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Testing the Hypothesis that Selenium Deficiency is a Risk Factor for Clozapine-Induced Agranulocytosis in Rats Julia Ip and Jack P. Uetrecht* Leslie Dan Faculty of Pharmacy, UniVersity of Toronto, Toronto, Ontario M5S 3M2, Canada ReceiVed NoVember 9, 2007

Clozapine is an effective atypical antipsychotic associated with a relatively high incidence of druginduced agranulocytosis. It forms a reactive nitrenium ion metabolite upon oxidation by peripheral neutrophils and their precursors in the bone marrow. Although the mechanism of this idiosyncratic drug reaction is still unknown, the observation that it does not occur rapidly on rechallenge of patients with a history of clozapine-induced agranulocytosis suggests that it is not immune-mediated. Previous studies by other research groups had found that patients on clozapine had lower plasma and red blood cell levels of selenium. The reactive metabolite of clozapine reacts with glutathione, and therefore, it is likely that it also binds to selenocysteine-containing proteins, such as glutathione peroxidase, thioredoxin reductase, and protein disulfide isomerase. We set out to test the hypothesis that clozapine-induced agranulocytosis is associated with selenium deficiency with rats on a selenium-deficient diet. We studied the effects of clozapine on selenium levels and the effect of selenium deficiency on leukocyte and neutrophil counts and clozapine covalent binding. We did not observe any significant difference between clozapine-treated rats given a selenium-adequate or deficient diet. Therefore, it is unlikely that selenium deficiency is a major risk factor for clozapine-induced agranulocytosis. Introduction Clozapine is an atypical antipsychotic used in the treatment of refractory schizophrenia. Although highly effective, its use has been limited because of its propensity to cause agranulocytosis in 0.8% of patients (1). Clozapine has also been found to be associated with other adverse reactions including myocarditis, cardiomyopathy, hepatotoxicity, and nephritis (2–7). The mechanisms of these reactions, including agranulocytosis, are still not understood; however, it is believed to be due to a reactive nitrenium ion generated by neutrophils and their precursors (8). In a small study conducted by Linday et al. that measured the free radical scavenging enzyme activity and levels of related trace metals, clozapine-treated patients had lower plasma selenium concentrations than normal healthy controls (9). However, a significant difference was not observed between those that developed agranulocytosis while on clozapine and those that did not. Another study carried out by Vaddadi et al. to assess the difference in selenium levels between schizophrenic patients that were or were not on clozapine also found lower plasma and red cell selenium concentrations in those treated with this drug (10). It is plausible to hypothesize that the observed lower levels of selenium in clozapine-treated patients could be a result of the binding of the nitrenium ion to the selenocysteine of those selenium-containing proteins (selenoproteins). Known as the 21st amino acid, selenocysteine is an analogue of cysteine with a selenium atom replacing the sulfur. We have shown in our previous studies that clozapine covalently binds to nucleophiles such as glutathione upon bioactivation by activated neutrophils or hypochlorous acid (8). Selenium displays many similarities with sulfur, yet it is different enough * To whom correspondence should be addressed. Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada. Tel.: 416-978-8939. E-mail: [email protected].

that a selenocyteine residue is usually more reactive than the cysteine analogue, by 2 orders of magnitude, because it has a much lower pKa and is ionized at physiological pH (11, 12). Therefore, it is very likely that clozapine will covalently bind to selenocyteine in a fashion similar to the thiol group in cysteine (Figure 1). Such covalent binding could alter the structure and/ or function of selenoproteins leading to their destruction and depletion of selenium, which can potentially be a risk factor for clozapine-induced agranulocytosis. The importance of selenoproteins to human health and its antioxidant properties had been well described in recent reviews (13–18). They are essential to many enzymatic functions in thyroid hormone metabolism, antioxidant defense systems, and the immune system (13, 19, 20). Selenoproteins such as glutathione peroxidases and thioredoxin reductases are among the key antioxidant enzymes needed to prevent excessive oxidative stress (13). Therefore, alterations in the function of such selenoproteins and their removal would induce oxidative stress, which may be a risk factor of idiosyncratic drug reactions. The aim of this study is to determine whether selenium deficiency can increase the risk of clozapineinduced agranulocytosis in rats and also to determine its effect on the amount of clozapine-protein covalent binding and the effect of clozapine treatment on selenium levels.

