Intracellular GSH level is a factor in As4.1 juxtaglomerular cell death by arsenic trioxide

Arsenic trioxide has been known to regulate many biological functions such as cell proliferation, apoptosis, differentiation, and angiogenesis in various cell lines. We investigated the involvement of GSH and ROS such as H(2)O(2) and O(2)(*-) in the death of As4.1 cells by arsenic trioxide. The intracellular ROS levels were changed depending on the concentration and length of incubation with arsenic trioxide. The intracellular O(2)(*-) level was significantly increased at all the concentrations tested. Arsenic trioxide reduced the intracellular GSH content. Treatment of Tiron, ROS scavenger decreased the levels of ROS in 10 microM arsenic trioxide-treated cells. Another ROS scavenger, Tempol did not decrease ROS levels in arsenic trioxide-treated cells, but slightly recovered the depleted GSH content and reduced the level of apoptosis in these cells. Exogenous SOD and catalase did not reduce the level of ROS, but did decrease the level of O(2)(*-). Both of them inhibited GSH depletion and apoptosis in arsenic trioxide-treated cells. In addition, ROS scavengers, SOD and catalase did not alter the accumulation of cells in the S phase induced by arsenic trioxide. Furthermore, JNK inhibitor rescued some cells from arsenic trioxide-induced apoptosis, and this inhibitor decreased the levels of O(2)(*-) and reduced the GSH depletion in these cells. In summary, we have demonstrated that arsenic trioxide potently generates ROS, especially O(2)(*-), in As4.1 juxtaglomerular cells, and Tempol, SOD, catalase, and JNK inhibitor partially rescued cells from arsenic trioxide-induced apoptosis through the up-regulation of intracellular GSH levels.     

A superoxide anion generator, pyrogallol induces apoptosis in As4.1 cells through the depletion of intracellular GSH content

We investigated the involvement of ROS such as H2O2 and O2*-, and GSH in As4.1 cell death induced by pyrogallol. The intracellular H2O2 levels were decreased or increased depending on the concentration and incubation time of pyrogallol. The levels of O2*- were significantly increased. Pyrogallol reduced the intracellular GSH content. And ROS scavengers, Tempol, Tiron, Trimetazidine and NAC could not significantly down-regulate the production of H2O2 and O2*-. However, these ROS scavengers slightly inhibited apoptosis. Interestingly, Tempol showing the recovery of GSH depletion induced by pyrogallol significantly decreased apoptosis without the significant reduction of intracellular O2*- levels. SOD and catalase did not change the level of H2O2 but decreased the level of O2*-. The inhibition of GSH depletion by these was accompanied with the decrease of apoptosis, as evidenced by sub-G1 DNA content, annexin V staining, mitochondria membrane potential (DeltaPsi(m)) and Western data. In addition, ROS scavengers and SOD did not alter a G2 phase accumulation of the cell cycle induced by pyrogallol. However, catalase changed the cell cycle distributions of pyrogallol-treated cells to those of pyrogallol-untreated cells. In summary, we have demonstrated that pyrogallol potently generates ROS, especially O2*-, in As4.1 JG cells, and Tempol, SOD and catalase could rescue to a lesser or greater extent cells from pyrogallol-induced apoptosis through the up-regulation of intracellular GSH content.     

Apoptosis in arsenic trioxide-treated Calu-6 lung cells is correlated with the depletion of GSH levels rather than the changes of ROS levels

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation in various cells. We investigated an involvement of ROS such as H(2)O(2) and O(2)(*-), and GSH in ATO-treated Calu-6 cell death. The levels of intracellular H(2)O(2) were decreased in ATO-treated Calu-6 cells at 72 h. However, the levels of O(2)(*-) were significantly increased. ATO reduced the intracellular GSH content. Many of the cells having depleted GSH contents were dead, as evidenced by the propidium iodine staining. The activity of CuZn-SOD was strongly down-regulated by ATO at 72 h while the activity of Mn-SOD was weakly up-regulated. The activity of catalase was decreased by ATO. ROS scavengers, Tiron and Trimetazidine did not reduce levels of apoptosis and intracellular O(2)(*-) in ATO-treated Calu-6 cells. Tempol showing a decrease in intracellular O(2)(*-) levels reduced the loss of mitochondrial transmembrane potential (DeltaPsi(m)). Treatment with NAC showing the recovery of GSH depletion and the decreased effect on O(2)(*-) levels in ATO-treated cells significantly inhibited apoptosis. In addition, BSO significantly increased the depletion of GSH content and apoptosis in ATO-treated cells. Treatment with SOD and catalase significantly reduced the levels of O(2)(*-) levels in ATO-treated cells, but did not inhibit apoptosis along with non-effect on the recovery of GSH depletion. Taken together, our results suggest that ATO induces apoptosis in Calu-6 cells via the depletion of the intracellular GSH contents rather than the changes of ROS levels.     

Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death

Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death.     

An ROS generator, antimycin A, inhibits the growth of HeLa cells via apoptosis

Antimycin A (AMA), an inhibitor of electron transport in mitochondria, has been used as a reactive oxygen species (ROS) generator in biological systems. Here, we investigated the in vitro effect of AMA on apoptosis in HeLa cells. AMA inhibited the growth of HeLa cells with an IC(50) of about 50 microM. AMA efficiently induced apoptosis, as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)), Bcl-2 down-regulation, Bax up-regulation, and PARP degradation. All caspase inhibitors used in this experiment, especially pan-caspase inhibitor (Z-VAD), could rescue some HeLa cells from AMA-induced cell death. When we examined the changes of the ROS, H(2)O(2) or O(2) (.-), in AMA-treated cells, H(2)O(2) and O(2) (.-) were markedly increased. In addition, we detected the depletion of GSH content in AMA-treated cells. Pan-caspase inhibitor showing the efficient anti-apoptotic effect significantly reduced GSH depletion by AMA. Superoxide dismutase (SOD) and catalase did not reduce intracellular ROS, but these could strongly rescue the cells from apoptosis. However, these anti-apoptotic effects were not accompanied by the recovery of GSH depletion. Interestingly, catalase significantly decreased the CMF negative (GSH depletion) and propidium iodide (PI) positive cells, indicating that catalase strongly maintained the integrity of the cell membrane in CMF negative cells. Taken together, these results demonstrate that AMA potently generates ROS, induces the depletion of GSH content in HeLa cells, and strongly inhibits the growth of HeLa cells throughout apoptosis.     

Suppression of arsenic trioxide-induced apoptosis in HeLa cells by N-acetylcysteine

Arsenic trioxide (ATO) can affect many biological functions such as apoptosis and differentiation in various cells. We investigated the involvement of ROS and GSH in ATO-induced HeLa cell death using ROS scavengers, especially N-acetylcysteine (NAC). ATO increased intracellular O(2)(*-) levels and reduced intracellular GSH content. The ROS scavengers, Tempol, Tiron and Trimetazidine, did not significantly reduce levels of ROS or GSH depletion in ATO-treated HeLa cells. Nor did they reduce the apoptosis induced by ATO. In contrast, treatment with NAC reduced ROS levels and GSH depletion in the ATO-treated HeLa cells and prevented ATO-induced apoptosis. Treatment with exogenous SOD and catalase reduced the depletion of GSH content in ATO-treated cells. Catalase strongly protected the cells from ATO-induced apoptosis. In addition, treatment with SOD, catalase and NAC slightly inhibited the G1 phase accumulation induced by ATO. In conclusion, NAC protects HeLa cells from apoptosis induced by ATO by up-regulating intracellular GSH content and partially reducing the production of O(2)(*-).     

The changes of reactive oxygen species and glutathione by MG132, a proteasome inhibitor affect As4.1 juxtaglomerular cell growth and death

The proteasome inhibitor MG132 has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we evaluated the effects of MG132 on the growth and death of As4.1 juxtaglomerular cells in relation to ROS and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells with an IC₅₀ of approximately 0.3–0.4 μM at 48 h and induced cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, activation of caspase-3 and -8, and PARP cleavage. MG132 increased intracellular ROS levels including O₂^? and GSH depleted cell numbers. N-acetyl cysteine (NAC, a well-known antioxidant) significantly decreased ROS level and GSH depleted cell numbers in MG132-treated As4.1 cells, along with the prevention of cell growth inhibition, cell death and MMP (ΔΨ_m) loss. NAC also decreased the caspase-3 activity of MG132. l-Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) or diethyldithiocarbamate (DDC; an inhibitor of Cu/Zn-SOD) did not affect cell growth, death, ROS and GSH levels in MG132-treated As4.1 cells. Conclusively, MG132 reduced the growth of As4.1 cells via apoptosis. The changes of ROS and GSH by MG132 were involved in As4.1 cell growth and 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.     

