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ABSTRACT: Although estrogen receptor (ER)α agonists, such as estradiol and ethinylestradiol (EE2), cause cholestasis in mice, they also reduce the degree of liver injury caused by hepatotoxicants as well as ischemia-reperfusion. The functional mechanisms of ERα have yet to be elucidated in drug-induced or chemical-induced liver injury. The present study investigated the effects of an ERα agonist, selective ER modulators (SERMs) and an ER antagonist on drug-induced and chemical-induced liver injuries caused by acetaminophen, bromobenzene, diclofenac, and thioacetamide (TA). We observed hepatoprotective effects of EE2, tamoxifen (TAM) and raloxifene pretreatment in female mice that were exposed to a variety of hepatotoxic compounds. In contrast, the ER antagonist did not show any hepatoprotective effects. DNA microarray analyses suggested that monocyte to macrophage differentiation-associated 2 (Mmd2) protein, which has an unknown function, is commonly increased by TAM and RAL pretreatment, but not by pretreatment with the ER antagonist. In ERα-knockout mice, the hepatoprotective effects of TAM and the increased expression of Mmd2 mRNA were not observed in TA-induced liver injury. To investigate the function of Mmd2, the expression level of Mmd2 mRNA was significantly knocked down to approximately 30% in mice by injection of siRNA for Mmd2 (siMmd2). Mmd2 knockdown resulted in a reduction of the protective effects of TAM on TA-induced liver injury in mice. This is the first report of the involvement of ERα in drug-induced or chemical-induced liver injury. Upregulation of Mmd2 protein in the liver was suggested as the mechanism of the hepatoprotective effects of EE2 and SERMs.
Toxicology and Applied Pharmacology 07/2012; 264(1):42-50. · 4.45 Impact Factor
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ABSTRACT: Drug-induced liver injury is a major safety concern in drug development and clinical pharmacotherapy; however, advances in the understanding of the mechanisms of drug-induced liver injury are hampered by the lack of animal models. Carbamazepine (CBZ) is a widely used antiepileptic agent. Although the drug is generally well tolerated, only a small number of patients prescribed CBZ develop severe hepatitis. In the present study, we developed a mouse model of CBZ-induced liver injury and elucidated the mechanisms accounting for the hepatotoxicity of CBZ. Male BALB/c mice were orally administered CBZ for 5 days. The plasma levels of alanine aminotransferase and aspartate aminotransferase were prominently increased, and severe liver damage was observed via histological evaluation. The analysis of the plasma concentration of CBZ and its metabolites demonstrated that 3-hydroxy CBZ may be relevant in CBZ-induced liver injury. The hepatic glutathione levels were significantly decreased, and oxidative stress markers were significantly altered. Mechanistic investigations found that hepatic mRNA levels of toll-like receptor 4, receptor for advanced glycation end products, and their ligands were significantly increased. Moreover, the plasma concentrations of proinflammatory cytokines were also increased. Prostaglandin E(1) administration ameliorated the hepatic injury caused by CBZ. In conclusion, metabolic activation followed by the stimulation of immune responses was demonstrated to be involved in CBZ-induced liver injury in mice.
Toxicological Sciences 07/2012; 130(1):4-16. · 4.65 Impact Factor
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ABSTRACT: Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical drug therapy. However, the underlying mechanism of DILI is little known. It is difficult to predict DILI in humans due to the lack of experimental animal models. Diclofenac, a non-steroidal anti-inflammatory drug rarely causes severe liver injury in human, but there is some evidence for immunoallergic idiosyncratic reactions. In this study, the mechanism of diclofenac-induced liver injury in mice was investigated. First, we established the dosing condition for liver injury in normal mice. Plasma ALT and AST levels were significantly increased in diclofenac-administered (80 mg/kg, i.p.) mice in a dose- and time-dependent manner. Among several interleukins (ILs) and chemokines, mRNA expression of helper T (Th) 17 cell-mediated factors, such as retinoid orphan receptor (ROR)-γt, and signal transducers and activators of transcription factor (STAT) 3 in the liver, and the plasma IL-17 level were significantly increased. Neutralization of IL-17 tended to suppress the hepatotoxicity of diclofenac, suggesting that IL-17 was partly involved. Gadolinium chloride (GdCl₃) administration demonstrated that Kupffer cells are not likely to be involved in diclofenac hepatotoxicity. Hepatic expressions of IL-1β mRNA and plasma IL-1β were significantly increased soon after the diclofenac administration. Then, the results of an in vivo neutralization study of IL-1β suggested that IL-1β was involved early in the time of pathogenesis of the diclofenac-induced liver injury. In conclusion, we firstly developed a diclofenac-induced acute liver injury model in normal mice, and the involvement of IL-17 and IL-1β was clarified.
