The Impact of the Combined Effect of Inhalation Anesthetics and Iron Dextran on Rats' Systemic Toxicity.

Disruption of any stage of iron homeostasis, including uptake, utilization, efflux, and storage, can cause progressive damage to peripheral organs. The health hazards associated with occupational exposure to inhalation anesthetics (IA) in combination with chronic iron overload are not well documented. This study aimed to investigate changes in the concentration of essential metals in the peripheral organs of rats after iron overload in combination with IA. The aim was also to determine how iron overload in combination with IA affects tissue metal homeostasis, hepcidin-ferritin levels, and MMP levels according to physiological, functional, and tissue features. According to the obtained results, iron accumulation was most pronounced in the liver (19×), spleen (6.7×), lungs (3.1×), and kidneys (2.5×) compared to control. Iron accumulation is associated with elevated heavy metal levels and impaired essential metal concentrations due to oxidative stress (OS). Notably, the use of IA increases the iron overload toxicity, especially after Isoflurane exposure. The results show that the regulation of iron homeostasis is based on the interaction of hepcidin, ferritin, and other proteins regulated by inflammation, OS, free iron levels, erythropoiesis, and hypoxia. Long-term exposure to IA and iron leads to the development of numerous adaptation mechanisms in response to toxicity, OS, and inflammation. These adaptive mechanisms of iron regulation lead to the inhibition of MMP activity and reduction of oxidative stress, protecting the organism from possible damage.

Intraperitoneal injection of iron dextran induces peripheral iron overload and mild neurodegeneration in the nigrostriatal system in C57BL/6 mice.

AIMS: Elevated iron levels in the affected areas of brain are linked to several neurodegenerative diseases including Parkinson's disease (PD). This study investigated the influence of peripheral iron overload in peripheral tissues, as well as its entry into the brain regions on lysosomal functions. The survival of dopaminergic neurons in the nigrostriatal system and motor coordination were also investigated. MAIN METHODS: An intraperitoneal injection of iron dextran (FeDx) mouse model was established. Western blot was used to detect iron deposition and lysosomal functions in the liver, spleen, hippocampal (HC), striatum (STR), substantia nigra (SN) and olfactory bulb (OB). Iron in serum and cerebrospinal fluid (CSF) was determined by an iron assay kit. Immunofluorescence and immunohistochemical staining were applied to detect dopaminergic neurons and fibers. Motor behavior was evaluated by gait analysis. KEY FINDINGS: Iron was deposited consistently in the liver and spleen, and serum iron was elevated. While iron deposition occurred late in the HC, STR and SN, without apparently affecting CSF iron levels. Although cathepsin B (CTSB), cathepsin D (CTSD), glucocerebrosidase (GCase) and lysosome integrated membrane protein 2 (LIMP-2) protein levels were dramatically up-regulated in the liver and spleen, they were almost unchanged in the brain regions. However, CTSB was up-regulated in acute iron-overloaded OB and primary cultured astrocytes. The number of dopaminergic neurons in the SN remained unchanged, and mice did not exhibit significant motor incoordination. SIGNIFICANCE: Intraperitoneal injection of FeDx in mice induces largely peripheral iron overload while not necessarily sufficient to cause severe disruption of the nigrostriatal system.

Ferritin But Not Iron Increases in Retina Upon Systemic Iron Overload in Diabetic and Iron-Dextran Injected Mice.

Purpose: Iron overload causes oxidative damage in the retina, and it has been involved in the pathogeny of diabetic retinopathy, which is one of the leading causes of blindness in the adult population worldwide. However, how systemic iron enters the retina during diabetes and the role of blood retinal barrier (BRB) in this process remains unclear. Methods: The db/db mouse, a well-known model of type 2 diabetes, and a model of systemic iron overload induced by iron dextran intraperitoneal injection, were used. Perls staining and mass spectrophotometry were used to study iron content. Western blot and immunohistochemistry of iron handling proteins were performed to study systemic and retinal iron metabolism. BRB function was assessed by analyzing vascular leakage in fundus angiographies, whole retinas, and retinal sections and by studying the status of tight junctions using transmission electron microscopy and Western blot analysis. Results: Twenty-week-old db/db mice with systemic iron overload presented ferritin overexpression without iron increase in the retina and did not show any sign of BRB breakdown. These findings were also observed in iron dextran-injected mice. In those animals, after BRB breakdown induced by cryopexy, iron entered massively in the retina. Conclusions: Our results suggested that BRB protects the retina from excessive iron entry in early stages of diabetic retinopathy. Furthermore, ferritin overexpression before iron increase may prepare the retina for a potential BRB breakdown and iron entry from the systemic circulation.

