Rapid Hypoxia-Induced Upregulation of Glutathione-Related Genes May Protect Elephant Seal Endothelial Cells Against Oxidative Stress.

Northern elephant seals (Mirounga angustirostris) experience extreme fluctuations in blood oxygen levels during repeated bouts of deep diving. Such fluctuations characterize human cardiovascular pathologies including myocardial infarction and ischemic stroke, though seals do not incur vascular injury during the repetitive hypoxia/reoxygenation cycles associated with diving. Marine mammals diving at sea remain inaccessible for most real-time in vivo biochemical investigations, thus we developed a proliferative arterial endothelial cell culture system from expelled placentae from elephant seals and humans to assess the molecular and biochemical response to oxidative stress. Seal endothelial cells generate oxidants when pharmacologically stimulated but remain protected against lipid peroxidation when treated with tert-Butyl hydroperoxide (t-BOOH, 300 uM), while human cells exposed to the same conditions display a doubling in lipid peroxidation levels (p=0.0002). Seal endothelial cells maintain viability at moderate (100 uM) doses of t-BOOH while human cells display significantly reduced viability at the same dose. Interestingly, protein expression of the only two enzymes capable of repairing lipid peroxidation - glutathione peroxidase 4 (Gpx4) and peroxiredoxin 6 (Prdx6) - decreases in both seal and human endothelial cells after t-BOOH treatment (Human: Gpx4 17% decrease, p=0.08; Prdx6 37% decrease, p=0.007; Seal: Gpx4 53% decrease, p=0.036; Prdx6 35% decrease, p=0.007). Additionally, seal endothelial cells display lower baseline glutathione peroxidase activity than human cells (p<0.0001) and activity in human (p=0.0002) but not seal cells declines after t-BOOH treatment. Seal endothelial cells exposed to 1% O for up to 6 h upregulate expression of genes in the KEGG glutathione metabolism pathway within 30 minutes of hypoxia onset, while expression of these genes returns to baseline levels beyond 1 h in hypoxia. Among those genes contributing to the overrepresentation of the glutathione pathway are several glutathione S-transferases (GSTM3, GSTK1, GSTO1, MGST2), suggesting that glutathione S-transferases may limit oxidative damage in seal endothelial cells during hypoxia/reoxygenation bouts. That this upregulation occurs absent the reintroduction of oxygen into the system may suggest that the seal glutathione system is "primed" for rapid response to protect against oxidative injury during the repeated hypoxia/reoxygenation cycles associated with diving.