The effect of the flavonol rutin on serum and liver iron content in a genetic mouse model of iron overload

The flavonol rutin has been shown to possess antioxidant and iron chelating properties in vitro and in vivo. These dual properties are beneficial as therapeutic options to reduce iron accumulation and the generation of reactive oxygen species (ROS) resultant from excess free iron. The effect of rutin on iron metabolism has been limited to studies performed in wildtype mice either injected or fed high-iron diets. The effect of rutin on iron overload caused by genetic dysregulation of iron homoeostasis has not yet been investigated. In the present study we examined the effect of rutin treatment on tissue iron loading in a genetic mouse model of iron overload, which mirrors the iron loading associated with Type 3 hereditary haemochromatosis patients who have a defect in Transferrin Receptor 2 (TFR2). Male TFR2 knockout (KO) mice were administered rutin via oral gavage for 21 continuous days. Following treatment, iron levels in serum, liver, duodenum and spleen were assessed. In addition, hepatic ferritin protein levels were determined by Western blotting, and expression of iron homoeostasis genes by quantitative real-time PCR. Rutin treatment resulted in a significant reduction in hepatic ferritin protein expression and serum transferrin saturation. In addition, trends towards decreased iron levels in the liver and serum, and increased serum unsaturated iron binding capacity were observed. This is the first study to explore the utility of rutin as a potential iron chelator and therapeutic in an animal model of genetic iron overload.     

Iron accumulation in tissues of magnesium-deficient rats with dietary iron overload

The mineral imbalances in magnesium-deficient rats with dietary iron overload were studied. Forty-four male Wister rats were divided into six groups and fed six diets, two by three, fully crossed: magnesium adequate or deficient, and iron deficient, adequate, or excess. The concentrations of iron, magnesium, calcium, and phosphorus in tissues of the rats were measured. The results were as follows: (1) The excess iron intake reinforced the iron accumulation in liver and spleen of magnesium deficient rats; (2) The saturation of iron binding capacity was enormously elevated in the magnesium deficient rats fed excess iron; and (3) Dietary iron deprivation diminished the degree of calcium deposition in kidney of magnesium deficient rats. These results suggest that magnesium-deprived-rats have abnormal iron metabolism losing homeostatic regulation of plasma iron, and magnesium deficient rats with dietary iron overload may be used as an experimental hemochromatosis model.     

Iron and zinc status in rats with diet-induced marginal deficiency of vitamin A and/or copper

The hypothesis was tested that there are interactions of marginal copper and vitamin A deficiency regarding iron and zinc status. Copper restriction (1 vs 5 mg Cu/kg diet) significantly lowered copper concentrations in plasma and tissues of rats and reduced blood hemoglobin, hematocrit, and iron concentrations in tibia and femur, but raised iron concentrations in liver. Vitamin A restriction (0 vs 4000 IU vitamin A/kg diet) reduced plasma retinol concentrations and induced a fall of blood hemoglobin and hematocrit. Neither copper nor vitamin A restriction for up to 42 d affected feed intake and body wt gain. There were no interrelated effects of vitamin A and copper deficiency on iron status. Copper deficiency slightly depressed liver, spleen, and kidney zinc concentrations. Vitamin A deficiency lowered zinc concentrations in heart, but only when the diets were deficient in copper.     

Iron deficiency stress can induce MxNAS1 protein expression to facilitate iron redistribution in Malus xiaojinensis

