Tin triggers suicidal death of erythrocytes

Research Article Received: 27 June 2008, Revised: 25 August 2008, Accepted: 27 August 2008, Published online 20 October 2008 in Wiley Interscience (www.interscience.wiley.com) DOI 10.1002/jat.1390 Tin triggers suicidal death of erythrocytes John Wiley & Sons, Ltd. Tan Thanh Nguyen, Michael Föller and Florian Lang* Tin triggers suicidal death of erythrocytes ABSTRACT: Suicidal erythrocyte death or eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the erythrocyte surface. Triggers of eryptosis include increase in cytosolic Ca2+ activity, formation of ceramide and energy depletion. Excessive eryptosis contributes to several anemic conditions. Intoxication with inorganic tin(II) may lead to anemia. The present study therefore explored whether tin influences eryptosis. To this end, erythrocytic phosphatidylserine exposure was estimated from annexin V-binding, cell volume from forward scatter, cytosolic Ca2+ activity from Fluo3 fluorescence, ceramide formation from binding of fluorescent antibodies and cytosolic ATP utilizing a luciferin–luciferase assay kit. Under control conditions, eryptosis was observed in less than 5% of the erythrocytes. Exposure to tin (1–100 mM) significantly increased the percentage of PS-exposing erythrocytes and decreased cell volume. The effect was paralleled by an increase in the cytosolic Ca2+ concentration, ceramide formation and a decrease of intracellular ATP concentration. In conclusion, tin triggers eryptosis, an effect at least partially due to Ca2+ entry, ceramide formation and ATP depletion. The effect could contribute to tin-induced anemia. Copyright © 2008 John Wiley & Sons, Ltd. Keywords: apoptosis; eryptosis; phosphatidylserine; calcium; energy depletion; cell volume; osmotic shock; oxidative stress Introduction J. Appl. Toxicol. 2009; 29: 79–83 Materials and methods Erythrocytes, Solutions and Chemicals Experiments were performed at 37 °C with isolated erythrocytes drawn from healthy volunteers. The erythrocytes were kindly provided by the blood bank of the University of Tübingen. The volunteers provided informed consent. The study has been approved by the ethics committee of the University of Tübingen (184/2003V). Ringer solution contained (in mM): 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4. Tin was purchased from Sigma as SnCl2 (Schnelldorf, Germany). FACS Analysis of Annexin V Binding and Forward Scatter FACS analysis was performed as described (Lang et al., 2003a). After incubation at 37 °C in Ringer solution in the absence or presence of 1–100 μM SnCl2 for 48 h, cells were washed in Ringer solution containing 5 mM CaCl2. Erythrocytes were stained with Annexin V-Fluos (Roche, Mannheim, Germany) at a 1 : 500 dilution. * Correspondence to: F. Lang, Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany. E-mail: florian.lang@uni-tuebingen.de Copyright © 2008 John Wiley & Sons, Ltd. 79 Tin is an environmental toxin (Darbre, 2006), which has been shown to cause anemia (Pies, 1940; Stewart and Lassiter, 1999). Moreover, organotin compounds may trigger hemolysis (Gray et al., 1986). Thus, tin and organotin compounds may stimulate erythrocyte death. Mature circulating erythrocytes may undergo suicidal death or eryptosis (Lang et al., 2005), which is characterized by cell shrinkage and phosphatidylserine exposure at the cell surface (Allen et al., 1988; Connor et al., 1994; Schroit et al., 1985). Eryptosis could be triggered by activation of Ca2+-permeable erythrocyte cation channels (Lang et al., 2003a) The entry of Ca2+ is followed by activation of Ca2+-sensitive K+ channels, KCl exit, osmotic loss of cellular water and cell shrinkage (Lang et al., 2003b; Myssina et al., 2004). Ca2+ further elicits phospholipid scrambling of the cell membrane (Woon et al., 1999) with subsequent phosphatidylserine exposure at the cell surface. Further stimulators of cell membrane scrambling include ceramide (acylsphingosine) (Lang et al., 2004a) and protein kinase C activation (de Jong et al., 2002; Klarl et al., 2006), whereas activation of protein kinase G inhibits eryptosis (Foller et al., 2008). Eryptosis is enhanced in a wide variety of anemic conditions such as iron deficiency (Kempe et al., 2006), phosphate depletion (Birka et al., 2004), Hemolytic Uremic Syndrome (Lang et al., 2006a), sepsis (Kempe et al., 2007), malaria (Brand et al., 2003), Wilson’s disease (Lang et al., 2007), sickle cell disease (Hebbel 1991; Lang et al., 2002; Wood et al., 