Mackenzie Nalepa, Brett Smith, Samantha Wargo

11/24/15
Cell Biology Lab BL 248

Effect of Phenol on the Expression of BAX in Mouse Lung Fibroblasts

Introduction

Much research has been conducted on cigarette smoke, its component compounds, and

their harmful effects on humans. Although the public knowledge of cigarette smoke has been

increasing, recent studies report that cigarette smoke is still responsible for 480,000 American

deaths each year. Cigarette smoking is linked to numerous diseases, including but not limited to

various types of cancers, pneumonia, stroke, coronary heart disease, and chronic obstructive

pulmonary disease (U.S. Department of Health and Human Services, 2014). Statistics display

that smoking is the leading cause of preventable death in the United States. Not only is cigarette

smoke responsible for destruction within the population, but studies also show that cigarettes are

responsible for $289 billion in health care spending each year (Jamal et al., 2014).

Not only is cigarette smoking directly related to health complications, but inhaling

second-hand smoke can also produce the same effects. Each year there are approximately 7,000

deaths from lung cancer and 33,950 deaths from heart disease in adults that resulted from

exposure to secondhand smoke (American Lung Association, 2014). These deaths are a result of

the exposure of many toxic chemicals found in cigarettes. When isolated, tobacco smoke is

comprised of over 7,000 chemicals, 70 of which are known to be cancer-causing agents. Some of

these compounds include formaldehyde, benzene, hydrogen cyanide, and arsenic. These

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compounds are also found in resins, gasoline, chemical weapons, and pesticides, respectively

(Centers for Disease Control and Prevention, 2011).

Our colleagues in the biochemistry laboratory extracted various chemicals from cigarette

smoke, one of which is phenol. Phenol is composed of one hydroxyl group attached to an

aromatic benzene ring and is one of the most common representatives of toxic organic

compounds. Studies have already displayed the harmful effects of phenol in mice embryos

injected through the placenta. These harmful effects were displayed through decreased

embryonic weight, increased placental thickness, and higher concentrations and sizes of

spongiotrophoblasts and trophoblastic giant cells (Monfared et al., 2013). Some substituted

phenol derivatives are known to have apoptosis inducing effects on leukemia cells in mice

(Tsygankova, I. M. & Zhenodarova, S. M., 2008).

Apoptosis is a mechanism of programmed cell death in which the contents of the cell are

compartmentalized into apoptotic bodies. The release of these apoptotic bodies destroys the cell

in a way that does not cause inflammation in the surrounding tissue (Reed, 2000). In this respect,

apoptosis occurs as a mechanism of homeostasis or immunologic defense for colonies within

tissue. This pathway can occur when cells are genetically programmed to undergo apoptosis

continually or when the cell is stimulated by ligand binding. Two common characteristics of

apoptosis are overall cellular shrinkage and pyknosis. Pyknosis involves the condensation of

chromatin in the nucleus (Elmore, 2007). Caspases are proteases that are involved in these

processes and the overall breaking apart of the cellular components with ATP hydrolysis (Reed,

200).

Another form of cellular death is necrosis, which does not require energy and is more

toxic and inflammatory to the surrounding tissue. In the necrotic pathway, the cell swells and

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bursts, breaking down the chromatic material through karyolysis instead of condensing it.

Necrosis is therefore induced when the cell has low levels of caspases or ATP, both of which

must be present for apoptosis to occur (Elmore, 2014). When the cell bursts, intracellular leakage

from the cell occurs because the plasma membrane is broken down. It was previously thought

that necrosis was accidental, but recent studies have shown that necrosis can also be planned,

like apoptosis (Chan et al., 2013).

