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or copper ions
Journal of Inorganic Biochemistry 104 (2010) 1084–1090
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Journal of Inorganic Biochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o
Redox-active complexes formed during the interaction between glutathione and mercury and/or copper ions Margarita E. Aliaga a,⁎, Camilo López-Alarcón a, Germán Barriga b, Claudio Olea-Azar b, Hernán Speisky b,c a b c
Facultad de Química, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Santiago 6094411, Chile Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Sergio Livingstone 1007, Independencia, Santiago, Chile Nutrition and Food Technology Institute, University of Chile, Macul 5540, Macul, Santiago, Chile
a r t i c l e
i n f o
Article history: Received 15 January 2010 Received in revised form 15 June 2010 Accepted 16 June 2010 Available online 28 June 2010 Keywords: Superoxide radicals Hydrogen peroxide Reduced glutathione GSH-binding metals Cu(I)–glutathione complex Hg(II)–glutathione complex
a b s t r a c t Prompted by the recently reported capacity of the physiologically occurring Cu(I)–[glutathione]2 complex (Cu(I)–[GSH)]2) to reduce oxygen, the effect of various GSH-binding metals (Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ and Ni2+) on the superoxide-generating capacity of such complex was investigated. Amongst all tested metals, only Hg2+ was able to substantially affect the capacity of Cu(I)–[GSH]2 to generate superoxide. When Hg2+ and Cu(I)–[GSH]2 were mixed equimolarly, the superoxide formation, assessed through the cytochrome c reduction and dihydroethidium oxidation, was increased by over 50%. Such effect was totally inhibitable by SOD. Based on the reportedly higher affinity of Hg2+ for GSH and the observed ability of Hg2+ to lower the concentration of Cu(I)–[GSH]2 (spectroscopically assessed), we suggest that Hg2+ displaces Cu (I) from Cu(I)–[GSH]2, to release Cu(I) ions and form a Hg(II)–[GSH]2 complex. The latter species would account for the Hg2+-induced exacerbation of the superoxide production. In fact, the present study provides first time evidence that a preformed Hg(II)–[GSH]2 complex is able to concentration-dependently reduce oxygen. Such redox-activity was evidenced using cytochrome c and confirmed by EPR studies using DMPO (5,5-dimethyl-1-pyrroline N-oxide, a spin-trapping agent). Considering this novel ability of Hg(II)–[GSH]2 to generate superoxide, a further characterization of its redox-activity and its potential to affect superoxidesusceptible biological targets appears warranted. © 2010 Elsevier Inc. All rights reserved.
1. Introduction The interaction between some physiologically occurring metals and certain proteins may increase the structural stability and the ability of the latter to optimally accomplish their biological function [1,2]. These effects may, however, be no longer seen when the concentration of such metals largely exceeds their biologically optimal level [3]. On the other hand, in the case of heavy metals, biologically deleterious effects can be often seen even at relatively low concentrations given their ability to readily bind (non-specifically) to functionally relevant sulfhydryl and/or amino groups, and to displace metals that are endogenously bound to such type of sites [4,5]. Many metal-binding ligands can be found in mammalian cells. However, the reduced glutathione molecule (GSH; γL-glutamyl-L-cysteinyl-glycine), which occurs within cells in millimolar concentrations and accounts for over 90% of the total non-protein sulfhydryl groups [6], stems as a major metal-binding ligand [7]. Several studies have described the ability of GSH to bind metals through its cysteine-contained sulfhydryl group (SH). Amongst these, GSH has been shown to bind the physiologically occurring Cu and Zn
⁎ Corresponding author. Tel.: + 56 2 354 7126; fax: + 56 2 686 4744. E-mail address: [email protected] (M.E. Aliaga). 0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2010.06.006
metals [8,9] and also the potentially toxic heavy metals Cd, Pb, Hg, Al and Ni [10–18]. Regarding the former metals, their binding to GSH results in the formation of complexes which are believed to serve as carriers to metal-dependent proteins. The latter function has been mainly described for copper ions; as such this metal is readily complexed by GSH after entering the cell, for its subsequent transferring to methalotionein, a protein which is involved in its storage [19,20]. A consequence of the interaction between various metals ions and GSH is the formation of complexes. The latter has been considered to function, particularly in the case of potentially toxic metals, as a mechanism to sequester them protecting the cell from their otherwise harmful effects [21]. Most complexes formed between metals and GSH exhibit a stoichiometry of 1:2 [10,12]. In the case of copper, the ability of GSH molecules to initially reduce Cu(II) ions and then complex Cu(I) [19,20,22] leads to the formation of Cu(I)–[GSH]2 [23]. Studies conducted in cellular systems, in which human hepatoma cells (HAC) [19,20] and intestinal epithelial cell line (Caco-2) [24] were exposed to high copper concentrations, have shown that most of the metal ions are complexed with GSH molecules and found within the cells as Cu(I)– [GSH]2. In non-cellular systems, such complex has been characterized by means of 1H-NMR and EPR (electron paramagnetic resonance) [25]. Recent work conducted in an oxygen-containing solution, revealed that even at low micromolar concentrations, the Cu(I)–[GSH]2 complex is
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able to continuously react with molecular oxygen leading to the formation of superoxide radicals [26]. Taking into account the potential toxicological implications associated with the redoxactivity of Cu(I)–[GSH]2 complex, in the present study we addressed the effects that, as metals capable of binding GSH, Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ and Ni2+ could have on the capacity of the Cu(I)– [GSH]2 complex to generate superoxide radicals. 2. Experimental section 2.1. Materials Cupric chloride (CuCI2·2H2O), reduced glutathione (GSH), DMPO (5,5-dimethyl-1-pyrroline N-oxide), hydrogen peroxide, ferrous sulfate (FeSO4·7H2O), DMSO (dimethyl sulfoxide), cytochrome c (Cyt c; bovine heart), superoxide dismutase (SOD; EC 1.15.1.1 from bovine erythrocytes) and catalase (CAT; EC 1.11.1.6 from bovine liver) were all purchased from Sigma-Aldrich. Dihydroethidium (DHE) was purchased from Calbiochem. Solutions of Hg2+ and Pb2+ were prepared from nitrate salts (supplied from Merck Titrisol solutions). Solutions of Co2+, Cd2+, Zn2+, Al3+ and Ni2+ were prepared from chloride salts (supplied from Merck Titrisol solutions). All aqueous solutions were prepared in Chelex-100-treated buffer (20 mM; pH 7.4). 2.2. Preparation of metal–glutathione complexes The Cu(I)–[GSH]2 complex was prepared as previously described [26] by mixing CuCI2 and GSH in a 1:3 molar ratio, respectively. The Hg(II)– [GSH]2 and the Ni(II)–[GSH]2 complexes were prepared by direct mixing either Hg(NO3)2 or NiCl2 with GSH, in a 1:2 molar ratio, as described previously [8,12]. Whenever referring to a given concentration of such complexes, it should be understood that it reflects the concentration of metal used in its preparation. Unless indicated otherwise, all complexes were prepared and used always immediately after being prepared. 2.3. Cytochrome c reduction assay The superoxide-dependent reduction of Cyt c was assessed as described before [27], monitoring the increase in optical density (OD) at 550 nm in a 96-well plate using a Multi-Mode Microplate Reader (Synergy™ HT). In brief, Cyt c (50 μM) was added to wells containing a concentration 10 μM of different metals salts and a fixed concentration of Cu(I)–[GSH]2 (10 μM). Incubations were carried out at 30 °C and readings at 550 nm were obtained after 2 min. SOD (250 U/well) was used as control to imply superoxide-dependent effects. Results, in Figs. 1 (a) and 2(a) were expressed as percentage, over control done with Cu (I)–[GSH]2 complex alone, 100% corresponds to ΔOD550nm of such complex 10 μM. Results in Figs. 4 and 6 were expressed as ΔOD550nm (ODCyt c/Cu(I)–[GSH]2 − ODCyt c alone). 2.4. Dihydroethidium oxidation assay The formation of superoxide radicals was assessed as described by [28], employing the dihydroethidium (DHE) oxidation assay. DHE oxidation was monitored fluorimetrically in a 96-well plate using a Multi-Mode Microplate Reader (SynergyTM HT). Excitation and emission wavelengths were 470 nm and 590 nm, respectively. Freshly prepared DHE, dissolved in DMSO, was added (50 μM) to wells containing a concentration 10 μM of different metal salts and a fixed concentration of Cu(I)–[GSH]2 (10 μM). Incubations were carried out at 30 °C and readings of fluorescence were obtained after 30 min. When employed (as referred in the text), SOD was added to the wells at 250 U/ well. Results were expressed as percentage, where 100% corresponds to delta relative fluorescence units (ΔRFU = RFU DHE/Cu(I)–[GSH]2 − RFUDHE alone) generated by 10 μM of the Cu(I)–[GSH]2 complex. Controls, which
Fig. 1. Effect of addition of metal salt (a) on the reduction of Cyt c induced by the Cu(I)– [GSH]2 complex and (b) on the oxidation of DHE induced by the Cu(I)–[GSH]2 complex. In (a), Cyt c (50 μM) was added to solutions containing several metal salt (10 μM) plus a fixed concentration of Cu(I)–[GSH]2 (Cx; 10 μM). Results were expressed as described in Section 2.3. In (b), DHE (50 μM) was added to solutions containing several metal salt (10 μM) plus a fixed concentration of Cu(I)–[GSH]2 (Cx; 10 μM). Results were expressed as described in Section 2.4.
