Increased reactive oxygen species (ROS) have been reported to be at the centre of various diseases. Although several reports have implicated elevated levels of ROS in the pathogenesis of diabetes mellitus, the early detection of ROS is still not attainable. This limitation causes difficulty in the early diagnosis of ROS related disorders. The presence of high levels of ROS was reported to result in differential expression of antioxidant genes involved in protecting cells from their deleterious effects. Among the antioxidant genes that are expressed, it was postulated that expression of metallothioneins (MTs) are also induced. MTs are low molecular weight, cysteine-rich proteins involved in metal homeostasis and reported to harbour antioxidant function. The aim of this investigation was to explore MTs as biomarkers for elevated levels of ROS in whole blood of type 2 diabetic (T2D) individuals. The level of ROS in diabetic, non-diabetic as well as individuals at risk of developing T2D was determined via the use of biochemical assays. Real-Time PCR was utilised to analyse the expression of MTs and the presence of MT proteins was analysed via the ELISA. In this study it was observed that diabetic individuals had elevated levels of ROS. However, no significant difference in the expression of MTs and the presence of MT proteins between the diabetic and non-diabetic individuals was observed. In vitro experimental conditions indicated that MT expression is induced by elevated levels of ROS. In pathological conditions the ROS-dependent induction of MT expression needs to be elucidated further. It therefore can be suggested that MTs can not yet be utilised as biomarkers for the detection of elevated levels of ROS in pathological conditions with ROS aetiology. This investigation also highlights the fact that blood is not an optimal medium in which this objective can be attained. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2005.

Increased reactive oxygen species (ROS) have been reported to be at the centre of various diseases. Although several reports have implicated elevated levels of ROS in the pathogenesis of diabetes mellitus, the early detection of ROS is still not attainable. This limitation causes difficulty in the early diagnosis of ROS related disorders. The presence of high levels of ROS was reported to result in differential expression of antioxidant genes involved in protecting cells from their deleterious effects. Among the antioxidant genes that are expressed, it was postulated that expression of metallothioneins (MTs) are also induced. MTs are low molecular weight, cysteine-rich proteins involved in metal homeostasis and reported to harbour antioxidant function. The aim of this investigation was to explore MTs as biomarkers for elevated levels of ROS in whole blood of type 2 diabetic (T2D) individuals. The level of ROS in diabetic, non-diabetic as well as individuals at risk of developing T2D was determined via the use of biochemical assays. Real-Time PCR was utilised to analyse the expression of MTs and the presence of MT proteins was analysed via the ELISA. In this study it was observed that diabetic individuals had elevated levels of ROS. However, no significant difference in the expression of MTs and the presence of MT proteins between the diabetic and non-diabetic individuals was observed. In vitro experimental conditions indicated that MT expression is induced by elevated levels of ROS. In pathological conditions the ROS-dependent induction of MT expression needs to be elucidated further. It therefore can be suggested that MTs can not yet be utilised as biomarkers for the detection of elevated levels of ROS in pathological conditions with ROS aetiology. This investigation also highlights the fact that blood is not an optimal medium in which this objective can be attained. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2005.

