RESUMO
Tricaine methanesulfonate (MS-222) is a commonly used anaesthetic agent for immobilization of aquatic species. However, delayed development and malformations have been observed in 24 hpf (hours post-fertilization) zebrafish embryos after long-term immobilization. Still, no comprehensive study has been described regarding zebrafish exposure to MS-222 during the first hours of development, which are one of the most sensitive life stages to toxicants. Therefore, this research aimed to assess the toxicity of a 24 h exposure to MS-222 on zebrafish embryonic development. Based on the MS-222 LC50, early blastula stage embryos (~2 hpf) were exposed to 0, 12.5, 25 and 50 mg L-1 for 24 h and then allowed to develop up to 144 hpf. The chromatographic analysis showed that this anaesthetic agent bioaccumulates in 26 hpf zebrafish larvae in a concentration-dependent manner. In addition, increased mortalities and skeletal abnormalities were observed at 144 hpf, namely in the highest tested concentration. Yet, no craniofacial anomalies were observed either by alcian blue or calcein staining methods. Independently of the tested concentration, decreased speed and distance travelled were perceived in 144 hpf larvae. At the biochemical level, decreased in vivo reactive oxygen species (ROS) generation and apoptosis was observed. Additionally, catalase activity was increased at 26 hpf while results of mRNA expression showed a decreased gclc transcript content at the same time-point. Overall, data obtained highlight the toxicological risk of MS-222 and support ROS-mediated cell death signalling changes through the elevation of catalase activity as an adaptative or protective response.
Assuntos
Aminobenzoatos/toxicidade , Anestésicos/toxicidade , Catalase/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Aminobenzoatos/metabolismo , Anestésicos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Catalase/genética , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento , Locomoção/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Regulação para Cima , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Ketamine, one anesthetic used as an illicit drug, has been detected both in freshwater and marine ecosystems. However, knowledge of its impact on aquatic life is still limited. This study aimed to test its effects in zebrafish embryos by analyzing its time- and dose-dependent developmental toxicity and long-term behavioral changes. The 24h-LC50 was calculated from percent survival using probit analysis. Based on the 24h-LC50 (94.4mgL-1), embryos (2hour post-fertilization - hpf) were divided into four groups, including control, and exposed for 24h to ketamine concentrations of 50, 70 or 90mgL-1. Developmental parameters were evaluated on the course of the experimental period, and anatomical abnormalities and locomotor deficits were analyzed at 144hpf. Although the portion of ketamine transferred into the embryo was higher in the lowest exposed group (about 0.056±0.020pmol per embryo), the results showed that endpoints such as increased mortality, edema, heart rate alterations, malformation and abnormal growth rates were significantly affected. At 144hpf, the developmental abnormalities included thoracic and trunk abnormalities in the groups exposed to 70 and 90mgL-1. Defects in cartilage (alcian blue) and bone (calcein) elements also corroborated the craniofacial anomalies observed. A significant up-regulation of the development-related gene nog3 was detected by qRT-PCR at 8 hpf. Early exposure to ketamine also resulted in long-term behavioral changes, such as an increase in thigmotaxis and disruption of avoidance behavior at 144 hpf. Altogether, this study provides new evidence on the ketamine teratogenic potential, indicating a possible pharmacological impact of ketamine in aquatic environments.
Assuntos
Anestésicos Dissociativos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/patologia , Desenvolvimento Embrionário/efeitos dos fármacos , Ketamina/toxicidade , Locomoção/efeitos dos fármacos , Animais , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Locomoção/fisiologia , Masculino , Comportamento Social , Peixe-ZebraRESUMO
Ketamine, a widely used anesthetic, has been shown to have NMDA receptor dependent and independent actions during zebrafish (Danio rerio) embryogenesis. Notwithstanding, the effects of developmental toxicity and the mechanisms of ketamine action on fish embryos are still not well understood, and its implications for early vertebrate development remains to be clarified. In this work, zebrafish embryos were exposed to ketamine (0.2, 0.4, and 0.8 mg mL(-1)) in order to study the stage-developmental toxicity of this pharmaceutical. During 256-cell (2.5 h post-fertilization, hpf), 50% epiboly (5.5 hpf) and 1-4 somites (10.5 hpf), embryos were exposed to the referred ketamine concentrations for a period of 20 min and were allowed to grow until 144 hpf. Both lethal and nonlethal parameters were evaluated. Skeletal development was assessed by alcian blue and calcein staining. Additionally, the expression of the developmental genes sonic hedgehog a (shh a) and noggin 3 (nog3) was evaluated. Similar to our previous work, bone and cartilage malformations were observed after 256-cell exposure. During 50% epiboly, ketamine exposure induced concentration-dependent mortality and malformations, such as lordosis and/or kyphosis and microcephaly, namely, at higher concentrations. Conversely, exposure during 1-4 somites showed the induction of nonspecific effects with no rise in mortality. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed differences in shh a and nog3 expressions comparatively to the control group. Overall, this study shows that the ketamine toxic profile is developmental phase-dependent with 256-cell being the most susceptible phase. The effects observed may result from ketamine interaction with cellular signaling pathways that merits further investigation.
