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The effect of various concentrations of thiourea on the synthesis of thyroxine by the starry flounder,… Kinnear, John Edward 1959

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THE EFFECT OP VARIOUS CONCENTRATIONS OP THIOUREA ON THE SYNTHESIS OP THYROXINE BY THE STARRY FLOUNDER, PLATICHTHYS STELLATUS JOHN EDWARD KINNEAR B.Sc, University of Alberta, 1956 A THESIS SUBMITTED IN PARTIiUj FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We -accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA September, 1959 i ABSTRACT _ k The e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f t h i o u r e a on t h e p r o d u c t i o n o f t h y r o i d hormones was i n v e s t i g a t e d i n t h e s t a r r y f l o u n d e r , P l a t i c h t h y s s t e l l a t u s . . F l o u n d e r were immersed i n s e a w a t e r s o l u t i o n s o f t h i o u r e a i n c o n c e n t r a t i o n s o f . 0 . 0 0 0 1 $ , 0.0005$, 0.001$ and 0.0025$ f o r p e r i o d s o f 10 - 11 d a y s and i n c o n c e n t r a t i o n s 0.005$, 0.01$, 0.02$ and 0.03$ f o r as l o n g as 74 d a y s . A f t e r i n j e c t i o n s o f t r a c e r d o s e s o f r a d i o i o d i n e , e x t r a c t s o f t h e t h y r o i d g l a n d s were s u b j e c t e d t o a u t o r a d i o c h r o m a t o g r a p h y . I t was e s t a b l i s h e d t h a t c o n c e n t r a t i o n s o f t h i o u r e a r a n g i n g f r o m 0.0025$ - 0.03$ c o m p l e t e l y i n h i b i t e d t h e s y n t h e s i s o f the t h y r o i d hormones t h r o u g h o u t t h e c o u r s e o f t h e e x p e r i m e n t s . A l t h o u g h t h e s y n t h e s i s o f t h e t h y r o i d hormones was a b o l i s h e d i n f l o u n d e r e x p o s e d t o 0.0025$ and h i g h e r c o n c e n t r a t i o n s o f t h i o u r e a , f l o u n d e r i n some i n s t a n c e s were a b l e t o s y n t h e s i z e m o n i o d o t y r o s -i n e and d i i o d o t y r o s i n e . The s i g n i f i c a n c e o f t h i s phenomenon i s d i s c u s s e d i n c o n n e c t i o n w i t h b i o s y n t h e s i s o f t h e t h y r o i d hormones and t h e mechanism o f a n t i t h y r o i d a c t i v i t y o f t h i o u r e a . Measurement o f the p e r c e n t a g e u p t a k e o f t h e i n j e c t e d d o se o f r a d i o i o d i n e r e v e a l e d t h a t i n h i b i t e d f l o u n d e r c o n s i s t e n t l y h a d l o w e r v a l u e s i n t h e t h y r o i d when compared t o c o n t r o l s . In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia, Vancouver Canada. Department of. i i TABLE OP CONTENTS I INTRODUCTION A. Historical 1 B. Nature of Antithyroid Compounds 2 1. "Interfering ions" affecting the iodide concentrating mechanism 2 2. Antithyroid compounds preventing organic synthesi s 2 a. The thiocarhamides 2 b. The aromatic antithyroid compounds 3 C. Mechanism of Action of Antithyroid Compounds .... 3 D. ' Effectiveness of Antithyroid Compounds 5 E. Toxicity and Side Effects 5 II MATERIALS AND METHODS A. Experimental Animal 8 B. Collection and Care of Pish 8 C. Treatment by Thiourea 9 131 D. Injection with Radioactive I ' 9 E. Determination of Percent Dose i n Thyroid 10 P. Digestion and Extraction Procedures for Labelled l!31 Compounds i n Thyroid 10 1. Digestion 10 2. Extraction 11 3. Serum Extraction 11 G. Chromatography ..' 