Ameliorative effects and mechanisms of salvianic acid A on retinal iron overload in vivo and in vitro.

Excessive iron can be accumulated in the retina and lead to retinal iron overload. Salvianic acid A (SAA) has a variety of pharmacologic effects, but there is only a limited understanding of its benefits for retinal iron overload. The aim of this study was to examine the protective effects and latent mechanisms of SAA on retinal iron overload. SAA reduced iron in the serum and retina, attenuated pathophysiological changes, and reduced retinal iron deposition in the retinas of iron-overloaded mice. It also reduced intracellular iron in ARPE-19 cells by regulating iron-handling proteins and chelating with iron. It also significantly inhibited cellular oxidative and inflammatory damage by increasing the nuclear translocation of nuclear erythroid 2-related factor 2 (Nrf2) while decreasing nuclear factor-kappa B (NF-κB), protecting the ARPE-19 cells from apoptosis by suppressing the Bax/Bcl-2 ratio, cytochrome c release, caspase activation, and poly ADP-ribose polymerase cleavage. The ability of SAA to inhibit apoptosis, increase nuclear Nrf2 expression, and decrease nuclear NF-κB expression was further confirmed in the retinas of iron-overloaded mice. This study demonstrates that SAA shows significant protective effects against retinal iron overload; its mechanisms might be associated with iron chelation; regulation of iron-handling proteins; and inhibition of oxidative stress, inflammation and apoptosis.

Systemic iron overload exacerbates osteoarthritis in the strain 13 guinea pig.

OBJECTIVE: Iron is emerging as a key player in aging-associated diseases due to its propensity for driving free radical formation. Studies examining the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. Our objective was to establish a direct relationship between excess iron and OA by administering iron dextran to a guinea pig strain with decreased propensity for developing this disease. DESIGN: Twenty, 12-week-old Strain 13 guinea pigs received either iron dextran or dextran control intraperitoneally once weekly for 4 weeks; termination occurred at 16 weeks of age. Iron levels were determined systemically (serum and liver) and within diarthrodial joints [femoral head articular cartilage and infrapatellar fat pads (IFPs) of knee joints]. One knee was collected to score structural changes associated with OA via microcomputed tomography (microCT) and histology using published grading schemes. Articular cartilage and IFPs were harvested from contralateral knees for gene expression analyses. RESULTS: Iron overload was confirmed systemically via increased serum iron and liver iron concentration. Articular cartilage and IFPs in the iron dextran group also had higher levels of iron. Excess iron worsened knee OA using both microCT and histologic scoring systems. Gene analyses revealed that exogenous iron altered the expression of iron trafficking proteins, select cytokines, and structural components of cartilage. CONCLUSION: These results demonstrate that systemic iron overload caused cellular iron accumulation in the knee joint. This excess iron is associated with increased expression of local inflammatory mediators and early onset and progression of knee joint OA in Strain 13 animals.

T2 ∗ Relaxation Time Obtained from Magnetic Resonance Imaging of the Liver Is a Useful Parameter for Use in the Construction of a Murine Model of Iron Overload.

Aim: Iron overload is a life-threatening disorder that can increase the risks of cancer, cardiovascular disease, and liver cirrhosis. There is also a risk of iron overload in patients with chronic kidney disease. In patients with renal failure, iron storage is increased due to inadequate iron utilization associated with decreased erythropoiesis and also to the inflammatory status. To evade the risk of iron overload, an accurate and versatile indicator of body iron storage in patients with iron overload is needed. In this study, we aimed to find useful iron-related parameters that could accurately reflect body iron storage in mice in order to construct a murine model of iron overload. Methods: To select an appropriate indicator of body iron status, a variety of parameters involved in iron metabolism were evaluated. Noninvasively measured parameters were R1, R2, and R2 ∗ derived from magnetic resonance imaging (MRI). Invasively measured parameters included serum hepcidin levels, serum ferritin levels, and liver iron contents. Histopathological analysis was also conducted. Results/Conclusion: Among the several parameters evaluated, the MRI T2 ∗ relaxation time was able to detect iron storage in the liver as sensitively as serum ferritin levels. Moreover, it is expected that using an MRI parameter will allow accurate evaluation of body iron storage in mice over time.

