BioMetals (v.23, #1)

EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport by Mohan B. Rajasekaran; Sanjay Nilapwar; Simon C. Andrews; Kimberly A. Watson (1-17).
The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component—the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved ‘HxxE’ motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu2+ using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe3+ and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the ‘EfeO-Cup’ family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology.
Keywords: Iron transport; Homology modelling; Cupredoxin; Electron transfer; GRID prediction; Metal binding site; DUF451; Peptidase-M75

Zinc is the second-most abundant transition metal within cells and an essential micronutrient. Although adequate zinc is essential for cellular function, intracellular free zinc (Zn2+) is tightly controlled, as sustained increases in free Zn2+ levels can directly contribute to apoptotic endothelial cell death. Moreover, exposure of endothelial cells to acute nitrosative and/or oxidative stress induces a rapid rise of Zn2+ with mitochondrial dysfunction and the initiation of apoptosis. This apoptotic induction can be mimicked through addition of exogenous ZnCl2 and mitigated by zinc-chelation strategies, indicating Zn2+-dependent mechanisms in this process. However, the molecular mechanisms of Zn2+-mediated mitochondrial dysfunction are unknown. Here we report that free Zn2+ disrupts cellular redox status through inhibition of glutathione reductase, and induces apoptosis by redox-mediated inhibition of the mitochondrial adenine nucleotide transporter (ANT). Inhibition of ANT causes increased mitochondrial oxidation, loss of ADP uptake, mitochondrial translocation of bax, and apoptosis. Interestingly, pre-incubation with glutathione ethyl ester protects endothelial cells from these observed effects. We conclude that key mechanisms of Zn2+-mediated apoptotic induction include disruption of cellular glutathione homeostasis leading to ANT inhibition and decreases in mitochondrial ATP synthesis. These pathways could represent novel therapeutic targets during acute oxidative or nitrosative stress in cells and tissues.
Keywords: Mitochondrial dysfunction; Apoptosis; Redox status

Zinc activates neutrophils’ oxidative burst by Marisa Freitas; Graça Porto; José L. F. C. Lima; Eduarda Fernandes (31-41).
Zinc has been shown to disturb the innate host defense response by interfering in the activation of neutrophils and subsequent oxidative burst, although the exact role of this metal, either as an activator or inhibitor, remains a matter of controversy among research groups. These apparent discrepancies may be due to experimental settings, through modification of zinc availability to neutrophils, or to inaccurate detections of reactive species. Thus, the main objective of the present study was to provide clarification on the role of zinc on the activation of human neutrophils and the subsequent oxidative burst. For that purpose, different detection methods and incubation media were used. The obtained results showed that phosphate buffers (PBS and HBSS) complex with zinc and interfere with the results obtained with this metal. By using Tris-G, it was clearly demonstrated that zinc, at low concentrations (5–12.5 μM), activates NADPH oxidase, mainly via protein kinase C, leading to the formation of superoxide radical (O2 •−). Higher concentrations of zinc results on a rapid dismutation of O2 •− to oxygen and hydrogen peroxide, which in turn is used by myeloperoxidase to generate hypochlorous acid (HOCl).
Keywords: Human neutrophils; Oxidative burst; Zinc; Protein kinase C; NADPH oxidase

