Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

Influence of decreased intracellular glutathione level on the mutagenicity of patulin in cultured mouse lymphoma cells

The mutagenicity of the mycotoxin patulin was assessed by the thymidine kinase mutation assay, which is based on point mutations and deletions. Patulin was mutagenic in cultured mouse lymphoma cells and the mutagenicity was significantly increased in cells pretreated with buthionine sulfoximine, which reduces intracellular glutathione levels. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004 Financial support Deutsche Forschungsgemeinschaft (Grant Me 574/14-2)     

Glutathione depletion greatly reduces neocarzinostatin cytotoxicity in Chinese hamster V79 cells

The role of the intracellular thiol glutathione in the reductive activation of neocarzinostatin was investigated in Chinese hamster V79 cells. The cells were pretreated with agents that either lower (buthionine sulfoximine or diethyl maleate) or elevate (oxothiazolidine carboxylate) intracellular glutathione levels. These cells were then exposed to 1-5 micrograms/ml neocarzinostatin for 1 h and assayed for survival. Depletion of glutathione to levels at or below the limit of detection resulted in a marked reduction in neocarzinostatin cytotoxicity, while increasing glutathione levels to 250% of control values had little or no effect on neocarzinostatin toxicity. High performance liquid chromatography analysis of cysteine in untreated and glutathione-depleted cells showed cysteine levels lower than 0.2 microM, indicating that cysteine does not play a major role in the reductive activation of neocarzinostatin in untreated or glutathione-depleted cells. When intracellular cysteine levels were artificially elevated by oxothiazolidine carboxylate treatment of glutathione-depleted cells, neocarzinostatin toxicity was about two-thirds that seen in cells with normal glutathione levels. In cell-free systems, others have shown that reducing agents such as 2-mercaptoethanol are necessary for the activation of neocarzinostatin to a species that will cleave DNA. In this study, we have identified glutathione as the major cellular reducing agent for the activation of neocarzinostatin in a mammalian cell line.     

The effect of glutathione on development in wild carrot suspension cultures

The role of reduced and oxidized glutathione in plant development was investigated using wild carrot suspension cultures. Concentrations of GSH are lower in developing than in proliferating carrot cultures. Addition of 0.3 mM buthionine sulfoximine (a glutathione synthesis inhibitor) to developing cultures decreased the cellular GSH levels and enhanced somatic embryogenesis while addition of 0.6 mM GSH increased the cellular GSH levels and inhibited embryogenesis. Additions of GSH and buthionine sulfoximine to developing cultures also indicated that buthionine sulfoximine is acting specifically to lower GSH levels and not through some nonspecific toxic effect. These results provide evidence that the levels of GSH are important in determining whether carrot cells develop into somatic embryos or grow proliferatively.     

The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

Background

Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.

Principal Findings

We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.

Conclusions

Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.     

Glutathione, a first line of defense against cadmium toxicity

Experimental modulation of cellular glutathione levels has been used to explore the role of glutathione in cadmium toxicity. Mice treated with buthionine sulfoximine [an effective irreversible inhibitor of gamma-glutamylcysteine synthetase (EC 6.3.2.2) that decreases cellular levels of glutathione markedly] were sensitized to the toxic effects of CdCl2. Mice pretreated with a sublethal dose of Cd2+ to induce metallothionein synthesis were not sensitized to Cd2+ by buthionine sulfoximine. Mice sensitized to Cd2+ by buthionine sulfoximine were protected against a lethal dose of Cd2+ by glutathione mono isopropyl ester (L-gamma-glutamyl-L-cysteinylglycylisopropyl ester), but not by glutathione. These results are in accord with studies that showed that glutathione mono esters (in contrast to glutathione) are efficiently transported into cells and converted intracellularly to glutathione. The findings indicate that intracellular glutathione functions in protection against Cd2+ toxicity, and that this tripeptide provides a first line of defense against Cd2+ before induction of metallothionein synthesis occurs. The experimental approach used here in which cellular levels of glutathione are decreased or increased seems applicable to investigation of other types of metal toxicity and of other glutathione-dependent biological phenomena.     

Influence of changes in glutathione concentration on body temperature and tolerance to cerebral ischemia

Two compounds that deplete glutathione (buthionine sulfoximine and diethyl maleate) with different mechanisms of action decrease body temperature and increase tolerance to complete global cerebral ischemia, both correlating closely with the glutathione concentration decrease. Glutathione apparently participates in the regulations of these functional parameters. GSH diethyl ester does not influence the latter, though it increases moderately the GSH concentration. Injection of GSH ester into the cerebral ventricles or subcutaneously selectively increases the GSH level in the brain and liver. An influence of the brain on the glutathione system in the liver was revealed. Diethyl maleate and GSH ester increase the activity of glutathione metabolizing enzymes under certain conditions.     

