Caracterização do potencial genotóxico de sedimentos estuarinos em sistemas experimentais in vitro e in vivo

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2013O estuário do Sado (Oeste de Portugal) está sujeito a várias fontes de poluição associadas a centros urbanos, industrias pesadas, actividades mineiras e agricultura. Este estuário permanece um local importante de actividade pesqueira para consumo próprio e para mercados locais e externos, assim com para actividades recreativas e turísticas. No entanto, estudos anteriores revelaram vários contaminantes presentes nos sedimentos aquáticos, maioritariamente metais, pesticidas e hidrocarbonetos aromáticos policíclicos. Estes compostos podem ser absorvidos e acumulados nas partes comestíveis das espécies estuarinas e nos produtos agrícolas locais entrando, deste modo, na cadeia alimentar humana, colocando em risco a saúde humana. Neste contexto, o presente estudo tem como objectivo analisar os efeitos genotóxicos, in vitro e in vivo, de extractos de sedimentos do estuário do Sado. Para esse fim, os sedimentos foram recolhidos de quatro locais de pesca (P, C, A e E) do estuário e os contaminantes orgânicos e inorgânicos totais foram extraídos com uma mistura de metanol:diclorometano. Células HepG2 foram expostas aos extractos e a um agente antimutagénico, para a análise da genotoxicidade utilizando o ensaio do cometa, a fim de analisar a possível reversão de danos oxidativos induzidos pelos contaminantes. Adicionalmente, recorreu-se a um modelo de ratinhos transgénicos baseados no plasmídeo lacZ expostos por via oral a duas doses de extracto P para testes de genotoxicidade in vivo. Neste modelo foi realizado o ensaio do micronúcleo, do cometa e de mutações em vários órgãos de ratinho. Nos resultados in vitro não se verificou a reversão dos danos oxidativos induzidos pelos extractos de sedimentos por parte da quercetina, nas condições testadas. In vivo, verificou-se a indução de micronúcleos, embora o ensaio do cometa não revelasse resultados positivos. Aparentemente, não se verificou mutagenecidade in vivo, embora sejam necessários mais estudos para concluir a análise de mutações. Globalmente, os resultados obtidos neste estudo reflectem o potencial efeito adverso para a saúde humana associado aos contaminantes encontrados nos sedimentos do estuário, contribuindo para a avaliação de risco.The Sado Estuary (West Portugal) is subject to several sources of pollution associated with an urban center, heavy-industries, mining activities and agriculture. It remains a site for fishing activity for personal consumption and for local and external market, and also for recreational and touristic activities. Previous studies revealed several contaminants in the aquatic sediments, mostly metals, pesticides and polycyclic aromatic hydrocarbons. These compounds can be absorbed and accumulated in edible parts of estuarine species and in local agriculture products, thus entering the human food chain and posing a risk to human health. In this context, this study aims at analyzing the genotoxic effects, in vitro and in vivo, of sediment extracts from the Sado Estuary. For this end, sediments were collected from four fishing sites (P, C, A and E) of the Estuary and total organic and inorganic contaminants were extracted with a mixture of methanol:dichloromethane. HepG2 cells were exposed to the extracts and an antimutagenic agent, for genotoxicity analysis using the comet assay, in order to see whether there was a reversion of the contaminant-induced oxidative damage. Furthermore, a lacZ plasmid-based model of transgenic mice was orally exposed to two doses of extract P for in vivo genotoxicity testing. For the in vivo analysis, the micronucleus assay, comet assay and mutant frequency analysis were performed in several mouse tissues. In vitro results failed to elicit a potential reversion of the contaminant-induced oxidative damage by quercetin, in the tested conditions. In vivo, there was a positive induction of micronuclei, however there was no positive results in comet assay. Overall, the results obtained in the present study reflect the potential hazard effect to human health associated with the contamination of estuarine sediments, contributing to risk assessment

