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Evening and morning peroxiredoxin-2 redox/oligomeric state changes in obstructive sleep apnea red blood cells: Correlation with polysomnographic and metabolic parameters

Publication . Feliciano, Amélia; Vaz, Fátima; Torres, Vukosava M.; Valentim-Coelho, Cristina; Silva, Rita; Prosinecki, Vesna; Alexandre, Bruno M.; Carvalho, Ana S.; Matthiesen, Rune; Malhotra, Atul; Pinto, Paula; Bárbara, Cristina; Penque, Deborah

We have examined the effects of Obstructive Sleep Apnea (OSA) on red blood cell (RBC) proteome variation at evening/morning day time to uncover new insights into OSA-induced RBC dysfunction that may lead to OSA manifestations. Dysregulated proteins mainly fall in the group of catalytic enzymes, stress response and redox regulators such as peroxiredoxin 2 (PRDX2). Validation assays confirmed that at morning the monomeric/dimeric forms of PRDX2 were more overoxidized in OSA RBC compared to evening samples. Six month of positive airway pressure (PAP) treatment decreased this overoxidation and generated multimeric overoxidized forms associated with chaperone/transduction signaling activity of PRDX2. Morning levels of overoxidized PRDX2 correlated with polysomnographic (PSG)-arousal index and metabolic parameters whereas the evening level of disulfide-linked dimer (associated with peroxidase activity of PRDX2) correlated with PSG parameters. After treatment, morning overoxidized multimer of PRDX2 negatively correlated with fasting glucose and dopamine levels. Overall, these data point toward severe oxidative stress and altered antioxidant homeostasis in OSA RBC occurring mainly at morning time but with consequences till evening. The beneficial effect of PAP involves modulation of the redox/oligomeric state of PRDX2, whose mechanism and associated chaperone/transduction signaling functions deserves further investigation. RBC PRDX2 is a promising candidate biomarker for OSA severity and treatment monitoring, warranting further investigation and validation.

2016-11-15Journal article

Open access

Cyto- and Genotoxicity assessment of manufactured nano cerium dioxide in the A549 cell line

Publication . Saruga, Andreia; Louro, Henriqueta; Silva, Maria João

In the past decades the growing application of nanomaterials (NMs) in diverse consumer products has raised various concerns in the field of toxicology. They have been extensively used in a broad range of applications and cover most of the industrial sectors as well as the medicine and the environmental areas. The most common scenarios for human exposure to NMs are occupational, environmental and as consumers and inhalation is the most frequent route of exposure, especially in occupational settings. Cerium dioxide NMs (nano-CeO2) are widely used in a number of applications such as in cosmetics, outdoor paints, wood care products as well as fuel catalysts. For such reason, nano-CeO2 is one of the selected NMs for priority testing within the sponsorship program of the Working Party of Manufactured Nanomaterials of the OECD. In this context, the aim of this study is to assess the safety of nano-CeO2 (NM-212, Joint Research Center Repository) through the characterization of its cytotoxicity and genotoxicity in a human alveolar epithelial cell line. A dispersion of the NM in water plus 0.05% BSA was prepared and sonicated during 16 minutes, according to a standardized protocol. DLS analysis was used to characterize the quality of the NM dispersion in the culture medium. To evaluate the cytotoxicity of nano-CeO2 in the A549 cell line, the colorimetric MTT assay was performed; the capacity of cells to proliferate when exposed to CeO2 was also assessed with the Clonogenic assay. The genotoxicity of this NM was evaluated by the Comet Assay (3 and 24h of exposure) to quantify DNA breaks and the FPG-modified comet assay to assess oxidative DNA damage. The Cytokinesis-Block Micronucleus (CBMN) assay was used to further detect chromosome breaks or loss. The nano-CeO2 particles are spherical, displaying a diameter of 33 nm and 28 m2/g of surface area. The results of the MTT assay did not show any decreased in cells viability following treatment with a dose-range of nano-CeO2 during 24h. Nevertheless, the highest concentrations of this NM were able to significantly reduce the colony forming ability of A549 cells, suggesting that a prolonged exposure may be cytotoxic to these cells. Data from both genotoxicity assays revealed that nano-CeO2 was neither able to induce DNA breaks nor oxidative DNA damage. Likewise, no significant micronucleus induction was observed. Taken together, the present results indicate that this nano-CeO2 is not genotoxic in this alveolar cell line under the tested conditions, although further studies should be performed, e.g., gene mutation in somatic cells and in vivo chromosome damage (rodent micronucleus assay) to ensure its safety to human health.