• Iron (Fe) is a vital trace element in plants, and deficiency of this element in apple trees can reduce fruit quality. Nicotianamine (NA) is known to play an important role in Fe transport and endogenous hormone balance. In the present study, we investigated the role of a nicotianamine synthase 1 gene (MxNas1) in an apple species, Malus xiaojinensis, that has a more Fe‐efficient genotype than other apple species and ecotypes.
• To characterise the response of M. xiaojinensis to Fe deficiency, we used quantitative Q‐PCR to determine the level of expression of MxNas1 and Western blot to measure protein levels. Immunohistochemical staining and GFP fluorescence localisation of the MxNAS1 protein were also carried out. HPLC and polarised absorption spectrophotometry were performed to investigate the effects of overexpression of MxNas1 in order to elucidate the role of MxNAS1 in the cellular uptake of active Fe in tobacco suspension cells.
• We found that MxNas1 expression and protein levels were higher under Fe deficiency stress than under Fe sufficiency. Immunohistochemical staining showed that MxNAS1 was localised mainly in the epidermal and vascular tissues of the roots, vascular tissues of the stem and palisade cells of mature leaves, and in parenchyma cells of young leaves. MxNAS1 was mainly localised in the plasma membranes and vesicles of protoplasts. In addition, overexpression of MxNas1 in stable transgenic tobacco cells increased NA and active Fe content under Fe sufficiency.
• The results suggest that MxNas1 expression in M. xiaojinensis is induced in response to Fe deficiency stress, resulting in higher levels of the protein. MxNAS1 may be involved in the redistribution of Fe in M. xiaojinensis under Fe deficiency.

     

Effects of iron deficiency and iron overload on manganese uptake and deposition in the brain and other organs of the rat

Managanese (Mn) is an essential trace element at low concentrations, but at higher concentrations is neurotoxic. It has several chemical and biochemical properties similar to iron (Fe), and there is evidence of metabolic interaction between the two metals, particularly at the level of absorption from the intestine. The aim of this investigation was to determine whether Mn and Fe interact during the processes involved in uptake from the plasma by the brain and other organs of the rat. Dams were fed control (70 mg Fe/kg), Fe-deficient (5–10 mg Fe/kg), or Fe-loaded (20 g carbonyl Fe/kg) diets, with or without Mn-loaded drinking water (2 g Mn/L), from day 18–19 of pregnancy, and, after weaning the young rats, were continued on the same dietary regimens. Measurements of brain, liver, and kidney Mn and nonheme Fe levels, and the uptake of⁵⁴Mn and⁵⁹Fe from the plasma by these organs and the femurs, were made when the rats were aged 15 and 63 d. Organ nonheme Fe levels were much higher than Mn levels, and in the liver and kidney increased much more with Fe loading than did Mn levels with Mn loading. However, in the brain the increases were greater for Mn. Both Fe depletion and loading led to increased brain Mn concentrations in the 15-d/rats, while Fe loading also had this effect at 63 d. Mn loading did not have significant effects on the nonheme Fe concentrations.⁵⁴Mn, injected as MnCl₂ mixed with serum, was cleared more rapidly from the circulation than was⁵⁹Fe, injected in the form of diferric transferrin. In the 15-d-rats, the uptake of⁵⁴Mn by brain, liver, kidneys, and femurs was increased by Fe loading, but this was not seen in the 63-d rats. Mn supplementation led to increased⁵⁹Fe uptake by the brain, liver, and kidneys of the rats fed the control and Fe-deficient diets, but not in the Fe-loaded rats. It is concluded that Mn and Fe interact during transfer from the plasma to the brain and other organs and that this interaction is synergistic rather than competitive in nature. Hence, excessive intake of Fe plus Mn may accentuate the risk of tissue damage caused by one metal alone, particularly in the brain.     

哺乳动物铁稳态分子机制研究进展

铁是机体必需微量元素,参与机体合成血红蛋白、肌红蛋白及多种酶的组成和功能发挥,对维持生命和健康至关重要。近四分之一的世界人口遭受铁缺乏或缺铁性贫血的威胁。此外,部分人群还存在铁过载问题,以脏器铁离子蓄积为主要病理改变的遗传性血色病,其在欧美发病率高达1/200,在中国也有报道。血色病后期多诱发肝脏、胰腺及心脏的功能衰退。铁过少或过多对健康都会造成严重危害,机体需要复杂而精密的调控体系维持铁稳态平衡。铁代谢主要包括小肠吸收、肝脏储存、血液转运、巨噬细胞再循环以及周身细胞利用。过去十多年是铁代谢研究的黄金时期,先后发现众多铁稳态代谢相关基因。该文综述了近年来哺乳动物铁代谢领域的研究进展,并对铁稳态代谢中存在的问题进行了初步讨论,为理解和进一步深入研究铁代谢分子机制提供参考。     

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model

Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.     

