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 (H₂O₂) to generate the reactive metabolite and compared reactive metabolite-induced 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 H₂O₂. 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.

INTRODUCTION

Clozapine is an atypical antipsychotic with several advantages over conventional antipsychotics, in addition to its well-known efficacy in treatment-resistant schizophrenia.^1,2) It lacks extrapyramidal side effects, such as parkinsonism, tardive dyskinesia, and dystonia; however, agranulocytosis is a well-known side effect associated with clozapine therapy that reportedly occurs in 0.8% of patients.³⁾ Notably, agranulocytosis induces life-threatening infections.³⁾ Thus, the use of clozapine is strictly controlled under well-monitored systems. More importantly, the mechanisms underlying clozapine-induced hematopoietic toxicity remain poorly understood. It has been suggested that hematopoietic toxicity, such as agranulocytosis, is an immune-mediated response⁴⁾ that is influenced by patient characteristics⁵⁾ or genetic background.⁶⁾

Oxidative stress is characterized by an imbalance between the production of reactive oxygen species and antioxidant defense mechanisms, which may lead to tissue injury.⁷⁾ Clozapine undergoes bioactivation to a reactive metabolite oxidized by activated neutrophils or hypochlorous acid. This unstable metabolite, identified as a nitrenium intermediate, covalently binds to neutrophils and induces cytotoxicity.^8,9) Glutathione (γ-glutamyl–cysteinyl–glycine) is one of the most important defenses against oxidative stress, which is mediated via the fine-tuned regulation of redox homeostasis.^10–12) In a previous report, patients taking clozapine reportedly exhibited lower levels of reduced glutathione in neutrophils.¹³⁾ In addition, reactive metabolite-induced cytotoxicity was found to be attenuated following the addition of reduced glutathione.¹⁴⁾

It should be noted that clozapine itself does not exhibit direct toxicity toward granulocytes, but toward hematopoietic cell.¹⁴⁾ We previously reported that clozapine (25 µM) itself decreased the survival rate of HL-60 cells undergoing differentiation, but not of undifferentiated and differentiated HL-60 cells.¹⁵⁾ The reactive metabolite induces considerably stronger cytotoxicity toward neutrophils than clozapine itself.¹⁶⁾ However, the impact of the metabolite on myeloid precursor cells compared to that on granulocytes remains unclear. In the present study, we used hydrogen peroxide (H₂O₂) to generate the reactive metabolite and compared reactive metabolite-induced 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. Olanzapine, an atypical antipsychotic drug structurally similar to clozapine, was used for comparison, as it possesses a low risk of agranulocytosis.^15,17)

MATERIALS AND METHODS

Cell Culture

HL-60 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Cells were incubated in RPMI 1640 (Sigma-Aldrich, MO, U.S.A.) containing 10–30% heat-inactivated fetal bovine serum (FBS: Thermo Scientific, MA, U.S.A.) and 1% penicillin/streptomycin. The cells were cultured in 100 × 20 mm circle dishes (BD Bioscience, CA, U.S.A.) at 37 °C in a humidified atmosphere with 5% CO₂ in the air. The medium was changed 2–3 times a week.

Materials

Clozapine and olanzapine (Sigma-Aldrich) were mixed with dimethyl sulfoxide (DMSO) (Kanto Chemical Co., Inc., Tokyo, Japan) to a final DMSO concentration of less than 0.1%. H₂O₂ (FUJIFILM Wako Pure Chemical Corporation [Wako], Osaka, Japan) and reduced glutathione (GSH; Wako) were separately dissolved in distilled water. All-trans retinoic acid (ATRA; Sigma-Aldrich), a differentiation inducer,¹⁸⁾ was mixed with ethanol. When exposed to ATRA (1 µM) for 5 d, HL-60 cells are gradually differentiated into mature neutrophils.^15,18)

Experimental Schedule

HL-60 cells were treated with ATRA (1 µM) plus metabolites or unchanged forms of clozapine and olanzapine to examine their effects on HL-60 cells undergoing granulocytic differentiation. We also examined the effects of the drugs in differentiated HL-60 cells as follows: HL-60 cells were treated with ATRA (1 µM) for 5 d, and then, metabolites or unchanged forms of clozapine and olanzapine were added to the cells.

