Medicinal plants have always played an important role in the treatment of human diseases all over the world. However, it is a fact that conventional use of medicinal plants cause poisoning and death on human and animals. Datura stramonium is one of the well-known traditional plant from the Solanacea family with both poisonous and medicinal properties [1]. It has been used as a herbal medicine for relieving coughing, asthma and controlling pain for a long time [2]. D. stramonium originated from the tropical areas of Central and South America [3] and it is now a cosmopolitan weed in temperate regions. Datura plant own toxic and poisonous features [4]. All parts of Datura are toxic, but highest concentration of toxic compound stored in ripe seeds [5].

D. stramonium contains a variety of alkaloids, including atropine and scopolamine, that can cause anticholinergic poisoning, if taken in large dosages [6,7]. Atropine is found to have more exciting properties, while scopolamine has more relaxing and hallucinogenic properties [8]. The concentration of these components may vary with species and the environment where it was grown. So the range of toxicity is unpredictable from plant to plant. The seeds are responsible for the anticholinergic toxicity of the plant [9]. Atropine and scopolamine are competitive antagonists of muscarinic cholinergic receptors and central nervous system depressants [6]. The estimated lethal dosages of atropine and scopolamine in adults are by order of 10 mg or more than 2-4 mg [10]. Symptoms of D. stramonium toxicity include both peripheral (dry mucosa, flushed skin, mydriasis and blurred vision, thirst, swallowing difficulty, urinary retention and tachycardia) and central (agitation, combative behavior, hallucination, delirium, seizure and coma) [2]. Lately, this plant has become an increasingly widespread narcotic and local anesthetic drug in many countries. This and more, young people use its leaves by smoking for hallucinogenic effects which causes serious poisoning [11]. The 2002 annual report of the American Association of Poison Control Centers reported 1072 cases of exposure to anticholinergic plants, 523 of these being deliberate [12]. Diverse cases have been reported Datura poisoning occurs because of contamination of food and decoction prepared from herbal prescriptions or when these plants species were eaten accidentally [13].

Atropine and scopolamine are commonly described as anticholinergic compounds, due to their tendency to bind to muscarinic acetylcholine receptors and hence acting as competitive antagonists at these receptors [14]. Acetylcholine (ACh) is a classical neurotransmitter in both central and peripheral nervous systems in which lymphocytes and accessory cells express muscarinic and nicotinic receptors [15].

In normal cell cycle ACh binds to M1 type muscarinic receptor. This receptor is found mediating slow excitatory postsynaptic potential (EPSP) at the ganglion in the postganglionic nerve [16], is common in exocrine glands and in the CNS [17]. It is predominantly found as bound to G proteins of class Gg which use upregulation of phospholipase C and therefore inositol triphosphate and intracellular calcium as a signaling pathway. Activation can induce calcium-release from intracellular stores, presence of Ca⁺² ions allows the binding of myosin motor protein to actin filament. Therefore, cleavage furrow formation is induced. If Atropine or scopolamine binds to muscarinic receptor instead of ACh, the mechanism of cleavage is inhibited [18] in Figure 1.

Figure 1: Ach, atropine and scopolamine pathway on human lymphocytes.

Former study at cytotoxic and genotoxic potential of DE invitro is limited. A few pharmacological investigations have been studied on Datura. Indeed, different type of extraction of Datura have been reported to exhibit antimicrobial [19,20], antifungal activities [21,22], hypoglycemic [23] and antimutagenic properties [24]. In addition, Datura aqueous leaf extract-induced cytotoxicity and oxidative stress in human cancer cell lines [25] and Datura extract effectively inhibits HeLa cell proliferation in vitro [26]. Severe toxicity has been associated with coma and seizures, although death is rare [27].

In the light of these works the aim of this study is determination of cytotoxic and genotoxic effects of methanolic seed extract of Datura stramonium on cultured human lymphocyte cells by confirming our results with four different methods: LDH, WST-1, TUNEL and SCE assays.

