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. 2025 Jan 11;204(1):70–78. doi: 10.1093/toxsci/kfae162

Inhaled ozone induces distinct alterations in pulmonary function in models of acute and episodic exposure in female mice

Jordan M Lee 1,†, Jaclynn A Meshanni 2,†, Kinal N Vayas 3, Vasanthi R Sunil 4, Jared Radbel 5, Jeffrey D Laskin 6, Debra L Laskin 7,✉, Andrew J Gow 8,✉
PMCID: PMC11879009  PMID: 39798125

Abstract

Ozone is an urban air pollutant known to cause lung injury and altered function. Using established models of acute (0.8 ppm, 3 h) and episodic (1.5 ppm, 2 h, 2 times/wk, 6 wk) inhalation exposure, we observed distinct structural changes in the lung; whereas acutely, ozone primarily disrupts the bronchiolar epithelial barrier, episodic exposure causes airway remodeling. Herein we examined how these responses altered pulmonary function. A SCIREQ small animal ventilator was used to assess lung function; impedance was used to conditionally model resistance and elastance. Episodic, but not acute ozone exposure reduced the inherent and frequency-dependent tissue recoil (elastance) of the lung. Episodic ozone also increased central and high-frequency resistance relative to air control after methacholine challenge, indicating airway hyperresponsiveness. Pressure-volume (PV)-loops showed that episodic ozone increased maximum lung volume, whereas acute ozone decreased lung volume. Episodic ozone-induced functional changes were accompanied by increases in alveolar circularization; conversely, minimal histopathology was observed after acute exposure. However, acute ozone exposure caused increases in total phospholipids, total surfactant protein D (SP-D), and low-molecular weight SP-D in bronchoalveolar lavage fluid. Episodic ozone exposure only increased total SP-D. These findings demonstrate that acute and episodic ozone exposure caused distinct alterations in surfactant composition and pulmonary function. Whereas loss in PV-loop area following acute ozone exposure is likely driven by increases in SP-D and inflammation, emphysematous pathology and airway hyperresponsiveness after episodic ozone appear to be the result of alterations in lung structure.

Keywords: ozone, pulmonary mechanics, surfactant protein, lung lipids

Acute exposure to elevated environmental levels of ozone is associated with increases in lung disease-related hospitalizations, risk of asthma exacerbations, and mortality (Galizia and Kinney 1999; Fauroux et al. 2000; Zhang et al. 2019). Ozone has also been shown to exacerbate inflammation in allergic airway disease and, in healthy subjects, can cause a transient 20% to 30% loss in forced expiratory volume (FEV) (McDonnell et al. 1991, 1997; Peden et al. 1995; Scannell et al. 1996). Importantly, these responses are more prominent after chronic ozone exposure, which is also correlated with increased mortality across multiple age groups (Tager et al. 2005; Turner et al. 2016). Of particular concern is the observation that chronic ozone exposure results in significant pulmonary dysfunction including a loss in forced expiratory flow rate, and forced expiratory volume (FEV1), suggesting obstructive lung disease (Galizia and Kinney 1999; Tager et al. 2005). As a consequence of rising temperature, ozone levels are increasing especially in highly populated polluted environments, resulting in more intense and frequent exposures and intensifying concerns about public health (East et al. 2024). Thus, it is essential to elucidate mechanisms underlying the adverse effects of ozone on pulmonary functioning and the impact of episodic or chronic exposure on this response.

In animal models, acute and episodic ozone inhalation cause distinct histopathologic changes in the lung (Sunil et al. 2012, 2015; Michaudel et al. 2018). Hence, while acute exposure causes transient alveolar endothelial barrier dysfunction, damages type I alveolar epithelial cells, and stimulates type II alveolar epithelial cell proliferation and surfactant production; episodic ozone exposure causes irreversible destruction of alveolar structure accompanied by thinning of the bronchial epithelium and peribronchial collagen deposition (Stephens et al. 1974; Connor et al. 2012; Sunil et al. 2013; Kim et al. 2018; Sokolowska et al. 2019; Francis et al. 2020). This is associated with persistent inflammation and structural alterations in the lung consistent with emphysema (Chang et al. 1992; Triantaphyllopoulos et al. 2011; Michaudel et al. 2018; Wiegman et al. 2020).

Functional studies using a single-compartment model have shown that both acute and episodic ozone exposure are associated with airway hyperresponsiveness (AHR), with no major effects on inherent airway resistance or smooth muscle mass (Triantaphyllopoulos et al. 2011; Pinart et al. 2013; Michaudel et al. 2018; Birukova et al. 2019). However, the effects of episodic ozone exposure on parenchymal lung function beyond the 16th airway generation are unknown. Herein, we assessed lung function in established murine models of acute and episodic ozone exposure (Triantaphyllopoulos et al. 2011; Sunil et al. 2012, 2013; Zhang et al. 2016; Michaudel et al. 2018) and assessed underlying physiological mechanisms. For these studies, we performed forced oscillation maneuvers and analyzed the resultant impedance data using a constant phase model, which we previously developed to distinguish lung function at low and high frequencies, an approach advantageous for analysis of heterogeneous lung injury such as that induced by ozone (Groves et al. 2012, 2013). Based on the distinct pathologies caused by acute and episodic exposure to ozone (Michaudel et al. 2018), we predicted that the different exposure models would be associated with unique changes in lung function and indeed this is what we observed. Our findings provide novel mechanistic insights into ozone-induced pulmonary toxicity, which may be useful for mitigating injury and disease pathogenesis in humans caused by exposure to oxidant air pollutants.

