ToxSci Advance Access originally published online on February 29, 2008
Toxicological Sciences 2008 103(2):311-324; doi:10.1093/toxsci/kfn044
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Khat (Catha edulis) Induces Reactive Oxygen Species and Apoptosis in Normal Human Oral Keratinocytes and Fibroblasts
,*,1
* Section for Pathology, The Gade Institute
Centre for International Health
Department of Clinical Dentistry
Department of Pathology, The Gade Institute, Haukeland University Hospital, University of Bergen, Bergen, Norway
1 To whom correspondence should be addressed at Department of Pathology, The Gade Institute, Haukeland University Hospital, University of Bergen, N-5021 Bergen, Norway. Fax: +47-55-97-3158. E-mail: Olav.Vintermyr{at}helse-bergen.no.
Received November 12, 2007; accepted February 26, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Khat chewing is widely practiced in Eastern Africa and the Middle East. Khat is genotoxic to cells within the oral mucosa, and several studies have suggested an association between khat use and oral lesions like hyperkeratosis and oral cancer. This study investigated the mechanism of khat-induced cytotoxicity using primary normal human oral keratinocytes (NOK) and fibroblasts (NOF). Khat induced rounding up of cells, plasma membrane blebbing, and condensation of nuclear chromatin within 3–6 h of exposure. The cells also showed externalization of phosphatidylserine and fragmentation of DNA. Morphological and biochemical features were compatible with cell death by apoptosis. Khat also induced an increase in cytosolic reactive oxygen species (ROS) and a depletion of intracellular glutathione (GSH) within 1 h of exposure. Antioxidants reduced ROS generation, GSH depletion and delayed the onset of cytotoxicity in both cell types. Generally, NOF cells were more sensitive to khat-induced cytotoxicity than NOK cells. These effects were elicited at concentrations of khat expected to occur in the oral cavity during khat chewing. In summary, khat induced apoptotic cell death in primary normal oral keratinocytes and fibroblasts by an early effect on mechanisms that regulate oxidative stress.
Key Words: khat; oral keratinocytes; oral fibroblasts; reactive oxygen species; apoptosis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Khat (Catha edulis) is an evergreen shrub of the Celastraceae family. Cultivation of khat is concentrated in parts of the Middle East and Eastern Africa where its use is believed to contribute to the social and economic well being of the communities (Al-Motarreb et al., 2002
). Fresh leaves and shoots of the khat plant contain the chemical cathinone, which has a psychoactive effect comparable to amphetamine (Kalix et al., 1990). When the leaves and shoots are chewed, cathinone is sufficiently extracted in saliva and absorbed into the blood stream to elicit a stimulant effect. Habitual chewing of khat is widespread in Yemen and the horn of Africa, and its use as a stimulant is gradually spreading to other parts of the world especially in immigrant communities (Al-Motarreb et al., 2002). Khat use has been reported to have adverse effects on various organ systems in humans (Al-Habori, 2005). In the oral cavity, khat chewing has been associated with histopathological changes like hyperkeratosis, epithelial hyperplasia and mild dysplasia (Ali et al., 2004, 2006). A higher incidence of head and neck cancer has been reported among khat chewers compared with nonchewers (Nasr and Khatri, 2000; Soufi et al., 1991). Khat has been found to be genotoxic to cells of the oral mucosa (Kassie et al., 2001), to inhibit de novo synthesis of proteins, RNA and DNA (Al-Ahdal et al., 1988; De Hondt et al., 1984), to reduce free radical metabolizing enzymes (Al-Qirim et al., 2002) and to induce a caspase dependent apoptotic cell death in various leukemic cells (Dimba et al., 2004). However, studies on the toxicological potential of khat remain scarce (Carvalho, 2003) especially regarding the mechanism(s) by which khat could induce the reported oral lesions.
Apoptosis is a regulated form of cell death that is distinguishable from necrosis by its distinct morphological features which include cytoplasmic shrinkage, plasma membrane blebbing and nuclear chromatin condensation (Kerr et al., 1972), as well as biochemical features like externalization of phosphatidylserine, fragmentation of DNA and activation of specific caspases (Gavrieli et al., 1992; Martin et al., 1995). During carcinogenesis, cells make use of defects in the regulation of apoptosis to generate immortal cells with an ability to evade apoptosis. Many plant-derived substances induce apoptosis in mammalian cells (Horie et al., 2005; Lai and Lee, 2006; Ramage et al., 2006).