The protein of interest for this study is BAX, which is in the Bcl2 family of proteins. In

this family, some of the proteins inhibit apoptosis, while some of them activate apoptosis

(Alberts et al., 2014). When BAX is activated, it acts to make the outer membrane of

mitochondria more permeable, which releases cytochrome c into the cytosol. This process takes

under 10 minutes to occur (Green, 2005). Cytochrome c then binds to a protein that forms an

apoptosome, which has seven arms in a pinwheel shape (Alberts et al., 2014). These

apoptosomes then become dispersed through phagocytotic mechanisms, a process that takes

hours or days (Green, 2005). Some members of the Bcl2 family are apoptotic inhibitors. For

instance, Bcl-xL is an inhibitor and regulator for BAX (Edlich et al., 2011). Some research

shows the relationship between phenol and triazoles moietes and their ability to break the

interactions of BAX and Bcl-xL in a way that promotes cell death (Vo, D. D., et al., 2014).

In this study, the goal of the experiment was to test the toxicity of phenol and the level of

BAX expression on normal fibroblast mouse lung cells. The cell lines used for this study were

CCL-206 fibroblast mouse lung cells and CCL-196 epithelial adenoma mouse lung cells, studied

by a partner group, purchased from ATCC (ATCC, 2014). The fibroblast lung cells are referred

to as normal mouse lung cells within the context of the experiment. The normal lung cells

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were cultured and dosed with various levels of phenol in order to perform the necessary

experiments needed for analysis.

Trypan blue assay is an analysis performed to determine cell survival after chemical

treatment. When cells are dead, or on the verge of dying, their membrane becomes damaged;

therefore, the membrane is more permeable. This characteristic allows the trypan blue to diffuse

across the membranes of dead cells, giving the cell a dark blue tint. The cells that appear blue are

classified as dead, and the cells that are still transparent are classified as living.

A LIVE/DEAD assay is performed using a LIVE/DEAD Viability/Cytotoxicity Kit. The

chemically treated cells are stained with chemical compounds known as fluorochromes that

absorb and emit different wavelengths. Cells that are alive and maintain an intact membrane emit

green-fluorescent calcein-AM. Cells that are presumed dead due to dysfunctional and permeable

membranes emit red-fluorescent ethidium homodimer-1 (Eth-D1). Fluorescent microscopy is

used to examine the cells.

qRT-PCR is used to analyze the BAX gene expression in isolated mRNA from the treated

cells. This assay uses the Ambion TaqMan PreAmp Cells-to-CT Kit from Life Technologies for

the analysis. This kit allows for quick RNA purification without the risk of contaminating or

losing any of the sample. The cell is lysed, the mRNA is isolated, any genomic DNA is

degraded, and RNases within the cell are deactivated through this process. Only the purified

mRNA remains, which is then converted to cDNA through reverse transcription. The cDNA is

then amplified for the target protein expression to be assessed through quantitative real time

PCR.

The Western blot technique was the final analysis used in this study. Sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to confirm the levels of gene

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expression performed by qRT-PCR. Electrophoresis allows proteins to be separated based on

molecular weight, as protein folding and charge are eliminated. The separation means that the

larger proteins will locate near the bottom and the smaller at the top. LumiGLO allows the

presence of probed antibodies to capture light on an X-ray film and result in the formation of

black bands that correspond to the protein of interest. Stains such as Ponceau and Coomassie

blue are also used in this type of analysis to intensify the bands shown in the Western blot.

We hypothesized that that the treatment of normal mouse fibroblast lung cells with

various doses of phenol will decrease the survival compared to adenoma mouse epithelial lung

cells and increase the expression of BAX compared to adenoma mouse epithelial lung cells.

Materials and Methods

Cell Culture

To maintain the cell line of normal mouse lung cells (catalog number CCL-206

purchased from ATCC), the cells were periodically cultured and passaged to a more optimal in

vitro environment. This passaging ensured that the samples did not become confluent and perish.

