Extraction of Caffeine from Tea Purpose is to learn some of the basic techniques of organic chemistry: extraction, filtration, evaporation of a solvent and drying methods-in the context of working with a chemical known to all, caffeine. Pure caffeine is a white, tasteless substance that makes up as much as 5% of the weight of tea leaves. By structure, caffeine is closely related to the
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Olanzapine protects pc12 cells from oxidative stress induced by hydrogen peroxideJournal of Neuroscience Research 73:364 –368 (2003)
Olanzapine Protects PC12 Cells From
Oxidative Stress Induced by Hydrogen
Zelan Wei,1 Ou Bai,1 J. Steven Richardson,1,2 Darrell D. Mousseau,1 and Xin-Min Li1*
1Neuropsychiatric Research Unit, Department of Psychiatry, University of Saskatchewan, Saskatoon,
2Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon,
Neuroanatomical studies suggest that neuronal atrophy many other pharmacologic properties that may contribute and destruction occur over the course of many years in to their superior therapeutic actions in schizophrenia and neurodegenerative conditions such as schizophrenia and that could be the basis for their usefulness in controlling Alzheimer’s disease. In schizophrenia, early intervention psychotic symptoms in other disorders as well (Tran et al., with atypical neuroleptics such as olanzapine has been 1997; Stoppe and Staedt, 1999; Wolfgang, 1999). Olan- shown to prevent development of some of the more zapine, one of the atypical antipsychotics, has been shown serious and debilitating symptoms in many patients. The to be safe and effective in reducing behavioral disturbances mechanisms whereby olanzapine slows or prevents and psychotic symptoms in patients with Alzheimer’s dis- symptom progression in schizophrenia remain unclear. A ease (Street et al., 2000). The mechanisms by which olan- previous study found that olanzapine increased mRNA zapine exerts its antipsychotic actions in schizophrenia and for the copper/zinc isoform of the superoxide dismutase in Alzheimer’s disease likely involve the blockade of do- enzyme (SOD-1). We investigated the effects of olanza- pamine and serotonin receptors; however, the molecular pine in PC12 cells exposed to hydrogen peroxide. We mechanisms by which olanzapine and the other atypical measured cell viability, observed evidence of necrosis antipsychotics prevent symptom progression remain to be and apoptosis, checked the SOD-1 mRNA by Northern blot analyses, and determined SOD-1 enzyme activity.
Neuroanatomical studies suggest that neuronal atro- We found that: 1) the decrease in cell viability induced by phy and cell loss occur over many years during the pro- hydrogen peroxide was attenuated in PC12 cells pre- gression of neurodegenerative diseases. Neuronal cell treated with olanzapine; 2) olanzapine increased SOD death can be induced in many ways. Apoptosis, which is enzyme activity in PC12 cells; 3) inhibiting SOD activity involved in normal embryological development of the with diethyldithiocarbamic acid prevented the cytopro- central nervous system, also seems to play roles in the tective actions of olanzapine; and 4) the decrease in process of normal aging and in the pathophysiology of SOD-1 mRNA level induced by hydrogen peroxide was neurodegenerative diseases (Jesberger and Richardson, blocked by pretreatment with olanzapine. These data 1991; Simonian and Coyle, 1996; Tong and Perez-Polo, indicate that the neuroprotective action of olanzapine 1996). The apoptotic cell death pathway can be activated 2003 Wiley-Liss, Inc.
by oxidative stress (Jacobson, 1996), and this contributes tothe neurodegenerative process in Alzheimer’s disease Key words: SOD-1; neuroleptics; hydrogen peroxide;
(Subbarao et al., 1990; Coyle and Puttfarcken, 1993; Richardson, 1993; Markesbery, 1997). Hydrogen perox-ide, formed as a natural byproduct of enzymatic oxidaseaction, is an endogenous source of free radicals that con- Although classic antipsychotic drugs such as haloper- tributes to the background level of cellular oxidative stress idol produce a marked reduction in positive symptoms ofschizophrenia, they do not improve the negative symp-toms such as apathy, confusion, and social withdrawal, nor Contract grant sponsor: Canadian Institutes of Health Research (Rx&DResearch Program); Contract grant number: 200104DFE-91579-112515.
