【作者】 王琳；

【导师】 梁旭方；

【作者基本信息】 暨南大学 ， 水生生物学， 2010， 博士

【摘要】 赤潮已成为一种全球性的海洋灾害,其频繁发生不仅威胁着渔业和水产业,也危害着海洋动物和人类的安全。软骨藻酸(domoic acid, DA)是由海洋硅藻产生的一种兴奋性神经毒素,常常伴随赤潮发生,可在鱼贝类中富集,人类摄入后能够引起记忆缺失性中毒(amnesic shellfish poison, ASP)。为从分子水平探讨海洋鱼类肝脏软骨藻酸毒素的去毒分子机理,本研究在获得真鲷(Pagrus major)肝脏芳香烃受体核转位蛋白(ARNT)和Ⅰ相异生素代谢酶细胞色素P4501A1(CYP1A1)基因cDNA核心序列和Ⅰ、Ⅱ相异生素代谢酶基因5’侧翼调控区序列的基础上,分别从氨基酸序列同源性、氨基酸保守功能域位点、3D分子模型、基因系统进化、5’侧翼调控区反应元件预测、基因组学等不同水平,对经济鱼类、模式生物(斑马鱼)和哺乳动物(人、大鼠、小鼠)肝脏芳香烃受体(AHR)、ARNT、CYP1A1,Ⅱ相异生素去毒酶alpha1、2和rho-型谷胱甘肽S-转移酶(GSTA1、2和GSTR)及热休克蛋白70(HSP70)基因进行全面综合的比较分析,为去毒相关基因功能研究提供基础资料。为从分子水平证实这些基因在鱼类肝脏软骨藻酸代谢中的作用,我们采用活体腹腔注射和半定量RT-PCR的方法研究了真鲷暴露于DA(2μg/g湿体重)24 h,肝脏各去毒相关基因表达水平的诱导改变。结果表明,DA暴露组中AHR和ARNT基因转录本水平较对照组分别增加约1倍(P>0.05),CYP1A1转录本水平显著性增加了约1.5倍(P<0.05)。Ⅱ相异生素代谢酶和HSP70基因表达水平则无显著性改变。因此,推测真鲷的DA暴露可显著诱导其肝脏Ⅰ相异生素代谢酶CYP1A1基因的表达,且可能是通过AHR/ARNT信号通路发挥作用。我们认为DA在海洋鱼类中积累,可能与其积累后只有Ⅰ相异生素代谢酶CYP1A1(解毒作用)的诱导表达而没有Ⅱ相异生素代谢酶GSTs(排毒作用)的诱导表达有关。因此,是否存在某种调控物质可通过刺激DAⅠ相生物转化后的Ⅱ相异生素代谢酶的加合和排泄,对水生生物肝脏中DA代谢起关键作用,成为我们进一步研究的重点。首先,通过基于反式因子及其信号通路的离体细胞实验体系筛选出增强毒素代谢中起显著作用的调控物质。采用荧光定量PCR方法研究了0、40和60μM叔丁基对苯二酚(tert-butylhydroquinone, tBHQ)体外诱导24 h,对真鲷原代肝细胞去毒相关基因转录水平的诱导改变。结果表明,tBHQ对真鲷原代肝细胞中Ⅱ相异生素代谢酶基因(GSTA1、GSTA2和GSTR)有诱导作用；且高浓度tBHQ对HSP70也有一定诱导作用。其次,在对鱼类离体细胞培养体系诱导的基础上,我们又通过活体灌胃实验研究了调控物质tBHQ对海水鱼活体肝脏组织中赤潮毒素DA的积累、代谢及去毒相关基因表达水平变化的影响。结果表明,在灌胃100 mgtBHQ/kg饲料24和72 h后,tBHQ对真鲷肝脏AHR、ARNT、CYP1A、GSTA1、GSTA2、GSTR及HSP70基因有不同程度的诱导影响,且对Ⅱ相异生素代谢酶的诱导作用较Ⅰ相异生素代谢酶作用更明显。同时发现饲料中添加tBHQ可有效地增强肝脏DA的代谢。综上所述,在对真鲷肝脏Ⅰ、Ⅱ相异生素代谢酶等去毒相关基因结构分析基础上,通过活体注射实验验证这些基因在真鲷肝脏软骨藻酸代谢过程中可能的作用；在对Ⅰ、Ⅱ相异生素代谢酶基因5’侧翼区序列研究基础上,推测可能影响海水鱼类肝脏去毒相关基因表达水平的反式因子,然后通过基于反式因子及其信号通路的离体细胞实验体系筛选出可能在增强毒素代谢中起显著作用的调控物质。再通过活体灌胃实验验证调控物质tBHQ对海水鱼活体组织肝脏中赤潮毒素DA的积累、代谢及去毒相关基因表达水平变化的影响。结果表明,Ⅰ相异生素代谢酶CYPIA是DA代谢的关键的酶,它可通过生物转化降低DA的毒性；虽然Ⅱ相异生素代谢酶GSTs在DA代谢过程中的作用不显著,不能有效地将毒素排出体外。但我们选择的基于反式因子及其信号通路的调控物质tBHQ,可增强Ⅱ相异生素代谢酶GSTs的表达水平,且对毒素积累的代谢有显著作用。该研究结果为海洋藻毒素在海洋鱼类中的积聚及代谢去毒分子机制的研究提供了大量的理论依据,并为研发可激活海水鱼贝类赤潮毒素、违禁渔药等毒害物质去毒基因表达的饵料添加剂提供了新的思路。更多还原