Reactive oxygen species (ROS) are unstable molecules, ions or radicals generated through normal cellular metabolic processes. They include free radical species like superoxide anion and hydroxyl radical as well as nonradical species like hydrogen peroxide. These molecules are involved in various normal cellular processes like gene expression (Fialkow et al., 2007), proliferation and differentiation (Dumont et al., 2000). Exogenous and endogenous stress may generate excessive amounts of ROS that can damage molecules like DNA, proteins and lipids. This in turn can induce cell cycle arrest and premature senescence (Dumont et al., 2000; Macip et al., 2002) as well as activation of pathways leading to cell death (Huang et al., 2000). Glutathione (GSH) is an essential tripeptide found in mammalian cells where it maintains the intracellular thiol redox status, and detoxifies exogenous and endogenous reactive molecules. Depletion of intracellular GSH predisposes cells to proapoptotic stimuli and can also activate apoptosis in the absence of such stimuli (Valko et al., 2007). Cellular antioxidant defense systems including superoxide dismutase, catalase, and GSH may thus prevent disturbances in ROS homeostasis, or reduce the effect of oxidative stress in cells (Valko et al., 2007).
Epithelial cells lining the oral cavity, like epithelial cells of the skin, normally express high levels of cytokeratins and are termed keratinocytes (Presland and Dale, 2000). Oral keratinocytes build a stratified epithelium that is nonkeratinized or keratinized depending on the location within the oral cavity (Presland and Dale, 2000; Squier and Kremer, 2001). In this study, normal human oral keratinocytes (NOK) and fibroblasts (NOF) were derived from the nonkeratinized buccal (cheek) mucosa. Culture and characterization of NOK and NOF cells in monolayer and organotypic cocultures have been described in previous studies (Costea et al., 2005; Merne et al., 2004; Moharamzadeh et al., 2007). Cells of the oral mucosa are exposed to high doses of khat constituents during khat chewing rendering them susceptible to its potentially toxic effects. In our previous work (Lukandu et al., 2008) normal oral cells exposed to low (sublethal) levels of khat in vitro showed an increased expression of p53, p21, and p16 proteins and a subsequent cell cycle arrest at the G1 phase. In the present study, higher concentrations of organic and aqueous extracts of khat were used to determine the effects on NOK and NOF cells in vitro. At an early time point, cellular GSH was depleted and the levels of ROS increased prior to onset of cell death having morphological and biochemical features of apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Materials
Dulbecco's Modified Eagle's Medium (DMEM), trypsin–ethylenediaminetetracacetic acid (EDTA), Trypan blue dye, dimethyl sulphoxide (DMSO), N-acetlyl-L-cysteine (NAC), 4,5-dihydroxyl-1,3-benzededisulfonic acid (Tiron) and cycloheximide (CHX) were acquired from Sigma (St Louis, MO). Caspase inhibitor (z-VAD-fmk) was bought from Nordic Biosite AB (Taby, Sweden). Serum-free keratinocyte medium (KSFM), human recombinant epidermal growth factor (EGF), bovine pituitary extract (BPE), fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin, and amphotericin B were acquired from GibcoBRL (Grand Island, NY). Cell culture chambers slides (Lab-Tek Permanox) were acquired from Electron Microscopy Sciences (Hatfield, PA). Cell culture flasks and plates were from Nunc (Napervile, IL). Fluorescent DNA stains (Hoechst 33342, propidium iodide [PI] and YO-PRO-1), the cell-permeable GSH-sensitive dye 5-chromomethylfluorescein diacetate (CMF-DA) as well as the ROS-sensitive dyes dihydroethidium (DHE) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) were from Molecular Probes Europe (Leiden, Netherlands). In situ Cell Death Detection kit, AP (terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling [TUNEL] assay), Annexin-V Biotin and the Cell Proliferation Reagent WST-1 were from Roche Diagnostics GmbH (Mannhein, Germany). The aqueous extract of khat tested in this study was donated by Dr Nezar Al-Hebshi, Institute of Oral Sciences—Oral Microbiology, University of Bergen, Norway.