Advanced glycation end products and antioxidant status in nondiabetic and streptozotocin induced diabetic rats: effects of copper treatment by S. Civelek; R. Gelişgen; G. Andican; A. Seven; S. H. Küçük; M. Özdoğan; G. Burçak (43-49).
The effects of Cu(II) supplementation on glycemic parameters, advanced glycation end products (AGEs), antioxidant status (glutathione; GSH and total antioxidant capacity; TAOC) and lipid peroxidative damage (thiobarbituric acid-reactive substances, TBARS) were investigated in streptozotocin (STZ) induced diabetic rats. The study was carried out on Wistar albino rats grouped as control (n = 10), CuCl2 treated (n = 9), STZ (n = 10) and STZ,CuCl2 treated (n = 9). STZ was administered intraperitoneally at a single dose of 65 mg/kg and CuCl2, 4 mg copper/kg, subcutaneously, every 2 days for 60 days. At the end of this period, glucose(mg/dl), Cu(μg/dl), TBARS(μmol/l), TAOC(mmol/l) were measured in plasma, GSH(mg/gHb) in erythrocytes and glycated hemoglobin (GHb)(%) in blood. Plasma AGE-peptides(%) were measured by HPLC flow system with spectrofluorimetric and spectrophotometric detectors connected on-line. Data were analyzed by the non-parametric Kruskal–Wallis and Mann–Whitney U test. In the STZ group glucose, GHb and AGE-peptide levels were all significantly higher than the control group (P < 0.01, P < 0.05, and P < 0.01, respectively). CuCl2 treated group had significantly lower glucose but significantly higher GHb, TAOC and TBARS levels than the control group (P < 0.05, P < 0.001, P < 0.05 and P < 0.001, respectively). STZ,CuCl2 treated group had significantly higher GHb, TAOC and TBARS levels compared with the control group (P < 0.001, P < 0.05 and P < 0.05, respectively); but only TAOC level was significantly higher than the STZ group (P < 0.01). This experimental study provides evidence that copper intake increases total antioxidant capacity in both nondiabetic and diabetic states. However despite the potentiated antioxidant defence, lipid peroxidation and glycation enhancing effects of CuCl2 are evident under nondiabetic conditions.
Keywords: STZ; Diabetes mellitus; Advanced glycation end products; Glycation; Copper

Zinc transporter expression profiles in the rat prostate following alterations in dietary zinc by Yang Song; Valerie Elias; Carmen P. Wong; Angus G. Scrimgeour; Emily Ho (51-58).
Zinc plays important roles in numerous cellular activities and physiological functions. Intracellular zinc levels are strictly maintained by zinc homeostatic mechanisms. Zinc concentrations in the prostate are the highest of all soft tissues and could be important for prostate health. However, the mechanisms by which the prostate maintains high zinc levels are still unclear. In addition, the response of the prostate to alterations in dietary zinc is unknown. The current study explored cellular zinc levels and zinc transporter expression profiles in the lobes of the prostate during dietary marginal zinc depletion. Rats were given either zinc-adequate (ZA, 30 mg Zn/kg) or marginal zinc-deficient (MZD, 5 mg Zn/kg) diet for 9 weeks. In addition, a subgroup of the MZD rats was supplemented with phytase (1,500 unit/kg diet) to improve zinc bioavailability. We found that both zinc concentrations and ZnT2 expression in the prostate dorsolateral lobes were substantially higher than in the ventral lobes (P < 0.05). Marginal zinc depletion significantly decreased ZnT2 expression in the dorsolateral lobes (P < 0.05), and phytase supplementation had a trend to increase ZnT2 expression. In addition, of all measured zinc transporters, only ZnT2 mRNA abundance was significantly correlated to the zinc concentrations in the dorsolateral lobe. No correlations were found between zinc transporter expression and zinc concentrations in the ventral lobes. These results indicate that ZnT2 may play a significant role in the maintenance of zinc homeostasis in the prostate.
Keywords: Zinc transporter; ZnT2; Prostate; Marginal zinc deficiency

