AMP-activated protein kinase-farnesoid X receptor pathway contributes to oleanolic acid-induced liver injury.

Natural pentacyclic triterpenoid oleanolic acid (OA) is used as an over-the-counter drug for acute and chronic hepatitis. However, clinical use of OA-containing herbal medicines has been reported to cause cholestasis, and the specific mechanism is unknown. The purpose of this study was to explore how OA causes cholestatic liver injury via the AMP-activated protein kinase (AMPK)-farnesoid X receptor (FXR) pathway. In animal experiments, it was found that OA treatment activated AMPK and decreased FXR and bile acid efflux transport proteins expression. When intervened with the specific inhibitor Compound C (CC), it was observed that AMPK activation was inhibited, the reduction of FXR and bile acid efflux transport protein expression was effectively alleviated, serum biochemical indicators were significantly reduced, and liver pathological damage brought about by OA was effectively ameliorated. In addition, OA was found to downregulate the expression of FXR and bile acid efflux transport proteins by activating the ERK1/2-LKB1-AMPK pathway in cellular experiments. The ERK1/2 inhibitor U0126 was used to pretreat primary hepatocytes, and this drastically reduced the phosphorylation levels of LKB1 and AMPK. The inhibition effects of OA on FXR and bile acid efflux transport proteins were also effectively alleviated after pretreatment with CC. In addition, OA-induced downregulation of FXR gene and protein expression levels was significantly prevented after silencing AMPKα1 expression in AML12 cells. Our study demonstrated that OA inhibited FXR and bile acid efflux transporters through the activation of AMPK, thus leading to cholestatic liver injury.

Suppressed farnesoid X receptor by iron overload in mice and humans potentiates iron-induced hepatotoxicity.

BACKGROUND AND AIMS: Iron overload (IO) is a frequent finding in the general population. As the major iron storage site, the liver is subject to iron toxicity. Farnesoid X receptor (FXR) regulates bile acid metabolism and is implicated in various liver diseases. We aimed to determine whether FXR plays a role in regulating iron hepatotoxicity. APPROACH AND RESULTS: Human and mouse hepatocytes were treated with ferric ammonium citrate or iron dextran (FeDx). Mice were orally administered ferrous sulfate or injected i.p. with FeDx. Wild-type and Fxr-/- mice were fed an iron-rich diet for 1 or 5 weeks. Mice fed an iron-rich diet were coadministered the FXR agonist, GW4064. Forced expression of FXR was carried out with recombinant adeno-associated virus 1 week before iron-rich diet feeding. Serum levels of bile acids and fibroblast growth factor 19 (FGF19) were quantified in adults with hyperferritinemia and children with ß-thalassemia. The data demonstrated that iron suppressed FXR expression and signaling in human and mouse hepatocytes as well as in mouse liver and intestine. FXR deficiency potentiated iron hepatotoxicity, accompanied with hepatic steatosis as well as dysregulated iron and bile acid homeostasis. FXR negatively regulated iron-regulatory proteins 1 and 2 and prevented hepatic iron accumulation. Forced FXR expression and ligand activation significantly suppressed iron hepatotoxicity in iron-fed mice. The FXR agonist, GW4064, almost completely restored dysregulated bile acid signaling and metabolic syndrome in iron-fed mice. Conjugated primary bile acids were increased and FGF19 was decreased in serum of adults with hyperferritinemia and children with ß-thalassemia. CONCLUSIONS: FXR plays a pivotal role in regulating iron homeostasis and protects mice against iron hepatotoxicity. Targeting FXR may represent a therapeutic strategy for IO-associated chronic liver diseases.

Sex-, Age-, and Race/Ethnicity-Dependent Variations in Drug-Processing and NRF2-Regulated Genes in Human Livers.

