Development of chicken embryos in a pulsed magnetic field

Six independent experiments of common design were performed in laboratories in Canada, Spain, Sweden, and the United States of America. Fertilized eggs of domestic chickens were incubated as controls or in a pulsed magnetic field (PMF); embryos were then examined for developmental anomalies. Identical equipment in each laboratory consisted of two incubators, each containing a Helmholtz coil and electronic devices to develop, control, and monitor the pulsed field and to monitor temperature, relative humidity, and vibrations. A unipolar, pulsed, magnetic field (500-microseconds pulse duration, 100 pulses per s, 1-microT peak density, and 2-microseconds rise and fall time) was applied to experimental eggs during 48 h of incubation. In each laboratory, ten eggs were simultaneously sham exposed in a control incubator (pulse generator not activated) while the PMF was applied to ten eggs in the other incubator. The procedure was repeated ten times in each laboratory, and incubators were alternately used as a control device or as an active source of the PMF. After a 48-h exposure, the eggs were evaluated for fertility. All embryos were then assayed in the blind for development, morphology, and stage of maturity. In five of six laboratories, more exposed embryos exhibited structural anomalies than did controls, although putatively significant differences were observed in only two laboratories (two-tailed Ps of .03 and less than .001), and the significance of the difference in a third laboratory was only marginal (two-tailed P = .08). When the data from all six laboratories are pooled, the difference in incidence of abnormalities in PMF-exposed embryos (approximately 25 percent) and that of controls (approximately 19 percent), although small, is highly significant, as is the interaction between incidence of abnormalities and laboratory site (both Ps less than .001). The factor or factors responsible for the marked variability of inter-laboratory differences are unknown.     

Hatching in zebrafish (Danio rerio) embryos exposed to a 50 Hz magnetic field

Zebrafish embryos were exposed intra ovo to a 50 Hz AC magnetic field of 1000 microT rms, and the progress of asynchronous hatching was monitored. A statistically significant delay was observed when field exposure started 48 h after fertilization. In contrast, when exposure started 2 h after fertilization, no statistically significant effect was seen. When field exposure was administered together with submaximal doses of progesterone at 48 h postfertilization, the two treatments appeared to delay hatching in an additive manner. Evaluating the progress of hatching in zebrafish embryos seems relevant for exploration of EMF effects on reproduction.     

The effects of minoxidil on the development of the chick embryo

The effects of minoxidil were studied on chick embryos of 24 and 48 hours of incubation. Minoxidil (3%) was injected into the air sacs of the eggs at doses of 20, 30, 40, and 50 microliters per egg. The controls received 100 microliters of physiological saline. All the embryos, including controls, were examined at day 13. The total number of eggs used in this study was 300. At 24 hours incubation, the percentage of survival ranged from 87 to 21 as the dosages of minoxidil were increased from 20 microliters to 50 microliters per egg (controls = 87%). The survival of the embryos ranged from 79% to 9% after the 48-hour treatment with the similar dosages of minoxidil utilized for the 24-hour group (controls = 83%). A low incidence of gross malformations such as twisted limbs, abnormal beak, short neck and everted viscera were observed; however, the increased incidence was not statistically significant when compared to controls. Body hemorrhage and edema were of high occurrence among the treated embryos. These effects are probably secondary to the known pharmacological effects of minoxidil. The frequency and types of gross malformations did not vary much in the 24 or 48-hour treated groups.     

Development of chicken embryos exposed to an intermittent horizontal sinusoidal 50 Hz magnetic field

The effects of intermittent exposure (2 h on/22 h off) to a 200 μT horizontal, sinusoidally oscillating (50 Hz) magnetic field were studied in 210 fertilized chicken eggs. Two hundred ten control eggs (sham-exposed) were incubated in the same chamber as the experimental eggs. Chick embryos were examined for developmental anomalies and maturity stage after 48 h of incubation. Immunohistochemical analysis of extracellular membrane components (laminin, fibronectin, and type IV collagen) were conducted on day 7 and histological examinations for malformations of brain, liver, and heart, on days 7, 12, and 18 of incubation. Furthermore, egg fertility and egg weights were evaluated on days 2, 7, 12, and 18. The investigation also measured the body weight of chickens for 90 days from hatching and included histological analysis of body organs. Each variable was investigated blind. Statistical comparison between exposed and sham-exposed values did not show significant differences in any of the variables investigated. Thus, it appears that the exposure of embryos to an intermittent 200 μT magnetic field at 50 Hz does not cause developmental anomalies, changes in maturity stage, alterations in distribution of extracellular membrane components, or malformations in the brain, liver, or heart. Moreover, there were no differences in body weight, morphology, or histology of central nervous system, liver, heart, or testis in 90-day-old chickens hatched from exposed in comparison to sham-exposed eggs. © 1996 Wiley-Liss, Inc.     

