Mineral-Enriched Deep-Sea Water Modulates Lactate Metabolism via PGC-1α-Mediated Metabolic Reprogramming.

Metabolic disorders such as diabetes and obesity are serious global health issues. These diseases are accelerated by mineral deficiencies, emphasizing the importance of addressing these deficiencies in disease management plans. Lactate metabolism is fundamentally linked to glucose metabolism, and several clinical studies have reported that blood lactate levels are higher in obese and diabetic patients than in healthy subjects. Balanced deep-sea water contains various minerals and exhibits antiobesity and antidiabetic activities in mice; however, the impact of balanced deep-sea water on lactate metabolism is unclear. Thus, we evaluated the effects of balanced deep-sea water on lactate metabolism in C2C12 myotubes, and found that balanced deep-sea water mediated lactate metabolism by regulating the gene expression levels of lactate dehydrogenases A and B, a monocarboxylate transporter, and a mitochondrial pyruvate carrier. The activities of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and signaling molecules involved in PGC-1α activation were also upregulated by treatment with balanced deep-sea water. These results suggest that balanced deep-sea water, which can mediate lactate metabolism, may be used to prevent or treat obesity and diabetes mellitus.

Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants.

A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me2SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me2SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me2SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3weeks) and long-term (1year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media.

Anti-diabetic effect of balanced deep-sea water and its mode of action in high-fat diet induced diabetic mice.

In this study, we investigated the effects of balanced deep-sea water (BDSW) on hyperglycemia and glucose intolerance in high-fat diet (HFD)-induced diabetic C57BL/6J mice. BDSW was prepared by mixing deep-sea water (DSW) mineral extracts and desalinated water to give a final hardness of 500-2000. Mice given an HFD with BDSW showed lowered fasting plasma glucose levels compared to HFD-fed mice. Oral and intraperitoneal glucose tolerance tests showed that BDSW improves impaired glucose tolerance in HFD-fed mice. Histopathological evaluation of the pancreas showed that BDSW recovers the size of the pancreatic islets of Langerhans, and increases the secretion of insulin and glucagon in HFD-fed mice. Quantitative reverse transcription polymerase chain reaction results revealed that the expression of hepatic genes involved in glucogenesis, glycogenolysis and glucose oxidation were suppressed, while those in glucose uptake, ß-oxidation, and glucose oxidation in muscle were increased in mice fed HFD with BDSW. BDSW increased AMP-dependent kinase (AMPK) phosphorylation in 3T3-L1 pre- and mature adipocytes and improved impaired AMPK phosphorylation in the muscles and livers of HFD-induced diabetic mice. BDSW stimulated phosphoinositol-3-kinase and AMPK pathway-mediated glucose uptake in 3T3-L1 adipocytes. Taken together, these results suggest that BDSW has potential as an anti-diabetic agent, given its ability to suppress hyperglycemia and improve glucose intolerance by increasing glucose uptake.

Cryopreservation of amniotic fluid-derived stem cells using natural cryoprotectants and low concentrations of dimethylsulfoxide.

Amniotic fluid-derived stem cells (AFSCs) are a potential cell source for therapeutic applications. They can be easily mass produced, cryopreserved and shipped to clinics for immediate use. However, one major obstacle to the manufacturing of clinical grade stem cells is the need for current good manufacturing practices for cryopreservation, storage, and distribution of these cells. Most current cryopreservation methods used for stem cells include the potentially toxic cryoprotectant (CPA) dimethylsulfoxide (Me(2)SO) in the presence of animal serum proteins that prevent direct use of these cells in human therapeutic applications. To avoid any potential cryoprotectant related complications, it will be essential to develop non-toxic CPAs or reduce CPA concentration in the freezing media used. In this study, we assessed the use of disaccharides, antioxidants and caspase inhibitors for cryopreservation of AFSCs in combination with a reduced concentration of Me(2)SO. The thawed cells were tested for viability with MTT assays and a growth curve was created to measure population doubling time. In addition, we performed flow cytometry analysis for cell surface antigens, RT-PCR for mRNA expression of stem cell markers, and assays to determine the myogenic differentiation potential of the cells. A statistically significant (p<0.05) increase in post-thawed cell viability in solutions containing trehalose, catalase and (Z)VAD-fmk with 5% Me(2)SO was observed. The solutions containing trehalose and catalase with 5% or 2.5% (v/v) Me(2)SO produced results similar to those for the control (10% (v/v) Me(2)SO and 30% FBS) in terms of culture growth, expression of cell surface antigens and mRNA expression of stem cell markers in AFSCs cryopreserved for a minimum of 3 weeks. Thus, AFSCs can be cryopreserved with 1/4 the standard Me(2)SO concentration with the addition of disaccharides, antioxidants and caspase inhibitors. The use of Me(2)SO at low concentrations in cell freezing solutions may support the development of clinical trials of AFSCs.

