Anat . Anz.

, Jena 161 (1986) 23-26

VEB Gustav Fischer Verlag Jena

Chick Embryonic Development Following Exposure

to Caffeine and Nicotine
By S .

H.

GILA`Il and T .

V. X.

PERSAUD2

1Department of Anatomy, University of Medicine and Dentistry of New Jersey, New

Jersey Medical School, Newark, N .J ., U.S .A ., and 2Department of Anatomy, University
of Manitoba, Basic Medical Sciences Building, Winnipeg, Canada

With

Tables

(Received

July

4,

1985)

Key words : teratogenesis, caffeine, nicotine, chick

Abstract
Because cigarette smoking is considered to be deleterious to the fetus and caffeine is held in
suspect as a potential human teratogen,the combined effects of caffeine and nicotine on early
chick embryos were investigated . Treatrnent of the embryos at 48 h incubation with both caffeine
(1 mg) and nicotine (1 mg) resulted in a high incidence of embryonic death and developmental
defects . At 72 h incubation teratogenicity was potentiated following the same treatment . Embryonic growth was not affected . Embryotoxic interactions of this nature might account for congenital anomalies of doubtful etiology .

Introduction
There is increasing concern as to whether caffeine is a potential human teratogen .
Although no case of human lnalformations, attributed to caffeine, has been reported, the results of several animal studies suggest that caffeine might be harmful
to the embryo, so much so that the Food and Drug Administration of the United
States urged recently that "pregnant women should avoid caffeine containing foods
and drugs, if possible, or consume them only sparingly" (FDA 1981 ; DEws et al .
1984) .
It is now established that smoking during pregnancy reduces significantly the
birth weight of the offspring and leads to a higher incidence of fetal and perinatal
death . There is also some evidence that maternal smoking may lead to an increased
incidence of congenital malformations (ABEL 1980) .
For the screening of drugs for possible embryotoxicitv and teratogenicity the
early chick embryo is considered a highly sensitive system (FISaER, SCHOENWOLF
1983 ; GEBIIARDT 1972) . The technique is inexpensive, simple to perform and exposes
the rapidly developing embryo to direct contact with the test substance . Furthermore, the influences of the maternal placenta, nutrition and metabolism are conveniently avoided .
Significant embryopathic effects have been observed following exposure of chick
embryos to either caffeine (GILANI, GIOVIxAzzo, PERSAUD 1983) or nicotine (LAxDAUER 1960 ; GATLING 1964) . Of particular interest is the potentiative embryotogic
interactions of caffeine with a wide spectrum of other teratogenic substances at mini-

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S . H . GILANI and T . V. X . PEasnun

24

mallv effective doses (RITTER et al . 1982) . Because of the widespread use of both
caffeine and nicotine we have studied their combined effects on the early developulent of chick embrvos .

Materials and Methods

Fertile White Leghorn chick eggs (Shamrock Farins, New Brunswick, N .J .) were used in these
studies . The eggs were incubated in a forced-draft incubator at a temperature of 38 C . Caffeine
(1 mg) and nicotine (1 mg) were injected into the air sacs, separately or in combination, at either
48 or 72 h incubation, as shown in Tables 1 and 2 . Physiological saline was used as the solvent
and the total volume of injected material was 0 .1 ml/egg . 2 batches of controls were maintained,
one was treated with 0.1 ml and the other with 0.2 nil of the solvent
; 112 at 48 .-Thetoalnumbrfegsdinthuywas20 and 108 at 72 h incubation,
respectively . On d 13 the live embryos were removed from the eggs, and after staging these were
weighed and examined for the presence of gross malformations . The embryos were fixed in 10
neutral formalin .

:~tatistical analysis of the data was carried out using the chi-square test with Yates' correction
for contiuuitv .

Results
The incidence of etubryonic mortalitv and anomalies following treatment with
caffeine at 48 or 72 h incubation was not significantly increased compared to the
saline treated controls . However, treatment of the etnbryos with nicotine at 48 h
incubation significantly affected their survival and development .
At 48 h incubation, significantly fewer embryos survived the combined treatment
with caffeine and nicotine, compared to those treated with only one of these substances . The incidence of malforlned enibryos following treatment .vith both caffeine
i
~ Table 1 . Effect of caffeine (1 mg) and nicotine (1 mg) on developing chick embryos at 48 h incubation
Treatment

Caffeine

_\"umber of

Live embryos

Survival

Abnormal

eggs treated

on d 13

(%)

(%)

13

10

76

20

Malformations

Short limbs,
Abnormal tail

\icotine

15

53*

88*

Hemorrhage,
Short neck,
Reduced body,
Everted viscera

affeine and

9*

Edema, Twisted
limb, Short neck,

Nicotine

Everted viscera,
Abnormal tai1,

Reduced body
Controls

(0.9

0 10

--eC1)

0 .1 ml/egg
0 .2 ml/egg

26
30

21
26

* P< 0 .05, chi-square test.

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s0
87

10
4

Short beak
Twisted limbs

25

Teratology

Table 2 . Effect of caffeine (1 mg) and nicotine (1 mg) on developing chick embryos at 72 h incubation
Treatment

Number of
eggs treated

Live embryos
on d 13

Survival
(%)

Abnormal
(%)

Malformations

Caffeine

13

11

85

18

Micromelia
Twisted limbs

Nicotine

13 -

10

77

30

Everted viscera,

Reduced body,
Hemorrhage
Caffeine and

'_6

11

42*

55*

Nicotine

Short neck,
Twisted limbs,

Reduced.body
Controls

(0 .9 o _\'aCl)
0 .1 mI/egg
0.2 m]/egg

8
_'8

25

89

4
8

Short neclc
Short limbs

* P < 0 .03, chi-equare test .

