Un model d'estrès oxidatiu en embrió de peix zebra. Aplicació per a l'avaluació de l'activitat protectora de substàncies antioxidants.

[cat] L'estrès oxidatiu és el resultat del desequilibri entre la producció d'espècies reactives i l'activitat dels sistemes de defensa antioxidants. Les estratègies químiques i cultius cel•lulars utilitzats habitualment per a l'avaluació de l'activitat antioxidant de compostos presents en els aliments, no tenen la capacitat de predir clarament la seva eficàcia in vivo, ni de determinar els mecanismes d'acció pels quals actuen. L'embrió de peix zebra és un model animal que ha estat utilitzat per a l'estudi de processos oxidatius, ja que presenta gens i enzims antioxidants anàlegs als sistemes dels mamífers. Els objectius de la tesi doctoral han inclòs el desenvolupament d'un model d'estrès oxidatiu basat en l'embrió de peix zebra per a l'avaluació de l'activitat antioxidant de diferents compostos i l'avaluació de l'efecte antioxidant in vivo i l'efecte neuroprotector de compostos polifenòlics presents en el vi negre, mitjançant el model d'estrès oxidatiu establert. Així mateix, s'han estudiat els efectes de l'estrès oxidatiu i d'alguns antioxidants en el model de neuroesferes humanes. Primerament, s'han caracteritzat els efectes de diferents compostos inductors d'estrès oxidatiu en el creixement i el desenvolupament embrionari del peix zebra. La confirmació del mecanisme oxidatiu en els efectes observats en l'exposició als inductors d'estrès oxidatiu, s'ha realitzat mitjançant la modulació de la resposta a estrès oxidatiu amb compostos que poden augmentar o disminuir ('estatus antioxidant. Si el compost estudiat actua per un mecanisme d'estrès oxidatiu s'observen diferències estadísticament significatives en la comparació de les corbes concentració-resposta de la modulació i de l'exposició única als inductors d'estrès oxidatiu. S'han seleccionat els inductors tert-butil hidroperòxid (tBOOH) i 6- hidroxidopamina (6-ONDA) per a ser utilitzats en el model d'estrès oxidatiu. La verificació de la capacitat del model per a detectar l'efecte antioxidant dels compostos s'ha portat a terme amb diferents compostos amb activitat antioxidant coneguda. S'ha confirmat l'efecte antioxidant dels compostos estudiats i la capacitat del model d'estrès oxidatiu establert per a identificar efectes antioxidants in vivo. La identificació dels compostos polifenòlics presents en una mostra de vi negre desalcoholitzada i en una mostra d'homogenat d'embrions de peix zebra que havien estat exposats a aquesta s'ha realitzat mitjançant la tècnica HPLC-LTQ-Orbitrap-MS. El model d'estrès oxidatiu amb tBOOH s'ha aplicat als compostos polifenòlics identificats amb major intensitat en la mostra de vi negre i s'ha observat un efecte antioxidant in vivo en tots ells. Els compostos polifenòlics de la mostra de vi negre també han produït un augment significatiu de la distància total recorreguda pels embrions i un augment significatiu del nombre de neurones dopaminèrgiques quan s'ha aplicat el model d'estrès oxidatiu amb 6-ONDA, indicant un efecte neuroprotector d'aquests compostos en els embrions de peix zebra. Els efectes de la TCHQ com a inductor d'estrès oxidatiu i dels antioxidants hesperetina i kaempferol s'han estudiat en el model de neuroesferes humanes. La teoria de l'hormesi com a possible estratègia antioxidant, s'ha aplicat en les neuroesferes i s'ha observat que la pre-exposició d'aquestes a una concentració baixa de TCHQ protegeix enfront als efectes oxidants d'aquest compost en les neuroesferes. Així mateix, aquesta teoria s'ha reproduït en el model d'embrió de peix zebra. La pre-exposició dels embrions a una concentració baixa de TCHQ produeix un efecte protector hormètic antioxidant enfront als efectes oxidants d'aquest compost en els embrions de peix zebra. En resum, mitjançant la integració i combinació de diferents procediments experimentals, s'ha establert un model d'estrès oxidatiu en embrió de peix zebra que permet l'avaluació in vivo dels efectes antioxidants dels compostos i d'altres estratègies antioxidants. El model establert s'ha aplicat a l'avaluació dels efectes antioxidants i neuroprotectors de compostos polifenòlics del vi negre.[eng] Oxidative stress is the result of an imbalance between reactive species production and antioxidant defence systems activity. Chemical strategies usually used for the evaluation of antioxidant activity are not able to predict antioxidant efficiency in vivo. The zebrafish embryo is a biological model which has been used in the study of oxidative processes. In the present work, an oxidative stress model in vivo based on the zebrafish embryo technique has been developed. The effects of oxidative stress inducers on the zebrafish embryo development have been characterised and its oxidative mechanism has been confirmed by strategies of modulation of the oxidative stress response. Tert-butyl hydroperoxide (tBOOH) and 6-hydroxy dopamine (6-OHDA) were selected as the inducers to be used in the oxidative stress model. The capacity of the model to detect in vivo antioxidant effects of the compounds was performed with well-known antioxidant compounds. Polyphenolic compounds present in a red wine sample and in the homogenate of zebrafish homogenate were identified by the HPLC-LTQ-Orbitrap-MS technique. The compounds identified with major intensity were evaluated with the oxidative stress model and an in vivo antioxidant effect in all of them was established. The polyphenolic compounds also produced a neuroprotective effects in dopaminergic neurons and protected them against the neurotoxic effects of 6-OHDA. The human neurosphere model was applied to the study of the effects of oxidative stress inducers and antioxidants. The hormesis paradigm was studied and confirmed as an antioxidant strategy. Pre-exposure of neurospheres to a low concentration of an oxidative stress inducer protected against oxidant effects of the same compound at higher concentrations. This paradigm was also applied and confirmed in the zebrafish embryo model. In summary, an oxidative stress model using the zebrafish embryo model has been established with the integration and combination of different experimental procedures. This model allows the in vivo evaluation of antioxidant effects of compounds and other antioxidant strategies. It has been applied to the evaluation of antioxidant and neuroprotective effects of polyphenolic compounds present in a red wine sampl

