In vitro characteristics of 1-phenyl-3-methyl-4-acylpyrazol-5-ones iron chelators

Iron chelators represent a group of structurally different compounds sharing the ability of iron binding. The group has been evolving in recent years mainly due to novel experimental indications associated with variable requirements for iron chelators. A group of synthetic 1-phenyl-3-methyl-4-acyl-pyrazol-5-ones has been known for many years but data on their potential biological activity are rather limited.In this study, we analysed a series of these compounds for their iron-chelating properties as well as for their effects on iron based Fenton chemistry. For the former ferrozine spectrophotometric method and for the latter HPLC method with salicylic acid were used.All of the tested compounds were very efficient ferric chelators but their ferrous-chelating effects differed according to the acyl substitution. Notwithstanding various ferrous chelation activities, the individual Fe²⁺-affinities were not significantly different through pathophysiologically relevant pH conditions and some of the tested substances were more potent ferrous chelators at pH 4.5 than clinically used standard deferoxamine. Of particular interest is H₂QpyQ /2,6-bis[4(1-phenyl-3-methylpyrazol-5-one)carbonyl]pyridine/ which iron-chelating affinity increased when pH was decreasing. In spite of ferrous chelation differences, most of the tested acylpyrazolones were similarly active powerful inhibitors of Fenton chemistry as deferoxamine.Conclusively, acylpyrazolones are efficient iron chelators and H₂QpyQ may represent a prototype of novel specific chelators designated particularly for chelation at acidic conditions.     

Zinc content, metal chelator inactivation and catalytic inhibition of D-lactate dehydrogenase from the barnacle, Balanus nubilus Darwin.

Purified NAD-linked d-lactate dehydrogenase from the depressor muscle of the giant barnacle, Balanus nubilus Darwin, is inactivated when incubated with the metal chelators o-phenanthroline and EDTA. M-Phenanthroline and p-phenanthroline, which lack metal chelating ability, are ineffective in inactivating the enzyme. Inactivated enzyme can be reactivated by the addition of zinc ions to the assay mixture. Atomic absorption spectrophotometric analysis of purified B. nubilusd-lactate dehydrogenase revealed that this enzyme contains stoichiometric amounts of zinc (2 g-atoms per mol of subunit), unlike other lactate dehydrogenases, which lack zinc. Zinc appears to be required for maximal catalytic activity. Aromatic, nitrogen-containing metal chelators and their nonchelating analogs are effective instantaneous inhibitors of B. nubilusd-lactate dehydrogenase. These compounds bind at the coenzyme binding site, as the mode of inhibition is distinctly competitive with respect to NADH. The different effects of metal chelators and their nonchelator analogs suggest that time-dependent inactivation (chelation of the enzyme zinc ions) and instantaneous inhibition (competition with NADH binding) have independent mechanisms.     

Heavy metal biosorption by fungal mycelial by-products: mechanisms and influence of pH

Summary Mycelial wastes of Rhizopus arrhizus, used in fermentation industries to produce lipases, were studied for their ability to absorb various heavy metal ions (Ni, Zn, Cd and Pb). Chelation of all these ions occurs by a chemical, equilibrated and saturatable mechanism, following the Langmuir adsorption model. Data transformation allowed us to calculate maximum uptake and dissociation constants of the sorption reaction. We also investigated the influence of pH on metal accumulation. Sorption capacity variations between different biosorbent types (Rhizopus, Mucor, Penicillium, and Aspergillus), could be related to their acidity. pH neutralisation during the sorption reaction considerably enhanced zinc chelation (up to 56 mg/g). Previous NaOH treatment of mycelial wastes also increased their capacity for metal sorption. We report R. arrhizus metal uptake curves versus pH, using a pH-stat system. Optimal adsorption was achieved at neutral pH for nickel and zinc, pH 5.0 for lead, and inhibition of chelation was observed when the pH decreased. These results illustrate the importance of pH during the adsorption process, indicating a competitive mechanism for chelation between heavy metal ions and protons at cell wall adsorption sites. Correspondence to: J.-C. Roux     

Clawing back: broadening the notion of metal chelators in medicine

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Highlights► Not all chelators are the same: more than metal sequestration and elimination. ► Metal chelates can redistribute metals across biological membranes. ► Metal-chelating fragments provide leads for metalloenzyme inhibition. ► Metal chelation can enhance metal reactivity and thus cytotoxicity. ► Prochelators provide site-activated metal chelation and passivation.     

