Aim: 

Mammalian cells require a constant supply of oxygen to maintain active mitochondrial oxidative phosphorylation. Hypoxia causes mitochondrial dysfunctions and an overproduction of ROS which are involved in apoptosis and necrosis. The aim of this study was to evaluate the role of hypoxia on the physiological behaviour of a line of neuroblastoma cells IMR-32.

Methods: 

IMR-32 cells were cultured in DMEM supplemented with 10% foetal calf serum, antibiotics and 2 mM L-glutamine, were distributed in 96 well plates at a concentration of 5 × 10⁴ cells/ml (0.2 ml/well) in a humidified incubator at 37 °C in 95% air : 5% CO₂ atmosphere and were differentiated for 2 weeks with BrdU (10 mM). Then were placed for 12 h in hypoxia with a gas concentration of 2% O₂: 5% CO₂: 93% N₂. Control cells were placed in a different incubator with 95% air: 5% CO₂ atmosphere. At the end of hypoxia period cells were incubated in normoxia for 24 h.

Results: 

Immediately after hypoxia we observed a significant increase of NO and [Ca²⁺]i, a decrease of proliferation and a mitochondrial membrane depolarization. The concentration of ERK 1 and ERK 2 proteins remained fairly constant and we observed a significant increase of free iron release and F₂-isoprostanes production. After the following 24 h of re-oxygenation the values returned similar to controls.

Conclusions: 

The increases of NO and [Ca²⁺]_i after hypoxia were responsible for an overproduction of ROS and mitochondrial membrane depolarization which caused an increase of Fe²⁺ release and F₂-isoprostanes production.