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Impact des hydrocarbures aromatiques polycycliques sur le métabolisme lipidique et le transport du phosphore chez le champignon mycorhizien à arbuscules Rhizophagus irregularis / Polycyclic aromatic hydrocarbons impact on lipid metabolism and phosphorus transport in the arbuscular mycorrhizal fungus Rhizophagus irregularisCalonne, Maryline 04 December 2012 (has links)
Les hydrocarbures aromatiques polycyliques (HAPs) figurent parmi les polluants organiques persistants majeurs des sols pollués et présentent une toxicité avérée vis-à-vis de l'homme et des écosystèmes. Parmi les méthodes de remédiation des sols pollués par les HAPs, la phytoremédiation assistée par les champignons mycorhiziens à arbuscules (CMA), pourrait représenter une alternative innovante, écologique et économique. L'utilisation des mycorhizes comme outil de phytoremédiation des sols pollués présente plusieurs avantages dont une meilleure tolérance à la toxicité des HAPs, une meilleure nutrition hydrique et minérale ainsi qu'une meilleure dissipation des HAPs. De rares études ont décrit l'impact des HAPs sur le développement des CMA en lien avec une péroxydation lipidique et une perturbation des teneurs en lipides du CMA, mais ni les cibles d'action de ces polluants au niveau du métabolisme lipidique, ni le rôle de ces modifications dans sa tolérance aux HAPs et dans leur dissipation n'ont été étudiés. C'est pourquoi, le premier objectif de ce travail vise tout d'abord à comprendre l'impact des HAPs sur le métabolisme lipidique. Le radiomarquage par l'acétate [1-¹⁴C] a permis de montrer une perturbation de la biosynthèse des lipides membranaires du CMA extra-racinaire. D'autre part, nos résultats montrent que les HAPs affectent la nutrition phosphatée. Par ailleurs, la capacité des mycorhizes à dégrader et à bioaccumuler le benzo[a]pyrène est démontrée. Enfin, l'implication du métabolisme des lipides de réserve (les triacylglycérols) du mycélium extra-racinaire dans la régénération des membranes altérées, la lutte contre le stress oxydant induit par les HAPs et dans leur métabolisation/bioaccumulation est discutée. / Polycyclic aromatic hydrocarbons (PAHs) are among the major persistent organic pollutant frequently found in the polluted soils and are harmful for human health and its environment. To clean-up the PAHs polluted soils, phytoremediation assisted by arbuscular mycorrhizal fungi (AMF) could represent an innovative, ecological and cost-effective alternative. The use of mycorrhizas, as phytoremediation tool, has several advantages including increased tolerance to the pollutant toxicity, improved water and mineral nutrition as well as a better pollutant dissipation. Few studies have described the impact of PAHs on the AMF development related with lipid peroxidation and total lipid content disturbance. However, so far neither the target action of these pollutants on the metabolism, nor the role of these lipid changes in PAH tolerance and in their dissipation have been studied. Therefore, the present work aims firstly to improve our understanding of the PAHs impact on the CMA lipid metabolism. Thanks to radiolabeling experiments with [1-¹⁴C] acetate, our results showed a disruption of the membrane lipid biosynthesis pathways in the AMF extraradical mycelium, grown in the presence of PAHs. Secondly, it was highlighted that the PAHs affectef the phosphate nutrition. Finally, the mycorrhizas abilities to degrade and to bioaccumulate the benzo[a]pyrene, were pointed out. The involvement of extraradical mycelium storage lipid (triacyglycerols) metabolism in the membrane regeneration, the fight against the PAH induced-oxidative stress and the PAH metabolism/bioaccumulation is discussed.
