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Apoptotic cell death in rat lung following mustard gas inhalation
Devon K Andres1, Brian M Keyser2, Ashley A Melber2
1Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Aberdeen, Maryland devon.k.andres.ctr@mail.mil.
Abstract:
To investigate apoptosis as a mechanism of sulfur mustard (SM) inhalation injury in animals, we studied different caspases (caspase-8, -9, -3, and -6) in the lungs from a ventilated rat SM aerosol inhalation model. SM activated all four caspases in cells obtained from bronchoalveolar lavage fluid (BALF) as early as 6 h after exposure. Caspase-8, which is known to initiate the extrinsic Fas-mediated pathway of apoptosis, was increased fivefold between 6 and 24 h, decreasing to the unexposed-control level at 48 h. The initiator, caspase-9, in the intrinsic mitochondrial pathway of apoptosis as well as the executioner caspases, caspase-3 and -6, all peaked (P < 0.01) at 24 h; caspase-3 and -6 remained elevated, but caspase-9 decreased to unexposed-control level at 48 h. To study further the Fas pathway, we examined soluble as well as membrane-bound Fas ligand (sFas-L and mFas-L, respectively) and Fas receptor (Fas-R) in both BALF cells and BALF. At 24 h after SM exposure, sFas-L increased significantly in both BALF cells (P < 0.01) and BALF (P < 0.05). However, mFas-L increased only in BALF cells between 24 and 48 h (P < 0.1 and P < 0.001, respectively). Fas-R increased only in BALF cells by 6 h (P < 0.01) after SM exposure. Apoptosis in SM-inhaled rat lung specimens was also confirmed by both immunohistochemical staining using cleaved caspase-3 and -9 antibodies and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining as early as 6 h in the proximal trachea and bronchi, but not before 48 h in distal airways. These findings suggest pathogenic mechanisms at the cellular and molecular levels and logical therapeutic target(s) for SM inhalation injury in animals.
Insights
Sulfur mustard inhalation activates caspases and Fas pathway proteins in rat lungs, confirming apoptosis as a key injury mechanism. These findings reveal cellular and molecular pathways for potential therapeutic targets in sulfur mustard lung injury.
Area of Science:
- Toxicology
- Cellular Biology
- Respiratory Medicine
Background:
- Sulfur mustard (SM) is a chemical warfare agent causing severe lung injury.
- Apoptosis, or programmed cell death, is a suspected mechanism in SM-induced lung damage.
- Understanding the molecular pathways of SM lung injury is crucial for developing treatments.
Purpose of the Study:
- To investigate apoptosis as a mechanism of sulfur mustard (SM) inhalation injury in rat lungs.
- To analyze the activation of key caspases and Fas pathway components following SM exposure.
- To identify potential therapeutic targets for SM inhalation injury.
Main Methods:
- Utilized a ventilated rat model exposed to sulfur mustard (SM) aerosol.
- Measured caspase activity (caspase-8, -9, -3, -6) in bronchoalveolar lavage fluid (BALF) cells and BALF.
- Assessed soluble and membrane-bound Fas ligand (sFas-L, mFas-L) and Fas receptor (Fas-R) levels.
- Confirmed apoptosis using immunohistochemical staining (cleaved caspase-3, -9) and TUNEL assay.
Main Results:
- SM exposure activated all four studied caspases in BALF cells within 6 hours.
- Caspase-8, -9, -3, and -6 levels showed distinct temporal activation patterns.
- Fas pathway components (sFas-L, mFas-L, Fas-R) were significantly altered post-SM exposure.
- Apoptosis was confirmed in proximal airways by 6 hours and distal airways by 48 hours.
Conclusions:
- Apoptosis is a significant mechanism contributing to sulfur mustard inhalation lung injury in rats.
- The extrinsic (Fas-mediated) and intrinsic (mitochondrial) apoptotic pathways are activated by SM.
- Specific caspases and Fas pathway molecules represent potential therapeutic targets for SM lung injury.