Diosbulbin B Research Articles

In Vitro DNA Adduction Resulting from Metabolic Activation of Diosbulbin B and 8-Epidiosbulbin E Acetate.

Diosbulbin B (DBB) and 8-epidiosbulbin E acetate (EEA), belonging to furan-containing diterpenoid lactones, are the primary components of Dioscorea bulbifera L. (DB), a traditional Chinese medicine herb. Our earlier studies indicated that consumption of DBB or EEA induced acute hepatotoxicities. Both DBB and EEA were bioactivated by P450 3A4 to generate the corresponding cis-enedial reactive metabolites which are associated with the hepatotoxicities. It has been proposed that the electrophilic intermediates attack cellular nucleophiles such as protein or DNA, thought to be a mechanism of triggering toxicities. The purposes of our present study were to define the interaction of the electrophilic reactive metabolites originating from DBB and EEA with 2'-deoxyguanosine (dGuo), 2'-deoxycytidine (dCyd), and 2'-deoxyadenosine (dAdo) and to characterize DNA adducts arising from the reactive metabolites of DBB and EEA. The reactive metabolites of DBB and EEA were found to covalently bind to the exocyclic and endocyclic nitrogens of dCyd, dGuo, and dAdo to generate oxadiazabicyclo[3.3.0]octaimine adducts. The reactive metabolites of DBB and EEA also attacked dGuo, dAdo, and dCyd of calf thymus DNA. The DNA adducts possibly contribute to the toxicologies of DBB and EEA.

Long-term diosbulbin B treatment induced liver fibrosis in mice

Airpotato yam is a traditional Chinese medicine used for treating thyroid disease and cancer in China. Diosbulbin B (DB) is reported to be the main hepatotoxic compound isolated from Airpotato yam. A variety of reports have shown the acute liver injury induced by DB in vivo. However, whether long-term administration of DB will cause liver fibrosis in mice is unknown. This study aims to investigate the liver fibrosis induced by long-term DB treatment in mice. C57BL/6 mice were orally given with DB (25, 50 mg/kg) for 1 or 2 month, respectively. Liver hydroxyproline content, hepatic collagen deposition and immune cells infiltration were increased in mice treated with DB (50 mg/kg) for 2 months. Serum amounts of hyaluronic acid and laminin were increased in mice treated with DB for 1 or 2 months. DB (50 mg/kg) induced hepatic stellate cells (HSCs) activation when mice were treated with DB for 2 months. Liver mRNA expression of Col1a1, Col1a2, Col3a1, fibronectin (Fn1), vimentin (Vim) and fibroblast-specific protein 1 (FSP1) were all increased in DB-treated mice. Hepatic protein expression of Vim, FSP1 and collagen 1 (COL1) were increased in DB-treated mice. Additionally, DB induced nuclear factor κB (NFκB) activation and increased the expression of pro-inflammatory molecules including tumor necrosis factor (TNF)-α, interleukin (IL)-6, intercellular cell adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS) in mice. In conclusion, long-term administration of DB induced liver fibrosis in mice. HSCs activation, epithelial-mesenchymal transition (EMT) and liver inflammation contributed to DB-induced liver fibrosis in mice.

Diosbulbin B induced G2/M cell cycle arrest in hepatocytes by miRNA-186-3p and miRNA-378a-5p-mediated the decreased expression of CDK1