Pagrindinis tyrimo tikslas – įvertinti švino ir nikelio jonų įtaką baltymų ir metalotioneinų sintezei laboratorinių pelių kepenyse bei cinko apsauginį poveikį, veikiant švinui ir nikeliui. Tyrimams naudotos 4-6 savaičių nelinijinės baltosios laboratorinės pelės, sveriančios nuo 20 iki 25 gramų. Pelių laikymo sąlygos atitiko reikalavimus. Eksperimentams pelės buvo suskirstytos į 6 grupes: I – kontrolinė, II – gaunančios Pb(CH3COO)2 (dozė 10 mg Pb/kg), III – gaunančios ZnSO4 (1,56 mg Zn/kg), IV – ZnSO4 ir po 20 minučių Pb(CH3COO)2 (anksčiau nurodytos dozės), V – NiCl2 (1,12 mg Ni/kg) , VI – ZnSO4 ir po 20 minučių NiCl2 (anksčiau nurodytos dozės). Druskų tirpalai į pilvo ertmę švirkšti 14 dienų. Praėjus 14 dienų, pelės buvo anestezuotos, joms atlikta cervikalinė dislokacija. Tyrimams naudotos pelių kepenys. Baltymų koncentracijai nustatyti naudojome Beer/Lambert metodą, spektrofotometriškai matavome sugertį ties 260 ir 280 nm banga. Metalotioneinų koncentraciją nustatėme pagal Peixoto metodiką, spektrofotometriškai sugertį matavome ties 412 nm banga. Nustatėme, jog metalai skirtingai veikia bendrų baltymų ir metalotioneinų sintezę. Švinas neturėjo statistiškai reikšmingo poveikio bendrų baltymų sintezei, o nikelis skatino bendrą baltymų sintezę. Nikelis metalotioneinų sintezę taip pat aktyvino labiau nei švinas. Padidėjus metalotioneinų koncentracijai bei suaktyvėjus bendrų baltymų sintezei, užfiksuotas cinko jonų apsauginis poveikis. / The present study was conducted to investigate the effects of lead and nickel ions on total proteins and metallothioneins synthesis in mice liver and evaluate protective effects of zinc ions. Experiments were done on 4-6 weeks old white laboratory outbreed mice weighing 20-25 g. All experiments performed according to the Republic of Lithuania Law on the Care, Keeping and Use of animals (License of State Veterinary Service for working with laboratory animals No 0221). For experiment mice were sorted out into 6 groups: I group – control; II group – Pb(CH3COO)2 (10 mg Pb per kg of body mass), III group – ZnSO4 (1,56 mg Zn per kg of body mass), IV – ZnSO4 and after 20 minutes Pb(CH3COO)2 (the same doses), V – NiCl2 (1,12 mg Ni per kg of body mass) , VI – ZnSO4 and after 20 minutes NiCl2 (the same doses). Concentration of protein was determined by Lowry method. MTs were assayed in mice liver according to the method of Peixoto N. C. There is no statistically significant effect of lead on protein synthesis in mice liver. ZnSO4, injected 20 minutes before Pb(CH3COO)2, increased protein synthesis. After injection of NiCl2 solution, marked amino acid actuation to new synthesized protein has increased, but ZnSO4, injected 20 minutes before NiCl2, decreased protein synthesis. Obtained data showed, that in mice liver treated with NiCl2 and Pb(CH3COO)2 solutions, MTs content was increased. According to the data, ZnSO4 injected 20 minutes before NiCl2 or Pb(CH3COO)2, decreased MTs content... [to full text]

Este trabalho apresenta a pesquisa de interação de organismos aquáticos com metais, especificamente cobre e cádmio assim como da combinação deles. Para tanto, descreve-se um protocolo baseado em ensaios de toxicidade com tilápias Oreochromis niloticus, desenvolvido com a finalidade de estabelecer a concentração letal 50 (CL50) 96h. Como recurso adicional, focando a translocação de metais, foram utilizadas soluções enriquecidas isotopicamente de 65Cu (99,7%) e 111Cd (95,5%), as quais foram aplicadas através de injeções intraperitoneais. As concentrações dos metais nos tecidos e nas frações citosólicas do fígado, brânquias e músculo foram determinadas a partir dos extratos preparados em solução tampão tris-HCl 50 mmolL-1, agentes redutores e antiproteolíticos, na proporção 3:1 (m/v). Adicionalmente, foram determinados os teores de proteínas totais seguidos da etapa de isolamento das metalotioneínas. As concentrações letais (CL50) 96h calculadas pelo método Trimmed Spearman-Karber (95% de confiança), foram iguais a 20,13, 3,53 e 1,36 mg L-1 para cádmio, cobre e cobre+cádmio respectivamente. Estes valores indicam uma significativa diferença na sensibilidade dos organismos aos diferentes tratamentos. Observou-se uma redução na bioconcentração dos metais em função da concentração do metal e do tecido analisado, esta é maior para o Cu do que para o Cd e menor para o musculo em relação a brânquia e ao fígado. Os teores de proteínas totais nos tecidos foram determinados e os valores encontrados estiveram entre 3,25 mg/mL para fígados e 11,83 mg/mL para músculo. A presença de MTs nos diferentes tecidos e respectivos tratamentos, foi pesquisada empregando-se eletroforese capilar e separação de proteínas utilizando a eletroforese em gel de poliacrilamida. Por ambas as técnicas eletroforéticas foi possível