Khat Extraction
Khat extraction and analysis by LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) was done as previously described (Dimba et al., 2004). Briefly, 200 g of fresh khat shoots from the Meru district of Kenya were chopped into small (5 mm) pieces and dissolved in 50 ml methanol. The mixture was sonicated at room temperature (RT) while shielding from light for 15 min, and filtered through an 11-µm filter (grade 1, Whatman, Kent, UK). The nonfiltered plant residue was re-extracted in 50 ml of fresh methanol and sonicated for 24 h. The mixture was filtered and admixed with the initial 50 ml of filtrate. The resultant solution was concentrated at 337 millibar in a Rotorvapor vacuum drier (Büchi, Switzerland) for 4–5 h into an oily paste. The 200 g fresh plant material yielded about 12.6 g of this oily paste. This was dissolved in 40 ml of DMSO (0.315 g/ml). Aliquots (each of 200 µl) were stored at –80°C. The quality of the extraction procedure was verified by confirmation of the presence of khat-specific phenylpropylamines (cathinone, cathine, norephedrine) in the alkaloid fraction using differential thin layer chromatography. A droplet of the methanolic extract was spotted onto a silica plate (Kieselgel F-254, Merck, Darmstadt, Germany) and developed in ethyl acetate:methanol:ammonia (85:10:5). Detection of these compounds was done by development of the plate in a 0.5% ninhydrin (Merck, Darmstadt, Germany) solution in ethanol and heating it (110°C, 5 min) to demonstrate a clear separation between cathinone (retardation factor 0.6) and the other two alkaloids (retardation factor 0.45). The organic extract was also evaluated for the amount of khat-specific phenylpropylamines by LC/MS/MS as previously described (Dimba et al., 2004). Prior to each experiment, a new batch of khat stock solution was thawed at RT, diluted in cell culture media to a concentration of 10 mg/ml and centrifuged at 3000 x gav. (average gravity) (10 min, RT). An alternative extraction protocol based on an aqueous extraction of khat was also performed. In brief, fresh khat was air dried and extracted in water at 37°C for 4 h. The extract was then lyophilized and stored as dry powder at –20°C as previously described (Al-Hebshi et al., 2005). Immediately prior to each experiment, the dry khat powder was dissolved in culture media to a concentration of 10 mg/ml and centrifuged (3000 x gav. 10 min, RT). In both organic and aqueous extracts, the supernatant fraction was collected after centrifugation and adjusted to the appropriate concentrations to be tested in each experiment (range 1000–1 µg/ml khat) by performing logarithmic serial dilution.
Isolation and Cultivation of Primary Human Oral Cells
NOK and NOF cells were isolated from superfluous tissues of the buccal mucosa from clinically healthy adult volunteers undergoing surgical removal of wisdom teeth. There were 35 donors in total (19 male, 16 female), with a mean age of 22 years and a history of smoking among 7 of them, but a potential effect(s) by gender, age and smoking habits on cell responses was not addressed in this study. All patients included were informed of the purpose of the study and signed consent forms. The study was approved by the Regional Committee for Medical Ethics in Research. Cells were isolated through a combination of enzymatic digestion and mechanical separation of cells, as previously described (Costea et al., 2002). NOK cells were cultured in serum-free media (KSFM) supplemented with 1 ng/ml EGF, 25 µg/ml BPE, 20 µg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. NOF cells were cultured in DMEM supplemented with 10% FBS, 20 µg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. All cultures were kept in a humidified atmosphere at 37°C and supplemented with 5% CO2. Cells used in the experiments were in their first to third passage or second to fifth passage for NOK and NOF cells, respectively. Control cultures were routinely supplemented with DMSO equal to the amount in the highest khat concentration tested, and this never exceeded 0.1%.
Determination of Cell Viability
Cells were seeded in 25-cm2 flasks and allowed to grow to approximately 50% confluency, then treated with various concentrations of khat. At indicated times, the cells were detached (0.25% trypsin/0.05% EDTA, 5 min) and centrifuged (200 x gav., 7 min) along with free floating cells in the culture media. The cells were then resuspended in phosphate buffered saline (PBS) pH 7.4, and equal volumes of the cell suspension and 0.4% trypan blue dye mixed. The fraction of cells able to exclude trypan blue was determined using a hemocytometer. A minimum of 200 cells was counted in each determination. In separate experiments the effect of NAC, Tiron, z-VAD-fmk, and CHX were tested on NOK and NOF cells in absence and presence of khat. At least three separate concentrations of each of these compounds were used with respect to induction of cell death and potential inhibition of khat-induced cell death. Cell viability was carried out using the Cell Proliferation Reagent WST-1 (Roche Diagnostics) as described by the manufacturer, whereas analysis was done using a microplate reader (Infinite 200, Tecan Schweiz AG, Männedorf, Switzerland).
