Efeitos da exposição crónica de microplásticos ao nível de stresse oxidativo, comportamento e função mitocondrial de peixe zebra adulto (Danio rerio)
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2020-07-20
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A poluição plástica é uma das principais preocupações ambientais a nível mundial, com destaque para os microplásticos – partículas menores que 5 mm. Não é ainda claro a dimensão dos efeitos prejudiciais que estas podem ter ou as suas reais concentrações no ambiente. Assim, com o objetivo de esclarecer um pouco mais acerca dos possíveis efeitos destas micropartículas, neste trabalho foram avaliados os efeitos causados pela exposição crónica a microplásticos ao nível do fígado e cérebro de peixes-zebra adultos. O ensaio decorreu durante 21 dias com diferentes concentrações de microplásticos – 0,1 mg/L (LMP) e 1 mg/L (HMP). Os microplásticos foram obtidos através da trituração de objetos usados do dia-a-dia e o produto final apresentou-se, maioritariamente, como uma mistura polipropileno e poli(etileno-co-propileno), de forma irregular e com diâmetro entre os 79 e 220 nm. A análise das consequências destas partículas foi centrada no metabolismo da mitocôndria – avaliação do potencial membranar, da respiração mitocondrial e da atividade dos complexos da cadeia transportadora de eletrões (CTE) – contudo também foram realizados testes comportamentais e análises de diversas enzimas e metabolitos associados ao stresse oxidativo – ROS, SOD, CAT, GPx, GR, GST, CarE, GSH, GSSG, OSI, LPO, AChE, LDH. No cérebro, foram verificados sinais de que os animais sofreram stresse ambiental e um consequente aumento do stresse oxidativo, com aumentos da respiração em estado 3 do complexo IV e da atividade do complexo I da mitocôndria no grupo HMP, para além da diminuição da GPx e LDH (HMP) e aumento da GSSG (LMP). No entanto, a inexistência de alterações no comportamento e na atividade de AChE apontam para que tenha existido uma adaptação aos microplásticos. No fígado, os sinais de toxicidade dos MP foram mais evidentes, com uma grande descida no potencial de membrana mitocondrial nos dois grupos – especialmente no HMP (inibição de 66% em relação ao controlo) – para além de uma queda do estado 3 da respiração mitocondrial nos complexos I e II, e de alterações em vários parâmetros de stresse oxidativo no grupo HMP – descida de GPx, GR e GST, e subida da GSSG. As descidas nos valores de CarE (LMP) e de AChE e LDH (HMP) reforçam também a existência de danos neste órgão. Todos estes são sinais claros de que existiu toxicidade no fígado. No entanto, os níveis de ROS desceram significativamente nos dois grupos experimentais, apontando para outro mecanismo de toxicidade que não através do stresse oxidativo. O desacoplamento da CTE por parte das micropartículas ou a interferência com componentes móveis da CTE são possíveis causas avançadas, mas outros testes complementares teriam de ser realizados de forma a apurar, com certezas, o mecanismo de origem desta toxicidade e a real dimensão dos danos nos órgãos testados. Concluindo, os microplásticos tiveram efeitos prejudiciais na fisiologia dos animais, principalmente no fígado. Isto pode causar dificuldades ao nível da saúde e eventual sobrevivência dos animais, algo que pode ser preocupante visto a ubiquidade dos microplásticos no ambiente. No entanto, o mecanismo pelo qual estes danos ocorreram não é claro e seriam necessários estudos complementares.
