Shinzo Kato

Tumour necrosis factor α signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice

Background: While tumour necrosis factor α (TNF-α) appears to be associated with the development of non-alcoholic steatohepatitis (NASH), its precise role in the pathogenesis of NASH is not well understood. Methods: Male mice deficient in both TNF receptors 1 (TNFR1) and 2 (TNFR2) (TNFRDKO mice) and wild-type mice were fed a methionine and choline deficient (MCD) diet or a control diet for eight weeks, maintaining isoenergetic intake. Results: MCD dietary feeding of TNFRDKO mice for eight weeks resulted in attenuated liver steatosis and fibrosis compared with control wild-type mice. In the liver, the number of activated hepatic Kupffer cells recruited was significantly decreased in TNFRDKO mice after MCD dietary feeding. In addition, hepatic induction of TNF-α, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 was significantly suppressed in TNFRDKO mice. While in control animals MCD dietary feeding dramatically increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in both whole liver and hepatic stellate cells, concomitant with enhanced activation of hepatic stellate cells, both factors were significantly lower in TNFRDKO mice. In primary cultures, TNF-α administration enhanced TIMP-1 mRNA expression in activated hepatic stellate cells and suppressed apoptotic induction in activated hepatic stellate cells. Inhibition of TNF induced TIMP-1 upregulation by TIMP-1 specific siRNA reversed the apoptotic suppression seen in hepatic stellate cells. Conclusions: Enhancement of the TNF-α/TNFR mediated signalling pathway via activation of Kupffer cells in an autocrine or paracrine manner may be critically involved in the pathogenesis of liver fibrosis in this NASH animal model.

Pathogenesis of alcoholic liver disease with particular emphasis on oxidative stress.

Oxidative stress is well recognized to be a key step in the pathogenesis of ethanol‐associated liver injury. Ethanol administration induces an increase in lipid peroxidation either by enhancing the production of oxygen reactive species and/or by decreasing the level of endogenous antioxidants. Numerous experimental studies have emphasized the role of the ethanol‐inducible cytochrome P450 in the microsomes and the molybdo‐flavoenzyme xanthine oxidase in the cytosol. This review shows the putative role of ethanol‐induced disturbances in iron metabolism in relation to iron as a pro‐oxidant factor. Ethanol administration also affects the mitochondrial free radical generation. Many previous studies suggest a role for active oxygens in ethanol‐induced mitochondrial dysfunction in hepatocytes. Recent studies in our laboratory in the Department of Internal Medicine, Keio University, using a confocal laser scanning microscopic system strongly suggest that active oxidants generated during ethanol metabolism produce mitochondrial membrane permeability transition in isolated and cultured hepatocytes. In addition, acetaldehyde, ethanol consumption‐associated endotoxaemia and subsequent release of inflammatory mediators may cause hepatocyte injury via both oxyradical‐dependent and ‐independent mechanisms. These cytotoxic processes may lead to lethal hepatocyte injury. Investigations further implicate the endogenous glutathione—glutathione peroxidase system and catalase as important antioxidants and cytoprotective machinery in the hepatocytes exposed to ethanol.

Oxidative stress on mitochondria and cell membrane of cultured rat hepatocytes and perfused liver exposed to ethanol.

BACKGROUND & AIMS The precise pathogenic significance of oxidative injury in the evolution of alcohol-induced liver disease is still obscure. The present report was designed to investigate whether ethanol alters the production of active oxidants and biological activities of hepatocytes. METHODS The following parameters in rat hepatocytes were investigated by using fluorescence probes in vitro and ex vivo: (1) mitochondrial membrane potential and membrane permeability transition, (2) oxygen radicals generation, (3) membrane barrier function, and (4) glutathione level. RESULTS Ethanol (50 mmol/L) increased oxidative stress in hepatocytes and subsequently induced an increased mitochondrial permeability transition and a decreased membrane potential. These ethanol-induced alterations were attenuated by an inhibitor of alcohol dehydrogenase and an intracellular oxidant scavenger, whereas they were enhanced by diethyl maleic acid, a glutathione depletor. Ethanol plus diethyl maleic acid but not ethanol alone increased the number of hepatocytes with membrane barrier dysfunction. A continuous infusion of ethanol (50 mmol/L) increased oxidative stress and decreased mitochondrial membrane potential in the pericentral area of isolated perfused rat liver. CONCLUSIONS Active oxidants generated during ethanol metabolism increase mitochondrial permeability transition and modulate mitochondrial energy synthesis in hepatocytes. Reduction of glutathione level enhances mitochondrial dysfunction and impairs membrane barrier function of hepatocytes.