were carried out using only the metal salts (10 μM), had no reducing effect on Cyt c (not shown). 2.5. Quantification of the Cu(I)–[GSH]2 complex Cu(I)–[GSH]2 was assessed as described [23], taking advantage of its spectroscopic properties. The decrease in OD associated with its disappearance was monitored at 267 nm using an Unicam Heλios α spectrophotometer. The assay was initiated after addition of samples containing the Cu(I)–[GSH]2 complex (90 μM), incubated in presence or in absence of Hg2+ or Ni2+ (90 μM), to a cuvette containing Tris buffer (20 mM; pH 7.4). Results were obtained and expressed as optical density at 267 nm. Controls were carried out using GSH (180 μM); Hg2+ (90 μM); Ni2+ (90 μM) and two preformed complexes: Hg(II)–[GSH]2 and Ni(II)–[GSH]2 (90 μM). 2.6. Electron paramagnetic resonance (EPR) studies EPR signals associated with the adduct formed between DMPO and superoxide radical were assessed in solutions containing the Hg(II)–
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plate using a Multi-Mode Microplate Reader (Synergy™ HT). The assay was initiated after mixing 1 mL of acetaminophen (0.01 M), 0.6 mL of sulfuric acid (2 M) and 5 mL of a solution containing either the preformed Hg(II)–[GSH]2 complex (5–50 μM) or H2O2 (2–50 μM). The mixture was boiled during 40 min. After cooling, the solution was brought to a 10 mL volume with distilled water and the fluorescence measured. Results were expressed as micromolar concentration of H2O2 (Insert to Fig. 6). 2.8. Data expression and analysis Data points represent the means of at least 3 independent experiments, each experiment conducted in triplicate. The standard deviation of such data represented less than 10% of the means. Statistical significance between points was assessed using the Student's t test (differences at p b 0.05 were considered significant). GraphPad Prism 4 was used as statistical software. 3. Results and discussion
Fig. 2. Effect of the addition of increasing concentrations of mercury and nickel (a) on the reduction of Cyt c induced by the Cu(I)–[GSH]2 complex and (b) on the oxidation of DHE induced by the Cu(I)–[GSH]2 complex. In (a), Cyt c (50 μM) was added to solutions containing increasing concentration of Hg2+ and Ni2+ (2.5–40 μM) plus a fixed concentration of Cu(I)–[GSH]2 (Cx; 10 μM). Results were expressed as described in Section 2.3. In (b), DHE (50 μM) was added to solutions containing increasing concentration of Hg2+ (2.5–40 μM) and Ni2+ (10–40 μM) plus a fixed concentration of Cu(I)–[GSH]2 (Cx; 10 μM). Results were expressed as described in Section 2.4.
[GSH]2 complex (1.5 mM; prepared as in Section 2.2) in absence or presence of SOD (1000 U/mL). EPR spectra were obtained at X-band (9.5 GHz) on a Bruker ECS 106 EPR spectrometer equipped with rectangular cavity and 50 kHz field modulation at room temperature, under the following conditions: microwave frequency, 9.79 GHz; center field, 3480 G; modulation amplitude, 0.9. Simulations of the EPR spectra were made using the software Simphonia 1.25 version. The hyper-fine splitting constants were estimated to be accurate within 0.05 G [29]. 2.7. Acetaminophen oxidation assay Hydrogen peroxide (H2O2) was quantified as described [30], employing acetaminophen as hydrogen peroxide reacting agent. The fluorescence obtained (excitation and emission wavelengths were 298 nm and 333 nm, respectively), associated with the oxidation of acetaminophen in acidic medium, was monitored at 30 °C in a 96-well
The reduction of Cyt c, a highly sensitive superoxide-reducible probe [31], was used to evaluate the effect of the addition of various GSH-binding metals on the ability of the Cu(I)–[GSH]2 complex to generate superoxide radicals. Fig. 1(a) shows the results obtained after adding identical concentrations of the Cu(I)–[GSH]2 complex (10 μM) to solutions containing Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ or Ni2+ salts. For the metals: Co2+, Cd2+, Zn2+, Pb2+ or Al3+, their addition produced no significant effect on the reduction of Cyt c induced by the Cu(I)–[GSH]2 complex (Fig. 1(a)). After the addition of Hg2+ (10 μM), however, the magnitude of the reduction of Cyt c induced by such complex was increased by near 50%. In the case of Ni2+, contrasting with the effect of Hg2+, the reduction of Cyt c was decreased by approximately 25%. Noteworthy, the increase in Cyt c reduction induced by Hg2+ as well as the decrease in such parameter induced by Ni2+ were completely inhibited by SOD (250 U/well). This latter effect indicates that, despite being opposite, the mechanism(s) by which Hg2+ and Ni2+ induce their effects would depend on the free occurrence of superoxide radicals in the Cu(I)–[GSH]2added media. The results observed using Cyt c were also evaluated employing DHE as superoxide-sensitive probe [28,32]. As shown in Fig. 1(b), the mixture obtained after adding a 10 μM concentration of Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ or Ni2+ to the Cu(I)–[GSH]2 complex (10 μM), generated changes on its capacity to oxidize DHE that were almost identical to those seen previously using Cyt c. The sole exceptions were Hg2+ and Ni2+. Also, as previously described in the Cyt c reduction experiments (Fig. 1(a)), the addition of SOD to an equimolar mixture of Cu(I)–[GSH]2 and Hg2+ or Ni2+ totally prevented DHE oxidation (Fig. 1(b)). The lack of effect of Co2+, Cd2+, Zn2+, Pb2+ or Al3+, observed in Fig. 1(a) and (b), suggest that despite the presence of these known GSH-binding metals, the structure as well as the capacity of the Cu(I)–[GSH]2 complex to generate superoxide radicals remained unaltered. Most likely, the inability of Co2+, Cd2+, Zn2+, Pb2+ or Al3+ to bind GSH, when the tripeptide is already compromised in binding Cu(I), reflect their very low-affinity constants to bind GSH compared to Cu+ [8,33]. On the other hand, regarding the increment in superoxide production observed after addition of Hg2+ to the Cu(I)–[GSH]2 complex (10 μM) (Fig. 1(a) and (b)), it could be suggested that such effect reflect the ability of Hg2+ ions to displace the Cu(I) bound to GSH. In fact, the affinity constant of Hg2+ for GSH (log K = 42) [10,34] is greater than that of Cu(I) for GSH (log K = 39) [33]. Thus, an expected first consequence would be the removal of Cu(I) from Cu(I)–[GSH]2 complex by Hg2+ and its subsequent binding to the GSH molecules to form, the presumably more stable, Hg(II)–[GSH]2 complex (Rx. 1). The latter interpretation is in line with the previously reported
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[12,13] ability of Hg2+ ions to directly bind GSH forming the Hg(II)– [GSH]2 complex. CuðIÞ−½GSH2 + HgðIIÞ→HgðIIÞ−½GSH2 + CuðIÞ
ðRx:1Þ
In addition to the putative formation of the Hg(II)–[GSH]2 complex, the binding of Hg2+ to GSH would result, upon their displacement from the Cu(I)–[GSH]2 complex, in the generation of free Cu+ ions. The latter species could interact either with molecular oxygen (Rx. 2) [35] or with itself (Rx. 3) [36] leading, in both cases, to Cu2+ ions. •−
CuðIÞ + O2 →CuðIIÞ + O2
ðRx:2Þ
2CuðIÞ⇄CuðIIÞ + Cu
ðRx:3Þ