do they alter the progressive deterioration in the mentalabilities of the patient. In recent years, several new drugs *Correspondence to: Dr. X-M Li, Neuropsychiatric Research Unit, 103 have been shown to improve both positive and negative Wiggins Road, Saskatoon, SK, Canada S7N 5E3.
symptoms of schizophrenia, and seem to prevent further worsening of psychotic symptoms (Buckley, 1997; Blin, Received 15 January 2003; Revised 9 April 2003; Accepted 10 April 2003 1999). These atypical antipsychotics are potent antagonistsof both dopamine and serotonin receptors, and also have 2003 Wiley-Liss, Inc.
Olanzapine Reduces Oxidative Stress
(Halliwell, 1992; Richardson et al., 1992). Exogenous from the ﬂask surface with a ﬂow of medium from a pipette, and hydrogen peroxide can elevate oxidative stress beyond the dispersing them through a 22-gauge needle. The dispersed protective capacity of endogenous antioxidant defenses PC12 cells were then seeded on collagen-coated 96-well plates and can induce apoptosis and necrosis in cultured cortical at a cell density of 2 ϫ 104 cells/well, or at the same density (2 ϫ neurons (Koh et al., 1995; Whittemore et al., 1995) and 105 cells/mL) in T75 tissue ﬂasks. After 24 hr, the cells in the PC12 cell lines (Tong and Perez-Polo, 1996; Maroto and 96-well plates were treated with olanzapine to give ﬁnal con- centrations of 0, 6, 12, 25, 50, 100, or 200 M. After 24 hr of The rat pheochromocytoma (PC12) cell line is a exposure to olanzapine, hydrogen peroxide was added to give well-established model for studying many aspects of the ﬁnal concentrations of 0, 100, 200, or 400 M. Other PC12 cellular biology of neurons. PC12 cells have been widely cells were treated with 100 M olanzapine plus 1 mM diethyl- used to investigate the mechanisms involved in neurotox- dithiocarbamic acid for 24 hr, followed by 200 M hydrogen icity, neuroprotection, and neuronal repair. Oxidative peroxide. In both cases, after exposure to hydrogen peroxide for stress induced by hydrogen peroxide (Jackson et al., 1992) 2 hr, cell viability was determined by the MTT assay as de- or by ␤-amyloid (Zhou et al., 1996) reduces the viability scribed below. The PC12 cells in T75 ﬂasks were treated with of PC12 cells, and these effects can be attenuated by nerve olanzapine (0 or 100 M) for 24 hr and then were exposed to growth factor or by antioxidant drugs, respectively. Su- hydrogen peroxide (0 or 100 M). Cells were harvested 1 hr peroxide dismutase-1 (SOD-1), one of the endogenous later and examined with the Annexin-V-Fluos Kit. Other cells, antioxidant enzymes, is also important in protecting cells exposed to hydrogen peroxide for 2 hr, were harvested and from oxidative stress. Reducing SOD-1 activity, either by SOD-1 mRNA levels were determined by Northern blot anal- downregulating SOD-1 with antisense oligonucleotides or ysis (see below). SOD activity was determined in PC12 cells by inhibiting SOD-1 with diethyldithiocarbamic acid, in- treated with 100 M olanzapine, with 1 mM diethyldithiocar- duces apoptotic degeneration in neuronal cultures (Roth- bamic acid, or with both, for 24 hr. In all cases, the olanzapine stein et al., 1994; Pappolla et al., 1997). Conversely, and hydrogen peroxide solutions were freshly prepared in augmenting endogenous SOD-1 activity by administering RPMI 1640 medium immediately before use.