1996), thalassemia (Lang et al., 2002) and glucose-phosphate dehydrogenase deficiency (Lang et al., 2002). Moreover, eryptosis may be triggered by oxidative stress (Cimen 2008), cordycepin (Lui et al., 2007), methylglyoxal (Nicolay et al., 2006), amyloid (Nicolay et al., 2007), hemolysin (Lang et al., 2004c), listeriolysin (Föller et al., 2007), Bay-5884 (Shumilina et al., 2006), amantadine (Föller et al., 2008a), curcumin (Bentzen et al., 2007), paclitaxel (Lang et al., 2006b), valinomycin (Schneider et al., 2007), chlorpromazine (Akel et al., 2006), cyclosporine (Niemoeller et al., 2006a), aluminum (Niemoeller et al., 2006b), lead (Kempe et al., 2005; Slobozhanina et al., 2005), mercury (Eisele et al., 2006), gold (Sopjani et al., 2008), vanadium (Föller et al., 2008b), zinc (Kiedaisch et al., 2008) and copper (Lang et al., 2007). The present study explored whether eryptosis is modified by tin. To this end, erythrocytes were exposed to inorganic tin(II), and phosphatidylserine exposure, cell volume, intracellular Ca2+ concentration, ceramide formation and intracellular ATP concentration were quantified. T. T. Nguyen et al. After 15 min, samples were measured by flow cytometric analysis (FACS-Calibur from Becton Dickinson; Heidelberg, Germany). Cells were analysed by forward scatter, and annexin-V-fluorescence intensity was measured in fluorescence channel FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. Measurement of Intracellular Ca2+ Intracellular Ca2+ measurements were performed as described previously (Lang et al., 2003a). After incubation at 37 °C in Ringer solution in the absence or presence of 1–100 μM SnCl2 for 48 h, erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM (Calbiochem; Bad Soden, Germany) in Ringer solution containing 5 mM CaCl2 and 2 μM Fluo-3/AM. The cells were incubated at 37 °C for 20 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 μl Ringer. Then, Ca2+-dependent fluorescence intensity was measured in FL-1. Measurement of Hemolysis After incubation at 37 °C in Ringer solution in the absence or presence of 1–100 μM SnCl2 for 48 h, the samples were centrifuged (3 min at 400g, room temperature), and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis. Determination of Ceramide Formation To determine formation of ceramide, which is exposed at the cell surface, a monoclonal antibody-based assay was used (Bieberich et al., 2003; Grassme et al., 2002) in FACS analysis. After incubation at 37 °C in Ringer solution in the absence or presence of 1–100 μM SnCl2 for 48 h, cells were stained for 1 h at 37 °C with 1 μg ml−1 anti-ceramide antibody (clone MID 15B4; Alexis, Grünberg, Germany) in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:5. After two washing steps with PBS-BSA, cells were stained for 30 min with polyclonal fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse IgG and IgM specific antibody (Pharmingen, Hamburg, Germany) diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. Samples were then analysed by flow cytometric analysis on a FACS-Calibur in FL-1. Statistics Data are expressed as arithmetic means ± SEM and statistical analysis was made by ANOVA. Results Phosphatidylserine-exposing erythrocytes were identified by determination of annexin V binding at the cell surface (Lang et al., 2005). The percentage of erythrocytes exposing annexin V following incubation for 48 h in Ringer solution remained below 5% (Fig. 1). A 48 h exposure to tin (1–100 μM) significantly increased the percentage of annexin V-binding erythrocytes [Fig. 1(B)]. To explore whether the effect of tin on erythrocytes is the consequence of tin-dependent damage of the cell membrane, the percentage of hemolysed erythrocytes was determined after a 48 h incubation with or without tin. As shown in Fig. 2(A), no appreciable rate of hemolysis was induced by tin. Furthermore, fluorescence microscopy of annexin V-stained erythrocytes after exposure to tin revealed that tin does not impair the integrity of the cell membrane [Fig. 2(B)]. Alterations of cell volume, another hallmark of eryptosis (Allan and Thomas 1981; Crespo et al., 1987; Lang et al., 2003b; Li et al., 1996; Myssina et al., 2004), were estimated from erythrocyte forward scatter, which decreases upon cell shrinkage. As a result, exposure to 3 μM tin significantly shrunk the erythrocytes, an effect becoming even more prominent at higher tin concentrations [Fig. 3(A, B)]. An increase in the intracellular Ca2+ concentration is known to stimulate