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Zinc stimulates the production of toxic reactive oxygen species (ROS) and inhibits glutathione reductase in astrocytes
Free Radical Biology & Medicine 42 (2007) 1222 – 1230 www.elsevier.com/locate/freeradbiomed
Original Contribution
Zinc stimulates the production of toxic reactive oxygen species (ROS) and inhibits glutathione reductase in astrocytes Glenda M. Bishop a,⁎, Ralf Dringen a,b , Stephen R. Robinson a a
School of Psychology, Psychiatry and Psychological Medicine, Monash University, Wellington Rd, Clayton, Victoria, Australia b Center for Biomolecular Interactions Bremen, Faculty 2 (Biology/Chemistry), University of Bremen, Bremen, Germany Received 10 May 2006; revised 1 December 2006; accepted 10 January 2007 Available online 17 January 2007
Abstract The release of zinc (Zn) from glutamatergic synapses contributes to the neuropathology of ischemia, traumatic brain injury, and stroke. Astrocytes surround glutamatergic synapses and are vulnerable to the toxicity of Zn, which impairs their antioxidative glutathione (GSH) system and elevates the production of reactive oxygen species (ROS). It is not known whether one or both of these actions are the primary cause of Zninduced cell death in astrocytes. Using primary rat astrocyte cultures we have examined whether Zn-mediated impairment of GSH redox cycling is the main source of its toxicity. Zn acetate at concentrations of 100 μM or greater were found to inactivate glutathione reductase (GR) via an NADPH-dependent mechanism, while concentrations of 150 μM and above caused substantial cell death. Furthermore, Zn increased the ratio of GSSG:GSH in astrocytes, increased their export of GSSG, slowed their clearance of exogenous H2O2, and promoted the intracellular production of ROS. In contrast, the GR inhibitor, carmustine, did not induce cell death, cause the production of ROS, or alter the GSH thiol redox balance. Taken together these results indicate that Zn toxicity in astrocytes is primarily associated with the generation of intracellular ROS, rather than the inhibition of GR. © 2007 Elsevier Inc. All rights reserved. Keywords: Astrocyte; Carmustine; Epileptiform; Glutathione reductase; Hydrogen peroxide; Ischemia; Reactive oxygen species; Stroke; Traumatic brain injury; Zinc
Introduction Zinc (Zn) has been demonstrated to inhibit the activity of enzymes associated with glutathione (GSH) metabolism, particularly glutathione reductase (GR) [1]. This enzyme is responsible for cellular GSH redox cycling, which is crucial for the detoxification of endogenous peroxides [2]. In this process GSH acts as an electron donor for the glutathione peroxidase (GPx)-dependent reduction of H2O2 and of organic hydroperoxides to water and the corresponding alcohol, respectively. The Abbreviations: 3AT, 3-amino-1,2,4-triazole; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DHR123, dihydrorhodamine 123; DMEM, Dulbecco's modified Eagle medium; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione disulfide; GSx, total glutathione (GSH + 2 × GSSG); PBS, phosphate-buffered saline; LDH, lactate dehydrogenase; NAC, N-acetyl-L-cysteine; PB, phosphate buffer; Rh123, rhodamine 123; ROS, reactive oxygen species; Zn, zinc. ⁎ Corresponding author. Fax: +61 3 990 53948. E-mail address: [email protected] (G.M. Bishop). 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.01.022
glutathione disulfide (GSSG) that is generated by the GPx reaction is reduced to GSH in the reaction catalyzed by the NADPH-dependent GR, which is the rate-limiting step [3,4]. Studies in fibroblast- and alveolar-like cell lines derived from lung have shown that Zn can inactivate GR, promoting cell death [5,6]. Zn is present in the synaptic vesicles of glutamatergic neurons [7–9], and during synaptic transmission is released into the synaptic cleft at concentrations of 100–300 μM [10,11]. Under normal circumstances, such high concentrations of Zn are transient and are rapidly cleared from the synapse. However, in pathological conditions that involve sustained neuronal depolarisation (e.g., ischemic stroke, traumatic brain injury, epileptiform activity), elevated levels of extracellular zinc are thought to contribute to the resulting neuropathology [12–14]. Indeed, Zn is toxic to both neurons and astrocytes (e.g., [15,16]) at concentrations that can be reached during excitotoxic episodes [8,17]. Cultured neurons appear to be more sensitive to the toxicity of Zn than cultured astrocytes [18], but the basis for this difference is unknown.
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Astrocytes enclose glutamatergic synapses [19] and rapidly accumulate Zn [18,20], via L-type calcium channels [20]. Zinc toxicity in astrocytes appears to involve an increase in oxidative stress and/or a decrease in antioxidant protection, because in astrocytes cultured from rat or mouse brains Zn decreases the GSH content of cells [16,21] and increases their GSSG content [21]. One possibility is that Zn prevents the reduction of GSSG to GSH by inactivating GR. Extracellular Zn has been shown to decrease GR activity in homogenates of rat brain [22], but it is not known whether zinc inhibits GR activity in intact astrocytes. Moreover, inhibition of GR (if it occurs) may not account for the toxicity of Zn in astrocytes since the GR inhibitor, carmustine, is reported not to be toxic to astrocytes [23]. In order to resolve this important issue, the present study has examined the effect of Zn and carmustine on GR activity and on GSH redox cycling in primary cultures of rat astrocytes. Furthermore, we have compared the incidence of cell death, the production of ROS, and the rate of detoxification of H2O2 as a consequence of exposure to Zn or carmustine.
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Incubation protocol Cultures were used for experiments after 14–20 days in vitro and the culture media were replaced 24–48 h prior to experimentation. Astrocytes were washed twice with 1 ml of prewarmed (37 °C) DMEM and were then incubated with 1 ml of DMEM containing 0–250 μM Zn, 200 μM carmustine (to inhibit GR), or 10 mM 3AT (to inhibit catalase), as indicated in the figure legends. When astrocytes were incubated with H2O2, they were washed once with 2 ml of prewarmed (37 °C) incubation buffer (20 mM Hepes, 145 mM NaCl, 0.8 mM Na2HPO4, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM glucose, pH 7.4), and then incubated with 500 μl of 100 μM H2O2 in incubation buffer. The cells and media were processed as indicated for each individual experiment. Cell viability
Materials and methods
Cell viability was assessed by measuring the activity of cytosolic lactate dehydrogenase (LDH) in the media, using a method to determine the enzymatic activity of LDH [25]. The cellular protein content of the cultures was determined according to the Lowry method [26].
Reagents
Glutathione content
Dulbecco's modified Eagle medium (DMEM), fetal calf serum, and penicillin/streptomycin were purchased from Gibco. Dihydrorhodamine 123 (DHR123; Cat. No. D632) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Cat. No. D21490) were obtained from Molecular Probes. Paraformaldehyde was from ProSciTech and Permafluor aqueous mounting media was from Beckman Coulter. Zn acetate, carmustine (1,3-bis-[2-chloroethyl]-1-nitrosourea), purified yeast GR, 3-amino-1,2,4-triazole (3AT), N-acetyl-Lcysteine (NAC), Trolox, and other chemicals were obtained from Sigma.
The cellular contents of GSx (amount of GSH plus twice the amount of GSSG) and GSSG were determined from cell lysates in microtiter plates as described previously [27], using a modification of the method originally described by Tietze [28]. The content of GSSG in the media was determined in a similar manner. The presence of Zn did not affect the quantitation of GSx or GSSG (data not shown). The intracellular contents of GSx and GSSG after incubation with Zn were normalized using cellular protein values determined from equivalently treated culture wells. Glutathione reductase activity
Primary astrocyte cell cultures Primary astrocyte cell cultures were derived from newborn Wistar rats (< 24 h old, obtained from Monash Animal Services) by the method of Hamprecht and Löffler [24]. Immunocytochemical analysis for the astrocyte-specific marker glial fibrillary acidic protein confirmed that the cultures contain approximately 95% astrocytes (data not shown). All animal experimentation was approved by the Monash University Psychology Animal Ethics Committee and met the guidelines defined by the National Health and Medical Research Council. Viable cells were seeded at 300,000 cells per well in 24-well culture plates and incubated in culture medium (90% DMEM, 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin). For Rh123 fluorescence, cells were grown on 13-mm glass coverslips in 24-well plates and were seeded at 150,000 cells per well. Cultures were maintained at 10% CO2 and the culture media were replaced every 7 days.
