Fatty acid-mediated intracellular iron translocation: a synergistic mechanism of oxidative injury

Fatty acid has been reported to be associated with cardiovascular diseases and cancer, but the possible mechanism remains unclear. Here, we reported a novel mechanism for the permissive role of fatty acid on iron intracellular translocation and subsequent oxidative injury. In vitro study from endothelial cells showed that iron alone had little effect, whereas in combination with PA (palmitic acid), iron-mediated toxicity was markedly potentiated, as reflected in mitochondrial dysfunction, cell death, apoptosis, and DNA mutation. We also showed that PA not only facilitated iron translocation into cells through a transferrin-receptor (TfR)-independent mechanism, but also translocated iron into mitochondria; the subsequent intracellular iron overload resulted in reactive oxygen species (ROS) overgeneration and lipid oxidation. Further investigation revealed that PA-facilitated iron translocation is due to Fe/PA-mediated extracellular oxidative stress and the subsequent membrane damage with increased membrane permeability. Fe/PA-mediated toxic effects were reduced in rho0 cells lacking mitochondrial DNA or by antioxidant enzyme SOD, especially mitochondrially localized MnSOD, suggesting a permissive role of PA for iron deposition on the vascular wall and its subsequent toxicity via mitochondrial oxidative stress. This observation was confirmed in vivo in mice, wherein higher vascular iron deposition and accompanying superoxide release were observed in the presence of a high-fat diet with iron administration.     

Effect of common sage plant treatment with polyamines on the contents of proline and free and conjugated polyamines under oxidative stress

Common sage (Salvia officinalis L.) plants grown in water culture to the stage of 4–5 true leaves were treated with paraquat (PQ) ( 1 ml of the solution containing 0.1 μM PQ in 0.05% Tween 80), putrescine (Put), spermidine (Spd), or Spermine (Spm) (all polyamines (PA) at the concentration of 0.5 mM in 0.05% Tween 80) or PA plus PQ for 6, 12, 24, 36, or 48 h. Under normal conditions, treatment with individual PA did not induce any changes in the content of free proline. Under oxidative stress induced by PQ, oxidation of proline and free PA by ROS resulted in their spending and demanded restoration of free PA due to hydrolysis of their insoluble conjugates. As distinct from treatment with PQ only, its combination with PA in the light was accompanied by proline accumulation. Treatment with Put plus PQ induced accumulation of intracellular Put and its soluble and insoluble conjugates. Treatments with Spd and Spm in combination with PQ resulted similarly in the increase in the levels of their intracellular soluble and insoluble conjugates. In treatments with these high-molecular PA, polyamine oxidase was activated and diaminopropan formation was observed. It seems likely that, for restoration of high-molecular PA homeostasis, their degradation by polyamine oxidase or production of insoluble conjugates is induced. Thus, the amount of free and conjugated particular PA is under strict control and is maintained at a definite level. A decrease in the content of a particular free PA induces primarily a decrease in the content of its soluble and then insoluble conjugates. The data obtained demonstrate the effects of exogenous PA on the content of intracellular proline under oxidative stress but do not allow a conclusion about direct regulation of its content by PA.     

Growth-stimulating effect of transferrin on a hybridoma cell line: Relation to transferrin iron-transporting function

The relation of the growth-stimulating capacity of transferrin to its iron-transporting function was investigated in mouse hybridoma PLV-01 cells cultivated in a chemically defined medium. The cells were precultivated in protein-free medium supplemented either with ferric citrate (cells with a high intracellular iron level) or with iron-saturated transferrin (cells with a low intracellular iron level). Iron uptake was monitored after the application of 59Fe-labeled ferric citrate or pig transferrin. Cultivation of the cells at the optimum growth-stimulating concentration (500 microM) of ferric citrate resulted in an intracellular iron level about 100-fold higher than that of cells cultivated at the optimum transferrin concentration (5 micrograms/ml). Replacement of pig transferrin with bovine transferrin resulted in similar intracellular iron levels, but the growth-stimulating effect of bovine transferrin was more than one order of magnitude lower. Cells with a high intracellular iron level grew equally well when cultivated with iron-saturated transferrin or with apotransferrin + deferoxamine (2 micrograms/ml). On the other hand, cells with a low intracellular iron level required iron-saturated transferrin for further growth and apotransferrin + deferoxamine was ineffective. The results suggest that transferrin can act as a cell growth factor only in the iron-saturated form. However, several findings of this work indicate that supplying cells with iron cannot be accepted as the full explanation of the transferrin growth-stimulating effect.     

