Single dose intravenous thioacetamide administration as a model of acute liver damage in rats

Thioacetamide (TAA) has been used extensively in the development of animal models of acute liver injury. Frequently, TAA is administered intraperitoneally to induce liver damage under anaesthesia. However, it is rarely administered by intravenous injection in conscious rats. The experiments in this study were designed to induce acute liver damage by single intravenous injection of TAA (0, 70 and 280 mg/kg) in unrestrained rats. Biochemical parameters and cytokines measured during the 60-h period following TAA administration, included white blood cells (WBC), haemoglobulin (Hb), platelet, aspartate transferase (GOT), alanine transferase (GPT), total bilirubin (TBIL), direct bilirubin (DBI), albumin, ammonia (NH3), r-glutamyl transpeptidase (r-GT), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Rats were sacrificed by decapitation 60 h after TAA administration and livers were removed immediately for pathology and immunohistochemical (IHC) examination. Another group of rats were sacrificed by decapitation 1, 6 and 24 h after TAA administration and livers were removed immediately for time course change of pathology and IHC examination. TAA significantly increased blood WBC, GOT, GPT, TBIL, DBIL, NH3, r-GT, TNF-α and IL-6 levels but decreased the blood Hb, platelet and albumin level. The levels of histopathological damage in the liver after intravenous TAA administration were also increased with a dose-dependent trend and more increased at 60 h after TAA administration. The levels of inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) detected by IHC in the liver after intravenous TAA administration were also increased with a dose-dependent trend and more increased at 1 h after TAA administration. Single intravenous TAA administration without anaesthesia is a restorable animal model which may be used to investigate acute liver damage.     

Apoptosis, oncosis, and necrosis. An overview of cell death.

The historical development of the cell death concept is reviewed, with special attention to the origin of the terms necrosis, coagulation necrosis, autolysis, physiological cell death, programmed cell death, chromatolysis (the first name of apoptosis in 1914), karyorhexis, karyolysis, and cell suicide, of which there are three forms: by lysosomes, by free radicals, and by a genetic mechanism (apoptosis). Some of the typical features of apoptosis are discussed, such as budding (as opposed to blebbing and zeiosis) and the inflammatory response. For cell death not by apoptosis the most satisfactory term is accidental cell death. Necrosis is commonly used but it is not appropriate, because it does not indicate a form of cell death but refers to changes secondary to cell death by any mechanism, including apoptosis. Abundant data are available on one form of accidental cell death, namely ischemic cell death, which can be considered an entity of its own, caused by failure of the ionic pumps of the plasma membrane. Because ischemic cell death (in known models) is accompanied by swelling, the name oncosis is proposed for this condition. The term oncosis (derived from ónkos, meaning swelling) was proposed in 1910 by von Reckling-hausen precisely to mean cell death with swelling. Oncosis leads to necrosis with karyolysis and stands in contrast to apoptosis, which leads to necrosis with karyorhexis and cell shrinkage.     

The hippocampal region of rats and mice after a single i.p. dose of clioquinol: loss of synaptic zinc, cell death and c-Fos induction

Clioquinol (CQ) is able to chelate synaptic zinc, which can modulate excitatory and inhibitory neurotransmission. In humans, CQ was associated with cases of transient global amnesia (TGA) and with the neurodegenerative syndrome subacute myelo-optico-neuropathy (SMON). We examined the CQ induced loss of synaptic zinc, cell death and c-Fos induction in rats and mice. In rats, we found a strong reduction of histochemically reactive synaptic zinc no later than 4 h after the injection of the lowest dose of CQ (50 mg/kg) and, for all doses used, a return to control levels after 48 h. There was no evidence of cell death for any dose and up to 1 week after CQ injections. Only a slight induction of c-Fos was seen in the hippocampus for the higher doses used (100-200 mg/kg). In mice injected with 100 mg/kg, CQ also resulted in a fast loss of synaptic zinc. c-Fos was induced after 4 h in cell populations of the hippocampal region and other parts of the telencephalon, and substantially increased after 24 h. One day after the injection we found a pattern of cell loss (hilus, parts of CA3, CA1 and layer III of the medial entorhinal cortex) reminiscent of that seen in models of temporal lobe epilepsy. In conjunction with published data on the behavioral effects of zinc chelation and the modulatory effects of zinc in excitatory neurotransmission, our results indicate that the loss of synaptic zinc may have been involved in TGA and the neuropathology associated with SMON.     

