Worldwide, schizophrenia is a significant public health concern because it affects neurotransmitter systems with changing brain microstructure, physiology, and connectivity [1]. Schizophrenia is not limited to religious, ethnic, geographic, or socioeconomic boundaries, so many people are affected directly or indirectly by this mental disorder, and it is a challenging global issue. Beyond health problems, schizophrenic patients are suffering from homelessness, joblessness, public discrimination, social isolation, and suicide. Providing support, their families and relatives also face emotional, social, financial, and physical hardship that extend to doctors, healthcare providers, and caregivers to navigate complex treatment regimens [2]. Clozapine (Clozaril®) is the only evidence-based medication for treatment-resistant schizophrenia [3] (Fig. 1). However, it is currently considered underutilized due to its boxed warning, including the risk of agranulocytosis (severe neutrophil depletion), an idiosyncratic drug reaction (IDR) [[4], [5], [6]]. IDRs are potentially life-threatening and present a significant problem for drug development as they are usually not detected during clinical trials [7]. Immune-mediated toxicity is a proposed mechanism of most IDRs, but small-molecule drugs are not immunogenic as they are too small to induce an immune response. However, an immune response can initiate if the drug binds to a larger molecule via metabolism to a reactive metabolite. This electrophilic species (hapten) can sensitize the immune system to produce a form of delayed hypersensitivity [8,9].

Activated human neutrophils are known to oxidize clozapine to reactive metabolites, including the clozapine nitrenium ion (CNI). The CNI, in turn, can cause oxidative and electrophilic stress or covalently bind to neutrophil proteins to induce neutrophil apoptosis in patients [[10], [11], [12], [13]]. The NAD(P)H-oxidase (NOX) subunit, CYBA, has a 640A > G mutation that is associated with lower NOX activity (and presumably less superoxide and sequentially H2O2 generation), and the mutation of 463G > A in the promoter region of myeloperoxidase (MPO) leads to lower MPO expression. It was expected that these mutations would result in less clozapine bioactivation, although other studies have not shown any significant differences [14,15]. Moreover, previous studies have demonstrated that NQO2 mRNA levels are low in clozapine-treated patients experiencing agranulocytosis due to NQO2 polymorphisms in the coding regions [16]. Moreover, NQO2 1541AA (a homozygous mutant genotype) was present at a higher frequency in agranulocytosis patients and associated with a greater odds ratio for developing clozapine-induced agranulocytosis. Thus, a lower expression of wild-type NQO2 or a higher expression of mutat NQO2 (1541AA) appears to be associated with a risk for clozapine-induced agranulocytosis [17]. Similarly, NQO1 C609T is associated with a greater risk of neutropenia in benzene-exposed adults, which suggests a protective role of NQO1 in human neutrophils [18,19]. These studies generated a rationale for studying clozapine with respect to NQO1 and NQO2. We hypothesized that NQO1 and NQO2 induction is a potential protective response upon exposure to clozapine. Furthermore, sulforaphane, a well-known phytochemical, has chemopreventive activity and is present in cruciferous vegetables like broccoli, cabbage, and Brussels sprouts (Fig. 1). Sulforaphane is a well-known inducer of NQO1 expression [20]. Although NQO1 and NQO2 are homologs and members of the quinone oxidoreductases family, it is unknown whether sulforaphane induces NQO2 expression or activity. In this study, we investigated the response of cellular NQO1 and NQO2 from clozapine treatment and also determined sulforaphane-mediated protection mechanisms against acute clozapine cytotoxicity.