TERATOLOGY 43:609-614 (1991)
Lethal and Teratogenic Effects of Long-Term Low-Intensity Radio Frequency Radiation at 428 MHz on Developing Chick Embryo KENICHI SAITO, KATSUSHI SUZUKI, AND SHIGEKATSU MOTOYOSHI Nippon Veterinary and Zootechnical Colkge, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180, Japan
ABSTRACT Exposure of developing chick embryos to 428 MHz radio frequency (RF) radiation at a power density of 5.5 mW/cm2for more than 20 days resulted in embryolethal andlor teratogenic effects and delayed hatching. These adverse biological effects were not due to any thermal effect of the RF radiation. We have demonstrated teratogenicity in the chick embryo as a result of protracted low-dose RF irradiation. Lary and Conover ('87) have pointed out that "over the past four decades, the use of radiofrequency (RF) radiation sources has increased dramatically. Because of the increasing prevalence of occupational, medical, and environmental exposure to RF radiation, there is growing concern about the potential effects of such radiation on the unborn child." This is also true in Japan. Teratogenic effects of RF radiation have been demonstrated in animals, although many investigators concluded that RF-induced teratogenic and embryolethal effects are due t o hyperthermia produced by high-intensity RF fields and not to field-specific effects unrelated to heating. Actually, it has been known that heat causes birth defects and prenatal death when the body temperature reaches 40-44"C, regardless of the species (Lary et al., '82; Lary, '86; Lary and Conover, '87). The authors have previously reported adverse biological effects of low-intensity RF radiation at 433 and 906 MHz on experimental animals, including prolongation of the gestational period in RF-exposed maternal mice and reduction of thymus weight in pups born from treated mothers (Saito et al., '86, '87). These adverse effects were considered to be due to nonthermal effects of RF radiation, since the rise in rectal temperature was not clearly detected. Maternal body temperature fluctuated considerably according to the activities and metabolic status. Furthermore, maternal mice moved around in the RF field. Therefore, it was 0 1991 WILEY-LISS, INC.
very diffciilt to estimate the actual specific absorption rate (SAR) in the fetuses. The developing: chick embryo would be a more suitable model for detection of the adverse effects of low-intensity RF radiation. The RF of 428 MHz was selected because the band has been frequently used in Japan by amateur radio stations, and hence the parts and equipment necessary for the experiment are readily available. The present article describes embryolethal andlon teratogenic effects of low-dose RF exposure of developing chick embryos during precisely known developmental stages (Parkhurst and Mountney, '88). It was determined that the embryolethality and teratosgenicity of RF radiation were associated with nonthermal effects of the radiation. MATERIALS AND METHODS
The fertilized eggs of white leghorn chickens, laid within 24 hr, were purchased from Tomaru Poultry Farm (Maebashi, Japan). The eggs were incubated and exposed to RF radiation in an incubator (model p-01; Showa Incubator Co., Ltd., Saitama, Japan) at 37°C 5 0.5"C, with a relative humidity of 80%. The eggs were automatically turned once each hour. Viability of the eggs was conventionally checked once every day during the entire period of incubation. The pre-
Received March 15, 1990; accepted December 11, 1990.
610
K. SAITO ET AL.
'*
9
0
8
0
7
6
0
.
0
5 0
4 .
3
2
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'Position c electric
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O P o s i t i o n of eggs
Electrode
1 1
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Wooden rack
, Elect rode
Fig. 1. Schematic representation of the positions of the eggs on the wooden rack placed between the electrodes in a n incubator.
cise inspections of eggs were carried out on days 10, 20, 21, and 22 after incubation so that it might be possible to determine if death occurred before or after day 10. Each of ten eggs was subjected to an experiment. Seven experiments were repeated in the same apparatus. The first and the fourth experiments were designated as controls, in which the eggs received sham exposure to RF radiation. In total, 50 eggs were used in the RF radiation group and 19 eggs in the control group (an egg was broken and lost). The inner appearance of the incubator and positions of the eggs on the wooden rack are shown in Figure 1. The apparatus for continuous-wave RF radiation was designed by one of the authors. A block diagram of the apparatus is shown in Figure 2. The linear amplifier was made using the hybrid integrated circuits, M57704M (Mitsubishi Electric Co. Ltd., Tokyo, Japan). A radio frequency field of 428 MHz was generated by a crystal oscillator (Yakumo Crystal Co. Ltd, Tokyo, Japan).
