats démontrent que la machine à fumer Borgwaldt RM20S® est un outil fiable pour générer et acheminer de façon répétitive et linéaire la fumée de cigarette aux cultures cellulaires in vitro. Notre approche consiste en l’élaboration d’une méthodologie permettant de travailler avec un composé unique du tabac, pouvant être appliqué à des échantillons plus complexes par la suite ; ex : la phase vapeur de la fumée de cigarette. La méthodologie ainsi développée peut potentiellement servir de méthode de standardisation pour l’évaluation d’instruments ou de l’identification de produits dans l’industrie de tabac. / Tobacco smoke is an extremely complex aerosol composed of thousands of constituents distributed amongst the particulate and vapor phases. Toxicological effects have been linked to compounds present in both of these phases. Many biologically active compounds have been identified within tobacco smoke; however, there is a lack of studies correlating specific in vitro or in vivo biological responses to components within whole tobacco smoke. The goal of this research was to develop reliable and robust smoke fractionation methods using analytical separation and detection techniques in combination with in vitro toxicological assays. In a previous study by our collaborators, toxicological assessment of the particulate phase combustion products of twelve individual tobacco components revealed that the combustion products of chlorogenic acid were the most cytotoxic using the in vitro micronucleus test. Therefore, a preparative liquid chromatography method was developed in this work to fractionate the combustion products of chlorogenic acid to assess the bioactivity of these fractions and to identify the compounds responsible for the toxicity observed. The sub-fraction responsible for the most cytotoxic response comprised catechol, which was identified by liquid chromatography/time-of-flight mass spectrometry. Emerging studies have highlighted the toxicological significance of whole tobacco smoke and specifically the vapor phase, which shifted our focus to whole smoke analyses. The Borgwaldt RM20S® smoking machine in combination with British American Tobacco’s in vitro cell exposure chamber allow for the generation of fresh cigarette smoke in various doses and delivery to cell cultures. In vitro biological assays have a high degree of variability, thus, all other sources of variability must be accounted for to accurately assess toxicological endpoints; however, the reliability of dose delivery of the instrument had not been assessed until now. We have determined the reliability (RSD from 0.7-12%) of smoke generation and dilution by quantifying two reference standard gases (CH4 by flame ionization detection and CO by infrared absorption) and the tobacco particulate phase marker, solanesol (by high performance liquid chromatography-ultraviolet absorption detection). The relationship between dose and diluted vapor phase components found within the exposure chamber was then characterized by developing a headspace stir-bar sorptive extraction-gas chromatography/mass spectrometry method. The method repeatability gave an RSD from 10-13% for five reference compounds identified in the vapor phase of cigarette smoke. The maximal peak area response was obtained using the following experimental conditions: exposure-to-desorption time interval of 10  0.5 min, desorption temperature of 200 °C for 2 min, and a cryofocussing temperature of -75 °C. The dilution precision was found to yield a linear response of analyte abundance and was observed to be a function of concentration (RSD from 6.2-17.2 %) with quantities of 6-450 ng for the reference compounds. The findings obtained suggest the Borgwaldt RM20S® is a reliable tool to generate and deliver repeatable and linear doses of cigarette smoke to in vitro cell cultures. Our approach began with designing the methodology to work with an individual tobacco component, which could then be applied to a more complex sample, e.g., the vapor phase of cigarette smoke. The methodology developed can potentially serve as standardized methods for the assessment of instrumentation or screening of products for the Tobacco Industry.