BioMetals (v.23, #2)

Can iron be teratogenic? by E. D. Weinberg (181-184).
Several kinds of evidence indicate that elevated iron during the 3–8 week embryonic (organogenesis) period of human gestation may be teratogenic. (1) In the embryonic period, the natural maternal absorption of food iron is 30% below the estimated daily iron loss. (2) As compared with maternal serum, embryonic fetal coelomic fluid contains only one-fourth as much iron but nearly six times the quantity of the iron withholding protein, ferritin. (3) In the embryonic period, intraplacental oxygen pressure is 2–3 times lower than in the subsequent fetal growth period. (4) Iron is a strong inducer of emesis which peaks in the embryonic period. (5) In a murine gestation model, iron was neurotoxic at a sharp peak of 8–9 days. Thus it would be prudent, in human pregnancy, to delay any needed iron supplementation until the embryonic period has been completed.
Keywords: Embryonic period; Iron; Pregnancy; Teratogen

HIV therapeutic possibilities of gold compounds by Pascaline N. Fonteh; Frankline K. Keter; Debra Meyer (185-196).
Highly active antiretroviral therapy (HAART) has resulted in decreased mortality and morbidity from the acquired immune deficiency syndrome caused by the human immunodeficiency virus (HIV). Drug resistance and toxicity of HAART has led to the search for novel inhibitors of HIV infection. Gold-based compounds have shown promising activity against a wide range of clinical conditions and microorganism infections including HIV-1. A typical example is auranofin which resulted in an elevated CD4+ T-cell count in an HIV patient being treated for psoriatic arthritis. In addition, reports exist on gold-based inhibitors of reverse transcriptase (RT), protease (PR) and viral entry of host cells. These and other characteristics of gold-based HIV drugs are reviewed here.
Keywords: Gold; Inhibitors; HIV; AIDS

Potential of lithium to reduce aluminium-induced cytotoxic effects in rat brain by Punita Bhalla; Neha Singla; D. K. Dhawan (197-206).
The present study was aimed to explore the potential of an antidepressant drug lithium (Li) in reducing aluminium (Al) induced neurotoxicity. To carry out the investigations, Al was administered orally (100 mg AlCl3/Kg b wt/day) whereas, Li was administered through diet (1.1 g Li2CO3/Kg diet, daily) for a total duration of 2 months. Al treatment resulted in a significant increase in the activity of enzyme nitric oxide synthase and the levels of l-citrulline which, however, were decreased appreciably following lithium supplementation. Al treatment also revealed an increase in DNA fragmentation as evidenced by an increase in number of comets. Interestingly, Li supplementation to Al treated rats reduced the damage inflicted on DNA by Al. Ultrastructural studies revealed an increase in chromatin condensation with discontinuity in nuclear membrane in both the cerebrum and cerebellum of Al treated rats which showed improvement following Li supplementation. Alterations in the structure of synapse and mitochondrial swelling were also seen. The present study shows the potential of Li in containing the damage inflicted by Al on rat brain.
Keywords: Aluminium toxicity; DNA damage; Lithium; Nitric oxide; Ultrastructural studies

Arsenic accumulation and thiol status in lichens exposed to As(V) in controlled conditions by Tanja Mrak; Zvonka Jeran; Franc Batič; Luigi Sanità di Toppi (207-219).
Thalli of epiphytic lichen Hypogymnia physodes (L.) Nyl. and terricolous Cladonia furcata (Huds.) Schrad., collected from an area with background arsenic concentrations, were exposed to 0, 0.1, 1 and 10 μg mL−1 arsenate (As(V)) solutions for 24 h. After exposure they were kept in the metabolically active state for 0, 24 and 48 h in a growth chamber. In the freeze dried samples glutathione (GSH), glutathione disulphide (GSSG), cysteine (Cys) and cystine were analysed and induction of phytochelatin (PC) synthesis measured by reversed-phase high-performance liquid chromatography in combination with fluorescence detection or UV spectrometry. Total arsenic content in thalli was measured by instrumental neutron activation analysis (INAA). In H. physodes, which contained higher amounts of arsenic compared to C. furcata, total glutathione content significantly decreased in samples exposed to 10 μg mL−1 As(V), whereas in C. furcata a significant increase was observed. In both species PC synthesis was induced in thalli exposed to 10 μg mL−1.
Keywords: Lichens; Arsenic; Glutathione; Phytochelatins; Half-cell reduction potential; Hypogymnia physodes ; Cladonia furcata

