Disruption of the Intracellular Sulfhydryl Homeostasis by Cadmium-induced Oxidative Stress Leads to Protein Thiolation and Ubiquitination in Neuronal Cells*

Maria E. Figueiredo-PereiraDagger §, Svetlana Yakushin, and Gerald Cohen

From the Dagger  Department of Biological Sciences, Hunter College of City University of New York, New York 10021 and  Department of Neurology, Mount Sinai School of Medicine of City University of New York, New York 10029

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Cadmium is a potent cell poison known to cause oxidative stress by increasing lipid peroxidation and/or by changing intracellular glutathione levels and to affect the ubiquitin/ATP-dependent proteolytic pathway. However, the cellular mechanisms involved in cadmium toxicity are still not well understood, especially in neuronal cells. To investigate the relationship between cadmium-induced oxidative stress and the ubiquitin/ATP-dependent pathway, we treated cultures of neuronal cells with different concentrations of the metal ion. In addition to decreases in glutathione levels, we observed marked increases in protein-mixed disulfides (Pr-SSGs) after exposure of HT4 cells (a mouse neuronal cell line) or rat primary mesencephalic cultures to Cd2+. The increases in intracellular levels of Pr-SSGs were concurrent with increases in the levels of ubiquitinated proteins (Ub proteins) when the HT4 cells were subjected to lower (25 µM or less) concentrations of cadmium. However, higher concentrations of cadmium (50 µM), which were toxic, led to increases in Pr-SSGs but inhibited ubiquitination, probably reflecting inhibition of ubiquitinating enzymes. The cadmium-induced changes in Pr-SSGs and Ub proteins were not affected when more than 85% of intracellular glutathione was removed from the cells by the glutathione synthetase inhibitor L-buthionine-(S,R)-sulfoximine. However, the reducing agent dithiothreitol, which prevented the build up of Pr-SSGs in the cell, also blocked the accumulation of Ub proteins induced by cadmium. In addition, dithiothreitol blocked the effects of the higher toxic (50 µM) concentrations of cadmium on cytotoxicity and on glutathione, Pr-SSGs, and Ub proteins. Together, these results strongly suggest that changes in the levels of intracellular Pr-SSGs and ubiquitin-protein conjugates in neuronal cells are responses closely associated with the disruption of intracellular sulfhydryl homeostasis caused by cadmium-mediated oxidative stress.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

One of the hallmarks of neurodegeneration is the appearance of intraneuronal inclusions consisting of ubiquitin-protein conjugates (1, 2). The mechanisms generating such abnormal inclusions remain unknown. Ubiquitination of proteins occurs posttranslationally and is a complex ATP-dependent process in which ubiquitin is sequentially activated, transferred to ubiquitin-conjugating enzymes, and then ligated to protein substrates (reviewed in Ref. 3). Very often, more than one ubiquitin is attached to the target proteins, forming polyubiquitin chains (4). Ubiquitin can be removed from the ubiquitin-protein conjugates by deubiquitinating enzymes (5).

Covalent binding of ubiquitin to proteins in the cytosol and in the nucleus is frequently viewed as a means by which proteins are marked for subsequent degradation by the ubiquitin/ATP-dependent proteinase, commonly known as the 26 S proteasome (reviewed in Refs. 6 and 7). In general, ubiquitinated proteins (Ub proteins)1 do not accumulate in healthy cells. They are rapidly degraded by the 26 S proteasome (reviewed in Ref. 7). The failure to eliminate the ubiquitin-protein deposits in the degenerating neurons may result either from a malfunction of the ubiquitin/ATP-dependent proteolytic pathway or from structural changes in the protein substrates rendering them inaccessible to the proteolytic machinery. The accumulation of Ub proteins can then lead to proteotoxicity.

Oxidative stress is one of the mechanisms that contributes to structural changes or misfolding of proteins. Substantial evidence has accumulated showing that oxidative stress may play an important role in neurodegeneration (reviewed in Refs. 8-10). The reactive oxygen species resulting from episodes of oxidative stress promote the modification of cellular proteins (11). Cells possess a protective mechanism to overcome the potentially toxic accumulation of oxidatively modified proteins, namely an increase in proteolysis (12, 13). More recently, Davies and co-workers (14) demonstrated that oxidative stress in cultured liver epithelial cells led to measurable changes in intracellular proteolysis. The degradation of oxidatively modified proteins was postulated to occur via ubiquitin-independent and ATP-independent mechanisms (15, 16).

On the other hand, studies with yeast showed that overexpression of the polyubiquitin gene conferred resistance to oxidative stress in cells grown by respiration (17). In addition, Taylor and co-workers (18-20) detected significant increases in ubiquitin-protein conjugates, ubiquitin-activating and ubiquitin-conjugating enzyme activity, and intracellular proteolysis in lens epithelial cells recovering from episodes of oxidative stress induced by H2O2. Together, these studies suggest that the ubiquitin/ATP-dependent proteolytic system may play a role in the removal of oxidatively modified proteins.

