BioMetals (v.21, #2)

Mercuric ion resistance in bacteria requires transport of mercuric ions (Hg2+) into the cytoplasmic compartment where they are reduced to the less toxic metallic mercury (Hg0) by mercuric reductase (MR). The long-established model for the resistance mechanism predicts interactions between the inner membrane mercuric ion transporter, MerT, and the N-terminal domain of cytoplasmic MR, but attempts to demonstrate this interaction have thus far been unsuccessful. A recently developed bacterial two-hybrid protein interaction detection system was used to show that the N-terminal region of MR interacts with the cytoplasmic face of MerT. We also show that the cysteine residues on the cytoplasmic face of the MerT protein are required for maximal mercuric ion transport but not for the interaction with mercuric reductase.
Keywords: Protein–Protein interactions; Mercuric ion transport; Mercuric ion reductase

Metal Cu(II) and Zn(II) bipyridyls as inhibitors of lactate dehydrogenase by Raj Kumar Koiri; Surendra Kumar Trigun; Santosh Kumar Dubey; Santosh Singh; Lallan Mishra (117-126).
Metal complex–protein interaction is an evolving concept for determining cellular targets of metallodrugs. Lacatate dehydrogenase (LDH) is critically implicated in tumor growth and therefore, considered to be an important target protein for anti-tumor metal complexes. Due to efficient biocompatibility of copper (Cu2+) and zinc (Zn2+), we synthesized CubpyAc2 · H2O (Cu-bpy) and ZnbpyAc2 · H2O (Zn-bpy; where bpy = 2,2′ bipyridine, Ac = CH3COO) complexes and evaluated their interaction with and modulation of LDH in mouse tissues. The increasing concentration of both the complexes showed a significant shift in UV–Vis spectra of LDH. The binding constant data (Kc = 1 × 103 M−1 for Cu-bpy and 7 × 106 M−1 for Zn-bpy) suggested that Zn-bpy-LDH interaction is stronger than that of Cu-bpy-LDH. LDH modulating potential of the complexes were monitored by perfusing the mice tissues with non-toxic doses of Cu-bpy and Zn-bpy followed by activity measurement and analysis of LDH isozymes on non-denaturing polyacrylamide gel electrophoresis (PAGE). As compared to the control sets, Cu-bpy caused a significant decline (P < 0.05–0.001) in the activity of LDH in all the tissues studied. However, Zn-bpy showed inhibition of LDH only in liver (P < 0.01), kidney (P < 0.001) and heart (P < 0.01), but with no effect in spleen, brain and skeletal muscle tissues. PAGE analysis suggested that all the five LDH isozymes are equally sensitive to both the complexes in the respective tissues. The results suggest that Cu- and Zn-bpy are able to interact with and inhibit LDH, a tumor growth supportive target protein at tissue level.
Keywords: Lactate dehydrogenase; Metal bipyridyl complexes; Glycolysis; Tumor growth; LDH isozymes

Sulfide increases labile iron pool in RD4 cells by Jonas Hälldin; Tiit Land (127-131).
A linkage between sulfur and iron metabolism has been suggested since sulfide has the ability to release iron from ferritin in the presence of iron acceptors in vitro. Nevertheless, this linkage is still lacking evidence in vivo as well as in cellular models. In this study we have treated human RD4 skeletal muscle cells with sodium sulfide and measured the level of the labile iron pool (LIP) as well as the intracellular sulfide concentration. We have also detected the amounts of L-ferritin protein as well as the iron regulatory protein 2 (IRP2). The sulfide treatment resulted in a 100% increase in the amount of LIP after 1 and 2 h. We also found that the raise of the LIP levels was coupled to an elevation of the amounts of intracellular sulfide that increased by 60%. The bioavailability of the released iron was confirmed by a 100% increase in L-ferritin protein as well as a 60% decrease of the IRP2 protein levels. These results suggest that there is a linkage between sulfur metabolism and intracellular iron regulation in mammalian cells.
Keywords: Sulfide; Ferritin; Labile iron pool; Iron regulation