The activity of GR in primary astrocyte cultures was determined using a modification of the protocol described previously [29]. Cells were washed once with 10 mM phosphate-buffered saline (PBS; 10 mM potassium phosphate buffer, 150 mM NaCl; pH 7.4) and were lysed with 100 μl of 20 mM potassium phosphate buffer (pH 7.0) for 10 min on ice. The lysates were collected and centrifuged at 15,000g for 10 min at 4 °C. The supernatants were tested for GR activity by measuring the decrease of absorbance at 340 nm due to oxidation of NADPH in a total volume of 360 μl in 96-well microtiter plates. The reaction mixture contained 100 mM potassium phosphate buffer, 1 mM EDTA, 1 mM GSSG, and 0.2 mM NADPH (pH 7.0, room temperature). The activity of GR was calculated using the extinction coefficient for NADPH (ε = 6.22 mM− 1 cm− 1). The specific activity is expressed as nmol/(minUmg protein), using the cellular protein content that had been obtained from equivalently treated culture wells.
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Extracellular H2O2 concentration The concentration of H2O2 in the media was determined using a colorimetric method described previously [25]. To ensure an accurate determination of H2O2 concentration in the media, fluid in the plates was gently swirled prior to the collection of the 10-μl samples. The half-life (t1/2) of H2O2 was calculated from the semilogarithmic representation of the data obtained for the first 8 min of incubation with H2O2. To standardize for potential differences in the protein content in each well following the specific pretreatment, and prior to addition of H2O2, the specific detoxification rate constant D was determined as previously described [30], where D = (t1/2 × mg protein)− 1. Intracellular ROS To detect ROS, cells were incubated with 2.5 μg/ml DHR123 in PBS for 30 min in the CO2 incubator, as described previously [31]. DHR123 is rapidly taken up by cells and is converted to rhodamine 123 (Rh123) in the presence of ROS. Cells were then washed 3 times in PBS for 5 min, fixed for 30 min in 4% paraformaldehyde in phosphate buffer (PB; 0.1 M sodium phosphate buffer, pH 7.2), and washed in PB (3 × 10 min). Counterstaining of nuclei was performed by incubating cells with 0.2 μg/ml DAPI in PBS for 5 min, washed in PBS (2 × 5 min), and mounted onto glass slides using Permafluor aqueous mounting media. Rh123 and DAPI fluorescence was examined by epifluorescence microscopy with fluorescein and UV filters, respectively. Statistical analysis All experiments were performed on a minimum of three independent cultures, with triplicate samples within each culture. The data are presented as means ± SD for the three cultures. Statistical analysis was performed by independent samples t tests, with statistical significance set at α = 0.05. Results To examine the toxicity of Zn to astrocyte cultures, cells were incubated with 0–250 μM Zn acetate for 4–8 h and cell death was determined by measuring extracellular LDH activity (Fig. 1). After 4 h of incubation, cell viability was largely unchanged by any concentration of Zn examined. However after 6 h incubation, toxicity was observed for concentrations of Zn ≥ 150 μM as indicated by the significant increase in extracellular LDH. The extent of cell death was maximal at 8 h, with extracellular LDH activities of 21.9 ± 5.2% (P < 0.01) or 26.3 ± 9.0% (P < 0.05), of the initial cellular LDH activity for 200 and 250 μM Zn, respectively. Unlike 200 μM Zn, incubation with 200 μM carmustine for up to 8 h did not increase extracellular LDH compared to control conditions (Fig. 1). Similarly, concentrations of ≤100 μM Zn did not differ from control conditions which lacked Zn, even after incubation periods as long as 48 h (data not shown).
Fig. 1. Cell viability after incubation with Zn. Primary astrocyte cultures were incubated with 0–250 μM Zn or 200 μM carmustine in DMEM and the activity of extracellular LDH in the media after 4–8 h was determined as a percentage of total initial LDH in untreated cultures. *P < 0.05, **P < 0.01 compared to 0 μM Zn.
To determine whether Zn alters GSH metabolism in cultured astrocytes, the cellular contents of total glutathione (GSx = GSH + [2 × GSSG]) and GSSG were determined following incubation with Zn (Fig. 2). After application of Zn for 4 or 6 h, there was no change in cellular GSx content for any Zn concentration; however, after 8 h incubation the GSx content was increased in cells incubated with ≥ 100 μM Zn (Fig. 2A). Following 6 h incubation, cellular GSSG content (expressed as GSx) was increased by Zn concentrations ≥ 150 μM (Fig. 2B). When the cellular GSSG content was expressed as a percentage of the GSx content of the respective culture well, control cells incubated without Zn contained approximately 2.5% GSSG (Fig. 2C). Following incubation with Zn the GSSG:GSx ratio in astrocytes increased dramatically, such that after 8 h incubation with 150 or 250 μM Zn, GSSG accounted for 8.3 ± 2.2% (P < 0.001) and 24.6 ± 7.6% (P < 0.001), respectively, of the GSx content. In contrast, incubation of astrocytes with 200 μM carmustine for 8 h only slightly increased cellular GSx and GSSG contents (Figs. 2A and B), and the GSSG:GSx ratio was only 2.1-fold greater than control conditions. Zn was also observed to stimulate the release of GSSG into the media in a concentration-dependent manner, while carmustine did not induce GSSG release from astrocytes (Fig. 2D). The continual accumulation of GSSG in astrocytes following incubation with Zn suggested a disruption of the GSH redox cycle. Since Zn has been reported to inhibit purified GR [1], the GR activity in astrocytes was determined following incubation with Zn (Fig. 3). The basal GR activity of untreated primary astrocyte cultures was on average 5.4 ± 1.0 nmol/(minUmg protein) (range = 4.7–6.6 nmol/(minUmg protein)). Incubation with Zn for 4 h caused a concentration-dependent decrease in cellular GR activity, with a substantial loss induced by 150 μM Zn and 250 μM Zn (37.7 ± 6.1 and 11.9 ± 11.5% of initial GR activity, respectively). The extent of inhibition of GR by Zn was comparable to that induced by the GR inhibitor carmustine,
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Fig. 2. Glutathione content in cells and media after incubation with Zn. Primary astrocyte cultures were incubated with 0–250 μM Zn or 200 μM carmustine in DMEM for 4–8 h. The cellular contents of GSx (A) and GSSG (B) were determined for each incubation condition. The ratio of GSSG to GSx was calculated for each well (C). The amount of GSSG released into the media was determined following 4 h incubation (D). *P < 0.05, **P < 0.01 compared to 0 μM Zn.
