ROS release and Hsp70 expression after exposure to 1,800 MHz radiofrequency electromagnetic fields in primary human monocytes and lymphocytes.

The aim of this study is to investigate if 1,800 MHz radiofrequency electromagnetic fields (RF-EMF) can induce reactive oxygen species (ROS) release and/or changes in heat shock protein 70 (Hsp70) expression in human blood cells, using different exposure and co-exposure conditions. Human umbilical cord blood-derived monocytes and lymphocytes were used to examine ROS release after exposure to continuous wave or different GSM signals (GSM-DTX and GSM-Talk) at 2 W/kg for 30 or 45 min of continuous or intermittent (5 min ON/5 min OFF) exposure. The cells were exposed to incubator conditions, to sham, to RF-EMF, or to chemicals in parallel. Cell stimulation with the phorbol ester phorbol-12-myristate-13-acetate (PMA; 1 microM) was used as positive control for ROS release. To investigate the effects on Hsp70 expression, the human monocytes were exposed to the GSM-DTX signal at 2 W/kg for 45 min, or to heat treatment (42 degrees C) as positive control. ROS production and Hsp70 expression were determined by flow cytometric analysis. The data were compared to sham and/or to control values and the statistical analysis was performed by the Student's t-test (P<0.05). The PMA treatment induced a significant increase in ROS production in human monocytes and lymphocytes when the data were compared to sham or to incubator controls. After continuous or intermittent GSM-DTX signal exposure (2 W/kg), a significantly different ROS production was detected in human monocytes if the data were compared to sham. However, this significant difference appeared due to the lowered value of ROS release during sham exposure. In human lymphocytes, no differences could be detected if data were compared either to sham or to incubator control. The Hsp70 expression level after 0, 1, and 2 h post-exposure to GSM-DTX signal at 2 W/kg for 1 h did not show any differences compared to the incubator or to sham control.

Hsp70 expression and free radical release after exposure to non-thermal radio-frequency electromagnetic fields and ultrafine particles in human Mono Mac 6 cells.

The contemporary urban environment has become increasingly complex in its composition, leading to discussions regarding possible novel health effects. Two factors that recently have received considerable attention are ultrafine particles (UFP; <0.1 microm) produced by combustion processes and emissions from wireless communication devices like mobile phones that emit in the radio-frequency (RF) part of the spectrum. Several studies have shown biological effects of both these exposures in various cell systems. Here we investigate if exposure to UFP (12-14 nm, 100 microg/ml) and RF-electromagnetic fields (EMF; 2 W/kg specific absorption rate (SAR); continuous wave (CW) or modulated (217Hz or GSM-nonDTX)), alone or in combination influences levels of the superoxide radical anion or the stress protein heat-shock protein (Hsp70) in the human monocyte cell line Mono Mac 6. Heat treatment (42-43 degrees C, 1h) was used as positive control for both stress reaction and for heat development in the RF exposure setup. Our results clearly show that Mono Mac 6 cells are capable to internalise UFP, and that this phagocytic activity is connected to an increased release of free radicals. This increase (40-45% above negative control) is stronger than the effect of heat treatment. On the other hand, none of the employed RF exposures showed any effects on free radical levels. Co-exposure of RF and UFP did not potentiate the UFP effect either. Our investigations showed a significantly increased Hsp70 expression level by heat treatment in a time-dependent manner, whereas UFP, RF, or UFP+RF were without any effect. Therefore, we conclude that in the investigated Mono Mac 6 cells, RF exposure alone or in combination with UFP cannot influence stress-related responses.

[Ca2+](i) rise in Jurkat E6-1 cell lines from different sources as a response to 50 Hz magnetic field exposure as a reproducible effect and independent of poly-L-lysine treatment.

Jurkat E6-1 cells obtained from three different sources were compared with respect to intracellular calcium response to a 50 Hz, 0.15 mT, magnetic field, to treatment with poly-L-lysine and to protein expression at the cell surface. The fura-2 single cell measurements were a replication study performed by three members of our group. The cells responded to the applied magnetic fields, although the percentage of responding cells was lower than in earlier studies. The geomagnetic field was backed off without changing the outcome of the intracellular calcium measurements. Fluorometric analyses showed no difference between the E6-1 cells obtained from three sources with respect to the expression of cell surface marker molecules. The addition of the cell adhesive peptide, poly-L-lysine, did not itself cause any effects on the intracellular calcium concentration.

ELF magnetic fields initiate protein tyrosine phosphorylation of the T cell receptor complex.