The RF radiated power was 6.8 W at the RF power meter (model SP-300, Welz Co. Ltd., Tokyo, Japan). The area of the two aluminum plate electrodes was 1,224 cm2 (34 x 36 cm). The theoretical power density was 5.5 mW/cm2 between the electrodes, which had a gap of 14 cm. The RF electrical field and RF magnetic field were measured by an RF survey meter (model HI3002, Holaday Co. Ltd., Minnesota) at each position of the eggs. The electric field could be accurately measured by the meter (operating range 0.5-6,000 MHz). However, the values of the magnetic field may be inaccurate since the meter operating range for magnetic fields is only 5-300 MHz. No meters for detection of magnetic fields produced by 428 MHz RF radiation are currently available. The specific absoption rate (SAR) was calculated according to the method of Fujiwara and Amemiya ('82) with the assumption that the egg is an ellipsoidal body 6 cm on its major axis and 4.5 cm on its minor one. To determine the increase in temperature of the egg caused by RF radiation, the contents of four eggs were replaced by 25% gelatin and a fluoroptic thermometer (model FG-1000, Luxtron Corp., Mountain View, CA) was fixed through a hole of the eggshell by paraffin (Fig. 3). Before measuring the temperature, the gelatin-replaced egg was placed in the incubator set at 37°C for 1hr. The increase in temperature of the gelatin per unit of time was repeatedly measured in different eggs with the fluoroptic thermometer for 20 min during exposure to RF at a position where the maximum SAR was obtained (position 3). The longitudinal axis of the eggs was oriented parallel to the electric field, and the eggs received RF radiation continuously during incubation. Right after the chicks hatched (0 to -6 hr after actual hatching), their body weights were measured. The chicks were then anesthesized with an overdose of ether and necropsied. The heart, liver, kidney, adrenal glands, thymus, lungs, thyroid gland, pancreas, bursa of Fabricius, gonads, and yolk sac were excised and weighed to the nearest 0.1 mg. The organs were then fixed with 4% neutral formalin, embedded in paraffin, cut at 3 km, stained with hematoxylin and eosin, and observed under a light microscope. Student's t test and the x2 test were used for the statistical analyses, with a significance level of P < 0.05.
MICROWAVE TERATOGENICITY IN CHICK EMBRYOS
611
Electrodes
Fig. 2. Block diagram of the apparatus for RF irradiation.
the RF-exposed group was associated with cessation O F development within 10 days after the start of incubation, whereas the remainder (76.6%) was due to death within the eggshell following inability to hatch (Figs. 5 and 6). In the latter case, the yolk was almost entirely absorbed by the chick. The incubation period necessary for hatching differed significantly between the groups (P 0.05, x2 test). The control eggs hatched on days 21 or 22, and the exposed eggs hatched on days 21-23 (Table 3). Therefore, it was clear that prolongation of Fig. 3. Gelatin-replaced egg with a fluoroptic ther- the incubation period occurred in the exmometer fixed through a hole of the eggshell by paraf- posed group. fin. Changes in hatchability according to the position of the rack in the RF-irradiated RESULTS group are shown in Table 4. It is noteworthy Regional distributions of the strength of that the severest effect was seen in positions the electric and magnetic field and of the 8-10, where the SARs were relatively low calculated SAR are shown in Table 1. The (see Table 1). In contrast, all chick embryos highest values were obtained at position 3 hatched at position 1, where the SAR was and the lowest at position 5. The calculated relatively high. One case of death in the SAR using both values of the electric and shell occurred at each of positions 3, 5, and magnetic fields ranged between 3.1 and 47.1 6 in the control group. mWIkg, whereas the power density ranged A functional abnormality consisting of between 0.05 and 0.42 mW/cm2 using the creeping movement and inability to stand data of electric field alone. The latter values (Fig. 7) was found in 89% of the hatched suggest that the actual radiation was less embryos in the exposed group but was not than one-tenth theoretical power density found in the controls. In these abnormal (5.5 mW/cm2). chicks, bending or shortening of the bone or The increase in temperature of 25%gela- abnormal ;uticulation was not detected. A tin during RF radiation is shown in Figure chick with a featherless lower trunk was 4. The average gelatin temperature was found when an eggshell was manually bro37.7"C and very stable during measurement ken to pull out a chick of the exposed group for 20 min. No increase in temperature by on day 22 :Fig. 8). The chick was a female, RF radiation was detected. and for 3 days after artificial hatching it The average hatchability was 84.2% in could not skand by itself. However, it grew control eggs and 38.0%in exposed eggs (Ta- normally until it was sacrificed on day 30 ble 2). Almost one-fourth of the mortality in (Fig. 9). The weight of the organs in male :c
