1. Cigarettes vs. E-cigarettes – the acute effects | Paige Heath
A COMPARISON OF THE
EFFECTS OF E-
CIGARETTE VAPOUR AND
CIGARETTE SMOKE ON
THE VIABILITY OF HELA
CELLS
CIGARETTES VS. E-CIGARETTES – THE ACUTE
EFFECTS
Paige Heath, 2016
BSc Biomedical Science
Leeds Beckett University
A thesis submitted in partial fulfilment of the requirements of Leeds Beckett
University for the degree BSc (Hons) in Biomedical Science with Human Biology
2. Leeds Beckett University.
Faculty of Health
Health Sciences
Research Project Module,
Statement of authenticity
I, Paige Heath, declare that this project is my own original work
(except where referenced) and does not infringe copyright.
Signed………………………………………………..
Date…………………………………………………..
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Acknowledgements
I would firstly like to thank my tutor John Skamarauskas and the lab technicians at Leeds Beckett
University, for their continued support throughout the months that have been dedicated to this
research. I would also like to thank my parents, family and friends for their constant interest in
my work and supporting me throughout.
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Abstract
The use of electronic cigarettes (e-cigarettes) has rapidly increased since their introduction to
the UK in 2008. They are now the most common method used in the aid of smoking cessation,
with the product being viewed as a safer option than mainstream cigarettes by many. However,
regulations surrounding the production of the liquids used in e-cigarettes are not currently in
place, leaving the safety of the devices questionable - both to the user and surrounding people
that may be subjected to inhalation of the product. I exposed HeLa cells to a variety of
concentrations of vapours collected from an electronic cigarette using a mechanical smoking
system designed specifically for the study. HeLa cells were also exposed to a variety of
concentrations of cigarette smoke, collected in the same way, and different concentrations of
pure nicotine to compare the results on cell viability for each variable. The extracted
condensates were applied to a monolayer of cultured HeLa cells by contaminating with fresh
media and carrying out a standard media change. Cells were exposed for 24 hours in a 96-well,
flat bottomed, culture plate and incubated at 37°C, 5% CO2. An MTT viability assay was carried
out after this time period. The viability of cells exposed to mainstream cigarette smoke was
found to be lower than that of cells exposed to e-cigarette vapour but higher than the viability
of cells exposed to pure nicotine. Many factors can effect this; the following paper will discuss
these factors and what the results can suggest about the effects of e-cigarettes in the real world.
6. PAGE | 1
Keywords:
Electronic Cigarette; Smoking simulation system; viability; passaging; MTT assay; nicotine.
1. Introduction
The research carried out in this study aims to compare cigarette smoke and e-cigarette vapours
on a quantitative level, based solely on their effects on cell survival. Health risks associated with
e-cigarettes are currently slightly ambiguous which can prove worrying for the general public –
this emphasises the need for further research in this area and shows just how useful this data
could be. It can provide results focusing on the acute effects of side stream smoke, that allow a
determination as to whether a transition from cigarettes to e-cigarettes is a beneficial one.
The prevalence of cigarette smoking in England is now (Nov 2015) at around a sixth of the UK
adult population, compared with a value of around 25% in January 2008. (Action on Smoking
and Health, Nov 2015) Smoking holds a massive strain on the National Health Service – In
2013/14 the cost of the net ingredients used in the prescription of items aiding smoking
cessation was almost £48.8 million. There was also over 1.6million hospital admissions where
primary diagnosis of diseases directly related to smoking were made. (Eastwood, P., 2013)The
figure below from March 2015 shows how much of a positive impact that e-cigarettes could have
in aiding the revival of the NHS if proven to be as beneficial as they are currently thought to be
in comparison to cigarettes. In this figure it can be seen that the use of e-cigarettes in quit
attempts has taken over Nicotine Replacement Therapy (NRT) and ‘NHS stop smoking service’
with a massive 30.1% compared with 11% and 2% respectively. (Fig 1. (West, R. and Brown, J.,
2012)). Such a statistic suggests that quitting smoking with the use of e-cigarettes is a method
preferred by many and could also be suggested as the most effective due to this. However, the
confirmation that e-cigarettes are safer than cigarettes in all aspects has still not been provided.
