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/. Embryol. exp. Morph. Vol. 29, 1, pp. 209-219, 1973
209
Printed in Great Britain
The effects of isonicotinic acid hydrazide on the
early chick embryo
By M. A. CASTELLANO, J. L. T 6RTORA, N. I. GERMINO,
F. RAMA AND C. OHANIAN 1
From the Facultad de Veterinaria, Montevideo
SUMMARY
1. Unincubated White Leghorn fertile eggs were injected with isonicotinic acid hydrazide
(INH) through a hole in the shell. A control group was injected with normal saline, a second
group with INH plus vitamin B6, and a third group was left unopened.
2. INH, in the doses we used, proved to be lethal for more than 50% of the early chick
embryos, and also produced important developmental alterations.
3. Alterations produced by INH were primarily observed at the level of the neural epithelium, particularly at its cephalic portion. The most important ones were a degenerative and
necrotic process of the neural epithelium, and a distortion of the normal anatomical relations
of the cephalic structures.
4. The embryonic mortality and developmental alterations induced by INH were prevented
to a considerable extent by the concurrent injection of pyridoxine hydrochloride and INH.
5. The preventive action of vitamin B6 suggests that toxicity of INH in the chicken embryos
is due to the antivitamin-B6 action of INH. Through this mechanism INH would block,
specially, the amino acid metabolism. The developing nervous system was the embryonic area
most sensitive to such metabolic alterations.
INTRODUCTION
It has been demonstrated that hydrazines induce a block in the metabolism
of amino acids (Lewis & Izume, 1926), and that this blockade can be raised by
the administration of vitamin B6 (McCormick & Snell, 1961; Simonsen &
Roberts, 1967).
Studies in vitro (Killam & Bain, 1957) indicated that a number of hydrazines
(thiosemicarbazide, furoyl hydrazine and isonicotinic acid hydrazide) inhibit
the enzymic activities of the amino acid metabolism, catalysed by pyridoxal.
The blockade of pyridoxal enzymes by isonicotinic acid hydrazide (INH) has
been reported by Yoneda, Kato & Okajuna (1952) and Yoneda & Asano (1953).
INH combines with pyridoxal phosphate to form a hydrazone and thereby
causes a competitive inhibition of aminopherases, decarboxilases, diamine
oxidase and other pyridoxal enzymes (Braunstein, 1960).
In the chick embryo, it has been demonstrated that vitamin B6 is essential for
1
Authors' address: Facultad de Veterinaria, Instituto de Anatomia Normal, Casilla de
Correo 2351 (distrito 6) Montevideo, Uruguay.
14
EMB 29
210
M. A. CASTELLANO AND OTHERS
the development of the early stages (Cravens & Snell, 1949; Cravens, 1952;
Conti & Milio, 1965, 1966a, b). The pyridoxine analogs (deoxypyridoxine and
methoxypyridoxine) have an inhibitory effect in development, producing 100%
mortality during the first 4 days of incubation (Cravens, 1952).
The purpose of this investigation is to study the action of INH in the early
chick embryo during the first 4 days of development and to test the preventive
action of pyridoxine hydrochloride.
MATERIAL AND METHODS
Fertilized White Leghorn eggs, purchased from a local hatchery, were used
throughout all the experiments. A forced-draft incubator maintained at 99 °F
(37-2 °C) was used. All experimental specimens were treated prior to incubation, using a slight modification of the method described by Landauer (1945):
eggs were opened after being wiped with alcohol, and the shell was cut with a
sterile saw-blade; a small rectangle (0-5 x 1 cm2) of the shell was removed. Experimental specimens were treated with 1 mg INH dissolved in 0-05 ml of 0-85 %
NaCl, injected into the yolk sac at 5 mm of the end of blastodisc just beneath
the vitelline membrane, using a sterile needle (0-5 mm external diameter).
Another group was injected using the same procedure with 1 mg INH and 1 mg
pyridoxine hydrochloride dissolved together in 0-05 ml of normal saline. Eggs
meant to be used as saline-injected controls were opened accordingly and injected with 0-05 ml of saline. The eggs were sealed with Parafilm and melted
paraffin. Unopened controls were handled but were not treated further.
After injection the eggs were incubated for 24, 48 or 72 h, and at the end of
these periods the embryos were dissected under a low-power binocular microscope, and we observed presence or absence of the embryonic heart beat. Only
those embryos exhibiting both heart beat and circulation in the vessels of the
area vasculosa were registered as living embryos and processed. After they had
been macroscopically examined and photographed under the low-power
binocular microscope, they were fixed in Bouin's fluid for histological study.