Oncogenic ras mediates apoptosis in response to protein kinase C inhibition through the generation of reactive oxygen species

Ras is a well established modulator of apoptosis. Suppression of protein kinase C (PKC) activity can selectively induce apoptosis in cells expressing a constitutively activated Ras protein. We wished to determine whether reactive oxygen species serve as an effector of Ras-mediated apoptosis. Ras-transformed NIH/3T3 cells contained higher basal levels of intracellular H(2)O(2) compared with normal NIH/3T3 cells, and PKC inhibition up-regulated ROS to 5-fold greater levels in Ras-transformed cells than in normal cells. Treatment with N-acetyl-l-cysteine reduced both the basal and inducible levels of intracellular H(2)O(2) in NIH/3T3-Ras cells and antagonized the induction of apoptosis by PKC inhibition. Culturing NIH/3T3-Ras cells in low oxygen conditions, which prevents ROS generation, also inhibited the apoptotic response to PKC inhibition. These results suggest that reactive oxygen species are necessary as downstream effectors of the Ras-mediated apoptotic response to PKC inhibition. However, the generation of ROS alone is not sufficient to induce apoptosis in Ras-transformed cells because inhibition of cell cycle progression prevented the induction of apoptosis in NIH/3T3-Ras cells without inhibiting the generation of intracellular H(2)O(2) observed after PKC inhibition. These findings suggest that continued cell cycle progression of Ras-transformed cells during PKC inhibition is also necessary for the induction of apoptosis.     

N-acetylcysteine increases apoptosis induced by H(2)O(2) and mo-antiFas triggering in a 3DO hybridoma cell line

N-Acetylcysteine (NAC) has been used as an antioxidant to prevent apoptosis triggered by different stimuli in different cell types. It is common opinion that cellular redox, which is largely determined by the ratio of oxidized and reduced glutathione (GSH), plays a significant role in the propensity of cells to undergo apoptosis. However, there are also contrasting opinions stating that intracellular GSH depletion or supplemented GSH alone are not sufficient to lead cells to apoptosis or conversely protect them. Unexpectedly, this study shows that NAC, even if it maintains the peculiar characteristics of an agent capable of reducing cell proliferation and increasing intracellular GSH content, increases apoptosis induced by H(2)O(2) treatment and mo-antiFas triggering in a 3DO cell line. We found that 24 h of NAC pre-treatment can shift cellular death from necrotic to apoptotic and determine an early expression of FasL in a 3DO cell line treated with H(2)O(2).     

Low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin-1 expression

The redox regulator thioredoxin-1 (Trx-1) is required for the redox potential of the cell and exerts important functions in cell growth and apoptosis. Severe oxidative stress has been implicated in the oxidation of proteins and cell death. However, the role of low doses of reactive oxygen species (ROS) is poorly understood. Here, we show that 10 and 50 microM H2O2 and short-term exposure to shear stress significantly increased Trx-1 mRNA and protein levels in endothelial cells. Since it is known that Trx-1 exerts anti-apoptotic functions, we next investigated whether low doses of ROS can inhibit basal and serum-depletion induced endothelial cell apoptosis. Indeed, treatment of endothelial cells with 10 and 50 microM H2O2 significantly reduced apoptosis induction. Reduction of Trx-1 expression using an antisense oligonucleotide approach resulted in the induction of apoptosis and abolished the inhibitory effect of low doses of H2O2. Taken together, our results demonstrate that low doses of ROS act as signaling molecules and exert anti-apoptotic functions in endothelial cells via upregulation of the redox-regulator Trx-1.     

Salvicine triggers DNA double-strand breaks and apoptosis by GSH-depletion-driven H2O2 generation and topoisomerase II inhibition