Individual variations in xenobiotic metabolism affect the sensitivity to diseases. In this study, the impacts of sex, age, and race/ethnicity on drug-processing genes and nuclear factor erythroid 2-related factor 2 (NRF2) genes in human livers were examined via QuantiGene multiplex suspension array (226 samples) and quantitative polymerase chain reaction (qPCR) (247 samples) to profile the expression of nuclear receptors, cytochrome P450s, conjugation enzymes, transporters, bile acid metabolism, and NRF2-regulated genes. Sex differences were found in expression of about half of the genes, but in general the differences were not large. For example, females had higher transcript levels of catalase, glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase 1 (HO-1), Kelch-like ECH-associated protein 1 (KEAP1), superoxide dismutase 1, and thioredoxin reductase-1 compared with males via qPCR. There were no apparent differences due to age, except children had higher glutamate-cysteine ligase modifier subunit (GCLM) and elderly had higher multidrug resistance protein 3. African Americans had lower expression of farnesoid X receptor (FXR) but higher expression of HO-1, Caucasians had higher expression of organic anion transporter 2, and Hispanics had higher expression of FXR, SULT2A1, small heterodimer partner, and bile salt export pump. An examination of 34 diseased and control human liver samples showed that compared with disease-free livers, fibrotic livers had higher NAD(P)H-quinone oxidoreductase 1 (NQO1), GCLC, GCLM, and NRF2; hepatocellular carcinoma had higher transcript levels of NQO1 and KEAP1; and steatotic livers had lower GCLC, GCLM, and HO-1 expression. In summary, in drug-processing gene and NRF2 genes, sex differences were the major findings, and there were no apparent age differences, and race/ethnicity differences occurred for a few genes. These descriptive findings could add to our understanding of the sex-, age-, and race/ethnicity-dependent differences in drug-processing genes as well as NRF2 genes in normal and diseased human livers. SIGNIFICANCE STATEMENT: In human liver drug-processing and nuclear factor erythroid 2-related factor 2 genes, sex differences were the main finding. There were no apparent differences due to age, except children had higher glutamate-cysteine ligase modifier subunit, and elderly had higher multidrug resistance protein 3. African Americans had lower expression of farnesoid X receptor (FXR) but higher expression of heme oxygenase 1, Caucasians had higher expression of organic anion transporter 2, and Hispanics had higher expression of FXR, small heterodimer partner, SULT2A1, and bile salt export pump.

LC-MS-Based Metabolomic Study of Oleanolic Acid-Induced Hepatotoxicity in Mice.

Oleanolic acid (OA), a natural triterpenoid, which has the development prospects in anti-tumor therapy is a widely used hepatoprotective drug in China. It has been reported that OA can cause liver toxicity after higher doses or longer-term use. Therefore, the study aims to explore the possible hepatotoxicity mechanism based on liver metabolic profiles. Liver metabolic profiles were obtained from untargeted ultrahigh performance liquid chromatography (UHPLC)-Q Exactive Orbitrap mass spectrometry (MS) technique. It was found that altered bile acid, amino acid, and energy metabolism might be at least partly responsible for OA-induced hepatotoxicity. Bile acid metabolism, as the most important pathway, was verified by using UHPLC-TSQ-MS, indicating that conjugated bile acids were the main contributors to OA-induced liver toxicity. Our findings confirmed that increased bile acids were the key element of OA hepatotoxicity, which may open new insights for OA hepatotoxicity in-depth investigations, as well as provide a reference basis for more hepatotoxic drug mechanism research.

Automatic recognition of breast invasive ductal carcinoma based on terahertz spectroscopy with wavelet packet transform and machine learning.

We demonstrate an automatic recognition strategy for terahertz (THz) pulsed signals of breast invasive ductal carcinoma (IDC) based on a wavelet entropy feature extraction and a machine learning classifier. The wavelet packet transform was implemented into the complexity analysis of the transmission THz signal from a breast tissue sample. A novel index of energy to Shannon entropy ratio (ESER) was proposed to distinguish different tissues. Furthermore, the principal component analysis (PCA) method and machine learning classifier were further adopted and optimized for automatic classification of the THz signal from breast IDC sample. The areas under the receiver operating characteristic curves are all larger than 0.89 for the three adopted classifiers. The best breast IDC recognition performance is with the precision, sensitivity and specificity of 92.85%, 89.66% and 96.67%, respectively. The results demonstrate the effectiveness of the ESER index together with the machine learning classifier for automatically identifying different breast tissues.

Metformin Disrupts Bile Acid Efflux by Repressing Bile Salt Export Pump Expression.