Hepatic injury in 12 patients taking the herbal weight loss aids Chaso or Onshido

Context Herbal weight loss aids are marketed as quick, safe fixes for obesity. Contribution This report describes 12 Japanese patients who developed liver injury while taking the herbal weight loss aids Chaso or Onshido. Chemical analysis of these products showed N-nitroso-fenfluramine, the active compound in a weight loss drug that the U.S. Food and Drug Administration withdrew from the U.S. market. Cautions This case series cannot tell us how frequently herbal weight loss aids lead to liver injury, nor can it prove that N-nitroso-fenfluramine is the toxic agent. Conclusion This report suggests that herbal weight loss aids are unsafe in some people. The Editors Although dietary supplements for weight loss (weight loss aids) are widely available, the evidence for their efficacy is weak (1). The weight loss aids can be purchased on the Internet and have become popular in Japan. Product labels rarely include safety information and often do not list potentially harmful ingredients. Several ingredients, such as ephedra alkaloids (ma huang), thyroid hormone, and fenfluramine, have been identified in some of the weight loss aids and have been reported to cause adverse side effects (2-4). Acute hepatotoxicity associated with herbal weight loss aids has been recently reported (5-7). We describe 12 patients who developed acute liver injury, including 2 patients who developed fulminant hepatic failure, after taking the Chinese herbal weight loss aids Chaso (Meikang Health Products, Hunan, China) and Onshido (Yuzhitang Health Products, Guangzhou, China). Methods We reviewed the records of 6 patients hospitalized at Keio University Hospital, Tokyo, Japan, or its affiliated hospitals and clinics from April to July 2002 because of acute hepatitis after ingesting Chaso or Onshido. In addition, we reviewed the records of 6 patients provided by physicians at other hospitals in Japan. Twelve Japanese patients (6 patients who took Chaso and 6 patients who took Onshido) are documented. One of the cases (patient 2) has been published as a single-case report (8). We classified fulminant hepatic failure according to international criteria (9). We analyzed the ingredients of the Chaso and Onshido products that 5 patients used (patients 1, 4, 6, 7, and 11) by using Agilent 1100 Series Liquid Chromatograph/Mass Selective Detector (LC/MSD) (Agilent Technologies, Palo Alto, California). We assessed contamination with heavy metals by using a spectrophotometer (U-1100, Hitachi Co., Tokyo, Japan). Results Patients Eleven of 12 patients were hospitalized (Table). All patients reported taking the manufacturers recommended dose, and no patients had chronic medical conditions or were extremely obese. No patient reported taking prescription or over-the-counter medications, birth control pills, estrogen therapy, or other alternative therapies in addition to Chaso or Onshido. Laboratory evaluation did not identify viral infection, autoimmune diseases, alcoholic liver diseases, or other metabolic liver diseases. Clinical symptoms, including fatigue and appetite loss, appeared 5 to 40 days after the weight loss aid was ingested. Most patients mistook their symptoms for weight loss effect and did not seek immediate care after the onset of symptoms. Table. Patient Characteristics The clinical presentation of these 12 patients was consistent with hepatocellular-type or mixed-type liver injury (10). No patient exhibited signs of allergy except for increased eosinophil count (up to 10% in leukocyte counts) in patient 2. Moreover, lymphocyte stimulation test results, which are useful in diagnosing drug-induced immunoallergic liver injury (11), were negative in 8 patients tested (patients 1 to 3, 6 to 8, and 10 to 12). Patients did not exhibit extrahepatic disease. Two patients (patients 1 and 7) had hepatic encephalopathy and met diagnostic criteria for fulminant hepatic failure. Patient 1 received a liver transplant on hospital day 8. Patient 7 died on hospital day 45, subsequent to intestinal bleeding and infection. The other 10 patients improved after discontinuing use of the products and had normalization of liver test results after hospital discharge. Pathologic findings of the removed liver from patient 1 showed massive hemorrhagic necrosis of the liver with a nonspecific inflammatory infiltrate by lymphocytes and neutrophils (Figure, top). Pathologic findings of liver needle biopsy from patient 7 showed submassive necrosis with a nonspecific inflammatory infiltrate (Figure, middle). Pathologic findings of the autopsied liver from patient 7 showed diffuse hepatocellular necrosis, infiltration of lymphocytes and macrophages, formation of prominent ductular proliferation with bile thrombi, and bridging fibrosis (Figure, bottom). Pathologic findings of liver needle biopsy from patient 2 showed necrosis with an inflammatory infiltrate by lymphocytes and