the effects of isonicotinic acid hydrazide on the early chick embryo · 48 72 72 72 72 no. of...

/ . Embryol. exp. Morph. Vol. 29, 1, pp. 209-219, 1973 2 0 9

Printed in Great Britain

The effects of isonicotinic acid hydrazide on theearly chick embryo

By M. A. CASTELLANO, J. L. T 6RTORA, N. I. GERMINO,F. RAMA AND C. OHANIAN1

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 secondgroup 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 chickembryos, and also produced important developmental alterations.

3. Alterations produced by INH were primarily observed at the level of the neural epithe-lium, particularly at its cephalic portion. The most important ones were a degenerative andnecrotic process of the neural epithelium, and a distortion of the normal anatomical relationsof the cephalic structures.

4. The embryonic mortality and developmental alterations induced by INH were preventedto 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 embryosis 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 areamost sensitive to such metabolic alterations.

INTRODUCTION

It has been demonstrated that hydrazines induce a block in the metabolismof amino acids (Lewis & Izume, 1926), and that this blockade can be raised bythe 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) inhibitthe enzymic activities of the amino acid metabolism, catalysed by pyridoxal.The blockade of pyridoxal enzymes by isonicotinic acid hydrazide (INH) hasbeen reported by Yoneda, Kato & Okajuna (1952) and Yoneda & Asano (1953).INH combines with pyridoxal phosphate to form a hydrazone and therebycauses a competitive inhibition of aminopherases, decarboxilases, diamineoxidase and other pyridoxal enzymes (Braunstein, 1960).

In the chick embryo, it has been demonstrated that vitamin B6 is essential for1 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 andmethoxypyridoxine) 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 earlychick embryo during the first 4 days of development and to test the preventiveaction of pyridoxine hydrochloride.

MATERIAL AND METHODS

Fertilized White Leghorn eggs, purchased from a local hatchery, were usedthroughout all the experiments. A forced-draft incubator maintained at 99 °F(37-2 °C) was used. All experimental specimens were treated prior to incu-bation, 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 asterile saw-blade; a small rectangle (0-5 x 1 cm2) of the shell was removed. Experi-mental 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 beneaththe 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 mgpyridoxine hydrochloride dissolved together in 0-05 ml of normal saline. Eggsmeant to be used as saline-injected controls were opened accordingly and in-jected with 0-05 ml of saline. The eggs were sealed with Parafilm and meltedparaffin. 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 ofthese periods the embryos were dissected under a low-power binocular micro-scope, and we observed presence or absence of the embryonic heart beat. Onlythose embryos exhibiting both heart beat and circulation in the vessels of thearea vasculosa were registered as living embryos and processed. After they hadbeen macroscopically examined and photographed under the low-powerbinocular 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 controlgroup (74%) and the unopened controls (84%). The mortality in the 1NH-treated 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 %).

Effects of isonicotinic acid hydrazide 111

Table 1. Results of yolk-sac injection ofJNH, INH+vitamin BG, and salinein fertilized White Leghorn eggs, prior to the incubation

Treatment

INH .1 mgINH 1 mgINH + vit. B6SalineUnopened

Age ofthe

embryos(h)

4872727272

No. ofembryos

recovered

6770729376

Mortalityi—

0/

/o57633326.16

*—,No.

3844242412

Total,—*—,% No.

43 2937 2667 4874 6984 64

Living embryos

No.malformed

,—*—,% No.

100 2977 2025 1220 14

5 3

No.normal,—K

% 1

023758095

—\vfo.

06

365561

But the mortality after 2 days of incubation was 57 % and there was no increasein the mortality after 3 days of incubation (63%; P > 0-05).

In the group injected with 1 mg INH together with 1 mg pyridoxine hydro-chloride the embryonic lethality (33%) was lower (P < 0-005) than in theINH-treated group (63 %). There was no significant difference (P > 0-05) in theproportion of dead embryos in INH + vitamin B6-injected group (33 %) and thesaline-injected control group (26%). However, the mortality in INH + vitaminB6-treated group (33%) was significantly higher (P < 0-025) than in the un-opened group (16%).

Statistically there was no difference (P > 0-05) in the proportion of mal-formed embryos between the saline-injected group (20%) and the unopenedgroup (5%). Alterations by administering INH were produced in the chickembryos; the percentage of abnormal embryos was 50 % after 24 h of incubationand over 75 % after 3 days of development.

