Differential regulation of mouse kidney sodium-dependent transporters mRNA by cadmium.

Chronic exposure to cadmium can result in renal glycosuria. Previously, we reported that cadmium reduced the relative abundance of the sodium-glucose cotransporter mRNA (Blumenthal et al., Toxicol. Appl. Pharmacol.149, 49-54, 1998). To investigate this phenomenon further, we isolated full-length cDNA clones encoding both high- and low-affinity sodium-dependent glucose transporters SGLT1 and SGLT2, respectively, from cultured mouse kidney cortical cells. We also amplified a fragment of another putative sodium-glucose cotransporter with homology to the known SAAT1/pSGLT2 or SGLT3 from our cultured cells and named it SGLT3. In order to examine the effect of cadmium on these transporters, primary cultures of mouse kidney cortical cells were exposed to micromolar concentrations of cadmium for 24 h and levels of SGLT1, SGLT2, and SGLT3 mRNA were determined by semiquantitative RT-PCR. Five to 10 microM of cadmium inhibited sodium-dependent uptake of the glucose analog, alpha-methyl D-glucopyranoside and progressively reduced the level of SGLT1. Cadmium also inhibited SGLT2 mRNA by 37%, but no further decline was observed at concentrations of cadmium greater than 5 microM. While cadmium inhibited SGLT1 and SGLT2, it significantly stimulated the expression of SGLT3 by fivefold. These results imply that individual sodium-glucose cotransporter mRNA species are not regulated in a similar fashion. In addition, the isolation of three separate SGLT species from these cultures suggests that, in addition to SGLT1 and SGLT2, glucose reabsorption by renal epithelial cells might involve additional glucose transporters such as SGLT3.

Model reactions of Cr (VI) with DNA mediated by thiol species.

Model reactions were devised to investigate the capacity of physiologically interesting thiol compounds to mediate reactions between CrO4(2-) (Cr (VI)) and DNA. The sulfhydryl containing reagents included cysteine, glutathione, apo-metallothionein (apoMT). Zinc finger 3 of transcription factor IIIA (Zn-F3) of Xenopus laevis was investigated as a potential redox active site of reaction of Cr (VI) and thiol compounds. The DNA samples were calf thymus DNA and two oligomers, one of them specific for binding Zn-F3. Results showed that in the presence of Cr (VI) apoMT readily participated in damaging DNA in a reaction that appeared to be hydroxyl radical dependent. It also became cross-linked to oligomer and native DNA samples. In comparison, the other two thiol donors were largely inactive in these assays even though they, like apoMT, were able to reduce Cr (VI) to Cr (III) under the conditions of the experiments. Direct attempts to cross link thiols with DNA in the presence of Cr3+ were unsuccessful at pH 7.4. Together, the results indicate that apoMT can effectively collaborate with Cr (VI) in reactions that are deleterious to DNA.

Interactions of iron bleomycin, phosphate or cyanide, and DNA: sequence-dependent conformations and reactions.

The hypothesis was investigated that axial ligands bound to Fe(III)-bleomycin [Fe(III)Blm] are destabilized at specific 5'-guanine-pyrimidine-3' binding sites but are stable at nonselective dinucleotides. DNA oligomers and calf-thymus DNA were used in reactions with L-Fe(III)Blm, where phosphate and cyanide served as examples of large and small ligands (L). Both ligands underwent dissociation when L-Fe(III)Blm was bound to d(GGAAGCTTCC)2 (I) but not d(GGAAATTTCCC)2 (II) and at large ratios of calf-thymus DNA to drug. Fe(III)Blm is high spin in 20 mM phosphate buffer, signifying the presence of a phosphate adduct. In the titration of HPO4-Fe(III)Blm with calf-thymus DNA, a large excess of DNA was needed to reach the low-spin state, consistent with an equilibrium competition between phosphate and DNA for Fe(III)Blm. Equilibrium constants for binding Fe(III)Blm and CN-Fe(III)Blm to calf-thymus DNA (6.8x10(5) M(-1) and 5.9x10(4) M(-1), respectively, in HEPES buffer at 25 degrees C and pH 7.4) showed that the CN- ligand also reduced the affinity of DNA for the drug. The kinetics of dissociation of CN- from CN-Fe(III)Blm-DNA were slow and first order in bound drug. The reversible nature of these dissociation reactions was shown using 1H NMR spectroscopy of Fe(III)Blm-I in the absence and presence of large excesses of CN- or phosphate. The results are discussed in terms of a two-state hypothesis for the binding of L-Fe(III)Blm to specific and nonspecific dinucleotides. It is proposed that steric restrictions at specific sites inhibit binding of these ligands.