Evaluation of Morphology by Light Microscopy
Cells were seeded in 12-well plates and allowed to grow to 50% confluency. The cells were then exposed to different concentrations of khat for various time points. The evaluation of morphology was done using phase contrast microscopy (Axiovert 25 inverted microscope, Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Serial photographs were taken in randomly selected areas at 400-fold magnification and at regular time intervals after exposure to khat using a Powershot G2 digital camera (Canon, Tokyo, Japan) mounted on the microscope. Quantification of cell death was based on the number of apparently normal cells in these photographs relative to the number of morphologically altered cells. Morphological changes used for this assessment included rounding up of cells, plasma membrane blebbing and detachment of cells. The cells that presented with intermediate morphological changes were scored as normal. A minimum of 200 cells were counted in each separate determination.
Evaluation of Morphology by Electron Microscopy
Cells were seeded in 25-cm2 flasks and allowed to grow to approximately 50% confluency, then treated with various concentrations of khat for different time periods. The cells were then detached (0.25% trypsin/0.05% EDTA for 5 min at 37°C), mixed with the medium containing floating cells and centrifuged (200 x gav. 37°C, 7 min). The cell pellet was fixed overnight at 4°C in 0.1M Na-cacodylate buffer, pH 7.4, containing 2% glutaraldehyde. Samples were then rinsed three times in PBS, postfixed in 1% osmium tetroxide in PBS (30 min), dehydrated using graded ethanols, embedded in epoxy resin, ultra-thin sectioned and double stained with uranyl acetate and lead citrate as previously described (Boe et al., 1991). Specimens were examined using a transmission electron microscope (JEOL 1230, Jeol Ltd, Tokyo, Japan) and the micrographs processed using an Arcus II scanner (Agfa-Gevaert N.V, Mortsel, Belgium) and Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA).
Chromatin Condensation Assay
Cells were seeded in 12 well plates and allowed to grow to approximately 50% confluency. The cells were then treated with various concentrations of khat for specified time periods. Hoechst 33342 (8.1µM) was added to each well and the plates incubated for 15 min at 37°C in a humidified atmosphere supplemented with 5% CO2. The cells were then analyzed for fluorescence under a Leica IRB inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany). Cells with intense fluorescence and condensed or fragmented nuclei were scored as nonviable, whereas those with diffuse and weak fluorescence in their nuclei were scored as normal. Very few cells expressed an intermediate fluorescence pattern and these were scored as normal. At least 200 cells were counted in randomly selected areas in each well, and the proportion of cells with increased nuclear staining determined.
Evaluation of Apoptosis
Annexin-V assay.
Cells were seeded in 8 well Permanox chamber slides and allowed to grow to approximately 50% confluency prior to khat exposure. The cells were then exposed to annexin-V biotin for 20 min, rinsed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer (10mM Hepes/NaOH, pH 7.4,140mM NaCl, 5mM CaCl2 in distilled water) and fixed (50% aqueous ice-cold acetone, 30 s, then 100% ice-cold acetone, 5 min). The cells were further processed using streptavidin-alkaline phosphatase and Fast Red substrate as described in a protocol provided by the manufacturer (Roche Diagnostics). The cells were counterstained with hematoxylin, mounted in immunomount medium (Shandon, Pittsburgh, PA), and evaluated under a light microscope (Leica DMLB, Leica Microsystems).
TUNEL assay.
NOF cells (104 per well) were seeded in 8 well chamber slides and allowed to grow for 48 h. The cells were treated with khat for various time points. The cells were fixed (4% paraformaldehyde in PBS, 30 min), air dried (20 min), and permeabilized (0.2% Triton X-100 in freshly prepared 0.1% sodium citrate, 5 min). Further processing with the TUNEL reaction mixture, anti-fluorescein conjugated with alkaline phosphatase and Fast Red substrate was carried out as described by the manufacturer of the kit (Roche). The slides were then mounted in Immunomount medium (Shandon) and analyzed under a light microscope (Leica DMLB). Photographs were taken using a camera (Leica DC 300, Leica Microsystems) attached on the microscope. A technical problem was experienced with permeabilization of keratinocytes for the TUNEL assay, therefore, for NOK cells an alternative apoptosis assay using YO-PRO-1 that did not require permeabilization was performed (see below).
Plasma membrane permeability.
NOK cells (104 per well) were seeded in 12 well plates and allowed to grow for 48 h. The cells were treated with various concentrations of khat for various times. The cells were then treated with three fluorescent DNA stains; Hoechst 33342 (8.1µM), PI (1.5µM), and YO-PRO-1 (0.1µM) for 20 min on ice and then analyzed for fluorescence (Leica IRB inverted microscope). Cells staining positive with all three dyes were considered necrotic, those staining with YO-PRO-1 and Hoechst 33342 were considered apoptotic, whereas cells stained only with Hoechst 33342 were considered normal.
