A proton nuclear magnetic resonance study of the binding of methylmercury in human erythrocytes

The binding of methylmercury, CH₃Hg(II), by small molecules in the intracellular region of human erythrocytes has been studied by ¹H-NMR spectroscopy. To suppress or completely eliminate interfering resonances from the much more abundant hemoglobin protons, spectra were measured by a technique based on the transfer of saturation throughout the envelope of hemoglobin resonances following a selective presaturation pulse or by the spin-echo Fourier transform method. With these techniques, ¹H-NMR spectra were measured for the more abundant intracellular small molecules, including glycine, alanine, creatine, lactic acid, ergothioneine and glutathione, in both intact and hemolyzed erythrocytes to which CH₃Hg(II) had been added. The results for intact erythrocytes indicate that part of the CH₃Hg(II) is complexed by intracellular glutathione. These results also indicate that exchange of CH₃Hg(II) among glutathione molecules is fast, with the average lifetime of a CH₃Hg(II)-glutathione complex estimated to be less than 0.01 s. From exchange-averaged chemical shifts of the resonance for the proton on the α-carbon of the cysteine residue of glutathione, it is shown that, in hemolyzed erythrocytes, the sulfhydryl group of glutathione binds CH₃Hg(II) more strongly than the sulfhydryl groups of hemoglobin.     

Biological evaluation of a cytotoxic 2-substituted benzimidazole copper(II) complex: DNA damage,antiproliferation and apoptotic induction activity in human cervical cancer cells

Exploring novel chemotherapeutic agents is a great challenge in cancer medicine. To that end, 2-substituted benzimidazole copper(II) complex, [Cu(BMA)Cl₂]·(CH₃OH) (1) [BMA = N,N′-bis(benzimidazol-2-yl-methyl)amine], was synthesized and its cytotoxicity was characterized. The interaction between complex 1 and calf thymus DNA was detected by spectroscopy methods. The binding constant (K _b = 1.24 × 10⁴ M^?1) and the apparent binding constant (K _app = 6.67 × 10⁶ M^?1) of 1 indicated its moderate DNA affinity. Complex 1 induced single strand breaks of pUC19 plasmid DNA in the presence of H₂O₂ through an oxidative pathway. Cytotoxicity studies proved that complex 1 could inhibit the proliferation of human cervical carcinoma cell line HeLa in both time- and dose-dependent manners. The results of nuclei staining by Hoechst 33342 and alkaline single-cell gel electrophoresis proved that complex 1 caused cellular DNA damage in HeLa cells. Furthermore, treatment of HeLa cells with 1 resulted in S-phase arrest, loss of mitochondrial potential, and up-regulation of caspase-3 and -9 in HeLa cells, suggesting that complex 1 was capable of inducing apoptosis in cancer cells through the intrinsic mitochondrial pathway.     

1H nmr study of the effectiveness of various thiols for removal of methylmercury from hemolyzed erythrocytes

The effectiveness of eight thiol ligands for removing methylmercury (CH₃Hg(II)) from its glutathione and hemoglobin complexes in hemolyzed erythrocytes has been studied by ¹H nuclear magnetic resonance spectroscopy. These complexes are the predominant methylmercury species in human erythrocytes. The effectiveness was determined from the exchange-averaged chemical shift of the resonance for the proton on the α-carbon of the cysteinyl residue and from the intensity of the resonance for the methylene protons of the glycine residue of reduced glutathione (GSH), both of which provide a measure of the amount of glutathione in the CH₃Hg(II)-complexed form. The thiol ligands were found to release GSH from its CH₃Hg(II) complex in the order 2, 3-dimercap-tosuccinic acid > mercaptosuccinic acid > cysteine > mercaptoacetic acid > D-penicillamine > 2, 3-dimercaptopropanesulfonic acid > N-acetyl-D,L-penicillamine > D.L-homocysteine.     

