| Mycotoxins were toxic secondary metabolites produced by fungi(molds), wildly contaminated in raw material and finished product. It would result in mycotoxins poisoning when animals were exposed the feed contaminated the mycotoxins.Aflatoxin B1 is a compound that produced during the growth of Aspergillus flavus and Aspergillus parasiticus. As we know it is the most power toxi city and stabization toxin in the world. Deoxynivalenol(DON)was a metabolism produced during the growth of Fusarium graminearum. It was a member of trichothecene family. DON is less toxicity than Aflatoxin B1.The aim of our research is to study the toxic effect of AFB1 and DON to cyprinus carpio at normal dosage.Test I The level which contaminated the aquatic animal feeds of AFB1 and DON in Jiangsu province.We collected the aquatic animal feed of Jiangsu province (Yangzhou, Yancheng et al.,).Among these 15 samples are powder feed and 15 samples are Pellet diet. We used the HPLC to detect the level of the toxin after they have been extracted and purified.The detection rate of AFB1 is 40% in the power feed and 26.7% in the pellet diet, and the detection rate of DON is 100% whatever in anyone feed. The maximum content of AFB1 is 16.4μg kg-1 which is less than the government standard of AFB1 in animal feed(20μg kg-1),and the maximum content of DON is 7046ug kg-1 which is more higher than the government standard(1000μg kg-1).Test II Toxic effect of AFB1 and DON to cyprinus carpioTwo hundred and seventy cyprinus carpio(middle weight was 18±0.1g) were devided into 9 groups and each group has 10 fishes. Three parallel were designed for every group. One parallel work for the anatomy during the study and two parallel group for the observation and analysis. Basic feed, basic feed and AFB1(0.05mg kg-1), basic feed and AFB1(0.1mg kg-1), basic feed and DON (1mg kg-1), basic feed and DON(5mg kg-1), basic feed and AFB1(0.05mg kg-1)+DON(1 mg kg-1), basic feed and AFB1(0.05mg kg-1)+DON(5 mg kg-1), basic feed and AFB1(0.1 mg kg-1)+DON(1 mg kg-1),basic feed and AFB1(0.1 mg kg-1)+DON(5mg kg-1)were designed for 9 groups. The experiment lasted for 40 days. The body length, body weight, fullness, liver weight/body weight, blood biochemical index and the pathologic histology of the liver, spleen and intestinal tract were checked every 20 days. It showed that whatever the toxin was separated or combined, it could depress the performance of the fish. And this injury is more serious with the higher concentration of the toxin. This toxic effect showed the significant dose-effect relationship.TestⅢTo Optimize the Isolation and Cultivation of cyprinus carpio primary hepatocytesIn this study, the cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion and pancreatin digestion, respectively. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll grade centrifugation). The harvesting hepatocytes were suspended in DMEM, M199(cultured in 5%CO2) and L-15 (cultured without 5%CO2) medium, then cultured at 17℃,27℃and 37℃, respectively. The cell yield was counted by hemocytometer, and the viability of the cells were assessed by Trypan blue exclusion test. Results from these studies showed that the best isolation method was pancreatin digestion [the cell yield was 2.7×108 per g(liver weight) and the viability was 98.4%] and the best medium was M199(cultured in 5%CO2) or L-15(cultured without 5%CO2), respectively. The optimal culture temperature was 27℃. The primary hepatocytes culture of cyprimus carpio grew well and satisfied the requirement of most of toxicological experiments in this condition.Test IV Toxic effect of AFB1 and DON to cyprinus carpio primary culture hepatocytesIsolation and culture the hepatocytes base on the test III,design the experiment group as AFB1(0.01μg mL-1 and 0.02μg mL-1),DON(125ng mL-1、250 ng mL-1、500 ng mL-1、1000 ngmL-1and2000ngmL-1), conbination group(0.01+125、0.01+250、0.01+500、0.02+125、0.02+250 and 0.02+500,μg mL-1, ng mL-1), reagent controI(DMSO:2μL mL-1)and the control. Check the cell shape, enzyme activity and the cytotoxicity every 2 hours to study the toxic effect of the toxin. The experiment lasted for 24 hours. From the experiment it showed that all of the toxin could induce a great quality of cells death. Alsothere was no significant difference between the experimental groups, but the difference between each experimental groups and the control is significant (P<0.05).We could saw the inequality apoptosis cells under the TEM in experimental groups contrast to the control. And the results of enzyme activity and the cytotoxicity support the viewpoint.Test V The DNA injury of cyprinus carpio primary cultured hepatocytes by AFB1 and DONWe have used SCGE and DNA ladder to detect the cell apoptosis induced by the toxin. When chose the SCGE to do the research, we found that the group of the highest rate of the apoptosis is the combination group[AFB1+DON:0.02 (μg mL-1)+125 (ng mL-1)], the second is DON 500ng mL-1.and when we choose the DNA ladder to do the research, it showed that DON(≤500ng mL-1)and the low concentration of combination [AFB,+DON:0.01(μg mL-1)+125(ng mL-1)'AFB1+DON:0.02(μg mL-1)+125(ng mL-1)] could induce significant the hepatocyte apoptosis ly, and showed the obvious "ladder" on 2.5% agarose gel electrophoresis. |
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