Effect of Aminoglutethimide on Neutrophils in Rats: Implications for

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Effect of Aminoglutethimide on Neutrophils in Rats: Implications for Idiosyncratic Drug-Induced Blood Dyscrasias Winnie Ng† and Jack Uetrecht*,†,‡ †

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2 ‡ Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8 ABSTRACT: Aminoglutethimide (AMG) is an aromatic amine aromatase inhibitor associated with a high incidence of idiosyncratic blood dyscrasias, especially agranulocytosis. Animal models of idiosyncratic drug reactions (IDRs) represent essential tools to study these reactions; however, there is currently no valid model of idiosyncratic drug-induced agranulocytosis. Although AMG does not cause agranulocytosis in most animals or humans, drugs associated with serious IDRs generally cause a higher incidence of mild reactions that resolve despite continued treatment. Therefore, the effects of AMG on neutrophils and bone marrow in rats were studied to understand the mechanisms of more serious IDRs. An increase in peripheral blood neutrophils occurred as early as 24 h after AMG treatment with minimal changes to the total leukocyte count. Further investigation using 5-bromo-2′-deoxyuridine (BrdU) found an increased release of neutrophils from the bone marrow. Histologically, this corresponded to an increase in myeloid cells in the bone marrow, which was confirmed by differential staining with CD45 and CD71. AMG treatment stimulated an increase in colony forming unit granulocyte-macrophage and colony forming unit granulocyte ex vivo. There was also a marked increase in the number of activated neutrophils in the circulation expressing the extravasation marker CD62L. These findings indicate that AMG affects neutrophil production, release, and function. Similar effects on neutrophil kinetics in clozapine-treated rats have previously been found, and transient neutrophilia has been observed in patients taking other drugs associated with idiosyncratic agranulocytosis; therefore, the changes observed with AMG may be biomarkers to predict the risk that a drug will cause agranulocytosis.

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INTRODUCTION Aminoglutethimide (AMG) is a first-generation aromatase inhibitor used primarily for the treatment of hormoneresponsive breast and prostate cancers. Structurally, AMG contains a primary aromatic amine. This chemical moiety is almost always associated with a high incidence of idiosyncratic drug reactions (IDRs); therefore, this is a notorious structural alert for drug development.1 The toxicity of aromatic amines is presumably due to the ease with which they can be oxidized to reactive electrophilic metabolites, which can covalently bind to cellular macromolecules and potentially induce damage.2 However, the exact mechanisms of IDRs are still poorly understood. More importantly, it is unknown why only a subset of patients develops these reactions, whereas most do not. Animal models, with similar pathogenesis as occurs in humans, are essential for studying the underlying mechanisms of IDRs; yet, valid animal models of IDRs are rare and quite difficult to develop.3 AMG is associated with a wide range of IDRs including serious skin rashes, cholestasis, and hematological toxicities.4 Of these reactions, the most commonly reported are blood dyscrasias such as agranulocytosis, which is characterized by a decrease in neutrophils to below 500/μL of blood; and if sustained for a prolonged period of time, it usually results in a fatal infection. Agranulocytosis occurs in approximately 1.9% of © 2013 American Chemical Society

AMG-treated patients and typically manifests after more than 4 weeks of treatment.5 In these patients, bone marrow aspirates are usually hypocellular with decreased myeloid cells, which return to normal after discontinuation of the drug. However, white blood cell counts (WBC) sometimes return to normal without cessation of AMG therapy,6 which suggests the induction of tolerogenic mechanisms that may be why most patients do not develop agranulocytosis. In fact, it is a common characteristic that agents that can induce serious IDRs always cause a much higher incidence of mild reactions that often resolve despite continued treatment. This suggests that such drugs have biological effects that are quite common and asymptomatic even if the severe reactions are rare. Nevertheless, data is lacking on the effects in most patients, in terms of the early events leading up to agranulocytosis because blood monitoring of AMG-treated patients does not usually occur before symptoms arise. In general, aromatic amines can be oxidized to a Nhydroxylamine metabolite by cytochrome P450 enzymes or myeloperoxidase, which is relatively stable and unreactive. However, the hydroxylamine can auto-oxidize to a nitrosoamine metabolite that is electrophilic and reactive and can Received: June 21, 2013 Published: July 26, 2013 1272

