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Development of method for assessing species-specific risk of Ah receptor-mediated dioxin toxicity

开发评估 Ah 受体介导的二恶英毒性的物种特异性风险的方法

基本信息

批准号：
13480170
负责人：
IWATA Hisato
金额：
$ 4.03万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480170/
关键词：
dioxin Ah receptor cormorant albatross red seabream 水鳥ゼニガタアザラシバイカルアザラシ

项目摘要

We identified two distinct AhR cDNAs (AhR1 and AhR2) from the livers of black-footed albatross (Diomedea nigripes) and common cormorant (Phalacrocorax carbo). The objective of this study is to investigate the functional characterization of the AhRs and their expression levels related to planar halogenated aromatic hydrocarbons (PHAHs) exposure and CYP1A expression in aquatic birds. In velocity sedimentation analysis using AhR proteins expressed by in vitro transcription and translation, both AhRs exhibited specific binding to [^3H]TCDD. Focusing on the amino acid residues corresponding to Ile^<325> and Ser^<381> in chicken AhR1, which may contribute to the differential TCDD-binding affinity, the amino acid residues in albatross and cormorant AhR1 were Ile-Ala and Val-Ala, respectively. The [^3H]TCDD binding affinity measured was in the order of chicken > albatross > cormorant, and agreed with the order that may be expected from the amino acids. In contrast, the binding affinity of avia … More n AhR2 appeared to be unrelated to the corresponding amino acids.To investigate the molecular mechanism of TCDD toxicity in a marine fish species, red seabream (Pagrus major), we identified two distinct AHR isoform cDNAs (rsAHR1 and rsAHR2), which shared only 32% identity in full-length amino acid sequence. Quantitative analyses of both rsAHR mRNAs revealed that their tissue expression profiles were isoform-specific in different tissues of adult fishes ; rsAHR1 mRNA expressed primarily in brain, heart and ovary, while rsAHR2 mRNA was observed in all tissues examined. The expression of rsAHR1,rsAHR2 and CYP1A mRNAs were also determined in different developmental stages treated either with TCDD or solvent control at 10 hpf (hours post fertilization). Both rsAHR transcript levels increased prior to the stage at which heart beat and blood circulation were observable, but expression patterns were slightly different ; maximal expression of rsAHR1 and rsAHR2 mRNA were 96 hpf and 190 hpf, respectively. The rsAHR2 mRNA expression increased following TCDD exposure compared with solvent control, while TCDD did not affect the rsAHR1 mRNA level. Although basal expression of CYP1A mRNA was detectable, TCDD exposure further enhanced CYP1A mRNA level. The maximal expression of CYPIA mRNA was recorded before the onset of the first signs of TCDD toxicities such as yolk sac edema. Less

我们从黑脚信天翁(Diomedea Nigripe)和普通蝎(Phalacrocorax Caro)的肝脏中鉴定出两个不同的AhR cDNAs(AHR1和AHR2)。本研究的目的是研究AHRs的功能特征及其与平面卤代芳香烃(PHAHs)暴露和CyP1A在水禽中表达相关的水平。在体外转录和翻译表达的AhR蛋白的速度沉降分析中，这两种AHRs都显示与[~3H]TCDD的特异性结合。针对鸡AHR1中可能与TCDD结合亲和力不同的Ile^&lt；325&gt；和Ser^&lt；381&gt；氨基酸残基，信天翁AHR1中的氨基酸残基分别为Ile-Ala和Val-Ala。测得的[~3H]TCDD结合亲和力的大小顺序为鸡和信天翁，与氨基酸的预期顺序一致。相比之下，Avia…的结合亲和力为了研究海水鱼TCDD毒性的分子机制，我们鉴定了两个不同的AHR亚型cDNAs(rsAHR1和rsAHR2)，它们在全长氨基酸序列上只有32%的同源性。对rsAHR和rsAHRmRNAs的定量分析表明，它们在成鱼不同组织中的表达谱具有异型性，rsAHR1mRNA主要在脑、心脏和卵巢表达，而rsAHR2mRNA在所有被检测组织中都有表达。在受精后10hpF，分别用TCDD和溶剂对照处理不同发育阶段的受精卵，检测rsAHR1、rsAHR2和CYP1AmRNAs的表达。RsAHR和rsAHR的转录水平均在可观察到心跳和血液循环的阶段之前升高，但表达模式略有不同，rsAHR1和rsAHR2mRNA的最大表达分别为96HPF和190HPF。与溶剂对照组相比，TCDD暴露后rsAHR2基因表达增加，而TCDD对rsAHR1基因表达水平无影响。尽管可检测到CYP1AmRNA的基础表达，但TCDD暴露进一步增强了CYP1AmRNA的水平。在TCDD毒性的第一个症状(如卵黄囊水肿)出现之前，CyPIA mRNA的最高表达被记录下来。较少