Isabel M. Moreno

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.

Dose-dependent antioxidant responses and pathological changes in tenca (Tinca tinca) after acute oral exposure to Microcystis under laboratory conditions

The effects of cyanobacterial cells containing microcystins (MCs), toxins from cyanobacteria, on oxidative stress biomarkers from liver and kidney of Tenca fish (Tinca tinca) were investigated under laboratory conditions. Moreover, a histopathological study of liver, kidney, heart and intestine tissues was performed. Fish were orally exposed to cyanobacterial cells dosing 0, 5, 11, 25 and 55 microg MC-LR/fish mixed with the food. Results showed a dose-dependent decrease of superoxide dismutase (SOD) activity, and also of catalase (CAT) in the liver. Glutathione levels and protein oxidation, however, were not altered by the exposure to the cyanobacterial material. The microscopic study revealed tissue alterations even at the lower cyanobacterial cells doses. Onion-like hepatocytes in the liver, glomerulopathy in the kidney, loss of myofibrils in the heart and vacuolated enterocytes in the gastrointestinal tract were the main changes observed. These findings suggest that this fresh water fish can be adversely affected by cyanobacterial blooms in their natural habitats.

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.

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain).

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.

Presence and bioaccumulation of microcystins and cylindrospermopsin in food and the effectiveness of some cooking techniques at decreasing their concentrations: a review.

Microcystins (MCs) and cylindrospermopsin (CYN) are among the cyanotoxins which occur naturally, produced by different cyanobacteria species when they grow or proliferate under favorable environmental conditions. From a toxicological point of view, their relevance is due to the deleterious effects that they have been reported to induce in a wide range of organisms, including humans. Cyanotoxins intake from contaminated water and food is an important source of human exposure. Various edible aquatic organisms, plants, and food supplements based on algae, can bioaccumulate these toxins. A thorough review of the scientific data available on this topic is provided, the studies on MCs being much more numerous than those focused on CYN. The scientific literature suggests that these cyanotoxins can be accumulated at concentrations higher than their respective recommended tolerable daily intake (TDI). Finally, the influence of different cooking procedures on their levels in food has been considered. In this regard, again studies on the matter dealing with CYN have been not yet raised. MCs contents have been reported to be reduced in muscle of fish after boiling, or cooking in a microwave-oven, although the effect of other traditional cooking processes such as frying, roasting or grilling have not been demonstrated.

Differentiation of two Canary DO red wines according to their metal content from inductively coupled plasma optical emission spectrometry and graphite furnace atomic absorption spectrometry by using Probabilistic Neural Networks

The metal content of 54 commercialized wines (30 samples from Tacoronte-Acentejo DO (class T) and 24 Valle de la Orotava DO (class O) wines) was performed by ICP-OES (Al, Ba, Cu, Fe, Mn, Sr, Zn, Ca, K, Na and Mg) and GF-AAS (Ni and Pb). Wine samples were processed by dry ashing followed by solution with 5% nitric acid. Metals were considered as suitable descriptors to differentiate between T and O classes. Supervised learning pattern recognition procedures were applied. Linear discriminant analysis (LDA) led to good results up to about 90% of correct classification. In order to improve the results, another kind of algorithms able to model non-linear separation between classes was considered: Probabilistic Neural Networks. Accordingly, excellent results were obtained, leading to sensitivities and specificities higher than 95% for the two classes.

Influence of microcystin-LR on the activity of membrane enzymes in rat intestinal mucosa.