Materials and Methods Animals. Sixteen female Sprague–Dawley rats weighing approximately 50 g were purchased from Charles River (Montreal, QC). All rats were housed in pairs in plastic cages with corncob chip bedding in a 12:12 h light-dark cycle at 22 °C. They were acclimatized and given access to regular rodent powder diet (Harlen Teklad, Madison, WI) and tap water ad libitum for 1 week prior to the beginning of the experiment. The experimental protocol was approved by University of Toronto’s animal care committee. Chemicals. Clozapine was provided by Novartis Pharmaceuticals Inc. (Dorval, QC). DTT and Ponceau S solution were purchased from Sigma-Aldrich (Oakville, ON). Stock acrylamide solution

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Figure 1. Clozapine is oxidized by the myeloperoxidase/H2O2/Cl- system of neutrophils forming the reactive nitrenium ion, which covalently binds to proteins. These may include selenoproteins with selenocysteine residues.

Figure 2. Glutathione peroxidase activity after 116 days of seleniumdeficient (SD) or adequate diet (SN) in the presence (CL) and absence of clozapine cotreatment. Values are expressed as the mean ( SD from 4 animals. * p < 0.05.

(30%) and nitrocellulose were purchased from Bio-Rad (Mississuaga, ON). Horseradish peroxidase-conjugated goat antirabbit IgG (H+L chains) was purchased from Cedarlane (Burlington, ON). SuperSignal West Pico Chemiluminescent Substrate was purchased from Pierce (Rockford, IL). Selenium-Deficient Diet and Clozapine Treatment. Half of the rats were placed on a selenium-deficient (SD1) purified powder rodent diet (TD. 92163) containing 0.03 ppm of background selenium from Harlan Teklad (Madison, WI). The rest were given a selenium-adequate (SN) diet supplemented with 0.1 ppm of selenium. All rats were given deionzed water filtered through a reverse osmosis system. Fifty-four days after the commencement of the selenium-deficient diet, four of the eight rats were subjected to clozapine treatment at 50 mg/kg body weight/day by addition of the drug to the diet (SDCL). This dose is approximately 5 times the therapeutic dose, and higher doses are not well tolerated by the animals. Four of the eight rats on the selenium supplemented diet were given the same dose of clozapine in their diet (SNCL). The rats were treated with clozapine for 62 days. Blood Collection and Leukocyte Counts. Blood samples were collected from each rat once a week. A sample of blood (200 µL) was obtained with a 25 G needle from the tail vein and collected into Microvettes CB 300 Kalium-EDTA capillary tubes (Sarstedt, Montreal, QC). Total leukocyte counts were performed by mixing 10 µL of blood with Turk Blood Diluting Fluid (Ricca Chemical Co., Arlington, TX) at 1:9 and using a hemacytometer. Leukocyte differential counts were obtained from blood smears on slides stained with Wright-Giemsa stain (Fisher Scientific Co., Middletwon, VA). Peripheral neutrophil counts were calculated by multi1 Abbreviations: SD, selenium deficient diet; SN, diet containing a normal amount of selenium; CL, clozapine cotreatment.