Materials

All products used for cell culture (RPMI-1640, phytohaemaglutinin (PHA), fetal bovine serum (FBS), phosphate buffer saline (PBS), Ficoll, L-glutamine and penicillin–streptomycin (PS) were purchased from Biochrom AG (Mannheim, Germany) and Biological Industries (Kibbutz Beit Haemek, Israel). Cytotoxicity Detection (LDH) kit, Cell Proliferation Reagent (WST-1) kit and TUNEL kit were purchased from Roche (Mannheim, Germany). Furthermore, colcemide solution, 5-bromo-2-deoksiuridin (BrdU) and Hoechst 33258 were purchased from Sigma (St Louis, Missouri, USA). All other chemicals were of reagent grade.

Methods

Plant collection and extraction: Seeds of D. stramonium were collected in Konya (southwest Turkey) between August and September 2010 from its natural habitat. Seeds were air-dried in dark at ambient temperature and ground to a fine powder with an electrical mortar. 20 grams of cleaned D. stramonium seeds extracts (DE) were crushed and mixed 1 to 4 with methyl alcohol. The mixed complex was set aside for 24 h in laboratory temperature. Then it was filtered three times through a mesh. The methanol was removed by vacuum rotary at 500ºC until DE dried. Then this portion was dissolved with 20 ml methyl alcohol. Finally, the extract was filtered through a GF/PET (glass fiber/ polyethyleneterephtalate) 1.0/0.45 μm micro filter, and prepared for PBMC culture [28]. The concentration of stock solution was 400 mg/ mL. DE was prepared freshly before every experiment.

HPLC assay for atropine and scopolamine in Datura stramonium

Standards and reagents: (L)-Atropine (99%), (L)-scopolamine hydrochloride (98%), potassium chloride, sodium chloride, potassium dihydrogen phosphate and sodium monohydrogen phosphate (>99.5%) were obtained from Sigma Chemical Company, USA. Acetonitrile (gradient quality 200 nm UV cutoff) and methanol (gradient quality 205 nm UV cutoff) were obtained from Romil, England. HPLC grade water (20 MO) was obtained from a Milli-Q/reversed osmosis system (Millipore, USA). The phosphate-buffered saline solution (PBS) was prepared according to the prescribed method supplied by Waters, Milford, USA. According to this method, PBS is prepared by adding the anhydrous salts of KCl (200 mg), NaCl (8000 mg), KH₂PO₄ (200 mg) and Na₂HPO₄ (1150 mg) to a one liter flask. One liter of deionized water is added and stirred to dissolve. Finally the pH is adjusted to 7.0 with 10% citric acid. HCL-PBS was prepared as described above, but the pH was lowered to 5.0 with hydrochloric acid.

HPLC Instrumentation and conditions: HPLC is one of the most commonly used techniques to analyze alkaloids. The HPLC analyses were made on the instrument LC20AT, of Shimadzu brand. The mobile phase was set at 85:15 phosphate buffer (pH: 3.8) and an isocratic pump was set up at the flow rate of 1000 μl/min while using acetonitrile. Sample injection with a volume of 20 μl was done while the separation was achieved in a 35°C column oven using C18 column (5 μm 10 x 0,46) of Teknokroma medaiterranea sea18 HPLC columns. The final reading of the results was achieved on the PDA detector at 215 nm in Figure 2.

Figure 2: HPLC chromatograms of scopolamine and atropine standards and of extract from Datura stramonium.

Standard solutions and sample preparations: From methanolic stock solution of atropine and scopolamine (each 10 mg/ml), standard solutions were prepared for the calibration.

Lymphocyte isolation and culture of PMBC: The peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by centrifugation on a Ficoll-Hypaque (Sigma Aldrich, St Louis, Missouri,USA) density gradient at 2500 rpm for 25 min at room temperature. The undisturbed lymphocyte layer was carefully transferred out. Lymphocytes were washed two times with PBS, and pellet resuspended RPMI-1640 media. Cell counting was performed to determine the PMBC cell number with equal volume of trypan blue. Adjusted cell suspension be equal to 1x106 cell /ml [29]. The PBMC were used depend on the 1x106 cell /mL equality and incubated with D. stramonium methanolic extract at concentrations (0.25, 0.5, 12.5, 25, 50, 125 μg/mL) at 37°C and 5% CO₂ in RPMI-1640 supplemented with 2 mM/L L-glutamine, 10% FBS and antibiotics (penicillin and streptomycin) for 4 days. This proportion estimated with cytotoxicity assays (LDH and WST-1 assays).