Plastic pollution has been a growing problem throughout the last few decades, currently being a major worldwide environmental issue. Microplastics – plastic particles with less than 5 mm diameter – specifically, are one of the main concerns, as their actual concentrations on the environment and their effects on the organism are still not entirely clear. This study was performed with the intent of enlightening a little more about the dangers and characteristics of these particles. We evaluated the effects caused by the chronic exposure to microplastics on liver and brain of adult zebrafish. The exposure lasted for 21 days using two different concentrations of microplastics – 0.1 mg/L (LMP) and 1 mg/L (HMP). The microplastics used were obtained by blending day-to-day objects, with the final product consisting predominantly of a polypropylene and poly(ethylene-co-propylene) mixture, with particles of irregular shape and sizes between 79 and 220 nm. The analysis of the effects of these particles was mainly focused on the metabolism of the mitochondria – membrane potential, mitochondrial respiration and electron transport chain (ETC) complexes activity – but behavioural tests and analysis of several enzymes and metabolites associated with oxidative stress were also performed – ROS, SOD, CAT, GPx, GR, GST, CarE, GSH, GSSG, OSI, LPO, AChE, LDH. In the brain, there were signs that the animals suffered from environmental stress and a consequent increase of oxidative stress, with the promotion of state 3 respiration of complex IV and complex I activity on the mitochondria of the HMP group, in addition to the inhibition of GPx and LDH (HMP) and the increase of GSSG levels (LMP). Nevertheless, the lack of significant alterations on the behaviour and neurotoxicity of tested animals indicate an adequate response to the microplastics. On the liver, the signs of toxicity were more prominent, with a significant inhibition of the mitochondrial membrane potential on both experimental groups – especially on HMP (a 66% inhibition) – in addition to a reduction of the state 3 respiration rate associated to complex I and II substrates, and several changes on oxidative stress parameters on the HMP group – inhibition of GPx, GR and GST, and an increase of GSSG. The inhibition of CarE (LMP), AChE and LDH (HMP) also support that damages have occurred on this organ. All of these are clear signs of toxicity on this organ. However, the levels of ROS dropped significantly on both experimental groups, indicating that oxidative stress wasn‟t the cause. Uncoupling of the ETC or inference of ETC‟s mobile components are suggested causes to these alterations, but only subsequent tests could determine the definite mechanism and the true nature of the damages caused on the tested organs. In conclusion, microplastics had detrimental effects on the physiology of the tested animals, especially on the liver. This can lead to health complications and threaten the subject‟s survival, which can be worrying given microplastics‟ worldwide presence and persistence on the environment. However the mechanism through which these damages have occurred is not entirely clear and subsequent studies would be necessary.
Plastic pollution has been a growing problem throughout the last few decades, currently being a major worldwide environmental issue. Microplastics – plastic particles with less than 5 mm diameter – specifically, are one of the main concerns, as their actual concentrations on the environment and their effects on the organism are still not entirely clear. This study was performed with the intent of enlightening a little more about the dangers and characteristics of these particles. We evaluated the effects caused by the chronic exposure to microplastics on liver and brain of adult zebrafish. The exposure lasted for 21 days using two different concentrations of microplastics – 0.1 mg/L (LMP) and 1 mg/L (HMP). The microplastics used were obtained by blending day-to-day objects, with the final product consisting predominantly of a polypropylene and poly(ethylene-co-propylene) mixture, with particles of irregular shape and sizes between 79 and 220 nm. The analysis of the effects of these particles was mainly focused on the metabolism of the mitochondria – membrane potential, mitochondrial respiration and electron transport chain (ETC) complexes activity – but behavioural tests and analysis of several enzymes and metabolites associated with oxidative stress were also performed – ROS, SOD, CAT, GPx, GR, GST, CarE, GSH, GSSG, OSI, LPO, AChE, LDH. In the brain, there were signs that the animals suffered from environmental stress and a consequent increase of oxidative stress, with the promotion of state 3 respiration of complex IV and complex I activity on the mitochondria of the HMP group, in addition to the inhibition of GPx and LDH (HMP) and the increase of GSSG levels (LMP). Nevertheless, the lack of significant alterations on the behaviour and neurotoxicity of tested animals indicate an adequate response to the microplastics. On the liver, the signs of toxicity were more prominent, with a significant inhibition of the mitochondrial membrane potential on both experimental groups – especially on HMP (a 66% inhibition) – in addition to a reduction of the state 3 respiration rate associated to complex I and II substrates, and several changes on oxidative stress parameters on the HMP group – inhibition of GPx, GR and GST, and an increase of GSSG. The inhibition of CarE (LMP), AChE and LDH (HMP) also support that damages have occurred on this organ. All of these are clear signs of toxicity on this organ. However, the levels of ROS dropped significantly on both experimental groups, indicating that oxidative stress wasn‟t the cause. Uncoupling of the ETC or inference of ETC‟s mobile components are suggested causes to these alterations, but only subsequent tests could determine the definite mechanism and the true nature of the damages caused on the tested organs. In conclusion, microplastics had detrimental effects on the physiology of the tested animals, especially on the liver. This can lead to health complications and threaten the subject‟s survival, which can be worrying given microplastics‟ worldwide presence and persistence on the environment. However the mechanism through which these damages have occurred is not entirely clear and subsequent studies would be necessary.
Descrição
Dissertação de Mestrado em Bioquímica
Palavras-chave
Peixe-zebra , microplásticos