Phospholipid (PL) composition and accumulation of phospholipid hydroperoxides in lung of mouse exposed to hyperoxia. A: phospholipid composition of control and hyperoxic lung. Mice were exposed to hyperoxia (100% of oxygen) for 72 h and killed thereafter. Lipids were extracted and separated by 2-dimensional high-performance thin-layer chromatography (2D-HPTLC). Spots of phospholipids were scraped, and lipid phosphorus was determined. Inset: typical 2D-HPTLC chromatogram of total lipids extracted from control mouse lung. NL, neutral lipids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; CL, cardiolipin; Sph, sphingomyelin. Open bars or number 1 are control; closed bars or number 2 are hyperoxia. Data are means ± SD; n = 3 (for control); n = 4 (for hyperoxia). B: accumulation of phospholipid hydroperoxides (PL-OOH) in lung of mice exposed to hyperoxia (100% of oxygen for 72 h). Lipids were extracted and separated by 2D-HPTLC. PL-OOH were detected using Amplex Red protocol. Open bars are control; closed bars are hyperoxia. Data are means ± SD. *P < 0.05 vs. control; n = 3 (for control); n = 4 (for hyperoxia).

Oxidation of PS in lung of mice exposed to hyperoxia. A: typical full negative LC/ESI-MS spectrum of PS isolated from control mouse lung. Major molecular ions of PS with m/z 788.5, 810.5, and 834.5 were detectable in spectrum. B: typical full negative LC/ESI-MS spectrum of PS isolated from hyperoxic mouse lung. A decrease of intensity of 2 molecular ions with m/z 810.5 (C_18:0/C_20:4) and 838.5 (C_18:0/C_22:4) in hyperoxic lung was detected. C: quantitative assessment of PS molecular species in normal and hyperoxic lung. Open bars are control; closed bars are hyperoxia. Data are means ± SD. *P < 0.05 vs. control; n = 3 (for control); n = 4 (for hyperoxia).

ESI-MS² analysis of PS isolated from control and hyperoxic mouse lungs. Aa: MS² fragmentation of PS species with m/z 810.5 from control lung. b: MS² fragmentation of oxidized PS molecular species with m/z 842.5 from hyperoxic lung. c: Part of MS² spectrum of molecular ion with m/z 842.5 shown in the range of m/z 270–350. The ion with m/z 153.0 corresponded to glycerophosphate without molecule of water, a common ion formed during phospholipid fragmentation. Molecular ions with m/z 283.3 and m/z 303.3 corresponding to C_18:0 and C_20:4 fatty acids, respectively, were also formed during fragmentation of parent ion with m/z 810.5. Molecular ions with m/z 335.3 corresponding to C_20:4 fatty acid with 2 oxygen added was formed during fragmentation of parent ion with m/z 842.5. The loss of serine group of PS yielded the fragments with m/z 723.6 and 751.6. Product ions with m/z 419.3 originated from fragments with m/z 723.6 and 755.5 after loss of arachidonic acid C_20:4 and oxidized arachidonic acid C_20:4-OOH, respectively. Thus the molecular ion at m/z 810.5 corresponds to molecular species of PS-C_18:0/C_20:4, whereas the molecular ion with m/z 842.5 corresponds to molecular species of oxidized PS-C_18:0/C_20:4-OOH. Ba: MS² fragmentation of PS species with m/z 838.5 from control lung. b: MS² fragmentation of oxidized PS molecular species with m/z 870.6 from hyperoxic lung. c: Part of MS² spectrum of molecular ion with m/z 870.5 shown in the range of m/z 270–370. The ions corresponded to glycerophosphate without molecules of water (m/z 153.0) were detected on MS² spectra. Fragmentation of parent ion with m/z 838.5 results in formation of molecular ions with m/z 283.3 and m/z 331.3 corresponding to C_18:0 and C_22:4 fatty acids, respectively. MS² analysis of molecular ion with m/z 870.5 revealed the presence of molecular ions with m/z 363.3 corresponding to C_22:4 after addition of 2 oxygens. The molecular ion with m/z 365.3 was formed during fragmentation process of nonoxidized molecular species of PS with m/z 870.5 (PS-C_18:1/C_24:1) and corresponds to nervonic acid (C_24:1). The loss of serine group of PS yielded the fragments with m/z 755.7 and 783.6. Product ion with m/z 419.3 originated from fragments with m/z 751.6 and 783.6 after loss of C_22:4 fatty acid and C_20:4-OOH, respectively. Therefore, molecular ions at m/z 838.5 and m/z 870.5 correspond to molecular species of PS-C_18:0/C_22:4 and product of its oxidation C_18:0/C_22:4-OOH.

