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Projeto de investigação
Mixtures of Environmental Carcinogens: a molecular approach to improve environmental risk assessment strategies.
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Interaction of Polycyclic Aromatic Hydrocarbon compounds in fish primary hepatocytes
Publication . Bramatti, Isabella; Matos, Beatriz; Figueiredo, Neusa; Pousão-Ferreira, Pedro; Branco, Vasco; Martins, Marta; MARE - Centro de Ciências do Mar e do Ambiente; DCEA - Departamento de Ciências e Engenharia do Ambiente; Elsevier
Polycyclic Aromatic Hydrocarbons (PAHs) are persistent pollutants normally found in the environment as complex mixtures. Although several individual PAHs are classified as mutagenic and carcinogenic pollutants, the interaction effects between compounds in a mixture may trigger different toxicological mechanisms and, consequently, yield different effects to organisms which are not accounted for in risk assessment guidelines. Given the ubiquity of PAHs, understanding the mechanistic features of their mixtures is a pressing research need. Therefore, the present work aimed to disclose the interaction effects of three PAHs with different carcinogenic potential and chemical structure, in primary hepatocyte cells of gilt-headed seabreams (Sparus aurata). Hepatocytes were exposed to Phenanthrene (Phe), Benzo[a]pyrene (B[a]P) and Benzo[b]fluoranthene (B[b]F) and their mixtures at different proportions and several cellular responses were analyzed: cellular viability, CYP1A1 activity (EROD assay) and protein expression level (Western blot); transcript (mRNA) levels of CYP1A1, EPXH1 and GST-3 (qRT-PCR); genotoxic effects (DNA strand breakage) by the Comet assay. Results show that B[a]P induced CYP1A1 gene and protein expression increasing its activity and, therefore, increasing the production of metabolites that trigger genotoxic DNA damage (%). Most importantly, mixtures containing Phe and B[a]P increased even further CYP1A1 mRNA levels and DNA damage (up to 70 %) which suggests that, although Phe is considered a non-carcinogenic PAH, it potentiates CYP1A1 synthesis induced by B[a]P, increasing its genotoxicity. These findings indicate that the upregulation of CYP1A1 by carcinogenic PAHs will not weaken even when in mixtures with non-carcinogenic PAHs. On contrary, non-carcinogenic PAHs may potentiate the genotoxic effect of carcinogenic PAH and therefore mixture composition should be taken in account when assessing PAH toxicity. In fact, our results point to the need of redefining Environmental Risk Assessment protocols for mixtures of carcinogenic pollutants.
Nanoplastics activate a TLR4/p38-mediated pro-inflammatory response in human intestinal and mouse microglia cells
Publication . Antunes, Joana; Sobral, Paula; Martins, Marta; Branco, Vasco; MARE - Centro de Ciências do Mar e do Ambiente; DCEA - Departamento de Ciências e Engenharia do Ambiente; Elsevier
The crescent presence of nanoplastics in the environment raises concerns regarding their potential impact on health. This study exposed human colon adenocarcinoma cells (HT29) and microglia cells (N9) to nanoplastics (25 nm, 50 nm, and 100 nm Polystyrene) to investigate their inflammatory responses, which are vital for body's defence. Although cytotoxicity remained generally low, HT29 cells exhibited a notable upregulation of p50 and p38 expression, concomitant with elevated TLR4 expression, in contrast with N9 cells that showed a less pronounced upregulation of these proteins. Additionally, nanoplastic exposure increased IL-1ß levels, partially attenuated by pre-exposure to TLR4 or p38 inhibitors. Intriguingly, N9 cells exposed to nanoplastics exhibited substantial increases in iNOS mRNA. This effect was entirely prevented by pre-exposure to TLR4 or p38 inhibitors, while TNF-α mRNA levels remained relatively stable. These findings underscore the potential of nanoplastics to activate inflammatory pathways, with response kinetics varying depending on the cell type.
In vitro Methodologies for Environmental Risk Assessment: Comparison between methods for the isolation of marine fish primary hepatocytes
Publication . Morais, João Pedro Robalo; Martins, Marta; Figueiredo, Neusa
Interest of in vitro methods has grown exponentially throughout the years especially due to the ethical problems surrounding the use of animals in in vivo studies. Cell-based assays for the isolation of hepatocytes using enzymatic methods have been credited in fish toxicology and play a substantial role in the propagation of in vitro methodologies, having been implemented with various degrees of success to a variety of fish species, especially in freshwater species. Isolated hepatocytes provide an optimal system to study the role of hepatic metabolism in the activation of environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs). For these reasons, the present work focused on the optimization of the isolation of two marine fish hepatocytes (P. maxima and S. aurata) using two enzymatic methods of isolation, pancreatin digestion and 2-step collagenase perfusion, for posterior validation in cytotoxicity testing. The two main parameters studied were cell viability and yield after isolation. Cell viability was assessed via the Trypan blue exclusion test and cell yield was counted with the use of a hemocytometer. The pancreatin method accounted for better overall cell yield and also produced the highest viability out of all experiments. Pancreatin digestion isolations also produced purer cultures, largely with 100% viability, which was made possible by way of modifications made during isolations, such as the use of histopaque for blood cell separation and addition of another smaller mesh filter (60 μm) to achieve better purity and fewer cell debris, respectively. A cytotoxicity assay was also performed using the MTT colorimetric assay to access cell viability after contamination with Phenanthrene and Benzo[b]fluoranthene, two polycyclic aromatic hydrocarbons (PAHs) of interest for cytotoxicity studies. Viabilities after contamination with both compounds only exhibited small variability demonstrating no dose-response curve, likely due to factors such as intra-species individual variability and the precipitation of contaminants in the highest concentrations, which might have affected absorbance readings and could cause misleading cell viabilities. Therefore, we call for further studies to access the cytotoxic effect on P. maxima and S. aurata by Phe and B[b]f. In conclusion, the research showed that the best method for isolation was the pancreatin digestion and the marine fish primary hepatocyte cultures obtained are suitable for the application in cytotoxicity assays.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/CTA-AMB/29173/2017