Clozapine (25 µM) and olanzapine (25 µM) were mixed with H₂O₂ (5 or 10 µM) or H₂O in the medium, immediately before addition to the cells to prepare their metabolites or their unchanged forms. The condition was adapted previous reports^19–21) with minor modification. Treated HL-60 cells were immediately centrifuged at 380× g (2000 rpm; Himac CT13R; HITACHI, Tokyo, Japan) for 4 min at 4 °C. As half-life of reactive metabolite of clozapine was 1 min,²²⁾ the total exposure time of cells to the different drugs (metabolites or unchanged forms of clozapine and olanzapine, GSH, and solvent controls) was 4 min in centrifugation. Then, the cells were washed with phosphate-buffered saline (PBS), centrifuged at 380× g for 5 min at 4 °C twice to remove all drugs including metabolites, and seeded at a density of 2 × 10⁵/mL in medium containing ATRA (1 µM). Thereafter, the cells were incubated for 1, 2, 3, or 4 h. The effects of clozapine were compared with those of olanzapine.

Determination of Reduced and Oxidized Glutathione Concentration

The concentrations of oxidized and reduced glutathione (GSSG/GSH) were determined using specific quantification kits (Dojindo Molecular Technologies, Kumamoto, Japan). The reagent 5.5′-dithio-bis(2-nitrobenzoic acid) (DTNB) contains disulfide groups, which oxidize glutathione while DTNB simultaneously undergoes reduction to 5′-thio-2-nitrobenzoic acid (TNB). GSH undergoes oxidation by DTNB to form the yellow derivative TNB, determined at 412 nm in a spectrophotometric microplate reader. Glutathione is found in two forms, reduced and oxidized. Under physiological conditions, only the reduced form is present in cells, while the oxidized form is less than 1% of the total glutathione.²³⁾ Data were analyzed using the following equations:

1. (1)    Total glutathione and GSSG = absorption sample − absorption blank
2. (2)    GSH = total glutathione − GSSG × 2

Cell Survival Determination

We determined cell survival using TC-20 (Bio-Rad, CA, U.S.A.). The cell suspension and an equal volume of trypan blue solution (Wako) were mixed thoroughly. The trypan blue dye is absorbed by nonviable cells, which are stained blue.²⁴⁾ The number of total and stained cells were counted using TC-20.

Statistical Analysis

All experiments were performed in duplicates or triplicates for each group and repeated two to four times. The results are indicated as the mean ± standard error. One-way or two-way ANOVA, followed by Tukey’s post-hoc tests, were performed for multi-group comparisons. The Student’s t-test was performed for comparing two groups. Statistical significance was set at p < 0.05.

RESULTS

Effect of Hydrogen Peroxide on the Survival of HL-60 Cells Undergoing Granulocytic Differentiation

The reactive metabolite is generated by incubating clozapine with H₂O₂. Ishisaka et al. reported that H₂O₂ induces cytotoxicity in HL-60 cells.²⁵⁾ Herein, we investigated the effects of H₂O₂, focusing on concentration and exposure time, on the survival of HL-60 cells undergoing granulocytic differentiation. The HL-60 cells were exposed to H₂O₂ (10 µM) for 4 min, washed to remove the H₂O₂, and then incubated for 3 h. The survival rate of the HL-60 cells significantly decreased (p < 0.01; Fig. 1A), after exposure to 10 µM H₂O₂. In addition, we exposed four other batches of HL-60 cells to 5 µM H₂O₂ for 4 min, washed the cells to remove H₂O₂, and then incubated them for either 1, 2, 3, or 4 h. The cells that were incubated for 4 h, after exposure to 5 µM H₂O₂, exhibited a significant decrease in the survival rate (p < 0.05; Fig. 1B). In contrast, the batch of HL-60 cells that was incubated for 3 h, after exposure to 5 µM H₂O₂, did not exhibit a significant decrease in the survival rate. This showed that if we prepared the reactive metabolites at this concentration and used an incubation time of 3 h, then the H₂O₂ used in the metabolite preparation would not influence the results of our investigation. Thus, we investigated the effects of the reactive metabolites, which were prepared in 5 µM H₂O₂, clozapine, and olanzapine by exposing the cells to the drugs for 4 min, washing the cells to remove the drugs and then incubating them for 3 h, in the subsequent experiments.