Synthesis and antiproliferating activity of iron chelators of hydroxyamino-1,3,5-triazine family

We synthesized and evaluated new specific tridentate iron(III) chelators of 2,6-bis[hydroxyamino]-1,3,5-triazine (BHT) family for use in iron deprivation cancer therapy. Physical properties of BHT chelators are easily customizable allowing easy penetration through cellular membranes. Antiproliferative activity of new BHT chelators was studied on MDA-MB-231 and MiaPaCa cells and compared to a clinically available new oral iron chelator, deferasirox (DFX). The antiproliferative activity of new chelators was found to correlate with iron(III) chelation ability and some of analogs showed substantially higher antiproliferative activity than DFX.     

Age as a major factor associated with zinc and copper deficiencies in pediatric thalassemia

BackgroundPatients with thalassemia encounter increased consumption of zinc (Zn) and copper (Cu) from chronic hemolysis and increased excretion from iron chelation. Iron-enriched diet restriction may result in low Zn and Cu intakes. Recent data on Zn and Cu status among Thai pediatric patients with thalassemia are lacking. This study aimed to identify frequencies and determine risk factors of Zn and Cu deficiencies among patients with thalassemia.MethodsPatients with transfusion-dependent thalassemia (TDT) receiving iron chelation ≥12 months and nonTDT (NTDT) aged 2–20 years were recruited. Serum Zn and Cu were measured. Dietary intakes were ascertained by interviews.ResultsA total of 209 patients (TDT = 126, NTDT = 83) were enrolled. Zn deficiency seemed to be associated with disease severity as median (IQR) Zn level of TDT was lower than that of NTDT [77 (69−85) vs. 80 (72−88) mcg/dL, p = 0.05], while higher frequency of Zn deficiency was identified in the former (24 % vs. 14 %). In TDT, Zn deficiency was associated with patients >10 years (OR 4.6; 95 %CI 1.1–6.4, p = 0.03), which likely resulted from combined low dietary Zn intake, prolonged exposures to hemolysis and iron chelators. Frequencies of Cu deficiency were similarly low in TDT and NTDT (8% and 7%) with comparable median (IQR) Cu levels of 103 (90−124) and 110 (92−132) mcg/dL, respectively (p = 0.13). Cu levels were inversely associated with age (r=-0.65 and r=-0.62 in TDT and NTDT, respectively; p < 0.001).ConclusionCompared with younger patients, Zn and Cu deficiencies were more common among patients with thalassemia >10 years. Age was a major factor associated with both Zn and Cu deficiencies.     

Metallobiology and therapeutic chelation of biometals (copper,zinc and iron) in Alzheimer’s disease: Limitations,and current and future perspectives

BackgroundAlzheimer's disease (AD) is the most prevalent cause of cognitive impairment and dementia worldwide. The pathobiology of the disease has been studied in the form of several hypotheses, ranging from oxidative stress, amyloid-beta (Aβ) aggregation, accumulation of tau forming neurofibrillary tangles (NFT) through metal dysregulation and homeostasis, dysfunction of the cholinergic system, and to inflammatory and autophagic mechanism. However, none of these hypotheses has led to confirmed diagnostics or approved cure for the disease.ObjectiveThis review is aimed as a basic and an encyclopedic short course into metals in AD and discusses the advances in chelation strategies and developments adopted in the treatment of the disease. Since there is accumulating evidence of the role of both biometal dyshomeostasis (iron (Fe), copper (Cu), and zinc (Zn)) and metal-amyloid interactions that lead to the pathogenesis of AD, this review focuses on unraveling therapeutic chelation strategies that have been considered in the treatment of the disease, aiming to sequester free and protein-bound metal ions and reducing cerebral metal burden. Promising compounds possessing chemically modified moieties evolving as multi-target ligands used as anti-AD drug candidates are also covered.Results and ConclusionSeveral multidirectional and multifaceted studies on metal chelation therapeutics show the need for improved synthesis, screening, and analysis of compounds to be able to effectively present chelating anti-AD drugs. Most drug candidates studied have limitations in their physicochemical properties; some enhance redistribution of metal ions, while others indirectly activate signaling pathways in AD. The metal chelation process in vivo still needs to be established and the design of potential anti-AD compounds that bi-functionally sequester metal ions as well as inhibit the Aβ aggregation by competing with the metal ions and reducing metal-induced oxidative damage and neurotoxicity may signal a bright end in chelation-based therapeutics of AD.     

