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  • Published on: 15th October 2019
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Introduction

Background information

According to the 2014 Surgeon General’s Report, an estimated more than 480,000 deaths per year in the United States are caused my smoking. Nonetheless, this number may be an underestimate because it qualifies only 21 diseases as caused by smoking, so there may be others, not formally established, causing these deaths. (Carter et al., 2015)

Nicotine found in cigarette and in e-cigarettes is proven to have a range of effects following the exposure to it. Some effects that follow acute exposure to nicotine from smoking tobacco include an increased heart rate, elevated blood pressure, and constricted blood vessels within the skin. Some even more serious effects include lung cancer, cardiovascular disease, diabetes, and pneumonia including influenza. (Carter et al., 2015) Altogether, the long-term exposure to smoking tobacco include chronic pulmonary disease, cardiovascular disease, and malignancies of the lungs and upper airways and other cancers. (Luttrell and Vogel, 2014, 39)

Researchers have been studying the impact of cigarettes on our health over the years and many social campaigns and advertisements have been in use in order to decrease the number of smokers.

   Over the years, cigarette smoking decreased, while the use of e-cigarettes has been significantly rising among teenagers and adults. It has been shown that e-liquids are more dangerous than tobacco because nicotine in its liquid state is absorbed more quickly even in diluted concentrations. Some effects that follow over-exposure to nicotine are nausea, abdominal pain, salivation, vomiting, dizziness, headache, and weakness. Death is also a possible result if the respiratory muscles get paralyzed. A conclusion has been made about an ingredient found in both cigarettes and e-cigarette liquid, nicotine, that it is very addictive and harmful over long term use. (Chaturvedi et al., 2015)

The organism Tetrahymena thermophila is a unicellular eukaryote that is found is temperate freshwater environment. It moves around its coat of cilia and phagocytosis food that is propelled into its gullet. Tetrahymena has played a major role in discovering insights about cilia, histone variants, properties of chromatin that are now seen to be general features of eukaryotic biology, the discovery of self-splicing RNA, and even describing avoidance behavior, and the complexity of microtubule and membrane systems. These organisms are considered to be a part of the model organism pantheon because they provide methods for strain storage, developing designer gene crosses, gene targeting to mention a few, and lack complexity of their cells, are low cost, and grow quickly. (Collins, 2012, 1)

In this specific experiment, the effects on ciliary structures in Tetrahymena are studied because they vastly depend on them to eat and move. Near the oral cavity there are cilia which are dedicated to stimulating feeding. After food particles enter through the oral cavity, they make their way to the cytostome. Next, they become enclosed in a vacuole that is bound by a membrane and are able to travel the cell. This process known as phagocytosis ends when lysosomes digest the food and the cytoproct discharges the undigested remnants. (Bozzone and Denise, 2000) Phagocytosis processes are observable thanks to phase contrast microscopy where structures like cilia and flagella will also be visible.

There have been recent studies that have tested the effects of e-cigarette liquid on the motility of Tetrahymena cilia. A study done by Graf and Wenz showed that e-cigarette smoke affected the cilia in the respiratory tract, toxifying it significantly, no matter how long the cilia were exposed to it. (1999)

Purpose

The purpose of this experiment was to determine if nicotine found in e-cigarette liquid has an effect on cilia mobility in Tetrahymena. Further, the experiment was an effective way to learn about Tetrahymena, how to perform experiments with these organisms, how to effectively use phase contrast microscopy, and how to determine correlation from a set of data and graphs.

Hypothesis

If nicotine found in e-cigarette liquid inhibits cilia mobility, then the number of visible ink-filled vacuoles will decline, meaning that the phagocytosis processes in Tetrahymena have been reduced.

METHODS

The purpose of these procedures was to determine the number of ink-filled vacuoles at four different time periods and at three different conditions. This was done by conducting an experiment that exposed Tetrahymena to e-cigarette liquid with (25 mg/ml nicotine) and without nicotine (0 mg/ml nicotine), and observing its effects on their cilia mobility.

