Cold-induced apoptosis in isolated rat hepatocytes: protective role of glutathione

Liver conservation for transplantation is usually made at 2-4 degrees C. We studied the effect of rewarming to 37 degrees C for up to 3 h of rat hepatocytes kept at 4 degrees C for 20 h, modulating intracellular glutathione (GSH) concentration either with a GSH precursor (N-acetyl-L-cysteine, NAC), or with GSH depleting agents (diethylmaleate and buthionine sulfoximine, DEM/BSO). Untreated hepatocytes showed time-dependent production of reactive oxygen species (ROS), lipid peroxidation, chromatin condensation and membrane blebbing, decrease in GSH concentration, and protein sulfhydryl groups. Fluorochromatization with Propidium Iodide (PI) and Annexin V (AnxV) of cells rewarmed for 1 h caused an increase of AnxV-positive cells without PI staining and any observed lactate dehydrogenase leakage. TUNEL and DNA-laddering tests were negative for all times and treatments, indicating that apoptosis may occur without DNA fragmentation. Cold preservation and rewarming in the presence of NAC induced a significant improvement in the morphology, less oxidative stress and apoptosis. Conversely, DEM/BSO caused a marked deterioration of morphology, increase of oxidative stress and apoptosis. These results suggested that marked changes in GSH status might play a critical role in triggering apoptosis during cold preservation of isolated rat hepatocytes. NAC, added before rewarming, might represent a therapeutic approach for preventing the early events of apoptosis during cold storage.     

Smokeless tobacco, oxidative stress, apoptosis, and antioxidants in human oral keratinocytes

We have investigated the effects of a smokeless tobacco extract (STE) on lipid peroxidation, cytochrome c reduction, DNA fragmentation and apoptotic cell death in normal human oral keratinocyte cells, and assessed the protective abilities of selected antioxidants. The cells, isolated and cultured from human oral tissues, were treated with STE (0-300 microl;g/ml) for 24 h. Superoxide anion production was determined by cytochrome c reductase. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, whereas apoptotic cell death was assessed by flow cytometry. STE-induced fragmentation of genomic DNA was also determined by gel electrophoresis. The comparative protective abilities of vitamin C (75 microM), vitamin E (75 microM), a combination of vitamins C & E (75 microM each), and a novel grape seed proanthocyanidin (IH636) extract (GSPE) (100 microg/ml) against STE induced oxidative stress and tissue damage were also determined. Following treatment of the cells with 300 microg STE/ml 1.5-7.6-fold increases in lipid peroxidation, cytochrome c reduction and DNA fragmentation were observed. The addition of the antioxidants to cells treated with STE provided 10-54% decreases in these parameters. Approximately 9, 29, and 35% increases in apoptotic cell death were observed following treatment with 100, 200, and 300 microg STE/ml, respectively, and 51-85% decreases in apoptotic cell death were observed with the antioxidants. The results demonstrate that STE produces oxidative tissue damage and apoptosis, which can be attenuated by antioxidants including vitamin C, vitamin E, a combination of vitamins C plus E and GSPE. GSPE exhibited better protection against STE than vitamins C and E, singly and in combination.     

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.     

Assessment of phosphamidon-induced apoptosis in human peripheral blood mononuclear cells: protective effects of N-acetylcysteine and curcumin

The molecular mechanism for noncholinergic toxicity of phosphamidon, an extensively used organophosphate pesticide, is still not clear. The aim of the present study is to find the possible molecular mechanism of this pesticide to induce apoptosis and the role of different drugs for attenuation of such effects. Human peripheral blood mononuclear cells (PBMC) were incubated with increasing concentrations of phosphamidon (0-20 μM) for 6-24 h. The MTT assay reveals that phosphamidon induces cytotoxicity in a dose-dependent manner. Cellular glutathione (GSH) is depleted in a dose-dependent manner from 55% to 70% at concentrations between 10 and 20 μM. The percentage of cells that bind to Annexin-V, which is a representative of cells either undergoing apoptosis or necrosis during 24 h incubation, increases in a dose-dependent manner. Above 5 μM, significant necrosis of cells was observed. DNA fragmentation assay revealed that at low concentration of phosphamidon (1 μM), no appreciable change in DNA fragmentation was seen; however, distinct fragmentation was observed beyond 2.5 μM. Phosphamidon was found to cause significant depletion of GSH, which correlates well with the percentage of cells undergoing apoptosis. An increasing trend in levels of cytochrome c was observed with increasing concentration of phosphamidon, indicating that the apoptotic effect of phosphamidon is mediated through cytochrome c release. Coadministration of the antioxidants N-acetylcysteine and curcumin attenuated phosphamidon-induced apoptosis. This further supports our hypothesis that oxidative stress, as indicated by GSH depletion, results in the induction of apoptosis by release of cytochrome c.     

