Psoralen and Isopsoralen, Two Estrogen-Like Natural Products from Psoraleae Fructus, Induced Cholestasis via Activation of ERK1/2.

With the increasing use of oral contraceptives and estrogen replacement therapy, the incidence of estrogen-induced cholestasis (EC) has tended to rise. Psoralen (P) and isopsoralen (IP) are the major bioactive components in Psoraleae Fructus, and their estrogen-like activities have already been recognized. Recent studies have also reported that ERK1/2 plays a critical role in EC in mice. This study aimed to investigate whether P and IP induce EC and reveal specific mechanisms. It was found that P and IP increased the expression of esr1, cyp19a1b and the levels of E2 and VTG at 80 µM in zebrafish larvae. Exemestane (Exe), an aromatase antagonist, blocked estrogen-like activities of P and IP. At the same time, P and IP induced cholestatic hepatotoxicity in zebrafish larvae with increasing liver fluorescence areas and bile flow inhibition rates. Further mechanistic analysis revealed that P and IP significantly decreased the expression of bile acids (BAs) synthesis genes cyp7a1 and cyp8b1, BAs transport genes abcb11b and slc10a1, and BAs receptor genes nr1h4 and nr0b2a. In addition, P and IP caused EC by increasing the level of phosphorylation of ERK1/2. The ERK1/2 antagonists GDC0994 and Exe both showed significant rescue effects in terms of cholestatic liver injury. In conclusion, we comprehensively studied the specific mechanisms of P- and IP-induced EC and speculated that ERK1/2 may represent an important therapeutic target for EC induced by phytoestrogens.

Evidence for exposure biomarkers in dictamnine-induced liver injury resulting from metabolic activation in vitro and in mice.

Dictamnine (DTN), a furoquinoline alkaloid isolated from Dictamni Cortex, is responsible for the liver injury caused by Dictamni Cortex and the preparations. Discovering new biomarkers with high specificity and sensitivity for diagnosis and tracing the source of DTN-induced liver injury is urgently needed. Considering that metabolic activation of DTN has been suggested as a primary trigger initiating hepatotoxicity, the present study aimed to investigate the bio-activation process of DTN in vitro and in mice and to explore whether the adducts could be developed as exposure biomarkers. When trapping with N-acetyl-cysteine (NAC) and glutathione (GSH) in mouse liver microsomes and CYP3A4 overexpressed L02 cells, two isomers of DTN-NAC adducts were detected in both systems and one DTN-GSH adduct was found in mouse liver microsomes. As expected, one DTN-NAC adduct was also found in plasma and bile of mice with liver injury after DTN exposure. Moreover, mouse liver microsomes were used to simulate the conjugation of serum albumin with metabolically activated DTN. The sole modified peptide 25 DAHKSEVAHR34 was found, and the oxidative metabolites of DTN might bind to the side chain amino of albumin at Arg34. The above findings not only provided confirmative evidence that DTN was metabolically activated to induce liver injury but also suggested that the adducts had the potential to be developed as exposure biomarkers of DTN-induced liver injury.

Integrated Metabolomics and Proteomics Approach To Identify Metabolic Abnormalities in Rats with Dioscorea bulbifera Rhizome-Induced Hepatotoxicity.

Previous studies have shown that Dioscorea bulbifera rhizome (DBR) can induce hepatotoxicity in clinical practice. However, its underlying mechanisms remain largely unexplored. In the present study, we investigated the global effect of DBR exposure on the proteomic and metabolomic profiles in rats over a 12-week administration using an integrated proteomics and metabolomics approach. The abundance of 1366 proteins and 58 metabolites in the liver of rats after subchronic exposure to DBR was dose-dependently altered. The results indicated that DBR mainly damaged hepatic cells through the aberrant regulation of multiple systems mainly including purine metabolism, pyrimidine metabolism, taurine and hypotaurine metabolism, and bile acid metabolism. Notably, the deregulated proteins including Pnp, Dpyd, Upp1, and Tymp and the differential metabolites including uridine, uracil, cytidine, thymine, adenine, adenosine, adenosine 3'-monophosphate, and deoxycytidine were well correlated to purine and pyrimidine metabolism, which might be novel pathways involved in metabolic abnormalities in rats with DBR-induced liver damage. Collectively, these findings not only contributed to understanding the mechanisms underlying the hepatotoxicity of DBR, but also illustrated the power of integrated proteomics and metabolomics approaches to improve the identification of metabolic pathways and biomarkers indicative of herb-induced liver injury.

Investigation of Dioscorea bulbifera Rhizome-Induced Hepatotoxicity in Rats by a Multisample Integrated Metabolomics Approach.

