Exposure of neonatal mice to bromine impairs their alveolar development and lung function

Abstract

The halogen bromine (Br₂) is used extensively in industry and stored and transported in large quantities. Its accidental or malicious release into the atmosphere has resulted in significant casualties. The pathophysiology of Br₂-induced lung injury has been studied in adult animals, but the consequences of Br₂ exposure to the developing lung are completely unknown. We exposed neonatal mouse littermates on postnatal day 3 (P3) to either Br₂ at 400 ppm for 30 min (400/30), to Br₂ at 600 ppm for 30 min (600/30), or to room air, then returned them to their dams and observed until P14. Mice exposed to Br₂ had decreased survival (S) and had decreased weight (W) at P14 in the 400/30 group (S = 63.5%, W = 6.67 ± 0.08) and in the 600/30 group (S = 36.1%, W = 5.13 ± 0.67) as compared with air breathing mice (S = 100%, W = 7.96 ± 0.30). Alveolar development was impaired, as evidenced by increased mean linear intercept at P14. At P14, Br₂ exposed mice also exhibited a decrease of arterial partial pressure of oxygen, decreased quasi-static lung compliance, as well as increased alpha smooth muscle actin mRNA and protein and increased mRNA for IL-1β, IL-6, CXCL1, and TNFα. Global gene expression, evaluated by RNA sequencing and Ingenuity Pathway Analysis, revealed persistent abnormalities in gene expression profiles at P14 involving pathways of “formation of lung” and “pulmonary development.” The data indicate that Br₂ inhalation injury early in life results in severe lung developmental consequences, wherein persistent inflammation and global altered developmental gene expression are likely mechanistic contributors.

INTRODUCTION

The lungs of term infants have ~15% of the number of alveoli compared with adult lungs. In addition, neonates have lower lung compliance, higher respiratory rate, relatively increased dead space, and undeveloped antioxidant and immune systems compared with adults (5, 16). Because of these inherent developmental differences, neonates react differently to hyperoxia, hypoxia, airway infections, and inhalation of toxic gases than adults. Injury to developing lungs carries the particular risk of derailed development and permanent airway and alveolar remodeling, resulting in death in childhood or lifelong increased risk of morbidity. For instance, the most common cause of death for premature neonates surviving beyond postnatal day 60 is bronchopulmonary dysplasia (BPD), which is characterized by persistent abnormalities in lung structure and function (29). Additionally, survivors with BPD have a lifetime increased risk for pulmonary infections (15), poor baseline pulmonary function and asthma (12), pulmonary artery hypertension (11), and poor neurodevelopmental outcome (35). Exposure of the developing lung to high levels of oxygen, and consequent redox injury, plays a leading role in the etiology of BPD (18, 20). Although BPD is a special condition because it is a disease of premature neonates requiring high levels of oxygen for survival, exposure of term neonates to oxidant gases also elicits lung injury and subsequent alterations of development. For example, neonatal exposure to ozone, nitrogen oxides, or fine particulate matter has been shown to affect human neonates in epidemiological studies and to alter lung development in neonatal animal models (2, 9, 13, 14, 23, 32).

The halogens bromine (Br₂) and chlorine (Cl₂) are oxidant gases, and exposure to them presents a significant threat to public health. Both halogens are manufactured, stored, and transported in large quantities. Over fifty million tons of halogens are produced each year worldwide and used in the manufacture of water disinfectants, flame retardants, medicinal compounds, gasoline additives, and dyes. High-level, accidental exposure during transit of halogens has occurred in Geneva, Switzerland (1988, Br₂) (27), in Graniteville, SC (2005, Cl₂) (7, 8), and in Chelyabinsk, Russia (2011, Br₂), exposing thousands to inhalation injury and causing a number of fatalities. Although the health effects attributed to the incident in Graniteville are well studied (4, 6–8), there is virtually no information available regarding injury to newborns either by Cl₂ or by Br₂. Therefore, the effects and mechanisms of halogen toxicity early in lung development are completely unknown.

