Browsing by Author "Costa, Pedro M."
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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.
- Avaliação integrada dos efeitos genotóxicos de nanomateriais manufaturados no ratinho transgénico LacZPublication . Louro, Henriqueta; Tavares, Ana; Vital, Nádia; Costa, Pedro M.; Alverca, Elsa; Lavinha, João; Silva, Maria JoãoNum estudo recente, demonstrámos que um nanomaterial de dióxido de titânio na forma cristalina designada anatase, o NM-102 (do repositório do Joint Research Center; Ispra, Itália), induziu um aumento significativo de quebras cromossómicas - detetáveis na forma de micronúcleos - em linfócitos humanos expostos ex vivo(3) não se tendo, porém, observado um efeito dose-resposta. No sentido de prosseguir e aprofundar a avaliação da genotoxicidade do NM-102, o presente estudo teve por objetivo caracterizar os seus efeitos genotóxicos, num modelo animal, utilizando uma abordagem integrada, a qual abrangeu a análise de vários parâmetros de genotoxicidade no mesmo animal.
- Environmental exposure to toxicants mixtures from a multi-purpose estuary: perspectives for a biomonitoring study in PortugalPublication . Silva, Maria João; Pinto, Miguel; Costa, Pedro M.; Louro, Henriqueta; Castanheira, Isabel; Machado, Ausenda; Dias, Carlos; Fernandes, Ana Paula; Martinho, AP; Costa, MH; Caeiro, Sandra; Lavinha, JoaoThis work was focused on a multi-purpose estuarine environment (river Sado estuary, SW Portugal) around which a number of activities (e.g., fishing, farming, heavy industry, tourism and recreational activities) coexist with urban centres with a total of about 200 000 inhabitants. Based on previous knowledge of the hazardous chemicals within the ecosystem and their potential toxicity to benthic species, this project intended to evaluate the impact of estuarine contaminants on the human and ecosystem health. An integrative methodology based on epidemiological, analytical and biological data and comprising several lines of evidence, namely, human contamination pathways, human health effects, consumption of local produce, estuarine sediments, wells and soils contamination, effects on commercial benthic organisms, and genotoxic potential of sediments, was used. The epidemiological survey confirmed the occurrence of direct and indirect (through food chain) exposure of the local population to estuarine contaminants. Furthermore, the complex mixture of contaminants (e.g., metals, pesticides, polycyclic aromatic hydrocarbons) trapped in the estuary sediments was toxic to human liver cells exposed in vitro, causing cell death, oxidative stress and genotoxic effects that might constitute a risk factor for the development of chronic-degenerative diseases, on the long term. Finally, the integration of data from several endpoints indicated that the estuary is moderately impacted by toxicants that affect also the aquatic biota. Nevertheless, the human health risk can only be correctly assessed through a biomonitoring study including the quantification of contaminants (or metabolites) in biological fluids as well as biomarkers of early biological effects (e.g., biochemical, genetic and omics-based endpoints) and genetic susceptibility in the target population. Data should be supported by a detailed survey to assess the impact of the contaminated seafood and local farm products consumption on human health and, particularly, on metabolic diseases or cancer development.
- 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 approach to the in vivo genotoxic effects of a titanium dioxide nanomaterial using LacZ plasmid-based transgenic micePublication . Louro, Henriqueta; Tavares, Ana; Vital, Nadia; Costa, Pedro M.; Alverca, Elsa; Zwart, Edwin; de Jong, Wim H.; Féssard, Valerie; Lavinha, João; Silva, Maria JoãoTitanium dioxide (TiO2 ) nanomaterials (NMs) are widely used in a diversity of products including cosmetics, pharmaceuticals, food, and inks, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. Previous studies on the genotoxicity of TiO2 have reported discrepant or inconclusive findings in both in vitro and in vivo systems. This study explores the in vivo genotoxic potential of a well-characterized uncoated TiO2 NM with an average diameter of 22 nm (NM-102, from JRC repository) using several genotoxicity endpoints in the LacZ plasmid-based transgenic mouse model. Mice were exposed by intravenous injection to two daily doses of NM-102: 10 and 15 mg/kg of body weight/day. Micronuclei were analyzed in peripheral blood reticulocytes 42 hr after the last treatment. DNA strand breaks (comet assay) and gene mutations were determined in the spleens and livers of the same animals 28 days after the last treatment. Histopathological and cytological analyses were also performed in liver samples. Genotoxic effects were not detected in mice exposed to the nanosized TiO2 under the experimental conditions used, despite a moderate inflammatory response that was observed in the liver. Considering the biopersistence of TiO2 in mouse liver and the moderate inflammatory response, the possibility of a secondary genotoxic effect at higher doses and in conditions that result in a stronger inflammatory response, for example, within a longer time window, should be investigated further.
- 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.
- The LacZ plasmid-based transgenic mouse model: an integrative approach to study the genotoxicity of nanomaterialsPublication . Louro, Henriqueta; Pinto, Miguel; Vital, Nádia; Tavares, Ana; Costa, Pedro M.; Silva, Maria JoãoNumerous in vitro studies have been performed to address the potential genotoxicity of chemicals and of emerging products, e.g., nanomaterials. Although valuable for hazard assessment, the in vitro assays do not reflect the complexity of an organism, including, bioavailability, toxicokinetics and immune responses. Moreover, the biological effects at the target organs are known to be greatly influenced by factors as cell proliferative rate, metabolic and DNA repair capacities. In this sense, data from suitable in vivo assays are useful to evaluate the performance of in vitro assays and to strengthen the knowledge about the genotoxicity of chemicals and nanomaterials, using several routes of exposure, at a whole organism level. This chapter provides an overview of an integrated experimental design, based on the use of a LacZ-plasmid based transgenic mouse model to investigate multiple genotoxicity endpoints in several organs, towards the safety evaluation of nanomaterials. This approach includes the analysis of chromosome instability, assessed by the micronucleus assay in blood or bone marrow cells and by sister chromatid exchanges in splenocytes, the analysis of DNA breaks and oxidative DNA damage by the comet assay, and the quantification of gene mutations in multiple organs. Furthermore, histological markers e.g., of inflammation and apoptosis, can add information about other relevant cell responses. A key point is that all assays are performed on the same animal, therefore increasing the efficiency while reducing the cost and the number of animals under experimentation, in compliance with the EU recommendations. Overall, gathering the data from the several endpoints and organs of the same animal depicts the complex response of a whole-organism to nanomaterials exposure, thereby providing a better prediction of the effects on humans.