Roles Of CYP2E1Enzyme In Norcantharidin Metabolism And Cyclosporine Induced Liver Injury

Cytochrome P4502E1(CYP2E1) isozyme can mediate the metabolism of endogenous and exogenous compounds, and plays important roles in the studies on pharmacology and toxicology. In order to investigate the roles of CYP2E1enzyme in drug metabolism and drug-induced liver injury, we undertook this study with norcantharidin and cyclosporine as entry points.In this study, the MetaPrint2D software was firstly used to predict metabolism sites and possible metabolites of norcantharidin and its hydrolysis product, norcantharidin acid, mainly based on the atomic fingerprint characteristics of the compounds, data mining and statistical analysis techniques by calculating the metabolism occurrence ratio; Then norcantharidin acid was docked with CYP1A2, CYP2A6, CYP2B6, CYP3A4, CYP2D6, CYP2C9, CYP2E1and CYP2C19isozymes respectively. Five of them with the top scores were chosen to be further metabolism studied in vitro with human recombined isozymes. The result showed that CYP2E1, CYP2C19and CYP2C9enzymes are mainly involved in norcantharidin acid metabolism. The metabolic prosess of norcantharidin acid mediated by recombinant human CYP2E1enzyme can be explained by Michaelis-Menten equation, with Km of23.64μM and CL of0.689ml/mmol CYP2E1/min. The results of prediction by software, metabolic experiment in vitro and metabolites speculation in vivo all confirmed that norcantharidin is firstly hydrolysed into norcantharidin acid, followed by oxygen bridge hydrolysis, six-membered ring broken by different ways or decarboxylation. Further oxidation can transform it to smaller molecules, which is easy to be eliminated, or combined with glucuronic acid, glycine or glutathione to increase the solubility; The same results of primary metabolic processe were found in rats and in vitro experiment, and the secondary metabolism similar.The character of rotating in CYP2E1stereo structure is necessary to make its substrates such as medium-long fatty acid entering, and also can easily make the enzyme-drug complexes unstable, then lead to oxidation stress and further induce the occurrence of liver injury. The incidence of Cyclosporine-induced liver injury is about30%, but its mechanism is still unknown. A total of322subjects who were orally taking Cyclosporine to prevent rejective reaction post organ-transplanting or to cure the autoimmune disease were recruited from Qilu hospital of Shandong University (P. R. China). All the subjects were divided into drug-induced liver injury (DILI) and control group according to the results of liver function examination,200of which were genotyped for thirteen CYP2E1SNPs, followed by Hardy-Weinberg equilibrium test, linkage disequilibrium analysis and tSNP selection in the first stage. The others were analyzed only for tSNP in the next stage. Results of tSNP genotyping from two stages were merged for statistic analysis. The results demonstrated that the SNP rs3813866(-1563T>A) showed a higher risk for developing DILI (OR:2.325,95%CI:1.491-3.626).Further study was conducted on the level of cell molecule to explore how the susceptible SNP regulate the transcription, expression and activity of CYP2E1. The mutated plasmid vector carrying rs3813866by site mutation technology was transferred into HepG-2cell, and the promoter activity, mRNA, protein expression and enzyme activity of CYP2E1were detected and compared with those of wild-type by dual-reporter gene, real-time PCR, western blot and substrate-probe methods. Besides, the same strategy was also conducted with or without Cyclosporine treatment. The results illustrated that the promoter activity of mutated-type of CYP2E1was2.46fold of wild type, and the mRNA increased by1.64times. Slight increasing of promoter activity, protein expression (1.72folder by20μM Cyclosporine) and enzyme activity (27.28%increasing by20μM Cyclosporine) can be made by Cyclosporine, but significant decreasing of mRNA was also found. The mRNA level of wild type of CYP2E1declined about2times and that of mutate-type even slipped20times after traetment by20μM Cyclosporine.To sum up, CYP2E1play an important role in norcantharidin metabolism. The rs3813866(-1563T>A) is susceptible SNP of Cyclosporine-induced liver injury through up-regulate the promoter activity and increasing the protein expression. Further study on the feedback regulation mechanism of CYP2E1transcriptional expression during Cyclosporine interference will be conducted.