Previously, we have shown that exposure of pregnant mice to 600 ppm Br₂ for 30 min results in significantly higher maternal mortality than the mortality observed by the same level of exposure in nonpregnant mice. Surviving Br₂-exposed pregnant mice exhibited severe fetal growth restriction, and their newborn pups had decreased neonatal viability (22). These findings highlight the need to identify specific populations that may have increased vulnerability to Br₂ toxicity. We hypothesized that exposure of newborns to Br₂ would result in altered lung development, resulting in chronic deterioration of lung function. To test this hypothesis, we exposed mouse pup littermates at postnatal day 3 (P3) to either air or to Br₂ at 400 ppm or 600 ppm for 30 min in environmental chambers, then returned them to their litters to observe survival and weight gain until P14. We then assessed alveolar development, pulmonary inflammation, and function at P14. The concentrations and timing of Br₂ exposure were based on atmospheric modeling experiments done on the occasion of a Cl₂ release disaster in Graniteville, SC, where the Cl₂ concentration was estimated between a 1,000 ppm and 100 ppm for 30 to 60 min within a 0.5–1.0 km radius of the release (19). Br₂ is heavier than Cl₂, and, consequently, it is likely to maintain higher concentrations near ground level than Cl₂; however, actual data are not available. Furthermore, we performed a global analysis of pulmonary gene expression by RNA sequencing at P14, followed by pathway analysis to identify genes and pathways that are persistently altered post-Br₂ inhalation injury. Our data show that a brief exposure to Br₂ at an early stage of lung development results in lasting changes of gene expression and inflammation in the lung that are accompanied by a remodeling and deficient function of the lung.

METHODS

Animals.

Specific pathogen-free, pregnant female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA) and housed in the University of Alabama at Birmingham Animal Facility under standard conditions with food and water access ad libitum. Pregnant females were housed two/cage and delivered together. On P2, littermate pups were marked for identification in separate experimental groups. On P3, groups of pups were placed in paper-lined plastic trays, were transferred into specially designed glass environmental chambers to Br₂ at 400 or to 600 ppm for 30 min as previously described (1). Control mice were exposed to air for 30 min under identical conditions. Br₂ is administered from gas cylinders filled with certified concentrations of Br₂ (400 ppm or 600 ppm ± 2%) and medical grade compressed air (Airgas, Birmingham, AL) at a flow rate of 5 l/min throughout the length of exposure. The concentration of Br₂ in the exposure chamber was measured with a Br₂ analyzer (InterScan Corporation, Simi Valley, CA).

Following exposure, mouse pups were returned to their litters. Weights were measured, and survival was monitored daily. At P14, pups were anesthetized, arterial blood was drawn from the abdominal aorta, and lung compliance were measured by flexiVent (Scireq, Montreal, Quebec, Canada) as described previously (17, 18). Blood gas analysis was performed immediately after collection using an Element POC analyzer (Heska, Loveland, CO). The lungs were then collected for RNA isolation. RNA was isolated using the RNAeasy mini kit (Qiagen, Germantown, MD) from the left lung and quantified using the RiboGreen kit (Thermo Fisher, Grand Island, NY) and was reverse transcribed using Primescript RT master mix (Takara/Clontech, Mountain View, CA). Real-time PCR was performed by using Premix EX Taq 2x master mix (Takara/Clontech), Taqman MGB primer, and probe sets (Life Technologies/Thermo Fisher) in duplex reactions with two replicates for each sample. Primer probe sets were as follows: interleukin-1β (IL-1β) Mm00434228_m1, tumor necrosis factor-α (TNF-α) Mm00443258_m1, IL-6 Mm00446190_m1, C-X-C motif chemokine ligand 1 (CXCL1, KC/GRO) Mm00433859_m1, and α-smooth muscle actin (α-SMA) Mm00725412_s1. All procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC no. 20343) . In a second group of animals, following termination, the lungs were inflated at a constant pressure (25 cm water) using alcoholic formalin, and, following embedding in paraffin and sectioning, hematoxylin and eosin (H and E)-stained histological sections were used for measurements of mean linear intercepts as described before (18). Other sections were used to immunohistochemically visualize α-SMA as described before (31).

