The Mechanism Underlying Bile Acid Metabolism-mediated Hepatotoxicity From Valproic Acid

Objectives:Valproic acid(VPA)is a widely prescribed antiepileptic drug(AED)with applications to multiple types of epilepsy by single or combination therapy.More recently,VPA has been an adjuvant drug for cancer treatment,due to its anti-histone deacetylase(HDAC)activity.For this reason,the side effect associated with VPA hepatotoxicity has aroused widespread and close concern among clinicians.Till now,several mechanisms,such as inhibition of mitochondrial?oxidation enzymes,carnitine and coenzyme A deficiencies,oxidative stress,and hyperammonemia,have been implicated in VPA hepatotoxicity,but the underlying mechanisms still remain debated.Metabolomics is dedicated to explore the final responses to stressors caused by drug intervention,diseases,environment,and genetic modification,and provides new insights into the exploration mechanisms of drug-induced liver injury(DILI)and the discovery of potential biomarkers.In this study,a serum nontargeted metabolomic analysis of children with epilepsy exhibiting VPA hepatotoxicity was performed,aimed to explore of differential metabolites and metabolic pathways,and the underlying mechanism was explored in animals and cells.Methods:1.Collection of serum samples from children with epilepsy exhibiting VPA hepatotoxicity:Serum samples and medical records of children with epilepsy(age?16 years old)who were admitted to Shengjing Hospital of China Medical University were collected.Based on liver function indicators,these children were divided into 2groups,normal liver function(NLF)group and abnormal liver function(ABLF)group.2.Screening of differential metabolites and metabolic pathways in serum of children with epilepsy exhibiting VPA hepatotoxicity:Metabolites in serum samples were extracted by protein precipitation using cold acetonitrile,separated by reversed-phase liquid chromatography(RPLC)and hydrophilic interaction liquid chromatography(HILIC)and detected in electrospray ion(ESI)positive and negative modes,respectively.According to the data collected from each mode,multivariate statistical analysis methods,such as principal component discriminant analysis(PCA-DA)and orthogonal projections to latent structures discriminant analysis(OPLS-DA),were used to analyze endogenous metabolites.Variable importance in projection(VIP)values?1.0,|p(corr)|?0.4(loadings scaled as correlation coefficient between model and original data)and P<0.05 were used for differential metabolites selection.KEGG(Kyoto encyclopedia of genes and genomes)analysis was performed on these differential metabolites to match differential metabolic pathways.3.Liver bile acid profiles in mice treated with VPA:Male C57BL/6 mice(20–22 g)treated with 250 or 500 mg/kg VPA and the same volume of saline(5 mL/kg)once daily by intragastric administration for 14 days.Biochemical and histopathological analysis were performed to evaluate hepatotoxicity induced by VPA in mice.Eighteen specific bile acids were quantified in mouse livers by HPLC-QTOF/MS,and analyzed by multivariate statistical analysis methods,such as principal component analysis(PCA)and OPLS-DA.The differential bile acids were selected based on VIP?1.2 and confirmed by one-way analysis of variance(ANOVA)or Kruskal-Wallis H test.These specific bile acids were further classified and analyzed statistically.4.The effects of VPA on the expression of genes and proteins related to bile acid metabolism:The mRNA and protein levels of genes involved in bile acid synthesis,transport and regulation were measured by Real-time PCR and Western Blot,respectively.5.The effect of farnesoid X receptor(FXR)in hepatotoxicity induced by VPA:After treated with VPA at different concentrations(0,1,2 and 5 mM)for 24 h,the expression of FXR,small heterodimer partner(SHP),HDAC1,HDAC2,HDAC3 and acetylated proteins in HepG2 cells were detected by Real-time PCR,Western Blot or Immunoprecipitation.FXR selective agonist(GW4064)were used to investigated whether the activation of FXR could alleviate the toxic effects of VPA on HepG2 cells.Results:1.The selection of