Fig. 1. Effect of Hydrogen Peroxide on the Survival of HL-60 Cells Undergoing Granulocytic Differentiation

(A) HL-60 cells were treated with H₂O or H₂O₂ (1, 5, or 10 µM) for 4 min. Thereafter, H₂O or H₂O₂ were washed off and the cells were incubated for 3 h. (B) HL-60 cells were treated with H₂O₂ (5 µM) for 4 min. Afterwards, the H₂O₂ (5 µM) was washed off and the cells were incubated for either 0, 1, 2, 3, or 4 h, cell survival was evaluated using trypan blue dye. The experiment was repeated twice (n = 4). One-way ANOVA: (A) F_{(3, 12)} = 29.39, p < 0.01; (B) F_{(4, 15)} = 8.13, p < 0.05. * p < 0.05, ** p < 0.01 vs. H₂O or time (0 h) (Tukey–Kramer test). H₂O₂, hydrogen peroxide.

Effects of the Reactive Metabolite on HL-60 Cell Survival

We next determined the effects of the reactive metabolite of clozapine on the survival of HL-60 cells undergoing granulocytic differentiation and differentiated HL-60 cells, and compared the observed effects with those demonstrated by clozapine, olanzapine, or the reactive metabolite of olanzapine. The survival rate of HL-60 cells undergoing granulocytic differentiation was significantly lower upon exposing with the clozapine reactive metabolite than upon exposing with clozapine (p < 0.01; Fig. 2A). In contrast, no cytotoxicity was observed in differentiated HL-60 cells after exposing with either clozapine or its reactive metabolite (Fig. 2B). Moreover, olanzapine did not induce cytotoxicity even after treatment with H₂O₂.

Fig. 2. Effect of the Reactive Metabolite of Clozapine on the Survival of HL-60 Cells Undergoing Granulocytic Differentiation and Differentiated HL-60 Cells

Clozapine, olanzapine, and H₂O₂ (5 µM) were added to (A) HL-60 cells undergoing granulocytic differentiation and (B) differentiated HL-60 cells for 4 min. After washing off the drugs, the cells were incubated for 3 h, cell survival was evaluated using trypan blue dye. The experiment was repeated four times (n = 8). (A) ** p < 0.01 vs. H₂O/CLZ (Student’s t-test). Two-way ANOVA: F_{H2O2 (1, 42)} = 15.11, p < 0.01; F_{Drug (1, 42)} = 30.78, p < 0.01; F_{H2O2×Drug (1, 42)} = 11.43, p < 0.05, (B) Two-way ANOVA: F_{H2O2 (1, 42)} = 2.07, p = 0.14; F_{Drug (1, 42)} = 2.76, p = 0.10; F_{H2O2×Drug (1, 42)} = 0.31, p = 0.73. H₂O₂, hydrogen peroxide; DMSO, dimethyl sulfoxide; CLZ, clozapine; OLZ, olanzapine; n.s., not significant.

The Role of Oxidative Stress in Reactive Metabolite-Induced Cytotoxicity

The concentration of reduced glutathione as well as the ratio of GSH and GSSG (GSSG/GSH), are indices of oxidative stress.²⁶⁾ We investigated the effect of the reactive metabolite of clozapine on glutathione metabolism in HL-60 cells undergoing granulocytic differentiation.

After exposing the cells with H₂O₂-treated clozapine, the concentration of reduced glutathione, but not of oxidized glutathione, significantly decreased when compared with that after exposing with DMSO (p < 0.01; Fig. 3A). The ratio of GSSG to GSH was significantly increased, thus indicating oxidative stress (p < 0.05; Fig. 3C). Furthermore, the addition of reduced glutathione significantly reversed the decreased survival rate induced by H₂O₂-treated clozapine (p < 0.01; Fig. 3D).