Toxicology 03/2012; 293(1-3):107-14. · 3.68 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are small RNA molecules that function to modulate the expression of target genes, playing important roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA expressions by acute liver injury (hepatocellular injury or cholestasis) and chronic liver injury (steatosis, steatohepatitis and fibrosis) using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found that the levels of a large number of miRNAs (121-317 miRNAs) were increased over 2-fold and the levels of a small number of miRNAs (6-35 miRNAs) were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level, reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis) and identified the miRNAs that could be specific and sensitive biomarkers of liver injury.
PLoS ONE 01/2012; 7(2):e30250. · 4.09 Impact Factor
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ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation that starts with steatosis and progresses to non-alcoholic steatohepatitis (NASH). Recently, the number of patients with such liver diseases has increased, but the understanding of the fundamental mechanisms and appropriate therapies are lacking. Tamoxifen (TAM) is a selective estrogen receptor modulator. We previously reported that TAM plays a protective role against drug-induced and chemical-induced acute liver injuries. However, the effects of TAM on chronic liver injury, including steatosis and NASH, remain to be addressed. We first found that the administration of TAM to mouse models of steatosis and NASH significantly decreased the plasma ALT and AST levels. The administration of TAM decreased the accumulated fat and inflammation in the livers in both mouse models. In addition, we observed decreased hepatic mRNA levels of triglyceride synthesis, acyl-CoA: diacylglycerol acyltransferase 2 (DGAT2), proinflammatory cytokines, tumor necrosis factor (TNF) α, and chemokines, monocyte chemoattractant protein (MCP) -1. TAM increased the extracellular signal-regulated kinase (ERK) phosphorylation, which is related to the proliferation and regeneration of liver and to decreased DGAT2 gene expression. Furthermore, a decrease in eukaryotic translational initiation factor (eIF2α), which is involved in apoptosis, was observed in both models. These findings suggest that TAM treatment exerts a hepatoprotective effect against steatosis and NASH, presumably via up-regulation of the ERK pathways and attenuation of eIF2α activation. These pathways represent a potential therapeutic target for steatosis and NASH in drug development.
The Journal of Toxicological Sciences 01/2012; 37(5):931-42. · 1.52 Impact Factor
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ABSTRACT: Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical drug therapy. However, the underlying mechanism of DILI is little known. It is generally believed that women exhibit worse outcomes from DILI than men. Recently, we found that pretreatment of mice with estradiol attenuated halothane (HAL)-induced liver injury, whereas pretreatment with progesterone exacerbated it in female mice. To investigate the mechanism of sex difference of DILI, we focused on progesterone in this study. We found the exacerbating effect of progesterone in thioacetamide (TA), α-naphthylisothiocyanate, and dicloxacillin-induced liver injury only in female mice. Higher number of myeloperoxidase-positive mononuclear cells infiltrated into the liver and increased levels of Chemokine (C-X-C motif) ligand 1 and 2 (CXCL1 and CXCL2) and intercellular adhesion molecule-1 in the liver were observed. Interestingly, CXCL1 was slightly increased by progesterone pretreatment alone. Progesterone pretreatment increased the extracellular signal-regulated kinase (ERK) phosphorylation in HAL-induced liver injury. Pretreatment with U0126 (ERK inhibitor) significantly suppressed the exacerbating effect of progesterone and the expression of inflammatory mediators. In addition, pretreatment with gadolinium chloride (GdCl(3): inhibitor of Kupffer cells) significantly suppressed the exacerbating effect of progesterone pretreatment and the expression of inflammatory mediators. Moreover, posttreatment of RU486 (progesterone receptor antagonist) 1 h after the HAL or TA administration ameliorated the HAL- or TA-induced liver injury, respectively, in female mice. In conclusion, progesterone exacerbated the immune-mediated hepatotoxic responses in DILI via Kupffer cells and ERK pathway. The inhibition of progesterone receptor and decrease of the immune response may have important therapeutic implications in DILI.
