FIGURE 5.

Production of secondary mediators A: exposure to Cl₂ increases mitochondrial superoxide/hydrogen peroxide. I–VI: H441 cells immersed in 50 µl of artificial epithelial lining fluid were exposed to 95% air-5% CO₂ as control (I–III) or Cl₂ (100 ppm for 15 min) (IV–VI) and returned to 95% air-5% CO₂ for 1 h. Before imaging with confocal laser microscopy, the cells were incubated with MitoSOX (red) for 15 min and MitoTracker (green). II and V: most of the Cl₂-exposed H441 cells showed higher levels of red fluorescence compared with controls. VI: MitoSOX fluorescence localized with MitoTracker (yellow), consistent with the presence of reactive species in mitochondria. Nuclei were counterstained with DAPI (blue). Quantitative analysis showed increased production of reactive intermediates at 6 h post-exposure (modified from Ref. 43, with permission from the publisher). B: plasmalogen-derived Cl₂ and Br₂ oxidation products. The vinyl ether bond of plasmalogens is targeted by Cl₂, Br₂, HOCl, and HOBr resulting in 2-chlorofatty(bromo)aldehyde production including 2-Cl(Br)-Pald and 2-Cl(Br)-Sald. The 2-chloro(bromo)fatty aldehydes are either oxidized to the 2-chloro(bromo)fatty acids and 2-Cl(Br)-PA and 2-Cl(Br)l-SA or reduced to the 2-chloro(bromo)fatty alcohols, 2-chloro(bromo)palmitoyl alcohol, and 2-chloro(bromo)stearoyl alcohol. Alternatively, nucleophilic attack of 2-chloro(bromo)fatty aldehydes by GSH results in either palmitaldehyde or stearaldehyde GSH adduct formation. R1 = C14H29 or C16H33 (modified from Ref. 147). C: effects of Cl-lipids on lung permeability and inflammation. C57bl/6 male mice were exposed to saline, ethanol, palmitate (PA), palmitic aldehyde (Pald), 2-Cl-PA, or 2-Cl-Pald by intranasal administration and lung injury was assessed by measuring total protein in the bronchoalveolar lavage levels of protein. Data are means ± SE (n = 4–6). *P < 0.05 relative to corresponding native fatty acid (from Ref. 147). BAL, bronchoalveolar lavage. D: plasma cell-free heme (CFH) and chlorinated lipids are elevated in humans and mice exposed to Cl₂ gas. Blood was collected from patients (pts) exposed to Cl₂ gas in the emergency room of the University of Alabama at Birmingham, 3–4 h post-exposure, stored for 72 h at 4°C at which time it was analyzed. Blood from human volunteers as controls was treated in the same fashion. I: plasma CFH levels in persons exposed to Cl₂ were higher than the age- and sex-matched human controls. II and III: Cl₂-exposed individuals also had elevated levels of 16ClFA (n = 4–5) (II) and 18ClFA (III). Similarly, adult male C57BL/6 mice exposed to Cl2 gas (400 ppm, 30 min) had increased levels of heme in plasma 24 h post-exposure. Individual values and means ± SE. *P < 0.05 vs. unexposed humans or air exposed mice; by unpaired t test (from Ref. 74). E: cardiac dysfunction and cardiomyocyte death with 2-bromohexadecanal (Br-HDA). I: left ventricular (LV) diastolic dysfunction was reflected by the decrease in mitral valve (MV) early and late wave velocities 4 h after injection of Br-HDA into the LV cavity. II: Br-HDA caused an increase in LV end-diastolic dimension and end-systolic dimension, resulting in a decrease in fractional shortening. Image demonstrates M-mode echocardiography of the LV before and 4 h after intracardiac injection of Br-HDA. III: Br-HDA caused extensive disruption of the cardiac cytoskeleton and loss of the normal highly organized linear mitochondrial sarcomere integrity. Transmission electron microscopy (×3,200 at top and middle and ×8,000 at bottom) demonstrated contraction band necrosis (red arrows), loss of I bands, and disruption of z-disk (yellow arrowheads) in the LV of rats administered Br-HDA as well as mitochondrial swelling (yellow arrows) and cristae lysis (red asterisk) (from Ref. 54). F: Br₂-exposed pregnant mice exhibit diminished cardiac function at gestational day 18.5 (E18.5). Nonpregnant and pregnant (E14.5) mice were exposed to air or 600 ppm Br₂ for 30 mi and returned to room air. Representative echosonography left ventricular (LV) and right ventricular (RV) traces at E18.5 of pregnant mice exposed to air (left) or Br₂ with demarcation of ventricular sizes (ED, end diastole; ES, end systole) (from Ref. 55).