thioacetamide and Necrosis

Embelin is an active component isolated from Embelia ribes Burm. In this study, we explored the protective effects of embelin on acute liver injury.. An animal model of acute liver injury was established via administration of a single injection of thioacetamide (TAA) (300 μg/g body weight) to adult mice. Embelin was administered by intragastric gavage at 50 μg/g body weight starting 2 days before TAA administration and continuing throughout the study. Survival of the mice was analyzed by the Kaplan-Meier method using the log-rank test. The acute liver injury protocol was repeated and the remaining mice were analyzed at indicated times. Hematoxylin and eosin staining and picrosirius red staining were used to examine necrosis/inflammation and liver healing, respectively. Liver function was assessed by serum alanine aminotransferase/alkaline phosphatase activity. Hepatic cleaved caspase-3 and F4/80 expression levels were examined via immunostaining. Statistical analysis was performed with GraphPad Software.. The survival and liver function of the mice were markedly better in the group treated with embelin prior to TAA toxication than in the TAA toxication-only group. Embelin significantly reduced TAA-induced hepatic necrosis/apoptosis. Massive inflammatory cell infiltration, which is consistent with hepatic fibrogenesis (a healing process), occurred earlier in the embelin-treated recovery group than in the spontaneous recovery group. Moreover, macrophage activities increased more rapidly with embelin treatment.. In summary, embelin can protect against acute liver injury. Its therapeutic value warrants further exploration.

Topics: Acute Disease; Animals; Apoptosis; Benzoquinones; Female; Inflammation; Liver; Macrophages; Mice, Inbred C57BL; Necrosis; Protective Agents; Survival Analysis; Thioacetamide; Wound Healing

Injecting MSCs via blood vessel is most commonly used method, which has a major drawback of safety. The aim of our study was to evaluate efficacy using scaffold-loaded MSCs in acute liver failure model.. Acute liver failure was induced in mice using thioacetamide (TAA) (200 mg/kg, i.p) once a day for two consecutive days. The animals were divided in four acute liver failure groups: (1) TAA; (2) empty scaffold; (3) MSCs injected through tail vein; (4) MSC + Scaffold, scaffold loaded with MSCs, to evaluate the mortality and changes in liver function. Polylactic-co-glycolic acid scaffold alone and loaded with human MSCs was implanted on mice dorsum.. TAA dose was titrated until one-third mortality rate was achieved. TAA (200 mg/kg) once daily for two consecutive days was injected to establish the acute liver failure model. The mortality of TAA and scaffold groups was 55.9% and 63.2%, respectively. Although, mortality of MSC-TV group decreased 14.7% as compared to TAA group (p = 0.200), MSC + Scaffold group had the lowest mortality (31.4%) (p = 0.013). Cells implanted in PLGA biomaterial were survived until 3 weeks, and their function was increased. Area of hepatic inflammation and necrosis was significantly reduced in MSC-TV and MSC + Scaffold groups; but there was no difference between the two groups. Gene expressions related to inflammation were significantly decreased in MSC-TV and MSC + Scaffold groups compared to TAA group. In MSC + Scaffold group, no migration of stem cells to liver tissue was observed. Although, not all cells in scaffold were stained, some of them were differentiated into hepatocyte-like cells which stained positive for PAS and CYP2E1 antibody.. Scaffold loaded with MSCs showed protective effects via paracrine signaling on acute liver failure model.

Topics: Animals; Biomarkers; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP2E1; Disease Models, Animal; Humans; Liver; Liver Failure, Acute; Liver Regeneration; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice, Inbred C57BL; Necrosis; Paracrine Communication; Phenotype; Polylactic Acid-Polyglycolic Acid Copolymer; Thioacetamide; Tissue Scaffolds

Clinically, liver fibrosis and cholestasis are two major disease entities, ultimately leading to hepatic failure. Although autophagy plays a substantial role in the pathogenesis of these diseases, its precise mechanism has not been determined yet.. Mouse models of liver fibrosis or cholestasis were obtained after the serial administration of thioacetamide (TAA) or surgical bile duct ligation (BDL), respectively. Then, after obtaining liver specimens at specific time points, we compared the expression of makers related to apoptosis (cleaved caspases), inflammation (CD68), necrosis (high-mobility group box 1), phospho-c-Jun N-terminal kinase (p-JNK), and autophagy (microtubule-associated protein light chain 3B and p62) in the fibrotic or cholestatic mouse livers, by polymerase chain reaction, Western blot analysis, immunohistochemistry, and immunofluorescence.. Although cholestatic livers exhibited the tendency of progressively increasing the expression of most apoptosis-related markers (cleaved caspases), it was not prominent when it was compared with the tendency found in the livers of TAA-treated mice. Contrastingly, the necrosis-related factor (high-mobility group box 1) was significantly increased in the livers of BDL mice over time, reaching their peak values on day 7 after BDL. In addition, the inflammation-related factor (CD68) was highly expressed in BDL mice compared with TAA-treated mice over time. Autophagy marker studies indicated that autophagy was upregulated in fibrotic livers, whereas it was downregulated in cholestatic livers. We also observed mild to moderate activation of p-JNK in the livers of TAA-treated mice, whereas significantly higher p-JNK activation was detected in the livers of BDL mice.. Unlike TAA-treated mice, BDL mice exhibited higher expression of the markers related with inflammation and necrosis, especially including p-JNK, while maintaining low levels of autophagic process. Therefore, obstructive cholestasis is characterized by higher p-JNK activation, which could be related with marked necrotic cell death resulting from extensive inflammation and little chance of compensatory autophagy.

Topics: Animals; Autophagy; Bile Ducts; Cholestasis; Hepatocytes; Humans; JNK Mitogen-Activated Protein Kinases; Ligation; Liver; Liver Cirrhosis, Experimental; Male; Mice; Necrosis; Phosphorylation; Thioacetamide

Hepatic macrophages play crucial roles in hepatotoxicity. We investigated immunophenotypes of macrophages in liver injury induced in rats by thioacetamide (TAA; 300 mg/kg, intraperitoneal) after hepatic macrophage depletion; hepatic macrophages were depleted by liposomal clodronate (CLD; 10 ml/kg, i.v.) one day before TAA injection. Samples were obtained on post-TAA injection days 0, 1, 2, 3, 5, and 7. TAA injection induced coagulation necrosis of hepatocytes on days 1 through 3 and subsequent reparative fibrosis on days 5 and 7 in the centrilobular area, accompanied by increased numbers of M1 macrophages (expressing cluster of differentiation [CD]68 and major histocompatibility complex class II) and M2 macrophages (expressing CD163 and CD204) mainly on days 1 through 3. TAA + CLD treatment markedly decreased the numbers of M1 and M2 macrophages mainly on days 1 through 3; CD163(+) Kupffer cells were most sensitive to CLD depletion. In TAA + CLD-treated rats, interestingly, coagulation necrosis of hepatocytes was prolonged with more increased levels of hepatic enzymes (aspartate transaminase, alanine transaminase, and alkaline phosphatase) to TAA-treated rats; reparative fibrosis was incomplete and replaced by dystrophic calcification in the injured area, indicating the aggravated damage. Furthermore, in TAA + CLD-treated rats, inflammatory factors (monocyte chemoattractant protein [MCP]-1, interferon-γ, tumor necrosis factor-α, and interleukin-10) and fibrosis-related factors (transforming growth factor-β1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1) were decreased at messenger RNA levels, indicating abnormal macrophage functions. It was clearly demonstrated that hepatic macrophages have important roles in tissue damage and remodeling in hepatotoxicity.

Topics: Animals; Chemical and Drug Induced Liver Injury; Hepatocytes; Immunohistochemistry; Liver; Macrophages; Male; Myofibroblasts; Necrosis; Rats; Rats, Inbred F344; Thioacetamide

The geographic map of cancer prevalence differs due to environmental and dietary factors in various populations. High prevalence of a number of cancers in some regions is thought to be attributed to local dietary habits. Dorema aucheri (Bilhar) is used commonly as an herbal medicine in some regions including Iran. The aim of this study was to evaluate whether Dorema aucheri has carcinogenic effects in albino mice or not.. The Dorema aucheri leaves were extracted by Soxhlet method and were injected intraperitoneally and randomly into 28 healthy albino mice which were divided into seven groups. One was put aside as the non-injected control group. The second control group was chosen to be injected by a known carcinogen. Another group was injected by carcinogen and then, Bilhar extract. The left four groups were injected the extracts in a dose- dependent manner, increasingly in the range of 0.4 - 3.2mL/kg. Extract injections were repeated every 48- hour intervals for three times. Then, liver and serum samples were analyzed biochemically and pathologically.. The pathologic and biochemical studies showed that the injection of plant extracts caused necrosis, inflammation of the liver tissue, cell proliferation, cholestasis, and there were significant increases in release of liver enzymes [ALP, ALT (SGPT) and AST (SGOT)] and bilirubin compared to the non-injected control group. The level of liver damage was dose dependent.. Dorema aucheri has potential hepatotoxic capacities and possibly this may be related to the high prevalence of cancer in some regions of Iran.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Apiaceae; Aspartate Aminotransferases; Bilirubin; Carcinogens; Cell Proliferation; Chemical and Drug Induced Liver Injury; Cholestasis; Hepatitis; Injections, Intraperitoneal; Liver; Mice; Necrosis; Plant Leaves; Plant Preparations; Thioacetamide

To date, considerable progress has been made both in the mechanisms driving liver fibrosis and in the prevention of disease progression. Resolution of liver fibrosis is an emerging field in hepatology; yet, the mediators involved remain elusive. Earlier work from our laboratory demonstrated that the matricellular cytokine osteopontin (OPN) is pro-fibrogenic by promoting hepatic stellate cell (HSC) activation and extracellular matrix (ECM) deposition in vitro and in vivo and specifically by governing fibrillar collagen-I expression, the key pro-fibrogenic protein. Here we hypothesized that OPN could also delay the resolution of liver fibrosis by sustaining collagen-I synthesis or by preventing its degradation. To demonstrate this, wild-type (WT) and OPN-knockout (Opn(-/-)) mice were administered thioacetamide (TAA) in the drinking water for 4 months. Half of the mice were killed at 4 months to assess the extent of fibrosis at the peak of injury, and the rest of the mice were killed 2 months after TAA withdrawal to determine the rate of fibrosis resolution. Following TAA cessation, livers from Opn(-/-) mice showed no centrilobular and parenchymal necrosis along with faster ECM remodeling than WT mice. The latter was quantified by less fibrillar collagen-I immunostaining. Western blot analysis demonstrated a significant decrease in fibrillar collagen-I and in tissue inhibitor of metalloproteinase-1 (TIMP-1) in Opn(-/-) mice undergoing fibrosis resolution compared with WT mice. In conclusion, these results suggest that OPN delays liver fibrosis resolution due to sustained fibrillar collagen-I deposition; hence, inhibiting OPN could be an effective therapeutic strategy for resolving liver fibrosis.

