Elsevier

Environmental Pollution

Volume 287, 15 October 2021, 117540
Environmental Pollution

Phenotypic and transcriptomic changes in the corneal epithelium following exposure to cigarette smoke

https://doi.org/10.1016/j.envpol.2021.117540Get rights and content

Highlights

  • We established a novel method that could qualitatively and quantitatively characterize alterations in vivo induced by CSE.

  • CSE destroyed the structure and barrier function of the corneal epithelium in mice in a dose-dependent manner.

  • CSE activated glutathione metabolism and cytochrome P450-mediated metabolism of xenobiotics in corneal epithelium.

  • CSE enhanced the antioxidant activity of corneal epithelium by upregulating antioxidant-related genes.

Abstract

Cigarette smoke extract (CSE), a complex mixture of compounds, contributes to a range of eye diseases; however, the underlying pathophysiological responses to tobacco smoke remain ambiguous. The purpose of the present study was to evaluate the cigarette smoke-induced phenotypic and transcriptomic changes in the corneal epithelium with a view to elucidating the likely underlying mechanism. Accordingly, for the first time, we characterized the genome-wide effects of CSE on the corneal epithelium. The ocular surface of the mice in the experimental groups was exposed to CSE for 1 h per day for a period of one week, while mice in the control group were exposed to preservative-free artificial tears. Corneal fluorescein staining, in vivo confocal microscopy and scanning electron microscopy were performed to examine the corneal ultrastructure. Transcriptome sequencing and bioinformatics analysis were performed followed by RT-qPCR to validate gene expression changes. The results indicate that CSE exposure disrupted the structural integrity of the superficial epithelium, decreased the density of microvilli, and compromised the corneal epithelial barrier intactness. RNA-seq revealed 667 differentially expressed genes, and functional analysis highlighted the enhancement of several biological processes such as antioxidant activity and the response to oxidative stress. Moreover, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that glutathione metabolism and drug metabolism cytochrome P450 were the most relevant pathways contributing to the effects of CSE on the corneal epithelium. Protein–protein interaction (PPI) network analysis illustrated that GCLC, NQO1, and HMOX1 were the most relevant nodes. In conclusion, the present study indicates that CSE exposure induces changes in the phenotype and genotype of the corneal epithelium. The antioxidant response element is essential for counteracting the effects of cigarette smoke on this tissue layer. These results shed novel insights into how cigarette smoke damages this ocular surface.

Introduction

Tobacco use is a globally recognized risk factor threatening public health, which causes more than 8 million deaths annually (eport on the Global, 2019). Second-hand smoke refers to exposure to side-stream smoke from the burning cigarette and mainstream smoke exhaled by the smoker (Apelberg et al., 2013); it is a mixture of numerous genotoxic and carcinogenic compounds including nicotine, particulate matter, carbon monoxide, 3-ethenylpyridine, polycyclic aromatic hydrocarbons, nitrosamine, nitrogen oxide, aldehydes, and other organic compounds (Apelberg et al., 2013; Lofroth, 1989). Second-hand smoke is not only strongly associated with chronic respiratory diseases and lung cancer but also initiates or exacerbates cardiovascular diseases and can shorten the average life expectancy (Lois et al., 2008).

The cornea is a highly specialized structure on the anterior surface of the eye that forms the main refractive element and accounts for approximately two-thirds of the total optical power (Casaroli-Marano et al., 2015). Corneal clarity depends on the maintenance of a well-defined intact five to six layered functionally coupled syncytium. It is the most superficial layer of the cornea and is in direct contact with the external environment. This layer is made up of a non-keratinized, stratified squamous epithelial sheet consisting of three differentiating central epithelial cell layers and superficial outermost top layers containing, wing cells as well as proliferating basal cells that are situated in intimate contact with an underlying basement membrane surface (Ehlers et al., 2010; Yazdanpanah et al., 2017). Functionally, the corneal epithelium acts as the frontline ocular defense system to form a critical barrier against various environmental stressors and external pathogen invasion. Loss of the corneal epithelium increases the risk of infection, which can lead to scarring or melting of the corneal stroma and even severe vision loss. Moreover, the corneal epithelium plays an essential role in regulating the passage of solutes and macromolecules into the underlying deeper layers of this tissue. The integrity and homeostasis of the corneal epithelium are essential for preserving corneal transparency, visual acuity, and the integrity of the ocular surface.