To further investigate the mechanisms underlying the removal of oxidatively modified proteins in mammalian cells, we chose to induce oxidative stress with cadmium in a neuronal cell line (HT4) and in rat mesencephalic primary cultures. Cadmium is a potent cell poison known to cause oxidative stress (reviewed in Ref. 21) and to affect the ubiquitin/ATP-dependent proteolytic pathway (22, 23). Our results show that the heavy metal decreased intracellular glutathione concentrations and increased the levels of protein-mixed disulfides (Pr-SSGs) and of ubiquitin-protein conjugates in a time- and concentration-dependent manner. In addition, we demonstrate that only a small pool of glutathione (less than 15% of the total) is sufficient to produce significant increases in Pr-SSGs levels in response to cadmium. Most importantly, we show that the thiol-reducing agent dithiothreitol blocks the increases in Pr-SSGs and Ub protein levels produced by cadmium, indicating that one of the mechanisms responsible for cadmium toxicity is the perturbation of intracellular sulfhydryl homeostasis.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
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References

Cell Cultures-- HT4 cells were derived from a mouse neuroblastoma cell line containing a recombinant temperature-sensitive mutant of SV40 large T antigen. When grown at 39 °C (nonpermissive temperature), HT4 cells differentiate with neuronal morphology, express neuronal antigens, synthesize and secrete nerve growth factor, and express receptors for nerve growth factor (24) and for glutamate (25). The cells were maintained at 33 °C in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and 100 units/ml penicillin, 100 µg/ml streptomycin in 5% CO2. To induce differentiation, the temperature was changed to 39 °C, at which the cells were kept for 3 days. Following the period of differentiation, the cells were maintained at 37 °C, at which they were kept for at least 7 h prior to treatment with the heavy metal.

Cultures of embryonic rat mesencephalon were prepared as described in Mytilineou et al. (26). Briefly, on day 14 of gestation, the mesencephalon was surgically removed, and the cells were dissociated mechanically and plated at a density of ~100,000 cells/cm2 on polyornithine-coated 35-mm tissue culture dishes. The feeding medium was minimum Eagle's medium supplemented with glucose (30 mM), sodium bicarbonate (44.6 mM), 10% fetal calf serum, and 10% horse serum. Experiments were run after 4 or 5 days in culture.

Glutathione Assay-- Total glutathione was quantified as described previously (26) by a modification of the standard recycling assay based on the reduction of 5,5-dithiobis-(2-nitrobenzoic acid) in the presence of glutathione reductase and NADPH. The assay measures both GSH and GSSG; normally, GSSG constitutes less than 5% of the total glutathione in control cell cultures. The medium was first aspirated, and then the cells were rinsed twice with PBS and harvested in 500 µl of 0.4 N perchloric acid, followed by sonication for 10 s (setting of 4; Vibra-Cell model V1A; Sonics and Materials, Danbury, CT) and centrifugation at 16,000 × g for 15 min. Glutathione and protein assays were performed on the supernatant and pellet, respectively. In several experiments, GSSG was separately measured after removal of GSH with N-ethylmaleimide (26).

Assay for Protein-Mixed Disulfides-- Pr-SSGs were quantified from pellets of the same samples prepared for the glutathione assays, following a modification of the procedure of Akerboom and Sies (27). The protein pellet was rinsed and sonicated twice with 0.4 N perchloric acid and resuspended in 0.01 M Tris-HCl, pH 7.5. The solution was then treated for 45 min at 41 °C with 0.25% sodium borohydride at neutral pH to reduce the disulfide linkage. Excess borohydride was subsequently decomposed by acidification. Liberated GSH was then measured as described above. The liberated GSH reflected that which was previously linked to Pr-SSGs. GSH standards were assayed in a similar medium.

Cytotoxicity Studies-- Cell viability was assessed by a modification of the method described in Mosmann (28). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide at a final concentration of 0.5 mg/ml was dissolved in Dulbecco's modified Eagle's medium (with 1 g/liter of D-glucose) containing 5% fetal bovine serum and 100 units/ml penicillin, 100 µg/ml streptomycin. The incubation medium was removed, and then each well of a 24-well plate was incubated with 0.5 ml of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution for 1 h at 37 °C in 5% C02. The supernatant was then aspirated, and 1 ml of a solution of 0.04 M HCl in isopropanol was added and gently shaken to dissolve the precipitated dye. The solution was transferred into 1.5-ml microcentrifuge tubes and centrifuged at 16,000 × g for 5 min, and the absorbance of the supernatant was read at 550 and 620 nm with a plate reader (ATCC model 340; SLT Laboratory Instruments, Hillsborough, NC). The results were expressed as the difference between the values obtained at the two wavelengths.

Preparation of Cell Extracts for Western Blotting-- Following the indicated treatments, cell extracts were prepared as described previously (29). Proteins were separated by SDS-polyacrylamide gel electrophoresis (following the method of Laemmli (30)) on 8% gels for ubiquitin-protein conjugate detection. Identification of the Ub proteins was by Western blotting, and the antigens were visualized by a horseradish peroxidase method (Bio-Rad) utilizing the substrate 3,3',5,5'-tetramethylbenzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Quantitative analysis of the immunostaining was by image analysis as described previously (31).

