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Shurubor YI, Keskinov AA, Yudin VS, Krasnikov BF. The Balance of Ketoacids α-Ketoglutarate and α-Ketoglutaramate Reflects the Degree of the Development of Hepatoencephalopathy in Rats. Int J Mol Sci 2024; 25:13568. [PMID: 39769330 PMCID: PMC11677448 DOI: 10.3390/ijms252413568] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2024] [Revised: 12/10/2024] [Accepted: 12/13/2024] [Indexed: 01/03/2025] Open
Abstract
Hepatoencephalopathy (HE) is a liver disease that can lead to brain pathology and the impairment of human cognitive abilities. The objective assessment of HE disease severity is difficult due to the lack of reliable diagnostic markers. This paper examines the background to the emergence of HE markers and provides a brief overview of research results indicating the diagnostic value of potential markers isolated from a wide range of metabolites analyzed. It has been suggested that metabolites of the glutamate-glutamine (Glu-Gln) cycle, α-ketoglutarate (αKG), and α-ketoglutaramate (αKGM) can act as such markers of HE. The informative value of these markers was revealed during a comparative analysis of the distribution of αKG and αKGM in samples of the blood plasma and tissues (liver, kidneys, and brain) of rats exposed to the strong hepatotoxin thioacetamide (TAA). A comparative analysis of the balance of αKG and αKGM, as well as their ratio (αKG/αKGM) in the examined samples of blood plasma and animal tissues in these models, revealed their diagnostic value for assessing the severity of HE and/or monitoring the recovery process.
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Affiliation(s)
- Yevgeniya I. Shurubor
- Centre for Strategic Planning of FMBA of the Russian Federation, Pogodinskaya St., Bld. 10, 119121 Moscow, Russia; (A.A.K.); (V.S.Y.)
| | - Anton A. Keskinov
- Centre for Strategic Planning of FMBA of the Russian Federation, Pogodinskaya St., Bld. 10, 119121 Moscow, Russia; (A.A.K.); (V.S.Y.)
| | - Vladimir S. Yudin
- Centre for Strategic Planning of FMBA of the Russian Federation, Pogodinskaya St., Bld. 10, 119121 Moscow, Russia; (A.A.K.); (V.S.Y.)
| | - Boris F. Krasnikov
- Centre for Strategic Planning of FMBA of the Russian Federation, Pogodinskaya St., Bld. 10, 119121 Moscow, Russia; (A.A.K.); (V.S.Y.)
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, N.I. Pirogov Russian National Research Medical University, 1 Ostrovitianova Str., 117997 Moscow, Russia
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Ezhilarasan D. Molecular mechanisms in thioacetamide-induced acute and chronic liver injury models. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2023; 99:104093. [PMID: 36870405 DOI: 10.1016/j.etap.2023.104093] [Citation(s) in RCA: 41] [Impact Index Per Article: 20.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/06/2022] [Revised: 02/26/2023] [Accepted: 02/28/2023] [Indexed: 06/18/2023]
Abstract
Thioacetamide (TAA) undergoes bioactivation in the liver by the CYP450 2E1 enzyme, resulting in the formation of TAA-S-oxide and TAA-S-dioxide. TAA-S-dioxide induces oxidative stress via lipid peroxidation of the hepatocellular membrane. A single TAA dose (50-300 mg/kg) administration initiates hepatocellular necrosis around the pericentral region after its covalent binding to macromolecules in the liver. Intermittent TAA administration (150-300 mg/kg, weekly thrice, for 11-16 weeks) activates transforming growth factor (TGF)-β/smad3 downstream signaling in injured hepatocytes, causing hepatic stellate cells (HSCs) to acquire myofibroblast like phenotype. The activated HSCs synthesize a variety of extracellular matrix, leading to liver fibrosis, cirrhosis, and portal hypertension. The TAA induced liver injury varies depending on the animal model, dosage, frequency, and routes of administration. However, TAA induces hepatotoxicity in a reproducible manner, and it is an ideal model to evaluate the antioxidant, cytoprotective, and antifibrotic compounds in experimental animals.
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Affiliation(s)
- Devaraj Ezhilarasan
- Department of Pharmacology, Molecular Medicine and Toxicology Lab, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu 600 077, India.
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Shurubor YI, Rogozhin AE, Isakova EP, Deryabina YI, Krasnikov BF. Residual Amino Acid Imbalance in Rats during Recovery from Acute Thioacetamide-Induced Hepatic Encephalopathy Indicates Incomplete Healing. Int J Mol Sci 2023; 24:ijms24043647. [PMID: 36835059 PMCID: PMC9967446 DOI: 10.3390/ijms24043647] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2023] [Revised: 02/06/2023] [Accepted: 02/07/2023] [Indexed: 02/16/2023] Open
Abstract
The delayed consequences of the influence of hepatic encephalopathy (HE) on the metabolism of animals have not been studied enough. We have previously shown that the development of acute HE under the influence of the thioacetamide (TAA) toxin is accompanied by pathological changes in the liver, an imbalance in CoA and acetyl CoA, as well as a number of metabolites of the TCA cycle. This paper discusses the change in the balance of amino acids (AAs) and related metabolites, as well as the activity of glutamine transaminase (GTK) and ω-amidase enzymes in the vital organs of animals 6 days after a single exposure to TAA. The balance of the main AAs in blood plasma, liver, kidney, and brain samples of control (n = 3) and TAA-induced groups (n = 13) of rats that received the toxin at doses of 200, 400, and 600 mg/kg was considered. Despite the apparent physiological recovery of the rats at the time of sampling, a residual imbalance in AA and associated enzymes persisted. The data obtained give an idea of the metabolic trends in the body of rats after their physiological recovery from TAA exposure and may be useful for prognostic purposes when choosing the necessary therapeutic agents.
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Affiliation(s)
| | - Alexander E. Rogozhin
- Valiev Institute of Physics and Technology of the Russian Academy of Sciences, Moscow 117218, Russia
| | - Elena P. Isakova
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia
| | - Yulia I. Deryabina
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia
| | - Boris F. Krasnikov
- Centre for Strategic Planning of FMBA of Russia, Moscow 119121, Russia
- Correspondence: ; Tel.: +7-(985)-095-5445
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4
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Shurubor YI, Rogozhin AE, Isakova EP, Deryabina YI, Krasnikov BF. Tricarboxylic Acid Metabolite Imbalance in Rats with Acute Thioacetamide-Induced Hepatic Encephalopathy Indicates Incomplete Recovery. Int J Mol Sci 2023; 24:ijms24021384. [PMID: 36674898 PMCID: PMC9861856 DOI: 10.3390/ijms24021384] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2022] [Revised: 12/20/2022] [Accepted: 01/05/2023] [Indexed: 01/13/2023] Open
Abstract
Exposure to the toxin thioacetamide (TAA) causes acute hepatic encephalopathy (HE), changes in the functioning of systemic organs, and an imbalance in a number of energy metabolites. The deferred effects after acute HE development are poorly understood. The study considers the balance of the tricarboxylic acid (TCA) cycle metabolites in the blood plasma, liver, kidneys, and brain tissues of rats in the post-rehabilitation period. The samples of the control (n = 3) and TAA-induced groups of rats (n = 13) were collected six days after the administration of a single intraperitoneal TAA injection at doses of 200, 400, and 600 mg/kg. Despite the complete physiological recovery of rats by this date, a residual imbalance of metabolites in all the vital organs was noted. The results obtained showed a trend of stabilizing processes in the main organs of the animals and permit the use of these data both for prognostic purposes and the choice of potential therapeutic agents.
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Affiliation(s)
- Yevgeniya I. Shurubor
- Centre for Strategic Planning and Management of Medical and Biological Health Risks, Federal Medical Biological Agency of The Russian Federation, Moscow 119121, Russia
| | - Alexander E. Rogozhin
- Valiev Institute of Physics and Technology of the Russian Academy of Sciences, Moscow 117218, Russia
| | - Elena P. Isakova
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia
| | - Yulia I. Deryabina
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia
| | - Boris F. Krasnikov
- Centre for Strategic Planning and Management of Medical and Biological Health Risks, Federal Medical Biological Agency of The Russian Federation, Moscow 119121, Russia
- Correspondence: ; Tel.: +7-(985)-095-5445
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5
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Huang W, Han N, Du L, Wang M, Chen L, Tang H. A narrative review of liver regeneration-from models to molecular basis. ANNALS OF TRANSLATIONAL MEDICINE 2021; 9:1705. [PMID: 34988214 PMCID: PMC8667151 DOI: 10.21037/atm-21-5234] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/14/2021] [Accepted: 11/04/2021] [Indexed: 12/11/2022]
Abstract
Objective To elucidate the characteristics of different liver regeneration animal models, understand the activation signals and mechanisms related to liver regeneration, and obtain a more comprehensive conception of the entire liver regeneration process. Background Liver regeneration is one of the most enigmatic and fascinating phenomena of the human organism. Despite suffering significant injuries, the liver still can continue to perform its complex functions through the regeneration system. Although advanced topics on liver regeneration have been proposed; unfortunately, complete regeneration of the liver has not been achieved until now. Therefore, increasing understanding of the liver regenerative process can help improve our treatment of liver failure. It will provide a new sight for the treatment of patients with liver injury in the clinic. Methods Literatures on liver regeneration animal models and involved basic research on molecular mechanisms were retrieved to analyze the characteristics of different models and those related to molecular basis. Conclusions The process of liver regeneration is complex and intricate, consisting of various and interactive pathways. There is sufficient evidence to demonstrate that liver regeneration is similar between humans and rodents. At the same time, many of the same cytokines, growth factors, and signaling pathways are relevant. There are many gaps in our current knowledge. Understanding of this knowledge will provide more supportive clinical treatment strategies, including small-scale liver transplantation and high-quality regenerative process after surgical resection, and offer possible targets to treat the dysregulation of regeneration that occurs in chronic hepatic diseases and tumors. Current research work, such as the use of animal models as in vivo vectors for high-quality human hepatocytes, represents a unique and significant cutting edge in the field of liver regeneration.
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Affiliation(s)
- Wei Huang
- Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China.,Division of Infectious Diseases, State Key Laboratory of Biotherapy and Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China
| | - Ning Han
- Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China.,Division of Infectious Diseases, State Key Laboratory of Biotherapy and Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China
| | - Lingyao Du
- Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China.,Division of Infectious Diseases, State Key Laboratory of Biotherapy and Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China
| | - Ming Wang
- Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China.,Division of Infectious Diseases, State Key Laboratory of Biotherapy and Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China
| | - Liyu Chen
- Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China.,Division of Infectious Diseases, State Key Laboratory of Biotherapy and Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China
| | - Hong Tang
- Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China.,Division of Infectious Diseases, State Key Laboratory of Biotherapy and Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China
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6
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Shurubor YI, Cooper AJL, Krasnikov AB, Isakova EP, Deryabina YI, Beal MF, Krasnikov BF. Changes of Coenzyme A and Acetyl-Coenzyme A Concentrations in Rats after a Single-Dose Intraperitoneal Injection of Hepatotoxic Thioacetamide Are Not Consistent with Rapid Recovery. Int J Mol Sci 2020; 21:E8918. [PMID: 33255464 PMCID: PMC7727790 DOI: 10.3390/ijms21238918] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2020] [Revised: 11/19/2020] [Accepted: 11/20/2020] [Indexed: 12/12/2022] Open
Abstract
Small biomolecules, such as coenzyme A (CoA) and acetyl coenzyme A (acetyl-CoA), play vital roles in the regulation of cellular energy metabolism. In this paper, we evaluated the delayed effect of the potent hepatotoxin thioacetamide (TAA) on the concentrations of CoA and acetyl-CoA in plasma and in different rat tissues. Administration of TAA negatively affects liver function and leads to the development of hepatic encephalopathy (HE). In our experiments, rats were administered a single intraperitoneal injection of TAA at doses of 200, 400, or 600 mg/kg. Plasma, liver, kidney, and brain samples were collected six days after the TAA administration, a period that has been suggested to allow for restoration of liver function. The concentrations of CoA and acetyl-CoA in the group of rats exposed to different doses of TAA were compared to those observed in healthy rats. The results obtained indicate that even a single administration of TAA to rats is sufficient to alter the physiological balance of CoA and acetyl-CoA in the plasma and tissues of rats for an extended period of time. The initial concentrations of CoA and acetyl-CoA were not restored even after the completion of the liver regeneration process.