identificar as metalotioneínas. Contudo, as análises dos extratos dos tecidos por espectrometria de massas, MALDI-TOF/TOF (matrix-assisted laser desorption/ionization) e ESI-MS (electrospray tandem mass spectrometry ) não mostraram-se adequadas para identificação de metalotioneínas / This paper discuss the interactions of Cd, Cu and it mixture upon aquatic organisms. To reach for these goals lethal 50 (LC50) acute toxicity 96h assays were carried out. In order to assess for the metals translocation in fish, isotopically enriched solutions of 65Cu (99,7%) and 111Cd (95,5%), were used, through an intra-peritoneal injections. Metals concentrations in tissue and in the citossolic fractions of liver, and muscular tissue were analysed in the 50 mmol-1 tris-HCl buffer solutions, reducing and un-proteolitic (3:1) solutions. In addition the total protein content were determinate, followed by the metalothyonein isolation. Lethal concentrations were calculated by the Spearman-Karber method at 95% confidence interval, as 20.13, 3.53 and 1.36 mgL-1 for Cd, Cu and Cd+Cu respectively, which denote differences in organisms sensitivity according to the treatment. Metal bio-concentration was reduced depending on the analyte concentration and kind of tissue, being higher to Cu than to Cd. Total protein concentration varied from 3.35 mg L-1 for liver to 11.83 mg L-1 in the muscular tissue. The occurrence of MTs in tissues and in all treatments were investigated by using both, capillary electrophoresis and gel of polyacrilamide. In all situations the occurrence and identification of Mts were verified, but even with MALDI-TOF/TOF (matrix-assisted laser desorption/ionization) and ESI-MS (electronspray tandem mass spectrometry), or actual instrumental facilities no satisfactory results were obtained, being not possible the identification of MTs in the analysed samples

Dans les écosystèmes littoraux, l‘activité anthropique, mais aussi le contexte naturel induisent chez les organismes aquatiques des situations de « multistress ». Parmi les sources potentielles de perturbation, trois d‘entre elles ont été étudiées : contamination métallique, infection bactérienne et infestation parasitaire. Une approche intégrée de leurs interactions sur la réponse génétique, protéique, cellulaire et populationnelle chez la coque Cerastoderma edule et la palourde japonaise Ruditapes philippinarum a été entreprise sur le terrain et en laboratoire. In situ, un suivi de deux ans a été mené sur quatre populations de bivalves issues d‘environnements différents afin d‘estimer la santé des populations (paramètres de dynamique de population), de la mettre en rapport avec les niveaux de base des stress subis (métaux, parasites) et d‘évaluer les réponses adaptatives mises en place par les bivalves en termes de détoxication et de défenses immunitaires. Cette approche a permis de décrire un certain nombre de scenarii concernant les relations entre des populations de bivalves et leur environnement. En laboratoire, des expériences de co-infestation contrôlées (parasite trématode Himasthla elongata, bactérie Vibrio tapetis) en milieu indemne de métaux ou contaminé au cadmium ont été menées sur certaines de ces populations et montrent que la réponse des bivalves à ces agressions multiples dépend des espèces, des combinaisons subies, et ne se déduit pas intuitivement à partir des réponses « mono-stress », soulignant ainsi la notion d‘interactions. Malgré la complexité et la diversité de ces dernières, certains mécanismes semblent prédominer quelle que soit l‘espèce étudiée. Alors que l‘infestation par le parasite ne semble pas modulée par la présence du métal ou de la bactérie dans le milieu, la bioaccumulation métallique est fortement influencée par la présence d‘un ou plusieurs agents pathogènes. En plus de perturber l‘accumulation du polluant, la présence d‘organismes pathogènes interfère dans les mécanismes de détoxication cellulaire avec notamment une perturbation de la synthèse des métallothionéines (MT). Des travaux menés plus particulièrement sur la réponse des MT chez la coque exposée à un métal, à travers l‘expression de l‘isoforme Cemt1 et la synthèse de la protéine, confirment la complémentarité des observations (génétique et protéique) ainsi que la nécessité d‘observer les réponses des organismes à différentes échelles afin d‘avoir une vision globale des processus interactifs existants entre polluants et pathogènes. Des interactions fortes sont révélées au niveau génétique, protéique et immunitaire: « cadmium x trématode » chez la coque et « bactérie x trématode » chez la palourde. Enfin ces expériences, en parallèle du suivi in situ, mettent en évidence le rôle majeur de l‘histoire de vie dans la sensibilité des organismes aux interactions polluants-pathogènes. / In