Towards an understanding of ketamine teratogenicity in zebrafish early development
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2016
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A cetamina é um antagonista não-competitivo do recetor N-metil D-aspartato
(NMDA). Este composto é um anestésico dissociativo e analgésico, utilizado
clinicamente em seres humanos e em animais há mais de 40 anos. Devido a várias das
suas propriedades clínicas, a cetamina é ainda amplamente utilizada, quando comparada
com outros agentes anestésicos, com especial ênfase em situações de emergência em
países em desenvolvimento. No entanto, e apesar de potenciais novos usos clínicos
estarem atualmente a ser estudados, a utilização da cetamina tem sido limitada pelos seus
potenciais efeitos secundários. Dados recentes sugerem que a cetamina pode causar a
morte neuronal durante fases iniciais do desenvolvimento cerebral. Além disso, os riscos
da utilização indevida da cetamina, nomeadamente a sua utilização como droga de abuso,
têm sido relacionados com contaminação ambiental e potenciais riscos ecotoxicológicos.
Tudo isto tem gerado preocupações sobre o uso e consumo de cetamina, que são revistas
na introdução desta tese (capítulo I).
Os trabalhos desenvolvidos nesta tese têm por base a utilização de peixe-zebra
(Danio rerio) que tem mostrado grande potencial em estudos sobre processos de
desenvolvimento e de toxicidade de compostos. O peixe-zebra tornou-se um organismo
modelo no âmbito da política dos 3Rs e uma alternativa à experimentação animal em
investigação biomédica e ecotoxicologia. A presente tese apresenta uma visão geral sobre
a utilização de fases iniciais de desenvolvimento de embriões de peixe-zebra, para
elucidar os mecanismos subjacentes associados à toxicidade induzida por cetamina.
Com base em ocorrências farmacológicas, os embriões de peixe-zebra foram
expostos a cetamina (0,2; 0,4 e 0,8 mg mL-1), durante três fases diferentes do
desenvolvimento, por um período de 20 minutos. Os resultados deste trabalho são
apresentados no capítulo II, divididos em cinco estudos. No capítulo II.1, são abordados
os efeitos da exposição a cetamina durante a fase de blástula (2,5 horas após a
fecundação - hpf). Verificou-se que a exposição a cetamina durante esta fase de
desenvolvimento induz distúrbios nos processos normais de crescimento, induzindo
malformações nos embriões e mortalidade. O capítulo II.2 prossegue o trabalho anterior
e acrescenta o estudo dos efeitos da exposição a cetamina durante as fases de gástrula
(5,5 hpf) e segmentação (10,5 hpf). Além dos parâmetros de desenvolvimento, a absorção
de cetamina, o desenvolvimento de cartilagem e osso e a expressão de genes de
desenvolvimento foram também avaliados. Em geral, este estudo mostra que o perfil de
toxicidade da cetamina é dependente do momento de exposição, tendo sido identificada
a fase de blástula como a mais sensível. O estudo, com recurso a técnicas de biologia
molecular e bioquímica, dos parâmetros bioquímicos, como biomarcadores da toxicidade
induzida por cetamina, é apresentado no capítulo II.3. A capacidade de resistência à
desregulação de processos de stresse oxidativo foi avaliada nas diferentes fases de
desenvolvimento. Apesar de não afetar o estado redox geral, a cetamina é capaz de
interferir com diversos processos de stresse oxidativo, que foram relacionados com o
desenvolvimento gradual do sistema de defesa antioxidante no embrião. Foram também
analisados os efeitos da cetamina ao nível de processos de apoptose durante o
desenvolvimento embrionário, resultados estes apresentados no capítulo II.4. Neste
estudo, diferentes padrões de expressão de genes foram observados e relacionados com a
ativação direta e indireta de apoptose durante períodos embrionários de suscetibilidade
diferencial. Por fim, no capítulo II.5 são descritos os efeitos ao nível do comportamento,
após exposição a cetamina. Este estudo mostrou uma tendência dependente da dose para
afetar as respostas relacionadas com medo e/ou ansiedade após exposição em fases mais
avançadas, tais como a gástrula e segmentação, indicando possíveis distúrbios neurocomportamentais.