The cells were kept in a media containing nutritional fetal bovine serum, antibiotics, and a pH

indicator. For each passage, this media was aspirated, and the cells were then washed with 5 mL

of 1X PBS buffer solution. Following the buffer wash, the cells were treated with 1 mL of

trypsin-EDTA and placed in a 37C incubator for 5 minutes. The cells were examined under a

microscope to confirm detachment from the bottom of the plate. The detached cells were then

mixed with 3 mL of media and transferred to a 15 mL storage container. 10 uL of sample were

pipetted to count the concentration of cells in the media, using a hemocytometer. The

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concentration of cells needed for an optimal concentration of cells/mL in a new plate was

calculated. This concentration was determined to be 7x105 cells/mL. The calculated volume of

media solution to obtain this optimal concentration was added to a new plate, and media was

added so the total volume of the solution was between 5-7 mL.

Trypan Blue & Hemocytometry

Following the trypsinization of the plates, 3 ml of media was added to the cell plate and

mixed with the trypsin-EDTA. This mixture was placed in a test tube, from which 100 ul was

transferred to an eppendorf. To count the cells stained with trypan blue, 10 ul of the cells and 10

ul of trypan blue were placed in a new eppendorf. From this mixture, 10 ul were placed on a

hemacytometer and counted under the microscope.

Seeding and Dosing

The cell line used for chemical treatment with phenol was maintained by cell passaging

and culturing. After the cells were passaged into two plates, those plates were each passaged into

two plates for a total of four plates. The cells were dosed with phenol 16 hours before analysis.

The four plates accounted for each of the four concentrations of phenol used: 0.0mM, 0.12mM,

0.3mM, and 0.6mM of a 60M stock solution. The cells were counted using a hemocytometer and

the appropriate amount of phenol was calculated to correspond to the correct concentration for

each plate. In plate with the 0.0mM concentration, 0.0ul of phenol was added. For the 0.12mM

concentration, 8.3ul of phenol was added. In the plate intended for the 0.3mM of phenol, 25ul

was added. For the 0.6mM concentration, 10ul was added to each plate. In order for each plate to

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contain 5000ul of total volume, the same amount of media as the amount of phenol added was

pipetted out of the plate before chemical dosing.

For the preparation of the fluorescent microscopy analysis, the cells were seeded into a

four chamber well rather than four individual plates. The cells were seeded into the wells two

days before the microscopy was completed. The amount of cells used for the wells was a ten-fold

difference less than the amount used for the plates. The amount of phenol added to the wells was

0.0ul, 1.6ul, 5ul, and 10ul respective to the 0.0mM, .12mM, 0.3mM, and 0.6mM phenol

concentrations of each well. The final volume of the wells was 1000ul. In order for each well to

contain 1000ul of total volume, the same amount of media as the amount of phenol added was

pipetted out of the well before chemical dosing.

Fluorescent Microscopy

To prepare the wells for microscopy, the media was first aspirated. In each well, 150 ul of

Eth-D1 and calcein AM solution were added. The cells were incubated with this for 45 minutes.

The chamber was removed from the slide and the edges around each block were scraped. The

cells were observed with two different filters to see the live and dead cells that emitted the

fluorochromes.

RNA Extraction

The cells were counted with the hemocytometer and centrifuged for five minutes. The

PBS was aspirated from the tube. The cells were re-suspended in PBS to make 1x10^6 cells for

every 500 ul of PBS. 50 ul of cells were transferred to a micro-centrifuge tube. The supernatant

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was removed and the cells of each concentration were resuspended in 50 ul lysis solution. Each

sample was mixed five times with the micropipette. The samples were incubated at room

temperature for two minutes, after which 6 ul of stop solution was added and mixed five times

with the micropipette. The samples were incubated at room temperature for two minutes again,

and then they were placed in the freezer.

qRT-PCR

The tube with the cDNA was first centrifuged, very briefly, to bring the condensation

down. The master mixes for the BAX and GAPDH were prepared in microcentrifuge tubes as

follows: 10 ul of the probe (specific to either BAX or GAPDH), 100 ul Taqman master mix, and

50 ul PCR grade water. The wells of a 96 well reaction plate were then prepared. Each

concentration had 2 wells of BAX and 2 wells of GAPDH. Each well had 16 ul of the master mix

(BAX or GAPDH) and 4 ul of the cDNA corresponding to that concentration. The plate was

briefly centrifuged and then loaded.

