Indian Journal of Experimental Biology Vol. 38, July, 2000, pp. 692-699

Teratogenic effects of sinusoidal extremely low frequency electromagnetic fields on morphology of 24 hr chick embryos

Maryam Shams Lahijani & Mahmoud Ghafoori

Department of Biology, Faculty of Science, University of Shahid- Beheshti, Tehran, Iran

Received 28 May 1999; revised 20 January 2000

To examine the potential teratogenicity of electromagnetic fields (EMF; sinusoidal and rectangular) on development of chick embryos (white leghorn), 221 freshly fertilized chicken eggs (55-65g) were exposed during first 24hr of postlaying in­

cubation (38° ± 0.5°C) to 24 different EMFs, with 50Hz repetition rate and 8.007-10.143 mT flux density. Following expo­sure, the exposed fertilized chicken eggs (n=8-l 0) and sham-exposed fertilized chicken eggs (n= 15) were incubated simulta­neously for 8 more days and unexposed control fertilized chicken eggs (n=20) for 9 days in absence of EMFs.The embryos were removed from egg shells and studied blind. All 24 EMF exposed-groups (inside the coil with exposure) showed an in­crease in the percentage of developmental anomalies compared to sham - exposed (inside the coil with no exposure) and control groups (outside the coil). Further, egg's weight was evaluated on day 9. This variable did not show significant dif­ference between control and exposed-groups. The investigation also covered the measurement of body weight , length of crown to rump, length of tip of the beak to occipital bone, heart and liver weight. Statistical comparison between sham­exposed and control values did not show significant differences, but comparison between 8.007, 8.453 and 8.713 mT ex­posed- groups and control groups showed significant differences; in other exposed-groups, the changes were not significant. These results revealed that 50Hz electromagnetic fields can induce irreversible developmental alterations in 24hr chick em­bryos and confirm that its strength could be a determinant factor for the embryonic response to extremely low frequency electromagnetic fields (window effects).

Very weak low frequency pulse magnetic fields (PMFs) can induce significant effect on the development of chick embryos, exposed at first 48hr of incubation .These effects are dependent on the frequency, intensity and wave form' -5. In other series of experiments, effects of PMFs were found to be dependent on the stage of development. First 24hr of incubation was reported to be crucial and could have some effects on the orientation of embryo in relation to the direction of the field6

•4

; but others did not find any differences between exposed and control unexposed chicken eggs7

-9

.

Effects of PMFs, particularly within the first 48hr of incubation, have been reported, but less attention has been paid to 50Hz sinusoidal magnetic fields4

·5

200J..LT magnetic fields induced a significant effect on embryological development of chicken eggs, exposed for 48hr. Exposure to 50Hz magnetic field (MF) induces adverse effects on development of chick embryos2

•10

·1'. In contrast, others did not observe any

such changes m the developmental anomalies, maturity stage, distribution of extracellular (membrane) components, egg weight and egg fertility, after exposure to intermittent horizontal sinusoidal 50Hz magnetic fields7

·12

.

Chick embryo, in the present study was selected because: a) as a whole, its early development is fundamentally similar to that of most vertebrates; b) its ovo embryo is an independent system and is not influenced by the condition of a materna[ host, therefore, teratogenic agents can react directly on the embryo; c) its embryonic development has been carefully staged and well documented allowing the indentification of abnormalities after exposure to electromagnetic field (EMF); and d) earlier studies, about chick embryos exposed to EMFs, showed some positive results although they have been examined at the end of 2-days exposure. Present study allowed us to specify even slight malformations occurred at early or advance developmental stages, as was hypothesized by Ubeda et a/ 5

• Thus, fresh fertile chicken eggs (55-65 g) were exposed to extremely low frequency (ELF) EMFs, during first 24hr of incubation, and then, incubated for 8 more days, in absence of EMFs; at the end of this period, all embryos were investigated blind for detection of possible developmental anomalies .

The specific aims of the present study were : a) to determine whether EMFs exposure (flux density 8.007-10.143 mT) during early development (first 24

SHAMS LAHIJANI & GHAFOORI : TERATOGENIC EFFECTS OF EMF 0 CHICK EMBRYOS 693

hr of incubati on) has any effect on the incidence of developmental abnormalities and death , and b) to examine if biological effects of EMFs are of "wi ndow" or "dose" effects.

