GSH-depleting Agent Research Articles

Protein glutathionylation: coupling and uncoupling of glutathione to protein thiol groups in lymphocytes under oxidative stress and HIV infection

We show here that exposure to oxidative stress induces glutathione (GSH) modification of protein cysteinyl residues (glutathionylation) in T cell blasts. Treating the cells with the oxidant diamide induces thiolation of a series of proteins that can be detected by 2D electrophoresis when 35 S -cysteine is used to label the intracellular GSH pool. This thiolation is reversible, proteins are rapidly dethiolated and GSH is released from proteins once the oxidants are washed and the cells are allowed to recover. Dethiolation is dependent on the availability of GSH and thiols, since it is inhibited by GSH-depleting agents and improved by N-acetyl- l-cysteine (NAC). The capacity of these agents to reverse glutathionylation is diminished in T cell blasts infected in vitro with HIV, which is known to cause oxidative stress. Consistent with these findings, the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme known to be inhibited by glutathionylation, is inhibited in diamide-treated cells and recovers rapidly when cells are allowed to dethiolate. Further, GAPDH activity is diminished by GSH-depleting agents and augmented by NAC. Thus, reversible glutathionylation of proteins can rapidly shift the activity of a key metabolic enzyme and thereby result in dramatic, reversible changes in cellular metabolism.

Regulation of p21waf1/cip1 expression by intracellular redox conditions.

Reactive oxygen species (ROS) have been considered for a long time only as molecules for inducing oxidative damage to proteins, lipids, and nucleic acids. However, in the last few years some physiological effects of ROS have been hypothesized, consisting of the redox regulation of several biological processes, including the transduction of mitogenic signals. This means that intracellular generation of ROS could be necessary to maintain homeostasis, as well as that their formation/scavenging should be controlled processes. We developed an experimental procedure that causes redox perturbations in intact cells, based on the exposure of living cells to diethylmaleate (DEM), a GSH-depleting agent. By this procedure we demonstrated that ROS generated following DEM treatment induces a G1 arrest, that is accompanied by several redox-dependent changes in cell cycle-related proteins. One of these is the p53-independent accumulation of p21waf1/cip1, which requires the integrity of the ras-MAPK pathway. Accordingly, DEM treatment strongly activates ERK2. On the other hand, redox perturbations provoked by DEM induce several early phenomena, including p21waf1/cip1 and Rb dephosphorylation.

The role of intracellular oxidation in death induction (apoptosis and necrosis) in human promonocytic cells treated with stress inducers (cadmium, heat, X-rays)

Treatment of U-937 human promonocytic cells with the stress inducers cadmium chloride (2 h at 200 microM), heat (2 h at 42.5 C) or X-rays (20 Gy), followed by recovery, caused death by apoptosis and stimulated caspase-3 activity. In addition, all stress agents caused intracellular oxidation, as measured by peroxide and/or anion superoxide accumulation. However, while pre-incubation with antioxidants (N-acetyl-L-cysteine or butylated hydroxyanisole) inhibited the induction of apoptosis by cadmium and X-rays, it did not affect the induction by heat-shock. Pre-incubation for 24 h with the GSH-depleting agent L-buthionine-[S,R]-sulfoximine (BSO) switched the mode of death from apoptosis to necrosis in cadmium-treated cells. By contrast, BSO only caused minor modifacions in the rate of apoptosis without affecting the mode of death in heat- and X-rays-treated cells. BSO potentiated peroxide accumulation in cells treated with both cadmium and X-rays. However, while the accumulation of peroxides was stable in the case of cadmium, it was transient in the case of X-rays. Moreover, the administration of antioxidants during the recovery period sufficed to prevent necrosis and restore apoptosis in BSO plus cadmium-treated cells. Cadmium and X-rays caused a decrease in intracellular ATP levels, but the decrease was similar in both apoptotic and necrotic cells. Taken together, these results demonstrate that (i) stress inducers cause intracellular oxidation, but oxidation is not a general requirement for apoptosis; and (ii) the duration of the oxidant state seems to be critical in determining the mode of death.

