A new macrocyclic diterpenoid from Anisomeles indica.

A new macrocyclic diterpenoid, 4ß,5ß-dihydroxyovatodiolide (1), together with twenty-two known compounds (2-23) were isolated from the MeOH extract of the dried aerial parts of Anisomeles indica (L.) O. Kuntze (Labiatae). The structure of 1 was established on the basis of spectral evidence. Phenylethanoids, acteoside (5) and isoacteoside (6) showed significant inhibitory to IL-2 secretion of with respect to phorbol myristate acetate and anti-CD28 monoclonal antibody co-stimulated activation of human peripheral blood T cells.

Astragaloside VI and cycloastragenol-6-O-beta-D-glucoside promote wound healing in vitro and in vivo.

BACKGROUND: Astragalus genus includes most of the common, historical herbal medicines that have various applications in Asian countries. However, clinical data and mechanistic insights into their actions are still lacking. PURPOSE: In this study, we aimed to examine the effects of astragalosides on wound healing in vitro and in vivo, as well as the underlying mechanisms of these actions. METHODS: The wound healing activity of astragalosides was investigated in human HaCaT keratinocytes, human dermal fibroblast (HDF) cells, and murine models of wound healing. RESULTS: All eight astragalosides studied enhanced epidermal growth factor receptor (EGFR) activity in HaCaT cells. Among them, astragaloside VI (AS-VI) showed the strongest EGFR activation. Consistently, AS-VI and cycloastragenol-6-O-beta-D-glucoside (CMG), which is the major metabolite of astragalosides, enhanced extracellular signal-regulated kinase (ERK) activity in a concentration-dependent manner. In agreement, both compounds induced EGFR-dependent cell proliferation and migration in HaCaT and HDF cells. In addition, we showed that AS-VI and CMG accelerated the healing of both sterile and infected wounds in vivo. These effects were associated with increased angiogenesis in the scar tissue. CONCLUSION: AS-VI and CMG increased the proliferation and migration of skin cells via activation of the EGFR/ERK signalling pathway, resulting in the improvement of wound healing in vitro and in vivo. These findings indicate the therapeutic potential of AS-VI and CMG to accelerate wound healing; additionally, they suggest the mechanistic basis of this activity.

Danshen extract circumvents drug resistance and represses cell growth in human oral cancer cells.

BACKGROUND: Danshen is a common traditional Chinese medicine used to treat neoplastic and chronic inflammatory diseases in China. However, the effects of Danshen on human oral cancer cells remain relatively unknown. This study investigated the antiproliferative effects of a Danshen extract on human oral cancer SAS, SCC25, OEC-M1, and KB drug-resistant cell lines and elucidated the possible underlying mechanism. METHODS: We investigated the anticancer potential of the Danshen extract in human oral cancer cell lines and an in vivo oral cancer xenograft mouse model. The expression of apoptosis-related molecules was evaluated through Western blotting, and the concentration of in vivo apoptotic markers was measured using immunohistochemical staining. The antitumor effects of 5-fluorouracil and the Danshen extract were compared. RESULTS: Cell proliferation assays revealed that the Danshen extract strongly inhibited oral cancer cell proliferation. Cell morphology studies revealed that the Danshen extract inhibited the growth of SAS, SCC25, and OEC-M1 cells by inducing apoptosis. The Flow cytometric analysis indicated that the Danshen extract induced cell cycle G0/G1 arrest. Immunoblotting analysis for the expression of active caspase-3 and X-linked inhibitor of apoptosis protein indicated that Danshen extract-induced apoptosis in human oral cancer SAS cells was mediated through the caspase pathway. Moreover, the Danshen extract significantly inhibited growth in the SAS xenograft mouse model. Furthermore, the Danshen extract circumvented drug resistance in KB drug-resistant oral cancer cells. CONCLUSION: The study results suggest that the Danshen extract could be a potential anticancer agent in oral cancer treatment.

Astragaloside II promotes intestinal epithelial repair by enhancing L-arginine uptake and activating the mTOR pathway.