Fig. 3. GR activity following incubation with Zn. Primary astrocyte cultures were incubated with 0–250 μM Zn or 200 μM carmustine in DMEM for 4 h. The specific activity of GR was determined as the rate of GSSG-dependent oxidation of NADPH in cell lysates. To control for differences in basal GR activity levels between the three independent cultures, GR activity in treated wells was expressed as a percentage of the basal GR activity for the respective culture. 100% GR activity corresponds to 5.4 ± 1.0 nmol/(minUmg protein) (n = 18). **P < 0.01 compared to 0 μM Zn.
which lowered the specific GR activity to 28.3 ± 9.5% of the initial value. This inhibition of GR by Zn or carmustine continued when cells were incubated for up to 8 h (data not shown). To determine whether Zn and carmustine inhibit GR activity directly by interacting with GR, purified yeast GR was incubated with Zn or carmustine and the activity of the enzyme was determined. In Eppendorf tubes, 10 mU (45 μl) of yeast GR in 20 mM K-PB was incubated with 15 μl of a mixture containing 33 nmol Zn acetate or carmustine plus 33 nmol NADPH for 1 h at RT in the dark. The activity of yeast GR was decreased by this incubation with Zn or carmustine to 29.8 ± 1.5 or 58.7 ± 2.5% of the initial GR activity, respectively. The GR activity was not inhibited by either Zn or carmustine if they were not coincubated with NADPH. To examine the consequences of Zn-induced impairment of GR activity in living astrocytes, the efficiency of GSH redox cycling was measured after application of 100 μM H2O2 to cells that had been pretreated with or without Zn. Application of H2O2 caused a rapid increase in the GSSG:GSx ratio, such that in the control condition (0 μM Zn), GSSG accounted for approximately 50% of the cellular GSx content within 2 min of H2O2 application (Fig. 4), and then quickly recovered toward basal levels. A similar pattern was observed in cells that had
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Fig. 4. GSSG recycling in astrocytes following application of H2O2 to Znpretreated cells. Primary astrocyte cultures were pretreated with 0–250 μM Zn or 200 μM carmustine for 4 h, and then incubated with 100 μM H2O2. The cellular contents of GSSG and GSx were determined for each culture well and the proportion of GSSG to GSx was calculated. * P < 0.05, **P < 0.01 compared to 0 μM Zn. The proportion of GSSG that was present at different time points after H2O2 application for specific Zn concentrations was compared. †P < 0.05, ‡P < 0.01 for each respective pretreatment condition compared to 2 min after H2O2 application.
been pretreated with 100 μM Zn, although the proportion of GSSG was 1.3- to 1.8-fold greater at all time points after application of H2O2. By contrast, in cells that had been pretreated with 250 μM Zn, GSSG accounted for more than 80% of GSx within 2 min of application of H2O2, and this proportion did not decrease over the time points examined. A similar pattern was observed in cells pretreated with carmustine, although the proportion of GSSG began to decline after 8 min. The capacity of astrocytes to detoxify H2O2 depends on the GSH and catalase systems [2]. To test whether Zncompromised GSH redox cycling impairs the capacity of astrocytes to detoxify H2O2, astrocytes were pretreated with Zn for 4 h and then incubated with 100 μM H2O2. Normal astrocytes detoxify 100 μM H2O2 very rapidly with a halftime of 5.8 ± 0.3 min, and preincubation of astrocytes with 100 μM Zn did not alter this clearance rate (Fig. 5A). In contrast, cells treated with 250 μM Zn detoxified H2O2 more slowly (Fig. 5A). Following pretreatment with increasing concentrations of Zn, the capacity of the cells to clear exogenous H2O2 was systematically lowered. This impairment was demonstrated by the significant increase in the half-time of H2O2 from 5.8 ± 0.3 min in controls, to 8.2 ± 0.9, 9.6 ± 0.1, or 10.8 ± 0.4 min in cells pretreated with 150, 200, or 250 μM Zn, respectively (P < 0.05 compared to controls), as well as by the decrease in the specific detoxification rate constant D (Fig. 6). The half-life for H2O2 in cells pretreated with 200 μM carmustine (10.5 ± 1.8 min) was identical to that in cells pretreated with 250 μM Zn. To verify that Zn inhibits the GSH system and not catalase, catalase was inactivated in astrocytes with 3AT prior to assessing the effect of Zn on the capacity of astrocytes to detoxify H2O2. Inhibition of catalase with 10 mM 3AT significantly decreased the capacity of astrocytes to clear
H2O2 as demonstrated by the increased half-time of H2O2 from 5.8 ± 0.3 min (controls) to 8.5 ± 0.8 min (3AT treated, P < 0.01). Incubation of cells with Zn plus 3AT slowed the clearance of H2O2 even further (Fig. 5B). Comparison of the Zn concentration with the specific detoxification rate highlights this additive effect (Fig. 6). For all Zn concentrations applied, concurrent inhibition of catalase lowered the D values by 0.3 to 0.5 (min × mg protein)− 1. This additive effect indicates that Zn inhibits the glutathione system and does not decrease H2O2 detoxification via the inhibition of catalase. To detect intracellular ROS in astrocytes after 4 h exposure to Zn, cells were incubated with DHR123, which is oxidized to Rh123 in the presence of ROS. The cells were then counterstained with DAPI so that the proportion of astrocytes labeled with Rh123 could be observed (Fig. 7). Incubation of astrocytes with concentrations of up to 100 μM Zn did not result in Rh123 fluorescence (Figs. 7A and C), even though numerous healthy cells were present on the coverslips, as determined by the lack of condensed nuclei (Figs. 7B and D). Only a small number of Rh123-positive cells were detected
Fig. 5. Detoxification of H2O2 by astrocytes pretreated with Zn. Primary astrocyte cultures were preincubated for 4 h with the indicated concentrations of Zn in DMEM in the absence (A) or the presence (B) of the catalase inhibitor 3AT (10 mM) and then incubated with 100 μM H2O2 in incubation buffer.
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Fig. 6. Specific detoxification rate of H2O2 in the media after preincubation of astrocytes with Zn and/or 3AT. Primary astrocyte cultures were incubated for 4 h with 0–250 μM Zn and/or 10 mM 3AT in DMEM, and then incubated with 100 μM H2O2 in incubation buffer. For each incubation condition, the specific detoxification rate D of H2O2 was determined. *P < 0.05, **P < 0.01 compared to 0 μM Zn for each respective Zn concentration (with or without 3AT). To compare D between cells incubated with or without 3AT but with the same Zn concentration, independent samples t tests were performed for specific Zn concentrations, ‡P < 0.01 compared to controls with no 3AT.
following incubation with 150 μM Zn (Fig. 7E), whereas this number was substantially greater in astrocytes incubated with 200 μM Zn (Fig. 7G). Incubation of astrocytes with 250 μM Zn resulted in the majority of cells being Rh123 positive (Figs. 7I and J). In contrast, incubation of astrocytes with 200 μM carmustine did not result in the generation of intracellular ROS (Fig. 7K). To determine whether ROS are responsible for the toxicity of Zn, astrocytes were pretreated for 1 h with 1000 μM NAC, 500 μM Trolox, or DMEM. Subsequently, Zn or carmustine was added directly to each culture well and LDH release and GR activity of cell lysates were determined. Pretreatment of astrocytes with NAC or Trolox decreased the release of LDH into the media following 8 h incubation with 250 μM Zn from 41.6 ± 6.3 to 5.5 ± 8.9 and 21.2 ± 10.8%, respectively (P < 0.05). The activity of GR in astrocytes incubated with 250 μM Zn for 4 h was decreased to 2.7 ± 0.4 nmol/(minUmg protein), while pretreatment with NAC prevented this inhibition of GR activity (5.9 ± 1.2 nmol/(minUmg protein) (P < 0.05). In contrast, preincubation with Trolox did not prevent the zinc-induced inhibition of GR activity (2.7 ± 1.2 nmol/(minUmg protein)). None of the antioxidant pretreatments altered the LDH release from cultured astrocytes following incubation with 200 μM carmustine, nor did they prevent the inhibition of GR activity by carmustine (data not shown). To determine if antioxidant pretreatment would decrease intracellular ROS formation by Zn, additional cultures of astrocytes were incubated with DHR123 as described above. It was observed that Zn-induced ROS formation was completely prevented by pretreatment with NAC, while Trolox substantially reduced but did not eliminate ROS formation (data not shown).
Fig. 7. ROS generation in astrocytes incubated with Zn. Primary astrocyte cultures were incubated for 4 h with 0 μM Zn (A,B), 100 μM Zn (C,D), 150 μM Zn (E,F), 200 μM Zn (G,H), 250 μM Zn (I,J), or 200 μM carmustine (K,L) in DMEM. They were then incubated with DHR123 to test for the generation of Rh123 fluorescence that indicates the presence of ROS (A,C,E,G,I,K). Subsequently the nuclei were counterstained with DAPI (B,D,F,H,J,L). Arrows indicate cells counterstained for both Rh123 and DAPI. The scale bar in K represents 50 μm and applies to all panels.
