Combined chelation of bi-functional bis-hydroxypiridinone and mono-hydroxypiridinone: Synthesis, solution and in vivo evaluation

3-Hydroxy-4-pyridinones (3,4-HP) are well known iron-chelators with applications in medicinal chemistry, mainly associated with their high affinity towards trivalent hard metal ions (e.g. M³⁺, M = Fe, Al, Ga) and use as decorporating agents in situations of metal accumulation. The polydenticity and the extra-functionality of 3,4-HP derivatives have been explored, aimed at improving the chelating efficacy and the selectivity of the interaction with specific biological receptors. However, the ideal conjugation of both features in one molecular unity usually leads to high molecular weight compounds which can have crossing-membrane limitations.Herein, a different approach is used combining a arylpiperazine-containing bis-hydroxypyridone (H₂L¹) with a biomimetic mono-hydroxypyridinone, ornithine-derivative (HL²), to assess the potential coadjuvating effect that could result from the administration of both compounds for the decorporation of hard metal ions. This work reports the results of solution and in vivo studies on their chelating efficacy either as a simple binary or a ternary system (H₂L¹:HL²:M³⁺), using potentiometric and spectrophotometric methods. The solution complexation studies with Fe(III) indicate that the solubility of the complexes is considerably increased in the ternary system, an important feature for the metal complex excretion, upon the metal sequestration. The results of the in vivo studies with ⁶⁷Ga-injected mice show differences on the biodistribution profiles of the radiotracer, upon the administration of each chelating agent, that are mainly ascribed to the differences of their extra-functional groups and lipo/hydrophilic character. However, administration of both chelating agents leads to a more steady metal mobilization, which may be attributed to an improved access to different cellular compartments.     

Biliary excretion of excess iron in mice requires hepatocyte iron import by Slc39a14

Iron is essential for erythropoiesis and other biological processes, but is toxic in excess. Dietary absorption of iron is a highly regulated process and is a major determinant of body iron levels. Iron excretion, however, is considered a passive, unregulated process, and the underlying pathways are unknown. Here we investigated the role of metal transporters SLC39A14 and SLC30A10 in biliary iron excretion. While SLC39A14 imports manganese into the liver and other organs under physiological conditions, it imports iron under conditions of iron excess. SLC30A10 exports manganese from hepatocytes into the bile. We hypothesized that biliary excretion of excess iron would be impaired by SLC39A14 and SLC30A10 deficiency. We therefore analyzed biliary iron excretion in Slc39a14-and Slc30a10-deficient mice raised on iron-sufficient and -rich diets. Bile was collected surgically from the mice, then analyzed with nonheme iron assays, mass spectrometry, ELISAs, and an electrophoretic assay for iron-loaded ferritin. Our results support a model in which biliary excretion of excess iron requires iron import into hepatocytes by SLC39A14, followed by iron export into the bile predominantly as ferritin, with iron export occurring independently of SLC30A10. To our knowledge, this is the first report of a molecular determinant of mammalian iron excretion and can serve as basis for future investigations into mechanisms of iron excretion and relevance to iron homeostasis.     

A New Chelation Method for Determining ATPase Activity in Skeletal Muscle

Traditional methods for visualizing ATPase in sections use heavy metals that generate visible metal salfide products. These methods use unpleasant and toxic reagents. We report a safer method using a novel ferric ion chelating agent to produce highly specific, low background, and permanent staining of muscle fiber enzymes.     

Inhibition of membrane-associated methyltransferases by a cholesterol-based metal chelator

We have designed, synthesized, and characterized a metal chelating compound that is based on the structure of cholesterol and contains the high affinity metal chelating group, lysine nitrilotriacetic acid (Lys-NTA). Using the enzyme isoprenylcysteine carboxylmethyltransferase (Icmt) from yeast as a model integral membrane metalloenzyme, we find that this agent potently inhibits Icmt activity with an IC(50) value between 35 and 75 microM, which is at least 40 times more potent than the best known Icmt metal chelating inhibitor, Zincon. We propose that the rigid hydrophobic cholesterol moiety promotes partitioning into the membrane, enabling the metal-binding NTA group(s) to inactivate the enzyme by metal chelation. Because this compound is based on a naturally occurring membrane lipid and appears to chelate metals buried deeply within water insoluble environments, this agent may also be useful as a general tool for identifying previously unappreciated metal dependencies of other classes of membrane proteins.     