Determination of Effects of Khat on Cellular Functions
Cells (2 x 104 per well) were seeded in 6 well plates and allowed to grow for 4 days (NOK) or 5 days (NOF). The cells were then briefly (0.5–24 h) exposed to 100 µg/ml khat after which the medium containing khat was discarded. The cells were then washed three times in prewarmed (37°C) PBS and supplemented with conditioned media obtained from parallel cultured cells not treated with khat. Cell numbers were determined daily by detachment of cells from selected wells using 0.25% trypsin/0.05% EDTA at 37°C for 5 min, centrifugation (200 x gav. 7 min), resuspension in PBS and counting using a hemocytometer. Growth curves were plotted based on cell numbers. In other experiments, cells were seeded in 25 cm2 flasks and allowed to grow to approximately 50% confluency. They were then treated with various concentrations of khat for 24 h, detached as described above and washed twice in prewarmed (37°C) culture medium and counted using a hemocytometer. The cells were then reseeded in fresh culture medium and allowed to plate. After 24 h, floating and loosely attached cells were removed by washing three times in PBS. The remaining attached cells (considered viable) were trypsinized (as described above) and counted again using a hemocytometer. The proportion of attached cells (seeding efficiency) was determined as a measure of survival for both khat exposed cells and controls.
Determination of Intracellular ROS and GSH
Intracellular levels of superoxide radicals, hydrogen peroxide, and GSH were assessed using DHE, DCF-DA and CMF-DA respectively. Cells were grown in 48 well plates to reach half confluency. They were then treated with khat for various time periods, washed three times in PBS and incubated in medium containing 20µM DCF-DA, 5µM DHE or 5µM CMF-DA for 20 min. The cells were then washed again in PBS, replenished with fresh culture medium and analyzed for fluorescence under a Leica IRB inverted microscope (Leica Microsystems GmbH). In separate experiments, half confluent cell cultures in 25 cm2 flasks were preincubated for 30 min with the antioxidants NAC (range 10µM–1mM) or Tiron (range 5–500µM) then exposed to khat for various time points, and later supplemented with 20µM DCF-DA, 5µM DHE or 5µM CMF-DA for 15 min. The cells were then trypsinized (0.25% trypsin/0.05% EDTA for 5 min at 37°C), mixed with any free floating cells in the culture media from the respective flasks and pooled by centrifugation (200 x gav., 7 min). The cells were thereafter washed twice in cold PBS (4°C) and analyzed using a fluorescence-activated cell sorter, FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) and CellQuest Pro software (Becton Dickinson). A minimum of 30,000 events (cells) were collected in each analysis.
Statistical Analysis
All experiments were carried out in duplicates and repeated separately three or more times. Data analysis was conducted using SPSS version 13 statistical program (SPSS, Inc., Chicago, IL). For each data set, means were compared using analysis of variance (ANOVA) with Bonferroni multiple comparisons. To determine khat concentrations causing 50% (LC50) and 90% (LC90) cell death, plots and curves were done by nonlinear regression sigmoidal curve-fitting with standard slope using Sigma Plot software (Systat Software, Inc., San Jose, CA). Whenever statistical significance was calculated, p values less than 0.05 were considered significant. Data were presented as means ± standard error of the means (SEM).
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RESULTS |
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Morphological Features of Khat-Induced Cell Death
Primary oral keratinocytes and fibroblasts treated with khat (
100 µg/ml) showed morphological features of cell death. Early effects were characterized by rounding up of cells and blebbing of the plasma membrane (Fig. 1). Using a fluorescent DNA stain (Hoechst 33342) a proportion of NOK and NOF cells treated with khat showed intensely stained nuclei indicating chromatin condensation. Generally, the affected nuclei appeared smaller; some had peripherally condensed or clumped chromatin whereas others had fragmented nuclear chromatin (Fig. 1). Blebbing was more prominent in NOK cells, whereas the nuclear changes were more typical of NOF cells. When observed under electron microscopy, khat treated cells showed a consistent loss of microvilli, prominent vacuolization in the cytoplasm, shrinkage and chromatin condensation (Fig. 2)
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Fibroblasts were More Sensitive to Khat than were Keratinocytes
On exposure to 316 µg/ml khat, 21.0 ± 5.6% NOK and 28.0 ± 6.1% NOF cells had abnormal morphology after 4 h compared with 4.5 ± 1.0% and 7.0 ± 1.7% in controls respectively. The proportion of cells taking up trypan blue dye was lower when compared with the proportion of morphologically altered cells. Thus, 39.5 ± 4.5% NOK cells and 48.5 ± 5.8% NOF cells had abnormal morphological features 8 h after exposure to 316 µg/ml, whereas only 16.2 ± 4.0% and 22.0 ± 2.3%, respectively, did not exclude trypan blue dye (p < 0.05) (Fig. 3). Cells exposed to khat underwent a concentration dependent nuclear chromatin condensation. On exposure to 316 µg/ml khat for 8 h, a higher number of NOF cells (47.5 ± 3.5%) had condensed nuclear chromatin when compared with NOK cells (29.0 ± 4.1%) (p < 0.05) (Fig. 4A). After 24 h exposure, the number of cells with condensed nuclei increased to 76.8 ± 5.7% in NOF cells and to 50.3 ± 7.1% in NOK cells (p < 0.05). When exposed to 100 µg/ml khat for 24 h, 32.0 ± 4.5% of the NOK cells had condensed nuclear chromatin compared with 53.8 ± 4.3% in NOF cells (p < 0.05). NOF cells were therefore, in general, more sensitive to khat than were NOK cells.
