Reduced glutathione esters—antidotes to toxicity. Cytotoxicity induced by hydrogen peroxide, 1-chloro-2,4-dinitrobenzene,and menadione in murine P388D1 macrophages in vitro

Repletion of depleted cellular reduced glutathione (GSH) levels in oxidative stress and exposure to arylating agents is a strategy for the development of antidotes to chemical toxicity. The effect of GSH, reduced glutathione ethyl monoester (GSHEt), and reduced glutathione ethyl diester (GSHEt₂) on the cytotoxicity of hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (CDNB), and menadione to P388D₁ macrophages in vitro was investigated. The median toxic concentration TC₅₀ values of the toxicants were hydrogen peroxide 24 ± 2 mM (N = 19), CDNB 63 ± 6 μM (N = 18), and menadione 30 ± 4 μM (N = 22). Reduced glutathione, GSHEt, and GSHEt₂ were poor antidotes to hydrogen peroxide toxicity. Indeed, the observed antidote effects were attributed to the nonenzymatic reaction of the GSH derivatives with hydrogen peroxide in the extracellular medium. Reduced glutathione ethyl diester was a more potent antidote of CDNB- and menadione-mediated toxicity than GSHEt and GSH. For cell incubations with the approximate median toxic concentration TC₅₀ values of hydrogen peroxide, CDNB, and menadione, the respective median effective antidote concentration EC₅₀ values were GSHEt 23.8 ± 4.1 mM (N = 9), 3.6 ± 0.6 mM (N = 11), and 226 ± 93 μM (N = 12); and GSHEt₂ 20.4 ± 1.9 mM (N = 6), 603 ± 2 μM (N = 9), and 7.6 ± 2.3 μM (N = 12). Reduced glutathione ethyl diester was a potent antidote to CDNB- and menadione-induced toxicities but not to hydrogen peroxide-induced toxicity under acute intoxication conditions. © 1996 John Wiley & Sons, Inc.     

The role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe.

The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated. A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain. The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells. The growth rate of the disruptant is not different from that of the parent strain. Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wildtype cells with normal catalase activity. These results indicate that the catalase of S. pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.     

Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings

Here, the kinetics of oxidative stress responses of alfalfa (Medicago sativa) seedlings to cadmium (Cd) and mercury (Hg) (0, 3, 10 and 30 microm) exposure, expanding from a few minutes to 24 h, were studied. Intracellular oxidative stress was analysed using 2',7'-dichlorofluorescin diacetate and extracellular hydrogen peroxide (H(2)O(2)) production was studied with Amplex Red. Growth inhibition, concentrations of ascorbate, glutathione (GSH), homoglutathione (hGSH), Cd and Hg, ascorbate peroxidase (APX) activity, and expression of genes related to GSH metabolism were also determined. Both Cd and Hg increased cellular reactive oxygen species (ROS) production and extracellular H(2)O(2) formation, but in different ways. The increase was mild and slow with Cd, but more rapid and transient with Hg. Hg treatments also caused a higher cell death rate, significant oxidation of hGSH, as well as increased APX activity and transient overexpression of glutathione reductase 2, glutamylcysteinyl synthetase, and homoglutathione synthetase genes. However, Cd caused minor alterations. Hg accumulation was one order of magnitude higher than Cd accumulation. The different kinetics of early physiological responses in vivo to Cd and Hg might be relevant to the characterization of their mechanisms of toxicity. Thus, high accumulation of Hg might explain the metabolism poisoning observed in Hg-treated seedlings.     

Oxidative Stimulation of Glutathione Synthesis in Arabidopsis thaliana Suspension Cultures

A system based on Arabidopsis thaliana suspension cultures was established for the analysis of glutathione (GSH) synthesis in the presence of hydrogen peroxide. Mild oxidative stress was induced by use of the catalase inhibitor, aminotriazole, and its development was monitored by measurement of the oxidative inactivation of aconitase. Addition of 2 mM aminotriazole resulted in a 25% decrease in activity of aconitase over 4 h. During the subsequent 10 h, no further decrease in aconitase activity was measured despite a sustained inhibition of catalase. In combination with our failure to detect significant increases in the level of lipid peroxidation, another marker indicative of oxidative injury, these data suggest that although hydrogen peroxide initially leaked into the cytosol, its accumulation was limited by a cytosolic catalase-independent mechanism. A 4-fold increase in the level of GSH, which was almost exclusively in the reduced form, was observed under the same treatment. To determine to what extent this increase in reduced GSH played a role in limiting the accumulation of hydrogen peroxide in the cytosol, we inhibited GSH synthesis with buthionine sulfoximine (BSO), a specific inhibitor of [gamma]-glutamylcysteine synthetase. No significant oxidative injury was detected as a result of treatment with 50 [mu]M BSO alone, and furthermore, this treatment had no effect on cell viability, However, addition of 2 mM aminotriazole to cells preincubated with 50 [mu]M BSO for 15 h led to a rapid loss of aconitase activity (75% in 4 h), and significant accumulation of products of lipid peroxidation. Within 72 h, cell viability was lost completely. After removal of BSO from the growth medium, GSH levels recovered to normal over a period of 20 h. Addition of 2 mM aminotriazole to cells at different time points during this recovery period demonstrated a strong correlation between the level of reduced GSH and the degree of protection against oxidative injury. These data strongly suggest that the induction of GSH synthesis by an oxidative stimulus plays a crucial role in determining the susceptibility of cells to oxidative stress.     

Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus

The viable but nonculturable (VBNC) suppression mutant formed platable cells at low temperature stress after inoculation in artificial seawater (ASW). Suppression subtractive hybridization was used to identify differentially expressed genes among cDNAs of the VBNC suppression mutant and the wild-type Vibrio vulnificus strain. Glutathione S-transferase was identified as a responsive gene of the VBNC suppression mutant in our assay, and was highly expressed from the VBNC suppression mutant at low temperature stress. Culturability tests revealed that the wild-type cells were sensitive to oxidative stress in the hydrogen peroxide (H(2)O(2)) and to 1-chloro-2,4-dinitrobenzene (CDNB) compared with the VBNC suppression mutant cells. Adding glutathione showed that many wild-type V. vulnificus cells maintained culturability in cold ASW. These results suggest that non-nutritional growth inhibitors, such as peroxide that accumulates at low temperatures, influence VBNC in V. vulnificus cells.     

A parallel proteomic and metabolomic analysis of the hydrogen peroxide- and Sty1p-dependent stress response in Schizosaccharomyces pombe

Using an integrated approach incorporating proteomics, metabolomics and published mRNA data, we have investigated the effects of hydrogen peroxide on wild type and a Sty1p-deletion mutant of the fission yeast Schizosaccharomyces pombe. Differential protein expression analysis based on the modification of proteins with matched fluorescent labelling reagents (2-D-DIGE) is the foundation of the quantitative proteomics approach. This study identifies 260 differentially expressed protein isoforms from 2-D-DIGE gels using MALDI MS and reveals the complexity of the cellular response to oxidative stress and the dependency on the Sty1p stress-activated protein kinase. We show the relationship between these protein changes and mRNA expression levels identified in a parallel whole genome study, and discuss the regulatory mechanisms involved in protecting cells against hydrogen peroxide and the involvement of Sty1p-dependent stress-activated protein kinase signalling. Metabolomic profiling of 29 intermediates using 1H NMR was also conducted alongside the protein analysis using the same sample sets, allowing examination of how the protein changes might affect the metabolic pathways and biological processes involved in the oxidative stress response. This combined analysis identifies a number of interlinked metabolic pathways that exhibit stress- and Sty1-dependent patterns of regulation.     

Oxidative stress-activated zinc cluster protein Stb5 has dual activator/repressor functions required for pentose phosphate pathway regulation and NADPH production

In Saccharomyces cerevisiae, zinc cluster protein Pdr1 can form homodimers as well as heterodimers with Pdr3 and Stb5, suggesting that different combinations of these proteins may regulate the expression of different genes. To gain insight into the interplay among these regulators, we performed genome-wide location analysis (chromatin immunoprecipitation with hybridization to DNA microarrays) and gene expression profiling. Unexpectedly, we observed that Stb5 shares only a few target genes with Pdr1 or Pdr3 in rich medium. Interestingly, upon oxidative stress, Stb5 binds and regulates the expression of most genes of the pentose phosphate pathway as well as of genes involved in the production of NADPH, a metabolite required for oxidative stress resistance. Importantly, deletion of STB5 results in sensitivity to diamide and hydrogen peroxide. Our data suggest that Stb5 acts both as an activator and as a repressor in the presence of oxidative stress. Furthermore, we show that Stb5 activation is not mediated by known regulators of the oxidative stress response. Integrity of the pentose phosphate pathway is required for the activation of Stb5 target genes but is not necessary for the increased DNA binding of Stb5 in the presence of diamide. These data suggest that Stb5 is a key player in the control of NADPH production for resistance to oxidative stress.     

Cellular functions and transcriptional regulation of a third thioredoxin from Schizosaccharomyces pombe

The structural gene encoding a third thioredoxin (Trx) homologue, TRX3, of the fission yeast Schizosaccharomyces pombe was characterized and its regulation was studied. The determined DNA sequence encoded a putative 290 amino acid sequence of Trx with a molecular mass of 31,889 Da. The TRX3 mRNA level was increased in S. pombe cells harboring plasmid pTRX3, suggesting that the cloned TRX3 gene was functional. Yeast cultures harbouring plasmid pTRX3 exhibited shorter generation times and higher survival on solid minimal media plates incorporating mercury chloride (0.01 mmol/L) or hydrogen peroxide (1 mmol/L) compared with control cultures. Yeast cells containing extra copies of TRX3, but not TRX1 and TRX2, gave rise to lower reactive oxygen species levels than control cells. Oxidative stress owing to hydrogen peroxide and menadione enhanced the synthesis of beta-galactosidase from the TRX3-lacZ fusion gene in Pap1-positive cells but not in Pap1-negative cells. The TRX3 mRNA level was increased by oxidative stress only in Pap1-positive cells. Basal expression of the TRX3 gene also depended on Pap1. We concluded that S. pombe TRX3 is linked with yeast growth and oxidative stress response, with its expression being regulated by oxidative stress in a Pap1-dependent manner.