Evaluation of metallothionein induction as a biological marker of environmental pollution

在本课题中，我主要研究了(1)哺乳动物金属硫蛋白（MT）酶联免疫吸附测定（ELISA）系统的建立，(2)鱼类金属硫蛋白特异性ELISA系统的建立，(3)环境因素诱导下鲫鱼金属硫蛋白动力学的评估。(1)由于其免疫原性较弱，目前还没有有效的ELISA系统可用于测定鱼类mt .首先，我开发了一种灵敏的ELISA系统，通过产生针对大鼠mt，马mt和同源n端肽的抗体。采用马MT-I特异性豚鼠抗血清构建竞争性ELISA，灵敏度高，最低可测限为3 ng/ml。该酶联免疫吸附测定系统可用于评价大鼠肠上皮细胞系IEC-6细胞重金属毒性与MT诱导的关系。(2)从注射了镉的鲫鱼肝脏中，纯化出了镉结合MT的2种同工型I和II，均相状态。虽然针对鲫鱼MT- ii的抗血清与鲫鱼MT- i的交叉反应性较弱，但所建立的鲫鱼MT- ii特异性ELISA系统具有与哺乳动物MT- i一样的敏感性，今后该检测系统可用于江河湖泊鲫鱼MT水平的分析。(3)研究了镉（1 mg/l）对鲫鱼组织MT的诱导作用。在镉的作用下，肝脏和肾脏有效地诱导了MT，随后在肠道中也有显著的诱导。这种组织特异性的MT诱导与镉的分布剖面密切相关。镉和锌主要诱导鱼肝脏中的MT-II，而地塞米松和应激（剧烈通气）诱导两种亚型。由此得出结论，鲫鱼MT水平的升高是一种有用的环境污染生物标志物。少

In this research project, I have studied on (1) the development of enzyme-linked immunosorbent assay (ELISA) system for mammalian metallothioneins (MT), (2) the establishment of ELISA system specific to fish MT,and (3) the assessment of MT kinetics in crucian carps induced by environmental factors.(1) Because of its weak immunogenicity, there has been no useful ELISA system available for the determination of fish MT.First, I have developed a sensitive ELISA system by producing antibodies against rat MTs, horse MTs, and homologous N-terminal peptide. The proposed competitive ELISA was constructed using guinea pig antiserum specific to horse MT-I,being highly sensitive with the lowest measurable limit of 3 ng/ml. This ELISA system made it possible to evaluate the relation between heavy metal toxicity and MT induction in rat intestinal epithelial cell line, IEC-6 cells. However, this method was not available for determination of fish MT.(2) From the liver of crucian carp injected with cad … More mium chloride, 2 isoforms of cadmium-bound MT,I and II,were purified to the homogeneous state. Although the antiserum raised against crucian carp MT-II had little cross-reactivity with crucian carp MT-I,Crucian carp MT-II-specific ELISA system established was as sensistive as that for mammalian MTs. In the future, this assay system should be applied to analyze the MT levels in crucian carps living in the river and lake.(3) The induction profiles of tissue MT were assessed in crucian carps exposed to cadmium (1 mg/liter) in their aquarium water. MT was efficiently induced in the liver and kidney in response to cadmium, followed by the significant induction in the intestine. Such a tissue-specific induction of MT closely related to the distribution profile of cadmium. Cadmium and zinc predominantly induced MT-II in the fish liver, while dexamethasone and stress (vigorous airation) induced both isoforms. From these results, I conclude that the elevated levels of crucian carp MT is a useful biological marker for environmental pollution. Less