The Impact of Polyphenolic Compounds on Apoptosis in Epithelial Cells

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1 The Impact of Polyphenolic Compounds on Apoptosis in Epithelial Cells Mayson Husband, 12 th grade, Latta High School Abstract The purpose of this investigation was to determine whether polyphenolic compounds present in common foods and beverages facilitate the process of apoptosis in epithelial cells. It was hypothesized that epithelial cells exposed to polyphenolic compounds would exhibit increased rates of apoptosis. It was also hypothesized that full strength solutions of polyphenolic compounds would have greater numbers of apoptotic cells than half strength solutions. It was further hypothesized that green tea beverages would produce higher percentages of apoptotic cells than other polyphenolic containing substances. Epithelial cells were treated with seven different substances known to contain polyphenolic compounds. After exposure, the slides were prepared using the Fragmentation Detection Kit and examined microscopically. To verify the cellular results, a DNA extraction test was also conducted using liver tissue which was exposed to the polyphenolic compounds. Twenty-four slides were covered in both full and half strength solutions. Extracts from the steeped green tea had the highest percentage of apoptotic cells. (11.61%, full strength; 6.18, half strength) The lowest percentage of apoptotic cells was found in the cells exposed to the cocoa solution. (5.99%, full strength; 1.75%, half strength) The results supported the hypothesis that cells exposed to polyphenolic compounds would exhibit increased rates of apoptosis (P= and ). It was also supported that full strength solutions would produce more apoptosis than the half strength solutions. The results supported the hypothesis that green tea beverages would produce higher percentages of apoptotic cells since the steeped green tea and Fuze green tea had the highest percentages of cells undergoing apoptosis exposed to full and half strength solutions. Introduction Polyphenols are chemicals that may protect against some common health problems and possibly forestall the effects of aging. They act as antioxidants and protect cells and body chemicals against damage caused by free radicals, reactive atoms that contribute to tissue damage in the body (Sahelian 2009). Polyphenolic compounds are common in the diet and have been suggested to have a number of beneficial health effects including prevention of cancer, cardiovascular disease,

2 diabetes, and others. Among the many kinds of polyphenols are flavonoids and catechins. Notable sources of polyphenols include berries, tea, beer, grapes/wine, olive oil, chocolate and cocoa, coffee, walnuts, peanuts, pomegranates, and other fruits and vegetables (Douglas 2002). Studies from the Department of Dermatology at Case Western Reserve University, Cleveland, have indicated that polyphenolic compounds present in tea may reduce the risk of a variety of illnesses, including cancer and coronary heart disease. Most of those studies involved green tea. Results from rodent studies have shown that green tea consumption protects against lung, stomach, esophagus, duodenum, pancreas, liver, breast, colon, and skin cancers (Mukhtar 2000). Other studies hold that green tea consumption protects against coronary heart disease, high blood cholesterol concentrations, and high blood pressure. Because the studies and research findings in laboratory animals have shown the protective potential of tea polyphenols in cancer, the usefulness of tea polyphenols for humans should be evaluated in clinical trials. The usefulness of tea polyphenols may be extended by combining them with other consumer products such as food items and vitamins. Further studies are required to see if this benefits the human population. The human body contains trillions of cells. These cells die and are replaced continually during normal functioning. There are two methods of cell death: necrosis and apoptosis. Apoptosis is sometimes called programmed cell death. Apoptosis is a way organisms remove unwanted cells. During apoptosis, cellular contents are not released and inflammation does not occur. Programmed cell death is a normal and necessary event of normal development (Giovannini et al. 2007). Necrosis is death due to unexpected and accidental cell damage. A number of toxic chemical or physical

3 events can cause necrosis: toxins, radiation, heat, trauma, or lack of oxygen due the blockage of blood flow. These physical or chemical insults can lead to the lethal disruption of cell structure and activity. As necrotic cells begin to die, they swell and holes appear in the plasma membrane and intracellular materials spill out into the surrounding environment. Cancerous cells lose the ability to undergo apoptosis and continue to divide uncontrollably. The purpose of this investigation was to determine whether polyphenolic compounds present in common foods and beverages facilitate the process of apoptosis in epithelial cells. The null hypothesis was that there would be no difference in the amount of apoptosis observed in the cells treated with each of the polyphenolic compounds and no difference would be seen between the full strength versus the half strength solutions. The research hypothesis was that epithelial cells exposed to polyphenolic compounds would exhibit increased rates of apoptosis. It was also hypothesized that full strength solutions of polyphenolic compounds would have greater numbers of apoptotic cells than half strength solutions. It was further hypothesized that green tea beverages would produce higher percentages of apoptotic cells than other polyphenolic containing substances. Methods Cheek cells were obtained by scraping the inside of the cheek with a flat toothpick. The cells were spread onto glass slides which had been sprayed with a fixative and allowed to adhere for two to three minutes. The slides were rinsed in TBS for five minutes, and was poured off slide. Slides were placed in containers for a thirty minute exposure treatment using full strength and half strength beverages which have

