BioMetals (v.23, #4)

Siderophore uptake in bacteria and the battle for iron with the host; a bird’s eye view by Byron C. Chu; Alicia Garcia-Herrero; Ted H. Johanson; Karla D. Krewulak; Cheryl K. Lau; R. Sean Peacock; Zoya Slavinskaya; Hans J. Vogel (601-611).
Siderophores are biosynthetically produced and secreted by many bacteria, yeasts, fungi and plants, to scavenge for ferric iron (Fe3+). They are selective iron-chelators that have an extremely high affinity for binding this trivalent metal ion. The ferric ion is poorly soluble but it is the form of iron that is predominantly found in oxygenated environments. Siderophore uptake in bacteria has been extensively studied and over the last decade, detailed structural information for many of the proteins that are involved in their transport has become available. Specifically, numerous crystal structures for outer membrane siderophore transporters, as well as for soluble periplasmic siderophore-binding proteins, have been reported. Moreover, unique siderophore-binding proteins have recently been serendipitously discovered in humans, and the structures of some of their siderophore-complexes have been characterized. The binding pockets for different ferric-siderophores in these proteins have been described in great molecular detail. In addition to highlighting this structural information, in this review paper we will also briefly discuss the relevant chemical properties of iron, and provide a perspective on our current understanding of the human and bacterial iron uptake pathways. Potential clinical uses of siderophores will also be discussed. The emerging overall picture is that iron metabolism plays an extremely important role during bacterial infections. Because levels of free ferric iron in biological systems are always extremely low, there is serious competition for iron and for ferric-siderophores between pathogenic bacteria and the human or animal host.
Keywords: Siderophore; Iron transport; Hepcidin; TonB; Host defense; Siderocalin

Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g|| = 2.24, A|| = 18.4 mT and g = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g|| = 2.29, A|| = 16.1 mT and g = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu2+ with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu2+ and three diamagnetic Cu1+ per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per αβγ-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe3+ was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu2+-EPR signal.
Keywords: Nitrosomonas europaea ; Soluble ammonia monooxygenase; Electron paramagnetic resonance; Copper and iron enzyme; Nitrogen oxide; Ferricyanide

Tellurite-induced oxidative stress leads to cell death of murine hepatocarcinoma cells by Juan M. Sandoval; Philippe Levêque; Bernard Gallez; Claudio C. Vásquez; Pedro Buc Calderon (623-632).
Data regarding tellurium (Te) toxicity are scarce. Studies on its metabolism, performed mainly in bacteria, underline a major role of reactive oxygen species (ROS). We investigated whether tellurite undergoes redox cycling leading to ROS formation and cancer cell death. The murine hepatocarcinoma Transplantable Liver Tumor (TLT) cells were challenged with tellurite either in the presence or in the absence of different compounds as N-acetylcysteine (NAC), 3-methyladenine, BAPTA-AM, and catalase. NAC inhibition of tellurite-mediated toxicity suggested a major role of oxidative stress. Tellurite also decreased both glutathione (GSH) and ATP content by 57 and 80%, respectively. In the presence of NAC however, the levels of such markers were almost fully restored. Tellurite-mediated ROS generation was assessed both by using the fluorescent, oxidation-sensitive probe dichlorodihydrofluorescein diacetate (DCHF-DA) and electron spin resonance (ESR) spectroscopy to detect hydroxyl radical formation. Cell death occurs by a caspase-independent mechanism, as shown by the lack of caspase-3 activity and no cleavage of poly(ADP-ribose)polymerase (PARP). The presence of γ-H2AX suggests tellurite-induced DNA strand breaking, NAC being unable to counteract it. Although the calcium chelator BAPTA-AM did show no effect, the rapid phosphorylation of eIF2α suggests that, in addition to oxidative stress, an endoplasmic reticulum (ER) stress may be involved in the mechanisms leading to cell death by tellurite.
Keywords: Cancer cell death; Necrosis; Oxidative stress; Tellurite