The suitability of gallium as a substitute for aluminum in tracing experiments by Rachel C. Walton; Keith N. White; Francis Livens; Catherine R. McCrohan (221-230).
Aluminum is a toxic metal whose complex aquatic chemistry, mechanisms of toxicity and trophic transfer are not fully understood. The only isotope of Al suitable for tracing experiments in organisms—26Al—is a rare, costly radioisotope with a low emission energy, making its use difficult. Gallium shares a similar chemistry with Al and was therefore investigated as a potential substitute for Al for use in aquatic organisms. The freshwater snail, Lymnaea stagnalis was exposed to either Al or Ga (0.0135 mM) under identical conditions for up to 40 days. Behavioural toxicity, metal accumulation in the tissues, and sub-cellular partitioning of the metals were determined. Al was more toxic than Ga and accumulated to significantly higher levels in the soft tissues (P < 0.05). The proportion of Al in the digestive gland (DG; detoxificatory organ) relative to other tissues was significantly lower than that of Ga (P < 0.05) from day 14 onwards. There were also differences in the proportions of Al and Ga associated with heat stable proteins (HSPs) in the digestive gland, with significantly more HSP present in the DGs of snails exposed to Al, but significantly less Al than Ga associated with the HSP per unit mass protein present. From this evidence, we conclude that Ga may be of limited use as a tracer for Al in animal systems.
Keywords: Metal toxicity; Lymnaea stagnalis ; Aluminum; Gallium; Bioaccumulation; Freshwater snail; Aquatic toxicology

Effects of deferasirox and deferiprone on cellular iron load in the human hepatoma cell line HepaRG by François Gaboriau; Anne-Marie Leray; Martine Ropert; Lucie Gouffier; Isabelle Cannie; Marie-Bérengère Troadec; Olivier Loréal; Pierre Brissot; Gérard Lescoat (231-245).
Two oral chelators, CP20 (deferiprone) and ICL670 (deferasirox), have been synthesized for the purpose of treating iron overload diseases, especially thalassemias. Given their antiproliferative effects resulting from the essential role played by iron in cell processes, such compounds might also be useful as anticancer agents. In the present study, we tested the impact of these two iron chelators on iron metabolism, in the HepaRG cell line which allowed us to study proliferating and differentiated hepatocytes. ICL670 uptake was greater than the CP20 uptake. The iron depletion induced by ICL670 in differentiated cells increased soluble transferrin receptor expression, decreased intracellular ferritin expression, inhibited 55Fe (III) uptake, and reduced the hepatocyte concentration of the labile iron pool. In contrast, CP20 induced an unexpected slight increase in intracellular ferritin, which was amplified by iron-treated chelator exposure. CP20 also promoted Fe(III) uptake in differentiated HepaRG cells, thus leading to an increase of both the labile pool and storage forms of iron evaluated by calcein fluorescence and Perls staining, respectively. In acellular conditions, compared to CP20, iron removing ability from the calcein-Fe(III) complex was 40 times higher for ICL670. On the whole, biological responses of HepaRG cells to ICL670 treatment were characteristic of expected iron depletion. In contrast, the effects of CP20 suggest the potential involvement of this compound in the iron uptake from the external medium into the hepatocytes from the HepaRG cell line, therefore acting like a siderophore in this cell model.
Keywords: Iron chelation; Iron metabolism; Siderophore; Hepatocytes; HepaRG cell line; Cell proliferation

Thiamine reduces tissue lead levels in rats: mechanism of interaction by Srinivasa Y. Reddy; Raghu Pullakhandam; B. Dinesh Kumar (247-253).
Lead (Pb) toxicity has been a serious concern in industrialized societies because of its association with functional deficits in nervous, haematopoietic and renal systems. Several studies have shown beneficial effects of thiamine on Pb toxicity. It is speculated that Pb chelation by thiamine may be a possible mechanism. However, the exact nature of these interactions remained elusive. In the present study we have characterized the interaction of Pb with thiamine using UV–Vis as well as fluorescence spectroscopic methods and studied the effect of thiamine treatment on blood and tissue Pb levels during simultaneous or post-exposure to Pb in rat model. The spectroscopic studies revealed that Pb interacts with the pyrimidine ring of thiamine, leading to its solubilization at physiological pH. Further, thiamine reduced the Pb levels in blood, kidney and bone during both simultaneous and post-exposure Pb treatment. Interestingly, thiamine appears to prevent the accumulation of Pb in bone during simultaneous treatment. Together these results suggest that pyrimidine ring of thiamine mediates its interaction with Pb, leading to the prevention of its accumulation and/or increased clearance from tissues.
Keywords: Lead toxicity; Thiamine; Metal chelation; Pyrimidine ring; Absorption; Solubilization