eosinophils. Figure. Pathologic findings of patients 1 and 7. Top. Middle. Bottom. Analysis of Ingredients in the Weight Loss Aids According to the manufacturer, Chaso contains the following botanicals: green tea, cassia torae semen, leaves of lotus, Fructus lycii, F. crataegi, and chrysanthemum flowers. Onshido contains extract of Gynostemma pentaphyllum makino, green tea, aloe, F. crataegi fructus, and raphani semen. The analysis revealed that both products contained N-nitroso-fenfluramine. Fenfluramine and heavy metals (copper, lead, bismuth, cadmium, arsenic, stibium, stannum, mercury, and chromium) were not detected. Discussion We describe 12 patients with acute liver injury after taking the herbal weight loss aids Chaso or Onshido, suggesting that use of these products can be associated with hepatotoxicity. In cooperation with the Ministry of Health, Labour and Welfare in Japan, we warned the Japanese public of the potential adverse effects of Chaso and Onshido in July 2002 (12). Subsequently, 474 cases of hepatotoxicity induced by herbal weight loss aids (including 21 Chaso-induced cases and 135 Onshido-induced cases) were reported to the Ministry by the end of that year. Most cases occurred from April to August 2002. We cannot ascertain how many people used these products, but we speculate that more than 10 000 Onshido products were distributed in Japan. Chaso was distributed much less than Onshido. Women most often used these supplements; more than 90% of the 474 patients were women. Drug-induced liver injury is mainly classified as toxic or immunoallergic. In this case series, pathogenesis of liver injury seems to be toxic rather than immunoallergic. We saw little evidence of allergy and a relatively high incidence of hepatotoxicity in the users. In addition, analysis revealed that the products contained N-nitroso-fenfluramine. However, patient 2 had an increased eosinophil count in both peripheral blood cells and pathologic findings, suggesting that an allergic reaction to these weight loss aids may also contribute to the pathogenesis of liver injury. Severity of hepatotoxicity does not seem to correlate with duration of use or the amount of product ingested, and not all patients who took these products developed liver injury. Thus, unidentified specific host factors may alter the individual susceptibility to the products. It is possible that the amount of toxic ingredients may vary among the lots because of inferior manufacturing processes. The Japan Society of Hepatology is currently investigating the nationwide survey of the detailed characteristics. Because Chinese herbal remedies usually consist of botanical and herbal components, it is difficult to identify a specific toxic ingredient (13, 14). The clinical features in patients who had taken Chaso or Onshido were similar; however, all the herbal components labeled on these products have not been reported to be hepatotoxic. Although the manufacturers of these weight loss aids have claimed that they are made only of Chinese herbal remedies, we detected a synthetic chemical, N-nitroso-fenfluramine, in both products. Fenfluramine was once prescribed for weight loss but was withdrawn from clinical use because of cardiac complications (2). Because N-nitroso compounds have been linked with carcinogenesis in the liver (15), N-nitroso-fenfluramine may be a potential hepatotoxic ingredient. Further toxicologic analysis should be done to determine its hepatotoxicity. Although it is not clear why these products contained N-nitroso-fenfluramine, it might have been added by the manufacturers to enhance the weight loss effect of fenfluramine or to mask the presence of fenfluramine. In addition to Chaso and Onshido, 38 other herbal weight loss aids were found to contain fenfluramine or N-nitroso-fenfluramine. Dietary supplements are considered health foods rather than drugs, despite their potential risks (16). In Japan, Chinese herbal remedies are widely used in both medicinal drugs and dietary supplements. Many users consider herbal products to be harmless. A survey indicates that users of herbal remedies would consult their general practitioner for adverse effects of over-the-counter medicine but not for those of herbal remedies (17). Overconfidence in the safety of herbal remedies might have contributed to the occurrence of series of cases. Previous information on the adverse effect of drugs is often important to diagnose drug-induced liver injury. As the safety test for dietary supplements is not required, diagnosing adverse effects of dietary supplements may be difficult and delayed. The manufacturers are primarily responsible for ensuring safety of their products, but adequate surveillance systems for monitoring the safety of dietary supplements should also be established. We highlight the need for increased awareness among individuals, including medical professionals, of the potential toxicity risks of some herbal weight loss aids.