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-05-03T13:16:12Z No. of bitstreams: 2 Tese - Wanessa Fernandes Carvalho - 2018.pdf: 4178094 bytes, checksum: 12ab926257d567aaefec72a5d4d53bf7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-05-03T13:23:49Z (GMT) No. of bitstreams: 2 Tese - Wanessa Fernandes Carvalho - 2018.pdf: 4178094 bytes, checksum: 12ab926257d567aaefec72a5d4d53bf7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-03T13:23:49Z (GMT). No. of bitstreams: 2 Tese - Wanessa Fernandes Carvalho - 2018.pdf: 4178094 bytes, checksum: 12ab926257d567aaefec72a5d4d53bf7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The increased use of pesticides, the risks and disruptions that these compounds can cause to organisms and the environment has been much discussed in recent years. 2,4-D and glyphosate herbicides are applied post-emergence in large-scale crops and play an important role in the optimization of agricultural production worldwide. Environmental quality bioindicators are used in environmental impact assessment studies because of their interaction with the environment and their ease of absorption and accumulation of xenobiotic compounds (Younes, 2000). Therefore, the objective of this study was to evaluate the genotoxic and mutagenic effects of herbicides on environmental quality bionicators. Acute toxicity of Credit® at 48%, Herbifen Super® at 97%, Weedar Full® at 83.5%, Dedalo elite® at 30%, and Imazethapyr and their binary combinations were tested on Cnesterodon decemmaculatus, Rhinella arenarum and Dendropsophus minutus. The lethal effect was determined from experiments with mortality and the sublethal was used the single cell gel electrophoresis (SCGE) bioassay and the micronucleus test. The LC5096h value for Herbifen Super® was 2.668 mg / L, Weedar Full® was 678.04 mg / L, Dedalo elite® was 0.463 mg / L and for Credit® was 91.73 mg / L and Imazethapyr® was 0.99 mg / L. The results of this study demonstrated that the comet assay and the micronucleus test are highly sensitive methods for the detection of herbicide-induced DNA damage in aquatic organisms. The herbicides tested showed different behaviors when combined at their concentrations of 5% and 10% of LC5096h. The toxic effect of the combination of Herbifen Super® and Credit® was basically due to the action of the active principle glyphosate present in Credit®. For Weedar Full® and Dedalo elite® herbicides, synergism was observed in combinations of 5% and 10% of LC5096h values. Our study is the first report on the induction of lethal acute and sublethal effects of Credit® binary combinations; Herbifen Super®, Weedar Full®, Dedalo elite®; Imazethapyr® in aquatic organisms. / O aumento da utilização dos pesticidas, os riscos e perturbações que esses compostos podem provocar aos organismos e ao meio ambiente está sendo bastante discutido nos últimos anos. Os herbicidas 2,4-D e glifosato são aplicados em pós-emergência em culturas de larga escala e desempenham um papel importante na otimização da produção agrícola em todo o mundo. Os bioindicadores de qualidade ambiental são utilizados em estudos de avaliações de impacto ambiental devido sua interação com o meio ambiente e a sua facilidade de absorção e acumulação de compostos xenobióticos (Younes, 2000). Sendo assim, o objetivo deste estudo foi avaliar os efeitos genotóxicos e mutagênicos de herbicidas em biondicadores de qualidade ambiental. A toxicidade aguda do Credit® a 48%, Herbifen Super® a 97%, Weedar Full® a 83.5%, Dedalo elite® a 30% e Imazethapyr e suas combinações binárias foram testadas em Cnesterodon decemmaculatus, Rhinella arenarum e Dendropsophus minutus. O efeito letal foi determinado a partir de experiências com mortalidade e o subletal foi utilizado o bioensaio de eletroforese em gel de célula única (SCGE) e oteste do micronúcleo. O valor LC5096h para Herbifen Super® foi de 2.668 mg / L, Weedar Full® foi de 678,04 mg/L, Dedalo elite® foi de 0,463 mg/L e para Credit® foi de 91.73 mg /L e Imazethapyr® foi de 0.99 mg/L. Os resultados deste estudo demontraram que o ensaio cometa e o teste do micronúcleo são métodos altamente sensíveis para a detecção de danos ao DNA induzidos por herbicidas em organismos aquáticos. Os herbicidas testados revelaram diferentes comportamentos ao serem combinados em suas concentrações de 5% e 10% da CL5096h. O efeito tóxico da combinação entre Herbifen Super® e Credit® deu-se basicamente pela ação do princípio ativo glifosato presente no Credit®. Para os herbicidas Weedar Full® e Dedalo elite® foi observado um sinergismo nas combinações de 5% e 10% dos valores de CL5096h. Nosso estudo constitui o primeiro relatório sobre a indução de efeitos agudos letais e subletal de combinações binárias de Credit® ; Herbifen Super® , Weedar Full® , Dedalo elite® ; Imazethapyr® em organismos aquáticos.