‘Biochemical Responses to Heavy Metals in Oreochromis mossambicus with special reference to Metal Detoxifying Mechanism

The present work is focused on the organelle and biochemical responses to heavy metal exposure in the fish Oreochromis mossambicus giving particular importance to the metal detoxifying machinery of the organism. The thesis is an outcome of the effort aimed at developing practicable monitoring techniques to deliver guidelines for biological effect monitoring and the need for specific biochemical methods to detect biological effects of heavy metals that can be interpreted in terms of the health status of the individual organism and eventually alterations in vital processes as growth and reproduction. The efficiency of the metal detoxifying metallothioneins which is an attractive tool for biological monitoring, their role as scavengers of trace metal ions and thus in relieving the biological machinery from their toxicity effects are important themes of this study. Efforts have also been made to test the reliability of the spill over hypothesis of the action of metallothioneins (Winge et a1.,1973) and their use as a biological barometer of heavy metal stress.

Spectral, Structural And Biological Studies Of Some Metal Complexes Of ‘Nsubstituted 2-Benzoylpyridine Thiosemicarbazones

Aqua complex ions of metals must have existed since the appearance of water on the earth, and the subsequent appearance of life depended on, and may even have resulted from the interaction of metal ions with organic molecules. Studies on the coordinating ability of metal ions with other molecules and anions culminated in the theories of/\lfred Werner. Thereon the progress in the studies of metal complex chemistry was rapid. Many factors, like the utility and economic importance of metal chemistry, the intrinsic interest _in many of the compounds and the intellectual challenge of the structural problems to be solved, have contributed to this rapid progress. X—ray diffraction studies further accelerated the progress. The work cited in this thesis was carried out by the author in the Department of Applied Chemistry during 2001-2004. The primary aim of these investigations was to synthesise and characterize some transition metal complexes of 2-benzoylpyridine N(4)-substituted thiosemicarbazones and to study the antimicrobial activities of the ligands and their metal complexes. The work is divided into eight chapters

Design Of Metal Architectures: Synthesis And Study Of Their Photophysical And Biomolecular Recognition Properties

Design and study of molecular receptors capable of mimicking natural processes has found applications in basic research as well as in the development of potentially useful technologies. Of the various receptors reported, the cyclophanes are known to encapsulate guest molecules in their cavity utilizing various non–covalent interactions resulting in significant changes in their optical properties. This unique property of the cyclophanes has been widely exploited for the development of selective and sensitive probes for a variety of guest molecules including complex biomolecules. Further, the incorporation of metal centres into these systems added new possibilities for designing receptors such as the metallocyclophanes and transition metal complexes, which can target a large variety of Lewis basic functional groups that act as selective synthetic receptors. The ligands that form complexes with the metal ions, and are capable of further binding to Lewis-basic substrates through open coordination sites present in various biomolecules are particularly important as biomolecular receptors. In this context, we synthesized a few anthracene and acridine based metal complexes and novel metallocyclophanes and have investigated their photophysical and biomolecular recognition properties.

Metal complexes of a tetraazacyclophane: solution and molecular modelling studies

An alternative synthetic approach to yield the compound 2,3,5,6,8,9,11,14-octahydrobenzo[1][ 1,4,7,10]tetraazacyclotetradecine (bz[14]N-4) is presented. The protonation constants of bz[14]N-4 and the stability constants of its complexes with Ni2+, Cu2+, Zn2+, Cd2+, Pb2+ were determined in H2O at 25degreesC with ionic strength 0.10 mol dm(-3) in KNO3 and they were compared with structurally related macrocycles cyclam (1,4,8,11-tetraazacyclotetradecane) and cyclen (1,4,7,10-tetraazacyclododecane). These studies indicate that only 1 : 1 ( M : L) species are formed in solution, and the ligand exhibits a high affinity for larger ions such as Cd2+ and Pb2+. The X-ray study of [bz[14]N4H3](3+) shows that an inclusion compound with a chloride counter-anion is formed through NH...Cl hydrogen bonds. Spectroscopic data in solution ( electronic and NMR spectra) showed that the macrocycle adopts a planar arrangement upon metal complexation. Molecular mechanics calculations reveal that in spite of the presence of the benzene ring in the macrocyclic framework this ligand can encapsulate metal ions with different stereo-electronic sizes in square planar arrangements. Our results indicate that the presence of the benzene ring in the backbone of the bz[14]N-4 confers a coordination behaviour intermediate between that of cyclam and cyclen.

