
==== Front
BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-341537739110.1186/1471-2202-5-34Research ArticleHeme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin Regan Raymond F 1Raymond.Regan@jefferson.eduChen Jing 1Jing.Chen@jefferson.eduBenvenisti-Zarom Luna 1Luna.Benvenisti-zarom@jefferson.edu1 Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, 239 Thompson Building, Philadelphia, PA 19107, USA2004 17 9 2004 5 34 34 30 6 2004 17 9 2004 Copyright © 2004 Regan et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 μM 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 μM; 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 μM 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.
==== Body
Background
Hemin is a potent oxidant that accumulates in intracranial hematomas and may contribute to neural cell injury [1,2]. It is also the preferred substrate for heme oxygenase-2, the constitutively-expressed isoform that accounts for most CNS heme oxygenase (HO) under normal conditions [3]. In pathologic states, HO frequently has an antioxidant effect, putatively due to the protection provided by increased cellular bilirubin, decreased heme, and up-regulation of other antioxidants [4-7]. However, in models that are relevant to CNS hemorrhage, HO inhibitors have surprisingly been found to be protective [8-10].

All HO inhibitors that are currently available have numerous non-specific actions that may complicate the interpretation of experimental results, including inhibition of nitric oxide synthase and guanyl cyclase, and modification of voltage-gated calcium currents [11-14]. Some may also have a direct antioxidant effect that is unrelated to HO inhibition [15]. In order to investigate HO-2 in heme-mediated injury more specifically, we have cultured neurons and astrocytes derived from HO-2 knockout mice and genetically-similar wild type controls. In recent studies, we observed that astrocytes derived from mutant mice were more vulnerable to hemin [16]. Conversely, HO-2 gene deletion decreased the vulnerability of neurons to hemoglobin [17]. Neither wild type nor knockout astrocytes were injured by hemoglobin at the micromolar concentrations that are feasible in vitro. HO-2 gene deletion per se did not result in a compensatory increase in HO-1 in these cultures, and produced minimal or no change in other cellular antioxidants [16,17].

The disparate effects of HO-2 gene deletion on hemin toxicity to astrocytes and hemoglobin toxicity to neurons may reflect the inability of neurons to tolerate the products of heme metabolism, i.e. iron, carbon monoxide, and bilirubin. Alternatively, it may reflect the different oxidant properties of hemin and hemoglobin. Although the oxidant effect of hemoglobin may be due in part to hemin release to membrane lipids [18], other mechanisms may also contribute. Extracellular hemoglobin undergoes autoxidation, which produces superoxide [19]. In addition to being an oxidant, superoxide reacts with globin amino acids in a complex fashion to generate a variety of reactive species, including thiyl radicals, hydroxyl radicals, and hydrogen peroxide [20,21]. It is also noteworthy that hemoglobin is highly water soluble while hemin is quite lipophilic; their accumulation in separate cellular compartments may lead to a different pattern of site-specific oxidative damage [22,23].

The present study was designed to test the effect of HO-2 gene deletion on the oxidative neuronal injury produced by extracellular hemin. We specifically tested the hypothesis that targeted deletion of the HO-2 gene attenuated oxidative cell injury in a primary cell culture model of hemin toxicity.

Results
Effect of HO-2 gene deletion on hemin neurotoxicity
In preliminary experiments, we observed that overnight (14 h) exposure to low micromolar concentrations of hemin consistently produced morphologic evidence of neuronal injury in wild-type cultures (Fig. 1). This time interval was therefore used for cytotoxicity studies. Consistent with prior observations in pure astrocyte cultures [24], no morphologic evidence of injury was observed in the astrocyte monolayer at hemin concentrations up to 10 μM. In order to specifically assess neuronal injury in this study, the concentrations used were limited to this range. In wild-type cultures, cell injury as quantified by LDH release was observed at 1 μM hemin and then increased exponentially, to release of 69.7 ± 8.6% of neuronal LDH at 3 μM (Fig 2A). The calculated EC50 was 1.85 μM. LDH release was significantly reduced in knockout cultures subjected to the same treatment. At 3 μM hemin, only 12.6 ± 4.1% of LDH had been released at this time point. Control experiments demonstrated that these low hemin concentrations do not interfere with the LDH assay.

