BioMetals (v.24, #6)

Mechanisms underlying the protective effect of zinc and selenium against cadmium-induced oxidative stress in zebrafish Danio rerio by Mohamed Banni; Lina Chouchene; Khaled Said; Abdelhamid Kerkeni; Imed Messaoudi (981-992).
The present study was designed to elucidate the protective effect mechanism of Zinc (Zn) and Selenium (Se) on cadmium (Cd)-induced oxidative stress in zebrafish. For this purpose we investigate the response of oxidative stress markers, metallothionein accumulation and gene expression in liver and ovary of female zebrafish exposed to 0,4 mg/l Cd in water and supplemented with Zn (5 mg kg−1) and/or Se (2 mg kg−1) for 21 days in their diet. Liver and ovary Cd uptake was evaluated after the exposure period. Cd exposure significantly inhibited the antioxidant enzyme activities termed as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxydase (GPx) and caused a pronounced malondialdehyde (MDA) accumulation in both organs. Co-administration of Zn and Se reversed the Cd-induced toxicity in liver and ovary measured as MDA accumulation. Interestingly, gene expression patterns of Cat, CuZnSod and Gpx were up-regulated when related enzymatic activities were altered. Zebrafish metallothionein transcripts (zMt) significantly decreased in tissues of fish supplemented with Zn and/or Se when compared to Cd-exposed fish. Our data would suggest that Zn and Se protective mechanism against Cd-induced oxidative stress is more depending on the correction of the proteins biological activities rather than on the transcriptional level of related genes.
Keywords: Oxidative stress; Gene expression; Cd; Zn; Se; Zebrafish; Protective effect

Potent inhibition of protein tyrosine phosphatases by copper complexes with multi-benzimidazole derivatives by Ying Li; Liping Lu; Miaoli Zhu; Qingming Wang; Caixia Yuan; Shu Xing; Xueqi Fu; Yuhua Mei (993-1004).
A series of copper complexes with multi-benzimidazole derivatives, including mono- and di-nuclear, were synthesized and characterized by Fourier transform IR spectroscopy, UV–Vis spectroscopy, elemental analysis, electrospray ionization mass spectrometry. The speciation of Cu/NTB in aqueous solution was investigated by potentiometric pH titrations. Their inhibitory effects against human protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2), srchomology phosphatase 1 (SHP-1) and srchomology phosphatase 2 (SHP-2) were evaluated in vitro. The five copper complexes exhibit potent inhibition against PTP1B, TCPTP and PTP-MEG2 with almost same inhibitory effects with IC50 at submicro molar level and about tenfold weaker inhibition versus SHP-1, but almost no inhibition against SHP-2. Kinetic analysis indicates that they are reversible competitive inhibitors of PTP1B. Fluorescence study on the interaction between PTP1B and complex 2 or 4 suggests that the complexes bind to PTP1B with the formation of a 1:1 complex. The binding constant are about 1.14 × 106 and 1.87 × 106 M−1 at 310 K for 2 and 4, respectively.
Keywords: Copper complex; Protein tyrosine phosphatases; Inhibition; Fluorescence

Role of aluminum in red-to-blue color changes in Hydrangea macrophylla sepals by Henry D. Schreiber; Andrew H. Jones; Corinne M. Lariviere; Kelly M. Mayhew; Judith B. Cain (1005-1015).
Red, purple, and blue sepals on selected cultivars of Hydrangea macrophylla were analyzed for their aluminum content. This content was determined to be a function of the sepal color with red sepals possessing 0–10 μg Al/g fresh sepal, purple sepals having 10–40 μg Al/g fresh sepal, and blue sepals containing greater than 40 μg Al/g fresh sepal. Accordingly, the threshold aluminum content needed to change H. macrophylla sepals from red to blue was about 40 μg Al/g fresh sepal. Higher aluminum concentrations were incorporated into the sepals, but this additional aluminum did not affect the intensity or hue of the blue color. These observations agreed with a chemical model proposing that the concentration of the blue Al3+-anthocyanin complex reached a maximum when a sufficient excess of aluminum was present. In addition, the visible absorbance spectra of harvested red, purple, and blue sepals were duplicated by Al3+ and anthocyanin (delphinidin-3-glucoside) mixtures in this model chemical system.
Keywords: Aluminum; Bluing; Delphinidin-3-glucoside; Hydrangea macrophylla ; Sepal color

