Influence of glutathione levels and heat-shock on the steady-state levels of oxidative DNA base modifications in mammalian cells.
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The effects of thiols, ascorbic acid and thermal stress on the basal (steady-state) levels of oxidative DNA base modifications were studied. In various types of untreated cultured mammalian cells, the levels of total glutathione were found to be inversely correlated with the levels of DNA base modifications sensitive to the repair endonuclease Fpg protein, which include 8-hydroxyguanine (8-oxoG). A depletion of glutathione by treatment with buthionine sulphoximine increased the steady-state level in AS52 Chinese hamster cells by approximately 50%. However, additional thiols in the culture medium did not reduce the level of Fpg-sensitive base modifications: 0-10 mM N-acetylcysteine had no effect, whereas cysteine ethylester even increased the oxidative DNA damage at concentrations >0.1 mM. Similarly, ascorbic acid (0-20 mM) failed to reduce the steady-state levels. When AS52 cells were grown at elevated temperature (41 degrees C), the steady-state level of the oxidative DNA modifications increased by 40%, in spite of a concomitant 1.6-fold increase of the cellular level of total glutathione. Depletion of glutathione at 41 degrees C nearly doubled the already elevated level of oxidative damage. A constitutive expression of the heat-shock protein Hsp27 in L929 mouse fibrosarcoma cells at 37 degrees C increased the glutathione level by 60%, but had little effect on the level of oxidative DNA damage. (+info)
Role of antioxidant defenses against ethanol-induced damage in cultured rat gastric epithelial cells.
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Reactive oxygen species appears to be involved in the pathogenesis of ethanol-induced gastric mucosal injury in vivo. Because ingested ethanol diffuses into the gastric mucosa, targeting both epithelium and endothelium, in the present study we examined the possible protective effect of antioxidants on ethanol damage in gastric epithelial cells and endothelial cells in vitro. Cytotoxicity by ethanol was quantified by measuring 51Cr release. The effects of impairment of the glutathione redox cycle and of inhibition of cellular catalase were examined. The generation of superoxide was assessed by the reduction in cytochrome c. Ethanol caused a time- and dose-dependent increase in 51Cr release from epithelial cells. Incubation of cells with DL-buthionine-(S,R)-sulfoximine, while reducing glutathione production, dose dependently enhanced ethanol-induced injury. 1,3-Bis(chloroethyl)-nitrosourea, while inhibiting glutathione reductase activity, also sensitized cells to ethanol. In contrast, the inhibition of catalase with 3-amino-1,2, 4-triazole did not alter the susceptibility of epithelial cells to ethanol. Ethanol induced damage to endothelial cells in a similar fashion. In endothelial cells, however, neither impairment of the glutathione cycle nor inhibition of catalase influenced ethanol-induced damage. Epithelial cells, when exposed to ethanol, increased superoxide production as a function of ethanol concentration, whereas endothelial cells did not. The glutathione redox cycle, but not cellular catalase, plays a critical role in protecting epithelial cells against ethanol damage, whereas neither antioxidant seems to play a role in protection of endothelial cells. The distinct difference in antioxidant protection against ethanol appears to depend on the capability of each cell to produce cytotoxic oxygen species in response to ethanol exposure. (+info)
Involvement of N-acetylcysteine-sensitive pathways in ricin-induced apoptotic cell death in U937 cells.
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We have found that the antioxidant N-acetylcysteine (NAC) strongly inhibited ricin-induced apoptotic cell death in U937 cells (human myeloid leukemia), as judged by cytotoxicity, nuclear morphological change, and DNA fragmentation. Consistent with these observations, a significant depletion of cellular glutathione was observed in ricin-treated cells, and NAC prevented the decrease in cellular glutathione. On the other hand, among the caspase inhibitors tested, Z-Asp-CH2-DCB, which inhibited ricin cytotoxicity, also suppressed ricin-mediated glutathione depletion, while NAC did not affect the generation of caspase-3 like activity in ricin-treated cells. These results suggest that glutathione loss takes place downstream from caspase activation during the ricin-induced apoptotic process. Treatment with a specific inhibitor of glutathione biosynthesis, buthionine sulfoximine (BSO) failed to induce apoptosis, and had no effect on the overall extent of ricin-induced apoptosis, even though the glutathione level was decreased to less than 5% of the control level. However, NAC still protected against ricin-induced apoptosis in the BSO-treated cells. We conclude that glutathione loss is one of several apoptotic changes caused by ricin, but is not a sufficient factor for the progress of apoptosis. NAC may prevent ricin-induced apoptosis through maintaining an intracellular reducing condition by acting as a thiol supplier. (+info)
Apoptosis in hematopoietic cells (FL5.12) caused by interleukin-3 withdrawal: relationship to caspase activity and the loss of glutathione.