Since the corneal epithelium is one of the cell types in direct contact with tobacco smoke, tobacco smoke may have a direct impact on the corneal epithelium. Smoking is one of the primary causes of a range of eye diseases, such as Graves’ ophthalmopathy (Hegedius et al., 2004), dry eye disease (Titiyal et al., 2018), age-related macular degeneration (Woodell and Rohrer, 2014), and cataract (Chowdhury et al., 2018); however, few studies have focused on clarifying underlying mechanism that accounts for how exposure to tobacco smoke damages the cornea. Jetton et al. surveyed patients with corneal abrasions or keratitis and found that the time required for completion of epithelial healing was longer in smokers (Jetton et al., 2014). Thomas et al. demonstrated that smoking causes a series of adverse effects including decreased tear film stability, impaired corneal sensitivity, and enlarged fluorescein staining (Thomas et al., 2012). An investigation of occupational tobacco exposure by Mittal et al. showed that workers in a smoky environment displayed a range of ocular complications such as superficial punctate keratitis, punctate epithelial erosion, eye irritation, and conjunctival hyperplasia (Mittal et al., 2008). While these clinical investigations indicated that tobacco smoke has harmful effects on the ocular surface and leads to many ocular symptoms, the underlying molecular mechanism is still not well defined and requires further exploration. In the present study, we aimed to investigate the phenotypic and transcriptomic impacts of cigarette smoke on the corneal epithelium with a view to clarifying the probable mechanism.

Section snippets

Animals

C57/BL6J mice (aged 6–8 weeks) were obtained from the Experimental Animal Center of Xiamen University. The mice were housed at a temperature of 23 ± 1 °C and a humidity of 60 ± 5% under a 12 h/12 h light/dark cycle. Food and water were provided ad libitum. All experiments were performed based on the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Experimental Animal Ethics Committee of Xiamen

Chemical characteristics of CSE

The generation of mainstream cigarette smoke is a complex and dynamic process with a variety of chemical, physical, and physiological characteristics. Mainstream cigarette smoke contains is a complex aerosol composed of a vapor and a particulate phase containing more than 4000 harmful chemical substances including nicotine, particulate matter, carbon monoxide, polycyclic aromatic hydrocarbons (PAHs), nitrosamine, nitrogen oxide, and other organic compounds. Therefore, it is difficult to analyze

Discussion

The corneal epithelium is the main barrier against exogenous substances, which helps to maintain the homeostasis of the cornea and is one of the first sites of exposure to cigarette smoke. Previous studies have demonstrated that chronic exposure to cigarette smoke has deleterious effects on the ocular surface, typically inducing lipid layer changes, apoptosis, inflammation, ROS-mediated DNA oxidation and autophagy impairment (Miao et al., 2019; Higuchi et al., 2011; Li et al., 2020; Ejaz et

Conclusion

Our results demonstrate that CSE had obvious toxic phenotypic and genetic effects on the corneal epithelium. In order to counter the toxicity of CS, the corneal epithelium developed a series of genetic changes. The antioxidant activity was enhanced and several oxidoreductase enzymes, including GPX, GCL, and GST, were activated to reduce excessive oxidative damage. After sensing a signal, it is likely that the corneal epithelial cells activate Nrf-2, a transcription factor, which binds to

Author statement

Cheng Li and Yuhua Xue are responsible for conceptualization and funding acquisition. Mengyi Jin drafted the manuscript. Cheng Li, Peter S Reinach and Zuguo Liu contributed to original draft, review and editing. Shuiping Wu made contribution to review and editing. Mengyi Jin, Xiaoya An, Honghua Kang, Yixin Wang, Guoliang Wang and Yang Gao performed the experiment. Mengyi Jin and Yanzi Wang made contribution to statistical analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported in part by grants from The National Key R&D Program of China (2020YFA0908103, 2018YFA0107301), the National Natural Science Foundation of China (NSFC No. 82070931, 81770891, 81672955, 81900822), and the Huaxia Translational Medicine Fund for Young Scholars (No. 2017-A-001), and the Fundamental Research Funds for the Central Universities (No. 20720200044). The authors thank Jing-Ru Huang from the Biomedical Science Core Facility of Xiamen University for technical

References (80)

  • G. Lofroth

    Environmental tobacco smoke: overview of chemical composition and genotoxic components

    Mutat. Res.

    (1989)
  • D.R. Mans

    Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213)

    Biochem. Pharmacol.

    (1991)
  • Q. Miao et al.

    Cigarette smoke induces ROS mediated autophagy impairment in human corneal epithelial cells

    Environ. Pollut.

