Caffeic acid phenethyl ester suppresses intestinal FXR signaling and ameliorates nonalcoholic fatty liver disease by inhibiting bacterial bile salt hydrolase activity.

Propolis is commonly used in traditional Chinese medicine. Studies have demonstrated the therapeutic effects of propolis extracts and its major bioactive compound caffeic acid phenethyl ester (CAPE) on obesity and diabetes. Herein, CAPE was found to have pharmacological activity against nonalcoholic fatty liver disease (NAFLD) in diet-induced obese mice. CAPE, previously reported as an inhibitor of bacterial bile salt hydrolase (BSH), inhibited BSH enzymatic activity in the gut microbiota when administered to mice. Upon BSH inhibition by CAPE, levels of tauro-ß-muricholic acid were increased in the intestine and selectively suppressed intestinal farnesoid X receptor (FXR) signaling. This resulted in lowering of the ceramides in the intestine that resulted from increased diet-induced obesity. Elevated intestinal ceramides are transported to the liver where they promoted fat production. Lowering FXR signaling was also accompanied by increased GLP-1 secretion. In support of this pathway, the therapeutic effects of CAPE on NAFLD were absent in intestinal FXR-deficient mice, and supplementation of mice with C16-ceramide significantly exacerbated hepatic steatosis. Treatment of mice with an antibiotic cocktail to deplete BSH-producing bacteria also abrogated the therapeutic activity of CAPE against NAFLD. These findings demonstrate that CAPE ameliorates obesity-related steatosis at least partly through the gut microbiota-bile acid-FXR pathway via inhibiting bacterial BSH activity and suggests that propolis enriched with CAPE might serve as a promising therapeutic agent for the treatment of NAFLD.

St. John's Wort alleviates dextran sodium sulfate-induced colitis through pregnane X receptor-dependent NFκB antagonism.

St. John's wort (SJW), from traditional herbs, activates the pregnane X receptor (PXR), a potential drug target for treating inflammatory bowel disease (IBD). However, how SJW alleviates dextran sodium sulfate (DSS)-induced experimental IBD by activating PXR is unknown. To test this, PXR-humanized, wild-type (WT) and Pxr-null mice, primary intestinal organoids cultures, and the luciferase reporter gene assays were employed. In vivo, a diet supplemented with SJW was found to activate intestinal PXR both in WT and PXR-humanized mice, but not in Pxr-null mice. SJW prevented DSS-induced IBD in PXR-humanized and WT mice, but not in Pxr-null mice. In vitro, hyperforin, a major component of SJW, activated PXR and suppressed tumor necrosis factor (TNF)α-induced nuclear factor (NF) κB translocation in primary intestinal organoids from PXR-humanized mice, but not Pxr-null mice. Luciferase reporter gene assays showed that hyperforin dose-dependently alleviated TNFα-induced NFκB transactivation by activating human PXR in Caco2 cells. Furthermore, SJW therapeutically attenuated DSS-induced IBD in PXR-humanized mice. These data indicate the therapeutic potential of SJW in alleviating DSS-induced IBD in vivo, and TNFα-induced NFκB activation in vitro, dependent on PXR activation, which may have clinical implications for using SJW as a herbal drug anti-IBD treatment.

Rutaecarpine inhibits KEAP1-NRF2 interaction to activate NRF2 and ameliorate dextran sulfate sodium-induced colitis.

Inflammatory bowel disease (IBD) represents a group of chronic relapsing intestinal disorders. Rutaecarpine (RUT), isolated from the Traditional Chinese Medicine (TCM) of Evodia rutaecarpa, was reported to suppress IBD. However, the mechanism by which RUT ameliorates dextran sulfate sodium (DSS)-induced IBD is largely unknown. By use of nuclear factor-erythroid 2-related factor 2 (NRF2) knockout mice, cell-based studies, surface plasmon resonance (SPR), western blotting analysis, and molecular docking studies, the mechanism by which RUT affects DSS-induced colitis was explored. In DSS-treated wild-type mice but not in Nrf2-null mice, RUT significantly improved colitis as revealed by rescued body weight loss, improved histology and inflammation, and induced expression of NRF2 target genes in colon and ileum. Cell-based studies showed that RUT significantly increased the LD50 for hydrogen peroxide (H2O2)-induced cell damage, activated NRF2 nuclear translocation, and suppressed the production of reactive oxygen species in H2O2-treated HCT116 cells, activated NRF2 luciferase reporter activities in HCT116 cells and HepG2 cells, and induced expression of NRF2 target genes in primary intestinal epithelial cells. Molecular docking in silico and SPR assays indicated that RUT interacted with kelch-like ECH-associated protein 1 (KEAP1), and extracellular incubation studies revealed that RUT bound to the KEAP1 kelch domain with a calculated equilibrium dissociation constant Kd of 19.6 µM. In conclusion, these results demonstrate that RUT ameliorates DSS-induced colitis, dependent on NRF2, and could be a potential therapeutic option for IBD patients. Mechanistically, RUT potentiates NRF2 nuclear translocation to upregulate NRF2-mediated antioxidant response by directly inhibiting KEAP1-NRF2 interaction.

