| Selenium plays crucial roles in maintaining the growth and development of vertebrates and humans.Selenium plays an important role in antioxidant,diabetes prevention,and prevention of common cancers such as breast,lung,and colon cancer.Selenium supplementation is beneficial in the body.However,it is hard to distinguish selenium beneficial concentrations from harmful concentrations for most vertebrates due to its narrow concentration range.Excessive selenium in cells will lead to developmental defects and diseases.Selenium has been reported to cause severe malformation in zebrafish embryos,but there are few studies on the mechanism of selenium excess-induced cardiovascular defects.In this study,zebrafish embryos were selected to evaluate the cardiovascular toxicity as well as the disorders of lipid metabolism and selenium transport caused by selenium,and fertilized embryos were exposed to L-selenomethionine until 96 hours post fertilization(hpf).In the meantime,lrp2 b knockdown and overexpression experiments were performed to verify the key role of the lrp2 b gene in selenium-induced cardiovascular defects.The main results of this paper are as follows:1.To assess the toxic effects of excess selenium on zebrafish development,fertilized embryos were exposed with selenium in different concentrations(0.25 μM,0.5 μM,1 μM,and 2 μM).The exposed embryos exhibited abnormal phenotype,including pericardial edema,scoliosis,and developmental delays when the selenium concentration was 0.5 μM.Almost all embryos died before 96 hpf when the selenium concentration was over 1 μM.Results showed that the mortality and deformity rate were increased with the increase of selenium concentration.The embryos treated with0.5 μM selenium existed lower mortality than 1 μM or 2 μM selenium and higher deformity rate than 0.25 μM selenium.2.Under selenium stress,wild-type embryos showed pericardial edema,decreased heart rate,an increase in the number of the tail circulating,and ectopic accumulation of hemoglobin flowing to the heart.The fli1-e GFP transgenic zebrafish displayed vascular malformations including an intersegmental vascular rupture with loss of integrity,incomplete intersegmental vascular growth,and intersegmental vascular malformations under selenium exposure.The myl7-e GFP transgenic zebrafish exhibited abnormal cardiac morphology,and increased ratio of atrial to ventricular area under selenium exposure.The q RT-PCR results indicated that the expressions of cardiovascular development genes were changed in selenium-stressed embryos.3.To evaluate the effect of selenium on lipid metabolism and selenium transport,we firstly performed Oil Red O staining to label neutral fats in zebrafish embryos.The results showed that the selenium-stressed embryos exhibited liver steatosis,and the content of triglycerides and total cholesterol were significantly increased.The expression of lipid metabolism-related and selenium metabolism-related genes were also evaluated.Among the tested lipid metabolism-related genes,there were no significant differences in the expression of lipid synthesis-related genes(hmgcra,hmgcs1,fasn,scd,and acaca)among control and selenium-treated group.The expression of lipidolysis-related genes(pnpla2,cpt1 aa,and acox1)were downregulated in selenium-treated embryos.Among the selenium-transport genes,lrp2 b gene was down-regulated,while lrp8 and sepp1 gene were up-regulated in seleniumstressed embryos at 24 hpf.Lrp2 b and lrp8 exhibited a reduced expression in seleniumstressed embryos at 96 hpf.Gene lrp2 b and lrp8 are not only the members of the lowdensity lipoprotein receptor(LDLR)family,but also related to selenium transport.4.To further verify the key role of lrp2 b gene in selenium-induced cardiovascular defect,we performed lrp2 b knockdown and overexpression experiments.The results showed that lrp2b-knockdown embryos exhibited the cardiac defects similar to selenium-stress embryos,and over-expression of lrp2 b rescued the selenium-induced defects,indicating that lrp2 b might play a key role in regulating selenium cardiotoxicity.In summary,our research evaluates the cardiotoxicity as well as the disorders of lipid metabolism and selenium transport caused by excessive selenium,and reveals the molecular mechanism of cardiovascular defects in selenium-exposed zebrafish embryos. |
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