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Biological Evaluation of the Environmental Pollution Based on the Induction of ECOD Activity in Human Hepatoma HepG2 Cells

基于诱导人肝癌HepG2细胞ECOD活性的环境污染生物学评价

基本信息

批准号：
13672350
负责人：
INOUE Yoshio
金额：
$ 1.79万
依托单位：
Toho University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

The extents of pollution of the rivers flowing into Tokyo Bay as well as the Sagami River draining into Sagami Bay were studied comparatively using river sediment extracts in an assay system based on the aryl hydrocarbon receptor (AhR)-dependent induction of 7-ethoxycoumarin O-deethylase (ECOD) activity in HepG2 cells. The Tsurumi River located in the middle of Keihin Industrial District was considered most polluted. The extract of heavily polluted river sediments sampled at Namamugi on the Tsurumi River showed a reverse U-shaped dose-response curve, for which polycyclic aromatic hydrocarbons (PAHs) might be mainly responsible, in a range of non-toxic concentrations. The pollution of the Namamugi sample was roughly estimated to be 2 to 20 μg as PAHs/g soil. Based on the longitudinal study on the pollution of the Tsurumi River, the former open-air industrial waste incineration site located in the midstream was found not to be a cause for the heavy pollution at the river mouth.The reverse U-shaped dose-response curve in terms of ECOD activity shown by the river sediment extracts as well as 3-methylcholanthrene (3-MC) was reflected in the dose-response curve for the CYP1A protein, whereas the expression of CYP1A1 mRNA in HepG2 cells remained roughly stationary at higher concentrations, implying translational suppression at higher AhR agonist levels.The XRE-luciferase gene reporter assay based on the transient transfection was found to be superior to the ECOD assay, in which time-wasting deglycosylation step was unavoidable, with respects to quantitativeness and reliability.

利用河流沉积物提取物，通过芳烃受体（AhR）依赖性诱导HepG2细胞7-乙氧基香豆素o -去乙基酶（ECOD）活性的测定系统，对比研究了东京湾入流河流和相模河入流相模湾的污染程度。位于京浜工业区中部的鹤见河被认为是污染最严重的。鹤见河Namamugi重度污染河流沉积物提取液在一定浓度范围内呈反u型剂量响应曲线，多环芳烃（PAHs）可能是主要原因。Namamugi样品的污染大致为2 ~ 20 μg （PAHs/g）。通过对鹤见河污染的纵向研究，发现原位于鹤见河中游的露天工业垃圾焚烧场并不是造成鹤见河河口重污染的原因。河流沉积物提取物和3-甲基胆蒽（3-MC）在ECOD活性方面的反向u型剂量-反应曲线反映在CYP1A蛋白的剂量-反应曲线上，而CYP1A1 mRNA在HepG2细胞中的表达在较高浓度下基本保持稳定，这意味着在较高的AhR激动剂水平下，翻译受到抑制。基于瞬时转染的xre -荧光素酶基因报告试验在定量和可靠性方面优于ECOD试验，ECOD试验不可避免地要进行耗时的去糖基化步骤。