Canadian researchers have shown that even low levels of exposure to vapour in the lungs can have significant health impacts.
The study, published 25 January 2023 in The FASEB Journal, found that e-cigarettes cause cellular and molecular changes in the lungs of lab mice, with prolonged inhalation of vapour resulting in changes in the animals’ pulmonary immune cell composition and altered gene and protein levels in the lungs.
Their analysis also revealed significant changes in the proteomic profile between the sexes in response to a specific brand of vaporizers and aerosols – JUUL.
JUUL, a puff-activated, pod-style e-cigarette that can deliver the approximate nicotine content of one pack of cigarettes, rapidly gained in popularity, representing nearly three quarters of the US e-cigarette market in 2018, and remains one of the most popular e-cigarette brands among youth and young adults around the world today.
However, JUUL was recently banned in the US, with the FDA issuing JUUL Labs with a marketing denial order on 23 June 2022, as their premarket tobacco product application “lacked sufficient evidence regarding the toxicological profile of the products.”
JUUL’s study findings “raised concerns due to insufficient and conflicting data regarding genotoxicity and potentially harmful chemical leaching from their proprietary e-liquid pods.”
Lead author, Dr Carolyn Baglole, from the Departments of Pharmacology, Pathology and Experimental Medicine at McGill University, Quebec, Canada, explained that although many ingredients in e-liquids are ‘generally recognized as safe’ for oral consumption, there is little data on their safety when aerosolized and subsequently inhaled.
“E-liquids contain varying amounts of humectants, most often a mix of propylene glycol (PG) and vegetable glycerine (VG), flavourings, and nicotine,” Dr Baglole said.
“There are thousands of different flavours and combinations available and as such, it may take decades to fully understand the effects of e-cigarette use in humans.
“Moreover, each device has a heating coil with varying metal contents, and these metals can be found in both the liquid and aerosol… JUUL uses a nichrome heating element and stainless-steel vapor path that can lead to leaching of metals such as chromium, iron, nickel, copper, and even lead into the emitted vapours.
“While there are limited health data regarding the significance of the presence of metals in e-cigarette aerosols, there is cause for concern with notable concentrations of toxic chemicals being inhaled by chronic users, where these metals may build up to toxic concentrations.”
However, comparison between experimental studies, and therefore the ability to draw conclusions, is limited partly by the lack of correlation with human puff topography, such as puff volume and puff interval, as well as objective measurements of exposure.
To replicate human exposures, 8-to 12-week-old male and female C57BL/6J mice were exposed to a puff regime of commercially available JUUL products (containing 59 mg/ml nicotine), consisting of three 20-min exposures per day for four weeks – a length of exposure roughly equivalent to three years in humans.
During exposure, each mouse was given one puff per minute with a 78 ml puff volume, 2.4 s puff duration, with three hours between sessions, and mice in the air-exposed control group placed in the exposure apparatus for the same length of time as the experimental ones.
After the exposure period, inflammatory markers were assessed via qRT-PCR, multiplex cytokine assays, and differential cell count, with proteomic and transcriptomic analyses also performed on samples isolated from the lavage of the lungs – including an unbiased analysis of the proteins and RNA contained within extracellular vesicles (EVs).
The team found that mice exposed to JUUL aerosols for four weeks had significantly increased neutrophil and lymphocyte populations in their lung lavage and several changes in cytokine mRNA expression, even though the frequency of BAL cytokines did not change.
“Neutrophils are one of the first cell types recruited upon infection or insult and thus, are a hallmark of acute inflammation, and Leucocytosis is the main respiratory immune alteration in traditional tobacco smokers,” the authors explained.
“Our observation that neutrophils are increased in the BAL of both PG/VG and JUUL suggests that the neutrophilia observed is independent of nicotine or flavouring chemicals but may be the result of the solvent itself (PG/VG).
Proteomic and transcriptomic analysis also revealed significant changes in many biological pathways including neutrophil degranulation, PPAR signalling, and xenobiotic metabolism.
“The pro-inflammatory genes assessed included tnfα, il1β, and il6, however only the mRNA expression of il6 was significantly increased in response to JUUL,” the authors said.
“We also assessed were the mRNA levels of Muc5ac and Muc5b, genes which encode major mucus-producing proteins in the airways, and although there was a trend toward increased expression of Muc5ac and Muc5b in response to JUUL, this did not reach statistical significance.
“There was, however, a significant decrease in the expression of Ace2, a key anti-inflammatory component of the renin-angiotensin system and the receptor for the SARS-Cov-2 virus responsible for the current COVID pandemic, in response to both PG/VG and JUUL aerosols.”
Ptgs2 mRNA, which encodes cyclooxygenase-2 (Cox-2) protein, was not altered by the exposure regime, however, the expression of Nqo1 and Sod2, genes that are part of the antioxidant response, were also increased in response to PG/VG.
To better understand sex-specific differences in the impact of vaping, the researchers performed a further analysis of the proteins in the EV-enriched BAL between male versus female mice, which revealed that the proteins Cyp4b1, Cyp2f2, and Cyp2b10 were significantly higher in JUUL-exposed males.
“Collectively these data highlight that JUUL exposure significantly alters proteins present in EV-enriched BAL fluid in a sex-dependent manner,” the authors said.
“Our results show that inhalation of the vapor generated by a popular brand of e-cigarette causes widespread changes inside the lungs, data that further highlight that these products are not inert and may lead to lung damage if used long term,” Dr Baglole concluded.
“Prolonged inhalation of e-cigarette aerosols cause changes in pulmonary immune cell composition and alters gene and protein levels in the lungs… Even low exposure to aerosols from JUUL had significant impacts.”