Compartmentalization and ultrastructural alterations induced by chromium in aquatic macrophytes by Pedro A. Mangabeira; Aluane S. Ferreira; Alex-Alan F. de Almeida; Valéria F. Fernandes; Emerson Lucena; Vânia L. Souza; Alberto J. dos Santos Júnior; Arno H. Oliveira; Marie F. Grenier-Loustalot; Fréderique Barbier; Delmira C. Silva (1017-1026).
The aim of the present study was to identify the sites of accumulation of Cr in the species of macrophytes that are abundant in the Cachoeira river, namely, Alternanthera philoxeroides, Borreria scabiosoides, Polygonum ferrugineum and Eichhornia crassipes. Plants were grown in nutritive solution supplemented with 0.25 and 50 mg l−1 of CrCl3·6H2O. Samples of plant tissues were digested with HNO3/HCl in a closed-vessel microwave system and the concentrations of Cr determined using inductively-coupled plasma mass spectrometry (ICP-MS). The ultrastructure of root, stem and leaf tissue was examined using transmission electron microscopy (TEM) and secondary ion mass spectrometry (SIMS) in order to determine the sites of accumulation of Cr and to detect possible alterations in cell organelles induced by the presence of the metal. Chromium accumulated principally in the roots of the four macrophytes (8.6–30 mg kg−1 dw), with much lower concentrations present in the stems and leaves (3.8–8.6 and 0.01–9.0 mg kg−1 dw, respectively). Within root tissue, Cr was present mainly in the vacuoles of parenchyma cells and cell walls of xylem and parenchyma. Alterations in the shape of the chloroplasts and nuclei were detected in A. philoxeroides and B. scabiosoides, suggesting a possible application of these aquatic plants as biomarkers from Cr contamination.
Keywords: Aquatic plants; Heavy metals; Chromium; Cell ultrastructure; Transmission electron microscopy; Secondary ion mass spectrometry

Supplementation of zinc mitigates the altered uptake and turnover of 65Zn in liver and whole body of diabetic rats by Ashima Pathak; Vishawjyoti Sharma; Sanjeev Kumar; D. K. Dhawan (1027-1034).
Diabetes is a life threatening disease and its onset is linked with both environmental and genetic factors. Zinc metabolism gets altered during diabetes and results in many complications. The present study was designed to elucidate the effects of zinc supplementation on the biokinetics of 65Zn in whole body, liver and its biodistribution in diabetic rats. The animals were divided into four groups viz; normal control; diabetic (single intraperitoneal injection of alloxan 150 mg/kg body weight); zinc treated (227 mg/l in drinking water); and diabetic + zinc treated. To carry out biokinetics study, each rat was injected intraperitoneally with 0.74 MBq radioactivity of 65Zn following 4 weeks of different treatments and the radioactivity was determined by using a suitably shielded scintillation counter. Alloxan induced diabetic rats showed a significant decrease in both the fast (Tb1) and slow (Tb2) components of biological half-life of 65Zn which, however, were normalized in whole body (P > 0.05) following zinc supplementation. In case of liver, Tb2 component was brought back to the normal but Tb1 component was not increased significantly. The present study indicates that the paucity of zinc in the tissues of the diabetic animals was due to decreased retention of tissue zinc as evidenced by increased serum Zn, hyperzincuria and increased rate of uptake of 65Zn by the liver. Zinc supplementation caused a significant improvement in the retention of zinc in the tissues and is therefore likely to be of benefit in the treatment of diabetes.
Keywords: Diabetes; Zinc supplementation; Biokinetics of 65Zn; 65Zn uptake; Hyperzincuria