Materials and methods

Animal care and use

Female C57Bl6/J mice (13 to 19 wk) were obtained from The Jackson Laboratories (Bar Harbor, ME, United States). Mice were exposed to air or ozone in a whole-body Plexiglas chamber. An ultraviolet light ozone generator (Gilmont Instruments, Barrington, IL, United States) was used to produce ozone from oxygen gas. Ozone was mixed with air, then released into the chamber; the concentration was continuously monitored using a Photometric ozone analyzer (model 202, 2B Instruments, Boulder, CO, United States). For acute studies, mice were exposed to air or ozone (0.8 ppm) for 3 h (Sunil et al. 2012, 2013); for episodic studies, mice were exposed to air or ozone (1.5 ppm) for 2 h, 2 times per week for 6 wk; this exposure model has been reported to induce pathology consistent with chronic lung disease in mice (Triantaphyllopoulos et al. 2011; Zhang et al. 2016; Michaudel et al. 2018). Animals received humane care in compliance with the institution’s guidelines, as outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Measurement of pulmonary mechanics

Forty-eight hours after acute exposure or 24 h after the final episodic exposure, mice were anesthetized by intraperitoneal injection of ketamine (115 mg/kg) and xylazine (30 mg/kg). A tracheotomy was performed using an 18-gauge cannula; the animals were then connected to a flexiVent small animal ventilator (SCIREQ, Montreal) via the cannula. Respiratory mechanics in response to increasing doses of methacholine (MCh, 0 to 96 mg/mL) were measured at a physiological positive end-expiratory pressure (PEEP) of 3 cm H2O as previously described (Groves et al. 2012; Sunil et al. 2013; Massa et al. 2014). After each MCh dose, 9 to 12 maneuvers of Snapshot-15 and Quick Prime 3 were repeated. The sixth, seventh, and eighth measurements were used for analysis of the maximal dose response. Deep inflation maneuvers were performed between each MCh dose. Input impedance was used in a single-compartment constant phase model to calculate central airway resistance (Rn). Submaximal quasi-static pressure-volume (PV)-loops were generated in a stepwise inflation as previously described (Massa et al. 2014). Briefly, the lung was inflated in 8 equal pressure-regulated steps over 16 s to a peak pressure of 10 cm H2O. PV-loop areas were calculated as the area between the inspiratory and expiratory curves. Data were analyzed using flexiVent software version 8. Note that no sample collection was performed following pulmonary mechanic measurements; separate groups of mice were used for all other endpoints.

Resistance and elastance spectra were generated from the real impedance (resistance R) and imaginary impedance (reactance X) at each MCh dose using a constant phase model. A line of best fit was generated using nonlinear regression. Parameters of resistance were calculated as: RL=(a+bf)/(c+f), where a/c denotes low-frequency in which tissue resistance and viscosity predominate, and b is high-frequency in which airway resistance predominates, and ƒ is frequency.

Parameters of elastance were calculated as: EL=E0+ΔE(1-e-βf), where E0 is theoretical elastance at baseline (0 Hz) and represents baseline tissue stiffness, ΔE is the magnitude of frequency-dependent change, and β is the rate that elastance changes with frequency.

Sample collection

Animals were euthanized 48 h after acute exposure or 24 h after the final episodic exposure session by intraperitoneal injection of ketamine (135 mg/kg) and xylazine (30 mg/kg). Bronchoalveolar lavage (BAL) fluid was collected by slowly instilling and withdrawing 1 mL of ice-cold PBS into the lung thrice through a cannula in the trachea. BAL fluid was centrifuged (300×g, 8 min) and supernatant collected and stored at −80°C until analysis. For preparation of histological sections, following lavage the lung was removed, instilled with 3% ice-cold paraformaldehyde, and suspended in 3% paraformaldehyde. After 24 h, the tissue was resuspended in 50% ethanol, embedded in paraffin, and cut longitudinally into 4 μm sections. After staining with hematoxylin and eosin, images of the tissue sections were captured by light microscopy at 40× using a VS-120 slide scanner (Olympus Corporation; Center Valley, PA, United States). Airway circularization was quantified by blindly scoring 10 fields per slide using ImageJ as previously described (Golden et al. 2021).

Phospholipid analysis

BAL (0.8 to 1 mL) was centrifuged for 1 h (17,000×g, 4 °C). The pellet containing lipid-enriched large aggregate fractions was collected and resuspended in 30 μL PBS. Total inorganic phosphates were measured as previously described (Massa et al. 2014). Briefly, inorganic phosphates were precipitated from the lipid and then heated in a sulfuric acid mixture to produce a colorimetric reaction. Total phospholipids were measured at 830 nm by light absorbance and quantified using a standard curve ranging from 0 to 3.1 µg phosphate.