Glutathione (GSH), as the major small-molecule antioxidant in cells, has been implicated in the regulation of cell proliferation and apoptosis. Salvicine (SAL), a novel diterpenoid quinone compound, exhibits potent antitumor activities both in vitro and in vivo by poisoning topoisomerase II (Topo II) and has entered Phase II clinical trials for cancer therapy. Herein, we provide further evidence that SAL-induced DNA double-strand breaks (DSBs) and apoptosis by GSH depletion drives H2O2 generation and Topo II inhibition. Our data reveal that treatment with SAL results in a pronounced increase in intracellular H2O2 and is accompanied by the occurrence of DNA DSBs and apoptosis in epithelial HeLa cells. Furthermore, SAL was also noted to trigger a dramatic depletion of intracellular GSH via its direct reaction with GSH. Importantly, the introduction of GSH and overexpression of catalase antagonized SAL-mediated DNA DSBs and apoptosis, and the GSH synthesis inhibitor dl-buthionine-[S,R]-sulfoximine reduced SAL-mediated H2O2 generation, indicating that SAL-mediated H2O2 generation is derived from intracellular GSH depletion. Notably, SAL-mediated Topo II inhibition was also concentration-dependently reversed by GSH. Furthermore, we found that Topo II-defective HL-60/MX2 cells were almost completely resistant to SAL-induced DNA DSBs, suggesting that, in addition to its direct inhibitory effect on Topo II, SAL-mediated H2O2 generation may also trigger DNA DSBs via poisoning of Topo II. All these findings together suggest that GSH-depletion-driven H2O2 generation and Topo II inhibition are both critical for SAL-induced DNA DSBs and apoptosis.     

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.     

Expression of gamma-glutamyl transpeptidase protects ramos B cells from oxidation-induced cell death

The ectoenzyme, gamma-glutamyl transpeptidase (GGT, EC ) cleaves glutathione (GSH) to facilitate the recapture of cysteine for synthesis of intracellular GSH. The impact of GGT expression on cell survival during oxidative stress was investigated using the human B cell lymphoblastoid cell line, Ramos. Ramos cells did not express surface GGT and exhibited no GGT enzyme activity. In contrast, Ramos cells stably transfected with the human GGT cDNA expressed high levels of surface GGT and enzymatic activity. GGT-transfected Ramos cells were protected from apoptosis when cultured in cyst(e)ine-deficient medium. The GGT-expressing cells also had lower levels of intracellular reactive oxygen species (ROS). Homocysteic acid and alanine, inhibitors of cystine and cysteine uptake, respectively, caused increased ROS content and diminished viability of GGT expressing cells. Exogenous GSH increased the viability of the GGT-transfected cells more effectively than that of control cells, whereas the products of GSH metabolism prevented death of both the control and GGT-transfected cells comparably. These data indicate that GGT cleavage of GSH and the subsequent recapture of cysteine and cystine allow cells to maintain low levels of cellular ROS and thereby avoid apoptosis induced by oxidative stress.     

Selenoprotein W as molecular target of methylmercury in human neuronal cells is down-regulated by GSH depletion

Methylmercury (MeHg) is well known as a neurotoxic chemical. However, little is mentioned about its neurotoxic mechanism or molecular target in human neuronal cells in particular. We show in this study that exposure of human neuronal cell line, SH-SY5Y, to MeHg dose- and time-dependently impairs viability and mRNA expression of selenoprotein W (SeW) with a significant difference, unlike other selenoenzymes such as, SeP, GPX4, 5DI, and 5'DI. Using real-time RT PCR, the influence of selenium (Se) and glutathione (GSH) on SeW expression was also investigated. While Se depletion caused a weakly reduced SeW mRNA levels, additional Se caused an increase of SeW mRNA levels. Although 2 mM GSH had induced a weak shift on SeW level, the expression of SeW mRNA was down-regulated in SH-SY5Y cells treated with 25 microM BSO, an inhibitor of GSH synthesis. To understand the relationship between a decrease of SeW expression and intracellular GSH and ROS, we measured the concentration of intracellular GSH and ROS in cells treated to 1.4 microM MeHg using fluorescence based assays. A positive correlation was found between SeW mRNA level and intracellular GSH but no significant correlation was observed between intracellular ROS and SeW mRNA level or intracellular GSH contents. Therefore, we suggest that SeW is the novel molecular target of MeHg in human neuronal cells and down-regulation of this selenoenzyme by MeHg is dependent not on generation of ROS but on depletion of GSH.     

Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation

Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via a GSH transporter. We now evaluate the relationship between GSH depletion, the generation of reactive oxygen species (ROS), and the progression of apoptosis. Simultaneous single cell analysis of changes in GSH content and ROS formation by multiparametric FACS revealed that loss of intracellular GSH was paralleled by the generation of different ROS including hydrogen peroxide, superoxide anion, hydroxyl radical, and lipid peroxides. However, inhibition of ROS formation by a variety of antioxidants showed that GSH loss was independent from the generation of ROS. Furthermore, GSH depletion was observed to be necessary for ROS generation. Interestingly, high extracellular thiol concentration (GSH and N-acetyl-cysteine) inhibited apoptosis, whereas, inhibition of ROS generation by other non-thiol antioxidants was ineffective in preventing cell death. Finally, GSH depletion was shown to be a necessary for the progression of apoptosis activated by both extrinsic and intrinsic signaling pathways. These results document a necessary and critical role for GSH loss in apoptosis and clearly uncouple for the first time GSH depletion from ROS formation.     