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University of Toronto Faculties of Medicine and Pharmacy Animal Care Committee. Treatments. Rats were treated with 80, 125, or 160 mg/kg/day of AMG in 0.5% methylcellulose through oral gavage. Control animals were given methylcellulose vehicle only. Each group had 4 rats each unless otherwise stated. The 80 mg/kg AMG dose was chosen as a starting dose based on previous rat studies14 to mimic the plasma concentrations found in patients taking therapeutic doses of AMG.15 Measurement of AMG Blood Levels. Blood levels of AMG were determined 1 and 24 h after a single 80 or 160 mg/kg dose of AMG, and analysis was performed similar to Adam et al.16 Briefly, blood was collected from the tail vein of rats into heparinized tubes, and plasma was obtained by centrifugation. To 20 μL of plasma, 10 μL of phenacetin (10 μg/mL stock in methanol) was added as an internal standard and 80 μL of methanol. The solution was vortexed vigorously for 30 s and then incubated at −20 °C for 30 min. The sample was centrifuged at 16000g for 10 min, and the supernatant was transferred to a clean tube and evaporated to dryness under nitrogen. Samples were then reconstituted in 50 μL of mobile phase (42% methanol/58% water), and 10 μL was injected into the HPLC (Hewlett-Packard Series 1050) for analysis with an Ultracarb column (5 μm ODS (30) 100 × 2.0 mm; Phenomenex) at a flow rate of 0.2 mL/min. AMG was detected by UV absorption at 245 nm, and a standard curve was produced to quantify AMG. The retention times were 3.8 and 7.3 min for AMG and phenacetin, respectively, and a standard curve was produced with a correlation coefficient greater than 0.99. The limit of detection was 0.96 μg/mL. Leukocyte Counts. Blood was collected from the tail vein into EDTA-coated tubes. Whole blood was diluted 20-fold in Turks solution (Ricca Chemical Company, Arlington, TX) to lyse red blood cells (RBCs), and the total WBC count was obtained manually using a hemocytometer. A 5 μL aliquot of whole blood was also smeared on a slide, fixed, and Wright−Giemsa stained using CAMCO Stain Pak (Cambridge Diagnostic Products Inc., Fort Lauderdale, FL) as per the manufacturer’s instructions, and the WBC differential was determined manually under a light microscope by characterizing 100 leukocytes per slide. Measurement of Cytokines. Peripheral blood cytokines, chemokine (C-X-C) 1 (Cxcl1), and tumor necrosis factor alpha (TNF-α) were measured using the Rat Quantikine ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Granulocyte colony stimulating factor (G-CSF) was also measured in the peripheral blood with a Mouse Quantikine ELISA kit (R&D Systems). Although the G-CSF ELISA was not specifically developed for use in rats, a BLAST search shows greater than 80% gene sequence identity to rat G-CSF. Leukocyte Isolation from Peripheral Blood. Collected whole blood was mixed with equal volumes of 3% dextran (prepared in saline) and incubated for 18 min at room temperature. The strawcolored upper layer was collected and centrifuged at 350g for 5 min and the supernatant discarded. RBCs were lysed with red cell lysis buffer by incubation for 10 min. Cells were washed twice in florescence-activated cell sorting (FACS) buffer (5% FBS in PBS) before final resuspension in FACS buffer, and cell counting was performed using trypan blue and Countess Automated Cell Counter (Invitrogen, Life Technologies). Measurement of New Neutrophil Release Using 5-Bromo2′-deoxyuridine (BrdU). Rats were treated with either methylcellulose vehicle control or AMG for 10 days. BrdU is known to be carcinogenic and was handled using proper procedures for carcinogenic substances as outlined by the University of Toronto Faculties of Medicine and Pharmacy Animal Care Committee. On day 4 of treatment, BrdU (100 mg/kg dissolved in warm saline) was injected intraperitoneally, and blood samples were taken from the tail vein to monitor neutrophil incorporation of BrdU after leukocyte isolation using FITC BrdU Flow Kit (BD Biosciences) as per the manufacturer’s protocol. Antirat granulocyte PE antibody was included as a surface marker for granulocytes. Cells were analyzed by BD FACSCalibur (BD Biosciences) at a flow rate of no more than 400 events/s.

covalently bind to proteins. Compared to other aromatic amine-containing drugs, such as the more common sulfonamides, AMG has a higher electron density, which allows it to be even more easily oxidizable (Figure 1). N-Hydroxyl-AMG has

