Ammonia toxicity in zebrafish (Danio rerio)
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2017
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Nos peixes, a amónia é um composto tóxico produzido durante o catabolismo dos
aminoácidos mas, no meio aquático, a sua acumulação deve-se a várias fontes naturais e
antropogénicas. Nos teleósteos, a amónia é produzida no fígado, sendo posteriormente
eliminada pelas brânquias através do transporte transcelular realizado pelas glicoproteínas Rh,
mas também através do transporte paracelular. Independentemente do mecanismo, a amónia
atravessa a membrana branquial seguindo o gradiente de difusão que é mantido pela camada
acídica presente junto do epitélio. Devido à sua toxicidade, a acumulação de amónia no
organismo desencadeia vários efeitos negativos sendo a sua maioria no sistema nervoso
central. Para evitar esses efeitos, os peixes desenvolveram mecanismos para reduzir a
intoxicação da amónia e garantir a sua sobrevivência.
O peixe-zebra (Danio rerio) é uma espécie modelo com características particulares e
cujo genoma se encontra sequenciado, sendo por isso uma espécie muito usada em
investigação. Contudo, muito ainda está por se saber sobre a toxicidade e excreção da amónia
nesta espécie. Posto isto, os principais objetivos desta tese são determinar 1) as concentrações
letais durante o desenvolvimento do peixe-zebra, 2) as alterações no transcriptoma da
brânquia, fígado e cérebro deste peixe após exposição a uma concentração sub-letal durante
96 h, 3) quais os mecanismos usados pelos adultos para evitar a intoxicação pela amónia e,
finalmente, 4) qual o papel do sistema endócrino na excreção e/ou toxicidade da amónia.
Assim, foram realizadas experiências de LC50 para todo o ciclo de vida desta espécie, sendo
que nos adultos também se testou a sua tolerância perante diferentes condições ambientais.
Posteriormente, realizou-se uma experiência de exposição à amónia, durante 96 h, sendo que
no final, a brânquia, fígado e cérebro dos peixes foram recolhidos e analisados num 44k
Microarray da Agilent. Os genes com diferenças estatísticas foram utilizados numa análise da
ontologia dos genes e das via metabólicas recorrendo a ferramentas on-line, mas também
numa análise detalhada da expressão de genes envolvidos em mecanismos que evitam a
intoxicação da amónia. Por fim, e considerando a resposta do sistema endócrino à exposição à
amónia, experiências sobre a taxa de excreção de amónia após a administração da prolactina e
do cortisol foram também realizadas, tanto em condições controlo como após administração
de amónia.
Os resultados mostraram que as fases do desenvolvimento embrionário do peixe-zebra
são os mais tolerantes à amónia, e que os adultos, quando comparados com os de água salobra (6 ‰) ou tamponada, apresentam menor sensibilidade do que quando expostos a água doce.
Quanto à exposição dos adultos à amónia, verificou-se que estes alteraram o transcriptoma do
fígado (2,169 sondas) e cérebro (669 sondas), mas principalmente da brânquia (7,001 sondas).
Uma das respostas desta espécie consistiu na alteração das vias de fornecimento de energia,
tendo sido os carboidratos preferencialmente usados aos lípidos. Dentro do metabolismo dos
carboidratos, a via da glicólise/gluconeogénese foi usada para produzir glucose no fígado e
energia na brânquia, o que permitiu a continua excreção de amónia contra gradiente
principalmente através de transportadores que consomem energia e cuja expressão também se
encontrou aumentada (p.ex. H+-ATPase e Na+/K+-ATPase). Por outro lado, a entrada da
amónia no organismo foi impedida, uma vez que se verificou um aumento da expressão dos
elementos do transporte paracelular (p.ex. claudinas, ocludinas e tight junctions) e,
consequentemente, redução da permeabilidade branquial. Para além disto, o ciclo,
crescimento, proliferação e diferenciação celular também foram alterados, para reduzir a
energia gasta na replicação celular e para eliminar as células danificadas pela amónia. No
peixe-zebra, enquanto que o fígado recorreu à incorporação da amónia em novos
aminoácidos, a brânquia reduziu a sua produção através da diminuição da expressão das
enzimas envolvidas no mesmo metabolismo. Relativamente ao sistema endócrino, a hormona
somatolactina e vários receptores hormonais (receptor da prolactina, da hormona do
crescimento, da tiroide, da paratiroide e péptido natriurético) responderam à exposição à
amónia, o que sugere que este sistema é importante para repor a homeostasia do peixe. No
entanto, ao contrário do esperado, a prolactina não induziu taxas de excreção de amónia
similares ou inferiores às do cortisol que, por sua vez, permitiu taxas de excreção muito
baixas. De facto, e embora ambas as hormonas tenham alterado a permeabilidade da brânquia,
aumentando a expressão das ocludinas, apenas o tratamento com cortisol aumentou os níveis
de transportadores de amónia. A disparidade destes resultados são discutidos nesta tese.