Iron-induced oxidative stress contributes to α-synuclein phosphorylation and up-regulation via polo-like kinase 2 and casein kinase 2.

α-Synuclein plays a central role in synucleinopathies pathogenesis such as Parkinson's disease (PD). Phosphorylation is the most common and important protein modification linked to α-synuclein pathologies. There is mounting evidence suggested iron and α-synuclein are closely related in PD. We previously reported iron up-regulated α-synuclein mRNA levels and induced α-synuclein aggregation. In the present study, we aimed to investigate whether and how phosphorylation was involved in iron-induced α-synuclein regulations. The results showed that iron could induce pS129 α-synuclein (phosphorylation at Ser129) and α-synuclein upregulation in the substantia nigra of iron-overloaded rats and iron-treated SH-SY5Y cells, accompanied by the elevated levels of polo-like kinase 2 (PLK2) and casein kinase 2 (CK2). Over-expression of CK2 or PLK2 induced pS129 α-synuclein up-regulation and inhibitors of CK2 or PLK2 could suppress iron-induced α-synuclein phosphorylation. Antioxidant NAC could fully block iron-induced upregulation of CK2, PLK2 and pS129 α-synuclein levels, indicating oxidative stress plays a critical role in iron-induced α-synuclein phosphorylation. However, iron-induced α-synuclein up-regulation could only be partially blocked by CK2/PLK2 inhibitor or NAC. These findings demonstrate that iron-induced oxidative stress is largely responsible for α-synuclein phosphorylation and upregulation via CK2 and PLK2, and α-synuclein upregulation is not fully phosphorylation-dependent.

CCL2 is Upregulated by Decreased miR-122 Expression in Iron-Overload-Induced Hepatic Inflammation.

BACKGROUND/AIMS: Iron overload (IO) is accompanied by hepatic inflammation. The chemokine (C-C motif) ligand 2 (CCL2) mediates inflammation, and its overexpression is associated with IO. However, whether IO results in CCL2 overexpression in the liver and the underlying mechanisms are unclear. METHODS: We subjected mice to IO by administering intraperitoneal injections of dextran-iron or by feeding mice a 3% dextran-iron diet to observe the effects of IO on miR-122/CCL2 expression through real-time qPCR and Western blot analysis. We also used indicators, including the expression of the inflammatory cytokine, the inflammation score based on H&E staining and the serum content of ALT and AST to evaluate the effects of IO on hepatic inflammation. Meanwhile, we observed the effects of vitamin E on IO-induced hepatic inflammation. In cells, we used 100 µΜ FeSO4 or 30 µΜ Holo-Tf to produce IO and observed the roles of miR-122 in regulating CCL2 expression by using miR-122 mimics or inhibitors to overexpress or inhibit miR-122. Then, we used a dual-luciferase reporter assay to prove that miR-122 regulates CCL2 expression through direct binding to its complementary sequence in the CCL2 mRNA 3'UTR. RESULTS: IO induces the downregulation of miR-122 and the upregulation of CCL2, as well as inflammatory responses both in vitro and in vivo. Although IO-induced oxidative stress is eliminated by the antioxidant vitamin E, IO-induced hepatic inflammation still exists, which probably can be explained by the fact that vitamin E has no effects on the miR-122/CCL2 pathway. In in vitro experiments, the overexpression and inhibition of miR-122 significantly reduced and increased CCL2 expression, respectively. The dual-luciferase reporter assay indicates that miR-122 binds CCL2 mRNA 3'UTR. CONCLUSION: We propose the roles of miR-122/CCL2 in IO-induced hepatic inflammation. Our studies should provide a new clue for developing clinical strategies for patients with IO.