Figure 1. Structure of AMG and the formation of its reactive metabolites. AMG can be detoxified through N-acetylation (NAT). Alternatively, AMG can be oxidized by cytochrome P450 (CYP450) or myeloperoxidase (MPO) to a hydroxylamine, which can auto-oxidize to a highly electrophilic and reactive nitrosoamine that can covalently bind to protein thiols. The nitrosoamine can also be reduced back to the hydroxylamine via ascorbate or glutathione (GSH) and subsequently to the parent drug by reductase, which may lead to redox cycling to cause oxidative stress.

been detected in both humans and mice.7,8 Neutrophils contain high concentrations of myeloperoxidase;9 therefore, it is quite likely that AMG can be oxidized by this cell type and lead to cell damage. Alternatively, myeloperoxidase protein free radicals have been detected in vitro upon incubation with AMG,10 which further suggests a role for oxidation by neutrophils in the pathogenesis of agranulocytosis. To date, very few studies, if any, have focused on the effect of AMG on neutrophils, and a true animal model of AMGinduced agranulocytosis has not yet been established. Leucopenia was observed in both ICI-derived and B6C3F1 hybrid mice as early as after 2 weeks of AMG treatment;11,12 however, these mouse strains are uncommon, and this model was not reproducible in CD-1 mice.13 Given our negative results in mice and our previous experience with Brown Norway rats, we investigated the effects of AMG on neutrophils and bone marrow in this rat strain.

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MATERIALS AND METHODS

Chemicals and Reagents. AMG was purchased from Toronto Research Chemicals (North York, ON). Phenacetin, dextran-500, BrdU, and 10% neutral buffered formalin were obtained from Sigma (Oakville, ON). Heat-inactivated fetal bovine serum (FBS), Iscove’s modified Dulbecco’s medium (IMDM), and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Life Technologies (Burlington, ON). Antibodies. Antirat granulocyte phycoerythrin (PE; clone: RP-1), 7-amino-actinomycin D (7-AAD), anti-BrdU fluorescein isothiocyanate (FITC), annexin V FITC, propidium iodide (PI), antirat CD18 FITC (clone: WT.3), and antirat CD11b allophycocyanin (APC; clone, WT.5) were acquired from BD Biosciences (San Jose, CA). Antirat CD71 FITC (clone: OX-26), and antirat CD62L FITC (clone: OX-85) were purchased from Cedarlane (Burlington, ON). Antirat CD45 PE (clone: OX-1) was obtained from BioLegend (San Diego, CA). Animals. Male Brown Norway rats (176−200 g) were purchased from Charles River (Montreal, QC). Rodents were housed doubly under standard conditions with automatic watering and a 12:12 h light/dark cycle at 22 °C. Food was provided ad libitum, and all animals were given standard rodent chow. Animals were acclimatized for one week before the start of experiments. The experimental protocol was approved by and performed in accordance with the 1273