Protein Sample Preparation

A 3X Reducing SDS Loading Buffer was diluted to a 1X Reducing SDS Loading Buffer.

After the media was aspirated and the samples were washed with 1X PBS solution, 175 mL of

the 1X Reducing SDS Loading Buffer was added to the plate. A rubber policeman was used to

scrape the samples from the bottom of the plate and into a microcentrifuge tube. The samples

were kept on ice throughout this preparation. The samples were mixed by being passed through a

26 G needle 10 times and then heated to 95-100 for five minutes. The samples were cooled

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and centrifuged for five minutes. These protein samples were then put in the freezer until the

Western blotting was performed.

Western Blotting

The glass plates of the gel mold were cleaned with water and then ethanol, then placed

together. The larger rectangle was placed with the spacers facing up and then the smaller plate

was placed on top, with the bottom edges lining up. The plates were placed in the green casting

frame with the short plate facing the clamp side. This was placed into the casting stand with the

clamps facing outward. The resolving gel was prepared by adding 3.1 ml of Acryl:bis (30%), 3

ml of 1.0 M Tris-CL pH 8.8, 38 ul of 20% SDS, and 1.3 ml dH2O. After inverting the tube

slowly 3 times, the other 2 ingredients were added: 36 ul 10% APS and 5 ul TEMED. The tube

was inverted 2 additional times and then pipetted immediately between the plates. The plates

were filled approximately three-fourths full and then 1-2 ml of water was added on top of the gel.

After polymerization occurred, indicated by a sharp line between the gel and water, the water

was poured out and blotted with filter paper. The stacking gel was prepared by adding 1.1 ml of

Acryl:bis (30%), 630 ul of 1.0 M Tris-CL pH 6.8, 25 ul of 20% SDS, and 3.5 ml of dH2O. The

tube was inverted 3 times slowly, then 25 ul of 10% APS and 5 ul of TEMED was added. The

tube was inverted again 2 times and immediately added to the plates on top of the resolving gel.

The comb was placed into the resolving gel, gently, so bubbles were not introduced. After the

stacking gel was solidified, the comb was gently removed and the wells were rinsed with water.

The previously prepared protein samples were boiled with parafilm wrapped around the top, for

10 minutes. While the samples were boiling, the electrode assembly was prepared by placing the

plates with the gel inside. When the protein samples were ready, they were placed into the wells.

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The gel was run for approximately 1 hour until the bromophenol blue dye had migrated to the

bottom.

After the gel was run, it had to be blotted. Two pieces of filter paper and one piece of

membrane, with a corner cut off, were soaked in transfer buffer for 15 minutes. The gel was

scraped off the glass and into transfer buffer to soak for 5 minutes. In the clamp, the set-up was

in this order: sponge, filter paper, nitrocellulose membrane, gel, filter paper, sponge. The clamp

was placed in the chamber along with an ice pack and transfer buffer. Transfer took place for one

hour after which the membrane was taken out and washed in 1XTBS-T for five minutes. At

room temperature, the membrane was placed in blocking buffer for one hour. The membrane was

then stored in 1XTBS-T until ready to proceed to immunodetection the following week.

The membrane was cut into two different pieces with actin on one piece and BAX on the

other piece, in respect to where the bands for each would show up. TBS-T was used to wash the

membrane three times with just enough to cover the membrane. When washing, the containers

were shook for five minutes each time. The membranes were placed in 10 ml of fresh blocking

solution that contained the primary antibody at a dilution of 1:1000. The primary antibodies used

were actin and BAX respectively. The containers were placed on the shaker for one hour. The

antibody solution was discarded and the membranes were washed 3 times. Five ml of the milk

solution and five ul of the secondary antibody were added to the membranes. The secondary

antibody used was rabbit mAb, for both. Again, they were shook for one hour. The membranes

were washed three more times. The substrate solution, 0.5 ml LumiGLO, 0.5 ml peroxide, and

9.0 ml water, was added to the membranes which were laid on plastic wrap. The substrate and

membranes were incubated for one minute. The excess solution was removed and the

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membranes were wrapped in plastic. The X-ray film was exposed to the membrane for one

minute and one hour and then the film was developed.