12 H. Autoradiography 12 I. Identification of Radioactive Compounds Detected by Autoradiochromatography 13 J. Rf Values of I 1 5 1 Compounds 14 i i i III RESULTS A. Inhibiting Concentrations of Thiourea 15 B. Percent Dose in the Thyroid 15 C. Serum Results 17 IV DISCUSSION A. Effects of Inhibiting Concentrations of Thiourea on Percent Dose in the Thyroid 29 B. Completeness of Inhibition 31 C. Escapement 33 D. Dosage and Toxicity 34 V CONCLUSIONS 37 VI APPENDIX 38 VII LITERATURE CITED 61 i v L I S T OP FIGURES 1. Average percent dose i n thyroid for groups of starry flounder exposed to 0.03$ thiourea 23 2. Average percent dose i n thyroid for groups of starry flounder exposed to 0.02% thiourea .. 24 3. Average percent dose i n thyroid for groups of starry flounder exposed to 0.01% thiourea 25 4* Average percent dose in thyroid for groups of starry flounder exposed to 0.005% thiourea 26 5. Average percent in the thyroid for groups of starry flounder exposed to 0.0025% and 0.001% thiourea . 27 6. Average percent in the thyroid for groups of starry flounder exposed to 0.0005% and 0.0001% thiourea . 28 7. An i l l u s t r a t i o n of an autoradiogram from a group of starry flounder exposed to 0.03% thiourea 28a V L I S T OF TABLES I A u t o r a d i o c h r o m a t o g r a p h i c r e s u l t s f r o m g r o u p s o f s t a r r y f l o u n d e r e x p o s e d t o 0.J03$ t h i o u r e a 19 I I A u t o r a d i o c h r o m a t o g r a p h i c r e s u l t s f r o m g r o u p s o f s t a r r y f l o u n d e r e x p o s e d t o 0.02$ t h i o u r e a 19 I I I A u t o r a d i o c h r o m a t o g r a p h i c r e s u l t s f r o m g r o u p s o f s t a r r y f l o u n d e r e x p o s e d t o 0.01$ t h i o u r e a 20 I V A u t o r a d i o c h r o m a t o g r a p h i c r e s u l t s f r o m g r o u p s o f s t a r r y f l o u n d e r e x p o s e d t o 0.3005$ t h i o u r e a 20 V A u t o r a d i o c h r o m a t o g r a p h i c r e s u l t s f r o m g r o u p s o f s t a r r y f l o u n d e r e x p o s e d t o 0.'0025$ t h i o u r e a 21 V I A u t o r a d i o c h r o m a t o g r a p h i c r e s u l t s f r o m g r o u p s o f s t a r r y f l o u n d e r e x p o s e d t o 0.001$ t h i o u r e a 21 V I I A u t o r a d i o c h r o m a t o g r a p h i c r e s u l t s f r o m g r o u p s o f s t a r r y f l o u n d e r e x p o s e d t o 0.0005$ t h i o u r e a 22 V I I I A u t o r a d i o c h r o m a t o g r a p h i c r e s u l t s f r o m g r o u p s o f s t a r r y f l o u n d e r e x p o s e d t o 0.0001$ t h i o u r e a 22 v i ACKNOWLEDGEMENTS The w r i t e r i s i n d e b t e d t o D r . ¥ . S . H o a r , D e p a r t m e n t o f Z o o l o g y , who s u g g e s t e d t h e p r o b l e m and u n d e r whose g u i d a n c e t h i s s t u d y was made. H i s v a l u a b l e c r i t i c i s m o f the m a n u s c r i p t i s a p p r e c i a t e d . I am d e e p l y g r a t e f u l t o D r . C P . Hickman, D e p a r t m e n t o f Z o o l o g y , U n i v e r s i t y o f A l b e r t a , f o r h i s i n t e r e s t , a d v i c e and h e l p o f f e r e d u n s e l f i s h l y d u r i n g t h e i n i t i a l s t a g e s o f t h i s work. The a d v i c e o f D r . W.N. Holmes i s a c k n o w l e d g e d . To M i s s E . B a r r e t t o f t h e H e a l t h S e r v i c e , Wesbrook B u i l d i n g , U n i v e r s i t y o f B r i t i s h C o l u m b i a , I am e s p e c i a l l y g r a t e f u l . Her a d v i c e and p r o v i s i o n o f t h e d a r k room f a c i l i t i e s f o r t h e d e v e l o p m e n t o f X - r a y f i l m s i s d e e p l y a p p r e c i a t e d . The V a n c o u v e r P u b l i c A q u a r i u m k i n d l y p r o v i d e d f a c i l i t i e s f o r c o l l e c t i o n and c a r e o f f i s h . The c o - o p e r a t i o n o f Mr. M. Newman, C u r a t o r , Mr. G. D i b b l e , A q u a r i u m B i o l o g i s t , and Mr. B. Duncan i s a c k n o w l e d g e d . Mr. J . B a u e r , o f S t e v e s t o n , was most h e l p f u l i n a i d i n g t h e w r i t e r i n the c