Astragaloside II (AS II) extracted from Astragalus membranaceus has been reported to promote tissue wound repair. However, the effect of AS II on inflammatory bowel disease is unknown. We investigated the effects and mechanism of AS II on intestinal wound healing in both in vitro and in vivo models. Human intestinal Caco-2 cells were treated with multiple concentrations of AS II to assess cell proliferation, scratch wound closure, L-arginine uptake, cationic amino acid transporter activity, and activation of the mTOR signaling pathway. These effects were also measured in a mouse model of colitis. AS II promoted wound closure and increased cell proliferation, L-arginine uptake, CAT1 and CAT2 protein levels, total protein synthesis, and phosphorylation of mTOR, S6K, and 4E-BP1 in Caco-2 cells. These effects were suppressed by lysine or rapamycin treatment, suggesting that the enhanced arginine uptake mediates AS II-induced wound healing. Similar results were also observed in vivo. Our findings indicate that AS II can contribute to epithelial barrier repair following intestinal injury, and may offer a therapeutic avenue in treating irritable bowel disease.

Antiangiogenic activity of phthalides-enriched Angelica Sinensis extract by suppressing WSB-1/pVHL/HIF-1α/VEGF signaling in bladder cancer.

The hypoxia-inducible factor-1α (HIF-1α) plays a critical role in tumor angiogenesis. It has been reported that the acetone extract of Angelica sinensis (AE-AS) rich in phthalides is able to inhibit cancer cell proliferation. However, whether AE-AS reduces cancer angiogenesis remains unknown. In this study, we demonstrated that AE-AS significantly inhibited the angiogenesis in vitro and in vivo evidenced by attenuation of the tube formation in hypoxic human umbilical vascular endothelial cells (HUVECs), and the vasculature generation in Matrigel plug, the chicken chorioallantoic membrane, and tumors. Treatment with AE-AS markedly decreased the protein accumulation and transcriptional activity of HIF-1α, vascular endothelial growth factor (VEGF) expression/secretion, and VEGFR2 phosphorylation in hypoxic human bladder cancer (T24) cells and tumor tissues accompanied by a reduction of tumor growth. Notably, AE-AS-induced HIF-1α protein degradation may, at least partly, attribute to inhibition of WSB-1-dependent pVHL degradation. Moreover, VEGFR2-activated PI3K/AKT/mTOR signaling pathway in hypoxic T24 cells was greatly inhibited by AE-AS. Collectively, AE-AS may be a potential anticancer agent by attenuating cancer angiogenesis via suppression of WSB-1/pVHL/HIF-1α/VEGF/VEGFR2 cascade.

In Vitro Anticancer Activity and Structural Characterization of Ubiquinones from Antrodia cinnamomea Mycelium.

Two new ubiquinones, named antrocinnamone and 4-acetylantrocamol LT3, were isolated along with six known ubiquinones from Antrodia cinnamomea (Polyporaceae) mycelium. The developed HPLC analysis methods successfully identified eight different ubiquinones, two benzenoids, and one maleic acid derivative from A. cinnamomea. The ubiquinones 1-8 exhibited potential and selective cytotoxic activity against three human cancer cell lines, with IC50 values ranging from 0.001 to 35.883 µM. We suggest that the different cytotoxicity levels were related to their chemical structures, especially the 4-hydroxycyclohex-2-enone ring and the presence of a free hydroxyl group in the side chain. The suppression by 4-acetylantrocamol LT3 stopped the cell cycle at the beginning of the G2-M phase thus making the cell cycle arrest at the sub-G1 phase as compared with control cells.

Sesquiterpenes from Baizhu Stimulate Glucose Uptake by Activating AMPK and PI3K.

Baizhu, the dried rhizome of Atractylodes Macrocephala Koidz (Compositae), is one of the most important traditional Chinese herbal medicines. Baizhu is generally used to treat digestive disorders and diabetes in Asian countries. This study investigates the activity of two sesquiterpenes isolated from Baizhu, atractylenolide I (AT-I) and atractylenolide II (AT-II), for their effects on glucose uptake in mouse skeletal muscle C2C12 cells, and the corresponding mechanism. These compounds show a significant stimulatory effect on glucose uptake in C2C12 myotubes. Both AT-I and AT-II significantly increased GLUT4 but not GLUT1 protein levels, and promoted GLUT4 translocation to the plasma membrane. The increased glucose uptake induced by these compounds is associated with activation of AMP-activated protein kinase (AMPK) and PI3K/Akt pathways in these cells. Further studies have indicated that AT-I and AT-II ameliorate TNF-[Formula: see text]-induced insulin resistance in C2C12 myotubes. In summary, our findings highlight the insulin mimetic activity of Baizhu in myotubes, and provide insights into the action mechanism underlying these effects. Our findings may also prove highly relevant to the development of novel therapeutic applications for these compounds.

Corrigendum to "Ferulic Acid, an Angelica sinensis-Derived Polyphenol, Slows the Progression of Membranous Nephropathy in a Mouse Model".

