Apoptotic Cell Death Of Hepatocytes Research Articles

Apoptotic Cell Death in an Animal Model of Virus-Induced Acute Liver Failure-Observations during Lagovirus europaeus/GI.2 Infection.

Lagovirus europaeus/GI.2 causes severe and highly fatal Rabbit Hemorrhagic Disease (RHD). Because of its characteristics, this infection is used as an animal model for acute liver failure (ALF). Apoptosis is one of the key processes underlying ALF and has been described as one of the mechanisms of RHD pathogenesis. Apoptotic cell death has been quite well characterized in infection with different variants of GI.1 strains, but so far, the GI.2 genotype has not been widely studied. In this study, we performed an evaluation of apoptotic cell death in hepatocytes of rabbits infected with Lagovirus europaeus/GI.2. We analyzed the expression of genes involved in apoptotic cell death by real-time PCR and performed immunohistochemical (IHC) assays. We showed a significant increase in the expression of caspase-3 and the proapoptotic Bax and anti-apoptotic Bcl-2 in infected animals. In addition, we recorded increased Bax/Bcl-2 ratios. IHC analyses showed the presence of morphological signs of apoptosis in the hepatocytes of infected rabbits. Our results indicate that caspase-3 and proteins from the Bcl-2 families play a key role in apoptosis induced by Lagovirus europaeus/GI.2 infection.

Autophagic degradation of IRF3 induced by the small-molecule auranofin inhibits its transcriptional and proapoptotic activities

The ubiquitously expressed transcription factor interferon (IFN) regulatory factor 3 (IRF3) is critical for the induction of antiviral genes, e.g., type-I IFN. In addition to its transcriptional function, IRF3 also activates a nontranscriptional, proapoptotic signaling pathway. While the proapoptotic function of IRF3 protects against viral infections, it is also involved in harmful immune responses that trigger hepatocyte cell death and promote liver disease. Thus, we hypothesized that a small-molecule inhibitor of the proapoptotic activity of IRF3 could alleviate fatty-acid-induced hepatocyte cell death. We conducted a high-throughput screen, which identified auranofin as a small-molecule inhibitor of the proapoptotic activity of IRF3. In addition to the nontranscriptional apoptotic pathway, auranofin also inhibited the transcriptional activity of IRF3. Using biochemical and genetic tools in human and mouse cells, we uncovered a novel mechanism of action for auranofin, in which it induces cellular autophagy to degrade IRF3 protein, thereby suppressing IRF3 functions. Autophagy-deficient cells were unable to degrade IRF3 upon auranofin treatment, suggesting that the autophagic degradation of IRF3 is a novel approach to regulate IRF3 activities. Using a physiologically relevant in vitro model, we demonstrated that auranofin inhibited fatty-acid-induced apoptotic cell death of hepatocytes. In summary, auranofin is a novel inhibitor of IRF3 functions and may represent a potential therapeutic option in diseases where IRF3 is deleterious.

Apoptosis of Hepatocytes: Relevance for HIV-Infected Patients under Treatment.

Due to medical advances over the past few decades, human immunodeficiency virus (HIV) infection, once a devastatingly mortal pandemic, has become a manageable chronic condition. However, available antiretroviral treatments (cART) cannot fully restore immune health and, consequently, a number of inflammation-associated and/or immunodeficiency complications have manifested themselves in treated HIV-infected patients. Among these chronic, non-AIDS (acquired immune deficiency syndrome)-related conditions, liver disease is one of the deadliest, proving to be fatal for 15–17% of these individuals. Aside from the presence of liver-related comorbidities, including metabolic disturbances and co-infections, HIV itself and the adverse effects of cART are the main factors that contribute to hepatic cell injury, inflammation, and fibrosis. Among the molecular mechanisms that are activated in the liver during HIV infection, apoptotic cell death of hepatocytes stands out as a key pathogenic player. In this review, we will discuss the evidence and potential mechanisms involved in the apoptosis of hepatocytes induced by HIV, HIV-encoded proteins, or cART. Some antiretroviral drugs, especially the older generation, can induce apoptosis of hepatic cells, which occurs through a variety of mechanisms, such as mitochondrial dysfunction, increased production of reactive oxygen species (ROS), and induction of endoplasmic reticulum (ER) stress and unfolded protein response (UPR), all of which ultimately lead to caspase activation and cell death.

