Of many closely related compounds investigated recently from the point of view of cytotoxity and tumour-inhibiting properties 2,4,6-tri-(ethyleneimino)-1,3,5-triazine (called also triethanomelamine or TEM) proved to be the most effective substance against the Walker rat carcinoma. First descriptions of this fact were published in 1950 (Buckley et al. and Burchenal et al.). Also in 1950 appeared the publication of the first results of the clinical application of TEM at the First International Cancer Congress (Mueller & Miller). Later on Karnovsky et al. (1951) and Petersen et al. (1951) recommended procedures for parenteral and oral administration of this substance in clinical treatment. Very recently TEM has been introduced in a commercial form for oral and parenteral administration in cases of leukaemia, Hodgkin’s disease, polycythaemia vera, and other neoplastic conditions.

The cytotoxic activity of TEM is very similar to that of the nitrogen mustards (Philips & Thiersch, 1950), chromosome breakage with simultaneous chromosomal rearrangements, chromosome bridges and other malformations being observed by Fahmy & Fahmy (1953,1954, 1955), Lüers (1953), and Herskowitz (1955). In somewhat higher doses TEM appears to act as an antimitotic agent (Hendry et al., 1951).

The present work was undertaken to investigate the influence of TEM on early stages of the embryonic development of chick embryos and on the formation of the first anlagen of some organs.

In the first part of this work a solution of TEM in 0 9 per cent. NaCl was injected into the albumen of fertilized chicken eggs, which had been incubated at 37·5° C. for 22 hours until the stage of the primitive streak was reached. The puncture in the shell was closed with wax and the eggs left for approximately 20 hours to allow the injected solution to diffuse throughout the albumen. The eggs were afterwards reincubated for another 24 hours, the embryos taken out, rinsed in saline solution, and fixed. The whole period of the incubation in ovo was consequently about 46 hours with a break of about 24 hours for injection and diffusion.

In the second part of this work we used chick embryos cultivated in vitro according to the method described by New (1955). The fertilized eggs were incubated for 22–23 hours, then the embryos transferred to watch-glasses; those which were in the stage of the primitive streak were selected and treated with TEM or left as controls. TEM was applied in solution in the liquid egg albumen put round the plastic ring used for this method. The embryos were then incubated in vitro for different periods of time, usually about 24 hours, but always till the controls reached the stage of 12-16 somites.

In the experiments in the first part of this work, after preliminary tests, three concentrations of TEM were used: 10−5, 2 × 10−5, and 5 × 10−5. Embryos cultivated in vitro were treated with lower doses: 2 × 10−6, 3 × 10−6, arid 5 × 10−6. Higher concentrations were used in ovo because of the additional dilution of TEM by the albumen.

For the comparison of the developmental stages the works of Patten (1950) and Hamburger & Hamilton (1951) were used.

The embryos were examined histologically and cytologically. For histological purposes the embryos were fixed in alcoholic Bouin solution, embedded in wax, and after sectioning stained with haematoxylin and eosin. For cytological purposes the embryos were fixed in Carnoy’s fluid and stained with methyl green pyronin. The whole mounts were stained with Mayer’s haematoxylin.

The TEM used was supplied by Imperial Chemical (Pharmaceuticals) Ltd., Manchester.

In both groups of experiments, i.e. whether TEM was applied in ovo or in vitro, the results were very similar and the intensity of changes depended roughly on the concentration used. However, there was a considerable variation in response between embryos treated with the same concentrations, this variation being much greater between embryos treated in ovo than between embryos cultivated in vitro.

Macroscopical changes

The lowest concentrations, 10−5 and 2 × 10−6in ovo and in vitro respectively, caused a distinct retardation of the growth and development of embryos. Whereas the control embryos injected with saline, or treated with the corresponding mixture of the liquid albumen and saline solution, were normally in the stage of 12–16 somites by the end of the experiment, the experimental embryos had only 6–9 somites. Many of the experimental embryos appeared to have peculiar hollows inside the somites, more particularly in the posterior ones, though in some embryos many somites were damaged. The embryos in this group also showed some changes in the head, which was considerably widened, probably due to increased accumulation of the head mesenchyme cells, whereas the development of the brain appeared to be slightly retarded (Plate 1, compare figs. 1 and 2).

The intermediate concentrations, 2 × 10−5 and 3 × 10−6 respectively, caused more distinct changes in development. The retardation was stronger, often with almost complete atrophy or considerable under-development of the somites. The neural tube, whether closed or not, was almost normal in appearance and shape. In the head there was usually much less mesenchyme than in embryos given the lower concentrations (Plate 1, fig. 3).

