Absence of geomagnetic field does not influence the chick embryo development

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1 Absence of geomagnetic field does not influence the chick embryo development Richard Jelinek, Jiri Blaha, and Jitka Janoutova Institute of Histology and Embryology, 3 rd Faculty of Medicine, Charles University (R.J., J.J.), Geophysical Institute, Academy of Sciences of Czech Republic, Prague, Czech Republic (J.B.) Effects of incubation in the absence of geomagnetic field was studied embryos of White Leghorns in a series of repeated experiments comprising about 500 treated and approximately the same number of control specimens. No adverse effects were detected after 2- and 9-day lasting incubation in zero magnetic field yielded by compensation method in a special computer-controlled space. Prevalence of malformed and dead embryos, malformation spectra as well as stage distribution and mean weight of normal foetuses were found similar in control and experimental groups. We may conclude that magnetic field deprivation exerts no influence upon the chick embryo development. Key words: geomagnetic field, embryogenesis, chick embryo INTRODUCTION Biological effects of electromagnetic fields (EMF) has been studied in nowadays hardly comprehensible number of papers extensively reviewed for by Dubrov (1978), Chernoff et al. (1992), and Carpenter and Arapetyan (1994). Experiments were performed using systems varying from cells to whole animals under great variety of conditions, and manifold effects were recorded. Now it seems evident that least effects are exerted from static and more remarkable ones from pulsed magnetic fields (MF) while the original intensity of electric fields (EF), due to their poor penetrance, is reduced within the living systems by at least six orders of magnitude. However effects of EMF upon animal reproduction still remain controversial (for review see Dubrov, 1978, Chernoff et al., 1992). A well founded exact analytic paper of Brent et al. (1993) summarises the findings of both animal and human studies and concludes that the reproductive risk of EMF seems under the current real conditions negligible. This statement is based upon the fact that EMF do not fit several criteria defining human teratogens. Among the criteria we find the requirement of positive results provided by in vivo and in vitro experimental systems. Most striking in the case of EMF is that the results are generally inconsistent even when using one and the same experimental system. As most of the reports concern the developing chick we will focus on this biological model which at the first sight seems very convenient. The frequently quoted disadvantage of this experimental system, i.e. the lack of maternal organism does not play in the case of MF a part comparable to the complex pharmacokinetics and biotransformation of chemicals. As mentioned above, MF is not significantly altered when penetrating maternal tissues. In spite of that Nguyen et al. (1995) claim that avian embryo does not represent a good model for mammalian teratogenesis and therefore they prefer to investigate the insect Drosophila melanogaster. Notwithstanding, the findings after the chick embryo exposure to MF have been really 1

2 controversial. While Zervins (1973), Krueger et al. (1975), and Durfee et al. (1975) did not obtain any positive result, Joshi et al. (1978), Delgado et al. (1982) and Ubeda et al. (1983) recorded an increase of abnormal embryos equalling, in the most dramatic case, 62.5% over control values. Since that time nobody was able to demonstrate such an unequivocal effect (Martucci et al., 1984, Maffeo et al., 1988). The following years were very productive as one concerns investigations, however failed to make the situation more clear. Positive results after exposure to MF of low intensity and frequency were newly described by Juutilainen (1986), Juutilainen and Saali (1986), Juutilainen et al., 1986, 1987), and Martin (1988), however denied again by Sisken et al. (1986), Bardosano et al. (1986), Sandstrõm et al. (1987), and Maffeo et al. (1988). The equilibrium between pros and cons did remain unchanged. The moment of truth should have brought the study by Berman et al. (1990) which evaluated results of experiments of six independent laboratories carried out under same conditions (unipolar pulse 500µs, 10 mg for the first 48 h of incubation). The conclusion seemed catastrophic for the model used. Four of the laboratories found no difference between control and experimental groups, the resting two reported a remarkably increased incidence of malformed specimens at the end of incubation, equalling 200 and 400% respectively. Pooling all the data brought about a small (25%) but highly significant difference. Statistical evaluation of these results was revisited and improved by Handcock and Kolassa (1992) who no doubt confirmed the effect of exposure, however demonstrated at the same time that undefined laboratory conditions were similarly effective. This uncertainty is, however, not specific for the chick embryo model. Mevissen et al. (1994) exposed pregnant Wistar rat females to static (30 mt) or pulsed (50 Hz) MF of the same intensity. No gross malformations were found in either group, however in the first group they registered a lower number foetuses per litter and in the second one an increased incidence of small skeletal defects. The authors concluded from these rather inexpressive and from the point of principles of teratogenesis inconsistent results that the static MF might have an embryotoxic effect. Many investigators feel that it is necessary too bring some decisive demonstration whether MF can deviate embryonic development or not. While chick embryo has not yet been considered useful for routine embryotoxicity testing, its value for disclosing basic mechanisms of development is seldom denied. According to our experience the chick embryo may serve as a suitable object even in the former case (Jelínek, 1982) provided that several rules are followed: 1. The basic condition is standardisation of the material. As the incubation loss depends upon genotype and housing conditions, season, time and storing conditions of fertile eggs before incubation and, of course, incubation conditions, it is recommended both to perform a thorough standardisation of embryos entering the experiment and to use parallel controls. 2. As any category of toxic action, the proof of teratogenicity requires to demonstrate positive dose-response relationships. This can be generally achieved when and only when respecting that embryotoxicity manifests itself not only by malformation but also by death and growth retardation. These three phenomena follow any embryotoxic exposure and transform into each other along dose axis. 3. To inhibit influences of seasonal (in a broad sense) character, it is recommended to plan experiments as repeated assays. Repeat testing provides greater confidence in the final interpretation of results (Carr et al., 1996). 2