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Induction of Cell Death by an Aqueous Extract of Khat
An aqueous extract, closely mimicking the extraction of khat in saliva during khat chewing, was also evaluated for its cytotoxicity. The aqueous extract induced cell death in NOF cells in a manner very similar to the organic extract (Fig. 4C).
Khat-Induced Cell Death Showed Biochemical Features of Apoptosis
Both NOK and NOF cells exposed to khat became annexin-V positive (Figs. 5A and 5C). Annexin-V positive cells had a rounded contour and showed other morphological characteristics of dying cells as described above. In NOK cells exposed to khat, the morphologically altered cells excluded PI, but were permeable to the fluorescent dye YO-PRO-1, in contrast to cells not exposed to khat which remained impermeable to both dyes (Fig. 5B). In NOF cells, the morphologically altered cells were positive with the TUNEL assay, indicating chromatin degradation in khat exposed cells (Fig. 5D).
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Short Term Exposure to Khat—Effects on Cell Death and Cell Substrate Attachment
Primary oral cells exposed to 100 µg/ml khat for less than 0.5 h did not show signs of cell death nor effects on the cell proliferation (Fig. 6). A small proportion of cells exposed to khat for an intermediate time period (0.5–2 h) underwent cell death, and the remaining morphologically unaffected cells regained their ability to proliferate. Cells exposed to 100 µg/ml khat for more than 4 h did not recover growth and most of these died. These results showed a critical maximal exposure time of 0.5 h during which all effects on cell death and cell proliferation induced by 100 µg/ml khat were fully reversible.
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In a functional bioassay, the ability of khat exposed oral cells to attach to a substrate was tested. In this assay, the ability of the cells to attach was used as a parameter to determine cell survival. It was found that cells not able to attach to a growth surface were committed to cell death. Khat concentration leading to 50% cell death (LC50) and 90% cell death (LC90) for NOK cells after 24 h of exposure was 83 µg/ml and 245 µg/ml respectively (Fig. 7A) versus 46 µg/ml and 117 µg/ml khat respectively for NOF cells (Fig. 7B). The ability of cells to attach after khat exposure (seeding efficiency) was inversely related with exposure time and concentration of khat used in the experiments. A detailed association between seeding efficiency, exposure time and khat concentration is shown in Figure 7C. Other consistent observations were that individually growing cells in culture were more prone to undergo cell death than cells clustered together, and cells seeded at higher densities were more resistant to khat-induced cytotoxicity than those seeded at lower densities.
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Effects of Khat on Intracellular ROS and GSH in Oral Cells
Primary oral cells exposed to DCF-DA in the presence of 200 µg/ml khat showed a progressive increase in green fluorescence with time of exposure indicating increasing levels of hydrogen peroxide in these cells (Fig. 8). In control cells, the intensity of green fluorescence remained low and basically unchanged during the experimental period. NOK and NOF cells stained with the DHE showed an increased number of nuclei showing red fluorescence in khat (200 µg/ml) exposed cells. In control cultures, only few cells stained with DHE showed red fluorescence and no apparent change was noted during the experiment. Increased fluorescence labeling was observed for both DCF-DA and DHE in cells exposed to khat for at least 45 min. Thus, effects on fluorescence labeling with these probes occurred prior to any morphological and biochemical effects observed in khat treated cells. Maximal staining of cells with DCF-DA and DHE was found after 4 h exposure to khat. Cells stained with the probe CMF-DA showed a progressive decrease in green fluorescence signal during the time of khat exposure (Fig. 8). No change in CMF-DA fluorescence staining was observed during the first 0.5 h of khat exposure. Unlike DCF-DA and DHE staining in which all cells treated with 200 µg/ml khat for 4 h were affected, about 20% of the cells stained with CMF-DA remained unaffected at 4 h (Fig. 8).
