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.

Keywords: bronchopulmonary dysplasia; halogen; lung development; lung injury.

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.

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.

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.

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.