Determination of cytotoxicity: Cells were seeded into 96-well plates at a density of 1x106 cells/mL. After 24 h of seeding, cells were treated with different concentrations of D. stramonium extracts and only media for the controls. Cytotoxicity of D. stramonium on human cultured lymphocytes was assayed at 24 and 48 h using two methods.

LDH activity assay: According to the manufacturer instructions, Cytotoxicity Detection Kit (LDH; Roche) is used to measure the amount of LDH released by dead cells. The conversion of tetrazolium salt into a red formazan dye product was measured calorimetrically. For this assay, a positive control, leading to 100% cytotoxicity by lysing the cells completely, was included in the assay. The positive control was 2% Triton X-100 solution in the assay medium, as proposed by the manufacturer. After preincubation of the cells, before addition of the test compounds, the growth medium was exchanged from medium containing 5% FBS to medium containing only 1% FBS. For testing the released LDH activity, 100 mL of culture medium was transferred to a new 96-well plate at 24^th and 48^th hour. The reaction solutions of 100 mL from the kit, containing the detection dye and the catalyst, were added to the wells and kept in the dark at room temperature for 30 min at 490 nm with 690 nm as a reference wavelength in an enzyme linked immunosorbent assay (ELISA) microplate reader (BioTek-Power Wave, Winooski, USA). A culture medium without cells was used as a blank. As for the other assays, background values from wells without cells were subtracted and average values for the six times calculated. Means +SD values are represented in Figure 3 cytotoxicity (%) was then calculated according to the following equation:

Figure 3: Comparison of Cytotoxicity (%) and Cell Viability (%) of DE on Cultured Human Lymphocytes ^ap<0.05 compare with control group, ^bp<0.05 compare with DE-1 group, ^cp<0.05 compare with DE-2 group ^dp<0.05 compare with DE-3 group ^ep<0.05 compare with DE-4 group at 24^th and 48^th hours.

Cytotoxicity (%) = (experimental value – negative control)/ (positive control-negative control) x100

Cell proliferation by WST-1 assay: Proliferation assays have become available for analyzing the number of viable cells. The cell viability was assessed using WST-1 assay kit (Roche). For WST-1 assay, cells were cultured in microplates (tissue culture grade, 96 wells and flat bottom) in a final volume of 100 mL/well culture medium in a humidified atmosphere (37ºC, 5% CO₂). Cell proliferation reagent WST-1 was added in a 10 μL/well volume at 24^th and 48^th hour. Cells were incubated for 4 h in a humidified atmosphere (37ºC, 5% CO₂). The absorbances of samples were measured at 450 nm with ELISA reader.

Genotoxicity assays

SCE assay: Blood samples were obtained by vein puncture from three (aged 25–35) healthy, nonsmoking men volunteer donors. This study was approved by the local ethics committee. Experiments were also conformed to the guidelines of the World Medical Assembly (Declaration of Helsinki). Lymphocyte cultures were set up by adding 0.5 mL of heparinized whole blood to RPMI-1640 chromosome medium supplemented with 15% heat-inactivated fetal calf serum, 100 IU/mL streptomycin, 100 IU/mL penicillin and 1% L-glutamine. Lymphocytes were stimulated to divide by 1% PHA. CCl4 (in concentration of 5 mM) and the extract of D. stramonium (DE) were added to the cultures. The experiments were performed on six groups. The positive control was 5 μM CCl4. The DE compounds were applied at concentrations: (0.25 (DE-1), 0.5 (DE-2), 12.5 (DE-3), 25 (DE-4), 50 (DE-5), 125 (DE-6) μg/ mL) respectively.

