Research On The Effect Of Ochratoxin A-induced Nephrotoxicity On DNA Methylation

Ochratoxin A(OTA)is a mycotoxin widely distributed in a variety of food products.In various food safety issues,OTA has been focused due to its nephrotoxicity and carcinogenicity.A previous study in our laboratory indicated that OTA exposure induced cytotoxicity by decreasing global DNA methylation in HepG2 cells.Kidney is the main target of OTA.However,there were little reports about the relationship between the mechanisms of OTA nephrotoxicity and DNA methylation.In this study,the OTA-induced DNA methylation in kidney tissues of rat and human embryonic kidney cells(HEK 293)has been investigated,providing new insights into the mechanisms of nephrotoxicity induced by OTA.The main results and conclusions were as follows:(1)Dynamic and dose-dependent changes of global DNA methylation were observed during OTA-induced nephrotoxicity.Significant loss of global methylation in the kidney occurred after the first 4 weeks of OTA exposure.Genomic hypermethylation,rather than global DNA hypomethylation,was observed in the kidneys of rats treated with OTA for 13 weeks.The expression levels of DNA methyltransferases were changed by OTA.The mRNA expression of DNA methyltransferase 1(DNMT1)was consistent with the changes of global DNA methylation.Aberrant hypermethylation and down-regulation of E-cadherin and N-cadherin genes was occurred after 13 weeks of OTA exposure.The relevant intracellular signal transduction pathways,including Wit and PI3K/Akt pathway,were activated.(2)MS-AP-PCR(Methylation-sensitive Arbitrarily Primed PCR)was used to screen the differentially methylated genes when cells were treated with 5 μM OTA.A total of eight differentially methylated fragments were identified,including six hypermethylated fragments and two hypomethylated fragments.According to GenBank web set,five of eight differentially methylated fragments were highly homologous with human genome sequence and they were located in chromosome 1,10,12,X and Y,respectively.Their functions were associated with transcriptional regulation.Results of the BSP showed that Hyper-1 and Hyper-2 were methylated when cells were treated with OTA.(3)Possible mechanisms of the toxicity induced by OTA through DNA methylation were explored using an Infinium HD Methylation 450K Array in the present study.According to the changes of methylation status,differentially methylated positions in 1 μM OTA vs control and 5 μM OTA vs control were divided into five group:hypermethylation-hypermethylation-hypomethylation,hypermethylation-hypermethylation-hypermethylation,hypomethylation-relative moderate methylation-hypermethylation,hypomethylation-hypermethylation-relative moderate methylation,relative moderate methylation-hypermethylation-hypermethylation.(4)The levels of cellular autophagy and apoptosis were found to be upregulated by treatment of low(1 μM)and high(5 μM)concentration of OTA,respectively.There were 1811 differentially methylated positions when cells were treated with low concentration of OTA(1 μM),and they were involved in 9 metabolic pathways.In addition,there were 1780 differentially methylated positions when cells were treated with high concentration of OTA(5 μM),and they were involved in 16 metabolic pathways.According to KEGG analysis,DNA methylation status of related genes in MAPK signaling pathway was affected by OTA,which was possibly involved in the regulation of autophagy and apoptosis.