Tadashi Negishi

Lack of chick embryotoxicity after 20 kHz, 1.1 mT magnetic field exposure.

This investigation was undertaken because biological studies to evaluate the effects of intermediate frequency magnetic fields are insufficient. White Leghorn fertile eggs (60/group) were either exposed to a 20 kHz, 1.1 mT(rms) sinusoidal magnetic field or sham-exposed during the first 2, 7, or 11 days of embryogenesis. Lower dose exposures at 0.011 and 0.11 mT(rms) for 2 days were also conducted to elucidate possible dose-response relationships. Additional eggs given all-trans-retinoic acid, a teratogen, were exposed to the 1.1 mT(rms) magnetic field for the same periods to investigate the modification of embryotoxicity. After exposure, embryos were examined for mortality and developmental abnormalities. Developmental stage, number of somite pairs, and other developmental endpoints were also evaluated. Experiments were triplicated and conducted in a blind fashion. No exposure-related changes were found in any of the endpoints in intact embryos exposed to 1.1 mT(rms) or to the lower doses of 0.11 and 0.011 mT(rms) magnetic fields. Retinoic acid administration produced embryotoxic responses, which were embryonic death and developmental abnormalities, in 40-60% of embryos in the sham-exposed groups. The magnitude of these responses was not changed significantly by the magnetic field exposures. Under the present experimental conditions, exposure to 20 kHz magnetic field up to 1.1 mT(rms) was not embryotoxic in the chick and did not potentiate the embryotoxic action of retinoic acid.

Absence of reproductive and developmental toxicity in rats following exposure to a 20-kHz or 60-kHz magnetic field.

The use of intermediate frequency (IF) magnetic fields (MFs) in occupational equipment and domestic appliances, such as inductive heating cookers, is increasing. The WHO indicated a lack of scientific evidence needed to assess the health risk of exposure to IF MFs. Male and female rats (24/group) were exposed to a 20 kHz, 0.2 mT(rms) or 60 kHz, 0.1 mT(rms) sinusoidal MF for 22 h/day from 14 days prior to and during mating. Copulated females were exposed until gestation day 7 and sacrificed thereafter. Mated males were sacrificed to examine MF exposure effects on spermatogenesis. Reproductive examinations were blinded, and experiments were duplicated per frequency to ensure reproducibility. No statistically significant, exposure-related changes were found in the estrous cycle, copulation and fertility indices, numbers of corpora lutea and implantation sites, or pre- and postimplantation loss. No reproducible changes were observed in sperm count, motility, or morphological abnormality, or in the weights of testes and epididymides after MF exposure. No significant abnormalities were observed in gross pathology or histopathology of the uterus, ovary, testis, and epididymis in the MF- or sham-exposed groups. MF exposure during the preimplantation period was not toxic to fertility or early embryogenesis under the experimental conditions.

A newly designed and constructed 20 kHz magnetic field exposure facility for in vivo study.

Exposure to man-made electromagnetic fields has increased over the past century. As a result of exposure to these fields, concerns have been raised regarding the relationship between electromagnetic fields and human health. Interest in the biological and health effects of intermediate frequency (IF) magnetic fields has grown recently because of the increase in public concern. In order to investigate whether IF magnetic fields have biological effects, we have developed a 20 kHz (IF) magnetic field exposure system for in vivo studies. The exposure facility was designed to study the biological effects of IF magnetic field on laboratory animals. The facility consists of a 9 m x 9 m x 5 m high room containing seven separate rooms including a 5.3 m x 4.5 m x 3 m high specific-pathogen free exposure room. The dimensions of the exposure system are 1.6 m x 1.6 m x 1.616 m high located inside this exposure room. The system is designed to provide magnetic fields up to 200 microT at 20 kHz with the uniformity within +/-5% over the space occupied by animals. After constructing the facility, performance tests were carried out. As a result, it was confirmed that our facility met requirements for evaluation of the biological effects of IF magnetic field on small animal experiments. In this paper, the design, construction, and results of evaluation of an animal exposure facility for the in vivo biological effects of an IF magnetic field are described.

Hepatocyte nuclear factor 1β induced by chemical stress accelerates cell proliferation and increases genomic instability in mouse liver.