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The mechanism of cell death caused by cytokine deprivation remains largely unknown. FL5.12 cells (a murine prolymphocytic cell line), following interleukin-3 (IL-3) withdrawal, undergo a decrease in intracellular glutathione (GSH) that precedes the onset of apoptosis. In the present study, the induction of apoptosis following IL-3 withdrawal or GSH depletion with DL-buthionine-[S,R,]-sulfoximine (BSO) was examined. Both conditions caused time-dependent increases in phosphatidylserine externalization, acridine orange and ethidium bromide staining, decreases in mitochondrial membrane potential, processing and activation of caspase-3 and proteolysis of the endogenous caspase substrate poly(adenosine diphosphate ribose)polymerase (PARP). Apoptosis induced by IL-3 deprivation but not BSO also caused lamin B1 cleavage, suggesting activation of caspase-6. Despite a more profound depletion of GSH after BSO than withdrawal of IL-3, the extent of apoptosis was somewhat lower. Benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone (z-VAD.fmk) blocked this caspase activity and prevented cell death after BSO exposure but not after IL-3 deprivation. Following IL-3 withdrawal, the caspase inhibitors z-VAD.fmk and boc-asp(OMe)fluoromethylketone (boc-asp.fmk) prevented the cleavage and activation of caspase-3, the breakdown of lamin B1 and partially mitigated PARP degradation. However, the externalization of phosphatidylserine, the fall in mitochondrial membrane potential and subsequent apoptotic cell death still occurred. These results suggest that IL-3 withdrawal may mediate cell death by a mechanism independent of both caspase activation and the accompanying loss of GSH. (+info)
ATP-Dependent efflux of CPT-11 and SN-38 by the multidrug resistance protein (MRP) and its inhibition by PAK-104P.
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Non-P-glycoprotein-mediated multidrug-resistant C-A120 cells that overexpressed multidrug resistance protein (MRP) were 10.8- and 29. 6-fold more resistant to 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) and SN-38, respectively, than parental KB-3-1 cells. To see whether MRP is involved in CPT-11 and SN-38 resistance, MRP cDNA was transfected into KB-3-1 cells. The transfectant, KB/MRP, which overexpressed MRP, was resistant to both CPT-11 and SN-38. 2-[4-Diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1,3 , 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) and MK571, which reversed drug resistance in MRP overexpressing multidrug-resistant cells, significantly increased the sensitivity of C-A120 and KB/MRP cells, but not of KB-3-1 cells, to CPT-11 and SN-38. The accumulation of both CPT-11 and SN-38 in C-A120 and KB/MRP cells was lower than that in KB-3-1 cells. The treatment with 10 microM PAK-104P increased the accumulation of CPT-11 and SN-38 in C-A120 and KB/MRP cells to a level similar to that found in KB-3-1 cells. The ATP-dependent efflux of CPT-11 and SN-38 from C-A120 and KB/MRP cells was inhibited by PAK-104P. DNA topoisomerase I expression, activity, and sensitivity to SN-38 were similar in the three cell lines. Furthermore, the conversion of CPT-11 to SN-38 in KB-3-1 and C-A120 cell lines was similar. These findings suggest that MRP transports CPT-11 and SN-38 and is involved in resistance to CPT-11 and SN-38 and that PAK-104P reverses the resistance to CPT-11 and SN-38 in tumors that overexpress MRP. (+info)
Protection against hydrogen peroxide cytotoxicity in rat-1 fibroblasts provided by the oncoprotein Bcl-2: maintenance of calcium homoeostasis is secondary to the effect of Bcl-2 on cellular glutathione.
