| T-2 toxin is a secondary metabolite produced by fungi such as Fusarium.It belongs to the trichothecenes class A compound in mycotoxins.It is stable in nature,strong in toxicity,widely distributed in nature and frequently pollutes food crops.Livestock and poultry are more likely to be exposed to mold than humans.Once animals ingested contaminated food crops can cause poisoning or even death,causing significant economic losses to agriculture and animal husbandry.Cytochrome P450 oxidase is a major class of enzymes that metabolize endogenous and exogenous compounds and participate in more than 75% of metabolic activities in humans.Many studies have confirmed that most of the CYP450 enzyme metabolites are inducers of CYP450,which can be regulated by transcription,expression and activity of CYP450.Among them,the CYP1 A subfamily has attracted extensive attention due to its obvious induction by exogenous compounds.In the previous study of our laboratory,it was found that T-2 toxin can significantly induce the increase of CYP1 A family transcription level,and CYP1A5 is involved in the hydroxylation metabolism of T-2 toxin.There are only two subtypes in the chicken CYP1 A family,namely CYP1A4 and CYP1A5,but the molecular mechanism of T-2 toxin regulating CYP1 A family expression in chicken was unclear.By revealing T-2 toxin-induced chicken CYP1 A subfamily regulation mechanism of family expression can not only enrich the regulation mode of avian CYP1 A family in response to trichothecenes,but also provide a theoretical basis for the prevention and control of subsequent mycotoxins.Therefore,in order to further explore the CYP1 A family in response to the metabolic regulatory network of T-2 toxin,this study will explore the two aspects of basal and induction regulation.In the study of the basal regulation mechanism of CYP1A5,the aryl hydrocarbon receptor binding was found between-311 and-253 upstream of the transcription start site of CYP1A5 promoter by dual luciferase assay.The xenobiotic response element(XRE)confirmed that Ah R can bind to the XRE site of-311 to-253 by mutating the key site of the XRE element and the DNA mobility shift assay(EMSA).The overexpression vector of Ah R and the reporter vector of-311/-1 were simultaneously transfected into COS-7 cells,and it was found that the promoter activity of CYP1A5 was significantly up-regulated.In the expression of CYP1A5 induced by T-2 toxin,the previous experiment found that Resveratrol,an antagonist of Ah R,inhibited the induction of CYP1A5 transcription by T-2 toxin,suggesting that Ah R may be involved in the expression of CYP1A5 induced by T-2 toxin.First,we treated the knockdown of Ah R cells with T-2 toxin and found that T-2 toxin could not revert to its induction of CYP1A5.Next,using the method of nuclear separation,it was found that T-2 toxin can promote the enrichment of Ah R into the nucleus.To explore the binding region of Ah R on the CYP1A5 promoter,we treated LMH cells transfected with the CYP1A5 truncation promoter reporter vector with T-2 toxin and found a significant decrease in promoter activity in the-311 to-253.Further,using the DAPA(DNA Affinity pull down assay)method,it was found that the T-2 toxin can promote the binding of Ah R to the XRE site of-311 to-253.The above results indicate that the T-2 toxin induces the expression of CYP1A5 by promoting the enrichment of Ah R into the nucleus.In order to investigate the effect of high expression of CYP1A5 on LMH cells,we treated T-2 toxin in overexpress CYP1A5 LMH cell and found that T-2 toxin can increase cell death rate and accompany DNA damage and apoptosis.Therefore,this study found that Ah R is involved in both the basal expression of CYP1A5 and induction process of T-2 toxin,and that T-2 toxin can increase cytotoxicity by inducing the expression of CYP1A5.Previous studies have found that the background promoter activity of CYP1A4 is significantly higher than that of CYP1A5,but the background expression is lower than CYP1A5,and we suspect that it may be due to the difference in epigenetic modifications.In this work,we first predicted the promoter of CYP1A4 and found that there are four CpG islands in the 5’UTR region of CYP1A4 promoter,suggesting that DNA methylation may be involved in the regulation of CYP1A4 expression.Next,we found that methylation can inhibit the promoter activity of CYP1A4 by in vitro mock methylation,the addition of methylation inhibitors can induce a significant increase in CYP1A4 transcription levels.In the transcriptional level of CYP1A4 induced by T-2 toxin,it was found that the transcription level of CYP1A4 induced by T-2 toxin was significantly higher than that of CYP1A5.We hypothesized that the difference in induction of chicken CYP1 A family is also related to DNA methylation,methylation-sensitive restriction enzymes-based quantitatire PCR(MSRE-q PCR)method was used.After treatment of cells with T-2 toxin at different times,the methylation level of CpG3 island of CYP1A4 was opposite to that of CYP1A4 m RNA levels.The methylation level of CpG3 island was minimized when T-2 toxin was treated for 12 h,and CYP1A4 was highest level of transcription.The presence of the Ah R binding element XRE in the CpG3 island was further revealed by truncation and mutation of the promoter of CYP1A4,and the methylation level near the element decreased by 10% after the T-2 toxin treatment.The Ch IP assay found that the T-2 toxin reduced the enrichment of the DNA methyltransferase Dnmt1 located in the vicinity of XRE.Taken together,this study found that the background expression of CYP1A4 is regulated by methylation and Ah R,and that the induction of CYP1A4 by T-2 toxin is caused by demethylation of CpG3. |
