Antipsychotics have been the mainstay of treatment for major psychiatric disorders, especially schizophrenia, since the introduction of chlorpromazine, the first phenothiazine derivative antipsychotic agent, in 1952. Typical or first-generation antipsychotics (FGAs), due to their high affinity for D2 dopamine receptors, are associated with increased risk of extrapyramidal symptoms such as tardive dyskinesia.1 Atypical or second generation antipsychotics (SGAs) have relatively lower affinity for dopamine D2 receptors and higher affinities for other receptors, including the serotonin, muscarinic, and histamine receptors.2 The reasons for the widespread shift toward use of SGAs rather than FGAs are fewer extrapyramidal symptoms (EPS),3 effectiveness in treating negative,4 cognitive,5 and mood6 symptoms, and improvement in quality of life.7–9 Although it has been known for over a century that there is an association between glucose dysregulation and mental illness,10 a series of human11,12 and animal studies13–17 that were done following the introduction of typical antipsychotics reported metabolic disturbances due to FGA use. However, these disturbances are most likely related to the use of chlorpromazine, which behaves more like an SGA than an FGA. Low-potency antipsychotics, such as chlorpromazine, have many similarities to SGAs. Both medications have multiple mechanism of actions,18 low risk of causing acute EPS, tardive dyskinesia, or dystonia, low risk of causing elevated serum prolactin levels, and activity against mood symptoms and cognitive deficits.19 High-potency antipsychotics such as haloperidol have a specific and selective mechanism of action, dopamine D2 receptor blockade, and a high propensity to cause EPS.19
With the introduction of clozapine, a prototype of atypical antipsychotics, and other SGAs, there has been a sharp rise in reports of severe metabolic derangements.20–23 Although the biological mechanisms underlying these side effects are still unknown, recent studies have shown differential risks with different SGA medications. Clozapine and olanzapine are associated with greater effects on glucose and more lipid abnormalities compared with other SGAs, and ziprasidone and aripiprazole are associated with the least.24,25 It has been shown that a single dose of olanzapine or clozapine affects insulin release, which suggests a direct effect on glucose metabolism in the absence of weight gain.26,27
Oxidative stress and the concomitant production of reactive oxygen species (ROS) have been implicated in the clinical side effects of antipsychotics. ROS are chemically-reactive oxygen-containing molecules. ROS contribute to pathological processes such as apoptosis,28 and aging or cellular injury during ischemia29 or reperfusion.30 Both haloperidol and clozapine induce oxidative stress in rat brain.31 Decreased levels of antioxidant enzymes and increased membrane lipid peroxidation (caused by ROS) occur following treatment with these drugs.32–34 These same alterations have been found to occur after long-term treatment with other atypical antipsychotics (risperidone and ziprasidone) in rats.35 It has been shown that clozapine induces oxidative stress in neutrophils of patients with schizophrenia.36 Oxidative stress has been implicated in the pathogenesis of both type 1 and type 2 diabetes.37 Mitochondrial dysfunction is considered a factor in the pathogenesis of insulin resistance, lipid accumulation, and diabetes.38,39 In addition, dysfunctional mitochondria can result in decreased fatty acid oxidation, which can lead to ectopic lipid accumulation and insulin resistance.40,41 Treatment with atypical antipsychotics such as olanzapine is associated with decreased fatty acid oxidation, lipid accumulation, and increased insulin levels (considered a precursor to insulin resistance).42 Taken together, these findings indicate that mitochondrial dysfunction is associated with increased levels of ROS as well as lipid accumulation, insulin resistance, and diabetes—all of which are also associated with the use of atypical antipsychotics such as clozapine. Therefore, it is possible that antipsychotic-induced oxidative stress is an underlying factor in the mechanism by which antipsychotics elicit increased risk for adverse metabolic side effects.
An earlier study by our group showed, using a proteomic approach, that clozapine treatment of neuroblastoma (SKNSH) cells induced oxidation of enzymes involved in energy metabolism, which suggests that protein oxidation may be the mechanism by which atypical antipsychotics induce increased risk of metabolic syndrome and diabetes.43 In the study presented in this paper, we used the same proteomic approach to search for proteins that are irreversibly oxidized by treatment with clozapine in lymphoblastoid cell lines of patients with schizophrenia and normal controls.
MATERIALS AND METHODS
Lymphoblastoid cell lines from individuals with schizophrenia (N=6) and healthy control subjects (not diagnosed with a DSM-IV psychiatric disorder, N=6), matched for age and gender, were selected for the experiments in this study. Patients and subjects were recruited from the Central Valley of Costa Rica in accordance with the principles of the Declaration of Helsinki and with approval from the Institutional Review Boards of the University of Costa Rica and the University of Texas Health Science Center at San Antonio, as previously described.44 Control cell lines were chosen from healthy individuals who were from the same Costa Rican population as, but who were not related to, the individuals with schizophrenia whose cell lines were used.
