O efeito do chumbo sobre a fisiologia celular bramquial de crustáceos

Abstract

Resumo: As branquias de crustaceos constituem uma interface seletiva entre o ambiente externo e o eio interno e sua funcao e localizacao expoe suas celulas a alteracoes do ambiente aquatico, como, por exemplo, variacoes de salinidade e presenca de contaminantes. Metais como o chumbo (Pb) tem sido registrados em ecossistemas aquaticos em niveis acima do estabelecido pela legislacao brasileira. Alguns estudos indicam que crustaceos decapodes acumulam metais em orgaos como as branquias, sendo que a toxicidade do Pb esta associada a um desequilibrio osmotico e ionico em animais aquaticos. Portanto, o objetivo deste trabalho e avaliar in vitro a interferencia deste metal sobre celulas branquiais de especies de crustaceos com distintos padroes de osmorregulacao. Para tanto, antes de analisar a interferencia do metal foi realizada uma caracterizacao osmo-ionica de duas especies (Hepatus pudibundus . marinha e Callinectes ornatus . marinha/estuarina) diante de diluicao da agua do mar. Os animais foram expostos a condicao controle (33 ñ 0,5h) e a agua do mar diluida (25 ñ 0,5h e 25 ñ 8h), e apos exposicao foi retirada a hemolinfa para medida da osmolalidade e dosagem de ions (Na+, Cl-, K+ e Mg++). Tambem foi retirada uma amostra do musculo da quela e as branquias. No musculo e nas branquias foram medidos o teor de hidratacao e a concentracao de NPS (substancias positivas a ninidrina). Nas branquias tambem foi medida a atividade das enzimas Na+K+ATPase (NaK),e Anidrase Carbonica (AC). Conforme esperado H. pudibundus osmoconforma quando em agua diluida enquanto C. ornatus osmorregula. Ambas as especies mantiveram a hidratacao de seus tecidos sendo que o musculo de H. pudibundus utiliza a liberacao e NPS para a manutencao da agua tecidual. Nao houve alteracao na atividade da AC e a atividade da NaK diminuiu significativamente ja em 0,5 h de exposicao a agua diluida em ambas as especies. Os resultados indicam que os animais utilizam diferentes estrategias de regulacao osmotica e ionica para lidar com a diminuicao de salinidade, enquanto H. pudibundus depende mais de IIR (regulacao isosmotica intracelular) para tolerar a diluicao, C. ornatus tampona parcialmente a variacao do meio extracelular atraves de AER (regulacao anisosmotica extracelular), dependendo menos de IIR. Apos a caracterizacao osmo-ionica, foram realizados os experimentos in vitro da toxicidade do Pb. Foi analisada a toxicidade de quatro concentracoes de chumbo - Pb(NO3)2 (1, 10, 20 e 40ƒÊM) - sobre a viabilidade celular branquial em 3 diferentes tempos experimentais (1, 3 e 6 horas). Os resultados indicam que a toxicidade do Pb sobre as celulas branquiais e dose- e tempo- dependente, e que as celulas de H. pudibundus parecem ser mais sensiveis ao chumbo do que as celulas de C. ornatus. Alem disso, foi analisada a capacidade das celulas branquiais de regular volume diante de hiposmoticidade (choque hipo de 75 e 50%) e a influencia do Pb sobre a manutencao de volume celular em condicoes isosmoticas (isosmoticidade associada a duas ncentracoes de Pb . 10 e 40ƒÊM). Sem a presenca do Pb, as celulas branquiais de ambas as especies sao perfeitamente capazes de regulacao de volume celular diante do choque hiposmotico 75%. Celulas branquiais de C. ornatus entretanto apresentaram um pequeno inchaco durante o choque hiposmotico de maior intensidade (50%), enquanto celulas de H. pudibundus ainda apresentaram capacidade de regulacao de volume. Na presenca de Pb, mesmo que sob isosmoticidade, as celulas branquiais de ambas as especies perderam volume. Celulas de H. pudibundus apresentaram cerca de 15% de reducao de volume em ambas concentracoes de Pb testadas (10 e 40ƒÊM), celulas de C. ornatus foram menos sensiveis a concentracao de 10ƒÊM e apresentaram cerca de 10% de reducao de volume, enquanto na concentracao de 40ƒÊM o volume perdido foi tambem de cerca de 15%. Esses resultados indicam que o Pb promove de alguma forma um desequilibrio osmotico fazendo com que as celulas percam agua. Quando o Pb 10ƒÊM foi associado ao choque hiposmotico de 75% e 50%, celulas de C. ornatus perderam ainda mais volume enquanto celulas de H. pudibundus se mostraram capazes de regular volume celular. Foram entao analisadas as possiveis vias de atuacao do Pb sobre os mecanismos de regulacao de volume celular atraves da utilizacao de inibidores dessas vias associados a isosmoticidade, a isosmoticidade + 0ƒÊMPb e a hiposmoticidade 75% + 10ƒÊMPb. Para inibir canais de Ca2+, Na+K+ATPase e aquaporinas foram utlizados: verapamil, ouabaina e cloreto de mercurio, respectivamente. Os resultados indicam que as diferencas da influencia do Pb encontradas na regulacao de volume celular entre as duas especies se referem ao padrao de regulacao osmotica e ionica e grau de capacidade de regulacao de volume que as especies apresentam. C. ornatus parece ser mais suscetivel a alteracoes de volume celular por infl encia do Pb devido a maior entrada do metal atraves de fluxos ionicos para a absorcao de sal inerentemente presentes nas celulas branquiais, comuns a um organismo osmorregulador. Canais de Ca2+ parecem ser via de entrada de Pb, e aquaporina parece ser alvo de toxicicidade do Pb pelo menos em H. pudibundus. O Pb parece nao interferir na atividade da NaK em nenhuma das especies. Por fim foi realizada a identificacao de um possivel mecanismo de defesa branquial, o mecanismo MXR, assim como sua inducao pelas 4 concentracoes de Pb testadas. As branquias de ambos os animais apresentam esse mecanismo que possui maior atividade nas concentracoes mais baixas de Pb (1 e 10ƒÊM), e nao existe inducao do mecanismo nas concentracoes mais altas (20 e 40ƒÊM). Nossos resultados indicam que os efeitos toxicos do chumbo podem ser diferentes conforme a estrategia osmotica e ionica adotada por animais aquaticos e sugerem ainda que celulas de organismos osmorreguladores sao mais suscetiveis a contaminacao por metal do que celulas de organismos smoconformadores, principalmente em situacoes de reducao de salinidade.