Cell death was also quantified by analysis of fluorescence intensity after staining cultures with propidium iodide. Using this method, widespread neuronal death was also observed at 3–10 μM hemin in wild type cultures, and the calculated EC50 was 1.05 μM. Propidium staining of nuclei was significantly reduced in cultures prepared from HO-2 knockout mice (Fig. 2B). The maximal fluorescence was produced by exposure to 10 μM hemin, which was 37.0 ± 3.2% of that observed in control sister cultures treated with NMDA to kill all neurons.

Effect of HO-2 gene deletion on markers of cell oxidation
In order to assess reactive oxygen species formation after hemin exposure, cultures were stained with 20 μM dihydrorhodamine 123 after 4 hour hemin exposure. This time interval was used because it preceded cell lysis, and therefore allowed cell retention of the reduced fluorophore. A marked increase in fluorescence was observed in cultures prepared from wild type mice (Fig. 3). This signal was concentrated in neuronal cell bodies and processes. In contrast, fluorescence in cultures prepared from HO-2 knockout mice was minimal, and did not exceed that observed in cultures subjected to medium exchanges only.

In order to further investigate the effect of HO-2 gene deletion on oxidative stress produced by hemin, cells were harvested after 4 hour hemin exposure. Protein carbonyl groups (i.e. aldehydes and ketones), which are markers of oxidation, were then derivatized and detected with a dinitrophenylhydrazone antibody. Increased immunoreactivity was apparent in lysates of wild type cultures treated with hemin (Figure 4). A prominent band was present at approximately 44 kDa, along with a higher molecular weight smear. At 10 μM hemin, the carbonyl signal intensity in wild type cultures was 2.3-fold higher than in cultures exposed to culture medium only, compared with only 1.3-fold higher in knockout cultures.

Hemin neurotoxicity is attenuated by iron chelators
Based on our prior observations in astrocytes and neuroblastoma cells [24,25], we hypothesized that the toxic product produced by hemin breakdown in primary murine neurons was iron. In order to test this hypothesis, the effect of iron chelators on hemin neurotoxicity in wild type cultures was assessed. Most cell death, as detected by both LDH release and PI staining, was prevented by concomitant treatment with phenanthroline, a lipid-soluble iron chelator (Fig. 5). Deferoxamine, which is water soluble, was less potent; its effect when applied at a concentration tenfold greater than that of hemin reached statistical significance only when injury was assessed by PI staining.

Discussion
In prior experiments, we demonstrated that targeted deletion of the HO-2 gene in primary neuron/astrocyte cultures did not alter expression of HO-1, and had little or no effect on other cellular antioxidants [17]. This simplified system therefore permits investigation of HO-2 without the confounding compensatory effects that have been observed in whole animal models [26,27]. We have previously reported that HO-2 gene deletion increased the vulnerability of astrocytes to hemin, the preferred substrate of HO, in cultures containing only astrocytes. The present study targeted neurons in mixed neuron/astrocyte cultures by using hemin concentrations that did not injure astrocytes [24]. In this model, the opposite was observed. HO-2 deletion attenuated hemin-induced ROS formation and reduced levels of oxidized proteins. Consistent with the oxidative nature of hemin toxicity, neuronal death was reduced in knockout cultures.

Although hemin is a highly reactive pro-oxidant, its breakdown as catalyzed by the heme oxygenases generates biologically active and potentially toxic products. Prior in vitro studies suggest that neurons are particularly vulnerable to these, i.e. iron, carbon monoxide, and bilirubin [28-30]. The present results suggest that when neurons are provided with an excess of HO substrate, the toxicity of breakdown products outweighs any benefit provided by hemin removal. The protective effect of iron chelators suggests that this phenomenon is at least partly due to iron neurotoxicity. Inorganic iron is toxic to cultured cortical neurons, with an EC50 of approximately 10 μM [17]. The lower EC50 for hemin is not surprising, given its lipophilicity and accumulation in cell membranes [31]. The latter phenomenon likely accounts for the greater efficacy of phenanthroline, which unlike deferoxamine is lipophilic. Deferoxamine is quite effective against hemoglobin neurotoxicity in this culture system [32], suggesting that hemoglobin releases its iron either in the medium or in an aqueous cellular compartment.

