Ralf Kriehuber

Stimulation of phagocytosis and free radical production in murine macrophages by 50 Hz electromagnetic fields.

Effects of 50 Hz electromagnetic fields on phagocytosis and free radical production were examined in mouse bone marrow-derived macrophages. Macrophages were in vitro exposed to electromagnetic fields using different magnetic field densities (0.5-1.5 mT). Short-time exposure (45 min) to electromagnetic fields resulted in significantly increased phagocytic uptake (36.3% +/- 15.1%) as quantified by measuring the internalization rate of latex beads. Stimulation with 1 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) showed the same increased phagocytic activity as 1 mT electromagnetic fields. However, co-exposure to electromagnetic fields and TPA showed no further increase of bead uptake, and therefore we concluded that because of the absence of additive effects, the electromagnetic fields-induced stimulation of mouse bone marrow-derived macrophages does not involve the protein kinase C signal transduction pathway. Furthermore, a significant increased superoxide production after exposure to electromagnetic fields was detected.

Micronucleus formation in human amnion cells after exposure to 50 Hz MF applied horizontally and vertically

Micronucleus (MN) induction as a genotoxic effect of extremely-low-frequency electromagnetic fields (ELF-EMF, 50 Hz, 1 mT) was studied in human amniotic fluid cells (AFC) after continuous exposure to magnetic fields (MF), oriented horizontally and vertically with respect to the surface of the culture medium, at different time points. To compare the effectiveness of different exposure systems, a Helmholtz-coil system and a so-called Merritt-coil system was used. A statistically significant increase in MN frequency could be detected in exposed cells compared to controls after 72 h continuous exposure to MF applied vertically in the Merritt-coil system, while no effect was found after exposure in the Helmholtz-coil system. Furthermore, a significant increase in MN induction occurred after 24, 48 and 72 h exposure to MF applied horizontally in the Helmholtz-coil system in comparison to controls, whereas horizontally MF generated in the Merritt-coil system induced no genotoxic effects. To exclude suppression of indirect EMF-induced DNA-lesions, we studied MN formation in the presence of N-Acetyl-p-aminophenol (APAP, Paracetamol(R)), which is an inhibitor of DNA-repair mechanisms. We found a dose-dependent increase of MN formation in APAP-treated AFC cells, but no significant further increase in MN frequency after additional MF exposure. Therefore we conclude, that EMF-induced MN formation is not caused by directly or indirectly induced clastogenic mechanisms. The obtained results show that the orientation of MF with respect to the cell culture dish and the physical condition of the exposure system is of major importance for the induction of micronuclei in certain cell types. Therefore, the reason for inconsistent results published in the literature may be caused by the variability of exposure systems, the exposure conditions and the cell types used.

Influence of 50 Hz electromagnetic fields in combination with a tumour promoting phorbol ester on protein kinase C and cell cycle in human cells

It still is an unsolved issue whether exposure to power-line frequency electromagnetic fields (EMF) may promote carcinogenesis and if so whether it does so by influencing the proliferation, the survival, and the differentiation of cells. Since the family of protein kinases C (PKC) takes part in these processes by interacting with signal transduction pathways at several levels including the activation of transcription factors, we evaluated in the present study the effects of exposure of human amniotic fluid cells (AFC) to 50Hz, 1 mT electromagnetic fields (EMF) alone and in combination with the tumour promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on the subcellular localization of PKC protein, on PKC enzyme activity, and on the cell cycle distribution.Quantitative analyses of the PKC expression pattern demonstrated the translocation of PKC from the cytosolic to the membrane fraction after exposure to 10, 50, 100nM, and 1 μM TPA. EMF exposure alone showed no effect on PKC translocation. Co-exposure to 10, 50, and 100nM TPA and 1 mT EMF revealed a significant additive effect (25 ± 50, 66 ± 29, 22 ± 50%, respectively) with the most prominent increase at the concentration of 50nM TPA. At the highest concentration of TPA used (1 μM) no additive effect of EMF could be observed. Data on enzymatic activity indicate that EMF modulate the PKC activity, showing a significant increase of 10 ± 16% in total PKC activity after co-exposure to 50nM TPA and 1 mT EMF when compared to 50nM TPA alone. Flow cytometric analyses showed a transient cell cycle arrest in G0/G1-phase followed by a delayed transit through S-phase in response to TPA, which was, however, not enhanced by co-exposure with EMF. We conclude that in AFC cells TPA at lower concentrations (≤ 100nM) induces a less than maximum effect on the PKC pathway, which can be enhanced by the applied EMF.

Glycogen synthase kinase-3beta regulates differentiation-induced apoptosis of human neural progenitor cells.

Glycogen synthase kinase‐3beta is a multifunctional key regulator enzyme in neural developmental processes and a main component of the canonical Wnt signaling pathway. It is already known that the Wnt‐driven differentiation of neural progenitor cells is accompanied by an increase of apoptosis at which the pro‐apoptotic function of GSK‐3beta is still discussed. The aim of the present study was to investigate whether the phosphorylation level of GSK‐3beta at serine 9 is the primary regulatory mechanism of differentiation‐induced apoptosis.

Apoptosis induction and micronucleus formation after exposure to the Auger electron emitter zinc-65 in a human cell line.

Induction of apoptosis and micronucleus formation has been studied in a transformed human squamous cell carcinoma cell line (SCL-II) after exposure to the Auger electron emitter Zinc-65 (65Zn) and after external low-LET radiation. Exposure to non-radioactive Zn and unirradiated cells served as controls. Studies on the cellular uptake of 65Zn2+ have been carried out in vitro and conventional dosimetric models have been applied to derive the absorbed radiation dose. Auger electrons, generated during decay of 65Zn2+, are strong inducers of micronuclei as well as of apoptosis, in comparison to external low-LET irradiation. The relative biological effectiveness has been determined and was found to be in the range of 1.2-2.7 for the two investigated biological endpoints, depending on which mathematical model for describing the dose-effect curves was used. A non-uniform distribution of intracellular Zn2+ was observed, showing a strong signal in the perinuclear region. We conclude that separate radiation weighting factors for Auger electrons should be established depending on the nuclide and its ability to interact with the DNA (e.g. 65Zn by Zinc-finger proteins).

Inhibition of BCL-2 leads to increased apoptosis and delayed neuronal differentiation in human ReNcell VM cells in vitro.

BCL‐2 is a multifunctional protein involved in the regulation of apoptosis, cell cycle progression and neural developmental processes. Its function in the latter process is not well understood and needs further elucidation. Therefore, we characterized the protein expression kinetics of BCL‐2 and associated regulatory proteins of the intrinsic apoptosis pathway during the process of neuronal differentiation in ReNcell VM cells with and without functional inhibition of BCL‐2 by its competitive ligand HA14‐1.