612
K. SAITO ET AL.
TABLE 1 , Regional distributions of electric and magnetic field strength, and of calculated S A R i n a wooden rack placed between electrodes in a n incubator' E2 (V2/m2) H2 (A2/m2) Position ( x lo3) ( x 10.') SAR (mW/kg) 1 1.1 1.4 11.0 2 1.2 4.2 33.0 3 1.6 6.6 47.1 4 0.2 0.7 5.5 5 0.2 0.4 3.1 6 0.2 0.5 3.9 7 0.2 0.6 4.7 8 0.2 0.6 4.7 9 0.2 0.7 5.5 10 0.2 1.1 8.6 'Positions correspond to those shown in Figure 1.
I S.D. 'C
38
37
-
-
TABLE 2. Comparison of hatchability between control and radio frequency exposed eggs Control Exposed (%I (%) Number of eggs Incubated 19 50 Hatched 16 (84.2) 19 (38.0)* Dead 3 (15.8) 30 (60.0)* .~ Within 10 days 0 7* In shell 3 23* Artificial hatching 0 1 (2.0)** ~~
,
*Significantly different from control at P < 0.01. **A case of undeveloped feather around lower trunks and legs.
TABLE 3. Incubation period for hatched eggs treated with RF radiation' Incubation period (days) 21 (%) 22(%) 23(%) 24(%) Group Control Number 12 (75) 4 (25) hatched Sex F8,M4 F3,Ml Exposed Number hatched 4 (16.7) 11 (61) 3 (16.7) l ( 5 . 6 ) Sex F2,M2 F5,M6 F2,Ml F1 'Eighty-nine percent of the hatched chicks in the exposed group showed a functional anomaly in their inability to stand on their legs and by creeping movement.
,
and female chicks in the control and exposed groups varied considerably (data not shown). This might be because of the variation of the incubation periods for hatching and of egg size at the start of incubation. There were no abnormal histological findings in any of the organs examined. DISCUSSION
TABLE 4. Positional distribution of hatched and dead embryos in radio frequency exposed group Number Number of dead Number of Posiwithin of dead Artificial tion 10 days hatched in shell hatching 1 5 0 0 0 2 2 0 3 0 3 3 1 1 0 4 2 0 3 0 5 3 0 2 0 6 2 0 3 0 7 2 0 3 0 8 0 2 2 1 9 0 2 3 0 10 0 2 3 0
Embryo-lethal and teratogenic effects of RF irradiation have been reported in various species, including the rat, mouse, monkey, and man (Lary and Conover, '87). In SAR was smaller than the basal metabolic those experiments, much higher powers rate reported in the chick (3.4 W/kg; Mount, were applied t o the animals for only a short '79). On the other hand, calculated SARs by time in comparison to the irradiation condi- both electric field and magnetic field data tions in the present study. They concluded and by electric field data alone were unexthat the teratogenic effect of the RF was due pectedly low compared with the theoretical invariably to its inherent thermal effect, value of 165 mW/kg. The Radiofrequency since the irradiated animals had a n ele- Radiation Dosimetry Handbook (Durney et vated body temperature, and hyperthermia al., '78) indicates that, at a power density of is a well known teratogen in the rat (Lary et 5.5 mW/cm2, the maximum SAR of a al., '82). chicken egg exposed at 428 MHz should be In the present study, the maximum SAR about 165 mW/kg. Based on the data meavalue was 47 mWlkg, and the minimum was sured, the power density at the source was 3.1 mW/kg. This maximum value for the 0.42 mW/cm2,which is less than one-tenth
MICROWAVE TERATOGENICITY IN CHICK EMBRYOS
613
Fig. 8. Chick with featherless trunk manually pulled from a n egg exposed to RF radiation for 22 days. Fig. 9. Same chick as in Figure 8 aged 30 days after artificial hatching. Note that lower trunk remains featherless.
