Download Doyle 1 The Cellular Response Of Neural And Kidney Cells After

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
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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
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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
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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
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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)
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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
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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
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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).
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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.
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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.
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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.
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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
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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.
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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.
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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. A Series of Normal Stages in the Development of the Chick Embryo.
Developmental Dynamics (1951) vol. 195 (4)
7. Adibhatla et al. Citicoline: neuroprotective mechanisms in cerebral ischemia. J Neurochem
(2002) vol. 80 (1) pp. 12-23
8. Winston et al. Neuropsychiatric effects of caffeine. Advances in Psychiatric Treatment (2005)
vol. 11 (6) pp. 432-439
9. Safety. Opinion on Caffeine, Taurine and D-Glucurono- g -Lactone as constituents of so-called
"energy" drinks . (2010) pp. 1-12
10. Timbrell et al. The in vivo and in vitro protective properties of taurine. Gen Pharmacol (1995)
vol. 26 (3) pp. 453-62
11. Stevenson and Begg. Concentration-dependent effects of cytochalasin D on tight junctions and
actin filaments in MDCK epithelial cells. Journal of Cell Science (1994) vol. 107 ( Pt 3) pp. 367-75
12. Fey and Penman. Tumor promoters induce a specific morphological signature in the nuclear
matrix-intermediate filament scaffold of Madin-Darby canine kidney (MDCK) cell colonies. Proc Natl
Acad Sci USA (1984) vol. 81 (14) pp. 4409-13
13. Leviton. Caffeine consumption and the risk of reproductive hazards. J Reprod Med (1988) vol.
33 (2) pp. 175-8
14. Scholey and Kennedy. Cognitive and physiological effects of an "energy drink": an evaluation
of the whole drink and of glucose, caffeine and herbal flavouring fractions. Psychopharmacology (Berl)
(2004) vol. 176 (3-4) pp. 320-30
16. Behrens et al. Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal
antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to
uvomorulin. J Cell Biol (1985) vol. 101 (4) pp. 1307-15
17. Riesenhuber et al. Diuretic potential of energy drinks. Amino Acids (2006) vol. 31 (1) pp. 81-3
18. Ragsdale et al. Effect of Red Bull energy drink on cardiovascular and renal function. Amino
Acids (2009) pp. 1-8
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19. Nawrot et al. Effects of caffeine on human health. Food Addit Contam (2003) vol. 20 (1) pp.
1-30
20. Baskin et al. Effects of taurine on psychomotor activity in the rat. Neuropharmacology (1974)
vol. 13 (7) pp. 591-4
20. Mantovani and DeVivo. Effects of taurine on seizures and growth hormone release in epileptic
patients. Arch Neurol (1979) vol. 36 (11) pp. 672-4
21. GAO. GAO-09-250 Dietary Supplements: FDA Should Take Further Actions to Improve
Oversight and Consumer Understanding. Report to Congressional Requesters (2009) pp. 1-77
22. Bestervelt. Raising the Red Flag on Some Energy Drinks. NSF International, The Public
Health and Safety Company (2009) pp. 1-3
23. Dlugosz and Bracken. Reproductive effects of caffeine: a review and theoretical analysis.
Epidemiol Rev (1992) vol. 14 pp. 83-100
24. Albrecht and Schousboe. Taurine interaction with neurotransmitter receptors in the CNS: an
update. Neurochem Res (2005) vol. 30 (12) pp. 1615-21
25. Specterman et al. The effect of an energy drink containing glucose and caffeine on human
corticospinal excitability. Physiol Behav (2005) vol. 83 (5) pp. 723-8
26. Kuhlmann et al. The mutagenic action of caffeine in higher organisms. Cancer Res (1968) vol.
28 (11) pp. 2375-89
27. Ernst. The risk-benefit profile of commonly used herbal therapies: Ginkgo, St. John's Wort,
Ginseng, Echinacea, Saw Palmetto, and Kava. Ann Intern Med (2002) vol. 136 (1) pp. 42-53
28. Sved et al. Absorption, tissue distribution, metabolism and elimination of taurine given orally
to rats. Amino Acids (2007) vol. 32 (4) pp. 459-66
Doyle 16