Western blot analysis

SP-D and SP-B in BAL were analyzed using denaturing SDS–PAGE as previously described (Massa et al. 2014). Briefly, for denaturing SDS–PAGE, cell-free BAL (SP-D, 1 μL/well) or large aggregate fractions of BAL (SP-B, 1.5 μg phospholipid/well) were loaded onto a NuPAGE 4% to 12% Bis-Tris gel (BioRad; Hercules, CA, United States). For native SDS–PAGE, equal SP-D in cell-free BAL, as determined by SP-D blot integrated density under reducing conditions, was loaded onto a NuPAGE 3% to 8% Tris-Acetate gel (BioRad). Proteins were transferred using a wet transfer chamber (BioRad) onto PVDF membranes. Blots were blocked with 10% milk and incubated for 1 h at room temperature with primary anti-SP-D antibody DU117 for denaturing gels (1:10,000), anti-SP-D (AB3434; MilliporeSigma Burlington, MA; 1:10,000), or anti-SP-B (M.F. Beers, University of Pennsylvania) followed by incubation with secondary goat anti-rabbit horseradish peroxidase-conjugated antibodies. Proteins were detected using BioRad ECL prime (BioRad) and densitometry performed using ImageJ (National Institutes of Health).

Statistical analysis

Data were analyzed using GraphPad Prism. If parametric, 2-way ANOVA with Tukey’s post hoc analysis or unpaired 2-tailed t test were used to assess statistical significance. If data were not parametric, a Mann–Whitney U test was used. A P-value ≤ 0.05 or nonoverlapping 95% CIs were considered significant.

Results

Effects of ozone on histopathology and pulmonary surfactant

Initially, we examined the effects of acute and episodic ozone exposure on lung histopathology. Consistent with previous studies (Bhalla 1999; Sunil et al. 2013; Michaudel et al. 2018), only minimal structural changes were detected in the lung parenchyma after acute ozone exposure (Fig. 1). By comparison, episodic ozone exposure resulted in a significant increase in circularization, indicative of alveolar degeneration (Fig. 1) (Bodduluri et al. 2021). These findings are in line with previous studies from our laboratory and others (Triantaphyllopoulos et al. 2011; Sunil et al. 2012; Michaudel et al. 2018; Francis et al. 2020).

Fig. 1.

Fig. 1.

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Effects of acute and episodic ozone exposure on lung histopathology. Mice were exposed to ozone as described in the Materials and Methods section. Tissue sections were prepared 48 h after acute exposure or 24 h after the final episodic exposure session and stained with hematoxylin and eosin. The alveolar area to perimeter ratio in 10 random fields/animal was analyzed using ImageJ, shown in the chart (mean alveolar space [ratio area/perimeter]±SE). Representative sections from 1 mouse/treatment group (n = 3 to 4 mice/treatment group) are shown. Original magnification, 40×. Data were analyzed using 2-way ANOVA. *Significantly different (P ≤ 0.05) from matched air controls.

We next evaluated the effects of ozone on pulmonary phospholipids, which play a key role in pulmonary functioning (Groves et al. 2012; Sunil et al. 2013). Following acute, but not episodic ozone exposure, total phospholipids in BAL increased significantly (Fig. 2A). Increased BAL levels of SP-D were also noted after acute ozone exposure (Fig. 2B, upper panels), along with conformational changes in SP-D, as evidenced by the appearance of small molecular weight proteins in the native gels (Fig. 2B, lower panel). Whereas SP-D levels also increased after episodic ozone exposure, there were no conformational changes in SP-D. Ozone also had no effects on levels of SP-B in BAL after acute or episodic ozone exposures (Fig. 2C).

Fig. 2.

Fig. 2.

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Effects of acute and episodic ozone exposure on pulmonary surfactants. BAL was collected from mice 48 h after acute exposure or 24 h after the final episodic exposure session. (A) Large aggregate fractions of BAL were prepared, and total phospholipids measured. Bars, mean±SE (n = 4 to 5 mice/treatment group). Data were analyzed using 2-way ANOVA. (B) Unmodified SP-D and modified SP-D monomers in cell-free BAL were assessed by western blotting using denatured (upper panels) or native gels (lower panels), respectively. One representative gel is shown. Data were analyzed using a Mann–Whitney U test. Bars, mean±SE (n = 4 to 7 mice/treatment group). (C) Total SP-B was analyzed in large aggregate fractions of BAL by western blotting using denaturing gels. Bars, mean±SE (n = 3 to 4 mice/treatment group). Data were analyzed using a Mann–Whitney U test. *Significantly different (P ≤ 0.05) from matched air controls. #Significantly different (P ≤ 0.05) from acute ozone. Original gel images are available in Fig. S5.

Effects of ozone on lung volume and compliance

In further studies, we evaluated changes in lung volume and compliance following ozone exposure by analyzing PV-loops. Acute exposure of mice to ozone had no significant effect on PV-loops (Fig. 3A). In contrast, episodic ozone exposure caused a leftward shift in the PV-loop, consistent with increased static compliance (Cst) and decreased elastance (E0) (Figs 3A and D and 4B). Episodic, but not acute ozone exposure, was also associated with an increase in maximum lung volume (Fig. 3C). PV-loop area (hysteresis), measured by the area between the inspiratory and expiratory curves, was significantly reduced by exposure to acute ozone, consistent with dysfunctional surfactant; episodic ozone had no effect on this response (Fig. 3B).

Fig. 3.

Fig. 3.

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Effects of acute and episodic ozone exposure on pressure (P) volume (V)-loops. Pulmonary function was assessed 48 h after acute exposure or 24 h after the final episodic exposure session using a SCIREQ flexiVent. (A) PV-loop measurements. Data are mean±SE. (B) Hysteresis was calculated as the area between the inspiration and expiration curves. Bars, mean±SE. Data were analyzed using 2-way ANOVA. (C) Maximum lung volume (mL) is the plateau of the nonlinear regression curve fitted to the expiratory arm of the PV-loop. Bars, mean±95% CI. Data were considered statistically significant if 95% CI did not overlap. (D) Static compliance (Cst) was derived from the Salazar–Knowles model. Bars, mean±SE. Data were analyzed using 2-way ANOVA. *Significantly different (P ≤ 0.05) from matched air controls. #Significantly different (P ≤ 0.05) from acute ozone. For all data, n = 8 to 11 mice/treatment group.

