Effects of sulfur dioxide derivatives on four asthma-related gene expressions in human bronchial epithelial cells

Abstract

Sulfur dioxide (SO₂) is a common air pollutant, and inhaled SO₂ in airway epithelium easily forms its soluble derivatives in vivo (bisulfite and sulfite), which are toxic to the respiratory system and related to the exacerbation of asthma. In order to study the possible asthmatic molecular mechanism of SO₂ and its derivatives, the dose-response and time-response relationships of SO₂ derivatives on gene expressions of some asthma-related genes in human bronchial epithelial cells (BEP2D) were investigated. The mRNA and protein levels of EGF, EGFR, ICAM-1 and COX-2 were analyzed in BEP2D cells using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assay, radio-immunoassay (RIA) method and Western blot analysis, respectively. The results showed that SO₂ derivatives caused the dose-dependent inductive expressions of four gene mRNA and protein in BEP2D cells. Moreover, SO₂ derivatives significantly increased the mRNA and protein levels at 0, 0.5, 1, 4 and 24 h post-exposure, along with the highest inductions at 0.5 h post-exposure for EGFR and COX-2 and at 4 h post-exposure for EGF and ICAM-1. It was suggested that SO₂ derivatives could increase the expressions of EGF, EGFR, ICAM-1 and COX-2 on the transcription and translation levels in BEP2D cells, and result in mucus over-production and inflammation responses. This might be one of the possible mechanisms that SO₂ aggravates asthma disease.

Introduction

Bronchial asthma is a chronic inflammatory disease characterized by airway hyper-reactivity (AHR), mucus over-secretion, and inflammation responses. Mortality and morbidity rates for asthma have been rapidly increasing in recent years, and asthma has become a major public health problem in various countries. It has been confirmed that the pathogenesis of asthma involves a combination of genetic and environmental factors (Blumenthal, 2005). Sulfur dioxide (SO₂) is a ubiquitous air pollutant, presents in low concentrations in the urban air, and in higher concentrations in the working environment. SO₂ inhalation could affect the respiratory system in children and elders (Tseng and Li, 1990, Derya, 2003), and especially might aggravate asthma symptoms in asthmatic subjects (Linn et al., 1987, Grella et al., 2002). Inhaled SO₂ can easily be hydrated to produce sulfurous acid in the respiratory tract and subsequently forms bisulfite and sulfite derivatives (1:3 M/M, in neutral fluid), the toxicity of SO₂, actually, is affected by this two derivatives (Shapiro, 1977). In addition, its derivatives (bisulfite and sulfite) have been widely used in food industry and drug production. Recent years, many studies on the effects of SO₂ and its derivatives on the genetic toxicity, oxidative damage, DNA damage, changes of the ion channels, signal transduction, membrane damage, etc., had been detected (Meng, 2003, Meng et al., 2005, Bai and Meng, 2005, Nie and Meng, 2005, Qin and Meng, 2005, Qin and Meng, 2006), and the results showed that the toxicological effects of SO₂ and its derivatives were abroad and complex. However, so far few studies about asthmatic molecular mechanisms have been reported.

Among plenty of asthma-related genes, epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), intercellular adhesion molecule-1 (ICAM-1) and cyclooxygenase-2 (COX-2) play an important role in the pathogenesis of asthma. EGF and EGFR might be involved in inflammatory repair and mucin synthesis. Ligand (such as EGF) binding to the EGFR in epithelium activates an EGFR signal transduction pathway leading to mucin synthesis (Nadel, 2001, Burgel and Nadel, 2004). ICAM-1 is an adhesion molecule expressed on vascular endothelial cells and epithelial cells and plays an important role in establishing and maintaining inflammation and AHR in asthma (Wegner et al., 1990). COX-2 is a key inducible enzyme that regulates the production of prostaglandin E₂ (PGE₂), and high levels of PGE2 may induce the constriction of airway smooth muscle resulting in plugged bronchi and enhance inflammatory cell survival (Cai et al., 2006, Park and Christman, 2006).

Recently, the animal models had been applied in the experiments and therapies of asthma (Wang and Yang, 2003, Kirsten and Charlotte, 2004, Wu et al., 2006), but few cell models in vitro had been used to investigate the mechanisms of asthma disease. Bronchial epithelium is a metabolically active barrier in the human airways that encounters any noxious agent, and changes in the structure and function of bronchial epithelial cells had relations with pathogenesis of asthma (Velden and Versnel, 1998, John and Fahy, 2001). Thus, in order to study the possible asthmatic molecular mechanism of SO₂ and its derivatives, The HPV-18 immortalized human bronchial epithelial cell line (BEP2D) was used in our study to analyze EGF, EGFR, ICAM-1 and COX-2 mRNA and protein expressions using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assay, Western blot analysis and radio-immunoassay (RIA) method, respectively.