RESUMEN
The objective of this study was to determine the structure of the helminth fauna and identify the macroscopic and histopathological alterations associated with parasitic infections in Phrynops geoffroanus. Freshwater turtles of both sexes were captured during the dry and rainy seasons in four municipalities along the Capibaribe River. The study included 63 animals, of which 79.37% (50/63) were parasitized by one or more helminths. In total, 933 helminths of seven taxa were recovered: Serpinema monospiculatus, Spiroxys figueiredoi, Nematophila grandis, Polystomoides brasiliensis, Cheloniodiplostomum testudinis, Telorchis birabeni, and Prionosomoides scalaris. Monogeneans and digenetic trematodes were more sensitive to environmental pressures, since the prevalences varied significantly between areas. Nematodes proved to be more resistant to environmental pressure and caused severe injuries to their hosts: nodules in the stomach and small intestine, adhesions in the liver capsule, and pulmonary emphysema. Parasitic granulomas were recorded at the infection sites and in the lungs and liver, the latter caused by migration of S. figueiredoi larvae. This is the first record of P. brasiliensis, N. grandis, C. testudinis, and T. birabeni parasitizing P. geoffroanus in the state of Pernambuco. Histopathology proved to be an important tool for studies on the impact of parasites at the individual, population, and ecosystem levels. Considering the use of the Capibaribe River for public water supply, fishing, and other activities, within the One Health perspective, this study demonstrates that the anthropogenic impact affects parasites and their hosts, in addition to the human population that uses this ecosystem.
Asunto(s)
Helmintos , Enfermedades Parasitarias , Tortugas , Animales , Efectos Antropogénicos , Ecosistema , Femenino , Masculino , Ríos , Tortugas/parasitologíaRESUMEN
We recently demonstrated that HepaRG cells encapsulated into 1.5% alginate beads are capable of self-assembling into spheroids. They adequately differentiate into hepatocyte-like cells, with hepatic features observed at Day 14 post-encapsulation required for external bioartificial liver applications. Preliminary investigations performed within a bioreactor under shear stress conditions and using a culture medium mimicking acute liver failure (ALF) highlighted the need to reinforce beads with a polymer coating. We demonstrated in a first step that a poly-l-lysine coating improved the mechanical stability, without altering the metabolic activities necessary for bioartificial liver applications (such as ammonia and lactate elimination). In a second step, we tested the optimized biomass in a newly designed perfused dynamic bioreactor, in the presence of the medium model for pathological plasma for 6 h. Performances of the biomass were enhanced as compared to the steady configuration, demonstrating its efficacy in decreasing the typical toxins of ALF. This type of bioreactor is easy to scale up as it relies on the number of micro-encapsulated cells, and could provide an adequate hepatic biomass for liver supply. Its design allows it to be integrated into a hybrid artificial/bioartificial liver setup for further clinical studies regarding its impact on ALF animal models.
Asunto(s)
Alginatos/química , Células Inmovilizadas/metabolismo , Hepatocitos/metabolismo , Hígado Artificial , Hígado/metabolismo , Polilisina/química , Reactores Biológicos , Línea Celular , HumanosRESUMEN
Marine neurotoxins accumulate in seafood and therewith represent a threat for consumers. At the European level, the use of in vivo bioassays is banned from 2015 onwards, except for the control of production areas. Cytotoxicity in the neuro-2a assay has been shown a promising in vitro alternative. However, given that cytotoxicity may be sensitive to confounding factors the current study investigates the suitability of functional endpoints as alternatives to cytotoxicity for the detection of marine neurotoxins. Microarray analyses were performed following exposure of neuro-2a cells to three marine neurotoxins (palytoxin (PlTx), saxitoxin (STX) and tetrodotoxin (TTX)) to identify genes up- or down-regulated that can be used as biomarkers for screening purposes. In addition to microarrays, the voltage dependent fluorescent probe bisoxonol was used to assess changes in cellular membrane potential. Biomarkers based on mRNA expression were detected for PlTx but not for STX and TTX. STX and TTX decreased the fluorescence of bisoxonol while PlTx showed no effect. When using cytotoxicity as the read out the neuro-2a assay detects these three neurotoxins at similar concentrations. Therefore it is concluded that the newly investigated endpoints in the neuro-2a assay are not preferred over cytotoxicity in a suitable broad and sensitive bioassay for the detection of marine neurotoxins in real practice.
