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Action of Acetazolamide on the Chick Embryo During Late Development1 KosER'ro NARBAITZ A N D LEONARD F. B ~ L A N G E R Received September 13. 1974 Department of'frlisbology mnd Embryology, University of'Otbawa, O t t u ~ ~C'anutla a, K I N 6x5
NARUAITZ. R., and B&I,ANC;ER, L. F. 1975. Action of acetazolamide on the chick embryo during late development. Can. J . Ph ysiol. Pharmacol. 53,397-402. Acet:izolamide was injected into chick embryos on the 14th or 15th day of incubation. Doses ranging between 5 and 10 mg per egg produced a retardation in the growth of long bones. 'I'he affected bones contained a normal proportion of mineral as determined by ashing and presented a normal histological picture. On the basis of these findings, it is suggested that the alterations were not due to a specific direct effect of the drug on bones. The incorporation of 13'1 by the thyroid glands of acetazolamide-injected embryos was analyzed r:idioautogr:iphically and quantitated on the same 6 pm-paraffin sections, with a thin window Geiger counter. The incorporation appeared notably reduced 3 h after the injection of acetazolamide and the reduction persisted for at least 24 h. The electron microscopical obsenration of thyroid fo'ollicularcells from similarly treated embryos showed that the cytological characteristics indicating an active protein synthesis were unmodified with respect to those found in control embryos. These results may indicate that acet-azolamide inhibits the iodination of the thyroid hormone without interfering with the synthesis of the globulin. It is suggested that the growth retardation observed in the embryos treated with acetazolamide may be secondary to the action of the drug on the thyroid @and, although this action appears to be a transitory one. NARBAIIZ, R. et BEKA N G E R , L. F. 1975. Action of acetazolamide on the chick embryo during late development. Can. J . Physiol. Pharmacol. 53,397-402. On injecte de l'acetazoiamide a des embryons de poulets au 14e ou 15e jour d'incubation. Des doses comprises entre 5 et 10 mg par ceuf produisent un retard de croissance des o s longs. Les o s touches csntiennent une proportion normale de mineraux, comme le montre le dosage des cendres, et presentent un aspect histologique normal. Sur labase de ces decouvertes, on suggere que les altkrations ne sont pas dGes B un effet specifique direct du medicament sur les os. L'inco~poriitiond'lflI par les glandes thyroi'des d'embryons injectes a l'acetazolamide est analysee radioautographiquement, et mesuree sur les mkmes sections de 6 p m d'epaisseur faites apres inclusion d a m la paraffine, avec un compteur Geiger h fenEtre itroite. L'incorpordtion est notable~nentreduite 3 h apres injection d'acetazolamide, et cette diminution persiste au moins 24 h. L'observation en microscopie electronique des cellules folliculaires thyroidiennes d'ernbryons traites cie f a ~ o nsemblable rnontre une absence de modifications cytologiques des caracteres propres 5 une synthkse proteique active, par rapport ceux trouves chez les temoins. Ces resultats indiquent que l'acetzolamide inhibe I'iodation de l'hormone thyroi'dienne, sans intervenir sur la synthese de la globuline. On suggere que le retard de croissance observe chez les embryons traitis a l'acitazolamide est secondaire a l'action du medicament sur la glande thyroide, bien que cette action semble itr-e transitoire. [Traduit par le journal]
Introductisn During the second half of its development, the chick embryo obtains from the egg shell the calcium it needs to build its skeleton (see review by Simkiss 1961). It has been suggested that the enzyme carbonic anhydrase plays an important role in the resorption of shell mineral by catalyzing the hydration of C 0 2 into carbonic acid which would, i~aturn, solubilize the shell's calcium carbonate (Simkiss 196% ; 'Supported by the Medical Research Council of Canada (MA 4845 and MT 799).
Owczarzak 1971 ) . Heckey and Owczalzak ( 1972) supplied preliminary histochemical evidence indicating that the enzyme is localized in specialized cells of the chorionic epithelium in close contact with the shell membrane. In order to explore further the role of this enzyme in mineral resorption, we conducted experiments in which the calcium content was determined in bones from chick embryos previously injected with acetazolamide, a sulfamide which inhibits specifically carbonic anhydrase (Marcn 1967). In the course of the above mentioned experiments, it was found that the drug inhibits the
398
CAN. J. PWYSIOL. PHARMACOL. VOL. 53, 1975
TABLE 1.
Mortality and body weight of 20 day embryos injected with acetarolamide on the 15th day
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Dose
Number injected Number of survivors Average weight of survivors (g)
Control
1 mg
5 mg
10 mg
15mg
20mg
40 36
12 12
12 10
40 21
12 1
12 0
32k2.9
29_+3.8*
2623.6-1
23.5L4.lt
-.
