BioMetals (v.25, #6)

Development of copper based drugs, radiopharmaceuticals and medical materials by Paweł Szymański; Tomasz Frączek; Magdalena Markowicz; Elżbieta Mikiciuk-Olasik (1089-1112).
Copper is one of the most interesting elements for various biomedical applications. Copper compounds show vast array of biological actions, including anti-inflammatory, anti-proliferative, biocidal and other. It also offers a selection of radioisotopes, suitable for nuclear imaging and radiotherapy. Quick progress in nanotechnology opened new possibilities for design of copper based drugs and medical materials. To date, copper has not found many uses in medicine, but number of ongoing research, as well as preclinical and clinical studies, will most likely lead to many novel applications of copper in the near future.
Keywords: Copper; Nuclear medicine; Nanotechnology; Drug development

Influence of siderophore pyoverdine synthesis and iron-uptake on abiotic and biotic surface colonization of Pseudomonas putida S11 by Paramasivan Ponraj; Manoharan Shankar; Devaraj Ilakkiam; Paramasamy Gunasekaran (1113-1128).
Fluorescent pseudomonads produce a characteristic fluorescent pigment, pyoverdines as their primary siderophore for iron acquisition under iron-limiting conditions. Here, we report the identification of a random transposon mutant IST3 of Pseudomonas putida S11 showing tolerance to iron starvation stress condition and increased pyoverdine production. The insertion of the Tn5 transposon was found to be in pstS gene of pstSR operon encoding sensor histidine kinase protein of the two-component signal transduction system. A pyoverdine negative derivative of IST3 mutant constructed was sensitive to iron stress condition. It indicated that increased survival of IST3 under iron-limiting condition was due to higher pyoverdine production. The iron starvation tolerant mutant (IST3) exhibited enhanced pyoverdine-mediated iron uptake in minimal medium which significantly improved its biofilm formation, seed adhesion and competitive root colonization.
Keywords: Pyoverdine production; Iron uptake; Biofilm; Seed adhesion; Root colonization

Effect of 17ß-estradiol on zinc content of hippocampal mossy fibers in ovariectomized adult rats by E. Padilla-Gómez; V. Beltrán-Campos; S. Montes; A. Díaz-Ruíz; G. L. Quirarte; C. Ríos; S. Diaz-Cintra (1129-1139).
Sex hormones such as estrogen (17ß-estradiol) may modulate the zinc content of the hippocampus during the female estrous cycle. The mossy fiber system is highly plastic in the adult brain and is influenced by multiple factors including learning, memory, and stress. However, whether 17ß-estradiol is able to modulate the morphological plasticity of the mossy fibers throughout the estrous cycle remains unknown. Ovariectomized (Ovx) female 70- to 90-day-old Sprague-Dawley rats without or with estrogen supplement (OvxE) were compared with control rats in three stages of the estrous cycle: diestrus, proestrus, and estrus. The brain tissue from each of the five groups was processed with Timm’s silver sulfide technique using the Image J program to measure the mossy fiber area in the stratum lucidum of CA3. Total zinc in the hippocampus was measured using Graphite Furnace Atomic Absorption Spectrophotometry. Two additional (Ovx and OvxE) groups were examined in spatial learning and memory tasks using the Morris water maze. Similar increases in total zinc content and mossy fiber area were observed. The mossy fiber area decreased by 26 ± 2 % (difference ± SEM percentages) in Ovx and 23 ± 4 % in estrus as compared to the proestrus group and by 18 ± 2 % in Ovx compared to OvxE. Additionally, only the OvxE group learned and remembered the task. These results suggest that estradiol has a significant effect on zinc content in hippocampal CA3 during the proestrus stage of the estrous cycle and is associated with correct performance in learning and memory.
Keywords: Zinc; Hippocampus; Mossy fibers; Estrous cycle; Estrogen

