Remedios Guzmán-Guillén

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.

Cylindrospermopsin determination in water by LC‐MS/MS: Optimization and validation of the method and application to real samples

A new method for determining dissolved cylindrospermopsin (CYN) in waters using solid-phase extraction (SPE) with graphitized carbon cartridges and quantification by liquid chromatography coupled with tandem mass spectrometry is described and discussed. The method has been suitably validated: the linear range covered is from 0.900 to 125 µg CYN/L. Limits of detection and quantification were 0.5 and 0.9 µg CYN/L, respectively, and allow CYN determination at concentrations below the guideline proposed of 1 µg CYN/L in natural waters. The method exhibits mean recoveries from 83 to 95%, and intermediate precision (relative standard deviation (%)) values from 5 to 12%, ensuring adequacy against the Association of Official Analytical Chemists guidelines. The method is robust against the following three influential factors considered in the cleanup stage: the batch of the graphitized carbon cartridges, the flow rate of the water sample through the cartridge, and the final redissolved water volume after SPE treatment. The method has been successfully applied to detection and quantification of CYN in water samples from aquaria of a toxicological in vivo laboratory experiment.

Exposure of Lycopersicon Esculentum to Microcystin-LR: Effects in the Leaf Proteome and Toxin Translocation from Water to Leaves and Fruits

Natural toxins such as those produced by freshwater cyanobacteria have been regarded as an emergent environmental threat. However, the impact of these water contaminants in agriculture is not yet fully understood. The aim of this work was to investigate microcystin-LR (MC-LR) toxicity in Lycopersicon esculentum and the toxin accumulation in this horticultural crop. Adult plants (2 month-old) grown in a greenhouse environment were exposed for 2 weeks to either pure MC-LR (100 μg/L) or Microcystis aeruginosa crude extracts containing 100 μg/L MC-LR. Chlorophyll fluorescence was measured, leaf proteome investigated with two-dimensional gel electrophoresis and Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)/TOF, and toxin bioaccumulation assessed by liquid chromatography-mass spectrometry (LC-MS)/MS. Variations in several protein markers (ATP synthase subunits, Cytochrome b6-f complex iron-sulfur, oxygen-evolving enhancer proteins) highlight the decrease of the capacity of plants to synthesize ATP and to perform photosynthesis, whereas variations in other proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and ribose-5-phosphate isomerase) suggest an increase of carbon fixation and decrease of carbohydrate metabolism reactions in plants exposed to pure MC-LR and cyanobacterial extracts, respectively. MC-LR was found in roots (1635.21 μg/kg fw), green tomatoes (5.15–5.41 μg/kg fw), mature tomatoes (10.52–10.83 μg/kg fw), and leaves (12,298.18 μg/kg fw). The results raise concerns relative to food safety and point to the necessity of monitoring the bioaccumulation of water toxins in agricultural systems affected by cyanotoxin contamination.

Development and optimization of a method for the determination of Cylindrospermopsin from strains of Aphanizomenon cultures: Intra-laboratory assessment of its accuracy by using validation standards

The occurrence of cyanobacterial blooms in aquatic environments is increasing in many regions of the world due to progressive eutrophication of water bodies. Because of the production of toxins such as Cylindrospermopsin (CYN), contamination of water with cyanobacteria is a serious health problem around the world. Therefore it is necessary to develop and validate analytical methods that allow us to quantify CYN in real samples in order to alert the public of this toxin. In this work, an analytical method has been developed an optimized for the determination of CYN from Aphanizomenon ovalisporum cultures. The analytical procedure is based on solvent extraction followed by a purification step with graphitized cartridges and CYN quantification by LC-MS/MS. The extraction and purification steps were optimized using a two-level full factorial design with replications. A suitable and practical procedure for assessing the trueness and precision of the proposed method has been applied by using validation standards. The method has been suitably validated: the regression equation was calculated from standards prepared in extracts from lyophilized M. aeruginosa PCC7820 (r(2)≥0.9999) and the linear range covered is from 5 to 500 μg CYN/L, equivalent to 0.18-18.00 μg CYN/g dry weight lyophilized cells. Limits of detection and quantification were 0.04 and 0.15 μg CYN/g, respectively, the recovery range (%) oscillated between 83 and 94% and intermediate precision (RSD %) values from 5.6 to 11.0%. Moreover, the present method showed to be robust for the three factors considered: the batch of the graphitized carbon cartridges, the flow rate of the sample through the cartridge, and the final re-dissolved water volume after SPE treatment, which permits its validation. The validated method has been applied to different lyophilized cultures of A. ovalisporum (LEGE X-001) to evaluate CYN content. This procedure can be used for determining CYN in lyophilized natural blooms samples in environmental studies.

The protective role of l-carnitine against cylindrospermopsin-induced oxidative stress in tilapia (Oreochromis niloticus)