It could also be said that such research is reluctant to be carried out due to the growing
popularity of e-cigarettes across the globe, ‘It’s a booming, billion-dollar industry -- on track to
outsell tobacco products within a decade.’ (Griffin, 2016)
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Figure 1. ‘Support used in quit attempts’ (West, R. and Brown, J., 2012)
One of the biggest dangers of smoking that is often forgotten about, is the danger associated
with the smoke or vapour as a concern for surrounding people – especially children – that are
subjected to inhalation of the substances on a secondary basis. Since 1964, around 2.5 million
people have been killed as a result of second hand smoke. (US Department of Health and
Human Services, 2014.)
Tobacco smoke contains over 4000 chemicals, some of which have marked irritant properties
and over fifty are known to be carcinogenic (US Department of Health and Human Services,
2006.) These chemicals are known to have a fairly obvious acute effect on cells – the presence of
carbon monoxide in tobacco smoke leads to a decreased function of the alveoli, leading to a
decreased amount of oxygen available in the blood and therefore the chance of premature cell
death due the lack of oxygen needed for respiration. (Abpischools.org.uk) ‘Studies show that
nicotine, present in tobacco smoke is the most potent stimulant and addictive agent in the
world’. It is already common knowledge that increased blood pressure and heart rate are results
0%
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support
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8. Cigarettes vs. E-cigarettes – the acute effects | Paige Heath
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of the presence of nicotine, however this study will provide an insight to the effects of nicotine
on a cellular level and also a suggestion as to whether it is simply the presence of nicotine
which results in an effect or if it is due to the combination of nicotine with other harmful
substances in the cigarette/e-cigarette. (US Department of Health and Human Services, 2010.)
The harmful effects of toxicants found in cigarette smoke are quite well documented which has
led to restrictions put in place such as no smoking in public places in 2007, and the recently
established law which states the smoking of a cigarette in a car containing anybody under the
age of 18 became illegal as of 1st October 2015. These laws are based upon facts such as the
recent statement that passive smoking kills up to 11,000 people a year in the UK. (Moore et al.
2015) There are currently no laws in place that prohibit the use of electronic cigarettes in public
places. (Gov.uk, 2015) However there has been little research conducted regarding the health-
risks associated with vapour from e-cigarettes. With an estimated 2.6 million e-cigarette users
in Great Britain as of March 2015, better documentation of the health risks associated with the
use of e-cigarettes is becoming vital. (Action on Smoking and Health, May 2015) Although e-
cigarette vapour is thought to be less toxic than cigarette smoke, e-cigarette vapour ‘increase(s)
the exposure of non-smokers and bystanders to nicotine and a number of toxicants’ (World
Health Organization, 2014.) The research I intend to carry out is proposed to extend the current
research based knowledge on the acute effects of e-cigarette vapour.
A lack in knowledge proves to be one 0f the main issues associated with the use of e-cigarettes
– there is currently little known about the liquid component of the e-cigarette, specifically
when inhaled. This recently been questioned due to the common use of food additives and
flavourings in the electronic cigarette liquids. It is currently known that the flavourings used in
e-cigarettes are perfectly safe for consumption in food, however when inhaled the agents could
prove to have a very different effect and be harmful to users. The first case where this was
recognised was with the use of the flavouring agent Diacetyl, which is used in popcorn to give
the buttery taste, was found to be the cause of a respiratory system now known as ‘popcorn
lung’. This was characterised by irreversible scarring of the alveoli. (Kovacic, P. and Cooksy,
A.L., 2010) Further research into the flavouring agent showed that diacetyl was found in over
75% of e-cigarette liquids tested by researchers in a study. (Harvard Gazette, 2015)
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The study will look directly into the effect of different concentrations of extracted cigarette
smoke condensate, extracted e-cigarette vapour condensate and nicotine dilutions and how the
application of each variable directly effects the viability of cells, allowing a determination of the
toxicity of each variable. It will also discuss other ways in which the effects of each variable on
cells can be analysed and how these results could further the results already being discussed.
