Made available in DSpace on 2015-04-14T14:51:20Z (GMT). No. of bitstreams: 1
442001.pdf: 1823261 bytes, checksum: 10761416aa58c3d7680e6ff4e42ce20f (MD5)
Previous issue date: 2012-08-23 / Microcystins (MCs) constitute a family of cyanobacterial toxins, with more than 80 variants. These toxins are able to induce hepatotoxicity in several organisms, mainly through the inhibition of protein phosphatases PP1 and PP2A and oxidative stress generation. Recent evidence shows that MCs can either accumulate in brain or alter behavior patterns of fish species. Thus, this thesis aimed to study the effects of MC-LR (with the variable amino acids leucine (L) and arginine (R)) exposure on biochemical parameters in zebrafish, emphasizing the cholinergic and purinergic signaling, as well as to evaluate the behavioral patterns and whole-body cortisol levels. In vivo studies showed that 100 μg/L MC-LR for 24 h led to a significant increase in the AChE activity (27%) when zebrafish were exposed to the toxin dissolved in water, but did not cause any significant changes when injected intraperitoneally. In addition, semiquantitative RT-PCR analysis demonstrated that 100 μg/L MC-LR exposure also increased ache mRNA levels in zebrafish brain. The in vitro assays did not reveal any significant changes in AChE activity. We also assessed behavioral patterns and whole-body cortisol levels of adult zebrafish exposed to cell culture of the microcystin-producing cyanobacterium Microcystis aeruginosa. MC-LR exposure (100 μg/L) decreased by 63% the distance traveled and increased threefold the immobility time when compared to the control group. Interestingly, no significant alterations in the number of line crossings were found at the same MC-LR concentration and time of exposure. When animals were exposed to 50 and 100 μg/L, MC-LR promoted a significant increase (around 93%) in the time spent in the bottom portion of the tank, suggesting an anxiogenic effect. In addition, the results also showed that none of the MC-LR concentrations tested promoted significant alterations in absolute turn angle, path efficiency, social behavior, or whole-body cortisol level. Moreover, we evaluated the acute effects of different concentrations of MC-LR on NTPDases (nucleoside triphosphate diphosphohydrolases) and 5 - nucleotidase in adult zebrafish (Danio rerio) brain membranes. The results have shown no significant changes in ATP, ADP and AMP hydrolysis in zebrafish brain membranes. MC-LR in vitro also did not alter ATP, ADP and AMP hydrolysis in the concentrations tested. These findings show that acute exposure to MC-LR did not modulate ectonucleotidases activity in the tested conditions. Taken together these findings provide the first evidence that brain AChE is another potential target for MCs and that MC-LR exposure significantly impairs animal's exploratory performance. Nevertheless, further studies including long-time exposure should be performed in order to achieve a better understanding about MCLR toxicity mechanisms in the central nervous system / Microcistinas (MCs) constituem uma fam?lia de toxinas, com mais de 80 variantes. Estas toxinas s?o capazes de induzir hepatotoxicidade em diversos organismos, principalmente atrav?s da inibi??o das fosfatases PP1 e PP2A e gera??o de estresse oxidativo. Evid?ncias recentes mostram que MCs podem acumular no c?rebro e alterar padr?es comportamentais em diferentes esp?cies de peixes. Portanto, a presente tese teve por objetivo estudar os efeitos da exposi??o ? MC-LR (com os amino?cidos vari?veis leucina (L) e arginina (R)) sobre par?metros bioqu?micos em zebrafish, enfatizando os sistemas colin?rgicos e purin?rgicos, bem como, avaliar padr?es comportamentais e n?veis de cortisol corporal. Resultados do estudo in vivo mostraram que a exposi??o a 100 μg/L de MC-LR durante 24 h causaram um aumento significativo na atividade da AChE (27%) quando zebrafish foi exposto ? toxina dissolvida em ?gua; por?m, a toxina n?o causou mudan?as significativas quando injetada intraperitonealmente. Al?m disso, a an?lise semiquantitativa de RT-PCR demonstrou que a exposi??o ? 100 μg/L de MC-LR tamb?m aumentou os n?veis de RNAm da ache em c?rebro de zebrafish. Os ensaios in vitro n?o revelaram nenhuma altera??o significativa na atividade da AChE. N?s tamb?m avaliamos padr?es comportamentais e n?veis de cortisol corporal de zebrafish adultos expostos ? cultura de c?lulas de Microcystis aeruginosa produtoras de MC-LR. A exposi??o ? MC-LR (100 μg/L) diminuiu em 63% a dist?ncia viajada e aumentou tr?s vezes o tempo de imobilidade quando comparado ao grupo controle. Interessantemente, n?o houve altera??o no n?mero de linhas cruzadas na mesma concentra??o e tempo de exposi??o ? MC-LR. Quando animais foram expostos a 50 e 100 μg/L, a MCLR promoveu um aumento significante (aproximadamente 93%) no tempo gasto na por??o inferior do aqu?rio teste, sugerindo um efeito ansiog?nico. Adicionalmente, os resultados tamb?m mostraram que nenhuma das concentra??es de MC-LR testadas promoveu altera??es significativas nas mudan?as de ?ngulo, efici?ncia de rota e intera??o social ou, no n?vel de cortisol corporal total. Al?m disso, tamb?m foi avaliado o efeito de diferentes concentra??es de MC-LR na atividade das NTPDases (nucleos?deo trifosfato difosfoidrolase) e 5 nucleotidase em membranas cerebrais de zebrafish (Danio rerio) adultos. Os resultados mostraram que n?o houve altera??o nas hidr?lises de ATP, ADP e AMP. Nos experimentos in vitro tamb?m n?o houve altera??o nas hidr?lises dos nucleot?deos nas concentra??es testadas. Estes achados mostram que exposi??o aguda ? MC-LR n?o modulou a atividade das ectonucleotidases nas condi??es testadas. Ademais, fornecem a primeira evid?ncia que a AChE cerebral ? um outro alvo potencial das MCs e que exposi??o ? MC-LR prejudica significativamente a performance explorat?ria do animal. Estudos futuros incluindo exposi??o de longo prazo dever ser feitos para um melhor entendimento dos mecanismos de toxicidade das MCs no Sistema Nervoso Central
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/5445 |
| Date | 23 August 2012 |
| Creators | Kist, Luiza Wilges |
| Contributors | Bogo, Maur?cio Reis |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Biologia Celular e Molecular, PUCRS, BR, Faculdade de Bioci?ncias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 8198246930096637360, 600, 600, 36528317262667714 |
Page generated in 0.0025 seconds