Nerium indicum leaf alleviates iron-induced oxidative stress and hepatic injury in mice.

CONTEXT: Nerium indicum Mill. (Apocynaceae) was reported for its efficient in vitro antioxidant and iron-chelating properties. OBJECTIVE: This study demonstrates the effect of 70% methanol extract of N. indicum leaf (NIME) towards in vitro DNA protection and ameliorating iron-overload-induced liver damage in mice. MATERIALS AND METHODS: Phytochemical and HPLC analyses were carried out to standardize the extract and the effect of Fe(2+)-mediated pUC18 DNA cessation was studied. Thirty-six Swiss Albino mice were divided into six groups of blank, negative control (iron overload only), and iron-overloaded mice receiving 50, 100, and 200 mg/kg b.w. doses of NIME and desirox (20 mg/kg b.w.). The biochemical markers of hepatic damage, various liver and serum parameters, and reductive release of ferritin iron were studied. RESULTS AND DISCUSSION: The presence of different phytocomponents was revealed from phytochemical and HPLC analyses. A substantial supercoiled DNA protection, with [P]50 of 70.33 ± 0.32 µg, was observed. NIME (200 mg/kg b.w.) significantly normalized the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and bilirubin by 126.27, 125.25, 188.48, and 45.47%, respectively. NIME (200 mg/kg b.w.) was shown to alleviate the reduced levels of superoxide dismutase, catalase, glutathione-S-transferase, and non-enzymatic-reduced glutathione, by 48.95, 35.9, 35.42, and 13.22%, respectively. NIME also lowered raised levels of lipid peroxidation, protein carbonyl, hydroxyproline, and liver iron by 32.28, 64.58, 136.81, and 83.55%, respectively. CONCLUSION: These findings suggest that the active substances present in NIME may be capable of lessening iron overload-induced toxicity, and possibly be a useful drug for iron-overloaded diseases.

Iron toxicity mediated by oxidative stress enhances tissue damage in an animal model of diabetes.

Although iron is a first-line pro-oxidant that modulates clinical manifestations of various systemic diseases, including diabetes, the individual tissue damage generated by active oxidant insults has not been demonstrated in current animal models of diabetes. We tested the hypothesis that oxidative stress is involved in the severity of the tissues injury when iron supplementation is administered in a model of type 1 diabetes. Streptozotocin (Stz)-induced diabetic and non-diabetic Fischer rats were maintained with or without a treatment consisting of iron dextran ip at 0.1 mL day(-1) doses administered for 4 days at intervals of 5 days. After 3 weeks, an extensive increase (p < 0.001) in the production of reactive oxygen species (ROS) in neutrophils of the diabetic animals on iron overload was observed. Histological analysis revealed that this treatment also resulted in higher (p < 0.05) tissue iron deposits, a higher (p < 0.001) number of inflammatory cells in the pancreas, and apparent cardiac fibrosis, as shown by an increase (p < 0.05) in type III collagen levels, which result in dysfunctional myocardial. Carbonyl protein modification, a marker of oxidative stress, was consistently higher (p < 0.01) in the tissues of the iron-treated rats with diabetes. Moreover, a significant positive correlation was found between ROS production and iron pancreas stores (r = 0.42, p < 0.04), iron heart stores (r = 0.54, p < 0.04), and change of the carbonyl protein content in pancreas (r = 0.49, p < 0.009), and heart (r = 0.48, p < 0.02). A negative correlation was still found between ROS production and total glutathione content in pancreas (r = -0.50, p < 0.03) and heart (r = -0.45, p < 0.04). In conclusion, our results suggest that amplified toxicity in pancreatic and cardiac tissues in rats with diabetes on iron overload might be attributed to increased oxidative stress.

[Establishment of an mouse model of iron-overload and its impact on bone marrow hematopoiesis].

