Teratogenic and lethal effects of long-term hyperthermia and hypothermia in the chick embryo

ReproductiveToxicology,Vol. 10,No. 4, pp. 327-332, 1996 Copyright0 1996Elsevier Science Inc. Printed in the USA. All rights reserved 0890-6238/96 $15.00 + .OO

ELSEVIER

PI1 SOS%-6238(%)00062-7

TERATOGENIC AND LETHAL EFFECTS OF LONG-TERM HYPERTHERMIA AND HYPOTHERMIA IN THE CHICK EMBRYO MIROSLAV PETERKA, RENATA PETERKOV~, and ZBYN~K LIKOVSK? Institute of Experimental

Medicine,

Academy of Sciences of the Czech Republic, Prague, Czech Republic

Abtsract - The teratogenic effect of maternal hyperthermla is well known in laboratory animals and is presumed to exist also in humans. The aim of our study was to describe the embryotoxlc effect of long-term higher and lower incubation temperatures on the chick embryo. Chick embryos were incubated within days 1 to 9 at 12 different incubation temperatures ranging from 31 to 42°C. On the basis of our results, we estimated that there are three upper and lower critical thresholds of the incubation temperature: the first thresholds are 31 and 42”C, at which all embryos died; the second thresholds are 32 and 41”C, at which all living embryos were malformed; the third thresholds are 33 and 4O”C,at which some of the living embryos were without structural malformations, but their weight was shifted down and up with lower and higher temperature, respectively. The incubation temperature of 37 to 38°C was optimal. Typical malformations detected on day 9 of incubation were mlcrophthahnia, gastroschisis, caudal regression syndrome, and hyperlordosis, all of which occurred in dead embryos several times more frequently than in living embryos. CNS malformations were only sporadically present on day 9, as most of specimens bearing CNS defects died during the first days of incubation. Key Words: chick embryo; hypothermia;

hyperthermia;

teratogenic

and lethal effect.

been reported after 24 h of exposure to an incubation temperature of 41 to 42T at the end of the first incubation day (8). Deuchar (9) prevented gastrulation in the chick embryo by a high-temperature shock (45.5”C) for 3 or 5 h. Delphia and Elliot (10) found that continuous incubation of chick embryos at 40°C resulted in coelosomia of the heart, liver, ventriculus, and small intestines in 63% of the specimens. Nilsen (11) incubated chick embryos at 41 and 42°C during the first 3 d of development and demonstrated intraembryonic and extraembryonic vascular abnormalities. Harrison (12) showed that chick embryos incubated at a very low incubation temperature of 25°C for 10 to 14 d in ovo had the ability to form the nervous and circulatory systems. A differential harmful effect upon the morphogenetic processes of neurulation and the formation of the circulatory system was shown; the nervous system being more susceptible. According to our experience with the embryotoxic effect of chemical factors, for example, drugs, agricultural chemicals, and heavy metals (13-17), we formulated the following hypothesis: the nonphysiologic incubation temperature, representing a teratogen of a physical nature, affects the development not only of the chick embryos vascular system, but also of its other organs, as chemical teratogens do in common laboratory mammals and in humans. Therefore, the aim of the present study was to de-

INTRODUCTION

Several retrospective and prospective epidemiologic studies have concluded that hyperthermia in women during early pregnancy probably causes children to be born with malformations of the CNS or severe brain dysfunction (l-3). Lipson (4) showed a significant causal relationship between congenital intestinal aganglionosisHirschsprung disease-and a history of maternal hyperthermia during pregnancy. Maternal hyperthermia has been shown to be a teratogen in all common laboratory animals (5,6) and even in Macaca radiata (7). The effect of low and high incubation temperature on the development of the primitive streak and somites of the chick embryo was studied by several authors in the last century. An increase of embryonic mortality has

Address correspondence to Miroslav Peterka, M.D., Ph.D., Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, VfdeiIsk6 1083, CZ 142 20 Prague 4, Czech Republic. Ackrtowledgments-The authors are indebted to Mrs. Alena Jelinkovh and Mrs. Jana Fialova for technical assistance and for help during the preparation of the manuscript. This work was supported in part by the Ministry of Health of the Czech Republic (Grant 1054-3) and Grant Agency of the Czech Republic (Grant 304/93/0594). Received 26 January 1996; Revision received 23 February 1996; Accepted 1 March 1996. 321