To asses if Cu2+ ions are generated by the interaction between Cu(I)– [GSH]2 complex and Hg(II), we conducted additional experiments employing EDTA as Cu2+-chelating agent [37]. In fact, when an equimolar mixture of Cu(I)–[GSH]2 complex and Hg(II) was added to an EDTA solution, an increase in the OD at 750 nm was observed (data not shown). Such absorption is characteristic of the formation of Cu(II)– EDTA complex. Control experiments, using Hg2+ or Cu2+ plus ascorbate (as a form of generating Cu+ ions) in the presence of EDTA results only in a minimal increase in the OD at 750 nm. Thus, taken together, the formation of Cu2+ ions and the Hg2+induced exacerbation of the superoxide production (presented in Fig 1) we postulate that the formation of Cu2+ would occur mainly through Rx. 2. On the other hand, assuming that the Cu(I)–[GSH]2 complex and free Cu+ ions have the same efficiency to reduce molecular oxygen, the sole generation of such ions in Rx. 1 would not suffice to explain over 50% increment in superoxide formation as evidenced through both superoxide assays (Cyt c reduction and DHE oxidation, Fig. 1(a) and (b)). Thus, we suggest that such increment imply that a redoxactive, presumably the Hg(II)–[GSH]2, complex has been formed. In the case of Ni2+, its decreasing effect of the initial capacity by the Cu (I)–[GSH]2 complex to generate superoxide cannot be explained in terms of a displacement of Cu(I) from the Cu(I)–[GSH]2 complex. Although Ni2+ has been reported to react directly with GSH, forming a Ni(II)–[GSH]2 complex [8], based on its relatively low-affinity constant for GSH, it is unlikely that Ni2+ could displace Cu(I) from Cu(I)–[GSH]2. However, since Ni2+ has been reported to simultaneously coordinate with Cu+ and the thiol moiety of cysteine [38,39], it cannot be discarded that upon their addition to the Cu(I)–[GSH]2 complex, the former ions form “mixed metal complexes”. Thus, to possibly explain the observed descend in superoxide-generating capacity associated to Ni2+, such purported “mixed metal complexes” would have to be assumed as having a redox-activity that is either nil or considerably lower than that of Cu(I)–[GSH]2. To gain further insights into the effects of Hg2+ and Ni2+ addition, we studied the effect of increasing concentrations of these metal ions on the production of superoxide (assessed through the Cyt c reduction and DHE oxidation assays) by the Cu(I)–[GSH]2 complex (Fig. 2(a) and (b)). At a 2.5 μM concentration of Hg2+ the percentages of Cyt c reduction (Fig. 2(a)) and DHE oxidation (Fig. 2(b)) were slightly lower (by near 15%) than control. Yet, greater concentrations of Hg2+ (5 and 10 μM) were associated, depending on the assay, with a 10– 25% and 50–65% increase in superoxide production, respectively. Higher concentrations of Hg2+ concentration-dependently lowered the capacity of Cu(I)–[GSH]2 (10 μM) to generate superoxide, to almost totally abolish it at 40 μM. If Hg2+ ions are assumed to equimolarly displace Cu(I) from its complex, it could be expected that from concentrations of Hg2+ greater than 10 μM, an excess of Hg2+ ions co-occur with the free Cu(I) ions. Since free Hg2+ ions have been shown to favor the autodismutation of superoxide anions [40], we
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suggest that the observed decline in Cyt c reduction and DHE oxidation result from a mercury-dependent accelerated autodismutation of such radicals. In the case of Ni2+, its addition to the Cu(I)– [GSH]2 complex in concentrations greater than 10 μM (and up to 40 μM) were associated with proportionally lower degrees of Cyt c reduction and DHE oxidation (Fig. 2(a) and (b)). Since no superoxidecatalyzing effect of Ni2+ has been reported, it is likely that the increasingly lower formation of superoxide could be the result of an increasing formation of “mixed metal complexes” with either nil or considerably lower redox-activity than that of Cu(I)–[GSH]2. Prompted by the over 50% increment in the capacity of the Cu(I)– [GSH]2 complex to generate superoxide induced by equimolar concentrations of Hg2+, (Fig. 1(a) and (b)), and taking advantage of the capacity of such complex to absorb at 265 nm [23], we assessed its possible disappearance upon the addition of Hg2+. As shown in the insert to Fig. 3(a), the Cu(I)–[GSH]2 complex (90 μM) absorbs at 267 nm with a comparatively minimal interference. A mixture of equimolar concentrations of Hg2+ and Cu(I)–[GSH]2 shows approximately 50% of the OD267nm depicted by the complex alone (Fig. 3(a)). Such optic density is three-fold higher than that of a preformed Hg (II)–[GSH]2 complex (90 μM). The fact that the summation of the OD of the preformed Hg(II)–[GSH]2 complex and that of the Cu(II) (as in Rx. 2) is identical to that obtained for the Hg2+ plus Cu(I)–[GSH]2 mixture, supports the previous interpretation that Hg2+ total displaces Cu(I) from the latter complex (as in Rx. 1). Fig. 3(b) (and its insert) depicts the results from evaluating whether the addition of Ni2+ to the Cu(I)–[GSH]2 complex also leads to the disappearance of the latter. As shown in the insert, a preformed Ni(II)–[GSH]2 complex (90 μM) did not absorb at 267 nm. Fig. 3(b) shows that the addition of an equimolar concentration of Ni2+ to the Cu(I)–[GSH]2 complex did not modify the OD267nm. This result strongly indicates, as suggested above through the comparison of the affinity constants, that Ni2+ is unable to displace Cu(I) from the Cu (I)–[GSH]2 complex. The same result, however, cannot be taken as evidence to discard the possibility that its addition led to the formation of “mixed metal complexes”. The addition of a three-fold larger concentration of Ni2+ respect to that of the Cu(I)–[GSH]2 complex led to an only slight decrease in the OD267nm. To evaluate the possibility that the Hg(II)–[GSH]2 complex formed after the interaction between Hg2+ and Cu(I)–[GSH]2 could also be redox-active (as suggested by results from Figs. 1 and 2), we evaluated the ability of a preformed Hg(II)–[GSH]2 to reduce Cyt c. As shown in Fig. 4, the Hg (II)–[GSH]2 complex (10 μM) was able to reduce Cyt c and such effect was totally prevented by SOD (250 U/well). Thus, the Cyt c reduction induced by the Hg(II)–[GSH]2 complex is clearly mediated by superoxide radicals. This first time reported redox-activity of Hg (II)–[GSH]2 was found to be approximately 40% that of Cu(I)–[GSH]2 (10 μM); the formation of the latter complex within cells has been established [19,23] and its superoxide-generating capacity postulated to be of potential biological and toxicological relevance [26]. Thus, addressing the occurrence of the Hg(II)–[GSH]2 might also be of interest. Unlike the Hg(II)–[GSH]2 complex, no redox-activity was detected for a preformed Ni(II)–[GSH]2 complex (10 μM). In order to complement the previously demonstrated redoxactivity of the Hg(II)–[GSH]2 complex toward molecular oxygen, EPR studies were conducted using DMPO as a spin-trap [41]. Fig. 5(a) depicts the EPR spectrum resulting from adding DMPO (100 mM) to a preformed Hg(II)–[GSH]2 complex (1.5 mM). The spectrum obtained for such mixture reveals the presence of a typical signal for the DMPO–OH adduct (Fig. 5(b)), suggesting that rather than superoxide, hydroxyl radicals [42–44] have been generated. It should be noted, however, that the formation of such adduct originates, most likely, from the decomposition of a DMPO–OOH adduct [42–44] formed previously from the interaction between DMPO and O•− 2 . Noteworthy, the signal observed in Fig. 5(a) was almost totally prevented in the presence of SOD (1000 U/mL), thus confirming the generation of
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Fig. 4. Reduction of Cyt c induced by different complexes, formed between metals and GSH. Time-dependence. Cyt c (50 μM) was added to solutions containing a concentration of 10 μM of the following preformed complexes: Ni(II)–[GSH]2 (△), Cu(I)–[GSH]2 (□) and Hg(II)–[GSH]2 (○). The closed symbols represent the complexes in the presence of SOD (250 U/well). Results were expressed as described in Section 2.3.
