| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| 1.45 MB | Adobe PDF |
Authors
Abstract(s)
Sendo a poluição dos recursos hídricos um problema a nível global, é de extrema importância o estudo e controlo dos seus poluentes. No caso do Estuário do rio Sado, situado na zona litoral Oeste de Portugal, estudos prévios indicaram a existência de hidrocarbonetos aromáticos policíclicos, pesticidas e metais pesados nos sedimentos, com distribuição heterogénea. Relativamente à genotoxicidade dos sedimentos, verificou-se que a mistura de poluentes extraídos das amostras da área Sul tinha a capacidade de induzir lesões na molécula do ADN enquanto os poluentes das amostras da área Norte causavam, principalmente, alterações permanentes nos cromossomas.
Um dos ensaios mais utilizados para a caracterização do potencial mutagénico de amostras ambientais, incluindo águas superficiais, é o teste de Ames que permite identificar substâncias com potencial para induzir mutações génicas do tipo frameshift ou por substituição dos pares de base. Este teste consiste na utilização de estirpes de Salmonella typhimurium com mutações em vários genes do operão da histidina, sendo incapazes de crescer e formar colónias na ausência deste aminoácido. Quando expostas a uma substância (ou mistura) que possa induzir a formação de mutações, a função do operão é restaurada, tornando-se assim as bactérias independentes da presença de histidina no meio de cultura. Um aumento do número de colónias formadas relativamente ao controlo negativo será pois indicativo de uma atividade mutagénica.
O presente estudo teve como objetivo a implementação do teste de Ames para a caracterização do potencial mutagénico das águas do Estuário do Sado. Para esse fim desenvolveu-se, numa primeira fase, um método de concentração baseado numa extração em fase sólida (SPE) dos compostos orgânicos e implementou-se o teste de Ames no laboratório, com recurso a compostos reconhecidamente mutagénicos (controlos positivos). Foram colhidas 6 amostras de água distribuídas pelas zonas norte e sul do referido Estuário. Essas amostras (5000 mL), juntamente com uma amostra de água fortificada, foram filtradas e submetidas ao método de concentração, sendo os compostos depois eluídos com diclorometano:metanol (50:50). De seguida, as propriedades mutagénicas dos extratos rediluídos em DMSO foram analisadas através do teste de Ames usando as estirpes TA98 e TA100, com e sem adição do sistema exógeno de metabolização S9 mix. A gama de concentrações utilizada foi de 10, 20 e 40 μL de extrato por placa nas amostras de água reais e 1, 5, 10, 20 e 40 μL de extrato por placa na amostra fortificada.
Os resultados mostraram que, nas condições utilizadas, quer os extratos obtidos a partir das seis amostras de água do Estuário do Sado, quer o da amostra fortificada, não foram capazes de induzir um aumento significativo na frequência de mutantes, pelo que nenhuma das amostras revelou um potencial mutagénico, com recurso ao teste de Ames. Apesar das diversas variáveis a considerar na realização deste estudo que poderão ter influenciado os resultados, considera-se que a implementação do teste de Ames permitiu a realização de uma primeira abordagem à problemática da poluição estuarina e seu impacto na saúde humana, sendo ponto de partida para estudos futuros mais completos.
