Uso de biomarcadores na avaliaçao dos efeitos do metilmercúrio em Hoplias malabaricus (Block, 1794) (Traíra)

Orientador : Ciro Alberto de Oliveira RibeiroDissertaçao (mestrado) - Universidade Federal do Paraná, Setor de Ciencias Biológicas, Programa de Pós-Graduaçao em Biologia Celular e Molecular. Defesa: Curitiba, 2004Inclui bibliografiaO mercúrio está entre os poucos poluentes que se acumula em organismos topo de cadeia, através dos níveis tróficos inferiores, e sendo assim os organismos predadores são os mais expostos aos efeitos tóxicos deste metal. Neste trabalho a espécie Hoplias malabaricus (traíra) foi utilizada para avaliar experimentalmente o efeito subcrônico do metilmercúrio (MeHg) por via trófica. Nove exemplares foram utilizados como controles e nove foram expostos ao organometal através de doses consecutivas de 0,075 µgMeHg/g peixe, onde exemplares vivos de Astyanax sp. foram utilizados como veículo. Após 14 doses administradas a cada 5 dias, totalizando 70 dias de exposição, os peixes foram anestesiados e amostras foram coletadas para análises químicas, hematológicas, bioquímicas e morfológicas (Microscopia de Luz e Microscopia Eletrônica de Transmissão). Os dados histopatológicos mostraram a presença de infiltração leucocitária e um aumento centro de melanomacrófagos no fígado. Além disso, através da ultraestrutura foi possível identificar outros tipos de alterações como: a presença de vacuolização celular nas células endoteliais dos sinusóides, grânulos eletrodensos no citosol dos hepatócitos, bem como áreas de necrose. A diminuição do número de microvilos dos hepatócitos no espaço de Disse e necroses nesta região também foram alterações observadas. O rim anterior exibiu áreas de necrose e um aumento do número de centro de melanomacrófagos. Houve também um aumento do espaço entre as células do parênquima e das áreas fagocíticas. Foi observado ainda a presença de células atípicas no grupo exposto ao contaminante. Os parâmetros hematológicos apresentaram algumas variações. O grupo exposto ao MeHg mostrou um aumento no número de eritrócitos, leucócitos, neutrófilos, trombócitos e também nos valores do hematócrito, hemoglobina e Volume Corpuscular Médio, no entanto o número de linfócitos diminuiu no grupo tratado. Com as análises bioquímicas, foi observado uma inibição da atividade colinesterásica no músculo dos peixes expostos. A concentração total de MeHg em músculo e fígado mostrou uma tendência de acúmulo de metilmercúrio, porém esta não foi estatisticamente significativa. Através do presente trabalho, verificou-se que a dose trófica e subcrônica do metilmercúrio é potencialmente tóxico para a espécie Hoplias malabaricus, acrescentando dados que auxiliam a compreensão dos mecanismos de toxicidade deste importante organometalMercury (Hg) is a toxic heavy metal able to concentrate in aquatic food chains, mainly in predatory fish, where the effects are more evident. In the present work a fish species, Hoplias malabricus (thraíra), was used to evaluate the effects of subcronic and throphic exposure to methylmercury (MeHg). A group of nine individuals was used as control and another group received an organometal dose each five days (0,075 µgMeHg/g w.w.) through living Astyanax sp. After 14 doses have been administrated, corresponding to 70 days of exposure time, the fishes were anesthetized and samples were collected to perform chemical, haematological, biochemical and morphological (light and transmission electron microscopy) analyses. Some morphological findings were evident in liver tissue as: Leukocytes infiltration, melano-macrophages centers and the presence of necrosis area. The ultrastructure investigation showed some alterations on cells from liver as: the vacuolization of endothelial cells in the sinusoids vessels, elecron-dense granules within the citosol of hepatocytes and a decrease in the number of hepatocytes membrane projections near to the sinusoids barrier. Also, the head kidney presented necrosis areas and an increase of melano-macrophages centers. The space between parenquimal cells increase in the organ, as well as the phagocytic areas. The presence of necrosis areas were also found at head kidney of exposed individuals. In addition, atypical cells were observed in head kidney of individuals from treated group. The hematological parameters presented some interesting variations. Individuals from the exposed group showed an increase of erythrocytes, leukocytes, neutrophils and trombocytes numbers. Also, the hematocrit, hemoglobin and MCV values increased the respective values, despite of the lymphocytes number that decreased in the individuals from the treated group. The biochemical analyses showed an inhibition of acetilcolinesterase activity in muscle confirm the neurotoxicity of the organometal. The chemical analysis presented a tendency of bioconcentration in muscle and liver also confirming the characteristic of methylmercury accumulation in fish organs and in muscle. According to the present results, we confirm the toxicity of metylmercury and in addition, this work was the first to show experimentally the effects of trophic and subchronic doses in tropical fish species Hoplias malabaricus