Assuntos
Ketamina/toxicidade , Teratogênicos/toxicidade , Peixe-Zebra/embriologia , Animais , Cromatografia Líquida de Alta Pressão , Espectrofotometria UltravioletaRESUMO
Apart from morphological, biochemical, and genetic alterations induced by teratogen compounds, there is an increased interest in characterizing behavioral alterations. Behavior is a sensitive parameter that can provide information regarding developmental disruptions non-invasively. Behavioral disturbances interfere with animals' capacity to cope with the environment, having an impact on the organism's life. Hereby, behavioral assays consisting of recording larvae in multi-well plates, Petri dishes, or cuvettes and video analysis using adequate software, allowing teratogen screening of behavior, are proposed. Examples of how to evaluate locomotor, anxiety-like and avoidance-like behaviors, and the integrity of sensory-motor functions and learning are discussed in this chapter.
Assuntos
Perciformes , Peixe-Zebra , Animais , Reflexo de Sobressalto , Teratogênicos , Ansiedade , LarvaRESUMO
Cortisol is the main stress biomarker used for zebrafish. However, zebrafish small size made it challenging to extract cortisol without harming or killing the fish. Thus, researchers adopted a terminal method, the trunk cortisol, as standard practice. Here, we developed and validated an alternative and minimally invasive technique for measuring cortisol in the skin mucus of adult zebrafish, using a commercial enzyme-linked immunosorbent assay (ELISA). For this, AB zebrafish were randomly assigned to a precision, accuracy, and specificity test. Each sample contained the skin mucus of five to ten fish or one fish trunk. The cortisol was extracted using methanol as organic solvent. The results obtained showed an adequate precision (intra-assay coefficient of variation (CV) <15%; inter-assay CV = 26%), accuracy (CV <120%), and specificity (r2 =0.96-0.98) for skin mucus cortisol levels, as well as for trunk cortisol.â¢A commercial ELISA was analytically validated to measure cortisol in the skin mucus of zebrafish.â¢Skin mucus cortisol is a non-terminal method that reduce the number of animals used and allows longitudinal studies.
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Numerous therapeutic and diagnostic approaches used within a clinical setting depend on the administration of compounds via systemic delivery. Biomaterials at the nanometer scale, as dendrimers, act as delivery systems by improving cargo bioavailability, circulation time, and the targeting of specific tissues. Although evaluating the efficacy of pharmacological agents based on nanobiomaterials is crucial, conducting toxicological assessments of biomaterials is essential for advancing clinical translation. Here, a zebrafish larvae model was explored to assess the biocompatibility of poly(amido amine) (PAMAM), one of the most exploited dendrimers for drug delivery. We report the impact of a systemic injection of polyethylene glycol (PEG)-modified G4 PAMAM conjugated with rhodamine (Rho) as a mimetic drug (PEG-PAMAM-Rho) on survival, animal development, inflammation, and neurotoxicity. A concentration- and time-dependent effect was observed on mortality, developmental morphology, and innate immune system activation (macrophages). Significant effects in toxicological indicators were reported in the highest tested concentration (50 mg/mL PEG-PAMAM-Rho) as early as 48 h post-injection. Additionally, a lower concentration of PEG-PAMAM-Rho (5 mg/mL) was found to be safe and subsequently tested for neurotoxicity through behavioral assays. In accordance, no significative signs of toxicity were detected. In conclusion, the dose response of the animal was assessed, and the safe dosage for future use in theragnostics was defined. Additionally, new methodologies were established that can be adapted to further studies in toxicology using other nanosystems for systemic delivery.
