Multi-endpoint analysis of human 3D airway epithelium following repeated exposure to whole electronic vapor product aerosol or cigarette smoke

Lukasz Czekala; Roman Wieczorek; Liam Simms; Fan Yu; Jessica Budde; Edgar Trelles Sticken; Kathryn Rudd; Thomas Verron; Oleg Brinster; Matthew Stevenson; Tanvir Walele

doi:10.1016/j.crtox.2021.02.004

Multi-endpoint analysis of human 3D airway epithelium following repeated exposure to whole electronic vapor product aerosol or cigarette smoke

Curr Res Toxicol. 2021 Feb 20:2:99-115. doi: 10.1016/j.crtox.2021.02.004. eCollection 2021.

Authors

Affiliations

¹ Imperial Brands PLC, 121 Winterstoke Road, Bristol BS3 2LL, United Kingdom.
² Reemtsma Cigarettenfabriken GmbH, An Imperial Brands PLC Company, Albert-EinsteinRing-7, D-22761 Hamburg, Germany.

Abstract

Smoking is a cause of serious diseases in smokers including chronic respiratory diseases. This study aimed to evaluate the tobacco harm reduction (THR) potential of an electronic vapor product (EVP, myblu™) compared to a Kentucky Reference Cigarette (3R4F), and assessed endpoints related to chronic respiratory diseases. Endpoints included: cytotoxicity, barrier integrity (TEER), cilia function, immunohistochemistry, and pro-inflammatory markers. In order to more closely represent the user exposure scenario, we have employed the in vitro 3D organotypic model of human airway epithelium (MucilAir™, Epithelix) for respiratory assessment. The model was repeatedly exposed to either whole aerosol of the EVP, or whole 3R4F smoke, at the air liquid interface (ALI), for 4 weeks to either 30, 60 or 90 puffs on 3-exposure-per-week basis. 3R4F smoke generation used the ISO 20778:2018 regime and EVP aerosol used the ISO 20768:2018 vaping regime. Exposure to undiluted whole EVP aerosol did not trigger any significant changes in the level of pro-inflammatory mediators, cilia beating function, barrier integrity and cytotoxicity when compared with air controls. In contrast, exposure to diluted (1:17) whole cigarette smoke caused significant changes to all the endpoints mentioned above. To our knowledge, this is the first study evaluating the effects of repeated whole cigarette smoke and whole EVP aerosol exposure to a 3D lung model at the ALI. Our results add to the growing body of scientific literature supporting the THR potential of EVPs relative to combustible cigarettes and the applicability of the 3D lung models in human-relevant product risk assessments.

Keywords: 2D, Two Dimensional; 3D, Three Dimensional; 3R4F, Scientific Reference Tobacco Cigarette (University of Kentucky); ALI, Air-Liquid Interface; ANOVA, Analysis of Variance; AOP, Adverse Outcome Pathway; CAA, Cilia Active Area; CBF, Cilia Beat Frequency; COPD, Chronic Obstructive Pulmonary Disease; CYP450, Cytochrome P450; Cigarette; Cilia; DPBS, Dulbecco's phosphate-buffered saline containing Ca2+ and Mg2+; EGFR, Epidermal Growth Factor Receptor; EVP, Electronic Vapor Product; Electronic vapor product; FOX-J1, Forkhead Box J1 protein; H&E, Hematoxylin and Eosin; IIVS, Institute for In Vitro Sciences; IL-13, Interleukin 13; IL-1β, Interleukin 1 Beta; IL-6, Interleukin-6; IL-8, Interleukin-8; ISO, International Organization for Standardization; Immunohistochemistry; KERs, Key Event Relationships; KEs, Key Events; LDH, Lactate Dehydrogenase; MIE, Molecular Initiating Event; MMP-1, Matrix Metalloproteinase-1; MMP-3, Matrix Metalloproteinase-3; MMP-9, Matrix Metalloproteinase-9; MUC5AC, Mucin 5AC Protein; MWP, Multi-Well Plate; NKT, Natural Killer T Cells; Organotypic tissue model; PBS, Phosphate Buffered Saline; PMN, polymorphonuclear; Pro-inflammatory markers; SAEIVS, Smoke Aerosol Exposure In Vitro System; TEER, Transepithelial Electrical Resistance; THR, Tobacco Harm Reduction; TNF-α, Tumor Necrosis Factor Alpha; TPM, Total Particulate Matter.