The human T cell line Jurkat registers a sinusoidal extremely low frequency (ELF), 0.10 mT magnetic fields (MFs) at the level of the plasma membrane. In this study, the protein tyrosine phosphorylation (PY) of two membrane-associated proteins in Jurkat cells were examined following a short-term MFs exposure, the zeta chains and the Src kinases p56lck. These proteins are interesting to study since the earliest biochemical event upon T cell receptor (TcR) activation is PY of the zeta chains. These signalling chains in the TcR complex was assessed using Western blotting and the activation of the p56lck kinase was analysed by in vitro kinase assay. The MFs exposure of Jurkat for 5 min activated p56lck and resulted in PY of zeta. These findings are in line with earlier reports on how MFs exposure affects signal transduction in Jurkat.

DNA damage, cell kinetics and ODC activities studied in CBA mice exposed to electromagnetic fields generated by transmission lines.

CBA mice were exposed outdoors to 50 Hz electromagnetic fields (EMF), with a flux density of about 8 microT rms (root mean square), generated by a 220 kV transmission line. Assays were performed in order to investigate, the possible genotoxic effects after 11, 20 and 32 days of exposure, as well as the effects on body weight, leukocytes, erythrocytes, and the level of ornithine decarboxylase (ODC) activity in spleen and testis. DNA migration was studied on brain cells by single cell electrophoresis (comet assay). After 32 days of exposure a highly significant change of the tail/head ratio of the comets was observed (p < 0.001), showing DNA-damage. Further, a decreased number of mononuclear leukocytes (0.02 < p < 0.05) was observed in mice EMF-exposed for 20 days. In summary, our data indicate that transmission lines of this type may induce genotoxic effects in mice, seen as changes in the DNA migration. These results might have an important implication for health effects.

Expression pattern of glutamate decarboxylase (GAD) in the developing cortex of the embryonic chick brain.

The development of the GABAergic system in the chick embryo telencephalon has been studied. Special emphasis was placed on the development of glutamate decarboxylase (GAD) between embryonic day 8 (E8) and E17. The GABA immunoreactivity and neuron-specific enolase expression was detected simultaneously in glutardialdehyde fixed sections, which confirmed that GABAergic cells exhibit neuronal phenotype. The GAD expression was studied by means of immunohistochemistry on cryo-sectioned material both at the light and electron microscopic levels. Furthermore, the presence and localization of GAD65 and GAD67 mRNAs were studied with an in situ hybridization technique with digoxigenin-labeled RNA probes. Protein expression as well as mRNA appearance mostly coincided both temporally and spatially. In the parahippocampal area, as well as in other regions of the developing cortex, GAD staining was seen from E8 onwards. The number of positive cells increased as did the intensity of staining up to E14. As observed in the electron microscope, the GAD protein was co-localized with GABA in most cases, although some GAD-positive cells devoid of GABA-staining also were observed. The pattern of GAD mRNA expression was in general similar to that of GAD immunostaining. Both GAD65 and GAD67 mRNA were detected during the entire period. Furthermore, GAD67 mRNA localization spatially was more correlated with GAD protein expression. The study provides evidence for the notion that development of the GABAergic system occurs rapidly during embryogenesis and, as suggested from mRNA data, that two forms of GAD with slight difference in distribution can contribute to this.

Two glutamate decarboxylase forms corresponding to the mammalian GAD65 and GAD67 are expressed during development of the chick telencephalon.

The gamma-aminobutyric acid (GABA)-synthesizing enzyme glutamate decarboxylase (GAD) was studied during development of the chick telencephalon. By means of reverse-phase HPLC analysis, we showed that GABA indeed accumulates during embryogenesis, whereas the levels of glutamate, the substrate for GAD, are more or less unchanged up to later developmental stages. The enzyme activity increased approximately 25-fold from embryonic day 3 to embryonic day 17. Immunoblotting data revealed that two GAD proteins, of approximately 65 and 67 kDa, were present during the period investigated. Furthermore, Northern blot analysis with probes obtained from rat cDNA sequences, as well as a chicken-specific probe for GAD65 generated by means of reverse transcriptase-polymerase chain reaction (RT-PCR), strengthened the interpretation that the chick embryo expresses genes corresponding to GAD65 and GAD67. The rat probes recognized transcript sizes of 3.9 kb (GAD65) and 5.6 kb (GAD67), sizes which are different from those of the rat brain (Erlander et al., Neuron, 7, 91-100, 1991). Sequencing of the RT-PCR products revealed a high level of homology (82% at the nucleotide level) between the mammalian and chick GAD65 genes. Taken together, these findings suggest that the chick embryo expresses two GAD genes during embryogenesis. The functional properties of each gene product remain to be investigated.

