Percorrer por autor "Costa, Pedro"
A mostrar 1 - 9 de 9
Resultados por página
Opções de ordenação
- Are standard genotoxicity tests useful for the safety evaluation of nanomaterials?Publication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Antunes, Susana; Costa, Pedro; Alverca, Elsa; Lavinha, João; Silva, Maria JoãoNanomaterials (NMs) are widely used in a diversity of consumer products, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. One of the major concerns is the potential to induce cancer. To analyze in a short term the carcinogenic properties of a compound, genotoxicity assays in mammalian cell lines or animal models are frequently used. In the context of an EU Joint Action, in the present work we have used standard genotoxicity assays (comet, micronucleus and mutation assays) to investigate the effects associated with the exposure to titanium dioxide nanomaterials (TiO2), following standardized dispersion and assay procedures, in three types of human cells and in a mouse model. The results showed slight but significant increases in the frequencies of micronuclei after exposure to some of the NMs, as compared to controls. No clear dose-response relationships could be disclosed. One of the tested TiO2 yielded equivocal results in vitro micronucleus assay and was positive in the comet assay in pulmonary cells. In view of the inconclusive results,it was further analyzed in vivo, using the lacZ transgenic mouse model. It did not induce genotoxic effects in mice, 28 days after injection, despite the accumulation of the NM observed in the mouse liver. Regarding safety assessment, the different genotoxicity observed for closely related NMs, but that differ in some physicochemical characteristics, highlights the importance of investigating the toxic potential of each NM individually, instead of assuming a common mechanism and equal genotoxic effects for a set of similar NMs. The equivocal genotoxicity of the nanosized TiO2 in human cells in vitro was not confirmed in vivo, and therefore the predictive value of these in vitro tests for identifying genotoxic (and potentially carcinogenic) NMs in vivo should be clarified, before extrapolating the conclusions for human health.
- Avaliação do risco ambiental e para a saúde pública de um ambiente estuarino contaminadoPublication . Pinto, Miguel; Louro, Henriqueta; Caeiro, Sandra; Costa, Pedro; Costa, Maria Helena; Lavinha, João; Silva, Maria JoãoO estuário do rio Sado é um ecossistema de elevado valor ecológico e económico. Apesar de ter vindo a ser afetado ao longo dos anos por várias fontes de poluição de origem urbana, industrial e agrícola, continua ainda a ser, para a população local, um local privilegiado para atividades piscícolas e agrícolas. Contudo, existe uma preocupação relacionada com a potencial bioacumulação de contaminantes nas partes edíveis de espécies estuarinas ou de produtos agrícolas locais que entrarão assim na cadeia alimentar humana, podendo representar um problema de saúde pública. O presente estudo integra-se num projeto mais amplo que visa avaliar os riscos ambientais e para a saúde humana, associados a esse 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. Observaram-se efeitos citotóxicos e genotóxicos diferentes em amostras de sedimentos derivadas da margem esquerda vs. direita do estuário que se podem correlacionar com diferenças nos níveis e tipos de contaminantes identificados nessas amostras. Estes refletem, provavelmente, as diversas pressões no ecossistema derivadas de uma área urbana e industrializada (margem esquerda) vs. uma área de intensa atividade agrícola (margem direita). A observação de que amostras de sedimentos estuarinos induzem efeitos citotóxicos e genotóxicos em células humanas, em consonância com os efeitos também detetados em espécies estuarinas, enfatiza a preocupação sobre o impacto do consumo de espécies contaminadas na saúde das populações locais e reforça a necessidade de serem tomadas medidas no sentido de reduzir e remediar a poluição desta área estuarina.
