Contribution of Glutathione and Metallothioneins to Protection against

Jul 25, 2002 - Effect of BSO and Zn on the Redox-Cycling Activity of Copper in MT+/+ ...... Conners, D. E., and Ringwood, A. H. (2000) Effects of glut...
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Chem. Res. Toxicol. 2002, 15, 1080-1087

Contribution of Glutathione and Metallothioneins to Protection against Copper Toxicity and Redox Cycling: Quantitative Analysis Using MT+/+ and MT-/- Mouse Lung Fibroblast Cells Jianfei Jiang, Claudette M. St. Croix, Nancy Sussman, Qing Zhao, Bruce R. Pitt, and Valerian E. Kagan* Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received March 15, 2002

Glutathione (GSH) and metallothioneins (MT) are believed to play important roles in protecting cells against high copper (Cu) concentrations. Little is known, however, about their specific intracellular interactions and the coordination of protective functions. We investigated contributions of GSH and MT to protection against Cu toxicity in fibroblasts derived from wild-type (MT+/+) and knockout (MT-/-) mice that were challenged with cupric nitrilotriacetate (Cu-NTA). Endogenous levels of GSH and MT were manipulated using an inhibitor of γ-glutamylcysteine synthetase, buthionine sulfoximine (BSO, 5 µM), as GSH depletor and ZnCl2 (100 µM) as inducer of MT expression. BSO pretreatment markedly decreased cellular GSH levels in MT+/+ and MT-/- cells, by 65% and 70%, respectively, which resulted in Cu cytotoxicity accompanied by its elevated redox-cycling activity and enhanced Cu-induced membrane phospholipid peroxidation. BSO-pretreated MT-/- cells were markedly more sensitive to Cu despite the fact that the residual levels of GSH were similar in both BSOpretreated MT+/+ and MT-/- cells. Zn pretreatment resulted in more than 10-fold induction of MT in MT+/+ cells but not in MT-/- cells. Accordingly, Zn pretreatment afforded significant protection of MT+/+ cells against Cu cytotoxicity, likely associated with MT-dependent suppression of Cu redox-cycling activity and phospholipid peroxidation, but it exerted no protection in MT-/- cells (as compared to naive cells). To determine whether MT functions specifically in Cu regulation or rather acts as a nonspecific Cu-binding cysteine-rich nucleophile, experiments were performed using MT+/+ and MT-/- cells pretreated with both BSO and Zn. BSO pretreatment did not affect Zn-induced MT expression in MT+/+ cells. As compared with BSO pretreatment alone, exposure to Cu of MT+/+ cells after Zn/BSO pretreatment resulted in the following: (i) a significantly higher viability; (ii) attenuated Cu-dependent redoxcycling activity; and (iii) a lower level of phospholipid peroxidation. In BSO/Zn-pretreated MT-/- cells, the redox-cycling activity of Cu and the level of phospholipid peroxidation remained remarkably higher than in naive cells and were not significantly different from those in cells pretreated with BSO alone. Cu-induced toxicity was remarkably higher in BSO/Znpretreated MT-/- cells than in naive or Zn-pretreated cells, although slightly lower than in the MT-/- cells pretreated with BSO alone.

Introduction Copper (Cu) is an essential metal for the structural and catalytic properties of many proteins such as Cu/ Zn1 superoxide dismutase, cytochrome c oxidase, and Curesponsive transcription factors (1, 2). Excessive Cu associated with Cu overload due to metabolic disorders (e.g., Wilson’s disease) or environmental and occupational factors may result in Cu-induced geno- and cytotoxicity (3). These toxic effects of Cu are believed to be mainly * Correspondence should be addressed to this author at the Department of Environmental and Occupational Health, University of Pittsburgh, 3343 Forbes Ave., Pittsburgh, PA 15260. Tel: (412) 383 2159; Fax: (412) 383 2123; E-mail: [email protected]. 1 Abbreviations: GSH, glutathione; MT, metallothionein; Zn, zinc; BSO, buthionine sulfoximine; Cu-NTA, copper nitrilotriacetate; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PnA, cis-parinaric acid [(9Z,11E,13E,15Z)-octadecatetraenoic acid]; FBS, fetal bovine serum.

due to its participation in Fenton-type reactions that include redox-cycling of Cu and univalent reduction of molecular oxygen to superoxide to yield a variety of other reactive oxygen species (ROS) (4) capable of causing lipid peroxidation and DNA damage and impairing calcium homeostasis (5, 6). It is therefore essential that the content and trafficking of intracellular Cu be carefully regulated and maintained at an extremely low level compatible with minimal Cu-dependent redox cycling. Intracellular delivery of Cu to its target proteins is performed by special chaperones such as Atx1 (7), Cox15 (8), Cox17 (9), and CCS (copper chaperone for Cu,Znsuperoxide dismutase) (10) whose high affinity and binding of Cu not only maintain negligibly low levels of free Cu but also prevent its redox-cycling activity. In biological fluids, ceruloplasmin, albumin, and transcupreine act as extracellular Cu-binding proteins regulating its redox interactions (11-13).