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Oxidace benzo(a)pyrenu cytochromem P450 1A1 exprimovaným v prokaryotickém a eukaryotickém systému / Oxidation of benzo(a)pyrene by cytochrome P450 1A1 expressed in prokaryotic and eukaryotic systemsKroftová, Natálie January 2013 (has links)
Benzo[a]pyrene (BaP) is a human carcinogen, which is metabolized by a variety of enzyms such as cytochrome P450 (CYP) and epoxide hydrolase. The aim of this work was to study BaP metabolism in vitro by the hepatic microsomal system of rats treated with CYP inducers and by human cytochrome P450 1A1 (CYP1A1) expressed in eukaryotic and prokaryotic systems. An eukaryotic expression system consisted of microsomes isolated from insect cells, whereas a prokaryotic expression system was formed by the membrane fragments of E. coli. In the case of recombinant human CYP1A1, we investigated the influence of cytochrome b5, NADPH:cytochrome P450 reductase (CPR) and epoxide hydrolase in BaP oxidation. Isolation and purification of rabbit hepatic CPR was another aim of this work. BaP metabolites were separated by HPLC. The results found in this work demostrate the fact that hepatic microsomal systems of rats treated with an inducer of CYP1A (Sudan I), an inducer of CYP2B (phenobarbital) and an inducer of CYP3A (PCN) exhibit higher efficiency of BaP oxidation than microsomes of control rats. BaP is oxidized by human CYP1A1 expressed in the eukaryotic system to six metabolites (BaP-9,10-dihydrodiol, BaP metabolite with unknown structure, BaP-7,8-dihydrodiol, BaP-1,6-dion, BaP-3,6-dion, BaP-3-ol), whereas by human...
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A aplicabilidade de combinações seletivas de linhagens S. typhimurium na caracterização da mutagenicidade de amostras de ar / The applicability of selective combinations of strains S. typhimurium in the characterization of mutagenicity of airborne samplesAlves, Débora Kristina Magalhães 03 October 2011 (has links)
O material particulado atmosferico é reconhecido como mutagênico. Embora vários compostos já tenham sido identificados nessa matriz, os mesmos não explicam o efeito observado na maioria dos bioensaios realizados. O ensaio de mutagenicidade Salmonella/microssoma tem sido amplamente utilizado para a avaliação de amostras atmosféricas o qual é muito sensível na detecção de vários tipos de mutágenos, pois é realizado com diferentes linhagens de Salmonella typhimurium na presença e ausência de ativação metabólica exógena. O principal objetivo deste trabalho foi verificar a aplicabilidade da utilização de combinações seletivas de linhagens S. typhimurium na caracterização da mutagenicidade de amostras de material particulado total de ar coletado em Limeira, uma cidade do interior do estado de São Paulo sob influência de intenso tráfego, emissões industriais e da agricultura, incluindo o plantio de cana de açúcar. Foram avaliados seis extratos orgânicos obtidos com diclorometano por ultrasonicação, utilizando o método Salmonella/microssoma em microssuspensão na presença e ausência de ativação metabólica (mistura S9) em experimentos de dose-resposta, com quantidade máxima de 50 µg de material orgânico extraído (MOE) por placa. Os pares de linhagens utilizados foram: YG1041/TA98, para verificar a presença de nitrocompostos, como 1- nitropireno e/ou aminas aromáticas; YG5161/TA1538, altamente sensivel aos hidrocarbonetos policíclicos aromáticos (HPA) não substituídos como o benzo[a]pireno (BaP); e YG7108/TA1535, para a indicação de agentes alquilantes. Os resultados indicaram que os compostos que podem melhor explicar os efeitos observados são os nitro e/ou aminas aromáticas e não os HPA não substituídos como o BaP. O par YG7108/TA1535 forneceu respostas negativas para todas as amostras. As combinações seletivas de linhagens S. typhimurium (YG1041/TA98 e YG5161/TA1538) mostraram-se eficientes na caracterização da mutagenicidade de amostras de ar coletadas na cidade de Limeira, podendo