Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-alpha-mediated apoptosis

Tumour necrosis factor-alpha (TNF-alpha) was found to be a cell cycle-independent apoptogenic cytokine in cultured fibroblast L929 cells. This assertion is based on the observations (1) TNF-alpha increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytometry, and (2) TNF-alpha induced DNA fragmentation as resolved by agarose gel electrophoresis. When cells were exposed to TNF-alpha (50 ng/ml), a slow rise in intracellular free Ca2+ level and a delayed increase in the production of reactive oxygen species (ROS) (both observed 3 h after the addition of TNF-alpha) were observed in fluo-3 and fura-red or dichlorofluorescein loaded cells, respectively. Interestingly, challenge of cells with TNF-alpha in the presence of BAPTA/AM, an intracellular Ca2+ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidinooxy (4OH-TEMPO) blocked the TNF-alpha-mediated Ca2+ rise. Moreover, when cells were exposed to TNF-alpha with both 4OH-TEMPO and BAPTA/AM, more viable cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca2+ are two cross-talk messengers important in TNF-alpha-mediated apoptosis.     

Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not on NADPH oxidase

Calcium ions (Ca(2+)) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca(2+) in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca(2+) induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death.     

Radiation-induced long-lived extracellular radicals do not contribute to measurement of intracellular reactive oxygen species using the dichlorofluorescein method

The dichlorofluorescein method has become a standard technique for measuring reactive oxygen species (ROS) formed in cells by ionizing radiation. A recent report (Korystov et al., Radiat. Res. 168, 226-232, 2007) has suggested that the method is subject to an artifact in that it erroneously reports hydrogen peroxides generated in the extracellular medium as ROS formed intracellularly by ionizing radiation. It was hypothesized that radiation-induced extracellular peroxides enter cells in the minutes after radiation exposure and subsequently oxidize the intracellular dichlorofluorescin probe and that dichlorofluorescein fluorescence is not due to ROS formed intracellularly by ionizing radiation. We tested this hypothesis by measuring the contribution of long-lived radicals formed in medium by ionizing radiation on intracellular dichlorofluorescein fluorescence. We found no evidence that this artifact contributes significantly to intracellular dichlorofluorescein fluorescence. These results and those of Korystov et al. are discussed in view of cellular dichlorofluorescin leakage and radiation chemistry. We conclude that the dichlorofluorescein method is effective for quantifying intracellular ROS induced by ionizing radiation.     

Reactive oxygen species modulates the intracellular level of HBx viral oncoprotein

HBx (hepatitis B virus X) viral oncoprotein is a multifunctional protein of which the cellular level may be one of the important factors in determining HBV-mediated pathological progression of liver diseases, chronic hepatitis, and hepatocellular carcinoma. Our previous work revealed that adriamycin, a chemotherapeutic agent, caused a marked increase in the intracellular level of HBx by retarding its rapid degradation. In the present study, modulation of HBx expression was found to be confined to adriamycin but not to other chemotherapeutic agents, cisplatin and 5-fluorouracil. Interestingly, adriamycin caused a rapid increase of reactive oxygen species (ROS) and its accumulation continued until 24h. In contrast, two other agents had little effect on ROS generation, suggesting the possible involvement of ROS in the HBx regulation. In fact, direct addition of H(2)O(2) to the cells significantly increased the level of HBx protein in HBx-expressing ChangX-34 cells as well as in hepatitis B virus-related hepatoma cells, PLC/PRF/5 and HepG2.2.15 cells. Furthermore, antioxidants, N-acetyl-cysteine and pyrrolidinedithiocarbamate (PDTC), completely abolished the increase of HBx protein induced by adriamycin, indicating that adriamycin modulates the intracellular HBx level via ROS generation. Together, these findings provide a novel aspect of HBx regulation by cellular ROS level. Therefore, intracellular microenvironments generating ROS such as severe inflammation may aggravate the pathogenesis of liver disease by accumulating the HBx level.     