The Zebrafish Embryo as a Model to Test Protective Effects of Food Antioxidant Compounds

t: The antioxidant activity of food compounds is one of the properties generating the most interest, due to its health benefits and correlation with the prevention of chronic disease. This activity is usually measured using in vitro assays, which cannot predict in vivo effects or mechanisms of action. The objective of this study was to evaluate the in vivo protective effects of six phenolic compounds (naringenin, apigenin, rutin, oleuropein, chlorogenic acid, and curcumin) and three carotenoids (lycopene B, β-carotene, and astaxanthin) naturally present in foods using a zebrafish embryo model. The zebrafish embryo was pretreated with each of the nine antioxidant compounds and then exposed to tert-butyl hydroperoxide (tBOOH), a known inducer of oxidative stress in zebrafish. Significant differences were determined by comparing the concentration-response of the tBOOH induced lethality and dysmorphogenesis against the pretreated embryos with the antioxidant compounds. A protective effect of each compound, except β-carotene, against oxidative-stressinduced lethality was found. Furthermore, apigenin, rutin, and curcumin also showed protective effects against dysmorphogenesis. On the other hand, β-carotene exhibited increased lethality and dysmorphogenesis compared to the tBOOH treatment alone

Evaluation of anti-inflammatory activity of food compounds using zebrafish

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/128014The principal aim of this work was to optimize and apply a zebrafish experimental model for the screening of anti-inflammatory substances present in the Mediterranean diet. The zebrafish is an organism widely used in various fields of experimental biology. The inflammation is easily inducible, reproducible and visualized in their early stages of development. Specifically, the migration of neutrophils to the injured caudal fin, one of the first steps of the inflammatory response, is quantitatively measured by image analysis. The anti-inflammatory effect of natural compounds can be evaluated as a decrease of migration. Adverse effects triggered by inflammation are mainly mediated by reactive oxygen species. The anti-oxidant activity of compounds was evaluated in zebrafish embryo measuring their protective effect against tert-butyl hydroperoxide toxicity. Several phenolic compounds have been assayed. Our results showed that the compounds with the greatest decrease on neutrophil migration were chlorogenic acid and cyanidin. The activity of these two polyphenols was quite similar to that observed with anti-inflammatory drugs (indomethacin, piroxicam) and NADPH oxidase inhibitor compounds (dibenzoidolium, apocynin). The anti-inflammatory and the anti-oxidant activity of the assayed polyphenols did not show a clear correlation