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Bone Marrow Histology. The femurs of the rats were isolated using standard procedures and fixed in neutral buffered formalin (Sigma) for 2−5 days. Tissues were submitted to the Toronto Centre for Phenogenomics Histology Laboratory (Toronto, ON) for paraffin embedding and H&E stained slide preparation. Microscopic pictures were taken at the Microscopy Imaging Lab (Faculty of Medicine, University of Toronto) on a Zeiss fluorescence microscope with deconvolution. Characterization of Bone Marrow Cells. The extracted rat femur was flushed with 5 mL of IMDM containing 10% FBS with a 22 gauge needle. Aggregates were broken up through repeated aspiration, and cells were passed through a 40 μm filter to make a single cell suspension. Cells were centrifuged at 300g for 5 min and resuspended in 5.0 mL of FACS buffer before cell counting as described above. Bone marrow cells were characterized using a protocol similar to that of Saad et al.17 For surface staining, 5 μL of antirat CD71 FITC and 1.25 μL of antirat CD45 PE antibodies were added to 1 × 106 cells in a total volume of 100 μL and incubated in the dark for 30 min at 4 °C. Cells were washed twice with FACS buffer before cells were fixed with IC Fixation Buffer (eBioscience) for 20 min, washed, and left overnight in FACS buffer. Cells were then refixed and permeablized with Fixation/Permeablization Buffer (eBioscience) for 20 min, washed once in FACS buffer, and then resuspended in 200 μL of staining buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.1% Tween20, and 0.5 mM MgCl2). DAPI was added to the sample in a final concentration of 3 μM in 100 μL volume and incubated in the dark for 15 min at room temperature before samples were analyzed immediately using flow cytometry. Investigation of Bone Marrow Progenitor Cells. Bone marrow cells were extracted as previously described and resuspended in IMDM containing 2% FBS. Cells were counted manually with a hemocytometer using Turk’s solution. Progenitor cells in the bone marrow were studied in a colony forming cell (CFC) assay for rat cells using the MethoCult GF R3774 medium (Stemcell Technologies, Vancouver, BC) according to the manufacturer’s protocols. Briefly, cells were plated at 1.5 × 104 per 1.1 mL culture in 35 mm dishes and incubated at 37 °C, 5% CO2, with ≥95% relative humidity for 10 days before colony forming unit-granulocyte-macrophage (CFU-GM), colony forming unit-granulocyte (CFU-G), and colony forming unitmacrophage (CFU-M) were manually assessed under a light microscope. All colonies on the plate were counted, and each sample was duplicated in a separate dish. Determination of Neutrophil Apoptosis. Apoptosis was measured using Annexin V Apoptosis Kit (BD Biosciences) as per the manufacturer’s protocol. Specifically, isolated blood leukocytes were washed twice with PBS and then reconstituted at 2 × 105 cells per 100 μL of binding buffer. Cells were then incubated with 5 μL each of annexin V FITC and PI for 15 min in the dark at room temperature. Binding buffer (400 μL) was added to each sample to stop the reaction, and cells were analyzed by flow cytometry within 2 h. Determination of Neutrophil Activation. Leukocytes were isolated as described previously, and cell surface markers were stained using standard flow cytometry procedures. Antirat CD62L FITC, antirat CD11b APC, antirat CD18 FITC, and 7-AAD were incubated in the dark for 30 min at 4 °C, washed twice in FACS buffer, and then analyzed by flow cytometry. Flow Cytometry Analysis. Unless otherwise stated, flow cytometry experiments were run at the Faculty of Medicine Flow Cytometry Facility (University of Toronto) on BD FACSCanto II (BD Biosciences) using BD FACSDiva Software (BD Biosciences) with a flow rate of no more than 400 events/s. Compensation was corrected using BD Compensation Beads (BD Biosciences). FlowJo Software (Tree Star, Inc., Ashland, OR) was used for detailed analysis of the flow cytometry results. Data Analysis. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). The Mann−Whitney U test or two-way ANOVA with Bonferroni post-tests were used depending on the constraints of the data to determine statistical

significance between AMG and control groups. Error is represented as standard error of the mean (SEM).

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RESULTS Effect of AMG on Peripheral Blood Leukocyte Counts. Plasma levels of AMG 1 h after a single gavage dose of 80 and 160 mg/kg AMG were 15.0 (±2.0) μg/mL and 18.4 (±2.1) μg/mL, respectively (Figure 2), which is within the therapeutic

Figure 2. Blood levels of AMG in rats. Blood was obtained from the tail vein of rats at 1 and 24 h after a single oral dose of 80 or 160 mg/ kg of AMG. Values expressed are the mean ± SEM for each group (n = 4).

range (4.7−32.4 μg/mL) found in humans during chronic AMG therapy.18 Upon AMG treatment at a dose of 80 mg/kg/ day, an increase in peripheral blood neutrophils was observed as early as 24 h and remained elevated at 48 h with minimal changes to the total WBC count, although not statistically significant (Figure 3). Further investigation of the dose−

Figure 3. Acute AMG-induced changes on peripheral blood neutrophils in rats given an oral dose of 80 mg/kg/day. Blood was taken from the tail vein of rats, and the WBC differential was determined by Wright−Giemsa staining. (A) No major changes were observed in the total WBC count; however, (B) an increase in neutrophils was observed as early as 24 h after AMG treatment. Values expressed are the mean ± SEM for each group (n = 4). None of the results was statistically significant by the Mann−Whitney U test. 1274