Fig. 4.

Fig. 4.

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Effects of acute and episodic ozone exposure on baseline resistance (b, a/c) and elastance (E0, ΔE, β) parameters. Pulmonary function was assessed 48 h after acute exposure or 24 h after the final episodic exposure session using a SCIREQ flexiVent. Real and imaginary impedances were measured at baseline (vehicle control) using a SCIREQ flexiVent. Parameters (b, a/c, E0, ΔE, β) were extracted from resistance/real (A) and elastance/imaginary (B) spectra using a multi-compartment constant phase model. Bars, mean±SE (n = 6 to 8 mice/treatment group). Data were analyzed using 2-way ANOVA. *Significantly different (P ≤ 0.05) from matched air controls. #Significantly different (P ≤ 0.05) from acute ozone.

Effects of ozone on inherent pulmonary mechanics

We next measured real and imaginary impedance in the absence of a bronchoconstrictor to assess inherent pulmonary resistance and elastance, respectively (Figs S1 and S2). No changes in inherent/baseline overall resistance were observed after acute or episodic ozone exposure (Fig. S2). Using the constant phase model, resistance was further broken down into low- (a/c) and high- (b) frequency components. Low-frequency resistance is primarily determined by the respiratory portion of the lung, whereas high-frequency factors are reflective of conduit airway function (Groves et al. 2013). Exposure of mice to acute or episodic ozone had no significant effect on small airway resistance (a/c) or large airway resistance (b) at baseline (Fig. 4A).

Episodic ozone exposure resulted in a decrease in the imaginary impedance, indicative of overall pulmonary elastance, with no change after acute ozone exposure (Fig. S1). To characterize components of elastance including tissue recoil at theoretical 0 Hz (E0), magnitude of frequency-dependent change in elastance (ΔE), and rate of frequency-dependent change in elastance (β), we used a constant phase model. Acute ozone exposure had no effect on the properties of elastance (E0, ΔE, or β) (Fig. 4B). However, episodic ozone reduced both E0 and ΔE, with no effect on β (Fig. 4B).

Effects of ozone on methacholine-dependent pulmonary resistance

To assess differences in airway hyperreactivity or hypersensitivity following acute and episodic ozone exposure, we measured real impedance following administration of increasing doses of MCh (0 to 96 mg/ml) (Fig. S2). Nonlinear regression of the impedance data was used to calculate central resistance (Rn), the resistance at theoretical 0 Hz (Fig. S3). MCh-induced Rn was unchanged by acute ozone exposure when compared with air control (Fig. S3). However, episodic ozone enhanced the magnitude of the Rn response to MCh (Fig. S3). In addition, whereas acute ozone exposure did not significantly alter low- (a/c) or high- (b) frequency resistance at any dose of MCh, episodic ozone caused high-frequency resistance (b) to reach a greater maximum response, with the peak response occurring at the same dose of MCh (12 mg/mL) after both exposures (Fig. 5). Episodic ozone had no effect on MCh-dependent effects on low-frequency resistance (a/c) (Fig. 5).

Fig. 5.

Fig. 5.

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Effects of acute and episodic ozone exposure on maximum resistance (b, a/c) parameters following methacholine challenge. Pulmonary function was assessed 48 h after acute exposure or 24 h after the final episodic exposure session using a SCIREQ flexiVent. Real impedance was measured following administration of 0 to 96 mg/mL methacholine. A multi-compartment constant phase model was used to generate b and a/c from real impedance data. (A) The dose-related a/c and b responses to increasing doses of methacholine (mg/mL). (B) The maximal response of a/c and b to methacholine stimulation. Data, mean±SE (n = 6 to 8 mice/treatment group). Data were analyzed using 2-way ANOVA. *Significantly different (P ≤ 0.05) from matched air controls. #Significantly different (P ≤ 0.05) from acute ozone.

Discussion

As ambient ozone represents a significant public health concern, elucidating functional alterations associated with acute and episodic exposure are essential in order to develop efficacious strategies for treating injury and diseases caused by this pulmonary toxicant. The present studies demonstrate that acute and episodic ozone exposure cause distinct alterations in pulmonary mechanics which are likely driven by surfactant dysfunction and structural alterations, respectively. Thus, while acute ozone resulted in surfactant dysfunction and decreased compliance, episodic ozone-related histopathology was characterized by emphysematous changes in the lung and associated with impaired elastance. These findings suggest that different exposure-related pathologic evaluations and clinical approaches will be required to adequately mitigate ozone toxicity.