3 We decided to apply these general rules in revisiting the problem of MF action upon embryonic development. The present paper deals with the development in hypomagnetic environment. MATERIAL AND METHODS The experiments were performed in the period between December 1994 and May 1995 on chick embryos yielded from fresh-laid eggs of randombred population of White Leghorn Fowl (farm Dominant, Dobøenice). Incubation The eggs were put into incubators 6 hrs after delivery, the while stored at room temperature. For each experiment 90 eggs were selected at random into experimental and control groups of equal size, and incubated parallely at C and 40-50% air humidity in zero and natural geomagnetic fields, respectively. Special forced-draught thermostatic ovens built of transparent plastic materials were heated by remote fans through ruckled high-capacity tubing. The temperature was computer regulated using special software and kept within practical limits +/ C. Automatic 5min record allowed precise control of the standard conditions for both incubators. Device Procedure Following 48h incubation in horizontal position eggs were candled to discard the few infertile ones and, in the rest, to mark position of the embryo on the shell. Then the eggs were opened just above the embryo using the conventional window technique (see, for instance, Jelínek and Peterka, 1983). Under preparation microscope (Olympus) embryos were examined for normality and staged according to Hamburger and Hamilton (1951). By comparing the distribution of early embryonic death, abnormal specimens and HH stages, we have got an initial information on the possible influence of hypomagnetic environment in course of the first 48 hrs of incubation and, moreover, we gained the particular group characteristics as a starting point for assessing the further embryonic development. Dead, abnormal, as well as normal looking embryos <HH 8 stage (four somites) were discarded After moistening the vascular area by 0.7% saline and closing windows in the shell with glass slides on paraffin frames, the eggs were put back into the appropriate oven. During the whole procedure the eggs were left outside the incubator and, therefore, out of the hypomagnetic environment for about half an hour. A control experiment with unopened eggs was designed to recognise how this condition might influence the results. In course of the 7-day-lasting reincubation period embryos were checked every day through windows and transparent walls of the incubator without disturbing any more the exposure conditions. Dead specimens were removed, examined under preparation microscope for visible malformations and probable cause of death, and the findings recorded. Embryos that died in consequence of technical fault during manipulation were discarded (<1%). Evaluation On day 9 of overall incubation the eggs were reopened, embryos gently removed from fetal membranes and their umbilical cords were cut at proximal ends. Then the embryos were weighed and checked under preparation microscope for defects of the following organs : central nervous system (neural dysrhaphia [NTD], exencephaly, microencephaly, brain herniation, eyes (microphthalmia, buphthalmia, coloboma), face (clefts, defects of pharyngeal arches), limbs (polydactylia and reduction deformities), 3