Toxicological Sciences 12/2011; 126(1):16-27. · 4.65 Impact Factor
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ABSTRACT: Drug-induced liver injury is a growing concern for pharmaceutical companies and patients because numerous drugs have been linked to hepatotoxicity and it is the most common reason for a drug to be withdrawn. Flutamide rarely causes liver dysfunction in humans, and immune allergic reactions have been suggested in some cases. In this study, we investigated the mechanisms of flutamide-induced liver injury in BALB/c mice. Plasma alanine aminotransferase and aspartate aminotransferase levels were significantly increased 3, 6 and 9 h after flutamide (1500 mg kg(-1) , p.o.) administration. The biomarker for oxidative stress was not changed, but Th2-dominant immune-related factors, such as interleukin (IL)-4, IL-5, STAT6 and GATA-binding protein (GATA)-3, were induced in flutamide-administered mice. The pre-administration of monoclonal-IL-4 antibody suppressed the hepatotoxicity of flutamide. In addition, we investigated the effect of 13,14-dihydro-15-keto-PGD(2) (DK-PGD(2) ; 10 µg per mouse, i.p.) administration on flutamide-induced acute liver injury. Coadministration of DK-PGD(2) and flutamide resulted in a significant increase in alanine aminotransferase and a remarkable increase of macrophage inflammatory protein-2. In conclusion, we demonstrated that flutamide-induced acute liver injury is mediated by Th2-dominant immune responses in mice.
Journal of Applied Toxicology 07/2011; 32(10):815-22. · 2.48 Impact Factor
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ABSTRACT: Drug-induced liver injury (DILI) is one of the major problems in drug development and clinical drug therapy. In general, it is believed that women exhibit worse outcomes from DILI than men. It is known that halothane (HAL), an inhaled anesthetic, rarely induces severe liver injury. The risk factors for severe HAL-induced liver injury (HILI) are female sex, genetics and adult age. To investigate the underlying mechanism by which women are more susceptible to HILI, we focused on two major female sex hormones, estradiol (E2) and progesterone (Prog). In this study, we first found that pretreatment of mice with E2 attenuated HILI, whereas pretreatment with Prog exacerbated HILI. E2 and Prog had no effects on the degree of metabolic activation, the ratio of GSH/GSSG or oxidative stress in the liver. We observed higher numbers of neutrophils infiltrated into the liver and increased hepatic mRNA levels of proinflammatory cytokines, tumor necrosis factor (TNF) α, interleukin (IL)-1β and IL-6 and chemokines, CXCL1 and CXCL2 by pretreatment with Prog, whereas E2 pretreatment resulted in the opposite effects. These results suggest that E2 and Prog play a critical role in HILI via immune-related responses and female sex hormone balance might represent a risk factor for HILI.
