Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency

This study investigated the role of cellular antioxidant defense mechanisms in modulating the neurotoxicity of domoic acid (DomA), by using cerebellar granule neurons (CGNs) from mice lacking the modifier subunit of glutamate-cysteine ligase (Gclm). Glutamate-cysteine ligase (Glc) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis. CGNs from Gclm (-/-) mice have very low levels of GSH and are 10-fold more sensitive to DomA-induced toxicity than CGNs from Gclm (+/+) mice. GSH ethyl ester decreased, whereas the Gcl inhibitor buthionine sulfoximine increased DomA toxicity. Antagonists of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors and of N-methyl-D-aspartate (NMDA) receptors blocked DomA toxicity, and NMDA receptors were activated by DomA-induced l-glutamate release. The differential susceptibility of CGNs to DomA toxicity was not due to a differential expression of ionotropic glutamate receptors, as evidenced by similar calcium responses and L-glutamate release in the two genotypes. A calcium chelator and several antioxidants antagonized DomA-induced toxicity. DomA caused a rapid decrease in cellular GSH, which preceded toxicity, and the decrease was primarily due to DomA-induced GSH efflux. DomA also caused an increase in oxidative stress as indicated by increases in reactive oxygen species and lipid peroxidation, which was subsequent to GSH efflux. Astrocytes from both genotypes were resistant to DomA toxicity and presented a diminished calcium response to DomA and a lack of DomA-induced L-glutamate release. Because polymorphisms in the GCLM gene in humans are associated with low GSH levels, such individuals, as well as others with genetic conditions or environmental exposures that lead to GSH deficiency, may be more susceptible to DomA-induced neurotoxicity.     

Glutathione status of isolated rat hepatocytes affects bile acid-induced cellular necrosis but not apoptosis

An accumulation of hydrophobic bile acids is implicated in the pathogenesis of cholestatic liver diseases. In the present study, we determined if hydrophobic bile acid-induced cellular injury compromised hepatocyte glutathione (GSH) status, and if modulating intracellular GSH levels prevented or facilitated bile acid-induced cellular cytotoxicities. Freshly isolated rat hepatocytes incubated with >/=125 microM of the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC), underwent a time- and dose-dependent decrease of intracellular GSH levels by 4-h incubation. This loss of intracellular GSH was not associated with an increase of intracellular GSH disulfide (GSSG). Rather, GCDC stimulated the dose-dependent accumulation of extracellular GSSG. The mechanism for extracellular GSSG accumulation by GCDC was through increased efflux of reduced GSH from hepatocytes into the media, where it subsequently oxidized to GSSG. Treatment of hepatocytes with GCDC (0-750 microM) did not directly alter GSH-dependent enzyme activities. The reduction of intracellular GSH with 125 microM GCDC correlated with extensive apoptosis at this concentration as determined by fluorescence microscopy of DAPI (4, 6-diamindino-2-phenylindole hydrochloride)-stained nuclei. Higher concentrations of GCDC (>/=500 microM) favored cellular necrosis and lipid peroxidation. Depleting GSH by treating hepatocytes with 1-bromoheptane increased their sensitivity toward GCDC-induced cellular necrosis, but not apoptosis. However, enhancing the hepatocyte GSH content by supplementation with GSH-ethylester (GSH-EE) failed to protect hepatocytes against either mode of cellular death. In conclusion, while GCDC-induced cytotoxicities were associated with an increased efflux of GSH from rat hepatocytes, GSH status modulated GCDC-induced necrosis, but not apoptosis.     

Glutathione-deficient mice are susceptible to TCDD-Induced hepatocellular toxicity but resistant to steatosis