Limited effects of combined dietary copper deficiency/iron overload on oxidative stress parameters in rat liver and plasma

Copper (Cu) deficiency decreases the activity of Cu-dependent antioxidant enzymes such as Cu,zinc-superoxide dismutase (Cu,Zn-SOD) and may be associated with increased susceptibility to oxidative stress. Iron (Fe) overload represents a dietary oxidative stress relevant to overuse of Fe-containing supplements and to hereditary hemochromatosis. In a study to investigate oxidative stress interactions of dietary Cu deficiency with Fe overload, weanling male Long–Evans rats were fed one of four sucrose-based modified AIN-93G diets formulated to differ in Cu (adequate 6 mg/kg diet vs. deficient 0.5 mg/kg) and Fe (adequate 35 mg/kg vs. overloaded 1500 mg/kg) in a 2×2 factorial design for 4 weeks prior to necropsy. Care was taken to minimize oxidation of the diets prior to feeding to the rats. Liver and plasma Cu content and liver Cu,Zn-SOD activity declined with Cu deficiency and liver Fe increased with Fe overload, confirming the experimental dietary model. Liver thiobarbituric acid reactive substances were significantly elevated with Fe overload (pooled across Cu treatments, 0.80±0.14 vs. 0.54±0.08 nmol/mg protein; P<.0001) and not affected by Cu deficiency. Liver cytosolic protein carbonyl content and the concentrations of several oxidized cholesterol species in liver tissue did not change with these dietary treatments. Plasma protein carbonyl content decreased in Cu-deficient rats and was not influenced by dietary Fe overload. The various substrates (lipid, protein and cholesterol) appeared to differ in their susceptibility to the in vivo oxidative stress induced by dietary Fe overload, but these differences were not exacerbated by Cu deficiency.     

Retrospective evaluation of the incidence and severity of hemosiderosis in a large captive lemur population

Significant concern has been generated about the susceptibility of captive lemurs to iron storage disease, which has led some researchers to propose husbandry changes regarding dietary iron. In the current study we sought to determine the history, severity, and prevalence of iron storage disease within a large captive lemur population. Iron concentration and hemosiderin accumulation in a target organ, the liver, were assessed in necropsy specimens from 15 different species (n=153) of lemurs over a 12-yr period at the Duke University Primate Center. Banked liver tissue was used to quantify liver iron concentration (LIC) via neutron activation analysis (NAA). Prussian blue staining was used to accentuate the presence of liver iron for evaluation using an established scoring system. Of the 153 reports examined, 49 (32%) of the animals were considered positive for the presence of hemosiderin in the liver, lymph node, duodenum, and kidney, with 36 of the 49 (73%) showing deposition of iron in the liver. Total iron scores (TIS) ranged from 0.3+/-0.3 in Lemur catta to 33.3+/-1.7 in Cheirogaleus medius. The mean LIC ranged from 209+/-1.4 microg/g wet weight in L. catta to 2957+/-414 microg/g in C. medius. Management practices may have contributed to some of the results observed in this study. Although evidence of excess iron deposition in the liver was present across several species studied, the levels were not as pervasive as previously reported in other captive lemur populations. Hemochromatosis was not observed, and excess iron was not related to the cause of death in any of the animals studied. The current findings suggest that iron overload in lemurs may be more complex than was previously believed.     

Rat brain iron concentration is lower following perinatal copper deficiency

Experiments performed with Holtzman rats demonstrated that brain iron (Fe) was lower by postnatal day 13 (P13) in pups born and nursed by dams that began copper-deficient (-Cu) treatment at embryonic day 7. Transcardial perfusion of P24-P26 males and females to remove blood Fe contamination revealed that brain Fe was still 20% lower in -Cu than +Cu rats. Estimated blood content of brain for -Cu rats was greater than for +Cu rats; for all groups, values ranged between 0.43 and 1.03%. Using group-specific data and regression analyses, r = 0.99, relating blood Fe to hemoglobin, brain Fe in non-perfused rats in a replicate study was lower by 33% at P13 and 39% at P24 in -Cu rats. Brain extracts from these rats and from P50 rats from a post-weaning model were compared by immunoblotting for transferrin receptor (TfR1). P24 brain -Cu/+Cu TfR1 was 3.08, suggesting that brains of -Cu rats were indeed Fe deficient. This ratio in P13 rats was 1.44, p < 0.05. No change in P50 -Cu rat brain TfR1 or Fe content was detected despite a 50% reduction in plasma Fe. The results suggest that brain Fe accumulation depends on adequate Cu nutriture during perinatal development.     

