Design and Dosimetric Characterization of a Broadband Exposure Facility for In Vitro Experiments in the Frequency Range 18-40.5 GHz.

A novel exposure facility for exposing cell monolayers to centimeter and millimeter waves (18-40.5 GHz) used by future 5G mobile communication technology and similar applications has been developed. A detailed dosimetric characterization of the apparatus for frequencies of 27 and 40.5 GHz and 60 mm petri dishes, used in a presently ongoing study on human dermal fibroblasts and keratinocytes, was carried out. The exposure facility enables a well-defined, randomized, and blinded application of sham exposure and exposure with selectable values of incident power flux density, and additionally provides the possibility of continuous monitoring of the sample temperature during exposure while it does not require significant deviations from routine in vitro handling procedures, i.e. petri dishes are not required to be placed inside waveguides or TEM cells. Mean specific absorption rate (SAR) values inside the cell monolayer of 115 W/kg (27 GHz) and 160 W/kg (40.5 GHz) per watt antenna input power and corresponding transmitted power density (St ) values at the bottom of the cell monolayer of 65 W/m2 (27 GHz) and 70 W/m2 (40.5 GHz) per watt antenna input power can be achieved, respectively. For reasonable amounts of harvested cells (80% of petri dish bottom area), the variation (max/min) of SAR and St over the cell monolayer remains below 3.7 dB (27 GHz) and 3.0 dB (40.5 GHz), respectively. © 2021 Bioelectromagnetics Society.

Effects of Long-Term Exposure of Intermediate Frequency Magnetic Fields (20 kHz, 360 µT) on the Development, Pathological Findings, and Behavior of Female Mice.

The use of magnetic fields in the intermediate-frequency (IF) range to wirelessly charge electric cars with power transfer in the kilowatt range has become increasingly widespread, leading to unavoidable stray fields in the microtesla range. Only a handful of studies have assessed the potential biological risks associated with exposure to such fields. We exposed female mice (n = 80 per group) to either 20 kHz, 360 µT (rms), or sham in Helmholtz coils to conduct a blind design study. Exposure started at 3 months of age (24 h/day). Body mass was recorded every 1-2 weeks. At 10 months of age, three behavioral tests were performed on 24 animals per group. Three months later, the mice were sacrificed and organs (brain, liver, kidney, spleen, and lung) were removed and prepared for microscopic analysis. Our findings demonstrate no differences in the development of body mass and survival rates (96% and 89%, respectively). Similarly, no significant differences were observed in tumor incidence rates. When it comes to behavioral tests, the 8-arm maze results revealed no significant differences. In contrast, the Rotarod data were significantly (P < 0.001) different with longer retention times seen in the exposed mice. In the open field, the number of supported rears was significantly lower (P < 0.01), whereas the other endpoints did not show any differences. Overall, our data reveal no adverse effects of exposure to 20 kHz, 360 µT on the development and tumor incidences, while the significant differences in the behavioral tests may indicate higher levels of alertness in mice.

No Increased DNA Damage Observed in the Brain, Liver, and Lung of Fetal Mice Treated With Ethylnitrosourea and Exposed to UMTS Radiofrequency Electromagnetic Fields.

The widespread use of mobile phones and Wi-Fi-based communication devices makes exposure to radiofrequency electromagnetic fields (RF-EMF) unavoidable. Previous experiments have revealed the tumor-promoting effects of non-ionizing RF-EMF in adult carcinogen-treated mice in utero. To extend these investigations, we tested whether these effects are due to the co-carcinogenicity of RF-EMF which would manifest as elevated DNA damage. Similar to previous experiments, pregnant mice were exposed to RF-EMF (Universal Mobile Telecommunication System [UMTS] standard, approximately 1,960 MHz) from day 7 post-conception (p.c.) at 0 (sham), 0.04, and 0.4 W/kg SAR. At day 14 p.c., the mice were injected with the carcinogen ethylnitrosourea (ENU, 40 mg/kg). At three time-points specifically 24, 36, and 72 h later, the pregnant females were sacrificed and the fetuses (n = 24-57) were removed. A dye (cy3) specific for adenyl adducts was used to detect DNA damage by fluorescence microscopy in the brain, liver, and lung of each fetus. Compared to control (0 W/kg SAR), exposure to RF-EMF had no effect on the formation of DNA adducts in the inspected tissues. We conclude that increased adenyl formation of DNA by RF-EMF exposure is not a valid explanation for the previously reported tumor-promoting effects of RF-RMF. Our findings may help to gain a deeper insight into the biological effects of RF-EMF exposure in the context of malignancy. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.