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compensated for by a slight decrease in the lymphocyte count, most noticeable after 14 days AMG treatment (Figure 5C). Because the most significant neutrophil response was observed at 125 mg/kg/day of AMG, this dose was used for subsequent experiments. To provide further evidence for the effect of AMG on neutrophils, blood cytokines associated with neutrophil production (G-CSF), chemotaxis (Cxcl1), and activation (TNF-α) were measured; they were all found to be noticeably elevated within a day of AMG treatment (Figure 6). Effect of AMG on Bone Marrow. Given that all neutrophils originate from bone marrow and that BrdU is a thymidine analogue that gets incorporated into newly formed DNA, BrdU was used to track new neutrophils released from the bone marrow into the circulation by flow cytometry and double staining with an antirat granulocyte antibody. Consistent with the increase in peripheral blood neutrophils, an increase in newly formed neutrophils labeled with BrdU was observed in the AMG-treated rats with significant changes occurring at 120 h post-BrdU injection (Figure 7). Furthermore, the increase in newly formed neutrophils was sustained throughout AMG treatment, which suggests that AMG prolongs the half-life of circulating neutrophils. H&E staining of bone marrow from the femurs of rats found hypercellularity induced by AMG that was attributed to an increase in the myeloid cells and was not observed in the controls (Figure 8). To confirm this, nucleated cells of the bone marrow, detected with DAPI, were phenotyped by flow cytometry through differential staining of CD45 and CD71 to identify changes in the myeloid, lymphoid, and erythroid cell populations. A significant increase in the myeloid cell population was found with AMG treatment, and this corresponded to a simultaneous decrease in the lymphoid cell population (Figure 9). The myeloid to erythroid ratio of the bone marrow in AMG-treated rats was also significantly elevated compared to that in controls (2.36 ± 0.31 vs 1.48 ± 0.13, respectively, p = 0.03). To determine which myeloid cell populations were affected by AMG, a MethoCult assay was performed ex vivo to evaluate the proliferative capacity of granulocyte and macrophage colony forming units (CFUs) in response to AMG treatment. The effect of AMG seemed to be more specific to granulocytes because there was an increase in the number of CFU-granulocyte-macrophage (CFU-GM) and CFU-granulocyte (CFU-G) colonies, but there was no change in CFU-macrophage (CFU-M) colonies (Figure 10). Effect of AMG on Neutrophils. Since AMG induced a sustained increase in newly formed neutrophils in rats, further investigation into the effect of AMG on neutrophil survival was warranted. Neutrophil apoptosis was investigated through annexin V and PI staining using flow cytometry whereby neutrophils were gated based on forward and side scatter, and cells that were annexin V positive/PI negative were considered early apoptotic cells. It is interesting to note the increase in density of the neutrophil population in the AMG-treated rats (Figure 11A,B), which corresponds to the increase in blood neutrophils that was previously observed. Using this approach, AMG treatment induced a significant reduction in the number of early apoptotic neutrophils compared to that in controls (62.5% at 7 days treatment, p = 0.01; and 55.6%, after 12 days of treatment, p = 0.03) as shown in representative flow plots (Figure 11C,D). In terms of activation, AMG significantly down-regulated the expression of CD62L in neutrophils over time (Figure 12), while a simultaneous increasing trend was observed in CD11b expression in neutrophils, most noticeably

response relationship found no clear changes in the WBC differential of rats treated with 160 mg/kg/day of AMG (data not shown). This was presumably due to direct toxicity because the rats did not tolerate the high dose of AMG well, and treatment had to be withdrawn. However, at a lower dose of 125 mg/kg/day of AMG, a similar elevation in peripheral blood neutrophils was found compared to the dose of 80 mg/kg/day; this was also significantly increased after 24 h (Figure 4) and

Figure 4. Acute AMG-induced changes on peripheral blood neutrophils in rats given an oral dose of 125 mg/kg/day. Blood was taken from the tail vein of rats, and the WBC differential was determined by Wright−Giemsa staining. (A) No differences were observed in the total WBC count between AMG-treated and control rats, whereas (B) significant increases in peripheral blood neutrophils were observed after both 1 and 7 days of AMG treatment, which seemed to be compensated for by (C) a slight decrease in peripheral blood lymphocytes. Open bars represent the control group; solid bars represent the AMG-treated group. Values expressed are the mean ± SEM (n = 4). *p < 0.05 and ***p < 0.001 compared to controls by two-way ANOVA.

sustained through 14 days of treatment (Figure 5). Again, changes in total WBC counts were minimal in the AMG-treated animals because the neutrophil increase seemed to be 1275

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Figure 5. Changes in peripheral leukocyte cell counts induced by the treatment of rats with AMG at a dose of 125 mg/kg/day for 14 days. (A) No changes were observed in total WBC between control and AMG-treated rats, whereas (B) a noticeable increase in the neutrophil population was found after both 7 and 14 days of AMG treatment and (C) a corresponding slight decrease in lymphocyte counts. Open circles and dotted lines represent the control group; solid squares and lines represent the AMG-treated group. Values expressed are the mean ± SEM (n = 4). None of the results was significant by two-way ANOVA.

