Apoptosis in hematopoietic cells (FL5.12) caused by interleukin-3 withdrawal: relationship to caspase activity and the loss of glutathione

The mechanism of cell death caused by cytokine deprivation remains largely unknown. FL5.12 cells (a murine prolymphocytic cell line), following interleukin-3 (IL-3) withdrawal, undergo a decrease in intracellular glutathione (GSH) that precedes the onset of apoptosis. In the present study, the induction of apoptosis following IL-3 withdrawal or GSH depletion with DL-buthionine-[S,R,]-sulfoximine (BSO) was examined. Both conditions caused time-dependent increases in phosphatidylserine externalization, acridine orange and ethidium bromide staining, decreases in mitochondrial membrane potential, processing and activation of caspase-3 and proteolysis of the endogenous caspase substrate poly(adenosine diphosphate ribose)polymerase (PARP). Apoptosis induced by IL-3 deprivation but not BSO also caused lamin B1 cleavage, suggesting activation of caspase-6. Despite a more profound depletion of GSH after BSO than withdrawal of IL-3, the extent of apoptosis was somewhat lower. Benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone (z-VAD.fmk) blocked this caspase activity and prevented cell death after BSO exposure but not after IL-3 deprivation. Following IL-3 withdrawal, the caspase inhibitors z-VAD.fmk and boc-asp(OMe)fluoromethylketone (boc-asp.fmk) prevented the cleavage and activation of caspase-3, the breakdown of lamin B1 and partially mitigated PARP degradation. However, the externalization of phosphatidylserine, the fall in mitochondrial membrane potential and subsequent apoptotic cell death still occurred. These results suggest that IL-3 withdrawal may mediate cell death by a mechanism independent of both caspase activation and the accompanying loss of GSH.     

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity.     

Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells

Intracellular glutathione (GSH) depletion induced by buthionine sulfoximine (BSO) caused cell death that seemed to be apoptosis in C6 rat glioma cells. Arachidonic acid (AA) promoted BSO-induced cell death by accumulating reactive oxygen species (ROS) or hydroperoxides. AA inhibited caspase-3 activation and internucleosomal DNA fragmentation during the BSO-induced GSH depletion. Furthermore, AA reduced intracellular ATP content, induced dysfunction of mitochondrial membrane and enhanced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) production. There was significant increase of 12-lipoxygenase activity in the presence of AA under the BSO-induced GSH depletion in C6 cells. These results suggest that AA promotes cell death by changing to necrosis from apoptosis through lipid peroxidation initiated by lipid hydroperoxides produced by 12-lipoxygenase under the GSH depletion in C6 cells. Some ROS such as hydroperoxide produced by unknown pathway make hydroxy radicals and induce 8-OH-dG formation in the cells. The conversion of apoptosis to necrosis may be a possible event under GSH depleted conditions.     

The role of glutathione in lymphocyte activation. I. Comparison of inhibitory effects of buthionine sulfoximine and 2-cyclohexene-1-one by nuclear size transformation

The role of glutathione (GSH) in lectin-induced lymphocyte activation can be studied by quantitating lectin-induced nuclear size transformation in the presence of variable degrees of GSH depletion. Buthionine sulfoximine (BSO) inhibits intracellular GSH synthesis by inhibition of the enzyme gamma-glutamyl-cysteine synthetase. By combining endogenous GSH depletion in cell cultures with BSO-induced inhibition of GSH synthesis, lectin-induced lymphocyte activation can be studied at various concentrations of soluble intracellular GSH. With this approach, the percentage of lymphocytes undergoing a nuclear size transformation is minimally affected despite depletion of soluble intracellular GSH to 0.27 nmol/10(7) cells (PBL), which represents approximately 95% depletion of intracellular GSH. When soluble intracellular GSH is depleted to undetectable levels (less than 0.10 nmol/10(7) cells) there is a 10 to 12% reduction in the number of cell nuclei transformed. However, in all BSO-pretreated cultures the lectin-induced nuclear size transformation is intermediate between resting and blast-transformed lymphocytes, suggesting only partial (or aborted) activation. The partial activation response observed in BSO-pretreated cultures may be due to mobilization of the protein-bound pool of GSH, which is relatively resistant to depletion by BSO. That the inhibition of full blast transformation is truly due to GSH depletion was proven by experiments in which GSH was repleted exogenously and a full blast transformation was restored. The results of previous work in our laboratory had shown that the sulfhydryl-reactive agent 2-cyclohexene-1-one (2-CHX) was a potent inhibitor of activation at soluble intracellular GSH concentrations well above 0.27 nmol/10(7) PBL. In the present study, the dose-dependent inhibition of activation by 2-CHX was confirmed, but it was shown that the degree of inhibition caused by 2-CHX could be at least partially dissociated from the level of intracellular GSH present at the time of lectin addition and that the inhibitory potential of 2-CHX exceeded that of BSO at comparable levels of soluble intracellular GSH. Thus, the inhibitory properties of 2-CHX cannot be accounted for solely on the basis of GSH depletion.     

