The mechanism of the elevation of intracellular glutathione induced by low-dose g g-rays was examined in RAW 264.7 cells. The expression of mRNA for g g-glutamylcysteine synthetase (g g-GCS) increased soon after g g-ray (0.5 Gy) irradiation, and peaked between 3 h and 6 h post-irradiation. A dose of 0.25 to 0.5 Gy was optimum for induction of g g-GCS mRNA expression at 3 h post-irradiation. The effect of inhibitors of activator protein-1 (AP-1) and nuclear factor k kB (NF-k kB) on the radiation-induced g g-GCS gene expression was then examined. The induction of g g-GCS mRNA expression was significantly suppressed when AP-1 DNA binding, but not NF-k kB DNA binding, was inhibited. Finally, electrophoretic mobility shift assay showed that the low-dose radiation markedly increased the DNA binding of AP-1, but not NF-k kB, soon after irradiation. These results suggest that the increase of glutathione levels in RAW 264.7 cells by low-dose g g-ray irradiation is mediated by transcriptional regulation of the g g-GCS gene, predominantly through the AP-1 binding site in its promoter.

Cells respond to oxidative stress including nitric oxide (NO) by increasing cellular glutathione concentration, as a part of adaptive response against oxidative injury. To elucidate the mechanism by which NO induces glutathione we investigated the reactive oxygen species (ROS) generated in the cell. Treatment of RAW264.7 cells with NO donor, sodium nitroprusside (SNP), resulted in a temporary increase in glutathione in a dose-dependent manner, which peaked between 6 h and 12 h after treatment, whereas expression of g g-glutamylcysteine synthetase (g g-GCS) mRNA peaked around 3 h after treatment. The increase was inhibited by NO scavengers, oxyhemoglobin and carboxyl-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). N-Acetyl-Lcysteine (NAC) also reduced the increase in glutathione to some extent, whereas both peroxynitrite scavenger ebselen and hydroxyl radical scavenger DMSO inhibited the increase in glutathione in a dose-dependent manner and complete inhibition was observed. Hydrogen peroxide exogenously added to the cell did not increase either glutathione or g g-GCS expression at any concentration, indicating that involvement of hydrogen peroxide is not likely. Flow cytometric analysis showed that SNP induced a marked dose-dependent increase in Rhodamine123 fluorescence, which was completely inhibited by ebselen in a dose-dependent manner, whereas, little increase in 2,7-dichlorofluorescin (DCF) fluorescence was observed. Generation of peroxynitrite in mitochondria by SNP was confirmed by elevated level of nitrotyrosine in a mitochondria fraction isolated from SNP-treated cells, and the elevation was completely inhibited by ebselen as well. These results suggest that induction of glutathione (GSH) synthesis by SNP treatment is mediated by peroxynitrite generated in mitochondria.

The mechanism underlying the elevation of intracellular glutathione (GSH) in RAW 264.7 cells exposed to low concentrations of sodium nitroprusside (SNP), a well-known nitric oxide (NO) donor, was investigated. The peak of intracellular GSH was reached at 6 h after exposure of the cells to SNP (0.1-0.5 mM), and this was preceded by the induction of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS; the rate-limiting enzyme of de novo GSH synthesis), which peaked at 3 h. N-alpha-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) and caffeic acid phenethyl ester (CAPE), specific inhibitors of NF-kappaB, significantly suppressed the SNP-induced elevation of GSH protein and gamma-GCS mRNA, while curcumin, an inhibitor of AP-1, was less effective. Electrophoretic mobility shift assay (EMSA) showed that SNP exposure markedly increased the DNA binding of NF-kappaB, but not that of AP-1. Deletion or mutagenesis of the NF-kappaB site in the gamma-GCS gene promoter abolished the SNP-induced up-regulation of GSH protein and gamma-GCS mRNA. These results suggest that the elevation of intracellular GSH in RAW 264.7 cells exposed to low concentrations of SNP occurs through the operation of the de novo GSH pathway, and is mediated by transcriptional up-regulation of the gamma-GCS gene, predominantly at the NF-kappaB binding site in its promoter.

The mechanism underlying the elevation of intracellular glutathione (GSH) in RAW 264.7 cells exposed to low-level sodium nitroprusside (SNP) was investigated by measuring the expression of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of de novo GSH synthesis, and the GSH content. A significant elevation of expression of mRNA for gamma-GCS was observed at 3 h after exposure of the cells to SNP at a concentration of 0.25 mM. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), N-acetylcysteine (NAC), or ebselen (Ebs) significantly suppressed the elevations induced by SNP, suggesting that hydrogen peroxide or peroxynitrite (ONOO(-)) is involved in this event as a triggering molecule. Hydrogen peroxide itself, however, did not induce the elevation of gamma-GCS mRNA and glutathione. Chemiluminescenses induced by SIN-1, a chemical ONOO(-) donor, and ONOO(-) itself were completely blocked by Ebs. SIN-1 also significantly elevated the cellular glutathione level, and the elevation was absolutely blocked by Ebs. These results suggest that the elevation of intracellular GSH in RAW 264.7 cells exposed to low-level SNP occurs via the de novo GSH pathway through transcriptional up-regulation of the gamma-GCS gene induced by peroxynitrite molecule.

The elevation of cellular glutathione (GSH) level induced by low concentrations of an nitric oxide (NO)-donor, sodium nitroprusside (SNP), and its effect on oxidant-induced cell injury were examined in RAW264.7 cells. The cellular GSH level increased 6 hr after exposure of the cells to SNP at low concentrations ranging from 0.1 to 0.5 mM, and the elevation followed the induction of mRNA coding for γ-glutamylcysteine synthetase, the ratelimiting enzyme of the de novo glutathione synthesis pathway. Pre-treatment of cells with low concentration of SNP (less than 0.25 mM) at 12 hr prior to exposure to menadione (MEND), an superoxide anion (O 2 -)-donor, significantly suppressed the cell injury induced by MEND alone. Simultaneous treatment with a higher concentration of SNP (1.0 mM or more) also blunted the MEND-induced cell injury. Low and high doses of NO both seem to show a preventive effect against oxidant injury: NO may protect against oxidant injury by up-regulating GSH synthesis at low concentrations, while at high concentrations it may directly react with radical oxygen species (ROS), thus acting as a free radical scavenger and blunting oxidant injury. These results suggest that modulation of the cellular glutathione metabolism through intracellular NO is a potential mechanism for enhancing the antioxidant defense of cells.