10.1021/tx020022u CCC: $22.00 © 2002 American Chemical Society Published on Web 07/25/2002

Metallothioneins vs GSH in Protection against Copper

An extensive literature clearly indicates that glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) is an intracellular Cu chelator that may play an important role in Cu detoxification. GSH is the most abundant nonprotein thiol, and it is usually present in different compartments of cells in millimolar concentrations (1-10 mM) (14, 15). GSH binding of Cu has been shown to inhibit free radical formation from Cu/H2O2 (16). Viarengo et al. (17) found a significant reverse correlation between the GSH concentration and Cu-induced lipid peroxidation in digestive glands of mussels. Conners et al. (18) showed that GSH depletion increased the potential for adverse effects of Cu in oysters. Metallothioneins (MT), stress-inducible thiol-rich proteins, comprise a second important pool of Cu and are essential for its intracellular regulation and trafficking (19). The 20 cysteines in MT protein form 2 thiolate clusters, termed the carboxyl-terminal R domain and the amino-terminal β domain, capable of binding various metals including Cu with high affinity [stability constant of 1019-1017 based on pH stability and ligand substitution experiments (20)]. Notably, the MT-bound Cu is redoxinactive in the absence of oxidative or nitrosative stress (21). Therefore, MT may be ideally suited for the antioxidant function as effective Cu chelators when cells are challenged with high Cu concentrations. We previously demonstrated that Zn-induced MT were able to bind intracellular Cu, quench redox-cycling activity of Cu, inhibit Cu-dependent oxidative stress in membrane phospholipids, and prevent Cu-dependent apoptosis in HL-60 cells (22). While it is likely that both MT and GSH participate in protecting cells against toxic Cu concentrations, the relationship between endogenous GSH and MT in the detoxification of Cu is not well understood. The results of several studies (23, 24) indicate that immediately after entering the cell, Cu is initially chelated by GSH from which it is further transferred to MT, for longer-term storage. In the present study, we quantitatively explored the contribution of GSH and MT to Cu detoxification. For this purpose, we utilized mouse lung fibroblasts derived from MT I/II knockout mice (MT-/-) and wild-type (MT+/+) mice in which we were able to independently manipulate the endogenous GSH and MT levels using buthionine sulfoximinine (BSO) and ZnCl2, respectively, and assessed their sensitivity to cupric nitrilotriacetate (Cu-NTA) as well as biomarkers of oxidative stress (phospholipid peroxidation) and Cu redox-cycling activity.

Experimental Procedures Materials and Reagents. All tissue culture media and additives were obtained from Invitrogen Co. (Carlsbad, CA) except fetal bovine serum (FBS), which was from Sigma Chemical Co. (St. Louis, MO). Chloroform, methanol, hexane, and 2-propanol (HPLC grade), and Tween-20 were purchased from Aldrich Chemical Co. (Milwaukee, WI). LIVE/DEAD kit and cisparinaric acid [(9Z,11E,13E,15Z)-octadecatetraenoic acid] were obtained from Molecular Probes (Eugene, OR). ThioGlo-1, a fluorogenic thiol reagent, was purchased from Calbiochem (San Diego, CA). Protein assay kit was obtained from Bio-Rad (Hercules, CA). CuSO4 and disodium nitrilotriacetic acid were purchased from Sigma Chemical Co. The Cu-NTA solution was prepared according to the method described by Toyokuni et al. (25), and the CuSO4 to nitrilotriacetic acid disodium molar ratio used was 1:2. The pH was adjusted to 7.4 with sodium bicarbonate. All other chemicals and reagents used were molecular biology grade.