Tumor promotion by exposure to radiofrequency electromagnetic fields below exposure limits for humans.

The vast majority of in vitro and in vivo studies did not find cancerogenic effects of exposure to electromagnetic fields (RF-EMF), i.e. emitted by mobile phones and base stations. Previously published results from a pilot study with carcinogen-treated mice, however, suggested tumor-promoting effects of RF-EMF (Tillmann et al., 2010). We have performed a replication study using higher numbers of animals per group and including two additional exposure levels (0 (sham), 0.04, 0.4 and 2 W/kg SAR). We could confirm and extend the originally reported findings. Numbers of tumors of the lungs and livers in exposed animals were significantly higher than in sham-exposed controls. In addition, lymphomas were also found to be significantly elevated by exposure. A clear dose-response effect is absent. We hypothesize that these tumor-promoting effects may be caused by metabolic changes due to exposure. Since many of the tumor-promoting effects in our study were seen at low to moderate exposure levels (0.04 and 0.4 W/kg SAR), thus well below exposure limits for the users of mobile phones, further studies are warranted to investigate the underlying mechanisms. Our findings may help to understand the repeatedly reported increased incidences of brain tumors in heavy users of mobile phones.

Systematic review of wireless phone use and brain cancer and other head tumors.

We conducted a systematic review of scientific studies to evaluate whether the use of wireless phones is linked to an increased incidence of the brain cancer glioma or other tumors of the head (meningioma, acoustic neuroma, and parotid gland), originating in the areas of the head that most absorb radiofrequency (RF) energy from wireless phones. Epidemiology and in vivo studies were evaluated according to an agreed protocol; quality criteria were used to evaluate the studies for narrative synthesis but not for meta-analyses or pooling of results. The epidemiology study results were heterogeneous, with sparse data on long-term use (≥ 10 years). Meta-analyses of the epidemiology studies showed no statistically significant increase in risk (defined as P < 0.05) for adult brain cancer or other head tumors from wireless phone use. Analyses of the in vivo oncogenicity, tumor promotion, and genotoxicity studies also showed no statistically significant relationship between exposure to RF fields and genotoxic damage to brain cells, or the incidence of brain cancers or other tumors of the head. Assessment of the review results using the Hill criteria did not support a causal relationship between wireless phone use and the incidence of adult cancers in the areas of the head that most absorb RF energy from the use of wireless phones. There are insufficient data to make any determinations about longer-term use (≥ 10 years).

Temporal control of spermatogenesis is independent of the central circadian pacemaker in Djungarian hamsters (Phodopus sungorus).

In mammals, the duration of the cycle of the seminiferous epithelium (DCSE) largely differs between species, but is remarkably stable within a species, usually showing variations of 1%-3%. It is difficult to change the DCSE, e.g., by hormones or chemicals. Initial experiments, employing quantitative RT-PCR, aimed at investigating the diurnal profiles of the clock genes Arntl (previously called Bmal1) and Per1 in testes and kidneys of Djungarian hamsters (Phodopus sungorus). While the testicular levels of Arntl were almost constant, clear diurnal variations were identified for Per1. In order to clarify whether day length (T-cycle) is a factor for DSCE, adult male hamsters (n = 20 per group) were exposed to normal (T = 24 h), prolonged (T = 25 h), or shortened (T = 23 h) T-cycles, with cycles thus being longer or shorter by 4.2% compared to the normal condition. Exposure lasted for 43 days, during which the activity of the animals was recorded to confirm entrainment. DCSE was estimated by incorporation of bromodeoxyuridine in dividing cells and the immunohistochemical localization of labeled cells in stages I-XII of the seminiferous epithelium. Despite the low variability of the results and the close agreement with previously published data, no effects of prolonged or shortened T-cycles on DCSE could be identified (24 h: 7.98 ± 0.05 days; 23 h: 7.94 ± 0.04 days; 25 h: 7.91 ± 0.03 days; P > 0.05). The results strongly indicate that the high temporal precision of spermatogenesis is independent of the central circadian clock.

Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervous system that are associated with learning and memory deficits.

BACKGROUND: Cathepsin K is a cysteine peptidase known for its importance in osteoclast-mediated bone resorption. Inhibitors of cathepsin K are in clinical trials for treatment of osteoporosis. However, side effects of first generation inhibitors included altered levels of related cathepsins in peripheral organs and in the central nervous system (CNS). Cathepsin K has been recently detected in brain parenchyma and it has been linked to neurobehavioral disorders such as schizophrenia. Thus, the study of the functions that cathepsin K fulfils in the brain becomes highly relevant. RESULTS: Cathepsin K messenger RNA was detectable in all brain regions of wild type (WT) mice. At the protein level, cathepsin K was detected by immunofluorescence microscopy in vesicles of neuronal and non-neuronal cells throughout the mouse brain. The hippocampus of WT mice exhibited the highest levels of cathepsin K activity in fluorogenic assays, while the cortex, striatum, and cerebellum revealed significantly lower enzymatic activities. At the molecular level, the proteolytic network of cysteine cathepsins was disrupted in the brain of cathepsin K-deficient (Ctsk⁻/⁻) animals. Specifically, cathepsin B and L protein and activity levels were altered, whereas cathepsin D remained largely unaffected. Cystatin C, an endogenous inhibitor of cysteine cathepsins, was elevated in the striatum and hippocampus, pointing to regional differences in the tissue response to Ctsk ablation. Decreased levels of astrocytic glial fibrillary acidic protein, fewer and less ramified profiles of astrocyte processes, differentially altered levels of oligodendrocytic cyclic nucleotide phosphodiesterase, as well as alterations in the patterning of neuronal cell layers were observed in the hippocampus of Ctsk⁻/⁻ mice. A number of molecular and cellular changes were detected in other brain regions, including the cortex, striatum/mesencephalon, and cerebellum. Moreover, an overall induction of the dopaminergic system was found in Ctsk⁻/⁻ animals which exhibited reduced anxiety levels as well as short- and long-term memory impairments in behavioral assessments. CONCLUSION: We conclude that deletion of the Ctsk gene can lead to deregulation of related proteases, resulting in a wide range of molecular and cellular changes in the CNS with severe consequences for tissue homeostasis. We propose that cathepsin K activity has an important impact on the development and maintenance of the CNS in mice.

Spermatogenesis in the Roborovski hamster (Phodopus roborovskii) and the Chinese hamster (Cricetulus griseus).

BACKGROUND: Spermatogenesis is an elaborately organized and tightly regulated differentiation process. The spermatogenesis duration is stable within a certain species but highly variable between species of the same family. OBJECTIVES: In this study, the spermatogenesis duration of the Roborovski hamster was measured for the first time, and the spermatogenesis duration of the Chinese hamster was re-assessed. MATERIALS AND METHODS: Stage classification and cycle length measurement were carried out by labeling the dividing cells with bromodeoxyuridine and an antibody-based chromogen as well as with the periodic acid-Schiff/hematoxylin stain. Analysis was conducted using reference calculation and linear regression. Morphological measurements completed our set of methods. RESULTS: The mean duration of one seminiferous epithelium cycle was 8.58 ± 0.34 days (mean ± SEM; Phodopus roborovskii) and 16.59 ± 0.47 days (Cricetulus griseus) based on the reference calculation. Slightly higher results were obtained using linear regression analysis: 9.72 ± 0.41 days for P. roborovskii and 17.64 ± 0.61 days for C. griseus. Additionally, a newly developed exemplary flowchart was proposed for the Roborovski hamster to facilitate spermatogenesis stage classification also in other species. The Chinese hamster presented an unexpectedly high paired epididymides weight of 1.701 ± 0.046 g (mean ± SEM) although having a body weight of only 40.5 ± 0.7 g. However, no significant correlation between the relative epididymis weight and spermatogenesis duration in mammals (Spearman rank correlation: r = -0.119, p = 0.607, n = 21) or rodents could be found (r = 0.045, p = 0.903, n = 11). CONCLUSION: Our data emphasize the stability of the spermatogenesis duration within species and its remarkable variability between species. Further research is needed to identify the principal mechanisms and selection drivers that are responsible for such stability within species and the variability between species.

