Studies Of G₂/M Cell Cycle Arrest Caused By Aflatoxin B₁ And Protective Effect Of Se In Jejunum Of Broilers

Aflatoxin B₁（AFB₁）,a powerful mycotoxin,is produced by different strains of Aspergillus flavus and Aspergillus parasiticus,which is found in moldy food and feed.AFB₁ can induce hepatocellular carcinoma,immune-suppression and oxidative stress of animals.However,only a few studies of the effect on the digestive tract caused by AFB₁ were available,and the results were not conclusive.At present,researchers have paid more attention to the studies on the protective roles of selenium against the toxicity of AFB₁.Our previous studies suggest that AFB₁ in the diet caused the G2/M cell cycle arrest in broiler’s jejunum,and selenium had protective action against the toxicity of AFB₁.But it should be further clarified the molecular mechanism of AFB₁-induced G2/M cell cycle arrest and protective role of Se on the cell cycle arrest of jejunum caused by AFB₁.Sodium selenite,the source of selenium,was used to feed broilers which ingested AFB₁ in this study.The dynamical changes of the jujunal histological morphology,the cell cycle and the G2/M cell cycle regulatory molecules will be investigated by using histopathology,immunohistochemistry,flow cytometry and Quantitative real time-polymerase chain reaction（qRT-PCR）methods.A total of 240 one-day-old healthy cobb broilers were randomly divided into four groups and fed with basal diet（control group）,0.6 mg/kg AFB₁（AFB₁ group）,0.4 mg/kg Se（+Se group）,and 0.6 mg/kg AFB₁+0.4 mg/kg Se（AFB₁+Se group）for 21 days respectively.Histological observation of jejunum showed the shedding of the mucosa epithelium in the apical region of villi,the decreased villus/crypt ratio in the AFB₁ group.However,no obvious histological changes occurred in the control group,+ Se group and AFB₁,Se group during the experiment.The results of flow cytometry showed that significant changes in the cell percentage of G0/G1 phase were only seen between the control group and AFB₁ group at 21 days of age（p<0.05）,the AFB₁ group and AFB₁,Se group at 7 days of age（p<0.05）.Furthermore,the cell percentage of G2/M phase in the AFB₁ group was significantly higher than that in the control group,+ Se group and AFB₁+ Se group at 7,14 and 21 days of age（p<0.01 or p<0.05）,respectively.And the cell percentage of G2/M phase was not significantly changed in the +Se group（p>0.05）,when separately compared with the control group and AFB₁ + Se group.The immunohistochemical tests revealed that the integrated optical density（IOD）value of p21 and p53 proteins were significantly increased（p<0.01）in the AFB₁ group at 7,14 and 21 days of age,when compared with the control group.Moreover,no significant differences in the value were seen between the + Se group and control group（p>0.05）.The IOD values of p-cdc25C,Cyclin B and PCNA proteins were significantly decreased（p<0.01 or 0.05）in the AFB₁ group at 7,14 and 21 days of age,compared with the control group.Meanwhile,in comparison to the AFB₁ group,these values increased in the AFB₁ + Se group.In addition,these values showed no significant changed between the + Se group and control group（p>0.05）.Real-time PCR analysis indicated that the mRNA expression levels of ATM,Chk2,p53 and p21 in the AFB₁ group were obviously increased at 7,14 and 21 days of age（p<0.05 or 0.01）,when compared with the control group.In comparison to the AFB₁ group,these values in the AFB₁ + Se group were also obviously decreased（p<0.05 or 0.01）,except for that of ATM at 7 days（p>0.05）.In addition,no significant differences in these values were found between the + Se group and control group（p>0.05）.When compared with the control group,the mRNA expression of Cdc25,Cdc2,CyclinB3,Mdm2 and PCNA in the AFB₁ group were decreased at 7,14 and 21 days of age（p<0.01 or 0.05）,except for the value of Cdc25 at 7 days and Cdc2 at 14 days（p>0.05）.Furthermore,when compared with the AFB₁ group,the significant increased values were observed in Cdc25 and CyclinB3 at 21 days（p<0.01 or 0.05）,Cdc2 at 7 days（p<0.05）,Mdm2 at 7,14 and 21 days and PCNA at 14,21 days in the AFB₁+ Se group.Besides,no significant differences in the mRNA expression of Cdc25,Cdc2,CyclinB3,Mdm2 and PCNA were found between the + Se group and control group during the experiment（p>0.05）.In summary,0.6 mg/kg AFB₁ in the diet inhibited the development of broiler’s jejunum by causing G2/M cell cycle arrest.This was accompanied by the increase of ATM,p53,p21 and Chk2 protein and mRNA expression,and the decrease of cdc25,cdc2,cyclin B,Mdm2 and PCNA protein and mRNA expression.And supplementation of dietary sodium selenite at the concentration of 0.4 mg/kg selenium could effectively restore these parameters to be close to those in the control group.This showed that AFB₁ blocked G2/M cell cycle by ATM-Chk2-Cdc25-Cyclin B/cdc2 and ATM-Chk2-p53 pathway,and 0.4 mg/kg selenium could effectively protect the broiler’s jejunum from the G2/M arrest induced by AFB₁ through those two pathways.This may be the molecular mechanism of AFB₁-induced G2/M cell cycle arrest and protective role of Se on the cell cycle arrest of jejunum caused by AFB₁.