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

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

doi:10.1016/j.crtox.2021.02.004

Cited by 15 publications

(27 citation statements)

References 89 publications

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“…Overall, it was clear that the effects of 1R6F smoke (diluted to a higher level) were substantially greater, and generally occurred earlier, than those of the p-HTPs. Generally greater effects of combustible cigarette smoke, compared to other test articles, in repeated exposure studies with such models have also been previously observed ( Czekala et al, 2021 ; Haswell et al, 2021 ).…”

Section: Discussionmentioning

confidence: 56%

“…Furthermore, upregulation of responses to exogenous agents may have protective effects in the event of any subsequent exposures, or may result in increased susceptibility and instability of cellular processes ( Chapman et al, 2015 ). Czekala et al (2021) recently compared the effects of repeat exposures of diluted fresh whole 3R4F reference cigarette smoke to whole fresh undiluted e-cigarette aerosol and found that whilst 3R4F induced strong declines in cellular functionality and integrity over a 28 days repeated exposure period compared to Sham, the e-cigarette did not. At lower exposures repeated over a 6 week period, diluted (1:80, then later 1:100) 1R6F reference cigarette smoke induced increases in the population of mucin-producing cells, or goblet cell hyperplasia, a pathology associated with development of COPD ( Haswell et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

“…Goblet cell hyperplasia is another process associated with respiratory disease pathologies, and has recently been modelled in vitro during sub-cytotoxic repeated exposures to diluted combustible cigarette smoke (but was not induced by HTP aerosol exposure) ( Haswell et al, 2021 ); Muc5AC is often used as a marker for mucin gene expression ( Behrsing et al, 2016 ; Bedford et al, 2022 ). Additionally, changes in inflammatory markers are used as a proxy of inflammatory responses in vitro , and a number of mediators, including the cytokines, TNFα and interleukins (ILs), and matrix metalloproteinases (MMPs), are associated with exposure to inhaled toxicants, including combustible cigarette smoke ( Behrsing et al, 2016 ; Czekala et al, 2021 ; Bedford et al, 2022 ; Langel et al, 2022 ). It is for the above reasons that these cellular processes (cilia activity (beat frequency and active area), FoxJ1 expression, changes in goblet cells, inflammatory readouts) are often used as in vitro indicators of potential responses to inhaled test articles ( Behrsing et al, 2016 ; Iskandar et al, 2017 ; Czekala et al, 2021 ; Bedford et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, changes in inflammatory markers are used as a proxy of inflammatory responses in vitro , and a number of mediators, including the cytokines, TNFα and interleukins (ILs), and matrix metalloproteinases (MMPs), are associated with exposure to inhaled toxicants, including combustible cigarette smoke ( Behrsing et al, 2016 ; Czekala et al, 2021 ; Bedford et al, 2022 ; Langel et al, 2022 ). It is for the above reasons that these cellular processes (cilia activity (beat frequency and active area), FoxJ1 expression, changes in goblet cells, inflammatory readouts) are often used as in vitro indicators of potential responses to inhaled test articles ( Behrsing et al, 2016 ; Iskandar et al, 2017 ; Czekala et al, 2021 ; Bedford et al, 2022 ). Therefore in the current study, we looked to assess these endpoints.…”

Section: Introductionmentioning

confidence: 99%

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Twenty-eight day repeated exposure of human 3D bronchial epithelial model to heated tobacco aerosols indicates decreased toxicological responses compared to cigarette smoke

Chapman

Pour²,

Wieczorek³

et al. 2023

Front. Toxicol.

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Tobacco harm reduction (THR) involves providing adult smokers with potentially reduced harm modes of nicotine delivery as alternatives to smoking combustible cigarettes. Heated tobacco products (HTPs) form a category with THR potential due to their ability to deliver nicotine and flavours through heating, not burning, tobacco. By eliminating burning, heated tobacco does not produce smoke but an aerosol which contains fewer and lower levels of harmful chemicals compared to cigarette smoke. In this study we assessed the in vitro toxicological profiles of two prototype HTPs’ aerosols compared to the 1R6F reference cigarette using the 3D human (bronchial) MucilAir™ model. To increase consumer relevance, whole aerosol/smoke exposures were delivered repeatedly across a 28 day period (16, 32, or 48 puffs per exposure). Cytotoxicity (LDH secretion), histology (Alcian Blue/H&E; Muc5AC; FoxJ1 staining), cilia active area and beat frequency and inflammatory marker (IL-6; IL-8; MMP-1; MMP-3; MMP-9; TNFα) levels were assessed. Diluted 1R6F smoke consistently induced greater and earlier effects compared to the prototype HTP aerosols across the endpoints, and in a puff dependent manner. Although some significant changes across the endpoints were induced by exposure to the HTPs, these were substantially less pronounced and less frequently observed, with apparent adaptive responses occurring over the experimental period. Furthermore, these differences between the two product categories were observed at a greater dilution (and generally lower nicotine delivery range) for 1R6F (1R6F smoke diluted 1/14, HTP aerosols diluted 1/2, with air). Overall, the findings demonstrate the THR potential of the prototype HTPs through demonstrated substantial reductions in toxicological outcomes in in vitro 3D human lung models.

