Enhanced Eryptosis in Glucose-6-Phosphate Dehydrogenase Deficiency.

BACKGROUND/AIMS: Defects in the Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme enhance cellular oxidative damage, thus impairing erythrocytes and radically shortening their lifespan. We aimed to study programmed erythrocyte cell death in G6PD-deficient patients, describe the molecular genetics basis of G6PD and investigate phenotype-genotype correlations. METHODS: We explored eryptosis using the annexin V-binding assay, taken as an indicator of PS exposure at the erythrocyte surface. We assessed reactive oxygen species (ROS) production, intracellular calcium concentrations and ceramide formation at the cell surface. Prior to and following treatments, cells were analyzed by flow cytometry. Finally, we explored G6PD gene mutations through PCR-Sanger sequencing. RESULTS: Before stimulation, PS-exposing erythrocytes were significantly higher in G6PD-deficient patients than in healthy volunteers. This was paralleled by a significant increase in reactive oxygen species production, suggesting that oxidative stress is the main trigger of PS exposure in G6PD-deficient erythrocytes. Five previously described mutations were detected in our patients. Two genotypes correlated with a significantly higher percentage of PS-exposing cells. CONCLUSION: Our study uncovers a novel effect detected in G6PD-deficient erythrocytes which is cell membrane scrambling with PS translocation to the erythrocyte surface. Our findings shed a light on the mechanisms of premature erythrocyte clearance in G6PD deficiency.

Biochemical, Cellular, and Proteomic Characterization of Hereditary Spherocytosis Among Tunisians.

BACKGROUND/AIMS: Hereditary Spherocytosis (HS) is the most common erythrocyte membrane disorder causing hemolytic anemia. The wide heterogeneity of both clinical and laboratory manifestations of HS contributes to difficulties associated with the diagnosis of this disorder. Although massive data previously reported worldwide, there is yet no data on HS among the Tunisian population. Here we aim to characterize HS in Tunisian patients at biochemical and cellular levels, identify the membrane protein deficiency, and compare the accuracy of the diagnostic tests to identify the most appropriate assay for HS diagnosis. METHODS: We investigated 81 patients with hemolytic anemia and 167 normal controls. The exploration of HS based on clinical and family history, physical examination, and the results of laboratory tests: blood smear, osmotic fragility test (OFT), cryohemolysis test (CT), pink test (PT), eosine-5'-maleimide (EMA) test, and erythrocyte membrane protein electrophoresis. RESULTS: We identified 21 patients with HS, classified as severe (6/21;28.5%), moderate (10/21;47.6%), and mild (5/21;23.8%). The most prevalent protein deficiency was the band 3 protein detected in ten Tunisian HS patients. The EMA test showed a high specificity (97.5%) and sensitivity (94.7%) for HS diagnosis compared to the other screening tests. Interestingly, fourteen among sixteen patients presenting with homozygous sickle cells HbSS showed an increase of EMA fluorescence intensity compared to other anemic patients. CONCLUSION: Our study highlights the efficiency of the EMA dye for the detection of HS whatever the nature of the involved protein deficiency. We report for the first time, the most prevalent protein deficiency among Tunisians with HS. Moreover, we found that the combination of the EMA-binding test with PT or incubated OFT improves the diagnosis sensitivity while maintaining a good specificity.

Regulatory network analysis of microRNAs and genes in imatinib-resistant chronic myeloid leukemia.

Targeted therapy in the form of selective breakpoint cluster region-abelson (BCR/ABL) tyrosine kinase inhibitor (imatinib mesylate) has successfully been introduced in the treatment of the chronic myeloid leukemia (CML). However, acquired resistance against imatinib mesylate (IM) has been reported in nearly half of patients and has been recognized as major issue in clinical practice. Multiple resistance genes and microRNAs (miRNAs) are thought to be involved in the IM resistance process. These resistance genes and miRNAs tend to interact with each other through a regulatory network. Therefore, it is crucial to study the impact of these interactions in the IM resistance process. The present study focused on miRNA and gene network analysis in order to elucidate the role of interacting elements and to understand their functional contribution in therapeutic failure. Unlike previous studies which were centered only on genes or miRNAs, the prime focus of the present study was on relationships. To this end, three regulatory networks including differentially expressed, related, and global networks were constructed and analyzed in search of similarities and differences. Regulatory associations between miRNAs and their target genes, transcription factors and miRNAs, as well as miRNAs and their host genes were also macroscopically investigated. Certain key pathways in the three networks, especially in the differentially expressed network, were featured. The differentially expressed network emerged as a fault map of IM-resistant CML. Theoretically, the IM resistance process could be prevented by correcting the included errors. The present network-based approach to study resistance miRNAs and genes might help in understanding the molecular mechanisms of IM resistance in CML as well as in the improvement of CML therapy.