The strongest concentrations (5 × 10−5 or 5 × 10−6) caused also strong retardation of development. The somites were often completely atrophied. In some embryos vesicles were found on both sides of the axis (Plate 1, fig. 4). They were usually located a short distance away from the axis, not right against it as is typical for the vesicles seen in embryos treated with purine analogues (Waddington, Feldman, & Perry, 1955).

Microscopic changes

According to histological observations it appears that the influence of TEM on these embryos is directed mainly to the mesodermal organs, primarily against somites. In fig. 5 (Plate 1) is shown a transverse section of the 8th pair of somites of a control embryo. In fig. 6 is a similar section of the 5th pair of an experimental embryo treated with TEM at a concentration of 10 −5 injected in ovo. Comparison shows that the development in this case was nearly the same, but the somites of the experimental embryo are empty with an absence of the core of the somites, which normally consists of irregularly arranged cells.

After treatment of embryos in vitro with TEM of 3 × 10−6 concentration or in ovo with a concentration of 2 × 10−5 the somites of some embryos were further damaged. In some cases there was an extreme retardation of development with very marked effects on the somites, which appeared to be completely hollow (Plate 1, figs. 7 and 8). In other cases the cells of the somites have been more or less separated, and the whole organ to some extent disintegrated, the somitic cells becoming more like mesenchymal cells. In such embryos changes could be found in other tissues and organs, for instance in the neural tube and notochord, though these seem to be more resistant to TEM than the somites (Plate 2, fig. 9).

Many of the embryos in the second and third group, i.e. those treated with TEM in ovo using concentrations of 2 × 10−5 or 5 × 10−5 and in vitro using concentrations of 3 × 10−6 or 5 × 10−6, appeared to have very large vacuoles or bubbles lying along both sides of the central organs of the embryo. In the transverse sections of such embryos a distinct disintegration of the somites and a marked widening of the coelom could be seen. These widenings were visible in the whole mounts as vesicles, which appeared to be present not only between both somato- and splanchnopleure, but also between splanchnopleure and the entoderm layer (Plate 2, fig. 10). The vesicles were thus of quite a different kind to those resulting from purine analogues, which are due to the disintegration of the mesoderm being formed from the primitive streak (Waddington et al., 1955).

The changes in the amount of the head mesenchyme caused by the lowest doses of TEM usually appeared distinctly, but did not occur in all the embryos examined. A moderate accumulation of mesenchyme cells is seen in fig. 11 (Plate 2) in comparison with the control head of fig. 12.

After treatment with any of the concentrations of TEM, both in ovo and in vitro, pronounced changes in nuclei and nucleoli of cells of the somites, neural tube, and notochord could be seen. By staining with methyl green pyronin after fixation with Carnoy’s fluid the nuclei of experimental embryos appeared to be nearly three times larger than those of the controls, the nucleoli showing still greater hypertrophy, and the cells were as a whole hyperplastically changed (Plate 2, figs. 13 and 14).

2,4,6-Tri-(ethyleneimino)-l,3,5-triazine is an extremely toxic compound, as can be seen from the concentrations used in the experiments in this and other works. In medicine it is being increasingly used against leukaemia, Hodgkin’s disease, polycythemia vera, i.e. against neoplastic conditions of particular organs of mesodermal origin. It has a very toxic effect on the bone marrow, where it affects the haemopoetic activity selectively (Gellhorn, Klingerman, & Jaffe, 1952). In stronger doses it produces rapid and widespread damage in lymphoid tissues (Hendry et al., 1951). The bone marrow and lymphatic glands are typical organs of mesodermal origin and this seems congruent with the rather selective activity of TEM in relation to the somites shown in this work. However, in amphibian embryos it is not the mesoderm but the neural tissue which is most sensitive to the drug (Waddington, 1958). Presumably this sensitivity depends on the precise chemical constitution of the cells; but since the mode of action of TEM on biological materials is not fully understood, it is not clear precisely what the relevant factors are.

Enlargement of the nuclei and nucleoli are frequent effects of the action of cytotoxic agents; after treatment with TEM these changes have been observed mostly in the tissues of the somites and the neural tube. They are presumably a consequence of the inhibition of mitosis. Similar enlargements of nucleoli and the production of giant cells were observed in a quite different material, planaria, in the process of regeneration after treatment with TEM (Pedersen, 1957), and also in amphibian embryos by Töndury (1955).

  1. Triethanomelamine (TEM), applied to chick embryos in the stage of the primitive streak both in ovo and in vitro, produces retardation of development which is the greater the higher the dose used.

  2. The embryonic organs most sensitive to TEM appeared to be the somites, which become empty through the disappearance of the central core.

  3. In higher doses the somites undergo more or less complete disintegration, the somitic cells being separated and resembling mesenchyme cells.

