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The Cellular Response Of Neural And Kidney Cells After Exposure To Commonly Consumed Energy Drinks Wayne Doyle, Vidya Chandrasekaran (Mentor) Department of Biology, Saint Mary’s College of California, Moraga, CA 94556, USA Abstract This study attempted to examine the possible effects of energy drink consumption on different cells of the body. Neurons from the forebrain of embryonic chick and MadinDarby kidney cells (MDCK) from a canine cell line were treated with varying concentrations of two commonly consumed energy drinks and then were examined for alterations in morphology and behavior. It was discovered that neurons responded to energy drink exposure by a reduction of processes and MDCK cells had apparent degradation in the actin cytoskeleton structure. Further research is needed to examine other alterations to cell behavior as well as other alterations to cell behavior. Introduction Energy drinks have become a prevalent aspect of modern society as people attempt to find an endless source of energy at the bottom of a can. Due to their marketing as a “health supplement,” energy drinks are not regulated by the Food and Drug Administration (FDA) under the 1994 Health Supplement Act (1). This lack of regulation has resulted in the inclusion of many pharmaceutical and plant-based products, many of which are potentially harmful to the human body or under-researched. Examples of these ingredients include caffeine, guarana, yohimbine, citicoline, and taurine. These compounds, presumably selected due to their energy-imparting effects, may in fact be Doyle 1 extraordinarily detrimental to the normal activity or even survival of different cells within the body. The physiological effects of energy drinks are a sorely under-researched area, and almost no data exists for the cellular effects of these beverages. This is particularly interesting due to the reported medical cases that have come forth about patients consuming energy drinks and then developing medical conditions. There have been reported cases of numbness, headaches, epileptic episodes, cerebral ischemia, myocardial infarction, and death. (1,3,4,5) Little to no research has been done to examine the cellular changes that could be accompanying consumption of energy drink that could lead to these conditions. The aim of this study was to look for possible cellular responses to energy drink exposure, such as changes to morphology or behavior. Two cell types were chosen: neurons from the forebrain of embryonic chickens, and kidney cells from a canine cell line. Neurons were chosen due to their connection to many of the observed medical conditions (i.e. seizures, numbness) as well as the fact that energy drinks are consumed in order to induce an increase in nervous system activity (energy). In the body the kidneys function as the primary filtration device for the blood, which would mean that kidney cells would be one of the cell groups most exposed to energy drink ingredients. The MDCK cells were selected to see if the beverages have an effect on the cellular components of these filtration organs. Two energy drinks were chosen for the study based on their apparent popularity on a typical college campus as well as their ingredients, common to most energy drinks. The two beverages chosen were 5 Hour Energy and Monster Nitrous. 5 Hour Energy is in Doyle 2 a concentrated shot and Monster Nitrous is in a traditional diluted can, thus allowing a comparison of the two common forms that energy drinks can come in. Procedure: Cells Neurons were obtained from dissection of the embryonic chick forebrain (E8-E10 under Hamburger-Hamilton staging) (6). The eggs were obtained from the University of California at Davis Hopkins Avian Facility. Cells were plated on coverslips pre-treated with poly-l-lysine (100µg/mL) overnight and left for 24 hours before treatment. Neurons were grown in M199 Media with 2% B27, 10% FBS, 1% NGF, and 1% Penn-Strep. MDCK cells were received from ATCC and plated according to ATCC protocols. Treatment The energy drinks were obtained from local convenience stores. The beverages were filter-sterilized through a .2 micron filter and then diluted in the media to final concentrations of .003, .01, .05, .1, and .2 mL per mL of media. The concentrations were chosen to provide a logarithmic scale for a dose response curve as well as their similarities to blood concentrations upon consumption of an energy drink. A higher concentration then .2 mL/mL would have provided too great of a dilution factor fo cell survival. Energy drinks are acidic (pH range around 3-4) so the media/energy drink solutions were neutralized with NaOH to bring the media to a pH slightly above 7. Controls received a small volume of NaOH that did not break the buffer, allowing the maintenance of a slightly basic