Results

The trypan blue analysis shows that cell survival remained relatively high, between 95-

100% survival, for the normal mouse lung cells. The numbers fluctuate and show no general

trend. In comparison, the survival of the adenoma cells showed a slight increase between 0.0mM

and 0.12mM, and then a decrease between 0.12mM and the higher concentrations. Overall, the

normal mouse cells showed more survival than the adenoma cells. The data in Figure 1 shows

the average data from two trypan blue analyses.

Figure 1. Quantitative analysis of percent survival of normal and adenoma mouse

lung cells treated with concentrations of 0.0 mM, 0.12 mM, 0.3 mM, and 0.6 mM

phenol. The percent survival was tested for cells that remained attached to the petri dish

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 following aspiration of media after treatment. Cells that died and detached from the petri

dish would not have factored into this calculation for survival rate. For the normal cells

(shown in blue), the cells were treated with phenol for 16 hours. For the adenoma cells

(shown in red), the cells were treated with phenol for 24 hours. The error bars denote

standard deviation of the samples after two independent runs.

To take account for any dead cells that may have come detached from the plate and

aspirated during cell culture, percent attachment of cells to the petri dish was analyzed. The

percent attachment is based on the control plate (0.0 mM) being the normalized amount. The

normal cells show a general decreasing trend in attachment. This is in exception of the highest

concentration, 0.6mM that shows a slight increase from the 0.3mM concentration. In

comparison, the adenoma cells show a much higher percent attachment. Their trend is the

opposite, where the percent attachment has a general increase with the exception of the highest

dosage. The normal cells were exposed to the phenol for 16 hours while the adenoma cells were

exposed for 24 hours.

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 Figure 2. Quantitative analysis of percent attached of normal and adenoma

mouse lungs cells treated with concentrations of 0.0 mM, 0.12 mM, 0.3 mM,

and 0.6 mM phenol. The percent attachment was tested for both samples of cells at

varying concentrations of phenol. For the normal cells (shown in blue), the cells were

treated with phenol for 16 hours. For the adenoma cells (shown in red), the cells were

treated with phenol for 24 hours. The adenoma cells displayed a much higher percent

attachment and range. The error bars denote standard deviation of the samples after two

independent runs.

The qualitative effects of percent survival were analyzed using a LIVE/DEAD assay

shown through fluorescent microscopy. Survival and death are represented by the reflection of

fluorochromes. The cells that appear green represent the live cells that reflect the calcein-AM

and the cells that appear red represent the dead cells that reflect the Eth-HD 1. The photographs

(Figure 3) show that as the concentration of phenol increases, the amount of surviving cells also

decreases, shown by a lack of green fluorescence. In the highest concentration, it appears that the

majority of the cells are dead. Figure 4 shows a magnified, 1000x, image of the cells dosed in

0.6mM phenol to show in more detail the effects the chemical had on cell survival.

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Figure 3. Fluorescent microscopy and LIVE/DEAD assay qualitative analysis of normal

fibroblast mouse lung cells dosed in concentrations of 0.0 mM, 0.12 mM, 0.3 mM, and

0.6mM of phenol over a 16 hour time period. In the figure above, living cells are represented

by a green color obtained by the reflection of fluorescent-green calcein-AM; dead cells are

represented by a red color obtained by the reflection of red-fluorescent ethidium homodimer-1.

The photos were taken through the eyepiece of a fluorescent microscope zoomed in to 400x

magnification on an iphone 6 camera. (A) represents the control group and shows mostly living

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cells. (B) represents the cells dosed with 0.12 mM phenol. Most of the cells shown are living,

despite the few dead cells marked with a glowing red color. (C) shows slightly less survival with

the appearance of mostly dead cells in the 0.3 mM concentration. (D) shows significant death at

the 0.6 mM concentration. There is little to no life represented by the lack of green cells present

in this photograph.