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Khat-Induced Cytotoxicity was Delayed, but not Prevented by Antioxidants
It was further tested whether the observed cytotoxic effects of khat in oral cells could be chemically modulated by antioxidants. NOK and NOF cells treated with 200 µg/ml khat in the presence of the antioxidants Tiron and NAC showed decreased DCF-DA and DHE staining as well as preserved CMF-DA staining (Fig. 9). Tiron was more effective than NAC in protecting against accumulation of ROS and depletion of GSH as evaluated by DCF-DA, DHE, and CMF-DA staining in both NOK and NOF cells (Fig. 9). In the viability assay, Tiron protected against cell death in both NOK cells and NOF cells (p < 0.05) after exposure to khat (200 µg/ml) for 8 h (Fig. 10). A possible protective effect by NAC (100µM) was not found to be significant in either cell types (Fig. 10). A higher concentration of NAC (1mM) was also tested, but did show cell death by itself and was not further tested. Tiron (concentration range 5–500µM) was not found to induce cell death by itself. Upon long-term exposure to khat, the cytoprotective effects of Tiron decreased, and were not observed after 24 h of exposure. A pan-caspase inhibitor, z-VAD-fmk (range 1–100µM) did not protect against cell death in NOK or NOF cells at concentrations previously reported to inhibit khat-induced cell death in a promyelocytic leukemic (HL-60) cell line (Dimba et al., 2004
). CHX (range 0.1–10 ng/ml) did not protect against khat-induced cell death although the highest concentration tested (10 ng/ml) was cytotoxic by itself, and a lower concentration (1 ng/ml) was routinely used in the experiments (Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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This study focused on the mechanism of khat-induced cytotoxicity in primary normal oral cells in vitro. Oral keratinocytes and fibroblasts exposed to khat underwent morphological changes like plasma membrane blebbing, loss of microvilli, cell shrinkage and condensation of nuclear chromatin (Fig. 1). In the early phase of cell death, the cells remained impermeable to vital dyes like trypan blue and showed externalization of the plasma membrane phosphatidylserine. The cells also showed fragmentation of nuclear DNA and a selective uptake of the apoptosis marker YO-PRO-1 (Fig. 5). Taken together, the mode of cell death observed in this study was therefore supportive of apoptosis.
For induction of cell death to occur, oral cells had to be exposed to khat for at least 0.5 h. Thus, the commitment to a regulated cell death, and initiation of specific irreversible cell death pathways occurred within a critical exposure time period of 0.5 h, prior to onset of any obvious morphological and biochemical signs of cytotoxicity. This was corroborated by the observation found in the functional bioassay in which cells were allowed to attach onto a substrate after treatment with 100 µg/ml khat for varying time periods. In this assay, more cells were observed to be functionally affected at an earlier time point than normally observed using standard biochemical and morphological criteria for assessing cell death. This observation would also support the view of a specific (regulated) rather than a nonspecific type of cell death induced by khat. The identification of a critical exposure time would be relevant in clinical terms because it could highlight the relationship between the duration of exposure to khat and the potential hazardous effects on the oral mucosa especially of prolonged khat chewing sessions.
In a previous study on rats, intragastric administration of an alkaloid fraction of khat caused a decrease in circulating free radical metabolizing enzymes (Al-Qirim et al., 2002). Free radical generation (Tan et al., 1998) and GSH depletion (Armstrong et al., 2002) have previously been found to play an important role in programmed cell death due to their potential damaging effect on macromolecules like DNA and due to their effect on cell death signaling mechanisms. A possible role of ROS and GSH in khat-induced cytotoxicity was therefore tested. Significant effects on levels of intracellular ROS and GSH were observed after 45 min of khat exposure. These effects preceded morphological and biochemical features of cell death, suggesting that ROS generation and GSH depletion could be early events in the induction of cell death following khat exposure. Moreover, addition of known antioxidants like NAC and Tiron prior to khat exposure reduced the accumulation of ROS and depletion of GSH within khat exposed cells. Tiron also showed significant inhibition of cell death induced by khat. One possible mechanism for the observed reversibility or recovery of cells after a limited exposure to khat could therefore be related to an effect of intracellular free radical scavengers like GSH, superoxide dismutase or catalase to re-establish the redox homeostasis. According to this hypothesis, prolonged exposure to khat leads to irreversible oxidative stress and cell death. Khat use is strongly associated with oral hyperplasia and hyperkeratinization (Ali et al., 2006), but also a few cases of oral ulceration and oral cancer have been reported among khat chewers (Fasanmade et al., 2007) A putative role of ROS in khat-induced oral lesions has not been reported previously. It is, however, known that khat chewing can cause genetic damage in oral cells (Kassie et al., 2001), but whether ROS have a role in the process has not yet been studied. In oral cells, the rapid increase in ROS following exposure to khat could point to sources like mitochondria and lysosomes rather than through activation of ROS-sensitive genes.