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Affiliation(s)
- Yevgeniya I. Shurubor
- Center for Strategic Planning and Management of Medical and Biological Health Risks, Federal Medical-Biological Agency of The Russian Federation, 119121 Moscow, Russia;
| | - Arthur J. L. Cooper
- Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA
| | | | - Elena P. Isakova
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia; (E.P.I.); (Y.I.D.)
| | - Yulia I. Deryabina
- Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia; (E.P.I.); (Y.I.D.)
| | - M. Flint Beal
- Department of Neurology, The Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY 10021, USA;
| | - Boris F. Krasnikov
- Center for Strategic Planning and Management of Medical and Biological Health Risks, Federal Medical-Biological Agency of The Russian Federation, 119121 Moscow, Russia;
- Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA
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7
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S. Hamed S, Abdel Sala S, F. El-Khad M, A. AL-Megr W, K. Hassan Z, M. Shuker E. Chlorpheniramine Maleate Induced Cardiotoxicity, Hepatotoxicity and Antioxidant Gene Expression Changes in Male Wistar Rats. INT J PHARMACOL 2020. [DOI: 10.3923/ijp.2020.351.366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
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8
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Bahçecıoğlu IH, Ispiroglu M, Tuzcu M, Orhan C, Ulas M, Demirel U, Yalniz M, Özercan IH, Ilhan N, Sahin K. Pistacia Terebinthus Coffee Protects against Thioacetamide-Induced Liver Injury in Rats. ACTA MEDICA (HRADEC KRÁLOVÉ) 2015; 58:56-61. [DOI: 10.14712/18059694.2015.94] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
Aim/background: Pistacia terebinthus is used as a coffee substitute in the East and Southern Anatolia regions of Turkey. It contains unsaturated fatty acids, tocopherols, polyphenols and carotenoids. P. terebinthus has anti-inflammatory and potential antioxidant activity. In this study we evaluated the protective effects of P. terebinthus coffee (PTC) on thioacetamide (TAA)-induced liver injury in rats. Materials and methods: Twenty-eight male Sprague-Dawley rats were equally randomized into four groups. Chronic liver injury was induced with TAA (100 mg/kg i.p. three times weekly). The first group of rats served as control and received only tap water (G1), and the remaining groups of rats received PTC, p.o (G2); TAA (G3); TAA plus PTC, p.o (G4), respectively. Results: After 8 weeks, PTC intake significantly reduced fibrosis/inflammation scores (p < 0.05) in the livers of TAA-treated group. Compared to control group, PTC intake reduced transforming growth factor beta (TGF-β) concentrations in the liver (p < 0.05). Compared to the TAA group, TGF-β, nuclear factor kappa B (NF)-κB (p < 0.05), tumor necrosis factor alpha (TNF-α) concentrations in the liver tissue were reduced by PTC intake. Discussion and conclusion: PTC intake provided beneficial effects against TAA-induced liver injury in rats. PTC probably suppresses the proinflammatory cytokines through NF-κB signaling pathway.
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9
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Kabil NN, Seddiek HA, Yassin NA, Gamal-Eldin MM. Effect of ghrelin on chronic liver injury and fibrogenesis in male rats: possible role of nitric oxide. Peptides 2014; 52:90-7. [PMID: 24333973 DOI: 10.1016/j.peptides.2013.11.022] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/25/2013] [Revised: 11/25/2013] [Accepted: 11/26/2013] [Indexed: 12/11/2022]
Abstract
Recent studies have revealed that ghrelin may be an antioxidant and anti-inflammatory agent in many organs, however its role in chronic liver injury (CLI) remains unclear. The role of nitric oxide (NO) in CLI is controversial as evidence suggests that NO is either a primary mediator of liver cell injury or exhibits a protective effect against injurious stimuli. Recent evidence demonstrated that the therapeutic potential for ghrelin was through eNOS activation and increase in NO production. However, its role on NO production in the liver has not been previously investigated. The aim of this study was to investigate the role of ghrelin in treatment of CLI, and whether this action is mediated through NO. Forty male rats were divided into four groups: Group I: Control; Group II: chronic liver injury (CLI); Group III: CLI+Ghrelin; and Group IV: CLI+Ghrelin+l-NAME. Liver enzymes and tumor necrosis factor alpha (TNF-α), were measured to assess hepatocellular injury. Liver tissue collagen content, malondialdehyde (MDA), gene expression of Bax, Bcl-2, and eNOS were assessed to determine the mechanism of ghrelin action. Results showed that ghrelin decreased serum liver enzymes and TNF-α levels. Ghrelin also reduced liver tissue collagen, MDA, and Bax gene expression, and increased Bcl-2 and eNOS gene expression. The effects on TNF-α, collagen, MDA, Bax, and eNOS were partially reversed in Group IV, suggesting that ghrelin's action could be through modulation of NO levels. Therefore, ghrelin's hepatoprotective effect is partially mediated by NO release.
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Affiliation(s)
- Nashwa N Kabil
- Department of Physiology, Faculty of Pharmacy & Biotechnology, German University in Cairo, Egypt.
| | - Hanan A Seddiek
- Department of Physiology, Kasr Al Aini Faculty of Medicine, Cairo University, Egypt.
| | - Nadia A Yassin
- Department of Physiology, Faculty of Pharmacy & Biotechnology, German University in Cairo, Egypt; Department of Physiology, Kasr Al Aini Faculty of Medicine, Cairo University, Egypt.
| | - Maha M Gamal-Eldin
- Department of Physiology, Faculty of Pharmacy & Biotechnology, German University in Cairo, Egypt; Department of Physiology, Kasr Al Aini Faculty of Medicine, Cairo University, Egypt.
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10
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Bautista M, Del Rio MÁG, Benedí J, Sánchez-Reus MI, Morales-González JA, Téllez-López AM, López-Orozco M. Effect of dichloromethylene diphosphonate on liver regeneration following thioacetamide-induced necrosis in rats. World J Hepatol 2013; 5:379-386. [PMID: 23898371 PMCID: PMC3724966 DOI: 10.4254/wjh.v5.i7.379] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/30/2013] [Revised: 06/17/2013] [Accepted: 06/18/2013] [Indexed: 02/06/2023] Open
Abstract
AIM To study the effect of dichloromethylene diphosphonate (DMDP), a selective Kupffer cell toxicant in reference to liver damage and postnecrotic liver regeneration in rats induced by sublethal dose thioacetamide (TA). METHODS Rats, intravenously (iv) pre-treated with a single dose of DMDP (10 mg/kg), were intraperitoneally (ip) injected with TA 6.6 mmol/kg (per 500 mg/kg body weight). Hepatocytes were isolated from rats at 0, 24, 48 and 72 h following TA intoxication and blood and liver samples were obtained. To evaluate the mechanisms involved in the postnecrotic regenerative state, DNA distribution and ploidy time course were assayed in isolated hepatocytes. Circulating cytokine tumor necrosis factor-alpha (TNF-α) was assayed in serum and determined by reverse transcriptase-polymerase chain reaction in liver extract. RESULTS The effect of DMDP induced noticeable changes in postnecrotic regeneration, causing an increased percentage of hepatocytes in the cell cycle S phase. The increase at 24 h in S1 population in rats pretreated with DMDP + TA was significantly (P < 0.05) different compared with that of the TA group (18.07% vs 8.57%). Hepatocytes increased their proliferation as a result of these changes. Also, TNF-α expression and serum level were increased in rats pre-treated with DMDP. Thus, DMDP pre-treatment reduced TA-induced liver injury and accelerated postnecrotic liver regeneration. CONCLUSION These results demonstrate that Kupffer cells are involved in TA-induced liver, as well as in postnecrotic proliferative liver states.
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Affiliation(s)
- Mirandeli Bautista
- Mirandeli Bautista, Ana María Téllez-López, Maricela López-Orozco, Área Académica de Farmacia, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Pachuca, Hidalgo, CP 42000, México
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11
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Duncan AW. Aneuploidy, polyploidy and ploidy reversal in the liver. Semin Cell Dev Biol 2013; 24:347-56. [PMID: 23333793 DOI: 10.1016/j.semcdb.2013.01.003] [Citation(s) in RCA: 133] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2012] [Accepted: 01/09/2013] [Indexed: 12/30/2022]
Abstract
Polyploidy has been described in the liver for over 100 years. The frequency of polyploid hepatocytes varies by age and species, but up to 90% of mouse hepatocytes and approximately 50% of human hepatocytes are polyploid. In addition to alterations in the entire complement of chromosomes, variations in chromosome copy number have been recently described. Aneuploidy in the liver is pervasive, affecting 60% of hepatocytes in mice and 30-90% of hepatocytes in humans. Polyploidy and aneuploidy in the liver are closely linked, and the ploidy conveyor model describes this relationship. Diploid hepatocytes undergo failed cytokinesis to generate polyploid cells. Proliferating polyploid hepatocytes, which form multipolar spindles during cell division, generate reduced ploidy progeny (e.g., diploid hepatocytes from tetraploids or octaploids) and/or aneuploid daughters. New evidence suggests that random hepatic aneuploidy can promote adaptation to liver injury. For instance, in response to chronic liver damage, subsets of aneuploid hepatocytes that are differentially resistant to the injury remain healthy, regenerate the liver and restore function. Future work is required to elucidate the mechanisms regulating dynamic chromosome changes in the liver and to understand how these processes impact normal and abnormal liver function.
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Affiliation(s)
- Andrew W Duncan
- Department of Pathology, McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA 15219, United States.
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12
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de David C, Rodrigues G, Bona S, Meurer L, González-Gallego J, Tuñón MJ, Marroni NP. Role of quercetin in preventing thioacetamide-induced liver injury in rats. Toxicol Pathol 2011; 39:949-57. [PMID: 21885874 DOI: 10.1177/0192623311418680] [Citation(s) in RCA: 83] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
In hepatic toxicity induced in rats by two injections of thioacetamide (TAA, 350 mg/kg with an interval of 8 hr), the action of quercetin was investigated. After 96 hr, TAA administration resulted in hepatic necrosis, significant increases in serum transaminase activity, and increases in hepatic lipoperoxidation. Thioacetamide-induced hepatotoxicity also showed changes in antioxidant enzymes in the liver of rats, with alterations in p-ERK 1/2 (phosphorylated extracellular-signal related kinase 1/2) as well as an imbalance between proapototic protein Bax and anti-apoptotic protein Bcl-2 expression. With administration of the flavonoid quercetin (50 mg/Kg i.p.) for four consecutive days following TAA, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity were close to normal values in rats. Histological findings suggested that quercetin had a preventive effect on TAA-induced hepatic necrosis. Quercetin treatment caused significant decreases in lipid peroxide levels in the TAA-treated rats, with some changes in antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Quercetin also inhibited the change of the p-ERK1/2 by TAA and significantly prevented the increase in Bax/Bcl-2 ratio, thus preventing apoptosis. Findings indicate that quercetin may have a preventive effect on TAA-induced hepatotoxicity by modulating the oxidative stress parameters and apoptosis pathway.