In order to provide a quantitative result, an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide), assay will be used to provide a cell viability value in the form of
absorbance. (Riss, T.L., 2004) The MTT assay is based on redox potential and uses a dye called
formazan – the quantity of which is measured using an absorbance reading at 570nm and is
directly proportional to the number of viable cells. (Johnson, M.D., Schilz, J., Djordjevic, M.V.,
Rice, J.R. and Shields, P.G., 2009) Viable cells are able to actively metabolise MTT into
formazan which after an incubation period of 1-4 hours appears a deep purple colour. Cells that
have died, or in this case been killed due to toxicity, are unable to metabolise MTT and this
therefore results in a reduced purple colour and a lower absorbance reading. The formazan is
produced as a precipitate on the surface of the cells and therefore must be solubilised in order
for the colour to show. In this study isopropanol will be used as a solubilising agent as it is
convenient and requires a short incubation time of just 1 hour.
2. Methodology
2.1 Cigarette and E-Cigarette vapour collection
For the purpose of this study, e-cigarette liquids from VIP Electronic Cigarette were used with a
standard ‘ego style’ Electronic Cigarette. The chosen flavour was USA Kentucky Tobacco, which
was also purchased from the VIP Electronic Cigarettes, in order to reduce the number of
variables when comparing with mainstream cigarettes. The vapours were collected from a
liquid with 0% nicotine. VIP is quality approved company – they use UKAS accredited
laboratories to detect harmful compounds including Ethylene glycol, Diethylene glycol,
Diacetyl, Acetoin and Acetyl propionyl. None of which were detected in the liquid used in this
10. Cigarettes vs. E-cigarettes – the acute effects | Paige Heath
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study. (Vipelectroniccigarette.co.uk, 2015) The cigarettes used were Lambert and Butler
original which were purchased from Morrisons supermarket.
In order to collect condensate from the vapours of the e-cigarette and smoke of the cigarettes, a
mechanical smoking system was set up. This had been designed in a pilot study that was carried
out prior to this research. The system consisted of a quick fit 100ml V condenser flask
submerged in a salt water and ethanol mix at a temperature of between -15°C and -5 °C.
Connected to each exit of the flask were silicone tubes to allow transportation of smoke and
vapours produced and a small KNF LABOPORT® vacuum pump used to demonstrate the act of
inhalation. (Fig 2.) Note that in this specific study, the induced air flow rate of the pump was
not recorded with a flow meter.
The quick fit 100ml V condenser flasks were weighed before condensate collection to allow a
calculation of the amount collected. For collection of the e-cigarette condensate, the pump was
used on full power for 15 minutes. The e-cigarette was connected to one tube leading to the
submerged quick fit 100ml V condenser flask, and another tube going from the flask to the
pump allowing the vacuum pump to pull the vapours from the e-cigarette through the
condenser flask demonstrating the act of inhalation. The low temperature created by the
beaker with a salt water – ethanol mixture allows the vapours ‘inhaled’ from the e-cigarette to
condense in the condenser flask for use in dilutions at a later stage in the study. The
temperature of the water-bath was maintained at an average of -7°C/-8°C throughout. A total of
0.79g of condensate was collected in the quick fit 100ml V condenser flask. The flask was then
washed with 50ml Phosphate Buffered Saline (PBS) – this was used to form the start of the
serial dilution. PBS was made up of 160g NaCl, 4.0g KCl, 28.8g Na2HPO4 ad 4.8g KH2PO4. This
produces a 10x solution which was diluted to 1x before use. This composition is the same for all
solutions of PBS used throughout. All of the above was carried out inside a MACH-AIRE
CRITERION® fume cupboard for health and safety reasons.
In the case of the collection of cigarette smoke condensate, a total of 10 cigarettes were burnt
using the mechanical smoking system until the cigarette was completely burnt out – in this
case each cigarette took an average of 2 minutes and 40 seconds. The filter end of the cigarette
was placed into the rubber tubing connected to the condenser flask. The opposite end of the
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cigarette was lit with a candle lighter and the induced air flow pump was turned on to simulate
the act of inhalation. A trial run was carried out and it was discovered that the induced air flow
pump must be used at a reduced rate due to the quick burning of the cigarette making the trial
unrealistic. The burning of 10 cigarettes resulted in the collection of 3.65g of condensate from
the cigarettes. The condenser flask was then washed with 50ml PBS and transferred to a falcon
tube – this was used to form the start of the serial dilution and served as the 10-1 concentration.
