BioMetals (v.30, #1)

The prevalence of neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease are increased by age. Alleviation of their symptoms and protection of normal neurons against degeneration are the main aspects of the research to establish novel therapeutic strategies. Iron as the one of most important cation not only play important role in the structure of electron transport chain proteins but also has pivotal duties in cellular activities. But disruption in iron hemostasis can make it toxin to neurons which causes lipid peroxidation, DNA damage and etc. In patients with Alzheimer and Parkinson misbalancing in iron homeostasis accelerate neurodegeneration and cause neuroinflmmation. mTOR as the common signaling pathway between cancer and neurodegenerative disorders controls iron uptake and it is in active form in both diseases. Anti-cancer drugs which target mTOR causes iron deficiency and dual effects of mTOR inhibitors can candidate them as a therapeutic strategy to alleviate neurodegeneration/inflammation because of iron overloading.
Keywords: Alzheimer’s disease; Parkinson’s disease; Iron; Neuroinflammation; mTOR; Cancer

Four mononuclear platinum(II) complexes: synthesis, DNA/BSA binding, DNA cleavage and cytotoxicity by Qingming Wang; Lei Yang; Jiahui Wu; Hua Wang; Jialiang Song; Xinhui Tang (17-26).
Four new platinum(II) complexes: PtII L1·H2O (C1, H2 L1 = C20H16N2O2), PtII L2Cl2 (C2, L2 = C22H16N2O2), PtII L3Cl2·H2O (C3, L3 = C20H16N2), PtII L4Cl2·0.4H2O (C4, L4 = C18H14N4) have been synthesized and characterized by using various physico-chemical techniques. The binding interaction of the four platinum(II) complexes C1C4 with calf thymus (CT)-DNA has been investigated by UV–Vis and fluorescence emission spectrometry. The apparent binding constant (K app) values follow the order: C3 > C1 > C2 > C4. In addition, fluorescence spectrometry of bovine serum albumin (BSA) with the four platinum(II) complexes C1C4 showed that the quenching mechanism might be a static quenching procedure. For C1C4, the number of binding sites was about one for BSA and the binding constants follow the order: C3 (7.08 × 105M−1) > C1 (2.82 × 105M−1) > C2 (0.85 × 105M−1) > C4 (0.15 × 105M−1). With the single condition change such as absence of an external agent, the DNA cleavage abilities of C3 exhibit remarkable changes. In addition, the cytotoxicity of C3 in vitro on tumor cells lines (MCF-7, HepG2 and HT29) were examined by MTT and showed better antitumor effects on the tested cells.
Keywords: Platinum(II) complexes; DNA binding; Bovine serum albumin (BSA) binding; Binding modes

Disease-causing point-mutations in metal-binding domains of Wilson disease protein decrease stability and increase structural dynamics by Ranjeet Kumar; Candan Ariöz; Yaozong Li; Niklas Bosaeus; Sandra Rocha; Pernilla Wittung-Stafshede (27-35).
After cellular uptake, Copper (Cu) ions are transferred from the chaperone Atox1 to the Wilson disease protein (ATP7B) for incorporation into Cu-dependent enzymes in the secretory pathway. Human ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologues in other organisms, has six similar cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of enzymatic activity and it is thus intriguing that point mutations in MBDs can promote Wilson disease. We here investigated, in vitro and in silico, the biophysical consequences of clinically-observed Wilson disease mutations, G85V in MBD1 and G591D in MBD6, incorporated in domain 4. Because G85 and G591 correspond to a conserved Gly found in all MBDs, we introduced the mutations in the well-characterized MBD4. We found the mutations to dramatically reduce the MBD4 thermal stability, shifting the midpoint temperature of unfolding by more than 20 °C. In contrast to wild type MBD4 and MBD4D, MBD4V adopted a misfolded structure with a large β-sheet content at high temperatures. Molecular dynamic simulations demonstrated that the mutations increased backbone fluctuations that extended throughout the domain. Our findings imply that reduced stability and enhanced dynamics of MBD1 or MBD6 is the origin of ATP7B dysfunction in Wilson disease patients with the G85V or G591D mutation.
Keywords: Wilson disease; ATP7B; Metal-binding domain; Thermal stability; Circular dichroism; Molecular dynamics