Natural Magnesium-Enriched Deep-Sea Water Improves Insulin Resistance and the Lipid Profile of Prediabetic Adults: A Randomized, Double-Blinded Crossover Trial.

Previous in vitro and in vivo studies have shown that the antidiabetic effect of balanced deep-sea water (BDSW) works through the suppression of hyperglycemia and improvement of glucose tolerance. Based on these promising results, we conducted an eight week randomized, double-blinded crossover trial of the effects of BDSW in prediabetic adults. The subjects consumed 440 mL of BDSW (hardness 4000) per day, and maintained an otherwise normal lifestyle and diet throughout. Efficacy assessments were made by measuring fasting glucose, postprandial glucose, fasting insulin, homeostasis model assessment for insulin resistance (HOMA-IR), C-peptide, glycosylated hemoglobin, lipid metabolism indicators, and physical metrics, along with safety assessments. Fasting insulin and HOMA-IR values of the BDSW group were significantly lower than those of the placebo group after eight weeks of BDSW ingestion. Total cholesterol and low-density lipoprotein-cholesterol were also significantly decreased in the BDSW group after eight weeks of BDSW ingestion compared with the placebo group. There were no statistically and clinically meaningful changes in adverse events, physical examination, laboratory medicine examination, or vital signs of the BDSW intake group. These results suggested that the intake of BDSW in prediabetic adults can improve glucose metabolism and lipid profiles and is safe for human consumption.

Inhibitory effect of deep-sea water on differentiation of 3T3-L1 adipocytes.

Currently, the utilization of deep-sea water (DSW) is receiving much attention due to its high productivity, large quantity, and potential for biological application. The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days, confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. In this study, the effects of DSW on differentiation adipocyte 3T3-L1 cells were studied. DSW significantly decreased lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. DSW of hardness 1,000 was the most effective for inhibiting adipocyte differentiation without any cytotoxicity. DSW significantly reduced expression mRNA levels of PPARgamma and C/EBPalpha and protein levels of fatty-acid-binding protein and adiponectin. Our results suggest a potential role for DSW as anti-obesity agents by inhibiting adipocyte differentiation mediated through the down-regulated expression of adipogenic transcription factors and adipocyte-specific proteins.

Suppression of metastasis of human breast cancer cells by chitosan oligosaccharides.

The present study investigated the antimetastatic property of chitosan oligosaccharides (COS) by evaluating motility, invasion, and the amount and activity of MMP-9 in MDA-MB-231 human breast carcinoma cells. Treatment of MDA-MB-231 cells with increasing concentrations of COS led to a concentration-dependent decrease in cell migration. COS significantly inhibited the invasion of MDA-MB-231 cells through a Matrigel-coated membrane. The treatment of MDA-MB-231 cells with COS reduced the amounts of secreted MMP-9. The activity and amount of MMP-9 protein in MDA-MB-231 cells were decreased by treatment with COS and occurred in a concentration-dependent manner. Our data indicated that COS can serve as a potential novel therapeutic candidate for the treatment of metastatic breast cancer.

Chemopreventive effect of chitosan oligosaccharide against colon carcinogenesis.

Chitosan oligosaccharide (COS, 3 kDa

Inhibition of proinflammatory cytokine-induced invasiveness of HT-29 cells by chitosan oligosaccharide.