For SCE demonstration, the cultures were incubated at 37ºC for 72 h, and 5-bromo-2- deoxyuridine was added at 8 μg/mL at the initiation of cultures. All cultures were maintained in darkness, and then 0.1 mg/ml of colcemide was added 1 h before harvesting to arrest the cells at metaphase. The cultures were centrifuged at 800 g for 10 min. Cells were harvested and treated for 30 min with hypotonic solution (0.075 M KCl) and fixed in a 1:3 mixture of acetic acid/methanol (v/v). Bromodeoxyuridine incorporated metaphase chromosomes were stained with the fluorescence plus Giemsa technique, as described by [30]. In the SCE study, by selecting 20 satisfactory metaphases, the results of SCE were recorded on the evaluation table. For each treatment condition, well-spread second division metaphases containing 42-46 chromosomes in each cell were scored, and the values obtained were calculated as SCEs per cell. (Table 1).

Table 1: SCE study.

TUNEL assay: DNA fragmentation is an indicator for detecting the late apoptosis. TUNEL (Roche) is an assay that is composed of an enzyme solution and labeling solution, which detects the nicks (single strand breaks) in the DNA and binds to free 3’-OH ends. Adding of dUTPs to 3’-OH ends by terminal deoxynucleotidyl transferase causes the labeling of DNA. Fluorescently labeled ends were detected using fluorescent microscopy. Blood samples were obtained by vein puncture of the healthy nonsmoking volunteer donors. Lymphocyte cultures were set up by adding 0.5 mL of heparinized whole blood to RPMI 1640 chromosome medium supplemented with 15% heat inactivated fetal calf serum, 100 IU/mL streptomycin, 100 IU/mL penicillin and 1% L-glutamine. Lymphocytes were stimulated to divide by 1% phytohaemaglutinin. Carbontetra chloride (CCI4) used as a positive control (in concentrations of 5 μM) and the different concentrations (0.25 (DE-1), 0.5 (DE-2), 12.5 (DE-3), 25 (DE-4), 50 (DE-5), 125 (DE-6) μg/mL) of DE were added to the cultures. The cultures were incubated at 37ºC for 48 h. The cultures were centrifuged at 800g for 10 min. The following fixation protocols were investigated: (1) acetic acid–methanol (3:1) for 10 min at 4°C at 1200 r/min; (2) acetic acid–methanol (2:1) for 10 min at 4ºC at 1200 r/min; (3) 4% phosphate-buffered saline (PBS, 0.01 M, pH 7.4) for 5 min at room temperature followed by washing in PBS. DNA fragmentation of cells was determined with the In Situ Cell Death Detection Kit (Fluorescein, Roche) according to the manufacturer’s instructions. The results were analyzed by fluorescence microscopy.

Positive control was prepared as described above with CCI₄ for 15 min at 37°C before the labeling reaction. Green fluorescence (TUNELpositive cells) was measured using a 530 nm band-pass filter, and blue fluorescence (DAPI-4’,6-diamidino-2-phenylindole) labeled cells using a 500 nm long-pass filter. The two colors were separated by a 560 nm dichronic filter. A total of 10 000 events were accumulated for each measurement at a flow rate of 200–300 cells/s.

Statistical analysis

The statistical analyses of SCE, LDH and WST-1 values Mann– Whitney U-test were used. Also for TUNEL assay values One-way ANOVA was used. Statistical differences between time, dose and extract were analyzed. A value of P less than 0.05 was accepted as statistically significant. Results were expressed as mean ± SE. For these procedures, SPSS 11.5 version for Windows (SPSS Inc, Chicago, Illinois, USA) was used.

HPLC analysis

For the HPLC analyses, the instrument was first calibrated by feeding the atropine and scopolamine standards of magnitudes 5, 10, 50, 100, 500 and 1000 μg/ml into the HPLC. The scopolamine (Retention time: 3.9) and atropine (Retention time: 6.2) peaks resulting from the standard solutions were introduced to the instrument. Then, the peaks of the same retention time resulting from the samples were identified as scopolamine and atropine. (Figure 2) The instrument has given the following values after the calibration samples were fed into it: scopolamine: 63.9 μg/ml and atropine: 935 μg/ml. We have R2: 0.9929 in the curves resulting from the calibration of the instrument.

