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In JoVE (1)
Other Publications (20)
- Talanta
- Toxicon : Official Journal of the International Society on Toxinology
- Environmental Toxicology
- Toxicon : Official Journal of the International Society on Toxinology
- Environmental Toxicology and Chemistry / SETAC
- Toxicon : Official Journal of the International Society on Toxinology
- Toxicology in Vitro : an International Journal Published in Association with BIBRA
- Toxicology in Vitro : an International Journal Published in Association with BIBRA
- Toxicology in Vitro : an International Journal Published in Association with BIBRA
- Toxicon : Official Journal of the International Society on Toxinology
- Archives of Toxicology
- Mutation Research
- Ecotoxicology (London, England)
- Ecotoxicology and Environmental Safety
- Toxicology in Vitro : an International Journal Published in Association with BIBRA
- Ecotoxicology (London, England)
- Aquatic Toxicology (Amsterdam, Netherlands)
- Ecotoxicology and Environmental Safety
- Water Research
- Environmental Toxicology
Articles by Josè Jalife in JoVE
JoVE Clinical and Translational Medicine
High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation
Center for Arrhythmia Research. Internal Medicine, University of Michigan
This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.
Other articles by Josè Jalife on PubMed
Differentiation of Sparkling Wines (cava and Champagne) According to Their Mineral Content
The metal content of a number of sparkling wines was determined by atomic spectrometry techniques. Al, Ba, Ca, Cu, Fe, K, Mg, Mn, Na, P, Sr and Zn by using inductively coupled plasma atomic emission spectrometry (ICP-AES); Cd, Ni and Pb by graphite furnace atomic absorption spectrometry (GFAAS) and As from hydride generation AAS (HGAAS). Two kinds of sparkling wines were studied with D.O. trademark: cava and champagne. 18 samples of "brut" cava and 17 samples of "brut" champagne of different brands were analyzed following the procedure described in the paper. By using the metal concentrations as chemical descriptors the two classes of samples (cava and champagne) are perfectly discriminated, when applying pattern supervised learning recognition techniques such as linear discriminant analysis (LDA) and soft independent modeling of class analogie (SIMCA). The number of false positives and negatives were zero, which indicates a remarkable authentication power of the descriptors used.
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.
Time-dependent Protective Efficacy of Trolox (vitamin E Analog) Against Microcystin-induced Toxicity in Tilapia (Oreochromis Niloticus)
Microcystins (MCs), hepatotoxins from cyanobacteria, induce oxidative stress and pathological changes in fish that can be ameliorated with chemoprotectants such as vitamin E (vit E). This study investigated the time period after MCs exposure in which Trolox, a vitamin E analog, is effective against oxidative and histological damage in different organs of tilapia (Oreochromis niloticus). Fish were fed Trolox supplement (700 mg/kg diet) for 7 days, or received only commercial fish food, and then were exposed to a single oral dose of 120 microg/fish microcystin-LR, and sacrificed in 24, 48, or 72 h. The Trolox protective efficacy was evaluated based on lipid peroxidation (LPO), protein oxidation, enzymatic and non-enzymatic antioxidants, and a morphologic study. Regarding the oxidative stress biomarkers altered by MCs, the higher protective action of Trolox was observed 24 h post toxin exposure, although it extends also until 48 h in gills (superoxide dismutase (SOD), catalase (CAT)), and liver, where glutathione reductase (GR) backed to control values 48 and 72 h after the toxin application. Glutathione-S- transferase (GST) activity in the liver was ameliorated by the chemoprotectant after 24 and 48 h, although control values were not recovered. Trolox modulation of these biomarkers and its ability to quench free radicals explain the recovery of LPO values in all organs at 24 h and also in gills at 48 h. Histopathologically, Trolox efficacy was more evident after 72 h.
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.
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.
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.
The Antioxidant Glutathione in the Fish Cell Lines EPC and BCF-2: Response to Model Pro-oxidants As Measured by Three Different Fluorescent Dyes
Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.
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.