4 been cited in literature to contain polyphenolic compounds. The tested substances were: red wine, pomegranate juice, cranberry juice, grape juice, Fuze green tea, steeped green tea, and cocoa. A 0.9% saline solution was used as a control. Slides were prepared according to the protocol included with the Fragmentation Detection Kit and then examined microscopically. Apoptotic cells were identified by their characteristically dark nuclei and ovoid shape. To verify the results of the cell treatment, a DNA extraction was conducted after exposing liver cells from poultry to the polyphenolic compounds. The DNA of cells undergoing apoptosis disintegrates due to enzyme action which causes the DNA to fragment. Liver tissue was ground and mixed with the polyphenlic compounds for a thirty minute exposure. DNA was isolated using a standard alcohol precipitation method developed by Cold Spring Harbor Laboratory. The extracted DNA was loaded into agarose gels and electrophoresed for forty five minutes at 100 V. After staining with ethidium bromide the gels were viewed to find evidence of DNA fragmentation. Results Epithelial cells were treated with seven different substances known to contain polyphenolic compounds. After exposure, the slides were prepared using the Fragmentation Detection Kit and examined microscopically. Twenty-four slides were covered in full strength solutions. Extracts from the steeped green tea had the highest percentage of apoptotic cells. A mean of cells had undergone apoptosis out of a mean cells (11.61%). This was followed closely by the Fuze green tea beverage with 10.10% apoptotic and red wine with 9.20% apoptotic. The lowest percentage was found in the cells exposed to the cocoa solution with only 9.33 apoptotic cells out of (5.99%).

5 Data Table #1: Mean Number of Cells Treated with Full Strength Solutions Polyphenolic Compounds Apoptotic Cells Number of Cells Percentage Red Wine Cocoa Fuze Green Tea Steeped Green Tea Pomegranite Juice Grape Juice Cranberry Juice Mean Saline Control The cells exposed to the extract from the steeped green tea had the highest percentage of apoptotic cells (6.18%). Similar to the full strength results, Fuze green tea beverage was second with 4.28% and red wine produced 3.60% apoptotic cells. The cocoa solution had the lowest percentage once again with 1.75%.

6 Data Table #2: Mean Number of Cells Treated with Half Strength Solutions Apoptotic Cells Number of Cells Percentage Red Wine Cocoa Fuze Green Tea Steeped Green Tea Pomegranate Juice Grape Juice Cranberry Juice Polyphenolic Compounds Mean When the DNA gels were examined, fragmentation to some degree was observed in all samples except for the control (see sample gel to the right). Lane 2 contains the saline control, and lane 7 is the grape juice. The polyphenolic compounds that were present in the grape,

7 pomegranate, and green tea produced the greatest amount of fragmentation with less evident with the half strength solutions. Virtually no fragmentation was seen in the control DNA which was exposed to saline solution. Discussion of Results A statistically significant difference was found to exist between the number of cells exposed to the polyphenolic compounds at both the full strength and half strength concentrations. An ANOVA test was performed using Microsoft Excel. The calculated P- values were and for the full and half strength solutions respectfully. Thus, the null hypothesis can be rejected. The research hypothesis was that epithelial cells exposed to polyphenolic compounds would exhibit increased rates of apoptosis and full strength solutions would have greater numbers than half strength solutions. Additionally, it was hypothesized that green tea beverages would produce more apoptotic cells than the other polyphenolic compounds containing substances. The results of this experiment supported the research hypothesis that epithelial cells exposed to polyphenolic compounds would exhibit increased rates of apoptosis. The results also supported the prediction that full strength solutions would produce more apoptosis than the half strength solutions. The mean number of apoptotic in the half strength solutions was 54.5% fewer apoptotic cells than the full strength mean. The results supported the hypothesis that green tea beverages would produce higher percentages of apoptotic cells since the steeped green tea and Fuze green tea had the highest percentages of cells undergoing apoptosis exposed to full and half strength solutions.

8 It should be noted that the exposure time was only thirty minutes for the epithelial cells and liver tissue. The apoptotic cell numbers would most likely increase if exposure time was lengthened. Possible sources of error might include duplicate counting of cells or misidentification of cells. However, since the DNA analysis seemed to substantiate the cell count results, any duplication would be small. The results of this experiment can be beneficial to individuals who seek to prevent diseases such as cancer and coronary heart disease. It is recommended that it would be wise for individuals to include the consumption of these beverages in their daily diets to promote good health and longevity. Acknowledgements Thanks go out to the following people: Mrs. Stevens for her hours spent at the school helping me finish my project and my mom for her patience all these years during science fair. Calbiochem for providing a discounted price on the DNA fragmentation kit. References 1. Sahelian, Ray M.D. Polyphenols Health benefit of polyphenol compounds. Accessed on 1/19/09. p Carrreiro, Elizabeth, Griffin, Leanne, and Hart, Peter. A TUNEL Assay for the Detection of Apoptosis in Cheek Cells, pg Giovannini, Claudio et al. Apoptosis in cancer and atherosclerosis: polyphenol activities. Published on October p Skovenborg, Erik. Red wine polyphenols in cancer research. Published on November p. 1-2

9 5. Douglas, David. Food polyphenols show action against pancreatic cancer. Published on April pg 6. Polyphenols stops cancer, heart disease, depending on does. [on-line]. Available from: (Accessed 1/19/09) 7. Polyphenolic compounds inhibit pancreatic cancer. [on-line]. Available From: (Accessed 1/19/2009) 8. Mukhtar, Hasan and Ahmad, Nihal. Tea polyphenols: prevention of cancer and optimizing health. Published in 2000, p. 1-5.