Carcinogenic Potential of E-cigarettes: Vapor Profile and Cellular Effects

E-cigarettes are devices that vaporize a liquid made of polyglycerol, glycol, flavorings, and nicotine, for inhalation. Initially created for smoking cessation, the health risks of these devices are still not clear. This literature review compiles data on the chemical profile of e-vapor and cell exposure studies to formulate conclusions regarding cancer risk and provide suggestions for future research. The reviewed studies identified a large range of potentially harmful compounds, namely formaldehyde, acrolein, and acetaldehyde, which were found in all studies. Metabolites of these compounds were then identified in exposed patients, showing bodily absorption. In vitro studies found evidence for cellular damage, including DNA mutations, reduced cell viability, and differentiated protein expression which may increase user’s cancer risk. Though the evidence is inconclusive given the heterogeneity of the field. Future studies should focus on the human effects of vaping, testing bronchial brushings and lavage fluid from users to determine the in vivo effects of exposure. Closely monitoring e-cigarette users for early warning signs of cancer would also help us understand future risk and answer questions about the safety of these devices.


Introduction
E-cigarettes have been growing in popularity among North Americans since their introduction in the late 2000s and have risen in popularity since (especially among young people 1 ).
The process of smoking an e-cigarette involves vaporizing a liquid with a heating coil so it can be inhaled into the lungs 2 . The liquid vaporized in an e-cigarette (e-liquids) are typically a mixture of propylene, glycol, glycerin, nicotine, tetrahydrocannabinol (THC), and flavorings 3 .
There are also many different types of devices, with different rates of air flow, heating coils, and materials, and many different types of liquids, with a variety of flavors, ratios, and nicotine levels 4,5 . This variety has made it complicated to study e-cigarettes, as it is difficult to pinpoint specific issues or components of concern. This was especially true in the 2019 E-cigarette and Vaping Associated Lung Injury (EVALI) outbreak, where it took several months for the dangerous component to be isolated, as patients used an incredible variety of products 4 .
Currently there is limited data on the carcinogenic effects of e-cigarettes in humans, due in part to their relative novelty. The link between cigarettes and lung cancer took several decades to be identified, and several more to broadly accepted. and the fact that a rise in cancer rates takes years to decades to be detected in the population 6 . This mistake has been learned from, and already there are studies determining the chemical profile of e-cigarette vapor to identify aerosol compositions and potential for chronic toxic exposure. There also is some data on the effects of vapor on mouse lungs, human explant tissue, or in vitro cells. In this review we collect and synthesize this data on chemical composition and in vitro effects to formulate conclusions about cancer risk from e-cigarette use.

Methods
Google Scholar database was reviewed for studies containing information on the chemical profile of E-cigarettes and cellular effects. Studies were then full text reviewed for final acceptance, meeting the above criteria. Finally, data analysis and synthesis was carried out using the chart shown below. After extraction, data was written up and presented in the report shown below.

Results
The following table outlines the chemical profile of e-cigarettes from reviewed studies.
Xcompound was identified in 50% of devices *-These studies only tested for metals. ^Found in 25% of devices **Conklin et al and Hecht et al did not test for formaldehyde or acetaldehyde metabolites Few compounds were identified in all studies, and a large variation in the compounds was identified in e-cigarettes with most being found in only one study, and not in all e-cigarettes.
For a compound to be included in the table, it had to be found in over 50% of devices and there was significant variation in chemical profiles found within the same study. Showing not only interstudy variation but also interstudy differences. The only compounds consistently found were formaldehyde, acrolein, and acetaldehyde.
To understand the potential for inter-study confounding, Table 3 shows study methods and materials. Studies employed similar methods to analyze the vapors, though there were differences in the preparation of samples that may have affected outcomes. There also was no overlap in the types of devices and liquids used.
In general, e-cigarettes had lower levels of harmful compounds compared to combustion cigarettes. Though certain compounds may be higher in e-cigarettes, due to the nature of these devices and their liquids. One study found aldehydes (including formaldehyde) in higher concentrations in e-cigarette vapor compared to cigarette smoke 25 . Notably, several studies found With one study finding a 1.8mg/mL liquid to a half nicotine cigarette 26 .
Even when compounds were at lower concentrations, they still raised concerns. The Geiss study 18 found that concentrations of identified compounds exceeded the World Health Organization's short term exposure limits. They also have health concerns with cancer, skin, and respiratory specificity, as shown in Table 5.
Organ toxicity: single exposure Cascade impactor data has shown that nicotine and menthol particles could be deposited in the oropharynx, trachea, bronchioles, and alveoli 19 . This may help us understand how bioavailable these compounds are. As the greatest limitation of these studies is their inability to provide concrete answers to questions about human risk.
To further understand this, a study from Hecht et al 7

Cellular Damage
Several studies have exposed human cells to vapor to understand their effects on cellular activities. Liquids containing nicotine and flavorings were found to have the greatest effect on cells while humectants (propylene glycol/glycerol) alone had little to no effect 47 .
There was a wide variation in the exposure scenarios employed and the devices/liquids used were found as with the chemical profile studies listed above. There also were variations in the exposed cell types which can affect outcomes.