Phospholipid composition and accumulation of phospholipid hydroperoxides in mouse pulmonary endothelial cells (MLEC) exposed to hyperoxia. A: phospholipid composition of control and hyperoxic MLEC. MLEC were exposed to hyperoxia (95% O₂-5% CO₂ for 72 h in a 37°C). Lipids were extracted and separated by 2D-HPTLC, spots of phospholipids were scraped, and lipid phosphorus was determined. B: accumulation of PL-OOH in MLEC exposed to hyperoxia. Lipids were extracted and separated by 2D-HPTLC. Phospholipid hydroperoxides were detected using Amplex Red protocol. Open bars are control; closed bars are hyperoxia. Data are means ± SD. *P < 0.05 vs. control; n = 3.

Activation of caspase-3 and -7 in MLEC exposed to hyperoxia. MLEC (15,000 per well) were treated with XJB-5-131 (10–40 uM, for 4 h). After that, the drug was removed and cells were exposed to hyperoxia (95% O₂-5% CO₂ for 72 h). At the end of the exposure, caspase-3 and -7 activity was measured. *P < 0.05, significantly different from control. #P < 0.05, significantly different from hyperoxia alone. Open bars are control; closed bars are hyperoxia. Data are means ± SD; n = 3.

Oxidation of CL in lung of mice exposed to hyperoxia. A: typical full negative liquid chromatography (LC)/electrospray ionization mass spectrometry (ESI-MS) spectrum of CL isolated from control mouse lung. Major cluster contains molecular ions with a mass-to-charge ratio (m/z) of 1,447.8 and 1,449.8 along with clusters at m/z 1,421.8, 1,473.8, and 1,497.9 were detectable in the spectrum. B: typical full negative LC/ESI-MS spectrum of CL isolated from hyperoxic mouse lung. MS analysis revealed significant reduction of CL molecular ions with m/z 1,495.8, 1,497.8, and 1,499.8. C: quantitative assessment of CL molecular species in control lung and lung isolated from mice exposed to hyperoxia. Open bars are control; closed bars are hyperoxia. Data are means ± SD. *P < 0.05 vs. control; n = 3 (for control); n = 4 (for hyperoxia).