The lymphoblastoid cells from the patients with schizophrenia and the normal controls were treated with either clozapine 0 or 20 μM for 24 hours. The 20 μM concentration was chosen based on our previous results,43 which showed that this dosing level would induce generation of ROS. Protein extracts were obtained and proteins were labeled by incubation with the fluorescent probe 6-iodoacetamide fluorescein (6-IAF). The lack of incorporation of 6-IAF into the thiol group of cysteine residues is an indicator of protein oxidation. The labeled proteins were exposed to two-dimensional gel electrophoresis, after which an image of the gels was obtained. Gels were then treated with SYPRO Ruby to assess differential protein labeling, and a second gel image was obtained. A total of 190 spots labeled with SYPRO Ruby were matched across all 24 gels (6 treated and 6 untreated schizophrenia cell lines, and 6 treated and 6 untreated control cell lines). Differences in 6-IAF incorporation in individual proteins were then assessed by calculating the ratio of 6-IAF/SYPRO Ruby fluorescence in each of the spots seen in the gels from untreated or clozapine-treated schizophrenia and control cell lines. (For a more detailed description of the. methods used in this study, see the Appendix to this article p. 330).
Differences in Protein Oxidation
Figure 1 shows gel images for 6-IAF and SYPRO Ruby fluorescence in a representative two dimensional gel electrophoresis separation of proteins isolated from lymphoblastoid cells. Statistical analyses using t-tests for independent samples, comparing results from treated vs. untreated schizophrenia cell lines and treated vs. untreated control cell lines, revealed significant changes in fluorescence intensity in six protein spots (P<0.05), three spots (5203, 6305 and 8611) in samples from schizophrenia cell lines and 3 spots (4201, 6102 and 7402) in normal control cell lines. All of these spots indicated greater irreversible protein oxidation (reduced 6-IAF fluorescence) in treated compared with untreated cells. Four spots (2304, 4201, 5605 and 9501) exhibited significant oxidation after treatment with clozapine in schizophrenia cells compared with healthy control cells. Figure 2 shows an expanded view of a selected protein spot that exhibited altered 6-IAF labeling after treatment with clozapine.
Identification of Oxidized Proteins
We identified 9 different spots that exhibited altered oxidation when treated with clozapine (as assessed by 6-IAF labeling) by using HPLC-ESI-MS/MS. Seven different proteins were identified in these 9 spots, with enolase identified in spots 8611 and 5605 and triosephosphate isomerase (TPI) identified in spots 6305 and 7402. The proteins that were identified are listed in Table 1.
The results of this study support our previous findings in SKNSH cells43 and may help to elucidate the molecular pathways by which clozapine causes its metabolic side effects. We used a proteomic approach to identify proteins in lymphoblastoid cells of patients with schizophrenia and normal controls that were oxidized after treatment with the antipsychotic clozapine. The overall concept was to identify irreversible protein oxidation by using a modification of a cysteine-labeling method initially described by Baty et al.45 Cysteine residues are remarkably sensitive markers of cellular oxidation status. In the approach used for our studies, the level of formation of irreversibly oxidized thiols (conversion of thiols in proteins to either sulfinic or sulfonic acids) is measured by quantifying the incorporation of 6-IAF into the thiol group of cysteine residues of proteins. The reaction chemistry of 6-IAF is specific for free thiol (−SH), and therefore 6-IAF cannot react with any oxidized form of cysteines. The loss of incorporation of 6-IAF into the cysteine residues of proteins, after all disulfide bonds and sulfenic acids have been reduced, is the indication of irreversible oxidation. Labeling of cysteine residues has previously been used to detect protein oxidation.45,46
For this study, we used cultured lymphoblastoid cells from patients with schizophrenia and normal controls to determine differential effects of the drug clozapine on protein oxidation. Increased protein oxidation (decreased 6-IAF fluorescence) was observed in 9 protein spots (P<0.05). Using HPLC-ESIMS/MS, we identified seven different proteins corresponding to these 9 spots (Table 1). Several of these proteins are involved in energy metabolism and mitochondrial function. Enolase functions in glycolysis by catalyzing conversion of 2-phosphoglycerate to phosphoenolpyruvate. It has been suggested that it plays a role in glucose metabolism in patients with schizophrenia.47 TPI catalyses the reversible interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate and plays an important role in several metabolic pathways (glycolysis, gluconeogenesis, and triglyceride synthesis) and is essential for efficient energy production. TPI deficiency in humans is manifested as a rare autosomal recessive multisystemic disorder characterized by lifelong hemolytic anemia and severe progressive