Nature-based tourism elicits a phenotypic shift in the coping abilities of fish

Nature-based tourism is gaining extensive popularity, increasing the intensity and frequency of human-wildlife contacts. As a consequence, behavioral and physiological alterations were observed in most exposed animals. However, while the majority of these studies investigated the effects of punctual exposure to tourists, the consequences of constant exposition to humans in the wild remains overlooked. This is an important gap considering the exponential interest for recreational outdoor activities. To infer long-term effects of intensive tourism, we capitalized on Odontostilbe pequira, a short-lived sedentary Tetra fish who spends its life close to humans, on which it feeds on dead skin. Hence, those fish are constantly exposed to tourists throughout their lifecycle. Here we provide an integrated picture of the whole phenomenon by investigating, for the first time, the expression of genes involved in stress response and neurogenesis, as well as behavioral and hormonal responses of animals consistently exposed to tourists. Gene expression of the mineralocorticoid (and cortisol) receptor (mr) and the neurogenic differentiation factor (NeuroD) were significantly higher in fish sampled in the touristic zone compared to those sampled in the control zone. Additionally, after a simulated stress in artificial and controlled conditions, those fish previously exposed to visitors produced more cortisol and presented increased behavioral signs of stress compared to their non-exposed conspecifics. Overall, nature-based tourism appeared to shift selection pressures, favoring a sensitive phenotype that does not thrive under natural conditions. The ecological implications of this change in coping style remain, nevertheless, an open question

Uso de biomarcadores na avaliaçao dos efeitos do metilmercúrio em Hoplias malabaricus (Block, 1794) (Traíra)

