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

Abstract

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.

Keywords: Agranulocytosis; HL60 cells; Hemin; Iron; Metamizole; N-methyl-4-aminoantipyrine.