Isolation and characterization of iron chelators from turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis>): selective metal binding by curcuminoids

Iron overload disorders may be treated by chelation therapy. This study describes a novel method for isolating iron chelators from complex mixtures including plant extracts. We demonstrate the one-step isolation of curcuminoids from turmeric, the medicinal food spice derived from Curcuma longa. The method uses iron-nitrilotriacetic acid (NTA)-agarose, to which curcumin binds rapidly, specifically, and reversibly. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin each bound iron-NTA-agarose with comparable affinities and a stoichiometry near 1. Analyses of binding efficiencies and purity demonstrated that curcuminoids comprise the primary iron binding compounds recovered from a crude turmeric extract. Competition of curcuminoid binding to the iron resin was used to characterize the metal binding site on curcumin and to detect iron binding by added chelators. Curcumin-Iron-NTA-agarose binding was inhibited by other metals with relative potency: (>90% inhibition) Cu²⁺ ~ Al³⁺ > Zn²⁺ ≥ Ca²⁺ ~ Mg²⁺ ~ Mn²⁺ (<20% inhibition). Binding was also inhibited by pharmaceutical iron chelators (desferoxamine or EDTA) or by higher concentrations of weak iron chelators (citrate or silibinin). Investigation of the physiological effects of iron binding by curcumin revealed that curcumin uptake by cultured cells was reduced >80% by addition of iron to the media; uptake was completely restored by desferoxamine. Ranking of metals by relative potencies for blocking curcumin uptake agreed with their relative potencies in blocking curcumin binding to iron-NTA-agarose. We conclude that curcumin can selectively bind toxic metals including iron in a physiological setting, and propose inhibition of curcumin binding to iron-NTA-agarose for iron chelator screening.     

Photochemical redox transformations of dimers of NAD+ and N'-methylnicotinamide

Electrochemically generated dimers of N′-methylnicotinamide and NAD⁺ both undergo photooxidation on irradiation at 254 nm in aqueous medium to yield the respective parent monomers. For the NAD dimer the quantum yield for photodissociation was about 0.01, whether irradiated at 254 nm or at wavelengths to the red of 320 nm. Irradiation at the latter wavelengths, where the NAD⁺ monomer itself does not absorb and is not photosensitive, led to quantitative regeneration of coenzyme activity in the alcohol dehydrogenase system. The photodissociation reaction exhibited no oxygen effect. The photochemically generated dimer of N′-methylnicotinamide was also photooxidized to the parent monomer by irradiation at 254 nm at pH 9.5. The foregoing process of electrochemical (or photochemical) reduction and photochemical oxidation, comprising a closed cycle of electron and proton transport, is similar to that previously observed for a number of pyrimidine analogs. Furthermore, the NAD dimer is a substrate of snake venom nucleotide pyrophosphatase and is hydrolyzed to release NMN dimer which, on irradiation at 254 nm, also undergoes photooxidation to the parent NMN⁺ monomer. A mechanism for the photooxidation reaction is formulated and relevant biological implications of the foregoing are presented.     