Nitrogen Monoxide (NO) and Glucose: UNEXPECTED LINKS BETWEEN ENERGY METABOLISM AND NO-MEDIATED IRON MOBILIZATION FROM CELLS

Nitrogen monoxide (NO) affects cellular iron metabolism due to its high affinity for this metal ion. Indeed, NO has been shown to increase the mRNA binding activity of the iron-regulatory protein 1, which is a major regulator of iron homeostasis. Recently, we have shown that NO generators increase (59)Fe efflux from cells prelabeled with (59)Fe-transferrin (Wardrop, S. L., Watts, R. N., and Richardson, D. R. (2000) Biochemistry 39, 2748-2758). The mechanism involved in this process remains unknown, and in this investigation we demonstrate that it is potentiated upon adding d-glucose (d-Glc) to the reincubation medium. In d-Glc-free or d-Glc-containing media, 5.6 and 16.5% of cellular (59)Fe was released, respectively, in the presence of S-nitrosoglutathione. This difference in (59)Fe release was observed with a variety of NO generators and cell types and was not due to a change in cell viability. Kinetic studies showed that d-Glc had no effect on the rate of NO production by NO generators. Moreover, only the metabolizable monosaccharides d-Glc and d-mannose could stimulate NO-mediated (59)Fe mobilization, whereas other sugars not easily metabolized by fibroblasts had no effect. Hence, metabolism of the monosaccharides was essential to increase NO-mediated (59)Fe release. Incubation of cells with the citric acid cycle intermediates, citrate and pyruvate, did not enhance NO-mediated (59)Fe release. Significantly, preincubation with the GSH-depleting agents, l-buthionine-[S,R]-sulfoximine or diethyl maleate, prevented NO-mediated (59)Fe mobilization. This effect was reversed by incubating cells with N-acetyl-l-cysteine that reconstitutes GSH. These results indicate that GSH levels are essential for NO-mediated (59)Fe efflux. Hence, d-Glc metabolism via the hexose monophosphate shunt resulting in the generation of GSH may be essential for NO-mediated (59)Fe release. These results have important implications for intracellular signaling by NO and also NO-mediated cytotoxicity of activated macrophages that is due, in part, to iron release from tumor target cells.

Effect of glutathione (GSH) depletion on the serum levels of triiodothyronine (T3), thyroxine (T4), and T3/T4 ratio in allyl alcohol-treated male rats and possible protection with zinc.

The current study is an attempt to elucidate the link between glutathione (GSH) as a major endogenous antioxidant and the thyroid hormone levels. Rats were treated with a single intraperitoneal (IP) dose of either ZnCl2 (5 mg/kg) or allyl alcohol (AIAI) (1.5 mmol/kg), which acts as a GSH-depleting agent. ZnCl2 and AIAI were administered either alone (Zn- and AlAl-treated groups) or in combination (AIAI + Zn-treated group). Blood and liver samples were collected 5 hours post treatment in all groups. Zinc was used because of its potential intracellular regulatory effect as a calcium antagonist. The data indicate a decrease in the serum levels of triiodothyronine (T3) and thyroxin (T4), the T3/T4 ratio, and serum and liver total protein in AlAl-treated rats. Increases in the serum levels of aminotransferases, hepatic calcium, and lipid peroxidation products were observed. The decrease in T3 and the T3/T4 ratio indicates a reduced capacity of the microsomes to convert T4 into T3. Rats treated with AlAl + Zn had replenished hepatic GSH and showed a marked decrease in the serum levels of aminotransferases and in the liver calcium contents and lipid peroxidation products compared to AlAl-treated rats. In contrast, zinc treatment failed to normalize the serum levels of total protein, T3 and T4, and the T3/T4 ratio in the same rats. Rat treated with ZnCI2 alone tended to have a lower serum protein level that was accompanied with a significant decrease in both serum T3 and the T3/T4 ratio. The effect of zinc in increasing capillary permeability with the probable leakage of some serum proteins including the thyroid-binding proteins could possibly be the reason behind this finding. Possible covalent binding of AIAI metabolites to some cellular proteins may explain the persistence of reduced liver protein levels in zinc-protected rats.