Cytotoxic effects of des

Figure 3 represents the results of the cytotoxic and cell viability assays of the present study. Although significant cytotoxic effects of DEs on cultured human lymphocytes were not observed at 24^th and 48th hours after treatment with different concentrations of DEs, cell viability decreased at the same times. Significant reduction in the cell proliferation were found in the DE-treated group especially, 50 and 125 μg/mL concentrations of DEs when compared with untreated group as seen in the Figure 3 (p<0.05).

Apoptotic effects of des

The apoptotic effect of DEs on the human lymphocyte cells were given in Figures 4-6. According to our TUNEL assay results; there was a significant increasing in the DNA fragmentation in the DE-treatment groups. Increasing in the DNA fragmentation was observed after treatment with DEs significantly (p<0.05). Although apoptotic effects of DE-1 and DE-2 were not different with control group statistically, other DEs increased the apoptotic effect on cultured human lymphocytes.

Figure 4: Apoptotic cells (%) in the control and different DE treated groups in human lymphocytes. ^ap<0.05 compare with control(Ngroup, ^bp<0.05 compare with CCl₄ group, ^cp<0.05 compare with DE-1 group ^dp<0.05 compare with DE-2 group ^ep<0.05 compare with DE-3, ^fp<0.05 compare with DE-4, ^gp<0.05 compare with DE-5 group.

Figure 5: TUNEL Assay of lymphocytes cells which are treated nothing (negative control) ;A, treated (positive control) CCI₄: B, treated DE-1:C , DE-2:D observed fluorescence microscope. Image is at 100X.

Figure 6: TUNEL Assay of lymphocytes cells which are treated: DE-3: E, DE- 4: F, DE-5:G, DE-6:H, observed fluorescence microscope. Image is at 100X.

Genotoxic effects of des

SCE frequency of the control and experimental groups are given in Figures 7-10. SCE frequency in CCl₄-treated (positive control) group was higher than the frequency in the control group and DE-treated groups. There was a significant increase in the SCE frequency in DE-4 (25 μg/mL), DE-5 (50 μg/mL), and DE-6 (125 μg/mL), groups when compared with the control group significantly (p<0.05).

Figure 7: Comparison the effects on the number of sister chromatid exchanges (SCEs) different concentrations of DE.

Figure 8: SCE on human peripheral lymphocyte cell which are treated nothing (negative control) ;A, treated (positive control) CCI4: B, observed fluorescence microscope. Image is at 100X .

Figure 9: SCE on human peripheral lymphocyte cell which are treated: DE- 1:C , DE-2:D, DE-3: E, DE-4:F, observed fluorescence microscope. Image is at 100X.

Figure 10: SCE on human peripheral lymphocyte cell which are treated: DE-5:G, DE-6:H, observed fluorescence microscope. Image is at 100X.

Most plant groups produce, tropane alkaloids as secondary metabolites, which act in mammals as antagonists of central and peripheral muscarinic acetylcholine receptors and hence they can induce a distinct toxic syndrome. Intoxications with tropane alkaloids are characterized by dryness of the mucosa in the upper digestive and respiratory tract, constipation, pupil dilation (mydriasis) and disturbance of vision, photophobia and changes in heart rate, dosedependent hyper- or hypotension, bradycardia or tachycardia as well as arrhythmias, nervousness, restlessness, irritability, disorientation, ataxia, seizures and respiratory depression.

According to EFSA report (2008), amounts of scopolamine and atropine is 100-900 and 1900 mg/kg dry weight; respectively in D. stramonium seeds. In another study, alkaloid content of seeds grown in different parts of the United States ranged from 1.69 to 2.71 mg/g for atropine and 0.36-0.69 mg/g for scopolamine [31]. The amount of the tropane alkaloids including scopolamine and atropine varies over the lifetime of an individual plant. For instance, it has been reported that in very young D. stramonium plants scopolamine dominates, but at the stage of flowering the atropine content increases when scopolamine content decreases gradually [32]. In our results the concentrations of scopolamine and atropine were 63.9 and 935 μg/ml respectively.