    (2019)
  • H. Sakurai

    Sulforaphane ameliorates steroid insensitivity through an Nrf2-dependent pathway in cigarette smoke-exposed asthmatic mice

    Free Radic. Biol. Med.

    (2018)
  • C.J. Smith et al.

    IARC carcinogens reported in cigarette mainstream smoke and their calculated log P values

    Food Chem. Toxicol.

    (2003)
  • S.P. Sugrue et al.

    ZO1 in corneal epithelium: association to the zonula occludens and adherens junctions

    Exp. Eye Res.

    (1997)
  • R. Tayyarah et al.

    Comparison of select analytes in aerosol from e-cigarettes with smoke from conventional cigarettes and with ambient air

    Regul. Toxicol. Pharmacol.

    (2014)
  • P. Xiang

    Molecular mechanisms of dust-induced toxicity in human corneal epithelial cells: water and organic extract of office and house dust

    Environ. Int.

    (2016)
  • M.A. Abegg et al.

    Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide

    Rev. Soc. Bras. Med. Trop.

    (2012)
  • S. Aktas

    Impact of smoking on the ocular surface, tear function, and tear osmolarity

    Curr. Eye Res.

    (2017)
  • D.D. Altinors

    Smoking associated with damage to the lipid layer of the ocular surface

    Am. J. Ophthalmol.

    (2006)
  • B.J. Apelberg

    Environmental monitoring of secondhand smoke exposure

    Tobac. Contr.

    (2013)
  • H. Carp et al.

    Possible mechanisms of emphysema in smokers. In vitro suppression of serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants

    Am. Rev. Respir. Dis.

    (1978)
  • A. Carrano

    Amyloid Beta induces oxidative stress-mediated blood-brain barrier changes in capillary amyloid angiopathy

    Antioxidants Redox Signal.

    (2011)
  • R.P. Casaroli-Marano et al.

    Edel, M. & A, B. A.-P. Potential role of induced pluripotent stem cells (IPSCs) for cell-based therapy of the ocular surface

    J. Clin. Med.

    (2015)
  • G. Cennamo et al.

    Anterior-segment optical coherence tomography and scanning electron microscopy to evaluate corneal epithelial changes in patients undergoing glaucoma therapy

    Cornea

    (2018)
  • W.H. Chang

    Cigarette smoke regulates the competitive interactions between NRF2 and BACH1 for heme oxygenase-1 induction

    Int. J. Mol. Sci.

    (2017)
  • X. Cheng et al.

    Impaired redox signaling and antioxidant gene expression in endothelial cells in diabetes: a role for mitochondria and the nuclear factor-E2-related factor 2-Kelch-like ECH-associated protein 1 defense pathway

    Antioxidants Redox Signal.

    (2011)
  • M. Chwa

    Increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts

    Invest. Ophthalmol. Vis. Sci.

    (2006)
  • S.P. Collin et al.

    A comparative SEM study of the vertebrate corneal epithelium

    Cornea

    (2000)
  • C.E. Cross

    Cigarette smoke oxidation of human plasma constituents

    Ann. N. Y. Acad. Sci.

    (1993)
  • N. Ehlers

    Morphological evaluation of normal human corneal epithelium

    Acta Ophthalmol.

    (2010)
  • WHO Report on the Global Tobacco Epidemic

    (2019)
  • C.B. Gilks et al.

    Antioxidant gene expression in rat lung after exposure to cigarette smoke

    Am. J. Pathol.

    (1998)
  • K.K. Griendling et al.

    NAD(P)H oxidase: role in cardiovascular biology and disease

    Circ. Res.

    (2000)
  • P.L. Grover et al.

    Enzyme-catalysed reactions of polycyclic hydrocarbons with deoxyribonucleic acid and protein in vitro

    Biochem. J.

    (1968)
  • N.R. Hackett

    Variability of antioxidant-related gene expression in the airway epithelium of cigarette smokers

    Am. J. Respir. Cell Mol. Biol.

    (2003)
  • S.S. Hecht

    Tobacco carcinogens, their biomarkers and tobacco-induced cancer

    Nat. Rev. Canc.

    (2003)
  • L. Hegedius et al.

    Relationship between cigarette smoking and Graves' ophthalmopathy

    J. Endocrinol. Invest.

    (2004)
  • H. Hu

    RNA sequencing analysis shows that titanium dioxide nanoparticles induce endoplasmic reticulum stress, which has a central role in mediating plasma glucose in mice

    Nanotoxicology

    (2018)
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