Regulation of constitutive neutrophil apoptosis by the alpha,beta-unsaturated aldehydes acrolein and 4-hydroxynonenal

Reactive alpha,beta-unsaturated aldehydes are major components of common environmental pollutants and are products of lipid oxidation. Although these aldehydes have been demonstrated to induce apoptotic cell death in various cell types, we recently observed that the alpha,beta-unsaturated aldehyde acrolein (ACR) can inhibit constitutive apoptosis of polymorphonuclear neutrophils and thus potentially contribute to chronic inflammation. The present study was designed to investigate the biochemical mechanisms by which two representative alpha,beta-unsaturated aldehydes, ACR and 4-hydroxynonenal (HNE), regulate neutrophil apoptosis. Whereas low concentrations of either aldehyde (<10 microM) mildly promoted apoptosis in neutrophils (reflected by increased phosphatidylserine exposure, caspase-3 activation, and mitochondrial cytochrome c release), higher concentrations prevented critical features of apoptosis (caspase-3 activation, phosphatidylserine exposure) and caused delayed neutrophil cell death with characteristics of necrosis/oncosis. Inhibition of caspase-3 activation by either aldehyde occurred despite increases in mitochondrial cytochrome c release and occurred in close association with depletion of cellular GSH and with cysteine modifications within caspase-3. However, procaspase-3 processing was also prevented, because of inhibited activation of caspases-9 and -8 under similar conditions, suggesting that ACR (and to a lesser extent HNE) can inhibit both intrinsic (mitochondria dependent) and extrinsic mechanisms of neutrophil apoptosis at initial stages. Collectively, our results indicate that alpha,beta-unsaturated aldehydes can inhibit constitutive neutrophil apoptosis by common mechanisms, involving changes in cellular GSH status resulting in reduced activation of initiator caspases as well as inactivation of caspase-3 by modification of its critical cysteine residue.     

Role of extracellular signal-regulated protein kinase in neuronal cell death induced by glutathione depletion in neuron/glia mesencephalic cultures

To date, glutathione (GSH) depletion is the earliest biochemical alteration shown in brains of Parkinson's disease patients, but the role of GSH in dopamine cell survival is debated. In this study we show that GSH depletion, produced with GSH synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), induces selectively neuronal cell death in neuron/glia, but not in neuronal-enriched midbrain cultures and that cell death occurs with characteristics of necrosis and apoptosis. BSO produces a dose- and time-dependent generation of reactive oxygen species (ROS) in neurons. BSO activates extracellular signal-regulated kinases (ERK-1/2), 4 and 6 h after treatment. MEK-1/2 and lipoxygenase (LOX) inhibitors, as well as ascorbic acid, prevent ERK-1/2 activation and neuronal loss, but the inhibition of nitric oxide sintase (NOS), cyclo-oxygenase (COX), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) does not have protective effects. Co-localization studies show that p-ERK-1/2 expression after BSO treatment increased in astrocytes and microglial cells, but not in neurons. Selective metabolic impairment of glial cells with fluoroacetate decreased ERK activation. However, blockade of microglial activation with minocycline did not. Our results indicate that neuronal death induced by GSH depletion is due to ROS-dependent activation of the ERK-1/2 signalling pathway in glial cells. These data may be of relevance in Parkinson's disease, where GSH depletion and glial dysfunction have been documented.     