eryptosis (Lang et al., 2003a). Therefore, the Ca2+-sensitive dye Fluo3 was employed in FACS analysis to determine whether tin induces an increase in the cytosolic Ca2+ level of erythrocytes. As shown in Fig. 4(A, B), higher tin concentrations led to a significant increase in the intracellular Ca2+ concentration of erythrocytes. Another trigger of eryptosis is ceramide, which sensitizes the erythrocyte to the eryptotic effects of Ca2+ (Lang et al., 2004a). Therefore, the formation of ceramide was investigated upon exposure to tin. As shown in Fig. 5(A, B), tin also stimulated Determination of the Intracellular ATP Concentration 80 Ninty microliters of erythrocyte pellet were incubated for 48 h at 37 °C in Ringer solution with or without 1–100 μM tin (final hematocrit 5%). All manipulations were then performed at 4 °C to avoid ATP degradation. Cells were lysed in distilled water, and proteins were precipitated by addition of HClO4 (5%). After centrifugation, an aliquot of the supernatant (400 μl) was adjusted to pH 7.7 by addition of saturated KHCO3 solution. After dilution of the supernatant, the ATP concentration of the aliquots was determined utilizing a luciferin–luciferase assay kit (Roche Diagnostics) on a luminometer (Berthold Biolumat LB9500, Bad Wildbad, Germany) according to the manufacturer’s protocol. ATP concentrations are expressed as mmol l−1 packed erythrocyte volume. www.interscience.wiley.com/journal/jat Figure 1. Stimulation of phosphatidylserine exposure by tin(II). (A) Histogram of erythrocyte annexin V-binding in a representative experiment of erythrocytes from healthy volunteers incubated for 48 h in the absence ( − , black line) or presence ( + , red line) of 100 μM tin. (B) Arithmetic means ± SEM (n = 45–49) of the percentage of annexin V-binding erythrocytes following incubation for 48 h in the absence (white bar) or presence (black bars) of tin (1–100 μM). ***Significant difference from the absence of tin (P ≤ 0.001). This figure is available in colour online at www.interscience.wiley.com/journal/jat Copyright © 2008 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2009; 29: 79–83 Tin triggers suicidal death of erythrocytes Figure 2. Analysis of the integrity of the erythrocyte membrane under the influence of tin(II). (A) Arithmetic means ± SEM (n = 8) of the percentage of hemolysed erythrocytes exposed for 48 h to Ringer solution without (white bar) or with (black bars) tin chloride at the indicated concentrations. (B) Transmission microphotograph (left panel) and fluorescence microphotograph (right panel) of an erythrocyte stained with fluorescent annexin V. Prior to microscopy, the erythrocytes were exposed for 48 h to 100 μM tin chloride in Ringer solution. Figure 3. Decrease of forward scatter by tin(II). (A) Histogram of erythrocytic forward scatter in a representative experiment of erythrocytes from healthy volunteers incubated for 48 h in the absence ( −, black line) or presence ( +, red line) of 100 μM tin. (B) Arithmetic means ± SEM (n = 44–48) of the normalized erythrocyte forward scatter following incubation for 48 h in the absence (white bar) or presence (black bars) of tin (1–100 μM). *,***Significant difference from the absence of tin (P ≤ 0.05, P ≤ 0.001). This figure is available in colour online at www.interscience. wiley.com/journal/jat Figure 5. Effect of tin(II) on ceramide abundance in erythrocytes. (A) Histogram of ceramide-dependent FITC-fluorescence in a representative experiment of erythrocytes from healthy volunteers incubated for 48 h in the absence ( −, black line) or presence ( +, red line) of 100 μM tin. (B) Arithmetic means ± SEM (n = 8) of the normalized geo means of ceramidedependent FITC-fluorescence in erythrocytes incubated for 48 h in Ringer solution in the absence (white bar) or presence (black bars) of tin (1–100 μM). ***Significant difference from the absence of tin (P ≤ 0.001). This figure is available in colour online at www.interscience.wiley.com/journal/jat ceramide formation in erythrocytes, an effect presumably contributing to tin-induced eryptosis. Eryptosis is further known to be stimulated by energy depletion, which is evident from a decreased intracellular ATP concentration (Klarl et al., 2006). Exposure of erythrocytes to tin indeed significantly reduced the intracellular ATP concentration (Fig. 6). Exposure to glucose-free Ringer, serving as a positive control, similarly decreased cytosolic ATP concentration (Fig. 6). Discussion J. Appl. Toxicol. 