The importance of liver lesions and changes to biochemical and coagulation factors in the pathogenesis of RHD.

RHDV (rabbit haemorrhagic disease virus) is an etiologic factor of RHD (rabbit haemorrhagic disease), which is highly morbid and mortal viral infection of an adult European rabbit. Although three decades have passed since the first outbreak of rabbit haemorrhagic disease, the pathogenesis of RHD has still not been fully elucidated. It is known that RHDV replicates in the liver within the first hours following infection, causing necrotic and apoptotic cell death of hepatocytes. Anatomopathological changes are also observed in other organs of infected rabbits, i.e. lungs, spleen, kidneys, heart, as well as central nerve system. These changes leading to animals death are predominantly caused by systemic hemorrhagic diathesis with disseminated intravascular coagulation (DIC), appearing most likely as a consequence of liver cell loss through RHDV-induced apoptosis. In this paper, we presented previously described changes in biochemical and coagulation factors in RHDV infection.

Ochratoxin A causes oxidative stress and cell death in rat liver

The effect of ochratoxin A (OTA) on oxidant/antioxidant status and on histopathological changes and apoptotic cell death in livers of male Sprague-Dawley rats has been investigated. OTA (0.5 mg/kg body weight/day) was administered by oral route for 14 days. Plasma biochemical parameters, activities of liver selenoenzymes (glutathione peroxidase-1, thioredoxin reductase) and antioxidant enzymes (catalase, superoxide dismutase, glutathione S-transferase), and levels of total glutathione and thiobarbituric acid reactive substance in hepatic tissue were measured. In addition, histopathological examinations were performed and apoptotic cell death of hepatocytes was evaluated by the TdT-mediated dUTP nick-end labelling (TUNEL) assay. OTA exposure was found to induce focal necrosis of hepatocytes and mononuclear cell infiltration. Besides, exposure to OTA caused an imbalance in oxidant and antioxidant parameters in the rat liver, as evidenced by significant decreases in glutathione S-transferase activity and glutathione levels, and marked increases in concentrations of thiobarbituric acid reactive substances. Furthermore, TUNEL analysis revealed a significant ~2.7-fold increase in the number of TUNEL-positive liver cells of rats exposed to OTA compared to the control group. The results of this study showed that oxidative stress is at least one of the mechanisms underlying the hepatic toxicity of OTA, and that both necrosis and apoptosis are types of cell death in the hepatic toxicity of this mycotoxin.

Local interaction of apoptotic hepatocytes and Kupffer cells in a rat model of systemic endotoxemia

There is strong evidence that hepatocellular apoptosis is not only initiated by circulating blood cells which become adherent within the endotoxemic liver, but also contributes to further sustain the inflammatory cell-cell response. Because previous studies assumed the importance of the role of cellular cross-talk in mediating inflammatory liver injury, we herein examined the activation of Kupffer cells (KCs) and their spatial coincidence with intrahepatic leukocyte adherence and hepatocellular apoptosis at 6 h after intraperitoneal exposure of rats with lipopolysaccharide (10 mg/kg). In vivo multifluorescence microscopy revealed liver injury including nutritive perfusion failure, tissue hypoxia, leukocyte accumulation, as well as KC activation and parenchymal apoptotic cell death. Detailed spatial analysis revealed frequent colocalization of activated KCs with apoptotic hepatocytes. Colocalization was absent in saline-treated controls.Colocalization was confirmed by histochemistry, which showed ED1-positive KCs neighboring and engulfing TUNEL-positive hepatocytes. Colocalization of KCs with leukocytes ranged between 4% and 5% and did not increase in endotoxemic animals. Taken together, the present results indicate that apoptotic cell death of hepatocytes may stimulate phagocytosis by neighboring KCs. Direct KC-leukocyte contact seems not to be mandatory for cellular communication in the process of hepatocellular apoptosis. With respect to the fundamental importance of cell apoptosis, improved knowledge of these cell-cell interactions might allow the development of new therapeutic strategies through the regulation of apoptotic cell death.

Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes

Diclofenac is a widely used nonsteroidal anti-inflammatory drug that has been associated with rare but serious hepatotoxicity. Experimental evidence indicates that diclofenac targets mitochondria and induces the permeability transition (mPT) which leads to apoptotic cell death in hepatocytes. While the downstream effector mechanisms have been well characterized, the more proximal pathways leading to the mPT are not known. The purpose of this study was to explore the role of free cytosolic calcium (Ca(2+)(c)) in diclofenac-induced cell injury in immortalized human hepatocytes. We show that exposure to diclofenac caused time- and concentration-dependent cell injury, which was prevented by the specific mPT inhibitor cyclosporin A (CsA, 5 microM). At 8 h, diclofenac caused increases in [Ca(2+)](c) (Fluo-4 fluorescence), which was unaffected by CsA. Combined exposure to diclofenac/BAPTA (Ca(2+) chelator) inhibited cell injury, indicating that Ca(2+) plays a critical role in precipitating mPT. Diclofenac decreased the mitochondrial membrane potential, DeltaPsi(m) (JC-1 fluorescence), even in the presence of CsA or BAPTA, indicating that mitochondrial depolarization was not a consequence of the mPT or elevated [Ca(2+)](c). The CYP2C9 inhibitor sulphaphenazole (10 microM) protected from diclofenac-induced cell injury and prevented increases in [Ca(2+)](c), while it had no effect on the dissipation of the DeltaPsi(m). Finally, diclofenac exposure greatly increased the mitochondria-selective superoxide levels secondary to the increases in [Ca(2+)](c). In conclusion, these data demonstrate that diclofenac has direct depolarizing effects on mitochondria which does not lead to cell injury, while CYP2C9-mediated bioactivation causes increases in [Ca(2+)](c), triggering the mPT and precipitating cell death.