O capítulo III está subdividido em dois trabalhos nos quais se descrevem os
impactos de concentrações de cetamina, relevantes do ponto de vista ambiental, (50; 70
e 90 mg L-1, LC50=94,4 mg L-1), após um período de exposição de 24 horas com início na
fase de blástula. No capítulo III.1 descrevem-se as malformações graves dependentes da
dose à exposição da cetamina bem como os seus efeitos a longo prazo a nível
comportamental, tais como um aumento da tigmotaxia. O capítulo III.2 prolonga os
estudos iniciados no capítulo anterior demonstrando que além das alterações
morfológicas, a exposição à cetamina foi capaz de induzir respostas de stresse oxidativo
e apoptose através do envolvimento de vias dependentes do p53.
Por fim, no capítulo IV apresentam-se as conclusões gerais da tese acerca dos
efeitos teratogénicos e embriotóxicos da exposição a cetamina em fases iniciais do
desenvolvimento de peixe-zebra e os seus possíveis impactos. Orientações para futuros
trabalhos são também discutidos.
Ketamine is a non-competitive antagonist of the receptor N-methyl-D-aspartate (NMDA). This compound is a dissociative anaesthetic and analgesic, used clinically in humans and animals for over 40 years. Due to several of its clinical properties, ketamine is still widely used, when compared to other anaesthetic agents, with special emphasis on emergency situations in developing countries. However, despite new potential clinical uses are currently being studied, the use of ketamine has been limited by its potential side effects. Recent data suggest that ketamine may cause neuronal death during the early stages of brain development. Moreover, the risks of improper use of ketamine, including its use as a drug of abuse, have been linked to environmental contamination and potential ecotoxicological risks. All this has raised concerns about the use and consumption of ketamine, which are reviewed in the introduction of this thesis (chapter I). The work in this thesis are based on the use of zebrafish (Danio rerio) that has shown to have a great potential in studies on development processes and compound toxicity. The zebrafish has become a model organism in the context of the 3Rs policy and an alternative to animal testing in biomedical research and ecotoxicology. This thesis provides an overview on the use of the early stages of zebrafish development to elucidate the mechanisms related to the toxicity induced by ketamine. Based on pharmacological events, zebrafish embryos were exposed ketamine (0.2, 0.4 and 0.8 mg mL-1) during three different stages of development, for a period of 20 minutes. The results of this work are presented in chapter II, which is divided into five studies. In chapter II.1, the effects of ketamine exposure at blastula stage (2.5 hours after fertilization - hpf) are addressed. It was found that ketamine exposure during this development phase induces disorders in normal growth processes, inducing defects in embryos and mortality. Chapter II.2 continues the previous work and adds the study of the effects of exposure to ketamine during the stages of gastrula (5.5 hpf) and segmentation (10.5 hpf). In addition to the development parameters, the absorption of ketamine, bone and cartilage development and the expression of developmental genes were also evaluated. Overall, this study shows that the toxicity profile of ketamine is dependent on the time of exposure, being blastula identified as the most sensitive phase. The study, with resource to molecular biology and biochemistry techniques, of biochemical parameters, as biomarkers of the toxicity induced by ketamine is shown in chapter II.3. The resilience of the deregulation of oxidative stress processes was evaluated at different stages of development. Although not affecting the overall redox state, ketamine is capable of interfering with oxidative stress processes which were associated with the progressive development of the embryo antioxidant defence system. Evaluation of ketamine effects at the level of apoptosis processes during embryonic development were also evaluated and the results presented in chapter II.4. In this study, different gene expression patterns were observed and related with the direct and indirect activation of embryonic apoptosis during periods of differential susceptibility. Finally, chapter II.5 describes the effects on behaviour after exposure to ketamine. This study showed a dose-dependent tendency to affect behavioural responses related with anxiety and fear after exposure in more developed stages, such as gastrula and segmentation, indicating possible neurobehavioural disorders. Chapter III is divided in two studies describing the effects of environmental relevant ketamine concentrations (50, 70 and 90 mg L-1,LC50 = 94.4 mg L-1) after a period of exposure of 24 hours starting on blastula stage. In chapter III.1 describes the serious dose-dependent malformations of ketamine exposure and its long term effects at the behavioral level, such as increased thigmotaxis. Chapter III.2 extends the studies initiated in the previous chapter and shows that in addition to the morphological changes, exposure to ketamine was able to induce oxidative stress responses and apoptosis through engagement of the p53-dependent pathways. Finally, chapter IV presents the general conclusions of this thesis about the embryotoxic and teratogenic effects of exposure to ketamine in the early stages of zebrafish development and its potential impacts. Guidelines for future work are also discussed.