Figure 6. AMG-induced changes in neutrophil-associated cytokines in the blood of rats. (A) G-CSF, involved in stimulating neutrophil production, was significantly elevated after 7 days of AMG treatment. (B) The neutrophil chemoattractant Cxcl1 was noticeably but not statistically significantly increased after both 1 and 7 days of AMG treatment, and (C) TNF-α, involved with neutrophil activation, was up-regulated after both 1 and 7 days of AMG treatment. Baseline values are similar to vehicle-treated values. Values are expressed as the mean ± SEM (n = 4). *p < 0.05 and ***p < 0.001 compared to baseline measurements by the Mann−Whitney U test.

at days 7 and 12 (data not shown). However, no changes were found in CD18 expression (data not shown). Both CD62L and

CD11b are involved with the infiltration of leukocytes into tissues, and these changes suggest neutrophil activation. 1276

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Figure 7. Effect of AMG on new neutrophil release from the bone marrow. Rats were treated with 125 mg/kg/day of AMG for 10 days. On the fourth day, 100 mg/kg BrdU was injected i.p., and blood from the tail vein was monitored for changes in neutrophils by flow cytometry. Neutrophils were gated by forward and side scatter, and newly formed neutrophils were characterized as cells double positive for antigranulocyte antibody and BrdU. Both the (A) percent BrdUstained neutrophils and the (B) absolute number of newly formed neutrophils were increased by AMG treatment. Open circles and the dotted line are from control animals; solid squares and lines represent AMG-treated animals. The values expressed are the mean ± SEM (n = 5 control, n = 6 AMG). *p < 0.05 compared to the control group by two-way ANOVA.

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Figure 8. Bone marrow changes induced by AMG. Representative H&E staining of bone marrow from (A) control rat femur and (B) rat femur after 14 days of AMG at a dose of 125 mg/kg/day. The magnification is 40×.

DISCUSSION To date, there have been no detailed studies on the effect of AMG on neutrophils despite the many reports of AMGinduced agranulocytosis. Although the doses of AMG given to rats in this study were high, they produced blood levels of AMG similar to therapeutic concentrations in humans who are also given high doses of about 1 g/day. The IDRs, and in particular agranulocytosis, induced by AMG are not surprising given the fact that it is an aromatic amine drug, and aromatic amines are readily oxidized by neutrophil myeloperoxidase.19,20 Consistent with the involvement of the aromatic amine group in the mechanism of AMG-induced agranulocytosis, the AMG analogue without the amino group, glutethimide, is not associated with agranulocytosis. Unfortunately, glutethimide is a controlled substance and not readily available, so we were unable to compare its effects on neutrophils with those of AMG. In the current studies, the major finding was that AMG increased the number of peripheral blood neutrophils. The effect on neutrophils was observed as early as 24 h, which suggests that AMG mobilized the marginal zone neutrophils; however, this increase was sustained by an increase in granulocyte production for at least 14 days. This change was compensated for by a slight decrease in peripheral blood

lymphocytes such that no significant changes were found in the total WBC count. In rodents, the neutrophils make up only approximately 10−30% of the total WBCs in the blood,21 whereas in humans the majority of peripheral WBCs are neutrophils; thus, this is consistent with a subtle compensatory effect by the lymphocyte population. It is interesting that the effect of AMG on neutrophils in rats is opposite to the agranulocytosis that is observed in some patients6 and the leucopenia that occurs in mice.12 However, in general, AMG patients are not monitored until blood dyscrasias arise; therefore, it is unknown whether patients experience earlier changes in their WBC differential. In the previous mouse models, leucopenia was observed as early as after 2 weeks of treatment,12 and it may be possible that if we had continued treatment for a much longer period of time it would result in agranulocytosis. A previous study with AMG-treated rats found no changes in the WBC differential even after 3 weeks of treatment;11 however, the dose was much lower (50 mg/kg/ day) than the dose currently used. In addition, rats had more sustained levels of AMG in the blood 24 h after a single dose than mice treated with similar doses (data not shown). For 1277

