title: Enhanced Eryptosis Following Exposure to Dolutegravir.
abstract: OBJECTIVE
  The viral integrase enzyme inhibitor dolutegravir is utilized for the treatment of immunodeficiency virus (HIV) infection. Knowledge on cytotoxicity of dolutegravir is limited. The present study thus explored, whether dolutegravir is able to trigger suicidal erythrocyte 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 triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, and activation of protein kinase C, p38 kinase, casein kinase, and caspases. The present study explored, whether Dolutegravir induces eryptosis and, if so, to gain insight into cellular mechanisms involved.
  
  
  METHODS
  Utilizing flow cytometry, 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. Hemolysis was quantified from haemoglobin concentration in the supernatant.
  
  
  RESULTS
  A 48 hours exposure of human erythrocytes to dolutegravir significantly increased the percentage of annexin-V-binding cells (≥ 4.8 µM), significantly increased hemolysis (19.1 µM), but did not significantly modify forward scatter. Dolutegravir significantly increased Fluo3-fluorescence (≥ 4.8 µM), DCFDA fluorescence (19.1 µM) and ceramide abundance (19.1 µM). The effect of dolutegravir on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, but was not significantly modified by protein kinase C inhibitor staurosporine (1 µM), p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM).
  
  
  CONCLUSIONS
  Dolutegravir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, ceramide formation and oxidative stress.
authors.affiliation: Departments of Cardiology, Vascular Medicine and Physiology, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
authors.forename: Abdulla
authors.initials: A
authors.lastname: Al Mamun Bhuyan
authors.forename: Elena
authors.initials: E
authors.lastname: Signoretto
authors.forename: Rosi
authors.initials: R
authors.lastname: Bissinger
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D000072817:Eryptosis / Q000187:drug effects*; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D019428:HIV Integrase Inhibitors / Q000494:pharmacology; D006461:Hemolysis / Q000187:drug effects; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology*; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D010078:Oxazines; D018384:Oxidative Stress / Q000187:drug effects; D010718:Phosphatidylserines / Q000378:metabolism; D010879:Piperazines; D011728:Pyridones; D017382:Reactive Oxygen Species / Q000378:metabolism; D000099226:Dolutegravir
chemical_list: D002118:Calcium; D002518:Ceramides; D019428:HIV Integrase Inhibitors; D006575:Heterocyclic Compounds, 3-Ring; D010078:Oxazines; D010718:Phosphatidylserines; D010879:Piperazines; D011728:Pyridones; D017382:Reactive Oxygen Species; D000099226:Dolutegravir