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.

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.

Germinated Soybean Embryo Extract Ameliorates Fatty Liver Injury in High-Fat Diet-Fed Obese Mice.

Soybean is known to have diverse beneficial effects against human diseases, including obesity and its related metabolic disorders. Germinated soybean embryos are enriched with bioactive phytochemicals and known to inhibit diet-induced obesity in mice, but their effect on non-alcoholic fatty liver disease (NAFLD) remains unknown. Here, we germinated soybean embryos for 24 h, and their ethanolic extract (GSEE, 15 and 45 mg/kg) was administered daily to mice fed with a high-fat diet (HFD) for 10 weeks. HFD significantly increased the weight of the body, liver and adipose tissue, as well as serum lipid markers, but soyasaponin Ab-rich GSEE alleviated these changes. Hepatic injury and triglyceride accumulation in HFD-fed mice were attenuated by GSEE via decreased lipid synthesis (SREBP1c) and increased fatty acid oxidation (p-AMPKα, PPARα, PGC1α, and ACOX) and lipid export (MTTP and ApoB). HFD-induced inflammation (TNF-α, IL-6, IL-1ß, CD14, F4/80, iNOS, and COX2) was normalized by GSEE in mice livers. In adipose tissue, GSEE downregulated white adipose tissue (WAT) differentiation and lipogenesis (PPARγ, C/EBPα, and FAS) and induced browning genes (PGC1α, PRDM16, CIDEA, and UCP1), which could also beneficially affect the liver via lowering adipose tissue-related circulating lipid levels. Thus, our results suggest that GSEE can prevent HFD-induced NAFLD via inhibition of hepatic inflammation and restoration of lipid metabolisms in both liver and adipose tissue.

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.

Concentration- and Time-Dependent Effects of Benzalkonium Chloride in Human Lung Epithelial Cells: Necrosis, Apoptosis, or Epithelial Mesenchymal Transition.

Benzalkonium chloride (BAC), an antimicrobial agent in inhalable medications and household sprays, has been reported to be toxic to pulmonary organs. Although cell membrane damage has been considered as the main cytotoxic mechanism of BAC, its concentration- and time-dependent cellular effects on lung epithelium have not been fully understood. In the present study, human lung epithelial (H358) cells were exposed to 0.2-40 µg/mL of BAC for 30 min or 21 days. Cell membranes were rapidly disrupted by 30 min exposure, but 24 h incubation of BAC (4-40 µg/mL) predominantly caused apoptosis rather than necrosis. BAC (2-4 µg/mL) induced mitochondrial depolarization, which may be associated with increased expression of pro-apoptotic proteins (caspase-3, PARP, Bax, p53, and p21), and decreased levels of the anti-apoptotic protein Bcl-2. The protein expression levels of IRE1α, BiP, CHOP, and p-JNK were also elevated by BAC (2-4 µg/mL) suggesting the possible involvement of endoplasmic reticulum stress in inducing apoptosis. Long-term (7-21 days) incubation with BAC (0.2-0.6 µg/mL) did not affect cell viability but led to epithelial-mesenchymal transition (EMT) as shown by the decrease of E-cadherin and the increase of N-cadherin, fibronectin, and vimentin, caused by the upregulation of EMT transcription factors, such as Snail, Slug, Twist1, Zeb1, and Zeb2. Therefore, we conclude that apoptosis could be an important mechanism of acute BAC cytotoxicity in lung epithelial cells, and chronic exposure to BAC even at sub-lethal doses can promote pulmonary EMT.

Metabolomic Analysis of the Liver of a Dextran Sodium Sulfate-Induced Acute Colitis Mouse Model: Implications of the Gut-Liver Connection.

The incidence of ulcerative colitis (UC) is increasing worldwide, and it has become a growing problem in Asia. Previous research on UC has focused on serum, plasma, urine, gut tissues, and fecal metabolic profiling, but a comprehensive investigation into the correlation between the severity of colitis and changes in liver metabolism is still lacking. Since the liver and gut exchange nutrients and metabolites through a complex network, intestinal diseases can affect both the liver and other organs. In the present study, concentration-dependent dextran sodium sulfate (DSS)-induced ulcerative colitis was employed to examine changes in liver metabolism using a proton nuclear magnetic resonance spectroscopy (1H-NMR)-and ultra-performance liquid chromatography time of flight mass spectroscopy (UPLC-TOF MS)-based metabolomics study. Using the multivariate statistical analysis method orthogonal projections to latent structures discriminant analysis (OPLS-DA), changes in metabolites depending on the DSS dose could be clearly distinguished. Specifically, hepatic metabolites involved in one-carbon metabolism, carnitine-related metabolism, and nucleotide synthesis were found to be affected by intestinal inflammation, implying the existence of a metabolic connection between the gut and liver. We are currently investigating the significance of this metabolic condition in UC.

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.

Comparison of toxic responses to acetaminophen challenge in ICR mice originating from different sources.

Acetaminophen (APAP) is the most common antipyretic analgesic worldwide. However, APAP overdose causes severe liver injury, especially centrilobular necrosis, in humans and experimental animals. At therapeutic dosage, APAP is mainly metabolized by sulfation and glucuronidation, and partly by cytochrome P450-mediated oxidation. However, APAP overdose results in production of excess reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), by cytochromes P450; NAPQI overwhelms the level of glutathione (GSH), which could otherwise detoxify it. NAPQI binds covalently to proteins, leading to cell death. A number of studies aimed at the prevention and treatment of APAP-induced toxicity are underway. Rats are more resistant than mice to APAP hepatotoxicity, and thus mouse models are mainly used. In the present study, we compared the toxic responses induced by APAP overdose in the liver of ICR mice obtained from three different sources and evaluated the usability of the Korl:ICR stock established by the National Institute of Food and Drug Safety Evaluation in Korea. Administration of APAP (300 mg/kg) by intraperitoneal injection into male ICR mice enhanced CYP2E1 protein expression and depleted hepatic GSH level 2 h after treatment accompanied with significantly increased level of hepatic malondialdehyde, a product of lipid peroxidation. Regardless of the source of the mice, hepatotoxicity, as evidenced by activity of serum alanine aminotransferase, increased from 8 h and peaked at 24 h after APAP treatment. In summary, hepatotoxicity was induced after the onset of oxidative stress by overdose of APAP, and the response was the same over time among mice of different origins.