Spontaneous and benzo[a]pyrene-induced micronuclei in the embryos of the black-headed gull (Larus ridibundus L.)

The spontaneous levels of micronuclei in erythrocytes were established in embryos of the black-headed gull of two natural populations. In total 216 blood samples from the same number of individuals were examined. A statistically significant decrease in the number of spontaneous micronucleated erythrocytes was found after 13 days of incubation. We found no statistically significant difference in the spontaneous frequencies of micronucleated erythrocytes in the embryos of the two colonies studied, although they differed in anthropogenic load. Results of analysis of variance indicated that egg incubation time was the only variable significantly (P=0.0001) affecting spontaneous frequency of micronucleated erythrocytes in the embryos of black-headed gulls. We also took 78 eggs of different developmental stages from both colonies and exposed them for a further 24h to a dose of benzo[a]pyrene (30 microg per egg). After exposure to benzo[a]pyrene, the frequency of micronucleated erythrocytes was not increased in the embryos incubated for a total period of 13 days. A statistically significant increase in the number of micronucleated erythrocytes was recorded in the benzo[a]pyrene-treated embryos incubated for a total period of 14 days. Decrease in numbers of spontaneous micronucleated erythrocytes after the 13 day of incubation and increased levels of benzo[a]pyrene-induced micronuclei after the 13 day of incubation were discussed to be caused by changes in spleen and liver function in advanced developmental stages of the embryo.     

Copper-deficient rat embryos are characterized by low superoxide dismutase activity and elevated superoxide anions

The teratogenicity of copper (Cu) deficiency may result from increased oxidative stress and oxidative damage. Dams were fed either control (8.0 microg Cu/g) or Cu-deficient (0.5 microg Cu/g) diets. Embryos were collected on Gestational Day 12 for in vivo studies or on Gestational Day 10 and cultured for 48 h in Cu-deficient or Cu-adequate media for in vitro studies. Superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione reductase (GR) activities were measured in control and Cu-deficient embryos as markers of the oxidant defense system. Superoxide anions were measured as an index of exposure to reactive oxygen species (ROS). No differences were found in GPX or GR activities among treatment groups. However, SOD activity was lower and superoxide anion concentrations higher in Cu-deficient embryos cultured in Cu-deficient serum compared to control embryos cultured in control serum. Even so, Cu-deficient embryos had similar CuZnSOD protein levels as controls. In the in vitro system, Cu-deficient embryos had a higher frequency of malformations and increased staining for superoxide anions in the forebrain, heart, forelimb, and somites compared to controls. When assessed for lipid and DNA oxidative damage, conjugated diene concentrations were similar among the groups, but a tendency was observed for Cu-deficient embryos to have higher 8-hydroxy-2'-deoxyguanosine concentrations than controls. Thus, Cu deficiency resulted in embryos with malformations and reduced SOD enzyme activity. Increased ROS concentrations in the Cu-deficient embryo may cause oxidative damage and contribute to the occurrence of developmental defects.     

Blood groups and affective disorders

Frequencies of ABO, Rh, MNSs, P, Kell, Lewis and Duffy blood groups were studied in a total of 219 patients with affective disorders. The patients were classified into four groups: (1) bipolar (manic-depressive) psychosis; (2) unipolar recurrent depressive psychosis; (3) nonpsychotic 'reactive' depression, and (4) 'unclassifiable'. The following statistically significant results were found: an increased frequency of the blood group factor B among psychotic (bipolar and unipolar) patients compared to nonpsychotic patients, a decreased frequency of the SS phenotype in the unclassifiable group and an increased frequency of the K(+) phenotype among the nonpsychotic patients. Previous results concerning differences between bipolar and unipolar patients with respect to the A and O blood types were not confirmed in this investigation.     

Parameters determining isotretinoin teratogenicity in rat embryo culture

At the in vitro threshold serum concentration of 500 ng/ml, isotretinoin induces defects of visceral arch development in 9.5-day rat embryos grown in culture for 48 h. Experiments were performed to determine the minimum period of exposure necessary to induce these arch defects and whether an increase in concentration of isotretinoin could compensate for reduced exposure time. The results showed that a minimum 6-h exposure to 500 ng/ml immediately prior to cranial neural crest migration was necessary to induce severe defects of the second visceral arch in a majority of embryos. Maximal increase in isotretinoin concentration to 16,000 ng/ml did not compensate for shorter exposure periods. These results suggest that to cause malformations of the visceral arches, the embryo must be exposed to isotretinoin for a minimum period of time regardless of the concentration of isotretinoin above the threshold.     