Topics: Actins; Animals; Biomarkers; Collagen Type I; Crosses, Genetic; Disease Models, Animal; Extracellular Matrix; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Osteopontin; Protein Stability; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1

To evalueate hepatoprotective effects Feronia elephantum (F. elephantum) correa against thioacetamide (TA) induced liver necrosis in diabetic rats.. Male wistar rats were made diabetic with alloxan (160 mg/kg) on day 0 of the study. They were intoxicated with hepatotoxicant (thioacetamide, 300 mg/kg, ip) on day 9 of study to produce liver necrosis. Effects of 7 day daily once administration (day 2 to day 9) of EF (400 and 800 mg/kg, po) were evaluated on necorosis of liver in terms of mortality, liver volume, liver weight, serum aspartate aminotransferase (AST) and serum alanine transaminase (ALT), and histopathology of liver sections (for signs of necorosis and inflammation) on day-9 of the study. Separate groups of rats with treated only with alloxan (DA control), thioacetamide (TA control) and both (TA+DA control) were maintained.. FE significantly lowered the mortality rate and showed improvement in liver function parameters in TA-induced diabetic rats without change in liver weight, volume and serum glucose levels.. FE showed promising activity against TA-induced liver necorsis in diabetic rats and so might be useful for prevention of liver complications in DM.

Topics: Animals; Blood Glucose; Chemical and Drug Induced Liver Injury; Diabetes Mellitus, Experimental; Disease Models, Animal; Liver Function Tests; Male; Necrosis; Plant Extracts; Protective Agents; Rats; Rutaceae; Thioacetamide

The present study was designed to examine the effect of the grape seed proanthocyanidin extract (GSPE) on developing hepatic fibrosis that was induced by thioacetamide (TAA) in mice. Administration of TAA for 9 weeks led to a serious necrosis and apoptosis of the parenchymal cells, which resulted in an accumulation of excessive collagen in the liver and an increase of transformed hepatic stellate cells (HSCs). In addition, the mRNA expression of transforming growth factor β1 (TGF-β1), α-smooth muscle actin (α-SMA), as the marker of the activated HSCs, and α1-(I)-collagen were all up-regulated significantly when compared with the control. However, combined oral administration of GSPE at 100 mg/kg suppressed the mRNA expression of TGF-β1 and α-SMA, with decreased collagen accumulation as demonstrated by histomorphological evaluation and quantitative RT-PCR. The mRNA expression of the pro-inflammatory factors, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), was remarkably enhanced by TAA treatment. However, their levels displayed a down-regulated trend beyond simultaneous GSPE treatment. Moreover, GSPE administration markedly suppressed lipid peroxidation. In conclusion, as a plant antioxidant, GSPE manifested effective hepatocellular protective action to ameliorate the developing liver fibrosis induced by chronic TAA administration in mice.

Topics: Actins; Alanine Transaminase; Animals; Antioxidants; Apoptosis; Aspartate Aminotransferases; Collagen Type I; Collagen Type I, alpha 1 Chain; Cyclooxygenase 2; Cytoprotection; Female; Gene Expression Regulation; Grape Seed Extract; Hepatic Stellate Cells; Immunohistochemistry; Inflammation Mediators; Lipid Peroxidation; Liver; Liver Cirrhosis, Experimental; Mice; Mice, Inbred ICR; Necrosis; Nitric Oxide Synthase Type II; Proanthocyanidins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thioacetamide; Time Factors; Transforming Growth Factor beta1

To date liver transplantation is the only effective treatment for end-stage liver diseases. Considering the potential of pluripotency and differentiation into tridermal lineages, induced pluripotent stem cells (iPSCs) may serve as an alternative of cell-based therapy. Herein, we investigated the effect of iPSC transplantation on thioacetamide- (TAA-) induced acute/fulminant hepatic failure (AHF) in mice. Firstly, we demonstrated that iPSCs had the capacity to differentiate into hepatocyte-like cells (iPSC-Heps) that expressed various hepatic markers, including albumin, α-fetoprotein, and hepatocyte nuclear factor-3β, and exhibited biological functions. Intravenous transplantation of iPSCs effectively reduced the hepatic necrotic area, improved liver functions and motor activity, and rescued TAA-treated mice from lethal AHF. 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate cell labeling revealed that iPSCs potentially mobilized to the damaged liver area. Taken together, iPSCs can effectively rescue experimental AHF and represent a potentially favorable cell source of cell-based therapy.

Topics: Animals; Biomarkers; Cell Differentiation; Disease Models, Animal; Gene Expression Profiling; Hepatocytes; Immunohistochemistry; Liver Failure, Acute; Liver Function Tests; Male; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Necrosis; Pluripotent Stem Cells; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide

It is well known that gadolinium chloride (GD) attenuates drug-induced hepatotoxicity by selectively inactivating Kupffer cells. In the present study the effect of GD in reference to cell cycle and postnecrotic liver regeneration induced by thioacetamide (TA) in rats was studied. Two months male rats, intraveously pretreated with a single dose of GD (0.1 mmol/Kg), were intraperitoneally injected with TA (6.6 mmol/Kg). Samples of blood and liver were obtained from rats at 0, 12, 24, 48, 72 and 96 h following TA intoxication. Parameters related to liver damage were determined in blood. In order to evaluate the mechanisms involved in the post-necrotic regenerative state, the levels of cyclin D and cyclin E as well as protein p27 and Proliferating Cell Nuclear Antigen (PCNA) were determined in liver extracts because of their roles in the control of cell cycle check-points. The results showed that GD significantly reduced the extent of necrosis. Noticeable changes were detected in the levels of cyclin D1, cyclin E, p27 and PCNA when compared to those induced by thioacetamide. Thus GD pre-treatment reduced TA-induced liver injury and accelerated the postnecrotic liver regeneration. These results demonstrate that Kupffer cells are involved in TA-induced liver and also in the postnecrotic proliferative liver states.

Topics: Animals; Cell Cycle; Cell Cycle Checkpoints; Cell Proliferation; Chemical and Drug Induced Liver Injury; Cyclin D; Cyclin E; Gadolinium; Kupffer Cells; Liver; Liver Regeneration; Male; Necrosis; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Thioacetamide

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.

Topics: Animals; Anti-Inflammatory Agents; Cell Proliferation; DNA; Gadolinium; Hepatocytes; Kupffer Cells; Liver; Liver Regeneration; Male; Necrosis; Ploidies; Rats; Rats, Wistar; Thioacetamide; Tumor Necrosis Factor-alpha

Macrophages are crucial in hepatic fibrogenesis. In acute hepatic necrosis induced in rats by a single injection of 300 mg/kg body weight (BW) of thioacetamide (TAA), macrophage properties were investigated using single or double immunohistochemistry. Macrophages reacting with anti-CD68, anti-CD163, or major histocompatibility complex (anti-MHC) class II antibody appeared in injured centrilobular areas on days 1-5 after injection. Increased expression of CD68 and CD163 reflect phagocytosis and production of pro-inflammatory factors, respectively. There were also macrophages double-positive to CD68/CD163, CD68/MHC class II, or CD163/MHC class II; of these, macrophages double-positive to CD68/MHC class II were most frequent, indicating that macrophages with enhanced phagocytic activity came to express MHC class II. The appearance of these macrophages corresponded to increased expression of mRNAs of monocyte chemoattractant protein-1 (MCP-1), a chemokine, on day 1, and TGF-beta1, a fibrogenic factor, on day 3. Some hepatic stellate cells (HSCs) in injured areas reacted with anti-MCP-1 antibody. To investigate the effects of MCP-1, we added MCP-1 to HS-P, a rat macrophage line. Addition of MCP-1 increased immunoexpression for CD68 and CD163 and up-regulated TGF-beta1 mRNA expression. Collectively, macrophages in acute hepatic necrosis may express different properties such as phagocytosis, MHC class II expression, and TGF-beta1 production; such expression may be influenced by MCP-1 produced by HSCs.

Topics: Analysis of Variance; Animals; Antigens, CD; Antigens, Differentiation; Antigens, Differentiation, Myelomonocytic; Cells, Cultured; Chemical and Drug Induced Liver Injury; Chemokine CCL2; Gene Expression Regulation; Hepatic Stellate Cells; Histocompatibility Antigens Class II; Immunohistochemistry; Liver; Liver Diseases; Macrophages; Male; Necrosis; Rats; Rats, Inbred F344; Receptors, Cell Surface; Thioacetamide; Transforming Growth Factor beta1

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.