Glutathione depletion induces giant DNA and high-molecular-weight DNA fragmentation associated with apoptosis through lipid peroxidation and protein kinase C activation in C6 glioma cells

Glutathione (GSH) depletion caused by l-buthionine-(S,R)-sulfoximine (BSO) induced apoptosis that was recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endo-labeling (TUNEL), nuclear DNA staining with fluorescence dye, and internucleosomal DNA fragmentation in C6 rat glioma cells. The BSO-induced cell death was associated with caspase-3 activation. Lipid peroxidation and protein kinase C (PK-C) activation were observed during the apoptosis of C6 cells, and these events were inhibited by antioxidants and iron chelators without affecting BSO-induced GSH depletion. Furthermore, approximately 2 Mbp giant DNA fragments were observed in the BSO-treated cells. The giant DNA fragmentation were followed by approximately 30-700 kbp and then less than 100 kbp, including internucleosomal DNA fragmentations. Such serial DNA degradation was prevented by the antioxidants, the iron chelators, and the PK-C inhibitors. These results suggest that during apoptosis induced by GSH-depletion caused by BSO, reactive oxygen species endogenously produced cause lipid peroxidation and that the lipid peroxidation induced PK-C activation, processes which are thought to be involved in the giant DNA, high-molecular-weight DNA, and the internucleosomal DNA fragmentations.     

Acrolein causes inhibitor kappaB-independent decreases in nuclear factor kappaB activation in human lung adenocarcinoma (A549) cells

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in various situations. In the present study, the effects of sublethal doses of acrolein on nuclear factor kappaB (NF-kappaB) activation in A549 human lung adenocarcinoma cells were investigated. Immediately following a 30-min exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA synthesis and NF-kappaB binding were reduced by more than 80%. All parameters returned to normal or supranormal levels by 8 h post-treatment. Pretreatment with acrolein completely blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of NF-kappaB. Cells treated for 1 h with 1 mM diethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA synthesis, respectively. DEM also reduced NF-kappaB activation by 64% at 2 h post-treatment, with recovery to within 22% of control at 8 h. Both acrolein and DEM decreased NF-kappaB function approximately 50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h after DEM treatment, but proliferation remained significantly depressed for 24 h. Interestingly, DEM caused a profound decrease in NF-kappaB binding, even at doses as low as 0.125 mM that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of phosphorylated or nonphosphorylated inhibitor kappaB-alpha (IkappaB-alpha). Furthermore, acrolein decreased NF-kappaB activation in cells depleted of IkappaB-alpha by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-kappaB activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-kappaB in the cytosol prior to chemical dissociation from IkappaB with detergent. Together, these data support the conclusion that the inhibition of NF-kappaB activation by acrolein and DEM is IkappaB-independent. The mechanism appears to be related to direct modification of thiol groups in the NF-kappaB subunits.     

Activation of the death receptor pathway of apoptosis by the aldehyde acrolein

Reactive alpha,beta-unsaturated aldehydes such as acrolein are major components of common environmental pollutants. As a toxic by-product of lipid peroxidation, acrolein has been implicated as a possible mediator of oxidative damage to cells and tissues in a wide variety of disease states, including atherosclerosis and neurodegenerative and pulmonary diseases. Although acrolein can induce apoptotic cell death in various cell types, the biochemical mechanisms are not understood. This study investigates the implication of the death receptor pathway in acrolein-induced apoptosis. Exposure of Chinese hamster ovary cells to acrolein caused translocation of adaptor protein Fas associated with death domain to the cytoplasmic membrane and caspase-8 activation. Kp7-6, an antagonist of Fas receptor activation, blocked apoptotic events downstream of caspase-8, such as caspase-7 activation and nuclear chromatin condensation. Acrolein activated the cross-talk pathway between the death receptor and mitochondrial pathways. Bid was cleaved to truncated-Bid, which was translocated to mitochondria. Activation of the mitochondrial pathway by acrolein was confirmed by caspase-9 activation. Inhibition of activation of either the Fas receptor or caspase-8 partially decreased acrolein-induced caspase-9 activation. These findings indicate that acrolein activates the Fas receptor pathway, which occurs upstream of the mitochondrial pathway. Caspase-9 activation still occurred despite inhibition of the Fas receptor pathway, suggesting that acrolein could also trigger the mitochondrial pathway independent of the receptor pathway. These findings improve our understanding of mechanisms of toxicity of the reactive aldehyde acrolein, which has widespread implications in multiple disease states which seem to be mediated by oxidative stress and lipid peroxidation.     

