Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity

Chronic ethanol consumption causes oxidative damage in the liver, and induction of cytochrome P450 2E1 (CYP2E1) is one pathway involved in oxidative stress produced by ethanol. The hepatic accumulation of iron and polyunsaturated fatty acids significantly contributes to ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment. The objective of this study was to analyze the effect of the green tea flavanol epigallocatechin-3-gallate (EGCG), which has been shown to prevent alcohol-induced liver damage, on CYP2E1-mediated toxicity in HepG2 cells overexpressing CYP2E1 (E47 cells). Treatment of E47 cells with arachidonic acid plus iron (AA + Fe) was previously reported to produce synergistic toxicity in E47 cells by a mechanism dependent on CYP2E1 activity and involving oxidative stress and lipid peroxidation. EGCG protected E47 cells against toxicity and loss of viability induced by AA+Fe; EGCG had no effect on CYP2E1 activity. Prevention of this toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species, a decrease in lipid peroxidation, and maintenance of intracellular glutathione in cells challenged by AA+Fe in the presence of EGCG. AA+Fe treatment caused a decline in the mitochondrial membrane potential, which was also blocked by EGCG. In conclusion, EGCG exerts a protective action on CYP2E1-dependent oxidative stress and toxicity that may contribute to preventing alcohol-induced liver injury, and may be useful in preventing toxicity by various hepatotoxins activated by CYP2E1 to reactive intermediates.     

Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs

Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective iron chelators in vivo and in vitro, and are of interest for the treatment of secondary iron overload. Because previous work has implicated the Fe(3+)-chelator complexes as a determinant of toxicity, the role of iron-based oxidative stress in the toxicity of PIH analogs was assessed. The Fe(3+) complexes of PIH analogs were reduced by K562 cells and the physiological reductant, ascorbate. Depletion of the antioxidant, glutathione, sensitized Jurkat T lymphocytes to the toxicity of PIH analogs and their Fe(3+) complexes, and toxicity of the chelators increased with oxygen tension. Fe(3+) complexes of pyridoxal benzoyl hydrazone (PBH) and salicyloyl isonicotinoyl hydrazone (SIH) caused lipid peroxidation and toxicity in K562 cells loaded with eicosapentenoic acid (EPA), a readily oxidized fatty acid, whereas Fe(PIH)(2) did not. The lipophilic antioxidant, vitamin E, completely prevented both the toxicity and lipid peroxidation caused by Fe(PBH)(2) in EPA-loaded cells, indicating a causal relationship between oxidative stress and toxicity. PBH also caused concomitant lipid peroxidation and toxicity in EPA-loaded cells, both of which were reversed as its concentration increased. In contrast, PIH was inactive, while SIH was equally toxic toward control and EPA-loaded cells, without causing lipid peroxidation, indicating a much smaller contribution of oxidative stress to the mechanism of toxicity of these analogs. In summary, PIH analogs and their Fe(3+) complexes are redox active in the intracellular environment. The contribution of oxidative stress to the overall mechanism of toxicity varies across the series.     

Activation of survival and apoptotic signaling pathways in lymphocytes exposed to palmitic acid

The toxicity of palmitic acid (PA) towards a human T-lymphocyte cell line (Jurkat) has been previously investigated but the mechanism(s) of PA action were unknown. In the current study, Jurkat cells were treated with sub-lethal concentrations of PA (50-150μM) and the activity of various signaling proteins was investigated. PA-induced apoptosis and mitochondrial dysfunction in a dose-dependent manner as evaluated by DNA fragmentation assay and depolarization of the mitochondrial membrane, respectively. PA treatment provoked release of cytochrome c from the inner mitochondrial membrane to the cytosol, activated members of the MAPK protein family JNK, p38, ERK, activated caspases 3/9, and increased oxidative/nitrosative stress. Exposure of cells to PA for 12 h increased insulin receptor (IR) and GLUT-4 levels in the plasma membrane. Insulin treatment (10 mU/ml/30 min) increased the phosphorylation of the IR β-subunit and Akt. A correlation was found between DNA fragmentation and expression levels of both IR and GLUT-4. Similar results were obtained for PA-treated lymphocytes from healthy human donors and from mesenteric lymph nodes of 48-h starved rats. PA stimulated glucose uptake by Jurkat cells (in the absence of insulin), stimulated accumulation of neutral lipids (triglyceride), and other lipid classes (phospholipids and cholesterol ester) but reduced glucose oxidation. Our results suggest that parameters of insulin signaling and non-oxidative glucose metabolism are stimulated as part of a coordinated response to prompt survival in lymphocytes exposed to PA but at higher concentrations, apoptosis prevails. These findings may explain aspects of lymphocyte dysfunction associated with diabetes.     