Differences in the release of L-glutamate and D-aspartate from primary neuronal chick cultures.

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release of D-[3H]aspartate and L-[3H]glutamate. The D[3H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependent L-[3H]glutamate release was calcium dependent, and furthermore L-[3H]glutamate release was optimal at potassium concentrations < 30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1 -aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux of D-[3H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulated L-glutamate release. We believe that L-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture. D-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.

Down-regulation of ornithine decarboxylase by an increased degradation of the enzyme during gastrulation of Xenopus laevis.

The present study was designed to analyze the regulation of the levels of the polyamines and their biosynthetic enzymes during embryonic development of Xenopus laevis. The activity of ornithine decarboxylase (ODC), a rate-controlling enzyme in polyamine biosynthesis, is elevated until, during gastrulation, there is a precipitous drop in activity. This is not attributable to a decrease in ODC mRNA content and polysome profiles reveal no apparent decrease in ODC message associated with polysomes. ODC synthesis seems to be maintained at a low, relatively constant rate until neurulation whereupon ribosome loading of ODC mRNA increases. During gastrulation the rate of ODC degradation increases dramatically, which can account for the decrease in ODC. S-Adenosylmethionine decarboxylase (AdoMetDC), another rate-controlling enzyme in polyamine biosynthesis, shows a low and constant activity from cleavage to neurulation. Subsequently, the AdoMetDC activity increases dramatically. The changes in AdoMetDC activity parallel the changes in AdoMetDC mRNA levels, suggesting a transcriptional control of AdoMetDC expression during this development period. The activities of ODC and AdoMetDC produce a steady increase in putrescine and spermidine content of the embryo. The spermine content also increases until gastrulation, but then decreases until the tailbud stage.

Morphological and GABA-immunoreactive development of the embryonic chick telencephalon.

The development of neurons utilizing gamma-aminobutyric acid (GABAergic neurons) in prosencephalon and telencephalon from chicken embryonic days 4-14 (E4-E14) was studied by means of immunohistochemistry. Furthermore, routine histology and transmission electron microscopy. respectively, were performed in order to study the morphological development in the designated area. The main finding is that development of GABAergic neurons in the chick telencephalon is rapid; the GABA neurons are appearing in bulk at day 8, being "overexpressed" at days 10-11, decreasing in numbers thereafter and achieving mature morphology on day 14, which is considerably faster than in the rodent. Morphological analysis revealed that the prosencephalon mainly consisted of a thin layer of undifferentiated neuroblasts in the E4 embryo. By E6, the prosencephalon had increased in thickness and occasional cells outside the neuroepithelium showed a more mature morphology with a few cells weakly staining positive for GABA. At E8, the prospective granular and subventricular layers had developed. At E14, the appearance of the telencephalon is approximating that of the adult since both ependymal cells and morphologically mature neurons can be seen.

Presence of the intermediate filaments cytokeratins and vimentin in the rat corpus luteum during luteal life-span.

The presence of the intermediate filament (IF) proteins cytokeratins and vimentin in corpus luteum (CL) and other parts of the ovary from adult pseudopregnant rats was investigated using immunohistochemistry and immunoblot analysis. To induce pseudopregnancy, female rats were mated with sterile male rats. The mating procedure induces a prolonged luteal life-span of 13 +/- 1 days. Positive staining for cytokeratin could be seen in CL, in the theca layer of follicles, and the ovarian surface epithelium with the broad-spectrum monoclonal antibody cocktail AE1/AE3. Weak staining was also seen in CL with antibodies against cytokeratins 8 and 18. A similar distribution was also seen for vimentin, which furthermore was detected in blood vessels. No changes in staining intensity was seen in CL of different luteal age. The strong staining for vimentin in CL was confirmed by immunoblot analysis, where one main band of 57 kDa was observed from day 1 to day 19 of pseudopregnancy. Expression of the IF proteins investigated seems to start in the newly formed CL and the continuous expression indicates that they are not directly regulated by luteal steroids.

Inhibition of SKF 89976-A of the gamma-aminobutyric acid release from primary neuronal chick cultures.

Neuronal cultures were made from the 8-d-old embryonic chick telencephalon. The primary culture model was further improved, the medium composition was modified, and the cells grown for 10 d, which allowed the development of relatively differentiated neurones. A superfusion protocol was developed and applied to study the release of [3H]-gamma-aminobutyric acid ([3H]GABA). High endogenous activity levels of glutamate decarboxylase (GAD) and of a Ca-dependent potassium stimulated [3H]GABA release were used as criteria for GABAergic differentiation. The influence of the non-substrate inhibitor of GABA transport, SKF 89976-A, on the GABA release, was studied using the primary neuronal culture. The release was found to be inhibited by SKF 89976-A at higher concentrations (= 400 microM).