Effect of glutathione on phytochelatin synthesis in tomato cells

Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably [γ-Glu-Cys]₂-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little [³⁵S]cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione.     

Influence of glutathione on zinc-mediated cellular toxicity

The effect of zinc on various pulmonary cell lines has been studied by measuring the depletion of total cellular glutathione after exposure to zinc(II) chloride at different concentrations. Total cellular glutathione (cGS) was measured at 31 ± 3 nmol/mg, 3.8 ± 0.6 nmol/mg, and 3.7 ±1.2 nmol/mg protein in A549, L2, and 11Lu cells, respectively. After treatment with buthionine sulfoximine (BSO), the cGS levels decreased by 20% in A549 cells and below 0.2 nmol/mg in L2 and 11Lu cells. Exposure of A549 cells to 25–200 μM ZnCl₂ for 4 h alone decreased the cGS content to 60–80%. There was little additional effect in BSO-pretreated cells. In L2 and 11Lu cells, the decrease of cGS was 70–85% following exposure to 15–150 μM ZnCl₂ for 2 h. If BSO was also used, the decrease in cGS was 85–95% in L2 cells and 75–85% in 11Lu cells. Exposure to 25–250 μM ZnCl₂ for 2 h diminished protein synthesis as determined by radiolabeled methionine incorporation, with half-maximum inhibition (EC₅₀) from 40–160 μM ZnCl₂. To attain similar EC₅₀ values in BSO-pretreated cells, only about half the zinc concentrations were required as compared to cells without pretreatment. The decrease of cGS was accompanied by an increased ratio of oxidized : reduced glutathione that was more pronounced in cells with low glutathione content.     

Alterations of the glutathione redox cycle status in fumonisin B1-treated pig kidney cells

Fumonisin B₁ (FB₁) causes equine leukoencephalomalacia, porcine pulmonary edema, and liver tumors and chronic nephritis in rats. To investigate mechanisms by which FB₁ induces toxicity, effects of FB₁ on cellular glutathione (GSH) redox status and GSH depletion on FB₁ toxicity in pig kidney (LLC-PK₁) cells were studied. Treatment of LLC-PK₁ cells with 50 μM FB₁ for 24 hours significantly decreased cellular GSH contents from 56 ± 3.2 to 42.7 ± 4.4 nmol/mg protein (p < 0.05) and increased the activities of glutathione reductase (GR) from 25.7 ± 2.4 to 35.7 ± 5.0 μmol NADPH/mg protein (p < 0.05). The activities of glutathione peroxidase (GSHpx), catalase, and Cu,Zn-superoxide dismutase (SOD) were not changed by this treatment. Treatment of LLC-PK₁ cells for 12 hours with 0.1. mM buthionine sulfoximine (BSO), a selective inhibitor of the enzyme γ-glutamylcysteine synthetase that catalyzes the rate-limiting reaction in de novo GSH synthesis, decreased cellular GSH levels to about 20% of that found in the control cells. The cells pretreated with 0.1 mM BSO for 12 hours were significantly sensitized to the FB₁ cytotoxicity as determined by a long-term survival assay (p < 0.05). The results demonstrate that FB₁ changes GSH redox cycle status in LLC-PK₁ cells, and GSH may play a role in cytoprotection against FB₁ toxicity. © 1997 John Wiley & Sons, Inc.     

Cadmium produces a delayed mitogenic response and modulates the EGF response in quiescent NRK cells

Recent studies have shown that cadmium, at subtoxic levels, may induce a response characteristic of that elicited by a type of growth factor that supports the anchorage independent growth of cells that are not fully transformed. That is, Cd⁺⁺ was found to replace transforming growth factor beta in supporting soft agar growth of NRK-49F cells. To tes the extent to which Cd⁺⁺ further mimics transforming growth factor beta in its effects and to establish response patterns that suggest possible molecular mechnisms of action, we have determined the effects of Cd⁺⁺ and/or epidermal growth factor (EGF) on DNA synthesis in quiescent NRK-49F cells. We found that subtoxic doses of Cd⁺⁺ modulate EGF-induced DNA synthesis in a dose-dependent fashion. Although Cd⁺⁺ effects on early (16–24 hr) EGF-induced DNA synthesis are primarily inhibitory, later effects involve stimulation as well. Subtoxic doses of Cd⁺⁺ did not stimulate DNA synthesis in quiescent cells within 24 hr of addition. At later times (40 or 64 hr), however, an increase in DNA synthesis of up to threefold was induced by 0.25 M Cd⁺⁺. This pattern of mitogenic response, involving inhibition of early growth-factor induced DNA synthesis and stimulation of late DNA synthesis, is consistent with that reported to be effected in some instances by transforming growth factor beta. Because a defined pattern of gene expression also is associated with the mitogenic responses to transforming growth factor beta, future studies at the molecular level can definitively test the degree to which Cd⁺⁺ and transforming growth factor beta effects are common.Abbreviations CFE colony forming efficiency - EGF epidermal growth factor - MT metallothionein - PGDF paltelet derived growth factor - TGF transforming growth factor     

