The Differences Of Emodin UGT Metabolism In Liver/Kidney And The Related Detoxification Pathway

Background and ObjectivesEmodin is a very important compound of the rhubarb anthraquinone,which is one of the main active and also toxic ingredients of rhubarb/polygonum multiflorum.The acute renal and liver toxicity of emodin have received significant attention worldwide.Although processing-driven toxicity attenuating and drug combination-driven toxicity attenuating were the most common strategies in clinical practice,there is little known about the mechanism.Clarifying the metabolic mechanism of emodin in liver and kidney has become the key role to the detoxification.UDP-glucuronosyltransferases(UGT)is one of the most important phase Ⅱ enzymes in the metabolic detoxification processes.Emodin was rapidly glucuronidated after oral and intravenous administration.Although studies has proved that glucuronidation is the main metabolic pathway for emodin,metabolism mechanism in liver and kidney has not yet clear.Further research is necessary in order to delineate the difference of emodin UGT metabolism in liver and kidney and also the related detoxification pathway.Methods1.Human liver and kidney microsomes were prepared by homogenizing and differential centrifugal sedimentation.Genistein,mycophenolic acid and zidovudine as probes were selected to detect the enzyme activities of UGTs,UGT1A9 and UGT2B7 respectively.2.In vitro glucuronidation incubation via 55 human liver microsomes(HLMs),36 human kidney microsomes(HKMs)and 12 recombinant human UGTs isoforms were investigated for the glucuronidation characteristics of emodin.Then contribution of UGTs to the glucuronidation of emodin in liver and kidney were calculated.The proposed study delineated organ-dependent differences in metabolism of emodin in liver and kidney.3.MTT assay was employed to inspect the cytotoxicity of emodin and its metabolite on HepG2.Western blotting and RT-qPCR were used to determine the gene and protein expression of UGT2B7 on siRNA-UGT2B7-HepG2.We detected the cytotoxicity and IC50 value of emodin on siRNA-UGT2B7-HepG2.Furthermore,enzyme kinetics of emodin in siRNA-UGT2B7-HepG2 cell lysates were conducted.Kinetic parameters were then obtained using the XL-Kinetics program.Results and Conclusion1.The expression level of UGT isoform transcripts was determined in human liver and kidney.Here,UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A7,UGT1A9,UGT2B4,UGT2B7,UGT2B15,and UGT2B17 have been localized in human liver.Only three UGT enzymes(UGT1A6,UGT1A9 and UGT2B7)were detectably expressed in human kidney.2.A panel of recombinant human UGTs was used to prove that UGT1A1,UGT1A7,UGT1A8,UGT1A9,UGT1A10 and UGT2B7 were involved in the metabolism of emodin.The average metabolize rate in HLM was 1.55-fold higher than that in HKM(10.14±0.537 nmol/min/mg vs.6.53 士0.208 nmol/min/mg).The biggest contributor of emodin glucuronidation in HLM was UGT2B7(51.71%),followed by UGT1A1(24.39%)and UGT1A9(13.84%).The enzyme activities of UGT1A9 and UGT2B7 were statistically significant correlated with the glucuronidation rates of emodin in HLM(r UGT1A9=0.4369,P UGT1A9=0.0019;r UGT2B77=0.6384,P UGT2B7<0.0001).In human kidney,the predominant UGTs involved in emodin glucuronidation were UGT2B7(50.49%)and UGT1A9(47.87%),which the enzyme activity were also significant correlated with the glucuronidation rates of emodin in HKM(r UGT1A9=0.7678,P UGT1A9<0.0001;r UGT2B7=0.6338,P UGT2B7<0.0001).Our result showed that the most important enzymes in emodin glucuronidation were UGT1A9 and UGT2B7.3.The emodin glucuronidation differences between individuals were varied enormously,meanwhile the expression variations can explain 15.9%total metabolic variation in human liver.The metabolic rate of emodin were statistically correlated with the expression of UGT1A9(P=0.044).In human kidney,expression variations of UGT1A9/2B7 can explain up to 57.0%total variation of emodin glucuronidation.The expression of transcription factor HNF4a was positively correlated with the expression of UGT1A9/2B7 as well as emodin metabolism.The expression of NF-κB was negatively correlated with UGT1A9/2B7 expression as well as emodin metabolism.4.The cytotoxicity of emodin glucuronide in HepG2 cells was weaker than that of emodin.The gene expression and protein expression of UGT2B7 were significantly lower in siRNA-UGT2B7-HepG2 cells than in control cells.The cytotoxicity of emodin in siRNA-UGT2B7-HepG2 cells was significantly higher than control cells(P=0.022).The enzyme kinetics of emodin siRNA-UGT2B7-HepG2 cell lysates followed autoactivation kinetics,and the enzyme was proved to have lower affinity(Km(siRNA-UGT2B7)= 116.39±13.28μM;Km(control)= 71.75±2.38μM)and clearance rate than the control(CL(siRNA-UGT2B7)= 0.0021ml/min/mg;CL(control)＝0.0060ml/min/mg).