Figure 2. Set up of smoking simulation system
2.2 Cell Culture
HeLa cells were cultured in VWR® 25ml tissue culture flasks from a stock of cell lines available
in the lab with 5ml of complete media containing Dulbecco’s Modified Eagle Medium
(DMEM), 10% fetal bovine serum, 1% non-essential amino acids and 2Mm Glutamine. From
here cells had continuous media changes and passaging until a sufficient amount of healthy
cells were available for exposure to extracted condensates.
Cells were observed under an inverted microscope to check for confluency. A simple media
change was carried out every 1-2 days in order to ensure cells had plentiful nutrients (available
Pear shaped flask
Silicone tubing
Salt-water ice bath
Vacuum pump
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in the complete medium) to grow and multiply continuously. In order to carry out a simple
media change, the appropriate amount of media for the number of tissue culture flasks is
heated in a water bath at 37°C for 5 minutes, with enough for 5ml of fresh media in each t-25
flask or 15ml for a t-75 flask. This is to ensure that the application of cold media does not shock
the cells and kill them. T-75 flasks were used when t-25 flasks became confluent as one t-75
flask can provide enough cells for a 96 well plate. Flasks containing the first culture of HeLa
cells are removed from incubation tank ensuring aseptic technique is used at all times, this
included techniques such as spraying gloves and anything else that enters the cell culture hood
(the hood used in this study was ESCO® AirstreamMAX Class 2 Biosafety cabinet) with a light
mist of 70% ethanol, and ensuring every step of culturing is carried out under a cell culture
hood. Media change requires removal of cell culture medium with a pipette, followed by
washing of the monolayer culture with 5Ml of pre heated 1x PBS (also heated for 5 minutes at
37°C in a water bath). Fresh, pre-heated media is added to the flask, volume of 5ml, using a 10ml
pipette and the flasks are then incubated in a Forma ™ Scientific CO2 incubator at 37°c and 5%
CO2 to increase enzyme activity and optimise cell growth.
Passaging takes place once a confluent monolayer (70-80%) of cells can be observed under an
inverted microscope. The process involves the same steps of removing media and washing of
cell monolayer, followed by the addition of 1ml 0.25% trypsin-EDTA solution which has been
heated in a water bath at 37°C to allow ‘lifting’ of cells from the flask. The culture flask
containing trypsin-EDTA is incubated for 5 minutes in the CO2 incubator to allow this process
to occur efficiently. The flasks are then observed under an inverted microscope to observe the
lifting of the cells where they appear to move around if the flask is tapped gently. This is
opposed to them being firmly attached to the bottom of the flask with no movement before
trypsinisation. 5ml of complete media is then added to deactivate the trypsin ensuring no
further break down of the cells occurs and all of the media (containing the cells) is removed
into a 50ml falcon tube using a 10ml pipette. Cells are counted using a haemocytometer and the
media is diluted into 2 new tissue culture flasks to give a concentration of 10-100 thousand cells
per ml. This process is repeated until enough flasks of cells are available for the study. In the
case of this study, two costar® 96-well cell culture cluster plates (flat bottomed with lid) were
needed which means 2-3 t-75 flask are required with a confluent culture of HeLa cells. The cells
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are then passaged using the same technique as above but this time transferred the media
containing the cells into 96 well, flat-bottomed, culture plates to allow for ease of exposure to
extracted condensate at different concentrations. Each well contains 100µl of complete media.