RESULTS

The effects of Br₂ exposure on the developing lung was assessed by exposing mouse littermates at P3 to air, or to Br₂ at 400 ppm or to Br₂ at 600 ppm to 30 min, then returning them to their litters for an additional 11 days (P14). Weights were recorded, and pups were counted daily. As shown in Fig. 1A, all pups gained weight at linear rates; however, Br₂-exposed pups had significantly lower rates of weight gain compared with air controls. At P14, weights were as follows (all data are means ± SE, stats by ANOVA): for air, 7.96 ± 0.30 g (n = 14), Br₂ 400, 6.67 ± 0.08 g (n = 10, P < 0.05), and Br₂ 600 ppm, 5.13 ± 0.67 g (n = 6, P < 0.0001). As shown in Fig. 1B, over the 11-day observation period, the survival of Br₂-exposed mice at 400 ppm was 62.5% (P < 0.05; Mantel-Cox; n = 16) and at 600 ppm was 36.1% (P < 0.001; Mantel-Cox; n = 38), which were significantly lower than the survival of the air-exposed mice (100%; n = 16). These data indicate that even the brief (30 min) exposure of neonatal mice to Br₂ has an exposure-level-dependent and long-lasting effect on survival and development.

Fig. 1.

The consequences of neonatal Br₂ exposure on survival, growth, and lung development. On postnatal day 3 (P3), neonatal mice were exposed to Br₂ at 400 ppm or 600 ppm for 30 min or air (control) and then returned to their litters. Weight and survival were monitored daily; all other variables were measured at P14 as described in methods. Data shown are mouse weights as a function of time (A), survival (B), micrographs of hematoxylin and eosin (H and E)-stained histological sections of the lung periphery (C), mean linear intercepts measured on images such as shown in C (D), and arterial partial pressure of oxygen (E). Br₂-exposed mouse pups exhibited decreased rate of weight gain, decreased survival, decreased arterial oxygenation, and alveolar simplification. A: individual points are means ± SE, dashed lines indicate linear regression; &, # and % indicate that all 3 slopes were significantly different from each other. Slopes: air = 0.56 ± 0.03 g/day, 400 ppm = 0.49 ± 0.02 g/day, 600 ppm = 0.34 ± 0.03 g/day. Brackets and ** indicate statistical significance of mean weights at P14 with ANOVA. Groups sizes: air n = 14, 400 ppm n = 16, 600 ppm n = 16. B: Mantel-Cox. Group sizes: air n = 16, 400 ppm n = 16, 600 ppm n = 38. D: individual points and means ± SE. ANOVA. Groups sizes: air n = 4, 400 ppm n = 5, 600 ppm n = 3. E: t-test. Means ± SE. Group sizes: air n = 3, 600 ppm n = 3. In each case *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

The lung of a mouse pup at P3 is at the approximate developmental stage of a full-term human newborn, and it has ~15% the number of alveoli compared with the adult mouse lung. In a normally developing mouse lung, most of the alveolar development is completed by P14. To evaluate the effect of Br₂ exposure on alveolar development, we assessed morphology by histological examination and performed blood-gas analysis as a measure of gas exchange. Images obtained from H and E-stained histological sections depicting areas of the alveolar space indicated an incomplete development of alveoli with large open spaces between alveolar septa (Fig. 1C). To quantitatively assess this incomplete alveolar development, we measured mean linear intercepts (MLI) on images of H and E-stained histological sections that are illustrated in Fig. 1C (Fig. 1D). MLI in air-exposed control mice was 44.50 ± 0.29 μm (n = 4), which is similar to MLI reported in P14 mice (18, 28). MLI were significantly increased in mice exposed to 400 ppm Br₂ to 61.58 ± 2.17 μm (P < 0.01; n = 5) and 600 ppm Br₂ to 74.87 ± 4.65 μm (P < 0.0001; n = 3). We also found that, in mice exposed at P3 to 600 ppm Br₂ for 30 min, arterial partial pressure of oxygen at P14 was significantly reduced (Fig. 1E), corresponding to a significant level of arterial hypoxemia. Because of the technically challenging nature of arterial blood collection from P14 mice, our sample numbers for the groups shown are limited, and we did not have enough samples from mice exposed at 400 ppm. To our knowledge, this is the first report on arterial blood gas analysis in P14 mice. In contrast, there were no changes in arterial partial pressure of carbon dioxide and pH (data not shown).