coastal ecosystems, man-mediated activity and natural context induce a ?multistress? situation in aquatic organisms. Amongst potential perturbation sources, three of them were studied: metal contamination, bacterial infection and parasite infestation. An integrated approach of their interactions on genetic, protein, cellular and population dynamics responses in the cockle (Cerastoderma edule) and the Manila clam (Ruditapes philippinarum) was undertaken through field and laboratory studies. A two-years monitoring was conducted in four bivalve populations from different environments to estimate the fitness of populations (parameters of population dynamics), in relation with the baseline levels of stressors (metals, parasites) and with the adaptive responses implemented by bivalves in terms of detoxification and immunity. This approach allowed describing different scenarii concerning the relationship between bivalve populations and their environment. In laboratory, co-infection experiments (trematode parasite Himasthla elongata, bacteria Vibrio tapetis) were conducted on these populations in controlled condition with or without metal contamination (cadmium). They showed that the response of bivalves to stress is species-dependent. Combinations were not intuitively deduced from the "mono - stress" responses underscoring the concept of interactions. Despite the complexity and diversity of these interactions, some mechanisms predominated regardless of the studied species. While parasite infestation was not modulated by the presence of metal or bacteria in the environment, metal bioaccumulation in turn was strongly influenced by the presence of one or several pathogens. Beyond disrupting the accumulation of pollutants, the presence of pathogens interfered with the cellular detoxification mechanisms including impairment of the metallothionein (MT) synthesis. A focus on the response of MT in cockles exposed to a metal through the expression of isoform Cemt1 and protein synthesis confirmed the complementary of these observations (gene and protein). They also highlighted the need to analyze responses at different scales to obtain an overview of existing interactive process between pollutants and pathogens. Strong interactions were found: ?Cd x parasite? in the cockle and" bacteria x parasite "in the clam, at the genetic, protein and immune levels. Finally, these experiments highlighted the important role of life history on the sensitivity of organisms to pollutant-pathogen interactions.

No description available.

Depuis plusieurs dizaines d’années, la population des anguilles européennes a souffert d’un dramatique déclin et est classée parmi les espèces hors de leurs limites biologiques depuis 1998. Différents phénomènes, tels que la pêche, les obstacles aux migrations, ou le réchauffement climatique, en sont à l’origine. Cependant, les perturbations environnementales, telles que les contaminations métalliques et organiques ou les épisodes d’hypoxie, participent probablement à la vulnérabilité de cette espèce. Or, l’estuaire de la Gironde est soumis à des contaminations poly-métalliques historiques, ainsi qu’à des contaminations organiques de différentes origines, et à des épisodes hypoxiques réguliers. Les travaux menés au cours de cette thèse, se sont donc composés d’études de terrain visant à identifier les contaminants majeurs chez les anguilles jaunes de l’estuaire de la Gironde, ainsi que leur voie de bio-accumulation et leurs impacts au niveau physiologique, biochimique et moléculaire. Ces études ont nécessité la mise en place d’expériences préliminaires ayant pour objectifs de vérifier les impacts de certaines procédures, telles que l’anesthésie et la mise en cage des anguilles jaunes. D’autre part, les impacts des deux principaux contaminants métalliques et organiques des anguilles européennes de l’estuaire de la Gironde, ont été testés, de façon individuelle et combinée, ainsi que ceux de l'hypoxie sur des anguilles pré-contaminées ou non, lors d’une étude expérimentale.Ainsi, ces travaux ont mis en évidence que les anguilles européennes installées dans la zone avale de l’estuaire, étaient susceptibles d’être soumises à des contaminations poly-métalliques plus importantes, essentiellement d’origine trophique, responsables d’une croissance pondérale moins élevée et de perturbations transcriptionnelles hépatiques et cérébrales. De plus, bien que les contaminations métalliques de ces anguilles ne mettent pas ne danger la santé humaine, les contaminations en PCB sont, en revanche, supérieures aux normes de consommation. D’autre part, ces travaux ont également mis en évidence des dérèglements mitochondriaux ainsi qu’un stress oxydant, au niveau branchial et cérébral chez des anguilles contaminées au Cd, et au