Caracterização e modelação de reservatórios carbonatados: estudo de caso baseado em análogos do Jurássico Médio da região de Vale Florido, Maciço Calcário Estremenho (MCE)

Tese de mestrado em Geologia, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016O presente trabalho visou dois objectivos: i) caracterizar uma formação do Jurássico Médio na região do Vale Florido (Maciço Calcário Estremenho), como análoga de um reservatório carbonatado, analisando as unidades a diversas escalas tendo em especial atenção as litofácies, petrografia, diagénese, estratonomia, geometria dos corpos sedimentares e heterogeneidades (em especial das propriedades petrofísicas do reservatório – porosidade e permeabilidade); ii) ensaiar a conjugação da análise à escala da rocha com a construção de modelos de reservatório que prevejam a distribuição dos geobodies e das suas propriedades petrofísicas, contribuindo para os aferir e aperfeiçoar. Este reservatório no seu todo tem baixa porosidade e permeabilidade, com excepções em certos intervalos, tendo sido amplamente influenciado pela diagénese. A maioria dos níveis classificam-se segundo Ahr (2008) como reservatório diagenéticos ou híbridos 1. Observam-se três litofácies definidas por Azerêdo (1993, 1998) no afloramento do Vale Florido: litofácies 1 (L1) – calcários oolíticos (que se subdividiu neste trabalho em L1a - sem estratificação ou com estratificação planar e L1b – com estratificação grosseira de vário tipos, como seja, ripple marks, estratificação cruzada, etc); litofácies 2 (L2) – calcários calciclásticos e litofácies 3 (L3) – biostromas ou biolititos de corais. A litofácies com maior porosidade associada é a litofácies 1 (em especial 1b). A maior permeabilidade não se relaciona com a fácies nem com a porosidade mais comum, mas sim com determinados processos de dissolução tardia, mais especificamente conducente à dissolução ao longo de estilólitos e à criação de fracturas. O afloramento foi dividido em sete sectores (A, B, C, D, E, F e G). Conclui-se deste estudo que os níveis com maior potencial seriam o D3 – nível oolítico com ripple marks e estratificação obliqua (L1b), o F2,75 – nível oolítico com estratificação oblíqua (L1b) e o E2 – nível biostrómico (L3), observando-se porosidade de canal em todos eles, e possivelmente o nível D5 – nível biostrómico (L3), onde é possível observar em lâmina delgada poros vacuolares (porosidade vuggy) de dimensão razoável e que parecem conectados. A maioria dos níveis seriam também excelentes selos, uma vez que a sua porosidade e permeabilidade são reduzidas. A geometria dos geobodies vista em campo, e posteriormente introduzida e extrapolada no software de modelação (Petrel), permite também verificar o potencial como armadilhas estratigráficas, tendo os corpos biostrómicos muitas vezes configurações lenticulares e os corpos oolíticos variações laterais/interdigitações para níveis mais clásticos/tempestíticos, isolando-se assim os níveis com maior permeabilidade.The present work aimed at two objectives: i) characterize a Middle Jurassic formation in Vale Florido region (Maciço Calcário Estremenho) as an anologue for a carbonate reservoir, analyzing the units at different scales, with particular focus in lithofacies, petrography, diagenesis, stratonomy, geometry of sedimentary bodies and heterogeneities (especially of the petrophysical properties of the reservoir - porosity and permeability); ii) evaluate the combination of analysis at rock scale and construction of reservoir models that estimate the distribution of geobodies and their petrophysical properties, contributing to their improvement. This reservoir as a whole has low porosity and permeability, with a few exception, having been widely influenced by diagenesis. Most levels are classified according to Ahr (2008) as diagenetic reservoir or hybrid 1. Three lithofacies defined by Azerêdo (1993, 1998) are observed in Vale Florido outcrop: lithofacies 1 (L1) – oolitic limestones (which was divided in this work in L1a – without stratification or with planar layering and L1b – with coarse stratification of various types, such as, ripple marks, cross bedding, etc); lithofacies 2 (L2) – calciclastic limestones and lithofacies 3 (L3) – biostromes or coral biolithites. The lithofacies with higher associated porosity is lithofacies 1 (especially 1b). The highest permeability is not related to facies nor the most common porosity, but with certain late diagenetic processes, more specifically processes that lead to dissolution along stylolites and fracture creation. The outcrop was divided into seven sectors (A, B, C, D, E, F and G). From this study it can be concluded that the levels with the greatest potential as reservoir would be the D3 – oolitic level with ripple marks and cross sets (L1b), F2,75 – oolitic level with cross bedding and E2 – biostrome level (L3), in all of which channel porosity is observed, and possibly D5 – biostrome level (L3) where one can observe, in thin section, vuggy pores of reasonable size and that seem connected. Most levels would also be excellent seals since their porosity and permeability is low. The geometry of the geobodies seen in the outcrop, and then introduced and extrapolated in the modeling software (Petrel), also allows to verify the potential as stratigraphic traps. The biostrome bodies often have lenticular configuration and the oolitic bodies display lateral variations/interfingering with more clastic/tempestite levels, thus isolating the levels with increased permeability