OBJECTIVE: To establish a mouse model of iron overload by intraperitoneal injection of iron dextran and investigate the impact of iron overload on bone marrow hematopoiesis. METHODS: A total of 40 C57BL/6 mice were divided into control group, low-dose iron group (12.5 mg/ml), middle-dose iron group (25 mg/ml), and high-dose iron group (50 mg/ml). The control group received normal saline (0.2 ml), and the rest were injected with intraperitoneal iron dextran every three days for six weeks. Iron overload was confirmed by observing the bone marrow, hepatic, and splenic iron deposits and the bone marrow labile iron pool. In addition, peripheral blood and bone marrow mononuclear cells were counted and the hematopoietic function was assessed. RESULTS: Iron deposits in bone marrow, liver, and spleen were markedly increased in the mouse models. Bone marrow iron was deposited mostly within the matrix with no significant difference in expression of labile iron pool.Compared with control group, the ability of hematopoietic colony-forming in three interventional groups were decreased significantly (P<0.05). Bone marrow mononuclear cells counts showed no significant difference. The amounts of peripheral blood cells (white blood cells, red blood cells, platelets, and hemoglobin) in different iron groups showed no significant difference among these groups;although the platelets were decreased slightly in low-dose iron group [(780.7±39.60)×10(9)/L], middle dose iron group [(676.2±21.43)×10(9)/L], and high-dose iron group [(587.3±19.67)×10(9)/L] when compared with the control group [(926.0±28.23)×10(9)/L], there was no significant difference(P>0.05). CONCLUSIONS: The iron-overloaded mouse model was successfully established by intraperitoneal administration of iron dextran. Iron overload can damage the hepatic, splenic, and bone marrow hematopoietic function, although no significant difference was observed in peripheral blood count.

Deferasirox shows in vitro and in vivo antileukemic effects on murine leukemic cell lines regardless of iron status.

Numerous studies have shown the antiproliferative effect of iron chelating agents (ICAs), which have been used traditionally in patients with secondary iron overload (SIO). Because the in vivo model for these studies has been animals with normal iron status, the antileukemic effect of ICAs in the SIO condition has not been determined clearly. We investigated the in vitro and in vivo effects of ICAs in murine leukemic cell lines regarding the iron status. The viability of both EL4 cells and L1210 cells incubated with either deferoxamine (DFO) or deferasirox (DFX) decreased in a concentration-dependent manner. This effect was most prominent in L1210 cells treated with DFX. The viability of L1210 cells incubated with both ICAs did not change regardless of the presence of ferric chloride. The percentage of apoptosis in L1210 cells treated with DFO or DFX increased in a concentration-dependent manner; however, the expression of Fas showed no significant change. The non-SIO mice and SIO mice bearing L1210 cells showed longer survival than other groups when treated with DFX, whereas the SIO mice treated with DFO showed shorter survival than the control group. The tumor was significantly smaller in the SIO mice treated with DFX or DFO compared with the control group. The iron content of the liver or the tumor in SIO mice decreased after ICA treatment. This study indicates an antileukemic effect of DFX regardless of iron status and suggests that the use of DFX has a survival benefit for SIO leukemia murine model in terms of iron chelation and antileukemic therapy.

Nifedipine prevents iron accumulation and reverses iron-overload-induced dopamine neuron degeneration in the substantia nigra of rats.

The mechanisms of iron accumulation in substantia nigra (SN) of Parkinson's diseases remain unclear. The objective of this study was to investigate effects of nifedipine on iron-overload-induced iron accumulation and neurodegeneration in SN of rats. By high performance liquid chromatography-electrochemical detection, tyrosine hydroxylase (TH) immunohistochemistry, and iron content array, we first quantified iron content and the number of dopamine neurons in SN of experimental rats treated with iron dextran. We further assessed effects of treatment with nifedipine. Our results showed that nifedipine treatment prevents iron dextran-induced dopamine depletion in the striatum. Consistently, we found that nifedipine restores the number of TH-positive neurons reduced by iron dextran overload and prevents increase of iron content in the SN. These results suggested that nifedipine may suppress iron toxicity in dopamine neurons and prevent neurodegeneration.

Evaluation of toxicity and oxidative stress induced by intravenous iron isomaltoside 1000 in a nonclinical model.