Inhibition of Cell Proliferation and Antitumor Activity of a Novel Organotin Compound

Organotin compounds showed more antineoplastic effect against P388 leukemia in mice than any other class of compounds. However, they have not received as much attention as the platinum compounds. The present compound, Et2SnCl2.L [L = N-(2-pyridylmethylene)-4-toluidine] (OTC), showed an Sn-N bond length of 2.46 A which, because it is bigger than 2.39 A, is expected to achieve better formation of tin-DNA complexes. We previously reported on the synthesis and biological activity of this compound and on its ability to cause a delay in cell proliferation and sister chromatid exchanges in mouse bone marrow cells. In this study, we carried out further investigations on the antiproliferative and antitumor activity of OTC in relation to the cellular glutathione (GSH) level, which plays an important role in the cellular defense mechanism. OTC induced significant delay in the cell cycle in mouse bone marrow cells and, when the GSH level was low, the extent of the delay was reduced. The antitumor activity was determined in accordance with the US National Cancer Institute (NCI) standard protocol for primary screening in Dalton's lymphoma (DL) that was maintained by serial intraperitoneal transplantation. The T/C (treated/control) value was 146% when organotin was treated after transplantation; this improved significantly when buthionine sulfoximine (BSO), a GSH-depleting agent, was added 24 hours before the tin treatment. Our data suggest that the present tin compound has antiproliferative ability and can increase the survival of mice bearing DL. The endogenous GSH level influences the effect of the tin compound.

Mutagenicity of mercury chloride and mechanisms of cellular defence: the role of metal-binding proteins.

The mechanisms of toxicity and, particularly, the potential carcinogenicity of inorganic mercury are still under debate. Results of mutagenicity and genotoxicity testing with mercury have been inconsistent: mercury induces DNA single-strand breaks at low concentrations in mammalian cells but has not proved mutagenic in several bacterial mutagenicity assays. We investigated the mutagenicity of subtoxic concentrations of inorganic mercury and the role of metal-binding proteins and free radicals in this process. A mutagenicity assay using NIH 3T3 cells, transfected with a vector containing lacZ' as a reporter for mutational events, was applied. In this model, inorganic mercury significantly increased the mutation frequency in the lacZ gene, even at the lowest concentration tested. The mutation frequency was greatest at an Hg(2+) concentration of 0.5 microM. To identify the mechanisms involved, different cellular responses to non-cytotoxic concentrations of HgCl(2) were measured. Hg(2+) increased the intracellular amount of reactive oxygen species. This induction of oxidative stress was observed, although the intracellular glutathione (GSH) and metallothionein (MT) concentrations were increased significantly. Mercury-induced MT expression was even more pronounced after GSH depletion. Correspondingly, radical formation was more evident in the presence of the GSH-depleting agent L-buthioneine-[S:,R:]-sulfoximine. These findings suggest that the observed mutations might be a consequence of oxidative processes, rather than due to a direct interaction of mercury with nuclear DNA. The results also indicate that the auto-induction of MT by Hg(2+) fails to prevent these mutational events.

Target cells for methylsulphonyl-2,6-dichlorobenzene in the olfactory mucosa in mice