Cellular toxicity of cadmium ions and their detoxification by heavy metal-specific plant peptides, phytochelatins, expressed in Mammalian cells

The apoptotic cell death of Jurkat cells due to Cd(2+) toxicity was studied by fluorescence microscopic observation and DNA fragmentation assaying. It was suggested that the apoptotic response to Cd(2+) was less clear than that to a typical apoptosis inducer, ultraviolet light (254 nm). Examination of MAP kinase phosphorylation (p38, JNKs, and c-Jun) due to Cd(2+) toxicity indicated that the phosphorylation was very slowly activated (4 h after stimulation), while UV light could activate the phosphorylation immediately. Therefore, it was suggested that Cd(2+) may not be a typical apoptosis inducer. Antioxidants [glutathione (GSH) and N-acetylcysteine (NAC)] could detoxify Cd(2+), indicating that the toxicity is a kind of oxidative stress. The detoxification effect of antioxidants showed cooperation with Bcl-2, suggesting that Cd(2+)-treatment causes diversified toxic signals including oxidative stress. On the addition of a plant-specific peptide, phytochelatin [PC(7), (gammaGlu-Cys)(7)-Gly], to the medium, the detoxification of Cd(2+) and cooperation with Bcl-2 were more intense than in the cases of GSH and NAC. Using an appropriate vector, a PC synthase gene was transferred from Arabidopsis thaliana to the Jurkat cell. The transfectant exhibited resistance to Cd(2+) and production of plant-specific PC (PC(2-6)).     

Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts

Cigarette smoke is a mixture of chemicals having direct and/or indirect toxic effects on different lung cells. We investigated the effect of cigarette smoke on human lung fibroblasts (HFL-1) oxidation and apoptosis. Cells were exposed to various concentrations (1, 5, and 10%) of cigarette smoke extract (CSE) for 3 h, and oxidative stress and apoptosis were assessed by fluorescence-activated cell sorting and confocal laser fluorescence microscopy. Both oxidative stress and apoptosis exhibited a dose-response relationship with CSE concentrations. Lung fibroblasts also showed marked DNA fragmentation at the Comet assay after exposure to 10% CSE. Coincubation of HLF-1 cells with N-acetylcysteine (1 mM) during CSE exposure significantly reduced oxidative stress, apoptosis, and DNA fragmentation, whereas preincubation (3 h) with the glutathione-depleting agent buthionine sulfoximine (125 microM) produced a significant increase of oxidative stress. Cigarette smoke is a potent source of oxidative stress, DNA damage, and apoptosis for HFL-1 cells, and we speculate that this could contribute to the development of pulmonary emphysema in the lungs of smokers.     

Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells.

Recent studies have demonstrated that induction of apoptosis is related to the cell growth inhibition potential of Salvia Miltiorrhiza (SM), a traditional herbal medicine. In the present study, we further explore the mechanistic pathway involved in SM-induced apoptosis in human hepatoma HepG2 cells. A rapid decline of intracellular glutathione (GSH) and protein thiol content was found in SM-treated cells. Moreover. SM exposure resulted in mitochondrial dysfunction as demonstrated by: (i) the onset of mitochondrial permeability transition (MPT); (ii) the disruption of mitochondrial membrane potential (MMP); and (iii) the release of cytochrome c from mitochondria into the cytosol. Subsequently, elevated level of intracellular reactive oxygen species (ROS) was observed prior to the onset of DNA fragmentation. However, no caspase-3 cleavage was observed throughout the whole period of SM treatment, while a caspase-3-independent poly(ADP-ribose) polymerase (PARP) cleavage was noted at the late stage in SM-induced apoptosis. Pretreatment of cells with N-acetylcysteine (NAC), the GSH synthesis precursor, conferred complete protection against MMP loss, ROS generation and apoptosis induced by SM. MPT inhibitors, cyclosporin A plus trifluoperazine, partially restored intracellular GSH content, and reduced SM-induced ROS formation and subsequently inhibited cell death. Moreover, antioxidants NAC, deferoxamine and catalase had little effect on GSH depletion and mitochondrial dysfunction, yet still were able to completely protect cells from SM-induced apoptosis. Taken together, our results suggest that SM deplete intracellular thiols, which, in turn, causes MPT and subsequent increase in ROS generation, and eventually apoptotic cell death.     

