Effects Of CYP2E1-dependent Presynaptic Plastic Damage Induced By Acrylamide

Objective:ACR(acrylamide,ACR)metabolized into GA(glycidamide,GA)by CYP2E1(Cytochrome P450 2E1,CYP2E1).Studies have shown that there are high concentrations of GA and ACR in muscle and nerve tissue.Therefore,we should not only pay attention to the prototype of ACR,but also explore the mechanism and weight of the toxic effect of its metabolite GA in this process.Based on the co-culture cells model of HEPG2 E47/C34 human hepatocytes with high/low expression of the target metabolic enzyme CYP2E1 and SH-SY5Y which can differentiate into mature neuroblastoma cells,in vitro platform for testing ACR-induced synaptic plastic damage was build.The toxic effects and the effects of the weight for both the ACR prototype and main metabolites GA were measured.Results of the study can provide certain clues and evidence for elaborating the mechanism of ACR’s neurotoxicity.Methods:1.Construction and verification of HepG2 E47 and HepG2 C34 cell lines The p WSLV-07-cyp2el over-expression plasmid was constructed and the vector pWSLV-07 without the cyp2e1 gene fragment was inserted as an empty plasmid control.The recombinant plasmid was amplified and collected,and the above plasmids were transfected into 293T cells.HepG2 cells were infected with virus containing different plasmids.After puromycin screening,monoclonal cells were selected for culture and amplification.The construction of the cell line was verified at the mRNA and protein levels of target gene CYP2E1 through fluorescence quantitative PCR and Western blot methods.2.Establishment of co-culture cells model of HepG2 E47/SH-SY5Y and HepG2 C34/SH-SY5Y and study on neurotoxicity of ACR SH-SY5Y in logarithmic phase was seeded in a 6-well plate.HepG2 E47/C34 was inoculated into the Transwell co-culture systemto construct HepG2 E47/SH-SY5Y and HepG2 C34/SH-SY5Y co-culture system.①CCK-8 method was used to detect the cells survival rate of SH-SY5Y cells induced by ACR gradient concentrations(0,50,100,150,200,250 and 500μg/ml)at different exposure time(24 h,48 h and 72 h).②HepG2 E47,HepG2 C34 and SH-SY5Y cells were co-cultured indirectly,HepG2 E47 or HepG2 C34 were cultured in the upper chamber of the Transwell,and SH-SY5Y was seeded in the lower chamber.CCK-8 method was used to detect the cells survival rate of SH-SY5Y cells induced by ACR gradient concentrations(0,50,100,150,200,250 and 500μg/ml)at the exposure time of 24 h.HPLC method was used to detect the content of ACR prototype and its main metabolite GA in different ACR treatment groups(0,50,100 and 150μg/ml).3.ACR and its metabolite GA on the structure and functional plastic damage in the co-culture systems the two groups of co-culture systems were treated with gradient concentrations of ACR(0,50,100 and 150μg/ml,24h).The changes of synaptic microstructure and mitochondria were detected by electron microscopy.Ca2+-ATPase and Na+-K+ATPase activities were detected by the chemical colorimetry.Western blot method was performed to detect the protein levels of Synapsin Ⅰ,GAP-43 and SNAREs complex including SNAP-25,Syntaxin 1A and VAMP-2.Results:1.Construction and verification of HepG2 E47 and HepG2 C34 cell lines Comparing HepG2 E47 with HepG2 and HepG2 C34 groups,the mRNA expression of cyp2e1 was higher than the other two groups,the difference was statistically significant(P<0.05).No statistical difference of cyp2e1 expression was found between HepG2 and HepG2 C34 groups(P>0.05).Comparing HepG2E47 with HepG2 and HepG2 C34 groups,the CYP2E1 protein expression was higher than the other two groups,the difference was statistically significant(P<0.05).No significant difference of CYP2E1 expression in HepG2 and HepG2 C34 groups was found(P>0.05).2.Effects of plastic damage induced by ACR in HepG2 