the complex increased from 4 to 10 μM; concentrations greater than 10 μM (and up to 50 μM) were associated with proportionally lower degrees of Cyt c reduction. For concentrations greater than 10 μM, Cyt c reduction started to decline. The latter is, presumably, because the greater flux of superoxide radicals expected to be generated by the higher concentrations of the Hg(II)–[GSH]2 complex favor the autodismutation of superoxide (Eq. (1)) over its reaction with Cyt c (Eq. (2)). A similar behavior was recently observed [46] when increasing concentrations of the Cu(I)–[GSH]2 complex was assessed through the oxidation of DHE. •− O2 VAutodismut: = k1 O•− 2 k1 = 2:4 × 105 M−1 s−1 (Zhao et al.) [28] VCyt Fig. 3. Effect of (a) Hg2+ addition and (b) Ni2+ addition to a Cu(I)–[GSH]2 complex on its spectroscopic. In (a), Hg2+ (90 μM) was added to a solution containing the Cu(I)– [GSH]2 complex (90 μM). Results were expressed as described in Section 2.5. Insert: Scan (optical density spectra) obtained for: the Cu(I)–[GSH]2 complex (90 μM) (□); the Cu(I)–[GSH]2 complex (90 μM) plus Hg2+ (90 μM) (■); a solution containing Cu(II) ions (90 μM) (△) and the Hg(II)–[GSH]2 complex (90 μM) (●). In (b), Ni2+ (90 and 270 μM) was added to a solution containing the Cu(I)–[GSH]2 complex (90 μM). Results were expressed as described in Section 2.5. Ni(II)–[GSH]2 (90 μM), Ni2+ (90 and 270 μM) were used as control. Insert: Scan (optical density spectra) obtained for: the Cu (I)–[GSH]2 complex (90 μM) (□); the Cu(I)–[GSH]2 complex (90 μM) plus Ni2+ (90 μM) (△) and the Ni(II)–[GSH]2 complex (90 μM) (■).
superoxide by the Hg(II)–[GSH]2 complex. Computer simulations of the EPR spectra are presented in Fig. 5(a), (b) and (c) (down) and were obtained using the hyper-fine splitting constants resulting from to analyze the experimental spectra. Such simulations, showed a characteristic hyper-fine pattern for the oxidation of DMPO/OH• (minor signal in Fig. 5(a) and (c)) [45], confirmed the presence of a DMPO–OH adduct (Fig. 5(a)) and the disappearance of signals of such adduct in presence of SOD (Fig. 5(c)). To further characterize the ability of a preformed Hg(II)–[GSH]2 complex to generate superoxide radicals, we evaluated the existence of a possible relationship between increasing concentrations of this complex and its capacity to reduce Cyt c. Fig. 6 shows an apparently biphasic behavior of the Hg(II)–[GSH]2 complex, as a function of its concentration. Cyt c reduction increased linearly as the concentration of
cRed:
= k2 O•− ½Cyt c 2
k2 = 2:6 × 105 M−1 s−1 (Butler et al.) [47]
ð1Þ
ð2Þ
The contention that higher concentrations of the Hg(II)–[GSH]2 complex are associated with a higher rate of production and subsequent dismutation of superoxide radicals, was also supported by the observation that (in the absence of Cyt c) increasing concentrations of such complex led to an increased production of hydrogen peroxide (insert to Fig. 6). In conclusion, the present study shows that amongst the seven (Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ and Ni2+) GSH-binding metals only Hg2+ affects (exacerbating) the ability of the Cu(I)– [GSH]2 complex to generate superoxide radicals. Based on the reportedly higher affinity of Hg2+ for GSH and on the observed ability of Hg2+ to lower the concentration of the Cu(I)–[GSH]2 complex, we suggest that Hg2+ displaces Cu(I) from such complex to form Hg(II)–[GSH]2. The present study provides, for first time, evidence that a preformed Hg(II)–[GSH]2 complex is concentration-dependently capable of reducing molecular oxygen. Such redox-activity was evidenced using Cyt c and DMPO as superoxide probe and spin-trapping agents, respectively. Considering the ability of a preformed Hg(II)–[GSH]2 complex to generate superoxide anions, a further characterization of its redox-activity and its potential to affect superoxide-susceptible biological targets appears warranted.
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Fig. 5. EPR spectra of DMPO-radical adducts formed from solutions containing a preformed Hg(II)–[GSH]2 complex and SOD-treated complex. (a) Spectrum of a solution containing the preformed Hg(II)–[GSH]2 complex (1.5 mM) (up) and simulation of the same spectrum (down). (b) Spectrum of DMPO–OH radical adduct, generated by mixing FeSO4 (1 mM) with H2O2 (1%) (Fenton's reaction) (up) and simulation of the same spectrum (down). (c) Spectrum obtained 1 h after the addition of SOD (1000 U/mL) to a solution containing the Hg(II)–[GSH]2 complex (arising from in a) (up) and simulation of the same spectrum (down).
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Fig. 6. Effect of increasing concentration of the Hg(II)–[GSH]2 complex on the Cyt c reduction. Cyt c (50 μM) was added to solutions containing increasing concentration of Hg(II)–[GSH]2 (2.5–50 μM) in the presence (●) or absence (o) of SOD (250 U/mL). Results were expressed as described in Section 2.3. Insert: Relationship between increasing concentrations of the Hg(II)–[GSH]2 complex (5–50 μM), in the presence (♦) or absence (◊) of CAT (50 U/mL), and the formation of hydrogen peroxide.