Chem. Res. Toxicol., Vol. 15, No. 8, 2002 1081 Cell Culture and Treatments. MTI/II null (MT-/-) and wild-type (MT+/+) mice were obtained by mating heterozygous mice (26) of mixed 129 Ola and C57 Bl/6 backgrounds. MT mutants were identified through a genotyping protocol using the polymerase chain reaction strategy to detect a neomycin sequence inserted within the MT2 altered gene. Mouse lung fibroblasts were isolated essentially as described by Harvey et al. (27) except that lung tissue was utilized instead of dissected embryos. MT-/- and MT+/+ cells were grown in DMEM medium supplemented with 15% FBS at 37 °C under a 5% CO2 atmosphere, and split at a ratio of 1:3 every 48 h. Cells from passages 5-12 were used for the experiment. MT+/+ and MT-/- cells were plated and allowed to adhere overnight, and then incubated with 5 µM BSO and 100 µM ZnCl2 alone or in combination for 20 h. These cells and naive (nonpretreated) cells were then exposed to different concentrations of Cu-NTA at 37 °C. Viability Assay. Cell viability was determined by measuring esterase activity using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes). Briefly, cells were seeded in 96-well plates at a density of 8000 cells/well and allowed to adhere overnight. Then, cells were incubated with 100 µM ZnCl2 and 5 µM BSO alone or in combination for 20 h. Naive cells were incubated during the same period of time without ZnCl2 or BSO. Cells were exposed to Cu at various concentrations (0, 1, 2, 3, 4, and 6 mM) for 3 h. At the end of incubation, cells were stained with the LIVE/DEAD kit reagent according to the protocol. The plates were incubated at 37 °C for 30 min, and then fluorescence was determined using a Cytofluor 2350 fluorescence plate reader (Millipore, Bedford, MA) with an excitation filter of 485 ( 10 nm and an emission filter of 530 ( 25 nm. Assay for Metallothioneins in Cells. MT levels in MT+/+ and MT-/- cells with and without BSO or ZnCl2 pretreatment were determined using the 109Cd-binding assay as described by Eaton and Toal (29). The cellular MT content was calculated based on the assumption that 7 mol of Cd is bound to 1 mol of MT, and was normalized to total cellular protein content. Assay for Glutathione in Cells. ThioGlo-1 was used to determine the GSH content in cells as previously described by Shvedova et al. (28). GSH content was estimated by an immediate fluorescence response registered upon addition of 10 µM ThioGlo-1 to the cell homogenate. Fluorescence was measured by a Cytofluor 2350 fluorescence plate reader (Millipore, Bedford, MA) using an excitation filter of 360 ( 40 nm and an emission filter of 530 ( 25 nm. A standard curve was established by the addition of GSH (0.25-3 µM) to phosphate buffer, pH 7.4 containing 10 µM ThioGlo-1. Determination of Cu-NTA-Induced Phospholipid Peroxidation in Cells. cis-Parinaric acid (PnA) was incorporated into naive or pretreated cells (2 × 105 cells/well) by addition of PnA-human serum albumin complex to give a final concentration of 4.0 µg in serum-free DMEM medium without phenol red as previously described (30). PnA-labeled cells were treated with Cu-NTA (3.0 mM) for 1 h at 37 °C in serum-free DMEM medium. Cells were scraped at the end of the incubation, and total lipids were extracted using the Folch procedure (31) in the presence of butylated hydroxytoluene to prevent subsequent oxidations. The lipid extract was dried under N2, dissolved in 0.25 mL of a 2-propanol/hexane/water mixture (4:3:0.16, v/v/v), and separated by normal-phase HPLC using a 5 µm MicrosorbMV Si column (4.6 mm × 250 mm) and an ammonium acetate gradient. The separations were performed using a Shimadzu HPLC system (LC-600) (Kyoto, Japan) equipped with an in-line RF-551 fluorescence detector. The fluorescence of PnA-phospholipids was measured at 420 nm (emission) after excitation at 324 nm. Data were processed and stored in digital form with Shimadzu EZchrom software. The amount of lipid phosphorus was determined using a micro method (32). EPR of Ascorbate Radical. Cells were collected after 1 h incubation with 3 mM Cu-NTA. Ascorbate (2 µM) was added to cell homogenates in 50 µL of PBS (pH 7.4), and EPR spectra of ascorbate radicals were recorded immediately. EPR measure-

