BioMetals (v.28, #4)

Listeria monocytogenes is a Gram-positive bacterium that causes a rare but severe human disease with high mortality rate. The microorganism is widespread in the natural environment where it shows a saprophytic lifestyle. In the human body it infects many different cell types, where it lives intracellularly, however it may also temporarily live extracellularly. The ability to survive and grow in such diverse niches suggests that this bacterium has a wide range of mechanisms for both the acquisition of various sources of iron and effective management of this microelement. In this review, data about the mechanisms of transport, metabolism and regulation of iron, including recent findings in these areas, are summarized with focus on the importance of these mechanisms for the virulence of L. monocytogenes. These data indicate the key role of haem transport and maintenance of intracellular iron homeostasis for the pathogenesis of L. monocytogenes. Furthermore, some of the proteins involved in iron homeostasis like Fri and FrvA seem to deserve special attention due to their potential use in the development of new therapeutic antilisterial strategies.
Keywords: Listeria monocytogenes ; Iron transport; Antilisterial strategies

Selenium (Se) is an essential trace element for humans. It is found in the enzyme glutathione peroxidase. This enzyme protects the organism against certain types of damage. Some data suggest that Se plays a role in the body’s metabolism of mercury (Hg). Selenium has in some studies been found to reduce the toxicity of Hg salts. Selenium and Hg bind in the body to each other. It is not totally clear what impact the amount of Se has in the human body on the metabolism and toxicity of prolonged Hg exposure.
Keywords: Selenium; Mercury; Interaction; Metal toxicology

Physico-chemical properties of the new generation IV iron preparations ferumoxytol, iron isomaltoside 1000 and ferric carboxymaltose by Susann Neiser; Daniel Rentsch; Urs Dippon; Andreas Kappler; Peter G. Weidler; Jörg Göttlicher; Ralph Steininger; Maria Wilhelm; Michaela Braitsch; Felix Funk; Erik Philipp; Susanna Burckhardt (615-635).
The advantage of the new generation IV iron preparations ferric carboxymaltose (FCM), ferumoxytol (FMX), and iron isomaltoside 1000 (IIM) is that they can be administered in relatively high doses in a short period of time. We investigated the physico-chemical properties of these preparations and compared them with those of the older preparations iron sucrose (IS), sodium ferric gluconate (SFG), and low molecular weight iron dextran (LMWID). Mössbauer spectroscopy, X-ray diffraction, and Fe K-edge X-ray absorption near edge structure spectroscopy indicated akaganeite structures (β-FeOOH) for the cores of FCM, IIM and IS, and a maghemite (γ-Fe2O3) structure for that of FMX. Nuclear magnetic resonance studies confirmed the structure of the carbohydrate of FMX as a reduced, carboxymethylated, low molecular weight dextran, and that of IIM as a reduced Dextran 1000. Polarography yielded significantly different fingerprints of the investigated compounds. Reductive degradation kinetics of FMX was faster than that of FCM and IIM, which is in contrast to the high stability of FMX towards acid degradation. The labile iron content, i.e. the amount of iron that is only weakly bound in the polynuclear iron core, was assessed by a qualitative test that confirmed decreasing labile iron contents in the order SFG ≈ IS > LMWID ≥ FMX ≈ IIM ≈ FCM. The presented data are a step forward in the characterization of these non-biological complex drugs, which is a prerequisite to understand their cellular uptake mechanisms and the relationship between the structure and physiological safety as well as efficacy of these complexes.
Keywords: Intravenous iron; Iron sucrose; Ferric carboxymaltose; Iron isomaltoside 1000; Ferumoxytol; Low molecular weight iron dextran