Changes of growth, photosynthesis and alteration of leaf antioxidative defence system of tea [Camellia sinensis (L.) O. Kuntze] seedlings under aluminum stress by Mainaak Mukhopadyay; Pranay Bantawa; Akan Das; Bipasa Sarkar; Biswajit Bera; Parthadeb Ghosh; Tapan Kumar Mondal (1141-1154).
Tea [Camellia sinensis (L.) O. Kuntze] is an aluminum (Al) hyperaccumulator plant and is commercially important due to its high content of antioxidants. Although Al induced growth is well-known for the plants growing in acid soil, yet the cause underlying the stimulatory effect of Al has not been fully understood. To investigate the possible role of Al in growth induction, we studied morphological, physiological as well as biochemical changes of tea plant under different Al concentrations (0–4,000 μM). In hydroponics, Al (15 μM), enhanced shoot and root growth, but at higher concentrations, it caused oxidative damage which culminated in a cascade of biochemical changes, Al content increased concurrently with the maturity of the leaf as well as stem tissues than their younger counterparts. Hematoxylin staining indicated that Al accumulation started after 6 h of exposure in the tips of young roots and accumulation was dose dependent. The physiological parameters such as pigments, photosynthetic rate, transpiration and stomatal conductance were declined due to Al toxicity. Alteration in activated oxygen metabolism was also evidenced by increasing lipid peroxidation, membrane injury, evolution of superoxide anions and accumulation of H2O2. Contents of phenols initially exhibited an acceleration which gradually plummeted at higher levels whereas total sugar and starch contents decimated beyond 15 μM of Al concentration. Activities of antioxidant defense enzymes were increased with the elevated concentration of Al. Expression of citrate synthase gene was up-regulated in the mature leaves, young as well as old roots simultaneously with increased concentration of Al in those parts; indicating the formation of Al-citrate complex. These results cooperatively specified that Al concentration at lower level promoted growth but turned out to be a stressor at elevated stages indicating the sensitivity of the cultivar (T-78) to Al.
Keywords: Aluminum toxicity; Antioxidative enzymes; Camellia sinensis ; Citrate synthase; Pigments; Reactive oxygen species

The role of salicylic acid in the prevention of oxidative stress elicited by cadmium in soybean plants by Guillermo Noriega; Ethel Caggiano; Manuel López Lecube; Diego Santa Cruz; Alcira Batlle; María Tomaro; Karina Beatriz Balestrasse (1155-1165).
The protective action of salicylic acid (SA) pre-treatment on soybean plants before cadmium (Cd) addition was tested. Oxidative stress parameters, such as TBARS formation, glutathione and chlorophyll content, were altered by Cd, instead no differences were observed in plants only pre-treated with SA. Antioxidant enzymes were affected by Cd treatment, while SA protected against these effects. These findings indicated that SA could act as a protector against oxidative stress induced by Cd. Taking into account the fact that heme-oxygenase-1 (HO-1) has been previously described as a novel antioxidant enzyme, experiments were carried out to determine whether it was involved in the protection exerted by SA. As expected, Cd brought about an enhancement of 57 % in HO-1 activity and 150 % in protein content (150 %), SA also increased both the enzyme activity and its protein content (28 and 75 %, respectively). Surprisingly, the observed rise of HO activity and protein content under SA treatment was lower than that produced by Cd alone. These lower values indicated, that HO-1 could not be directly involved in the protection of SA against Cd effects. In order to shed light in the mechanisms involved in SA effects, Cd content was determined in the tissues of Cd treated plants with and without SA pre-treatment. Results indicated that, in the presence of SA, Cd uptake was inhibited, thus avoiding its deleterious effects. Moreover, the observed HO-1 activity enhancement by SA indicates that this phytohormone could be engaged in the signalling pathway of heme degradation.
Keywords: Cadmium; Heme oxygenase; Oxidative stress; Salicylic acid; Soybean plants