plying the total leukocyte counts by the percentage of neutrophils in each blood sample. Selenium Status Assessment. The selenium status of each rat was obtained using the Ransel kit by Randox Laboratories (Crumlin, UK) which is based on the principle of NADPH oxidation. Glutathione peroxidase is a selenoprotein in which selenium is present in the form of selenocysteine (16). Glutathione peroxidase activity decreases in response to selenium deficiency in most animals (16, 21–23). Therefore, whole blood glutathione peroxidase activities in the rats provided an index of their selenium status and were measured in this experiment. Collection of Bone Marrow and Liver. At the end of the study, rats were sacrificed with an overdose of anesthetic (ketamine, 50 mg/rat)/xylazine, 5 mg/rat). The femurs and tibia were removed, and bone marrows were collected by flushing them with 20 mL of RPMI 1640 culture medium (University of Toronto, Tissue Culture). The bone marrow cells were resuspended by a five times passage through a 1 mL serological pipet tip. The cell suspension was centrifuged at 125g for 6 min. Red blood cells were then removed by resuspension of the cell pellet in red cell lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM EDTA) for 6 min and centrifugation at 125g for 6 min. Tissue debris was removed by passing the cell suspension through a 70 µm nylon cell strainer (BD Biosciences, Bedford, MA) upon resuspension in PBS (University of Toronto, Tissue Culture). The bone marrow cells were washed again in PBS and resuspended in 500 µL of cell lysis buffer (10 mM Tris-HCl at pH 7.4, 1 mM EDTA, 0.2% Triton X-100, and protease inhibitor cocktail). Livers were excised from the rats and stored at -80 °C. Liver tissue homogenate was prepared by homogenizing a small aliquot of the frozen liver in cell lysis buffer using a tissue homogenizer (9500 rpm, 3 bursts of 10 s). Bone marrow cell lysate and liver tissue homogenate samples were analyzed for protein concentration using a BCA protein assay kit from Pierce (Rockford, IL). SDS-PAGE and Immunoblotting. Bone marrow cell lysate and liver tissue homogenate samples were diluted to a final protein concentration of 1 µg/µL with cell lysis buffer. One part of a 6× SDS-PAGE sample buffer (0.35 M Tris-Cl, 10% SDS, 4% glycerol, 0.02% bromophenol blue, and 18 mg/mL DTT) was added to 5 parts of sample and then heated at 90 °C for 10 min. SDS-PAGE was performed using a mini-gel system (MiniPROTEAN II, Bio-Rad). Stacking and resolving gels were 4% and 10% acrylamide, respectively. Prestained broad range molecular mass makers were used (Bio-Rad). A sample (20 µL) was loaded into each well. Gels were run at 120 V for 90 min until the dye front reached the bottom of the resolving gel. Electrophoretic transfer to nitrocellulose membrane was carried out at 100 V for 60 min using a mini Trans-Blot transfer cell (Bio-Rad) in a transfer buffer (25 mM Tris-HCl, 0.19 M glycine, 20% methanol, and 0.1% SDS). The nitrocellulose membrane was stained with Ponceau S solution for 5 min to assess the efficiency of the transfer. Lane densitometry was performed on the Ponceau S-stained blots. Only blots with all lanes having a net arbitrary lane density differing no more than 10% of the mean density of all lanes were used. The membrane was destained with wash buffer (100 mM Tris-Cl, 0.9% NaCl, and 0.1% Tween 20). The subsequent steps were conducted at room temperature with gentle shaking on a rocker. The membrane was blocked with 5%

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Figure 3. Peripheral leukocyte counts of rats during clozapine treatment. Values are expressed as the mean ( SD of 4 animals for each treatment group.

Figure 4. Peripheral neutrophil counts of rats during clozapine treatment. Values are expressed in mean ( SD of 4 animals for each treatment group.

the blot was immediately captured using the FluorChem8800 imaging system by Alpha Innotech (San Leandro, CA) by exposing it for 10 min to visualize the bound antibodies. Statistical Analysis. Statistical analyses were performed using the GraphPad Prism 4 software (GraphPad Software Inc.). Unpaired t-tests (two tailed, 95% confidence interval) were used to compare between treatment groups.

Results

Figure 5. Western blot detection of clozapine-modified hepatic proteins from selenium-deficient (SDCL) and adequate (SNCL) rats given a daily dose of 50 mg/kg clozapine for 62 days. Lane SN represents sample from a rat on the selenium-adequate diet without clozapine treatment. Thirty micrograms of total protein from liver tissue homogenate were loaded per lane, and the primary antiserum was used at a dilution of 1:3000. Each lane represents a sample from an individual animal.

(w/v) skimmed milk powder in 100 mM Tris-HCl buffer (pH 7.5) containing 0.9% NaCl and 0.1% Tween 20 for 1 h. The blocked membrane was then incubated for 15 h with an anticlozapine antibody diluted (1:3000 for liver tissue homogenate blots, 1:2000 for bone marrow cell lysate blots) in the Tris-HCl buffer; production of the anticlozapine antibody has been described previously (24). The membrane was washed with wash buffer for 10 min 5 times to remove any unbound antibodies. It was then incubated for 2 h with horseradish peroxidase-conjugated goat antirabbit IgG (H + L chain) antiserum diluted 1: 20 000 with wash buffer. After washing 5 times with wash buffer to remove any unbound antibodies, the membrane was incubated in SuperSignal West Pico Chemiluminescent Substrate for 10 min. Chemiluminescence on