2.3 Exposure of condensate to cells
A 1 in 10 serial dilution is carried out to dilute the extracted cigarette smoke condensate,
extracted e-cigarette vapour condensate and nicotine. The collection of cigarette condensate
resulted in a yield of 3.654g in the flask which when washed with 50ml PBS provided a cigarette
smoke condensate with a concentration of 0.731mg/ml. When looking at the e-cigarette
condensate that had been collected, the total yield was 0.207g, which when also washed in 50ml
PBS provided a condensate with a concentration of 0.0415mg/ml. The initial concentration of
the pure nicotine was 990mg/ml. Serial dilutions were carried out in microfuge tubes with
0.45ml PBS in each tube. 50µl of the original condensate was added to the 10-2 microfuge tube
for each variable and this was repeated to 10-6 ensuring the solution was mixed well after each
step. Once cell containing media has been passaged into 96-well plates and left for 24 hours
after a fresh media change, another media change is carried out in the 96- well plates. This
involves complete removal of media in all wells, after which each well is filled with 90µl of
media and 10µl of the appropriately diluted variable. The different concentrations of the
different condensates are added to a full column of well which are pre-labelled A-H. It can be
advised to not use wells A and H as experimental wells, therefore providing 6 replicates of each
dilution in wells B-G which can later provide an average result and therefore increases
reliability.
Cells exposed to the condensate were incubated for 24 hours at 37°C, 5% CO2.
2.4 Analysis of cell viability.
In order to evaluate the effects of each variable on cell viability, an assay using Thiazolyl Blue
Tetrazolium Blue (MTT) was performed. MTT reagent was prepared according to protocol,
preparing a 12Mm stock of MTT by adding 1ml PBS to a 5mg vile of MTT. After cells had been
14. Cigarettes vs. E-cigarettes – the acute effects | Paige Heath
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incubated with media and condensate, 10µl of MTT reagent was added to each well. The cells
were then incubated for 4 hours at 37°C, 5% CO2. After incubation, all media including MTT
reagent was removed and discarded and the cells in each well were washed with 50µl PBS. PBS
was then removed and 50µl 2-propanol (isopropanol) was added to each well and left at room
temperature for a further 2 hours. The isopropanol acts as a solubilisation solution, reducing
the MTT and giving the visibly purple colour. Plates were then inserted to a Thermo
Varioskan™ LUX multimode microplate reader and absorbance was read at 550n using SkanIt™
software. (Riss et al. 2004)
2.5 Statistical tests
Unpaired t-tests were used throughout with statistical significance being portrayed with * and #
that resemble different values in each figure.
3. Results
A variety of concentrations of each variable were added to HeLa cells and viability was recorded
as a % of the control variable (DMEM only) when looking at absorbance readings at 550nm. In
order to compare the effects of cigarette smoke condensate, e-cigarette vapour condensate and
nicotine with positive and negative controls, a graph was constructed which looked at the
concentration of each variable that were most similar to each other. Although this does not
provide a conclusive result due to the differences in concentration, the comparison represents
the truest conclusion that can be drawn with the data produced.
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Figure 3. The effects of extracted cigarette smoke condensate concentration (diluted in PBS) on
HeLa cell viability as a % of the control variable (DMEM alone). Results were provided that when
using an unpaired t-test, show that the concentration values marked ‘#’ are similar to each other
with p < 0.02. A second test compared all values to a cigarette smoke condensate of 0.0731mg/ml
where statistical significance was defined by: * p<0.1; ** p<0.5; ***p>0.5.
As the concentration of extracted cigarette smoke condensate that was applied to the HeLa
cells changes, overall there appears to be very little change in cell viability between 1x10-
6
mg/mL and 1x10-1
mg/mL but at concentrations higher than this there appears to be a
significant decrease in cell viability with the probability of 0.0196 that marked values ‘#’ are
similar. This suggests that condensate of concentrations over this value are particularly toxic to
HeLa cells and that here a difference in concentration provides a change in viability that is
significant. Using a t-test also allows statistical evidence that there is little change in viability
between 1x10-6mg/mL and 1x10-1mg/mL due to the probability of values being different >0.5
which suggests that the difference in concentration between these two values is insignificant to
the viability of HeLa cells.
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Figure 4. The effects of extracted e-cigarette vapour condensate concentration (diluted in PBS)
on HeLa cell viability as a % of the control variable (DMEM alone). An unpaired t-test was carried
out comparing all results to the viability of HeLa cells when the above condensate of
concentration 0.0415mg/ml was applied where, in this figure only, statistical significance is
defined by: # p < 0.01; ## p < 0.1; ### p < 0.3
Here a clearer pattern can be seen with an increase in extracted e-cigarette condensate
concentration generally following a trend of reduced cell viability. The use of an unpaired t-test
shows that for concentrations below 1x10-4mg/ml, a reduction in concentration is statistically
significant to an increase in cell viability. This is compared with concentrations above 1x10-
4mg/ml where there seems to be little change in viability and a probability >0.2 suggests that
between these values, a change in concentration is statistically insignificant to HeLa cell
viability.