Ritonavir-Induced Suicidal Death of Human Erythrocytes.

The antiviral drug ritonavir has been shown to trigger suicidal death or apoptosis of tumour cells and has thus been considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca(2+) entry with increase in cytosolic Ca(2+) activity ([Ca(2+) ]i ), oxidative stress and ceramide. The present study explored whether and how ritonavir induces eryptosis. To this end, flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from Annexin-V binding, [Ca(2+) ]i from Fluo3 fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing specific antibodies. As a result, a 48-hr exposure of human erythrocytes to ritonavir significantly increased the percentage of Annexin-V-binding cells (≥5 µg/ml), significantly decreased forward scatter (≥5 µg/ml), significantly increased Fluo3 fluorescence (20 µg/ml), slightly, but significantly increased DCFDA fluorescence (20 µg/ml) and slightly, but significantly increased ceramide abundance (20 µg/ml). The effect of ritonavir on Annexin-V binding was significantly blunted, but not fully abolished by the removal of extracellular Ca(2+) . In conclusion, ritonavir triggers erythrocyte shrinkage and phosphatidylserine translocation at the erythrocyte cell membrane, an effect in part due to the stimulation of Ca(2+) entry, oxidative stress and ceramide.

Triggering of Suicidal Erythrocyte Death by Regorafenib.

BACKGROUND/AIMS: The multikinase inhibitor regorafenib is utilized for the treatment of malignancy. The substance is effective in part by triggering suicidal death or apoptosis of tumor cells. Side effects of regorafenib include anemia. At least in theory, regorafenib induced anemia could result from stimulated suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether regorafenib induces eryptosis and, if so, whether it is effective up- and/or downstream of Ca2+. METHODS: To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to regorafenib (≥ 0.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (≥ 1.25 µg/ml), but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of regorafenib on annexin-V-binding and forward scatter was not significantly blunted by removal of extracellular Ca2+. Regorafenib (5 µg/ml) significantly augmented the increase of annexin-V-binding, but significantly blunted the decrease of forward scatter following treatment with the Ca2+ ionophore ionomycin. CONCLUSIONS: Regorafenib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+.

Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin.

The K(+),H(+) ionophore and antibiotic nigericin has been shown to trigger apoptosis and is thus considered for the treatment of malignancy. Cellular mechanisms involved include induction of oxidative stress, which is known to activate erythrocytic Ca(2+)-permeable unselective cation channels leading to Ca(2+) entry, increase in cytosolic Ca(2+) activity ([Ca(2+)]i) and subsequent stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. This study explored whether and how nigericin induces eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3 fluorescence, pHi from BCECF fluorescence, ceramide abundance utilizing antibodies and reactive oxygen species (ROS) formation from DCFDA-dependent fluorescence. A 48-hr exposure of human erythrocytes to nigericin significantly increased the percentage of annexin-V-binding cells (0.1-10 nM), significantly decreased forward scatter (0.1-1 nM), significantly decreased cytosolic pH (0.1-1 nM) and significantly increased Fluo3 fluorescence (0.1-10 nM). Nigericin (1 nM) slightly, but significantly, increased ROS, but did not significantly modify ceramide abundance. The effect of nigericin on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca(2+). The nigericin-induced increase in [Ca(2+)]i and annexin V binding was again significantly blunted but not abolished by the Na(+)/H(+) exchanger inhibitor cariporide (10 µM). Nigericin triggers eryptosis, an effect paralleled by ROS formation, in part dependent on stimulation of Ca(2+) entry, and involving the cariporide-sensitive Na(+)/H(+) exchanger.

Enhanced Eryptosis Following Exposure to Lopinavir.

