Massimiliano Peana

Toxicity of Nanoparticles

Nowadays more than thousands of different nanoparticles are known, though no well-defined guidelines to evaluate their potential toxicity and to control their exposure are fully provided. The way of entry of nanoparticles together with their specificities such as chemistry, chemical composition, size, shape or morphology, surface charge and area can influence their biological activities and effects. A specific property may give rise to either a safe particle or to a dangerous one. The small size allows nanoparticles to enter the body by crossing several barriers, to pass into the blood stream and lymphatic system from where they can reach organs and tissues and strictly interact with biological structures, thus damaging their normal functions in different ways. This review provides a summary of what is known on the toxicology related to the specificity of nanoparticles, both as technological tools or ambient pollutants. The aim is to highlight their potential hazard and to provide a balanced update on all the important questions and directions that should be focused in the near future.

Copper and nickel binding in multi-histidinic peptide fragments

Multi-histidinic peptides have been investigated for Cu(II) and Ni(II) binding. We present spectroscopic evidence that, at low pH and from sub-stoichiometric to stoichiometric amounts of metals, macrochelate and multi-histidinic Cu(II) and Ni(II) complexes form; but, from neutral pH and above, both copper and nickel bind to individual histidine residues. NMR, EPR, UV-Visible (UV-Vis) and UV-Visible CD spectroscopy were used to understand about the variety of complexes obtained at low pHs, where amide deprotonation and coordination is unfavoured. A structural transition between two coordination geometries, as the pH is raised, was observed. Metal binds to N(delta) of histidine imidazole when main-chain coordination is involved and coordinates via N(epsilon) under mildly acidic conditions and sub-stoichiometric amounts of metals. From EPR results a distortion from planarity has been evidenced for the Cu(II) multi-histidinic macrochelate systems, which may be relevant to biological activity. The behaviour of our peptides was comparable to the pH dependent effect on Cu(II) coordination observed in octapeptide repeat domain in prion proteins and in amyloid precursor peptides involved in Alzheimers disease. Changes in pH and levels of metal affect coordination mode and can have implications for the affinity, folding and redox properties of proteins and peptide fragments.

Copper(II) binding to Cap43 protein fragments

The C-terminal 20 and 30 amino acid sequences of Cap43 protein were chosen as models to study their interactions with Cu(II) ions. The behaviour of the 20 amino acid Ac-TRSRSH6TSEG-TRSRSH16TSEG and 30 amino acid Ac-TRSRSH6TSEG-TRSRSH16TSEG-TRSRSH26TSEG peptides towards Cu(II) ions at different pH values and different ligand-to-metal molar ratios, was examined. Spectroscopic (EPR, UV-Vis) and potentiometric techniques were performed to understand the details of metal binding to the peptides. The study showed that, starting from pH 4.0, each 10 amino acid fragment T1R2S3R4S5H6T7S8E9G10 was able to independently coordinate a single Cu(II) ion. The coordination mode involved the imidazole nitrogen of histidine H6 residue, and three amidic nitrogens from histidine H6, serine S5, and arginine R4 residues, respectively.

Mn(II) and Zn(II) interactions with peptide fragments from Parkinson's disease genes

Two peptide sequences from PARK9 Parkinsons disease gene, ProAspGluLysHisGluLeu, (P(1)D(2)E(3)K(4)H(5)E(6)L(7)) (1) and PheCysGlyAspGlyAlaAsnAspCysGly (F(1)C(2)G(3)D(4)G(5)A(6)N(7)D(8)C(9)G(10)) (2) were tested for Mn(II), Zn(II) and Ca(II) binding. The fragments are located from residues 1165 to 1171 and 1184 to 1193 in the PARK9 encoded protein. This protein can protect cells from poisoning of manganese, which is an environmental risk factor for a Parkinsons disease-like syndrome. Mono- and bi-dimensional NMR spectroscopy has been used to understand the details of metal binding sites at different pH values and at different ligand to metal molar ratios. Mn(II) and Zn(II) coordination with peptide (1) involves imidazole N(ε) or N(δ) of His(5) and carboxyl γ-O of Asp(2), Glu(3) and Glu(6) residues. Six donor atoms participate in Mn(II) binding resulting in a distorted octahedral geometry, possibly involving bidentate interaction of carboxyl groups; four donor atoms participate in Zn(II) binding resulting in a tetracoordinate geometry. Mn(II) and Zn(II) coordination involves the two cysteine residues with peptide (2); Mn(II) accepts additional ligand bonds from the carboxyl γ-O of Asp(4) and Asp(8) to complete the coordination sphere; the unoccupied sites may contain solvent molecules. The failure of Ca(II) ions to bind to either peptide (1) or (2) appears to result, under our conditions, from the absence of chelating properties in the chosen fragments.

