Non-immunological toxicological mechanisms of metamizole-associated neutropenia in HL60 cells

Metamizole is an  analgesic and  antipyretic , but can cause  neutropenia and  agranulocytosis . We investigated the toxicity of the metabolites N-methyl-4-aminoantipyrine (MAA),  4-aminoantipyrine (AA), N-formyl-4-aminoantipyrine (FAA) and N-acetyl-4-aminoantipyrine (AAA) on neutrophil granulocytes and on HL60 cells (granulocyte precursor cell line). MAA, FAA, AA, and AAA (up to 100 µM) alone were not toxic for HL60 cells or granulocytes. In the presence of the  myeloperoxidase substrate H2O2, MAA reduced cytotoxicity for HL60 cells at low concentrations (<50 µM), but increased cytotoxicity at 100 µM H2O2. Neutrophil granulocytes were resistant to H2O2 and MAA. Fe2+ and Fe3+ were not toxic to HL60 cells, irrespective of the presence of H2O2 and MAA. Similarly, MAA did not increase the toxicity of  lactoferrin ,  hemoglobin or  methemoglobin for HL60 cells.  Hemin (hemoglobin degradation product containing a  porphyrin ring and Fe3+) was toxic on HL60 cells and cytotoxicity was increased by MAA.  EDTA , N-acetylcystein and  glutathione prevented the toxicity of hemin and hemin/MAA. The absorption spectrum of hemin changed concentration-dependently after addition of MAA, suggesting an interaction between Fe3+and MAA. NMR revealed the formation of a stable MAA reaction product with a reaction pathway involving the formation of an electrophilic intermediate. In conclusion, MAA, the principle metabolite of metamizole, increased cytotoxicity of hemin by a reaction involving the formation of an electrophilic metabolite. Accordingly, cytotoxicity of MAA/hemin could be prevented by the  iron chelator EDTA and by the electron donors NAC and glutathione. Situations with increased production of hemin may represent a risk factor for metamizole-associated  granulocytopenia.