Artemia is a tiny marine crustacean, serves as an excellent tool in both basic and applied aspects of stress biology research. In the current manuscript, we report that Artemia parthenogenetica embryos (cysts), in diapause stage, undergo iron transition changes when exposed to chemical diapause deactivation stimulus (hydrogen peroxide). X-ray surface analysis of A. parthenogenetica embryos exposed to H2O2 showed significant transitional changes in iron, as seen in cyst cross-sections. Electron paramagnetic resonance study revealed that upon H2O2 exposure, increased nitric oxide (NO) production was observed in non-decapsulated cysts (ND), but not in decapsulated cysts (DC) (shell-removed cysts). Spin trapping studies also showed an increase in hydroxyl radical formation in NDs exposed to H2O2 through Fenton-like reaction. On the contrary, exposure of DCs to H2O2 did not induce hydroxyl radical formation. Taken together, results from the present study indicate a key role of cyst shell-bound iron and reactive oxygen species on successful diapause termination in eukaryotic extremophile animal model, such as Artemia.
Keywords: Artemia parthenogenetica ; Diapause; Hydrogen peroxide; Nitric oxide; Shell-bound Iron

Acute and chronic metal exposure impairs locomotion activity in Drosophila melanogaster: a model to study Parkinsonism by Leonardo Bonilla-Ramirez; Marlene Jimenez-Del-Rio; Carlos Velez-Pardo (1045-1057).
The biometals iron (Fe), manganese (Mn) and copper (Cu) have been associated to Parkinson’s disease (PD) and Parkinsonism. In this work, we report for the first time that acute (15 mM for up to 5 days) or chronic (0.5 mM for up to 15 days) Fe, Mn and Cu exposure significantly reduced life span and locomotor activity (i.e. climbing capabilities) in Drosophila melanogaster. It is shown that the concentration of those biometals dramatically increase in Drosophila’s brain acutely or chronically fed with metal. We demonstrate that the metal accumulation in the fly’s head is associated with the neurodegeneration of several dopaminergic neuronal clusters. Interestingly, it is found that the PPL2ab DAergic neuronal cluster was erode by the three metals in acute and chronic metal exposure and the PPL3 DAergic cluster was also erode by the three metals but in acute metal exposure only. Furthermore, we found that the chelator desferoxamine, ethylenediaminetetraacetic acid, and d-penicillamine were able to protect but not rescue D. melanogaster against metal intoxication. Taken together these data suggest that iron, manganese and copper are capable to destroy DAergic neurons in the fly’s brain, thereby impairing their movement capabilities. This work provides for the first time metal-induced Parkinson-like symptoms in D. melanogaster. Understanding therefore the effects of biometals in the Drosophila model may provide insights into the toxic effect of metal ions and more effective therapeutic approaches to Parkinsonism.
Keywords: Copper; Drosophila melanogaster ; Manganese; Parkinson; Parkinsonism; Iron

Pseudomonas siderophores in the sputum of patients with cystic fibrosis by Lois W. Martin; David W. Reid; Katrina J. Sharples; Iain L. Lamont (1059-1067).
The lungs of patients with cystic fibrosis become chronically infected with the bacterium Pseudomonas aeruginosa, which heralds progressive lung damage and a decline in health. Iron is a crucial micronutrient for bacteria and its acquisition is a key factor in infection. P. aeruginosa can acquire this element by secreting pyoverdine and pyochelin, iron-chelating compounds (siderophores) that scavenge iron and deliver it to the bacteria. Siderophore-mediated iron uptake is generally considered a key factor in the ability of P. aeruginosa to cause infection. We have investigated the amounts of pyoverdine in 148 sputum samples from 36 cystic fibrosis patients (30 infected with P. aeruginosa and 6 as negative controls). Pyoverdine was present in 93 samples in concentrations between 0.30 and 51 μM (median 4.6 μM) and there was a strong association between the amount of pyoverdine and the number of P. aeruginosa present. However, pyoverdine was not present, or below the limits of detection (~0.3 μM), in 21 sputum samples that contained P. aeruginosa. Pyochelin was also absent, or below the limits of detection (~1 μM), in samples from P. aeruginosa-infected patients with little or no detectable pyoverdine. Our data show that pyoverdine is an important iron-scavenging molecule for P. aeruginosa in many cystic fibrosis patients, but other P. aeruginosa iron-uptake systems must be active in some patients to satisfy the bacterial need for iron.
Keywords: Pyoverdine; Pyochelin; Iron and infection; Pseudomonas aeruginosa ; Cystic fibrosis