[This corrects the article DOI: 10.1155/2012/161235.].

Effects of Salvia miltiorrhiza ethanolic extract on lipopolysaccharide-induced dental alveolar bone resorption in rats.

BACKGROUND/PURPOSE: Salvia miltiorrhiza (SM) Bunge (Labiatae/Lamiaceae; common name danshen) is a Chinese medicine that improves blood circulation and inhibits inflammatory response. Thus, it is used for the treatment of cardiac diseases and inflammation. In this study, we aimed to evaluate the effect of an ethanolic extract of SM (SME) on the dental alveolar bone resorption induced by bacterial lipopolysaccharide (LPS) in rats. MATERIALS AND METHODS: An ethanolic extract was prepared from roots of SM. The major constituents of this extract were determined by high-performance liquid chromatography. The activity of the extract was evaluated in a rat model in which the dental alveolar bone resorption was induced by injection of bacterial LPS into the palatal gingiva around the maxillary molar teeth. The effect of SME on the bone resorption was studied by histologic and histomorphometric analysis. RESULTS: The number of osteoclasts and the percentage of osteoclasts covering the alveolar bone surfaces were significantly increased in the LPS group compared with those in the phosphate-buffered saline (PBS) group. The number and percentage of the osteoclasts on the bony surfaces were significantly reduced in the SME group in comparison with the LPS group, although it was still higher than the numbers observed in the PBS group. CONCLUSION: Because SME reduced bone resorption caused by the injections of bacterial LPS in rats, we suggest that SME might have a protective effect on dental alveolar bone resorption in periodontitis.

[Correlation between the expression of Pim-1 and androgen-deprivation therapy for prostate cancer].

OBJECTIVE: To investigate the expression of the Pim-1 gene in the LNCaP cells of the animal model of orthotopically implanted prostate cancer by surgical castration simulating androgen-deprivation therapy. METHODS: We equally allocated 32 male BALBc-nu mice into 4 groups, androgen-dependent prostate cancer (ADPC), androgen-deprivation therapy (ADT) , castration-resistant prostate cancer (CRPC) and blank control, and established the models of orthotopically implanted tumor using human prostate cancer LNCaP cells. We detected and ,compared the expressions of Pim-1, PSA, and androgen receptor (AR) in the tumor tissues of different groups by RT-PCR. qRT-PCR, ELSIA and immunohistochemistry. RESULTS: The relative gray scales in the ADPC and CRPC groups were 0.59 ± 0.01 and 1.14 ± 0.02, with statistically significant differences from 0.62 ± 0.03 in the ADT group (P < 0.05), and the Δ Ct values of Pim-1 were 6.15 ± 0.34 and 4.56 ± 0.23 in the former two groups, also with significant differences from 5.11 ± 0.21 in the latter (P < 0.05). The results of 2-ΔΔ Ct relative quantification analysis showed that the amplification products of Pim-1 in the ADT and CRPC groups increased 2.05 and 3.01 times respectively that of the ADPC group. The concentration of PSA was significantly higher in the ADPC ([480 ± 25] pg/ml) and CRPC ([870 ± 23] pg/ml) than in the ADT ([170 ± 32] pg/ml) and blank control groups (0 µg/L) (P < 0.01). The mean optical densities of Pim-1 and AR proteins were 0.017 ± 0.002 and 0.032 ± 0.009 in the ADPC group and 0.024 ± 0.002 and 0.040 ± 0.011 in the CRPC group, both with significant differences from those in the ADT group (0.018 ± 0.001 and 0.019 ± 0.006) (P < 0.01). CONCLUSION: Pim-1 is highly expressed in nude mice with prostate cancer receiving androgen-deprivation therapy and plays an important role in the progression and metastasis of prostate cancer.

An Essential Role of cAMP Response Element Binding Protein in Ginsenoside Rg1-Mediated Inhibition of Na+/Glucose Cotransporter 1 Gene Expression.