o l l e c t i o n o f f i s h . To t h o s e s t u d e n t s who so o f t e n a s s i s t e d me c o l l e c t i n g f i s h , I w i s h t o e x t e n d my t h a n k s . I am i n d e b t e d t o Mr. J . G l a s s , who u n s e l f i s h l y a s s i s t e d t h e w r i t e r many t i m e s d u r i n g t h e c o u r s e o f t h i s s t u d y . F i n a n c i a l s u p p o r t f o r t h i s r e s e a r c h was r e c e i v e d f r o m t h e N a t i o n a l R e s e a r c h C o u n c i l o f Canada t h r o u g h g r a n t s - i n - a i d o f r e s e a r c h t o W.S. H o a r . 1 INTRODUCTION Chester Jones (1957) writes - "The f i r s t step i n exploring the function of a gland i s to remove i t experimentally and observe the consequences." Such a logical approach i s denied the physiologist studying the function of the thyroid i n teleost fishes. The thyroid i n most boney fishes i s a diffuse, non-incapsulated gland scattered about the ventral aorta (Gudernatsch, 1911). Removal by surgical means i s impossible. As a consequence, other means of achieving thyroidectomy have been employed and among them i s the use of several different antithyroid compounds. A. HISTORICAL The discovery that certain compounds possess antithyroid activity was fortuitous. Plants of the genus Brassica (turnip, cauliflower, cabbage, brussel sprouts) when fed continuously to animals caused goiter (Goodman and Gillman, 1956). Efforts were then made to isolate the antithyroid agents from these foods, and soon cyanogens and substances related to thiourea became implicated (Charipper and Gordon, 1947). Mackenzie _et a l . (1941) extended the l i s t of known antithyroid agents when i t was observed that sulfaguanidine caused thyroid enlargement. Other sulfa drugs were also shown to be powerful goitrogens by the same workers. Cl i n i c a l applications of antithyroid compounds for the control of hyperthyroidism became widespread, and the search for more effective and less toxic agents con-tributed to the l i s t of known goitrogens. Today the number of known antithyroid compounds i s in the hundreds. 2' B. NATURE OF ANTITHYROID COMPOUNDS 1. "Interfering Ions" Affecting the Iodide Concentrating Mechanism The term antithyroid compound (goitrogen, antithyroid drug, and antithyroid agent are synonyms) i s usually restricted to those compounds which directly interfere with the synthesis of the thyroid hormones (Astwood, 1955). There are other agents however, which interfere with the production of the thyroid hormones, but do so i n an indirect manner. Certain anions — thiocyanate, perchlorate, nitrate, iodide and others -affect the iodide concentrating mechanism i n some unknown way. The net result i s that the thyroid becomes deficient i n iodine, an essential component of the thyroid hormones, and production of the hormones i s inhibited. The anions are only effective when the dietary intake of iodine i s low and being relatively toxic, are seldom used as inhibiting agents (Charipper and Gordon, 1947). 2. Antithyroid Compounds Preventing Organic Synthesis (a) The Thiocarbamides The thiocarbamides i.e. thiourea and i t s derivatives,, constitute the largest group of known antithyroid agents (Rawson e_t a l . ) . Members of this group are phenyl thiourea, allythiourea,thiouracil, methyl thiouracil and propylthiouracil. They owe their antithyroid activity to the sulfur or thio group, for without sulfur, the activity disappears. The thio-carbamides are strong reducing agents and this property has 3 been linked with their antithyroid activity (Astwood, 1955). The straight-chained derivatives of thiourea are less active than the cyclic derivatives. Incorporation of thiourea into a cyclic