Study On Metalloproteins Related To Selenium-mercury Antagonism In Cells

The antagonism between mercury(Hg)and selenium(Se)was firstly reported in 1960s in rats exposed to mercuric chloride(Hg(?))and sodium selenite(Se(?)).Se(IV)is considered to be a potential prevention therapy for Hg poisoning.Further studies have substantiated this protective effect in animal and plant individuals,bacterias,cells and populations of Se/Hg contaminated areas.It is confirmed that Se(IV)has a significant effect on the content,distribution and biotransformation of Hg in the body.Currently,most efforts have been put toward the evaluation of the Se-Hg antagonism mechanism at the molecular level.However,most previous studies concerning Se-Hg antagonism have been described in animals and plants,but rarely in cells.Besides,the underlying mechanism for the Se-Hg antagonism remains elusive and requires further investigation on the chemical species and complete metabolic pathways of Se and Hg.Considering the diversity and complexity of biological samples,especially precious and rare cells,high-resolution and high-sensitive analytical techniques are needed to unravel the Se-Hg antagonism mechanism.Metallomics is defined as a systematic scientific field of biometals focusing on the distribution,species,content,and function of biometals in a cellular compartment or overall in an organism.So far,a series of analytical techniques have been developed and applied for the extraction and separation of metalloproteins,quantitative analysis of metallomes,spatial distribution of metallomes and identification of metal-biomolecule complexes.In addition,the hyphenation of different techniques is favorable to study metallomes systematically along with their biotransformation and bioavailability mechanism in organisms.Hyphenated techniques in metallomics can be used to comprehensively investigate the content,speciation,distribution and metabolism of Se and Hg,with an emphasis on the Se-Hg antagonism mechanism at the molecular level.The aim of this dissertation is to investigate the metalloproteins related to Se-Hg antagonism using a variety of hyphenated techniques with HepG2 cells as an experimental model and to explore the metabolic pathways and antagonism mechanism of Se and Hg at the molecular level.The main contents of this dissertation are summarized as follows:(1)The molecular weight distribution and relative content of biomolecules containing Se and Hg in HepG2 cells exposed to Hg(II)alone and co-exposed to Se(IV)and Hg(II)with various Hg(II)/Se(IV)molar ratios were investigated preliminarily by size exclusion chromatography-inductively coupled plasma mass spectrometry(SEC-ICP-MS).According to the MTT assay,median lethal concentration(MLC)of Hg(?)and Se(?)were 50 ?M and 120 ?M,respectively.Besides,no obvious toxicity of Hg(?)and Se(IV)was observed within the tested concentration 0-25 ?M for Hg(?)and 0-30?M for Se(?).Thus,the exposure concentration of Hg(?)was chosen as 5 ?M,and the Hg(?)/Se(?)molar ratios were chosen as 1:1,1:3 and 1:6,respectively.When HepG2 cells were exposed to Hg(?)alone,Hg(?)was metabolized into two species:macromolecular species(>67 kDa)and small molecular species(1 kDa->7 kDa).The small molecular species of Hg decreased gradually with the addition of Se(IV)and were undetectable until the Hg(?)/Se(?)molar ratio was 1:3.The macromolecular species of Hg increased gradually with the addition of Se(?),and the macromolecular species of Se with the same retention time also increased.It exhibits a significant correlation among the macromolecular species of Se,the macromolecular species of Hg and the small molecular species of Hg.It is speculated that macromolecular species of Se and Hg is Hg-selenoproteins or(HgSe)n-Proteins with core-shell structures.(2)Hg-MTs in the small molecular species(1 kDa->7 kDa)characterized above and their metal binding properties were systematically investigated by multiple hyphenated techniques,including capillary reversed-phase liquid chromatography-inductively coupled plasma mass spectrometry(capRPLC-ICP-MS),capillary reversed-phase liquid chromatography-electrospray ionization mass spectrometry capRPLC-ESI-MS,and electrospray ionization tandem mass spectrometry(capRPLC-ESI-MS/MS).First of all,four Hg-MTs were detected by capRPLC-ICP-MS and capRPLC-ESI-MS,and their mass charge ratios(m/z)were 1691.1056(z=5),1637.0824(z=5),1710.6929(z=5)and 1730.2909(z=5),respectively.Then,four Hg-MTs were confirmed as respective MTs subisoforms by using shotgun method,namely capRPLC-ESI-MS/MS hyphenated technique,Mascot software retrieval and UniProt protein database retrieval.The information of corresponding Hg-MTs is summarized as follows:m/z 1637.0824(z=5)was corresponding to Rg9Zn2-MT-1G or Hg9Cu3Zn-MT-1M,m/z 1691.1056(z=5)was corresponding to Hg11-MT-1G,m/z 1710.6929(z=5)was corresponding to Hg11Zn3-MT-2,and m/z 1730.2909(z=S)was corresponding to Hg12Zn-MT-1G isoform 1 or Hg11Zn3MT-1G.In other words,MTs within HepG2 cells exposed to Hg(?)bound with 9-12 Hg.The obtained results show the important metabolic pathways of Hg(?)in HepG2 cells at the molecular level.(3)The macromolecular species(Hg-1 and 1:3-1,>67 kDa)characterized above in HepG2 cells exposed to Hg(?)alone and co-exposed to Se(?)and Hg(?)(Hg(?):Se(?)=1:3)were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis-laser ablation inductively coupled plasma mass spectrometry(SDS-PAGE-LA-ICP-MS)in parallel.The band containing Hg remained at the top of the resolving gel,indicating that the target species containing Hg were not separated.In subsequent work,the oxidation state,atom type and arrangement and direct structural information of Se and Hg can be characterized by using X-ray absorption near-edge structure(XANES)and Extended X-ray absorption fine structure(EXAFS).Accordingly,it will be concluded whether the products of Se-Hg antagonism in 1:3-1 group are the putative(HgSe)n-Proteins complexes.