Toxicity of E-liquid Flavors and Base Components on the AGS and A549 Cell Lines

Shyleen Frost

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https://hdl.handle.net/2142/95986 Copy

Description

Title

Toxicity of E-liquid Flavors and Base Components on the AGS and A549 Cell Lines

Author(s)

Shyleen Frost

Issue Date

2017

Keyword(s)

• biology
• chemistry

Abstract

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’ alternatives. Studies that have focused on other aspects of e-liquid indicate the flavoring is one of the most toxic components, yet these liquids are generally thought of as safe because they are FDA food grade. Understanding the effects of these often overlooked components of e-liquid becomes more important as more people switch over to using e-cigarette products. The goal of this experiment was to study how the liquid base components and flavorings affected the stomach human cell line, AGS cells, 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 flavor and base components were tested individually as well as testing the e-liquid as an unheated complete mixture, and as a heated complete mixture. To test the toxicity and viability of these components and the complete e-liquid, two different cell assays were employed. The first was an MTT assay in which cells were treated for two time points, one at 4 hours and one at 24 hrs. 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. Once the MTT is introduced to a sample the living cells will take up the chemical and it is then 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, indicating a positive result. At the 4 hour and 24 hour time points the flavorings were shown to be the most harmful part of the e-liquid on AGS and A549 cells. The second assay was a CytoTox 96® Non-Radioactive Cytotoxicity Assay to measure the release of lactate dehydrogenase (LDH) from dead cells into the media surrounding the cell. 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%. 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). In this assay, cells were treated with different concentrations of the flavorings at 1, 4 and 24 hour time points. Lactate dehydrogenase (LDH) release is measured in the Cytotox96® assay and is a measure of cell toxicity. The results were similar to the results obtained through the MTT assays. Again, the flavorings were indeed the most toxic individual component, and agreed with current literature that PG is more toxic than VG. This study showed that similar flavorings vary in toxicity between manufacturers and contribute significantly to the overall toxicity of an e-liquid. This could be due to the components of the flavorings themselves. The LA Strawberry contained PG, VG, and Triacetin, most of which are already found in most e-liquids. On the other hand TFA strawberry contained PG and ethyl alcohol, this alcohol may have caused the difference in pH, and also toxicity. 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 used. This research can help consumers be more aware of the toxicity of their components and perhaps alter their e-liquid recipes to include a smaller percentage of the more toxic components. There needs to be more research done into the safety of flavorings and components of e-liquids, as well as what make the heated e-liquid more toxic.

Type of Resource

text

Language

en

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http://hdl.handle.net/2142/95986

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