新精神活性物质氯胺酮对斑马鱼幼鱼的神经毒性
Neurotoxicity of A New Psychoactive Substance Ketamine on Zebrafish Larvae
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摘要: 氯胺酮(ketamine, KET)是一种新精神活性物质，使用后会产生致幻、视听分离感和欣快感。KET已经在地表水中广泛检出，浓度高达420 ng·L-1。已有大量研究表明KET对人、鼠类等哺乳动物具有强烈的神经毒性，但KET对鱼类的神经毒性及潜在机制尚不清晰。目前研究多关注KET对非竞争性N-甲基-D-天门冬氨酸(N-methyl-D-aspartate, NMDA)受体的影响，较少考虑多巴胺(dopamine, DA)和γ-氨基丁酸(GABA)通路的影响。为研究KET对斑马鱼幼鱼早期神经毒性及其作用机制，将2 hpf (hours post-fertilization)的斑马鱼胚胎分为对照组和3个暴露组(10、100和1 000 ng·L-1)，分析KET对斑马鱼胚胎和幼鱼行为、活性氧水平以及神经相关基因转录水平的影响。结果表明，10 ng·L-1 KET显著降低7 dpf斑马鱼心率(P<0.05)。行为分析显示，100 ng·L-1和1 000 ng·L-1 KET显著增加斑马鱼胚胎自发摆尾运动频率(P<0.001)。1 000 ng·L-1 KET显著升高27 hpf斑马鱼对触碰反应的敏感程度、而降低48 hpf斑马鱼的敏感程度(P<0.01)。10 ng·L-1 KET显著降低斑马鱼在黑暗状态下的趋触性(P<0.05)。活性氧水平检测结果表明，1 000 ng·L-1 KET显著增加斑马鱼幼鱼腹部ROS荧光信号强度(P<0.05)。qPCR分析结果表明，10 ng·L-1 KET下调多巴胺受体基因drd3转录水平，100 ng·L-1 KET上调钾内向整流通道亚家族J成员6基因kcnj6转录水平，1 000 ng·L-1 KET上调γ-氨基丁酸转运体基因slc6a1b、钾内向整流通道亚家族J成员5基因kcnj5转录水平。上述结果表明，KET通过影响DA和GABA通路来显著影响斑马鱼幼鱼行为和神经相关基因转录水平，产生神经毒性。Abstract: Ketamine (KET) is a new psychoactive substance that can produce hallucinations, audiovisual separation and euphoria. KET has been widely detected in surface water at concentrations up to 420 ng·L-1. Some studies show that KET has strong neurotoxicity to human, rodents and other mammals, but the neurotoxicity of KET to fish and its potential mechanism are not known. Current studies have focused more on the effect of KET on non-competitive N-methyl-D-aspartate (NMDA) receptors, and less on the effects of dopamine (DA) and γ-aminobutyric acid (GABA) pathways. To investigate the neurotoxicity of KET on zebrafish larvae and its mechanism, 2 hpf (hours post-fertilization) zebrafish embryos were divided into control group and three KET groups (10, 100 and 1 000 ng·L-1). The effects of KET on the behavior, reactive oxygen species level and nerve-related gene transcription of zebrafish embryos and larvae were analyzed. The results showed that 10 ng·L-1 KET apparently reduced the heart rate of 7 dpf zebrafish (P<0.05). Behavioral analysis revealed that 100 ng·L-1 and 1 000 ng·L-1 KET significantly enhanced the frequency of spontaneous tail contraction in embryos (P<0.001). 1 000 ng·L-1 KET significantly elevated the sensitivity of touch response at 27 hpf zebrafish, while decreased the sensitivity at 48 hpf zebrafish (P<0.01). 10 ng·L-1 KET obviously reduced the degree of thigmotaxis of zebrafish in dark (P<0.05). The results of ROS showed that 1 000 ng·L-1 KET markedly increased the ROS fluorescence signal intensity in the abdomen of zebrafish larvae (P<0.05). The qPCR data showed that 10 ng·L-1 KET reduced the transcription of dopamine receptor gene drd3, 100 ng·L-1 KET enhanced the transcription of potassium inwardly rectifying channel subfamily J member 6 gene kcnj6, 1 000 ng·L-1 KET increased the transcriptions of GABA transporter gene slc6a1b and potassium inwardly rectifying channel subfamily J member 5 gene kcnj5. The above results indicated that KET could affect the behavior and the transcriptions of nerve-related genes of zebrafish larvae by affecting the DA and GABA pathways, which resulted in neurotoxicity in fish.
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Key words:
- ketamine /
- zebrafish /
- neurotoxicity /
- cardiotoxicity
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