The use of herbal medicines continues to expand globally, meanwhile, herb-associated hepatotoxicity is becoming a safety issue. As a conventional Chinese medicinal herb, Dioscorea bulbifera rhizome (DBR) has been documented to cause hepatic toxicity. However, the exact underlying mechanism remains largely unexplored. In the present study, we aimed to profile entire endogenous metabolites in a biological system using a multisample integrated metabolomics strategy. Our findings offered additional insights into the molecular mechanism of the DBR-induced hepatotoxicity. We identified different metabolites from rat plasma, urine, and feces by employing gas chromatography-mass spectrometry in combination with multivariate analysis. In total, 55 metabolites distributed in 33 metabolic pathways were identified as being significantly altered in DBR-treated rats. Correlation network analysis revealed that the hub metabolites of hepatotoxicity were mainly associated with amino acid, bile acid, purine, pyrimidine, lipid, and energy metabolism. As such, DBR affected the physiological and biological functions of liver via the regulation of multiple metabolic pathways to an abnormal state. Notably, our findings also demonstrated that the multisample integrated metabolomics strategy has a great potential to identify more biomarkers and pathways in order to elucidate the mechanistic complexity of toxicity of traditional Chinese medicine.

[Absorption mechanism of neobavaisoflavone in Caco-2 cell monolayer mode].

Neobavaisoflavone is one of flavonoids of traditional Chinese medicine Psoralea corylifolial. It has numerous biological properties such as antibacterial, anti-inflammatory, anti-cancer, and anti-osteoporosis effects. This paper aimed to investigate the absorption mechanism of neobavaisoflavone in Caco-2 cell monolayer model. The analyte and osalmide were separated on Thermo Syncronis C18 column with methanol-0.1% formic acid solution (90∶10) as the mobile phase, at a flow rate of 0.2 mL•min⁻¹. The concentration of neobavaisoflavone was determined in eletrospray ionization(ESI) positive ion mode with osalmide as an the internal standard. The effects of time, concentration, P-gp inhibitor verapamil, MRP-2 inhibitor MK-571 and BCRP inhibitor Ko143 on the absorption of neobavaisoflavone were investigated. According to the results, neobavaisoflavone showed a good linearity within the concentration of 10-2 000 µg•L⁻¹, and the results of its specificity, matrix effect, extraction recovery, precision, accuracy and stability all met the requirements. In the Caco-2 cell monolayer model, the transport volume of neobavaisoflavone was correlated positively with the time and concentration. The ER values of 15, 30, 50 µmol•L⁻¹ neobavaisoflavone were 1.64, 1.94,0.99, respectively. As compared with the control group, all of verapamil hyduochloride, MK-571 and Ko143 could promote the transportation of neobavaisoflavone, and the effect was more obvious in verapamil hyduochloride and Ko143. The absorption of neobavaisoflavone may be mainly of active transport in Caco-2 cell monolayer model, and also involve passive transport. Excretion mechanism of intestinal transport protein may be also involved.

[Effect and Mechanism of Foliar Application Nano-MnO2 on Cadmium Enrichment of Rice].

Cadmium (Cd) is easily enriched in rice, resulting in an excessive Cd content in the grain, which seriously threatens human health. Manganese (Mn) is an essential element of plants. In a field experiment on Cd-contaminated acid soils, we investigated the effectiveness and mechanism of Mn in minimizing Cd accumulation in rice via foliar spraying using 0.1%, 0.3%, and 0.5% nano-MnO2 solutions at an early stage of heading. Compared with a control treatment, foliar spraying effectively reduced the Cd content of rice leaves, husks, and brown rice; increased the Mn content of all rice organs; but had no effect on rice yield. Foliar application of nano-MnO2 alleviated the plant stress induced by Cd by improving leaf photosynthesis, inhibiting lipid peroxidation, and increasing the content of the oxidative stress protein kinase. In addition, foliar application of nano-MnO2 limited the absorption of Cd by roots by increasing the content of iron-manganese plaque on the surfaces of rice roots and strengthening its adsorption/co-precipitation of Cd. Therefore, foliar application of nano-MnO2 during the early stages of rice heading is an effective measure to increase the Mn content and reduce the Cd content of brown rice.

Integration of transcriptomics and metabolomics profiling reveals the metabolic pathways affected in dictamnine-induced hepatotoxicity in mice.

