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. 2016 Jan;52:58-65.
doi: 10.1016/j.oraloncology.2015.10.018. Epub 2015 Nov 4.

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

Vicky Yu  1 , Mehran Rahimy  1 , Avinaash Korrapati  1 , Yinan Xuan  1 , Angela E Zou  1 , Aswini R Krishnan  1 , Tzuhan Tsui  2 , Joseph A Aguilera  3 , Sunil Advani  3 , Laura E Crotty Alexander  4 , Kevin T Brumund  1 , Jessica Wang-Rodriguez  5 , Weg M Ongkeko  1
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Free PMC article

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

Vicky Yu et al. Oral Oncol. 2016 Jan.
Free PMC article

Abstract

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.

Keywords: DNA damage; Electronic cigarettes; Head and neck squamous cell carcinoma (HNSCC); Nicotine; Oral cancer; Smoking; Strand breaks.

Conflict of interest statement

Conflict of Interest Statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1. E-cigarette exposure increases DNA damage as measured by neutral comet assay
A, HaCaT, UMSCC10B, and HN30 were treated with 1% by volume vaporized e-cigarette liquid with brands V2 and VaporFi, and compared to untreated controls. HaCaT cells were treated for 8 weeks, and UMSCC10B and HN30 treated for 1 week each. E-cigarette containing nicotine at 1% by volume treatment was calculated to contain 0.5 mM nicotine, so cells treated at 0.5 mM nicotine directly are shown for comparison. Results are given as mean tail length ± SEM, with at least 35 cells per sample. B, representative images of cells captured following neutral comet assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001
Figure 2
Figure 2. E-cigarette exposure increases DNA double strand breaks as measured by γ-H2Ax immunofluorescence
A, HaCaT cells were treated for one week at 1% by volume vaporized e-cigarette liquid with brands V2 and VaporFi. E-cigarette containing nicotine at 1% by volume calculated to be equivalent to 0.5 mM nicotine; cultures treated with cigarette smoke at 0.5 mM nicotine and with 0.5 mM nicotine directly are shown for comparison. Graphed results are given as mean foci count per cell ± SEM, with at least 35 cells per sample. Representative images of γ-H2Ax foci formation (green) are shown with nuclei stained with DAPI (blue). B, UMSCC10B and HN30 were treated with the brand V2 at the same concentration and duration. Graphed results are given as mean foci count per cell ± SEM, with at least 35 cells per sample. *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001
Figure 3
Figure 3. HNSCC cell line exposure to e-cigarette vapor results in altered cell cycle profiles
UMSCC10B and HN30 were treated for one week at 1% by volume vaporized e-cigarette liquid with brands V2 and VaporFi, and analyzed by flow cytometry for DNA content. Results are given as mean percentage ± SEM. Peaks generated by flow cytometry analysis are shown for each of the permutations. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 4
Figure 4. E-cigarette treated cells exhibit increased apoptosis and necrosis
HaCaT cells were treated for one week at 1% by volume vaporized e-cigarette liquid with brands V2 and VaporFi, and analyzed by flow cytometry after staining with Annexin V-FITC and counterstaining with PI. Results for cultures treated with cigarette smoke at 0.5 mM nicotine and with 0.5 mM nicotine directly are shown for comparison. Positioning of quadrants on Annexin V/PI dot plots was performed and live cells (Annexin V−/I−, P3), early/primary apoptotic cells (Annexin V+/PI−, P4), late/secondary apoptotic cells (Annexin V+/PI+, P2) and necrotic cells (Annexin V−/PI+, P1) were distinguished.
Figure 5
Figure 5. E-cigarette exposure induces cell death
A, HaCaT, UMSCC10B, and HN30 cells were treated at 1% by volume vaporized e-cigarette liquid with brands V2 and VaporFi for 48 hours before trypan blue staining. Cultures treated with cigarette smoke at 0.5 mM nicotine and with 0.5 mM nicotine directly are shown for comparison. Cell death was normalized to the untreated control cell cultures. Results are shown as mean percentage of cell death per sample ± SEM. B, representative images of the cell cultures treated at 2% by volume vaporized e-cigarette or 1.0 mM nicotine for 2 weeks taken at 20X to show cell death and changes in cell morphology.
Figure 6
Figure 6. E-cigarette exposure decreases clonogenic survival in both normal and HNSCC cell lines
A, HaCaT cells were treated for 10 days at 0.5, 1.0, and 2.0% by volume vaporized e-cigarette liquid. Colony counts were normalized to the untreated control cell cultures. Graphed results are given as mean colony count ± SEM. B and C, the same treatments were replicated for 12 days using UMSCC10B and HN30 respectively. ****P<0.0001
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