Cytotoxic, genotoxic, and toxicogenomic effects of heated tobacco products and cigarette smoke in human primary keratinocytes

Abstract

Introduction Heated tobacco products (HTPs) appear to be less harmful to health than conventional cigarettes (CCs). However, limited analytical data are available to support this claim. This study aimed to compare the cytotoxic, genotoxic, and toxicogenomic effects of HTPs and CCs in carcinogenesis via multistep gene mutations in the oral mucosal cells. Methods Cigarette smoke extract (CSE) was obtained from HTPs and CCs. Primary human oral keratinocytes (HOKs) were treated with 5% and 20% CSE from HTPs and CCs. Cell survival rate assays were performed after 6, 12, and 24 h. After 6 h, DNA double-strand breaks (DSBs) were evaluated using anti-γH2AX antibodies with immunohistochemistry. mRNAs expressions of mediator of DNA damage checkpoint 1 (MDC1) and ataxia telangiectasia and Rad3-related protein (ATR), were analyzed. Expressions of miR-22 and miR-185 were analyzed because miR- 22 targets MDC1 and miR-185, ATR. Results The HOKs had equivalent survival rates after exposure to the same concentrations of CSE from CCs and HTPs. HTPs increased foci formation of γH2AX in HOKs, as did CCs (without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of MDC1 and ATR decreased in cells exposed to CSE from CCs and HTPs (MDC1: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05; ATR: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of miR-22 and miR-185 were not significantly increased when exposed to CSE from CCs or HTPs. Conclusions HTPs and CCs had similar cytotoxic effects. HTPs are genotoxic, can cause DSBs, and have toxicogenomic damage because they inhibit the MDC1 and ATR-CHK1 DNA repair pathways in the oral mucosa. The miRNA-mRNA axis was not related to these inhibitions.

Keywords

• tobacco
• smoking
• carcinogenesis
• heated tobacco products