Copper(II) complexes of methimazole, an anti Grave’s disease drug. Synthesis, characterization and its potential biological behavior as alkaline phosphatase inhibitor by Nora M. Urquiza; Silvia G. Manca; María A. Moyano; Raquel Arrieta Dellmans; Luis Lezama; Teófilo Rojo; Luciana G. Naso; Patricia A. M. Williams; Evelina G. Ferrer (255-264).
Methimazole (MeimzH) is an anti-thyroid drug and the first choice for patients with Grave’s disease. Two new copper(II) complexes of this drug: [Cu(MeimzH)2(NO3)2].0.5H2O and [Cu(MeimzH)2(H2O)2](NO3)2·H2O were synthesized and characterized by elemental analysis, dissolution behavior, thermogravimetric analysis and UV-vis, diffuse reflectance, FTIR and EPR spectroscopies. As it is known that copper(II) cation can act as an inhibitor of alkaline phosphatase (ALP), the inhibitory effect of methimazole and its copper(II) complexes on ALP activity has also been investigated.
Keywords: Methimazole; Copper; Biometals; Grave’s disease; EPR; FTIR; UV-visible; ALP inhibition

Oxidative damage to DNA by 1,10-phenanthroline/l-threonine copper (II) complexes with chlorogenic acid by Yong Wang; Xiaoyan Zhang; Qianru Zhang; Zhousheng Yang (265-273).
The oxidative DNA damage by copper (II) complexes in the presence of chlorogenic acid was explored using agarose gel electrophoresis. The extent of pBR322 DNA damage was enhanced significantly with increasing concentration of [Cu-phen-Thr] complex and incubation time. A fluorescence quenching activity of calf thymus DNA–EB was observed more remarkably with chlorogenic acid than without chlorogenic acid. The fluorescence measurements suggested that [Cu-phen-Thr] complex not only can bind to DNA by intercalation but also can damage the double strand DNA in the presence of chlorogenic acid. Further, 8-hydroxy-2′-deoxyguanosine, a biomarker of DNA oxidative damage was determined by electrochemical method. The control experiments revealed that the structure of copper (II) complexes affected capability of complex to DNA damage. The planar structure copper (II) complex showed high efficiency to DNA damage. The chlorogenic acid as biological reductant could improve copper (II) complex to DNA damage. A mechanism on [Cu-phen-Thr] complex to DNA damage in the presence of chlorogenic acid was proposed.
Keywords: Copper (II) complexes; Chlorogenic acid; DNA damage; Oxidative stress

Yersinia ironomics: comparison of iron transporters among Yersinia pestis biotypes and its nearest neighbor, Yersinia pseudotuberculosis by Stanislav Forman; James T. Paulley; Jacqueline D. Fetherston; Yi-Qiang Cheng; Robert D. Perry (275-294).
Although Yersinia pestis epidemic biovars and Yersinia pseudotuberculosis are recently diverged, highly related species, they cause different diseases via disparate transmission routes. Since iron transport systems are important for iron acquisition from hosts and for survival in the environment, we have analyzed potential iron transport systems encoded by epidemic and non-epidemic or endemic strains of Y. pestis as well as two virulent Y. pseudotuberculosis strains. Computational biology analysis of these genomes showed a high degree of identity/similarity among 16 proven or possible iron/heme transporters identified. Of these, 7 systems were essentially the same in all seven genomes analyzed. The remaining 9 loci had 2–6 genetic variations among these genomes. Two untested, potential siderophore-dependent systems appear intact in Y. pseudotuberculosis but are disrupted or absent in all the endemic Y. pestis strains as well as the epidemic strains from the antiqua and mediaevalis biovars. Only one of these two loci are obviously disrupted in Y. pestis CO92 (epidemic orientalis biovar). Experimental studies failed to identify a role for hemin uptake systems in the virulence of pneumonic plague and suggest that Y. pestis CO92 does not make a siderophore other than Ybt.
Keywords: Iron transport; Plague; Siderophore; Heme transport; Yersinia pestis ; Yersinia pseudotuberculosis ; Yersiniabactin