Lactoferricin B (LfcinB), a 25 residue peptide derived from the N-terminal of bovine lactoferrin (bLF), causes depolarization of the cytoplasmic membrane in susceptible bacteria. Its mechanism of action, however, still needs to be elucidated. In the present study, synthetic LfcinB (without a disulfide bridge) and LfcinB (C–C; with a disulfide bridge) as well as three derivatives with 15-, 11- and 9-residue peptides were prepared to investigate their antimicrobial nature and mechanisms. The antimicrobial properties were measured via minimum inhibitory concentration (MIC) determinations, killing kinetics assays and synergy testing, and hemolytic activities were assessed by hemoglobin release. Finally, the morphology of peptide-treated bacteria was determined by atomic force microscopy (AFM). We found that there was no difference in MICs between LfcinB and LfcinB (C–C). Among the derivatives, only LfcinB15 maintained nearly the same level as LfcinB, in the MIC range of 16–128 μg/ml, and the MICs of LfcinB11 (64–256 μg/ml) were 4 times more than LfcinB, while LfcinB9 exhibited the lowest antimicrobial activity. When treated at MIC for 1 h, many blebs were formed and holes of various sizes appeared on the cell surface, but the cell still maintained its integrity. This suggested that LfcinB had a major permeability effect on the cytoplasmic membrane of both Gram-positive and Gram-negative bacteria, which also indicated it may be a possible intracellular target. Among the tested antibiotics, aureomycin increased the bactericidal activity of LfcinB against E. coli, S. aureus and P. aeruginosa, but neomycin did not have such an effect. We also found that the combination of cecropin A and LfcinB had synergistic effects against E. coli.
Keywords: Bovine lactoferricin; Antimicrobial properties; Hemolytic activity; Mechanism of action; Synergistic effects

Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus by Martina Höckner; Karin Stefanon; Annette de Vaufleury; Freddy Monteiro; Sílvia Pérez-Rafael; Òscar Palacios; Mercè Capdevila; Sílvia Atrian; Reinhard Dallinger (1079-1092).
Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C. aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80–90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30–50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.
Keywords: Cadmium; Copper; Zinc; Metallothionein; Mollusca; Pulmonata

Metal chelation therapy in rheumathoid arthritis: a case report by Fabrizia Bamonti; Alessandro Fulgenzi; Cristina Novembrino; Maria Elena Ferrero (1093-1098).
Toxic metals are involved in the pathogenesis of some neurodegenerative and vascular diseases and are known to impair the immune system functions. We report here the case of a patient affected by heavy metal intoxication, who had developed an autoimmune disease. There was evidence of aluminium, cadmium and lead intoxication in a 63-year old Italian woman affected by rheumatoid arthritis (RA). We treated the patient with calcium disodium edetate (EDTA) once a week for a year in order to remove traces of heavy metal intoxication. Oxidative status profile was carried out at the beginning and after 6 months’ EDTA chelation. At the end of the treatment, the patient did not show any signs of metal intoxication, RA symptoms and oxidative status improved.
Keywords: Chelation therapy; Rheumathoid arthritis; EDTA; Metal intoxication