The effect of chitosan oligosaccharide (COS, 1 kDa

Effects of proton beam irradiation on mitochondrial biogenesis in a human colorectal adenocarcinoma cell line.

Proton beam therapy has recently been used to improve local control of tumor growth and reduce side-effects by decreasing the global dose to normal tissue. However, the regulatory mechanisms underlying the physiological role of proton beam radiation are not well understood, and many studies are still being conducted regarding these mechanisms. To determine the effects of proton beams on mitochondrial biogenesis, we investigated: mitochondrial DNA (mtDNA) mass; the gene expression of mitochondrial transcription factors, functional regulators, and dynamic-related regulators; and the phosphorylation of the signaling molecules that participate in mitochondrial biogenesis. Both the mtDNA/nuclear DNA (nDNA) ratio and the mitochondria staining assays showed that proton beam irradiation increases mitochondrial biogenesis in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced aggressive HT-29 cells. Simultaneously, proton beam irradiation increases the gene expression of the mitochondrial transcription factors PGC-1α, NRF1, ERRα, and mtTFA, the dynamic regulators DRP1, OPA1, TIMM44, and TOM40, and the functional regulators CytC, ATP5B and CPT1-α. Furthermore, proton beam irradiation increases the phosphorylation of AMPK, an important molecule involved in mitochondrial biogenesis that is an energy sensor and is regulated by the AMP/ATP ratio. Based on these findings, we suggest that proton beam irradiation inhibits metastatic potential by increasing mitochondrial biogenesis and function in TPA-induced aggressive HT-29 cells.

Effective chemopreventive activity of genistein against human breast cancer cells.

Chemopreventive and cytotoxic effect of genistein against human breast cancer cell lines was investigated. Genistein inhibited cell proliferation in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) human breast carcinoma cell lines. Cytochrome P450 (CYP) 1A1-mediated ethoxyresorufin O-deethylase (EROD)activity was inhibited by genistein in a concentration-dependent manner. Genistein significantly inhibited 12-Otetradecanoylphorbol-13-acetate (TPA)-induced cyclooxygenase-2 activity and protein expression at the concentrations of 10 (p < 0.05), 25 (p < 0.05) and 50 mM (p < 0.01). In addition, ornithine decarboxylase (ODC) activity was reduced to 53.8 % of the control after 6 h treatment with 50 mM genistein in MCF-7 breast cancer cells. These results suggest that genistein could be of therapeutic value in preventing human breast cancer.

Chemopreventive effect of protein extract of Asterina pectinifera in HT-29 human colon adenocarcinoma cells.

We investigated the effect of protein extract of Asterina pectinifera on the activity of 4 enzymes that may play a role in adenocarcinoma of the colon: quinone reductase (QR), glutathione S-transferase (GST), ornithine decarboxylase (ODC), and cyclooxygenase (COX)-2. QR and GST activity increased in HT-29 human colon adenocarcinoma cells increased that had been exposed to 4 concentrations of the protein extract (80, 160, 200, and 240 microg/mL). Additionally, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC activity decreased significantly in cells exposed to the extract in concentrations of 160 microg/mL (p<0.05), 200 microg/mL (p<0.005), and 240 microg/mL (p<0.005). TPA-induced COX-2 activity also decreased in cells exposed to extract concentrations of 10, 20, 40, and 60 microg/mL. COX-2 expression was also inhibited in cells exposed to this extract. These results suggest that this protein extract of A pectinifera has chemopreventive activity in HT-29 human colon adenocarcinoma cells, and therefore, may have the potential to function as a chemopreventive agent in human colorectal cancer.

Stimulatory Effect of Balanced Deep-Sea Water Containing Chitosan Oligosaccharides on Glucose Uptake in C2C12 Myotubes.