Metal complexes of a dipyridine octaazamacrocycle: stability constants, structural and modelling studies

Two 28-membered octaazamacrocycles, [28]py(2)N(6) and Me-2[28]py(2)N(6), have been synthesized. The protonation constants of the N-methyl. derivative and the stability constants of its complexes with Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ were determined at 25degreesC in 0.10 mol dm(-3) KNO3. The high overall basicity of Me-2[28]py(2)N(6) is ascribed to the weaker repulsion between protonated contiguous charged ammonium sites separated by propyl chains. These studies together with NMR, UV-vis and EPR spectroscopies indicated the presence of mono- and di-nuclear species, The single crystal structure of the complex [Ni-2([28]py(2)N(6))(H2O)(4)]Cl-4.3H(2)O was determined, and showed each nickel centre in a distorted octahedral co-ordination environment. The nickel centres are held within the macrocycle at a large distance of 6.991(g) Angstrom from each other. The formation of mononuclear complexes was evaluated theoretically via molecular mechanics (MM) and molecular dynamics (MD) calculations and showed that these large macrocycles have sufficient flexibility to encapsulate metal ions with different stereo-electronic sizes. Structures for small and large metal ions are proposed.

Cyclam derivatives containing three acetate pendant arms: synthesis, acid-base, metal complexation and structural studies

The ligands 1,4,8,11-tetraazacyclotetradecane-1,4,8-triacetic-11-methylphosphonic acid (H(5)te3a1p) and 1,4,8,11-tetraazacyclotetradecane-1,4,8-triacetic acid (H(3)te3a) were synthesized, the former one for the first time. The syntheses of these ligands were achieved from reactions on 1,4,8,11-tetraazacyclotetradecane-1,4,8-tris( carbamoylmethyl) hydroiodide (te3am center dot HI), and compounds (Hte3am)(+), 1, and (H(7)te3a1p)(2+), 4, were characterized by X-ray diffraction. Structures of two other compounds resulting from side-reactions, (H(2)te2lac)(2+), 2, and (H(4)te2a2p(OEt2))(2+), 3, were also determined by X-ray diffraction. Potentiometric titrations of H(5)te3a1p and H(3)te3a were performed at 298.2 K and ionic strength 0.10 mol dm(-3) in NMe4NO3 to determine their protonation constants. H-1 and P-31 NMR titrations of H(5)te3a1p were carried out in order to determine the very high first protonation constant of this ligand and to elucidate the sequence of protonation. Potentiometric studies of the two ligands with Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Pb2+ metal ions performed in the same experimental conditions showed that the complexes of H5te3a1p present very high thermodynamic stability while complexes of H(3)te3a, particularly Co2+ and Zn2+, are even more stable. P-31 NMR spectra of the cadmium(II) complex of H(5)te3a1p showed that the phosphonate moiety was coordinated to the metal ion. The UV-vis-NIR spectroscopic data and magnetic moment values of Co2+ and Ni2+ complexes of H(5)te3a1p and H(3)te3a together with the EPR of the corresponding Cu2+ complexes indicated that all these complexes adopt distorted octahedral coordination geometries in solution. This was confirmed by the single crystal structure of [Cu-2(Hte3a)(H2O)(3)Cl]Cl-0.5(ClO4)(0.5) center dot 2H(2)O that showed two distorted octahedral copper centres bridged by a N-acetate pendant arm with a Cu center dot center dot center dot Cu distance of 4.890(1) angstrom. The first one is encapsulated into the macrocyclic cavity surrounded by four nitrogen and two oxygen donors from the macrocycle, whereas the second one is on the periphery of the macrocycle and is coordinated to two oxygen atoms of one acetate pendant arm in chelating fashion, one chloride and three water molecules.

A Metal-Repressed Promoter From Gram-Positive Bacillus subtilis Is Highly Active and Metal-Induced in Gram-Negative Cupriavidus metallidurans

A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation. Biotechnol. Bioeng. 2010; 107: 469-477. (C) 2010 Wiley Periodicals, Inc.

PREDICTION OF METAL CATION TOXICITY TO THE BIOLUMINESCENT FUNGUS GERRONEMA VIRIDILUCENS

A correlation between the physicochemical properties of mono- [Li(I), K(I), Na(I)] and divalent [Cd(II), Cu(II), Mn(II), Ni(II), Co(II), Zn(II), Mg(II), Ca(II)] metal cations and their toxicity (evaluated by the free ion median effective concentration. EC50(F)) to the naturally bioluminescent fungus Gerronema viridilucens has been studied using the quantitative ion character activity relationship (QICAR) approach. Among the 11 ionic parameters used in the current study, a univariate model based on the covalent index (X(m)(2)r) proved to be the most adequate for prediction of fungal metal toxicity evaluated by the logarithm of free ion median effective concentration (log EC50(F)): log EC50(F) = 4.243 (+/-0.243) -1.268 (+/-0.125).X(m)(2)r (adj-R(2) = 0.9113, Alkaike information criterion [AIC] = 60.42). Additional two- and three-variable models were also tested and proved less suitable to fit the experimental data. These results indicate that covalent bonding is a good indicator of metal inherent toxicity to bioluminescent fungi. Furthermore, the toxicity of additional metal ions [Ag(I), Cs(I), Sr(II), Ba(II), Fe(II), Hg(II), and Pb(II)] to G. viridilucens was predicted, and Pb was found to be the most toxic metal to this bioluminescent fungus (EC50(F)): Pb(II) > Ag(I) > Hg(I) > Cd(II) > Cu(II) > Co(II) Ni(II) > Mn(II) > Fe(II) approximate to Zn(II) > Mg(II) approximate to Ba(II) approximate to Cs(I) > Li(I) > K(I) approximate to Na(I) approximate to Sr(II)> Ca(II). Environ. Toxicol. Chem. 2010;29:2177-2181. (C) 2010 SETAC

