The Toxicological Effects of E-Cigarette Use in the Upper Airway: A Scoping Review

Abstract

Objective:

While evidence continues to emerge on the negative health effects of electronic cigarettes (e-cigarettes) on the lungs, little is known regarding their deleterious effects on the upper airway. The purpose of this review is to summarize the toxicological effects of e-cigarettes, and its components, on the upper airway.

Data Sources:

PubMed, SCOPUS, EMBASE databases

Review Methods:

Systematic searches were performed in accordance with PRISMA guidelines from 2003–2023. Studies were included if they investigated toxicological effects of e-cigarette exposure on human or animal upper airway tissue. Two authors independently screened, reviewed, and appraised all included articles.

Results:

822 unique articles were identified, of which 53 met inclusion criteria and spanned subsites including the oral cavity (22/53 studies), nasal cavity/nasopharynx (13/53), multiple sites (10/53), larynx (5/53), trachea (2/53), and oropharynx (1/53). The most commonly observed consequences of e-cigarette use on the upper airway included: pro-inflammatory (15/53 studies), histological (13/53), cytotoxicity (11/53), genotoxicity (11/53), and pro-carcinogenic (6/53). E-cigarette humectants independently induced toxicity at multiple upper airway subsites, however, effects were generally amplified when flavoring(s) and/or nicotine were added. Across almost all studies, exposure to cigarette smoke exhibited increased toxicity in the upper airway compared with exposure to e-cigarette vapor.

Conclusion:

Current data suggests that while e-cigarettes are generally less harmful than traditional cigarettes, they possess a distinct toxicological profile that is enhanced upon addition of flavoring(s) and/or nicotine. Future investigations into under-examined subsites such as the oropharynx and hypopharynx are needed to comprehensively understand the effects of e-cigarettes on the upper airway.

INTRODUCTION

Since their introduction into the United States market in the mid-2000s, electronic cigarettes, e-cigarettes or “vapes” have gained significant popularity. Much of the early wave of e-cigarette popularity was among the younger age groups. In 2018, 14.8% of all adults in the United States reported having used an e-cigarette, with the highest percentage (25.8%) in the 18 to 24 year-old range.¹ In fact, in 2014, e-cigarettes surpassed conventional combustible cigarettes in popularity for this age range.² This could be due to the fact that until 2016, e-cigarettes were left largely unregulated in the United States, and lacked the same advertisement and promotional restrictions placed on conventional cigarettes. E-cigarette companies benefited greatly from this delay, as made evident by the roughly $2.35 billion generated from e-cigarette sales in the United States in 2016.³

Among the many reasons that patients choose to use e-cigarettes, the one most commonly cited is smoking cessation and the resulting health benefits.⁴ In fact, the idea of utilizing e-cigarettes as a tool to stop smoking has been lauded among those in the harm reduction community and promoted by numerous governing health agencies, such as the British Royal College of General Practitioners.⁵ A 2021 Cochrane review comprising 61 separate studies found moderate evidence that nicotine-containing e-cigarettes increased quit rates of conventional cigarettes when compared to non-nicotine e-cigarettes and other nicotine replacement therapies.⁶ Nevertheless, despite the perceived benefits of e-cigarettes, their use is accompanied by their own unique risks.

Due to the heterogeneous composition of ingredients in these vaporized liquids and the mechanics of delivery, the overall health effects from using e-cigarettes are distinct from those associated with using conventional cigarettes and can be difficult to predict. Briefly, e-liquids (typically made of a combination of propylene glycol, glycerol, and flavoring with or without nicotine) are heated on a coiled wire, which creates a vapor/aerosol that can be inhaled through the mouthpiece. Compared to traditional tobacco smoke, particle size distribution in e-cigarette vapor is more wide ranging. In a recent study, aerosol particle size generated from e-cigarette use was shown to be tri-modal, with peaks at 40 nm, 200 nm, and 1 μm, and size distribution primarily impacted by the power of the vaping device being used.⁷ Based on particle deposition studies, it is estimated that smaller sized particles (40–200 nm) are more likely to deposit in the trachea and lower airway compared to large particles (1–2 μm) which primarily deposit in the oral cavity and oropharynx. The large distribution in particle sizes, primarily due to user device power levels, creates differential harmful effects of e-cigarette use at sites within the upper and lower airway. Additionally, e-cigarette users have been shown to practice exclusive nasal exhalation patterns at significantly higher rates than traditional cigarette users.⁸ These behavioral differences predispose the nasal cavity to toxicological impacts of e-cigarette use. However, while there has been significant research over the past 10 years regarding the effects of e-cigarette use on the lower airways, including in the context of Acute Respiratory Distress Syndrome (ARDS), diffuse alveolar hemorrhage, and lipid pneumonia, few studies have examined the effects on the upper airway.⁹ To fill this void, we examined the available evidence regarding the effect of e-cigarette use on the different subsites of the upper airway.

METHODS

Search Methodology, Inclusion Criteria, and Data Collection

A scoping review of the literature was performed following PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses)¹⁰ guidelines (Figure 1, Supplemental Data). The study did not require IRB approval from the authors’ institution. This scoping review was not registered online and a publically-available study protocol was not generated. A query of publications written in English between 2003, when the e-cigarette was invented, to November 2023 was performed in PubMed, SCOPUS, and EMBASE databases with the assistance of a research librarian. The complete search strategy for all three databases with included MeSH (Medical Subject Heading) terms is included (Supplemental Data). References from the retrieved articles were also analyzed for any missed studies that were relevant to and could be included in our review. Two authors independently screened all identified articles in Covidence (https://www.covidence.org/). A consensus was achieved based on pre-determined inclusion and exclusion criteria. Inclusion criteria encompassed studies that analyzed the toxicological effects of e-cigarette aerosol/vapor, condensate, or e-liquid exposure on human or animal upper airway tissue. Exclusion criteria included non-original research (e.g. review articles, editorials), case reports, inaccessible articles, Non-English studies, and articles investigating only dental subsites (e.g. dental enamel, periodontal ligament, gingiva) or saliva.

Figure 1.

PRISMA Flow Diagram of Literature Search and Study Inclusion

Data Extraction, Analysis, and Quality Assessment

A schematic flow diagram highlighting the PRISMA search methodology is shown in Figure 1. Data were extracted from the included studies and reviewed independently by two investigators. A spreadsheet in Microsoft Excel (Redmond, WA, USA) was used to denote manuscript and author information, country, year of publication, study design, tissue/cell type, e-cigarette component or flavor studied (if applicable), research findings, and associated toxicological findings. Toxicological findings were classified as cytotoxicity, genotoxicity, pro-inflammatory, histological, pro-carcinogenic, immunosuppression/increased infectivity, ciliary dysfunction, and/or other according to pre-specific criteria outlined in Figure 2. Finally, each article was assigned a “level of evidence” for its study design based on the Oxford Centre for Evidence-based Medicine- 2011 Levels of Evidence guidelines¹¹.

Figure 2.

Toxicological Classification of Results

RESULTS

The literature search yielded 1588 total records. After removal of duplicates, 822 unique abstracts were screened, resulting in the inclusion of 53 studies (Figure 1, Table 1).^12–64 Out of the 53 included articles, 34 (64 %) analyzed the effects of e-cigarette use on human subjects or cell lines, 16 (30%) utilized an animal model to investigate the in-vivo effects of e-cigarette aerosol exposure, and 3 (6%) studied both human and animal models. Human studies included: 1) in vitro assessments of e-cigarette aerosol, condensate, or e-liquid exposure on primary cultured cells or immortalized cell lines derived from upper airway tissue, or 2) clinical studies using samples obtained from current or former e-cigarette users. Using the Oxford Centre for Evidence-Based Medicine: Levels of Evidence guidelines, 36 of the included studies were graded as level 5 evidence, whereas 17 studies were graded as level 2b evidence (Table 1).

Table 1.

