Ozone-induced injury and oxidative stress in bronchiolar epithelium are associated with altered pulmonary mechanics

Abstract

In these studies, we analyzed the effects of ozone on bronchiolar epithelium. Exposure of rats to ozone (2 ppm, 3 h) resulted in rapid (within 3 h) and persistent (up to 72 h) histological changes in the bronchiolar epithelium, including hypercellularity, loss of cilia, and necrotizing bronchiolitis. Perivascular edema and vascular congestion were also evident, along with a decrease in Clara cell secretory protein in bronchoalveolar lavage, which was maximal 24 h post-exposure. Ozone also induced the appearance of 8-hydroxy-2'-deoxyguanosine, Ym1, and heme oxygenase-1 in the bronchiolar epithelium. This was associated with increased expression of cleaved caspase-9 and beclin-1, indicating initiation of apoptosis and autophagy. A rapid and persistent increase in galectin-3, a regulator of epithelial cell apoptosis, was also observed. Following ozone exposure (3-24 h), increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and arginase-1 was noted in bronchiolar epithelium. Ozone-induced injury and oxidative stress in bronchiolar epithelium were linked to methacholine-induced alterations in pulmonary mechanics. Thus, significant increases in lung resistance and elastance, along with decreases in lung compliance and end tidal volume, were observed at higher doses of methacholine. This indicates that ozone causes an increase in effective stiffness of the lung as a consequence of changes in the conducting airways. Collectively, these studies demonstrate that bronchiolar epithelium is highly susceptible to injury and oxidative stress induced by acute exposure to ozone; moreover, this is accompanied by altered lung functioning.

Keywords: bronchiole; epithelium; oxidative stress; ozone; pulmonary mechanics.

FIG. 1.

Effects of ozone on terminal airway histology. Tissue sections, prepared 3–72h after exposure of rats to air or ozone, were stained with H & E. Images were acquired using a VS120 Virtual Microscopy system. One representative lung section from four rats per treatment group is shown. Left panels: Original magnification, ×10. Right panels: Original magnification, ×40. ndl, necrotic debris in lumen; pve, perivascular edema; vc, vascular congestion; am, alveolar macrophage; hc, hypercellularity of bronchiolar epithelium; boxes show areas of higher magnification.

FIG. 2.

Effects of ozone on CCSP. BAL, collected 3–72h after exposure of rats to air or ozone, was analyzed for CCSP levels by Western blotting. Each lane represents BAL from one rat.

FIG. 3.

Effects of ozone on markers of oxidative stress. Lung sections, prepared 3–72h after exposure of rats to air or ozone, were stained with antibody to 8-OHdG, Ym1, or HO-1. Binding was visualized using a peroxidase DAB substrate kit. One representative section from three rats per treatment group is shown (original magnification, ×20).

FIG. 4.

Effects of ozone on markers of apoptosis and autophagy. Lung sections, prepared 3–72h after exposure of rats to air or ozone, were stained with antibody to cleaved caspase-9, galectin-3, or beclin-1. Binding was visualized using a peroxidase DAB substrate kit. One representative section from three rats per treatment group is shown (original magnification, ×20).

FIG. 5.

Effects of ozone on iNOS, COX-2, and arginase-1 expression. Lung sections, prepared 3–72h after exposure of rats to air or ozone, were stained with antibody to iNOS, COX-2, or arginase-1. Binding was visualized using a peroxidase DAB substrate kit. One representative section from three rats per treatment group is shown (original magnification, ×20).

FIG. 6.

Effects of ozone on methacholine-induced pulmonary mechanics. Total lung resistance, total lung compliance, static compliance, and end tidal volume were evaluated in response to increasing doses of methacholine 24h following exposure of rats to air or ozone. Values were normalized and expressed as percentage change from baseline. Each point is the mean ± SE (n = 6 rats). *Significantly different (p ≤ 0.05) from air-exposed animals.

FIG. 7.

Effects of ozone on methacholine-induced resistance and elastance spectra. Total respiratory resistance (R_RS) and elastance (E_RS) were evaluated in response to increasing doses of methacholine (0–96mg/ml), 24h following exposure of rats to air or ozone. Data are cm H₂O/ml/s (for R_RS) and cm H₂O/ml (for E_RS). Top and middle panels: Best fit lines of the primary model fitting of the Z_L spectra to the constant-phase model. Each point is the average value of three to four rats per treatment. Bottom panels: a, low-frequency resistance; ΔE, frequency dependence of elastance. *Significantly different (p ≤ 0.05) from air-exposed animals.