Platinum(II) binding to metallothioneins

The reaction of equine renal metallothionein (MT) with excess K2PtCl4 at pH 2 results in a polymeric adduct containing 17 +/- 2 mol Pt/mol MT. A monomeric adduct containing 7 mol Pt/mol MT is obtained at neutral pH. Rates of reaction of Pt7MT with DTNB and iodoacetic acid are consistent with Pt2+ to cysteine thiolate coordination, and the extent of reaction in both cases is 11 +/- 2 mol cys/mol MT. Adducts from the reaction of K2PtCl4 with apoMT chemically modified at the N-terminal methionine residue, Cd7MT, and native MT are also reported. A structural model of Pt7MT is proposed in which the square planar tetrathiolate Pt(II) unit is incorporated into a three-metal beta cluster. Implications for the metabolism of platinum anticancer drugs are discussed.     

Crystal structure of (theophyllinato)methylmercury(II) monohydrate

The title compound belongs to space group P2₁/c, a = 10.884 Å, b = 9.187 Å, c = 14.458 Å, β = 131.02°, Z = 4. The structure was refined on 1355 nonzero reflections to an R factor of 0.059. The crystal contains discrete [CH₃Hg(theophyllinate)] molecules in which the proton initially bound to N7 is replaced by the CH₃Hg⁺ ion. The water molecule forms hydrogen bonds with both carbonyl oxygens, whereas an intermolecular contact of 2.98 Å is established between mercury and N9. The intramolecular Hg?O6 distance of 3.18 Å is consistent with the absence of significant Hg?carbonyl bonding interactions in the present structure.     

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.     

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.     

Mercury(II) binding to metallothioneins. Variables governing the formation and structural features of the mammalian Hg-MT species.

With the aim of extending our knowledge on the reaction pathways of Zn-metallothionein (MT) and apo-MT species in the presence of Hg(II), we monitored the titration of Zn7-MT, Zn4-alphaMT and Zn3-betaMT proteins, at pH 7 and 3, with either HgCl2 or Hg(ClO4)2 by CD and UV-vis spectroscopy. Detailed analysis of the optical data revealed that standard variables, such as the pH of the solution, the binding ability of the counter-ion (chloride or perchlorate), and the time elapsed between subsequent additions of Hg(II) to the protein, play a determinant role in the stoichiometry, stereochemistry and degree of folding of the Hg-MT species. Despite the fact that the effect of these variables is unquestionable, it is difficult to generalize. Overall, it can be concluded that the reaction conditions [pH, time elapsed between subsequent additions of Hg(II) to the protein] affect the structural properties more substantially than the stoichiometry of the Hg-MT species, and that the role of the counter-ion becomes particularly apparent on the structure of overloaded Hg-MT.     

Chemical cleavage of plasmid DNA by Cu(II), Ni(II) and Co(III) desferal complexes.

It is demonstrated that the Cu(II), Co(III) and Ni(II) complexes of a siderophore chelating drug desferal cleave DNA, in contrast to the corresponding Fe(II) complex which does not bring about DNA scission. Hydroxy radical scavengers inhibit the cleavage reaction.     

Chemical probes of the conformation of DNA modified by cis-diamminedichloroplatinum(II)

The purpose of this work was to analyze at the nucleotide level the distortions induced by the binding of cis-diamminedichloroplatinum(II) (cis-DDP) to DNA by means of chemical probes. In order to test the chemical probes, experiments were first carried out on two platinated oligonucleotides. It has been verified by circular dichroism and gel electrophoresis that the binding of cis-DDP to an AG or to a GTG site within a double-stranded oligonucleotide distorts the double helix. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers strongly suggests that the platinated oligonucleotides are bent. The reactivity of the oligonucleotide platinated at the GTG site with chloroacetaldehyde, diethyl pyrocarbonate, and osmium tetraoxide, respectively, suggests a local denaturation of the double helix. The 5'G residue and the T residue within the adduct are no longer paired, while the 3'G residue is paired. The double helix is more distorted (but not denatured) at the 5' side of the adduct than at the 3' side. In the case of the oligonucleotide platinated at the AG site, the double helix is also more distorted at the 5' side of the adduct than at the 3' side. The G residue within the adduct is paired. The reactivities of the chemical probes with six platinated DNA restriction fragments show that even at a relatively high level of platination only a few base pairs are unpaired but the double helix is largely distorted. No local denaturation has been detected at the GG sites separated from the nearest GG or AG sites by at least three bases pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro lymphocyte cytotoxicity. II. Unstable lymphotoxins (beta-LT) secreted and inactivated by mitogen-stimulated human lymphocytes.

