Microcystin-LR induced apoptosis and mRNA expression of p53 and cdkn1a in liver of whitefish (Coregonus lavaretus L.)
Microcystin-LR induced apoptosis and mRNA expression of p53 and cdkn1a in liver of whitefish (Coregonus lavaretus L.)
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DOI：
10.1016/j.toxicon.2009.03.032
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发表时间：
2009-08-01
期刊：
影响因子：
2.8
通讯作者：
Dobosz, Stefan
中科院分区：
文献类型：
--
作者：
Brzuzan, Pawel;Wozny, Maciej;Dobosz, Stefan
越来越多的证据表明，微囊藻毒素-LR（MC-LR）的不良反应与氧化应激过程、自由基和DNA损伤密切相关，并涉及主要基因转录的变化。这项研究，利用基因表达分析和血浆化学是第一次测量MC-LR的影响白鱼（Coregonus lavaretus L），一个可行的生物体在水生系统中的污染监测。给鱼注射不同浓度的MC-LR（0、10和100 μ g/kg体重），然后在0、8、24、48或72 h后处死，收获它们的肝组织用于详细研究。具体而言，我们感兴趣的是MC-LR是否能够：（i）调节两个基因的表达，肿瘤抑制基因p53和cdkn 1a，p53直接转录靶，和（ii）诱导白鱼肝脏细胞凋亡。为了研究这些影响，我们开发了一种实时qPCR检测方法，可用于测量肝脏中p53和cdkn 1a基因转录水平。为了获得研究所需的信息，我们利用分子克隆和cDNA末端快速扩增（RACE）技术获得了白鲑p53全长cDNA（Wf-p53），或者根据鱼类cdkn 1a的高度保守区域设计了Wf-cdkn 1a的特异性引物。发现Wf-p53与其他脊椎动物的已知p53 mRNA序列具有相同的特征。白鱼p53氨基酸序列与鱼类、两栖类和哺乳类的序列具有高度的同源性。注射研究表明，较高剂量（即100 μ g/kg体重）的MC-LR上调了白鱼肝脏中p53和cdkn 1a基因的表达，这反映在其mRNA水平在整个实验中持续增加。此外，在暴露于MC-LR（100 μ g/kg）24小时后，在白鱼的肝细胞中观察到DNA片段化，这表明细胞凋亡的可能性。最后，该研究证实了先前观察到的肝脏严重损伤和正常器官功能丧失，如血液AspAT、AlaAT和肝体指数（HSI）水平升高所揭示的。(C)2009爱思唯尔有限公司版权所有。
There is growing evidence that adverse effects of microcystin-LR (MC-LR) are closely related to oxidative stress processes, free radicals and DNA damage, and involve major gene transcript changes. This study, utilizing gene expression analysis and plasma chemistries was the first to measure the effects of MC-LR in whitefish (Coregonus lavaretus L), a feasible organism for pollution monitoring in aquatic systems. Fish were injected with different concentrations of MC-LR (0, 10 and 100 mu g/kg of body weight) and then sacrificed at either 0, 8, 24, 48 or 72 h later, and their liver tissue were harvested for detailed investigation. Specifically, we were interested whether MC-LR is capable of: (i) modulating expression of two genes, tumor suppressor gene p53 and cdkn1a, p53 direct transcription target, and (ii) inducing apoptosis in whitefish liver. To study these effects, we developed a real-time qPCR assays useful for measuring both p53 and cdkn1a gene transcript levels in liver. To obtain necessary information for the study, either full-length p53 cDNA of whitefish (Wf-p53) was determined, using molecular cloning and rapid amplification of cDNA ends (RACE), or as for Wf-cdkn1a, specific primers were designed based on highly conserved regions of cdkn1a in fish. The Wf-p53 was found to share the same characteristics with a known p53 mRNA sequence of other vertebrates. Whitefish p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals. The injection study showed that MC-LR at a higher dose, i.e. 100 mu g/kg body weight, up-regulated expression of p53 and cdkn1a genes in whitefish liver, as reflected by the continuous increase in their mRNA levels through the whole experiment. Furthermore, DNA fragmentation was observed in liver cells of whitefish after 24 h of exposure to MC-LR (100 mu g/kg) that suggests the possibility of apoptosis. Finally, the study confirmed previous observations of severe injury of the liver and loss of normal organ functions as revealed by elevated levels of blood AspAT, AlaAT, and hepatosomatic index (HSI). (C) 2009 Elsevier Ltd. All rights reserved.