The chromate resistance phenotype of some yeast mutants correlates with a lower level of Cr(V)-species generated in the extra-cellular medium by Helena Ksheminska; Taras Honchar; Yuriy Usatenko; Galina Gayda; Mykhailo Gonchar (633-642).
The paper describes the selection of chromate-resistant mutants of the yeast Pichia guilliermondii with a higher chromate-reducing activity and reports the EPR-study of Cr(V)-generation in the extra-cellular medium during the reduction of chromate by the yeast culture. It is shown that the reduction of chromate to Cr(III) species runs through the extra-cellular generation of Cr(V)-intermediate(s), thus supporting the assumption about the existence of an extra-cellular pathway of Cr(VI)-reduction. Furthermore, it is demonstrated that the chromate-resistance phenotype of tested mutants correlates with a lower stationary level of Cr(V)-species in the medium. It is thus suggested that isolated mutants can be used as sources of Cr(III)-biocomplexes due to their ability to effectively reduce chromate to Cr(III)-chelates with potential pharmacological applications.
Keywords: Chromate reduction; Yeast; Chromium(V); Chromate-resistant mutants; Chromium(III)-biocomplexes

The ZIP (ZRT-, IRT-like Protein) protein ZupT from Escherichia coli is a transporter with a broad substrate range. Phenotypic and transport analysis showed that ZupT, in addition to Zn(II), Fe(II) and Co(II) uptake, is also involved in transport of Mn(II) and Cd(II). Competition experiments with other substrate cations suggested that ZupT has a slight preference for Zn(II) and kinetic parameters for Zn(II) in comparison to Co(II) and Mn(II) transport support this observation. Metal uptake into cells by ZupT was optimum at near neutral pH and inhibited by ionophores. Bicarbonate or other ions did not influence metal-uptake via ZupT. Amino acid residues of ZupT contributing to substrate specificity were identified by site directed mutagenesis. ZupT with a H89A exchange lost Co(II) and Fe(II) transport activity, while the S117V mutant no longer transported Mn(II). ZupT with E152D was impaired in overall metal uptake but completely lost its ability to transport the substrates Zn(II) and Mn(II). These experimental findings expand our knowledge on the substrate specificity of ZupT and provide further insight into the function of ZupT as a bacterial member of the vastly distributed and important ZIP family.
Keywords: Escherichia coli ; Metal; Transport; Site directed mutagenesis; ZIP protein

Deficiency in the divalent metal transporter 1 increases bleomycin-induced lung injury by Funmei Yang; Jacqueline G. Stonehuerner; Judy H. Richards; Ngoc-Bich Nguyen; Kimberly D. Callaghan; David J. Haile; Andrew J. Ghio (657-667).
Exposure to bleomycin can result in an inflammatory lung injury. The biological effect of this anti-neoplastic agent is dependent on its coordination of iron with subsequent oxidant generation. In lung cells, divalent metal transporter 1 (DMT1) can participate in metal transport resulting in control of an oxidative stress and tissue damage. We tested the postulate that metal import by DMT1 would participate in preventing lung injury after exposure to bleomycin. Microcytic anemia (mk/mk) mice defective in DMT1 and wild-type mice were exposed to either bleomycin or saline via intratracheal instillation and the resultant lung injury was compared. Twenty-four h after instillation, the number of neutrophils and protein concentrations after bleomycin exposure were significantly elevated in the mk/mk mice relative to the wild-type mice. Similarly, levels of a pro-inflammatory mediator were significantly increased in the mk/mk mice relative to wild-type mice following bleomycin instillation. Relative to wild-type mice, mk/mk mice demonstrated lower non-heme iron concentrations in the lung, liver, spleen, and splenic, peritoneal, and liver macrophages. In contrast, levels of this metal were elevated in alveolar macrophages from mk/mk mice. We conclude that DMT1 participates in the inflammatory lung injury after bleomycin with mk/mk mice having increased inflammation and damage following exposure. This finding supports the hypothesis that DMT1 takes part in iron detoxification and homeostasis in the lung.
Keywords: Iron; Lung diseases; Ferritin; Mouse

Exposure to lanthanum compound diminishes LPS-induced inflammation-associated gene expression: involvements of PKC and NF-κB signaling pathways by Fei Guo; Yuanlei Lou; Nianhua Feng; Guohui Li; An Xie; Xueming Huang; Yang Wang (669-680).
Lanthanum chloride, a rare earth compound, possesses antibacterial and cellular immunity regulating properties. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the effects of lanthanum chloride on the production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α), the expression of inducible NO synthase (iNOS) and TNF-α in RAW 264.7 cells, a mouse macrophage cell line. We found that the LPS-elicited excessive production of NO and TNF-α in RAW 264.7 cells was inhibited significantly in the presence of lanthanum chloride, and the attenuation of iNOS and TNF-α occurred at mRNA level. Furthermore, the possible signaling components affected by lanthanum chloride in the pathway that lead to LPS-induced iNOS and TNF-α expression were explored. The results indicated the involvements of PKC/Ca2+ and NF-κB in the attenuation of NO and pro-inflammatory cytokine production by lanthanum chloride. Our observations suggest a possible therapeutic application of this agent for treating inflammatory diseases.
Keywords: Lanthanum chloride; Inducible nitric oxide synthase (iNOS); Tumor necrosis factor-α (TNF-α); Nuclear factor-κ B (NF-κ B); Protein kinase C (PKC)