Topics: Animals; Ascorbic Acid; Aspartate Aminotransferases; Blotting, Western; Dimethyl Sulfoxide; Extracellular Signal-Regulated MAP Kinases; Free Radical Scavengers; JNK Mitogen-Activated Protein Kinases; Liver; Male; Necrosis; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Rats; Rats, Wistar; Thioacetamide; Vitamin E

Due to the loss of cell-cell and cell-matrix interactions, cell culture models poorly mimic the in vivo situation. Therefore, we tested the applicability of precision-cut liver slices (PCLS) to study the early activation of the two main liver fibrogenic cell subpopulations: hepatic stellate cells (HSC) and portal fibroblasts (PF). PCLS were treated with thioacetamide or acetaminophen to induce HSC activation. In PCLS culture, both were able to trigger centrolobular lesion and HSC activation as observed in vivo. However, thioacetamide also presented a toxic effect on portal tract cells. In this PCLS model of centrolobular lesion, the antioxidant N-acetylcysteine was able to prevent acetaminophen-induced injury. To induce a specific activation of PF, PCLS were treated with epidermal growth factor or beta-oestradiol. As in vivo, epidermal growth factor and beta-oestradiol induced bile duct epithelial cell proliferation accompanied by PF activation; however, beta-oestradiol also triggers sinusoidal cell proliferation. We demonstrated that treatments usually used in vivo to induce liver fibrosis allow, in cultured PCLS, the specific activation of the two main liver fibrogenic cell subpopulations, making this model very useful to study the mechanisms involved in early fibrogenic cell activation.

Topics: Acetaminophen; Acetylcysteine; Animal Use Alternatives; Animals; Antioxidants; Bile Ducts, Intrahepatic; Cell Survival; Disease Models, Animal; Drug Antagonism; Epidermal Growth Factor; Estradiol; Fibroblasts; Hepatocytes; Kupffer Cells; Liver; Liver Cirrhosis; Male; Necrosis; Organ Culture Techniques; Portal System; Rats; Rats, Wistar; Thioacetamide

S-adenosylmethionine (SAMe) has been shown to protect hepatocytes from toxic injury, both experimentally-induced in animals and in isolated hepatocytes. The mechanisms by which SAMe protects hepatocytes from injury can result from the pathways of SAMe metabolism. Unfortunately, data documenting the protective effect of SAMe against mitochondrial damage from toxic injury are not widely available. Thioacetamide is frequently used as a model hepatotoxin, which causes in vivo centrilobular necrosis. Even though thioacetamide-induced liver necrosis in rats was alleviated by SAMe, the mechanisms of this protective effect remain to be verified. The aim of our study was to determine the protective mechanisms of SAMe on thioacetamide-induced hepatocyte injury by using primary hepatocyte cultures. The release of lactate dehydrogenase (LDH) from cells incubated with thioacetamide for 24 hours, was lowered by simultaneous treatment with SAMe, in a dose-dependent manner. The inhibitory effect of SAMe on thioacetamide-induced lipid peroxidation paralleled the effect on cytotoxicity. A decrease in the mitochondrial membrane potential, as determined by Rhodamine 123 accumulation, was also prevented. The attenuation by SAMe of thioacetamide-induced glutathione depletion was determined after subsequent incubation periods of 48 and 72 hours. SAMe protects both cytoplasmic and mitochondrial membranes. This effect was more pronounced during the development of thioacetamide-induced hepatocyte injury that was mediated by lipid peroxidation. Continuation of the SAMe treatment then led to a reduction in glutathione depletion, as a potential consequence of an increase in glutathione production, for which SAMe is a precursor.

Topics: Animal Testing Alternatives; Animals; Carcinogens; Cell Survival; Cells, Cultured; Drug Antagonism; Glutathione; Hepatocytes; L-Lactate Dehydrogenase; Lipid Peroxidation; Male; Membrane Potential, Mitochondrial; Necrosis; Protective Agents; Rats; Rats, Wistar; S-Adenosylmethionine; Thioacetamide

Many chemical compounds and infectious agents such as viruses induce liver damage like necrosis or fulminant hepatic failure which is sometimes difficult to manage by medical therapies. The induced liver necrosis by carbon tetrachloride (CCl4) and thioacetamide (TAA) are exemplary models for experimental liver necrosis caused by oxygen free radicals. The aim of this study was to investigate the effects of tungstophosphoric acid (TPA) and sodium tungstate (ST) on liver injury induced by CCl4 or TAA.. Hepatoprotective effects of TPA and ST on acute liver necrosis, chemically induced, were evaluated by the activity of serum enzymes (alkaline phosphatase, alanine transaminase and aspartate transaminase), oxidative stress parameters (activity of xanthine oxidase, concentrations of malondialdehyde and production of superoxide anion), antioxidative defence markers (concentration of reduced glutathione), and histopathology in Wistar rats. Liver necrosis was induced by administering a single intraperitoneal (i.p.) injection of CCl4 (1.0 ml/kg b.wt. of 80% CCl4 in corn oil) or a single i.p. injection of TAA (400 mg/kg b.w. dissolved in normal saline). TPA and ST were administrated to rats orally for 7 weeks (50 mg/kg b.wt.) prior to induction of liver necrosis.. Induced liver necrosis caused significant elevation of activity of liver enzymes, parameters of oxidative stress and marked changes in histopathology, like necrosis of hepatocytes, hepatocyte degeneration and infiltration of inflammatory cells. In TPA and ST pretreated rats histopathological changes were almost absent, serum enzymes and oxidative stress parameters were decreased, while at the same time the concentration of reduced gluthathione was increased.. The present findings suggest that treatment with TPA and ST for 7 weeks could be useful for the prevention of hepatic injury in rats.

Topics: Acute Disease; Administration, Oral; Animals; Antioxidants; Carbon Tetrachloride; Disease Models, Animal; Female; Glutathione; Liver; Liver Function Tests; Necrosis; Oxidative Stress; Phosphoric Acids; Rats; Rats, Wistar; Thioacetamide; Tungsten Compounds

A previous study (Oliver, J.R., Mara, T.W., Cherian, M.G. 2005. Impaired hepatic regeneration in metallothionein-I/II knockout mice after partial hepatectomy. Exp. Biol. Med. 230, 61-67) has shown an impairment of liver regeneration following partial hepatectomy (PH) in metallothionein (MT)-I and MT-II gene knockout (MT-null) mice, thus suggesting a requirement for MT in cellular growth. The present study was undertaken to investigate whether MT may play a similar role in hepatic injury and regeneration after acute treatment with thioacetamide (TAA). Hepatotoxicity of TAA is caused by the generation of oxidative stress. TAA was injected ip to both wild-type (WT) and MT-null mice. Mice were killed at 6, 12, 24, 48, 60, and 72 h after injection of TAA (125 mg/kg) or 48 h after injection of saline (vehicle control), and different parameters of hepatic injury were measured. The levels of hepatic lipid peroxidation were increased at 12 h in both types of mice; however, lipid peroxidation was significantly less in WT mice than MT-null mice at 48 h after injection of TAA. Analysis of hepatic glutathione (GSH) levels after TAA injection showed depletion of GSH at 12 h in WT mice and at 6 h in MT-null mice; however, significantly more GSH was depleted early (6-24 h) in MT-null mice than WT mice. An increase in hepatic iron (Fe) levels was observed in both types of mice after injection of TAA, but Fe levels were significantly higher in MT-null mice than WT mice at 6-60 h. The levels of hepatic copper (Cu) and zinc (Zn) were significantly higher in WT mice than MT-null mice at 6-60 h for Cu, and at 24 h and 60 h for Zn, respectively. Histopathological examination showed hemorrhagic necrosis in the liver of both types of mice at 12-72 h, with hepatic injury being more prominent in MT-null mice than WT mice. The hepatic MT levels were increased in WT mice after injection of TAA, and were highest at 24-72 h. Immunohistochemical staining for MT in WT mice indicated the presence of MT in both nucleus and cytoplasm of hepatocytes at 24-72 h after TAA injection. Cell proliferation, as assessed by immunohistochemical staining for proliferating cell nuclear antigen, was detected mainly in the livers of WT mice at 48-72 h after TAA treatment. Hepatic proliferation index in MT-null mice was very low as compared to WT mice during liver regeneration after injection of TAA. These results show that the liver cells of MT-null mice with no functional MT are unable to regenerate afte

Topics: Acute Disease; Animals; Cell Proliferation; Chemical and Drug Induced Liver Injury; Glutathione; Iron; Lipid Peroxidation; Liver; Liver Regeneration; Metallothionein; Mice; Mice, Knockout; Necrosis; Thioacetamide

The hexane extract of oleo-gum-resin of Boswellia serrata (BSHE) was evaluated for its effect on liver injury induced by carbon tetrachloride, paracetamol or thioacetamide. The BSHE was given in two different doses (87.5 mg/kg p.o. and 175 mg/kg p.o.). Silymarin, a known hepatoprotective agent was used as standard. The lower dose of BSHE (87.5 mg/kg p.o.) significantly reduced the elevated levels of serum marker enzymes and prevented the increase in liver weight in all three models of liver injury, while the higher dose showed mild hepatoprotective activity. The hepatoprotective effect of lower dose of BSHE was supported by changes in histopathology. It was concluded that hexane extract of oleo-gum-resin of Boswellia serrata plant in lower doses possess hepatoprotective activity.

Topics: Acetaminophen; Animals; Anti-Inflammatory Agents; Boswellia; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Dose-Response Relationship, Drug; Hexanes; Liver; Liver Diseases; Necrosis; Organ Size; Plant Extracts; Protective Agents; Rats; Rats, Wistar; Resins, Plant; Silymarin; Thioacetamide

Gingko biloba (GB) has antioxidant and platelet-activating factor (PAF) antagonistic effects. We investigated the protective effects of GB on thioacetamide (TAA)-induced fulminant hepatic failure in rats. Fulminant hepatic failure was induced in treatment groups by three intraperitoneal (ip) injections of TAA (350 mg/kg) at 24-hour intervals. Treatments with GB (100 mg/kg per day, orally) and N-acetylcysteine (20 mg/kg twice daily, sc) were initiated 48 hours prior to TAA administration. The liver was removed for histopathological examinations. Serum and liver thiobarbituric acid-reactive substance (TBARS) levels were measured for assessment of oxidative stress. Liver necrosis and inflammation scores and serum and liver TBARS levels were significantly higher in the TAA group compared to the control group (P < 0.001, < 0.001, 0.001, < 0.001, respectively). Liver necrosis and inflammation scores and liver TBARS levels were significantly lower in the GB group compared to the TAA group (P < 0.001, < 0.001 and 0.01, respectively). GB ameliorated hepatic damage in TAA-induced fulminant hepatic failure. This may be due to the free radical-scavenging effects of GB.