PURPOSE: The bile salt export pump (BSEP), a key player in hepatic bile acid clearance, has been the center of research on drug-induced cholestasis. However, such studies focus primarily on the direct inhibition of BSEP, often overlooking the potential impact of transcriptional repression. This work aims to explore the disruption of bile acid efflux caused by drug-induced BSEP repression. METHODS: BSEP activity was analyzed in human primary hepatocytes (HPH) using a traditional biliary-clearance experiment and a modified efflux assay, which includes a 72-h pretreatment prior to efflux measurement. Relative mRNA and protein expressions were examined by RT-PCR and Western blotting, respectively. RESULTS: Metformin concentration-dependently repressed BSEP expression in HPH. Although metformin did not directly inhibit BSEP activity, longer metformin exposure reduced BSEP transport function in HPH by down-regulating BSEP expression. BSEP repression by metformin was found to be AMP-activated protein kinase-independent. Additional screening of 10 reported cholestatic non-BSEP inhibitors revealed that the anti-cancer drug tamoxifen also markedly repressed BSEP expression and reduced BSEP activity in HPH. CONCLUSIONS: Repression of BSEP alone is sufficient to disrupt hepatic bile acid efflux. Metformin and tamoxifen appear to be prototypes of a class of BSEP repressors that may cause drug-induced cholestasis through gene repression instead of direct BSEP inhibition.

Glucocorticoids Increase Renal Excretion of Urate in Mice by Downregulating Urate Transporter 1.

Both nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids have been widely used for the treatment of gout, a disease promoted by an excess body burden of uric acid (UA); however, their effects on the homeostasis of UA remain poorly understood. The present study showed that 1-week treatments with three NSAIDs (ibuprofen, diclofenac, and indomethacin) had little effect on UA homeostasis in mice, whereas 1-week low doses (1 and 5 mg/kg) of dexamethasone (DEX) significantly decreased serum UA by about 15%. Additionally, low doses of DEX also resulted in increases in hepatic UA concentration and urinary UA excretion, which were associated with an induction of xanthine oxidoreductase (XOR) in the liver and a downregulation of urate transporter 1 (URAT1) in the kidney, respectively. Neither 75 mg/kg DEX nor 100 mg/kg pregnenolone-16α-carbonitrile altered UA concentrations in serum and livers of mice, suggesting that the effect of DEX on UA homeostasis was not due to the pregnane X receptor pathway. Further in vitro studies demonstrated that glucocorticoid receptor (GR) was involved in DEX-mediated downregulation of URAT1. Knockdown of both p65 and c-Jun completely blocked the effect of DEX on URAT1, suggesting that GR regulates URAT1 via its interaction with both nuclear factor κB and activator protein 1 signaling pathways. To conclude, the present study identifies, for the first time, a critical role of glucocorticoids in regulating UA homeostasis and elucidates the mechanism for GR-mediated regulation of URAT1 in mice. SIGNIFICANCE STATEMENT: This study demonstrates, for the first time, a critical role of glucocorticoid receptor in regulating urate transporter 1 in mouse kidney.

Role of intestinal microbiota-mediated genipin dialdehyde intermediate formation in geniposide-induced hepatotoxicity in rats.

Geniposide is a natural hepatotoxic iridoid glycoside. Its hydrolysate of intestinal microbiota, genipin, is thought to be responsible for the hepatotoxicity. However, the underlying mechanism that genipin contributes to the hepatotoxicity of geniposide is not well understood. In this study, we found that genipin spontaneously converted into a reactive dialdehyde intermediate and covalently bond to the primary amine group of free amino acids in both of the phosphate buffers and geniposide-treated rats. Furthermore, genipin dialdehyde can form the covalent linkage to the primary amino group (ε) of lysine side chains of the hepatic proteins in geniposide-treated rats. Pretreatment with ß-glucosidase or antibiotics significantly modulated the genipin dialdehyde formation and protein modification, revealing the essential role of microbial glycosidases. The levels of protein adduct were that mapped onto the hepatotoxicity of geniposide. In summary, this study demonstrates that the intestinal microbiota mediated covalent modification of the hepatic protein by genipin dialdehyde may play a crucial role in the liver injury of geniposide. The study is also helpful for understanding the contribution of intestinal microbiota to the metabolic activation of xenobiotics.