Combined chelation of lead (II) by deferasirox and deferiprone in rats as biological model

In order to investigate the capability of two chelators deferasirox (DFX or ICL670) and deferiprone (L₁) in removing lead from the body, the present research was performed. Two does levels of 40 and 80 mg/kg body weight of lead (II) chloride was given to rats as biological model for 45 days. After 45 days, some toxicity symptoms were observed in rats such as loss of hair and weight, appearance of red dots around eyes, weakness and irritability. After lead application, chelation therapy with DFX and L₁ as mono and combined (DFX, L₁ and DFX + L₁) was done for 10 days. After chelation therapy, lead level in different tissues reduced. The combined chelation therapy results showed that these chelators are able to remove lead from the body and toxicity symptoms decreased. The combined therapy results (DFX + L1) show higher efficacy and lower toxicity compared to single therapies.     

Iron chelators of the pyridoxal isonicotinoyl hydrazone class

Summary Formation constants for the calcium(II), magnesium(II) and zinc(II) complexes of the orally effective iron chelator, pyridoxal isonicotinoyl hydrazone (PIH) and three analogues, pyridoxal benzoyl hydrazone (PBH), pyridoxalp-methoxybenzoyl hydrazone (PpMBH) and pyridoxalm-fluorobenzoyl hydrazone (PmFBH) have been determined by potentiometry at 25\dg C andI=0.1 M [KNO₃]. The four ligands bind calcium(II) weakly and magnesium(II) only slightly more strongly, as a l: l complex which is formed at pH \s> 8. The chelation of zinc(II) for all the ligands studied was greater than that for calcium(II) and magnesium(II), with complexation generally becoming significant at about pH 5. Thus, chelation of zinc(II) but not calcium(II) or magnesium(II) at physiological pH, 7.4 may be expected. Calculated values of the concentration of uncomplexed metal ion indicate that the selectivity of these ligands towards Fe(III) is comparable to that of the clinically used chelator desferrioxamine.     

Effect of chaotropes on the kinetics of iron release from ferritin by flavin nucleotides

BackgroundFerritins are ubiquitous multi-subunit iron storage and detoxification proteins that play a critical role in iron homeostasis. Ferrous ions that enter the protein's shell through hydrophilic channels are rapidly oxidized at dinuclear centers on the H-subunit before transfer to the protein's cavity for storage. The mechanisms of iron loading have been extensively studied, but little is known about iron mobilization. Fe(III) reduction can occur via rapid reduction by suitable reducing agents followed by chelation of Fe(II) ions or via direct and slow Fe(III) chelation. Here, the iron release kinetics from ferritin by FMNH₂ in the presence of various chaotropic agents are studied and their in-vivo physiological significance discussed.MethodsThe iron release kinetics from horse and human ferritins by FMNH₂ were monitored at 522 nm where the Fe(II)–bipyridine complex absorbs. The experiments were performed in the presence of different concentrations of three chaotropic agents, urea, guanidine HCl, and triton.Results and conclusionsUnder our experimental conditions, iron reductive mobilization by the non-enzymatic FMN/NAD(P)H system is limited by the concentration of FMNH₂ and is independent on the type or amount of chaotropes present. Diffusion of FMNH₂ through the ferritin pores is an unlikely mechanism for ferritin iron reduction. An iron mobilization mechanism involving rapid electron transfer through the protein shell is discussed.General significanceCaution must be exercised when interpreting the kinetics of iron mobilization from ferritin using the FMN/NAD(P)H system. The kinetics are highly dependent on the amount of dissolved oxygen and the concentration of reagents used.     

Gradient ion-pair high-performance liquid chromatographic method for analysis of 3-hydroxypyridin-4-one iron chelators

A gradient ion-pair HPLC separation of highly hydrophilic 3-hydroxypyridin-4-one (HPO) iron chelators is described. The separation of HPOs was performed using a reversed-phase polymer HPLC column (PLRP-S 100 Å, 15×0.46 cm ID, 5 μm). The ion-pair buffer contained 1-heptanesulfonic acid (sodium salt) (5 mM) and the pH was adjusted to 2.0 using HCl. The gradient was 2%–35% CH₃CN in 20 min and post-run was followed for 5 min using 2% CH₃CN and 98% buffer. The flow-rate was 1 ml/min and the analytes were monitored at 280 nm. The retention times of 30 hydrophilic HPOs fell in the range of 10–18 min with sharp peak shapes, although these iron chelators possess various functional groups and distribution coefficients. The application of this HPLC method in the analysis of HPO chelators and their metabolites in rat bile and urine is described.     