E47/SH-SY5Y and HepG2 C34/SH-SY5Y co-culture system After treated with gradient concentrations of ACR(0,50,100,150,200,250 and 500 μg/ml)at 24,48 and 72 hours,the proliferation of single culture SH-SY5Y cells was detected.Survival rate of 0,50,100,150 and 200μg/ml groups at 24 hours was below 50%.Survival rate of 0,50,100 and 150μg/ml groups at 48 hours was below 50%.Survival rate of 0,50 and 100μg/ml groups at 72 hours was below 50%.The linear fitting graph was drawn.Results indicated that when the exposure time was 24 hours,the value of R2 was the largest,and the degree of linear fitting was the best.Comprehensive analysis of the above results and other important factors,the exposure time for subsequent experiments was set as 24 hours.The gradient concentrations of ACR were set for 0,50,100,150,200,250 and 500μg/ml.In the two co-culture systems,when the ACR concentration was 0,50,100 and 150μg/ml,the cell survival rate of SH-SY5Y was below 50%.Therefore,the ACR exposure concentration was set for 0,50,100 and 150μg/ml,and the exposure time was 24h in the subsequent experiments.With the increase of ACR concentration(0,50,100,150μg/ml),the ACR and GA contents in SH-SY5Y cells in the two co-culture systems was significantly increased(P<0.05).3.ACR and its metabolite GA on the structure and functional plastic damage in the co-culture systems In the control group,the synaptic structure was normal and clear,the synaptic space was obvious,and the synapse was visible.The mitochondria were round or long rod-shaped with clear mitochondrial peak structure.With the increase of the ACR exposure concentration,compared with the HepG2 C34 co-culture group,the synapses in the HepG2 E47 group were blurred,the synaptic structure was empty and the degree of synaptic vesicles were decreased.The degree of mitochondrial swelling was increased in the HepG2 E47 group,and the mitochondrial membrane and mitochondrial structure was disappeared.In the two co-culture systems,the Ca2+-ATPase and Na+-K+-ATPase enzyme activities of the 0,50,100 and 150μg/ml dose groups were statistically decreased(P<0.05).The enzyme activity of Ca2+-ATPase and Na+-K+-ATPase were significantly decreased compared with the HepG2 C34 group.In the two co-culture systems,the Synapsin Ⅰ and GAP-43 proteins in the 0,50,100,and 150 dose groups were statistically decreased,and the Syntaxin 1A,SNAP-25,and VAMP-2 proteins were statistically increased(P<0.05).The protein levels of Synapsin Ⅰ and GAP-43 in HepG2 E47/SH-SY5Y co-culture system were significantly lower than the HepG2 C34/SH-SY5Y group.The protein levels of Syntaxin 1A,SNAP-25,and VAMP-2 proteins were significantly higher than the HepG2 C34/SH-SY5Y group(P<0.05).Conclusion:1.The HepG2 E47 cell line was successfully obtained on the facts of continuously stable and high expression of CYP2E1.The HepG2 E34 cell line with empty plasmid was set as the blank control to eliminate the effect of plasmids on the expression of cell protein.The results of this study provide a in vitro cells model for further study the CYP2E1-dependent neuropathy and molecular mechanism of ACR.2.Using HepG2 E47 and HepG2 E34 as the cell basis,the co-culture system models of HepG2 E47/SH-SY5Y and HepG2 E34/SH-SY5Y were constructed.This co-culture system can be used as an in vitro platform to further explore the neurotoxic difference of ACR and its metabolite GA.3.Under the ACR-induced structural and functional synaptic damage,the changes of synaptic micro-structure and the key protein levels of synapsin Ⅰ,GAP-43 and SNAREs complex(VAMP-2,syntax 1 and SNAP-25)were measured.Current results indicated that GA,the main metabolite of ACR,might be one of the principal factors in of ACR neurotoxicity.Results of the current study provide significant clues for the follow-up mechanism study.