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Inhalation Exposure to Cigarette Smoke and Inflammatory Agents www.nature.com/scientificreports OPEN Inhalation exposure to cigarette smoke and infammatory agents induces epigenetic changes in the lung Christopher L. Seiler1,9, J ung Min Song2,9, Delshanee Kotandeniya1,9, Jianji Chen3, Thomas J. Y. Kono4, Qiyuan Han3, Mathia Colwell5, Benjamin Auch6, Aaron L. Sarver7,9, Pramod Upadhyaya9, Yanan Ren8, Christopher Faulk5, Silvio De Flora10, Sebastiano La Maestra10, Yue Chen3, Fekadu Kassie2,9 & Natalia Y. Tretyakova1,9* Smoking-related lung tumors are characterized by profound epigenetic changes including scrambled patterns of DNA methylation, deregulated histone acetylation, altered gene expression levels, distorted microRNA profles, and a global loss of cytosine hydroxymethylation marks. Here, we employed an enhanced version of bisulfte sequencing (RRBS/oxRRBS) followed by next generation sequencing to separately map DNA epigenetic marks 5-methyl-dC and 5-hydroxymethyl-dC in genomic DNA isolated from lungs of A/J mice exposed whole-body to environmental cigarette smoke for 10 weeks. Exposure to cigarette smoke signifcantly afected the patterns of cytosine methylation and hydroxymethylation in the lungs. Diferentially hydroxymethylated regions were associated with infammatory response/disease, organismal injury, and respiratory diseases and were involved in regulation of cellular development, function, growth, and proliferation. To identify epigenetic changes in the lung associated with exposure to tobacco carcinogens and infammation, A/J mice were intranasally treated with the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the infammatory agent lipopolysaccharide (LPS), or both. NNK alone caused minimal epigenetic alterations, while exposure either to LPS or NNK/LPS in combination led to increased levels of global cytosine methylation and formylation, reduced cytosine hydroxymethylation, decreased histone acetylation, and altered expression levels of multiple genes. Our results suggest that infammatory processes are responsible for epigenetic changes contributing to lung cancer development. Lung cancer is responsible for 30% of all cancer deaths worldwide and is expected to kill 154,050 Americans this year, with over 80% of cases directly attributable to smoking1. Cigarette smoke contains over 60 known carcinogens, such as the tobacco specifc nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 75 ng/cigarette)2,3, as well as non-genotoxic co-carcinogens including the infammatory agent lipopolysaccharide (LPS, 120 ng/cigarette)4. Chronic infammation plays a central role in the pathogenesis of smoking-induced lung cancer 5. Smoking is characterized by neutrophilic infammation and reduced mucociliary clearance in the lung, which, at least in part, can be explained by exposure to LPS and other endotoxins 6. When administered intranasally to laboratory 1Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. 2Department of Veterinary Medicine, University of Minnesota, Minneapolis, MN 55455, USA. 3Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. 4Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN 55455, USA. 5Department of Animal Science, University of Minnesota, Minneapolis, MN 55455, USA. 6Genomics Center, University of Minnesota, Minneapolis, MN 55455, USA. 7Institute for Health Informatics, University of Minnesota, Minneapolis, MN 55455, USA. 8Biostatistics Core, University of Minnesota, Minneapolis, MN 55455, USA. 9Masonic Cancer Center, University of Minnesota, 2231 6th Street SE, 2-147 CCRB, Minneapolis 55455, USA. 10Department of Health Sciences, University of Genoa, 16132 Genoa, Italy. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:11290 | https://doi.org/10.1038/s41598-020-67502-8 1 Vol.:(0123456789) www.nature.com/scientificreports/ mice, LPS induces an infammatory response mimicking chronic obstructive pulmonary disease (COPD), a major risk factor for lung cancer development in smokers7. Furthermore, long-term exposure to LPS increases lung tumor size and multiplicity in A/J mice following treatment with NNK, supporting a role for infammation in lung cancer etiology 7. Anti-infammatory agents such as aspirin and other NSAIDS have been suggested as potential chemopreventive agents for lung cancer8. Smoking-induced lung tumors are characterized by genetic alterations in tumor suppressor genes and pro- tooncogenes, as well as epigenetic changes which include deregulated DNA methylation, altered histone acety- lation, and aberrant microRNA expression9. Aberrant DNA methylation patterns in malignant cells result in silencing of tumor suppressor genes, activation of protooncogenes, and decreased chromosomal stability10. Tese “epimutations” are thought