Catalytic enhancement of the heme-based oxygen-sensing phosphodiesterase EcDOS by hydrogen sulfide is caused by changes in heme coordination structure by Fang Yan; Veronika Fojtikova; Petr Man; Martin Stranava; Markéta Martínková; Yongming Du; Dongyang Huang; Toru Shimizu (637-652).
EcDOS is a heme-based O2-sensing phosphodiesterase in which O2 binding to the heme iron complex in the N-terminal domain substantially enhances catalysis toward cyclic-di-GMP, which occurs in the C-terminal domain. Here, we found that hydrogen sulfide enhances the catalytic activity of full-length EcDOS, possibly owing to the admixture of 6-coordinated heme Fe(III)–SH and Fe(II)–O2 complexes generated during the reaction. Alanine substitution at Met95, the axial ligand for the heme Fe(II) complex, converted the heme Fe(III) complex into the heme Fe(III)–SH complex, but the addition of Na2S did not further reduce it to the heme Fe(II) complex of the Met95Ala mutant, and no subsequent formation of the heme Fe(II)–O2 complex was observed. In contrast, a Met95His mutant formed a stable heme Fe(II)–O2 complex in response to the same treatment. An Arg97Glu mutant, containing a glutamate substitution at the amino acid that interacts with O2 in the heme Fe(II)–O2 complex, formed a stable heme Fe(II) complex in response to Na2S, but this complex failed to bind O2. Interestingly, the addition of Na2S promoted formation of verdoheme (oxygen-incorporated, modified protoporphyrin IX) in an Arg97Ile mutant. Catalytic enhancement by Na2S was similar for Met95 mutants and the wild type, but significantly lower for the Arg97 mutants. Thus, this study shows the first isolation of spectrometrically separated, stable heme Fe(III)–SH, heme Fe(II) and heme Fe(II)–O2 complexes of full-length EcDOS with Na2S, and confirms that external-ligand–bound, 6-coordinated heme Fe(III)–SH or heme Fe(II)–O2 complexes critically contribute to the Na2S-induced catalytic enhancement of EcDOS.
Keywords: Heme; O2 sensor; Phosphodiesterase; c-di-GMP; Hydrogen sulfide

Label free impedance technology enables the monitoring of cell response patterns post treatment with drugs or other chemicals. Using this technology, a correlation between the lipophilicity of metal complexes and the degree of cytotoxicity was observed. Au(L1)Cl (1), AuPd(L1)(SC4H8)Cl3 (1a) and Au(L2)Cl (2) [L1 = diphenylphosphino-2-pyridine; L2 = 2-(2-(diphenylphosphino)ethyl)-pyridine] were synthesised, in silico drug-likeness and structure–activity relationship monitored using impedance technology. Dose dependent changes in cytotoxicity were observed for the metal complexes resulting in IC50s of 12.5 ± 2.5, 18.3 ± 8.3 and 16.9 ± 0.5 µM for 1, 1a and 2 respectively in an endpoint assay. When a real time impedance assay was used, dose-dependent responses depicting patterns that suggested slower uptake (at a toxic 20 µM) and faster recovery of the cells (at the less toxic 10 µM) of the bimetallic complex (1a) compared to the monometallic complexes (1 and 2) was observed. These data agreed with the ADMET findings of lower aqueous solubility of 1a and non-ideal lipophilicity (AlogP98 of 6.55) over more water soluble 1 and 2 with ideal lipophilicity (4.91 and 5.03 respectively) values. The additional coordination of a Pd atom to the nitrogen atom of a pyridine ring, the sulfur atom of a tetrahydrothiophene moiety and two chlorine atoms in 1a could be contributing to the observed differences when compared to the monometallic complexes. This report presents impedance technology as a means of correlating drug-likeness of lipophilic phosphine complexes containing similar backbone structures and could prove valuable in filtering drug-like compounds in a drug discovery project.
Keywords: Impedance; Cytotoxicity; Uptake; Recovery; Lipophilicity; Phosphine gold complexes

