High Levels of Hydrogen
Peroxide Reduce Cell Count and Protein Concentration through Planarian
Regeneration
Authored By:
Caitlin Fehlinger
Amy Lebeis
William Schneider
Ian Troutman
Abstract
ÊÊÊÊÊ The effects of variable
concentrations of hydrogen peroxide ranging from 30% to 0.003%, on
first-generation embryonic fibroblast mice cells and planarian were
examined.Ê Hydrogen peroxide was used
because of its ability to diffuse across the cell membrane and the damage it
does to the cell (Jang and Imlay, 2006).Ê
It is commonly used as an antiseptic to clean wounds.Ê If hydrogen peroxide is helpful for wounds,
can it cause damage to mice cells and hinder planarian regeneration
growth?Ê Cell cultures were used to see
the effect of hydrogen peroxide on mice cells.Ê
The organism Platyhelminthes
was also tested because of its ability to regenerate.Ê Different concentrations of our reagent were
used in mice fibroblast cell cultures and the planarian to see if the planarian
could regenerate in those conditions.Ê
Antioxidants, flavonoid in green tea, were
also used in different dilutions of hydrogen peroxide, too, because hydrogen
peroxide is an oxidant.Ê Flavonoid as an antioxidant only had a higher cell count in
3% and 0.03%, while vitamin C killed cells.Ê
In the 3% there were 19 more cells in the flavonoid
solution.Ê There were no cells in 30%
hydrogen peroxide and many cells were found in 0.03% and 0.003% hydrogen
peroxide.Ê With the different dilutions
of hydrogen peroxide, the
Most Interesting Result
FigureÊ 1: Displays the relationship between the concentration of H2O2
and the related Ph values obtained in Lab 0. As the concentration
decreases, the Ph value becomes increases and therefore becomes more basic.
Discussion
Cells Cells require raw materials, time, and external signals to allow
for growth and replication.Ê These
external signals are involved in social control, meaning they come from other
cells, in the cell cycle.Ê Cells can also
receive signals that inhibit growth and modulate cell responsiveness to normal
signals.Ê Exogenous chemicals such as
hydrogen peroxide, fresh squeezed orange juice, and Lipton green tea can
interrupt the cells normal signals causing an alteration in growth,
replication, and/or cell life (Fata-Hartley, et al. 2007).
The hydrogen peroxide is
an oxidizing agent which disrupts the membrane of the cells leading to
apoptosis.Ê Adding natural antioxidants
such as vitamin C found in oranges and flavonoid
found in green tea are tested to try to decrease the apoptotic effect hydrogen
peroxide has in high concentrations like 30%.Ê
High concentrations of hydrogen peroxide are known killers of cells,
whereas lower concentrations have a minimal effect.Ê This combined with the anti-oxidants should
allow the cells to resist the apoptotic effect of the oxidizing agent and
display more cell life and growth (
In lab 2I the effect of
the addition of reactive oxygen species was tested due to the fact that free
radicals will damage different biological targets such as lipids, DNA, and
proteins. Also, the cellular metabolism will be disturbed, unless corrective
defenses intervene. (Sahnoun, et al. 1998)
Varying concentrations of
hydrogen peroxide were added to the cell wells.Ê
One set of the five concentrations (30%, 3%, 0.3%, 0.03%, and 0.003%)
was left without anti-oxidants and one cell plate was left with just
water.Ê The cells with 30% and 3%
hydrogen peroxide showed very minimal cell life, whereas the cell wells with
lower concentrations and the well with water still exhibited plentiful cell
life observed by the attachment of the cells to the monolayer. ÊAs expected, the higher concentrations of
hydrogen peroxide acted as a positive control and killed the cells due to the
increase in acidity, visible by the cell media turning clear representing a
much lower pH value, and the determined apoptotic factor of reactive oxygen
species (
The addition of vitamin C
in the form of orange juice allowed for more of the cells to resist the
apoptotic factor of hydrogen peroxide due to the reducing properties of vitamin
C.Ê Hydrogen peroxide is a reactive
oxygen species and the water-soluble vitamin has reduction properties by
scavenging for free radicals dispersed by the oxidizing agent, hydrogen
peroxide.Ê Flavonoids,
found in Lipton green tea, were also added to the wells.Ê Flavonoids have the
ability to enhance catalase activity when introduced
to hydrogen peroxide.Ê Catalase is an enzyme that breaks down hydrogen peroxide
into oxygen and water.Ê The introduction
of flavonoids to the cell wells allowed for the
decomposition of hydrogen peroxide into components that did not harm the cell
or the cellâs growth cycle.Ê Vitamin C
and flavonoids therefore, preserved the cellular
integrity of the cells in the well plates at lower concentrations of hydrogen
peroxide, supporting the hypothesis that the addition of an anti-oxidant to the
cell well plates along with hydrogen peroxide will allow for an increase in
cell life as opposed to hydrogen peroxide exposure alone (Doronicheva, et
al. 2007).