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Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o
Redox-active complexes formed during the interaction between glutathione and mercury and/or copper ions Margarita E. Aliaga a,⁎, Camilo López-Alarcón a, Germán Barriga b, Claudio Olea-Azar b, Hernán Speisky b,c a b c
Facultad de Química, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Santiago 6094411, Chile Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Sergio Livingstone 1007, Independencia, Santiago, Chile Nutrition and Food Technology Institute, University of Chile, Macul 5540, Macul, Santiago, Chile
a r t i c l e
i n f o
Article history: Received 15 January 2010 Received in revised form 15 June 2010 Accepted 16 June 2010 Available online 28 June 2010 Keywords: Superoxide radicals Hydrogen peroxide Reduced glutathione GSH-binding metals Cu(I)–glutathione complex Hg(II)–glutathione complex
a b s t r a c t Prompted by the recently reported capacity of the physiologically occurring Cu(I)–[glutathione]2 complex (Cu(I)–[GSH)]2) to reduce oxygen, the effect of various GSH-binding metals (Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ and Ni2+) on the superoxide-generating capacity of such complex was investigated. Amongst all tested metals, only Hg2+ was able to substantially affect the capacity of Cu(I)–[GSH]2 to generate superoxide. When Hg2+ and Cu(I)–[GSH]2 were mixed equimolarly, the superoxide formation, assessed through the cytochrome c reduction and dihydroethidium oxidation, was increased by over 50%. Such effect was totally inhibitable by SOD. Based on the reportedly higher affinity of Hg2+ for GSH and the observed ability of Hg2+ to lower the concentration of Cu(I)–[GSH]2 (spectroscopically assessed), we suggest that Hg2+ displaces Cu (I) from Cu(I)–[GSH]2, to release Cu(I) ions and form a Hg(II)–[GSH]2 complex. The latter species would account for the Hg2+-induced exacerbation of the superoxide production. In fact, the present study provides first time evidence that a preformed Hg(II)–[GSH]2 complex is able to concentration-dependently reduce oxygen. Such redox-activity was evidenced using cytochrome c and confirmed by EPR studies using DMPO (5,5-dimethyl-1-pyrroline N-oxide, a spin-trapping agent). Considering this novel ability of Hg(II)–[GSH]2 to generate superoxide, a further characterization of its redox-activity and its potential to affect superoxidesusceptible biological targets appears warranted. © 2010 Elsevier Inc. All rights reserved.
1. Introduction The interaction between some physiologically occurring metals and certain proteins may increase the structural stability and the ability of the latter to optimally accomplish their biological function [1,2]. These effects may, however, be no longer seen when the concentration of such metals largely exceeds their biologically optimal level [3]. On the other hand, in the case of heavy metals, biologically deleterious effects can be often seen even at relatively low concentrations given their ability to readily bind (non-specifically) to functionally relevant sulfhydryl and/or amino groups, and to displace metals that are endogenously bound to such type of sites [4,5]. Many metal-binding ligands can be found in mammalian cells. However, the reduced glutathione molecule (GSH; γL-glutamyl-L-cysteinyl-glycine), which occurs within cells in millimolar concentrations and accounts for over 90% of the total non-protein sulfhydryl groups [6], stems as a major metal-binding ligand [7]. Several studies have described the ability of GSH to bind metals through its cysteine-contained sulfhydryl group (SH). Amongst these, GSH has been shown to bind the physiologically occurring Cu and Zn
⁎ Corresponding author. Tel.: + 56 2 354 7126; fax: + 56 2 686 4744. E-mail address: [email protected] (M.E. Aliaga). 0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2010.06.006
metals [8,9] and also the potentially toxic heavy metals Cd, Pb, Hg, Al and Ni [10–18]. Regarding the former metals, their binding to GSH results in the formation of complexes which are believed to serve as carriers to metal-dependent proteins. The latter function has been mainly described for copper ions; as such this metal is readily complexed by GSH after entering the cell, for its subsequent transferring to methalotionein, a protein which is involved in its storage [19,20]. A consequence of the interaction between various metals ions and GSH is the formation of complexes. The latter has been considered to function, particularly in the case of potentially toxic metals, as a mechanism to sequester them protecting the cell from their otherwise harmful effects [21]. Most complexes formed between metals and GSH exhibit a stoichiometry of 1:2 [10,12]. In the case of copper, the ability of GSH molecules to initially reduce Cu(II) ions and then complex Cu(I) [19,20,22] leads to the formation of Cu(I)–[GSH]2 [23]. Studies conducted in cellular systems, in which human hepatoma cells (HAC) [19,20] and intestinal epithelial cell line (Caco-2) [24] were exposed to high copper concentrations, have shown that most of the metal ions are complexed with GSH molecules and found within the cells as Cu(I)– [GSH]2. In non-cellular systems, such complex has been characterized by means of 1H-NMR and EPR (electron paramagnetic resonance) [25]. Recent work conducted in an oxygen-containing solution, revealed that even at low micromolar concentrations, the Cu(I)–[GSH]2 complex is
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able to continuously react with molecular oxygen leading to the formation of superoxide radicals [26]. Taking into account the potential toxicological implications associated with the redoxactivity of Cu(I)–[GSH]2 complex, in the present study we addressed the effects that, as metals capable of binding GSH, Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ and Ni2+ could have on the capacity of the Cu(I)– [GSH]2 complex to generate superoxide radicals. 2. Experimental section 2.1. Materials Cupric chloride (CuCI2·2H2O), reduced glutathione (GSH), DMPO (5,5-dimethyl-1-pyrroline N-oxide), hydrogen peroxide, ferrous sulfate (FeSO4·7H2O), DMSO (dimethyl sulfoxide), cytochrome c (Cyt c; bovine heart), superoxide dismutase (SOD; EC 1.15.1.1 from bovine erythrocytes) and catalase (CAT; EC 1.11.1.6 from bovine liver) were all purchased from Sigma-Aldrich. Dihydroethidium (DHE) was purchased from Calbiochem. Solutions of Hg2+ and Pb2+ were prepared from nitrate salts (supplied from Merck Titrisol solutions). Solutions of Co2+, Cd2+, Zn2+, Al3+ and Ni2+ were prepared from chloride salts (supplied from Merck Titrisol solutions). All aqueous solutions were prepared in Chelex-100-treated buffer (20 mM; pH 7.4). 2.2. Preparation of metal–glutathione complexes The Cu(I)–[GSH]2 complex was prepared as previously described [26] by mixing CuCI2 and GSH in a 1:3 molar ratio, respectively. The Hg(II)– [GSH]2 and the Ni(II)–[GSH]2 complexes were prepared by direct mixing either Hg(NO3)2 or NiCl2 with GSH, in a 1:2 molar ratio, as described previously [8,12]. Whenever referring to a given concentration of such complexes, it should be understood that it reflects the concentration of metal used in its preparation. Unless indicated otherwise, all complexes were prepared and used always immediately after being prepared. 2.3. Cytochrome c reduction assay The superoxide-dependent reduction of Cyt c was assessed as described before [27], monitoring the increase in optical density (OD) at 550 nm in a 96-well plate using a Multi-Mode Microplate Reader (Synergy™ HT). In brief, Cyt c (50 μM) was added to wells containing a concentration 10 μM of different metals salts and a fixed concentration of Cu(I)–[GSH]2 (10 μM). Incubations were carried out at 30 °C and readings at 550 nm were obtained after 2 min. SOD (250 U/well) was used as control to imply superoxide-dependent effects. Results, in Figs. 1 (a) and 2(a) were expressed as percentage, over control done with Cu (I)–[GSH]2 complex alone, 100% corresponds to ΔOD550nm of such complex 10 μM. Results in Figs. 4 and 6 were expressed as ΔOD550nm (ODCyt c/Cu(I)–[GSH]2 − ODCyt c alone). 2.4. Dihydroethidium oxidation assay The formation of superoxide radicals was assessed as described by [28], employing the dihydroethidium (DHE) oxidation assay. DHE oxidation was monitored fluorimetrically in a 96-well plate using a Multi-Mode Microplate Reader (SynergyTM HT). Excitation and emission wavelengths were 470 nm and 590 nm, respectively. Freshly prepared DHE, dissolved in DMSO, was added (50 μM) to wells containing a concentration 10 μM of different metal salts and a fixed concentration of Cu(I)–[GSH]2 (10 μM). Incubations were carried out at 30 °C and readings of fluorescence were obtained after 30 min. When employed (as referred in the text), SOD was added to the wells at 250 U/ well. Results were expressed as percentage, where 100% corresponds to delta relative fluorescence units (ΔRFU = RFU DHE/Cu(I)–[GSH]2 − RFUDHE alone) generated by 10 μM of the Cu(I)–[GSH]2 complex. Controls, which
Fig. 1. Effect of addition of metal salt (a) on the reduction of Cyt c induced by the Cu(I)– [GSH]2 complex and (b) on the oxidation of DHE induced by the Cu(I)–[GSH]2 complex. In (a), Cyt c (50 μM) was added to solutions containing several metal salt (10 μM) plus a fixed concentration of Cu(I)–[GSH]2 (Cx; 10 μM). Results were expressed as described in Section 2.3. In (b), DHE (50 μM) was added to solutions containing several metal salt (10 μM) plus a fixed concentration of Cu(I)–[GSH]2 (Cx; 10 μM). Results were expressed as described in Section 2.4.
