Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin.
BACKGROUND
Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin.


RESULTS
Continuous exposure of wild-type cultures to 1-10 microM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1-2 microM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 microM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR) staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect.


CONCLUSIONS
These results suggest that HO-2 gene deletion protects neurons in mixed neuron-astrocyte cultures from heme-mediated oxidative injury. Selective inhibition of neuronal HO-2 may have a beneficial effect after CNS hemorrhage.
Regan
Raymond F
RF
Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, 239 Thompson Building, Philadelphia, PA 19107, USA. Raymond.Regan@jefferson.edu.
Chen
Jing
J
Benvenisti-Zarom
Luna
L
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism* / Q000633:toxicity; D007502:Iron Chelating Agents / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000235:genetics / Q000502:physiology*
D007502:Iron Chelating Agents; D007527:Isoenzymes; D006427:Hemin; D006419:Heme Oxygenase (Decyclizing); C105245:heme oxygenase-2