The main alkololoids of D. stramonium, atropine and scopolamine, which are inhibitors of muscarinic receptors, caused a slight but significant increasing of the frequency of binuclear interphase cells and also of the frequency of cells in late telophase and early G1 that had not completed cleavage. Binuclear cells are prone to inhibition of next generation of cells with highly aberrant chromosome numbers convenient to acetylcholine blocking agents as atropine and scopolamine [33]. Both atropine and scopolamine selectively block certain acetlycholine receptors; atropines are anti-inflammatory, and hyoscine prevents secretions and is a component of commercial preanaesthetic drugs and Omnopon-Scopolamine. Small doses of these chemicals, present in most Datura species, may produce a sedative effect and cause submissive behavior and memory loss.

It has been reported that a 90-day feeding study with ground D. stramonium seeds containing 2710 mg atropine and 660 mg scopolamine/kg seed mixed into the feed at 0, 0.5, 1.58, and 5.0% corresponding to 6.85, 53.25 and 168.5 mg tropane alkaloids per kg was conducted with 20 male and female weanling Sprague- Dawley rats. Body weight, serum albumin and serum calcium were decreased, however liver and testes weights, and serum alkaline phosphatase and blood urea nitrogen levels were increased [30]. In another study in rats showed lower body weights and liver weights with diet containing 0.5% ground D. stramonium seeds [34,35].

According to Ducan et al., there is no difference in micronucleus frequency in bone marrow of rats when rats were fed with Datura seeds [31]. On the other hand, 0.01 g/ml concentrations of water and methanol extracts of D. stramonium caused DNA damaged in human peripheral blood mononuclear cells. Many studies showed that Datura extracts have inhibition effect on different cancer cell lines. Ahmad et al. [25] reported that four different cancer cell lines was treated with the Datura extracts for 48 hours, proliferation of all cancer cell lines were inhibited by Datura aqueous leaf extract [25]. In other studies, researchers found antiproliferative activity of DEs on glioma and HeLa cell lines. The inhibition effects of DEs could due to Datura agglutinin [26,31].

In another studies, both atropine and scopolamine have been intensively tested and found to be non-mutagenic in the Salmonella assay [36,37]. No DNA-binding potential was detected for atropine at concentrations of 10-100 μM [36]. On the other hand, Steenkamp et al. reported that Datura plant extracts damaged DNA [38]. The DNAdamaging properties of D. stramonium may also be one of the reasons that the toxic effects atropine and scopolamine [39,40].

There is not sufficient information in literature about the genotoxic and cytotoxic effects of D. stramonium seeds extract on cultured human lymphocytes. In our work, we have demonstrated that D. stramonium methanolic seed extracts have cytotoxic and genotoxic effects on human cultured lymphocytes by confirming with four different methods: LDH, WST-1, TUNEL and SCE assay. WST-1 results show that cell proliferation was inhibited by DE at concentrations 50 and 125 μg/mL. TUNEL assay and SCE results showed DNA damage in the cells due to the increasing D. stramonium concentrations. Previous studies suggested that D. stramonium can be used as a new anticancer drug in cancer theraphy. However; based on our results, it is risky to use it as a drug in cancer theraphy due to its cytototix and genotoxic effects on human healthy lymphocytes in vitro.

In this study, we demonstrate that different DE concentrations have slight cytotoxic effect on human cultured lymphocytes. In addition all DE concentrations inhibite cell proliferation on human cultured lymphocytes as it is significantly seen on statistics. Genotoxic and apoptotic effects of DE concentrations especially at concentrations 50 and 125 μg/mL caused DNA fragmentation on cultured human lymphocytes. Tunnel assay confirmed the SCE result on chromosome level, shows the gren fluorescence signals in the cells due to the increasing D. stramonium concentrations. Chromosomal damage and abnormal chromosome number are result of DNA fragmentation.

The authors are thankful to the Fatih University, Research Project Foundation (Contract no: P50031005_2 (1392) for financial support of this study.