Cysteine and iron handling characterize seal endothelial cell resistance to lipid peroxidation

Elephant seals (Mirounga angustirostris) tolerate repeated bouts of hypoxemia/reoxygenation secondary to their prolonged diving behavior. Oxidative stress associated with hypoxia/reoxygenation events derives from oxidant generation upon the return of O 2 to the depleted system. It is well accepted that seals do not sustain excessive oxidative injury derived from diving, but the cellular and molecular mechanisms which maintain redox balance in these animals remain undefined due to experimental limitations. We used functional assays and RNA-seq in an endothelial cell culture system from elephant seals to compare the responses to hypoxia (0.5% O 2 for 1 h) and hypoxia/reoxygenation (0.5% O 2 for 1 h / 21% O 2 for 30 min) with a chemical oxidant exposure ( tert- Butyl hydroperoxide, t- BOOH; 100 µM for 2 h) to identify potential protective strategies in these cells. We compared these responses to those of two known redox-sensitive species (human and sheep) to separate generalized cellular responses from those that may be specific to seal. Seal endothelial cells tolerated high (up to 200 µM) concentrations of t- BOOH without increases in lipid peroxidation or cytotoxicity, though human and sheep endothelial cells were susceptible to these insults. For each species, we identified genes differentially expressed (DE) in each treatment compared to control, as well as pairwise between treatments. Gene set enrichment analysis (GSEA) on DE genes in seal cells identified downregulation of several sulfur metabolism-related pathways in t- BOOH treatment compared to hypoxia. These pathways include sulfur amino acid metabolism, degradation of cysteine and homocysteine, and selenocysteine synthesis. Among the 54 hit genes in all three pathways, only TXNRD1, SECISBP2, and EPRS1 were upregulated; downregulated genes were involved in the methionine salvage pathway, H 2 S metabolism, amino acid metabolism, redox balance, and formation of mitochondrial iron-sulfur complexes. t- BOOH treatment enriched expression of genes in the ferroptosis pathway in seal cells compared to hypoxia/reoxygenation, but not in any other species or pairwise comparison. Ferroptosis is an iron-dependent form of cell death characterized by excessive lipid peroxidation. The enrichment comprised six genes; three of these are involved in glutathione synthesis (glutamate-cysteine ligase catalytic subunit, GCLC; glutamate-cysteine ligase modifier subunit, GCLM; sodium-independent cystine-glutamate antiporter, SLC7A11/XCT), one is involved in lipid synthesis (acyl-CoA synthetase long-chain family member 3, ACSL3), and two are involved in iron homeostasis (ferroportin-1, SLC40A1/FPN1; heme oxygenase 1, HMOX1/HO-1). Seal cells upregulated expression of SLC40A1, the only known exporter of cellular iron, ~4-fold in t- BOOH treatment compared to all other conditions. SLC40A1 was never DE in sheep cells, and all instances of SLC40A1 DE in human cells were downregulated. Together with our functional data demonstrating lipid peroxidation resistance in seal cells, these results suggest that seal endothelial cells may avoid oxidant-induced injury and ferroptosis by regulating cellular iron levels. KA is supported by Woods Hole Oceanographic Institution. This work was supported in part by a NSF GRFP to KA. 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.