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Figure 5. The effect of nicotine concentration (diluted in PBS) on HeLa cell viability as a % of the
control variable (DMEM only). An unpaired t-test was carried out which compared all results to
the viability of HeLa cells when nicotine of concentration 990mg/ml was applied where, in this
figure only, statistical significance was defined by: # =p < 0.01; ##= p > 0.5
When looking at the concentration of nicotine it can be seen that a difference in concentrations
between 0.01mg/ml and 1mg/ml has little effect of the viability of HeLa cells. However, it can be
seen that after this point an increase in concentration leads to a visible decrease in cell viability
with a probability <0.01 which suggests that nicotine concentrations higher than 1mg/ml have
an effect on HeLa cell viability that is statistically significant. Although a concentration of
100mg/ml provides a HeLa cell viability that is seen to follow the trend observed above, there is
a probability >0.5 that this value are similar to those seen when a concentration of 990mg/ml
was used. This suggests that between a nicotine concentration of 100mg/ml and 1000mg/ml
there is little change in the effect on HeLa cell viability and there is no significant difference
between the two variances in concentration.
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Figure 6 - A comparison of cell viability for each variable using the concentration in mg/mL that
are most similar to each other. Here the most similar concentrations of e-cigarette vapour
condensate and cigarette smoke (10-4 dilution and 10-5 dilution respectively) condensate are
compared with a 10-6
dilution of pure nicotine, and also compared with complete media and
distilled water. An unpaired t-test was carried out where an asterisk provides a statistical
significance value when compared to cigarette smoke condensate and the use of a hash mark
provides a statistical significance when values are compared with e-cigarette vapour condensate.
For figure 6 these values are defined as: #,* p<0.01; ##,** p<0.025; ###,*** p<0.05; ####,****
p<0.25.
When looking at all variables together, it can be seen that extracted cigarette smoke
condensate leads to a lower cell viability when applied to HeLa cells compared with the
application of e-cigarette vapour condensate on average. However when looking at results of an
unpaired t-test, the results of the two tests are similar with a p=0.208. This suggests that
although cells exposed to extracted e-cigarette vapour generally showed higher viability, the
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result cannot be conclusive as the difference between the values is not significant as p is not
<0.05 and therefore there is a more than 1% probability that the results are due to chance.
Figure 6 also shows that the application of nicotine to HeLa cells reduces viability to a lower %
of the control value than both cigarette smoke condensate and e-cigarette vapour condensate.
When compared to cigarettes, the effect of nicotine had little statistical significance, with a p
value of 0.205, suggesting the effect on viability between the two variables is insignificant.
However, when comparing nicotine to the effect of extracted e-cigarette vapour condensate on
HeLa cells, the two variables were similar with a p = 0.022 which suggests that the difference in
viability of HeLa cells exposed to nicotine and e-cigarette vapour condensate separately is
statistically significant and that the difference between variables has a <1% probability of being
due to chance.
When comparing the negative control (distilled water) to both cigarette smoke condensate and
e-cigarette vapour condensate, a significant difference can be seen with both variables where
p=0.004 and p=0.001 respectively. The graph shows the addition of distilled water only to HeLa
cells to provide the lowest cell viability when compared to all other variables.
4. Discussion
Although the results discussed above provide a basis upon which conclusions can be made
there are many aspects to the study that reduce its validity in stating that extracted cigarette
smoke has a more toxic effect on HeLa cells then extracted e-cigarette vapour.
The methods used in the study provided the development of a simple system that allowed the
collection of condensate from both cigarettes and e-cigarettes very easily and quickly. The
system was cheap to run and used basic equipment that can be found easily in university
science lab. However the simplicity of the system resulted in some variables being very difficult
to control. This included things such as maintaining a constant temperature in the water bath
and the ability to reproduce this exact temperature again when repeating the study with the
different variables.