Redox activation of DUSP4 by N-acetylcysteine protects endothelial cells from Cd2+-induced apoptosis

Redox imbalance is a primary cause of endothelial dysfunction (ED). Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source in cells, can significantly regulate cell function via reversible protein thiol modification. N-acetylcysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. From HPLC analysis, NAC significantly increases both cellular GSH and tetrahydrobiopterin levels. Immunoblotting of endothelial NO synthase (eNOS) and DUSP4, a dual-specificity phosphatase with a cysteine as its active residue, revealed that both enzymes are upregulated by NAC. EPR spin trapping further demonstrated that NAC enhances NO generation from cells. Long-term exposure to Cd²⁺ contributes to DUSP4 degradation and the uncontrolled activation of p38 and ERK1/2, leading to apoptosis. Treatment with NAC prevents DUSP4 degradation and protects cells against Cd²⁺-induced apoptosis. Moreover, the increased DUSP4 expression can redox-regulate the p38 and ERK1/2 pathways from hyperactivation, providing a survival mechanism against the toxicity of Cd²⁺. DUSP4 gene knockdown further supports the hypothesis that DUSP4 is an antioxidant gene, critical in the modulation of eNOS expression, and thus protects against Cd²⁺-induced stress. Depletion of intracellular GSH by buthionine sulfoximine makes cells more susceptible to Cd²⁺-induced apoptosis. Pretreatment with NAC prevents p38 overactivation and thus protects the endothelium from this oxidative stress. Therefore, the identification of DUSP4 activation by NAC provides a novel target for future drug design.     

Selenite-induced inhibition of colony formation by buthionine sulfoximine-sensitive and resistant cell lines

We previously demonstrated that treatment of HeLa cells with buthionine sulfoximine (BSO), which decreases the level of cellular glutathione, resulted in a decrease in the potency of selenite in inhibiting cell colony formation. We have now examined the effect of selenite on normal human lung fibroblast (CCL-210) cells, which resemble HeLa cells in their sensitivity to BSO, and on human lung adenocarcinoma (A549) cells, which are relatively insensitive to BSO. We have found that BSO treatment caused an approximately fourfold decrease in selenite potency in the CCL-210 cells, but had no significant effect on its potency in A549 cells. These results support the hypothesis that for selenite to exert its cytotoxic effect, it must under-go the reaction with an SH compound to form the selenotrisulfide. As a result of the lower sensitivity of the tumor cells to BSO, it was possible to achieve a large differential sensitivity to the cytotoxic effect of selenite.     

Nitric oxide production by cultured aortic endothelial cells in response to thiol depletion and replenishment

The requirements and influence of thiols on the production of nitric oxide (NO) were examined in cultured porcine aortic endothelial cells. NO production was diminished when cells were pretreated with thiol-depleting agents (IC50: N-ethylmaleimide, 30 microM; 1-chloro-2,4-dinitrobenzene, 200 microM; diamide, 1.5 mM; diethyl maleate, 20 mM). The depletion of glutathione (45-99% loss at the various IC50 values) and protein thiols (3-25% loss at IC50) showed no consistent relationship to decreased NO production. The effects of the agents on NO production were not linked to altered sensitivity to the stimulant (calcium ionophore A23187; maximal effect at 10 microM), but roughly paralleled the appearance of cell damage (17-44% lactate dehydrogenase release at IC50). The decrease in NO production due to 1-chloro-2,4-dinitrobenzene was partially reversed by cysteine, dithioerythritol, and dihydrolipoate, whereas cystine partially reversed the decrease due to diamide or diethyl maleate. On the other hand, several thiols diminished NO production in control cells. Overall, alterations of NO production did not parallel the depletion or replenishment of either glutathione, protein thiol, or soluble thiol pools, and so the results argue against hypotheses that cellular thiols are either substrates or necessary cofactors in the pathway of NO synthesis in endothelial cells.     

