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Biomedical subjects

D Beyersmann

Publications and source records attributed to D Beyersmann.

At least 19 recordsLinked to original sources
PubMed2001

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Nitric oxide inhibits the cochaperone activity of the RING finger-like protein DnaJ.

As a consequence of bacterial infection and the ensuing inflammation, expression of the inducible NO synthase results in prolonged synthesis of NO in high concentrations, which among other functions, contributes to the innate defense against the infectious agent. Here we show that NO inhibits the ability of the bacterial cochaperone DnaJ containing a RING finger-like domain to cooperate with the Hsp70 chaperone DnaK in mediating correct folding of denatured rhodanese. This inhibition is accompanied by S-nitrosation of DnaJ as well as by Zn2+ release from the protein. In contrast, NO has no effect on the activity of GroEL, a bacterial chaperone without zinc sulfur clusters. Escherichia coli cells lacking the chaperone trigger factor and thus relying on the DnaJ/DnaK system are more susceptible toward NO-mediated cytostasis than are wild-type bacteria. Our studies identify the cochaperone DnaJ as a molecular target for NO. Thus, an encounter of bacterial cells with NO can impair the protein folding activity of the bacterial chaperone system, thereby increasing bacterial susceptibility toward the defensive attack by the host.

Cell Division↗
PubMed2001

Cadmium-induced apoptosis in murine fibroblasts is suppressed by Bcl-2.

We investigated the induction of apoptosis by cadmium in NIH 3T3 murine fibroblasts. Apoptosis was triggered effectively by 10 microM CdCl2 within 24 h, under which conditions cell viability was reduced by 50%. Cadmium-induced apoptosis was demonstrated by both morphological and biochemical analysis. We have shown that cadmium concentrations of 5-20 microM caused nuclear fragmentation. Moreover, internucleosomal DNA fragmentation was evoked by 10-25 microM CdCl2 within 24 h, as detected by the formation of ladder patterns in DNA electrophoresis. Since the induction of programmed cell death occurs together with modifications in the cell cycle, we examined the ability of cadmium to block cell divisions by using a 5-bromo2-deoxy-uridine incorporation assay. Our results indicate that about 40% of treated cells are blocked in G0-G1 phase when exposed to 10 microM cadmium for 27 h. Finally, we addressed the question of whether the effect of cadmium could be prevented by suppressing apoptosis. Over-expression of the anti-apoptotic protein Bcl-2 in NIH 3T3 cells protects against cadmium toxicity, thus suggesting a role for Bcl-2 in the regulation of cadmium-induced apoptosis.

3T3 Cells↗
PubMed2001

Zinc induces apoptosis that can be suppressed by lanthanum in C6 rat glioma cells.

Zinc ions have both essential and toxic effects on mammalian cells. Here we report the ability of zinc to act as an inducer of apoptosis in C6 rat glioma cells. Incubation with 150 to 300 microM ZnCl2 caused cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, nuclear fragmentation and breakdown of the mitochondrial membrane potential. On the other hand, zinc deprivation by the membrane permeable chelator TPEN [N,N,N',N',-tetrakis (2-pyridyl-methyl)-ethylenediamine] also induced programmed death in this cell line, indicating the existence of intracellular zinc levels below and above which apoptosis is induced. Zinc-induced apoptosis in C6 cells was independent of major signaling pathways (protein kinase C, mitogen activated protein kinase and guanylate cyclase) and protein synthesis, but was increased by facilitating zinc uptake with the ionophore pyrithione. Lanthanum(III)chloride was also able to increase the net zinc uptake, but nevertheless apoptotic features and zinc toxicity were reduced. Remarkably, lanthanum suppressed the zinc-induced breakdown of the mitochondrial membrane potential. We conclude that in C6 cells lanthanum acts in two different ways, as a promoter of net zinc uptake and as a suppressor of zinc-induced apoptosis.

Aminoquinolines↗
PubMed2001

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.

Adult↗
PubMed2000

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.

Alternative Splicing↗
PubMed1999

Transient peaks in zinc and metallothionein levels during differentiation of 3T3L1 cells.