Selenium Status. Glutathione peroxidase activities of whole blood in the rats given a selenium deficient diet for 116 days were significantly lower than the rats given an adequate level (p < 0.05) (Figure 2). However, clozapine treatment did not result in any significant changes in the level of glutathione peroxidase activities either in the rats given a selenium-deficient or a selenium-adequate diet. Peripheral Leukocyte Counts. Total leukocytes and peripheral neutrophil counts were within the normal ranges of Sprague–Dawley rats in this study (25). Changes in total leukocyte and peripheral neutrophil counts were not observed during the course of clozapine treatment in either the group of rats given a selenium-deficient diet or those given a seleniumadequate diet (Figures 3 and 4). Covalent Binding of Clozapine to Hepatic and Bone Marrow Proteins. In both the liver homogenates and bone marrow cell lysates from drug-treated rats, covalent binding of clozapine was detected. Immunoblots of the liver homogenate showed bands with molecular masses ranging from 30 to 240 kDa, whereas immunoblot of the bone marrow cell lysate blot showed only one prominent band at ∼75 kDa. Differences in terms of the amount of clozapine covalent binding in the rats

Selenium and Clozapine-Induced Agranulocytosis

Figure 6. Western blot detection of clozapine-modified bone marrow proteins from selenium-deficient (SDCL) and adequate (SNCL) rats given a dose of 50 mg/kg clozapine for 62 days. Lane SN represents a sample from a rat on the selenium-adequate diet without clozapine treatment. Thirty micrograms of total protein from bone marrow cell lysate were loaded per lane, and the primary antiserum was used at a dilution of 1:2000. Each lane represents a sample from an individual animal.

given an adequate selenium diet and those given a deficient diet were not observed in either the liver or bone marrow (Figures 5 and 6).

Discussion Plasma and red blood cell selenium levels have been found to be lower in clozapine-treated schizophrenic patients (9, 10). It is not clear whether this atypical antipsychotic depletes this essential trace element in these patients or whether the disease itself is associated with decreased selenium levels. Selenium has an important role in reducing oxidative stress; for example, glutathione peroxidase can reduce hydrogen peroxide and modulate the oxidative burst (13). Other selenium-containing proteins are also thought to be involved in a defense mechanism against arylating agents because they are the common target of the electrophilic metabolites of many chemicals (26). In mice, one of the proteins to which the reactive metabolite of acetaminophen covalently binds is a 56 kDa selenium-binding protein (26–28). Glutathione peroxidase activity was significantly lower in the selenium-deficient rats, dropping to 12% of the adequate control groups after 116 days of selenium-deficient diet. These results confirm that this commercial selenium-deficient diet induced selenium-deficiency in these rats. However, clozapine treatment did not result in a significant decrease in whole blood selenium level in either selenium-deficient or adequate rats as measured in the glutathione peroxidase activity. Although there is a suggestion of a decrease in selenium levels associated with clozapine treatment in the selenium deficient animals but even with a one-tailed t-test, it did not quite reach statistical significance (p ) 0.059); it is difficult to know if this represents a biologically significant difference. However, this is only with a relatively extreme selenium deficiency that is unlikely to occur in humans. In Linday’s study, plasma glutathione peroxidase levels in the postclozapine agranulocytosis group were lower than healthy controls as well as those that did not develop agranulocytosis while on clozapine (9). From our study, it did not appear that clozapine had a direct effect on selenium status. Recent findings showed a possible association between the pathogenesis of schizophrenia and increased oxidative stress and cellular injuries (29–31). It is possible that schizophrenic patients, especially those that require clozapine treatment, are predisposed to lower selenium levels because of poor diet.

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Despite prolonged treatment with a combination of a seleniumdeficient diet and clozapine, trends suggesting a decrease in white blood cell or neutrophil counts were not observed. Agranulocytosis clearly was not induced. It appeared that there may be slightly more covalent binding of clozapine to bone marrow proteins in the deficient animals; however, using densitometry these differences were not statistically significant. Differences in the amount of covalent binding in hepatic tissue between selenium-deficient and adequate rats were not observed. In summary, although there may have been subtle changes in selenium levels caused by clozapine treatment in animals already very selenium deficient as determined by glutathione peroxidase activity, there were no effects on neutrophil numbers. These results do not support the hypothesis that clozapine treatment causes selenium deficiency nor that selenium deficiency is the major risk factor for clozapineinduced agranulocytosis. Acknowledgment. J.P.U. is the recipient of the Canada Research Chair in Adverse Drug Reactions. This work was supported by grants from the Canadian Institutes of Health Research.

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