Zebrafish as a model for developmental toxicity assessment

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/67430The zebrafish embryo has emerged as promising alternative model for traditional in vivo developmental toxicological screening due to their advantageous characteristics as their small size and transparency. In this paper, we reviewed the applicability of the zebrafish embryo model in some relevant areas to human t oxicology as developmental toxicity, cardiovascular toxicity and neurotoxicity (behavioral assessment). Despite the promising results, further optimization and testing of more substances as well as a harmonized methodology is needed to streamline the metho ds and make the assay conducive to medium - throughput

¿La presencia de cannabis en saliva es suficiente para confirmar la conducción bajo sus efectos?

In Spain today, the presence of drugs in saliva is considered sufficient to confirm driving under its effects, although the concentration obtained is near the threshold limits of the analytic system. This legal approach entails accepting that there is no concentration threshold below which the drug does not affect driving capacity, and that oral fluid concentration is a reliable parameter to value its effect. In the case of cannabis, some experimental studies in humans and in intercepted drivers have suggested blood tetrahydrocannabinol concentrations from which effects that can affect driving are observed. Furthermore, correlation between tetrahydrocannabinol concentration in oral fluid and in blood shows a high variability. To resolve disagreement between these types of samples, some countries have harmonized their legislation establishing presumptive oral fluid cannabis values at the same time as blood concentration limit values from which it can be clearly considered that driving capacities are affected. In this work, we consider that the Spanish road safety law should, following trends adopted by other countries, establish a limit value for cannabis and other abused drugs, which allows evidence that the driver has not only consumed the drug, but is also driving under its effects

Modulation and Protection Effects of Antioxidant Compounds against Oxidant Induced Developmental Toxicity in Zebrafish

The antioxidant effect of compounds is regularly evaluated by in vitro assays that do not have the capability to predict in vivo protective activity or to determine their underlying mechanisms of action. The aim of this study was to develop an experimental system to evaluate the in vivo protective effects of different antioxidant compounds, based on the zebrafish embryo test. Zebrafish embryos were exposed to tert-butyl hydroperoxide (tBOOH), tetrachlorohydroquinone (TCHQ) and lipopolysaccharides from Escherichia coli (LPS), chemicals that are known inducers of oxidative stress in zebrafish. The developmental toxic effects (lethality or dysmorphogenesis) induced by these chemicals were modulated with n-acetyl l-cysteine and Nω-nitro l-arginine methyl ester hydrochloride, dimethyl maleate and dl-buthionine sulfoximine in order to validate the oxidant mechanism of oxidative stress inducers. The oxidant effects of tBOOH, TCHQ, and LPS were confirmed by the determination of significant differences in the comparison between the concentration-response curves of the oxidative stress inducers and of the modulators of antioxidant status. This concept was also applied to the study of the effects of well-known antioxidants, such as vitamin E, quercetin, and lipoic acid. Our results confirm the zebrafish model as an in vivo useful tool to test the protective effects of antioxidant compounds

La enseñanza de la Toxicología en Farmacia: los seminarios como herramienta para la evaluación continuada

Con la finalidad de adaptarnos al EEES, desarrollamos una herramienta que nos permitiera realizar un proceso de evaluación continua de la asignatura troncal de Toxicología. En el presente trabajo presentamos los resultados de este modelo en el que utilizamos los seminarios como elementos básicos de este proceso. Describimos cómo se estructuran y desarrollan estos seminarios, así como el modelo de evaluación de los mismos. Los seminarios fueron evaluados con una puntuación máxima del 30 % sobre la nota final de la signatura, y la participación en los mismos con un máximo del 10 %. Algunos de estos seminarios incorporaban evaluaciones realizadas antes del desarrollo de los mismos, que denominábamos «pre», y otras justo al final del desarrollo de los mismos, que denominábamos «post». Esta herramienta de evaluación continua se ha mostrado muy eficaz en lo que respecta al grado de participación y preparación de los alumnos. Además, ha supuesto un cambio significativo en el grado de implicación de los profesores, y una mejora de la comunicación alumno-profesor

