Iron overload inhibits cell proliferation and promotes autophagy via PARP1/SIRT1 signaling in endometriosis and adenomyosis.

Emerging evidence suggests that excess iron accumulates in endometriotic and adenomyotic lesions. However, the role iron overload plays in the pathogenesis of endometriosis or adenomyosis remains unknown. Primary human eutopic endometrial stromal cells (EuESCs) from endometriosis or adenomyosis patients were used as the in vitro model of endometriosis or adenomyosis in this study. We found that iron, manifesting as ferric ammonium citrate (FAC; 0.05-4.8 mM), significantly inhibited cell growth, induced oxidative stress through the Fenton reaction, and functionally activated autophagy in EuESCs, as measured by 5-ethynyl-2'-deoxyuridine incorporation assay, MitoSOX™ Red staining, LC3 turnover assay, and tandem mCherry-eGFP-LC3B fluorescence microscopy. Immunohistochemistry analysis of Ki67 expression in proliferative-phase endometrial tissues revealed that cell proliferation in ectopic tissues was dramatically compromised, suggesting that iron overload may play a role in cell growth inhibition in vivo. We observed that autophagy may alleviate the FAC-induced inhibition of endometrial stromal cell proliferation. Furthermore, sequential FAC (0.8 mM, 24 h) and hydrogen peroxide (H2O2; 300 µM, 2 h) treatment successfully induced the Fenton reaction in EuESCs and caused extensive apoptosis, whereas the disruption of autophagy by the knockdown of BECN1 further aggravated cell death. MitoSOX™ Red staining showed that autophagy may promote the survival of EuESCs by decreasing of the Fenton reaction-induced reactive oxygen species generation. In addition, we observed that the Fenton reaction-induced oxidative stress significantly suppressed iron overload-induced autophagy. Moreover, we found that FAC treatment impaired poly(ADP-ribose)-polymerase 1 (PARP1) expression while simultaneously upregulating SIRT1 expression in EuESCs. Our data further showed that PARP1 expression decreased in endometriotic lesions, which may partially result from iron overload. We also found that PARP1 inhibition aggravated iron overload-induced cell growth suppression, and was implicated in iron overload-induced autophagy. In addition, SIRT1 silencing alleviated iron overload-induced PARP1 downregulation and autophagy activation. Overall, our data suggest that iron overload in endometrial stromal cells of endometriotic or adenomyotic lesions may be involved in the inhibition of cell proliferation, simultaneously with the activation of protective autophagy via PARP1/SIRT1 signaling.

Chronic iron overload induces functional and structural vascular changes in small resistance arteries via NADPH oxidase-dependent O2- production.

Iron overload leads to excessive free radical formation and induces cardiovascular dysfunction. Thus, our aim was to investigate the structural and endothelial modulation of vascular tone induced by chronic iron overload in mesenteric arteries. Rats were divided into two groups: the control (vehicle) group and the group treated with iron dextran for 28days (100mg/kg, 5days a week). Chronic iron overload altered the following morpho-physiological parameters of third-order mesenteric resistance arteries: decreased lumen and external diameters; increased wall/lumen ratio and wall thickness; decreased distensibility and increased stiffness; and increased pulse wave velocity. Additionally, iron overload increased the vasoconstrictor response in mesenteric arterial rings in vitro but did not affect the relaxation induced by acetylcholine and sodium nitroprusside. It is suggested that iron overload reduces nitric oxide bioavailability by increasing free radicals, because L-NAME did not shift the concentration-response curve to phenylephrine, but L-NAME plus superoxide dismutase shifted the curve to the left. In vitro assays with DAF-2 and DHE indicated reduced NO production and increased superoxide anion (O2-) generation in the iron-overloaded group. Furthermore, tiron, catalase, apocynin and losartan induced reduced reactivity only in iron-overloaded rats. Moreover, increased ACE activity was observed in the mesenteric resistance arteries of iron-overloaded rats accompanied by an increase in gp91phox, catalase, ERK1/2 and eNOS protein expression. In conclusion, these findings show that chronic iron overload induces structural and functional changes in resistance arteries, most likely due to a decrease in NO bioavailability resulting from an increase in O2- production by NADPH oxidase.

En Route to New Therapeutic Options for Iron Overload Diseases: Matriptase-2 as a Target for Kunitz-Type Inhibitors.