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Figure 9. AMG-induced changes in bone marrow cells. Bone marrow cells were extracted from rats treated for 14 days with 125 mg/kg/day of AMG and analyzed through FACS analysis. Representative flow plots are shown for (A,B) control and (C,D) AMG-treated rats. (A,C) From the nucleated cells, stained as DAPI positive, nucleated erythroid cells were defined as CD71 positive, CD45 medium expression, whereas the myeloid and lymphoid cells were defined as CD71 low, CD45 medium-high. (B,D) From this, myeloid and lymphoid cells were further separated based on side scatter where myeloid cells were more granular than lymphoid cells. (E) No change was observed in the erythroid population between treated and control animals; however, in the AMG-treated rats there was a significant increase in myeloid cells with a simultaneous significant decrease in the lymphoid population. Open bars represent the control group; solid bars represent the AMG-treated group. The values expressed are the mean ± SEM (n = 4). **p < 0.01 compared to the control group by two-way ANOVA.

Interestingly, clozapine, a drug that is notorious for its ability to cause agranulocytosis,23 is often associated with neutrophilia before the onset of agranulocytosis,24 and increases in CD34+ hematopoietic stem and progenitor cells have also been reported within 2 weeks of initiating therapy,25 suggesting the mobilization of neutrophil precursors. Analogous to AMG, clozapine also induces neutrophilia in rabbits26 as well as in rats (unpublished observation). Clozapine appears to decrease neutrophil half-life in rabbits;26 in contrast, the kinetic profile of the BrdU-labeled neutrophils suggest that AMG may increase neutrophil survival and half-life. Although further studies using annexin V and PI found a decrease in apoptotic

these reasons, the rat may be a better model of AMG-induced blood dyscrasias and be more useful as a tool for mechanistic studies. The mechanism of AMG-induced neutrophilia in rats is unknown. Certainly inflammation or stress can induce neutrophilia, and part of the stress response is the release of corticosteroids, which can cause neutrophilia. However, because of its effects on the adrenal gland, AMG is known to decrease corticosterone levels in rats.22 Moreover, inflammation may be a central feature to induce immune activation, and if these reactions are immune-mediated, this may lead to an immune response to drugs associated with agranulocytosis. 1278

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fraction of newly released neutrophils in the circulation that express less phosphatidylserine. Furthermore, in vitro studies on neutrophil survival in the presence of AMG resulted in no difference in neutrophil apoptosis from vehicle-treated (data not shown). Therefore, more detailed studies are required to determine how AMG alters neutrophil survival, although the rapid turnover rate and clearance of apoptotic neutrophils may make such studies difficult. In the bone marrow of the AMGtreated rats, there was also an increase in CFU-GM and CFU-G colonies, which are precursors to neutrophils, and this is consistent with an increase in the production of progenitor cells from the bone marrow. Although most patients treated with AMG or clozapine do not develop agranulocytosis, about half of the clozapine-treated patients develop fever, and this is associated with increases in IL-6, TNF-α, and sIL-2r.27 These changes suggest an immune response to the drug. Clozapine has also been found to transiently increase the levels of G-CSF in patients that was independent of the development of fever.28 AMG treatment is also associated with a variety of IDRs that include drug fever.29 In our study, AMG treatment led to an increase in neutrophilassociated cytokines, G-CSF and TNF-α. Moreover, TNF-α can stimulate the neutrophil respiratory burst by activating NADPH oxidase, and this could increase the formation of reactive metabolites.30 Thus, there are similarities in the effects of AMG and clozapine, and such effects may be biomarkers that predict whether a drug candidate can cause agranulocytosis.

Figure 10. Effect of AMG on bone marrow granulocyte and macrophage CFUs. Bone marrow cells were extracted from rats treated with 125 mg/kg/day AMG for 14 days. Cells were then seeded into culture dishes at 1 × 105 cells per 1.1 mL MethoCult medium and incubated at 37 °C, 5% CO2, and ≥95% humidity. After 10 days, colonies were counted under a light microscope and classified as CFUGM, CFU-G, or CFU-M. AMG significantly increased the total number of CFUs and CFU-GM. An increase in the CFU-G was also observed with AMG treatment; however, no change was observed in CFU-M. Open bars represent the control group; solid bars represent the AMG-treated group. Samples were performed in duplicate. The values expressed are the mean ± SEM (n = 4). *p < 0.05 and **p < 0.001 compared to the control by two-way ANOVA.

