Thirdhand smoke exposure causes replication stress and impaired transcription in human lung cells

Abstract

Thirdhand cigarette smoke (THS) is a newly described toxin that lingers in the indoor environment long after cigarettes have been extinguished. Emerging results from both cellular and animal model studies suggest that THS is a potential human health hazard. DNA damage derived from THS exposure could have genotoxic consequences that would lead to development of diseases. However, THS exposure-induced interference with fundamental DNA transactions such as replication and transcription, and the role of DNA repair in ameliorating such effects, remain unexplored. Here we found that THS exposure increased the percentage of cells in S-phase, suggesting impaired S-phase progression. Key DNA damage response proteins including RPA, ATR, ATM, CHK1 and BRCA1 were activated in lung cells exposed to THS, consistent with replication stress. In addition, THS exposure caused increased 53BP1 foci, indicating DNA double-strand break (DSB) induction. Consistent with these results, we observed increased micronuclei formation, a marker of genomic instability, in THS-exposed cells. Exposure to THS also caused a significant increase in phosphorylated RNA Polymerase II engaged in transcription elongation, suggesting an increase in transcription-blocking lesions. In agreement with this conclusion, ongoing RNA synthesis was very significantly reduced by THS exposure. Loss of nucleotide excision repair (NER) exacerbated the reduction in RNA synthesis, suggesting that bulky DNA adducts formed by THS are blocks to transcription. The adverse impact on both replication and transcription supports genotoxic stress as a result of THS exposure, with important implications for both cancer and other diseases.

Tobacco-specific nitrosamine 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA) causes DNA damage and impaired replication/transcription in human lung cells

“…We previously reported the formation of DNA strand breaks in THS-exposed human cell lines by the alkaline Comet assay [ 7 ] and γ-H2AX immunofluorescence [ 8 ]. Consistent with these results, we observed that THS exposure increased micronuclei (MN) formation [ 9 ], a marker of genomic instability. These cellular effects are consistent with THS exposure induced adverse effects in vivo [ 10 ].…”

“…For measuring DSBs, immunofluorescence analysis was carried out as described previously [ 9 , 13 ]. For 53BP1 immunostaining, cells grown in 4-well chamber slides (Nunc Lab-Tek) were exposed to NNA (1 and 10 μM) or mock exposure (DMEM only) for 24 h and then washed three times with PBS.…”

“…These cellular effects are consistent with THS exposure induced adverse effects in vivo [ 10 ]. Furthermore, THS exposure also induced replication and transcription stress that are linked with an elevated risk of cancer and premature aging, respectively [ 9 ].…”

“…We also investigated the consequences of cellular replication and transcription from exposure of lung cells to NNA. We chose lung epithelial BEAS-2B cells since the lungs would be a major target site for NNA-containing THS and we could compare the effect of NNA with our recently reported similar studies using whole THS samples [ 9 ]. Finally, we also examined the role of nucleotide excision repair (NER), a major DNA repair pathway involved in repair of bulky DNA lesions, in the defense of NNA-derived cellular effects.…”

The effects of thirdhand smoke on reproductive health

“…Chinese school-aged children exposed to SHS have reduced lung function compared with that of unexposed children (He et al, 2011). An in vitro study shows that THS exposure can impair DNA replication and transcription in human lung cells (Sarker et al, 2020). In addition, low-dose nicotine in tobacco smoke can induced EGFR phosphorylation via nicotine acetylcholine receptor subunit 5 (5-nAChR) and promote lung adenocarcinoma progression (Wang et al, 2020).…”