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BACKGROUND: Ketamine is an anaesthetic and analgesic drug used in research and clinical practice. Little is known about the effects of different doses of this drug on memory and brain cellular death. OBJECTIVE: To study the effects of different doses of ketamine on working and reference memory, and neurodegeneration in adult mice. DESIGN: A randomised study. SETTINGS: The study was carried out in a basic science laboratory, between March 2011 and August 2012. ANIMALS: Forty-eight 7-month-old, male C57BL/6 mice were used. INTERVENTION: Animals received a single intraperitoneal injection of physiological saline solution or one of three doses of ketamine (25, 75 or 150 mg kg(-1)). Each group consisted of 12 animals (seven animals for behavioural tests and five animals for histopathological and immunohistochemical studies). The animals used for histopathology studies were sacrificed 3 h after anaesthesia. MAIN OUTCOME MEASURES: Working and reference memories were assessed using the radial-maze test over 12 consecutive days. The equilibrium was tested using the vertical pole (4 and 24 h after injection), whereas locomotion was assessed using the open field (24, 48 and 72 h after injection). Histopathological (haematoxylin-eosin staining) and immunohistochemical analyses (procaspase-3 and activated caspase-3 detections) were performed 3 h after injection to assess neurodegeneration in the retrosplenial and visual cortices, pyramidal cell layer of the cornu Ammonis 1 and cornu Ammonis 3 areas of the hippocampus, in the granular layer of the dentate gyrus, in the laterodorsal thalamic nucleus, striatum and accumbens nucleus. RESULTS: No significant differences were observed between the groups regarding the number of dead cells and cells showing positive immune-reactivity in the different regions of the brain studied. The performance in the vertical pole test and the number of reference and working memory errors in the radial-maze were similar in all groups. Nevertheless, the animals treated with ketamine 75 mg kg(-1) were transiently more active, walking a greater total distance at a greater speed in the open field than other groups (power of 0.96). CONCLUSION: These data indicate that a single intraperitoneal injection of ketamine at subanaesthetic and anaesthetic doses does not impair working memory, reference memory or neurodegeneration in adult mice, but an intermediate dose of ketamine produces transitory hyperlocomotion.
Assuntos
Anestésicos Dissociativos/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Ketamina/administração & dosagem , Memória de Curto Prazo/efeitos dos fármacos , Degeneração Neural , Memória Espacial/efeitos dos fármacos , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Injeções Intraperitoneais , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Fatores de TempoRESUMO
Zebrafish is a valuable model for neuroscience research, but the housing conditions to which it is exposed daily may be impairing its welfare status. The use of environmental enrichment and the refinement of methodology for cortisol measurement could reduce stress, improving its welfare and its suitability as an animal model used in stress research. Thus, this study aimed to evaluate (I) the influence of different housing conditions on zebrafish physiology and behavior, and (II) skin mucus potential for cortisol measurement in adult zebrafish. For this, AB zebrafish were raised under barren or enriched (PVC pipes and gravel image) environmental conditions. After 6 months, their behavior was assessed by different behavioral paradigms (shoaling, white-black box test, and novel tank). The physiological response was also evaluated through cortisol levels (whole-body homogenates and skin mucus) and brain oxidative stress markers. The results revealed that enriched-housed fish had an increased nearest neighbors' distance and reduced activity. However, no effect on body length or stress biomarkers was observed; whole-body and skin mucus cortisol levels had the same profile between groups. In conclusion, this study highlights the skin mucus potential as a matrix for cortisol quantification, and how housing conditions could influence the data in future studies.
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The use of zebrafish (Danio rerio) as an animal model is growing and occurs in a wide range of scientific areas. Therefore, researchers need better and more appropriate anaesthetics for stressful and/or painful procedures to prevent unpleasant experiences. Thus, we aimed to study if adult zebrafish displayed aversion-associated behaviours (conditioned place aversion) and alterations in cortisol levels when exposed to equipotent concentrations of MS222, propofol/lidocaine, clove oil, or etomidate. Adult AB zebrafish (mixed-sex, N = 177) were randomly assigned to MS222 (150 mg/L), Propofol/Lidocaine (5 mg/L propofol + 150 mg/L lidocaine), Clove Oil (45 mg/L), or Etomidate (2 mg/L) groups. The conditioned place aversion test was used to assess behavioural aversion. Only etomidate resulted in a similar aversion to the positive control group (HCl; pH = 3). Cortisol levels were measured 5 and 15 min after loss of equilibrium. Etomidate induced low levels of cortisol by impairing its synthesis, whereas all the other groups had similar cortisol levels. Based on our data, etomidate was ruled out as an alternative to MS222, as it showed an aversive profile. The remaining protocols were not innocuous, displaying a weak aversive profile when compared to the positive control. In conclusion, a combination of propofol with lidocaine, clove oil, and MS222 were valid candidates for use as anaesthetic protocols.