Directed evolution of a Bacillus chitinase

Chitinases have potential in various industrial applications including bioconversion of chitin waste from crustacean shells into chito-oligosaccharide-based value-added products. For industrial applications, obtaining suitable chitinases for efficient bioconversion processes will be beneficial. In this study, we established a straightforward directed evolution method for creating chitinase variants with improved properties. A library of mutant chitinases was constructed by error-prone PCR and DNA shuffling of two highly similar (99% identical) chitinase genes from Bacillus licheniformis. Activity screening was done in two steps: first, activity towards colloidal chitin was screened for on culturing plates (halo formation). This was followed by screening activity towards the chitotriose analogue p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose at various pH in microtiter plates. From a medium-throughput screening (517 colonies), we were able to isolate one mutant that demonstrated improved catalytic activity. When using p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose as substrate, the overall catalytic efficiency, k_cat/K_m of the improved chitinase was 2.7- and 2.3-fold higher than the average k_cat/K_m of wild types at pH 3.0 and 6.0, respectively. The mutant contained four residues that did not occur in either of the wild types. The approach presented here can easily be adopted for directed evolution of suitable chitinases for various applications.     

The effects of chelators on zinc levels in patients with thalassemia major

Zinc which is an essential element has very important effects on growth and immune system in patients with thalassemia major (TM). The effects of two oral iron chelator agents, desferrioxamine (DFO) and deferiprone (DFP), on zinc levels were investigated in previous studies and they were found to cause zinc deficiency. Zinc level alteration by the new chelator deferasirox (DFX) is not present in the literature. The aim of this study was to examine the effects of different oral chelators on serum and urine zinc levels in TM patients. Zinc levels are compared in the patients who received different chelators: only DFX, combined chelation with DFO plus DFP and the healthy control group. A total of 56 patients with TM were involved in this study: 39 patients received only DFX and 17 patients were given combined treatment DFO + DFP between August 2008 and August 2009. In addition, a control group was established from the healthy population. Blood was taken from all the patients for serum zinc levels and 24 hour-urine samples were collected for urine zinc levels. Serum zinc levels were found to be 64.8 ± 14.8 μg/dL in DFX group and 66.5 ± 15.1 μg/dL in DFO + DFP group. These levels were statistically lower than that in the control group (149 ± 54.3 μg/dL) (p < 0.05), but there was no statistically difference between the two different chelation groups (p > 0.05). The urine zinc levels of DFX and DFO + DFP group were 662.2 ± 428.2 μg/day and 1182.3 ± 980.3 μg/day respectively (p < 0.05). Urinary zinc excretion in the chelation groups (DFX and DFO + DFP) was significantly higher than the control group (395.1 ± 208.9 μg/day) (p < 0.05). As a conclusion, the new chelation agent, DFX, also leads to zinc deficiency, though its urinary zinc excretion is lower. New studies are required to examine the effects of DFX on zinc extensively. Zinc levels of patients with TM should be followed up regularly and zinc supply should be given at early ages.     

