Heme oxygenase-2 knockout neurons are less vulnerable to hemoglobin toxicity.
When cortical neurons are exposed to hemoglobin, they undergo oxidative stress that ultimately results in iron-dependent cell death. Heme oxygenase (HO)-2 is constitutively expressed in neurons and catalyzes heme breakdown. Its role in the cellular response to hemoglobin is unclear. We tested the hypothesis that HO-2 attenuates hemoglobin neurotoxicity by comparing reactive oxygen species (ROS) formation and cell death in wild-type and HO-2 knockout cortical cultures. Consistent with prior observations, hemoglobin increased ROS generation, detected by fluorescence intensity after dihydrorhodamine 123 or dichlorofluorescin-diacetate loading, in wild-type neurons. This fluorescence was significantly attenuated in cultures prepared from HO-2 knockout mice, and cell death as determined by propidium iodide staining was decreased. In other experiments, hemoglobin exposure was continued for 19 h; cell death as quantified by LDH release was decreased in knockout cultures, and was further diminished by treatment with the HO inhibitor tin protoporphyrin IX. In contrast, HO-2 knockout neurons were more vulnerable than wild-type neurons to inorganic iron. HO-1, ferritin, and superoxide dismutase expression in HO-2 -/- cultures did not differ significantly from that observed in HO-2 +/+ cultures; cellular glutathione levels were slightly higher in knockout cultures. These results suggest that heme breakdown by heme oxygenase accelerates the oxidative neurotoxicity of hemoglobin, and may contribute to neuronal injury after CNS hemorrhage.
Rogers
Bret
B
Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA
Yakopson
Vladimir
V
Teng
Zhi-Ping
ZP
Guo
Yaping
Y
Regan
Raymond F
RF
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D005260:Female; D005293:Ferritins / Q000378:metabolism; D005452:Fluoresceins; D005978:Glutathione / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051547:Heme Oxygenase-1; D006454:Hemoglobins / Q000633:toxicity*; D007501:Iron / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins; D008665:Metalloporphyrins / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology*; D018384:Oxidative Stress; D011524:Protoporphyrins / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012235:Rhodamines; D013482:Superoxide Dismutase / Q000378:metabolism
D005452:Fluoresceins; D006454:Hemoglobins; D008565:Membrane Proteins; D008665:Metalloporphyrins; D011524:Protoporphyrins; D017382:Reactive Oxygen Species; D012235:Rhodamines; C058319:dihydrorhodamine 123; C037631:2',7'-dichlorofluorescein; D005293:Ferritins; C032628:tin protoporphyrin IX; D007501:Iron; D007770:L-Lactate Dehydrogenase; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; C105245:heme oxygenase-2; D013482:Superoxide Dismutase; D005978:Glutathione