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ments were performed on a JEOX-RE1X spectrometer (Kyoto, Japan) using gas-permeable Teflon tubing (0.8 mm internal diameter, 0.013 mm thickness) obtained from Alpha Wire Corp. (Elizabeth, NJ). The tube (∼8 cm in length) was filled with 50 µL of mixed sample, folded in fourths, and placed in an open 3 mm internal diameter EPR quartz tube so that the entire sample was within the effective microwave area. Spectra of ascorbate radicals were recorded under the following instrumental conditions: 335.5 mT center field, 20 mW power, 0.05 mT field modulation, 5 mT sweep width, 4000 receiver gain, and 0.1 s time constant. Protein Assay. The protein concentration in MT+/+ and MT-/- cell homogenates was determined with the Bio-Rad protein assay kit. A standard curve was established by addition of bovine serum albumin to the reagent, and the protein content was calculated. Statistical Analysis. We investigated how manipulations of MT and/or GSH levels in MT+/+ or MT-/- cells affect cell viability, redox-cycling activity of copper (using surrogate ESR measurements of Cu-induced ascorbate radical production), and copper-dependent peroxidation of major phospholipid classes such as PE, PS, and PC. We statistically analyzed the results of each of these five experiments by means of a balanced analysis of variance (ANOVA), imposing a linear model. The model included each of the main effects (cell type, zinc, BSO, and, in the case of cell viability, the concentration of copper) and all two-way and three-way interactions. For the cell viability model, all main effects and all but two of the interactions were highly statistically significant (p ) 0.000). The model explained 98% of the variability in viability. In the redox-cycling activity of copper model, the main effects and the cell type-zinc interaction were all highly statistically significant (p ) 0.000). The model explained 88% of the variability observed in the ascorbate radical ESR measurements. In the analysis of phospholipid peroxidation, the main effects on PC peroxidation (cell type, zinc, BSO, and the cell typezinc interaction) were all highly statistically significant (p ) 0.000, p ) 0.004, p ) 0.000, p ) 0.032, respectively). The model explained 91% of the variability observed in the PC peroxidation measurements. For the PS peroxidation model, all main effects and the cell type-zinc interactions were highly statistically significant (p ) 0.000). The cell type-BSO interaction was also statistically significant (p ) 0.003). The model explained 98% of the variability in the phosphatidylserine peroxidation measurement. In the PE peroxidation, the main effects were statistically significant for cell type, zinc, and BSO (p ) 0.007, p ) 0.003, p ) 0.000, respectively). None of the interactions was statistically significant. The model explained 87% of the variability observed in the PE peroxidation measurement. To prevent a large probability of falsely declaring statistical significance due to the application of many statistical tests, we used Tukey’s procedure to perform our pairwise comparisons of selected group means following the ANOVA.

Results Effect of BSO and Zn Pretreatment on GSH and MT Levels. MT+/+ and MT-/- cells were treated with 5 µM BSO, 100 µM ZnCl2 singly or in combination to manipulate the intracellular levels of GSH and MT. Twenty hours later, the GSH and MT levels were determined. BSO treatment significantly decreased cellular GSH levels in MT+/+ cells from 27.4 ( 5.1 to 9.6 ( 1.5 nmol/ mg of protein (p < 0.01) (Figure 1). MT-/- cells had a higher constitutive level of GSH (38.5 ( 2.1 nmol/mg of protein) that was also decreased following BSO (11.4 ( 1.3 nmol/mg of protein, p < 0.01). Pretreatment with ZnCl2 increased GSH in both MT+/+ and MT-/- cells by 33.5% (p < 0.05) and 15.6%, respectively, resulting in

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Figure 1. Effect of ZnCl2 and BSO on GSH levels in MT+/+ and MT-/- cells. Glutathione content was estimated by an immediate fluorescence response registered upon addition of 10 µM ThioGlo-1 to cell homogenates. Values are expressed as mean ( SD, n ) 3. Changes in variables for different pretreatments were analyzed by the Student’s t-test. (a) indicates statistically significant differences as compared to naive (nonpretreated) cells (p < 0.05); (b) indicates statistically significant differences from Zn-pretreated cells (p < 0.01).

Figure 2. Effect of ZnCl2 and BSO on metallothionein levels in MT+/+ and MT-/- cells. MT content was determined using the 109Cd-binding assay and calculated on the basis of the assumption that 7 mol of Cd is bound to 1 mol of MT. MT concentration was normalized to total cellular protein content. Data are means ( SD, n ) 3. Changes in variables for different pretreatments were analyzed by the Student’s t-test. (a) indicates statistically significant differences from naive (nonpretreated) cells (p < 0.01); (b) indicates statistically significant differences from BSO-pretreated cells (p < 0.01).

GSH levels that were not significantly different from each other in these cells. In the presence of BSO, ZnCl2 did not elevate GSH concentration; levels of GSH were not significantly different after pretreatment with BSO alone or with a combination of BSO and ZnCl2 in both MT+/+ and MT-/- cells (Figure 1). As expected, constitutive levels of MT were significantly higher in MT+/+ cells (0.017 ( 0.003 nmol/mg of protein) than in MT-/- cells (0.007 ( 0.002 nmol/mg of protein) (Figure 2). In MT-/- cells, the amount of MT was within the error of the assay. No changes in MT levels were detected after pretreatment of the MT-/cells with either Zn or BSO singly or in combination. In contrast, the MT levels were remarkably enhanced up to 12-fold by Zn pretreatment of MT+/+ cells [the MT concentration was increased to 0.202 ( 0.029 nmol/mg of protein in Zn-pretreated cells (p < 0.01)]. BSO pretreatment did not affect constitutive levels of MT in either MT+/+ or MT-/- cells. Moreover, BSO did not change Zn induction of MT in MT+/+ cells; the MT content was increased from 0.021 ( 0.007 nmol/mg of protein (in the presence of BSO alone) to 0.194 ( 0.023 nmol/mg of protein in the presence of BSO and Zn.