Alkaline-earth metal complexes of the monoanionic form of the polyether ionophore monensin A were isolated for the first time in solid state and were structurally characterized using various spectroscopic methods (IR, NMR, FAB-MS). The stoichiometric reaction of monensic acid (MonH) with M2+ (M = Mg, Ca) in the presence of an organic base leads to the formation of mononuclear complexes of composition [M(Mon)2(H2O)2]. The structures of magnesium (1) and calcium (2) monensin complexes in the solid state were established by single crystal X-ray crystallography. The complexes crystallize as [Mg(Mon)2(H2O)2]·5MeCN (1) and [Ca(Mon)2(H2O)2]·H2O·5MeCN (2) in the monoclinic P21 space group. The alkaline-earth metal ion is placed in a distorted octahedral environment, defined by two monensin anions acting as bidentate ligands in the equatorial plane of the complex as well as by two water molecules occupying the axial positions of the inner coordination sphere. The bactericidal activity of 1 and 2 was evaluated against aerobic Gram-positive microorganisms applying the double layer agar hole diffusion method.
Keywords: Monovalent carboxylic ionophore; Alkaline-earth monensin complexes; Crystal structure; IR; NMR; FAB-MS; Bactericidal activity

d-Hydantoinase (HDT) is a metal-dependent enzyme that is widely used in industrial bioconversion to d-amino acids as valuable intermediates in the fields of food, pharmaceutical industry and agriculture. In this report, we prepared apo-HDT (metal-removed HDT) and Zn2+-HDT (Zn2+-added HDT) in vitro from a recombinant HDT (re-HDT) expressed in E. coli. The Zn2+-HDT and re-HDT contain 2.17 and 0.95 mol Zn2+ per mol subunit, respectively, and they have comparable enzymatic activities. In contrast, the apo-HDT only retains 0.04 mol Zn2+ per mol subunit with less than 10% activity, compared with the re-HDT. When the apo-HDT was reconstituted with ZnCl2, the enzymatic activity recovery was about 75%. Moreover, the fluorescence intensity, circular dichroism spectra and thermo-stability of the apo-HDT and Zn2+-HDT are quite different from those of the re-HDT. These data suggest that the re-HDT may have two Zn2+-binding sites, one is an intrinsic or tight-binding site (zinc-α) essential for its activity and the other is a vacant or loose-binding site (zinc-β) possibly non-essential for the activity.
Keywords: d-Hydantoinase; Zn2+ analysis; Functional evaluation

Cadmium effects on p38/MAPK isoforms in MDA-MB231 breast cancer cells by Caterina Casano; Maria Agnello; Rosalia Sirchia; Claudio Luparello (83-92).
Emerging evidence seems to indicate that the heavy metal cadmium (Cd) is able to regulate gene expression, drastically affecting the pattern of transcriptional activity in normal and pathological eukaryotic cells, also affecting intracellular signalization events. Human p38 is a family of mitogen-activated protein kinases consisting of four isoforms (α, β, γ and δ) which mediate signal transduction cascades controlling several aspects of cell physiology. In this study we examined whether exposure of MDA-MB231 tumor cells from the human breast to Cd may exert some effect on p38 isoform expression and accumulation, as well as on p38 activation. Employing a combination of proliferation tests, conventional and semiquantitative multiplex (SM)-polymerase chain reaction (PCR) and Western blot assays, we report that the treatment of breast cancer cells with 5 μM CdCl2 induces a diversified modulation of the transcription patterns of p38 isoform genes and of the accumulation of the related protein products, which are, on the other hand, also affected by α and β isoform functional inactivation induced by SB203580. Our findings suggest the existence of so far unexplored mechanisms of gene regulation in our model system and validate that MDA-MB231 cell line is a suitable in vitro model for further and more detailed studies on the intracellular mechanisms underlying the control of p38 expression, synthesis and activation in mammary tumor cells exposed to different stresses.
Keywords: Cadmium; SB203580; p38 isoforms; p38 activation; Gene expression