Antibodies-- Ubiquitin-protein conjugates were detected with a rabbit polyclonal antibody (1:600) raised against ubiquitin conjugated to gamma -globulins with glutaraldehyde, obtained from Dako Corp. (Carpinteria, CA).

Protein Determination-- Protein determination was by a bicinchoninic acid assay kit (Pierce) and by the method of Lowry et al. (32) using bovine serum albumin as a standard.

Statistical Analysis-- Statistical comparisons were performed with the Tukey-Kramer multiple comparison test (Instat 2.0, Graphpad Software, San Diego, CA).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cadmium Induces a Time- and Dose-dependent Decrease in Intracellular Glutathione-- To determine the time and concentration dependence of the effect of the metal ion on the intracellular levels of glutathione, we treated confluent differentiated HT4 cells with a range of concentrations between 1 and 100 µM CdSO4. After incubations for 1-8 h, the cells were harvested, and intracellular concentrations of glutathione were measured as described under "Experimental Procedures." Fig. 1A represents the time course of the changes in glutathione levels measured after treatment with 10, 25, and 50 µM CdSO4. It shows that the decrease in glutathione induced by all three concentrations of the metal ion achieved statistical significance after 2 or 4 h of treatment. At the highest concentration tested in these experiments (50 µM), the glutathione level was decreased to almost 0 after 8 h of treatment.


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Fig. 1.   Cadmium decreases glutathione levels in a time-dependent (A) and dose-dependent (B) fashion in HT4 cells. Intracellular glutathione levels were determined as described under "Experimental Procedures." Data represent the mean and S.E. from at least eight determinations in A and six determinations in B. All levels are expressed relative to no cadmium treatment, which corresponds to an average of 28.9 ± 2.6 nmol of glutathione/mg of protein (100%). The cadmium concentrations (A) were as follows: black-square, 10 µM; open circle , 25 µM; bullet , 50 µM. The times of incubation (B) were as follows: triangle , 1 h; black-triangle, 4 h. The * identifies the values that are significantly different from control, with p < 0.05 or less for all values marked.

Based on these results we chose two time points (1 and 4 h) to establish a dose-dependent curve for the cadmium effect. No significant change was detected after 1 h of incubation with the various concentrations of the heavy metal (Fig. 1B). On the other hand, 4 h of incubation with 25 µM decreased the glutathione levels sharply to 56% of control levels (Fig. 1B). Higher concentrations, however, failed to further reduce the intracellular levels of glutathione.

Cadmium Induces a Time- and Dose-dependent Increase in the Levels of Protein-Mixed Disulfides in HT4 Cells-- Exposure of HT4 cells to 50 µM Cd2+ for 4 h induced an elevation in GSSG from 0.17 ± 0.03 to 0.41 ± 0.07 nmol/mg of protein (p < 0.1; two-tailed Student's t test; n = 8 per group).

The oxidation of GSH to GSSG may lead concomitantly to a thiolation of protein-SH groups to form protein-mixed disulfides (33). To verify this paradigm, we evaluated the effect of Cd2+ on intracellular levels of protein-mixed disulfides by determining the reductive release of GSH from perchloric acid-precipitated proteins.

Fig. 2 shows that Pr-SSGs is elevated by treatment with cadmium. The increase was significant even after 1 h of treatment with the highest concentration of Cd2+ (50 µM) and reached 3.2-, 4.0-, and 5.8-fold of control levels after 1, 2, and 4 h, respectively (Fig. 2A). Maximum levels (6.8-fold of control) were observed after 6 h of exposure to 50 µM Cd2+ (Fig. 2A). Increases in Pr-SSGs levels were also observed when the cells were treated with 25 or 10 µM heavy metal up to 6 h. Significant changes in Pr-SSG levels were detected with 10 µM metal ion after 4 h of treatment (Fig. 2A). Dose-dependent studies indicated that when cells were exposed to increasing concentrations of CdSO4, the greatest elevation in Pr-SSGs (7.6-fold) was seen after treatment with 100 µM (Fig. 2B). Due to its cytotoxic effect, higher concentrations of the divalent metal were not tested. Exposures of 1 h elicited significant increases in the levels of Pr-SSGs at concentrations of 50 µM Cd2+ or higher (Fig. 2B).


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Fig. 2.   Cadmium increases Pr-SSG levels in a time-dependent (A) and dose-dependent (B) fashion in HT4 cells. Intracellular Pr-SSGs levels were determined as described under "Experimental Procedures." Data represent the mean and S.E. from at least eight determinations in A and six determinations in B. The intracellular levels of Pr-SSGs were expressed as pmol of Pr-SSGs/mg of protein. The cadmium concentrations (A) were as follows: black-square, 10 µM; open circle , 25 µM; bullet , 50 µM. The times of incubation (B) were as follows: triangle , 1 h; black-triangle, 4 h. The * identifies the values that are significantly different from control, with p < 0.05 or less for all values marked.