-
*Difference with controls was not statistically significant. *Differences with controls were statistically sigllificant ( p < 0.001).
growth of the embryos. Since most sulfanlides appear to affect the function of the thyroid gland in adult animals (Astwood 19701, it was suspected that the action of acetazolamide on embryonic growth might be secondary to an interference with the function of the embryonic thyroid gland. To investigate this aspect of the problem, additional experiments were conducted analyzing the rate of 1311-incorporation and the electron microscopical aspect of thyroid glands from embryos injected with acetazolamide.
Material and Methods Eggs from White Leghorn hens obtained from a comn~ercialsource were used in all our experiments. Several experimental series were conducted as detailed in Tables 1 and 2. Aeetazolamide (2-acetarnino1,3,4-thiadiazole-5 sulfonamide) dissolved in distilled water (Diamox, Ixderle C y a n m i d of Canada Ltd., Montreal, parenteral solution) was injected in the air chamber of the eggs on the 14th or 15th day of incubation. Blood samples were obtained frona a branch of the vitelline artery and the concentration of calcium in the serum was determined Wuorimetrically with calcein and ethyleneglycolbis (@-aminoethyl ether)-N,N1-tetraacetic acid (EGTA) titration in a Corning automatic calcium analyzer. The embryos were extracted from the eggs, blotted carefully with a towel and weighed after eliminating the yolk sacs. In the first series of experiments (Table I ) the embryos were fixed in alcohol at 95 "C, eviscerated, stained with alizarin, and cleared according to Karnofsky (1965). Both tibiae were dissected from a second series of experiments. The left oixes were dried overnight at 105 "C and then weighed (dry weight Table 2). They were then ashed at 650 "C for 4 h and the ashes were weighed (Table 2 ) . The right tibiae of the same embryos were fixed in a mixture containing 75 ml of 95 " C ethyl alcohol, 28 ml formalin, and 5 an1 acetic acid (A.F.A.) and embedded in paraffin. Sections were stained with phloxine - oxalic acid - methylene blue according to Bilanger and Jande ( 1974). An additional experimental series was carried out to analyze '''I-incorporation by the thyroid glands.
Thirty embryos were injected with 10 mg acetazolamide on the 14th day of incubation. They were sacrificed 3, 7, 24, 48, or 42 h after the injection, together with a similar number of uninjected controls. In both experimental and control embryos, I3lI in the form of NaI (20 @Ci per egg) was injected into the air chamber 2 h prior to sacrifice. Thyroid glands were dissected, fixed in A.F.A. for 24 h, embedded in paraffin, and sectioned at 6 ,urn. Five slides containing one section of each thyroid gland were made from each embryo. The amount of @-emission from each section was recorded with a thin window Geiger counter at a constant distalace of 2 mm. The surface of each section was calculated by projecting its image on paper of uniform weight, tracing the projected section contour, and then weighing the cut-out paper area. The radioactivity was finally expressed as counts per minute per square rnillirnetre. The sanae sections were coated then for radioautography with liquid emulsion (BTB2, Eastman Kodak & Co., Rochester, N.Y.) following a modification of the original procedure of Belanger and keblond (1944), as described by Bogoroch (1972). Thyroid glands. from six embryos injected with 10 nag acetazolamide on day 14, were dissected on day 15 together with six controls of the same age. They were fixed in half-strength Karnovsky's fixative (Karnovsky 19651, for 6 h, post-fixed for 2 h in 1 % osmium tetroxide in 8.1 A4 cacodylate b~afler at pH 7.3, dehydrated with ethyl alcohol, and embedded in araldite. Thin sections were ~tained with uranyl acetate and lead citrate according to Reynolds ( 1963 ) and examined with a Philips 300 electron microscope. The statistical significance s f differences, between the data obtained from experimental and control embryos and inclaided in Table 2, was verified through the Lase of Student's "'r" test.