Selection and characterization of cadmium-resistant suspension cultures of the wild tomato Lycopersicon peruvianum

Suspension cultures of Lycopersicon peruvianum were selected for resistance to cadmium by stepwise exposure to increasing concentrations of cadmium sulfate. Resistant cells grow in 1500 micromolar Cd⁺⁺. This resistance was retained for thirty generations without selection. Both resistant and parental sensitive cultures take up Cd⁺⁺ at similar rates and to the same final levels. Exposure of sensitive or resistant cultures to Cd⁺⁺, Cu⁺⁺, or Zn⁺⁺ leads to the intracellular accumulation of a low molecular weight, cysteine-rich, cadmium-binding protein. This metallothionein is induced over fifteen fold by 100 M cadmium and builds up to about five fold higher levels in the resistant cultures.Abbreviations Cd⁺⁺ divalent cadmium ion - Cu⁺⁺ divalent copper ion - Zn⁺⁺ divalent zinc ion - BA benzyl adenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-acetic acid     

Biotransformation and cytotoxic effects of hydroxychavicol, an intermediate of safrole metabolism, in isolated rat hepatocytes

The biotransformation and cytotoxic effects of hydroxychavicol (HC; 1-allyl-3,4-dihydroxybenzene), which is a catecholic component in piper betel leaf and a major intermediary metabolite of safrole in rats and humans, was studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to HC caused not only concentration (0.25-1.0 mM)- and time (0-3 h)-dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione, and protein thiols, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. At a concentration of 1 mM, the cytotoxic effects of safrole were less than those of HC. The loss of mitochondrial membrane potential and generation of oxygen radical species assayed using 2′,7′-dichlorodihydrofluoresein diacetate (DCFH-DA) in hepatocytes treated with HC were greater than those with safrole. HC at a weakly toxic level (0.25 and/or 0.50 mM) was metabolized to monoglucuronide, monosulfate, and monoglutathione conjugates, which were identified by mass spectra and/or ¹H nuclear magnetic resonance spectra. The amounts of sulfate rather than glucuronide or glutathione conjugate predominantly increased, accompanied by a loss of the parent compound, with time. In hepatocytes pretreated with either diethyl maleate or salicylamide, HC-induced cytotoxicity was enhanced, accompanied by a decrease in the formation of these conjugates and by the inhibition of HC loss. Taken collectively, our results indicate that (a) mitochondria are target organelles for HC, which elicits cytotoxicity through mitochondrial failure related to mitochondrial membrane potential at an early stage and subsequently lipid peroxidation through oxidative stress at a later stage; (b) the onset of cytotoxicity depends on the initial and residual concentrations of HC rather than those of its metabolites; (c) the toxicity of HC is greater than that of safrole, suggesting the participation of a catecholic intermediate in safrole cytotoxicity in rat hepatocytes.     

Role of Endogenous Glutathione in the Oxidation of Dopamine

Abstract: Intrastriatal injection of dopamine causes the selective degeneration of tyrosine hydroxylase-containing terminals and an increase in content of cysteinylcatechols, an index of dopamine oxidation. Both of these effects can be attenuated by coadministration of antioxidants such as glutathione. Therefore, we investigated the effects of decreased endogenous glutathione on the neurotoxic potential of dopamine. We observed that pretreatment with either l -buthionine sulfoximine, a specific inhibitor of glutathione synthesis, or diethyl maleate, which forms adducts with glutathione, caused significant decreases in endogenous glutathione levels at the time of dopamine injection. Pretreatment with l -buthionine sulfoximine potentiated the formation of protein cysteinyl-dopamine after intrastriatal injection of 1.0 µmol of dopamine. We also observed that intrastriatal injection of 1.0 µmol of dopamine decreased striatal glutathione content in all pretreatment conditions. However, injection of a low dose (0.05 µmol of dopamine) caused a decrease in striatal glutathione levels only in the l -buthionine sulfoximine-pretreated rats. Diethyl maleate pretreatment was not effective in potentiating either cysteinyl-catechol formation or glutathione loss after dopamine injection. We conclude that dopamine contributes to cellular oxidative stress and that this can be exacerbated, or at least unmasked, if glutathione synthesis is compromised.     

Induction of cystine and glutamate transport activity in human fibroblasts by diethyl maleate and other electrophilic agents

The transport activity for cystine and glutamate in cultured human diploid fibroblasts is enhanced in response to diethyl maleate treatment. The enhancement is time- and dose-related, with a lag of about 3 h, and maximum enhancement (approximately 3-fold increase in the rate of uptake) is attained after 1 to 2 days of incubation of the cells with 0.1 mM diethyl maleate. The enhancement of the transport activity is accompanied by an increase in the Vmax and little change in the Km, and it requires RNA and protein synthesis. Other electrophilic agents, such as cyclohex-2-en-1-one, ethacrynic acid, 1,2-epoxy-3-(p-nitro-phenoxy)propane, and sulfobromophthalein, similarly enhance the transport activity. These electrophiles are known as agents that interact with glutathione. For example, diethyl maleate at high concentrations, i.e. 1 mM, depletes intracellular glutathione and injures the cells. However, at relatively low concentrations diethyl maleate and other electrophilic compounds do cause increases in the intracellular levels of glutathione which we attribute to the enhanced uptake of cystine. It is suggested that the transport system for cystine and glutamate is involved in a protective mechanism of cells against an electrophilic attack.     

Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides

The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.