A total of 3000 µl Tetrahymena stock was collected and then distributed equally amongst three 15 ml conical tubes, 1000 µl in each. Four timepoints were chosen: 0, 5, 10, 15 minutes and they were used for the three conditions performed in the experiment; first condition being the control, second being with e-cigarette liquid with 0 mg/ml of nicotine, and the third condition with e-cigarette liquid with 25 mg/ml of nicotine. Next, 12 1.5 ml microfuge tubes were collected and labelled accordingly to their condition, e.g., “C_0” meaning that this was the microfuge tube from the control for timepoint 0 minutes.

Keeping the 3.7% formaldehyde stock in the fume hood, 100 µl of it were pipetted into each of the 12 labeled microfuge tubes. It kills the cells and freezes them in the state they were in at the time. Following, the first condition which was the control was prepared and 1000 µl of India Ink and 25 µl of deionized water were mixed together into the first conical tube containing 1000 µl of the Tetrahymena stock and the timer was started immediately. This step was true for the second run of the experiment because in the first run 200 µl of deionized water and the e-cigarette liquids was used instead. This marked the first timepoint which was 0 minutes.

Fort that first timepoint, t=0, 200 µl were instantly withdrawn and into the first microfuge tube containing formaldehyde labelled accordingly. This was repeated for the remaining timepoints of 5, 10, and 15 minutes. While waiting for the time intervals, slides were prepared by pipetting 10 µl of the mixture from each microfuge tube onto a slide and covered with a coverslip. The slide was placed under the microscope for viewing, where the cell counting procedure was done. There 20 cells were counted and the number of ink-filled vacuoles in each was recorded in Table 1.

Further, the second condition was prepared similarly to the control, but instead of 25 µl of deionized water, there was 25 µl of the e-cigarette liquid containing 0 mg/ml of nicotine added. The timepoints stayed the same, the pipetting of 200 µl of the mixture into the respective formaldehyde-filled microfuge tubes was performed, the slides were prepared, and the cell counting procedure was done. The third condition was also conducted this way, but once again the variable being switched out was the now present 25 µl of the e-cigarette liquid containing 25 mg/ml of nicotine instead of the deionized water in the control and the e-cigarette liquid with no nicotine in the second condition. The volumes of the stock mixtures in each conical tube were made sure to be made consistent.

Calculations were performed for the data pooled from each one of the counts of 20 cells. The ratio of ink-filled vacuoles to the number of cells was calculated.

RESULTS

Table 1 Ratio of the Number of Ink-filled Vacuoles to the Number of Cells Observed during the first run of the experiment with 200 µl of e-cig liquid with and without nicotine and deionized water for control.

Condition Ratio of Number of Ink-filled Vacuoles

to the Number of Cells Observed

T=0 T=5 T=10 T=15

Control 2/20=0.1 76/20=3.8 199/20=9.95 262/20=13.1

E-cigarette liquid with 0 mg/ml of Nicotine 0 7/20=0.35 6/20=0.3 6/20=0.3

E-cigarette liquid with 25 mg/ml of Nicotine 0 1/20=0.05 0 1/20=0.05

As can be seen in Table 1, twenty randomly chosen cells were observed and the number of ink-filled vacuoles was recorded. The cells were looked at through a 60x lens on a phase contrast microscope. The numbers of vacuoles were recorded at each timepoint and each condition. In the control the number was constantly rising, ranging from 0.1, 3.8, 9.95, and 13.1. While for the condition with e-cigarette liquid with 0 mg/ml of nicotine and with 25 mg/ml of nicotine the number stayed significantly under one or remained zero.

Table 2 Ratio of the Number of Ink-filled Vacuoles to the Number of Cells Observed during the second run of the experiment with 25 µl of e-cig liquid with and without nicotine and deionized water for control.

