Neuroprotective Effects of Theaflavins Against Oxidative Stress-Induced Apoptosis in PC12 Cells

Oxidative stress can induce neuronal apoptosis via the production of superoxide and hydroxyl radicals. This process is as a major pathogenic mechanism in neurodegenerative disorders. In this study, we aimed to clarify whether theaflavins protect PC12 cells from oxidative stress damage induced by H₂O₂. A cell model of PC12 cells undergoing oxidative stress was created by exposing cells to 200 μM H₂O₂ in the presence or absence of varying concentrations of theaflavins (5, 10, and 20 μM). Cell viability was monitored using the MTT assay and Hoechst 33258 staining, showing that 10 μM theaflavins enhanced cell survival following 200 μM H₂O₂ induced toxicity and increased cell viability by approximately 40?%. Additionally, we measured levels of intracellular reactive oxygen species (ROS) and antioxidant enzyme activity. This suggested that the neuroprotective effect of theaflavins against oxidative stress in PC12 cells is derived from suppression of oxidant enzyme activity. Furthermore, Western blot analyses indicated that theaflavins downregulated the ratio of pro-apoptosis/anti-apoptosis proteins Bax/Bcl-2. Theaflavins also downregulated the expression of caspase-3 compared with a H₂O₂-treated group that had not been treated with theaflavins. Interestingly, this is the first study to report that the four main components of theaflavins found in black tea can protect neural cells (PC12) from apoptosis induced by H₂O₂. These findings provide the foundations for a new field of using theaflavins or its source, black tea, in the treatment of neurodegenerative diseases caused by oxidative stress.     

Antioxidant system activation by mercury in Pfaffia glomerata plantlets

Oxidative stress caused by mercury (Hg) was investigated in Pfaffia glomerata plantlets grown in nutrient solution using sand as substrate. Thirty-day-old acclimated plants were treated for 9 days with four Hg levels (0, 1, 25 and 50 μM) in the substrate. Parameters such as growth, tissue Hg concentration, toxicity indicators (δ-aminolevulinic acid dehidratase, δ-ALA-D, activity), oxidative damage markers (TBARS, lipid peroxidation, and H₂O₂ concentration) and enzymatic (superoxide dismutase, SOD, catalase, CAT, and ascorbate peroxidase, APX) and non-enzymatic (non-protein thiols, NPSH, ascorbic acid, AsA, and proline concentration) antioxidants were investigated. Tissue Hg concentration increased with Hg levels. Root and shoot fresh weight and δ-ALA-D activity were significantly decreased at 50 μM Hg, and chlorophyll and carotenoid concentration were not affected. Shoot H₂O₂ concentration increased curvilinearly with Hg levels, whereas lipid peroxidation increased at 25 and 50 μM Hg, respectively, in roots and shoots. SOD activity showed a straight correlation with H₂O₂ concentration, whereas CAT activity increased only in shoots at 1 and 50 μM Hg. Shoot APX activity was either decreased at 1 μM Hg or increased at 50 μM Hg. Conversely, root APX activity was only increased at 1 μM Hg. In general, AsA, NPSH and proline concentrations increased upon addition of Hg, with the exception of proline in roots, which decreased. These changes in enzymatic and non-enzymatic antioxidants had a significant protective effect on P. glomerata plantlets under mild Hg-stressed conditions.     

The octadecaneuropeptide ODN prevents 6‐hydroxydopamine‐induced apoptosis of cerebellar granule neurons through a PKC‐MAPK‐dependent pathway

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6‐hydroxydopamine (6‐OHDA)‐induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central‐type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10^?18 to 10^?12 M) inhibited 6‐OHDA‐evoked cell death in a concentration‐dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo_1–8[DLeu⁵]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6‐OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6‐OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro‐apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase‐3 activity. Exposure of 6‐OHDA‐treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6‐OHDA‐induced oxidative stress and apoptotic cell death.     

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H₂O₂), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H₂O₂-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury.     