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) generates both hepatocellular injury and steatosis, processes that involve oxidative stress. Herein, we evaluated the role of the antioxidant glutathione (GSH) in TCDD-induced hepatotoxicity. Glutamate-cysteine ligase (GCL), comprising catalytic (GCLC) and modifier (GCLM) subunits, is rate limiting in de novo GSH biosynthesis; GCLM maintains GSH homeostasis by optimizing the catalytic efficiency of GCL holoenzyme. Gclm(-/-) transgenic mice exhibit 10-20% of normal tissue GSH levels. Gclm(-/-) and Gclm(+/+) wild-type (WT) female mice received TCDD for 3 consecutive days and were then examined 21 days later. As compared with WT littermates, Gclm(-/-) mice were more sensitive to TCDD-induced hepatocellular toxicity, exhibiting lower reduction potentials for GSH, lower ATP levels, and elevated levels of plasma glutamic oxaloacetic transaminase (GOT) and γ-glutamyl transferase (GGT). However, the histopathology showed that TCDD-mediated steatosis, which occurs in WT mice, was absent in Gclm(-/-) mice. This finding was consistent with cDNA microarray expression analysis, revealing striking deficiencies in lipid biosynthesis pathways in Gclm(-/-) mice; qrt-PCR analysis confirmed that Gclm(-/-) mice are deficient in expression of several lipid metabolism genes including Srebp2, Elovl6, Fasn, Scd1/2, Ppargc1a, and Ppara. We suggest that whereas GSH protects against TCDD-mediated hepatocellular damage, GSH deficiency confers resistance to TCDD-induced steatosis due to impaired lipid metabolism.     

Butylhydroquinone protects cells genetically deficient in glutathione biosynthesis from arsenite-induced apoptosis without significantly changing their prooxidant status.

Arsenic, first among the top environmentally hazardous substances, is associated with skin, lung, liver, kidney, prostate, and bladder cancer. Arsenic is also a cardiovascular and a central nervous system toxicant, and it has genotoxic and immunotoxic effects. Paradoxically, arsenic trioxide is used successfully in the treatment of acute promyelocytic leukemia and multiple myeloma. Arsenic induces oxidative stress, and its toxicity is decreased by free thiols and increased by glutathione depletion. To further characterize the role of glutathione and oxidative stress in the toxicity of arsenic, we have used fetal fibroblasts from Gclm(-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis. Gclm(-/-) mouse embryo fibroblasts (MEFs) are eight times more sensitive to arsenite-induced apoptotic death. Because of a dramatic decrease in glutathione levels, Gclm(-/-) MEFs have a high prooxidant status that is not significantly relieved by treatment with the phenolic antioxidant tBHQ; however, tBHQ blocks arsenite-induced apoptosis in both Gclm(+/+) and Gclm(-/-) cells, although it raises a significant antioxidant response only in Gclm(+/+) cells. Global gene expression profiles indicate that tBHQ is significantly effective in reversing arsenite-induced gene deregulation in Gclm(+/+) but not in Gclm(-/-) MEFs. This effect of tBHQ is evident in the expression of metalloproteases and chaperones, and in the expression of genes involved in DNA damage and repair, protein biosynthesis, cell growth and maintenance, apoptosis, and cell cycle regulation. These results suggest that regulation of glutathione levels by GCLM determines the sensitivity to arsenic-induced apoptosis by setting the overall ability of the cells to mount an effective antioxidant response.     

Organophosphorus insecticides chlorpyrifos and diazinon and oxidative stress in neuronal cells in a genetic model of glutathione deficiency

Over the past several years evidence has been accumulating from in vivo animal studies, observations in humans, and in vitro studies, that organophosphorus (OP) insecticides may induce oxidative stress. Such effects may contribute to some of the toxic manifestations of OPs, particularly upon chronic or developmental exposures. The aim of this study was to investigate the role of oxidative stress in the neurotoxicity of two commonly used OPs, chlorpyrifos (CPF) and diazinon (DZ), their oxygen analogs (CPO and DZO), and their "inactive" metabolites (TCP and IMP), in neuronal cells from a genetic model of glutathione deficiency. Cerebellar granule neurons from wild type mice (Gclm +/+) and mice lacking the modifier subunit of glutamate cysteine ligase (Gclm -/-), the first and limiting step in the synthesis of glutathione (GSH), were utilized. The latter display very low levels of GSH and are more susceptible to the toxicity of agents that increase oxidative stress. CPO and DZO were the most cytotoxic compounds, followed by CPF and DZ, while TCP and IMP displayed lower toxicity. Toxicity was significantly higher (10- to 25-fold) in neurons from Gclm (-/-) mice, and was antagonized by various antioxidants. Depletion of GSH from Gclm (+/+) neurons significantly increased their sensitivity to OP toxicity. OPs increased intracellular levels of reactive oxygen species and lipid peroxidation and in both cases the effects were greater in neurons from Gclm (-/-) mice. OPs did not alter intracellular levels of GSH, but significantly increased those of oxidized glutathione (GSSG). Cytotoxicity was not antagonized by cholinergic antagonists, but was decreased by the calcium chelator BAPTA-AM. These studies indicate that cytotoxicity of OPs involves generation of reactive oxygen species and is modulated by intracellular GSH, and suggest that it may involve disturbances in intracellular homeostasis of calcium.     

Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats

Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis.     

Mechanisms of the apoptotic and necrotic actions of trimethyltin in cerebellar granule cells.

In evaluating mechanisms of trimethyltin (TMT)-initiated neuronal damage, the present study focused on involvement of reactive oxygen species, protein kinase C (PKC), and glutamate receptors. Exposure of cerebellar granule cells to TMT (0.01-0.1 microM) produced primarily apoptosis, but higher concentrations were associated with cellular lactate dehydrogenase efflux and necrosis. TMT increased generation of cellular reactive oxygen species, which was inhibited by either L-NAME (inhibitor of nitric oxide synthase, NOS) or catalase, indicating that both NO and H(2)O(2) are formed on TMT exposure. Since chelerythrine (selective PKC inhibitor) also inhibited oxidative species generation, PKC appears to play a significant role in TMT-induced oxidative stress. The metabotropic glutamate receptor antagonist, MCPG, (but not MK-801) prevented oxidative species generation, indicating significant involvement of metabotropic receptors (but not NMDA receptors) in TMT-induced oxidative stress. NOS involvement in the action of TMT was confirmed through measurement of nitrite, which increased concentration dependently. Nitrite accumulation was blocked by L-NAME, chelerythrine, or MCPG, showing that NO is generated by TMT and that associated changes in NOS are regulated by a PKC-mediated mechanism. Oxidative damage by TMT was demonstrated by detection of elevated malondialdehyde levels. It was concluded that low concentrations of TMT (0.01-0.1 microM) cause apoptotic cell death in which oxidative signaling is an important event. Higher concentrations of TMT initiate necrotic death, which involves both an oxidative and a non-oxidative component. TMT-induced necrosis but not apoptosis in granule cells is mediated by glutamate receptors.     

Modulating GSH Synthesis Using Glutamate Cysteine Ligase Transgenic and Gene-Targeted Mice

Glutathione (GSH) is an important antioxidant and cofactor for glutathione S-transferase conjugation. GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), composed of catalytic (GCLC) and modifier (GCLM) subunits. Transgenic mice that conditionally over express GCL subunits are protected from acetaminophen induced liver injury. Gclm null mice exhibit low GSH levels and enhanced sensitivity to acetaminophen. When Gclm expression and GCL activity are restored in Gclm conditional transgenic X Gclm null mice, they become resistant to APAP-induced liver damage. These animal models are a valuable resource for investigating the role of GSH synthesis in modulating oxidative damage and drug-induced hepatotoxicity.     

Dosage effects of resveratrol on ethanol-induced cell death in the human K562 cell line

Previous studies have established that ethanol induces cell apoptosis and necrosis. However, the precise molecular mechanisms are currently unclear. Here, we show that higher concentrations of ethanol (250-400 mM) induced a shift from apoptotic to necrotic cell death in human K562 cells, and that resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties, inhibited or enhanced ethanol-induced apoptosis/necrosis depending on the treatment dosage. Using the cell permeable dye 2',7'-dichlorofluorescin diacetate (DCF-DA) as an indicator of reactive oxygen species (ROS) generation, we showed that ethanol treatment directly increased intracellular oxidative stress. This intracellular oxidative stress increased in response to high concentrations (100-200 microM) of resveratrol, but remained unchanged following treatment with low concentrations (10-25 microM) of resveratrol. Further studies showed that resveratrol could attenuate or enhance ethanol-induced intracellular oxidative stress generation-dependent on treatment dosage, and that this effect could be correlated with cell apoptosis or necrosis. Importantly, ethanol-induced changes in intracellular ATP levels were also correlated with resveratrol dosage. Taken together, these results indicate that the treatment dosage may determine the effect of resveratrol on ethanol-induced ROS generation, intracellular ATP levels, and cell apoptosis or necrosis. Thus our findings support the possibility that appropriate dosage of resveratrol aids in decreasing the toxic effect of ethanol.     