Given that the pollution of hydric resources is a global problem, the study and control of the pollutants is of utmost importance. In the case of the Sado River estuary, located in the west coastal area of Portugal, previous studies have indicated the existence of polycyclic aromatic hydrocarbons, pesticides and heavy metals in sediment, with a heterogeneous distribution. Regarding the genotoxicity of sediments, it was found that the mixture of pollutants extracted from the samples collected in the South area had the ability to induce lesions in the DNA molecule, while the pollutants from the samples collected in the North area caused, mainly, permanent chromosome alterations. One of the most used assays for the characterization of the mutagenic potential of environmental samples, including surface waters, is the Ames Test, that allows the identification of substances with potential to induce gene mutations, including frameshift and base substitutions. This test consists of using Salmonella typhimurium strains with mutations in various genes within the histidine operon, being unable to grow and form colonies in the absence of this amino acid. When exposed to a substance (or mixture) that can induce the formation of mutations, the operon function is restored, thus making bacteria independent of the presence of histidine in the culture medium. An increase in the number of colonies formed relative to the negative control is therefore indicative of a mutagenic activity. This study aimed to implement the Ames Test for the characterization of the mutagenic potential of the Sado Estuary waters. For this purpose on a first phase, a method of water concentration based on solid phase extraction (SPE) of organic compounds was developed, and the Ames Test was implemented in laboratory, using compounds proven to be mutagenic (positive controls). As to the water samples, six samples were collected, distributed by the north and south areas of Sado Estuary. These samples (5000 mL), together with a fortified water sample, were filtered and subjected to the concentration method, being the compounds then eluted with dichloromethane:methanol (50:50). Then, the mutagenic properties of the extracts rediluted in DMSO were analyzed through the Ames Test using strains TA98 and TA100, with and without the metabolizing exogenous system, S9 mix, addition. The concentration range used was 10, 20 and 40 μL of extract per plate from the real water samples, and 1, 5, 10, 20 and 40 μL of extract per plate from the fortified sample. The results showed that, under the conditions used, both the extracts obtained from the six water samples from the Sado Estuary and the extracts obtained from the fortified sample, were not able to induce a significant increase in the mutant frequency and thus, none of the samples showed a mutagenic potential through the use of Ames Test. Despite the many variables to consider in this study that may have influenced the results, it is considered that the implementation of the Ames Test allowed for a first approach to the estuarine pollution and its impact on human health problem, and therefore can be a starting point for more complete future studies.
Given that the pollution of hydric resources is a global problem, the study and control of the pollutants is of utmost importance. In the case of the Sado River estuary, located in the west coastal area of Portugal, previous studies have indicated the existence of polycyclic aromatic hydrocarbons, pesticides and heavy metals in sediment, with a heterogeneous distribution. Regarding the genotoxicity of sediments, it was found that the mixture of pollutants extracted from the samples collected in the South area had the ability to induce lesions in the DNA molecule, while the pollutants from the samples collected in the North area caused, mainly, permanent chromosome alterations. One of the most used assays for the characterization of the mutagenic potential of environmental samples, including surface waters, is the Ames Test, that allows the identification of substances with potential to induce gene mutations, including frameshift and base substitutions. This test consists of using Salmonella typhimurium strains with mutations in various genes within the histidine operon, being unable to grow and form colonies in the absence of this amino acid. When exposed to a substance (or mixture) that can induce the formation of mutations, the operon function is restored, thus making bacteria independent of the presence of histidine in the culture medium. An increase in the number of colonies formed relative to the negative control is therefore indicative of a mutagenic activity. This study aimed to implement the Ames Test for the characterization of the mutagenic potential of the Sado Estuary waters. For this purpose on a first phase, a method of water concentration based on solid phase extraction (SPE) of organic compounds was developed, and the Ames Test was implemented in laboratory, using compounds proven to be mutagenic (positive controls). As to the water samples, six samples were collected, distributed by the north and south areas of Sado Estuary. These samples (5000 mL), together with a fortified water sample, were filtered and subjected to the concentration method, being the compounds then eluted with dichloromethane:methanol (50:50). Then, the mutagenic properties of the extracts rediluted in DMSO were analyzed through the Ames Test using strains TA98 and TA100, with and without the metabolizing exogenous system, S9 mix, addition. The concentration range used was 10, 20 and 40 μL of extract per plate from the real water samples, and 1, 5, 10, 20 and 40 μL of extract per plate from the fortified sample. The results showed that, under the conditions used, both the extracts obtained from the six water samples from the Sado Estuary and the extracts obtained from the fortified sample, were not able to induce a significant increase in the mutant frequency and thus, none of the samples showed a mutagenic potential through the use of Ames Test. Despite the many variables to consider in this study that may have influenced the results, it is considered that the implementation of the Ames Test allowed for a first approach to the estuarine pollution and its impact on human health problem, and therefore can be a starting point for more complete future studies.
Description
Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016
Keywords
Mutagenicidade Teste de Ames Estuário Água Teses de mestrado - 2016