Major Characteristics of the 53 Included Studies

Study	Year	City, Country	Study Type	Tissue Type	Comparison to cigarette smoke	Sample, Participant, or Animal	Duration of Exposure	Methodology	Site		Flavors Examined (if stated)	E-Cigarette Components (if stated)	Oxford Level of Evidence
Oral Cavity
Ji EH, et al12	2016	Multi-national (USA, China)	Basic Science	Human	No	Oral keratinocyte cell line	24 hours of e-cigarette aerosol exposure	ATP assay, GSH assay, qPCR	Oral mucosa		Not stated	PG/VG + Nicotine + Flavor	5
Franco T, et al13	2016	Magna Graecia, Italy	Clinical	Human	Yes	Buccal mucosa scrape biopsy from 23 cigarette smokers, 22 e-cigarette users, and 20 non-smokers	Not stated	Exfoliative cytology, Optical microscopy	Buccal mucosa		Not stated	Not stated	2b
Ganapathy V et al14	2017	Multi-national (USA, India)	Basic Science	Human	Yes	Dysplastic oral keratinocyte cell line (POE9n), Oral SCCa cell line (UM-SCC-1)	1, 10, or 100 puffs/5 L, 1 hour/day, every other day for 2 weeks of e-cigarette aerosol condensate exposure	q-PADDA assay, ELISA, MTT assay, TAC assay, DCFDA assay, RT-PCR, Western Blot	Oral mucosa		Traditional, Desert Sands	PG/VG +/− Nicotine + Flavor	5
Tommasi S, et. al.15	2019	Los Angeles, CA, USA	Clinical	Human	Yes	Buccal mucosa scrape biopsy from 42 e-cigarette users, 24 cigarette smokers, and 27 non-smokers	Not stated	RNA-sequencing, qRT-PCR, IPA	Buccal mucosa		Not stated	Not stated	2b
Iskander AR, et al16	2019	Multi-national (Switzerland, USA)	Basic Science	Human	Yes	Organotypic buccal epithelial cultures (MatTek)	28 minutes of e-cigarette aerosol exposure	Histopathology, Multiplex immunoassay, qRT-PCR, IPA	Buccal mucosa		Not stated	PG/Glycerol +/− Nicotine +/− Flavor	5
Ureña JF, et al17	2020	University Park, PA, USA	Basic Science	Human	No	Oral SCCa cell line (SCC-25)	45 total puffs of 4 seconds duration of e-cigarette aerosol exposure	MTT assay, Immunofluorescence	Oral mucosa, Gingiva		Lava Flow, Very Cool, Hawaiian Pog, American Patriots	PG/VG +/− Nicotine + Flavor	5
Tsai KYF, et al18	2020	Provo, UT, USA	Basic Science	Human	No	Oral SCCa cell line (Ca9-22), Tongue SCCa cell line (CAL-27)	24 hours of e-liquid exposure	Real-Time Cell Invasion, Immunofluorescence, ELISA	Oral mucosa, Tongue		Red Hot, Green Apple	PG/VG +/− Nicotine + Flavor	5
Pop AM, et. al.19	2021	Tirgu Mures, Romania	Clinical	Human	Yes	Buccal mucosa scrape biopsy from 25 cigarette smokers, 23 e-cigarette users and 20 non-smokers	≥ 12 months of daily e-cigarette use	Exfoliative cytology, Optical microscopy	Buccal mucosa		Not stated	Not stated	2b
Guo J, et. al.20	2021	Minneapolis, MN, USA	Clinical	Human	Yes	Buccal mucosa scrape biopsy from 35 non-smokers, 30 cigarette smokers, and 30 e-cigarette users	≥ 3 months of daily e-cigarette use; ≥ 12 months of daily cigarette use	LC-NSI-HRMS/MS	Buccal mucosa		Not stated	Not stated	2b
Muqawwi et al21	2021	Bandung, Indonesia	Clinical	Human	Yes	Buccal mucosa scrape biopsy from 20 non-smokers, 20 cigarette smokers, and 15 e-cigarette users	≥ 12 months of e-cigarette use	Micronucleus assay	Buccal mucosa		Not stated	Not stated	2b
Tellez CS, et al22	2021	Multi-national (USA, Japan)	Basic Science	Human	Yes	3 immortalized oral epithelial cell lines	20 minutes of e-cigarette aerosol exposure	Neutral red uptake assay, ROS-Glo assay, TBARS assay, Alkaline comet assay, Micronuclei assay	Oral mucosa		Arctic Blast, Blue Pucker, Jamestown, Love Potion, Mardi Gras, Midnight Splash, Port Royale, Tobacco Row, Tortuga, Uptown	PG/VG +/− Nicotine +/− Flavor	5
Schwarzmeier LAT, et. al.23	2021	Sao Jose dos Campos, Brazil	Clinical	Human	Yes	Oral cavity scrape biopsy from 20 e-cigarette users, 22 current cigarette smokers, 22 former cigarette smokers, and 27 non-smokers	≥ 5 months of e-cigarette use	Exfoliative cytology, Optical microscopy	Lateral tongue, floor of mouth		Not stated	Not stated	2b
Hamad S, et. al.24	2021	Chapel Hill, NC, USA	Clinical	Human	No	Buccal mucosa scrape biopsy from 3 healthy e-cigarette users	≥ 2 months of daily e-cigarette use; Scripted vaping of 20 puffs over 20 minutes	qRT-PCR, IPA	Buccal mucosa		Not stated	PG/VG + Nicotine	2b
Cátala-Valentín AR, et al25	2022	Orlando, FL, USA	Basic Science	Human	No	Oral epithelial cell line (OKF6)	30 minutes of e-cigarette condensate exposure	EdU cell proliferation assay, Biofilm measurement, ELISA, qRT-PCR, Immunofluorescence	Oral mucosa		None	PG/VG +/− Nicotine	5
Marinucci L et al26	2022	Perugia, Italy	Basic Science	Human	Yes	Oral keratinocyte cell line (PSC-200-01)	24 hours of e-cigarette aerosol condensate exposure	MTT assay, Scanning electron microscopy, Scratch assay, Flow cytometry, qRT-PCR	Oral mucosa		Tobacco	PG/VG + Nicotine + Flavor	5
Cheng G et al27	2022	Minneapolis, MN, USA	Clinical	Human	Yes	Buccal mucosa scrape biopsy from 8 cigarette smokers, 20 e-cigarette users and 20 non-smokers	≥ 3 months of 4 days/week of e-cigarette use; ≥ 12 months of daily cigarette use	LC-NSI-HRMS/MS	Buccal mucosa		Not stated	Not stated	2b
Robin H et al28	2022	South Jordan, UT, USA	Basic Science	Human	No	Oral SCCa cell line (Ca9-22), Tongue SCCa cell line (CAL-27)	6 hours of e-cigarette aerosol condensate exposure	ELISA, Western Blot, Real time cell invasion	Oral mucosa		Red Hot, Green Apple	PG/VG +/− Nicotine + Flavor	5
Reeve G et al29	2022	New York, NY, USA	Clinical	Human	No	Non-smokers and e-cigarette users	Active e-cigarette users	Histopathology, Transcriptomics	Oral mucosa		Not stated	Not stated	2b
Mandour D et al30	2023	Zagazig, Egypt	Basic Science	Murine	No	30 adult male albino rats	1 hour/day, 5 days/week, for 4 weeks of e-cigarette aerosol exposure	ELISA, Histopathology, Immunohistochemistry	Submandibular gland		Not stated	VG/PG + Nicotine + Flavor	5
Elmahdi F et al31	2023	Madinah, Saudi Arabia	Clinical	Human	No	Buccal mucosa scape biopsy from 250 non-smokers, 250 e-cigarette users	≥ 6 months of daily e-cigarette use	Papanicolaou staining, Immunohistochemistry	Buccal mucosa		Not stated	Not stated	2b
Tommasi S et al32	2023	Los Angeles, CA, USA	Clinical	Human	Yes	Oral mucosa scrape biopsy from 24 non-smokers, 24 e-cigarette users, 24 cigarette smokers	≥ 3x week for 6 months of e-cigarette use; ≥ 3x week for 12 months of cigarette use	LA-QPCR	Oral epithelium		Fruit, Sweet, Mint/Menthol, Tobacco	VG/PG + Nicotine + Flavor	2b
de Lima JM, et al33	2023	Multi-national (Brazil, Canada)	Basic Science	HumanMurine	No	Human oral mucosal keratinocyte and tongue epithelial cell lines, human tongue SCCa cell lines (CAL-27, HSC3), and mouse oral cancer cell line (AT84)	7, 24, or 48 hours of e-liquid exposure	MTT assay, Colony formation assay, Wound healing migration assay, Cell invasion assay, Western Blot, qRT-PCR, Immunocytochemistry	Oral mucosa, Tongue		Not stated	PG/Glycerine + Nicotine + Flavor	5
Oropharynx
Welz C, et. al.34	2016	Munich, Germany	Basic Science	Human	No	Primary oropharyngeal epithelial cells from healthy adult donors	24 hours of e-liquid exposure	MTT assay, Alkaline microgel electrophoresis	Oropharynx mucosa		Apple. Cherry, Tobacco	PG/VG + Nicotine + Flavor	5
Nasal Cavity/Nasopharynx
Martin EM, et. al.35	2016	Chapel Hill, NC, USA	Clinical	Human	Yes	Nasal biopsy and lavage fluid from 13 non-smokers, 14 cigarette smokers, 12 e-cigarette users	≥ 6 months of e-cigarette use	Liquid chromatography tandem mass spectrometry, Nanostring based gene expression analysis, IPA, ELISA	Nasal mucosa, Nasal lavage fluid	Not stated		Not stated	2b
Kumral TL, et. al.36	2016	Istanbul, Turkey	Clinical	Human	No	72 current smokers in cessation clinic randomized to e-cigarette smoking group (n=42) or non e-cigarette group (n=30).	≥ 5 years of daily cigarette use	Mucociliary clearance (via saccharin transit time), SNOT-22	Nasal epithelium	Not stated		Not stated	2b