neutrophils in AMG-treated rats, which is consistent with an increase in neutrophil survival, this may be caused by a higher

Figure 11. AMG-induced changes in neutrophil apoptosis. Blood taken from the tail vein of rats treated with AMG at a dose of 125 mg/kg/day was incubated with annexin V and PI to determine the number of apoptotic cells. As compared to (A) the control, representative flow plots show (B) an increase in the percentage of neutrophils upon AMG treatment. From the neutrophil population, (C) the percent annexin V positive/PI negative early apoptotic cells are high in the control rats, whereas (D) there is a decrease in the number of apoptotic neutrophils in the AMG-treated rats. 1279

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Figure 12. Changes in neutrophil CD62L expression induced by AMG. Blood taken from the tail vein of rats treated with 125 mg/kg/day of AMG were analyzed by flow cytometry for CD62L expression on neutrophils. Neutrophils were identified by their distinct forward and side scatter characteristics as shown previously in Figure 11. Representative flow plots from (A) control and (B) AMG-treated rats show that CD62 is expressed in neutrophils under normal physiological conditions; however, upon AMG treatment there is an increase in the number of neutrophils that have decreased CD62L expression, as shown by the increase in cells in the CD62L low gate. (C) The mean florescence intensity (MFI) for the CD62L channel is significantly decreased at both days 7 and 12 in the AMG-treated as compared to the controls. (D) A significant increase was also observed in the percentage of cells in the CD62L low gate in the AMG-treated at days 7 and 12. Open bars represent the control; solid bars represent AMGtreated rats. The values expressed are the mean ± SEM (n = 4). *p < 0.05, **p < 0.01 compared to the control by two-way ANOVA.

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One possible reason that AMG and clozapine do not cause agranulocytosis in most patients even though they commonly induce an immune response could be immune tolerance. Evidence for this is provided by the resolution of AMG- and clozapine-induced agranulocytosis without discontinuing treatment.6,31 If the agranulocytosis is immune-mediated, then the resolution must involve immune tolerance. From the current studies, AMG appears to stimulate the bone marrow to release new neutrophils that are activated in terms of the extravasation marker CD62L. In humans challenged with endotoxin, CD62Ldim neutrophils were found to be more hypersegmented than the CD62Lbright neutrophils, and this population was also able to suppress T-cell activation,32 which may be a mechanism of immune tolerance. However, more studies will be required to determine the link between neutrophil activation and AMGinduced IDRs including agranulocytosis. Nevertheless, numerous studies have shown additional effects of neutrophils on the adaptive immune system in addition to their traditional role in innate immunity; specifically, their ability to interact and regulate a variety of different immune cells.33 Thus, it is imperative to consider the role of neutrophils when investigating the mechanism of IDRs.

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Funding

This work was supported by the Canadian Institutes of Health Research. J.U. is the Canada Research Chair in Adverse Drug Reactions. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Alexandra Lobach for assistance with the BrdU injections and Imir Metushi for assistance in bone marrow extraction.

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ABBREVIATIONS IDRs, idiosyncratic drug reactions; AMG, aminoglutethimide; BrdU, 5-bromo-2′-deoxyuridine; FBS, fetal bovine serum; IMDM, Isocove’s modified Dulbecco’s medium; DAPI, 4′,6diamidino-2-phenylindole dihydrochloride; PE, phycoerythrin; FITC, fluorescein isothiocyanate; PI, propidium iodide; APC, allophycocyanin; RBCs, red blood cells; Cxcl1, chemokine (CX-C) 1; TNF-α, tumor necrosis factor alpha; G-CSF, granulocyte colony stimulating factor; FACS, fluorescenceactivated cell sorting; SEM, standard error of the mean; CFUs, colony forming units; NAT, N-acetylation; MPO, myeloperoxidase; GSH, glutathione; H&E, hematoxylin and eosin; MFI, mean fluorescence intensity

AUTHOR INFORMATION

Corresponding Author

*University of Toronto, Leslie Dan Faculty of Pharmacy, 144 College Street, Room 1002, Toronto, Ontario, Canada M5S 1280

dx.doi.org/10.1021/tx400224j | Chem. Res. Toxicol. 2013, 26, 1272−1281

Chemical Research in Toxicology

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Article

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dx.doi.org/10.1021/tx400224j | Chem. Res. Toxicol. 2013, 26, 1272−1281