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The pro-oxidant role of protein SH groups of hemoglobin in rat erythrocytes exposed to menadione
Chemico-Biological Interactions 139 (2002) 97 – 114
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The pro-oxidant role of protein SH groups of hemoglobin in rat erythrocytes exposed to menadione Lorenzo Lusini, Ranieri Rossi, Daniela Giustarini, Paolo Di Simplicio * Department of Neuroscience, Pharmacology Unit, Uni6ersity of Siena, Via A. Moro 4, 53100 Siena, Italy Received 31 July 2001; received in revised form 4 September 2001; accepted 5 November 2001
Abstract Menadione is selectively toxic to erythrocytes. Although GSH is considered a primary target of menadione, intraerythrocyte thiolic alterations consequent to menadione exposure are only partially known. In this study alterations of GSH and protein thiols (PSH) and their relationship with methemoglobin formation were investigated in human and rat red blood cells (RBC) exposed to menadione. In both erythrocyte types, menadione caused a marked increase in methemoglobin associated with GSH depletion and increased oxygen consumption. However, in human RBC, GSH formed a conjugate with menadione, whereas, in rat RBC it was converted to GSSG, concomitantly with a loss of protein thiols (corresponding to menadione arylation), and an increase in glutathione– protein mixed disulfides (GS– SP). Such differences were related to the presence of highly reactive cysteines, which characterize rat hemoglobin (cys b125). In spite of the greater thiol oxidation in rat than in human RBC, methemoglobin formation and the rate of oxygen consumption elicited by menadione in both species were rather similar. Moreover, in repeated experiments under N2 or CO-blocked heme, it was found that menadione conjugation (arylation) in both species was not dependent on the presence of oxygen or the status of heme. Therefore, we assumed that GSH Abbre6iations: CO, carbon monoxide; CO-Hb, carboxy hemoglobin; DTNB, 5,5%-dithiobis (2-nitrobenzoic acid); f-PSH, fast reacting protein-SH groups; GSH, glutathione; GSSG, glutathione disulfide; GS–SP, glutathione–protein mixed disulfides; met-Hb, methemoglobin; NEM, N-ethylmaleimide; NPSH, non-protein SH groups; oxy-Hb, oxy-hemoglobin; PSH, protein-SH groups; RBC, red blood cells; ROS, reactive oxygen species; TCA, trichloroacetic acid. * Corresponding author. Tel.: + 39-057723-4097; fax: + 39-057723-4098. E-mail address: [email protected] (P.Di. Simplicio). 0009-2797/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 1 ) 0 0 2 9 6 - 4
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(human RBC) and protein (rat RBC) arylation was equally responsible for increased oxygen consumption and Hb oxidation. Moreover, thiol oxidation of rat RBC was strictly related to methemoglobin formation. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Menadione; Rat hemoglobin; Glutathione; Redox cycling; Protein arylation; Oxidative stress
1. Introduction Menadione, a widely studied quinone, is an electrophilic agent utilized either as an experimental oxidant [1– 4] or for various clinical purposes to prevent vitamin K deficiencies, myopathies, or used to treat malaria [5– 8]. Menadione or other quinones are also used to treat neoplastic diseases [9–12]. For some time much attention has been focused on cellular targets and on the number of toxic mechanisms evoked by quinones, such as redox cycling, arylation, intercalation, protein crosslinks and disruption of mitochondrial electron transport [13–16]. Menadione is metabolized by redox and arylation reactions. One-electron reduction, mediated by flavoenzymes [17] or by interaction with oxy-Hb [18,19], leads to the production of the highly reactive semiquinone form, which autoxidizes producing redox cycling reactions with oxygen consumption, sustained formation of reactive oxygen species (ROS) and a rapid drop in levels of reducing agents. This would cause various effects, such as, ATP depletion, Ca2 + entry, enzyme activation (phospholipases, proteases and endonucleases), bleb formation or cytoskeletal alterations up to cell death [20,21]. Arylation of different cellular nucleophiles, is an additional toxic event which disrupts protein functions and cellular structures [3,4,15,16]. The potential toxicity of quinones is controlled by detoxification processes. For example, hydroquinones, obtained by two electron reduction of quinones, may in turn be conjugated to stable sulfonated or glucuronidated adducts that are unable to redox cycle [15,16]. By contrast, the hydroquinones are per se able to evoke redox cycling phenomena [16,22]. Thiols are considered to be the best antioxidant defense against electrophiles (oxidizing or conjugating agents). For example, GSH, the richest cell nucleophile that forms detoxifying conjugates with electrophiles (menadione), prevents S-arylation of important cell structures [1,2,16,23]. However, this role of GSH must be considered to some extent to be restrictive of more complex actions played by thiols altogether, i.e. by the sum of protein (PSH) and non-protein thiols (NPSH). More precisely, we assume that PSH are not a simple molecular target to be protected by GSH, but a normal biochemical site that like GSH intervenes in redox and conjugating reactions. By virtue of this functional similarity of PSH and NPSH, we assume that the menadione – protein conjugates (PS-quinone thioethers) could cause redox cycle reactions as the conjugates with small thiols are capable of doing [24–27]. It is therefore important to clarify under which conditions PSH, a potential antioxidant site, are able to become a pro-oxidant risk factor and how cells defend themselves
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against this risk. All of these events may depend on the concerted action of intracellular thiols towards electrophiles, whose mode of function is only partially known [28]. Moreover, given the therapeutic usage of menadione, an understanding of these aspects may be of interest in preventing the damage of selective targets such as erythrocytes (hemolytic anemia) [6,11,19,29,30]. In this study, the biological role of thiols in the menadione metabolism was analyzed in human and rat erythrocytes which were selected as models which display great differences in PSH reactivity (i.e. the b125 cysteinyl residue of rat Hb is about 1000 times more reactive than the b93 of human Hb and 30 times more reactive than GSH) [31]. The aim of the study is to elucidate the pro-oxidant contribution of PSH in the rat. Human and rat RBC were exposed to increasing doses of menadione and the metabolism of cell thiols and the toxic effects (oxygen consumption and Hb oxidation) were assayed under various redox conditions (nitrogen- or CO-pretreatment). Arylated proteins of rat RBC by menadione were found to be able to elicit redox cycling reactions. We have already investigated some preliminary aspects of this study [32].
2. Materials and methods
2.1. Chemicals Reagents HPLC grade were obtained from BDH; all chemicals of analytical grade were from SIGMA.
2.2. Blood preparation and treatment Sprague – Dawley rats were obtained from Charles River, Como, Italy. Blood was withdrawn from the abdominal aorta under chloralium hydrate anesthesia (400 mg/kg b.w.) using K3EDTA (1.5 mg/ml blood) as an anticlotting agent. Human blood was obtained from healthy donors, volunteers. Erythrocytes were separated from the other blood components and resuspended at 37% hematocrit with a PBS solution, pH 7.4 containing 10 mM glucose. All experiments were carried out at 37 °C using freshly prepared menadione (60 mM stock solution in ethanol). Anaerobic experiments were performed in sealed vials after prolonged 100% N2 bubbling. CO-treated RBC were prepared by bubbling CO into sealed vials for 15 min, shaking the cell suspension to saturate hemoglobin. Erythrocytes were then aerated and treated with menadione (at 37 °C). No change in all parameters was observed in erythrocytes treated with the same volume of the veichle (95% ethanol).
2.3. Glutathione measurements Human and rat RBC samples were collected at specified times and deproteinized with TCA (5% final concentration). GSH, GSSG and GS– SP were measured
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spectrophotometrically at room temperature in the supernatant of acid deproteinized samples and on the corresponding protein pellets [28,33]. On the same acidic supernatant menadione– GSH conjugate was detected by reverse phase HPLC (4×250 mm C18 column; isocratic run 60% 50 mM acetate buffer, pH 3.1, 40% ethanol) with spectrophotometric detector (420 nm). Calibration curves were performed by reacting menadione with an excess amount of GSH in 0.1 M phosphate buffer, pH 7.4, for 20 min at room temperature. A retention time of 16.9 min for the menadione–glutathione conjugate was observed. HPLC determinations were performed using a Hewlett– Packard (Palo-Alto, CA) 1100 series HPLC apparatus.