硫氧还蛋白-过氧化物氧还蛋白与过氧化氢构成环路参与肿瘤的发生与发展

组织细胞可经过多种途径产生氧自由基（ROS）,而肿瘤组织由于多种应激因素会产生大量ROS,其中最重要的是过氧化氢（H2O2).H2O2对细胞发挥着致损伤及亚毒性信使的双重作用,作为信使其不仅参与调节正常细胞信号通路,重要的是促进肿瘤的发生及进展. ROS作为一种应激刺激信号激活细胞内的AP-1（activator protein 1）、Nrf-2（NF-E2-related factor 2）等核转录因子,活化后的AP-1、Nrf-2会结合到硫氧还蛋白（sulfiredoxin, SRX）基因启动子上游的调控序列,促进SRX基因的表达.SRX的表达上调则影响其下游的抗氧化蛋白,即特定亚型的过氧化物氧还蛋白（peroxiredoxin, PRX）的活性状态,最终使细胞内H2O2浓度受到调节. 由SRX-PRX轴与H2O2形成1个环路,通过调节H2O2含量来参与细胞众多信号通路.本文对H2O2、SRX及PRX各自的功能进行综述,还进一步探讨三者构成的信号环路对肿瘤的调控机制,从而了解该环路在肿瘤发生发展中所发挥的作用.     

Lead‐induced DNA damage and cell apoptosis in human renal proximal tubular epithelial cell: Attenuation via N‐acetyl cysteine and tannic acid

This study investigates the exposure of lead‐induced reactive oxygen species (ROS) generation, DNA damage, and apoptosis and also evaluates the therapeutic intervention using antioxidants in human renal proximal tubular cells (HK‐2 cells). Following treatment of HK‐2 cells with an increasing concentration of lead nitrate (0–50 μM) for 24 h, the intracellular ROS level increased whereas the GSH level decreased significantly in a dose‐dependent manner. Comet assay results revealed that lead nitrate showed the ability to increase the levels of DNA strand breaks in HK‐2 cells. Lead exposure also induced apoptosis through caspase‐3 activation at 30 μg/mL. Pretreatment with N‐acetylcysteine (NAC) and tannic acid showed a significant ameliorating effect on lead‐induced ROS, DNA damage, and apoptosis. In conclusion, lead induces ROS, which may exacerbate the DNA damage and apoptosis via caspase‐3 activation. Additionally, supplementation of antioxidants such as NAC and tannic acid may be used as salvage therapy for lead‐induced DNA damage and apoptosis in an exposed person.     

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.     

Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

Airway epithelial cells are constantly exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen and reactive oxygen species. Previous work from our laboratory demonstrated that the reactive oxygen species (ROS), hydrogen peroxide (H(2)O(2)), specifically activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in airway epithelial cells. In the current study we examine the biological consequence of exposure of human airway epithelial (HAE) cells to reactive nitrogen species (RNS). Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. We found that nitric oxide (NO) exposure via the NO donor papa-NONOate, failed to induce apoptosis in HAE cells. However, when papa-NONOate was combined with a superoxide anion donor (DMNQ) to generate peroxynitrite (ONOO(-)), apoptosis was observed. Similarly pure ONOO(-)-induced apoptosis, and ONOO(-)-induced apoptosis was associated with an increase in cellular ceramide levels. Pretreatment with the antioxidant glutathione did not prevent ONOO(-)-induced apoptosis, but did prevent H(2)O(2)-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. We confirmed our findings that H(2)O(2) specifically activated a neutral sphingomyelinase (nSMase2). However, ONOO(-) exposure did not affect neutral sphingomyelinase activity; rather, ONOO(-) specifically activated an acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H(2)O(2)-induced apoptosis, but had no effect on ONOO(-)-induced apoptosis. On the other hand, silencing of aSMase markedly impaired ONOO(-)-induced apoptosis, but did not affect H(2)O(2)-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different enzymes of the ceramide generating machinery to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury.