Fasting potentiates diclofenac-induced liver injury via inductions of oxidative/endoplasmic reticulum stresses and apoptosis, and inhibition of autophagy by depleting hepatic glutathione in mice.

Diclofenac, a widely used non-steroidal anti-inflammatory drug, can cause liver damage via its metabolic activation by hepatic CYP450s and UGT2B7. Fasting can affect drug-induced liver injury by modulating the hepatic metabolism, but its influence on diclofenac hepatotoxicity is unknown. Thus, we investigated diclofenac-induced liver damage after fasting in mice, and the cellular events were examined. Male ICR mice fasted for 16 h showed the elevation of CYP3A11, but the decreases of UGT2B7, glutathione (GSH), and GSH S-transferase-µ/-π levels in the livers. Diclofenac (200 mg/kg) injection into the mice after 16-h fasting caused more significant liver damage compared to that in the diclofenac-treated fed mice, as shown by the higher serum ALT and AST activities. Diclofenac-promoted hepatic oxidative stress (oxidized proteins, 4-hydroxynonenal, and malondialdehyde), endoplasmic reticulum (ER) stress (BiP, ATF6, and CHOP), and apoptosis (cleaved caspase-3 and cleaved PARP) were enhanced by fasting. Autophagic degradation was inhibited in the diclofenac-treated fasting mice compared to that of the corresponding fed mice. The results suggest that fasting can make the liver more susceptible to diclofenac toxicity by lowering GSH-mediated detoxification; increased oxidative/ER stresses and apoptosis and suppressed autophagic degradation may be the cellular mechanisms of the aggravated diclofenac hepatotoxicity under fasting conditions.

Role of autophagy in betaine-promoted hepatoprotection against non-alcoholic fatty liver disease in mice.

Betaine, a compound found in plants and sea foods, is known to be beneficial against non-alcoholic fatty liver disease (NAFLD), but its hepatoprotective and anti-steatogenic mechanisms have been not fully understood. In the present study, we investigated the mechanisms underlying betaine-mediated alleviation of NAFLD induced by a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) in mice, with special focus on the contribution of betaine-stimulated autophagy to NAFLD prevention. Male ICR mice were fed a CDAHFD with or without betaine (0.2-1% in drinking water) for 1 week. Betaine ameliorated the CDAHFD-induced fatty liver by restoring sulfur amino acid (SAA)-related metabolites, such as S-adenosylmethionine and homocysteine, and the phosphorylation of AMPK and ACC. In addition, it reduced the CDAHFD-induced ER stress (BiP, ATF6, and CHOP) and apoptosis (Bax, cleaved caspase-3, and cleaved PARP); however, it induced autophagy (LC3II/I and p62) which was downregulated by CDAHFD. To determine the role of autophagy in the improvement of NAFLD, chloroquine (CQ), an autophagy inhibitor, was injected into the mice fed a CDAHFD and betaine (0.5 % in drinking water). CQ did not affect SAA metabolism but reduced the beneficial effects of betaine as shown by the increases of hepatic lipids, ER stress, and apoptosis. Notably, the betaine-induced improvements in lipid metabolism determined by protein levels of p-AMPK, p-ACC, PPARα, and ACS1, were reversed by CQ. Thus, the results of this study suggest that the activation of autophagy is an important upstream mechanism for the inhibition of steatosis, ER stress, and apoptosis by betaine in NAFLD.

Doxorubicin Attenuates Free Fatty Acid-Induced Lipid Accumulation via Stimulation of p53 in HepG2 Cells.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of fat in the liver, and there is a global increase in its incidence owing to changes in lifestyle and diet. Recent findings suggest that p53 is involved in the development of non-alcoholic fatty liver disease; however, the association between p53 expression and the disease remains unclear. Doxorubicin, an anticancer agent, increases the expression of p53. Therefore, this study aimed to investigate the role of doxorubicin-induced p53 upregulation in free fatty acid (FFA)-induced intracellular lipid accumulation. HepG2 cells were pretreated with 0.5 µg/mL of doxorubicin for 12 h, followed by treatment with FFA (0.5 mM) for 24 h to induce steatosis. Doxorubicin pretreatment upregulated p53 expression and downregulated the expression of endoplasmic reticulum stress- and lipid synthesis-associated genes in the FFA -treated HepG2 cells. Additionally, doxorubicin treatment upregulated the expression of AMP-activated protein kinase, a key modulator of lipid metabolism. Notably, siRNA-targeted p53 knockdown reversed the effects of doxorubicin in HepG2 cells. Moreover, doxorubicin treatment suppressed FFA -induced lipid accumulation in HepG2 spheroids. Conclusively, these results suggest that doxorubicin possesses potential application for the regulation of lipid metabolism by enhance the expression of p53 an in vitro NAFLD model.

Dramatic reduction of toxicity of Poly(hexamethylene guanidine) disinfectant by charge neutralization.

The current study aimed to investigate the toxicity of positively charged polyhexamethylene guanidine (PHMG) polymer and its complexation with different anionic natural polymers such as k-carrageenan (kCG), chondroitin sulfate (CS), sodium alginate (Alg.Na), polystyrene sulfonate sodium (PSS.Na) and hydrolyzed pectin (HP). The physicochemical properties of the synthesized PHMG and its combination with anionic polyelectrolyte complexes (PECs) namely PHMG:PECs were characterized using zeta potential, XPS, FTIR, and TG analysis. Furthermore, cytotoxic behavior of the PHMG and PHMG:PECs, respectively, were evaluated using human liver cancer cell line (HepG2). The study results revealed that the PHMG alone had slightly higher cytotoxicity to the HepG2 cells than the prepared polyelectrolyte complexes such as PHMG:PECs. The PHMG:PECs showed a significant reduction of cytotoxicity to the HepG2 cells than the pristine PHMG alone. A reduction of PHMG toxicity was observed may be due to the facile formation of complexation between the positively charged PHMG and negatively charged anionic natural polymers such as kCG, CS, Alg. Na, PSS.Na and HP, respectively, via charge balance or neutralization. The experimental results indicate that the suggested method might significantly lower PHMG toxicity while improving biocompatibility.

Effect of Isoquercitrin on Free Fatty Acid-Induced Lipid Accumulation in HepG2 Cells.

Liver metabolic disorders and oxidative stress are crucial factors in the development of nonalcoholic fatty liver disease (NAFLD); however, treatment strategies to combat NAFLD remain poorly established, presenting an important challenge that needs to be addressed. Herein, we aimed to examine the effect of isoquercitrin on lipid accumulation induced by exogenous free fatty acids (FFA) using HepG2 cells and elucidate the underlying molecular mechanism. The cells were exposed to 0.5 mM FFA to induce intracellular lipid accumulation, followed by co-treatment with isoquercitrin to confirm the potential inhibitory effect on FFA-induced lipid production. HepG2 cells exposed to FFA alone exhibited intracellular lipid accumulation, compromised endoplasmic reticulum (ER) stress, and enhanced expression of proteins and genes involved in lipid synthesis; however, co-treatment with isoquercitrin decreased the expression of these molecules in a dose-dependent manner. Furthermore, isoquercitrin could activate AMP-activated protein kinase (AMPK), a key regulatory protein of hepatic fatty acid oxidation, suppressing new lipid production by phosphorylating acetyl-CoA carboxylase (ACC) and inhibiting sterol regulatory element-binding transcription factor 1 (SREBP-1)/fatty acid synthase (FAS) signals. Overall, these findings suggest that isoquercitrin can be employed as a therapeutic agent to improve NAFLD via the regulation of lipid metabolism by targeting the AMPK/ACC and SREBP1/FAS pathways.

Effects of dietary restriction on hepatic sulfur-containing amino acid metabolism and its significance in acetaminophen-induced liver injury.