ESI-MS² analysis of CL isolated from control and hyperoxic mouse lungs. Aa: MS² fragmentation of CL species with m/z 1,499.8 from control lung. Ions [a]⁻ and [b]⁻ with m/z 721.8 corresponding to C_18:1/C_20:4-PA as well as characteristic daughter [a+136]⁻ and [b+136]⁻ (m/z 857.0); ions [a-(C_18:1)]⁻ and [b-(C_18:1)]⁻ (m/z 440.6); and [a-(C_20:4)]⁻ and [b-(C_20:4)]⁻ (m/z 419.5) were formed during fragmentation of the molecular ion with m/z 1,499.5 corresponding to molecular species of CL (C_18:1/C_20:4/C_20:4/C_18:1). Fragmentation of molecular species of CL C_18:1/C_18:2/C_18:2/C_22:5 revealed the presence of [a]⁻ and [b]⁻ ions with m/z 697.7 and 746.8, mainly corresponding to C_18:1/C_18:2-PA and C_18:2/C_22:5-PA. In addition, ions with m/z 753.6 ([a+56]⁻); 833.6 ([a+136]⁻); 802.6 ([b+56]⁻); and 882.6 ([b+136]⁻) were detected on MS² spectrum. Moreover, [a]⁻ and [b]⁻ ions with m/z 699.5 and 744.6 corresponded to C_18:0/C_18:2-PA and C_18:2/C_22:6-PA. In addition, ions with m/z 755.6 ([a+56] ⁻); 835.6 ([a+136]⁻); 800.4 ([b+56]⁻); and 880.7 ([b+136]⁻) were formed after fragmentation of molecular ion corresponding to molecular species of CL C_18:0/C_18:2/C_18:2/C_22:6. b: Part of MS² spectrum of molecular ion with m/z 1,499.8 shown in the range of m/z 270–350. Molecular ions of C_18:1 (m/z 281.3), C_18:2 (m/z 279.3), C_22:5 (m/z 329.3), and C_22:6 (m/z 327.3) were presented in MS² spectrum as well. Thus MS² analysis of the singly charged ion (m/z 1,499.8) revealed the copresence of molecular species of CL (C_18:1/C_20:4/C_20:4/C_18:1) along with CLs (C_18:1/C_18:2/C_18:2/C_22:5 and C_18:0/C_18:2/C_18:2/C_22:6). Ba: MS² fragmentation of CL species with m/z 1,532.1 from hyperoxic lung. Fragments {[a+136+16 (one oxygen)] and [b+136+16 (one oxygen)]} of oxidized molecular species of CL (C_18:1/C_20:4-OH/C_20:4-OH/C_18:1) with m/z 873.5 corresponding to [C_18:1/C_20:4-OH-PA+136] were detected on MS² spectrum. In addition, fragmentation of CL molecular species C_18:1/C_18:2-OOH/C_18:2/C_22:5 resulted in appearance of fragments {[a+32 (two oxygen)]} with m/z 729.6 corresponding to C_18:1/C_18:2-OOH-PA. Moreover, fragments with m/z 760.8 {[a+16 (one oxygen)]} corresponding to C_18:0/C_18:2-OH-PA; m/z 896.6 {[a+136+16 (one oxygen)]} corresponding to C_18:0/C_18:2-OH-PA+136; m/z 771.6 {[b+56+16 (one oxygen)]} corresponding to C_18:2/C_22:6-OH-PA; and 851.5 {[b+136+16 (one oxygen)]} corresponding to C_18:2/C_22:6-OH-PA were detected in the MS² spectrum and originated from a molecular species of CL , C_18:0/C_18:2-OH/C_18:2/C_22:6-OH. b: Part of MS² spectrum of molecular ion with m/z 1,532.1 shown in the range of m/z 270–370. Molecular ions of oxygenated fatty acids C_18:2-OH (m/z 295.3), C_18:2-OOH (m/z 311.3), C_20:4-OH (m/z 319.3), and C_22:6-OH (m/z 343.3) alone with nonoxidized fatty acids C_18:0 (m/z 283.3), C_18:1 (m/z 281.3), C_18:2 (m/z 279.3), C_22:5 (m/z 329.3), and C_22:6 (m/z 327.3) were presented in the MS² spectrum of CL molecular ion with m/z 1,532.1.

Exposure of mice to hyperoxia results in activation of caspase-3 and -7 activity in lung. A: assessment of caspase-3 and -7 activity in mouse lung using Caspase-Glo assay. Open bars are control; closed bars are hyperoxia. AU, arbitrary units. Data are means ± SD. *P < 0.05 vs. control; n = 3 (for control); n = 4 (for hyperoxia). B: exposure of mice to hyperoxia for 72 h results in apoptosis of endothelial cell origin. Lungs were inflated with 2% paraformaldehyde, and frozen sections were used for immunofluorescent detection of apoptosis using an Alexa 488-labeled (green) nick end labeling kit. There were very few apoptotic cells in lungs of animals breathing room air as evident at low (×10) and higher (×40) magnification (top). In contrast, numerous apoptotic cells were apparent (×10 and ×40) in the hyperoxic group and many of these were associated with CD31-positive (e.g., endothelium) cells as was evident in the inset at ×100 (bottom right). Boxed areas at low magnification indicate the subsequent area at high magnification.

Oxidation of CL and PS in MLEC exposed to hyperoxia. Quantitative assessments of CL (A) and PS (B) molecular species in MLEC. Open bars are control; closed bars are hyperoxia. Data are means ± SD. *P < 0.05 vs. control; n = 3.

Mitochondria-targeted small-molecule, XJB-5-131, protect both CL (A) and PS (B) against oxidation induced by hyperoxia in MLEC (95% oxygen, 72 h). Open bars are control; closed bars are hyperoxia. Data are means ± SD. *P < 0.05 vs. control; n = 3.