Evaluation of heme oxygenase 1 (HO 1) in Cd and Ni induced cytotoxicity and crosstalk with ROS quenching enzymes in two to four leaf stage seedlings of <Emphasis Type="Italic">Vigna radiata</Emphasis>

Research on heme oxygenase in plants has received consideration in recent years due to its several roles in development, defense, and metabolism during various environmental stresses. In the current investigation, the role of heme oxygenase (HO) 1 was evaluated in reducing heavy metal (Cd and Ni) uptake and alleviating Cd and Ni toxicity effects in the hydroponically grown seedlings of Vigna radiata var. PDM 54. Seedlings were subjected to Cd- and Ni-induced oxidative stress independently at different concentrations ranging from 10 to 100 μM. After 96 h (fourth day) of treatment, the stressed plants were harvested to study the cellular homeostasis and detoxification mechanism by examining the growth, stress parameters (LPX, H₂O₂ content), and non-enzymatic and enzymatic parameters (ascorbate peroxidase (APX), guaicol peroxidase (GPX), and catalase (CAT)) including HO 1. At 50 μM CdCl₂ and 60 μM NiSO₄, HO 1 activity was found to be highest in leaves which were 1.39 and 1.16-fold, respectively. The greatest HO 1 activity was reflected from the reduction of H₂O₂ content at these metal concentrations (50 μM CdCl₂ and 60 μM NiSO₄) which is correlated with the increasing activity of other antioxidant enzymes (CAT, APX). Thus, HO 1 works within a group that generates the defense machinery for the plant’s survival by scavenging ROS which is confirmed by a time-dependent study. Hence, it is concluded that seedlings of V. radiata were more tolerant towards metal-induced oxidative stress in which HO 1 is localized in its residential area (plastids).     

Neuroprotective Effects of Hydroalcoholic Extract of Ocimum sanctum Against H2O2 Induced Neuronal Cell Damage in SH-SY5Y Cells via Its Antioxidative Defence Mechanism

Oxidative stress mediates the cell damage in several ailments including neurodegenerative conditions. Ocimum sanctum is widely used in Indian ayurvedic medications to cure various ailments. The present study was carried out to investigate the antioxidant activity and neuroprotective effects of hydroalcoholic extract of O. sanctum (OSE) on hydrogen peroxide (H₂O₂)-induced oxidative challenge in SH-SY5Y human neuronal cells. The extract exhibited strong antioxidant activity against DPPH, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical and hydroxyl radicals with IC₅₀ values of 395 ± 16.2, 241 ± 11.5 and 188.6 ± 12.2 μg/ml respectively, which could be due to high amount of polyphenols and flavonoids. The observed data demonstrates 41.5 % cell survival with 100 μM H₂O₂ challenge for 24 h, which was restored to 73 % by pre-treatment with OSE for 2 h. It also decreased the lactate dehydrogenase leakage and preserved the cellular morphology. Similarly OSE inhibited lipid peroxidation, DNA damage, reactive oxygen species generation and depolarization of mitochondrial membrane. The extract restored superoxide dismutase and catalase enzyme/protein levels and further downregulated HSP-70 over-expression. These findings suggest that OSE ameliorates H₂O₂ induced neuronal damage via its antioxidant defence mechanism and might be used to treat oxidative stress mediated neuronal disorders.     

Heavy metal-induced oxidative damage is reduced by β-estradiol application in lentil seedlings

The protective effect of β-estradiol (E) application against heavy metal (HM) toxicity in lentil (Lens culinaris) seedlings was investigated. Seeds were treated with distilled water (control) or aqueous solutions of 100 μM CdCl₂, 200 μM CuCl₂ and 1 μM E singly or in combinations (1 μM E+100 μM CdCl₂ and 1 μM E+200 μM CuCl₂). HM treatments resulted in increase in the activities of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), guaicol peroxidase and ascorbate peroxidase. In a similar manner, Cd and Cu affected significantly oxidative injury indicators measured as electrolyte leakage (electrical conductivity of germination medium), lipoxygenase (LOX) activity and contents of malondialdehyde (MDA; lipoperoxidation marker), carbonyl groups (protein oxidation marker) and hydrogen peroxide (a reactive oxygen species). However, E was effective in reducing HM-induced toxicity. The steroid (1) alleviated HM-induced increase in the electrolyte leakage, LOX activity and contents of MDA, carbonyl and H₂O₂ and (2) improved the activities of SOD and CAT, but not the peroxidase ones, as compared to treatments with HM singly. In addition, E application prevented HM-induced decrease in dry weight production, but did not reduce the accumulation of Cd and Cu in tissues. Results of the present study suggest that E is able to protect lentil from HM-induced oxidative damage most likely by avoidance of H₂O₂ generation and improving antioxidative enzyme activities and, thereby, decreasing oxidative stress injury, but not by reducing Cd and Cu uptake.     

