Mice forced to inhale e-cigarette vapour during pregnancy

Research Paper:

Maternal E‑Cigarette Exposure Results in Cognitive and Epigenetic Alterations in Offspring in a Mouse Model (2018)
Tara Nguyen, Gerard E. Li, Hui Chen, Charles G. Cranfield, Kristine C. McGrath, and Catherine A. Gorrie


Funded by the UTS Faculty of Science and UTS Center for Health Technologies.


Novel Object Recognition (NOR) test- The Novel Object Recognition (NOR) task is used to evaluate cognition, particularly recognition memory, in rodent models of CNS disorders. This test is based on the spontaneous tendency of rodents to spend more time exploring a novel object than a familiar one.
Elevated plus maze (EPM)- The Elevated Plus Maze (EPM) test is used to assess anxiety-related behavior in rodent models of CNS disorders. The EPM apparatus consists of a “+”-shaped maze elevated above the floor with two oppositely positioned closed arms, two oppositely positioned open arms, and a center area. As subjects freely explore the maze, their behavior is recorded by means of a video camera mounted above the maze and analyzed using a video tracking system. The preference for being in open arms over closed arms (expressed as either as a percentage of entries and/or a percentage of time spent in the open arms) is calculated to measure anxiety-like behaviour
Epigenetic- external modifications to DNA that turn genes “on” or “off.” These modifications do not change the DNA sequence, but instead, they affect how cells “read” genes
DNA methylation- an epigenetic mechanism that occurs by the addition of a methyl (CH3) group to DNA, thereby often modifying the function of the genes.
Chromatin modification enzymes– Chromatin modification involves enzymes that affect DNA transcription. Chromatin modifying enzymes essentially work to make the condensed chromatin accessible for transcription factors, DNA replication, recombination, and repair.


Electronic cigarettes (e-cigarettes) are battery operated devices that heat a liquid (called ‘e-liquid’) to produce a vapour that users inhale. Although their composition varies, e-liquids typically contain a range of chemicals, which may include nicotine (1) and vitamin e acetate which, when inhaled, may interfere with normal lung functioning (2).

The first electronic nicotine delivery systems first began development in 1963, however it was not until 2003 that the modern e-cigarette was invented, by Chinese pharmacist Hon Lik (3).

In a 2010 review (4) of the evidence regarding the safety of e-cigarettes, it was suggested “a preponderance of the available evidence shows them to be much safer than tobacco”. Indeed, they were a tobacco industry disruptor and originally thought to be an alternate and safer option for regular smokers.

However, concerns have emerged involving the link between e-cigarette use and the reports of associated lung disease. Beginning in 2019, the United States experienced an outbreak of ‘e-cigarette, or vaping, product use-associated lung injury’ (EVALI) in which 2,807 hospitalised cases or deaths were reported.(2)

The Experiment:

Researchers from the School of Life Sciences at the University of Technology in Sydney studied the effect in the offspring of mice of e-cigarette vapour inhalation by mothers for 6 weeks before pregnancy, throughout the pregnancy, and during lactation.

Twenty-four female mice were housed in groups of four on a 12- hour light, 12- hour dark cycle before being randomly separated in to three groups of eight. One group was exposed only to ambient air, one group to e-cigarette vapour without nicotine, and the last group to vapour with nicotine. During the experiment, mice were placed in a 9 L chamber filled with e-cigarette aerosols Inhalation exposure occurred twice daily, in fifteen-minute exposures totalling 30 minutes a day.

Following the collective birth of 126 babies, the offspring were randomly divided into three groups and killed at intervals: one group on their first day of life, one group at twenty days, and the final group at 13 weeks following behavioural trials. Once killed, the first two groups were dissected, and their brains removed and stored until the conclusion of the third group’s trials.

Those in the third group began behavioural assessments at twelve weeks of age. The first was to examine short-term memory and mice were given the challenge of identifying familiar objects they had previously been exposed to against unfamiliar objects. It was expected that unimpaired mice spend less time exploring a familiar object than they would a new object.

The group exposed to vapour containing nicotine did not identify familiar objects in a reduced timeframe as expected and as shown in the other two groups, indicating “offspring from maternal exposure to e-cigarette aerosols with nicotine may have short-term memory deficits”.

Following this, the final group of mice were tested for anxiety by being placed on an elevated plus maze (EPM) and allowed to navigate the maze for two minutes.

Both groups who were exposed to the vapour displayed “reduced anxiety levels” by exploring the exposed sections of the elevated platform more than the group who was not exposed to vapour.

Brain tissue was analysed to investigate neurological changes during gestation (D1), lactation (D20), and at maturity (week 13).

DNA was assessed for global DNA methylation levels. 1 Ecig(−nic) and D20 Ecig(−nic) samples showed significantly higher global DNA methylation compared to the sham group. At week 13, global DNA methylation was investigated only in the hippocampus, and no significant global DNA methylation change between the control and treatment groups in these older-aged mice was observed. The changes in global DNA methylation in the whole brain indicates that e-cigarette aerosols induce epigenetic changes. Global DNA methylation and alterations in chromatin modification enzymes were found at all stages of the developing offspring. It is speculated that this can potentially cause developmental delays and cognitive deficits in offspring.