Diosbulbin B (DB) is the main hepatotoxic compound in Airpotato yam, which is traditionally used for treating thyroid disease and cancer in China, and its hepatotoxic mechanism still remains unclear. This study aims to investigate its hepatotoxic mechanism by focusing on regulating microRNA (miRNA). DB induced hepatotoxicity both in vivo and in vitro. Results of miRNA chip analysis showed that the expression of eleven miRNAs was up-regulated and twelve miRNAs was down-regulated in livers from DB-treated mice. The altered expression of seven miRNAs was further validated by using real-time polymerase chain reaction (RT-PCR) assay. DB induced G2/M arrest in L-02/cytochrome P450 3A4 (CYP3A4) cells in both concentration- and time-dependent manner. A total of eleven predicted target genes was related with cell cycle regulation of those above seven miRNAs, among which the mRNA and protein expression of cyclin-dependent kinase 1 (CDK1) decreased both in vivo and in vitro. Both miR-378a-5p and miR-186-3p have binding sites in the 3′-untranslated region (UTR) of CDK1. With the use of CDK1 3′-UTR luciferase reporter assay, miR-378a-5p and miR-186-3p was found to down-regulate the luciferase activity. The mimics of miR-378a-5p or miR-186-3p reduced CDK1 expression in L-02/CYP3A4 cells, but their inhibitors reversed the decreased CDK1 expression induced by DB. Moreover, overexpression of miR-186-3p inhibitor reversed the G2/M cell cycle arrest induced by DB in L-02/CYP3A4 cells. Taken together, our results showed that DB induced hepatotoxicity by inducing G2/M cell cycle arrest in hepatocytes via miR-186-3p or miR-378a-5p-mediated the reduced CDK1 expression.

Development of Polyclonal Antibodies for Detection of Diosbulbin B-Derived cis-Enedial Protein Adducts.

Diosbulbin B (DSB), a major component of herbal medicine Dioscorea bulbifera L. (DB), can be metabolized to an electrophilic intermediate, DSB-derived cis-enedial (DDE). DDE was suggested to contribute to the hepatotoxicity observed in experimental animals and humans after their exposure to DSB. Our previous work found that DDE reacted with primary amino and/or sulfhydryl groups of hepatic protein. The objective of the study was to develop polyclonal antibodies that can recognize DDE-derived protein adducts. Immunogens synthesized from DDE and keyhole limpet hemocyanin were employed to raise polyclonal antibodies in rabbits. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titers of antisera obtained from immunized rabbits. Immunoblot analysis showed that DDE-modified bovine serum albumin (BSA) was recognized by the obtained polyclonal antibodies in a concentration-dependent manner and without cross-reaction to native BSA. Competitive ELISA and competitive immunoblot analyses defined the specificity of the antibodies to recognize BSA modified by DDE. Immunoblot analysis also detected a multitude of chemiluminescent bands with a variety of molecular weights in liver homogenates that were harvested from mice treated with DSB. In summary, we have successfully raised polyclonal antibodies to detect protein adducts derived from DDE.

Discovery of Hepatotoxic Equivalent Combinatorial Markers from Dioscorea bulbifera tuber by Fingerprint-Toxicity Relationship Modeling

Due to extremely chemical complexity, identification of potential toxicity-related constituents from an herbal medicine (HM) still remains challenging. Traditional toxicity-guided separation procedure suffers from time- and labor-consumption and neglects the additive effect of multi-components. In this study, we proposed a screening strategy called “hepatotoxic equivalent combinatorial markers (HECMs)” for a hepatotoxic HM, Dioscorea bulbifera tuber (DBT). Firstly, the chemical constituents in DBT extract were globally characterized. Secondly, the fingerprints of DBT extracts were established and their in vivo hepatotoxicities were tested. Thirdly, three chemometric tools including partial least squares regression (PLSR), back propagation-artificial neural network (BP-ANN) and cluster analysis were applied to model the fingerprint-hepatotoxicity relationship and to screen hepatotoxicity-related markers. Finally, the chemical combination of markers was subjected to hepatotoxic equivalence evaluation. A total of 40 compounds were detected or tentatively characterized. Two diterpenoid lactones, 8-epidiosbulbin E acetate (EEA) and diosbulbin B (DIOB), were discovered as the most hepatotoxicity-related markers. The chemical combination of EEA and DIOB, reflecting the whole hepatotoxicity of original DBT extract with considerable confidential interval, was verified as HECMs for DBT. The present study is expected not only to efficiently discover hepatotoxicity-related markers of HMs, but also to rationally evaluate/predict the hepatotoxicity of HMs.