The lung morphology of Br₂-exposed mice at P14 resembles the morphology observed in those in a murine model of BPD at P14, in which mice are exposed to 85% oxygen from P1 to P14 (28). The increased presence of α-SMA is a hallmark of air alveolar remodeling noted in BPD in humans and in animal models of BPD and is considered to be a marker of myofibroblast differentiation and a prefibrotic change, which may account for the decreased compliance observed in BPD. Analysis of α-SMA by immunohistochemistry revealed increased intensity of staining in the alveolar septa (Fig. 2D; white arrow), around the bronchioles (Fig. 2D; black arrow), and around the peribronchial arteries (Fig. 2D; blue arrow). To quantify these changes, we assessed mRNA levels of α-SMA in lung homogenates and found a 2.45 ± 0.28-fold increase (P < 0.001; ANOVA) in lungs of mice exposed to 600 ppm Br₂ vs. air-exposed mice, whereas the levels of α-SMA mRNA were not significantly elevated in lungs of mice exposed to 400 ppm Br₂ (Fig. 2E). To assess lung mechanics, quasistatic lung compliance was evaluated by flexiVent (Fig. 2F). Because the Br₂-exposed mice were significantly smaller, compliance was corrected for total lung capacity. We found that lung compliance of mice at P14 was significantly decreased (P < 0.01; ANOVA) in mice exposed to Br₂ at 600 ppm (n = 5) compared with mice that were exposed to air (n = 10). The lung compliance of mice exposed to 400 ppm Br₂ did not change significantly (n = 10).

Fig. 2.

The effect of Br₂ exposure on α-smooth muscle actin (α-SMA) content and compliance of the lungs. On postnatal day 3 (P3), neonatal mice were exposed to Br₂ at 600 ppm or 400 ppm for 30 min or air (control) and then returned to their litters. α-SMA immunohistochemistry (A–D), quantification of α-SMA mRNA (individual values and means ± SE; E), and lung function testing with flexiVent (individual values and means ± SE; F) were performed on P14 as described in methods. Compared with air-exposed lungs (A and B), α-SMA immunoreactivity was increased in lungs of pups exposed to Br₂ at 600 ppm for 30 min (C and D), particularly in the alveolar septa (D; white arrow), in the bronchiolar walls (D; black arrow), and in the vessel walls (D; blue arrow). mRNA levels for α-SMA (E) were increased in lungs of pups exposed to Br₂ at 600 ppm for 30 min (n = 5) compared with air-exposed lungs (n = 9), whereas there was no statistically significant difference at 400 ppm for 30 min (n = 6). Quasistatic compliance in lungs (F) of pups exposed to Br₂ at 600 ppm for 30 min (n = 5) was decreased compared with air-exposed lungs (n = 10), whereas there was no statistically significant difference at 400 ppm for 30 min (n = 10). TLC, total lung capacity. **P < 0.01, ***P < 0.001 by ANOVA.

A chronic inflammatory response plays a role in the remodeling of the developing lung in BPD and in animal models of BPD. To assess whether there is chronic inflammation in the lungs in Br₂-exposed mice, we tested mRNA levels of common inflammatory cytokines that are associated with BPD (Fig. 3, A–E). We found that the levels of IL-1β (Fig. 3A), TNF-α (Fig. 3B), IL-6 (Fig. 3C), and CXCL1 (Fig. 3D) were significantly increased at P14 in the lungs of mice that were exposed to 600 ppm Br₂ at P3 compared with mice that were exposed to air. Among these, IL-1β mRNA was also significantly elevated compared with air controls at Br₂ exposure level of 400 ppm.

Fig. 3.