Free iron in bacteria

Free iron content has been estimated in autotrophic and heterotrophic bacteria. It constituted 40-50 micrograms/g dry weight as compared to 15 micrograms/g dry weight in animal cells. A method for estimation of free iron has been proposed. It is based on formation of paramagnetic dinitrosyl iron complexes by free iron and protein thiol groups or low molecular weight thiol ligands. The reasons for high iron content in bacteria have been discussed.     

Cytological changes and alterations in polyamine contents induced by cadmium in tobacco BY-2 cells.

Changes in cell viability, proliferation, cell and nuclear morphology including nuclear and DNA fragmentation induced by 0.05 and 1 mM CdSO4 (Cd2+) in tobacco BY-2 cell line (Nicotiana tabacum L.) were studied in the course of 7 days. Simultaneously changes in endogenous contents of both free and conjugated forms of polyamines (PAs) were investigated for 3 days. The application of 0.05 mM Cd2+ evoked decline of cell viability to approximately 60% during the first 24 h of treatment. Later on degradation of cytoplasmic strands, formation of the stress granules and vesicles, modifications in size and shape of the nuclei, including their fragmentation, were observed in the surviving cells. Their proliferation was blocked and cells elongated. Beginning the first day of treatment TUNEL-positive nuclei were detected in cells cultivated in medium containing 0.05 mM Cd2+. Treatment with highly toxic 1 mM Cd2+ induced fast decrease of cell viability (no viable cells remained after 6-h treatment) and cell death occurred before DNA cleavage might be initiated. The exposure of tobacco BY-2 cells to 0.05 mM Cd2+ resulted in a marked accumulation of total PAs (represented by the sum of free PAs and their perchloric acid (PCA)-soluble and PCA-insoluble conjugates) during 3-day treatment. The increase in total PA contents was primarily caused by the increase in putrescine (Put) concentration. The accumulation of free spermidine (Spd) and spermine (Spm) at 12 and 24 h in 0.05 mM Cd2+ treated BY-2 cells and high contents of Spd and especially Spm determined in dead cells after I mM Cd2+ application was observed. The participation of PA conjugation with hydroxycinnamic acids and PA oxidative deamination in maintaining of free PA levels in BY-2 cells under Cd2+-induced oxidative stress is discussed.     

Uptake and handling of iron from transferrin, lactoferrin and immune complexes by a macrophage cell line.

The murine macrophage-like cell line P388D1 has been used as a model to investigate whether iron acquired simultaneously from different sources (transferrin, lactoferrin, and ovotransferrin-anti-ovotransferrin immune complexes) is handled in the same way. P388D1 cells bound both lactoferrin and transferrin, but over a 6 h incubation period only the latter actually donated iron to the cells. When the cells were incubated with [55Fe]transferrin and [59Fe]ovotransferrin-anti-ovotransferrin immune complexes iron was acquired from both sources. However, there was a difference in the intracellular distribution of the two isotopes, proportionally more 55Fe entering haem compounds and less entering ferritin. When the cells were precultured in a low-iron serum-free medium almost no transferrin-iron was incorporated into ferritin, whereas the proportion of immune complex-derived iron incorporated into ferritin was unchanged. Lactoferrin enhanced the rate of cellular proliferation, as measured by [3H]thymidine incorporation, despite its inability to donate iron to the cells, suggesting a stimulatory effect independent of iron donation. In contrast immune complexes inhibited cell proliferation. These findings indicate that iron acquired from transferrin and iron acquired by scavenging mechanisms are handled differently, and suggest that more than one intracellular iron transit pool may exist.     