Topics: Acetylcysteine; Alanine Transaminase; Ammonia; Animals; Aspartate Aminotransferases; Bilirubin; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Drugs, Chinese Herbal; Free Radical Scavengers; Ginkgo biloba; Hepatic Encephalopathy; Liver; Male; Necrosis; Oxidative Stress; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Severity of Illness Index; Thioacetamide; Thiobarbituric Acid Reactive Substances

The hepatic lesion produced as a result of oxidative stress is of wide occurrence. In the present study, the effect of tungsten on liver necrosis and fulminant hepatic failure (FHF) has been studied in rats treated with various compounds known to produce oxidative stress. Supplementation of animals with sodium tungstate for 7 weeks before the induction of liver injury by chemicals including thioacetamide (TAA), carbon tetrachloride (CCl(4)), or chloroform (CHCl(3)) could protect progression of hepatic injury. Various biochemical changes associated with liver damage and oxidative stress were measured. Hepatic malondialdehyde content, endogenous tripeptide, and reduced glutathione were measured as oxidative stress markers. The activity of xanthine oxidase, which generates reactive oxygen species (ROS) as a by-product, was also determined and found to be perturbed. Tungsten supplementation to rats caused a significant decrease in lipid peroxidation and lowered the levels of the biochemical markers of hepatic lesions produced by TAA, CCl(4) (CCl(4)), or CHCl(3). Tungsten could also cause an increase in the survival rate in rats receiving lethal doses of TAA, CCl(4), or CHCl(3). The protective effect of tungsten, however, is suggested to be limited to the conditions where the hepatic lesion is reported to be due to the generation of ROS. The progression of liver injury produced by the compounds causing oxidative stress without initiating the generation of free radicals such as bromobenzene (BB), or acetaminophen (AAP), could not be inhibited by tungsten. The possible mechanism explaining the role of oxyanionic form of tungsten in free radical-induced hepatic lesions is discussed.

Topics: Acetaminophen; Alkaline Phosphatase; Allopurinol; Animals; Bromobenzenes; Carbon Tetrachloride Poisoning; Chloroform; Dimethyl Sulfoxide; Female; Lethal Dose 50; Liver; Liver Failure; Necrosis; Oxidative Stress; Rats; Rats, Wistar; Thioacetamide; Transaminases; Tungsten Compounds

This study examines how treatment with a single dose of thioacetamide, a known experimental hepatotoxin, alters the content of dolichol, dolichol isoprene units and retinol in isolated rat parenchymal and non-parenchymal liver cells at different times and when the animals are supplemented with Vitamin A. Thioacetamide (300 mg/kg i.p.) was administered in a single injection to rats, sacrificed at intervals of 0.5, 1, 2, 3, 4, 15 and 30 days. Rats supplemented, following thioacetamide, with Vitamin A, 3 days before sacrifice showed increased mortality and cellular necrosis on the third and fourth days. Parameters indicating tissue necrosis returned to normal values in surviving animals. Dolichol and retinol content showed a variable, reversible decrease, with normal levels being restored in 15-30 days. After Vitamin A, dolichol content only in hepatic stellate cells (HSC) was lower then the controls 3 and 4 days after thioacetamide treatment, in parallel with the decrease of retinol storage. The percentage of dolichol-18 is not modified by thioacetamide alone. When supplemented with Vitamin A the percentage of dolichol-18 always decreased after thioacetamide, showing that damage was still present. Mechanisms that might be operative in liver cells are briefly discussed. This approach would provide an indication to investigate how the length of the dolichol chain is determined.

Topics: Animals; Dolichols; Injections, Intraperitoneal; Liver; Male; Necrosis; Rats; Rats, Wistar; Terpenes; Thioacetamide; Time Factors; Vitamin A

Hepatic stimulator substance (HSS) is a known liver-specific but species-nonspecific growth factor. In the present study we examined the activity of the endogenously produced HSS in an established experimental model of fulminant hepatic failure (FHF) and encephalopathy, induced by repeated injections of thioacetamide (TAA). FHF was induced by three consecutive intraperitoneal injections of TAA (400 mg/kg body weight) in rats, at time intervals of 24 hr. The animals were killed at 0, 6, 12, or 18 hr following the last injection of TAA. The rate of tritiated thymidine incorporation into hepatic DNA, the enzymatic activity of liver thymidine kinase (EC 2.7.1.21), and the assessment of mitotic index in hepatocytes were used to estimate liver regeneration. HSS extract obtained from the livers of TAA-treated rats, sacrificed at the above-mentioned time points was tested for its activity. Increased HSS activity was noted in all TAA-treated animals, presenting a peak at 12 hr following the third TAA dose, suggesting active participation of this growth factor in hepatocyte replication in this animal model of FHF and encephalopathy. It may also be suggested that up-regulation of HSS activity could be used in future as a therapeutic approach in FHF.

Topics: Animals; Disease Models, Animal; Growth Substances; Hepatic Encephalopathy; Humans; Injections, Intraperitoneal; Intercellular Signaling Peptides and Proteins; Liver; Liver Failure; Liver Function Tests; Liver Regeneration; Male; Necrosis; Peptides; Rats; Rats, Wistar; Thioacetamide; Up-Regulation

Xanthine oxidase (XO) generates reactive oxygen metabolites (ROM) as a by-product while catalyzing their reaction. The present study implicates these ROM in the pathogenesis of liver necrosis produced in rats by the intraperitoneal administration of thioacetamide (TAA; 400 mg/kg b.wt.). After 16 h of TAA administration, the activity of rat liver XO increased significantly compared to that of the control group. At the same time, the level of serum marker enzymes of liver necrosis (aminotransferases and alkaline phosphatase) and tissue malondialdehyde content also increased in TAA treated rats. Tissue malondialdehyde concentration is an indicator of lipid peroxidation and acts as a useful marker of oxidative damage. Pretreatment of rats with XO inhibitor (4-hydroxypyrazolo[3,4-d]pyrimidine; allopurinol (AP)) followed by TAA could lower the hepatotoxin-mediated rise in malondialdehyde level as well as the level of marker enzymes associated with liver necrosis. The survival rate also increased in rats given AP followed by the lethal dose of TAA. In either case, the effect of AP was dose-dependent. Results presented in the paper indicate that increased production of XO-derived ROM contributes to liver necrosis, which can be protected by AP.

Topics: Alkaline Phosphatase; Allopurinol; Animals; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Glutathione; Lipid Peroxidation; Liver; Malondialdehyde; Necrosis; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Thioacetamide; Transaminases; Xanthine Oxidase

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.

Topics: Animals; Apoptosis; Ascorbic Acid; Aspartate Aminotransferases; Caspase 3; Caspases; Chemical and Drug Induced Liver Injury; Enzyme Activation; Enzyme Induction; Free Radicals; Lipid Peroxidation; Liver; Male; Necrosis; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Wistar; Thioacetamide; Thiobarbituric Acid Reactive Substances; Vitamin E

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.

Topics: Age Factors; Animals; Aspartate Aminotransferases; Bilirubin; Biotransformation; Drug Synergism; Enzyme Induction; gamma-Glutamyltransferase; Liver; Male; Microsomes, Liver; Necrosis; Oxygenases; Phenobarbital; Rats; Rats, Wistar; Thioacetamide

Diabetes is known to potentiate thioacetamide (TA)-induced liver injury via enhanced bioactivation. Little attention has been given to the role of compensatory tissue repair on ultimate outcome of hepatic injury in diabetes. The objective of this study was to investigate the effect of diabetes on TA-induced liver injury and lethality and to investigate the underlying mechanisms. We hypothesized that hepatotoxicity of TA in diabetic rats would increase due to enhanced bioactivation-mediated liver injury and also due to compromised compensatory tissue repair, consequently making a nonlethal dose of TA lethal. On day 0, male Sprague-Dawley rats (250-300 g) were injected with streptozotocin (STZ, 60 mg/kg ip) to induce diabetes. On day 10 the STZ-induced diabetic rats and the nondiabetic rats received a single dose of TA (300 mg/kg ip). This normally nonlethal dose of TA caused 90% mortality in the STZ-induced diabetic rats. At various times (0-60 h) after TA administration, liver injury was assessed by plasma alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), and liver histopathology. Liver function was evaluated by plasma bilirubin. Cell proliferation and tissue repair were evaluated by [(3)H]thymidine ((3)H-T) incorporation and proliferating cell nuclear antigen (PCNA) assays. In the nondiabetic rat, liver necrosis peaked at 24 h and declined thereafter toward normal by 60 h. In the STZ-induced diabetic rat, however, liver necrosis was significantly increased from 12 h onward and progressed, culminating in liver failure and death. Liver tissue repair studies showed that, in the liver of nondiabetic rats, S-phase DNA synthesis was increased at 36 h and peaked at 48 h following TA administration. However, DNA synthesis was approximately 50% inhibited in the liver of diabetic rats. PCNA study showed a corresponding decrease of cell-cycle progression, indicating that the compensatory tissue repair was sluggish in the diabetic rats. Further investigation of tissue repair by employing equitoxic doses (300 mg TA/kg in the non-diabetic rats; 30 mg TA/kg in the diabetic rats) revealed that, despite equal injury up to 24 h following injection, the tissue repair response in the diabetic rats was much delayed. The compromised tissue repair prolonged liver injury in the diabetic rats. These studies suggest that the increased TA hepatotoxicity in the diabetic rat is due to combined effects of increased bioactivation-mediated liver injury of TA and compromised

Topics: Alanine Transaminase; Animals; Bilirubin; Chemical and Drug Induced Liver Injury; Diabetes Mellitus, Experimental; DNA; Drug Synergism; L-Iditol 2-Dehydrogenase; Liver; Liver Regeneration; Male; Necrosis; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Survival Rate; Thioacetamide; Thymidine; Time Factors

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.