Results A first series of experiments was conducted to determine the optimal dose of acetazolamide. Its results are summarized in Table 1 , shows that doses over 15 mg were lethal for most c9f the embryos and doses between 5 and 10 mg produced a significant reduction in body weight. The embryos injected with 10
NARBAI'TZ AND RGL,AVGER: ACETAZO1,AMIDE EFFECTS ON DEVE1,OPMENT
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TABLE2. Length and weight of tibiae from 20 day embryos injected on 15th day with 10 mg acetazolamide
Experimental Controls
Length (mm)
Dry weight (mg)
21.3+ 1 . 7 23.8k 0.8
42.959.2 57.5k4.1
Weight of ashes / dry weight (%)
Ashes (mg) 13.6652 8 16.91_+1.3
31.7 29.5
N o r @: ,411 figures are averages from 20 embryos. With the exception of the weight of ashes 1 drv weight rela< Q.001). tionship, all differences between expcrirnental and control groups were statistically significkt
(p
mg, together with a similar number of controls, were staiilcd with alizarine for study of their skeletons. Their examination showed that the long bones were especially affected, appearing shorter and more slender than those of controls. In the second series of experiments, embryos injected with 10 mg of acetazolamide on day 15 and sacrificed on day 20 were used. Both tibiae werc dissectcd from each embryo. Table 2 shows that the left tibiae from the injected embryos were somewhat shorter and notably lighter than those from controls. In spite of this, the weight of ashes per 100 mg of dry weight was similar in both groups. 'The histological study of the right tibiae of the same embryos failed to disclose any difference between injected and control embryos. The thickness of trabeculae and the size of vascular spaces appeared to be within normal limits (Fig. I ), although no precise quantitation was made. The presently used histological technique (Elanger and Jande 1974) allows the identification of osteoid as a lightly stained band on the surface of trabeculae. In the present experiments a significant increase in the amount of osteoid was never found in the bones of experimental embryos (Fig. I ) . TweIve embryos injected with 20 mg of acetazolamide on day 14 were sacrificed on day 15, together with a similar number of controls of the same age. The determination of the calcium content of their sera showed no significant difference between injected and control embryos (averages 9.18 mg/100 ml, experimental, and 9.23 mg/100 ml, control embryos). The incorporation of %"I, as demonstrated by radioautographs, was reduced clearly during the first day after injection of acetazolamide but not later periods. Figure 3 shows a radio-
autograph of a section of the thyroid gland from a chick embryo sacrificed 3 h after thc injection of acetazolamide. Few of the numerous follicles present (as shown in the stained section of the same material in Fig. 2 ) have incorporated iodine. Figure 4 shows the radioautograph from a section of a control embryo, which shows a striking amount of isotope incorporation in controls. The results of quantitative determinations agreed with the concIusions drawn from thc analysis of radioautographs. Thus, 24 h after acetazolamide injection the incorporation of I 3 l I by sections of the thyroid gland (average of six embryos: 15 c.p.rn./mm" was much lower than the one observed in the corresponding controls (32 c.p.m./n~m". This difference was statistically significant ( p < 0.001 ) . In the embryos sacrificed 48-72 h (both groups were pooled) after acetazolamide injection, the incorporation (average of six experimental embryos 49 c.p.rn./mm" did not differ significantly froin the one found in controls (average of six embryos 45 c.p.m./mm" Figure 3 is an electron micrograph from a section of a thyroid fixed 24 h after the injection of acetazolamide. 'The cytoplasm of the follicular cells contains a well developed Colgi complex, numerous dilated cisternae of granular endoplasmic reticulum, and few secretory granules. Although no precise quantitative approach was followed, the comparison of sections from six injected embryos with those of a similar nuraaber of controls f ailcd to disclose differences in the amount of rough endoplasmic reticulum or secretory granules, or in the degree of development of the Golgi complexes. ).
Discussion Layton and Hallesy (1965) described the
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400 CAN. 3. PHYSIOL. PHARMACOL. VOL. 53, 1975
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NAKBAHTZ AND BELANGER: ACETAZOLAMIDE EFFECTS ON DEVELOPMENT
presence of limb malformations in the offspring of rats given a diet containing a large concentration of acetazolamide (during pregnancy). Landauer and Wakasugi (1 967, 1968) showed that the drug is also teratogenic for chick embryos when administered early in develspinent. Doses between 2.5 and 10 mg injected between 24 and 120 h of incubation produced various anomalies consisting in shortening of the uppcr beak, rumplessness, bending of the tibiotarsus shaft, etc. They suggested (Landauer and Wakasugi 1967, 1968) that the malformations were not due to an inhibition of carbonic anhydrase but to interference with NAD functions. In the presently described experiments, the drug affected mainly the long bones, which were reduced both in length and weight. The mechanisms underlying these changes are not clear. Although acetazolamide is known to inhibit bone resorption in adult birds (Siegmund and Bulce 1960; Bulce and Siegmund 1960; Brubaker and Mueller 1971 ), the fact that in our experiments the shape and histological structure of the bones appeared unaltered tends to suggest that the action of acetazolamide was not a direct one on bone tissue but a more general one on whole body growth. About 80% of the calcium in bones originates from the shell mineral (Simkiss 196 1 ) and it has been suggested that carbonic anhydrase is needed for its resorption (Simkiss 1961; Bwczarzak 1971) . Since the bones of embryos injected with acetazolamide contained an amount of mineral (per dry weight) similar to the one found in controls and since their histological analysis did not disclose signs of rickets, it appears that the retardation in bone growth was not secondary to a caIcium deficit produced by inhibition of carbonic anhydrase at the choris-allantoic membrane level.