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Affiliation(s)
- Cíntia de David
- Laboratory of Experimental Hepatology and Physiology, Porto Alegre Clinical Hospital, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil
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13
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Bautista M, Andres D, Cascales M, Morales-González JA, Sánchez-Reus MI. Effect of gadolinium chloride on liver regeneration following thioacetamide-induced necrosis in rats. Int J Mol Sci 2010; 11:4426-4440. [PMID: 21151447 PMCID: PMC3000091 DOI: 10.3390/ijms11114426] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2010] [Revised: 10/19/2010] [Accepted: 10/19/2010] [Indexed: 02/07/2023] Open
Abstract
Gadolinium chloride (GD) attenuates drug-induced hepatotoxicity by selectively inactivating Kupffer cells. The effect of GD was studied in reference to postnecrotic liver regeneration induced in rats by thioacetamide (TA). Rats, intravenously pretreated with a single dose of GD (0.1 mmol/Kg), were intraperitoneally injected with TA (6.6 mmol/Kg). Hepatocytes were isolated from rats at 0, 12, 24, 48, 72 and 96 h following TA intoxication, and samples of blood and liver were obtained. Parameters related to liver damage were determined in blood. In order to evaluate the mechanisms involved in the post-necrotic regenerative state, the time course of DNA distribution and ploidy were assayed in isolated hepatocytes. The levels of circulating cytokine TNFα was assayed in serum samples. TNFα was also determined by RT-PCR in liver extracts. The results showed that GD significantly reduced the extent of necrosis. The effect of GD induced noticeable changes in the post-necrotic regeneration, causing an increased percentage of hepatocytes in S phase of the cell cycle. Hepatocytes increased their proliferation as a result of these changes. TNFα expression and serum level were diminished in rats pretreated with GD. Thus, GD pre-treatment reduced TA-induced liver injury and accelerated postnecrotic liver regeneration. No evidence of TNFα implication in this enhancement of hepatocyte proliferation and liver regeneration was found. These results demonstrate that Kupffer cells are involved in TA-induced liver damage, as well as and also in the postnecrotic proliferative liver states.
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Affiliation(s)
- Mirandeli Bautista
- Área Académica de Farmacia, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Ex-Hacienda de la Concepción, Tilcuautla, 42080 Pachuca de Soto, Hgo, Mexico; E-Mail: (J.A.M.-G.)
| | - David Andres
- Instituto de Bioquímica (CSIC–UCM), Facultad de Farmacia, Ciudad Universitaria, Plaza de Ramón y Cajal S/N, 28040 Madrid, Spain; E-Mail: (M.I.S.-R.)
| | - María Cascales
- Instituto de Bioquímica (CSIC–UCM), Facultad de Farmacia, Ciudad Universitaria, Plaza de Ramón y Cajal S/N, 28040 Madrid, Spain; E-Mail: (M.I.S.-R.)
| | - José A. Morales-González
- Área Académica de Farmacia, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Ex-Hacienda de la Concepción, Tilcuautla, 42080 Pachuca de Soto, Hgo, Mexico; E-Mail: (J.A.M.-G.)
| | - María Isabel Sánchez-Reus
- Instituto de Bioquímica (CSIC–UCM), Facultad de Farmacia, Ciudad Universitaria, Plaza de Ramón y Cajal S/N, 28040 Madrid, Spain; E-Mail: (M.I.S.-R.)
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14
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Karantonis HC, Gribilas G, Stamoulis I, Giaginis C, Spiliopoulou C, Kouraklis G, Demopoulos C, Theocharis SE. Platelet-activating factor involvement in thioacetamide-induced experimental liver fibrosis and cirrhosis. Dig Dis Sci 2010; 55:276-84. [PMID: 19242794 DOI: 10.1007/s10620-009-0745-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/04/2008] [Accepted: 01/27/2009] [Indexed: 12/13/2022]
Abstract
Platelet-activating factor (PAF) is a potent lipid inflammatory mediator acting on cells through its specific receptor. Plasma PAF-acetylhydrolase (PAF-AH) is the main enzyme that inactivates PAF in blood, participating in its homeostasis. The objective of this study was to investigate the involvement of PAF in the liver fibrotic process using an experimental animal model. Liver fibrosis was induced in adult male Wistar rats by administration of thioacetamide (TAA) in drinking water (300 mg/l) for three months. The animals were sacrificed at time 0 (control group) and after 1, 2, and 3 months. PAF levels in liver and blood and PAF-AH activity in plasma were determined. Liver histopathological examination was also performed. TAA administration resulted in progressively increased liver fibrosis, leading finally to the formation of cirrhotic nodules in the liver. Throughout the experiment PAF levels in liver tissue remained stable. "Total" ("free" plus "bound") PAF levels in blood decreased, reaching statistically significant differences in the first and third months compared with the control group (P < 0.05). "Free" PAF levels in blood were higher at one month (P < 0.05) and decreased gradually thereafter. In all treated groups, "bound" PAF levels in blood decreased whereas plasma PAF-AH activity increased (P < 0.05) compared with the control group. Our data indicated alterations of PAF levels in blood and PAF-AH activity during fibrosis induction, implicating participation of PAF in the liver fibrotic process.
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Affiliation(s)
- Haralabos C Karantonis
- Department of Forensic Medicine and Toxicology, School of Medicine, National and Kapodistrian University of Athens, 75 Mikras Asias str, 11527 Goudi, Athens, Greece
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15
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Nishikawa M, Hashida M, Takakura Y. Catalase delivery for inhibiting ROS-mediated tissue injury and tumor metastasis. Adv Drug Deliv Rev 2009; 61:319-26. [PMID: 19385054 DOI: 10.1016/j.addr.2009.01.001] [Citation(s) in RCA: 143] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Reactive oxygen species (ROS) have been suggested to be involved in a variety of human diseases. Catalase, an enzyme degrading hydrogen peroxide, can be used as a therapeutic agent for such diseases, but its successful application will depend on the distribution of the enzyme to the sites where ROS are generated. Chemical modification techniques have been used to control the tissue distribution of catalase, and delivery to hepatocytes (galactosylation), liver nonparenchymal cells (mannosylation or succinylation), kidney (cationization) and the blood pool (PEGylation) has been achieved. The effectiveness of catalase delivery has been demonstrated in animal models for hepatic ischemia/reperfusion injury, chemical-induced tissue injuries and tumor metastasis to the liver, lung and peritoneal organs. Significant inhibition was observed in the ROS-mediated oxidative tissue damages and ROS-mediated upregulation of expression of genes responsible for recruitment of inflammatory cells and for metastatic growth of tumor cells. Because oxygen plays a fundamental key role in our life and oxidative stress is implicated in a wide variety of human diseases, catalase delivery could have wide application in the near future.
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16
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Duncan AW, Hickey RD, Paulk NK, Culberson AJ, Olson SB, Finegold MJ, Grompe M. Ploidy reductions in murine fusion-derived hepatocytes. PLoS Genet 2009; 5:e1000385. [PMID: 19229314 PMCID: PMC2636893 DOI: 10.1371/journal.pgen.1000385] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2008] [Accepted: 01/20/2009] [Indexed: 12/12/2022] Open
Abstract
We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH), and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ß-galactosidase and the Y-chromosome. Approximately 2–5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ß-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ß-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by ≥50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis. The liver comprises many different types of cells, including hepatocytes. Hepatocytes perform numerous physiological functions, such as detoxification, metabolism, and protein synthesis. Hepatocytes have the ability to fuse with blood cells, generating hybrid hepatocytes that contain nuclei from both fusion partners. In cases of genetic liver disease, fusion between diseased hepatocytes and normal blood cells can result in the formation of hybrid hepatocytes that function normally. In this series of experiments, we show that fusion hepatocytes produce daughter cells with one-half the amount of DNA found in the parental fusion hepatocyte. Furthermore, we show that the daughter cells are genetically distinct from each other. The increase in genetic diversity within the liver could give rise to hepatocytes lacking proper growth control, potentially resulting in tumor formation and cancer.
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Affiliation(s)
- Andrew W Duncan
- Oregon Stem Cell Center, Oregon Health and Science University, Portland, Oregon, United States of America.
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17
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Chen HN, Fan S, Weng CF. Down-regulation of TGFbeta1 and leptin ameliorates thioacetamide-induced liver injury in lipopolysaccharide-primed rats. ACTA ACUST UNITED AC 2007; 13:176-88. [PMID: 17621560 DOI: 10.1177/0968051907081102] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Pretreatment with a low dose of bacterial endotoxin (lipopolysaccharide, LPS) caused the reduction of cytochrome P450 (CYP) enzymes and inflammatory factors which are capable of protecting the liver from a lethal LPS challenge. However, the effects of LPS pretreatment on the expression of transforming growth factor beta1 (TGFbeta1) and leptin in thioacetamide (TAA)-induced liver fibrosis remain unknown. In this study, Sprague-Dawley rats were pretreated intraperitoneally with LPS (5 mg/kg body weight) for 24 h, and subsequently treated with TAA (200 mg/kg body weight/ 3 days) for 1 month to examine the effects of LPS on TAA-injured rats. LPS pretreatment was associated with lower granulation and lower (P < 0.05) GOT/GPT than in TAA-injured rats. The LPS-pretreated group had less collagen (Sirius red histochemical staining). Semiquantitative RT-PCR showed that the levels of collagen 3 and TGFbeta1 mRNAs were lower (P < 0.05) in the liver of LPS-pretreated rats than in TAA-injured rats. TGFbetaRI mRNA in the liver of LPS-pretreated rats exceeded (P < 0.05) that in TAA-injured rats. LPS pretreatment reduced the leptin content (Western blot) below that of TAA-injured rats. These results imply that LPS pretreatment (endotoxin tolerance) alleviates the TAA-induced liver fibrosis of rats by reducing TGFbeta1 and leptin content.
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Affiliation(s)
- Huan-Nan Chen
- Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan
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18
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Kishioka T, Iida C, Fujii K, Nagae R, Onishi Y, Ichi I, Kojo S. Effect of dimethyl sulphoxide on oxidative stress, activation of mitogen activated protein kinase and necrosis caused by thioacetamide in the rat liver. Eur J Pharmacol 2007; 564:190-5. [PMID: 17395177 DOI: 10.1016/j.ejphar.2007.03.001] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2006] [Revised: 02/18/2007] [Accepted: 03/01/2007] [Indexed: 01/13/2023]
Abstract
Thioacetamide (400 mg/kg body weight, i.p.) was administered to rats. After 12 h the activity of plasma glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) was significantly higher than that of the control group, and after 24 h plasma GOT and GPT activities strongly increased. These results indicated that the necrotic process was initiated at about 12 h and developed thereafter. By co-administration of dimethyl sulphoxide (DMSO, 18 and 1 h before, and 8 h after administration of thioacetamide: each time, 2.5 ml/kg body weight, p.o.), plasma GOT and GPT were significantly decreased and were even comparable to the control group, showing that DMSO totally prevented the necrotic action of thioacetamide. After 12 and 24 h of thioacetamide administration, the hepatic level of vitamin C, the most sensitive chemical indicator of oxidative stress, decreased significantly, indicating that oxidative stress was significantly enhanced 12 h after thioacetamide intoxication and thereafter. DMSO totally restored the liver vitamin C level, demonstrating that DMSO effectively ameliorated the oxidative stress caused by thioacetamide, resulting in the prevention of necrosis of the liver. Phosphorylated c-Jun NH(2)-terminal kinase (JNK) significantly increased transiently 12 h after treatment with thioacetamide. These results indicated that oxidative stress and the activation of JNK took place almost simultaneously. Phosphorylated extracellular signal-related kinase (ERK) 2 was significantly increased 6-12 h after thioacetamide injection. Phosphorylated p38 MAPK (mitogen activated protein kinase) was significantly decreased 24 h after administration of thioacetamide. DMSO treatment inhibited the change of these MAPKs by thioacetamide, corresponding with the prevention of the liver necrosis as well as the attenuation of oxidative stress.