2.4. Fast reacting protein SH groups f-PSH of rat erythrocytes were determined by DTNB with the Ellman’s method [34] modified as follows. Briefly, 20 ml RBC were hemolyzed with 1.4 ml 10 mM phosphate buffer. After centrifugation (10 min at 15000× g) samples were passed through a desalting Sephadex G-25 column (Pharmacia-Biotech, Sweden), to remove low molecular weight thiols. On the protein eluates PSH were titrated with DTNB (100 pM, final concentration) at 450 nm (m= 7.0 mM − 1 cm − 1). f-PSH were calculated from the increase in absorbance after 5 s of reaction. Values were expressed as nanomoles/ml of RBC.
2.5. Methemoglobin formation In hemolysates of menadione treated RBC the percentage of met-Hb formed was calculated by spectral deconvolution by comparing the spectrum of the sample with standard spectra of reduced and oxidized Hb in the range 500– 700 nm. The reduced standard Hb spectrum was obtained by the addition of a few grains of sodium dithionite to RBC hemolysate and subsequent elution with Sephadex G-25 column; the spectrum of oxidized Hb standard (met-Hb) was obtained by adding K3Fe(CN)6 in excess. Deconvolution was performed by importing sample spectra into the computer and fitting them by non linear minimization (Sigma Plot program, version 2.01, Jandel Scientific). In experiments carried out in the presence of CO-Hb, the percentage of met-Hb was derived from the comparison of sample spectra with CO-Hb and oxidized Hb standard spectra. In all experiments no evidence of hemichrome formation was found. All spectrophotometric determinations were carried out with Jasco (Tokio, Japan) UV–vis V-550 spectrophotometer.
2.6. Oxygen consumption RBC (10%), prepared as above, was treated with 0.3 mM menadione to maintain similar experimental conditions to those of 37% hematocrit and 1 mM menadione. After 5 min of preincubation menadione was added with syringe to RBC in magnetically stirred chambers maintained at 37 °C; the rate of oxygen consumption was measured polarographically with a Clark oxygen electrode (Yellow Spring Instruments Co., USA).
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2.7. Experiments with purified Hb Rat Hb was purified as previously described in Rossi et al. [31]. Solutions of equal amounts (0.3 mM) of GSH and f-PSH of rat Hb (corresponding to 1 mM heme concentration in 0.1 M phosphate buffer, pH 7.4) were treated with 0.5 mM menadione at room temperature. At specified times samples were collected and passed onto Sephadex G-25 column or precipitated with TCA. f-PSH were assayed on protein eluates, whereas, GSH and GSSG were measured on supernatants of TCA treated samples (see above). In experiments of rat Hb deprived of f-PSH, 0.3 mM f-PSH were mixed with 0.3 mM NEM for 15 min at room temperature in 0.1 M phosphate buffer, pH 7.4 and then 0.3 mM GSH and 0.5 mM menadione were successively added. At specified times samples were collected and GSH and GSSG measured as above. No change was induced by the treatment with veichle (95% ethanol).
2.8. Statistical analysis Statistical tests were performed using the Student’s t-test.
3. Results
3.1. Alterations of cellular glutathione in RBC In both erythrocyte models, menadione treatment induced a marked alteration of glutathione over time. Responses of human RBC differed from those of rat RBC (Fig. 1). Total glutathione (sum of GSH, GSSG and GS–SP) decreased in human RBC in a dose dependent manner, whereas it was unchanged in rat RBC. This difference was due to the prevailing conversion of GSH into a GSH–menadione adduct in human (see Fig. 5, panel D) or into GS– SP in rat RBC (Fig. 1). In both species, GSSG was transiently increased, being more modified in rat than in human RBC, as the experiment of Table 1 illustrates typically.
3.2. Formation of methemoglobin and oxygen consumption in RBC The complex variations caused by menadione to the glutathione system of rat and human RBC, were matched by a persistent dose-dependent increase in Hb oxidation (up to 3 h of observation) (Fig. 2). Met-Hb was the major oxidation form detected. Although different rates in the formation of met-Hb were observed in rat and human RBC, the final extent (180 min) of oxidation was similar in both species, except at the lowest oxidant dose (0.25 mM), that in rat caused a lower met-Hb formation than in human RBC. Considering that 1% of Hb corresponds to approximately 80 mM heme concentration for a 37% hematocrit, it was deduced that the amount of Hb oxidation in each experiment was far higher than the corresponding menadione dose.
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Moreover, RBC of both species exposed to menadione were subjected to equal redox cycling phenomena as suggested by the similar rates of oxygen consumption in experiments reported in Table 2.
Fig. 1. The redox state of glutathione in human and rat RBC exposed to increasing menadione concentrations. Human (left) and rat (right) RBC resuspended at 37% in PBS plus 10 mM glucose, pH 7.4, were treated with indicated doses of menadione at 37 °C. GSH, GSSG and GS– SP were assayed spectrophotometrically as described in Section 2. GSSG was expressed as GSH moles. All data represent the mean of three to five independent experiments; the S.D. was omitted for clarity and was below 15% of the mean.
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Table 1 GSSG increases in human and rat RBC exposed to increasing menadione Menadione (mM)
0 0.25 0.5 1.0
GSSG Human
Rat
70.6 9 18 120 925 180 928 305 951
80.7 915.1 250 9 23a 315 9 31a 430 9 62a
GSSG, expressed as nanomoles/ml of RBC, was assayed at 5 min from RBC treatment. Mean 9S.D. of three independent experiments. The experimental conditions were equal to those reported in the legend of Fig. 1. a Statistically significant with respect to human RBC; PB0.01 Student’s t-test.
3.3. Thiol reaction of rat hemoglobin: competition between fast PSH and GSH In comparison to the human, rat Hb possesses fast reactive PSH (f-PSH; see Section 1) which is able to condition the GSH metabolism with electrophiles (Fig. 1) [28,32]. In rat RBC menadione caused a rapid decrease in f-PSH levels that was attributed to the formation of menadione-Hb S-adducts (Fig. 3). An increase in GS– SP that was reversible at the lowest menadione dose (0.25 mM) was observed. Moreover, the GS–SP maximum (Fig. 3) occurred slightly later (at 30 min) than the f-PSH drop (at 1–3 min). The decrease in the sum of f-PSH plus GS – SP was dose-related and in each case reflected approximately a 1:1 stoichiometry with menadione. As rat Hb is unable to form inter-chain disulfide links [31], we suggest that the decrease in f-PSH in rat RBC exposed to menadione is related to the formation of thioether compounds with proteins. Other authors arrived at similar conclusions (formation of thioether derivatives with b93 cysteine of hemoglobin) [35]. We next evaluated the competition between thiols (i.e. GSH and fast PSH) in attacking menadione (Fig. 4). Equal concentrations of GSH and f-PSH (0.3 mM) of rat Hb were mixed at room temperature with 0.5 mM menadione and GSH, GSSG, f-PSH and GSH –menadione adducts were measured over time (Fig. 4). The drop of f-PSH levels (panel C) occurred faster (see the value at 1 min) than that of GSH (panel B). Moreover, the formation of GSH–menadione adducts (data not shown) was minor, whereas the GSSG increase (panel B) was higher than in controls, i.e. in experiments with GSH alone (panel A) or in those with GSH plus Hb deprived of f-PSH (NEM-pretreated Hb, panel D). Therefore, the greater GSSG production in samples with f-PSH suggests that S-arylated proteins by menadione are able to redox cycle at the expense of GSH and are responsible for the different glutathione alterations in rat and human RBC.
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Fig. 2. Hemoglobin oxidation in human and rat RBC exposed to increasing menadione concentrations. At specified times RBC samples of the same experiment of Fig. 1 were hemolyzed in 10 mM phosphate buffer, pH 7.4. Percentage of met-Hb formation was measured by spectral deconvolution as described in Section 2. Symbols, rat (black) and human (white); menadione doses, 0.25 mM circles, 0.5 mM triangles, 1 mM squares. Mean values of three independent experiments whose standard deviation (omitted for clarity) did not exceed 20% of the mean. Values at 60, 120 and 180 min in 0.25 mM menadione treatments were found to be significantly different (P B0.05, Student’s t-test).
