1. Toxicity of E-liquid Flavors and Base Components on AGS and A549 Cell Lines
Shyleen R. Frost and Teresa A. Liberati, DVM, PhD
Department of Internal Medicine, SIU Medicine, Springfield, IL
Introduction
There are few studies performed on the toxicity of e-liquid components particularly without a focus on nicotine. Many studies focus on cigarette vs. e-cigarette nicotine levels especially as e-cigarettes are examined as ‘safer’ alternatives1. 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 smokers3 and are generally thought of as safe because the flavorings are FDA food grade4. The goal of this experiment was to study how the base components propylene glycol (PG) and vegetable glycerin (VG) and flavorings affected the human stomach cell line, AGS, that would normally be exposed to these FDA approved flavorings, as compared to the human lung cell line, A549, that would be exposed with e-cigarette use. 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.
Results
About the Tests
This study showed that similar flavorings vary in toxicity between manufacturers and contribute significantly to the overall toxicity of an e-liquid. As the graphs below show the flavorings were generally the most harmful component of the e-liquid and the heated complete e-liquid was more toxic compared to the unheated complete e-liquid.
All 6 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.
Graphs 1 and 2 are the results of the two strawberry flavorings, TFA and LorAnn’s, at both the 4 hour and 24 hour time points. These results are from the Promega Cytotox 96® assay.
The MTT Assay
MTT is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.
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.
Promega Cytotox96® assay
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).
Cell Culture
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.
MTT Assay
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
CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega)
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
Treatments
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.
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.
Cytotox96® treatments were similar including only the two strawberry flavorings (LorAnn’s and TFA) tested at concentrations of 0.1%, 0.3%, 0.5%, 1%, and 3%
Methods
Discussion
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 a higher toxicity in the MTT tests, however the A549 cells were most affected by the TFA Strawberry according to the Promega Cytotox96® Assay.
Graphs 3 and 4 show the results of all three flavorings (TFA and LorAnn’s Strawberry and TFA cinnamon) after 24 hours of exposure, while Graphs 5 and 6 show 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.
This poster needs to be printed as “oversized” since images go out to the edges. Prints as 36x48 at 100%.
Title:90 pt Name: 60pt Affiliation:50 pt
Headers: 56pt Body text:40pt
Figure header: 36 pt Figure text: 33 pt
References: 45pt Text:32pt PG VG
Flavoring
Complete Liquid
0.01%
0.1%
0.5% 1% 3% 5%
10%
20%
50%
Table 1. Concentrations of Components Tested via MTT Assay
Acknowledgements
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.
Farsalinos, K., et al. (2013). International Journal of Environmental Research and Public Health, 10(10), pp
Cervellati, F., et al. (2014). Toxicology in Vitro, 28(5), pp
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
Kosmider, L., et al. (2014). Nicotine & Tobacco Research, 16(10), pp