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Anat . Anz., Jena 161 (1986) 23-26

VEB Gustav Fischer Verlag Jena

Chick Embryonic Development Following Exposureto Caffeine and Nicotine

By S . H . GILA`Il and T . V . X. PERSAUD2

1Department of Anatomy, University of Medicine and Dentistry of New Jersey, NewJersey Medical School, Newark, N.J., U.S .A., and 2Department of Anatomy, Universityof Manitoba, Basic Medical Sciences Building, Winnipeg, Canada

With 3 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 insuspect as a potential human teratogen,the combined effects of caffeine and nicotine on earlychick 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 developmentaldefects. At 72 h incubation teratogenicity was potentiated following the same treatment . Embryo-nic growth was not affected. Embryotoxic interactions of this nature might account for con-genital 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 re-ported, the results of several animal studies suggest that caffeine might be harmfulto the embryo, so much so that the Food and Drug Administration of the UnitedStates urged recently that "pregnant women should avoid caffeine containing foodsand drugs, if possible, or consume them only sparingly" (FDA 1981 ; DEws et al .1984) .

It is now established that smoking during pregnancy reduces significantly thebirth weight of the offspring and leads to a higher incidence of fetal and perinataldeath . There is also some evidence that maternal smoking may lead to an increasedincidence of congenital malformations (ABEL 1980).

For the screening of drugs for possible embryotoxicitv and teratogenicity theearly chick embryo is considered a highly sensitive system (FISaER, SCHOENWOLF1983 ; GEBIIARDT 1972) . The technique is inexpensive, simple to perform and exposesthe rapidly developing embryo to direct contact with the test substance . Further-more, the influences of the maternal placenta, nutrition and metabolism are con-veniently avoided .

Significant embryopathic effects have been observed following exposure of chickembryos to either caffeine (GILANI, GIOVIxAzzo, PERSAUD 1983) or nicotine (LAx-DAUER 1960 ; GATLING 1964) . Of particular interest is the potentiative embryotogicinteractions of caffeine with a wide spectrum of other teratogenic substances at mini-

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

mallv effective doses (RITTER et al . 1982) . Because of the widespread use of bothcaffeine and nicotine we have studied their combined effects on the early develop-ulent 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 either48 or 72 h incubation, as shown in Tables 1 and 2. Physiological saline was used as the solventand 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

. - The total number of eggs used in this study was 220; 112 at 48 and 108 at 72 h incubation,respectively. On d 13 the live embryos were removed from the eggs, and after staging these wereweighed and examined for the presence of gross malformations . The embryos were fixed in 10neutral formalin .

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

Results

The incidence of etubryonic mortalitv and anomalies following treatment withcaffeine at 48 or 72 h incubation was not significantly increased compared to thesaline treated controls . However, treatment of the etnbryos with nicotine at 48 hincubation significantly affected their survival and development .

At 48 h incubation, significantly fewer embryos survived the combined treatmentwith caffeine and nicotine, compared to those treated with only one of these sub-stances. 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 incu-

bation

Treatment _\"umber ofeggs treated

Live embryoson d 13

Survival

(%)

Abnormal

(%)

Malformations

Caffeine 13 10 76 20 Short limbs,

\icotine 15 8 53* 88*

Abnormal tail

Hemorrhage,

affeine and 8 1 9* 4•

Short neck,Reduced body,Everted viscera

Edema, Twisted

Nicotine limb, Short neck,

-Everted viscera,Abnormal tai1,

Controls(0.9 0 10 --eC1) -

Reduced body

0.1 ml/egg 26 21 s0 10 Short beak0.2 ml/egg 30 26 87 4 Twisted limbs

* P< 0.05, chi-square test.


Teratology 25

Table 2. Effect of caffeine (1 mg) and nicotine (1 mg) on developing chick embryos at 72 h incuba-tion

Treatment Number ofeggs treated

Live embryoson d 13

Survival(%)

Abnormal(%)

Malformations

Caffeine 13 11 85 18 Micromelia

Nicotine 13 - 10 77 30Twisted limbs

Everted viscera,

Caffeine and '_6 11 42* 55*

Reduced body,HemorrhageShort neck,

Nicotine

Controls(0.9 °o _\'aCl)0.1 mI/egg 8 6 3 4

Twisted limbs,Reduced.body

Short neclc0.2 m]/egg _'8 25 89 8 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 ± 0.24Nicotine 4.19 = 0.10 4.55 f 0.53Caffeine 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 ± 0.150.2 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 treatedgroup. At 72 11 incubation, both embryolethality and teratogenicity were signifi-cantly increased from the combined treatment compared to any other group (Tables1 and 2) .

The different types of developmental defects observed in the embryos are listedin 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 andnicotine at both incubation periods . As shown in Table 3, the mean weight of em-bryos exposed to caffeine, nicotine, caffeine and nicotine, did not differ significantlyfrom each other nor from the saline-treated controls .

Discussion

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


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

with the caffeine treatment at 48 h incubation and with either caffeine or nicotineat 72 h incubation . Thus, the older embryos were less susceptible to damage followingexposure to either of the substances but not when subjected to the combined treat-ment.

Teratological studies in laboratory rodents have shown that high doses of caffeinemay lead to embryonic growth retardation, limb malformations, cleft palate andbleeding disturbances (see DEws et al . 1984) . Similar observations were made inthe chick embryo following treatment with caffeine (Gir.aNi et al . 1983) . A co-terato-genic activity for caffeine and nicotine is evident at 72 h incubation because thecombined treatment led to significant embryopathic effects whereas exposure of theembryos to either one was relatively ineffective . Embryotoxic interactions of thisnature might account for developmental defects where the etiology is not certain .From animal studies caffeine is known to enhance the teratogenic potential of se-veral chemicals, including metabolic inhibitors, but the mechanisms involved arenot clear and have been discussed elsewhere (TlatsoN 1977 ; THasER, PALM 1975 ;RlTr m et al . 1982) .

AcknowledgementThis 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 assist-ance of \ir. S. -l1AITRA is gratefully acknowledged .

References

AREL, E. L . : Smoking during pregnancy : A review of effects on growth and development of off-spring. 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 Publi-

cation, Washington No. (FDA) 81 (1981) 1081 .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)65-72.

GATLIXG, R . R . : Effect of nicotine on chick embryo. Arch. Path., Chicago ^rS (1964) 652-657.GEBHARDT, D. O. E. : The use of the chick embryo in applied teratology . In: Advances in Terato-

logy, Vol. 5., ed. R'ooLLAaI, D. H. _li. Academic Press, New York 1972, 97-111 .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 effectsof caffeine. CRC Critical Reviews in Toxicology, Florida 3(1975) 345-369 . -

Tlatso., J. : Caffeine. Mutation Res., New York 47 (1977) 1-52 .

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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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