Hemin induces an iron-dependent, oxidative injury to human neuron-like cells.
Hemin is released from hemoglobin after CNS hemorrhage and is present at high micromolar concentrations in intracranial hematomas. This highly reactive compound is potentially cytotoxic via a variety of oxidative and nonoxidative mechanisms. However, despite its clinical relevance, little is known of its effect on neuronal cells. In this study, we tested the hypotheses that hemin is toxic to human neurons at physiologically relevant concentrations and that its toxicity is iron dependent and oxidative. A homogeneous population of neuron-like cells was produced by sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor, using the protocol of Encinas et al. Hemin exposure for 24 hr resulted in cell death that progressively increased between 3 and 30 microM (EC(50) approximately 10 microM); protoporphyrin IX, the iron-free congener of hemin, was not toxic. Cell death commenced at 14 hr and was preceded by a marked increase in cellular reactive oxygen species (ROS). Most injury and ROS production were prevented by concomitant treatment with an equimolar concentration of the lipid-soluble iron chelator phenanthroline; the water-soluble chelator deferoxamine was also effective at concentrations of 0.1 mM or higher. Heme oxygenase-2 was constitutively expressed by these cells, and heme oxygenase-1 was induced by hemin. Heme oxygenase inhibition attenuated ROS generation and reduced injury by about one-third. Cell death was also prevented with the sulfhydryl reducing agents glutathione and mercaptoethanol. Nuclear morphology in the hours prior to cell lysis revealed a predominantly homogenous staining pattern; the percentage of fragmented nuclei was increased only at 4 hr and then accounted for only 1.45% +/- 0.25% of cells. The general caspase inhibitor zVAD-fmk had no effect on cell viability. These results suggest that hemin is toxic to human neuron-like cells at concentrations that are less than 3% of those observed in intracranial hematomas. In this model, its toxicity is iron dependent, oxidative, and predominantly necrotic.
Goldstein
Laurel
L
Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Teng
Zhi-Peng
ZP
Zeserson
Eli
E
Patel
Monica
M
Regan
Raymond F
RF
D000970:Antineoplastic Agents / Q000494:pharmacology; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects; D002543:Cerebral Hemorrhage / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000037:antagonists & inhibitors / Q000378:metabolism; D006427:Hemin / Q000633:toxicity*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D019208:Brain-Derived Neurotrophic Factor; D005609:Free Radicals; D007502:Iron Chelating Agents; D017382:Reactive Oxygen Species; D013439:Sulfhydryl Reagents; D014212:Tretinoin; D006427:Hemin; D007501:Iron; D006419:Heme Oxygenase (Decyclizing)