Comparative binding properties of metallobleomycins with DNA 10-mers.

Properties of the interaction of bleomycin (Blm) and metallobleomycins [M = Zn, Cu(II), Fe(III), and HO(2)-Co(III)] with site-specific and nonspecific DNA oligomers, d(GGAAGCTTCC)(2) (I) and d(GGAAATTTCC)(2) (II), respectively, were investigated. With both 10-mers association constants increased in the series Blm A(2), ZnBlm A(2), Cu(II)Blm A(2), Fe(III)Blm A(2), and HO(2)-Co(III)Blm A(2). Generally, the metallobleomycins were bound with a modestly higher affinity to I. One-dimensional (1)H NMR spectra of the imino proton region of I in the presence of this series of compounds revealed that Blm and Zn- and CuBlm bind in fast exchange on the NMR time scale, while the Fe and Co complexes bind in slow exchange. Blm, ZnBlm, and Cu(II)Blm caused little perturbation of the UV circular dichroism spectrum of I or II. In contrast, Fe(III)Blm and HO(2)-Co(III)Blm induced hypochromic effects in the CD spectrum of I and altered the spectrum of II to a smaller extent. On the basis of these results, the DNA binding structures and properties of Blm A(2), ZnBlm A(2), and CuBlm A(2) differ substantially from those of Fe(III)Blm A(2) and HO(2)-Co(III)Blm A(2).

Raman spectroscopy of an O(2)-Co(II)bleomycin-calf thymus DNA adduct: alternate polymer conformations.

Bleomycin (Blm) is an antitumor agent which binds to specific sequences of DNA and as HO(2)-Fe(III)Blm causes single and double strand cleavage. In the present investigation, binding of O(2)-Co(II)Blm to a native DNA polymer, calf thymus DNA, was examined using conventional Raman spectroscopy. O(2)-Co(II)Blm is a model for O(2)-Fe(II)Blm, the direct precursor of HO(2)-Fe(III)Blm. Although the DNA polymer retained a predominant B-form structure, Raman spectral evidence was obtained for localized structural changes to A, C and Z-DNA forms. The presence of these alternate DNA forms within B-DNA implied the presence of B/A, B/C and B/Z junctions. The observed changes in DNA secondary structure were attributed to perturbation of structural water resulting from binding of O(2)-Co(II)Blm within the minor groove.

Structure-reactivity relationships among metallothionein three-metal domains: role of non-cysteine amino acid residues in lobster metallothionein and human metallothionein-3.