Effect of aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells.

Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin.     

The flavonoid quercetin induces hypoxia-inducible factor-1α (HIF-1α) and inhibits cell proliferation by depleting intracellular iron

Quercetin, a flavonoid with anti-oxidant, metal chelating, kinase modulating and anti-proliferative properties, can induce hypoxia-inducible factor-1α (HIF-1α) in normoxia, but its mechanism of action has not been determined. In this study we characterized the induction of HIF-1α and the inhibition of cell proliferation caused by quercetin in HeLa and ASM (airway smooth muscle) cells and examined the effect of iron on these processes. Furthermore, we investigated the relevance of the intracellular levels of quercetin to HIF-1α expression and cell proliferation. Our data demonstrate that quercetin depletes intracellular calcein–chelatable iron and that supplying additional iron from extracellular or intracellular pools abrogates the induction of HIF-1α by quercetin. Moreover, addition of iron reverses the quercetin-induced inhibition of DNA synthesis, cell proliferation and cycle progression, but to different extents, depending on cell type. We propose that quercetin stabilises HIF-1α and inhibits cell proliferation predominantly by decreasing the concentration of intracellular iron through chelation.     

The flavonoid quercetin induces hypoxia-inducible factor-1alpha (HIF-1alpha) and inhibits cell proliferation by depleting intracellular iron

Quercetin, a flavonoid with anti-oxidant, metal chelating, kinase modulating and anti-proliferative properties, can induce hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia, but its mechanism of action has not been determined. In this study we characterized the induction of HIF-1alpha and the inhibition of cell proliferation caused by quercetin in HeLa and ASM (airway smooth muscle) cells and examined the effect of iron on these processes. Furthermore, we investigated the relevance of the intracellular levels of quercetin to HIF-1alpha expression and cell proliferation. Our data demonstrate that quercetin depletes intracellular calcein-chelatable iron and that supplying additional iron from extracellular or intracellular pools abrogates the induction of HIF-1alpha by quercetin. Moreover, addition of iron reverses the quercetin-induced inhibition of DNA synthesis, cell proliferation and cycle progression, but to different extents, depending on cell type. We propose that quercetin stabilises HIF-1alpha and inhibits cell proliferation predominantly by decreasing the concentration of intracellular iron through chelation.     

Role of HIF-1 in iron regulation: potential therapeutic strategy for neurodegenerative disorders

A disruption in optimal iron levels within different brain regions has been demonstrated in several neurodegenerative disorders. Although iron is an essential element that is required for many processes in the human body, an excess can lead to the generation of free radicals that can damage cells. Iron levels are therefore stringently regulated within cells by a host of regulatory proteins that keep iron levels in check. The iron regulatory proteins (IRPs) have the ability to sense and control the level of intracellular iron by binding to iron responsive elements (IREs) of several genes encoding key proteins such as the transferrin receptor (TfR) and ferritin. Concurrently, the hypoxia-inducible factor (HIF) has also been shown in previous studies to regulate intracellular iron by binding to HIF-responsive elements (HREs) that are located within the genes of iron-related proteins such as TfR and heme oxygenase-1 (HO-1). This review will focus on the interactions between the IRP/IRE and HIF/HRE systems and how cells utilize these intricate networks to regulate intracellular iron levels. Additionally, since iron chelation has been suggested to be a therapeutic treatment for disorders such as Parkinson's and Alzheimer's disease, understanding the exact mechanisms by which iron acts to cause disease and how the brain would be impacted by iron chelation could potentially give us novel insights into new therapies directed towards preventing or slowing neuronal cell loss associated with these disorders.     