The histological study of the embryos recovered after 24 h of incubationshowed that 50 % were abnormal. The study of the gross morphology of 2-dayembryos injected with INH showed that 81 % were abnormal, but the micro-scopical examination demonstrated that all the embryos had histological altera-tions. When the gross morphology of 3-day embryos injected with INH wasstudied, 54% were abnormal, but the microscopical examination showed a77 % of malformed embryos. According to the statistical analysis by the chi-square method, the proportion of malformed embryos did not increase from thesecond (100%) to the third day of development (77%) (P > 0-05). Both per-centages (77 and 100%) were significantly higher than the proportion of mal-formed embryos in the saline-injected control group (20%) (P < 0-005).

A significantly lower proportion (P < 0-005) of the embryos from those in-jected simultaneously with INH and vitamin B6 were considered abnormal(25 %) compared to the proportion of those abnormal from the group injectedwith INH (77%). There was no difference (P > 0-05) between the 1NH +

14-2


vitamin B6-injected group (25%) and the normal saline-injected group (20%)in the proportion of malformed embryos. However, the INH + vitamin Bo-injected group demonstrated a higher percentage (25 %) of malformed embryoscompared with the unopened group (5 %) (P < 0-025).

Finally, some of the INH-treated embryos, that in gross anatomy had beenconsidered normal, in the histological study demonstrated, in all cases, varyingdegrees of developmental alterations, mainly a necrotic and degenerativeprocess in the neural epithelium.

Morphological data

The macroscopical and microscopical study of 24 h chick embryos treated with1 mg INH demonstrated in the abnormal cases (50 %) the absence or the rudi-mentary organization of the primitive streak (Fig. 1 A), or in some cases theirregular invagination of the mesoblast at the primitive groove.

The macroscopical examination of 2- and 3-day chicken embryos showed thatalmost every INH-injected embryo had a growth and developmental retar-dation; they were approximately two-thirds the size of the corresponding con-trols. The most severe effects were found in the cranial portion of the neuraltube (Fig. 1B-E, Fig. 2G-K): some of them showed the absence of an ence-phalic nervous system (Fig. 1B-E) and sometimes partial absence of closure ofthe neural folds (Fig. 1F). The macroscopical analysis also demonstrated thescanty amount of circulating blood in INH-treated embryos; whereas thevascular system did not show any remarkable anomalies.

The histological study of 2- and 3-day chick embryos showed that the mostsubstantial 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 treatedwith INH. Note a rudimentary primitive streak formed in a blastoderm whichbelongs to experimental series, x 250.(B) 48 h chick embryo treated with J mg INH. The forebrain is suppressed andrepresented only by a mass of neural tissue with a tiny neural canal. Note the failureto 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 fore-brain is suppressed and represented by a tiny neural tube (n) located under theprimitive pharynx (ph) and surrounded by the embryonic heart (h). x 130.(D) 48 h chick embryo treated with INH, showing disorganized brain and theirregular 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 thissection. Note the disorganized brain, the defect of the floor (arrow) of neuraltube and numerous degenerate cells in the neural epithelium and surroundingtissues, x 85.(F) Transverse section through the caudal portion of the same embryo (D),showing a disorganized neural epithelium with picnotic cells and without thecharacteristic histological pattern of this epithelium, x 200.


group were anomalies of the developing nervous system, while the other em-bryonic tissues only occasionally showed alterations. The most frequentanomalies 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 consistschiefly of picnosis and cariorrexis (Fig. 2, I; Fig. 3, M-P); the degeneratecells are frequently seen desquamated and occasionally fill the lumen of theneural tube (Fig. 2H, K, L; Fig. 3N, P). In some embryos the floor of theneural tube showed an intense degeneration which occasionally produced therupture of this sector (Fig. IE, arrow). Only from time to time does the noto-chord participate in this degenerative process (Fig. I F ; Fig. 3O, P). In all thesecases 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 theINH-treated embryos showed degeneration of the neural epithelium, while only20% 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 portionof nervous system developed between the primitive pharynx and the cardiacregion.

DISCUSSION

In order to study the action of INH in early chick embryos, eggs were in-jected 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 saline-injected 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); notethe irregular forebrain formation, and the degenerative and necrotic process in thehigher magnification, x 130 and x 340.(J) 48 h chick embryo treated with INH; note the neural tube open at theposterior level (arrow) and the disorganized forebrain. x 18.(K) Transverse section through the forebrain of the embryo of (J), showing theintense degenerative and necrotic process of the neural epithelium and the lumenfilled with desquamated cells, x 140.(L) Section through the embryo of (J) at the level of primitive pharynx; note thefloor of the neural tube disorganized and the cells desquamated in the lumen,x 140.