Dietary restriction (DR) has been revealed to have health benefits as it induces reduction in oxidative stress. Glutathione (GSH), an important cellular antioxidant, is increased in rodent livers owing to DR; however, the exact mechanism and clinical relevance of DR are yet to be fully understood. In this study, male C57BL/6 mice were administered a 50% restricted diet for 7 d, and the hepatic sulfur-containing amino acid (SAA) metabolism was determined to assess the biosynthesis of GSH. The hepatic methionine level was found to decrease, while the homocysteine, cysteine, and GSH levels were increased owing to decreased betaine-homocysteine methyltransferase (BHMT) and increased CßS, CγL, and glutamate cysteine ligase catalytic subunit (GCLC) proteins in the livers of mice subjected to DR. To determine the effects of DR on drug-induced oxidative liver injury, mice subjected to DR were injected with a toxic dose (300 mg/kg) of acetaminophen (APAP). DR significantly alleviated APAP-induced liver damage and oxidative stress, which might be attributed to the higher levels of GSH and related antioxidant enzyme (GPx, GSTα, and GSTµ) in the livers. The decrease in the levels of hepatic CYP1A, 2E1, and 3A, which imply the inhibition of APAP metabolic activation, could contribute to the lower hepatotoxicity in mice subjected to DR. Overall, our findings revealed that DR stimulated the hepatic transsulfuration pathway and GSH synthesis. The consequent elevation of GSH could thus serve as an important mechanism of DR-mediated liver protection against APAP intoxication.

Taurine Ameliorates Tunicamycin-Induced Liver Injury by Disrupting the Vicious Cycle between Oxidative Stress and Endoplasmic Reticulum Stress.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver dysfunction characterized by excess lipid accumulation; non-alcoholic steatohepatitis can transform into more severe conditions, such as cirrhosis and hepatocellular carcinoma. Although several pharmacologic approaches have been evaluated in clinical trials, there are no approved therapies for NAFLD. Previous studies have suggested that taurine supplementation alleviates fatty liver; however, the underlying mechanism remains obscure. In this study, we investigated the beneficial effects of taurine on fatty liver injury in vivo induced by tunicamycin, a chemical endoplasmic reticulum (ER) stressor. The mice were administered 2% taurine for 2 weeks prior to intraperitoneal tunicamycin injection; after 72 h of treatment, the mice were euthanized. Tunicamycin treatment significantly increased the levels of serum ALT and AST and hepatic triglycerides. Notably, these changes were alleviated by taurine supplementation. Taurine normalized the protein and/or mRNA levels involved in ER stress signaling (IRE1a, p-IRE1a, ATF6, XBP1, BiP, and CHOP) and lipid metabolism (CD36, MTTP, and ApoB), which were dysregulated by tunicamycin treatment. The stimulation of hepatic lipid export by taurine was evidenced by the recovery of blood VLDL levels. Furthermore, taurine supplementation prevented tunicamycin-induced lipid peroxidation and decreased glutathione (GSH) levels by correcting abnormal cysteine catabolism involved in the production of both taurine and GSH. Therefore, taurine supplementation can prevent tunicamycin-induced liver injury by counteracting oxidative and ER stress.

Downregulation of Glutathione-Mediated Detoxification Capacity by Binge Drinking Aggravates Acetaminophen-Induced Liver Injury through IRE1α ER Stress Signaling.