ser utilizadas como uma importante ferramenta de auxílio do ensaio Salmonella/microssoma em microssuspensão em estudos de análises quimicas direcionadas pelo efeito biológico (ADEB). As potências mutagênicas observadas com a linhagem TA98 para a cidade de Limeira foram similares aquelas verificadas na Região Metropolitana de São Paulo. A comparação das respostas de mutagenicidade observadas para as amostras analisadas com o perfil de compostos puros, encontrados na literatura, não forneceu candidatos que podem representar a mistura. Mais estudos de mutagenicidade com compostos puros utilizando os pares de linhagens propostos (TA98/YG1041 e TA1538/YG5161) e análises quimicas de extratos fracionados são necessários para verificar se esta abordagem pode ser útil em estudos de ADEB, e consequentemente na descoberta de novos poluentes atmosféricos. / The atmospheric particulate matter is recognized as mutagenic. Although several compounds have been identified in this matrix, they do not explain the observed effect in most bioassays that have been performed. The Salmonella/microsome assay has been widely used for the evaluation of atmospheric samples, The assay is very sensitive in the detection of various types of mutagens, because it is performed with different strains of Salmonella typhimurium in the presence and absence of exogenous metabolic activation. The main objective of this work was to verify the applicability of selective combination of strains in the characterization of mutagenicity of total airborne particulate matter collected in Limeira, a São Paulo State city in a site influenced by heavy traffic, industrial and agricultural emissions, including sugar cane plantations. We tested six extracts obtained with methylene chloride by ultrasonication using the Salmonella/microsome microsuspension protocol in the presence and absence of rat liver S9 in dose response experiments, using 50 µg of extractable organic matter (EOM) as a maximum dose per plate. The following pairs of strains were used: YG1041/TA98, to verify the presence of nitro-compounds, as 1-nitropyrene and/or aromatic amines; YG5161/TA1538, highly sensitive to non-substituted polycyclic aromatic hydrocarbons (PAHs) as benzo[a]pyrene (BaP); and YG7108/TA1535, for the indication of alkylating agents. The results indicated that the compounds that can better explain the observed effect are the nitro and/or aromatic amines and not nonsubstituted PAHs such as BaP. The pair YG7108/TA1535 provided negative responses for all the samples. Selective combinations of S. typhimurium strains (YG1041/TA98 and YG5161/TA1538) proved efficient in the characterization of mutagenicity of airborne samples collected in the city of Limeira and can be used as an important tool for the Salmonella/microsome microsuspension assay in effect directed chemical analysis (EDA) studies. The mutagenic potencies observed with TA98 strain for the city of Limeira were similar to those verified in the Metropolitan Region of São Paulo. The comparison of the observed mutagenicity responses for the samples analyzed with the profiles of pure compounds found in the literature did not provide any candidates that could alone represent the mixture. More mutagenicity studies with pure compounds using the proposed pair of strains (TA98/YG1041 and TA1538/YG5161) along with chemical analysis of fractioned extracts are needed to verify if this approach can be useful in EDA studies, and consequently in the discovery of new air priority pollutants.
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Ensaio cometa em microalgas marinhas: danos no DNA de Dunaliella tertiolecta Butcher 1959 causados pela exposição à 4-nitroquinolina-N-óxido e ao benzo [a] pireno / Comet assay in marine microalgae: DNA damage in Dunaliella tertiolecta Butcher 1959 caused by exposure to 4-nitroquinoline-N-oxide and benzo[a]pyreneUssami, Keyi Ando 03 October 2007 (has links)
Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.