Ketamine is a non-competitive antagonist of the receptor N-methyl-D-aspartate (NMDA). This compound is a dissociative anaesthetic and analgesic, used clinically in humans and animals for over 40 years. Due to several of its clinical properties, ketamine is still widely used, when compared to other anaesthetic agents, with special emphasis on emergency situations in developing countries. However, despite new potential clinical uses are currently being studied, the use of ketamine has been limited by its potential side effects. Recent data suggest that ketamine may cause neuronal death during the early stages of brain development. Moreover, the risks of improper use of ketamine, including its use as a drug of abuse, have been linked to environmental contamination and potential ecotoxicological risks. All this has raised concerns about the use and consumption of ketamine, which are reviewed in the introduction of this thesis (chapter I). The work in this thesis are based on the use of zebrafish (Danio rerio) that has shown to have a great potential in studies on development processes and compound toxicity. The zebrafish has become a model organism in the context of the 3Rs policy and an alternative to animal testing in biomedical research and ecotoxicology. This thesis provides an overview on the use of the early stages of zebrafish development to elucidate the mechanisms related to the toxicity induced by ketamine. Based on pharmacological events, zebrafish embryos were exposed ketamine (0.2, 0.4 and 0.8 mg mL-1) during three different stages of development, for a period of 20 minutes. The results of this work are presented in chapter II, which is divided into five studies. In chapter II.1, the effects of ketamine exposure at blastula stage (2.5 hours after fertilization - hpf) are addressed. It was found that ketamine exposure during this development phase induces disorders in normal growth processes, inducing defects in embryos and mortality. Chapter II.2 continues the previous work and adds the study of the effects of exposure to ketamine during the stages of gastrula (5.5 hpf) and segmentation (10.5 hpf). In addition to the development parameters, the absorption of ketamine, bone and cartilage development and the expression of developmental genes were also evaluated. Overall, this study shows that the toxicity profile of ketamine is dependent on the time of exposure, being blastula identified as the most sensitive phase. The study, with resource to molecular biology and biochemistry techniques, of biochemical parameters, as biomarkers of the toxicity induced by ketamine is shown in chapter II.3. The resilience of the deregulation of oxidative stress processes was evaluated at different stages of development. Although not affecting the overall redox state, ketamine is capable of interfering with oxidative stress processes which were associated with the progressive development of the embryo antioxidant defence system. Evaluation of ketamine effects at the level of apoptosis processes during embryonic development were also evaluated and the results presented in chapter II.4. In this study, different gene expression patterns were observed and related with the direct and indirect activation of embryonic apoptosis during periods of differential susceptibility. Finally, chapter II.5 describes the effects on behaviour after exposure to ketamine. This study showed a dose-dependent tendency to affect behavioural responses related with anxiety and fear after exposure in more developed stages, such as gastrula and segmentation, indicating possible neurobehavioural disorders. Chapter III is divided in two studies describing the effects of environmental relevant ketamine concentrations (50, 70 and 90 mg L-1,LC50 = 94.4 mg L-1) after a period of exposure of 24 hours starting on blastula stage. In chapter III.1 describes the serious dose-dependent malformations of ketamine exposure and its long term effects at the behavioral level, such as increased thigmotaxis. Chapter III.2 extends the studies initiated in the previous chapter and shows that in addition to the morphological changes, exposure to ketamine was able to induce oxidative stress responses and apoptosis through engagement of the p53-dependent pathways. Finally, chapter IV presents the general conclusions of this thesis about the embryotoxic and teratogenic effects of exposure to ketamine in the early stages of zebrafish development and its potential impacts. Guidelines for future work are also discussed.
Descrição
Tese de Doutoramento em Ciências Químicas e Biológicas
Palavras-chave
Anestesia , Cetamina , Comportamento , Desenvolvimento , Expressão génica , Peixe-Zebra (Danio rerio) , Stresse oxidativo