Overdose of acetaminophen (APAP) can cause severe liver injury. Although alcohol is considered a risk factor for APAP toxicity, the mechanism underlying the interaction between alcohol and APAP remains unclear. Binge alcohol (5 g/kg every 12 h, 3 doses) reduced the concentration of cysteine and glutathione (GSH) and decreased expression of cystathionine ß-synthase (CßS), cystathionine γ-lyase (CγL), and glutamate cysteine ligase catalytic subunit (GCLC) in the livers of male C57BL/6 mice. Furthermore, the levels of GSH S-transferase (GST) and GSH peroxidase (GPx) were decreased. To evaluate the effect of binge drinking on APAP-induced liver injury, 300 mg APAP was administered following alcohol binges. APAP in the binge group significantly amplified the serum ALT more than two fold and enhanced the pro-apoptotic proteins with a severe centrilobular necrosis compared to APAP alone. APAP treatment after alcohol binges caused lower levels of hepatic cysteine and GSH than APAP alone over 24 h, indicating that alcohol binges reduced GSH regenerating potential. Exposure to APAP after binge treatment significantly increased oxidative stress (lipid peroxidation) and endoplasmic reticulum (ER) stress (Grp78 and ATF6) markers at 6 h after treatment. Notably, the IRE1α/ASK1/MKK4/JNK pathway was activated, whereas CHOP expression was reduced by APAP administration in mice with pre-exposed alcohol binges compared with APAP alone. Thus, pretreatment with binge alcohol decreases GSH-mediated antioxidant capacity and contributes to augmentation of liver injury caused by subsequent APAP administration through differential ER stress signaling pathway.

Synthesis and Biological Evaluation of (S)-2-(Substituted arylmethyl)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide Analogs and Their Synergistic Effect against PTEN-Deficient MDA-MB-468 Cells.

A series of twenty-six compounds of furfuryl or benzyl tetrahydropyrazino[1,2-a]indole analogs were synthesized and evaluated for cytotoxic activity against the estrogen receptor (ER)-positive breast cancer cell line (MCF-7) and the epidermal growth factor receptor (EGFR) over-expressed triple-negative breast cancer cell line (MDA-MB-468). Among them, compounds 2b, 2f and 2i showed more potent activity and selectivity against MDA-MB-468 cells than gefitinib, as an EGFR- tyrosine kinase inhibitor. In addition, it was confirmed by means of isobologram analysis of combinational treatment with gefitinib that they have a synergistic effect, especially compounds 2b and 2f, which inhibit Akt T308 phosphorylation. Moreover, it was confirmed that 2-benzyl-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs (2b, 2f, and Ref 2) tend to selectively inhibit PI3Kß, which is involved in the phosphorylation of Akt.

Simple Host-Guest Assembly for High-Resolution Magnetic Resonance Imaging of Microvasculature.

Magnetic resonance angiography (MRA) is an important imaging technique that can be used to identify and characterize various types of vascular diseases. However, currently used molecular contrast agents are unsuitable for MRA due to the short intravascular retention time, the whole-body distribution, and the relatively low contrast effect. In this study, we developed a vascular analysis contrast agent (i.e., VasCA) for MRA, which is a simple and biocompatible 1:1 host-guest assembly of PEGylated ß-cyclodextrin and gadolinium chelate with renal clearable size and high relaxivity (r1 = 9.27 mM-1 s-1). Its biocompatibility was confirmed by in vivo animal studies as well as in vitro 3D cell culture. In a tumor-bearing rat model, VasCA circulated in the blood vessels much longer (4.3-fold increase) than gadoterate meglumine (Dotarem) and was mainly excreted by the renal system after intravenous injection. This feature of VasCA allows characterization of tumor microvasculature (e.g., feeding and draining vessels) as well as visualization of small vessels in the brain and body organs. Furthermore, after treatment with an angiogenesis inhibitor (i.e., sorafenib), VasCA revealed the vessel normalization process and allowed the assessment of viable and necrotic tumor regions. Our study provides a useful tool for diverse MRA applications, including tumor characterization, early-stage evaluation of drug efficacy, and treatment planning, as well as diagnosis of cardiovascular diseases.

N-Phenyl Cinnamamide Derivatives Protect Hepatocytes against Oxidative Stress by Inducing Cellular Glutathione Synthesis via Nuclear Factor (Erythroid-Derived 2)-Like 2 Activation.