Carson JL, et al.37	2017	Chapel Hill, NC, USA	Basic Science	Human	Yes	Primary nasal epithelial cells from healthy non-smoking adults	20 consecutive e-cigarette “puffs”	Electron microscopy, Nitric oxide production, Ciliary beat frequency	Nasal epithelium	Tobacco		PG/VG + Nicotine + Flavor	5
Miyashita L et al38	2018	Multi-national (UK, South Africa)	Basic Science	Human, Murine	No	11 adult vaping males; Primary nasal epithelial cells from a healthy never-smoking, never-vaping adult; Female CD1 mice	≥ 1 week of e-cigarette use; 2x/day of e-cigarette aerosol condensate exposure	Immunostaining, Flow cytometry, Adhesion assay, Miles and Misra method	Nasal epithelium	Not stated		PG/VG +/− Nicotine + Flavor	5
Jabba S et al39	2020	Durham, NC, USA	Basic Science	Human	No	Primary nasal epithelial cells (PromoCell)	24 hours of e-liquid flavor exposure	LIVE/DEAD Viability/Cytotoxicity assay	Nasal epithelium	Benzaldehyde, Vanillin		PG + Flavor	5
Rouabhia M, et al40	2020	Quebec, Canada	Basic Science	Human	Yes	Primary nasal epithelial cells from healthy non-smoking adults	15 minutes, 2x/day, for 3 days of e-cigarette aerosol exposure	Tryptan blue exclusion assay, LDH assay, MTT assay, Ki67 immunostaining, ELISA assay	Nasal epithelium	Smooth Canadian Tobacco		PG/VG +/− Nicotine + Flavor	5
Rebuli ME, et al41	2021	Chapel Hill, NC, USA	Clinical	Human	Yes	Nasal lavage and epithelial lining fluid from 20 non-smokers, 14 cigarette smokers, and 15 e-cigarette users	Not stated	Viral load, NanoString nCounter, qRT-PCR, ELISA, Influenza-specific IgA quantification	Nasal epithelial-lining fluid, Nasal lavage fluid, Nasal mucosa	Not stated		Not stated	2b
Escobar YN et al42	2021	Chapel Hill, NC, USA	Basic Science	Human	No	Primary nasal epithelial cells from adult smokers and nonsmokers	20 consecutive e-cigarette “puffs” each of 4 secs duration	Western blot, ELISA	Nasal epithelium	None		PG/VG +/− Nicotine	5
Kwak S et al43	2021	Daegu, Republic of Korea	Basic Science	Human	No	Primary nasal epithelial cells (Epithelix)	24 hours of Glyoxal or Methylglyoxal exposure	WST-1 assay, Western blot, ELISA	Nasal epithelium	None		Glyoxal or Methylglyoxal	5
Hinds D et al44	2022	Iowa City, IA, USA	Basic Science	Murine	No	Male golden Syrian Hamsters	2 hours/day for 2 days of e-cigarette aerosol exposure	Histopathology, qRT-PCR	Nasal epithelium	None		PG/VG +/− Nicotine	5
Karey E et al45	2022	Chapel Hill, NC, USA	Clinical	Human	Yes	Nasal epithelial lining fluid from 37 never-smokers, 16 cigarette smokers, and 20 e-cigarette users	E-cigarette or cigarette use within last 7 days	ELISA	Nasal epithelial lining fluid	Not stated		Not stated	2b
Pozuelos et al46	2022	Riverside, CA, USA	Clinical	Human	Yes	Nasal biopsy from 3 never-smokers, 3 cigarette smokers, and 3 e-cigarette users	Active e-cigarette or cigarette use	RNA sequencing, Gene Ontology, IPA	Nasal epithelium	Not stated		Not stated	2b
Nicholas BD, et. Al.47	2022	Syracuse, NY, USA	Basic Science	Murine	No	Male and female BALB/cJ mice	15 minutes, 3x/day for 5 days/week for 8 weeks of e-cigarette aerosol exposure	Histopathology	Eustachian tube mucosa	Tobacco		PG/VG + Flavor	5
Larynx
Salturk Z, et al.48	2015	Istanbul, Turkey	Basic Science	Murine	No	Female Wistar albino rats	1 hour/day for 4 weeks of e-cigarette aerosol exposure	Histopathology, Ki67 immunostaining	Vocal fold epithelium		None	PG/VG + Nicotine	5
Ha TN, et. Al.49	2019	Houston, TX, USA	Basic Science	Murine	Yes	C57BL/6 mice	31 minutes/day, 5 days/week for 16 weeks of e-cigarette aerosol exposure	ELISA	Larynx		None	PG/VG +/− Nicotine	5
Hassan NH, et al50	2022	Zagazig, Egypt	Basic Science	Murine	No	Adult male Wistar albino rats	1 hour/day, 5 days/week for 4 weeks of e-cigarette aerosol exposure	Histopathology, Ki67 and p53 immunostaining, MDA and TAC levels, Transmission electron microscopy	Larynx		Not stated	PG/VG + Nicotine + Flavor	5
Martinez JD, et al.51	2022	Palo Alto, CA, USA	Basic Science	Human	Yes	Immortalized vocal fold fibroblast cell line	24 hours of e-cigarette condensate exposure	MTT assay, qRT-PCR, Immunofluorescence	Vocal fold epithelium		None	PG/VG +/− Nicotine	5
Easwaran M et al52	2023	Stanford, CA, USA	Basic Science	Murine	No	Male C57BL6/J mice	2 hour/day for 1, 5, and 10 days of e-cigarette aerosol exposure	Histopathology, Immunohistochemistry, immunofluorescence, Scanning electron microscopy	Larynx		Mint, Mango	PG/VG + Nicotine + Flavor	5
							Trachea
Wu Q et al53	2014	Denver, CO, USA	Basic Science	Human	No	Primary tracheobronchial epithelial cells	24 or 48 hours of e-liquid exposure	LDH assay, ELISA, qRT-PCR	Trachea		Tobacco	PG/VG +/− Nicotine	5
El-Merhie et al54	2021	Borstel, Germnay	Basic Science	Drosophilia melanogaster	No	L3 larvae	2 min, every hour x 8 sessions of e-cigarette aerosol exposure	Stereomicroscope, NeuronJ	Trachea		None	PG/VG + Nicotine	5
Multi-site
Manyanga J, et al55	2021	Oklahoma City, OK, USA	Basic Science	Human	Yes	Floor of mouth SCCa cell line (UM-SCC-1), Tongue SCCa cell line (WSU-HN6), and Pharyngeal SCCa cell line (WSU-HN30)	48 hours of e-cigarette condensate exposure	Tryptan blue exclusion assay, MTT assay, qRT-PCR, Western Blot	Tongue, Floor of mouth, and Pharynx		Not stated	PG/VG +/− Nicotine	5
Werley MS, et al56	2016	Richmond, VA USA	Basic Science	Murine	No	Male and female Sprague-Dawley rats	16, 48, or 160 minutes/day for 13 weeks of e-cigarette aerosol exposure	Histopathology	Nasal epithelium, Larynx		Not stated	PG/VG +/− Nicotine +/− Flavor	5
Phillips B, et al57	2017	Multi-national (Singapore, Germany, Switzerland)	Basic Science	Murine	No	Male and female Sprague-Dawley rats	6 hours/day, 5 days/week for 13 weeks of e-cigarette aerosol exposure	Histopathology, DNA microarray, Proteomics	Nasal epithelium, Larynx		None	PG/VG +/−Nicotine	5
Lee KM, et al58	2018	Multi-national (Switzerland, USA)	Basic Science	Murine	Yes	Female C57BL/6 mice	4 hours/day for 13 days of e-cigarette aerosol exposure	Histopathology	Nasal epithelium, Larynx, trachea		Not stated	PG/VG + Nicotine +/− Flavor	5
Ho J, et al59	2020	Multi-national (Singapore, Switzerland)	Basic Science	Murine	No	Male and female Sprague-Dawley rats	6 hours/day, 5 days/week for 13 weeks of e-cigarette aerosol exposure	Histopathology, DNA microarray	Nasal epithelium, Larynx		Not stated	PG/VG +/− Nicotine +/− Flavor	5
Ni F, et. al.60	2020	Baltimore, MD, USA	Basic Science	Murine	No	Male and female C57BL/6 mice	30 minutes of e-cigarette aerosol exposure	Immunohistochemistry, Event-related potential recordings	Nasal epithelium, Trachea		Vanilla, Menthol, Cinnamon	PG/VG + Nicotine + Flavor	5
Wong ET et al61	2021	Multi-national (Singapore, Switzerland, USA, Germany)	Basic Science	Murine	Yes	Female ApoE−/− mice	3 hours/day, 5 days/week for 6 months of e-cigarette aerosol exposure	Histopathology, Transcriptomics	Nasal epithelium, Larynx, Trachea		Not stated	PG/VG +/− Nicotine +/− Flavor	5
Kim M et al62	2022	Kansas City, KS, USA	Clinical & Basic Science	Human, Sheep	No	Primary nasal epithelial cells and epithelial lining fluid from healthy non-smoking adults; Tracheal secretions from adult female sheep	Humans vaped 80 puffs/day for 8 days; Sheeps exposed to 80 puffs/day for 5 days of e-cigarette aerosols	Nasal ion transport assay, ELISA	Nasal epithelium, Nasal epithelial lining fluid, Tracheal secretions		None	VG +/− PG	2b
Wong ET, et al63	2022	Multi-national (Singapore, Switzerland, USA)	Basic Science	Murine	Yes	Male and female A/J mice	6 hours/day for 5 weeks of e-cigarette aerosol exposure	Histopathology	Nasal epithelium, Larynx, Trachea		Not stated	PG/VG + Nicotine +/− Flavor	5
Desai R et al64	2023	Multi-national (USA, Greece)	Basic Science	Murine	Yes	Male and female Sprague-Dawley rats	6 hours/day, 5 days/week for 90 days of e-cigarette aerosol exposure	Histopathology	Nasal epithelium, Larynx, Trachea		Virginia Tobacco, Menthol	PG/VG + Nicotine + Flavor	5