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Discussion The present study investigated whether Zn inhibits GR activity in astrocytes, and if so, whether such inhibition can account for the toxicity of this metal. The toxicity of Zn was compared to that of the GR inhibitor, carmustine. Our results indicate that both Zn and carmustine inhibit GR activity, yet only Zn is toxic to cells and induces oxidative stress. The significance of these findings will be discussed below. We observed a threshold effect of Zn toxicity whereby 100 μM Zn did not cause LDH release even after 48 h, yet concentrations greater than 100 μM produced substantial cell death from 6 h onward. This pattern of toxicity is comparable to that previously reported for astrocytes [16,20]. By contrast, 200 μM carmustine did not induce cell death in astrocytes even after 8 h incubation, which is consistent with a previous report showing that 300 μM carmustine is not toxic after 24 h [32]. Thus astrocytes differ from other cell types, including neuronlike cells, in which the EC50 of carmustine has been reported to range from 10 to 280 μM (see [33]). Treatment of astrocytes with ≥ 150 μM Zn inhibited the activity of GR. This observation is consistent with reports from studies using fibroblast- and alveolar-like cell lines derived from lung [5,6]. The degree of inhibition of astrocytic GR by Zn was similar to that caused by carmustine, which is known to quickly and irreversibly inhibit GR [34]. The Zninduced inhibition of GR was confirmed by examining the production of GSSG after activating GSH redox cycling by exposure of the cells to H2O2. In control cells, a spike in cellular GSSG content was observed after H2O2 application, which was quickly resolved as GR recycled GSSG back to GSH, and is consistent with our previous reports [4]. Zn treatment, in a concentration-dependent manner, increased the content of GSSG following incubation with H2O2, and 250 μM Zn prevented recycling of GSSG within the 15-min time period examined. Carmustine also increased the content of GSSG but some recycling did occur within the 15-min period, indicating either a partial inhibition of GR or a reversible inactivation of the enzyme. The latter possibility seems more likely since both carmustine and 250 μM Zn initially inhibited GR to a similar extent (Fig. 4). Furthermore, the presence of EDTA in the GR activity assay in at least 4fold excess of Zn indicates that the inhibition of GR in astrocyte lysates or purified yeast GR is irreversible, otherwise the EDTA would have chelated the Zn during the 20-min enzyme activity assay. While the precise mechanism by which Zn inhibits GR is unknown, the inhibition is direct and does not require second messengers, since we have shown that purified GR is inhibited effectively by Zn. Since the carbamoylation reaction by which carmustine inhibits GR is NADPH dependent [35], it is noteworthy that our data demonstrate that the inhibition of GR by Zn is also dependent on the availability of NADPH. In view of this similarity, we suggest that NADPH may reduce the disulfide bond at Cys-45 and Cys-50 in the active site [36], thereby allowing Zn to bind to the exposed sulfhydryl groups, causing an irreversible inactivation of the enzyme.
When astrocytes were pretreated with Zn, their capacity to detoxify H2O2 was greatly reduced, such that the half-life of H2O2 was almost doubled after incubation with 250 μM Zn. Inactivation of catalase activity via 3AT treatment, which removed a key cellular mechanism used to detoxify H2O2, further slowed the detoxification of H2O2 to the extent that the specific detoxification rate curve was shifted relative to the Zn concentration. This shift indicates that the effect of Zn was primarily on the GSH system. This conclusion is supported by the fact that the cells treated with either 250 μM Zn or 200 μM carmustine were equally impaired in their capacity to detoxify H2O2. At concentrations of 150 μM and above, at exposure durations of 6 h or more, Zn greatly increased the intracellular GSSG content, which is consistent with previous reports [21]. The fact that 200 μM carmustine did not greatly increase the intracellular GSSG content demonstrates that inhibition of GR by Zn is not the primary cause of the cellular GSSG accumulation. The present study also found that GSSG is exported from astrocytes following incubation with Zn. While we are the first to report the export of GSSG in response to Zn, cultured astrocytes in addition to other cells and tissues have been reported to export GSSG in response to prooxidants [37–40]. Such export is thought to overcome the shift in thiol balance produced by an accumulation of cellular GSSG. These observations, combined with the present data, suggest that Zn causes the production of oxidative stress in astrocytes via a mechanism that is independent of the inhibition of GR. It is notable that we observed an increase in cellular GSx content after 8 h incubation with Zn, indicating that the astrocytes may have been synthesizing additional GSH in order to restore their thiol balance. We found that Zn induced Rh123 fluorescence in astrocytes in a concentration-dependent manner, whereas no Rh123 fluorescence was observed in carmustine-treated cells. This difference further supports the notion that ROS generation in Zn-treated cells is separate from the inhibition of GR. There are several potential sources of Zn-induced ROS production including the autooxidation of sulfhydryl groups on cell membranes [41], dysregulation of the mitochondrial membrane potential [18], inhibition of thioredoxin reductase [42] and thiol oxidoreductase [43], and increased activity of NADPH oxidase [44]. Preincubation with either NAC or Trolox prevented the generation of intracellular ROS by Zn, as detected by Rh123 fluorescence (data not shown). The prevention of cell death following preincubation with NAC or Trolox indicates that oxidative mechanisms are responsible for the toxicity of Zn. The fact that NAC and Trolox prevented both the production of ROS and the cell death by Zn, yet only NAC prevented the inhibition of GR, provides additional evidence that the toxicity of Zn in astrocytes is not directly due to the inhibition of GR. In conclusion, the present results indicate that Zn is a highly effective inhibitor of GR activity in astrocytes, and that this inhibition impairs their capacity to detoxify H2O2. When astrocytes are subsequently exposed to prooxidants, which is likely to occur in pathological conditions, they will be less able to protect the brain from oxidation. Furthermore, pathological
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conditions are often associated with elevations in extracellular Zn, and we have found that elevated Zn leads to the production of intracellular ROS, oxidative stress, and cell death. We have shown that Zn, like carmustine, inhibits GR via an NADPHdependent mechanism, yet carmustine, unlike Zn, is not associated with the production of ROS, oxidative stress, or cell death. Taken together, these results suggest that the inhibition of GR by Zn is not sufficient to kill astrocytes, and that the toxicity of Zn is due instead to a separate mechanism which involves the production of ROS. Thus, antioxidant therapy may be efficacious in conditions such as ischemia, traumatic brain injury, and epileptiform activity, since all involve elevations in extracellular Zn.
