BioMetals (v.30, #3)

Human topoisomerase inhibition and DNA/BSA binding of Ru(II)–SCAR complexes as potential anticancer candidates for oral application by Rone A. De Grandis; Mariana S. de Camargo; Monize M. da Silva; Érica O. Lopes; Elias C. Padilha; Flávia A. Resende; Rosângela G. Peccinini; Fernando R. Pavan; Alessandro Desideri; Alzir A. Batista; Eliana A. Varanda (321-334).
Three ruthenium(II) phosphine/diimine/picolinate complexes were selected aimed at investigating anticancer activity against several cancer cell lines and the capacity of inhibiting the supercoiled DNA relaxation mediated by human topoisomerase IB (Top 1). The structure–lipophilicity relationship in membrane permeability using the Caco-2 cells have also been evaluated in this study. SCAR 5 was found to present 45 times more cytotoxicity against breast cancer cell when compared to cisplatin. SCAR 4 and 5 were both found to be capable of inhibiting the supercoiled DNA relaxation mediated by Top 1. Interaction studies showed that SCAR 4 and 5 can bind to DNA through electrostatic interactions while SCAR 6 is able to bind covalently to DNA. The complexes SCAR were found to interact differently with bovine serum albumin (BSA) suggesting hydrophobic interactions with albumin. The permeability of all complexes was seen to be dependent on their lipophilicity. SCAR 4 and 5 exhibited high membrane permeability (P app  > 10 × 10−6 cm·s−1) in the presence of BSA. The complexes may pass through Caco-2 monolayer via passive diffusion mechanism and our results suggest that lipophilicity and interaction with BSA may influence the complexes permeation. In conclusion, we demonstrated that complexes have powerful pharmacological activity, with different results for each complex depending on the combination of their ligands.
Keywords: Anticancer agent; Ruthenium(II) complexes; Topoisomerase; Binding affinity; Caco-2 permeability

Zinc inhibition of pyruvate kinase of M-type isozyme by Keiko Murakami; Masataka Yoshino (335-340).
Inhibitory effect of Zn on the pyruvate kinase of M (muscle)-type isozyme was analyzed for the purpose of elucidating the cytotoxicity of Zn. Zn inhibited pyruvate kinase uncompetitively with respect to the substrate PEP, and competitively with respect to ADP. Quotient velocity plot calculated from the Zn-inhibition curves showed that Zn2+ as a ZnADP complex acted as competitive and uncompetitive inhibitors of the enzyme with respect to the substrate ADP and PEP, respectively: Zn2+ forms a ZnADP complex, which may bind to the ADP-binding site of the free enzyme with the Ki value of 1.4 μM causing competitive inhibition, or to the ADP-site of the enzyme-PEP complex with 2.6 μM resulting in uncompetitive inhibition. The inhibition of pyruvate kinase by Zn2+ may be responsible for the cytotoxicity of this metal by decreasing glycolytic flux.
Keywords: Pyruvate kinase; Zn; Inhibition; Muscle isozyme

Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations by Ekaterina L. Nemashkalova; Eugene A. Permyakov; Sergei E. Permyakov; Ekaterina A. Litus (341-353).
Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer’s disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn2+, Cu2+, Mg2+ and Ca2+ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn2+ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1–2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn2+-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca2+/Mg2+-binding, Zn2+ or Cu2+ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca2+-binding to HSA synergistically support each other. Zn2+ and Cu2+ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.
Keywords: Serum albumin; Linoleic acid; Metal cations; Protein-lipid interaction; Metal-protein interaction; Alzheimer’s disease

Blood trace metals in a sporadic amyotrophic lateral sclerosis geographical cluster by Stefano De Benedetti; Giorgio Lucchini; Cristian Del Bò; Valeria Deon; Alessandro Marocchi; Silvana Penco; Christian Lunetta; Elisabetta Gianazza; Francesco Bonomi; Stefania Iametti (355-365).
Amyotrophic lateral sclerosis (ALS) is a fatal disorder with unknown etiology, in which genetic and environmental factors interplay to determine the onset and the course of the disease. Exposure to toxic metals has been proposed to be involved in the etiology of the disease either through a direct damage or by promoting oxidative stress. In this study we evaluated the concentration of a panel of metals in serum and whole blood of a small group of sporadic patients, all living in a defined geographical area, for which acid mine drainage has been reported. ALS prevalence in this area is higher than in the rest of Italy. Results were analyzed with software based on artificial neural networks. High concentrations of metals (in particular Se, Mn and Al) were associated with the disease group. Arsenic serum concentration resulted lower in ALS patients, but it positively correlated with disease duration. Comet assay was performed to evaluate endogenous DNA damage that resulted not different between patients and controls. Up to now only few studies considered geographically well-defined clusters of ALS patients. Common geographical origin among patients and controls gave us the chance to perform metallomic investigations under comparable conditions of environmental exposure. Elaboration of these data with software based on machine learning processes has the potential to be extremely useful to gain a comprehensive view of the complex interactions eventually leading to disease, even in a small number of subjects.
Keywords: Amyotrophic lateral sclerosis; Geographic cluster; Serum metallome; Artificial neural networks

