BioMetals (v.29, #6)

Synthesis and evaluation of copper(II) complexes with isoniazid-derived hydrazones as anticancer and antitubercular agents by Gisele S. S. Firmino; Marcus V. N. de Souza; Claudia Pessoa; Maria C. S. Lourenco; Jackson A. L. C. Resende; Josane A. Lessa (953-963).
In this study, the N,N,O metal chelator 2-pyridinecarboxaldehydeisonicotinoyl hydrazone (HPCIH, 1) and its derivatives 2-acetylpyridine-(HAPIH 2), 2-pyridineformamide-(HPAmIH, 3) and pyrazineformamide-(HPzAmIH, 4) were employed in the synthesis of four copper(II) complexes, [Cu(HPCIH)Cl2]·0.4H2O (5), [Cu(HAPIH)Cl2]·1.25H2O (6), [Cu(HPAmIH)Cl2]·H2O (7) and [Cu(HPzAmIH)Cl2]·1.25H2O (8). The compounds were assayed for their action toward Mycobacterium tuberculosis H37Rv ATCC 27294 strain and the human tumor cell lines OVCAR-8 (ovarian cancer), SF-295 (glioblastoma multiforme) and HCT-116 (colon adenocarcinoma). All copper(II) complexes were more effective in reducing growth of HCT-116 and SF-295 cells than the respective free hydrazones at 5 µg/mL, whereas only complex 7 was more cytotoxic toward OVCAR-8 lines than its ligand HPAmIH. 6 proved to be cytotoxic at submicromolar doses, whose IC50 values (0.39–0.86 µM) are similar to those ones found for doxorubicin (0.23–0.43 µM). Complexes 5 and 6 displayed high activity against M. tuberculosis (MIC = 0.85 and 1.58 µM, respectively), as compared with isoniazid (MIC = 2.27 µM), which suggests the compounds are attractive candidates as antitubercular drugs.
Keywords: Copper(II) complexes; Hydrazones; Isoniazid; Anticancer agent; Antitubercular agent

Impact of different environmental conditions on the aggregation of biogenic U(IV) nanoparticles synthesized by Desulfovibrio alaskensis G20 by S. Sevinç Şengör; Gursharan Singh; Alice Dohnalkova; Nicolas Spycher; Timothy R. Ginn; Brent M. Peyton; Rajesh K. Sani (965-980).
This study investigates the impact of specific environmental conditions on the formation of colloidal U(IV) nanoparticles by the sulfate reducing bacteria (SRB, Desulfovibrio alaskensis G20). The reduction of soluble U(VI) to less soluble U(IV) was quantitatively investigated under growth and non-growth conditions in bicarbonate or 1,4-piperazinediethanesulfonic acid (PIPES) buffered environments. The results showed that under non-growth conditions, the majority of the reduced U nanoparticles aggregated and precipitated out of solution. High resolution transmission electron microscopy revealed that only a very small fraction of cells had reduced U precipitates in the periplasmic spaces in the presence of PIPES buffer, whereas in the presence of bicarbonate buffer, reduced U was also observed in the cytoplasm with greater aggregation of biogenic U(IV) particles at higher initial U(VI) concentrations. The same experiments were repeated under growth conditions using two different electron donors (lactate and pyruvate) and three electron acceptors (sulfate, fumarate, and thiosulfate). In contrast to the results of the non-growth experiments, even after 0.2 μm filtration, the majority of biogenic U(IV) remained in the aqueous phase resulting in potentially mobile biogenic U(IV) nanoparticles. Size fractionation results showed that U(IV) aggregates were between 18 and 200 nm in diameter, and thus could be very mobile. The findings of this study are helpful to assess the size and potential mobility of reduced U nanoparticles under different environmental conditions, and would provide insights on their potential impact affecting U(VI) bioremediation efforts at subsurface contaminated sites.
Keywords: Biogenic uranium; Mobile uraninite; Nanoparticles; Sulfate reducing bacteria; Uranium aggregates

Three novel copper(II), cobalt(II), and nickel(II) complexes of juglone (Jug) containing 1,10-phenanthroline (phen) ligand, [M(Jug)2(phen)] (M = Cu(II), 1, Co(II), 2, and Ni(II), 3), have been synthesized and characterized using, elemental analysis and spectroscopic studies. Their interactions with calf thymus DNA were investigated using viscosity measurements, UV–visible and fluorescence spectrophotometric methods. The catalytic activities on DNA cleavage of the complexes 13 were studied, which copper complex 1 showed better catalyst activity in the DNA cleavage process than complexes 2 and 3. The in vitro cytotoxic potential of the complexes 13 against human cervical carcinoma (HeLa), human liver hepatocellular carcinoma (HepG-2), and human colorectal adenocarcinoma (HT-29) cells indicated their promising antitumor activity with quite low IC50 values in the range of 0.09–1.89 μM, which are 75 times lower than those of cisplatin.
Keywords: Metal complex; DNA binding; Cytotoxicity; Juglone