Withaferin A Improves Nonalcoholic Steatohepatitis in Mice.

Nonalcoholic steatohepatitis (NASH) is the progressive stage of nonalcoholic fatty liver disease that highly increases the risk of cirrhosis and liver cancer, and there are few therapeutic options available in the clinic. Withaferin A (WA), extracted from the ayurvedic medicine Withania somnifera, has a wide range of pharmacological activities; however, little is known about its effects on NASH. To explore the role of WA in treating NASH, two well defined NASH models were used, the methionine-choline-deficient diet and the 40 kcal% high-fat diet (HFD). In both NASH models, WA treatment or control vehicle was administered to evaluate its hepatoprotective effects. As assessed by biochemical and histologic analyses, WA prevented and therapeutically improved liver injury in both models, as revealed by lower serum aminotransaminases, hepatic steatosis, liver inflammation, and fibrosis. In the HFD-induced NASH model, both elevated serum ceramides and increased hepatic oxidative stress were decreased in the WA-treated group compared with the control vehicle-treated group. To further explore whether WA has an anti-NASH effect independent of its known action in leptin signaling associated with obesity, leptin signaling-deficient ob/ob mice maintained on an HFD were used to induce NASH. WA therapeutically reduced NASH in HFD-treated leptin-deficient ob/ob mice, thus demonstrating a leptin-independent hepatoprotective effect. This study revealed that WA treatment could be an option for NASH treatment.

Glycyrrhizin Alleviates Nonalcoholic Steatohepatitis via Modulating Bile Acids and Meta-Inflammation.

Nonalcoholic steatohepatitis (NASH) is the progressive stage of nonalcoholic fatty liver disease that may ultimately lead to cirrhosis and liver cancer, and there are few therapeutic options for its treatment. Glycyrrhizin (GL), extracted from the traditional Chinese medicine liquorice, has potent hepatoprotective effects in both preclinical animal models and in humans. However, little is currently known about its effects and mechanisms in treating NASH. To explore the effects of GL on NASH, GL or its active metabolite glycyrrhetinic acid (GA) was administered to mice treated with a methionine- and choline-deficient (MCD) diet-induced NASH model, and histologic and biochemical analyses were used to measure the degree of lipid disruption, liver inflammation, and fibrosis. GL significantly improved MCD diet-induced hepatic steatosis, inflammation, and fibrosis and inhibited activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. GL significantly attenuated serum bile acid accumulation in MCD diet-fed mice partially by restoring inflammation-mediated hepatic farnesoid X receptor inhibition. In Raw 264.7 macrophage cells, both GL and GA inhibited deoxycholic acid-induced NLRP3 inflammasome-associated inflammation. Notably, both intraperitoneal injection of GL's active metabolite GA and oral administration of GL prevented NASH in mice, indicating that GL may attenuate NASH via its active metabolite GA. These results reveal that GL, via restoration of bile acid homeostasis and inhibition of inflammatory injury, can be a therapeutic option for treatment of NASH.

Structure-Activity Relationships of the Main Bioactive Constituents of Euodia rutaecarpa on Aryl Hydrocarbon Receptor Activation and Associated Bile Acid Homeostasis.