Table 2 Rates of oxygen consumption in human and rat RBC treated with menadione in the presence of carbon mono-oxide No pretreatment
CO-pretreatment
Rat
Human
Rat
Human
3.22 9 0.35*
2.87 90.31**
4.91 90.79*
4.32 90.75**
RBC at 10% hematocrit were treated with 0.3 mM menadione at 37 °C and the oxygen consumption measured by Clark electrode. The experiment was repeated in rat RBC saturated with CO (see Section 2 for further details). The rate of oxygen consumption is expressed as nmoles (ml per min). Mean 9 S.D. of three independent experiments. *, **, Significantly different (PB0.05, Student’s t-test).
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Fig. 3. Time – courses of f-PSH and GS – SP levels in rat RBC exposed to increasing menadione concentrations. Rat RBC were treated at 37 °C with 0.25, 0.5 and 1.0 mM menadione. At specified times samples were collected, hemolyzed and passed through a Sephadex G-25 column for f-PSH measurement (see Section 2). GS –SP were measured on the acid pellet derived from the sample precipitation with TCA (see Section 2). Data, expressed as nmoles/ml RBC, are the mean of three independent experiments whose standard deviation (omitted for clarity) did not exceed 15% of the mean.
3.4. The relationships between thiol and Hb oxidations in RBC exposed to menadione in absence of oxygen or in presence of carbon monoxide Although thioethers of quinones with proteins (for a presumably slow turnover) were expected to cause a more pronounced pro-oxidant effect than those of NPSH, no difference in the final extent of met-Hb formation in rat versus human RBC exposed to menadione was found (Fig. 2). Consequently, further experiments were performed to discover how the modulation of factors involved in redox cycling processes, such as oxygen and iron two of oxy-Hb, could modify the oxidation of thiols and Hb. RBC were exposed to 1.0 mM menadione, in the absence of oxygen (pretreatment with nitrogen) or in the presence of oxygen but with iron two of Hb blocked by CO (pretreatment with CO). The thiol metabolism, the oxygen consumption and the Hb oxidation were then measured. Under anaerobic conditions (Fig. 5), menadione caused a modest and reversible oxidation of hemoglobin which in human RBC was less pronounced than in rat RBC (Fig. 5, panel A). The small changes in rat were accompanied by corresponding reversible increases in GSSG and GS – SP (Fig. 5, panel B). This would confirm that the presence of oxygen is important for the more persistent effects of menadione. By contrast the decrease in the sum of f-PSH and GS – SP (total f-PSH decrease) was oxygen-independent, whereas the extent of GS– SP formation was reversible and much lower in the absence than in the presence of oxygen (Fig. 5, panel C). The GSSG formation in deoxygenated human RBC was almost absent (not shown) and GSH was consumed only in conjugations with menadione, the initial extent of this process not being influenced by oxygen (Fig. 5, panel D).
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The most important aspects of all these results were that S-arylation by menadione occurs independently from oxygen, and that iron two of deoxy-Hb is oxidized in the presence of menadione. In particular, results of arylation were in agreement with those of Wilson et al. [36] who showed that menadione forms prevalently GSH-adducts in human RBC in the absence of oxygen. On the contrary, the Hb oxidation in RBC exposed to menadione was unexpected, because other authors [35] reported differing results when they used purified Hb. The refractory nature of Hb was confirmed by us when the rat protein was exposed to menadione in the absence of oxygen (data not shown). The Hb oxidation by menadione (Fig. 6, panel A) was much lower in CO-pretreated RBC than in normal (Fig. 2) or in deoxygenated RBC (Fig. 5, panel A),
Fig. 4. Time –courses of GSH, GSSG and f-PSH after treatment with menadione of reconstituted systems with GSH and rat hemoglobin. Panel A, reaction of 0.3 mM GSH with 0.5 mM menadione. The reaction was performed in 0.1 M phosphate buffer, pH 7.4, at room temperature. GSH (black), GSSG (grey) were titrated spectrophotometrically after TCA acidification (see Section 2 for further details). Panels B and C, 0.3 mM GSH and 0.3 mM f-PSH of purified rat Hb (1 mM heme concentration) were treated with 0.5 mM menadione in 0.1 M phosphate buffer, pH 7.4, at room temperature. B, GSH (black) and GSSG,(grey); C, f-PSH. GSSG values at 30 and 60 min were significantly different from the corresponding values of panel A and D (P B0.05, Student’s t-test). Panel D, rat Hb deprived of f-PSH (0. 3 mM f-PSH was pretreated for 15 min with 0.3 mM NEM to block f-PSH) was successively mixed with 0.3 mM GSH and 0.5 mM menadione. GSH (black) and GSSG (grey) were titrated on supernatants after TCA precipitation (see Section 2 for further details). Mean values of three independent experiments whose S.D. (omitted for clarity) did not exceed 18% of the mean.
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Fig. 5. Time-courses of met-Hb, GSH, GSSG, GS – SP, f-PSH and GSH– menadione adducts in human and rat de-oxygenated RBC exposed to menadione. Hunan and rat RBC, resuspended at 37% in PBS plus 10 mM glucose, pH 7.4, were deoxygenated by prolonged 100% N2 bubbling in sealed vials. Cells were subsequently exposed to 1 mM menadione at 37 °C in nitrogen atmosphere. At specified times samples were collected and precipitated with TCA for GSH, GSSG and GS –SP measurements. Alternatively cells were hemolyzed and passed through Sephadex G-25 column for the f-PSH assay (see Section 2 for further details). Panel A, time – course of met-Hb in human and rat RBC. Panel B, time– course of glutathione in rat RBC. Panel C, time – course of f-PSH and GS – SP in rat RBC in the absence and presence of oxygen. Panel D, menadione – GSH conjugate formation in human RBC in the absence and presence of oxygen. The menadione – GSH conjugate was detected by reverse phase HPLC as described in Section 2. Mean values, expressed as nanomoles/ml RBC of three independent experiments whose standard deviation (omitted for clarity) did not exceed 22% of the mean. *, statistically significant with respect to human RBC; PB 0.01 Student’s t-test.
indicating an effective protection of CO in heme oxidation. In particular, the protein oxidation was absent in human and negligible in rat RBC (Fig. 6, panel A) (starting only after 15 min from the oxidant addition in rat). In contrast, CO pretreated cells of both species exhibited a significant increase in the rate of oxygen consumption with respect to the corresponding controls (Table 2). Moreover, whereas the GSH oxidation in CO-pretreated rat RBC was surprisingly slower (Fig.
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6, panel B) than that observed in experiments under anaerobiosis (see results at 1 min of Fig. 5, panel B), the attack of menadione to proteins (f-PSH) was rapid and equal in the presence or absence of CO (Fig. 6, panel C). The decrease over time in total f-PSH (sum of f-PSH and GS–SP) was not influenced by CO but a lower GS – SP production was obtained in CO-treated rat RBC (Fig. 6, panel C). The formation of GS – menadione adducts in human RBC in the presence or absence of CO was rather similar (Fig. 6, panel D). However, in the presence of CO the levels of GSH– menadione conjugate were stable 15 min after menadione addition, whereas they decreased in the absence of CO.
Fig. 6. Time –courses of met-Hb, GSH, GSSG, GS– SP, f-PSH and GSH – menadione adducts in human and rat CO-pretreated RBC exposed to menadione. Human and rat RBC, resuspended at 37% in PBS plus 10 mM glucose, pH 7.4, were saturated with CO, as described in Section 2, and then treated with 1 mM menadione at 37 °C in air atmosphere. Panel A, time – course of met-Hb in human and rat RBC. Panel B, time –course of glutathione in rat RBC. Panel C, time – course of f-PSH and GS – SP in rat RBC in the absence or presence of CO. Panel D, time – course of menadione – GSH conjugate in the absence or presence of CO mean values, expressed as nanomoles/ml RBC, of three independent experiments whose standard deviation (omitted for clarity) did not exceed 15% of the mean. *, statistically significant with respect to human RBC; PB0.01 Student’s t-test.