Fig. 3. The Role of Oxidative Stress in the Cytotoxicity Induced by the Reactive Metabolite of Clozapine in HL-60 Cells Undergoing Granulocytic Differentiation

(A–C) HL-60 cells undergoing granulocytic differentiation were treated with clozapine and H₂O₂ (5 µM) for 4 min. After washing off the drugs and incubating the cells for 3 h, GSH concentration, GSSG concentration, and the GSSG/GSH ratio were evaluated using the GSSG/GSH quantification kit, (D) HL-60 cells undergoing granulocytic differentiation were treated with clozapine, GSH, and H₂O₂ (5 µM) for 4 min. After washing off the drugs and incubating the cells for 3 h, cell survival was evaluated using trypan blue dye. The experiment was repeated twice (n = 6). (A) ** p < 0.01 vs. DMSO/H₂O₂ (Student’s t-test). Two-way ANOVA: F_{H2O2 (1, 20)} = 4.36, p < 0.05; F_{CLZ (1, 20)} = 5.95, p < 0.05; F_{H2O2×CLZ (1, 20)} = 6.69, p < 0.05. (B) Two-way ANOVA: F_{H2O2 (1, 20)} = 1.81, p = 0.19; F_{CLZ (1, 20)} = 1.00, p = 0.33; F_{H2O2×CLZ (1, 20)} = 0.37, p = 0.55. (C) * p < 0.05 vs. DMSO/H₂O₂ (Student’s t-test). Two-way ANOVA: F_{H2O2 (1, 20)} = 8.13, p < 0.01; F_{CLZ (1, 20)} = 5.18, p < 0.05; F_{H2O2×CLZ (1, 20)} = 8.53, p < 0.01. (D) ** p < 0.01 vs. H₂O/CLZ/H₂O₂ (Student’s t-test). Two-way ANOVA: F_{GSH (1, 30)} = 10.21, p < 0.01; F_{CLZ (1, 30)} = 6.55, p < 0.05; F_{GSH×CLZ (1, 30)} = 8.79, p < 0.01. DMSO, dimethyl sulfoxide; CLZ, clozapine; H₂O₂, hydrogen peroxide; n.s., not significant; GSSG, glutathione disulfide (oxidized glutathione); GSH, reduced glutathione.

DISCUSSION

The reactive metabolite of clozapine is an unstable intermediate generated by the exposure of clozapine with H₂O₂.¹⁹⁾ As H₂O₂ induces cytotoxicity,²⁵⁾ we first confirmed that exposure of cells with 5 µM H₂O₂ for 4 min followed by a 3 h incubation after washing off the H₂O₂, did not result in any cytotoxicity. Therefore, we exposed the cells to clozapine or H₂O₂-treated clozapine for 4 min to determine and compare the effect of clozapine and its reactive metabolite on cell survival. The reactive metabolite induced more rapid cytotoxicity in HL-60 cells undergoing granulocytic differentiation than clozapine (Fig. 2A). In contrast, differentiated HL-60 cells were not affected by ether of the drugs (Fig. 2B). Similar results have been observed in neutrophils¹⁶⁾ or HL-60 cells undergoing granulocytic differentiation exposed to clozapine.¹⁵⁾ Our findings suggest that the reactive metabolite has more potent cytotoxicity on myeloid precursor cells undergoing granulocytic differentiation than clozapine itself.

The reactive metabolite exhibits stronger covalent bonds with the neutrophil membrane surface than clozapine and acts on intracellular proteins^9,16); however, its action on myeloid precursor cells undergoing granulocytic differentiation remains unclear. The half-life of the reactive metabolite is shorter than that of clozapine.²²⁾ Geib et al. have investigated the formation and protein binding of the reactive metabolite using liquid chromatography-tandem mass spectrometry,²⁷⁾ but the concentration of the reactive metabolite also remains unclear. Further investigations are needed to clarify the mechanisms of hematopoietic toxicity.

Notably, olanzapine is effective for the treatment of schizophrenia at lower doses than clozapine. The reactive metabolite of olanzapine, presenting a similar chemical structure to that of clozapine, evoked no cytotoxicity on HL-60 cells undergoing granulocytic differentiation. It has been reported that the reactive metabolite of olanzapine forms weaker covalent bonds with neutrophils than the reactive metabolite of clozapine,^22,28,29) thus exhibiting lower toxicity toward neutrophils.^22,28) Therefore, our findings suggest that the reactive metabolite of clozapine has a specific toxicity for myeloid precursor cells undergoing granulocytic differentiation. On the other hand, the effects of clozapine itself and olanzapine itself on the survival rate (94.1% and 95.9%, Fig. 2A) and live cell counts (2.1 × 10⁵/mL and 2.0 × 10⁵/mL) of HL-60 cells undergoing granulocytic differentiation did not differ. Although these results were different from previous report that incubation with clozapine itself for 2 d affected live cell counts,¹⁵⁾ we found that exposure to clozapine itself and olanzapine itself for 4 min had no cytotoxicity.