Metallothionein (MT) domains of different origins, exhibiting distinct, highly conserved cysteine positions, show differences in metal-cysteine coordination and reactivity. Lobster MT, which includes two Cd3S9 beta domains, was chosen as a basic model to study the structure-function relationship among the clusters. The possible influence of (1) the position of the cysteine residues and (2) the steric and electrostatic effects of neighboring amino acids on the folding and stability of MT clusters have been examined with the native lobster beta C and beta N domains, each having nine cysteines and binding three M2+ ions, and a modified domain beta C-->N, in which the cysteines of the C-terminal domain are relocated so they are spaced as in the N-terminal domain. Each has been synthesized and characterized by UV, CD, 113Cd NMR, and 1H NMR spectroscopies. The synthetic native domains (Cd3 beta C and Cd3 beta N) displayed spectroscopic properties, metal-binding affinities, and kinetic reactivity similar to those of the holo protein. In contrast, the modified Cd3 beta C-->N domain was unusually reactive and, in the presence of Chelex, a metal-ion chelating resin, was converted to a Cd5(beta C-->N)2 dimer. These differences in structure and reactivity demonstrate that the requirements for formation of a stable type-B, Cd3S9, beta cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and include specific interactions with neighboring amino acids. Molecular mechanics calculations suggest that changes of even a single amino acid in lobster Cd3 beta N toward lobster Cd3 beta C-->N or in mammalian MT1 or MT2 toward Cd3 beta-MT3 (GIF) can destabilize their structures.

Orientation of iron bleomycin and porphyrin complexes on DNA fibers.

Bleomycin (Blm) is an antitumor agent that requires iron and oxygen for strand cleavage of DNA. In this study, ferric bleomycin, Fe(III)Blm, or the nitric oxide adduct of ferrous bleomycin, ON-Fe(II)Blm, were bound to one-dimensionally oriented DNA fibers. Reductive nitrosylation of Fe(III) complexes took place in situ on B-form DNA fibers. Electron paramagnetic resonance (EPR) spectra were obtained as a function of the angle phi between the magnetic field B and the fiber axis Zf. For comparison, EPR spectra were acquired for ON-Fe(II)TMpyP and ON-Fe(II)TMpyP-Im on oriented DNA fibers, where TMpyP is 5,10,15,20-tetrakis(1-methyl-4-pyridino)porphyrin and Im is imidazole. EPR spectra showed both low-spin Fe(III)Blm and ON-Fe(II)Blm bound to B-form DNA in two slightly different binding orientations in the ratio of 1:0.2. With A-form DNA, a fraction of bound Fe(III)Blm was high spin. Specifically, the angle beta between the fiber axis Zf and the g axis, gz, perpendicular to or nearly perpendicular to the equatorial plane of the iron complex was estimated as 20 degrees and 25 degrees for ON-Fe(II)Blm and 30 degrees and 25 degrees for Fe(III)Blm, respectively. The angle gamma that determines the orientation of gx and gy axes was estimated as 90 degrees for the two ON-Fe(II)Blm species and 10 degrees for the two Fe(III)Blm species, respectively. The NO was held rigidly in place as the temperature increased from 123 K to room temperature for ON-Fe(II)Blm but not for ON-Fe(II)TMpyP or ON-Fe(II)TMpyP-Im. It is hypothesized that the NO is structurally oriented by hydrogen bonding like the peroxide is held in HO2(-)-Co(III)Blm (Wu et al. J. Am. Chem. Soc. 1996, 118, 1281-1294). The EPR parameters are consistent with a six-coordinate complex for ON-Fe(II)Blm, although the superhyperfine structure from the trans nitrogen was not detected. The increase in g value anisotropy upon binding ON-Fe(II)Blm to DNA fiber may be caused by an increase in the overlap of d pi and 2p pi* orbitals induced by an interaction of NO with DNA and/or by a perturbation of d orbitals due to the pyrimidine-guanine interaction. It is concluded that the EPR parameters of ON-Fe(II)Blm and Fe(III)Blm bound to oriented DNA support the hypothesis that FeBlm species bind to DNA with adduct structures similar to those formed by related CoBlm species and DNA.

The requirements for stable metallothionein clusters examined using synthetic lobster domains.

