María Puerto

Cytotoxicity of carboxylic acid functionalized single wall carbon nanotubes on the human intestinal cell line Caco-2.

The unique properties of carbon nanotubes (CNTs) have been explored for their use in biomedical sciences and in biotechnological fields; however, their possible toxic effects are of concern. The sources of human exposure to nanomaterials include inhalation, ingestion, dermal contact and injection. The pulmonary and dermal effects of CNTs in vitro have been previously studied with contradictory results, but data on intestinal cells are scarce. The aim of this study was to evaluate the cytotoxicity of single wall CNTs functionalized with carboxylic acid on differentiated and non-differentiated Caco-2 cells, derived from a human intestinal adenocarcinoma. Biomarkers assessed were neutral red uptake (NR), protein content (PT), a tetrazolium salt (MTS) metabolization, LDH leakage (LDH) and cell viability by means of the trypan blue exclusion test (TBET). Moreover, a morphological study was performed. Cells were exposed to concentrations between 5 and 1,000 microg/ml CNTs and toxic effects were studied after 24h of exposure. NR and MTS results showed a concentration-dependent trend with an inhibitory response from 100 microg/ml CNT, together with an increase in LDH leakage. TBET resulted in an 80% reduction at higher concentrations, and finally PT was only modified at higher concentrations. Overall, results indicated cytotoxic effects on the Caco-2 cells with differentiated cultures showing a higher sensitivity. Thus, a hazard assessment of CNTs is necessary as the nanotechnology industry grows, and more nanoscale wastes are released into the environment.

Differential protein expression in two bivalve species; Mytilus galloprovincialis and Corbicula fluminea; exposed to Cylindrospermopsis raciborskii cells

The cyanobacteria Cylindrospermopsis raciborskii is considered a threat to aquatic organisms due to the production of the toxin cylindrospermopsin (CYN). Despite the numerous reports evidencing the toxic effects of C. raciborskii cells and CYN in different species, not much is known regarding the toxicity mechanisms associated with this toxin and the cyanobacteria. In this work, a proteomics approach based in the two-dimensional gel electrophoresis and mass spectrometry was used to study the effects of the exposure of two bivalve species, Mytilus galloprovincialis and Corbicula fluminea, to CYN producing (CYN+) and non-producing (CYN-) C. raciborskii cells. Additionally the activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx) were determined. Alterations in actin and tubulin isoforms were detected in gills of both bivalve species and digestive gland of M. galloprovincialis when exposed to CYN- and CYN+ cells. Moreover, GST and GPx activities changed in gills and digestive tract of bivalves exposed to both C. raciborskii freeze dried cells, in comparison to control animals exposed to the green alga Chlorella vulgaris. These results suggest the induction of physiological stress and tissue injury in bivalves by C. raciborskii. This condition is supported by the changes observed in GPx and GST activities which indicate alterations in the oxidative stress defense mechanisms. The results also evidence the capacity of CYN non-producing C. raciborskii to induce biochemical responses and therefore its toxicity potential to bivalves. The heat shock protein 60 (HSP60), extrapallial (EP) fluid protein and triosephosphate isomerase homologous proteins from gills of M. galloprovincialis were down-regulated specifically with the presence of CYN+ C. raciborskii cells. The presence of CYN may lead to additional toxic effects in M. galloprovincialis. This work demonstrates that proteomics is a powerful approach to characterize the biochemical effects of C. raciborskii and to investigate the physiological condition of the exposed organisms.

In vitro toxicological evaluation of essential oils and their main compounds used in active food packaging: A review

Essential oils (EOs) and their main constituent compounds have been extensively investigated due to their application in the food industry for improving the shelf life of perishable products. Although they are still not available for use in food packaging in the market in Europe, considerable research in this field has been carried out recently. The safety of these EOs should be guaranteed before being commercialized. The aim of this work was to review the scientific publications, with a primary focus on the last 10 years, with respect to different in vitro toxicological aspects, mainly focussed on mutagenicity/genotoxicity. In general, fewer genotoxic studies have been reported on EOs in comparison to their main components, and most of them did not show mutagenic activity. However, more studies are needed in this field since the guidelines of the European Food Safety Authority have not always been followed accurately. The mutagenic/genotoxic activities of these substances have been related to metabolic activation. Therefore, in vivo tests are required to confirm the absence of genotoxic effects. Considering the great variability of the EOs and their main compounds, a case-by-case evaluation is needed to assure their safe use in food packaging.