Correlation between liver cell necrosis and circulating alanine aminotransferase after ischaemia/reperfusion injuries in the rat liver

Circulating liver enzymes such as alanine transaminase are often used as markers of hepatocellular damage. Ischaemia/reperfusion (I/R) injury is an inevitable consequence of prolonged liver ischaemia. The aim of this study was to examine the correlation between liver enzymes and volume of liver cell necrosis after ischaemia/reperfusion injuries, using design‐unbiased stereological methods. Forty‐seven male Wistar rats were subjected to 1 h of partial liver ischaemia, followed by either 4 or 24 h of reperfusion. Within each group, one‐third of animals were subjected to ischaemic preconditioning and one‐third to ischaemic postconditioning. At the end of reperfusion, blood and liver samples were collected for analysis. The volume of necrotic liver tissue was subsequently correlated to circulating markers of I/R injury. Correlation between histological findings and circulating markers was performed using Pearson's correlation coefficient. Alanine transferase peaked after 4 h of reperfusion; however, at this time‐point, only mild necrosis was observed, with a Pearson's correlation coefficient of 0.663 (P = 0.001). After 24 h of reperfusion, alanine aminotransferase was found to be highly correlated to the degree of hepatocellular necrosis R = 0.836 (P = 0.000). Furthermore, alkaline phosphatase (R = 0.806) and α‐2‐macroglobulin (R = 0.655) levels were also correlated with the degree of necrosis. We show for the first time that there is a close correlation between the volume of hepatocellular necrosis and alanine aminotransferase levels in a model of I/R injury. This is especially apparent after 24 h of reperfusion. Similarly, increased levels of alkaline phosphatase and α‐2‐macroglobulin are correlated to the volume of liver necrosis.     

Histopathology of the rat liver following a single autoprotective dose of 3'-methyl-4-dimethylaminoazobenzene.

Male inbred Leeds strain rats were given single intragastric doses of 1200 mg/kg of 3'-methyl-4-dimethylaminoazobenzene, autoprotective against liver necrosis and then sacrificed at intervals up to 14 months. About 40 per cent of the animals died, apparently as a result of pancreatic necrosis, between 9 and 13 days after treatment while the remainder survived until they were killed. The liver changes induced included an early intense ductular cell reaction, presistent hepatocellular abnormalities and at later stages, cholangiofibrosis and increasing numbers of parenchymal clear cells. Although no tumours arose as a result of treatment, the observed histopathological changes are similar to those seen during liver carcinogenesis induced by a variety of carcinogens and may be due specifically to the carcinogenic action of the azo dye.     

细胞凋亡、胀亡和坏死--关于细胞死亡的新认识

一、一种不同于凋亡的细胞死亡方式———胀亡细胞死亡和细胞坏死代表的是两个完全不同的概念 ,这一点往往被人们忽视。近 2 0年来 ,人们习惯将凋亡(ａｐｏｐｔｏｓｉｓ)和坏死 (ｎｅｃｒｏｓｉｓ)进行比较 ,把凋亡和坏死看作为两种不同的细胞死亡方式。事实上 ,细胞死亡是生物学上表示细胞不可逆损伤的功能性定义 ,凋亡是细胞死亡方式之一 ,而坏死代表了活体内局部组织、细胞死亡后所发生的终末变化的总和 ,并不管其死亡前过程如何 ,是一形态学诊断。当今很多文献所提及的“坏死性细胞死亡”并非真正的坏死[1] ,实际上是另一种不同于凋亡的…     

Tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro.