The present results are consistent with observations that heme oxygenase inhibitors are protective in models of CNS hemorrhage [2,8,9], in contrast to the beneficial effect of HO in ischemia [33]. In a recent study, Koeppen et al. [2] observed that repeated administration of tin mesoporphyrin protected thalamic neurons from the delayed degeneration that occurred in tissue adjacent to injected autologous blood. Similarly, Huang et al. [9] observed that tin protoporphyrin attenuated edema formation after stereotactic hemoglobin injection into the rat striatum. It is noteworthy that the number of astrocytes per neuron is significantly higher in the human brain than in rodents [34]. The deleterious effect of HO inhibition on heme mediated injury to astrocytes may therefore be less prominent in animal models than in clinical intracerebral hemorrhage [35].

The disparate effect of HO on neurons and astrocytes exposed to extracellular hemin suggests that it may be somewhat difficult to target it effectively after CNS hemorrhage. All currently available HO inhibitors inhibit both HO-2, which is predominantly neuronal in vivo [36], and HO-1, which is induced mainly in glial cells [37]. The protection that these non-selective agents provide to neurons may be negated by their deleterious effect on astrocytes. Further investigation is needed for the development of strategies that would permit the selective inhibition or down-regulation of HO-2 in neurons.

Conclusions
Targeted deletion of the heme oxygenase-2 gene mitigates oxidative stress in cultured neurons exposed to hemin, and is cytoprotective. Selective inhibition of neuronal heme oxygenase may have a beneficial effect after CNS hemorrhage.

Methods
Cell cultures
The HO-2 knockout mice which were used in this study are descended from mutants produced by Poss et al. [38], and have a C57BL/6 X 129/Sv genetic background. All mice were obtained from our local breeding colony, and were provided with food and water ad libitum and a 12 hour light/dark cycle. All breeding mice were the offspring of heterozygotes. Genotype was determined by polymerase chain reaction (PCR) using genomic DNA isolated from tail clippings; primers were previously published [17].

Cortical cell cultures were prepared from fetal mice at gestational age 15–17 d as previously described [39]. Under a dissecting microscope, cortices were dissected free from other brain tissue, minced with forceps, and incubated in medium containing 0.075%-acetylated trypsin at 37°C for one hour. Tissue was then collected by low speed centrifugation, and was dissociated by trituration through a flamed Pasteur pipette in plating medium containing Eagle's minimal essential medium (MEM), 5% fetal bovine serum (Hyclone, Logan, UT), 5% heat inactivated equine serum (Hyclone), glutamine (2 mM), and glucose (23 mM). The cell suspension was diluted with additional plating medium, and cells were plated on confluent astrocyte cultures in 24-well plates (Primaria, Falcon) at a density of 3 hemispheres/plate. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2/95% air. Two-thirds of the culture medium was replaced at days 4 and 8 in vitro with MEM containing 10% equine serum, 2 mM glutamine, and 23 mM glucose. After ten days in vitro this feeding procedure was performed daily.

Hemin exposure
Hemin was freshly prepared as a 1 mM stock solution and was diluted to the desired concentration with minimal essential medium containing 10 mM glucose (MEM10). Experiments were conducted at 12–16 days in vitro. Serum was washed out of cultures with MEM10 (> 1000X dilution) prior to addition of hemin. Cultures were incubated at 37°C in a 5% CO2 atmosphere for the entire exposure interval.