Substituted N-phenyl cinnamamide derivatives were designed and synthesized to confirm activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway by the electronic effect on beta-position of Michael acceptor according to introducing the R1 and R2 group. Compounds were screened using the Nrf2/antioxidant response element (ARE)-driven luciferase reporter assay. Compound 1g showed desirable luciferase activity in HepG2 cells without cell toxicity. mRNA and protein expression of Nrf2/ARE target genes such as NAD(P)H quinone oxidoreductase 1, hemeoxygenase-1, and glutamate-cysteine ligase catalytic subunit (GCLC) were upregulated by compound 1g in a concentration-dependent manner. Treatment with 1g resulted in increased endogenous antioxidant glutathione, showing strong correlation with enhanced GCLC expression for synthesis of glutathione. In addition, tert-butyl hydroperoxide (t-BHP)-generated reactive oxygen species were significantly removed by 1g, and the results of a cell survival assay in a t-BHP-induced oxidative cell injury model showed a cytoprotective effect of 1g in a concentration dependent manner. In conclusion, the novel compound 1g can be utilized as an Nrf2/ARE activator in antioxidative therapy.

Inflammatory responses of C57BL/6NKorl mice to dextran sulfate sodium-induced colitis: comparison between three C57BL/6 N sub-strains.

BACKGROUND: Inflammatory bowel disease (IBD), including both Crohn's disease and ulcerative colitis, are chronic human diseases that are challenging to cure and are often unable to be resolved. The inbred mouse strain C57BL/6 N has been used in investigations of IBD as an experimental animal model. The purpose of the current study was to compare the inflammatory responsiveness of C57BL/6NKorl mice, a sub-strain recently established by the National Institute of Food and Drug Safety Evaluation (NIFDS), with those of C57BL/6 N mice from two different sources using a dextran sulfate sodium (DSS)-induced colitis model. RESULTS: Male mice (8 weeks old) were administered DSS (0, 1, 2, or 3%) in drinking water for 7 days. DSS significantly decreased body weight and colon length and increased the colon weight-to-length ratio. Moreover, severe colitis-related clinical signs including diarrhea and rectal bleeding were observed beginning on day 4 in mice administered DSS at a concentration of 3%. DSS led to edema, epithelial layer disruption, inflammatory cell infiltration, and cytokine induction (tumor necrosis factor-α, interleukin-6, and interleukin-1ß) in the colon tissues. However, no significant differences in DSS-promoted abnormal symptoms or their severity were found between the three sub-strains. CONCLUSIONS: These results indicate that C57BL/6NKorl mice responded to DSS-induced colitis similar to the generally used C57BL6/N mice, thus this newly developed mouse sub-strain provides a useful animal model of IBD.

Weaning Mice and Adult Mice Exhibit Differential Carbon Tetrachloride-Induced Acute Hepatotoxicity.

Age is a risk factor for drug-induced liver injury (DILI). However, there is a limited understanding of pediatric DILI. Here, 2-week-old weaning and 8-week-old adult male ICR mice were intraperitoneally injected with CCl4 (0.1 mmol/kg equal to 15.4 mg/kg) to comparatively evaluate the time-dependent liver damage and cellular events. CCl4 significantly enhanced the serum alanine aminotransferase/aspartate aminotransferase levels and hepatic centrilobular necrosis in the weaning mice, whereas it induced mild liver injury in the adult mice. CCl4-treated weaning mice exhibited higher hepatic levels of pro-apoptotic proteins (Bax, cleaved caspase-3, -7, and -9), activated MAPKs (p-JNK and p-Erk), and endoplasmic reticulum stress indicators (ATF6 and CHOP) and lower hepatic anti-apoptotic Bcl-2 levels than the adult mice. The weaning mice exhibited enhanced basal hepatic glutathione (GSH) levels due to high glutamate cysteine ligase (GCL) and low anti-cysteine dioxygenase (CDO) enzyme levels. However, CCl4 markedly reduced the hepatic GSH levels only in the weaning mice. Furthermore, higher hepatic levels of oxidative stress-induced malondialdehyde, 4-hydroxynonenal, nitrotyrosine-protein adducts, and oxidized proteins were observed in CCl4-treated weaning mice than in CCl4-treated adult mice. The enhanced levels of hepatic cytochrome P450 (CYP) 2E1 and CYP3A, and decreased hepatic GSH S-transferase (GST)-π and GSH reductase (GR) levels in the weaning mice may contribute to their enhanced susceptibility to liver damage.