six membered ring may increase i t s activity as i n thiouracil (2-thio-4-oxypyrimidine) which i s 10 times more effective than thiourea in the rat (Charipper and Gordon, 1947). (b) The Aromatic Antithyroid Compounds. The aromatic antithyroid compounds as a group are less potent goitrogens than the thiocarbamides. Many are deriva-tives of aniline and some are extremely complex molecules. Examples of this group are p-aminobenzoic acid, the sulfa drugs, resorcinol and aminothiazole. Numerous polyphenols have been shown to be active particularly when metasubstitution on the benzene ring i s achieved. Phloroglucinol i s one of the more active polyphenols (Astwood, 1955). Apparently, polar groups of more than one kind can confer antithyroid activity to the benzene nucleus. C. MECHANISM OF ACTION OF ANTITHYROID COMPOUNDS The biosynthesis of the thyroid hormones, triiodothyronine and thyroxine, has been discussed in an extensive review by Roche and Michel (1955). The process involves several distinct steps, the f i r s t being the accumulation of iodide ions by the iodide concentrating mechanism. Iodide ions are oxidized to elemental iodine and then combine with the amino acid tyrosine to form moniodotyrosine. A second atom of iodine combines with 4 moniodotyrosine to form diiodotyrosine, following which, two > molecules of diiodotyrosine "couple" or join together to form thyroxine (tetraiodothyronine). Triiodothyronine i s most li k e l y formed by the coupling of moniodotyrosine and diiodo-tyrosine. The precise mechanism of 4iow antithyroid compounds interfere with the synthesis of the thyroid hormones i s not f u l l y understood. Antithyroid drugs do not affect the trapping of iodide, but in some way interfere with the iodination of tyrosine. According to Astwood (1955), a l l effective anti-thyroid agents interfere with peroxidase reactions, the enzyme which i s thought to be responsible for the oxidation of iodide to iodine. The thiocarbamides could serve as a substrate for the enzyme and thus compete with iodide. It has also been suggested that thiourea and i t s derivatives could reduce iodine to iodide as rapidly as i t i s formed. Many of the aromatic antithyroid agents are thought to combine with iodine as i t i s formed, forming an iodine complex. Recent work with rats, by Slingerland e_fc a l . (1959), and Richards and Ingbar (1959), have revealed that the second iodination reaction (i.e. conversion of moniodotyrosine to diiodotyrosine), i s more sensitive to the inhibiting action of propylthiouracil, than the iodination of tyrosine to form moniodotyrosine. Richards and Ingbar (1959) present evidence which indicates that the "coupling" reaction i s also affected. In this investigation, similar results were observed with thiourea (see Results and discussion). Summarizing the 5 available evidence, i t appears that antithyroid compounds, for the most part, interfere with the biosynthesis of the thyroid hormones by preventing the iodination of tyrosine. D. EFFECTIVENESS OF ANTITHYROID COMPOUNDS Not a l l antithyroid are equally effective." Species differences have been shown to exist (Charipper and Gordon, 1947). Thiourea i s not an effective inhibitor i n the rat, but i s potent in man, the chick, and f i s h . Thiouracil i s more potent than thiourea i n both man and the rat, but thiourea at certain dosages i s equally as effective as thiouracil i n the domestic fowl. Wollman (1955) has noted that no matter how high a dosage of propylthiouracil i s administered to a strain of mice some organic synthesis s t i l l occurs. Some strains of mice are quite resistant to many goitrogens (Wilson, 1955). In certain instances some antithyroid drugs