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Ensaio cometa em microalgas marinhas: danos no DNA de Dunaliella tertiolecta Butcher 1959 causados pela exposição à 4-nitroquinolina-N-óxido e ao benzo [a] pireno / Comet assay in marine microalgae: DNA damage in Dunaliella tertiolecta Butcher 1959 caused by exposure to 4-nitroquinoline-N-oxide and benzo[a]pyreneKeyi Ando Ussami 03 October 2007 (has links)
Dunaliella tertiolecta, uma alga verde fitoplanctônica de ampla distribuição no ambiente marinho, foi escolhida como organismo teste para estudar a possibilidade de ser utilizada no ensaio cometa, um teste de detecção de danos no DNA em células individualizadas muito utilizado na ecotoxicologia. Essas algas foram facilmente lisadas pela solução de lise alcalina iônica, seus cometas foram corados eficientemente pelo brometo de etídio e pela prata, e a análise por índice de danos apresentou boa correlação com momento de cauda, comprimento de cauda e porcentagem de DNA na cauda. As algas foram expostas a concentrações crescentes de 4-nitroquinolina-N-óxido (4NQO) e benzo[a]pireno (BAP) por 1, 2 e 4 h no escuro. Após somente 1 h de exposição, observou-se um aumento significativo de danos no DNA das algas expostas a 0,25 µM de 4NQO, demonstrando a sensibilidade das mesmas em relação a células de animais. Os dados obtidos da exposição ao BAP não foram consistentes e necessitam de verificação. A metabolização de BAP em compostos tóxicos pelas algas e o efeito das condições de luminosidade antes e durante as exposições são discutidos. Os resultados indicam que D. tertiolecta pode ser utilizada em laboratório para avaliação de genotoxicidade na água através do ensaio cometa. / D. tertiolecta, a phytoplanktonic green algae with ubiquitous distribution in the marine environment, was chosen as a test organism to study the possibility of being used in the comet assay, a frequently used test in ecotoxicology to detect DNA damage in single cells. These algae were easily lised by the alkaline ionic lysis solution, their comets were efficiently stained by ethidium bromide and silver and the damage index was well correlated to tail moment, tail length and percentage of DNA in the tail. The algae were exposed to increasing concentrations of 4-nitroquinoline-N-oxide (4NQO) and benzo[a]pyrene (BAP) for 1, 2 and 4 h in the dark. After 1 h exposure, a significant increase in the DNA damage of algae exposed to 0,25 µM of 4NQO was observed, demonstrating their sensitivity in relation to cells from animals. The data of BAP exposure were not consistent and need further verification. The metabolization of BAP to toxic compounds by algae and the light conditions before and during exposure are discussed. The results indicate that D. tertiolecta can be used in laboratory to evaluate water genotoxicity with comet assay.
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A aplicabilidade de combinações seletivas de linhagens S. typhimurium na caracterização da mutagenicidade de amostras de ar / The applicability of selective combinations of strains S. typhimurium in the characterization of mutagenicity of airborne samplesDébora Kristina Magalhães Alves 03 October 2011 (has links)
O material particulado atmosferico é reconhecido como mutagênico. Embora vários compostos já tenham sido identificados nessa matriz, os mesmos não explicam o efeito observado na maioria dos bioensaios realizados. O ensaio de mutagenicidade Salmonella/microssoma tem sido amplamente utilizado para a avaliação de amostras atmosféricas o qual é muito sensível na detecção de vários tipos de mutágenos, pois é realizado com diferentes linhagens de Salmonella typhimurium na presença e ausência de ativação metabólica exógena. O principal objetivo deste trabalho foi verificar a aplicabilidade da utilização de combinações seletivas de linhagens S. typhimurium na caracterização da mutagenicidade de amostras de material particulado total de ar coletado em Limeira, uma cidade do interior do estado de São Paulo sob influência de intenso tráfego, emissões industriais e da agricultura, incluindo o plantio de cana de açúcar. Foram avaliados seis extratos orgânicos obtidos com diclorometano por ultrasonicação, utilizando o método Salmonella/microssoma em microssuspensão na presença e ausência de ativação metabólica (mistura S9) em experimentos de dose-resposta, com quantidade máxima de 50 µg de material orgânico extraído (MOE) por placa. Os pares de linhagens utilizados foram: YG1041/TA98, para verificar a presença de nitrocompostos, como 1- nitropireno e/ou aminas aromáticas; YG5161/TA1538, altamente sensivel aos hidrocarbonetos policíclicos aromáticos (HPA) não substituídos como o benzo[a]pireno (BaP); e YG7108/TA1535, para a indicação de agentes alquilantes. Os resultados indicaram que os compostos que podem melhor explicar os efeitos observados são os nitro e/ou