Halogen exposure injury in the developing lung

Abstract

Owing to a high-volume industrial usage of the halogens chlorine (Cl₂ ) and bromine (Br₂ ), they are stored and transported in abundance, creating a risk for accidental or malicious release to human populations. Despite extensive efforts to understand the mechanisms of toxicity upon halogen exposure and to develop specific treatments that could be used to treat exposed individuals or large populations, until recently, there has been little to no effort to determine whether there are specific features and or the mechanisms of halogen exposure injury in newborns or children. We established a model of neonatal halogen exposure and published our initial findings. In this review, we aim to contrast and compare the findings in neonatal mice exposed to Br₂ with the findings published on adult mice exposed to Br₂ and the neonatal murine models of bronchopulmonary dysplasia. Despite remarkable similarities across these models in overall alveolar architecture, there are distinct functional and apparent mechanistic differences that are characteristic of each model. Understanding the mechanistic and functional features that are characteristic of the injury process in neonatal mice exposed to halogens will allow us to develop countermeasures that are appropriate for, and effective in, this unique population.

Keywords: alveolar simplification; bromine; bronchopulmonary dysplasia; chlorine; halogen exposure; lung development; newborn.

Figure 1.

Infant treated after a chemical attack in the Syrian civil war. Published in the Washington Post on February 17, 2019. A quote from the article: “Medical workers and first responders in opposition-held areas say they have treated more than 5000 people for suspected chemical exposure since 2012, adding strain to an already buckling health system. At least 188 people have died after chlorine attacks, according to estimates by medical workers and first responders.”

Figure 2.

The survival of adult and neonatal mice following exposure to Br₂. Adult male and female mice were exposed in environmental chambers to Br₂ at 600 ppm for 30 minutes. Neonatal mouse littermates were exposed to Br₂ in environmental chambers on P3 at exposure levels indicated. The overall mortality is comparable in adult and neonatal mice exposed to 400 ppm for 30 min or to 600 ppm for 30 min of Br₂. The majority of mortality occurs earlier after exposure in neonatal mice.

Figure 3.

Increased alveolar size and decreased alveolar density as indicated by increased MLI in neonatal mice exposed to Br₂ (A), to chronic hyperoxia (B), and in adult mice exposed to Br₂ (C). Panel A was adapted from Jilling et al., panel B was adapted from Nardiello et al., and panel C was adapted from Aggarwal et al. (A) Neonatal mouse littermates were exposed to air or Br₂ in environmental chambers on P3 at exposure levels indicated and lungs were collected and inflated/fixed on P14. (B) Neonatal mice were exposed to chronic hyperoxia at FiO₂ levels or at intermittent or oscillating levels, as indicated on the x axis from P3 to P14, and lungs were collected and inflated/fixed on P14. (C) Adult male and female mice were exposed in environmental chambers to Br₂ at 400 ppm for 30 minutes. Lungs were collected and inflated/fixed at 1, 7, 14, or 21 days postexposure as indicated. MLI for all three are determined by placing fixed length lines over images of lung sections occupied by alveoli, the number of alveolar walls crossing the line is counted, and the length of line is divided by the number of the alveolar walls intercept with the line. The relative enlargements of alveoli are comparable between neonatal mice exposed to Br₂ at 600 ppm for 30 min, and to 85% oxygen for 11 days and to adult mice exposed to 400 ppm Br₂ for 30 min at 21 days postexposure. Statistical significance between groups was analyzed by ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Lung compliance is differentially altered in neonatal (A and B) and adult (C) mice. (A) Quasistatic compliance in neonates exposed to Br₂ was decreased in dose-dependent manner with maximal and significant decrease at 600 ppm for 30 minutes. Lung compliances in (A) were normalized to the total lung capacity (TLC). (B) A significant reduction in lung compliance in neonates exposed to hyperoxia compared with neonates raised in normoxia similar to the reduction we see in Br₂-exposed neonates. (C) In sharp contrast, adult mice exposed to Br₂ show an increase in compliance 14 and 21 days postexposure. Measurements of lung compliance are done using flexiVent®. Neonatal mice measurements are done at P14. Br₂ exposures are done at P3 and exposure at hyperoxia is from P3 to P14. Adult mice were exposed to Br₂ 600 ppm for 30 min and lung compliance was measured at day 7, 14, or 21 postexposure. For panels A and C, statistical significance between groups was analyzed by ANOVA. For panel B, two-tailed Student’s t-test was used. *P < 0.05, **P < 0.01, ***P < 0.001. Panel A was adapted from Jilling et al., panel B was adapted from Nardiello et al., and panel C was adapted from Aggarwal et al.

Figure 5.

Fibrotic changes in the neonatal (A and B) and adult (5C–E) lungs. (A) Representative images of peripheral lung tissue staining for α-SMA in the neonatal lungs of air and Br₂ (600 ppm/30 min) exposed animals. (B) α-SMA mRNA expression in neonatal lungs is significantly increased in animals exposed to 600 ppm for 30 min compared with their corresponding littermate air controls. (C) Representative images of peripheral lung tissue staining for α-SMA in adult mice show a build-up at 14 and 21 days in Br₂-exposed mice compared with air controls. (D) Representative images of Masson’s trichrome stain demonstrate collagen deposition (blue stain) primarily around airways on days 14 and 21 after Br₂ exposure. (E) Quantification of collagen by measuring lung hydroxyproline levels showed significant increases at 14 and 21 days after Br₂ inhalation. Values are means ± SEM. Statistical significance between groups was analyzed by ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars are 100 μM. Panels A and B were adapted from Jilling et al. and panels C–E were adapted from Aggarwal et al.

Figure 6.

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