Prevention of toxicity of metal ions to Aeromonas and other bacteria in drinking-water samples using nitrilotriaceticacid (NTA) instead of ethylenediaminetetraaceticacid (EDTA)

The addition of chelating agent to drinking-water samples reduces die-off, due to the toxic effect of metal ions, of bacteria such as Aeromonas. The use of ethylenediaminetetraaceticacid (EDTA) is undesirable since it is a highly persistent chemical which contributes to environmental pollution. This study shows that the less persistent nitrilotriaceticacid (NTA) is a suitable alternative. No significant differences ( P < 0.001) were detected between EDTA and NTA in protecting bacteria.     

The effect of some chelating agents on the biliary and urinary excretion of manganese in rats

The biliary excretion of manganese, in rats which have been loaded with manganese via their drinking water, can be significantly enhanced by the administration of the chelating agents: desferrioxamine (DSF), sodium bis(hydroxyethyl) dithiocarbamate (DEDTC), and sodium isonipecotamidedithiocarbamate (INADTC). The effect of these chelating agents on the urinary excretion of manganese was more complex and was found to be dependent upon the level of loading of manganese as well as the individual chelating agent. For animals with drinking water containing 400 mg/liter of manganese, the administration of the chelating agents led to a decrease in the sum of the biliary plus urinary manganese excretion. The results are of special interest in that they show that under some conditions the administration of chelating agents can lead to changes other than those expected.     

Effects of heavy metals on Tetraselmis suecica: Ultrastructural and energy-dispersive X-ray spectroscopic studies

The influence of metal contamination on the marine alga Tetraselmis suecica was investigated at physiological and ultrastructural levels. For this analysis, the growth response of this microalga was studied after the addition of various concentrations of heavy metals (Cd, Cu). The concentration corresponding to 50% growth inhibition (IC₅₀) and the number of days per cell cycle (Td) studied, revealed that the toxic effects of copper are heavier than those of cadmium. In the case of copper contamination, the Td grows with increasing metal concentration in the culture medium, while it remains unchanged during the cadmium contamination. The toxicity of cadmium, only observed in the latency phase of growth, suggests an adaptation phenomenon of T suecica to this metal. Ultrastructural changes in response to pollutants were investigated; copper induced cytoplasmic vacuolisation, organelle changes, appearance of cells with multilayered cell walls and excretion of organic matter. In the case of cadmium contamination, ultrastructural changes mainly affected the osmiophilic vesicles, of which both number and volume increased with increasing metal concentration in the culture medium. The results of X-ray microanalysis revealed that Cd and Cu were strongly present in excreted organic matter and osmiophilic vesicles. The latter can be excreted during cell division, thus participating in detoxification processes. Intracellular cadmium incorporation proved that some toxic effects of this metal are a result of interaction with endogenous cellular constituents. In the case of copper contamination, the presence of copper in walls of a multilayered cell suggests that these structures constitute an additionnal adsorbing area for this element, reducing metal free concentration in the medium. Mechanisms of metal detoxification of Tetraselmis suecica are discussed.     

Modelling root-induced solubilization of nutrients

Theory for coupled diffusion processes in soil is briefly described and three examples of its application to understand root-induced solubilization of nutrients given. The examples are: (1) solubilization of P through root-induced pH changes in the rhizosphere of rice plants growing in flooded soil; (2) solubilization of P through excretion of organic chelating agents from rice roots growing in aerobic soil; and (3) the effects of root geometry on P solubilization, particularly cylindrical versus planar geometry and the effect of excretion of a solubilizing agent being localized along the root axis. The theory is tested by comparing measured concentration profiles of P near roots with the predictions of the theory made using independently measured parameter values. In the examples given, the agreement between the observed and predicted concentration profiles is very good, indicating that the theory is sound and the processes involved well understood.     