Modulating GSH synthesis using glutamate cysteine ligase transgenic and gene-targeted mice

Glutathione (GSH) is an important antioxidant and cofactor for glutathione S-transferase conjugation. GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), composed of catalytic (GCLC) and modifier (GCLM) subunits. Transgenic mice that conditionally over express GCL subunits are protected from acetaminophen induced liver injury. Gclm null mice exhibit low GSH levels and enhanced sensitivity to acetaminophen. When Gclm expression and GCL activity are restored in Gclm conditional transgenic X Gclm null mice, they become resistant to APAP-induced liver damage. These animal models are a valuable resource for investigating the role of GSH synthesis in modulating oxidative damage and drug-induced hepatotoxicity.     

Induction of oxidative stress and TNF-alpha secretion by dichloroacetonitrile, a water disinfectant by-product, as possible mediators of apoptosis or necrosis in a murine macrophage cell line (RAW).

The water disinfectant by-product dichloroacetonitrile (DCAN) is a direct-acting mutagen and induces DNA strand breaks in cultured human lymphoblastic cells. Cellular activation by environmental agents may exert detrimental effects to the cells. Activated macrophages produce reactive oxygen intermediates such as H(2)O(2), (-)OH and O(2). Therefore, the effect of various concentrations of DCAN (100-400 microM) on the activity macrophage cells (RAW 264.7) was studied. In these cells, DCAN-induced oxidative stress was characterized by the production of reactive oxygen intermediates (ROI). Also, the ratios of intracellular GSH/GSSG was assessed and used as a biomarker for oxidative stress. The secretion of TNF-alpha was assessed since macrophages are known to secrete TNF-alpha as a result of cellular oxidative stress. Electrophoretic detection of DNA degradation and light microscopy was utilized for the characterization of DCAN-induced apoptosis. Lactate dehydrogenase (LDH) leakage and trypan blue exclusion were used as markers of cellular necrosis. Following exposure to DCAN (200 microM and 400 microM), intracellular GSSG was increased (2.5-fold of control, P<0. 05). DCAN activation of RAW cells was detected by elevated levels of intracellular ROI (1.9-2.5-fold than control, P<0.05) and increased secretion of TNF-alpha (4.5 fold-than control, P <0.05). Elecrophoresis of genomic DNA of treated cells indicated a dose-dependent increase in degradation of genomic DNA. Morphological studies also indicated that exposure of RAW cells to 100 microM or 200 microM DCAN incites apoptotic cell death. At higher concentrations (400 microM), however, significant (P<0.05) increase in LDH leakage and decrease in cell viability (55% of control) indicative of cellular necrosis, were observed. These studies indicate that DCAN induces dose-dependent apoptosis or necrosis in RAW cells that could be due to the disturbance in intracellular redox status and initiation of ROI-mediated oxidative mechanisms of cellular damage.     

Mecamylamine prevents neuronal apoptosis induced by glutamate and low potassium via differential anticholinergic-independent mechanisms

Neuronal loss via apoptosis caused by various stimuli may be the fundamental mechanism underlying chronic and acute neurodegenerative diseases. A drug inhibiting neuronal apoptosis may lead to a practical treatment for these diseases. In this study, treatment with mecamylamine, a classical antagonist of nicotinic acetylcholine receptors (nAChRs), prevented neuronal apoptosis induced by 75 microM glutamate and by low potassium (LK) in cerebellar granule neurons (CGNs) with EC(50)s of 35 and 293 microM, respectively. Two other antagonists of nAChRs, dihydro-beta-erythroidine and tubocurarine, failed to inhibit these two kinds of apoptosis. Mecamylamine inhibited the NMDA (30 microM)-evoked current and competed with [(3)H]MK-801. Furthermore, two inhibiters of the c-Jun N-terminal kinase (JNK) pathway prevented LK-induced apoptosis. Mecamylamine reversed the phosphorylation levels of JNK and c-Jun as well as the expression of c-Jun caused by LK in a Western blot assay. In addition, the JNK/c-Jun pathway was not involved in glutamate-induced cell death of CGNs. Our results suggest that mecamylamine prevents glutamate-induced apoptosis by blocking NMDA receptors at the MK-801 site and LK-induced apoptosis by inhibiting the activation of the JNK/c-Jun pathway.     