Tumor necrosis factor (TNF) is cytotoxic for many tumoral cell lines, whereas normal cells generally are considered resistant to this action. This study shows that this cytokine causes massive death of bovine endothelial cells in primary culture in a concentration- and time-dependent manner. Dying cells exhibit all the ultrastructural changes and the inter-nucleosome cleavage of DNA associated with apoptosis or 'programmed cell death.' This is the first report clearly showing a direct toxicity of TNF on endothelial cells and demonstrating that this results from the induction of the program of apoptotic death. Our observation raises the possibility that hemorrhagic necrosis in vivo, after treatment with TNF, might involve a direct cytocidal action on endothelial cells of the tumor neovasculature.     

Early liver cell lesions in rats induced by thioacetamide. An ultrastructural, cytophotometric and autoradiographic study

A morphological, cytophotometrical and autoradiographical study was carried out on the early liver cell lesions present after one month of thioacetamide (TAA) exposure. We wanted to determine the extent of cell damage in the hepatocytes in relation to the later occurring cholangiocarcinoma. Cytoplasmic and nuclear alterations were present in the hepatocytes. They were mainly due to the toxic influence of the metabolites of TAA. No Feulgen-DNA changes have been observed in the hepatocytes in any of the studied zones. The increased TdR H3+ uptake and mitotic activity in the periportal and midzonal areas represented regenerative activity. In addition to these hepatocytic changes, the centrolobular area was infiltrated by oval cells. These cells appeared at first singly, later on they formed clusters. The electron microscopy of these cells revealed phenotypic characteristics, which were different from the hepatocytes. When separately, these cells resembled undifferentiated cells with cytoplasmic extensions and absent basement membrane. When arranged in clusters, a definite canalicular arrangement was present with characteristics of bile canalicular cells with microvillous extensions at the apical border of the cytoplasm and the presence of a basement membrane. A transition from the first oval cell type to the second oval cell type was suggested. This transition might represent a differentiation process of cells, which are regarded as the target cell and the precursor cell in the development of the cholangiocarcinoma. This is the first study reporting oval cell proliferation in the centrolobular area in a multistep model of livercarcinogenesis in rats.     

The sequential analysis of liver cell necrosis: inhibition of diethylnitrosamine- and dimethylnitrosamine-induced acute liver cell death by posttreatment with diethyldithiocarbamate.

Posttreatment with diethyldithiocarbamate (DEDTC) largely prevented the development of acute hepatocellular necrosis induced by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in male Fischer rats as monitored by the release of glutamate-pyruvate transaminase and sorbitol dehydrogenase into the serum and by histologic examination. Liver cell necrosis was evident with a dose of 25 mg of DEN/kg and was progressive with increasing doses of DEN. DEDTC (50 mg/kg; three times at 4-hour intervals) was given at 4 or 8 hours after the administration of DEN (100 mg/kg), time points at which at least 50% and 75%, respectively, of the administered DEN had disappeared from both the serum and liver. Under these conditions, DEDTC prevented liver cell necrosis, except for a few isolated cells. Similar inhibition was also observed when DEDTC was given 4 hours after the administration of a necrogenic dose of DMN (20 mg/kg). DEDTC, when administered 4 hours after DEN, delayed the rate of clearance of DEN and of ethylation of DNA and RNA but did not significantly affect the total extent of ethylation of rat liver nucleic acids. These results offer further support for the multistep hypothesis for the development of liver cell necrosis.     

Microtubule antagonists activate programmed cell death (apoptosis) in cultured rat hepatocytes.

We investigated the mechanism of lethal injury following the disruption of microtubules in cultured hepatocytes treated with vinblastine (VBL) or colchicine (COL). These agents kill hepatocytes by a process readily distinguished from two well-known pathways that lead to a loss of viability, namely, oxidative stress and inhibition of mitochondrial electron transport. Cell killing with VBL and COL was accompanied by fragmentation of DNA. Both the loss of viability and the fragmentation of DNA were prevented by the inhibition of protein synthesis within 6 hours following exposure to VBL or COL. Cell death and the fragmentation of DNA were also prevented when Ca2+ was removed from the culture medium. By contrast, the inhibition of protein kinase C prevented cell killing by VBL or COL, but did not alter the extent of DNA fragmentation. The requirements here for protein synthesis, extracellular Ca2+, and protein kinase C activity define a model of apoptosis, or programmed cell death, that seems to involve mechanisms that can be dissociated from the fragmentation of DNA.     