Icariin protects rotenone-induced neurotoxicity through induction of SIRT3.

Sirtuin-3 (SIRT3) is a mitochondrial NAD + -dependent deacetylase that is essential in regulating mitochondrial proteins and maintaining cellular antioxidant properties. It has been reported that icariin (ICA) is neuroprotective over various neurotoxicant induced oxidative stress. This study aimed to determine whether ICA exerts neuroprotective effects on rotenone (ROT)-induced neurotoxicity through activation of SIRT3. Rats treated with ROT exhibited a marked loss of dopamine (DA) neurons and a decline in motor function, along with a decrease in protein expressions of SIRT3 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the substantia nigra (SN). Administration of ICA significantly alleviated the loss of DA neurons, improved behavioral function, and concomitantly enhanced SIRT3 and PGC-1α expressions. The neuroprotective effect of ICA on ROT-induced cytotoxicity was further confirmed in the PC12 cell model, which showed significant improvement in the survival of ROT-treated cells with ICA pretreatment. The cytoprotective effect of ICA was abolished in ROT-treated cells by SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP), along with a resultant decrease in PGC-1α expression. In addition, knockdown of PGC-1α by siRNA suppressed ICA-mediated protective effects but did not affect SIRT3 expression, indicating the role of regulation of PGC-1α by SIRT3 in the protective action of ICA. Furthermore, we showed that ICA improved mitochondrial respiration, oxidative status, enhanced antioxidant enzyme SOD activity and GSH/GSSG ratio in cells treated with ROT. However, these protective effects of ICA on ROT-treated cells was markedly abolished by SIRT3 inhibitor 3-TYP. Our findings demonstrate that ICA exerts a neuroprotective role through upregulation of SIRT3.

Trabid inhibits hepatocellular carcinoma growth and metastasis by cleaving RNF8-induced K63 ubiquitination of Twist1.

TRAF-binding domain (Trabid), one of deubiquitination enzymes, was recently reported to activate Wnt/ ß-catenin signaling pathway. However, the role of Trabid in tumors including hepatocellular carcinoma (HCC) and the underlying mechanisms controlling its activity remain poorly understood. Here, we report that Trabid is significantly downregulated in HCC tumor samples and cell lines compared with normal controls and that its expression level is negatively correlated with HCC pathological grading, recurrence, and metastasis. The reintroduction of Trabid expression in tumor cells significantly decreases HCC progression as well as pulmonary metastasis. The effect of Trabid on HCC development occurs at least partially through regulation of Twist1 activity. Mechanistically, Trabid forms a complex with Twist1 and specifically cleaves RNF8-induced K63-linked poly-ubiquitin chains from Twist1, which enhances the association of Twist1 with ß-TrCP1 and allows for subsequent K48-linked ubiquitination of Twist1. Knockdown of Trabid increases K63-linked ubiquitination, but abrogates K48-linked ubiquitination and degradation of Twist1, thus enhancing HCC growth and metastasis. Interestingly, Twist1 negatively regulates the promoter activity of Trabid, indicating that a double-negative feedback loop exists. Our findings also identify an essential role for activation of Trabid by AKT-mediated phosphorylation at Ser78/Thr117 in negatively regulating Twist1 signaling, which further provides insights into the mechanisms by which Trabid regulates Twist1 ubiquitination. Our results reveal that Trabid is a previously unrecognized inhibitor of HCC progression and metastasis, which sheds light on new strategies for HCC treatment.

Dictamnine-induced hepatotoxicity in mice: the role of metabolic activation of furan.

