Effects of depuration on histopathological changes in tilapia (Oreochromis niloticus) after exposure to cylindrospermopsin.

Cylindrospermopsin (CYN) is a highly water-soluble cytotoxin produced by several species of freshwater cyanobacteria and it is considered the second most studied cyanotoxin worldwide. CYN acts as a potent protein and glutathione synthesis inhibitor, as well as inducing genotoxicity, oxidative stress and histopathological alterations. Studies concerning the depuration of cyanobacterial toxins in aquatic organisms, especially in fish, are of great interest for fish economy and public health, but are scarce in the case of CYN. This is the first study reporting the ability of depuration (3 - 7 days) in reversing or ameliorating the histopathological lesions induced in liver, kidney, heart, intestines, and gills of tilapia (Oreochromis niloticus) due to exposure by immersion to repeated doses of a CYN-containing culture of A. ovalisporum for 14 days. The main histopathological changes induced by CYN were glucogenic degeneration and loss of the normal hepatic cord-structure (liver), hyperemia, dilated Bowman's capsule and cellular tumefaction (kidney), myofibrolysis, hemorrhages and edema (heart), necrosis and partial loss of microvilli (gastrointestinal tract), and hyperemia and inflammatory cells infiltrates (gills). After 3 days of depuration, gills were totally recovered, while the liver, kidney, and gastrointestinal tract required 7 days, and longer depuration periods may be needed for a full recovery of the heart. In addition, the morphometric study indicated that depuration managed to reverse the affectation in the hepatocytes nuclear diameters and cross sections of the proximal and distal convoluted tubules induced in CYN-exposed fish. In general, these results validate depuration as an effective practice for detoxification of fish contaminated with CYN. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1318-1332, 2017.

Vitamin E pretreatment prevents histopathological effects in tilapia (Oreochromis niloticus) acutely exposed to cylindrospermopsin.

Cylindrospermopsin (CYN) is a cyanotoxin frequently involved in blooms with a predominantly extracellular availability, which makes it easily taken up by a variety of aquatic organisms. CYN is a potent protein and glutathione synthesis inhibitor, and also induces genotoxicity, oxidative stress and several histopathological lesions. The present study investigates the protective role of a vitamin E pretreatment (700 mg vit E/kg fish bw/day, for 7 days) on the histopathological alterations induced in different organs of tilapia (Oreochromis niloticus) acutely exposed to a single oral dose of 400 µg pure CYN/kg bw fish. The major histological changes observed were degenerative glucogenic process and loss of the hepatic structure in the liver, glomerulopathy and tubular tumefaction in the kidney, myofibrolysis and edema in the heart, catarrhal enteritis and necrosis in the gastrointestinal tract, hyperemic processes in the gill lamellae, and high basophilia, degeneration and tumefaction of granular neurons in the brain. Vitamin E pretreatment was effective in preventing or ameliorating the abovementioned alterations induced by CYN. In addition, a morphometric study indicated that the average nuclear diameter of hepatocytes, and cross-sections of proximal and distal convoluted tubules, together with the cardiac fiber and capillaries diameters represent a useful tool to evaluate the damage induced by CYN. This is the first study reporting vitamin E prevention of histopathological damage in tissues (liver, kidney, heart, gastrointestinal tract, gills and brain) of fish intoxicated with CYN. Therefore, vitamin E can be considered a useful chemoprotectant in the treatment of histopathological changes induced in CYN-intoxicated fish. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1469-1485, 2016.

Hyperoxia but not AOX expression mitigates pathological cardiac remodeling in a mouse model of inflammatory cardiomyopathy.