aminas aromáticas e não os HPA não substituídos como o BaP. O par YG7108/TA1535 forneceu respostas negativas para todas as amostras. As combinações seletivas de linhagens S. typhimurium (YG1041/TA98 e YG5161/TA1538) mostraram-se eficientes na caracterização da mutagenicidade de amostras de ar coletadas na cidade de Limeira, podendo ser utilizadas como uma importante ferramenta de auxílio do ensaio Salmonella/microssoma em microssuspensão em estudos de análises quimicas direcionadas pelo efeito biológico (ADEB). As potências mutagênicas observadas com a linhagem TA98 para a cidade de Limeira foram similares aquelas verificadas na Região Metropolitana de São Paulo. A comparação das respostas de mutagenicidade observadas para as amostras analisadas com o perfil de compostos puros, encontrados na literatura, não forneceu candidatos que podem representar a mistura. Mais estudos de mutagenicidade com compostos puros utilizando os pares de linhagens propostos (TA98/YG1041 e TA1538/YG5161) e análises quimicas de extratos fracionados são necessários para verificar se esta abordagem pode ser útil em estudos de ADEB, e consequentemente na descoberta de novos poluentes atmosféricos. / The atmospheric particulate matter is recognized as mutagenic. Although several compounds have been identified in this matrix, they do not explain the observed effect in most bioassays that have been performed. The Salmonella/microsome assay has been widely used for the evaluation of atmospheric samples, The assay is very sensitive in the detection of various types of mutagens, because it is performed with different strains of Salmonella typhimurium in the presence and absence of exogenous metabolic activation. The main objective of this work was to verify the applicability of selective combination of strains in the characterization of mutagenicity of total airborne particulate matter collected in Limeira, a São Paulo State city in a site influenced by heavy traffic, industrial and agricultural emissions, including sugar cane plantations. We tested six extracts obtained with methylene chloride by ultrasonication using the Salmonella/microsome microsuspension protocol in the presence and absence of rat liver S9 in dose response experiments, using 50 µg of extractable organic matter (EOM) as a maximum dose per plate. The following pairs of strains were used: YG1041/TA98, to verify the presence of nitro-compounds, as 1-nitropyrene and/or aromatic amines; YG5161/TA1538, highly sensitive to non-substituted polycyclic aromatic hydrocarbons (PAHs) as benzo[a]pyrene (BaP); and YG7108/TA1535, for the indication of alkylating agents. The results indicated that the compounds that can better explain the observed effect are the nitro and/or aromatic amines and not nonsubstituted PAHs such as BaP. The pair YG7108/TA1535 provided negative responses for all the samples. Selective combinations of S. typhimurium strains (YG1041/TA98 and YG5161/TA1538) proved efficient in the characterization of mutagenicity of airborne samples collected in the city of Limeira and can be used as an important tool for the Salmonella/microsome microsuspension assay in effect directed chemical analysis (EDA) studies. The mutagenic potencies observed with TA98 strain for the city of Limeira were similar to those verified in the Metropolitan Region of São Paulo. The comparison of the observed mutagenicity responses for the samples analyzed with the profiles of pure compounds found in the literature did not provide any candidates that could alone represent the mixture. More mutagenicity studies with pure compounds using the proposed pair of strains (TA98/YG1041 and TA1538/YG5161) along with chemical analysis of fractioned extracts are needed to verify if this approach can be useful in EDA studies, and consequently in the discovery of new air priority pollutants.
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Dietary exposure to contaminants during pregnancy and fetal growthDuarte Salles, Talita, 1985- 31 July 2012 (has links)
Introduction: Prenatal exposure to polycyclic aromatic hydrocarbons (PAH) and to acrylamide has been suggested to reduce fetal growth. The role of diet, the main source of exposure to these compounds in the general population, remains uncertain. This thesis aimed to characterize women’s exposure during pregnancy to dietary acrylamide and the genotoxic PAH benzo(a)pyrene [B(a)P], and to assess the effects of prenatal exposure to these compounds on fetal growth indicators.
Methods: This thesis was done within two large European population-based cohort studies conducted in Spain and Norway: the INfancia y Medio Ambiente (INMA; n=657) and The Norwegian Mother and Child Cohort (MoBa; n=50651). Dietary B(a)P and acrylamide intakes were estimated based on information from food frequency questionnaires and the concentrations of these compounds in foods.