Restored vulnerability of cultured endothelial cells to high glucose by iron replenishment.

High glucose (HG) concentrations are toxic to various cells in vivo, but cells become insensitive to HG toxicity when they are subcultured serially in vitro. Oxidative stress is involved in HG toxicity, and metal ions, especially iron, mediate some oxidative stress. To investigate mechanisms involved in the insensitiveness of cultured cells to HG toxicity, we focused on the level of intracellular iron. Freshly prepared human umbilical vein endothelial cells contained a substantial amount of iron, whereas its level decreased rapidly during the course of culture (to less than 10%). The iron content was restored by incubation of the cells with Fe(III)/8-hydroxyquinoline, and the iron-supplemented cells were more susceptible to both oxidant- and HG-induced injury. Under the HG conditions, the iron-loaded cells were subjected to higher levels of oxidative stress. The enhanced HG toxicity by iron was attenuated by the treatment with several antioxidants including catalase, ascorbic acid, and pyruvate. These data suggested that the insensitiveness of subcultured cells to HG toxicity is, at least in part, due to rapid and dramatic loss of intracellular iron. Supplementation with iron is useful to restore the vulnerability of cultured cells to HG that is normally observed in in vivo situations.     

DNA base modifications and membrane damage in cultured mammalian cells treated with iron ions

We investigated DNA base damage in mammalian cells exposed to exogenous iron ions in culture. Murine hybridoma cells were treated with Fe(II) ions at concentrations of 10 μM, 100 μM, and 1 mM. Chromatin was isolated from treated and control cells and analyzed by gas chromatography/mass spectrometry for DNA base damage. Ten modified DNA bases were identified in both Fe(II)-treated and control cells. The quantification of modified bases was achieved by isotope-dilution mass spectrometry. In Fe(II)-treated cells, the amounts of modified bases were increased significantly above the background levels found in control cells. Dimethyl sulfoxide at concentrations up to 1 M in the culture medium did not significantly inhibit the formation of modified DNA bases. A mathematical simulation used to evaluate the plausibility of DNA damage upon Fe(II) treatment predicted a dose-dependent response, which agreed with the experimental results. In addition, Fe(II) treatment of cells increased the cell membrane permeability and caused production of lipid peroxides. The nature of DNA base lesions suggests the involvement of the hydroxyl radical in their formation. The failure of dimethyl sulfoxide to inhibit their formation indicates a site-specific mechanism for DNA damage with involvement of DNA-bound metal ions. Fe(II) treatment of cells may increase the intracellular iron ion concentration and/or cause oxidative stress releasing metal ions from their storage sites with subsequent binding to DNA. Identified DNA base lesions may be promutagenic and play a role in pathologic processes associated with iron ions.     

The role of oxidative stress in the toxicity of pyridoxal isonicotinoyl hydrazone (PIH) analogues

Pyridoxal isonicotinoyl hydrazone (PIH) analogues are effective iron chelators in vivo and in vitro, and may be of value for the treatment of secondary iron overload. The sensitivity of Jurkat cells to Fe-chelator complexes was enhanced several-fold by the depletion of the antioxidant glutathione, indicating the role of oxidative stress in their toxicity. K562 cells loaded with eicosapentaenoic acid, a fatty acid particularly susceptible to oxidation, were also more sensitive to the toxic effects of the Fe complexes, and toxicity was proportional to lipid peroxidation. Thus Fe-chelator complexes cause oxidative stress, which may be a major component of their toxicity. As was the case for their Fe complexes, the toxicity of PIH analogues was enhanced by glutathione depletion of Jurkat cells and eicosapentaenoic acid-loading of K562 cells. Thus the toxicity of the chelators themselves is also enhanced by compromised cellular redox status. In addition, the toxicity of the chelators was diminished by culturing Jurkat cells under hypoxic conditions, which may limit the production of the reactive oxygen species that initiate oxidative stress. A significant part of the toxicity of the chelators may be due to intracellular formation of Fe-chelator complexes, which oxidatively destroy the cell.     