To evaluate histopathologic responses to ozone, we selected 48 h after acute exposure, as we previously found that peak inflammation measured by BAL cell number and macrophage activation occurs at this time; moreover, this post-exposure time coincides with the greatest alterations in lung function (Tighe et al. 2018). The exposure regimen for the episodic treatment followed a model reported to induce inflammatory disease (Triantaphyllopoulos et al. 2011; Michaudel et al. 2018; Tighe et al. 2018). To assess lung function, we conducted forced oscillation maneuvers and collected measurements of PV responses and forced expiration using a small animal ventilator. Using the constant phase model, elastance and resistance spectra were generated from impedances, then separated based on frequency into physiological parameters of resistance (a/c, b) and elastance (E0, ΔE, β). Initially, we assessed lung mechanics at baseline in the absence of a bronchoconstrictor. In mice exposed to ozone acutely, PV-loop area was significantly decreased, consistent with surfactant dysfunction. This is in line with our findings that BAL levels of total phospholipids, total SP-D protein, and low-MW SP-D were increased after acute ozone exposure. SP-D is a pulmonary collectin synthesized mainly by alveolar type II cells that play a central role in regulating innate immune defenses (Stephens et al. 1974; Connor et al. 2012). Under homeostatic conditions, SP-D functions as an anti-inflammatory protein, in part by suppressing NF-κB-mediated transcription of inflammatory genes (Sunil et al. 2013). However, post-translational modification of critical cysteines in SP-D by nitric oxide, which is known to be produced after acute ozone exposure, leads to the formation of SP-D trimers and a change in its activity to a proinflammatory mediator (Triantaphyllopoulos et al. 2011). Findings of persistent localization of activated macrophages in the lung and increased production of nitric oxide by these cells in mice lacking native SP-D are consistent with its anti-inflammatory function (Chang et al. 1992; Wiegman et al. 2020). Low-MW SP-D trimers are known to promote inflammation by increasing macrophage accumulation and activation in injured lung (Reid 1998; Von Bredow et al. 2006; Guo et al. 2008; Gaunsbaek et al. 2013). In previous studies, we demonstrated that impaired lung function and inflammation caused by acute ozone exposure are due, in part, to loss of normal SP-D activity (Groves et al. 2012, 2013). The present studies show that surfactant function was normal after episodic ozone exposure, as evidenced by findings that PV-loop area and total phospholipid content were at control levels and that there was no evidence of low-MW SP-D monomers. Of note, total SP-D levels remained elevated despite alveolar destruction following episodic ozone exposure. This may be a consequence of type II alveolar epithelial cell proliferation which has been observed in rodents exposed chronically to ozone (Chang et al. 1992). Increases in native SP-D are considered a marker of inflammation (Forbes and Haczku 2010). In line with this, we found that episodic ozone exposure was associated with persistent increases in BAL cell number (Fig. S4). In earlier studies, we demonstrated that these cells contribute to acute ozone toxicity and we speculate that they play a similar role in the lung injury and oxidative stress induced by episodic ozone exposure (Sunil et al. 2012, 2013). Previously, we showed that acute ozone exposure increases the frequency-dependent change in elastance (ΔE) at low PEEP/hypoinflation, a response markedly exacerbated by loss of SP-D (Forbes and Haczku 2010; Groves et al. 2012). Taken together, these data indicate that SP-D is critical for proper tissue recoil after acute ozone exposure (Groves et al. 2012, 2013). In contrast, we did not observe alterations in resistance or elastance after acute ozone exposure, in accord with our published studies using a constant phase model at a PEEP of 3 cm H2O, or a single-compartment model (Groves et al. 2012, 2013).

Following episodic ozone exposure, the PV-loop was shifted to the left, indicating an increase in distensibility and a corresponding decrease in recoil. Inherent elastance (E0) and the frequency-dependent change in elastance (ΔE) were consequently reduced. The leftward shift of the PV-loop and decreases in E0 and ΔE, along with alveolar degeneration are consistent with emphysematous-like lung pathology (Knudsen et al. 2015). Notably, episodic ozone did not significantly alter low- or high-frequency resistance at baseline, suggesting that there are no appreciable effects on airway obstruction, which would be expected to occur as a consequence of altered basal tone, inflammatory cell accumulation, or debris from tissue destruction. It may be that changes in resistance were not detected due to limitations of our constant phase model.

In further studies, we analyzed the effects of ozone exposure on AHR after challenge with the bronchoconstrictor MCh. Acute ozone exposure had no effect on AHR, as assessed using the constant phase model. This is in contrast to other studies using a single-compartment model, which reported increases in AHR up to 72 h post-ozone exposure in mice (Sunil et al. 2012, 2015; Pinart et al. 2013; Tighe et al. 2018). Differences in our findings could be attributed to the constant phase model method of compartmentalizing resistive components. However, it is unlikely that it is due to variations in the model, as our analysis of Rn using the single-compartment model revealed AHR similar to previous studies (Groves et al. 2013). Although acute ozone did not cause AHR in our studies, PV-loop area decreased, a characteristic shared with asthma, but absent in some animal models of allergic asthma (Vanoirbeek et al. 2010; Li et al. 2019b). These findings are notable as the effects of acute ozone are often compared with asthma due to the common outcome of AHR and the positive association between ozone levels and asthma-related hospital visits (Cody et al. 1992; Fauroux et al. 2000; Li et al. 2019a). Ultimately, findings of AHR are limited following acute ozone exposure; however, in the context of human health, ozone exposure may play a more significant pathologic role in individuals with preexisting sensitivities such as asthma.