  4. TEM causes a considerable enlargement and widening of the coelom and of the space between splanchnopleure and the entoderm layer.

  5. After treatment by lower doses there appears a slight accumulation of the head mesenchyme cells.

  6. TEM causes a considerable enlargement of the cells, nuclei and nucleoli of the somitic, neural tube, and notochord cells.

This work was done during the tenure of a British Council studentship, for which grateful acknowledgement is made. I should like to thank Professor C. H. Waddington for suggesting this problem and for helpful advice.

Buckley
,
S. M.
,
Stock
,
C. C.
,
Crossley
,
M. L.
, &
Rhoads
,
C. P.
(
1950
).
Inhibition of Crocker Mouse Sarcoma 180 by certain ethyleneimine derivatives and related compounds
.
Cancer Res
.
10
,
207
.
Burchenal
,
J. H.
,
Johnston
,
S. F.
,
Stock
,
C. C.
,
Crossley
,
M. L.
, &
Rhoads
,
C. P.
(
1950
).
The effect of 2,4,6-triethyleneimino-s-triazine and related compounds on transplanted mouse leukaemia
.
Cancer Res
.
10
,
208
.
Fahmy
,
O. G.
, &
Fahmy
,
M. J.
(
1953
).
The effect of dose on mutagenicity and chromosome breakage induced by 2,4,6-tri(ethyleneimino)-l,3,5-triazine
.
Drosophila Inform. Serv
.
27
,
89
.
Fahmy
,
O. G.
, &
Fahmy
,
M. J.
(
1954
).
Cytogénie analysis of the action of carcinogens and tumour inhibitors in Drosophila melanogaster. II. The mechanism of induction of dominant lethals by 2,4,6-tri-(ethyleneimino)-l,3,5-triazine
.
J. Genet
.
52
,
603
15
.
Fahmy
,
O. G.
, &
Fahmy
,
M. J.
(
1955
).
Cytogénie analysis of the action of carcinogens and tumour inhibitors in Drosophila melanogaster. III. Chromosome structural changes induced by 2,4,6,-tri(ethyleneimino)-1,3,5-triazine
.
J. Genet
.
53
,
181
99
.
Gellhorn
,
A.
,
Klingerman
,
M. M.
, &
Jaffe
,
I.
(
1952
).
Triethylene melamine in clinical cancer chemotherapy
.
Amer. J. Med
.
13
,
428
31
.
Hamburger
,
O.
, &
Hamilton
,
H. L.
(
1951
).
A series of normal stages in the development of the chick embryo
.
J. Morph
.
88
,
55
92
.
Hendry
,
J. A.
,
Homer
,
R. F.
,
Rose
,
F. L.
, &
Walpole
,
A. L.
(
1951
).
Cytotoxic agents. III. Derivatives of ethyleneimine
.
Brit. J. Pharmacol
.
6
,
357
410
.
Herskowitz
,
I. H.
(
1955
).
The incidence of chromosomal rearrangements and recessive lethal mutations following treatment of mature Drosophila sperm with 2,4,6-tri(ethyleneimino)-1,3,5-triazine
.
Genetics
,
40
,
574
.
Karnovsky
,
D. A.
,
Burchenal
,
J. H.
,
Armistead
,
G. C.
Jr.
,
Southam
,
C. M.
,
Bernstein
,
J. L.
,
Craver
,
L. F.
, &
Rhoads
,
C. P.
(
1951
).
Triethylene melamine in the treatment of neoplastic disease
.
Arch, intern. Med
.
87
,
477
516
.
Lüers
,
H.
(
1953
).
Untersuchung iiber die Mutagenitât des Triathylenmelamin (TEM) on Drosophila melanogaster
.
Arch. Geschwulstforsch
.
6
,
77
83
.
Mueller
,
G. C.
, &
Miller
,
J. A.
(
1950
).
Acta Un. int. Cancr
.
7
,
134
.
New
,
D. A. T.
(
1955
).
A new technique for the cultivation of the chick embryo in vitro
.
J. Embryol. exp. Morph
.
3
,
326
31
.
Patten
,
B. M.
(
1950
).
Early Embryology of the Chick
.
London
:
H. K. Lewis
.
Pedersen
,
K. J.
(
1957
).
Morphogenetic activities during planarian regeneration as influenced by triethylenemelamine
.
A paper read at the S.E.B. Conference
,
Cambridge
, 1957.
Petersen
,
E.
, &
Boland
,
F.
(
1951
).
Trisethyleneimino-s-triazine in human malignant disease. A preliminary trial
.
Brit. J. Cancer
,
5
,
28
37
.
Philips
,
F. S.
, &
Thiersch
,
J. B.
(
1950
).
The nitrogen mustard-like actions of 2,4,6-trz’s(ethyleni-mino)-s-triazine and other tó(ethylenamines)
.
J. Pharmacol
.
100
,
398
407
.
Töndury
,
G.
(
1955
).
Einfluß chemischer Stoffe auf die embryonale Zelle
.
Bull, schweiz. Akad. med. Wiss
.
11
,
332
45
.
Waddington
,
C. H.
(
1958
).
A note on the effects of some cytotoxic substances on amphibian embryos
.
J. Embryol. exp. Morph
.
6
,
363
4
.
Feldman
,
M.
, &
Perry
,
M.
(
1955
).
Some specific developmental effects of purine antagonists
.
Exp. Cell Res
.,
Suppl. 3
,
366
80
.
Plate 1