Relevance to Humans:

The lack of translation between results in mice and humans is a key concern. Researchers often use animals with shorter life spans, such as mice, to extrapolate data into human relevant timespans. For example, a mouse gestation period is typically between 19-21 days compared to 37-42 weeks for a human.

Mice develop and age very differently to humans, and major developmental life events occur at different rates. A human reaches puberty at around 11.5 years of age, whereas a mouse reaches puberty at 42 days, the approximate equivalent of 5 years of age in humans.

There are also specific challenges to using mice in smoking studies. Whether an animal is an obligate nose breather, the structure of the nasal turbinates, respiration rate, etc. influence the size and number of particles reaching the alveoli. For example, rodents are obligate nose breathers with more convoluted nasal passages than humans, potentially resulting in test substances being deposited in the nasal passages before they can cause lung injury (6).

Calculating and comparing inhalation rates is also problematic. The inhalation calculations are based on previous referenced studies in which mice were exposed to cigarette smoke, the researchers concluded that the nicotine dosage in two fifteen-minute vapour exposures “is equivalent to smoking two tobacco cigarettes twice daily”. However, human inhalation of one cigarette takes approximately 5-7 minutes depending on the manufacturer and ‘quality’ of the cigarette, the presence or absence of a filter, the inhalation speed of the user, and environmental factors such as humidity and air temperature.

During the experiment, mice “were placed in a 9 L chamber filled with e-cigarette aerosols” for a total of 30 minutes each day in two 15- minute exposures with a five minute “washout interval” where the mice were provided a break in the exposure. The sourced e-liquid contained a nicotine “concentration of 18 mg/mL”. One average cigarette contains 10-12mg of nicotine (5). In contrast, typically, when a human smokes, they inhale deeply once on the cigarette, exhale the smoke, then take a few breaths of ambient air before their next cigarette inhalation.

Results from using mice in laboratory research can be impacted by unpredictable stress causing factors including, but not limited to, the way they are handled, the size and discomfort of their cages, and noise levels. It cannot be reliably determined which of these may have impacted the results of research in addition to the direct research methods. “The cumulative effect is that these stressors cause animals to be less reliable and less representative of human biology (7)”.

Additionally, a recent study in the journal Nature (8) demonstrated that differences in the brains of mice and humans result in a tenfold difference in the synthesis of neurotransmitter receptors. As some neurotransmitters regulate anxiety, these species differences could impact on the conclusions drawn.

Animal Welfare:

The researchers observed that the expectant mothers “tolerated the aerosol exposure without discomfort”. It is unclear exactly what methods were used in determining this aside from assuming basic observation.

The mice were housed in groups of four with enrichment, but it is unclear as to how they were grouped for the experimental inhalation procedure.

Alternatives to Animals:

In the field of vaping research, VITROCELL® has developed cigarette-smoking machines and robots, including an adaptor for e-cigarettes, that can be used to study the impact of gases, environmental atmospheres, nanoparticles and complex mixtures on the lungs. Whilst not directly relevant to this research, it is an indication of the need for and existence of human-relevant research to overcome species differences.

There have been significant and numerous studies into the effects of inhalant use during pregnancy by observing and researching voluntary human participants. For example, the Norwegian Mother and Child Cohort Study (9) in 2017 performed a human volunteer trial to identify the link between maternal and paternal smoking during pregnancy. The trial involved over 100,000 maternal smoking volunteers and included records of associations with paternal smoking, grandmother’s smoking when pregnant with the mother, and maternal smoking in previous pregnancies.

Due to their relatively recent introduction, it is not possible to obtain similar data for e-cigarette use. However, it is reported that 11% of Australians aged 14 and over have used e-cigarettes (10), giving a significant amount of potential human volunteers with which to conduct direct human experience research. It is promising to see that since this research was conducted, human relevant research is progressing, including UK research into the effects of prenatal cigarette and e-cigarette exposure on infant neurobehaviour (11).

The effects of vaping are already being researched in Australia using human volunteers. The University of Southern Queensland is beginning research into the effects of e-cigarettes on respiratory function and exercise capacity on otherwise healthy 18-35 year- old human volunteers (12)


1 https://www.nhmrc.gov.au/health-advice/all-topics/electronic-cigarettes
2 https://www.cdc.gov/tobacco/basic_information/e-cigarettes/severe-lung-disease.html
3 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432409/
4 https://link.springer.com/article/10.1057/jphp.2010.41
5 https://www.healthline.com/health/how-much-nicotine-is-in-a-cigarette#nicotine-in-cigarettes
6. https://www.pcrm.org/news/good-science-digest/physicians-committee-urges-cdc-use-human-relevant-models- study-lung
7 https://muse.jhu.edu/book/57527
8 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919571/
9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5260151/
10 https://www.tobaccoinaustralia.org.au/chapter-18-harm-reduction/indepth-18b-e-cigarettes/18b-3-extent
11 https://www.thelancet.com/action/showPdf?pii=S2589-5370%2820%2930346-1
12 https://www.usq.edu.au/news/2020/09/ecigarettes-exercise-study

For an example of a non-animal case study researching the impact of vaping on bronchial epithelial cells read here.

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