Hydroquinone Effect on Mus Musculus Mouse Lung Fibroblast Cells and p53 Expression
Viktoria Farian and Hannah Judy
Division of Natural and Health Sciences, Seton Hill University, Greensburg, PA
Introduction:
The 158 chemicals found in cigarettes smoke can cause toxicity to an
individual's cells when the smoke is inhaled (Fowles & Dybing, 2003).
These chemicals mostly affect the lung cells due to the inhalation of
toxins. The cells that are being examined in this research are normal
Mus musculus mouse lung fibroblast, acquired from ATCC. These
cells were treated with a chosen chemical from a cigarette. The
chemical used in this study is hydroquinone. This chemical is
hazardous, as it is highly suspected to be carcinogenic and toxic to
several organs (Hydroquinone n.d.). To observe the levels of gene
expression, a protein of interest was chosen to be p53. When DNA is
damaged, the cell will attempt to repair the DNA or activate
apoptosis, which is regulated by the p53 protein (Alberts, et al 2014).
When the cell undergoes stress, p53 expression levels are increased
(Gibbons, Byers, Kurie 2014). Hydroquinone is known to cause stress
Percent Survival (%)
upon the cell which will increase the need for p53 expression
(Levine, A.J., 2001). We will be using cell culture to grow and treat
the normal Mus musculus mouse lung fibroblast cells with different
doses of hydroquinone. Our hypothesis is that the treatment of
normal Mus musculus mouse lung fibroblast cells with various doses
of hydroquinone will decrease cell survival and increase the
expression of p53.
Methods:
Cell Culture: Mus musculus mouse lung fibroblast cells were passaged in order to
transfer the cells to new plates to avoid overcrowding and contact inhibition, with the
use of standard aseptic tissue culture technique. The cells were placed in a 5% CO 2
incubator at 37°C
Dosing: The cells were dosed with concentrations of 2.0µM hydroquinone in order to
observe the chemical effect occurring on the cells at various concentrations.
Cytotoxicity Assay:
Qualitative: The cells were counted in a hemocytometer to calculate the amount of
cells per milliliter of solution. Trypan blue was used to aid in determining cell
survival.
Quantitative: The cells were seeded in the chamber wells, dosed with
concentrations of hydroquinone, and incubated at 37°C. The cells were stained
with EthD1 and Calcein AM, incubated for 30 min at 37°C, and observed under a
fluorescent microscope.
qRT-PCR: The mRNA was isolated by centrifugation and lysing of the cells. The cells
were incubated at room temperature, and a stop solution was added to the cells. The
cells were incubated again at room temperature before being placed in a fridge at 4°C.
cDNA was created and added to each of the master mixes, GAPDH master mix and
p53 master mix, to obtain the specific concentration levels. The cDNA , master mix
concentration solutions were centrifuged and analyzed with a qRT-PCR to determine
the levels of p53 expression.
SDS-PAGE : Protein Electrophoresis and Western Blotting:
The proteins were isolated with the use of a lysis solution and centrifuged to obtain a
supernatant for each concentration. Half of the supernatant for each concentration
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was transferred to new microcentrifuge tubes and boiled for 4 min. All the
microcentrifuge tubes were placed in a freezer box. Protein electrophoresis was
conducted using a gel, membrane, and filter papers in a cassette. After the
electrophoresis, the membrane was placed in a blocking solution and situated on a
rocker. The membrane was cut at the band marker; the bottom half was placed in a
solution
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goat primary
antibody, and the top half was placed in a solution for p53 that contained a blocking
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solution and a goat anti rabbit secondary antibody. (The gel was created prior to
experimentation).
A B
Figure 5: SDS-PAGE: Protein Electrophoresis and Western Blotting with p53 expression results from Mus
musculus mouse lung fibroblast cells treated with hydroquinone concentrations. Phospho-p53 was used for
the protein expression marker. The band present in the 3rd lane indicates that the lowest dose 20 µM exhibit
p53 expression. No expression results were present for doses 40 µM and 50µM. As the dose concentrations of
Figure 1: A. Hydroquinone chemical structure (Hydroquinone n.d.) B. p53 protein structure (PDB101) hydroquinone increased,  p53 expression decreased. The smear at the bottom right of the image cannot be
confirmed actin. Thus, no actin was presented on this blot. The gel was treated with a rabbit anti goat
primary antibody and a goat anti rabbit secondary antibody.   
Results: Conclusions:
• Our hypothesis was rejected due to the increase in
A Average Percent Survival [Hydroquinone] Average Percent Attachment [Hyroquinone]
120
B
120
hydroquinone dose concentrations causing a decrease in the
100
p53 protein expression (Figures 4a-b).
• Percent survival and percent attachment results indicate that
100
Percent Attachment (%)
80 80
as the concentrations of hydroquinone doses increase, the
60 60 percent survival and percent attachment of the cells decrease
(Figure 2a-b). This suggests that the chemical was toxic to the
40 40
cells.    