Rutaecarpine (RUT), evodiamine (EOD), and dehydroevodiamine (DHED) are the three main bioactive indoloquinazoline alkaloids isolated from Euodia rutaecarpa, a widely prescribed traditional Chinese medicine. Here, the structure-activity relationships of these analogs for aryl hydrocarbon receptor (AHR) activation were explored by use of Ahr-deficient (Ahr-/-) mice, primary hepatocyte cultures, luciferase reporter gene assays, in silico ligand-docking studies, and metabolomics. In vitro, both mRNA analysis of AHR target genes in mouse primary hepatocytes and luciferase reporter assays in hepatocarcinoma cell lines demonstrated that RUT, EOD, and DHED significantly activated AHR, with an efficacy order of RUT > DHED > EOD. Ligand-docking analysis predicted that the methyl substitute at the N-14 atom was a key factor affecting AHR activation. In vivo, EOD was poorly orally absorbed and failed to activate AHR, whereas RUT and DHED markedly upregulated expression of the hepatic AHR gene battery in wild-type mice, but not in Ahr-/- mice. Furthermore, RUT, EOD, and DHED were not hepatotoxic at the doses used; however, RUT and DHED disrupted bile acid homeostasis in an AHR-dependent manner. These findings revealed that the methyl group at the N-14 atom of these analogs and their pharmacokinetic behaviors were the main determinants for AHR activation, and suggest that attention should be given to monitoring bile acid metabolism in the clinical use of E. rutaecarpa.

Chemical inhibition and stable knock-down of efflux transporters leads to reduced glucuronidation of wushanicaritin in UGT1A1-overexpressing HeLa cells: the role of breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in the excretion of glucuronides.

Active efflux transport of glucuronides out of cells is a critical process in elimination of drugs and food-derived compounds. Wushanicaritin, a natural polyphenol from Epimedium species, has shown many biological activities. However, the transporters responsible for excretion of wushanicaritin glucuronides still remain undefined. Herein, chemical inhibitors (Ko143, MK571, dipyridamole and leukotriene C4) and single stable knocked-down efflux transporters (BCRP, MRP1, MRP3 and MRP4) were used to determine the contributions of efflux transporters to glucuronide efflux and cellular glucuronidation in UGT1A1-overexpressing HeLa cells (HeLa1A1). Knock-down of transporters was performed by stable transfection of short hairpin RNA (shRNA) using lentiviral vectors. The HeLa1A1 cell lysate catalyzed wushanicaritin glucuronidation, generating wushanicaritin-3-O-glucuronide and wushanicaritin-7-O-glucuronide. Ko143 (a dual inhibitor of BCRP, 5-20 µM) caused a marked decrease in excretion rate (maximal 53.4%) and increase of intracellular glucuronides (maximal 86.0%), while MK-571 (an inhibitor of MRPs, 5-20 µM) resulted in a significant reduction in excretion rate (maximal 64.6%) and rise of intracellular glucuronides (maximal 98.0%). By contrast, dipyridamole and leukotriene C4 showed no inhibitory effects on glucuronide excretion. Furthermore, shRNA-mediated silencing of a target transporter led to a marked reduction in the excretion rate of wushanicaritin glucuronides (maximal 33.8% for BCRP; 25.9% for MRP1; 26.7% for MRP3; 39.3% for MRP4). Transporter silencing also led to substantial decreases in efflux clearance (maximal 61.5% for BCRP; 48.7% for MRP1; 35.1% for MRP3; 63.1% for MRP4). In conclusion, chemical inhibition and gene silencing results suggested that BCRP, MRP1, MRP3 and MRP4 were significant contributors to excretion of wushanicaritin glucuronides.

Glycyrrhizin Protects against Acetaminophen-Induced Acute Liver Injury via Alleviating Tumor Necrosis Factor α-Mediated Apoptosis.