pH. Staining Cells were stained with neurofilament or MAP2 antibodies (for neural cells) in order to visualize cellular morphology. Neurofilament antibodies are specific for intermediate filaments found in axons, while MAP2 are specific for microtubules found Doyle 3 in dendrites. The cells were visualized with an Alexa Fluor 488. MDCK cells were stained with rhodamine-phalloidin, which fluoresces when bound to actin, in order to visualize kidney cell morphology. All images were taken with a MicroPublisher microscope camera from QImaging. Monster Nitrous and 5 Hour Energy Reduce Neurite Numbers In neural cells, energy drinks induced a shift from large number of processes (≥4 processes) to a smaller amount of processes (≤3 processes) after one day of treatment. In control cells the number of cells with three or fewer processes was only at 18% for MAP2 staining, and 20±8% for neurofilament staining. When treated with 5 Hour Energy at a concentration of .1 mL/mL, these numbers increased to 59±2% (MAP2) and 50±12% (neurofilament). Monster Nitrous treatment at .1 mL/mL induced a similar effect with the percentage of cells expressing 3 or fewer processes increasing to 58±11% (MAP2) and 59±3% (neurofilament). The decrease in neurites shown by the two different stains indicate that axons and dendrites are both withdrawing when exposed to the energy drinks (Tables 1 and 2, Figure 1, 2, and 3). With 5 Hour Energy treatments the percentage of cells with decreased processes gradually rises from .003 mL/mL to .01 mL/mL. For the MAP2 antibody stain, the percentage of four or greater processes then increases to .03 mL/mL followed by an increase in three or fewer processes. The neurofilament stain shows an increase in processes from .01 mL/mL until .05 mL/mL, at which point the percentages of cells with lesser processes then begins to rise again. After a concentration of .1 mL/mL the neurofilament stain shows a continuation in the trend of decreased processes while the Doyle 4 MAP2 stain indicates there is a slight rise in the percentage of cells with four or more processes (Table 1, Figure 4). Monster Nitrous exposure had different effects based on which stain the neurons were treated with. Neurofilament antibodies had a decrease in the number of processes at a concentration of .003 mL/mL followed by a slight increase in the number of processes at a concentration of .01 mL/mL The percentage of cells with four or greater processes proceeded to slowly increase, although the percentages of three or fewer cells are far above control levels (~30% increase). The MAP2 antibody stain showed a gradual increase in the percentages of three or fewer cells along with the increase in energy drink concentration (Table 2, Figure 5). Figure 1: Effect of Energy Drink Exposure on the Number of Cells Expressing 3 of Fewer Neurites (neurofilament antibody stain) Figure 2: Effect of Energy Drink Exposure on the Number of Cells Expressing 3 of Fewer Neurites (neurofilament antibody stain) Doyle 5 Table 1: Effect of Varying Concentrations of 5 Hour Energy on the Number of Neurons Expressing Three or Fewer Neurites Table 2: Effect of Varying Concentrations of Monster Nitrous on the Number of Neurons Expressing Three or Fewer Neurites Doyle 6 A dose response curve shows that in response to energy drink exposure in the media cells first respond by a sharp decrease in the percentages of cells expressing four or more processes. Some of the treatments did have a rebound effect with an increase in the number of neurites, followed a decrease once again. Even with this trend the percentage of cells with three or fewer processes are well above those of control, and in general there is a decrease in the number of neurites. This effect was seen in both 5 Hour Energy and Monster Nitrous treatments (Figures 4 and 5). Figure 4: 5 Hour Energy Dose Response Curve for Chick Forebrain Neurons Doyle 7 Figure 5: Monster Nitrous Dose Response Curve for Neurons MDCK Cells Respond With Actin Disorganization Madin-Darby Canine Kidney cells originate from a healthy Cocker Spaniel kidney and act as a representative model of normal kidney cells in culture. The treatment of MDCK cells with both Monster Nitrous and 5 Hour Energy resulted in changes to the actin cytoskeleton after one day of treatment. Control cells have nicely arranged, parallel rays of actin within the cell body (Figure 6A). Both 5 Hour Energy (Figure 6B) and Monster Nitrous (Figure 6C) treated cells have a loss of ordered actin arrays. Instead the actin is in a disorganized state with clusters at apparently random locations throughout the cell. In some of the cells treated with Monster Nitrous actin projections are visible from the cell periphery (arrows in Figure 7). Doyle 8 Figure 6: MDCK Cells. (A) Cells treated with Control at a concentration of .1 mL/mL. Arrows point to parallel actin