Browsing by Author "Costa, Pedro Manuel"
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- An integrative assessment to determine the genotoxic hazard of estuarine sediments: combining cell and whole-organism responsesPublication . Costa, Pedro Manuel; Pinto, Miguel; Vicente, Ana M.; Gonçalves, Cátia; Rodrigo, Ana P.; Louro, Henriqueta; Costa, Maria Helena; Caeiro, Sandra; Silva, Maria JoãoThe application of the Comet assay in environmental monitoring remains challenging in face of the complexity of environmental stressors,e.g.,when dealing with estuarine sediments,that hampers the drawing of cause-effect relationships. Although the in vitro The application of the Comet assay in environmental monitoring remains challenging in face of the complexity of environmental stressors, e.g., when dealing with estuarine sediments, that hampers the drawing of cause-effect relationships. Although the in vitro Comet assay may circumvent confounding factors, its application in environmental risk assessment (ERA) still needs validation. As such, the present work aims at integrating genotoxicity and oxidative DNA damage induced by sediment-bound toxicants in HepG2 cells with oxidative stress-related effects observed in three species collected from an impacted estuary. Distinct patterns were observed in cells exposed to crude mixtures of sediment contaminants from the urban/industrial area comparatively to the ones from the rural/riverine area of the estuary, with respect to oxidative DNA damage and oxidative DNA damage. The extracts obtained with the most polar solvent and the crude extracts caused the most significant oxidative DNA damage in HepG2 cells, as measured by the formamidopyrimidine-DNA glycosylase (FPG)-modified Comet assay. This observation suggests that metals and unknown toxicants more hydrophilic than polycyclic aromatic hydrocarbons may be important causative agents, especially in samples from the rural part of the estuary, where oxidative DNA damage was the most significant. Clams, sole, and cuttlefish responded differentially to environmental agents triggering oxidative stress, albeit yielding results accordant with the oxidative DNA damage observed in HepG2 cells. Overall, the integration of in vivo biomarker responses and Comet assay data in HepG2 cells yielded a comparable pattern, indicating that the in vitro FPG-modified Comet assay may be an effective and complementary line-of-evidence in ERA even in particularly challenging, natural, scenarios such as estuarine environments.
- Avaliação do potencial citotóxico e genotóxico de contaminantes de sedimentos do Estuário do Sado numa linha celular humanaPublication . Pinto, Miguel; Costa, Pedro Manuel; Louro, Henriqueta; Costa, Maria Helena; Lavinha, João; Caeiro, Sandra; Silva, Maria JoãoO presente estudo integra-se num projeto mais amplo que visa avaliar o risco ambiental – que inclui os riscos ecológicos e para a saúde humana - associado a este ambiente estuarino contaminado. Em particular, este estudo teve como objetivo caracterizar o potencial citotóxico e genotóxico de sedimentos colhidos em vários locais de pesca do Estuário do Sado numa linha celular humana, tendo em vista uma avaliação de eventuais efeitos nefastos para a saúde humana.
- Can Estuary Sediment Contaminants Interfere with the DNA Repair Capacity of HEPG2 Cells?Publication . Pinto, Miguel; Louro, Henriqueta; Costa, Pedro Manuel; Caeiro, Sandra; SIlva, Maria JoãoEstuarine sediments tend to act as reservoirs of pollutants, many of which are acknowledged genotoxicants and potential carcinogens for humans. In addition, many of these environmental contaminants, particularly metals, have the potential to interfere with DNA repair mechanisms. Taking an impacted estuary as a case study (the Sado, SW Portugal), previous studies showed that human hepatoma cells (HepG2) exposed to extracts of sediments collected from two areas (urban/industrial and riverine/agricultural), both contaminated by distinct mixtures of organic and inorganic toxicants, revealed differential cytotoxic and genotoxic effects, consistent with contamination indices. In this context, the present study aimed at determining whether contaminants present in sediment extracts are able to interfere with the DNA repair mechanisms of HepG2 cells. Organic and inorganic contaminants were extracted (methanol:dichloromethane) from sediment samples collected in different sites of the Sado Estuary, either heavily impacted by an urban/industrial environment (site P) or by agriculture (sites E and A); a sediment collected in a potentially “clean” site was also included (site C). The repair capacity of HepG2 cells towards ethyl methanesulfonate (EMS, 5 - 40 mM, 1h) induced DNA lesions was assessed after cells recovery in the presence of non-genotoxic concentrations of each sediment extract, during 24h and 48h, using the comet assay. Negative and solvent controls were included. Preliminary results show that at 24h and 48h after exposure, 60% and 85% of EMS-induced DNA strand breakage, respectively, was spontaneously repaired by HepG2 cells. However, exposure to extracts P and A after EMS challenge, partially inhibited DNA repair given that, relatively to the spontaneous DNA repair level, 20 and 30%, of DNA damage remained unrepaired, after 24h whereas 30 and 40% remained unrepaired after 48h. Extract E slightly interfered with HepG2 DNA repair capacity (10% unrepaired damage), independently of the period of exposure. Extract C and solvent control did not interfere with DNA damage recovery, 24 and 48h after EMS exposure. Our results suggest that extracts P and A, which display the highest concentrations of PAHs and metals, respectively, produce a clear interference with the DNA repair capacity of HepG2 cells while less or no contaminated extracts (E and C, respectively) resulted in a low or no influence on DNA repair mechanisms. These results, together with the previous observations that extracts P and A were genotoxic, raise more concern on the potential hazard of estuarine contaminants on the health of exposed populations.