Phytic acid was extracted from sweet potato (Ipomoea batatas) and fed to Wistar rats with or without zinc for 3 weeks. Animals were then sacrificed and bone and faecal minerals were assessed. The ultra-structure of the bones was examined via scanning electron microscopy. Phytic acid extract or commercial phytic acid supplemented diets (D + Zn + PE or D + PE) displayed reduced bone calcium levels (101.27 ± 59.11 and 119.27 ± 45.36 g/kg) compared to the other test groups. Similarly, reduced calcium were observed in the control groups (D + Zn and D) fed formulated diets with or without zinc supplementation (213.14 ± 15.31 and 210 ± 6.88 g/kg) compared to the other test groups. The group fed supplemented commercial phytic acid diet (D + CP) demonstrated the lowest femur magnesium (3.72 ± 0.13 g/kg) while the group fed phytic acid extract supplementation (D + PE) recorded the highest level (4.84 ± 0.26 g/kg) amongst the groups. Femur iron was highest in the group fed commercial phytic acid supplemented diet (D + CP −115.74 ± 2.41 g/kg) compared to the other groups. Faecal magnesium levels were significantly higher in the two test groups fed phytic acid extract with or without zinc (D + Zn + PE or D + PE) compared to all other groups. All the groups which had phytic acid supplemented diets had significantly thinner bone in the trabecular region, compared to the groups fed formulated diet or zinc supplemented formulated diet (D or D + Zn). These observations suggest that the consumption of foods high in phytic acid may contribute to a reduction in the minerals available for essential metabolic processes in rats.
Keywords: Phytic acid; Minerals; Bone; Sweet potato

Experimental study on the estrogen-like effect of mercuric chloride by Xiaojuan Zhang; Yadong Wang; Yingzheng Zhao; Xiaoyu Chen (143-150).
Although mercuric chloride has toxicity on reproductive system, it is uncertain if such toxicity is induced by estrogen-like effect. To study whether mercuric chloride has the estrogen-like effect and its relevant mechanism, proliferation assay of MCF-7 human breast cancer cells, uterotrophic assay, peroxidase activity assay and estrogen receptor competitive binding assay were conducted to screen the estrogen-like effect of mercuric chloride. The MCF-7 cells proliferated in the stimulation of mercuric chloride and got to the peak at 10−7 mol/l concentration. And this proliferation could be completely blocked by estrogenic antagonist ICI182.780. In addition, mercuric chloride could increase the weight of uterus of ovariectomized SD rats and the peroxidase activity of uterus complying with dose-effect relationship. However, mercuric chloride could not affect the binding of estradiol (E2) to estrogen receptor (ER). So mercuric chloride exhibits the estrogen-like effect through binding and activating ER rather than bind to ER by competing with E2.
Keywords: Mercuric chloride; Estrogen-like effect; Proliferation of MCF-7 cells; Uterotrophic assay; Peroxidase activity; ER competitive binding assay

Depletion of vesicular zinc in dorsal horn of spinal cord causes increased neuropathic pain in mice by Seung Mook Jo; Gorm Danscher; Henrik D. SchrØder; Sang Won Suh (151-158).
Zinc enriched (ZEN) neurons and terminals are abundant in the rodent spinal cord. Zinc ions have been suggested to modulate the excitability of primary afferent fibers believed to be important in nociceptive transmission. To test the hypothesis that vesicular zinc concentration is related to neuropathic pain we applied Chung’s rodent pain model on BALB/c mice, and traced zinc transporter 3 (ZnT3) proteins and zinc ions with immunohistochemistry and autometallography (AMG), respectively. Under anesthesia the left fifth lumbar spinal nerve was ligated in male mice in order to produced neuropathic pain. The animals were then sacrificed 5 days later. The ZnT3 immunoreactivity was found to have decreased significantly in dorsal horn of fourth, fifth, and sixth lumbar segments. In parallel with the depressed ZnT3 immunoreactivity the amount of vesicular zinc decreased perceptibly in superficial gray matters of especially layer I-IV of the same segments. The transection-induced reduction of vesicular zinc in ZEN terminals of the dorsal horn was synchronic to reduced pain threshold, as measured by von Frey method. In a separate study, we observed intensive zinc selenite precipitation in somata of the smaller spinal ganglion cell, but 5 days after spinal nerve transection zinc precipitation was also found in the lager ganglion cells. The present results indicate that zinc may be involved in pain mechanism in the spinal ganglion level. These results support the hypothesis that vesicular zinc might have a modulatory role for neuropathic pain. Thus, increased pain sensitivity might be related to reduce vesicular zinc level in the dorsal spinal gray matter.
Keywords: Zinc; Spinal cord; Pain; Zinc transporter; Autometallography