Anti-Oxidant Activity of Gallotannin-Enriched Extract of Galla Rhois Can Associate with the Protection of the Cognitive Impairment through the Regulation of BDNF Signaling Pathway and Neuronal Cell Function in the Scopolamine-Treated ICR Mice.

The antibacterial, anti-inflammatory, anti-metastatic/anti-invasion activities and laxative activity of Galla Rhois (GR) are well-known, although the neuropreservation effects of their extracts are still to be elucidated. To investigate the novel therapeutic effects and molecular mechanism of GR on alleviation of cognitive impairment, two different dosages of gallotannin-enriched GR (GEGR) were administered to Korl:ICR mice for three weeks, and to induce memory impairment, scopolamine (SP) was administered during the last seven days of the GEGR treatment period. GEGR showed the high level of the free radical scavenging activity to DPPH and suppressive activity to reactive oxygen species (ROS) in B35 cells as well as enhanced SOD and CAT activity in brains of the SP-induced model. Latency time for memory impairment assessed by the passive avoidance test significantly protected in the SP+GEGR treated group as compared to the SP+Vehicle treated group. Moreover, similar protective effects were observed on the secretion of BDNF in SP+GEGR treated mice. The expression of TrkB receptor, and phosphorylation of PI3K on the TrkB receptor signaling pathway were dramatically protected in the SP-induced model after GEGR treatment, whereas the expression of p75NTR receptor, the phosphorylation of JNK, and expression of Bax/Bcl-2 on the p75NTR receptor signaling pathway was significantly protected in the same group. Furthermore, the GEGR treated SP-induced model showed decreased number of dead neural cells and suppressed acetylcholine esterase (AChE) activity and inhibited inflammatory responses. Taken together, these results indicate that the anti-oxidant activity of GEGR contributes to improving the neuronal cell function and survival during cognitive impairment in the SP-induced model through regulation of BDNF secretion and their receptor signaling pathway.

Polyhexamethyleneguanidine Phosphate-Induced Cytotoxicity in Liver Cells Is Alleviated by Tauroursodeoxycholic Acid (TUDCA) via a Reduction in Endoplasmic Reticulum Stress.

Polyhexamethyleneguanidine phosphate (PHMG-P) is a widely used polymeric antimicrobial agent known to induce significant pulmonary toxicity. Several studies have reported that the liver also can be a target organ of polyhexamethyleneguanidine (PHMG) toxicity, but the exact effect of this compound on liver cells is not well understood. To identify the mechanism of PHMG hepatotoxicity, HepG2 cells were exposed to PHMG-P for 72 h. The cell viability was significantly decreased by PHMG-P in a time- and concentration-dependent manner. The mitochondrial membrane potential was markedly reduced by PHMG-P and the apoptotic signaling cascade was activated. The increases observed in C/EBP homologous protein (CHOP), p-IRE, and p-JNK levels in PHMG-P-treated cells indicated the induction of endoplasmic reticulum stress. To verify the role of ER stress in PHMG-P-induced cytotoxicity, HepG2 cells were pretreated with the chemical chaperone, tauroursodeoxycholic acid (TUDCA) and then co-treated with TUDCA and PHMG-P for 24 h. Interestingly, TUDCA inhibited PHMG-P-induced ER stress and cytotoxicity in a dose-dependent manner. The apoptotic cell death and mitochondrial depolarization were also prevented by TUDCA. The proteins involved in the apoptotic pathway were all normalized to their control levels in TUDCA-treated cells. In conclusion, the results suggest that PHMG-P induced significant cytotoxicity in liver cells and ER stress-mediated apoptosis, which may be an important mechanism mediating this hepatotoxicity.

Discovery and SAR studies of novel 2-anilinopyrimidine-based selective inhibitors against triple-negative breast cancer cell line MDA-MB-468.