The cell-surface serine protease matriptase-2 is a critical stimulator of iron absorption by negatively regulating hepcidin, the key hormone of iron homeostasis. Thus, it has attracted much attention as a target in primary and secondary iron overload diseases. Here, we have characterised Kunitz-type inhibitors hepatocyte growth factor activator inhibitor 1 (HAI-1) and HAI-2 as powerful, slow-binding matriptase-2 inhibitors. The binding modes of the matriptase-2-HAI complexes were suggested by molecular modelling. Different assays, including cell-free and cell-based measurements of matriptase-2 activity, determination of inhibition constants and evaluation of matriptase-2 inhibition by analysis of downstream effects in human liver cells, demonstrated that matriptase-2 is an excellent target for Kunitz inhibitors. In particular, HAI-2 is considered a promising scaffold for the design of potent and selective matriptase-2 inhibitors.

Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane.

Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1), iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 µM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5%) than in women and was associated with an increase (446%) in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS) and an increase (327%) in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 µM FeCl3 for 24 h showed an increase (132%) in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels.

Phytochemical profile of a microalgae Euglena tuba and its hepatoprotective effect against iron-induced liver damage in Swiss albino mice.

AIMS: This study was aimed to evaluate different phytochemical constituents and the ameliorating effect of 70% methanol extract of Euglena tuba (ETME) on iron overload-induced liver injury, along with its in vitro iron-chelating and DNA protection effects. METHODS AND RESULTS: Phytochemicals of ETME were identified by GC-MS analysis. Iron chelation and protection of Fenton reaction-induced DNA damage was conducted in vitro. Post oral administration of ETME to iron-overloaded mice, the levels of serum parameters, antioxidant enzymes, liver iron, lipid peroxidation, protein carbonyl and hydroxyproline contents were measured. ETME showed inhibition of lipid peroxidation, protein oxidation and liver fibrosis. The serum markers and liver iron were lessened, whereas enhanced levels of liver antioxidant enzymes were detected in ETME-treated group. Furthermore, the histopathological observations also substantiated the protective effects of the extract. CONCLUSIONS: Several bioactive compounds identified by GC-MS may be the basis of hepatoprotective as well as antioxidant and iron-chelating effect of ETME. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently available iron-chelating agents show several side effects and limitations which may be overcome by ETME, which suggest its benefit against pathology of iron overload-linked diseases. Hence, ETME can be used as a promising hepatoprotective agent.

Pyrimidine-5'-nucleotidase Campinas, a new mutation (p.R56G) in the NT5C3 gene associated with pyrimidine-5'-nucleotidase type I deficiency and influence of Gilbert's Syndrome on clinical expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Stimulation of gene expression and activity of antioxidant related enzyme in Sprague Dawley rat kidney induced by long-term iron toxicity.

The trace elements such as iron are vital for various enzyme activities and for other cellular proteins, but iron toxicity causes the production of reactive oxygen species (ROS) that causes alterations in morphology and function of the nephron. The present study was designed to determine the effect of long-term iron overload on the renal antioxidant system and to determine any possible correlation between enzymatic and molecular levels. Our data showed that reduced glutathione (GSH) levels, which is a marker for oxidative stress, strikingly decreased with a long-term iron overload in rat kidney. While renal mRNA levels of glucose 6-phosphate dehydrogenase (G6pd), 6-phosphogluconate dehydrogenase (6pgd) and glutathione peroxidase (Gpx) were significantly affected in the presence of ferric iron, no changes were seen for glutathione reductase (Gsr) and glutathione S-transferases (Gst). While the iron affected the enzymatic activity of G6PD, GSR, GST, and GPX, it had no significant effect on 6PGD activity in the rat kidney. In conclusion, we reported here that the gene expression of G6pd, 6pgd, Gsr, Gpx, and Gst did not correlate to enzyme activity, and the actual effect of long-term iron overload on renal antioxidant system is observed at protein level. Furthermore, the influence of iron on the renal antioxidant system is different from its effect on the hepatic antioxidant system.

The effect of the Gly139His, Gly143His, and Ser142His mouse heme oxygenase-1 mutants on the HO reaction in vivo and in vitro.

Heme oxygenase-1 (HO-1), which catalyzes the degradation of heme to iron, carbon monoxide, and biliverdin, performs a cytoprotective function. Previous studies on the crystal structure of the human and rat HO-1 in complex with heme showed that Gly139His (G139H) and Gly143His (G143H) mutants have no HO activity. In the present study, we reported the effect of the G139H, G143H, and Ser142His mutants of mouse HO-1 on the HO reaction in vivo and in vitro. In vitro, of the mutant transfectants, only Ser142His catalyzed degradation of heme, retaining 31.7% of the wild-type mouse HO-1 activity, whereas G139H and G143H mutants exhibited no activity. In vivo, only Tg HO-1 G143H females presented with anemia, enlarged spleen and tissue iron overload, which was similar to HO-1(-/-) mice. The results suggested the critical role of Gly139 and Gly143 in maintaining HO-1 activity in vitro and the critical role of Gly143 in maintaining HO-1 activity in vivo.

