The Effects Of Seven Transporters On Ochratoxin A Nephrotoxicity Using Cell Models

Ochratoxin A(OTA)is one of mycotoxin,a secondary metabolite from fungi Aspergillus.sp and Penicillium.sp.It mainly pollutes grains.OTA is the strongest renal carcinogen in mycotoxins and also has hepatotoxicity,reproductive toxicity and other toxicity.OTA toxicity is related to its transporters to a great extent.At present,many researches have been done on organic anion transporters(OATs)and OTA.No reports have been made on the interaction between OTA and organic cation transporters(OCTs)so far.In our research,the role of organic cation transporter 2(OCT2)protein in OTA nephrotoxicity and the role of breast caner resistance protein(BCRP),multidrug resistance protein 2(MRP2),organic anion-transporting polypeptides(OATPs)in OTA transport and absorption were studied to further improve the understanding of OTA nephrotoxicity.First part.Objective:Digital gene expression(DGE)method was used to get a general way to think OTA-induced rat nephrotoxicity.Methods:F344 rats were administered with OTA(0,70,210?g/kg b.w.)for 13 weeks.The kidneys were analyzed by DGE method.Results:There were significantly different genes belonging to Slc22 family in both high-dose and low-dose groups compared to control group.Slc22a2(OCT2)was one of them.In KEGG pathway,ABC transporters pathway was enriched in high-dose group.Abcg2(BCRP)gene was one of the differential genes in this pathway.In the function enrichment of Gene Ontology(GO),transport,organic anion transport,plasma membrane,plasma membrane part were enriched in high-dose group.Conclusion:OTA changed transport related pathways in rat kidney.Second part.Objective:The influence of OTA and TEA(inhibitor of OCTs)on NRK-52E cells and OCTs of rat kidney.Methods:NRK-52E cells were treated with OTA for 24 h.CCK-8 method was used to detect cell viability.Flow cytometry methods were used to detect cell apoptosis and ROS production.DNA damage was analyzed by comet assay.Gene expression was detected by RT-PCR.Protein expression was detected by western blot.Rats were administered with OTA for 1,4 and 13 weeks.The kidneys were acquired to detect change of OCTs.Results:TEA could alleviate OTA-induced cell death,cell apoptosis,the production of ROS and DNA damage.OTA treatment significantly increased cell OCT2 expression,while OCT2 expression were all decreased in rat kidneys administered with OTA at all time points.Conclusion:OCTs may be involved in OTA-induced cytotoxicity.OTA treatment changed the expression of OCTs both in vitro and in vivo.Third part.Objective:Study OTA-induced cytotoxicity with Slc22a2 knockout NRK-52E cells.Methods:Slc22a2 knockout NRK-52E cells were constructed by plasmid pX458.Flow cytometry methods were used to detect cell apoptosis.Protein expression was detected by western blot.Results:The degree was lower of OTA-induced cell apoptosis in Slc22a2 knockout cells.OTA treatment significantly increased the levels of Caspase 3 and CDK1 in wild-type NRK-52E cells,but not the knockout cells.Conclusion:Slc22a2 knockout alleviated OTA-induced cell apoptosis.Fourth part.Objective:Study OTA-induced cytotoxicity with Slc22a2-overexpressing NRK-52E cells.Methods:rOCT2 overexpression plasmid was constructed by pcDNA3.1(+)plasmid.rOCT2 overexpression plasmid was transiently transfected to NRK-52E cells.CCK-8 method was used to detect cell viability.Protein expression was detected by western blot.Results:The transfected NRK-52E cell line had an increased expression of rOCT2 protein compared with the control cell line.Cell viability was lower in pcDNA3.1(+)-Slc22a2 transfected cell line compared with control group with OTA treatment.Caspase 3 was increased much more in the overexpressing cell line than that in the control group.Conclusion:pcDNA3.1(+)-Slc22a2 transfected cell line was more sensitive to OTA toxicity.OCT2 overexpression increased OTA-induced cell apoptosis.Fifth part.Objective:Study the transport of OTA by other transporters.Methods:Transwell and uptake experiments were applied to detect the transport and uptake of OTA with several cell lines at pH 7.4 and pH 6.4.Results:Both mBcrp and hBCRP clearly transported OTA at pH 6.4.There was modest transport of OTA by mMrp2 and hMRP2 only at pH 6.4.OATP1A2 and OATP2B1 mediated uptake of OTA both at pH 7.4 and 6.4,but OATP1B1 only at pH 7.4.Conclusion:hBCRP and hMRP2 can mediate elimination of OTA from cells thus reducing OTA toxicity.Human OATP1A2,OATP1B1,and OATP2B1 can mediate cellular uptake of OTA.