- freely available
Toxins 2014, 6(3), 1096-1108; doi:10.3390/toxins6031096
Published: 18 March 2014
Abstract: Background: Mushroom tyrosinase, a copper containing enzyme, modifies growth and survival of tumor cells. Mushroom tyrosinase may foster apoptosis, an effect in part due to interference with mitochondrial function. Erythrocytes lack mitochondria but are able to undergo apoptosis-like suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and activation of sphingomyelinase with subsequent formation of ceramide. The present study explored, whether tyrosinase stimulates eryptosis. Methods: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 24 h exposure to mushroom tyrosinase (7 U/mL) was followed by a significant increase of [Ca2+]i, a significant increase of ceramide abundance, and a significant increase of annexin-V-binding. The annexin-V-binding following tyrosinase treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca2+. Tyrosinase did not significantly modify forward scatter. Conclusions: Tyrosinase triggers cell membrane scrambling, an effect, at least partially, due to entry of extracellular Ca2+ and ceramide formation.
Mushroom tyrosinase has been suggested for the use in malignancy . When applied with appropriate substrates, it may generate cytostatic products effective in vivo [2,3,4]. Tyrosinase may at least in part be effective by interference with mitochondrial function . On the other hand, mushroom tyrosinase generates products leading to mutagenesis and carcinogenesis [5,6,7,8,9,10]. As a matter of fact, tyrosinase has been shown to trigger the suicidal death of nucleated cells or apoptosis .
Even though lacking mitochondria and nuclei, erythrocytes are still able to undergo apoptosis-like suicidal death or eryptosis . Eryptosis may be elicited by increase of cytosolic Ca2+ concentration ([Ca2+]i) resulting at least partially from Ca2+ entry through Ca2+-permeable cation channels . An increase of [Ca2+]i may shrink erythrocytes due to activation of Ca2+-sensitive K+ channels leading to K+ exit, hyperpolarization, Cl- exit and, thus, cellular loss of KCl and osmotically obliged water . Increased [Ca2+]i further stimulates cell membrane scrambling with translocation of phosphatidylserine to the erythrocyte surface . The Ca2+ sensitivity of cell membrane scrambling is increased by ceramide . Signaling of eryptosis further includes caspases [14,15,16,17,18] and several kinases including AMP activated kinase AMPK , casein kinase 1α [20,21], cGMP-dependent protein kinase , Janus-activated kinase JAK3 , protein kinase C , p38 kinase , PAK2 kinase , as well as sorafenib  and sunifinib  sensitive kinases.
Eryptosis is stimulated by a wide variety of xenobiotics [12,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63] and excessive eryptosis is observed in several clinical disorders , such as diabetes [18,64,65], renal insufficiency , hemolytic uremic syndrome , sepsis , malaria , sickle cell disease , Wilson’s disease , iron deficiency , malignancy , phosphate depletion , and metabolic syndrome .
The present study explored, whether tyrosinase influences [Ca2+]i, cell volume and phosphatidylserine translocation to the erythrocyte surface. The observations disclose that exposure to tyrosinase stimulates erythrocyte cell membrane scrambling, an effect paralleled by and at least in part secondary to increase of [Ca2+]i.
2. Results and Discussion
The present study addressed the effect of tyrosinase on eryptosis. A hallmark of eryptosis is the breakdown of phosphatidylserine asymmetry of the erythrocyte cell membrane, which increases the phosphatidylserine abundance at the cell surface. Phosphatidylserine exposing erythrocytes were identified by annexin-V-binding in FACS analysis. As illustrated in Figure 1, a 24-h exposure to tyrosinase increased the percentage of annexin-V-binding erythrocytes, an effect reaching statistical significance at 5 U/mL tyrosinase activity.
A second hallmark of eryptosis is cell shrinkage. Accordingly, cell volume was estimated utilizing forward scatter, which was determined by flow cytometry. As shown in Figure 2, a 24-h exposure to tyrosinase tended to increase erythrocyte forward scatter, an effect, however, not reaching statistical significance. Further experiments were performed to elucidate whether tyrosinase abrogates the effect of the Ca2+ ionophore inomomycin (1 µM) on erythrocytes forward scatter. As a result, a 30-min exposure of erythrocytes was followed by a decrease of forward scatter from 518 ± 6 (n = 4) to 134 ± 5 (n = 4) in the absence of tyrosinase and from 557 ± 8 (n = 4) to 194 ± 16 (n = 4) in the presence of 7 U/mL tyrosinase. Thus, tyrosinase did not abrogate the shrinking effect of excessive Ca2+ entry.