Constitutive expression of the chemokine Mcp1 in mouse cardiomyocytes creates a model of inflammatory cardiomyopathy, with death from heart failure at age 7-8 months. A critical pathogenic role has previously been proposed for induced oxidative stress, involving NADPH oxidase activation. To test this idea, we exposed the mice to elevated oxygen levels. Against expectation, this prevented, rather than accelerated, the ultrastructural and functional signs of heart failure. This result suggests that the immune signaling initiated by Mcp1 leads instead to the inhibition of cellular oxygen usage, for which mitochondrial respiration is an obvious target. To address this hypothesis, we combined the Mcp1 model with xenotopic expression of the alternative oxidase (AOX), which provides a sink for electrons blocked from passage to oxygen via respiratory complexes III and IV. Ubiquitous AOX expression provided only a minor delay to cardiac functional deterioration and did not prevent the induction of markers of cardiac and metabolic remodeling considered a hallmark of the model. Moreover, cardiomyocyte-specific AOX expression resulted in exacerbation of Mcp1-induced heart failure, and failed to rescue a second cardiomyopathy model directly involving loss of cIV. Our findings imply that mitochondrial involvement in the pathology of inflammatory cardiomyopathy is multifaceted and complex.

Estimation of dietary intake and target hazard quotients for metals by consumption of wines from the Canary Islands.

This paper describes the impact of mineral content on wines and assesses the potential health risk from consuming these wines from Canary Islands. The metal content (B, Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Zn) of red wines belonging to different regions in the Canary Islands was determined by ICP-OES. The studied wine regions were Valle de la Orotava, Tacoronte-Acentejo, Ycoden-Daute-Isora, Abona and Valle de Güimar in Tenerife Island and only one in La Gomera and La Palma Islands. According to the content found, elements could be classified in two categories: the main group including Ca, K, Mg, Na, and the ''minor'' set consisting of B, Co, Cr, Cu, Fe, Li, Mn, Mo, Ni, Pb and Zn. Once calculated the metal intake through red wines consumption, we can conclude that Canarian drinkers are not exposed to unsafe levels of the metals studied, actually, the safety intake limits (daily) ranges between 0.9% in Zn and 2% in Cu, for normal drinkers. And also it has been demonstrated the good quality of Canarian red wines and there is no reason for health concern through the THQ calculation being the highest values determined in La Gomera wines.

Cylindrospermopsin induces neurotoxicity in tilapia fish (Oreochromis niloticus) exposed to Aphanizomenon ovalisporum.

Cylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of freshwater cyanobacteria, such as Aphanizomenon ovalisporum. CYN is a tricyclic alkaloid known for its ability to inhibit both protein and glutathione synthesis, and the alteration of different oxidative stress biomarkers in mammals and vertebrates. Although the liver and kidney appear to be the main CYN targets for this toxin, it also affects other organs. In fish, there is no evidence about the neurotoxicity of CYN yet. In the present study, we aimed to study the potential neurotoxicity of CYN, based on the measure of Acetylcholinesterase (AChE) activity, lipid peroxidation (LPO) levels and histopathological studies in brain of tilapia (Oreochromis niloticus) subchronically exposed to repeated concentrations of 10µg CYN/L by immersion in an A.ovalisporum culture for 14 days. The results showed significant inhibition of AChE activity and increases in LPO levels, as well as relevant histopathological alterations in the brain of fish (O. niloticus) subchronically exposed to the toxin. Moreover, we also investigated the potential recovery of these parameters by subjecting the fish to two depuration periods (3 and 7 days) in clean uncontaminated water, showing a recovery of the biochemical parameters since 3 days of depuration, and being necessary 7 days to recover the histopathological changes. In order to support these results, CYN was detected and quantified by enzyme-linked immunosorbent assay (ELISA) in brain of all the exposed fish and the effects of the depuration periods were also observed. Based on these results, it was demonstrated for the first time the neurotoxicity of CYN and its presence in brain of tilapia fish subchronically exposed to CYN.

Decomposition of microcystin-LR, microcystin-RR, and microcystin-YR in water samples submitted to in vitro dissolution tests.

