| As one of the polyether ionophore antibiotic,maduramicin is widely used for the prevention and treatment of coccidiosis in livestock and poultry industry.However,the safe dosage range of this antibiotic is narrow.If the mixture of fodder and drug was not uniform,it may probably cause the poisoning of chicken.In the actual application process,there are indeed various cases of animal poisoning caused by maduramicin.On the other hand,maduramicin is poorly absorbed in chicken and its metabolism process is fast,thus some drugs will be excreted primarily unchanged in the urine and feces.Moreover,maduramicin may be introduced into the soil and water environment when animal manure containing the residual maduramicin were used as fertilizer for farmland,which may pose a great threat to aquatic organisms such as fish,algae and so on.In our study,zebrafish was used as the experimental object to evaluate the toxic effects of maduramicin on zebrafish,and its toxic mechanisms were also studied.The specific results are as follows:1 Study on acute toxicity of maduramicin to zebrafishBased on the result of preliminary test,the formal acute toxicity test was designed according to modified Kaber method.Zebrafish were randomly exposed to 10.00,11.89,14.14,16.81,20.00 mg·L-1 maduramicin solutions,and blank control and solvent control group were set at the same time.We adopted the semi-static method to treat zebrafish,and the toxic symptoms and death of zebrafish were observed during 96 h.The median lethal concentration（96 h LC50）and the 95%confidence interval were calculated.In addition,water samples of each group were collected and the concentrations of maduramicin in these samples were determined by high performance liquid chromatography（HPLC）.The obtained results showed that zebrafish in the maduramicin group showed acute poisoning symptoms within 6 h after treatment started,including irregular swimming,losing body balance.The abdomen of the dead zebrafish was upward,and there were white substances at the edge of the gills.The 96 h LC50 of zebrafish death caused by maduramicin was 13.33 mg·L-1 with a 95%confidence interval ranged from 12.64 to 14.05 mg·L-1.The deviation between the measured concentration and the corresponding theoretical concentration was less than 20%.Taken together,the acute exposure protocols of maduramicin for zebrafish was reasonable.The zebrafish treated with maduramicin for a short time may showed obvious acute poisoning symptoms and lethal effects.According to the grading criteria for acute toxicity of fish in Water and Wastewater Monitoring and Analysis,the toxicity of maduramicin to zebrafish was moderate.2 Study on genetic toxicity of maduramicin to zebrafishAfter treating with maduramicin for 14 days,six zebrafish were randomly selected from each group and then euthanized in ice-water.After that,the liver of zebrafish was excised to prepare hepatocyte suspension.The degree of DNA damage in hepatocytes was determined by single cell gel electrophoresis（SCGE）.In addition,four zebrafish from each group were selected,and then the fish tail was cut and peripheral blood was collected.Then,the blood was dropped on glass slide to prepare the blood smear.The blood smear was then stained with giemsa stain solution and observed under the light microscope.Results showed that the DNA comet images of zebrafish hepatocytes in the control group were perfectly round,while the "comet tail" appeared in the DNA comet images of zebrafish hepatocytes in the maduramicin group.Furthermore,the number of micronucleus cells in the erythrocytes of zebrafish in the maduramicin group was significantly higher than that of zebrafish in the control group.Taken together,maduramicin may cause serious damage to DNA of zebrafish hepatocytes and lead to the aberration of chromosome in erythrocytes of zebrafish.In conclusion,maduramicin has strong genetic toxic effects on zebrafish.3 Study on the effect of maduramicin on the histological structure of zebrafish organs and the antioxidant system of zebrafishZebrafish were randomly exposed to 0.1,0.5,2.5 mg·L-1 maduramicin for 14 days.At the end of the 14-day exposure test,four zebrafish were randomly selected from each treatment group.Then,the liver,gill and intestine were dissected to make paraffin sections,and stained with hematoxylin-eosin（HE）.The morphological structures were observed under a light microscope.On days 3,6,9 and 14,six zebrafish were selected from each repeat and then quickly euthanized in ice-water.After which,the gill and liver of zebrafish were excised and homogenized separately.Then,the activities of SOD,CAT,GPX,GST and the contents of MDA were evaluated with commercial kits.In addition,the mRNA expression levels of sod1,cat,gpx1a,gstr,nrf2 and keap1 that involved in the oxidative stress response of zebrafish were determined by RT-qPCR.Results showed that an induction in histopathological lesion was observed in the organs after 14 days of exposure,including lamellar fusion of gill and vacuole formation in the liver as well as the fracture of intestinal villus in the intestine.It turned out that 0.1 mg·L-1 maduramicin significantly elevated the activities of SOD,CAT,GPX and GST in the liver of zebrafish,while the activities of these antioxidant enzymes in the liver were significantly restrained by 2.5 mg·L-1 maduramicin.Compared with the control group,the MDA contents in the liver of zebrafish treated by maduramicin for 14 days were significantly increased,while the MDA contents in the gill of zebrafish were significantly elevated after only 3 days of treatment with maduramicin.Moreover,the mRNA expression levels of genes involved in oxidative stress in the liver of zebrafish were significantly up-regulated and then significantly down-regulated by maduramicin on days 6,9 and 14 compared with the control group.The mRNA expression levels of those genes in the gill of zebrafish were significantly down-regulated by maduramicin during the whole 14-day period.Taken together,2.5 mg·L-1 maduramicin can cause serious tissue damage to major organs of zebrafish and induce severe oxidative stress in both liver and gill of zebrafish,may resulting in oxidative stress injury.4 Effects of maduramicin on the mRNA expression levels of genes related in immune response and cell apoptosis in liver of zebrafishZebrafish with different genders were randomly exposed to 0.1,0.5,2.5 mg·L-1 maduramicin for 14 days.On days 3,6,9 and 14,six zebrafish were chosen from each repeat group and the liver were excised separately.RT-qPCR were used to evaluated the mRNA expression levels of il-1β,il-8,il-10,lyz and c3 which involved in the immune response of zebrafish.In addition,the gene expression levels of p53,caspase-3,caspase-8,caspase-9,bcl-2,bax and apaf-1 were also investigated.Results revealed that gene expression levels of il-1β,i1-8,il-10 and lyz in liver of zebrafish were significantly up-regulated by maduramicin on days 3 and 6 compared with controls.Compared with control group,0.5 and 2.5 mg·L-1 maduramicin significantly up-regulated the mRNA levels of p53,caspase-3,caspase-8 and bax in the liver of zebrafish on days 3 and 6.In conclusion,2.5 mg·L-1 maduramicin can significantly induce inflammatory immune response and cell apoptosis in zebrafish liver,may causing damage to the liver of zebrafish. |
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