Figure 4. Fluorescent microscopy qualitative analysis of cells dosed in 0.6 mM phenol over

a 16-hour time period. This is a zoomed in photo at 1000x magnification to show in more detail

the effects of the phenol.

The results from the qRT-PCR show the expression level of BAX in normal cells

exposed to phenol for 16 hours. Our results of the qRT-PCR are closely related to the second

analysis of the percent survival graph, which shows mastery of the technique. The graph below is

an average of the first and second trial for both cell types. There was an overall decrease in

expression of BAX except for the concentration, 0.3mM, that showed a higher expression than

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the rest of the concentrations. Relative to the control, the 0.12 mM concentration had a 0.482

fold difference, the 0.3 mM concentration had a 0.78 fold difference, and the 0.6 mM had a

0.198 fold difference.

Figure 5. Relative quantification of BAX expression in cells chemical treated with 0.0 mM,

0.12 mM, 0.3 mM, and 0.6 mM. We performed a qRT-PCR analysis to determine the

expression of BAX of untreated normal mouse lung cells in comparison to normal fibroblast

mouse lung cells treated with 0.12 mM, 0.3 mM, and 0.6 mM phenol. The BAX expression was

quantified through calculations to find the fold difference in reference to the GAPDH control.

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The error bars signify standard deviations between two separate runs of each concentration. Note

that the bars in the graph do not go in increasing order of phenol dosage.

The Western blot analysis showed the qualitative results of the qRT-PCR. The results of

the Western blot are inconclusive due to lack of visible actin band markers. In the position of

0.12 mM concentration well, there is a band located at 21 kDa indicating BAX expression. There

is also a faint band located at 21 kDa in the position of the 0.3 mM concentration. The marker

well is labeled in the figure below.

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Figure 9. Western blot qualitative analysis of BAX and actin expression. (A) shows the

membrane after it was stained with ponceau stain to indicate the BAX expression. (B) shows the

membrane after it was stained with coomassie blue stain. The arrows indicate the row where

BAX expression bands appear.

Discussion

The hypothesis was rejected based upon the general decrease in BAX expression with

increasing dosage of phenol. Notably, the results from the adenoma samples showed opposite

effects, with increasing BAX expression with more phenolic effects. Cell death increased with

higher phenol dosages. This trend was more strongly evident through the percent attachment

results (Figure 2) than the percent survival results (Figure 1). The normal cells displayed higher

survivability than adenoma cells with increasing dosages. The percent survival results for normal

cells showed high numbers of survival throughout the increasing dosages, although much less

cells were reported. We believe this was because the cells that died during dosing were detached

from the bottom of the plate, and were thus aspirated with the media before trypsinization and

hemocytometry. With this in mind, the percent attachment results displayed a more accurate

insight into cell death, as the detached cells were presumed dead. The general decreasing trend in

the amount of cells attached is indicative of increasing levels of cell death. Cell death was also

reported similarly through qualitative means in fluorescent microscopy (Figures 3-4).

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 The qRT-PCR results (Figure 5) showed a general decreasing trend in BAX expression

through higher dosages as well. One interesting aspect of this analysis was the increase in BAX

expression from the 0.12 mM dosed group to the 0.3 mM dosed group, which is against the

general trend of the data. This slight increase is believed to be the result of performing only one

run of our data. If the qRT-PCR was performed multiple times, it would be expected that the

increase would be lessened or nonexistent, and the general downward trend of BAX expression

would be consistent.

Actin was used as the control marker for the Western blot because it is present in all

eukaryotic cells in abundance. Actin bands should have been present in the Western blot, but

they were not. One reason that this could have happened was that the actin dimerized with itself.

If the actin dimerized, then it would not form a band at 45 kDa. The bands would instead appear

at a different molecular weight marker because the dimers are heavier than the monomers. This

dimerization would have happened only if the actin was defective (Fornsaglio, 2015).