niveau cérébral, branchial, hépatique et rénal chez les anguilles contaminées aux PCB. Enfin, la concomitance de ces deux contaminants et/ou d’un épisode d’hypoxie, réduit et/ou retarde les capacités de réponses transcriptionnelles de ces anguilles. Il semble donc que les différentes perturbations chimiques subies par les anguilles européennes au stade jaune au sein de l’estuaire de la Gironde participent de façon non négligeable à la vulnérabilité de cette espèce. / For several decades, the European eel has been suffering from a dramatic decline and has been classified among species beyond their biological limit since 1998. Different phenomena, as fishing, migration barriers, or global warming, are to blame. Environmental perturbations, as metallic and organic contaminations, or hypoxic episodes, probably take part to the vulnerability of this species. The Gironde estuary has been submitted to historic poly-metallic contaminations, to organic contaminations from different origins, and to regular hypoxic episodes. This thesis work is composed of field studies aimed to identify the main contaminants in yellow eel living in the Gironde estuary, their major bioaccumulation way and impacts on physiological, biochemical and molecular parameters. These studies needed preliminary experiments aimed to verify whether some field and handling procedures are consistent with ecotoxicological analyses. Moreover, impacts of the two main contaminants in European eels from the Gironde estuary have been assessed individually or combined, with those of hypoxia on pre-contaminated eels.Thus, these studies have demonstrated that European eels installed in the downstream area of the estuary, were likely to be subject to largest poly-metallic contaminations, mostly by food web, and responsible for a less weight gain and high liver and brain transcriptional disturbances. Furthermore, although the metal contamination of eels does not endanger human health, PCB contaminations are higher than consumption standards. On the other hand, these studies have also revealed oxidative stress and mitochondrial disorders in gills and brain of Cd-contaminated eels, and in brain, gills, liver and kidneys of PCB-contaminated eels. The combination of these two contaminants and/or an episode of hypoxia, reduces and/or delays the transcriptional responses ability of these eels. It seems that the different chemical disturbances, suffered by the yellow eels in the estuary of the Gironde, participate significantly to this species vulnerability.

Zinc, cadmium, and mercury are divalent metals and constitute same group of the periodic table. While zinc is an essential metal, the others are toxic metals. The most important common feature among these metals is the ability to induce the synthesis of metallothioneins (MT), which occurs in two vital organs involved in detoxification, the liver and kidney. The main role of MT is the detoxification of heavy metals and the regulation of homeostasis of essential trace metals, such as copper and zinc. There are several studies about mercury toxicity and the role of MT in adult animals. However, the sensitivity of developing animals to various compounds differs from that observed in adults and may to related to different posnatal phases of the development. The aim of this investigation was to verify the effects of CdCl2 and ZnCl2 pretreatments on the deleterious effects of HgCl2 in young rats and to investigate whether MT were involved in this protection mechanism. When pups were three days old, they received five consecutive injections (s.c.) of saline, CdCl2 (3.7 mg/kg/day) or ZnCl2 (27.0 mg/kg/day). On the five subsequent days, the animals were injected daily with one dose (s.c.) of saline or HgCl2 (5.0 mg/kg). Pups were sacrificed 24 h after the last dose and samples were collected (blood, liver and kidneys). The body and renal weights, hepatic and renal porphobilinogen synthase (PBG-synthase) activity, alanine aminotransferase activity, creatinine, urea, glycemia, and the retention of heavy metal in tissues were significantly altered by HgCl2. Prior exposure to CdCl2 prevented the effect of mercury on renal PBG-synthase, but did not alter mercury levels in the tissues. In general, the effects of mercury were prevented or lessened by zinc, except that the zinc pre-treatment increased the retention of mercury in the kidneys and did not modify the increase of renal weight induced by mercury. MT contents were increased by treatments with mercury and zinc and the greatest increase was induced by latter. The metal distribution in subcellular fractions showed that in both the insoluble fraction (IF) and heat treated cytosolic fraction (HTC), the contents were modified by the treatments. Although the HTC fraction is rich in