Spontaneous tolerance: experience with the rat liver transplant model

Soluble major histocompatibility complex (MHC) class I antigens released from hepatocytes and the passenger leukocyte population of the liver allograft have both been considered as important contributors for spontaneous liver tolerance upon allogeneic transplantation into fully MHC-mismatched hosts. This study was conducted to delineate the role of “passenger leukocytes” (PL) as well as local intra-graft defence mechanisms of long-term accepted liver allografts in more detail. Orthotopic liver transplantation was performed in male inbred rats as follows ( n = 4–6): (i) Lewis (LEW; RT1. l) → LEW; (ii) DA (RT1. av1) → DA; (iii) DA → LEW; (iv) LEW → DA; (v) LEW (10-Gy whole body irridiation [WBI], d-7) → DA; (vi) LEW (10-Gy WBI, d-7) → LEW; (vii) LEW (10-Gy WBI, d-7) → LEW (parked for 36 hours) → DA; and (viii) LEW (10-Gy WBI, d-7) → DA (parked for 36 hours) → DA. The model specifically investigated the role of PLs as potential contributors to liver tolerance as WBI destroys this nonresident liver population. Characterization of Fas/FasL expression and the frequency of apoptotic cell death was performed by immunohistochemistry and TUNEL staining. Reverse transcriptase—polymerase chain reaction, by the use of Fas and FasL-specific cDNA probes, was performed on isolated hepatocytes of tolerized and rejected livers at various time points after transplantation. Animal survival in the various experimental groups was calculated using Kaplan-Meier survival statistics and related log-rank statistics as follows: (i) < 100 days; (ii) ≥ 100 days; (iii) > 100 days; (iv) 11.3 ± 1.0 day; (v) 9.0 ± 0.5 days; (vi) > 100 days; (vii) 5/6 > 100 days; and (viii) 8.0 ± 1.5 days. Immunohistochemistry revealed high numbers of proliferating cells in tolerized liver allografts. Apoptotic cell death of hepatocytes could be detected in both rejecting and to a lower extent in tolerized animals. Conversely, only tolerized but not rejected liver allografts revealed upregulation of FasL-expression on hepatic parenchymal cells from day 3 onwards. Irridiated LEW livers, in turn, loose their ability to survive in allogeneic DA hosts (group v) whereas they survive in syngeneic hosts (group vi), indicating that irridiation itself does not destroy the liver parenchyma. Reconstitution of irridiated LEW livers with syngeneic (group vii) but not with allogeneic (group viii) PLs restored tolerance induction. The underlying mechanisms of immune-privilege observed with liver allografts appear to share characteristics of clonal exhaustion suggesting that alloreactive lymphocytes are depleted by AICD via the FasL/Fas signal transduction pathway. The high frequency of apoptotic lymphocytes found in the portal tract of tolerized (but not rejected) LEW grafts supposes that functional FasL expression on graft hepatocytes mediates specific elimination of graft-directed effector lymphocytes. This mechanism constitutes peripheral deletion as one of the possible tolerogenic mechanisms involved. Chimerical liver grafts consisting of donor (LEW) parenchyma and host (DA) passenger leukocytes loose their tolerogenic capacity. In contrast, syngeneic reconstitution with LEW-PLs, restores liver graft acceptance upon transplantation into allogeneic DA hosts. This phenomenon is not relying on the induction of micro- or macrochimeric hosts, as no LEW PLs were found in spleen, thymus or the blood compartment from long-term surviving DA rats. Thus, non-resident liver cells contribute significantly to liver graft acceptance. Subsequently, liver tolerance appears to be mainly induced in the graft itself.

Fas-mediated hepatocyte apoptosis is increased by hepatitis C virus infection and alcohol consumption, and may be associated with hepatic fibrosis: mechanisms of liver cell injury in chronic hepatitis C virus infection.

Epidemiological studies have established that heavy alcohol consumption in persons with chronic hepatitis C virus (HCV) infection is associated with advanced liver disease, including cirrhosis. The aims of this study were to evaluate the relationship between alcohol consumption and hepatocyte apoptosis in HCV-infected patients and to determine the role of Fas in HCV-mediated apoptosis. Liver tissue from 44 HCV-infected patients with variable alcohol consumption, and 10 normal control subjects who did not consume alcohol was examined for hepatocyte apoptosis, proliferation and Fas expression. Alcohol consumption was assessed using the 'Lifetime Drinking History' alcohol questionnaire. HCV RNA, alanine aminotransferase (ALT) and ferritin were also assessed in addition to demographic data. Hepatocyte apoptosis was significantly greater in HCV-infected patients compared to controls. Expression of Fas (CD95) was found in HCV patients but not in controls. The degree of Fas expression correlated with hepatocyte apoptosis as detected by terminal UTP nick end labelling (TUNEL). Active ethanol consumption led to a significant increase in hepatocyte apoptosis. Fas expression correlated with fibrosis in HCV-infected patients who were not actively drinking ethanol. In summary, HCV leads to increased apoptotic cell death in hepatocytes. Programmed cell death can be further up-regulated by active ethanol consumption. The correlation between Fas expression and TUNEL supports the hypothesis that the Fas-Fas ligand interaction is the major mechanism for HCV-induced hepatocyte apoptosis.