Deep-sea water (DSW) and chitosan oligosaccharides (COS) have recently drawn much attention because of their potential medical and pharmaceutical applications. Balanced DSW (BDSW) was prepared by mixing DSW mineral extracts and desalinated water. This study investigated the effects of BDSW, COS, and BDSW containing COS on glucose uptake and their mode of action in mature C2C12 myotubes. BDSW and COS increased glucose uptake in a dose-dependent manner. BDSW containing COS synergistically increased glucose uptake; this was dependent on the activation of insulin receptor substrate 1 and protein kinase C in insulin-dependent signaling pathways as well as liver kinase B1, AMP-activated protein kinase, and mammalian target of rapamycin in insulin-independent signaling pathways. Quantitative real-time polymerase chain reaction revealed that the expressions of the following genes related to glucose uptake were elevated: glucose transporter 4 (GLUT4), insulin-responsive aminopeptidase, and vesicle-associated membrane protein 2 for abundant proteins of GLUT4 storage vesicles (GSVs); syntaxin 4 and soluble N-ethylmaleimide-sensitive factor attachment protein 23 for trafficking between the plasma membrane and GSVs; and syntaxin 6 and syntaxin 16 for trafficking between GSVs and the trans-Golgi network. Taken together, these results suggest BDSW containing COS has a greater stimulatory effect on glucose uptake than BDSW or COS alone. Moreover, this effect is mediated by the stimulation of diverse signaling pathways via the activation of main signaling molecules related to GSV trafficking.

Magnesium and calcium-enriched deep-sea water promotes mitochondrial biogenesis by AMPK-activated signals pathway in 3T3-L1 preadipocytes.

Recent studies showed that deficiencies of essential minerals including Mg, Ca, and K, and trace minerals including Se, Zn, and V, have implications for the development, prevention, and treatment of several chronic diseases including obesity and type 2 diabetes. Our previous studies revealed that balanced deep-sea water (BDSW), which is composed of desalinated water enriched with Mg and Ca, has potential as a treatment for diabetes and obesity. In this study, to determine whether BDSW regulates mitochondrial biogenesis and function, we investigated its effects on mitochondrial DNA (mtDNA) content, mitochondrial enzyme activity, expression of key transcription factors and mitochondria-specific genes, phosphorylation of signaling molecules associated with mitochondrial biogenesis, and mitochondrial function in 3T3-L1 preadipocytes. BDSW increased mitochondrial biogenesis in a dose-dependent manner. Quantitative real-time PCR revealed that BDSW enhances expression of PGC1-α, NRF1, and TFAM genes. Upregulation of these genes was supported by increased mitochondria staining, CytC oxidase activity, and AMPK phosphorylation. The stimulatory effect of BDSW on mitochondrial biogenesis and function suggests a novel mechanism for BDSW-induced anti-diabetic and anti-obesity action.

Effect of Cnidii Rhizoma on nitric oxide production and invasion of human colorectal adenocarcinoma HT-29 cells.

Colorectal adenocarcinoma is the most common type of gastrointestinal cancer. Colon adenocarcinoma is a major health problem worldwide due to the high prevalence and mortality rates associated with the disease. The majority of colorectal carcinomas are adenocarcinomas, which originate from the epithelial cells of the colorectal mucosa. HT-29 cells, which originate from human colon adenocarcinoma, are used as an in vitro model to investigate the effect of malignant transformation on the expression of cellular constituents and functions of the intestinal epithelium. Nitric oxide (NO) is a signaling molecule, which is involved in inflammation and carcinogenesis. It has been reported that enhanced inducible NO synthase (iNOS) activity and the resulting NO concentrations in human colon carcinoma contribute to tumor progression and vascular invasion. The present study investigates the effect of pro-inflammatory cytokine-induced nitric oxide (NO) production and iNOS expression on the invasion of human colorectal adenocarcinoma HT-29 cells, and the effect of extract from Cnidii Rhizoma on NO production and the invasiveness of HT-29 cells. Treatment of HT-29 cells with cytokines, 100 U/ml interferon γ, 10 ng/ml interleukin-1 α and 25 ng/ml tumor necrosis factor α was found to increase NO production. Pretreatment of the cells with Cnidii Rhizoma (0.1-5 mg/ml) resulted in an inhibition of cytokine-induced NO production and iNOS expression. The invasiveness of HT-29 cells through Matrigel was significantly increased by treatment with cytokines. Cnidii Rhizoma inhibited the invasiveness of cytokine-treated HT-29 cells through the Matrigel-coated membrane in a concentration-dependent manner. Matrix metalloproteinase (MMP) activity in HT-29 cells increased following the treatment with cytokines, and pretreatment of the cells with Cnidii Rhizoma inhibited cytokine-induced MMP-2 activity. These results provide sufficient information for the further development of Cnidii Rhizoma as an antitumor metastatic agent for the treatment of colon cancer.