Describing the holistic toxicokinetics of hepatotoxic Chinese herbal medicines by a novel integrated strategy: Dioscorea bulbifera rhizome as a case study.

It is vital to monitor the holistic toxicokinetics of toxic Chinese herbal medicines (CHMs) for safety. Although an integrated strategy based on the area under the curve (AUC) has been proposed to characterize the pharmacokinetic/toxicokinetic properties of CHMs, improvement is still needed. This study attempted to use 50% inhibitory concentration (IC50) as weighting coefficient to investigate holistic toxicokinetics of the major diosbulbins i.e. diosbulbin A (DA), diosbulbin B (DB), and diosbulbin C (DC) after oral administration of Dioscorea bulbifera rhizome (DBR) extract. Firstly, the cytotoxicities of the three diosbulbins on human hepatic L02 cells were evaluated and the IC50 values were calculated. Then, integrated toxicokinetics of multiple diosbulbins based on AUC and IC50 were determined. Finally, correlations between integrated plasma concentrations and hepatic injury biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bile acid (TBA) were analyzed. As a result, integrated plasma concentrations were correlated well with TBA and the correlation between TBA and IC50-weighting integrated plasma concentrations was better than that of AUC-weighting integrated plasma concentrations. In conclusion, the newly developed IC50-weighting method is expected to generate more reasonable integrated toxicokinetic parameters, which will help to guide the safe usage of DBR in clinical settings.

Serum microRNA-122-3p, microRNA-194-5p and microRNA-5099 are potential toxicological biomarkers for the hepatotoxicity induced by Airpotato yam

Airpotato yam (the rhizome of Dioscorea bulbifera L.) is traditionally used to treat thyroid disease and various cancers in China. However, it was found to cause hepatotoxicity during clinical practice. This study aims to identify candidate serum microRNAs (miRNAs) as diagnostic biomarkers for the liver injury induced by Airpotato yam. The results of serum alanine/aspartate aminotransferase (ALT/AST) showed the remarkable hepatotoxicity induced by ethyl acetate fraction of Airpotato yam (EF) (450mg/kg) and diosbulbin B (DB) (300mg/kg) in mice. The results of miRNAs chip analysis showed that the expression of 28 and 37 serum miRNAs was obviously altered in EF- and DB-treated mice, respectively. Among these miRNAs, miRNA-122-3p, miR-194-3p and miR-5099 have passed the further validation in serum from both EF- and DB-treated mice. Moreover, the expression of miRNA-122-3p and miRNA-194-5p was significantly increased in EF (375mg/kg)-treated mice with no significant elevation of serum ALT/AST activity. Only the expression of serum miRNA-5099 was not altered in the liver injury induced by acetaminophen (APAP), monocrotaline (MCT) or toosendanin (TSN). In conclusion, this study demonstrated that miR-122-3p and miRNA-194-5p were two sensitive biomarkers, and miR-5099 might be a specific biomarker for reflecting the liver injury induced by Airpotato yam.

Chemical Identity of Interaction of Protein with Reactive Metabolite of Diosbulbin B In Vitro and In Vivo.

Diosbulbin B (DIOB), a hepatotoxic furan-containing compound, is a primary ingredient in Dioscorea bulbifera L., a common herbal medicine. Metabolic activation is required for DIOB-induced liver injury. Protein covalent binding of an electrophilic reactive intermediate of DIOB is considered to be one of the key mechanisms of cytotoxicity. A bromine-based analytical technique was developed to characterize the chemical identity of interaction of protein with reactive intermediate of DIOB. Cysteine (Cys) and lysine (Lys) residues were found to react with the reactive intermediate to form three types of protein modification, including Cys adduction, Schiff’s base, and Cys/Lys crosslink. The crosslink showed time- and dose-dependence in animals given DIOB. Ketoconazole pretreatment decreased the formation of the crosslink derived from DIOB, whereas pretreatment with dexamethasone or buthionine sulfoximine increased such protein modification. These data revealed that the levels of hepatic protein adductions were proportional to the severity of hepatotoxicity of DIOB.