neuromuscular degeneration.48Glyceraldehyde-3-phosphate dehydrogenase (GAPD) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, a step in glycolysis which is an important pathway of energy supply from breakdown of glucose. Recent studies have also demonstrated that GAPD has non-glycolytic functions in physiologic and pathologic processes, including integration of energy metabolism and cell-cycle regulation; the inhibition of GAPD has been linked to neuronal apoptosis.49GDP dissociation inhibitor (GDI) participates in insulinstimulated GLUT4 translocation by facilitating Rab10 recycling.50 The actin-binding protein cofilin is a key target of oxidation. Oxidation of cofilin causes it to lose affinity for actin and to translocate to the mitochondria, where it induces swelling and release of cytochrome c by mediating the opening of the permeability transition pore (PTP).51Uridine monophosphate/cytidine monophosphate (UMP-CMP) kinase, a ubiquitous enzyme, catalyses an important step in the phosphorylation of uridine triphosphate (UTP), cytidine triphosphate (CTP), and deoxycytidine triphosphate (dCTP), nucleotides involved in energy metabolism. It is involved in both the de novo and salvage pathways of nucleosides, and is necessary for the phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies.52,53
As seen in Table 1, both schizophrenia and control cells revealed three spots each that were oxidized due to drug treatment. In addition, four spots showed increased oxidation in cells from treated patients with schizophrenia compared with those from treated controls, although we did not observe differential oxidation in untreated patients compared with untreated controls. This suggests that cells of patients with schizophrenia are more prone to oxidative stress, which may be the reason for the increased risk of metabolic syndrome and diabetes in patients with schizophrenia.
We have previously directly identified energy metabolism proteins in SKNSH cells that are oxidized by treatment with antipsychotic drugs.43 The results presented here support our hypothesis that alterations in protein oxidation could be caused by generation of free radicals and ROS by clozapineinduced oxidative stress, and that this could be a mechanism by which antipsychotics induce increased risk for metabolic side effects.
Because no sophisticated statistical analyses were done to control for multiple comparisons, it is possible that some of the proteins identified in this study were false positives. However, given the exploratory nature of the present study, we preferred to err on the side of Type I errors (false positives), rather than risk Type II errors (false negatives) which might have led to proteins of importance being overlooked. Further studies should be performed to corroborate the present results.
To summarize, this study used a novel proteomic approach to identify proteins that are irreversibly oxidized by clozapine treatment in lymphoblastoid cell lines. We found that clozapine causes oxidation of several proteins that are involved in energy metabolism and mitochondrial function, and that patients with schizophrenia appear to be more susceptible to clozapine-induced protein oxidation. Our results support the hypothesis that oxidative stress may be a mechanism by which antipsychotics increase the risk for metabolic syndrome. Additional studies examining the effects of other SGAs on protein oxidation are underway. It is hoped that the identification of specific proteins that are structurally altered by treatment with antipsychotics may lead to the development of treatments that could mitigate or prevent the serious metabolic side effects associated with a number of antipsychotics.
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Appendix: Detailed Description of Materials and Methods
Peripheral blood leucocytes were isolated from subjects from the Central Valley of Costa Rica using LeucoPREP brand cell separation tubes (Becton Dickinson Labware, Lincoln Park, NJ, USA) and transformed using Epstein- Barr virus (EBV) as previously described by Anderson and Gusella.54 Cells were grown in RPMI 1640 medium with 2 mM L-glutamine and 15% bovine growth serum at 37°C in a humidified 5% CO2 chamber to a density of approximately 2×106 cells/ml, collected by centrifugation and cryo-preserved for future use. These cell lines were generated from subjects with schizophrenia and their unaffected parents and/or siblings, recruited in accordance with the principles of the Declaration of Helsinki and with approval from the Institutional Review Boards of the University of Costa Rica and the University of Texas Health Science Center at San Antonio, as previously described.44 Lymphoblastoid cell lines from individuals with schizophrenia (N=6) and healthy controls (not diagnosed with a DSM-IV psychiatric disorder, N=6), matched for age and gender, were selected for the experiments described below. Control cell lines were chosen from unaffected individuals from the same Costa Rican population, who were not related to the schizophrenia individuals whose cell lines were used for this study.