differential metabolites and metabolic pathways in serum of children with epilepsy exhibiting VPA hepatotoxicity:A total of 23 children with epilepsy treated with VPA were enrolled in this study,and 13 children in ABLF group and 10 children in NLF group.PCA-DA analysis suggested that,regardless of the ionization mode and chromatographic system,samples were clearly clustered into two groups,the ABLF and NLF groups,indicating that serum metabolic patterns were significantly different between the two groups.After univariate and multivariate analyses,25 differential metabolites were obtained.KEGG pathway analysis indicated that dozens of biological processes and pathways were altered in ABLF group,including fatty acid pathway,amino acid metabolism,citrate cycle,urea cycle,and bile acid pathway.2.The effects of VPA on liver bile acid profiles in mice:(1)After treatment for 14 days,the total cholesterol levels in plasma and triglycerides in livers were significantly higher in mice treated with VPA(250 and 500 mg/kg),while the plasma levels of aminotransferases(ALP,ALT,and AST)did not differ among groups.H&E and oil red O staining results indicated that lipid accumulation was observed in mice treated with VPA.(2)A heat map and PCA model(three components,R~2X=0.944,R~2Y=0.797)analysis showed that a clear separation was observed among the control,250,and 500 mg/kg groups,bile acid profiles in mouse livers were perturbed by VPA.Most bile acids increased in mice of 500mg/kg group compared to control group.The difference in bile acid profiles between the250 mg/kg group and control group was not as great as the difference between the 500mg/kg group and control group.(3)Further,an OPLS-DA model(R~2X=0.854,R~2Y=0.706,Q~2Y=0.549)was established,the R~2Y and Q~2 above suggested that the model interpretation rate and prediction rate were effective.The permutation tests duplicated for200 times and showed no over-fitting of this model.(4)According to VIP?1.2,deoxycholic acid(DCA)and chenodeoxycholic acid(CDCA)showed a higher correlation with hepatotoxicity induced by VPA.ANOVA or Kruskal-Wallis H analysis indicated that the two bile acids increased in a dose-dependent manner and appeared to potential biomarker for prediction of hepatotoxicity induced by VPA.(5)The total bile acids and unconjugated bile acids in mice of 500 mg/kg group increased significantly.3.The effects of VPA on the expression of genes and proteins related to bile acid metabolism:(1)Real-time PCR results showed that,in mice treated with VPA,especially the 500 mg/kg group,the expressions of bile acid synthetase(CYP7A1,CYP8B1 and CYP7B1)and transporters(BSEP and MRP2)increased,the expression of regulatory proteins(FXR and SHP)decreased,while the expressions of amino acid conjugated enzymes(BAAT and BACS)did not changed obviously.(2)Western Blot results showed that,in mice of 500mg/kg group,the expressions of bile acid synthetase(CYP7A1 and CYP8B1)increased,the expressions of amino acid conjugated enzymes(BAAT and BACS)and regulatory proteins(FXR and SHP)decreased,while the expressions of transporters(BSEP and MRP2)did not changed obviously.4.The effect of FXR on hepatotoxicity induced by VPA:After treated with VPA at different concentrations(0,1,2 and 5 mM)for 24 h,the expression of FXR,SHP and HDAC1-3 in HepG2 cells decreased in a concentration-dependent manner,and protein acetylation was significantly increased.The activation of FXR induced by GW4064 could not reversed the inhibition of VPA on FXR activity,and alleviate the toxic effects of VPA on HepG2 cells.Conclusions:1.Children with epilepsy exhibiting VPA hepatotoxicity displayed metabolic profile shifts,and the fatty acid pathway,amino acid metabolism,citrate cycle,urea cycle,and bile acid pathway were altered,thereinto,the perturbation of bile acid homeostasis was found for the first time.2.Bile acid homeostasis was perturbed by VPA via affecting bile acid production and conjugation.Impaired FXR signaling might be involved in VPA hepatotoxicity.3.The activation of FXR via GW4064 could not alleviate hepatotoxicity induced by VPA,which might be related to the acetylation of FXR.