the theoretical power density value. Since RF radiation at 428 MHz is about 70 cm in wavelength, a considerable amount of energy was lost because of the size of the electrode, which was about one-half of the wavelength. In the piresent study, no thermal effect of the RF radiation was detected when using a fluoroptic thermometer, which gives accurate temperature measurements in an RF field. In the low-intensity field of this study, no measurable temperature increase would be expected. This suggests that chick embryos in this study did not show any inFig. 5. Dead chick embryo whose development ter- crease in tlody temperature during RF radiminated within 10 days after incubation under RF exation. Therefore, the leg abnormality and posure. high mortality seen in the present study Fig. 6. Embryo that died about 17-18 days after in- were not likely to have been caused by the cubation started under the same RF irradiation condi- thermal etTect of the RF radiation. This is tions as used for Figure 5. the first study to detect direct teratogenicity Fig. 7. Chick, hatched from the RF-exposed egg on following long-term low-dose RF irradiation day 21 after incubation started, unable to stand on its of a n animal. However, Delgado et al. ('82) legs and showing creeping movement. reported abnormalities in chick embryo development from exposure to a very-low-in-
614
K. SAITO ET AL.
tensity (nonthermal) pulsed magnetic field at 10,100, or 1,000 Hz. The teratogenic dose of the magnetic field was reported t o range between 0.12 and 12 pT. Prolongation of the gestation period by long-term low-dose RF radiation has been reported in mice (Saito et al., '86). Similar effects were found in mice housed in a magnetic field (Nakagawa, '70). In the present study, the period necessary for hatching was prolonged in RF irradiated eggs. It is clear that these biological effects are not caused by the thermal effect of the RF but by its direct action. There is little information on the mechanisms governing the time periods necessary for birth or hatching. Growth retardation seen in some organs at necropsy (data not shown) may relate to the prolonged hatching period, although the mechanism is unknown. We speculate that RF radiation may have an effect on regulation of the growth rate by influencing the rate of cellular proliferation in the embryo. The pearl chain phenomenon seen in lower animals such as protozoanspecies during RF exposure (Schwan, '83) is thought to result from changes in electronic charges on the cell surface. Electrical charges on the cellular surface may interfere with cell separation. Another possibility is that RF radiation might affect cell-cell interaction during development and/or cellular migrations through interactions with the extracellular matrix as discussed by Delgado et al. ('82). Further study will be necessary to determine the actual mechanisms causing the adverse effects on development observed in this study.
LITERATURE CITED Delgado, J.M, J . Leal, J.L. Monteagudo, and M.G. Garcia-Gracia (1982) Embryological changes induced by weak, extremely low frequency electromagnetic fields. J . Anat., 134:533-551. Durney, C., C. Johnson, P. Barber, et al. (1978) The Radiofrequency Radiation Dosimetry Handbook (2nd edition). Report SAM-TR-78-22, U.S. Air Force School of Aerospace Medicine, Brooks Air Force Base, Texas, p. 101. Fujiwara, O., and Y. Amemiya (1982) Microwave power absomtion in a sDecimen inside a standine-wave irradiatidn waveguide. IEEE Trans. Microwave Theory Tech, MTT-23:2008-2012. Lary, J.M. (1986) Hyperthermia and teratogenicity. In: Hyperthermia in Cancer Treatment, Vol. 1. A. Anghileri and D. Robert, eds. CRC Press. Boca Raton, FL, pp. 107-126. Lary, J.M., and D.L. Conover (1987) Teratogenic effects of radio freauencv radiation. IEEE E n ~ Med. n Biol. Mag., Ma&, pp.h2-46. Lary, J.M., D.L. Conover, E.D. Foley, and P.L. Hanser (1982) Teratoeenic effects of 27.12 MHz radiofrequency radiathn in rats. Teratology, 26t299-309. Mount, E.L. (1979) Adaptation to Thermal Environment. Edward Arnold, London, p. 22. Nakagawa, M. (1970) Experimental studies on effect of magnetic fields on fertility, general reproductive performance and growth of mice. J . Nagoya City Univ. Med. School, 29:323-341 (in Japanese). Parkhurst, C.R., and G.J. Mountney (1988) Poultry Meat and Egg Production. Van Nostrand Reinhold Co., New York, pp. 16-74. Saito, K., N. Goto, T. Ogasawara, K. Imamura, and M. Isoda (1987) Growth pattern of several organs in mice under low-level RF exposure. J. Growth 2669-73 (in Japanese). Saito, K., H. Kajigaya, K. Imamura, N. Goto, and M. Isoda (1986)The effect of low-level RF exposure on the growth and reproduction in mice. J. Growth, 25:3945 (in Japanese). Schwan, H.P. (1983) Biophysics of the interaction of electromagnetic energy with cells and membranes. In: Biological Effects and Dosimetry of Nonionizing Radiation. Plenum Press, New York, pp. 213-230.