Materials and Methods Freshly fertilized white leghorn eggs (256),

obtained from Bonyad Mostaza ffan farm (Karaj, Tehran, Iran) were transported to the laboratory immediate ly after co llection. Eggs, weighing 55-65 g were !>-elected and placed at !5°± O.SOC with thei r long axis horizontal , for less than 48 hr .

In 24 different experiments, 24 different flux densities of EMFs were used (n=8- l 0) . There were also sham - exposed (n= I 5) and contro l (unexposed) groups (n=20) available si multaneously (Table I ).

During first day of incubation (38° ± O.SOC, 65% RH), the experimental eggs were exposed to sinusoi­dal EMp-4·14

"1:;, with thei r long axis towards north­

south geomagnetic direction. In each ex periment, the exposed, sham-ex posed and control eggs were trans­ferred to the incubator (with no exposure) for 8 and 9 days respectivel/.w. At the end of th is period, the e mbryos were removed from their shells and im­mersed in Tymde solution 16 and studied blind . Em­bryo~ were ~;,.::o!"ed for several gross anatomical fea­tures like: eyes, beak, developmental stage, tail, cen­tral nervous system, limbs, heart and liver. Criteria for normality required normal embryological develop­ment of each of these features as described and i llus­trated by Hamburger and 1-Jamil ton u ; according to this scale, the embryos shou ld ha ,,e reached to devel­opmental stage 35 at the end of 9 days.

Thus, eggs were weighed just before incubation as well as before and after removing embryos from their she ll s. Their liver and heart weights were recorded . Lengths of crown to rump (CR) and top of the beak to occipital bone (BO) were also measured. The fre­quencies of abnormal embryos in the exposed, sham­exposed and control groups were compared, using c hi square test. The differences between the va lues for the different variables of exposed, sham-exposed and control eggs were compared by one way ANOV A test. Values are given as a mean ± SE. Differences having P<O.o:'i were regarded as significant.

l{csults The most frequent external malformations seen in

the present ~tudy have been monomicrophthal mia, exencephalia , thoracogastroschisis, tai l malformation

and crossed beak (Figs 1-5). Internal abnormali ties included formation of large heart and liver (Figs 6 and 7); however, always internal abnormalities were accompanied with thoracogastro~c hi s is.

Sham-exposed a:1d centro! samples have equivalent percentages of abnormalities, either for tota l percentage of abnormal embryos or for different types of abnormalities (dead or alive) (Table!; Fig.8). Exposure to EMFs (E I , E5 and E9) led to significant increase in the percentage of abnormal embryos (dead and alive) compared to control groups (X 2=4 .171 ; ?<0.05). Other exposed groups showed an increase, though not significant , in the percentage of abnormal (dead and alive) embryos compared to the control groups .

The mean eggs' weights were decreased after 9 days of incubatio11; this dec rease was approximately 5 g in all groups (Table 2 ; Fig.9) . However, the decrease in eggs' weights , during incubation , was similar in a ll groups and stati stical ana lysis did not reveal any differences in eggs' wei ghts decrease between sham-exposed, control and exposed groups (Fig.9).

The mean weights of living embryos at day 9th of incubation in E I , E5 (?<0.05) and E9 (P<O.O I) , were significantly more than the control and s ham-expo~ed embryos. No statistically significant inc rease (in the weight of embryos) was seen in other ex posed -grou ps, compared to the control and sham-exposed e!~~~ryos (Fig. I 0).

hE I and E9, the living embryos were significantl y shorter in length and h..:ad s ize (P<O.O I) compared to the control and sham-exposed. embryos (Figs 2, II and 12; Table 2). None of the differences (between control, sham - exposed and other exposed groups) were statistically signi ficant. In E 1, E5 and E9, the heart and liver weigh ts were s ignificantlly more than control and sham-exposed; one of the differences (between sham-exposed, control and other ex posed -groups) was stati sti ca ll y significant (Table 2; Figs 13 and 14).