Abbreviations: ATP, Adenosine; GSH, Glutathione; qPCR, quantitative polymerase chain reaction, PG, propylene glycol; VG, vegetable glycerin; SCCa, squamous cell carcinoma; q-PADDA, quantitative primer-anchored DNA damage detection assay; ELISA, enzyme-linked immunosorbent assay; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; TAC, total antioxidant capacity; DCFDA, 2’,7’–dichlorofluorescin diacetate; qRT-PCR, reverse transcription quantitative polymerase chain reaction; IPA, Ingenuity Pathway Analysis; LC-NSI-HRMS/MS, Liquid Chromatography-Nanoelectrospray Ionization-High-Resolution Tandem Mass Spectrometry; ROS, reactive oxidative species; TBARS, Thiobarbituric Acid Reactive Substances; EdU, 5-ethynyl-2’-deoxyuridine; LA-QPCR, long-amplicon quantitative polymerase chain reaction; SNOT-22, Sino-nasal Outcome Test; LDH, lactatae dehydrogenase; WST-1, Water-soluble tetrazolium salt-1, MDA, malondialdehyde

Across included clinical studies, duration of e-cigarette use by study participants was highly variable, ranging from a minimum of seven days⁴⁵ to over a year of daily use¹⁹ (Table 1). For human in vitro studies, exposure time to e-cigarette aerosols, condensate, or e-liquid also varied greatly, ranging from seconds³⁷ to over two weeks¹⁴ (Table 1). Animal in-vivo studies were generally designed to simulate sub-chronic e-cigarette aerosol exposures and, as such, were typically multi-week in duration.

The majority of the assessed studies analyzed the effects of e-cigarette use on the oral cavity (22/53 studies), followed by the nasal cavity/nasopharynx (13/53), multiple sites (10/53), larynx (5/53), trachea (2/53), and oropharynx (1/53). The toxicological impact of e-cigarette use on the upper airway included: pro-inflammatory (15/53 studies), histological (13/53), cytotoxicity (11/53), genotoxicity (11/53), pro-carcinogenic (6/53), immunosuppression or infectivity (4/53), ciliary dysfunction (4/53), and other findings (3/53) (Table 2). In six studies, no toxicity was observed following exposure to e-cigarettes (Table 2).

Table 2.