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Figure 3. Comparison of Cu-NTA toxicity to MT+/+ and MT-/- cells pretreated with ZnCl2 and BSO. Cu cytotoxicity was assayed using the LIVE/DEAD assay Kit. Values represent means ( SD (n ) 3). (a) denotes a statistically significant difference from control cells (in the absence of Cu). (b) denotes a statistically significant difference from naive (nonpretreated) cells under the same condition. (c) denotes a statistically significant difference from BSO-pretreated cells under the same condition. (d) denotes a statistically significant difference from Zn/BSO-pretreated cells under the same condition. Results were statistically analyzed by means of a balanced analysis of variance (ANOVA), imposing a linear model.

Effect of BSO and Zn on Cu Toxicity in MT+/+ and MT-/- Cells. After 3 h incubation, Cu caused a concentration-dependent toxicity both in MT+/+ and in MT-/- naive (nonpretreated) cells. As shown in Figure 3A, Cu cytotoxicity was not detectable in naive MT+/+ until at 3 mM Cu-NTA, where there was a significant change in viability compared to control cells (in the absence of Cu, p ) 0.0136). Cytotoxicity increased with increasing concentration of Cu. Cell viability was reduced to 19.9% at 6 mM Cu-NTA (p ) 0.0000). The decreased GSH level (BSO pretreatment) had no effect on Cu toxicity at 1 and 2 mM Cu-NTA, while sharp viability changes took place at 3 mM Cu-NTA in BSO-pretreated cells as compared to control cells (p ) 0.0001) and naive cells under the same conditions (p ) 0.0000). The viability of BSO-pretreated cells approached 0% at 6 mM CuNTA. Pretreatment of MT+/+ cells with Zn exerted protection against Cu toxicity over a wide range of Cu concentrations; the cells showed no significant toxicity even at 6 mM Cu-NTA, and 86.4% of the cells were still viable. After combined pretreatment with BSO and Zn (Figure 3A), the latter protected the MT+/+ cells against Cu toxicity enhanced by GSH depletion. The viability of Zn/ BSO-pretreated cells was significantly higher than that of BSO-pretreated MT+/+ cells at 3 mM (p ) 0.0000), and this protective effect of Zn pretreatment remained significant at higher concentrations of Cu (p ) 0.0000). However, the protection of Zn against Cu cytotoxicity was reduced after its combined pretreatment with BSO (yielding lowered GSH levels) as compared to the protection afforded by Zn alone in MT+/+ cells. Zn/BSOpretreated cells showed a trend toward decreased viability compared to Zn-pretreated cells at 1, 2, and 3 mM Cu-NTA. At 4 mM Cu, Zn/BSO-pretreated MT+/+ cells were significantly less viable than those pretreated with Zn alone (p ) 0.0000) and became substantially less viable at 6 mM Cu (p ) 0.0000). When compared to naive cells, however, Zn/BSO-pretreated cells showed a significantly higher resistance to Cu at 4 mM (p ) 0.0001) and similarly at 6 mM Cu-NTA (p ) 0.0002). In MT-/- naive cells (Figure 3B), significant viability changes occurred when the concentration of Cu-NTA reached 3 mM (p ) 0.0003). The viability of naive cells was reduced to 32% at 6 mM Cu-NTA (p ) 0.0000). The Cu toxicity that was enhanced by BSO-mediated GSH

