BioMetals (v.28, #6)

Magnetic susceptibility and isothermal remanent magnetization in human tissues: a study case by H. Sant’Ovaia; G. Marques; A. Santos; C. Gomes; A. Rocha (951-958).
This study evaluated the magnetic properties, magnetic susceptibility and isothermal remanent magnetization (IRM) of tissue samples from the brain, liver, spleen, pancreas, heart and lungs, resected from human corpses, with the aim of identifying the magnetic mineral structures and understanding their possible connection to diseases, professional activity, age and gender of the individual, smoking habits and the environment. The heart was the organ with the highest values of magnetic susceptibility and the pancreas showed the lowest values. No relationship was found between magnetic susceptibility, IRM values and ages of the individuals. However the samples obtained in females showed lower values of magnetic susceptibility than those resected from males. The samples collected from the lungs of smokers have higher values of magnetic susceptibility and IRM indicating the presence of magnetic particles with an anthropic origin. Moreover, the complexity of the magnetic behaviour of these tissues may suggest a contribution of both biogenic and anthropogenic magnetic particles also due to some professional activities. In the brain a heterogeneous distribution of the magnetic susceptibility values was found, which might be related mainly to the diamagnetic behaviour of myelin-rich structures. This study suggests that although the diamagnetic and paramagnetic behaviour is common to all structures, magnetite-type structures are always present in the tissues and hematite-type structures may also contribute to the magnetic signal of the sample. IRM values are only dependent on the presence of magnetite or hematite-type magnetic structures and so this technique seems more suitable to achieve the characterization of biomagnetic structures than magnetic susceptibility.
Keywords: Human tissues; Isothermal remanent magnetization; Magnetic susceptibility; Magnetite

Zinc affects miR-548n, SMAD4, SMAD5 expression in HepG2 hepatocyte and HEp-2 lung cell lines by Arthur Grider; Richard D. Lewis; Emma M. Laing; Abhijeet A. Bakre; Ralph A. Tripp (959-966).
MicroRNAs affect disease progression and nutrient status. miR-548n increased 57 % in Zn supplemented plasma from adolescent females (ages 9 to 13 years). The purpose of this study was to determine the effects of Zn concentration in cell culture on the expression of miR-548n, SMAD4 and SMAD5 in hepatocyte (HepG2) and lung epithelium (HEp-2) cell lines. Cells were incubated for 48 h in media containing 10 % Chelex 100-treated FBS (0 μM Zn), or with 15 or 50 μM Zn, before isolation of total RNA and cDNA. Expression of miR-548n, SMAD4 and SMAD5 was measured by qPCR. The ΔΔCT method was used to calculate the fold-change, and 15 µM expression levels were used as reference values. HepG2 miR-548n expression decreased 5-fold, and SMAD4 expression increased 4-fold in the absence of Zn, while HEp-2 miR-548n expression increased 10.5-fold, and SMAD5 expression increased 20-fold in the absence of Zn. HEp-2 miR-548n expression increased 23-fold, while SMAD4 expression decreased twofold, in 50 μM Zn-treated cells. However, SMAD4 and SMAD5 expression was not correlated. These data indicate that miR-548n expression is in part regulated by Zn in a cell-specific manner. SMAD4 and SMAD5 are genes in the TGF-β/BMP signaling pathway, and SMAD5 is a putative target for miR-548n; Zn participates in regulating this pathway through controlling SMAD4 and SMAD5 expression. However, SMAD5 expression may be more sensitive to Zn than to miR-548n since SMAD5 expression was not inversely correlated with miR-548n expression.
Keywords: HepG2; HEp-2; miRNA-548n; SMAD4; SMAD5; Zinc concentration

While the effects of systemic zinc ion deficiency and toxicity on animal health are well documented, the impacts of localized, tissue-specific disturbances in zinc homeostasis are less well understood. Previously we have identified zinc dyshomeostasis scenarios caused by the targeted manipulation of zinc transport genes in the Drosophila eye. Over expression of the uptake transporter dZIP42C.1 (dZIP1) combined with knockdown of the efflux transporter dZNT63C (dZNT1) causes a zinc toxicity phenotype, as does over expression of dZIP71B or dZNT86D. However, all three genotypes result in different morphologies, responses to dietary zinc, and genetic interactions with the remaining zinc transport genes, indicating that each causes a different redistribution of zinc within affected cells. dZNT86D eGFP over expression generates a completely different phenotype, interpreted as a Golgi zinc deficiency. Here we assess the effect of each of these transgenes when targeted to a range of Drosophila tissues. We find that dZIP71B is a particularly potent zinc uptake gene, causing early developmental lethality when targeted to multiple different tissue types. dZNT86D over expression (Golgi-only zinc toxicity) is less deleterious, but causes highly penetrant adult cuticle, sensory bristle and wing expansion defects. The dZIP42C.1 over expression, dZNT63C knockdown combination causes only moderate adult cuticle defects and sensitivity to dietary zinc when expressed in the midgut. The Golgi-only zinc deficiency caused by dZNT86D eGFP expression results in mild cuticle defects, highly penetrant wing expansion defects and developmental lethality when targeted to the central nervous system and, uniquely, the fat bodies.
Keywords: Drosophila ; Zinc homeostasis; ZIP; ZNT; Ion transport