Incorporación de pequeñas secuencias de cine comercial en la enseñanza de las drogodependencias. Ensayo piloto en la asignatura de Toxicología

El Grupo de Innovación Docente Orfila, en su propósito por mejorar la calidad de la docencia, está ensayando la utilización del cine con fines didácticos. El material desarrollado en este proyecto consiste en secuencias cortas de películas comerciales de 3 a 5 minutos de duración, que son utilizadas como elementos ilustrativos del proceso de adicción a las a drogas. Se seleccionan escenas de la filmografía y se adecúan al programa docente de la asignatura Toxicología. Se recoge la opinión de los profesores participantes, así como la de los alumnos, mediante una entrevista personal y una encuesta de opinión, respectivamente, de las que se deduce un alto grado de satisfacción

Triclabendazole sulfoxide causes stage-dependent embryolethality in zebrafish and mouse in vitro

Background Fascioliasis and paragonimiasis are widespread foodborne trematode diseases, affecting millions of people in more than 75 countries. The treatment of choice for these parasitic dis- eases is based on triclabendazole, a benzimidazole derivative which has been suggested as a promising drug to treat pregnant women and children. However, at the moment, this drug is not approved for human use in most countries. Its potential adverse effects on em- bryonic development have been scarcely studied, and it has not been assigned a pregnan- cy category by the FDA. Thus, to help in the process of risk-benefit decision making upon triclabendazole treatment during pregnancy, a better characterization of its risks during ges- tation is needed. Methodology The zebrafish embryo test, a preimplantation and a postimplantation rodent whole embryo culture were used to investigate the potential embryotoxicity/teratogenicity of triclabenda- zole and its first metabolite triclabendazole sulfoxide. Albendazole and albendazole sulfox- ide were included as positive controls. Principal Findings Triclabendazole was between 10 and 250 times less potent than albendazole in inducing dysmorphogenic effects in zebrafish or postimplantation rodent embryos, respectively. However, during the preimplantation period, both compounds, triclabendazole and tricla- bendazole sulfoxide, induced a dose-dependent embryolethal effect after only 24 h ofexposure in rodent embryos and zebrafish (lowest observed adverse effect concentra- tions = 10 μ M). Conclusions/Significance In humans, after ingestion of the recommended doses of triclabendazole to treat fascioliasis and paragonimiasis (10 mg/kg), the main compound found in plasma is triclabendazole sulf- oxide (maximum concentration 38.6 μ M), while triclabendazole concentrations are approxi- mately 30 times lower (1.16 μ M). From our results it can be concluded that triclabendazole, at concentrations of the same order of magnitude as the clinically relevant ones, does not entail teratogenic potential in vitro during the organogenesis period, but its first metabolite triclabendazole sulfoxide has a high embryotoxic capacity in vitro during the preimplantation stag

Risk assessment for human embryonic development of triclabendazole residues in milk and cheese in the diet of a rural population in Cajamarca (Peru): A preliminary approach

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/103042Triclabendazole (TCBZ) is a veterinary drug used against Fasciola hepatica in cattle. The Cajamarca Valley in Peru is an endemic area of fascioliasis with a high infection rate in animals producing milk for human consumption. The administration of TCBZ during the lactating period can lead to TCBZ derivative residues in milk and cheese entering the human food chain. Milk-derivatives from treated animals have been found positive for TCBZ metabolites. One of these metabolites, triclabendazole sulfoxide (TCBZSO), is embryolethal during early developmental stages in vitro in mouse and zebrafish. In this study, we have calculated the estimated daily intake (EDI) of TCBZSO due to milk and cheese consumption among a rural population in Cajamarca in order to evaluate the associated risk for human embryonic development. Although the expected maximum plasma concentration of TCBZSO after a worst-case scenario simulation would be below the reported lowest observed adverse effect concentration (LOAEC) for embryolethality in vitro (10 μM), several limitations on the available information for exposure, bioavailability and interspecies differences still impede the accomplishment of an accurate risk assessment