The physicochemical characteristics of intravenous iron complexes affect the extent of weakly-bound iron and thus the degree of oxidative stress. The new preparation iron isomaltoside 1000 (IIM) was compared to iron sucrose (IS) and a control group in terms of biochemistry, oxidative stress, inflammatory markers and iron deposition in the liver, heart and kidneys of healthy rats. Renal function was significantly impaired in the IIM group versus both IS and controls. Liver enzymes were also significantly higher in IIM-treated animals versus the other groups, indicative of hepatic injury. Systolic blood pressure was significantly lower following IIM administration compared to IS or control animals. Oxidative stress in the liver, heart and kidneys was greater in the IIM group, as indicated by significantly increased levels of malondialdehyde and antioxidant enzyme activity, accompaniedby a significantly lower ratio of reduced to oxidized glutathione. Microscopy demonstrated more extensive positive staining for iron, and a smaller area of ferritin staining, in the liver, heart and kidneys of rats treated with IIM versus IS.Levels of the inflammatory markers TNF-alpha and IL6 were both significantly higher in the IIM group versus IS in all assessed tissues. These findings indicate that IIM has a less favorable safety profile than IS in healthy rats, adversely affecting iron deposition, oxidative stress and inflammatory responses, with impaired liver and renal function.

Assessment of the extent of oxidative stress induced by intravenous ferumoxytol, ferric carboxymaltose, iron sucrose and iron dextran in a nonclinical model.

Intravenous (i.v.) iron is associated with a risk of oxidative stress. The effects of ferumoxytol, a recently approved i.v. iron preparation, were compared with those of ferric carboxymaltose, low molecular weight iron dextran and iron sucrose in the liver, kidneys and heart of normal rats. In contrast to iron sucrose and ferric carboxymaltose, low molecular weight iron dextran and ferumoxytol caused renal and hepatic damage as demonstrated by proteinuria and increased liver enzyme levels. Higher levels of oxidative stress in these tissues were also indicated, by significantly higher levels of malondialdehyde, significantly increased antioxidant enzyme activities, and a significant reduction in the reduced to oxidized glutathione ratio. Inflammatory markers were also significantly higher with ferumoxytol and low molecular weight iron dextran rats than iron sucrose and ferric carboxymaltose. Polarographic analysis suggested that ferumoxytol contains a component with a more positive reduction potential, which may facilitate iron-catalyzed formation of reactive oxygen species and thus be responsible for the observed effects. Only low molecular weight iron dextran induced oxidative stress and inflammation in the heart.

Protective effects of baicalin and quercetin on an iron-overloaded mouse: comparison of liver, kidney and heart tissues.

Excessive accumulation of iron in the body can lead to organ injury. Some flavonoids which possess high affinity to iron can be used as iron chelators for curing iron overload diseases such as haemochromatosis. In this article, the effects of baicalin and quercetin on different mouse tissues (liver, kidney and heart) afflicted with iron overload-induced injury were studied. It was found that after administration of 500 mg kg⁻¹ iron dextran for 45 days, the degree of oxidative injury in the three organs was different. When iron dextran-induced iron-overloaded mice were fed a diet containing baicalin or quercetin (1% w/w), both flavonoids showed significant effects in suppressing iron overload-induced injury. The efficiencies of both flavonoids on different tissues varied. Both flavonoids caused a significant decrease of iron content in liver and kidney tissues but not in the heart, whereas the flavonoids were more efficient in inhibiting the increase of carbonyl content and the thiobarbituric acid reactive substance levels in heart tissues. The results indicate that the protective effects of baicalin and quercetin on different tissues of iron-overloaded mice are different.

Establishment of secondary iron overloaded mouse model: evaluation of cardiac function and analysis according to iron concentration.