Iron, chromium, nickel, and manganese released from gas-atomized AISI 316L stainless steel powders (sized <45 and <4 μm) were investigated in artificial lysosomal fluid (ALF, pH 4.5) and in solutions of its individual inorganic and organic components to determine its most aggressive component, elucidate synergistic effects, and assess release mechanisms, in dependence of surface changes using atomic absorption spectroscopy, Raman, XPS, and voltammetry. Complexation is the main reason for metal release from 316L particles immersed in ALF. Iron was mainly released, while manganese was preferentially released as a consequence of the reduction of manganese oxide on the surface. These processes resulted in highly complexing media in a partial oxidation of trivalent chromium to hexavalent chromium on the surface. The extent of metal release was partially controlled by surface properties (e.g., availability of elements on the surface and structure of the outermost surface) and partially by the complexation capacity of the different metals with the complexing agents of the different media. In general, compared to the coarse powder (<45 μm), the fine (<4 μm) powder displayed significantly higher released amounts of metals per surface area, increased with increased solution complexation capacity, while less amounts of metals were released into non-complexing solutions. Due to the ferritic structure of lower solubility for nickel of the fine powder, more nickel was released into all solutions compared with the coarser powder.
Keywords: Stainless steel; Powder; Complexation; Metal release; Dissolution; Inhalation

Interaction of inorganic mercury with CoA-SH and acyl-CoAs by Robert Gradinaru; Alin Ionas; Aurel Pui; Gheorghita Zbancioc; Gabi Drochioiu (1115-1121).
Sulfur containing biomolecules are involved in complexes with mercury. CoA is an important cofactor for many enzymes involved in metabolic processes. Fatty acyl-CoA-thioesters, substrates of mitochondrial ß-oxidation, are sulfur containing compounds and potential mercury ligands. The CoA-Hg2+ complex can be easily assessed by UV-Vis spectroscopy or indirectly by antibacterial tests that reconfirmed the protective role of CoA on E. coli. The characteristics of these complexes were determined by means of FTIR spectroscopy. The reverse phase liquid chromatography combined with electrospray ionization tandem mass spectrometry was used for detection of the side-product that resulted through the cleavage of thioesters in the presence of mercury. An unexpected result was the detection of octathioic acid as a product. Our study shows that mitochondrial β-oxidation can be affected by thioesters depletion assisted by Hg2+. The GC-MS technique could be used to detect some possible mitochondrial injuries due to the heavy metal ions.
Keywords: Mercury; Coenzyme A; E. coli ; Fatty acid thioesters; FTIR; GC-MS

Effect of diquat-induced oxidative stress on iron metabolism in male Fischer-344 rats by Masashi Higuchi; Yasunaga Yoshikawa; Koichi Orino; Kiyotaka Watanabe (1123-1131).
Diquat toxicity causes iron-mediated oxidative stress; however, it remains unclear how diquat affects iron metabolism. Here, we examined the effect of diquat-induced oxidative stress on iron metabolism in male Fischer-344 rats, with particular focus on gene expression. Hepatic nonheme iron content was unchanged until 20 h after diquat treatment. Hepatic free iron levels increased markedly in the early stages following treatment and remained elevated for at least 6 h, resulting in severe hepatotoxicity, until returning to control levels at 20 h. The level of hepatic ferritin, especially the H-subunit, increased 20 h after diquat treatment due to elevated hepatic ferritin-H mRNA expression. These results indicate that early elevated levels of free iron in the liver of diquat-treated rats cause hepatotoxicity, and that this free iron is subsequently sequestered by ferritin synthesized under conditions of oxidative stress, thus limiting the pro-oxidant challenge of iron. The plasma iron concentration decreased at 6 and 20 h after diquat treatment, whereas the level of plasma interleukin-6 increased markedly at 3 h and remained high until 20 h. In the liver of diquat-treated rats, expression of hepcidin mRNA was markedly upregulated at 3 and 6 h, whereas ferroportin mRNA expression was downregulated slightly at 20 h. Transferrin receptor 1 mRNA expression was significantly upregulated at 3, 6, and 20 h. These results indicate that inhibition of iron release from iron-storage tissues, through stimulation of the interleukin-6-hepcidin-ferroportin axis, and enhanced iron uptake into hepatocytes, mediated by transferrin receptor 1, cause hypoferremia.
Keywords: Diquat; Iron metabolism; Oxidative stress; Rat