Results: (i) Smokers had higher dietary intakes of B(a)P and acrylamide compared to non-smokers; (ii) the main predictors of B(a)P intake were shellfish and processed/cured meats while the main predictors of acrylamide intake were snacks, fried potatoes, and crisp bread; (iii) higher prenatal exposure to dietary B(a)P and acrylamide may reduce birth weight and increase the risk of small for gestational age, independently of smoking-related exposure and (iv) stronger effects of dietary B(a)P on fetal growth were observed among women with low vitamin C intake.
Conclusions: Dietary B(a)P and acrylamide intakes during pregnancy may impair fetal growth. Therefore, reducing the intake of these compounds from the diet should be recommended in dietary guidelines for pregnant women. Likewise, increasing intakes of fruits and vegetables rich in vitamin C should be recommended given its potential to prevent adverse effects from exposure to such contaminants. / Introducción: La exposición prenatal a los hidrocarburos aromáticos policíclicos (HAP) y a la acrilamida ha sido asociada con la reducción del crecimiento fetal. El papel de la dieta, la principal fuente de exposición a estos compuestos en la población general, sigue siendo incierto. Los objetivos de esta tesis son caracterizar la exposición a través de la dieta a la acrilamida y a los HAP, específicamente el compuesto genotóxico benzo(a)pireno [B(a)P], durante el embarazo, y evaluar los efectos de la exposición prenatal a estos compuestos sobre indicadores del crecimiento fetal.
Métodos: Esta tesis se realizó dentro del marco de dos grandes estudios europeos de cohortes de base poblacional realizados en España y Noruega: INfancia y Medio Ambiente (INMA; n=657) y The Norwegian Mother and Child Cohort (MoBa; n=50651). La ingesta de B(a)P y acrilamida fue estimada a partir de información de cuestionarios de frecuencia alimentaria y las concentraciones de estos compuestos en los alimentos.
Resultados: (i) Las fumadoras tuvieron mayor ingesta de B(a)P y acrilamida a través de la dieta en comparación con las no fumadoras; (ii) los principales predictores de la ingesta de B(a)P fueron los mariscos y los embutidos, mientras que los principales predictores de la ingesta de acrilamida fueron los aperitivos, las patatas fritas y el pan crujiente; (iii) la exposición prenatal al B(a)P y la acrilamida pueden reducir el peso al nacer y aumentar el riesgo de pequeño para la edad gestacional, independientemente de la exposición relacionada con el tabaco y (iv) los efectos de la ingesta de B(a)P a través de la dieta sobre el crecimiento fetal fueron más fuertes entre las mujeres con baja ingesta de vitamina C.
Conclusiones: La ingesta de B(a)P y acrilamida a través de la dieta durante el embarazo, puede perjudicar el crecimiento fetal. Consecuentemente, recomendaciones para la reducción de la ingesta de estos compuestos a través de la dieta deberían ser incluidas en las guías dietéticas para mujeres embarazadas. Asimismo, debería recomendarse un aumento en la ingesta de frutas y verduras con alto contenido en vitamina C por su potencial para prevenir efectos relacionados con la exposición a estos contaminantes.
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Towards Development of an Immunoassay Utilizing Circularly Permutated Proteins to Detect Environmental ContaminantsZunnoon Khan, Sara 29 August 2013 (has links)
A fusion protein composed of antibody fragments and β-lactamase was earlier created by Kojima et al. (2011), with antigen specificities against a bone disease marker and a pesticide. The enzyme was circularly permutated and fused to the variable heavy and light chain antibody fragments, thereby ensuring inactivity until binding of the target antigen triggered enzyme
activation. Upon activation, the β-lactamase produced a colorimetric signal, which indicated antigen presence. In this work, a similar strategy was used to create two novel fusion proteins composed of circularly permuted β-lactamase and superfolder green fluorescent protein with anti-benzo[a]pyrene variable antibody fragments. The fusion proteins were designed and expressed in E. coli for the development of a single-step visual immunoassay. It was hypothesized that the cp reporter proteins would be activated once the binding of B[a]P to the variable antibody fragments occurred, and this interaction was expected to produce a detectable colorimetric or fluorescent signal. Although positive results were obtained in one instance, substantial supportive evidence in favour of the hypothesis could not be obtained. / SENTINEL Bioactive Paper Network, Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Research Chairs Program.