Critical comments on DNA breakage by mobile-phone electromagnetic fields [Diem et al., Mutat. Res. 583 (2005) 178-183].

In a publication that appeared in 2005 (Diem et al., Mutat. Res. 583:178-183) [10] harmful effects (DNA breakage) were reported to occur in rat and human cells after exposure to mobile-phone electromagnetic fields. The extremely low standard deviations in this paper, and in another publication by the same group of authors, prompted Vijayalaxmi to write a critical comment [Mutat. Res. 603 (2006) 104-106] [16]. An investigation by the Medical University of Vienna (Austria) was initiated by a letter by the first author of the present paper, based on the data contained in the reply by the authors [Rüdiger et al., Mutat. Res. 603 (2006) 107-109] [17]. The University published three press releases, stating that "the data were not measured experimentally, but fabricated" and that the Mutation Research paper and another, published by the International Archives of Occupational and Environmental Health (IAOEH) in 2008, should be retracted. So far, neither of these papers has been retracted. Only a Letter of Concern by the Editors of IAOEH, and an Editorial by Mutation Research were published. Here we describe the statistical methods used to identify the evidence of data fabrication. The major point is the small variation in the reported data, which is below the theoretical lower limit derived from multinomial distributions and also lower than those derived from detailed simulations. Another reason for doubt was the highly significant non-equal distribution of last digits, a known hint towards data fabrication. In view of the results of the University's investigation and the evidence presented in this paper, the Diem et al. (2005) [10] publication should be retracted, with or without the authors' agreement.

Effects of radiofrequency electromagnetic fields (UMTS) on reproduction and development of mice: a multi-generation study.

Male and female mice (C57BL) were chronically exposed (life-long, 24 h/day) to mobile phone communication electromagnetic fields at approximately 1966 MHz (UMTS). Their development and fertility were monitored over four generations by investigating histological, physiological, reproductive and behavioral functions. The mean whole-body SARs, calculated for adult animals at the time of mating, were 0 (sham), 0.08, 0.4 and 1.3 W/kg. Power densities were kept constant for each group (0, 1.35, 6.8 and 22 W/m(2)), resulting in varying SARs due to the different numbers of adults and pups over the course of the experiment. The experiment was done in a blind fashion. The results show no harmful effects of exposure on the fertility and development of the animals. The number and the development of pups were not affected by exposure. Some data, albeit without a clear dose-response relationship, indicate effects of exposure on food consumption that is in accordance with some data published previously. In summary, the results of this study do not indicate harmful effects of long-term exposure of mice to UMTS over several generations.

Indirect blue light does not suppress nocturnal salivary melatonin in humans in an automobile setting.

In 2007, the International Agency for Research on Cancer (IARC) classified shift work that involves circadian disruption as being probably carcinogenic to humans (Group 2A). In this context, light exposure during the night plays a key role because it can suppress nocturnal melatonin levels when exposures exceed a certain threshold. Blue light around 464 nm is most effective in suppressing melatonin because of the spectral sensitivity of melanopsin, a recently discovered photopigment in retinal ganglion cells; the axons of these cells project to the suprachiasmatic nucleus, a circadian master clock in the brain. Due to advances in light technologies, normal tungsten light bulbs are being replaced by light-emitting diodes which produce quasi-monochromatic or white light. The objective of this study was to assess whether the light-melanopsin-melatonin axis might be affected in automobiles at night which employ the new generation diodes. To this end, we have tested in an experimental automobile setting whether indirect blue light (lambda(max) = 465 nm) at an intensity of 0.22 or 1.25 lx can suppress salivary melatonin levels in 12 male volunteers (age range 17-27 years) who served as their own controls. Daytime levels were low (2.7 +/- 0.5 pg/mL), and night-time levels without light exposure were high (14.5 +/- 1.1 pg/mL), as expected. Low-intensity light exposures had no significant effect on melatonin levels (0.22 lx: 17.2 +/- 2.8 pg/mL; P > 0.05; 1.25 lx: 12.6 +/- 2.0 pg/mL; P > 0.05). It is concluded that indirect blue light exposures in automobiles up to 1.25 lx do not cause unintentional chronodisruption via melatonin suppression.