were carried out using only the metal salts (10 μM), had no reducing effect on Cyt c (not shown). 2.5. Quantification of the Cu(I)–[GSH]2 complex Cu(I)–[GSH]2 was assessed as described [23], taking advantage of its spectroscopic properties. The decrease in OD associated with its disappearance was monitored at 267 nm using an Unicam Heλios α spectrophotometer. The assay was initiated after addition of samples containing the Cu(I)–[GSH]2 complex (90 μM), incubated in presence or in absence of Hg2+ or Ni2+ (90 μM), to a cuvette containing Tris buffer (20 mM; pH 7.4). Results were obtained and expressed as optical density at 267 nm. Controls were carried out using GSH (180 μM); Hg2+ (90 μM); Ni2+ (90 μM) and two preformed complexes: Hg(II)–[GSH]2 and Ni(II)–[GSH]2 (90 μM). 2.6. Electron paramagnetic resonance (EPR) studies EPR signals associated with the adduct formed between DMPO and superoxide radical were assessed in solutions containing the Hg(II)–
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plate using a Multi-Mode Microplate Reader (Synergy™ HT). The assay was initiated after mixing 1 mL of acetaminophen (0.01 M), 0.6 mL of sulfuric acid (2 M) and 5 mL of a solution containing either the preformed Hg(II)–[GSH]2 complex (5–50 μM) or H2O2 (2–50 μM). The mixture was boiled during 40 min. After cooling, the solution was brought to a 10 mL volume with distilled water and the fluorescence measured. Results were expressed as micromolar concentration of H2O2 (Insert to Fig. 6). 2.8. Data expression and analysis Data points represent the means of at least 3 independent experiments, each experiment conducted in triplicate. The standard deviation of such data represented less than 10% of the means. Statistical significance between points was assessed using the Student's t test (differences at p b 0.05 were considered significant). GraphPad Prism 4 was used as statistical software. 3. Results and discussion
Fig. 2. Effect of the addition of increasing concentrations of mercury and nickel (a) on the reduction of Cyt c induced by the Cu(I)–[GSH]2 complex and (b) on the oxidation of DHE induced by the Cu(I)–[GSH]2 complex. In (a), Cyt c (50 μM) was added to solutions containing increasing concentration of Hg2+ and Ni2+ (2.5–40 μM) plus a fixed concentration of Cu(I)–[GSH]2 (Cx; 10 μM). Results were expressed as described in Section 2.3. In (b), DHE (50 μM) was added to solutions containing increasing concentration of Hg2+ (2.5–40 μM) and Ni2+ (10–40 μM) plus a fixed concentration of Cu(I)–[GSH]2 (Cx; 10 μM). Results were expressed as described in Section 2.4.
[GSH]2 complex (1.5 mM; prepared as in Section 2.2) in absence or presence of SOD (1000 U/mL). EPR spectra were obtained at X-band (9.5 GHz) on a Bruker ECS 106 EPR spectrometer equipped with rectangular cavity and 50 kHz field modulation at room temperature, under the following conditions: microwave frequency, 9.79 GHz; center field, 3480 G; modulation amplitude, 0.9. Simulations of the EPR spectra were made using the software Simphonia 1.25 version. The hyper-fine splitting constants were estimated to be accurate within 0.05 G [29]. 2.7. Acetaminophen oxidation assay Hydrogen peroxide (H2O2) was quantified as described [30], employing acetaminophen as hydrogen peroxide reacting agent. The fluorescence obtained (excitation and emission wavelengths were 298 nm and 333 nm, respectively), associated with the oxidation of acetaminophen in acidic medium, was monitored at 30 °C in a 96-well
The reduction of Cyt c, a highly sensitive superoxide-reducible probe [31], was used to evaluate the effect of the addition of various GSH-binding metals on the ability of the Cu(I)–[GSH]2 complex to generate superoxide radicals. Fig. 1(a) shows the results obtained after adding identical concentrations of the Cu(I)–[GSH]2 complex (10 μM) to solutions containing Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ or Ni2+ salts. For the metals: Co2+, Cd2+, Zn2+, Pb2+ or Al3+, their addition produced no significant effect on the reduction of Cyt c induced by the Cu(I)–[GSH]2 complex (Fig. 1(a)). After the addition of Hg2+ (10 μM), however, the magnitude of the reduction of Cyt c induced by such complex was increased by near 50%. In the case of Ni2+, contrasting with the effect of Hg2+, the reduction of Cyt c was decreased by approximately 25%. Noteworthy, the increase in Cyt c reduction induced by Hg2+ as well as the decrease in such parameter induced by Ni2+ were completely inhibited by SOD (250 U/well). This latter effect indicates that, despite being opposite, the mechanism(s) by which Hg2+ and Ni2+ induce their effects would depend on the free occurrence of superoxide radicals in the Cu(I)–[GSH]2added media. The results observed using Cyt c were also evaluated employing DHE as superoxide-sensitive probe [28,32]. As shown in Fig. 1(b), the mixture obtained after adding a 10 μM concentration of Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ or Ni2+ to the Cu(I)–[GSH]2 complex (10 μM), generated changes on its capacity to oxidize DHE that were almost identical to those seen previously using Cyt c. The sole exceptions were Hg2+ and Ni2+. Also, as previously described in the Cyt c reduction experiments (Fig. 1(a)), the addition of SOD to an equimolar mixture of Cu(I)–[GSH]2 and Hg2+ or Ni2+ totally prevented DHE oxidation (Fig. 1(b)). The lack of effect of Co2+, Cd2+, Zn2+, Pb2+ or Al3+, observed in Fig. 1(a) and (b), suggest that despite the presence of these known GSH-binding metals, the structure as well as the capacity of the Cu(I)–[GSH]2 complex to generate superoxide radicals remained unaltered. Most likely, the inability of Co2+, Cd2+, Zn2+, Pb2+ or Al3+ to bind GSH, when the tripeptide is already compromised in binding Cu(I), reflect their very low-affinity constants to bind GSH compared to Cu+ [8,33]. On the other hand, regarding the increment in superoxide production observed after addition of Hg2+ to the Cu(I)–[GSH]2 complex (10 μM) (Fig. 1(a) and (b)), it could be suggested that such effect reflect the ability of Hg2+ ions to displace the Cu(I) bound to GSH. In fact, the affinity constant of Hg2+ for GSH (log K = 42) [10,34] is greater than that of Cu(I) for GSH (log K = 39) [33]. Thus, an expected first consequence would be the removal of Cu(I) from Cu(I)–[GSH]2 complex by Hg2+ and its subsequent binding to the GSH molecules to form, the presumably more stable, Hg(II)–[GSH]2 complex (Rx. 1). The latter interpretation is in line with the previously reported
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[12,13] ability of Hg2+ ions to directly bind GSH forming the Hg(II)– [GSH]2 complex. CuðIÞ−½GSH2 + HgðIIÞ→HgðIIÞ−½GSH2 + CuðIÞ
ðRx:1Þ
In addition to the putative formation of the Hg(II)–[GSH]2 complex, the binding of Hg2+ to GSH would result, upon their displacement from the Cu(I)–[GSH]2 complex, in the generation of free Cu+ ions. The latter species could interact either with molecular oxygen (Rx. 2) [35] or with itself (Rx. 3) [36] leading, in both cases, to Cu2+ ions. •−
CuðIÞ + O2 →CuðIIÞ + O2
ðRx:2Þ
2CuðIÞ⇄CuðIIÞ + Cu
ðRx:3Þ