Heavy metal resistance in Cupriavidus metallidurans CH34 is governed by an intricate transcriptional network by Pieter Monsieurs; Hugo Moors; Rob Van Houdt; Paul J. Janssen; Ann Janssen; Ilse Coninx; Max Mergeay; Natalie Leys (1133-1151).
The soil bacterium Cupriavidus metallidurans CH34 contains a high number of heavy metal resistance genes making it an interesting model organism to study microbial responses to heavy metals. In this study the transcriptional response of strain CH34 was measured when challenged to sub-lethal concentrations of various essential or toxic metals. Based on the global transcriptional responses for each challenge and the overlap in upregulated genes between different metal responses, the sixteen metals were clustered in three groups. In addition, the transcriptional response of already known metal resistance genes was assessed, and new metal response gene clusters were identified. The majority of the studied metal response loci showed similar expression profiles when cells were exposed to different metals, suggesting complex interplay at transcriptional level between the different metal responses. The pronounced redundancy of these metal resistant regions—as illustrated by the large number of paralogous genes—combined with the phylogenetic distribution of these metal response regions within either evolutionary related or other metal resistant bacteria, provides important insights on the recent evolutionary forces shaping this naturally soil-dwelling bacterium into a highly metal-resistant strain well adapted to harsh and anthropogenic environments.
Keywords: Transcriptomics; Cupriavidus metallidurans CH34; Transcriptional regulation; Metal resistance; Regulatory networks

Cyto- and genotoxicity of a vanadyl(IV) complex with oxodiacetate in human colon adenocarcinoma (Caco-2) cells: potential use in cancer therapy by Ana L. Di Virgilio; Josefina Rivadeneira; Cecilia I. Muglia; Miguel A. Reigosa; Nataliya Butenko; Isabel Cavaco; Susana B. Etcheverry (1153-1168).
The complex of vanadyl(IV) cation with oxodiacetate, VO(oda) caused an inhibitory effect on the proliferation of the human colon adenocarcinoma cell line Caco-2 in the range of 25–100 μM (P < 0.001). This inhibition was partially reversed by scavengers of free radicals. The difference in cell proliferation in the presence and the absence of scavengers was statistically significant in the range of 50–100 μM (P < 0.05). VO(oda) altered lysosomal and mitochondria metabolisms (neutral red and MTT bioassays) in a dose–response manner from 10 μM (P < 0.001). Morphological studies showed important transformations that correlated with the disassembly of actin filaments and a decrease in the number of cells in a dose response manner. Moreover, VO(oda) caused statistically significant genotoxic effects on Caco-2 cells in the low range of concentration (5–25 μM) (Comet assay). Increment in the oxidative stress and a decrease in the GSH level are the main cytotoxic mechanisms of VO(oda). These effects were partially reversed by scavengers of free radicals in the range of 50–100 μM (P < 0.05). Besides, VO(oda) interacted with plasmidic DNA causing single and double strand cleavage, probably through the action of free radical species. Altogether, these results suggest that VO(oda) is a good candidate to be evaluated for alternative therapeutics in cancer treatment.
Keywords: Vanadyl(IV) cation; Multidentate ligands; Cytotoxicity; Genotoxicity; Caco-2 tumoral cells

To account for the observed anticancer properties of plant polyphenols, we have earlier proposed a mechanism which involves the mobilization of endogenous copper ions by polyphenols leading to the generation of reactive oxygen species (ROS) that serve as proximal DNA cleaving agents and lead to cell death. Over the last decade we have proceeded to validate our hypothesis with considerable success. As a further confirmation of our hypothesis, in this paper we first show that oral administration of copper to rats leads to elevated copper levels in lymphocytes. When such lymphocytes with a copper overload were isolated and treated with polyphenols EGCG, genistein and resveratrol, an increased level of DNA breakage was observed. Further, preincubation of lymphocytes having elevated copper levels with the membrane permeable copper chelator neocuproine, resulted in inhibition of polyphenol induced DNA degradation. However, membrane impermeable chelator of copper bathocuproine, as well as iron and zinc chelators were ineffective in causing such inhibition in DNA breakage, confirming the involvement of endogenous copper in polyphenol induced cellular DNA degradation. It is well established that serum and tissue concentrations of copper are greatly increased in various malignancies. In view of this fact, the present results further confirm our earlier findings and strengthen our hypothesis that an important anticancer mechanism of plant polyphenols could be the mobilization of intracellular copper leading to ROS-mediated cellular DNA breakage. In this context, it may be noted that cancer cells are under considerable oxidative stress and increasing such stress to cytotoxic levels could be a successful anticancer approach.
Keywords: Cancer chemoprevention; Copper; EGCG; Genistein; Polyphenols; Prooxidant DNA breakage