The presence of copper in water environment may have detrimental effects on aquatic organisms, including algae, where different enzymatic systems can be affected. Algae acid phosphatase plays important roles in metabolic processes such as decomposition of organic phosphate, autophagic digestive process, recycling cellular materials and zygote formation during reproduction. This work describes an in vitro activation effect of copper on the acid phosphatase of the green algae Pseudokirchneriella subcapitata (formely Selenastrum capricornutum) under preincubation condition. Apparent Michaelis constant values of 1.21 and 0.37 mM, and activation energy values of 26.8 and 13.6 kJ mol−1 were determined in the absence and in the presence of 0.2 mM Cu2+, respectively. The dissociation constant value for Cu2+ binding to the enzyme was determined to be 22.04 μM. The decrease of the apparent Michaelis constant (Km) and activation energy values in the presence of Cu2+ correlates well with its activating effect on the acid phosphatase activity. This propriety could be used as a sensitive bioindicator for copper in environmental samples.
Keywords: Enzyme; Phytoplankton; Toxicity; Fungicide; Metal; Selenastrum capricornutum

Crystal structure, DNA binding studies, nucleolytic property and topoisomerase I inhibition of zinc complex with 1,10-phenanthroline and 3-methyl-picolinic acid by Hoi-Ling Seng; Sze-Tin Von; Kong-Wai Tan; Mohd Jamil Maah; Seik-Weng Ng; Raja Noor Zaliha Raja Abd Rahman; Ignez Caracelli; Chew-Hee Ng (99-118).
Crystal structure analysis of the zinc complex establishes it as a distorted octahedral complex, bis(3-methylpicolinato-κ2 N,O)2(1,10-phenanthroline-κ2 N,N)-zinc(II) pentahydrate, [Zn(3-Me-pic)2(phen)]·5H2O. The trans-configuration of carbonyl oxygen atoms of the carboxylate moieties and orientation of the two planar picolinate ligands above and before the phen ligand plane seems to confer DNA sequence recognition to the complex. It cannot cleave DNA under hydrolytic condition but can slightly be activated by hydrogen peroxide or sodium ascorbate. Circular Dichroism and Fluorescence spectroscopic analysis of its interaction with various duplex polynucleotides reveals its binding mode as mainly intercalation. It shows distinct DNA sequence binding selectivity and the order of decreasing selectivity is ATAT > AATT > CGCG. Docking studies lead to the same conclusion on this sequence selectivity. It binds strongly with G-quadruplex with human tolemeric sequence 5′-AG3(T2AG3)3-3′, can inhibit topoisomerase I efficiently and is cytotoxic against MCF-7 cell line.
Keywords: Zinc(II) ternary complex; Duplex and quadruplex DNA binding; Nucleolytic; Topo I inhibition; Molecular modeling; Docking

Pentavalent methylated arsenicals are substrates of human AQP9 by Joseph R. McDermott; Xuan Jiang; Lauren C. Beene; Barry P. Rosen; Zijuan Liu (119-127).
Liver aquaglyceroporin AQP9 facilitates movement of trivalent inorganic arsenite (AsIII) and organic monomethylarsonous acid (MAsIII). However, the transport pathway for the two major pentavalent arsenic cellular metabolites, MAsV and DMAsV, remains unknown in mammals. These products of arsenic metabolism, in particular DMAsV, are the major arsenicals excreted in the urine of mammals. In this study, we examined the uptake of the two pentavalent organic arsenicals by human AQP9 in Xenopus laevis oocytes. Xenopus laevis oocytes microinjected with AQP9 cRNA exhibited uptake of both MAsV and DMAsV in a pH-dependent manner. The rate of transport was much higher at acidic pH (pH5.5) than at neutral pH. Hg(II), an aquaporin inhibitor, inhibited transport of AsIII, MAsIII, MAsV and DMAsV via AQP9. However, phloretin, which inhibits water and glycerol permeation via AQP9, can only inhibit transport of pentavalent MAsV and DMAsV but not trivalent AsIII and MAsIII, indicating the translocation mechanisms of these arsenic species are not exactly the same. Reagents such as FCCP, valinomycin and nigericin that dissipate transmembrane proton potential or change the transmemebrane pH gradient did not significantly inhibit all arsenic transport via AQP9, suggesting the transport of pentavalent arsenic is not proton coupled. The results suggest that in addition to the initial uptake of trivalent inorganic AsIII inside cells, AQP9 plays a dual role in the detoxification of arsenic metabolites by facilitating efflux from cells.
Keywords: AQP9; Liver; Urine; Methylation; Arsenite; Arsenate; Monomethylarsonous acid; Monomethylarsonate; Dimethylarsinate