Supernatants from phytohemagglutinin (PHA)-activated human lymphocytes contain two major classes of cytotoxins (α-LT and β-LT). While α-LT appears to be stable, and the major component in 5-day culture supernatants, the majority of cytolytic activity at earlier intervals in these cultures is due to a “family” of highly unstable cytotoxins which are both secreted and destroyed at a rapid rate. The inactivation of the unstable LT molecules appears to be due to: (a) inherent instability o β-LT molecules, and (b) a lymphocyte-mediated inactivation mechanism(s) which involves serum.     

Chelation therapy for methylmercury(II) poisoning. Synthesis and determination of solubility properties of MeHg(II) complexes of thiol and dithiol antidotes

Methylmercury(II) complexes of the most widely studied antidotes for mercury poisoning have been prepared, and both the water solubility and 1-octanol/water partition coefficients determined for these complexes and the L-cysteine complex. New complexes of N-acetyl-D,L-penicillamine, 2-mercaptosuccinic acid, meso-dimercaptosuccinic acid, and Unithiol have been synthesized and characterized, and are found to have the formulations MeHgSCMe2CH(NHCOMe)CO2H, MeHgSCH(CO2H)CH2CO2H, MeHgSCH(CO2H)CH(CO2H)SHgMe, and Na[MeHgSCH2CH-(SHgMe)CH2SO3], respectively. Trends in octanol/water partition coefficients are consistent with reported studies of the effectiveness of antidotes for MeHg(II) poisoning and redistribution of MeHg(II) on administration of antidotes, particularly for British anti-Lewisite, Unithiol, and meso-dimercaptosuccinic acid.     

Binding of methylmercury(II) by HeLa S3 suspension-culture cells: Intracellular methylmercury levels and their effect on DNA replication and protein synthesis

HeLa S3 cells were exposed to varied concentrations of methylmercury over varied periods of time and its binding by the cells was studied using ²⁰³Hg-labeled methylmercuric chloride as radioactive marker. Also studied was the effect of cell-bound methylmercury on DNA replication and protein synthesis and on the growth rate of the cells. The results show that methylmercury binding is a rapid process, with much of the organomercurial bound within the the first 60 min of incubation, and that considerable quantities of organic mercury become affixed to the cells. The amounts of bound methylmercury, [CH₃Hg(II)]_bound, given in mol/cell, range from 2 × 10^?16 (at 1 h of incubation and at 1 μM CH₃Hg(II) in the medium) to almost 4 × 10^?14 (at 24 h of incubation and at 100 μM CH₃Hg(II) in the medium). A [CH₃Hg(II)]_bound value of about 30 × 10^?16 mol/cell appears to be the threshold below which cells display a normal growth pattern and below which metabolic events such as DNA replication or protein synthesis are affected only to a minor degree but above which major changes in cell metabolism and cell growth take place. Methylmercury binding by the cells is tight so that only 20% of the bound material is released from the cells over a 3-h incubation period when the cells are placed into fresh, methylmercury-free growth medium. Analysis of the binding data in terms of binding to identical and completely independent sites yields an association constant K of 7.92 × 10⁴ l/mol and for the maximum concentration of cellular binding sites the value 2.40 × 10^?14 mol/cell or 1.45 × 10¹⁰ sites/cell. Evidence is presented which shows that cellular sulfhydryl groups do not suffice to provide all the sites taken up by methylmercury and that binding, in all likelihood, involves basic nitrogen, too. The levels of cell-bound methylmercury are such that binding to HeLa DNA and HeLa chromatin, for instance, can readily take place. Methylmercury binding data obtained by using the technique of particle-induced X-ray emission (PIXE) are in good agreement with the data obtained via isotope dilution.     