Length of abnormal heart was longer than normal (Fig.7). Thi s abnormality was always accompanied with thoracogastroschi s is .

Discussion According to the previous 14

"15 as well as the present

findings , it has been established that left side of the head, including brain and eye, are very sensit ive to the EMFs . Embryos with microphthalmia had also crossed beak and asymmetrical skeletal

694 INDIAN J EXP BIOL, JULY 2000

Oil

SHAMS LAHIJANI & GHAFOORI : TERATOGENIC EFFECTS OF EMF ON CHI C K E IBRYOS 695

structures.Neural crest cells are engaged in the formation of different parts of neurocranium and viscerocranium; it is possible that EMFs have affected the charac teri stics of neural crest cells and disturbed their behaviour.

Embryos wi th anencephalia and exencephalia (in affected embryos) could have been created because of the existence of large and abnormal interce llular gaps, dense nucle i in the neural ectoderm and open neural folds1

• Degraded glycosaminoglycans, abnormal gaps, necrotic zones and reduction in the number of head

~

mesenchyme cells have also been proposed for the abnormalities in the head, beak and face regions5

·6

.

Defects in the tail region (atrophied tail) could be due to the influence of EMFs on the number of somites, which confirms previous results4.7. Embryos with gastroschisis and thoracogastroschi sis have demonstrated that EMFs have some effects on the ectomesodermal wall of gut regioRs; and large size of liver and heart could be casuative factors .

Comparing weights, thoracograstroschisis embryos were much heavier than the normal embryos. It has

Fig. !-Head region of 9- days old chick embryo (x 6) . A: normal eye; B: monomicrophthalmia. Left eye is much smaller than the nor­mal eye. There are less pigmentation around the left eye (arrow); Fig. 2-Head region of 9 - days old chick embryo (x 6). A: normal brain ; B: exencephali a. Length of BO and upper beak are shorter than normal size; Fig. 3-Trunk region of 9 - days old chick embryo with thoracogastroschi sis (x 6). c=crop; h= heart ; i= intestine; I =li ver; Fig. 4--Tail region of 9 -days old chi ck embryo (x 6). A: normal tail ; B: atrophied short tail

Fig. S-9 - days old chick embryo with crossed beak (x 7). a: cerebral hemisphere; b: crossed beak (U pper beak is shorter than lower beak); Fig. 6-Liver of 9- days old chick embryo (x 7). A: normal liver; 8 and C: abnormal liver. Notice the size and border of left lobe IR . ~rrnw). It is n~rt itinn~d into twn l r . <~ rrnw): FiP . 7-H~;~rt nf 9 - d~ vs nld c:hic:k ~mhrvo lx 7). A: nnrm~ l h ~a rt : R: ahnorrnal l ar11~

696 1:--:0IAN J EXP BIOL, JULY 2000

100 ® l!lll Abnormal ,alive ~ Dead 0 Normal. alive

75

~ . I 50 . . .

U1 0 >. .... D

E 25 :il

0 u X ;;; "' M ~ "' ::: .... "' "' 0 "' M ... "' "' .... "' "' ~ OJ "' M ~

"' t.l t.l t.l "' "' "' .., ;;; ;;; ;;; ;;; ;;; ;;; ;;; ;;; ;;; "' "' "' "' "' "' w "' "'

0 "' 0 0 ~ 0 "' "' "' 0 "' "' N

' ' ;; il u . I I I II c c c c c c c c c c c c c c c

6 ® ~ ~ r-

-·~~. I I ..c: .~ 4 cu ~

fi~l p btl

I ~ btl

11· l:il

I :;.; ~

0 ~

u :r ;;; "' M ... "' "' .... "' ~ 0 N '" .. "' "' .... .. "' 0 OJ N M ~

U1 w "" "' "' "" "' "' ;;; "' ;;; ;;; ;;; ;;; ;;; ;;; ;;; ;;; N N N N

"' "' w w "' 2 ~ ~ ~ 0 ~ "' "' "' 0 "' "' "' "' "' C> "' . I I I I I 'i' I I II

c c c c c c c c c c c c c c c c

~ 2.5 @ .. ~

c: btl

2.0

;:; ~ 1.5 >, ~

" [l

0.5

u :r ;;; N M .. "' "' r- "' "' 0 N M .. "' "' r- "' "' 0 N M ... U1 . .., "" t.l "' "' "' "' "' ;;; ;;; ;;; w ;;; ;;; ;;; ;;; w ;;; N OJ N N N

