The Metabolic Activation And The Mechanism Study Of Hepatotoxicity Of Diosbulbin B And 8-epidiosbulbin E Acetate

Dioscorea bulbifera L.(DB),known as Huang-Yao-Zi in Chinese,belongs to a member of the yam family Dioscoreaceae.It is wildly distributed in the tropical and subtropical regions of Asia,and exists in parts of Africa.DB is more widely distributed in China,and mainly produced in Hubei,Hunan,Jiangsu,Henan,Shandong,Zhejiang,Anhui,Fujian,Yunnan,Guizhou,Sicuan,Guangxi.It is cold and bitter taste,and it has the role of eliminating gall,cooling blood and hemostasis,sending fire and detoxification.DB is widely used in clinical,and it is used to treat a variety of tumors and struma,which has been a common medcine.Despite this,the safety of DB use has been questioned,and ingestion of DB was reportedly associated with high incidence of liver injury.Diterpene lactones isolated from DB are the primary components responsible for DB induced hepatotoxicity.The covalent modification of proteins by reactive metabolites of diosbulbin B(DIOB)and 8-epidiosbulbin E acetate(EEA)belonging to diterpene lactones is facilitated to illuminate the molecular mechanism of toxicology.The studies performed are summarized as below.1.The identification of the reactive metabolites of Diosbulbin B(DIOB)DIOB,a furan-containing deterpenoid lactone,is the most abundant component of DB.a traditional Chinese medicine herb.Administration of purified DIOB or DB extracts has been reported to cause liver injury in animals.The mechanisms of DIOB-induced hepatotoxicity remain unknown.The major objective of the present study was to identify reactive metabolites of DIOB.A DIOB-derived cis-enedial was trapped by N-acetyl lysine(NAL)and glutathione(GSH)or N-acetyl cysteine(NAC)in rat and human liver microsomal incubation systems after exposure to DIOB.Four metabolites(2-3-2-6)associated with GSH were detected by LC-MS/MS.Apparently,2-3 was derived from both NAL and GSH.2-4 and 2-5 resulted from the reaction of GSH without the involvement of NAL.Two molecules of GSH participated in the formation of 2-6.2-4 and 2-5 were also detected in bile and urine of rats given DIOB.2-6,a DIOB-derived NAC/NAL conjugate,was detected in microsomal incubations with DIOB fortified with NAC and NAL as trapping agents.2-3-2-7 were all chemically synthesized for metabolite identification.Microsomal incubation study demonstrated that KTC inhibited the production of the cis-enedial in a concentration-dependent manner,and P450 3A4 was found to be the enzyme responsible for the metabolic activation of DIOB.2.The identification of the reactive metabolites of 8-epidiosbulbin E acetate(EEA)8-epidiosbulbin E acetate(EEA)was actually the most abundant diterpenoid lactone in some DB,which has never been reported.Our studies demonstrated that administration of EEA caused acute hepatotoxicity in mice and the observed toxicity required cytochromes P450-mediated metabolism.Metabolic activation studies of EEA were performed in vitro and in vivo.Microsomal incubations of EEA supplemented with NAL and GSH generated six metabolites(3-4-3-9).3-4-3-7 were characterized as pyrrole derivatives,and 3-8 and 3-9 were pyrrolinones.3-5-3-9 were detected in bile and/or urine of rats given EEA.A dimethyldioxirane-mediated oxidation of EEA in the presence of NAL and GSH produced 3-4-3-9,all of which were generated in microsomal incubations.The structures of 3-6 and 3-9 were confirmed by 1H-and 13C-NMR.The findings of the conjugates provided the evidence for the metabolic activation of EEA to the corresponding cis-enedial intermediate both in vitro and in vivo.KTC inhibited the microsomal production of the cis-enedial,and P450 3A4 was found to be the primary enzyme involved in the bioactivation of EEA.3.The correlation of the reactive metabolites of EEA with EEA induced hepatotoxicity.EEA was actually the most abundant diterpenoid lactone in some DB,and we found that the occurrence of EEA in DB depended on the commercial source.EEA was hepatotoxic and more toxic than DIOB.EEA-containing DB is more toxic than EEA-free DB.In the study,we investigated the mechanism of EEA induced hepatotoxicity and explored the correlation of the reactive metabolites of EEA with EEA induced hepatotoxicity.The present study showed that EEA exhibited time-and dose-dependent liver injury in mice.Pretreatment with KTC prevented the animals from EEA-induced liver injury,caused 7-and 13-fold increases in plasma Cmax and AUC of EEA,and decreased urinary excretion of GSH conjugates derived from EEA.Pretreatment with BSO exacerbated EEA-induced hepatotoxicity.In order to define the role of furan moiety of EEA in EEA-induced hepatotoxicity,we synthesized the tetrahydro-EEA by catalytic hydrogenation of the furan moiety of EEA.No liver injury was observed in the animals given the same doses of tetrahydro-EEA.The results indicate that EEA itself does not appear to be hepatotoxic,but the electrophilic intermediate generated by the metabolic activation of the furan ring mediated by cytochromes P450 was responsible for EEA-induced liver injury.4.The interaction of the reactive metabolites derived from EEA with proteins Our early studies demonstrated that administration of EEA caused acute hepatotoxicity in mice and the observed toxicity required P450-mediated metabolic activation.Protein modification by reactive metabolites of EEA has been suggested to be an important mechanism of EEA-induced hepatotoxicity.The objectives of the present study were to investigate the interaction of the electrophilic reactive metabolites derived from EEA with lysine and cysteine residues of proteins and to define the correlation of protein adductions of EEA and the hepatotoxicity induced by EEA.EEA-derived cis-enedial was found to modify both lysine and cysteine residues of proteins.EEA was found to modify both cysteine and lysine residues of protein and form crosslinks,which were detected in liver microsomal incubation and the liver homogenate.In addition to the crosslinks,EEA derived cis-enedial reacted with amino group of lysine residues of protein to form the pyrrolines,and reacted with amino group of lysine residues of protein and sulfhydryl group of GSH to form the pyrroles.The observed modifications increased with the increase in doses administered in the animals.The protein adductions reached the peak at the at 6 h after the administration.The formation of protein adductions derived from the reactive metabolites of EEA were potentiated by BSO,but were attenuated by KTC.5.The interaction of the reactive metabolites derived from DIOB and EEA with DNAWe found the cis-enedial reactive intermediates of DIOB and EEA derived from the oxidation of DMDO could bind with dAdo,dGuo,and dCyd of DNA.The ctDNA was used as a model of DNA.The cis-enedial reactive intermediates of DIOB and EEA derived from the oxidation of DMDO were mixed with ctDNA,and hydrolyzed by DNA hydrolase to produce the adducts of dAdo,dGuo,and dCyd derived from EEA and DIOB.These results were suggested that DIOB and EEA could modify DNA.