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Lethal and Teratogenic Effects of Long-Term Low-Intensity Radio Frequency Radiation at 428 MHz on Developing Chick Embryo KENICHI SAITO, KATSUSHI SUZUKI, AND SHIGEKATSU MOTOYOSHI Nippon Veterinary and Zootechnical Colkge, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180, Japan
ABSTRACT Exposure of developing chick embryos to 428 MHz radio frequency (RF) radiation at a power density of 5.5 mW/cm2for more than 20 days resulted in embryolethal andlor teratogenic effects and delayed hatching. These adverse biological effects were not due to any thermal effect of the RF radiation. We have demonstrated teratogenicity in the chick embryo as a result of protracted low-dose RF irradiation. Lary and Conover ('87) have pointed out that "over the past four decades, the use of radiofrequency (RF) radiation sources has increased dramatically. Because of the increasing prevalence of occupational, medical, and environmental exposure to RF radiation, there is growing concern about the potential effects of such radiation on the unborn child." This is also true in Japan. Teratogenic effects of RF radiation have been demonstrated in animals, although many investigators concluded that RF-induced teratogenic and embryolethal effects are due t o hyperthermia produced by high-intensity RF fields and not to field-specific effects unrelated to heating. Actually, it has been known that heat causes birth defects and prenatal death when the body temperature reaches 40-44"C, regardless of the species (Lary et al., '82; Lary, '86; Lary and Conover, '87). The authors have previously reported adverse biological effects of low-intensity RF radiation at 433 and 906 MHz on experimental animals, including prolongation of the gestational period in RF-exposed maternal mice and reduction of thymus weight in pups born from treated mothers (Saito et al., '86, '87). These adverse effects were considered to be due to nonthermal effects of RF radiation, since the rise in rectal temperature was not clearly detected. Maternal body temperature fluctuated considerably according to the activities and metabolic status. Furthermore, maternal mice moved around in the RF field. Therefore, it was 0 1991 WILEY-LISS, INC.
very diffciilt to estimate the actual specific absorption rate (SAR) in the fetuses. The developing: chick embryo would be a more suitable model for detection of the adverse effects of low-intensity RF radiation. The RF of 428 MHz was selected because the band has been frequently used in Japan by amateur radio stations, and hence the parts and equipment necessary for the experiment are readily available. The present article describes embryolethal andlon teratogenic effects of low-dose RF exposure of developing chick embryos during precisely known developmental stages (Parkhurst and Mountney, '88). It was determined that the embryolethality and teratosgenicity of RF radiation were associated with nonthermal effects of the radiation. MATERIALS AND METHODS
The fertilized eggs of white leghorn chickens, laid within 24 hr, were purchased from Tomaru Poultry Farm (Maebashi, Japan). The eggs were incubated and exposed to RF radiation in an incubator (model p-01; Showa Incubator Co., Ltd., Saitama, Japan) at 37°C 5 0.5"C, with a relative humidity of 80%. The eggs were automatically turned once each hour. Viability of the eggs was conventionally checked once every day during the entire period of incubation. The pre-
Received March 15, 1990; accepted December 11, 1990.
610
K. SAITO ET AL.
'*
9
0
8
0
7
6
0
.
0
5 0
4 .
3
2
1
0
0
0
Ok
'Position c electric
supply
O P o s i t i o n of eggs
Electrode
1 1
>--T
Wooden rack
, Elect rode
Fig. 1. Schematic representation of the positions of the eggs on the wooden rack placed between the electrodes in a n incubator.
cise inspections of eggs were carried out on days 10, 20, 21, and 22 after incubation so that it might be possible to determine if death occurred before or after day 10. Each of ten eggs was subjected to an experiment. Seven experiments were repeated in the same apparatus. The first and the fourth experiments were designated as controls, in which the eggs received sham exposure to RF radiation. In total, 50 eggs were used in the RF radiation group and 19 eggs in the control group (an egg was broken and lost). The inner appearance of the incubator and positions of the eggs on the wooden rack are shown in Figure 1. The apparatus for continuous-wave RF radiation was designed by one of the authors. A block diagram of the apparatus is shown in Figure 2. The linear amplifier was made using the hybrid integrated circuits, M57704M (Mitsubishi Electric Co. Ltd., Tokyo, Japan). A radio frequency field of 428 MHz was generated by a crystal oscillator (Yakumo Crystal Co. Ltd, Tokyo, Japan).