depletion in MT-/- cells was much more severe than in MT+/+ cells. Significant cytotoxicity was already observed in BSO-pretreated MT-/- cells at 2 mM Cu-NTA (p ) 0.0000), and viability decreased dramatically with increasing Cu concentrations. A significant viability difference between BSO-pretreated cells and naive cells was also demonstrated at 3 mM Cu-NTA (p ) 0.0001). Zn provided no protection against the toxic effects of CuNTA in MT-/- cells, as compared to naive cells. In fact, the viability profiles were indistinguishable for naive and Zn-pretreated MT-/- cells. While Zn did not completely protect against Cu toxicity enhanced by BSO pretreatment, it caused a significantly lower viability loss in the range of Cu concentrations (2-3 mM) in MT-/- cells that were pretreated with Zn/BSO. Effect of BSO and Zn on the Redox-Cycling Activity of Copper in MT+/+ and MT-/- Cells. One of the mechanisms through which Cu realizes its toxic effects depends on its redox-cycling activity. Since Cu can catalyze one-electron oxidation of ascorbate to produce ascorbate radicals, we took advantage of EPR measurements of ascorbate radicals to evaluate the redox-cycling activity of intracellular Cu. These measurements were conducted at the Cu concentration at which cytotoxicity was markedly pronounced, and viability differed significantly between cells with different levels of GSH and MT (3 mM). As shown in Figure 4, addition of Cu in the presence of ascorbate yielded a typical doublet signal with a splitting constant of 1.7 G in both MT+/+ and MT-/cells. The magnitude of the EPR signal was dependent on both MT and GSH levels in cells. Quantitatively, the data on the redox-cycling activity of intracellular Cu are summarized as the magnitude of ascorbate radical EPR signals. Predictably, the ascorbate radical EPR signals were essentially undetectable from control cells (naive cells in the absence of Cu): 61.1 ( 3.1 AU/mg of protein in MT+/+ and 71.5 ( 2.9 AU/mg of protein in MT-/cells, respectively. Exposure of naive cells to 3.0 mM CuNTA for 1 h yielded a pronounced EPR signal of ascorbate radicals in both MT+/+ (373.4 ( 13.8 AU/mg of protein, p ) 0.000, compared to MT+/+ control cells) and MT-/cells (314.9 ( 6.4 AU/mg of protein, significantly different from that in MT-/- control cells, p ) 0.000). Ascorbate radicals were significantly suppressed by Zn pretreatment in MT+/+ cells (96.1 ( 4.1 AU/mg of protein, p )

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Figure 4. Effect of ZnCl2 and BSO on the redox-cycling activity of Cu in MT+/+ and MT-/- cells assayed by ascorbate radical production. The inset shows typical EPR spectra of ascorbate radicals from MT+/+ control cells (nonpretreated cells, in the absence of Cu) (A) and MT+/+ naive cells exposed to 3 mM CuNTA for 1 h (B). Data are means ( SD (n ) 3). (a) denotes a statistically significant difference from control cells. (b) denotes a statistically significant difference from naive (nonpretreated) cells exposed to 3 mM Cu-NTA. (c) denotes a statistically significant difference from Zn/BSO-pretreated cells. Results were statistically analyzed by means of a balanced analysis of variance (ANOVA), imposing a linear model.

0.0000 vs Cu-loaded naive MT+/+ cells), but not in MT-/- cells (337.2 ( 14.6 AU/mg of protein). BSO pretreatment increased the radical signals to 505.6 ( 17.6 AU/mg of protein in MT+/+ cells (p ) 0.0000 vs Culoaded MT+/+ naive cells) and 586.5 ( 16.0 AU/mg of protein in MT-/- cells (p ) 0.0000 vs Cu-loaded naive cells). When MT+/+ cells were pretreated with Zn/BSO, the ascorbate radical signal was significantly attenuated as compared to BSO-pretreated cells (257.6 ( 22.0 AU/ mg of protein, p ) 0.0000). This signal, however, was greater than that from Zn-pretreated cells (p ) 0.0000) and lower than that from naive cells (p ) 0.0000). In BSO/Zn-pretreated MT-/- cells, the redox-cycling activity of Cu remained remarkably higher (572.8 ( 19.1 AU/ mg of protein) than that in naive cells (p ) 0.0000) and was not significantly different from that in BSO-pretreated cells. Effect of BSO and Zinc on Cu-Dependent Phospholipid Peroxidation in MT+/+ and MT-/- Cells.

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Redox cycling of Cu is associated with the production of reactive oxygen species (ROS), capable of initiating oxidative damage to membrane phospholipids, ultimately causing cytotoxicity. To assess Cu-induced phospholipid peroxidation in MT+/+ and MT-/- cells with different levels of GSH and MT, a sensitive fluorescence-HPLC technique based on metabolic integration of oxidationsensitive fluorescent fatty acid, cis-PnA, into membrane phospholipids was used. We demonstrated that exposure of MT+/+ and MT-/- naive cells to 3 mM Cu-NTA for 1 h resulted in a pronounced oxidation of all major classes of membrane phospholipids including phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) (Figure 5). PnA levels were expressed as a percentage of control levels (in the absence of Cu). BSO-pretreated/Cu-loaded cells underwent a remarkably greater membrane phospholipid peroxidation than Culoaded naive MT+/+ and MT-/- cells. In MT+/+ cells, Zn significantly inhibited BSO-enhanced phospholipid peroxidation as evidenced by comparison of the effects in Zn/BSO-pretreated cells with BSO-pretreated cells. However, phospholipid peroxidation was greater in BSO/ Zn-pretreated cells than in Zn-pretreated cells. In MT-/cells, Zn pretreatment did not cause any inhibition of Cuinduced oxidation compared to naive cells. In addition, Zn did not attenuate the BSO-enhanced Cu-dependent phospholipid oxidation in Zn/BSO-pretreated MT-/cells.