Glutathione: a key component of the cytoplasmic labile iron pool by Robert C. Hider; Xiao L. Kong (1179-1187).
The cytoplasmic labile iron pool supplies iron to the mitochondrion for heme and iron sulfur cluster synthesis and to many cytoplasmic enzymes, thereby controlling numerous metabolic reactions. Surprisingly the chemical nature of this pool has never been convincingly characterised. Here we provide evidence for iron(II)glutathione being the dominant component of this pool. We report for the first time the affinity constant for the glutathione–iron(II) interaction and use this value to study the cytoplasmic speciation of iron(II). The formation of this complex is a major determinant of the electrode potential of the cytoplasmic ferrous iron pool, a means of selecting between iron(II) and manganese(II) and it provides a substrate for glutaredoxin/iron clusters at the dimer interface of glutaredoxins involved in the synthesis of Fe–S cluster proteins.
Keywords: Iron; Glutathione; Cytoplasmic labile iron pool

The metal-coordinated Casiopeína IIIEa induces the petite-like phenotype in Saccharomyces cerevisiae by Angélica López-Rodríguez; Alfonso Cárabez-Trejo; Fernando Rosas-Sánchez; Carmen Mejía; Lena Ruiz-Azuara; Ricardo Miledi; Ataúlfo Martínez-Torres (1189-1196).
The Casiopeínas® are mixed chelate copper (II) complexes and promising antineoplastics agents against cancer cells and tumors in vitro and in vivo. However, the action mode of these compounds is poorly characterized. In this work the effect of the antineoplastic Casiopeína IIIEa on the metabolism and ultrastructure of the yeast Saccharomyces cerevisiae was investigated. Exposure of cells growing in rich or in low-iron medium to 5 μM of the compound decreased duplication time and reduced oxygen consumption. Those cells formed smaller colonies when growing in a non-fermentable carbon source and low-iron medium, and under the light microscope, multiple folds were observed along the plasma membrane accompanied with a reduction in the diameter of the yeast. These observations were confirmed under the electron microscope, which also revealed a slight reduction of the mitochondrial size. A correlation was found with smaller colonies exhibiting lower rates of oxygen consumption, and yeast labelled with fluorescent MitoTrackerTM consistently exhibited reduced mitochondrial activity. It appears that Casiopeína IIIEa gives rise to smaller yeast and petite-like colonies by reducing the mitochondrial respiratory activity without significantly affecting the mitochondrial structure.
Keywords: Yeast; Petite colonies; Mitochondria; Respiratory activity; Casiopeínas; Copper compounds

Uranium (U) as a redox-active heavy metal can cause various redox imbalances in plant cells. Measurements of the cellular glutathione/glutathione disulfide (GSH/GSSG) by HPLC after cellular U contact revealed an interference with this essential redox couple. The GSH content remained unaffected by 10 μM U whereas the GSSG level immediately increased. In contrast, higher U concentrations (50 μM) drastically raised both forms. Using the Nernst equation, it was possible to calculate the half-cell reduction potential of 2GSH/GSSG. In case of lower U contents the cellular redox environment shifted towards more oxidizing conditions whereas the opposite effect was obtained by higher U contents. This indicates that U contact causes a consumption of reduced redox equivalents. Artificial depletion of GSH by chlorodinitrobenzene and measuring the cellular reducing capacity by tetrazolium salt reduction underlined the strong requirement of reduced redox equivalents. An additional element of cellular U detoxification mechanisms is the complex formation between the heavy metal and carboxylic functionalities of GSH. Because two GSH molecules catalyze electron transfers each with one electron forming a dimer (GSSG) two UO2 2+ are reduced to each UO2 + by unbound redox sensitive sulfhydryl moieties. UO2 + subsequently disproportionates to UO2 2+ and U4+. This explains that in vitro experiments revealed a reduction to U(IV) of only around 33% of initial U(VI). Cellular U(IV) was transiently detected with the highest level after 2 h of U contact. Hence, it can be proposed that these reducing processes are an important element of defense reactions induced by this heavy metal.
Keywords: Uranium; GSH/GSSG; Half-cell reduction potential; Redox process; Complex formation