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Therefore, all these results evidence that the heme iron is important in the thiolic oxidative effects of menadione. On the contrary, the conjugative effects of menadione are scarcely affected by the presence of heme iron, and similar levels of menadione conjugates were formed in erythrocytes pretreated or not with CO. 4. Discussion Thiols are generally represented by NPSH (i.e. GSH) and PSH. Whereas the variation of reactivity among various NPSH is known to be minimal, that of protein thiols may range within several orders of magnitude [37]. The case of rat Hb, possessing fast reacting SH groups in position b125 which are far more reactive than GSH (see Section 1) [28,32], is paradigmatic. Consequently, in rat erythrocytes, f-PSH rather than GSH are the most important factors which scavenge noxious compounds [32]. It is well known that various reductants (such as thiols, vitamin C, or NADPH) in particular conditions behave as pro-oxidant factors [16,38] increasing the ROS formation and oxygen consumption when coupled to the redox cycling agents (e.g. quinones). However, the in vivo conditions by which, thiols and also some their by-products (catechol thioethers) [39] may favor or prevent the toxic effects of quinones are unknown. The aim of this study was to demonstrate the pro-oxidant contribution of proteins after rat RBC treatment with menadione that forms S-adducts with fast PSH of Hb [32]. Two biological models (rat and human RBC) were chosen to emphasize better the pro-oxidant contribution of rat proteins. Moreover, the choice of menadione was dictated by its interesting property of acting as an oxidizing and arylating agent, a condition that modulates the endogeneous competition of thiols, which per se is different in the two species. We found that the pro-oxidant response of rat RBC exposed to menadione was mainly related to the formation of S-arylated Hb f-PSH (decrease in f-PSH levels in Figs. 3 and 4, panel C). Moreover, the GSSG formation, an index of redox cycling reactions, was higher in rat RBC than in human RBC (Table 1), and GSSG was rapidly and irreversibly transformed into GS–SP (Figs. 1 and 3). The lack of reversibility of the GS– SP process confirmed the high levels of oxidative stress reached in rat RBC. Therefore, protein S-arylation by menadione may represent a sign of pro-oxidant risk in RBC. The rapid decrease in f-PSH observed in experiments of Figs. 3 and 4, also confirms the strong nucleophilic feature of rat Hb. It was previously shown in rat RBC that the Hb reactions with 1-chloro-2,4-dinitrobenzene and ethacrynic acid, specific substrates of glutathione trasferases, are faster than those with GSH [32]. Therefore, the notion that GSH and its enzymes may serve to defend important macromolecular structures from the attack of electrophilic compounds [1,2,16,23,35] seems to be not always valid in rat RBC. Results presented here of quinones that arylate and redox cycle to form a mixture of GS–SP and S-adducts on the same target (f-PSH) (Figs. 1 and 3), have clearly indicated the complexity of the GSH/PSH interplay during the metabolism of these agents.
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Although in terms of thiol oxidation (formation of GSSG and GS– SP) rat RBC displayed more signs of oxidative stress than human RBC, this difference did not result when the met-Hb formation (Fig. 2) and oxygen consumption (Table 2) were compared. Further experiments with RBC deprived of thiols were carried out to prove a decrease in oxygen consumption in rat RBC. Human and rat blood were pretreated with an NEM excess, that was removed by washing and exposed to menadione under conditions equal to those reported in Table 2. All attempts at assaying oxygen consumption were unsuccessful, because in both species menadione caused a massive Hb oxidation (and oxygen delivery from heme) which impeded any measurements (data not shown). No useful information was obtained from these experiments, with the exception that both human and rat RBC deprived of thiols became equally sensitive to menadione (data not shown). From all these data we concluded that erythrocytes of different species have specific oxidative behavior as already underlined in a previous study [28]. Results of RBC treatment with menadione under conditions of absence of oxygen (Fig. 5; treatments in nitrogen atmosphere) or in presence of CO (Fig. 6), confirmed the importance of oxygen in redox cycling reactions [40,41], but also stressed the involvement of oxy-Hb or oxygen by itself in these reactions. However, the met-Hb formation by menadione in de-oxygenated erythrocytes (Fig. 5, panel A) was in part unexpected because it was not seen with deoxy-Hb of rat (data not shown) or human [35]. Thus, the role of Hb and/or other non-thiolic factors must be further studied in order to clarify whether menadione oxidizes Hb directly or indirectly. Experiments of Figs. 5 and 6 gave additional information on the series of biochemical events elicited by menadione in rat erythrocytes under normal oxygen conditions. Although menadione is prevalently and rapidly blocked by f-PSH, we assume that part of it escapes from this fate and interacts with oxy-Hb or other factors (reducing agents) [25]. As indicated by experiments of Fig. 5 (panels A and B), showing the parallel and easy oxidation of deoxy-Hb and GSH, the semiquinone radicals, as widely inferred by the literature and generated in these reactions, can prime redox cycling processes with formation of ROS, GSSG, GS – SP and so on. The oxidative stress is further increased by the contribution of S-arylated proteins. According to the literature [35,36,42], a mixture of quinone/hydroquinone may form conjugates of menadione with small thiols. We speculate that the same may occur with f-PSH of rat Hb. Analogously, we assume that the protein adducts would produce semiquinone radicals during redox reactions as adducts of small thiols are able to do [24,25,43]. Since, from experiments of Figs. 5 and 6, the formation of S-arylated proteins would be independent of the presence of oxygen or the Hb state, we can readily suggest that only the PSH reactivity would govern the S-arylation process by quinones and the metabolic consequences of it. The present results are in agreement with those of other authors, who however, studied only different aspects of erythrocytic thiols [22,44,45]. For example, according to Miller and Smith [45] redox cycling phenomena elicited by menadione caused 40– 60% of GSH depletion in human erythrocytes, whereas this was almost total in the rat [44].
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In conclusion, our data suggest that within few minutes from erythrocyte exposure, menadione is conjugated to cellular thiols and oxidative reactions contemporaneously occur (GSSG and met-Hb formation). As a consequence of the formation of menadione adducts by cell thiols, oxidative reactions are further generated, involving oxygen consumption and leading to a massive and persistent hemoglobin oxidation, as well as a thiolic unbalance. Moreover, our data demonstrate that highly reactive protein thiols can undergo the same conjugative and redox cycling reactions as GSH. Therefore, they are not only a simple target of menadione toxicity, but are actively involved in primary toxic reactions (i.e. oxygen dependent redox cycling). It has previously been shown that rat platelets treated with menadione exhibit protein arylation and no increase in GSSG levels [46]. As a consequence of the no GSSG change, it was also suggested that protein arylation may be an injure mechanism different from redox cycling [46]. Experiments of rat RBC (Fig. 1) and the oxidant treatment of human platelets (that are characterized by having fast PSH) have shown that the GSSG increase may be only transient or negligible in comparison with that of GS– SP [47]. Consequently, GSSG may not always be considered a good index of redox cycling phenomena. In agreement with other authors, the alkylation reactions are important metabolic events that increase the toxicity of quinones exerted by redox cycling processes [24,48]. Data here presented on rat RBC have emphasized the pro-oxidant role of PSH. However, the generalization of the protein action in other cell models seems to be rather difficult because it is linked to the occurrence of other events related to the protein accessibility to oxygen and reducing agents. In plasma, that is constitutively poor in NPSH and relatively rich in PSH (albumin), the contribution of PSH to redox cycling phenomena by menadione has been clearly evidenced [49]. Acknowledgements Fruitful discussions and helpful suggestions by Dr Christine Winterbourn are gratefully acknowledged. This study was supported in part by MURST and PAR programs of research. References [1] H. Thor, M.T. Smith, P. Hartzell, G. Bellomo, S.A. Jewell, S. Orrenius, The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes, J. Biol. Chem. 257 (1982) 12419–12425. [2] D. Di Monte, D. Ross, G. Bellomo, L. Eklo¨ w, S. Orrenius, Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes, Arch. Biochem. Biophys. 235 (1984) 334 –342. [3] D. Di Monte, G. Bellomo, H. Thor, P. Nicotera, S. Orrenius, Menadione-induced cytotoxicity is associated with protein thiol oxidation and alteration in intracellular Ca2 + homeostasis, Arch. Biochem. Biophys. 235 (1984) 343 –350.
