Hepatotoxicity Of Emodin And The Relevant ADME Mechanism Research

Background and ObjectivesEmodin is a natural anthraquinone compound which mainly exists in Chinese traditional medicines including Polygonum multiflorum and Rhubarb.In recent years,emodin has drawn rising and extensive attention because it exhibits remarkable pharmacological effects including purgative,antibacterial,anti-inflammatory,anti-tumor,and hepatoprotective activities.However,emodin has bidirectional effects on the liver.Short-term use of low doses of emodin results in liver protection,whereas long-term use of high doses of emodin causes liver toxicity.At present,the relationship between hepatotoxicity and metabolism still remain unclear.Hence,the purpose of our study is to figure out the following four points:1)The hepatotoxic of emodin and the effect of CYPs-mediated metabolism on emodin hepatotoxicity;2)The effect of UGTs-mediated glucuronidation on emodin hepatotoxicity and the regulation of predominant UGT isoform by transcription factors;3)The potential mechanism of gender differences in the hepatotoxicity of emodin;4)The effect of co-administration of emodin and probenecid on the hepatotoxicity and the relative mechanism.This study hopes to provide fundamental basis and scientific evidence for the safety clinical use of emodin.Methods1.The hepatotoxicity of emodin was studied by HepG2 cell model.In HepG2 cells,CYP1A2/3A4 inhibitors and inducers were used to investigate the effect of CYPs-mediated metabolism on emodin cytotoxicity.2.The genome and transcriptome sequencing of 55 human liver/36 human kidney samples combined with humanized recombinant UGTs-mediated metabolisms were used to investigate the predominant UGT isoform responsible for the metabolism of emodin in vivo.The main metabolic enzymes UGT2B7 and the transcription factor HNF4A were knocked down in HepG2 cells,respectively,to investigate the relationship between the UGTs-mediated metabolism and toxicity as well as the regulation of HNF4A on the UGT2B7.3.Male and female rats were randomly divided into control group,model group,high-dose emodin group,and low-dose emodin group.Gender differences of hepatotoxicity were estimated and the related mechanisms were explored in rats after oral administration of emodin for consecutive 28 days.4.The effect of probenecid on the hepatotoxicity and cytotoxicity of emodin and the related mechanism were investigated by combined use of emodin and probenecid in rats(28 days)and cells(48 h).Results and Conclusions1.High concentrations of emodin could inhibit cell growth while inducing oxidative stress and depleting reduced glutathione.Supplementation of reduced glutathione and n-acetylcysteine could reduce the inhibition of emodin on cell growth.CYP1A2/3A4 inhibitors could enhance the cytotoxicity of emodin,and CYP1A2/3A4 inducers could decrease the cytotoxicity of emodin.Hence,CYP1A2/3A4-mediated metabolisms played an important role in detoxification of emodin.2.UGT2B7 was the predominant enzyme for emodin glucuronidation.Four novel genetic variants(rs12186237,rs56724509,rs135297,and rs11726899)that affected the UGT2B7-catalyzed emodin glucuronidation were identified in our study.The results showed that the toxic effect of emodin was higher than its glucuronide.Hence,UGTs-mediated glucuronidation played an important role in detoxification of emodin.High-dose emodin could reduce HNF4A and UGT2B7 expression.In addition,the expression of UGT2B7 was decreased after HNF4A was knocked down in HepG2 cells.It is confirmed that emodin down-regulated UGT2B7 by inhibiting HNF4A and thus decreased phase ? metabolism and increased toxicity.3.Long-term use of high-dose emodin could cause gender differences in the hepatotoxicity,which were potentially mediated by UGT2B7 and MRP2.4.Combined use of emodin and probenecid for a long time could cause severe liver damage,as demonstrated by the 33.3%rats died in combination group.This finding was attributed to the ability of probenecid to increase exposures of emodin and its glucuronides by inhibiting UGTs and MRP2 expression and thus exacerbated emodin-induced hepatotoxicity and cytotoxicity in vivo and in vitro.