Previously we reported that methylsulphonyl-2,6-dichlorobenzene, 2,6-(diCl-MeSO 2-B), was irreversibly bound to the olfactory mucosa of mice and induced necrosis of the Bowman's glands with subsequent neuroepithelial degeneration and detachment. In this study, autoradiography and histopathology were used to determine tissue-localization and toxicity of 2,6-(diCl-MeSO 2-B) in the olfactory mucosa of control mice and animals pretreated with cytochrome P450 (CYP) and glutathione (GSH) modulators. The Bowman's glands of the olfactory mucosa were the major target sites of non-extractable binding of 2,6-(diCl- 14C-MeSO 2-B), whereas the olfactory neuroepithelium and nerve bundles showed only background levels of silver grains. Metyrapone pretreatment slightly decreased binding in the Bowman's glands and markedly decreased toxicity in the olfactory mucosa after 2,6-(diCl-MeSO 2-B) administration. These results support that a CYP-mediated activation of 2,6-(diCl-MeSO 2-B) takes place in the Bowman's glands giving rise to toxic reactive intermediates. In mice pretreated with the GSH-depleting agent phorone, a marked increase of irreversible binding of 2,6-(diCl- 14C-MeSO 2-B) in the Bowman's glands was observed. Tape-section autoradiograms also revealed a significant increase of uptake of radioactivity in the olfactory bulb. As determined by histopathology, GSH-depletion increased both the extent and severity of the lesion in the mucosa. These results imply that 2,6-(diCl-MeSO 2-B)-reactive intermediates are conjugated with GSH. The amount of irreversible binding and toxicity in the olfactory mucosa seems to be associated with the level of 2,6-(diCl-MeSO 2-B)-reactive intermediates.

Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4-hydroxynonenal

4-Hydroxynonenal (4HNE) is the most prevalent toxic lipid peroxidation product formed during oxidative stress. It exerts its cytotoxicity mainly by the modification of intracellular proteins. The detection of 4HNE-modified proteins in several degenerative disorders suggests a role for 4HNE in the onset of these diseases. Efficient protection mechanisms are required to prevent the intracellular accumulation of 4HNE. The toxicity of 4HNE was tested with the small cell lung cancer cell lines GLC(4) and the multidrug-resistance-protein (MRP1)-overexpressing counterpart GLC(4)/Adr. In the presence of the MRP1 inhibitor MK571 or the GSH-depleting agent buthionine sulphoximine, both cell lines became more sensitive and showed decreased survival. Transport experiments were performed with the (3)H-labelled glutathione S-conjugate of 4HNE ([(3)H]GS-4HNE) with membrane vesicles from GLC(4)-derived cell lines with different expression levels of MRP1. [(3)H]GS-4HNE was taken up in an ATP-dependent manner and the transport rate was dependent on the amount of MRP1. The MRP1 inhibitor MK571 decreased [(3)H]GS-4HNE uptake. MRP1-specific [(3)H]GS-4HNE transport was demonstrated with membrane vesicles from High Five insect cells overexpressing recombinant MRP1. Kinetic experiments showed an apparent K(m) of 1.6+/-0.21 microM (mean+/-S.D.) for MRP1-mediated [(3)H]GS-4HNE transport. In conclusion, MRP1 has a role in the protection against 4HNE toxicity and GS-4HNE is a novel MRP1 substrate. MRP1, together with GSH, is hypothesized to have a role in the defence against oxidative stress.

In vitro cytotoxic effect of wheat gliadin-derived peptides on the Caco-2 intestinal cell line is associated with intracellular oxidative imbalance: implications for coeliac disease

Coeliac disease (CD) is an inflammatory disorder of the upper small intestine in which gluten acts as an essential factor in its pathogenesis. Although it is generally accepted that cereal protein activation of the immune system is involved in CD progression, a non-immunomediated cytotoxic activity of gliadin-derived peptides on the jejunal/duodenal tract cannot be excluded. In this work, considering that (a) little has been reported about the intracellular metabolic events associated with gliadin toxicity, and (b) an important role for free radicals in a number of gastrointestinal disease has been demonstrated, we investigated the in vitro effects of gliadin-derived peptides on redox metabolism of Caco-2 intestinal cells during a kinetic study in which cells were exposed to peptic–tryptic digest of bread wheat up to 48 h. We found that the antiproliferative effects displayed by gliadin exposure was associated with intracellular oxidative imbalance, characterised by an increased presence of lipid peroxides, an augmented oxidised (GSSG)/reduced (GSH) glutathione ratio and a loss in protein-bound sulfhydryl groups. Significant structural perturbations of the cell plasma membrane were also detected. Additional experiments performed by using the specific GSH-depleting agent buthionine sulfoximine provide evidence that the extent of gliadin-induced cell growth arrest critically depends upon the ‘basal’ redox profile of the enterocytes. On the whole, these findings seem to suggest that, besides the adoption of a strictly gluten-free diet, the possibility for an adjuvant therapy with antioxidants may be considered for CD patients.

