Experimental Acute Exposure to Thirdhand Smoke and Changes in the Human Nasal Epithelial Transcriptome

Key Points Question Does acute inhalation of thirdhand smoke alter the transcriptome of the human nasal epithelium? Findings This randomized clinical trial exposed 4 healthy, nonsmoking women to clean air, which altered the expression of only 2 genes. When the same women were exposed to thirdhand smoke at least 21 days later, 389 genes associated with cell stress and survival pathways were differentially expressed, and many affected genes were associated with increased mitochondrial activity, oxidative stress, DNA repair, cell survival, and inhibition of cell death. Meaning These results suggest that acute exposure to thirdhand smoke stresses the human nasal epithelium, a finding that may be valuable to physicians treating exposed patients.

Introduction Thirdhand smoke (THS) is a subset of chemicals in secondhand cigarette smoke (sidestream smoke emitted by a burning cigarette and exhaled mainstream smoke) that sticks to indoor surfaces and persists after active smoking has occurred 1,2 . THS chemicals accumulate and can react with other compounds or can be re-emitted into the environment 1,2,3 . Nonsmokers can be exposed to chemicals in THS months or even years after smoking has stopped 3 . Many THS chemicals are toxic volatile and semi-volatile organic compounds 2,3,4 . Nicotine, a major chemical in THS, has a high affinity for surfaces 3 and can react with ambient nitrous acid to form tobacco-specific nitrosamines (TSNAs), some of which are carcinogens 5,6 . Nicotinederived nitrosamines in THS include 4-(methylnitrosamino)-1-(3-pyridinyl)-1butanone (NNK), and N-nitrosonornicotine (NNN) 5,6 , which are also found in secondhand smoke and have been associated with the development of lung cancer 7 . Ozone can also react with nicotine to form formaldehyde, a known human carcinogen 8 .
Due to the presence of these and other hazardous chemicals, such as acrolein, in THS, it is important to understand if there is a correlation between exposure to THS and human health, especially in nonsmokers. Previous studies have demonstrated that exposure of human cell lines to THS extracts for 24 hours increased DNA strand breaks and oxidative DNA damage 9,10 . Mouse neural stem cells undergo blebbing, fragmentation, cytoskeletal disruption, and vacuolization when treated with extracts of THS 11 . THS also causes stress-induced mitochondrialhyperfusion (SIMH), which is accompanied by increased mitochondrial membrane potential, ATP production, and reactive oxygen species (ROS) 12  137 molecules including mitochondrial DNA as a survival mechanism 13 . Acrolein has been identified as a THS chemical that inhibits cell proliferation 11 . In a metabolomics study using male germ cells, THS exposure is correlated with down-regulation of several molecular pathways, including nucleic acid metabolism, ammonia metabolism and up-regulation of glutathione metabolism 14 .
THS also causes adverse health effects in mice. Three-week old mice that were housed for 6 months in cages containing a THS-impregnated fabric and bedding showed an increase in inflammatory cytokines in lung tissue, impaired wound healing, and were hyperactive compared to controls 15 . Adult mice developed insulin resistance as a consequence of oxidative stress caused by THS and showed increased blood glucose, increased serum insulin, and accumulation of fat in viscera 16 . Oxidative stress in skeletal muscle and accumulation of H 2 O 2 accompanied by low catalase activity was observed in chronically exposed mice 17 .
After THS exposure, neonatal mice had significantly more eosinophils, increased platelet volume, lower hematocrit, and decreased mean cell volume than controls, while adult exposed mice had a significant increase in the percentage of B-cells and a decrease in myeloid cells 18 .
Elimination of THS can be challenging, as it persists in houses previously owned by smokers even after 2 months of vacancy 19 . Cars previously owned by smokers also retain THS, and new owners may be at risk of exposure 20 . Common household fabrics retained THS chemicals 19 months after smoking had occurred 4 .
Individuals absorb nicotine through their skin while wearing THS exposed clothes 21 .
Moreover, infants whose mothers smoked outdoors had much higher levels of urine cotinine, a nicotine metabolite, than infants of nonsmoking parents 22  Although these prior studies demonstrate humans are at risk of exposure to THS, the molecular effects of such exposure on humans have not been investigated.
The purpose of this study was to evaluate the effects of inhalation of THS chemicals on gene expression in humans. Nasal epithelial cells were collected from nonsmokers before and after 3 hours of exposure to either clean air or to THS, subjected to mRNA sequencing (mRNA-seq), and analyzed for differential expression of genes (DEG). Significant changes in gene expression were found following THS exposure, but not exposure to clean air. healthy nonsmokers who were not exposed to secondhand cigarette smoke (SHS) in daily life, were exposed, head-only to THS aerosol and to conditioned, filtered air for 3 hours, using an exposure chamber described previously 23  Riverside. The RNA-Seq analysis workflow implemented by systemPipeR 24 was used to perform all the downstream data processing. Briefly, adapter sequences and low quality tails were removed from the raw reads using the Trimmomatic package 25 .