Temporal production of the two Bacillus anthracis siderophores, petrobactin and bacillibactin by Melissa K. Wilson; Rebecca J. Abergel; Jean E. L. Arceneaux; Kenneth N. Raymond; B. Rowe Byers (129-134).
Bacillus anthracis secretes two siderophores, petrobactin (PB) and bacillibactin (BB). These siderophores were temporally produced during germination and outgrowth of spores (the usual infectious form of B. anthracis) in low-iron medium. The siderophore PB was made first while BB secretion began several hours later. Spore outgrowth early in an infection may require PB, whereas delayed BB production suggests a role for BB in the later stages of the infection. Incubation of cultures (inoculated as vegetative cells) at 37°C, as compared to 2°C, increased PB production and decreased secretion of BB, suggesting that the production of PB and BB responded to the host temperature signal. The dual siderophores of B. anthracis may fulfill independent roles in the life cycle of B. anthracis.
Keywords: Siderophores; Petrobactin; Bacillibactin; Iron; Anthrax

Cadmium exposure modifies lactotrophs activity associated to genomic and morphological changes in rat pituitary anterior lobe by Ana María Calderoni; Verónica Biaggio; Mariano Acosta; Liliana Oliveros; Fabian Mohamed; María Sofía Giménez (135-143).
Cadmium (Cd) is widely used in industrial applications and is an important contaminant of agricultural products. As an endocrine disruptor, Cd modifies the hormone release of pituitary anterior lobe (PAL). This work was undertaken to evaluate a possible association between phospholipase D (PLD) and prolactin mRNA expressions and the activity of lactotrophs and folliculostellate cells (FSC) in PAL of Cd exposed adult male Wistar rats (Cd, 0.133 mM per liter for 2 months). The PALs were submitted to immunohistochemical and morphometric analysis to determine the percentage of lactotrophs (PRL-ir) and FSC (S-100-ir). Cultured PAL cells were stained with Hoechst 33258 to determine the presence of alterations in nuclear morphology consistent with apoptosis. The expressions of PLD and prolactin mRNA were assessed by RT-PCR. Cd treated rats showed a decrease of PLD mRNA levels that can be associated to both high number of apoptotic cells and increase of S-100 protein expression in FSC. Cd decreased prolactin mRNA expression, number of lactotrophs and percentage of PRL-ir suggesting a low availability of prolactin to be secreted from PAL. Cd modifies the lactotrophs activity of pituitary gland through biochemical, genomic and morphological changes and contributes directly or indirectly to the levels of serum prolactin.
Keywords: Cadmium; Pituitary anterior lobe; Prolactin; Phospholipase D; Lactotrophs; Folliculostellate cells