Comments on "Radiofrequency electromagnetic fields (UMTS, 1,950 MHz) induce genotoxic effects in vitro in human fibroblasts but not in lymphocytes" by Schwarz et al. (Int Arch Occup Environ Health 2008: doi: 10.1007/s00420-008-0305-5).

BACKGROUND: A recent publication by Schwarz et al. describes the effects of exposure of human fibroblast and lymphocytes to radiofrequency-electromagnetic fields at frequencies used for communication with mobile phones. Even at very low specific absorption rates of 0.05 W/kg, thus well below internationally accepted exposure limits, significant effects were seen in fibroblasts whose DNA molecules were damaged as assessed by their comet tail factor (CTF) in the comet assay. AREAS OF CONCERN: The CTF mean values and the standard deviations of the replicates revealed very low coefficients of variation, ranging from 1.2 to 4.9% (average 2.9%), which are in contrast to much higher variations reported by others. Moreover, inter-individual differences of the CTF values strongly disagree with the previously published data from the same group of researchers. CONCLUSION: The critical analysis of the data given in the figures and the tables furthermore reveal peculiar miscalculations and statistical oddities which give rise to concern about the origin of the reported data.

Effects of mobile phone electromagnetic fields at nonthermal SAR values on melatonin and body weight of Djungarian hamsters (Phodopus sungorus).

In three experiments, adult male Djungarian hamsters (Phodopus sungorus) were exposed 24 hr/day for 60 days to radio frequency electromagnetic fields (RF-EMF) at 383, 900, and 1800 MHz, modulated according to the TETRA (383 MHz) and GSM standards (900 and 1800 MHz), respectively. A radial waveguide system ensured a well defined and uniform exposure at whole-body averaged specific absorption rates of 80 mW/kg, which is equal to the upper limit of whole-body exposure of the general population in Germany and other countries. For each experiment, using two identical waveguides, hamsters were exposed (n = 120) and sham-exposed (n = 120) in a blind fashion. In all experiments, pineal and serum melatonin levels as well as the weights of testes, brain, kidneys, and liver were not affected. At 383 MHz, exposure resulted in a significant transient increase in body weight up to 4%, while at 900 MHz this body weight increase was more pronounced (up to 6%) and not transient. At 1800 MHz, no effect on body weight was seen. The results corroborate earlier findings which have shown no effects of RF-EMF on melatonin levels in vivo and in vitro. The data are in accordance with the hypothesis that absorbed RF energy may result in metabolic changes which eventually cause body weight increases in exposed animals. The data support the notion that metabolic effects of RF-EMFs need to be investigated in more detail in future studies.

Lymphoma development in mice chronically exposed to UMTS-modulated radiofrequency electromagnetic fields.