Mechanism of cytotoxicity of methylmercury

Mechanism of methylmercury cytotoxicity was investigated with special reference to its preferential action on microtubules and protein biosynthesis in cultured cells. The tubulin synthesis analyzed by autoradiography of two-dimensional electropherogram using³⁵S-methionine was inhibited by 50–70% in mouse glioma cells exposed to 5×10^?6 M methylmercury for 3 h, which almost completely depolymerized microtubules. Total protein synthesis monitored by incorporation of labeled methionine into acid insoluble fraction was decreased slightly but significantly and the protein bands other than tubulin on gradient urea-PAGE gel appeared to remain unchanged under the experimental condition used. These results suggest that the inhibition of protein synthesis observed on exposure to methylmercury can be ascribed, at least partly, to a possible autoregulatory depression in tubulin synthesis owing to the increase in the pool of tubulin subunits resulted from microtubule depolymerization by methylmercury.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

Interactions between selenium and methylmercury in rat brain

The interaction of selenium with methylmercury was investigated in brain of animals labeled with ⁷⁵SeO₃^2? and CH₃²⁰³Hg⁺. Brains were fractionated into subcellular components and the cytosol was further fractionated by chromatography on Sephadex G-150 and G-200. The main result of these studies was evidence suggesting a shift of ⁷⁵Se from the cytosol to the mitochondrial fraction in brain when CH₃Hg⁺ was given. Concurrent equimolar (10 μmoles/kg) selenite injections increased the uptake of Hg but did not alter ²⁰³Hg distribution in brain. Changing the dose of CH₃Hg⁺ from 1 to 38 μmoles/kg had little effect on Hg uptake (% of dose per g). Gel filtrations on Sephadex G-150 and G-200 revealed that ²⁰³Hg in cytosol followed a pattern more closely related to protein (A₂₈₀) than to ⁷⁵Se, although a considerable portion of both isotopes eluted with proteins in the void volume. Assays of whole brain homogenates revealed a slight reduction in glutathione peroxidase activity in CH₃Hg⁺-treated rats which was not seen when equimolar selenite was injected with the CH₃Hg⁺.     

Peptidic models for the binding of Pb(II), Bi(III) and Cd(II) to mononuclear thiolate binding sites

Herein, we evaluate the binding of Pb(II) and Bi(III) to cysteine-substituted versions of the TRI peptides [AcG-(LKALEEK)₄G-NH₂] which have previously been shown to bind Hg(II) and Cd(II) in unusual geometries as compared with small-molecule thiol ligands in aqueous solutions. Studies of Pb(II) and Bi(III) with the peptides give rise to complexes consistent with the metal ions bound to three sulfur atoms with M–S distances of 2.63 and 2.54 Å, respectively. Competition experiments between the metal ions Pb(II), Cd(II), Hg(II) and Bi(III) for the peptides show that Hg(II) has the highest affinity, owing to the initial formation of the extremely strong HgS₂ bond. Cd(II) and Pb(II) have comparable binding affinities at pH > 8, while Bi(III) displays the weakest affinity, following the model, M(II) + (TRI LXC)₃ ^3? → M(II)(TRI LXC)₃ ^?. While the relevant equilibria for Hg(II) binding to the TRI peptides corresponds to a strong first step forming Hg(TRI LXC)₂(HTRI LXC), followed by a single deprotonation to give Hg(TRI LXC)₃ ^?, the binding of Cd(II) and Pb(II) is consistent with initial formation of M(II)(TRI LXC)(HTRI LXC)₂ ⁺ at pH < 5 followed by a two-proton dissociation step (pK _a2) yielding M(II)(TRI LXC)₃ ^?. Pb(II)(TRI LXC)(HTRI LXC)₂ ⁺ converts to Pb(II)(TRI LXC)₃ ^? at slightly lower pH values than the corresponding Cd(II)–peptide complexes. In addition, Pb(II) displays a lower pK _a of binding to the “d”-substituted peptide, (TRI L12C, pK _a2 = 12.0) compared with the “a”-substituted peptide, (TRI L16C, pK _a2 = 12.6), the reverse of the order seen for Hg(II) and Cd(II). Pb(II) also showed a stronger binding affinity for TRI L12C (K _bind = 3.2 × 10⁷ M^?1) compared with that with TRI L16C (K _bind = 1.2 × 10⁷ M^?1) at pH > 8.     

