Genotoxic effects of metal compounds.
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Animals↗
Biomedical subjects
D Beyersmann
Publications and source records attributed to D Beyersmann.
Interactions in metal carcinogenicity.
The carcinogenicity and genotoxicity of cadmium, chromium, cobalt and nickel strongly depend on their chemical ligands (speciation) which modulate their bioavailability and reactivity with biochemical targets. With the exception of hexavalent chromium, carcinogenic metal compounds are only weakly genotoxic. However, the ions of the carcinogenic metals cadmium, cobalt and nickel, and also the noncarcinogenic lead, inhibit the repair of DNA damaged by direct genotoxic agents like UV irradiation and alkylating substances, thereby enhancing the effects of the latter agents. These effects are interpreted by the interference of the toxic metal ions with biochemical functions of magnesium, calcium and zinc ions.
Animals↗
Mechanisms in nickel genotoxicity: the significance of interactions with DNA repair.
Even though nickel compounds are strong carcinogens, the underlying mechanism is still unclear. In contrast to their weak mutagenic potential, they enhance the cytotoxicity and genotoxicity of UV light, X-rays and cytostatic agents like cis-platinum, trans-platinum and mitomycin C. Studies in combination with UV light indicate an inhibition of DNA repair, presumably at the incision step of nucleotide excision repair. Possible reasons for repair inhibition are structural changes of the DNA or direct interactions with repair enzymes or proteins, possibly by competition with essential metal ions.
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Effects of cadmium on nuclear protein kinase C.
Cadmium is a carcinogen whose genotoxicity is only weak. Besides its tumor-initiating capacity, cadmium may be tumor-promoting, since it interferes with several steps of cellular signal transduction. We have investigated effects of cadmium(II) on protein kinase C (PKC), which is a key enzyme in the control of cellular growth and differentiation. Tumor-promoting phorbol esters cause an activation and translocation of PKC from the cytosol to the plasma membrane and to the nucleus of mammalian cells. In mouse 3T3/10 T 1/2 fibroblasts, cadmium(II) potentiated the effect of phorbol ester on nuclear binding and activation of PKC. Furthermore, in a reconstituted system consisting of rat liver nuclei and rat brain PKC, cadmium stimulated the binding of the enzyme to a 105-kDa protein. We propose a model in which cadmium(II) substitutes for zinc(II) in the regulatory domain of PKC, thus rendering the putative protein-protein binding site exposed. Further work is required to elucidate the potential role of the nuclear PKC binding protein(s) in the control of cell proliferation.
3T3 Cells↗
Interference of cadmium with ATP-stimulated nuclear calcium uptake.
The spatial and temporal regulation of intracellular free Ca2+ serves as a modulator of signal transduction pathways involved in cell growth and differentiation. Thus, interference of metals with intracellular Ca2+ homeostasis has been considered as a target of toxic action. We used the fluorescence indicator fura-2 to monitor the level of free Ca2+ in isolated bovine liver nuclei. Nuclei accumulated Ca2+ by an ATP-stimulated Ca2+ uptake system, which is sensitive to inhibition by thapsigargin, a specific inhibitor of intracellular Ca(2+)-ATPases. Preincubation of nuclei with nanomolar concentrations of free Cd2+ resulted in a dose-dependent inhibition of ATP-dependent nuclear Ca2+ uptake. We conclude that impairment of nuclear Ca2+ regulation caused by Cd2+ provokes alterations in nuclear events related to gene expression and cell proliferation.
Adenosine Triphosphate↗
Effects of zinc and cadmium on apoptotic DNA fragmentation in isolated bovine liver nuclei.
Isolated nuclei from mammalian cells contain a calcium-dependent endonuclease. The produced DNA fragmentation is a necessary step in the sequence of events resulting in apoptosis (programmed cell death). We report here that zinc and cadmium inhibit the calcium-dependent endonuclease. The essential metal ion zinc may counterbalance the calcium-mediated apoptosis. In contrast to zinc, cadmium alone stimulates the endonuclease by replacing calcium. Thus cadmium exerts a dual effect: micromolar concentrations inhibit the apoptotic endonuclease in the presence but activate the enzyme in the absence of calcium.
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Modulation of bradykinin-induced calcium signals by oxidative stress in PC12 cells.