[15]
[16]
[17]
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Acknowledgments G.M.B. is supported by a National Health and Medical Research Council Peter Doherty Fellowship (ID:284393), and R.D. was supported by a NeuroSciences Victoria Senior Research Fellowship. We also thank the Department of Psychology, Monash University, for providing generous funding of this work. References [1] Mize, C. E.; Langdon, R. G. Hepatic glutathione reductase. I. Purification and general kinetic properties. J. Biol. Chem. 237:1589–1595; 1962. [2] Dringen, R.; Pawlowski, P. G.; Hirrlinger, J. Peroxide detoxification by brain cells. J. Neurosci. Res. 79:157–165; 2005. [3] Dringen, R.; Gutterer, J. M. Glutathione reductase from bovine brain. Methods Enzymol. 348:281–288; 2002. [4] Dringen, R.; Hamprecht, B. Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells. Brain Res. 759:67–75; 1997. [5] Walther, U. I.; Czermak, A.; Muckter, H.; Walther, S. C.; Fichtl, B. Decreased GSSG reductase activity enhances cellular zinc toxicity in three human lung cell lines. Arch. Toxicol. 77:131–137; 2003. [6] Wilhelm, B.; Walther, U. I.; Fichtl, B. Effects of zinc chloride on glutathione and glutathione synthesis rates in various lung cell lines. Arch. Toxicol. 75:388–394; 2001. [7] Frederickson, C. J.; Kasarskis, E. J.; Ringo, D.; Frederickson, R. E. A quinoline fluorescence method for visualizing and assaying the histochemically reactive zinc (bouton zinc) in the brain. J. Neurosci. Methods 20:91–103; 1987. [8] Choi, D. W.; Koh, J. Y. Zinc and brain injury. Annu. Rev. Neurosci. 21:347–375; 1998. [9] Lee, J. Y.; Mook-Jung, I.; Koh, J. Y. Histochemically reactive zinc in plaques of the Swedish mutant beta-amyloid precursor protein transgenic mice. J. Neurosci. 19:RC10; 1999. [10] Assaf, S. Y.; Chung, S. H. Release of endogenous Zn2+ from brain tissue during activity. Nature 308:734–736; 1984. [11] Howell, G. A.; Welch, M. G.; Frederickson, C. J. Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308:736–738; 1984. [12] Koh, J. Y.; Suh, S. W.; Gwag, B. J.; He, Y. Y.; Hsu, C. Y.; Choi, D. W. The role of zinc in selective neuronal death after transient global cerebral ischemia. Science 272:1013–1016; 1996. [13] Suh, S. W.; Chen, J. W.; Motamedi, M.; Bell, B.; Listiak, K.; Pons, N. F.; Danscher, G.; Frederickson, C. J. Evidence that synaptically-released zinc contributes to neuronal injury after traumatic brain injury. Brain Res. 852:268–273; 2000. [14] Frederickson, C. J.; Hernandez, M. D.; Goik, S. A.; Morton, J. D.; McGinty, J. F. Loss of zinc staining from hippocampal mossy fibers during
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Original Contribution
Zinc stimulates the production of toxic reactive oxygen species (ROS) and inhibits glutathione reductase in astrocytes Glenda M. Bishop a,⁎, Ralf Dringen a,b , Stephen R. Robinson a a
School of Psychology, Psychiatry and Psychological Medicine, Monash University, Wellington Rd, Clayton, Victoria, Australia b Center for Biomolecular Interactions Bremen, Faculty 2 (Biology/Chemistry), University of Bremen, Bremen, Germany Received 10 May 2006; revised 1 December 2006; accepted 10 January 2007 Available online 17 January 2007
Abstract The release of zinc (Zn) from glutamatergic synapses contributes to the neuropathology of ischemia, traumatic brain injury, and stroke. Astrocytes surround glutamatergic synapses and are vulnerable to the toxicity of Zn, which impairs their antioxidative glutathione (GSH) system and elevates the production of reactive oxygen species (ROS). It is not known whether one or both of these actions are the primary cause of Zninduced cell death in astrocytes. Using primary rat astrocyte cultures we have examined whether Zn-mediated impairment of GSH redox cycling is the main source of its toxicity. Zn acetate at concentrations of 100 μM or greater were found to inactivate glutathione reductase (GR) via an NADPH-dependent mechanism, while concentrations of 150 μM and above caused substantial cell death. Furthermore, Zn increased the ratio of GSSG:GSH in astrocytes, increased their export of GSSG, slowed their clearance of exogenous H2O2, and promoted the intracellular production of ROS. In contrast, the GR inhibitor, carmustine, did not induce cell death, cause the production of ROS, or alter the GSH thiol redox balance. Taken together these results indicate that Zn toxicity in astrocytes is primarily associated with the generation of intracellular ROS, rather than the inhibition of GR. © 2007 Elsevier Inc. All rights reserved. Keywords: Astrocyte; Carmustine; Epileptiform; Glutathione reductase; Hydrogen peroxide; Ischemia; Reactive oxygen species; Stroke; Traumatic brain injury; Zinc
Introduction Zinc (Zn) has been demonstrated to inhibit the activity of enzymes associated with glutathione (GSH) metabolism, particularly glutathione reductase (GR) [1]. This enzyme is responsible for cellular GSH redox cycling, which is crucial for the detoxification of endogenous peroxides [2]. In this process GSH acts as an electron donor for the glutathione peroxidase (GPx)-dependent reduction of H2O2 and of organic hydroperoxides to water and the corresponding alcohol, respectively. The Abbreviations: 3AT, 3-amino-1,2,4-triazole; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DHR123, dihydrorhodamine 123; DMEM, Dulbecco's modified Eagle medium; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione disulfide; GSx, total glutathione (GSH + 2 × GSSG); PBS, phosphate-buffered saline; LDH, lactate dehydrogenase; NAC, N-acetyl-L-cysteine; PB, phosphate buffer; Rh123, rhodamine 123; ROS, reactive oxygen species; Zn, zinc. ⁎ Corresponding author. Fax: +61 3 990 53948. E-mail address: [email protected] (G.M. Bishop). 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.01.022
glutathione disulfide (GSSG) that is generated by the GPx reaction is reduced to GSH in the reaction catalyzed by the NADPH-dependent GR, which is the rate-limiting step [3,4]. Studies in fibroblast- and alveolar-like cell lines derived from lung have shown that Zn can inactivate GR, promoting cell death [5,6]. Zn is present in the synaptic vesicles of glutamatergic neurons [7–9], and during synaptic transmission is released into the synaptic cleft at concentrations of 100–300 μM [10,11]. Under normal circumstances, such high concentrations of Zn are transient and are rapidly cleared from the synapse. However, in pathological conditions that involve sustained neuronal depolarisation (e.g., ischemic stroke, traumatic brain injury, epileptiform activity), elevated levels of extracellular zinc are thought to contribute to the resulting neuropathology [12–14]. Indeed, Zn is toxic to both neurons and astrocytes (e.g., [15,16]) at concentrations that can be reached during excitotoxic episodes [8,17]. Cultured neurons appear to be more sensitive to the toxicity of Zn than cultured astrocytes [18], but the basis for this difference is unknown.
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Astrocytes enclose glutamatergic synapses [19] and rapidly accumulate Zn [18,20], via L-type calcium channels [20]. Zinc toxicity in astrocytes appears to involve an increase in oxidative stress and/or a decrease in antioxidant protection, because in astrocytes cultured from rat or mouse brains Zn decreases the GSH content of cells [16,21] and increases their GSSG content [21]. One possibility is that Zn prevents the reduction of GSSG to GSH by inactivating GR. Extracellular Zn has been shown to decrease GR activity in homogenates of rat brain [22], but it is not known whether zinc inhibits GR activity in intact astrocytes. Moreover, inhibition of GR (if it occurs) may not account for the toxicity of Zn in astrocytes since the GR inhibitor, carmustine, is reported not to be toxic to astrocytes [23]. In order to resolve this important issue, the present study has examined the effect of Zn and carmustine on GR activity and on GSH redox cycling in primary cultures of rat astrocytes. Furthermore, we have compared the incidence of cell death, the production of ROS, and the rate of detoxification of H2O2 as a consequence of exposure to Zn or carmustine.
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Incubation protocol Cultures were used for experiments after 14–20 days in vitro and the culture media were replaced 24–48 h prior to experimentation. Astrocytes were washed twice with 1 ml of prewarmed (37 °C) DMEM and were then incubated with 1 ml of DMEM containing 0–250 μM Zn, 200 μM carmustine (to inhibit GR), or 10 mM 3AT (to inhibit catalase), as indicated in the figure legends. When astrocytes were incubated with H2O2, they were washed once with 2 ml of prewarmed (37 °C) incubation buffer (20 mM Hepes, 145 mM NaCl, 0.8 mM Na2HPO4, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM glucose, pH 7.4), and then incubated with 500 μl of 100 μM H2O2 in incubation buffer. The cells and media were processed as indicated for each individual experiment. Cell viability
Materials and methods
Cell viability was assessed by measuring the activity of cytosolic lactate dehydrogenase (LDH) in the media, using a method to determine the enzymatic activity of LDH [25]. The cellular protein content of the cultures was determined according to the Lowry method [26].
