Iron-Chelating Potential of Novel Phytochemicals in Poplar and Cedar Trees

Introduction: Iron overload is not only a consequence of diseases such as thalassemia and hereditary hemochromatosis (HFE), but also of neurodegeneration with brain iron accumulation (NBIA). In HFE, iron increases the risk of developing hepatocellular and colorectal cancers. Excess iron resulting from disruptions in normal iron homeostasis can accumulate in major organs including liver, heart and brain, and has devastating effects if left untreated. Currently, treatment includes using iron chelators, which at higher concentrations can have significant adverse effects and require constant medical supervision. Therefore, the search for alternative unique or adjuvant iron chelators that have reduced toxicity could be of significant benefit. Plants that grow in alkaline soils may be a good source of chelators for this purpose. Since iron is generally unavailable in such soils, plant roots have evolved mechanisms to solubilize iron for uptake, such as soil acidification, but need additional strategies to overcome high alkalinity. This may include producing secondary metabolites that are exuded into the soil and can chelate iron directly, including phenolic acids that may chelate iron at physiological pH in humans. This project is focused on finding, isolating, and testing bioactivity of compounds from western red cedar (alkaline tolerant) and poplar (rich in phenolics). Methods: Plants are grown in iron-normal and -deficient conditions in an innovative aeroponic system to stimulate the production of secondary metabolites related to Fe deficiency. Plant tissue extracts and root washings are collected and concentrated with solid phase extraction chromatography to form plant-derived concentrates (PDC) that are analyzed by UPLC-MS and colourimetric assays to isolate, identify, and characterize compounds induced by iron-deficiency. For bio-activity testing, PDCs are introduced to cultures of THP-1 cells, a model human monocytic cell line, to study their effect on Fe homeostasis. Prior to treatment with chelators, cells are cultured under normal (Con) and Fe-overload (CrFe) conditions (produced by treatment with 10 and 20 µM Fe-citrate) for one week to model human chronic iron overload. Deferoxamine (DFO), a well-known clinical iron chelator, model phenolics like caffeic (CafA) and chlorogenic acid (CGA), and PDCs have been applied to cultures as potential chelators. Results: Leaf compared to root tissues from poplar vary greatly in their CGA and phenolic content. Leaf extracts contained 5 times more phenolics than root extracts, and root extracts from iron-deficient plants produced 66% more phenolic compounds than those from iron-normal plants. Compared to leaf extracts, root extracts showed a 4-fold increase in iron-binding activity in vitro. PDCs including these extracts were found to contain compounds responsive to iron deficiency, which are semi-polar and low in molecular weight (140 - 340 m/z). Distinct iron-responsive compounds were also identified from cedar. Following acute dosage with Fe-citrate, THP-1 cells showed a moderate reduction in iron content after treatment with CafA, CGA, and PDCs from roots, with no influence on cell viability. On-going work includes dose-dependency with CafA and PDCs and co-operative effects of PDCs with DFO. Iron-loading in THP-1 cells is time sensitive, with maximum iron uptake measured at 8 hours following delivery of 20 µM Fe-citrate. Detailed kinetics of cellular iron-loading in the presence of iron-chelators is currently being investigated. Conclusions: We found that low-molecular weight and water-soluble PDCs from iron-deficient plants had excellent iron-binding capacity in vitro, and inhibited iron uptake in THP-1 cells. Effects of chelators on cellular iron uptake is both dose and time dependent. Screening plants for novel chelators provides an abundance of opportunity to search for new chelators for human medicinal use. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Marginally excessive iron loading transiently blocks mucosal iron uptake in iron-deficient rats

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00305.2013 ◽

2014 ◽

Vol 307 (1) ◽

pp. G89-G97 ◽

Cited By ~ 6

Author(s):

Shoko Shinoda ◽

Shiho Yoshizawa ◽

Eriko Nozaki ◽

Kouki Tadai ◽

Anna Arita

Keyword(s):