Antioxidant system activation by mercury in Pfaffia glomerata plantlets by N. S. Calgaroto; G. Y. Castro; D. Cargnelutti; L. B. Pereira; J. F. Gonçalves; L. V. Rossato; F. G. Antes; V. L. Dressler; E. M. M. Flores; M. R. C. Schetinger; F. T. Nicoloso (295-305).
Oxidative stress caused by mercury (Hg) was investigated in Pfaffia glomerata plantlets grown in nutrient solution using sand as substrate. Thirty-day-old acclimated plants were treated for 9 days with four Hg levels (0, 1, 25 and 50 μM) in the substrate. Parameters such as growth, tissue Hg concentration, toxicity indicators (δ-aminolevulinic acid dehidratase, δ-ALA-D, activity), oxidative damage markers (TBARS, lipid peroxidation, and H2O2 concentration) and enzymatic (superoxide dismutase, SOD, catalase, CAT, and ascorbate peroxidase, APX) and non-enzymatic (non-protein thiols, NPSH, ascorbic acid, AsA, and proline concentration) antioxidants were investigated. Tissue Hg concentration increased with Hg levels. Root and shoot fresh weight and δ-ALA-D activity were significantly decreased at 50 μM Hg, and chlorophyll and carotenoid concentration were not affected. Shoot H2O2 concentration increased curvilinearly with Hg levels, whereas lipid peroxidation increased at 25 and 50 μM Hg, respectively, in roots and shoots. SOD activity showed a straight correlation with H2O2 concentration, whereas CAT activity increased only in shoots at 1 and 50 μM Hg. Shoot APX activity was either decreased at 1 μM Hg or increased at 50 μM Hg. Conversely, root APX activity was only increased at 1 μM Hg. In general, AsA, NPSH and proline concentrations increased upon addition of Hg, with the exception of proline in roots, which decreased. These changes in enzymatic and non-enzymatic antioxidants had a significant protective effect on P. glomerata plantlets under mild Hg-stressed conditions.
Keywords: Antioxidant system; Brazilian ginseng; Mercury; Oxidative stress; Pfaffia glomerata ; Plant growth

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine, which is produced by the action of hemolytic phospholipase C on phosphatidylcholine or sphyngomielin, to generate choline and inorganic phosphate. Among divalent cations, its activity is dependent on Mg2+ or Zn2+. Mg2+ produced identical activation at pH 5.0 and 7.4, but Zn2+ was an activator at pH 5.0 and became an inhibitor at pH 7.4. At this higher pH, very low concentrations of Zn2+ inhibited enzymatic activity even in the presence of saturating Mg2+ concentrations. Considering experimental and theoretical physicochemical calculations performed by different authors, we conclude that at pH 5.0, Mg2+ and Zn2+ are hexacoordinated in an octahedral arrangement in the PchP active site. At pH 7.4, Mg2+ conserves the octahedral coordination maintaining enzymatic activity. The inhibition produced by Zn2+ at 7.4 is interpreted as a change from octahedral to tetrahedral coordination geometry which is produced by hydrolysis of the $$ left[ {{ ext{Zn}}^{ 2+ } { ext{L}}_{ 2}^{ - 1} { ext{L}}_{ 2}^{0} left( {{ ext{H}}_{ 2} { ext{O}}} ight)_{ 2} } ight] $$ complex.
Keywords: Pseudomonas aeruginosa ; Phosphorylcholine phosphatase; Zn2+ ; Mg2+