The liver has a considerable capacity of regeneration against the damage. The regulatory factors and molecular mechanism for the capacity are not fully appreciated. In developmental processes, hepatocyte nuclear factor 1β (HNF1β) is a cooperative factor for HNF6, which is a known stimulatory factor for hepatocyte proliferation after partial hepatectomy. We showed that carbon tetrachloride (CCl4)-induced liver injury up-regulated HNF1β, whereas the expression of HNF6 was not affected by the chemical stress, indicating unknown physiological roles of HNF1β against the chemical stress, not in cooperation with HNF6. To determine whether HNF1β has a novel function in the liver regeneration, we overexpressed HNF1β in the mouse liver by adenoviral gene delivery. We revealed that overexpression of HNF1β resulted in accelerated cell proliferation with the protein level up-regulation of plasminogen and plasmin, a converted active form of plasminogen, which play a pivotal role in liver regeneration inducing hepatocyte proliferation. Despite this stimulatory effect for the liver regeneration, HNF1β overexpression significantly increased genomic instability with decreased protein level of mediator of DNA damage checkpoint 1 (MDC1) and dephosphorylation of SP1 transcription factor. The increased expression of HNF1β is associated with several types of hepatocyte carcinomas, indicating possible involvement of the factor in carcinogenesis. Our data extend the current understanding of the mechanism underlying liver regeneration against chemical stress, and identified HNF1β as a novel regulatory factor in this mechanism and as a potential initiator for carcinogenesis.

Chronic inhalation exposure and phospholipids in lung surfactant and tissue

Golden hamsters were exposed to 2 mg/m3 coal fly ash for 180 days. The exposure raised the phospholipid level in the tissue, whereas no such elevation was observed in the surfactant. Increased phospholipid in the tissue is thought to reflect the accumulated surfactant in type II cells. Fatty acid composition analysis indicated an increase of arachidonic acid in the surfactant and increases of palmitic acid and arachidonic acid in the tissue. In conclusion, the pulmonary surfactant high in fluidity was stored in the lung tissue more than control, and the surfactant was secreted into the alveoli normally.

Adaptive gene regulation of pyruvate dehydrogenase kinase isoenzyme 4 in hepatotoxic chemical-induced liver injury and its stimulatory potential for DNA repair and cell proliferation

The processes involved in the adaptation of animals to environmental factors remain unclear. We examined the mechanisms underlying the adaptive potential of the mouse against hepatotoxic chemical-induced injury. Microarray analysis revealed that ethylbenzene, a hepatotoxic chemical, upregulated PDK4 (encoding pyruvate dehydrogenase kinase isoenzyme 4) in mouse livers and that the upregulation was enhanced by previous exposure to the chemical. Although PDK4 is an energy resource regulator induced by starvation, expression of other fasting-inducible genes was unaffected. PDK4 induced by chemical stress developed hepatic accumulation of sirtuin 1 by regulating pyruvate concentration and activated the Nbn and ATM, which are critical for DNA repair and checkpoint activation. PDK4 overexpression on carbon tetrachloride (CCl4)-induced liver injury resulted in delayed necrotic tissue recovery with cell cycle arrest and decreased γH2AX foci and micronucleus formation. PDK4 silencing on CCl4-induced liver injury accelerated necrotic tissue recovery and increased γH2AX foci and micronucleus formation, indicating the essential role of PDK4 in DNA repair and checkpoint activation. PDK4 overexpression induced pancreas-specific transcription factor 1a (Ptf1a) upregulation and transcriptional activation of several pancreatic genes in the liver. Ptf1a overexpression by adenoviral gene delivery resulted in accelerated tissue recovery on CCl4-induced liver injury. Our data identified PDK4 as a novel pivotal factor in adaptation to chemical stress.

