BioMetals (v.31, #6)

Exposure to Cd and Pb reduces the activity of antioxidant enzymes, which points to a decrease in the antioxidant potential of the body as a result of supplying factors which enhance cellular oxidation processes. Man is exposed to the effects of toxic metals because they are present in the environment, including in food. Since no effective ways to reduce the concentrations of Cd an Pb in food exist, studies are undertaken to develop methods of reducing their toxic effect on the body through chelating these metals using nutrients (which reduces their absorption by tissues) or increasing the oxidative capacity of the body (which decreases the possibility of inducing oxidative damage to internal organs). Studies performed on laboratory animals have shown that the use of tea infusions fulfil both functions.
Keywords: Tea; Antioxidants; Protective effect; Oxidative stress; Cadmium; Lead

Iron chelator deferiprone rescues memory deficits, hippocampal BDNF levels and antioxidant defenses in an experimental model of memory impairment by Luisa Azambuja Alcalde; Betânia Souza de Freitas; Gustavo Dalto Barroso Machado; Pedro Castilhos de Freitas Crivelaro; Victoria Campos Dornelles; Henrique Gus; Ricardo Tavares Monteiro; Luiza Wilges Kist; Mauricio Reis Bogo; Nadja Schröder (927-940).
Brain-derived neurotrophic factor (BDNF) plays a key role in neural development and physiology, as well as in pathological states. Post-mortem studies demonstrate that BDNF is reduced in the brains of patients affected by neurodegenerative diseases. Iron accumulation has also been associated to the pathogenesis of neurodegenerative diseases. In rats, iron overload induces persistent memory deficits, increases oxidative stress and apoptotic markers, and decreases the expression of the synaptic marker, synaptophysin. Deferiprone (DFP) is an oral iron chelator used for the treatment of systemic iron overload disorders, and has recently been tested for Parkinson’s disease. Here, we investigated the effects of iron overload on BDNF levels and on mRNA expression of genes encoding TrkB, p75 NTR , catalase (CAT) and NQO1. We also aimed at investigating the effects of DFP on iron-induced impairments. Rats received iron or vehicle at postnatal days 12–14 and when adults, received chronic DFP or water (vehicle). Recognition memory was tested 19 days after the beginning of chelation therapy. BDNF measurements and expression analyses in the hippocampus were performed 24 h after the last day of DFP treatment. DFP restored memory and increased hippocampal BDNF levels, ameliorating iron-induced effects. Iron overload in the neonatal period reduced, while treatment with DFP was able to rescue, the expression of antioxidant enzymes CAT and NQO1.
Keywords: Deferiprone; Iron; Memory; BDNF; Catalase; Neurodegeneration

LFchimera protects HeLa cells from invasion by Yersinia spp. in vitro by Tjitske Sijbrandij; Antoon J. Ligtenberg; Kamran Nazmi; Petra A. M. van den Keijbus; Enno C. I. Veerman; Jan G. M. Bolscher; Floris J. Bikker (941-950).
Yersinia pestis is the causative agent of plague. As adequate antibiotic treatment falls short and currently no effective vaccine is available, alternative therapeutic strategies are needed. In order to contribute to solving this problem we investigated the therapeutic potential of the peptide construct LFchimera against the safer-to-handle Y. pestis simulants Yersinia enterocolitica and Yersinia pseudotuberculosis in vitro. LFchimera is a heterodimeric peptide construct mimicking two antimicrobial domains of bovine lactoferrin, i.e. lactoferrampin and lactoferricin. LFchimera has been shown to be a potent antimicrobial peptide against a variety of bacteria in vitro and in vivo. Also Y. enterocolitica and Y. pseudotuberculosis have been shown to be susceptible for LFchimera in vitro. As Yersiniae spp. adhere to and invade host cells upon infection, we here investigated the effects of LFchimera on these processes. It was found that LFchimera has the capacity to inhibit host-cell invasion by Yersiniae spp. in vitro. This effect appeared to be host-cell mediated, not bacteria-mediated. Furthermore it was found that exposure of human HeLa epithelial cells to both LFchimera and the bacterial strains evoked a pro-inflammatory cytokine release from the cells in vitro.
Keywords: Antimicrobial peptide; Biowarfare simulants; Cellular adhesion and invasion; Lactoferrin; LFchimera