BACKGROUND/AIMS: The protease inhibitor lopinavir, used for the treatment of HIV infections, triggers suicidal death or apoptosis of nucleated cells. Side effects of lopinavir include anemia, which could in theory result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and by phospholipid scrambling of the cell membrane leading to phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide. The present study explored, whether lopinavir induces eryptosis. METHODS: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide abundance utilizing labelled specific antibodies. Hemolysis was estimated from haemoglobin concentration of the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to lopinavir significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥15 µg/ml), significantly increased hemolysis (≥ 15 µg/ml), significantly increased Fluo3-fluorescence (20 µg/ml), and significantly increased DCFDA fluorescence (20 µg/ml) but did not significantly modify ceramide abundance. The effect of lopinavir on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSION: Lopinavir treatment of erythrocytes from healthy volunteers is followed by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca2+ entry.

Efavirenz Induced Suicidal Death of Human Erythrocytes.

BACKGROUND/AIMS: The reverse transcriptase inhibitor efavirenz utilized for the treatment of human immunodeficiency virus (HIV)-1 infection, triggers suicidal cell death or apoptosis, an effect in part due to interference with mitochondrial potential. Side effects of efavirenz include anemia. Causes of anemia include accelerated clearance of circulating erythrocytes. Even though lacking mitochondria, erythrocytes may enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry and increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of p38 kinase, casein kinase 1α and/or cyclooxygenase. The present study explored, whether and how efavirenz induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing selective antibodies. RESULTS: A 48 hours exposure of human erythrocytes to efavirenz (≥ 2 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (2 µg/ml), significantly increased Fluo3-fluorescence (≥ 2 µg/ml), but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of efavirenz on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. The effect of efavirenz on annexin-V-binding was further significantly blunted by p38 kinase inhibitor SB203580 (2 µM) and casein kinase 1α inhibitor D4476 (10 µM), but not by cyclooxygenase inhibitor aspirin (50 µM). CONCLUSIONS: Efavirenz triggers cell shrinkage and phosphatidylserine translocation to the erythrocyte surface, an effect in part due to stimulation of Ca2+ entry as well as activation of p38 kinase and casein kinase 1α.

Enhanced Eryptosis Following Exposure to Carnosic Acid.

BACKGROUND/AIMS: The phenolic abietane diterpene component of rosemary and sage, carnosic acid, may either induce or inhibit apoptosis of nucleated cells. The mechanisms involved in the effects of carnosic acid include altered mitochondrial function and gene expression. Human erythrocytes lack mitochondria and nuclei but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide formation. The present study explored, whether and how carnosic acid induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to carnosic acid significantly increased the percentage of annexin-V-binding cells (2.5 µg/ml), significantly decreased forward scatter (10 µg/ml), significantly increased Fluo3 fluorescence (10 µg/ml), significantly increased ceramide abundance (10 µg/ml), significantly increased hemolysis (10 µg/ml), but significantly decreased DCFDA fluorescence (10 µg/ml). The effect of carnosic acid on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSION: Carnosic acid triggers cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane, an effect paralleled by and/or in part due to Ca2+ entry and increased ceramide abundance.

Stimulation of Eryptosis by Narasin.

BACKGROUND/AIMS: Narasin, an ionophore used for the treatment of coccidiosis, has been shown to foster apoptosis of tumor cells. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by Ca2+ entry with subsequent increase of cytosolic Ca2+ activity ([Ca2+]i), and by ceramide. The present study explored, whether and how narasin induces eryptosis. METHODS: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to narasin (10 and 25 ng/ml) significantly increased the percentage of annexin-V-binding cells. Forward scatter was decreased by 1 ng/ml narasin but not by higher narasin concentrations (10 and 25 ng/ml). Narasin significantly increased Fluo3-fluorescence (10 and 25 ng/ml) and slightly, but significantly increased ceramide abundance (25 ng/ml). The effect of narasin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Narasin triggers phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled and partially dependent on Ca2+ entry. Narasin further leads to cell shrinkage and slight increase of ceramide abundance.

Saquinavir Induced Suicidal Death of Human Erythrocytes.

BACKGROUND/AIMS: The antiretroviral protease inhibitor saquinavir is used for the treatment of HIV infections. Effects of saquinavir include induction of apoptosis, the suicidal death of nucleated cells. Saquinavir treatment may further lead to anemia. In theory, anemia could result from accelerated erythrocyte loss by enhanced suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress with increase of reactive oxygen species (ROS) and ceramide. The present study explored, whether and how saquinavir induces eryptosis. METHODS: To this end, flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ROS abundance from DCFDA fluorescence and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to saquinavir significantly decreased forward scatter (≥ 5 µg/ml), significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly increased Fluo3-fluorescence (15 µg/ml), significantly increased DCFDA fluorescence (15 µg/ml), but did not significantly modify ceramide abundance. The effect of saquinavir on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Saquinavir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca2+ entry.