Photoinduced cytotoxicity mediated by a triphenylenamine-modified platinum diimine complex in human breast adenocarcinoma cells has been studied by cell viability assay. The triphenylenamine-modified platinum diimine complex showed more potent cytotoxicity in light than its carboxylate-modified analogue. To gain insights into the mechanism of photodynamic activity of this class of platinum diimine complexes, flow cytometric analyses were performed. The results suggest that upon irradiation the two platinum diimine complexes studied could induce cell cycle arrest in G2/M or S phase, and both of them could induce cancer cell death by apoptosis.
Keywords: Platinum diimine complex; Photodynamic therapy; Cytotoxicity; Cell cycle arrest; Apoptosis

The mitochondrial apoptotic pathway is induced by Cu(II) antineoplastic compounds (Casiopeínas®) in SK-N-SH neuroblastoma cells after short exposure times by Juan Carlos García-Ramos; Anllely Grizett Gutiérrez; Adriana Vázquez-Aguirre; Yanis Toledano-Magaña; Ana Luisa Alonso-Sáenz; Virginia Gómez-Vidales; Marcos Flores-Alamo; Carmen Mejía; Lena Ruiz-Azuara (43-58).
The family of Copper(II) coordination compounds Casiopeínas® (Cas) has shown antiproliferative activity in several tumour lines by oxidative cellular damage and mitochondrial dysfunction that lead to cell death through apoptotic pathways. The goal of this work is looking for the functional mechanism of CasIIgly, CasIIIia and CasIIIEa in neuroblastoma metastatic cell line SK-N-SH, a paediatric extra-cranial tumour which is refractory to several anti-carcinogenic agents. All Cas have shown higher antiproliferative activity than cisplatin (IC50 = 123 μM) with IC50 values of 18, 22 and 63 µM for CasIIgly, CasIIIEa and CasIIIia, respectively. At low concentrations and early times (4 h), these compounds cause a disruption of the mitochondrial transmembrane potential (Δψm). Concomitantly, an important depletion of intracellular glutathione and an increase of reactive oxygen species (ROS) hydrogen peroxide and radical superoxide were observed. On the other side, the lower cytotoxic effect of Casiopeínas on cultures of human peripheral blood lymphocytes (IC50 CasIIgly  = 1720 µM, IC50 CasIIIEa  = 3860 µM and IC50 CasIIIia  = 4700 µM) show the selectivity of these compounds over the tumour cells compared with the non-transformed cells. Chemically, glutathione (GSH) interacts with Casiopeínas® through the coordination of sulphur atom to the metal centre, process which facilitates the electron transfer to get Cu(I), GSSG and the posterior production of ROS. Additionally, the molecular structure of CasIIIia as nitrate is reported. These results have shown that the anticarcinogenic activity of Casiopeínas® on neuroblastoma SK-N-SH is through mitochondrial apoptosis due to the enhanced pro-oxidant environment promoted by the presence of the coordination copper compounds.
Keywords: Casiopeínas; Antitumour copper(II) compounds; Reactive oxygen species; Neuroblastome SK-N-SH; Lymphocytes cytotoxicity

Three new platinum(II) complexes of lidocaine and phenylcyanamide derivative ligands of formula K[Pt(3,5-(NO2)2pcyd)2(LC)], 1, K[Pt(3,5-(CF3)2pcyd)2(LC)], 2, K[Pt(3,5-Cl2pcyd)2(LC)], 3 (LC: lidocaine, 3,5-(NO2)2pcyd: 3,5-dinitro phenylcyanamide, 3,5-(CF3)2pcyd: 3,5-bis(trifluoromethyl) phenylcyanamide, 3,5-Cl2pcyd: 3,5-dichloro phenylcyanamide) have been synthesized and fully characterized. Cellular uptake, DNA platination and cytotoxicity against a panel of human tumor cell lines were evaluated. The complexes 13 revealed a significant in vitro antiproliferative activity against human ovarian carcinoma (A2780), colorectal adenocarcinoma (HT29), breast (MCF-7), liver hepatocellular carcinoma (HepG-2) and lung adenocarcinoma (A549) cancer cell lines. All the complexes are more active than cisplatin and follow the trend 1 > 2 > 3. Mechanistic studies showed that the trend in cytotoxicity of the Pt(II) complexes is mainly consistent with their ability to accumulate into cancer cells and to increase intracellular basal reactive oxygen species levels, which consequently results in the loss of mitochondrial membrane potential and apoptosis induction. The complex 1 caused to approximately 80-fold higher DNA platination level with respect to cisplatin. The complexes 13 can considerably stimulate the production of hydrogen peroxide in a time-dependent manner. Also, the complexes 13 induced an increase in reactive oxygen species (ROS) production that was superior to that induced by antimycin. The complex 1 had the most effect on ROS production in comparison with other complexes.
Keywords: Pt(II) complex; Lidocaine; Phenylcyanamide; Cytotoxicity; Apoptosis