Early midzonal oxidative stress preceding cell death in hypoperfused rat liver

Intralobular heterogeneity of oxidative stress and its topographic relationship with cell death during low-flow hypoxia were shown in perfused rat liver by digital microfluorography using dichlorofluorescin diacetate, a fluorochrome sensitive to intracellular hydroperoxide formation, and propidium iodide, which labels the nuclei of nonviable cells. The surface of the liver loaded with two precursors was microscopically visualized, and the fluorescence of dichlorofluorescein, a highly fluorescent molecule generated by hydroperoxide-mediated dichlorofluorescin oxidation, was digitally processed. Dichlorofluorescein fluorescence significantly increased in midzonal regions as early as 20 minutes after starting the 25% low-flow hypoxia. At 40 minutes the fluorograph showed multiple dotted patterns, and the intensity peaked at 60 minutes. The onset of cell death studied by propidium iodide was observed at 40 minutes, and its topographic distribution corresponded to the dichlorofluorescein-enhanced midzonal regions. Allopurinol diminished the early midzonal oxidative stress and retarded the onset of cell death. The current findings show that xanthine oxidase-dependent oxidative stress and the resultant cell death during low-flow hypoxia are spatially restricted in the intermediate zone between the periportal and pericentral regions.

Prostaglandin E1 abrogates early reductive stress and zone-specific paradoxical oxidative injury in hypoperfused rat liver

This study was designed to investigate the effects of prostaglandin E1 on reductive stress and the subsequent oxidative cell injury in hypoperfused rat liver. The intralobular heterogeneity of hepatocellular redox state, mitochondrial dysfunction, and intracellular hydroperoxide formation were visually monitored by digital microfluorography of pyridine nucleotide autofluorescence, rhodamine 123, and dichlorofluorescein fluorescence, respectively. Under the 25% low flow perfusion, pyridine nucleotide autofluorescence increased time-dependently and reached a steady state at 10 min among the entire lobules. The decrease in mitochondrial membrane potential was > 20 mV in all portions of the lobules at 60 min. The onset of hydroperoxide formation was observed at 40 min in the marginally oxygenated proximal portion of anoxic pericentral regions and the oxidative impact reached a maximum level at 60 min. Sodium (-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazo [1,5-a]-1,3,5-triazine-4-olate monohydrate (BOF 4272), a novel xanthine oxidase inhibitor, suppressed the zone-specific oxidative changes without attenuating the increase in pyridine nucleotide autofluorescence and mitochondrial dysfunction. Pretreatment with prostaglandin E1 not only abrogated an early increase in pyridine nucleotide fluorescence and mitochondrial dysfunction induced by hypoperfusion but also diminished the subsequent midzonal oxidative injury. Since prostaglandin E1 has no oxyradical-scavenging action, the preventive effect of this reagent on the hypoxia-induced oxidative cell injury is attributable to the attenuation of mitochondrial dysfunction. These results suggest that, in low flow hypoxia, early reductive stress plays a key role in the initiation of xanthine oxidase-mediated midzonal oxidative changes, which may lead to subsequent centrilobular necrosis.