cease to be effective during long term treatment. Gish and Gatz (1951) found that rats fed thiouracil over a year f i r s t showed signs of thyroid inactivation, but later i t became , evident that a return to a euthyroid state had occurred. Freiders (1949) using phenylthiourea on several species of fi s h observed a similar phenomenon in the case of one species.1 Pickford and Atz (1957) l i s t other cases which imply that the thyroid can become refractory to an antithyroid compound. E. TOXICITY AND SIDE EFFECTS The toxicity of many antithyroid compounds i s discussed i n detail by Charipper and Gordon (1947) and w i l l only be briefly discussed here. In man, reactions to antithyroid drugs include 6 fever, skin rash, edema, leucopenia, and granulocytopenia. Granulocytopenia has proven to be fa t a l i n some cases. Salter (1950) points out that high dosages of antithyroid drugs can k i l l by inhibiting general cellular oxidations.- There has only been one toxicity study carried out involving f i s h so far as i t i s known to this writer. Chambers (1953) found that injections of thiourea into teleost had a profound effect on the l i v e r . Liver size increased,depletion of fat and glycogen was observed and cytological changes were apparent. In a study on cold resistance of goldfish, thiourea prolonged survival time, while an equivalent dose of thiouracil decreased survival time (Hoar, 1959).1 It was suggested that a pharmacological effect was being exerted by thiourea. Smith et^ ajL. (1953) working with f i s h has demonstrated that thiourea retards growth.: It i s obvious then, that the above examples i l l u s t r a t e that antithyroid compounds can affect tissues other than thyroid.. Much remains to be learned regarding the pharmacological effects of antithyroid compounds. This i s especially so i n fi s h . Thiourea and thiouracil have been widely used for thyroid inactivation in f i s h , but frequently the results have been conflicting and inconclusive. Often i t i s not evident whether or not the observed results are due to thyroid i n -activation or due to the effects of the goitrogen. The side effects are poorly known, and nobody to the knowledge of this writer has ever attempted to establish for f i s h effective threshold dosages to minimize the side effects of these agents. 7 The p o s s i b i l i t y t h a t t h e t h y r o i d may e s c a p e t h e i n h i b i t i n g a c t i o n o f a g o i t r o g e n must a l w a y s be c o n s i d e r e d . S p e c i e s d i f f e r e n c e s a r e l i k e l y t o e x i s t . I n b r i e f , i f c e r t a i n a n t i -t h y r o i d compounds a r e t o be t h e s o l u t i o n t o t h e p e r p l e x i n g p r o b l e m o f t h y r o i d e c t o m y i n f i s h e s , a f u l l e r u n d e r s t a n d i n g o f t h e i r e f f e c t i v e n e s s , t o x i c i t y , s i d e e f f e c t s and m e t a b o l i s m must be known. I n v i e w o f t h e s i t u a t i o n o u t l i n e d a b o v e , t h i s i n v e s t i g a t i o n was u n d e r t a k e n i n an a t t e m p t t o e s t a b l i s h t h e e f f e c t i v e n e s s o f v a r i o u s d o s a g e s o f t h i o u r e a i n p r e v e n t i n g t h e s y n t h e s i s o f t h y r o i d hormones by a m a r i n e t e l e o s t s I n a d d i t i o n an e f f o r t was made t o d e t e r m i n e w h e t h e r o r n o t t h e t h y r o i d " e s c a p e s " t h e i n h i b i t i n g e f f e c t s o f t h i o u r e a o v e r a p e r i o d o f time.- The r e s u l t s a r e d i s c u s s e d and compared w i t h t h e l i t e r a t u r e p e r t i n e n t t o t h e s u b j e c t . 