Expression of the zinc importer protein ZIP9/SLC39A9 in glioblastoma cells affects phosphorylation states of p53 and GSK-3β and causes increased cell migration by Nico Münnich; Simon Wernhart; Christer Hogstrand; Uwe Schlomann; Christopher Nimsky; Jörg W. Bartsch (995-1004).
Zinc importer proteins (ZIPs) have been proven as important molecular regulators in different cancers. As a member of the solute carrier family, ZIP9/SLC39A9 is overexpressed in prostate and breast cancer and affects B-cell receptor signaling. Here, we present data indicating that changes in intracellular zinc levels in glioblastoma cells can cause enhanced cell survival and cell migration, both hallmarks of the disease process. In particular, treatment of human glioblastoma cells with sublethal doses of cell-permeable heavy metal (Zn2+ > Fe2+ > Mn2+) chelator (N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN)) induced ZIP9 expression. Either TPEN treatment or expression of ZIP9 cDNA causes enhanced migration behavior of glioblastoma cells. Compared to untreated glioblastoma cells TPEN treatment or expression of ZIP9 results in activation of the tumor suppressor p53 by phosphorylation at serine residue 46 (Ser46) and in inactivation of the migration relevant glycogen synthase kinase 3 beta (GSK-3β) by phosphorylation at serine residue 9 (Ser9). Whilst p53 activation affects cell survival in response to TPEN, GSK-3β inactivation directly affects glioblastoma cell migration. Therefore, ZIP9 expression could regulate the migratory behavior of glioblastoma cells, so that ZIP9 may be of biological, but not of clinical relevance for glioblastomas, since in GBM tumor tissues, ZIP9 expression is not significantly increased compared to normal brain.
Keywords: Zinc metabolism; TPEN; ZIP9; Glioblastoma; p53; GSK-3beta; Cell migration

After oxidation, zinc nanoparticles lose their ability to enhance responses to odorants by Samantha Hagerty; Yasmine Daniels; Melissa Singletary; Oleg Pustovyy; Ludmila Globa; William A. MacCrehan; Shin Muramoto; Gheorghe Stan; June W. Lau; Edward E. Morrison; Iryna Sorokulova; Vitaly Vodyanoy (1005-1018).
Electrical responses of olfactory sensory neurons to odorants were examined in the presence of zinc nanoparticles of various sizes and degrees of oxidation. The zinc nanoparticles were prepared by the underwater electrical discharge method and analyzed by atomic force microscopy and X-ray photoelectron spectroscopy. Small (1.2 ± 0.3 nm) zinc nanoparticles significantly enhanced electrical responses of olfactory neurons to odorants. After oxidation, however, these small zinc nanoparticles were no longer capable of enhancing olfactory responses. Larger zinc oxide nanoparticles (15 nm and 70 nm) also did not modulate responses to odorants. Neither zinc nor zinc oxide nanoparticles produced olfactory responses when added without odorants. The enhancement of odorant responses by small zinc nanoparticles was explained by the creation of olfactory receptor dimers initiated by small zinc nanoparticles. The results of this work will clarify the mechanisms for the initial events in olfaction, as well as to provide new ways to alleviate anosmia related to the loss of olfactory receptors.
Keywords: Olfaction; Olfactory receptors; Electroolfactogram; Perception; XPS; TEM; AFM

The impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation by Grzegorz Majka; Grażyna Więcek; Małgorzata Śróttek; Klaudyna Śpiewak; Małgorzata Brindell; Joanna Koziel; Janusz Marcinkiewicz; Magdalena Strus (1019-1033).
Translocation of bacteria, primarily Gram-negative pathogenic flora, from the intestinal lumen into the circulatory system leads to sepsis. In newborns, and especially very low birth weight infants, sepsis is a major cause of morbidity and mortality. The results of recently conducted clinical trials suggest that lactoferrin, an iron-binding protein that is abundant in mammalian colostrum and milk, may be an effective agent in preventing sepsis in newborns. However, despite numerous basic studies on lactoferrin, very little is known about how metal saturation of this protein affects a host’s health. Therefore, the main objective of this study was to elucidate how iron-depleted, iron-saturated, and manganese-saturated forms of lactoferrin regulate intestinal barrier function via interactions with epithelial cells and macrophages. For these studies, a human intestinal epithelial cell line, Caco-2, was used. In this model, none of the tested lactoferrin forms induced higher levels of apoptosis or necrosis. There was also no change in the production of tight junction proteins regardless of lactoferrin metal saturation status. None of the tested forms induced a pro-inflammatory response in Caco-2 cells or in macrophages either. However, the various lactoferrin forms did effectively inhibit the pro-inflammatory response in macrophages that were activated with lipopolysaccharide with the most potent effect observed for apolactoferrin. Lactoferrin that was not bound to its cognate receptor was able to bind and neutralize lipopolysaccharide. Lactoferrin was also able to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect. Therefore, we hypothesize that lactoferrin supplementation is a relevant strategy for preventing sepsis.
Keywords: Lactoferrin; Sepsis; Bacterial translocation; Iron; Manganese

