Primary hepatocyte apoptosis is unlikely to relate to caspase-3 activity under sustained endogenous oxidative stress

We previously showed that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in endogenous oxidative stress and apoptosis. For the present study, we determined whether this apoptosis involved activation of caspase-3, which is known to execute apoptosis in many cell types. ATZ and MS increased levels of reactive oxygen species (ROS) from 3-9 h, just before the onset of chromatin condensation (apoptosis) and decreases in protein thiols. Pretreatment with either SKF, a cytochrome P450 inhibitor, or L-ascorbic acid, an antioxidant, completely suppressed the increase in ROS levels and apoptosis, suggesting that the sustained ROS increases may cause the apoptosis. SKF also abolished the decrease in protein thiol content, further supporting the contribution of the P450 system to increased ROS levels. DEVD-CHO, a caspase-3 inhibitor, even at 1 mM had no effect on apoptosis. Caspase-3 activity remained unchanged and pro-caspase-3 processing was not detected during 18 h incubation with ATZ and MS. Moreover, the amount of unoxidized pro-caspase-3 decreased even below the level of untreated hepatocytes. These findings suggest that the sustained oxidative stress is a major cause for the hepatocyte apoptosis, which occurs independently of the caspase-3 related pathway.     

Involvement of endonuclease G in nucleosomal DNA fragmentation under sustained endogenous oxidative stress

We have previously shown that inhibition of catalase and glutathione peroxidase activities by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively, in rat primary hepatocytes caused sustained endogenous oxidative stress and apoptotic cell death without caspase-3 activation. In this study, we investigated the mechanism of this apoptotic cell death in terms of nucleosomal DNA fragmentation. Treatment with ATZ+MS time-dependently increased the number of deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive nuclei from 12 h, resulting in clear DNA laddering at 24 h. The deoxyribonuclease (DNase) inhibitor, aurintricarboxylic acid (ATA), completely inhibited nucleosomal DNA fragmentation but the pan-caspase inhibitor, z-VAD-fmk was without effects; furthermore, the cleavage of inhibitor of caspase-activated DNase was not detected, indicating the involvement of DNase(s) other than caspase-activated DNase. Considering that endonuclease G (EndoG) reportedly acts in a caspase-independent manner, we cloned rat EndoG cDNA for the first time. Recombinant EndoG alone digested plasmid DNA and induced nucleosomal DNA fragmentation in isolated hepatocyte nuclei. Recombinant EndoG activity was inhibited by ATA but not by hydrogen peroxide, even at 10 mm. ATZ+MS stimulation elicited decreases in mitochondrial membrane potential and EndoG translocation from mitochondria to nuclei. By applying RNA interference, the mRNA levels of EndoG were almost completely suppressed and the amount of EndoG protein was decreased to approximately half the level of untreated cells. Under these conditions, decreases in TUNEL-positive nuclei were significantly suppressed. These results indicate that EndoG is responsible, at least in part, for nucleosomal DNA fragmentation under endogenous oxidative stress conditions induced by ATZ+MS.     

Critical role of exposure time to endogenous oxidative stress in hepatocyte apoptosis

We have previously shown that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in sustained oxidative stress, followed by apoptosis. To examine the effects of duration of oxidative stress, ATZ and MS were removed from culture medium at 3, 6 and 9 h after treatment with both inhibitors. Oxidative stress was induced for periods of time by ATZ and MS exposures in primary hepatocytes. Treatment with ATZ and MS reduced catalase (CAT) and glutathione peroxidase (GPx) activities, and decreased CAT and GPx activities recovered to normal values upon withdrawal. Although oxidative stress of up to 6 h duration did not cause cell death, sustained oxidative stress (over 9 h) induced apoptosis. The increase in the glutathione disulfide/reduced glutathione ratio under oxidative stress up to 6 h was transient and reversible, while that due to sustained oxidative stress was irreversible. These results suggest that irreversible redox shifts resulting from sustained oxidative stress play a critical role in the induction of hepatocyte apoptosis in this experimental system.     

Critical role of exposure time to endogenous oxidative stress in hepatocyte apoptosis

Abstract

We have previously shown that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in sustained oxidative stress, followed by apoptosis. To examine the effects of duration of oxidative stress, ATZ and MS were removed from culture medium at 3, 6 and 9 h after treatment with both inhibitors. Oxidative stress was induced for periods of time by ATZ and MS exposures in primary hepatocytes. Treatment with ATZ and MS reduced catalase (CAT) and glutathione peroxidase (GPx) activities, and decreased CAT and GPx activities recovered to normal values upon withdrawal. Although oxidative stress of up to 6 h duration did not cause cell death, sustained oxidative stress (over 9 h) induced apoptosis. The increase in the glutathione disulfide/reduced glutathione ratio under oxidative stress up to 6 h was transient and reversible, while that due to sustained oxidative stress was irreversible. These results suggest that irreversible redox shifts resulting from sustained oxidative stress play a critical role in the induction of hepatocyte apoptosis in this experimental system.     