2009; 29: 79–83 The present study shows that tin increases cytosolic Ca2+ activity, stimulates the formation of ceramide and decreases cytosolic ATP concentration. Each increase in cytosolic Ca2+ activity (Lang et al., 2003a), ceramide (Lang et al., 2004a) and ATP depletion (Klarl et al., 2006) is known to stimulate phospholipid scambling of the cell membrane. Accordingly, tin exposure indeed stimulates phosphatidylserine exposure at the cell surface. Tin exposure is further followed by a decrease in forward scatter, an indicator of cell shrinkage. The erythrocyte shrinkage following exposure to tin is at least partially due to activation of Ca2+- Copyright © 2008 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jat 81 Figure 4. Effect of tin(II) on the cytosolic Ca2+ concentration of erythrocytes. (A) Histogram of Fluo3-dependent fluorescence in a representative experiment of erythrocytes from healthy volunteers incubated for 48 h in the absence ( − , black line) or presence ( + , red line) of 100 μM tin. (B) Arithmetic means ± SEM (n = 43–48) of the normalized geo means of Fluo3-dependent fluorescence in erythrocytes incubated for 48 h in Ringer solution in the absence (white bar) or presence (black bars) of tin (1–100 μM). *** Significant difference from the absence of tin (P ≤ 0.001). This figure is available in colour online at www.interscience. wiley.com/journal/jat T. T. Nguyen et al. Acknowledgements The authors acknowledge the meticulous preparation of the manuscript by Tanja Loch and Lejla Subasic. This study was supported by the Deutsche Forschungsgemeinschaft, Nr. La 315/4-3 and La 315/13-1 and the Carl-Zeiss-Stiftung. References Figure 6. Effect of tin(II) on the ATP content of erythrocytes. Arithmetic means ± SEM (n = 4) of the intracellular ATP concentration of erythrocytes incubated for 48 h in Ringer solution in the absence (white bar) or presence (black bars) of tin (1–100 μM). Incubation of erythrocytes in glucose-deprived Ringer served as a positive control (grey bar). *,**Significant difference from control (P ≤ 0.05, P ≤ 0.01). 82 sensitive K+ channels, which leads to hyperpolarization of the cell membrane, imposing an electrical driving force for exit of Cl− (Lang et al., 2003b; Myssina et al., 2004). The cellular loss of K+, Cl− and osmotically obliged water then leads to cell shrinkage (Lang et al., 2003b; Myssina et al., 2004). On the other hand, organometallic tin compounds have been shown to inhibit Na+/ K+-ATPase activity (Samuel et al., 1998), an effect, which eventually leads to loss of the K+ gradient across the cell membrane and depolarization with entry of Cl− and subsequent cell swelling. The concentrations required to induce eryptosis are well in the range of concentrations of tin determined in subjects with tin intoxication (Ohhira and Matsui 2003; Stewart and Lassiter 1999). Accordingly, the observed stimulation of eryptosis could participate in the in vivo toxicity of tin and could explain the observed tin-induced anemia (Pies, 1940; Stewart and Lassiter 1999). Erythrocytes exposing phosphatidylserine at their surface are engulfed by macrophages and thus eliminated from the blood stream (Allen et al., 1988; Boas et al., 1998; Connor et al., 1994; Kempe et al., 2006; Schroit et al., 1985). Thus, similar to erythrocyte senescence (Arese et al., 2005; Barvitenko et al., 2005; Bosman et al., 2005) and neocytolysis (Rice and Alfrey 2005), eryptosis leads to the clearance of erythrocytes from circulating blood. Phosphatidylserine (PS)- exposing erythrocytes could further bind to the vascular wall and thus impede microcirculation (Andrews and Low 1999; Closse et al., 1999; Gallagher et al., 2003) and trigger hemostasis (Andrews and Low 1999). For instance, enhanced trapping of annexin V-binding erythrocytes may occur in renal medulla following renal ischemia (Lang et al., 2004b). Alkyl-di-organotincompounds have been shown to inhibit cell proliferation and exert antitumor activity (Koch et al., 2008). It is tempting to speculate that tin similarly induces suicidal cell death of nucleated cells. Possibly, tin similarly increases cytosolic Ca2+ activity and stimulates the formation of ceramide in nucleated cells. Excessive Ca2+ entry (Orrenius et al., 2003) and ceramide (Gulbins and Li 2006) are both well-known triggers of suicidal death of nucleated cells or apoptosis. In conclusion, tin triggers eryptosis, the suicidal death of erythrocytes. 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