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
substances such as formaldehyde, lead, arsenic, ammonia, and
nicotine. Nicotine considered one of the worst of these chemicals
due to its addictive properties (ACS, 2017). Nicotine is a popular
drug in the United States due to its availability, lack of legal
consequences, and marketing techniques (“Nicotine”, 2017). Bax is
a protein within the Bcl2 family and is partially responsible for
apoptosis by opening an anion channel which releases cytochrome
c, which directly triggers apoptosis (“BAX Gene”, 2019). Nicotine
has shown to inhibit Bax expression, while also increasing the
expression of Bcl2, thus inhibiting apoptosis (Heusch, 1998 and
Zeidler, 2007). Bax expression was determined in mouse lung
fibroblasts using both quantitative and qualitative means. Nicotine
was selected due to the interest in its addictive properties and how
it would affect the mouse lung fibroblast’s apoptotic pathway. 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 as a
whole. The hypothesis is 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 fibroblasts were cultured and passaged using
standard aseptic tissue culture techniques. The cells were incubated with media in a
5% CO2 incubator at 37OC.
LIVE/DEAD Viability/Cytotoxicity Assay: Samples of mouse fibroblasts with 0, 300,
500, and 700 uM concentrations of nicotine were fluorescently labelled and
observed using fluorescent calcein-Am and fluorescent ethidium homodimer-1.
Real Time qRT-PCR/Western Blotting: Samples of mouse fibroblasts with 0, 300,
500, and 700 uM concentrations of nicotine were run through a TaqMan in order to
determine the expression of Bax for each sample.
Results:
Figure 1: 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 2: 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 3: 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 4a: qRT-PCR amplification plot for GAPDH and the protein of
interest, Bax. The red, blue, green, and pink lines correspond to the
0 uM, 300 uM, 500 uM, and 700 uM nicotine samples, respectively.
Figure 4b: This bar graph represents the change in the protein of
interest, Bax, when compared to the amount of Bax in the
control group. The graph shows three of samples having a fold of
1, indicating no fold increase. But, the 500 uM sample
experiences a fold decrease.
Conclusions:
• Accept or reject hypothesis
• The concentration of nicotine had little effect on cell
survival, though it did cause a decrease in cell
attachment as concentration increases (Figure 1 and 2).
• The 300 uM and 700 uM samples showed no change in
Bax expression but the 500 uM sample showed a
decrease in Bax, meaning…
• The results of the fluorescence microscopy show that
the cells are sending signals to cause them to become
irregularly shaped and clump together as the
concentration of nicotine increases
• Necrosis could have been cause of death, not apoptosis
(when and where)
• Data is inconsistent (reference figures and give reasons)
• Inconclusive
Future Studies:
• Isolate cells from 500 uM nicotine concentration and
perform further testing and analysis (say what further
tests)
• Observe and analyze the cells that were detached by
isolating them from the aspirator
• Test dosages closer to 500 uM nicotine concentration
• Look at Bcl2 expression with concentrations of nicotine
• Look at other assays from papers
References:
1. “Harmful Chemicals in Tobacco Products.” American Cancer Society,
American Cancer Society, 5 Apr. 2017, www.cancer.org/cancer/cancer-
causes/tobacco-and-cancer/carcinogens-found-in-tobacco-products.html.
2. “Nicotine.” Psychology Today, Sussex Publishers, 26 Mar. 2019,
www.psychologytoday.com/us/conditions/nicotine.
3. “BAX Gene.” Genecards.org, Weizmann Institute of Science, 2019,
www.genecards.org/cgi-bin/carddisp.pl?gene=BAX.
4. Heusch, & Maneckjee. (1998, April 01). Signalling Pathways Involved in
Nicotine Regulation of Apoptosis of Human Lung Cancer Cells. Retrieved
January 25, 2019, from
https://academic.oup.com/carcin/article/19/4/551/2365442
5. Zeidler, R., Albermann, K., & Lang, S. (2007, September 11). Nicotine and
Apoptosis. Retrieved January 25, 2019, from
https://link.springer.com/article/10.1007/s10495-007-0102-8