Episodic ozone exposure caused AHR in conducting airways as evidenced by greater maximum effects of Rn and high-frequency resistance (b) in response to MCh. Our findings that Rn peaked at the same MCh concentration (12 mg/mL) in control and ozone-treated mice suggest that episodic exposure induces AHR, but not hypersensitivity. Episodic ozone exposure also resulted in emphysematous-like changes in lung structure and function, including losses in E0 and ΔE, and increased Cst. Other studies have shown that episodic ozone causes AHR as well as features of emphysema, but that they seem to arise largely via independent mechanisms (Pinart et al. 2013; Li et al. 2018; Michaudel et al. 2018). Whereas episodic ozone-induced AHR requires interleukin 17-mediated inflammation, the development of emphysematous-like injury and airway remodeling involves oxidative stress and apoptosis via the NLRP3-caspase-1 pathway (Pinart et al. 2013; Zhang et al. 2016; Li et al. 2018). We speculate that after 6 wk of repeated ozone exposure, a combination of these mechanisms leads to the emphysematous tissue structure and airway remodeling which drive functional losses (i.e. decreased elastance and AHR). Elastance, which we measured at baseline, was not considered in the MCh responses as in the presence of a bronchoconstrictor, elasticity may be artificially altered.

There are some limitations to our studies including an inability to evaluate complex lung pathologies. Using a small animal ventilator, we measured overall changes in pulmonary function. In the presence of 2 conflicting pathologies such as fibrosis and emphysema, the output will mainly reflect the disease process. Although there is some histological evidence that prolonged exposure to ozone results in both fibrosis and emphysema, advanced technologies such as CT and MRI imaging are needed to distinguish these diverse pathologies (Cross et al. 1981; Chang et al. 1992; Michaudel et al. 2018). We also recognize that measuring impedances at a range of PEEPs would provide more insight into elastic properties in the parenchyma versus airways and these studies are in progress. Another limitation of our studies is the fact that the 2 models of exposure are not directly comparable as they involve different doses delivered over different time periods and with distinct post-analysis time points. However, the main purpose of our study was to understand the functional effects of ozone in established models of acute and prolonged exposure. In this context, the 3 h exposure has been used extensively to model acute ozone-induced injury, whereas the repeat dosing model over a 6 wk period has been used to estimate the effects of prolonged exposure (Triantaphyllopoulos et al. 2011; Sunil et al. 2012, 2013; Zhang et al. 2016; Michaudel et al. 2018).

In summary, the present studies demonstrate that losses in lung function following acute ozone exposure are primarily driven by increases in SP-D, whereas episodic ozone exposure causes functionally distinct alterations largely driven by structural changes in the lung. Moreover, the acute response to ozone did not result in permanent structural alterations in the context of this study design (Michaudel et al. 2018). Conversely, episodic exposure to ozone causes histologic pathology and decreases in PV-loop area and elastance; these data indicate that episodic ozone exposure leads to permanent emphysematous-like alterations in lung structure. We also found that episodic, but not acute ozone exposure significantly increases the severity of AHR relative to air exposure. Another important observation is that the constant phase model is useful for assessing compartmentalized resistive and elastic properties of lung injury but may be less sensitive for evaluating changes in AHR. In humans, the culmination of recurrent episodic ozone exposures over time poses a significant risk for emphysema development and asthma exacerbation. These events may occur in the general population; however, they have more severe implications for the elderly and individuals with preexisting lung disease. Further understanding of mechanisms contributing to aberrant lung structure and function following acute and episodic ozone exposure are necessary for limiting oxidant-induced injury and disease.

Supplementary Material

kfae162_Supplementary_Data
kfae162_supplementary_data.docx (1.5MB, docx)

Acknowledgments

The authors would like to thank Dr. Marianne Polunas of the Rutgers Histology Core for their assistance.

Contributor Information

Jordan M Lee, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, United States.

Jaclynn A Meshanni, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, United States.

Kinal N Vayas, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, United States.

Vasanthi R Sunil, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, United States.

Jared Radbel, Division of Pulmonary and Critical Care Medicine, Department of Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08901, United States.

Jeffrey D Laskin, Department of Environmental and Occupational Health and Justice, School of Public Health, Rutgers University, Piscataway, NJ 08854, United States.

Debra L Laskin, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, United States.

Andrew J Gow, Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, United States.

Supplementary material

Supplementary material is available at Toxicological Sciences online.

Funding

This work was supported by the National Institutes of Health (grant numbers: T32ES01984 ES004738, ES033698, K08ES031678, and ES005022).

Conflicts of interest. None declared.

Data availability

The data that support the findings of this study are available from the corresponding author, D.L.L., upon reasonable request. The code used for modeling in this publication is previously published and available from https://doi.org/10.1165/rcmb.2011-0433OC (Groves et al. 2012).