Dictamnine (DTN), a major furoquinoline alkoloid from Dictamni Cortex, was reported to induce hepatotoxicity. However, the underlying mechanism is unclear. In the present study, integrated transcriptomic and metabolomics analysis of mouse liver was performed in combination with serum biochemical and histopathological evaluation to investigate the potential mechanism. The results suggested that 640 mg/kg DTN significantly increased serum alanine transaminase and aspartate transaminase levels and induced serious cellular degeneration, with no changes occurring with 4 mg/kg DTN. Integrated analysis suggested that the metabolism of xenobiotics by cytochrome P450, drug metabolism-other enzymes, bile secretion and glutathione metabolism were the major metabolic pathways involved in DTN-induced hepatotoxicity. Notably, 640 mg/kg DTN exposure increased hepatic GSH, GSH peroxidase, superoxide dismutase and malondialdehyde, and decreased ROS, together with altered expression of Idh2 and Nedd9. Representative genes, including Mup12, Lipc, NTCP, MRP3, MRP4, CYP2E1, CYP2D9 and UGT1A9, in altered pathways were verified through PCR and Western blot. Collectively, the combined strategy of transcriptomics and metabolomics profiling could facilitate a better understanding for the discovery of metabolic pathways and that oxidative damage, ABC transporters and lipid metabolism might be the mechanisms linked to DTN-induced hepatotoxicity in mice. SIGNIFICANCE: Dictamnine (DTN) was reported to induce hepatotoxicity. Nevertheless, the underlying mechanism is unknown. This study is the first to utilize integrated transcriptomics and metabolomics in combination with general toxicity evaluation to characterize the potential molecular mechanism in DTN-induced hepatotoxicity in mice. We found that acute exposure to higher dose of DTN induced hepatocellular liver injury with more changes in biochemical parameters, genes and metabolites. Gene expression and metabolite profiles were more sensitive than general toxicity studies for detecting earlier hepatotoxicity. Integrated analysis suggested that oxidative damage, ABC transporters and lipid metabolism were closely correlated with DTN-induced hepatotoxicity. Overall, our results provide insights into the mechanism responsible for DTN-induced hepatotoxicity.

The crucial role of metabolic regulation in differential hepatotoxicity induced by furanoids in Dioscorea bulbifera.

Diterpenoid lactones (DLs), a group of furan-containing compounds found in Dioscorea bulbifera L. (DB), have been reported to be associated with hepatotoxicity. Different hepatotoxicities of these DLs have been observed in vitro, but reasonable explanations for the differential hepatotoxicity have not been provided. Herein, the present study aimed to confirm the potential factors that contribute to varied hepatotoxicity of four representative DLs (diosbulbins A, B, C, F). In vitro toxic effects were evaluated in various cell models and the interactions between DLs and CYP3A4 at the atomic level were simulated by molecular docking. Results showed that DLs exhibited varied cytotoxicities, and that CYP3A4 played a modulatory role in this process. Moreover, structural variation may cause different affinities between DLs and CYP3A4, which was positively correlated with the observation of cytotoxicity. In addition, analysis of the glutathione (GSH) conjugates indicated that reactive intermediates were formed by metabolic oxidation that occurred on the furan moiety of DLs, whereas, GSH consumption analysis reflected the consistency between the reactive metabolites and the hepatotoxicity. Collectively, our findings illustrated that the metabolic regulation played a crucial role in generating the varied hepatotoxicity of DLs.

Metabolomic-transcriptomic landscape of 8-epidiosbulbin E acetate -a major diterpenoid lactone from Dioscorea bulbifera tuber induces hepatotoxicity.

Studies have shown that 8-epidiosbulbin E acetate (EEA), a major diterpenoid lactone in the tuber of Dioscorea bulbifera, can induce hepatotoxicity in vivo. However, the underlying mechanisms remain unknown. Using the integrated transcriptomic and metabolomics method, in this study we investigated the global effect of EEA exposure on the transcriptomic and metabolomic profiles in mice. The abundance of 7131 genes and 42 metabolites in the liver, as well as 43 metabolites in the serum were altered. It should be noted that EEA mainly damaged hepatic cells through the aberrant regulation of multiple systems primarily including bile acid metabolism, and taurine and hypotaurine metabolism. In addition, an imbalance of bile acid metabolism was found to play a key pat in response to EEA-triggered hepatotoxicity. In summary, these findings contributed to understanding the underlying mechanisms of EEA hepatotoxicity.

Transplantation of a Novel Recombinant Adeno-Associated Virus (pAAV-HE1B19K-TE1A) Demonstrates Higher Anti-Tumor Effects in Tumor Cells.