Cortex Dictamni is extensively used as an herbal medicine worldwide, but is believed to induce hepatotoxicity and even causes mortality in many Asian and European countries. As the most abundant furoquinoline alkaloid ingredient of Cortex Dictamni, dictamnine (DIC) can be metabolically activated by CYP3A to an epoxide metabolite, which possesses the potential to induce hepatotoxicity by covalent binding with proteins. As yet, the hepatotoxicity of DIC and the role played by metabolic activation remain unknown. Here, we found that DIC caused acute liver injury in a time- and dose-dependent manner in mice. The hepatic and urinary DIC epoxide intermediates were observed in DIC-treated mice. Ketoconazole, a CYP3A inhibitor, significantly reduced the hepatotoxicity of DIC and inhibited the formation of reactive metabolites of DIC. Moreover, treatment with 2,3-dihydro-DIC, a DIC analog synthesized by selective reduction of the furan moiety, produced no hepatotoxicity in mice, and no reactive metabolite was formed, suggesting a structural necessity of furan moiety in DIC hepatotoxicity. A time course of gradual hepatic glutathione consumption was observed in DIC-treated mice, while depletion of hepatic glutathione by L-buthionine-S,R-sulfoximine enhanced the hepatotoxicity of DIC. Collectively, this study demonstrates that DIC induces acute hepatocellular injury in mice, and that metabolic activation of furan plays a crucial role in DIC-induced hepatotoxicity.

Autophagy plays a protective role in Mn-induced toxicity in PC12 cells.

Excessive environmental or occupational exposure to manganese (Mn) is associated with increased risk of neuron degenerative disorders. Oxidative stress and mitochondrial dysfunction are the main mechanisms of Mn mediated neurotoxicity. Selective removal of damaged mitochondria by autophagy has been proposed as a protective mechanism against neuronal toxicant-induced neurotoxicity. Whether autophagic flux plays a role in Mn-induced cytotoxicity remains to be fully elucidated. The present study was designed to investigate the effect of Mn exposure on autophagy, and how modulation of autophagic flux alters the sensitivities of cells to Mn-elicited cytotoxicity. Rat adrenal pheochromocytoma PC12 cells were treated with Mn for 24h to establish a cellular mode of Mn toxicity. Treatment of cells with Mn resulted in increased expression of autophagic marker LC3-II protein, as well as accumulation of p62, indicating an interference of autophagy flux caused by Mn. Pre-incubation of cells with antioxidant N-acetyl-l-cysteine (NAC) or resveratrol improved cell survival, accompanied by decreased LC3-II expression and increased expression level of p62, suggesting a down regulation of autophagy flux. To further determine the role of autophagy in Mn-induced cytotoxicity, the effect of chloroquine and rapamycin on cell viability was examined. Inhibition of autophagy flux by chloroquine exacerbated Mn-induced cytotoxicity, while induction of autophagy by rapamycin significantly reduced cell death caused by Mn. Furthermore, it was found that rapamycin, NAC and resveratrol improved cellular oxygen consumption accompanied by a decrease in cellular ROS generation and increase in GSH level, while chloroquine suppressed cellular respiration and deteriorated cellular oxidative stress. Collectively, these results demonstrate that autophagy plays a protective role in Mn-induced cell toxicity. Antioxidants NAC and resveratrol confer protective role in Mn toxicity mainly through maintaining mitochondrial dynamics and function, other than a modulation of autophagy flux.

Activation of intestinal GR-FXR and PPARα-UGT signaling exacerbates ibuprofen-induced enteropathy in mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs)-induced small intestinal injury (enteropathy) occurs in about two-thirds of regular NSAID users. To date, there is no proven-effective treatment for NSAID enteropathy, and its underlying mechanism remains obscure. The present study showed that glucocorticoids are an important determinant of NSAID enteropathy. High dose dexamethasone (DEX, 75 mg/kg) markedly exacerbated the acute toxicity of ibuprofen (IBU, 200 mg/kg) in the small intestine of mice, which was not due to the pregnane-X-receptor pathway. Instead, glucocorticoid receptor (GR) mediated the effect of DEX (5 mg/kg) on both the acute (200 mg/kg) and 7-day repeated-dose (50 mg/kg) toxicity of IBU in the small intestine. Combined treatment of DEX (5 mg/kg) and IBU (50 mg/kg) synergistically repressed the intestinal farnesoid X receptor (FXR)-cystathionine-γ-lyase signaling, which was accompanied with an elevation in the biliary excretion of bile acids, especially the FXR antagonist tauro-ß-muricholic acid. DEX (5 mg/kg) also activated intestinal peroxisome proliferator-activated receptor α (PPARα)-UDP-glucuronosyltransferase (UGT) pathway, which increased the formation and enterohepatic circulation of IBU-acyl glucuronide. Furthermore, DEX (5 mg/kg) and IBU (50 mg/kg) altered the intestinal microbial composition, characterized with a marked decrease in Actinobacteria. To conclude, the present study for the first time suggests that glucocorticoids play vital roles in control of IBU enteropathy via intestinal GR-FXR and PPARα-UGT signaling.