Orientador : Ciro Alberto de Oliveira RibeiroDissertaçao (mestrado) - Universidade Federal do Paraná, Setor de Ciencias Biológicas, Programa de Pós-Graduaçao em Biologia Celular e Molecular. Defesa: Curitiba, 2004Inclui bibliografiaO mercúrio está entre os poucos poluentes que se acumula em organismos topo de cadeia, através dos níveis tróficos inferiores, e sendo assim os organismos predadores são os mais expostos aos efeitos tóxicos deste metal. Neste trabalho a espécie Hoplias malabaricus (traíra) foi utilizada para avaliar experimentalmente o efeito subcrônico do metilmercúrio (MeHg) por via trófica. Nove exemplares foram utilizados como controles e nove foram expostos ao organometal através de doses consecutivas de 0,075 µgMeHg/g peixe, onde exemplares vivos de Astyanax sp. foram utilizados como veículo. Após 14 doses administradas a cada 5 dias, totalizando 70 dias de exposição, os peixes foram anestesiados e amostras foram coletadas para análises químicas, hematológicas, bioquímicas e morfológicas (Microscopia de Luz e Microscopia Eletrônica de Transmissão). Os dados histopatológicos mostraram a presença de infiltração leucocitária e um aumento centro de melanomacrófagos no fígado. Além disso, através da ultraestrutura foi possível identificar outros tipos de alterações como: a presença de vacuolização celular nas células endoteliais dos sinusóides, grânulos eletrodensos no citosol dos hepatócitos, bem como áreas de necrose. A diminuição do número de microvilos dos hepatócitos no espaço de Disse e necroses nesta região também foram alterações observadas. O rim anterior exibiu áreas de necrose e um aumento do número de centro de melanomacrófagos. Houve também um aumento do espaço entre as células do parênquima e das áreas fagocíticas. Foi observado ainda a presença de células atípicas no grupo exposto ao contaminante. Os parâmetros hematológicos apresentaram algumas variações. O grupo exposto ao MeHg mostrou um aumento no número de eritrócitos, leucócitos, neutrófilos, trombócitos e também nos valores do hematócrito, hemoglobina e Volume Corpuscular Médio, no entanto o número de linfócitos diminuiu no grupo tratado. Com as análises bioquímicas, foi observado uma inibição da atividade colinesterásica no músculo dos peixes expostos. A concentração total de MeHg em músculo e fígado mostrou uma tendência de acúmulo de metilmercúrio, porém esta não foi estatisticamente significativa. Através do presente trabalho, verificou-se que a dose trófica e subcrônica do metilmercúrio é potencialmente tóxico para a espécie Hoplias malabaricus, acrescentando dados que auxiliam a compreensão dos mecanismos de toxicidade deste importante organometalMercury (Hg) is a toxic heavy metal able to concentrate in aquatic food chains, mainly in predatory fish, where the effects are more evident. In the present work a fish species, Hoplias malabricus (thraíra), was used to evaluate the effects of subcronic and throphic exposure to methylmercury (MeHg). A group of nine individuals was used as control and another group received an organometal dose each five days (0,075 µgMeHg/g w.w.) through living Astyanax sp. After 14 doses have been administrated, corresponding to 70 days of exposure time, the fishes were anesthetized and samples were collected to perform chemical, haematological, biochemical and morphological (light and transmission electron microscopy) analyses. Some morphological findings were evident in liver tissue as: Leukocytes infiltration, melano-macrophages centers and the presence of necrosis area. The ultrastructure investigation showed some alterations on cells from liver as: the vacuolization of endothelial cells in the sinusoids vessels, elecron-dense granules within the citosol of hepatocytes and a decrease in the number of hepatocytes membrane projections near to the sinusoids barrier. Also, the head kidney presented necrosis areas and an increase of melano-macrophages centers. The space between parenquimal cells increase in the organ, as well as the phagocytic areas. The presence of necrosis areas were also found at head kidney of exposed individuals. In addition, atypical cells were observed in head kidney of individuals from treated group. The hematological parameters presented some interesting variations. Individuals from the exposed group showed an increase of erythrocytes, leukocytes, neutrophils and trombocytes numbers. Also, the hematocrit, hemoglobin and MCV values increased the respective values, despite of the lymphocytes number that decreased in the individuals from the treated group. The biochemical analyses showed an inhibition of acetilcolinesterase activity in muscle confirm the neurotoxicity of the organometal. The chemical analysis presented a tendency of bioconcentration in muscle and liver also confirming the characteristic of methylmercury accumulation in fish organs and in muscle. According to the present results, we confirm the toxicity of metylmercury and in addition, this work was the first to show experimentally the effects of trophic and subchronic doses in tropical fish species Hoplias malabaricus

Evaluation of toxic effects of methylmercury in the retina of fotoreceptores Hoplias malabaricus (BLOCK, 1794), trahira, through histopathological techniques

