Chemical background of silver nanoparticles interfering with mammalian copper metabolism.

The rapidly increasing application of silver nanoparticles (AgNPs) boosts their release into the environment, which raises a reasonable alarm for ecologists and health specialists. This is manifested as increased research devoted to the influence of AgNPs on physiological and cellular processes in various model systems, including mammals. The topic of the present paper is the ability of silver to interfere with copper metabolism, the potential health effects of this interference, and the danger of low silver concentrations to humans. The chemical properties of ionic and nanoparticle silver, supporting the possibility of silver release by AgNPs in extracellular and intracellular compartments of mammals, are discussed. The possibility of justified use of silver for the treatment of some severe diseases, including tumors and viral infections, based on the specific molecular mechanisms of the decrease in copper status by silver ions released from AgNPs is also discussed.

Properties of recombinant extracellular N-terminal domain of human high-affinity copper transporter 1 (hNdCTR1) and its interactions with Cu(II) and Ag(I) ions.

High-affinity copper transporter 1 (CTR1) is a key link in the transfer of copper (Cu) from the extracellular environment to the cell. Violation in the control system of its expression, or mutations in this gene, cause a global copper imbalance. However, the mechanism of copper transfer via CTR1 remains unclear. It has been shown that transformed bacteria synthesizing the fused GB1-NdCTR become resistant to toxic silver ions. According to UV-Vis spectrophotometry and isothermal titration calorimetry, electrophoretically pure GB1-NdCTR specifically and reversibly binds copper and silver ions, and binding is associated with aggregation. Purified NdCTR1 forms SDS-resistant oligomers. The link between nontrivial properties of NdCTR1 and copper import mechanism from extracellular space, as well as potential chelating properties of NdCTR1, are discussed.

Aggregation Properties of Albumin in Interacting with Magnetic Fluids.

In this study, interactions of Fe3O4 magnetic nanoparticles with serum albumin biomolecules in aqueous solutions were considered. The studies were conducted with the laser correlation spectroscopy and optical analysis of dehydrated films. It was shown that the addition of magnetite to an albumin solution at low concentrations of up to 10-6 g/L led to the formation of aggregates with sizes of up to 300 nm in the liquid phase and an increase in the number of spiral structures in the dehydrated films, which indicated an increase in their stability. With a further increase in the magnetite concentration in the solution (from 10-4 g/L), the magnetic particles stuck together and to albumin, thus forming aggregates with sizes larger than 1000 nm. At the same time, the formation of morphological structures in molecular films was disturbed, and a characteristic decrease in their stability occurred. Most stable films were formed at low concentrations of magnetic nanoparticles (less than 10-4 g/L) when small albumin-magnetic nanoparticle aggregates were formed. These results are important for characterizing the interaction processes of biomolecules with magnetic nanoparticles and can be useful for predicting the stability of biomolecular films with the inclusion of magnetite particles.

Size-Dependent Bioactivity of Silver Nanoparticles: Antibacterial Properties, Influence on Copper Status in Mice, and Whole-Body Turnover.

PURPOSE: The ability of silver nanoparticles (AgNPs) of different sizes to influence copper metabolism in mice is assessed. MATERIALS AND METHODS: AgNPs with diameters of 10, 20, and 75 nm were fabricated through a chemical reduction of silver nitrate and characterized by UV/Vis spectrometry, transmission and scanning electronic microscopy, and laser diffractometry. To test their bioactivity, Escherichia coli cells, cultured A549 cells, and C57Bl/6 mice were used. The antibacterial activity of AgNPs was determined by inhibition of colony-forming ability, and cytotoxicity was tested using the MTT test (viability, %). Ceruloplasmin (Cp, the major mammalian extracellular copper-containing protein) concentration and enzymatic activity were measured using gel-assay analyses and WB, respectively. In vitro binding of AgNPs with serum proteins was monitored with UV/Vis spectroscopy. Metal concentrations were measured using atomic absorption spectrometry. RESULTS: The smallest AgNPs displayed the largest dose- and time-dependent antibacterial activity. All nanoparticles inhibited the metabolic activity of A549 cells in accordance with dose and time, but no correlation between cytotoxicity and nanoparticle size was found. Nanosilver was not uniformly distributed through the body of mice intraperitoneally treated with low AgNP concentrations. It was predominantly accumulated in liver. There, nanosilver was included in ceruloplasmin, and Ag-ceruloplasmin with low oxidase activity level was formed. Larger nanoparticles more effectively interfered with the copper metabolism of mice. Large AgNPs quickly induced a drop of blood serum oxidase activity to practically zero, but after cancellation of AgNP treatment, the activity was rapidly restored. A major fraction of the nanosilver was excreted in the bile with Cp. Nanosilver was bound by alpha-2-macroglobulin in vitro and in vivo, but silver did not substitute for the copper atoms of Cp in vitro. CONCLUSION: The data showed that even at low concentrations, AgNPs influence murine copper metabolism in size-dependent manner. This property negatively correlated with the antibacterial activity of AgNPs.