Material was double embedded in celloidin and paraffin, serially sectioned
(10 jtim) in a transverse plane, and stained with haematoxylin and eosin.
RESULTS
Quantitative data
According to data summarized in Table 1, there was no significant difference
(P > 0-05) between the percentages of survivors in the saline-injected control
group (74%) and the unopened controls (84%). The mortality in the 1NHtreated group at 2 days and 3 days of incubation (57 % and 63 % respectively)
was significantly higher (P < 0-005) than in the saline-injected group (26 %).
111
Effects of isonicotinic acid hydrazide
Table 1. Results of yolk-sac injection ofJNH, INH+vitamin BG, and saline
in fertilized White Leghorn eggs, prior to the incubation
Living embryos
Age of
the
(h)
No. of
embryos
recovered
48
72
72
72
72
67
70
72
93
76
embryos
Treatment
INH .1 mg
INH 1 mg
INH + vit. B6
Saline
Unopened
Mortality
i—
0/
/o
57
63
33
26
.16
*—,
No.
38
44
24
24
12
Total
,—*—,
% No.
43
37
67
74
84
29
26
48
69
64
No.
malformed
,—*—,
% No.
100
77
25
20
5
29
20
12
14
3
No.
normal
,—K —\
% 1vfo.
0 0
23 6
75 36
80 55
95 61
But the mortality after 2 days of incubation was 57 % and there was no increase
in the mortality after 3 days of incubation (63%; P > 0-05).
In the group injected with 1 mg INH together with 1 mg pyridoxine hydrochloride the embryonic lethality (33%) was lower (P < 0-005) than in the
INH-treated group (63 %). There was no significant difference (P > 0-05) in the
proportion of dead embryos in INH + vitamin B6-injected group (33 %) and the
saline-injected control group (26%). However, the mortality in INH + vitamin
B6-treated group (33%) was significantly higher (P < 0-025) than in the unopened group (16%).
Statistically there was no difference (P > 0-05) in the proportion of malformed embryos between the saline-injected group (20%) and the unopened
group (5%). Alterations by administering INH were produced in the chick
embryos; the percentage of abnormal embryos was 50 % after 24 h of incubation
and over 75 % after 3 days of development.
The histological study of the embryos recovered after 24 h of incubation
showed that 50 % were abnormal. The study of the gross morphology of 2-day
embryos injected with INH showed that 81 % were abnormal, but the microscopical examination demonstrated that all the embryos had histological alterations. When the gross morphology of 3-day embryos injected with INH was
studied, 54% were abnormal, but the microscopical examination showed a
77 % of malformed embryos. According to the statistical analysis by the chisquare method, the proportion of malformed embryos did not increase from the
second (100%) to the third day of development (77%) (P > 0-05). Both percentages (77 and 100%) were significantly higher than the proportion of malformed embryos in the saline-injected control group (20%) (P < 0-005).
A significantly lower proportion (P < 0-005) of the embryos from those injected simultaneously with INH and vitamin B6 were considered abnormal
(25 %) compared to the proportion of those abnormal from the group injected
with INH (77%). There was no difference (P > 0-05) between the 1NH +
14-2
212
M. A. CASTELLANO AND OTHERS
Effects of isonicotinic acid hydrazide
213
vitamin B6-injected group (25%) and the normal saline-injected group (20%)
in the proportion of malformed embryos. However, the INH + vitamin Boinjected group demonstrated a higher percentage (25 %) of malformed embryos
compared with the unopened group (5 %) (P < 0-025).
Finally, some of the INH-treated embryos, that in gross anatomy had been
considered normal, in the histological study demonstrated, in all cases, varying
degrees of developmental alterations, mainly a necrotic and degenerative
process in the neural epithelium.
Morphological data
The macroscopical and microscopical study of 24 h chick embryos treated with
1 mg INH demonstrated in the abnormal cases (50 %) the absence or the rudimentary organization of the primitive streak (Fig. 1 A), or in some cases the
irregular invagination of the mesoblast at the primitive groove.
The macroscopical examination of 2- and 3-day chicken embryos showed that
almost every INH-injected embryo had a growth and developmental retardation; they were approximately two-thirds the size of the corresponding controls. The most severe effects were found in the cranial portion of the neural
tube (Fig. 1B-E, Fig. 2G-K): some of them showed the absence of an encephalic nervous system (Fig. 1B-E) and sometimes partial absence of closure of
the neural folds (Fig. 1F). The macroscopical analysis also demonstrated the
scanty amount of circulating blood in INH-treated embryos; whereas the
vascular system did not show any remarkable anomalies.