Acetaminophen (APAP) overdose is the leading cause of drug-induced acute liver failure in Western countries. Glycyrrhizin (GL), a potent hepatoprotective constituent extracted from the traditional Chinese medicine liquorice, has potential clinical use in treating APAP-induced liver failure. The present study determined the hepatoprotective effects and underlying mechanisms of action of GL and its active metabolite glycyrrhetinic acid (GA). Various administration routes and pharmacokinetics-pharmacodynamics analyses were used to differentiate the effects of GL and GA on APAP toxicity in mice. Mice deficient in cytochrome P450 2E1 enzyme (CYP2E1) or receptor interacting protein 3 (RIPK3) and their relative wild-type littermates were subjected to histologic and biochemical analyses to determine the potential mechanisms. Hepatocyte death mediated by tumor necrosis factorα(TNFα)/caspase was analyzed by use of human liver-derived LO2 cells. The pharmacokinetics-pharmacodynamics analysis using various administration routes revealed that GL but not GA potently attenuated APAP-induced liver injury. The protective effect of GL was found only with intraperitoneal and intravenous administration and not with gastric administration. CYP2E1-mediated metabolic activation and RIPK3-mediated necroptosis were unrelated to GL's protective effect. However, GL inhibited hepatocyte apoptosis via interference with TNFα-induced apoptotic hepatocyte death. These results demonstrate that GL rapidly attenuates APAP-induced liver injury by directly inhibiting TNFα-induced hepatocyte apoptosis. The protective effect against APAP-induced liver toxicity by GL in mice suggests the therapeutic potential of GL for the treatment of APAP overdose.

St. John's Wort Attenuates Colorectal Carcinogenesis in Mice through Suppression of Inflammatory Signaling.

Despite widespread use as well as epidemiologic indications, there have been no investigations into the effect of St. John's wort (SJW) extract on colorectal carcinogenesis in vivo. This study reports a systematic evaluation of the impact of dietary supplementation of SJW extract on azoxymethane-induced colorectal carcinogenesis in mice. Mice were fed with either AIN-93G (control) diet or SJW extract-supplemented diet (SJW diet) prior to azoxymethane treatment. SJW diet was found to significantly improve the overall survival of azoxymethane-treated mice. Pretreatment with the SJW diet significantly reduced body weight loss as well as decrease of serum albumin and cholesterol levels associated with azoxymethane-induced colorectal tumorigenesis. SJW diet-fed mice showed a significant decrease in tumor multiplicity along with a decrease in incidence of large tumors and a trend toward decreased total tumor volume in a dose-dependent manner. A short-term study, which examined the effect of SJW prior to rectal bleeding, also showed decrease in colorectal polyps in SJW diet-fed mice. Nuclear factor kappa B (NF-κB) and extracellular signal-regulated kinase (ERK1/2) pathways were attenuated by SJW administration. SJW extract resulted in early and continuous attenuation of these pathways in the colon epithelium of SJW diet-fed mice under both short-term and long-term treatment regimens. In conclusion, this study demonstrated the chemopreventive potential of SJW extract against colorectal cancer through attenuation of proinflammatory processes.

Modulation of colon cancer by nutmeg.

Colon cancer is the most common cancer and the third leading cause of cancer mortality in humans. Using mass spectrometry-based metabolomics, the current study revealed the accumulation of four uremic toxins (cresol sulfate, cresol glucuronide, indoxyl sulfate, and phenyl sulfate) in the serum of mice harboring adenomatous polyposis coli (APC) gene mutation-induced colon cancer. These uremic toxins, likely generated from the gut microbiota, were associated with an increase in the expression of the proinflammatory cytokine IL-6 and a disorder of lipid metabolism. Nutmeg, which exhibits antimicrobial activity, attenuated the levels of uremic toxins and decreased intestinal tumorigenesis in Apc(min/+) mice. Nutmeg-treated Apc(min/+) mice had decreased IL-6 levels and normalized dysregulated lipid metabolism, suggesting that uremic toxins are responsible, in part, for the metabolic disorders that occur during tumorigenesis. These studies demonstrate a potential biochemical link among gut microbial metabolism, inflammation, and metabolic disorders and suggest that modulation of gut microbiota and lipid metabolism using dietary intervention or drugs may be effective in colon cancer chemoprevention strategies.

Saikosaponin d protects against acetaminophen-induced hepatotoxicity by inhibiting NF-κB and STAT3 signaling.

