Ana I. Prieto

Time-dependent oxidative stress responses after acute exposure to toxic cyanobacterial cells containing microcystins in tilapia fish (Oreochromis niloticus) under laboratory conditions.

Microcystins (MCs) have been reported to induce oxidative stress in aquatic organisms including fish. The effect of acute exposure to toxic cyanobacterial material containing MCs on antioxidant enzymes and lipid peroxidation has been studied in liver, kidney and gills of tilapia fish (Oreochromis niloticus). Fish were orally exposed to a single dose of cyanobacterial cells containing 120 microg/fish MC-LR and sacrificed at 24 and 72 h. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) enzymes in the studied organs decreased in general 24 and 72 h after the dose application, although elevation of CAT and GR was found in liver at 72 h post exposure in comparison to 24h values. In contrast, the lipid peroxidation level increased significantly in all the studied organs with the liver (3.6-fold) proving to be the most affected. Protein oxidation was also increased 1.5-fold in the liver. However, recovery in these parameters was observed in liver 72 h after exposure. The results show that an acute dose of MCs does not induce an adaptative response of the antioxidant enzymes, as a sub-chronic exposure to MCs in tilapia fish does, but a general decrease in them with an initial recovery of the oxidative damage after 72 h, expressed as enhancement of CAT and GR activities and a reduction of LPO and protein oxidation in comparison to 24h values.

Role for Salmonella enterica enterobacterial common antigen in bile resistance and virulence.

Passage through the digestive tract exposes Salmonella enterica to high concentrations of bile salts, powerful detergents that disrupt biological membranes. Mutations in the wecD or wecA gene, both of which are involved in the synthesis of enterobacterial common antigen (ECA), render S. enterica serovar Typhimurium sensitive to the bile salt deoxycholate. Competitive infectivity analysis of wecD and wecA mutants in the mouse model indicates that ECA is an important virulence factor for oral infection. In contrast, lack of ECA causes only a slight decrease in Salmonella virulence during intraperitoneal infection. A tentative interpretation is that ECA may contribute to Salmonella virulence by protecting the pathogen from bile salts.

Repair of DNA Damage Induced by Bile Salts in Salmonella enterica

Exposure of Salmonella enterica to sodium cholate, sodium deoxycholate, sodium chenodeoxycholate, sodium glychocholate, sodium taurocholate, or sodium glycochenodeoxycholate induces the SOS response, indicating that the DNA-damaging activity of bile resides in bile salts. Bile increases the frequency of GC → AT transitions and induces the expression of genes belonging to the OxyR and SoxRS regulons, suggesting that bile salts may cause oxidative DNA damage. S. enterica mutants lacking both exonuclease III (XthA) and endonuclease IV (Nfo) are bile sensitive, indicating that S. enterica requires base excision repair (BER) to overcome DNA damage caused by bile salts. Bile resistance also requires DinB polymerase, suggesting the need of SOS-associated translesion DNA synthesis. Certain recombination functions are also required for bile resistance, and a key factor is the RecBCD enzyme. The extreme bile sensitivity of RecB−, RecC−, and RecA− RecD− mutants provides evidence that bile-induced damage may impair DNA replication.

Effects of dietary selenium on the oxidative stress and pathological changes in tilapia (Oreochromis niloticus) exposed to a microcystin-producing cyanobacterial water bloom

The present study investigates the role of selenium (Se) supplementation (as sodium selenite) on the oxidative stress and histopathological changes induced by cyanobacterial cells containing microcystins (MCs) in tilapia fish (Oreochromis niloticus). Variation in lipid peroxidation (LPO) levels and carbonyl groups content, reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, and catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities in liver and kidney of tilapia fish exposed to a single oral dose of 120 microg MC-LR/fish and sacrificed in 24 h, were investigated in the absence and presence of 1.5, 3.0 and 6.0 microg Se/g diet. Results showed a protective role of Se depending on the dose and the biomarker considered. Thus, the lower Se dose made CAT, liver GR and kidney SOD converged to basal values, whereas LPO and liver SOD and GST needed the higher dose. Kidney GR, however, was not protected at any Se dose. Moreover, Se has also shown to have a pro-oxidant effect with increased kidney LPO values and liver and kidney GPx activities in MC-free fish. The microscopic study revealed tissue alterations induced by cyanobacterial cells in the liver, kidney, heart and gastrointestinal tract that were ameliorated by the highest Se dose assayed. The level of Se supplementation must be therefore carefully selected to provide beneficial effects and to avoid potential negative consequences.

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.