The role of low molecular weight thiols in T lymphocyte proliferation and IL-2 secretion

Glutathione (GSH) is an abundant intracellular tripeptide that has been implicated as an important regulator of T cell proliferation. The effect of pharmacological regulators of GSH and other thiols on murine T cell signaling, proliferation, and intracellular thiol levels was examined. l-Buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH synthesis, markedly reduced GSH levels and blocked T cell proliferation without significant effect on cell viability. N-acetylcysteine markedly enhanced T cell proliferation without affecting GSH levels. Cotreatment of T cells with N-acetylcysteine and BSO failed to restore GSH levels, but completely restored the proliferative response. Both 2-ME and l-cysteine also reversed the BSO inhibition of T cell proliferation. Intracellular l-cysteine levels were reduced with BSO treatment and restored with cotreatment with NAC or l-cysteine. However, 2-ME completely reversed the BSO inhibition of proliferation without increasing intracellular cysteine levels. Therefore, neither GSH nor cysteine is singularly critical in limiting T cell proliferation. Reducing equivalents from free thiols were required because oxidation of the thiol moiety completely abolished the effect. Furthermore, BSO did not change the expression of surface activation markers, but effectively blocked IL-2 and IL-6 secretion. Importantly, exogenous IL-2 completely overcame BSO-induced block of T cell proliferation. These results demonstrate that T cell proliferation is regulated by thiol-sensitive pathway involving IL-2.     

TNFalpha-induced glutathione depletion lies downstream of cPLA(2) in L929 cells.

Both glutathione (GSH) depletion and arachidonic acid (AA) generation have been shown to regulate sphingomyelin (SM) hydrolysis and are known components in tumor necrosis factor alpha (TNFalpha)-induced cell death. In addition, both have hypothesized direct roles in activation of N-sphingomyelinase (SMase); however, it is not known whether these are independent pathways of N-SMase regulation or linked components of a single ordered pathway. This study was aimed at differentiating these possibilities using L929 cells. Depletion of GSH with L-buthionin-(S,R)-sulfoximine (BSO) induced 50% hydrolysis of SM at 12 h. In addition, TNF induced a depletion of GSH, and exogenous addition of GSH blocked TNF-induced SM hydrolysis as well as TNF-induced cell death. Together, these results establish GSH upstream of SM hydrolysis and ceramide generation in L929 cells. We next analyzed the L929 variant, C12, which lacks both cytosolic phospholipase A(2) (cPLA(2)) mRNA and protein, in order to determine the relationship of cPLA(2) and GSH. TNF did not induce a significant drop in GSH levels in the C12 line. On the other hand, AA alone was capable of inducing a 60% depletion of GSH in C12 cells, suggesting that these cells remain responsive to AA distal to the site of cPLA(2). Furthermore, depleting GSH with BSO failed to effect AA release, but caused a drop in SM levels, showing that the defect in these cells was upstream of the GSH drop and SMase activation. When cPLA(2) was restored to the C12 line by expression of the cDNA, the resulting CPL4 cells regained sensitivity to TNF. Treatment of the CPL4 cells with TNF resulted in GSH levels dropping to levels near those of the wild-type L929 cells. These results demonstrate that GSH depletion following TNF treatment in L929 cells is dependent on intact cPLA(2) activity, and suggest a pathway in which activation of cPLA(2) is required for the oxidation and reduction of GSH levels followed by activation of SMases.     

Nonxenobiotic manipulation and sulfur precursor specificity of human endothelial cell glutathione

Confluent human umbilical vein endothelial (HUVE) cells were readily (within 1 h) depleted of their glutathione (GSH) by diethylmaleate (0.1-1.0 mM), but dose-dependent cell detachment was noted. Buthionine sulfoximine (BSO, 25 microM) depleted cell GSH with sigmoidal kinetics, showing an initial half-life of depletion of 4-6 h and greater than 95% depletion by 48 h without morphological changes to the cells. However, BSO-dependent depletion of cell GSH was only partially reversible by cell washing and reincubation with complete medium. Likewise, incubation of the cells in sulfur-free medium depleted cell GSH again without morphological changes to the cells. However, unlike with BSO, these cells readily resynthesized GSH when resupplied with complete medium, fresh plasma, or whole blood, with a characteristic overloading of cell GSH (up to 200%) by 12 h. By use of the sulfur-free medium, it was shown that both cystine and cysteine are effective precursors to GSH synthesis in HUVE cells in culture and that cystine is the most likely precursor in vivo. During cystine-supported resynthesis of GSH, high levels of cysteine accumulated in the cells (up to 10% of total soluble free thiol). Physiologically relevant concentrations of extracellular GSH were not as effective as cystine or cysteine in stimulating GSH biosynthesis, whereas nonphysiologically high (mM) concentrations resulted in substantial elevation of GSH levels above those of control cells in a BSO-insensitive manner. These findings provide a simple methodology for the manipulation of HUVE cell GSH in studies of endothelial-specific oxidant toxicity and the sulfur dependence of the biochemistry and turnover of GSH in these human cells.     