RESUMO
The rapid increase in zebrafish use needs to be accompanied by research into the refinement of procedures. The European (EU) Directive lists three possible euthanasia methods for fish: anesthetic overdose, electrical stunning, and concussion. However, for small fish such as zebrafish, concussion and electrical stunning are difficult to perform, leaving anesthetic overdose as the most used method. Our aim was to test the efficacy and side effects of anesthesia overdose using different anesthetics and the rapid cooling method to euthanize adult zebrafish. Adult mixed-sex AB zebrafish were randomly assigned to: 250 mg/L MS222; 20 mg/L propofol +100 mg/L lidocaine; 6 mg/L etomidate; 50 mg/L clove oil; and rapid cooling (water at 2°C-4°C). Two minutes after opercular movement ceased, animals were transferred into clean water for 20 min and recovery assessed, or decapitated and used for biochemical analysis of the gills, muscle, liver, and brain; for the histological analysis of the gills and muscle; or for the assessment of cortisol levels. No animal recovered; rapid cooling was the quickest and etomidate overdose was the slowest method to cease the opercular movements. There were no major differences between euthanasia methods regarding the biochemical or histological data. Cortisol levels were higher in the rapid cooling group, but only when compared with the propofol/lidocaine group. The use of a physical method of euthanasia, such as rapid cooling, is essential when chemicals, such as anesthetics, may interfere with postmortem analyses. Although anesthetic overdose can be used without major effects on the analyses conducted in this work, rapid cooling can be another option with the advantage of being simple to administer, easily available, affordable, and very quick; this decreases the potential duration of suffering, being more humane. Therefore, a change in EU legislation should be considered to include additional humane options for euthanasia, such as rapid cooling, for zebrafish and other small tropical fish.
Assuntos
Anestesia , Anestésicos , Etomidato , Propofol , Anestesia/métodos , Anestesia/veterinária , Animais , Eutanásia Animal/métodos , Hidrocortisona , Lidocaína , Água , Peixe-Zebra/fisiologiaRESUMO
The use of proper anesthesia in zebrafish research is essential to ensure fish welfare and data reliability. However, anesthesia long-term side effects remain poorly understood. The purpose of this study was to assess anesthesia quality and recovery in adult zebrafish using different anesthetic protocols and to determine possible long-term effects on the fish activity and anxiety-like behaviors after anesthesia. Mixed-sex adult AB zebrafish were randomly assigned to five different groups (Control, 175 mg/L of tricaine methanesulfonate [MS222], 45 mg/L of clove oil, 2 mg/L of etomidate, and 5 mg/L of propofol combined with 150 mg/L of lidocaine) and placed in the respective anesthetic bath. Time to lose the equilibrium, response to touch and to caudal fin pinch stimuli, and recovery after anesthesia administration were evaluated. In addition, after stopping anesthesia, respiratory rate, activity, and anxiety-like behaviors in the novel tank test were studied. Overall, all protocols proved to be adequate for zebrafish anesthesia research as they showed full recovery at 1 h, and only etomidate had minor effects on fish behavior in the novel tank, a validated test for anxiety.