The influence of oxidative stress on agonist-stimulated changes of intracellular free calcium and inositol trisphosphate in the neurosecretory PC12 cell line was investigated. The oxidant H2O2 modulated the bradykinin-induced calcium signal by decreasing the initial peak and the plateau phase in the same manner as tetraphorbolacetate, an activator of protein kinase C. Inositol trisphosphate formation, induced by bradykinin was also decreased by oxidative stress. Thiol protecting agents were able to restore the altered signal. In contrast to this, radical quenching substances had no influence on calcium signals in stressed cells. Inhibitors of several protein kinases, such as protein kinase C, protein kinase A, or cyclic GMP-dependent protein kinase showed the ability to protect the plateau phase of calcium signals against oxidative stress, but not the peak response. These results indicate that under the influence of oxidative stress multiple targets within the signal transduction cascades are affected.
Adenosine Triphosphate↗
Cadmium and zinc mediated changes of the Ca(2+)-dependent endonuclease in apoptosis.
Isolated nuclei from mammalian cells contain a Ca(2+)-dependent endonuclease [1]. The produced DNA fragmentation is a necessary step in the sequence of events resulting in apoptosis [2]. We report here that zinc inhibits the DNA fragmentation in dependence of the free Ca2+ concentrations, suggesting that a balance between zinc and calcium might regulate the Ca(2+)-dependent endonuclease. Incubation of nuclei with different free calcium concentrations combined with cadmium shows a stronger inhibition of the DNA fragmentation than zinc. Cadmium inhibits the endonuclease in a calcium-independent way. Surprisingly cadmium alone is able to stimulate the endonuclease, thus to replace Ca2+.
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Differential control of free calcium and free zinc levels in isolated bovine liver nuclei.
Modulation of intracellular signal transduction by Ca2+ and possibly Zn2+ is based on an effective homoeostatic control of the corresponding free ion concentrations. We used the fluorescent indicator fura-2 to monitor concentrations of free Ca2+ and free Zn2+ in nuclei isolated from bovine liver. The nuclei showed an ATP-stimulated accumulation of intranuclear free Ca2+, which was inhibited in the presence of the Ca(2+)-pump inhibitor thapsigargin. Furthermore, uptake and intranuclear levels of free Zn2+ were measured after incubation with different extranuclear Zn2+ concentrations. There was no stimulating effect of ATP on Zn2+ uptake. Our data suggest that the levels of intranuclear free Ca2+ and free Zn2+ are controlled differentially. A distinct regulation of free ion levels in the nucleus may contribute to the specific control of nuclear events associated with gene transcription and cell differentiation.
Adenosine Triphosphate↗
Cellular damage by ferric nitrilotriacetate and ferric citrate in V79 cells: interrelationship between lipid peroxidation, DNA strand breaks and sister chromatid exchanges.
Ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-citrate) were used to study the cellular damage mediated by iron overload with respect to cytotoxicity, lipid peroxidation, DNA strand breaks and sister chromatid exchanges (SCE). At non-toxic concentrations, Fe-NTA induced lipid peroxidation, DNA strand breaks and SCE in a dose- and time-dependent manner. Comparing the time courses of the different events, the increase in lipid peroxidation seems to be associated with the generation of DNA strand breaks, since both types of cellular damage were observed after 1-3 h of incubation. In contrast, the induction of SCE was low after 24 h and increased after 48 and 72 h treatment, indicating the requirement of other types of DNA damage. Fe-citrate was inactive in the induction of lipid peroxidation and SCE, and no significant number of DNA strand breaks were generated, as determined by the alkaline unwinding method. Our results suggest that the induction of lipid peroxidation and DNA strand breaks by iron overload depend on special features of the iron complex applied, while the chromosomal and genetic effects require site specific DNA damage dependent on intracellular iron metabolism. It is concluded that iron overload is able to induce genetic damage in intact cells provided that iron is present in a bioavailable form.
Animals↗
Role of cadmium in activating nuclear protein kinase C and the enzyme binding to nuclear protein.