Another reason that the bands for actin might not have shown up was because the

membrane was dropped face down on a paper towel. The compounds on the nitrocellulose

membrane can be easily removed for reprobing purposes (20X LumiGLO Reagent and 20X

Peroxide #7003). When the membrane was dropped, this could have affected the LumiGLO

and hydrogen peroxide solution. If some of the solution was wiped off, then the light emission

would have been lower than it should have or nonexistent. With the lumiGLO and hydrogen

peroxide, the maximum emittance happens right after the membranes are exposed to it, so length

of time prior to development may have also been a factor in the minimal expression. The light

emission typically continues for 30 minutes to one hour (20X LumiGLO Reagent and 20X

19
Peroxide #7003). If we had not done the procedure quick enough at this step, then it is possible

we would not have gotten the maximum effect of light emittance.

The decrease in BAX expression was most likely due to differences between apoptosis

and necrosis. Previous studies have shown that phenols have inhibitory effects on mitochondrial

ATP production through the inhibition of dehydrogenases, phosphokinases, and ATPases. These

enzymes all have important functions in the ATP production pathway, and so their inhibition

would greatly reduce ATP production and hydrolysis within the cell (Stockdale & Selwyn,

1971). With reduced ATP production, and an inability to hydrolyze ATP molecules to use

energy, the cells may have insufficient energy for the caspases to form apoptosomes. BAX

would not be expressed without apoptotic organization and instead the cells would undergo a

necrotic pathway. Through this reasoning, as the concentration of phenol increased, less ATP

would be produced and the cells would be unable to undergo apoptosis, resulting in less BAX

expression. Research also shows that apoptosis and necrosis may not be mutually exclusive, but

exist on a continuum in which morphological evidence for both pathways exist simultaneously

(Zeiss, 2003). This evidence supports our data, in that the lower concentrations of phenol could

have allowed for slight apoptotic action and BAX expression, which would have dissipated with

higher concentrations as the spectrum shifted more toward necrosis.

Another proposal for decreasing BAX expression is that the normal cells were dosed with

phenol for only 16 hours, while the adenoma cells were dosed for 24 hours. We previously

believed that the cells did not have enough time for BAX to be expressed in higher quantities

with the 16 hour dosing. This belief, however, is refuted with evidence from other studies

showing that the mitochondrial aspect of apoptosis, which is associated with BAX expression,

only takes about ten minutes to occur (Green, 2005). With this short time frame, the majority of

20
apoptosis occurs after BAX has released cytochrome c into the cytosol, and therefore the shorter

dosing time would not affect the BAX expression.

The research conducted in this study only contributes to the present knowledge on how

chemicals isolated from cigarette smoke can have detrimental effects on the health of organisms

that are exposed. Eventually, in continuation of research, the goal is to understand how these

isolated chemicals affect humans. One way to clarify doubt in a future experiment could be to

study necrosis in cells after being dosed with phenol. If apoptosis is not the pathway the cells are

taking, then it could be necrosis that is occurring. The number of assays to help study necrosis in

cells are limited. One way to do so is to measure lactate dehydrogenase. Lactate dehydrogenase

is an enzyme that leaks from cells after necrosis takes place (Chan et al., 2013). Another further

experiment that could be performed is using concentrations of phenol lower than 0.12 mM. If the

concentrations are too high for the normal cells, then they might take a different pathway instead

of apoptosis. Less concentrated dosages may be more conducive to inducing apoptosis in the

cells. More research can be done to determine how much phenol is too much for the cell to have

apoptotic tendencies. In the continuation of research pertaining to phenol induced cell death, the

hope is to more completely understand the harmful cellular effects of toxins found in cigarette

smoke.

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References

20X LumiGLO Reagent and 20X Peroxide #7003 (n.d.). Retrieved November 22, 2015 from

Cell Signaling Technology: http://www.cellsignal.com/products/wb-ip-reagents/20x-

lumiglo-reagent-and-20x-peroxide/7003

Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts, Walter (2014). The Cell Division

Cycle. In Essential Cell Biology (pp 603-644). New York, NY: Garland Science, Taylor

& Francis Group, LLC.