Evaluation of Genotoxicity of Sediments from the Sado-River Estuary Using Solvent Extractions of Diferent Polarities

Located in the western coast of Portugal, our case study, the river Sado Estuary, is affected by various sources of pollution, such as heavy-industry, urbanism, mining, agriculture and maritime traffic. Recent studies showed that sediment samples were contaminated with a mixture of different groups of contaminants related to the different anthropogenic pressures along the estuary. The present study aims to assess the cytotoxic and genotoxic potential of these sediments following a fractioning method, in order to elucidate whether their toxicity can be attributed to a particular group of contaminants, or is rather the result of the complex interaction of contaminants. Sediment samples were collected from four distinct and contaminated sites of the Sado Estuary: sites C and P from the urban/industrial area and sites E and A from the riverine/agriculture area. Organic and inorganic contaminants were extracted with solvents of increasing polarity n-hexane < dichloromethane < methanol and a mixture (DCM:methanol), and recovered in DMSO. Cytotoxicity and genotoxicity were evaluated through the neutral red and comet (coupled with FPG) assays, respectively, in HepG2 cells. Cells were exposed for 48h to concentrations of each extract ranging from 1 to 200 mg SEQ/ml. Cytotoxicity was only observed for extracts PDCM/met, EDCM/met and Pmet. Sediment sample C failed to induce genotoxicity. A significant increase in the level of DNA damage was observed for sub-cytotoxic concentrations of PDCM/met, EDCM/met and Pmet. DNA damage was accentuated following treatment with FPG, suggesting oxidative DNA damage, mainly for DCM/met extractions of all samples except C, as well as Phex and Pmet. Complex toxicant mixtures are present in estuarine sediments which often makes the association between surveyed contaminants and toxicity difficult to establish. In this study, genotoxic effects were observed in extracts obtained with the mixture of solvents (DCM:met). Nevertheless, when different extractions were performed with solvents of different polarities, the effects of the different fractions were mainly diluted, or more weakly expressed, suggesting that the interaction between contaminants, and not a set of particular contaminants, might be responsible for the observed effects. Also, we suggest that oxidative DNA damage, revealed by the FPG enzyme, might be a common effect of the exposure to these environmental contaminants. Together with contamination analysis, these results are expected to disclose the genotoxic potential of sediment sample extracts in a human cell line, in order to derive a potential risk to human health

HERA - Environmental Risk Assessment of a contaminated estuarine environment: a case study