Abstract: The crustacean gills constitute a selective interface between the external and internal environment, and their location and function expose their cells to alterations in the aquatic environment such as, for example, salinity variations and presence of contaminants. Metals such as lead (Pb) have been reported in aquatic ecosystems at levels above the established by Brazilian legislation. Some studies indicate that decapod crustaceans accumulate metals in organs like gills, and lead toxicity is associated with an osmotic and ionic disequilibrium in aquatic animals. Therefore, the objective of this work was to evaluate in vitro the influence of this metal on gill cells of crustacean species with different patterns of osmoregulation. For this, before considering lead interference, an osmoionic characterization of two species (Hepatus pudibundus- marine and Callinectes ornatus- marine/estuarine) in face of sea water dilution was performed. Animals were exposed to control condition (33‰ 0.5h) and diluted sea water (25‰ 0.5h and 25‰ 8h). After the exposure haemolymph was sampled to measure osmolality and ions Na+, Cl-, K, and Mg. A fragment of chelipod muscle and the gills were also removed for quantitative evauation of tissue hydration and the concentration of NPS (ninhydrin positive substances). In the gills Na+K+ATPase (NaK) and carbonic anhydrase (AC) activities were also measured. As expected, H. pudibundus osmoconforms when in diluted seawater and C. ornatus osmorregulates. Both species maintained the degree of hydration of their tissues, and H. pudibundus uses the release of NPS for tissue water maintenance. There was no change in CA activity and the activity of NaK has decreased significantly after 0.5 h of exposure to diluted seawater in both species. The results indicate that the animals use different strategies for osmotic and ionic regulation to eal with decreasing salinity, while H. pudibundus depends more on IIR (isosmotic intracellular regulation) to tolerate dilution, Callinectes ornatus partially buffers the extracellular fluid by AER (anisosmotic extracellular regulation), depending less on IIR. After osmo-ionic characterization of both species, the in vitro experiment of Pb toxicity was performed. The toxicity of four lead concentrations was evaluated - Pb(NO3)2 (1, 10, 20 and 40ìM) - on gill cell viability in three different experimental times (1, 3 and 6 h). The results indicate that Pb toxicity on gill cells is dose- and time-dependent, and cells of H. pudibundus seem to be more sensitive to lead than the cells of C. ornatus. In addition, we analyzed the capacity of gill cells to regulate volume in hyposmotic conditions (hypo shock of 75 and 50%) and the influence of Pb on the maintenance of cell volume in isosmotic conditions (isosmotic associated with two concentrations of Pb - 10 and 40ìM ). Without the presence of Pb, gill cells of both species are quite capable of cell volume regulation in a 75% hyposmotic shock. Gill cells of C. ornatus, however, showed a small swelling during hyposmotic shock of greater intensity (50%), while cells of H. pudibundus still showed the ability of volume regulation. In the presence of Pb, even under isosmotic condition, the gill cells of both species lost volume. Cell of H. pudibundus showed about 15% of volume reduction in both tested Pb concentrations (10 and 40ìM), cells of C. ornatus were less sensitive to the concentration of 10ìM and showed about 10% in volume reduction, while the concentration of 40ìM the volume lost was also about 15%. These results indicate that Pb somehow promotes an osmotic imbalance leading to the loss of cell water. When 10ìM Pb was associated with hyposmotic shock of 75% and 50%, cells of C. ornatus lost even more volume, while cells of H. pudibundus were able to regulate cell volume. We then analyzed the possible pathways of Pb action on the mechanisms of cell volume regulation by using inhibitors of these pathways linked to isosmoticity, isosmoticity plus 10ìMPb, and 75% hyposmoticity plus 10ìM Pb. To inhibit Ca2+ channels, Na+K+ATPase and aquaporins were utilized: verapamil, ouabain and mercuric chloride, respectively. The results indicate that the differences of Pb influence found in cell volume regulatory capacity between the two species refer to the pattern of osmotic and ionic regulation and the capacity of cell volume regulation displayed by the species. C. ornatus seems to be more susceptible to changes in cell volume by Pb influence due to higher inflow of metal through ionic fluxes for salt absorption, putatively present in gill cells, common to osmoregulators. Ca2+ channels seem to be the Pb inflow pathway, and aquaporin seems to be target of Pb toxicity at least in H. pudibundus. Pb does not appear to interfere in the NaK activity in either species. Finally, the possible role of a gill defense mechanism, the MXR mechanism, was also evaluated. Its induction by four Pb concentrations was tested. The gills of both animals indeed show this mechanism, that has higher activity in lower concentrations of Pb (1 and 10ìM), and there is no induction of mechanism in higher Pb concentrations (20 and 40ìM). Our results indicate that the toxic effects of lead may be different depending on osmotic and ionic strategy dopted by aquatic animals, and further suggest that osmoregulators are more susceptible to contamination by metal than osmoconformers, especially in situations of salinity reduction.