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Another variable that was difficult to control given the simplicity of the system was the induced
air flow rate of the pump that was used to simulate inhalation of the cigarette and e-cigarette. It
was found that when the vacuum pump was used with its maximum flow rate, cigarettes burnt
out completely within 5-10 seconds which was very unrealistic. This meant that the pipe
attaching the vacuum pump to the rubber tubing (as seen in figure 2) had to be slightly
restricted with a clamp in order to slow down the burning process of the cigarette. Because of
this, the induced flow rate used during the ‘inhalation’ of the cigarettes was different to that of
e-cigarettes and this is something that I would focus on controlling in further studies by
introducing apparatus that allows the measurement of induced flow rate produced by the
vacuum pump such as a flow meter.
This difficulty in the ‘inhalation’ process made the prediction and measurement of the mass of
condensate collected from each variable difficult as there was a large range in the yield of
condensate from each cigarette. It was found that in the same amount of time, cigarettes
produced a much larger mass of condensate compared with e-cigarettes. This meant that when
the flasks used to collect condensates from both variables were washed with PBS, the
concentration of the cigarette smoke condensate was over 17 times more concentrated than
that of the e-cigarette vapour condensate. Timing restrictions meant that condensates could
not be re-collected and it was therefore necessary to perform a serial dilution for each variable
and use the most similar concentrations for comparison purposes. This again provides a
problem for the reproducibility of the study and reduces the validity of conclusions made.
In order to further this study I would repeat the application process but ensure that the
condensates that the cells are being exposed to are of equal concentrations for both variables
and also of equal concentration to the nicotine that was applied to the HeLa cells.
It is also possible that the method used to collect e-cigarette vapours was not similar to that of a
typical ‘vaper’. The e-cigarette liquid was burnt for 10 minutes under constant suction from the
vacuum pump. This could affect the results as the levels of pyrolysis within the e-cigarette are
at a very high level due to the constant burning compared to the burning of liquid in short puffs
when used by the public. In order to make this process more accurate I would carry out further
works in which the average ‘puff length’ for an e-cigarette user is discovered and use this to
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create a more realistic system. It should also be noted that the condensates that were applied to
the cells were of a diluted form (cigarette condensate was diluted to 10-5 and e-cigarette
condensate was diluted to 10-4) which could suggest that the toxicity of the substances from
both variables could be increased in a real life situation due to the inhalation of a more
concentrated product than is discussed in this study.
In order to further this study I would look into the possibility of selecting a specific component
found in both e-cigarette vapour and cigarette smoke and base the concentrations of each
variable applied to cells on the specific concentration of that component to provide a more
specific conclusion when comparing . This could provide results with a conclusion that is more
detailed and also show which components of the smoke and/or vapour have the most toxic
effect. This is similar to the comparison of the effects of pure nicotine when compared with the
e-cigarette vapour condensate and the cigarette smoke condensate where a statistically
significant difference can be seen between nicotine and e-cigarette vapour but not between
nicotine and cigarette smoke. This is likely due to the 0% nicotine content of the e-liquid used
in the study when compared to 0.9mg of nicotine found in one lambert and butler cigarette
that was used. (Statistic available on packaging of cigarette) In order to investigate the
significance of nicotine presence further I would repeat the same study but also including an e-
liquid with higher concentrations of nicotine and if possible expose cells to e-liquid vapour
condensate and to cigarette smoke condensate and ensure that each variable contains the same
concentration of nicotine. Other studies that have looked into the effect of nicotine levels on
cells have provided results which suggest the application to endothelial cells of nicotine
containing e-liquid, results in cellular changes that suggest an increase in stress levels when
compared to the application of nicotine free e-liquid. (Lerner et al. 2015)It could be suggested
that to continue this study, further tests be carried out that use a variety of intercellular assays
to look for the different effects on cells such as oxidative stress levels. This is something that
would have been completed in this study if more time was available.