Anti-proliferative effect of Fe(III) complexed with 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone in HepG2 cells by Takeshi Fukushima; Erina Taniguchi; Hiroshi Yamada; Kiyomasa Kato; Ayako Shimizu; Yoshikazu Nishiguchi; Mayu Onozato; Hideaki Ichiba; Yutaro Azuma (669-677).
We previously developed a chelating ligand, 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone (HMB–ASH), which can chelate Fe(III) to form a complex. The HMB–ASH–Fe(III) complex exhibits a dose-dependent anti-proliferative effect in HepG2 cells, whereas the ligand, HMB–ASH, and Fe(III) alone had no considerable effect. The HMB–ASH–Fe(III) complex was composed of Fe(III):HMB–ASH (1:2), as determined by high-performance liquid chromatography with high-resolution mass spectrometry. The IC50 value was approximately 20 μM, which was comparable to those of the anti-cancer drugs oxaliplatin (OXP) and etoposide (ETP) under the same conditions. Similar to OXP and ETP, HMB–ASH–Fe(III) induced apoptosis in HepG2 cells, as revealed by terminal deoxynucleotidyl transferase fluorescein-12-dUTP nick end labeling assay.
Keywords: 1-(2-Hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone; Metal complex; Anti-proliferation; Fe(III); HepG2 cells

Both human and horse fibrinogen are heme-binding proteins, and horse fibrinogen also exhibits heme-mediated ferritin binding. This study found that bovine and human fibrinogen are heme-mediated ferritin-binding proteins and demonstrated direct binding of bovine ferritin to protoporphyrin (PPIX) and its derivatives or to Zn ions. Binding of bovine and human fibrinogen to bovine spleen ferritin coated on microtiter plate wells was detected using an anti-human fibrinogen antibody, and this binding was inhibited in a dose-dependent manner by hemin (iron-PPIX) and also inhibited by Zn-PPIX. PPIX showed less of an inhibitory effect on the binding of bovine and human fibrinogen to bovine ferritin. The inhibitory effect of Sn-PPIX was similar to that of PPIX, but with respect to human fibrinogen, PPIX did not inhibit the binding of human fibrinogen to ferritin. Bovine fibrinogen immobilized on CNBr-activated Sepharose 4B beads showed affinity for hemin, Sn-PPIX, Zn-PPIX, and iron-free PPIX in the order Sn-PPIX < iron-free PPIX < hemin < Zn-PPIX. The fibrinogen beads also directly bound to zinc ions. These results suggest that bovine fibrinogen is a heme- and zinc-binding protein and that binding of circulating mammalian fibrinogen to ferritin is heme mediated.
Keywords: Ferritin-binding protein; Fibrinogen; Hemin; Protoporphyrin; Zinc

Role of fructose 1,6-bisphosphate-mediated iron oxidation in the generation of reactive oxygen species was analyzed. Aconitase the most sensitive enzyme to oxidative stress was inactivated potently by fructose 1,6-bisphosphate in the presence of ferrous ion, and further by ADP and PEP to a lesser extent. The inactivation requires cyanide, suggesting that the superoxide radical is responsible for the inactivation. Addition of ascorbic acid and dithiothreitol prevented aconitase from the inactivation. Fructose 1,6-bisphosphate, ADP and PEP stimulated the oxidation of ferrous ion causing one-electron reduction of oxygen molecule. Superoxide radical formed with iron oxidation participates in the oxidative inactivation of aconitase and the citric acid cycle, resulting in the induction of the Crabtree effect, that is, high glucose-mediated inhibition of oxidative metabolism in mitochondria.
Keywords: Fructose 1,6-bisphosphate; Superoxide radical; Aconitase inactivation; Ferrous ion; Crabtree effect

Alterations in zinc binding capacity, free zinc levels and total serum zinc in a porcine model of sepsis by Janine Hoeger; Tim-Philipp Simon; Sabine Doemming; Christoph Thiele; Gernot Marx; Tobias Schuerholz; Hajo Haase (693-700).
Zinc is crucial for immune function. In addition, the redistribution of zinc and other nutrients due to infection is an integral part of the host immune response to limit availability to pathogens. However, the major zinc binding protein albumin is down regulated during the acute phase response, implicating a decrease in zinc binding capacity. A prospective animal study with eight female German landrace pigs was conducted to investigate alterations in zinc binding capacity, total serum zinc and free zinc levels in the initial phase of sepsis. Sepsis was induced by instillation of autologous feces via midline laparotomy. Total serum zinc declined significantly after 1 h (10.89 ± 0.42 µM vs. 7.67 ± 0.41 µM, p < 0.001), total serum copper and iron reached a significant reduction at 4 h. Urinary excretion of zinc declined in line with total serum zinc. In comparison to total serum zinc, free zinc levels declined to a lesser, though significant, extent. Zinc binding capacity of serum decreased over time, whereby free zinc levels after addition of zinc correlated negatively with total serum protein and albumin levels. In addition IL-6 and TNF-α concentrations were measured and increased significantly 2 h after induction of sepsis. Hence, total serum zinc was the first marker of inflammation in our experiment, and might therefore be a promising biomarker for the early diagnosis of sepsis. Furthermore the observation of a substantially different serum free zinc homeostasis during sepsis provides valuable information for a potential therapeutic zinc supplementation, which has to take buffering capacity by serum proteins into account.
Keywords: Zinc; Sepsis; Albumin; Inflammation; Copper; Intensive care