Cell membrane scrambling is stimulated by increase of cytosolic Ca2+ activity ([Ca2+]i). Thus, [Ca2+]i was determined utilizing Fluo3 fluorescence. To this end, erythrocytes were loaded with Fluo3-AM and Fluo3 fluorescence determined in FACS analysis following prior incubation in Ringer solution without or with tyrosinase. As shown in Figure 3, a 24-h exposure of human erythrocytes to tyrosinase was followed by an increase of Fluo3 fluorescence, an effect reaching statistical significance at 5 U/mL of tyrosinase concentration.
An additional series of experiments was performed testing whether extracellular Ca2+ entry was required for the effect of tyrosinase on cell membrane scrambling. Erythrocytes were exposed to 7 U/mL tyrosinase for 24 h, either in the presence of 1 mM Ca2+, or in the absence of Ca2+, and the presence of Ca2+ chelator EGTA (1 mM). As illustrated in Figure 4, the effect of tyrosinase on annexin-V-binding was significantly decreased in the nominal absence of Ca2+. However, even in the absence of extracellular Ca2+ tyrosinase still increased the percentage annexin V binding erythrocytes.
In order to test whether tyrosinase increased the formation of ceramide, which is known to trigger eryptosis even without increase of [Ca2+]i, ceramide abundance at the erythrocyte surface was determined utilizing an anti-ceramide antibody. As illustrated in Figure 5, exposure of erythrocytes to 7 U/mL tyrosinase significantly increased the abundance of ceramide at the erythrocyte surface.
The present study uncovers that tyrosinase stimulates cell membrane scrambling leading to phosphatidylserine translocation to the erythrocyte surface. Treatment of human erythrocytes with 5 U/L tyrosinase is further followed by increase of cytosolic Ca2+ activity ([Ca2+]i) and ceramide formation.
Despite its effect on [Ca2+]i, tyrosinase did not decrease but tended to increase the erythrocyte forward scatter. The increase of [Ca2+]i were expected to activate Ca2+ sensitive K+ channels  with subsequent K+ exit, cell membrane hyperpolarization, Cl− exit and, thus, cellular loss of KCl with osmotically obliged water . Possibly, tyrosinase inhibits the Ca2+ sensitive K+ channels, blocks the Cl− channels or stimulates some other mechanism increasing cell volume. Notably, the ionomycin induced erythrocyte shrinkage was not abrogated in the presence of tyrosinase. Excessive erythrocyte swelling may eventually result in rupture of the cell membrane leading to release of cellular hemoglobin, which is filtered in renal glomerula and subsequently occludes renal tubules .
Eryptosis is followed by removal of the defective erythrocytes, Phosphatidylserine at the surface of eryptotic cells binds to the respective receptors of phagocytosing cells leading to subsequent engulfment of the affected erythrocytes . Accordingly, eryptotic cells are rapidly cleared from circulating blood . If the accelerated loss of erythrocytes during stimulated eryptosis is not compensated by enhanced formation of new erythrocytes, the clearance of eryptotic erythrocytes from circulating blood results in anemia .
Phosphatidylserine exposing erythrocytes may further adhere to the vascular wall by binding of phosphatidylserine at the erythrocyte surface to endothelial CXCL16/SR-PSO . The adherence of suicidal erythrocytes to the vascular wall is expected to interfere with microcirculation [76,77,78,79,80,81]. Phosphatidylserine exposing erythrocytes have further been shown to trigger blood clotting and, thus, foster thrombosis [77,82,83].
3. Experimental Section
3.1. Erythrocytes, Solutions and Chemicals
Leukocyte-depleted erythrocytes were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003V). Erythrocytes were incubated in vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4 at 37 °C for 48 h. Where indicated, erythrocytes were exposed to mushroom tyrosinase (Sigma, Aldrich, Germany) at the indicated concentrations. In Ca2+-free Ringer solution, 1 mM CaCl2 was substituted by 1 mM glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA).
3.2. FACS Analysis of Annexin-V-Binding and Forward Scatter
After incubation under the respective experimental condition, 50 µL cell suspension was washed in Ringer solution containing 5 mM CaCl2 and then stained with Annexin-V-FITC (1:200 dilution; ImmunoTools, Friesoythe, Germany) in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD, Heidelberg, Germany). Following treatment with Ca2+-free Ringer solution, care was taken to measure annexin V binding rapidly enough to avoid triggering of cell membrane by the addition of 5 mM Ca2+.
