A potential carcinogen is present in electronic cigarette liquids and smokeless tobacco products

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A potential carcinogen that has been banned as a food additive is present in concerningly high levels in electronic cigarette liquids and smokeless tobacco products, according to a new study from Duke Health.

The chemical – called pulegone (pronounced pju-leh-goan) – is contained in menthol and mint flavored e-cigarettes and smokeless tobacco products.

Because of its carcinogenic properties, the U.S. Food and Drug Administration banned pulegone as a food additive last year in response to petitions from consumer groups.

Yet the agency does not regulate the chemical’s presence in e-cigarettes and smokeless tobacco, which are promoted as safer alternatives to regular cigarettes.

“Our findings suggest that the FDA should implement measures to mitigate pulegone-related health risks before suggesting mint- and menthol-flavored e-cigarettes and smokeless tobacco products as alternatives for people who use combustible tobacco products,” said Sven-Eric Jordt, Ph.D., a professor of the Department of Anesthesiology at Duke and lead author of a study publishing online Sept. 16 in JAMA Internal Medicine.

Jordt and research partner Sairam V. Jabba became interested in the topic because the U.S. Centers for Disease Control and Prevention published studies showing that mint- and menthol-flavored e-cigarette liquids and smokeless tobacco products marketed in the U.S. contain substantial amounts of pulegone.

The two researchers analyzed whether several top brands of regular menthol cigarettes, three e-cigarette brands, and one smokeless tobacco brand contain enough pulegone to be a cause for concern.

They compared the CDC-reported amounts of pulegone with the FDA’s exposure risk data – the levels at which exposure-related tumors were reported in animal studies.

Their analysis found that the levels in the e-cigarettes and smokeless tobacco exceeded the thresholds of concern. Regular menthol cigarettes contained levels below the thresholds.

“Our analysis suggests that users of mint- and menthol-flavored e-cigarettes and smokeless tobacco are exposed to pulegone levels higher than the FDA considers acceptable for intake in food, and higher than in smokers of combustible menthol cigarettes,” Jordt said.

“The tobacco industry has long known about the dangers of pulegone and has continuously tried to minimize its levels in menthol cigarette flavorings, so the levels are much lower in menthol cigarettes than in electronic cigarettes,” Jordt said.

E-cigarette manufacturers may be less familiar with the dangers and use cheaper ingredients to lower costs.

One limitation of the study is that the FDA’s exposure risk calculations are based on oral exposure in animal studies.

These risks may apply to the oral exposure through smokeless tobacco but may differ from inhalation exposure through e-cigarette vapor.

There is no toxicity data available on exposure via inhalation. This is concerning because toxicologists consider the lung to be more sensitive to toxic chemicals than the digestive tract.


The diffusion of e-cigarette (e-CIG) opens a great scientific and regulatory debate about its safety. The huge number of commercialized devices, e-liquids with almost infinite chemical formulations and the growing market demand for a rapid and efficient toxicity screen system that is able to test all of these references and related aerosols. A consensus on the best protocols for the e-CIG safety assessment is still far to be achieved, since the huge number of variables characterizing these products (e.g., flavoring type and concentration, nicotine concentration, type of the device, including the battery and the atomizer).

This suggests that more experimental evidences are needed to support the regulatory frameworks. T

he present study aims to contribute in this field by testing the effects of condensed aerosols (CAs) from three main e-liquid categories (tobacco, mint, and cinnamon as food-related flavor), with (18 mg/mL) or without nicotine.

Two in vitro models, represented by a monoculture of human epithelial alveolar cells and a three-dimensional (3D) co-culture of alveolar and lung microvascular endothelial cells were used.

Cell viability, pro-inflammatory cytokines release and alveolar-blood barrier (ABB) integrity were investigated as inhalation toxicity endpoints. Results showed that nicotine itself had almost no influence on the modulation of the toxicity response, while flavor composition did have.

The cell viability was significantly decreased in monoculture and ABB after exposure to the mints and cinnamon CAs.

The barrier integrity was significantly affected in the ABB after exposure to cytotoxic CAs.

With the exception of the significant IL-8 release in the monoculture after Cinnamon exposure, no increase of inflammatory cytokines (IL-8 and MCP-1) release was observed. These findings point out that multiple assays with different in vitro models are able to discriminate the acute inhalation toxicity of CAs from liquids with different flavors, providing the companies and regulatory bodies with useful tools for the preliminary screening of marketable products.

Comparative Effects of CAs on Cells Monoculture

The results obtained by testing CAs belonging to six e-liquid samples with and without nicotine on the monoculture A549 cells, suggest that they induce different cellular responses.

In the case of Base, the e-liquid sample without flavor, no cytotoxic and pro-inflammatory effects were seen at both no and 18 mg/mL nicotine content (Figure 2 and Figure 3). Similar results were obtained for Tobacco 1 and Tobacco 2. This suggests that their CAs do not interact with cell viability and that no contribution to the toxicity was furnished by humectants and nicotine.