The RF radiated power was 6.8 W at the RF power meter (model SP-300, Welz Co. Ltd., Tokyo, Japan). The area of the two aluminum plate electrodes was 1,224 cm2 (34 x 36 cm). The theoretical power density was 5.5 mW/cm2 between the electrodes, which had a gap of 14 cm. The RF electrical field and RF magnetic field were measured by an RF survey meter (model HI3002, Holaday Co. Ltd., Minnesota) at each position of the eggs. The electric field could be accurately measured by the meter (operating range 0.5-6,000 MHz). However, the values of the magnetic field may be inaccurate since the meter operating range for magnetic fields is only 5-300 MHz. No meters for detection of magnetic fields produced by 428 MHz RF radiation are currently available. The specific absoption rate (SAR) was calculated according to the method of Fujiwara and Amemiya ('82) with the assumption that the egg is an ellipsoidal body 6 cm on its major axis and 4.5 cm on its minor one. To determine the increase in temperature of the egg caused by RF radiation, the contents of four eggs were replaced by 25% gelatin and a fluoroptic thermometer (model FG-1000, Luxtron Corp., Mountain View, CA) was fixed through a hole of the eggshell by paraffin (Fig. 3). Before measuring the temperature, the gelatin-replaced egg was placed in the incubator set at 37°C for 1hr. The increase in temperature of the gelatin per unit of time was repeatedly measured in different eggs with the fluoroptic thermometer for 20 min during exposure to RF at a position where the maximum SAR was obtained (position 3). The longitudinal axis of the eggs was oriented parallel to the electric field, and the eggs received RF radiation continuously during incubation. Right after the chicks hatched (0 to -6 hr after actual hatching), their body weights were measured. The chicks were then anesthesized with an overdose of ether and necropsied. The heart, liver, kidney, adrenal glands, thymus, lungs, thyroid gland, pancreas, bursa of Fabricius, gonads, and yolk sac were excised and weighed to the nearest 0.1 mg. The organs were then fixed with 4% neutral formalin, embedded in paraffin, cut at 3 km, stained with hematoxylin and eosin, and observed under a light microscope. Student's t test and the x2 test were used for the statistical analyses, with a significance level of P < 0.05.
MICROWAVE TERATOGENICITY IN CHICK EMBRYOS
611
Electrodes
Fig. 2. Block diagram of the apparatus for RF irradiation.
the RF-exposed group was associated with cessation O F development within 10 days after the start of incubation, whereas the remainder (76.6%) was due to death within the eggshell following inability to hatch (Figs. 5 and 6). In the latter case, the yolk was almost entirely absorbed by the chick. The incubation period necessary for hatching differed significantly between the groups (P 0.05, x2 test). The control eggs hatched on days 21 or 22, and the exposed eggs hatched on days 21-23 (Table 3). Therefore, it was clear that prolongation of Fig. 3. Gelatin-replaced egg with a fluoroptic ther- the incubation period occurred in the exmometer fixed through a hole of the eggshell by paraf- posed group. fin. Changes in hatchability according to the position of the rack in the RF-irradiated RESULTS group are shown in Table 4. It is noteworthy Regional distributions of the strength of that the severest effect was seen in positions the electric and magnetic field and of the 8-10, where the SARs were relatively low calculated SAR are shown in Table 1. The (see Table 1). In contrast, all chick embryos highest values were obtained at position 3 hatched at position 1, where the SAR was and the lowest at position 5. The calculated relatively high. One case of death in the SAR using both values of the electric and shell occurred at each of positions 3, 5, and magnetic fields ranged between 3.1 and 47.1 6 in the control group. mWIkg, whereas the power density ranged A functional abnormality consisting of between 0.05 and 0.42 mW/cm2 using the creeping movement and inability to stand data of electric field alone. The latter values (Fig. 7) was found in 89% of the hatched suggest that the actual radiation was less embryos in the exposed group but was not than one-tenth theoretical power density found in the controls. In these abnormal (5.5 mW/cm2). chicks, bending or shortening of the bone or The increase in temperature of 25%gela- abnormal ;uticulation was not detected. A tin during RF radiation is shown in Figure chick with a featherless lower trunk was 4. The average gelatin temperature was found when an eggshell was manually bro37.7"C and very stable during measurement ken to pull out a chick of the exposed group for 20 min. No increase in temperature by on day 22 :Fig. 8). The chick was a female, RF radiation was detected. and for 3 days after artificial hatching it The average hatchability was 84.2% in could not skand by itself. However, it grew control eggs and 38.0%in exposed eggs (Ta- normally until it was sacrificed on day 30 ble 2). Almost one-fourth of the mortality in (Fig. 9). The weight of the organs in male :c