Acute and subchronic toxicity of 20 kHz and 60 kHz magnetic fields in rats

Despite increasing use of intermediate frequency (IF) magnetic fields (MFs) in occupational and domestic settings, scientific evidence necessary for health risk assessments of IF MF is insufficient. Male and female Crl:CD(SD) rats (12 per sex per group) were exposed to 20 kHz, 0.20 mT(root mean square, rms) or 60 kHz, 0.10 mT(rms) sinusoidal MFs for 22 h day−1 for 14 days (acute) or 13 weeks (subchronic). Experiments were duplicated for each frequency to ensure outcome reproducibility, and examinations were blinded for quality assurance. All rats survived without significant clinical signs until the end of experiments. Some changes in body weight between the MF‐exposed and control groups were observed over the course of exposure, although the directions of the changes were inconsistent and not statistically significant after subchronic exposure. There were significant differences between MF‐exposed and control groups in some organ weights and parameters in hematology and clinical chemistry, but these were minor in magnitude and not repeated in duplicate experiments. Histopathological findings reflecting toxicity were sporadic. Frequencies of other findings were similar to historic data in this rat strain, and findings had no specific relationship to changes in organ weight or parameters of hematology and clinical chemistry in each animal. The changes observed throughout this study were considered biologically isolated and were attributable to chance associations rather than to MF exposure. The results, in particular the histopathological evidence, indicate an absence of toxicity in IF MF‐exposed rats and do not support the hypothesis that IF MF exposure produces significant toxicity. Copyright

Intermediate frequency magnetic field and chick embryotoxicity.

Intermediate frequency magnetic fields (MFs) have widely been used in industrial machines and home appliances, such as induction heating cookers, although toxicity studies to evaluate the potential health risks of such fields are insufficient. In induction heating cookers, the MF source (i.e. hobs), is located near the abdominal position of a person cooking. Hence, developmental effects on the fetus may be a concern in case the person is a pregnant woman. Fertile White Leghorn eggs (60/group) were either exposed to 20 kHz, 1.1 mT(rms) or 60 kHz, 0.11 mT(rms) sinusoidal MFs for 19 days during embryogenesis. The same number of eggs served as a control group. In addition, a sham‐sham experiment was conducted to validate the equality between exposure and control facilities. After exposure, embryos were examined for mortality rate and stage. Live embryos were evaluated for developmental stage and gross and skeletal anomalies. Length of upper beak and leg digits was also measured. Examinations were conducted in a blinded fashion to ensure quality assurance; experiments were triplicated for each frequency to confirm the outcome reproducibility. Mortality rate and stage, incidence of malformed embryos, and developmental variables in live embryos were found to be similar between the MF‐exposed and corresponding control group. Incidence of gross anomalies such as mandibular edema and skeletal anomalies such as coccyx defects were low across the experiments, and no significant group differences were noted. In conclusion, exposure to 20 kHz or 60 kHz MF did not produce any significant teratogenic developmental effects in chick embryos.

Intermediate frequency magnetic fields did not have genotoxic and promotion potentials in in vitro tests

In recent years, the use of new electrical appliances, that use or emit intermediate frequency (IF; 300 Hz to 10 MHz) electromagnetic fields (EMFs), has been increasing. We have investigated genotoxic and promotion potentials of a vertical and sinusoidal IF MF of 0.91mT (rms) at 2 kHz, 1.1mT (rms) at 20 kHz and 0.11mT (rms) at 60 kHz. We used microbial mutagenicity tests, gene conversion tests, micronucleus tests or mouse lymphoma assay for genotoxicity, and Bhas 42 promotion tests for promotion. The results indicate that the IF MFs did not have genotoxic nor promotion potentials in the experimental conditions.

A Study on Progression of Spontaneous Leukemia in AKR Mice Exposed to 50-Hz, Circularly Polarized Magnetic Field for 24 Weeks

There have been animal studies investigating MF effect on leukemia based on the suggestive association between magnetic field (MF) exposure and increased risk of leukemia in epidemiology. In a rat model, large granular lymphocyte leukemia did not show the late stage progression of the disease by 60-Hz, linearly polarized MF exposure of 1 mT1. In an ongoing study, a transgenic mouse strain of Em-Piml that predisposed to be T-cell lymphoma, is used to examine the effect of 50-Hz MF exposure2. AKR mouse spontaneously develops lymphatic leukemia originating in the thymus; in females the life time is about 55 weeks. Bellossi found that pulsed MF exposure for 5 generations did not affect the leukemic lethality in AKR mouse3. That study evaluates organ weights and survival time, however, not examining the progression process of leukemia with histopathology. The objective of our study is to evaluate the possible MF effect on the progression of spontaneous leukemia in AKR/J female mice.