Relationship between elevated liver enzyme with iron overload and viral hepatitis in thalassemia major patients in Northern Iran.

OBJECTIVE: To determine the relationship between elevated liver enzymes with iron overload and viral hepatitis in thalassemic patients. METHODS: This descriptive cross-sectional study was carried out in the thalassemic ward of Tonekabon Hospital, Mazandaran, Northern Iran from 20 April to 20 September of 2006. Patients were studied with respect to age, liver enzymes, anti-hepatitis C virus (anti-HCV) antibody, and hepatitis B surface antigen (HBsAg), transferrin saturation (TSAT), and blood transfusion index (multiplication of frequency and units of transfusion). Alanine aminotransferase (ALT) > or =40 U/L was considered elevated. RESULTS: Sixty-five patients were evaluated (median age 19.51+/-8.9 years, range 4-54). Eleven patients were anti-HCV positive (16.9%). The mean serum ferritin was significantly higher in patients with ALT > or =40 (2553.08 ug/L versus 1783.7750 microg/L) (p=0.012). The mean ALT was significantly higher in patients with TSAT > or = 60% (41.26 U/L versus 28.82 U/L) (p=0.021). The relationship between ALT > or =40 and anti-HCV positivity was statistically significant. The mean ALT was 60.91 U/L in anti-HCV positive patients and 39.29 U/L in the negative group (p=0.001). The mean serum iron and transfusion index were significantly higher in anti-HCV positive versus negative patients (234.0 versus 195.4815; p=0.02), (1693.6 versus 1036.29, p=0.014). CONCLUSION: Close association between elevated ALT with iron overload, transfusion index, age, and anti-HCV positivity in thalassemic patients of Tonekabon is recommended to re-evaluate transfusion and Desferal doses and therapies other than blood transfusion.

Increased hepatic telomerase activity in a rat model of iron overload: a role for altered thiol redox state?

Telomeres are repeated sequences at chromosome ends that are incompletely replicated during mitosis. Telomere shortening caused by proliferation or oxidative damage culminates in replicative arrest and senescence, which may impair regeneration during chronic liver injury. Whereas the effects of experimental liver injury on telomeres have received little attention, prior studies suggest that telomerase, the enzyme complex that catalyzes the addition of telomeric repeats, is protective in some rodent liver injury models. Thus, the aim of this study was to determine the effects of iron overload on telomere length and telomerase activity in rat liver. Mean telomere lengths were similar in iron-loaded and control livers. However, telomerase activity was increased 3-fold by iron loading, with no change in levels of TERT mRNA or protein. Because thiol redox state has been shown to modulate telomerase activity in vitro, hepatic thiols were assessed. Significant increases in GSH (1.5-fold), cysteine (15-fold), and glutamate cysteine ligase activity (1.5-fold) were observed in iron-loaded livers, whereas telomerase activity was inhibited by treatment with N-ethylmaleimide. This is the first demonstration of increased telomerase activity associated with thiol alterations in vivo. Enhanced telomerase activity may be an important factor contributing to the resistance of rodent liver to iron-induced damage.

Enhanced NTPDase and 5'-nucleotidase activities in diabetes mellitus and iron-overload model.

The activity of the enzymes NTPDase and 5'-nucleotidase was studied in both diabetes mellitus and an associated model of iron-overload. Rats were divided in five groups: citrate (CC), saline (S), diabetic (D), iron-overload (IO), and diabetic iron-overload (DIO). Diabetes was induced with alloxan (150 mg/kg), and iron-overload was induced with iron-dextran (10 intramuscular applications of +/-80 mg/kg). The enzymatic activities were evaluated in the platelets. The results demonstrated an increase in the activity of NTPDase with substrates ATP and ADP (60% and 120%, respectively; P<0.001), and 5'-nucleotidase (60%, P<0.001). This increase was more intense in the IO and DIO groups. The results obtained in vitro showed an activation in ATP, ADP, and AMP hydrolysis between 1 microM and 1,000 microM ferric nitrate concentrations, being more pronounced at 100 microM and decreasing at 1,000 microM. We concluded that diabetes mellitus in association with iron-overload increased the hydrolysis of adenine nucleotides in platelets, contributing to the abnormalities found in these pathological conditions.