The Na(+)/glucose cotransporter 1 (SGLT1) is responsible for glucose uptake in intestinal epithelial cells. It has been shown that the intestinal SGLT1 level is significantly increased in diabetic individuals and positively correlated with the pathogenesis of diabetes. The development of targeted therapeutics that can reduce the intestinal SGLT1 expression level is, therefore, important. In this study, we showed that ginsenoside Rg1 effectively decreased intestinal glucose uptake through inhibition of SGLT1 gene expression in vivo and in vitro. Transient transfection analysis of the SGLT1 promoter revealed an essential cAMP response element (CRE) that confers the Rg1-mediated inhibition of SGLT1 gene expression. Chromatin immunoprecipitation assay and targeted CRE-binding protein (CREB) silencing demonstrated that Rg1 reduced the promoter binding of CREB and CREB binding protein associated with an inactivated chromatin status. In addition, further studies showed that the epidermal growth factor receptor (EGFR) signaling pathway also plays an essential role in the inhibitory effect of Rg1; taken together, our study demonstrates the involvement of the EGFR-CREB signaling pathway in the Rg1-mediated downregulation of SGLT1 expression, which offers a potential strategy in the development of antihyperglycemic and antidiabetic treatments.

An essential role of cAMP response element-binding protein in epidermal growth factor-mediated induction of sodium/glucose cotransporter 1 gene expression and intestinal glucose uptake.

The sodium/glucose cotransporter 1 (SGLT1) is responsible for glucose uptake in intestinal epithelial cells. Its expression is decreased in individuals with intestinal inflammatory disorders and is correlated with the pathogenesis of disease. The aim of this study was to understand the regulatory mechanism of the SGLT1 gene. Using the trinitrobenzene sulfonic acid-induced mouse models of intestinal inflammation, we observed decreased SGLT1 expression in the inflamed intestine was positively correlated with the mucosal level of epidermal growth factor (EGF) and activated CREB. Overexpression of EGF demonstrated that the effect of EGF on intestinal glucose uptake was primarily due to the increased level of SGLT1. We identified an essential cAMP binding element (CRE) confers EGF inducibility in the human SGLT1 gene promoter. ChIP assay further demonstrated the increased binding of CREB and CBP to the SGLT1 gene promoter in EGF-treated cells. In addition, the EGFR- and PI3K-dependent CREB phosphorylations are involved in the EGF-mediated SGLT1 expression. This is the first report to demonstrate that CREB is involved in EGF-mediated transcription regulation of SGLT1 gene in the normal and inflamed intestine, which can provide potential therapeutic applications for intestinal inflammatory disorders.

Anti-cancer agents derived from solid-state fermented Antrodia camphorata mycelium.

Three new ubiquinone derivatives, antrocamol LT1, antrocamol LT2, and antrocamol LT3, along with two known compounds, were isolated from Antrodia camphorata (Polyporaceae) mycelium. The structures of these compounds were established on the basis of extensive 1D and 2D NMR spectroscopic analyses. These ubiquinones exhibited selective cytotoxicities against five human cancer cell lines (CT26, A549, HepG2, PC3 and DU-145) with IC50 values ranging from 0.01 to 1.79µΜ.

A gut microbial metabolite of ginsenosides, compound K, induces intestinal glucose absorption and Na(+) /glucose cotransporter 1 gene expression through activation of cAMP response element binding protein.

SCOPE: The Na(+) /glucose cotransporter 1 (SGLT1) plays a crucial role in glucose uptake in intestinal epithelial cells (IECs), which has been shown essential in ameliorating intestinal inflammation. Ginseng has historically been used to treat inflammatory disorders. Understanding the regulatory mechanism of ginseng-mediated induction of SGLT1 gene expression in human intestinal cells is therefore important. METHODS AND RESULTS: We demonstrate that ginsenoside compound K (CK) enhances SGLT1-mediated glucose uptake in mice and human intestinal Caco-2 cells. Transient transfection analysis using SGLT1 promoter-luciferase reporters demonstrated that the presence of an essential cAMP response element (CRE) is required for CK-mediated induction of SGLT1 gene expression. The ChIP assays indicated that increased CRE-binding protein (CREB) and CREB-binding protein (CBP) binding to the SGLT1 promoter in CK-treated cells is associated with an activated chromatin state. Our result showed that the increased CREB phosphorylation is directly correlated with SGLT1 expression in IECs. Further studies indicated that the epidermal growth factor receptor (EGFR) signaling pathway is involved in the CK-mediated effect. CONCLUSION: These findings provide a novel mechanism for the CK-mediated upregulation of SGLT1 expression through EGFR-CREB signaling activation, which could contribute to reducing gut inflammation.

Cytotoxic effect of triterpenoids from the root bark of Hibiscus syriacus.

