Elsevier

Oral Oncology

Volume 52, January 2016, Pages 58-65
Oral Oncology

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines

https://doi.org/10.1016/j.oraloncology.2015.10.018Get rights and content

Highlights

  • Normal and HNSCC cell lines were cultured in e-cigarette vapor pulled through media.

  • Cell cultures exposed to e-cigarette vapor show increased DNA double strand breaks.

  • E-cigarette vapor induces increased cell death as compared to control.

  • E-cigarette vapor decreases clonogenic survival as compared to control.

Summary

Objectives

Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines.

Materials and methods

HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining.

Results

E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks.

Conclusion

E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed.

Introduction

Electronic cigarettes, or e-cigs, are battery-operated devices that allow users to inhale an aerosolized “e-liquid” in lieu of traditional tobacco smoke. E-liquid typically contains varying compositions of propylene glycol (PG), vegetable glycerin (VG), flavorings, and/or nicotine. Since their introduction to the U.S. in 2007 [1], e-cigs have experienced an exponential surge in popularity, with overall use increasing from 3.3% to 8.5% between 2010 and 2013 [2], and usage among adolescents doubling between 2011 and 2012 alone [3]. E-cigs have gained traction not only among current smokers as a replacement or supplement to traditional cigarettes, but also among non-smokers who may not have otherwise developed smoking habits and nicotine addiction [4]. The rapid rise of e-cigs is often attributed to advertisements portraying e-cigs as a smoking cessation tool or as a completely safe alternative to traditional smoking. However, these claims have been widely found to be controversial and unfounded in scientific evidence [5], [6].

Much remains to be elucidated about the health effects of e-cigs, as existing research is collectively inconclusive. Several studies have substantiated e-cig companies’ claims of negligible e-cig toxicity, with a recent report finding only one e-cig brand marginally cytotoxic to mammalian fibroblasts, and still nearly eight times less potent than conventional cigarettes [7]. E-cigs have also been reported to contain toxicants at trace levels 9–450 times lower than in traditional cigarettes [8] and no toxicological synergies among their compounds [9]. However, numerous other studies suggest otherwise, concluding that e-cigs are hazardous and should be regulated similarly to traditional cigarettes [10], [11], [12]. Heating of e-liquids to high temperatures has been found to release carcinogenic carbonyl compounds, such as formaldehyde, acetaldehyde, and acrolein [13]. The lungs have been shown to be especially vulnerable to e-cig exposure, as e-vapor particles deposit in lungs in a pattern similar to that of regular cigarette smoke particles [11], producing changes in bronchial gene expression [10] and increased oxidative stress and inflammation [12].

Among studies demonstrating the health risks of e-cigs, there is still a paucity of data regarding the cytotoxicity and genotoxicity of e-cig vapor and the role of e-liquid nicotine content in mediating the harmful effects of e-cig exposure. With e-cig usage becoming increasingly prevalent, it is critical to comprehensively evaluate the safety and potency of these devices. In this paper, we sought to investigate the cytotoxic and DNA strand break-inducing effects of nicotine-containing and non-nicotine-free e-cig vapor on a panel of epithelial cell lines. We also assess the contribution of e-cigs to the pathogenesis and progression of head and neck squamous cell carcinoma (HNSCC), a disease for which traditional cigarette smoking is a well-established risk factor yet the potential role of e-cigs has remained entirely unexplored.

Section snippets

Cell culture

To explore the effects of e-cigs on the oropharynx, in vitro experiments were performed on normal epithelial cells as well as head and neck squamous cell carcinoma (HNSCC) cell lines. We chose to use the widely available cell line HaCat, a spontaneously transformed immortal keratinocyte, to determine the potential effects of e-cig on normal epithelium [14]. We also chose to use the HNSCC cell lines HN30 and UMSCC10B for two reasons. First, these cell lines were originally derived from the

E-cigarette vapor extract increases DNA damage via single- and double-strand breaks

One of the most pertinent questions regarding the relative safety of e-cigs is whether or not e-cigs have the potential to cause DNA damage in human cells. To evaluate the ability of e-cigs to induce DNA strand breaks, the normal human epithelial cell line HaCaT and two HNSCC cell lines UMSCC10B and HN30 were treated for one week with two brands of e-cigarette vapor at 1% by volume. Experiments were performed both in normal and cancer cells to assess the effects of e-cigs on healthy cells as

Discussion

Although e-cigs have skyrocketed in popularity and have been widely marketed as a safe alternative to traditional cigarettes, their safety and long-term effects have remained shrouded in controversy. In this paper, we have demonstrated cytotoxicity of short-term e-cig vapor exposure on a panel of normal epithelial and HNSCC cell lines. Through Annexin V staining, trypan blue exclusion, and colony forming assays, we have shown that the cytotoxic effects of e-cig vapor are mediated through

Conclusion

In conclusion, our study strongly suggests that electronic cigarettes are not as safe as their marketing makes them appear to the public. Our in vitro experiments employing 2 brands of e-cigs show that at biologically relevant doses, vaporized e-cig liquids induce increased DNA strand breaks and cell death, and decreased clonogenic survival in both normal epithelial and HNSCC cell lines independently of nicotine content. Further research is needed to definitively determine the long-term effects

Conflict of interest statement

The authors have no conflicts of interest to disclose.

Acknowledgements

This work was supported by funding from the National Institutes of Health, Grant number DE023242 to W.M.O.; by funding from the Department of Veterans Affairs (VA) BLR&D Career Development Award (CDA)-2, award number 1IK2BX001313 to L.C.A.; and by a grant from the Brandon C. Gromada Head and Neck Cancer Foundation to W.M.O. This work was also performed with the support of the Flow Cytometry Core at the UC San Diego Center for AIDS Research (P30 AI036214). The HaCaT cell line was a generous gift

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