328

Reproductive

Toxicology

termine, in populations of chick embryos incubated during the whole organ critical period at different incubation temperatures from 3 1 to 42°C: 1) the incidence of dead and malformed living embryos: 2) malformation spectra in living and dead embryos. MATERIALS

AND METHODS

Fertilized eggs of the domestic fowl, White Leghorn randombred population (Dobrenice Farm), were incubated in a horizontal position at a relative humidity of 40 to 60%. The eggs were not stored in our laboratory and were used the same day they were supplied from the farmer. Twelve experimental groups were used, corresponding to incubation temperatures differing by 1°C. from 31 to 42°C (100 eggs per one incubation temperature were used). Temperature was monitored by a minimo-maximal thermometer installed in the incubator near the eggs. The temperature in the incubator did not fluctuate by more than plus or minus 0S”C. The common window technique for opening the egg shell was used on day 3 of incubation (16). During this procedure, nonfertilized eggs were removed from the experimental groups. The exact numbers of eggs for each experimental group are shown in Table 1. The windows of the eggs containing living embryos were covered with a slide sealed with paraffin. These eggs were further incubated without rotation until day 9. During incubation, the embryos were checked daily through the window and dead embryos were discarded and their external structural malformations determined (provided an embryo was not destroyed by autolysis). The embryos were harvested on day 9. The living embryos were weighed, and both living and dead embryos were checked for the presence of external structural malformations (CNS, neural tube, eye, beak, trunk, extremities) under a stereomicroscope. After microdis-

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10, Number 4, 1996

section, which involved opening the ventral body wall and anterior wall of the right heart ventricle, the presence or absence of a defect of the interventricular septum and great arteries was determined. The heart defects and malformations of the great arteries could be determined only in those embryos incubated at 37 to 42°C; at lower incubation temperatures (33 to 36”(Z), the embryos exhibited growth retardation resulting in a decrease in body weight to below 1000 mg. According to our experience, the interventricular septal “defect” is physiologically present in control embryos incubated at 37S”C. the weights of which are lower than 1000 mg. RESULTS Teratogenic and lethal @ects of various incubation temperatures (Figure I, Table I). The optimal incubation temperature was 37 to 38°C. Chicks incubated at this temperature included the lowest proportion of malformed (1 to 2%) and dead embryos (24 to 26%). At lower (34 to 36°C) or higher (39 to 40°C) incubation temperatures, both the teratogenic and the lethal effects increased. Most of the living malformed embryos were harvested on day 9 after incubation at 33 and 41 “C. All embryos were affected, for instance, were either malformed or dead, after 9 d of incubation at the lowest (3 1 to 32°C) and highest (41 to 42°C) temperatures. Only one embryo incubated at 42°C survived until day 9. Weight of living normal and malformed embryos (Figure 2, Table 2). An extremely low incubation temperature (31 “C) induced death in all embryos before day 9 of incubation. Only the developing area vasculosa (extraembryonal vascularization) without the embryo could be detected under the stereomicroscope on day 9.

1. The percentage of normal, malformed, and dead chick embryos on day 9 after exposure to different incubation temperatures

Table

Temperature (“C) 31 32 33 34 35 36 37 38 39 40 41 42

Living embryos n 46 100 91 93 82 83 94 90 87 93 87 99

Normal

Malformed

Dead

0

0

IOO.0 81.0 64.8 33.3 45.1 33.1 26.6 24.4 39. I 38.7 62. I 99.0

12.1 59. I 51.2 60.2 72.3 73.3 51.7 44. I 0 0

19.0 23.1 7.5 3.6 6.1 I.1 2.2 9.2 17.2 37.9 I.0

s

42

31 32333435363738394041 Incubation

-*-total

--A--

temperature

dead

(“C) -*-

malformed

Fig. I. Percentage of malformed and dead embryos after 9 d of incubation at temperatures of 31 to 42°C.

Hyperthermia and hypothermia 0 M.

329

PETERKA ET AL.

The highest proportions of embryos with caudal regression syndrome were living specimens incubated at temperatures of 32 or 40°C (16 and 15%, respectively). The highest proportion of dead specimens with caudal regression syndrome was 9% after incubation at a lower temperature (at 33°C) and 67% after incubation at a higher temperature (at 39°C).

31 32 33 34 35 36 37 38 39 40 incubation temperature (“C)

4 1 42

embryos after 9 d of incubation at temperatures of 31 to 42°C.