Asunto(s)
Acrilamidas/toxicidad , Saxitoxina/toxicidad , Tetrodotoxina/toxicidad , Animales , Bioensayo , Biomarcadores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Venenos de Cnidarios , Determinación de Punto Final , Fluorescencia , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones , Neuroblastoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on either the Na(+), K(+), or Ca(2+) channels or the Na(+)/K(+) ATP-ase pump, on the beating was assessed. Diphenhydramine, veratridine, isradipine, verapamil and ouabain induced specific beating arrests that were reversible and none of the concentrations tested induced cytotoxicity. Three K(+) channel blockers, amiodarone, clofilium and sematilide, and the Na(+)/K(+) ATPase pump inhibitor digoxin had no specific effect on the beating. In addition, two marine neurotoxins i.e. saxitoxin and tetrodotoxin elicited specific beating arrests in cardiomyocytes. Comparison of the results obtained with cardiomyocytes to those obtained with the neuroblastoma neuro-2a assay revealed that the cardiomyocytes were generally somewhat more sensitive for the model compounds affecting Na(+) and Ca(2+) channels, but less sensitive for the compounds affecting K(+) channels. The stem cell-derived cardiomyocytes were not as sensitive as the neuroblastoma neuro-2a assay for saxitoxin and tetrodotoxin. It is concluded that the murine stem cell-derived beating cardiomyocytes provide a sensitive model for detection of specific neurotoxins and that the neuroblastoma neuro-2a assay may be a more promising cell-based assay for the screening of marine biotoxins.
Asunto(s)
Alternativas a las Pruebas en Animales , Cardiotoxinas/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Neurotoxinas/toxicidad , Saxitoxina/toxicidad , Tetrodotoxina/toxicidad , Animales , Canales de Calcio/fisiología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Ratones , Miocitos Cardíacos/fisiología , Canales de Potasio/fisiología , Canales de Sodio/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiologíaRESUMEN
SCOPE: At the European level, detection of marine neurotoxins in seafood is still based on ethically debated and expensive in vivo rodent bioassays. The development of alternative methodologies for the detection of marine neurotoxins is therefore of utmost importance. We therefore investigated whether and to what extent a multielectrode array (MEA) approach can be used as an in vitro alternative for screening of marine neurotoxins potentially present in seafood. METHODS: This MEA approach utilizes rat cortical neurons comprising a wide range of ion channels/pumps and neurotransmitter receptors targeted by marine neurotoxins. We tested the effects of neurotoxic model compounds, pure marine neurotoxins, and extracts from contaminated seafood on neuronal activity of rat cortical neurons cultured on commercial 48-well plates to increase throughput. CONCLUSION: We demonstrate that the MEA approach has a sensitivity of 88% (7/9 model compounds, 6/6 pure marine neurotoxins, and 2/2 marine neurotoxins present in seafood extracts were correctly identified) and a good reproducibility compared to existing in vitro alternatives. We therefore conclude that this MEA-based approach could be a valuable tool for future food safety testing.
Asunto(s)
Contaminación de Alimentos/análisis , Inocuidad de los Alimentos/métodos , Neuronas/efectos de los fármacos , Neurotoxinas/análisis , Alimentos Marinos/análisis , Acrilamidas/análisis , Alternativas al Uso de Animales , Animales , Células Cultivadas , Ciguatoxinas/análisis , Venenos de Cnidarios , Peces , Ácido Kaínico/análogos & derivados , Ácido Kaínico/análisis , Toxinas Marinas/análisis , Oxocinas/análisis , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Tetrodotoxina/análisisRESUMEN
Marine biotoxins can accumulate in fish and shellfish, representing a possible threat for consumers. Many marine biotoxins affect neuronal function essentially through their interaction with ion channels or receptors, leading to different symptoms including paralysis and even death. The detection of marine biotoxins in seafood products is therefore a priority. Official methods for control are often still using in vivo assays, such as the mouse bioassay. This test is considered unethical and the development of alternative assays is urgently required. Chemical analyses as well as in vitro assays have been developed to detect marine biotoxins in seafood. However, most of the current in vitro alternatives to animal testing present disadvantages: low throughput and lack of sensitivity resulting in a high number of false-negative results. Thus, there is an urgent need for the development of new in vitro tests that would allow the detection of marine biotoxins in seafood products at a low cost, with high throughput combined with high sensitivity, reproducibility, and predictivity. Mode of action based in vitro bioassays may provide tools that fulfil these requirements. This review covers the current state of the art of such mode of action based alternative assays to detect neurotoxic marine biotoxins in seafood.
Asunto(s)
Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Neurotoxinas/análisis , Alimentos Marinos/análisis , Animales , Bioensayo/métodos , Cadena Alimentaria , Ratones , Neurotoxinas/toxicidad , RatasRESUMEN
In order to have a general view of metabolic requirements during swimming, in vitro aerobic and anaerobic fluxes were measured in red and white muscles from silver eels and yellow eels which differ in activity levels and nutritional states. These measurements were performed in control eels and after a 4 day swimming session (70% U(crit) in yellow eels, 80% U(crit) in silver eels). A swimming session significantly increases U(crit) from 12% to 18%, depending on the stage, with a significantly higher in vitro energy cost during the yellow stage at the muscle level. In vitro, the swimming session brings about a gain in anaerobic capacities rather than in aerobic ones. Some in vivo hypotheses are proposed.