Optimal copper supply is required for normal plant iron deficiency responses

Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. Understanding crosstalk between Fe and Cu nutrition could lead to strategies for improved growth on soils with low or excess metals, with implications for agriculture and phytoremediation. Here, we show that Cu and Fe nutrition interact to increase or decrease Fe and/or Cu accumulation in leaves and Fe uptake processes. Leaf Cu concentration increased under low Fe supply, while high Cu lowered leaf Fe concentration. Ferric reductase activity, an indicator of Fe demand, was inhibited at insufficient or high Cu supply. Surprisingly, plants grown without Fe were more susceptible to Cu toxicity.     

铁代谢红细胞系调节因子与铁稳态

红细胞合成是人类和其他脊椎动物最耗铁的生理过程,对机体铁稳态具有重要调节作用。Erythroferrone（ERFE）是红细胞系来源的调节铁调素的主要激素。当机体存在应激性红细胞合成时,ERFE合成增加,铁调素表达受抑,可促进机体铁吸收和储铁动员,满足红细胞合成对铁的需求,但在无效红细胞生成疾病中,通过此作用也导致了铁过载的发生。ERFE抑制肝细胞合成铁调素的作用机制尚不清楚,但至少部分地依赖BMP/SMAD信号通路。ERFE对铁代谢障碍性疾病和红细胞生成紊乱性贫血有重要的诊断及治疗价值。     

Iron overload induces cerebral endothelial senescence in aged mice and in primary culture in a sex-dependent manner

Iron imbalance in the brain negatively affects brain function. With aging, iron levels increase in the brain and contribute to brain damage and neurological disorders. Changes in the cerebral vasculature with aging may enhance iron entry into the brain parenchyma, leading to iron overload and its deleterious consequences. Endothelial senescence has emerged as an important contributor to age-related changes in the cerebral vasculature. Evidence indicates that iron overload may induce senescence in cultured cell lines. Importantly, cells derived from female human and mice generally show enhanced senescence-associated phenotype, compared with males. Thus, we hypothesize that cerebral endothelial cells (CEC) derived from aged female mice are more susceptible to iron-induced senescence, compared with CEC from aged males. We found that aged female mice, but not males, showed cognitive deficits when chronically treated with ferric citrate (FC), and their brains and the brain vasculature showed senescence-associated phenotype. We also found that primary culture of CEC derived from aged female mice, but not male-derived CEC, exhibited senescence-associated phenotype when treated with FC. We identified that the transmembrane receptor Robo4 was downregulated in the brain vasculature and in cultured primary CEC derived from aged female mice, compared with those from male mice. We discovered that Robo4 downregulation contributed to enhanced vulnerability to FC-induced senescence. Thus, our study identifies Robo4 downregulation as a driver of senescence induced by iron overload in primary culture of CEC and a potential risk factor of brain vasculature impairment and brain dysfunction.     

Iron and cadmium uptake by duodenum of hypotransferrinaemic mice

Absorption from food is an important route for entry of the toxic metal, cadmium, into the body. Both cadmium and iron are believed to be taken up by duodenal enterocytes via the iron regulated, proton-coupled transporter, DMT1. This means that cadmium uptake could be enhanced in conditions where iron absorption is increased. We measured pH dependent uptake of ¹⁰⁹Cd and ⁵⁹Fe by duodenum from mice with an in vitro method. Mice with experimental (hypoxia, iron deficiency) or hereditary (hypotransferrinaemia) increased iron absorption were studied. All three groups of mice showed increased ⁵⁹Fe uptake (p<0.05) compared to their respective controls. Hypotransferrinaemic and iron deficient mice exhibited an increase in ¹⁰⁹Cd uptake (p<0.05). Cadmium uptake was not, however, increased by lowering the medium pH from 7.4 to 6. In contrast, ⁵⁹Fe uptake (from ⁵⁹FeNTA₂) and ferric reductase activity was increased by lowering medium pH in control and iron deficient mice (p<0.05). The data show that duodenal cadmium uptake can be increased by hereditary iron overload conditions. The uptake is not, however, altered by lowering medium pH suggesting that DMT1-independent uptake pathways may operate.     