Cadmium Induces Changes in Glutathione and Protein-Mixed Disulfides in Primary Cultures of Fetal Rat Mesencephalon-- To establish that the Cd2+ effect was not restricted to transformed neuronal cells, such as the HT4 cell line, we studied the effect of the heavy metal on primary cultures of embryonic rat mesencephalon. As with HT4 cells, incubations for 1 h with all Cd2+ concentrations tested led to no statistically significant changes in the cellular levels of glutathione in the mesencephalic cultures (Fig. 3A). However, longer (4 h) incubations with the divalent metal induced significant changes in the cellular glutathione levels. The lowest concentration of CdSO4 tested (5 µM) produced a transient increase in intracellular glutathione, but the highest concentration tested (100 µM) caused a drop to 45% of control levels (Fig. 3A).


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Fig. 3.   Cadmium decreases glutathione levels (A) and increases Pr-SSG levels (B) in a dose-dependent fashion in mesencephalic primary cell cultures. Intracellular glutathione and Pr-SSG levels were determined as described under "Experimental Procedures." Data represent the mean and S.E. from at least four determinations. Glutathione levels are expressed relative to no cadmium treatment, which corresponds to an average of 23.6 ± 2.6 nmol of glutathione/mg of protein (100%). Pr-SSG levels are expressed as pmol of Pr-SSG/mg of protein. The times of incubation were as follows: A, open circle , 1 h, and bullet , 4 h; B, triangle , 1 h, and black-triangle, 4 h. The * identifies the values that are significantly different from control, with p < 0.05 or less for all values marked.

The dose-dependent elevation in Pr-SSGs induced by the heavy metal in the mesencephalic cultures (Fig. 3B) was parallel to but not as great as that detected in HT4 cells. After 4 h of treatment, the highest concentration tested (100 µM) induced an 8-fold increase in Pr-SSGs in the HT4 cells as compared with controls, but only a 4-fold increase was observed in the treated mesencephalic cultures (compare Figs. 2B and 3B).

Cadmium Decreases HT4 Cell Viability-- The time-dependent cytotoxicity of 10, 25, and 50 µM cadmium was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (Fig. 4). Treatment with 10 and 25 µM Cd2+ was not cytotoxic up to 8 and 6 h of incubation, respectively. However, there was a sharp drop in viability in cells treated for 8 h with 25 µM heavy metal. Treatment with 50 µM Cd2+ significantly reduced cell viability even after 1 h of incubation (Fig. 4).


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Fig. 4.   Effect of cadmium on the viability of HT4 cells assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay as described under "Experimental Procedures." Data represent the mean and S.E. of eight determinations and are shown as percentage of cell viability measured with no Cd2+ (control, 100%). The cadmium concentrations were as follows: black-square, 10 µM; open circle , 25 µM; bullet , 50 µM. The * identifies the values that are significantly different from control, with p < 0.05 or less for all values marked.

Cadmium Induces a Time- and Dose-dependent Change in the Levels of Ubiquitin-Protein Conjugates in HT4 Cells-- The cadmium-induced thiolation of protein-SH groups leading to the formation of Pr-SSGs may provoke the misfolding of proteins, which may then be targeted for degradation by the ubiquitin/ATPdependent proteolytic pathway. Therefore, we examined whether cadmium ions led to an accumulation of Ub proteins in the treated cells. As shown in Figs. 5 and 6, the levels of ubiquitin conjugates were significantly increased after 1 and 2 h of treatment with 25 and 10 µM CdSO4, respectively. The greatest levels (4.3-fold of control) were detected after 8 h of exposure to 10 µM heavy metal. The highest dose of cadmium tested (50 µM) caused no detectable increases in the accumulation of Ub proteins. In fact, the levels of ubiquitin-protein conjugates in cells treated with 50 µM Cd2+ for 6 and 8 h were significantly below the control levels (Figs. 5 and 6). The latter immunoblot was overstained to show this decrease (Fig. 5, right panel).


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Fig. 5.   Immunoblots showing Ub proteins in HT4 cell extracts (8 µg of protein/lane) prepared as described under "Experimental Procedures." Cells were incubated at 37 °C for 1-8 h without (C) or with 10, 25, or 50 µM Cd2+. The intact blots are presented, showing the ubiquitin-conjugates, and are representative of one of at least four identical experiments for each condition tested.


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Fig. 6.   Quantitative analysis of the immunoblots prepared in Fig. 5. Shown are the changes in the levels of Ub-proteins, detected in cells treated for different periods of time (x axis) with concentrations of Cd2+ as follows: black-square, 10 µM; open circle , 25 µM; bullet , 50 µM. The values (y axis) are shown as fold changes compared with no added Cd2+ (100%). Data represent the mean and S.E. of four identical experiments for each condition tested. Quantification of the immunostaining was by image analysis as described under "Experimental Procedures." The * identifies the values that are significantly different from control, with p < 0.05 or less for all values marked.