Tetrandrine cytotoxicity and its dual effect on oxidative stress-induced apoptosis through modulating cellular redox states in Neuro 2a mouse neuroblastoma cells

Tetrandrine (TET), a plant alkaloid, is known primarily as a non-selective Ca(2+) channel blocker. On the contrary to the cytoprotective effect on ischemia/reperfusion injury, TET has also been reported to cause cytotoxicity. In this study, we wished to understand the apparently disparate effects of this potential drug and thus investigated molecular mechanisms on proliferation and apoptosis and its effect on oxidative stress-induced apoptosis in Neuro 2a mouse neuroblastoma cells. We showed that TET, at high concentrations, induced cell cycle arrest and apoptosis through oxidative stress with following observations. Firstly, 10 microM TET elevated the reactive oxygen species (ROS) level and accordingly depleted glutathione (GSH) content. Secondly, pretreatment with antioxidants (NAC or GSH) protected cells from TET-induced apoptosis. We also demonstrated that treatment with 10 microM TET caused not only induction of p53, p21(waf1), and Bax, but also nuclear translocation of p53 and hypo-phosphorylation of pRb concurrently. Our important finding is that the concentration-dependent dual effect of TET, either inhibiting or promoting cell death induced by H(2)O(2) was observed, probably through regulating redox balance, which was well reflected on the GSH content in each condition. Besides, inhibition of Ca(2+) influx protected cells from H(2)O(2)-induced apoptosis even in the presence of 10 microM TET. Taken together, our data suggest that TET regulation of cellular redox states may play a major role in its dual action of cytotoxicity and cytoprotection.     

Endosulfan decreases cell growth and apoptosis in human HaCaT keratinocytes: partial ROS-dependent ERK1/2 mechanism

Endosulfan is an organochlorine insecticide described as a potential carcinogen in humans. This insecticide was recently reported to alter the mitogen-activated protein (MAP) kinase signaling pathways and is suspected to affect cell growth and differentiation in human keratinocytes. This study was designed to assess the mitogenic, apoptogenic, and genotoxic effects of endosulfan on the HaCaT cell line. We first found that 25 microM endosulfan led to persistent extracellular signal-regulated kinase (ERK)1/2 phosphorylation with an accumulation of the phosphorylated form in the nucleus, probably caused by MAP kinase phosphatase (MKP) inhibition. As previously described under sustained ERK1/2 activation, cell growth was decreased: delayed confluency and 35% decrease of BrdU incorporation was demonstrated in endosulfan-treated keratinocytes. In addition, endosulfan has been shown to generate transient reactive oxygen species (ROS), and blocking this oxidative stress by N-acetyl cysteine (NAC) strongly prevented both persistent nuclear ERK1/2 phosphorylation and cell growth decrease. Additional experiments demonstrated that unchanged endosulfan rather than its metabolites has mutagenic effects (Ames positive without S9) and increased DNA strand breaks (Comet assay) in HaCaT cells, via a ROS-dependent mechanism. Therefore, to assess the putative pro-apoptotic response of damaged cells, caspases 3/7 activity and poly(ADP-ribose)-polymerase (PARP) cleavage were measured. The results clearly indicated that endosulfan inhibited both spontaneous and staurosporine-induced apoptosis. Taken together, these findings strongly support that endosulfan induces ROS generation leading to sustained ERK1/2 phosphorylation and decrease in cell growth. Moreover, endosulfan was found to inhibit apoptosis and this could contribute to mutant cell survival and therefore have possible carcinogenic effects.     