Specific effects of cadmium on nuclear protein kinase C activity were found with 3T3/10T1/2 mouse fibroblast and rat liver nuclei. Treatment of the mouse fibroblasts in culture with 12-O-tetradecanoylphorbol-13-acetate resulted in the stimulation of nuclear protein kinase C activity in a "fixed" pool which is defined by its resistance to chelator extraction, whereas the chelator extractable enzyme activity, defined as the "labile" pool was unaffected. Cadmium was found to potentiate the effect of the phorbol ester, directed specifically to nuclei, since the particulate protein kinase C activity was not changed under similar treatment. In a reconstituted system consisting of rat liver nuclei and rat brain protein kinase C, cadmium stimulated the binding of the enzyme to a 105-kDa nuclear protein. The binding of a 105-kDa protein to protein kinase C is attributed strictly due to the cadmium effect, whereas a 50-kDa protein binding to protein kinase C was only enhanced by cadmium. We propose a mechanistic model, where cadmium substitutes zinc in the regulatory domain of protein kinase C rendering the putative protein-protein binding site exposed.
3T3 Cells↗
Mechanisms of cobalt(II) uptake into V79 Chinese hamster cells.
V79 Chinese hamster cells were used as a model for the characterization of the Co(II) uptake into mammalian cells as well as the mechanisms involved. Co(II) was taken up in a dose and time dependent manner. The uptake was exponential without saturation in the tested concentration range up to 400 microM CoCl2. Furthermore, there was a high intracellular cobalt accumulation at elevated extracellular Co(II) doses (up to 16 fold at 200 microM). The time course of Co(II) uptake showed a maximum after about 8-12 h with no further change after the longest tested incubation time (24 h). The uptake of Co(II) into V79 cells seems to be mediated by multiple mechanisms: active, energy consuming transport like ion pumps and endocytosis, since the Co(II) uptake was significantly reduced by ouabain (an inhibitor of the Na+/K+ATPase), N-ethylmaleinimide (an inhibitor of the Ca2+/Mg2+ATPase and the Na+/K+ATPase), chlorpromazine (a calmodulin antagonist and inhibitor of the Ca2+/Mg2+ ATPase) as well as by the endocytosis inhibitor chloroquine. Furthermore, the two agents iodoacetate and potassium cyanide, which produce ATP depletion, resulted in a diminution of the intracellular cobalt concentration. An uptake through anion channels could be excluded, since 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid was not inhibitory.
Animals↗
The genetic toxicology of cobalt.
Genetic and related effects of cobalt compounds are reviewed and discussed with respect to mechanisms. In prokaryotic assays, Co(II) salts generally are nonmutagenic. In Saccharomyces cerevisiae, CoCl2 is mutagenic to mitochondrial genes and weakly mutagenic or nonmutagenic to chromosomal genes. In plants, Co(II) salts induced gene mutations and chromosomal aberrations. In mammalian cells in vitro, Co(II) compounds caused DNA strand breaks, sister-chromatid exchanges and aneuploidy, but not chromosomal aberrations. In two cell lines, CoCl2 was weakly mutagenic. Interestingly, the poorly soluble compound CoS caused DNA strand breaks and morphological transformation of mammalian cell lines. In contrast to its weak clastogenic and mutagenic properties, cobalt(II) exerts pronounced antimutagenicity in bacteria and mostly comutagenic effects in mammalian cells. In Escherichia coli CoCl2 lowered the frequency of mutations induced by MNNG, uv or X rays. In Chinese hamster V79 cells, CoCl2 enhanced the mutagenicity and clastogenicity of uv light but not of gamma rays. Regarding direct genotoxic mechanisms, Co(II) induces the formation of reactive oxygen species when combined with hydrogen peroxide in cell-free systems. At high (i.e., millimolar) concentrations, Co(II) also decreases the fidelity of DNA synthesis. Regarding anti- and co-mutagenic mechanisms, evidence for the interference of Co(II) with DNA repair processes is discussed. These mechanisms are regarded as relevant for the risk assessment of human exposure to cobalt in combination with other agents.
Animals↗
Modulation by Co(II) of UV-induced DNA repair, mutagenesis and sister-chromatid exchanges in mammalian cells.
In bacterial test systems, Co(II) has been shown to be antimutagenic in combination with several chemical and physical agents. To investigate whether such modulations also apply to mammalian cells, the effect of Co(II) on UV-induced mutagenesis, sister-chromatid exchanges as well as DNA damage and its removal was determined. Co(II) itself is weakly mutagenic at the HPRT locus and increases the frequency of sister-chromatid exchanges. Additionally, at both endpoints the metal ions enhance the genotoxicity of UV light. To discriminate between an enhancement of DNA damage and an interference with repair processes, the number of pyrimidine cyclobutane dimers was determined by HPLC. While the induction of these DNA lesions is not affected by Co(II), their removal is inhibited at concentrations of 75 microM Co(II) and higher. Analysis of the kinetics of strand-break induction and closure after UV irradiation by nucleoid sedimentation reveals an accumulation of strand breaks in the presence of Co(II). This indicates that either the polymerization or the ligation step in excision repair is affected. Since similar interactions with the processing of UV-induced DNA damage have been observed with other carcinogenic and/or mutagenic metal ions, this appears to be a common mechanism of metal genotoxicity.