3.3. Measurement of Intracellular Ca2+
After incubation erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM (Biotium, Hayward, USA) in Ringer solution containing 5 mM CaCl2 and 5 µM Fluo-3/AM. The cells were incubated at 37 °C for 30 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 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur.
3.4. Determination of Ceramide Formation
For the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 h at 37 °C with 1 µg/mL anti ceramide antibody (clone MID 15B4, Alexis, Grünberg, Germany) in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:5. The samples were washed twice with PBS-BSA. Subsequently, the 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. The samples were then analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.
Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and t-test as appropriate. n denotes the number of different erythrocyte specimens studied. As different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.
Tyrosinase stimulates Ca2+ entry, which in turn triggers cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface.
The authors acknowledge the meticulous preparation of the manuscript by Ali Soleimanpour and Tanja Loch. The study was supported by the Deutsche Forschungsgemeinschaft and the Open Access Publishing Fund of Tuebingen University.
Leonie Frauenfeld, Kousi Alzoubi, Majed Abed: have done the experiments, collected the data, prepared the figures. Florian Lang: conception and writing of the paper. All authors have read and approved the final version of the paper.
Conflicts of Interest
The authors declare no conflict of interest.
- Seo, S.Y.; Sharma, V.K.; Sharma, N. Mushroom tyrosinase: Recent prospects. J. Agric. Food Chem. 2003, 51, 2837–2853, doi:10.1021/jf020826f.
- Vogel, F.S.; Kemper, L.A.; Jeffs, P.W.; Cass, M.W.; Graham, D.G. Gamma-l-glutaminyl-4-hydroxybenzene, an inducer of cryptobiosis in agaricus bisporus and a source of specific metabolic inhibitors for melanogenic cells. Cancer Res. 1977, 37, 1133–1136.
- Wick, M.M.; Fitzgerald, G. Inhibition of reverse transcriptase by tyrosinase generated quinones related to levodopa and dopamine. Chem. Biol. Interact. 1981, 38, 99–107, doi:10.1016/0009-2797(81)90156-3.
- Wick, M.M.; Rosowsky, A.; Ratliff, J. Antitumor effects of L-glutamic acid dihydroxyanilides against experimental melanoma. J. Invest. Dermatol. 1980, 74, 112–114, doi:10.1111/1523-1747.ep12520030.
- Walton, K.; Walker, R.; Ioannides, C. Effect of baking and freeze-drying on the direct and indirect mutagenicity of extracts from the edible mushroom agaricus bisporus. Food Chem. Toxicol. 1998, 36, 315–320, doi:10.1016/S0278-6915(97)00161-0.
- Toth, B. Mushroom toxins and cancer (review). Int. J. Oncol. 1995, 6, 137–145.
- Walton, K.; Coombs, M.M.; Walker, R.; Ioannides, C. Bioactivation of mushroom hydrazines to mutagenic products by mammalian and fungal enzymes. Mutat. Res. 1997, 381, 131–139, doi:10.1016/S0027-5107(97)00160-7.
- Walton, K.; Coombs, M.M.; Walker, R.; Ioannides, C. The metabolism and bioactivation of agaritine and of other mushroom hydrazines by whole mushroom homogenate and by mushroom tyrosinase. Toxicology 2001, 161, 165–177, doi:10.1016/S0300-483X(00)00430-3.
- Papaparaskeva, C.; Ioannides, C.; Walker, R. Agaritine does not mediate the mutagenicity of the edible mushroom agaricus bisporus. Mutagenesis 1991, 6, 213–217, doi:10.1093/mutage/6.3.213.
- Papaparaskeva-Petrides, C.; Ioannides, C.; Walker, R. Contribution of phenolic and quinonoid structures in the mutagenicity of the edible mushroom agaricus bisporus. Food Chem. Toxicol. 1993, 31, 561–567, doi:10.1016/0278-6915(93)90205-D.
- Russo, G.L.; De Nisco, E.; Fiore, G.; Di Donato, P.; d’Ischia, M.; Palumbo, A. Toxicity of melanin-free ink of sepia officinalis to transformed cell lines: Identification of the active factor as tyrosinase. Biochem. Biophys. Res. Commun. 2003, 308, 293–299, doi:10.1016/S0006-291X(03)01379-2.
- Lang, E.; Qadri, S.M.; Lang, F. Killing me softly—Suicidal erythrocyte death. Int. J. Biochem. Cell Biol. 2012, 44, 1236–1243, doi:10.1016/j.biocel.2012.04.019.