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Affiliation(s)
- Terumi Kishioka
- Department of Food Science and Nutrition, Nara Women's University, Nara 630-8506, Japan
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19
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Hyoudou K, Nishikawa M, Kobayashi Y, Kuramoto Y, Yamashita F, Hashida M. Analysis of in vivo nuclear factor-kappaB activation during liver inflammation in mice: prevention by catalase delivery. Mol Pharmacol 2007; 71:446-53. [PMID: 17105872 DOI: 10.1124/mol.106.027169] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays crucial roles in inflammation, immunity, cell proliferation, and apoptosis. Until now, there have been few studies of NF-kappaB activation in whole animals because of experimental difficulties. Here, we show that mice receiving a simple injection of plasmid vectors can be used to examine NF-kappaB activation in the liver. Two plasmid vectors, pNF-kappaB-Luc (firefly luciferase gene) and pRL-SV40 (Renilla reniformis luciferase gene), were injected into the tail vein of mice by the hydrodynamics-based procedure, an established method of gene transfer to mouse liver. Then, the ratio of the firefly and R. reniformis luciferase activities (F/R) was used as an indicator of the NF-kappaB activity in the liver. Injection of thioacetamide or lipopolysaccharide plus d-galactosamine increased the F/R ratio in the liver, and this was significantly (P<0.001) inhibited by an intravenous injection of catalase derivatives targeting liver nonparenchymal cells. Imaging the firefly luciferase expression in live mice clearly demonstrated that the catalase derivatives efficiently prevented the NF-kappaB-mediated expression of the firefly luciferase gene. Plasma transaminases and the survival rate of mice supported the findings obtained by the luminescence-based analyses. Thus, this method, which requires no genetic recombination techniques, is highly sensitive to the activation of NF-kappaB and allows us to continuously examine the activation in live animals. In conclusion, this novel, simple, and sensitive method can be used not only for analyzing the NF-kappaB activation in the organ under different inflammatory conditions but also for screening drug candidates for the prevention of liver inflammation.
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Affiliation(s)
- Kenji Hyoudou
- Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
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20
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Chen LH, Hsu CY, Weng CF. Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells. World J Gastroenterol 2006; 12:5175-81. [PMID: 16937528 PMCID: PMC4088015 DOI: 10.3748/wjg.v12.i32.5175] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Thioacetamide (TAA) has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death.
METHODS: The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAA-induced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAA-induced clone 9 cells were measured by Western blot.
RESULTS: The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died (MTT assay) within 24 h after 100 mg/L TAA was applied. Apoptotic cell percentage (TUNEL assay) and caspase 3 activities were highest after 100 mg/L TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspase-dependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment.
CONCLUSION: These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents, perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.
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Affiliation(s)
- Li-Hsuen Chen
- Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, China
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21
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Roomi MW, Gaal K, Yuan QX, French BA, Fu P, Bardag-Gorce F, French SW. Preneoplastic liver cell foci expansion induced by thioacetamide toxicity in drug-primed mice. Exp Mol Pathol 2006; 81:8-14. [PMID: 16729998 DOI: 10.1016/j.yexmp.2006.02.006] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2006] [Accepted: 02/07/2006] [Indexed: 01/16/2023]
Abstract
Mice primed by feeding griseofulvin or diethyl 1,4-dihydro 1,4,6-trimethyl 3,5-pyridine decarboxylate for 5 months followed by drug withdrawal for 1 month (drug-primed mice) were given thioacetamide intraperitoneally, and the livers were subsequently studied at intervals up to 7 days. The hepatocellular proliferative response was measured by immunostaining for proliferative cell nuclear antigen. Necrosis was followed by measuring ALT. Mallory bodies were identified by immunoperoxidase stains for ubiquitin and cytokeratin. Preneoplastic foci were localized using immunofluorescence stain for glutathione S-transferase (GST mu) and histochemical stain for gamma glutamyl transpeptidase (GGT). The results showed that the preneoplastic foci selectively proliferated and expanded and formed nodules as indicated by quantitation of nuclei stained positive for proliferating cell nuclear antigen after thioacetamide treatment. Data support the hypothesis that the preneoplastic foci consisted of clones of hepatocytes which preferentially express GST mu, GGT and Mallory bodies. These preneoplastic cells selectively proliferate in response to the promoter effects of necrosis-induced liver cell regeneration ("chemical partial hepatectomy").
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Affiliation(s)
- M Waheed Roomi
- Department of Pathology, Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, CA 90509, USA
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22
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Ming Z, Fan YJ, Yang X, Lautt WW. Synergistic protection by S-adenosylmethionine with vitamins C and E on liver injury induced by thioacetamide in rats. Free Radic Biol Med 2006; 40:617-24. [PMID: 16458192 PMCID: PMC2925887 DOI: 10.1016/j.freeradbiomed.2005.09.034] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/22/2005] [Revised: 08/18/2005] [Accepted: 09/12/2005] [Indexed: 01/19/2023]
Abstract
Free radicals are involved in the pathogenesis of acute liver injury induced by thioacetamide (TAA). We investigated the effects of S-adenosylmethionine (SAMe) combined with/without vitamins C and E on TAA-induced acute liver injury in rats. TAA was given intraperitoneally (200 mg kg-1). Antioxidant treatments (SAMe, 25 mg kg-1; vitamin C, 100 mg kg-1; vitamin E, 200 mg kg-1, intraperitoneal) were given 1 h later. Liver histology, enzymology, and ability to release hepatic insulin-sensitizing substance (HISS) were assessed. TAA caused liver tissue injury, increased liver enzymes, and decreased insulin sensitivity (p<0.01). Blockade of HISS release by atropine did not further decrease insulin sensitivity in rats with TAA insult, indicating that the decrease in insulin sensitivity was HISS dependent. Treatment with SAMe alone or vitamins C+E slightly improved liver histology but not the changes in liver enzymes and insulin sensitivity. Combined treatment with SAMe plus vitamins C+E greatly protected the liver from tissue injury, the increase in liver enzymes, and the decrease in insulin sensitivity. In conclusion, acute liver injury causes HISS-dependent insulin resistance (HDIR). There are synergistic antioxidative effects among the antioxidants, SAMe and vitamins C and E, that protect the liver from TAA-induced HDIR, suggesting that antioxidant treatment may best be done using a balanced "cocktail."
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Affiliation(s)
- Zhi Ming
- Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, A210–753, McDermot Avenue, Winnipeg, Manitoba, Canada R3E 0T6
| | - Yi-jun Fan
- Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3
| | - Xi Yang
- Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3
| | - W. Wayne Lautt
- Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, A210–753, McDermot Avenue, Winnipeg, Manitoba, Canada R3E 0T6
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23
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Pallottini V, Martini C, Bassi AM, Romano P, Nanni G, Trentalance A. Rat HMGCoA reductase activation in thioacetamide-induced liver injury is related to an increased reactive oxygen species content. J Hepatol 2006; 44:368-74. [PMID: 16140414 DOI: 10.1016/j.jhep.2005.06.011] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/05/2005] [Revised: 05/11/2005] [Accepted: 06/13/2005] [Indexed: 12/15/2022]
Abstract
BACKGROUND/AIMS In thioacetamide-induced liver injury a modification of isoprenoid content and an increase of reactive oxygen species has been described. We have examined how reactive oxygen species influence the 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate limiting enzyme of the isoprenoid biosynthetic pathway, to verify if changes of that enzyme activity are involved in the changed lipid composition of the liver. METHODS In chronic and acute thioacetamide-treated rat liver we measured the reactive oxygen species content, the activation state and K(M), the level and degradation rate of the hepatic reductase, its short term regulatory enzymes and the liver lipid profile. RESULTS In thioacetamide-treated rat liver, the reactive oxygen species content is high and the reductase is fully activated with no modifications in its K(M) and its short term regulatory enzymes. The reductase level is reduced in chronic thioacetamide treated rats and its degradation rate is altered. CONCLUSIONS The data show a relationship between reactive oxygen species production and altered 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. It is suggested that reducing the levels of reactive oxygen species may improve the altered lipid profile found in liver injury.
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Affiliation(s)
- Valentina Pallottini
- Department of Biology, University of Rome Roma Tre, Viale Marconi 446, 00146-Rome, Italy.
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24
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Novosyadlyy R, Dargel R, Scharf JG. Expression of insulin-like growth factor-I and insulin-like growth factor binding proteins during thioacetamide-induced liver cirrhosis in rats. Growth Horm IGF Res 2005; 15:313-323. [PMID: 16098781 DOI: 10.1016/j.ghir.2005.06.015] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/04/2005] [Revised: 05/27/2005] [Accepted: 06/08/2005] [Indexed: 11/23/2022]
Abstract
OBJECTIVE The liver plays a central role in insulin-like growth factor (IGF) homeostasis providing the majority of circulating IGF-I and some of its binding proteins (IGFBPs). In liver cirrhosis the IGF axis is severely disturbed, and these alterations are associated with reduced IGF-I, IGFBP-3 but elevated IGFBP-1 serum levels. METHODS By Northern blotting and in situ hybridization (ISH), hepatic expression of IGF-I and of IGFBP was studied in a rat model of liver cirrhosis induced by thioacetamide. RESULTS ISH revealed a homogeneous distribution of IGFBP-1, IGFBP-4 and IGF-I mRNA over hepatic parenchyma in normal and cirrhotic liver. Fibrous septa of cirrhotic liver were IGFBP-1 mRNA negative, whereas IGFBP-4 and IGF-I transcripts were detected in single cells. In normal liver, IGFBP-3 mRNA was distributed within nonparenchymal cells of the hepatic lobule and in the wall of the portal vein. In cirrhotic liver, IGFBP-3 transcripts were abundant in mesenchymal cells of fibrous tissue. IGFBP-3 mRNA expression was also prominent in cells at the septal-nodular interface most likely representing monocyte infiltration. IGFBP-3 mRNA expression was reduced in nonparenchymal liver cells located more distantly from the septal-nodular interface in the cirrhotic nodule that correlated with reduced IGFBP-3 mRNA expression observed in Kupffer cells (KC) and sinusoidal endothelial cells (SEC) isolated from macronodular cirrhotic livers. CONCLUSION Cirrhosis is accompanied by an altered spatial expression of IGFBP-3 in liver tissue, which is characterized by decreased levels of IGFBP-3 mRNA in KC and SEC, but elevated IGFBP-3 expression in myofibroblast-like cells and inflammatory infiltrate.
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Affiliation(s)
- R Novosyadlyy
- Department of Internal Medicine, Georg-August-Universität, Göttingen, Germany
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25
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Low TY, Leow CK, Salto-Tellez M, Chung MCM. A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers. Proteomics 2005; 4:3960-74. [PMID: 15526343 DOI: 10.1002/pmic.200400852] [Citation(s) in RCA: 105] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Thioacetamide (TAA) administration is an established technique for generating rat models of liver fibrosis and cirrhosis. Oxidative stress is believed to be involved as TAA-induced liver fibrosis is initiated by thioacetamide S-oxide, which is derived from the biotransformation of TAA by the microsomal flavine-adenine dinucleotide (FAD)-containing monooxygense (FMO) and cytochrome P450 systems. A two-dimensional gel electrophoresis-mass spectrometry approach was applied to analyze the protein profiles of livers of rats administered with sublethal doses of TAA for 3, 6 and 10 weeks respectively. With this approach, 59 protein spots whose expression levels changed significantly upon TAA administration were identified, including three novel proteins. These proteins were then sorted according to their common biochemical properties and functions, so that pathways involved in the pathogenesis of rat liver fibrosis due to TAA-induced toxicity could be elucidated. As a result, it was found that TAA-administration down-regulated the enzymes of the primary metabolic pathways such as fatty acid beta-oxidation, branched chain amino acids and methionine breakdown. This phenomenon is suggestive of the depletion of succinyl-CoA which affects heme and iron metabolism. Up-regulated proteins, on the other hand, are related to oxidative stress and lipid peroxidation. Finally, these proteomics data and the data obtained from the scientific literature were integrated into an "overview model" for TAA-induced liver cirrhosis. This model could now serve as a useful resource for researchers working in the same area.