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Étude du continuum exposition-effet cancérogène du benzo[a]pyrèneMoreau, Marjory 12 1900 (has links)
Le benzo[a]pyrène (BaP) est un contaminant environnemental de la famille des hydrocarbures aromatiques polycycliques ayant été classé cancérogène chez l’humain. Cependant, la relation entre l’exposition et les effets est toujours mal documentée. L’objectif de cette thèse était de mieux documenter la relation quantitative entre l’exposition au BaP, l’évolution temporelle des biomarqueurs d’exposition et l’apparition d’altérations biologiques précoces, à partir d’études expérimentales chez le rat. Dans un premier temps, nous avons déterminé l’effet de 4 doses de BaP (0.4, 4, 10 et 40 µmol/kg) sur plusieurs biomarqueurs d’exposition (3- et 7-OHBaP, 4,5- et 7,8-diolBaP, BaPtétrol et 1,6-, 3,6- et 7,8-diones-BaP), les adduits à l’ADN et l’expression de gènes impliqués dans le métabolisme du BaP, la réparation de l’ADN et le stress oxydatif. Le BaP et ses métabolites ont été mesurés dans le sang, les tissus et les excrétas, 8 h et 24 h après l’administration intraveineuse de BaP par chromatographie liquide à ultra haute performance (UHPLC) couplée à la fluorescence. Les adduits à l’ADN ont été quantifiés dans les poumons par immuno-essai en chémoluminescence. L’expression des gènes dans les poumons a été réalisée par PCR quantitative en temps réel (qRT-PCR). Les résultats ont révélé une bonne relation dose-excrétion pour le 3-OHBaP, le 4,5-diolBaP et le 7,8-diolBaP et ils ont également renforcé l’utilité du 4,5-diolBaP comme potentiel biomarqueur du BaP en plus du 3-OHBaP. De plus, l’augmentation dose-dépendante de la formation des adduits et de l’expression de certains gènes impliqués dans le métabolisme et le stress oxydatif a suggéré l’intérêt de ces derniers comme biomarqueurs d’effet précoce. Dans un second temps, nous avons évaluer le profil cinétique des biomarqueurs en lien avec la modulation temporelle de la formation des adduits à l’ADN et de l’expression génique, en utilisant la dose de 40 µmol/kg de BaP telle qu’établie dans l’étude précédente, avec une série de mesures sur une durée de 72 h après l’injection intraveineuse de BaP. Il est apparu que le 3- et le 7-OHBaP ainsi que le 4,5- et le 7,8-diolBaP semblaient être de bons biomarqueurs d'exposition; les hydroxyBaP et diolBaP présentaient des cinétiques différentes, mais tous ces métabolites étaient corrélés de façon significative aux adduits BaPDE dans les poumons. L’expression de certains gènes et l’étude de leur profil cinétique a également permis de faire des liens avec la formation des adduits et de mieux comprendre le métabolisme du BaP. Après ces résultats, il semblait alors logique de s’intéresser à l’effet de la voie d’exposition dans un context d’exposition multiple de la population. Les données mesurées dans le sang et les excréta, après administration de 40 µmol/kg de BaP par voie intraveineuse, intratrachéale, orale, et cutanée, ont encore une fois montré l'intérêt de mesurer plusieurs métabolites pour l’évaluation de l’exposition en raison des différences en fonction de la voie d’administration du composé et des différences dans la cinétique de plusieurs biomarqueurs, notamment entre les hydroxy (3- et 7-OHBaP) et les diols-BaP (4,5- et 7,8-diolBaP). Les résultats suggèrent aussi que la mesure de ratios de concentrations de différents métabolites pourrait aider à indiquer le moment et la principale voie d’exposition. Ces données ont permis une meilleure compréhension du continuum entre l’exposition et les effets. / Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that has been classified as carcinogenic to humans. However, the relationship between exposure and effect is still poorly documented. The aim of this thesis was to document the quantitative relationship between exposure to BaP, the temporal evolution of biomarkers of exposure and the appearance of early biological alterations, by conducting experimental studies in rats. First, we determined the effect of four doses of BaP (0.4, 4, 10 and 40 µmol / kg) on several biomarkers of exposure (3- and 7-OHBaP, 4,5- and 7,8 -diolBaP, BaPtetrol and 1,6 -, 3,6 - and 7,8-dione-BaP), on DNA adducts and the expression of genes