Inhibitory effects of proton beam irradiation on integrin expression and signaling pathway in human colon carcinoma HT29 cells.

Proton radiotherapy has been established as a highly effective modality used in the local control of tumor growth. Although proton radiotherapy is used worldwide to treat several types of cancer clinically with great success due to superior targeting and energy deposition, the detailed regulatory mechanisms underlying the functions of proton radiation are not yet well understood. Accordingly, in the present study, to assess the effects of proton beam on integrin-mediated signaling pathways, we investigated the expression of integrins related to tumor progression and integrin trafficking, and key molecules related to cell adhesion, as well as examining phosphorylation of signaling molecules involved in integrin-mediated signaling pathways. Proton beam irradiation inhibited the increase in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced integrin ß1 protein expression and the gene expression of members of the integrin family, such as α5ß1, α6ß4, αvß3, and αvß6 in human colorectal adenocarcinoma HT-29 cells. Simultaneously, the gene expression of cell adhesion molecules, such as FAK and CDH1, and integrin trafficking regulators, such as RAB4, RAB11, and HAX1, was decreased by proton beam irradiation. Moreover, proton beam irradiation decreased the phosphorylation of key molecules involved in integrin signaling, such as FAK, Src, and p130Cas, as well as PKC and MAPK, which are known as promoters of cell migration, while increased the phosphorylation of AMPK and the gene expression of Rab IP4 involved in the inhibition of cell adhesion and cell spreading. Taken together, our findings suggest that proton beam irradiation can inhibit metastatic potential, including cell adhesion and migration, by modulating the gene expression of molecules involved in integrin trafficking and integrin-mediated signaling, which are necessary for tumor progression.

Stimulatory Effects of Balanced Deep Sea Water on Mitochondrial Biogenesis and Function.

The worldwide prevalence of metabolic diseases, including obesity and diabetes, is increasing. Mitochondrial dysfunction is recognized as a core feature of these diseases. Emerging evidence also suggests that defects in mitochondrial biogenesis, number, morphology, fusion, and fission, contribute to the development and progression of metabolic diseases. Our previous studies revealed that balanced deep-sea water (BDSW) has potential as a treatment for diabetes and obesity. In this study, we aimed to investigate the mechanism by which BDSW regulates diabetes and obesity by studying its effects on mitochondrial metabolism. To determine whether BDSW regulates mitochondrial biogenesis and function, we investigated its effects on mitochondrial DNA (mtDNA) content, mitochondrial enzyme activity, and the expression of transcription factors and mitochondria specific genes, as well as on the phosphorylation of signaling molecules associated with mitochondria biogenesis and its function in C2C12 myotubes. BDSW increased mitochondrial biogenesis in a time and dose-dependent manner. Quantitative real-time PCR revealed that BDSW enhances gene expression of PGC-1α, NRF1, and TFAM for mitochondrial transcription; MFN1/2 and DRP1 for mitochondrial fusion; OPA1 for mitochondrial fission; TOMM40 and TIMM44 for mitochondrial protein import; CPT-1α and MCAD for fatty acid oxidation; CYTC for oxidative phosphorylation. Upregulation of these genes was validated by increased mitochondria staining, CS activity, CytC oxidase activity, NAD+ to NADH ratio, and the phosphorylation of signaling molecules such as AMPK and SIRT1. Moreover, drinking BDSW remarkably improved mtDNA content in the muscles of HFD-induced obese mice. Taken together, these results suggest that the stimulatory effect of BDSW on mitochondrial biogenesis and function may provide further insights into the regulatory mechanism of BDSW-induced anti-diabetic and anti-obesity action.