The role of Ntcp, Oatp2, Bsep and Mrp2 in liver injury induced by Dioscorea bulbifera L. and Diosbulbin B in mice.

Dioscorea bulbifera L. (DB) is a traditional Chinese herb used in thyroid disease and cancer. However, the clinical use of DB remains a challenge due to its hepatotoxicity, which is caused, in part, by the presence of Diosbulbin B (DIOB), a toxin commonly found in DB extracts. As abnormal expression of hepatobiliary transporters plays an important role in drug-induced liver injury, we assessed the hepatotoxicity induced by DB and DIOB, and explored their impacts on hepatobiliary transporter expression levels. Following liquid chromatography-tandem mass analysis of the DIOB content of DB extract, male ICR mice were randomly orally administered DB or DIOB for 14days. Liver injury was assessed by histopathological and biochemical analysis of liver fuction. The levels of transporter protein and mRNA were determined by western blotting and real-time PCR. Liver function and histopathological analysis indicated that both DB and DIOB could induce liver injury in mice, and that DIOB might be the primary toxic compound in DB. Moreover, down-regulation of Mrp2 blocked the excretion of bilirubin, glutathione disulfide, and bile acids, leading to the accumulation of toxic substrates in the liver and a redox imbalance. We identified down-regulated expression of Mrp2 as potential factors linked to increased serum bilirubin levels and decreased levels of glutathione in the liver and increased liver injury severity. In summary, our study indicates that down-regulation of Mrp2 represents the primary mechanism of DB- and DIOB-induced hepatotoxicity, and provides insight into novel therapies that could be used to prevent DB- and DIOB-mediated liver injury.

Metabolic-induced cytotoxicity of diosbulbin B in CYP3A4-expressing cells

As a candidate antitumor agent, diosbulbin B (DB) can induce serious liver toxicity and other adverse reactions. DB is mainly metabolized by CYP3A4 in vitro and in vivo, but the cytotoxicity and anti-tumor mechanisms of DB have yet to be clarified. This study aimed to determine whether the cytotoxicity and anti-tumor effects of DB are related to the metabolism-induced activation of CYP3A4 in various cell models, including CYP-free NIH3T3 cells, primary rat hepatocytes, HepG2 and L02 cells of high CYP3A4 expression and wild-type. Results showed that DB did not markedly decrease the viability of NIH3T3 cells. DB metabolites, obtained from the metabolism by mouse liver microsomes, did not elicit cytotoxicity on NIH3T3 cells either. By contrast, DB could induce significant cytotoxicity on primary rat hepatocytes. The DB induced cytotoxicity on HepG2 or L02 cells with high CYP3A4 expression were stronger than those on wild-type cells. As a metabolic biomarker, the metabolite conjugate (M31) of DB with GSH was detected in the incubation system. A higher amount of M31 was generated in the transfected HepG2 and L02 cells than in the wild-type cells at different time points. Ketoconazole, however, could restrain DB induced cytotoxicity on primary rat hepatocytes and in CYP3A4 transfected HepG2 and L02 cells. Therefore, the cytotoxicity of DB was closely related to CYP3A4-metabolized reactive DB metabolites.

Ferulic acid prevents liver injury induced by Diosbulbin B and its mechanism.