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Figure 2
Cytotoxic effects of condensed aerosols (CAs) from e-liquid samples on monoculture A549 cells. MTT viability test was performed after 24 h of exposure to CAs belonging to e-liquids with (18 mg/mL, black bars) and without (grey bars) nicotine. Bars represent the percentage of viable cells with respect to the control (unexposed cells, white bar), considered as 100%. Data are presented as mean±SEM of at least 5 independent experiments. * p < 0.05; unpaired t-test over the control; @ p < 0.05; unpaired t-test over Base 0; § p < 0.05; unpaired t-test over Base 18.
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Figure 3
Pro-inflammatory cytokines released by monoculture A549 cells exposed to CAs from e-liquid samples with (18 mg/mL, black bars) and without (grey bars) nicotine; white bars represent control samples. (A), IL-8 release; (B), MCP-1 release. Bars represent the concentration (pg/mL) of the released protein. Data are presented as mean ±SEM of at least five different independent experiments. * p < 0.05; unpaired t-test over the control; @ p < 0.05; unpaired t-test over Base 0; § p < 0.05; unpaired t-test over Base 18.

Conversely, slight to strong decrease in cell viability was observed after exposure to samples Cinnamon and Menthol 2 containing nicotine (Figure 2). In particular, the exposure to Menthol 2 induced cell death in the 90% of the cells.

In absence of nicotine, these two flavors did not induce cytotoxic effects, suggesting that a synergistic action between flavor molecules and nicotine may take place.

The synergistic effect of nicotine and flavors in e-CIG vapors is actually an open field of research and no literature is yet available.

It is anyway known that the menthol contained in the mainstream smoke from conventional cigarettes may enhance the cellular uptake of PAHs and other tobacco-related carcinogens, finally producing a synergistic effect in altering metabolism [28] and promoting lung cancer [29].

These studies have been done on mentholated conventional cigarettes, where the tobacco component is determinant for the biological responses.

Although no tobacco products are of course present in mentholated e-CIG, the capability of menthol to increase the bioavailability and cytotoxicity of nicotine and/or other molecules, deriving from the heating of the e-liquids, should not be excluded.

Since there are many concerns over the potential of e-CIGs to promote pulmonary inflammation, the capability of CAs to induce an inflammatory response was investigated.

In the lung tissues of patients affected by chronic obstructive pulmonary disease (COPD), neutrophil infiltration is increased, as well as IL-8 (CXCL8, a key neutrophil chemoattractant) levels, and this may positively correlate with disease severity [30].

Furthermore, lung epithelial cells were seen to respond to several external stimuli by secreting specific chemo-attractants and pro-inflammatory cytokines, like MCP-1, in addition to IL-8, to activate the secondary response, consisting in neutrophils and macrophage recruitment.

Finally, IL-8 is involved in endothelial dysfunction and MCP-1 contributes to the recruitment of monocytes to the endothelium, which may contribute in the development of vascular atherosclerotic processes [31]. For all these reasons, in this study we measured the upstream pro-inflammatory cytokines, IL-8 and MCP-1.

Our data showed that only the Cinnamon CA was able to induce a slight increase in the release of the pro-inflammatory cytokines IL-8 and MCP-1 in A549 cells. Specifically, Cinnamon with nicotine (Figure 3A) induced an increased secretion of IL-8 in A549 with respect to control cells and cells treated with CAs of Base with nicotine e-liquid. This suggests that the flavor occurring in Cinnamon could be responsible of the inflammatory response.

The cinnamon flavor in e-liquids was previously reported to be cytotoxic [8], and it has been here confirmed that also its derived CAs are able to significantly decrease cell viability, while increasing inflammatory mediators release.

The strong decrease of IL-8 and MCP-1 release after exposure to Menthol 2 with nicotine was likely the consequence of the very high cell mortality registered by MTT.

This result support the hypothesis that in alveolar epithelial cells, Cinnamon flavor is able to induce significant cytotoxic and pro-inflammatory response. Menthol 2, consisting in a combination of menthol with other flavoring molecules, such as carvone, (Table 1), displayed strong primary cytotoxic action (Figure 2A), which mined the capability of the cells to secrete inflammatory signals (Figure 3).

Leigh and co-workers [9] obtained similar results testing e-CIG CAs on NCI-H292 lung cells exposed at the air-liquid interface (ALI). In that case, strawberry flavor caused the highest cell mortality and release of inflammatory mediators.

Similarly, our results, showed that CAs of Menthol 2 flavor cause significant reduction in cell metabolic activity and viability, without increasing the levels of cytokines release.

Our results are in line with the findings of Putzhammer et al. [16], who showed that herbal flavors contain a higher number of toxic compounds, which are likely responsible of the enhanced cytotoxicity.

To verify this assumption, we used our experimental system to obtain and test CAs from e-liquids derived from plant extracts. The results highlighted the extremely high cytotoxic and pro-inflammatory effects of these compounds on A549 cells (see Supplementary Materials, Figures S1A and S2).


Journal information: JAMA Internal Medicine
Provided by Duke University Medical Center

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