The histological study of 2- and 3-day chick embryos showed that the most
substantial morphological changes in the abnormal cases of the INH-injected
FIGURE 1
(A) Transverse section of a 24 h chick embryo at the primitive-streak stage treated
with INH. Note a rudimentary primitive streak formed in a blastoderm which
belongs to experimental series, x 250.
(B) 48 h chick embryo treated with J mg INH. The forebrain is suppressed and
represented only by a mass of neural tissue with a tiny neural canal. Note the failure
to close of the neural tube, h, Embryonic heart, x 18.
(C) The same embryo as (B), section made at the level of the arrows in B. The forebrain is suppressed and represented by a tiny neural tube (n) located under the
primitive pharynx (ph) and surrounded by the embryonic heart (h). x 130.
(D) 48 h chick embryo treated with INH, showing disorganized brain and the
irregular outline of the neural groove at the caudal level, h, Heart, x 18.
(E) Transverse section of the embryo of D, the arrows point out the level of this
section. Note the disorganized brain, the defect of the floor (arrow) of neural
tube and numerous degenerate cells in the neural epithelium and surrounding
tissues, x 85.
(F) Transverse section through the caudal portion of the same embryo (D),
showing a disorganized neural epithelium with picnotic cells and without the
characteristic histological pattern of this epithelium, x 200.
214
M. A. CASTELLANO AND OTHERS
Effects of isonicotinic acid hydrazide
215
group were anomalies of the developing nervous system, while the other embryonic tissues only occasionally showed alterations. The most frequent
anomalies observed in the nervous system of the INH-treated embryos were:
(a) Foci of degenerate cells and necrosis affecting a variable number of cells
(Fig. 1 E, F; Fig. 2H, I, K, L; Fig. 3 M-P). This degenerative process consists
chiefly of picnosis and cariorrexis (Fig. 2, I; Fig. 3, M-P); the degenerate
cells are frequently seen desquamated and occasionally fill the lumen of the
neural tube (Fig. 2H, K, L; Fig. 3N, P). In some embryos the floor of the
neural tube showed an intense degeneration which occasionally produced the
rupture of this sector (Fig. IE, arrow). Only from time to time does the notochord participate in this degenerative process (Fig. I F ; Fig. 3O, P). In all these
cases the neural epithelium lost its characteristic histological pattern (Fig. I F ;
Fig. 3 M-P) and the cells appeared with no special orientation. Over 50 % of the
INH-treated embryos showed degeneration of the neural epithelium, while only
20% of INH + vitamin B6-injected embryos showed the same process.
(b) Failure of the neural tube closure, with persistence of the neural groove
(Fig.lD,F;Fig.2J;Fig.3P).
(c) Multiple accessory lumen or duplication of the neural tube (Fig. 3R).
(d) Asymmetric folding of the neural groove (Fig. 2H).
(e) Complete anatomical disorganization in the cephalic structures as shown
in Fig. 1C and H. In this embryo (Fig. 1B, C) the rudimentary cranial portion
of nervous system developed between the primitive pharynx and the cardiac
region.
DISCUSSION
In order to study the action of INH in early chick embryos, eggs were injected prior to incubation. Some authors, such as Carter (1964) and Gebhardt
(1968), found that the insertion of a needle in the yolk sac, prior to incubation,
causes a high mortality even in the controls. Our results indicate that the salineinjected controls developed until the third day of incubation with no significant
FIGURE 2
(G) 48 h chick embryo treated with INH, showing defects in forebrain (/). x 15.
(H, I) Transverse section through the forebrain of the same embryo as (G); note
the irregular forebrain formation, and the degenerative and necrotic process in the
higher magnification, x 130 and x 340.
(J) 48 h chick embryo treated with INH; note the neural tube open at the
posterior level (arrow) and the disorganized forebrain. x 18.
(K) Transverse section through the forebrain of the embryo of (J), showing the
intense degenerative and necrotic process of the neural epithelium and the lumen
filled with desquamated cells, x 140.
(L) Section through the embryo of (J) at the level of primitive pharynx; note the
floor of the neural tube disorganized and the cells desquamated in the lumen,
x 140.
216
M. A. CASTELLANO AND OTHERS
R
Effects of isonicotinic acid hydrazide
217
difference from the unopened controls. The different results we obtained could
be due either to the brief incubation period we used or to the differences in the
genetic background of the White Leghorn eggs used. On the other hand,
McLaughlin et ah (1963) injected thousands of eggs prior to incubation without
apparent loss in the controls.
