Nicotine Impact on Mouse Lung Fibroblast Survival and Bax Expression
Kyle Lovisone, Sierra Megonnell, and Daneiris Mejias
Division of Natural and Health Sciences, Seton Hill University, Greensburg, PA
Introduction:
Cigarettes are created from dried tobacco leaves and contain a
variety of chemicals. Although all of these chemicals can be
harmful, nicotine is known to be the worst chemical due to its
addictive properties (ACS, 2017). Nicotine is a popular drug in the
United States due to availability, lack of legal consequences, and
marketing techniques (“Nicotine”, 2017). Nicotine has been shown
to have inhibitory effects on apoptosis in cells. It does this by
increasing the amount of Bcl2 in a cell and decreasing the amount
of Bad, Bak, and Bax (Heusch, 1998 and Zeidler, 2007). Bax is a
protein within the Bcl2 family and is partially responsible for
apoptosis, by opening an anion channel that releases cytochrome
c, apoptosis is triggered (“BAX Gene”, 2019). The objectives of the
study are to determine cell survival and attachment after exposure
to different nicotine concentrations, determine the impact nicotine
has on expression of Bax, and conclude the impact nicotine has on
the mouse fibroblast overall. This was done using both quantitative
and qualitative techniques. The hypothesis was that the treatment
of normal mouse lung fibroblasts with increasing doses of nicotine
will increase the survival of the cells and decrease the expression
of Bax in the cells.
Methods:
Cell Culture: A sample of mouse lung fibroblasts were cultured and passaged using
standard aseptic tissue culture techniques. The cells were incubated with media in a 5%
CO2 incubator at 37OC.
Cytotoxicity Assay:
• Trypan Blue: A sample of mouse lung fibroblasts were treated with various nicotine
concentrations; 0 uM, 300 uM, 500 uM, and 700 uM. Following cell passaging they
were quantitatively analyzed with 1:1 trypan blue and counted using standard
hemocytometer techniques. Cell survival was determined.
• Live/Dead Assay and Fluorescence Microscopy: A sample of mouse lung fibroblasts
were seeded into a chamber, dosed, and incubated at 37°C for 22 hrs. Calcein-AM and
ethidium-homodimer-1 solution were added to the cells and incubated at 37°C for 30
mins. The stain solution was removed and the slide was placed under a fluorescent
microscope. The live cells showed green fluorescence due to esterase activity. The
dead cells showed red fluorescence due to a loss of membrane integrity.
qRT-PCR Data Analysis: Master mixes were made following company suggested
procedures. 16 uL of PCR master mix and 4 uL of cDNA was added to each of the wells
and the plate was centrifuged. Plate was loaded into the instrument and the TaqMan
Expressions Cells-to-CT Kit program was conducted. The results were analyzed to
determine the qRT-PCR spectrum and the Bax fold expression which had been normalized
to the housekeeping gene, GAPDH.
Western Blotting: A sample of treated mouse lung fibroblasts were analyzed to Bax
expression using Western Blot analysis. In order to analyze, an SDS-PAGE gel
electrophoresis was performed. The denatured proteins were transferred to a membrane
and immunoblotted by blocking, where the membrane was placed in a milk-based buffer
to allow occupation of any regions in the membrane to avoid non-specific binding of the
antibodies. The membrane was washed and 7 uL of the primary antibody, rabbit anti-
goat, were added followed by incubation. The primary antibody was then washed off of
the membrane followed by the treatment of 3.5 uL of the secondary antibody, goat anti-
rabbit. The secondary antibody contained an enzyme known as horseradish peroxidase
that was used for protein detection.
Results:
Figure 1: Chemical structure of nicotine. Image retrieved from www.scifinder.cas.org.
A B
Figure 2: This graph represents the average cell survival following exposure to various
concentrations of nicotine. As the concentration of nicotine increases, the percentage of
cell survival increases. The first two error bars for represent standard error and the second
two represent standard deviation. The line represents the line of best fit.
Figure 3: This graph represents the average cell attachment following exposure to various
concentrations of nicotine. As the concentration of nicotine is increased, the percentage of
cells attached decreases. The error bars for the 0 uM and 500 uM concentrations of
nicotine represent standard error and the 300 uM and 700 uM error bars represent
standard deviation. The line represents the line of best fit.
Figure 4: These images are captured at 100x magnification using a LIVE/DEAD assay and
fluorescence microscopy. Images A, C, D, and F show the esterase activity and intact
plasma membranes of the live cells at 0 uM (A), 300 uM (C), 500 uM (D), and 700 uM
(F). Images B, E, and G, show the red-fluorescent dye for the cells in the control group
(B), 500 uM (E), and 700 uM (G) of nicotine at 100X magnification.
Figure 5: The qRT-PCR amplification plot for GAPDH is labeled with the star and Bax is
represented by the unlabeled lines. The orange line is the GAPDH threshold and the
blue line is the Bax threshold line (A). The bar graph represents the increase in Bax
after normalization to the control. There are three concentrations that had a fold of 1,
but the expression of Bax when treated with 500 uM nicotine shows a fold decrease
(B).
Conclusions:
• Hypothesis is inconclusive
• The concentration of nicotine had little effect on cell
survival, though it did cause a decrease in cell
attachment as concentration increases (Figures 2 and
3).
• The 300 uM and 700 uM samples showed no change in
Bax expression while the 500 uM sample showed a
decrease in Bax, indicating nicotine had little impact on
the living cell expression of Bax (Figure 5B).
• The results of the LIVE/DEAD assay show that the cells’
morphology is being affected by nicotine. The nicotine
is also causing the cells to become more densely
packed (Figure 4).
• Necrosis could have been cause of death, not
apoptosis. This is due to the cells’ experiencing
morphologic changes, increasing detachment, and
consistent survival (Figures 2, 3, and 4).
Future Studies:
• Repeat the same experiment for statistical significance.
• Analyze the cells that were detached by isolating them
from the aspirator and determine Bax expression using
qRT-PCR.
• Repeat the same experiment with dosages closer to
500 uM nicotine concentration (e.g. 450-550 uM).
• Analyze the Bcl2 expression using a Western Blot with
the same concentrations of nicotine.
• Analyze cells used in this experiment by measuring the
release of lactate dehydrogenase in order to determine
if the cells died by necrosis (Chan, 2013).
References:
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smoking/smoking-facts/whats-in-a-cigarette.html
“Nicotine.” Psychology Today, Sussex Publishers, 26 Mar. 2019,
www.psychologytoday.com/us/conditions/nicotine.
4. “BAX Gene.” Genecards.org, Weizmann Institute of Science, 2019, www.genecards.org/cgi-
bin/carddisp.pl?gene=BAX.
5. Heusch, & Maneckjee. (1998, April 01). Signalling Pathways Involved in Nicotine Regulation of Apoptosis
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https://academic.oup.com/carcin/article/19/4/551/2365442
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25, 2019, from https://link.springer.com/article/10.1007/s10495-007-0102-8
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dehydrogenase activity. Retrieved April 25, 2019, from
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