Comparison of the Effects of Glutathione Depletion and Protein Thiol Reduction on the Intracellular Changes Induced by Cadmium-- Glutathione is considered the most important intracellular thiol involved in the formation of protein-mixed disulfides. Therefore, we determined whether depletion of intracellular GSH would interfere with the cadmium-induced formation of protein-mixed disulfides. HT4 cells were incubated with 5 µM glutathione synthetase inhibitor L-buthionine-(S,R)-sulfoximine (L-BSO) for 24 h followed by an additional 4 h of incubation with fresh reagent, preceding the cadmium treatment. Under these conditions, the concentrations of glutathione (20-30 nmol/mg of protein), as well as those of Pr-SSGs (0.1-0.2 nmol of GSH equivalent/mg of protein), were reduced to 14 and 30% of control levels, respectively (Fig. 7A). In contrast, the increase in the levels of Pr-SSG resulting from treatment with cadmium was not blocked by depletion of more than 85% of glutathione (Fig. 7A). Total depletion of intracellular glutathione was never accomplished, even when the cells were treated with higher concentrations of L-BSO for longer periods of time (results not shown). Therefore, the data demonstrate that the residual 3-4 nmol of glutathione/mg of protein left in the cell are sufficient for the formation of Pr-SSGs in response to the cadmium treatment. The cadmium-induced accumulation of Ub proteins was also not affected by the glutathione deficiency (Fig. 7A).


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Fig. 7.   Effect of 5 µM L-BSO (A) and 1 mM DTT (B) on levels of glutathione (square ), Pr-SSGs (black-square), and Ub proteins () in HT4 cells treated with 20 µM cadmium. Where indicated in A, cells were exposed consecutively to 5 µM L-BSO for 24 h, a fresh solution of 5 µM L-BSO for 4 h, and/or 20 µM cadmium for 4 h. Where indicated in B, cells were preincubated without or with 20 µM cadmium for 1 h followed by 1 mM DTT for an additional 3 h. After the indicated treatments, the cells were harvested and prepared for measurements of glutathione, Pr-SSGs, and Ub proteins as indicated under "Experimental Procedures." All levels are expressed relative to no cadmium treatment (100%). Data represent the mean and S.E. of three identical experiments for each condition tested. The * identifies the values that are significantly different from control, with p < 0.05 or less for all values marked.

When, in separate experiments, all of the intracellular glutathione was removed by subsequent addition of N-ethylmaleimide (60 µM for 1 h), no Pr-SSGs were formed in response to cadmium (not shown).

One of the mechanisms involved in protein S-thiolation is the oxidation of sulfhydryl groups of cysteine residues in proteins by glutathione disulfide (GSSG). The protein-bound glutathione (Pr-SSG) should be reduced by dithiothreitol (DTT), an effective reducing agent useful in the study of thiol-disulfide exchange reactions. To determine whether there was an association between Pr-SSG formation and the accumulation of ubiquitinated proteins, we incubated cells with DTT (1 mM). Exposure of the neuronal cells to the reducing agent alone significantly (p < 0.05) decreased the control levels of Pr-SSGs, without affecting the control levels of glutathione and Ub proteins. However, addition of the reducing agent to cadmium-treated cells concomitantly decreased the levels of Pr-SSGs and of Ub proteins (Fig. 7B).

Prevention of the Cytotoxic Effect of Cadmium by the Thiol-reducing Agent DTT-- As noted earlier, incubations with 50 µM cadmium significantly diminished cell viability (Fig. 4) and suppressed Ub proteins (Figs. 5 and 6). To determine whether one of the mechanisms involved in cadmium toxicity is the oxidation of protein thiols, we attempted to block the toxic effect of 50 µM cadmium by treating cells with increasing concentrations of DTT.

As shown in Fig. 8, concentrations of the reducing agent up to 1 mM prevented the decreases in glutathione (Fig. 8A) and Ub proteins (Fig. 8C) and the increases in Pr-SSGs (Fig. 8B) and cytotoxicity (Fig. 8D) observed in the presence of 50 µM Cd2+. Nevertheless, Pr-SSG was still increased by 37% over control under these experimental conditions.


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Fig. 8.   Effect of different concentrations of DTT on levels of glutathione (A, square ), Pr-SSGs (B, black-square), Ub proteins (C, ), and cytotoxicity (D, ) in HT4 cells treated with 50 µM cadmium. The cells were preincubated with 50 µM cadmium for 1 h followed by different concentrations of DTT for an additional 3 h. After the indicated treatments, the cells were harvested and prepared for measurements of glutathione, Pr-SSGs, Ub proteins, and cytotoxicity as indicated under "Experimental Procedures." All levels are expressed relative to no cadmium treatment (100%). Data represent the mean and S.E. of three identical experiments for each condition tested.