Fig. 2. Weight of living chick

At an incubation temperature of 32”C, 80% of embryos died by day 9. In those specimens, a connection between extra- and intraemblyonic vascularization was not formed. All embryos still living on day 9 exhibited severe growth retardation (their developmental morphologic stage, according to Hamburger and Hamilton (18) corresponded to 13 to 23 HH, for instance, to the second to fourth day of incubation). On day 9, we first weighed specimens that had been incubated at 33°C. The embryos incubated at 33°C corresponded morphologically to 5-d-old embryos, for instance, they were developmentally retarded by approximately 4 d. The mean weight of 9-d embryos increased with elevated incubation temperature ranging from 132 mg at 33°C to 2272 mg at 40°C. The mean weight of the embryos after incubation at 41°C was the same as in the embryos incubated at 40°C. The weight of the only living specimen incubated at 42°C was 1165 mg. Malformations in both living and dead embryos on day 9. Caudal Regression Syndrome (Figure 3a, Table 3). Table 2. The mean weight of normal and malformed living chick embryos on day 9 after exposure to different incubation temperatures Temperature (“C)

n

mg (Mean + SE)

33 34 35 36 37 38 39 40 41 42

32 62 45 55 69 68 53 57 33

132.4 f 6.7 308.2 f 11.3 410.9 f 17.1 878.2 f 18.5 1205.7 f 12.7 1613.3 f 16.6 1634.0 f 38.7 2272.6 f 26.5 2265.0 f 53.5 1165.0

Microphthalmia (Figure 3b, Table 3). The proportion of living embryos with microphthalmia never exceeded 10%. Microphthalmia was detected in 42 to 67% of dead embryos. The most adverse incubation temperatures were 34”C, 35”C, and 39°C. Hyperlordosis (Figure 3c, Table 3). Hyperlordosis occurred in living embryos only after incubation at 33°C (in 56% of cases). Among dead embryos incubated at 33 and 39”C, hyperlordosis reached maximal values of 93 and 33%, respectively. Gastroschisis (Figure 3c, Table 3). Gastroschisis occurred only in specimens incubated at temperatures higher than 38°C. This type of malformation was found in 11 to 14% of living embryos after incubation at temperatures of 39 and 40°C. Incubation at 41°C produced gastroschisis in 100% of living embryos. Gastroschisis occurred in 60 to 67% of dead embryos with an incubation temperature of 39 to 4O”C, and in nearly 92% of specimens after an incubation temperature of 41°C.

CNS malformations. Only two living embryos on day 9 exhibited exencephaly: one after an incubation temperature of 35°C and one other after an incubation temperature of 41°C. Many more malformations of the CNS were found on days 5 and 6 in the dead embryos incubated at 42°C. All embryos that died during incubation were examined, but most of them were morphologically destroyed by autolysis. A reliable examination could be performed only on 17 dead embryos. Among them, 11 specimens (65%) exhibited exencephaly or myeloschisis. DISCUSSION According to Co&oft and New (19), procedures employed in experiments investigating the effect of hyperthermia on prenatal development in mammalian models lead to unavoidable severe distress on the maternal organism. Consequently, the final effect of hyperthermia on the offspring can result from the combined effect of the disturbance of the maternal organism and the direct effect of heat on the embryo itself. On the other hand, several studies have compared the effects of heat exposure in vivo and in vitro on the rat, where embryos exhibited a similar significant de-

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Volume 10, Number 4, 1996

Table 3. Percentage of living and dead malformed

embryos examined on day 9 after exposure to different incubation temperatures Percent embryos with malformation

Temperature

Embryo status

(“C)

32

Caudal regression

33 34 35 36 37 38 39 40 41

Embryos

Microphthalmia

16 4 6 9 3 0 0 0 0 0 0 0 2 22 0 67 15 0 3

living dead living dead living dead living dead living dead living dead living dead living dead living dead living dead

Hyperlordosis

0

0

0 6 5 8 33 4 42

9 56 93 2 19 2 0 0 0 0 0 0 0 0 33 0 0 0 0

0 0 0 0

0 3 67 2 10 3 0

Gastroschisis 0 0 0 0 0 0 0

0 0 0

I 0 0

0 11 67 14 60 100 92

that died before day 8 are excluded. 1001

a

b

80-

80

t :: :: : :