Iron incorporation in biosilica of the marine diatom Stephanopyxis turris: dispersed or clustered? by Janine Kaden; Stephan I. Brückner; Susanne Machill; Christoph Krafft; Andreas Pöppl; Eike Brunner (71-82).
Iron incorporation into diatom biosilica was investigated for the species Stephanopyxis turris. It is known that several “foreign” elements (e.g., germanium, titanium, aluminum, zinc, iron) can be incorporated into the siliceous cell walls of diatoms in addition to silicon dioxide (SiO2). In order to examine the amount and form of iron incorporation, the iron content in the growth medium was varied during cultivation. Fe:Si ratios of isolated cell walls were measured by ICP-OES. SEM studies were performed to examine of a possible influence of excess iron during diatom growth upon cell wall formation. The chemical state of biosilica-attached iron was characterized by a combination of infrared, 29Si MAS NMR, and EPR spectroscopy. For comparison, synthetic silicagels of variable iron content were studied. Our investigations show that iron incorporation in biosilica is limited. More than 95% of biosilica-attached iron is found in the form of iron clusters/nanoparticles. In contrast, iron is preferentially dispersedly incorporated within the silica framework in synthetic silicagels leading to Si–O–Fe bond formation.
Keywords: Iron incorporation; Biosilica; Infrared spectroscopy; 29Si MAS NMR; EPR

Flow of essential elements in subcellular fractions during oxidative stress by Larissa Lago; Emilene A. Nunes; Aryane A. Vigato; Vanessa C. O. Souza; Fernando Barbosa Jr.; João R. Sato; Bruno L. Batista; Giselle Cerchiaro (83-96).
Essential trace elements are commonly found in altered concentrations in the brains of patients with neurodegenerative diseases. Many studies in trace metal determination and quantification are conducted in tissue, cell culture or whole brain. In the present investigation, we determined by ICP-MS Fe, Cu, Zn, Ca, Se, Co, Cr, Mg, and Mn in organelles (mitochondria, nuclei) and whole motor neuron cell cultured in vitro. We performed experiments using two ways to access oxidative stress: cell treatments with H2O2 or Aβ-42 peptide in its oligomeric form. Both treatments caused accumulation of markers of oxidative stress, such as oxidized proteins and lipids, and alteration in DNA. Regarding trace elements, cells treated with H2O2 showed higher levels of Zn and lower levels of Ca in nuclei when compared to control cells with no oxidative treatments. On the other hand, cells treated with Aβ-42 peptide in its oligomeric form showed higher levels of Mg, Ca, Fe and Zn in nuclei when compared to control cells. These differences showed that metal flux in cell organelles during an intrinsic external oxidative condition (H2O2 treatment) are different from an intrinsic external neurodegenerative treatment.
Keywords: Metal quantification; Metal flow; Oxidative stress; Neuroscience

Methane alleviates copper-induced seed germination inhibition and oxidative stress in Medicago sativa by Muhammad Kaleem Samma; Heng Zhou; Weiti Cui; Kaikai Zhu; Jing Zhang; Wenbiao Shen (97-111).
Recent results discovered the protective roles of methane (CH4) against oxidative stress in animals. However, the possible physiological roles of CH4 in plants are still unknown. By using physiological, histochemical and molecular approaches, the beneficial role of CH4 in germinating alfalfa seeds upon copper (Cu) stress was evaluated. Endogenous production of CH4 was significantly increased in Cu-stressed alfalfa seeds, which was mimicked by 0.39 mM CH4. The pretreatment with CH4 significantly alleviated the inhibition of seed germination and seedling growth induced by Cu stress. Cu accumulation was obviously blocked as well. Meanwhile, α/β amylase activities and sugar contents were increased, all of which were consistent with the alleviation of seed germination inhibition triggered by CH4. The Cu-triggered oxidative stress was also mitigated, which was confirmed by the decrease of lipid peroxidation and reduction of Cu-induced loss of plasma membrane integrity in CH4-pretreated alfalfa seedlings. The results of antioxidant enzymes, including ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (POD) total or isozymatic activities, and corresponding transcripts (APX1/2, Cu/Zn SOD and Mn-SOD), indicated that CH4 reestablished cellular redox homeostasis. Further, Cu-induced proline accumulation was partly impaired by CH4, which was supported by the alternation of proline metabolism. Together, these results indicated that CH4 performs an advantageous effect on the alleviation of seed germination inhibition caused by Cu stress, and reestablishment of redox homeostasis mainly via increasing antioxidant defence.
Keywords: Copper stress; Methane; Oxidative stress; Seed germination; Medicago sativa