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The reduction of chromate is a prerequisite of chromium binding to cell nuclei.
Isolated calf thymus nuclei bound a chromium(III) glutathione complex in a time-dependent manner. In contrast chromium(VI) (sodium chromate) did not bind. However, when chromate was incubated with the nuclei in the presence of glutathione, chromium adducts were detected. These observations indicate that the reduction of chromate, by a reducing agent such as glutathione, is a prerequisite for the generation of bonds between the metal and constituents of the cell nuclei in vitro. Chromium adducts with nuclei are probably one cause of DNA lesions and mutations.
Animals↗
Inhibition of sarcoplasmic reticulum Ca(2+)-ATPase activity by cadmium, lead and mercury.
The effect of Cd2+, Pb2+ and Hg2+ on the Ca(2+)-ATPase activity of sarcoplasmic reticulum from rabbit muscle was studied. The concentration of relevant free and complex species for the assay conditions have been computed. As a result, ATP hydrolysis was found to be inhibited with an IC50 value of 950 nmol/l free Cd2+ or 95 nmol/l free Pb2+. Although calculation of the free Hg2+ was not possible, the comparison of the IC50 values for total metal ions show that Hg2+ is the strongest inhibitor of enzyme activity. The inhibition by Cd2+ seems to be independent of substrate concentration, whereas the inhibitory effect of Pb2+ is lowered in the presence of higher MgATP concentrations. Our data illustrate that the three heavy metals are potent inhibitors of the Ca2+ pump. Therefore low concentrations of these metal ions may disturb intracellular Ca2+ homeostasis and act on Ca(2+)-mediated cell functions.
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Two different zinc sites in bovine 5-aminolevulinate dehydratase distinguished by extended X-ray absorption fine structure.
The zinc coordination in 5-aminolevulinate dehydratase was investigated by extended X-ray absorption fine structure (EXAFS) associated with the zinc K-edge. The enzyme binds 8 mol of zinc/mol of octameric protein, but only four zinc ions seem sufficient for full activity. We have undertaken a study on four forms of the enzyme: (a) the eight-zinc native enzyme; (b) the enzyme with only the four zinc sites necessary for full activation occupied; (c) the enzyme with the vacant sites of (b) occupied by four lead ions; (d) the product complex between (b) and porphobilinogen. We have shown that two structurally distinct types of zinc sites are available in the enzyme. The site necessary for activity has an average zinc environment best described by two/three histidines and one/zero oxygen from a group such as tyrosine or a solvent molecule at 2.06 +/- 0.02 A, one tyrosine or aspartate at 1.91 +/- 0.03 A, and one cysteine sulfur at 2.32 +/- 0.03 A with a total coordination of five ligands. The unoccupied site in (b), obtained by taking the difference spectrum between the spectra from samples (a) and (b), is dominated by a single contribution of four cysteinyl sulfur atoms at 2.28 +/- 0.02 A. Spectra from samples (c) and (d) show only small changes from that of (b), reflecting a slight rearrangement of the ligands around the zinc atom.
Animals↗
Accumulation of chromium in Chinese hamster V79-cells and nuclei.
The bioavailability of carcinogenic compounds plays an important role in the process of oncogenesis. Chromium in its hexavalent oxidation state is classified as carcinogen to humans. Therefore we studied the uptake of chromate(VI) into Chinese hamster V79 cells and nuclei isolated after the incubation of the whole cells with chromate. The chromium content of cells and nuclei was determined by atomic absorption spectroscopy. Chromate is taken up in a concentration dependent manner and accumulates to about 30 fold over the extracellular concentration of 0.1 mmol/l. Incubating whole cells with the same concentration results in an intranuclear concentration of up to 6 mmol/l after 3 h. The kinetic parameters (KM = 0.34 mmol/l, Vmax = 0.12 fmol per cell and minute) are in the same order of magnitude as previously published data. The consequences of the high intracellular and intranuclear concentrations are discussed in terms of the genotoxic effects.
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