Mitochondrial calcium uniporter blocker prevents cardiac mitochondrial dysfunction induced by iron overload in thalassemic mice by Sirinart Kumfu; Siriporn Chattipakorn; Suthat Fucharoen; Nipon Chattipakorn (1167-1175).
Iron-overload induced cardiomyopathy is a major cause of morbidity and mortality in thalassemic patients. Previous studies suggest that cardiac mitochondrial dysfunction may be involved in the pathogenesis of cardiomyopathy in thalassemia. We tested the hypothesis that iron overload causes dysfunction of cardiac mitochondria isolated from thalassemic mice. Cardiac mitochondria were isolated from the heart tissue of genetically-altered, β-thalassemic mice (HT) and adult wild-type mice (WT). Ferrous iron (Fe2+) at various concentrations (0–5 μg/ml) was applied to induce iron toxicity. Pharmacological interventions, facilitated by mitochondrial permeability transition pore (mPTP) blocker, CsA, and mitochondrial Ca2+ uniporter (MCU) blocker, Ru360, were used to study their respective effects on cardiac mitochondrial dysfunction. Cardiac mitochondrial ROS production, mitochondrial membrane potential changes, and mitochondrial swelling were determined. Iron overload caused increased ROS production, mitochondrial depolarization, and mitochondrial swelling in a dose-dependent manner in WT and HT cardiac mitochondria. CsA decreased only ROS production in WT and HT cardiac mitochondria, whereas Ru360 completely prevented the development of cardiac mitochondrial dysfunction by decreasing ROS, mitochondrial depolarization, and swelling in both WT and HT cardiac mitochondria. Ru360, an MCU blocker, provides protective effects by preventing ROS production and mitochondrial depolarization as well as attenuating mitochondrial swelling caused by Fe2+ overload. These findings indicate that the MCU could be a major portal for Fe2+ entry into cardiac mitochondria. Therefore, blocking MCU may be an effective therapy to prevent iron-overload induced cardiac mitochondrial dysfunction in patients with thalassemia.
Keywords: Cardiac mitochondria; Iron overload; Thalassemia; Mitochondrial dysfunction; MCU

Competition of zinc ion for the [2Fe–2S] cluster binding site in the diabetes drug target protein mitoNEET by Guoqiang Tan; Aaron P. Landry; Ruili Dai; Li Wang; Jianxin Lu; Huangen Ding (1177-1184).
Human mitochondrial protein mitoNEET is a novel target of type II diabetes drug pioglitazone, and contains a redox active [2Fe–2S] cluster that is hosted by a unique ligand arrangement of three cysteine and one histidine residues. Here we report that zinc ion can compete for the [2Fe–2S] cluster binding site in human mitoNEET and potentially modulate the physiological function of mitoNEET. When recombinant mitoNEET is expressed in Escherichia coli cells grown in M9 minimal media, purified mitoNEET contains very little or no iron–sulfur clusters. Addition of exogenous iron or zinc ion in the media produces mitoNEET bound with a [2Fe–2S] cluster or zinc, respectively. Mutations of the amino acid residues that hosting the [2Fe–2S] cluster in mitoNEET diminish the zinc binding activity, indicating that zinc ion and the [2Fe–2S] cluster may share the same binding site in mitoNEET. Finally, excess zinc ion effectively inhibits the [2Fe–2S] cluster assembly in mitoNEET in E. coli cells, suggesting that zinc ion may impede the function of mitoNEET by blocking the [2Fe–2S] cluster assembly in the protein.
Keywords: Human mitoNEET; Type II diabetes drug pioglitazone; Iron–sulfur cluster; Zinc binding

An emergent property of exopolysaccharides (EPS) produced by marine bacteria is their net negative charge, predominantly conferred by their high uronic acids content. Here, we investigated the EPS produced by an algal-associated marine bacterium, Halomonas sp. strain TG39, for its capacity to sequester trace metals and mediate their bioavailability to eukaryotic phytoplankton. Metal analysis of the purified EPS revealed that it contained high levels of K, Ca, Mg and several essential trace metals, including Zn, Cu, Fe and the metalloid Si. Desorption experiments with marine sediment showed that the EPS possessed a specific binding capacity for Ca, Si, Fe, Mn, Mg and Al. Depending on the ionic conditions, Fe was the third or fourth most highly-adsorbed metal out of 27 elements analyzed. Experiments employing Fe-limited synthetic ocean seawater showed that growth of the marine diatom Thalassiosira weissflogii (axenic strain) was enhanced when incubated in the presence of either purified EPS or EPS that had been pre-exposed to marine sediment, compared to non-EPS amended controls. This growth enhancement was attributed to the EPS binding and increasing the bioavailability of key trace metal elements, such as Fe(III). Since the bacterium used in this study was originally isolated from a marine micro-alga, this work highlights the possibility that bacterial associates of eukaryotic algae could be influencing the bioavailability of Fe(III) to phytoplankton via their production of polyanionic EPS. More widely, this work reinforces the potential importance of marine bacterial EPS in trace metal biogeochemical cycling.
Keywords: Exopolysaccharide (EPS); Iron; Metal ions; Eukaryotic phytoplankton; Marine bacteria