Dental amalgam affects urinary selenium excretion

Selenium may have a protective effect against mercury toxicity. The aim of the present study was to investigate if selenium excretion in urine was affected in persons with dental amalgam fillings. The reason for this study is that dental amalgam is the most important source of inorganic mercury exposure in the general population, although the potential toxic effects of this exposure remain a subject for debate. The chelating agent 2,3 dimercaptopropane-1-sulfonate (DMPS) was injected intravenously (2 mg/kg) to provoke metal excretion. Urine samples were subsequently collected at intervals over a 24-h period. Selenium concentration was determined by hydride-generation atomic absorption spectrometry. The study was comprised of 20 persons who claimed symptoms from dental amalgam and 21 healthy persons with amalgam fillings. There were two control groups without amalgam. One control group had amalgam replaced because of concern about illness resulting from mercury release (n=20), whereas the other control group never had amalgam (n=19). Individuals with amalgam excreted less selenium (36.4 μg, median value) over 24 hours than those without amalgam (47.5 μg) (p=0.016). There was no difference in selenium excretion between groups with (42.4 μg) and without (39.4 μg) amalgam-related symptoms (p=0.15). The findings indicate that individuals exposed to low levels of elemental mercury from dental amalgam excrete less selenium to urine than unexposed individuals.     

Role of Oxalic Acid Overexcretion in Transformations of Toxic Metal Minerals by Beauveria caledonica

The fungus Beauveria caledonica was highly tolerant to toxic metals and solubilized cadmium, copper, lead, and zinc minerals, converting them into oxalates. This fungus was found to overexcrete organic acids with strong metal-chelating properties (oxalic and citric acids), suggesting that a ligand-promoted mechanism was the main mechanism of mineral dissolution. Our data also suggested that oxalic acid was the main mineral-transforming agent. Cadmium, copper, and zinc oxalates were precipitated by the fungus in the local environment and also in association with the mycelium. The presence of toxic metal minerals often led to the formation of mycelial cords, and in the presence of copper-containing minerals, these cords exhibited enhanced excretion of oxalic acid, which resulted in considerable encrustation of the cords by copper oxalate hydrate (moolooite). It was found that B. caledonica hyphae and cords were covered by a thick hydrated mucilaginous sheath which provided a microenvironment for chemical reactions, crystal deposition, and growth. Cryo-scanning electron microscopy revealed that mycogenic metal oxalates overgrew parental fungal hyphae, leaving a labyrinth of fungal tunnels within the newly formed mineral matter. X-ray absorption spectroscopy revealed that oxygen ligands played a major role in metal coordination within the fungal biomass during the accumulation of mobilized toxic metals by B. caledonica mycelium; these ligands were carboxylic groups in copper phosphate-containing medium and phosphate groups in pyromorphite-containing medium.     

Determination by ICP-MS and multivariate data analysis of elemental urine excretion profile during the EDTA chelation therapy: A case study

BackgroundBased on the medical history and laboratory analytical tests, a patient presenting symptoms compatible with Chronic Fatigue Syndrome was suspected of metal intoxication; therefore, a chelating therapy was attempted. In parallel, the profile of elemental excretion in urine was determined.MethodsChelation therapy by CaNa₂EDTA was administered every two weeks and urine samples were routinely collected for 17 months. The samples were mineralized with HNO₃ 69 % and analyzed by Inductively-Coupled Plasma – Mass Spectrometry. Data were processed by multivariate statistical methods.ResultsMost of the toxic elements showed a peak of excretion in 12−24 h after EDTA administration, which returned to basal level by 24−36 h after the treatment. Yet, the excretion of some trace elements persisted in the urine collected 26 h after the treatment.ConclusionsThe analysis of excreted metals following the CaNa₂EDTA infusion allowed to monitor dynamically the chelation therapy. The chelation therapy was effective in mobilizing and eliminating the principal heavy metals present from the body. However, since such clearance almost vanished 24 h after the treatment, a protocol with more frequent and low-dose administrations is advisable to improve the metal excretion.     