Oxidative Stress Induced by Microcystin-LR on PLHC-1 Fish Cell Line
Increasing evidences suggest that oxidative stress may play a significant role in microcystins (MCs) toxicity not only in mammals, but also in fish. In this regard, many in vivo studies have been performed but little is still known about the alteration of oxidative stress biomarkers on fish cell lines so far. In this study, the toxic effects of MC-LR were investigated in the fish cell line PLHC-1, derived from a hepatocellular carninoma of the topminnow Poeciliosis lucida, after 48 h of exposure. The different response of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST), as well as lipid peroxidation (LPO) as a biomarker of oxygen-mediated toxicity, were assessed in PLHC-1 cells. The increases in the antioxidant enzymatic activities (SOD, GPx, and GST) as well as in LPO values observed evidenced the oxidative stress induced by MC-LR exposure. Moreover, the enhancements of these enzymes could suggest an adaptative response to combat oxidative injure induced by MC-LR, confirming that this mechanism is involved in the damage induced by MCs on fish cells.
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.
Microcystin-LR Induces Toxic Effects in Differentiated and Undifferentiated Caco-2 Cells
Microcystins (MCs) are toxins of heptapeptidic structure produced by toxic cyanobacteria in surface eutrophic waters. MCs are known to be hepatotoxic in humans, but they are also able to induce gastrointestinal alterations, allergic reactions, irritation, and pneumonia-like symptoms. The impact of MC-LR, one of the most common cyanobacterial toxins, was studied on the Caco-2 cell line, a commonly used enterocytic model, established from a human colon carcinoma. Caco-2 cells were differentiated in order to compare the effect of MC-LR in differentiated and non-differentiated cells. They were seeded in a 96-well microtiter plate and treated with MC-LR pure standard (98% purity). The effects of different concentrations of this cyanotoxin (50, 100, 150, and 200 microM) were investigated at 24 and 48 h of exposure by morphological observation and biochemical changes (total protein content, neutral red uptake, and MTS metabolization). Differentiated Caco-2 cells were slightly more sensitive than undifferentiated cells. Moreover, toxic effects induced by MCs were higher at 48 h compared to those observed at 24 h. The most sensitive endpoint for the cell line was the reduction of total protein content. Morphological changes induced by MC-LR were reduction in the cell number and hydropic degeneration, being these alterations more evident 48 h after the exposure to MC-LR.
In Vitro Evaluation of Cytotoxicity and Genotoxicity of a Commercial Titanium Alloy for Dental Implantology
Titanium and its alloys have many applications in dentistry, being used in orthodontics, endodontics, prosthetics and implantology. But the use in the biomedical field depends on its biocompatibility, as the Council Directive 93/42/EEC of 14 June 1993 concerning medical devices has established. The aim of this study was to investigate the cytotoxicity and genotoxicity of a commercial titanium/aluminium/vanadium alloy (Ti-6Al-4V) developed by an innovative sand-blast process with aluminium oxide, and nitric-acid passivation. This procedure created a material with an average surface roughness of 1.73±0.16μm with applications in dental implants. International Organization for Standardization (ISO) procedures 7405:2008 and 10993-5:2009 were used to perform the cytotoxicity tests, and bacterial and cell-mutation assays to evaluate genotoxicity. The results show that this titanium alloy (Ti-6Al-4V) was neither cytotoxic nor genotoxic in any of the tests performed. It can be concluded that this new Ti-6Al-4V material with the roughness characteristics specified shows good biocompatibility and can be considered of choice in dental implantology.
Subchronic Effects of Cyanobacterial Cells on the Transcription of Antioxidant Enzyme Genes in Tilapia (Oreochromis Niloticus)
The increasing occurrence of toxic cyanobacterial blooms in eutrophic water bodies is nowadays of worldwide concern due to their ability to produce toxins such as microcystins (MCs). These cyanobacterial toxins have been shown to affect aquatic organisms such as fish, resulting in oxidative stress. Among the antioxidant enzymes, glutathione peroxidase (GPx) and soluble glutathione-S-transferases (sGST) play an important role in the detoxification of MCs. In the present work tilapia (Oreochromis niloticus) were orally exposed to cyanobacterial cells containing MCs and non-containing MCs for 21 days. The activity and relative mRNA expression by real-time PCR of both enzymes and the GST protein abundance by Western blot analysis were evaluated in liver and kidney. Also the induction of lipid peroxidation (LPO) was assayed. MCs containing cyanobacterial cells induced an increase of LPO products in both organs, and MCs containing and MCs non-containing cyanobacterial cells altered the activity, gene expression and protein abundance of the enzymes, indicating the importance of GPx and sGST in MCs detoxification. Moreover, liver, the main organ involved in biodegradation and biotransformation, experienced an adaptative response to the toxic insult. These results show for the first time that the subchronic exposure to cyanobacterial cells causes changes in antioxidant and detoxification enzymes and that GPx and GST gene expression are good markers of these alterations in tilapia.