Oxidative stress responses to carboxylic acid functionalized single wall carbon nanotubes on the human intestinal cell line Caco-2

Carbon nanotubes (CNT) are among the more promising nanomaterials due to their potential applications. In this sense, the adverse effects that CNT can induce are of concern. In particular, carboxylic acid functionalized single wall carbon nanotubes (COOH-SWCNT) have shown to reduce cell viability and induce morphological effects on the human intestinal cell line Caco-2, but little is known about the toxic mechanisms involved. The aim of the present study was to investigate the oxidative stress responses of this cell line after 24h exposure to COOH-SWCNT. Biomarkers assayed included lipid peroxidation (LPO), reactive oxygen species (ROS) generation, and enzymatic and non-enzymatic antioxidant defences. Results showed an increase in ROS from 100μg/mL reaching 5.2-fold the basal value at the highest concentration assayed. An induction of catalase, superoxide dismutase and glutathione peroxidase activities was also observed, meanwhile glutathione reductase showed a reduced activity at 1000μg/mL. Glutathione (GSH) levels also decreased (2.5-fold) at the highest level of exposure. Therefore, the antioxidant defences could not overwhelm the oxidative insult caused by COOH-SWCNT and LPO products increased in a concentration-dependent manner. We can conclude that oxidative stress plays a role in the pathogenicity induced by COOH-SWCNT on Caco-2 cells at the concentrations assayed.

Differential oxidative stress responses to pure Microcystin-LR and Microcystin-containing and non-containing cyanobacterial crude extracts on Caco-2 cells

Cyanobacterial blooms are a worldwide problem due to the production of cyanotoxins such as microcystins (MCs), causing serious water pollution and public health hazard to humans and livestock. Oxidative stress plays a significant role in MCs toxicity. In the present work the differential oxidative stress responses to pure MCs, and Microcystin-containing and non-containing cyanobacterial crude extracts on the human colon carcinoma cell line Caco-2 has been studied for the first time. After exposure, cells were collected and the antioxidant enzymes activities superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) were measured. Moreover, lipid peroxidation (LPO) induction, reactive oxygen species (ROS) and reduced glutathione (GSH) content were also analyzed. The oxidative stress biomarkers that experienced higher alterations were ROS, CAT, SOD and GR activities. The MC containing cyanobacterial extract showed the higher toxic effects, followed by pure MC-LR. The non-MC containing cyanobacterial extract showed limited effects mainly in SOD activity, GSH content, and GP and GR activities only at the highest concentration used. These results suggest that MC-LR is the responsible of the oxidative stress responses observed in Caco-2 cells, but other compounds contained in the cyanobacterial extracts can contribute to the toxic effects.

In vitro toxicological assessment of clays for their use in food packaging applications

Montmorillonite based clays have a wide range of applications that are going to contribute to increase human exposure to these materials. One of the most promising uses of clays is the development of reinforced food contact materials that results in nanocomposites with improved barrier properties. Different organoclays have been developed introducing modifiers in the natural clay which is commercially available. However, the toxicological aspects of these materials have been scarcely studied so far. In the present study, the cytotoxic effects of a non-modified clay (Cloisite Na+) and an organoclay (Cloisite 30B) have been investigated in the hepatic cell line HepG2. Only Cloisite 30B showed cytotoxicity. In order to elucidate the toxic mechanisms underlying these effects, apoptosis, inflammation, oxidative stress and genotoxicity biomarkers were assayed. Moreover, a morphology study with light and electron microscopy was performed. Results showed genotoxic effects and glutathione decrease. The most relevant ultraestructural alterations observed were mitochondrial degeneration, dilated endomembrane systems, heterophagosomes formation, fat droplets appearance and presence of nuclear lipid inclusions. Cloisite 30B, therefore, induces toxic effects in HepG2 cells. Further research is needed to assess the risk of this clay on the human health.

Comparison of the toxicity induced by microcystin-RR and microcystin-YR in differentiated and undifferentiated Caco-2 cells.

Cyanobacterial toxins, especially microcystins (MCs), are found in eutrophized waters throughout the world. Acute poisonings on animals and humans have been reported following MC exposure. Around 80 MCs variants have been isolated in surface waters worldwide so far. The toxicity of the most frequent MC congener, MC-LR, is well known; however, studies dealing with MC-RR and MC-YR are less abundant. In this present work, the toxic effects of MC-RR and MC-YR at concentrations of 50, 100, 150 and 200 microM have been investigated in the human colon carcinoma cell line Caco-2 both undifferentiated and differentiated after 24 and 48 h exposure. Toxicity endpoints assessed were cell number by quantification of total protein content of the cell cultures; cell viability by means of neutral red uptake, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) metabolization to detect mitochondrial changes. Moreover, morphological alterations were also investigated. Results showed that protein content was the most sensitive endpoint for MC-RR with reductions of 45% after 48 h exposure to 200 microM MC-RR in differentiated cells (EC(50)>200 microM); whereas for MC-YR is the inhibition of neutral red uptake with reductions higher than 80% at 100 microM in undifferentiated cells after 48 h (EC(50) of 57.3 microM). Furthermore, alteration in the cells was shown in the morphological studies, particularly at high concentrations, undergoing general reduction in cell number and hydropic degeneration. The sensitivity of the cultures to these toxins was highly affected by the exposure time and in a lesser extent by the differentiation state, with MC-YR showing higher toxicity than MC-RR.