Dosage effects of curcumin on cell death types in a human osteoblast cell line.

Curcumin, the yellow pigment of Curcuma longa, is known to have antioxidant and anti-inflammatory properties, as well as their ability to either induce or prevent cell apoptosis. However, the precise molecular mechanisms of these effects are unknown. Here, we demonstrate that curcumin can induce apoptotic changes, including JNK activation, caspase-3 activation, and cleavage of PARP and PAK2, at treatment concentrations lower than 25 microM in human osteoblast cells. In contrast, treatment with 50-200 microM of curcumin does not induce apoptosis, but rather triggers necrotic cell death in human osteoblasts. Using the cell permeable dye 2',7'-dichlorofluorescin diacetate (DCF-DA) as an indicator of reactive oxygen species (ROS) generation, we found that while treatment with 12.5-25 microM curcumin directly increased intracellular oxidative stress, 50-200 microM curcumin had far less effect. Pretreatment of cells with N-acetyl cysteine or alpha-tocopherol, two well known ROS scavengers, attenuated the intracellular ROS levels increases and converted the apoptosis to necrosis induced by 12.5-25 microM curcumin. Moreover, we observed a dose-dependent decrease in intracellular ATP levels after treatment of osteoblast cells with curcumin and pretreatment of cells with antimycin or 2-deoxyglucose to cause ATP depletion significantly converted 12.5-25 microM curcumin-induced apoptosis to necrosis, indicating that ATP (a known mediator of apoptotic versus necrotic death) is most likely involved in the switching mechanism. Overall, our results signify that curcumin dosage treatment determines the possible effect on ROS generation, intracellular ATP levels, and cell apoptosis or necrosis in osteoblast cells.     

Protection of cells from menadione-induced apoptosis by inhibition of lipid peroxidation

Menadione is a commonly used compound that causes oxidative stress. We investigated the influence of lipid peroxidation on the apoptotic response of mouse myogenic C2C12 cells following menadione-induced oxidative stress. The presence of hypodiploid cells and phosphatidylserine translocation were assayed to detect apoptotic cells. Menadione at 10-40 micro M induced cell apoptosis. Menadione at dose of 80 micro M induced both apoptosis and necrosis. At a 160 micro M dosage, menadione induced cell necrosis. Caspase 3 activation is required for menadione-induced apoptosis. Incubation of cells with 40 micro M menadione resulted in the depletion of cellular glutathione and increased lipid peroxidation. Pre-treatment of cells with cysteine suppressed the menadione-induced apoptosis and prevented changes in reactive oxygen species levels, glutathione levels and lipid peroxidation. Pre-treatment of cells with deferoxamine mesylate, an iron chelator, also reduced both menadione-induced apoptosis and lipid peroxidation. However, this did not prevent menadione-induced glutathione depletion. Thus, the inhibition of lipid peroxidation by deferoxamine mesylate prevented apoptosis even though cellular glutathione remained depleted. Our data suggest that menadione-induced apoptosis is directly linked to iron-dependent lipid peroxidation.     

Damage to lung epithelial cells and lining fluid antioxidant defense by humic acid

Humic acid causes diseases including lung emphysema and fibrosis. Emerging evidence indicates that oxidative stress is involved in humic acid-induced effects. In the present study, we investigated generation of hydroxyl radicals from humic acid, as well as the effects of humic acid to lung epithelial cells and artificial alveolar lining fluid antioxidant mixture. The involvement of iron in humic acid-induced effects was also determined. We found that humic acid (concentration and time dependently) reduced the cell survival, increased caspase-3 activity, depleted GSH and raised lipid peroxidation in epithelial cells. Humic acid reduced antioxidant levels in the lining fluid antioxidant mix, which could be prevented by adding metal ion chelators. These findings suggest that humic acid causes oxidative stress in lung cells and alveolar lining fluid, which is most likely triggered by hydroxyl radicals produced directly from humic acid. Iron is probably involved in humic acid toxicity.     