A novel role for zinc mediated by metallothionein (MT) is found in the process of differentiation of 3T3L1 mouse fibroblasts to adipocytes. Twenty-four hours after the stimulation of differentiation by hormones, the cells enter into a phase of synchronous proliferation. In this phase the cellular contents of zinc and metallothionein rise rapidly to fivefold and threefold levels, respectively. Simultaneously MT is translocated from the cytoplasm to the nucleus. The rise of intracellular zinc is essential for the transition from G0/G1- to S-phase of the cell cycle. Deprivation of zinc with N,N,N', N'-tetrakis[2-pyridyl]ethylenediamine, a membrane-permeable zinc chelator, inhibited hormonal induced proliferation. After the short phase of proliferation a slower stage of actual differentiation to adipocytes begins. The elevated levels of MT and zinc decline quickly to start levels, and a rapid redistribution of MT to the cytoplasm occurs. We propose that the nuclear translocation of MT mediates the transfer of zinc to nuclear factors in the mitogenic process. The redistribution of MT to the cytoplasm and the decrease of the zinc content are postulated to be required for the start of the actual differentiation.

3T3 Cells↗
PubMed1999

Uptake and intracellular distribution of labile and total Zn(II) in C6 rat glioma cells investigated with fluorescent probes and atomic absorption.

The uptake, intracellular distribution and cytotoxicity of high doses of extracellular zinc was investigated in C6 rat glioma cells. Net zinc uptake occurred only above certain thresholds in time and concentration, below them no alterations of the intracellular zinc level were observed. These results were obtained by measurements with the fluorescent dye Zinquin and by atomic absorption spectrometry, yielding similar results with both methods. Sequestration of zinc in intracellular vesicles was observed by fluorescence microscopy. A protective effect of vesicular sequestration is indicated, because increased levels of intracellular zinc located in vesicles did not necessarily lead to an increase in cytotoxicity. We were able to show that in C6 cells, in contrast to other cell lines, zinc that is released from proteins by the NO donor SNOC is also sequestered in vesicular structures. These zinc-carrying vesicles showed to be constitutive and are assumed to have a function in the maintainance of the cytosolic content of Zn2+ ions.

Animals↗
PubMed1998

Nickel(II) inhibits the repair of O6-methylguanine in mammalian cells.

Nickel compounds are widespread carcinogens, and although only weakly mutagenic, interfere with nucleotide excision repair and with the repair of oxidative DNA base modifications. In the present study we investigated the effect of nickel(II) on the induction and repair of O6-methylguanine and N7-methylguanine after treatment with N-methyl-N-nitrosourea (MNU). We applied Chinese hamster ovary cells stably transfected with human O6-methylguanine-DNA methyltransferase (MGMT) cDNA (CHO-AT), and compared the results with the MGMT-deficient parental cell line. As determined by high-performance liquid chromatography/electrochemical detection (HPLC/ECD), there was a slight but mostly not significant reduction in the formation of both types of DNA lesions by MNU in the presence of nickel(II). Although nickel(II) did not markedly affect the repair of N7-methylguanine, it decreased the repair of O6-methylguanine in a dose-dependent manner, starting at concentrations as low as 50 microM. While the MGMT protein level was not altered in the presence of nickel(II), the MGMT activity was diminished as demonstrated in cell extracts form nickel-treated cells. This repair inhibition was accompanied by an increase in MNU-induced cytotoxicity in nickel-treated CHO-AT cells but not in MGMT-deficient control cells. There is strong evidence that O6-methylguanine is involved in tumour formation after exposure to alkylating agents. Thus, the finding that nickel(II) inhibits the repair of this lesion could be of major importance for risk assessment in case of combined exposures at work places and in the general environment.

Animals↗
PubMed1998

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.

Animals↗
PubMed1997

Study of the interactions of cadmium and zinc ions with cellular calcium homoeostasis using 19F-NMR spectroscopy.

