Investigation of Cytotoxic Effects of Thallium acetate and Ellagic acid on C6 cell line

1. Investigation of Cytotoxic Effects of Thallium acetate and Ellagic acid on C6 cell line Öge Basoglan1¹, Filiz Alanyalı¹, Zerrin Cantürk² ¹Anadolu University, Faculty of Science, Department of Biology ²Anadolu University, Faculty of Pharmacy, Department of Phar. Microbiology 26470, Eskisehir, TURKEY INTRODUCTION Teel 1986 and Thresiamma 1996 demostrated that oral administration of ellagic acid significantly reduced the level of lipid peroxidase and liver dihydroxy proline in animal models (1). Thresiamma 1996 indicated that oral administration of ellagic acid can overcome carbon tetrachloride toxicity and subsequent lung fibrosis (1). Thallium (Tl) is a Group IIIA metal which has been used in medicine to treat ringworm, gonorrhea, syphilis and tuberculosis, diagnosis of myocardial infarction or ischemia, detection of coronary diseases, sexually transmitted diseases (6) in addition to these usages it has been applied as a rodenticide(4). The application of thallium is now increasingly used in the manufacture of crystals, imitation jewellery, dyes, pigments, electric and electronic equipment, semiconductors, optical systems, infrared spectrometers, low-temperature thermometers, scintillation counters, fibreglass cables for communication and in cement industry(4). The aim of the present study was to investigate the cytotoxic effect of ellagic acid and thallium on NIH3T3 cell line by using MTT and Neutral Red Method. Table 2. Effects of thallium acetate on C6 cell line (MTT method ) (n:8 p≤0,001 ***)‏ Table 1. Effects of ellagic acid on C6 cell line (MTT method ) (n:8 p≤0,001 ***)‏ MATERIALS AND METHODS Cell Culture C6 (glioma cell) cell line was obtained from American Type Culture Collection (Rockville, USA). C6 cells were cultured in DMEM:F12 containing 2 mmol/L glutamine, 100 IU/ml penicilin, 100µg/ml streptomycin and 10% fetal bovine serum. Incubation was carried out at 37 °C in a humidified atmosphere of 5% CO2and 95% air. Viability of cells was determined by using the trypan-blue exclusion test. C6 cells were treated with thallium acetate and ellagic acid for 4 days. Mitochondrial and lysosomal activities were evaluated with MTT and Neutral Red methods. All experiments were performed in plastic flasks. The cells (50000 cells/well) were seeded in 96-well plates and treated with various concentrations of ellagic acid and thallium acetate for 24 h at 37 °C. After the exposure period, media from all treatments were withdrawn. MTT ASSAY The cytotoxicity effect of thallium and ellagic acid on C6 cell line was performed according to the MTT colorimetric assay (3) with different concentrations of thallium and ellagic acid. Thallium and ellagic dissolved in steril distile water and concentration-dependent cytotoxic effect was studied, which 1, 10, 25, 250, 500, 750, 1000 uL ellagic acid and thallium. Briefly, 100 uL of cell suspension, ellagic acid or thallium were seeded in 96-well plate . Then 24 h of incubation, 10 uL of MTT solution (5 mg/ml) was added to each well and te plates were incubated for a further 4h. The viable cell number/flask is directly proportional to the production of formazan, which following solubilization with DMSO, can be measured by Elisa at 540 nm. Each assay was performed in triplicate. NEUTRAL RED ASSAY The assay depends on the uptake of a vital stain, neutral red. Most of the cells will accumulate neutral red in lysosomes. The process requires intact membranes and active metabolism in the cell. 100 μl of the cell suspension and test compounds were seeded in the 96-well plate . After 24 h of incubation, 10 μl neutral red (0,5 %solution) was added to each well and the plates were incubated for a further 4h. Destaining process was used for 1% glacial acetic acid, 49% dH20 and 50% ethanol. The culture plate was placed on a Biotek Model micro-plate reader and the absorbance was measured at 540 nm. Each assay was performed in triplicate. This process was repeated during four days. STATISTICAL ANALYSIS Proliferation values were compared by ANOVA, followed by Tukey's-b tests for determination of statistical differences. Table 4. Effects of thallium acetate on C6 cell line (Neutral Red method ) (n:8 p≤0,001 ***)‏ Table 3. Effects of ellagic acid on C6 cell line (Neutral Red method ) (n:8 p≤0,001 ***)‏ RESULTS and DISCUSSION Application of concentrations of 750 µM and 1000 µM thallium acetate on C6 cell line, caused decrease in mitochondrial activity at the rates of 30 % and 42 % at the end of the first day, respectively. In the second and the third days, again applying the concentrations of 750 µM and 1000 µM thallium acetate on C6 cell line, resulted in 65 % and 72 % decline in mitochondrial activity, respectively. In the fourth day, upto 40 % decrease in mitochondrial activity was observed when even 100 µM concentration of thallium acetate was applied on C6 cell line (table 2). 100 µM concentration of Ellgic acid applied on C6 cell line was observed to be highly effective. In the first day, mitochondrial activity decreased at the rate of 65 % for 100 µM concentration of Ellgic acid. When 250 µM concentration of Ellgic acid applied on C6 cell line, rate of decrease in mitochondrial activity was 80 %. But for higher concentrations (500, 750, 1000 µM) of Ellagic acid did not caused a decrease higher than 80 % in mitochondrial activity. During the experiment period there was no reasonable difference among the lower concentrations of Ellagic acid (1,10,25,50 µM). In the third and the fourth days, the most effective concentration was found to be 250 µM and higher concentrations did not increase this effect (table 1). We have studied in vitro cytotoxicity of ellagic acid and thallium acetate on C6 cell line. It was observed that thallium acetate applied on C6 cell line by means of Neutral Red methods, caused a decrease in the lysosomal activity at the rate of 62% in the 750 µL; whereas ellagic acid caused a decrease at the rate of 80%. While application of 1000 µM concentration of thallium acetate on C6 cell line caused a 65 % decrease in lysosomal activity in the first day, the same concentrastion of Ellagic acid applied on C6 cell line resulted in 65 % decline in in lysosomal activity in the first day. In the first, second, third and fourth days, for concentrations of 1, 10, 25, 50, 100 and 250 µM, there was no statistical sign from the point of decreasing lysosomal activity. (table 3 and 4). REFERENCES 1-Narayanana B. A., Geoffroya O., Willinghama M.C., Re G.G., Nixona D. W. p53/p21(WAF1/CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells 2-Induction of apoptosis in rat C6glioma cells by panaxydol 3-Hai J., Lin Q, Lu Y., Zhang H, Yi J., Induction of apoptosis in rat C6 glioma cells by panaxydol. 4- Le´onard A., Gerber G.B., Mutagenicity, carcinogenicity and teratogenicity of thallium compounds. Mutation Research 387 1997 47–53 5-Leung K.M., Ooi V.E.C., Studies on thallium toxicity, its tissue distribution and histopathological effects in rats. Chemosphere 41 (2000) 155-159. 6-Chia C.F., Chen S.C., Chen C.S., Shih C.M., Lee H.M., Wu C.H., Thallium Acetate Induces C6 Glioma Cell TRACE ELEMENTS IN DIET, NUTRITION AND HEALTH ESSENTIALLY AND TOXICITY (ISTERH-2007) CRETE,GREECEA, 21-26.07.2007)‏