a metalloporphyrin compound that mimics the action ofSOD protects neurons from staurosporine-induced apo- Cell Viability Studies
ptosis (Patel, 1998). Previous reports from this laboratory The viability of PC12 cells in 96-well plates treated as indicated that olanzapine increases SOD-1 mRNA ex- above was determined by an MTT assay modiﬁed from Mos- pression in PC12 cells (Li et al., 1999). In the present mann (1983) and Shearman et al. (1994). In this assay, a yellow study, we investigate the ability of olanzapine to protect dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- PC12 cells from oxidative stress induced by hydrogen mide (MTT), is metabolized by active mitochondria in healthy cells to a deeply-colored formazan product. Thus, the accumu-lation of formazan is directly proportional to the viability of the MATERIALS AND METHODS
cells. After the experimental manipulations outlined above, theold medium was removed from each well and replaced with 50 Materials
L of RPMI-1640 with 1% newborn calf serum containing 0.5 The PC 12 cell line, obtained from American Type Cul- mg/mL MTT in 0.1 M phosphate-buffered saline (PBS). The ture Collection (Rockville, MD), was grown in RPMI 1640 96-well plates were incubated at 37°C for 4 hr, and then 100 L culture medium obtained from local sources. Newborn calf of acid-isopropanol (0.04 M HCl) was added to each well. After serum, horse serum, diethyldithiocarbamic acid and all other an additional 4 hr at 37°C to insure that all formazan crystals had chemicals were purchased from Sigma Chemical Co. (St. Louis, been dissolved, the optical density of each well was determined MO). The Annexin-V-Fluos Staining Kit for detecting apopto- with a microELISA reader at a wavelength of 570 nm. In this sis was purchased from Roche Molecular Biochemicals (Laval, assay, cell viability is proportional to the optical density of each PQ, Canada). TRIZOL reagent was purchased from Life Tech- well. After exposure to olanzapine (0 or 100 M) for 24 hr, and nologies (Rockville, MD). The SOD Assay Kit-WST was pur- hydrogen peroxide (0 or 100 M) for 1 hr, cells were examined chased from Dojindo Molecular Technologies (Kumamoto, Ja- with the Annexin-V-Fluos Staining Kit, a histological procedure pan), and the BCA Protein Assay Reagent kit for measuring that differentially labels necrotic cells versus cells undergoing protein concentration was from Pierce Inc. (Rockford, IL).
apoptosis. Although the MTT assays were done after 2 hr of Olanzapine was a gift from Lilly Research Laboratories (India- exposure to H O , preliminary studies showed that 1 hr of exposure to H O produced optimal resolution with Annexin- Cell Culture and Experimental Treatment
The PC12 cells were cultured in RPMI 1640 medium SOD Studies
containing 5% newborn calf serum and 10% horse serum plus Northern Blot.
100 IU/mL penicillin G sodium salt and 3% glutamine. Cells Dr. J.T. Coyle (Harvard Medical School, Boston, MA). The were fed with fresh medium every 2 or 3 days and maintained cDNA probes were labeled by random primer synthesis with in collagen-coated T75 tissue culture ﬂasks at 37°C in a humid- [␣-32P] dCTP as described previously (Li et al., 1999). Brieﬂy, iﬁed atmosphere containing 5% CO and 95% air. When cells total cellular RNA was prepared from treated cells by extraction became 80% conﬂuent, they were harvested by dislodging them with TRIZOL reagent according to supplier’s instructions. The Wei et al.
RNA was resuspended in DEPC-treated water and stored at Ϫ70°C until use. The total RNA, denatured at 65°C for 15 minin MOPS buffer containing 50% formamide and 2.2 M form-aldehyde, was separated by electrophoresis in a 1.0% agarose gelcontaining MOPS buffer and 2.2 M formaldehyde. After elec-trophoresis, RNA was transferred to a nylon membrane andcross-linked to the membrane with a UV Stratalinker 2400.
The nylon membranes were incubated at 65°C for 2 hr in a prehybridization solution containing 10% dextran sulfate, 5ϫSSPE, 5ϫ Denhardt’s solution, 0.5% SDS, and denaturedsalmon sperm DNA (200 g/mL). The cDNA probe was addedand hybridization was carried out at 65°C for 18 hr. Afterhybridization, membranes were washed twice at room temper-ature in 2ϫ SSPE and 0.1% SDS, and once at 60°C in 0.1ϫSSPE and 0.1% SDS. The membranes were then exposed atroom temperature to X-Omat AR ﬁlm with intensifying screens Fig. 1. Effects of olanzapine on H O -induced neurotoxicity in PC12 to obtain autoradiograms of the membranes. The autoradio- cells. PC12 cells were treated with olanzapine for 24 hr in different grams were scanned with a computerized densitometer (Du 640, concentrations, 0 (solid black), 50 (cross-hatched), 100 (diagonal), or Beckman Inc., Fullerton, CA) for quantitative analysis and the 200 (blank) M, then exposed to 0, 100, 200, or 400 M H O for signals were adjusted according to the original signal in trans- 2 hr. Cell viability was measured with the MTT assay. Data (mean Ϯ SEM of ﬁve separate experiments) are expressed as a percentage of the SOD Enzyme Activity
control value. *P Ͻ 0.05, ***P Ͻ 0.001, compared to the untreated harvested and lysed in a hypotonic buffer (1% NP-40, 50 mM control value. #P Ͻ 0.05, ##P Ͻ 0.01, compared to correspondingH O concentrations without olanzapine.