The physiological concentration of ferrous iron (II) alters the inhibitory effect of hydrogen peroxide on CD45, LAR and PTP1B phosphatases by Alicja Kuban-Jankowska; Magdalena Gorska; Lukasz Jaremko; Mariusz Jaremko; Jack A. Tuszynski; Michal Wozniak (975-986).
Hydrogen peroxide is an important regulator of protein tyrosine phosphatase activity via reversible oxidation. However, the role of iron in this reaction has not been yet elucidated. Here we compare the influence of hydrogen peroxide and the ferrous iron (reagent for Fenton reaction) on the enzymatic activity of recombinant CD45, LAR, PTP1B phosphatases and cellular CD45 in Jurkat cells. The obtained results show that ferrous iron (II) is potent inhibitor of CD45, LAR and PTP1B, but the inhibitory effect is concentration dependent. We found that the higher concentrations of ferrous iron (II) increase the inactivation of CD45, LAR and PTP1B phosphatase caused by hydrogen peroxide, but the addition of the physiological concentration (500 nM) of ferrous iron (II) has even a slightly preventive effect on the phosphatase activity against hydrogen peroxide.
Keywords: Ferrous iron; Hydrogen peroxide; CD45; LAR; PTP1B

Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells by Tai C. Holland; David W. Killilea; Swapna V. Shenvi; Janet C. King (987-996).
A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N′,N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.
Keywords: Lymphocyte; Metallothionein; Metal chelator; Biomarkers

Aluminium stress disrupts metabolic performance of Plantago almogravensis plantlets transiently by Tomás Grevenstuk; Annick Moing; Mickaël Maucourt; Catherine Deborde; Anabela Romano (997-1007).
Little is known about how tolerant plants cope with internalized aluminium (Al). Tolerant plants are known to deploy efficient detoxification mechanisms, however it is not known to what extent the primary and secondary metabolism is affected by Al. The aim of this work was to study the metabolic repercussions of Al stress in the tolerant plant Plantago almogravensis. P. almogravensis is well adapted to acid soils where high concentrations of free Al are found and has been classified as a hyperaccumulator. In vitro reared plantlets were used for this purpose in order to control Al exposure rigorously. The metabolome of P. almogravensis plantlets as well as its metabolic response to the supply of sucrose was characterized. The supply of sucrose leads to an accumulation of amino acids and secondary metabolites and consumption of carbohydrates that result from increased metabolic activity. In Al-treated plantlets the synthesis of amino acids and secondary metabolites is transiently impaired, suggesting that P. almogravensis is able to recover from the Al treatment within the duration of the trials. In the presence of Al the consumption of carbohydrate resources is accelerated. The content of some metabolic stress markers also demonstrates that P. almogravensis is highly adapted to Al stress.
Keywords: Aluminium tolerance; Accumulation; Metabolomics; Nuclear magnetic resonance; In vitro culture

Effect of manganese treatment on the accumulation on biologically relevant metals in rat cochlea and brain by inductively coupled plasma mass spectrometry by Elizabeth J. Mullin; Stacia R. Wegst-Uhrich; Dalian Ding; Senthilvelan Manohar; Vijaya Prakash Krishnan Muthaiah; Richard Salvi; Diana S. Aga; Jerome A. Roth (1009-1016).
Manganese (Mn), iron (Fe), zinc (Zn), and copper (Cu) are essential transitions metals that are required in trace amounts, however chronic exposure to high concentrations can cause severe and irreversible neurotoxicity. Since prolonged exposure to Mn leads to manganism, a disorder exhibiting a diverse array of neurological impairments progressing to a debilitating and irreversible extrapyramidal condition symptomatically similar to Parkinson’s disease, we measured the concentration of Mn as well as Fe, Zn and Cu in three region of the brain (globus pallidus, striatum and inferior colliculus) and three regions in the cochlea (stria vascularis, basilar membrane and modiolus) under normal conditions or after 30 or 60 days of oral administration of Mn (10 mg/ml ad libitum). Under normal conditions, Mn, Zn and Fe were typically higher in the cochlea than in the three brain regions whereas Cu was equal to or lower. Oral treatment with Mn for 30 or 60 days resulted in 20–75 % increases in Mn concentrations in both cochlea and brain samples, but had little effect on Cu and Fe levels. In contrast, Zn levels decreased (20–80 %) with Mn exposure. Our results show for the first time how prolonged oral Mn-ingestion affects the concentration of Mn, Cu, Zn and Fe, in the three regions of the cochlea, the inferior colliculus in auditory midbrain and the striatum and globus pallidus, two regions implicated in Parkinson’s disorder. The Mn-induced changes in the concentration of Mn, Cu, Zn and Fe may provide new insights relevant to the neurotoxicity of Mn and the transport and accumulation of these metals in cochlea and brain.
Keywords: Manganese; ICP-MS; Trace analysis; Cochlea; Iron; Copper; Zinc