Envenomation by vipers with hemotoxic enzymes continues to be a worldwide source of morbidity and mortality. The present work examined the effects of exposure of venom enzymes to carbon monoxide and O-phenylhydroxylamine, agents that modulate the biometal heme, by forming carboxyheme and metheme, respectively. Four venoms obtained from medically important, diverse snake venom found in Africa, Asia and Australia were analyzed. The species that had venom tested in human plasma with thrombelastography and heme modulating agents were Deinagkistrodon acutus, Protobothrops mucrosquamatus, Dispholidus typus and Pseudonaja textilis. These venoms varied four hundred-fold in potency (ng-µg/ml) to exert procoagulant effects on human plasma; further, there was species specific variability in venom inhibition after exposure to carboxyheme or metheme agents. Lastly, using a wide range of carbon monoxide concentrations, it was determined that the factor V component of P. textilis venom was likely inhibited before the factor X component. Further investigation using this thrombelastograph-based, venom “kinetomic” methodology involving heme modulation will demonstrate in time its laboratory and clinical utility.
Keywords: Hemotoxic venom; Prothrombin activator; Thrombin-like activity; Heme; Carbon monoxide; Metheme

Effect of a new anti-T. cruzi metallic compound based on palladium by M. Florencia Mosquillo; Lucía Bilbao; Fabricio Hernández; Ignacio Machado; Dinorah Gambino; Beatriz Garat; Leticia Pérez-Díaz (961-974).
Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. It is estimated that 6 million people are infected in Latin America. Current treatment is not effective due to the severe side effects and the limited efficacy towards the chronic phase of the disease. Considering the growing need for specific anti-Trypanosoma cruzi drugs, organometallic Pt and Pd based compounds were previously synthesized. Although the Pt-based compound effects on T. cruzi death have been reported, no mechanism of action has been proposed for the Pd-based analogous compound. In this work, we determined excellent to very good values of IC50 and SI. To analyze the compound mode of action, we measured Pd uptake and its association to the macromolecules of the parasite by electrothermal atomic absorption spectrometry. We found a poor uptake, which reaches only 16% after 24 h of incubation using 10× IC50, being the scarce incorporated metal preferentially associated to DNA. However, this compound has a trypanocidal effect, leading to morphological changes such as shortening of the parasite cell body and inducing necrosis after 24 h of treatment. Furthermore, this compound impairs the parasite development in the host both at the trypomastigote infection process and the intracellular amastigotes replication. In conclusion, our findings support that Pd-dppf-mpo compound constitutes a promising anti-T. cruzi compound effective against the chronic phase of the disease.
Keywords: Trypanosoma cruzi ; Pd-based compound; Cell death mechanism; Morphological changes

Silver in the meat and organs of broiler chickens in case of using colloidal silver as an alternative to antibiotics by Aleksandrova Svetlana Sergeevna; Simonov Oleg Anatolevich; Shigabaeva Gulnara Nurchallaeva; Bakharev Alexey Aleksandrovich; Renev Evgeniy Petrovich; Shabaldin Sergey Vladimirovich; Grigoreva Marina Alexeevna; Ivanov Igor Diadorovitch (975-980).
The search for new antibacterial products, the mechanisms of action of which differ from conventional antibiotics is a current a topical issue. The objective of our research is to identify the presence of silver in meat and organs of broiler chicks that had been given colloidal silver. The results show that the broiler chick meat contains silver in quantities safe for humans regardless of the use of colloidal silver. Comparison of meat analysis results in experimental and control groups indicate that the ratio of parameters distribution variance for all birds to the mean variance by group for each measured no statistical differences in the chemical composition of bird’s meat of experimental and control groups. The analysis also confirmed the existing difference in chemical composition of leg muscle meat and chest muscle meat (P < 0.05), whereas leg muscle contains more fat (6.81% vs. 2.85%) and less protein (20.25% vs. 22.81%).
Keywords: Colloidal silver; Broiler; Chicken meat