To asses if Cu2+ ions are generated by the interaction between Cu(I)– [GSH]2 complex and Hg(II), we conducted additional experiments employing EDTA as Cu2+-chelating agent [37]. In fact, when an equimolar mixture of Cu(I)–[GSH]2 complex and Hg(II) was added to an EDTA solution, an increase in the OD at 750 nm was observed (data not shown). Such absorption is characteristic of the formation of Cu(II)– EDTA complex. Control experiments, using Hg2+ or Cu2+ plus ascorbate (as a form of generating Cu+ ions) in the presence of EDTA results only in a minimal increase in the OD at 750 nm. Thus, taken together, the formation of Cu2+ ions and the Hg2+induced exacerbation of the superoxide production (presented in Fig 1) we postulate that the formation of Cu2+ would occur mainly through Rx. 2. On the other hand, assuming that the Cu(I)–[GSH]2 complex and free Cu+ ions have the same efficiency to reduce molecular oxygen, the sole generation of such ions in Rx. 1 would not suffice to explain over 50% increment in superoxide formation as evidenced through both superoxide assays (Cyt c reduction and DHE oxidation, Fig. 1(a) and (b)). Thus, we suggest that such increment imply that a redoxactive, presumably the Hg(II)–[GSH]2, complex has been formed. In the case of Ni2+, its decreasing effect of the initial capacity by the Cu (I)–[GSH]2 complex to generate superoxide cannot be explained in terms of a displacement of Cu(I) from the Cu(I)–[GSH]2 complex. Although Ni2+ has been reported to react directly with GSH, forming a Ni(II)–[GSH]2 complex [8], based on its relatively low-affinity constant for GSH, it is unlikely that Ni2+ could displace Cu(I) from Cu(I)–[GSH]2. However, since Ni2+ has been reported to simultaneously coordinate with Cu+ and the thiol moiety of cysteine [38,39], it cannot be discarded that upon their addition to the Cu(I)–[GSH]2 complex, the former ions form “mixed metal complexes”. Thus, to possibly explain the observed descend in superoxide-generating capacity associated to Ni2+, such purported “mixed metal complexes” would have to be assumed as having a redox-activity that is either nil or considerably lower than that of Cu(I)–[GSH]2. To gain further insights into the effects of Hg2+ and Ni2+ addition, we studied the effect of increasing concentrations of these metal ions on the production of superoxide (assessed through the Cyt c reduction and DHE oxidation assays) by the Cu(I)–[GSH]2 complex (Fig. 2(a) and (b)). At a 2.5 μM concentration of Hg2+ the percentages of Cyt c reduction (Fig. 2(a)) and DHE oxidation (Fig. 2(b)) were slightly lower (by near 15%) than control. Yet, greater concentrations of Hg2+ (5 and 10 μM) were associated, depending on the assay, with a 10– 25% and 50–65% increase in superoxide production, respectively. Higher concentrations of Hg2+ concentration-dependently lowered the capacity of Cu(I)–[GSH]2 (10 μM) to generate superoxide, to almost totally abolish it at 40 μM. If Hg2+ ions are assumed to equimolarly displace Cu(I) from its complex, it could be expected that from concentrations of Hg2+ greater than 10 μM, an excess of Hg2+ ions co-occur with the free Cu(I) ions. Since free Hg2+ ions have been shown to favor the autodismutation of superoxide anions [40], we
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suggest that the observed decline in Cyt c reduction and DHE oxidation result from a mercury-dependent accelerated autodismutation of such radicals. In the case of Ni2+, its addition to the Cu(I)– [GSH]2 complex in concentrations greater than 10 μM (and up to 40 μM) were associated with proportionally lower degrees of Cyt c reduction and DHE oxidation (Fig. 2(a) and (b)). Since no superoxidecatalyzing effect of Ni2+ has been reported, it is likely that the increasingly lower formation of superoxide could be the result of an increasing formation of “mixed metal complexes” with either nil or considerably lower redox-activity than that of Cu(I)–[GSH]2. Prompted by the over 50% increment in the capacity of the Cu(I)– [GSH]2 complex to generate superoxide induced by equimolar concentrations of Hg2+, (Fig. 1(a) and (b)), and taking advantage of the capacity of such complex to absorb at 265 nm [23], we assessed its possible disappearance upon the addition of Hg2+. As shown in the insert to Fig. 3(a), the Cu(I)–[GSH]2 complex (90 μM) absorbs at 267 nm with a comparatively minimal interference. A mixture of equimolar concentrations of Hg2+ and Cu(I)–[GSH]2 shows approximately 50% of the OD267nm depicted by the complex alone (Fig. 3(a)). Such optic density is three-fold higher than that of a preformed Hg (II)–[GSH]2 complex (90 μM). The fact that the summation of the OD of the preformed Hg(II)–[GSH]2 complex and that of the Cu(II) (as in Rx. 2) is identical to that obtained for the Hg2+ plus Cu(I)–[GSH]2 mixture, supports the previous interpretation that Hg2+ total displaces Cu(I) from the latter complex (as in Rx. 1). Fig. 3(b) (and its insert) depicts the results from evaluating whether the addition of Ni2+ to the Cu(I)–[GSH]2 complex also leads to the disappearance of the latter. As shown in the insert, a preformed Ni(II)–[GSH]2 complex (90 μM) did not absorb at 267 nm. Fig. 3(b) shows that the addition of an equimolar concentration of Ni2+ to the Cu(I)–[GSH]2 complex did not modify the OD267nm. This result strongly indicates, as suggested above through the comparison of the affinity constants, that Ni2+ is unable to displace Cu(I) from the Cu (I)–[GSH]2 complex. The same result, however, cannot be taken as evidence to discard the possibility that its addition led to the formation of “mixed metal complexes”. The addition of a three-fold larger concentration of Ni2+ respect to that of the Cu(I)–[GSH]2 complex led to an only slight decrease in the OD267nm. To evaluate the possibility that the Hg(II)–[GSH]2 complex formed after the interaction between Hg2+ and Cu(I)–[GSH]2 could also be redox-active (as suggested by results from Figs. 1 and 2), we evaluated the ability of a preformed Hg(II)–[GSH]2 to reduce Cyt c. As shown in Fig. 4, the Hg (II)–[GSH]2 complex (10 μM) was able to reduce Cyt c and such effect was totally prevented by SOD (250 U/well). Thus, the Cyt c reduction induced by the Hg(II)–[GSH]2 complex is clearly mediated by superoxide radicals. This first time reported redox-activity of Hg (II)–[GSH]2 was found to be approximately 40% that of Cu(I)–[GSH]2 (10 μM); the formation of the latter complex within cells has been established [19,23] and its superoxide-generating capacity postulated to be of potential biological and toxicological relevance [26]. Thus, addressing the occurrence of the Hg(II)–[GSH]2 might also be of interest. Unlike the Hg(II)–[GSH]2 complex, no redox-activity was detected for a preformed Ni(II)–[GSH]2 complex (10 μM). In order to complement the previously demonstrated redoxactivity of the Hg(II)–[GSH]2 complex toward molecular oxygen, EPR studies were conducted using DMPO as a spin-trap [41]. Fig. 5(a) depicts the EPR spectrum resulting from adding DMPO (100 mM) to a preformed Hg(II)–[GSH]2 complex (1.5 mM). The spectrum obtained for such mixture reveals the presence of a typical signal for the DMPO–OH adduct (Fig. 5(b)), suggesting that rather than superoxide, hydroxyl radicals [42–44] have been generated. It should be noted, however, that the formation of such adduct originates, most likely, from the decomposition of a DMPO–OOH adduct [42–44] formed previously from the interaction between DMPO and O•− 2 . Noteworthy, the signal observed in Fig. 5(a) was almost totally prevented in the presence of SOD (1000 U/mL), thus confirming the generation of
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Fig. 4. Reduction of Cyt c induced by different complexes, formed between metals and GSH. Time-dependence. Cyt c (50 μM) was added to solutions containing a concentration of 10 μM of the following preformed complexes: Ni(II)–[GSH]2 (△), Cu(I)–[GSH]2 (□) and Hg(II)–[GSH]2 (○). The closed symbols represent the complexes in the presence of SOD (250 U/well). Results were expressed as described in Section 2.3.