Ca(2+)]-dependent generation of intracellular reactive oxygen species mediates maitotoxin-induced cellular responses in human umbilical vein endothelial cells

Maitotoxin (MTX) is known as one of the most potent marine toxins involved in Ciguatera poisoning, but intracellular signaling pathways caused by MTX was not fully understood. Thus, we have investigated whether intracellular reactive oxygen species (ROS) are involved in MTX-induced cellular responses in human umbilical vein endothelial cells. MTX induced a dose-dependent increase of intracellular [Ca(2+)]. MTX stimulated the production of intracellular ROS in a dose- and time-dependent manner, which was suppressed by BAPTA-AM, an intracellular Ca(2+) che-lator. Ionomycin also elevated the ROS production in a dose-dependent manner. MTX elevated transamidation activity in a time-dependent manner and the activation was largely inhibited by transfection of tissue transglutaminase siRNA. The activation of tissue transglutaminase and ERK1/2 by MTX was sup-pressed by BAPTA-AM or ROS scavengers. In addition, MTX-induced cell death was significantly de-layed by BAPTA-AM or a ROS scavenger. These results suggest that [Ca(2+)]-dependent generation of in-tracellular ROS, at least in part, play an important role in MTX-stimulated cellular responses, such as activation of tTGase, ERK phosphorylation, and in-duction of cell death, in human umbilical vein endothelial cells.     

Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-α-mediated apoptosis

Tumour necrosis factor-α(TNF-α) was found to be a cell cycle-independent apoptogenic cytokine in cultured fibroblast L929 cells. This assertion is based on the observations (1) TNF-α increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytometry, and (2) TNF-α induced DNA fragmentation as resolved by agarose gel electrophoresis. When cells were exposed to TNF-α (50ng/ml), a slow rise in intracellular free Ca²⁺ level and a delayed increase in the production of reactive oxygen species (ROS) (both observed 3h after the addition of TNF-α) were observed in fluo-3 and furared or dichlorofluorescein loaded cells, respectively. Interestingly, challenge of cells with TNF-α in the presence of BAPTA/AM, an intracellular Ca²⁺ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidinooxy (4OH-TEMPO) blocked the TNF-α-mediated Ca²⁺ rise. Moreover, when cells were exposed to TNF-α with both 4OH-TEMPO and BAPTA/AM, more viable cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca²⁺ are two cross-talk messengers important in TNF-α-mediated apoptosis.     

HBx or HCV core gene expression in HepG2 human liver cells results in a survival benefit against oxidative stress with possible implications for HCC development

Hepatitis virus replication in the liver is often accompanied by inflammation resulting in the formation of reactive oxygen species (ROS) and nitric oxide (NO) and these may induce cell death. We investigated whether the expression of HBx or HCV core protein in HepG2 cells has an influence on the sensitivity of these cells for oxidative radicals. Our previous study, using the inducible HBV model of HepAD38, revealed that oxidative-stress-related genes are upregulated by virus replication. In the present study, we examined the intracellular pro-oxidant status with dichlorofluorescein (DCF) in HepG2 cell lines transfected with HBx, HbsAg and HCV core. Baseline intracellular oxidative levels were not different in the cell lines expressing viral proteins as compared to control. However, when these cells were exposed to H(2)O(2), the viral protein expressing cells, especially those expressing HBx, showed a reduced level of ROS. This suggests that HBx and HCV core transfected cells can convert H(2)O(2) to less reactive compounds at a higher rate than the control cells. When HBx or HCV core expressing cells were exposed to peroxynitrite (a highly reactive product formed under physiological conditions through interaction of superoxide (O(2)(-)) with NO) these cells were less sensitive to induction of cell death. In addition, these cell lines were less prone to cell death when exposed to H(2)O(2) directly. In conclusion, HBx and HCV core expression in HepG2 cells leads to a survival benefit under oxidative stress which in vivo can be induced during inflammation.     

植物细胞中蓝光诱导ROS生成的识别和亚细胞定位检测

以2’,7’-二氯二氢荧光素二乙酯(dichlorofluorescein diacetate,H2DCF-DA)为荧光探针孵育拟南芥叶表皮条,利用荧光光谱和激光共聚焦扫描显微技术,对高辐照蓝光诱导下叶肉细胞活性氧(reactive oxygen spe-cies,ROS)的生成,进行了分子识别和亚细胞定位检测。结果表明:植物细胞在蓝光诱导下,可以产生大量的ROS。过氧化氢酶清除实验表明:高辐照蓝光诱导产生的ROS,主要成分是H2O2,并且主要定位在叶绿体和细胞膜上。     