Vanadium complex: an appropriate candidate for killing hepatocellular carcinoma cancerous cells by Hamid Bakhshi Aliabad; Soudeh Khanamani Falahati-pour; Hadis Ahmadirad; Maryam Mohamadi; Mohammad Reza Hajizadeh; Mehdi Mahmoodi (981-990).
Hepatocellular carcinoma (HCC) is a prevalent human malignancy which its drug resistance is increasing world-wide. This project was designed to assess the anti-cancer effects of 4-bromo-2-(((5-chloro-2-hydroxyphenyl) imino) methyl) phenol ([IV(L)] complex) on the HepG2 cell line and also L929 cells, as normal cells. HepG2 and L929 cells were cultured in RPMI culture medium and the survival rates of the cells were determined after 24 and 48 h using MTT assay to find IC50 concentration of vanadium m, [IV(L)] complex. The early apoptosis and necrosis/late apoptosis were determined by means of annexin V/PI apoptosis detection kit. The results revealed that vanadium m, [IV(L)] complex induce early apoptosis higher in HepG2 cell line than L929 cells. The rates of necrosis/late apoptosis were also induced in HepG2 cells more than L929 cells. Based on the results, vanadium m, [IV(L)] complex might be considered as a safe new drug for treatment of HCC with low side effects on control liver cells.
Keywords: Vanadium complex; HepG2 cells; L929 cells; Hepatocellular carcinoma; Cytotoxicity

Studies on the activation of isocitrate dehydrogenase kinase/phosphatase (AceK) by Mn2+ and Mg2+ by Yanxia Yin; Yadan Gao; Shanze Li; Guohua Jiang; Qun Wei (991-1002).
Isocitrate dehydrogenase kinase/phosphatase (AceK) is a bifunctional enzyme with both kinase and phosphatase activities that are activated by Mg2+. We have studied the interactions of Mn2+and Mg2+ with AceK using isothermal titration calorimetry (ITC) combined with molecular docking simulations and show for the first time that Mn2+ also activates the enzyme activities. However, Mn2+ and Mg2+ exert their effects by different mechanisms. Although they have similar binding constants (of 1.11 × 105 and 0.98 × 105 M−1, respectively) for AceK and induce conformational changes of the enzyme, they do not compete for the same binding site. Instead Mn2+ appears to bind to the regulatory domain of AceK, and its effect is transmitted to the active site of the enzyme by the conformational change that it induces. The information in this study should be very useful for understanding the molecular mechanism underlying the interaction between AceK and metal ions, especially Mn2+ and Mg2+.
Keywords: AceK; Kinase; Phosphatase; Binding; Activation; ITC

In vitro leishmanicidal activity and theoretical insights into biological action of ruthenium(II) organometallic complexes containing anti-inflammatories by Victor M. Miranda; Monica S. Costa; Silvana Guilardi; Antonio E. H. Machado; Javier A. Ellena; Kelly A. G. Tudini; Gustavo Von Poelhsitz (1003-1017).
Leishmaniasis, a neglected tropical disease caused by protozoans of the genus Leishmania, kills around 20–30 thousand people in Africa, Asia, and Latin America annually and, despite its potential lethality, it can be treated and eventually cured. However, the current treatments are limited owing to severe side effects and resistance development by some Leishmania. These factors make it urgent to develop new leishmanicidal drugs. In the present study, three ruthenium(II) organometallic complexes containing as ligands the commercially available anti-inflammatories diclofenac (dic), ibuprofen (ibu), and naproxen (nap) were synthesized, characterized, and subjected to in vitro leishmanicidal activity. The in vitro cytotoxicity assays against Leishmania (L.) amazonensis and Leishmania (L.) infantum promastigotes have shown that complexes [RuCl(dic)(η6-p-cymene)] (1) and [RuCl(nap)(η6-p-cymene)] (3) were active against both Leishmania species. Complex [RuCl(ibu)(η6-p-cymene)] (2) has exhibited no activity. The IC50 values for the two active complexes were respectively 7.42 and 23.55 μM, for L. (L.) amazonensis, and 8.57 and 42.25 μM, for L. (L.) infantum. Based on the toxicological results and computational analysis, we proposed a correlation between the complexes and their activity. Our results suggest both complexation to ruthenium(II) and ligands structure are key elements to leishmanicidal activity.
Keywords: Ruthenium(II); p-Cymene; Leishmaniasis; Anti-inflammatories

