title: Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study.
abstract: BACKGROUND
  Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C.
  
  
  RESULTS
  No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress.
  
  
  CONCLUSIONS
  From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.
authors.affiliation: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. lisarepsold@yahoo.com.
authors.forename: Lisa
authors.initials: L
authors.lastname: Repsold
authors.affiliation: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. thandi.mqoco@up.ac.za.
authors.forename: Thandi
authors.initials: T
authors.lastname: Mqoco
authors.affiliation: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. elizewol@yahoo.com.
authors.forename: Elize
authors.initials: E
authors.lastname: Wolmarans
authors.affiliation: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. sandra.djeukam@up.ac.za.
authors.forename: Sandra
authors.initials: S
authors.lastname: Nkandeu
authors.affiliation: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. joji.theron@up.ac.za.
authors.forename: Joji
authors.initials: J
authors.lastname: Theron
authors.affiliation: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. tomek.piorkowski@up.ac.za.
authors.forename: Tomek
authors.initials: T
authors.lastname: Piorkowski
authors.affiliation: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. peet.dutoit@up.ac.za.
authors.forename: Peet du
authors.initials: Pd
authors.lastname: Toit
authors.affiliation: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. dirk.vanpapendorp@up.ac.za.
authors.forename: Dirk van
authors.initials: Dv
authors.lastname: Papendorp
authors.affiliation: Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. annie.joubert@up.ac.za.
authors.forename: Annie Margaretha
authors.initials: AM
authors.lastname: Joubert
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D001682:Biological Availability; D024402:Carbonic Anhydrase II / Q000187:drug effects; D002257:Carbonic Anhydrase Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology*; D002352:Carrier Proteins / Q000493:pharmacokinetics / Q000494:pharmacology; D003198:Computer Simulation; D055808:Drug Discovery; D004912:Erythrocytes / Q000187:drug effects* / Q000648:ultrastructure; D004958:Estradiol / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology / Q000633:toxicity; D004963:Estrenes / Q000493:pharmacokinetics / Q000494:pharmacology*; D005260:Female; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D036301:Qualitative Research; D013449:Sulfonamides / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000633:toxicity; D013696:Temperature
chemical_list: C588027:2-ethyl-3-O-sulfamoylestra-1,3,5(10)16-tetraene; C581967:2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol; D000970:Antineoplastic Agents; D002257:Carbonic Anhydrase Inhibitors; D002352:Carrier Proteins; D004963:Estrenes; D013449:Sulfonamides; D004958:Estradiol; D024402:Carbonic Anhydrase II