Iron-induced oxidative stress in haemodialysis patients: a pilot study on the impact of diabetes by Ann Van Campenhout; Christel Van Campenhout; Albert Lagrou; Begoña Manuel-y-Keenoy (159-170).
Background: Administration of intravenous iron preparations in haemodialysis patients may lead to the appearance of non-transferrin bound iron which can catalyse oxidative damage. We investigated this hypothesis by monitoring the oxidative stress of haemodialysis patients and the impact of iron and diabetes mellitus herein. Materials and methods: Baseline values of serum iron and related proteins, transferrin glycation, non-transferrin bound iron, antioxidant capacity and lipid peroxidation (malondialdehyde) of 11 haemodialysis patients (six non-diabetic and five type 2 diabetes) were compared to those of non-haemodialysis control subjects (non-diabetic and type 2 diabetes). Changes in these parameters were monitored during haemodialysis before and after iron administration. Results: Baseline values of malondialdehyde correlated with ferritin concentration (r = 0.664, P = 0.036) and were elevated to the same extent in non-diabetic and diabetic haemodialysis patients (median of 1.09 compared to 0.60 μmol/l in control persons, P < 0.02). After iron infusion, transferrin saturation increased more markedly in non-diabetic subjects from 28% to 185% vs. from 33% to 101% in diabetic patients (P = 0.008). This increase was accompanied by the appearance of non-transferrin bound iron (5.91 ± 1.33 μmol/l), a loss in plasma iron-binding antioxidant capacity and a further increase in malondialdehyde which was more pronounced in diabetic patients (from 0.93 ± 0.30 μmol/l to 2.21 ± 0.69 μmol/l vs. from 1.21 ± 0.42 μmol/l to 1.86 ± 0.56 μmol/l in the non-diabetic subjects, P = 0.046). Conclusions: In haemodialysis patients, higher lipid peroxidation is determined by higher body iron stores. The increase induced by iron infusion is accompanied by a loss in iron-binding antioxidant capacity and is more pronounced in diabetes mellitus.
Keywords: Haemodialysis; Iron; Oxidative stress; Diabetes

Liver iron overload induced by tamoxifen in diabetic and non-diabetic female Wistar rats by Carlos André Nunes Jatobá; Adriana Augusto de Rezende; Sarah Jane de Paiva Rodrigues; Maria Margareth de Almeida Câmara; Maria das Graças Almeida; Francisco Freire-Neto; Luiz Reginaldo Menezes da Rocha; Aldo Cunha da Medeiros; José Brandão-Neto; Maria Célia de Carvalho Formiga; Ítalo Medeiros de Azevedo; Ana Maria de Oliveira Ramos (171-178).
Tamoxifen (TX), a drug used in the treatment of breast cancer, may cause hepatic changes in some patients. The consequences of its use on the liver tissues of rats with or without diabetes mellitus (DM) have not been fully explored. The purpose of this study was to evaluate the correlation between plasma hepatic enzyme levels and the presence of iron overload in the hepatic tissue of female Wistar rats with or without streptozotocin-induced DM and using TX. Female rats were studied in control groups: C-0 (non-drug users), C-V (sorbitol vehicle only) and C-TX (using TX). DM (diabetic non-drug users) and DM-TX (diabetics using TX) were the test groups. Sixty days after induced DM, blood samples were collected for glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST) alkaline phosphatase (ALP) and bilirubin measures. Hepatic fragments were processed and stained with hematoxylin and eosin, Masson’s trichrome, Perls. The hepatic iron content was quantified by atomic absorption spectrometry. AST, ALT and ALP levels were significantly elevated in the DM and DM-TX groups, with unchanged bilirubin levels. Liver iron overload using Perls stain and atomic absorption spectrometry were observed exclusively in groups C-TX and DM-TX. There was positive correlation between AST, ALT and ALP levels and microscopic hepatic siderosis intensity in group DM-TX. In conclusion, TX administration is associated with liver siderosis in diabetic and non-diabetic rats. In addition, TX induced liver iron overload with unaltered hepatic function in non-diabetic rats and may be a useful tool for investigating the biological control of iron metabolism.
Keywords: Liver; Hemosiderin; Siderosis; Iron metabolism; Diabetes mellitus