Major Findings and Toxicities in the 53 Included Studies

Upper Airway Site	Study	Major Findings	Toxicity
Oral Cavity	Ji EH, et al12	Dose-dependent decrease in intracellular GSH, decreased cell viability, and increased gene expression of HO-1 in oral keratinocytes exposed to flavored e-cigarette aerosols (+ nicotine) vs air controls.	Cytotoxicity
	Franco T, et al13	Decreased micronuclei in oral epithelial cells of e-cigarette users compared to cigarette smokers; no difference between non-smoking controls and e-cigarette users.	None
	Ganapathy V et al14	Dose-dependent increase in DNA damage, increased intracellular ROS, decreased intracellular TAC, and decreased gene expression of the DNA repair proteins ERCC1 and OGG1 in oral keratinocytes exposed to flavored e-cigarette aerosol condensate (+/− nicotine) vs vehicle controls. Cigarette smoke exerted increased DNA damage but similar intracellular oxidative stress and protein expression changes compared to e-cigarettes	Genotoxicity, Cytotoxicity
	Tommasi S, et al15	Significantly increased numbers of differentially expressed genes in oral epithelial cells of cigarette smokers and e-cigarette users (1726 genes, 1152 genes; respectively) vs non-smokers. IPA showed that “Cancer” was the top disease pathway associated with differentially expressed genes in both e-cigarette users and cigarette smokers. The most enriched canonical pathway was “Wnt/Ca+” in e-cigarette users, and “integrin signaling” in cigarette smokers. Oral epithelial cells from e-cigarette users and cigarette smokers exhibited downregulation of tumor suppressors NOTCH1 and HERC2, and upregulation of BCL-2 and its binding heat-shock protein HSPA1B compared to non-smokers.	Pro-carcinogenic
	Iskander AR, et al16	No changes in histopathology in buccal epithelial cells exposed to e-cigarette aerosols (+/− nicotine) compared to air controls. Increased secretion of IL-1α, decreased secretion of IL-13, and increased expression of genes associated with “inflammatory process” network family in buccal epithelial cells exposed to e-cigarette aerosols (+/− nicotine) vs air controls. Cigarette smoke exposure resulted in buccal epithelial cell morphological changes, increased secretion of IL-1β, and increased number of differentially expressed genes compared to e-cigarettes.	Pro-inflammatory
	Ureña JF, et al17	Increased intracellular ROS and decreased cell viability in SCC-25 cells that were exposed to Lava Flow flavored e-cigarette aerosols (+/− nicotine) vs air controls. No cytotoxicity seen following exposure to other flavored e-cigarette aerosols.	Cytotoxicity
	Tsai KYF, et al18	Increased Ca9-22 invasion and decreased Cal-27 invasion following exposure to Red Hot flavored e-liquid (+/− nicotine) vs media-only treated controls. Decreased Ca9-22 invasion following exposure to Green Apple flavored e-liquid (+/− nicotine) vs controls. Increased RAGE expression and differential expression of secreted cytokines IL-1α, IL-8, MMP-13 in both cell lines following exposure to Red Hot or Green Apple. Nicotine addition to both e-liquid flavors enhanced RAGE expression, however reduced secretion of IL-1α, IL-8.	Pro-carcinogenic, Pro-inflammatory
	Pop AM, et al19	Increased micronuclei in oral epithelial cells of e-cigarette users and cigarette smokers vs non-smoking controls; no difference between e-cigarette users and cigarette smokers.	Genotoxicity
	Guo J, et al20	Significantly lower apurinic/apyramidinic sites in e-cigarette users vs nonsmokers and cigarette smokers.	None
	Muqawwi et al21	Decreased number of micronucleated cells and micronucleous in oral epithelial cells of e-cigarette users and non-smoking controls vs cigarette smokers (no difference between e-cigarette users and non-smoking controls).	None
	Tellez CS, et al22	Significant heterogeneity across different flavors; however, oral epithelial cells exposed to flavored e-cigarette aerosols (+/− nicotine) generally displayed increased intracellular oxidative stress, lipid peroxidation, cellular toxicity, and micronuclei formation compared to cells exposed to unflavored e-cigarette aerosols. Cigarette smoke exposure induced dose-dependent cytotoxicity, intracellular oxidative stress, and DNA damage, which exceeded any flavored or unflavored e-cigarette aerosol.	Cytotoxicity, Genotoxicity
	Schwarzmeier LAT, et. al23	Oral epithelial cells from e-cigarette users displayed increased metanuclear abnormities as evidenced by: increased karolysis (vs never-smokers and former cigarette smokers), increased binucleation (vs never-smokers and former smokers), increased “broken egg” (vs cigarette smokers, never-smokers, and former cigarette smokers), and increased “nuclear bud” (vs never-smokers and former smokers).	Genotoxicity
	Hamad S, et. al24	Increased expression of genes related to DNA damage (TP53, FEN1, TREX1, XRCC2, AIFM1) in oral epithelial cells following scripted vaping session. Specifically, TP53 expression was puff volume and flow rate dependent. IPA showed that “cancer”, “cell cycle”, and “DNA repair” pathways were the most enriched pathways following e-cigarette exposure.	Genotoxicity, Pro-carcinogenic
	Cátala-Valentín AR, et al25	Oral epithelial cells exposed to e-cigarette condensate (+/− nicotine) showed a dose-dependent decrease in cell viability, increased COX2 expression, increased pro-inflammatory pERK1/2 and NF-kB nuclear translocation and signaling, increased immunofluorescence of DNA damage marker pH2AX, and reduced secreted cytokines (IL-8, IL-6, IL-1β) vs controls who were treated with media exposed to air.	Cytotoxicity, Genotoxicity, Pro-inflammatory
	Marinucci L et al26	Oral epithelial cells exposed to e-cigarette aerosol condensate (+nicotine) displayed no changes in cell viability, morphology, apoptosis rate, or gene expression compared to media-treated controls. Oral epithelial cells exposed to cigarette smoke condensate showed significant alterations in cellular morphology, increased apoptosis rate and cellular migration vs media-treated controls.	None
	Cheng G et al27	Oral epithelial cells from e-cigarette users displayed higher levels of acrolein-DNA adducts compared to non-smokers, but less than cigarette smokers.	Genotoxicity
	Robin H et al28	Human oral squamous cell carcinoma cells exposed to Green Apple flavored e-cigarette aerosol condensate (+ nicotine) displayed increased protein expression of NF-kB, TNF-α, ERK, JNK, MMP-13 and increased cell invasion vs media-exposed controls. Human tongue squamous cell carcinoma cells exposed to Green Apple or Red Hot flavored-cigarette aerosol condensate (+ nicotine) showed increased protein expression of TNF-α and JNK vs media-exposed controls.	Pro-inflammatory, Pro-carcinogenic
	Reeve G et al29	There were no statistically significant histological or transcriptomic changes in the oral epithelium of e-cigarettes users vs non-smokers.	None
	Mandour D et al30	Albino rats exposed to nicotine-containing e-cigarette aerosols demonstrated widening of submandibular acini and ducts along with epithelial degeneration, cytoplasm vacuolization, connective tissue septa thickening, and increased TNF-α immunostaining compared to submandibular glands from air controls.	Histological, Pro-inflammatory
	Elmahdi F et al31	E-cigarette users displayed higher rates of oral epithelial cytological atypia (4.8 vs 0.4%) and HPV infection rates (3.2 vs 0.8%) vs non-smokers. Among e-cigarette users, higher atypia rates and HPV infection rates were seen in individuals with a longer duration of e-cigarette use (> 7 years).	Histological, Immunosupression or Infectivity
	Tommasi S et al32	Oral epithelial cells from e-cigarette users displayed a dose-dependent increase in DNA damage vs non-smoking controls. There was no difference in DNA damage levels between e-cigarette users and smokers. Nicotine content was not associated with increased DNA damage. Among e-cigarette flavors, fruit, sweet, and mint/menthol showed the highest amount of DNA damage.	Genotoxicity
	Muniz de Lima J, et al33	Increased cytotoxicity in normal oral epithelial cells treated with e-liquid (+ nicotine) vs untreated control. Human and murine oral SCCa cells treated with e-liquid (+ nicotine) showed increased cell proliferation, cell migration and invasion, as well as gene expression changes consistent with an epithelial to mesenchymal transition when compared to untreated controls.	Cytotoxicity, Pro-carcinogenic
Oropharynx	Welz C, et al34	Increased cytotoxicity and DNA fragmentation in oropharyngeal epithelial cells exposed to e-cigarette condensates (fruit flavors > tobacco) vs controls.	Cytotoxicity, Genotoxicity
Nasal Cavity/ Nasopharynx	Martin EM, et al35	Decreased expression of immune-related genes (ZBTB16, PIGR, PTGS2, FKBP5) and transcription factors (NFKB1, ETS1, NOTCH1, BCL3, XBP1, BCL6, EGR1) in nasal epithelial cells from current e-cigarette users and cigarette smokers compared to non-smokers (> suppression in e-cigarette users vs. cigarette smokers). Top IPA canonical pathways in both e-cigarette users and cigarette smokers included: “cytokine-cytokine receptor interaction,” “apoptosis,” “Toll-like receptor signaling pathway,” and “NOD-like receptor signaling pathway.”	Immunosuppression or Infectivity, Pro-inflammatory
	Kumral TL, et al36	After 3 months of cigarette smoking cessation, both e-cigarette users and non-smokers displayed lower SNOT-22 scores vs baseline (SNOT-22 of non-smokers < e-cigarette users), whereas after 3 month of smoking cessation, only non-smokers had significantly lower mucociliary clearance times.	Ciliary dysfunction
	Carson JL, et al37	Human nasal epithelial cells exposed to cigarette smoke or e-cigarette aerosols displayed similar declines in ciliary beat frequency with gradual return of function within 1 hour. Nitric oxide production was far greater in nasal epithelial cells exposed to cigarette smoke vs e-cigarette aerosol or unexposed controls. Following exposure to e-cigarette aerosols, nasal epithelial cells displayed secretory material overlying ciliary beds indicating mucous hypersecretion; however, this finding was much more prominent in nasal epithelial cells exposed to cigarette smoke.	Histological, Ciliary dysfunction
