Elsevier

Reproductive Toxicology

Volume 34, Issue 4, December 2012, Pages 529-537
Reproductive Toxicology

Comparison of electronic cigarette refill fluid cytotoxicity using embryonic and adult models

https://doi.org/10.1016/j.reprotox.2012.08.001Get rights and content

Abstract

Electronic cigarettes (EC) and refill fluids are distributed with little information on their pre- and postnatal health effects. This study compares the cytotoxicity of EC refill fluids using embryonic and adult cells and examines the chemical characteristics of refill fluids using HPLC. Refill solutions were tested on human embryonic stem cells (hESC), mouse neural stem cells (mNSC), and human pulmonary fibroblasts (hPF) using the MTT assay, and NOAELs and IC50s were determined from dose–response curves. Spectral analysis was performed when products of the same flavor had different MTT outcomes. hESC and mNSC were generally more sensitive to refill solutions than hPF. All products from one company were cytotoxic to hESC and mNSC, but non-cytotoxic to hPF. Cytotoxicity was not due to nicotine, but was correlated with the number and concentration of chemicals used to flavor fluids. Additional studies are needed to fully assess the prenatal effect of refill fluids.

Highlights

► Electronic cigarette refill fluids were examined for cytotoxicity. ► Embryonic cells were more sensitive to refill fluid than adult lung cells. ► Flavorings, but not nicotine, were linked to toxicity. ► Refill products varied from non-cytotoxic to highly cytotoxic. ► Products from two companies were cytotoxic to embryonic cells but not lung cells.

Introduction

Electronic cigarettes (EC) are nicotine delivery devices that are rapidly gaining acceptance as an alternative to conventional cigarettes with little knowledge regarding their effects on prenatal development or adult health [1], [2], [3]. EC have a mouthpiece containing a fluid-filled cartridge, an atomizer used to vaporize the cartridge fluid, and a battery that powers the atomizer [3]. The cartridge fluid usually contains nicotine, flavorings, and a humectant that when heated by the atomizer creates an inhalable aerosol. In some EC, the cartridge and atomizer are combined into a single unit called a “cartomizer” [3], [4]. Refill fluid, also known as E-juice or E-liquid, contains flavoring, nicotine, and a humectant(s), such as propylene glycol (PG) and/or vegetable glycerin (VG). Used EC cartridges or cartomizers can be refilled with drops of refill fluid, which is readily available often from third party vendors on the Internet or in shopping malls.

While the detrimental effects of conventional cigarette smoke on both adult and prenatal health are well documented [5], [6], [7], [8], [9], little direct work has been done on the health effects of EC products, in spite of a recognized need for such information [10]. It has been proposed that EC are less harmful than conventional tobacco products due to their lower total number of chemicals and lower concentration of carcinogens [11], [12]. EC refill fluids are often sold by vendors other than the EC manufacturers, and they have received even less evaluation than EC devices themselves. As a step toward better understanding the health effects of EC, we evaluated the cytotoxicity of 40 samples of EC refill fluid using cells that model both embryonic and adult stages of the life cycle. With the introduction of human embryonic stem cells (hESC) [13], it is now possible to examine effects of consumer products and environmental chemicals on cells that model an early stage of prenatal development [14]. Recent studies have shown that hESC when cultured in vitro have the characteristics of the epiblast cells present in young implantation embryos [15], [16]. Although some toxicological work has been done previously using hESC [17], [18], adaptation of these cells to standard toxicological studies has been slow because they grow in colonies that are difficult to count and plate accurately. We recently developed a method that is amenable to studying hESC in 96-well plate assays, such as the MTT assay. In the current study, we have taken advantage of this method to perform dose–response cytotoxicity experiments using: (1) hESC, which model the epiblast stage of development [15], [16], (2) mouse neural stem cells (mNSC) isolated from the brain of a newborn, and (3) human pulmonary fibroblasts (hPF), which represent an adult cell from one of the initial points of contact for inhaled EC aerosol. The purpose of our study was to compare the sensitivity of embryonic and adult cells to a range of EC refill products and to test the hypothesis that embryonic cells are more sensitive to EC product exposure than adult lung cells. The study included two humectants, 29 different flavors of refill fluid, products from four vendors, five concentrations of nicotine, and six samples that may have caused adverse health effects in users. HPLC spectral analysis was also done to determine if chemicals varied between products with the same flavor or between bottles of the same product.