Hepcidin deficiency undermines bone load-bearing capacity through inducing iron overload

Osteoporosis is one of the leading disorders among aged people. Bone loss results from a number of physiological alterations, such as estrogen decline and aging. Meanwhile, iron overload has been recognized as a risk factor for bone loss. Systemic iron homeostasis is fundamentally governed by the hepcidin–ferroportin regulatory axis, where hepcidin is the key regulator. Hepcidin deficiency could induce a few disorders, of which iron overload is the most representative phenotype. However, there was little investigation of the effects of hepcidin deficiency on bone metabolism. To this end, hepcidin-deficient (Hamp1^−/−) mice were employed to address this issue. Our results revealed that significant iron overload was induced in Hamp1^−/− mice. Importantly, significant decreases of maximal loading and maximal bending stress were found in Hamp1^−/− mice relative to wildtype (WT) mice. Moreover, the levels of the C-telopeptide of type I collagen (CTX-1) increased in Hamp1^−/− mice. Therefore, hepcidin deficiency resulted in a marked reduction of bone load-bearing capacity likely through enhancing bone resorption, suggesting a direct correlation between hepcidin deficiency and bone loss. Targeting hepcidin or the pathway it modulates may thus represent a therapeutic for osteopenia or osteoporosis.     

Iron and neoplasia

Normal and neoplastic cells (like nonpathogenic and pathogenic microorganisms) apparently have similar needs and tolerances for iron, but neoplastic cells (like pathogenic microorganisms) may exhibit altered mechanisms of iron acquisition that permit continued growth in host iron-restricted tissues. Excess iron tends to interfere with host defense against malignant cells (as well as against microbial invaders); severe iron deficiency may likewise be detrimental. Elevated temperature is more toxic towards neoplastic than to normal host cells; it is not yet known whether the site of action of heat might be associated with iron acquisition (as has been demonstrated for gram negative bacteria). Persons or animals with iron overload tend to be at greater risk than normal hosts in the development of neoplasms. Construction of animal models of iron overload, although difficult, is strongly indicated at this time. Based on such models, decisions then can be made about the extent to which (a) nutritional immunity against neoplastic cells is practiced by vertebrate hosts and (b) clinical procedures could be employed to strengthen such immunity as an adjunct to radiotherapy, chemotherapy, and surgery.     

Serum iron and copper status and oxidative stress in severe and mild preeclampsia

Our aim was to investigate parameters of iron and copper status and oxidative stress and antioxidant function in women with healthy pregnancy, mild and severe preeclampsia with a view to exploring the possible contribution of these parameters to the aetiology. Thirty healthy, 30 mild preeclamptic and 30 severe preeclamptic pregnant women were included. Serum and placental lipid peroxides, and serum vitamin E and total carotene levels were measured by colorimetric assay. Cholesterol, copper, iron, total iron binding capacity (TIBC), ceruloplasmin and transferrin concentrations were measured by commercially available procedures. Data were analysed statistically using one-way analysis of variance and Pearson correlation test. Logistic regression procedures were used to calculate odds ratios. Lipid peroxides in serum and placental tissue, and iron, copper and ceruloplasmin levels in serum were significantly increased, and transferrin, TIBC, vitamin E/total cholesterol and total carotene/total cholesterol ratios in serum were significantly decreased especially in women with severe preeclampsia. Significant correlations were detected between serum iron and lipid peroxides in serum and placental tissue and between serum iron and vitamin E/total cholesterol in severe preeclamptic pregnancy. Furthermore, there were significant correlations between serum malondialdehyde and ceruloplasmin and vitamin E/total cholesterol in women with severe preeclampsia, and changes in serum and placental lipid peroxides and serum iron concentrations were significantly associated with preeclampsia. In conclusion, ischaemic placental tissue may be a primary source of potentially toxic iron in preeclampsia and the released iron species may contribute to the aetiology and would exacerbate lipid peroxidation and endothelial cell injury, which may be abated by antioxidant supplementation.