The effect of heavy metal cations on the mitochondrial ornithine/citrulline transporter was tested in proteoliposomes reconstituted with the protein purified from rat liver. The transport activity was measured as [3H]ornithine uptake in proteoliposomes containing internal ornithine (ornithine/ornithine antiport mode) or as [3H]ornithine efflux in the absence of external substrate (ornithine/H+ transport mode). 0.1 mM Cu2+, Pb2+, Hg2+, Cd2+ and Zn2+ strongly inhibited (more than 85%) the antiport; whereas Mn2+, Co2+ and Ni2+ inhibited less efficiently (25, 47 and 69%, respectively). The IC50 values of the transporter for the different metal ions ranged from 0.71 to 350 μM. Co2+ and Ni2+ also inhibited the [3H]ornithine efflux whereas Cu2+, Pb2+, Hg2+, Cd2+ and Zn2+ stimulated the [3H]ornithine efflux. The stimulation of the [3H]ornithine efflux by Cu2+ and Cd2+ (as well as by Pb2+, Hg2+ and Zn2+) was not prevented by NEM and was reversed by DTE. These features indicated that the inhibition of the antiport was due to the interaction of the Cu2+, Pb2+, Hg2+, Cd2+ and Zn2+ with a population of SH groups, of the transporter, responsible for the inhibition of the physiological function; whereas the stimulation of [3H]ornithine efflux was due to the induction of a pore-like function of the transporter caused by interaction of cations with a different population of SH groups. Differently, the inhibition of the ornithine transporter by Ni2+, Co2+ or Mn2+ was caused by interaction with the substrate binding site, as indicated by the competitive or mixed inhibition.
Keywords: Transport; Heavy metals; Liposomes; Ornithine

Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron by Aya Usami; Masakazu Tanaka; Yasunaga Yoshikawa; Kiyotaka Watanabe; Hiromichi Ohtsuka; Koichi Orino (1217-1224).
Bovine milk α-casein was identified as a ferritin-binding protein, and ferritin is known to be a heme-binding protein. In this study, we found that the binding of α-casein to bovine spleen ferritin in vitro was blocked by hemin, but not by iron-free hemin (protoporphyrin IX) or zinc-protoporphyrin IX, suggesting that the presence of iron in heme play a key role in this interaction. Indeed, the binding of α-casein to ferritin and biotinylated hemin was inhibited by adding excess ferrous ammonium sulfate (FAS). To further elucidate the binding mechanism of α-casein to biotinylated hemin, Ferrozine and nitrilotriacetic acid (NTA) were used as ferrous and ferric iron chelators, respectively. FAS-mediated inhibition of α-casein to biotinylated hemin was neutralized with Ferrozine, but not NTA, while FAS- as well as ferric chloride-mediated inhibition in their interaction was neutralized by NTA. The following ions also inhibited α-casein-biotinylated hemin binding in order of potency of inhibition: FAS (Fe2+) ≪ ferric chloride (Fe3+) < copper sulfate (Cu2+) < zinc sulfate (Zn2+) < manganese chloride (Mn2+) < calcium chloride (Ca2+) < magnesium sulfate (Mg2+). These results suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-casein to iron in the heme on the surface of ferritin molecule, and that α-casein preferentially binds Fe2+ compared with any other metal ions, including Fe3+.
Keywords: α-Casein; Ferritin; Heme; Iron; Milk