40 1
A pronounced inhibition of 1311-incorporation by the thyroid glands was observed after the injection of acetazolamide. This inhibition was very notable 24 h after the injection but disappeared later. Electron microscopical observations were made on the thyroid glands of similarly treated embryos. Special attention was paid to cytological signs of protein synthesis and no reduction in the amount of granular endoplasmic reticulum, Golgi elements, or secretion granules was detected in the follicular epithelium. These observations tend to suggest that the drug did not inhibit significantly the production of protein by the thyroid epithelial cell, although more sophisticated quantitative techniques would be needed to prove this point. If these suggestions would be confirmed, the action of acetazolamide on the thyroid gland would consist of inhibition of the iodination of the thyroid hormone without interfering with the synthesis of the globulin. A similar conclusion has been reached for thiourea and its derivatives (Astwood 1970). It has been demonstrated (Grossowicz 1946; Adams and Bull 1 949; Adams and Buss 1952; Romanoff and kaufer 1956) that the inhibition of thyroid function produced by the injection of antithyroid drugs such as thiourea and thiouracil during the second half of incubation is always accompanied by growth retardation. The growth retardation observed in the embryos treated with acetazolamide may also be secondary to the inhibition of thyroid function, although in this case the inhibition appeared to be a transitory one. The authors wish to acknowledge the skillflll collaboration of Mrs. Ckcile Btlanger and Mrs. AndrCe Boucher in the histological and radiaautograghic techniques and Mr. Vijay Kapal in the electron microscopical preparation.
FIG. 1. Cross section of a tibia from a 20 day embryo injected with acetazolarnide on day 15. Bklanger and Jande9s ( 1 974) strain, 68 X . FIG. 2. Thyroid gland from a 14 day embryo injected with acetazolarnides 3 h before sncrifice. Hernatoxylin alld Eosin stain, 130 X . FIG.3 . Thyroid gland from a 14 day ernbrays injected with acetazalarnide 3 h before sacrifice. Unstained integrated radioautograph, 130 X . FIG. 4. Thyroid gland from an uninjected control 14 day embryo sacrificed simultaneously with that of Fig. 3. Unstained integrated radioautograph, 130 X. FIG.5. Electron micrograph of part of thyroid follicle from a 15 day embryo injected with acetazolamide 24 h before sacrifice. G , Golgi complex; L, Lumen, 24 000 X.
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402
CAN. J . PHYSIOL. PHARMACOL. VOL. 53, 1995
ADAMS,.4. E., and BULL, A. IA. 1949. The effects of antithyroid drugs on chick embryos. Anat. Rcc. 104, 421-433. ADAMS,A. E., and Buss. J . M. 1952. The effect of a singJe in-jection of an antithyroid drug on hyperplasia in the thyroid of the chick embryo. Endocrinology, 50, 23.1-253. A s r w o o ~ E. , B. 1970. Thyroid and antithyroid drugs. In The pharmacological basis of therapeutics. 4th Edn. Edited& IT,.S. Guodnman and A. Gilman. TheMacmilIan Company, New York. N.Y. pp. 1466-1500. B~IAWC;ER, I,. F., and JANDE,S. S. 1974. Staining of an ?rganic fraction of bone related to initial mineralizat ~ o nAnat. . Rec. 178,307-308. (Abstr.j Bkn A N G E R . L. F., and LEBI-OND, C. P. 1946. A method for locating radioactive elements in tissues by covering histological sections with a photographic emulsion. Endocrinology, 39,8-13. ROUOROCH, K. 1972. L i q ~ ~emulsion id autoradiography. I n Atitoradiography for l?iologists. Edited by P. B. (;ahan. Acadenmic Press Inc.. London and New York. BRUB A K E R . R. L.. and M r ; ~ r a _ ~W. w , J. 8971. Blood flow. blood volume and carbonic anhydrase activity of the avian femur during bone resorption and accretion. Fed. hcrc. 30,346. (Abstr.). Dur.ce, M. J . , and SYEGMUND, P. 1960. Znr Biochemie der Knschenausflosung, II, Der Einflub von Diamox aa~fdas PHasma-Salciunm Ostron-behandelter Hahrrae. Moppe-SeyBer's %. Physiol. Chem. 320, 168-162. GKOSSOWICZ, N. 1946. Infliaence of thiourea on development of the chick embryo. Proc. Soc. Exp. Biol. Med. 63. 151-152. HECKEY,W. P . , and OWCZAKZAK, A. 1972. The chorioalBantoic membrane: histochenlistry and electron microscopy of carbonic anhydrase. 9. Cell Biol. 55, 118A. (Abs1r.j KARNOFSKY, D. A. 1965. 'The chick embryo in drug screen-
ing; survey of teratc8logical effects observed in the 4-day chick embryo. Hn Teratslogy. Principles and techniques. Edited by J . G. Wilson and J. Wtrkany . The University of Chicago Press, Chicago, 118. pp. 194-2 13. KARNCPVSKY. M. .I.1965. .4 formaldehyde-glutaraldehyde fixative of high osrnolality for use in electron microscopy. J. Cell Biol. 27,137A-B38A. I J ~W., and ~ WAKASUGI, ~ ~ PJ.~ 1%7.~ FBTc~blenis ~ of , acetazolaniide and n -ethylnicotinamide as teratogens. 9. Exp. Zool. 164,499-516. 1968. Terntological studies with sulphonamides and their implications. J. Embryo!. Exp. blorphol. 20: 261-284. I,AYTON,W. hi.,JR.. and HAEY.ESY, 11. W. 1965. Deformity of forelimb in rats: association with high doses of acetazolamide. Science, 149, 306-308. MAKEN,T . H. 1967. Carbonic anhydrdse: chemistry, physiology and inhibition. Physiol. Rev. 47, 595-781. OWCZAWZAK, A. 1978. Calcium-absorbing cell of the chick chorioallantc9ic mernisr:ine. I. Morphology. distribution and cellular interactions. Exp. Cell Res. 68. 113-129. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. 9. Cell B i d . 17,208-212 Wtaw\~orls,A. I,., and I,AUFI:K,H. 1956. The effect of injected thiourea on the development of some organs of the chick embryo. Endocrinc~logy,59,611-619. SIEGMUND, P., ;ind DUI~CE, H. J. 1960. Zrsr Biochemie der Knochenauflosung, I. Einflub des CarboanydrataseInhibitors 2-Acetamino-1.3.4-thiodiazol-sulfonamid65) (Diamox) auf den Calciumstoff~vechsd vom Legehenne. Moppe-Seyler's Z. Physiol. Chem. 320, 149-159. SIMKISS,K. 1961. Calcium metabolism and avian reproduction. Biol. Rev. 36, 321-367.