arrays. (B) Cells treated with 5 Hour Energy at a concentration of .1 mL/mL. Arrows point to disorganization of the actin network. (C) Cells treated with Monster Nitrous at a concentration of .1 mL/mL. Arrows point to signs of actin disorganization and actin clusters. Figure 7: MDCK Cell Treated with Monster Nitrous at a concentration of .1 mL/mL. Arrows point to actin extensions from the cell periphery. Doyle 9 Discussion In order to impart the energy that consumers crave, an excess of different ingredients have been added to energy drinks. The Food and Drug Administration (FDA) however does not regulate the addition of these ingredients, due to their marketing as herbal supplements rather than as a beverage or as a drug. Many of these ingredients have varying effects in the body, such as citicholine’s neuroprotective properties (7), caffeine’s psychostimulant properties (8), or taurine’s antitoxicity properties (9). Although many of these ingredients have been studied alone, some however have been poorly researched with a lack of knowledge in either their toxicity or even their effects in the body (9, 10). For example, it is known that taurine plays roles in the maintenance of osmoticity and has some role in the brain as a possible neurotransmitter, but it is not known if it can cause damage to the body in high amounts or after repeated ingestion. The combinatorial effects of ingesting many of these compounds have been barely researched, if at all. A possible benign compound alone could obtain vastly different properties when it acts in concert with another compound. The lack of research into many of the compounds and compound-compound interactions is especially disconcerting considering many of the reported medical cases that have occurred after the consumption of an energy drink. There have been reported cases of seizures, myocardial infarction, cerebral hemorrhaging, bilateral numbness and many other conditions since the rise of energy drink consumption (1,2,3,4,5). Some of the reported cases have looked into possible reasons for these albeit rare, but severe, reactions to consuming energy drinks on a physiological level. There has been a great lack of research into what could be occurring at the cellular level however. Doyle 10 As shown in Figure 3, the number of processes, as well as their relative lengths, decrease with exposure to energy drinks within the media. These results are supported by the dose-response curve data, which overall shows that the percentage of cells with three or fewer neurites increases dramatically. The withdrawal of neural processes is a common response of neurons to either noxious stimuli or in preparation of a major cellular event. These cellular events could include apoptosis, mitosis, or some other unknown process. Since it is currently unknown at this point exactly what is driving the reduction of processes in the observed cells, further research will need to examine this important topic. The components of many of these energy drinks have roles in the nervous system, either as precursors to neurotransmitters (such as N-Acetyl L-Tyrosine) or functioning as neurotransmitters (taurine). Other compounds found in the beverages mimic neurotransmitters (caffeine) inducing many of the same pathways that a neurotransmitter would induce. This barrage of stimuli could lead to a steady state of depolarization causing the cell to pull back its processes in order to conserve resources or even be inducing apoptosis. It is also possible that the combination of the ingredients could be inducing neurons to leave their permanent G0 phase and reenter the cell cycle. In order to examine these possibilities it would be necessary to complete cell counts from before and after treatments to look for either decreased or increased cell numbers. It would also be useful to stain for markers specific to either mitosis or apoptosis. As shown by the dose response curves (Figures 4,5), the concentration of the energy drink in the media does play a role in the level of neuronal response. Increasing the amount of energy drink in the media led to an overall increase in the observed effects. Doyle 11 There is an increase in the percentages of cells with three or fewer processes, although individual concentrations produce different effects within that general trend. The most classical dose response curve can be seen with the Monster Nitrous MAP2 stain data with the standard logarithmic curve of decreasing numbers of processes. MAP2 antibody’s selective binding to dendrites means that the reduction of dendrites occurs in a classical dose-response fashion when neurons are treated with Monster Nitrous. The non-classical curves are more likely to be explained by a currently unknown cellular process rather than a switch between either reducing or gaining processes in response to different