Role of phospholipase D signaling in ethanol-induced inhibition of carbachol-stimulated DNA synthesis of 1321N1 astrocytoma cells

Inhibition of astrocyte proliferation has been suggested to be an important event in the developmental neurotoxicity associated with ethanol. We have previously shown that the acetylcholine analog carbachol induces astroglial cell proliferation through activation of muscarinic M3 receptors, and that ethanol strongly inhibits this effect by inhibiting activation of protein kinase C (PKC) zeta and its down-stream effector 70-kDa ribosomal S6 kinase (p70S6K). In this study, we investigated whether inhibition by ethanol of this signal transduction pathway in 1321N1 human astrocytoma cells may be due, at least in part, to inhibition of the formation of the PKC zeta activator phosphatidic acid (PA), which is formed by hydrolysis of phosphatidylcholine by phospholipase D (PLD). 1-Butanol, which is a substrate for PLD and inhibits PA formation, inhibited carbachol-induced cell proliferation and the underlying intracellular signaling, whereas its analog tert-butanol, which is a poor substrate for PLD, was much less effective. In addition, exogenous PAs were able to increase DNA synthesis and to activate PKC zeta and p70S6K. Furthermore, in carbachol-stimulated cells, ethanol increased the formation of phosphatidylethanol and inhibited the formation of PA. Taken together, these results indicate that PLD activation plays an important role in carbachol-induced astroglial cell proliferation by generating the second messenger PA, which activates PKC zeta. Moreover, the effect of ethanol on carbachol-induced proliferation appears to be mediated, at least in part, by its ability to interact with PLD leading to a decreased synthesis of PA.     

A nuclear microscopic study of elemental changes in the rat hippocampus after kainate-induced neuronal injury

The effect of intracerebroventricular kainate injection on the elemental composition of the hippocampus was studied in adult Wistar rats, at 1 day and 1, 2, 3, and 4 weeks postinjection, using a nuclear microscope. An increase in calcium concentration was observed on the injected side from 1 day postinjection. The increase peaked at 3 weeks postinjection, reaching a concentration of 18 times normal. Large numbers of glial cells but no neurons were observed in the lesioned CA fields at this time, suggesting that an increased calcium level was present in glial cells. This was confirmed by high-resolution elemental maps of the lesioned areas, which showed very high intracellular calcium concentrations in almost all glial cells. It is possible that the high intracellular calcium level could activate calcium-dependent enzymes, including calpain II and cytosolic phospholipase A2, shown to be expressed in reactive glial cells after kainate injections. In addition to calcium, an increase in iron content was also observed at the periphery of the glial scar at 4 weeks postinjection. Because free iron could catalyze the formation of free radicals, the late increase in iron content may be related to oxygen radical formation during neurodegeneration.     

Role of iron (II)-2-oxoglutarate-dependent dioxygenases in the generation of hypoxia-induced phosphatidic acid through HIF-1/2 and von Hippel-Lindau-independent mechanisms

Hypoxia-inducible factors (HIF-1/HIF-2) govern the expression of critical genes for cellular adaptation to low oxygen tensions. We have previously reported that the intracellular level of phosphatidic acid (PA) rises in response to hypoxia (1% O(2)). In this report, we have explored whether components of the canonical HIF/von Hippel-Lindau (VHL) pathway are involved in the induction of PA. We found that hypoxia induces PA in a cell line constitutively expressing a stable version of HIF-1alpha. PA induction was also found in HIF-1alpha- and 2alpha-negative CHO Ka13 cells, as well as in HIF-beta-negative HepaC4 cells. These data indicate that HIF activity is neither sufficient nor necessary for oxygen-dependent PA accumulation. PA generation was also detected in cells deficient for the tumor suppressor VHL, indicating that the presence of VHL was not required for the induction of PA. Here we show that PA accumulation also occurs at moderate hypoxia (5% O(2)), although to a lesser extent to that seen at 1% O(2), revealing that PA is induced at the same hypoxia range required to activate HIF-1. Prolyl hydroxylases (PHD) and asparaginyl hydroxylase (FIH) belong to the iron (II) and 2-oxoglutarate-dependent dioxygenase family and have been proposed as oxygen sensors involved in the regulation of HIFs. Chemical inhibition of these activities by treatment with iron chelators or 2-oxoglutarate analogs also results in a marked PA accumulation similar to that observed in hypoxia. Together these data show that PA accumulation in response to hypoxia is both HIF-1/2- and VHL-independent and indicate a role of iron (II)-2-oxoglutarate-dependent dioxygenases in the oxygen-sensing mechanisms involved in hypoxia-driven phospholipid regulation.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