Apoptotic signals induce specific degradation of ribosomal RNA in yeast

Organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. Stress response can either repair the damage or activate one of the programmed cell death (PCD) mechanisms, for example apoptosis, and finally end in cell death. One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. This process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rRNA is less susceptible to degradation in respiring cells with functional defence against oxidative stress. In addition, RNA fragmentation is independent of two yeast apoptotic factors, metacaspase Yca1 and apoptosis-inducing factor Aif1, but it relies on the apoptotic chromatin condensation induced by histone H2B modifications. These data describe a novel phenotype for certain stress- and ageing-related PCD pathways in yeast.     

Docosahexaenoic acid and tetracyclines as promising neuroprotective compounds with poly(ADP-ribose) polymerase inhibitory activities for oxidative/genotoxic stress treatment

The human genome is exposed to oxidative/genotoxic stress by several endogenous and exogenous compounds. These events evoke DNA damage and activate poly(ADP-ribose) polymerase-1 (PARP-1), the key enzyme involved in DNA repair. The massive stress and over-activation of this DNA-bound enzyme can be responsible for an energy crisis and neuronal death. The last data indicated that product of PARP-1, i.e. poly(ADP-ribose) (PAR), acts as a signalling molecule and plays a significant role in nucleus-mitochondria cross-talk. PAR translocated to the mitochondria can be involved in mitochondrial permeability, the release of an apoptosis-inducing factor (AIF). Its translocation into the nucleus leads to chromatin condensation, fragmentation and cell death. The exact mechanism of this novel death pathway has not yet fully been understood.     

Arachidonic acid cytotoxicity in leukocytes: implications of oxidative stress and eicosanoid synthesis

Arachidonic acid (AA)-induced cytotoxicity was evaluated in leukocytes: the human leukemia cell lines HL-60, Jurkat and Raji and in rat lymphocytes. Such cytotoxicity was dose- and time-dependent. At concentrations below 5 microM, AA was not toxic; at 10-400 microM, AA induced apoptosis and at concentrations beyond 400 microM, necrosis. The minimum exposure time to trigger cell death was of around 1 h, but the effect was increased by longer exposure times until 6-24 h. Apoptosis was morphologically characterized by a decrease in cell and nuclear volume, chromatin condensation and DNA fragmentation and the presence of lipid bodies, without changes in organelle integrity. Biochemically, AA-induced apoptosis was associated with internucleosomal fragmentation and caspase activation, evaluated by PARP cleavage and the use of a caspase inhibitor. Necrosis was characterized by increased cell volume, presence of loose chromatin, appearance of vacuoles, loss of membrane integrity and of the definition of organelles. The apoptotic effect of AA was studied as to oxidative-reductive imbalance and the participation of eicosanoids. Apoptotic AA treatment was accompanied by an increase in the quantity of thiobarbituric acid reactive substances (TBARS), low-level chemiluminescence and in the glutathione disulfide/reduced glutathione ratio, indicating oxidative stress. The addition of tocopherol, ascorbate, prostaglandin E2 and lipoxygenase inhibitors delayed cell death, whereas the inhibition of cyclooxygenase promoted AA-induced cell death. Cell treatment with AA was accompanied by increased cellular production of LTB4. AA, therefore, is cytotoxic at physiological and supraphysiological concentrations, causing apoptosis and necrosis. Cell treatment with apoptotic concentrations of AA involves oxidative stress and changes in eicosanoid biosynthesis.     

Starvation for an essential amino acid induces apoptosis and oxidative stress in yeast

Protracted starvation of auxotrophic Saccharomyces cerevisiae strains for an essential amino acid is commonly used to allow investigation of adaptive mutation mechanisms during starvation-induced cell cycle arrest. Under these conditions, the majority of cells dies during the first 6 days. We investigated starving cells for markers of programmed cell death and for the production of reactive oxygen species (ROS). We observed that protracted starvation for lysine or histidine resulted in an increasing number of cells exhibiting DNA fragmentation and chromatin condensation, thus an apoptotic phenotype. Not only respiration-competent cells but also respiratory deficient rho0 cells were able to undergo programmed cell death. In addition the starving cells rapidly exhibited indicators of oxidative stress, independently of their respiratory competence. These results indicate that starvation for an essential amino acid results in severe cell stress, which may finally be the trigger of programmed cell death.     