Hematological indices and activity of NTPDase and cholinesterase enzymes in rats exposed to cadmium and treated with N-acetylcysteine by Jamile F. Gonçalves; Marta M. M. F. Duarte; Amanda M. Fiorenza; Roselia M. Spanevello; Cinthia M. Mazzanti; Roberta Schmatz; Margarete D. Bagatini; Fabiane G. Antes; Pauline Costa; Fátima H. Abdalla; Valderi L. Dressler; Vera M. Morsch; Maria Rosa C. Schetinger (1195-1206).
The present study aimed to investigate the influence of N-acetylcysteine (NAC) on cadmium (Cd) poisoning by evaluating Cd concentration in tissues, hematological indices as well as the activity of NTPDase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes of rats exposed to Cd and co-treated with NAC. For this purpose, the rats received Cd (2 mg/kg) and NAC (150 mg/kg) by gavage every other day for 30 days. Animals were divided into four groups (n = 6–8): control/saline, NAC, Cd, and Cd/NAC. Cd exposure increased Cd concentration in plasma, spleen and thymus, and NAC co-treatment modulated this augment in both lymphoid organs. Cd exposure reduced red blood cell count, hemoglobin content and hematocrit value. Cd intoxication caused a decrease in total white blood cell count. NAC treatment per se caused an increase in lymphocyte and a decrease in neutrophil counts. On contrary, Cd exposure caused a decrease in lymphocyte and an increase in neutrophil and monocyte counts. NAC reversed or ameliorated the hematological impairments caused by Cd poisoning. There were no significant alterations in the NTPDase activity in lymphocytes of rats treated with Cd and/or NAC. Cd caused a decrease in the activities of lymphocyte AChE, whole blood AChE and serum BChE. However, NAC co-treatment was inefficient in counteracting the negative effect of Cd in the cholinesterase activities. The present investigation provides ex vivo evidence supporting the hypothesis that Cd induces immunotoxicity by interacting with the lymphoid organs, altering hematological parameters and inhibiting peripheral cholinesterase activity. Also, it highlights the possibility to use NAC as adjuvant against toxicological conditions.
Keywords: AChE; Cd; Ectonucleotidase; Immunotoxicology; NAC

Evaluating metal ion salts as acid hydrolase mimics: metal-assisted hydrolysis of phospholipids at lysosomal pH by Sarah S. Cepeda; Dominique E. Williams; Kathryn B. Grant (1207-1219).
Niemann-Pick disease and drug-induced phospholipidosis are lysosomal storage disorders in which there is an excessive accumulation of sphingomyelin in cellular lysosomes. Here we have explored the possibility of developing metal-based therapeutic agents to reverse phospholipid build-up through phosphate ester bond hydrolysis at lysosomal pH (~4.8). Towards this end, we have utilized a malachite green/molybdate-based colorimetric assay to quantitate the inorganic phosphate released upon the hydrolysis of sphingomyelin by twelve d- and f-block metal ion salts. In reactions conducted at 60 °C, the yields produced by the cerium(IV) complex Ce(NH4)2(NO3)6 were superior. An Amplex® Red-based colorimetric assay and mass spectrometry were then employed to detect choline. The data consistently showed that Ce(IV) hydrolyzed sphingomyelin more efficiently at lysosomal pH: i.e., yields of choline and phosphate were 54 ± 4 and 22 ± 5 % at pH ~ 4.8, compared to 8 ± 1 and 5 ± 2 % at pH ~ 7.2. Hydrolysis at 60 °C could be significantly increased by converting sphingomyelin vesicles to mixed lipid vesicles and mixed micelles of Triton X-100. We then utilized cerium(IV) to cleave sphingomyelin at 37 °C (no Triton X-100). Although choline and phosphate levels were relatively low, hydrolysis continued to be considerably more efficient at lysosomal pH. A side by side comparison to phosphatidylcholine was then made. While the yields of choline and phosphate produced by phosphatylcholine were higher, the ratio of pH ~ 4.8 hydrolysis to pH ~ 7.2 hydrolysis was usually more favorable for sphingomyelin (37 and 60 °C).
Keywords: Cerium(IV); Lysosomal storage disease; Membrane lipids; Phosphate diester

