Toxicity of E-liquid Flavors and Base Components on AGS and A549 Cell Lines Shyleen Frost Department of Biology, University of Illinois Springfield Internal Medicine, SIU Medicine

Introduction Too many studies focus on nicotine levels especially as e-cigarettes are examined as ‘safer’ alternatives than conventional cigarettes1. Studies that have focused on other aspects of e-liquid indicate the flavorings are one of the most toxic components2, yet these liquids are a main selling point to adults, children, and ‘never smokers’3 and are generally thought of as safe because the flavorings are FDA food grade4.  At this point we probably all know someone who vapes. There are many arguments that go back and forth about the safety of e-cigarettes and the literature reflects both sides of these arguments. However the majority of studies focus on the nicotine content. http://ukvapers.org/Thread-Zamplebox-subscribers

goals The goal of this experiment was to study the toxicity of the base components that make up e- liquid including: Propylene glycol (PG) Vegetable glycerin (VG) Flavorings We did not use nicotine At this time we already know that nicotine is bad for us, and so we aimed to look into the toxicity of the other components of e-liquid beyond nicotine such as propylene glycol (PG) and vegetable glycerin (VG) and the flavorings that make up the base of e-liquids. In this study the flavoring and base components were tested individually as well as testing complete e-liquid as an unheated mixture, and as a heated mixture, the way it would be exposed to the lungs when vaping. https://www.dreamstime.com/stock-illustration-e-liquid-components-vector-illustration-image72383439

AGS GI (Stomach) Cell Line Cell culture Methods AGS GI (Stomach) Cell Line A549 Lung Cell Line AGS and A549 cells were acquired from ATCC Maintained in Ham’s F-12K Nutrient Mixture, Kaighn’s Mod, 10% fetal bovine serum, and penicillin-streptomycin. Incubated in a T75 flask at 37˚ C with 5% CO2 Subcultured with the use of trypsin when they reached 80-90% confluency as determined visually with microscope. https://www.phe-culturecollections.org.uk/products/celllines/generalcell/detail.jsp?refId=89090402&collection=ecacc_gc https://www.phe-culturecollections.org.uk/products/celllines/generalcell/detail.jsp?refId=86012804&collection=ecacc_gc#medImg

Promega Cytotox96® assay Assays used MTT Assay A darker color indicates that more cells that are alive Promega Cytotox96® assay A darker color indicates that more cells that are dead Once introduced to a sample the living cells will take up the MTT and the chemical is reduced by the cell’s metabolism to create a purple crystal. The more living cells (i.e. viability), the darker purple color that will be produced. Lactate dehydrogenase (LDH) release is measured in the Cytotox96® assay and is a measure of cell toxicity. LDH is released upon cell death due to a “leaky” cytoplasm. The reporter chemical INT is converted to a red formazan creating a pink/red color which is more intense and darker in the presence of dead cells (i.e. more LDH released).

Table 1. Concentrations of Components Tested via MTT Assay Methods of MTT Assay MTT treatments included PG, VG, flavorings (LorAnn’s and TFA) Strawberry, (TFA) Cinnamon Spice, and e-liquid as used for human vaping (70/30/3 mix of VG/PG/LA Strawberry) tested in both heated and unheated forms. PG VG Flavoring Complete Liquid   0.01% 0.1% 0.5% 1% 3% 5% 10% 20% 50% Cells were plated in 24-well cell culture plates and incubated for 48 h Cells were treated with components of the e-liquid (Table 1) Two hours prior to the time point, (4 or 24 h) 50 μL of MTT (Sigma) was added to each well, and the cultures were incubated. At the time point, the MTT medium was removed and the cells were solubilized using 0.5 mL/well of solubility buffer containing 10% Triton X-100, 10% 1 N HCl, and 80% isopropanol. 200 μL was removed x2 from each well and transferred to a 96 well plate (solubility buffer as a blank). Absorbance was read at 570 and 690 nm Biotech Synergy Plate Reader Unheated complete e-liquid was steeped in water bath at 37˚ C for 30 minutes. Heated e-liquid was heated in the chamber for 30 min, by repeatedly ‘vaping’ for 3-5 seconds maintaining condensation within the chamber and the chamber being hot to-the-touch using a Nautilus mini chamber (1.8 ohm BVC atomizer) and an Eleaf™ (iStick 30W) battery. Table 1. Concentrations of Components Tested via MTT Assay

Methods of CytoTox 96® AssAy Cytotox96® treatments included only the two strawberry flavorings (LorAnn’s and TFA) tested at concentrations of: 0.1% 0.3% 0.5% 1% 3% Cells were plated in 12-well cell culture plates and incubated for 48 h Cells were treated with different concentrations of flavorings. At 1, 4 and 24 h post treatment, 120 μL of media was removed and transferred to individual tubes and stored at 4˚ C. To complete the assay, 50 μL (x2) from each tube was transferred into a 96 well plate. 50 μL of reagent was added to each well and the plate was covered with aluminum foil and allowed to rest for 30 min at RT. 50 μL of Stop solution was added to each well. Absorbance was read at 490 nm Biotech Synergy Plate Reader

Cytotox flavor results All graphs are presented with the same axes. The horizontal X-axis shows the concentration of the substance being tested, while the vertical Y-axis shows how many cells are alive (shown as percentage of control). The graphs on the left are results using the AGS, gastrointestinal cell line. The graphs on the right are results of the same assays using the A549, lung cell line. These graphs show the results of the Cytox Assay of the two different strawberry flavorings over the time points of 1 hour, 4 hours, and 24 hours. You can see with both cell lines the TFA Strawberry was more toxic.