In this study, 4 new triterpenoids-3ß- acetoxy-olean-11-en,28,13ß-olide (1), 3ß- acetoxy-11α,12α-epoxy-olean-28,13ß-olide (2), 19α-epi-betulin (3), and 20, 28-epoxy-17ß,19ß-lupan-3ß-ol (4)-and 12 known compounds, were isolated from the root bark of Hibiscus syriacus L. by using acetone extraction. Their structures were characterized by extensive spectroscopic analysis. To investigate cytotoxicity, A549 human lung cancer cells were exposed to the extract and the compounds identified from it. Significantly reduced cell viability was observed with betulin-3-caffeate (12) (IC50, 4.3 µM). The results of this study indicate that betulin-3-caffeate (12) identified from H. syriacus L. may warrant further investigation for potential as anticancer therapies.

Alkaloids from Coptis chinensis root promote glucose uptake in C2C12 myotubes.

The root of Coptis chinensis Franch. (COCH) is regularly used for medicinal purposes, and has been prescribed alone or in combination with other traditional herbs for the treatment of diabetes. To investigate the effects of COCH on glucose utilization by skeletal muscles, we prepared an ethanol extract of COCH root (COCH-Et) partitioned with dichloromethane, n-butanol, and water and tested its effects on glucose uptake in differentiated C2C12 myotubes. We found that dichloromethane and n-butanol sub-fractions of COCH-Et promoted glucose uptake in differentiated C2C12 cells at 50 µg/mL. Further fractionation of these preparations by using column chromatography, analysis of their effects on glucose uptake and characterization using nuclear magnetic resonance, mass spectrometry, and thin layer chromatography helped identify two new alkaloids, 8,13-dioxocoptisine hydroxide (1) and coptisonine (2), together with eleven known compounds. These were isolated from the dichloromethane layer of COCH-Et. In particular, exposure of C2C12 cells to berberine (6) at 12.5 and 6.25 µg/mL for 24h resulted in significant promotion of glucose uptake. Coptisonine (2) and octadecyl caffeate (9) also stimulated glucose uptake at 25 and 50 µg/mL. These findings indicate that active constituents of COCH root may help alleviate hyperglycemia in diabetes by promoting glucose uptake by skeletal muscles.

Differential immunomodulatory effects by Tripterygium wilfordii Hook f-derived refined extract PG27 and its purified component PG490 (triptolide) in human peripheral blood T cells: potential therapeutics for arthritis and possible mechanisms explaining in part Chinese herbal theory "Junn-Chenn-Zuou-SS".

BACKGROUND: For thousands of years, it remains unclear why Chinese prefer complex herbal remedy and seldom try to purify it. One of the reasons is that they believe Chinese herbs compared to Western drugs are relatively less toxic and better tolerated. The so called "Junn-Chenn-Zuou-SS" theory illustrates a concept of coordinated effects from a combination of different Chinese herbs. PG27, a refined extract from a well-known Chinese antirheumatic herb Tripterygium wilfordii Hook f (TwHf), is effective in attenuating transplantation rejection and extending survival of cardiac xenografts. METHODS: Experiments were conducted in human primary T lymphocytes isolated from buffy coat. The activities of the inhibitor of kappaB alpha kinase-inhibitor of kappaB alpha-nuclear factor kappaB (IKK-IκBα-NF-κB) and mitogen activated protein kinase-activator protein-1 (MAPK-AP-1) signaling pathways were determined via electrophoretic mobility shift assays, immunoprecipitation kinase assays, Western blots, and transfection assays. RESULTS: We showed that PG27 inhibited IKKα-IκBα-NF-κB and MAPK-AP-1 signaling pathways; however, IKKß activity was less susceptible to inhibition by PG27. In contrast, the purified component of TwHf, PG490 (triptolide), reduced both MAPK-AP-1 and IKK-IκBα-NF-κB signaling pathways, including both IKKα and IKKß, with similar potency. By means of high performance liquid chromatography analysis, it was estimated that PG490 constituted 1.27 ± 0.06% of the total PG27 content. Further analysis demonstrated that compared to PG490 alone, PG27 that contained an equal amount of PG490 was less toxic and less immunosuppressive, suggesting the presence of cytoprotective ingredient(s) in the non-PG490 components of PG27. CONCLUSIONS: In addition to demonstrating the immunomodulatory capacity of PG27 as the potential therapeutics for arthritis and prevention of transplantation rejection, the differential regulatory effects and mechanisms by PG27 and PG490 further support in part a possibly-existing Chinese herbal theory "Junn-Chenn-Zuou-SS".

Ovatodiolide Targets ß -Catenin Signaling in Suppressing Tumorigenesis and Overcoming Drug Resistance in Renal Cell Carcinoma.

