The induction of quinone oxidoreductases NQO1 and NQO2 by clozapine: Potential implications for clozapine-induced agranulocytosis

Clozapine exhibits superior efficacy in the management of treatment-resistant schizophrenia. However, clozapine is currently considered under-prescribed as it carries a risk of idiosyncratic drug reactions (including severe neutropenia or agranulocytosis). The mechanisms of clozapine-induced agranulocytosis mechanisms are evolving. Reports of polymorphisms with NADPH: Quinone Oxidoreductase 2 (NQO2) being associated with clozapine-induced agranulocytosis prompted the studies described herein. Clozapine is known to produce reactive, electrophilic metabolites. It is not known if the latter can interact with NQO2 or with NQO1, its more well-characterized isoform, as quinoid electrophiles do. We hypothesized that clozapine or its metabolites can induce both NQO1 and NQO2 expression via the Nrf2 signaling pathway, as observed with quinoid electrophiles. HL-60 cells were used in this study as they are similar to granulocytes/neutrophils and contain enzymes to metabolize clozapine. A UV-Vis spectrophotometric assay was performed to determine NQO1 and NQO2 enzymatic activity using selective substrates and inhibitors. Immunoblotting was used to investigate NQO1, NQO2, and Nrf2 protein expression. NQO1 and NQO2 gene expression was determined using RT-PCR. Clozapine treatment induced NQO1 and NQO2 enzyme activity, mRNA, and protein expression significantly more than vehicle control accompanied by translocation of the transcription factor, Nrf2, from cytoplasm to nucleus. A structurally related antipsychotic, quetiapine, did not show these effects. The upregulation of both NQO1 and NQO2 enzymatic activity, protein, and gene expression indicated that these enzymes may be involved in the biological response to clozapine toxicity. The translocation of the Nrf2 suggested that the Nrf2 signaling pathway is involved in these response pathways. Further studies are required to determine if NQO2 expression levels and activity are protective mechanisms against clozapine-induced agranulocytosis or if NQO2 levels are a prognostic risk factor for clozapine-induced agranulocytosis.