Reagents
Glutathione content
Dulbecco's modified Eagle medium (DMEM), fetal calf serum, and penicillin/streptomycin were purchased from Gibco. Dihydrorhodamine 123 (DHR123; Cat. No. D632) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Cat. No. D21490) were obtained from Molecular Probes. Paraformaldehyde was from ProSciTech and Permafluor aqueous mounting media was from Beckman Coulter. Zn acetate, carmustine (1,3-bis-[2-chloroethyl]-1-nitrosourea), purified yeast GR, 3-amino-1,2,4-triazole (3AT), N-acetyl-Lcysteine (NAC), Trolox, and other chemicals were obtained from Sigma.
The cellular contents of GSx (amount of GSH plus twice the amount of GSSG) and GSSG were determined from cell lysates in microtiter plates as described previously [27], using a modification of the method originally described by Tietze [28]. The content of GSSG in the media was determined in a similar manner. The presence of Zn did not affect the quantitation of GSx or GSSG (data not shown). The intracellular contents of GSx and GSSG after incubation with Zn were normalized using cellular protein values determined from equivalently treated culture wells. Glutathione reductase activity
Primary astrocyte cell cultures Primary astrocyte cell cultures were derived from newborn Wistar rats (< 24 h old, obtained from Monash Animal Services) by the method of Hamprecht and Löffler [24]. Immunocytochemical analysis for the astrocyte-specific marker glial fibrillary acidic protein confirmed that the cultures contain approximately 95% astrocytes (data not shown). All animal experimentation was approved by the Monash University Psychology Animal Ethics Committee and met the guidelines defined by the National Health and Medical Research Council. Viable cells were seeded at 300,000 cells per well in 24-well culture plates and incubated in culture medium (90% DMEM, 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin). For Rh123 fluorescence, cells were grown on 13-mm glass coverslips in 24-well plates and were seeded at 150,000 cells per well. Cultures were maintained at 10% CO2 and the culture media were replaced every 7 days.
The activity of GR in primary astrocyte cultures was determined using a modification of the protocol described previously [29]. Cells were washed once with 10 mM phosphate-buffered saline (PBS; 10 mM potassium phosphate buffer, 150 mM NaCl; pH 7.4) and were lysed with 100 μl of 20 mM potassium phosphate buffer (pH 7.0) for 10 min on ice. The lysates were collected and centrifuged at 15,000g for 10 min at 4 °C. The supernatants were tested for GR activity by measuring the decrease of absorbance at 340 nm due to oxidation of NADPH in a total volume of 360 μl in 96-well microtiter plates. The reaction mixture contained 100 mM potassium phosphate buffer, 1 mM EDTA, 1 mM GSSG, and 0.2 mM NADPH (pH 7.0, room temperature). The activity of GR was calculated using the extinction coefficient for NADPH (ε = 6.22 mM− 1 cm− 1). The specific activity is expressed as nmol/(minUmg protein), using the cellular protein content that had been obtained from equivalently treated culture wells.
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Extracellular H2O2 concentration The concentration of H2O2 in the media was determined using a colorimetric method described previously [25]. To ensure an accurate determination of H2O2 concentration in the media, fluid in the plates was gently swirled prior to the collection of the 10-μl samples. The half-life (t1/2) of H2O2 was calculated from the semilogarithmic representation of the data obtained for the first 8 min of incubation with H2O2. To standardize for potential differences in the protein content in each well following the specific pretreatment, and prior to addition of H2O2, the specific detoxification rate constant D was determined as previously described [30], where D = (t1/2 × mg protein)− 1. Intracellular ROS To detect ROS, cells were incubated with 2.5 μg/ml DHR123 in PBS for 30 min in the CO2 incubator, as described previously [31]. DHR123 is rapidly taken up by cells and is converted to rhodamine 123 (Rh123) in the presence of ROS. Cells were then washed 3 times in PBS for 5 min, fixed for 30 min in 4% paraformaldehyde in phosphate buffer (PB; 0.1 M sodium phosphate buffer, pH 7.2), and washed in PB (3 × 10 min). Counterstaining of nuclei was performed by incubating cells with 0.2 μg/ml DAPI in PBS for 5 min, washed in PBS (2 × 5 min), and mounted onto glass slides using Permafluor aqueous mounting media. Rh123 and DAPI fluorescence was examined by epifluorescence microscopy with fluorescein and UV filters, respectively. Statistical analysis All experiments were performed on a minimum of three independent cultures, with triplicate samples within each culture. The data are presented as means ± SD for the three cultures. Statistical analysis was performed by independent samples t tests, with statistical significance set at α = 0.05. Results To examine the toxicity of Zn to astrocyte cultures, cells were incubated with 0–250 μM Zn acetate for 4–8 h and cell death was determined by measuring extracellular LDH activity (Fig. 1). After 4 h of incubation, cell viability was largely unchanged by any concentration of Zn examined. However after 6 h incubation, toxicity was observed for concentrations of Zn ≥ 150 μM as indicated by the significant increase in extracellular LDH. The extent of cell death was maximal at 8 h, with extracellular LDH activities of 21.9 ± 5.2% (P < 0.01) or 26.3 ± 9.0% (P < 0.05), of the initial cellular LDH activity for 200 and 250 μM Zn, respectively. Unlike 200 μM Zn, incubation with 200 μM carmustine for up to 8 h did not increase extracellular LDH compared to control conditions (Fig. 1). Similarly, concentrations of ≤100 μM Zn did not differ from control conditions which lacked Zn, even after incubation periods as long as 48 h (data not shown).
Fig. 1. Cell viability after incubation with Zn. Primary astrocyte cultures were incubated with 0–250 μM Zn or 200 μM carmustine in DMEM and the activity of extracellular LDH in the media after 4–8 h was determined as a percentage of total initial LDH in untreated cultures. *P < 0.05, **P < 0.01 compared to 0 μM Zn.
To determine whether Zn alters GSH metabolism in cultured astrocytes, the cellular contents of total glutathione (GSx = GSH + [2 × GSSG]) and GSSG were determined following incubation with Zn (Fig. 2). After application of Zn for 4 or 6 h, there was no change in cellular GSx content for any Zn concentration; however, after 8 h incubation the GSx content was increased in cells incubated with ≥ 100 μM Zn (Fig. 2A). Following 6 h incubation, cellular GSSG content (expressed as GSx) was increased by Zn concentrations ≥ 150 μM (Fig. 2B). When the cellular GSSG content was expressed as a percentage of the GSx content of the respective culture well, control cells incubated without Zn contained approximately 2.5% GSSG (Fig. 2C). Following incubation with Zn the GSSG:GSx ratio in astrocytes increased dramatically, such that after 8 h incubation with 150 or 250 μM Zn, GSSG accounted for 8.3 ± 2.2% (P < 0.001) and 24.6 ± 7.6% (P < 0.001), respectively, of the GSx content. In contrast, incubation of astrocytes with 200 μM carmustine for 8 h only slightly increased cellular GSx and GSSG contents (Figs. 2A and B), and the GSSG:GSx ratio was only 2.1-fold greater than control conditions. Zn was also observed to stimulate the release of GSSG into the media in a concentration-dependent manner, while carmustine did not induce GSSG release from astrocytes (Fig. 2D). The continual accumulation of GSSG in astrocytes following incubation with Zn suggested a disruption of the GSH redox cycle. Since Zn has been reported to inhibit purified GR [1], the GR activity in astrocytes was determined following incubation with Zn (Fig. 3). The basal GR activity of untreated primary astrocyte cultures was on average 5.4 ± 1.0 nmol/(minUmg protein) (range = 4.7–6.6 nmol/(minUmg protein)). Incubation with Zn for 4 h caused a concentration-dependent decrease in cellular GR activity, with a substantial loss induced by 150 μM Zn and 250 μM Zn (37.7 ± 6.1 and 11.9 ± 11.5% of initial GR activity, respectively). The extent of inhibition of GR by Zn was comparable to that induced by the GR inhibitor carmustine,
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Fig. 2. Glutathione content in cells and media after incubation with Zn. Primary astrocyte cultures were incubated with 0–250 μM Zn or 200 μM carmustine in DMEM for 4–8 h. The cellular contents of GSx (A) and GSSG (B) were determined for each incubation condition. The ratio of GSSG to GSx was calculated for each well (C). The amount of GSSG released into the media was determined following 4 h incubation (D). *P < 0.05, **P < 0.01 compared to 0 μM Zn.