MT, higher zinc and mercury contents were verified in the IF from all tissues analyzed. The relationships between MT and HTC metals showed that in the hepatic and renal tissues whenever there is an increase of metal levels there is increase of MT content. The reduction of hepatic and blood mercury levels and the increase of this metal in the kidneys induced by zinc suggests that the heavy metal contained in the liver is carried to the kidneys through the blood. This process also would transport MT from the liver to the kidneys. Moreover, it is important to emphasize that in cells in proliferation, which occur during rapid growth, there are nuclear and mitochondrial MT. Therefore, the high content of mercury found in the IF, enriched fraction in nucleus and mitochondria, would be bound to MT, as well. Considering that the zinc pretreatment induced an increase of renal MT of around 80% and the group treated with zinc and mercury presented a content of mercury in this protein that was 25% higher than for the group treated only with mercury, these results suggest that MT are, at least in part, responsible for the reduction of the toxicity of mercury seen in the various parameters analyzed in this work. / O zinco, o cádmio e o mercúrio são metais divalentes pertencentes ao mesmo grupo da tabela periódica. O primeiro é um metal essencial e os demais são metais tóxicos. A característica comum mais notável entre eles é a capacidade de induzir à síntese de metalotioneínas (MT), que ocorre em dois órgãos vitais envolvidos na destoxificação, fígado e rins. A principal função das MT é a destoxificação de metais pesados e a regulação da homeostase de metais essenciais, como cobre e zinco. Há muitos estudos sobre a toxicidade do mercúrio e sobre o papel das MT em animais adultos. Entretanto, a sensibilidade de animais em desenvolvimento a vários compostos difere daquela observada em adultos e pode estar relacionada a diferentes intervalos pós-natais de desenvolvimento. O objetivo desta investigação foi verificar os efeitos dos pré-tratamentos com CdCl2 e ZnCl2 sobre os efeitos deletérios do HgCl2 em ratos jovens e investigar se as MT estão envolvidas neste mecanismo de proteção. Ratos de três dias de idade foram injetados com uma dose diária (s.c.), nos cinco dias consecutivos, de salina, CdCl2 (3,7 mg/kg) ou ZnCl2 (27,0 mg/kg). Nos cinco dias subseqüentes os animais foram injetados com uma dose diária (s.c.) de salina ou HgCl2 (5,0 mg/kg). Os animais foram sacrificados 24 h após a última dose e as amostras foram coletadas (sangue, figado e rins). Os pesos corporal e renal, a atividade da porfobilinogênio sintase (PBG-sintase) hepática e renal, a atividade da alanina aminotransferase, a creatinina, a uréia, a glicemia e a retenção do metal tóxico pelos tecidos foram significativamente alterados pelo HgCl2. A exposição prévia ao CdCl2 preveniu o efeito do mercúrio sobre a PBG-sintase renal, mas não alterou os níveis de mercúrio nos tecidos. Em geral, os efeitos do mercúrio foram prevenidos ou atenuados pelo zinco, exceto que o pré-tratamento com zinco aumentou o acúmulo de metal pesado nos rins e não modificou o aumento do peso renal induzidos pelo mercúrio. O conteúdo de MT foi aumentado pelos tratamentos com mercúrio e zinco e a sua maior elevação foi induzida pelo zinco. A distribuição de metal nas frações subcelulares mostrou que em ambas, fração insolúvel (FI) e fração citosólica tratada a quente (CTQ), os conteúdos foram modificados pelos tratamentos. Embora a fração CTQ seja rica em MT, os maiores conteúdos de zinco e mercúrio foram verificados na FI de todos os tecidos analisados. As relações entre MT e metais na fração CTQ revelaram que nos tecidos hepático e renal sempre que há um aumento nos teores de metal, há um aumento no conteúdo de MT. A redução dos níveis de mercúrio hepático e sangüíneo e o aumento do conteúdo desse metal nos rins induzidos pelo zinco sugerem que o metal pesado contido no fígado é transportado para os rins pelo sangue. Esse processo também pode estar carreando MT do fígado para os rins. Além disso, é importante salientar que em células em proliferação, o que ocorre durante o crescimento acelerado, há MT nos núcleos e nas mitocôndrias. Desse modo, o alto conteúdo de mercúrio encontrado na FI, fração rica em núcleos e mitocôndrias, também estaria associado às MT. Considerando que o pré-tratamento com zinco induziu a um aumento de 80% no conteúdo de MT renal e o grupo tratado com zinco e mercúrio apresentou um conteúdo 25% maior de metal tóxico nessa proteína do que aquele verificado no grupo que foi tratado somente com mercúrio, esses resultados sugerem que as MT são, pelo menos em parte, responsáveis pela redução da toxicidade do mercúrio verificada em vários parâmetros analisados nesse trabalho.