Fibroblastic cell lines are commonly used as test cells in cytotoxic studies. In this study, primary normal oral fibroblasts and keratinocytes were used to assess khat cytotoxicity in an attempt to mimic the exposure during khat chewing. Fibroblasts appeared to be more sensitive to khat than keratinocytes. Some possible explanations for increased sensitivity in fibroblasts could be that keratinocytes are more likely (1) to grow in colonies of closely packed cells than fibroblasts, and (2) to differentiate in response to stress signals (Lukandu et al., 2008), which enhance survival.
An issue that was further considered in this study was whether the observed effects of khat could be of potential clinical relevance with respect to concentration of khat in saliva during khat chewing. The major components of khat are considered to be extracted in the oral cavity during chewing, but the exact concentration of such components in saliva has not been accurately assessed in previous studies. A previous study indicated that the amount of cathinone ingested after 1 h of khat chewing was on average 45.1 mg (Toennes et al., 2003). If one assumes that the whole amount of cathinone would remain in saliva without being absorbed into the blood stream, then the concentration of cathinone in stimulated saliva (Vissink et al., 2003) would reach close to 500 mg/l. However, it has been shown that cathinone is partly absorbed into the blood stream through the oral mucosa (Toennes et al., 2003), thus making this scenario unlikely to occur in a clinical situation. On the other hand, considering that the whole amount of cathinone would be immediately absorbed into the blood stream, the concentration of cathinone in saliva would be 0.9 mg/l, as in all other body fluids. The concentration of khat tested in this study contained cathinone at levels of 0.1–10 mg/l. This is within the range of the two extreme assumptions mentioned above, indicating that the concentrations of khat used in this study are within the range of concentrations that could be found in saliva among khat chewers. It should be pointed out, however, that the concentrations of other putative active ingredients in our khat extracts could be outside their normal concentration found during saliva extraction of khat in vivo.
The khat-specific components in an organic extract of khat could be different from those found in extracts produced in saliva during khat chewing. This issue was partly addressed by also testing an aqueous extract of khat. Cell death induction by an organic or an aqueous extract of khat was similar at higher concentration of khat. At lower concentrations, the effect of the aqueous khat extract was somewhat less potent (Fig. 4C). Principally, a simple aqueous extract of khat could also differ in the content of khat-specific biological active compound(s) normally extracted in saliva among khat chewers. It was also tested whether the khat-specific phenylpropylamines like cathinone, cathine and norephedrine, could separately or in combination induce cell death in primary oral cells. No such effects were observed at concentrations up to 0.1mM (data not shown), although the opposite has been reported for other cells types (Dimba et al., 2004; Tariq et al., 1987). There is currently no data available to adequately determine which of the many bioactive compounds in khat could be responsible for khat-induced cell death in primary cells. In this study, a whole extract of khat was used because it closely mimics the situation in vivo in which the oral cavity is exposed to all the bioactive compounds in khat.
In conclusion, an extract of khat-induced cell death by apoptosis in primary human oral cells in vitro. The cell death was preceded by oxidative stress characterized by a rapid increase in intracellular ROS and depletion of GSH. It is therefore proposed that oxidative stress could be a likely mechanism through which khat induces cell death. These effects could be of clinical relevance because they were observed at concentrations of khat obtainable in the oral cavity among people chewing khat.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Research Council of Norway to A.C.J.; L. Meltzer's Høgskolefond to O.M.L., A.C.J., and O.K.V.; University of Bergen, The Gade Institute and Centre for International Health to O.M.L.; and Norwegian State Education Loans Fund (Lånekassen) to O.M.L.
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ACKNOWLEDGMENTS |
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We thank the staff at the Institute of Oral Sciences—Oral Surgery and Oral Medicine, University of Bergen for their assistance in getting tissue samples. The technical support from Anne Marie Austarheim (electron microscopy), Kjell Ove Fossan (analysis of cathinone content in khat samples), and Raymond Lygre (flow cytometry) is highly appreciated.
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