- Lang, P.A.; Kaiser, S.; Myssina, S.; Wieder, T.; Lang, F.; Huber, S.M. Role of Ca2+-activated K+ channels in human erythrocyte apoptosis. Am. J. Physiol. Cell Physiol. 2003, 285, C1553–C1560, doi:10.1152/ajpcell.00186.2003.
- Bhavsar, S.K.; Bobbala, D.; Xuan, N.T.; Foller, M.; Lang, F. Stimulation of suicidal erythrocyte death by alpha-lipoic acid. Cell Physiol. Biochem. 2010, 26, 859–868, doi:10.1159/000323995.
- Foller, M.; Huber, S.M.; Lang, F. Erythrocyte programmed cell death. IUBMB Life 2008, 60, 661–668, doi:10.1002/iub.106.
- Foller, M.; Mahmud, H.; Gu, S.; Wang, K.; Floride, E.; Kucherenko, Y.; Luik, S.; Laufer, S.; Lang, F. Participation of leukotriene C(4) in the regulation of suicidal erythrocyte death. J. Physiol. Pharmacol. 2009, 60, 135–143.
- Lau, I.P.; Chen, H.; Wang, J.; Ong, H.C.; Leung, K.C.; Ho, H.P.; Kong, S.K. In vitro effect of ctab- and peg-coated gold nanorods on the induction of eryptosis/erythroptosis in human erythrocytes. Nanotoxicology 2012, 6, 847–856, doi:10.3109/17435390.2011.625132.
- Maellaro, E.; Leoncini, S.; Moretti, D.; Del Bello, B.; Tanganelli, I.; De Felice, C.; Ciccoli, L. Erythrocyte caspase-3 activation and oxidative imbalance in erythrocytes and in plasma of type 2 diabetic patients. Acta Diabetol. 2013, 50, 489–495, doi:10.1007/s00592-011-0274-0.
- Foller, M.; Sopjani, M.; Koka, S.; Gu, S.; Mahmud, H.; Wang, K.; Floride, E.; Schleicher, E.; Schulz, E.; Munzel, T.; et al. Regulation of erythrocyte survival by amp-activated protein kinase. FASEB J. 2009, 23, 1072–1080, doi:10.1096/fj.08-121772.
- Kucherenko, Y.; Zelenak, C.; Eberhard, M.; Qadri, S.M.; Lang, F. Effect of casein kinase 1alpha activator pyrvinium pamoate on erythrocyte ion channels. Cell Physiol. Biochem. 2012, 30, 407–417, doi:10.1159/000339034.
- Zelenak, C.; Eberhard, M.; Jilani, K.; Qadri, S.M.; Macek, B.; Lang, F. Protein kinase ck1alpha regulates erythrocyte survival. Cell Physiol. Biochem. 2012, 29, 171–180, doi:10.1159/000337598.
- Foller, M.; Feil, S.; Ghoreschi, K.; Koka, S.; Gerling, A.; Thunemann, M.; Hofmann, F.; Schuler, B.; Vogel, J.; Pichler, B.; et al. Anemia and splenomegaly in cgki-deficient mice. Proc. Natl. Acad. Sci. USA 2008, 105, 6771–6776, doi:10.1073/pnas.0708940105.
- Bhavsar, S.K.; Gu, S.; Bobbala, D.; Lang, F. Janus kinase 3 is expressed in erythrocytes, phosphorylated upon energy depletion and involved in the regulation of suicidal erythrocyte death. Cell Physiol. Biochem. 2011, 27, 547–556, doi:10.1159/000329956.
- Klarl, B.A.; Lang, P.A.; Kempe, D.S.; Niemoeller, O.M.; Akel, A.; Sobiesiak, M.; Eisele, K.; Podolski, M.; Huber, S.M.; Wieder, T.; et al. Protein kinase c mediates erythrocyte “programmed cell death” following glucose depletion. Am. J. Physiol. Cell Physiol. 2006, 290, C244–C253.
- Gatidis, S.; Zelenak, C.; Fajol, A.; Lang, E.; Jilani, K.; Michael, D.; Qadri, S.M.; Lang, F. P38 MAPK activation and function following osmotic shock of erythrocytes. Cell Physiol. Biochem. 2011, 28, 1279–1286, doi:10.1159/000335859.