The rhizome of Dioscorea bulbifera Linn, traditionally used to treat thyroid disease and cancer in China, is reported to induce serious liver injury during clinical practice. Diosbulbin B (DB), a diterpene lactone, has been found to be the main toxic compound in D. bulbifera. The present study aims to investigate the protection of ferulic acid (FA) against DB-induced acute liver injury and its engaged mechanism. Mice were orally administered FA (20, 40, 80 mg/kg) once daily for 6 consecutive days; and then orally given DB (250 mg/kg) on the last day. Daily FA (40, 80 mg/kg) decreased DB (250 mg/kg)-induced increase in serum levels of alanine/aspartate aminotransferase (ALT/AST) and alkaline phosphatase (ALP). Histological evaluation showed that FA (80 mg/kg) ameliorated DB-induced hepatocellular degeneration and lymphocyte infiltration. Results of terminal dUTP nick-end labeling (TUNEL) staining assay showed that FA (80 mg/kg) decreased the DB-increased number of apoptotic hepatocytes. FA (40, 80 mg/kg) reduced DB-increased liver malondialdehyde (MDA) amount. FA (40, 80 mg/kg) decreased DB-increased serum levels of tumor necrosis factor alpha (TNF-α) and interferon-γ (IFN-γ), and liver myeloperoxidase (MPO) activity. FA (80 mg/kg) reversed the DB-induced decrease in expression of inhibitor of kappa B (IκB) and the increase in nuclear translocation of the p65 subunit of nuclear factor kappa B (NFκBp65). Taken together, our results demonstrate that FA prevents DB-induced acute liver injury via inhibiting intrahepatic inflammation and liver apoptosis.

Metabolic activation of furan moiety makes Diosbulbin B hepatotoxic.

Diosbulbin B (DIOB), a furanoid, is a major constituent of herbal medicine Dioscorea bulbifera L. Exposure to DIOB caused liver injury in humans and experimental animals. The mechanisms of DIOB-induced hepatotoxicities remain unknown. The present study demonstrated that DIOB induced hepatotoxicities in a time- and dose-dependent manner in mice. H&E stained histopathologic image showed the occurrence of necrosis in the liver obtained from the mice treated with DIOB at dose of 200mg/kg. Pretreatment with KTC protected the animals from hepatotoxicities and hepatic GSH depletion induced by DIOB, increased area under the concentration-time curve of blood DIOB, decreased urinary excretion of GSH conjugates derived from DIOB, and increased urinary excretion of parent drug. Pretreatment with BSO exacerbated DIOB-induced hepatotoxicities. In order to define the role of furan moiety in DIOB-induced liver toxicities, we replaced the furan of DIOB with a tetrahydrofuran group by chemical hydrogenation of the furan ring of DIOB. No liver injury was observed in the animals given the same doses of tetrahydro-DIOB. The furan moiety was essential for DIOB-induced hepatotoxicities. The results implicate the cis-enedial reactive metabolite of DIOB was responsible for the observed toxicities. The observed modest depletion of hepatic GSH in DIOB-treated animals suggests the actions of one or more reactive metabolites, and the hepatic injury observed could be due at least in part to reactions of these metabolites with crucial biomolecules. Cytochrome P450 3A enzymes are implicated in DIOB-induced hepatotoxicities by catalyzing the formation of the reactive metabolite of DIOB.

The HPLC–MS/MS Method for Determination of Diosbulbin B in the Plasma of Rats Administered with Rhizoma Dioscoreae Bulbiferae Combinations: Application to Comparative Pharmacokinetic Study

Diosbulbin B (DB) is a diterpenoid lactone with a remarkable anticancer effect isolated from Rhizoma Dioscoreae Bulbiferae (RDB). The present study aimed to develop a new and sensitive method for determination of DB in rats after oral administration of single herb RDB decoction and its three combinations with Radix Angelicae Sinensis (RAS). A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method with a multiple-reaction monitoring (MRM) mode was employed for quantification of DB in the rat plasma. DB and spinosin (internal standard, IS) were separated on an Xtimate C18 column (4.6 mm × 150 mm, 5 µm) using a gradient mobile phase consisting of water and acetonitrile. The calibration curve was linear over the range of 0.5 to 500 ng/mL. The lower limit of quantification was 0.5 ng/mL. The accuracy and precision of DB in the rat plasma were between −4.2% and 8.1% relative error, and 3.2% to 9.1% relative standard deviation, respectively. The fully validated method was successfully applied to comparative pharmacokinetics (PK) of DB in RDB–RAS combinations. The results showed that components in RAS had a dramatic effect on the PK pattern of DB.