In addition, the reactive metabolite decreased the levels of reduced glutathione and increased the levels of GSSG/GSH. Therefore, the reactive metabolite was found to induce oxidative stress on myeloid precursor cells undergoing granulocytic differentiation. Clozapine reportedly causes a dose-dependent disruption of the antioxidant system by decreasing the endothelial glutathione level.³⁰⁾ Accordingly, it can be suggested that the presence of the reactive metabolite disrupts the balance between oxidation and the antioxidant systems, thus resulting in cytotoxicity. However, this study did not examine the detailed transduction pathway from oxidative stress to cytotoxicity. Oxidative stress increases the expression of Bax and p53, which trigger the internal pathway of apoptosis (mitochondrial pathway),¹³⁾ and the reactive metabolite induces oxidative stress through apoptosis in neutrophils.³¹⁾ Collectively, these findings suggest that oxidative stress can also play a role in reactive metabolite-induced cytotoxicity on myeloid precursor cells undergoing granulocytic differentiation.

In the present study, the addition of reduced glutathione attenuated reactive metabolite-induced cytotoxicity in HL-60 cells undergoing granulocytic differentiation. Reduced glutathione decreases the number of covalent bonds between the reactive metabolite and the neutrophil membrane surface.⁹⁾ Reactive metabolite-induced cytotoxicity can be attenuated by ascorbic acid and N-acetylcysteine.¹⁶⁾ N-Acetylcysteine is a known precursor of reduced glutathione³²⁾ and modulates glutamatergic, neurotropic, and inflammatory pathways.³³⁾ In patients with chronic schizophrenia taking clozapine, supplementation with N-acetylcysteine significantly improves the symptoms.³²⁾ Therefore, supplementation with exogenous reduced glutathione may be beneficial in reducing reactive metabolite-induced cytotoxicity.

It remains unclear whether glutathione is solely responsible for the reactive metabolite-induced cytotoxicity. Patients with psychiatric disorders, such as schizophrenia, are known to experience chronic inflammation and can produce greater levels of reactive oxygen species than people without this condition.³⁴⁾ In addition, glutathione S-transferase, which enhances the level of reduced glutathione, has individual differences in gene polymorphisms.³⁵⁾ Patients with these polymorphisms have shown a high risk for neutropenia and agranulocytosis.³⁶⁾ Thus, environmental or genetic factors may influence reactive metabolite-induced hematopoietic toxicity.

In conclusion, glutathione metabolism plays a role in the hematopoietic toxicity induced by the reactive metabolite of clozapine in HL-60 cells. Thus, oxidative stress and glutathione metabolism could be molecular therapeutic targets and provide a basis for designing novel antipsychotic agents geared toward preventing agranulocytosis.

Acknowledgments

This study was supported in part by following Grants: Grants-in-Aid for Scientific Research A [Grant No. 21H04815], Scientific Research C [Grant Nos. 21K06719, 22K06755], and Young Scientists B [Grant No. 17K16403] from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan; Grant-in-Aid for the Strategic Research Program for Brain Sciences from Japan Agency for Medical Research and development, AMED [Grant Nos. JP21dk0307103, JP21dm0207075]; The Adaptable and Seamless Technology Transfer Program Through Target-driven R&D, Japan Science and Technology Agency [Grant No. AS251Z03018]; Meijo University Research Institute Grant; The Encouragement of Scientific Research, Promoted Research Center Subsidy by Meijo University Research Institute.

Conflict of Interest

Yukihiro Noda had received funding of speaking fees from Sumitomo Dainippon. Norio Ozaki had received funding from Sumitomo Dainippon, Eisai, Otsuka, KAITEKI, Takeda, Mochida, Mitsubishi Tanabe, Shionogi, Eli Lilly, DAIICHI SANKYO, Nihon Medi-Physics; speaking fees from Sumitomo Dainippon, Eisai, Otsuka, Takeda, Mochida, Meiji Seika Pharma, EA Pharma, Pfizer, MSD, Lundbeck Japan; consultant fees from Sumitomo Dainippon, KAITEKI, Taisho Pharm. The other authors declare no conflict of interest.

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