the complex increased from 4 to 10 μM; concentrations greater than 10 μM (and up to 50 μM) were associated with proportionally lower degrees of Cyt c reduction. For concentrations greater than 10 μM, Cyt c reduction started to decline. The latter is, presumably, because the greater flux of superoxide radicals expected to be generated by the higher concentrations of the Hg(II)–[GSH]2 complex favor the autodismutation of superoxide (Eq. (1)) over its reaction with Cyt c (Eq. (2)). A similar behavior was recently observed [46] when increasing concentrations of the Cu(I)–[GSH]2 complex was assessed through the oxidation of DHE. •− O2 VAutodismut: = k1 O•− 2 k1 = 2:4 × 105 M−1 s−1 (Zhao et al.) [28] VCyt Fig. 3. Effect of (a) Hg2+ addition and (b) Ni2+ addition to a Cu(I)–[GSH]2 complex on its spectroscopic. In (a), Hg2+ (90 μM) was added to a solution containing the Cu(I)– [GSH]2 complex (90 μM). Results were expressed as described in Section 2.5. Insert: Scan (optical density spectra) obtained for: the Cu(I)–[GSH]2 complex (90 μM) (□); the Cu(I)–[GSH]2 complex (90 μM) plus Hg2+ (90 μM) (■); a solution containing Cu(II) ions (90 μM) (△) and the Hg(II)–[GSH]2 complex (90 μM) (●). In (b), Ni2+ (90 and 270 μM) was added to a solution containing the Cu(I)–[GSH]2 complex (90 μM). Results were expressed as described in Section 2.5. Ni(II)–[GSH]2 (90 μM), Ni2+ (90 and 270 μM) were used as control. Insert: Scan (optical density spectra) obtained for: the Cu (I)–[GSH]2 complex (90 μM) (□); the Cu(I)–[GSH]2 complex (90 μM) plus Ni2+ (90 μM) (△) and the Ni(II)–[GSH]2 complex (90 μM) (■).
superoxide by the Hg(II)–[GSH]2 complex. Computer simulations of the EPR spectra are presented in Fig. 5(a), (b) and (c) (down) and were obtained using the hyper-fine splitting constants resulting from to analyze the experimental spectra. Such simulations, showed a characteristic hyper-fine pattern for the oxidation of DMPO/OH• (minor signal in Fig. 5(a) and (c)) [45], confirmed the presence of a DMPO–OH adduct (Fig. 5(a)) and the disappearance of signals of such adduct in presence of SOD (Fig. 5(c)). To further characterize the ability of a preformed Hg(II)–[GSH]2 complex to generate superoxide radicals, we evaluated the existence of a possible relationship between increasing concentrations of this complex and its capacity to reduce Cyt c. Fig. 6 shows an apparently biphasic behavior of the Hg(II)–[GSH]2 complex, as a function of its concentration. Cyt c reduction increased linearly as the concentration of
cRed:
= k2 O•− ½Cyt c 2
k2 = 2:6 × 105 M−1 s−1 (Butler et al.) [47]
ð1Þ
ð2Þ
The contention that higher concentrations of the Hg(II)–[GSH]2 complex are associated with a higher rate of production and subsequent dismutation of superoxide radicals, was also supported by the observation that (in the absence of Cyt c) increasing concentrations of such complex led to an increased production of hydrogen peroxide (insert to Fig. 6). In conclusion, the present study shows that amongst the seven (Co2+, Cd2+, Zn2+, Pb2+, Al3+, Hg2+ and Ni2+) GSH-binding metals only Hg2+ affects (exacerbating) the ability of the Cu(I)– [GSH]2 complex to generate superoxide radicals. Based on the reportedly higher affinity of Hg2+ for GSH and on the observed ability of Hg2+ to lower the concentration of the Cu(I)–[GSH]2 complex, we suggest that Hg2+ displaces Cu(I) from such complex to form Hg(II)–[GSH]2. The present study provides, for first time, evidence that a preformed Hg(II)–[GSH]2 complex is concentration-dependently capable of reducing molecular oxygen. Such redox-activity was evidenced using Cyt c and DMPO as superoxide probe and spin-trapping agents, respectively. Considering the ability of a preformed Hg(II)–[GSH]2 complex to generate superoxide anions, a further characterization of its redox-activity and its potential to affect superoxide-susceptible biological targets appears warranted.
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Fig. 5. EPR spectra of DMPO-radical adducts formed from solutions containing a preformed Hg(II)–[GSH]2 complex and SOD-treated complex. (a) Spectrum of a solution containing the preformed Hg(II)–[GSH]2 complex (1.5 mM) (up) and simulation of the same spectrum (down). (b) Spectrum of DMPO–OH radical adduct, generated by mixing FeSO4 (1 mM) with H2O2 (1%) (Fenton's reaction) (up) and simulation of the same spectrum (down). (c) Spectrum obtained 1 h after the addition of SOD (1000 U/mL) to a solution containing the Hg(II)–[GSH]2 complex (arising from in a) (up) and simulation of the same spectrum (down).
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Fig. 6. Effect of increasing concentration of the Hg(II)–[GSH]2 complex on the Cyt c reduction. Cyt c (50 μM) was added to solutions containing increasing concentration of Hg(II)–[GSH]2 (2.5–50 μM) in the presence (●) or absence (o) of SOD (250 U/mL). Results were expressed as described in Section 2.3. Insert: Relationship between increasing concentrations of the Hg(II)–[GSH]2 complex (5–50 μM), in the presence (♦) or absence (◊) of CAT (50 U/mL), and the formation of hydrogen peroxide.
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