The NikR protein is a nickel-responsive regulator, which in the gastric pathogen Helicobacter pylori controls expression of nickel-transporters and the nickel-cofactored urease acid resistance determinant. Although NikR-DNA interaction has been well studied, the Helicobacter NikR operator site remains poorly defined. In this study we have identified the NikR operators in the promoters of two inversely nickel-regulated urease operons (ureAB and ureA2B2) in the ferret pathogen Helicobacter mustelae, and have used bioinformatic approaches for the prediction of putative NikR operators in the genomes of four urease-positive Helicobacter species. Helicobacter mustelae NikR bound to the ureA2 promoter to a sequence overlapping with the −35 promoter region, leading to repression. In contrast, NikR binding to a site far upstream of the canonical σ80 promoter in the H. mustelae ureA promoter resulted in transcriptional induction, similar to the situation in H. pylori. Using H. pylori NikR operators and the newly identified H. mustelae NikR operators a new consensus sequence was generated (TRWYA-N15-TRWYA), which was used to screen the genomes of four urease-positive Helicobacter species (H. mustelae, H. pylori, H. acinonychis and H. hepaticus) for putative NikR-regulated promoters. One of these novel putative NikR-regulated promoters in H. mustelae is located upstream of a putative TonB-dependent outer membrane protein designated NikH, which displayed nickel-responsive expression. Insertional inactivation of the nikH gene in H. mustelae resulted in a significant decrease in urease activity, and this phenotype was complemented by nickel-supplementation of the growth medium, suggesting a function for NikH in nickel transport accross the outer membrane. In conclusion, the H. mustelae NikR regulator directly controls nickel-responsive regulation of ureases and metal transporters. The improved consensus NikR operator sequence allows the prediction of additional NikR targets in Helicobacter genomes, as demonstrated by the identification of a new nickel-repressed outer membrane protein in H. mustelae.
Keywords: Nickel-responsive gene regulation; Nickel uptake; NikR repressor; Helicobacter

Response of antioxidant defense system to chromium (VI)-induced cytotoxicity in human diploid cells by Nino Asatiani; Marina Abuladze; Tamar Kartvelishvili; Nina Kulikova; Lali Asanishvili; Hoi-Ying Holman; Nelly Sapojnikova (161-172).
The aim of this study is to establish antioxidant indicators of chromium toxicity in fetal human lung fibroblasts (HLF). The results obtained corroborate and develop our earlier observation of low-dose and long-term action of Cr(VI) on human cells in culture. In the case of a nontoxic chromium dose, temporary oxidative stress is overcome by increased activity of the antioxidant system with correlation to cell cycle re-entry. The toxic concentrations misbalance the cell antioxidant defense systems and cause irreversible growth arrest and massive cell death by apoptosis. Sub-toxicity is defined as toxicity stretched in time. The activity of GPx (glutathione peroxidase) is proposed as a biomarker of oxidative stress caused by Cr(VI), and the GR (glutathione reductase) inhibition is considered as a marker of the toxicity developed under the complex Cr(VI) action. In HLF cells the glutathione dependent defense system is the first system destroyed in response to toxic chromium action. Only the balance between SOD (superoxide dismutase) and H2O2 degrading enzymes (catalase and GPx), should play an important role in the fate of a cell, not individual enzymes.
Keywords: Chromium (VI); Antioxidant enzymes; Cell cycle; ROS; Apoptosis

Serum zinc is decreased in Alzheimer’s disease and serum arsenic correlates positively with cognitive ability by Larry Baum; Iris Hiu Shuen Chan; Stanley Kwok-Kuen Cheung; William B. Goggins; Vincent Mok; Linda Lam; Vivian Leung; Elsie Hui; Chelsia Ng; Jean Woo; Helen Fung Kum Chiu; Benny Chung-Ying Zee; William Cheng; Ming-Houng Chan; Samuel Szeto; Victor Lui; Joshua Tsoh; Ashley I. Bush; Christopher Wai Kei Lam; Timothy Kwok (173-179).
Zinc, copper, and iron aggregate Aβ and accumulate in Alzheimer’s disease (AD) plaques. Some metals are increased in AD vs. control serum. The authors examined levels of 12 metals in serum of 44 AD and 41 control subjects. Zinc decreased from 12.3 to 10.9 μmol/L (means, p = 0.0007). Arsenic positively correlated with Mini-Mental State Examination score (p < 0.0001). Zinc deposition in brain amyloid might deplete zinc from other body compartments, such as serum. The arsenic correlation might be caused by the major contribution of seafood consumption to intake of both arsenic and docosahexaenoic acid, of which the latter may delay AD.
Keywords: Alzheimer’s disease; Aluminum; Arsenic; Metals; MMSE; Serum; Zinc