Discussion Cu is an essential micronutrient, and over 30 proteins in mammalian cells require Cu for their normal functions (1, 2, 33). As a transition metal, Cu is capable of adopting one of the two covalent states of Cu1+ and Cu2+, thus readily undergoing reversible redox conversions. This property, essential for the catalytic activity of Cu, can also trigger redox-cycling reactions in which Cu catalyzes one-electron reduction of dioxygen to sequentially produce superoxide, hydrogen peroxide, and hydroxyl radical (34, 35). These reactive oxygen species (ROS) can oxidatively modify membrane phospholipids, DNA, and proteins, ultimately causing geno- and cytotoxicity. Normally, the catalytic activity of Cu in cells and biological fluids is strictly regulated by its tight chelation and structural coordination within the proteins. In

Figure 5. Effect of BSO and ZnCl2 pretreatment on Cu-induced peroxidation of PnA-phospholipid in MT+/+ and MT-/- cells. PnA-labeled naive and pretreated cells were exposed to 3 mM Cu-NTA for 1 h, and lipids were extracted and resolved by HPLC. Contents of PnA-phospholipids are expressed as a percentage of control levels (in the absence of Cu). Data are means ( SD (n ) 3). (a) denotes a statistically significant difference from naive/Cu-loaded cells. (b) denotes a statistically significant difference from BSO-pretreated/Cu-loaded cells. (c) denotes a statistically significant difference from Zn/BSO-pretreated/Cu-loaded cells. PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine.

Metallothioneins vs GSH in Protection against Copper

contrast, redox-cycling activity is typically associated with free or loosely bound Cu. It is therefore essential that intracellular levels of redox-active unregulated Cu be maintained at a minimum in order to avoid its calamitous redox cycling. In plasma, ceruloplasmin, albumin, and trans-cupreine are the three major proteins involved in safe storage and hauling of Cu to cells (1113). Intracellular trafficking of copper, within physiological concentrations, is mainly accomplished by a recently identified and characterized group of Cu-chaperones that safely deliver Cu to its target proteins (7-10). As a result of high-affinity binding of Cu by the chaperones, intracellular concentrations of free redox-active copper are extremely low [4 mM), where GSH may play a prominent role in binding Cu. These results indicate that GSH may substitute for MT, at least to some extent, as a protector against Cu-induced toxicity. One may assume that the elevation of endogenous GSH in MT-/- cells was a compensatory response that afforded, in the absence of MT, coping with redoxregulation of Cu. Further, the EPR experiments revealed that the BSOinduced depletion of GSH brought about elevated redoxcycling activity of Cu and consequently substantial peroxidation of membrane phospholipid peroxidation in both MT+/+ and MT-/- cells as compared to naive cells. This clearly demonstrates that GSH is instrumental in protecting cells against Cu cytotoxicity. These results are consistent with previous observations that GSH is important in intracellular handling of Cu and preventing Cu-induced oxidative stress (18, 40, 41). Depletion of GSH, however, resulted in a remarkably enhanced sensitivity of MT-/- cells to Cu as compared to MT+/+ cells despite the fact that the residual levels of GSH in BSO-pretreated cells were similar in both types of cells. This emphasizes a special role that MT most likely plays in regulating intracellular Cu. A plethora of data indicates that MT plays a pivotal role in detoxification of different metals including Cu (42-44). Our previous work demonstrated that expression of MT in human leukemia HL-60 cells pretreated with Zn was associated with increased sequestration of intracellular Cu, quenching of its redox-cycling activity, inhibition of Cu-dependent oxidative stress in membrane phospholipids, and prevention of Cu-dependent apoptosis (22). This was confirmed in the present experiments with MT+/+ mouse lung fibroblasts. As expected, Zn-induced expression of MT afforded significant protection against Cu toxicity in MT+/+ cells, most of which retained their viability within the broad range of Cu concentrations tested. Apparently, elevated MT was able to effectively regulate the redox-cycling activity of Cu as only minimal EPR-detectable ascorbate radical signals were recorded from Zn-pretreated MT+/+ cells (but not MT-/- cells) challenged with Cu. The HPLC results also revealed that MT induction protected against Cu-induced phospholipid peroxidation in MT+/+ but not in MT-/- cells. It should be also noted that a significant part of MT in cells is represented by apo-thioneins that do not contain any metals and therefore have large Cu-binding capacity (45). In addition to the previously described Cu chaperones and intracellular thiols such as MT and GSH, two major types of Cu-transporting proteins are involved in the regulation of intracellular copper, and could therefore potentially protect cells from Cu-induced toxicity. Cu transporters (46, 47) are mainly responsible for delivery of exogenous Cu into cells, while Cu-ATPases (48-50) contribute to the regulation of intracellular Cu via its efflux. In the present studies, we utilized Cu-NTA