	Miyashita L et al38	Nasal epithelial PAFR expression increased following vaping session in e-cigarette users. Pneumococcal adhesion increased in human nasal epithelial cells following exposure to e-cigarette aerosol condensate (+/− nicotine) compared to vehicle controls. In CD1 mice, e-cigarette aerosol condensate (+ nicotine) exposure increased nasal PAFR expression and nasopharyngeal pneumococcal colonization vs vehicle controls.	Immunosupression or infectivity
	Jabba S et al39	Human nasal epithelial cells exposed to the benzaldehyde-propylene glycol adduct (berry/fruit e-cigarette flavoring) showed increased cell death vs benzaldehyde exposure alone.	Cytotoxicity
	Rouabhia M, et al40	Nasal epithelial cells exposed to e-cigarette aerosol (+/− nicotine) or cigarette smoke showed increased LDH levels, fewer Ki67+ cells, reduced cellular proliferation rate, and increased secretion of IL-6, IL-8, TNF-α, and MCP-1 compared to controls (Magnitude of effects: cigarette smoke > nicotine-rich e-cigarette aerosols > nicotine-free e-cigarette aerosols).	Pro-inflammatory, Cytotoxicity
	Rebuli ME, et al41	Compared to non-smokers, e-cigarette users and cigarette smokers showed suppression of live attenuated influenza vaccine (LAIV)-induced nasal immune response as evidenced by decreased gene expression, decreased cytokine/chemokine secretion, and decreased LAIV-specific IgA levels (Magnitude of effects: e-cigarette users > cigarette smokers > non-smokers)	Immunosuppression or infectivity
	Escobar YN et al42	Human nasal epithelial cells from non-smokers exposed to e-cigarette aerosols (+/− nicotine) demonstrated increased protein expression of MUC5AC vs air controls. Human nasal epithelial cells from non-smokers exposed to e-cigarette aerosol (+ nicotine) showed elevated MUC5AC and MUC5B protein levels vs air controls. Human nasal epithelial cells from smokers exposed to e-cigarette aerosols (+/− nicotine) showed elevated pro-inflammatory cytokine secretion.	Pro-inflammatory
	Kwak S et al43	Human nasal epithelial cells exposed to the e-cigarette compounds (glyoxal or methylglyoxal) demonstrated increased secretion of IL-1β, IL-6, MUC5AC, MUC5B, and increased activation of nuclear factors (ERK1/2, p38, and NF-κB) compared to culture-exposed controls.	Pro-inflammatory
	Hinds D et al44	Golden Syrian mice exposed to 2 days of e-cigarette aerosols displayed nicotine-dependent increases in nasal epithelial gene expression of CCL-5, CXCL-10, TNF-α, IL-1β, TGF-β, and nicotine-independent increases in gene expression of SOD-2 and decreases in gene expression of VEGF vs air controls. There were no changes in the histology of the nasal epithelium of e-cigarette aerosol exposed mice compared to air controls.	Pro-inflammatory
	Karey E et al45	Pro-inflammatory cytokine levels (IL-2, IL-4, IL-8, IL-10, IL-12p70, IL-13, TNF-α, IL-1β, IFN-γ) were significantly increased in the nasal fluid of e-cigarette users compared to cigarette users and non-smoking controls.	Pro-inflammatory
	Pozuelos et al46	Significantly increased number of differentially expressed genes in primary human nasal epithelial cells of cigarette smokers and e-cigarette users (407 genes, 1817 genes; respectively) vs non-smokers. Enriched gene ontology terms for the downregulated genes between e-cigarette users and non-smokers included “cilium assembly and function” and for the upregulated genes included “immune response”, “neutrophil activation”, “leukocyte degranulation”, and “granulocyte activation”.	Pro-inflammatory, Ciliary dysfunction, Immunosupression or Infectivity
	Nicholas BD, et al47	BALB/cJ mice exposed to 8 weeks of e-cigarette aerosols displayed decreased goblet cells in Eustachian tube mucosa vs controls (no significant differences in cilia, mucin, and squamous metaplasia). In BALB/cJ mice exposed to e-cigarette aerosol, trans-tympanic application of anti-IL-13 or AG1478 led to increased goblet cells in Eustachian tube mucosa.	Histological
Larynx	Salturk Z, et al48	Wistar albino rats exposed to 4 weeks of e-cigarette aerosols (+ nicotine) displayed increased vocal fold hyperplasia and metaplasia, however results were not statistically significant.	None
	Ha TN, et al49	C57BL/6 mice exposed to 16 weeks of e-cigarette aerosols (+ nicotine) or cigarette smoke displayed significantly elevated levels of IL-4 in larynx homogenates compared to control mice exposed to air or e-cigarette aerosols without nicotine.	Pro-inflammatory
	Hassan NH, et al50	Wistar albino rats exposed to 4 weeks of e-cigarette aerosols (+ nicotine) displayed increased MDA levels and decreased TAC levels in laryngeal tissue homogenates vs control rats exposed to air. Histologically in the larynx, e-cigarette-exposed rats showed increased cilia loss, increased epithelial hyperplasia, increased vascular dilation of the lamina propria, increased p53 immunostaining, and decreased Ki67 immunostaining compared to control rats exposed to air.	Cytotoxicity, Histological, Ciliary dysfunction
	Martinez JD, et al51	Human vocal fold fibroblasts (hVFFs) exposed to e-cigarette condensate (+/− nicotine) showed decreased cell viability vs untreated controls. Compared to e-cigarette condensate, hVFFs exposed to cigarette smoke condensate showed a greater reduction in cell viability and increased DNA damage. Gene expression was not significantly altered in hVFFs following either e-cigarette or cigarette smoke condensate exposure.	Cytotoxicity, Genotoxicity
	Easwaran M et al52	There were no differences in vocal fold epithelial thickness, cellular proliferation, glandular surface area, surface topography between C57BL6/J rats exposed to flavored e-cigarette aerosol (+ nicotine) or air controls. There was a slight increase in acidic mucus content in the subglottis and increase in macrophage and CD3+ T cell infiltration in the vocal cords of e-cigarette exposed mice vs air controls.	Histological
Trachea	Wu Q et al53	Tracheobronchial epithelial cells exposed to tobacco flavored e-liquid (+/− nicotine) showed increased IL-6 secretion, enhanced human rhinovirus infection, and decreased SPLUNC1 expression. There was no change in cytotoxicity of tracheobronchial epithelial cells exposed to flavored e-liquid (+/− nicotine) at 24 or 48 hours post exposure.	Pro-inflammatory, Immunosupression or Infectivity
	El-Merhie et al54	Maternal Drosophilia Melanogaster flies exposed to nicotine-containing e-cigarette aerosols prior to mating produced offspring with abnormalities in tracheal length compared to sham controls.	Other
Multisite	Manyanga J, et al55	E-cigarette (+/− nicotine) or cigarette smoke condensate exposure in various oral cavity and oropharyngeal cancer cell lines reduced cisplatin-induced cytotoxicity, reduced expression of DNA repair genes MMS19 and ERCC1, decreased expression of drug influx protein CTR1, and increased expression of drug efflux proteins ABCG2 and ATP7A compared to cells exposed to saline treated controls.	Pro-carcinogenic, Genotoxicity
	Werley MS, et al56	Sprague-Dawley rats chronically exposed to 13 weeks of e-cigarette aerosols (+/− nicotine or flavor) displayed increased nasal secretions, nasal mucous cell hyperplasia and epithelial vacuolization, and intraluminal mucin exudate in the larynx; histological changes did not resolve following 42-day recovery period.	Histological
	Phillips B, et al57	Sprague-Dawley rats chronically exposed to 13 weeks of e-cigarette aerosols (+ nicotine) displayed mild adaptive squamous cell metaplasia and basal cell hyperplasia in the larynx vs air controls. No statistically significant histological changes in nasal epithelium. There were no changes in nasal epithelial gene or protein expression between rats exposed to e-cigarettes or filtered air.	Histological
	Lee KM, et al58	C57BL/6 exposed to 13 days of e-cigarette aerosols had a minimal increase in microscopic squamous metaplasia of the nasal turbinates and epiglottis compared to sham controls. All histological findings were significantly more severe in cigarette smoke-exposed group.	Histological
	Ho J, et al59	Sprague-Dawley rats exposed to 13 weeks of e-cigarette aerosols (+ nicotine) displayed mild adaptive squamous cell metaplasia and lamina propria infiltration in the larynx. No statistically significant histological changes in nasal epithelium. No biologically significant gene expression differences in nasal epithelium of rats exposed to e-cigarettes aerosols vs PBS vehicle controls.	Histological
	Ni F, et al60	C57BL/6 mice exposed to flavored e-cigarette aerosols (+/− nicotine) displayed increased numbers of stimulated nociceptive neurons (nasal cavity > trachea) vs air controls.	Other
	Wong ET et al61	ApoE−/− mice exposed to 6 months of e-cigarette aerosols (+/− nicotine) displayed no differences in nasal or tracheal histology and only minimal squamous metaplasia at the epiglottis compare to the sham control group. All histological findings were significantly more severe in cigarette smoke-exposed group. E-cigarette exposed mice showed no significant changes in gene expression in the nasal epithelium or trachea following exposure.	Histological
	Kim M, et al62	Reduced nasal CFTR function was seen in human subjects after 7 days of vaping vegetable glycerol (VG)-containing e-cigarettes vs baseline. Primary human nasal epithelial cells exposed to VG-containing e-liquid displayed increased secretions of pro-inflammatory mediators (IL-6, IL-8, MMP-2, MMP-9, TGF-β) vs baseline. Tracheal secretions from sheep exposed to 5 days of VG-containing e-cigarette aerosols showed increased mucus concentrations and MMP-9 activity vs baseline	Pro-inflammatory, Other
	Wong ET, et al63	A/J mice exposed to 5 weeks of flavored e-cigarette aerosols (+ nicotine) displayed a mild adaptive increase in nasal and laryngeal epithelial hyperplasia and laryngeal squamous metaplasia. No significant histological changes in trachea. All histological findings were significantly more severe in cigarette smoke-exposed group.	Histological
	Desai R, et al64	Sprague-Dawley rats exposed to 13 weeks of e-cigarette aerosols (+ nicotine) displayed mild squamous cell metaplasia in the larynx vs air controls. No statistically significant histological changes were seen in the nasal epithelium. All histological findings were significantly more severe in cigarette smoke-exposed group	Histological