New tripodal hydroxypyridinone based chelating agents for Fe(III), Al(III) and Ga(III): Synthesis, physico-chemical properties and bioevaluation

Two new tris-hydroxypyridinone based compounds (KEMPPr(3,4-HP)₃ and KEMPBu(3,4-HP)₃) have been developed and studied as strong sequestering agents for iron and the group III of metal ions, aimed as potential pharmacological applications on metal-chelation therapy. Their structure is based on the KEMP acid scaffold to which three 3-hydroxy-4-pyridinone chelating moieties are attached via two different size spacers. After the preparation and characterization of the compounds their physico-chemical properties were studied, in relation with their metal binding affinity and lipophilicity. The KEMPPr(3,4-HP)₃ ligand was also bioassayed to evaluate its in vivo metal sequestering capacity from most organs using an animal model overload with ⁶⁷Ga. These studies showed that, for both in solution and in vivo conditions, the compounds have higher metal chelating efficacy than Deferriprone, the commercially available iron chelator in medical application, thus some perspectives are envisaged as potential pharmaceutical drug candidates for chelating therapy.     

Implications of reiterative DNA—Metal ion complexes in the induction and development of neoplastic cells

Experimental data on the content in metal ions of DNA preparations from various neoplastic and healthy tissues are summarized: metal ions are preferentially bound to reiterative DNA sequences, where they may induce conformational variations and thus modify the binding of effector molecules such as repressors and polymerases. A model is described where essential and toxic metals are successively loaded on ligand acceptor groups of increasing affinity and thus may reach the final active sites: enzymes and reiterative DNA sequences (equated at least partially to regulative DNA sequences). The effects of some molecules, including peptides, antibiotics, growth factors, hormones, and antineoplastic substances, on DNA conformation could be explained in part by their chelating ability. The neoplastic state may be induced by a modification of metal ion transfer chains: quantitatively by a continuous derepression of genes coding for metal ligands, genes that are only temporarily derepressed during development in normal cells, and qualitatively by modifications of the nucleotidic sequence of structural genes leading to an increase of the chelating ability of the coded metal ligand.     

Effect of EDTA and Tannic Acid on the Removal of Cd,Ni, Pb and Cu from Artificially Contaminated Soil by Althaea rosea Cavan

In this study an ornamental plant of Althaea rosea Cavan was investigated for its potential use in the removal of Cd, Ni, Pb and Cu from an artificially contaminated soil. Effect of two different chelating agents on the removal has also been studied by using EDTA (ethylenediaminetetracetic acid) and TA (tannic acid). Both EDTA and TA have led to higher heavy metal concentration in shoots and leaves compared to control plants. However EDTA is generally known as an effective agent in metal solubilisation of soil, in this study, TA was found more effective to induce metal accumulation in Althaea rosea Cavan under the studied conditions. In addition to this, EDTA is toxic to some species and restraining the growth of the plants. The higher BCF (Bio Concentration Factor) and TF (Translocation Factor) values obtained from stems and leaves by the effects of the chemical enhancers (EDTA and TA) show that Althaea rosea Cavan is a hyper accumulator for the studied metals and may be cultivated to clean the contaminated soils.     

Characterization of Lactobacillus brevis L62 strain, highly tolerant to copper ions

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stress-resistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non-toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.     

Activation of NADP-specific isocitrate dehydrogenase by chelating agents.

The effects of different metal chelating agents on the activity of the NADP-linked isocitrate dehydrogenase from pig heart have been studied. Addition of ethylene glycolbis(β-aminoethyleter) N,N′-tetraacetic acid, N-hydroxyethylenediamine triacetic acid, and ethylenediamine tetraacetic acid (EDTA) under certain conditions could enhance the activity by a factor of nearly 3. Moreover, the time lag occurring before the reaction rate approached a constant value at suboptimal metal-ion concentrations was abolished by the metal chelating agents. S^0.5 for isocitrate increased slightly in the presence of the metal-chelating agents. The substrate inhibition occurring at high NADP concentrations was abolished by the activator. The pH optimum was the same in the absence and presence of EDTA. The extent of activation increased on a relative basis with increasing pH. Studies of the sedimentation behavior of the enzyme under different conditions suggested that the effect of the metal-chelating agents could not be accounted for by aggregation or depolymerization of the enzyme. NADPH affects the enzyme activity in a similar way, although less efficiently than the metal chelating agents. The results indicate that most organic metal complexes can activate the enzyme. It has previously been suggested that isocitrate complexed with a metal ion is the real substrate for the enzyme. If this holds true, the activation found with other organic metal complexes can be accounted for by a reduction in the apparent K_m for the isocitrate metal complex and by an increase in the maximum rate of the reaction by removal of the substrate inhibition at high NADP concentrations.     

