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KMID : 0351619700110010161
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Kyungpook Medical Journal
1970 Volume.11 No. 1 p.161 ~ p.172
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^(3)H-thymidine Autoradiographic Studies on Trypan Blue Induced Congenital Malformations in Chick Embryos
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Abstract
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The teratogenic action of trypan blue on a variety of vertebrate embryos has been well established. Injection of this substance into fish eggs (Battle and Laale, 1960), rat embryos (Jensh, 1907) and rana pipiens embryos (Green-house, 1968) produces a wide spectrum "`. malformations.
The first investigators who treated chicly embryos with the potent teratogen, trypan blue, discovered high incidence of rumplessnes;s resulted (Ansel and Lallemand, 1941). Several others have since repeated this experiment an~i obtained similar results (Beandoir and Wilson;, 1958; Beaudoin, 1961, and Kaplan et al, 1967), However, little is known concerning the event occurring between dye-treatment at one to three days of incubation (24 to 72 hours)and abscence of rump tissues in 10-day (or older) chicle embryos. Ansel and Lallemand suggested that the dye acts directly on the elements of the caudal bud, leading to agenesis of the rump., Later, Ansel (1950) stated that dye treatment led first to the development of a caudal hematoma and that this hematoma vas responsible for tl~e observed rumplessness. Unfortunately, Ansel offerd no experimental evidence to substantiate this statement.
Stephan and Sutter (1961) also observed caudal he.-naiomas in trypan blue-treated chick embryos, but held these to be clear blisters which had secondarily become billed with blood.
According to these authors, the dye act in chick embryo ´oy inhibiting cellular proliferationin the somite-producing centers of the rump and´ it is this inhibition which results in failur of normal morphogenesis in the. caudal portion of the embryo. Still others have been unable to explain the pecaliar susceptibility of such tissues. as tail to trypan blue or to any of the other chemical teratogenes known to produce rumplessness.
Attempts to elucidate the mechanism of action of this teratogen in mamals and chick _ embryos are complicated by the observation that the dye does not penetrate the embryonic tissue properly (Gillman, Gilbert, 1948 and Hamburgh, 1954), but accumulates in the internal entodermal layer of the yol_K sac placenta (Wilson, 1955; germ 1958). Three hypothesis have been proposed to explain the: teratogenic action of the dye.
The first suggested that the dye or a part of " it enters the blastocyst and acts directly on the embryonic cells,
The second theory implies that maternal. metabolic changes may be responsible for fetal malformations. The third postulates leading of the yolk sac epitheLum with large dye molecules, thus inhibiting transfer of essential substance (Beck, Baxter, 1960; Johnson and. Chepnik, 1966).
The prewent study was undertaken to investigate this problem and specifically, to determine (i) whether the time of maximun concentration in the yolk sac epithelium is correlated with. the time of teratogenic action of the dye, (2) whether the yolk sac epithelium, subsequent to heavy dye accumulation, suffers any damage.
(3) whether the radioautographic an~~lysis of trypan treated cell or tissue has any I:ey, and
(4) whether the caudal hematoma car. produce and is responsible for tail malformation in chick embryos.
RESULTS AND DISCUSSION:
1) Fig.3 to Fig.B shows the results from radioautographic analysis of neuroepithelial cell cycle in 0.1~ trypan blue treated chick embryo.
The percentage of labeled mitosis figures is plotted aginst the time between 3H-tliymidine administration and acrifice of the animal. These curves were constructed for all of the other populations studied. The time intervals between 50 per cent mitosis labeling on the ascending and descending slopes of these curves were measured.
The time intervals is an estimate of the mean duration of DNA synthesis in neuroepithelial cells indicating the duration of DNA synthesis in neuroepithelial cells are constant through normal and are prolonged one hour through trypan treated groups.
2) The incidence and type of malformation obtained following treatment with trypan blue was similar to the pattern reported for chick embryo by other investigators.
The column. of table 1. lists a summary of the defects found in 290 abnormal trypan blue treated. embryos. Twelve days after injection of 0.1% and 2 o trypan blue at 12 day, a high incidence of various degrees of resorption was noted, while surviving embryos showed various degrees of leg defect and tail defect including gastroschisis. Injection of trypan blue on day 3 (72 hours) of incubation led eitheir to resorption of the embryos or retardation of the growth of the embryos, but no histological malformations.
3) No malformations were noted at any time among embryos of control group and only two out of one-hundred twenty control chick emb ryos.
4) Examination of the histological sections revealed that within twenty-four hours injection of the dye at eighteen hours, at daysly and 3 of incubation showed eye defects, abscence of tail bud and irregular neuroepithelial cell arrangement with ventricular thickness.
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