References

  1. Bhalla DK.  1999. Ozone-induced lung inflammation and mucosal barrier disruption: toxicology, mechanisms, and implications. J Toxicol Environ Health B Crit Rev. 2:31–86. [DOI] [PubMed] [Google Scholar]
  2. Birukova A, Cyphert-Daly J, Cumming RI, Yu Y-R, Gowdy KM, Que LG, Tighe RM.  2019. Sex modifies acute ozone-mediated airway physiologic responses. Toxicol Sci. 169:499–510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bodduluri S, Kizhakke Puliyakote A, Nakhmani A, Charbonnier J-P, Reinhardt JM, Bhatt SP.  2021. Computed tomography–based airway surface area–to-volume ratio for phenotyping airway remodeling in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 203:185–191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang L-Y, Huang Y, Stockstill BL, Graham JA, Grose EC, Menache MG, Miller FJ, Costa DL, Crapo JD.  1992. Epithelial injury and interstitial fibrosis in the proximal alveolar regions of rats chronically exposed to a simulated pattern of urban ambient ozone. Toxicol Appl Pharmacol. 115:241–252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cody RP, Weisel CP, Birnbaum G, Lioy PJ.  1992. The effect of ozone associated with summertime photochemical smog on the frequency of asthma visits to hospital emergency departments. Environ Res. 58:184–194. [DOI] [PubMed] [Google Scholar]
  6. Connor AJ, Laskin JD, Laskin DL.  2012. Ozone-induced lung injury and sterile inflammation. Role of toll-like receptor 4. Exp Mol Pathol. 92:229–235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cross CE, Hesterberg T, Reiser K, Last JA.  1981. Ozone toxicity as a model of lung fibrosis. Chest. 80:52–54. [DOI] [PubMed] [Google Scholar]
  8. East JD, Monier E, Saari RK, Garcia‐Menendez F.  2024. Projecting changes in the frequency and magnitude of ozone pollution events under uncertain climate sensitivity. Earth’s Future. 12:e2023EF003941. [Google Scholar]
  9. Fauroux B, Sampil M, Quénel P, Lemoullec Y.  2000. Ozone: a trigger for hospital pediatric asthma emergency room visits. Pediatr Pulmonol. 30:41–46. [DOI] [PubMed] [Google Scholar]
  10. Forbes L, Haczku A.  2010. SP‐D and regulation of the pulmonary innate immune system in allergic airway changes. Clin Exp Allergy. 40:547–562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Francis M, Guo G, Kong B, Abramova EV, Cervelli JA, Gow AJ, Laskin JD, Laskin DL.  2020. Regulation of lung macrophage activation and oxidative stress following ozone exposure by farnesoid x receptor. Toxicol Sci. 177:441–453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Galizia A, Kinney PL.  1999. Long-term residence in areas of high ozone: associations with respiratory health in a nationwide sample of nonsmoking young adults [dsee comments]. Environ Health Perspect. 107:675–679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gaunsbaek MQ, Rasmussen KJ, Beers MF, Atochina-Vasserman EN, Hansen S.  2013. Lung surfactant protein D (SP-D) response and regulation during acute and chronic lung injury. Lung. 191:295–303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Golden T, Murray A, Venosa A, Gow AJ.  2021. Comprehensive dataset to assess morphological changes subsequent to bleomycin exposure. Data Brief. 37:107270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Groves AM, Gow AJ, Massa CB, Hall L, Laskin JD, Laskin DL.  2013. Age-related increases in ozone-induced injury and altered pulmonary mechanics in mice with progressive lung inflammation. Am J Physiol Lung Cell Mol Physiol. 305:L555–L568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Groves AM, Gow AJ, Massa CB, Laskin JD, Laskin DL.  2012. Prolonged injury and altered lung function after ozone inhalation in mice with chronic lung inflammation. Am J Respir Cell Mol Biol. 47:776–783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guo CJ, Atochina-Vasserman EN, Abramova E, Foley JP, Zaman A, Crouch E, Beers MF, Savani RC, Gow AJ.  2008. S-nitrosylation of surfactant protein-D controls inflammatory function. PLoS Biol. 6:e266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kim BG, Lee PH, Lee SH, Park CS, Jang AS.  2018. Impact of ozone on claudins and tight junctions in the lungs. Environ Toxicol. 33:798–806. [DOI] [PubMed] [Google Scholar]
  19. Knudsen L, Atochina-Vasserman EN, Massa CB, Birkelbach B, Guo C-J, Scott P, Haenni B, Beers MF, Ochs M, Gow AJ.  2015. The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice. Am J Physiol Lung Cell Mol Physiol. 309:L959–L969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Li F, Xu M, Wang M, Wang L, Wang H, Zhang H, Chen Y, Gong J, Zhang JJ, Adcock IM, et al.  2018. Roles of mitochondrial ROS and NLRP3 inflammasome in multiple ozone-induced lung inflammation and emphysema. Respir Res. 19:230–212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li X, Chen Q, Zheng X, Li Y, Han M, Liu T, Xiao J, Guo L, Zeng W, Zhang J, et al.  2019a. Effects of ambient ozone concentrations with different averaging times on asthma exacerbations: a meta-analysis. Sci Total Environ. 691:549–561. [DOI] [PubMed] [Google Scholar]
  22. Li Y, Zhang L, Wang X, Wu W, Qin R.  2019b. Effect of syringic acid on antioxidant biomarkers and associated inflammatory markers in mice model of asthma. Drug Dev Res. 80:253–261. [DOI] [PubMed] [Google Scholar]