t.l "' "" "' "' 0 "' N n (EI - E24) = 8-10 I II

~ c

11 E JO

E ~ " 5 20

btl c: .,

...J 10

,~ 0 ~ '" ~· . " " 0 0

u ffi ;;; N !:l .. "' ~ ,.. "' .. 0 N M .. "' ~

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.., w w ;;; ;;; ;;; ;;; ;.; ;;; ;;; ~l N N

"' "' "' ~ "' I I n(EI-E24) c 8 -10 c c

Fi g. X-Percentage of abnormal dead and alive embryos in each group. Th ree exposed- groups (E 1. E; and E~) showed sign ilicant increase ( P<0.05) in the percentage o f abflorn1a li ti es compared to corresponding control (C) and sham- exposed (Sl-1) groups: Fig. 9- Mean of eggs' weigh ts dccre:1se after lJ day~ o f incubation. None of the differences be tween cont ro l (C). sham - exposed (SI-IJ and exrosed- groups (E)' were st:Hist 1ca ll y signifi cant; Fig. 10-Body wei ght o f li ving e mbryos at day 9 of incubation. Three ex­posed - grour~ tE 1• E; and E.1) show~d signi licant increase( * P<0.05; ** P<O.Ol) compared to sham - exposed (S II J :1 nd control (C) groups ; Fig. 11 --Length or crown- rump or living embryos en day 9 of incubation. Two expmed- grours (E1 and E.1) showed signi licant decr~ase ( P<(J.(J!) compared 1,1 sh ~un - eXJ)(>sed (SH) ~.nd control (C) groups.

SHAMS LAHIJANI & GHAFOORI : TERATOGENIC EFFECTS OF EMF ON CHICK EMBRYOS

E' 20

! 15@ •• Q) N 'iii 10

"0

~ .c X

0

lil ~ I I c c

';;030 ! @ .. ... .c 20 .!!!! ., ~

.... 10

!ii · Ill X o

QO 40 @4 ! ... 30 .c .,., 'ii ~ 20 ... ~ :3 10

0

I\! ~ I I

" c

•• 1

..

•• 1

••

n(EI-E24) • 11-10

•.•

n(El - E24) • 8-10

n(El-E24) • 8-10

697

Fig. 12-Head size (top of beak - occipital bone) of living embryos on day 9 of incubation. Two exposed -groups (E 1 and ~) showed significant decrease ( P<O.Ol) compared to sham- exposed (SH) and control (C) groups; Fig. 13-Heart weight of living embryos at day 9 of incubation. Three exposed -groups (Ei> E5 and~) showed significant increase ( P<O.OI) compared to sham- exposed (SH) and con­trol (C) groups; Fig. 14-Liver weight of living embryos at day 9 of incubation. Three exposed-groups (E 1, E5 and~) showed significant increase(* P<0.05; ** P<O.O/) compared to sham-exposed (SH) and control (C) groups.

Groups

Flux intensities (mT)

Groups

Flux intensities (mT)

Control (n=20)

0

Control (n=20)

0

Sham­exposed (n= I 5)

0

Sham­exposed (n= I 5)

0

Table I -Embryos exposed (E) to EMFs

El E2 E3 E4 E5 E6 E7 E8 E9 EIO Ell El2 (n= I 0) (n= I 0) (n=9) (n=9) (n= I 0) (n=9) (n=9) (n=9) (n= I 0) (n=9) (n=9) (n=9)

8.007 8.141 8.294 8.408 8.453 8.541 8.591 8.679 8.713 8.82 8.868 8.941

El3 El4 El5 El6 El7 El8 El9 E20 E21 E22 E23 E24 (n= I 0) (n=9) (n=9) (n=9) (n=9) (n=9) (n=9) (n=9) (n=9) (n=9) (n=9) (n=9)