Metallothionein, essential elements and lipid peroxidation in mercury-exposed suckling rats pretreated with selenium by Tatjana Orct; Maja Lazarus; Marija Ljubojević; Ankica Sekovanić; Ivan Sabolić; Maja Blanuša (701-712).
Detoxification of mercury (Hg) with selenium (Se) in the early postnatal period with regard to the expression of metallothionein protein (MT), essential element status, and lipid peroxidation level in tissues has not been studied. Seven-day-old Wistar pups were orally pretreated with Se [6 μmol Na2SeO3/kg body weight (b.w.)] for 3 days and then cotreated with Hg (6 μmol HgCl2/kg b.w.) for the following 4 days. This group (Se + Hg) was compared to the groups treated with Hg, Se, or vehicle (control). Compared to the Hg-group, Se + Hg-group exhibited lower renal MT expression, reduced accumulation of Hg, Cu and Zn, and reduced excretion of Se, Hg and Zn in urine. In the liver, MT was stimulated by Se treatment in both, Se and Se + Hg-group. Hepatic and brain levels of the endogenous essential elements Cu, Fe, Mg, and Zn remained unchanged in all of the studied groups. Brain Hg levels and oxidation of lipids measured as thiobarbituric acid reactive substances were diminished in Se + Hg-group of pups compared to the Hg-group. This study suggests that Se pretreatment can help reduce Hg in the tissues of suckling rats, simultaneously preventing impairment of essential element levels in the kidneys and their excessive excretion via urine. Also, Se was shown to prevent oxidative damage of lipids in the brain, which is particularly susceptible to Hg during the early postnatal period.
Keywords: Selenium supplementation; Mercury exposure; Suckling rat; Metallothionein; Essential element; Lipid peroxidation

The transport of heavy-metal ions across the plasma membrane is essential for mycobacterial intracellular survival; in this context, P-type ATPases are pivotal for maintenance of ionic gradients and the plasma membrane homeostasis of mycobacteria. To date, the copper ion transport that is mediated by P-type ATPases in mycobacteria is poorly understood. In this work, the ion-specific activation of CtpA, a putative plasma membrane Mycobacterium tuberculosis P-type ATPase, with different heavy-metal cations was assessed. Mycobacterium smegmatis mc2155 cells heterologously expressing the M. tuberculosis ctpA gene displayed an increased tolerance to toxic levels of the Cu2+ ion (4 mM) compared to control cells, suggesting that CtpA is possibly involved in the copper detoxification of mycobacterial cells. In contrast, the tolerance of M. smegmatis recombinant cells against other heavy-metal divalent cations, such as Co2+, Mn2+, Ni2+ and Zn2+, was not detected. In addition, the ATPase activity of plasma membrane vesicles that were obtained from M. smegmatis cells expressing CtpA was stimulated by Cu+ (4.9 nmol of Pi released/mg of protein.min) but not by Cu2+ ions; therefore, Cu2+ reduction to Cu+ inside mycobacterial cells is suggested. Finally, the plasma membrane vesicles of M. smegmatis that were enriched with CtpA exhibited an optimal activity at 37 °C and pH 7.9; the apparent kinetic parameters of the enzyme were a K 1/2 of 4.68 × 10−2 µM for Cu+, a V max of 10.3 U/mg of protein, and an h value of 1.91.
Keywords: Mycobacterium tuberculosis ; Mycobacterial plasma membrane; P-type ATPases; CtpA; Heavy metal tolerance