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Section: Discussionmentioning

confidence: 56%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Twenty-eight day repeated exposure of human 3D bronchial epithelial model to heated tobacco aerosols indicates decreased toxicological responses compared to cigarette smoke

Chapman

Pour²,

Wieczorek³

et al. 2023

Front. Toxicol.

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“…Furthermore, cell growth on porous membranes allows morphological and ultrastructural analyses after treatment ( [34], http://www.epithelix.com, accessed on 25 August 2021). The nasal MucilAir™ in vitro model has been used to study repeated exposure to smoke [50][51][52], gasoline engine emissions [53], formaldehyde [54], a novel hypertonic seawater solution for nasal lavage [55], and inhaled drugs or drug candidates [56]. To the best of our knowledge, our study is the first to investigate the relevance, value, and possible application of the nasal MucilAir™ in vitro model to predict chronic in vivo ciliotoxicity and cytotoxicity of topical nasal formulations.…”

Section: Applicability Of the Nasal Mucilair™ In Vitro Model For The Analysis Of Repeated Doses Ciliotoxicity And Cytotoxicity Of Topicalmentioning

confidence: 99%

In Vitro Ciliotoxicity and Cytotoxicity Testing of Repeated Chronic Exposure to Topical Nasal Formulations for Safety Studies

et al. 2021

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Certain active drugs and excipients of nasal formulations may impair ciliary function and mucociliary clearance. The ciliary beat frequency (CBF) is a key parameter for determining mucociliary clearance rate, and in vitro assessments of CBF have proven to be accurate and reproducible. Since topical nasal formulations are applied with repeated doses, it is essential to elucidate their chronic, as opposed to acute, effect on mucociliary clearance and nasal mucosa. The aim of this study was to assess for the first time the ciliotoxicity and cytotoxicity of nasal sprays intended for chronic treatment (with repeated doses) using a previously designed set-up for CBF measurements. For 2 weeks, the 3D nasal MucilAir™ in vitro models were treated daily with undiluted or clinically relevant doses of mometasone nasal spray, placebo nasal spray, culture medium, or they were untreated. We demonstrated a dose-dependent and time-dependent (cumulative) effect of the nasal sprays on ciliary activity and cytotoxicity using CBF measurements and ultrastructural analysis, respectively. Our results indicate that repeated administration of clinically relevant doses of mometasone nasal spray is safe for in vivo use, which is in good agreement with a previous clinical study. Overall, our study suggests that such in vitro assays have great potential for topical nasal drug screening.

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Advances in whole aerosol approaches for in vitro e‐cigarette testing

Bishop

Gaça

Thorne

2023

Drug Testing and Analysis

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Electronic‐cigarette regulation and risk assessment is a prominent and developing field, as the popularity and prevalence of this product category increases. Over the last 10 years since their emergence, there have been many advances and adaptations to current in vitro testing techniques to better assess and predict absolute consumer risk. However, there are still requirements to create a cross‐field harmonised approach to appropriate exposure and experimental design. With many assessments still being carried out using methods developed and optimised for cigarette smoke, there must first be an acknowledgement regarding the differences between cigarette smoke and tobacco‐free e‐cigarette aerosols before we can accurately assess these distinct products. Here, we discuss five published studies from within our own research to demonstrate how in vitro testing techniques have evolved to improve determination of risk by considering appropriate dosimetry and exposure for both e‐cigarette and cigarette aerosols and how we can contextualise the data through human consumption and dose extrapolation, ultimately giving more relevance to in vitro data. Furthermore, we have demonstrated the evolution of techniques, which has allowed us to bridge between platforms, simplify exposure set‐up, experimental design and demonstrate technology evolution within our products, thus fulfilling a responsible duty of care to consumers via an appropriate and robust in vitro product assessment.

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

Abstract: Graphical abstract

Cited by 15 publications

References 89 publications

Twenty-eight day repeated exposure of human 3D bronchial epithelial model to heated tobacco aerosols indicates decreased toxicological responses compared to cigarette smoke

Twenty-eight day repeated exposure of human 3D bronchial epithelial model to heated tobacco aerosols indicates decreased toxicological responses compared to cigarette smoke

In Vitro Ciliotoxicity and Cytotoxicity Testing of Repeated Chronic Exposure to Topical Nasal Formulations for Safety Studies

Advances in whole aerosol approaches for in vitro e‐cigarette testing

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