Induction of neutrophil death resembling neither apoptosis nor necrosis by ONO-AE-248, a selective agonist for PGE2 receptor subtype 3

An increase of intracellular cAMP mediated by prostaglandin E2 (PGE2) has been shown to delay spontaneous apoptosis of neutrophils. It has been demonstrated that a selective agonist for PGE2 receptor subtype 3 (the EP3 receptor) is capable of decreasing cAMP and stimulating phosphoinositide turnover in various types of cells. We investigated the effect of a selective EP3 receptor agonist, ONO-AE-248, on neutrophil viability. ONO-AE-248 rapidly caused a unique form of neutrophil death. The agonist primarily induced morphological changes of the nucleus, including fusion of the lobules, decreased compactness of the chromatin, and blebbing and rupture of the nuclear membrane. This was followed by an increase of plasma membrane permeability and cell lysis. During these processes, neither apoptotic changes such as nuclear condensation, DNA fragmentation, and expression of phospholipid phosphatidylserine on the plasma membrane nor necrotic changes such as chromatin clumping and organelle destruction were apparent in the treated neutrophils. The fatal effect of the agonist night be specific for neutrophils because it failed to promote the rapid death of other types of cells. Although activation of neutrophils by ONO-AE-248 was not evident, experiments using metabolic inhibitors demonstrated that the agonist caused neutrophil death via the activation of protein kinase C in the presence of intracellular ATP. These findings indicated that EP3 receptor-mediated signals might promote a novel form of neutrophil death, which differs from typical apoptosis or necrosis.     

Occurrence of cell death (apoptosis) in preneoplastic and neoplastic liver cells. A sequential study.

A sequential study was performed to investigate the occurrence of cell death in preneoplastic and neoplastic liver cells of F-344 rats. The animals were administered a single initiator dose of 1,2-dimethylhydrazine and were then subjected to a liver carcinogenesis promotion regimen, consisting of a diet containing 1% orotic acid. Cell death, morphologically similar to that described as apoptosis, was evident in foci of preneoplastic hepatocytes at 10 weeks after orotic acid feeding. An increased frequency of apoptotic bodies was observed in nodules, but not in the surrounding liver, 20 weeks after starting the dietary regimen, and in hepatocellular carcinomas that developed after 1 year of continuous promotion. Occurrence of this type of cell death was also observed in liver foci of rats subjected to two other promoting regimens, suggesting, thus, a possible relevance of apoptosis to the carcinogenic process in the liver.     

Involvement of multiple cell cycle aberrations in early preneoplastic liver cell lesions by tumor promotion with thioacetamide in a two-stage rat hepatocarcinogenesis model

Thioacetamide (TAA) induces oxidative stress and hepatocarcinogenicity in rats. We previously reported that TAA promotion caused various disruptions in cell cycle protein expression in rats, including downregulation of p16^Ink4a, which is associated with intraexonic hypermethylation in hepatocellular proliferative lesions. This study further investigated the contribution of cell cycle aberrations associated with early hepatocarcinogenic processes induced by TAA using antioxidants, enzymatically modified isoquercitrin (EMIQ) and α-lipoic acid (ALA), in a two-stage rat hepatocarcinogenesis model. TAA-promotion after initiation with N-diethylnitrosamine increased the number and area of hepatocellular foci immunoreactive for glutathione S-transferase placental form (GST-P) and the numbers of proliferating and apoptotic cells. Co-treatment with EMIQ and ALA suppressed these increases. TAA-induced formation of p16^Ink4a? foci in concordance with GST-P⁺ foci was not suppressed by co-treatment with EMIQ or ALA. TAA-promotion increased cellular distributions of cell proliferation marker Ki-67, G₂/M and spindle checkpoint proteins (phosphorylated checkpoint kinase 1 and Mad2), the DNA damage-related protein phosphorylated histone H2AX, and G₂-M phase-related proteins (topoisomerase IIα, phosphorylated histone H3 and Cdc2) within GST-P⁺ foci, and co-treatment with EMIQ or ALA suppressed these increases. These results suggest that downregulation of p16^Ink4a may allow selective proliferation of preneoplastic cells by TAA promotion. However, antioxidants did not counteract this gene control. Moreover, effective suppression of TAA-induced cellular population changes within preneoplastic lesions by antioxidants may reflect facilitation of cell cycling and accumulation of DNA damage causing the activation of cell cycle checkpoints, leading to G₂ and M phase arrest at the early stages of hepatocarcinogenesis promoted by TAA.     