Iron Uptake ◽

Iron Concentration ◽

Iron Loading ◽

Short Acting ◽

Excess Iron ◽

Rapid Induction ◽

Cellular Iron ◽

Iron Load ◽

Iron Deficient ◽

Cellular Iron Uptake

Regular “mucosal block” is characterized by decreased uptake of a normal iron load 3–72 h after the administration of excess iron (generally 10 mg) to iron-deficient animals. We found that short-acting mucosal block could be induced by much lower iron concentration and much shorter induction time than previously reported, without affecting levels of gene expression. A rapid endocytic mechanism was reported to decrease intestinal iron absorption after a high iron load, but the activating iron load and the time to decreased absorption were undetermined. We assessed the effects of 30–2,000 μg iron load on iron uptake in the duodenal loop of iron-deficient and iron-sufficient rats under anesthesia. One hour later, mucosal cellular iron uptake in iron-deficient rats administered 30 μg iron was 76.1%, decreasing 25% to 50.7% in rats administered 2,000 μg iron. In contrast, iron uptake by iron-sufficient rats was 63% (range 60.3–65.5%) regardless of iron load. Duodenal mucosal iron concentration was significantly lower in iron-deficient than in iron-sufficient rats. Iron levels in portal blood were consistently higher in iron-deficient rats regardless of iron load, in contrast to the decreased iron uptake on the luminal side. Iron loading blocked mucosal uptake of marginally excess iron (1,000 μg), with a greater effect at 15 min than at 30 min. The rapid induction of short-acting mucosal block only in iron-deficient rats suggests DMT1 internalization.

Download Full-text

The behavior of transferrin iron in the rat

Blood ◽

10.1182/blood.v57.2.218.218 ◽

1981 ◽

Vol 57 (2) ◽

pp. 218-228 ◽

Cited By ~ 16

Author(s):

H Huebers ◽

W Bauer ◽

E Huebers ◽

E Csiba ◽

C Finch

Keyword(s):

Binding Sites ◽

Specific Binding ◽

Ferrous Ammonium Sulfate ◽

Iron Binding ◽

Body Tissues ◽

Iron Deficient ◽

Diferric Transferrin ◽

Iron Exchange

Abstract The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.

Download Full-text

Characterization of Pit, a Streptococcus pneumoniae Iron Uptake ABC Transporter

Infection and Immunity ◽

10.1128/iai.70.8.4389-4398.2002 ◽

2002 ◽

Vol 70 (8) ◽

pp. 4389-4398 ◽

Cited By ~ 86

Author(s):

Jeremy S. Brown ◽

Sarah M. Gilliland ◽

Javier Ruiz-Albert ◽

David W. Holden

Keyword(s):

Streptococcus Pneumoniae ◽

Abc Transporter ◽

Iron Uptake ◽

Systemic Infection ◽

Iron Chelator ◽

Iron Deficient ◽

Deficient Medium ◽

Mutant Strains

ABSTRACT Bacteria frequently have multiple mechanisms for acquiring iron, an essential micronutrient, from the environment. We have identified a four-gene Streptococcus pneumoniae operon, named pit, encoding proteins with similarity to components of a putative Brachyspira hyodysenteriae iron uptake ABC transporter, Bit. An S. pneumoniae strain containing a defined mutation in pit has impaired growth in medium containing the iron chelator ethylenediamine di-o-hydroxyphenylacetic acid, reduced sensitivity to the iron-dependent antibiotic streptonigrin, and impaired virulence in a mouse model of S. pneumoniae systemic infection. Furthermore, addition of a mutation in pit to a strain containing mutations in the two previously described S. pneumoniae iron uptake ABC transporters, piu and pia, resulted in a strain with impaired growth in two types of iron-deficient medium, a high degree of resistance to streptonigrin, and a reduced rate of iron uptake. Comparison of the susceptibilities to streptonigrin of the individual pit, piu, and pia mutant strains and comparison of the growth in iron-deficient medium and virulence of single and double mutant strains suggest that pia is the dominant iron transporter during in vitro and in vivo growth.

Download Full-text

THE CARBOXYLASES OF LEAVES AND THEIR ROLE IN PHOTOSYNTHESIS

Canadian Journal of Botany ◽

10.1139/b52-031 ◽

1952 ◽

Vol 30 (4) ◽

pp. 395-409 ◽

Cited By ~ 20

Author(s):

K. A. Clendenning ◽

E. R. Waygood ◽

P. Weinberger

Keyword(s):