Labeling of Protein Thiols by 6-iodoacetamide Fluorescein
The thiol (-SH) group in the amino acid cysteine is particularly sensitive to oxidation and can be oxidized to several states including irreversible oxidation states [sulfinic- (SO2H) and sulfonic (SO3H) acids]. The cysteine residue can therefore be used as a marker for protein oxidation. Lymphoblastoid cells from patients with schizophrenia and normal controls were treated either with clozapine (20 μM) or vehicle (DMSO) for 24 hours. A 1 mM drug stock was prepared fresh by dissolving in DMSO. Protein extracts, which were obtained by sonication of cells in buffer containing 50 mM potassium phosphate (pH 7.9), 0.5 mM MgCl2, 1 mM EDTA, and Halt protease inhibitor cocktail (Pierce, Rockford, IL), were then centrifuged at 6000 g at 4°C for 1 hour.55 The supernatant was treated with 6 M urea at 37°C for 1 hour, followed by treatment with 2 mM dithiothreitol (DTT) and sodium arsenite (20 mM) to reduce the disulfide (S-S) and sulfenic acid (SOH), respectively.56 It should be noted that sodium arsenite cannot reduce sulfinic and sulfonic acids; thus, these modifications are considered to be irreversible. The extract was then treated with 5 mM 6-iodoacetamide fluorescein (6-IAF) (Invitrogen, Carslbad, CA), which binds to thiol groups, and incubated at 37°C for 1 hour in the dark for complete reaction between thiols and 6-IAF. The proteins were then precipitated with an equal volume of 20% trichloroacetic acid (TCA), centrifuged at 12,000 g for 20 minutes, and washed at least three times with ethanol/ethylacetate (1:1, v/v) to remove urea, DTT, sodium arsenite, and free 6-IAF. Protein pellets labeled with 6-IAF were then dissolved in 8M urea, 2% 3-[(3- cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS), 0.5% n-isopropyl glycine (IPG) buffer (GE Healthcare, Piscataway NJ), and DeStreak reagent (15 mg/ml, GE Healthcare); protein concentrations were assessed through the use of a bicinchoninic acid (BCA) assay (Pierce).
Two Dimensional Gel Electrophoresis
The labeled protein extracts were subjected to two dimensional gel electrophoresis as follows: 100 μg of protein was subjected to isoelectric focusing overnight on 13 cm strips (pH 3–10) using the IPGphor system (GE Healthcare). After the first dimension, strips were equilibrated for 15 minutes in buffer containing first DTT (100 mg/ml) and then iodoacetamide (250 mg/ml). The second dimension was then run using precast Criterion 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (BioRad, Hercules, CA), after which 6-IAF fluorescence was assessed using a Typhoon 9400 variable mode imager (GE Healthcare) with excitation at 490 nm. Gels were then stained with SYPRO Ruby (Invitrogen) overnight, after which they were washed twice with water and then soaked in water for 15 minutes. Gel images were then acquired using the Typhoon 9400 with excitation at 532 nm.
Quantitative Assessment of Gel Images
Spot intensities on 6-IAF and SYPRO Ruby gel images were quantified separately using Imagequant version 5.0 software (GE Healthcare). To obtain the 6-IAF fluorescence relative to total protein fluorescence, the pixel intensity/area of 6-IAF fluorescence was divided by the pixel intensity/area of SYPRO Ruby fluorescence for each spot. Because 6-IAF does not bind to irreversibly oxidized cysteine residues, a decrease in fluorescence intensity in protein spots of treated compared with untreated samples is an indication of greater oxidation (i.e., less labeling of 6-IAF). To identify protein spots that showed statistically significant (P<0.05) mean differences in fluorescence intensity, we compared gels from treated vs. untreated cells in patients and controls separately, using t tests for independent samples, with two-sided significance testing. We also performed t tests to compare gels from schizophrenia cells and normal control cells that had been treated with clozapine.
Protein Identification by Mass Spectrometry
Spots exhibiting significant differences in 6-IAF fluorescence were excised from the gels and digested in situ with trypsin. The resulting digests were subjected to capillary high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLCESI- MS/MS) using a Thermo Fisher LTQ ion trap mass spectrometer interfaced to an Eksigent NanoLC micro HPLC system (Eksigent, Dublin, CA) via a New Objective (Woburn, MA) PicoFrit nanospray interface. Online HPLC separation of the proteolytic peptides was accomplished as follows: column, PicoFrit (New Objective; 75 μm i.d.) packed to 10 cm with C18 adsorbent (Vydac; 218MSB5; 5 μm, 300 Å); mobile phase A, 0.5% acetic acid/0.005% trifluoroacetic acid (TFA); mobile phase B, 90% acetonitrile/0.5% acetic acid/0.005% TFA; linear gradient of 2% to 42% B in 30 minute flow rate, 0.4 μl/min. As part of the data-dependent acquisition protocol, the seven most intense ions in each survey scan were sequentially fragmented in the ion trap by collision-induced dissociation using an isolation width of 3.5 and relative collision energy of 35%. Uninterpreted tandem mass spectra were searched against published databases using Mascot (Matrix Science, Boston, MA; 10 processor in-house license). Cross-correlation of the Mascot results with X! Tandem and determination of probabilities of protein and peptide identifications were made by Scaffold (Proteome Software, Portland, OR). Assignment of the tandem MS fragments were verified by comparison with the predicted ions generated in silico by GPMAW (Lighthouse Data, Denmark).