Upon the addition of the
orange juice, the cell wells turned a bright yellowish color due to the acidic
properties of the orange juice.Ê This
drop in the pH level is believed to have lead to the drop in live cells and an
increase in cell death as opposed to the addition of the flavonoids
through green tea.Ê When viewed under a
microscope, the cell wells with the orange juice and hydrogen peroxide still
had more live cells than that of the hydrogen peroxide exposure alone, but less
visible cell life than that of the flavonoids
introduced from the green tea.Ê The last
set of wells contained both anti-oxidants as well as the hydrogen
peroxide.Ê These well plates showed more
life than those just exposed to orange juice, however,
less cell life was seen than the wells that just contained the flavonoid anti-oxidant.Ê
This supports the idea that the change in pH due to the acidic orange
juice increased cell death.Ê If repeated,
this section of the experiment should be altered, and instead of using orange
juice as the source of vitamin C, simply use powdered vitamin C to avoid the
acidity and unnecessary cell death simply due to experimental faults.
The protein assay in lab
3 is used to determine the protein concentration in a cell sample by means of a
ÊÊÊÊÊÊÊ The
SDS-page in lab 3 determines the molecular weight of a protein in a cell.Ê The lighter the protein, the further the
protein will travel. ÊThe Rf value is calculated by dividing
the distance the protein traveled by the distance the dye front traveled,
therefore, if the protein is light, the numerator will contain a higher number,
leading to a high Rf value.Ê If the molecular weight of the protein in the
cell is high, then the protein is expected to not travel as far which would
make the numerator a smaller number, giving heavier proteins a lower Rf value.
ÊÊÊÊÊÊÊ For
our independent research, planarians will be used to determine the amount of
wound healing/regenerative growth when exposed to the external factors
previously tested; hydrogen peroxide, vitamin C in the form of orange juice,
and flavonoids in the form of Lipton green tea.Ê It is predicted that the planarians in pure
water will have full regenerative growth due to the previous results seen in
lab 2I where the well with water had plentiful cell life and was not effected by the addition of water.Ê The planarian in the 30% hydrogen peroxide is
expected to die due to the apoptotic effect this chemical has when exposed at
this concentration especially.Ê The
addition of the anti-oxidants to the solutions that the planarians will be in
is expected to allow for faster wound healing/regenerative growth due to the
decomposition of hydrogen peroxide by the flavonoids,
the reducing properties of vitamin C.Ê
However, the regeneration rate is expected to follow the same results as
found in lab 2I where hydrogen peroxide alone yields death and minimal growth,
hydrogen peroxide and orange juice yield more cells, hydrogen peroxide, orange
juice and green tea yield even further cell life, and hydrogen peroxide and
green tea yield the best cell life of the previously mentioned combinations and
even the same or slightly better results than the water controls.
Troubleshooting
ÊÊÊÊÊÊÊ The first week of
laboratory was reserved to gain experience when keeping a laboratory notebook,
using a pipette, creating a dilution series and using Microsoft Excel to create
a graph based on findings from a spectrometer.Ê
Although these procedures were carefully explained and simplistically
demonstrated, it is possible the experimenters made a mistake during one of
these procedures in any of the future experiments.
ÊÊÊÊÊÊÊ During
the creation of the various dilution seriesâ used throughout the different
experiments, it is possible a pipette tip was not changed before moving to the
next test tube with the diluted solution.Ê
This would change the concentration of the dilutions than what is
recorded and could possibly skew the results.Ê
Whenever it was noticed that a pipette tip had not been changed prior to
creating a new dilution in the series, the new dilution made would be removed
and remade with a new pipette tip to ensure it was the proper recorded
concentration.Ê There is a slim chance,
however, that the experimenters missed this practice on one or more occasions.
ÊÊÊÊÊÊÊ During
counting of the cells it is possible the monolayer was not completely lifted
from the bottom and sides of the well plate.Ê
This would cause the living and dead cell averages to change and
possibly not be accurate.Ê Special care
was taken by the experimenters to ensure that the monolayer was completely
lifted from the well plates before counting, including using proper chemical
techniques along with visual cues.
ÊÊÊÊÊÊÊ The
laboratory ran out of Trypan blue dye to count the
cells with.Ê This created a particular
problem for the experimenters because part of the hypothesis formed was to
check the number of dead cells, which could no longer be done since without the
Trypan blue only living cells could be counted and
dead cells could not be seen.Ê This
problem definitely changed the results and could not be redone as the
laboratory was out of the dye for several weeks leading up to and through the
end of experimentation.
ÊÊÊÊÊÊÊ One
of the experiments performed was using a Bradford Assay to obtain protein
concentration information.Ê Although the
proper procedures were written, explained, and practiced prior to the actual
experimentation, it is possible that one of the aforementioned problems occurred
during this experiment as well and skewed the results.Ê Special care by the experimenters was taken
to ensure that the results were correct and properly obtained.
ÊÊÊÊÊÊÊ Lab
4 involving the regeneration of planarian involved precise cutting.Ê The head of the planarian was to be cut off
and a bilateral cut was to be made forming a y-shaped lower section.Ê Since the cutting was done under a dissection
scope and the planarian were no longer than half a
centimeter it was hard to make the cuts precise.Ê Even though the planarian
have regenerating abilities, a misplaced cut could prove fatal and
perhaps did.
ÊÊÊÊÊÊÊ ÊÊ
ÊÊÊÊÊÊÊ
ÊÊÊÊÊÊÊÊÊ