Metallothioneins (MTs) are small, cysteine-rich proteins which detoxify xenobiotic metals such as cadmium (Cd) and mercury (Hg). In crustaceans and mammals they consist of two independent domains which are folded around metal-thiolate clusters. MT clusters of different origins, exhibiting distinct, highly conserved cysteine positions on their sequences, show differences in metal-cysteine coordination and reactivity. Lobster-MT, containing two Cd3 beta domains, is an important model for structure-function relationships among the clusters. The influence of (1) the position of the cysteine residues and (2) steric and electrostatic effects of neighboring amino acids on the folding and stability of MT cluster were investigated. Thus, the native lobster beta C and beta N domains (each having nine cysteines and binding three M2+ ions) and a modified domain Cd3 beta C-->N, in which the cysteines of the C-terminal domain were relocated to match the positions of those in the N-terminal domain, were chemically prepared and characterized. The synthetic native domains (Cd3 beta C and Cd3 beta N) were found to exhibit spectroscopic properties, metal-binding affinities and kinetic reactivity similar to the holo-protein. However, the modified Cd3 beta C-->N domain was unusually reactive and in the presence of Chelex, metal chelation resin, aggregated to a Cd5(beta C-->N)2 dimer, which exhibited unusual structure as observed by its 113Cd-nuclear magnetic resonance. These differences in structure and reactivity demonstrated that the requirements for formation of a stable Cd3S9 beta-cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and must include interactions involving neighboring, noncysteine amino acids.

Cadmium and lead interactions with transcription factor IIIA from Xenopus laevis: a model for zinc finger protein reactions with toxic metal ions and metallothionein.

Zinc finger proteins comprise the largest class of eukaryotic transcription factors. The metal binding sites in these proteins have been proposed as plausible targets for exchange reactions between zinc and toxic metal ions that lead to the alteration of function of the proteins in gene transcription. According to the present work, both Cd2+ and Pb2+ displace Zn2+ from transcription factor IIIA (TFIIIA). Neither product binds to the internal control region (ICR) of the 5 S rRNA gene, the normal binding site for Zn-TFIIIA. Furthermore, the adduct of Zn-TFIIIA with ICR is also reactive with Cd2+ and Pb2+, leading to the dissociation of the DNA-protein complex. Cd-TFIIIA reacts with apometallothionein (apoMT) to form Cd-MT and apoTFIIIA. Similarly, Cd2+ and Zn2+ can be exchanged in the reaction of Cd-TFIIIA with Zn-MT. Zn-finger 3 of TFIIIA has also been examined to compare the reactivity of a single finger motif with fingers in the holoprotein. Zn-finger 3 reacts with much faster kinetics than the holoprotein.

Characterization of the cadmium complex of peptide 49-61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA.

The synthetic peptide fragment containing residues 49-61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH), which includes the only sequential four cysteines bound to the same metal ion in Cd7MT, forms a stable, monomeric Cd-peptide complex with 1:1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This represents the first synthesis of a single metal-binding site of MT independent of the domains. The 111Cd NMR chemical shift at 716 ppm indicates that the 111Cd2+ in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation for this Cd-peptide derivative, approximately 3.3, demonstrates an affinity similar to that for Cd7MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd2+ center has the same counterclockwise chirality (lambda or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced protein folding. However, the kinetic reactivity of the CdS4 structure toward 5,5'-dithiobis(5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (a-domain or holo-protein). The rate law for the reaction with DTNB is rate = (k(uf) + k(1,f) + k(2,f) [DTNB])[peptide], where k(uf) = 0.15 s(-1), k(1,f)= 2.59x10(-3) s(-1), and k(2,f) = 0.88 M(-1) s(-1). The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M7MT) and crustacean (M6MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination chemistry, including stoichiometries of M-peptide for Hg2+, Cd2+, and Zn2+, M2-peptide for Hg2+ and Au+, and (Et3PAu)2-peptide.

Interprotein metal ion exchange between cadmium-carbonic anhydrase and apo- or zinc-metallothionein.