Differential responses to cadmium induced oxidative stress in marine macroalga Ulva lactuca (Ulvales, Chlorophyta) by Manoj Kumar; Puja Kumari; Vishal Gupta; P. A. Anisha; C. R. K. Reddy; Bhavanath Jha (315-325).
This study describes various biochemical processes involved in the mitigation of cadmium toxicity in green alga Ulva lactuca. The plants when exposed to 0.4 mM CdCl2 for 4 days showed twofold increase in lipoperoxides and H2O2 content that collectively decreased the growth and photosynthetic pigments by almost 30% over the control. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione peroxidase (GPX) enhanced by twofold to threefold and that of catalase (CAT) diminished. Further, the isoforms of these enzymes, namely, Mn-SOD (~85 kDa), GR (~180 kDa) and GPX (~50 kDa) responded specifically to Cd2+ exposure. Moreover, the contents of reduced glutathione (3.01 fold) and ascorbate (1.85 fold) also increased substantially. Lipoxygenase (LOX) activity increased by two fold coupled with the induction of two new isoforms upon Cd2+ exposure. Among the polyunsaturated fatty acids, although n − 3 PUFAs and n − 6 PUFAs (18:3n − 6 and C18:2n − 6) showed relatively higher contents than control, the latter ones showed threefold increase indicating their prominence in controlling the cadmium stress. Both free and bound soluble putrescine increased noticeably without any change in spermidine. In contrast, spermine content reduced to half over control. Among the macronutrients analysed in exposed thalli, the decreased K content was accompanied by higher Na and Mn with no appreciable change in Ca, Mg, Fe and Zn. Induction of antioxidant enzymes and LOX isoforms together with storage of putrescine and n − 6 PUFAs in cadmium exposed thallus in the present study reveal their potential role in Cd2+ induced oxidative stress in U. lactuca.
Keywords: Antioxidant enzymes; Cadmium; LOX; Minerals; Oxidative stress; PUFAs; Ulva lactuca

Effect of cadmium on 24-hour pattern in expression of redox enzyme and clock genes in rat medial basal hypothalamus by Vanesa Jiménez-Ortega; Daniel P. Cardinali; María P. Fernández-Mateos; María J. Ríos-Lugo; Pablo A. Scacchi; Ana I. Esquifino (327-337).
The effect of cadmium (Cd) in the brain has been attributed to an increase in reactive oxygen species in cells, particularly when high amounts of the metal are given. In this study we examined the effect of a low dose of Cd (7.5 μg/day) on 24-h changes in expression of redox pathway enzyme and circadian genes in rat medial basal hypothalamus (MBH). Rats receiving CdCl2 (5 ppm in drinking water) or tap water for 1 month were killed at six different time intervals throughout a 24 h cycle. MBH mRNA levels were measured by real-time PCR analysis. In CdCl2 treated rats a disruption of 24-h pattern of hypothalamic gene expression of nitric oxide synthase (NOS)-1 and -2, heme oxygenase (HO)-1 and -2, Mn- superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione reductase was detectable. Mean levels of MBH mRNA for HO-2, Mn-SOD and catalase augmented after Cd intake, whereas those of NOS-2 decreased. After CdCl2 intake rats the 24-h pattern of clock gene expression in MBH seen in controls was significantly suppressed (Bmal1) or changed in phase (Per1, Per2, Cry2) while in the case of Clock significant 24-h variations were induced. The results are compatible with the view that a low amount of Cd given in tap water brought about significant changes in circadian expression of redox enzyme and clock genes in rat MBH.
Keywords: Cadmium; Circadian rhythms; Gene expression; Medial basal hypothalamus; Nitric oxide synthase; Heme oxygenase; Superoxide dismutase; Catalase; Glutathione peroxidase; Glutathione reductase; Clock genes

Increased intracellular free zinc concentrations are associated with activation of several stress signaling pathways, specific organelle injury and final cell death. In the present work we examined the involvement of mitochondria and lysosomes and their crosstalk in free zinc-induced cell demise. We report that treatment of cervical tumor Hep-2 cells with zinc pyrithione leads to an early appearance of cytoplasmic zinc-specific foci with corresponding accumulation of zinc first in mitochondria and later in lysosomes. Concomitant with these changes, upregulation of expression of metallothionein II A gene as well as the increased abundance of its protein occurs. Moreover, zinc activates p53 and its dependent genes including Puma and Bax and they contribute to an observed loss of mitochondrial membrane potential and activation of apoptosis. Conversely, lysosomal membrane permeabilization and its promoted cleavage of Bid occurs in a delayed manner in treated cells and their effect on decrease of mitochondrial membrane potential is limited. The use of specific inhibitors as well as siRNA technology suggest a crucial role of MT-IIA in trafficking of free zinc into mitochondria or lysosomes and regulation of apoptotic or necrotic cell demise.
Keywords: Zinc; Mitochondria; Lysosomes; Metallothionein; Apoptosis; Necrosis