Oeanolic acid protects against the hepatotoxicity of D-galactosame plus endotoxin in mice.

Oleanolic acid (OA) is a triterpenoid contained in many herbal medicines. The aim of this study was to investigate the protective effect of OA against D-galactosame plus lipopolysaccharides (D-GalN/LPS)-induced acute liver injury and the underling mechanisms. Mice were randomly divided into normal control with vehicles only (corn oil), D-GalN/LPS only (700mg/10µg/kg, ip), OA-po (200µmol/kg in corn oil, po) plus D-GalN/LPS, and OA-sc (50µmol/kg in 2% tween 80, sc) plus D-GalN/LPS groups. OA pretreatment was conducted twice daily for 4 consecutive days. Hepatotoxicity was evaluated by histopathology, serum enzyme activity, hepatic lipid peroxidation and GSH levels. To reveal the possible mechanisms of the protection, mRNA and protein expressions of toxicity-relevant genes and proteins were examined by real-time RT-PCR and western-blot analysis. Both OA-po and OA-sc at therapeutic doses successfully protected liver injury induced by D-GalN/LPS, as evidenced by reduced serum enzyme activities, prevented liver hemorrhage, massive necrosis, and reduced degenerative lesions. OA increased hepatic GSH contents and decreased lipid peroxidation (MDA) levels. Furthermore, OA significantly inhibited the mRNA expression of the tumor necrosis factor-α (TNF-α) and ER responsive gene Gadd45. D-GalN/LPS-induced activation of p-JNK and NF-κB p65, and protein overexpression of caspase-3, caspase-8, and COX2 were significantly suppressed by OA. These results clearly demonstrated OA-po is effective as OA-sc in protecting against D-GalN/LPS-induced liver injury, and the protection mechanisms are related to reduction of oxidative damage, suppression of TNFα-triggered signaling through the NF-kB and JNK pathways, thus reducing apoptosis and hepatocellular death.

Age-associated differences in transporter gene expression in kidneys of male rats.

Kidney transporters are involved in the secretion and reabsorption of endogenous and exogenous molecules. Numerous factors may influence their expression and affect drug disposition, efficacy and toxicity. The present study aimed to examine the development­ and age­associated variations in primary renal transporters in rats, including 6 uptake transporters: Organic anion transporter (OAT) 1 and 3, organic cation transporter (OCT) 1, 2 and 3 and organic anion­transporting polypeptide (OATP) 4C1, and 6 efflux transporters: Multidrug resistance protein 1 (MDR1), breast cancer resistance protein (BCRP), multidrug resistance­associated protein (MRP) 2 and 4, and multidrug and toxin extrusion protein (MATE) 1 and 2­K. Kidneys from male Sprague Dawley rats during development (­2, 1, 7, 14 and 21 days), maturation (28, 35 and 60 days) and aging (180, 540 and 850 days) were collected and total RNA was extracted, purified and subjected to reverse transcription­quantitative polymerase chain reaction analysis. Total proteins were extracted for western blot analysis. OAT1 and 3, OCT1, BCRP, MRP2 and 4 and MATE2­K expression levels were low in fetal kidneys, increased gradually following birth and markedly increased on maturation and adulthood. High levels were maintained until 850 days. OCT2, OATP4C1, Mdr1b and MATE1 expression levels were low in fetal kidneys, increased gradually following birth, and increased markedly on weaning, maturation and adulthood; however, levels were decreased on aging. OCT3 mRNA expression levels were low in fetal and newborn kidneys, and had two peaks at 35 and 850 days. The selected OAT1 and 3 and MDR1 protein expression levels revealed a similar expression pattern. Thus, kidney transporter expression is affected by ontogeny and aging, which may impact drug and toxicant disposition in children and the elderly.

