Effect Of Gene ABCG2 On Transport Of Aflatoxin M1 In Dairy Caw Mammary Epithelial Cells

The Aflatoxins(AF) are defined as group 1 carcinogens. Aflatoxin B1(AFB1) are the major AFs. This contaminant can significantly decreased the food safety level and have a risk for human health. Aflatoxin M1(AFM1) is the oxidized metabolite of AFB1. Cows fed crop contaminats containing AFB1 had detectable AFM1 in the blood as soon as 15 minutes and in the milk as soon as 8 hours from treatment. Some AFM1 discharge with the milk and urine. The others stock in the edible tissues and organs such as blood, kidney and liver. AFM1 is common in the milk. Milk and its by-products are easily contaminated with AFM1. To further study the mechanism of AFM1 transport by dairy cow mammary epithelial cells(DCMECs) contribute to decreased the transport of AFM1 from blood circulation by diary cow blood-milk barrier, and it would improve the quality security of the milk and its by-products in our country.In order to study the transport of AFM1 in diary cow blood-milk barrier and clarify the transport mechanism of AFM1, DCMECs were seeded on microporous membrance with the density of 1×106 cells/m L to form the blood milk barrier. Blood milk barrier was formed by a single DCMEC with proliferation in vitro. Laser scanning confocal microscopy showed that the tight junctions were made up by DCMECs. And the cells could synthesis and secretion milk protein as in vivo. The cells barrier in vitro could be as a model to study the transport of the drugs in the blood-milk barrier.In order to study the transport of AFM1 in DCMECs, AFM1 was added in the medium, The m RNA expression of ABCG2, ABCC5, ABCC1, ABCB1, ABCC3, 5-HT, SLC22A5 and SLC22A4 gene were detected by q RT-PCR. The results showed that ABCG2 and ABCB1 both significantly expressed in DCMECs. It stated ABCG2 and ABCB1 may be involved in the transport of AFM1.At the same time, AFM1 was added in the medium, the protein expression of ABCG2 and ABCB1 were detected by western blotting. The protein of them were expressed significantly(P<0.01)comparing to the cells cultured in medium without AFM1. This enhanced the possibility that ABCG2 and ABCB1 may be involved in the transport of AFM1.In order to explore the effect of ABCG2 on the transport of AFM1, Recombinant plasmid p GCMV-ABCG2-IRES-EGFP was construct. It was transient transfected into cell barrier to perform the ABCG2 gene overexpression experiment. At the same time, RNAi method was used to transfect ABCG2 si RNA in the ABCG2 gene inhibition experiment.AFM1 was added in the medium, The m RNA expression of ABCG2 was detected by q RT-PCR and the protein expression of ABCG2 was detected by western blotting. Meanwhile,DCMECs and the upper chamber medium were collected. The content of AFM1 in the cells and medium were respectively measured by HPLC method. The results of q RT-PCR and western blotting showed that the expression of ABCG2 revealed significant difference both in the ABCG2 protein overexpression and the ABCG2 gene inhibition experiments(P<0.01), and the expression of β-casein revealed no significant difference(P>0.05). The result of AFM1 by HPLC analysis showed: Overexpressed of ABCG2 up-regulated the content of AFM1 in the cells and the upper chamber medium(P<0.01) and ABCG2 gene inhibition down-regulated the content of AFM1(P<0.01).In summary, ABCG2 gene is involved in the transport of AFM1, but isn’t involved in regulating lactation and proliferation ability of DCMECs. The mechanism that ABCG2 gene is involved in the transport of AFM1 should be explored.