8.987 9.075 9.13 9.208 9.272 9.342 9.415 9.557 9.79 9.849 9.996 10.143

been suggested that EMFs have no influence on em­bryo's weight 12

, but in previous studies results were controversial 17

; some authors have proposed that

EMFs could effect on extraembry<;mic membranes 18

and consequently embroy's weight. Decrease in C-R length has been accompanied with

698 INDIAN J EXP BIOL, JULY 2000

Table 2- Eggs weights, embryos weights, embryos lengths, head size, li ver and heart weights in control (C), exposed (E) and sham-exposed (SH)chicks

c SH

El

E2

E3

E4

E5

E6

E7

E8

E9

EIO

E ll

E12

E 13

El4

E15

E16

E 17

E18

El9

E20

E21

E22

E23

E24

Dead

2

2

2

2

2

2

nil

0.0 19

4. 17 1

2.3

1.28

1.28

4.17 1

1.28

0.23

0.23

4. 17 1

0.23

0.23

1.28

0.94

0.23

0.23

0.23

1.28

1.28

1.28

0.23

0.23

0.23

0.23

0.23

Decrease of egg weight x (gr)=

5.1

5.056

5.06

5.1

5.0:B

5.0:B

5.08

5.077

5.044

5.044

5

5.oi I

5.022

5.033

4.98

4.988

5.oi i

4.966

5

4.933

4.9

4.9

4.933

4.933

4.877

4.9

Embryo weight x (gr)=

2.024

2.044

2. 173

2.133

2.089

2. 159

2.178

2.098

2.09

2. 102

2.219

2.1 08

2.097

2.077

2.133

2. 11 5

2.1 22

2.04

2. 127

1.962

2.1 3

2.095

2.055

2.076

2.097

2.037

exencephalia, microphthalmia and atrophied tail. It is possible that this decrease has been caused by mentioned abnormalities. EMFs have affected length of C-R m mice3

·19

; even length of beak to occipital bone has been reduced, which could have been created by the inducti ve effect of EMFs on forebrain and optic vesicles 19

•

But why EMF has affected organs wh ich have not yet been fo rmed in 24hr chick embryos? Answer to this question is not clear, but according to previous results, EMFs have some effects on intracellular DNA, RNAs, proteins20

-22

, metabolism, cell di vision and cell growthn 24

; with respect to morphogenetic fi elds, which play an important role in developmental processes, di sturbances in these fie lds could influence the development of organs later on.

As the results showed, the effects are not dependent on doses but E I, 'ES and E9 are the most

Embryo length

x(mm)=

28.56 1

28.384

27.062

28.207

28.405

28.407

27.575

27.6

27.8 1

28.735

26.875

28.6 12

28.84

28.427

28.185

28.957

28.64

27.9

27.792

27.997

27.755

28.422

28.406

28.225

28.86

28.82

Head size

x(mm)=

14.0 15

13.892

13.555

13.742

14. 11 2

13.6

13.99 1

13.637

13.837

13.762

13.725

14. 11 2

13 .862

13.687

13.6 12

13.8

14.1

13.575

13.875

13.582

13.8

13.8

14.013

13.625

13.675

13.968

Li ver weight x(mg)=

3 1.805

3 1.938

38.1 62

33 .287

35.375

35.075

38.11 2

35.683

34. 11 2

33.952

36.793

33.287

33.375

33.85

33 .3 12

35.075

33. 11 2

33 .062

33.825

30.862

35 .65

32.9

32.218

32.925

34.075

34.75

Heart weight x(mg)=

20.942

20.992

23 .9

2 1.45

22.4 12

22.287

24.367

22.28

2 1.875

22. 133

23.7

2 1.677

2 1. 1

22.56

20.762

2 1.642

2 1.52

20.45

22

20.45

22.475

20.675

22.1

21.087

21.43

20.762

affected exposed-groups, which confi rm Ubeda's "window effects" theory.

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cal changes induced by weak extremely low frequency elec­tromagnetic fi elds, 1 Anatomy, 134 (1982) 533 .

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SHAMS LAHIJANI & GHAFOORI: TERATOGENIC EFFECTS OF EMF ON CHICK EMBRYOS 699

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