Sulforaphane treatment of HL-60 cells protects against clozapine toxicity and protein covalent binding: implications for clozapine-induced agranulocytosis.

Clozapine is a critical drug for treatment-resistant schizophrenia patients. However, clozapine is currently considered under-prescribed as it carries a risk of idiosyncratic adverse drug reactions, including severe neutropenia (agranulocytosis). Some stuides reported that clozapine-treated patients that exhibited agranulocytosis had a lower frequency of wild-type NRH: Quinone Oxidoreductase 2 (NQO2) and a higher frequency of mutant NQO2 (NQO2-1541-AA). Sulforaphane is a well-known inducer for the expression of a related enzyme isoform, NQO1, that has protective effects; however, sulforaphane's role in modulating NQO2 expression is unknown, and sulforaphane-mediated protection via NQO1 and NQO2 activation against clozapine toxicity remains uninvestigated. We hypothesized that sulforaphane can induce NQO1 and NQO2 expression, and sulforaphane-mediated NQO1 & NQO2 activation play a protective role against clozapine-induced toxicity. Sulforaphane and clozapine HL-60 cell viability were determined to select appropriate treatment concentrations. Immunoblotting was used to investigate NQO1 and NQO2 protein expression and protection against clozapine-protein binding. Glutathione (GSH) was analyzed by microtiter plates, LCMS was used to quantify clozapine metabolites, and UV-Vis spectrophotometry was used to determine myeloperoxidase activity. Sulforaphane (1-10 μM) significantly induced NQO1 and NQO2 protein expression. Clozapine's LC5024h for viability was approximately 50 μM, and sulforaphane did not decrease viability up to 5 μM. Sulforaphane (2.5-5 μM) prevented the clozapine-induced decrease in viability. The formation of clozapine-N-oxide, a less reactive metabolite, was increased in sulforaphane pre-treated groups than in untreated groups. Also, intracellular myeloperoxidase activity increased significantly in the presence of sulforaphane (2.5-10 μM). GSH levels were significantly higher in sulforaphane pre-treated groups, and clozapine covalent protein binding was significantly attenuated by the pre-treatment of sulforaphane. Collectively, the results suggest that sulforaphane pre-treatment of HL-60 cells provides significant defenses against the reactive metabolites of clozapine. The induction of NQO2 is novel and requires further studies to demonstrate specifically how it is involved. Sulforaphane-induced increases in GSH in combination with enhanced clozapine-N-oxide formation appear to contribute to decreasing covalent protein binding, which is correlated with clozapine-induced decreases in cell viability and increased myeloperoxidase activity.