Sensitization effect of l-2-oxothiazolidine-4-carboxylate on tumor cells to melphalan and the role of 5-oxo- l-prolinase in glutathione modulation in tumor cells

5-Oxo- l-prolinase (5-OPase) (EC 3.5.2.9) links the synthesis and metabolism of glutathione (GSH) in the γ-glutamyl cycle. Previous studies showed that l-2-oxothiazolidine-4-carboxylate (OTZ), a 5-oxo- l-proline analog that is metabolized by 5-OPase, can preferentially decrease the cellular GSH levels in vivo in rat mammary tumors and sensitizes the tumors to the alkylating agent melphalan. The present study investigated the biochemical mechanism of this effect in a human breast cancer cell line, MCF7. We found that OTZ decreased the GSH levels in MCF7 cells. When the cells were treated with OTZ plus melphalan, the cytotoxicity of melphalan was increased as compared with that of melphalan alone, and this effect could be reversed by the addition of glutamate, which is the product of 5-OPase reaction and a critical substrate in GSH synthesis. We concluded that OTZ increases melphalan toxicity by limiting glutamate production from 5-OPase for GSH synthesis. We also observed that the expression of 5-OPase in the stably transfected MCF7 cells decreased the cellular GSH contents, sensitized the cells to melphalan toxicity, and diminished the sensitizing effect of OTZ. Furthermore, exposure to the GSH-depleting agent buthionine sulfoximine led to increased expression of 5-OPase in both MCF7 cells and the peripheral blood mononuclear cells of patients. These results indicate a critical interaction between cellular GSH levels and 5-OPase activity that could be important in GSH modulation in therapeutic settings.

Modulation of Intracellular Glutathione and Cysteine Metabolism in Bovine Oviduct Epithelial Cells Cultured In Vitro1

The objective of this study was to determine how alterations in intracellular thiol levels of oviduct epithelium occur in response to chemical or environmental conditions that could result in oxidative stress. Bovine oviducts were classified as follicular (F) or luteal (L) according to the reproductive stage of the ovary. Epithelial cells were harvested from the ampulla (AMP) and isthmus (ISTH) region of each oviduct, suspended in culture medium, and then plated into collagen-coated culture plates and grown to confluency. Basal levels of intracellular cysteine (Cys) and glutathione (GSH) were determined in oviduct epithelial cells and found to range from 0.36 to 0.46 pmol/ microg protein for Cys and from 5.3 to 6.4 pmol/ microg protein for GSH. Oxidized Cys values ranged from 21% to 39% of total Cys, whereas the oxidized GSH levels observed were from 21% to 28% of total GSH except in luteal ISTH, where they were significantly lower (6%). Confluent cells were exposed to GSH-depleting agents, <FONT SIZE=-1>L-buthionine-S,R-sulfoximine (BSO) or diethyl maleate (DEM), at doses ranging from 10 to 5000 microM. Both compounds depleted GSH in a dose-dependent manner, and 500 microM concentrations were chosen for subsequent studies with each compound. Cys levels in BSO (500 microM)-treated oviduct epithelial cells were transiently elevated over control values during the initial 5-h incubation; there was then a decrease in Cys levels by AMP but not ISTH oviduct epithelial cells. BSO-treated oviduct epithelial cells displayed a continued depletion of GSH over the incubation period and by 24 h were depleted by 38% to 61%. These results demonstrate a difference in GSH turnover in oviduct epithelial cells associated with reproductive stage. Exposure to DEM (500 microM) caused a decline in both Cys and GSH levels, which were partially restored after DEM removal. In general, L-staged oviduct epithelial cells were observed to be more competent at replenishing thiol stores than F-staged oviduct epithelial cells. Results from this study suggest that reproductive stage and region influence intracellular oviduct epithelium thiol status and therefore may affect how this tissue responds to chemicals or environmental conditions leading to oxidative stress.