The presence of cyanobacterial toxins (microcystins) in waters and food increases the risk of toxicity to animal and human health. These toxins can degrade in the human gastrointestinal tract before they are absorbed. To evaluate this possible degradation, water samples spiked with known concentrations of microcystins MC-LR, MC-RR, and MC-YR, which are the toxins most commonly produced by such toxic cyanobacteria as Microcystis aeruginosa, Oscillatoria spp., and Nostoc spp., were submitted to a dissolution test that used gastric and intestinal fluids according to U.S. Pharmacopeia conditions. HPLC with UV detection was used to determine the toxins before and after treatments. This study revealed enzymatic alterations in gastric conditions for all the toxins assayed. MC-RR was the toxin most affected: its range of inactivation was 49-64%. The percentage of degradation for MC-YR and MC-LR was around 30%. However, none was degraded by intestinal digestion.

Influence of two depuration periods on the activity and transcription of antioxidant enzymes in tilapia exposed to repeated doses of cylindrospermopsin under laboratory conditions.

The cyanobacterial toxin Cylindrospermopsin (CYN), a potent protein synthesis inhibitor, is increasingly being found in freshwater bodies infested by cyanobacterial blooms worldwide. Moreover, it has been reported to be implicated in human intoxications and animal mortality. Recently, the alteration of the activity and gene expression of some glutathione related enzymes in tilapias (Oreochromis niloticus) exposed to a single dose of CYN has been reported. However, little is known about the effects induced by repeated doses of this toxin in tilapias exposed by immersion and the potential reversion of these biochemical alterations after two different depuration periods (3 or 7 days). In the present study, tilapias were exposed by immersion to repeated doses of a CYN-containing culture of Aphanizomenon ovalisporum during 14 days, and then were subjected to depuration periods (3 or 7 days) in clean water in order to examine the potential reversion of the effects observed. The activity and relative mRNA expression by real-time polymerase chain reaction (PCR) of the antioxidant enzymes glutathione peroxidase (GPx) and soluble glutathione-S-transferases (sGST), and also the sGST protein abundance by Western blot analysis were evaluated in liver and kidney of fish. Results showed significant alterations in most of the parameters evaluated and their recovery after 3 days (GPx activity, sGST relative abundance) or 7 days (GPx gene expression, sGST activity). These findings not only confirm the oxidative stress effects produced in fish by cyanobacterial cells containing CYN, but also show the effectiveness of depuration processes in mitigating the CYN-containing culture toxic effects.

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.

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.

Determination of microcystins in natural blooms and cyanobacterial strain cultures by matrix solid-phase dispersion and liquid chromatography-mass spectrometry.

An analytical procedure based on matrix solid-phase dispersion (MSPD) and liquid chromatography-mass spectrometry (LC-MS) was developed for determining three microcystins (MCs) in natural water blooms and cyanobacteria strain cultures. The procedure involves sample homogenization with C(18), washed with dichloromethane to eliminate interfering compounds, and elution with acidic methanol. Results were compared to those achieved by using an organic solvent standard method. Mean recoveries of MCs with MSPD were 85-92% with intra-day relative standard deviation (RSDs) of 9-19%, whereas organic solvent extraction resulted in recovery rates of 92-105% with intra-day RSDs ranging from 8 to 18%. Limits of quantification (LOQs) were 1 microg g(-1) dry weight for the MCs either by MSPD or organic solvent extraction. The two analytical methods tested were specific and sensitive to the extraction of MCs and were applied to the detection of MCs in water blooms and culture strains. The concentration of MCs varied from 7 to 3,330 microg g(-1) of lyophilized cells with MC-LR always showing the highest concentration. MCs levels were higher in culture strains than in water blooms, except for MC-LR, whose concentration in blooms was slightly superior to that determined in culture strains.

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 mg mcyst-LR equivalentúL-1 in Valdelacalzada and 0.10-11.3 µg mcyst-LR equivalentúL-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.