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Affiliation(s)
- Teck Yew Low
- Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore
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26
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Bueno MTD, Spira B. Thioacetamide differentially affects the expression and activity of glutathione-S-transferase in the liver of Wistar rats. Hum Exp Toxicol 2004; 23:431-7. [PMID: 15497818 DOI: 10.1191/0960327104ht469oa] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Glutathione-S-transferase (GST) is a family of enzymes involved in the detoxification of toxic and carcinogenic compounds. In the present study, the effect of thioacetamide (TA), a hepatotoxic and hepatocarcinogenic compound, on the activity and expression of GST of Wistar female rats was tested. Animals were treated with a single dose of TA (250 mg/kg) for 12, 24, 48 and 72 hours. GST activity toward the broad substrate 1-chloro-2,4-dinitrobenzene was enhanced by TA. The protein level of the GST classes alpha and mu as well as the mRNA level of several GST subunits were also positively affected by the TA treatment. Female Wistar rats of the same age but from two other different colonies had their GST activity either inhibited or not affected by TA. The basal mRNA level of class alpha and class mu GST was also tested in female Wistar rats obtained from five different sources. Differences in the basal level of class alpha mRNA were observed in rats from at least three different sources, while class mu mRNA level was distinct in two groups of animals.
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Affiliation(s)
- Murilo Tadeu Domingues Bueno
- Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Sao Paulo, SP, Brazil
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27
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Sun D, Gong Y, Kojima H, Wang G, Ravinsky E, Zhang M, Minuk GY. Increasing cell membrane potential and GABAergic activity inhibits malignant hepatocyte growth. Am J Physiol Gastrointest Liver Physiol 2003; 285:G12-9. [PMID: 12799308 DOI: 10.1152/ajpgi.00513.2002] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Increasing hepatocyte membrane potentials by augmenting GABAergic activity inhibits nonmalignant hepatocyte proliferative activity. The objectives of this study were to document 1) potential differences (PDs) of four malignant hepatocyte cell lines, 2) GABAA receptor mRNA expression in the same cell lines, and 3) effects of restoring malignant hepatocyte PDs to levels approximating those of resting, nonmalignant hepatocytes. Hepatocyte PDs were documented in nonmalignant and malignant (Chang, HepG2, HuH-7, and PLC/PRF/5) hepatocytes with a fluorescent voltage-sensitive dye and GABAA receptor expression by RT-PCR and Western blot analyses. Compared with nonmalignant human hepatocytes, all four malignant cell lines were significantly depolarized (P < 0.0001, respectively). Only PLC/PRF/5 cells had detectable GABAA-beta3 receptor mRNA expression and all cell lines were negative for GABAA-beta3 receptor protein by Western blot analysis. Stable transfection of Chang cells with GABAA-beta3 receptor cDNA resulted in significant increases in PD and decreases in proliferative activity as manifest by decreased [3H]thymidine and bromodeoxyurieine incorporation rates, 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate activity, a lower mitotic index, prolongation of cell-doubling times, and attenuated growth patterns compared with cells transfected with vector alone. Colony formation in soft agar and the number of abnormal mitoses were also significantly decreased in GABAA-beta3 receptor transfected cells. The results of this study indicate 1) relative to healthy hepatocytes, malignant hepatocytes are significantly depolarized, 2) GABAA-beta3 receptor expression is absent in malignant hepatocyte cell lines, and 3) increasing the PD of malignant hepatocytes is associated with less proliferative activity and a loss of malignant features.
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Affiliation(s)
- D Sun
- Department of Medicine, Liver Diseases Unit, University of Manitoba Health Sciences Centre, Winnipeg, Manitoba, Canada R3E 3P4
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Hwang TH, Yoon BC, Jeong JS, Seo SY, Lee HJ. A single administration of adenoviral-mediated HGF cDNA permits survival of mice from acute hepatic failure. Life Sci 2003; 72:851-61. [PMID: 12479984 DOI: 10.1016/s0024-3205(02)02337-8] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Heptatocyte growth factor (HGF) having a variety of biological activity was suggested as a protective agent against acute toxic hepatic injury or a potentially therapeutic agent. For the efficient in vivo application of this factor, we employed adenoviral-mediated HGF gene delivery system. In this study, we constructed E1-deleted recombinant adenovirus carrying cDNA of human HGF (Ad.hHGF) and elucidated that HGF was efficiently expressed in the liver of C57/BL mice. A mouse model of acute hepatic failure was induced by high dose (1000mg/kg) of thioacetamide (TA) administration. Mice infected with Ad.hHGF showed a dramatic resistance to TA-induced acute hepatic injury. Serum ALT was increased transiently and then the level was normalized in Ad.hHGF-infected mice with TA administration. Furthermore, the survival rate was remarkably enhanced in the mice infected with Ad.hHGF. In the histological examination, massive hepatic necrosis induced by TA was almost completely protected by HGF produced by Ad.hHGF. Our results indicate that a single dose of HGF-encoding adenoviral vector maintained liver function and prevented the progression of liver necrosis in a mouse model of acute hepatic failure.
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Affiliation(s)
- Tae-Ho Hwang
- Department of Pharmacology, Dong-A University College of Medicine and Institute of Medical Science, Dongdaesin-Dong, Seo-Ku, Busan 602-103, South Korea
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29
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Díez-Fernández C, Andrés D, Cascales M. Attenuating effects of heat shock against TGF-beta1-induced apoptosis in cultured rat hepatocytes. Free Radic Biol Med 2002; 33:835-46. [PMID: 12208371 DOI: 10.1016/s0891-5849(02)00975-9] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Heat shock proteins (HSPs) induction confers protection against diverse forms of cellular injury. However, the mechanism by which HSPs exert cytoprotective effects remains unclear. Treatment of rat hepatocyte with transforming growth factor-beta1 (TGF-beta1) induces growth arrest followed by extensive cell death by apoptosis. In this study, the effects of preexposure to heat on TGF-beta1-induced apoptosis of cultured hepatocytes were examined. Treatment of hepatocytes for 24 h with TGF-beta1 resulted in significant apoptotic cell death, as demonstrated by DNA fragmentation, caspase activation, and hypodiploid DNA peak. Moreover, TGF-beta1-induced cell death was accompanied by an enhanced generation of reactive oxygen species and a loss of the mitochondrial membrane potential. These effects were attenuated when the hepatocytes were subjected to 43 degrees C for 20 min prior to the cytokine stimulation. The enhancement in HSP70 expression at mRNA and protein levels induced by heat preexposure was accompanied by an increase in mRNA levels of intracellular antioxidant enzymes. Heat treatment also prevented TGF-beta1-induced activation of nuclear factor kappa B (NF-kappaB) by preventing the degradation of the inhibitory protein kappa Balpha (IkappaBalpha). In conclusion, these data indicate that in the mechanism by which a mild heat pretreatment increases the resistance of hepatocytes to TGF-beta1-induced apoptotic cell death, the upregulation of catalase expression and a decrease in ROS generation are involved.
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Affiliation(s)
- Carmen Díez-Fernández
- Instituto de Bioqui;mica (Centro Mixto CSIC-UCM), Facultad de Farmacia, Madrid, Spain
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30
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Andrés D, Cascales M. Novel mechanism of Vitamin E protection against cyclosporine A cytotoxicity in cultured rat hepatocytes. Biochem Pharmacol 2002; 64:267-76. [PMID: 12123747 DOI: 10.1016/s0006-2952(02)01112-7] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Cyclosporine A (CsA) is the immunosuppressor most frequently used in transplant surgery and in the treatment of autoimmune diseases. It has been shown that CsA is able to generate reactive oxygen species and lipid peroxidation which are directly involved in the CsA hepatotoxicity. As antioxidant, Vitamin E (VitE) has been used to diminish the toxicity of CsA in vitro. Besides its direct action as the classical antioxidant implicated in preventing lipid peroxidation, we decided to investigate the effect of VitE on the endogenous antioxidant defense system, such as Mn and CuZn superoxide dismutase (MnSOD, CuZnSOD) catalase and glutathione peroxidase (GPx) on CsA cytotoxicity in primary cultures of rat hepatocytes. In cells incubated in the presence of CsA, there was an increase in the expression and activity of MnSOD and CuZnSOD but not in that of catalase and GPx. However, when hepatocytes were coincubated with CsA and VitE, an increase in the expression and activity in all antioxidant enzymes (MnSOD, CuZnSOD, catalase and GPx) was observed. In conclusion, we suggest (a) that the imbalance between SOD and catalase/GPx by the effect of CsA is the main mechanism responsible for peroxide accumulation and cell death in hepatocytes, and (b) that the presence of VitE in culture media reduces the oxidative stress through the inhibition of lipid peroxidation, but also through the increase of the expression and activity of catalase and GPx which allows the restoration of SOD and catalase/GPx coordination, indispensable for the correct cell defense against ROS.
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Affiliation(s)
- David Andrés
- Facultad de Farmacia, Instituto de Bioquímica (CSIC-UCM), Universidad Complutense, Plaza de Ramón y Cajal sn, 28040 Madrid, Spain.
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31
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Andrés D, Díez-Fernández C, Castrillo A, Cascales M. Relationship between the activation of heat shock factor and the suppression of nuclear factor-kappaB activity in rat hepatocyte cultures treated with cyclosporine A. Biochem Pharmacol 2002; 64:247-56. [PMID: 12123745 DOI: 10.1016/s0006-2952(02)01115-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
We investigated on primary cultures of rat hepatocytes the effect of cyclosporine A (CsA) on the activation of nuclear factor-kappaB (NF-kappaB), activator protein 1 (AP-1), and heat shock factor 1 (HSF1), three transcription factors involved in cellular response pathways. Hepatocytes were subjected to a time-course (1, 3, 6, and 22 hr) incubation and CsA treatment in the range 1-50 microM. NF-kappaB, AP-1, and HSF1 binding activities were established through electrophoretic mobility shift assay. Levels of HSP70 mRNA and protein were measured by Northern and Western blot analysis respectively. In cells incubated for 1 and 3 hr, electrophoretic mobility shift assay experiments showed a dose-dependent increase of the NF-kappaB binding activity; while following 22hr of incubation, a suppression of the positive effect of CsA at shorter times was detected. At all periods of incubation assayed, CsA induced the activation of AP-1 which was detected by DNA-binding activity of this transcription factor. A dose-dependent activation of HSF1 was observed at 22 hr of incubation. We conclude that in rat hepatocyte cultures, CsA induces the transcriptional activation of NF-kappaB, AP-1, and HSF1. However, the time point at which activation of each transcription factor occurs is different. Thus, at 22 hr of incubation, the CsA-induced activation of HSF1 is accompanied by the reduction of the positive effect of CsA on NF-kappaB activation at earlier time points.