Topics: 3T3 Cells; Aging; Animals; Aspartate Aminotransferases; Carcinogens; Cell Division; Chemical and Drug Induced Liver Injury; Glutathione; Liver; Liver Diseases; Liver Regeneration; Male; Mice; Necrosis; Nitric Oxide; Oxygenases; Rats; Rats, Wistar; Thioacetamide; Tumor Necrosis Factor-alpha

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.

Topics: Animals; Carcinogens; Catalase; Enzyme Activation; Glutathione; Liver; Liver Regeneration; Male; Necrosis; Rats; Rats, Wistar; Superoxide Dismutase; Thioacetamide

Age-associated changes in liver injury and post-necrotic regeneration were studied in rats aged 6 and 30 months in a period of 96 h following a dose of thioacetamide (6.6 mmol/kg body weight). Hepatocellular necrosis was detected in both groups by serum aspartate aminotransferase, but the severity of injury was significantly lower (one fourth, p < 0.001) in the oldest. Differences were observed in hepatocyte FAD monooxygenase activity between 6 and 30 months old rats at 24 h (278 versus 170%, p < 0.001, respectively) and also in GSH/GSSG ratio, in protein thiol groups and in malondialdehyde. Glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase activities rose markedly in both groups, this increase being slightly lower in the oldest. Superoxide dismutase and catalase did not show significant changes between both groups. At the end of the 96 h experimental period the restoration towards normal of GSG/GSSG, protein thiols malondialdehyde and the activities of Cu-Zn superoxide dismutase and catalase were significantly lower in hepatocytes from 30 months old rats. We summarize that the main age-related changes in the sequenced process of liver injury and regeneration occurred to a lesser extent in severity of injury and delayed response in the post-necrotic restoration of liver function, probably due to a lower increase in antioxidant enzyme system.

Topics: Aging; Animals; Aspartate Aminotransferases; Catalase; Cells, Cultured; Glucosephosphate Dehydrogenase; Glutathione; Glutathione Disulfide; Glutathione Peroxidase; Glutathione Reductase; Liver; Liver Cirrhosis, Experimental; Liver Regeneration; Male; Necrosis; Rats; Rats, Wistar; Superoxide Dismutase; Thioacetamide

1. The biological actions of nitric oxide (NO), a highly diffusible and short-lived radical, range from signal transduction to cytotoxicity. The present study investigated whether NO is released in the course of liver necrosis and regeneration induced by a single necrogenic dose of thioacetamide (6.6 mmol kg-1 body wt) to rats. Samples of liver were obtained at 0, 3, 12, 24, 48, 72 and 96 h after thioacetamide administration. 2. Inducible nitric oxide synthase (iNOS) activity was determined in purified liver homogenates and a sharp 6 fold increase (P < 0.001) in iNOS activity was recorded at 48 h of intoxication, followed by a slight but progressive increase at 72 and 96 h. Changes in the expression of iNOS, as detected by its mRNA levels, were parallel to the NOS enzyme activity. Hepatocyte NO synthesis showed a progressive increase at 24, 48 and 72 h, to 8 (P < 0.001), 13 (P < 0.001) and 13 (P < 0.001) times the initial values, respectively. 3. In isolated Kupffer cells, where initial NO release was ten fold higher than in hepatocytes, a progressive increase was detected from 48 h which reached two fold of initial at 72 h of intoxication (192%; P < 0.001). Hepatic cyclic GMP concentration did not change significantly. However, mitochondrial aconitase activity decreased markedly at 12 and 24 h of intoxication showing a sharp increase towards normal values at 48 h which was maintained at 72 and 96 h. 4. As protein kinase C (PKC) is one of the likely candidates to mediate iNOS expression, translocation (activation) of PKC was assayed in hepatocytes, and a significant two fold increase (P < 0.001) between 48 and 96 h after thioacetamide intoxication was observed. When peritoneal macrophages from control rats were incubated with serum from thioacetamide-treated rats, a sharp increase in NO release was detected with serum obtained at 48 h, reaching at 96 h a value four fold (P < 0.001) that of the control. 5. These results suggest that iNOS activity and NO release play a role in the pathophysiological mechanisms that trigger post-necrotic hepatocellular regeneration following thioacetamide administration.

Topics: Aconitate Hydratase; Animals; Carcinogens; Cyclic GMP; Kupffer Cells; Liver; Male; Necrosis; Nitric Oxide; Nitric Oxide Synthase; Protein Kinase C; Rats; RNA, Messenger; Thioacetamide; Tumor Necrosis Factor-alpha

We have shown that urinary taurine level may be used as a biomarker of pathological and biochemical lesions. Detection of changes in the urinary concentration of this low molecular weight metabolite indicates biochemical lesions which may also be associated with pathological damage. Hepatotoxic compounds such as CCl4, galactosamine and thioacetamide that cause hepatic necrosis and compounds such as hydrazine and ethionine that cause fatty liver all result in elevated urinary taurine levels in rats. However compounds which do not cause liver damage, such as cycloheximide, also raise urinary taurine levels. All of these substances are known to or are believed to inhibit protein synthesis. Conversely, compounds which increase protein synthesis, such as phenobarbital and clenbuterol, significantly decrease urinary taurine levels. Compounds which interfere with hepatic GSH synthesis will also change urinary taurine levels. Thus, depletion of GSH with diethyl maleate or phorone decreases urinary taurine whereas inhibition of GSH synthesis with compounds such as buthionine sulphoximine increases urinary taurine levels. In isolated hepatocytes in vitro, leakage of taurine occurs in response to cytotoxic compounds such as hydrazine and allyl alcohol. However, total taurine levels were increased by the hepatotoxicant CCl4. Taurine synthesis is decreased by depletion of GSH with allyl alcohol in isolated hepatocytes. Therefore taurine levels are an important potential biomarker for biochemical lesions induced by chemicals both in vivo and in vitro, in particular changes in protein and GSH synthesis.

Topics: Animals; Biomarkers; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Cells, Cultured; Clenbuterol; Galactosamine; Glutathione; Ketones; Liver; Liver Diseases; Male; Maleates; Necrosis; Phenobarbital; Rats; Rats, Sprague-Dawley; Taurine; Thioacetamide

The purpose of the present study was to establish a dose-response relationship for thioacetamide (TA), where tissue regeneration as well as liver injury were two simultaneous but opposing responses. Male Sprague-Dawley rats were injected intraperitioneally with a 12-fold dose range of TA, and both liver injury and tissue repair were measured. Liver injury was assessed by serum enzyme elevations. Serum alanine aminotransferase (ALT) elevation did not show any dose response over a 12-fold dose range up to 24 hr. A dramatic ALT elevation was evident after 24 hr and only for the highest dose (600 mg/kg). Tissue regeneration response was measured by 3H-thymidine (3H-T) incorporation into hepatocellular DNA and by proliferating cell nuclear antigen (PCNA) procedure during a time course (6, 12, 24, 36, 48, 72, and 96 hr). Tissue regeneration, as indicated by 3H-T incorporation, peaked at 36 hr after administration of a low dose of TA (50 mg/kg). With increasing doses, a greater but delayed stimulation of cell division was observed until a threshold was reached (300 mg/kg). Above the tissue repair threshold (600 mg/kg), because stimulated tissue repair as revealed by 3H-T incorporation in hepatonuclear DNA was significantly delayed and attenuated, injury assessed by serum enzyme elevations was remarkably accelerated, indicating unrestrained progression of injury leading to animal death. These findings suggest that, in addition to the magnitude of tissue repair response, the time at which this occurs is critical in restraining the progression of injury, thereby determining the ultimate outcome of toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alanine Transaminase; Animals; DNA; Dose-Response Relationship, Drug; Injections, Intraperitoneal; Liver; Liver Regeneration; Male; Necrosis; Rats; Rats, Sprague-Dawley; S Phase; Thioacetamide; Time Factors

Sequential changes of gap junctions (GJs), tight junctions (TJs) and desmosomes (DSs) between hepatocytes during restorative proliferation were studied in rats after a single intraperitoneal administration of 200 mg/kg thioacetamide (TAA). Antibody against connexin 32 was used to demonstrate GJs; simultaneously the changes in TJs and DSs were studied using antibodies against 7H6 protein and desmoplakins. Propidium iodide and bromodeoxyuridine were used to recognize necrotic and proliferative cells. GJs were evenly distributed in early necrotic hepatocytes at 16 h after TAA treatment, then disappeared from necrotic and surrounding cells at 24 h. At 48 h, GJs had disappeared completely from hepatocytes in whole liver lobules, while many hepatocytes were heavily labelled with BrdU. At 72 h, GJs reappeared, firstly in perinecrotic areas. At 96 h after treatment, when the injured areas had disappeared and restorative proliferation ceased, GJs were distributed evenly throughout the lobules. Immunohistochemical observation of GJs in centrilobular, perinecrotic and periportal areas after TAA-induced hepatic necrosis was confirmed by counting the number of connexin-32-positive spots in the respective areas. TJs and DSs disappeared from necrotic cells at 24 h, but then increased between 24 and 48 h in perinecrotic areas, though the increased intensity of these junctions was more evident at 48 h. At 72 h, localization of TJs and DSs returned to normal. These results suggest that during the course of acute hepatic injury, GJs (cell-cell communication) behave differently from other intercellular junctions.

