Cigarette smoke, but not novel tobacco vapor products, causes epigenetic disruption and cell apoptosis

Naoko Hattori,Takeya Nakagawa,Mitsuhiro Yoneda,Kaori Nakagawa,Hiromi Hayashida,Takashi Ito

doi:10.1016/j.bbrep.2020.100865

Abstract

Heat-Not-Burn (HNB) products, generating vapor without combusting tobacco leaves, have been developed with the expectation that the number and quantity of chemicals in the vapor of these products would be reduced compared with the smoke from conventional combustible cigarettes. However, whether the lower chemical levels correlate with lower toxicity remains to be determined.Here we examined differences in the biological effects of conventional cigarette smoke (CS) and two HNB products, Ploom TECH and Ploom TECH+, using the cultured cancer cell line A549 and the normal bronchial epithelium cell line BEAS-2B. The conventional CS 3R4F extract (0.5%) markedly decreased cell proliferation of both A549 and BEAS-2B cells; however, 0.5% extracts of these commercially available HNB products did not affect cell growth. To determine the cause of decreased cell proliferation, a TUNEL assay was performed, and the results indicated that apoptosis had occurred in both A549 and BEAS-2B cells at 24 h after exposure to 3R4F. To further explore the effect of CS on epigenetics, we performed western blotting to detect histone H2A phosphorylation, which is known to affect transcriptional regulation. Only the 3R4F extract decreased histone H2A phosphorylation in both A549 and BEAS-2B cells. Next, we examined alterations in gene expression after treatment of A549 cells with Ploom TECH, Ploom TECH+, or 3R4F extracts. It was found that 339, 107, and 103 genes were upregulated more than 2 fold in A549 cells treated with 3R4F, Ploom TECH, or Ploom TECH + extracts, respectively. Among the 339 genes that were upregulated in response to 3R4F, we focused on EGR1, FOS, and FOSB, since they were upregulated more than 100 fold, which was confirmed using RT-qPCR. These results suggest that CS, but not HNB products, cause epigenetic disruption and cell apoptosis, possibly by elevating transcription of genes such as EGR1.

Highlights

The use of tobacco-containing vapor products is increasing world wide
In this study it was found that conventional cigarette smoke (CS) but not the Ploom TECH or Ploom TECH + HNB products cause apoptosis, a change in histone H2A phosphorylation that is closely correlated with changes in gene expression, and upregulation of the EGR1, FOS, and FOSB genes
We focused on EGR1, since it has multiple functions relating to the development of cancer, cardio vascular disease, memory and psychiatric disorders, and apoptosis [13,14,15,16,17]

Summary

Introduction

In addition to e-cigarettes, which produce vapor by heating a nicotine-containing liquid, Heat-Not-Burn (HNB) products, in which the tobacco is heated without being combusted, have been developed [1]. Emissions from HNB products have been reported to contain lower levels of harmful chemicals than conventional cigarette smoke (CS), [2]. Whether this reduction of potentially harmful chemicals is correlated with decreased toxicity and lower risk for developing chronic obstructive pulmonary disease (COPD) or other disorders needs to be verified. Several mechanisms are possible for COPD development, such as an influx of inflammatory cells into the airway, resulting in chronic inflammation, or an imbalance between proteolysis and anti-proteolysis, resulting in lung tissue destruction and oxidative stress. An increase in apoptotic alveolar epithelial and endothelial cells is observed in the lungs of COPD patients, suggesting a role for this process in the destruction of lung tissue and the development of emphysema and COPD [5]

Methods

Results

Conclusion