Dinuclear copper(II) complexes with valsartan. Synthesis, characterization and cytotoxicity

Two novel dinuclear complexes involving the antihypertensive drug valsartan and copper(II) ion have been prepared in water and DMSO. The complex compositions were determined as: [Cu(vals)(H₂O)₃]₂.6H₂O and [Cu(vals)(H₂O)₂DMSO]₂.2H₂O. They were thoroughly characterized by elemental and thermal analysis, spectrophotometric titrations and UV-visible, diffuse reflectance, FTIR, Raman and EPR spectroscopies. No effect of the ligand on two tested osteoblastic cell lines in culture (one normal MC3T3E1 and one tumoral UMR106) was observed in concentrations up to 100 μM. Higher concentrations of Valsartan are required to induce cytotoxicity in both cell lines. The antiproliferative effect of the tested complex ([Cu(vals)(H₂O)₃]₂.6H₂O) in a dose-response manner, was higher in the UMR106 osteoblastic cell line than that of the MC3T3E1 normal line at concentrations ≥ 100 μM. Morphological alterations are in accordance with proliferative observations.     

Preparation and structural characterization of methylmercury(II) complexes of the minor tRNA-base 7-methylguanine

Methylmercury(II) complexes of 7-methylguanine (7mguaH) have been isolated from aqueous solution in the pH range 1-12 and structurally characterized. 1:1 complexes [(7mgua)HgCH₃]·2H₂O and [(7mguaH)HgCH₃][NO₃]· H₂O with respectively N1 - and N9-coordination (X-ray analyses) were obtained from solutions in the respective pH ranges 9–12 and 1–4. A 2:1 complex [(7mgua)(HgCH₃)₂][NO₃] with N1,N9-coordination (X-ray) may be prepared in the intermediate pH range 4–7. Two 3:1 complexes were isolated: [(7mgua)(HgCH₃)₃][NO₃]₂ from strongly acid solution (pH = 1–3), and [(7mguaH₋₁)(HgCH₃)₃][NO₃] in the pH range 7–9. Whereas an X-ray analysis establishes N1,N3,N9-coordination for the former species in the solid state, the ¹H NMR data suggest N2,N3,N9-coordination for the former and N2,N2,N9-coordination for the latter species in d₆-DMSO solution.     

Chemical prevention of colony foundation by Cryptotermes brevis (Isoptera: Kalotermitidae) in attic modules

Disodium octaborate tetrahydrate (DOT) dust, DOT aqueous solution, imidacloprid dust, and amorphous silica gel dust with synergized 1% pyrethrins were applied on wood surfaces to simulated attic modules. Modules (30 by 30 cm) with and without fiberglass insulation were exposed to dispersal flights of Cryptotermes brevis (Walker) in May and June of 1998 and 1999. Six months after flights, modules were disassembled and inspected for nuptial chamber location and contents. During both years, air and water control treatments contained 22.2+/-9.94 (mean +/- SD) nuptial chambers, 7.5+/-5.7 live imagos, and 2.0+/-1.4 chambers with brood. This survivorship indicated that the attic modules performed well as a colonizing platform for C. brevis. C. brevis dealates preferred constructing nuptial chambers in the crevices at the bases or tops of the modules instead of internal crevices. Modules treated in 1998 and 1999 with DOT or silica dusts contained no live termites, whereas zero of five modules treated with imidacloprid dust in 1998 and two of 20 modules treated with imidacloprid dust in 1999 contained single live incipient colonies. In 1998, 15% DOT solution, applied as a postconstruction treatment, yielded significantly fewer chambers and live termites than controls, but was not as effective as dusts in preventing successful colonization. In 1999, the DOT solution, applied as a construction-phase treatment, was equally as effective in preventing colonization as the dust treatments during that year. Results indicate that dust formulations of DOT, silica gel, and imidacloprid can be used to prevent drywood termite colonization in existing building voids and attics. Where the entire wood framing is exposed to treatment, such as during building construction, aqueous DOT solution can be equally effective as dusts in preventing colonization by C. brevis.     