Effects of amino acids from selenium-rich silkworm pupas on human hepatoma cells

Selenium (Se) plays an important role in cancer-prevention. Silkworm pupas have been used as a Chinese traditional medicine since ancient time. In order to find effective carcinostatic agents, Se-rich amino acids were extracted from Ziyang silkworm pupas. The Se content of Ziyang pupas was measured to be 215 times higher than that of Luoyang normal ones, and the majority of Se was stored in proteins. Composition analysis showed that Se-rich amino acids from Ziyang pupas had higher amounts of selenomethionine, methionine, cystine, and tyrosine than normal amino acids from Luoyang pupas which were rich in amino acids containing alkyl side chains. When cultured with human hepatoma cells SMMC-7721, Se-rich amino acids at concentrations of 0.5, 1.5, and 2.5 micromol L(-1) Se significantly and dose-dependently inhibited cell viability, induced changes in cell morphology and cycle, and caused cell apoptosis. On the contrary, normal amino acids did not show any inhibitory effect on SMMC-7721 cells. Sodium selenite or selenomethionine at the same Se concentrations only slightly inhibited the hepatoma cells. Mechanism study showed that selenium-rich amino acids could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. Those results suggested that Se-rich amino acids were effective carcinostatic agents compared with sodium selenite and selenomethionine. The mechanism for their hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation.     

Oxidation pathways underlying the pro-oxidant effects of apigenin

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (H₂DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H₂DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H₂DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H₂DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.     

Eckol isolated from Ecklonia cava attenuates oxidative stress induced cell damage in lung fibroblast cells

We have investigated the cytoprotective effect of eckol, which was isolated from Ecklonia cava, against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Eckol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, eckol reduced H(2)O(2) induced cell death in V79-4 cells. In addition, eckol inhibited cell damage induced by serum starvation and radiation by scavenging ROS. Eckol was found to increase the activity of catalase and its protein expression. Further, molecular mechanistic study revealed that eckol increased phosphorylation of extracellular signal-regulated kinase and activity of nuclear factor kappa B. Taken together, the results suggest that eckol protects V79-4 cells against oxidative damage by enhancing the cellular antioxidant activity and modulating cellular signal pathway.     

Role of reactive oxygen species in bradykinin-induced proliferation of vascular smooth muscle cells

In addition to the induction of cell proliferation and migration, bradykinin (BK) can increase c-fos mRNA expression, activate ERK 1/2 and generate reactive oxygen species (ROS) in vascular smooth muscle cells (VSMC). It is not known, however, whether BK can induce cellular proliferation and extracellular matrix production via redox-sensitive signaling pathways. We investigated the role(s) of ROS in proliferation, migration and collagen synthesis induced by BK in VSMC derived from Sprague Dawley rat aorta. BK (10 nM) increased VSMC proliferation by 30% (n=5); this proliferation was inhibited by the antioxidants N-acetylcysteine (20 mM) and alpha-lipoic acid (LA, 250 mM). In addition, BK induced an increase in cell migration and in collagen levels that were blocked by LA. ROS production induced by BK (n=10) was significantly inhibited by bisindolylmaleimide (4microM) and by PD98059 (40microM). These results suggest that: 1) ROS participate in the mechanism(s) used by bradykinin to induce cellular proliferation; 2) bradykinin induces ROS generation through a pathway that involves the kinases PKC and MEK; and 3) ROS participate in the pathways mediating cell migration and the production of collagen as a response to treatment with bradykinin. To our knowledge, this is the first report describing mechanisms to explain the participation of ROS in the cellular proliferation and extracellular matrix pathway regulated by BK.     

Requirement of intracellular free thiols for hydrogen peroxide-induced hypertrophy in cardiomyocytes

Reactive oxygen species (ROS) are by-products of aerobic metabolism and are implicated in the pathogenesis of several diseases. H(2)O(2) produces oxidative stress and acts as a second messenger in several cell types. We tested whether the effect of H(2)O(2) on cellular events could be altered by changes in the intracellular redox status in a cardiomyocyte cell line. Using flow cytometric measurements, we found that adding H(2)O(2) induced hypertrophy in control cells in a time-dependent manner. Pre-incubation of the cells with buthionine sulfoximine (BSO), an inhibitor of de novo GSH synthesis, induced increase in the number of cells of small sizes by the addition of H(2)O(2) as compared to non-BSO pre-incubated control cells, and exacerbated the decrease in viability. Total thiol and GSH levels in H9c2 cells pre-incubated with BSO were about 75 and 30% of control, respectively, and GSH levels fell to below the limitation of detection after the addition of H(2)O(2), although total thiol levels were not markedly decreased. In the cells pre-incubated with BSO, hypertrophy was not observed by the addition of H(2)O(2) at any level of concentration. N-acetyl-L-cysteine and cysteine not only prevented increase in the number of cells of small sizes caused by H(2)O(2) but also induced hypertrophy in cells pre-incubated with BSO. These results suggest that the intracellular free thiol levels determine whether cell death or hypertrophy occurs in cardiomyocytes in the presence of H(2)O(2). On the other hand, the hypertrophied cells did not become larger by adding H(2)O(2), but had high levels of cellular GSH, suggesting the possibility that the hypertrophied cells have tolerance to oxidative stress.     