Induction of apoptotic cell death specifically in rat and human cancer cells by pancratistatin

The major challenge in the battle against cancer is the specific targeting of cancer cells. Most chemotherapeutics and radiotherapies induce cancer cell death by inducing DNA damage. These treatments also cause severe side effects by affecting normal cells causing toxicity and mutations that may predispose them to become cancerous. Some non-genotoxic drugs such as tamoxifen are useful but are of limited applicability. Natural compounds such as paclitaxel have been useful in cancer treatment, but due to its effect as a general microtubule stabilizer and genotoxic agent, it also induces death of normal cells. Pancratistatin is a natural compound isolated from Pancratium littorale that has been shown to have anti-viral and anti-neoplastic activity. The objective in the present study was to elucidate the mechanism of the anti-neoplastic action of pancratistatin and evaluate the specificity of this compound for cancer cells. METHODS: We used cancer cell lines and normal human endothelial and fibroblast cells to investigate the effect of pancratistatin treatment. Further, we compared the toxic effects of paclitaxel and VP-16 to that of pancratistatin on non-cancerous cells. RESULTS: Pancratistatin induced apoptosis in all the cancer cell lines used in this study at sub-micromolar concentrations. Interestingly, normal human fibroblasts and endothelial cells remained unaffected by pancratistatin treatment under identical conditions whereas paclitaxel and VP-16 were both toxic to these two normal cell lines. CONCLUSION: The capability of pancratistatin to selectively induce apoptosis in cancer cells is an exciting finding and makes it a suitable anti-cancer agent. Since pancratistatin shows little structural similarity to any DNA intercalating drug or to paclitaxel derivatives, it appears to be non-genotoxic. Additionally, due to the unprecedented differential cytotoxicity observed in cancerous cells, we believe pancratistatin may act upon a novel target, allowing selective induction of apoptosis in cancer cells.     

Sequential observation of liver cell regeneration after massive hepatic necrosis in auxiliary partial orthotopic liver transplantation.

The morphogenesis of hepatocytes after massive hepatic necrosis to recovery through liver cell regeneration has not been fully understood. Sequential biopsies were performed on the native liver of a 22-year-old man who underwent auxiliary partial orthotopic liver transplantation 1 month after fulminant hepatitis. Auxiliary partial orthotopic liver transplantation was successful, and the biopsy samples permitted us to examine the regenerating process of hepatocytes after massive necrosis. At the time of auxiliary partial orthotopic liver transplantation (postoperative day 0), 95% of hepatocytes were lost and a few ductules were found in the portal areas. The ductules stained with cytokeratin 19. At postoperative day 7, the ductules began to increase in size and number and became dilated over a period of 1 month, when individual hepatocytes with clear cytoplasm appeared from the ductules. As the differentiation of hepatocytes increased, the expression of cytokeratin 19 was found to decrease. From 2 to 3 months, all of the ductules were transformed into hepatocytes, and they began to form round cell clusters. From 3 to 6 months, the round cell clusters became organized into trabecula with fibrosis. From 6 to 12 months, a lobular architecture was established, and by 14 months, the necrotic liver was fully recovered to normal. This study by examination of sequential biopsies demonstrates the progression of the regenerating process from total hepatic necrosis to complete recovery.