Em exposições especificamente relacionadas ao metilmercúrio (MeHg), o principal órgão alvo é o Sistema Nervoso Central (SNC). Os efeitos morfológicos do metilmercúrio nos fotorreceptores da retina de Hoplias malabaricus, traíra, foram investigados através de técnicas histopatológicas. Os exemplares de traíra foram distribuídos em três grupos: um grupo controle e dois grupos expostos ao MeHg. A contaminação trófica e subcrônica foi realizada através de exemplares vivos de Astyanax sp os quais receberam intraperitonealmente um volume de solução aquosa de MeHg correspondente a (0,075µg/g de traíra) ou (0,75µg/g de traíra). Quinze peixes por condição experimental foram sacrificados após 70 dias de exposição e os olhos foram coletados para as análises de microscopia de luz, microscopia eletrônica de transmissão e microscopia eletrônica de varredura. Através de análises químicas quantificamos o mercúrio em músculo e observamos em ambas as doses um acúmulo do metal. Para a maior dose do metal (0,75µg/g), as análises histopatológicas revelaram alterações nas membranas que unem os dois elementos de um cone pareado, degeneração da camada dos fotorreceptores e alterações morfológicas no segmento interno e segmento externo dos bastonetes. As análises também demonstraram alterações estruturais na membrana plasmática do segmento externo e alterações no processo de eliminação dos discos membranosos dos fotorreceptores. No momento da eliminação dos discos antigos foi observada a formação de vacúolos e também uma anormal eliminação dos discos membranosos pelas partes laterais do segmento externo. Para a menor dose do metal (0,075µg/g) além da alteração observada nas membranas que unem os cones pareados e da degeneração celular na camada dos fotorreceptores, observamos uma descontinuidade estrutural dos discos membranosos do segmento externo dos fotorreceptores. Todas estas alterações histopatológicas nos levam a concluir que o metilmercúrio induz alterações histopatológicas nas células da camada dos fotorreceptores podendo consequentemente trazer danos fisiológicos para toda a retina.In exposures specifically related to methylmercury (MeHg), the main target organ is the Central Nervous System (CNS). In this study, morphological effects of methylmercury in retinal photoreceptors of Hoplias malabaricus, trahira, through histopathological techniques were investigated. Some mature fish were divided in three groups - one control group and the other two which were exposed to MeHg. The trophic and subchronic contamination was performed by live specimen of Astyanax sp which received a volume of aqueous solution of MeHg corresponding to (0,075µg /g trahira) or (0,75µg /g trahira). In an experimental condition, fifteen fish were sacrificed after 70 days of exposure and their eyes were collected for analysis of light microscopy, electron transmission microscopy and scanning electron microscopy. Through chemical analysis the amount of mercury found in muscle was quantified. In both doses an accumulation of the metal in the muscle of these animals was observed. For the highest dose of metal (0,75µg /g), the histopathological analysis revealed changes in the membrane that connects the two elements of a paired cone, cellular degeneration in the layer of photoreceptors and morphological changes in the internal and outer rod segments. The analysis also showed structural changes in the plasma membrane of the outer segment and changes in the process of removal of membranous discs in the apical region of a photoreceptor. At the time of disposal of old discs, the formation of vacuoles and also an abnormal membranous discs removal through the sides of the outer segment were observed. For the smallest dose of the metal (0,075µg/g) besides the morphological changes observed in the membrane which unites the paired cones and cellular degeneration throughout the layer of photoreceptors, a structural discontinuity of the membranous discs of the outer segment were observed in radial sections of rods. All these histopathological changes lead us to conclude that methylmercury induces morphological changes in cells of the layer of photoreceptors, and therefore causing physiological damage to the smooth functioning of the retina

Critical Evaluation of Embedding Media for Histological Studies of Early Stages of Chick Embryo Development

A histological examination is an important tool in embryology, developmental biology, and correlated areas. Despite the amount of information available about tissue embedding and different media, there is a lack of information regarding best practices for embryonic tissues. Embryonic tissues are considered fragile structures, usually small in size, and frequently challenging to position correctly in media for the subsequent histological steps. Here, we discuss the embedding media and procedures that provided us with appropriate preservation of tissue and easier orientation of embryos at early development. Fertilized Gallus gallus eggs were incubated for 72 h, collected, fixed, processed, and embedded with paraplast, polyethylene glycol (PEG), or historesin. These resins were compared by the precision of tissue orientation, the preview of the embryos in the blocks, microtomy, contrast in staining, preservation, average time, and cost. Paraplast and PEG did not allow correct embryo orientation, even with agar–gelatin pre-embedded samples. Additionally, structural maintenance was hindered and did not allow detailed morphological assessment, presenting tissue shrinkage and disruption. Historesin provided precise tissue orientation and excellent preservation of structures. Assessing the performance of the embedding media contributes significantly to future developmental research, optimizing the processing of embryo specimens and improving results

Morphological evidence of neurotoxicity in retina after methylmercury exposure

The visual system is particularly sensitive to methylmercury (MeHg) exposure and, therefore, provides a useful model for investigating the fundamental mechanisms that direct toxic effects. During a period of 70 days, adult of a freshwater fish species Hoplias malabaricus were fed with fish prey previously labeled with two different doses of methylmercury (0.075 and 0.75 mu g g(-1)) to determine the mercury distribution and morphological changes in the retina. Mercury deposits were found in the photoreceptor layer, in the inner plexiform layer and in the outer plexiform layer, demonstrating a dose-dependent bioaccumulation. The ultrastructure analysis of retina revealed a cellular deterioration in the photoreceptor layer, morphological changes in the inner and outer segments of rods, structural changes in the plasma membrane of rods and double cones, changes in the process of removal of membranous discs and a structural discontinuity. These results lead to the conclusion that methylmercury is able to cross the blood-retina barrier, accumulate in the cells and layers of retina and induce changes in photoreceptors of H. malabaricus even under subchronic exposure. (c) 2012 Elsevier Inc. All rights reserved.Capes (Brazilian Agencies for Science and Technology) at University of Bordeaux-I, FranceCNRS (National Centre for Scientific Research) at University of BordeauxI, FranceCNRS (National Centre for Scientific Research) at University of Bordeaux-I, Franc