Liver toxicity and apoptosis: role of TGF-β 1, cytochrome c and the apoptosome

Transforming growth factor-β 1 (TGF-β 1), is involved in controlling liver size, by inducing apoptotic cell death in hepatocytes. However the mechanism by which TGF-β 1 induces caspase activation and cell death is unknown. Apoptosis can be initiated either by receptor-mediated (e.g. Fas/CD95) or non-receptor chemically mediated (stress-induced) processes. With Fas/CD95 receptor mediated cell death, a multi-protein complex (DISC) is assembled at the plasma membrane, which activates the downstream caspases and cell death. In stress-mediated apoptosis, a cytosolic DISC equivalent, the apoptosome is formed that activates the effector caspases. We have characterised this complex in THP.1 cells, and shown that this is a cytochrome c dependent process that induces the formation of an ∼700 kDa apoptosome caspase processing complex. This is formed by oligomerisation of apoptotic protease-activating factor 1 (Apaf-1), and recruitment and processing of caspase-9. We have now shown that TGF-β 1-induced apoptosis also occurs via the release of cytochrome c and the subsequent oligomerisation of Apaf-1 into an ∼700 kDa apoptosome complex. Our studies show that, even though TGF-β 1 induction of apoptosis is a receptor-mediated event, it operates through the mitochondrial/Apaf-1 caspase activation pathway that appears to act as a common execution pathway for many diverse apoptotic stimuli.

Microcystin-LR toxicodynamics, induced pathology, and immunohistochemical localization in livers of blue-green algae exposed rainbow trout (oncorhynchus mykiss).

With this retrospective study, we investigated the temporal pattern of toxin exposure and pathology, as well as the topical relationship between hepatotoxic injury and localization of microcystin-LR, a potent hepatotoxin, tumor promoter, and inhibitor of protein phosphatases-1 and -2A (PP), in livers of MC-gavaged rainbow trout (Oncorhynchus mykiss) yearlings, using an immunohistochemical detection method and MC-specific antibodies. H&E stains of liver sections were used to determine pathological changes. Nuclear morphology of hepatocytes and ISEL analysis were employed as endpoints to detect the advent of apoptotic cell death in hepatocytes. Trout had been gavaged with lyophilized cyanobacteria (Microcystis aeruginosa, strain PCC 7806) at acutely toxic doses of 5700 microg microcystin (MC) per kg of body weight (bw), as described previously (Tencalla and Dietrich, 1997). Briefly, 3 control and 3 test animal were killed 1, 3, 12, 24, 48, and 72 h after bolus dosing, and livers were fixed and paraffin embedded for histological analysis and later retrospective histochemical analyses. The results of the immunohistochemistry reported here revealed a time dependent, discernible increase in MC-positive staining intensity throughout the liver, clearly not concurring with the kinetics of hepatic PP inhibition observed in the same fish and reported in an earlier publication by Tencalla and Dietrich (1997). After 3 h, marked and increasing MC-immunopositivity was observed in the cytoplasm, as well as the nuclei of hepatocytes. Apoptotic cell death could be detected after 48 h, at the very earliest. These data suggest that accumulation of MC and subsequent changes in cellular morphology, PP inhibition, and hepatocyte necrosis represent the primary events in microcystin induced hepatotoxicity and appear to be associated with the reversible interaction of MC with the PP. In contrast, apoptotic cell death, as demonstrated here, seems to be of only secondary nature and presumably results from the covalent interaction of MC with cellular and nuclear PP as well as other thiol containing cellular proteins.