In this study, Ni–Ti alloy and stainless steal were exposed to artificial saliva containing fibrinogen, IgG or mucin, and the resultant corrosion behavior was studied. The purpose was to determine the mechanisms by which different types of protein contribute to corrosion. The effect of different proteins on the electrochemical resistance of Ni–Ti and SS was tested by potentiodynamic polarization, and the repair capacity of passivation film was tested by cyclic polarization measurements. The dissolved corrosion products were determined by ICP-OES, and the surface was analyzed by SEM and AFM. The results showed fibrinogen, IgG or mucin could have different influences on the susceptibility to corrosion of the same alloy. Adding protein lead to the decrease of corrosion resistance of SS, whereas protein could slow down the corrosion process of Ni–Ti. For Ni–Ti, adding mucin could enhance the corrosion stability and repair capacity of passivation film. The susceptibility to pitting corrosion of Ni–Ti and stainless steal in fibrinogen AS is not as high as mucin and IgG AS. There are different patterns of deposition formation on the metal surface by different types of protein, which is associated with their effects on the corrosion process of the alloys.
Keywords: Corrosion; Ni–Ti alloy; Stainless steal; Protein adsorption

Silver(I) complexes with chromone-derived hydrazones: investigation on the antimicrobial and cytotoxic effects by Lenka V. Tamayo; Ane F. Santos; Isabella P. Ferreira; Verlane G. Santos; Miriam T. P. Lopes; Heloisa Beraldo (379-392).
Complexes [Ag(HCrPh)2]NO3·2H2O (1) and [Ag(HCrpClPh)2]NO3 (2) were obtained with 3-formyl-6-methylchromone-phenyl hydrazone (HCrPh, HL1) and 3-formyl-6-methylchromone-para-chloro-phenyl hydrazone (HCrpClPh, HL2). Although the hydrazones were inactive, upon coordination to silver(I) antifungal activity significantly improved against several Candida strains. Complexes (12) revealed to be more active than silver nitrate, silver sulfadiazine and the reference drug nystatin against Candida parapsilosis. The cytotoxic activities of the hydrazones and their silver(I) complexes were evaluated in comparison with cisplatin on B16F10 (metastatic melanoma) and Melan-a (non-tumorigenic melanocyte) cells. The hydrazones showed low cytotoxicity against B16F10 cells, reducing only about 20% of cell viability at the concentration of 10 μM. Upon coordination to silver(I) the cytotoxic effect did not appreciably change in complex (1) while complex (2) proved to be as cytotoxic as cisplatin and much more cytotoxic than both the free ligand and silver nitrate at 1 μM. Both complexes (1) and (2) were less active than cisplatin on non-malignant Melan-a cells, indicating that these compounds might promote less damage on normal cells.
Keywords: Chromone; Hydrazones; Silver(I) complexes; Antifungal; Cytotoxic activity

Zinc transporter 3 (ZnT3) is one of the zinc transporters family. It is closely connected to the nervous system, where enables the transport of zinc ions from the cytoplasm to synaptic vesicles. This substance has been described within the central and peripheral nervous system, especially in the enteric nervous system (ENS). The aim of the present study was to describe the co-localization of ZnT3 with selected neuromediators and/or neuromodulators participating in sensory stimuli conduction in neurons of the ENS within the porcine esophagus. Co-localization of ZnT3 with substance P (SP), leucine enkephalin (LENK) and calcitonin gene-related peptide (CGRP) was studied using standard double-immunofluorescence technique. The obtained results show that ZnT3, SP and/or LENK may occur in the same enteric neurons, and the degree of co-localization of these substances clearly depends on the fragment of esophagus studied and the type of enteric ganglia. In contrast, the co-localization of ZnT3 with CGRP was not observed during the present investigation. The obtained results suggest that ZnT3 in the ENS may be involved in the conduction of sensory and/or pain stimuli.
Keywords: Enteric nervous system; Zinc transporters; Esophagus; Immunofluorescence technique; Pig

Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the constitutive expression of BCR-ABL tyrosine kinase. Although successful implementation of tyrosine kinase inhibitors for the treatment of CML remain a traditional choice for molecularly targeted therapy, some patients present primary or secondary resistance to such therapy. Therefore, alternative therapeutic strategies are required to treat resistant CML cells. Accordingly, new anti-proliferative and/or pro-apoptotic compounds would be needed for clinical treatment. In the present investigation, we demonstrate that TPEN (e.g. 3 μM), a lipid-soluble metal chelator, induces apoptosis in K562 cells via a molecular cascade involving H2O2 ≫ JNK, NF-κB > c-JUN, P73 > PUMA, BAX > loss of ΔΨm > CASPASE-3 > nuclei/DNA fragmentation. Fragmentation of the nuclei and DNA are indicative of cell death by apoptosis. Remarkably, the antioxidant N-acetyl-cysteine, and inhibitors of the transcription factors CASPASE 3 and (JNK) kinase, decreased oxidative stress (OS) and cell death in these cells. This is evidenced by fluorescence microscopy, flow cytometry and immunocytochemistry for OS markers (e.g. generation of H2O2 and DJ 1 oxidation) and nuclear expression of apoptotic markers (e.g. activated caspase 3 and JNK kinase). In addition, TPEN causes no detectable damage in human peripheral blood lymphocyte cells (hPBLCs). We conclude that TPEN selectively induces apoptosis in K562 cells via an OS-mechanism. Our findings may provide insight into more effective CML anticancer therapies.
Keywords: Apoptosis; Antileukemic; Chronic myeloid leukemia; K562; TPEN; Oxidative stress

Schiff base complexes of copper and zinc as potential anti-colitic compounds by Elaine M. Conner; John Reglinski; W. Ewen Smith; I. Jack Zeitlin (423-439).
The design, synthesis and activity of polymodal compounds for the treatment of inflammatory bowel disease are reported. The compounds, being based on a metal-Schiff base motif, are designed to degrade during intestinal transit to release the bioactive components in the gut. The compounds have been developed sequential with the biomodal compounds combining copper or zinc with a salicylaldehyde adduct. These compounds were tested in a formalin induced colonic inflammation model in BK:A mice. From these studies a trimodal compound based on a zinc Schiff base analogue of sulfasalazine was designed. This was tested against a trinitrobenzenesulfonic acid (TNB) induced colitic model in Wistar rats. The use of two models allows us to test our compounds in both an acute and a chronic model. The trimodal compound reported is observed to provide anticolitic properties in the chronic TNB induced colitis model commensurate with that of SASP. However, the design of trimodal compound still has the capacity for further development. This the platform reported may offer a route into compounds which can markedly outperform the anti-colitic properties of SASP.
Keywords: Ulcerative colitis; Polymodal drugs; Sulfasalazine; Metal Schiff base compounds; Formalin induced colonic inflammation; TNB induced colonic inflammation

An optical material for the detection of trace S2O3 2− in milk based on a copper complex by Qingming Wang; Huifang Sun; Weilin Sha; Juan Chen; Liuyue Gu; Dong Wang; Xinhui Tang (441-447).
A novel S2O3 2− luminescent sensor (Cu2+-p-CPIP) was developed and the presence of S2O3 2− caused an obvious fluorescence enhancement at 420 nm upon excitation at 330 nm, which could be distinguished with the naked eye under a UV lamp. Remarkably, the compound exhibited excellent selective and sensitive response to S2O3 2− over other common anions with a micromolar limit of detection (0.442 μM) in DMSO/H2O (v/v, 1:1) buffer. The absorbance intensity and the color of Cu2+-p -CPIP solution changed gradually with the increase of S2O3 2− concentration. The proposed method was applied to the determination of S2O3 2− in milk samples and the recoveries were 97.5–105%. The preparation of Cu2+-p -CPIP exhibited the quick, simple and facile advantages. The results showed that Cu2+-p -CPIP can be a good candidate for simple, rapid and sensitive colorimetric detection of S2O3 2− in aqueous solution.
Keywords: Chemodosimeter; S2O3 2− ; 1,10-Phenanthroline-5,6-dione; Copper complex

Glycolysis and Krebs cycle intermediates were incubated with Eu3+-tetracycline and separated using capillary electrophoresis utilizing post-column laser-induced luminescence detection in a sheath flow cuvette. 3-phopshoglycerate, phosphoenolpyruvate, adenosine diphosphate, phosphate, citrate and oxaloacetate were detected at a concentration of 100 μM or lower. When all these detected metabolites were contained within the same sample it was found that they interfered with one another. Of all the metabolites, oxaloacetate showed the highest detectability. The system was found to yield a linear response for oxaloacetate from 50 nM to 10 μM. The injected volume of sample was 400 pL. This corresponds to 2 × 10−17 mol of injected oxaloacetate from the 50 nM sample. As an application, the system was used to assay the enzyme aspartate aminotransferase, for whom oxaloacetate is a product. After a 1 h incubation period, 1.2 × 10−13 M (3.3 μU/mL) enzyme was sufficient to form a detectable product signal. Extension of this incubation to 18 h permitted the detection of the activity of 1.2 × 10−14 M (330 nU/mL) enzyme. This is the equivalent of 4.8 ymoles (2.9 molecules) of enzyme in the 400 pL injection volume. The enzyme’s catalytic rate was determined to be 240 s−1 under the conditions used. In a second application, homogenates of Drosophila melanogaster were analyzed for metabolites, providing several peaks, including one which had the same retention time as citrate.
Keywords: Aspartate aminotransferase; Capillary electrophoresis; Eu3+-tetracycline; Luminescence; Metabolites; Oxaloacetate