- Characterization of cytotoxic and genotoxic effects of contaminated sediments from the Sado Estuary and potential human health riskPublication . Pinto, Miguel; Louro, Henriqueta; Costa, Pedro; Costa, Maria Helena; Caeiro, Sandra; Lavinha, João; Silva, Maria JoãoThe river Sado Estuary (W Portugal) is affected by various sources of pollution, such as heavy-industry, urbanism, mining, agriculture and maritime traffic. Mostly classified as a natural reserve, it also remains a privileged site for fishing activities performed by the local population, who not only consume but distribute their fishery. The present study is part of a broader project whose objective is to evaluate the environmental and human health risks associated with the estuarine benthic environment. This study aims to assess the cytotoxic and genotoxic potential of sediments from several local fishing areas of the Sado Estuary. Sediments were collected from four geochemically distinct and potentially contaminated sites of the Sado Estuary: sites C and P from the northern shore and sites E and A from the southern shore. A previously characterized sample (F) from the northern shore was added as a positive control. Total organic and inorganic contaminants were extracted with a mixture of methanol:dichloromethane (1:2) and recovered in DMSO. Cytotoxicity and genotoxicity were evaluated through the neutral red uptake assay and by the alkaline comet (coupled with DNA repair endonucleases) and the micronucleus assays, respectively, in the human HepG2 cell line. Cells were exposed for 48h to concentrations of each extract ranging from 0.1 to 20µl/ml of culture medium. A dose-related decrease in cell viability was observed for extracts F, P and E, indicating sediment contaminant-driven cytotoxicity, whereas no effect was observed for extracts C and A. No significant genotoxicity was observed for extract C, while extract F was clearly genotoxic, as expected. A significant increase in the level of DNA and chromosome damage was observed, by the comet and micronucleus assays, respectively, for sub cytotoxic concentrations of extracts P and E. The level of DNA damage was accentuated following treatment with the DNA repair endonuclease FPG, suggesting the existence of oxidative DNA damage. Extract A was genotoxic in the micronucleus assay and in the comet assay only after FPG treatment. Negative results from sample C leads us to consider it as potential clean reference for further studies. Moreover, sediment contamination analysis revealed high levels of metals in all samples except C, whereas only sample P exhibited high levels of known genotoxic polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and DDTs, similar to the previously described for sample F. The differential cytotoxicity and genotoxicity observed in samples from the northern (P) and southern areas (E and A) of the Sado Estuary probably reflects different pressures from a urban and heavy industrialized area versus an intense agricultural area, respectively. The observation that sediment samples have cytotoxic and genotoxic effects, together with the knowledge that contaminants can be accumulated in the edible parts of estuarine species or local agricultural products entering the human food chain, raise concern about a hazardous impact on the health of exposed populations that must be assessed. Work supported by the Foundation for Science and Technology (ref. PTDC/SAU-ESA/100107/2008).
- Cytotoxic and genotoxic assessment of potentially contaminated sediments from the Sado EstuaryPublication . Pinto, Miguel; Louro, Henriqueta; Costa, Pedro; Costa, Maria Helena; Caeiro, Sandra; Silva, Maria JoãoPrevious studies have shown that the river Sado Estuary (W Portugal) is highly susceptible to contamination due to anthropogenic activity. In fact, sizable concentrations of metals, pesticides and polycyclic aromatic hydrocarbons have been measured in sediments from various points of the estuary. Those compounds may be absorbed by aquatic species, making it a public health issue when entering the human food chain. The purpose of this study was to assess the cytotoxic and genotoxic potential of sediment extracts in a human cell line, through the neutral red uptake assay and the alkaline comet assay, respectively. Three sediment samples were collected: two in potentially contaminated sites (samples C and F, Sado Estuary) and one in a putative control station (sample M, Mira Estuary). The overall contaminants were extracted from the three samples. HepG2 cells were exposed for 48h to serial dilutions of each extract (ranging from 0.01 to 2μl). A dose-related decrease in cell viability was observed for samples F and M (from 0.1 and 0.5μl, respectively, up to 2μl), indicating a toxic effect caused by the complex contaminants mixtures. However, no genotoxicity could be observed for any sample in the experimental conditions used. Further studies are underway with different exposure times and different endpoints for a more complete assessment of the extracts' genotoxic properties.
- Determining oxidative and non-oxidative genotoxic effects driven by estuarine sediment contaminants on a human hepatoma cell linePublication . Pinto, Miguel; Costa, Pedro; Louro, Henriqueta; Costa, Maria Helena; Lavinha, João; Caeiro, Sandra; Silva, Maria JoãoEstuarine sediments may be reservoirs of hydrophilic and hydrophobic pollutants, many of which are acknowledged genotoxicants, pro-mutagens and even potential carcinogens for humans. Still, studies aiming at narrowing the gap between ecological and human health risk of sediment-bound contaminant mixtures are scarce. Taking an impacted estuary as a case study (the Sado, SW Portugal), HepG2 (human hepatoma) cells were exposed in vitro for 48 h to extracts of sediments collected from two areas (urban/industrial and Triverine/agricultural), both contaminated by distinct mixtures of organic and inorganic toxicants, among which are found priority mutagens such as benzo[a]pyrene. Comparatively to a control test, extracts of sediments from both impacted areas produced deleterious effects in a dose-response manner. However, sediment extracts from the industrial area caused lower replication index plus higher cytotoxicity and genotoxicity (concerning total DNA strand breakage and clastogenesis), with emphasis on micronucleus induction. On the other hand, extracts from the rural area induced the highest oxidative damage to DNA, as revealed by the FPG (formamidopyrimidine-DNA glycosylase) enzyme in the Comet assay. Although the estuary, on its whole, has been classified as moderately contaminated, the results suggest that the sediments from the industrial area are significantly genotoxic and, furthermore, elicit permanent chromosome damage, thus potentially being more mutagenic than those from the rural area. The results are consistent with contamination by pro-mutagens like polycyclic aromatic hydrocarbons (PAHs), potentiated by metals. The sediments from the agriculture-influenced area likely owe their genotoxic effects to metals and other toxicants, probably pesticides and fertilizers, and able to induce reactive oxygen species without the formation of DNA strand breakage. The findings suggest that the mixtures of contaminants present in the assayed sediments are genotoxic to HepG2 cells, ultimately providing a useful approach to hazard identification and an effective line-of-evidence in the environmental monitoring of anthropogenically-impacted coastal ecosystems.
