Abigail Kronau

The Effects of JUUL-pod e-Cigarette Vaping Solution on Human Lung Fibroblast Cells Over a 72-Hour Period

Abigail Kronau

Biology 351L - Dr. Ann Zeeh

Abstract

Vaping and e-cigarette use have become increasingly popular and addicting activities among young people in the U.S. over the past ten years. In an attempt to help educate the public on the dangers of these products, we conducted a study to see how the chemicals in vaping solutions affected human lung fibroblast cells. Our results show that even after only 24 hours, a diluted version of the solution has detrimental effects on lung cell viability.

Introduction

Vaping and e-cigarettes have exponentially grown in popularity among young people in the U.S since their release to the market in 2007 (1). For many, vaping is an alternative to tobacco cigarettes but, contrary to common belief, vaping products are not necessarily any safer to inhale due to the many chemicals present in the vaping solutions (1). In fact, the vapor inhaled while using a vape pen contains many chemicals that are not found in traditional cigarette smoke (2). According to the Journal of the American Academy of Pediatrics, chemical toxicants and carcinogens have been found in a number of different vape pen products and these substances can have significant health risks associated with their use (2). The vape pens often are small and easily hidden, making them perfect for middle and high school students, who constitute 72% of the JUUL market, to use and hide from parents or teachers (1). The liquid contained in these e-cigarette pods generally contains a propylene glycol or vegetable glycerin base, varying concentrations of nicotine, and several other chemicals and enticing flavorings (i.e. Bubble Gum, Virginia Tobacco, Peach) (1). These products are often advertised as flavored and nicotine-containing in order to lure in this specific group

of young people, but they are also advertised as a tool to help cigarette smokers quit the habit (3). Nicotine is a highly addictive substance and companies like JUUL have begun using compounds like acidified nicotine salts, which deliver nicotine to the body at higher concentrations, making these vape products more addictive than traditional cigarettes (4). In a study published by the Tobacco Regulatory Science Journal, JUUL products can deliver up to eight times more nicotine to the bloodstream per puff compared to traditional cigarettes (4). A study done by Tsai et al. (2020), found that in human and animal models, chronic use of e-cigarettes caused a large number of cardiac and pulmonary changes in subjects (3). These changes ranged from increased blood pressure and heart rate in human models caused by acute exposure to the e-cigarette chemicals, to angiogenesis, airway obstruction, cardiorenal fibrosis, arterial stiffness, atherosclerotic plaque formation, and emphysema caused by chronic exposure to the ecigarette chemicals in animal models (3). After learning about the many adverse effects of vape products, we wanted to conduct our own study. For this experiment, we tested the effects of JUUL pod e-cigarette vaping solution on human lung fibroblast cells grown in culture. Lerner et al. (2015) reported that human fetal lung fibroblasts showed reduced cell counts and morphological changes associated with cellular stress when exposed to e-cigarette compounds. We referred to the paper by Lerner et al. (2015) to create our experimental plan and decide how to expose our cells to the pod solution (5).

Materials & Methods

To begin this experiment, we cultured and grew two groups of human lung fibroblast cells, a control population which contained cells and MEM+ culture medium, and the experimental population, which contained cells and MEM+ culture medium mixed with Virginia Tobacco-flavored JUUL pod vape juice. The medium for the experimental group contained 0.025% nicotine, which was diluted in the MEM+ culture medium from the 5.0% nicotine contained within the pure vape solution taken directly out of the pod. These cells were left to grow in a 37°C incubator injected with 5% CO2, then observed and photographed after 24 hours, 48 hours, and 72 hours.

Results

We had very clear results after the 24-hour period. Evident by Figures 1 through 3 shown below, the e-cigarette vaping solution had a negative effect on the lung fibroblast cells.

Figure 1: A: Control lung fibroblast cells after 24 hours growth in MEM+ medium. B: Experimental lung fibroblast cells after 24 hours growth in Virginia Tobacco JUUL solution plus MEM+ medium with a 0.025% nicotine concentration. C: Giemsa-stained control lung fibroblast cells after 24 hours growth in MEM+ medium. D: Giemsa-stained experimental lung fibroblast cells after 24 hours growth in Virginia Tobacco JUUL solution plus MEM+ medium with a 0.025% nicotine concentration. Panels A and B, photos taken under inverted phase contrast microscope at 200x magnification. Panels C and D, photos taken under compound light microscope at 40x magnification.