Action of Acetazolamide on the Chick Embryo During Late Development1 KosER'ro NARBAITZ A N D LEONARD F. B ~ L A N G E R Received September 13. 1974 Department of'frlisbology mnd Embryology, University of'Otbawa, O t t u ~ ~C'anutla a, K I N 6x5
NARUAITZ. R., and B&I,ANC;ER, L. F. 1975. Action of acetazolamide on the chick embryo during late development. Can. J . Ph ysiol. Pharmacol. 53,397-402. Acet:izolamide was injected into chick embryos on the 14th or 15th day of incubation. Doses ranging between 5 and 10 mg per egg produced a retardation in the growth of long bones. 'I'he affected bones contained a normal proportion of mineral as determined by ashing and presented a normal histological picture. On the basis of these findings, it is suggested that the alterations were not due to a specific direct effect of the drug on bones. The incorporation of 13'1 by the thyroid glands of acetazolamide-injected embryos was analyzed r:idioautogr:iphically and quantitated on the same 6 pm-paraffin sections, with a thin window Geiger counter. The incorporation appeared notably reduced 3 h after the injection of acetazolamide and the reduction persisted for at least 24 h. The electron microscopical obsenration of thyroid fo'ollicularcells from similarly treated embryos showed that the cytological characteristics indicating an active protein synthesis were unmodified with respect to those found in control embryos. These results may indicate that acet-azolamide inhibits the iodination of the thyroid hormone without interfering with the synthesis of the globulin. It is suggested that the growth retardation observed in the embryos treated with acetazolamide may be secondary to the action of the drug on the thyroid @and, although this action appears to be a transitory one. NARBAIIZ, R. et BEKA N G E R , L. F. 1975. Action of acetazolamide on the chick embryo during late development. Can. J . Physiol. Pharmacol. 53,397-402. On injecte de l'acetazoiamide a des embryons de poulets au 14e ou 15e jour d'incubation. Des doses comprises entre 5 et 10 mg par ceuf produisent un retard de croissance des o s longs. Les o s touches csntiennent une proportion normale de mineraux, comme le montre le dosage des cendres, et presentent un aspect histologique normal. Sur labase de ces decouvertes, on suggere que les altkrations ne sont pas dGes B un effet specifique direct du medicament sur les os. L'inco~poriitiond'lflI par les glandes thyroi'des d'embryons injectes a l'acetazolamide est analysee radioautographiquement, et mesuree sur les mkmes sections de 6 p m d'epaisseur faites apres inclusion d a m la paraffine, avec un compteur Geiger h fenEtre itroite. L'incorpordtion est notable~nentreduite 3 h apres injection d'acetazolamide, et cette diminution persiste au moins 24 h. L'observation en microscopie electronique des cellules folliculaires thyroidiennes d'ernbryons traites cie f a ~ o nsemblable rnontre une absence de modifications cytologiques des caracteres propres 5 une synthkse proteique active, par rapport ceux trouves chez les temoins. Ces resultats indiquent que l'acetzolamide inhibe I'iodation de l'hormone thyroi'dienne, sans intervenir sur la synthese de la globuline. On suggere que le retard de croissance observe chez les embryons traitis a l'acitazolamide est secondaire a l'action du medicament sur la glande thyroide, bien que cette action semble itr-e transitoire. [Traduit par le journal]
Introductisn During the second half of its development, the chick embryo obtains from the egg shell the calcium it needs to build its skeleton (see review by Simkiss 1961). It has been suggested that the enzyme carbonic anhydrase plays an important role in the resorption of shell mineral by catalyzing the hydration of C 0 2 into carbonic acid which would, i~aturn, solubilize the shell's calcium carbonate (Simkiss 196% ; 'Supported by the Medical Research Council of Canada (MA 4845 and MT 799).