The ligand substitution reactions of cadmium-carbonic anhydrase with EDTA and pyridine-2,6-dicarboxylic acid were compared with one in which rabbit apometallothionein was the competing metal-binding agent. This last reaction occurred more rapidly than the other two at a much smaller ratio of competing ligand to Cd-carbonic anhydrase. It was characterized as a second-order reaction, first-order in Cd-carbonic anhydrase and in apometallothionein, having a rate constant of 5.8 +/- 0.1 M-1 s-1 at 25 degrees C and pH 7.4 in Tris.HCl buffer and 0.1 M KCl. At 25 degrees C, Zn7-metallothionein also exchanged metal ions with Cd-carbonic anhydrase with a rate constant of 0.33 +/- 0.02 M-1 s-1 to reconstitute enzymatically active protein. Cd-carbonic anhydrase reacted within the time of mixing with the peptide sequence 49-61 of rabbit metallothionein 2 which contains four cysteinyl residues, leading to the exchange of most of the Cd2+ into the peptide. At pH 7.4 and 25 degrees C, Cd2+ has higher affinity for apometallothionein than for the apo-peptide.

A systematic approach toward the analysis of drug-DNA interactions using Raman spectroscopy: the binding of metal-free bleomycins A(2) and B(2) to calf thymus DNA.

Bleomycins A(2) and B(2) are the two active components in the antineoplastic drug Blenoxane. DNA is targeted by this drug in cancer cells and the mode of action of this drug involves DNA binding. Ambiguity exists as to the way in which bleomycin binds to DNA. Raman spectroscopy was used to examine both calf thymus DNA and a bleomycin/DNA complex at two temperatures. A curvefitting technique was applied to these spectra for a spectral region obscured by many overlapping bands associated with the nucleotide bases in order to derive information about frequencies, bandwidths, and intensities of the vibrational modes in this region. This allowed identification and analysis of bands associated with specific assigned nucleotide base residues. Upon binding of bleomycin, several significant changes in bandwidth, intensities, and frequencies relative to uncomplexed DNA were observed consistently at both higher (30 degrees C) and lower (19 degrees C) temperature. The data presented here support at least a partial intercalation mode of binding for bleomycin that is temperature dependent and more pronounced at the more physiologically relevant temperature of 30 degrees C.

The presence of two modes of binding to calf thymus DNA by metal-free bleomycin: a low frequency Raman study.

Double-stranded DNA is targeted by bleomycin in cancer cells and ambiguity exists as to its mode of DNA binding. A conventional Raman study was performed on drug/DNA complexes in which the low frequency spectral region (560-930 cm(-1)) was examined at two temperatures (19 and 30 degrees C). At 30 degrees C, a global Raman hypochromism was observed consistent with partial intercalation of the bithiazole moiety. At 19 degrees C, Raman hypochromism (increased base pair stacking) was detected for bands associated with GC base pairs while Raman hyperchromism (base pair destacking) was evident for bands associated with AT base pairs. These results suggest that intercalation of the bithiazole moiety occurs with greater disruption of the more efficiently stacked AT base pairs at the lower temperature. Evidence for minor groove binding was indicated by an increase in the population of bands corresponding to C3' endo sugar conformations resulting from drug induced local desolvation of the DNA polymer.

Protection of DNA in HL-60 cells from damage generated by hydroxyl radicals produced by reaction of H2O2 with cell iron by zinc-metallothionein.