The highest DTT concentration tested (10 mM) was as effective as 1 mM in reversing the decrement in glutathione (Fig. 8A). However, it was less effective than the lower concentration in blocking the changes in the other three parameters tested, namely Pr-SSGs, Ub proteins, and cytotoxicity, perhaps due to its inhibition of protein synthesis (34).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Cadmium accumulates in humans throughout their lives because of its very long half-life (35). The heavy metal is a substantial industrial and environmental pollutant that seriously injures a variety of organs, such as the brain, liver, testis, and kidneys (for a review, see Ref. 36). Recent studies demonstrated that cadmium toxicity was mediated by the oxidative damage of essential cellular macromolecules (reviewed in Ref. 21). For example, the heavy metal was shown to increase lipid peroxidation in the brain, an organ particularly sensitive to cadmium toxicity (37), and in hepatocytes and testicular Leydig cells (38, 39). In addition, Cd2+ increased cellular levels of hydrogen peroxide in Leydig cells (39) and inhibited SOD in the liver and kidneys (40). Cadmium ions were also shown to cause changes in intracellular glutathione concentrations and to induce the synthesis of metallothioneins, cysteine-rich proteins that avidly bind the metal ion (reviewed in Ref. 21).

The cellular mechanisms involved in cadmium toxicity are still not well understood. The heavy metal interacts with thiol groups of proteins with a greater affinity than Zn2+ and may therefore disrupt the structure of certain cellular proteins (41). In addition, Cd2+ forms complexes with reduced glutathione (GSH), binding mostly to the sulfhydryl group of the cysteinyl moiety (42). The heavy metal may therefore contribute to an imbalance of the sulfhydryl homeostasis in the cell.

To test this hypothesis, we investigated the effect of cadmium on a mouse neuronal cell line (HT4 cells) and on rat mesencephalic primary cultures. Our studies are the first to show that cadmium induced a time- (up to 8 h) and dose-dependent increase in protein-mixed disulfides reflecting decreases in glutathione. Many other studies with nonneuronal cells report similar decreases in glutathione (reviewed in Ref. 21) but did not measure protein S-thiolation.

To explain our findings we propose the following mechanism (Scheme 1) mediating cadmium action.
<AR><R><C><UP>GSH</UP> <LIM><OP><ARROW>→</ARROW></OP><UL><UP>Cd</UP><SUP><UP>2+</UP></SUP></UL></LIM><UP> GSSG</UP></C></R><R><C><UP>GSSG</UP>+<UP>Pr-SH</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>     </UL></LIM><UP> Pr-SSG</UP>+<UP>GSH</UP></C></R></AR>
<UP><SC>Scheme 1</SC></UP>
Although Cd2+ does not by itself facilitate the aerobic oxidation of GSH in solution at neutral pH,2 it induced oxidation of GSH to GSSG within cells by a yet unidentified mechanism. Cadmium may therefore stimulate other intracellular events leading to the oxidation of GSH to GSSG, which in turn promotes the oxidation of protein thiol groups.

A second possibility is that cadmium induces intracellular oxidation of GSH or protein thiols to thiyl radicals (43), which may in turn generate mixed disulfides.
<UP>GSH or Pr-SH</UP> <LIM><OP><ARROW>→</ARROW></OP><UL><UP>Cd</UP><SUP><UP>2+</UP></SUP></UL></LIM><UP> GS· or Pr-S·</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>     </UL></LIM><UP> Pr-SSG</UP>
<UP><SC>Scheme 2</SC></UP>
The cadmium-induced elevations in Pr-SSGs were not blocked by depletion of more than 85% of glutathione by the glutathione synthetase inhibitor (L-BSO), suggesting that less than 15% of intracellular GSH can sustain significant increases in Pr-SSGs. This small cellular pool of GSH consistently failed to be depleted by the L-BSO and cadmium treatment.

The effect of cadmium could also be mediated by a third mechanism, described in Scheme 3. The divalent metal may form complexes directly with the thiol groups of proteins, leading to oxidized aggregates, such as Pr-SS-Pr (disulfide-linked proteins).
<UP>Pr-SH</UP> <LIM><OP><ARROW>→</ARROW></OP><UL><UP>Cd</UP><SUP><UP>2+</UP></SUP></UL></LIM><UP> Pr-SS-Pr</UP>
<UP><SC>Scheme 3</SC></UP>
Both Pr-SSG and Pr-SS-Pr products were identified in cells treated with iodoacetamide, an alkylating reagent known to decrease glutathione levels and to induce oxidative stress (34).

Our study also shows that decreases in glutathione and accumulation of Pr-SSGs were detected in cadmium-treated mesencephalic cultures. These primary cultures contain neurons and glial cells, possibly explaining why the changes may not be as great as those observed in the pure neuronal HT4 cell cultures. For example, astroglia cells were shown to tolerate low levels of lead exposure, which could be toxic to neuronal cells (44).

In addition to producing increments in Pr-SSGs, we found that cadmium has a biphasic effect on the ubiquitin/ATP-dependent proteolytic pathway. Although low concentrations of Cd2+ (25 µM or less) increase the intracellular levels of Ub proteins, higher concentrations (50 µM or more) have the opposite effect. The accumulation of Ub proteins observed in the presence of low cadmium concentrations could result from: (i) a direct inhibition of the activity of the 26 S proteasome; (ii) an overload of the ubiquitin/ATP-dependent pathway due to an increase production of structurally damaged proteins, such as the Pr-SSGs or Pr-SS-Pr; and/or (iii) inhibition of the activity of deubiquitinating enzymes. The latter are thiol isopeptidases (45), and the essential active site sulfhydryl groups may become oxidized in the presence of Cd2+.