Detection of reactive oxygen species
ROS formation was quantified by staining with dihydrorhodamine 123 (DHR, Molecular Probes, Eugene, OR), which is a cell-permeable, non-fluorescent compound that is oxidized by cellular peroxides to fluorescent rhodamine [40]. Fluorescence intensity is directly proportional to cellular oxidative stress. In order to prevent oxidation of DHR by hemin in the medium, cultures were washed free of hemin prior to DHR addition. After incubation with 20 μM DHR in MEM10 for 15 min, the medium was replaced, and cultures were imaged using a Nikon inverted microscope with epifluorescence attachment. Images were captured immediately after illumination (25 msec exposure). Photomicrographs of random 100X fields were analyzed with IPLab image analysis software (Scanalytics, Inc., Fairfax, VA). The low fluorescence in control cultures exposed to experimental medium only was subtracted from mean values to define the signal associated with hemin exposure.

Detection of protein oxidation
Protein oxidation was assessed using the Oxyblot™ kit (Chemicon, Inc., Temecula, CA). At the end of the hemin exposure interval, culture medium was aspirated, and cells were washed and then harvested in 100 μl of lysis buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, and 0.1% sodium dodecyl sulfate. Carbonyl groups were derivatized to 2, 4-dintrophenylhydrazone (DNP-hydrazone) by reaction with 2, 4-dinitrophenylhydrazine, following the manufacturer's instructions. Proteins were then separated on a 12% polyacrylamide gel and were transferred onto a polyvinylidene difluoride Imobilon-P transfer membrane filter (Millipore, Billerica, MA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Carbonylated proteins were detected with rabbit anti-DNP (1:150) followed by goat anti-rabbit IgG (1:300). Immunoreactive proteins were visualized using Super Signal West Femto Reagent (Pierce Biotechnology, Rockford, IL) and Kodak ImageStation 400.

Quantification of cell death
After examination of cultures using phase contrast microscopy, cell death was quantified by measurement of LDH activity in the culture medium, as previously described [41]. To facilitate comparisons, values were scaled to the mean value in sister cultures exposed to NMDA 300 μM for 40 h. This treatment releases essentially all neuronal LDH in this system without injuring astrocytes [42]. Since the low micromolar concentrations of hemin that were used in this study do not injure cultured cortical astrocytes [24], the contribution of astrocyte LDH to the total signal is negligible.

Cell death was also quantified by staining with propidium iodide (13 μg/ml for 15 min). When viewed with a rhodamine filter, the nuclei of cells with disrupted membranes stain red, while cells with intact membranes exclude propidium. Random 100X fields were captured with a Nikon Diaphot epifluorescence microscope and were analyzed with IPLab image analysis software. As with LDH data, fluorescence intensity was scaled to that in sister cultures treated with NMDA 300 μM for 40 h, which kills all neurons. Propidium iodide fluorescence was not observed in cells that had an astrocyte phenotype after treatment with NMDA or hemin at the concentrations used in this study.

Abbreviations
DHR: dihydrorhodamine; DNP: dinitrophenylhydrazone; HO: heme oxygenase; LDH: lactate dehydrogenase; MEM10: minimal essential medium containing 10 mM glucose; NMDA: N-methyl-D-aspartate; PI: propidium iodide; ROS: reactive oxygen species.

Authors' contributions
RFR designed the study, collected and analyzed data, and wrote the manuscript. JC also participated in data collection and analysis, and edited the manuscript. LBZ participated in genotyping and edited the manuscript. All authors reviewed and approved the final manuscript.

Acknowledgements
This study was supported by a grant from the National Institutes of Health (1RO1NS042273) and from the Pennsylvania/Delaware affiliate of the American Heart Association. We thank Dr. Frank Sharp for providing the HO-2 knockout mice that were used to establish our colony.

Figures and Tables
Figure 1 Morphologic changes in wild type and knockout cultures exposed to hemin. Phase contrast photomicrographs of cultures exposed for 14 h to: A) Experimental medium (MEM10) only; neurons rest on a monolayer of astrocytes, and aggregate in groups which send out an array of processes; B) hemin 3 μM, wild type culture; most neurons and processes have degenerated; C) hemin 3 μM, knockout culture; neurons with intact processes persist.