Cylindrospermopsin (CYN) is one of the most important cyanotoxins in terms of both human health and environmental quality and is produced by several different species of cyanobacteria, including Aphanizomenon ovalisporum. The principal mechanisms of action of CYN involve inhibition of protein and glutathione synthesis. In addition, CYN-mediated genotoxicity results from DNA fragmentation. The results of both in vivo and in vitro studies suggest that oxidative stress also plays a significant role in CYN pathogenesis in fish. We investigated the protective effects of l-carnitine (LC) pre-treatment on A. ovalisporum-induced oxidative stress in cells containing CYN and deoxy-CYN, or pure standard CYN, in tilapia (Oreochromis niloticus) that had been acutely exposed via oral administration. Various oxidative stress markers, including lipid peroxidation (LPO), protein oxidation, DNA oxidation, and the ratio of reduced glutathione to oxidised glutathione (GSH/GSSG), and the activities of NADPH oxidase, superoxide dismutase (SOD), catalase (CAT), and gamma-glutamyl-cysteine synthetase (γ-GCS), were evaluated in the livers and kidneys of fish in the absence and presence of 400 or 880mgLC/kgfish/day during a 21 day period prior to CYN-intoxication. The results of our study demonstrated for the first time the beneficial antioxidant effects of LC dietary supplementation on oxidative stress status in fish. No pro-oxidant effects were detected at any of the LC doses assayed, suggesting that LC is a chemoprotectant that reduces hepatic and renal oxidative stress and may be effective when used for the prophylaxis and treatment of CYN-related intoxication in fish.

Effects of depuration on oxidative biomarkers in tilapia (Oreochromis niloticus) after subchronic exposure to cyanobacterium producing cylindrospermopsin

Cylindrospermopsin (CYN) is a cytotoxic polyketide-derived alkaloid produced by several freshwater cyanobacterial species. It is now considered the second most studied cyanotoxin worldwide. Among the toxic mechanisms suggested for CYN pathogenicity are inhibition of protein and glutathione synthesis, genotoxicity by DNA fragmentation, and oxidative stress. The study of depuration of cyanobacterial toxins by aquatic organisms, particularly by fish, is important for fish economy and public health, but in the case of CYN is practically nonexistent. In this work, we investigated the efficiency of two distinct depuration periods, 3 or 7d, in a clean environment, as a mean of restoring the levels of several oxidative stress biomarkers in tilapia (Oreochromis niloticus) subchronically exposed to CYN by immersion in an Aphanizomenon ovalisporum culture (by adding 10 μg CYN/L every two days during 14 d). Lipid peroxidation (LPO) and DNA oxidation returned to normal values after 7d of depuration, whereas the time needed for restoring of the oxidatively damaged proteins was longer. Superoxide dismutase (SOD) and gamma-glutamyl-cysteine-synthetase (γ-GCS) activities recovered after just 3d of depuration, while catalase (CAT) activity needed up to 7d to return to control values. Ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) returned to control levels after 7d of depuration in both organs. These results validate the depuration process as a very effective practice for detoxification in fish contaminated with these toxins.

CYN determination in tissues from freshwater fish by LC–MS/MS: Validation and application in tissues from subchronically exposed tilapia (Oreochromis niloticus)

Harmful cyanobacterial blooms are occurring in eutrophic freshwater lakes and reservoirs throughout the world and, because of the production of toxins such as cylindrospermopsin (CYN), they can present a public safety hazard through contamination of seafood and fish for human consumption. Therefore it is important to develop methods to determine CYN at trace levels in those organisms. A new method for unconjugated CYN determination in tissues (liver and muscle) of tilapia (Oreochromis niloticus) is herein described and discussed; it is based on solvent extraction and purification with C18 and graphitized carbon cartridges, and quantification by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The method was optimized and suitably validated, with a linear range from 0.125-12.5 µg CYN/g dry weight (dw) in the case of the liver, and 0.02-1 µg CYN/g dw for the muscle. Limits of detection and quantitation were 0.07 and 0.12 µg/g dw for the liver, and 0.002 and 0.007 µg/g dw for the muscle, respectively. Mean recoveries ranged 80-110% in liver, and 94-104% in muscle, and intermediate precision values from 6 to 11%. The method is robust against the three factors considered for purification (batch of the graphitized carbon cartridges, time for the sample to pass through the cartridge, and final dissolving water volume). Furthermore, it has been successfully applied to the extraction and quantification of CYN in tissue samples from tilapia subchronically exposed to CYN in the laboratory. This represents a sensitive, reproducible, accurate, and robust method for extraction and determination of unconjugated CYN in tissues of fish exposed to the toxin. This procedure can be used for confirmatory routine monitoring of CYN in fish samples in environmental studies.

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.

Cyanobacterium producing cylindrospermopsin cause histopathological changes at environmentally relevant concentrations in subchronically exposed tilapia (Oreochromis niloticus).

The acute toxicity of cylindrospermopsin (CYN) has been established in rodents, based on diverse intraperitoneal an oral exposure studies and more recently in fish. But no data have been reported in fish after subchronic exposure to cyanobacterial cells containing this cyanotoxin, so far. In this work, tilapia (Oreochromis niloticus) were exposed by immersion to lyophilized Aphanizomenon ovalisporum cells added to the aquaria using two concentration levels of CYN (10 or 100 μg CYN L−1) and deoxy‐cylindrospermopsin (deoxy‐CYN) (0.46 or 4.6 μg deoxy‐CYN L−1), during two different exposure times: 7 or 14 d. This is the first study showing damage in the liver, kidney, hearth, intestines, and gills of tilapia after subchronic exposure to cyanobacterial cells at environmental relevant concentrations. The major histological changes observed were degenerative processes and steatosis in the liver, membranous glomerulopathy in the kidney, myofibrolysis and edema in the heart, necrotic enteritis in the gastrointestinal tract, and hyperemic processes in gill lamellae and microhemorrhages. Moreover, these histopathological findings confirm that the extent of damage is related to the CYN concentration and length of exposure. Results from the morphometric study indicated that the average of nuclear diameter of hepatocytes and cross‐sections of proximal and distal convoluted tubules are useful to evaluate the damage induced by CYN in the main targets of toxicity.

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.