Mtt flavor results This set of graphs show the results of the three flavorings (TFA and LorAnn’s Strawberry and TFA cinnamon) after 24 hours of exposure measured by the MTT test. From these you can see again that the TFA Strawberry and Cinnamon were the most toxic and that the LorAnn’s Strawberry was the least toxic in both cell lines.

MTT Results of All components These graphs are a compilation of results from all the components tested as well as the complete heated and unheated e-liquid after 24 hours of exposure. Both sets of graphs are the results of the MTT assay. You can see that the red TFA Strawberry and Cinnamon resulted in the most cell death with both cell lines, followed by the complete heated e-liquid, which was made with the least toxic LorAnn’s Strawberry flavoring.

Conclusions and discussion Similar flavorings vary in toxicity between manufacturers and contribute significantly to the overall toxicity of an e-liquid. They are generally the most harmful component of the e-liquid. The heated complete e-liquid was more toxic compared to the unheated complete e-liquid. AGS cells showed less viability in the MTT tests and this was unexpected. This study showed that flavorings vary greatly in toxicity between flavors and manufacturers. Of the two strawberry flavorings tested, the LorAnn’s brand consistently tested lower in toxicity of all the components or complete e-liquid. This could be due to the components of the flavorings themselves. The LA Strawberry contained PG, VG, and Triacetin, while TFA strawberry contained PG and ethyl alcohol. Heated complete e-liquid also exhibited an increased toxicity. It is interesting to note that heated complete mix is more toxic than the same mix that had not been heated. This suggests that the mixture is altered in the heating process. Other chemicals or heavy metals leaching into the mix from the heating components, or chemical reactions of the components to produce such things as formaldehyde and acrolein. The appearance of these and other carbonyl compound in the heated liquid seems to increase in response to the voltage used5. Unexpectedly the AGS cells showed less viability in the MTT tests and this was unexpected due to the flavorings being FDA approved. We expected the AGS cell line to be more resistant to the effects of the flavorings and the A549 lung cells to show more damage. We can attribute some of this to the dose, in most foods these artificial flavorings aren’t making up a huge percentage of the total product and some of it may be broken down before it ever reaches these cells. In e-liquids flavorings can make up 3-5% of the product and it is being directly applied to the cells.

Where to go from here What exactly causes higher toxicity in heated e-liquid? Can this be changed or avoided? Look at comparisons with different brands of flavors for tighter regulation Discourage use of flavorings in e-liquids when in use as a cessation device There are many hypotheses and the literature varies as to why the heated e-liquid is more toxic. Common examples cite leeching of chemicals from the metals that make up the pens and tanks, or point to a chemical reaction occurring in the e-liquid as its exposed to heat. I cited an example where a study was shown to have an increased toxicity when it was heated at a higher voltage, if we know correlations like this, we can encourage safer use. Similarly this study demonstrated how much different flavors can vary in toxicity between brands and this could be due to the chemical makeup of the flavorings. If a larger study were to look at more brands, they might be able to connect the increased toxicity to a certain make-up and then we could encourage against using brands who hav that make up in e-liquid use. Or there could be legislation put into use. Examples of this are things like the butter flavoring which was shown to cause popcorn lung in those who inhale it. In addition more I would suggest that in people using e-cigarettes especially as an aid to quit smoking limit or avoid the use of flavorings in their e-cigs to increase the health benefits associated with quitting smoking.

Thanks and references I would like to thank Dr. Liberati for guidance and mentorship, and the William E. McElroy Charitable Foundation, the Ralph Engelstad Lung Cancer Research Fund, and SIU School of Medicine for supporting this position and study. Cover photo from http://riseofthevapers.tumblr.com/ Farsalinos, K., et al. (2013). International Journal of Environmental Research and Public Health, 10(10), pp.5146-5162. Cervellati, F., et al. (2014). Toxicology in Vitro, 28(5), pp.999-1005. U.S. Department of Health and Human Services. (2016). E-Cigarette Use Among Youth and Young Adults: A Report of the Surgeon General. Farsalinos, K., et al. (2014). Nicotine & Tobacco Research, 17(2), pp.168-174. Kosmider, L., et al. (2014). Nicotine & Tobacco Research, 16(10), pp.1319-1326.

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