title: Methemoglobin incites primaquine toxicity through single-electron oxidation and modification.
abstract: BACKGROUND
  Primaquine (Pq) metabolic products are responsible for drug-associated hemotoxicity and limit primaquine usage.
  
  
  METHODS
  Methemoglobin (MetHb)-Pq molecular modeling was used to identify the Pq binding pocket. UPLC, mass spectrometry, and other indirect analytical methods were used to predict the metabolite. MetHb generation, development of oxidative stress, inhibition of antioxidant enzymes, and scanning electron microscope (SEM) were used to characterize the hemotoxic potentials of oxidized Pq (Pqox).
  
  
  RESULTS
  MetHb binded Pq at the heme site with KD =6.4 μM as evidenced by a difference spectroscopy study. MetHb oxidized Pq through a single e-transfer mechanism to form Pqox. The analysis of Pq from MetHb-H2O2 peroxidase reaction mixture gave peaks at m/z 300.53 and m/z 243.42, corresponding to the hydroxyl and desamino derivative of Pq, respectively. Similar peaks were absent in Pq or Pq incubated with H2O2 in the same buffer system. A robust increase in MetHb formation, reactive oxygen species generation, and inhibition of antioxidant enzymes were found in red blood cells (RBCs) exposed to Pqox compared with a parent drug molecule. The RBC membrane exhibited visible damages to plasma membrane (holes) as evidenced by SEM analysis of Pqox-exposed RBCs.
  
  
  CONCLUSIONS
  The MetHb-H2O2 system transforms quiescent parent drug molecule to a highly reactive oxidative form to exhibit severe hemolysis. MetHb-H2O2-mediated Pq hemolytic potentiation that is sensitive to spin trap indicates the role of Pq* radical or other single e-species in the process. The result suggests that MetHb incites the molecular property of the Pq and peroxidase inhibitors can be explored to control drug-associated toxicity.
authors: Malaria Research Group, Department of Biotechnology, Indian Institute of Technology-Guwahati, Guwahati, Assam, India.
authors: S N
authors: SN
authors: Balaji
authors: Vishal
authors: V
authors: Trivedi
mesh_terms: D000962:Antimalarials / Q000633:toxicity*; D000975:Antioxidants / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004910:Erythrocyte Membrane / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects; D005982:Glutathione Transferase / Q000378:metabolism; D006400:Hematocrit; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008706:Methemoglobin / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D008958:Models, Molecular; D009997:Osmotic Pressure; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D011319:Primaquine / Q000633:toxicity*; D011485:Protein Binding; D013057:Spectrum Analysis; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D000962:Antimalarials; D000975:Antioxidants; D008706:Methemoglobin; D002374:Catalase; D013482:Superoxide Dismutase; D005982:Glutathione Transferase; D011319:Primaquine