There are public concerns regarding possible carcinogenic or cancer-promoting effects of electromagnetic fields (EMFs) from mobile phones and base stations. The objective of the present study was to investigate whether chronic exposure to EMFs of the UMTS (Universal Mobile Telecommunication System) influences the development of lymphoma in a lymphoma animal model, the AKR/J mouse. Unrestrained mice were chronically sham-exposed (n = 160) or exposed (n = 160) in identical exposure systems (radial waveguides) to a generic UMTS test signal (24 h per day, 7 days per week, 0.4 W/kg SAR). Additionally, 30 animals were kept as cage controls. Animals were checked visually each day and were weighed and palpated weekly to detect swollen lymph nodes. Starting at the age of 6 months, blood samples were taken from the tail every 2 weeks to perform differential leukocyte counts and to measure the hematocrit. Visibly diseased animals or those older than 43 weeks were killed humanely, and tissue slices were examined for metastatic infiltrations and lymphoma type. The study was performed in a blinded way. Cage control animals had a significantly lower growth rate than those kept in the radial waveguides. The number of ill animals, the mean survival time, and the severity code of the disease did not differ between the experimental groups. Therefore, the data show no negative effects from exposure and corroborate earlier findings in AKR/J mice exposed to GSM EMF (Sommer et al., BMC Cancer 4, 77-90, 2004).

Cognitive function in outbred house mice after 22 weeks of drinking oxygenated water.

Oxygen-enriched drinking water, which is increasingly sold worldwide, is claimed to "keep both the body and the mind healthy." However, currently there is no scientific evidence for such a statement. Therefore, we assessed the effect of 22 weeks of drinking oxygenated water on cognitive performance in healthy mice, using a spatial learning task and behavioral observations. Thirty-six female mice (age 3 to 6 months) received either hyperoxic or normal tap water (approximately 6.6 vs. 1.8 microg O(2) g(-1) day(-1), respectively) throughout the study period. Mice were weighed one to two times per month, and a blood sample was taken from the tail to determine the hematocrit. In addition, red blood cells were counted microscopically one and two months after the start of the experiment. Four weeks after the last blood sample (21 weeks after the start of the experiment), exploration behavior and locomotor activity were observed on a holeboard, and learning ability tests were performed using an elevated open maze. No significant differences were seen between groups for any of the parameters investigated. Thus, the study does not support the hypothesis that drinking oxygenated water improves cognitive function or hematological parameters in mice.

50 Hz magnetic fields of 1 mT do not promote lymphoma development in AKR/J mice.

Some epidemiological studies suggest that exposure to power-frequency magnetic fields increases the risk of leukemia, especially in children with high residential exposures. In contrast, most animal studies did not find a correlation between magnetic-field exposure and hematopoietic diseases. The present study was performed to investigate whether chronic, high-level (1 mT) magnetic-field exposure had an influence on lymphoma development in a mouse strain that is genetically predisposed to thymic lymphoblastic lymphoma. Three groups of 160 unrestrained female AKR/J mice were sham-exposed or exposed to sinusoidal 50 Hz magnetic fields beginning at the age of 12 weeks for 32 weeks, 7 days per week, either for 24 h per day or only during nighttime (12 h). Exposure was carried out in a blind design. Exposure did not affect survival time, body weight, lymphoma development or hematological parameters. The resulting data do not support the hypothesis that exposure to sinusoidal 50 Hz magnetic fields is a significant risk factor for hematopoietic diseases, even at this relatively high exposure level.

Predicted 3D-structure of melanopsin, the non-rod, non-cone photopigment of the mammalian circadian clock, from Djungarian hamsters (Phodopus sungorus).

Melanopsin is the photopigment of the retinal ganglion cells, which are involved in the synchronization of the biological clock in the suprachiasmatic nucleus (SCN) of mammals with the exogenous photoperiod. So far, no information about the three-dimensional (3D) structure of melanopsin is available. Here we report the predicted structure based on the protein-coding region of the nucleotide sequence of the gene for melanopsin, originating from isolated mRNA from the eyes of Djungarian hamsters (Phodopus sungorus). The nucleotide sequence shares the largest homologies with melanopsin of mice and rats (each 89%) and humans (84%). Based on the amino-acid sequence, and in comparison with the known structure of bovine rhodopsin, the three-dimensional melanopsin protein structure was modeled by using automated homology modeling approaches that were subsequently refined. Melanopsin consists of highly conserved seven-transmembrane domains and a long cytoplasmatic tail with multiple putative phosphorylation sites. In the binding site of the chromophore, a 11-cis-retinal is likely to be bound to lysine at position 336 as Schiff's base. The modeling results may indicate different photoisomerization within the melanopsin molecule compared with bovine rhodopsin.