撤除外源生长素诱发棉花胚性悬浮细胞程序性死亡

棉花胚性悬浮细胞在仅含生长素的MS培养基上培养时,生长良好;但当转入到不含生长素的MS培养基上培养时,大规模死亡.通过细胞学观察发现,在转入无生长素的MS培养基培养3～4 d后可见明显的核质浓缩、胞质收缩,而高温处理引起的细胞坏死无此现象.用抽提悬浮细胞基因组DNA进行凝胶电泳发现:这种细胞死亡还伴随有典型的DNA梯度出现,而坏死的细胞和对照无DNA梯度.表明这种由生长素撤除引起的细胞死亡是一种程序性死亡.这种细胞死亡能被水解酶抑制剂和半胱氨酸蛋白酶抑制剂抑制,表明水解酶和类半胱氨酸蛋白水解酶(CLP)参与细胞程序性死亡.     

Pyrrolidinium-type fullerene derivative-induced apoptosis by the generation of reactive oxygen species in HL-60 cells

The biological activities of C₆₀-bis(N,N-dimethylpyrrolidinium iodide), a water-soluble cationic fullerene derivative, on human promyeloleukaemia (HL-60) cells were investigated. The pyrrolidinium fullerene derivative showed cytotoxicity in HL-60 cells. The characteristics of apoptosis, such as DNA fragmentation and condensation of chromatin in HL-60 cells, were observed by exposure to the pyrrolidinium fullerene derivative. Caspase-3 and -8 were activated and cytochrome c was also released from mitochondria. The generation of reactive oxygen species (ROS) by the pyrrolidinium fullerene derivative was observed by DCFH-DA, a fluorescence probe for the detection of ROS. Pre-treatment with α-tocopherol suppressed cell death and intracellular oxidative stress caused by the pyrrolidinium fullerene derivative. The apoptotic cell death induced by the pyrrolidinium fullerene derivative was suggested to be mediated by ROS generated by the pyrrolidinium fullerene derivative.     

Chromatin condensation of ovarian nurse and follicle cells is regulated independently from DNA fragmentation during Drosophila late oogenesis

Programmed cell death constitutes a common fundamental incident occurring during oogenesis in a variety of different organisms. In Drosophila melanogaster, it plays a significant role in the maturation process of the egg chamber. In the present study, we have used an in vitro development system for studying the effects of inducers and inhibitors of programmed cell death during the late stages of oogenesis. Treatment of the developing egg chambers with two widely used inducers of cell death, etoposide and staurosporine, blocks further development and induces chromatin condensation but not DNA fragmentation in nurse and follicle cells, as revealed by propidium iodide staining and terminal transferase-mediated dUTP nick-end labeling assay. Moreover, incubation of the developing egg chambers with the caspase-3 inhibitor Z-DEVD-FMK significantly delays development, prevents DNA fragmentation, but does not affect chromatin condensation. The above results demonstrate, for the first time, that chromatin condensation in Drosophila ovarian nurse and follicle cells is a caspase-3-like independent process and is regulated independently from DNA fragmentation.     

Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis

Chromosomal DNA and mitochondrial dysfunctions play a role on mammalian cell death induced by oxidative stress. The major biochemical dysfunction of chromosome is the presence of an ordered cleavage of the DNA backborn, which is separated and visualized as an electrophoretic pattern of fragments. Oxidative stress provides chromatin dysfunction such as single strand and double strand DNA fragmentation leading to cell death. More than 1 Mb of giant DNA, 200-800 kb or 50-300 kb high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced during apoptosis or necrosis induced by oxidative stress such as glutathione (GSH) depletion in several types of mammalian cells. Reactive oxygen species (ROS)-mediated DNA fragmentation is enhanced by polyunsaturated fatty acids including arachidonic acid or their hydroperoxides, leading to necrosis. Mitochondrial dysfunction on decrease of trans membrane potential, accumulation of ROS, membrane permeability transition and release of apoptotic factors during apoptosis or necrosis has been implicated. This review refers to the correlation of chromosomal DNA fragmentation and apoptosis or necrosis induced by GSH depletion, and the possible mechanisms of oxidative stress-induced cell death.     

Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H₂O₂) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H₂O₂ preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H₂O₂ stimulated divergent pathways, with caspases being activated only by H₂O₂. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H₂O₂, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H₂O₂. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.     