Effects of secondary bile acids on the in vitro development of early somite rat embryos

Effects of secondary bile acids--lithocholic (LCA) and deoxycholic (DCA)--on the in vitro development of early somite (10.5 days old) rat embryos were studied. It was shown that an addition to the culture medium of 0.1 mM LCA (final concentration) resulted in 9% growth-retarded and 12% malformed embryos when the duration of exposure was 24 h. When treatment with LCA was prolonged to 48 h, the rate of growth retardation increased to 18% and that of malformations to 40% versus 0.5% for both parameters observed in controls. This could be interpreted as a reversible or time-dependent effect of LCA on the in vitro development of the mammalian embryo. Culture of embryos in medium with 0.5 mM DCA resulted in 22% of growth retardation and 50% of malformations. DCA in 0.1 mM final concentration had only slight and statistically nonsignificant effects. Retardation of growth development could be demonstrated by a decrease in crown-rump length and the number of somites. Among malformed embryos, abnormalities in the development of the neural tube and exencephaly were the most common types of malformations. Abnormalities as well as growth retardation were accompanied by significant pathological changes in structure and perhaps in function of the endodermal visceral yolk sac cells. It could be suggested that secondary bile acids when present in pathophysiological concentrations can affect the embryonic development by direct inhibitory effects and that these effects may be time and dose dependent.     

A genetic characterization of differences in the sensitivity to radiation-induced malformation frequencies in the mouse strains Heiligenberger,C57Bl,and Heiligenberger × C57Bl

We studied the frequency of malformations induced in two mouse strains (Heiligenberger, C57Bl/6J) by exposure to x-rays 3 h after conception. Whereas there was a high number of malformed fetuses in Heiligenberger mice (mostly gastroschises) on day 19 of pregnancy, C57Bl did not respond to radiation exposure shortly after conception with an increased frequency of malformed fetuses. Cross-breeding of both strains revealed that no statistically significant increase in radiation-induced malformations was obtained in the F₁ fetuses when the father was Heiligenberger and the mother C57Bl. In the opposite case (Heiligenberger mother, C57Bl father a small but statistically significant increase was observed.     

Effects of 100-Hz magnetic fields with various waveforms on the development of chick embryos

Summary Chick embroys were exposed during their 52 first hours of development to 100-Hz magnetic fields. Sinusoidal, square and pulsed waveforms were used at average field strengths from 0.1 A/m to 80 A/m. After exposure, the embryos were examined for abnormalities and classified by the developmental stages. When bipolar oscillations (oscillating at both sides of the zero-level) were used, the percentage of abnormal embryos was significantly increased above 1 A/m. In exposure to unipolar square waves, no significant effect on the percentage of abnormalities could be demonstrated. The developmental stage was possibly affected by unipolar square waves at 0.1 A/m, all other field strengths and wave-forms being apparently ineffective.     

Teratological studies with prostaglandin E2 in chick embryos

Prostaglandin E₂ (20–100 μg) was lethal to a relatively high percentage of chick embryos when administered at 48 hours incubation; no such effect was observed after 72 hours incubation. A relatively high incidence of abnormal embryos, which increased with the dose-levels of prostaglandin, was induced at both 48 and 72 hours incubation compared to the controls. However, this difference was statistically significant only in embryos treated with 100 μg prostaglandin at 48 hours incubation. The embryos showed no signs of growth retardation.     

Pulsed Electromagnetic Fields and Normal Chick Development

The effects of two clinical-type signals of pulsed electromagnetic fields (PEW) were tested on a large sample of developing chick embryos by two independent laboratories. The embryos were examined after seven days of incubation to avoid the early stages when most spontaneous abnormalities occur. The data were pooled and analyzed statistically. Neither signal, when administered continuously for the first seven days of development, or for the first 24 hours, produced a significant increase in the incidence of malformations. These results are discussed in relation to those of other investigators claiming PEW-related effects on normal development.     

Role of pulse shape in cell membrane electropermeabilization

The role of the amplitude, number, and duration of unipolar rectangular electric pulses in cell membrane electropermeabilization in vitro has been the subject of several studies. With respect to unipolar rectangular pulses, an improved efficiency has been reported for several modifications of the pulse shape: separate bipolar pulses, continuous bipolar waveforms, and sine-modulated pulses. In this paper, we present the results of a systematic study of the role of pulse shape in permeabilization, cell death, and molecular uptake. We have first compared the efficiency of 1-ms unipolar pulses with rise- and falltimes ranging from 2 to 100 μs, observing no statistically significant difference. We then compared the efficiency of triangular, sine, and rectangular bipolar pulses, and finally the efficiency of sine-modulated unipolar pulses with different percentages of modulation. We show that the results of these experiments can be explained on the basis of the time during which the pulse amplitude exceeds a certain critical value.     