612
K. SAITO ET AL.
TABLE 1 , Regional distributions of electric and magnetic field strength, and of calculated S A R i n a wooden rack placed between electrodes in a n incubator' E2 (V2/m2) H2 (A2/m2) Position ( x lo3) ( x 10.') SAR (mW/kg) 1 1.1 1.4 11.0 2 1.2 4.2 33.0 3 1.6 6.6 47.1 4 0.2 0.7 5.5 5 0.2 0.4 3.1 6 0.2 0.5 3.9 7 0.2 0.6 4.7 8 0.2 0.6 4.7 9 0.2 0.7 5.5 10 0.2 1.1 8.6 'Positions correspond to those shown in Figure 1.
I S.D. 'C
38
37
-
-
TABLE 2. Comparison of hatchability between control and radio frequency exposed eggs Control Exposed (%I (%) Number of eggs Incubated 19 50 Hatched 16 (84.2) 19 (38.0)* Dead 3 (15.8) 30 (60.0)* .~ Within 10 days 0 7* In shell 3 23* Artificial hatching 0 1 (2.0)** ~~
,
*Significantly different from control at P < 0.01. **A case of undeveloped feather around lower trunks and legs.
TABLE 3. Incubation period for hatched eggs treated with RF radiation' Incubation period (days) 21 (%) 22(%) 23(%) 24(%) Group Control Number 12 (75) 4 (25) hatched Sex F8,M4 F3,Ml Exposed Number hatched 4 (16.7) 11 (61) 3 (16.7) l ( 5 . 6 ) Sex F2,M2 F5,M6 F2,Ml F1 'Eighty-nine percent of the hatched chicks in the exposed group showed a functional anomaly in their inability to stand on their legs and by creeping movement.
,
and female chicks in the control and exposed groups varied considerably (data not shown). This might be because of the variation of the incubation periods for hatching and of egg size at the start of incubation. There were no abnormal histological findings in any of the organs examined. DISCUSSION
TABLE 4. Positional distribution of hatched and dead embryos in radio frequency exposed group Number Number of dead Number of Posiwithin of dead Artificial tion 10 days hatched in shell hatching 1 5 0 0 0 2 2 0 3 0 3 3 1 1 0 4 2 0 3 0 5 3 0 2 0 6 2 0 3 0 7 2 0 3 0 8 0 2 2 1 9 0 2 3 0 10 0 2 3 0
Embryo-lethal and teratogenic effects of RF irradiation have been reported in various species, including the rat, mouse, monkey, and man (Lary and Conover, '87). In SAR was smaller than the basal metabolic those experiments, much higher powers rate reported in the chick (3.4 W/kg; Mount, were applied t o the animals for only a short '79). On the other hand, calculated SARs by time in comparison to the irradiation condi- both electric field and magnetic field data tions in the present study. They concluded and by electric field data alone were unexthat the teratogenic effect of the RF was due pectedly low compared with the theoretical invariably to its inherent thermal effect, value of 165 mW/kg. The Radiofrequency since the irradiated animals had a n ele- Radiation Dosimetry Handbook (Durney et vated body temperature, and hyperthermia al., '78) indicates that, at a power density of is a well known teratogen in the rat (Lary et 5.5 mW/cm2, the maximum SAR of a al., '82). chicken egg exposed at 428 MHz should be In the present study, the maximum SAR about 165 mW/kg. Based on the data meavalue was 47 mWlkg, and the minimum was sured, the power density at the source was 3.1 mW/kg. This maximum value for the 0.42 mW/cm2,which is less than one-tenth
MICROWAVE TERATOGENICITY IN CHICK EMBRYOS
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Fig. 8. Chick with featherless trunk manually pulled from a n egg exposed to RF radiation for 22 days. Fig. 9. Same chick as in Figure 8 aged 30 days after artificial hatching. Note that lower trunk remains featherless.