Topics: Animals; Bromodeoxyuridine; Cell Division; Desmosomes; Gap Junctions; Immunohistochemistry; Intercellular Junctions; Kinetics; Liver; Male; Necrosis; Propidium; Rats; Rats, Inbred F344; Thioacetamide; Time Factors

Heat shock protein (hsp), which changes both its concentration and localization in reaction to stresses such as heating, ischemia, etc., is thought to protect protein structure and act as a chaperone in intracellular transportation. We examined one of the hsps, hsp 70, in rat liver with necrosis and regeneration produced by thioacetamide (TAA). Hsp 70 was determined by immunoblotting and detected histologically by immunostaining, using a specific antibody. Generally, hsp 70 moves from the cytosol to the nucleus, where it concentrates 15 min after TAA injection. After 15 min, hsp 70 was not detected in the nuclei of hepatocytes around the central vein, where the hepatocytes later became necrotic. However, hsp 70 immunostaining was increasingly strong in the nuclei of hepatocytes around the portal area, which did not become necrotic. These findings show that, in acute necrosis, hsp 70 seems to correlate with nuclear protection or with the transportation of some protein from the cytosol to the nucleus. Hepatocytes probably neither survive nor regenerate without hsp 70 in their nuclei.

Topics: Animals; Chemical and Drug Induced Liver Injury; Heat-Shock Proteins; Immunoblotting; Liver; Liver Regeneration; Male; Necrosis; Rats; Rats, Wistar; Thioacetamide; Time Factors

Thioacetamide proved to be a potent necrogenic agent when a single dose of 6.6 mmol/kg was administered intraperitoneally to rats. Its necrogenic ability was assessed on the basis of morphological and biochemical changes. The injury of centrilobular hepatocytes showed a peak of cell death 24 hr after thioacetamide administration; it was followed immediately by the regenerative response. Parallel increases of serum aminotransferases, isocitrate dehydrogenase and gamma-glutamyl transferase activities were observed. Severe liver damage was also evident at 24 hr on the basis of glutathione depletion (29% of control), malondialdehyde production (169%), cytochrome P-450 level decrease (26%) and increased activity of glutathione S-transferase (160%). We checked the regenerative response by determining nuclear DNA content in isolated hepatocytes 0, 6, 12, 18, 24, 36, 48 and 72 hr after thioacetamide administration. Changes in DNA cell distribution between G0-G1, S and G2 + M phases of the cell cycle were observed. The sharp decrease in the percentage of the tetraploid cell population (G2 + M phases) and the abrupt increase of the S-phase cells at 36 and 48 hr suggest transition from adult to fetal in hepatocyte populations obtained 24 and 36 hr after thioacetamide treatment. At 72 hr of treatment, hepatocyte populations showed recovery to adult state. In the shift from the adult to fetal, registered at 24, 36 and 48 hr after thioacetamide administration, mitosis seemed to precede the synthesis of DNA.

Topics: Animals; Cell Cycle; Cytochrome P-450 Enzyme System; DNA; Glutathione; Glutathione Transferase; Liver; Liver Regeneration; Male; Malondialdehyde; Necrosis; Ploidies; Polyploidy; Rats; Rats, Wistar; Thioacetamide

To determine whether lysosomal lipid composition is altered in hepatic necrosis, we studied this parameter in thioacetamide-induced necrosis and in its regenerating stage as well as in the recovery of thioacetamide-induced injury. Results showed that in liver necrosis there is an increase in the protein and phospholipid lysosomal contents. This effect may be related to an increased number of lysosomes. These organelles also suffered a decrease in cholesterol content. When liver necrosis was recovered either pharmacologically or spontaneously all three parameters recovered their normal values. These results suggest that lysosomes and their lipid composition play a role in progression of hepatic necrosis.

Topics: Acid Phosphatase; Alanine Transaminase; Animals; Aspartate Aminotransferases; Chemical and Drug Induced Liver Injury; Cholesterol; Lipid Metabolism; Lysosomes; Male; Membranes; Necrosis; Rats; Rats, Wistar; S-Adenosylmethionine; Thioacetamide

Hepatocytes isolated from the liver of rats after a necrotizing dose of thioacetamide (6.6 mmol/kg) were used to study the postnecrotic process of liver regeneration. Flow cytometry analysis revealed populations of dedifferentiated hepatocytes exhibiting physical properties (size and fluorescence emission at 530 nm) similar to those found in fetal (22 days old) liver cells. The percentage of these cells increased progressively from 24 to 48 and 72 hr after thioacetamide administration. In primary cultures of hepatocytes the effects of phorbol 12-myristate 13-acetate, bombesin and insulin were investigated on the 6-phosphofructo 2-kinase/fructose 2,6 bisphosphate system. Bombesin and insulin stimulated 6-phosphofructo 2-kinase activity and fructose 2,6-bisphosphate content both in control and in thioacetamide-treated hepatocytes. However, phorbol 12-myristate 13-acetate stimulated 6-phosphofructo 2-kinase activity and increased fructose 2,6-bisphosphate concentration in thioacetamide-treated liver cells, whereas no similar response was found in hepatocytes from control rats. The response of postnecrotic thioacetamide-treated hepatocytes to phorbol 12-myristate 13-acetate was similar to that obtained from 22-day-old fetal liver cells, which reveals that different methods might control fructose 2,6-bisphosphate content and therefore the mechanisms of glycolysis and gluconeogenesis at this regulatory step. The lack of response to glucagon of glycogen phosphorylase a and 6-phosphofructo 2-kinase from thioacetamide-treated hepatocytes may indicate that the expression of specific enzymes of carbohydrate metabolism undergoes transitions to less-differentiated isoenzymatic forms. Moreover, the isoenzyme pattern of hexokinases elicits a complete disturbance in glucokinase and hexokinases activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bombesin; Carbohydrate Metabolism; Cells, Cultured; Fetus; Fructosediphosphates; Glucagon; Growth Substances; Insulin; Isoenzymes; Liver; Liver Glycogen; Male; Necrosis; Phosphofructokinase-2; Phosphorylase a; Phosphotransferases; Protein Kinases; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Thioacetamide

Cytoprotective effects of the prostaglandins 16,16-dimethyl PGE2 (dmPGE2) and PGF2 alpha tromethamine (PGF2 alpha) were evaluated in the rat model of acute hepatocellular necrosis induced by thioacetamide (TAA). dmPGE2 (100 micrograms/kg SC 8 hourly) did not induce a significant increase in survival when started after the onset of TAA-induced fulminant hepatic failure. However, priming with dmPGE2 (100 micrograms/kg SC 30 min before TAA) reduced TAA-induced elevations in serum ALT (684 +/- 68 (SEM) vs 274 +/- 135 IU/1, p less than 0.01). This phenomenon did not occur if dmPGE2 was administered after TAA or by the IP route. Modulation of TAA-induced centrizonal hepatocellular necrosis by dmPGE2 was associated with a striking increase in centrizonal ballooning of hepatocytes (p less than 0.01), and, as assessed by stereology, less hepatocellular necrosis and degenerative changes. PGF2 alpha, which in contrast to dmPGE2 does not act via cAMP, had no effect on TAA-induced changes in serum ALT or hepatic histology. These findings suggest that dmPGE2 decreases hepatocellular necrosis by activating surface membrane adenylate cyclase and consequently stimulating cAMP. Ballooning of hepatocytes could occur secondary to these membrane events and appears to be a marker of dmPGE2-induced cytoprotection in this model.

Topics: 16,16-Dimethylprostaglandin E2; Alanine Transaminase; Animals; Dinoprost; Hepatic Encephalopathy; Liver; Male; Necrosis; Rats; Rats, Sprague-Dawley; Thioacetamide

A sequential study of the appearance of liver cell death after thioacetamide (TH) administration was performed in male Wistar rats. Within 3 hours of a single dose of TH, occurrence of cell death by apoptosis was evident around the centrilobular area. Light as well as electron microscopic examination demonstrated the presence of eosinophilic globules, often containing nuclear remnants (apoptotic bodies); they frequently were found within the cytoplasm of intact hepatocytes. The number of apoptotic bodies (ABs) was further enhanced at 6 hours, resulting in a 70-fold increase over the control values. Although necrosis or inflammation could not be observed at this time, as monitored by microscopic analysis as well as by determination of serum glutamate pyruvate transaminase levels, centrilobular necrosis accompanied by massive inflammatory reaction was evident at 12 hours and even more pronounced at 24 to 36 hours. Evidence of liver regeneration was found to occur at 48 hours, and the liver regained its normal architecture between 72 and 96 hours. Studies performed to analyze the activity of 'tissue' transglutaminase (tTG), a presumptive marker of apoptosis, showed that, 1 hour after treatment, TH caused a drastic dose-dependent inhibition of the enzyme activity. This early inhibition was followed by a rapid recovery in tTG activity that paralleled the induction of apoptosis in the liver. Treatment with cycloheximide (CH) 2 hours after TH partially inhibited the incidence of ABs at 6 hours (approximately 30% inhibition). The present study indicates that two different modes of cell death, apoptosis and necrosis, may be induced in a sequential fashion by a single dose of TH.