Imidacloprid impedes mitochondrial function and induces oxidative stress in cotton bollworm, <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> larvae (Hubner: Noctuidae)

Neonicotinoids have high agonistic affinity to insect nicotinic acetylcholine receptors (nAChR) and are frequently used as insecticides against most devastating lepidopteran insect pests. Imidacloprid influenced dose-dependent decline in the state III and IV respiration, respiration control index (RCI), and P/O ratios, in vitro and in vivo. The bioassay indicated its LD₅₀ value to be 531.24 μM. The insecticide exhibited a dose-dependent inhibition on F₀F₁-ATPase and complex IV activity. At 600 μM, the insecticide inhibited 83.62 and 27.13% of F₀F₁-ATPase and complex IV activity, respectively, and induced the release of 0.26 nmoles/min/mg protein of cytochrome c. A significant dose- and time-dependent increase in oxidative stress was observed; at 600 μM, the insecticide correspondingly induced lipid peroxidation, LDH activity, and accumulation of H₂O₂ content by 83.33, 31.51 and 223.66%. The stress was the maximum at 48 h of insecticide treatment (91.58, 35.28, and 189.80%, respectively). In contrast, catalase and superoxide dismutase were reduced in a dose- and time-dependent manner in imidacloprid-fed larvae. The results therefore suggest that imidacloprid impedes mitochondrial function and induces oxidative stress in H. armigera, which contributes to reduced growth of the larvae along with its neurotoxic effect.     

Insulin on hydrogen peroxide-induced oxidative stress involves ROS/Ca2+ and Akt/Bcl-2 signaling pathways

Abstract

Oxidative stress is induced by excess accumulation of reactive oxygen and nitrogen species (RONS). Astrocytes are metabolically active cells in the brain and understanding astrocytic responses to oxidative stress is essential to understand brain pathologies. In addition to direct oxidative stress, exogenous hydrogen peroxide (H₂O₂) can penetrate biological membranes and enhance formation of other RONS. The present study was carried out to examine the role of insulin in H₂O₂-induced oxidative stress in rat astrocytic cells. To measure changes in the viability of astrocytes at different concentrations of H₂O₂ for 3 h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay was used and 500 μM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 3 h of 500 μM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and calcium ion (Ca²⁺) in C6 cells, with insulin able to effectively diminish H₂O₂-induced oxidative damage to C6 cells. Western blotting studies showed that insulin treatment of astrocytes increased the levels of phosphorylated Akt and magnified the decrease in total Bcl-2 protein. The protective effect of insulin treatment on H₂O₂-induced oxidative stress in astrocytes by reducing apoptosis may relate to the PI3K/Akt pathway.     

Oxidative stress response of Blakeslea trispora induced by H2O2 during β-carotene biosynthesis

The cellular response of Blakeslea trispora to oxidative stress induced by H₂O₂ in shake flask culture was investigated in this study. A mild oxidative stress was created by adding 40 μm of H₂O₂ into the medium after 3 days of the fermentation. The production of β-carotene increased nearly 38 % after a 6-day culture. Under the oxidative stress induced by H₂O₂, the expressions of hmgr, ipi, carG, carRA, and carB involving the β-carotene biosynthetic pathway all increased in 3 h. The aerobic metabolism of glucose remarkably accelerated within 24 h. In addition, the specific activities of superoxide dismutase and catalase were significantly increased. These changes of B. trispora were responses for reducing cell injury, and the reasons for increasing β-carotene production caused by H₂O₂.     

Cell Hypertrophy and MEK/ERK Phosphorylation are Regulated by Glyceraldehyde-Derived AGEs in Cardiomyocyte H9c2 Cells

Diabetic cardiomyopathy has been shown to promote hypertrophy, leading to heart failure. Recent studies have reported a correlation between diabetic cardiomyopathy and oxidative stress, suggesting that the accumulation of advanced glycation end products (AGEs) induces the production of reactive oxygen species (ROS). In a clinical setting, AGEs have been shown to increase the risk of cardiovascular disease; however, the relationship between AGEs and cardiac hypertrophy remains unclear. This study sought to identify the role of AGEs in cardiac hypertrophy by treating H9c2 cells with glyceraldehyde-derived AGEs (200 μg/ml) or H₂O₂ (50 μM) for 96 h. Our results demonstrate that AGEs significantly increased protein levels and cell size. These effects were effectively blocked with PD98059 (10 μM; MEK/ERK inhibitor) pretreatment, suggesting that AGEs caused cell hypertrophy via the MEK/ERK pathway. We then treated cells with AGEs and H₂O₂ for 0–120 min and employed the Odyssey infrared imaging system to detect MEK/ERK phosphorylation. Our results show that AGEs up-regulated MEK/ERK phosphorylation. However, this effect was blocked by NAC (5 mM; ROS inhibitor), indicating that AGEs regulate MEK/ERK phosphorylation via ROS. Our findings suggest that glyceraldehyde-derived AGEs are closely related to cardiac hypertrophy and further identify a molecular mechanism underlying the promotion of diabetic cardiomyopathy by AGEs.     