8 MATERIALS AND METHODS A. EXPERIMENTAL ANIMAL The starry flounder, (Platichthys stellatus) a euryhaline pleuronectid was chosen as the experimental animal. Starry flounder are readily obtainable, easily maintained i n the laboratory, and withstand handling extremely well (Hickman, 1958). B. COLLECTION AND CARE OP PISH Plounder were collected at different times from January 0 1959 to June 1959, mainly by a small otter trawl near the mouth of the Fraser river at Steveston, British Columbia. Occasionally, a small beach seine was employed on Vancouver beaches usually near H.M.C.S. Discovery in Stanley Park, or at Lacarno beach. The f i s h were transported to the Vancouver Public Aquarium i n 10 gallon containers under aeration and held i n a sea water holding tank. At least one week elapsed following collection before the f i s h were used. Plounder were held in wooden I aquaria containing 100 l i t r e s of sea water or in circular f l a t bottom tanks f i l l e d to 200 l i t r e s . The tanks were constantly aerated and the temperature maintained at 10-12°C. The f i s h were fed twice weekly on a mixture (50-50) of horse heart and herring flesh ground to a suitable size. Aquarium specimens held for public display purposes thrive on this diet. 9 C. TREATMENT BY THIOUREA Pish were immersed i n sea water solutions of thiourea (0.0001%, 0.:0005%, 0.001% and 0.0025%) for 10-11 day periods and for periods up to 60-74 days i n concentrations of 0.005%, 0.01%, 0.02% and 0.03%. The water was completely changed every 3-5 days. Untreated control f i s h were held under similar conditions. During the course of the investigation, salinity of the water ranged from 20°/oo -27%>o. D., INJECTION WITH RADIO ACTIVE I 1 3 1 The method followed was identical with that developed by Hickman (1958). Plounder weighing from 25-300 grams were injected 96 hours before they were to be k i l l e d . Por example, f i s h to be k i l l e d after 10 days of treatment were injected on 131 the sixth day of exposure. Carrier free Nal •* i n tracer dosages of 20 or 25 microcuries, i n a solution of d i s t i l l e d water. (0.2-0.25 ml.), were injected intraperitoneally with a \ c c . tuberculin syringe and 27 gauge needle. Injection was from the blind side with the needle f i r s t passing through muscle tissue slightly caudad to the coelom. On withdrawal of the needle, the muscle acted as a seal preventing the loss of the tracer dose from the coelom. Two standards were made up representing 1/100 of the dose slightly prior to the time of injection. Injected f i s h were always held i n 100 l i t r e aquaria,and no food was offered following injection. a 10 E. PROCEDURE POR DETERMINATION OP PERCENT DOSE IN THE THYROID Pish were k i l l e d i n groups of 5 to 13 (usually 10). The animals were blotted dry, weighed and then k i l l e d by making a quick incision through the spinal cord just posterior to the coelom. This operation also severed the dorsal aorta liberat-ing a quantity of blood. In most cases, blood (0.5 ml. ' quantities depending on the size of the animal) was collected with an eyedropper from the wound and subsequently analyzed for thyroid hormones (see below).!» After bleeding, the lower jaw containing the thyroid gland was removed. Thyroid tissue in the starry flounder extends from the f i r s t g i l l arch to the third g i l l bar, with some tissue projecting outward from the midline along the hypo-branchial elements of the g i l l arches (Hickman, 1958). Care was taken not to infringe upon thyroidal tissue when trimming away extraneous tissue.: Each thyroid was placed i n a steel planchet (25 mm. x 7 mm.) for measurement of radioactivity within the gland. Counting was done by means of an end-probe s c i n t i l l a t i o n