Sugar Beet ◽

Hydrogen Cyanide ◽

Malic Enzyme ◽

Hill Reaction ◽

Leaf Extracts ◽

Root Extracts ◽

Phosphoglyceric Acid ◽

Reaction Capacity ◽

The Hill

"Malic" enzyme isolated from the cytoplasm of parsley and sugar beet leaves was linked with illuminated spinach chloroplast fragments to effect photosynthesis in vitro. The model photosynthesis system containing excess "malic" enzyme was not inhibited by 5 × 10−4 M hydrogen cyanide. The "malic" enzyme system was inhibited by cyanide, however, at very low enzyme concentrations. The richest source of "malic" enzyme found in this study was the mature parsley leaf. Expressed on the same basis, the enzymatic capacities of parsley leaf "malic" enzyme and the Hill reaction capacity of isolated spinach chloroplasts are of similar magnitude. Higher "malic" enzyme and oxalacetic carboxylase activities were found in purified extracts of parsley leaves than in the corresponding root extracts. Oxalacetic, oxalsuccinic, α-ketoglutaric, and pyruvic carboxylases were not inhibited by 10−3 M hydrogen cyanide. The α-ketoglutaric and pyruvic carboxylases were much less abundant in leaves than in other plant organs; formic dehydrogenase was not detected in leaves although it is abundant in seeds. Glutamic carboxylase was found in the cytoplasm of wheat and sugar beet leaves, and with the aid of C14O2 was shown to be only weakly reversible. No evidence was obtained for the presence in leaf extracts of an enzyme, or mixture of enzymes, capable of decarboxylating phosphoglyceric acid in vitro.

Download Full-text

Synthetic methods and in vitro iron binding studies of the novel 1-alkyl-2-ethyl-3-hydroxypyrid-4-one iron chelators

Inorganica Chimica Acta ◽

10.1016/s0020-1693(00)84951-8 ◽

1988 ◽

Vol 152 (3) ◽

pp. 195-199 ◽

Cited By ~ 26

Author(s):

G.J. Kontoghiorghes ◽

L. Sheppard ◽

J. Barr

Keyword(s):

Synthetic Methods ◽

Iron Chelators ◽

Iron Binding ◽

The Novel ◽

Binding Studies

Download Full-text

Eltrombopag (ELT) Reverses Iron Mediated Suppression of Insulin Secretion in Pancreatic Cells By Chelating Iron and Decreasing ROS

Blood ◽

10.1182/blood.v128.22.1278.1278 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1278-1278 ◽

Cited By ~ 1

Author(s):

Evangelia Vlachodimitropoulou Koumoutsea ◽

Pimpisid Koonyosying ◽

John B. Porter ◽

Nichola Cooper ◽

Bethan Psaila ◽

...

Keyword(s):

Insulin Secretion ◽

Iron Overload ◽

B Cell ◽

Cell Line ◽

Iron Chelators ◽

Ros Production ◽

Iron Loading ◽

Intracellular Iron ◽

Pancreatic Cell ◽

Cellular Iron

Abstract Introduction: Eltrombopag (ELT) is an orally active, nonpeptide, small-molecule thrombopoietin receptor agonist (TPO-R), used to treat chronic immune thrombocytopenic purpura (ITP). We have recently reported its ability to mobilise cellular iron, and act as an iron shuttle when combined with currently licensed chelation therapies (Vlachodimitropoulou et al, Blood 2014, Volume 124, 21). Tissue damage induced by ROS production in iron overload conditions includes endocrine dysfunction including type I diabetes. We have developed a model where iron overload of the pancreatic cell line (RINm5F) inhibits insulin secretion. We investigated the ability of ELT, compared with clinically licensed iron chelators, to reverse ROS production and concomitant suppression of insulin production by iron loading of these cells. Methods: Cell line: RINm5F is a clonal rat pancreatic b cell line (LGC ATCC Sales, UK). These cells secrete insulin following a glucose challenge (Praz et al., 1983, Biochemistry J). Intracellular Iron: Cellular iron loading and mobilisation were measured as a decrease in cellular iron content using the ferrozine assay (Vlachodimitropoulou et al. 2015, British Journal of Haematology). A four-fold increase in intracellular iron compared to control was obtained by serially treating cells with 10% Fetal Bovine Serum (FBS) RPMI media in pancreatic cells over two ten hour periods (Figure 1A). The cells were then exposed to iron chelators/ELT, lysed and intracellular iron concentration determined, normalised against protein content. Reactive oxygen species (ROS) estimation: A cell-permeable oxidation-sensitive fluorescent probe 5,6-carboxy-2',7'- dichlorofluorescein diacetate (DCFH-DA); (Molecular Probes, Leiden, Netherlands) was used to measure intracellular ROS. Following iron loading, the cells were pre-incubated with 6 mM H2DCF-DA for 30 minutes at 37°C. Chelators were added and the fluorescence of control and treated cells was read throughout the treatment period in the plate reader (excitation 504 nm, emission 526 nm). Insulin quantification: Following iron loading and chelator treatment, the cells were challenged with Kreb's Ringer Buffer twice, for one hour at a time, containing 2.8mM and 16.7mM glucose (Lu et al. 2010, Toxicology letter). The supernatant was then collected and insulin concentration determined using a standard rat insulin ELISA kit (Life Technologies Limited, UK). Viability: The Sulforhodamide B (SRB) viability assay was used to ensure viability >98% and assess the toxicity on the pancreatic cell line. It is commonly used to measure drug-induced cytotoxicity and is a colorimetric assay dependent on healthy adherent cells. Results: Pancreatic cell iron loading was achieved with serial changes of media containing 10% FBS. This loading method was comparable to treating cells with ferric ammonium citrate (FAC) for 24 hours, which was not adopted as FAC adheres to the extracellular surface and produces bias to our intracellular iron quantification system when using iron chelators (Figure 1A). When cells were then treated with increasing ELT concentrations, a dose-dependent cellular iron removal were demonstrated so that at 10μΜ for 8hours, approximately 40% of total cellular iron was mobilised (Figure 3A). Iron mobilisation by ELT was further enhanced by combination with DFO, DFX or DFP (Figure 3). For example, when 10μΜ DFP is combined with 3μΜ ELT, iron mobilisation increases by a further 17% when compared to DFP treatment alone (Figure 3C). ROS production was also decreased in iron-loaded cells in a concentration-dependent manner by increasing ELT concentrations (Figure 2). These reductions in ROS and cellular iron were associated with restoration of insulin secretion, which was reduced by 2.6 fold following iron loading (Figure 1B). The levels of insulin secretion returned back to higher than baseline levels (better than with DFX 1μΜ) (Figure 1C). Conclusions: This is the first demonstration of a link between cellular iron overload and reduced insulin secretion using pancreatic b-cell line. This is also the first demonstration of improved pancreatic b-cell function, evidenced by restoration of insulin secretion, when iron is chelated and ROS decreased by ELT and other iron chelators. ELT may be useful alone or in combination with other chelators for decreasing iron-mediated ROS induced damage to pancreatic b-cells. Disclosures Porter: Novartis: Consultancy, Honoraria, Research Funding; Bluebird Bio: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Celegene: Consultancy.