Dysregulation of metallothionein and circadian genes in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the major threat to human health, and disruption of circadian clock genes is implicated in hepatocarcinogenesis. This study examined the dysregulation of metallothioneins and circadian genes in achieved human HCC (n = 24), peri-HCC tissues (n = 24) as compared with normal human livers (n = 36). Total RNA was extracted and reverse transcribed. Real-time RT-qPCR was performed to determine the expression of genes of interest. The results demonstrated the downregulation of metallothionein-1 (MT-1), MT-2, and metal transcription factor-1 (MFT-1) in human HCC as compared with Peri-HCC and normal tissues. MTs are a biomarker for HCC and have typical circadian rhythms; the expression of major circadian clock genes was also determined. HCC produced a dramatic decrease in the expression of core clock genes, circadian locomotor output cycles kaput (Clock) and brain and muscle Arnt-like protein 1 (Bmal1), and decreased the expression of the clock feedback control genes, Periods (Per1, Per2) and Cryptochromes (Cry1, Cry2). On the other hand, the expression of clock target genes nuclear orphan receptor factor protein (Nr1d1) and D-box-binding protein (Dbp) was upregulated as compared with Peri-HCC and normal livers. Peri-HCC also had mild alterations in these gene expressions. In summary, the present study clearly demonstrated the dysregulation of MTs and circadian clock genes in human HCC, which could provide the information of targeting MT and circadian clock in HCC management.

Mercury sulfides are much less nephrotoxic than mercury chloride and methylmercury in mice.

Mercury sulfides (α-HgS, ß-HgS) are frequently included in traditional medicines. Mercury is known for nephrotoxicity, their safety is of concern. To address this question, mice were orally administrated with Zuotai (54% ß-HgS, 30mg/kg), α-HgS (HgS, 30mg/kg), HgCl2 (33.6mg/kg), or MeHgCl (3.1mg/kg) for 7days, and nephrotoxicity was examined. Animal body weights were decreased by HgCl2 and to a lesser extent by MeHg, but unaltered after Zuotai and HgS. HgCl2 and MeHg produced renal tubular vacuolation, interstitial inflammation and cell degeneration with protein cysts in the tubular lumen, while these pathological lesions were mild in Zuotai and HgS-treated mice. Electron microscopy showed that HgCl2 and MeHg produced spotted swelling endothelium reticulum, while these lesions were mild or absent in Zuotai and HgS-treated mice. Renal Hg contents reached 250-300ng/mg kidney in HgCl2 and MeHg groups as compared to 2-3ng/mg in Zuotai and HgS groups. The expression of kidney injury biomarkers, kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (Ngal), were increased after HgCl2 and MeHg, but unaltered after Zuotai and HgS. The expression of renal influx transporters Oat3 and Oatp4c1 was decreased, while the expression of renal efflux transporter such as Mrp2, Mrp4, and Mate2 was increased following HgCl2 and MeHg. These gene expressions were unchanged after Zuotai and HgS. In summary, both α-HgS and ß-HgS are less nephrotoxic than HgCl2 and MeHg, indicating that chemical forms of mercury are a major determinant of mercury disposition and toxicity.

Characterizing novel metabolic pathways of melatonin receptor agonist agomelatine using metabolomic approaches.

Agomelatine (AGM), an analog of melatonin, is a potential agonist at melatonin receptors 1/2 and a selective antagonist at 5-hydroxytryptamine 2C receptors. AGM is widely used for the treatment of major depressive episodes in adults. However, multiple adverse effects associated with AGM have been reported in clinical practice. It is little known about AGM metabolism in vitro and in vivo, although metabolism plays a pivotal role in its efficacy and safety. To elucidate metabolic pathways of AGM, we systemically investigated AGM metabolism and its bioactivation in human liver microsomes (HLM) and mice using metabolomic approaches. We identified thirty-eight AGM metabolites and adducts, among which thirty-two are novel. In HLM, we uncovered five GSH-trapped adducts and two semicarbazide-trapped aldehydes. Moreover, we characterized three N-acetyl cysteine conjugated-AGM adducts in mouse urine and feces, which were formed from the degradation of AGM_GSH adducts. Using recombinant CYP450 isoenzymes and chemical inhibitors, we demonstrated that CYP1A2 and CYP3A4 are primary enzymes contributing to the formation of AGM_GSH adducts and AGM_hydrazones. This study provided a global view of AGM metabolism and identified the novel pathways of AGM bioactivation, which could be utilized for further understanding the mechanism of adverse effects related to AGM and possible drug-drug interactions.