Core domain mutant Y220C of p53 protein has a key role in copper homeostasis in case of free fatty acids overload by Mario Arciello; Alessia Longo; Carmela Viscomi; Concetta Capo; Antonio Angeloni; Luisa Rossi; Clara Balsano (1017-1029).
Nonalcoholic fatty liver disease (NAFLD) is a pathology that includes a wide variety of clinical conditions ranging from simple steatosis to end-stage liver diseases. Despite the huge amount of researches, the molecular basis of NAFLD are still not fully understood. Recently, it was suggested a role for p53 in NAFLD pathogenesis. Among its targets there is Synthesis of Cytochrome c Oxidase 2 (SCO2), a copper chaperone, involved in both aerobic respiration and metal cellular excretion. Copper seems to play a role in NAFLD. It was demonstrated a low hepatic copper content in NAFLD patients, which correlates with metabolic syndrome parameters. Copper homeostasis deregulation, in fact, seems to be related to lipid metabolism alteration and insulin resistance. Here we provide evidence on the role of p53 in the modulation of copper homeostasis, in an experimental model of NAFLD. We used two different hepatoma cell lines, HepG2 and Huh 7.5.1, characterized by the presence of wt p53 and its Y220C mutant, respectively, treated with a free fatty acids (FFAs) solution. Interestingly, p53 activation correlated with the intracellular copper level maintenance. We demonstrated that, in hepatoma cell lines, core domain mutant Y220C of p53 affects the modulation of SCO2 and Copper transporter 1 (CTR1), influencing, in this way, intracellular copper homeostasis in presence of FFAs accumulation, and that the 220 residue of the protein is crucial for such control. The role of p53 we highlighted may have deep implications in clinical conditions where copper homeostasis is deregulated.
Keywords: P53; Copper; Metal homeostasis; Fatty acid; Hepatocyte

The interaction of a mononuclear monofunctional anticancer Pt(II) complex, [PtLCl]Cl (L = 4′-bis(pyridine-2-ylmethyl)amino-2-phenylbenzothiazole) (1), and human serum albumin (HSA) was investigated under physiological conditions using UV–Vis absorption, circular dichroism, fluorescence, and synchronous fluorescence. The experimental results suggested that the Pt(II) complex could bind to HSA, induce conformation and microenvironmental changes of HSA with a moderate binding affinity, and quench the intrinsic fluorescence of HSA through a static quenching mechanism. The thermodynamic parameters, ΔG°, ΔH°, and ΔS°, calculated at different temperatures, indicated that the binding reaction was spontaneous and hydrophobic forces and π–π stacking played major roles in the association. Based on the number of binding sites, it was considered that one molecule of complex 1 could bind to a single site of HSA. In view of the results of site marker competition experiments, the reactive site of HSA to complex 1 mainly located in subdomain IIA (site I). Moreover, the binding distance, r, between donor (HSA) and acceptor (complex 1) was 4.69 nm according to Förster nonradiation energy transfer theory. The present study provides relevant and useful information that can be used for the design and application of mononuclear monofuctional Pt(II) complexes in biomedical sciences.
Keywords: Monofunctional Pt(II) complex; Human serum albumin; Fluorescence quenching; Anticancer metallodrugs

Piperazine derivatives as iron chelators: a potential application in neurobiology by S. Abdelsayed; N. T. Ha Duong; C. Bureau; P. P. Michel; E. C. Hirsch; J. M. El Hage Chahine; N. Serradji (1043-1061).
Polysubstituted piperazine derivatives, designed as new iron chelators, were synthesized and fully characterized by nuclear magnetic resonance and mass spectroscopy. Their potential to prevent iron-induced neurotoxicity was assessed using a cellular model of Parkinson disease. We demonstrated their ability to provide sustained neuroprotection to dopaminergic neurons that are vulnerable in this pathology. The iron chelating properties of the new compounds were determined by spectrophotometric titration illustrating that high affinity for iron is not associated with important neuroprotective effects.
Keywords: Chelates; Iron; Parkinson; Piperazine; Fast kinetics