Antioxidative role of selenoprotein W in oxidant-induced chicken splenic lymphocyte death

To verify the antioxidative role of SelW in oxidant-induced chicken splenic lymphocyte, in this report, the influence of selenite supplementation and SelW gene silence on H₂O₂-mediated cell viability and cell apoptosis in cultured splenic lymphocyte derived from spleen of chicken were examined. The cultured cells were treated with sodium selenite and H₂O₂, or knocked down SelW with small interfering RNAs (siRNAs). The lymphocytes were examined for cell viability, cell apoptosis and mRNA expression levels of SelW and apoptosis-related genes (Bcl-2, Bax, Bak-1, caspase-3 and p53). The results show that the mRNA expression of SelW were effectively increased after treatment with sodium selenite, and H₂O₂-induced cell apoptosis was significantly decreased and cell viability was significantly increased. 20 μM H₂O₂ was found to induce cell apoptosis and decrease cell viability, which was alleviated obviously when cells were pretreated with sodium selenite before exposure to 20 μM H₂O₂. Meanwhile, H₂O₂ induced a significantly up-regulation of the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulation of Bcl-2 (P < 0.05). When lymphocytes were pretreated with Se before treated with H₂O₂, the Bax/Bcl-2 ratio and mRNA expression of those genes were significantly decreased, and Bcl-2 was increased (P < 0.05). SelW siRNA-transfected cells were more sensitive to the oxidative stress induced by treatment of H₂O₂ than control cells. Silencing of the lymphocyte SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. Silencing of SelW significantly up-regulated the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulated Bcl-2 (P < 0.05). The present study demonstrates that SelW plays an important role in protection of splenic lymphocyte of birds from oxidative stress.     

Olfactory Ensheathing Cell-Conditioned Medium Protects Astrocytes Exposed to Hydrogen Peroxide Stress

The purpose of this study was to observe the effects of olfactory ensheathing cell conditioned medium (OECCM) on damaged astrocytes after exposure to H₂O₂ in vitro. OECCM was used to treat astrocytes after injury, which was induced by exposure to 500 μmol/L H₂O₂ for 20 min. The cell morphology was then observed under a light microscope, cell viability assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell ultrastructure observed with transmission electron microscopy (TEM), and apoptosis assessed by Annexin V staining followed by cytometry and Western blot. H₂O₂ induced severe damage to astrocytes as evidenced by decreased cell number, pathological changes in cell morphology, and significantly elevated cell apoptosis. Cells incubated with OECCM displayed significantly improved cell viability and decreased cell apoptotic rate. Under TEM, H₂O₂-treated cells showed partially broken plasma membranes, swollen rough endoplasmic reticula, visible vacuoles, and swollen or deformed mitochondria with ruptured cristae. Incubation with OECCM significantly ameliorated these pathological changes in astrocytes. These results suggest that OECCM may protect astrocytes from oxidative damage by promoting cell survival while reducing apoptosis of the damaged cells.     

Cobalt-induced oxidative stress causes growth inhibition associated with enhanced lipid peroxidation and activates antioxidant responses in Indian mustard (Brassica juncea L.) leaves

The effect of 100 μM cobalt (Co) on plant growth and on biochemical parameters indicative of oxidative stress was investigated in a hydroponic experiment. The responses of antioxidant enzymes and compounds of the ascorbate–glutathione (AsA–GSH) cycle were also assessed on the hyperaccumulating plant, Indian mustard (Brasssica juncea L.). The effect of excess Co was associated with a significant increase in the levels of proline, carbonylated protein, malondialdehyde, superoxide anion (O ₂ ^·? ), and hydrogen peroxide (H₂O₂), and resulted in the accumulation of Co. Co toxicity was associated with an increase in the volume of palisade and spongy cells, and a reduction in the number of chloroplasts per cell. Co-induced cell death was characterized by DNA fragmentation and a 36 kDa DNase activity. Despite decreased catalase activity, peroxidase, superoxide dismutase, and AsA–GSH cycle-related enzymes including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase exhibited remarkable induction under Co stress. Furthermore, the contents of reduced and oxidized forms of ascorbate and glutathione were significantly increased with Co supplementation. Co treatment led to the activation of 44 and 46 kDa mitogen-activated protein kinase (MAPK) and indicated the role of the MAPK cascade in transducing Co-mediated signals. The present results suggest that excess Co reduces seedling growth by inducing oxidative stress related to lipid peroxidation and overproduction of O ₂ ^·? and H₂O₂. The stimulated activities of antioxidative enzymes and induction of MAPKs did not reverse the oxidative stress caused by Co-induced reactive oxygen species generation in Indian mustard seedlings.     