concentrations. A cellular event, such as apoptosis, could explain most if not all of the variations seen in the graphs. After the initial response of neurons to low concentrations of energy drinks (the initial reduction of processes) the cells that have already reduced processes have no other options other then dying. This would create a loss of cells that had three or fewer processes, leaving behind cells that respond at higher concentrations of energy drinks. This loss of already reduced cells would cause a decrease in the observed percentages leading to the above graphs. For example, if thirty percent of the cells responded at .003 mL/mL and then half of them were to die at a higher concentration, the total percentage of cells with reduced processes would be decreased by the fifteen percent that were lost. The validity of this hypothesis will need to be examined through the use of cell counts and stains for apoptosis markers. After treatment with energy drinks, at a concentration of 0.1 mL/mL, the MDCK kidney cells developed apparent degeneration in the actin cytoskeleton (Figure 4). In comparison to control cells, with normal parallel arrays of actin (Figure 4A), cells treated Doyle 12 with both Monster Nitrous and 5 Hour Energy have a disarray in the actin network (Figures 4B, 4C). The results of these treatments are very similar to previous work done with MDCK cell lines involving both cytochalasin D, an actin disruptor, and tumorpromoting factors (11, 12). The images from this study are strikingly similar to the results obtained by Stevenson et. al (11) implying that for a currently unknown reason, energy drinks induce a disruption of the actin network. The maintenance of the actin network is crucial for cell division and migration, as well as the retention of cellular shape. Any disruption to the actin cytoskeleton will lead to a disruption in cell function, and may lead to cell death. The observed cellular effects could be due to a multitude of reasons, each one requiring further study to help determine the mechanism and reason for actin disarrangement. The lack of parallel arrays could be due to the cells being in the middle of a cellular process at the time of their fixing, although there the parallel arrays that should be retained are not present. The division and death possibilities can be examined by mitosis or apoptosis specific stains. Cell movement is an intriguing possibility, especially considering that in some cells there was a congregation of actin projections along the cell periphery (Figure 7). It is conceivable that in response to noxious stimuli, such as energy drink exposure, the cells are attempting to leave the site of that exposure. Movement could be examined through staining individual cells and then watching their behavior with treatment to see if they remain in the same location or travel across the plate. A cell becoming motile would involve the termination of many cellular processes that make the cell vital to the tissue it is a part of, which would have dangerous implications for the body that has ingested an energy drink. Doyle 13 Conclusion Energy drinks, specifically Monster Nitrous and 5 Hour Energy induce a demonstrable cellular response in both neural and kidney cells. Forebrain neurons have a decrease in the number of cells with four or more processes, a possible indication of either cell division or death. Kidney cells of the MDCK cell line show a disarrangement of the actin cytoskeleton, similar to cells treated with compounds that break down actin networks. Further research is needed to examine the possible reasons for these results and to determine which ingredient(s) could be leading to the observed changes. Acknowledgements Vidya Chandrasekaran for her support, discussions, mentorship, and assistance. Amy Bockman for all the amazing work that she does. Valerie Burke for her mentorship. The Saint Mary’s College of California School of Science for this opportunity and financial support. The Robert J. Summers Scholarship for their financial support. Doyle 14 References 1. Reissig et al. Caffeinated energy drinks--a growing problem. Drug Alcohol Depend (2009) vol. 99 (1-3) pp. 1-10 2. Babu et al. Energy Drinks: The New Eye-Opener For Adolescents. Clinical Pediatric Emergency Medicine (2008) vol. 9 (1) pp. 35-42 3. Worrall et al. Herbal energy drinks, phenylpropanoid compounds, and cerebral vasculopathy. Neurology (2005) vol. 65 (7) pp. 1137-8 4. Iyadurai and Chung. New-onset seizures in adults: possible association with consumption of popular energy drinks. Epilepsy Behav (2007) vol. 10 (3) pp. 504-8 5. Clauson et al. Safety issues associated with commercially available energy drinks. Journal of the American Pharmacists Association : JAPhA (2008) vol. 48 (3) pp. 55-63 6. Hamburger and Hamilton. 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