Topics: Alanine Transaminase; Animals; Cell Death; Chemical and Drug Induced Liver Injury; Cycloheximide; Dose-Response Relationship, Drug; Immunohistochemistry; Liver; Liver Regeneration; Male; Microscopy, Electron; Nafenopin; Necrosis; Rats; Rats, Inbred Strains; Thioacetamide; Time Factors; Transglutaminases

Topics: Acetamides; Animals; Liver; Male; Methyltransferases; Microsomes, Liver; Necrosis; Phosphatidyl-N-Methylethanolamine N-Methyltransferase; Phosphatidylcholines; Phosphatidylethanolamine N-Methyltransferase; Phosphatidylethanolamines; Rats; Rats, Inbred Strains; S-Adenosylmethionine; Thioacetamide

Thioacetamide is a weak hepatocarcinogen. To determine whether alterations in lysophosphatidylcholine are implicated in thioacetamide-induced hepatic necrosis, rats were injected i.p. with this agent (50 mg/Kg body weight per day) or diluent for 1, 3, 8 and 30 days. Serum catalytic activities of aminotransferases were determined. Incorporation of (32P)-orthophosphate into hepatic lysophosphatidylcholine was also evaluated in animals killed 75 minutes or 13 hours after isotope administration. Results demonstrate that: A significant increase in hepatic lysolecithin concentration occurs when a maximum level of serum aminotransferases is present. An increase of (32P)-orthophosphate radioactive incorporation in lysolecithin was observed at the two assayed labelling periods, which suggest an activation of phospholipase A. The radioactivity present in lysolecithin after 13 h isotope injection showed a close correlation with serum level of aminotransferases. From these results it can be deduced that lysolecithin is implicated in TAA-induced necrosis and may be generated by increase in either phospholipase A activity and/or synthesis.

Topics: Acetamides; Animals; Liver; Lysophosphatidylcholines; Male; Necrosis; Rats; Rats, Inbred Strains; Thioacetamide

The appearance of a liver DNA synthesis promoter (HP) in rat plasma after dimethylnitrosamine (DMNA) or thioacetamide injection was investigated. After 48 h, DMNA (30 mg kg-1 body weight) produced liver (centrilobular) necrosis and intense hepatic regeneration, as assessed by microscopic observations of liver slices, as well as augmented transaminase levels; HP was detectable under these conditions. After 5 days, transaminases and HP returned to normal values (the latter undetectable), coinciding with a lack of necrotic zones. At 60 mg DMNA kg-1 body weight, necrotic areas were more marked and transaminases and HP levels higher after 48 h than with the lower dose; these increases were even more pronounced at 90 mg DMNA kg-1 body weight. After thioacetamide injection (200 mg kg-1 body wt) the situation at 48 h was very similar, with focal, centrilobular necrosis, frequent regenerative signs, high transaminases and detectable HP. Rats recovered after 7 days in a similar fashion as with DMNA. At 400 mg thioacetamide kg-1 body weight, necrotic areas and regeneration zones were more widespread and transaminases and HP higher after 48 h than with the lower dose. On account of the differing modes of action of DMNA and thioacetamide in rat liver, it is proposed that the appearance of HP activity in plasma could be related to the regenerative process that follows hepatotoxic damage.

Topics: Acetamides; Animals; Dimethylnitrosamine; DNA; Liver; Liver Regeneration; Necrosis; Rats; Rats, Inbred Strains; Thioacetamide; Transaminases

Thioacetamide, given intraperitoneally (1.4 mmol/kg body mass) to male Wistar rats 24 h before sacrifice promoted a marked elevation of serum aminotransferases, loss of microsomal cytochrome P-450 content and a significant reduction (about 50%) of the liver plasma membrane enzymatic activities (5'-nucleotidase; K+, Na+- and Mg2+-adenosine triphosphatases; and gamma-glutamyl transferase). Previous starvation for 48 h, immediately prior to thioacetamide administration, strongly potentiated the effects of thioacetamide on the serum, microsomal and liver plasma membrane parameters, while fasting itself did not affect them. The liver plasma membrane damage may be one of the reasons for the cell death in thioacetamide-intoxicated rat livers.

Topics: Acetamides; Animals; Cell Membrane; Cytochrome P-450 Enzyme System; Fasting; Liver; Male; Microsomes, Liver; Necrosis; Rats; Rats, Inbred Strains; Thioacetamide

Carbon tetrachloride, chloroform, dimethylnitrosamine, thioacetamide or acetaminophen was each administered to rats in a single hepatotoxic dose. Nifedipine, verapamil or chlorpromazine was administered in association with the hepatotoxic agents to determine if calcium channel blocking agents would prevent an increase in liver cell calcium associated with hepatotoxicity and to determine if these agents would protect against the development of centrilobular necrosis. Following a latent period different for each toxic agent, a 4- to 18-fold increase in liver cell calcium content had occurred by 24 hr. The calcium increase and the centrilobular necrosis (mean histologic score) were correlated. A relatively high calcium to necrosis ratio was obtained with dimethylnitrosamine, thioacetamide and acetaminophen. A lesser calcium to necrosis ratio was obtained with chloroform and carbon tetrachloride, the two toxic agents that destroyed the intracellular calcium sequestration activity of the liver endoplasmic reticulum. Nifedipine or chlorpromazine, administered prior to and 7 hr after the toxic agent, completely prevented the centrilobular necrosis caused by thioacetamide, carbon tetrachloride and acetaminophen; almost completely prevented necrosis with dimethylnitrosamine; and provided partial protection against chloroform toxicity. Two doses of verapamil provided partial protection against necrosis when carbon tetrachloride was the toxic agent and provided almost complete protection with dimethylnitrosamine. A reduction in liver cell calcium was associated with the protective action of the three calcium channel blocking agents. These findings are compared with earlier studies of the protective effects of calcium channel blocking agents in cardiac ischemia.

Topics: Acetaminophen; Animals; Calcium; Calcium Channel Blockers; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Chloroform; Dimethylnitrosamine; Liver; Male; Microsomes, Liver; Necrosis; Rats; Rats, Inbred Strains; Thioacetamide

To evaluate the different contributions of either microsomal FAD-containing ( FADM ) or cytochrome P-450 dependent monooxygenases in the bioactivation and liver toxicity of thioacetamide-S-oxide ( TASO ) (a proximate metabolite of the liver toxin and carcinogen thioacetamide), this compound: (i) was given to rats pretreated with methimazole (a substrate and inhibitor of FADM ), SKF 525-A (an inhibitor of cytochrome P-450) and cobalt protoporphyrin IX (a synthetic porphyrin which induces a long-lasting depletion of the hepatic cytochrome P-450); and (ii) was added to liver microsomes performing oxidation of model FADM or cytochrome P-450 substrates. Whereas the prior administration of methimazole alleviated the TASO induced liver necrosis, SKF 525-A was almost ineffective. Also pretreatment with cobalt protoporphyrin IX prevented liver necrosis. However, this porphyrin derivative was found to depress both cytochrome P-450 dependent and the FADM dependent biotransformations. On the other hand, addition of TASO to liver microsomes in vitro induced changes in the kinetics of S-oxidation of thiobenzamide and of N-oxidation of dimethylaniline, whereas the O-deethylation of ethoxycoumarin was unchanged. The overall results show the necessity of TASO bioactivation by mixed-function monooxygenases for the toxic action to be apparent; at the same time, the findings suggest FADM as the system mainly involved in TASO metabolism.

Topics: Acetamides; Animals; Biotransformation; Cytochrome P-450 Enzyme System; Liver; Male; Microsomes, Liver; Necrosis; Oxygenases; Proadifen; Rats; Rats, Inbred Strains; Thioacetamide

Prior administration of chlorpromazine (CPZ), imipramine (IMP), mercaptoethylamine (MEA), 1-(1-naphthyl)2-thiourea (ANTU) or phenyl-thiocarbamide (PTC) but not 1,4-dithio-1-threitol (DTT), was able effectively to prevent most of thioacetamide (TAC) -induced liver necrosis. These and previous observations suggest that liver microsomal flavin-containing monooxygenase critically controls the process of activation of TAC to the ultimate necrogen.

Topics: Acetamides; Animals; Biotransformation; Flavins; Liver; Male; Microsomes, Liver; Necrosis; Rats; Rats, Inbred Strains; Thioacetamide

Phenylmethylsulfonyl fluoride (PMSF) administration to rats, was effective in partially preventing liver necrosis induced by thioacetamide, dimethylnitrosamine or galactosamine, when given 6 hr after the hepatotoxins. In the case of galactosamine but not of the other necrogenic chemicals, protection was also observed when PMSF was given 10 hr after this compound. These results and previous studies from our laboratory suggest participation of protein degradation at late stages of liver injury by these chemicals.

Topics: Acetamides; Animals; Dimethylnitrosamine; Drug Evaluation, Preclinical; Galactosamine; Liver; Male; Necrosis; Phenylmethylsulfonyl Fluoride; Rats; Rats, Inbred Strains; Sulfones; Thioacetamide; Time Factors

Prior administration of aminoacetonitrile (AAN) or imidazole but not isoxazole to rats, was able partially to prevent thioacetamide (TAC)-induced liver necrosis at 24 h. AAN and isoxazole did not prolong the pentobarbital sleeping time of the rats, while imidazole did. These and previous observations suggest a possible participation of non-cytochrome P-450 (P-450)-dependent aminoxidases in TAC activation to a necrogenic metabolite.

Topics: Acetamides; Acetonitriles; Aminoacetonitrile; Animals; Chemical and Drug Induced Liver Injury; Imidazoles; Male; Necrosis; Pentobarbital; Rats; Rats, Inbred Strains; Sleep; Thioacetamide; Time Factors

Our previous studies showed that polybrominated biphenyl (PBB) induced hepatic microsomal cytochrome P-450 in dairy cattle but did not elevate hepatic cytosolic ornithine decarboxylase or serum isocitrate dehydrogenase. These enzymes would be expected to increase during hepatotoxic injury and regeneration. Thus, PBB appeared to be a hepatotoxin in rats but not in cattle. In order to identify and confirm the response capability of bovine liver to hepatotoxins, we administered thioacetamide, a hepatotoxin known to induce hepatonecrosis, to a dairy calf. A progression of clinical signs of toxicosis was evident until the animal was moribund by 23 hr postdosing. Histolopathologic alterations in the liver included centrilobular necrosis with congestion and subcapsular microhemmorrhage. Marked changes in serum protein profiles were not noted. However, distinct increases in serum Fe and bilirubin occurred with progressing toxicosis, as did sharp declines in glucose and triglycerides. Serum lactic dehydrogenase, alkaline phosphatase, glutamic-oxaloacetic transaminase, isocitrate dehydrogenase and glutamic-pyruvate transaminase were elevated. Elevation of ornithine decarboxylase was dramatic when compared to the level in normal fetal bovine liver. From studies of its kinetic properties, bovine liver ornithine decarboxylase appears to have an apparent Km for ornithine decarboxylase of .45 mM. Liver homogenates from PBB-treated animals did not form inhibitors to ornithine decarboxylase. Compared with the thioacetamide-treated calf, the normal adult bovine, pregnant adult and 6-month fetus had relative activities of .2 .4 and 5.8%, respectively. These studies show that ornithine decarboxylase is low in liver of normal cattle, but is elevated markedly by agents that cause hepatonecrosis.