Hepatic fibrosis produced in guinea pigs by chronic ethanol administration and immunization with acetaldehyde adducts

Experimental hepatic fibrosis was produced in the guinea pig. We produced hepatic necrosis associated with inflammatory cell infiltration in guinea pigs immunized with acetaldehyde adducts and fed ethanol for 40 days. Extending the period of these treatments to 90 days resulted in producing hepatic fibrosis developing around individual hepatocytes in the terminal hepatic venule areas and portal areas, accompanied by an increase in hepatic hydroxyproline content. In contrast, no fibrosis was observed in the livers of the control groups that had been exposed to nothing, ethanol alone, or a combination of ethanol and immunization with unmodified human hemoglobin. Minimal fibrotic changes were observed in animals immunized with human hemoglobin acetaldehyde adducts but not fed ethanol. These results indicate that the formation of acetaldehyde adducts and the acquisition of immunity against them can produce hepatic fibrosis. Immune mechanisms against acetaldehyde adducts may, in part, be involved in the pathogenesis of hepatic fibrosis seen in alcoholics.

Evaluation of platelet kinetics in patients with liver cirrhosis: Similarity to idiopathic thrombocytopenic purpura

Background:  Thrombocytopenia is a common manifestation of liver cirrhosis (LC), but its underlying mechanism is not fully understood. The purpose of the present paper was to evaluate the platelet kinetics in LC patients by examining several non‐invasive convenient markers.

Oxidative stress and mitochondrial damage precedes gastric mucosal cell death induced by ethanol administration

Although it has been speculated that active oxidants and mitochondrial membrane damages play roles in ethanol-induced gastric mucosal damages, its detail remains unknown. The present study was designed to investigate whether ethanol induces oxidative stress and mitochondrial permeability transition (MPT) before cell death of gastric mucosal cells. Rat gastric mucosal cells (RGM-1) were kept in serum-free Dulbeccos modified Eagles medium before addition of various concentrations of ethanol. Nuclear morphological aftemations and membrane barrier dysfunction of RGM-1 cells were assessed by staining with Hoechst 33342 and propidium iodide, respectively. To assess the contribution of oxygen-derived free radicals and intracellular glutathione, scavenger of hydrogen peroxide and the hydroxyl radical, N,N-dimethylthiourea, glutathione precursor, N-acetyl-L-cysteine, and an inhibitor of alcohol dehydrogenase, 4-methylpyrazole were added before treatment with ethanol. To investigate MPT, calcein and tetramethylrhodamine methyl ester were loaded before addition of ethanol, and the changes of fluorescence intensity were monitored using a laser scanning confocal microscope. Ethanol (>5% v/v) dose-dependently increased the number of propidium iodide-positive cells, suggesting a diminished barrier function of cell membrane. After addition of ethanol, mitochondria were filled quickly with calcein indicating MPT, which was accompanied by mitochondrial depolarization, as shown by loss of tetramethylrodamine methyl ester before cell death. Ethanol-induced cell death was significantly attenuated by simultaneous incubation with either N,N-dimethylthiourea or N-acetyl-L-cysteine, suggesting the importance of intracellular redox states in inducing cellular damage, whereas such change was not attenuated by 4-methylpyrazole. Present results suggest that ethanol treatment induces intracellular oxidative stress and produces MPT and mitochondrial depolarization, which are preceding cell death in gastric mucosal cells. Intracellular antioxidants, such as glutathione, may have a significant protective action against ethanol in gastric mucosal cells.

Potentiation of halothane hepatotoxicity by chronic ethanol administration in rat: An animal model of halothane hepatitis

To determine if chronic ethanol administration modifies the effect of halothane on the liver, fourteen male Wistar rats were pair-fed nutritionally adequate liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate (controls) for 6 weeks. After halothane anesthesia of these animals under different oxygen concentration, the livers were examined light microscopically as well as biochemically. The livers from rats fed ethanol which received halothane at low oxygen concentration showed multifocal or patchy necrosis primarily in the centrilobular regions with parenchymal lipid accumulation, whereas no such lesions were not observed in pair-fed controls. Hepatic necrosis was also seen after halothane anesthesia even at ambient oxygen concentrations, although the degree of necrosis was much milder. Hepatic microsomal cytochrome P450 content was increased by 30% after ethanol but was decreased following halothane anesthesia. These data suggest that halothane is hepatotoxic to liver of rats chronically pretreated with ethanol, especially under hypoxic condition.