Modulation of mitomycin C-induced sister chromatid exchanges and cell cycle delay by buthionine sulfoximine and reduced glutathione in mouse bone marrow cells in vivo

Mitomycin C (MMC) is a bifunctional alkylating agent and a chemotherapeutic drug, covalently binds to the DNA of cells and produces monoadducts and DNA–DNA, DNA–protein crosslinks, induces damages at chromosomal level and slows down the rate of cell proliferation. Reduced glutathione (GSH), a major non-protein thiol substance plays an important role in detoxification of cells against the toxic effect of exogenous compounds. In order to understand the role of factor which affects MMC sensitivity, we have made an attempt to establish a relationship between MMC-induced DNA damages and the endogenous GSH-status of the cells. MMC was treated to normal and buthionine sulfoximine (BSO; GSH-depleting agent)-treated mice. Exogenous GSH was also added to MMC-treated normal mice. Cells were fixed at 24 h and sister chromatid exchanges (SCEs) and average generation time (AGT) were scored. MMC-induced SCEs and cell cycle delay significantly with respect to control and the frequency of SCEs was increased considerably while MMC treatment combined with either GSH or BSO. The induction of cell cycle delay by MMC was reduced significantly when GSH or BSO was present along with MMC. These observations indicate that the factor responsible for inducing delay in cell cycle after MMC treatment may not be relevant for SCE-induction.

Influence of buthionine sulfoximine and reduced glutathione on arecoline-induced chromosomal damage and sister chromatid exchange in mouse bone marrow cells in vivo.

Arecoline (ARC), an alkaloid of the betel nut (Areca catechu), is a major ingredient of betel quid. The carcinogenic potentiality as well as its cell transformation ability has already been reported. Reduced glutathione (GSH), a major non-protein thiol substance plays an important role in protection of cells against the toxic effect of exogenous compounds. In order to understand the role of factors which affect ARC sensitivity, we have made an attempt to establish a relationship between ARC-induced DNA damage and the endogenous GSH status of the cells. ARC was administered to untreated and buthionine sulfoximine (BSO) (a GSH-depleting agent)-treated mice. Exogenous GSH was also added to ARC-administered mice. Cells were fixed at 20 h and both chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) were scored. Both CAs and SCEs were significantly induced by ARC and the frequency of both these parameters were increased further when ARC was given to BSO-treated mice. However, GSH reduced the frequency of CAs induced by ARC but failed to do so for SCEs. The data indicate that ARC-induced DNA damage is influenced by endogenous GSH level. The failure of GSH to reduce the frequency of SCEs indicates that the mechanism of induction of CAs and SCEs by ARC are different.

Alterations of DNA methylation by glutathione depletion

One of the most consistent findings in cancer cells is an overall decrease of 5-methylcytosine content in DNA. The causes that lead to this alteration are not known. We have shown in a recent study that the methyl-donor, methionine (Met), can easily be depleted and that O- and S-methylation can be impaired in response to glutathione (GSH) depletion. This is because mammalian cells are capable of resynthesizing GSH after GSH is depleted, and GSH turnover occurs at the expense of Met. An extensive utilization of Met for the resynthesis of GSH causes Met depletion and impairment in methylation. In the present study we now demonstrate that GSH depletion has a significant impact on DNA methylation. An i.p. dose of a model GSH-depleting hepatotoxin, bromobenzene (BB), caused a progressive impairment in genomic DNA methylation in the Syrian hamster. The administration of a single i.p. dose of Met labeled with [ 14CH 3]Met to BB-treated hamsters at either 1, 3, 5.5 or 9 h after BB resulted in an increase of methyl-group incorporation into liver genomic DNA at 24 h after BB. With respect to the time points chosen for Met administration, methyl-group incorporation found in the BB+Met groups were 1-, 2-, 4- and 12-fold of the controls that received only Met. We further employed an in vitro methylation assay using specific bacterial SssI CpG methylase as the catalyzing enzyme to demonstrate that BB caused a progressive increase of unmethylated CpG sites in genomic DNA. Interestingly, the time response curve of global DNA methylation in vitro showed an identical pattern to that observed in the in vivo experiment. The results provide strong evidence that GSH-depleting agents significantly impair cytosine methylation. Thus, alterations in gene expression could result from a high dose and/or prolonged exposure to GSH-depleting agents, e.g. medications, chemotherapeutic agents and environmental toxins.