The effects of the heavy-metal ions Cd2+ and Zn2+ on the homoeostasis of intracellular free Ca2+ in E367 neuroblastoma cells were examined using 19F-NMR spectroscopy with the fluorinated chelator probe 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N', N'-tetra-acetic acid (5F-BAPTA). First, the technique was used to quantify the uptake and intracellular free concentrations of the heavy metals after treatment of the cells with 20 microM CdCl2 or 100 microM ZnCl2. Secondly, metal-induced transients in intracellular free Ca2+ were recorded. Addition of 20 microM CdCl2, but not 100 microM ZnCl2, evoked a transient increase in Ca2+ from a resting level of 84 nM to approx. 190 nM within 15 min after addition of the metal. Zn2+ at 20 microM completely prevented the induction of a Ca2+ transient by Cd2+. Ca2+ was mobilized by Cd2+ from intracellular organelles, since depletion of these stores by thapsigargin abolished the effect of the toxic metal. Furthermore, 20 microM Cd2+ evoked a transient rise in cellular Ins(1,4,5)P3, reaching a maximum level within 5 min after addition of the metal. These results demonstrate that perturbation of the Ins(1,4,5)P3/Ca2+ messenger system is an early and discrete cellular effect of Cd2+.

Animals↗
PubMed1997

Cadmium, gene regulation, and cellular signalling in mammalian cells.

Effects of the carcinogenic metal cadmium on the regulation of mammalian gene expression are reviewed and discussed in the light of observations on interference with cellular signal transduction pathways. Cadmium ions are taken up through calcium channels of the plasma membrane of various cell types, and cadmium is accumulated intracellularly due to its binding to cytoplasmic and nuclear material. At elevated cytotoxic concentrations, cadmium inhibits the biosyntheses of DNA, RNA, and protein, and it induces lipid peroxidation, DNA strand breaks, and chromosome aberrations. Cadmium compounds as such are only weak mutagens and clastogens. However, cadmium at noncytotoxic doses interferes with DNA repair processes and enhances the genotoxicity of directly acting mutagens. Hence, the inhibition of repair and detoxifying enzymes by this metal may partially explain the observed weak genotoxic properties of this metal. Nongenotoxic mechanisms upregulating intracellular signalling pathways leading to increased mitogenesis are discussed as major mechanisms for the interpretation of the carcinogenic activity by chronic cadmium exposure. About 1 microM cadmium stimulates DNA synthesis and cell proliferation in various cell lines, whereas more elevated concentrations are inhibitory. Cadmium enhances the expression of several classes of genes at concentrations of a few microM. It stimulates the expression of immediate early genes (c-fos, c-jun, and c-myc), of the tumor suppressor gene p53, and of genes coding for the syntheses of protective molecules, including metallothioneins, glutathione, and stress (heat shock) proteins. The mechanisms underlying the modulation of gene activity by cadmium are discussed in terms of interference with cellular signalling at the levels of cell surface receptors, cellular calcium and zinc homeostases, protein phosphorylation, and modification of transcription factors. In considering the available evidence, the carcinogenic properties of cadmium are interpreted using a multifactorial approach involving indirect genotoxicity (interference with DNA repair) and the upregulation of mitogenic signalling pathways.

Animals↗
PubMed1997

Interaction of arsenic(III) with nucleotide excision repair in UV-irradiated human fibroblasts.

Even though epidemiological studies have identified arsenic compounds as carcinogenic to humans, they are not mutagenic in bacterial and mammalian test systems. However, they increase the mutagenicity and clastogenicity in combination with other DNA damaging agents and there are indications of inhibition of DNA repair processes. We investigated the effect of arsenic(III) on nucleotide excision repair (NER) after UV irradiation in human fibroblasts in detail by using two repair-proficient and one partly repair-deficient xeroderma pigmentosum group C human fibroblast cell lines. The results show that two steps of NER are affected by arsenite. Most severely, the incision frequency is reduced at concentrations as low as 2.5 microM arsenic(III); at higher, cytotoxic concentrations, the ligation of repair patches is also impaired. Furthermore, our results indicate that both the global genome repair pathway and the transcription-coupled repair pathway are affected by arsenite. Repair inhibition may well explain the potentiation of genotoxic effects by arsenic in combination with other DNA damaging agents and may thus be of high relevance for the carcinogenic action of arsenic compounds.

Arsenites↗
PubMed1996

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.

Journal Article↗
PubMed1996

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.

Adenosine Triphosphate↗
PubMed1995

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.

Alkalies↗
PubMed1994

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.

Colony-Forming Units Assay↗