Cadmium (Cd), a possible human carcinogen is a potent immunotoxicant. In rodents it causes thymic atrophy and splenomegaly, in addition to immuno-suppression and modulation of humoral and/or cellular immune response. Oxidative stress and apoptosis appear to be underlying mechanism of Cd induced thymic injury. To understand the involvement of reactive oxygen species (ROS), intracellular glutathione (GSH) and apoptosis in modulation of T-cell repertoire, we studied the effect of Cd (10, 25 and 50 μM) on primary T lymphocytes of BALB/c mice at different time intervals (6, 12 and 18 h). We observed a dose and time dependent decline in CD4+/CD8+ ratio (a bio-indicator of immunotoxicity) as a result of significant suppression of CD4+ subsets (helper T-cells) and enhancement in CD8+ cells (cytotoxic T-cells) At the same time, the CD4+CD8+ (DP) cell population was lowered while the CD4CD8 (DN) cells were increased. The oxidative stress and apoptotic data revealed almost similar ROS generation in both CD4+ and CD8+ cells, but relatively more marked GSH depletion and apoptosis in CD4+ than in CD8+ population. On further analysis of CD4+ T-subsets, cytokine release (IL-2 and IFNγ) by Th 1 cells and IL-4 by Th 2 cells were shown to be significantly suppressed in a dose responsive manner. The highest inhibition was observed in IFNγ, then IL-2 followed by IL-4. In conclusion, our data demonstrates that T-cell apoptosis by Cd, more in CD4+ than in CD8+ cells appear related to higher depletion of intracellular glutathione. Th 1 cells of CD4+ sub-population are more responsive to Cd than Th 2, leading to higher suppression of IL-2 and IFNγ than IL-4 and hence, the study unravels to some extend, the underlying events involved in Cd immunotoxicity.
Keywords: Cadmium; T-subsets; Apoptosis; Oxidative stress; Interleukin-2; Interleukin-4; Gamma-Interferon

Aggregation of the amyloid β-peptide (Aβ) into insoluble fibrils is a key pathological event in Alzheimer’s disease. Cu(II) and Zn(II) ions were reported to be able to induce Aβ aggregation at nearly physiological concentrations in vitro. In this study, the binding modes of Cu(II) and Zn(II) in this process were explored by molecular modeling. In the pre-associated Aβ, Nτ atom of imidazole ring of His14, O atom of carbonyl of main-chain and two O atoms of water occupied the four ligand positions of the complex. While in the aggregated form of Aβ, the His13(N)–Metals–His14(N) bridges were formed through metal cross-linking action. These results would be helpful to put insight on revealing the formation mechanism of pathogenic Aβ aggregates in brain.
Keywords: Molecular modeling; Amyloid β-peptide; Metal ions; Binding mode; Alzheimer’s disease

From the carbolithiation of 6-bis-N,N-dimethylamino fulvene (3a) and different ortho-lithiated heterocycles (furan, thiophene and N-methylpyrrole), the corresponding lithium cyclopentadienide intermediate (4a–c) was formed. These three lithiated intermediates underwent a transmetallation reaction with TiCl4 resulting in bis-N,N-dimethylamino-functionalised titanocenes 5a–c. When these titanocenes were tested against LLC-PK cells, the IC50-values obtained were of 240, and 270 μM for titanocenes 5b and 5c, respectively. The most cytotoxic titanocene in this paper, 5a with an IC50-value of 36 μM was found to be approximately six times less cytotoxic than its mono-N,N-dimethylamino substituted analogue Titanocene C (IC50 = 5.5 μM) and almost ten times less cytotoxic than cisplatin, which showed an IC50-value of 3.3 μM, when tested on the LLC-PK cell line. Bis-(bis- (N,N-dimethylamino)-2-(N′-methylpyrrolyl)methylcyclopentadienyl) titanium (IV) dichloride, {η5-C5H4-CH[N(CH3)2]2[C5H3NCH3]}2TiCl2 was synthesised starting from 6-bis-(N,N-dimethylamino) fulvene and 2-N-methylpyrrolyl lithium. Herein, we present the synthesis and DFT structure of the titanocene and two further derivatives followed by MTT-based cytotoxicity tests on pig kidney epithelial (LLC-PK) cells.
Keywords: Anti-cancer drugs; Cisplatin; Titanocene; Fulvene; LLC-PK

Adipogenic action of vanadium: a new dimension in treating diabetes by Ruchi Shukla; Ramesh R. Bhonde (205-210).
Vanadium is a well known anti-diabetic agent which mimics most of the actions of insulin on mature adipocytes. We report here the effect of vanadium on proliferation and differentiation of 3T3-L1 preadipocytes. Like insulin, vanadium treatment leads to increased proliferation as evidenced by H3thymidine uptake studies and differentiation of 3T3-L1 cells into adipocytes as evidenced by oil-red-O staining. Adipogenic potential of vanadium can be attributed to CREB activation, as documented by phospho-CREB antibody staining. This adipogenic potential is of significance in an in vivo scenario as the new adipocytes are likely to be insulin sensitive as against resistant existing mature adipocytes and thus indirectly may help in reduction of insulin resistance. Till today decrease in insulin resistance by vanadium treatment has been mainly attributed to its potential to inhibit PTP-1B, however the present study opens a new dimension in vanadium treatment for diabetes due to its novel role in adipogenesis.
Keywords: Vanadium; 3T3-L1 cells; Adipogenesis; CREB activation