C2H2 type of zinc finger transcription factors (C2H2-ZFP TFs) play crucial roles in plant developments and stress response. Regarding its importance, genome-wide study of C2H2-ZFs were performed in multiple important plant species, but any such investigation was not fulfilled in Triticum turgidum ssp. Durum (durum wheat) as an important nutritional crop. The present study identified 122 C2H2-ZFs in durum wheat and physically mapped them onto the genome. The phylogenetic analysis classified these TFs into six major groups. Genes structure and conserved motifs assay showed TtC2H2-ZF involvement in the important cellular functions. Comparative phylogeny between durum wheat TtC2H2-ZF genes and the orthologs in rice revealed the evolutionary relationships of C2H2-ZF proteins. The gene ontology and promoter cis-element analysis indicated that most of TtC2H2-ZF genes are involved in multiple molecular functions including metal ion-binding and various stimuli responses. Further, the miRNAs targeting TtC2H2-ZF transcripts, homology modeling and proteins interaction network were also demonstrated, suggesting the vital cellular functions of TtC2H2-ZFs during various circumstances. The expression heatmap demonstrated differential and tissue-specific expression patterns of these genes. Expression profiling of this gene family members in response to dehydration and heat stresses showed differential expression pattern of these genes at multiple time points of stresses. This study can prepare a comprehensive overview of the durum wheat C2H2-ZF gene family and may provide a new perspective on the evolution of them, which will form the basis for further investigation of the roles of this family members and future genetic engineering studies in crops.
Keywords: C2H2 zinc finger family; Metal ion-binding; Stimuli response; Systematic evaluation; Triticum turgidum ssp.; Durum

Citrate analysis using capillary electrophoresis and complexation with Eu3+-tetracycline by Douglas B. Craig; Jennifer R. Lischynski; Isabela C. C. Cardoso (1043-1049).
A sensitive assay for citrate was developed. Citrate was incubated with 50 μM Eu3+-tetracycline and the complex separated using capillary electrophoresis utilizing post-column laser-induced luminescence detection in a sheath flow cuvette. Signal was linear with citrate concentration from 10 μM to 200 nM. Injection volumes were 320 pL. For the 200 nM sample, this corresponds to the injection of 64 amoles of citrate. Separation time was < 90 s with a total run time of 5 min. As an application the method was used to analyze citrate in agricultural and medicinal products. The method was also used to develop an assay for the enzyme citrate synthase.
Keywords: Capillary electrophoresis; Citrate; Citrate synthase; Eu3+-tetracycline; Luminescence