Triplet state properties of the methylmercury(II)-tyrosine complex

CH₃Hg(II)OH forms complexes at pH 8 with tyrosine and with tyrosine ethyl ester (TEE) that are detected by ultraviolet difference absorption spectra. With K_f defined by CH₃HgOH + HB

CH₃HgB + H₂O, we find log K_f = 3.61 (tyrosine) and 3.36 (TEE). A heavy-atom effect is observed in frozen glasses of the complexes; this indicates a close interaction between Hg and the chromophore. No UV difference spectrum or heavy-atom effect is observed with N-acetyl tyrosine ethyl ester, indicating that complexing at the phenol O does not occur, and suggesting that binding occurs at the amine N. Zero field optically detected magnetic resonance (ODMR) measurements of the CH₃Hg(II)-tyrosine triplet state give (D, E) = (0.129, 0.047) or (0.134, 0.041) cm^?1 depending upon assignment of transitions. D of tyrosine is relatively unaffected, but E is reduced by CH₃Hg(II) complexing. Low-temperature kinetic measurements show that the shortest lived sublevel of the complex is T_z, where z lies along the phenol long axis in tyrosine. A dominant 11.6-msec component in the 77 K decay of the phosphorescence is consistent with the individual sublevel lifetimes obtained by ODMR.     

Interaction of Shigella Shigae Cytotoxin with Receptors on Sensitive and Insensitive Cells

Abstract

The effect of a cytotoxin isolated from Shigella shigae has been tested on different cell lines. HeLa S₃ cells, as well as some other human carcinoma cells, were killed by picomolar to femtomolar concentrations of the pure toxin, whereas certain other human carcinoma cells and a variety of non-epithelial cells from human tissue and from various animal tissues were resistant to nanomolar concentrations of the toxin. Binding studies with ¹²⁵I-labelled Shigella shigae cytotoxin showed that the sensitive HeLa S₃ cells contain 1.3 × 10 binding sites per cell, whereas in an insensitive HeLa cell line 2.6 × 10 sites per cell were measured. In all cases the apparent association constant, k_a, was found to be about 10₁₀ M^?1. The binding occurred fairly rapidly, whereas dissociation of bound toxin occurred at a very slow rate, even in the presence of excess unlabelled toxin. All toxin sensitive cell lines bound similar amounts of toxin as HeLa S₃ cells, whereas some of the resistant cell lines did not contain measurable amounts of toxin receptors.     

Purification and characterization of MerR, the regulator of the broad-spectrum mercury resistance genes in Streptomyces lividans 1326

Streptomyces lividans 1326 carries inducible mercury resistance genes on the chromosome, which are arranged in two divergently transcribed operons. Expression of the genes is negatively regulated by the repressor MerR, which binds in the intercistronic region between the two operons. The merR gene was expressed in E. coli using a T7 RNA polymerase/promoter expression system, and MerR was purified to around 95% homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration showed that the native MerR is a dimer with a molecular mass of 31?kDa. Two DNA binding sites were identified in the intercistronic mer promoter region by footprinting experiments. No evidence for cooperativity in the binding of MerR to the adjacent operator sequences was observed in gel mobility shift assays. The dissociation constants (K_D) for binding of MerR were: binding site I, 8.5?×?10^?9?M; binding site II, 1.2?×?10^?8?M; and for the complete promoter/operator region 1?×?10^?8?M. The half-life of the MerR-DNA complex was 19.4?min and 18.8?min for binding site I and binding site II, respectively. The K_D value for binding of mercury(II)chloride to MerR, again determined by mobility shift assay, was 1.1?×?10^?7?M.     

Effect of Inorganic and Organic Ligands on the Bioavailability of Methylmercury as Determined by Using a mer-lux Bioreporter

A mer-lux bioreporter was constructed to assess the bioavailability of methylmercury [CH₃Hg(II)] in Escherichia coli. The bioreporter was shown to be sensitive, with a detection limit of 2.5 nM CH₃Hg(II), and was used to investigate the effects of chlorides, humic acids, and thiols on the bioavailability of CH₃Hg(II) in E. coli. It was found that increasing the concentration of chlorides resulted in an increase in CH₃Hg(II) bioavailability, suggesting that there was passive diffusion of the neutral complex (CH₃HgCl⁰). Humic acids were found to reduce the bioavailability of CH₃Hg(II) in varying degrees. Complexation with cysteine resulted in increased bioavailability of CH₃Hg(II), while assays with equivalent concentrations of methionine and leucine had little or no effect on bioavailability. The mechanism of uptake of the mercurial-cysteine complexes is likely not passive diffusion but could result from the activities of a cysteine transport system. The bioavailability of CH₃Hg(II) decreased with increasing glutathione concentrations.     