Synthesis of a new chelating gel: removal of Ca2+ ions from parvalbumin

The synthesis of a chelating gel which contains the effective metal chelating agent ethylenediaminetetraacetic acid covalently linked to amino-agarose is described. This gel is shown to be a rapid and extremely effective material for the removal of tightly bound, but labile metal ions from proteins without introducing contaminants into the biological system. The synthesis involves the formation of an amide linkage between the dangling carboxylate arm of the [Co(EDTA)Cl]2-complex and amino-agarose using a standard carbodiimide coupling reaction. The chelating gel is shown to remove approximately 98.5% of the calcium from fully bound Ca2-parvalbumin and over 99% of the europium from Eu2-parvalbumin.     

Influence of Complexation on the Uptake by Plants of Iron, Manganese, Copper and Zinc: II. EFFECT OF DTPA IN A MULTI-METAL AND COMPUTER SIMULATION STUDY

Barley (Hordeum vulgare L.) plants were grown in nutrient solutionscontaining the chelating agent, DTPA. The experiments replicatedthose reported in the preceding paper in which EDTA was thechelating agent used. The concentrations of all the chemicalspecies of metals were stimulated using the program NUTRIENT.The concentrations of DTPA used were chosen to give a similarrange of complexation as used in the EDTA experiments. The effectof complexation by DTPA on the uptakes of the metal ions Fe³⁺,Mn²⁺, Cu²⁺, and Zn²⁺ and on plant growth were sufficiently differentfrom those with EDTA to indicate some dependence on the natureof the chelating agent used. The biggest difference betweenthe EDTA and DTPA experiments occurred in the solutions containingthe largest concentrations of these reagents. With DTPA, chlorosiswas seen only in the early stages; otherwise the plants showednormal growth. A simple chemical model for metal uptake is discussed. Key words: DTPA, EDTA, micronutrients, trace metals, computer simulation, plants, absorption, iron, manganese, copper, zinc     

A case of multiple sclerosis improvement following removal of heavy metal intoxication

Multiple sclerosis (MS) is a chronic progressive disease of the central nervous system (CNS) provoking disability and neurological symptoms. The exact causes of SM are unknown, even if it is characterized by focal inflammatory lesions in CNS accompanied by autoimmune reaction against myelin. Indeed, many drugs able to modulate the immune response of patients have been used to treat MS. More recently, toxic metals have been proposed as possible causes of neurodegenerative diseases. The objective of this study is to investigate in vivo the impact of heavy metal intoxication in MS progression. We studied the case of a patient affected by MS, who has been unsuccessfully treated for some years with current therapies. We examined his levels of toxic heavy metals in the urine, following intravenous "challenge" with the chelating agent calcium disodium ethylene diamine tetraacetic acid (EDTA).The patient displayed elevated levels of aluminium, lead and mercury in the urine. Indeed, he was subjected to treatment with EDTA twice a month. Under treatment, the patient revealed in time improved symptoms suggestive of MS remission. The clinical data correlated with the reduction of heavy metal levels in the urine to normal range values. Our case report suggests that levels of toxic metals can be tested in patients affected by neurodegenerative diseases as MS.     

A long way ahead: understanding and engineering plant metal accumulation

Some plants can hyperaccumulate metal ions that are toxic to virtually all other organisms at low dosages. This trait could be used to clean up metal-contaminated soils. Moreover, the accumulation of heavy metals by plants determines both the micronutrient content and the toxic metal content of our food. Complex interactions of transport and chelating activities control the rates of metal uptake and storage. In recent years, several key steps have been identified at the molecular level, enabling us to initiate transgenic approaches to engineer the transition metal content of plants.