Vapours of US and EU Market Leader Electronic Cigarette Brands and Liquids Are Cytotoxic for Human Vascular Endothelial Cells

Abstract

The present study was conducted to provide toxicological data on e-cigarette vapours of different e-cigarette brands and liquids from systems viewed as leaders in the e-cigarette market and to compare e-cigarette vapour toxicity to the toxicity of conventional strong high-nicotine cigarette smoke. Using an adapted version of a previously constructed cigarette smoke constituent sampling device, we collected the hydrophilic fraction of e-cigarette vapour and exposed human umbilical vein endothelial cells (HUVECs) to the mixture of compounds present in the vapour of 4 different single-use e-cigarettes, 6 different liquid vapours produced by the same refillable e-cigarette, and one e-cigarette with an exchangeable liquid cartridge. After incubation of cells with various concentrations and for various periods of time we analysed cell death induction, proliferation rates, the occurrence of intra-cellular reactive oxygen species, cell morphology, and we also measured e-cigarette heating coil temperatures. Overall, conventional cigarette smoke extract showed the most severe impact on endothelial cells. However, some e-cigarette vapour extracts showed high cytotoxicity, inhibition of cell proliferation, and alterations in cell morphology, which were comparable to conventional high-nicotine cigarettes. The vapours generated from different liquids using the same e-cigarette show substantial differences, pointing to the liquids as an important source for toxicity. E-cigarette vapour-mediated induction of oxidative stress was significant in one out of the 11 analysed vapours. There is a high variability in the acute cytotoxicity of e-cigarette vapours depending on the liquid and on the e-cigarettes used. Some products showed toxic effects close to a conventional high-nicotine cigarette. Liquid nicotine, menthol content, and the formation of acute intracellular reactive oxygen species do not seem to be the central elements in e-cigarette vapour toxicity.

Fig 1. E-cigarette vapours are cytotoxic and inhibit cell proliferation of primary human endothelial cells.

Fig 1 (A) shows the summary of FACS analyses of annexin V-propidium iodide stained HUVECs 48 hours after the addition of the indicated concentrations of conventional cigarette extract (TS) and different e-cigarette vapour extracts. Fig 1 (B) shows the effects of the same smoke extract and vapour extracts on the proliferation of endothelial cells after 48 hours of exposure analysed by CFSE staining and FACS analysis. A, B, C, and D reflect different market leader brands, and numbers label different products. Extracts labelled with “Cre” are vapour extracts from different liquids generated using the same liquid-refill vaporizer. A/1, A/2, B/1 and C/1 are single use e-cigarettes; D/1 is an e-cigarette with an exchangeable liquid cartridge. Data shown are mean values +/- S.D. of a representative performed in triplicates. The experiment was repeated three times. Asterisks indicate significant differences compared to the 0% (i.e. the control) p<0.05.

Fig 2. The induction of intracellular reactive oxygen species does not correlate with e-cigarette vapour toxicity.

Fig 2 shows an analysis of the formation of intracellular reactive oxygen species in HUVECs 1 hour after exposure to cigarette smoke extract (TS) and e-cigarette vapour extracts and concentrations indicated. MFI…mean fluorescence intensity of the oxidation-sensitive dye H2DCF-DA. A, C, and D reflect different market leader brands, and numbers label different products. Data shown are mean values +/- S.D. of a representative experiment performed in triplicates. The experiment was repeated three times. Asterisks indicate significant differences compared to the control (0%) p<0.05.

Fig 3. Vapour extracts alter the morphology of endothelial cells.

The images in Fig 3 show HUVECs after 48 hours of exposure to the indicated extracts and concentrations. TS…conventional cigarette smoke extract. Cre2, 5 and 6 are the most cytotoxic liquid vapours according to the results shown in Fig 1. The experiment was repeated three times. Representative images are shown.

Fig 4. Assessment of the temperature of the heat coil of a commercially available vaporizer.

The diagram in Fig 4 shows the two temperature curves of the heating element in ‘dry mode’ and ‘wet mode’; the broken line reflect the temperatures of the coil when surrounded by liquid (wet), the continuous line reflects the temperatures measured in the absence of a liquid (dry). The experiment was repeated three times. Data shown are mean values +/- S.D. Temperature was analysed using an infrared thermometer (max temperature (tmax) that can be measured using the device is 217°C). The image in the lower right of the diagram shows a photograph of different liquids, which remained in the liquid container during several times of e-cigarette vapour extract generation. All liquids were initially clear and colourless. Examples are shown.