Puerarin Improves Diabetic Aorta Injury by Inhibiting NADPH Oxidase-Derived Oxidative Stress in STZ-Induced Diabetic Rats.

OBJECTIVE: Puerarin is a natural flavonoid isolated from the TCM lobed kudzuvine root. This study investigated the effect and mechanisms of puerarin on diabetic aorta in rats. METHODS: Streptozotocin- (STZ-) induced diabetic rats were administered with puerarin for 3 weeks. Levels of serum insulin (INS), PGE2, endothelin (ET), glycated hemoglobin (GHb), H2O2, and nitric oxide (NO) in rats were measured by ELISA and colorimetric assay kits. The aortas were stained with H&E. Moreover, the mRNA expression of ICAM-1, LOX-1, NADPH oxidase 2 (NOX2), and NOX4 and the protein expression of ICAM-1, LOX-1, NF-κB p65, E-selectin, NOX2, and NOX4 in aorta tissues were measured by real-time PCR and Western blot, respectively. The localization of ICAM-1, NF-κB p65, NOX2, and NOX4 in the aorta tissues was also determined through immunohistochemistry. RESULTS: Puerarin treatment exerted no effect on fasting blood glucose levels but significantly reduced the serum levels of INS, GHb, PGE2, ET, H2O2, and NO. In addition, puerarin improved the pathological alterations and inhibited the expression of ICAM-1, LOX-1, NOX2, and NOX4 at both mRNA and protein levels. Puerarin also significantly reduced the number of cells showing positive staining for ICAM-1, NOX2, NOX4, and NF-κB p65. CONCLUSION: Puerarin demonstrated protective effect on the STZ-induced diabetic rat aorta. The protective mechanisms may include regulation of NF-κB and inhibition of NOX2 and NOX4 followed by inhibition of cell adhesion molecule expression.

Liver expression of Nrf2-related genes in different liver diseases.

BACKGROUND: The KEAP1-Nrf2 antioxidant signaling pathway is important in protecting liver from various insults. However, little is known about the expression of Nrf2-related genes in human liver in different diseases. METHODS: This study utilized normal donor liver tissues (n=35), samples from patients with hepatocellular carcinoma (HCC, n=24), HBV-related cirrhosis (n=27), alcoholic cirrhosis (n=5) and end-stage liver disease (n=13). All of the liver tissues were from the Oriental Liver Transplant Center, Beijing, China. The expressions of Nrf2 and Nrf2-related genes, including its negative regulator Kelch-like ECH-associated protein 1 (KEAP1), its targeted gene NAD(P)H-quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC) and modified subunit (GCLM), heme oxygenase 1 (HO-1) and peroxiredoxin-1 (PRDX1) were evaluated. RESULTS: The expression of Nrf2 was decreased in HCC, increased in alcoholic cirrhosis and end-stage liver disease. The expression of KEAP1 was increased in all of the liver samples. The most notable finding was the increased expression of NQO1 in HCC (18-fold), alcoholic cirrhosis (6-fold), end-stage liver disease (5-fold) and HBV-related cirrhosis (3-fold). Peri-HCC also had 4-fold higher NQO1 mRNA as compared to the normal livers. GCLC mRNA levels were lower only in HCC, as compared to the normal livers and peri-HCC tissues. GCLM mRNA levels were higher in HBV-related cirrhosis and end-stage liver disease. HO-1 mRNA levels were increased in all liver tissues except for HCC. Peri-HCC had higher PRDX1 mRNA levels compared with HCC and normal livers. CONCLUSION: Nrf2 and Nrf2-related genes are aberrantly expressed in the liver in different diseases and the increase of NQO1 was the most notable finding, especially in HCC.