Nickel(II) interferes with the incision step in nucleotide excision repair in mammalian cells.

Nickel compounds are carcinogenic to humans and experimental animals. However, the mechanisms leading to tumor formation are still not understood since the mutagenic potential is rather weak. In contrast, nickel(II) enhances the cytotoxicity and genotoxicity in combination with several other DNA-damaging agents. To elucidate possible interactions with DNA repair processes, the effect of nickel(II) on the nucleotide excision repair pathway has been investigated after UV irradiation in HeLa cells. Nickel(II) blocks the removal of cyclobutane pyrimidine dimers as determined by T4 endonuclease V-sensitive sites. When the alkaline unwinding technique was applied, significantly less transient DNA strand breaks after UV irradiation were detected in the presence of nickel(II) compared to UV alone, suggesting an inhibition of the incision step of nucleotide excision repair. Once incisions are made, the ligation of repair patches is delayed as well in nickel-treated cells, as observed by the alkaline unwinding and nucleoid sedimentation techniques. This inhibition of DNA repair is partly reversible by the addition of magnesium(II), indicating that the competition between Ni2+ and Mg2+ may provide an important mechanism for the disturbance of DNA-protein interactions involved in the repair process. Since the repair inhibition is observed at noncytotoxic concentrations of nickel(II), it may well be relevant for its carcinogenic action.

Affinity labelling of 5-aminolevulinic acid dehydratase with 2-bromo-3-(5-imidazolyl)propionic acid.

2-Bromo-3-(5-imidazolyl)propionic acid, a zinc-directed thiol reagent, inactivates the enzyme 5-aminolevulinic acid dehydratase from bovine liver (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing, EC 4.2.1.24). The substrate, 5-aminolevulinic acid, completely protects against inactivation. The reagent inhibits the zinc-containing enzyme to a greater extent than the zinc-deprived enzyme; and it competes with the zinc chelator 1,10-phenanthroline. The reagent alkylates essential sulfhydryl groups of the enzyme, since the extent of the inactivation depends on the reduction of the enzyme protein by thiol compounds. It is concluded that the zinc site, the substrate site and the essential sulfhydryl groups are in close proximity in the active site.

Agonist-stimulated calcium transients in PC12 cells are affected differentially by cadmium and nickel.

Rat pheochromocytoma (PC12) cells were loaded with the fluorescent indicator Fura-2 and the effects of cadmium and nickel on the mobilization of calcium elicited by bradykinin and external ATP were studied. Cadmium and nickel ions provoked a concentration-dependent decrease of the initial peak and/or the subsequent plateau phase of bradykinin-induced Ca2+ transients in a different manner: whereas cadmium (0.5-2.5 microM) diminished the calcium peak without modifying the sustained plateau, nickel (25-1000 microM) only slightly lowered the peak but markedly decreased the plateau phase. In the case of ATP-stimulated calcium transients, which are without a sustained plateau, both cadmium and nickel ions decreased the peak signal. Possible consequences are discussed in terms of a disturbance of hormone-stimulated cell activation by cadmium and nickel.

Biomonitoring on carcinogenic metals and oxidative DNA damage in a cross-sectional study.