Influence of autocrine factor deficit in culture on survival and energy metabolism of CTLL-2 cells under oxidative stress

A lot of data has shown recently that survival of mammalian cells is under a control of growth factors and autocrine survival factors (AF). We studied the influence of AF deficit on survival, intracellular ATP content, and transmembrane potential of mitochondria of IL-2-dependent CTLL-2 cells under oxidative stress. CTLL-2 cells cultivated under deficit of AF have been shown to be more susceptible to oxidative injury in comparison with the cells cultivated without deficit of AF (control); they died at smaller concentrations of H2O2 than control cells did. The ATP content in CTLL-2 cells was decreased under AF deficit conditions even without any stress and treatment of the cells by hydrogen peroxide resulted in additional large decrease of it. ATP depression was accompanied by disruption of cell membrane (blebbing) and drop of mitochondrial potential. Cell death under oxidative stress in the presence of AF deficit has been shown to proceed by both apoptosis and necrosis.     

Cytoprotective Effect of Short-Term Pretreatment with Proanthocyanidin on Human Gingival Fibroblasts Exposed to Harsh Environmental Conditions

Our previous study showed that exposing mouse fibroblasts to proanthocyanidin (PA) for only 1 min accelerated cell proliferation in a concentration-dependent manner. In this study, exposing human gingival fibroblasts (HGFs) to PA for 1 min similarly accelerated the proliferative response of the cells. Besides the accelerated proliferative response, PA showed a cytoprotective effect on HGFs exposed to harsh environmental conditions; short-term exposure of HGFs in the mitotic phase to pure water or physiological saline resulted in a lower recovery of viable cells. Pretreatment and concomitant treatment with PA improved the low recovery of cells exposed to pure water or physiological saline. In addition, HGFs exposed to PA for 1 min proliferated well even after being cultured in serum-free medium. In 100% confluent HGFs, being cultured in serum-free medium resulted in a high intracellular reactive oxygen species (ROS) level, but pretreatment with PA prevented the cells from increasing intracellular ROS. Thus, the results suggest that a short-term PA treatment exerts cytoprotective effects on HGFs exposed to harsh environmental conditions by improving the intracellular oxidative stress response.     

Formation of Palmitic Acid/Ca2+ Complexes in the Mitochondrial Membrane: A Possible Role in the Cyclosporin-Insensitive Permeability Transition

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.     

Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.     

Over-expressed human divalent metal transporter 1 is involved in iron accumulation in MES23.5 cells

Elevated iron accumulation has been reported in brain regions in some neurodegenerative disorders. However, the mechanism for this is largely unknown. Divalent metal transporter 1 (DMT1) is an important divalent cation transporter. The aim of the present study is to construct recombinant adenovirus encoding human DMT1 with iron responsive element (DMT1+IRE) and infect MES23.5 dopaminergic cells in order to investigate the relationship between increased DMT1+IRE expression and iron accumulation. The human DMT1 gene was obtained by RT-PCR from tissues of human duodenum. AdDMT1+IRE was successfully constructed and identified by PCR, restriction endonuclease analyses and DNA sequencing, respectively. It was able to efficiently infect MES23.5 cells, which was confirmed by RT-PCR and Western blots. When incubated with 100 microM ferrous iron for 6h, the intracellular iron levels dramatically increased in AdDMT1+IRE infected MES23.5 cells compared to the solely adenovirus infected cells. Meanwhile, the levels of hydroxyl free radicals and malondialdehyde (MDA) in these cells increased. This led to the activation of caspase-3. The apoptosis in AdDMT1+IRE infected cells was shown with hypercondensed nuclei using Hoechst staining. Analysis of DNA extracted from these cells showed the typical "ladder pattern", indicating the formation of mono- and oligonucleosomes. These results suggested that increased DMT1+IRE expression in MES23.5 cells caused the increased intracellular iron accumulation. This resulted in the increased oxidative stress leading to ultimate cell apoptosis.