Toxicity and Glutathione Implication in the Effects Observed by Exposure of the Liver Fish Cell Line PLHC-1 to Pure Cylindrospermopsin
Cylindrospermopsin (CYN), a cyanotoxin produced by several freshwater cyanobacteria species, has been reported to cause human and animal intoxications. CYN is a potent inhibitor of protein and glutathione synthesis. In order to study these effects, various in vitro models have been used, which are representative of the organs targeted by the toxin. However, studies concerning CYN toxicity to fish species, both in vivo and in vitro, are still very scarce. To our knowledge, this is the first work dealing with the effects of CYN in a fish cell line. In the present work, we tried to test the hypothesis that CYN could be hepatotoxic to fish causing cell damage and oxidative stress, which may lead to pathogenicity. To deal this purpose, PLCH-1 cells, derived from fish liver, were exposed to concentrations that ranged from 0.3 to 40 μg/mL CYN during 24 and 48 h for the cytotoxicity study, and 2, 4 and 8 μg/mL CYN for the oxidative stress assays. The basal cytotoxicity endpoints studied were protein content, neutral red uptake and the tetrazolium salt, MTS, reduction. The biomarkers used for the oxidative stress study were reactive oxygen species (ROS) content, reduced glutathione content and γ-glutamylcysteine synthetase activity. The cytotoxicity endpoints revealed a decrease in the cellular viability in a time and concentration-dependent way. Moreover, when cells were exposed to pure CYN, an increase in the ROS content was observed, being more marked at the higher concentrations used. Finally, the present work shows alterations in GSH content and synthesis due to CYN. Moreover, a relationship between cytotoxic effects and ROS production has been evidenced. The results obtained confirm the alteration on fish liver cells, which should be considered relevant to what it may happen in real scenarios since fish are frequently in contact with this cyanotoxin.
Influence of Carboxylic Acid Functionalization on the Cytotoxic Effects Induced by Single Wall Carbon Nanotubes on Human Endothelial Cells (HUVEC)
A vast variety of nanomaterials have been developed in the recent years, being carbon nanotubes (CNTs) the ones that have attracted more attention, due to its unique properties which make them suitable for numerous applications. Consequently, it is predicted that tons of CNTs will be produced worldwide every year, being its exposure of toxicological concern. Nanomaterials, once into the body, can translocate from the uptake sites to the blood circulation or the lymphatic system, resulting in distribution throughout the body. Thus, the vascular endothelium can be in contact with them and can suffer from their toxic effects. In this regard, the aim of this work was to investigate the cytotoxicity of single-walled carbon nanotubes (SWCNTs) on human endothelial cells evaluating the influence of acid carboxylic functionalization and also the exposure time (24 and 48 h). Biomarkers assessed were neutral red uptake, protein content, a tetrazolium salt metabolization and cell viability by means of the Trypan blue exclusion test. Cells were exposed to concentrations between 0 and 800 μg/mL SWCNTs for 24 and 48 h. Results have shown that both SWCNTs and carboxylic acid functionalized single-walled carbon nanotubes (COOH-SWCNTs) induce toxic effects in HUVEC cells in a concentration- and time-dependent way. Moreover, the carboxylic acid functionalization results in a higher toxicity compared to the SWCNTs.
Acute Effects of Pure Cylindrospermopsin on the Activity and Transcription of Antioxidant Enzymes in Tilapia (Oreochromis Niloticus) Exposed by Gavage
The cyanobacterial toxin cylindrospermopsin (CYN) is a widely distributed contaminant of freshwater systems with the consequent risk for human and wildlife, particularly fish. However, CYN toxicity data on fish are still scarce. It is known that CYN inhibits glutathione synthesis and this could contribute to oxidative damage. In the present work tilapia (Oreochromis niloticus) were exposed by gavage to 200 and 400 μg/kg bw of pure CYN and sacrificed after 24 h. The activity and relative mRNA expression by real-time PCR of antioxidant enzymes glutathione peroxidase (GPx) and soluble glutathione-S-transferases (sGST) and the sGST protein abundance by Western blot analysis were evaluated in liver and kidney. Also the induction of lipid peroxidation (LPO) was assayed. Results show an increase of LPO products in both organs. Moreover, CYN altered the activity, gene expression and protein abundance of the enzymes, indicating the importance of GPx and sGST in CYN pathogenicity. This is the first time that CYN is reported to affect these enzymes in fish and they have shown to be responsive biomarkers of CYN toxicity.