Oxidative DNA damage is mediated by reactive oxygen species and is supposed to play an important role in various diseases including cancer. The endogenous amount of reactive oxygen species may be enhanced by the exposure to genotoxic metals. A cross-sectional study was conducted from 1993 to 1994 in an urban population in Germany to investigate the association between metal exposure and oxidative DNA damage. The cross-sectional sample of 824 participants was recruited from the registry of residents in Bremen, comprising about two-third males and one-third females with an average age of 61.1 years. A standardized questionnaire was used to obtain the occupational and smoking history. The incorporated dose of exposure to metals was assessed by biological monitoring. Chromium, cadmium, and nickel were measured in 593 urine samples. Lead was determined in blood samples of 227 participants. As a biomarker for oxidative DNA damage, 7,8-dihydro-8-oxoguanine has been analyzed in lymphocytes of 201 participants. Oxidative lesions were identified by single strand breaks induced by the bacterial formamidopyrimidine-DNA glycosylase (Fpg) in combination with the alkaline unwinding approach. The concentrations of metals indicate a low body load (median values: 1.0 microg nickel/l urine, 0.4 microg cadmium/l urine, and 46 microg lead/l blood; 83% of chromium measures were below the technical detection limit of 0.3 microg/l). The median level of Fpg-sensitive DNA lesions was 0.23 lesions/10(6) bp. A positive association between nickel and the rate of oxidative DNA lesions (Fpg-sensitive sites) was observed (odds ratio, 2.15; tertiles 1 versus 3, P < 0.05), which provides further evidence for the genotoxic effect of nickel in the general population.

Regulation of mammalian gene expression.

The expression of mammalian genes is regulated primarily at the level of initiation of transcription. The regulatory structure of the mammalian genes consists of the sequence coding for a protein, a proximal upstream promoter sequence which binds the general (basal) transcription factors and a distant enhancer sequence which binds the inducible transcription factors. The general transcription factors are proteins which combine with the RNA polymerase at the promoter to form the initiation complex. Binding of the inducible transcription factors at short DNA sequences, named response elements, mostly enhances or rarely represses the formation of the initiation complex. Transcription factors share common structural motifs; the most frequent are zinc finger, leucine zipper and helix-loop-helix structures. Inducible transcription factors are activated to bind their target response elements on DNA by protein kinases, by binding of activating or removal of inhibitory factors, or by de novo protein synthesis. Inducible transcription factors are activated by hormones or growth factors addressing a number of genes which share common response elements. Steroid and thyroid hormones combine with intracellular receptors to form active transcription factors. Other transcription factors are activated by protein kinases which are themselves activated by hormones through cell membrane receptors and further cellular signaling paths. Whereas the main level of transcriptional control is the initiation of RNA synthesis, in some instances genes are also regulated by alternative splicing of the primary transcript or control of translation into proteins. Large-scale silencing of genes is mediated by the packing of DNA in highly condensed heterochromatin structures and DNA methylation at cytosines in defined guanine-cytosine (GC)-sequences.

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.

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.

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.

Zn2+ and Cd2+ increase the cyclic GMP level in PC12 cells by inhibition of the cyclic nucleotide phosphodiesterase.

In the present study, the influence of the heavy metal ions Cd2+ and Zn2+ on cGMP metabolism in the neurosecretory rat pheochromocytoma (PC12) cell line has been investigated. Cadmium and zinc ions showed a concentration-dependent increase of intracellular cGMP levels as determined by radioimmunoassay: a 20-fold increase in cGMP concentration was found after 15 min of incubation with 20 microM Cd2+, and a 7-fold increase in cGMP was found after incubation with 50 microM Zn2+ (control: 6.05+/-2.1 pmol cGMP/mg protein). To obtain further mechanistic informations, the effects of Cd2+ and Zn2+ on intracellular 3',5'-cyclic nucleotide phosphodiesterase have been studied by a high performance liquid chromatography-based phosphodiesterase-assay. The cellular cGMP hydrolysis was found to be inhibited by these ions with an IC(50) value of 6+/-0.7 microM for Cd2+ and 13+/-2.5microM for Zn2+ . Hence, dose-dependent increase in cellular cGMP content is due to an inhibition of cGMP hydrolysis and not due to an increase in cGMP synthesis. Cd2+ and Zn2+ were taken up by PC12 cells as determined by atomic absorption spectroscopy, all measurements were performed in a subtoxic concentration range. Our data illustrate that zinc and cadmium ions are efficient inhibitors of the cGMP-stimulated cyclic nucleotide PDEII in PC12 cells resulting in elevated cellular cGMP concentrations. Therefore, subtoxic doses of these metals may disturb intracellular cGMP/cAMP-signalling pathways leading to an impaired or altered gene expression.