Persistent inflammation on P14 in lungs of mouse pups exposed to Br₂ on postnatal day 3 (P3). On P3, neonatal mice were exposed to Br₂ at 600 ppm for 30 min, at 400 ppm for 30 min, or were exposed to air (control) and then returned to their litters. RNA was isolated from lungs at P14, and reverse-transcription real-time PCR was performed using Taqman MGB primer probe sets and 18S rRNA as housekeeping control as described in methods. Data shown are mRNA fold change (individual values and means ± SE) of the cytokines IL-1β (A), TNF-α (B), IL-6 (C), and the chemokine C-X-C motif chemokine ligand 1 (CXCL1) (D). All 3 cytokine mRNAs and the chemokine CXCL1 mRNA increased significantly in lungs of pups exposed to Br₂ at 600 ppm for 30 min vs. air-exposed lungs. Only IL-1β was significantly increased in 400 ppm vs. air. Group sizes: air n = 9, 400 ppm n = 6, 600 ppm n = 5. **P < 0.01, ***P < 0.001 with ANOVA.

To test whether there are long-lasting global changes of gene expression in the developing lung at P14 following a brief exposure to 600 ppm Br₂ at P3, we analyzed transcript levels at P14 by RNA sequencing. Figure 4A depicts the result of hierarchical clustering (dendogram) of the genes that exhibited the highest degree of changes. Clusters of related genes that were upregulated in Br₂-exposed lungs are boxed in magenta, whereas genes that were downregulated in Br₂-exposed lungs are boxed in blue. To further analyze and categorize the genes that were highly regulated, we performed Ingenuity Pathway Analysis (IPA) by evaluating the genes that were regulated up or down greater than twofold at a P value <0.5. As shown in Fig. 4B, the most prominent pathways identified included formation of lung, respiratory system development, development of cytoplasm, and expression of protein.

Fig. 4.

Persistent global gene expression changes at postnatal day 14 (P14) in lungs of mouse pups exposed to Br₂ at P3 compared with lungs of mice exposed to air. On P3, neonatal mice were exposed to Br₂ at 600 ppm for 30 min or were exposed to air (control) and then returned to their litters. RNA was isolated from lungs at P14 and RNA sequencing, and pathway analysis was performed as described in methods. A heat map generated by hierarchical clustering (A) identified groups of genes that were downregulated (blue outlines) or were upregulated (magenta outlines) in Br₂-exposed lungs compared with air-exposed lungs. Ingenuity pathway analysis (B) revealed that the regulated genes belong to pathways such as respiratory system development, formation of the lung, and hypoplasia of an organ, which all can be related to altered lung development in Br₂-exposed pups.

DISCUSSION

To assess developmental toxicity of halogen inhalation, we developed a novel murine model wherein neonatal mice are exposed to Br₂ at exposure levels that are reportedly sublethal in adult mice (1) and are similar to halogen exposure levels in the vicinity of a Cl₂ transportation accident (19). Subsequently, we monitored the mouse pups for growth and survival, followed by evaluation of lung development and function. The key finding of this study is that a brief whole body Br₂ exposure of mouse pups at P3 initiated a remodeling process that continued while the mouse pups were breathing room air for 11 days and resulted in persistently altered lung structure, persistent inflammation, and altered global lung gene expression at P14. The observed structural and genetic changes correlated with impaired arterial oxygenation, decreased lung compliance, failure to thrive, and substantial mortality. The changes that we observed are similar to those observed in models of arrested neonatal lung development, such as continuous exposure to 40–85% oxygen for 3–4 days (28) or repeated systemic administration of bleomycin (10). There are remarkable similarities in terms of morphology, functional changes, and lung mechanics among these classic BPD models and the remodeling process in lungs exposed to Br₂. Myofibroblast differentiation, indicated by increased expression of α-SMA in the lung parenchyma, is a hallmark finding in both samples from human BPD and in murine models of hyperoxia-induced airway remodeling (30), and it was observed in the present study in lungs exposed to Br₂. There is a concomitant decrease of lung compliance in BPD models (18) that was also seen in the present study. Finally, a chronic inflammatory response characterized by an increase in CXCL1 (aka KC/GRO), IL-1β, and TNF-α is seen in hyperoxia models of BPD (18) and is seen in Br₂-exposed lungs.

Alveolar development in mice recapitulates the main characteristics of alveolar development in humans, but the mouse is born with a lung that resembles the developmental stage of the lung of a human fetus at 23–24 wk of gestation. The lungs of mice at P3 approximate the lung developmental stage of a term human infant. Therefore, our model intends to mimic the pulmonary developmental effects of Br₂ inhalation injury to term human infants. As discussed in the introduction, the levels of Br₂ used in this study are likely to be encountered in the vicinity of industrial accidents and acts of terrorism (7, 8).

