Clarification of the mechanisms of mercury (Hg) toxicity in fish remains challenging. An innovative approach to this clarification is the combined assessment of metabolomic shifts, alterations in the antioxidant system and the risk of oxidative damage, and Hg bioaccumulation. This strategy was applied to the livers of golden grey mullet (Liza aurata) inhabiting an Hg-contaminated system in Aveiro Lagoon, Portugal. Marked changes in both the metabolic profile and the response to antioxidants were observed in fish exposed to Hg in the environment, indicating their compromised state of health. The metabolomics analysis showed a clear differentiation between the contaminated and uncontaminated areas, revealing that Hg has multiple levels of impact, including membrane stabilization/degradation/repair processes, osmoregulation, energy metabolism, gene expression and antioxidant protection. Research on oxidative stress biomarkers showed that Hg triggered adaptive responses in the antioxidant system as seen by an increase in the activities of glutathione-S-transferase and catalase, as well as the total glutathione content, which compensated for a decrease in glutathione peroxidase activity. The integration of metabolomics and oxidative stress endpoints allowed the identification of reduced glutathione as a first line of defence against Hg and evidenced oxidative insults in cell membranes, recognized through the increase in phosphatidylcholine degradation products (phosphocholine and glycerophosphocholine). Despite these effects, the induction of lipid peroxidation was efficiently prevented. The triad approach used here was demonstrated to be sensitive and effective towards a mechanistically based assessment of Hg hepatotoxicity in fish.

TiO 2 particles are widely used in products for everyday consumption, such as cosmetics and food; their possible adverse effects on human health must therefore be investigated. The aim of this study was to document in vitro impact of the food additive E171, i.e. TiO 2 , and of TiO 2 nanoparticles, on a co-culture of Caco-2 and HT29-MTX cells, which is an in vitro model for human intestine. Cells were exposed to TiO 2 particles three days after seeding, i.e. while they were not fully differentiated. Cell viability, reactive oxygen species (ROS) levels and DNA integrity were assessed, by MTT assay, DCFH-DA assay, alkaline and Fpg-modified comet assay and 8-oxo-dGuo measurement by HPLC-MS/MS. The mRNA expression of genes involved in ROS regulation, DNA repair via base-excision repair, and endoplasmic reticulum stress was assessed by RT-qPCR. Exposure to TiO 2 particles resulted in increased intracellular ROS levels, but did not impair cell viability and did not cause any oxidative damage to DNA. Only minor changes in mRNA expression were detected. Altogether, this shows that E171 food additive and TiO 2 nanoparticles only produce minor effects to this in vitro intestinal cell model.

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Superparamagnetic iron oxide nanoparticles (ION) have attracted great interest for use in several biomedical fields. In general, they are considered biocompatible, but little is known of their effects on the human nervous system. The main objective of this work was to evaluate the cytotoxicity of two ION (magnetite), coated with silica and oleic acid, previously determining the possible interference of the ION with the methodological procedures to assure the reliability of the results obtained. Human neuroblastoma SHSY5Y and glioblastoma A172 cells were exposed to different concentrations of ION (5-300 μg ml -1 ), prepared in complete and serum-free cell culture medium for three exposure times (3, 6 and 24 h). Cytotoxicity was evaluated by means of the MTT, neutral red uptake and alamar blue assays. Characterization of the main physical-chemical properties of the ION tested was also performed. Results demonstrated that both ION could significantly alter absorbance readings. To reduce these interferences, protocols were modified by introducing additional washing steps and cell-free systems. Significant decreases in cell viability were observed for both cell lines in specific conditions by all assays. In general, oleic acid-coated ION were less cytotoxic than silica-coated ION; besides, a serum-protective effect was observed for both ION studied and cell lines. These results contribute to increase the knowledge of the potential harmful effects of ION on the human nervous system. Understanding these effects is essential to establish satisfactory regulatory policies on the safe use of magnetite nanoparticles in biomedical applications.