Effects of phlebotomy therapy on cytochrome P450 2e1 activity and oxidative stress markers in dysmetabolic iron overload syndrome: a randomized trial.

AIM: To assess the effects of iron removal on cytochrome P450 2E1 activity and oxidative stress in dysmetabolic iron overload syndrome. METHODS: Forty-eight patients were randomized to phlebotomy therapy consisting of removal of 300-500 mL of blood every 14 days until serum ferritin levels dropped under 100 microg/L or to follow-up without phlebotomy therapy. Cytochrome P450 2E1 activity was measured at baseline and at the end of treatment by using the 6-hydroxychlorzoxazone/chlorzoxazone blood metabolic ratio, 2 h after the intake of 500 mg of chlorzoxazone. RESULTS: In the treatment group, a mean of 3.9 +/- 1.3 L of blood was removed and serum ferritin levels dropped from 715 +/- 397 to 74 +/- 34 microg/L. Variation of cytochrome P450 2E1 activity was not significantly different between the 2 groups (0.07 +/- 0.26 vs. 0.03 +/- 0.19, P = 0.36). In the treatment group, low-density lipoprotein cholesterol and vitamin E were lowered after treatment compared with control group (-0.15 +/- 0.51 vs. 0.24 +/- 0.58, P = 0.002 and -1.3 +/- 4.4 vs. 2.3 +/- 5.2, P = 0.03, respectively). Inversely, vitamin C was increased (0.5 +/- 3.5 vs. -1.8 +/- 3.9, P = 0.03). CONCLUSIONS: In dysmetabolic iron overload syndrome, reduction of iron stores does not significantly influence cytochrome P450 2E1 activity but is associated with a significant decrease of low-density lipoprotein cholesterol, suggesting that venesection therapy may be a suitable option in these patients.

Glutathione S-transferase M1 gene polymorphisms are associated with cardiac iron deposition in patients with beta-thalassemia major.

Patients with beta-thalassemia (thal) major are subject to peroxidative tissue injury by iron overload. Glutathione S-transferases work as antioxidants, and their activity is determined genetically. In this study, we used multiplex polymerase chain reaction (m-PCR) to analyze polymorphisms of two endogenous antioxidant agents, glutathione S-transferase M1 (GSTM1) and glutathione S-transferase T1 (GSTT1), and to determine their roles in 41 patients with beta-thal major. Our results showed that the GSTM1 and GSTT1 null genotypes were not associated with any incidence of endocrine dysfunction (including diabetes mellitus, hypogonadism, hypothyroidism, and growth hormone deficiency), liver function, or impaired left ventricular ejection fraction (LVEF). The GSTM1 null genotype, but not the GSTT1 null genotype, was associated with a decreased signal intensity ratio on cardiac magnetic resonance imaging (MRI). Our results suggest that genetic variations of the GSTM1 enzyme are associated with cardiac iron deposition in patients with beta-thal major.

Peroxidative stress and antioxidant enzymes in children with beta-thalassemia major.

OBJECTIVE: Regular blood transfusions and secondary iron overload make thalassemic erythrocytes prone to peroxidative injury. Although some reports suggest endogenous free radical damage in thalassemia, there remains discrepancy in the status of antioxidant enzymes. The aim of this study was to evaluate the extent of lipid peroxidation and status of antioxidant enzyme in children with beta-thalassemia. METHODS: Fifty transfusion-dependent beta-thalassemics were subjected to analysis of lipoperoxides as malondialdehyde (MDA), nitric oxide (NOx), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx) along with serum iron and ferritin, liver functions and uric acid. Plasma MDA was analyzed to indicate the oxidative parameters, whereas the erythrocyte SOD, GPx, and plasma NOx were measured to show the antioxidant status of the children. All these parameters in 30 non-anemic healthy controls attending the child health promotion clinic of hospital were also studied. RESULTS: All the patients were iron overloaded. Markers of free radical injury such as MDA and antioxidant enzyme SOD and NOx levels were significantly elevated in thalassemic children while mean GPx levels were decreased in patients compared to controls (P < 0.001). All these markers significantly correlated with serum ferritin levels. There was no significant difference in levels of GSH measured but it correlated with serum iron levels. CONCLUSION: Our study results suggest that iron overload causes peroxidative damage in beta-thalassemia and antioxidant systems try to compensate for reducing lipid peroxidation to lower tissue damage.

Chronic iron overload enhances inducible nitric oxide synthase expression in rat liver.