Fig. 3. GR activity following incubation with Zn. Primary astrocyte cultures were incubated with 0–250 μM Zn or 200 μM carmustine in DMEM for 4 h. The specific activity of GR was determined as the rate of GSSG-dependent oxidation of NADPH in cell lysates. To control for differences in basal GR activity levels between the three independent cultures, GR activity in treated wells was expressed as a percentage of the basal GR activity for the respective culture. 100% GR activity corresponds to 5.4 ± 1.0 nmol/(minUmg protein) (n = 18). **P < 0.01 compared to 0 μM Zn.
which lowered the specific GR activity to 28.3 ± 9.5% of the initial value. This inhibition of GR by Zn or carmustine continued when cells were incubated for up to 8 h (data not shown). To determine whether Zn and carmustine inhibit GR activity directly by interacting with GR, purified yeast GR was incubated with Zn or carmustine and the activity of the enzyme was determined. In Eppendorf tubes, 10 mU (45 μl) of yeast GR in 20 mM K-PB was incubated with 15 μl of a mixture containing 33 nmol Zn acetate or carmustine plus 33 nmol NADPH for 1 h at RT in the dark. The activity of yeast GR was decreased by this incubation with Zn or carmustine to 29.8 ± 1.5 or 58.7 ± 2.5% of the initial GR activity, respectively. The GR activity was not inhibited by either Zn or carmustine if they were not coincubated with NADPH. To examine the consequences of Zn-induced impairment of GR activity in living astrocytes, the efficiency of GSH redox cycling was measured after application of 100 μM H2O2 to cells that had been pretreated with or without Zn. Application of H2O2 caused a rapid increase in the GSSG:GSx ratio, such that in the control condition (0 μM Zn), GSSG accounted for approximately 50% of the cellular GSx content within 2 min of H2O2 application (Fig. 4), and then quickly recovered toward basal levels. A similar pattern was observed in cells that had
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Fig. 4. GSSG recycling in astrocytes following application of H2O2 to Znpretreated cells. Primary astrocyte cultures were pretreated with 0–250 μM Zn or 200 μM carmustine for 4 h, and then incubated with 100 μM H2O2. The cellular contents of GSSG and GSx were determined for each culture well and the proportion of GSSG to GSx was calculated. * P < 0.05, **P < 0.01 compared to 0 μM Zn. The proportion of GSSG that was present at different time points after H2O2 application for specific Zn concentrations was compared. †P < 0.05, ‡P < 0.01 for each respective pretreatment condition compared to 2 min after H2O2 application.
been pretreated with 100 μM Zn, although the proportion of GSSG was 1.3- to 1.8-fold greater at all time points after application of H2O2. By contrast, in cells that had been pretreated with 250 μM Zn, GSSG accounted for more than 80% of GSx within 2 min of application of H2O2, and this proportion did not decrease over the time points examined. A similar pattern was observed in cells pretreated with carmustine, although the proportion of GSSG began to decline after 8 min. The capacity of astrocytes to detoxify H2O2 depends on the GSH and catalase systems [2]. To test whether Zncompromised GSH redox cycling impairs the capacity of astrocytes to detoxify H2O2, astrocytes were pretreated with Zn for 4 h and then incubated with 100 μM H2O2. Normal astrocytes detoxify 100 μM H2O2 very rapidly with a halftime of 5.8 ± 0.3 min, and preincubation of astrocytes with 100 μM Zn did not alter this clearance rate (Fig. 5A). In contrast, cells treated with 250 μM Zn detoxified H2O2 more slowly (Fig. 5A). Following pretreatment with increasing concentrations of Zn, the capacity of the cells to clear exogenous H2O2 was systematically lowered. This impairment was demonstrated by the significant increase in the half-time of H2O2 from 5.8 ± 0.3 min in controls, to 8.2 ± 0.9, 9.6 ± 0.1, or 10.8 ± 0.4 min in cells pretreated with 150, 200, or 250 μM Zn, respectively (P < 0.05 compared to controls), as well as by the decrease in the specific detoxification rate constant D (Fig. 6). The half-life for H2O2 in cells pretreated with 200 μM carmustine (10.5 ± 1.8 min) was identical to that in cells pretreated with 250 μM Zn. To verify that Zn inhibits the GSH system and not catalase, catalase was inactivated in astrocytes with 3AT prior to assessing the effect of Zn on the capacity of astrocytes to detoxify H2O2. Inhibition of catalase with 10 mM 3AT significantly decreased the capacity of astrocytes to clear
H2O2 as demonstrated by the increased half-time of H2O2 from 5.8 ± 0.3 min (controls) to 8.5 ± 0.8 min (3AT treated, P < 0.01). Incubation of cells with Zn plus 3AT slowed the clearance of H2O2 even further (Fig. 5B). Comparison of the Zn concentration with the specific detoxification rate highlights this additive effect (Fig. 6). For all Zn concentrations applied, concurrent inhibition of catalase lowered the D values by 0.3 to 0.5 (min × mg protein)− 1. This additive effect indicates that Zn inhibits the glutathione system and does not decrease H2O2 detoxification via the inhibition of catalase. To detect intracellular ROS in astrocytes after 4 h exposure to Zn, cells were incubated with DHR123, which is oxidized to Rh123 in the presence of ROS. The cells were then counterstained with DAPI so that the proportion of astrocytes labeled with Rh123 could be observed (Fig. 7). Incubation of astrocytes with concentrations of up to 100 μM Zn did not result in Rh123 fluorescence (Figs. 7A and C), even though numerous healthy cells were present on the coverslips, as determined by the lack of condensed nuclei (Figs. 7B and D). Only a small number of Rh123-positive cells were detected
Fig. 5. Detoxification of H2O2 by astrocytes pretreated with Zn. Primary astrocyte cultures were preincubated for 4 h with the indicated concentrations of Zn in DMEM in the absence (A) or the presence (B) of the catalase inhibitor 3AT (10 mM) and then incubated with 100 μM H2O2 in incubation buffer.
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Fig. 6. Specific detoxification rate of H2O2 in the media after preincubation of astrocytes with Zn and/or 3AT. Primary astrocyte cultures were incubated for 4 h with 0–250 μM Zn and/or 10 mM 3AT in DMEM, and then incubated with 100 μM H2O2 in incubation buffer. For each incubation condition, the specific detoxification rate D of H2O2 was determined. *P < 0.05, **P < 0.01 compared to 0 μM Zn for each respective Zn concentration (with or without 3AT). To compare D between cells incubated with or without 3AT but with the same Zn concentration, independent samples t tests were performed for specific Zn concentrations, ‡P < 0.01 compared to controls with no 3AT.
following incubation with 150 μM Zn (Fig. 7E), whereas this number was substantially greater in astrocytes incubated with 200 μM Zn (Fig. 7G). Incubation of astrocytes with 250 μM Zn resulted in the majority of cells being Rh123 positive (Figs. 7I and J). In contrast, incubation of astrocytes with 200 μM carmustine did not result in the generation of intracellular ROS (Fig. 7K). To determine whether ROS are responsible for the toxicity of Zn, astrocytes were pretreated for 1 h with 1000 μM NAC, 500 μM Trolox, or DMEM. Subsequently, Zn or carmustine was added directly to each culture well and LDH release and GR activity of cell lysates were determined. Pretreatment of astrocytes with NAC or Trolox decreased the release of LDH into the media following 8 h incubation with 250 μM Zn from 41.6 ± 6.3 to 5.5 ± 8.9 and 21.2 ± 10.8%, respectively (P < 0.05). The activity of GR in astrocytes incubated with 250 μM Zn for 4 h was decreased to 2.7 ± 0.4 nmol/(minUmg protein), while pretreatment with NAC prevented this inhibition of GR activity (5.9 ± 1.2 nmol/(minUmg protein) (P < 0.05). In contrast, preincubation with Trolox did not prevent the zinc-induced inhibition of GR activity (2.7 ± 1.2 nmol/(minUmg protein)). None of the antioxidant pretreatments altered the LDH release from cultured astrocytes following incubation with 200 μM carmustine, nor did they prevent the inhibition of GR activity by carmustine (data not shown). To determine if antioxidant pretreatment would decrease intracellular ROS formation by Zn, additional cultures of astrocytes were incubated with DHR123 as described above. It was observed that Zn-induced ROS formation was completely prevented by pretreatment with NAC, while Trolox substantially reduced but did not eliminate ROS formation (data not shown).