Ni2+ effects on Nicotiana tabacum L. pollen germination and pollen tube growth by Maria Breygina; Natalie Matveyeva; Svetlana Polevova; Natalie Meychik; Yulia Nikolaeva; Anna Mamaeva; Igor Yermakov (1221-1233).
To investigate the mechanisms of Ni2+ effects on initiation and maintenance of polar cell growth, we used a well-studied model system—germination of angiosperm pollen grains. In liquid medium tobacco pollen grain forms a long tube, where the growth is restricted to the very tip. Ni2+ did not prevent the formation of pollen tube initials, but inhibited their subsequent growth with IC50 = 550 μM. 1 mM Ni2+ completely blocked the polar growth, but all pollen grains remained viable, their respiration was slightly affected and ROS production did not increase. Addition of Ni2+ after the onset of germination had a bidirectional effect on the tubes development: there was a considerable amount of extra-long tubes, which appeared to be rapidly growing, but the growth of many tubes was impaired. Studying the localization of possible targets of Ni2+ influence, we found that they may occur both in the wall and in the cytoplasm, as confirmed by specific staining. Ni2+ disturbed the segregation of transport vesicles in the tips of these tubes and significantly reduced the relative content of calcium in the aperture area of pollen grains, as measured by X-ray microanalysis. These factors are considered being critical for normal polar cell growth. Ni2+ also causes the deposition of callose in the tips of the tube initials and the pollen tubes that had stopped their growth. We can assume that Ni2+-induced disruption of calcium homeostasis can lead to vesicle traffic impairment and abnormal callose deposition and, consequently, block the polar growth.
Keywords: Nickel; Heavy metals; Pollen germination; Polar growth

Coordinating properties of uridine 5′-monophosphate with selected Ln3+ ions in ionic micellar media by M. Sudhiranjan Singh; Naorem Homendra; R. K. Lonibala (1235-1246).
Coordinating properties of uridine 5′-monophosphate (UMP) towards trivalent La, Pr, Nd, Sm, Eu and Gd ions in presence of cationic and anionic micelles have been investigated by potentiometric pH-titration and spectroscopic methods. Stability constants of the 2:1 complexes have been determined and the change in free energy, enthalpy and entropy associated with the complexation are also calculated. Nd(III) complexes isolated from aqueous and aqueous-micellar media do not show any significant structural difference. Formation of Ln(III) complexes in all cases completes below pH 7.5 showing that UMP best interacts with Ln3+ ions at the physiological pH range 7.3–7.5. The nucleobase is not involved in the complexation and the metal ion coordination of UMP is through the phosphate moiety only. Coordinating tendency of UMP with lanthanides, Nd(III) ion in particular, at different pH is also discussed. Luminescent properties of Eu(III) complex and its decay lifetime are also presented. This information may prove helpful regarding the use of lanthanides as biological probes for calcium/magnesium ions.
Keywords: Uridine 5′-monophosphate; Ln3+ ions; Ionic micellar media; Potentiometric titration; Spectroscopic; Luminescent