Zinc and cadmium in 5-aminolevulinic acid dehydratase. Equilibrium, kinetic, and 113Cd-nmr-studies.

5-Aminolevulinic acid dehydratase (ALAD) from bovine liver contains zinc that is partially lost during the isolation of the enzyme. ALAD has its maximal activity at 10(-5) M ZnCl2. It binds 7.4 Zn per octameric protein with an association constant of 5.3 X 10(6)M-1. ALAD is inactivated by 1,10-phenanthroline or ethylenediaminetetraacetic acid (EDTA) but not by monodentate anions like cyanide or sulfide. After removal of zinc by chelating agents, the enzyme activity may be restored by Zn2+ or Cd2+. Removal of zinc by EDTA increases KM 60-fold and decreases Vmax to about 1/2 of its original value. The 113Cd nuclear magnetic resonance spectrum of the enzyme reconstituted with 113Cd-acetate exhibits a single sharp resonance signal at 79 ppm. It does not change by the addition of substrate but disappears when the inhibitor lead acetate is added. Therefore, an immediate interaction between the metal ion of the enzyme and the substrate is excluded, whereas lead changes the environment of cadmium and probably of zinc too.

Analysis of metal-induced oxidative DNA damage in cultured mammalian cells.

Reactive oxygen species are continuously generated during oxygen metabolism, and a measurable amount of oxidative DNA damage exists in aerobic organisms. By the determination of Fpg-sensitive sites in mammalian cells in culture, we assessed the background level of oxidative DNA damage and its potential increase by extracellularly applied complexes of iron(III). In V79 Chinese hamster cells the endogenous level of Fpg-sensitive modifications is detectable, but the extent is much lower as compared with results derived from other analytical methods. In V79 cells, the frequency of Fpg-sensitive modifications is considerably enhanced by Fe-NTA in a time- and dose-dependent manner, while no increase is observed after treatment with Fe-citrate. These results indicate that the ability of transition metals to generate oxidative DNA damage in intact cells strongly depends on factors like uptake and intracellular distribution, which will affect the intracellular availability of redox-active metal ions close to critical targets.

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.

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.

Effect of cadmium(II) on the extent of oxidative DNA damage in primary brain cell cultures from Pleurodeles larvae.

Compounds of cadmium(II) are well-known human and animal carcinogens. Furthermore, they affect development. growth and brain functions at subacute environmental concentrations in experimental animals. We investigated the potential of cadmium(II) to induce oxidative DNA damage in brain cell cultures obtained from larvae of Pleurodeles waltl. As indicators of DNA lesions typical of oxygen free radicals, we determined the frequencies of DNA strand breaks and of DNA base modifications recognized by the bacterial formamidopyrimidine-DNA glycosylase (Fpg protein). DNA strand breaks were generated in a dose-dependent manner at concentrations of 1 microM and greater. In contrast, no significant increase in Fpg-sensitive sites was observed under our experimental conditions. However, the repair of Fpg-sensitive DNA lesions induced by visible light was slightly diminished at 1 microM and inhibited completely at 10 microM of cadmium(II), while the closure of DNA strand breaks was not affected. Our results show that, although cadmium is not able to induce oxidative DNA base modifications in larval brain cells directly, its capability to generate DNA strand breaks and to interfere with the repair of oxidative DNA damage could explain the early life stage neurotoxicity of this metal.

Enhancement of UV-induced mutagenesis and sister-chromatid exchanges by nickel ions in V79 cells: evidence for inhibition of DNA repair.