counter using a,l f " diameter x 1^" thick Nal (TI) crystal. Predetermined counts of 10,000 or 30,000 were recorded by a Philips scaler. Two standards were counted under the same conditions. DIGESTION AND EXTRACTION PROCEDURES POR M B B i J^r7Tl^WR7JIT? cTOoTTTO  i f T H E 1 T H Y R O I D G L A U S 1. Digestion The method followed was a modified version of the technique 11 described for mammals by Roche, Lissitzky, and Michel (1954). Following the determination of radioactivity i n the thyroids, 1 the glands for each group were pooled and macerated in a mortar with sand. The macerated tissue was transferred immediately to a 50 ml. test tube and digested with trypsin (B.D.H.) i n a 10 ml. borate buffer solution at a pH 8..6. Digestion was carried on for approximately 72 hours. Every 12 hours the pH was checked, and i f necessary, readjusted to pH 8.6 with 0.5 N. NaOH. Temperature was maintained at 39-40°C. i n a thermostatically controlled water bath. 2. Extraction After 72 hours, the digestate was acidified with concentrated HCl to a pH 1-2. It was then poured into a 50 ml. centrifuge tube and extracted by acid butanol (pH 1-2). The acid butanol contained added synthetic thyroxine (Na salt) at a concentration of 50 micrograms per ml. Five ml. portions of acid butanol were shaken with the digestate for 5-10 minutes and the mixture was centrifuged. This extraction procedure was repeated 4-5 times with the f i n a l extraction allowed to settle overnight. After each extraction, the butanol layer was removed by a pipette and expressed on to a watch glass. The combined extracts were evaporated over a water bath (temperature not exceeding 40°C.) un t i l approximately 1 ml. remained to be used for chromatography. 3. Serum Extraction In some experiments, sera from treated f i s h and controls were extracted for labelled hormones. The blood collected from each f i s h was allowed to clot i n a 2 ml. test tube, centrifuged 12 and the serum removed with a pipette. The serum was pooled in' a 15 ml. centrifuge tube and shaken with 5-5 mis. of acid butanol for 5-10 minutes. Serum was extracted 5-6 times - the extractions treated i n the same manner as described for the thyroid. G. CHROMATOGRAPHY Chromatography was by the ascending method using Whatman No. I or Whatman 5 MM f i l t e r paper i n sheets of 13" x 9". The solvent was a mixture n - butanol, dioxane and 2 N. NH^ OH in a 4:1:5 ratio shaken i n a separatory funnel using the butanol phase as the solvent. A rectangular glass chamber 12" x 10" x 16" served as a chromatogram chamber. The acid butanol extract was applied with a micro pipette, as a thin line 5 cm. long, I 2 " from the base of the chromatogram in quantities ranging from 50 to 200 microlitres depending on the activity of the extract. The solution was applied to the chromatogram i n 20 microlitre quantities in stages.- This process was hastened by drying the aliquots with a heat lamp between applications. Before dipping the chromatograms i n the solvent, they were allowed to become saturated i n the vapors of the chromatogram chamber for at least 6 hours. Development was overnight (12-14 hours) - the solvent travelling approximately 10 inches. On removal from the chamber, the solvent front was marked,, and the chromatogram prepared for au t or adi ogr aphy. H. AUTORADIOGRAPHY Ilford no screen X-ray films 12" x 10" individually packed 13 in light proof envelopes were found convenient to use. Before' "loading" the autoradiogram, a thin sheet of cellophane was placed over the chromatogram to prevent the development of chemoartifacts on the X-ray film. The loading took place i n the dark room under a "safe l i t e " , by placing the film over the chromatogram and returning the autoradiogram to the envelope which was subsequently sealed by adhesive tape. The autoradio-grams were placed between 2 weighted sheets of glass and le f t for a period of 8 days or longer. Films were developed by routine methods. J.