The Apoptosis Mechanism Induced by Aflatoxin B1 and Deoxynivalenol in Primary Hepatocyte of Cyprinus Carpio
|School||Nanjing Agricultural College|
|Course||Clinical Veterinary Medicine|
|Keywords||aflatoxin B1 deoxynivalenol carpio primary cullture hepatocyte apoptosis|
Mycotoxins are the toxic secondary metabolites produced by fungi. All kinds of the feed ingredients and finished products could be polluted by fungi and be contaminated by mycotoxin. So far, Aflatoxin B1 (AFB1) is one of the most toxic substances in the world, which can cause extensive damage to animal. Deoxynivalenol (DON) is contaminates worldwild in cereals. Although the toxicity of DON is not strang in mycotoxin, the polluted scope and content of DON are more than any other toxins. According to the survey on mycotoxins in feed and raw materials in China, the positive rate of DON was 93.0%, the mean concentration was 1140 mg·kg-1. The positive rate was higher than other mycotoxins. Most of current research focuse on the toxity of one mycotoxin, the study in toxity of serveral mycotoxins is important obviously because of which are often existence simultaneously in nature. So, this paper focus on the toxicity of AFB1 and DON in single and combined format to the primary hepatocyte of cyprinus carpio, including morphological changes, mitochondrial transmembrane potential response and genes changes of Bcl-2 family, to establish fundament for the further investigation in AFB1 and DON.Text I:Primary hepatocyte of cyprinus carpio in exponential phase were treated with AFB1 (10 ng·mL-1 or 20 ng·mL-1), DON (125 ng·mL-1,250 ng·mL-1,500 ng·mL-1,1000 ng·mL-1 or 2000 ng·mL-1) and AFB1+DON (10+125 ng·mL-1,10+250 ng·mL-1,10+500 ng·mL-1,20+125 ng·mL-1,20+250 ng·mL-1,or 20+500 ng·mL-1), seperately. Morphological changes of cells were observed under the inverted microscope and apoptosis rate was tested by flow cytometry. The results showed that the cell density and growth state were greatly change depended on the concentration of toxin; the apoptosis rate increased gradually depended on the concentration of DON; in the combined toxin group, under the existence of AFB1, the apoptosis rate of primary hepatocyte of cyprinus carpio increased with the increasing concentration of DON.TextⅡ:Primary hepatocyte of cyprinus carpio in exponential phase were treated with AFB, (10 ng·mL-1 or 20 ng·mL-1), DON (125 ng·mL-1,250 ng·mL-1,500 ng·mL-1,1000 ng·mL-1 or 2000 ng·mL-1) or AFB1+DON (10+125 ng·mL-1,10+250 ng·mL-1,10+500 ng·mL-1,20+125 ng·mL-1,20+250 ng·mL-1, or 20+500 ng·mL-1), separately. Those hepatocytes dealt were stained by JC-1, then mitochondrial transmembrane potential was detected by flow cytometry. The results showed that:the change of mitochondrial transmembrane potential in AFB1 group was not significant compared with the control group.Depended on the concentration increasing of DON (0-500 ng·mL-1), the mitochondrial transmembrane potential decreased. In the group treated with AFB1+ DON, mitochondrial transmembrane potential decreased with the increased concentration of DON, and was significantly decreased compared with the DON group.TextⅢ:Primary hepatocyte of cyprinus carpio in exponential phase were treated with DON (125 ng·mL-1,250 ng·mL-1,500 ng·mL-1 or 1000 ng·mL-1) or AFB1+DON (10+125 ng·mL-1,10+250 ng·mL-1,10+500 ng·mL-1,20+125 ng·mL-1 or 20+250 ng·mL-1), separately. Total RNA were extracted and studied by reverse transcription quantitative PCR. The results showed that with the increasing concentration of DON, the ratio of Bcl-2 to Bax decreased,while it reached the lowest in the 500 ng·mL-1, but it increased slightly in the 1000 ng·mL-1.In the AFB1+DON group, under the existence of AFB1, the ratio of Bcl-2 to Bax decreased with the increasing concentration of DON, while it reached the lowest in the concentration of AFB1+DON separately dealt with 10+500 n g·mL-1 and it was significantly decreased compared with the group dealt with DON alone.