Mitochondrial Reactive Oxygen Species Production in Lungs of Rats with Different Susceptibilities to Hyperoxia-InducedARDS

Objective: Rat exposure to hyperoxia is a well-established model of human Acute Respiratory Distress Syndrome (ARDS). Adult rats exposed to hyperoxia (100% O 2 ) die from respiratory failure within 60-72 hrs. However, rats preconditioned by exposure to >95% O 2 for 48 hrs followed by a 24-hr “rest period” in room air (H-T) acquire tolerance of the otherwise lethal effects of exposure to 100% O 2 . In contrast, rats preconditioned by exposure to 60% O 2 for 7 days (H-S) become more susceptible to 100% O 2 . The H-T and H-S rat models provide a platform for assessing differences in key cellular processes in animals that are more or less likely to develop ARDS. The objectives of this study were to quantify rates and identify sources of H 2 O 2 production in mitochondria isolated from lungs of normoxia (control), H-T, and H-S rats, and the rates and sources of H 2 O 2 emission from lungs of these groups of rats. Methods: Mitochondria were isolated from lungs of normoxia, H-S, and H-T rats, and the sources and rates of mitochondrial H 2 O 2 production and emission (production - scavenging) were quantified in the presence of NADH-linked substrate pyruvate + malate and FADH 2 -linked substrate succinate, with and without inhibitors of mitochondrial complex I (CI, rotenone), complex II (CII, TTFA), serine protease (phenylmethylsulfonyl fluoride), and/or the H 2 O 2 scavenging systems glutathione (dinitrochlorobenzene) and thioredoxin (auranofin). Lung rates of H 2 O 2 emission were quantified in isolated perfused lungs (IPLs) with and without CII inhibitor (TTFA) added to lung perfusate. Results: Isolated mitochondria studies showed that CII is the main source of mitochondrial H 2 O 2 for all rat groups in the presence of CI or CII substrates, with little contribution from CI, and that both rates of H 2 O 2 production and H 2 O 2 scavenging capacity were ~48% lower in H-S mitochondria compared to normoxia or H-T mitochondria. Additional results show that under state 1 conditions (no substrates), serine protease is the main source of H 2 O 2 emission. Results from IPLs studies showed that CII is also the dominant source of H 2 O 2 emission from lung tissue, and that H 2 O 2 emission rate was lower in H-T lungs compared to normoxia (32%) and H-S (29%) lungs. Discussion & Conclusions: CII is the dominant source of H 2 O 2 in both isolated lung mitochondria and IPLs, with little contribution from CI. For H-S rats, both mitochondrial rate of H 2 O 2 production and antioxidant capacity were significantly lower than those in normoxia mitochondria and may contribute to their increased susceptibility to hyperoxia. The lower rate of H 2 O 2 emission in lungs from H-T rats along with no change in the mitochondrial rate of H 2 O 2 production are consistent with previously measured higher tissue glutathione concentration in lung of H-T rats, which may contribute to their increased tolerance of hyperoxia. Understanding altered function of lung mitochondria from subjects preconditioned to be tolerant or sensitive to hyperoxia is a critical step in identifying patients at highest risk of ARDS due to “second hits” such as hyperoxia and provides a basis for the development of interventions that may protect them. This work was supported by NIH 2R15HL129209-03 (Audi, Clough), and NSF grant DMS 2153387 (Dash). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.