Reactive Oxygen Species-Mediated DNA Damage and Apoptosis in Human Skin Epidermal Cells After Exposure to Nickel Nanoparticles

Nickel nanoparticles (NiNPs) are increasingly used in various applications due to their unique properties. However, there is little information concerning the toxicity of NiNPs in the human skin cell (A431). The present study was designed to investigate the cytotoxicity, apoptosis, and DNA damage due to NiNPs in A431 cells. A cellular proliferative capacity test showed that NiNPs induce significant cytotoxicity in a dose- and time-dependent manner. NiNPs were also found to induce oxidative stress evidenced by the generation of reactive oxygen species (ROS) and depletion of glutathione (GSH). Further, co-treatment with the antioxidant N-acetylcysteine (NAC) mitigated the ROS generation due to NiNPs, suggesting the potential mechanism of oxidative stress. NiNPs also induced significant elevation of lipid peroxidation, catalase, and superoxide dismutase and caspase-3 activity in A431 cells. In addition, NAC suppressed NiNP-induced caspase-3 activity. DNA fragmentation analysis using the comet assay showed that the NiNPs cause genotoxicity in a dose- and time-dependent manner. Therefore, the study points out the capability of the NiNPs to induce oxidative stress resulting in apoptosis and genotoxicity. This study warrants more careful assessment of NiNPs before their industrial applications.     

PGE 1 Protection against Apoptosis Induced by d -galactosamine is Not Related to the Modulation of Intracellular Free Radical Production in Primary Culture of Rat Hepatocytes

d -galactosamine ( d -GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE 1 treatment reduced necrosis and apoptosis induced by d -GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by d -GalN (0-40 mM). Also, the present study investigated if PGE 1 (1 &#119 M) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H 2 O 2 ), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of d -GalN concentrations (2.5-10 mM) induced apoptosis in association with a moderate oxidative stress. High d -GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with d -GalN (2.5-10 mM)-treated cells. Although PGE 1 reduced apoptosis induced by d -GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.     

PGE1 protection against apoptosis induced by D-galactosamine is not related to the modulation of intracellular free radical production in primary culture of rat hepatocytes

d -galactosamine ( d -GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE 1 treatment reduced necrosis and apoptosis induced by d -GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by d -GalN (0-40 mM). Also, the present study investigated if PGE 1 (1 μM) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H 2 O 2 ), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of d -GalN concentrations (2.5-10 mM) induced apoptosis in association with a moderate oxidative stress. High d -GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with d -GalN (2.5-10 mM)-treated cells. Although PGE 1 reduced apoptosis induced by d -GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.     

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.     

Antioxidants-induced rearrangements of actin cytoskeleton in 3T3 and 3T3-SV40 fibroblasts

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.     

Effects of naringin on cytosine arabinoside (Ara-C)-induced cytotoxicity and apoptosis in P388 cells

Naringin (NG), a flavonoid in grapefruit and citrus, has been reported to exhibit antioxidant effects and pharmacological actions. Recently, we have reported that NG suppressed the cytotoxicity and apoptosis induced by H(2)O(2), a typical pro-oxidant, in mouse leukemia P388 cells. Cytosine arabinoside (1-beta-d-arabinofuranosylcytosine; Ara-C) is the most important antimetabolite chemotherapeutic drug used for acute leukemia. It has been suggested that Ara-C-induced cytotoxicity is caused by apoptosis, which is mediated by reactive oxygen species (ROS). In this study, we examined the effect of NG on the cytotoxicity and apoptosis in mouse leukemia P388 cells treated with Ara-C. Ara-C caused cytotoxicity in a concentration and time-dependent manner in the cells. N-Acetyl-L-cysteine (NAC), cystamine (CysA) or a reduced form of glutathione (GSH), typical antioxidants significantly blocked Ara-C-induced cytotoxicity. Similarly, Ara-C-induced cell death was completely prevented by NG. NG strongly reduced ROS production caused by Ara-C in the cells. NG slightly increased the activities of antioxidant enzymes, catalase and glutathione peroxidase. Ara-C caused apoptosis with nuclear morphological change and DNA fragmentation. NG remarkably attenuated the Ara-C-induced apoptosis. NG completely blocked the DNA damage caused by Ara-C treatment at 6 h using the Comet assay. Our data suggest that NG reduces Ara-C-induced oxidative stress through both an inhibition of the generation of ROS production and an increase in antioxidant enzyme activities. Consequently, NG blocked apoptosis caused by Ara-C-induced oxidative stress, resulting in the inhibition of the cytotoxicity of Ara-C.     