60S 0 40-

# ,#’

20-

,A

/

\ :f-++_ ==-/I,.L-..nG 31 32333435363738394041

0

incubation 31

32

33

34

incubation -o-

living

35

36

37

38

39

temperature

40

4 1 42

-o-

--.--

(OC)

&ad

IO0

80-

80

60-

60

31 32 33 34 35 36 37 38 39 40 4 1 42

31 32 33 34 35 36 37 38 39 40 4 1 42 living

42

temperature

IwIng

: : : : : : :

--•-- dead

C

-o-

: : : : : : _:

(‘C)

1001

incubation

: : : :

temperature --a-- dead

incubation

(OC) -o-

temperature

living

Fig. 3. Percentage of living and dead embryos with malformations after 9 d of incubation at temperatures regression. (b) Microphthalmia. (c) Hyperlordosis. (d) Gastroschisis.

--+-

(‘C)

dead

of 3 1 to 42°C. (a) Caudal

Hyperthermia

and hypothermia

crease in the growth parameters of head length and somite number (20,21). Our experimental conditions allowed us to investigate the direct influence of various long-term incubation temperatures on developing chick embryos while avoiding possible problems arising from disturbance to the maternal organism. In addition, we were able to follow the destiny of all embryos during the entire critical period for all organs. We estimated three upper and lower critical thresholds of incubation temperature during the first 9 d of incubation. The most extreme thresholds were 31 and 42°C at which all embryos died. The second thresholds were 32 and 41”C, at which all living embryos were malformed. The third thresholds were 33 and 40°C at which parts of the living embryos were without structural malformations, but the mean weight of all living embryos was shifted up (with higher temperature) or down (with lower temperature). The optimal incubation temperature for the chicken embryo was between 37 and 38°C. We can conclude that a long-term incubation temperature higher than 39°C (1.5”C above the optimum 37.5”C) has teratogenic effects. This result is in agreement with results from experiments with laboratory animals in which the threshold for adverse effects of maternal hyperthermia was 1.5 to 2°C (5). The frequencies of malformations in living and dead chick embryos were different. The number of malformed dead chick embryos was several times higher than the number of malformed living embryos. A similar phenomenon has been noted after maternal hyperthermia in guinea pigs, as well as in human embryos from the Kyoto Human Embryo Collection. It appears that embryos with neural tube defects are filtered out by resorption or abortion (22,23). Prenatal elimination of abnormal embryos and fetuses is a natural screening phenomenon called terathanasia (24). The results of several epidemiologic studies in humans should be corrected with regard to this phenomenon. Epidemiologic studies can analyze only one part of the embryotoxicity profile-living malformed newborns. For the epidemiology of developmental defects in humans, it is also extremely important to know the numbers of dead embryos and fetuses with or without malformations. For example, the relatively low incidence of CNS malformations in Finland, where the great majority of women visit saunas during early pregnancy (25), might be explained hypothetically by early spontaneous abortion of embryos with CNS malformations. The types of malformations produced by higher incubation temperatures in the chick embryo were similar in comparison with the known malformation spectra induced by hyperthermia in mammals, including humans.

331

0 M. PETERKA ET AL.

In our study, gastroschisis and microphthalmia in the chick embryo were typical malformations originating from higher incubation temperatures. In humans, a significant increase in the frequency of abdominal wall defects has been reported in offspring whose mothers had sustained high temperatures during the embryogenesis period (26). Hendrickx et al. (7) described umbilical cord malformations in Mucaca rudiatu after maternal hyperthermia. Microphthalmia after hyperthermia in laboratory animals has been described for mice (27), rats (28,29), and guinea pigs (22). In the chick embryo, early embryonic loss between embryonic days 1 and 5 eliminated CNS defects. Defects of the CNS in living chick embryos on day 9 were present only in two cases. CNS defects were described in guinea pigs following maternal hyperthermia by Cawdell-Smith et al. (22), and in mice after heat shock by Webster and Edwards (30), Chemoff and Golden (31), and Bennett et al. (32). Our results showed that hypothermia and hyperthemria in the chick embryo produced similar malformation spectra as in laboratory mammals. It seems that the influence of hyperthermia on the developing embryo has a universal character, with minimal interspecies differences. Acknowledgements -The

authors are indebted to Mrs. Alena Jelinkova and Mrs. Jana Fialova for technical assistance and for help during the preparation of the manuscript. This work was supported in part by the Ministry of Health of the Czech Republic (Grant 1054-3) and Grant Agency of the Czech Republic (Grant 304/93/0594).

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