to cooperate with genetic alterations to drive the malignant lung tumor phenotype 9. However, the nature and the mechanistic origins of smoking-induced epigenetic deregulation remain largely unknown, limiting our understanding of cancer etiology and hindering the development of future treatments. In the present work, we characterized epigenetic changes in the lungs of A/J mice exposed to cigarette smoke by inhalation for 10 weeks or intranasally treated with the tobacco carcinogen NNK and/or infammatory agent LPS. Previous studies have shown that this treatment leads to pulmonary infammation, atelectasis, emphysema, vascular alterations, bronchial hyperplasia, and alveolar bronchiolarization11. Our results revealed infammation- driven changes in cytosine methylation and hydroxymethylation patterns indicative of an imbalance of DNA methylation/demethylation dynamics, which in turn give rise to a shif in histone acetylation marks and gene expression patterns that could contribute to initiation of lung cancer. Results Animal studies. Our experimental design included several mouse studies aimed to characterize epigenetic changes in the lung induced by exposure to cigarette smoke and its components (Fig. 1a), all conducted in A/J mice. In the smoking (ECS) study (panel 1 in Fig. 1a), A/J mice were treated with cigarette smoke for 10 weeks starting at birth with or without oral co-administration of the nonsteroidal anti-infammatory agent acetylsali- cylic acid (aspirin) on weeks 4–108. In the acute treatment study (panel 2 in Fig. 1a), mice were exposed to the tobacco specifc nitrosamine NNK (25 mg/kg, in 0.3 ml physiological saline solution, every three days) and/or the infammatory agent lipopolysaccharide (LPS, intranasal instillation of 8.3 µg/mouse on days 1 and 5 and 4.15 µg/mouse on day 9) and either sacrifced on day 16 or allowed to recover for 1 week. In the sub-chronic study, animals were treated with NNK (25 mg/kg once a week) and/or LPS (4.15 µg/mouse) for 6 weeks as shown in panel 3 of Fig. 1a. Finally, Study 4 (panel 4 in Fig. 1a) examined epigenetic changes in lung tumors. Mice were treated with two weekly doses of NNK (100 mg/kg) and exposed to LPS every week beginning with the frst dose of NNK and until the termination of the study at week 27. Upon sacrifce of the mice, lungs were harvested, tumors on the surface of the lung counted, and some of them dissected for subsequent downstream assays. Histopathological analysis of the tumors harvested at week 27 showed that about 50% of the tumors had progressed to adenoma with dysplasia. Global epigenetic marks of DNA in lung DNA of A/J mice exposed to cigarette smoke. A quantitative isotope dilution HPLC-ESI+-MS/MS methodology developed in our laboratory (Supplementary Methods, Figs. S1–S3) was used to quantify global levels of cytosine methylation, hydroxymethylation, formyla- tion, and carboxylation in lung DNA of mice chronically exposed to cigarette smoke12. Total amounts of 5mC and 5hmC in genomic DNA isolated from lung tissues of A/J mice (2.8–3.0% of total cytosines being methyl- ated, ~ 0.3% of total cytosines being hydroxymethylated, and 0.02–0.03% being formylated—see Figs. 2a, 3a, Supplementary Fig. S4) were comparable to previously published values for mouse lung13. HPLC-ESI+-MS/MS revealed that global 5mC, 5hmC, and fC concentrations in DNA isolated from lung DNA of A/J mice following whole-body exposure to environmental cigarette smoke (ECS) or ECS and aspirin for 10 weeks were similar to those of unexposed controls, suggesting that these treatments do not afect the overall cytosine methyla- tion, hydroxymethylation, and formylation levels (see Figs. 2a, 3a, and Supplementary Fig. S4, respectively). Te amounts of 5-carboxylcytosined in lung tissues were below our method’s limit of quantitation. Genome-wide changes in cytosine methylation and hydroxymethylation revealed by oxRRBS—NGS. Although the total amounts of 5mC, 5hmC, and fC in mouse lung DNA were unafected by exposure to cigarette smoke (Figs. 2a, 3a, and Supplementary Fig. S4), this does not rule out potential localized changes at specifc genomic loci. Terefore, reduced representation bisulfte sequencing (RRBS) and oxidative- RRBS (oxRRBS) were used to probe for site specifc methylation and hydroxymethylation changes14. oxRRBS is a relatively new methodology that makes it possible to separately map 5mC and 5hmC in CpG rich regions of the genome (Fig. 1c)14. Standard RRBS employs bisulfte treatment to convert C to U, while 5mC resists deamination15,16. Unfortunately, this method does not distinguish between 5mC and 5hmC, therefore the major- ity of previously published studies actually report the sum of 5mC and 5hmC. In oxRRBS, separate mapping
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