Selenium-Rich Yeast protects against aluminum-induced peroxidation of lipide and inflammation in mice liver by Junchong Luo; Xiaowen Li; Xinran Li; Yongming He; Mengdan Zhang; Changyu Cao; Kai Wang (1051-1059).
To investigate the effect of Selenium Rich Yeast (SeY) on hepatotoxicity of Aluminium (Al), SeY (0.1 mg/kg) was orally administrated to aluminium-exposed mice (10 mg/kg) for 28 days. The risk of oxidative stress was assessed by detecting the total antioxidant capacity (T-AOC), catalase activity, H2O2 content, and mRNA levels of the Keap1/Nrf-2/HO-1 pathway. Inflammatory reactions were assessed by detecting the mRNA levels of inflammatory biomarkers. Our results showed that SeY protected against the liver histological changes induce by Al. The body weight gain of mice treated with SeY + Al restore to normal compare with mice exposed to Al alone. Al treatment significantly decreased the activities of antioxidant enzymes, reduced T-AOC levels, and up-regulated the mRNA level of Nrf2 and HO-1, thereby ultimately leading to peroxidation. SeY shown a significant protective effect against oxidative stress caused by Al. In addition, Al exposure induced inflammatory responses in rat liver by promoting the release of inflammatory cytokines (TNF-a, NF-kB, TNF-R1, IL-1, IL-6, and COX-2). SeY protected against changes in liver by regulating the mRNA expression levels of inflammatory factors. These results suggested that Se protected the liver from the Al-induced hepatotoxicity by regulating the mRNA level of Keap1/Nrf2/HO-1, and inhibited inflammatory responses by down-regulating the expression level of inflammatory cytokine.
Keywords: Selenium-Rich Yeast (SeY); Aluminum; Inflammation; Oxidative stress; Mice liver

We present further analyses of a previous experiment published in 2016 where the distribution, concentration and correlation of iron, zinc, copper and sulphur in the choroid of the eye in young and aged old world primates (Macaca fascicularis) was studied with synchrotron X-ray fluorescence with a 2 μm resolution. The results indicate that iron accumulates in hotspots in the choroid with age with fluorescence intensity ranging from 2- to 7-fold (1002–3752 ppm) the mean level in the choroidal stroma (500 ppm) and maximum iron levels in blood vessel lumina. Iron hotspots with iron ppm > 1000 preferentially contained Fe3+ as demonstrated by Perls staining. There was a strong spatial co-localisation and correlation between copper and zinc (Pearson’s correlation coefficient 0.97), and both elements with sulphur in the choroid of young animals. However, these are reduced in the choroid of aged animals and lost in the iron hotspots. The lack of proportional co-distribution suggests that iron accumulation does not induce a concomitant increase in zinc, copper or zinc-, copper-metalloproteins. It is possible that the iron hotspots are ferritin or hemosiderin molecules loaded with Fe3+ in stable, insoluble, non-toxic complexes without a significant oxidative environment.
Keywords: Age-related macular degeneration; Retina; Trace elements; Choroid; Iron; Zinc; Copper

Animals can greatly increase their fitness by choosing oviposition sites free from damaging substances such as the heavy metal cadmium (Cd). Previous research has shown that Drosophila melanogaster reared on uncontaminated media lay fewer eggs on substrates containing Cd. In this study, we examined the effects of prior exposure to Cd on oviposition site preference. We tested flies that had been exposed to Cd continuously from egg to adult, only during pre-adult stages, or for the 4 days of adulthood prior to testing. We found that flies avoid laying eggs on Cd-contaminated medium and also that flies exposed to Cd as adults laid significantly fewer eggs than controls never exposed to Cd. We did not observe consistent changes in oviposition as a result of pre-adult exposure alone. Our results suggest that the presence of cadmium in the environment, even at low doses and for short periods of time during adulthood, can harm the fitness of Drosophila melanogaster.
Keywords: Oviposition; Cadmium; Drosophila melanogaster ; Development