Periodic blood transfusion can lead to secondary iron overload in patients with hematologic and oncologic diseases. Iron overload can result in iron deposition in heart tissue, which decreases cardiac function and can ultimately lead to death due to dilated cardiomyopathy and cardiac failure. In this study, we established murine model of secondary iron overload, studied the changes in cardiac function with echocardiography, and examined the histopathologic changes. Three experimental groups of the six week-old C57/BL mice (H-2(b)) were injected intraperitoneally with 10 mg of iron dextran daily 5 days a week for 2, 4, and 6 weeks. Cumulative doses of iron for the three experimental groups were 100, 200, and 300 mg, while the control groups were injected with the same amounts of phosphate-buffered saline. We studied the cardiac function under anesthesia with echocardiography using a GE Vivid7 Dimension system. Plasma iron levels and liver iron contents were measured. The hearts and livers were harvested and stained with H&E and Perls Prussian blue for iron, and the levels of iron deposit were examined. We assessed the cardiac measurements after adjustment for weight. On echocardiography, thicknesses of the interventricular septum and posterior ventricular wall (PS) during diastole showed correlation with the amount of iron deposit (P < 0.01). End-diastolic volume showed dilatation of the left ventricle in the 300 mg group (P < 0.01). Changes in the fractional shortening were not statistically significant (P = 0.07). Plasma iron levels and liver iron contents were increased proportionally according to the amount of iron loaded. The histopathologic findings of PS and liver showed higher grade of iron deposit proportional to the cumulated iron dose. In this study, we present an animal model which helps understand the cardiac function changes in patients with secondary iron overload due to repeated blood transfusions. Our results may help characterize the pathophysiologic features of cardiomyopathy in patients with secondary iron overload, and our model may be applied to in vivo iron-chelating therapy studies.

Deferasirox protects against iron-induced hepatic injury in Mongolian gerbil.

Iron overload is associated with an increased risk of liver complications including fibrosis, cirrhosis, and hepatocellular carcinoma. Deferasirox is a new oral chelator with high iron-binding potency and selectivity. Here we investigate the ability of deferasirox to remove excessive hepatic iron and prevent iron-induced hepatic injury. Adult male Mongolian gerbils were divided into 3 groups (n=5/group)-control, iron overload (100 mg iron-dextran/kg body weight/5 days; intraperitoneal for 10 weeks), and iron overload followed by deferasirox treatment (100 mg deferasirox/kg body weight/d; pulse oral for 1 or 3 months). Compared with the nontreated iron overload group, deferasirox reduced hepatic iron concentration by 44% after 3 months of treatment (P<0.05). Histological analysis of hepatic tissue from the iron overloaded group detected frequent iron deposition, evidence of hepatic damage, and an accumulation of lipid vacuoles. Iron deposition was significantly diminished with deferasirox treatment, and no evidence of lipid accumulation was observed. Immunoblotting demonstrated that iron overload caused approximately 2-fold increase in hepatic ferritin expression (P<0.05), which was 48% lower after 3 months of deferasirox treatment (P<0.05). Deferasirox treatment also was associated with reduced hepatic protein oxidation, superoxide abundance, and cell death. The percentage of terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells in the deferasirox-treated livers was 41% lower than that of iron overloaded group (P<0.05). Similarly, an iron-related increase in the expression of Bax/Bcl2, Bad, and caspase-3 were significantly lower after deferasirox treatment. These findings suggest that deferasirox may confer protection against iron-induced hepatic toxicity.

Adiponectin-mediated heme oxygenase-1 induction protects against iron-induced liver injury via a PPARα dependent mechanism.

Protective effects of adiponectin (APN; an adipocytokine) were shown against various oxidative challenges; however, its therapeutic implications and the mechanisms underlying hepatic iron overload remain unclear. Herein, we show that the deleterious effects of iron dextran on liver function and iron deposition were significantly reversed by adiponectin gene therapy, which was accompanied by AMP-activated protein kinase (AMPK) phosphorylation and heme oxygenase (HO)-1 induction. Furthermore, AMPK-mediated peroxisome proliferator-activated receptor-α (PPARα) activation by APN was ascribable to HO-1 induction. Additionally, we revealed direct transcriptional regulation of HO-1 by the binding of PPARα to a PPAR-responsive element (PPRE) by various experimental assessments. Interestingly, overexpression of HO-1 in hepatocytes mimicked the protective effect of APN in attenuating iron-mediated injury, whereas it was abolished by SnPP and small interfering HO-1. Furthermore, bilirubin, the end-product of the HO-1 reaction, but not CO, protected hepatocytes from iron dextran-mediated caspase activation. Herein, we demonstrate a novel functional PPRE in the promoter regions of HO-1, and APN-mediated HO-1 induction elicited an antiapoptotic effect and a decrease in iron deposition in hepatocytes subjected to iron challenge.