With regard to contradictory results concerning the mutagenicity of nickel compounds in short-term assays, especially in bacterial test systems, Chinese hamster V79 cells were used to measure mutagenicity, comutagenicity and the induction of sister-chromatid exchanges (SCEs) by NiCl2. We confirmed the induction of mutations at the HGPRT locus as well as SCEs. In addition, NiCl2 shows a pronounced comutagenic effect towards UV. When using confluent cultures or resting cells due to serum deprivation, where more time is given for repair processes, the comutagenic effect is higher compared to logarithmically growing cells (10 and 4 times, respectively, compared to twice). Hence, we attribute this enhancement in mutagenicity to inhibition of DNA repair. Also the increase in induced SCEs after combined treatment with UV and NiCl2 supports this thesis. Furthermore, NiCl2 enhances the cyto-toxicity of cis-DDP about 12-fold. Since no comutagenic effect is observed in combination with MMS, we suggest that the inhibition of DNA repair by Ni(II) applies to all DNA changes that are repaired by the 'long-patch' excision repair system. This inhibition may occur via replacement of other divalent metal ions essential in repair and regulation processes.

The DNA cleavage induced by a chromium(V) complex and by chromate and glutathione is mediated by activated oxygen species.

The number of strand breaks induced by the combination of chromate and glutathione (GSH) in PM2 DNA was effectively reduced upon addition of the hydroxyl radical scavengers dimethyl sulphoxide (DMSO), formate and benzoate. Administration of catalase also led to a depression of DNA degradation whereas superoxide dismutase (SOD) had very little influence. Essentially the same results were obtained in experiments employing a chromium(V) complex Na4(GSH)4Cr.8H20, which is an intermediate chromium species isolated from the reduction of chromate by glutathione. DNA cleavage was dependent on the presence of iron (FeCl3). When compared with the number of breaks produced by FeCl3 and GSH alone, chromate stimulated the generation of single-strand breaks. These findings suggest that hydroxyl radicals are one ultimate DNA cleaving agent in both reactions. A reaction scheme for the production of hydroxyl radicals is proposed.

Indirect mechanism of lead-induced genotoxicity in cultured mammalian cells.

The data concerning the mutagenic, clastogenic and carcinogenic properties of inorganic lead compounds have been conflicting. To investigate whether the genotoxicity of lead is due to indirect effects such as interference with DNA-repair processes, the induction of mutations, sister-chromatid exchanges and strand breaks by lead ions alone as well as in combination with UV light as a standard mutagen were determined. Lead acetate alone does not induce DNA-strand breaks in HeLa cells or mutations at the HPRT locus and sister-chromatid exchanges in V79 Chinese hamster cells. However, at all endpoints tested, lead ions interfere with the processing of UV-induced DNA damage. They inhibit the closing of DNA-strand breaks after UV irradiation and enhance the number of UV-induced mutations and sister-chromatid exchanges, indicating an inhibition of DNA repair. These data point out the necessity to consider such indirect effects when assessing the genotoxicity of metal compounds. As possible mechanisms of repair inhibition we suggest either the interaction with repair enzymes such as polymerase or ligase or else the interaction with calcium-regulated processes, for example with calmodulin.

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.

Sensitive nonradioactive detection of UV-induced cyclobutane pyrimidine dimers in intact mammalian cells.

In this paper we present a sensitive procedure to determine specifically the induction as well as the removal of cyclobutane pyrimidine dimers in intact mammalian cells without radioactive labeling of the DNA. This technique allows the detection of DNA damage by UV doses as low as 0.1 J/m2. The method consists of gentle lysis of cell monolayers, high-salt treatment and incubation with the cyclobutane pyrimidine dimer-specific repair enzyme T4 endonuclease V, followed by alkaline unwinding, hydroxyapatite chromatography and fluorimetric DNA analysis. The number of T4 endonuclease V-sensitive sites correlates well with the amount of UV-induced cyclobutane pyrimidine dimers reported in the literature, indicating that these cyclobutane pyrimidine dimers are recognized quantitatively by the system. The assay is easily transferable to the detection of other types of DNA adducts by applying different damage-specific repair enzymes, providing a sensitive method to investigate the induction and the repair of DNA lesions without the use of radioactive labeling.

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione.

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.