Topics: Acetamides; Animals; Blood Glucose; Blood Proteins; Carboxy-Lyases; Cattle; Cattle Diseases; Chemical and Drug Induced Liver Injury; Isocitrate Dehydrogenase; Kidney; Liver; Liver Diseases; Male; Necrosis; Ornithine Decarboxylase; Thioacetamide

Several inhibitors of cytochrome P-450 mediated oxidative transformations, ethyl 2-diethylaminoethyl-2-phenyl-2-ethylmalonate, ethyl-2-diethyl-aminoethyl-2-ethyl-2-buthylmalonate, 2,4 dichloro-6-phenoxyethyl diethylamine, 2-diethylaminoethyl-2-phenyl- (2-propene)-4-penten-1-oate or 3-amino,1,2,4 triazole were not able to significantly prevent thiocetamide induced necrosis at 24 h as evidenced by isocitric acid dehydrogenase activity or histologically. In contrast, several other sulfur containing compounds, tetraethyl thiuramidisulfide, diethyldithiocarbamic acid, thiourea or 1-methyl-2-mercaptoimidazole, which are inhibitors of non-cytochrome P-450 dependent amine oxidase systems, significantly prevented thioacetamide induced liver necrosis at 24 h. Notwithstanding, diphenhydramine, nicotinamide, trimethylamine and imipramine, which are substrates of this amino oxidase system, do not protect. All the chemicals tested prolonged the pentobarbital sleeping time, but there is no correlation between the intensity of this effect and their ability for preventing thioacetamide liver necrosis. These observations suggest that cytochrome P-450 does not play a major role in the activation of thioacetamide to a proximal or an ultimate necrogenic metabolite. Other microsomal enzymes metabolizing sulfur compounds could be involved in the major activation process.

Topics: Acetamides; Animals; Chemical and Drug Induced Liver Injury; Cytochrome P-450 Enzyme System; Liver; Liver Diseases; Male; Necrosis; Pentobarbital; Rats; Thioacetamide

The results of an electron microscopic study of the changes in hepatocytes induced by chronic intoxication with thioacetamide are reported. During the poisoning aspecific toxic changes are intermingled with progressive, preneoplastic ones. The main cell subpopulations identified are: 1) large hepatocytes with smooth endoplasmic reticulum (SER) hypertrophy, with or without rough endoplasmic reticulum (RER) neoformation and glycogen storage, which is starvation resistant; 2) smaller hepatocytes, where RER hypertrophy and ribosome accumulation are the prominent features. Such a pattern persists for months. After the withdrawal of the drug most of the cell changes disappear. However, during this time a simplification of the liver structure and cell composition takes place, allowing a sequence of cell events which seem relevant for establishment of neoplastic progression. The SER-hypertrophied cell appears first and gives rise, via several intermediate stages, to the RER-hypertrophied one, which is believed to play a key role as the ultimate precursor of cancer cells.

Topics: Acetamides; Animals; Cell Transformation, Neoplastic; Endoplasmic Reticulum; Female; Hypertrophy; Liver; Liver Neoplasms; Microscopy, Electron; Necrosis; Precancerous Conditions; Rats; Thioacetamide; Time Factors

Using Mancini's single radial immunodiffusion technique, the excretion of a liver specific saline soluble antigen was semiquantitatively assayed in the urine of rats with thioacetamine induced hepatic necrosis. The relative amounts of antigen detected in the urine were found to roughly correlate with the extent of necrotic liver parenchyma. It is suggested that a quantitative approach to histuria may provide clinically relevant information on the activity of pathologic processes associated with release of organ specific antigens from degenerative and necrotic lesions into the circulation (histemia) and thence into the urine.

Topics: Animals; Antigens; Chemical and Drug Induced Liver Injury; Dose-Response Relationship, Drug; Immunodiffusion; Liver; Male; Necrosis; Organ Specificity; Rats; Thioacetamide; Time Factors

Topics: Acetamides; Animals; Chemical and Drug Induced Liver Injury; Kinetics; Liver; Male; Necrosis; Oxidation-Reduction; Rats; Sulfates; Thioacetamide; Tissue Distribution

Metabolic activation of thioacetamide (CH3CSNH2) to a toxic metabolite which is responsible for its hepatotoxicity and/or its carcinogenicity has been proposed by a number of investigators. In this investigation thioacetamide and one of its metabolites, thioacetamide sulfine (CH3CSONH2), have been compared for their ability to inhibit hepatic mixed-function oxidase enzymes as well as their ability to induce hepatic necrosis. Thioacetamide sulfine was found to decrease aminopyrine N-demethylation and aniline hydroxylation at a lower dose and at an earlier time after administration than was the case with thioacetamide. In addition, at all doses examined, thioacetamide sulfine produced a more severe centrilobular hepatic necrosis than equivalent doses of thioacetamide. To determine whether the hepatic mixed-function oxidase enzyme system was involved in the biotransformation of thioacetamide and/or thioacetamide sulfine to a hepatotoxic compound(s), the severity of liver damage was examined after the administration of an inducer or inhibitors of hepatic mixed-function oxidase enzyme activity. Phenobarbital pretreatment potentiated the hepatic necrosis produced by both thioacetamide and thioacetamide sulfine. In contrast, pyrazole, SKF 525-A, and cobaltous chloride protected against the hepatic necrosis caused by thioacetamide and thioacetamide sulfine. These data suggest that both thioacetamide and thioacetamide sulfine are activated by hepatic mixed-function oxidase enzymes to a hepatotoxic compound(s). These data also suggest that the hepatotoxicity may be mediated by its metabolism to thioacetamide sulfine which, in turn, is metabolized to an ultimate toxic metabolite.

Topics: Acetamides; Aminopyrine; Aniline Compounds; Animals; Chemical and Drug Induced Liver Injury; Drug Interactions; Liver; Liver Diseases; Male; Mixed Function Oxygenases; Necrosis; Oxidoreductases; Phenobarbital; Rats; Sulfonium Compounds; Thioacetamide; Time Factors

The temporal sequence of alpha-fetoprotein appearance in serum was determined in both necrogenic and nonnecrogenic liver injury. Ethionine, thioacetamide, and CCl4 were used to intoxicate male and female rats for evaluating serum enzyme levels, mitotic indices, and morphological reflections of impairment. Thioacetamide- and CCl4-induced cell death preceded the mitotic wave in residual hepatocytes, and, in the case of both agents, this intoxicant-mediated necrosis preceded the emergence of alpha-fetoprotein. Yet, although there was no evidence of either cell destruction or significant mitotic activity in ethionine-poisoned animals, serum alpha-fetoprotein levels progressively increased. Thus the temporal sequence of alpha-fetoprotein synthesis and/or release and cellular reorganization for regeneration suggests that reappearance of the protein macro-molecule is an expression of the altered phenotype observed during the "step-down" phase of liver regeneration.

Topics: Alanine Transaminase; alpha-Fetoproteins; Animals; Aspartate Aminotransferases; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Ethionine; Female; Liver Diseases; Liver Regeneration; Male; Mitosis; Necrosis; Rats; Thioacetamide; Time Factors

Topics: Acetamides; Animals; Cats; Chemical and Drug Induced Liver Injury; Liver; Models, Biological; Necrosis; Thioacetamide

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Body Temperature; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Female; Heparinoids; Liver; Liver Diseases; Male; Necrosis; Rats; Thioacetamide

Normal and adrenalectomized rats were given a single oral dose of thioacetamide (5, 10 or 20 mg/100 g body weight). The size, weight and histology of the thymus were observed for 3 weeks following intoxication. An initial rapid loss of thymic weight and size occurred during the first 3 days of intoxication; there was no significant recovery. This loss was associated with decreased cortical mass without significant change in medullary size or histology. The decrease of the cortex was associated with significant cortical cell death and the formation of a "starry sky" pattern. This response occurred in both adrenalectomized and nonadrenalectomized rats, suggesting that this phenomenon is not the adrenal-mediated stress response. Measurement of circulating mononuclear cells indicated that thymocyte release did not play a significant role in depletion changes. The basis for this prompt, at least temporarily irreversible, chemically induced thymic atrophy is not apparent.

Topics: Acetamides; Administration, Oral; Adrenalectomy; Animals; Body Weight; Hematocrit; Leukocyte Count; Male; Necrosis; Organ Size; Rats; T-Lymphocytes; Thioacetamide; Thymus Gland

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Cholestasis; Cholinesterases; Clinical Enzyme Tests; Diagnosis, Differential; Ethionine; Fatty Liver; Fructose-Bisphosphate Aldolase; Fructosephosphates; Necrosis; Phenobarbital; Proadifen; Rats; Thioacetamide

Topics: Amides; Bilirubin; Blood Chemical Analysis; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Hepatitis; Hepatitis A; Isocitrate Dehydrogenase; Necrosis; Ouabain; Pathology; Pharmacology; Promethazine; Pyruvates; Rats; Research; Sulfhydryl Compounds; Thioacetamide; Toxicology; Transaminases

Topics: Acetonitriles; Amides; Benzene; Bromobenzenes; Cyanides; Liver; Liver Diseases; Necrosis; Tannins; Thioacetamide