A New bis-3-hydroxy-4-pyrone as a potential therapeutic iron chelating agent: effect of connecting and side chains on the complex structures and metal ion selectivity

This work reports the synthesis, characterization and study of complex formation equilibria of the new ligand 6,6-(2-(diethylamino)ethylazanediyl)bis(methylene)bis(5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) with Fe(III), Al(III), Cu(II) and Zn(II). On the basis of previous encouraging results with tetradentate bis-kojic acid chelators, this ligand was designed to improve the pharmacokinetic properties: increase the solubility, neutral at physiological pH7.4, and enhancement of membrane crossing ability. Fe(III) and Al(III) complexation gave evidence of high metal-sequestering capacity of L9. Cellular assays showed that the ligand is capable of crossing cellular membranes and it does not present toxic effects. Complex formation equilibria with the essential metal ions Cu(II) and Zn(II) have been furthermore studied to evaluate disturbances of this chelator on the homeostatic equilibria of these essential metal ions. A variety of techniques (potentiometry, UV-visible spectrophotometry, 1D and 2D NMR spectroscopy, ESI-MS (electrospray ionization-mass spectrometry), quantum mechanical calculations and X-ray diffraction) have facilitated the characterization of the ligand, and the corresponding iron and zinc complexes, together with an exhaustive analysis of the protonation and complex equilibria.

The binding of Ni(II) and Cu(II) with the N-terminal tail of the histone H4

We have analyzed, for Ni(II) and Cu(II) binding, the sequence of the N-terminal tail of the histone H4, the 22-amino acid peptide Ac-SGRGKGGKGLGKGGAKRHRKVL-Am and, in addition, the 7- and 11-amino acid peptides Ac-AK(Ac)RHRK(Ac)V-Am, Ac-GK(Ac)GGAK(Ac)RHRK(Ac)V-Am where all side chains of lysines were blocked by acetylation. Potentiometric and spectroscopic studies (UV-Vis, CD, EPR, NMR) showed that histidine 18 acted as an anchoring binding site for metal ions in all the peptides investigated. The stability constants of the 3N and 4N complexes are higher than those obtained for simple peptides with glycine instead of arginine and lysine residues in the metal binding site. The coordination was not significantly affected by the acetylation of lysines. The behavior of the “tail” suggested a possible bent structure with organized side-chain orientation promoted by nNi(II).

Tungsten-induced carcinogenesis in human bronchial epithelial cells.

Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten (W), remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. Here, for the first time, we demonstrate tungstens ability to induce carcinogenic related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer-related pathways in transformed clones as determined by RNA sequencing. Human bronchial epithelial cell line (Beas-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA-sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. Taken together, our data show the carcinogenic potential of tungsten. Further tests are needed, including in vivo and human studies, in order to validate tungsten as a carcinogen to humans.

Coordination Environment of Cu(II) Ions Bound to N-Terminal Peptide Fragments of Angiogenin Protein

Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang–actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo (“wild-type”, wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein–copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1–17) and AcAng(1–17), which encompass the sequence 1–17 of angiogenin (QDNSRYTHFLTQHYDAK-NH2), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides.

The Involvement of Amino Acid Side Chains in Shielding the Nickel Coordination Site: An NMR Study

Coordination of proteins and peptides to metal ions is known to affect their properties, often by a change in their structural organization. Side chains of the residues directly involved in metal binding or very close to the coordination centre may arrange themselves around it, in such a way that they can, for instance, disrupt the protein functions or stabilize a metal complex by shielding it from the attack of water or other small molecules. The conformation of these side chains may be crucial to different biological or toxic processes. In our research we have encountered such behaviour in several cases, leading to interesting results for our purposes. Here we give an overview on the structural changes involving peptide side chains induced by Ni(II) coordination. In this paper we deal with a number of peptides, deriving from proteins containing one or more metal coordinating sites, which have been studied through a series of NMR experiments in their structural changes caused by Ni(II) complexation. Several peptides have been included in the study: short sequences from serum albumin (HSA), Des-Angiotensinogen, the 30-amino acid tail of histone H4, some fragments from histone H2A and H2B, the initial fragment of human protamine HP2 and selected fragments from prion and Cap43 proteins. NMR was the election technique for gathering structural information. Experiments performed for this purpose included 1D 1H and 13C, and 2D HSQC, COSY, TOCSY, NOESY and ROESY acquisitions, which allowed the calculation of the Ni(II) complexes structural models.

An NMR study on the 6,6'-(2-(diethylamino)ethylazanediyl)bis(methylene)bis(5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) interaction with Al(III) and Zn(II) ions.

Here we report about the complex formation among an amine-bearing bis-kojic acid, 6,6-(2-(diethylamino)ethylazanediyl)bis(methylene)bis(5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) and two metal ions, the trivalent hard and not essential metal ion Al(III) and the borderline and essential divalent metal ion Zn(II). We carried out a thorough NMR study in order to reach the indispensable structural information on the behavior of these complexes in solution. A combination of 1D, 2D total correlation spectroscopy, heteronuclear single quantum coherence spectroscopy, nuclear Overhauser enhancement spectroscopy and rotating-frame Overhauser effect spectroscopy experiments was used to assign the signals of both free and metal-bound ligand at different pH values. Our results highlighted the different coordination behaviors of the ligand towards the different metal ions, depending on their hard or borderline character. The trivalent metal ion, Al(III), mainly forms dinuclear helicate complexes of M2L3 stoichiometry, and the coordination only involves both hydroxypyrone (O,O)-donor atoms. NMR data are in agreement with the presence of a rigid and symmetric structure of L9-Al(III) complexes up to physiological pH. On the contrary, with the divalent metal ion, NMR data showed the coexistence of several species in solution though Zn(II) forms complexes of ML stoichiometry at physiological pH, where the metal coordination involves the nitrogen atoms of both the linker and the side-chain amine groups together with the oxygen atoms of phenolate groups. The in solution study will be of interest for providing an insight on the ligand bioavailability and on its behavior in the chelation treatments.