Tris-HCl, pH 7.5, 0.05 mM EDTA) for 20 minutes at 4°C. The lysates were centrifuged at 16,000 ϫ g for 10 minutes, and analiquot of the supernatant was used for determination of SODactivity with the SOD Assay Kit-WST following the manufac-turer’s instructions (Shimazaki et al., 2002). Protein concentra-tions of cell lysates were measured using the BCA (bicinchoninicacid) Protein Assay Reagent Kit. The SOD activity was adjustedby the amount of protein in each sample.
All data points are presented as means Ϯ SEM. Statistical signiﬁcance was assessed by ANOVA followed by Newman-Keul’s post-hoc comparisons using Prism software (GraphPadSoftware Inc., San Diego, CA).
Fig. 2. Effects of diethyldithiocarbamic acid on the action of olanzap- Effect of Olanzapine on Viability of PC12 Cells
ine and H O on PC12 cells. Cells were treated with 1 mM diethyl- Exposed to H O
dithiocarbamic acid plus 0 or 100 M olanzapine for 24 hr, and then The ANOVA indicated that the main effects of exposed to 0 or 200 M H O for 2 hr. Cell viability was measured hydrogen peroxide (F ϭ 187; df ϭ 3, 48; P Ͻ 0.001) and with the MTT assay. Data (mean Ϯ SEM of four separate experiments) of olanzapine (F ϭ 8.4; df ϭ 3, 48; P Ͻ 0.001) on the are expressed as a percentage of the diethyldithiocarbamic acid alone viability of PC12 cells were statistically signiﬁcant. As control value. ***P Ͻ 0.001, compared to the control value.
shown in Figure 1, the exposure of PC12 cells to increas-ing concentrations of H O 1 mM of the SOD inhibitor diethyldithiocarbamic acid, dependent decrease in cell viability, whereas olanzapine, at 100 M olanzapine did not attenuate the 60% reduction the concentrations used, did not alter cell viability. The in viability induced by 200 M hydrogen peroxide (Fig.
viability of PC12 cells exposed to 100 M hydrogen 2). Annexin-V-Fluos staining indicated that numerous peroxide was reduced by 25%. This was not altered by PC12 cells were undergoing apoptosis or necrosis after 50 M olanzapine, but was prevented by the 100 and treatment with H O , whereas there were fewer apoptotic 200 M concentrations of olanzapine. The 60% reduction cells in olanzapine pretreated cultures (data not shown).
in viability produced by 200 M hydrogen peroxide was attenuated by the two higher concentrations of olanzap- Investigation of SOD-1 in PC12 Cells Treated
ine. None of the olanzapine concentrations used was able With Olanzapine and H O
to alter the 90% reduction in viability produced by As shown in Figure 3, the expression of SOD-1 400 M hydrogen peroxide. In PC 12 cells treated with mRNA was reduced by 30% by 100 M hydrogen per- Olanzapine Reduces Oxidative Stress
Fig. 4. Effects of diethyldithiocarbamic acid (DETC) and olanzapineon SOD-1 activity in PC12 cells. Cells were treated with vehicle alone,100 M olanzapine, 1 mM diethyldithiocarbamic acid, or both, for24 hr. SOD-1 activity was measured according to the SOD AssayKit-WST. Data (mean Ϯ SEM of three to ﬁve separate experiments)are expressed as a percentage of the vehicle control value. *P Ͻ 0.05,**P Ͻ 0.01 compared to control value.