Sado River estuary is located in the west coast of Portugal. Previous environmental studies identified industrial contamination, non-point anthropogenic sources and contamination coming from the river, all promoting accumulation of polluted sediments with known impacts on the ecological system. Surrounding human populations have intense economic fishery activities. Together with agriculture, estuary fishing products are available to local residents. Food usage previously characterized through ethnographic studies suggests exposure to estuarine products, farming products, and water in daily activities, as potential routes of contamination. It is well established that long term exposure to heavy metals are associated with renal and neurological diseases, most heavy metals are classified as carcinogenic and teratogenic.Instituição Financiadora: FCT; Instituições participantes: IMAR -Instituto do Mar (coord.)e PRÓ-INSA, Associação para a Promoção da Investigação em Saúde, Instituto Nacional de Saúde Doutor Ricardo Jorg

Integrated investigation of the genotoxicity of a contaminated estuary sediment extract using LacZ plasmid-based transgenic mice

As targets for diverse anthropogenic activities, estuaries are reservoirs for a variety of pollutants. Recent studies showed that sediments from a Portuguese Estuary were cytotoxic and genotoxic in a human liver cell line and in local aquatic species, possibly due to the presence of PAHs and metals. However, the extrapolation of ecological risk to risk to human health is difficult and in vivo studies are crucial to better reflect human exposure and effects. This study aimed at investigating the genotoxic potential of a contaminated sediment sample from a local fishing area of the Estuary, combining the analysis of multiple endpoints in the LacZ plasmid-based transgenic mouse model. Groups of LacZ mice were exposed through drinking water to two dilutions of an estuarine sediment extract from an impacted site, as well as to solvent control, for 28 days. The DNA and chromosome damaging effects were monitored in peripheral blood at 7 day intervals using the Comet (plus DNA repair endonucleases) and Micronucleus (MN) assays in peripheral blood cells. After euthanasia, DNA and oxidative DNA damage were analyzed in several mouse organs, and LacZ mutant frequency was determined in liver. The results showed a time-dependent increase in MN frequency for each treated group. In contrast, no induction of DNA or oxidative DNA damage was observed in any organ, irrespectively of the dose; likewise, no mutation induction was detected in the liver of exposed mice. Overall, the increase in the MN frequency in mice orally exposed to the sediment extract is suggestive of adverse effects on human health, and points to the need of further research in the human population resident in the affected area

DNA and Chromosome Damaging Effects in Mice Exposed to an Estuary Sediment Extract

Previous studies have shown that an extract of a sediment sample collected in a fishing area of Sado Estuary, impacted by the urban and industrial pollution from the city of Setúbal, was able to induce cytotoxicity and genotoxicity in a human cell line (HepG2) and in local aquatic species, probably due to the presence of PAHs and metals. However, the assessment of the potential hazard of those contaminants to humans, through extrapolation of the in vitro data, is difficult and thereby in vivo studies are crucial to better reflect human exposure and effects. This study aims to assess the in vivo DNA and chromosome damaging potential of a contaminated sediment sample from a local fishing area of the Sado Estuary. Groups of mice (n=6) were exposed, through drinking water, to two concentrations of a sediment extract (1 and 2 g SEQ/ml), as well as to solvent control, for a 28 day period. The DNA and chromosome damaging effects were monitored at 7 day intervals by the comet assay (coupled with DNA repair endonucleases FPG and ENDO III) and the micronucleus assays, performed in blood leukocytes and immature erythrocytes, respectively. After sacrifice, DNA lesions, oxidative damage and histopathological biomarkers (apoptosis and inflammation) were analysed in liver, spleen and kidney cells. Preliminary results showed that at days 7, 14 and 21, mice exposure to the highest extract dose caused a significant induction on the frequency of micronuclei comparatively to the unexposed group (p < 0.001), while exposure to the lowest dose raised the micronucleus frequency only at day 14 (p = 0.003). No induction of DNA damage or oxidative DNA damage was observed in blood cells, at the 2 first timepoints. The results of DNA damage and micronucleus quantification at the 28 days timepoint, together with data from histopathological biomarkers, will be integrated and discussed in view of the contaminants present in the sediment sample under study. Overall, the results obtained in the present in vivo study are expected to reflect the potential hazard to human health associated to the contamination of estuary water and sediments and to contribute to risk assessment