The present study indicates that 1NH causes a high mortality in the early
chick embryos (over 50% at 2 or 3 days of incubation). The preventive effect of
pyridoxine hydrochloride suggests that this lethal action is due, at least partially,
to the antivitamin B6 effect of INH. These results agree with those developed
by Cravens (1952) and Conti & Milio (1965, 1966#, b) using vitamin B6 analogs.
Moreover, the present work indicates that INH can modify the processes of
development. The modifications produced by INH have some characteristic
features; this effect may be shown by early death or by arrested or delayed
development, and developmental abnormalities chiefly at the neural epithelium,
including the absence of cephalic structures of the nervous system. We wish to
emphasize the presence of a necrotic and degenerative process at the neural
epithelium, as well as a disorder of the normal pattern of cell distribution in this
embryonic epithelium. In the chick embryo, Duraiswami (1961) found that INH
administered on the 3rd, 4th and 5th days of development causes limb deformities, beak defects and ectopia viscerum. The disagreement between these
results and those presented in this paper could be attributed to the stage during
development of the embryo when the drug is administered, giving differential
susceptibility to INH (Murphy, 1967).
The simultaneous injection of pyridoxine hydrochloride together with the
INH prevented these developmental abnormalities, which suggests that they
are produced by a vitamin B6 deficiency.
The toxicity of hydrazines has been attributed to an acute vitamin B6 deficiency, and pyridoxal reverses this toxic effect (Cornish, 1969). A deficiency of
pyridoxine has been considered to be in the adult, the cause of the 1NH-
FIGURE 3
(M) INH-treated 48 h chick embryo. The transverse section shows the presence of
picnotic and degenerate cells, and disorganization of the neural epithelium, x 400.
(N) Transverse section through the forebrain of a 48 h INH-treated chick embryo;
note desquamated cells filling the lumen, x 340.
(O) INH-treated 48 h chick embryo; note the degenerative process affecting the
neural epithelium and the notochord. x 220.
(P) Failure of the caudal neural tube to close in a 48 h chick embryo INH-treated.
x200.
(Q) INH-treated 72 h chick embryo, showing the irregular rombencephalon formation and the desquamation to the lumen, x 85.
(R) A 72 h chick embryo treated with INH + vitamin B6; note the multiple neural
tube. x225.
218
M. A. CASTELLANO AND OTHERS
induced neuropathy. However, some of the neurological disorders produced
by INH are not prevented by pyridoxine (Lampert & Schochet, 1968; Yonezawa, Mori & Nakatani, 1969; Carlton & Kreutzberg, 1966). It has been demonstrated both in vitro and in vivo, that INH forms a hydrazone with pyridoxal
phosphate and thereby causes a competitive inhibition of several pyridoxal
enzymes (Braunstein, 1960). An additional explanation for the inhibitory
effect of INH on enzyme systems catalysed by vitamin B6 emerges from the
inhibition of pyridoxal kinase enzyme by INH, so that, while pyridoxal accumulates, there is insufficient pyridoxal phosphate to saturate the apoenzymes
(Levy, 1969). The interference by INH with the vitamin B6 enzymes produces
primarily a block in the amino acid metabolism.
Our results indicate that the normal development of the nervous system at its
early stages probably depends on a normal amino acid metabolism.
Regional differences in sensitivity to several metabolic inhibitors during
development have been described (Spratt, 1952, 1956), and particular events
of development such as brain formation, heart formation, etc., seem to be underlain by both quantitatively and qualitatively different metabolic processes. The
inhibition of amino acid metabolism by INH in our experiments indicates the
importance of this metabolism for the normal development of the nervous
system in early stages. On the contrary, the remaining embryonic structures with
no major alterations during the first days of incubation. However, the 1NHinduced disorders in the chick embryo are not totally prevented by supplemental
pyridoxine; therefore, the effect of INH cannot be interpreted purely as an
inhibitory mechanism acting on pyridoxine. Unrelated side effects of the INH
must be considered. This point needs further investigation.
Finally, we wish to emphasize the scanty amount of circulating blood in INHtreated embryos, which was interpreted as the result of a severe anaemia, and
which could probably lead to hypoxia. We cannot dismiss the fact that the
effects of INH, including the embryonic death, could be attributed to anaemia.
We attribute this anaemia to the antivitamin B6 action of INH, because it is
prevented by supplemental pyridoxine.
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(Manuscript received 25 April 1972)