Effect of Oxidative Stress on the Expression of Thin Filament-Associated Proteins in Gastric Smooth Muscle Cells

Thin filament-associated proteins such as calponin, caldesmon, and smoothelin are believed to regulate acto-myosin interaction and thus, muscle contraction. Oxidative stress has been found to affect the normal contractile behavior of smooth muscle and is involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the expression of smooth muscle contractile proteins. The aim of the current study is to investigate the effect of oxidative stress on the expression of thin filament-associated proteins in rat gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague–Dawley rats were used. Muscle cells were treated with hydrogen peroxide (H₂O₂) (500 μM) for 30 min or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) (1 mM) for 90 min to induce oxidative stress. Calponin, caldesmon, and smoothelin expressions were measured via specifically designed enzyme-linked immunosorbent assay. We found that exposure to exogenous H₂O₂ or incubation of dispersed gastric muscle cells with SIN-1 significantly increased the expression of calponin, caldesmon, and smoothelin proteins. In conclusion: oxidative stress increases the expression of thin filament-associated proteins in gastric smooth muscle, suggesting an important role in gastrointestinal motility disorders associated with oxidative stress.     

Adiponectin and colon cancer: evidence for inhibitory effects on viability and migration of human colorectal cell lines

Exogenous hydrogen peroxide (H₂O₂) induces oxidative stress and apoptosis in cancer cells. This study evaluated the antiapoptotic effects of pan-caspase and caspase-3, -8, or -9 inhibitors on H₂O₂-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). Treatment with 50–500 μM H₂O₂ inhibited the growth of Calu-6 and A549 cells at 24 h and induced apoptosis in these cells. All the tested caspase inhibitors significantly prevented cell death in H₂O₂-treated lung cancer cells. H₂O₂ increased intracellular ROS levels, including that of O ₂ ^·? , at 1 and 24 h. It also increased the activity of catalase but decreased the activity of SOD. In addition, H₂O₂ triggered GSH deletion in Calu-6 and A549 cells at 24 h. It reduced GSH levels in Calu-6 cells at 1 h but increased them at 24 h. Caspase inhibitors decreased O ₂ ^·? levels in H₂O₂-treated Calu-6 cells at 1 h and these inhibitors decreased ROS levels, including that of O ₂ ^·? , in H₂O₂-treated A549 cells at 24 h. Caspase inhibitors partially attenuated GSH depletion in H₂O₂-treated A549 cells and increased GSH levels in these cells at 24 h. However, the inhibitors did not affect GSH deletion and levels in Calu-6 cells at 24 h. In conclusion, H₂O₂ induced caspase-dependent apoptosis in Calu-6 and A549 cells, which was accompanied by increases in ROS and GSH depletion. The antiapoptotic effects of caspase inhibitors were somewhat related to the suppression of H₂O₂-induced oxidative stress and GSH depletion.     

Phenanthrolines Protect Astrocytes from Hemin Without Chelating Iron

Hemin, the degradation product of hemoglobin, contributes to the neurodegeneration that occurs in the weeks following a hemorrhagic stroke. The breakdown of hemin in cells releases redox-active iron that can facilitate the production of toxic hydroxyl radicals. The present study used 3-week old primary cultures of mouse astrocytes to compare the toxicity of 33 μM hemin in the presence of the iron chelator 1,10-phenanthroline or its non-chelating analogue, 4,7-phenanthroline. This concentration of hemin killed approximately 75 % of astrocytes within 24 h. Both isoforms of phenanthroline significantly decreased the toxicity of hemin, with the non-chelating analogue providing complete protection at concentrations of 33 μM and above. The decrease in toxicity was associated with less cellular accumulation of hemin. Approximately 90 % of the hemin accumulated was not degraded, irrespective of treatment condition. These observations indicate that chelatable iron is not the cause of hemin toxicity. Cell-free experiments demonstrated that hemin can inactivate a molar excess of hydrogen peroxide (H₂O₂), and that the rate of inactivation is halved in the presence of either isoform of phenanthroline. We conclude that phenanthrolines may protect astrocytes by limiting hemin uptake and by impairing the capacity of intact hemin to interact with endogenous H₂O₂.