Ethics
The preprocessed reads were then aligned against the UCSC hg19 human reference genome with Tophat2 (Version 2.0.14) 26,27 . Read counting was performed with the summarizeOverlaps function of the GenomicsAlignment package. Only unique reads overlapping the exonic gene regions were counted 28 . Using a cut-off value of at least 1 RPKM average across all samples, raw expression counts of the remaining 10,938 genes passing this filter were used for differential expression analysis with EdgeR 29 . Within each experimental group (Group 1, 2, 3, 4), the read counts from the four biological replicates were combined. For differential expression analysis, Groups 1 & 2 (before and after clean air) and Groups 3 & 4 (before and after THS) were treated as two separate experimental comparisons. Genes were considered to be DEGs if they had a false discovery rate (FDR) < 0.1 by EdgeR. Cluster-Profiler 30 and Reactome PA 31 (RPA) packages were used to identify over represented GO terms and enriched Reactome pathways, respectively, as described in the package manual. Additionally, enrichment analyses of pathways were performed using the Ingenuity Pathway Analysis (IPA) software (Qiagen, Germantown, MD, USA). Briefly, statistically significant transcripts were uploaded to IPA, and human homologs were automatically identified using NCBI's HomoloGene.

Exposure to THS Altered Gene Expression in Human Nasal Epithelium
The samples collected with this method were small and sufficient quantities of RNA for sequencing analysis could only be extracted from four women. By chance, these four participants had all been randomized to receive the clean air exposure first and THS exposure second, thus we were unable to determine the effect of order on RNA expression. After processing RNA-seq reads, data were analyzed to determine if there were differences in gene expression in the groups exposed to either clean air (Group 1 vs Group 2) or THS (Group 3 vs Group 4) (eFigure 1 in the Supplement). The dataset consisted of approximately 10,000 genes of which 2 and 389 were significantly differentially affected (FDR < 0.10) in clean air and THS-exposed subjects, respectively (eTable 1 in the Supplement). The data set is downloadable from SRA/GEO under submission number PRJNA514351/GSE129959. The two down-regulated genes (hemoglobin, alpha 1 and hemoglobin, alpha 2) identified when subjects were exposed to clean air had an absolute fold change of 8.2 and 8.7, respectively (eTable 2 in the Supplement). No genes were significantly up-regulated in the group exposed to clean air (eFigure 2 in the Supplement). Because these results showed that wearing the respirator for 3.5 hours and inhaling clean air did not significantly impact gene expression, clean air was not studied further.

GO Term Enrichment Analysis
We performed Gene Ontology (GO) enrichment analysis on the up-regulated DEGs to identify biological functions affected by THS (Figure 2A

Reactome Enrichment Analysis
The Reactome enrichment analysis was used to further evaluate the up-  Figure 2C shows the genes associated with each pathway and their overlap, when they belong to multiple pathways. Also shown are the approximate fold-change values of each gene.

Ingenuity Pathway Analysis
IPA was also performed using upregulated genes in the THS-exposed group.  (Table 2). These data were filtered and only functions with activated z-scores that predict transcriptional activation or inhibition based on literature reports are presented ( Table 2). The identified functions included decreased cell death and increased cell viability, homologous recombination, and cell proliferation. (overall process decreased) ( Table 2). Complementary to cell death, cell viability (zscore = 5.026) (eFigure 4 in the Supplement) and homologous recombination (z score = 2.828) (eFigure 5 in the Supplement) both had increased activation states (Table 2).