Assuntos
Anestesia , Anestésicos , Aminobenzoatos/farmacologia , Anestesia/métodos , Anestesia/veterinária , Anestésicos/farmacologia , Animais , Reprodutibilidade dos Testes , Peixe-Zebra/fisiologiaRESUMO
BACKGROUND: Volatile anesthetics such as isoflurane are widely used in clinical and research contexts. Concerns have been raised that the effects of these drugs on the central nervous system may result in long-term impairment after surgery or general anesthesia. Hence, this study aimed to detect how different isoflurane concentrations influence spatial learning and cell death in adult mice. METHODS: Fifty-two C57BL/6 mice were randomly divided in four groups. Mice in three groups were exposed to different concentrations of isoflurane (1, 1.5, and 2%) for 1 h; the control group was not exposed to anesthesia. Five mice per group were killed 3 h after anesthesia to perform histopathologic and immunohistochemical analyses (hematoxylin-eosin staining; caspase-3 activation). Eight mice per group were used for behavioral tests (open field, T-maze spontaneous alternation, and water maze) on subsequent days. RESULTS: There were no differences between groups in the T-maze spontaneous alternation test or in the open field (no confounding effects of stress or locomotion). The group anesthetized with 1% isoflurane performed worse in the water maze task on day 1 (550.4 ±162.78 cm) compared with the control group (400.1 ± 112.88 cm), 1.5% isoflurane (351.9 ± 150.67 cm), and 2% isoflurane (364.5 ± 113.70 cm; P ≤ 0.05) and on day 3 (305.0 ± 81.75 cm) compared with control group (175.13 ± 77.00 cm) and 2% isoflurane (204.11 ± 85.75 cm; P ≤ 0.038). In the pyramidal cell layer of the region cornu ammonis 1 of the hippocampus, 1% isoflurane showed a tendency to cause more neurodegeneration (apoptosis) (61.4 ± 26.40, profiles/mm) than the group with 2% of isoflurane (20.6 ± 17.77, profiles/mm; P = 0.051). CONCLUSION: Low isoflurane concentration (1%) caused spatial learning impairment and more neurodegeneration compared with higher isoflurane concentrations. Results for mice receiving the latter concentrations were similar to those of control mice.
Assuntos
Isoflurano/administração & dosagem , Isoflurano/efeitos adversos , Aprendizagem em Labirinto/efeitos dos fármacos , Degeneração Neural/induzido quimicamente , Comportamento Espacial/efeitos dos fármacos , Fatores Etários , Anestésicos Inalatórios/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/patologia , Distribuição Aleatória , Comportamento Espacial/fisiologiaRESUMO
MS-222, the most widely used anaesthetic in fish, has been shown to induce embryotoxic effects in zebrafish. However, the underlying molecular effects are still elusive. This study aimed to investigate the effects of MS-222 exposure during early developmental stages by evaluating biochemical and molecular changes. Embryos were exposed to 50, 100 or 150â¯mgâ¯L-1 MS-222 for 20â¯min at one of three developmental stages (256-cell, 50% epiboly, or 1-4 somite stage) and oxidative-stress, cell proliferation and apoptosis-related parameters were determined at two time-points (8 and 26 hpf). Following exposure during the 256-cell stage, the biochemical redox balance was not affected. The genes associated with glutathione homeostasis (gstpi and gclc) were affected at 8 hpf, while genes associated with apoptosis (casp3a and casp6) and cellular proliferation (pcna) were found affected at 26 hpf. An inverted U-shaped response was observed at 8 hpf for catalase activity. After exposure at the 50% epiboly stage, the gclc gene associated with oxidative stress was found upregulated at 8 hpf, while gstpi was downregulated and casp6 was upregulated later on, coinciding with a decrease in glutathione peroxidase (GPx) activity and a non-monotonic elevation of protein carbonyls and casp3a. Additionally, MS-222 treated embryos showed a decrease in DCF-staining at 26 hpf. When exposure was performed at the 1-4 somite stage, a similar DCF-staining pattern was observed. The activity of GPx was also affected whereas RT-qPCR showed that caspase transcripts were dose-dependently increased (casp3a, casp6 and casp9). The pcna mRNA levels were also found to be upregulated while gclc was changed by MS-222. These results highlight the impact of MS-222 on zebrafish embryo development and its interference with the antioxidant, cell proliferation and cellular death systems by mechanisms still to be explained; however, the outcomes point to the Erk/Nrf2 signalling pathway as a target candidate.
Assuntos
Aminobenzoatos/toxicidade , Anestésicos/toxicidade , Desenvolvimento Embrionário/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião não Mamífero , Perfilação da Expressão Gênica , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Apart from morphological, biochemical, and genetic alterations induced by teratogen compounds, there is an increase interest in characterizing behavioral alterations. Behavior is a sensitive parameter that can provide information regarding developmental disruptions noninvasively, as it is the result of brain processes. Behavioral disturbances interfere with animals' capacity to cope with the environment, having an impact on the organism's life. Hereby, it is proposed behavioral assays consisting on recording larvae in multiwell plates and video analysis with a proper software, allowing for teratogen screening of behavior. How to evaluate locomotor, anxiety-like and avoidance-like behaviors, and the integrity of sensory-motor functions and learning are discussed in this chapter.