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Affiliation(s)
- David Andrés
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal sn, 28040 Madrid, Spain
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32
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Nanni G, Majorani F, Bassi AM, Canepa C, Maloberti G, Casu A. Dolichol content in isolated sinusoidal liver cells after in vivo chronic treatment with thioacetamide. EXPERIMENTAL AND TOXICOLOGIC PATHOLOGY : OFFICIAL JOURNAL OF THE GESELLSCHAFT FUR TOXIKOLOGISCHE PATHOLOGIE 2002; 54:43-50. [PMID: 12180801 DOI: 10.1078/0940-2993-00237] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
The content of dolichol, an isoprenoid present in all biological membranes, was determined in isolated sinusoidal liver cells after treatment of rats for 2 and 4 months with a low dosage of the hepatotoxin thioacetamide. The significant decrease in dolichol observed in hepatocytes after 2 months might be explained by peroxidation of the isoprenoid. At the same time point, retinol was retained, and decreased only after 4 months of treatment. After 4 months of treatment therefore both lipids decreased. In a subfraction of hepatic stellate cells, Ito-1 cells, the main storage site of vitamin A, dolichol decreased significantly only after 4 months. A remarkable difference from hepatocytes is that in Ito-1 cells retinol content significantly decreased after 2 months of treatment. In another subfraction, Ito-2 cells, the content of the two isoprenoids decreased in parallel. This heterogeneous subfraction might represent those transitional hepatic stellate cells that, while losing retinol, are in the process of differentiating into myofibroblasts secreting extracellular matrix components. In Kupffer cells and sinusoidal endothelial cells, impairment of dolichol might be observed later, only after 4 months of treatment, while retinol decreases uniformly over time. Starting after two months of treatment, the decrease of dolichol and the increase of retinol in hepatocytes, at the same time as retinol decreases in hepatic stellate cells, might be taken as an early index of incipient liver injury due to thioacetamide. This hypothesis is discussed with regard to a role of dolichol in the modulation of membrane fluidity for intracellular and intercellular retinol transport.
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Affiliation(s)
- Giorgio Nanni
- Department of Experimental Medicine, University of Genoa, Italy.
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Sanz N, Díez-Fernández C, Andrés D, Cascales M. Hepatotoxicity and aging: endogenous antioxidant systems in hepatocytes from 2-, 6-, 12-, 18- and 30-month-old rats following a necrogenic dose of thioacetamide. BIOCHIMICA ET BIOPHYSICA ACTA 2002; 1587:12-20. [PMID: 12009419 DOI: 10.1016/s0925-4439(02)00048-0] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The influence of aging on the mechanisms of liver injury and regeneration was studied in a model of hepatotoxicity induced in 2-, 6-, 12-, 18- and 30-month-old rats by a sublethal dose of thioacetamide (500 mg/kg body weight), a soft nucleophilic and hepatotoxic compound metabolized by the hepatic microsomal FAD monooxygenase system. Samples-blood and hepatocytes-were obtained at 0, 12, 24, 48, 72 and 96 h following thioacetamide intoxication. Parameters of liver injury in serum (NADPH-isocitrate dehydrogenase (ICDH) activity) indicate that the severity of injury was significantly higher in the adult groups (6 and 12 months old) when compared either with the youngest (2 months old) or oldest (18 and 30 months old) groups. Parameters related to biotransformation, such as microsomal FAD monooxygenase, followed mainly the same pattern of age-dependent changes as those observed for injury. The profile of glutathione-S-transferase activity showed an initial induction parallel to liver injury and opposite to the levels of reduced glutathione and protein -SH groups. Enzyme activities and gene expression of the systems involved in the cell endogenous antioxidant defense, such as Mn- and Cu,Zn-superoxide dismutases (SOD), catalase and glutathione peroxidase (GPX) showed significant age-dependent changes that can be summarized as follows: an increase in all enzyme activities and gene expression and a decreased ability to restore the initial activities following 96 h of thioacetamide. We conclude, first, that the gene expression and activity of the enzymes involved in the intracellular antioxidant defense system increased with aging, which can be considered a consequence of the enhanced oxidative state of the cell (decreased in GSH level); and second, that the lower and delayed response in the aged groups significantly influenced the restoration towards normal of GSH and the antioxidant enzyme activities.
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Affiliation(s)
- Nuria Sanz
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, Plaza Ramón y Cajal sn, 28040 Madrid, Spain
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34
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French BA, van Leeuwen F, Riley NE, Yuan QX, Bardag-Gorce F, Gaal K, Lue YH, Marceau N, French SW. Aggresome formation in liver cells in response to different toxic mechanisms: role of the ubiquitin-proteasome pathway and the frameshift mutant of ubiquitin. Exp Mol Pathol 2001; 71:241-6. [PMID: 11733949 DOI: 10.1006/exmp.2001.2401] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Aggresomes form in cells when intracellular proteins undergo conformational changes, as in so-called conformational diseases. This phenomenon has been observed in the liver and brain and in cell culture in response to abnormal protein formation, such as mutant proteins. In the case of the brain the frameshift mutant ubiquitin (UBB+1) is involved. Mallory body formation in the liver is one example of this phenomenon in vivo. Mallory body formation is common in a variety of liver diseases of diverse pathogenesis. The study of the Mallory body forming model indicated that drug-conditioned hepatocytes form Mallory bodies when mice are given colchicine, ethanol, okadaic acid, or exposure to heat shock. These findings suggest that aggresome formation is a common pathway of liver injury due to diverse mechanisms. To further characterize the role of this common pathway, drug-primed mice were exposed to different types of liver injury, i.e., using such drugs as thioacetamide, galactosamine, tautomycin, and the proteasome inhibitor PS341. Mallory body formation was induced by treatment with all the toxins tested, giving credence to the proposal that aggresome formation in the liver is a common pathway in response to different primary mechanisms of liver injury. The frameshift mutant UBB+1 was invariably found to colocalize with ubiquitin in the Mallory body, indicating its essential involvement in the mechanism of MB formation.
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Affiliation(s)
- B A French
- Harbor-UCLA Medical Center, Torrance, California 90509-2910, USA
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35
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Andrés D, Díez-Fernández C, Zaragoza A, Alvarez A, Cascales M. Induction of cell proliferation by cyclosporine A in primary cultures of rat hepatocytes. Biochem Pharmacol 2001; 61:427-35. [PMID: 11226376 DOI: 10.1016/s0006-2952(00)00571-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Cyclosporine A (CsA) has been reported to be able to promote cell proliferation, although the precise mechanism by which CsA stimulates cell growth remains uncertain. In the present study, we examined, in hepatocyte cultures, the effect of CsA on parameters related to the cell cycle as well as the levels of proteins involved in the control and progression of the cycle. Flow cytometry analysis detected an increase in the percentage of cells involved in the S phase of the cycle, which correlated with increases in the levels of cyclins D1 and E (two G1-progression regulators), as well as in those of PCNA (proliferating cell nuclear antigen), and without modification in p27, an inhibitory protein of CDKs. We also examined in nucleus the levels of nuclear factor kappaB (a nuclear factor involved in the transcription of the cyclin D1 gene) and found that this transcription factor increased in the presence of CsA. We conclude that the increases in cyclin D1, PCNA, and cyclin E, together with the invariable level of p27, clearly show that CsA induces hepatocytes to proliferate. These results reinforce the idea of the growth-promoting effect of CsA in cultured hepatocytes.
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Affiliation(s)
- D Andrés
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal s/n, 28040, Madrid, Spain
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36
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Sun F, Hamagawa E, Tsutsui C, Ono Y, Ogiri Y, Kojo S. Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in rat liver. BIOCHIMICA ET BIOPHYSICA ACTA 2001; 1535:186-91. [PMID: 11342007 DOI: 10.1016/s0925-4439(00)00098-3] [Citation(s) in RCA: 111] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
After 12, 18, and 24 h of oral administration of carbon tetrachloride (as a 1:1 mixture with mineral oil: 4 ml/kg body weight) to rats, the activity of caspase-3-like protease in the liver increased significantly compared to that in the control group that was given mineral oil (4 ml/kg). In plasma, the activity of caspase-3 was barely detectable in the control rat, but increased significantly 24 h after drug administration along with a dramatic increase in glutamate oxaloacetate transaminase. These results indicate that carbon tetrachloride causes apoptosis in the liver by activating caspase-3, which is released to plasma by secondary necrosis. After 18 and 24 h of carbon tetrachloride administration, the liver concentration of hydrophilic vitamin C was decreased significantly, while that of hydrophobic vitamin E was not affected. The plasma concentration of vitamins C and E was not influenced significantly. These results suggest that carbon tetrachloride induces oxidative stress mainly in the aqueous phase of the liver cell.
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Affiliation(s)
- F Sun
- Department of Food Science and Nutrition, Nara Women's University, 630-8506, Nara, Japan
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37
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Spira B, Raw I. The effect of thioacetamide on the activity and expression of cytosolic rat liver glutathione-S-transferase. Mol Cell Biochem 2000; 211:103-10. [PMID: 11055553 DOI: 10.1023/a:1007114801362] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The effect of thioacetamide (TA), an hepatotoxic and hepatocarcinogenic compound, on the expression and activity of the cytosolic enzyme glutathione-S-transferase (GST) was studied in rat liver. Four h following the administration of 14C-labeled thioacetamide (50 mg/Kg), several subunits of GST were found to be radioactively labeled. A single sublethal dose of TA (250 mg/Kg) decreased by three-fold the expression of class alpha GST at 24-48 h of treatment, but did not significantly affect the transcription of class mu GST. The activity of the enzyme toward 1-chloro-2,4-dinitrobenzene was mildly inhibited (66% of the control) by a 24 h TA treatment and gradually increased thereafter. It is proposed that the covalent binding of TA or its derivative to the GST subunits does not affect the activity of the enzyme. Nevertheless, GST activity inhibition is due to the deleterious effect of TA on GST transcription.
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Affiliation(s)
- B Spira
- Centro de Biotecnologia, Instituto Butantan, São Paulo-SP, Brazil
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38
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Andrés D, Sanz N, Zaragoza A, Alvarez AM, Cascales M. Changes in antioxidant defence systems induced by cyclosporine A in cultures of hepatocytes from 2- and 12-month-old rats. Biochem Pharmacol 2000; 59:1091-100. [PMID: 10704938 DOI: 10.1016/s0006-2952(00)00233-1] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
The in vitro effect of cyclosporine A (CsA) was studied in reference to the production of reactive oxygen species (peroxides and superoxide anion) and to cell enzyme-mediated antioxidant defence in hepatocytes isolated from rats aged 2 and 12 months. Primary cultures of hepatocytes were incubated in the presence of concentrations of cyclosporine in the range of 0 to 50 microM for 24 hr, and the release of lactate dehydrogenase into the culture medium was evaluated as a parameter of cytotoxicity and membrane lysis. Peroxides were quantified by using 2',7'-dichlorodihydrofluorescein diacetate, and superoxide anion levels were evaluated by the fluorescence of dihydroethidium. Enzyme activity and gene expression of catalase and Mn- and Cu,Zn-superoxide dismutase were also assayed. CsA cytotoxicity was significantly higher in hepatocytes from rats aged 12 months when compared to those aged 2 months. Intracellular peroxide content resulted in a dose-dependent increase, while the anion superoxide intracellular level slightly decreased as CsA increased from 0-50 microM. The progressive increase in intracellular peroxides in cell cultures in the range from 0-50 microM CsA was associated with the loss of cell viability and accompanied by significantly higher levels of Mn- and Cu, Zn-superoxide dismutase enzyme activities and mRNAs, and slight increases in catalase activity and mRNA. We conclude that, in primary hepatocyte cultures, the cytotoxicity of CsA was dose-dependent in both age groups and significantly higher in cultures from 12-month-old rats when compared to those from 2-month-old animals. The non-coordinated regulation of the gene expression of antioxidant enzyme systems, i.e. catalase and Mn- and Cu,Zn-superoxide dismutases, evidenced to a greater extent in hepatocytes from the older group of rats, could be one of the mechanisms involved in CsA toxicity.