Toxicological Effects of Thimerosal and Aluminum in the Liver, Kidney, and Brain of Zebrafish (<i>Danio rerio</i>)

Vaccination programs in the first years of a child’s life are effective and extremely important strategies for the successful eradication of diseases. However, as no intervention is without risks, the metal-based components of some vaccines, such as thimerosal (TMS), a preservative composed of ethylmercury, and aluminum (Al), have begun to generate distrust on the part of the population. Therefore, this study evaluated the effects of exposure to thimerosal and aluminum hydroxide (alone or in mixture) on Danio rerio (zebrafish) specimens. The fish were exposed to thimerosal and/or aluminum hydroxide intraperitoneally. The liver, kidney, and brain were removed for a biochemical biomarker analysis, histopathological analysis, and metal quantification. As a result, we observed changes in the activity of the analyzed enzymes (SOD, GST, GPx) in the kidney and brain of the zebrafish, a reduction in GSH levels in all analyzed tissues, and a reduction in MT levels in the kidney and liver as well as in the brain. Changes in AChE enzyme activity were observed. The biochemical results corroborate the changes observed in the lesion index and histomorphology sections. We emphasize the importance of joint research on these compounds to increase the population’s safety against their possible toxic effects

Methylmercury localization in Danio rerio retina after trophic and subchronic exposure: A basis for neurotoxicology

Methylmercury is a known neurotoxic organometal which affects visual functions and few studies concerns to wild fish are available. The autometallography mercury distribution in the retina of Danio rerio was mapped using light and electron microscopy. Abundant mercury deposits were found in the photoreceptor layer (outer and inner segments of the photoreceptors) and in the inner and outer nuclear layers. Occasionally, the presence of mercury deposits in plexiform layers was observed and very rarely in the ganglion cell layer. Also the occurrence of mercury deposits in cells from the disc region was observed, but not in the nerve fiber layer. An interesting difference was found between mercury accumulation in the central and peripheral regions of the retina. These results demonstrate that mercury after trophic exposure to Danio rerio is able to cross the blood-retina barrier and accumulate in the cells of the retina even under subchronic exposure. (C) 2010 Elsevier Inc. All rights reserved.Capes (Brazilian Agencies for Science and Technology)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Centre National de la Recherche Scientifique (CNRS)CNRS (National Centre for Scientific Research) at University of Bordeaux-I, Franc

Nature-Based Tourism Elicits a Phenotypic Shift in the Coping Abilities of Fish

Nature-based tourism is gaining extensive popularity, increasing the intensity and frequency of human-wildlife contacts. As a consequence, behavioral and physiological alterations were observed in most exposed animals. However, while the majority of these studies investigated the effects of punctual exposure to tourists, the consequences of constant exposition to humans in the wild remains overlooked. This is an important gap considering the exponential interest for recreational outdoor activities. To infer long-term effects of intensive tourism, we capitalized on Odontostilbe pequira, a short-lived sedentary Tetra fish who spends its life close to humans, on which it feeds on dead skin. Hence, those fish are constantly exposed to tourists throughout their lifecycle. Here we provide an integrated picture of the whole phenomenon by investigating, for the first time, the expression of genes involved in stress response and neurogenesis, as well as behavioral and hormonal responses of animals consistently exposed to tourists. Gene expression of the mineralocorticoid (and cortisol) receptor (mr) and the neurogenic differentiation factor (NeuroD) were significantly higher in fish sampled in the touristic zone compared to those sampled in the control zone. Additionally, after a simulated stress in artificial and controlled conditions, those fish previously exposed to visitors produced more cortisol and presented increased behavioral signs of stress compared to their non-exposed conspecifics. Overall, nature-based tourism appeared to shift selection pressures, favoring a sensitive phenotype that does not thrive under natural conditions. The ecological implications of this change in coping style remain, nevertheless, an open question