Effects of dietary N‐acetylcysteine on the oxidative stress induced in tilapia (Oreochromis Niloticus) exposed to a microcystin‐producing cyanobacterial water bloom

Fish can be exposed to toxic cyanobacterial cells in natural waters and fish farms and suffer from oxidative damage. The present study investigates the effects of N-acetylcysteine (NAC), a glutathione (GSH) precursor, on the oxidative stress induced by Microcystis cyanobacterial cells containing microcystins (MCs) in tilapia fish (Oreochromis niloticus). Variation in lipid peroxidation (LPO) levels, carbonyl group content, reduced glutathione to oxidized glutathione ratio (GSH:GSSG), and catalase (Enzyme Commission [EC] 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1), glutathione reductase (GR; EC 1.8.1.7), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione S-transferase (EC 2.5.1.18) activities in liver and kidney of tilapia exposed to a single oral dose of 120 microg MC-LR (with leucine [L] and arginine [R])/fish and killed in 24 h were investigated in the absence and presence of 20.0, 44.0, and 96.8 mg NAC/fish/d. Results showed a protective role of NAC, depending on the dose and the biomarker considered. The increase in LPO (1.9- and 1.4-fold in liver and kidney, respectively) and the decreased protein content and GSH: GSSG in the liver induced by MCs were recovered mainly by the lower doses of NAC employed. Antioxidant enzyme activities increased (range, 1.4- to 1.7-fold) by MCs also were ameliorated by NAC, although the highest level used induced significant alteration of some enzymatic activities, such as SOD, GPx, and GR. Thus, NAC can be considered to be a useful chemoprotectant that reduces hepatic and renal oxidative stress in the prophylaxis and treatment of MC-related intoxications in fish when careful attention is given to its application dose because of its own pro-oxidant activity, as shown in the present study at 96.8 mg NAC/fish/d.

In vitro pro-oxidant/antioxidant role of carvacrol, thymol and their mixture in the intestinal Caco-2 cell line.

The food industry needs to provide consumers with fresh and healthy products. In this context, food packaging plays an important role. Thus, certain essential oils are being incorporated into plastic polymers to confer better preservative properties. The oregano essential oil contains carvacrol and thymol, two important polyphenols. Considering their increasing use in active food packaging, the evaluation of their suitability and safety is of great interest. In the present work, a concentration-dependent increase in the antioxidant effects of carvacrol, thymol, and their mixture (10:1) was determined using DPPH and ABTS assays. In addition, the safety of these compounds was tested in vitro. Reactive oxygen species and glutathione levels were measured after exposing cells for 24 and 48 h to different concentrations of carvacrol, thymol and their mixture. The abilities of these compounds to protect against or revert the effects of H2O2 on cells were also studied. The results showed that oxidative stress plays a role in the damage induced by carvacrol and the mixture at high concentrations. However, at lower concentrations, both compounds and their mixture were shown, for the first time, to protect cells against the damage induced by the H2O2.

Acute exposure to pure cylindrospermopsin results in oxidative stress and pathological alterations in tilapia (Oreochromis niloticus)

Cylindrospermopsin (CYN) is increasingly recognized as a potential threat to drinking water safety, due to its ubiquity. This cyanotoxin has been found to cause toxic effects in mammals, and although fish could be in contact with this toxin, acute toxicity studies on fish are nonexistent. This is the first study showing that single doses of CYN pure standard (200 or 400 μg CYN/kg fish bw) by oral route (gavage) generate histopathological effects in fish (Tilapia—Oreochromis niloticus) exposed to the toxin under laboratory condition. Among the morphological changes, disorganized parenchymal architecture in the liver, dilated Bowmans space in the kidney, fibrolysis in the heart, necrotic enteritis in the intestines, and hemorrhages in the gills, were observed. Moreover, some oxidative stress biomarkers in the liver and kidney of tilapias were altered. Thus, CYN exposure induced increased protein oxidation products in both organs, NADPH oxidase activity was significantly increased with the kidney being the most affected organ, and decreased GSH contents were also detected in both organs, at the higher dose assayed.