Human apo-lactoferrin as a physiological mimetic of hypoxia stabilizes hypoxia-inducible factor-1 alpha by Elena T. Zakharova; Valeria A. Kostevich; Alexey V. Sokolov; Vadim B. Vasilyev (1247-1259).
Apo-form of human lactoferrin (LF) is a potent iron chelator, this feature being similar to the iron-binding properties of a synthetic chelator desferoxamine (DFO). The latter stabilizes the principal adaptive transcriptional hypoxia-inducible factor-1 alpha (HIF-1α). Since DFO is known as a pharmacological mimetic of hypoxia it was decided to test whether apo-LF is able to perform as such. Mice either injected intraperitoneally or given per os apo-LF displayed HIF-1α in liver, lungs, heart, brain, spleen and kidneys, as judged by results of Western blotting. Similar administration of iron-saturated LF (75 mg/kg) did not bring forth such effect. Synthesis of erythropoietin and ceruloplasmin became increased in the first case, which is explained by the respective genes being targets for HIF-1α. Apo-LF, but not Fe-LF, injected intraperitoneally to hypoxia low-resistant mice 24 h before animals were subjected to normobaric hypoxia with hypercapnia caused a significant increase of life-time by 40 %. The results obtained show that, like DFO, apo-LF performs as a normoxic mimetic of hypoxia, capable of stabilizing HIF-1α. Protective features of LF and DFO and their pharmacological properties involving HIF-1α are discussed.
Keywords: Lactoferrin; Transferrin; Ceruloplasmin; Erythropoietin; Desferoxamin; Iron chelator; Hypoxia-inducible factor-1 alpha

Vanadium complexes are potent hypoglycemic agents and of great potential for therapeutical treatment of diabetes. In the present work, a novel vanadium compound, bis ((5-hydroxy-4-oxo-4H-pyran-2-yl)methyl benzoatato) oxovanadium (IV) (BBOV) has been synthesized. Treatment of STZ-induced diabetic rats with BBOV restored the blood glucose to normal level and ameliorated glucose tolerance. The hypoglycemic effect of BBOV is similar to that of bis (maltolato) oxovanadium but is less toxic in median lethal dose. Overall, the present work will provide useful information for further development of new anti-diabetic vanadium compounds.
Keywords: Bis ((5-hydroxy-4-oxo-4H-pyran-2-yl)methyl benzoatato) oxovanadium (IV); Diabetes mellitus; Blood glucose; Antioxidants

BjHO-1 is involved in the detoxification of heavy metal in India mustard (Brassica juncea) by Hua Li; Ming Jiang; Li Ling Che; Li Nie; Zhi Min Yang (1269-1279).
Heme oxygenase-1 (HO-1) is a stress-responsive gene coding for an enzyme catalyzing the catabolism of heme to yield biliverdin IXα, carbon monoxide (CO) and iron. However, its biological role in regulating metal homeostasis, particularly the tolerance to toxic heavy metals is poorly understood. In this study, a novel gene encoding a Brassica juncea heme oxygenase-1 (designated as BjHO-1) was cloned and functionally identified. Spatial expression of BjHO-1 showed that it was differentially expressed in cotyledon, hypocotyl, leaf and root. BjHO-1 was found to be induced significantly by heavy metal Hg. To understand whether BjHO-1 is able to regulate plant tolerance to Hg, we constructed transgenic B. juncea plants overexpressing HO-1, and showed that 35S::BjHO1 plants confer the plant resistance to Hg toxicity by improving plant dry mass, reducing Hg accumulation, and attenuating Hg-induced oxidative stress. We further cloned a 1,099 bp promoter sequence upstream of BjHO-1 using genome walking approach. Multiple stress-responsive elements were detected in the BjHO-1 promoter regions. The promoter can be activated by Zn, Cd, Hg and Pb exposure. Our results indicate that up-regulation of BjHO-1 is beneficial for limiting the uptake or accumulation of heavy metals into plants. This work also provides a new example for molecular breeding designed for plants that do not accumulate or minimizing accumulation of toxic trace metals growing on heavy metal-contaminated soils.
Keywords: Brassica juncea ; BjHO-1 ; Heavy metals; Accumulation; Detoxification