- Zelenak, C.; Foller, M.; Velic, A.; Krug, K.; Qadri, S.M.; Viollet, B.; Lang, F.; Macek, B. Proteome analysis of erythrocytes lacking amp-activated protein kinase reveals a role of pak2 kinase in eryptosis. J. Proteome Res. 2011, 10, 1690–1697, doi:10.1021/pr101004j.
- Lupescu, A.; Shaik, N.; Jilani, K.; Zelenak, C.; Lang, E.; Pasham, V.; Zbidah, M.; Plate, A.; Bitzer, M.; Foller, M.; et al. Enhanced erythrocyte membrane exposure of phosphatidylserine following sorafenib treatment: An in vivo and in vitro study. Cell Physiol. Biochem. 2012, 30, 876–888, doi:10.1159/000341465.
- Shaik, N.; Lupescu, A.; Lang, F. Sunitinib-sensitive suicidal erythrocyte death. Cell Physiol. Biochem. 2012, 30, 512–522, doi:10.1159/000341434.
- Abed, M.; Towhid, S.T.; Mia, S.; Pakladok, T.; Alesutan, I.; Borst, O.; Gawaz, M.; Gulbins, E.; Lang, F. Sphingomyelinase-induced adhesion of eryptotic erythrocytes to endothelial cells. Am. J. Physiol. Cell Physiol. 2012, 303, C991–C999, doi:10.1152/ajpcell.00239.2012.
- Abed, M.; Towhid, S.T.; Shaik, N.; Lang, F. Stimulation of suicidal death of erythrocytes by rifampicin. Toxicology 2012, 302, 123–128, doi:10.1016/j.tox.2012.10.006.
- Bottger, E.; Multhoff, G.; Kun, J.F.; Esen, M. Plasmodium falciparum-infected erythrocytes induce granzyme b by nk cells through expression of host-hsp70. PLoS One 2012, 7, e33774, doi:10.1371/journal.pone.0033774.
- Firat, U.; Kaya, S.; Cim, A.; Buyukbayram, H.; Gokalp, O.; Dal, M.S.; Tamer, M.N. Increased caspase-3 immunoreactivity of erythrocytes in stz diabetic rats. Exp. Diabetes Res. 2012, 2012, 316384.
- Ganesan, S.; Chaurasiya, N.D.; Sahu, R.; Walker, L.A.; Tekwani, B.L. Understanding the mechanisms for metabolism-linked hemolytic toxicity of primaquine against glucose 6-phosphate dehydrogenase deficient human erythrocytes: Evaluation of eryptotic pathway. Toxicology 2012, 294, 54–60, doi:10.1016/j.tox.2012.01.015.
- Gao, M.; Cheung, K.L.; Lau, I.P.; Yu, W.S.; Fung, K.P.; Yu, B.; Loo, J.F.; Kong, S.K. Polyphyllin D induces apoptosis in human erythrocytes through Ca2+ rise and membrane permeabilization. Arch. Toxicol. 2012, 86, 741–752, doi:10.1007/s00204-012-0808-4.
- Ghashghaeinia, M.; Cluitmans, J.C.; Akel, A.; Dreischer, P.; Toulany, M.; Koberle, M.; Skabytska, Y.; Saki, M.; Biedermann, T.; Duszenko, M.; et al. The impact of erythrocyte age on eryptosis. Br. J. Haematol. 2012, 157, 606–614, doi:10.1111/j.1365-2141.2012.09100.x.
- Jilani, K.; Lupescu, A.; Zbidah, M.; Abed, M.; Shaik, N.; Lang, F. Enhanced apoptotic death of erythrocytes induced by the mycotoxin ochratoxin A. Kidney Blood Press. Res. 2012, 36, 107–118, doi:10.1159/000341488.
- Jilani, K.; Lupescu, A.; Zbidah, M.; Shaik, N.; Lang, F. Withaferin a-stimulated Ca2+ entry, ceramide formation and suicidal death of erythrocytes. Toxicol. in Vitro 2013, 27, 52–58, doi:10.1016/j.tiv.2012.09.004.
- Kucherenko, Y.V.; Lang, F. Inhibitory effect of furosemide on non-selective voltage-independent cation channels in human erythrocytes. Cell Physiol. Biochem. 2012, 30, 863–875, doi:10.1159/000341464.
- Lang, E.; Qadri, S.M.; Jilani, K.; Zelenak, C.; Lupescu, A.; Schleicher, E.; Lang, F. Carbon monoxide-sensitive apoptotic death of erythrocytes. Basic Clin. Pharmacol. Toxicol. 2012, 111, 348–355.