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www.elsevier.com/locate/chembiont
The pro-oxidant role of protein SH groups of hemoglobin in rat erythrocytes exposed to menadione Lorenzo Lusini, Ranieri Rossi, Daniela Giustarini, Paolo Di Simplicio * Department of Neuroscience, Pharmacology Unit, Uni6ersity of Siena, Via A. Moro 4, 53100 Siena, Italy Received 31 July 2001; received in revised form 4 September 2001; accepted 5 November 2001
Abstract Menadione is selectively toxic to erythrocytes. Although GSH is considered a primary target of menadione, intraerythrocyte thiolic alterations consequent to menadione exposure are only partially known. In this study alterations of GSH and protein thiols (PSH) and their relationship with methemoglobin formation were investigated in human and rat red blood cells (RBC) exposed to menadione. In both erythrocyte types, menadione caused a marked increase in methemoglobin associated with GSH depletion and increased oxygen consumption. However, in human RBC, GSH formed a conjugate with menadione, whereas, in rat RBC it was converted to GSSG, concomitantly with a loss of protein thiols (corresponding to menadione arylation), and an increase in glutathione– protein mixed disulfides (GS– SP). Such differences were related to the presence of highly reactive cysteines, which characterize rat hemoglobin (cys b125). In spite of the greater thiol oxidation in rat than in human RBC, methemoglobin formation and the rate of oxygen consumption elicited by menadione in both species were rather similar. Moreover, in repeated experiments under N2 or CO-blocked heme, it was found that menadione conjugation (arylation) in both species was not dependent on the presence of oxygen or the status of heme. Therefore, we assumed that GSH Abbre6iations: CO, carbon monoxide; CO-Hb, carboxy hemoglobin; DTNB, 5,5%-dithiobis (2-nitrobenzoic acid); f-PSH, fast reacting protein-SH groups; GSH, glutathione; GSSG, glutathione disulfide; GS–SP, glutathione–protein mixed disulfides; met-Hb, methemoglobin; NEM, N-ethylmaleimide; NPSH, non-protein SH groups; oxy-Hb, oxy-hemoglobin; PSH, protein-SH groups; RBC, red blood cells; ROS, reactive oxygen species; TCA, trichloroacetic acid. * Corresponding author. Tel.: + 39-057723-4097; fax: + 39-057723-4098. E-mail address: [email protected] (P.Di. Simplicio). 0009-2797/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 1 ) 0 0 2 9 6 - 4
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(human RBC) and protein (rat RBC) arylation was equally responsible for increased oxygen consumption and Hb oxidation. Moreover, thiol oxidation of rat RBC was strictly related to methemoglobin formation. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Menadione; Rat hemoglobin; Glutathione; Redox cycling; Protein arylation; Oxidative stress
1. Introduction Menadione, a widely studied quinone, is an electrophilic agent utilized either as an experimental oxidant [1– 4] or for various clinical purposes to prevent vitamin K deficiencies, myopathies, or used to treat malaria [5– 8]. Menadione or other quinones are also used to treat neoplastic diseases [9–12]. For some time much attention has been focused on cellular targets and on the number of toxic mechanisms evoked by quinones, such as redox cycling, arylation, intercalation, protein crosslinks and disruption of mitochondrial electron transport [13–16]. Menadione is metabolized by redox and arylation reactions. One-electron reduction, mediated by flavoenzymes [17] or by interaction with oxy-Hb [18,19], leads to the production of the highly reactive semiquinone form, which autoxidizes producing redox cycling reactions with oxygen consumption, sustained formation of reactive oxygen species (ROS) and a rapid drop in levels of reducing agents. This would cause various effects, such as, ATP depletion, Ca2 + entry, enzyme activation (phospholipases, proteases and endonucleases), bleb formation or cytoskeletal alterations up to cell death [20,21]. Arylation of different cellular nucleophiles, is an additional toxic event which disrupts protein functions and cellular structures [3,4,15,16]. The potential toxicity of quinones is controlled by detoxification processes. For example, hydroquinones, obtained by two electron reduction of quinones, may in turn be conjugated to stable sulfonated or glucuronidated adducts that are unable to redox cycle [15,16]. By contrast, the hydroquinones are per se able to evoke redox cycling phenomena [16,22]. Thiols are considered to be the best antioxidant defense against electrophiles (oxidizing or conjugating agents). For example, GSH, the richest cell nucleophile that forms detoxifying conjugates with electrophiles (menadione), prevents S-arylation of important cell structures [1,2,16,23]. However, this role of GSH must be considered to some extent to be restrictive of more complex actions played by thiols altogether, i.e. by the sum of protein (PSH) and non-protein thiols (NPSH). More precisely, we assume that PSH are not a simple molecular target to be protected by GSH, but a normal biochemical site that like GSH intervenes in redox and conjugating reactions. By virtue of this functional similarity of PSH and NPSH, we assume that the menadione – protein conjugates (PS-quinone thioethers) could cause redox cycle reactions as the conjugates with small thiols are capable of doing [24–27]. It is therefore important to clarify under which conditions PSH, a potential antioxidant site, are able to become a pro-oxidant risk factor and how cells defend themselves
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against this risk. All of these events may depend on the concerted action of intracellular thiols towards electrophiles, whose mode of function is only partially known [28]. Moreover, given the therapeutic usage of menadione, an understanding of these aspects may be of interest in preventing the damage of selective targets such as erythrocytes (hemolytic anemia) [6,11,19,29,30]. In this study, the biological role of thiols in the menadione metabolism was analyzed in human and rat erythrocytes which were selected as models which display great differences in PSH reactivity (i.e. the b125 cysteinyl residue of rat Hb is about 1000 times more reactive than the b93 of human Hb and 30 times more reactive than GSH) [31]. The aim of the study is to elucidate the pro-oxidant contribution of PSH in the rat. Human and rat RBC were exposed to increasing doses of menadione and the metabolism of cell thiols and the toxic effects (oxygen consumption and Hb oxidation) were assayed under various redox conditions (nitrogen- or CO-pretreatment). Arylated proteins of rat RBC by menadione were found to be able to elicit redox cycling reactions. We have already investigated some preliminary aspects of this study [32].
2. Materials and methods
2.1. Chemicals Reagents HPLC grade were obtained from BDH; all chemicals of analytical grade were from SIGMA.
2.2. Blood preparation and treatment Sprague – Dawley rats were obtained from Charles River, Como, Italy. Blood was withdrawn from the abdominal aorta under chloralium hydrate anesthesia (400 mg/kg b.w.) using K3EDTA (1.5 mg/ml blood) as an anticlotting agent. Human blood was obtained from healthy donors, volunteers. Erythrocytes were separated from the other blood components and resuspended at 37% hematocrit with a PBS solution, pH 7.4 containing 10 mM glucose. All experiments were carried out at 37 °C using freshly prepared menadione (60 mM stock solution in ethanol). Anaerobic experiments were performed in sealed vials after prolonged 100% N2 bubbling. CO-treated RBC were prepared by bubbling CO into sealed vials for 15 min, shaking the cell suspension to saturate hemoglobin. Erythrocytes were then aerated and treated with menadione (at 37 °C). No change in all parameters was observed in erythrocytes treated with the same volume of the veichle (95% ethanol).
2.3. Glutathione measurements Human and rat RBC samples were collected at specified times and deproteinized with TCA (5% final concentration). GSH, GSSG and GS– SP were measured
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spectrophotometrically at room temperature in the supernatant of acid deproteinized samples and on the corresponding protein pellets [28,33]. On the same acidic supernatant menadione– GSH conjugate was detected by reverse phase HPLC (4×250 mm C18 column; isocratic run 60% 50 mM acetate buffer, pH 3.1, 40% ethanol) with spectrophotometric detector (420 nm). Calibration curves were performed by reacting menadione with an excess amount of GSH in 0.1 M phosphate buffer, pH 7.4, for 20 min at room temperature. A retention time of 16.9 min for the menadione–glutathione conjugate was observed. HPLC determinations were performed using a Hewlett– Packard (Palo-Alto, CA) 1100 series HPLC apparatus.