Discussion
The adverse health effects of THS have been studied in cultured cells and animal models 2 , but similar investigations have not been previously performed on human subjects. Our study provides the first insight into the transcriptional responses of human respiratory epithelium to acute THS exposure. Remarkably, we found changes in gene expression in healthy nonsmokers following a 3-hour exposure to THS. The absence of an effect following clean air exposure provides evidence that the changes in gene expression following THS exposure are caused by THS per se and are not by the respirator worn during exposure. Because gene expression in the nasal epithelium is similar to the bronchial epithelium 33  Our analyses demonstrate that brief exposure to THS affects mitochondrial activity. We previously reported that cultured mNSC undergo SIMH following exposure to THS extracts 12 . This process was originally described during treatment of mouse embryonic fibroblasts with UV light and cell cycle inhibitors, such as actinomycin D 13 . SIMH is characterized by fusion of mitochondria and subsequent increased production of ATP and superoxide 12 . We found an enrichment in pathways and biological processes related to increased mitochondrial activity and oxidative stress after THS exposure, such as mitochondrial ATP synthesis coupled electron transport chain (GO:0042773), respiratory electron transport (R-HSA-611105) and oxidative phosphorylation (IPA). Increased expression of these pathways is also consistent with an increase in ATP synthesis, as occurs in SIMH 12 . Some genes related to the TCA cycle were also upregulated, which could also increase ATP production. Several studies have shown that cigarette smoking also induces activation of mitochondrial pathways similar to those found in our study 34,35,36 .
While SIMH results in increased ATP production, it also increases ROS 12,13 .
Our IPA analysis showed that Glutathione Depletion Phase II Reactions were upregulated after THS exposure. Specifically, there was an increase in glutathione synthetase (GSS) expression, which was also increased in a male germ cell line exposed to THS 14 . This gene is part of the glutathione (GSH) synthetase pathway, which scavenges ROS 37 , suggesting the increase of the GSS gene is a cellular response to high levels of ROS.
In prior studies, increased ROS was associated with oxidative stress and damage of proteins, lipids and DNA 38 , while THS treatment was correlated with DNA damage in vitro 10 and RNA polymerase II. The former is essential for recognition of DNA damage and plays a role in the early steps of the Nucleotide Excision Repair Pathway 39 . Upregulation of RNA polymerase II has also been associated with a response to increased DNA damage 40 . IPA also identified an increased activation of homologous recombination. This pathway provides a repair mechanism for double stranded DNA breaks 41 . Activation of the DNA repair pathways is also a cellular mechanism to facilitate survival 42 . In addition, an in vitro study showed that THS induces oxidation of mitochondrial proteins 12 . The increase in ROS as evidenced by upregulation of ROS scavenging genes in our data could also result in oxidation of mitochondrial proteins by high local concentrations of superoxide.
Our data further demonstrate that there is an overall increase in processes related to cell viability, which includes some genes involved in cell proliferation. Our results are consistent with previous in vitro studies showing increased proliferation of cultured mouse neural stem cells and human lung cancer cells exposed to THS extract 12,43 . Nicotine, a major component of THS 4 and a chemical in our exposure chamber, can activate alpha nicotinic acetylcholine receptors (nAChRs) in normal human airway epithelial cells, leading to phosphorylation (activation) of serine/threonine kinase Akt, which is involved in many cellular survival pathways 44 .
Akt can be activated within minutes of exposure to nicotine or NNK 44 , further demonstrating that chemicals in THS could produce a rapid response. Nicotine is also associated with increased proliferation of human cancer cell lines by activating the 7α nAChR 45 . Considering that nicotine stimulates cell proliferation 45  Nicotine is also involved in inhibiting apoptosis 46 . In our study, the increased expression of genes involved in inhibiting cell death (IPA) may have been associated with nicotine, which was present in the THS at a concentration of 0.03 mg/m 3 .
Consistent with our study, cells exposed to THS in vitro showed decreased expression in pro-apoptotic genes 12 . The mechanism by which nicotine inhibits apoptosis has been studied in mouse liver cells 47 . Activation of 7α nAChRs in the mitochondrial outer membrane by nicotine inhibited hydrogen peroxide induced apoptosis by impairing Ca 2+ accumulation in mitochondria and cytochrome C release 47 . However, this suppression of cell death may be transitory. Bahl et al. 12 showed that cells exposed to THS for 30 days had a decrease in cell proliferation and lost mitochondrial membrane potential, indicating that cells were entering apoptosis.
Limitations. This is an initial study based on four participants. Future work should be done to determine if similar data are obtained with a larger number of subjects that includes both genders.