Conservation of copper-transporting P(IB)-type ATPase function by Adam Southon; Nickless Palstra; Nicholas Veldhuis; Ann Gaeth; Charles Robin; Richard Burke; James Camakaris (681-694).
Copper-transporting P(IB)-type ATPases are highly conserved, and while unicellular eukaryotes and invertebrates have only one, a gene duplication has occurred during vertebrate evolution. Copper-induced trafficking of mammalian ATP7A and ATP7B from the trans-Golgi Network towards the plasma membrane is critical for their role in copper homeostasis. In polarized epithelial cells ATP7A and ATP7B traffic towards the basolateral and apical membranes respectively. We examined the localization and function of DmATP7, the single Drosophila melanogaster orthologue, in cultured D. melanogaster and mammalian cells to explore the conservation of P(IB)-type ATPase function. Comparative genomic analysis demonstrated motifs involved in basolateral targeting and retention of ATP7A were conserved in DmATP7, whereas ATP7B targeting motifs were not. DmATP7 expression was able to correct the copper hyper-accumulation phenotype of cultured fibroblasts from a Menkes disease patient expressing a null ATP7A allele. DmATP7 was able to transport copper to the cupro-enzyme tyrosinase and under elevated copper conditions DmATP7 was able to traffic towards the plasma membrane and efflux copper, essentially phenocopying ATP7A. When expressed in polarized Madin-Darby Canine Kidney cells, DmATP7 translocated towards the basolateral membrane when exposed to elevated copper, similar to ATP7A. These results demonstrate DmATP7 is able to functionally compensate for the absence of ATP7A, with important trafficking motifs conserved in these distantly related orthologues.
Keywords: Menkes; Wilson; DmATP7; Copper; Drosophila

Testicular toxicity induced by dietary cadmium in cocks and ameliorative effect by selenium by Jin-Long Li; Rui Gao; Shu Li; Jin-Tao Wang; Zhao-Xin Tang; Shi-Wen Xu (695-705).
Cadmium (Cd) is an ubiquitous environmental pollutant that has been associated with male reproductive toxicity in animal models. However, little is known about the reproductive toxicity of Cd in birds. To investigate the toxicity of Cd on male reproduction in birds and the protective effects of selenium (Se) against subchronic exposure to dietary Cd, 100-day-old cocks received either Se (as 10 mg Na2SeO3 per kg of diet), Cd (as 150 mg CdCl2 per kg of diet) or Cd + Se in their diets for 60 days. Histological and ultrastructural changes in the testis, the concentrations of Cd and Se, amount of lipid peroxidation (LPO), the activities of the antioxidants superoxide dismutase (SOD) and glutathione peroxidase (GPx), and apoptosis and serum testosterone levels were determined. Exposure to Cd significantly lowered SOD and GPx activity, Se content in the testicular tissue, and serum testosterone levels. It increased the amount of LPO, the numbers of apoptotic cells and Cd concentration and caused obvious histopathological changes in the testes. Concurrent treatment with Se reduced the Cd-induced histopathological changes in the testis, oxidative stress, endocrine disorder and apoptosis, suggesting that the toxic effects of cadmium on the testes is ameliorated by Se. Se supplementation also modified the distribution of Cd in the testis.
Keywords: Cadmium; Selenium; Oxidative stress; Apoptosis; Cock testis

Clinical evaluation of Deferasirox for removal of cadmium ions in rat by Amir Shokooh Saljooghi; S. Jamil A. Fatemi (707-712).
An investigation was conducted to evaluate the ability of Deferasirox (ICL670 or Exjade) following the distribution of cadmium salt in male Wistar rats. Cadmium was introduced to several groups of weanling male Wistar rats through different means, by act of drinking, feeding. A control group was fed on a diet containing normal level of iron. After a period of 30 days, all the rats administered cadmium were severely anemic and showed toxicity symptoms through loss of hair and increasing in cadmium and reduction in iron levels in blood. Chelation therapy was carried out to remove the toxic element from the body. The ability of Deferasirox chelator in removing cadmium was investigated this chelator for 1 week to the remaining rats of similar groups. The results showed that the cadmium level present in blood was significantly reduced and at the same time, iron concentration returned to the normal level. It was concluded that Deferasirox chelator is able to remove cadmium from the body and could be used for the treatment of complications and eradication of symptoms of cadmium intoxication.
Keywords: Deferasirox; Cadmium toxicity; Chelation therapy; Rats