Metal Ion-Biomolecule Interactions. VIII: Methylmercury(II) Complexes of 8-Thioguanosine. Synthesis and Spectroscopic Investigations

The reaction of 8-thioguanosine (8-thioGuo^H₂ with methylmercury(II) has been shown to give rise to 1:1 (neutral and cationic), 1:2 (neutral and cationic), and 1:3 (cationic) complexes of the type [CH₃Hg(8-thioGuoH)], [(CH₃Hg(8-thioGuoH₂)]NO₃, [(CH₃Hg)₂(8-thioGuo)], [(CH₃Hg)₂(8-thioGuoH)]NO₃ and [(CH₃Hg)₃(8-thioGuo)]NO₃, depending upon the reactant stoichiometry and pH. ¹H NMR, ¹³C NMR, and IR, as well as analytical data were used to characterize the complexes. Coupling of methylmercury(II)-protons to mercury-199 has been observed in all compounds. The magnitude of the coupling, ²J(¹H-¹⁹⁹Hg), is strongly dependent on the nature of the ligand bonded to the methylmercury(II) group and correlates with the ¹³C chemical shifts of coordinated CH₃Hg(II) at the different binding sites.     

Multiple [3H]-Oxytocin Binding Sites in Rat Myometrial Plasma Membranes

Abstract

The affinity spectrum method has been used to analyse binding isotherms for [³H]-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (K_d) of 0.6–1.5 × 10^?9, 0.4–1.0 × 10^?7 and 7 × 10^?6 mol/l were identified and their existence verified by cluster analysis based on similarities between K_d, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTPrS (guanosine-5′-0-(3-thiotriphosphate) in the incubation medium. The binding parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.     

Novel naphthalimide polyamine derivatives as potential antitumor agents

A novel series of naphthalimide polyamine conjugates were designed, synthesized and evaluated for in vitro antiproliferative activity against human leukemia (Jurkat), human cervical adenocarcinoma (HeLa), human breast adenocarcinoma (MCF-7) and human lung adenocarcinoma (A549) cell lines. From the six derivatives, the new I1 and A3 exhibited highest antiproliferative activity with the IC₅₀ values of 5.67–11.02 μmol·L^?1. Cell cycle analysis of Jurkat cells exposed to I1 at a concentration of 30 μmol × L^?1 for 24 h exhibited a mild increase in S and G₂/M fraction caused by accumulation of cells. This arrest was followed by an increase in sub-G₀/G₁ after 48 h of incubation. Jurkat cells exposed to A3 at a concentration of 30 μmol × L^?1 for 24 h showed an increase in G₀/G₁ fraction and after 48 h an increase in G₂/M fraction followed by an increase in sub-G₀/G₁ after 72 h of incubation. Moreover, the A3 compound was observed to displace the intercalating agent ethidium bromide from calf thymus DNA using fluorescence spectroscopy. The apparent binding constant was estimated to be 3.1 × 10⁶ M^?1 what indicates non-intercalating mode of DNA binding. On the other hand, we found no inhibitory effect of studied compounds on topoisomerase I and topoisomerase II activity. Finally, the localization of these compounds in the cells due to their inherent fluorescence was investigated with the fluorescence microscopy. Our results suggest that the naphthalimide polyamine conjugates rapidly penetrate to the cancer cells. Further studies are necessary to investigate the precise mechanism of action and to find out the relationship between the structure, character and position of substituents of naphthalimide polyamine conjugates and their biological activities.     

Metal ion-biomolecule interactions. XII. 1H and 13C NMR evidence for the preferred reaction of thymidine over guanosine in exchange and competition reactions with Mercury(II) and Methylmercury(II)

Mercury(II) bridge complexes of the type [Nuc-Hg-Nuc] (Nuc = thymidine or guanosine), and methylmercury(II) complexes of thymidine and guanosine of the type [CH₃Hg(Nuc)], have been prepared under appropriate conditions of pH and reactant's stochiometry in acqueous soluton. The various complexes have been characterized by ¹H and ¹³C NMR and used as probes, in competition and exchange studies, to establish the relative affinities of Hg(II) and CH₃Hg(II) towards the nucleosides guanosine and thymidine. These studies have confirmed that Hg(II) and CH₃Hg(II) bind to N₃ of thymidine in preference to N₁ of guanosine. The studies further show that reactions of mercury(II) with the nucleosides are thermodynamically controlled; the preperential binding reflects the relative stabilities of the respective complexes.     