Ferric pyrophosphate citrate: interactions with transferrin by Raymond Pratt; Garry J. Handelman; Thomas E. Edwards; Ajay Gupta (1081-1089).
There are several options available for intravenous application of iron supplements, but they all have a similar structure:—an iron core surrounded by a carbohydrate coating. These nanoparticles require processing by the reticuloendothelial system to release iron, which is subsequently picked up by the iron-binding protein transferrin and distributed throughout the body, with most of the iron supplied to the bone marrow. This process risks exposing cells and tissues to free iron, which is potentially toxic due to its high redox activity. A new parenteral iron formation, ferric pyrophosphate citrate (FPC), has a novel structure that differs from conventional intravenous iron formulations, consisting of an iron atom complexed to one pyrophosphate and two citrate anions. In this study, we show that FPC can directly transfer iron to apo-transferrin. Kinetic analyses reveal that FPC donates iron to apo-transferrin with fast binding kinetics. In addition, the crystal structure of transferrin bound to FPC shows that FPC can donate iron to both iron-binding sites found within the transferrin structure. Examination of the iron-binding sites demonstrates that the iron atoms in both sites are fully encapsulated, forming bonds with amino acid side chains in the protein as well as pyrophosphate and carbonate anions. Taken together, these data demonstrate that, unlike intravenous iron formulations, FPC can directly and rapidly donate iron to transferrin in a manner that does not expose cells and tissues to the damaging effects of free, redox-active iron.
Keywords: Iron; Transferrin; Crystal structure; Ferric pyrophosphate citrate; Kinetics

Physicochemical characterization of ferric pyrophosphate citrate by Ajay Gupta; Raymond Pratt; Bhoopesh Mishra (1091-1099).
Iron deficiency is a significant health problem across the world. While many patients benefit from oral iron supplements, some, including those on hemodialysis require intravenous iron therapy to maintain adequate iron levels. Until recently, all iron compounds suitable for parenteral administration were colloidal iron–carbohydrate conjugates that require uptake and processing by macrophages. These compounds are associated with variable risk of anaphylaxis, oxidative stress, and inflammation, depending on their physicochemical characteristics. Ferric pyrophosphate citrate (FPC) is a novel iron compound that was approved for parenteral administration by US Food and Drug Administration in 2015. Here we report the physicochemical characteristics of FPC. FPC is a noncolloidal, highly water soluble, complex iron salt that does not contain a carbohydrate moiety. X-ray absorption spectroscopy data indicate that FPC consists of iron (III) complexed with one pyrophosphate and two citrate molecules in the solid state. This structure is preserved in solution and stable for several months, rendering it suitable for pharmaceutical applications in solid or solution state.
Keywords: Ferric pyrophosphate citrate; XAFS; Synthesis; Physicochemistry

Chromosomal Sil system contributes to silver resistance in E. coli ATCC 8739 by Mariana Blanco Massani; Jochen Klumpp; Madeleine Widmer; Christian Speck; Marc Nisple; Rainer Lehmann; Markus Schuppler (1101-1114).
The rise of antibiotic resistance in pathogenic bacteria is endangering the efficacy of antibiotics, which consequently results in greater use of silver as a biocide. Chromosomal mapping of the Cus system or plasmid encoded Sil system and their relationship with silver resistance was studied for several gram-negative bacteria. However, only few reports investigated silver detoxification mediated by the Sil system integrated in Escherichia coli chromosome. Accordingly, this work aimed to study the Sil system in E. coli ATCC 8739 and to produce evidence for its role in silver resistance development. Silver resistance was induced in E. coli ATCC 8739 by stepwise passage in culture media containing increasing concentrations of AgNO3. The published genome of E. coli ATCC 8739 contains a region showing strong homology to the Sil system genes. The role of this region in E. coli ATCC 8739 was assessed by monitoring the expression of silC upon silver stress, which resulted in a 350-fold increased expression. De novo sequencing of the whole genome of a silver resistant strain derived from E. coli ATCC 8739 revealed mutations in ORFs putative for SilR and CusR. The silver resistant strain (E. coli AgNO3R) showed constitutive expression of silC which posed a cost of fitness resulting in retarded growth. Furthermore, E. coli AgNO3R exhibited cross-resistance to ciprofloxacin and a slightly increased tolerance to ampicillin. This study demonstrates that E. coli is able to develop resistance to silver, which may pose a threat towards an effective use of silver compounds as antiseptics.
Keywords: Silver resistance; Escherichia coli ; De novo sequencing; Sil system; silC expression; qRT-PCR

Due to a technical error, the copyright line of the above mentioned article was incorrect. The original publication has been corrected.