Owczarzak 1971 ) . Heckey and Owczalzak ( 1972) supplied preliminary histochemical evidence indicating that the enzyme is localized in specialized cells of the chorionic epithelium in close contact with the shell membrane. In order to explore further the role of this enzyme in mineral resorption, we conducted experiments in which the calcium content was determined in bones from chick embryos previously injected with acetazolamide, a sulfamide which inhibits specifically carbonic anhydrase (Marcn 1967). In the course of the above mentioned experiments, it was found that the drug inhibits the
398
CAN. J. PWYSIOL. PHARMACOL. VOL. 53, 1975
TABLE 1.
Mortality and body weight of 20 day embryos injected with acetarolamide on the 15th day
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
Dose
Number injected Number of survivors Average weight of survivors (g)
Control
1 mg
5 mg
10 mg
15mg
20mg
40 36
12 12
12 10
40 21
12 1
12 0
32k2.9
29_+3.8*
2623.6-1
23.5L4.lt
-.
-
*Difference with controls was not statistically significant. *Differences with controls were statistically sigllificant ( p < 0.001).
growth of the embryos. Since most sulfanlides appear to affect the function of the thyroid gland in adult animals (Astwood 19701, it was suspected that the action of acetazolamide on embryonic growth might be secondary to an interference with the function of the embryonic thyroid gland. To investigate this aspect of the problem, additional experiments were conducted analyzing the rate of 1311-incorporation and the electron microscopical aspect of thyroid glands from embryos injected with acetazolamide.
Material and Methods Eggs from White Leghorn hens obtained from a comn~ercialsource were used in all our experiments. Several experimental series were conducted as detailed in Tables 1 and 2. Aeetazolamide (2-acetarnino1,3,4-thiadiazole-5 sulfonamide) dissolved in distilled water (Diamox, Ixderle C y a n m i d of Canada Ltd., Montreal, parenteral solution) was injected in the air chamber of the eggs on the 14th or 15th day of incubation. Blood samples were obtained frona a branch of the vitelline artery and the concentration of calcium in the serum was determined Wuorimetrically with calcein and ethyleneglycolbis (@-aminoethyl ether)-N,N1-tetraacetic acid (EGTA) titration in a Corning automatic calcium analyzer. The embryos were extracted from the eggs, blotted carefully with a towel and weighed after eliminating the yolk sacs. In the first series of experiments (Table I ) the embryos were fixed in alcohol at 95 "C, eviscerated, stained with alizarin, and cleared according to Karnofsky (1965). Both tibiae were dissected from a second series of experiments. The left oixes were dried overnight at 105 "C and then weighed (dry weight Table 2). They were then ashed at 650 "C for 4 h and the ashes were weighed (Table 2 ) . The right tibiae of the same embryos were fixed in a mixture containing 75 ml of 95 " C ethyl alcohol, 28 ml formalin, and 5 an1 acetic acid (A.F.A.) and embedded in paraffin. Sections were stained with phloxine - oxalic acid - methylene blue according to Bilanger and Jande ( 1974). An additional experimental series was carried out to analyze '''I-incorporation by the thyroid glands.
Thirty embryos were injected with 10 mg acetazolamide on the 14th day of incubation. They were sacrificed 3, 7, 24, 48, or 42 h after the injection, together with a similar number of uninjected controls. In both experimental and control embryos, I3lI in the form of NaI (20 @Ci per egg) was injected into the air chamber 2 h prior to sacrifice. Thyroid glands were dissected, fixed in A.F.A. for 24 h, embedded in paraffin, and sectioned at 6 ,urn. Five slides containing one section of each thyroid gland were made from each embryo. The amount of @-emission from each section was recorded with a thin window Geiger counter at a constant distalace of 2 mm. The surface of each section was calculated by projecting its image on paper of uniform weight, tracing the projected section contour, and then weighing the cut-out paper area. The radioactivity was finally expressed as counts per minute per square rnillirnetre. The sanae sections were coated then for radioautography with liquid emulsion (BTB2, Eastman Kodak & Co., Rochester, N.Y.) following a modification of the original procedure of Belanger and keblond (1944), as described by Bogoroch (1972). Thyroid glands. from six embryos injected with 10 nag acetazolamide on day 14, were dissected on day 15 together with six controls of the same age. They were fixed in half-strength Karnovsky's fixative (Karnovsky 19651, for 6 h, post-fixed for 2 h in 1 % osmium tetroxide in 8.1 A4 cacodylate b~afler at pH 7.3, dehydrated with ethyl alcohol, and embedded in araldite. Thin sections were ~tained with uranyl acetate and lead citrate according to Reynolds ( 1963 ) and examined with a Philips 300 electron microscope. The statistical significance s f differences, between the data obtained from experimental and control embryos and inclaided in Table 2, was verified through the Lase of Student's "'r" test.