Cobalt protoporphyrin represses osteoclastogenesis through blocking multiple signaling pathways by Yuka Yashima; Kuniaki Okamoto; Eiko Sakai; Mayumi Iwatake; Yutaka Fukuma; Kazuhisa Nishishita; Takayuki Tsukuba (725-732).
Cobalt protoporphyrin (CoPP) is a metallo-protoporphyrin that works as a powerful inducer of heme oxigenase-1 (HO-1) in various tissues and cells. Our recent studies have demonstrated that induction of HO-1 by several reagents inhibited differentiation and activation of osteoclasts (OCLs), which are multinucleated bone resorbing cells. However, the effects of CoPP on osteoclastogenesis remain to be elucidated. In this study, we report that CoPP inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced OCL formation in a dose dependent manner. Importantly, CoPP had little cytotoxicity, but rather enhanced cell proliferation of OCLs. CoPP suppressed the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) as well as those of OCLs markers such as Src and cathepsin K, which are transcriptionally regulated by NFATc1 in mature OCLs. Western blot analyses also showed that CoPP abolished RANKL-stimulated phosphorylation of several major signaling pathways such as IκB, Akt, ERK, JNK and p38 MAPKs in OCL precursor cells. Thus, our results show that CoPP represses osteoclastogenesis through blocking multiple signaling pathways.
Keywords: Osteoclasts; Cobalt protoporphyrin; Hemeoxigenase-1; RANKL

Mouse genetic background impacts both on iron and non-iron metals parameters and on their relationships by Thibault Cavey; Martine Ropert; Marie de Tayrac; Edouard Bardou-Jacquet; Marie-Laure Island; Patricia Leroyer; Claude Bendavid; Pierre Brissot; Olivier Loréal (733-743).
Iron is reported to interact with other metals. In addition, it has been shown that genetic background may impact iron metabolism. Our objective was to characterize, in mice of three genetic backgrounds, the links between iron and several non-iron metals. Thirty normal mice (C57BL/6, Balb/c and DBA/2; n = 10 for each group), fed with the same diet, were studied. Quantification of iron, zinc, cobalt, copper, manganese, magnesium and rubidium was performed by ICP/MS in plasma, erythrocytes, liver and spleen. Transferrin saturation was determined. Hepatic hepcidin1 mRNA level was evaluated by quantitative RT-PCR. As previously reported, iron parameters were modulated by genetic background with significantly higher values for plasma iron parameters and liver iron concentration in DBA/2 and Balb/c strains. Hepatic hepcidin1 mRNA level was lower in DBA/2 mice. No iron parameter was correlated with hepcidin1 mRNA levels. Principal component analysis of the data obtained for non-iron metals indicated that metals parameters stratified the mice according to their genetic background. Plasma and tissue metals parameters that are dependent or independent of genetic background were identified. Moreover, relationships were found between plasma and tissue content of iron and some other metals parameters. Our data: (i) confirms the impact of the genetic background on iron parameters, (ii) shows that genetic background may also play a role in the metabolism of non-iron metals, (iii) identifies links between iron and other metals parameters which may have implications in the understanding and, potentially, the modulation of iron metabolism.
Keywords: Iron; Metal; Genetic background; Mice; ICP/MS

Structural evidences for a secondary gold binding site in the hydrophobic box of lysozyme by Giarita Ferraro; Lara Massai; Luigi Messori; Maria Agostina Cinellu; Antonello Merlino (745-754).
A new crystal structure is reported here for the adduct formed in the reaction between NH4 [Au(Sac)2], AuSac2, a cytotoxic homoleptic gold(I) complex with the saccharinate ligand, and the model protein hen egg white lysozyme. To produce this adduct, AuSac2 breaks down and releases both saccharinate ligands. The resulting Au(I) ions bind the protein to ND1 and NE2 atoms of His15 but also to SD atom of the zero-solvent accessible Met105 side chain, which is located in the protein hydrophobic box. The unexpected existence of this secondary gold(I) binding site is confirmed by spectroscopic and spectrometric measurements in solution.
Keywords: Gold-based drugs; Metallodrugs; Protein–ligand interactions; Mechanisms of action; Gold complexes