Progressive apoptotic cell death triggered by transient oxidative insult in H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanisms

Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer that is known to cause rapid cell death. Here we show that a brief oxidative insult (5-min exposure to 400 microM H(2)O(2)), although it did not kill H9c2 rat ventricular cells during the exposure, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 h after the insult had been withdrawn, and this post-H(2)O(2) cell death cumulated gradually, reaching a maximum level 8 h after H(2)O(2) withdrawal. By comparison, sustained exposure to H(2)O(2) caused complete cell death within a narrow time frame (2 h). The time-dependent post-H(2)O(2) cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines, and caspase-3 activation). A dichlorofluorescein fluorescent signal showed a time-dependent endogenous increase of reactive oxygen species (ROS) production, which was almost abolished by inhibition of the mitochondrial electron transport chain. Application of antioxidants (vitamin E or DTT) before H(2)O(2) addition or after H(2)O(2) withdrawal prevented the H(2)O(2)-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with activation of the mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release, and late activation of caspase-3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.     

Programmed cell death of secretory cavity cells in fruits of Citrus grandis cv. Tomentosa is associated with activation of caspase 3-like protease

Previously, we found that secretory cell degradation typically occurred through programmed cell death during secretory cavity development in Citrus sinensis L. (Osbeck). This finding indicated that secretory cavities could be utilized as a new cell biology model for investigating the regulatory mechanisms of plant programmed cell death. To study further the programmed cell death during secretory cavity development in Citrus fruit, we studied the morphogenetic characteristics of secretory cavities during their development in Citrus grandis cv. Tomentosa. Using light microscope- and electron microscope-TUNEL assays, immunohistochemistry and immunocytochemistry, we described the precise spatial and temporal alterations in caspase 3-like distribution, chromatin condensation and DNA fragmentation during the programmed cell death of secretory cavity cells. Caspase 3-like was found to be significantly located in both the cytoplasm and the nucleus of secretory cavity cells undergoing programmed cell death, and caspase 3-like is closely associated with chromatin condensation and DNA fragmentation. Interestingly, both caspase 3-like and DNA fragmentation were detected in the nucleoli. Our findings suggest that caspase 3-like may be involved in the programmed cell death of secretory cavity cells, especially in chromatin condensation, DNA fragmentation, nuclear degradation and the degradation of certain organelles.     

Induced thermotolerance to apoptosis in a human T lymphocyte cell line.

A brief exposure to elevated temperatures elicits, in all organisms, a transient state of increased heat resistance known as thermotolerance. The mechanism for this thermotolerant state is unknown primarily because it is not clear how mild hyperthermia leads to cell death. The realization that cell death can occur through an active process of self destruction, known as apoptosis, led us to consider whether thermotolerance provides protection against this mode of cell death. Apoptosis is a common and essential form of cell death that occurs under both physiological and pathological conditions. This mode of cell death requires the active participation of the dying cell and in this way differs mechanistically from the alternative mode of cell death, necrosis. Here we show that mild hyperthermia induces apoptosis in a human leukemic T cell line. This is evidenced by chromatin condensation, nuclear fragmentation and the cleavage of DNA into oligonucleosome size units. DNA fragmentation is a biochemical hallmark of apoptosis and requires the activation of an endogenous endonuclease. The extent of DNA fragmentation was proportional to the severity of heat stress for cells heated at 43 degrees C from 30 to 90 minutes. A brief conditioning heat treatment induced a resistance to apoptosis. This was evident as a resistance to DNA fragmentation and a reduction in the number of apoptotic cells after a heat challenge. Resistance to DNA fragmentation developed during a recovery period at 37 degrees C and was correlated with enhanced heat shock protein (hsp) synthesis. This heat-induced resistance to apoptosis suggests that thermotolerant cells have gained the capacity to prevent the onset of this pathway of self-destruction. An examination of this process in heated cells should provide new insights into the molecular basis of cellular thermotolerance.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.     

Role of the mitochondrial permeability transition and cytochrome C release in hydrogen peroxide-induced apoptosis

We investigated the role of the mitochondrial inner membrane permeability transition and subsequent release of cytochrome c into the cytosol during oxidative stress-evoked apoptosis. Sublethal oxidative stress was applied by treating L929 cells with 0.5 mM H2O2 for 90 min. Then the cellular localization of cytochrome c was examined by immunofluorescent staining and Western blotting. H2O2 treatment caused the permeability transition and pore formation, resulting in membrane depolarization and translocation of cytochrome c from the mitochondria into the cytosol. Pretreatment with cyclosporin A and aristolochic acid (to inhibit pore formation) significantly attenuated a reduction of the mitochondrial membrane potential, as well as signs of apoptosis such as DNA fragmentation, increased plasma membrane permeability, and chromatin condensation. Therefore, exposure to H2O2 caused the opening of permeability transition pores in the inner mitochondrial membrane. An essential role of cytosolic cytochrome c in the execution of apoptosis was demonstrated by its direct microinjection into the cytosol, thus bypassing the need for cytochrome c release from the mitochondrial intermembrane space. Microinjection of cytochrome c caused caspase-dependent apoptosis.