4 body wall (coelosomia, umbilical hernia), and trunk (syndrome of caudal regression, rumplessness, defects of vertebral column curvature). A routine dissection of the heart allowed to include also defects of great vessels and of the interventricular septum. Statistics Beside the descriptive statistics and t-test, several non-parametric methods were used comprising the standard error of proportions, contingency table analysis and the distribution (Smirnov-Kolmogorov) test of good fit (STATGRAPHIC). RESULTS AND DISCUSSION Aiming to give greater confidence in the final interpretation of results, we used repeat testing. Any of the experiments represents an independent entity with its own control. In this way we were able to express the variability present and to extend the array of relevant statistical methods. As mentioned in the Introduction, manifestations of embryotoxicity comprise three distinct phenomena (malformation, death and growth retardation) that occur in different proportions depending on dose and nature of the inducing factor. While embryonic death is a most common appearance accompanying the prenatal development of any species, the other two phenomena occur only seldom and are generally more specific. At difference from the growth retardation, malformations are tied to exposures in the so called teratogenetical critical periods. In our study we took into consideration all the embryotoxicity manifestations observed in course of the main period of organogenesis (day 2-9). Results of exposure to the hypomagnetic field will be introduced as prevalences at two time horizons (48 h and day 9 of development) and as a dynamic parameter of out-dying rate. Effects after 48 hours As seen in Table 1 and Fig. 1 summarising results of 12 experiments, the absence of geomagnetic field did not influence significantly either prevalence of abnormal specimens on day 2 or degree of their development as evaluated by the distribution of HH stages (p=0.258 and p=0.434, respectively). Effects after 9 incubation days In three experiments the incubation in hypomagnetic environment proceeded over the whole period until day 9. In two of them (E1-2 and E1-4) embryos were selected on day 2 through windowing, in E1-5 eggs were left intact. As apparent from Table 2, no signs of adverse effects were recorded both in prevalence of embryotoxicity phenomena in general as same as in prevalence of particular malformation types (p=0,416 and p=0,476, respectively). Weight analysis The average weight of embryos carrying no developmental defects appeared similar regardless of they were incubated under normal or hypomagnetic conditions (p=0,390 - ANOVA, Table 3.) Basing upon these results may conclude that the absence of geomagnetic field does not influence the chick embryo development. This conclusion, however, does not accord with the observation of Asashima et al. (1991) who kept the developing larvae of the Japanese newt (Cynops pyrrhogaster) in a shielded environment in which the MF appeared as low as 5 nt. In larvae examined 20 days after 5-day-lasting shielding, the authors recorded an increased incidence in somatic defects as well as developmental retardation. The response varied with the stage of development when deprivation from the normal GMF took place. The most sensitive appeared embryos at gastrula stage and at very early stages of embryogenesis which seems consistent with the known 4

5 principles. No explanation for such difference in reaction to hypomagnetic field between chick and amphibians can be afforded excepting the method used. It is well known that shielding, contrary to compensation, affects a broader array of factors beyond MF (Dubrov, 1978). This work was supported by COST Grant No? REFERENCES -Asashima M, Shimada K, Pfeiffer CJ (1991) Magnetic shielding induces early developmental abnormalities in the newt, Cynops pyrrhogaster. Bioelectromagnetics 12: Bardosano JL, Meaer AJ, Picazo L (1986) Pineal cells with multipolar spindles in chicken embryos exposed to magnetic fields - first trials. Z Mikrosk Anat Forsch 100: Berman E, Chacon L, House D, Koch BA, Koch W, Leal J, Lovtrup S, Mantiply E, Martin AH, Martucci GI et al. (1990) Development of chicken embryos in a pulsed magnetic field. Bioelectromagnetics 11: Brent R, Gordon WE, Bennett WR, Beckmann DA (1993) Reproductive and teratologic effects of electromagnetic fields. Reprod Toxicol 7: Carpenter DO, Ayrapetyan S (eds) (1994) Biological Effects of Electric and Magnetic Fields. Vol. 1 Sources and Mechanisms. New York: Acad Press, pp Carr GJ, Gorelicks NJ (1996) A place for statistics in the generation and analysis of genetic toxicity data: A response to rodent mutation assay data presentation and statistical assessment. Mutation Res 357: Chernoff N, Rogers JM, Kavet R (1992) A review of the literature on potential reproductive and developmental toxicity of electric and magnetic fields. Toxicology 74: Dubrov AP (1978) The Geomagnetic Field and Life. Geomagnetobiology. (Transl ed Brown FA Jr) New York: Plenum Press, pp 318 (original in Russian, Gidrometeoizdat, Leningrad 1974) -Durfee WK, Glante PR, Muthukrishnan S (1975) Extremely low frequency electric and magnetic fields in domestic birds. Part I: The influence of ELF, magnetic and electric fields upon hatchability and early development of chicks. NMRDC Report: Compilation of navy sponsored ELF Biomedical and Ecological Reports. -Delgado JMR, Leal J., Monteagudo JL, Gracia MG (1982) Embryological changes induced by weak, extremely low frequency electromagnetic fields. J Anat 14: Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morph 88: Handcock MS, Kolassa JE (1992) Statistical review on the henhouse experiments: the effects of pulsed magnetic field on chick embryos. Bioelectromagnetics 13: Jelínek R (1982) Use of the chick embryo in screening for embryotoxicity. Teratogenesis Carcinogenesis Mutagenesis 2: Jelínek R, Peterka M (1983) Method used in our laboratory in Prague for the Chick Embryotoxicity Screening Test (CHEST). In Neubert D, Merker H-J (eds): Culture Techniques. Applicability for Studies on Prenatal Differentiation and Toxicity. Berlin, New York: Walter de Gruyter, pp Joshi MV, Khan MZ, Damle PS (1978) Effect of magnetic field on chick morphogenesis. Differentiation 10:

6 -Juutilainen J (1986) Effects of low frequency magnetic fields on chick embryos. Dependence on incubation temperature and storage of the eggs. Teratology 34: Juutilainen J, Saali K (1986) Development of chick embryos in 1 hz to 100 khz magnetic fields. Radiat Environ Biophys 25: Juutilainen J, Harri M, Saali K, Lahtinen T (1986) Effects of 100-Hz magnetic fields with various wave forms on the development of chick embryos. Radiat Environ Biophys 25: Juutilainen J, Laara E, Saali K (1987) Relationship between field strength and abnormal development in chick embryos exposed to 50 Hz magnetic fields. Int J Radiat Biol 52: Krueger WF, Giarola AJ, Bradley JW, Shrekenhamer A (1975) Effects of electromagnetic fields on fecundity in the chicken. Ann NY Acad Sci 247: Maffeo S, Miller MW, Carstensen EL (1984) Lack of effect of weak low frequency electromagnetic fields on chick embryogenesis. J Anat 4: Maffeo S, Brayman AA, Miller MW, Carstensen EL, Ciaravino V, Cox C (1988) Weak low frequency electromagnetic fields and chick embryogenesis: failure to reproduce positive findings. J Anat 157: Martin AH (1988) Magnetic fields and time dependent effects on development. Bioelectromagnetics 9: Martucci G, Gailey PC, Tell RA (1984) Investigation of possible effects of weak pulsed magnetic fields on the chick embryo. Abstract of the 6th Ann Meeting of the Bioelectromagnetic Soc. Atlanta, GA, USA. -Mevissen M, Buntenkõter S, Lõscher W (1994) Effects of static and time-varying (50-Hz) magnetic fields on reproduction and fetal development in rats. Teratology 50: Nguyen P, Bourniasvardiabasis N, Haggren W, Adey WR, Phillips JL (1995) Exposure of Drosophila melanogaster embryonic cell cultures to 60 Hz sinusoidal magnetic fields: Assessment of potential teratogenic effects. Teratology 51: Sandstrõm M, Mild KH, Lovtrup S (1987) Effects of weak pulsed magnetic fields on chick embryogenesis. In Nave B, Wideback PG (eds): Work with display units. B.V. North Holland: Elsevier Sci Publ, pp Sisken BF, Fowler I, Mayaud C, Ryaby J, Pilla AA (1986) Pulsed electromagnetic fields and normal chick development. Bioelectronics 5: Ubeda A, Leal J, Trillo MA, Jimanez MA, Delgado JMR (1983) Puls shape of magnetic field influences chick embryogenesis. J Anat, 139: Zervins A (1973) Chick embryo development in a 26 khz electromagnetic field. Am Ind Hygiene Assoc J 34:

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