Concluindo, apesar da amónia não causar nenhum efeito visível nos peixes, ela pode
causar efeitos a nível molecular, comprometendo a geral função do organismo e levando,
posteriormente, à sua morte. Neste sentido, e tal como tem sido descrito noutras espécies, o
peixe-zebra desenvolveu estratégias para ultrapassar a toxicidade da amónia e inclusivé
recorreu ao sistema endócrino para garantir a sua homeostase e sobrevivência. Este estudo é
portanto relevante especialmente para aquaculturas e para monitorização ambiental.
In fishes, ammonia is a toxic compound produced endogenously through the catabolism of amino acids, but it can also be taken up from the aquatic environment coming from different natural and anthropogenic sources. In teleosts, ammonia is produced primarily in liver and its elimination occurs directly through the gills. Ammonia excretion is dominated by transcellular transport facilitated by Rh glycoproteins; however, paracellular transport elements may also contribute to its elimination. Regardless of the mechanism, ammonia crosses the branchial membranes following the blood-to-water partial pressure gradient, with the presence of an acidic boundary layer relevant for maintaining its excretion. Due to its toxicity, the internal accumulation of ammonia may cause severe negative effects in several tissues but most profoundly in the central nervous system. In an attempt to avoid these negative effects, fishes have developed mechanisms to reduce ammonia intoxication and guarantee their survival. Zebrafish (Danio rerio) is a vertebrate model species whose particular features and sequenced genome, make it widely used in research; however, much remains unclear about ammonia toxicity and excretion in this species. Hereupon, the main objectives of this thesis were to determine the zebrafish 1) life history stage specific lethal concentrations of ammonia, 2) transcriptomic responses of gill, liver and brain after a 96 h sub-lethal exposure, 3) mechanisms applied by adults to avoid ammonia intoxication and, finally, 4) endocrine roles in ammonia excretion and/or toxicity. To address these goals, LC50 experiments were initially performed for all zebrafish life history stages and adult tolerance was also tested under different water conditions. A sub-lethal concentration was then used for an additional 96 h experiment where gill, liver and brain samples were analyzed using an Agilent 44k Zebrafish Microarray. The statistical different genes obtained in the microarray were then used to perform a gene ontology and pathway analysis using online tools, as well as in a detailed gene expression analysis involving mechanisms to avoid ammonia intoxication. In addition, endocrine responses to ammonia exposure were determined, followed by exogenous prolactin and cortisol experiments on ammonia excretion rates under control and ammonia loading conditions. My results clearly shows that early developmental stages were the most ammonia tolerant among the entire life cycle of zebrafish and adults were less susceptible in freshwater than in 6 ‰ brackish water or Tris buffered freshwater. The 96 h exposure of adults to a sublethal concentration of ammonia resulted in transcriptomic changes in liver (2,169 probes) and brain (669 probes), but most predominately in gill (7,001 probes). Zebrafish increased the energy-demanding pathways, with carbohydrates instead of lipids used as fuel to guarantee the internal homeostasis. Within carbohydrate metabolism, the glycolysis/gluconeogenesis pathway was used to produce glucose in liver, while in gill it was used to produce energy, probably to maintain the excretion of ammonia against the inward gradient, as determined by the upregulation of several branchial transporter proteins that are energy dependent (e.g. H+- ATPase and Na+/K+-ATPase). In addition, branchial permeability was likely altered as indicated by changes in tight junction elements (e.g. claudins, occludins and tight junctions) to impede ammonia entrance. Moreover, changes in pathways involved in cell cycle, growth, proliferation and differentiation were observed, likely in an attempt to reduce energy demanding cell replication and to eliminate ammonia exposure damaged cells. In liver, upregulation of genes involved in the incorporation of ammonia in de novo amino acids were also noted, probably to reduce the ammonia intoxication. However, in gill, decreased mRNA of amino acid metabolic enzymes, indicated reduced ammonia production. Regarding the endocrine system response, somatolactin and hormone receptor mRNA levels (e.g. prolactin receptor, growth hormone receptor, thyroid receptor, parathyroid receptor and natriuretic peptide receptor) changed after ammonia exposure, which indicate an endocrine response to overcome the ammonia challenge and to regain the internal homeostasis. However, in contrast to what was expected, exogenous prolactin did not result in lower ammonia excretion rates than cortisol that, in the present study, unexpectedly induced the lowest excretion rates. In fact, although both hormones affected the expression of occludins and thus presumably branchial paracellular permeability, only cortisol enhanced ammonia transporter mRNA levels. The discrepancies in the results are discussed in the thesis. Overall, although ammonia may not cause any overt effect on fish, it can have impacts at the molecular level, which will compromise the general functions of the organism and consequently impede it performance. However, zebrafish, similarly to other teleosts, developed strategies to overcome ammonia toxicity and mobilized the endocrine system to guarantee their homeostasis and survival. This study is then important, especially for aquaculture and environmental monitoring.