Genotoxicity and oxidative stress induced by sediments from the Sado Estuary and potential antimutagenic effects of quercetin

The Sado Estuary is affected by various sources of pollution, such heavy-industry, urbanism, mining, agriculture and maritime traffic and sizable amounts of organic and inorganic contaminants were identified in the sediments. These compounds can be accumulated in the edible parts of estuarine species and agricultural products, thus entering the human food chain and posing a public health problem. This study had two objectives: i) to study genotoxic effects of sediments from the Sado Estuary in a human liver-derived cell line; and ii) to analyze oxidative DNA damage (produced by the same samples) and try to reverse it by treatment with quercetin, as an antioxidant. Sediments were collected from four distinct sites of the Sado Estuary: Sites P and C from the northern area and sites A and E from the southern area. Contaminants were extracted with dichloromethane:methanol (2:1) and genotoxicity was evaluated by the comet assay; oxidative damage was quantified using the DNA repair endonuclease FPG-modified comet assay. HepG2 cells were exposed (48h) to concentrations of each extract (10 - 200 mg SEQ/ml). To confirm oxidative stress, cells were co-exposed for 48h to the concentrations of each extract that were able to produce oxidative DNA damage. A significant increase in total DNA strand breakage was observed following cells exposure to extract P (with and without FPG). Significant DNA damage was only observed following FPG treatment for extracts E and A, suggesting induction of oxidative DNA damage. Extract C failed to induce genotoxicity. Co-exposure to quercetin did not reverse the observed oxidative DNA damage, but rather increased it, suggesting a possible co-mutagenicity. The differential genotoxicity observed in samples from the northern (P) and southern areas (E and A) of the Sado Estuary probably reflects different pressures from an industrialized and urban area versus an agricultural area, respectively

Toxicity of environmental pollutants: from the bench research to human biomonitoring studies

Being subjected to diverse anthropogenic pressures, from industrial to agricultural activities, estuaries have long been regarded as ecosystems particularly sensitive to contamination. A recent study addressed the potential adverse effects of the Portuguese Sado river sediment contaminants on human and ecosystem health. Several complementary approaches were used to assess the effects on human health, including: (i) an epidemiological one to characterize the exposure pathway to the estuary environment; (ii) an in vitro characterization of the genotoxic potential of sediments’ contaminants in a human cell line; (iii) an in vivo study of gene and chromosome alterations induction in LacZ plasmid-based transgenic mice. The epidemiological survey confirmed the occurrence of direct and indirect (through food chain) exposure of the local population to estuarine contaminants. On the other hand, the complex mixture of contaminants extracted from sediments, which contain metals and polycyclic aromatic hydrocarbons, was toxic to human liver cells exposed in vitro, causing cell death, oxidative stress and genetic damage [3, 4]. Furthermore, the results of an in vivo study showed a time-dependent increase in chromosome damage in blood immature erythrocytes but did not show induction of mutations in liver cells or DNA damage in blood, spleen, kidney or liver cells of exposed comparatively to control mice. Although the histopathological analysis of liver tissues did not reveal specific alterations associated with exposure, changes observed in hepatocytes structures indicated that liver function related to carbohydrate metabolism and storage was compromised, therefore revealing an important chronic effect. Overall, the complementary approaches used in this study suggested that the exposure of local populations to the Sado river estuary contaminants may have a negative impact on their health. Nevertheless, there is still a need to perform a biomonitoring study, i.e., obtain and integrate data from exposure and from cellular and molecular biomarkers of early biological effects and susceptibility in the exposed comparatively to a control population, in order to try to derive an indicator of risk of estuary-associated chronic diseases.N/