In vivo effect of copper status on cisplatin-induced nephrotoxicity.

Cisplatin is a widely used antitumor agent; however, tumor resistance and severe side effects limit its use. It is well accepted that cisplatin toxicity can be modulated in vitro in cell cultures by copper salts. In the present work, mice with different blood serum copper status were treated with a single intraperitoneal cisplatin injection at a dose of 5 mg/kg, monitored for 3 days in metabolic cages and analyzed for renal function. Both copper-deficient and copper-overloaded mice displayed more severe early proteinuria and retarded platinum excretion than control mice. The effects of copper status on cisplatin-induced nephrotoxicity are discussed.

Non-hepatic tumors change the activity of genes encoding copper trafficking proteins in the liver.

To assess the statistical relationship between tumor growth and copper metabolism, we performed a metaanalysis of studies in which patients with neoplasms were characterized according to any of the copper status indexes (atomic copper serum concentration, serum oxidase activity, ceruloplasmin protein content). Our metaanalysis shows that in the majority of cases (more than 3100 patients), tumor growth positively correlates with the copper status indexes. Nude athymic CD-1 nu/nu mice with subcutaneous tumors of human origin, C57Bl/6J mice with murine melanoma and Apc(Min) mice with spontaneously developing adenomas throughout the intestinal tract were studied to experimentally determine the relationship between tumor progression, liver copper metabolism, and copper status indexes. We showed that the copper status indexes increased significantly during tumor growth. In the liver tissue of tumor-bearing mice, ceruloplasmin gene expression, as well as the expression of genes related to ceruloplasmin metallation (CTR1 and ATP7B), increased significantly. Moreover, the presence of an mRNA splice variant encoding a form of ceruloplasmin anchored to the plasma membrane by glycosylphosphatidyl inositol, which is atypical for hepatocytes, was also detected. The ATP7A copper transporter gene, which is normally expressed in the liver only during embryonic copper metabolism, was also activated. Depletion of holo-ceruloplasmin resulted in retardation of human HCT116 colon carcinoma cell growth in nude mice and induced DNA fragmentation in tumor cells. In addition, the concentration of cytochrome c increased significantly in the cytosol, while decreasing in the mitochondria. We discuss a possible trans-effect of developing tumors on copper metabolism in the liver.

Structural Features of the Peptide Homologous to 6-25 Fragment of Influenza A PB1 Protein.

A mirror-symmetry motif was discovered in the N-terminus of the influenza virus PB1 protein. Structure of peptide comprised of the corresponding part of PB1 (amino acid residues 6-25) was investigated by circular dichroism and in silico modeling. We found that peptide PB1 (6-25) in solution assumes beta-hairpin conformation. A truncated peptide PB1 (6-13), containing only half of the mirror-symmetry motif, appeared to stabilize the beta-structure of the original peptide and, at high concentrations, was capable of reacting with peptide to form insoluble aggregates in vitro. Ability of PB1 (6-13) peptide to interact with the N-terminal domain of PB1 protein makes it a potential antiviral agent that inhibits PA-PB1 complex formation by affecting PB1 N-terminus structure.

Serum depletion of holo-ceruloplasmin induced by silver ions in vivo reduces uptake of cisplatin.

There is an emerging link between extracellular copper concentration and the uptake of cisplatin mediated by copper transporter CTR1 in cell cultures and unicellular eukaryotes. To test the link between extracellular copper level and cisplatin uptake by organs in vivo we used mice with low copper status parameters induced by AgCl-containing diet (Ag-mice). In Ag-mice, serum copper status and liver copper metabolism were characterized. It was shown that the expression level of copper transporter genes and activity of ubiquitous intracellular cuproenzymes were not affected but the level of serum holo-ceruloplasmin was not detectable. Silver was selectively absorbed by liver and accumulated in the mitochondrial matrix. Silver was present in an exchangeable form and was excreted through bile. Ag-mice model is characterized by high reproducibility, reversibility, synchronicity, and definiteness of ceruloplasmin-associated copper deficiency. After cisplatin treatment Ag-mice, as compared to control mice, demonstrated the delay in platinum uptake by organs during first 30 min. This effect was not observed at later time points probably due to cisplatin induced copper release to blood, which resulted in the recovery of copper status. These data allowed us to conclude that cisplatin uptake was coupled to copper transport in vivo.