involved in the metabolism of BaP, DNA repair and oxidative stress. BaP and its metabolites were measured in blood, tissues and excreta, 8 h and 24 h after intravenous administration of BaP by ultra high performance liquid chromatography (UHPLC) coupled to fluorescence. The DNA adducts were quantified in lungs by chemiluminescence immunoassay. Gene expression in lungs was achieved by quantitative real time PCR (qRT-PCR). The results showed a good dose-excretion relationship for 3- OHBaP , 4,5- and 7,8- diolBaP and they also showed the usefulness of 4,5- diolBaP as a potential biomarker of BaP in addition to 3- OHBaP. Furthermore, the dose-dependent increase in the formation of adducts and the expression of certain genes involved in metabolism and oxidative stress highlighted the latter as potentially interesting biomarkers of effect. The following study was designed to evaluate the toxicokinetic profile of biomarkers of exposure related to the temporal modulation of the formation of DNA adducts and gene expression, using a dose of 40 µmol/kg BaP as set out in the previous study, but with regular measurements during the 72-h period following intravenous injection of BaP. It appeared that the 3 - and 7- OHBaP and 4,5 - and 7,8- diolBaP seemed to be good biomarkers of exposure; hydroxyBaPs and diolBaPs exhibited different kinetics but all the metabolites were significantly correlated with BaPDE adducts in the lungs. The expression of genes and the study of their kinetic profile also allowed to assess the relationship with the formation of adducts and better understand the metabolism of BaP. Based on these results, it seemed logical to focus on the effect of the route of exposure. The data measured in the blood and excreta after intravenous, intratracheal, oral, and cutaneous administration of 40 mmol/kg BaP have once again demonstrated the importance of measuring several metabolites due to kinetic differences depending on the route of administration of the compound and among biomarkers, in particular between OHBaPs (3 - and 7 - OHBaP) and diolBaPs (4,5 - and 7,8- diolBaP). Results also suggest that measurements of concentration ratios of different metabolites could help indicate the time and the main route of exposure. Overall, these data allowed a better understanding of the continuum between BaP exposure and early effects.
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The relationship between dietary factors, meat consumption, heterocyclic amines, Benzo[a]pyrene, meat-derived mutagenic activity and colorectal cancer in Western AustraliaTabatabaei, Seyed Mehdi January 2009 (has links)
The role of meat consumption in the development of cancer, including colorectal cancer (CRC), has been subject of much investigation in recent years. The observation of geographical variation in CRC incidence and increased CRC risks in populations consuming high levels of meat prompted researchers to hypothesise a link between meat and CRC. An area of particular interest in CRC pathogenesis is the meat-derived compounds such are heterocyclic amines (HCAs), polycyclic aromatic hydrocarbons (PAHs), and meatderived mutagenic activity. Australia is among the countries with high incidence of CRC and also high levels of per capita meat consumption. Hence, clarifying the possible link between meat consumption and the risk of CRC in order that this can be translated into preventive dietary recommendations for the public is important. The objective of this thesis was to examine whether meat consumption is related to risk of CRC in an Australian population. The term meat consumption in this thesis means meaures of consumption of red and white meat that incorporate frequency and cooking method. The following hypotheses were investigated: 1. Increasing intake of meat prepared by methods that involve higher cooking temperature and time is positively associated with the risk of CRC; 2. Increasing exposure to meat-derived heterocyclic amines (HCAs) is positively associated with the risk of CRC; 3. Higher levels of exposure to polycyclic aromatic hydrocarbons (PAHs) from meat consumption is a risk factor for CRC; 4. Exposure to meat-derived mutagens increases the risk of CRC.
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