Triple-negative breast cancers (TNBCs) are characterized as an invasive and intractable subtype of breast cancers. Overexpression of epidermal growth factor receptor (EGFR) has been considered to be an important target for TNBC therapy, but efficacies of EGFR inhibitors in clinical trials are elusive. In this study, novel series of 2-anilinopyrimidines were synthesized in an effort to identify selective inhibitors against an EGFR-overexpressing TNBC cell line. Biological evaluation demonstrated that compounds 21 and 38, with a 4-methylpiperidine group and a high ClogP value, exhibited good potency and selectivity for the TNBC cell line. This study has provided evidence to support further development of 2-anilinopyrimidine-based TNBC selective inhibitors and investigation of the targets of compounds 21 and 38.

Comparative study of liver injury induced by high-fat methionine- and choline-deficient diet in ICR mice originating from three different sources.

Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. It is characterized by the accumulation of lipids without alcohol intake and often progresses to non-alcoholic steatohepatitis (NASH), liver fibrosis, and end-stage liver diseases such as cirrhosis or cancer. Although animal models have greatly contributed to the understanding of NAFLD, studies on the disease progression in humans are still limited. In this study, we used the recently reported high-fat L-methionine-defined and choline-deficient (HFMCD) diet to rapidly induce NASH and compared the responses to HFMCD in ICR mice from three different countries: Korea (supplied by the National Institute of Food and Drug Safety Evaluation), USA, and Japan during 6 weeks. Feeding HFMCD did not cause significant differences in weight gain in comparison with mice fed control diet. Relative weight of the liver increased gradually, while the relative weight of the kidneys remained unchanged. The parameters of liver injury (serum activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase) increased rapidly from 1 week and remained elevated for as long as 6 weeks. Histopathological analysis showed that the accumulation of hepatic lipids induced by HFMCD was prominent at 1 week after diet supplementation and increased further at 6 weeks. Inflammatory markers were significantly increased in a time-dependent manner by HFMCD. The mRNA levels of TNF-α and IL-6 were elevated approximately 15-fold relative to control diet and that of IL-1ß was increased more than 20-folds at 6 week after the onset of HFMCD intake. In addition, mRNA expression of fibrosis markers such as α-SMA, TGFß1, and Col1a1 were also significantly increased at 6 week. In summary, the responses of Korl:ICR mice by intake of HFMCD diet were similar to those of ICR mice from other sources, which suggests that Korl:ICR mice is also a useful resource to study the pathogenesis of diet-induced NAFLD.

Tunicamycin-Induced ER Stress is Accompanied with Oxidative Stress via Abrogation of Sulfur Amino Acids Metabolism in the Liver.

Endoplasmic reticulum (ER) stress is involved in non-alcoholic fatty liver disease (NAFLD), but the relationship between oxidative stress, another well-known risk factor of NAFLD, and ER stress has yet to be elucidated. In this study, we treated mice with tunicamycin (TM) (2 mg/kg body weight) for 48 h to induce ER stress in the liver and examined the metabolic pathway that synthesizes the endogenous antioxidant, glutathione (GSH). Tunicamycin (TM) treatment significantly increased mRNA levels of CHOP and GRP78, and induced lipid accumulation in the liver. Lipid peroxidation in the liver tissue also increased from TM treatment (CON vs. TM; 3.0 ± 1.8 vs. 11.1 ± 0.8 nmol MDA/g liver, p < 0.001), which reflects an imbalance between the generation of reactive substances and antioxidant capacity. To examine the involvement of GSH synthetic pathway, we determined the metabolomic changes of sulfur amino acids in the liver. TM significantly decreased hepatic S-adenosylmethionine concentration in the methionine cycle. The levels of cysteine in the liver were increased, while taurine concentration was maintained and GSH levels profoundly decreased (CON vs. TM; 8.7 ± 1.5 vs. 5.4 ± 0.9 µmol GSH/g liver, p < 0.001). These results suggest that abnormal cysteine metabolism by TM treatment resulted in a decrease in GSH, followed by an increase in oxidative stress in the liver. In HepG2 cells, decreased GSH levels were examined by TM treatment in a dose dependent manner. Furthermore, pretreatment with TM in HepG2 cells potentiated oxidative cell death, by exacerbating the effects of tert-butyl hydroperoxide. In conclusion, TM-induced ER stress was accompanied by oxidative stress by reducing the GSH synthesis, which made the liver more susceptible to oxidative stress.

