Aim of the experiment: Genistein, a phyto-estrogen, is mainly found in foods that consist of soybeans and/or soy protein. It is thought that genistein has antioxidant properties. However, in the past toxic effects have been indicated. The aim of the present experiment was to investigate the cytotoxicity of genistein by means of quantifying the cell viability of Chinese Hamster Ovary (CHO) cells after an 24 hours exposure to different concentrations of genistein.
Principle: The viability of CHO cells after exposure to genistein was tested using a MTT assay. The MTT assay is a colorimetric assay. The principle of this assay is based on the conversion of the yellow MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium), which enters the cell and pass into the mitochondria, to insoluble purple formazan crystals. This reduction is catalyzed by the mitochondrial enzyme succinate-tetrazolium reductases. This reaction will only occur in metabolically active cells. Therefore, the level of MTT reduction to the formazan product is a measure of the viability of the cells; the less formazan crystals are formed, the more toxic the test compound is for the cells under the tested conditions.
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Materials and methods: CHO cells were grown in 96-wells plates for one day. Subsequently, they were incubated with different concentrations of genistein in a range of 0-25 µM (fc). Different concentrations of CuSO4 were used as a positive control (fc 0-0.50 µM). For each concentration of genistein or CuSO4, three replicates were included. After the incubation period, 50 µl of MTT reagent (0.5 mg/ml) was added to each well and incubated for 1 hour at 37°C in a humidified atmosphere of 5% CO2. Here after the medium was removed from the cells and DMSO was added to each well to solubilize the purple formazan crystals. Finally, the formation of formazan was determined by measuring the extinction using a spectrophotometer at a wavelength of 562 nm and 620 nm for the color intensity of formazan crystals and the background respectively. The obtained absorption data measured at 562 nm were corrected for absorption data measured at 620 nm. Results were expressed as a percentage of non-exposed cells with genistein or CuSO4. A more detailed description of the used materials and methods can be found in the reader Food Toxicology, TOX30306 Year 2009/2010, Period 3.
Results: Figure 1 represents the cell viability after exposure to genistein expressed as a percentage of non-exposed cells. Cell viability is slightly decreased in a dose-dependent manner. At the highest concentration tested, approximately 57% of the cells are viable. Figure 2 represents the cell viability after exposure to CuSO4 . Cell viability is markedly decreased after exposure in a dose-dependent manner. At a concentration of 0.05 µM the cell viability is decreased with almost 50%. At the highest concentration tested (fc 0.5 µM), cell viability is further decreased up to 15% compared to the non-exposed cells.
Figure 1. Cell viability of CHO cells after a 24 hours exposure to different concentrations of genistein (fc 0-25 µM). Data represent mean values ± STDEV of triplicate measurements expressed as a percentage of non-exposed cells.
Figure 2. Cell viability of CHO cells after a 24 hours exposure to different concentrations of CuSO4 (fc 0-0.5 µM). Data represent mean values ± STDEV of triplicate measurements expressed as a percentage of non-exposed cells.
Discussion/conclusion: CuSO4, as was used as a positive control for cytotoxicity, showed a severe decrease in cell viability as dose increased. As we compare this toxic effect to the effects that were seen for genistein, it can be concluded that genistein is less toxic towards CHO cells under the test conditions. The highest tested concentration of genistein results in higher cell viability than the highest tested concentration of CuSO4, while the highest concentration of CuSO4 is 50 times lower than the highest concentration of genistein. However, according to the results, exposure of CHO cells to genistein decreases cell viability, indicating a toxic effect.
As seen in figure 2, the bar indicating cell viability with exposure to 0.04 µM show a high standard deviation compared to the other bars. In addition, it does not follow the dose-response curve represented by the other bars. Investigating the raw data, this outliner was caused by only one absorption value corresponding to 0.04 µM of CuSO4. This can be caused by several factors. One is that the amount of seeded cells in that particular well was higher compared to the cells present in the other wells, leading to a higher final cell viability with the same fractional decrease (expressed in %). Another explanation may be that a pipetting error occurred, during the procedure; the test compound might not be added. However, this is unlikely, since the amount of viable cells is even higher than the non-exposed cells. Another more likely possibility is that more MTT was added due to leftovers in the tip; maybe not all the fluid was pressed out during addition of MTT to the adjacent wells to which MTT was already added using the same tips. It can be seen in the raw data that the two wells before the odd well give a slightly lower absorption level compared to the other two wells of the triplicates.
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When suboptimal culturing conditions are applied i.e. circumstances that do not resemble normal physiological conditions the cell growth can be reduced or cells can even die. Some examples of factors that could be able to affect cell growth and or cell death;
Inadequate percentage of CO2
Low atmospheric humidity
Lack or insufficient amount of nutrients and growth factors
Temperature; either a too high or too low temperature
pH; either a too high or too low pH
infections (yeast, mould, bacteria)
Presence of too much or too little cells in a culture flask
When interpreting the results of these cellular toxicity assays and extrapolation of the results to the in vivo situation there are several factors hampering the interpretation/extrapolation. This difficulty may be caused by the fact that in a cell culture system, hormonal and physiological regulation and feed-back mechanisms, which do occur in an in vivo situation, are absent.
MTT is light sensitive and will be broken down when exposed to light, therefore this compound should be protected from light. During the MTT assay this can be achieved by wrapping the 96-wells plate in aluminum foil for the incubation period of the cells with MTT.
For both genistein as for the positive control, CuSO4, the lowest concentration tested was 0 µM. 0 X and 0 CuSO4 as referred to in the reader contain different solvents; genistein and CuSO4 are dissolved in DMSO and demineralised water respectively.
During the experiment, the outer wells of the 96-well plate were not used for cell exposure. This was done for the reason that medium in the outer wells might evaporate, probably affecting test results.