Role of p38 MAPK in CYP2E1-dependent arachidonic acid toxicity

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 (CYP2E1) and in HepG2 E47 cells, which express CYP2E1. The possible role of mitogen-activated protein kinase (MAPK) members in this process was evaluated. SB203580, a p38 MAPK inhibitor, and PD98059, an ERK inhibitor, but not wortmannin a phosphatidylinositol 3-kinase (PI3K) inhibitor, prevented AA toxicity in pyrazole hepatocytes and E47 cells. SB203580 prevented the enhancement of AA toxicity by salicylate. SB203580 neither lowered the levels of CYP2E1 nor affected CYP2E1-dependent oxidative stress. The decrease in mitochondrial membrane potential produced by AA was prevented by SB203580. Treating CYP2E1-induced cells with AA activated p38 MAPK but not ERK or AKT. This activation was blocked by antioxidants. AA increased the translocation of NF-kappaB to the nucleus. Salicylate blocked this translocation, which may contribute to the enhancement of AA toxicity by salicylate. SB203580 restored AA-induced NF-kappaB translocation, which may contribute to protection against toxicity. In conclusion, AA toxicity was related to lipid peroxidation and oxidative stress, and to the activation of p38 MAPK, as a consequence of CYP2E1-dependent production of reactive oxygen species. Activation of p38 MAPK by AA coupled to AA-induced oxidative stress may synergize to cause cell toxicity by affecting mitochondrial membrane potential and by modulation of NF-kappaB activation.     

Wood smoke extract induces oxidative stress-mediated caspase-independent apoptosis in human lung endothelial cells: role of AIF and EndoG

Although a link between toxic smoke and oxidant lung vascular injury has been indicated, the cellular mechanisms of smoke-induced injury to lung endothelial cells are unknown. We investigated oxidative stress and apoptosis induced by wood smoke extract (SE) in human pulmonary artery endothelial cells (HPAECs) and delineated their relationship. We found that SE increased intracellular reactive oxygen species (ROS), depleted intracellular glutathione, and upregulated Cu/Zn superoxide dismutase and heme oxygenase-1 (2 antioxidant enzymes), but it failed to alter the expression of catalase and glutathione peroxidase. In addition, SE promoted apoptosis as indicated by the external exposure of membrane phosphatidylserine, the loss of mitochondrial membrane potential, an increase in the level of Bax (a proapoptotic protein), and enhanced DNA fragmentation. This apoptosis was associated with mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) (2 apoptogenic proteins) but was independent of caspase cascade activation. Whereas N-acetylcysteine (an antioxidant) effectively reversed the SE-induced increase in ROS and depletion of glutathione, it also suppressed SE-induced nuclear translocation of either AIF or EndoG and prevented the enhanced DNA fragmentation that would have resulted from this. We conclude that 1) although SE upregulates Cu/Zn superoxide dismutase and heme oxygenase-1, it nevertheless increases intracellular oxidative stress in HPAECs, and 2) SE promotes oxidative stress-mediated caspase-independent HPAEC apoptosis that involves mitochondrial-to-nuclear translocation of AIF and EndoG. Thus modulations of the expression of antioxidant enzymes and the caspase-independent apoptotic pathway are possible target choices for potential therapeutic regimes to treat smoke-induced lung injury.     

Hepatic response to aluminum toxicity: dyslipidemia and liver diseases

Aluminum (Al) is a metal toxin that has been implicated in the etiology of a number of diseases including Alzheimer's, Parkinson's, dialysis encephalopathy, and osteomalacia. Al has been shown to exert its effects by disrupting lipid membrane fluidity, perturbing iron (Fe), magnesium, and calcium homeostasis, and causing oxidative stress. However, the exact molecular targets of aluminum's toxicity have remained elusive. In the present review, we describe how the use of a systems biology approach in cultured hepatoblastoma cells (HepG2) allowed the identification of the molecular targets of Al toxicity. Mitochondrial metabolism is the main site of the toxicological action of Al. Fe-dependent and redox sensitive enzymes in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) are dramatically decreased by Al exposure. In an effort to compensate for diminished mitochondrial function, Al-treated cells stabilize hypoxia inducible factor-1α (HIF-1α) to increase ATP production by glycolysis. Additionally, Al toxicity leads to an increase in intracellular lipid accumulation due to enhanced lipogenesis and a decrease in the β-oxidation of fatty acids. Central to these effects is the alteration of α-ketoglutarate (KG) homeostasis. In Al-exposed cells, KG is preferentially used to quench ROS leading to succinate accumulation and HIF-1α stabilization. Moreover, the channeling of KG to combat oxidative stress leads to a reduction of l-carnitine biosynthesis and a concomitant decrease in fatty acid oxidation. The fluidity and interaction of these metabolic modules and the implications of these findings in liver-related disorders are discussed herein.     