Submitted by Biblioteca de Pós-Graduação em Geoquímica BGQ (bgq@ndc.uff.br) on 2017-04-26T16:59:53Z No. of bitstreams: 1 Dissert-LeonardoFrazão.pdf: 2516279 bytes, checksum: 48923c148348e036ced167489ad1b5d2 (MD5) / Made available in DSpace on 2017-04-26T16:59:53Z (GMT). No. of bitstreams: 1 Dissert-LeonardoFrazão.pdf: 2516279 bytes, checksum: 48923c148348e036ced167489ad1b5d2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Universidade Federal Fluminense. Instituto de Química. Programa de Pós-Graduação em Geociências- Geoquímica Ambiental. Niterói, RJ / O potencial de bioacumulação de metais; capacidade de expressar biomarcadores moleculares (como a indução de proteínas semelhantes a metalotioneínas, MTLPs); e biomarcadores histológicos (quantidade de canais aquíferos, câmaras coanocitárias e fibras de colágeno) foram estudados na esponja Hymeniacidon heliophila. Em aquário, foi realizada exposição da esponja a diferentes concentrações de cádmio (controle; 0,05; 0,4 e 4 mg L-1 de Cd) ao longo de determinados períodos de tempo (tempo zero, 24 horas, 7 dias e 14 dias). Também foi avaliada a ocorrência de MTLPs e as concentrações de metais potencialmente indutores da síntese dessas proteínas (Cd, Ni, Cu e Zn) em H. heliophila proveniente diretamente da praia da Boa Viagem, situada na Baía de Guanabara (RJ). No experimento em aquário, a esponja acumulou uma concentração máxima de 114 mg Cd Kg-1, após 14 dias de exposição a 4 mg L-1 deste metal. Este valor corresponde a 14 vezes o encontrado em esponjas do aquário controle. Por meio da técnica de eletroforese (SDS-Page) aliada a derivatização com monobromobimano, foi possível observar forte indução de MTLPs apenas nas amostras expostas a 0,4 mg L-1 de Cd nos períodos de 24 horas e 7 dias. As técnicas de histologia utilizadas para evidenciar os efeitos morfológicos causados pela exposição ao Cd revelaram que em 24 horas houve diminuição na quantidade das estruturas analisadas. A partir desse período, nas esponjas submetidas a 4 mg L-1 de Cd, houve tendência de diminuição (exceto para fibras colágenas) das estruturas, enquanto em 0,4 mg L-1 de Cd houve tendência de estabilização e, em 0,05 mg L-1 de Cd, recuperação. No estudo na praia da Boa Viagem, em nenhuma das cinco coletas realizadas, foi constatada indução de MTLPs. As médias das concentrações de metais encontradas no tecido das esponjas foram: 0,28 mg Cd kg-1, 46,3 mg Cu kg-1, 2 mg Ni kg-1 e 965 mg Zn kg-1. Estes valores são elevados quando comparados às concentrações reportadas para outros organismos da Baía de Guanabara. Assim, a alta eficiência em acumular metais (observada na praia da Boa Viagem) e a capacidade de induzir MTLPs e respostas histológicas (frente à exposição ao Cd), indicam possibilidades para utilização de H. heliophila em futuros programas de biomonitoramento. / The metal bioaccumulation potential, the expression of molecular biomarkers (induction of metallothionein-like proteins, MTLPs) and histological biomarkers (number of channels, choanocyte chambers and collagen fibers) were studied in the sponge Hymeniacidon heliophila. Exposure experiments to different concentrations of cadmium (<0.01, 0,05, 0,4 and 4 mg L-1 Cd) along different time intervals (t = 24 hours, 7 days and 14 days) were performed under controlled (aquarium) conditions. The occurrence of MTLPs and metal concentrations potentially involved in inducing the synthesis of these proteins (Cd, Ni, Cu and Zn) were also investigated in H. heliophila directly sampled from the Boa Viagem beach, located in Guanabara Bay (RJ). In the aquarium experiment, the sponge accumulated a maximum concentration of 114 mg Cd kg-1 after 14 days of exposure to 4 mg L-1 of this metal. This corresponds to 14 times the concentration found in sponges found in a control aquarium. Electrophoresis (SDS-PAGE) combined with monobromobimano derivatization indicated a strong induction MTLPs only in samples exposed to 0,4 mg L-1 Cd in periods of 24 hours and 7 days. The