Higher concentrations of cadmium (50 µM) decreased the intracellular levels of Ub proteins. This result may be explained by a decline in the catalytic activities of the ubiquitinating enzymes, including ubiquitin-activating (E1) and ubiquitin-conjugating (E2) enzymes. These enzymes contain sulfhydryl groups in the active sites (reviewed in Ref. 46) and may be oxidized in the presence of high cadmium concentrations. This hypothesis is supported by recent studies demonstrating that ubiquitin-activating and ubiquitin-conjugating enzymes are inactivated by S-thiolation under conditions of oxidative stress induced by exposure to hydrogen peroxide (19, 20). In addition, others have shown that in yeast, expression of the ubiquitin-conjugating enzymes and of the poly ubiquitin gene is highly increased after exposure to 100 µM cadmium for 30 min and that strains defective in proteasome activity are more susceptible to cadmium toxicity (22). However, ubiquitin overexpression did not increase yeast tolerance to cadmium toxicity (47).

Two effects of cadmium discussed above, namely increases in Pr-SSGs and Ub proteins, could be reversed by the thiol-reducing agent DTT (Fig. 7). These results conclusively show that the heavy metal perturbs the thiol-disulfide redox status of intracellular proteins.

In summary, cadmium induces the loss of glutathione, the oxidation of protein thiols to Pr-SSGs, and the accumulation of ubiquitinated proteins in the neuronal cells. These effects can be reversed by a thiol-reducing agent, indicating that they result from perturbations of the thiol-disulfide redox status of intracellular proteins. The formation of Pr-SSGs appears to be linked to inactivation of the Ub/ATP-dependent pathway, which leads to the accumulation of ubiquitinated proteins. This general mechanism may reflect cellular responses to other agents that promote the modification of the structure of intracellular proteins and inhibit the Ub/ATP-dependent pathway. Failure to overcome this inhibition may result in the proteotoxic accumulation of ubiquitinated proteins in intracellular inclusions and may lead to cell degeneration.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants NS34018 (to M. E. F.-P.) and NS23017 (to G. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Biological Sciences, Hunter College of City University of New York, 695 Park Ave., New York, NY 10021. Tel.: 212-650-3565; Fax: 212-772-5227; E-mail: pereira{at}genectr.hunter.edu.

1 The abbreviations used are: Ub protein; ubiquitinated protein; GSH, reduced glutathione; GSSG, glutathione disulfide; Pr-SSG, protein-mixed disulfide; L-BSO, L-buthionine-(S,R)-sulfoximine; DTT, dithiothreitol.