2016-06-22Conference object

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Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties

Publication . Louro, Henriqueta; Pinhão, Mariana; Santos, Joana; Tavares, Ana; Vital, Nádia; Silva, Maria João

To contribute with scientific evidence to the grouping strategy for the safety assessment of multi-walled carbon nanotubes (MWCNTs), this work describes the investigation of the cytotoxic and genotoxic effects of four benchmark MWCNTs in relation to their physicochemical characteristics, using two types of human respiratory cells. The cytotoxic effects were analysed using the clonogenic assay and replication index determination. A 48h-exposure of cells revealed that NM-401 was the only cytotoxic MWCNT in both cell lines, but after 8-days exposure, the clonogenic assay in A549 cells showed cytotoxic effects for all the tested MWCNTs. Correlation analysis suggested an association between the MWCNTs size in cell culture medium and cytotoxicity. No induction of DNA damage was observed after any MWCNTs in any cell line by the comet assay, while the micronucleus assay revealed that both NM-401 and NM-402 were genotoxic in A549 cells. NM-401 and NM-402 are the two longest MWCNTs analyzed in this work, suggesting that length may be determinant for genotoxicity. No induction of micronuclei was observed in Beas-2B cell line and the different effect in both cell lines is explained in view of the size-distribution of MWCNTs in the cell culture medium, rather than cell's specificities.

2016-10-01Journal article

Restricted access

Comparing the genotoxicity of a multiwalled carbon nanotube and crocidolite towards the evaluation of its potential impact on the workers’ health

Publication . Ventura, Célia; Sousa Uva, António; Silva, Maria João

Background: Multiwalled carbon nanotubes (MWCNT) are one of the most promising and widespread class of manufactured nanomaterials, with several industrial and biomedical applications. However, their unique physicochemical properties may have detrimental effects on human health upon unintentional exposure by inhalation. Although there is still no sufficient epidemiological and toxicological data on most MWCNT, several professional and scientific organizations adopted a precautionary principle and considered MWCNT as an occupational hazard. In vitro toxicological studies can contribute to fulfil the gaps on the knowledge about their potential health adverse effects and to identify biomarkers for human biomonitoring, particularly in the workplace. Aim- This study was aimed at characterizing the cytotoxic and genotoxic effects of NRCWE-006, a high aspect-ratio rigid MWCNT, comparatively to crocidolite, a well-known tumorigenic asbestos fiber causing mesothelioma, using a co-culture of alveolar epithelial cells (A549) and monocyte-derived macrophages (THP-1). Methods – The MTT, the comet and the micronucleus assays were performed on a co-culture of A549 and differentiated THP-1 cells following exposure to a concentration- range of NRCWE-006 or crocidolite. Results- Both NRCWE-006 and crocidolite revealed cytotoxicity by the MTT assay. NRCWE-006 did not induce a detectable level of DNA breaks under the comet assay conditions tested, while a significant increase in the micronucleus frequency was detected at 6.25 and 12.5 µg/cm2. In contrast, crocidolite revealed a clear dose-dependent increase in the level of DNA strand breaks (comet assay) and induced a significant increase in the micronucleus frequency at the highest concentrations tested (10 and 20 µg/cm2). Discussion and Conclusions – Our results suggest that NRCWE-006 is less cytotoxic than crocidolite to alveolar cells grown in co-culture with monocyte-derived macrophages. As expected, crocidolite was clearly genotoxic, given that it was able to induce DNA and chromosome damage, probably due to its known potential of ROS production. On the other hand, even though NRCWE-006 did not cause DNA damage, it demonstrated aneugenic/clastogenic effects at the two lowest concentrations, which are closer to the ones that may represent a concern in terms of occupational exposure.

2017-10-03Conference object

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Nanotoxicology and nanotechnology interplay towards a safe and responsible innovation

Publication . Silva, Maria João; Ventura, Célia; Dias, Kamila; Saruga, Andreia; Pinhão, Mariana; Louro, Henriqueta

The European Union Strategic and Action Plan for Nanosciences and Nanotechnologies emphasizes the need of ensuring the development of safe, integrated, and responsible nanotechnologies and nanotechnology products. Indeed, although the use of manufactured nanomaterials (NM) is widespread and has been considered greatly beneficial for several scientific and technological areas, uncertainties about their safety still remain, representing a challenge to scientists, risk assessors and regulators. Recent studies of the team have been aimed at assessing the potential deleterious effects, e.g., genotoxicity that is intimately associated with carcinogenicity, of several classes of NM, using complementary in vitro and in vivo approaches, nested within the conventional risk assessment paradigm. Overall, the results have suggested that specific physicochemical properties of NM are crucial to define their nano-bio interactions and their toxic potential. Furthermore, our findings reinforced previous reports suggesting that factors inherent to experimental conditions and methodologies may affect the effects observed. These and other nano-specific issues have been hindering the categorization of NM according to their toxicity and, consequently, their risk assessment and management. Nonetheless, considering the wide array of NM produced or under development, a case-by-case approach to the risks of each NM seems an unreasonably extensive task. Thus, the development of high-throughput omics-based tools adapted for the toxicity assessment of NM and, on the other hand, the elucidation of the cellular and molecular mechanisms underlying NM toxicity is of utmost importance to progress faster in the field of nanotoxicology. Such information will allow the development of cost-efficient screening strategies and promote predictive nanotoxicology, driving the synthesis of safer NM. The ultimate goal is fostering a sustainable and safe innovation in the field of nanotechnologies.

2017-07-04Conference object

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