A possible molecular link between the toxicological effects of arsenic,selenium and methylmercury: methylmercury(II) seleno bis(<Emphasis Type="Italic">S</Emphasis>-glutathionyl) arsenic(III)

Using a combination of As and Se K-edge and Hg L_III-edge X-ray absorption spectroscopy, ⁷⁷Se nuclear magnetic resonance spectroscopy, electrospray ionization mass spectrometry and molecular modeling, we have structurally characterized the novel species methylmercury(II) seleno bis(S-glutathionyl) arsenic(III). This species is formed in aqueous solution from CH₃HgOH and the seleno bis(S-glutathionyl) arsinium ion and constitutes an important first step towards characterizing the observed toxicologically relevant interaction between arsenite, selenite and methylmercury which has been previously reported in mammals.     

The effect of methylmercury (II) binding on the conformation of poly(L-glutamic acid) and poly(L-lysine: a Raman spectroscopic study

The binding of the methylmercury cation CH₃Hg⁺ by poly(L -glutamic acid) (PGA) and by poly(L -lysine) (PLL) has been investigated by Raman spectroscopy. Coordination on the side-chain COO^? and NH groups of these polypeptides gave characteristic ligand–Hg stretching modes at ca. 505 and 450 cm^?1, respectively. Precipitation generally occurred upon formation of the complexes and changes of conformation were common. The solid complex obtained from PGA at pH 4.6 was found to have a mostly disordered conformation, which differed from the respective α-helical and β-sheet structures of the dissolved and precipitated uncomplexed polypeptide in the same conditions. An α-helical structure was generally adopted by the complex formed with PLL, even in pH and temperature conditions where the free polypeptide normally exists in another conformation. The addition of a stronger complexing agent, glutathione, to the PLL/CH₃Hg⁺ complex caused a migration of the bound cations and a restoration of the polypeptide to its original state.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.     

Cardiolipin modulates allosterically the nitrite reductase activity of horse heart cytochrome c

Upon cardiolipin (CL) liposomes binding, horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential, binds CO and NO with high affinity, displays peroxidase activity, and facilitates peroxynitrite isomerization. Here, the effect of CL liposomes on the nitrite reductase activity of ferrous cytc (cytc-Fe(II)) is reported. In the absence of CL liposomes, hexa-coordinated cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO (k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4 and 20.0 °C). However, CL liposomes facilitate the NO₂ ^?-mediated nitrosylation of cytc-Fe(II) in a dose-dependent manner inducing the penta-coordination of the heme-Fe(II) atom. The value of k _on for the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO is 2.6 ± 0.3 M^?1 s^?1 (at pH 7.4 and 20.0 °C). Values of the apparent dissociation equilibrium constant for CL liposomes binding to cytc-Fe(II) are (2.2 ± 0.2) × 10^?6 M, (1.8 ± 0.2) × 10^?6 M, and (1.4 ± 0.2) × 10^?6 M at pH 6.5, 7.4, and 8.1, respectively, and 20.0 °C. These results suggest that the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO could play anti-apoptotic effects impairing lipid peroxidation and therefore the initiation of the cell death program by the release of pro-apoptotic factors (including cytc) in the cytoplasm.     

Factors Affecting Methylmercury Levels in Surficial Tailings from Historical Nova Scotia Gold Mines

Large quantities of Hg remain in tailings dumps from historical Nova Scotian gold mines. Depth profiles of total Hg (Hg_T) and methylmercury (MeHg) were compared with geochemical and microbiological variables, to identify factors influencing MeHg levels in tailings. Hg_T and MeHg were highly variable in tailings (0.2–73.5 μ mol kg^{? 1} and < dl-56.4 nmol kg^{? 1}, respectively), and were influenced by a complex set of in situ factors. Elevated MeHg was linked with > 5 μ mol kg^?1 Hg_T, organic matter, hydrology, abundance and activity of sulfate reducing bacteria, and demethylation processes. Methylmercury levels in tailings from a wet, bog-like site appeared to undergo seasonal fluctuations, with higher concentrations measured in September and October, and lower concentrations in May. Evaluations of amalgamation tailings should examine MeHg and Hg_T transport out of low-lying, saturated tailings dumps after snowmelt and major rainfall events, and should take into account the possibility of seasonal variation in MeHg levels in northern regions.