Results A first series of experiments was conducted to determine the optimal dose of acetazolamide. Its results are summarized in Table 1 , shows that doses over 15 mg were lethal for most c9f the embryos and doses between 5 and 10 mg produced a significant reduction in body weight. The embryos injected with 10
NARBAI'TZ AND RGL,AVGER: ACETAZO1,AMIDE EFFECTS ON DEVE1,OPMENT
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TABLE2. Length and weight of tibiae from 20 day embryos injected on 15th day with 10 mg acetazolamide
Experimental Controls
Length (mm)
Dry weight (mg)
21.3+ 1 . 7 23.8k 0.8
42.959.2 57.5k4.1
Weight of ashes / dry weight (%)
Ashes (mg) 13.6652 8 16.91_+1.3
31.7 29.5
N o r @: ,411 figures are averages from 20 embryos. With the exception of the weight of ashes 1 drv weight rela< Q.001). tionship, all differences between expcrirnental and control groups were statistically significkt
(p
mg, together with a similar number of controls, were staiilcd with alizarine for study of their skeletons. Their examination showed that the long bones were especially affected, appearing shorter and more slender than those of controls. In the second series of experiments, embryos injected with 10 mg of acetazolamide on day 15 and sacrificed on day 20 were used. Both tibiae werc dissectcd from each embryo. Table 2 shows that the left tibiae from the injected embryos were somewhat shorter and notably lighter than those from controls. In spite of this, the weight of ashes per 100 mg of dry weight was similar in both groups. 'The histological study of the right tibiae of the same embryos failed to disclose any difference between injected and control embryos. The thickness of trabeculae and the size of vascular spaces appeared to be within normal limits (Fig. I ), although no precise quantitation was made. The presently used histological technique (Elanger and Jande 1974) allows the identification of osteoid as a lightly stained band on the surface of trabeculae. In the present experiments a significant increase in the amount of osteoid was never found in the bones of experimental embryos (Fig. I ) . TweIve embryos injected with 20 mg of acetazolamide on day 14 were sacrificed on day 15, together with a similar number of controls of the same age. The determination of the calcium content of their sera showed no significant difference between injected and control embryos (averages 9.18 mg/100 ml, experimental, and 9.23 mg/100 ml, control embryos). The incorporation of %"I, as demonstrated by radioautographs, was reduced clearly during the first day after injection of acetazolamide but not later periods. Figure 3 shows a radio-
autograph of a section of the thyroid gland from a chick embryo sacrificed 3 h after thc injection of acetazolamide. Few of the numerous follicles present (as shown in the stained section of the same material in Fig. 2 ) have incorporated iodine. Figure 4 shows the radioautograph from a section of a control embryo, which shows a striking amount of isotope incorporation in controls. The results of quantitative determinations agreed with the concIusions drawn from thc analysis of radioautographs. Thus, 24 h after acetazolamide injection the incorporation of I 3 l I by sections of the thyroid gland (average of six embryos: 15 c.p.rn./mm" was much lower than the one observed in the corresponding controls (32 c.p.m./n~m". This difference was statistically significant ( p < 0.001 ) . In the embryos sacrificed 48-72 h (both groups were pooled) after acetazolamide injection, the incorporation (average of six experimental embryos 49 c.p.rn./mm" did not differ significantly froin the one found in controls (average of six embryos 45 c.p.m./mm" Figure 3 is an electron micrograph from a section of a thyroid fixed 24 h after the injection of acetazolamide. 'The cytoplasm of the follicular cells contains a well developed Colgi complex, numerous dilated cisternae of granular endoplasmic reticulum, and few secretory granules. Although no precise quantitative approach was followed, the comparison of sections from six injected embryos with those of a similar nuraaber of controls f ailcd to disclose differences in the amount of rough endoplasmic reticulum or secretory granules, or in the degree of development of the Golgi complexes. ).
Discussion Layton and Hallesy (1965) described the
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400 CAN. 3. PHYSIOL. PHARMACOL. VOL. 53, 1975
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NAKBAHTZ AND BELANGER: ACETAZOLAMIDE EFFECTS ON DEVELOPMENT
presence of limb malformations in the offspring of rats given a diet containing a large concentration of acetazolamide (during pregnancy). Landauer and Wakasugi (1 967, 1968) showed that the drug is also teratogenic for chick embryos when administered early in develspinent. Doses between 2.5 and 10 mg injected between 24 and 120 h of incubation produced various anomalies consisting in shortening of the uppcr beak, rumplessness, bending of the tibiotarsus shaft, etc. They suggested (Landauer and Wakasugi 1967, 1968) that the malformations were not due to an inhibition of carbonic anhydrase but to interference with NAD functions. In the presently described experiments, the drug affected mainly the long bones, which were reduced both in length and weight. The mechanisms underlying these changes are not clear. Although acetazolamide is known to inhibit bone resorption in adult birds (Siegmund and Bulce 1960; Bulce and Siegmund 1960; Brubaker and Mueller 1971 ), the fact that in our experiments the shape and histological structure of the bones appeared unaltered tends to suggest that the action of acetazolamide was not a direct one on bone tissue but a more general one on whole body growth. About 80% of the calcium in bones originates from the shell mineral (Simkiss 196 1 ) and it has been suggested that carbonic anhydrase is needed for its resorption (Simkiss 1961; Bwczarzak 1971) . Since the bones of embryos injected with acetazolamide contained an amount of mineral (per dry weight) similar to the one found in controls and since their histological analysis did not disclose signs of rickets, it appears that the retardation in bone growth was not secondary to a caIcium deficit produced by inhibition of carbonic anhydrase at the choris-allantoic membrane level.