Role of phospholipase A2 activation and calcium in CYP2E1-dependent toxicity in HepG2 cells

Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.     

Downregulation of sirtuin 3 by palmitic acid increases the oxidative stress,impairment of mitochondrial function,and apoptosis in liver cells

Elevated levels of saturated fatty acids show a strong cytotoxic effect in liver cells. Sirtuin 3 (SIRT3), a mitochondrially localized member of NAD⁺‐dependent deacetylase has been shown to protect hepatocytes against the oxidative stress. The role of SIRT3 on the cytotoxicity caused by fatty acids in liver cells is not fully understood. The aim of this study was to evaluate the expression level of SIRT3, oxidative stress, and mitochondrial impairments in human hepatoma HepG2 cells exposed to palmitic acid (PA). Our results showed that PA treatment caused the deposition of lipid droplets and resulted in an increased expression of tumor necrosis factor‐α in a dose‐dependent manner. Excessive accumulation of PA induces the reactive oxygen species formation and apoptosis while dissipating the mitochondrial transmembrane potential. The level of SIRT3 expression in both nuclear and mitochondrial fractions in HepG2 cells was decreased with the increase in PA concentrations. However, in the cytosolic fraction, the SIRT3 was undetectable. In conclusion, our results showed that PA caused an increase in inflammation and oxidative stress in HepG2 cells. The exposure of PA also resulted in the decline in transmembrane potential and an increase in apoptosis. The underexpression of nuclear and mitochondrial SIRT3 by PA suggests that the PA target the process that regulates the stress‐related gene expression and mitochondrial functions.     

Inhibition of deoxycholate-induced apoptosis in iron-depleted HCT-116 cells

The bile acid, deoxycholate (DOC), can induce apoptosis in cells containing adequate amounts of all key nutrients, but it is unknown whether DOC-induced apoptosis can occur in cells lacking a single key nutrient. The aim of this study was to determine if DOC is able to induce apoptosis in HCT-116 colon epithelial cells depleted of iron. The cells were made iron-deficient by pre-treating them with the iron chelator, deferoxamine (DFO), before subsequent exposure to DOC. Mitochondrial dysfunction was detected in control cells exposed to DOC, but not in iron-depleted cells exposed to DOC. Moreover, characteristic features of apoptosis, namely, membrane blebbing, formation of apoptotic bodies, cytochrome c release into cytosol, generation of the activated form of caspase-3, chromatin condensation and fragmentation, and also plasma membrane phospholipid translocation, were all induced by DOC in control cells but not in iron-depleted cells. Treating DFO-pretreated cells with ferrous sulfate to replenish cellular iron restored the ability of DOC to induce apoptosis. In relating these findings to oxidative stress, it was found that DOC also induced the formation of reactive oxygen species and caused DNA damage in control cells, but not in iron-depleted cells. Collectively, the results suggest that in order for HCT-116 cells to undergo apoptosis when exposed to DOC, adequate amounts of intracellular iron must be present.     

Role of the mitochondrial permeability transition and cytochrome C release in hydrogen peroxide-induced apoptosis

We investigated the role of the mitochondrial inner membrane permeability transition and subsequent release of cytochrome c into the cytosol during oxidative stress-evoked apoptosis. Sublethal oxidative stress was applied by treating L929 cells with 0.5 mM H2O2 for 90 min. Then the cellular localization of cytochrome c was examined by immunofluorescent staining and Western blotting. H2O2 treatment caused the permeability transition and pore formation, resulting in membrane depolarization and translocation of cytochrome c from the mitochondria into the cytosol. Pretreatment with cyclosporin A and aristolochic acid (to inhibit pore formation) significantly attenuated a reduction of the mitochondrial membrane potential, as well as signs of apoptosis such as DNA fragmentation, increased plasma membrane permeability, and chromatin condensation. Therefore, exposure to H2O2 caused the opening of permeability transition pores in the inner mitochondrial membrane. An essential role of cytosolic cytochrome c in the execution of apoptosis was demonstrated by its direct microinjection into the cytosol, thus bypassing the need for cytochrome c release from the mitochondrial intermembrane space. Microinjection of cytochrome c caused caspase-dependent apoptosis.     