Overdose of acetaminophen (APAP) can cause acute liver injury that is sometimes fatal, requiring efficient pharmacological intervention. The traditional Chinese herb Bupleurum falcatum has been widely used for the treatment of several liver diseases in eastern Asian countries, and saikosaponin d (SSd) is one of its major pharmacologically-active components. However, the efficacy of Bupleurum falcatum or SSd on APAP toxicity remains unclear. C57/BL6 mice were administered SSd intraperitoneally once daily for 5days, followed by APAP challenge. Biochemical and pathological analysis revealed that mice treated with SSd were protected against APAP-induced hepatotoxicity. SSd markedly suppressed phosphorylation of nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) and reversed the APAP-induced increases in the target genes of NF-κB, such as pro-inflammatory cytokine Il6 and Ccl2, and those of STAT3, such as Socs3, Fga, Fgb and Fgg. SSd also enhanced the expression of the anti-inflammatory cytokine Il10 mRNA. Collectively, these results demonstrate that SSd protects mice from APAP-induced hepatotoxicity mainly through down-regulating NF-κB- and STAT3-mediated inflammatory signaling. This study unveils one of the possible mechanisms of hepatoprotection caused by Bupleurum falcatum and/or SSd.

Role of white adipose lipolysis in the development of NASH induced by methionine- and choline-deficient diet.

Methionine- and choline-deficient diet (MCD) is a model for nonalcoholic steatohepatitis (NASH) in rodents. However, the mechanism of NASH development by dietary methionine/choline deficiency remains undetermined. To elucidate the early metabolic changes associated with MCD-NASH, serum metabolomic analysis was performed using mice treated with MCD and control diet for 3 days and 1 week, revealing significant increases in oleic and linoleic acids after MCD treatment. These increases were correlated with reduced body weight and white adipose tissue (WAT) mass, increased phosphorylation of hormone-sensitive lipase, and up-regulation of genes encoding carboxylesterase 3 and ß2-adrenergic receptor in WAT, indicating accelerated lipolysis in adipocytes. The changes in serum fatty acids and WAT by MCD treatment were reversed by methionine supplementation, and similar alterations were detected in mice fed a methionine-deficient diet (MD), thus demonstrating that dietary methionine deficiency enhances lipolysis in WAT. MD treatment decreased glucose and increased fibroblast growth factor 21 in serum, thus exhibiting a similar metabolic phenotype as the fasting response. Comparison between MCD and choline-deficient diet (CD) treatments suggested that the addition of MD-induced metabolic alterations, such as WAT lipolysis, to CD-induced hepatic steatosis promotes liver injury. Collectively, these results demonstrate an important role for dietary methionine deficiency and WAT lipolysis in the development of MCD-NASH.

Targeted Metabolomics of Serum Acylcarnitines Evaluates Hepatoprotective Effect of Wuzhi Tablet (Schisandra sphenanthera Extract) against Acute Acetaminophen Toxicity.

Possible prevention and therapeutic intervention strategies to counteract acetaminophen (APAP) hepatotoxicity would be of great value. Wuzhi tablet (WZ, extract of Schisandrae sphenanthera) possesses hepatoprotective effects against hepatitis and the hepatic dysfunction induced by various chemical hepatotoxins. In this study, the protective effect of WZ on APAP-induced hepatic injury was evaluated and targeted metabolomics by LC-MS-based metabolomics was used to examine whether WZ influences hepatic metabolism. The results demonstrated significant hepatoprotection of WZ against APAP-induced liver injury; pretreatment with WZ prior to APAP administration blocks the increase in serum palmitoylcarnitine and oleoylcarnitine and thus restores the APAP-impaired fatty acid ß-oxidation to normal levels. These studies further revealed a significant and prolonged upregulation of the PPARα target genes Cpt1 and Acot1 by WZ mainly contributing to the maintenance of normal fatty acid metabolism and thus potentially contributing to the hepatic protection of WZ against APAP-induced hepatic toxicity. Taken together, the current study provides new insights into understanding the hepatoprotective effect of WZ against APAP-induced liver toxicity.

Novel metabolites and roles for α-tocopherol in humans and mice discovered by mass spectrometry-based metabolomics.