the theoretical power density value. Since RF radiation at 428 MHz is about 70 cm in wavelength, a considerable amount of energy was lost because of the size of the electrode, which was about one-half of the wavelength. In the piresent study, no thermal effect of the RF radiation was detected when using a fluoroptic thermometer, which gives accurate temperature measurements in an RF field. In the low-intensity field of this study, no measurable temperature increase would be expected. This suggests that chick embryos in this study did not show any inFig. 5. Dead chick embryo whose development ter- crease in tlody temperature during RF radiminated within 10 days after incubation under RF exation. Therefore, the leg abnormality and posure. high mortality seen in the present study Fig. 6. Embryo that died about 17-18 days after in- were not likely to have been caused by the cubation started under the same RF irradiation condi- thermal etTect of the RF radiation. This is tions as used for Figure 5. the first study to detect direct teratogenicity Fig. 7. Chick, hatched from the RF-exposed egg on following long-term low-dose RF irradiation day 21 after incubation started, unable to stand on its of a n animal. However, Delgado et al. ('82) legs and showing creeping movement. reported abnormalities in chick embryo development from exposure to a very-low-in-
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tensity (nonthermal) pulsed magnetic field at 10,100, or 1,000 Hz. The teratogenic dose of the magnetic field was reported t o range between 0.12 and 12 pT. Prolongation of the gestation period by long-term low-dose RF radiation has been reported in mice (Saito et al., '86). Similar effects were found in mice housed in a magnetic field (Nakagawa, '70). In the present study, the period necessary for hatching was prolonged in RF irradiated eggs. It is clear that these biological effects are not caused by the thermal effect of the RF but by its direct action. There is little information on the mechanisms governing the time periods necessary for birth or hatching. Growth retardation seen in some organs at necropsy (data not shown) may relate to the prolonged hatching period, although the mechanism is unknown. We speculate that RF radiation may have an effect on regulation of the growth rate by influencing the rate of cellular proliferation in the embryo. The pearl chain phenomenon seen in lower animals such as protozoanspecies during RF exposure (Schwan, '83) is thought to result from changes in electronic charges on the cell surface. Electrical charges on the cellular surface may interfere with cell separation. Another possibility is that RF radiation might affect cell-cell interaction during development and/or cellular migrations through interactions with the extracellular matrix as discussed by Delgado et al. ('82). Further study will be necessary to determine the actual mechanisms causing the adverse effects on development observed in this study.
LITERATURE CITED Delgado, J.M, J . Leal, J.L. Monteagudo, and M.G. Garcia-Gracia (1982) Embryological changes induced by weak, extremely low frequency electromagnetic fields. J . Anat., 134:533-551. Durney, C., C. Johnson, P. Barber, et al. (1978) The Radiofrequency Radiation Dosimetry Handbook (2nd edition). Report SAM-TR-78-22, U.S. Air Force School of Aerospace Medicine, Brooks Air Force Base, Texas, p. 101. Fujiwara, O., and Y. Amemiya (1982) Microwave power absomtion in a sDecimen inside a standine-wave irradiatidn waveguide. IEEE Trans. Microwave Theory Tech, MTT-23:2008-2012. Lary, J.M. (1986) Hyperthermia and teratogenicity. In: Hyperthermia in Cancer Treatment, Vol. 1. A. Anghileri and D. Robert, eds. CRC Press. Boca Raton, FL, pp. 107-126. Lary, J.M., and D.L. Conover (1987) Teratogenic effects of radio freauencv radiation. IEEE E n ~ Med. n Biol. Mag., Ma&, pp.h2-46. Lary, J.M., D.L. Conover, E.D. Foley, and P.L. Hanser (1982) Teratoeenic effects of 27.12 MHz radiofrequency radiathn in rats. Teratology, 26t299-309. Mount, E.L. (1979) Adaptation to Thermal Environment. Edward Arnold, London, p. 22. Nakagawa, M. (1970) Experimental studies on effect of magnetic fields on fertility, general reproductive performance and growth of mice. J . Nagoya City Univ. Med. School, 29:323-341 (in Japanese). Parkhurst, C.R., and G.J. Mountney (1988) Poultry Meat and Egg Production. Van Nostrand Reinhold Co., New York, pp. 16-74. Saito, K., N. Goto, T. Ogasawara, K. Imamura, and M. Isoda (1987) Growth pattern of several organs in mice under low-level RF exposure. J. Growth 2669-73 (in Japanese). Saito, K., H. Kajigaya, K. Imamura, N. Goto, and M. Isoda (1986)The effect of low-level RF exposure on the growth and reproduction in mice. J. Growth, 25:3945 (in Japanese). Schwan, H.P. (1983) Biophysics of the interaction of electromagnetic energy with cells and membranes. In: Biological Effects and Dosimetry of Nonionizing Radiation. Plenum Press, New York, pp. 213-230.
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