The use of different assays would provide a more reliable result as there are several assays
available for cell viability. In the early stages of this study it was the aim that both an MTT assay
and a trypan blue assay be carried out to provide a more reliable value for cell viability, however
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timing restriction meant that this could not be done. MTT was chosen due to the cheap cost
and also due to the colour of the dye being of an advantage when used in tobacco smoke
studies. (Johnson et al. 2009)
In terms of cell culture, problems were encountered with obtaining cell lines which led to the
use of HeLa cells for this study opposed to epithelial cells which would have provided a more
accurate representation of the cells that are exposed to these vapours in the human body. The
use of HeLa cells made the cell culture process slightly easier for a first time cell culture
scientist due to their durability and ability to survive and resist apoptosis. (AccessScience
Editors, 2014) In order to provide a more accurate basis upon which conclusions can be made, I
would use bronchial endothelial cells in a further study allowing results to be more relevant to
the general public.
Looking at results in figure 3, it can be said that above concentrations of 0.1mg/ml, an increase
in cigarette smoke condensate is proportional to a decrease in cell viability and therefore
suggesting an increase in cytotoxicity as concentration increases. Another study carried out
involving the exposure of Human lung epithelial cells to commercial filtered cigarette smoke,
used an MTT assay to show that cytotoxicity was dependent upon dosage and exposure time.
(Das, A., Bhattacharya, A. and Chakrabarti, G., 2009.) This supports the conclusion that a
higher concentrations of cigarette smoke has a higher toxic effect on a variety of cells, not just
HeLa. It also suggests that exposure time can also lead to a change in toxicity and therefore
provides an area for this study to be furthered, investigating the exposure of cells for different
amounts of time and comparing the results.
When comparing this with the effects of e-cigarette vapour concentration, a much clearer trend
is seen with an increase in concentration leading to a reduction in cell viability. This suggests
that concentration is directly proportional to toxicity when applied to HeLa cells. It is also
important to note that the e-liquids used in this study were purchased from a company that
ensure several harmful compounds are not present in their liquids, as mentioned in the
methods section. It would therefore be possible that other liquids bought from cheaper high
street stores could have a different components and therefore have different effect on cell
viability. In further works I would look into a variety of e-liquid brands and compare their
23. Cigarettes vs. E-cigarettes – the acute effects | Paige Heath
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effects on epithelial cell viability whilst also comparing the difference in ingredients between
brands.
5. Conclusion
The data produced in this study suggests that when comparing e-cigarettes to cigarettes in
terms of their effect on cell viability, cigarette smoke condensate was seen to be more toxic to
HeLa cells than the condensate of extracted e-cigarette vapour. However, when looking at the
reliability and reproducibility of results, problems with concentration control and collection of
condensate mean the results are somewhat inconclusive. It can be seen that, other than DMEM
alone, e-cigarette vapour condensate led to the highest % HeLa cell viability, when compared to
other variables, the viability of HeLa cells exposed to cigarette smoke was 13.99% less. Whilst
this is a substantial difference, statistical values showed that in this study, the difference
between cigarette smoke condensate and e-cigarette smoke condensate was insignificant.
It must however be remembered that the concentration of each variable added to the HeLa
cells were not identical and the concentration of cigarette condensate was almost twice the
concentration of the e-cigarette condensate that was shown in figure 6.
Nicotine showed to be the variable that resulted in the lowest HeLa cell viability, excluding the
negative control distilled water, with statistics that show a higher degree of significance when
compared with the other two variables than when comparing cigarette condensate and e-
cigarette condensate with each other. This can lead to the conclusion that it is clearly visible
that high concentrations of nicotine are toxic to HeLa cells and this could be a main factor
when comparing cigarettes with e-cigarettes. The e-liquid used in this study contained 0mg/ml
of nicotine but many liquids on the market contain concentrations of up to 24mg/ml compared
with the 0.9mg of nicotine found in a standard lambert and butler cigarette. It could therefore
be suggested that the exposure of cells to nicotine containing e-cigarette condensate would
lead to lower viability than when compared with nicotine free e-cigarette condensate. From the
results in this study it can therefore be suggested that whilst e-cigarettes may show a lower
toxicity to cells when compared with cigarettes, an increase in nicotine levels in the e-liquids is
proportional to the toxicity of its vapours on human cells.
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