20 20
• The SDS-PAGE, protein electrophoresis and western blotting
0
0 10 20 30 40 50 60
0
0 10 20 30 40 50 60 results showed that p53 was expressed at the lowest dose, 20
[Hydroquinone] (µM) [Hydroquinone] (µM) µM. There was no expression results shown for 40 µM and 50
Figure 2: Average Percent Survival of the Cells following exposure to Hydroquinone. µM (Figure 5). The multiple bands that appeared may have
Three trials were completed for the control, while only two doses were performed for the 20µM, 40µM, and
been due to the presence of the phospho-p53 antibody. Lack
50µM concentrations. A. A high percent survival rate was shown for the control, but as the doses of
hydroquinone increased, a decrease trend in percent survival was observed based on the line of best fit. The of protein expression could have resulted from the loss of cells
standard error was only able to be taken for the three control trials and was shown to be small. The standard
deviation of the two trials of the concentrations of 20µM, and 50µM were also recognized as a small error. The during the scraping of the plates.     
40µM had a large standard deviation. B. The high percent attachment was observed for the control, but as the
doses of hydroquinone increased, a major decreasing trend was displayed by the line of best fit. The standard
• The results from the qRT-PCR were expected to indicate an
error for the three trials of the control was minimal. The standard deviation of the two trials of 20µM and increase in p53 expression as the doses increased in
40µM concentrations showed a small standard deviation, while the 50µM concentration should a large
standard deviation. concentration. The results revealed that only the 20 µM dose
had a 2X fold increase relative to control while the 40 µM had
no change and the 50 µM decreased (Figure 4a-b). These
results could have been due to the cells resorting to necrosis
instead of apoptosis in the higher levels of hydroquinone.         
Future Studies:
• A future study could be conducted to determine if hydroquinone
reacts with other chemicals within a cigarette to cause the same
cell survival rate, or if it would cause a greater increase or
decrease in cell survival. This could a help conclude the cell
toxicity variance levels that hydroquinone has on its own versus
Figure 3. Cytotoxicity Assay of Mus musculus mouse lung fibroblast cells treated with 0 µM, 20 µM, and 40 µM
when it reacts with other chemical (Peters, Jones, Monks, & Lau,
of hydroquinone and observed under fluorescent microscopy. The cells were stained with Ethidium
Homodimer-1 (Fig. D, E, F) and Calcein AM (Fig. A, B ,C) and viewed under 100x magnification. The cells were 1997).
dosed 24 hrs and incubated at 37°C prior to observations under a fluorescent microscope. In figures A and D, the
• Same experiment could be repeated to determine statistical
cells were treated with 0µM concentration of 2.0mM of hydroquinone. In figures B and E, the cells were dosed
with 20µM concentration of hydroquinone. In figures C and F, the cells were treated with 40µM concentration of significance on the effect of hydroquinone treated normal Mus
hydroquinone. As the dose concentrations of hydroquinone increased, the percent of cell survival was shown to
decrease when compared to the corresponding cytotoxicity assay concentration. musculus mouse lung fibroblast cells and p53 expression
• Hydroquinone effect on different proteins to observe if
Concentration levels of Hydroquinone and the
A B Concentration of Hydroquinone and Corresponding hydroquinone has this same toxic effect on other proteins
Corresponding p53 Protein Expression Fold Increase/Decrease of p53
(Enguita & Leitão, 2013).
2.5
• Due to the great amount of floating cells that were observed on
2
the plates after dosing with the concentrations of hydroquinone,
Fold Increase/Decrease (X)
1.5
further studies could be performed to determine p53
1 underwent apoptosis or necrosis (Yeung, 1998).
0.5
0 References:
0 20 40 50
• Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Robers, and Walter. (2014). Essential Cell
Concentration of Hydroquinone (µM) Biology (Fourth). New York, New York: Garland Science.
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Figure 4: qRT-PCR Amplification Plot of protein p53 expression with hydroquinone treated Mus musculus mouse • Gibbons, D. L., Byers, L. A., & Kurie, J. M. (2014). Smoking, p53 mutation, and lung
lung fibroblast cells. The qRT-PCR amplification plot displays a CT threshold value for the p53 expression (bright cancer. Molecular cancer research : MCR, 12(1), 3-13.
• Hydroquinone. (n.d.). Retrieved from
blue horizontal line). The qRT-PCR amplification plot was normalized to the GAPDH master mix. A. The red line https://pubchem.ncbi.nlm.nih.gov/compound/hydroquinone
displays a 0µM (control) concentration of hydroquinone, the blue line displays the 20µM concentration, the green • Levine, A. J. (2001, April 11). P53, the Cellular Gatekeeper for Growth and Division.
Retrieved from https://www.sciencedirect.com/science/article/pii/S0092867400818711?via=ihub
line displays the 40µM concentration, and the pink line displays the 50µM concentration. B. The fold increase and • PDB101: Molecule of the Month: P53 Tumor Suppressor. (n.d.). Retrieved April 26, 2019, from https://pdb101.rcsb.org/motm/31
decrease were displayed in a bar graph to represent the protein expression for the increasing concentrations of • Yeung, M. C. (1998, September 25). Michael C. Yeung. Retrieved from http://www.jbc.org/content/273/39/25198.full
• Enguita, F. J., & Leitão, A. L. (2013). Hydroquinone: Environmental pollution, toxicity, and microbial answers. Retrieved April 26, 2019, from
hydroquinone. With comparison to the control, the fold increased for the 20µM concentration by 2.14X. Minimal https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3727088/
change in fold was calculated at the 40µM concentration (1.07X). The fold decreased for the 50µM concentration • Peters, M. M., Jones, T. W., Monks, T. J., & Lau, S. S. (1997). Cytotoxicity and cell-proliferation induced by the nephrocarcinogen hydroquinone and its nephrotoxic
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by 0.307X.