Scavenging of hydroxyl radicals (.OH) by the zinc form of metallothionein (ZnMT) was studied in HL-60 cells and in nuclei from such cells previously treated with ZnCl2 (ZnMT cells). Cells were grown for 48 h to label DNA for alkaline elusion experiments. During the last 24 h 0.1 mM ZnMT was included to induce ZnMT. Generation of DNA single-strand breaks (SSBs) by H2O2 in cells (5 x 10(5)/ml) treated at 4 degrees was increased by approximately 70% in Zn-treated cells by comparison with control cells. These cells had grown from an initial concentration of 5 x 10(5)/ml to a concentration at harvest of 16 x 10(5)/ml. Cells started at 6 x 10(5)/ml and growing to a final concentration of 20 x 10(5)/ml did not exhibit a similar increase in SSBs. This elevation in SSBs was traced to an increase in cell Fe content which exhibited a sharp dependence upon concentrations of cells and of ZnCl2 at the time of addition. The diffusion distance (d) from Fe to DNA of ZnMT cells treated with H2O2 was found to be 3.4 nm. This compares with a distance of 6.1 nm in control cells. SSB generation by hydroxyl radicals formed by 137Cs-gamma rays in Zn-treated cells decreased by 12%, accompanied by a decrease in d from 4.8 nm to 2.9 nm. Thus, ZnMT preferentially reacts with OH formed at some distance from DNA. In nuclei isolated from ZnMT cells started at 5 x 10(5)/ml, SSB generation by H2O2 increased by 60%. The d in these nuclei was 4.9 nm, similar to the distance in control nuclei reported previously. These data suggest that, in addition to altering the scavenging environment, treatment of cells with Zn leads to an increase in reactive Fe in cells and in isolated nuclei which can generate DNA damage through reaction with H2O2.

Cadmium decreases SGLT1 messenger RNA in mouse kidney cells.

Mouse renal cortical tubule cells in primary culture exposed to cadmium (Cd2+) develop decreased Na(+)-glucose cotransport activity as measured by uptake of the glucose analogue alpha-methyl-glucoside. RNA was isolated from kidney cell cultures, and after reversed transcription, the DNA was amplified with primers to rat SGLT1 (the high affinity isoform of the sodium glucose cotransporter) and mouse beta-actin. Only one product was identified after amplification with the rat SGLT1 primers, which on sequencing was 96% identical to rat SGLT1. Compared to beta-actin, the intensity of the SGLT1 message declined progressively as CdCl2 concentration in the medium increased from 0 to 10 microM. Similar decreases in SGLT1 mRNA were also observed as media zinc (Zn2+) concentrations rose from 0 to 75 microM or as copper (Cu) concentrations increased from 0 to 150 microM. Exposure to 8 microM Cd as Cd-metallothionein (Cd7-MT) also caused a fall in relative SGLT1 mRNA abundance, and at nearly identical internal Cd concentrations of 40-43 pmol/microgram DNA, both Cd7-MT and CdCl2 reduced SGLT1 mRNA to 33% of control. In general, the fall in SGLT1 mRNA was more rapid than the decline in Na(+)-dependent glucose uptake after cells were exposed to Cd2+. These findings suggest that the effects of Cd2+ and other metals on renal glucose transport are related to decreased expression of SGLT1 message.

Fe- and Co-bleomycins bound to site specific and nonspecific DNA decamers: comparative binding and reactivity of their metal centers.

Co- and Fe-bleomycins (Blms) have been reacted with DNAa, d(GGAAGCTTCC)2, containing a specific site for cleavage, and DNAb, d(GGAAATTTCC)2, a closely related nonspecific 10-mer, to survey whether features of structure and reactivity of these adducts vary systematically as a function of the base sequence of the DNA oligomer. The ESR spectrum of NO-Fe(II)BlmDNAa is rhombically perturbed in comparison with that of NO-Fe(II)BlmDNAb, which is nearly identical to the spectrum of NO-Fe(II)Blm. The ESR spectrum of Fe(III)BlmDNAa in phosphate buffer is low-spin; that of Fe(III)BlmDNAb is high-spin as seen with Fe(III)Blm alone. According to absorbance spectroscopy, O2-Fe(II)BlmDNAa is stabilized in comparison with the DNAb adduct. Similar stabilization of O2-Co(II)Blm bound to DNAa but not to DNAb was also observed by ESR spectroscopy. HO2(-)-Co(III)Blm A2 binds in slow exchange on the NMR time scale to DNAa at its 5'-G-pyrimidine-3' site of cleavage. In contrast, fluorescence and NMR spectroscopy demonstrate that most of HO2(-)-Co(III)Blm A2 binds stoichiometrically in fast exchange to DNAb. The reactions of Fe(III)BlmDNAa and Fe(III)BlmDNAb with ascorbate and O2 reveal that the latter becomes activated and cleaves its 10-mer, producing base propenals, at a faster initial rate. Thus, in two series of metallobleomycins, (A) NO-Fe(II)Blm, O2-Fe(II)Blm, Fe(III)Blm in phosphate buffer, and HO2(-)-Fe(III)Blm and (B) O2-Co(II)Blm and HO2(-)-Co(III)Blm, the metal domain of each species interacts differently with DNA depending upon its base sequence.