Toxicology Letters 07/2011; 204(1):17-24. · 3.23 Impact Factor
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ABSTRACT: Drug-induced liver injury (DILI) is a major problem in drug development and clinical drug therapy. In most cases, the mechanisms are still unknown. It is difficult to predict DILI in humans due to the lack of experimental animal models. Dicloxacillin, penicillinase-sensitive penicillin, rarely causes cholestatic or mixed liver injury, and there is some evidence for immunoallergic idiosyncratic reaction in human. In this study, we investigated the mechanisms of dicloxacillin-induced liver injury. Plasma ALT and total-bilirubin (T-Bil) levels were significantly increased in dicloxacillin-administered (600 mg/kg, i.p.) mice. Dicloxacillin administration induced Th2 (helper T cells)-mediated factors and increased the plasma interleukin (IL)-4 level. Neutralization of IL-4 suppressed the hepatotoxicity of dicloxacillin, and recombinant mouse IL-4 administration (0.5 or 2.0 μg/mouse, i.p.) exacerbated it. Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) is a cognate receptor for prostaglandin (PG) D(2), and is suggested to be involved in Th2-dependent allergic inflammation. We investigated the effect of 13,14-Dihydro-15-keto-PGD(2) (DK-PGD(2); 10 μg/mouse, i.p.) administration on dicloxacillin-induced liver injury. DK-PGD(2)/dicloxacillin coadministration resulted in a significant increase of alanine aminotransferases and a remarkable increase of macrophage inflammatory protein 2 expression. In conclusion, to the best of our knowledge, this is the first report to demonstrate that dicloxacillin-induced liver injury is mediated by a Th2-type immune reaction and exacerbated by DK-PGD(2).
Toxicology Letters 02/2011; 200(3):139-45. · 3.23 Impact Factor
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ABSTRACT: Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical drug therapy. The pathogenesis of DILI usually involves the participation of the parent drug or metabolites that either directly affect the cell biochemistry or elicit an immune response. However, in most cases the mechanisms are unknown. Alpha-naphthylisothiocyanate (ANIT) is known as a hepatotoxicant that causes biliary cell and hepatocyte damage and induces intense neutrophil infiltration in the liver. To investigate whether an immune-mediated mechanism is involved in ANIT-induced liver injury, we examined the plasma AST, ALT and T-Bil levels, hepatic expression of transcriptional factors, cytokines and CXC chemokine genes, plasma IL-17 level and histopathological changes in liver after ANIT administration in mice. Hepatic mRNA expression of retinoid related orphan receptor gamma t (ROR gamma t) and macrophage inflammatory protein (MIP-2) and plasma IL-17 level was significantly increased in ANIT-administered mice as well as the plasma AST, ALT and T-Bil. Neutralization of IL-17 using anti-IL-17 antibody (100 microg/mouse, single i.p.) suppressed the hepatotoxic effect of ANIT. Co-administration of recombinant IL-17 (1 microg/mouse, single i.p.) to ANIT-administered mice resulted in a remarkable increase of the plasma AST, ALT and T-Bil levels. In conclusion, it was firstly demonstrated that IL-17 is involved in the ANIT-induced liver injury in mice.
Toxicology 09/2010; 275(1-3):50-7. · 3.68 Impact Factor
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ABSTRACT: Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. Previously, we reported the establishment of an adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA), and applied this to evaluate drug-induced cytotoxicity. In this study, infection of AdSOD2-shRNA to Fisher 344 rats resulted in a significant decrease of SOD2 mRNA, protein expression, and SOD2 enzyme activity to 28%, 35%, and 39%, respectively, 7 days after infection. Serum AST and ALT were significantly increased by single oral administration of acetaminophen (1000 mg/kg) to these SOD2-knockdown rats without fasting compared with the control adenovirus infected groups. Heme oxygenase-1 protein, known to be induced by oxidative stress, was detected in SOD2-knockdown rats administered acetaminophen. In addition, protein carbonyl and lipid peroxidation, also known to be induced by oxidative stress, were significantly increased in SOD2 knockdown rats. This is the first report of a SOD2-knockdown rat model that could be useful to evaluate the drug-induced hepatotoxicity with high sensitivity.