2 M. E. Figueiredo-Pereira, S. Yakushin, and G. Cohen, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Mayer, R. J., Arnold, J., László, L., Landon, M., and Löwe, J. (1991) Biochim. Biophys. Acta 1089, 141-157[Medline] [Order article via Infotrieve]
  2. Mayer, R. J., Lowe, J., and Landon, M. (1994) J. Pathol. 163, 279-281
  3. Hershko, A. (1991) Trends Biochem. Sci. 16, 265-268[CrossRef][Medline] [Order article via Infotrieve]
  4. Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583[Medline] [Order article via Infotrieve]
  5. Wilkinson, K. D., Deshpande, S., and Larsen, C. N. (1992) Biochem. Soc. Trans. 20, 631-637[Medline] [Order article via Infotrieve]
  6. Rechsteiner, M. (1987) Annu. Rev. Cell Biol. 3, 1-31[CrossRef]
  7. Ciechanover, A., and Schwartz, A. L. (1994) FASEB J. 8, 182-191[Abstract/Free Full Text]
  8. Coyle, J. T., and Puttfarcken, P. (1993) Science 262, 689-695[Medline] [Order article via Infotrieve]
  9. Cohen, G., and Werner, P. (1994) in Neurodegenerative Diseases (Calne, D., and Saunders, W. B., eds), pp. 139-161, Philadelphia, W. B. Saunders Co.
  10. Williams, L. R. (1995) Cerebrovasc. Brain Metab. Rev. 7, 55-73[Medline] [Order article via Infotrieve]
  11. Stadtman, E. R. (1992) Science 257, 1220-1224[Medline] [Order article via Infotrieve]
  12. Rivett, A. J. (1986) Curr. Top. Cell. Regul. 28, 291-337[Medline] [Order article via Infotrieve]
  13. Pacifici, R. E., and Davies, K. J. A. (1991) Gerontology 37, 166-180[Medline] [Order article via Infotrieve]
  14. Grune, T., Reinheckel, T., Joshi, M., and Davies, K. J. A. (1995) J. Biol. Chem. 270, 2344-2351[Abstract/Free Full Text]
  15. Fagan, J. M., and Waxman, L. (1992) J. Biol. Chem. 267, 23015-23022[Abstract/Free Full Text]
  16. Davies, K. J. A. (1993) Biochem. Soc. Trans. 21, 346-353[Medline] [Order article via Infotrieve]
  17. Cheng, L., Watt, R., and Piper, P. W. (1994) Mol. Gen. Genet. 243, 358-362[Medline] [Order article via Infotrieve]
  18. Shang, F., and Taylor, A. (1995) Biochem. J. 307, 297-303[Medline] [Order article via Infotrieve]
  19. Shang, F., Gong, X., and Taylor, A. (1997) J. Biol. Chem. 272, 23086-23093[Abstract/Free Full Text]
  20. Jahngen-Hodge, J., Obin, M. S., Gong, X., Shang, F., Nowell, T. R., Jr., Gong, J., Abasi, H., Blumberg, J., and Taylor, A. (1997) J. Biol. Chem. 272, 28218-28226[Abstract/Free Full Text]
  21. Stohs, S. J., and Bagchi, D. (1995) Free Radic. Biol. Med. 18, 321-336[CrossRef][Medline] [Order article via Infotrieve]
  22. Jungmann, J., Reins, H.-A., Schobert, C., and Jentsch, S. (1993) Nature 361, 369-371[CrossRef][Medline] [Order article via Infotrieve]
  23. Figueiredo-Pereira, M. E., Yakushin, S., and Cohen, G. (1997) Mol. Biology Reports 24, 35-38[CrossRef][Medline] [Order article via Infotrieve]
  24. Whittemore, S. R., Holets, V. R., Keana, R. W., Levy, D. J., and McKay, R. D. G. (1991) J. Neurosci. Res. 28, 156-170[Medline] [Order article via Infotrieve]
  25. Lerma, J., Paternain, A. V., Naranjo, J. R., and Mellström, B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11688-11692[Abstract]
  26. Mytilineou, C., Han, S. K., and Cohen, G. (1993) J. Neurochem. 61, 1470-1478[Medline] [Order article via Infotrieve]
  27. Akerboom, T. P. M., and Sies, H. (1981) Methods Enzymol. 77, 373-382[Medline] [Order article via Infotrieve]
  28. Mosmann, T. (1983) J. Immunol. Methods 65, 55-63[CrossRef][Medline] [Order article via Infotrieve]
  29. Figueiredo-Pereira, M. E., Berg, K. A., and Wilk, S. (1994) J. Neurochem. 63, 1578-1581[Medline] [Order article via Infotrieve]
  30. Laemmli, U. K. (1970) Nature 277, 680-685
  31. Pereira, M. E., Yu, B., and Wilk, S. (1992) Arch. Biochem. Biophys. 294, 1-8[Medline] [Order article via Infotrieve]
  32. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  33. Reed, D. J. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 603-631[CrossRef][Medline] [Order article via Infotrieve]
  34. Liu, H., Lightfoot, R., and Stevens, J. L. (1996) J. Biol. Chem. 271, 4805-4812[Abstract/Free Full Text]
  35. Savolainen, H. (1995) Renal Failure 17, 483-487[Medline] [Order article via Infotrieve]
  36. Morselt, A. F. W. (1991) Toxicology 70, 1-84[CrossRef][Medline] [Order article via Infotrieve]
  37. Acan, N. L., and Tezcan, E. F. (1995) Biochem. Mol. Med. 54, 33-37[CrossRef][Medline] [Order article via Infotrieve]
  38. Muller, L. (1986) Toxicology 40, 285-292[CrossRef][Medline] [Order article via Infotrieve]
  39. Koizumi, T., and Li, Z. G. (1992) J. Toxicol. Environ. Health 37, 25-36[Medline] [Order article via Infotrieve]
  40. Hussain, T., Shukla, G. S., and Chandra, S. F. (1987) Pharmacol. Toxicol. 60, 355-358[Medline] [Order article via Infotrieve]
  41. Vallee, B. L., and Ulmer, D. D. (1972) Annu. Rev. Biochem. 41, 91-129[CrossRef][Medline] [Order article via Infotrieve]
  42. Christie, N. T., and Costa, M. (1984) Biol. Trace Elem. Res. 6, 139-158
  43. Miller, R. M., Sies, H., Park, E.-M., and Thomas, J. A. Arch. Biochem. Biophys. 276, 355-363
  44. Opanashuk, L. A., and Finkelstein, J. N. (1995) J. Neurosci. Res. 42, 623-632[Medline] [Order article via Infotrieve]
  45. Wilkinson, K. D. (1997) FASEB J. 11, 1245-1256[Abstract/Free Full Text]
  46. Wilkinson, K. D. (1995) Annu. Rev. Nutr. 15, 161-189[CrossRef][Medline] [Order article via Infotrieve]
  47. Chen, Y., and Piper, P. W. (1995) Biochim. Biophys. Acta 1268, 59-64[CrossRef][Medline] [Order article via Infotrieve]


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