- Lupescu, A.; Jilani, K.; Zbidah, M.; Lang, E.; Lang, F. Enhanced Ca(2+) entry, ceramide formation, and apoptotic death of erythrocytes triggered by plumbagin. J. Nat. Prod. 2012, 75, 1956–1961, doi:10.1021/np300611r.
- Lupescu, A.; Jilani, K.; Zbidah, M.; Lang, F. Induction of apoptotic erythrocyte death by rotenone. Toxicology 2012, 300, 132–137, doi:10.1016/j.tox.2012.06.007.
- Lupescu, A.; Jilani, K.; Zelenak, C.; Zbidah, M.; Qadri, S.M.; Lang, F. Hexavalent chromium-induced erythrocyte membrane phospholipid asymmetry. Biometals 2012, 25, 309–318, doi:10.1007/s10534-011-9507-5.
- Polak-Jonkisz, D.; Purzyc, L. Ca influx versus efflux during eryptosis in uremic erythrocytes. Blood Purif. 2012, 34, 209–210, doi:10.1159/000341627.
- Qian, E.W.; Ge, D.T.; Kong, S.K. Salidroside protects human erythrocytes against hydrogen peroxide-induced apoptosis. J. Nat. Prod. 2012, 75, 531–537, doi:10.1021/np200555s.
- Shaik, N.; Zbidah, M.; Lang, F. Inhibition of Ca(2+) entry and suicidal erythrocyte death by naringin. Cell Physiol. Biochem. 2012, 30, 678–686, doi:10.1159/000341448.
- Vota, D.M.; Maltaneri, R.E.; Wenker, S.D.; Nesse, A.B.; Vittori, D.C. Differential erythropoietin action upon cells induced to eryptosis by different agents. Cell Biochem. Biophys. 2013, 65, 145–157, doi:10.1007/s12013-012-9408-4.
- Weiss, E.; Cytlak, U.M.; Rees, D.C.; Osei, A.; Gibson, J.S. Deoxygenation-induced and Ca(2+) dependent phosphatidylserine externalisation in red blood cells from normal individuals and sickle cell patients. Cell Calcium 2012, 51, 51–56, doi:10.1016/j.ceca.2011.10.005.
- Zappulla, D. Environmental stress, erythrocyte dysfunctions, inflammation, and the metabolic syndrome: Adaptations to CO2 increases? J. Cardiometab Syndr. 2008, 3, 30–34, doi:10.1111/j.1559-4572.2008.07263.x.
- Zbidah, M.; Lupescu, A.; Jilani, K.; Lang, F. Stimulation of suicidal erythrocyte death by fumagillin. Basic Clin. Pharmacol. Toxicol. 2013, 112, 346–351, doi:10.1111/bcpt.12033.
- Zbidah, M.; Lupescu, A.; Shaik, N.; Lang, F. Gossypol-induced suicidal erythrocyte death. Toxicology 2012, 302, 101–105, doi:10.1016/j.tox.2012.09.010.
- Zelenak, C.; Pasham, V.; Jilani, K.; Tripodi, P.M.; Rosaclerio, L.; Pathare, G.; Lupescu, A.; Faggio, C.; Qadri, S.M.; Lang, F. Tanshinone IIA stimulates erythrocyte phosphatidylserine exposure. Cell Physiol. Biochem. 2012, 30, 282–294, doi:10.1159/000339064.
- Abed, M.; Herrmann, T.; Alzoubi, K.; Pakladok, T.; Lang, F. Tannic acid induced suicidal erythrocyte death. Cell Physiol. Biochem. 2013, 32, 1106–1116, doi:10.1159/000354510.
- Ahmed, M.S.; Langer, H.; Abed, M.; Voelkl, J.; Lang, F. The uremic toxin acrolein promotes suicidal erythrocyte death. Kidney Blood Press. Res. 2013, 37, 158–167, doi:10.1159/000350141.
- Ghashghaeinia, M.; Cluitmans, J.C.; Toulany, M.; Saki, M.; Koberle, M.; Lang, E.; Dreischer, P.; Biedermann, T.; Duszenko, M.; Lang, F.; et al. Age sensitivity of NFκB abundance and programmed cell death in erythrocytes induced by NFκB inhibitors. Cell Physiol. Biochem. 2013, 32, 801–813, doi:10.1159/000354481.