Early ethanolamine phospholipid translocation marks stress-induced apoptotic cell death in oligodendroglial cells

The consequences of H(2)O(2)/Fe(2+)-induced oxidative stress on translocation of ethanolamine phosphoglyceride (EPG) and serine phosphoglyceride (SPG) were studied in an oligodendroglia-like cell line (OLN 93) following 3 days of supplementation with 0.1 mM docosahexaenoic acid (DHA) and a series of polar head group precursors, including N-monomethyl- and N,N-dimethylethanolamine at millimolar concentrations. Added DHA was predominantly esterified in EPG species and those cells enriched in DHA showed enhanced sensitivity to oxidative stress and eventually died by apoptosis. Co-supplements with ethanolamine and DHA resulted in a rapid, but transient, EPG translocation with a maximum at 30 min following stress, as characterized by a trinitrobenzenesulfonic acid reagent. There was no significant translocation of SPG as evidenced by annexin V binding. Unlike SPG, which is usually irreversibly translocated to subserve as a tag for phagocytosis, EPG acted as a signaling molecule with biphasic kinetic characteristics. N-Monomethyl- and N,N-dimethylethanolamine supplements reduced EPG synthesis, prevented its externalization and rescued cells from apoptotic death. Following stress, the fatty acid profile of the externalized EPG showed marked losses in polyunsaturated fatty acids and aldehydes compared with the remaining intracellular EPG. Prevention of EPG species selective translocation to the outer membrane leaflet by altering phospholipid asymmetry may be important in the mechanism of rescue from cell death.     

MDL1 is a high copy suppressor of ATM1: evidence for a role in resistance to oxidative stress

The yeast ATM1 gene is essential for normal cellular iron homeostasis. Deletion of ATM1 results in mitochondrial iron accumulation and increased sensitivity to oxidative stress and transition metal toxicity. Atm1p is an ATP-binding cassette (ABC) transporter localized to the mitochondrial inner membrane. The specific function of Atm1p has not been determined, though roles in both mitochondrial iron export and cytosolic Fe-S cluster assembly have been proposed. We undertook a screen for yeast genes capable of suppressing the abnormalities of cellular iron metabolism demonstrated by Deltaatm1 cells. One of the genes we identified was MDL1, which like ATM1, encodes a mitochondrial inner membrane ABC transporter. Mdl1p has previously been shown to function in the export of peptides from the mitochondrial matrix. We demonstrate that over-expression of MDL1 in Deltaatm1 cells results in a reduction of mitochondrial iron content, and decreased sensitivity to H(2)O(2) and transition metal toxicity. Additionally, in studies of the effect of over-expression and deletion of MDL1, we have identified a novel role for Mdl1p in the regulation of cellular resistance to oxidative stress.     

Supplementation of endothelial cells with mitochondria-targeted antioxidants inhibit peroxide-induced mitochondrial iron uptake, oxidative damage, and apoptosis

The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H(2)O(2) and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 microM) and MitoVit-E (1 microM) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H(2)O(2)- and lipid peroxide-induced 2',7'-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis.     

β-Amyloid peptide increases levels of iron content and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease

Recent studies indicate that the deposition of β-amyloid peptide (Aβ) is related to the pathogenesis of Alzheimer disease (AD); however, the underlying mechanism is still not clear. The abnormal interactions of Aβ with metal ions such as iron are implicated in the process of Aβ deposition and oxidative stress in AD brains. In this study, we observed that Aβ increased the levels of iron content and oxidative stress in SH-SY5Y cells overexpressing the Swedish mutant form of human β-amyloid precursor protein (APPsw) and in Caenorhabditis elegans Aβ-expressing strain CL2006. Intracellular iron and calcium levels and reactive oxygen species and nitric oxide generation significantly increased in APPsw cells compared to control cells. The activity of superoxide dismutase and the antioxidant levels of APPsw cells were significantly lower than those of control cells. Moreover, iron treatment decreased cell viability and mitochondrial membrane potential and aggravated oxidative stress damage as well as the release of Aβ1-40 from the APPsw cells. The iron homeostasis disruption in APPsw cells is very probably associated with elevated expression of the iron transporter divalent metal transporter 1, but not transferrin receptor. Furthermore, the C. elegans with Aβ-expression had increased iron accumulation. In aggregate, these results demonstrate that Aβ accumulation in neuronal cells correlated with neuronal iron homeostasis disruption and probably contributed to the pathogenesis of AD.