Figure 2 Heme oxygenase-2 gene deletion attenuates the neurotoxic effect of hemin. Cultures were treated with indicated concentrations of hemin for 14 h. Injury was assessed by A) LDH activity in the medium, which is scaled to that in sister cultures treated with 300 μM NMDA for 40 h (= 100), which releases essentially all neuronal LDH; B) fluorescence intensity of cultures stained with propidium iodide, again scaled to that in sister cultures treated with NMDA. *P < 0.05, ***P < 0.001 v. knockout cultures treated with same concentration of hemin, Bonferroni multiple comparisons test.

Figure 3 Heme oxygenase-2 gene deletion reduces production of cellular reactive oxygen species, assessed with dihydrorhodamine 123. Cultures were treated with 3 μM hemin for 4 h. Hemin was then washed out, and cultures were treated with 20 μM dihydrorhodamine 123 for 15 min and imaged. A) Wild type culture; fluorescence is localized to neuronal cell bodies and processes; B) Knockout culture; fluorescence is markedly diminished. Fluorescence intensity is quantified in arbitrary units.

Figure 4 Heme oxygenase-2 gene deletion reduces protein oxidation in hemin-treated cultures. Top: Representative immunoblot of protein lysates from wild type (WT) or HO-2 knockout (KO) neuron/astrocyte cultures treated for 4 h with indicated hemin concentrations, stained with anti-DNP antibody to detect derivatized carbonyl groups. M: molecular weight standard with attached DNP residues. Bottom: The mean protein carbonyl signal intensity (± SEM, n = 5/condition) was normalized to that in wild-type cultures exposed to culture medium only (= 1.0) *P < 0.05; ***P < 0.001 versus signal in wild type cultures exposed to the same hemin concentration, Bonferroni multiple comparisons test.