Magnetic fields and time dependent effects on development

Pulsed, extremely-low-frequency electromagnetic fields caused a significant increase in abnormalities in the developing chick embryo. The effect was observed when the field was presented during the first 24 h of incubation; no significant effect was observed with exposure from 24 to 48 h of incubation.     

Effects of nonionizing radiation on birds

1. With the ability to fly comes a greater probability of direct irradiation by nonionizing radiation. The effect of nonionizing radiation on birds is, therefore, of environmental significance. 2. Most biological effects of exposure to nonionizing radiation in avian species are a result of radiation-induced temperature increases. 3. The incubating avian egg provides a model to study nonthermal effects of microwave exposure since ambient incubation temperature can be adjusted to compensate for absorbed thermal energy. 4. Some studies have shown that exposure to nonthermal levels of nonionizing radiation affect a bird's ability to recover from acute physiological stressors. 5. Although earlier research indicated that modulated radiofrequency radiation increased calcium-ion efflux in chick forebrain tissue, criticism of experimental techniques and contradictory results between related studies have made final conclusions elusive. 6. Birds have been shown to be able to reliably detect magnetic fields in both the field and laboratory. Some researchers have reported malformations in chicken embryos exposed to a sinusoidal bipolar oscillating magnetic field.     

Influence of L.5-hydroxytryptophan (L.5-HTP) on the development of chick embryo]

L.5-hydroxytryptophan (L.5-HTP) injections provoke, in the chick embryo, some malformations of the nervous system, when treated at 24 hours of incubation. The same treatement after 48 hours of incubation does not lead to malformations, but to a reduction in size which is as much obvious as the embryos are treated at a later stage. It seems that there could be some relation between the serotonin metabolism and the growth hormon secretion.     

Chick embryo development can be irreversibly altered by early exposure to weak extremely-low-frequency magnetic fields

Several reports have shown that weak, extremely-low-frequency (ELF), pulsed magnetic fields (PMFs) can adversely affect the early embryonic development of the chick. In this study, freshly fertilized chicken eggs were exposed during the first 48 h of postlaying incubation to PMFs with 100 Hz repetition rate, 1.0 μT peak-to-peak amplitude, and 500 μs pulse duration. Two different pulse waveforms were used, having rise and fall times of 85 μs (PMF-A) or 2.1 μs (PMF-B). It has been reported that, with 2 day exposure, these fields significantly increase the proportion of developmental abnormalities. In the present study, following exposure, the eggs were allowed to incubate for an additional 9 days in the absence of the PMFs. The embryos were taken out of the eggs and studied blind. Each of the two PMF-exposed groups showed an excess in the percentage of developmental anomalies compared with the respective sham-exposed samples. This excess of anomalies was not significant for the PMF-A-treated embryos (P = 0.173), whereas it was significant for the PMF-B-exposed group (P = 0.007), which showed a particularly high rate of early embryonic death. These results reveal that PMFs can induce irreversible developmental alterations and confirm that the pulse waveform can be a determinant factor in the embryonic response to ELF magnetic fields. The data also validate previous work based on the study of PMFs' effects at day 2 of embryonic development under field exposure. © 1994 Wiley-Liss, Inc.     

Serological markers in unipolar and bipolar affective disorders

ABO blood groups and haptoglobin types have been studied with special reference to their association with unipolar and bipolar affective disorders. The associations of serogenetic markers show statistically significant differences between unipolars and controls, bipolars and controls, and unipolars and bipolars. The results are largely in agreement with those reported in previous studies.     

Teratogenic effects in early chick embryos of solanine and glycoalkaloids from potatoes infected with late-blight, Phytophthora infestans.

Solanine or a preparation of mixed glycoalkaloids from potatoes naturally infected with the late-blight fungus, Phytophthora infestans, was injected into fertile chicken eggs between 0 and 26 h of incubation, before formation of the neural tube. The embryos were examined after a total of 72 h of incubation. Various abnormalities were found, the most conspicuous being absence of the tail or trunk below the wing bud (rumplessness). A statistically significant proportion of the abnormal embryos showed malformations that seemed to be related to this condition; these included fluid- or blood-filled vesicles in the lower trunk or tail region on one or both sides of the neural tube. Such abnormalities were not observed in control embryos.