Iron is an essential micronutrient promoting oxidative stress in the liver of overloaded animals and human, which may trigger the expression of redox-sensitive genes. We have tested the hypothesis that chronic iron overload (CIO) enhances inducible nitric oxide synthase (iNOS) expression in rat liver by extracellular signal-regulated kinase (ERK1/2) and NF-kappaB activation. CIO (diet enriched with 3%(wt/wt) carbonyl-iron for 12 weeks) increased liver protein carbonylation and decreased reduced glutathione (GSH) content and the GSH/GSSG ratio after 6 weeks, parameters that are normalized after 8-12 weeks of treatment. These changes are paralleled by higher phosphorylated-ERK1/2 to non-phosphorylated-ERK1/2 ratios at 6 and 8 weeks, increased NF-kappaB DNA binding to the iNOS gene promoter at 8-12 weeks, and higher iNOS mRNA expression and activity at 8 and 12 weeks. It is concluded that CIO triggers liver oxidative stress at early times, with upregulation of iNOS expression involving the ERK/NF-kappaB pathway at later times, a finding that may represent a hepatoprotective mechanism against CIO toxicity in addition to the recovery of GSH homeostasis.

Reduced insulin growth factor I concentrations in iron-overloaded beta thalassaemic patients with normal growth hormone secretion and liver function.

We selected 92 subjects (46 females and 46 males), aged 10-15 years, from the Haematology and Endocrine Clinic of Shiraz University, Iran. Forty-six were beta thalassaemia patients (beta-Th) with short stature, 23 had idiopathic short stature (ISS) and 23 were healthy children with a standing height between the 10th and 95th percentile. Growth hormone (GH) secretion was normal in 23 beta-Th patients and reduced in the remaining 23 patients. A low insulin growth factor I (IGF-I) was found in 73.9% of beta-Th patients with GH deficiency, 56.5% of beta-Th patients with normal GH secretion to stimulation test and 8.7% of children with ISS. The reduced IGF-I concentration in beta-Th patients with normal GH secretion may be explained by partial insensitivity to GH (GHIS), neurosecretory dysfunction, low bioactive GH or increased proportion of circulating, non-22-kDa GH isoform. The possibility of GHIS in beta-Th patients with short stature indicates that higher doses of rechGH may be required to obtain an improvement in growth velocity in beta-Th patients.

GRACILE syndrome, a lethal metabolic disorder with iron overload, is caused by a point mutation in BCS1L.

GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death) syndrome is a recessively inherited lethal disease characterized by fetal growth retardation, lactic acidosis, aminoaciduria, cholestasis, and abnormalities in iron metabolism. We previously localized the causative gene to a 1.5-cM region on chromosome 2q33-37. In the present study, we report the molecular defect causing this metabolic disorder, by identifying a homozygous missense mutation that results in an S78G amino acid change in the BCS1L gene in Finnish patients with GRACILE syndrome, as well as five different mutations in three British infants. BCS1L, a mitochondrial inner-membrane protein, is a chaperone necessary for the assembly of mitochondrial respiratory chain complex III. Pulse-chase experiments performed in COS-1 cells indicated that the S78G amino acid change results in instability of the polypeptide, and yeast complementation studies revealed a functional defect in the mutated BCS1L protein. Four different mutations in the BCS1L gene have been reported elsewhere, in Turkish patients with a distinctly different phenotype. Interestingly, the British and Turkish patients had complex III deficiency, whereas in the Finnish patients with GRACILE syndrome complex III activity was within the normal range, implying that BCS1L has another cellular function that is uncharacterized but essential and is putatively involved in iron metabolism.

Decreasing effects of iron toxicosis on selenium and glutathione peroxidase activity.

Heart failure due to chronic iron overload is a leading cause of cardiovascular mortality in the second and third decades of life worldwide, but its mechanism is not known. Deficiencies of selenium have been shown to result in damage to the myocardium and to the development of various cardiomyopathies. In the current investigation, the dose-dependent effects of chronic iron toxicosis on heart tissue concentrations of selenium and the protective antioxidant enzyme glutathione peroxidase (GPx) were investigated in a murine model of iron-overload cardiomyopathy (n = 20). Significant dose-dependent decreases in heart tissue selenium concentrations (r = -0.95, p < 0.001) and selenium-dependent GPx activity (r = -0.93, p < 0.001) were observed in chronically iron-loaded mice in comparison with placebo controls. These results suggest that dietary supplementation with selenium may be beneficial in the clinical management of disorders of iron metabolism.

Differential effects of iron overload on GST isoform expression in mouse liver and kidney and correlation between GSTA4 induction and overproduction of free radicles.

We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.