Discussion
TSNAs are contested compounds of particular concern, as they pose significant lung cancer risk due to their pulmonary organ specificity 48 . TSNAs (such as NNN and NNK) have been in some studies 49,50,51 while being absent in others 52 . Small 2 or 3 ring PAHs were also found in one reviewed study 25 , though any presence is of concern given their carcinogenic potential.

Study Designs
Our findings demonstrate a pervasive issue in e-cigarette research, the heterogeneity of device design and liquid composition. This is likely the main source of the profile variation Another challenge to analyzing the current literature is the significant difference in employed study methods. Two main groups of study designs were identified in both cell and profile studies; "short-term exposure" that utilized a short but intense period of exposure, and "vaper-type" groups that modeled exposure after user behavior. Though there was significant heterogeneity within these classifications; with short term exposure times ranging from 24 or 48h in one study, to 50 minutes in another.
Despite this heterogeneity in design and materials, there were still trends in the summarized studies. Specifically, significant increases in formaldehyde, acetaldehyde, and acrolein. As well as some changes in DNA, though the full evidence on this was not extensively reviewed. It is interesting then, that conclusions were still identified when exposures were so varied. Potentially pointing to the intensity of the effects of e-cigarettes.
One of outcomes of this review, is evidence on the effects of glycol on vapor profile.
Several studies found that as glycerol percentage increased, so did the device's toxic profile. This provides an opportunity to restrict the amount of glycol in e-liquids for harm-reduction purposes.
Several studies also tested how glycol/glycerol ratios would affect toxic profiles. A study by Ooi et al 19 liquids with different ratios of propylene glycol and glycerol and found the presence of aldehydes in vapor were related to liquids with higher glycol ratios. This is corroborated by Conklin et al and Wang et al 53,54 . Another identified that the glycerol percentage in liquids had a positive correlation with metal concentration 19 .

Chemical Profile and Cellular Effects
The presence of TSNAs and PAHs in e-cigarettes is contested and cannot be concluded here. Given the carcinogenicity of these compounds, their presence or absence would greatly affect cancer risk. Evidence would point to the possibility of TSNAs and PAHs in at least some e-cigarettes, given the heterogeneity of device profiles seen. The production of these compounds is also heavily reliant on tobacco content and other specific conditions that vary in devices. A focused study testing or TSNA's and PAHs may provide insight into this issue.
Metals found in E-cigarettes correlated to device composition, and thus likely originate from the devices themselves. Though others have proposed that e-cigarettes become contaminated with metals during manufacturing. Our studies identified several device factors that increased metal transfer: a high liquid boiling temperature, high nicotine content, and increased device airflow. This poses an opportunity for design changes to protect users by reducing these factors. It may also be prudent to sell liquids separately from devices, as liquids purchased as "refills" did not contain significant amounts of metal in a study that tested both 22 . Future studies should focus on the effects of e-cigarettes on a select group of cell lines to identify links between device type, cell type, and biomarkers for DNA damage, viability, and pro-cancer protein expression. An analysis of the effects these devices have on human cells, respiratory functioning and symptoms, and respiratory disease prevalence is needed also needed to draw conclusions about the effects of the exposures stated here while offering the opportunity to protect users through concrete understanding and health regulations.

Conclusion
From the current review, e-cigarette vapor is confirmed to contain harmful compounds.
Formaldehyde, acetaldehyde, acrolein, and metals were consistently present in most e-cigarettes.
There was significant variation in the compounds identified in chemical profiles, making further conclusions impossible. There were no commonalities in the devices and liquids used in our reviewed studies and significant differences in the exposure levels used for analysis, which showing that these variations mostly likely originate from the liquids and devices, not study errors. Even with this variation, every study found potentially harmful and carcinogenic compounds, showing no liquid or device can be considered safe.
E-cigarettes contain lower levels of harmful compounds compared to combustion cigarettes, but in concentrations significantly above non-smoking exposure. These lower concentrations still pose health risks, as shown by in vitro studies that identified changes in cell viability, increased DNA mutations, and altered protein expression. Urine metabolites of these compounds have been found in users at significant levels, demonstrating the potential for bioabsorption. Pointing to the possibility that e-cigarette uses impacts cellular functioning and may harm human health.