Download Full-text

Do FeS clusters rule bacterial iron regulation?

Journal of Biological Chemistry ◽

10.1074/jbc.h120.016190 ◽

2020 ◽

Vol 295 (46) ◽

pp. 15464-15465

Author(s):

Roland Lill

Keyword(s):

Escherichia Coli ◽

Ferrous Iron ◽

Iron Uptake ◽

Iron Regulation ◽

Ferric Uptake Regulator ◽

Iron Binding ◽

Cellular Iron ◽

Open Question ◽

Exciting Possibility

For decades, the bacterial ferric uptake regulator (Fur) has been thought to respond to ferrous iron to transcriptionally regulate genes required for balancing iron uptake, storage, and utilization. Because iron binding to Fur has never been confirmed in vivo, the physiological iron-sensing mechanism remains an open question. Fontenot et al. now show that Fur purified from Escherichia coli binds an all-Cys-coordinated [2Fe-2S] cluster. This finding opens the exciting possibility that Fur may join numerous well-studied bacterial, fungal, and mammalian proteins that use FeS clusters for cellular iron regulation.

Download Full-text

Red cell iron uptake in hereditary microcytic anemia

Blood ◽

10.1182/blood.v46.3.381.bloodjournal463381 ◽

1975 ◽

Vol 46 (3) ◽

pp. 381-388 ◽

Cited By ~ 1

Author(s):

JA Edwards ◽

JE Hoke

Keyword(s):

Iron Uptake ◽

Binding Capacity ◽

Cell Receptor ◽

Microcytic Anemia ◽

Red Cells ◽

Red Cell ◽

Cellular Iron ◽

Receptor Sites ◽

Cellular Iron Uptake

The iron uptake in vitro of red cells from mice with hereditary microcytic anemia (gene symbol mk) was studied to examine the hypothesis of a generalized impairment of cellular iron uptake in this conidition. Reticulocyte-rich red cells from anemic (mk/mk) and acutely bled normal (+/+) mice were incubated in 59Fe-labeled mouse plasma and the radioiron uptake measured. The 59Fe uptake of the mk/mk and +/+ cells was related in the same way to the reticulocyte concentration, the duration of incubation, and the percentage saturation of the plasma iron-binding capacity. However, under the same conditions, the iron uptake of red cells from normal (+/+) mice was greater than that by red cells from anemic (mk/mk) mice. Furthermore, the cellular loss of radioiron on exposure to EDTA was greater for the mk/mk red cells, although the proportion of the radioiron taken up that was incorporated into heme was the same for mk/mk and +/+ red cells. These results support the hypothesis of a generalized impairment of cellular iron uptake in hereditary microcytic anemia and suggest that there might be a defect in red cell receptor sites for transferrin in this condition.