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Affiliation(s)
- D Andrés
- Instituto de Bioquímica (CSIC-UCM)Facultad de Farmacia, Universidad Complutense, Madrid, Spain
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39
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Zaragoza A, Díez-Fernández C, Alvarez AM, Andrés D, Cascales M. Effect of N-acetylcysteine and deferoxamine on endogenous antioxidant defense system gene expression in a rat hepatocyte model of cocaine cytotoxicity. BIOCHIMICA ET BIOPHYSICA ACTA 2000; 1496:183-95. [PMID: 10771087 DOI: 10.1016/s0167-4889(00)00036-7] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
In the present study we investigated on cultures of hepatocytes from phenobarbital-pretreated rats, the effect of the antioxidants, 0.5 mM N-acetylcysteine (NAC) or 1.5 mM deferoxamine (DFO), previously incubated for 24 h and coincubated with cocaine (0-1000 microM) for another 24 h. Cocaine cytotoxicity was monitored by either the lysis of the cell membranes or apoptosis. Lysis of the cell membranes was evidenced by lactate dehydrogenase leakage, apoptosis was observed by detecting a hypodiploid peak (<2C) in DNA histograms obtained by flow cytometry, peroxide production was quantified with 2', 7'-dichlorodihydrofluorescein diacetate and gene expression of the antioxidant enzymes: Mn- and Cu,Zn-superoxide dismutases, catalase and glutathione peroxidase were measured by Northern blot analysis. NAC and DFO significantly decreased the extent of lysis of cell membranes and apoptosis, and the antiapoptotic effect was parallel to peroxide generation. By the effect of NAC and DFO, significant increases were detected in the levels of mRNA of catalase, manganese superoxide dismutase and glutathione peroxidase. From these results we conclude that NAC or DFO, when incubated in the presence of cocaine, exerted a protective effect against cocaine toxicity at the level of both lysis of the membranes and apoptosis. This protective effect, in the case of NAC, was directed towards an increase in antioxidant enzyme expression, and in the case of DFO against reactive oxygen species generation.
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Affiliation(s)
- A Zaragoza
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal sn, 28040, Madrid, Spain
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40
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Sun F, Hayami S, Ogiri Y, Haruna S, Tanaka K, Yamada Y, Tokumaru S, Kojo S. Evaluation of oxidative stress based on lipid hydroperoxide, vitamin C and vitamin E during apoptosis and necrosis caused by thioacetamide in rat liver. BIOCHIMICA ET BIOPHYSICA ACTA 2000; 1500:181-5. [PMID: 10657587 DOI: 10.1016/s0925-4439(99)00100-3] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
After 12 h of thioacetamide (500 mg/kg body weight) administration to rats, the activity of caspase-3-like protease in the liver increased significantly compared to that in the control group. In plasma, the activity of caspase-3 was barely detectable in the control rat, but had increased significantly after 24 h of drug administration along with a dramatic increase in GOT. These results indicate that thioacetamide causes apoptosis in the liver by activating caspase-3, which is released to plasma by successive necrosis. At 24 h, the concentration of liver lipid hydroperoxides, a mediator of radical reaction, was 2.2 times as high as that of control rats. After 12 and 24 h of thioacetamide administration, the liver concentrations of vitamins C and E decreased significantly. The decrease of antioxidants and formation of lipid hydroperoxides 24 h after thioacetamide administration support the view that extensive radical reactions occur in the liver during the necrotic process.
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Affiliation(s)
- F Sun
- Department of Food Science and Nutrition, Nara Women's University, Nara, Japan
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41
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Zaragoza A, Andrés D, Sarrión D, Cascales M. Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment in rats. Inducibility of FAD monooxygenase system and age effect. Chem Biol Interact 2000; 124:87-101. [PMID: 10670821 DOI: 10.1016/s0009-2797(99)00147-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The ability of phenobarbital to induce the expression and activity of microsomal drug monooxygenases in the liver presents one of the most important issues in the field of chemical interactions and in the toxicity of xenobiotics. The model of rat liver injury induced by a single dose of thioacetamide (500 mg/kg intraperitoneally) was used to study the effect of phenobarbital (80 mg/kg/day intraperitoneally) for 5 days prior to thioacetamide. Serum parameters of liver injury such as aspartate aminotransferase activity, gamma-glutamyl transferase activity and the total bilirubin levels, as well as the activities of hepatic FAD and cytochrome P450 microsomal monooxygenases, were assayed in 2- and 12-month-old rats. Samples of blood and liver were obtained from controls (injected at 0 h with 0.5 ml of 0.9% NaCl) and at 12, 24, 48, 72 and 96 h of thioacetamide intoxication either to non-treated or phenobarbital pretreated rats. Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment was demonstrated at morphological level, and by significant increases in the activities of serum aspartate aminotransferase and gamma-glutamyl transferase, and in the levels of total bilirubin. The extent of potentiation of thioacetamide-induced liver injury by phenobarbital pretreatment was similar in both age groups. Microsomal FAD monooxygenase activity, the enzyme responsible for thioacetamide biotransformation, was significantly enhanced (twofold) by phenobarbital pretreatment, and also underwent a further increase following thioacetamide, preceding the peak of necrosis. Cytochrome P450 monooxygenases were induced by phenobarbital pretreatment more than sixfold, and sharply decreased when phenobarbital was withdrawn and thioacetamide administered, showing at 48 h intoxication values close to basal. Phenobarbital pretreatment potentiated thioacetamide necrogenicity, and this potentiation was parallel to the induction of the microsomal FAD monooxygenase system, both by phenobarbital and by thioacetamide itself. The extent of thioacetamide-induced liver injury was significantly higher in 12-month-old rats, but the effect of phenobarbital pretreatment was similar in both age groups.
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Affiliation(s)
- A Zaragoza
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, Madrid, Spain
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42
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Hayami S, Ikeda K, Sun F, Tanaka K, Kojo S. Increase of caspase-3 activity in rat liver and plasma by thioacetamide. Biochem Pharmacol 1999; 58:1941-3. [PMID: 10591148 DOI: 10.1016/s0006-2952(99)00295-6] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Twelve and twenty-four hours after intraperitoneal administration of thioacetamide (200 mg/kg body weight) to rats, caspase-3 activity in the liver and plasma of the treated rats increased significantly compared with that of the control group. These results demonstrated that thioacetamide caused apoptosis, which involved activation of caspase-3, although there may be a number of pathways to apoptosis in the liver that may or may not involve the activation of this protein. The results also indicated that the activated caspase-3 was released in plasma along with glutamate-oxaloacetate transaminase (GOT) by successive necrosis. This is the first study that shows an increase of caspase-3 activity in plasma.
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Affiliation(s)
- S Hayami
- Department of Food Science and Nutrition, Nara Women's University, Japan
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43
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Díez-Fernández C, Zaragoza A, Alvarez AM, Cascales M. Cocaine cytotoxicity in hepatocyte cultures from phenobarbital-induced rats: involvement of reactive oxygen species and expression of antioxidant defense systems. Biochem Pharmacol 1999; 58:797-805. [PMID: 10449189 DOI: 10.1016/s0006-2952(99)00168-9] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
The present study was designed to investigate whether cocaine modifies the production of reactive oxygen species, affects cellular enzyme-mediated antioxidant defense systems and, subsequently, promotes apoptosis and/or necrosis of hepatocytes. Primary cultures of hepatocytes isolated from phenobarbital-induced rats were exposed to cocaine (0-1000 microM) for 24 hr, and cell death (apoptosis or necrosis), antioxidant enzyme activities and mRNA levels, and peroxide generation were determined. Cocaine cytotoxicity by apoptosis was observed by detecting apoptotic nuclei using optic microscopy and by measurement of the hypodiploid peak (<2C) in DNA histograms obtained by flow cytometry. Necrosis was evidenced by lactate dehydrogenase (LDH) leakage, and peroxide production was quantified with 2',7'-dichlorodihydrofluorescein diacetate. Low concentrations of cocaine (less than 100 microM) resulted in an increase in dichlorofluorescein fluorescence, associated with an enhancement in apoptotic cell death and sharp decreases in the enzyme activities and RNAs of catalase and manganese-superoxide dismutase (Mn-SOD). The progressive decrease in peroxide production in cell cultures detected in the range of 250-1000 microM cocaine was associated with increases in LDH leakage and decreases in the percentage of apoptotic cells, accompanied by low levels in catalase and Mn-SOD enzyme activities and mRNAs, without apparent changes in apoptosis. These data indicate that oxygen radicals may contribute directly or indirectly to cocaine-induced apoptosis in cultured hepatocytes. We conclude that, in primary hepatocyte cultures, cocaine-induced cell death by necrosis was dependent on cocaine concentration, while cell death by apoptosis was parallel to peroxide concentration. The down-regulation of the gene expression of antioxidant enzyme systems should be one of the mechanisms involved in cocaine toxicity.
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Affiliation(s)
- C Díez-Fernández
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, Madrid, Spain
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Sigal SH, Rajvanshi P, Gorla GR, Sokhi RP, Saxena R, Gebhard DR, Reid LM, Gupta S. Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events. THE AMERICAN JOURNAL OF PHYSIOLOGY 1999; 276:G1260-72. [PMID: 10330018 DOI: 10.1152/ajpgi.1999.276.5.g1260] [Citation(s) in RCA: 76] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
In understanding mechanisms of liver repopulation with transplanted hepatocytes, we studied the consequences of hepatic polyploidization in the two-thirds partial hepatectomy model of liver regeneration. Liver repopulation studies using genetically marked rodent hepatocytes showed that the number of previously transplanted hepatocytes did not increase in the liver with subsequential partial hepatectomy. In contrast, recipients undergoing partial hepatectomy before cells were transplanted showed proliferation in transplanted hepatocytes, with kinetics of DNA synthesis differing in transplanted and host hepatocytes. Also, partial hepatectomy caused multiple changes in the rat liver, including accumulation of polyploid hepatocytes along with prolonged depletion of diploid hepatocytes, as well as increased senescence-associated beta-galactosidase and p21 expression. Remnant hepatocytes in the partially hepatectomized liver showed increased autofluorescence and cytoplasmic complexity on flow cytometry, which are associated with lipofuscin accumulation during cell aging, and underwent apoptosis more frequently. Moreover, hepatocytes from the partially hepatectomized liver showed attenuated proliferative capacity in cell culture. These findings were compatible with decreased proliferative potential of hepatocytes experiencing partial hepatectomy compared with hepatocytes from the unperturbed liver. Attenuation of proliferative capacity and other changes in hepatocytes experiencing partial hepatectomy offer novel perspectives concerning liver regeneration in the context of cell ploidy.