Progression of Mouse Buthionine Sulfoximine Cataracts in vitro is Inhibited by Thiols or Ascorbate

Mouse lens cultures were employed to study the progression of cataracts initiated by injection of buthionine sulfoximine, an inhibitor of glutathione (GSH) biosynthesis. Culture of lenses removed from untreated mice on postnatal day 7, for 48 hr in the presence of 4 mmBSO, resulted in only limited cataractous changes. To enable substantial progression of cataracts in vitro, it was therefore necessary to pretreat the mice with BSO prior to lens culture. A single injection of BSO (4 nmol mg−1lens), administered on day 7, resulted in >90% depletion of lens GSH within 3 days, but no visible cataractous changes. The clear lenses were incubated for 29±1 hr at 37°C in Medium HL-1, supplemented with EGF, insulin and Ca2+, in the presence or absence of BSO, and were scored for cataract development by previously described criteria. In the absence of BSO, only 4 of 10 lenses developed large opacities. However, in the presence of 4 mmBSO, 40 out of 45 experimental lenses developed opacities affecting at least 50% of the lens visual field and were scored as stages 1C-4, depending upon the extent and density of the cataracts. In addition, three lenses had opacities involving 20–50% of the field (stage 1B). By contrast, less than 10% of lenses from untreated mice incubated in the absence of BSO developed opacities. The cataracts developed in 4 mmBSO were accompanied by reduction of lens glutathione levels to <0·010 nmol mg−1lens. They were almost completely prevented by 1 mmascorbate, 2 mmGSH, 2 mmGSH monoethyl ester and 2 mmcysteamine. GSH and GSH ester maintained lens glutathione content between 0·1 and 0·2 nmol mg−1in the presence of BSO, whereas ascorbate did not prevent near-total GSH depletion. The prevention of cataracts by thiols and ascorbate was confirmed by lens Na/K ratios not significantly different from those in control lenses. The above combination of GSH depletion in vivo by a single injection of BSO, followed 3 days later with lens culture in the presence of BSO, may yield a useful system to elucidate and control the biochemical mechanisms involved in oxidative cataract induction by this GSH-depleting agent.

Effect of glucose-cysteine adduct as a cysteine prodrug in rats

Effect of intraperitoneal administration (5 mmol/kg of body weight) of glucose- cysteine adduct (glc-cys) as a cysteine prodrug in rat tissues was studied. Cysteine levels in liver and kidney increased to 1.08 and 1.98μmol per g or ml, respectively, at 2h after the administration. GSH levels did not change substantially. However, when glc-cys was injected to rats treated with diethyl maleate, a GSH-depleting agent, the decreased GSH levels were restored rapidly. The recoveries in liver and kidney were 72% at 1h and 66% at 2h, respectively, after glc-cys administration. Metabolism of glc-cys was assessed by urinary excretion of glc-cys, sulfate and taurine. Average excretion of glc-cys was 2.86mmol/kg/24h after glc-cys administration. Increased excretions of sulfate and taurine were 0.77 and 0.14mmol/kg/24h, respectively. Data show that, although glc-cys excretion was relatively rapid, glc-cys was effectively utilized for GSH synthesis in GSH-depleted tissues.

Arsenite efflux is inhibited by verapamil, cyclosporin A, and GSH-depleting agents in arsenite-resistant Chinese hamster ovary cells.