Download Full-text

The Use of Caco-2 Cells to Estimate Fe Absorption in Humans - a Critical Appraisal

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000038 ◽

2010 ◽

Vol 80 (45) ◽

pp. 307-313 ◽

Cited By ~ 17

Author(s):

Ann-Sofie Sandberg

Keyword(s):

Iron Uptake ◽

Iron Absorption ◽

Cell Model ◽

Iron Bioavailability ◽

Dietary Factors ◽

Cell Uptake ◽

Epithelial Cell Line ◽

Cellular Iron ◽

Cellular Iron Uptake

The Caco-2 cell model is widely used to assess the bioaccessibility/availability of iron from foods and diets. Analysis of iron uptake in this human epithelial cell line is usually preceded by a two-step digestion to simulate the conditions in the stomach and small intestine. Moreover, culturing the cells on inserts permits the measurement of iron transport. The cellular iron uptake is determined by direct measurements using radioisotopes, or indirectly by measurement of ferritin, the intracellular storage form of iron. There is a good correlation between Caco-2 cell uptake and human iron bioavailability for a number of dietary factors known to affect iron absorption. However, recent data suggest that in some cases there is no correlation. Possible reasons for such discrepancies, the benefits, and limitations of the Caco-2 cell model are discussed. In conclusion, in vitro experiments with Caco-2 cells are important tools for ranking foods with respect to bioavailability, for mechanistic studies of iron absorption, and for studies of dietary factors influencing absorption. The results need to be confirmed in humans.

Download Full-text

A COMPARATIVE STUDY OF THE ANTI-OXIDATIVE AND ANTI-DIABETIC POTENTIAL OF IN VITRO AND IN VIVO ROOT AND LEAF EXTRACTS OF WITHANIA SOMNIFERA ON STREPTOZOTOCIN INDUCED DIABETIC RATS

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2016v8i10.8103 ◽

2016 ◽

Vol 8 (10) ◽

pp. 85 ◽

Cited By ~ 1

Author(s):

Somanatha Jena ◽

Ram C. Jena ◽

Rasmita Bhol ◽

Khusbu Agarwal ◽

Ansuman Sarangi ◽

...

Keyword(s):

Blood Glucose ◽

Withania Somnifera ◽

Fasting Blood Glucose ◽

Density Lipoprotein ◽

Diabetic Rats ◽

Root Extract ◽

Leaf Extracts ◽

Root Extracts

Objective: The present investigation explores the possibilities of using the in vitro and in vivo root and leaf extracts of Withania somnifera for anti-diabetic and anti-hyperlipidaemic effects on streptozotocin-induced diabetic rats.Methods: In vitro shoot cultures of Withania somnifera were raised by the axillary proliferation in nodal explants from a garden grown plant using Murashige and Skoog medium then in vitro raised roots and shoots were used for the anti-hyperglycemic and anti-hyperlipidaemic experiment. After 72 h of STZ administration, the fasting blood glucose levels were measured and the rats showing FBG level>220 mg/dl were considered to be diabetic and were used for the hyperglycemic study. In vitro and in vivo methanolic root and leaf extracts were orally administered daily to diabetic rats for eight weeks. After the treatment period, blood glucose and serum enzymes like aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total cholesterol, triglycerides, HDL-c high density lipoprotein-bound cholesterol, LDL-c low density lipoprotein-bound cholesterol, LDH, serum protein level, total phenolics and anti-oxidative analysis (DPPH and FRAP) were determined.Results: The levels of blood glucose, AST, ALT, ALP, LDH, HDL-c significantly increased by the use of in vitro methanolic root extracts compared to normal control rats. However, remarkable loss of total protein, albumin, albumin: globulin (A: G) ratio was reported in streptozotocin-induced diabetic rats by using in vitro root extracts. Methanolic in vitro root extract at the dose levels of 300 mg/kg body weight produced a significant decrease in fasting blood glucose (FBG) level by 102.65 with respect to initial fasting blood glucose level after 30 d of the treatment. In vitro root extract demonstrated highest DPPH and FRAP free radical scavenging activity, i.e. 86.55±1.77 and 48.87±2.55 than other extracts.Conclusion: It may be concluded that methanolic in vitro root extract W. somnifera at the dose (300 mg/kg) has more potent anti-hyperglycaemic activity than the other in vitro and in vivo extracts of leaf and root on streptozotocin induced diabetic rats and was also found to be similar in effect to that of the standard drug ‘Glibenclamide’.

Download Full-text