Mycothiol serves as the primary reducing agent in Mycobacterium species, and is also a cofactor for the detoxification of xenobiotics. Mycothiol conjugate amidase (Mca) is a metalloamidase that catalyzes the cleavage of MS-conjugates to form a mercapturic acid, which is excreted from the mycobacterium, and 1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside. Herein we report on the metal cofactor preferences of Mca from Mycobacterium smegmatis and Mycobacterium tuberculosis. Importantly, results from homology models of Mca from M. smegmatis and M. tuberculosis suggest that the metal binding site of Mca is identical to that of the closely related protein N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase (MshB). This finding is supported by results from zinc ion affinity measurements that indicate Mca and MshB have comparable $$K_{ ext{D}}^{{{ ext{Zn}}left( { ext{II}} ight)}}$$ K D Zn II values (~10–20 pM). Furthermore, results from pull-down experiments using Halo-Mca indicate that Mca purifies with (stoichiometric) Fe2+ when purified under anaerobic conditions, and Zn2+ when purified under aerobic conditions. Consequently, Mca is likely a Fe2+-dependent enzyme under physiological conditions; with Zn2+-Mca an experimental artifact that could become biologically relevant under oxidatively stressed conditions. Importantly, these findings suggest that efforts towards the design of Mca inhibitors should include targeting the Fe2+ form of the enzyme.
Keywords: Mycothiol conjugate amidase; Metalloamidase; Zinc; Iron; MshB; Mycothiol

The effect of 1:2 Ag(I) thiocyanate complexes in MCF-7 breast cancer cells by Eloise Ferreira; Appollinaire Munyaneza; Bernard Omondi; Reinout Meijboom; Marianne J. Cronjé (765-781).
There is much interest currently in the design of metal compounds as drugs and various metal compounds are already in clinical use. These include gold(I) compounds such as auranofin and the anti-cancer platinum(II) complex, cisplatin. Bis-chelated gold(I) phosphine complexes have also shown great potential as anticancer agents, however, their efficacy has been limited by their high toxicity. In this study, silver(I) thiocyanate compounds linked to four specific ligands, were synthesized and characterized. These silver-phosphine adducts included [AgSCN{P(4-MeC6H4)3}2]2 (1); [AgSCN{P(4-ClC6H4)3}2]2 (2); [AgSCN{P(4-MeOC6H4)3}2]2 (3); [AgSCN(PPh3)2]2 (4). The compounds were found to be toxic to MCF-7 breast cancer cells while the ligands on their own were not toxic. Our findings further indicate that the silver(I) phosphine compounds induce apoptotic cell death in these breast cancer cells. In addition, the compounds were not toxic to nonmalignant fibroblast cells at the IC50 concentrations. This is an indication that the compounds show selectivity towards the cancer cells.
Keywords: Silver-phosphine complexes; Apoptosis; Flow-cytometry; MCF-7 breast cancer cells; Anti-cancer; Drug discovery

Fusaric acid induces a notochord malformation in zebrafish via copper chelation by Emily S. Yin; Malika Rakhmankulova; Kaury Kucera; Jose Guedes de Sena Filho; Carolina E. Portero; Alexandra Narváez-Trujillo; Scott A. Holley; Scott A. Strobel (783-789).
Over a thousand extracts were tested for phenotypic effects in developing zebrafish embryos to identify bioactive molecules produced by endophytic fungi. One extract isolated from Fusarium sp., a widely distributed fungal genus found in soil and often associated with plants, induced an undulated notochord in developing zebrafish embryos. The active compound was isolated and identified as fusaric acid. Previous literature has shown this phenotype to be associated with copper chelation from the active site of lysyl oxidase, but the ability of fusaric acid to bind copper ions has not been well described. Isothermal titration calorimetry revealed that fusaric acid is a modest copper chelator with a binding constant of 4.4 × 105 M−1. These results shed light on the toxicity of fusaric acid and the potential teratogenic effects of consuming plants infected with Fusarium sp.
Keywords: Copper chelation; Endophytic fungi; Fusaric acid; Natural products; Zebrafish notochord