Fig. 7. ROS generation in astrocytes incubated with Zn. Primary astrocyte cultures were incubated for 4 h with 0 μM Zn (A,B), 100 μM Zn (C,D), 150 μM Zn (E,F), 200 μM Zn (G,H), 250 μM Zn (I,J), or 200 μM carmustine (K,L) in DMEM. They were then incubated with DHR123 to test for the generation of Rh123 fluorescence that indicates the presence of ROS (A,C,E,G,I,K). Subsequently the nuclei were counterstained with DAPI (B,D,F,H,J,L). Arrows indicate cells counterstained for both Rh123 and DAPI. The scale bar in K represents 50 μm and applies to all panels.
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Discussion The present study investigated whether Zn inhibits GR activity in astrocytes, and if so, whether such inhibition can account for the toxicity of this metal. The toxicity of Zn was compared to that of the GR inhibitor, carmustine. Our results indicate that both Zn and carmustine inhibit GR activity, yet only Zn is toxic to cells and induces oxidative stress. The significance of these findings will be discussed below. We observed a threshold effect of Zn toxicity whereby 100 μM Zn did not cause LDH release even after 48 h, yet concentrations greater than 100 μM produced substantial cell death from 6 h onward. This pattern of toxicity is comparable to that previously reported for astrocytes [16,20]. By contrast, 200 μM carmustine did not induce cell death in astrocytes even after 8 h incubation, which is consistent with a previous report showing that 300 μM carmustine is not toxic after 24 h [32]. Thus astrocytes differ from other cell types, including neuronlike cells, in which the EC50 of carmustine has been reported to range from 10 to 280 μM (see [33]). Treatment of astrocytes with ≥ 150 μM Zn inhibited the activity of GR. This observation is consistent with reports from studies using fibroblast- and alveolar-like cell lines derived from lung [5,6]. The degree of inhibition of astrocytic GR by Zn was similar to that caused by carmustine, which is known to quickly and irreversibly inhibit GR [34]. The Zninduced inhibition of GR was confirmed by examining the production of GSSG after activating GSH redox cycling by exposure of the cells to H2O2. In control cells, a spike in cellular GSSG content was observed after H2O2 application, which was quickly resolved as GR recycled GSSG back to GSH, and is consistent with our previous reports [4]. Zn treatment, in a concentration-dependent manner, increased the content of GSSG following incubation with H2O2, and 250 μM Zn prevented recycling of GSSG within the 15-min time period examined. Carmustine also increased the content of GSSG but some recycling did occur within the 15-min period, indicating either a partial inhibition of GR or a reversible inactivation of the enzyme. The latter possibility seems more likely since both carmustine and 250 μM Zn initially inhibited GR to a similar extent (Fig. 4). Furthermore, the presence of EDTA in the GR activity assay in at least 4fold excess of Zn indicates that the inhibition of GR in astrocyte lysates or purified yeast GR is irreversible, otherwise the EDTA would have chelated the Zn during the 20-min enzyme activity assay. While the precise mechanism by which Zn inhibits GR is unknown, the inhibition is direct and does not require second messengers, since we have shown that purified GR is inhibited effectively by Zn. Since the carbamoylation reaction by which carmustine inhibits GR is NADPH dependent [35], it is noteworthy that our data demonstrate that the inhibition of GR by Zn is also dependent on the availability of NADPH. In view of this similarity, we suggest that NADPH may reduce the disulfide bond at Cys-45 and Cys-50 in the active site [36], thereby allowing Zn to bind to the exposed sulfhydryl groups, causing an irreversible inactivation of the enzyme.
When astrocytes were pretreated with Zn, their capacity to detoxify H2O2 was greatly reduced, such that the half-life of H2O2 was almost doubled after incubation with 250 μM Zn. Inactivation of catalase activity via 3AT treatment, which removed a key cellular mechanism used to detoxify H2O2, further slowed the detoxification of H2O2 to the extent that the specific detoxification rate curve was shifted relative to the Zn concentration. This shift indicates that the effect of Zn was primarily on the GSH system. This conclusion is supported by the fact that the cells treated with either 250 μM Zn or 200 μM carmustine were equally impaired in their capacity to detoxify H2O2. At concentrations of 150 μM and above, at exposure durations of 6 h or more, Zn greatly increased the intracellular GSSG content, which is consistent with previous reports [21]. The fact that 200 μM carmustine did not greatly increase the intracellular GSSG content demonstrates that inhibition of GR by Zn is not the primary cause of the cellular GSSG accumulation. The present study also found that GSSG is exported from astrocytes following incubation with Zn. While we are the first to report the export of GSSG in response to Zn, cultured astrocytes in addition to other cells and tissues have been reported to export GSSG in response to prooxidants [37–40]. Such export is thought to overcome the shift in thiol balance produced by an accumulation of cellular GSSG. These observations, combined with the present data, suggest that Zn causes the production of oxidative stress in astrocytes via a mechanism that is independent of the inhibition of GR. It is notable that we observed an increase in cellular GSx content after 8 h incubation with Zn, indicating that the astrocytes may have been synthesizing additional GSH in order to restore their thiol balance. We found that Zn induced Rh123 fluorescence in astrocytes in a concentration-dependent manner, whereas no Rh123 fluorescence was observed in carmustine-treated cells. This difference further supports the notion that ROS generation in Zn-treated cells is separate from the inhibition of GR. There are several potential sources of Zn-induced ROS production including the autooxidation of sulfhydryl groups on cell membranes [41], dysregulation of the mitochondrial membrane potential [18], inhibition of thioredoxin reductase [42] and thiol oxidoreductase [43], and increased activity of NADPH oxidase [44]. Preincubation with either NAC or Trolox prevented the generation of intracellular ROS by Zn, as detected by Rh123 fluorescence (data not shown). The prevention of cell death following preincubation with NAC or Trolox indicates that oxidative mechanisms are responsible for the toxicity of Zn. The fact that NAC and Trolox prevented both the production of ROS and the cell death by Zn, yet only NAC prevented the inhibition of GR, provides additional evidence that the toxicity of Zn in astrocytes is not directly due to the inhibition of GR. In conclusion, the present results indicate that Zn is a highly effective inhibitor of GR activity in astrocytes, and that this inhibition impairs their capacity to detoxify H2O2. When astrocytes are subsequently exposed to prooxidants, which is likely to occur in pathological conditions, they will be less able to protect the brain from oxidation. Furthermore, pathological
G.M. Bishop et al. / Free Radical Biology & Medicine 42 (2007) 1222–1230
conditions are often associated with elevations in extracellular Zn, and we have found that elevated Zn leads to the production of intracellular ROS, oxidative stress, and cell death. We have shown that Zn, like carmustine, inhibits GR via an NADPHdependent mechanism, yet carmustine, unlike Zn, is not associated with the production of ROS, oxidative stress, or cell death. Taken together, these results suggest that the inhibition of GR by Zn is not sufficient to kill astrocytes, and that the toxicity of Zn is due instead to a separate mechanism which involves the production of ROS. Thus, antioxidant therapy may be efficacious in conditions such as ischemia, traumatic brain injury, and epileptiform activity, since all involve elevations in extracellular Zn.
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