American Lung Association (2015). Health Effects of Secondhand Smoke. Retrieved

from http://www.lung.org/stop-smoking/smoking-facts/health-effects-of-secondhand-

smoke.html

Centers for Disease Control and Prevention. (2015). Cigarette Smoking in the United

States. Retrieved from

http://www.cdc.gov/tobacco/campaign/tips/resources/data/cigarette-smoking-in-united-

states.html

Centers for Disease Control and Prevention. Chemicals in Tobacco Smoke. (2011).

Retrieved from

22
 http://www.cdc.gov/tobacco/data_statistics/sgr/2010/consumer_booklet/chemicals_smok

e/

Chan, F. K.-M., Moriwaki, K., & De Rosa, M. J. (2013). Detection of Necrosis by Release of

Lactate Dehydrogenase (LDH) Activity. Methods in Molecular Biology, 979, 6570. Doi:

10.1007/978-1-62703-290-2_7

Edlich, F., Banerjee, S., Suzuki, M., Cleland, M.M., Arnoult, D., Wang, C., Neutzner, A.,

Tjandra, N., & Youle, R.J. (2011). Bcl-xL Retrotranslocates Bax from the Mitochondria

into the Cytosol. Cell, 145(1), 104-116. Retrieved from www.cell.com/abstract/S0092-

8674(11)00186-3.

Elmore, S. (2007). Apoptosis: A Review of Programmed Cell Death. Toxicologic Pathology,

35(4), 495516. http://doi.org/10.1080/01926230701320337

Green, D. (2005). Apoptotic Pathways: Ten Minutes to Dead. Cell, 121(5), 671-674.

Jamal, A., Agaku, I., O'Connor, E., King, B., Kenemer, J., & Neff, L. (2014). Current Cigarette

Smoking Among Adults United States, 20052013. Morbidity and Mortality Weekly

Report, 63(47), 1108-1112. Retrieved November 18, 2015, from

http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6347a4.htm?s_cid=mm6347a4_e

MLg [Mlg 2908] (ATCC CCL-206). (2014). Retrieved from

http://www.atcc.org/Products/All/CCL-206.aspx

Monfared, A. L., Tootian, Z., Fazelipour, S., Sheibani, M.T. (2013) "Morphological And

Structural Changes Of The Placenta In Mice Exposed To Phenol." Comparative Clinical

Pathology 22(6), 1219-1224.

Reed, J. C. (2000). Mechanisms of Apoptosis. The American Journal of Pathology, 157(5),

14151430.

23
Stockdale, M., & Selwyn, M. (1971). Influence of Ring Substituents on the Action of Phenols on

Some Dehydrogenases, Phosphokinases, and the Soluble ATPase from Mitochondria.

European Journal of Biochemistry, 21, 416-423.

Tsygankova, I. G. & Zhenodarova, S. M. (2008). Structure-Property Relationship for Description

of Apoptosis (Programmed Cell Death) Induction by Phenol Derivatives. Russian

Journal of General Chemistry 78(9), 1760-1766.

U.S. Department of Health and Human Services. The Health Consequences of Smoking: 50 Years

of Progress. A Report of the Surgeon General. Atlanta, GA: U.S. Department of Health

and Human Services, Centers for Disease Control and Prevention, National Center for

Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, 2014.

Printed with corrections, January 2014.

Vo, D. D., Gautier, F., Barill-Nion, S., Juin, P., Levoin, N., & Gre, R. (2014). Design, synthesis

and biological evaluation of new inhibitors of Bax/Bcl-xL interaction in cancer cells.

Bioorganic & Medicinal Chemistry Letters, 24(7), 1758-1761.

doi:10.1016/j.bmcl.2014.02.035

Zeiss, C. (2003). The Apoptosis-Necrosis Continuum: Insights from Genetically Altered Mice.

Veterinary Pathology, 481-495.

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