Figure 5 Effect of iron chelators on hemin toxicity. Wild-type cultures were treated with 3 μM hemin for 14 h, alone or with indicated concentrations of deferoxamine (DFO) or phenanthroline (PHE). Injury was assessed by A) LDH activity in the culture medium, which is scaled to that in sister cultures treated with 300 μM NMDA for 40 h (= 100), which releases essentially all neuronal LDH; B) fluorescence intensity of cultures stained with propidium iodide, again scaled to that in sister cultures treated with NMDA. ***P < 0.001 v. cultures treated with hemin only, Bonferroni multiple comparisons test.
==== Refs
Letarte PB Lieberman K Nagatani K Haworth RA Odell GB Duff TA  Hemin: levels in experimental subarachnoid hematoma and effects on dissociated vascular smooth muscle cells J Neurosurg 1993 79 252 255 8331409 
Koeppen AH Dickson AC Smith J  Heme oxygenase in experimental intracerebral hemorrhage: the benefit of
tin-mesoporphyrin J Neuropathol Exp Neurol 2004 63 587 597 15217087 
Trakshel GM Kutty RK Maines MD  Resolution of rat brain heme oxygenase activity: absence of detectable amount of the inducible form (HO-1) Arch Biochem Biophys 1988 260 732 739 3124761 
Stocker R Yamamoto Y McDonagh AF Glazer AN Ames BN  Bilirubin is an antioxidant of possible physiologic significance Science 1987 235 1043 1046 3029864 
Doré S Takahashi M Ferris CD Hester LD Guastella D Snyder SH  Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury Proc Natl Acad Sci USA 1999 96 2445 2450 10051662 10.1073/pnas.96.5.2445 
Taille C El-Benna J Lanone S Dang MC Ogier-Denis E Aubier M Boczkowski J  Induction of heme oxygenase-1 inhibits NAD(P)H oxidase activity by down-regulating cytochrome b558 expression via the reduction of heme availability J Biol Chem 2004 279 28681 28688 15123630 10.1074/jbc.M310661200 
Balla G Jacob HS Balla J Rosenberg M Nath K Apple F Eaton JW Vercellotti GM  Ferritin:  A cytoprotective strategem of endothelium J Biol Chem 1992 267 18148 18153 1517245 
Wagner KR Hua Y de Courten-Myers GM Broderick JP Nishimura RN Lu SY Dwyer BE  Tin-mesoporphyrin, a potent heme oxygenase inhibitor, for treatment of intracerebral hemorrhage: in vivo and in vitro studies Cell Mol Biol (Nolsy-le-grand) 2000 46 597 608 
Huang FP Xi G Keep. RF Hua Y Nemoianu A Hoff JT  Brain edema after experimental intracerebral hemorrhage: role of hemoglobin degradation products J Neurosurg 2002 96 287 293 11838803 
Wu J Hua Y Keep RF Nakemura T Hoff JT Xi G  Iron and iron-handling proteins in the brain after intracerebral hemorrhage Stroke 2003 34 2964 2969 14615611 10.1161/01.STR.0000103140.52838.45 
Linden DJ Narisimhan K Gurfel D  Protoporphyrins modulate voltage-gated calcium current in AtT-20 pituitary cells J Neurophysiol 1993 70 2673 2677 7907135 
Luo D Vincent SR  Metalloporphyrins inhibit nitric oxide-dependent cGMP formation in vivo Eur J Pharmacol 1994 267 263 267 7522180 10.1016/0922-4106(94)90149-X 
Meffert MK Haley JE Schuman EM Schulman H Madison DV  Inhibition of hippocampal heme oxygenase, nitric oxide synthase, and long-term potentiation by metalloporphyrins Neuron 1994 13 1225 1233 7524564 10.1016/0896-6273(94)90060-4 
Grundemar L Ny L  Pitfalls using metalloporphyrins in carbon monoxide research Trends Pharmacol Sci 1997 18 193 195 9226997 10.1016/S0165-6147(97)01065-1 
Wagner KR Dwyer BE  Hematoma removal, heme, and heme oxygenase following hemorrhagic stroke Ann NY Acad Sci 2004 1012 237 251 15105270 10.1196/annals.1306.020 
Chen J Regan RF  Heme oxygenase-2 gene deletion increases astrocyte vulnerability to hemin Biochem Biophys Res Commun 2004 318 88 94 15110757 10.1016/j.bbrc.2004.03.187 
Rogers B Yakopson V Teng ZP Guo Y Regan RF  Heme oxygenase-2 knockout neurons are less vulnerable to hemoglobin toxicity Free Rad Biol Med 2003 35 872 881 14556851 10.1016/S0891-5849(03)00431-3 
Chiu D Lubin B  Oxidative hemoglobin denaturation and RBC destruction: the effect of heme on red cell membranes Sem In Hematol 1989 26 128 135 
Misra HP Fridovich I  The generation of superoxide radical during the autoxidation of hemoglobin  J Biol Chem 1972 247 6960 6962 4673289 