【Abstract】 Harmful algal blooms (HABs) are a global phenomena and recent evidence indicates that their frequency and intensity are increasing. They are a serious threat to human health, aquaculture, fisheries, and ecosystem health. Amnesic shellfish poisoning (ASP) toxin belongs to a marine neurotoxin which may cause permanent short-term memory loss. The principal toxin responsible for ASP is domoic acid (DA).To investigate the molecular detoxification mechanism of marine algae toxin DA detoxinfication-related genes (AHR, ARNT, CYP1A1, GSTA1, GSTA2, GSTR and HSP70) in marine fish liver, the phaseⅠxenobiotic metabolizing enzymes (XMEs) CYP1A1 and ARNT cDNA sequences were cloned from the liver of red sea bream Pagrus major; the promoter regions of phaseⅠXMEs (CYP1A1) and phaseⅡXMEs (GSTA2 and GSTR) gene of red sea bream were also obtained and characterized; and finally, the putative regulatory elements were predicted. In addition, the comprehensive and comparative analysis of the amino acid sequence similarity, conserved domains, three-dimensional molecular model, gene phylogenetic tree, cis-acting elements, gene and genomics structure of the DA detoxinfication-related liver genes among the economic fishes, model animal (zebrafish) and mammals (human, mouse, and rat), which would provide abundant basic information on the functional research of detoxinfication-related genes.To elucidate the role of the detoxinfication-related liver genes in the DA toxin metabolism of the major cultivated carnivorous fish, red sea bream intracoelomically injected with DA, were investigated. Experimental fish were administered with one injection of DA (2 mg/kg wet weight) or PBS as control. After 24 h, fish were killed and hepatic RNA was isolated. Usingβ-actin as an external control, the relative liver AHR, ARNT, CYP1A1, GSTA1, GSTA2, GSTR and HSP70 mRNA abundance of red sea bream were determined by semi-quantitative RT-PCR within the exponential phase. The result showed the significant induction of hepatic CYP1A1 in response to DA indicates an important role for phase I XMEs in DA metabolism. In contrast, the transcription of three major phaseⅡXMEs GSTs as well as HSP70 was not affected significantly by DA exposure, which suggests that a possible role of CYP1A after DA exposure in the toxin metabolism of marine fish, possibly through the AHR/ARNT signaling pathway.We assume that the high induction of CYP1A1 may decrease the toxicity of DA in fish, whereas GSTs expression were not significant in response to DA, thus DA couldn’t be excreted out of fish effectively. If exsit some regulation materials, which could increase the DA excretion by inducing GSTs expression, was deserved further research. The influences of the food antioxidant, tert-butylhydroquinone (tBHQ), on DA metabolism and detoxification-related gene transcription were investigated both in vivo and in vitro. Oral administration of tBHQ (100 mg/kg) resulted in significant decreases of DA accumulations in liver tissues in our experimental fish which were fed with a single dose of 10 mg DA/kg body weight (bwt) 24 h before tBHQ treatment. Real-time RT-PCR further revealed that the mRNA level of GSTA2 and the mRNA levels of AHR, ARNT, CYP1A1, GSTA1 and GSTR were upregulated in the above liver tissues at 24 and 72 h post tBHQ treatment, respectively. In consistence, increased mRNA expressions of GSTA1, GSTA2 and GSTR were also observed in cultured hepatocytes that were exposed to 40 or 60μM tBHQ. Collectively, our findings in this research suggested that the dietary intake of tBHQ accelerated DA metabolism in fish, through mechanisms involving altered transcriptions of detoxification-related liver genes.In conclusion, the study has demonstrated a significant stimulatory effect of DA exposure on the transcription of phase I XMEs CYP1A1 possibly through AHR/ARNT signaling pathway in the liver of red sea bream. The dietary intake of tBHQ can stimulate DA metabolism effectively possibly by increasing the transcription of detoxification-related liver genes in fish. The metabolic adaptation may be based on coordinate regulation of a set of phase I and II XMEs, which would provide abundant evidences to develop new dietary additives to stimulate detoxification-related genes expression levels in aquaculture.更多还原