Browsing by Author "Sacadura, Joana"
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- Assessment of the Genotoxic Hazard of Estuarine Sediments Using an Integrative Approach With LacZ Plasmid‐Based Transgenic MicePublication . Pinto, Miguel; Sacadura, Joana; Costa, Pedro M.; Caeiro, Sandra; Louro, Henriqueta; Silva, Maria J.Under the influence of multiple anthropogenic pressures, from industrial to agricultural activities, estuaries have long been regarded as particularly sensitive ecosystems to contamination. The present study aimed at investigating the genotoxic potential of a contaminated sediment sample from an urban and industrial area of the Sado Estuary, by combining the analysis of multiple endpoints in the LacZ plasmid‐based transgenic mouse model exposed for 28 days to contaminated estuarine sediment extracts through drinking water. The DNA and chromosome damaging effects were monitored in peripheral blood at 7‐day intervals using the standard and enzyme‐modified Comet assay, as well as the micronucleus assays in peripheral blood cells. After euthanasia, DNA damage was analyzed in several mouse tissues, and LacZ mutant frequencies were determined in the liver. Livers were also surveyed for histopathological analysis. A time‐dependent increase in micronuclei frequency was seen at all tested doses, in spite of no induction of DNA damage in any organ or mutation induction in the liver of exposed mice. The liver from mice exposed to sediment extracts did not reveal major alterations besides evidence of inflammation. Overall, the integration of the endpoints analyzed in the mice is suggestive of potential chronic, rather than acute, adverse effects in vivo, and points to the need for further research in the resident human population in the area. This experimental design can be used to assess the genotoxicity of complex environmental mixtures, understand how they work, and reduce costs and resources while speeding up data collection and interpretation.
- DNA and Chromosome Damaging Effects in Mice Exposed to an Estuary Sediment ExtractPublication . Pinto, Miguel; Sacadura, Joana; Louro, Henriqueta; Costa, Pedro Manuel; Lavinha, João; Silva, Maria JoãoPrevious 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.
- Evaluation of Genotoxicity of Sediments from the Sado-River Estuary Using Solvent Extractions of Diferent PolaritiesPublication . Vicente, Ana Margarida; Sacadura, Joana; Pinto, Miguel; Louro, Henriqueta; Costa, Pedro Manuel; Lavinha, João; Caeiro, Sandra; Silva, Maria JoãoLocated 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.
- Genotoxicity and oxidative stress induced by sediments from the Sado Estuary and potential antimutagenic effects of quercetinPublication . Pinto, Miguel; Sacadura, Joana; Louro, Henriqueta; Costa, Pedro Manuel; Costa, Maria Helena; Caeiro, Sandra; Lavinha, João; Silva, Maria JoãoThe 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.
- HERA - Environmental Risk Assessment of a contaminated estuarine environment: a case studyPublication . Martinho, Ana Paula; Rodrigo, Ana; Vicente, Ana; Machado, Ausenda; Dias, Carlos Matias; Guiomar, Carla Sofia; Gonçalves, Cátia; Paixão, Eleonora; Santos, Fernanda; Louro, Henriqueta; Pinhal, Hermínia; Mateus, Inês; Coelho, Inês; Lopes, Inês; Castanheira, Isabel; Antunes, Joana; Sacadura, Joana; Lavinha, João; Toro, Joaquim; Lobo, Jorge; Nunes, Luís; Quintas, Maria do Carmo; Costa, Maria Helena; Silva, Maria João; Saraiva, Marina; Diniz, Mário; Martins, Marta; Pinto, Miguel; Fernandes, Paula Vaz; Costa, Pedro M.; Caeiro, Sandra; Gueifão, Sandra; carreira, Sara; Silva, Susana Pereira; Neuparth, TeresaSado 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.
- Integrated investigation of the genotoxicity of a contaminated estuary sediment extract using LacZ plasmid-based transgenic micePublication . Pinto, Miguel; Louro, Henriqueta; Sacadura, Joana; Costa, Pedro M.; Caeiro, Sandra; Silva, Maria JoãoAs 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.
- Toxicity of environmental pollutants: from the bench research to human biomonitoring studiesPublication . Pinto, Miguel; Sacadura, Joana; Louro, Henriqueta; Costa, Pedro Manuel; Lavinha, João; Costa, Maria Helena; Caeiro, Sandra; Silva, Maria JoãoBeing 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.