R


difference from the unopened controls. The different results we obtained couldbe due either to the brief incubation period we used or to the differences in thegenetic background of the White Leghorn eggs used. On the other hand,McLaughlin et ah (1963) injected thousands of eggs prior to incubation withoutapparent loss in the controls.

The present study indicates that 1NH causes a high mortality in the earlychick embryos (over 50% at 2 or 3 days of incubation). The preventive effect ofpyridoxine hydrochloride suggests that this lethal action is due, at least partially,to the antivitamin B6 effect of INH. These results agree with those developedby Cravens (1952) and Conti & Milio (1965, 1966#, b) using vitamin B6 analogs.

Moreover, the present work indicates that INH can modify the processes ofdevelopment. The modifications produced by INH have some characteristicfeatures; this effect may be shown by early death or by arrested or delayeddevelopment, and developmental abnormalities chiefly at the neural epithelium,including the absence of cephalic structures of the nervous system. We wish toemphasize the presence of a necrotic and degenerative process at the neuralepithelium, as well as a disorder of the normal pattern of cell distribution in thisembryonic epithelium. In the chick embryo, Duraiswami (1961) found that INHadministered on the 3rd, 4th and 5th days of development causes limb defor-mities, beak defects and ectopia viscerum. The disagreement between theseresults and those presented in this paper could be attributed to the stage duringdevelopment of the embryo when the drug is administered, giving differentialsusceptibility to INH (Murphy, 1967).

The simultaneous injection of pyridoxine hydrochloride together with theINH prevented these developmental abnormalities, which suggests that theyare produced by a vitamin B6 deficiency.

The toxicity of hydrazines has been attributed to an acute vitamin B6 defi-ciency, and pyridoxal reverses this toxic effect (Cornish, 1969). A deficiency ofpyridoxine 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 ofpicnotic 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 theneural 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 forma-tion and the desquamation to the lumen, x 85.(R) A 72 h chick embryo treated with INH + vitamin B6; note the multiple neuraltube. x225.


induced neuropathy. However, some of the neurological disorders producedby INH are not prevented by pyridoxine (Lampert & Schochet, 1968; Yone-zawa, Mori & Nakatani, 1969; Carlton & Kreutzberg, 1966). It has been demon-strated both in vitro and in vivo, that INH forms a hydrazone with pyridoxalphosphate and thereby causes a competitive inhibition of several pyridoxalenzymes (Braunstein, 1960). An additional explanation for the inhibitoryeffect of INH on enzyme systems catalysed by vitamin B6 emerges from theinhibition of pyridoxal kinase enzyme by INH, so that, while pyridoxal accumu-lates, there is insufficient pyridoxal phosphate to saturate the apoenzymes(Levy, 1969). The interference by INH with the vitamin B6 enzymes producesprimarily a block in the amino acid metabolism.

Our results indicate that the normal development of the nervous system at itsearly stages probably depends on a normal amino acid metabolism.

Regional differences in sensitivity to several metabolic inhibitors duringdevelopment have been described (Spratt, 1952, 1956), and particular eventsof development such as brain formation, heart formation, etc., seem to be under-lain by both quantitatively and qualitatively different metabolic processes. Theinhibition of amino acid metabolism by INH in our experiments indicates theimportance of this metabolism for the normal development of the nervoussystem in early stages. On the contrary, the remaining embryonic structures withno major alterations during the first days of incubation. However, the 1NH-induced disorders in the chick embryo are not totally prevented by supplementalpyridoxine; therefore, the effect of INH cannot be interpreted purely as aninhibitory mechanism acting on pyridoxine. Unrelated side effects of the INHmust be considered. This point needs further investigation.

Finally, we wish to emphasize the scanty amount of circulating blood in INH-treated embryos, which was interpreted as the result of a severe anaemia, andwhich could probably lead to hypoxia. We cannot dismiss the fact that theeffects of INH, including the embryonic death, could be attributed to anaemia.We attribute this anaemia to the antivitamin B6 action of INH, because it isprevented by supplemental pyridoxine.

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(Manuscript received 25 April 1972)

the effects of isonicotinic acid hydrazide on the early chick embryo · 48 72 72 72 72 no. of...

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