Pre-clinical efficacy and safety evaluation of human amniotic fluid-derived stem cell injection in a mouse model of urinary incontinence.

PURPOSE: Stem cell-based therapies represent new promises for the treatment of urinary incontinence. This study was performed to assess optimized cell passage number, cell dose, therapeutic efficacy, feasibility, toxicity, and cell trafficking for the first step of the pre-clinical evaluation of human amniotic fluid stem cell (hAFSC) therapy in a urinary incontinence animal model. MATERIALS AND METHODS: The proper cell passage number was analyzed with hAFSCs at passages 4, 6, and 8 at week 2. The cell dose optimization included 1×104, 1×105, and 1×106 cells at week 2. The in vivo cell toxicity was performed with 0.25×106, 0.5×106, and 1×106 cells at weeks 2 and 4. Cell tracking was performed with 1×106 cells at weeks 2 and 4. RESULTS: The selected optimal cell passage number was smaller than 6, and the optimal cell dose was 1×106 for the mouse model. In our pre-clinical study, hAFSC-injected animals showed normal values for several parameters. Moreover, the injected cells were found to be non-toxic and non-tumorigenic. Furthermore, the injected hAFSCs were rarely identified by in vivo cell trafficking in the target organs at week 2. CONCLUSION: This study demonstrates for the first time the pre-clinical efficacy and safety of hAFSC injection in the urinary incontinence animal model and provides a basis for future clinical applications.

Inhibitory effect of GBH on platelet aggregation through inhibition of intracellular Ca2+ mobilization in activated human platelets.

Geiji-Bokryung-Hwan (GBH) was studied on antiplatelet activity in human platelet suspensions. GBH consists of the 5 herbs Cinnamomi Ramulus, Poria Cocos, Mountan Cortex Radicis, Paeoniae Radix, and Persicae Semen, which have been used in herbal medicine for thousands of years for atherosclerosis. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH inhibited platelet aggregation and Ca2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. GBH had no inhibitory effect on thromboxane B2 (TXB2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca2+ mobilization is regulated by phospholipase C-2 (PLC-gamma2) activation. We evaluated the effect of GBH on tyrosine phosphorylation of PLC-gamma2 and the production of inositol-1,4,5-trisphosphate (IP3). GBH at concentrations that inhibited platelet aggregation and Ca2+ mobilization had no effects on tyrosine phosphorylation of PLC-gamma2 or on the formation of IP3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. GBH inhibited platelet aggregation and Ca2+ mobilization induced by thrombin without affecting the production of IP3. We then evaluated the effect of GBH on the binding of IP3 to its receptor. GBH at high concentrations partially blocked the binding of IP3 to its receptor. Therefore, the results suggested that GBH suppresses Ca2+ mobilization at a step distal to IP3 formation. GBH may provide a good tool for investigating Ca2+ mobilization.

Protective effect of Moutan Cortex extract on acetaminophen-induced hepatotoxicity in mice.

Previously, we demonstrated that Moutan Cortex prevents acetaminophen (AAP)-induced cytotoxicity in vitro. The present study examined the protective effect of Moutan Cortex on AAP induced hepatotoxicity and the possible mechanisms underlying this effect in mice. When Montan Cortex was administered to ICR mice, followed by hepatotoxic dose of AAP (400 mg/kg, i.p.), Moutan Cortex pre-exposure prevented liver injury as indicated by the decrease of serum alanine aminotransferase level. Moutan Cortex also protected AAP-induced hepatic glutathione depletion. Cytochrome P450 2E1-dependent aniline and p-nitrophenol hydroxylases activities in microsomal incubations were significantly inhibited by Moutan Cortex. Abrogation of toxicity was also mirrored in DNA fragmentation. These observations demonstrate that Moutan Cortex pre-exposure may attenuate AAP-induced GSH depletion, cytochrome P450 2E1 activity, and hepatic DNA damage in vivo.