Development of a heavy metal sorption system through the P=S functionalization of coconut (Cocos nucifera) fibers

Lignocellulosic residues are interesting materials for the production of heavy metal adsorbents for aquatic systems. Whole fibers taken from coconut (Cocos nucifera) husks were functionalized with the thiophosphoryl (P=S) group by means of the direct reaction with CI(3)P=S, (CH(3)O)(2)CIP=S or (CH(3)CH(2)O)(2)CIP=S in order to obtain an adsorptive system for `soft` metal ions, particularly Cd(2+). These functionalized fibers (FFs) were characterized by means of elemental analysis, infrared spectroscopy, thermal analysis and acid-base titration. Adsorption isotherms for Cd(2+) fitted the Langmuir model, with binding capacities of 0.2-5 mmol g(-1) of FF at 25 degrees C. (C) 2009 Elsevier Ltd. All rights reserved.

Automated two-dimensional separation flow system with electrochemical preconcentration, stripping, capillary electrophoresis and contactless conductivity detection for trace metal ion analysis

This paper describes the automation of a fully electrochemical system for preconcentration, cleanup, separation and detection, comprising the hyphenation of a thin layer electrochemical flow cell with CE coupled with contactless conductivity detection (CE-C(4)D). Traces of heavy metal ions were extracted from the pulsed-flowing sample and accumulated on a glassy carbon working electrode by electroreduction for some minutes. Anodic stripping of the accumulated metals was synchronized with hydrodynamic injection into the capillary. The effect of the angle of the slant polished tip of the CE capillary and its orientation against the working electrode in the electrochemical preconcentration (EPC) flow cell and of the accumulation time were studied, aiming at maximum CE-C(4)D signal enhancement. After 6 min of EPC, enhancement factors close to 50 times were obtained for thallium, lead, cadmium and copper ions, and about 16 for zinc ions. Limits of detection below 25 nmol/L were estimated for all target analytes but zinc. A second separation dimension was added to the CE separation capabilities by staircase scanning of the potentiostatic deposition and/or stripping potentials of metal ions, as implemented with the EPC-CE-C(4)D flow system. A matrix exchange between the deposition and stripping steps, highly valuable for sample cleanup, can be straightforwardly programmed with the multi-pumping flow management system. The automated simultaneous determination of the traces of five accumulable heavy metals together with four non-accumulated alkaline and alkaline earth metals in a single run was demonstrated, to highlight the potentiality of the system.

Development of a new analytical approach based on cellulose membrane and chelator for differentiation of labile and inert metal species in aquatic systems

A new procedure was developed in this study, based on a system equipped with a cellulose membrane and a tetraethylenepentamine hexaacetate chelator (MD-TEPHA) for in situ characterization of the lability of metal species in aquatic systems. To this end, the DM-TEPHA system was prepared by adding TEPHA chelator to cellulose bags pre-purified with 1.0 mol L-1 of HCl and NaOH solutions. After the MD-TEPHA system was sealed, it was examined in the laboratory to evaluate the influence of complexation time (0-24 h), pH (3.0, 4.0, 5.0, 6.0 and 7.0), metal ions (Cu, Cd, Fe, Mn and Ni) and concentration of organic matter (15, 30 and 60 mg L-1) on the relative lability of metal species by TEPHA chelator. The results showed that Fe and Cu metals were complexed more slowly by TEPHA chelator in the MD-TEPHA system than were Cd, Ni and Mn in all pH used. It was also found that the pH strongly influences the process of metal complexation by the MD-TEPHA system. At all the pH levels, Cd, Mn and Ni showed greater complexation with TEPHA chelator (recovery of about 95-75%) than did Cu and Fe metals. Time also affects the lability of metal species complexed by aquatic humic substances (AHS); while Cd, Ni and Mn showed a faster kinetics, reaching equilibrium after about 100 min, and Cu and Fe approached equilibrium after 400 min. Increasing the AHS concentration decreases the lability of metal species by shifting the equilibrium to AHS-metal complexes. Our results indicate that the system under study offers an interesting alternative that can be applied to in situ experiments for differentiation of labile and inert metal species in aquatic systems. (c) 2006 Elsevier B.V. All rights reserved.

Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions

Pyrophosphatase activity of rat osseous plate alkaline phosphatase was studied at different concentrations of calcium and magnesium ions. with the aim of characterizing the modulation of enzyme activity by these metals. In the absence of metal ions, the enzyme hydrolysed pyrophosphate following Michaelian kinetics with a specific activity of 36.7 U/mg and K-0.5 = 88 mu M. In the presence of low concentrations (0.1 mM) of magnesium (or calcium) ions, the enzyme also exhibited Michaclian kinetics for the hydrolysis of pyrophosphate, but a significant increase in specific activity (123 U/mg) was observed. K-m values remained almost unchanged. Quite different behavior occurred in the presence of 2 mM magnesium (or calcium) ions. In addition to low-affinity sites (K-0.5 = 40 and 90 mu M, for magnesium and calcium, respectively), high-affinity sites were also observed with K-0.5 values 100-fold lower. The high-affinity sites observed in the presence of calcium ions represented about 10% of those observed for magnesium ions. This was correlated with the fact that only magnesium ions triggered conformational changes yielding a fully active enzyme. These results suggested that the enzyme could hydrolyse pyrophosphate, even at physiological concentrations (4 mu M), since magnesium concentrations are high enough to trigger conformational changes increasing the enzyme activity. A model, suggesting the involvement of magnesium ions in the hydrolysis of pyrophosphate by rat osseous plate alkaline phosphatase is proposed. (C) 1998 Published by Elsevier B.V. Ltd. All rights reserved.

Approach combining on-line metal exchange and tangential-flow ultrafiltration for in-situ characterization of metal species in humic hydrocolloids

This paper deals with the development and optimization of an analytical procedure using ultrafiltration and a flow-injection system, and its application in in-situ experiments to characterize the lability and availability of metal species in humic-rich hydrocolloids. The on-line system consists of a tangential flow ultrafiltration device equipped with a 3-kDa filtration membrane. The concentration of free ions in the filtrate was determined by atomic-absorption spectrometry, assuming that metals not complexed by aquatic humic substances (AHS) were separated from the complexed species (M-AHS) retained by the membrane. For optimization, exchange experiments using Cu(II) solutions and AHS solutions doped with the metal ions Ni(II), Mn(II), Fe(III), Cd (II), and Zn(II) were carried out to characterize the stability of the metal-AHS complexes. The new procedure was then applied in-situ at a tributary of the Ribeira do Iguape river (Iguape, São Paulo State, Brazil) and evaluated using the ions Fe(III) and Mn(II), which are considered to be essential constituents of aquatic systems. From the exchange between metal-natural organic matter (M-NOM) and the Cu(II) ions it was concluded that Cu(II) concentrations > 485 mu g L(-1) were necessary to obtain maximum exchange of the complexes Mn-NOM and Fe-NOM, corresponding to 100% Mn and 8% Fe. Moreover, the new analytical procedure is simple and opens up new perspectives for understanding the complexation, transport, stability, and lability of metal species in humic-rich aquatic environments.

Attachment of 2,2-bipyridine onto a silica gel for application as a sequestering agent for copper, cadmium and lead ions from an aqueous medium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

New analytical procedure based on a cellulose bag and ionic exchanger with p-aminobenzoic acid groups for differentiation of labile and inert metal species in aquatic systems

A new procedure was developed for the in situ characterization of the lability of metal species in aquatic systems by using a system equipped with a diffusion membrane and cellulose organomodified with p-aminobenzoic acid groups (DM-Cell-PAB). To this end, the DM-Cell-PAB system was prepared by adding cellulose organomodified with p-aminobenzoic acid groups (Cell-PAB) to pre-purified cellulose bags. After the DM-Cell-PAB system was sealed, it was examined in the laboratory to evaluate the influence of complexation time, mass of exchanger, pH, metal ions (Cu, Cd, Fe, Mn, and Ni), and concentration of organic matter on the relative lability of metal species. It was found that the pH and kinetics strongly influence the process of metal complexation by the DM-Cell-PAB system. At all pH levels, Cd, Mn, and Ni showed lower complexation with Cell-PAB resin than Cu and Fe metals. Note that relative lability of metals complexed to aquatic humic substances (AHS) in the presence of Cell-PAB resin showed the following order: Cu congruent to Fe >> Ni > Mn=Cd. The results presented here also indicate that increasing the AHS concentration decreases the lability of metal species by shifting the equilibrium to AHS-metal complexes. Our results indicate that the system under study offers an interesting alternative that can be applied to in situ experiments for differentiation of labile and inert metal species in aquatic systems.