Heavy metal-nucleotide interactions. 15. Reactions of calf thymus DNA with the electrophiles methylmercury(II) nitrate, cis-dichlorodiammineplatinum(II), and trans-dichlorodiammineplatinum(II) studied using Raman difference spectroscopy. Evidence for the formation of c-DNA upon metalation

This study details the reactions of the electrophiles CH3Hg(NO3), cis-[PtCl2(NH3)2] (cis-DDP) and trans-[PtCl2(NH3)2] (trans-DDP) with calf thymus DNA using Raman and Raman difference spectroscopy. The order of CH3Hg(II) binding to calf thymus DNA is G > T > C > A. The electrophilic attack of cis- and trans-DDP on calf thymus DNA produces different orders of binding: cis-DDP-G>C approximately AT, trans-DDP-G approximately C approximately AT. The reaction of CH3Hg(II) with DNA results in a decrease in the percentage of B-form DNA. whereas the reactions of cis- and trans-DDP with DNA decrease the percentage of B-DNA and cause the formation of C-DNA structure.     

Enhancement of cytotoxicity of bleomycin by dithiocarbamates: formation of bis(dithiocarbamato) cu(II).

Properties of the reactions of dithiocarbamates and their Cu(II) or Fe(III) complexes with Ehrlich cells were determined and related to their effects on the inhibition of cell proliferation caused by bleomycin and Cu bleomycin. In complete culture medium containing Eagle's minimal essential medium plus Earles salts and 2.5% fetal calf serum, dimethyl- and diethyldithiocarbamates and their copper complexes inhibit cell proliferation and cause cell death. The copper complexes are more effective agents. Ferric tris-diethyldithiocarbamate is also a cytotoxic species. In contrast, when cells are exposed to dimethyldithiocarbamate or its copper complex in Ringer's buffer under metal-restricted condition, washed, and then placed in complete medium, the copper complex is much more active in inhibiting cell growth. The difference is magnified when dihydroxyethyldithiocarbamate and N-methylglucamine dithiocarbamate and their copper complexes are compared in complete media. Incubation of bleomycin or copper bleomycin with Ehrlich cells in Ringer's buffer with or without dimethyldithiocarbamate or bis-dimethyldithiocarbamato Cu(II) leads to no enhancement of cytotoxicity from combinations of agents, except when the two copper complexes are present. Diethyl- or dimethyldithiocarbamate readily extracts copper from Cu(II)bleomycin and iron from Fe(III)bleomycin when ethylacetate is present to remove the tris-dithiocarbamato Fe(III) complex from aqueous solution. When bis-dimethyldithiocarbamato Cu(II) is incubated with Ehrlich cells, copper is released from the complex and bound to high molecular weight and metallothionein fractions. A reductive mode of dissociation of the copper complexes in cells is supported by ESR experiments. Reactions of diethyl- and dimethyldithiocarbamato Cu(II) with thiol compounds demonstrates one possible mechanism of reduction of these complexes.     

Metal ion biomolecule interactions. VI: Synthesis and spectroscopic properties of methylmercury(II) complexes of xanthosine

The interaction of MeHg(II) with xanthosine (Xanth H₂, 1) in aqueous medium has been found to lead to several methylmercurated complexes depending on the reactant stoichiometries and the pH. The N-bound complexes [(MeHg)(Xanth H)] (2), [(MeHg)₂Xanth] (3), [(MeHg)₃(Xanth)]NO₃ (4), [(MeHg)(Xanth H₂)]NO₃ (5), and the N- and C-bound complex [(MeHg)₄(Xanth)]NO₃ (6) have thus been prepared. The complexes were characterized by means of ¹H and ¹³C nuclear magnetic resonance and infrared as well as elemental analysis. Formation of the carbon-bound methylmercurated species 6 is in accord with our previous results obtained with inosine and imidazole derivatives, thus substantiating our proposal that activation through electrophilic coordination at N₇ is a requirement for C₈-H abstraction. Correlations are drawn between ²J(¹H-¹¹⁹Hg) values and pK_a as well as ¹³C chemical shifts.