Rodent and human mast cells produce functionally significant intracellular reactive oxygen species but not nitric oxide

In immunity, reactive oxygen species (ROS) and nitric oxide (NO) are important antimicrobial agents and regulators of cell signaling and activation pathways. However, the cellular sources of ROS and NO are much debated. Particularly, there is contention over whether mast cells, key secretory cells in allergy and immunity, can generate these chemical species, and if so, whether they are of functional significance. We therefore examined directly by flow cytometry the capacity of mast cells to generate intracellular ROS and NO using the respective cell-permeable fluorescent probes dichlorodihydrofluorescein and diaminofluorescein and evaluated the effects of inhibitors of ROS and NO synthesis on cell degranulation. For each of three mast cell types (rat peritoneal mast cells, mouse bone marrow-derived mast cells, and human blood-derived mast cells), degranulation stimulated by IgE/antigen was accompanied by production of intracellular ROS but not NO. Inhibition of ROS production led to reduced degranulation, indicating a facilitatory role for ROS, whereas NO synthase inhibitors were without effect. Likewise, bacterial lipopolysaccharide and interferon-gamma over a wide range of conditions failed to generate intracellular NO in mast cells, whereas these agents readily induced intracellular NO in macrophages. NO synthase protein, as assessed by Western blotting, was readily induced in macrophages but not mast cells. We conclude that rodent and human mast cells generate intracellular ROS but not NO and that intracellular ROS but not intracellular NO are functionally linked to mast cell degranulation.     

Cooperation between p21 and Akt is required for p53‐dependent cellular senescence

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53‐induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53‐induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53‐induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H‐Ras‐induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt‐dependent NF‐κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53‐mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence‐associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

Screening assay for oxidative stress in a feline astrocyte cell line, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H(2)DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H(2)DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H(2)DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H(2)DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H(2)DCFDA fluorescence was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.     

Genotoxic risk and oxidative DNA damage in HepG2 cells exposed to perfluorooctanoic acid

Perfluorooctanoic acid (C8HF15O2, PFOA) is widely used in various industrial fields for decades and it is environmentally bioaccumulative. PFOA is known as a potent hepatocarcinogen in rodents. But it is not yet clear whether it is also carcinogenic in humans, and the genotoxic effects of PFOA on human cells have not yet been examined. In this study, the genotoxic potential of PFOA was investigated in human hepatoma HepG2 cells in culture using single cell gel electrophoresis (SCGE) assay and micronucleus (MN) assay. In order to clarify the underlying mechanism(s) we measured the intracellular generation of reactive oxygen species (ROS) using dichlorofluorescein diacetate as a fluorochrome. The level of oxidative DNA damage was evaluated by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG) in PFOA-treated HepG2 cells. PFOA at 50-400 microM caused DNA strand breaks and at 100-400 microM MN in HepG2 cells both in a dose-dependent manner. Significantly increased levels of ROS and 8-OHdG were observed in these cells. We conclude that PFOA exerts genotoxic effects on HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS.     

Changes in the antioxidant systems as part of the signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobacco Bright-Yellow 2 cells

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for the activation of the hypersensitive reaction, a defense response induced in the noncompatible plant-pathogen interaction. However, its involvement in activating programmed cell death (PCD) in plant cells has been questioned. In this paper, the involvement of the cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition to the culture medium of NO and/or ROS generators. The individual increase in NO or ROS had different effects on the studied parameters than the simultaneous increase in the two reactive species. NO generation did not cause an increase in phenylalanine ammonia-lyase (PAL) activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death when it was generated at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed. The involvement of ASC and GSH as part of the transduction pathway leading to PCD is discussed.