Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells

doi:10.1016/j.tiv.2014.04.012

Toxicology in Vitro

Volume 28, Issue 5, August 2014, Pages 999-1005

https://doi.org/10.1016/j.tiv.2014.04.012 Get rights and content

Highlights

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Users of electronic cigarette are increasing.
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The use of flavor in electronic cigarette affect cell viability and cell ultrastructure.
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Even the only vaping can stimulate proinflammatory cytokines release in cells.
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Electronic cigarette might not be harmless and affect cell physiology.

Abstract

Information about the harmful effects of vaping is sparse and inconsistent, therefore, since the use of electronic cigarettes (e-CIGs) has become increasingly popular as a tool to limit tobacco smoking, it is urgent to establish the toxicity of the commercial e-CIGs.

Skin (HaCaT) and lung (A549) cells, the main targets of cigarette smoke (CS), were exposed to e-CIG vapor and CS using an in vitro system. The cytotoxic effect of the exposure was analyzed in both cell types by ultrastructural morphology, Trypan Blue exclusion test and LDH assay. In addition, pro-inflammatory cytokines were measured by the Bio-Plex assay.

The cytotoxic components of e-CIG were restrained to the flavoring compound and, to a lesser extent, to nicotine although their effects were less harmful to that of CS. Humectants alone exhibited no cytotoxicity but induced the release of cytokines and pro-inflammatory mediators.

Based on our results, we can state that exposure to e-CIG vapors results in far less toxic than exposure to CS. In fact, besides the deleterious effect of flavor and nicotine, even the humectants alone are able to evocate cytokines release. This study will hopefully promote the development of safer e-CIGs to help people quit smoking.

Introduction

Developed in China in 2004, the electronic cigarette (e-CIG) has become increasingly popular in worldwide. Marketers of the e-CIG describe it as an aid to help people quit smoking. They claim that while using an e-CIG simulates tobacco cigarette smoking, the odor and risks associated with tobacco smoke are eliminated as no combustion products and no tobacco toxins are inhaled (Caponnetto et al., 2012).

In fact, in addition to variable doses of nicotine and different flavors, the base liquid typically includes propylene glycol (PG) and/or glycerol (also called vegetable glycerin or VG) and/or polyethylene glycol 400 (PEG400), all of which are widely used as additives in foods and personal care products, such as toothpaste (Fluhr et al., 2008).

Thus, the components of e-CIG vapors, inhaled in the act now called vaping, are assumed to be less harmful than the thousands of known and unknown toxicants in tobacco smoke. Nonetheless, this assumption does not entirely rule out potentially deleterious effects of inhaling the vapor of the nicotine/flavor mixture. Indeed, while The World Health Organization (WHO) has not excluded the e-CIG might be useful as a smoking cessation aid, it has stated that current research does not warrant the conclusion that the e-CIG is as safe and effective, in reducing nicotine-related withdrawal symptoms, as nicotine-replacement patches or gum (Barbeau et al., 2013). There is no specific legislation on the use of e-CIGs in Europe and currently member countries set their own regulations. Some countries, such as Belgium and Denmark, banned the sale of e-CIGs while Germany and Austria classified the e-CIG as a medical product. In the Netherlands, the use and purchase of e-CIGs is legal, but advertisement of them is banned. The European Union however, is currently debating banning all smokeless tobacco throughout Europe. Taking these products off the market however, would force thousands of users, who positively experienced vaping (Barbeau et al., 2013), to return to cigarette smoking, with the known deleterious effects. Therefore, it is urgent to establish the safety or the toxicity of the components of the vapors from commercial e-CIGs in order to provide legislators, manufacturers and smokers with the essential scientific information required to make informed decisions. In the current study, we compared the in vitro cytotoxicity of cigarette smoke and e-CIG vapors on cells from lung and skin, the organs directly targeted by tobacco cigarettes (Sticozzi et al., 2012, Valacchi et al., 2011).

Short term exposure of HaCaT cells (keratinocytes) and A549 cells (lung epithelial cells) to tobacco smoke and e-CIG vapors with and without aroma or nicotine were carried out. The results revealed that e-CIG vapors have some toxic effect on cell viability. In particular, the harmful component of the e-CIG seems to be confined to the flavoring compounds rather than to nicotine and humectants. In addition, screening of an array of cytokines released from the cells exposed to e-CIG vapors without additives showed that the basal components alone are able to induce the release of several cytokines and pro-inflammatory mediators, suggesting that even humectants might have a potential, although non-cytotoxic, harmful effect.

Section snippets

Cell culture

HaCaT cells, (a gift from Dr. F. Virgili), were grown in Dulbecco’s modified Eagle’s medium High Glucose (Lonza, Milan, Italy), supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/mL streptomycin and 2 mM l-glutamine. A549 cells were purchased from ATCC (Rockville, MD). Ham’s F-12, foetal bovine serum, RPMI-1640, penicillin/streptomycin and l-glutamine were obtained from Lonza (Milan, Italy). Cell suspension containing 5 × 10⁶ viable cells/ml were used. Cells were incubated at 37 °C for 24 h in 95%

LDH release and viability

As shown in Figs. 1A and 2A respectively, HaCaT and A549 cells viability does not change in controls over 24 h and, consistently, a steady low release of LDH was observed (Figs. 3A and 4A). In contrast, exposure to CS caused an early (6 h) and progressive decrease in cell viability (Figs. 1B and 2B) and increased LDH release (Figs. 3B and 4B) with a similar trend during the different time points in both cell lines, although keratinocytes seem to be more susceptible to CS induced toxicity after 24

Discussion

Although there have been few studies suggestive of harmful effects from vaping, the results thus far have been inconsistent. This is likely due to a lack of standardized assessment, but also to varying chemical composition of commercial refill fluids among brands. A recent study showed that using an e-CIG for 5 min has immediate adverse physiologic effects similar to those seen with tobacco smoking (Vardavas et al., 2012). In another recent study, where serum cotinine, lung function, exhaled

Funding

HJF is supported by NIH grant ES020942.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

Acknowledgements

The authors thank Marangoni’s Pharmacy, Argenta, Italy for the kind gift of e-CIG Mini Touch T-Fumo T-TEX srl.

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View full text

Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells

Highlights

Abstract

Introduction

Section snippets

Cell culture

LDH release and viability

Discussion

Funding

Conflict of Interest

Transparency Document

Acknowledgements

Reprod. Toxicol.

Int. Immunopharmacol.

Chest

Int. J. Biochem. Cell Biol.

Chest

Perceived efficacy of e-cigarettes versus nicotine replacement therapy among successful e-cigarette users: a qualitative approach

Addict Sci. Clin. Pract.

Release of sICAM-1 in oocytes and in vitro fertilized human embryos

PLoS ONE

Electronic cigarettes as a harm reduction strategy for tobacco control: a step forward or a repeat of past mistakes?

J. Public Health Policy

The emerging phenomenon of electronic cigarettes

Expert Rev. Respir. Med.