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Affiliation(s)
- S H Sigal
- Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA
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45
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Sanz N, Díez-Fernández C, Alvarez AM, Fernández-Simón L, Cascales M. Age-related changes on parameters of experimentally-induced liver injury and regeneration. Toxicol Appl Pharmacol 1999; 154:40-9. [PMID: 9882590 DOI: 10.1006/taap.1998.8541] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Age-dependent changes related to liver injury and regeneration were studied in rats aged 2, 12, and 30 months in a time period of 96 hr following a sublethal dose of thioacetamide (6.6 mmoles/kg body wt). Serum aspartate aminotransferase activity increased earlier in young rats, but the severity of injury was higher in those aged 12 months when compared to young and to old. Microsomal hepatocyte FAD monooxygenase activity was induced earlier in 2-month-old rats following intoxication and the increase was significantly lower both in the youngest and in the oldest groups when compared to adults. As a parameter of hepatocellular postnecrotic regeneration, DNA synthesis (2C --> 4C) was evaluated. The population of hepatocytes in S phase peaked more sharply and earlier in young rat hepatocytes, and was 8 to 12 times higher than the initial in hepatocytes from 2- and 12-month-old rats, while the rise was only 3 times in the oldest group. At 96 hr of intoxication the restoration towards normal in all these parameters was complete in young, incomplete in adult, and slightly detected in the oldest. Serum proliferative activity, assayed on mouse NIH 3T3 fibroblast cultures, increased preceding the necrosis and this increase was higher in 2- and 12-month-old (171% and 224%, respectively), while in the oldest the increase was only 110%. This mitogenic activity decreased in all groups during necrosis, showing a second peak, nondetectable in rats aged 30 months, parallel to regeneration. Serum TNFalpha level was absent in untreated animals and increased markedly following intoxication, the highest values being recorded at 72 hr of intoxication in serum from rats aged 12 months (347 +/- 30 pg/ml) and the lowest at 30 months (4.1 +/- 0.3 pg/ml). The serum ability to induce nitric oxide synthase activity on peritoneal macrophages ex vivo showed significant time- and age-dependent changes in nitric oxide release: a decrease throughout necrosis and an increase during regeneration. We conclude that the main age-related changes in the sequenced process of liver injury and regeneration are the delayed response in the development of cell killing and regeneration and the decreased regenerative ability, which significantly delays the restoration of liver function.
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Affiliation(s)
- N Sanz
- Facultad de Farmacia, Universidad Complutense, Madrid, 28040, USA
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46
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Rajvanshi P, Liu D, Ott M, Gagandeep S, Schilsky ML, Gupta S. Fractionation of rat hepatocyte subpopulations with varying metabolic potential, proliferative capacity, and retroviral gene transfer efficiency. Exp Cell Res 1998; 244:405-19. [PMID: 9806791 DOI: 10.1006/excr.1998.4223] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated that small diploid and large polyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.
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Affiliation(s)
- P Rajvanshi
- Division of Gastroenterology, Marion Bessin Liver Research Center, Bronx, New York, 10461, USA
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Díez-Fernández C, Sanz N, Alvarez AM, Zaragoza A, Cascales M. Influence of aminoguanidine on parameters of liver injury and regeneration induced in rats by a necrogenic dose of thioacetamide. Br J Pharmacol 1998; 125:102-8. [PMID: 9776349 PMCID: PMC1565582 DOI: 10.1038/sj.bjp.0702014] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
1. When aminoguanidine, a nucleophilic hydrazine compound, was administered to rats (50 mg kg(-1) body wt) 30 min before a necrogenic dose of thioacetamide (500 mg kg(-1) body wt), significant changes related to liver injury and hepatocellular regeneration were observed. 2. The extent of necrosis was noticeably less pronounced, as detected by the peak of serum aspartate aminotransferase activity. Depletion of hepatic glutathione (GSH) and the increase in malondialdehyde concentration as markers of oxidative stress, produced by thioacetamide metabolism, were significantly diminished. However, the activity of microsomal FAD monooxygenase, the system responsible for thioacetamide oxidation, did not show significant alterations. Antioxidant enzyme systems involved in the glutathione redox cycle, such as glutathione reductase and glutathione peroxidase activities, slightly decreased following aminoguanidine pretreatment. 3. Primary cultures of peritoneal macrophages from control rats, when incubated in the presence of serum collected following thioacetamide intoxication, showed a significant decrease in nitric oxide (NO) release at 24 h, that was more pronounced in the group pretreated with aminoguanidine. However, the sharp and progressive increase in macrophage NO release, when incubated in the presence of serum obtained at 48, 72 and 96 h, were increased by aminoguanidine-pretreatment. 4. The cell population involved in DNA synthesis sharply increased in both groups at 48 h of intoxication, although the values at 0, 24, 72 and 96 h were markedly higher in the group pre-treated with aminoguanidine. Polyploidy at 72 and 96 h of intoxication was delayed by the effect of aminoguanidine and a progressive increase in the hypodiploid hepatocyte population, which reached 16% of the total at 96 h, was observed. 5. These results indicate that a single dose of aminoguanidine before thioacetamide administration, markedly diminished the severity of the liver injury by decreasing oxidative stress and lipoperoxidation, but hepatocellular regeneration was apparently unaffected probably due to an enhanced mitogenic activity.
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Affiliation(s)
- C Díez-Fernández
- Instituto de Bioquímica (CSIC - UCM), Facultad de Farmacia, Universidad Complutense, Madrid, Spain
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48
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Ramaiah SK, Soni MG, Bucci TJ, Mehendale HM. Diet restriction enhances compensatory liver tissue repair and survival following administration of lethal dose of thioacetamide. Toxicol Appl Pharmacol 1998; 150:12-21. [PMID: 9630448 DOI: 10.1006/taap.1998.8365] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Diet restriction is known to prevent a plethora of age-associated diseases including cancer. However, the effects of diet restriction on noncancer end points are not known. The objective of this study was to investigate whether diet restriction protects against hepatotoxicity of thioacetamide (TA), and if so, to investigate the underlying mechanism. Male Sprague-Dawley rats (250-275 g) were maintained on 65% of their ad libitum (AL) food consumption for a period of 3 weeks and then treated with a single low dose of 50 mg TA/kg i.p.. Plasma enzymes (ALT and SDH), hepatic glycogen levels, and 3H-thymidine incorporation into hepatocellular nuclear DNA were measured during a time course (0-120 h) after TA administration. Liver sections were examined for histopathology, and cell-cycle progression was assessed by proliferating cell nuclear antigen (PCNA) immunohistochemistry. In AL rats hepatic necrosis was evident at 12 h, peaked at 36 h, persisted up to 72 h, and was resolved by 96 h. In the diet-restricted (DR) group hepatic necrosis was observed at 12 h, peaked at 24 h, persisted till 72 h, and was resolved by 96 h. Maximal injury indicated by enzyme elevation occurred in DR rats and was approximately sixfold greater than that observed in the AL group. Histopathological examination of the liver sections revealed liver injury concordant with plasma enzyme elevations. There was a higher and sustained S-phase synthesis in the DR rats compared to AL group. S-phase stimulation was evident at 36 h, peaked at 48 h, and persisted until 96 h in the DR rats, whereas in the AL rats peak S-phase stimulation occurred at 36 h and subsided by 72 h. PCNA studies revealed a corresponding stimulation of cell-cycle progression indicating highly stimulated compensatory tissue repair. The 14-day lethality experiments (600 mg TA/kg i.p.) indicated 70% survival in the DR rats compared to 10% survival in the AL group. Although diet restriction increases hepatotoxic injury of TA, it protects from the lethal outcome by enhanced liver tissue repair. Comparison of liver injury and tissue repair employing an equitoxic dose (600 mg TA/kg in AL rats yields similar liver injury as observed with 50 mg TA/kg in DR rats) revealed that in spite of near equal injury up to 36 h, tissue repair response in DR rats is much higher. The compensatory tissue repair allows the DR rats to escape death in contrast to much lower compensation in AL rats leading to progression of liver injury culminating in death.
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Affiliation(s)
- S K Ramaiah
- Division of Toxicology, College of Pharmacy and Health Sciences, North Louisiana University, Monroe 71209-0470, USA
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49
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Sanz N, Díez-Fernández C, Fernández-Simón L, Alvarez A, Cascales M. Necrogenic and regenerative responses of liver of newly weaned rats against a sublethal dose of thioacetamide. BIOCHIMICA ET BIOPHYSICA ACTA 1998; 1384:66-78. [PMID: 9602062 DOI: 10.1016/s0167-4838(97)00218-5] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The hepatocellular necrogenic and regenerative responses of newly weaned rats (21 days old) to a sublethal dose of thioacetamide (6.6 mmol kg-1) were studied in comparison to adult (6-month old rats), in terms of liver injury, antioxidant defense systems and cell proliferation. Hepatocellular necrosis, detected by serum aspartate aminotransferase, was less severe in newly weaned rats than in adult animals and was parallel to previous changes in the activity of microsomal FAD monooxygenase system responsible for thioacetamide biotransformation. Liver damage in hepatocytes from newly weaned rats was also detected by the decreased levels of glutathione and protein thiol groups (47%, p < 0.001 and 52%, p < 0.001 vs. untreated, respectively) and by the enhanced malondialdehyde production (334%, p < 0.001) and glutathione S-transferase activity (384%, p < 0.001). No significant differences were detected in these values when compared to adults. Changes in cytosolic and mitochondrial superoxide dismutase and catalase activities in hepatocytes from newly weaned rats at 24 h, following thioacetamide (49%, p < 0.001; 50% and 53%, p < 0.001 vs. untreated, respectively), were less severe against those in adult hepatocytes at 48 h of intoxication, and the increases in glutathione peroxidase and glutathione reductase activities were significantly lowered: 25% (p < 0.001) and 41% (p < 0.001), respectively. Post-necrotic DNA synthesis in hepatocytes from newly weaned rats peaked at 48 h of intoxication, while in adults a more intense peak appeared at 72 h preceded by a sharp decrease in tetraploid population. These differences indicate that the lower necrogenic response against the same dose of thioacetamide in newly weaned rats may be due to the lower rate of thioacetamide biotransformation and to the earlier onset of cell division. Accordingly, the growing liver from newly weaned rats presents advantages against the necrogenic aggression of thioacetamide, first, because the diminished activity of its specific microsomal detoxification system, and second because the earlier increase in the proliferative response prevents the progression of injury permitting an earlier restoration of liver function.
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Affiliation(s)
- N Sanz
- Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, Madrid, Spain
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50
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Bruck R, Oren R, Shirin H, Aeed H, Papa M, Matas Z, Zaidel L, Avni Y, Halpern Z. Hypothyroidism minimizes liver damage and improves survival in rats with thioacetamide induced fulminant hepatic failure. Hepatology 1998; 27:1013-20. [PMID: 9537441 DOI: 10.1002/hep.510270417] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Recent data from animal studies suggest that induced hypothyroidism prevents the hyperdynamic circulation in portal vein ligated rats, liver cirrhosis in rats chronically treated with thioacetamide (TAA), and immune-mediated acute liver injury induced in mice by concanavalin A. Therefore, the aim of this present study is to determine whether hypothyroidism would likewise prevent fulminant hepatic failure (FHF) in rats. FHF was induced by 3 consecutive ip injections of TAA (400 mg/kg) at 24-hour intervals. Hypothyroidism was induced in rats by either methimazole (MMI) or propylthiouracil (PTU) and surgical thyroidectomy and was confirmed by elevated serum thyroid stimulating hormone levels. Serum levels of liver enzymes, blood ammonia, and prothrombin time were significantly lower in all 3 groups of hypothyroid rats. The stage of hepatic encephalopathy (HE) and the survival rates were significantly improved in the hypothyroid rats (P < .01); the histologic examination of their livers showed less necrosis and inflammation (P < .01). In the hypothyroid rats, the serum levels of malondialdehyde 48 hours after thioacetamide (TAA) administration were lower than in control rats (P < .01). Exogenous supplementation of hypothyroid rats with L-thyroxine started 48 hours before TAA administration abrogated the protective effects of hypothyroidism. The serum levels of tumor necrosis factor alfa (TNF-alpha), interleukin (IL) 2 and IL-6 after 24 hours were slightly lower in the hypothyroid rats, but the administration of soluble receptor of TNF (10-1,000 microg/rat) did not prevent the induction of fulminant liver failure by TAA. Oxygen extraction, studied in isolated perfused liver preparation, was significantly lower in livers of hypothyroid rats (P < .01). These results suggest that induced hypothyroidism decreases the development of liver injury in a rat model of FHF. The mechanism may involve diminished oxidative cell injury caused by decreased oxygen utilization and hypometabolism associated with hypothyroidism.
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Affiliation(s)
- R Bruck
- Department of Gastroenterology, The E. Wolfson Medical Center, Holon, Israel
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