Watkins JA Kawanashi S Caughey WS  Autoxidation reactions of hemoglobin A free from other red cell components: a minimal mechanism Biochem Biophys Res Comm 1985 132 742 748 2998382 
Balagopalakrishna C Abugo OO Horsky J Manoharan PT Nagababu E Rifkind JM  Superoxide produced in the heme pocket of the beta-chain of hemoglobin reacts with the beta-93 cysteine to produce a thiyl radical Biochemistry 1998 37 13194 13202 9748326 10.1021/bi980941c 
Samuni A Aronivitch J Godinger G Chevion M Czapski G  On the cytotoxicity of vitamin C and metal ions. A site-specific Fenton mechanism. Eur J Biochem 1983 137 119 124 6317379 
Arouma OI Grootveld M Halliwell B  The role of iron in ascorbate-dependent deoxyribose degradation.  Evidence consistent with a site-specific hydroxyl radical generation caused by iron ions bound to the deoxyribose molecule J Inorg Chem 1987 29 289 299 
Regan RF Kumar N Gao F Guo YP  Ferritin induction protects cortical astrocytes from heme-mediated oxidative injury Neuroscience 2002 113 985 994 12182902 10.1016/S0306-4522(02)00243-9 
Goldstein L Teng ZP Zeserson E Patel M Regan RF  Hemin induces an iron-dependent, oxidative injury on human neuron-like cells J Neurosci Res 2003 73 113 121 12815715 10.1002/jnr.10633 
Dennery PA Spitz DR Yang G Tatarov A Lee CS Shegog ML Poss KD  Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2 J Clin Invest 1998 101 1001 1011 9486970 
Dennery PA Visner G Weng Y-H Nguyen X Lu F Zander D Yang G  Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron Free Radic Biol Med 2003 34 124 133 12498987 10.1016/S0891-5849(02)01295-9 
Liu R Liu W Doctrow SR M. Baudry  Iron toxicity in organotypic cultures of hippocampal slices: role of reactive oxygen species J Neurochem 2003 85 492 502 12675926 
Miro O Casademont J Urbanomarquez A Cardellach F  Mitochondrial cytochrome C oxidase inhibition during carbon monoxide poisoning Pharmacology and Toxicology 1998 82 199 202 9584335 
Silva RF Rodriguez CM Brites D  Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin Pediatr Res 2002 51 535 541 11919342 
Shaklai N Shviro Y Rabizadeh E Kirschner-Zilber I  Accumulation and drainage of hemin in the red cell membrane Biochim Biophys Acta 1985 821 355 366 4063370 10.1016/0005-2736(85)90106-3 
Regan RF Panter SS  Neurotoxicity of hemoglobin in cortical cell culture Neurosci Lett 1993 153 219 222 8327197 10.1016/0304-3940(93)90326-G 
Doré S Sampei K Goto S Alkayed NJ Guastella D Blackshaw S Gallagher M Traystman RJ Hurn PD Koehler RC Snyder SH  Heme oxygenase-2 is neuroprotective in cerebral ischemia Mol Med 1999 5 656 663 10602774 
Nedergaard M Ransom B Goldman SA  New roles for astrocytes: Redefining the functional architecture of the brain Trends Neurosci 2003 26 523 530 14522144 10.1016/j.tins.2003.08.008 
Regan RF Guo YP Kumar N  Heme oxygenase-1 induction protects murine cortical astrocytes from hemoglobin toxicity Neurosci Lett 2000 282 1 4 10713382 10.1016/S0304-3940(00)00817-X 
Ewing JF Maines MD  In situ hybridization and immunohistochemical localization of heme oxygenase-2 mRNA and protein in normal rat brain: differential distribution of isozyme 1 and 2 Mol Cell Neurosci 1992 3 559 570 
Matz P Turner C Weinstein PR Massa SM Panter SS Sharp FR  Heme-oxygenase-1 induction in glia throughout rat brain following experimental subarachnoid hemorrhage Brain Res 1996 713 211 222 8724993 10.1016/0006-8993(95)01511-6 
Poss KD Thomas MJ Ebralidze AK TJ OD Tonegawa S  Hippocampal long-term potentiation is normal in heme oxygenase-2 mutant mice Neuron 1995 15 867 873 7576635 10.1016/0896-6273(95)90177-9 
Regan RF Choi DW  The effect of NMDA, AMPA/kainate, and calcium channel antagonists on traumatic cortical neuronal injury in culture Brain Res 1994 633 236 242 7907934 10.1016/0006-8993(94)91544-X 
Royall JA Ischiropoulos H  Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells Arch Biochem Biophys 1993 302 348 355 8387741 10.1006/abbi.1993.1222 
Regan RF Rogers B  Delayed treatment of hemoglobin neurotoxicity J Neurotrauma 2003 20 111 120 12614593 10.1089/08977150360517236 
Koh JY Choi DW  Vulnerability of cultured cortical neurons to damage by excitotoxins: Differential susceptibility of neurons containing NADPH-diaphorase J Neurosci 1988 8 2153 2163 3385492

