LC/MS analysis of three-dimensional model cells exposed to cigarette smoke or aerosol from a novel tobacco vapor product

Abstract

A novel tobacco vapor product (NTV) contains tobacco leaves and generates nicotinecontaining aerosols using heating elements. Subchronic biological effects have been evaluated previously using three-dimensional bronchial epithelial model cells by repeated exposure to cigarette smoke (CS) and the NTV aerosols; however, the intracellular exposure characteristics have not been studied in detail. In this study, cells were initially exposed to an aqueous extract (AqE) of cigarette smoke (CS) at two concentration levels, and the cell lysate underwent untargeted analysis by LC-high resolution mass spectrometry to determine the exogenous compounds present in the cells. Among the thousands of peaks detected, four peaks showed a CS-dependency, which were reproducibly detected. Two of the peaks were nicotine and nicotine N-oxide, and the other two putative compounds were myosmine and norharman. The cells were then exposed to an AqE of CS in various combinations of exposure and post-exposure culture durations. Post-exposure culturing of cells with fresh medium markedly decreased the peak areas of the four compounds. The in-vitro switching study of CS to NTV aerosols was investigated by intermittently exposing cells to an AqE of CS four times, followed by exposure to either an AqE of CS, NTV aerosol or medium another four times. Switching to NTV reduced myosmine and norharman levels, which are known CS constituents. The results indicate that extracellular compounds inside cells reflect the exposure state outside cells. Thus, monitoring functional changes to cells in these exposure experiments is feasible.

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

“…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.…”