Apoptotic cell death of hepatocytes in rat experimental severe acute pancreatitis

Background: Apoptosis of hepatocytes has been reported to be involved in liver failure complicated with systemic manifestations such as endotoxemia. We hypothesized that hepatocyte apoptosis occurs in severe acute pancreatitis. Methods: Induction of apoptosis was evaluated in the liver from rats with necrotizing pancreatitis. Apoptosis-inducing activity of the pancreatitis-associated ascitic fluid on hepatocytes was evaluated in vivo by intraperitoneal injection of the ascitic fluid and in vitro using rat primary hepatocyte culture. Results: Apoptosis was detected in hepatocytes in the rats both with severe acute pancreatitis and with the intraperitoneal injection of the ascitic fluid by in situ nick-end labeling and DNA fragmentation. Apoptotic change and hepatic injury were ameliorated by administration of an interleukin-1β–converting enzyme inhibitor. The ascitic fluid exhibited cytocidal activity in rat primary hepatocyte culture via apoptosis, which was confirmed by DNA fragmentation, by cell cycle analysis, and by nuclear fragmentation. The neutralizing antibody for transforming growth factor–β1 partially blocked the apoptosis induction, but the antibody to tumor necrosis factor–α had no effect. Conclusions: Apoptotic cell death occurs in hepatocytes in severe acute pancreatitis partially via transforming growth factor–β1 in the pancreatitis-associated ascitic fluid. (Surgery 2000;127:55-64.)

Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model.

The apoptotic cell death induced in D-galactosamine-sensitized mice by administration of lipopolysaccharide was characterized. Administration of lipopolysaccharide caused apoptotic cell death in livers of D-galactosamine-sensitized mice. Apoptotic cells were also detected in the kidney, thymus, spleen, and lymph node. Severe hepatic apoptosis in D-galactosamine-sensitized mice was reproduced by transfer of the sera from mice injected with D-galactosamine and lipopolysaccharide. The hepatocyte apoptosis induced by lipopolysaccharide was completely prevented by an anti-tumor necrosis factor alpha antibody but not by an anti-gamma interferon antibody. Administration of recombinant tumor necrosis factor into D-galactosamine-sensitized mice also caused hepatocyte apoptosis. Lipopolysaccharide-induced hepatocyte apoptosis in D-galactosamine-sensitized mice did not seem to be mediated by Fas antigen. It was suggested that lipopolysaccharide- induced hepatic injury and failure in D-galactosamine-sensitized mice was due to the apoptotic cell death of hepatocytes caused by tumor necrosis factor alpha released in the circulation.

Transforming growth factor beta 1 preferentially induces apoptotic cell death in rat hepatocytes cultured under pericentral-equivalent conditions.

The cytotoxicity of transforming growth factor beta 1 (TGF beta 1) was assessed in rat hepatocytes cultured under periportal (PP)-or pericentral (PC)-equivalent conditions. TGF beta 1 induced a 5-fold greater DNA fragmentation and LDH release in PC cultures than in PP cultures. At low exposure level (1 ng/ml TGF beta 1), albumin secretion and mitochondrial activity (rhodamine-123 uptake) were selectively reduced in PP cultures, whereas the incidence of apoptotic cells in PC cultures was about 10-fold higher than that in PP cultures. The time profiles of TGF beta 1-induced apoptotic and necrotic events and the concentration-response relationship differed in PC and PP cultures. In PC cultures the early appearance of cells with apoptotic nuclei was not associated with DNA fragmentation nor with an increase in LDH release or impaired mitochondrial function. At a high exposure level (5 ng/ml TGF beta 1), again cells with apoptotic nuclei were much more strongly induced in PC cultures but DNA fragmentation, LDH release, and impairment of mitochondrial activity all increased in an exposure-time dependent manner in both PP and PC cultures. At this exposure level 48 and 72% of the apoptotic cells detected in PC cultures after continuous exposure for 24 hr were induced within an exposure of 1 and 4 hr, respectively. Aurintricarboxylic acid (50 microM), an inhibitor of endonucleases, significantly inhibited the appearance of apoptotic cells and the progression in apoptosis. Clearly, TGF beta 1 preferentially induced apoptotic cell death in hepatocytes with PC-equivalent metabolism at low exposure levels. High exposure levels or prolonged exposure periods produced both apoptosis and necrosis.