40 1
A pronounced inhibition of 1311-incorporation by the thyroid glands was observed after the injection of acetazolamide. This inhibition was very notable 24 h after the injection but disappeared later. Electron microscopical observations were made on the thyroid glands of similarly treated embryos. Special attention was paid to cytological signs of protein synthesis and no reduction in the amount of granular endoplasmic reticulum, Golgi elements, or secretion granules was detected in the follicular epithelium. These observations tend to suggest that the drug did not inhibit significantly the production of protein by the thyroid epithelial cell, although more sophisticated quantitative techniques would be needed to prove this point. If these suggestions would be confirmed, the action of acetazolamide on the thyroid gland would consist of inhibition of the iodination of the thyroid hormone without interfering with the synthesis of the globulin. A similar conclusion has been reached for thiourea and its derivatives (Astwood 1970). It has been demonstrated (Grossowicz 1946; Adams and Bull 1 949; Adams and Buss 1952; Romanoff and kaufer 1956) that the inhibition of thyroid function produced by the injection of antithyroid drugs such as thiourea and thiouracil during the second half of incubation is always accompanied by growth retardation. The growth retardation observed in the embryos treated with acetazolamide may also be secondary to the inhibition of thyroid function, although in this case the inhibition appeared to be a transitory one. The authors wish to acknowledge the skillflll collaboration of Mrs. Ckcile Btlanger and Mrs. AndrCe Boucher in the histological and radiaautograghic techniques and Mr. Vijay Kapal in the electron microscopical preparation.
FIG. 1. Cross section of a tibia from a 20 day embryo injected with acetazolarnide on day 15. Bklanger and Jande9s ( 1 974) strain, 68 X . FIG. 2. Thyroid gland from a 14 day embryo injected with acetazolarnides 3 h before sncrifice. Hernatoxylin alld Eosin stain, 130 X . FIG.3 . Thyroid gland from a 14 day ernbrays injected with acetazalarnide 3 h before sacrifice. Unstained integrated radioautograph, 130 X . FIG. 4. Thyroid gland from an uninjected control 14 day embryo sacrificed simultaneously with that of Fig. 3. Unstained integrated radioautograph, 130 X. FIG.5. Electron micrograph of part of thyroid follicle from a 15 day embryo injected with acetazolamide 24 h before sacrifice. G , Golgi complex; L, Lumen, 24 000 X.
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CAN. J . PHYSIOL. PHARMACOL. VOL. 53, 1995
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ing; survey of teratc8logical effects observed in the 4-day chick embryo. Hn Teratslogy. Principles and techniques. Edited by J . G. Wilson and J. Wtrkany . The University of Chicago Press, Chicago, 118. pp. 194-2 13. KARNCPVSKY. M. .I.1965. .4 formaldehyde-glutaraldehyde fixative of high osrnolality for use in electron microscopy. J. Cell Biol. 27,137A-B38A. I J ~W., and ~ WAKASUGI, ~ ~ PJ.~ 1%7.~ FBTc~blenis ~ of , acetazolaniide and n -ethylnicotinamide as teratogens. 9. Exp. Zool. 164,499-516. 1968. Terntological studies with sulphonamides and their implications. J. Embryo!. Exp. blorphol. 20: 261-284. I,AYTON,W. hi.,JR.. and HAEY.ESY, 11. W. 1965. Deformity of forelimb in rats: association with high doses of acetazolamide. Science, 149, 306-308. MAKEN,T . H. 1967. Carbonic anhydrdse: chemistry, physiology and inhibition. Physiol. Rev. 47, 595-781. OWCZAWZAK, A. 1978. Calcium-absorbing cell of the chick chorioallantc9ic mernisr:ine. I. Morphology. distribution and cellular interactions. Exp. Cell Res. 68. 113-129. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. 9. Cell B i d . 17,208-212 Wtaw\~orls,A. I,., and I,AUFI:K,H. 1956. The effect of injected thiourea on the development of some organs of the chick embryo. Endocrinc~logy,59,611-619. SIEGMUND, P., ;ind DUI~CE, H. J. 1960. Zrsr Biochemie der Knochenauflosung, I. Einflub des CarboanydrataseInhibitors 2-Acetamino-1.3.4-thiodiazol-sulfonamid65) (Diamox) auf den Calciumstoff~vechsd vom Legehenne. Moppe-Seyler's Z. Physiol. Chem. 320, 149-159. SIMKISS,K. 1961. Calcium metabolism and avian reproduction. Biol. Rev. 36, 321-367.