Metabolism of diosbulbin B in vitro and in vivo in rats: formation of reactive metabolites and human enzymes involved.

Diosbulbin B (DB), a major constituent of the furano-norditerpenes in Dioscorea bulbifera Linn, exhibits potential antineoplasmic activity and hepatotoxicity. The metabolism and reactive metabolites of DB in vitro (with human and animal liver microsomes) and in vivo in rats were investigated. The human enzymes involved in DB metabolism were identified. DB was first catalyzed into reactive metabolites of 2-butene-1,4-dial derivatives dependent on NADPH and then trapped by Tris base or oxidized to hemiacetal lactones (M12 and M13) in microsomal incubations. Tris base was used as buffer constituent and as a trapping agent for aldehyde. Methoxylamine and glutathione (GSH) were also used as trapping agents. DB metabolism in vivo in rats after oral administration was consistent with that in vitro. The structures of M12 and M13, as well as mono-GSH conjugates of DB (M31), were confirmed by nuclear magnetic resonance spectroscopy of the chemically synthesized products. The bioactivation enzymes of DB were identified as CYP3A4/5, 2C9, and 2C19. CYP3A4 was found to be the primary enzyme using human recombinant cytochrome P450 enzymes, specific inhibitory studies, and a relative activity factor approach for pooled human liver microsomes. Michaelis-Menten constants K(m) and V(max) were determined by the formation of M31. The reactive metabolites may be related to the hepatotoxicity of DB. The gender difference in CYP3A expression in mice and rats contributed to the gender-related liver injury and pharmacokinetics in mice and rats, respectively.

Cytochrome p450-mediated metabolic activation of diosbulbin B.

Diosbulbin B (DIOB), a furan-containing diterpenoid lactone, is the most abundant component of Dioscorea bulbifera L. (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 this 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 (M1-M4) associated with GSH were detected by liquid chromatography coupled to tandem mass spectrometry. Apparently, M1 was derived from both NAL and GSH. M2 and M3 resulted from the reaction of GSH without the involvement of NAL. Two molecules of GSH participated in the formation of M4. M2 and M3 were also detected in bile and urine of rats given DIOB. M5, a DIOB-derived NAC/NAL conjugate, was detected in microsomal incubations with DIOB fortified with NAC and NAL as trapping agents. Biomimetic M1-M5 were prepared by oxidation of DIOB with Oxone for metabolite identification. Microsomal incubation study demonstrated that ketoconazole inhibited the production of the enedial in a concentration-dependent manner, and CYP3A4 was found to be the enzyme responsible for the metabolic activation of DIOB. The metabolism study facilitates the understanding of the role of bioactivation of DIOB in its hepatotoxicity.

Diosbulbin B-induced liver injury in mice and its mechanism

Dioscorea bulbifera L., a commonly used medicinal plant in China, is reported to induce hepatotoxicity. The present study is undertaken to investigate the hepatotoxicity induced by diosbulbin B (DB), a diterpene lactone isolated from D. bulbifera L., and to further explore its underlying mechanism. DB was administered to mice at the doses of 0, 16, 32, and 64 mg/kg once daily for 12 consecutive days. Liver injury induced by DB was evidenced by the increased activity of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Liver histological evaluation showed that the mice treated with DB exhibited liver damage with the swelling of hepatocytes. Further results showed that the amount of malondialdehyde (MDA) in the liver was increased in mice treated with DB, while the glutathione amount and the enzymatic activity of glutathione peroxidase (GPx), glutathione-S-transferase (GST), copper/zinc-superoxide dismutase (CuZn-SOD), manganese-SOD (Mn-SOD), and catalase (CAT) were all decreased. DB also decreased the gene expression of CuZn-SOD and CAT. Taken together, our results indicate that oral administration of DB for 12 consecutive days can lead to the oxidative stress liver injury in mice.