The objective of the present study was to evaluate the effects of microcystin-LR (MCLR) on the activity of membrane enzymes from intestinal mucosa. In addition, serum chemistry and peroxidative status of both serum and intestinal homogenate were evaluated after treatment with MCLR. Wistar rats were treated with intraperitoneal injection of either 100 μg pure MCLR/Kg body weight or saline solution. A significant increase in liver weight and altered serum enzyme activities were found in MCLR-treated rats, indicating damage to the liver in these rats, as previously suggested. A higher specific activity of sucrase (1.5-fold) was observed after the administration of MCLR, whereas other intestinal apical membrane enzymes, such as lactase, maltase and alkaline phosphatase were not modified by the treatment. The specific activities of acid phosphatase and succinate dehydrogenase, markers for lysosomal and mitochondrial membranes, respectively, were also increased (32% and 60%, respectively) in treated rats. The analysis of lipid peroxidation showed that the peroxidative status was increased in both serum and intestinal mucosa from MCLR-treated rats, reflecting an excess production of oxygen free radicals induced by this cyanobacterial toxin. In conclusion, this study shows that acute exposure to MCLR affects the intestinal physiology by modifying the intestinal peroxidation status as well as the activity of membrane enzymes.ResumenEn este trabajo se analiza la acción de la toxina microcistina-LR (MCLR) sobre la actividad de diversas enzimas de membrana en la mucosa intestinal. Para ello, se utilizan ratas Wistar a las que se inyecta por vía intraperitoneal 100 μg MCLR/Kg peso corporal o bien solución salina (grupo control). Asimismo, se realiza un estudio bioquímico en suero, y se determina el grado de peroxidación lipídica en la mucosa intestinal y suero de estos animales tras el tratamiento con MCLR. La toxina induce daño hepático severo en las ratas tratadas, como lo demuestra el aumento del peso del hígado y diversas alteraciones de las enzimas hepáticas en suero. Por lo que respecta a las enzimas de membrana, las ratas tratadas con MCLR presentan un aumento en la actividad de la enzima sacarasa en la mucosa intestinal, no alterándose otras enzimas apicales como la lactasa, maltasa o fosatasa alcalina. MCLR también produce un aumento en la actividad de la fosfatasa ácida y succinato deshidrogenasa, marcadores respectivos de membranas lisosomales y mitocondriales. Además, los niveles de peroxidación lipídica en suero y mucosa intestinal aparecen anormalmente elevados tras el tratamiento, como consecuencia de la producción excesiva de radicales libres de oxígeno inducida por la toxina. Por consiguiente, la intoxicación aguda con MCLR afecta a la fisiología intestinal provocando una modificación del estado peroxidativo y alterando la actividad de las enzimas de membrana intestinales.

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.

Differentiation of Spanish brandies according to their metal content

Eleven metals, namely, aluminium, calcium, cadmium, copper, iron, lead, magnesium, manganese, potassium, sodium and zinc were determined in twenty samples of Sherry brandies and twelve samples of Penedés brandies by applying atomic spectrometry techniques. Flame atomic absorption spectrometry was used for quantitating calcium, copper, iron, magnesium, manganese and zinc; atomic emission spectrometry to determine potassium and sodium; and graphite furnace atomic absorption spectrometry to analyse aluminium, cadmium and lead. A chemometric approach was followed to study the discrimination between brandies from Sherry or Penedés according to the metal profile.

Protective role of vitamin E on the microcystin‐induced oxidative stress in tilapia fish (Oreochromis niloticus)

Microcystins (MCs) are potent hepatotoxins produced by cyanobacteria in water systems that induce oxidative stress in fish. The present study investigated the effect of vitamin E pretreatment on MC-induced oxidative damage in the liver, kidneys, and gills of tilapia fish (Oreochromis niloticus). Groups of fish were fed vitamin E supplements (200 or 700 mg per kilogram of diet) for 7 d or received only commercial fish food and then were exposed to a single oral dose of cyanobacterial cells (120 microg of MC-LR [2:Leu, 4:Arg] per fish), and were sacrificed in 24 h. The potential benefits of vitamin E were evaluated based on lipid peroxidation (LPO), protein oxidation, catalase (CAT, Enzyme Commission [EC] 1.11.1.6), superoxide dismutase (EC 1.15.1.1), glutathione peroxidase (EC 1.11.1.9), glutathione reductase (EC 1.8.1.7), and the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG). Microcystins induced a 1.6-fold increase in LPO values in liver, whereas vitamin E-pretreated fish showed no alteration on this biomarker. Enzyme activities levels were also ameliorated by the chemoprotectant, whereas protein oxidation and GSH/GSSG did not show any significant change. The higher vitamin E dose used proved to have the greater protective effects, particularly on the biomarkers LPO and CAT. The results show that vitamin E could have a potential use as a preventive or therapeutic measure in MC-exposed fish.