Embelin-Induced Phosphatidylserine Translocation in the Erythrocyte Cell Membrane.

BACKGROUND/AIMS: The antihelminthic, contraceptive, anti-inflammatory and anticancer phytochemical embelin is at least in part effective against malignancy by inducing suicidal death or apoptosis of tumor cells. Erythrocytes are similarly able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca(2+)-activity ([Ca2+]i), ceramide formation, oxidative stress as well as activation of p38 kinase and protein kinase C (PKC). The present study tested, whether and how embelin induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance utilizing specific antibodies and reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. RESULTS: A 48 hours exposure of human erythrocytes to embelin (≥25 µM) significantly increased the percentage of annexin-V-binding cells and hemolysis. Embelin did not significantly modify [Ca2+]i. The effect of embelin on annexin-V-binding was not blunted by removal of extracellular Ca2+, by p38 kinase inhibitor SB203580 (2 µM) or by PKC inhibitor staurosporine (1 µM). Embelin did, however, significantly increase the ceramide abundance. CONCLUSIONS: Embelin stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect involving ceramide formation.

Triggering of Suicidal Erythrocyte Death by Topotecan.

BACKGROUND/AIMS: The topoisomerase I inhibitor topotecan is used as treatment of various malignancies. The substance is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the outer face of the erythrocyte membrane. Signaling leading to eryptosis include Ca(2+)-entry and ceramide formation. The present study explored, whether and how topotecan induces eryptosis. METHODS: Phosphatidylserine abundance at the erythrocyte surface was estimated from annexin V binding, cell volume from forward scatter, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to topotecan significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. The effect of topotecan was paralleled by a significant increase of ceramide abundance. The effect of topotecan on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Topotecan stimulated cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by increase of ceramide abundance and partially dependent on entry of extracellular Ca2+.

Zopolrestat Induced Suicidal Death of Human Erythrocytes.

BACKGROUND/AIMS: The aldose reductase inhibitor zopolrestat has been shown to either decrease or increase apoptosis, the suicidal death of nucleated cells. Erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress, Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide formation. The present study explored, whether and how zopolrestat induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, oxidative stress from DCFDA dependent fluorescence, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to zopolrestat (≥ 150 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (≥ 125 µg/ml), significantly increased Fluo3-fluorescence (200 µg/ml), significantly increased ceramide abundance (150 µg/ml), but did not significantly modify DCFDA fluorescence. The effect of zopolrestat on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Exposure of human erythrocytes to zopolrestat triggers cell shrinkage and cell membrane scrambling, an effect in part due to Ca2+ entry and ceramide.

Blood parameters as biomarkers of cadmium and lead exposure and effects in wild wood mice (Apodemus sylvaticus) living along a pollution gradient.

Small mammal populations living on contaminated sites are exposed to various chemicals. Lead (Pb) and cadmium (Cd), two well-known nonessential trace metals, accumulate in different organs and are known to cause multiple adverse effects. To develop nonlethal markers in ecotoxicology, the present work aimed to study the relationships between blood parameters (hematocrit, leukocyte levels and granulated erythrocyte levels) and Cd and Pb concentrations in the soil and in the liver and kidneys of wood mice (Apodemus sylvaticus). Individuals were trapped along a pollution gradient with high levels of Cd, Pb and zinc (Zn) contamination. The results indicated that hematological parameters were independent of individual characteristics (age and gender). Blood parameters varied along the pollution gradient, following a pattern similar to the accumulation of Cd in the organs of the wood mice. No relationship was found between the blood parameters studied and Pb concentrations in the organs or in the environment. The hematocrit and leukocyte number decreased with increasing concentrations of Cd in the kidneys and/or in the liver. Moreover, the hematocrit was lower in the animals that were above the thresholds (LOAELs) for Cd concentrations in the liver. These responses were interpreted as a warning of potential negative effects of Cd exposure on the oxygen transport capacity of the blood (e.g., anemia). The present results suggest that blood parameters, notably hematocrit, may offer a minimally invasive biomarker for the evaluation of Cd exposure in further ecotoxicological studies.