Hypoxia-selective inhibition of angiogenesis development by NAMI-A analogues by Maria Oszajca; Guillaume Collet; Grażyna Stochel; Claudine Kieda; Małgorzata Brindell (1035-1046).
The antimetastatic ruthenium(III) complex (H2Im)[trans-RuCl4(HIm)(DMSO)] (NAMI-A) as well as its two analogues (H2Ind)[trans-RuCl4(HInd)(DMSO)] (Ru-Ind) and (HIsq)[trans-RuCl4(Isq)(DMSO)] (Ru-Isq) (HIm–imidazole, HInd–indazole, Isq–isoquinoline, DMSO–dimethyl sulfoxide) were tested for their effect on endothelial cell functions in vitro on human skin microvascular endothelial cells (HSkMEC) and human endothelial progenitor cells (HPEC-CB.2) under normoxic (21 % O2) and hypoxic (1 % O2) conditions. All studied complexes showed very low cytotoxicity profiles towards both mature microvascular and precursor endothelial cells (ECs), independently of oxygen concentration. Among tested compounds Ru-Ind exhibited the highest cytotoxicity. The antiangiogenic activity of ruthenium complexes was evaluated for their influence on pseudo-vessels formation by microvascular endothelial cells (HSkMEC) because of their involvement in melanoma progression. Our studies indicated that Ru-Ind and Ru-Isq exhibited hypoxia- and dose-dependent-inhibition of angiogenesis on Matrigel™. Significant hypoxia-selective downregulation of pseudo-vessels formation by Ru-Isq correlates with efficient inhibition of cell motility. Interestingly, in the applied concentration doses migration of endothelial cells was also inhibited by NAMI-A, but the pseudo-vessels formation on Matrigel™ was unaffected. Angiogenesis-related genes expression profile for both mature and precursor ECs indicated that inhibition of angiogenesis, mainly due to Ru-Isq, as compared to NAMI-A and Ru-Ind correlated with downregulation of CD31 and CD144 expression and upregulation of NOTCH4 expression in mature ECs, which is essential for endothelial cell motility and stalk cells organization control. The hypoxia-selective antiangiogenic activity of Ru-Ind and Ru-Isq, NAMI-A analogues makes them potent antimetastatic therapeutics for their selective action in hypoxia which controls tumor pathologic angiogenesis.
Keywords: Ruthenium complexes; NAMI-A analogues; Angiogenesis; Hypoxia; Endothelial cells

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. Four mutants (D37K, D73K, D110K and D146K) were created firstly to elucidate the importance of the first aspartic acid residues (Asp37, Asp73, Asp110 and Asp146) in the beginning of the four EF-loops of EoCen. Aromatic-sensitized Tb3+ fluorescence indicates that the aspartic acid residues are very important for the metal-binding of EoCen, except for Asp73 (in EF-loop II). Resonance light scattering (RLS) measurements for different metal ions (Ca2+ and Tb3+) binding proteins suggest that the order of four conserved aspartic acid residues for contributing to the self-assembly of EoCen is Asp37 > Asp146 > Asp110 > Asp73. Cross-linking experiment also exhibits that Asp37 and Asp146 play critical role in the self-assembly of EoCen. Asp37, in site I, which is located in the N-terminal domain, plays the most important role in the metal ion-dependent self-assembly of EoCen, and there is cooperativity between N-terminal and C-terminal domain (especially the site IV). In addition, the dependence of Tb3+ induced self-assembly of EoCen and the mutants on various factors, including ionic strength and pH, were characterized using RLS. Finally, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) binding, ionic strength and pH control experiments indicate that in the process of EoCen self-assembly, molecular interactions are mediated by both electrostatic and hydrophobic forces, and the hydrophobic interaction has the important status.
Keywords: Centrin; Aspartic acid; Terbium(III); Self-assembly