  23. Massa CB, Scott P, Abramova E, Gardner C, Laskin DL, Gow AJ.  2014. Acute chlorine gas exposure produces transient inflammation and a progressive alteration in surfactant composition with accompanying mechanical dysfunction. Toxicol Appl Pharmacol. 278:53–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McDonnell WF, Kehrl HR, Abdul-Salaam S, Ives PJ, Folinsbee LJ, Devlin RB, O’Neil JJ, Horstman DH.  1991. Respiratory response of humans exposed to low levels of ozone for 6.6 hours. Arch Environ Health. 46:145–150. [DOI] [PubMed] [Google Scholar]
  25. McDonnell WF, Stewart PW, Andreoni S, Seal E Jr, Kehrl HR, Horstman DH, Folinsbee LJ, Smith MV.  1997. Prediction of ozone-induced FEV1 changes: effects of concentration, duration, and ventilation. Am J Respir Crit Care Med. 156:715–722. [DOI] [PubMed] [Google Scholar]
  26. Michaudel C, Fauconnier L, Julé Y, Ryffel B.  2018. Functional and morphological differences of the lung upon acute and chronic ozone exposure in mice. Sci Rep. 8:10611–10610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Peden DB, Setzer RW Jr, Devlin RB.  1995. Ozone exposure has both a priming effect on allergen-induced responses and an intrinsic inflammatory action in the nasal airways of perennially allergic asthmatics. Am J Respir Crit Care Med. 151:1336–1345. [DOI] [PubMed] [Google Scholar]
  28. Pinart M, Zhang M, Li F, Hussain F, Zhu J, Wiegman C, Ryffel B, Chung KF.  2013. IL-17A modulates oxidant stress-induced airway hyperresponsiveness but not emphysema. PLoS One. 8:e58452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reid KB.  1998. Functional roles of the lung surfactant proteins SP-A and SP-D in innate immunity. Immunobiology. 199:200–207. [DOI] [PubMed] [Google Scholar]
  30. Scannell C, Chen L, Aris RM, Tager I, Christian D, Ferrando R, Welch B, Kelly T, Balmes JR.  1996. Greater ozone-induced inflammatory responses in subjects with asthma. Am J Respir Crit Care Med. 154:24–29. [DOI] [PubMed] [Google Scholar]
  31. Sokolowska M, Quesniaux VF, Akdis CA, Chung KF, Ryffel B, Togbe D.  2019. Acute respiratory barrier disruption by ozone exposure in mice. Front Immunol. 10:2169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stephens RJ, Sloan MF, Evans MJ, Freeman G.  1974. Early response of lung to low levels of ozone. Am J Pathol. 74:31–58. [PMC free article] [PubMed] [Google Scholar]
  33. Sunil VR, Francis M, Vayas KN, Cervelli JA, Choi H, Laskin JD, Laskin DL.  2015. Regulation of ozone-induced lung inflammation and injury by the β-galactoside-binding lectin galectin-3. Toxicol Appl Pharmacol. 284:236–245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sunil VR, Patel-Vayas K, Shen J, Laskin JD, Laskin DL.  2012. Classical and alternative macrophage activation in the lung following ozone-induced oxidative stress. Toxicol Appl Pharmacol. 263:195–202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sunil VR, Vayas KN, Massa CB, Gow AJ, Laskin JD, Laskin DL.  2013. Ozone-induced injury and oxidative stress in bronchiolar epithelium are associated with altered pulmonary mechanics. Toxicol Sci. 133:309–319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tager IB, Balmes J, Lurmann F, Ngo L, Alcorn S, Künzli N.  2005. Chronic exposure to ambient ozone and lung function in young adults. Epidemiology. 16:751–759. [DOI] [PubMed] [Google Scholar]
  37. Tighe RM, Birukova A, Yaeger MJ, Reece SW, Gowdy KM.  2018. Euthanasia-and lavage-mediated effects on bronchoalveolar measures of lung injury and inflammation. Am J Respir Cell Mol Biol. 59:257–266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Triantaphyllopoulos K, Hussain F, Pinart M, Zhang M, Li F, Adcock I, Kirkham P, Zhu J, Chung KF.  2011. A model of chronic inflammation and pulmonary emphysema after multiple ozone exposures in mice. Am J Physiol Lung Cell Mol Physiol. 300:L691–L700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Turner MC, Jerrett M, Pope CA, Krewski D, Gapstur SM, Diver WR, Beckerman BS, Marshall JD, Su J, Crouse DL, et al.  2016. Long-term ozone exposure and mortality in a large prospective study. Am J Respir Crit Care Med. 193:1134–1142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vanoirbeek JAJ, Rinaldi M, De Vooght V, Haenen S, Bobic S, Gayan-Ramirez G, Hoet PHM, Verbeken E, Decramer M, Nemery B, et al.  2010. Noninvasive and invasive pulmonary function in mouse models of obstructive and restrictive respiratory diseases. Am J Respir Cell Mol Biol. 42:96–104. [DOI] [PubMed] [Google Scholar]
  41. Von Bredow C, Hartl D, Schmid K, Schabaz F, Brack E, Reinhardt D, Griese M.  2006. Surfactant protein D regulates chemotaxis and degranulation of human eosinophils. Clin Exp Allergy. 36:1566–1574. [DOI] [PubMed] [Google Scholar]
  42. Wiegman CH, Li F, Ryffel B, Togbe D, Chung KF.  2020. Oxidative stress in ozone-induced chronic lung inflammation and emphysema: a facet of chronic obstructive pulmonary disease. Front Immunol. 11:1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhang J, Wei Y, Fang Z.  2019. Ozone pollution: a major health hazard worldwide. Front Immunol. 10:2518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang M, Fei X, Zhang G-Q, Zhang P-Y, Li F, Bao W-P, Zhang Y-Y, Zhou X.  2016. Role of neutralizing anti-murine interleukin-17A monoclonal antibody on chronic ozone-induced airway inflammation in mice. Biomed Pharmacother. 83:247–256. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

kfae162_Supplementary_Data
kfae162_supplementary_data.docx (1.5MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, D.L.L., upon reasonable request. The code used for modeling in this publication is previously published and available from https://doi.org/10.1165/rcmb.2011-0433OC (Groves et al. 2012).

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