2.4. Fast reacting protein SH groups f-PSH of rat erythrocytes were determined by DTNB with the Ellman’s method [34] modified as follows. Briefly, 20 ml RBC were hemolyzed with 1.4 ml 10 mM phosphate buffer. After centrifugation (10 min at 15000× g) samples were passed through a desalting Sephadex G-25 column (Pharmacia-Biotech, Sweden), to remove low molecular weight thiols. On the protein eluates PSH were titrated with DTNB (100 pM, final concentration) at 450 nm (m= 7.0 mM − 1 cm − 1). f-PSH were calculated from the increase in absorbance after 5 s of reaction. Values were expressed as nanomoles/ml of RBC.
2.5. Methemoglobin formation In hemolysates of menadione treated RBC the percentage of met-Hb formed was calculated by spectral deconvolution by comparing the spectrum of the sample with standard spectra of reduced and oxidized Hb in the range 500– 700 nm. The reduced standard Hb spectrum was obtained by the addition of a few grains of sodium dithionite to RBC hemolysate and subsequent elution with Sephadex G-25 column; the spectrum of oxidized Hb standard (met-Hb) was obtained by adding K3Fe(CN)6 in excess. Deconvolution was performed by importing sample spectra into the computer and fitting them by non linear minimization (Sigma Plot program, version 2.01, Jandel Scientific). In experiments carried out in the presence of CO-Hb, the percentage of met-Hb was derived from the comparison of sample spectra with CO-Hb and oxidized Hb standard spectra. In all experiments no evidence of hemichrome formation was found. All spectrophotometric determinations were carried out with Jasco (Tokio, Japan) UV–vis V-550 spectrophotometer.
2.6. Oxygen consumption RBC (10%), prepared as above, was treated with 0.3 mM menadione to maintain similar experimental conditions to those of 37% hematocrit and 1 mM menadione. After 5 min of preincubation menadione was added with syringe to RBC in magnetically stirred chambers maintained at 37 °C; the rate of oxygen consumption was measured polarographically with a Clark oxygen electrode (Yellow Spring Instruments Co., USA).
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2.7. Experiments with purified Hb Rat Hb was purified as previously described in Rossi et al. [31]. Solutions of equal amounts (0.3 mM) of GSH and f-PSH of rat Hb (corresponding to 1 mM heme concentration in 0.1 M phosphate buffer, pH 7.4) were treated with 0.5 mM menadione at room temperature. At specified times samples were collected and passed onto Sephadex G-25 column or precipitated with TCA. f-PSH were assayed on protein eluates, whereas, GSH and GSSG were measured on supernatants of TCA treated samples (see above). In experiments of rat Hb deprived of f-PSH, 0.3 mM f-PSH were mixed with 0.3 mM NEM for 15 min at room temperature in 0.1 M phosphate buffer, pH 7.4 and then 0.3 mM GSH and 0.5 mM menadione were successively added. At specified times samples were collected and GSH and GSSG measured as above. No change was induced by the treatment with veichle (95% ethanol).
2.8. Statistical analysis Statistical tests were performed using the Student’s t-test.
3. Results
3.1. Alterations of cellular glutathione in RBC In both erythrocyte models, menadione treatment induced a marked alteration of glutathione over time. Responses of human RBC differed from those of rat RBC (Fig. 1). Total glutathione (sum of GSH, GSSG and GS–SP) decreased in human RBC in a dose dependent manner, whereas it was unchanged in rat RBC. This difference was due to the prevailing conversion of GSH into a GSH–menadione adduct in human (see Fig. 5, panel D) or into GS– SP in rat RBC (Fig. 1). In both species, GSSG was transiently increased, being more modified in rat than in human RBC, as the experiment of Table 1 illustrates typically.
3.2. Formation of methemoglobin and oxygen consumption in RBC The complex variations caused by menadione to the glutathione system of rat and human RBC, were matched by a persistent dose-dependent increase in Hb oxidation (up to 3 h of observation) (Fig. 2). Met-Hb was the major oxidation form detected. Although different rates in the formation of met-Hb were observed in rat and human RBC, the final extent (180 min) of oxidation was similar in both species, except at the lowest oxidant dose (0.25 mM), that in rat caused a lower met-Hb formation than in human RBC. Considering that 1% of Hb corresponds to approximately 80 mM heme concentration for a 37% hematocrit, it was deduced that the amount of Hb oxidation in each experiment was far higher than the corresponding menadione dose.
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Moreover, RBC of both species exposed to menadione were subjected to equal redox cycling phenomena as suggested by the similar rates of oxygen consumption in experiments reported in Table 2.
Fig. 1. The redox state of glutathione in human and rat RBC exposed to increasing menadione concentrations. Human (left) and rat (right) RBC resuspended at 37% in PBS plus 10 mM glucose, pH 7.4, were treated with indicated doses of menadione at 37 °C. GSH, GSSG and GS– SP were assayed spectrophotometrically as described in Section 2. GSSG was expressed as GSH moles. All data represent the mean of three to five independent experiments; the S.D. was omitted for clarity and was below 15% of the mean.
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Table 1 GSSG increases in human and rat RBC exposed to increasing menadione Menadione (mM)
0 0.25 0.5 1.0
GSSG Human
Rat
70.6 9 18 120 925 180 928 305 951
80.7 915.1 250 9 23a 315 9 31a 430 9 62a
GSSG, expressed as nanomoles/ml of RBC, was assayed at 5 min from RBC treatment. Mean 9S.D. of three independent experiments. The experimental conditions were equal to those reported in the legend of Fig. 1. a Statistically significant with respect to human RBC; PB0.01 Student’s t-test.
3.3. Thiol reaction of rat hemoglobin: competition between fast PSH and GSH In comparison to the human, rat Hb possesses fast reactive PSH (f-PSH; see Section 1) which is able to condition the GSH metabolism with electrophiles (Fig. 1) [28,32]. In rat RBC menadione caused a rapid decrease in f-PSH levels that was attributed to the formation of menadione-Hb S-adducts (Fig. 3). An increase in GS– SP that was reversible at the lowest menadione dose (0.25 mM) was observed. Moreover, the GS–SP maximum (Fig. 3) occurred slightly later (at 30 min) than the f-PSH drop (at 1–3 min). The decrease in the sum of f-PSH plus GS – SP was dose-related and in each case reflected approximately a 1:1 stoichiometry with menadione. As rat Hb is unable to form inter-chain disulfide links [31], we suggest that the decrease in f-PSH in rat RBC exposed to menadione is related to the formation of thioether compounds with proteins. Other authors arrived at similar conclusions (formation of thioether derivatives with b93 cysteine of hemoglobin) [35]. We next evaluated the competition between thiols (i.e. GSH and fast PSH) in attacking menadione (Fig. 4). Equal concentrations of GSH and f-PSH (0.3 mM) of rat Hb were mixed at room temperature with 0.5 mM menadione and GSH, GSSG, f-PSH and GSH –menadione adducts were measured over time (Fig. 4). The drop of f-PSH levels (panel C) occurred faster (see the value at 1 min) than that of GSH (panel B). Moreover, the formation of GSH–menadione adducts (data not shown) was minor, whereas the GSSG increase (panel B) was higher than in controls, i.e. in experiments with GSH alone (panel A) or in those with GSH plus Hb deprived of f-PSH (NEM-pretreated Hb, panel D). Therefore, the greater GSSG production in samples with f-PSH suggests that S-arylated proteins by menadione are able to redox cycle at the expense of GSH and are responsible for the different glutathione alterations in rat and human RBC.
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Fig. 2. Hemoglobin oxidation in human and rat RBC exposed to increasing menadione concentrations. At specified times RBC samples of the same experiment of Fig. 1 were hemolyzed in 10 mM phosphate buffer, pH 7.4. Percentage of met-Hb formation was measured by spectral deconvolution as described in Section 2. Symbols, rat (black) and human (white); menadione doses, 0.25 mM circles, 0.5 mM triangles, 1 mM squares. Mean values of three independent experiments whose standard deviation (omitted for clarity) did not exceed 20% of the mean. Values at 60, 120 and 180 min in 0.25 mM menadione treatments were found to be significantly different (P B0.05, Student’s t-test).