Dysregulated ß -catenin signaling is intricately involved in renal cell carcinoma (RCC) carcinogenesis and progression. Determining potential ß -catenin signaling inhibitors would be helpful in ameliorating drug resistance in advanced or metastatic RCC. Screening for ß -catenin signaling inhibitors involved in silico inquiry of the PubChem Bioactivity database followed by TCF/LEF reporter assay. The biological effects of ovatodiolide were evaluated in 4 RCC cell lines in vitro and 2 RCC cell lines in a mouse xenograft model. The synergistic effects of ovatodiolide and sorafenib or sunitinib were examined in 2 TKI-resistant RCC cell lines. Ovatodiolide, a pure compound of Anisomeles indica, inhibited ß -catenin signaling and reduced RCC cell viability, survival, migration/invasion, and in vitro cell or in vivo mouse tumorigenicity. Cytotoxicity was significantly reduced in a normal kidney epithelial cell line with the treatment. Ovatodiolide reduced phosphorylated ß -catenin (S552) that inhibited ß -catenin nuclear translocation. Moreover, ovatodiolide decreased ß -catenin stability and impaired the association of ß -catenin and transcription factor 4. Ovatodiolide combined with sorafenib or sunitinib overcame drug resistance in TKI-resistant RCC cells. Ovatodiolide may be a potent ß -catenin signaling inhibitor, with synergistic effects with sorafenib or sunitinib, and therefore, a useful candidate for improving RCC therapy.

Association of the polymorphism of APE1 gene with the risk of prostate cancer in Chinese Han population.

BACKGROUND: DNA damage, caused by numerous carcinogens, contributes to the increased risk of different types of cancer. The base excision repair (BER) pathway including the apurinic/apyrimidic endonuclease (APE1, also known as APEX1) gene plays an important role in preventing the accumulation of DNA damage and maintaining genomic stability. The aim of the present study is to determine whether polymorphisms of APE1 are associated with the risk of prostate cancer (PCa) in the Chinese Han population. METHODS: This study consisted of 198 patients with PCa and 156 healthy controls. The polymorphisms of APE1, Asp148Glu (rs1130409) and -141T/G (rs1760944) were determined by the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The genotypic distributions of the two polymorphisms in controls were in Hardy-Weinberg equilibrium (p = 0.92 and p = 0.83, respectively). Logistic regression analysis indicated that the -141GG genotype was significantly associated with a decreased risk of PCa compared with the -141TT genotype (p = 0.03; OR 0.49; 95% CI 0.26 - 0.92). The G allele was also significantly associated with a reduced risk of PCa compared with the T allele (p = 0.02; OR 0.71; 95% CI 0.52 - 0.96). However, no association between Asp148Glu polymorphism and the risk of PCa was found. CONCLUSIONS: The -141GG genotype and G allele of the APE1 gene are associated with a decreased risk of PCa in the Chinese Han population.

A novel mechanism underlies the hepatotoxicity of pyrazinamide.

Relatively little is known about the hepatotoxicity of pyrazinamide (PZA). PZA requires activation by amidase to form pyrazinoic acid (PA). Xanthine oxidase then hydroxylates PA to form 5-hydroxypyrazinoic acid (5-OH-PA). PZA can also be directly oxidized to form 5-OH-PZA. Before this study, it was unclear which metabolic pathway or PZA metabolites led to hepatotoxicity. This study determines whether PZA metabolites are responsible for PZA-induced hepatotoxicity. PZA metabolites were identified and cytotoxicity in HepG2 cells was assessed. Potential PZA and PA hepatotoxicity was then tested in rats. Urine specimens were collected from 153 tuberculosis (TB) patients, and the results were evaluated to confirm whether a correlation existed between PZA metabolite concentrations and hepatotoxicity. This led to the hypothesis that coadministration of amidase inhibitor (bis-p-nitrophenyl phosphate [BNPP]) decreases or prevents PZA- and PZA metabolite-induced hepatotoxicity in rats. PA and 5-OH-PA are more toxic than PZA. Electron microscopy showed that PZA and PA treatment of rats significantly increases aspartate transaminase (AST) and alanine aminotransferase (ALT) activity and galactose single-point (GSP) levels (P < 0.005). PA and 5-OH-PA levels are also significantly correlated with hepatotoxicity in the urine of TB patients (P < 0.005). Amidase inhibitor, BNPP, decreases PZA-induced, but not PA-induced, hepatotoxicity. This is the first report of a cell line, animal, and clinical trial confirming that the metabolite 5-OH-PA is responsible for PZA-induced hepatotoxicity.