Melatonin is a conserved substance, which was discovered in the evolutionary distant organisms like bacteria, plants, invertebrates and vertebrates. Recent studies have shown that melatonin despite its possible role in photoperiod processes, has been found to be a direct free radical scavenger and an indirect antioxidant. In this report the impact of exogenous melatonin on the Bax inhibitor-1 (BI-1) expression level in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells exposed to lead was examined. BI-1 is a well-conserved protein in plants and animals that serves as the inhibitor of mammalian proapoptotic proteins as well as plant ROS-induced cell death. Our results showed that pretreatment with 200 nm melatonin, expressing BI-1 and fortified tobacco suspension cells against damages induced by lead. The obtained results revealed, that melatonin significantly increases BY-2 cells proliferation and protects BY-2 cells against death. Moreover, the conducted analyses showed for the first time that the protective effect of melatonin may be connected not only with its antioxidant properties but also with its direct impact on elevating BI-1 expression and lead-induced programmed cell death (PCD) restriction.
Keywords: Bax inhibitor-1; BI-1; Biostymulators; BY-2 tobacco cells; Melatonin

Intracellular uptake of an antitumor-active azole-bridged dinuclear platinum(II) complex in cisplatin-resistant tumor cells by Shuichi Kishimoto; Megumi Yasuda; Ryosuke Suzuki; Shoji Fukushima (1075-1083).
A cationic azolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH3)2}2(μ-OH)(μ-methyl-pyrazolate)]2+ (4M-PzPt), was developed to overcome resistance to cisplatin (CDDP). This study aimed to assess the cytotoxicity of 4M-PzPt against a CDDP-resistant cell line, H4-II-E/CDDP, and compare the intracellular accumulation of CDDP and 4M-PzPt. H4-II-E and H4-II-E/CDDP displayed similar sensitivity to 4M-PzPt; however, the sensitivity of H4-II-E/CDDP to CDDP was approximately 19-fold lower than that of H4-II-E. The difference in the sensitivity to both platinum complexes corresponded with the difference in the amount of intracellular platinum accumulation after exposure to CDDP or 4M-PzPt in both cell lines. In H4-II-E, HepG2, and HuH-7 cells, the intracellular uptake of CDDP and 4M-PzPt occurred via active transport and passive transport. Results of co-exposure with the transport inhibitors ouabain, tetraethylammonium, and cimetidine indicated that the intracellular uptake of CDDP was dependent on Na+/K+-ATPase and that of 4M-PzPt was dependent on organic cation transporters (OCTs), probably OCT1. This study suggested that 4M-PzPt could inhibit the growth of a CDDP-resistant tumor via an intracellular uptake mechanism different from that of CDDP.
Keywords: Cancer; Platinum; Cisplatin; Transporter; Resistance

Siderophores, a family of biogenic metal chelating agents, play critical roles in the biogeochemical cycling of Fe and other metals by facilitating their solubilization and uptake in circumneutral to alkaline oxic environments. However, because of their small concentrations (ca. nM) and large number of molecular structures, siderophore detection and quantification in environmental samples requires specialized equipment and expertise, and often requires pre-concentration of samples, which may introduce significant bias. The “universal” CAS assay, which was originally designed for use in bacterial cultures, quantifies the iron chelating function of a pool of siderophores but only at concentrations (>2 µM) well above the concentrations estimated to be present in marine, freshwater, and soil samples. In this manuscript, we present a high sensitivity modification of this universal assay (HS-CAS) suitable for detecting and quantifying siderophore activity in the nM concentration range, allowing for direct quantitation of siderophore reactivity in transparent aqueous samples.
Keywords: Siderophores; Environmental assay; Metal uptake; Biogeochemistry

A broad mercury resistant strain of Pseudomonas putida secretes pyoverdine under limited iron conditions and high mercury concentrations by Franco Baldi; Michele Gallo; Dario Battistel; Elena Barbaro; Andrea Gambaro; Salvatore Daniele (1097-1106).
The Pseudomonas putida FB1, known as a broad-spectrum mercury resistant strain, becomes yellow-green due to the secretion of pyoverdine (PVDs) under limited iron conditions and high mercury concentrations. Different modified Nelson’s media were obtained by adding mercury, iron, and the complexing agent nitrilotriacetic acid to demonstrate that the strain produces only the highest concentrations of PVDs due to the induction with 25 µM Hg2+. An amount of 250 mg PVDs was purified from the supernatant of 1 litre culture. The various forms of PVDs were characterized using different techniques such as fluorescence spectroscopy, high performance liquid chromatography coupled with high resolution mass spectrometry, and scanning electron microscope equipped with energy dispersive X-ray analyser. A set of “in vivo” experiments demonstrated that additions of Hg2+ to the cultures from 10 to 25 µM Hg2+ stimulate an over secretion of PVDs suggesting that the toxic cation strongly reduces the availability of apo-PVDs, because the complex mercuric-pyoverdine is very stable at neutral pH, and hinder the formation of PVDs-Fe(III).
Keywords: “In vivo” experiment; Cyclic voltammetry; Gaseous Hg(0); HPLC-HRMS method; Pyoverdine; Mercuric-pyoverdine