Changes in the ovarian intermediate filament desmin during the luteal phase of the adult pseudopregnant rat.

The occurrence of the intermediate filament desmin in ovary and corpus luteum of pseudopregnant rats was studied using Western blot analysis and immunohistochemistry. The luteal phase was induced by mating with vasectomized male rats and ovaries were studied after 6, 11 and 19 days. The findings from the Western blot analysis showed that desmin was present in the corpus luteum. Immunohistochemical localization of desmin showed two types of localization in the corpus luteum. The arteries around the corpus luteum, as well as arteries elsewhere in the ovary, had a high content of desmin in their muscle layer. Dispersed in the corpus luteum was an immunohistochemical staining of desmin that was localized mainly adjacent to the luteal cells. In the other part of the ovary a weak staining was registered in the theca layer, no staining in the granulosa layer and a streaky staining in the hilar region of the ovary. Desmin filaments are found in muscle cells of all types, including vascular smooth muscle cells. Probably, all desmin in the ovary is localized to smooth muscle cells with the possible exception of the corpus luteum where very few muscle cells have been identified. Localization to other vascular cells as endothelial is possible. In this study we found an increase in desmin content in the corpus luteum after day 6. If desmin is related to vascular resistance, our finding is consistent with the decrease in blood flow that occurs after day 6.

Inhibition of transporter mediated gamma-aminobutyric acid (GABA) release by SKF 89976-A, a GABA uptake inhibitor, studied in a primary neuronal culture from chicken.

The effect of SKF 89976-A, a lipophilic non-substrate inhibitor of the gamma-aminobutyric acid (GABA) transporter, on the release of radioactive GABA and D-aspartate has been studied. Neuronal cultures from 8 day old chick embryos, grown for six days, served as a model. The cultures were incubated with [3H] D-aspartate and [14C] GABA with the subsequent addition of high or low concentrations of SKF 89976-A. Finally the cultures were exposed to differently composed media for either 30 or 300 seconds. The release was quantified, using liquid scintillation counting. The efflux of [3H] D-aspartate and [14C] GABA was increased by [K+] and time, and a minimum value was obtained at [Ca2+] 1.05 mM. The release of both [3H] D-aspartate and [14C] GABA was inhibited by SKF 89976-A. The obtained results indicate that transporter mediated processes are the major mechanisms of transmitter release in the investigated model.

On the activity of gamma-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes.

The uptake of radioactive gamma-aminobutyric acid (GABA) and D-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P2-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [3H]GABA and for D-[3H]aspartate with an increasing Vmax during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P2-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake of D-aspartate. We found no non-saturable uptake of D-aspartate.

Factors involved in the formation and stabilization of cell aggregates obtained from amphibian embryonic explants.

The effect of factors influencing the formation and stability of animal and vegetal aggregates from Xenopus laevis and Ambystoma mexicanum was examined in the light and scanning electron microscopes. At extreme values of pH the surface coat covering the vegetal aggregates is dissolved and dissociation may take place. Animal aggregates are more resistant. At high tonicities vegetal aggregates may be dissociated, and in the animal aggregates the epidermal differentiation is suppressed. In the absence of Ca2+ the vegetal aggregates are dissociated, but the animal aggregates are not affected. The results obtained with the inhibitor selenate and from incorporation experiments indicate that sulfated glycosaminoglycans are involved in the formation of aggregates in both species. Corresponding observations with tunicamycin suggest that even glycoproteins may play a role in aggregate formation, particularly in the vegetal aggregates.

Autoneuralization in the amphibian ectoderm--a species-specific and stage-specific phenomenon.

Ectoderm from Ambystoma is especially prone to undergo 'autoneuralization'. This assertion has led to the maxim that ectoderm from this species is unsuitable for studying cell differentiation. Here we report that the degree of neuralization in cultured explants is stage-dependent. Control explants from blastulae (stage 8-9) show no neuralization, while explants treated with LiCl (10 mM) give rise to neuralization in about 70% of the cases. This difference between control and experiment decreases during gastrulation, in late gastrulae (stage 12) it is more or less negligible. Ectoderm from Cynops pyrrhogaster reacts like that of Ambystoma when exposed to LiCl, but like Triturus ectoderm it is insensitive to cyclic nucleotides.