Modulations Of Epigenetic Mechanism Are Involved In The OTA-induced Cytotoxicity In PK15 Cells

Since ochratoxin A(OTA)was discovered,it has been ubiquitous as a natural contaminant of moldy food and feed.The multiple toxic effects of OTA are a real threat for human beings and animal health.For example,OTA can cause porcine nephropathy.Humans exposed to OTA can develop a range of chronic disorders.OTA-induced toxicity mechanisms observed in vitro or in vivo primarily include oxidative stress,inhibition of protein synthesis,immune function regulation and energetic metabolism.It has been reported that oxidative stress could induce epigenetic modification by changing the expressions of methylation or acetylation.In addition,it has been reported that mytoxin could induce epigenetic modification.It reported that deoxynivalenol exposure induces epigenetic modification changes during porcine oocyte maturatio.However,whether epigenetic mechanism is involved in OTA-induced nephotoxicity has not been clarified until now.Thus,the objectives of the present study are to clarify OTA-induced cytotoxicity and to investigate the epigenetic mechanism affecting OTA-induced cytotoxicity.Experiment 1:Modulations of Epigenetic related enzymes are involved in the OTA-induced cytotoxicityOTA is reported to induce cytotoxicity and apoptosis through the mechanism of oxidative stress.Oxidative stress could induce the epigenetic enzymes modifications.However,whether epigenetic enzymes modifications are involved in OTA-induced cytotoxicity and apoptosis has not been reported until now.Therefore,the objectives of this study were to verify OTA-induced cytotoxicity and apoptosis and to investigate the potential role of epigenetic enzymes in OTA-induced cytotoxicity and apoptosis in PK15 cells.The results demonstrated that OTA at 4 ?g/mL treatment for 12 h and 24 h induced cytotoxicity and apoptosis as demonstrated by decreasing cell viability,increasing LDH release,Annexin V/PI staining,Bcl-2/Bax mRNA ratio and apoptotic nuclei in PK15 cells.OTA treatment up-regulated ROS production and down-regulated GSH levels.Moreover,OTA at 2.0-8.0 ?g/mL for 24 h induced cytotoxicity and apoptosis in a dose-dependent manner in PK 15 cells as demonstrated by decreasing cell viabilities,and increasing LDH release,Annexin V/PI staining cells and apoptotic nuclei.In addition,OTA treatment activated the epigenetics related enzymes DNA methyltransferase 1(DNMT1)and Histone deacetylase 1(HDAC1).Moreover,High concentration OTA increased DNMT1 and DNMT3a expressions and the effects of OTA on DNMT1 expression are larger than that on DNMT3a expression.In conclusion,the observed effects indicate that the critical modulation of DNMT1 and HD AC 1 is related to OTA-induced cytotoxicity and apoptosis.Experiment 2:Epigenetic enzymes are involved in OTA-induced cytotoxicity and the effects of SOCS3 hymethylationin in PK15 cellsOur previous Test 1 indicated that DNMT1 plays a key role in OTA-induced cytotoxicity in PK15 cells.However,the mechanism underlying OTA-induced cytotoxicity by DNMT1 has not been reported until now.The present study aims to demonstrate that DNMT1 plays a key role in OTA-induced cytotoxicity and to investigate its mechanism.Adding DNMT1 inhibitor(5-Aza-2dc)or HDAC1 inhibitor(LBH589)depressed the up-regulation of DNMT1 or HDAC1 expression,trespectively.Furthermore,inhibiting DNMT1 by 5-Aza-2dc reversed the decreases of cell viability and GSH levels,increases of LDH activity and ROS levels and apoptosis induced by OTA.Meanwhile,inhibiting HDAC1 by LBH589 reversed the decreases of cell viability,increases of ROS levels and apoptosis induced by OTA.GSH levels and LDH activity have no significant change.OTA induced SOCS3 promoter methylation and increased SOCS3 mRNA and protein expression.Further,DNMT1 inhibitor inhibited SOCS3 promoter methylation and promoted the increases of SOCS3 expression.The observed effects indicate that OTA induced nephrotoxicity by DNMT1-mediated SOCS3 promoter methylation,in turn,SOCS3 expression in PK15 cells,and provides a new explanation the mechanism underlying the toxicity of OTA.