We have previously demonstrated that arsenite-resistant SA7 cells can extrude arsenite more effectively and completely than their parental Chinese hamster ovary cells. Our present results show that arsenite efflux from SA7 cells is inhibited by chemosensitizing agents to multidrug resistant-associated protein: verapamil and cyclosporin A and by glutathione-depleting agents: dinitrofluorobenzene and diethyl maleate. These results suggest that arsenite extrusion in SA7 cells may be mediated by a GSH-dependent and verapamil- and cyclosporin A-sensitive membrane transport system. Since arsenite extrusion was found dose-dependently inhibited by energy poison [potassium cyanide (KCN)] and an ATPase inhibitor (sodium vanadate), ATP is apparently required for arsenite extrusion in SA7 cells.

Redox Regulation of GA-binding Protein-α DNA Binding Activity

We have investigated the reduction/oxidation (redox) regulation of the heteromeric transcription factor GA-binding protein (GABP). GABP, also known as nuclear respiratory factor 2, regulates the expression of nuclear encoded mitochondrial proteins involved in oxidative phosphorylation, including cytochrome c oxidase subunits IV and Vb, as well as the expression of mitochondrial transcription factor 1. GABP is composed of two subunits, the Ets-related GABP-alpha, which mediates specific DNA binding, and GABP-beta, which forms heterodimers and heterotetramers on DNA sequences containing the PEA3/Ets motif ((C/A)GGA(A/T)(G/A)). We demonstrate here that GABP DNA binding activity and GABP-dependent gene expression in 3T3 cells are inhibited by pro-oxidant conditions. DNA binding of recombinant GABP-alpha was activated by chemical reduction (dithiothreitol) and by thioredoxin; however, GSSG inhibited GABP DNA binding activity. Treatment of GABP-alpha, but not GABP-beta1, with sulfhydryl-alkylating agents also inhibited GABP DNA binding activity. Our results suggest that GABP DNA binding activity is redox-regulated in vivo, possibly by thioredoxin-mediated reduction and by GSSG-mediated oxidation of the GABP-alpha subunit. The regulation of GABP (nuclear respiratory factor 2) DNA binding activity by cellular redox changes provides an important link between mitochondrial and nuclear gene expression and the redox state of the cell.

Induction of Heme Oxygenase by Phorone (Diisopropylidene Acetone), a Potent Glutathione-Depleting Agent, Is Not Mediated by Glutathione Depletion and Lipid Peroxidation.

Oxidative stress is suggested to be involved in the induction of tissue microsomal heme oxygenase (HO). Phorone (diisopropylidene acetone) is a glutathione (GSH)-depleting agent as well as a potent inducer of HO. In this study, phorone and another GSH-depleting agent, buthionine sulfoximine (BSO), were compared in rats given a normal (7 IU /100 g diet) or high (100 IU/100 g diet) level of dietary vitamin E (VE) to determine whether HO induction is mediated through GSH depletion and/or lipid peroxidation. In Experiment 1, single (250 mg/kg B.W.) and three consecutive doses (250mg/kg B.W./day) of phorone to animals given normal dietary VE comparably decreased the liver GSH contents, but the liver microsomal HO was induced in a dose-dependent manner. Liver chemiluminescence intensity also increased in response to the dose level of phorone, suggesting an enhanced liver lipid peroxidation stimulated by phorone administration. In Experiment 2, phorone (250 mg/kg B.W./day) or BSO (1 mmol/kg B.W./day) was administered intermittently 4 times to animals given a normal or high level of dietary VE. Administration of phorone and BSO significantly decreased the GSH contents both in the liver and kidney, but no effect of high dietary VE on the contents was observed. HO was induced significantly in both the liver and kidney following phorone treatment, but not by BSO. High dietary VE also did not affect the phorone-induced HO activity. Phorone administration significantly enhanced the liver chemiluminescence intensity, but it was suppressed to the control level by high dietary VE. BSO administration did not promote lipid peroxidation in the liver. In the kidney, neither phorone nor BSO stimulated lipid peroxidation. From these results, we presume that induction of HO by phorone in the liver and kidney is not mediated by GSH depletion and lipid peroxidation.