histological techniques used to reveal the morphological effects caused by exposure to Cd revealed that within 24 hours there was a decrease in the quantity of the analyzed structures. In the study carried out in Boa Viagem beach, there was absence of MTLPs induction along the five sampling periods evaluated. The mean concentrations of metals found in the tissue of the sampled sponges were: 0,28 mg kg-1 Cd, 46,3 mg kg-1 Cu, Ni 2 mg kg-1 and 965 mg kg-1 Zn. These values are higher than concentrations reported for other organisms from Guanabara Bay. Thus, the high efficiency in accumulating metals (found in the Boa Viagem beach) and the ability to induce MTLPs and histological responses (under high Cd exposure) indicate possible uses of H. heliophila in future biomonitoring programs

Le bassin d’Arcachon est une lagune semi-fermée qui concentre de forts enjeux économiques grâce à la pratique de l’ostréiculture. Cette activité est affectée depuis une trentaine d’années par des phénomènes de mortalités estivales, et plus récemment par des surmortalités du naissain. Ces travaux se sont intéressés au rôle des polluants majeurs du bassin d’Arcachon dans ce contexte de crise en étudiant leurs effets sur la biologie de Crassostrea gigas. Une approche in situ a d’abord été adoptée afin d’identifier les contaminants les plus présents dans le milieu naturel. Des opérations de transplantation d’huîtres et des prélèvements de sédiments ont permis de quantifier divers contaminants et d’associer leur présence à des réponses biologiques. Les polluants ainsi identifiés ont ensuite été employés en conditions contrôlées au laboratoire. Trois expérimentations ont été réalisées : la première concerne l’étude des voies de contamination par le tributylétain ; la seconde concerne les effets des pesticides et du cuivre ; la troisième concerne l’effet des HAP sur des huîtres diploïdes et triploïdes. Nos résultats indiquent que les organismes transplantés au coeur de la lagune sont plus exposés aux polluants, en lien avec les caractéristiques hydrodynamiques du système. La plupart des paramètres biologiques étudiés sur le terrain ont par ailleurs montré une saisonnalité liée aux processus de gamétogenèse. Au laboratoire, la plupart des contaminants testés ont induit une réponse adaptative chez les huîtres exposées. Notre étude souligne l’importance de coupler approche de terrain et approche expérimentale pour comprendre le fonctionnement des écosystèmes côtiers. / The Arcachon Bay is a semi-enclosed lagoon and represents the core of strong economic stakes through the practice of oyster-farming. This activity has been affected for around thirty years by summer mortality events, and more recently by abnormally high death rates of juveniles. This work focused on the role of the Arcachon Bay’s main contaminants in this crisis, by studying their effects on the cupped oyster’s biology. Firstly, an in situ approach was adopted in order to identify the major pollutants of the bay: caged oysters were transplanted and sediments were sampled. The presence of contaminants in the samples was associated to biological responses. Secondly, the contaminants identified in situ were used in controlled conditions at the laboratory. Three experimentations were performed; the first dealt with the contamination pathways of tributyltin; the second focused on the biological effects of pesticides and copper; the third concerned the effects of PAH towards diploid and triploid oysters. Our results indicate that the inner stations present higher accumulation of metals and PAH, in accordance with the hydrodynamic features of the bay. The bioindicators used in situ exhibited seasonal trends related to the oysters’ gametogenesis. In the laboratory, most of the contaminants used at environmental levels induced an adaptive response of the exposed oysters. Our study highlights the importance of coupling in situ and laboratory approaches in order to understand the functioning of coastal ecosystems.