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The oncoprotein Bcl-2 protects cells against apoptosis, but the exact molecular mechanism that underlies this function has not yet been identified. Studying H2O2-induced cell injury in Rat-1 fibroblast cells, we observed that Bcl-2 had a protective effect against the increase in cytosolic calcium concentration and subsequent cell death. Furthermore, overexpression of Bcl-2 resulted in an alteration of cellular glutathione status: the total amount of cellular glutathione was increased by about 60% and the redox potential of the cellular glutathione pool was maintained in a more reduced state during H2O2 exposure compared with non-Bcl-2-expressing controls. In our cytotoxicity model, disruption of cellular glutathione homoeostasis closely correlated with the pathological elevation of cytosolic calcium concentration. Stabilization of the glutathione pool by Bcl-2, N-acetylcysteine or glucose delayed the cytosolic calcium increase and subsequent cell death, whereas depletion of glutathione by dl-buthionine-(S, R)-sulphoximine, sensitized Bcl-2-transfected cells towards cytosolic calcium increase and cell death. We therefore suggest that the protection exerted by Bcl-2 against H2O2-induced cytosolic calcium elevation and subsequent cell death is secondary to its effect on the cellular glutathione metabolism. (+info)
Significance of glutathione-dependent antioxidant system in diabetes-induced embryonic malformations.
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Hyperglycemia-induced embryonic malformations may be due to an increase in radical formation and depletion of intracellular glutathione (GSH) in embryonic tissues. In the past, we have investigated the role of the glutathione-dependent antioxidant system and GSH on diabetes-related embryonic malformations. Embryos from streptozotocin-induced diabetic rats on gestational day 11 showed a significantly higher frequency of embryonic malformations (neural lesions 21.5 vs. 2.8%, P<0.001; nonneural lesions 47.4 vs. 6.4%, P<0.001) and growth retardation than those of normal mothers. The formation of intracellular reactive oxygen species (ROS), estimated by flow cytometry, was increased in isolated embryonic cells of diabetic rats on gestational day 11. The concentration of intracellular GSH in embryonic tissues of diabetic pregnant rats on day 11 was significantly lower than that of normal rats. The activity of y-glutamylcysteine synthetase (gamma-GCS), the rate-limiting GSH synthesizing enzyme, in embryos of diabetic rats was significantly low, associated with reduced expression of gamma-GCS mRNA. Administration of buthionine sulfoxamine (BSO), a specific inhibitor of gamma-GCS, to diabetic rats during the period of maximal teratogenic susceptibility (days 6-11 of gestation) reduced GSH by 46.7% and increased the frequency of neural lesions (62.1 vs. 21.5%, P<0.01) and nonneural lesions (79.3 vs. 47.4%, P<0.01). Administration of GSH ester to diabetic rats restored GSH concentration in the embryos and reduced the formation of ROS, leading to normalization of neural lesions (1.9 vs. 21.5%) and improvement in nonneural lesions (26.7 vs. 47.4%) and growth retardation. Administration of insulin in another group of pregnant rats during the same period resulted in complete normalization of neural lesions (4.3 vs. 21.5%), nonneural lesions (4.3 vs. 47.4%), and growth retardation with the restoration of GSH contents. Our results indicate that GSH depletion and impaired responsiveness of GSH-synthesizing enzyme to oxidative stress during organogenesis may have important roles in the development of embryonic malformations in diabetes. (+info)
Molecular basis for hepatic detoxifying enzyme induction by 2-(allylthio)pyrazine in rats in comparison with oltipraz: effects on prooxidant production and DNA degradation.
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The expression of hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferases (GSTs) by 2-(allylthio)pyrazine (2-AP), an experimental chemopreventive agent, was investigated in rats. Northern blot analysis revealed that 2-AP caused increases in mEH, rGSTA2/3/5, and rGSTM1/2 mRNA levels. mEH and rGSTA2 proteins were also induced. Molecular basis of the enzyme induction by 2-AP was studied in comparison with oltipraz (Olt). Rats exposed to buthionine sulfoximine, a GSH-depleting agent, before treatment with either 2-AP or Olt exhibited greater increases in the mRNA levels than the individual treatment. Conversely, increases of the mRNAs were prevented by cysteine treatment, indicating that metabolic intermediates or reactive oxygens produced from the agents could be reduced by cysteine. Gel shift analysis revealed that nuclear factor-kappaB, which is associated with the altered cellular redox state, was not activated by the agents. Effects of these agents on the breakage of phix-174 DNA were compared in vitro. 2-AP effectively reduced the conversion of supercoiled DNA to the open circular form induced by benzenetriol and prevented benzenetriol- and iron-catalyzed degradation of DNA, whereas Olt failed to prevent strand breakage of DNA. These results provided evidence that: 1) 2-AP was effective in elevating the hepatic mEH and GST gene expression in rats, which might be mediated with the production of reactive oxygen species; 2) nuclear factor-kappaB activation was not involved in the induction of the detoxifying enzymes by either 2-AP or Olt in spite of their production of reactive oxygens in vivo; and 3) the antioxidant effect of 2-AP in vitro differed from that of Olt. (+info)