BACKGROUND: Contradictory results from clinical trials that examined the role of vitamin E in chronic disease could be a consequence of interindividual variation, caused by factors such as xenobiotic use. Cometabolism of vitamin E with other pharmaceutical products could affect the bioavailability of the drug. Thus, it is necessary to understand fully the metabolic routes and biological endpoints of vitamin E. OBJECTIVE: The objective was to uncover novel metabolites and roles of vitamin E in humans and mouse models. DESIGN: Human volunteers (n = 10) were fed almonds for 7 d and then an α-tocopherol dietary supplement for 14 d. Urine and serum samples were collected before and after dosing. C57BL/6 mice (n = 10) were also fed α-tocopherol-deficient and -enriched diets for 14 d. Urine, serum, and feces were collected before and after dosing, and liver samples were collected after euthanization. Ultraperformance liquid chromatography electrospray ionization time-of-flight mass spectrometry and multivariate data analysis tools were used to analyze the samples. RESULTS: Three novel urinary metabolites of α-tocopherol were discovered in humans and mice: α-carboxyethylhydroxychroman (α-CEHC) glycine, α-CEHC glycine glucuronide, and α-CEHC taurine. Another urinary metabolite, α-CEHC glutamine, was discovered in mice after α-CEHC gavage. Increases in liver fatty acids and decreases in serum and liver cholesterol were observed in mice fed the α-tocopherol-enriched diet. CONCLUSION: Novel metabolites and metabolic pathways of vitamin E were identified by mass spectrometry-based metabolomics and will aid in understanding the disposition and roles of vitamin E in vivo.

Disruption of phospholipid and bile acid homeostasis in mice with nonalcoholic steatohepatitis.

UNLABELLED: Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease that can develop into cirrhosis, hepatic failure, and hepatocellular carcinoma. Although several metabolic pathways are disrupted and endogenous metabolites may change in NASH, the alterations in serum metabolites during NASH development remain unclear. To gain insight into the disease mechanism, serum metabolite changes were assessed using metabolomics with ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry and a conventional mouse NASH model induced by a methionine- and choline-deficient (MCD) diet. Significant decreases in serum palmitoyl-, stearoyl-, and oleoyl-lysophosphatidylcholine (LPC) and marked increases in tauro-ß-muricholate, taurocholate and 12-hydroxyeicosatetraenoic acid (12-HETE) were detected in mice with NASH. In agreement with these metabolite changes, hepatic mRNAs encoding enzymes and proteins involved in LPC degradation (lysophosphatidylcholine acyltransferase [Lpcat] 1-4), basolateral bile acid excretion (ATP-binding cassette subfamily C member [Abcc] 1/4/5 and organic solute transporter ß), and 12-HETE synthesis (arachidonate 12-lipoxygenase) were significantly up-regulated. In contrast, the expression of solute carrier family 10 member 1 (Slc10a1) and solute carrier organic anion transporter family member (Slco) 1a1 and 1b2, responsible for transporting bile acids into hepatocytes, were markedly suppressed. Supplementation of the MCD diet with methionine revealed that the changes in serum metabolites and the related gene expression were derived from steatohepatitis, but not dietary choline deficiency or steatosis. Furthermore, tumor necrosis factor-α and transforming growth factor-ß1 induced the expression of Lpcat2/4 and Abcc1/4 and down-regulated Slc10a1 and Slco1a1 in primary hepatocytes, suggesting an association between the changes in serum LPC and bile acids and proinflammatory cytokines. Finally, induction of hepatitis in ob/ob mice by D-galactosamine injection led to similar changes in serum metabolites and related gene expression. CONCLUSION: Phospholipid and bile acid metabolism is disrupted in NASH, likely due to enhanced hepatic inflammatory signaling.

Pregnane X receptor-mediated induction of Cyp3a by black cohosh.

Black cohosh (BC) has been widely applied for the treatment of menopausal symptoms. However, increasing concerns about herb-drug interactions demand the need for studies on the influence of BC on cytochrome 450. Cyp3a11 in liver was induced by 7-fold in wild-type mice treated with 500 mg/kg black cohosh for 28 days compared with the control group as assessed by quantitative real-time PCR; no difference was found in small intestine and kidney, suggesting that up-regulation of Cyp3a11 by black cohosh was liver-specific. Western blot, activity assays, and pharmacokinetic analyses established dose- and time-dependent induction of Cyp3a11. To determine the mechanism of Cyp3a11 induction, including the role of pregnane X receptor (PXR) in vivo and in vitro, respectively, in Pxr-null, PXR-humanized, and double transgenic CYP3A4/hPXR mice, cell-based luciferase assays were employed revealing that mouse PXR played a direct role in the induction of Cyp3a11; human PXR was not activated by black cohosh. Overall, these findings demonstrate that induction of Cyp3a11 is liver-specific and involved only mouse PXR, not the human counterpart. Thus, the incidence of herb-drug interaction in patients administered black cohosh may not be mediated by human PXR and CYP3A4.