Ultraviolet disinfection of Schistosoma mansoni cercariae in water

BackgroundSchistosomiasis is a parasitic disease that is transmitted by skin contact with waterborne schistosome cercariae. Mass drug administration with praziquantel is an effective control method, but it cannot prevent reinfection if contact with cercariae infested water continues. Providing safe water for contact activities such as laundry and bathing can help to reduce transmission. In this study we examine the direct effect of UV light on Schistosoma mansoni cercariae using ultraviolet light-emitting diodes (UV LEDs) and a low-pressure (LP) mercury arc discharge lamp.MethodologyS. mansoni cercariae were exposed to UV light at four peak wavelengths: 255 nm, 265 nm, 285 nm (UV LEDs), and 253.7 nm (LP lamp) using bench scale collimated beam apparatus. The UV fluence ranged from 0–300 mJ/cm² at each wavelength. Cercariae were studied under a stereo-microscope at 0, 60, and 180 minutes post-exposure and the viability of cercariae was determined by assessing their motility and morphology.ConclusionVery high UV fluences were required to kill S. mansoni cercariae, when compared to most other waterborne pathogens. At 265 nm a fluence of 247 mJ/cm² (95% confidence interval (CI): 234–261 mJ/cm²) was required to achieve a 1-log₁₀ reduction at 0 minutes post-exposure. Cercariae were visibly damaged at lower fluences, and the log reduction increased with time post-exposure at all wavelengths. Fluences of 127 mJ/cm² (95% CI: 111–146 mJ/cm²) and 99 mJ/cm² (95% CI: 85–113 mJ/cm²) were required to achieve a 1-log₁₀ reduction at 60 and 180 minutes post-exposure at 265 nm. At 0 minutes post-exposure 285 nm was slightly less effective, but there was no statistical difference between 265 nm and 285 nm after 60 minutes. The least effective wavelengths were 255 nm and 253.7 nm. Due to the high fluences required, UV disinfection is unlikely to be an energy- or cost-efficient water treatment method against schistosome cercariae when compared to other methods such as chlorination, unless it can be demonstrated that UV-damaged cercariae are non-infective using alternative assay methods or there are improvements in UV LED technology.     

Quantitation of Cytidine 3′,5′-Cyclic Monophosphate in Aqueous Solution by UV Absorption Spectrophotometry Independent of Direct Determination of Solution pH

Abstract

uv absorbance spectrophotometry is the routine method of determining nucleotide concentrations in solution. To obviate the need for determining solution pH a method is described whereby cyclic CMP concentration in aqueous solution is calculated from absorbances at four wavelengths: the rationale is of general applicability to nucleosides and nucleotides.     

OBSERVATTVNS ON THE UNDERWATER LIGHT FIELD OF TWO HWERTROPHIC,SOUTH AFRICAN IMPOUNDMENTS

SUMMARY

The spectral composition of the underwater light field was examined in two hypertrophic South African Impoundments (Hartbeespoort and Roodeplaat Dams) under a range of inorganic turbidities and chlorophyll α concentrations. The data indicated that inorganic turbidity and gilvin were dominant to chlorophyll in regulating underwater light attenuation during the present study. Under all conditions the wavelengths between 405 and 510 nm were attenuated more rapidly than near UV and the wavelengths above 510 nm and the 623 nm component penetrated deepest. Under low turbidities the 546 nm wavelength was the next most penetrating component, but its attenuation increased with increasing turbidity. This characteristic of the underwater light field may be important to the cyanobacteria which dominate in these hypertrophic lakes.     

Zinc-aggravated M1 microglia regulate astrocytic engulfment via P2×7 receptors

BackgroundGlial cells such as astrocytes and microglia play an important role in the central nervous system via communication between these glial cells. Activated microglia can exhibit either the inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which influences astrocytic neuroprotective functions, including engulfment of cell debris. Recently, extracellular zinc has been shown to promote the inflammatory M1 phenotype in microglia through intracellular zinc accumulation and reactive oxygen species (ROS) generation.PurposeHere, we investigated whether the zinc-enhanced inflammatory M1 phenotype of microglia affects the astrocytic engulfing activity.MethodsEngulfing activity was assessed in astrocytes treated with microglial-conditioned medium (MCM) from lipopolysaccharide (LPS)-activated or from ZnCl₂-pretreated LPS-activated M1 microglia. The effect of zinc on microglia phenotype was also validated using the zinc chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the ROS scavenger Trolox.ResultsAlthough treatment of astrocytes with LPS showed no significant effect on the engulfing activity, MCM from LPS-induced M1 microglia increased the beads uptake by astrocytes. This increased uptake activity was suppressed when MCM from LPS-induced M1 microglia pretreated with ZnCl₂ was applied to astrocytes, which was further abolished by the intracellular zinc chelator TPEN and the ROS scavenger Trolox. In addition, expression of P2×7 receptors (P2×7R) was increased in astrocytes treated with MCM derived from M1 microglia but not in the M1 microglia pretreated with ZnCl₂.ConclusionThese findings suggest that zinc pre-treatment abolishes the ability of LPS-induced M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2×7R.