Studies of Cr(VI) toxicity are generally performed using chromate salts in solution, both when studying the effects on prokaryotes and eukaryotes. Some studies on human carcinogenesis and toxicology on bacteria were done using dichromate, but comparison with chromate was never reported before, and dichromate existence was never taken into consideration and usually overlooked. This paper studied comparatively the effect of dichromate and chromate on the physiology of Ochrobactrum tritici strain 5bvl1, a highly Cr(VI)-resistant and reducing microorganism. This study demonstrated that the addition of chromate or dichromate sodium salts to growth medium at neutral pH ended-up in two different solutions with a different balance of chemical species. Cr(VI) was toxic to O. tritici strain 5bvl1, as clearly shown on growth, reduction, respiration, glucose accumulation assays and by comparing cell morphology. Moreover, the addition of sodium dichromate was always more toxic to cells when compared to chromate and achieved a higher inhibition of every parameter studied. The toxicity differences between the two Cr(VI) oxyanions indicate the possibility of a different impact of Cr(VI) contamination on the environment. This may be of major importance, considering the slight acidity of most of the arable lands which favours the presence of dichromate, the more toxic species.
Keywords: Chromium; Chromate; Dichromate; Toxicity; Resistant bacteria

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction by Kouji Takeda; Junichi Sato; Kazuyuki Goto; Takanori Fujita; Toshihiro Watanabe; Mitsuru Abo; Etsuro Yoshimura; Junichi Nakagawa; Akira Abe; Shinji Kawasaki; Youichi Niimura (727-737).
Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP+ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k cat/K m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k cat/K m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.
Keywords: Ferredoxin-NADP+ reductase; Nitroreductase; Flavin reductase; Ferric reductase; Fenton reaction

A new ligand DBHIP and its two ruthenium(II) complexes [Ru(dmb)2(DBHIP)](ClO4)2 (1) and [Ru(dmp)2(DBHIP)](ClO4)2 (2) have been synthesized and characterized. The cytotoxicity of DBHIP and complexes 1 and 2 has been assessed by MTT assay. The apoptosis studies were carried out with acridine orange/ethidium bromide (AO/EB) staining methods. The binding behaviors of these complexes to calf thymus DNA (CT-DNA) were studied by absorption titration, viscosity measurements, thermal denaturation and photoactivated cleavage. The DNA-binding constants of complexes 1 and 2 were determined to be 8.64 ± 0.16 × 104 (s = 1.34) and 2.79 ± 0.21 × 104 (s = 2.17) M−1. The results suggest that these complexes interact with DNA through intercalative mode. The studies on the mechanism of photocleavage demonstrate that superoxide anion radical (O2 •–) and singlet oxygen (1O2) may play an important role in the DNA cleavage. The experiments on antioxidant activity show that these compounds also exhibit good antioxidant activity against hydroxyl radical (OH).
Keywords: Ruthenium(II) complexes; DNA; Cytotoxicity; Apoptosis; Antioxidant activity

Aluminium toxicity has been recognized as a primary growth-limiting factor in acid soil, resulting in a decrease in plant growth and production. In this experiment we have studied the induction of oxidative stress and changes in antioxidant gene expression in pea (var. ALASKA) under aluminium (Al) stress. We have found that Al treatment affected the growth of pea plant and induced oxidative stress with a change in antioxidant gene expression profile. While the expression of glutathione-s-transferase (GST) and catalase (CAT) was more in root, cytosolic Ascorbate peroxidase (cAPX) expression increased in shoot under aluminium stress. Copper- Zinc Superoxide dismutase (Cu-Zn SOD) gene expression was higher after 24 h but decreased after 48 h along with elevated expression of manganese superoxide dismutase (MnSOD) and iron-superoxide dismutase (FeSOD) at higher aluminium contentrations after 24 and 48 h. Aluminium stress elevated hydrogen peroxide (H2O2) level and affected the growth. The proline content did not change significantly, whereas glutathione content increased with a decreased ascorbate content under Al stress. The present study indicates that aluminium treatment affected the antioxidant gene expression and induced oxidative stress in pea plant.
Keywords: Aluminium; Oxidative stress; Antioxidants; Pea; Gene expression