- Environmental risk assessment in a contaminated estuary: An integrated weight of evidence approach as a decision support toolPublication . Caeiro, Sandra; Vaz-Fernandes, Paula; Martinho, Ana Paula; Costa, Pedro; Silva, Maria João; Lavinha, João; Matias-Dias, Carlos; Machado, Ausenda; Castanheira, Isabel; Costa, Maria HelenaEnvironmental risk assessment of complex ecosystems such as estuaries is a challenge, where innovative and integrated approaches are needed. The present work aimed at developing an innovative integrative methodology to evaluate in an impacted estuary (the Sado, in Portugal, was taken as case study), the adverse effects onto both ecosystem and human health. For the purpose, new standardized lines of evidence based on multiple quantitative data were integrated into a weight of evidence according to a best expert judgment approach. The best professional judgment for a weight of evidence approach in the present study was based on the following lines of evidence: i) human contamination pathways; ii) human health effects: chronic disease; iii) human health effects: reproductive health; iv) human health effects: health care; v) human exposure through consumption of local agriculture produce; vi) exposure to contaminated of water wells and agriculture soils; vii) contamination of the estuarine sedimentary environment (metal and organic contaminants); viii) effects on benthic organisms with commercial value; and ix) genotoxic potential of sediments. Each line of evidence was then ordinally ranked by levels of ecological or human health risk, according to a tabular decision matrix and expert judgment. Fifteen experts scored two fishing areas of the Sado estuary and a control estuarine area, in a scale of increasing environmental risk and management actions to be taken. The integrated assessment allowed concluding that the estuary should not be regarded as impacted by a specific toxicant, such as metals and organic compounds hitherto measured, but by the cumulative risk of a complex mixture of contaminants. The proven adverse effects on species with commercial value may be used to witness the environmental quality of the estuarine ecosystem. This method argues in favor of expert judgment and qualitative assessment as a decision support tool to the integrative management of estuaries. Namely it allows communicating environmental risk and proposing mitigation measures to local authorities and population under a holistic perspective as an alternative to narrow single line of evidence approaches, which is mandatory to understand cause and effect relationships in complex areas like estuaries.
- Exploring the potential interference of estuarine sediment contaminants with the DNA repair capacity of human hepatoma cellsPublication . Pinto, Miguel; Louro, Henriqueta; Costa, Pedro; Caeiro, Sandra; Silva, MariaEstuaries may be reservoirs of a wide variety of pollutants, including mutagenic and carcinogenic substances that may impact on the ecosystem and human health. A previous study showed that exposure of human hepatoma (HepG2) cells to extracts from sediment samples collected in two areas (urban/industrial and riverine/agricultural) of an impacted estuary (Sado, Portugal), produced differential cytotoxic and genotoxic effects. Those effects were found to be consistent with levels and nature of sediment contamination. The present study aimed at evaluating whether the mixtures of contaminants contained in those extracts were able to modulate DNA repair capacity of HepG2 cells. The residual level of DNA damage was measured by the comet assay in cells exposed for 24 or 48 h to different extracts,after a short preexposure to a challenging concentration range of ethyl methanesulfonate(EMS), as a model alkylating agent. The results suggested that the mixture of contaminants present in the tested samples, besides a potential direct effect on the DNA molecule, may also interfere with DNA repair mechanisms in HepG2 cells, thus impairing their ability to deal with genotoxic stress and, possibly, facilitating accumulation of mutations. Humans are environmentally/occupationally exposed to mixtures rather than to single chemicals. Thus, the observation that estuarine contaminants induce direct and indirect DNA strand breakage in human cells, the latter through the impairment of DNA repair, raises additional concerns regarding potential hazards from exposure and the need to further explore these endpoints in the context of environmental risk assessment.