- Cytotoxic and genotoxic potential of sediments from the Portuguese Mira-River EstuaryPublication . Pinto, Miguel; Louro, Henriqueta; Costa, Pedro Manuel; Costa, Maria Helena; Caeiro, Sandra; Lavinha, João; Silva, Maria JoãoThe Mira-River Estuary (SW Portugal) although being a protected natural area has been affected by urban pollution from the village of Vila Nova de Milfontes (VNMF) and by contaminants from farming and aquaculture activities. The present study is part of a broader project comprising the evaluation of environmental and human health risks from potentially contaminated estuaries and aims to assess the cytotoxic and genotoxic potential of sediments from the Mira-River Estuary. Sediments were collected from two sites: Mf located near VNMF, and M near an aquaculture discharge channel. Organic and inorganic contaminants were extracted as four fractions using: methanol:dichloromethane (F1), dichloromethane (F2), hexane (F3) and methanol (F4) and recovered in DMSO. Cytotoxicity and genotoxicity were evaluated through the neutral red assay and the comet (coupled with DNA repair endonucleases) and micronucleus assays, respectively, in the human hepatoma-derived (HepG2) cell line. Contaminants analysis was carried out by GC-MS and ICP-MS for organic and metallic contaminants, respectively. A dose-related decrease in cell viability indicating cytotoxicity was observed for sample M, extracts F1, F2 and F4. A significant increase in the level of DNA and chromosome damage was observed for sub-cytotoxic doses of extracts F1, F2 and F3, by the comet and micronucleus assays, respectively. Additionally, oxidative DNA damage was also detected in the same samples. All Mf fractions failed to induce significant cytotoxic or genotoxic effects. These data agree with contaminants characterisation, given that a higher level of known genotoxic metals and PAHs were found in sample M but not in Mf. Overall, our results suggest that urban pollution of Mira-River Estuary sediments might be less hazardous than contamination from the aquaculture and agriculture activities, raising the question of the safe consumption of aquatic species grown in aquacultures, and of aquacultures as potential sources of aquatic pollution.
- 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.
- Human hepatoma cells exposed to estuarine sediment contaminant extracts permitted the differentiation between cytotoxic and pro-mutagenic fractionsPublication . Pinto, Miguel; Costa, Pedro Manuel; Louro, Henriqueta; Costa, Maria Helena; Lavinha, João; Caeiro, Sandra; Silva, Maria JoãoComplex toxicant mixtures present in estuarine sediments often render contaminant screening unfeasible and compromise determining causation. HepG2 cells were subjected to bioassays with sediment extracts obtained with a series of progressively polar solvents plus a crude extract. The sediments were collected from an impacted area of an estuary otherwise regarded as pristine, whose stressors result mostly from aquaculture effluents and hydrodynamic shifts that enhance particle deposition. Compared to a reference scenario, the most polar extracts yielded highest cytotoxicity while higher genotoxicity (including oxidative damage) was elicited by non-polar solvents. While the former caused effects similar to those expected from biocides, the latter triggered effects compatible with known pro-mutagens like PAHs, even though the overall levels of toxicants were considered of low risk. The results indicate that the approach may constitute an effective line-of-evidence to infer on the predominant set of hazardous contaminants present in complex environmental mixtures.
- 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.