Direct reaction of H2O2 with sulfhydryl groups in HL-60 cells: zinc-metallothionein and other sites.

The reaction of the sulfhydryl groups in metallothionein with hydrogen peroxide was examined in HL-60 cells. Partial purification of cell cytosol using Sephadex G-75 chromatography showed that zinc-metallothionein (Zn-MT) was induced by 24-h treatment with 100 microM ZnCl2, but the cellular glutathione content and glutathione peroxidase and catalase activities were unaffected. The ratio of H202 concentrations needed to reduce cell survival 50% in Zn-induced cells compared to normal cells was 1.65 to 1. According to alkaline elution experiments, the average ratio of single-strand breaks caused by H202 at 37 degrees C in Zn-induced vs normal cells was 0.5 to 1. A similar reduction in strand breakage was seen in nuclei from Zn-treated cells exposed to H202; however, at 4 degrees C protection against DNA strand breakage by Zn pretreatment was not seen. Incubation of Zn-pretreated cells with H202 at 37 degrees C but not 4 degrees C was accompanied by loss of Zn bound to MT and a reduction in the number of MT sulfhydryl groups. In the absence or presence of Zn-MT, sulfhydryl groups from glutathione and protein fractions were also reduced by exposure of cells to H202. However, thiolate groups in the MT fraction were preferentially lost compared to the other pools of sulfhydryl residues. Zn-MT also spared glutathione sulfhydryl groups in vitro from oxidation by H202. Protection against strand breakage correlated with the ability of Zn-MT to react in vitro with H202 at 37 degrees C, but not at 4 degrees C. The reaction was slow and was not inhibited by the presence of an hydroxyl radical scavenger, dimethyl sulfoxide. Similarly, in cells dimethyl sulfoxide did not prevent the loss of sulfhydryl groups from glutathione or protein. Incubation of MT or higher molecular weight fractions from cells exposed to H202 with either 2-mercaptoethanol or dithiothreitol in the presence of Cd failed to regenerate any detectable, reduced MT, suggesting that MT sulfhydryl groups were oxidized by H202 beyond the disulfide oxidation state.

Surface chemistry and biological pathogenicity of silicates: an X-ray photoelectron spectroscopic study.

We extend our electron spectroscopy for chemical analysis studies of the chemistry of silicates to provide direct surface chemical information on the interactions involved in silicate-induced lung and tissue pathology. A total of five fibrous and non-fibrous silicate minerals, primarily amphiboles, have been studied: anthophyllite, tremolite, cummingtonite, hornblende and actinolite. We have followed the 'inlattice' surface chemistry of these materials and monitored features such as the simultaneous presence of four- and six-coordinate (with respect to oxygen) structural aluminium, and the presence of iron in the M4 octahedral positions. In vitro experiments involving contact of the silicate with cultured murine Ehrlich cells have identified modifications in the surface chemistry of Al, Mg and Fe in the silicates and changes in cellular iron content.

Characterization of calf liver Cu,Zn-metallothionein: naturally variable Cu and Zn stoichiometries.