Conjugated linoleic acid isomers have differential effects on triglyceride secretion in Hep G2 cells

Treatment for 2 h with 200 microM cadmium chloride, followed by recovery, caused apoptosis and induced heat-shock protein 70 (HSP70) expression in U-937 promonocytic cells. However, pre-incubation with the GSH depleting agent L-buthionine-[S,R]-sulfoximine (BSO, 1 mM for 24 h) caused necrosis instead of apoptosis and failed to induce HSP70 expression. This failure was a consequence of necrosis instead of GSH depletion, since BSO allowed or even potentiated HSP70 induction when used in combination with heat shock (2 h at 42.5 degrees C) or with 50 microM cadmium, which caused apoptosis. The administration of N-acetyl-L-cysteine (NAC) at the beginning of recovery after BSO/200 microM cadmium treatment prevented the execution of necrosis and restored the execution of apoptosis, but did not restore HSP70 induction, indicating that the inhibition by BSO of HSP70 expression is an early regulated event. This contrasted with the capacity of NAC to prevent the alterations caused by BSO/200 microM cadmium in other proteins, namely the suppression of Bax expression and the increase in Bcl-2 and HSP-60 expression. Finally, it was observed that treatment with 200 microM cadmium rapidly increased the HSP70 mRNA level and stimulated heat-shock factor 1 (HSF1) trimerization and binding, and that these effects were prevented by pre-incubation with BSO. Taken together, these results indicate that the stress response is compatible with apoptosis but not with necrosis in cadmium-treated promonocytic cells. The suppression of the stress response is specifically due to the early inhibition of HSF1 activation.     

Glial Cells Mediate Toxicity in Glutathione-Depleted Mesencephalic Cultures

We have examined the role of glial cells in the toxicity that results from inhibition of reduced glutathione (GSH) synthesis by L-buthionine sulfoximine (BSO) in mesencephalic cell cultures. We show that GSH depletion, to levels that cause total cell loss in cultures containing neurons and glial cells, has no effect on cell viability in enriched neuronal cultures. An increase in the plating cell density sensitizes glia-containing cultures to GSH depletion-induced toxicity. This suggests that cell death in this model is the consequence of events that are induced by GSH depletion and are mediated by glial cells. The antioxidant ascorbic acid and the lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid (1-10 microM) provide full protection from BSO toxicity, indicating that arachidonic acid metabolism through the LOX pathway and the generation of reactive oxygen species play a role in the loss of cell viability. In contrast, inhibition of nitric oxide (NO) synthase affords only partial protection from BSO toxicity, suggesting that increased NO production cannot entirely account for cell death in this model. Our data provide evidence that GSH depletion in the presence of glial cells leads to neuronal degeneration that can be prevented by inhibition of LOX. This may have relevance to the pathogenesis of Parkinson's disease, where glial activation and depletion of GSH have been found in the substantia nigra pars compacta.     

Altered differentiation in rat and rabbit limb bud micromass cultures by glutathione modulating agents

Glutathione (GSH) is the primary source of reducing equivalents in most cells, contributes significantly to the cellular redox potential and can control differentiation, proliferation, and apoptosis. Using limb bud micromass cultures from Sprague Dawley rats and New Zealand White rabbits, GSH modulating agents, L-buthionine-S,R-sulfoximine (BSO) and diethyl maleate (DEM) altered the formation of Alcian blue positive chondrogenic foci. Limb bud micromass cultures were treated for 5 d with BSO (50 or 100 μM) or DEM (5–25 μM). GSH content was determined by HPLC analysis. In rat cultures, BSO treatment did not affect differentiation but did show GSH depletion. In rabbit cultures, BSO completely inhibited differentiation and significantly depleted GSH. Treatment of rat cultures with DEM resulted in the dose-dependent decrease of chondrogenic foci, which correlated with a dose-dependent depletion of GSH. DEM completely inhibited rabbit limb bud cell differentiation and depleted GSH by 44%. Inhibition of differentiation was confirmed in rabbit cultures by the reduction in BMP-4 content. Addition of N-acetylcysteine to rabbit micromass cultures restored chondrogenic foci differentiation seen following treatment with both DEM and BSO. These results show species differences in GSH depletion in rat vs. rabbit limb bud cells and implicate GSH and cysteine in affecting pathways involved in chondrocyte differentiation.     