BACKGROUND Oncolytic viruses (OVs) can specifically infect and kill tumor cells. Adeno-associated virus (AAV) is a widely-studied OV. This study aimed to construct a tumor-targeted recombinant AAV using genetic engineering technology. MATERIAL AND METHODS The transgene plasmid pAAV-HE1B19K-TE1A was constructed with 4 genes (hTERT, E1A, HKII, and E1B19K) and co-transfected with pAAV-RC and pHelper to tumor cells (HepG2, A549, BGC-803) and normal cells (HUVEC). rAAV was verified with fluorescence microscopy. Quantitative PCR (qPCR) assay was used to test the titer of rAAV in each cell line. Apoptosis was analyzed using qPCR and Western blot assay. MTT was used to detect the effect of rAAV on cell viability. RESULTS The pAAV-HE1B19K-TE1A transgene plasmid was successfully structured. pAAV-HE1B19K-TE1A was highly expressed in all tumor cells. The titers of pAAV-HE1B19K-TE1A in HepG2, A549, and BGC-803 were 7.4×107, 1.4×108, and 1.1×108 gc/µl, respectively. pAAV-HE1B19K-TE1A significantly decreased cell viability of tumor cells compared to that in HUVEC (p<0.05). pAAV-HE1B19K-TE1A remarkably triggered cleaved caspase 3 (C-caspase 3) activity in tumor cells compared to that in untransfected tumor cells (p<0.05). pAAV-HE1B19K-TE1A significantly induced release of cytochrome C (Cyto C) in tumor cells compared to that in untransfected tumor cells (p<0.05). pAAV-HE1B19K-TE1A demonstrated no toxicity to vital tissues of animals. CONCLUSIONS Tumor-targeted rAAV was successfully produced using the Helper-free system with recombinant plasmid, demonstrating high efficacy in decreasing viability of tumor cells without adverse effects on normal cells.

Liver-specific metabolomics characterizes the hepatotoxicity of Dioscorea bulbifera rhizome in rats by integration of GC-MS and 1H-NMR.

ETHNOPHARMACOLOGICAL RELEVANCE: Dioscorea bulbifera rhizome (DBR), one type of herbal medicine, is extensively used in both Indian and Chinese system of traditional medicine. It has been effective in treating various diseases, such as sore throat, struma, and tumors. However, more and more clinical investigations have suggested that DBR can cause liver injury. AIM OF THE STUDY: In the present study, we aimed to characterize the corresponding molecular changes of liver dysfunction and reveal overall metabolic and physiological mechanisms of the subchronic toxic effect of DBR. MATERIALS AND METHODS: A liver-specific metabolomics approach integrating GC-MS and 1H-NMR was developed to assess the hepatotoxicity in rats after DBR exposure for 12 weeks. Multivariate statistical analysis and pattern recognition were employed to examine different metabolic profiles of liver in DBR-challenged rats. RESULTS: A total of 61 metabolites were screened as significantly altered metabolites, which were distributed in 43 metabolic pathways. The correlation network analysis indicated that the hub metabolites of hepatotoxicity could be mainly linked to amino acid, lipid, purine, pyrimidine, bile acid, gut microflora, and energy metabolisms. Notably, purine, pyrimidine, and gut microflora metabolisms might be novel pathways participating in metabolic abnormalities in rats with DBR-triggered hepatic damage. CONCLUSIONS: Our results primarily showed that the liver-specific metabolic information provided by the different analytical platforms was essential for identifying more biomarkers and metabolic pathways, and our findings provided novel insights into understand the mechanistic complexity of herb-induced liver injury.

The Modulatory Role of CYP3A4 in Dictamnine-Induced Hepatotoxicity.

Dictamni Cortex (DC) has been reported to be associated with acute hepatitis in clinic and may lead to a selective sub-chronic hepatotoxicity in rats. Nevertheless, the potent toxic ingredient and the underlying mechanism remain unknown. Dictamnine (DTN), the main alkaloid from DC, possesses a furan ring which was suspected of being responsible for hepatotoxicity via metabolic activation primarily by CYP3A4. Herein, the present study aimed to evaluate the role of CYP3A4 in DTN-induced liver injury. The in vitro results showed that the EC50 values in primary human hepatocytes (PHH), L02, HepG2 and NIH3T3 cells were correlated with the CYP3A4 expression levels in corresponding cells. Furthermore, the toxicity was increased in CYP3A4-induced PHH by rifampicin, and CYP3A4 over-expressed (OE) HepG2 and L02 cells. Contrarily, the cytotoxicity was decreased in CYP3A4-inhibited PHH and CYP3A4 OE HepG2 and L02 cells inhibited by ketoconazole (KTZ). In addition, the hepatotoxicity of DTN in enzyme induction/inhibition mice was further investigated in the aspects of biochemistry, histopathology, and pharmacokinetics. Administration of DTN in combination with KTZ resulted in attenuated liver injury, including lower alanine transaminase and aspartate transaminase activities and greater AUC and C max of serum DTN, whereas, pretreatment with dexamethasone aggravated the injury. Collectively, our findings illustrated that DTN-induced hepatotoxicity correlated well with the expression of CYP3A4, namely inhibition of CYP3A4 alleviated the toxicity both in vitro and in vivo, and induction aggravated the toxicity effects.

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.