Silver-based devices activated by electric current are of interest in biomedicine because of their broad-spectrum antimicrobial activity. This study investigates the in vitro antibacterial efficacy and cytotoxicity of a low intensity direct current (LIDC)-activated silver–titanium implant system prototype designed for localized generation and delivery of silver ions at the implantation site. First, the antibacterial efficacy of the system was assessed against methicillin-resistant Staphylococcus aureus (MRSA) over 48 h at current levels of 3 and 6 µA in Mueller–Hinton broth. The cytotoxicity of the system was then evaluated over 48 h in two phases using an in vitro model with in which the activated electrodes were suspended in growth medium in a cell-seeded tissue culture plate. In phase-1, the system was tested on human osteosarcoma (MG-63) cell line and compared to titanium controls. In phase-2, the cytotoxicity characteristics were validated with normal human diploid osteoblast cells. The LIDC-activated system demonstrated high antimicrobial efficacy against MRSA, but was also toxic to human cells immediately surrounding the electrodes. The statistical analysis showed that the cytotoxicity was a result of the presence of silver, and the electric activation did not make it worse.
Keywords: Low intensity direct current; Silver–titanium implant; Orthopaedic application; Antimicrobial efficacy; Cytotoxicity

Xanthomonas translucens pv. undulosa (Xtu) causes Bacterial Leaf Streak disease in the staple food crops such as wheat and barley. The survival strategies of pathogen and host are determined by the complex interactions occurring between the host plants and the pathogenic microbes. Iron binding proteins are important in the plant–microbe interactions as they are indulged in enzyme catalysis, virulence, metabolic and transport activities. In the presented study, we have identified that ~9.8% of Xtu proteome possess iron binding sequence motifs. Further, the analysis of Xtu proteome for secretory iron binding virulent proteins (IBVPs) revealed the fact that iron co-regulate the function of secretory proteins in virulence. We have found 26 secretory IBVPs and observed that these proteins are diverse in their biological functions ranging from transport to antimicrobial resistance, Reactive oxygen species detoxification and carbohydrate catabolism. The inferences may instigate to design the new strategies to combat and control the microbial diseases of staple food crops.
Keywords: Xanthomonas translucens pv. undulosa ; Iron binding proteins; Secretory iron binding virulent proteins; Ton-B dependent receptors

Effects of aluminum trichloride on the cartilage stimulatory growth factors in rats by Fan Zhang; Xudong Sun; Hongyan Yu; Xu Yang; Miao Song; Yanfei Han; Yanfei Li; Yanzhu Zhu (143-150).
Aluminum (Al) is considered to be a potentially toxic metal and inhibits cartilage formation. Transforming growth factor β1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2) are cartilage stimulatory growth factors, which play important roles in regulating the cartilage formation. To investigate the effects of aluminum trichloride (AlCl3) on the regulation of cartilage formation. Eighty Wistar rats were orally exposed to 0 (control group), 0.4 g/L (low-dose group), 0.8 g/L (mid-dose group) and 1.6 g/L (high-dose group) AlCl3 for 120 days, respectively. The rats body weight were decreased, the cartilage histological structure were disrupted, the cartilage and serum contents of Al and the serum level of C-telopeptide of type II collagen were all increased, the serum level of type II collagen (Col II) and alkaline phosphatase (ALP), and the mRNA expressions of TGF-β1, BMP-2, ALP and Col II were all decreased in the AlCl3-treated groups compared with those in control group. These results indicate that AlCl3 inhibits the cartilage formation through inhibition of the cartilage stimulatory growth factors expressions.
Keywords: Aluminum trichloride; Cartilage; Cartilage stimulatory growth factors; Rat