Section snippets

Sources of refill fluids

A convenience sampling procedure was adopted to select products for analysis. Products were manufactured by Freedom Smoke USA (Tucson, AZ), Global Smoke (Los Angeles, CA), Johnson Creek (Johnson Creek, WI), and Red Oak (a subsidiary of Johnson Creek). These manufacturers were chosen as they represent popular domestic companies whose products are readily available to e-cigarette users on the Internet. Thirty-six bottles of refill fluid containing various flavorings and nicotine concentrations

Dose–response of 35 refill products using the MTT assay

Refill solutions had various effects on cell survival in the MTT assay ranging from no evidence of cytotoxicity to high levels of toxicity (representative graphs are shown in Fig. 1; additional data are shown in Table 1 and Supplement Figs. 1–3). Products listed in Table 1 are grouped in a hierarchy of potency based on their IC50s for hESC, which, in general, were more sensitive to refill solutions than the other two cell types. Table 1 also gives information on the NOAELs for each cell type

Discussion

Understanding the health effects of EC refill fluid is important as these products have become widely distributed without much prior testing. Refill fluid is handled by users, manufacturers, and potentially by children living in homes where EC are used. As a step to understanding how EC products affect human health, we compared the cytotoxicity of 35 refill fluid samples using embryonic and adult cells. Refill products varied significantly in their potency over the dose range tested. In

Conclusions

Embryonic and neonatal stem cells were generally more sensitive to refill products than adult lung fibroblasts. Refill fluid users should be aware that: (1) the low doses and one time exposure used in our study may underestimate cytotoxicity, and (2) within a flavor, such as Butterscotch or Caramel, chemical composition and cytotoxicity were variable. The latter point demonstrates that it cannot be assumed that a specific flavor, such as Butterscotch, will always be non-cytotoxic. The results

Funding

Funding was provided by the California Tobacco-Related Disease Research Program (XT-0118), the California Institute for Regenerative Medicine (NE-A0005A-IE), the UCR Academic Senate, the UCR Graduate Division (Dean's Fellowship to V.B.), a STEM Pathway Project award to Barbara Davis, and a TRDRP Postdoctoral Fellowship (20FT-0084) to S.L.

Conflict of interest

None declared.

Acknowledgements

We are grateful to Dr. Evan Snyder (Stanford-Burnham Medical Institute, San Diego, CA) for providing the mNSC. We also thank Monique Williams, R. Talbot, Kamlesh Asotra, and Rachel Behar for their comments on the manuscript, Laurie Graham for her assistance with equipment, Frank Monreal for help preparing Fig. 4, Crystal Hua for performing literature searching, and Jisso Kim and Michael Dang for assisting with the HPLC.

References (28)

  • S. Higgins

    Smoking in pregnancy

    Current Opinion in Obstetrics and Gynecology

    (2002)
  • P. Talbot

    In vitro assessment of reproductive toxicity of tobacco smoke and its constituents

    Birth Defects Research Part C: Embryo Today

    (2008)
  • P. Talbot et al.

    Smoking and reproduction: the oviduct as a target of cigarette smoke

    Reproductive Biology and Endocrinology

    (2005)
  • J.-F. Etter et al.

    Electronic nicotine delivery systems: a research agenda

    Tobacco Control

    (2011)
  • Cited by (0)

    1

    These authors contributed equally to this paper.

    2

    Current address: University of Pennsylvania, Philadelphia, PA 19104, United States.

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