- Impact of Potentially Contaminated Sediments From The Sado Estuary in Human Health: Cytotoxic And Genotoxic Assays in a Human Cell LinePublication . Pinto, Miguel; Louro, Henriqueta; Costa, Pedro; Caeiro, Sandra; Lavinha, João; Silva, Maria JoãoAs many estuaries in industrialized countries, the river Sado Estuary (W Portugal) is affected by various sources of pollution, such as heavy-industry, urbanism, mining, agriculture and maritime traffic. Mostly classified as a natural reserve, it also remains a privileged site for fishing activities performed by the local population, who not only consume but distribute their fishery. Previous studies revealed sizable amounts of contaminants in the estuary sediments, namely metals, pesticides and polycyclic aromatic hydrocarbons. These compounds can be accumulated in the edible parts of estuarine species with commercial value or local agricultural products and enter the human food chain, posing a public health concern. The present study is part of a broader project whose objective is to evaluate the environmental risk, including ecologic and human health risk, associated with the estuarine benthic environment, complemented with the analysis of a target population from a small village located near the estuary shore. This study aims to assess the cytotoxic and genotoxic potential of sediments from the Sado Estuary through the neutral red uptake assay and the alkaline comet assay (coupled with DNA repair endonucleases) in a human cell line respectively, using multiple samples collected in various points of the Sado Estuary. Sediments were collected from two geochemically distinct and potentially contaminated sites of the Sado Estuary: site F and site C. Total organic and inorganic contaminants were extracted with a mixture of methanol:dicholomethane (1:2) and recovered in DMSO. HepG2 cells were exposed for 48h to concentrations of each extract ranging from 0.1 to 20ul/ml of culture medium. A dose-related decrease in cell viability was observed for extract F from 1ul/ml up to 20ul/ml, indicating sediment contaminant-driven cytotoxicity, whereas no cytotoxicity induction was observed for extract C. Genotoxicity was only found for extract F, collected near a heavy-industrialized site. Also, increased genotoxicity was observed in cells treated with the DNA repair endonuclease FPG, for extract F, suggesting oxidative DNA damage. No significant genotoxity was observed for extract C. Given the difference in cytotoxic and genotoxic effects between both samples, a larger number of samples is necessary to reflect the overall contamination status of the Sado Estuary. Therefore, other samples are currently being analysed in order to obtain a more complete evaluation of the cytotoxic and genotoxic potential of the sediment contaminants from the river Sado Estuary, and a sample from a non-contaminated site will be added as a reference. The results are expected to reflect the Sado Estuary contamination by different anthropogenic pressures.
- Investigation of the in vivo genotoxic effects of a titanium dioxide nanomaterial in LacZ plasmid-based transgenic micePublication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Costa, Pedro; Alverca, Elsa; Zwart, Edwin; de Jong, Wim H.; Fessard, Valérie; Lavinha, João; Silva, Maria JoãoTitanium dioxide nanomaterials (TiO2) are increasingly used in a diversity of products, including cosmetics, pharmaceuticals, food and inks, which contrasts with the existing uncertainties in respect to their risks for human and environment health. Previous studies on the genotoxicity of TiO2 reported discrepant findings, both in cellular and organismal systems. In a recent work, we showed that some nanosized TiO2 were able to induce a significant increase in the frequency of micronucleated human lymphocytes, whereas for anatase TiO2 (NM-102, JRC repository), this effect was observed with a single significant concentration, providing inconclusive evidence. To further investigate the genotoxic potential of NM-102, the LacZ plasmid-based transgenic mouse model was used allowing an integrated analysis of multiple genotoxicity endpoints at a whole-organism level. Following two administrations of 0, 10 or 15 mg/kg of NM-102 by intravenous route within a 24h period, the micronucleus frequency was determined in peripheral blood reticulocytes whereas DNA strand breaks (comet assay) and gene mutations were quantified in spleen and liver cells 28 days after exposure. Histopathological analyses were concomitantly performed in liver tissues, using both light and transmission electron microscopy (TEM). The results did not show any significant genotoxic effects after exposure to the TiO2 NM under the experimental conditions used, but a moderate inflammatory response was observed in liver. In addition, TEM evidenced the presence of TiO2 in liver cells. The overall integration of the data strengthens the weight of evidence of an absence of TiO2 genotoxicity in vivo, although the possibility of a secondary genotoxic effect driven by an inflammatory response within a longer time window or at higher doses cannot be excluded and should be further investigated.