Research On Expression Of HSP70 In Zebrafish Tissues Induced By Roxarsone

Roxarsone is an additive added to poultry and livestock feed for resisting bacteria and coccidiosis, improving feed conversion, stimulating growth and so on. Roxarsone is excreted mostly as its parent drug in animal manure. Poultry or livestock litter, which is a mixture of animal manure and bedding material, has widespread use as a fertilizer in the world. The roxarsone run into environment with the use of manure fertilizers, which usually be not treated by detoxicity technical process in China. Now about 50～70% intensive hogpens excluded to pounds and rivers directly. These may affect the quality of soil and surface or underground water seriously.Stress protein is also called heat shock proteins(HSPs), it is one kind of proteins which are induced by some stress factors such as heat, pathogenic organism, physics and chemisty complication, etc. It is highly conserved, and play an important role in the survival of stressed cells and stabililization of internal environment. HSPs may perform a series of biological functions, enhancing tolerance to stressors, and so on. In term of the size of molecular weight, HSPs are usually identified four classes, namely the HSP90 family, the HSP70 family, the HSP60 family, and the small HSPs family. In HSPs, the HSP70 family is the most conserved and the most important family. Because its quantity is the most in most organisms,and its synthesis is the most remarkable after being stressed. A majourity reseach has shown that many pollutants can induced HSP70 of the organnisms living in waters. All these pollutants can significantly induce HSP70, which implied HSP70 can serve as one of biomarkers in aquatics and hydrophytes when they are intimidated by pollutants. This implies that HSP70 appears important potential as one of early responser in the pollution of aqutic environment.In this paper, zebrafish is used for the experiment. By using SABC immunohistochemical method, RT-PCR, and fluorescence quantitative PCR (FQ- PCR), we try to study the mechamism of inducement of hsp70mRNA, hsc70mRNA and hsf1mRNA and the feasibility of HSP70 as a biomarker.The acute toxicity experiment of roxarsone to zebeafish(Danio rerio) was determined, the LC50 values of 96 hours is 249.80 mg/L (248.65~250.95 mg/L). The expression and distribution of HSP70 in zebrafish tissues (gills, livers, and gonad) exposed to roxarsone were observed with SABC immunohistochemical method. In gills of zebrafish, the positive signal of HSP70 was located significantly in the basilar membrane of branch leaves, gill arches, red blood cellulate cytoplast and epithelia of gill filament. In livers the positive signals of HSP70 distributed obviousely in vein blood vessel, which indicates HSP70 maybe closely related to the damages and functions of cadiovascular system. In stroma cellulate cytoplast of the tried zebrafishs'gonads HSP70 also erpressed strongly. By using RT-PCR,hsp70, hsc70 and hsf1 were amplified in zebrafish tissues.They are 412, 605 and 457bp respectively.By using FQ- PCR, the induction and expression of hsp70mRNA, hsc70mRNA and hsf1mRNA in tissues (gills, livers and gonad) of zebrafish exposed to100mg/l roxarsone were studied. As a result, the three genes of zebrafish changed didn't at the same level, but they all display some rules. Compared to the control goup, the expression of hsp70mRNA, hsc70mRNA and hsf1mRNA in tissues (gills, livers and gonad) of zebrafish exposed to roxarsone are different. The quantitative results displayed the peak of hsp70, hsc70 and hsf1 mRNA expression appeared at 12h exposed to roxarsone in gill, gonad and liver tissues. Comparing the mRNA expression of the same gene in normal control, change folds were 52.721, 34.925 and 59.727 fold of hsp70, hsc70 and hsf1 respectively in gill tissue, 26.590, 13.810 and 17.716 in gonad, 4.530, 3.796 and 3.275 in liver. Based on the relative abundance of hsp70, hsc70 and hsf1 mRNA at peak time, the express level of three genes was the highest in gill, moderate in gonad and the lowest in liver. From the time sequence, the induction expression of these genes in gill was start-up firstly, in liver secondly and in gonad lastly. In a word, all these results showed that HSP70 displays tremendous potential as a biomarker used in the application of enviroment pollution of roxsarsone.