Condition Ratio of Number of Ink-filled Vacuoles

to the Number of Cells Observed

T=0 T=5 T=10 T=15

Control 1/20=0.05 58/20=2.9 120/20=6 156/20=7.8

E-cigarette liquid with 0 mg/ml of Nicotine 0 11/20=0.55 19/20=0.95 14/20=0.7

E-cigarette liquid with 25 mg/ml of Nicotine 0 10/20=0.5 8/20=0.4 5/20=0.25

As can be seen in Table 2, the ratio calculated of the number of ink-filled vacuoles to the number of cells counted was presented. These ratios correlate with the findings from the second run of the experiment where 25 µl of the e-cigarette liquid and deionized water was used, opposed to the 200 µl used in the first run. The ratios for the control range from 0.05, 3.9, 6, 7.8 for the timepoints T=0, 5, 10, 15 minutes, respectively. For the second condition the ratios are: 0, 0.55, 0.95, 0.7, and for the third condition they are: 0, 0.5, 0.4, 0.25.

In Figure 1 we see the ratios calculated from the results of the second run of the experiment. The graph shows the steady increase of the number of ink-filled vacuoles per cell, while the remaining two conditions remain very minimal, at an average under one cell.

DISCUSSION

Conclusions

The results of this experiment clearly support the hypothesis that nicotine found in

e-cigarette liquid inhibits cilia mobility, the number of visible ink-filled vacuoles will decline, meaning that the phagocytosis processes in Tetrahymena have been reduced. The number of ink-filled vacuoles decreased significantly as shown in Table 2. Further, the graph in Figure 1 is consistent with the interpretation of the results. However, the results in Table 1 show the ratios obtained during the first run of this experiment that used 200 µl of deionized water and the e-cigarette liquids instead. The amount of this variable was significantly reduced to 25 µl in the second run because we observed instant death of the cells after they were exposed to the high volume in the first run. The cells appeared abnormal, misshaped, and some looked torn apart. The results from the first run for the condition with either e-cigarette liquid cannot be considered by us, are inconclusive, because the lack of ink-filled vacuoles may have been due to too much of the liquid and not the ingredient nicotine found in one of the liquids.

Explanation of conclusions

It was expected that the number of ink-filled vacuoles would decrease or be significantly less in the run with e-cigarette liquid with 25 mg/ml of nicotine, and this was observed in Table 2 and Figure 1 where the ratio of the number of ink-filled vacuoles to the number of cells counted was presented.

There are also many factors that may have impacted the number of ink-filled vacuoles that were observed. One of the conditions was a control that was conducted to avoid any results that could have been an effect of confounding variables. The function of phagocytosis processes is closely related to the mobility of cilia in Tetrahymena. If nicotine inhibits the cilia’s mobility as well as the cilia’s length, these processes will decline (Leopold et al., 2009, 1). According to the results, a decrease in the number of these vacuoles was due to the nicotine and even the e-cigarette liquid since it decreased the cilia’s motility, so it could be concluded that this observation is directly related to the decrease of phagocytosis processes in Tetrahymena.

The results of the earlier mentioned study and the results of this study are consistent with one another, hence there is substantiation concerning the effects on nicotine found in e-cigarette liquid on cilia.

Future experiment

This experiment should be repeated with several variations to further test the proposition that nicotine found in e-cigarette liquid inhibits cilia mobility, the number of visible ink-filled vacuoles will decline, meaning that the phagocytosis processes in Tetrahymena have been reduced. First, the experiment should be repeated with the test conditions having a very low amount of e-cigarette liquid with and without nicotine, and later the exact opposite- with larger amounts.

Another test could be done with altering the timepoints by either making shorter or elongating the intervals in between each. The trials can be replicated using the same procedure, but increasing the number of Tetrahymena cells being observed to give a bigger sample and stronger results.

Finally, another aspect of the study that can be altered is the e-cigarette liquid itself to an e-cigarette liquid with the different flavors that are offered like bubble gum or vanilla. They could be tested to see whether the flavor itself is as harmful as the nicotine found within the liquid.

The results of this study further the idea that nicotine is a very harmful ingredient found in normal cigarettes and e-cigarettes. Also, the malignance of e-cigarettes themselves could be proven because they are not shed in a light as bad as cigarettes, where we have campaigns and advertisements against their use. There are not many, if any, against the use of e-cigarettes, which is interesting to think about whether the e-cigarette industry is doing a good job of concealing the effects of smoking them.

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