Table 2 Rates of oxygen consumption in human and rat RBC treated with menadione in the presence of carbon mono-oxide No pretreatment
CO-pretreatment
Rat
Human
Rat
Human
3.22 9 0.35*
2.87 90.31**
4.91 90.79*
4.32 90.75**
RBC at 10% hematocrit were treated with 0.3 mM menadione at 37 °C and the oxygen consumption measured by Clark electrode. The experiment was repeated in rat RBC saturated with CO (see Section 2 for further details). The rate of oxygen consumption is expressed as nmoles (ml per min). Mean 9 S.D. of three independent experiments. *, **, Significantly different (PB0.05, Student’s t-test).
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Fig. 3. Time – courses of f-PSH and GS – SP levels in rat RBC exposed to increasing menadione concentrations. Rat RBC were treated at 37 °C with 0.25, 0.5 and 1.0 mM menadione. At specified times samples were collected, hemolyzed and passed through a Sephadex G-25 column for f-PSH measurement (see Section 2). GS –SP were measured on the acid pellet derived from the sample precipitation with TCA (see Section 2). Data, expressed as nmoles/ml RBC, are the mean of three independent experiments whose standard deviation (omitted for clarity) did not exceed 15% of the mean.
3.4. The relationships between thiol and Hb oxidations in RBC exposed to menadione in absence of oxygen or in presence of carbon monoxide Although thioethers of quinones with proteins (for a presumably slow turnover) were expected to cause a more pronounced pro-oxidant effect than those of NPSH, no difference in the final extent of met-Hb formation in rat versus human RBC exposed to menadione was found (Fig. 2). Consequently, further experiments were performed to discover how the modulation of factors involved in redox cycling processes, such as oxygen and iron two of oxy-Hb, could modify the oxidation of thiols and Hb. RBC were exposed to 1.0 mM menadione, in the absence of oxygen (pretreatment with nitrogen) or in the presence of oxygen but with iron two of Hb blocked by CO (pretreatment with CO). The thiol metabolism, the oxygen consumption and the Hb oxidation were then measured. Under anaerobic conditions (Fig. 5), menadione caused a modest and reversible oxidation of hemoglobin which in human RBC was less pronounced than in rat RBC (Fig. 5, panel A). The small changes in rat were accompanied by corresponding reversible increases in GSSG and GS – SP (Fig. 5, panel B). This would confirm that the presence of oxygen is important for the more persistent effects of menadione. By contrast the decrease in the sum of f-PSH and GS – SP (total f-PSH decrease) was oxygen-independent, whereas the extent of GS– SP formation was reversible and much lower in the absence than in the presence of oxygen (Fig. 5, panel C). The GSSG formation in deoxygenated human RBC was almost absent (not shown) and GSH was consumed only in conjugations with menadione, the initial extent of this process not being influenced by oxygen (Fig. 5, panel D).
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The most important aspects of all these results were that S-arylation by menadione occurs independently from oxygen, and that iron two of deoxy-Hb is oxidized in the presence of menadione. In particular, results of arylation were in agreement with those of Wilson et al. [36] who showed that menadione forms prevalently GSH-adducts in human RBC in the absence of oxygen. On the contrary, the Hb oxidation in RBC exposed to menadione was unexpected, because other authors [35] reported differing results when they used purified Hb. The refractory nature of Hb was confirmed by us when the rat protein was exposed to menadione in the absence of oxygen (data not shown). The Hb oxidation by menadione (Fig. 6, panel A) was much lower in CO-pretreated RBC than in normal (Fig. 2) or in deoxygenated RBC (Fig. 5, panel A),
Fig. 4. Time –courses of GSH, GSSG and f-PSH after treatment with menadione of reconstituted systems with GSH and rat hemoglobin. Panel A, reaction of 0.3 mM GSH with 0.5 mM menadione. The reaction was performed in 0.1 M phosphate buffer, pH 7.4, at room temperature. GSH (black), GSSG (grey) were titrated spectrophotometrically after TCA acidification (see Section 2 for further details). Panels B and C, 0.3 mM GSH and 0.3 mM f-PSH of purified rat Hb (1 mM heme concentration) were treated with 0.5 mM menadione in 0.1 M phosphate buffer, pH 7.4, at room temperature. B, GSH (black) and GSSG,(grey); C, f-PSH. GSSG values at 30 and 60 min were significantly different from the corresponding values of panel A and D (P B0.05, Student’s t-test). Panel D, rat Hb deprived of f-PSH (0. 3 mM f-PSH was pretreated for 15 min with 0.3 mM NEM to block f-PSH) was successively mixed with 0.3 mM GSH and 0.5 mM menadione. GSH (black) and GSSG (grey) were titrated on supernatants after TCA precipitation (see Section 2 for further details). Mean values of three independent experiments whose S.D. (omitted for clarity) did not exceed 18% of the mean.
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Fig. 5. Time-courses of met-Hb, GSH, GSSG, GS – SP, f-PSH and GSH– menadione adducts in human and rat de-oxygenated RBC exposed to menadione. Hunan and rat RBC, resuspended at 37% in PBS plus 10 mM glucose, pH 7.4, were deoxygenated by prolonged 100% N2 bubbling in sealed vials. Cells were subsequently exposed to 1 mM menadione at 37 °C in nitrogen atmosphere. At specified times samples were collected and precipitated with TCA for GSH, GSSG and GS –SP measurements. Alternatively cells were hemolyzed and passed through Sephadex G-25 column for the f-PSH assay (see Section 2 for further details). Panel A, time – course of met-Hb in human and rat RBC. Panel B, time– course of glutathione in rat RBC. Panel C, time – course of f-PSH and GS – SP in rat RBC in the absence and presence of oxygen. Panel D, menadione – GSH conjugate formation in human RBC in the absence and presence of oxygen. The menadione – GSH conjugate was detected by reverse phase HPLC as described in Section 2. Mean values, expressed as nanomoles/ml RBC of three independent experiments whose standard deviation (omitted for clarity) did not exceed 22% of the mean. *, statistically significant with respect to human RBC; PB 0.01 Student’s t-test.
indicating an effective protection of CO in heme oxidation. In particular, the protein oxidation was absent in human and negligible in rat RBC (Fig. 6, panel A) (starting only after 15 min from the oxidant addition in rat). In contrast, CO pretreated cells of both species exhibited a significant increase in the rate of oxygen consumption with respect to the corresponding controls (Table 2). Moreover, whereas the GSH oxidation in CO-pretreated rat RBC was surprisingly slower (Fig.
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6, panel B) than that observed in experiments under anaerobiosis (see results at 1 min of Fig. 5, panel B), the attack of menadione to proteins (f-PSH) was rapid and equal in the presence or absence of CO (Fig. 6, panel C). The decrease over time in total f-PSH (sum of f-PSH and GS–SP) was not influenced by CO but a lower GS – SP production was obtained in CO-treated rat RBC (Fig. 6, panel C). The formation of GS – menadione adducts in human RBC in the presence or absence of CO was rather similar (Fig. 6, panel D). However, in the presence of CO the levels of GSH– menadione conjugate were stable 15 min after menadione addition, whereas they decreased in the absence of CO.
Fig. 6. Time –courses of met-Hb, GSH, GSSG, GS– SP, f-PSH and GSH – menadione adducts in human and rat CO-pretreated RBC exposed to menadione. Human and rat RBC, resuspended at 37% in PBS plus 10 mM glucose, pH 7.4, were saturated with CO, as described in Section 2, and then treated with 1 mM menadione at 37 °C in air atmosphere. Panel A, time – course of met-Hb in human and rat RBC. Panel B, time –course of glutathione in rat RBC. Panel C, time – course of f-PSH and GS – SP in rat RBC in the absence or presence of CO. Panel D, time – course of menadione – GSH conjugate in the absence or presence of CO mean values, expressed as nanomoles/ml RBC, of three independent experiments whose standard deviation (omitted for clarity) did not exceed 15% of the mean. *, statistically significant with respect to human RBC; PB0.01 Student’s t-test.