Abbreviations: GSH, glutathione; ROS, reactive oxidative species, TAC, total antioxidant capacity; IPA, Ingenuity pathway analysis; RAGE, receptor for advanced glycation end-products; HPV, human papilloma virus; SNOT-22, Sino-nasal Outcome Test; MDA, malondialdehyde

Cytotoxicity was seen in all upper airway subsites, except the trachea, following e-cigarette exposure and was most commonly measured via changes in markers of cellular apoptosis rate, cellular metabolic activity, and/or intracellular oxidative species or antioxidant levels.^{12,14,17,22,25,33,34,39,40,50,51} Pro-inflammatory effects were seen in all upper airway subsites, except for the oropharynx. These included elevated levels of secreted soluble inflammatory mediators (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α, IFN-γ, CCL-5, CXCL-10, MCP-1, MMP-2, MMP-9, MMP-13, MUC5AC, MUC5B), increased expression of genes or proteins involved in inflammatory signaling (ERK1/2, p38, COX-2, JNK, and NF-kB), and enrichment of gene sets or pathways associated with inflammatory processes.^{16,18,25,28,30,35,40,42–46,49,53,62} Genotoxicity was seen primarily in the oral cavity, but also in the oropharynx and larynx, and was detected by increased DNA fragmentation, adducts, double- or single-strand breaks, appearance of micronuclei or metanuclear abnormalities, or increased expression of genes or proteins involved with DNA damage or repair pathways.^{14,19,22–25,27,32,51}

Histological changes associated with acute and sub-chronic e-cigarette aerosol exposure in animal models were seen in the oral cavity, submandibular gland, nasal cavity, larynx, and trachea; however, the majority of these changes were considered adaptive and resolved following a recovery period.^{30,31,37,47,50,52,56–59,61,64} Persistent histological changes in the nasal cavity and larynx included: mild epithelial hyperplasia and squamous metaplasia, and mucus hypersecretion. In the submandibular gland, ductal dilation, connective tissue thickening, and epithelial degeneration with cytoplasmic vacuolization was observed.³⁰ Pro-carcinogenic features such as downregulation of tumor suppression genes were seen in normal oral cavity epithelial cells following exposure to e-cigarette aerosols or e-liquid.^15,33 Additionally, in both human oral and oropharyngeal squamous cell carcinoma cell lines, exposure to e-cigarette aerosols and flavorings led to increased cellular proliferation/survival, migration, invasion, and resistance to cytotoxic chemotherapeutic agents.^18,28,55 Various mechanisms for e-cigarette-induced immunosuppression were seen in the nasal cavity, including suppression of an influenza-induced immune response and decreased expression of genes related to immune cell function.^35,41,46 Additionally, enhanced bacterial and viral infectivity following e-cigarette exposure were observed and included increased nasopharyngeal pneumococcal colonization³⁸, and increased human papillomavirus and rhinovirus infection rates in oral and tracheobronchial epithelial cells, respectively^31,53. Ciliary dysfunction was also seen in the nasal cavity of e-cigarette users and in primary nasal epithelial cells following exposure to e-cigarette aerosols, and was measured by increased mucociliary clearance times³⁶, decreased ciliary beat frequency³⁷, and downregulation of genes associated with cilium assembly and function⁴⁵. Other toxicological effects of e-cigarettes that were identified included: embryological abnormalities in tracheal development in Drosophilia Melanogaster flies⁵⁴, enhanced stimulation of nociceptive neurons in the nasal cavity and trachea of mice⁶⁰, and reduced nasal cystic fibrosis transmembrane conductance regulator protein function in human subjects⁶².

A variety of e-cigarette components were analyzed. In clinical studies, the brand of e-cigarettes used by subjects was generally not provided, and no information was included regarding whether devices contained added flavorings and/or nicotine. Thirteen human in-vitro studies^{14,16,17,18,22,25,28,38,40,42,51,53,55} analyzed the effects of e-cigarette aerosol/condensate exposure with and without nicotine and showed mixed toxicological effects: eight studies^{14,16,17,22,25,51,53,55} demonstrated similar effects with and without nicotine; whereas five studies^{18,28,38,40,42} showed aerosol/condensate exposure from nicotine-containing e-cigarettes had enhanced toxicity. In these five studies, the addition of nicotine to e-cigarettes enhanced cytotoxicity, increased secretion of inflammatory mediators, increased intracellular oxidative stress, increased bacterial infectivity, and enhanced cellular invasion and RAGE expression. In sub-chronic exposure studies in animal models^49,56,57,59, histological findings of squamous cell metaplasia in the larynx was predominately seen in those exposed to nicotine-containing e-cigarette aerosols as opposed to nicotine-free e-cigarette aerosols.

Twenty-six studies investigated the effects of flavored e-cigarettes.^{12,14,16–18,22,26,28,30,32–34,37–40,47,50,52,56,58,59,61,63,64} The most commonly studied flavors were tobacco and mint/menthol, which appeared in 9/26 and 4/26 studies, respectively. One study²² compared the effects of ten different flavored e-liquids vs. unflavored e-liquid of the same brand on oral epithelial cells and showed that, in general, flavored e-liquids (+/− nicotine) were associated with increased oxidative stress, lipid peroxidation, cytotoxicity, and micronuclei formation compared to unflavored e-liquid. Eleven studies^{14,17,18,22,28,32,34,39,52,60,64} directly compared various e-cigarette flavors to one another; however, the flavors varied between studies limiting the generalizability of results.

Twenty-five studies^{13–16,19–23,26,27,32,35,37,40,41,45,46,49,51,55,58,61,63,64} directly compared the effects of exposure to e-cigarette vapor vs. traditional cigarette smoke. In general, exposure to traditional cigarette smoke/condensate resulted in increased cytotoxicity, intracellular oxidative stress, DNA damage, and upregulated pro-inflammatory response compared with exposure to e-cigarette aerosol/condensate (+/− nicotine). Additionally, in sub-chronic animal exposure studies, cigarette smoke led to more severe and permanent histological changes in both the nasal cavity and larynx compared to e-cigarette aerosols (+/− nicotine).^58,61,63,64

DISCUSSION

This scoping review collates the current literature and highlights the effects of e-cigarettes on the upper airway, a topic that is becoming a greater public health concern. The studies we analyzed are heterogeneous and include: human clinical studies using samples from current or former e-cigarette users; in-vitro experiments utilizing primary cell cultures or immortalized cell lines derived from human or animal upper airway tissue; or in-vivo studies utilizing animal models to characterize sub-chronic e-cigarette aerosol exposure. A majority of studies primarily analyzed tissue from the oral cavity and were spearheaded by dental or basic science researchers. Few studies analyzed other subsites in the upper aerodigestive tract and even less were led by Otolaryngologist.