Fig. 1. Macroscopical view of a control chick embryo of 13 somite stage, × 20.

Fig. 2. Macroscopical view of a chick embryo treated in ovo with 0 5 ml. solution of TEM (concentration 10 −5). × 20.

Fig. 3. Macroscopical view of a chick embryo treated in ovo with 0 5 ml. solution of TEM (concentration 2 ×10 −5). × 20.

Fig. 4. Pronounced changes after injection into the egg of 0-5 ml. solution of TEM (concentration 5 × 10 −5). ×20.

Fig. 5. Transverse section of a control chick embryo through the 8th pair Of somites. × 280.

Fig. 6. Transverse section through the 5th pair of somites of a chick embryo treated in ovo with 0 5 c.c. solution of TEM (concentration 10 −5). ×280.

Fig. 7. Transverse section through somites of a chick embryo treated in ovo with 0 5 c.c. solution of TEM (concentration 2 x 10 −5). × 280.

Fig. 8. Transverse section through somites of a chick embryo treated in vitro with solution of TEM (concentration 3 × 10 −6). × 280.

Plate 1

Fig. 1. Macroscopical view of a control chick embryo of 13 somite stage, × 20.

Fig. 2. Macroscopical view of a chick embryo treated in ovo with 0 5 ml. solution of TEM (concentration 10 −5). × 20.

Fig. 3. Macroscopical view of a chick embryo treated in ovo with 0 5 ml. solution of TEM (concentration 2 ×10 −5). × 20.

Fig. 4. Pronounced changes after injection into the egg of 0-5 ml. solution of TEM (concentration 5 × 10 −5). ×20.

Fig. 5. Transverse section of a control chick embryo through the 8th pair Of somites. × 280.

Fig. 6. Transverse section through the 5th pair of somites of a chick embryo treated in ovo with 0 5 c.c. solution of TEM (concentration 10 −5). ×280.

Fig. 7. Transverse section through somites of a chick embryo treated in ovo with 0 5 c.c. solution of TEM (concentration 2 x 10 −5). × 280.

Fig. 8. Transverse section through somites of a chick embryo treated in vitro with solution of TEM (concentration 3 × 10 −6). × 280.

Plate 2

Fig. 9. Transverse section of somites of a chick embryo treated in vitro with solution of TEM (concentration 3 × 10 −6). × 280.

Fig. 10. Transverse section of a chick embryo after treatment with solution of TEM in ovo (concentration 5 × 10 −5). On the left of the neural tube there are vesicles between somatopleure and splanchnopleure and between splanchnopleure and endoderm, × 110.

Fig. 11. Transverse section of the head of a chick embryo treated with TEM in ovo (concentration 10 −5). × 100.

Fig. 12. Transverse section of the head of a control chick embryo at 13 somite stage, × 100.

Fig. 13. The cells of the neural tube of a control chick embryo at 13 somite stage, × 700.

Fig. 14. The cells of the neural tube of a chick embryo after treatment with TEM in vitro (concentration 3 × 10 −6), × 700.

Plate 2

Fig. 9. Transverse section of somites of a chick embryo treated in vitro with solution of TEM (concentration 3 × 10 −6). × 280.

Fig. 10. Transverse section of a chick embryo after treatment with solution of TEM in ovo (concentration 5 × 10 −5). On the left of the neural tube there are vesicles between somatopleure and splanchnopleure and between splanchnopleure and endoderm, × 110.

Fig. 11. Transverse section of the head of a chick embryo treated with TEM in ovo (concentration 10 −5). × 100.

Fig. 12. Transverse section of the head of a control chick embryo at 13 somite stage, × 100.

Fig. 13. The cells of the neural tube of a control chick embryo at 13 somite stage, × 700.

Fig. 14. The cells of the neural tube of a chick embryo after treatment with TEM in vitro (concentration 3 × 10 −6), × 700.