A Reactive Metabolite of Clozapine Induces Hematopoietic Toxicity in HL-60 Cells Undergoing Granulocytic Differentiation through Its Effect on Glutathione Metabolism

Abstract

Clozapine is an atypical antipsychotic with several advantages over conventional antipsychotics, in addition to its well-known efficacy in treatment-resistant schizophrenia. However, the high risk of agranulocytosis associated with clozapine therapy limits its clinical application. Clozapine bioactivation to an unstable protein-reactive metabolite, identified as a nitrenium intermediate, has been implicated in cytotoxicity toward neutrophils. Clozapine affects myeloid precursor cells rather than neutrophils; however, the impact of its reactive metabolite on myeloid precursor cells undergoing granulocytic differentiation remains unclear. Herein, we used hydrogen peroxide (H 2 O 2 ) to generate the reactive metabolite and compared reactive metaboliteinduced cytotoxicity between HL-60 cells undergoing granulocytic differentiation and differentiated HL-60 cells. In addition, we examined the role of oxidative stress in this type of cytotoxicity. The reactive metabolite of clozapine induced rapid cytotoxicity in HL-60 cells undergoing granulocytic differentiation, but not in differentiated HL-60 cells, with the metabolite exhibiting more potent cytotoxicity than clozapine. No cytotoxicity was observed following incubation with olanzapine, a structural analog of clozapine, even after exposure of the drug to H 2 O 2 . The reactive metabolite of clozapine decreased the levels of reduced glutathione, while addition of reduced glutathione attenuated the reactive metabolite-induced cytotoxicity. These findings indicate that glutathione metabolism plays a role in the hematopoietic toxicity induced by the reactive metabolite of clozapine. Oxidative stress may potentially increase susceptibility to the hematopoietic toxicity induced by the reactive metabolite of clozapine.

Possible nonimmunological toxicological mechanisms of vesnarinone-associated agranulocytosis in HL-60 cells: role of reduced glutathione as cytotoxic defense

“…In the case of clozapine, its reactive metabolite decreased the levels of GSH, whereas the addition of GSH attenuated the reactive metabolite-induced cytotoxicity. These findings indicate that GSH metabolism plays a role in hematopoietic toxicity and oxidative stress (Torii-Goto et al, 2022).…”

“…MPO is present in circulating neutrophil granulocytes (Maseneni et al, 2012) and in HL-60 cells, a human promyeloid cell line (Yamada et al, 1981). Therefore, HL-60 cells are frequently used in the investigation of agranulocytosis and neutropenia (Rudin et al, 2019;Torii-Goto et al, 2022).…”