The remodeling that we observed in the lungs post Br₂ inhalation resembles morphological and functional changes that are characteristic in murine models of BPD, including alveolar simplification, inflammation, increased levels of α-SMA, and decreased compliance. In the most common of these BPD models, mice are exposed to $\mathrm{F}{\mathrm{I}}_{{\mathrm{O}}_{\mathrm{2}}}$ = 80–85% from P3 to P14 continuously, covering the same window of development as the present study. Despite the very similar morphological and functional outcome, the transcripts with most altered expression (up or down) that we detected with RNASeq in the Br₂ exposure model at P14 show virtually no overlap with the top changed transcripts that we detected with microarray in the hyperoxia exposure model at P14 (our unpublished observations; data not shown). Similarly, the most regulated pathways by IPA were also different (our unpublished observations; data not shown). This may be due to technical differences (i.e., the present study was conducted by RNASeq, and our data and pathway analysis in the BPD model was conducted using microarray), but different mechanisms of injury are also plausible. Notably, pathway analysis in the present study revealed some key regulated genes in lungs exposed to Br₂ that may have potential relevance to lung development and may warrant follow-up investigation. Ataxin 1 (ATXN1) has been implicated in the regulation of matrix metalloproteinases and in lung development (25). As shown by both pathway analysis and by hierarchical clustering, a related gene, ataxin 2 (ATXN2), was one of the key upregulated genes in lung exposed to Br₂. Although the role of ATXN2 in the lung is unknown, it has been identified as a key molecule in the pathogenesis of amyotrophic lateral sclerosis via its role in propagating cellular stress response (3). Another critical upregulated gene, integrin β1 (ITGB1), has multiple potential mechanistic roles in lung remodeling. Pathological signaling via ITGB1 has been shown to promote fibroblast growth in idiopathic pulmonary fibrosis (36). Lung epithelial cells signal to macrophages to regulate migration via microvesicle-packaged micro-RNA-s targeting ITGB1 (24). The engraftment and survival of fibroblasts into decellularized mouse lung are mediated by ITGB1 (33). Testicular kinase 1 (TESK1) was the most downregulated gene, and it plays a role in assembly of focal adhesions via integrin signaling that involves cofilin and the spread of cells on fibronectin (21, 34). SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), also known by several other acronyms including brahma-related gene 1 (BRG1), is a major epigenetic regulator that has been implicated in regulation of embryonic lung development via jumonji D3 (26). Taken together, our transcriptome and network analysis identified several potential candidate genes for future mechanistic exploration, involving regulators of fibroblast migration, macrophage recruitment, cellular stress response, and epigenetic regulation, all of which are mechanisms well recognized in normal and abnormal lung development.

In summary, we developed a model that will enable the study of mechanisms that mediate acute and chronic injury caused by Br₂ inhalation in developing murine lungs, approximating the developmental stage of lungs in full-term human neonates. We report that, following Br₂ inhalation in the developing lung, there is exposure-level-dependent severe lung remodeling that results in functional impairment. Morphologically, and to some extent mechanistically, the lung remodeling observed in halogen inhalation injury resembles the altered lung development observed in murine models of BPD. Transcriptome and network analysis performed in chronically remodeling lungs at a time point that is far removed from the initial acute injury identified potential intriguing targets for future mechanistic exploration.

GRANTS

This research was supported by National Institute of Environmental Health Sciences Grants 5U01ES026458 02 (to S. Matalon) and 1-U01-ES027697–01 (to S. Matalon and T. Jilling).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

T.J., N.A., and S.M. conceived and designed research; T.J., C.R., A.J.Y., and B.H. performed experiments; T.J., C.R., A.J.Y., S.A., and B.H. analyzed data; T.J., S.A., B.H., N.A., and S.M. interpreted results of experiments; T.J. prepared figures; T.J. drafted manuscript; T.J., B.H., N.A., and S.M. edited and revised manuscript; T.J., C.R., A.J.Y., S.A., N.A., and S.M. approved final version of manuscript.