Effects on growth and oxidative stress status of rice plants (Oryza sativa) exposed to two extracts of toxin-producing cyanobacteria (Aphanizomenon ovalisporum and Microcystis aeruginosa)

Toxic cyanobacteria are considered emerging world threats, being responsible for the degradation of the aquatic ecosystems. Aphanizomenon ovalisporum produces the toxin Cylindrospermopsin (CYN) being a concern in fresh water habitats. This work aims to increase our knowledge on the effects of this toxic cyanobacterium in plants by studying the alterations in growth parameters and oxidative stress status of rice (Oriza sativa) exposed to the cyanobacteria cell extracts containing CYN. Significant increases in glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities were detected in the different experiments performed. The roots showed to be more sensitive than leaves regarding the enzyme activities. A reduction in the leaf tissue fresh weight was observed after 9 days of plant treatment suggesting a major physiological stress. The exposure of rice plants to a mixture of A. ovalisporum and Microcystis aeruginosa cell extracts containing CYN and microcystins including microcystin-LR, resulted in a significant increase in the GST and GPx activities, suggesting a synergistic effect of both extracts. Together these results point out the negative effects of cyanotoxins on plant growth and oxidative status, induced by A. ovalisporum cell extracts, raising also concerns in the accumulation of CYN.

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.

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.

Cyanobacterium producing cylindrospermopsin cause oxidative stress at environmentally relevant concentrations in sub-chronically exposed tilapia (Oreochromis niloticus)

Cylindrospermopsin (CYN) is a potent cyanobacterial cytotoxin produced by certain freshwater cyanobacteria. Structurally, it is an alkaloid with a tricyclic guanidine moiety combined with hydroxymethyluracil. It has proved to be a potent inhibitor of protein synthesis, and to deplete hepatic glutathione. Recently, some studies have shown that CYN produces changes in some oxidative stress biomarkers in fish acutely exposed to pure CYN by oral and intraperitoneal (i.p.) routes. In the present study tilapia (Oreochromis niloticus) were exposed by immersion to lyophilized Aphanizomenon ovalisporum cells added to the aquaria using two concentration levels, 10 or 100 μg CYN L(-1), during two different exposure times: 7 and 14 d. Fish were sacrificed and liver and kidney were extracted. The oxidative status of fish was evaluated by analyzing in both organs the following biomarkers: lipid peroxidation (LPO), protein oxidation, DNA oxidation, reduced-oxidized glutathione ratio (GSH/GSSG), and changes in the activity of Glutathione-S-transferase (GST), Glutathione Peroxidase (GPx), Superoxide dismutase (SOD), Catalase (CAT), and γ-Glutamyl-cysteine synthetase (GCS). In general, major changes were observed in tilapia treated with 100 μg CYN L(-1) after 14 d of exposure. However, some endpoints were altered at the lowest concentration assayed only after 7d of exposure, such as DNA oxidation and γ-GCS in kidney, and CAT and GSH/GSSG decrease in the liver and kidney. The kidney was the most affected organ. These findings confirm that the oxidative stress play a role in the pathogenicity induced by CYN in this fish species, and the results obtained could be useful for future ecotoxicological risks assessment studies, for the protection of fish and aquatic ecosystems. To our knowledge this is the first study dealing with the oxidative stress changes induced by cyanobacterial cells containing CYN and its derivative deoxy-CYN on fish exposed sub-chronically under laboratory conditions.

Acute Effects of Microcystins MC-LR and MC-RR on Acid and Alkaline Phosphatase Activities and Pathological Changes in Intraperitoneally Exposed Tilapia Fish (Oreochromis sp.)

Microcystins (MC) are frequently present in cyanobacterial blooms in rivers and lakes, increasing the risk of toxicity to both animals and humans. There more than eighty reported microcystins, and the present study was undertaken to determine whether MC-LR and MC-RR can induce different enzyme alterations and histopathological changes in tilapia fish (Oreochromis sp.) exposed to a single intraperitoneal (i.p.) injection of the pure standards (MC-LR and MC-RR) at a dose of 500 μg/kg; the tilapia fish were then observed for seven days. The two MC variants caused significant changes in the activities of acid and alkaline phosphatases (ACP and ALP) in vital organs, showing a different response pattern. The livers and kidneys of fish injected with MC-LR were particularly affected. MC-RR induced a very pronounced increase of ACP in the kidney and a significant increase of ALP in the liver. Both MC variants caused pathological lesions in hepatic tissues, such as megalocytosis, necrotic process, and microvesicular steatosis, particularly in fish treated with MC-LR, and degenerative renal changes, glomerulopathy, were more severe in tilapias exposed to MC-RR. In addition, both microcystins also caused significant myopathy in the heart. In contrast, the gills did not show any change in enzyme activity or histopathological injury.