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 LC50_24h 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.