In fishes, ammonia is a toxic compound produced endogenously through the catabolism of amino acids, but it can also be taken up from the aquatic environment coming from different natural and anthropogenic sources. In teleosts, ammonia is produced primarily in liver and its elimination occurs directly through the gills. Ammonia excretion is dominated by transcellular transport facilitated by Rh glycoproteins; however, paracellular transport elements may also contribute to its elimination. Regardless of the mechanism, ammonia crosses the branchial membranes following the blood-to-water partial pressure gradient, with the presence of an acidic boundary layer relevant for maintaining its excretion. Due to its toxicity, the internal accumulation of ammonia may cause severe negative effects in several tissues but most profoundly in the central nervous system. In an attempt to avoid these negative effects, fishes have developed mechanisms to reduce ammonia intoxication and guarantee their survival. Zebrafish (Danio rerio) is a vertebrate model species whose particular features and sequenced genome, make it widely used in research; however, much remains unclear about ammonia toxicity and excretion in this species. Hereupon, the main objectives of this thesis were to determine the zebrafish 1) life history stage specific lethal concentrations of ammonia, 2) transcriptomic responses of gill, liver and brain after a 96 h sub-lethal exposure, 3) mechanisms applied by adults to avoid ammonia intoxication and, finally, 4) endocrine roles in ammonia excretion and/or toxicity. To address these goals, LC50 experiments were initially performed for all zebrafish life history stages and adult tolerance was also tested under different water conditions. A sub-lethal concentration was then used for an additional 96 h experiment where gill, liver and brain samples were analyzed using an Agilent 44k Zebrafish Microarray. The statistical different genes obtained in the microarray were then used to perform a gene ontology and pathway analysis using online tools, as well as in a detailed gene expression analysis involving mechanisms to avoid ammonia intoxication. In addition, endocrine responses to ammonia exposure were determined, followed by exogenous prolactin and cortisol experiments on ammonia excretion rates under control and ammonia loading conditions. My results clearly shows that early developmental stages were the most ammonia tolerant among the entire life cycle of zebrafish and adults were less susceptible in freshwater than in 6 ‰ brackish water or Tris buffered freshwater. The 96 h exposure of adults to a sublethal concentration of ammonia resulted in transcriptomic changes in liver (2,169 probes) and brain (669 probes), but most predominately in gill (7,001 probes). Zebrafish increased the energy-demanding pathways, with carbohydrates instead of lipids used as fuel to guarantee the internal homeostasis. Within carbohydrate metabolism, the glycolysis/gluconeogenesis pathway was used to produce glucose in liver, while in gill it was used to produce energy, probably to maintain the excretion of ammonia against the inward gradient, as determined by the upregulation of several branchial transporter proteins that are energy dependent (e.g. H+- ATPase and Na+/K+-ATPase). In addition, branchial permeability was likely altered as indicated by changes in tight junction elements (e.g. claudins, occludins and tight junctions) to impede ammonia entrance. Moreover, changes in pathways involved in cell cycle, growth, proliferation and differentiation were observed, likely in an attempt to reduce energy demanding cell replication and to eliminate ammonia exposure damaged cells. In liver, upregulation of genes involved in the incorporation of ammonia in de novo amino acids were also noted, probably to reduce the ammonia intoxication. However, in gill, decreased mRNA of amino acid metabolic enzymes, indicated reduced ammonia production. Regarding the endocrine system response, somatolactin and hormone receptor mRNA levels (e.g. prolactin receptor, growth hormone receptor, thyroid receptor, parathyroid receptor and natriuretic peptide receptor) changed after ammonia exposure, which indicate an endocrine response to overcome the ammonia challenge and to regain the internal homeostasis. However, in contrast to what was expected, exogenous prolactin did not result in lower ammonia excretion rates than cortisol that, in the present study, unexpectedly induced the lowest excretion rates. In fact, although both hormones affected the expression of occludins and thus presumably branchial paracellular permeability, only cortisol enhanced ammonia transporter mRNA levels. The discrepancies in the results are discussed in the thesis. Overall, although ammonia may not cause any overt effect on fish, it can have impacts at the molecular level, which will compromise the general functions of the organism and consequently impede it performance. However, zebrafish, similarly to other teleosts, developed strategies to overcome ammonia toxicity and mobilized the endocrine system to guarantee their homeostasis and survival. This study is then important, especially for aquaculture and environmental monitoring.
Descrição
Tese de Doutoramento em Ciências Químicas e Biológicas
Palavras-chave
Amónia , Peixe-Zebra (Danio rerio) , Ontogenia , Hormonas , Transcriptoma