Oxidative Stress Responses in Tilapia (Oreochromis Niloticus) Exposed to a Single Dose of Pure Cylindrospermopsin Under Laboratory Conditions: Influence of Exposure Route and Time of Sacrifice
Cylindrospermopsin (CYN) is a toxin produced by various cyanobacterial species that are increasingly being found in freshwater systems. Although CYN can have toxic effects in humans, domestic animals and wildlife, it has been subject to very little investigation (particularly in fish). It has been reported to deplete the cellular glutathione content but the role of oxidative stress in the pathogenicity of CYN in fish is unknown. For this reason tilapia fish were exposed to 200 μg/kg pure CYN through two different exposure routes-gavage and intraperitoneal injection-and sacrificed after 24 h and 5 days. The results showed an increase in NADPH oxidase activity (a biomarker of reactive oxygen species formation), lipid peroxidation (LPO) and protein oxidation; no changes in DNA oxidation; and a reduction in glutathione levels (GSH) and γ-glutamylcysteine synthetase (GCS) activity, the limiting enzyme in glutathione synthesis. The time of sacrifice had a bigger influence on the results than the exposure route because after 5 days some of the biomarkers assayed had recovered their pre-intoxication levels, which was not the case after 24 h.
Time-dependent Histopathological Changes Induced in Tilapia (Oreochromis Niloticus) After Acute Exposure to Pure Cylindrospermopsin by Oral and Intraperitoneal Route
Although fish and aquatic organisms can be in contact with the cyanotoxin cylindrospermopsin (CYN), toxicological studies are practically nonexistent. CYN has a late and progressive acute toxicity in rodents, but no data have been reported in fish. In this work, tilapia (Oreochromis niloticus) were exposed for the first time to an acute dose of CYN (200 μg/kg fish) by intraperitoneal (i.p.) injection, and the effects were compared with the oral route (gavage). In both cases, fish were sacrificed after 24 h or 5 days of the toxin administration. CYN induced multiorganic damage, being the liver and kidney the main targets of toxicity. The histological findings were more pronounced after i.p. administration (in the liver, kidney, heart, gills) with the exception of the gastrointestinal tract. The time of sacrifice influenced the degree of histological damage in all organs studied, and was more severe after 5 d in comparison to 24 h. Moreover, CYN induced an increase in the average nuclear diameter of hepatocytes in the liver, and decreased cross sections of proximal and distal convoluted tubules in the kidney. The changes in these parameters were also more severe by i.p. route, and with the time of sacrifice, supporting the histopathological results obtained in these organs. Thus, both parameters could be useful for quantifying the extent of the damage in fish after CYN exposure.
Biochemical and Pathological Toxic Effects Induced by the Cyanotoxin Cylindrospermopsin on the Human Cell Line Caco-2
Cylindrospermopsin (CYN), a cyanotoxin produced by several freshwater cyanobacteria, causes human intoxications and animal mortalities. The present study focuses on the cytotoxic effects of CYN on Caco-2 cells at 24 and 48 h. The basal cytotoxicity endpoints studied were total protein content (TP), neutral red uptake (NR) and reduction of the tetrazolium salt (MTS). The effect of non-cytotoxic concentrations of CYN on the generation of intracellular reactive oxygen species (ROS), γ-glutamylcysteine synthetase (GCS) activity and glutathione (GSH) content was also studied and the morphological alterations in the Caco-2 cells subsequent to CYN exposure were recorded. The most sensitive endpoint - the reduction of MTS - showed that the viability of Caco-2 cells after exposure to the highest concentration assayed (40 μg/mL CYN) was reduced by about 90%. Intracellular ROS production increased only when exposed to a concentration of 1.25 μg/mL CYN, while GSH content and GCS activity increased when exposed to 2.5 μg/mL CYN. The main insights provided by the present study are the ultrastructural alterations, which reveal lipid degeneration, mitochondrial damage and nucleolar segregation with altered nuclei. Therefore, it has been demonstrated that CYN can induce toxic effects in Caco-2 cells in a time-concentration dependent manner. Moreover, unlike the cytotoxic and biochemical alterations, which were only evident at higher concentrations, morphological damage at the ultrastructural level was noticeable even at the lowest concentration used.
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 Bowman's 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. © 2012 Wiley Periodicals, Inc. Environ Toxicol, 2012.