Figure 2: A: Control lung fibroblast cells after 48 hours growth in MEM+ medium. B: Experimental lung fibroblast cells after 48 hours of growth in Virginia Tobacco JUUL solution plus MEM+ culture medium with a 0.025% nicotine concentration. C: Giemsa-stained control lung fibroblast cells after 48 hours growth in MEM+ medium. D: Giemsa-stained experimental lung fibroblast cells after 48 hours growth in Virginia Tobacco JUUL solution plus MEM+ medium with a 0.025% nicotine concentration. E: Giemsa-stained control lung fibroblast cells after 72 hours growth in MEM+ medium. F: Giemsa-stained experimental lung fibroblast cells after 72 hours growth in Virginia Tobacco JUUL solution plus MEM+ medium with a 0.025% nicotine concentration.

Table 1: Cell Count in Control and Experimental Groups at 24, 48, and 72 hours

Control: Number of Cells present Experimental: Number of Cells present 24 Hours 48 Hours 72 Hours 102.0 cells/mm2 292.0 cells/mm2 318.0 cells/mm2

72.5 cells/mm2 9.6 cells/mm2 7.5 cells/mm2

Figure 3: This graph shows the growth of our cells, both the control and experimental, over 24, 48, and 72 hours. It is apparent here that the growth of cells according to our experimental group, is much less than that of our control group. This also confirms that the JUUL pod vape solution is extremely damaging to human lung fibroblast cells.

Discussion

After 24 hours, nearly all of the cells in the experimental group had died, in contrast to the cells suspended in the MEM+ culture medium alone, which were growing well in culture. Healthy fibroblasts in culture appear as attached, spindle-shaped cells; dead cells detach and

lose their spindle shape, appearing instead more spherical in shape (compare living cells in Figure 1A and 1C to non-living cells in 1B and 1D). As time went on, the results continued to show us the same outcome, cell death after 48 hours and then 72 hours in the experimental cultures. At the same time, cells in our control cultures for the 48- and 72-hour marks both continued to divide as shown by the Giemsa-stained samples in Figures 2C and 2E. From these results we conclude thate-cigarette vaping solution causes the death of human lung fibroblast cells grown in culture, while the MEM+ culture medium allows continued growth of these cells. Our preliminary results are strong evidence that even with just a small amount of the e-cigarette solution present, there was a detrimental effect on the lung fibroblast cells’ viability. Even more shocking is that this effect only took 24 hours to occur. The toxic effects were also supported by the quantified cell counts shown in Table 1 and the graph shown in Figure 3. Future work should include repeated trials on the fibroblast cells with the same and varying concentrations of the vaping solution to confirm reliability of our preliminary results. Our findings are extremely important because of the percentage of individuals, especially young students, that are using these vape products every day. It is important to educate these individuals on the dangers of e-cigarettes and the detrimental effects that it can have on their lung cells.

Acknowledgments

This research was completed as part of a project for Cell Biology Laboratory (BIO 351L) in Fall 2019 at the College of Saint Rose with the help of my classmate, Morgan Percy. Dr. Ann Zeeh was an advisor on this project.

References

1) Richter, L. (2018, October 1). What is Vaping? Retrieved March 18, 2020, from https://www.centeronaddiction.org/ecigarettes/recreational-vaping/what-vaping

2) Walley, S., Wilson, K., Winickoff, J., & Groner, J. (2019, June 01). A Public Health Crisis: Electronic Cigarettes, Vape, and JUUL. Retrieved January 18, 2021, from https://pediatrics.aappublications.org/content/143/6/e2018274 1

3) Tsai, M., Byun, M. K., Shin, J., & Alexander, L. E. (2020). Effects of e‐cigarettes and vaping devices on cardiac and pulmonary physiology. The Journal of Physiology, 598(22), 5039-5062. doi:10.1113/jp279754

4) Maier, S. (2020, January 6). JUUL Delivers Substantially More Nicotine than Previous Generation E-Cigs and Cigarettes. Retrieved January 18, 2021, from https://www.ucsf.edu/news/2020/01/416371/juul-deliverssubstantially-more-nicotine-previous-generation-e-cigs-and

5) Lerner, C. A., Sundar, I. K., Yao, H., Gerloff, J., Ossip, D. J., Mcintosh, S., … Rahman, I. (2015). Vapors Produced by Electronic Cigarettes and E-Juices with Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. Plos One, 10(2). doi: 10.1371/journal.pone.0116732