Assuntos
Comportamento Animal/efeitos dos fármacos , Teratogênicos/farmacologia , Peixe-Zebra/fisiologia , Animais , Larva , Atividade Motora/efeitos dos fármacos , Fotoperíodo , Peixe-Zebra/embriologiaRESUMO
MS-222 has been widely used as an anaesthetic in fish, thus, raising the need to infer about its toxicological safety during development. In this study, MS-222 toxicity in zebrafish embryos was evaluated after a 20-min exposure at different stages of development. Embryos exposed during the 256-cell stage displayed an increase in mortality, associated with defective early developmental pathways. Following exposure during the 50% epiboly stage, an increase in mortality and abnormal cartilage development, as well as changes in noggin expression were observed. Locomotor deficits were detected and associated with changes in early signalling pathways through the involvement of noggin. When exposed at the 1-4 somites stage, zebrafish were phenotypically normal, although presenting changes in the expression pattern of developmental genes. These findings indicate a teratogenic impact, independent of sodium channels that should be taken into consideration when MS-222 toxicity is discussed.
Assuntos
Aminobenzoatos/toxicidade , Anestésicos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Teratogênicos/toxicidade , Peixe-Zebra , Animais , Comportamento Animal/efeitos dos fármacos , Cartilagem/anormalidades , Embrião não Mamífero/anormalidades , Embrião não Mamífero/fisiologia , Comportamento Exploratório/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Peixe-Zebra/anormalidades , Peixe-Zebra/fisiologiaRESUMO
Ketamine is a widely used pharmaceutical that has been detected in water sources worldwide. Zebrafish embryos were used in this study to investigate the oxidative stress and apoptotic signals following a 24h exposure to different ketamine concentrations (0, 50, 70 and 90â¯mgâ¯L-1). Early blastula embryos (â¼2â¯h post fertilisation-hpf) were exposed for 24â¯h and analysed at 8 and 26 hpf. Reactive oxygen species and apoptotic cells were identified in vivo, at 26 hpf. Enzymatic activities (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE)), glutathione levels (oxidised (GSSG) and reduced (GSH)), oxidative damage (lipid peroxidation (LPO) and protein carbonyls (CO)) as well as oxidative stress (gclc, gstp1, sod1 and cat), apoptosis (casp3a, casp6, casp8, casp9, aifm1 and tp53) and cell proliferation (pcna) related-genes were evaluated at 8 and 26 hpf. Caspase (3 and 9) activity was also determined at both time-points by colorimetric methods. Superoxide dismutase (SOD), catalase (CAT), glutathione levels (GSSG), caspase-9 and reactive oxygen species (ROS) were shown to be affected by ketamine exposure while in vivo analysis showed no difference in ROS. A significant up-regulation of superoxide dismutase (sod1) and catalase (cat) genes expression was also perceived. Ketamine-induced apoptosis was observed in vivo and confirmed by the apoptotic-related genes up-regulation. The overall results suggest that ketamine induced oxidative stress and apoptosis through the involvement of p53-dependent pathways in zebrafish embryos which could be important for the evaluation of the overall risk of ketamine in aquatic environments.
Assuntos
Apoptose/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Ketamina/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra , Animais , Antioxidantes/metabolismo , Apoptose/genética , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/genética , Peixe-Zebra/embriologiaRESUMO
RATIONALE: Ketamine has been associated with pediatric risks that include neurocognitive impairment and long-term behavioral disorders. However, the neurobehavioral effects of ketamine exposure in early development remain uncertain. OBJECTIVES: This study aimed to test stage- and dose-dependent effects of ketamine exposure on certain brain functions by evaluating alterations in locomotion, anxiety-like and avoidance behaviors, as well as socialization. METHODS: Embryos were exposed to different concentrations of ketamine (0, 0.2, 0.4, and 0.8 mg mL-1) for 20 min during the 256-cell (2.5 h post fertilization-hpf), 50% epiboly (5.5 hpf), and 1-4 somites (10.5 hpf) stages. General exploratory activities, natural escape-like responses, and social interactions were analyzed under continuous light or under a moving light stimulus. RESULTS: A dose-dependent decrease in the overall mean speed was perceived in the embryos exposed during the 256-cell stage. These results were related to previously observed head and eye malformations, following ketamine exposure at this stage and may indicate possible neurobehavioral disorders when ketamine exposure is performed at this stage. Results also showed that ketamine exposure during the 50% epiboly and 1-4 somites stages induced a significant increment of the anxiety-like behavior and a decrease in avoidance behavior in all exposed groups. CONCLUSIONS: Overall, the results validate the neurodevelopmental risks of early-life exposure to ketamine.