Combined stress of salinity and heavy metal is a serious problem for crop production; however, physiological mechanisms of tolerance to such condition remain elusive in cotton. Here, we used two cotton genotypes differing in salt tolerance, to understand their response to salinity (NaCl) and cadmium (Cd) either alone or in combination (Cd + Na) via hydroponics. Results showed that salinity and/or Cd drastically reduced plant growth, chlorophyll content and photosynthesis, with greater effect observed in Zhongmian 41 (sensitive) than Zhong 9806 (tolerant). Although salinity and/or Cd induced malondialdehyde (MDA) accumulation in Zhongmian 41 at 5 and 10 days after treatment, MDA content remained unchanged in Zhong 9806, implying that Zhongmian 41 but not Zhong 9806 faced oxidative stress following exposure to salinity and/or Cd. Differential responses of antioxidant enzymes such as superoxide dismutase, guaiacol peroxidase, catalase and ascorbate peroxidase to Cd, NaCl and Cd + Na indicate genotype- and time course- dependent variations. In both genotypes, Cd content was decreased while Na concentration was increased under combined stress compared with Cd alone. Importantly, NaCl addition in Cd-containing medium caused remarkable reduction in Cd concentration, with the extent of reduction being also dependent on genotypes. The salt-tolerant genotypes had lower Na concentration than sensitive ones. Furthermore, obvious changes in leaf and root ultrastructure was observed under Cd, Na and Cd + Na stress, however Zhong 9806 was less affected compared with Zhongmian 41. These results may provide novel insight into the physiological mechanisms of Cd + Na stress tolerance in various cotton genotypes.
Keywords: Antioxidant enzyme; Cadmium; Combined stress; Cotton; Salinity; Ultrastructure

Interactions of the organogold(III) compound Aubipyc with the copper chaperone Atox1: a joint mass spectrometry and circular dichroism investigation by Tiziano Marzo; Federica Scaletti; Elena Michelucci; Chiara Gabbiani; Gennaro Pescitelli; Luigi Messori; Lara Massai (1079-1085).
The so called “copper trafficking system” in mammalian cells is primarily devoted to the regulation of copper transport and homeostasis. This system, now well characterized, consists of a few strictly interconnected proteins that assist copper entrance inside cells and then promote metal transfer and delivery to essential copper-dependent cellular proteins (Boal and Rosenzweig 2009a; Banci et al., Mol Life Sci 67:2563–2589, 2010). Yet, the “copper trafficking system” may also facilitate the entrance inside cells of non-physiological metal species such as clinically established platinum drugs. ESI and MALDI MS methods are exploited here to characterize the interactions occurring between the experimental anticancer organogold(III) drug, Aubipyc, and the copper chaperone Atox1, a key protein of the copper trafficking system. The nature of the adducts that are formed when reacting Aubipyc with Atox1 is elucidated in detail. Characterization of the Aubipyc/Atox1 system is further supported by circular dichroism experiments. Binding competitions with mercury and bismuth ions were also explored. The relevance and the biological implications of the present results are discussed.
Keywords: Anticancer drugs; Gold compounds; ESI-MS; Circular dichroism; Protein melting; Protein metalation

Synergistic effect of copper and low temperature over Listeria monocytogenes by Mauricio Latorre; Ana María Quesille-Villalobos; Felipe Maza; Angel Parra; Angélica Reyes-Jara (1087-1092).
The capacity to grow at low temperatures has allowed Listeria monocytogenes to become one of the primary food pathogens to date, representing a major public health problem worldwide. Several works have described the homeostatic response of L. monocytogenes under different copper (Cu) treatments growing at mild temperature (30 °C). The aims of this report were to evaluate if changes in the external concentration of Cu affected viability and Cu homeostasis of L. monocytogenes growing at low temperature. Ours results showed that L. monocytogenes growing at 8 °C had a reduced viability relative to 30 °C when exposed to Cu treatments. This decrease was correlated with an increase in the internal concentration of Cu, probably linked to the transcriptional down-regulation of mechanisms involved in Cu homeostasis. This combined effect of Cu and low temperature showed a synergistic impact over the viability and homeostasis of L. monocytogenes, where low temperature exacerbated the toxic effect of Cu. These results can be useful in terms of the use of Cu as an antibacterial agent.
Keywords: Listeria monocytogenes ; Copper; Low temperature; Synergistic effect