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Therefore, all these results evidence that the heme iron is important in the thiolic oxidative effects of menadione. On the contrary, the conjugative effects of menadione are scarcely affected by the presence of heme iron, and similar levels of menadione conjugates were formed in erythrocytes pretreated or not with CO. 4. Discussion Thiols are generally represented by NPSH (i.e. GSH) and PSH. Whereas the variation of reactivity among various NPSH is known to be minimal, that of protein thiols may range within several orders of magnitude [37]. The case of rat Hb, possessing fast reacting SH groups in position b125 which are far more reactive than GSH (see Section 1) [28,32], is paradigmatic. Consequently, in rat erythrocytes, f-PSH rather than GSH are the most important factors which scavenge noxious compounds [32]. It is well known that various reductants (such as thiols, vitamin C, or NADPH) in particular conditions behave as pro-oxidant factors [16,38] increasing the ROS formation and oxygen consumption when coupled to the redox cycling agents (e.g. quinones). However, the in vivo conditions by which, thiols and also some their by-products (catechol thioethers) [39] may favor or prevent the toxic effects of quinones are unknown. The aim of this study was to demonstrate the pro-oxidant contribution of proteins after rat RBC treatment with menadione that forms S-adducts with fast PSH of Hb [32]. Two biological models (rat and human RBC) were chosen to emphasize better the pro-oxidant contribution of rat proteins. Moreover, the choice of menadione was dictated by its interesting property of acting as an oxidizing and arylating agent, a condition that modulates the endogeneous competition of thiols, which per se is different in the two species. We found that the pro-oxidant response of rat RBC exposed to menadione was mainly related to the formation of S-arylated Hb f-PSH (decrease in f-PSH levels in Figs. 3 and 4, panel C). Moreover, the GSSG formation, an index of redox cycling reactions, was higher in rat RBC than in human RBC (Table 1), and GSSG was rapidly and irreversibly transformed into GS–SP (Figs. 1 and 3). The lack of reversibility of the GS– SP process confirmed the high levels of oxidative stress reached in rat RBC. Therefore, protein S-arylation by menadione may represent a sign of pro-oxidant risk in RBC. The rapid decrease in f-PSH observed in experiments of Figs. 3 and 4, also confirms the strong nucleophilic feature of rat Hb. It was previously shown in rat RBC that the Hb reactions with 1-chloro-2,4-dinitrobenzene and ethacrynic acid, specific substrates of glutathione trasferases, are faster than those with GSH [32]. Therefore, the notion that GSH and its enzymes may serve to defend important macromolecular structures from the attack of electrophilic compounds [1,2,16,23,35] seems to be not always valid in rat RBC. Results presented here of quinones that arylate and redox cycle to form a mixture of GS–SP and S-adducts on the same target (f-PSH) (Figs. 1 and 3), have clearly indicated the complexity of the GSH/PSH interplay during the metabolism of these agents.
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Although in terms of thiol oxidation (formation of GSSG and GS– SP) rat RBC displayed more signs of oxidative stress than human RBC, this difference did not result when the met-Hb formation (Fig. 2) and oxygen consumption (Table 2) were compared. Further experiments with RBC deprived of thiols were carried out to prove a decrease in oxygen consumption in rat RBC. Human and rat blood were pretreated with an NEM excess, that was removed by washing and exposed to menadione under conditions equal to those reported in Table 2. All attempts at assaying oxygen consumption were unsuccessful, because in both species menadione caused a massive Hb oxidation (and oxygen delivery from heme) which impeded any measurements (data not shown). No useful information was obtained from these experiments, with the exception that both human and rat RBC deprived of thiols became equally sensitive to menadione (data not shown). From all these data we concluded that erythrocytes of different species have specific oxidative behavior as already underlined in a previous study [28]. Results of RBC treatment with menadione under conditions of absence of oxygen (Fig. 5; treatments in nitrogen atmosphere) or in presence of CO (Fig. 6), confirmed the importance of oxygen in redox cycling reactions [40,41], but also stressed the involvement of oxy-Hb or oxygen by itself in these reactions. However, the met-Hb formation by menadione in de-oxygenated erythrocytes (Fig. 5, panel A) was in part unexpected because it was not seen with deoxy-Hb of rat (data not shown) or human [35]. Thus, the role of Hb and/or other non-thiolic factors must be further studied in order to clarify whether menadione oxidizes Hb directly or indirectly. Experiments of Figs. 5 and 6 gave additional information on the series of biochemical events elicited by menadione in rat erythrocytes under normal oxygen conditions. Although menadione is prevalently and rapidly blocked by f-PSH, we assume that part of it escapes from this fate and interacts with oxy-Hb or other factors (reducing agents) [25]. As indicated by experiments of Fig. 5 (panels A and B), showing the parallel and easy oxidation of deoxy-Hb and GSH, the semiquinone radicals, as widely inferred by the literature and generated in these reactions, can prime redox cycling processes with formation of ROS, GSSG, GS – SP and so on. The oxidative stress is further increased by the contribution of S-arylated proteins. According to the literature [35,36,42], a mixture of quinone/hydroquinone may form conjugates of menadione with small thiols. We speculate that the same may occur with f-PSH of rat Hb. Analogously, we assume that the protein adducts would produce semiquinone radicals during redox reactions as adducts of small thiols are able to do [24,25,43]. Since, from experiments of Figs. 5 and 6, the formation of S-arylated proteins would be independent of the presence of oxygen or the Hb state, we can readily suggest that only the PSH reactivity would govern the S-arylation process by quinones and the metabolic consequences of it. The present results are in agreement with those of other authors, who however, studied only different aspects of erythrocytic thiols [22,44,45]. For example, according to Miller and Smith [45] redox cycling phenomena elicited by menadione caused 40– 60% of GSH depletion in human erythrocytes, whereas this was almost total in the rat [44].
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In conclusion, our data suggest that within few minutes from erythrocyte exposure, menadione is conjugated to cellular thiols and oxidative reactions contemporaneously occur (GSSG and met-Hb formation). As a consequence of the formation of menadione adducts by cell thiols, oxidative reactions are further generated, involving oxygen consumption and leading to a massive and persistent hemoglobin oxidation, as well as a thiolic unbalance. Moreover, our data demonstrate that highly reactive protein thiols can undergo the same conjugative and redox cycling reactions as GSH. Therefore, they are not only a simple target of menadione toxicity, but are actively involved in primary toxic reactions (i.e. oxygen dependent redox cycling). It has previously been shown that rat platelets treated with menadione exhibit protein arylation and no increase in GSSG levels [46]. As a consequence of the no GSSG change, it was also suggested that protein arylation may be an injure mechanism different from redox cycling [46]. Experiments of rat RBC (Fig. 1) and the oxidant treatment of human platelets (that are characterized by having fast PSH) have shown that the GSSG increase may be only transient or negligible in comparison with that of GS– SP [47]. Consequently, GSSG may not always be considered a good index of redox cycling phenomena. In agreement with other authors, the alkylation reactions are important metabolic events that increase the toxicity of quinones exerted by redox cycling processes [24,48]. Data here presented on rat RBC have emphasized the pro-oxidant role of PSH. However, the generalization of the protein action in other cell models seems to be rather difficult because it is linked to the occurrence of other events related to the protein accessibility to oxygen and reducing agents. In plasma, that is constitutively poor in NPSH and relatively rich in PSH (albumin), the contribution of PSH to redox cycling phenomena by menadione has been clearly evidenced [49]. Acknowledgements Fruitful discussions and helpful suggestions by Dr Christine Winterbourn are gratefully acknowledged. This study was supported in part by MURST and PAR programs of research. References [1] H. Thor, M.T. Smith, P. Hartzell, G. Bellomo, S.A. Jewell, S. Orrenius, The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes, J. Biol. Chem. 257 (1982) 12419–12425. [2] D. Di Monte, D. Ross, G. Bellomo, L. Eklo¨ w, S. Orrenius, Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes, Arch. Biochem. Biophys. 235 (1984) 334 –342. [3] D. Di Monte, G. Bellomo, H. Thor, P. Nicotera, S. Orrenius, Menadione-induced cytotoxicity is associated with protein thiol oxidation and alteration in intracellular Ca2 + homeostasis, Arch. Biochem. Biophys. 235 (1984) 343 –350.
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