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chelates for intracellular delivery of Cu. These methods have been successfully used by others, and by us, in earlier studies of Cu toxicity and Cu sequestration by metallothioneins both in cell culture and in vivo (22, 5154). Given that the rates of Cu transport afforded by Cu transporters are in the picomoles per minute per milligram of protein range (46), it is not likely that Cu transporters contributed to Cu-induced toxicity under the conditions used in our experiments. In addition, Cu transporters are saturable at Cu concentrations ≈10 µM (46), which is far below those used in the present study (2-6 mM). Recent reports indicate that members of the family of cation-transporting P-type ATPases, ATP7A and ATP7B, the products of the Menkes and Wilson disease genes, respectively, regulate Cu transport and bioavailability in cells (48-50). While the biological functions of the two proteins appear to be different, the data supporting a role for Cu-ATPase in protection from Cu-induced toxicity include the observation that cultured Chinese hamster ovary (CHO) cells that overexpressed ATP7A were resistant to toxic levels of Cu in the growth medium and showed enhanced copper efflux (55). The rates of Cu efflux achievable by Cu-ATPases, however, are very low [≈0.0002 pmol min-1 (mg of protein)-1] (49). This suggests that during relatively short-term incubations used in our experiments their role in Cu extrusion was likely minimal. Moreover, these transporters have not yet been characterized in primary cultures of pulmonary fibroblasts. Finally, our hypothesis that handling of intracellular Cu by MT and GSH plays an important role in protecting cells from Cu-induced cytotoxicity is supported by our previously published data (54) demonstrating that MT protection against Cu-induced cytotoxicity was reversed and pro-oxidant and pro-apoptotic effects were induced in HL-60 cells exposed to nitric oxide. In this model, the S-nitrosylation and oxidation of cysteine residues caused the release of redox-active Cu, enhanced the redox-cycling activity of Cu, and increased the peroxidation of major classes of membrane phospholipids. To determine whether MT functions specifically in Cu regulation or rather acts as a nonspecific carrier of ≈20 useful cysteines, thus fulfilling only a nonspecific Cubinding role, we performed experiments in which we used MT+/+ and MT-/- cells pretreated with both BSO and Zn. Not surprisingly, in MT-/- cells, this resulted in a dramatic loss of viability upon exposure to Cu, comparable to that observed after pretreatment of these cells with BSO alone. Obviously, Zn/BSO pretreatment was not accompanied by any increase of MT levels in MT-/cells. In MT+/+ cells, however, Zn/BSO pretreatment did enhance expression of MT to approximately the same level as Zn alone and attenuated BSO-enhanced Cu toxicity. Yet, in MT+/+ cells, the cytotoxicity of Cu was significantly more pronounced after Zn/BSO pretreatment than after Zn alone. These results suggest that BSO-induced GSH depletion (loss of 17.8 nmol of GSH/ mg of protein) could not be fully compensated by increased levels of MT cysteines (4.1 nmol of SH/mg of protein). Overall, comparison of our results on combined effects of Zn/BSO with those on the effects of Zn alone and BSO alone indicates that MT and GSH may be involved in synergistic interactions in which both of them seem to be essential for the accomplishment of their protective regulatory function against redox-cycling activity of Cu.

Jiang et al.

It should be noted that MT can bind up to 10-12 mol of Cu/mol of MT. This binding, however, may be considered as effective and safe in terms of controlled and regulated chelation of Cu only to a certain limit. In fact, only 5-6 Cu per mole of MT are bound in a way that is not accompanied by any significant redox-cycling activity of Cu (21). This is mainly Cu bound by thiolate clusters in the carboxyl-terminal R domain of MT (56). Loading of the amino-terminal β domain of MT with Cu may cause oxidation of some of the cysteines and formation of “loosely bound” copper that maintains high levels of redox-cycling activity. MT cysteines may be the target of this redox-cycling, implying that overloading of MT with Cu may convert the system into suicidal redox machinery that catalyzes autoxidation of its own cysteines, releases Cu, and enhances its own autoxidation. It is tempting to suggest that GSH may play a very important role of a reductant that can participate in recycling of MT cysteines essential for Cu binding. In this way, GSH may synergistically interact with MT to maintain its important Cu-binding capacity that is not associated with Cu redox-cycling.

Acknowledgment. Supported by NIH Grants 1RO1HL64145-01A1 and HL32154.

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