- Abed, M.; Feger, M.; Alzoubi, K.; Pakladok, T.; Frauenfeld, L.; Geiger, C.; Towhid, S.T.; Lang, F. Sensitization of erythrocytes to suicidal erythrocyte death following water deprivation. Kidney Blood Press. Res. 2013, 37, 567–578.
- Alzoubi, K.; Honisch, S.; Abed, M.; Lang, F. Triggering of suicidal erythrocyte death by penta-o-galloyl-beta-d-glucose. Toxins 2014, 6, 54–65, doi:10.3390/toxins6010054.
- Jilani, K.; Qadri, S.M.; Lang, F. Geldanamycin-induced phosphatidylserine translocation in the erythrocyte membrane. Cell Physiol. Biochem. 2013, 32, 1600–1609.
- Jilani, K.; Lang, F. Carmustine-induced phosphatidylserine translocation in the erythrocyte membrane. Toxins 2013, 5, 703–716, doi:10.3390/toxins5040703.
- Jilani, K.; Enkel, S.; Bissinger, R.; Almilaji, A.; Abed, M.; Lang, F. Fluoxetine induced suicidal erythrocyte death. Toxins 2013, 5, 1230–1243, doi:10.3390/toxins5071230.
- Bissinger, R.; Modicano, P.; Frauenfeld, L.; Lang, E.; Jacobi, J.; Faggio, C.; Lang, F. Estramustine-induced suicidal erythrocyte death. Cell Physiol. Biochem. 2013, 32, 1426–1436, doi:10.1159/000356580.
- Lupescu, A.; Jilani, K.; Zbidah, M.; Lang, F. Patulin-induced suicidal erythrocyte death. Cell Physiol. Biochem. 2013, 32, 291–299, doi:10.1159/000354437.
- Lupescu, A.; Bissinger, R.; Jilani, K.; Lang, F. Triggering of suicidal erythrocyte death by celecoxib. Toxins 2013, 5, 1543–1554, doi:10.3390/toxins5091543.
- Lang, E.; Modicano, P.; Arnold, M.; Bissinger, R.; Faggio, C.; Abed, M.; Lang, F. Effect of thioridazine on erythrocytes. Toxins 2013, 5, 1918–1931, doi:10.3390/toxins5101918.
- Calderon-Salinas, J.V.; Munoz-Reyes, E.G.; Guerrero-Romero, J.F.; Rodriguez-Moran, M.; Bracho-Riquelme, R.L.; Carrera-Gracia, M.A.; Quintanar-Escorza, M.A. Eryptosis and oxidative damage in type 2 diabetic mellitus patients with chronic kidney disease. Mol. Cell Biochem. 2011, 357, 171–179, doi:10.1007/s11010-011-0887-1.
- Nicolay, J.P.; Schneider, J.; Niemoeller, O.M.; Artunc, F.; Portero-Otin, M.; Haik, G., Jr.; Thornalley, P.J.; Schleicher, E.; Wieder, T.; Lang, F. Stimulation of suicidal erythrocyte death by methylglyoxal. Cell Physiol. Biochem. 2006, 18, 223–232, doi:10.1159/000097669.
- Myssina, S.; Huber, S.M.; Birka, C.; Lang, P.A.; Lang, K.S.; Friedrich, B.; Risler, T.; Wieder, T.; Lang, F. Inhibition of erythrocyte cation channels by erythropoietin. J. Am. Soc. Nephrol. 2003, 14, 2750–2757, doi:10.1097/01.ASN.0000093253.42641.C1.
- Lang, P.A.; Beringer, O.; Nicolay, J.P.; Amon, O.; Kempe, D.S.; Hermle, T.; Attanasio, P.; Akel, A.; Schafer, R.; Friedrich, B.; et al. Suicidal death of erythrocytes in recurrent hemolytic uremic syndrome. J. Mol. Med. (Berl.) 2006, 84, 378–388, doi:10.1007/s00109-006-0058-0.
- Kempe, D.S.; Akel, A.; Lang, P.A.; Hermle, T.; Biswas, R.; Muresanu, J.; Friedrich, B.; Dreischer, P.; Wolz, C.; Schumacher, U.; et al. Suicidal erythrocyte death in sepsis. J. Mol. Med. 2007, 85, 269–277.
- Foller, M.; Bobbala, D.; Koka, S.; Huber, S.M.; Gulbins, E.; Lang, F. Suicide for survival–death of infected erythrocytes as a host mechanism to survive malaria. Cell Physiol. Biochem. 2009, 24, 133–140, doi:10.1159/000233238.
