Toxicological Effects And Metallothionein Gene Xpression In Misgurnus Anguillicaudatus During Cadmium Exposure

Misgurnus anguillicaudatus belongs to Cypriniformes, Cobutudae, Cobitinae,Misgurnus. It has become an important aquaculture object for its tasty meat and strongmarket demand. Nowadays, cadmium has become the main factor of water heavy metalpollution. At the same time, cadmium is threatening pollution-free aquaculture ofMisgurnus anguillicaudatus, and harming environment and human health seriously. Inthis study,（1） the activity changes of antioxidant enzymes and ultrastructure damageduring cadmium exposure in Misgurnus anguillicaudatus were researched;（2）metallothionein cDNA of Misgurnus anguillicaudatus was cloned and analyzed;（3）metallothionein gene expression during cadmium exposure in Misgurnusanguillicaudatus was investigated. The major results are as following:1Study on toxicological effects during cadmium exposure in Misgurnusanguillicaudatus①The activity changes of superoxide dismutase（SOD）, catalase（CAT）, glutathioneperoxiase（GSH-PX） in Misgurnus anguillicaudatus during Cd²⁺（0.05mg/L,0.5mg/L,5mg/L） exposure in10days was researched. The results showed that:0.05mg/L Cd²⁺and0.5mg/L Cd²⁺had significant inductive effect to SOD activity in liver and gill ofMisgurnus anguillicaudatus; however, SOD activity had some decrease with exposuretime prolonging. The activity of SOD in liver and gill was induced first and theninhibited during5mg/L Cd²⁺exposure, and the activity was inhibited significantly withtime going.0.05mg/L Cd²⁺had no significant change to CAT in liver and gill. However,the CAT activity was increased first and then decreased in0.5mg/LCd²⁺and5mg/L Cd²⁺exposure; the significant inhibition was happened at last. GSH-PX activity was inducedfirst then inhibited in liver and gill and inhibited finally. All Cd²⁺had first inductioneffect and then inhibition effect to GSH-PX activity in liver and gill; finally, significant inhibition. With Cd²⁺concentration increased and exposure time prolonged, theinhibition happened earlier and was more significant.②The ultrastructure damage of liver and gill in Misgurnus anguillicaudatus wasobserved after10days exposure in three concentration Cd²⁺mentioned above. Theresults demonstrated that: chromatin in liver cell increased and gathered around nuclearmembrane after0.05mg/L Cd²⁺exposure.0.5mg/L Cd²⁺made plastosome enlarged,endoplasmic reticulum expanded, cell nucleus deformed and off-centerd. Cell nucleusshrinked and deformed seriously, chromatin gathered to edge, plastosome enlargeddeeply, endoplasmic reticulum expanded acutely, even some cell apoptosis in5mg/LCd²⁺. Gill lamellaes and filament appeared secretions, front-end gill lamellaes emergedsome breakage after0.05mg/L Cd²⁺exposure.0.5mg/L Cd²⁺made gill filament swollen,gill lamellaes deformed even damaged. Gill appeared pathological changes, gilllamellaes and gill filament emerged necrosis seriously. With Cd²⁺concentrationincreased, the damage in gill and liver seemed deeply, and the damage was irreversible.2Cloning and analysis of metallothionein cDNA of MisgurnusanguillicaudatusA fragement of metallothionein（MT） cDNA of Misgurnus anguillicaudatus wasobtained by PCR. This cDNA was434bp, incluing180bp ORF which coding60amimoacid residues. Misgurnus anguillicaudatus MT molecular weight is6035.9Da andlsoelectric point is8.05. This MT sequence is highly conservative comparing with otherfish. Its homology with Misgurnus mizolepis reaches98%, as the some time, it has thetypical structure（Cys-X-Cys or Cys-Cys） of MT in vertebrate. MT gene expression intissue of Misgurnus anguillicaudatus showed significant difference in this study.Expression in brain was1.5-30times higher than other tissues, intestine was12-20times than other. Liver, eyes and fin expressed faintly. These results may declear thatMT expression is relevant with the different structure and function of different tissues.3Effects of metallothionein gene expression during cadmium exposure inMisgurnus anguillicaudatusThe effects of MT gene expression in liver and gill of Misgurnus anguillicaudatusin24hours,48hours,72hours Cd²⁺（0mg/L,0.25mg/L,2.5mg/L）exposure were analyzedby real-time PCR. The results indicated that: MT gene expression in liver was moresensitive than gill during Cd²⁺exposure. With Cd²⁺concentration increased andexposure time prolonged, the MT gene expressed higher. It may has something to do with MT chelating Cd²⁺, regulating immune response and providing metal ions toimmune enzymes. When facing Cd²⁺stress, MT will express highly in order to protectorganism from damage.