Oxidative stress is not required for the induction of apoptosis upon glutamine starvation of Sp2/0-Ag14 hybridoma cells

L-glutamine (Gln) withdrawal rapidly triggers apoptosis in the murine hybridoma cell line Sp2/0-Ag14 (Sp2/0). In this report, we examined the possibility that Gln deprivation of Sp2/0 cells triggers an oxidative stress which would contribute to the activation of apoptotic pathways. Gln withdrawal triggered an oxidative stress in Sp2/0 cells, as indicated by an increased accumulation of reactive oxygen species (ROS) and an increase in the intracellular content in protein carbonyl groups. Gln starvation also caused a decrease in the intracellular levels of glutathione (GSH). However, a decrease in GSH was not sufficient to induce Sp2/0 cell death since reducing GSH levels with DL-buthionine-[S,R]-sulfoximine did not affect cell viability. The antioxidant N-acetyl-L-cysteine (NAC), while effective in inhibiting ROS accumulation and oxidative stress, did not prevent the loss in cell viability or the processing and activation of caspase-3 triggered by Gln starvation. On the other hand, NAC did reduce the formation of apoptotic bodies in dying cells. Altogether these results indicate that in Sp2/0 cells, Gln deprivation leads to the induction of an oxidative stress which, while involved in the formation of apoptotic bodies, is not essential to the activation of the cell death program.     

Dual role of glutathione in selenite-induced oxidative stress and apoptosis in human hepatoma cells

It is well known that glutathione, the major intracellular antioxidant, is closely involved in the metabolism and bioactivity of selenium. In the present study, glutathione was demonstrated to play a dual role on selenite (Se)-induced oxidative stress and apoptosis in human hepatoma HepG(2) cells. The experiment was carried out in two different modes to modulate intracellular reduced glutathione (GSH) content. In Mode A (pretreatment), cells were pretreated with N-acetylcysteine (NAC), buthionine sulfoximine (BSO), or GSH prior to Se exposure. In Mode B (simultaneous treatment), cells were treated with Se and NAC, BSO, or GSH simultaneously. It was found that Se-induced oxidative stress and apoptosis are closely related to the intracellular level of GSH. Both the increase and depletion of GSH content significantly enhanced Se-induced oxidative stress and apoptosis in HepG(2) cells. Results from this study clearly demonstrated that GSH has a dual role in the effects of Se on cancer cells: (i) GSH acts as a pro-oxidant, facilitating Se-induced oxidative stress, and (ii) GSH acts as an antioxidant, protecting against Se-induced oxidative stress and apoptosis. Understanding such a unique association between GSH and Se may help to explain the controversy in the literature over the complex relationship between selenium and glutathione, and ultimately the capability of selenium to prevent cancer.     

Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.     

Senecio latifolius induces in vitro hepatocytotoxicity in a human cell line

The objectives of this study were twofold: (i) to determine the mechanism(s) of Senecio-induced toxicity in human hepatoblastoma cells (HepG2) in vitro and whether such toxicity could be prevented using N-acetyl-cysteine (NAC), and (ii) to evaluate whether caspases are involved in Senecio-induced apoptosis. Cells were treated with aqueous extracts of Senecio (10 mg x mL-1) with and without NAC. Cytotoxicity was determined by using the MTT assay. Total glutathione (GSH) was measured by using the Tietze assay. Cells were also treated with aqueous extracts of Senecio in the presence or absence of 50 micromol/L caspase-3 inhibitor (IDN) for 24 h. Apoptosis was determined by transmission electron microscopy, and DNA fragmentation was determined by ELISA and terminal dUTP nick-end labelling (TUNEL). Senecio produced cytotoxicity and depleted GSH in a concentration- and time-dependent manner. A significant depletion in GSH was observed after 15 min (p < 0.001 vs. control), whereas significant cytotoxicity was only observed after 3 h (p < 0.001 vs. control). Treatment with NAC prevented Senecio-induced GSH depletion and resulted in a significant decrease in Senecio-induced cytotoxicity (p < 0.001 vs. NAC-untreated cells). Treatment with Senecio for 24 h resulted in 22% +/- 2.5% (p < 0.001) apoptosis (vs. control). Pretreatment with 50 mumol caspase inhibitor reduced Senecio-induced apoptosis significantly (vs. non-exposed to IDN) (12% +/- 1.5%; p < 0.05). Our results suggest the mechanism of Senecio-induced cytotoxicity in HepG2 cells in vitro involves depletion of cellular GSH. Cytotoxicity is reduced by supplementation with NAC, which thus prevents GSH depletion. Caspase activation is involved in Senecio-induced apoptosis.     

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.