Evaluation of the Biological Effects of Novel Tobacco Product Vapor Using Two- or Three-Dimensional Culture Systems of Human Bronchial Epithelia

Abstract

Introduction: Because of the rising popularity of novel tobacco products, there has been an increasing interest in the biological effects of these products. This study was performed to investigate the biological effects of vapor from a novel tobacco vapor product (NTV) in comparison with cigarette smoke (CS) from a 3R4F reference cigarette by using two types of in vitro model. Materials and Methods: A two-dimensional (2D) culture system composed of a submerged monolayer of BEAS-2B cells was used to detect cellular oxidative stress responses. A three-dimensional (3D) culture system composed of a pseudostratified air-liquid interface culture of normal human bronchial epithelial cells was used to detect tissue inflammation. In both culture systems, exposures were conducted with an aqueous extract (AqE) of NTV vapor or CS. Results: In the 2D culture system, exposure to CS AqE induced glutathione oxidization and antioxidant response element reporter gene activation. NTV vapor AqE also induced reporter gene activity, but the concentration required for activation was higher than that of CS AqE. In the 3D culture system, exposure to CS AqE increased the secretion of various inflammatory mediators. In contrast, the effect of NTV vapor AqE on tissue inflammation was limited, and only interferon-c showed a slight increase in expression. Conclusions: Overall, these results demonstrate that NTV vapor has fewer effects on cellular oxidative stress and tissue inflammatory responses as compared with CS in in vitro models using human cells.

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

“…Furthermore, the repeated exposure element of the study more accurately models likely human exposure patterns than a single, acute, in vitro exposure ( Mallock et al, 2019 ; Jones et al, 2020 ; Laverty et al, 2021 ). Interestingly, lower or absent responses in 3D respiratory tissue models exposed to heated tobacco aerosol, compared to cigarette smoke, in the short term (e.g., up to 72 h), following an acute exposure ( Iskandar et al, 2018 ; Mori et al, 2021 ) was a relative response observed to be maintained over the 28 days of the current study. The findings of the current study therefore indicate reduced harm potential of the p-HTPs over the longer period tested.…”

Chemical and in vitro toxicological comparison of emissions from a heated tobacco product and the 1R6F reference cigarette

“…Oxidative responses were also assessed using immortalized normal human bronchial epithelial cells in an ARE reporter assay that detects nuclear factor-erythroid 2-related factor-2-mediated oxidative stress responses [35] , [36] , like in previous studies [31] , [32] , [37] .…”

“…New approach methodologies (NAMs), which include in silico , in chemico , in vitro , and ex vivo approaches [47] , [48] , are being rapidly developed because of, while not yet fully validated their potential to reliably reflect human biology [49] . Recently, the 3D bronchial epithelial model has been applied as mechanistic follow-up in vitro toxicity comparison among CS and THS2.2 [50] , THP1.0 [51] , [52] , or the other type of HTP [31] , [53] , [54] . These assessments using NAMs such as human 3D tissue models, once fully validated, would also contribute to the examination of reduced risk potential of DT3.0a.…”

“…Two-dimensional culture assays were performed following a previously published method [31] using an immortalized normal human bronchial epithelial BEAS-2B cell line (American Type Culture Collection, Manassas, VA, USA). BEAS-2B cells were transfected with anti-oxidant responsive element (ARE) luciferase reporter vector (E3641; Promega, Madison, WI, USA) as previously reported [32] .…”