Antioxidant Effect of Barley Sprout Extract via Enhancement of Nuclear Factor-Erythroid 2 Related Factor 2 Activity and Glutathione Synthesis.

We previously showed that barley sprout extract (BSE) prevents chronic alcohol intake-induced liver injury in mice. BSE notably inhibited glutathione (GSH) depletion and increased inflammatory responses, revealing its mechanism of preventing alcohol-induced liver injury. In the present study we investigated whether the antioxidant effect of BSE involves enhancing nuclear factor-erythroid 2 related factor 2 (Nrf2) activity and GSH synthesis to inhibit alcohol-induced oxidative liver injury. Mice fed alcohol for four weeks exhibited significantly increased oxidative stress, evidenced by increased malondialdehyde (MDA) level and 4-hydroxynonenal (4-HNE) immunostaining in the liver, whereas treatment with BSE (100 mg/kg) prevented these effects. Similarly, exposure to BSE (0.1-1 mg/mL) significantly reduced oxidative cell death induced by t-butyl hydroperoxide (t-BHP, 300 µM) and stabilized the mitochondrial membrane potential (∆ψ). BSE dose-dependently increased the activity of Nrf2, a potential transcriptional regulator of antioxidant genes, in HepG2 cells. Therefore, increased expression of its target genes, heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase catalytic subunit (GCLC) was observed. Since GCLC is involved in the rate-limiting step of GSH synthesis, BSE increased the GSH level and decreased both cysteine dioxygenase (CDO) expression and taurine level. Because cysteine is a substrate for both taurine and GSH synthesis, a decrease in CDO expression would further contribute to increased cysteine availability for GSH synthesis. In conclusion, BSE protected the liver cells from oxidative stress by activating Nrf2 and increasing GSH synthesis.

Transformation of liver cells by 3-methylcholanthrene potentiates oxidative stress via the downregulation of glutathione synthesis.

Polycyclic aromatic hydrocarbons (PAHs) are widespread contaminants resulting from the incomplete combustion of organic materials in the environment. The primary concern for the hazardous effect of PAHs is their ability to activate the pathway linked to the aryl hydrocarbon receptor (AhR) and lead to carcinogenesis. While previous research has demonstrated that oxidative stress plays a key role in the AhR-dependent toxic response, the effect of PAHs on the biosynthesis of glutathione (GSH), which is a powerful endogenous antioxidant, has not been extensively investigated. In the present study, we utilized a global metabolomic approach, via high resolution magic angle spinning nuclear magnetic resonance spectroscopy, and identified significant metabolome differences between non-tumorigenic liver cells (BNL CL.2; CL2) and transformed liver cells (BNL 1MEA. 7R.1; 1MEA) chronically exposed to 3-methylcholanthrene (3MC), a well­known carcinogenic PAH. A significant change that was observed, was a lower GSH level in 1MEA cells compared with that in CL2 cells. This was contrasted by increased levels of precursor metabolites for GSH synthesis, such as S-adenosylmethionine and cysteine. These changes were accompanied by a significantly reduced expression of γ-glutamylcysteine ligase (GCL), known to be the rate­limiting step of GSH synthesis. Furthermore, the protein level of cysteine dioxygenase was downregulated; however, the concentration of taurine was unaltered. Therefore, the present study demonstrated that cells transformed by chronic exposure to 3MC exhibited inhibition of GSH biosynthesis by suppression of GCL protein expression and reduction of cysteine availability, which may subsequently render cells vulnerable to oxidative stress.