- Lang, P.A.; Kasinathan, R.S.; Brand, V.B.; Duranton, C.; Lang, C.; Koka, S.; Shumilina, E.; Kempe, D.S.; Tanneur, V.; Akel, A.; et al. Accelerated clearance of plasmodium-infected erythrocytes in sickle cell trait and annexin-A7 deficiency. Cell Physiol. Biochem. 2009, 24, 415–428, doi:10.1159/000257529.
- Lang, P.A.; Schenck, M.; Nicolay, J.P.; Becker, J.U.; Kempe, D.S.; Lupescu, A.; Koka, S.; Eisele, K.; Klarl, B.A.; Rubben, H.; et al. Liver cell death and anemia in wilson disease involve acid sphingomyelinase and ceramide. Nat. Med. 2007, 13, 164–170, doi:10.1038/nm1539.
- Kempe, D.S.; Lang, P.A.; Duranton, C.; Akel, A.; Lang, K.S.; Huber, S.M.; Wieder, T.; Lang, F. Enhanced programmed cell death of iron-deficient erythrocytes. FASEB J. 2006, 20, 368–370.
- Qadri, S.M.; Mahmud, H.; Lang, E.; Gu, S.; Bobbala, D.; Zelenak, C.; Jilani, K.; Siegfried, A.; Foller, M.; Lang, F. Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene. J. Cell Mol. Med. 2012, 16, 1085–1093, doi:10.1111/j.1582-4934.2011.01387.x.
- Birka, C.; Lang, P.A.; Kempe, D.S.; Hoefling, L.; Tanneur, V.; Duranton, C.; Nammi, S.; Henke, G.; Myssina, S.; Krikov, M.; et al. Enhanced susceptibility to erythrocyte “apoptosis” following phosphate depletion. Pflugers Arch. 2004, 448, 471–477.
- Harrison, H.E.; Bunting, H.; Ordway, N.K.; Albrink, W.S. The pathogenesis of the renal injury produced in the dog by hemoglobin or methemoglobin. J. Exp. Med. 1947, 86, 339–356, doi:10.1084/jem.86.4.339.
- Borst, O.; Abed, M.; Alesutan, I.; Towhid, S.T.; Qadri, S.M.; Foller, M.; Gawaz, M.; Lang, F. Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX. Am. J. Physiol. Cell Physiol. 2012, 302, C644–C651, doi:10.1152/ajpcell.00340.2011.
- Andrews, D.A.; Low, P.S. Role of red blood cells in thrombosis. Curr. Opin. Hematol. 1999, 6, 76–82, doi:10.1097/00062752-199903000-00004.
- Closse, C.; Dachary-Prigent, J.; Boisseau, M.R. Phosphatidylserine-related adhesion of human erythrocytes to vascular endothelium. Br. J. Haematol. 1999, 107, 300–302, doi:10.1046/j.1365-2141.1999.01718.x.
- Gallagher, P.G.; Chang, S.H.; Rettig, M.P.; Neely, J.E.; Hillery, C.A.; Smith, B.D.; Low, P.S. Altered erythrocyte endothelial adherence and membrane phospholipid asymmetry in hereditary hydrocytosis. Blood 2003, 101, 4625–4627, doi:10.1182/blood-2001-12-0329.
- Pandolfi, A.; Di Pietro, N.; Sirolli, V.; Giardinelli, A.; Di Silvestre, S.; Amoroso, L.; Di Tomo, P.; Capani, F.; Consoli, A.; Bonomini, M. Mechanisms of uremic erythrocyte-induced adhesion of human monocytes to cultured endothelial cells. J. Cell Physiol. 2007, 213, 699–709, doi:10.1002/jcp.21138.
- Wood, B.L.; Gibson, D.F.; Tait, J.F. Increased erythrocyte phosphatidylserine exposure in sickle cell disease: Flow-cytometric measurement and clinical associations. Blood 1996, 88, 1873–1880.
- Chung, S.M.; Bae, O.N.; Lim, K.M.; Noh, J.Y.; Lee, M.Y.; Jung, Y.S.; Chung, J.H. Lysophosphatidic acid induces thrombogenic activity through phosphatidylserine exposure and procoagulant microvesicle generation in human erythrocytes. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 414–421.
- Zwaal, R.F.; Comfurius, P.; Bevers, E.M. Surface exposure of phosphatidylserine in pathological cells. Cell Mol. Life Sci. 2005, 62, 971–988, doi:10.1007/s00018-005-4527-3.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).