Serum metabolomics reveals irreversible inhibition of fatty acid beta-oxidation through the suppression of PPARalpha activation as a contributing mechanism of acetaminophen-induced hepatotoxicity.

Metabolic bioactivation, glutathione depletion, and covalent binding are the early hallmark events after acetaminophen (APAP) overdose. However, the subsequent metabolic consequences contributing to APAP-induced hepatic necrosis and apoptosis have not been fully elucidated. In this study, serum metabolomes of control and APAP-treated wild-type and Cyp2e1-null mice were examined by liquid chromatography-mass spectrometry (LC-MS) and multivariate data analysis. A dose-response study showed that the accumulation of long-chain acylcarnitines in serum contributes to the separation of wild-type mice undergoing APAP-induced hepatotoxicity from other mouse groups in a multivariate model. This observation, in conjunction with the increase of triglycerides and free fatty acids in the serum of APAP-treated wild-type mice, suggested that APAP treatment can disrupt fatty acid beta-oxidation. A time-course study further indicated that both wild-type and Cyp2e1-null mice had their serum acylcarnitine levels markedly elevated within the early hours of APAP treatment. While remaining high in wild-type mice, serum acylcarnitine levels gradually returned to normal in Cyp2e1-null mice at the end of the 24 h treatment. Distinct from serum aminotransferase activity and hepatic glutathione levels, the pattern of serum acylcarnitine accumulation suggested that acylcarnitines can function as complementary biomarkers for monitoring the APAP-induced hepatotoxicity. An essential role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the regulation of serum acylcarnitine levels was established by comparing the metabolomic responses of wild-type and Ppara-null mice to a fasting challenge. The upregulation of PPARalpha activity following APAP treatment was transient in wild-type mice but was much more prolonged in Cyp2e1-null mice. Overall, serum metabolomics of APAP-induced hepatotoxicity revealed that the CYP2E1-mediated metabolic activation and oxidative stress following APAP treatment can cause irreversible inhibition of fatty acid oxidation, potentially through suppression of PPARalpha-regulated pathways.

Metabolism of melatonin by human cytochromes p450.

In humans, the pineal hormone melatonin (MEL) is principally metabolized to 6-hydroxymelatonin (6-HMEL), which is further conjugated with sulfate and excreted in urine. MEL O-demethylation represents a minor reaction. The exact role of individual human cytochromes P450 (P450s) in these pathways has not been established. We used a panel of 11 recombinant human P450 isozymes to investigate for the first time the 6-hydroxylation and O-demethylation of MEL. CYP1A1, CYP1A2, and CYP1B1 all 6-hydroxylated MEL, with CYP2C19 playing a minor role. These reactions were NADPH-dependent. CYP2C19 and, to some extent CYP1A2, O-demethylated MEL. The K(m) (microM) and V(max) (k(cat), pmol min(-1) pmol(-1) P450) for 6-hydroxylation were estimated as 19.2 +/- 2.01 and 6.46 +/- 0.22 (CYP1A1), 25.9 +/- 2.47 and 10.6 +/- 0.32 (CYP1A2), and 30.9 +/- 3.76 and 5.31 +/- 0.21 (CYP1B1). These findings confirm the suggestion of others that CYP1A2 is probably the foremost hepatic P450 in the 6-hydroxylation of MEL and a single report that CYP1A1 is also able to mediate this reaction. However, this is the first time that CYP1B1 has been shown to 6-hydroxylate MEL. The IC50 for the CYP1B1-selective inhibitor (E)-2,4,3',5'-tetramethoxystilbene was estimated to be 30 nM for MEL 6-hydroxylation by recombinant human CYP1B1. Comparison of brain homogenates from wild-type and cyp1b1-null mice revealed that MEL 6-hydroxylation was clearly mediated to a significant degree by CYP1B1. CYP1B1 is not expressed in the liver but has a ubiquitous extrahepatic distribution, and is found at high levels in tissues that also accumulate either MEL or 6-HMEL, such as intestine and cerebral cortex, where it may assist in regulating levels of MEL and 6-HMEL.