Cu,Zn-metallothioneins were purified from bovine calf liver in order to examine the stoichiometry of metal binding to the protein. Copper and zinc analyses were carried out by atomic absorption spectrophotometry. Consistent quantitative thiolate analyses were obtained spectrophotometrically with Ellman's reagent and amperometrically with phenylmercuric acetate. These were used to define protein concentration. A complementary method to assess the sum of the thiol and Cu(I) content of metallothionein involved titration of the reducing equivalents of the protein with ferricyanide. The stoichiometry of reaction was consistent with the oxidation of all the sulphydryl groups to disulphides and all of the bound Cu from the cuprous to the cupric oxidation state. Accordingly to these methods, total numbers of zinc plus copper ions bound to metallothionein isolated from a number of calf livers centred on about 7, 10-12, or 15 g-atoms of metal per mol of protein. The reactivity of ferricyanide and 4,7-phenylsulphonyl-2,9-dimethyl-1, 10-phenanthroline (BCS) with Cu,Zn-metallothioneins of various metal ratios was assessed. Zinc metallothionein reacted almost entirely in two slow steps with ferricyanide. As the Cu content of the protein increased, the fraction of reaction occurring in the time of mixing increased in parallel. BCS was able to remove 70-80% of metallothionein-bound Cu as Cu(I). The rest was resistant to reaction.

Stoichiometry and cluster specificity of copper binding to metallothionein: homogeneous metal clusters.

Experiments were done to define the stoichiometry of binding of Cu(I) to metallothionein (MT) and to determine its sites of binding in mixed-metal species. Spectrophotometric titrations of rabbit liver Cd7-MT 2, apoMT, and Cd4-alpha-domain with Cu(I) revealed endpoints of 3-4, 4 and 8, and 4 and 6-7 added Cu(I)/mol of MT for the three species respectively. Observed endpoints depended on conditions of the titration and the wavelength chosen for absorbance measurement. Nevertheless, from metal and sulphydryl analyses of titrated proteins that were pretreated with Chelex-100 to remove metal ions from solution, almost all of the cadmium was displaced from Cd7-MT by the addition of 7 Cu(I)/mol of MT. Similarly, 4 Cu(I)/mol of Cd4-alpha-domain completely displaced bound cadmium. The Cu4-alpha-domain was converted into a Cu6-alpha species upon addition of two equivalents of Cu(I)/mol of alpha-domain. Reaction of Cd7-MT with 7, 12 and 20 Cu/mol of MT, followed by reaction with Chelex resin, generated protein samples in each case with about 7 Cu/mol of MT. 111Cd-NMR analysis of the reaction of 111Cd7-MT with Cu(I) showed that nearly co-operative one-for-one replacement of 111Cd occurred and that the beta-domain cluster reacted before the alpha-domain cluster. Two mixed-metal MTs with Cu to Zn ratios approximating 3 to 4 and 6 to 4 were isolated from calf liver. After substitution of Zn with 111Cd, NMR spectra of each protein showed that 111Cd was confined almost completely to the alpha-domain. By inference, about 3 or 6 Cu were bound in the beta-domain of these proteins. Supporting this segregation of metal ions into domains, reaction of Cu6, Zn4-MT with nitrilotriacetate removed zinc exclusively, whereas reaction of Cu6,Cd4-MT with 4,7-phenylsulphonyl-2,9-dimethyl-1,10-phenanthroline extracted only Cu(I). Proteolytic digestion of both products followed by gel filtration demonstrated that Cu(I) and Cd were bound to fragments of the intact protein. Finally, reaction of rabbit liver 111Cd7-MT 2 with Cu10-MT 2 resulted in interprotein metal exchange in which 111Cd-moved from the beta- to the alpha-domain according to NMR analysis. In contrast with the prevalent view that six Cu(I) bind to each domain of MT, the present results show that Cu(I) binds to MT with a minimum stoichiometry of about 7 Cu(I)/mol of MT and can bind to the alpha-domain with stoichiometries of 4 or 6 Cu(I)/mol of MT. Although MTs interacting with 12 or 20 Cu(I)/mol of MT are less stable than that binding about 7 Cu(I)/mol, it appears that MT can bind Cu(I) in multiple stoichiometries.