Arsenic trioxide-induced apoptosis and its enhancement by buthionine sulfoximine in hepatocellular carcinoma cell lines

We treated four hepatocellular carcinoma cell lines, HLE, HLF, HuH7, and HepG2 with ATO and demonstrated that arsenic trioxide (ATO) at low doses (1--3 muM) induced a concentration-dependent suppression of cell growth in HLE, HLF, and HuH7. HLE cells underwent apoptosis at 2 microM ATO, which was executed by the activation of caspase-3 through the mitochondrial pathway mediated by caspase-8 activation and Bid truncation. When these cell lines were exposed to ATO in combination with l-S,R-buthionine sulfoximine (BSO) which inhibits GSH synthesis, a synergistic growth suppression was induced, even in HepG2 showing a lower sensitivity to ATO than other cell lines tested. The intracellular GSH levels after the treatment with ATO plus BSO were considerably decreased in HLE cells compared with those after the treatment with ATO or BSO alone. The production of reactive oxygen species (ROS) which was examined by 2' ,7' -dichlorodihydrofluorescein diacetate, increased significantly after the treatment with ATO plus BSO in HLE cells. These findings indicate that ATO at low concentrations induces growth inhibition and apoptosis, and furthermore that the ATO-BSO combination treatment enhances apoptosis through increased production of ROS in hepatocellular carcinoma cells.     

Early redox imbalance mediates hydroperoxide-induced apoptosis in mitotic competent undifferentiated PC-12 cells

Our recent study has demonstrated that cellular redox imbalance can directly initiate apoptosis in a mitotic competent PC-12 cell line without the involvement of reactive oxygen species (ROS). However, whether cell apoptosis induced by ROS is, in fact, mediated by a loss of redox balance caused by the oxidant is unresolved. The linkage between oxidant-mediated apoptosis and the induction of cellular redox was examined in PC-12 cells using the oxidant, tert-butylhydroperoxide (TBH). TBH caused cell apoptosis in 24 h that was preceded by an early increase (30 min) in oxidized glutathione (GSSG). Pretreatment with N-acetyl cysteine prevented TBH-induced GSSG increases and cell apoptosis. Altered Bax/BcL-2 expression and release of mitochondrial cytochrome c occurred post-redox imbalance and was kinetically linked to caspase-3 activation and poly ADP-ribose polymerase cleavage. Moreover, cell apoptosis was attenuated by inhibition of caspase-9, but not caspase-8, and blockade of mitochondrial ROS generation and permeability transition pore attenuated caspase 3 activation and cell apoptosis. Collectively, these results show that TBH-induced GSSG elevation is associated with the disruption of mitochondrial integrity, activation of caspase-3 and cell apoptosis. This redox induction of the apoptotic cascade was dissociated from cellular GSH efflux.     

Involvement of Oxidative Stress in Paraquat-Induced Metallothionein Synthesis Under Glutathione Depletion

The inhibition of glutathione (GSH) synthesis by -buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.     

Implications of altered glutathione metabolism in aspirin-induced oxidative stress and mitochondrial dysfunction in HepG2 cells

We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.     

The effect of glutathione depletion on sister-chromatid exchange induction by cytostatic drugs

Using sister-chromatid exchanges (SCEs) as an indicator for DNA damage, we investigated the role of glutathione (GSH) as a determinant of cellular sensitivity to the DNA-damaging effects of the cytostatic drugs adriamycin (AM) and cyclophosphamide (CP). Exposure of V79 cells to buthionine sulfoximine (BSO) resulted in a complete depletion of cellular GSH content without toxicity and without increasing the SCE frequency. Subsequent 3-h treatment of GSH-depleted cells with AM or S9-mix-activated CP caused a potentiation of SCE induction. In Chinese hamster ovary (CHO) cells, which showed a higher GSH level compared to V79 cells, BSO treatment led to a depletion of GSH to about 5% of the control and increased SCE induction by AM and CP. Compared to V79 cells, the effect of AM on SCE frequencies was less distinct in CHO cells, while CP exerted a similar effect in both cell lines. Pretreatment of V79 cells with GSH increased the cellular GSH content, but had no effect on the induction of SCEs by AM, and pretreatment with cysteine influenced neither GSH levels nor SCE induction by AM. The study shows that SCEs are a suitable indicator for testing the modulation of of drug genotoxicity by GSH. The importance of different GSH contents of cell lines for their response to mutagens is discussed.