Toxicology 08/2009; 264(1-2):89-95. · 3.68 Impact Factor
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ABSTRACT: Drug-induced liver injury is a major problem in drug development and clinical drug therapy. In most cases the mechanisms are still unknown, thus, it is difficult to predict or prevent these reactions. It has been known that halothane, an inhaled anesthetic, induces liver injury. To investigate the mechanisms of halothane-induced liver injury, we used a recently established mouse model of liver injury. The expression of transcription factors and cytokines specific for Th1 and Th2 (helper T cells), respectively, were compared between BALB/c and C57BL/6 mice. The mRNA expression ratios of mouse T-bet(a Th1-specific transcription factor)/GATA-binding protein (GATA-3, a Th2-specific transcription factor) and interferon gamma/interleukin (IL)-10 were lower in BALB/c mice compared with C57BL/6 mice, suggesting that a typical Th1 or Th2-dominant response could not be distinguished in halothane-induced liver injury. We observed increases of the plasma IL-17 level and hepatic macrophage inflammatory protein 2 expression in halothane-administrated BALB/c mice, as well as neutrophil infiltration. Neutralization of IL-17 suppressed the hepatotoxic effect of halothane. Administration of recombinant IL-17 (1 microg per mouse, single ip) to the halothane-treated mice resulted in a remarkable increase of alanine and aspartate aminotransferases. In conclusion, we demonstrated that IL-17 is involved in the halothane-induced liver injury.
Toxicological Sciences 08/2009; 111(2):302-10. · 4.65 Impact Factor
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ABSTRACT: Idiosyncratic drug-induced liver injury (DILI) is a major clinical problem for drug development. It is generally known that DILI is mainly caused by hepatic glutathione (GSH) depletion. The glutathione S-transferase activity of rodent is higher than that of human, which could make the prediction of DILI more difficult. Recently, we reported that an experimental rat model of GSH-depletion displayed high susceptibility to acetaminophen-induced hepatotoxicity. To deplete GSH, we used an adenovirus vector with short hairpin RNA against gamma-glutamylcysteine synthetase heavy chain subunit (AdGCSh-shRNA). In this study, we further investigated the usefulness of this rat model for determining drug-induced sensitive acute and subacute toxicity. Rats were administered diclofenac and flutamide which have been reported as idiosyncratic hepatotoxic drugs. In the acute (6 or 24h) or subacute (7 days) toxicity tests, rats were administered the drugs once or once a day for a week, respectively. Plasma biochemical markers for hepatotoxicity were measured. The 6 and 24h toxicity test of diclofenac, and the 24h and 7 days toxicity tests of flutamide showed significant ALT elevations. Additionally, the 24h toxicity test of flutamide showed a slight bilirubin elevation, and histological hepatotoxicity. The 7 days toxicity test of flutamide also demonstrated histological hepatotoxicity. In conclusion, this rat model would contribute to evaluating acute and subacute DILI in preclinical drug development.
Toxicology Letters 06/2009; 189(2):159-65. · 3.23 Impact Factor
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ABSTRACT: Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. Gamma-glutamylcysteine synthetase (gamma-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat gamma-GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 x 10(11) pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development.
Journal of Biological Chemistry 09/2007; 282(33):23996-4003. · 4.77 Impact Factor
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ABSTRACT: Idiosyncratic drug-induced liver injury (DILI) is a major clinical problem for drug development. It is generally known that DILI is mainly caused by hepatic glutathione (GSH) depletion. The glutathione S-transferase activity of rodent is higher than that of human, which could make the prediction of DILI more difficult. Recently, we reported that an experimental rat model of GSH-depletion displayed high susceptibility to acetaminophen-induced hepatotoxicity. To deplete GSH, we used an adenovirus vector with short hairpin RNA against γ-glutamylcysteine synthetase heavy chain subunit (AdGCSh-shRNA). In this study, we further investigated the usefulness of this rat model for determining drug-induced sensitive acute and subacute toxicity. Rats were administered diclofenac and flutamide which have been reported as idiosyncratic hepatotoxic drugs. In the acute (6 or 24 h) or subacute (7 days) toxicity tests, rats were administered the drugs once or once a day for a week, respectively. Plasma biochemical markers for hepatotoxicity were measured. The 6 and 24 h toxicity test of diclofenac, and the 24 h and 7 days toxicity tests of flutamide showed significant ALT elevations. Additionally, the 24 h toxicity test of flutamide showed a slight bilirubin elevation, and histological hepatotoxicity. The 7 days toxicity test of flutamide also demonstrated histological hepatotoxicity. In conclusion, this rat model would contribute to evaluating acute and subacute DILI in preclinical drug development.
Toxicology Letters.