E-liquids require solvents/humectants such as propylene glycol (PG) and glycerol (vegetable glycerin) to maintain moisture and the ability to vaporize. Studies in this review demonstrated toxicity from exposure to e-cigarette aerosols or e-liquids in the absence of nicotine or flavoring, which highlights the potential harmful effects of these solvents/humectants on the upper airway.^{16,22,25,42,44,49,51,53,55,56,57,59,61,62} Across human in-vitro studies, exposure to e-cigarette humectants alone increased the secretion of soluble inflammatory mediators in the oral cavity^22,25, nasal cavity^42,44, and trachea⁶²; induced cytotoxicity in oral epithelial cells²² and vocal fold fibroblasts⁵¹; promoted genotoxicity in oral epithelial cells^22,25; and enhanced human rhinovirus infection in tracheobronchial epithelial cells⁶². Additionally, exposing oral cavity and oropharyngeal cancer cell lines to humectant condensate reduced cisplatin-induced cytotoxicity.⁵⁵ This finding supports the idea that exposure to e-cigarette vapor promotes resistance to chemotherapy and may worsen the efficacy of oral cavity and oropharyngeal cancer treatment, similar to the effects of tobacco smoke.

Studies investigating histological changes in the upper airway following sub-chronic humectant aerosol exposure in animal models were mixed. Increased nasal secretions, nasal mucous cell hyperplasia and epithelial vacuolization, and increased intraluminal mucin exudate was observed in the larynx of Sprague-Dawley rats following a 90-day exposure⁵⁶; however, in two other murine exposure studies^57,59, histological changes were only seen when nicotine was added to humectants. The toxicological effects of humectants have been corroborated in lower airway studies as humectant aerosol exposure increased the secretion of inflammatory cytokines compared to air controls in human bronchial epithelial cells.⁶⁵ Further, humectant aerosol exposure disrupted lipid homeostasis in murine alveolar macrophages and airway epithelial cells resulting in decreased surfactant production and impaired immunological response to viral infections.⁶⁶

While the above studies demonstrate that e-cigarettes can cause damage to tissues of the upper airway, even if they only including humectants such as glycerol and propylene glycol, it is probable that the addition of flavorings that make e-cigarettes so popular contribute additional deleterious effects. In lower airway studies^65,67,68, e-cigarette flavorings directly influence cytotoxicity. However, only one study²² included in this review directly compared flavored vs. unflavored e-cigarettes of the same brand. In this study, out of the ten flavors tested only one, Arctic Blue, demonstrated lower combined toxicity scores compared to the unflavored e-liquid. While data from this singular study strongly suggest that the addition of flavorings to e-liquids has toxicological impact, additional future studies are needed to better characterize the specific effect in the upper airway.

One challenge of investigating the toxicological impact of flavorings is the heterogeneity of flavors between studies, which limits the generalizability of results. Additionally, flavoring compounds associated with common flavor names (e.g. fruit) often vary across brands, which precludes accurate comparisons of their toxicological impact. Our review found eleven studies^{14,17,18,22,28,32,34,39,52,60,64} that directly compared flavors against one another; however, only menthol and tobacco were included in more than two studies. Notably, tobacco flavoring appeared to be generally less harmful than the other flavors studied. Tobacco flavored e-cigarettes were less cytotoxic and genotoxic to oropharyngeal cells than fruit flavors³⁴ and were less cytotoxic to oral epithelial cells compared with other e-cigarette flavorings (sweet, mint/menthol, fruit)³². Given the wide variety of e-cigarette flavorings available on the market–from tobacco to chocolate to strawberry–and the different chemicals used to achieve those flavors, there is much to glean from specifically assessing the interactions of these additives with upper aerodigestive tract cells. Testing the effects of available flavorings should be prioritized in future studies given the allure that flavors hold in the public perception of these products.

E-cigarettes are promoted as a tool to stop smoking conventional cigarettes and are perceived as relatively safe; therefore, it is critically important to assess the effects of exposure to e-cigarettes on tissues of the upper aerodigestive tract compared to the effects of exposure to conventional cigarette smoke. A majority of studies in this review found that upper airway tissue that was exposed to cigarette smoke exhibited more severe toxicological effects compared to tissue exposed to e-cigarettes vapors. In particular, exposure to conventional cigarette smoke induced dose-dependent cytotoxicity, intracellular oxidative stress, and DNA damage in oral epithelial cells at levels that far exceeded the effects of any flavored or unflavored e-cigarette aerosol either with or without the addition of nicotine.²² Similar effects were shown across other upper airway subsites. Human vocal fold fibroblasts that were exposed to media treated with cigarette smoke extract demonstrated a greater reduction in cell viability and increased DNA damage compared to cells exposed to media treated with e-cigarette extract.⁵¹ In sub-chronic animal exposure studies, cigarette smoke consistently led to more severe and permanent histological changes in the nasal cavity and larynx compared to e-cigarette vapor either with or without the addition of nicotine.^58,61,63,64 In the nasal cavity, nasal epithelial cells exposed to e-cigarette aerosol (+/− nicotine) or cigarette smoke showed increased intracellular oxidative stress, reduced cellular proliferation rate, and increased secretion of inflammatory mediators compared to air controls, although the magnitude of effects were larger in the group exposed to cigarette smoke.⁴⁰ Of note, e-cigarettes seem to exert a comparable or possibly greater immunosuppressive effect in the nasal cavity compared to conventional tobacco cigarettes. In particular, nasal epithelial cells from current e-cigarette users and cigarette smokers exhibited decreased expression of immune-related genes and transcription factors compared to non-smokers, with greater suppression observed in the e-cigarette cohort.³⁵ More recent work has shown that following inoculation with the live-attenuated influenza virus, current e-cigarette users and conventional cigarette smokers demonstrated similar impairment in viral nasal immune responses, as evidenced by decreased gene expression, secretion of inflammatory cytokines and chemokines, and influenza-specific IgA as compared with non-smokers.⁴¹

This scoping review had several limitations. Firstly, clinical studies had varying inclusion criteria for the duration of e-cigarette use and many studies had limited information regarding the types of e-cigarettes used by participants. Future clinical studies should include information regarding the brand of e-cigarette used by subjects as well as whether the device contains nicotine and/or flavoring additives. An additional concern was the limited number of studies investigating the toxicological effects of e-cigarettes at upper airway sites outside of the oral and nasal cavities. There was only one study that investigated effects in the oropharynx and no studies examined effects in the hypopharynx. Future studies investigating these other subsites are needed to fully characterize the toxicological profile of e-cigarettes on the upper airway. Finally, across the in-vitro studies there was significant heterogeneity in the methods used to expose cells to e-cigarettes. These included exposing upper airway cells: 1) directly to e-liquid, 2) to e-cigarette aerosol condensate in culture media, and 3) to e-cigarette aerosols utilizing an exposure chamber. It is possible that there are differential toxicological effects following exposure to heated e-cigarette aerosols vs. e-liquid vs. e-cigarette condensate. As in-vivo exposure to e-cigarettes in the upper airway physiological occurs in aerosol form, priority should be place on using this method of exposure in future in vitro studies.

CONCLUSIONS

E-cigarette use has become increasingly mainstream in the last two decades, especially in younger populations; however, there is a lack of research investigating its deleterious effects, particularly in the upper airway. This scoping review compiled the current literature reporting the effects of e-cigarettes in the upper aerodigestive tract to better understand their harms and to identify avenues for future research. The consensus from our analysis is that exposure to e-cigarette aerosols, condensate, or e-liquids can increase cytotoxicity, pro-inflammatory effects, genotoxicity, histological changes, pro-carcinogenic effects, immunosuppression, healing impairment, ciliary dysfunction, microbiome changes, and neurotoxicity at multiple sites within the upper airway. While future investigations are needed to fully characterize the effects of e-cigarettes on less studied sites such as the oropharynx and hypopharynx, the otolaryngologist should counsel patients that emerging evidence indicates that vaping is toxic to upper airway tissue and should ideally be avoided. Patients should be advised that while e-cigarettes are likely less harmful to the upper airway compared to traditional cigarettes, they have an independent toxicological profile that is enhanced with the addition of nicotine or flavorings that often vary considerably between brands. If e-cigarettes are to being considered as a bridge for smoking cessation, the clinician should have a detailed discussion with patients about the potential risks and benefits of this approach and a shared decision should be made. Finally, patients should be made aware that due to their recency of introduction, data regarding the health effects of long-term e-cigarette use are not currently known. As the primary caregivers for diseases affecting the upper airway, otolaryngologists should take initiative in forwarding research to help identify the possible injurious effects of e-cigarettes so that they can appropriately counsel patients.