Was improvement of spinach growth by nano-TiO2 treatment related to the changes of Rubisco activase? by Fengqing Gao; Chao Liu; Chunxiang Qu; Lei Zheng; Fan Yang; Mingyu Su; Fashui Hong (211-217).
Characterized by a photo—catalysis property, nano-anatase TiO2 is closely related to photosynthesis of spinach. It could not only improve light absorbance, transformation from light energy to electron energy and active chemical energy, but also promote the activity of Rubiso activase of spinach. However, the relation between the activity of Rubiso activase and the growth of spinach promoted by nano-anatase TiO2 treatment remains largely unclear. In this study, we find that the amount and the activity of Rubiso activase are obviously increased by nano-anatase TiO2 treatment, which led to the great promotion of Rubsico carboxylation and the high rate of photosynthesis, thus improving of spinach growth. The significant enhancement of Rubiso activase activity of nano-anatase TiO2 treated spinach is also accompanied by conformational changes as determined by spectroscopic analysis. But bulk TiO2 effect is not as significant as nano-anatase TiO2, as the grain size of nano-anatase TiO2 (5 nm) is much smaller than that of bulk TiO2, which entered spinach cell more easily.
Keywords: Nano-anatase TiO2 ; Spinach; Rubsico activase; Spectral property

Curium(III) complexation with pyoverdins secreted by a groundwater strain of Pseudomonas fluorescens by Henry Moll; Anna Johnsson; Mathias Schäfer; Karsten Pedersen; Herbert Budzikiewicz; Gert Bernhard (219-228).
Pyoverdins, bacterial siderophores produced by ubiquitous fluorescent Pseudomonas species, have great potential to bind and thus transport actinides in the environment. Therefore, the influence of pyoverdins secreted by microbes on the migration processes of actinides must be taken into account in strategies for the risk assessment of potential nuclear waste disposal sites. The unknown interaction between curium(III) and the pyoverdins released by Pseudomonas fluorescens (CCUG 32456) isolated from the granitic rock aquifers at the Äspö Hard Rock Laboratory (Äspö HRL), Sweden, is the subject of this paper. The interaction between soluble species of curium(III) and pyoverdins was studied at trace curium(III) concentrations (3 × 10−7 M) using time-resolved laser-induced fluorescence spectroscopy (TRLFS). Three Cm3+P. fluorescens (CCUG 32456) pyoverdin species, MpHqLr, could be identified from the fluorescence emission spectra, CmH2L+, CmHL, and CmL, having peak maxima at 601, 607, and 611 nm, respectively. The large formation constants, log β121 = 32.50 ± 0.06, log β111 = 27.40 ± 0.11, and log β101 = 19.30 ± 0.17, compared to those of other chelating agents illustrate the unique complexation properties of pyoverdin-type siderophores. An indirect excitation mechanism for the curium(III) fluorescence was observed in the presence of the pyoverdin molecules.
Keywords: Curium; Pyoverdin; Fluorescence spectroscopy; TRLFS; Complexation

Trace element balance is changed in infected organs during acute Chlamydophila pneumoniae infection in mice by Marie Edvinsson; Peter Frisk; Ylva Molin; Eva Hjelm; Nils-Gunnar Ilbäck (229-237).
Most infectious diseases are accompanied by changed levels of several trace elements in the blood. However, sequential changes in trace elements in tissues harbouring bacterial infections have not been studied. In the present study the respiratory pathogen Chlamydophila pneumoniae (C. pneumoniae), adapted to C57BL/6J mice, was used to study whether the balance of trace elements is changed in infected organs. Bacteria were quantitatively measured by real-time PCR in the blood, lungs, liver, aorta, and heart on days 2, 5, and 8 of the infection. Concentrations of 13 trace elements were measured in the liver, heart, and serum by inductively coupled plasma mass-spectrometry (ICP-MS). Infected mice developed expected clinical signs of disease and bacteria were found in lungs, liver, and heart on all days. The number of bacteria peaked on day 2 in the heart and on day 5 in the liver. The copper/zinc (Cu/Zn) ratio in serum increased as a response to the infection. Cu increased in the liver but did not change in the heart. Iron (Fe) in serum decreased progressively, whereas in the heart it tended to increase, and in the liver it progressively increased. C. pneumoniae may thus cause a changed trace element balance in target tissues of infection that may be pivotal for bacterial growth.
Keywords: Bacterial infection; Heart; Liver; Serum; Trace elements