Table 3 . Effect of caffeine (1 mg) and nicotine (1 mg) on embryonic weight in the chick (d 13)'
Treatment

48 1t Incubation
(\fean S .E .)

72 Ir Incubation
(Mean S .E .)

Caffeine

4 .66 = 0 .55

5 .78 0.24

Nicotine

4 .19 = 0 .10

4 .55 f 0.53

Caffeine and \icotine

4 .45 = 0 .05

5 .12 0 .33

Controls (0 .9 o SaCI)
0 .1 ml!egg

4 .71 0 .37

5 .33 0 .15

0 .2 mllegg

5 .03 0 .33

5 .16 0.26

_\'o significant differences between treated and control groups .

and nicotine was significantly increased when compared with the caffeine treated
group . At 72 11 incubation, both embryolethality and teratogenicity were significantly increased from the combined treatment compared to any other group (Tables
1 and 2) .
The different types of developmental defects observed in the embryos are listed
in Tables 1 and 2 . Reduced body size and defective formation of the abdominal wall,
resulting in an everted viscera, were induced following treatment with caffeine and
nicotine at both incubation periods . As shown in Table 3, the mean weight of embryos exposed to caffeine, nicotine, caffeine and nicotine, did not differ significantly
from each other nor from the saline-treated controls .

Discussion
The results of the present investigation revealed a deleterious effect of caffeine
and nicotine on the development of the chick embryo . Compared to treatment with
caffeine or nicotine alone, the difference was statistically significant when compared

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26

S . H. GILANI and T. V. N. PERSAUD, Teratology

with the caffeine treatment at 48 h incubation and with either caffeine or nicotine
at 72 h incubation . Thus, the older embryos were less susceptible to damage following
exposure to either of the substances but not when subjected to the combined treatment .

Teratological studies in laboratory rodents have shown that high doses of caffeine
may lead to embryonic growth retardation, limb malformations, cleft palate and
bleeding disturbances (see DEws et al . 1984) . Similar observations were made in

the chick embryo following treatment with caffeine (Gir.aNi et al . 1983) . A co-teratogenic activity for caffeine and nicotine is evident at 72 h incubation because the

combined treatment led to significant embryopathic effects whereas exposure of the

embryos to either one was relatively ineffective . Embryotoxic interactions of this
nature might account for developmental defects where the etiology is not certain .
From animal studies caffeine is known to enhance the teratogenic potential of several chemicals, including metabolic inhibitors, but the mechanisms involved are

not clear and have been discussed elsewhere (TlatsoN 1977 ; THasER,
RlT r m et al . 1982) .

PALM

1975 ;

Acknowledgement
This investigation was supported by an Institutional Grant from the

University

of

Medicine

and Dentistry of New Jersey, New Jersey Medical School to S .H.G . The excellent technical assistance of \ir . S . -l1AITRA is gratefully acknowledged .

References
AREL, E. L . : Smoking during pregnancy : A review of effects on growth and development of offspring. Hum . Biol ., Detroit 52 (1980) 593-625 .
DEwB,

P ., H. C . GRICE, A . N'EI]35, J .

WILSON

and

R . WIIRT]IAN : Report of fourth international

caffeine workshop, Athens, 1982 . Fd . Chem . Toxicol ., Oxford 22 (1984) 163-169 .

FDA : Caffeine and Pregnancy . U .S . Department of Health and Human Services . HHS Publication, Washington No . (FDA) 81 (1981) 1081 .
I

FisHER, 31 ., and G . C. ScaoEx}voLl- : The use of early chick embryos in experimental embryology
and teratology : Improvements in standard procedures . Teratology, Philadelphia 27 (1983)
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GATLIXG, R . R . : Effect of nicotine on chick embryo. Arch . Path., Chicago ^rS (1964) 652-657.
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GILANI, S . H ., .1 . .I . GIoclxAzzo and T . V. N . PERSAVD : Embryopathic effects of caffeine in the
chick . Exper. Path ., lena 23 (1983) 79-33 .

LA1rDAtER, I4 . : Vicotine induced malformations of chick embryos and their bearing on the
phenocopy problem . .1 . exper. Zool ., Philadelphia 143 (1960) 107-122 .
RITTER, E . J., IV. J. SCOTT, J . G. RILSO_\-, P . R . >IATHIN09 and J . L. RANDALL : Potentiative

interactions between caffeine and various teratogenic agents . Teratology, Philadelphia 25

(1982) 95-100.
.
THAYER, P . S ., and P . E . PALM : A current assessment of the mutagenic and teratogenic effects
of caffeine . CRC Critical Reviews in Toxicology, Florida 3(1975) 345-369 . Tlatso ., J. : Caffeine . Mutation Res ., New York 47 (1977) 1-52 .
Prof. Dr . Dr. T. V . V. PERSArD, Department of Anatomy, University of Manitoba,
Basic Medical Sciences Building, Winnipeg, Manitoba, Canada R3E OW3 . .

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