Mediation of ionotropic glutamate receptors in domoic acid-induced striatal dopamine release in rats

Our objective was to characterize the mechanism of action of intrastriatal infusion of domoic acid on extracellular dopamine levels, using in vivo dialysis in conscious and freely moving rats. The local infusion of domoic acid (500 microM) caused an increase (567.9+/-142.5%, versus basal) in dopamine extracellular levels associated with a decrease in its metabolites: dihydroxyphenylacetate (DOPAC) and homovanillate (HVA) (47.3+/-4.4% and 33.8+/-4.2%, respectively, compared to basal). Infusion of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX; 200 microM) reversed the effect of domoic acid infusion on striatal dopamine levels. However, the infusion of the selective non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine (MK-801; 50 microM), did not change significantly the effect of domoic acid on dopamine extracellular levels. In conclusion, based on results with a microdialysis technique, we suggest that domoic acid may act through AMPA/kainate receptors in striatum.     

The aminopeptidase inhibitor,z-L-CMK,is toxic and induces cell death in Jurkat T cells through oxidative stress

The leucine aminopeptidase inhibitor, benzyloxycarbonyl-leucine-chloromethylketone (z-L-CMK), was found to be toxic and readily induce cell death in Jurkat T cells. Dose-response studies show that lower concentration of z-L-CMK induced apoptosis in Jurkat T cells whereas higher concentration causes necrosis. In z-L-CMK-induced apoptosis, both the initiator caspases (-8 and -9) and effector caspases (-3 and -6) were processed to their respective subunits. However, the caspases remained intact in z-L-CMK-induced necrosis. The caspase inhibitor, z-VAD-FMK inhibited z-L-CMK-mediated apoptosis and caspase processing but has no effect on z-L-CMK-induced necrosis in Jurkat T cells. The high mobility group protein B1 (HMGB1) protein was found to be released into the culture medium by the necrotic cells and not the apoptotic cells. These results indicate that the necrotic cell death mediated by z-L-CMK at high concentrations is via classical necrosis rather than secondary necrosis. We also demonstrated that cell death mediated by z-L-CMK was associated with oxidative stress via the depletion of intracellular glutathione (GSH) and increase in reactive oxygen species (ROS), which was blocked by N-acetyl cysteine. Taken together, the results demonstrated that z-L-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. The toxic side effects in Jurkat T cells mediated by z-L-CMK are associated with oxidative stress via the depletion of GSH and accumulation of ROS.     

Role of reactive oxygen species, glutathione and NF-kappaB in apoptosis induced by 3,4-methylenedioxymethamphetamine ("Ecstasy") on hepatic stellate cells

"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA), is a derivative of amphetamine with hepatotoxic effects that has been shown to induce apoptosis of cultured liver cells. In the present work, we studied the role played by oxidative stress in the apoptotic response caused by MDMA on a cell line of hepatic stellate cells (HSC). MDMA-treatment provoked oxidative stress determined as reactive oxygen species (ROS) accumulation and decrease of intracellular reduced glutathione levels. Pre-treatment with the antioxidant pyrrolidine dithiocarbamate blocked ROS production but did not prevent MDMA-induced apoptosis of HSC. The pro-oxidant menadione induced in HSC ROS production and apoptosis that were prevented by pyrrolidine dithiocarbamate, showing HSC to be susceptible to oxidative stress-induced apoptosis. Addition of exogenous GSH or its precursor NAC potentiated the apoptotic action of MDMA but blocked apoptosis induced by menadione. Pre-treatment of HSC with the cytochrome P450 inhibitor quinine diminished the extent of apoptosis caused by MDMA, suggesting the involvement of a metabolic derivative of MDMA on its apoptotic effect. Nuclear factor NF-kappaB was activated by MDMA in a oxidative stress independent fashion and played a protective role in the apoptotic response, since inhibition of NF-kappaB by treatment with parthenolide or by viral infection with a dominant-negative form of NIK (Ad5dnNIK) resulted in an increase of MDMA-induced cell death. In summary, MDMA-induced apoptosis of HSC is accompanied, but not caused by oxidative stress; a metabolic derivative of the drug is responsible for the apoptotic effect of MDMA, which is partially blocked by NF-kappaB activation.