Fig. 3. Effect of olanzapine on SOD-1 mRNA expression in PC12 sumption that predisposes to free radical formation, and, cells. Cells were pretreated with olanzapine (0 or 100 M) for 24 hr, compared to other organs, is relatively deﬁcient in anti- and then exposed for 2 hr to 0 or 100 M H O . A: The Northern blot
oxidant activity (Floyd, 1999). When the production of bands show SOD-1 mRNA expression in total cell lysates. B: Data,
free radicals exceeds the protective capacity of the cellular expressed as percentage of untreated control, are the means Ϯ SEM of antioxidant defenses, this leads to oxidative damage to the integrated density volumes of four separate Northern blot experi- neuronal proteins and lipids, which reduces the efﬁciency ments. *P Ͻ 0.05, compared to the untreated control, # P Ͻ 0.05, of neuronal communication, and, if severe enough, will induce neuronal apoptosis. Cu/Zn superoxide dismutase(SOD-1) is one of the key enzymes by which cells detox-ify free radicals and protect themselves from oxidative oxide. If cells were pretreated with 100 M olanzapine before exposure to hydrogen peroxide, however, SOD-1 Because olanzapine increases SOD activity (Fig. 4), mRNA expression was not signiﬁcantly decreased.
the neuroprotective effects of olanzapine (Fig. 1) may be SOD-1 enzyme activity (Fig. 4) was increased by 10% due to acceleration of metabolic processing of free radicals.
after treatment with 100 M olanzapine for 24 hr, and was As indicated by Northern blot studies, olanzapine not only reduced by 40% after 24 hr of exposure to 1 mM dieth- increases SOD-1 mRNA (Li et al., 1999), but also inhibits yldithiocarbamic acid. Olanzapine did not signiﬁcantly the decrease in SOD-1 mRNA caused by H O (Fig. 3).
alter the SOD inhibition produced by diethyldithiocar- When SOD-1 activity is inhibited by diethyldithiocar- bamic acid (Fig. 4), the protective effect of olanzapine islost. This indicates that elevated SOD-1 activity plays a DISCUSSION
role in the cytoprotective action of olanzapine; however, Although it is known that clinically efﬁcacious anti- the underlying mechanisms by which olanzapine increases psychotics block central dopamine D receptors, seroto- SOD-1 activity and how this reduces cell damage remain nergic 5-HT receptors, or both, the total clinical effect of to be determined. Because the annexin staining study these drugs may depend on other actions in addition to revealed less evidence of apoptosis, but no change in receptor blockade. The present study examines the effect necrosis, in cultures pretreated with olanzapine before of olanzapine on the regulation of SOD activity in PC12 exposure to hydrogen peroxide, it seems that olanzapine cells exposed to excessive levels of oxygen free radicals.
can protect cells from apoptosis, but not necrosis.
Oxidative stress contributes to neuronal destruction Future work will focus on how olanzapine regulates in a wide range of neurodegenerative disorders, such as posttranslational modiﬁcation of SOD-1 and will investi- cerebral ischemia, Alzheimer’s disease, Parkinson’s disease, gate the effects of olanzapine on other aspects of the and amyotrophic lateral sclerosis (Jesberger and Richard- cellular oxidative stress protection system.
son, 1991; Behl, 1997; Markesbery, 1997). A variety offactors make brain function especially vulnerable to dis- ACKNOWLEDGMENTS
ruption by oxidative stress. The brain has a high metabolic We thank Ms. G. Stegeman for her technical assis- rate with a correspondingly high level of oxygen con- tance. This work was supported in part by a postdoctoral Wei et al.
fellowship from the CIHR/Rx&D Research Program to Richardson JS. 1993. Free radicals in the genesis of Alzheimer’s disease.
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Behandeling spasticiteit Wat is spasticiteit? Spasticiteit is een snelheidsafhankelijke bewegingsstoornis van spieren, die een te hoge spanning hebben. Spasticiteit wordt veroorzaakt door beschadiging van de hersenen of het ruggenmerg, die het gevolg is van een letsel (bijvoorbeeld ongeval), een aangeboren afwijking of beschadiging bij de geboorte, maar ook door ziekte op latere leeftijd (