* IDENTIFICATION OF /RADIOACTIVE COMPOUNDS  DETECTED BY AUTORADIOCHROMATOGRAPHY In most experiments, a control group was followed in the same manner as described above. On each chromatogram, synthetic thyroxine (Na salt) which was "tagged" with I 1 3 1 by the method of Gleason (1955), was applied i n 20 microlitre quantities to act as a reference point for the identification of natural thyroxine synthesized by the gland. In addition, after the autoradiogram was developed, the chromatogram was sprayed with diazotized s u l f a n i l i c acid followed by ^  saturated Na2C0^. The su l f a n i l i c acid spray test imparts a pink to purple color to thyroxine and i t s analogues. The resulting pink and purple spots were matched with darkened areas of the autoradiogram. As a check to determine whether or not the method of Gleason (1955) was followed correctly, the "tagged" thyroxine spot on the autoradiogram was matched with the pink thyroxine spot on chromatogram_and was found to coincide. 14 J. Rf VALUES OP COMPOUNDS Compound Moniodotyrosine Diiodotyrosine Inorganic Thyroxine Rf 0.30 0.20 0.37 0.63 Triiodothyronine was not conclusively identified, hut i t s presence was suspected as the thyroxine areas on the autoradio-gram exhibited a tendency to s p l i t into two separate bands. When the darkened areas of the autoradiogram were matched with the spots imparted to the chromatogram by the sul f a n i l i c acid spray test, the lower darkened area coincided with the pink thyroxine spot. The upper band did not coincide with any spots on the chromatogram. No synthetic triiodothyronine was available to serve as a reference point for positive identification. 15 RESULTS A. I N H I B I T I N G 1 CONCENTRATIONS 03? THIOUREA The r e s u l t s show t h a t t h i o u r e a i n c o n c e n t r a t i o n s o f 0.0025%, 0.005%, 0.01%, 0.02% and 0.05% e f f e c t i v e l y p r e v e n t e d t h e s y n t h e s i s o f t h y r o x i n e ( s e e T a b l e s I - V ) , w h i l e l o w e r c o n c e n t r a t i o n s (0.001%, 0.0005%, and 0.0001%) d i d n o t c o m p l e t e l y a b o l i s h t h i s s y n t h e s i s , ( T a b l e s V I - V I I I ) . M o n i o d o t y r o s i n e and d i i o d o t y r o s i n e were d e t e c t e d i n t h o s e g r o u p s o f f i s h e x p o s e d t o 0.005%, 0.01% and 0102% c o n c e n t r a t i o n s f o r 32 d a y s ( T a b l e s I I - I V ) . M o n i o d o t y r o s i n e as j u d g e d b y t h e i n t e n s i t y o f t h e s p o t on t h e a u t o r a d i o g r a m , was p r e s e n t i n g r e a t e r q u a n t i t i e s t h a n d i i o d o t y r o s i n e . An u n i d e n t i f i e d compound was p r e s e n t on t h e a u t o r a d i o g r a m f r o m f i s h e x p o s e d t o 0.03% t h i o u r e a f o r 74 d a y s . I t v/as n o t i n o r g a n i c i o d i d e . The s p o t was most l i k e l y m o n i o d o t y r o s i n e . B. PERCENT DOSE IN THE THYROID F i g u r e s 1 - 8..are. h i s t o g r a m s i l l u s t r a t i n g t h e p e r c e n t d o s e o f r a d i o a c t i v e i o d i n e i n t h e t h y r o i d 96 h o u r s a f t e r i n j e c t i o n . The v a l u e s r e p r e s e n t t h e a v e r a g e p e r c e n t d o s e f o r t h e g r o u p . I n d i v i d u a l v a l u e s f o r e a c h f i s h a r e l i s t e d i n t h e a p p e n d i x . F i g u r e s 1 - 5 show t h a t g r o u p s o f f l o u n d e r , w h i c h were i n h i b i t e d , , r e t a i n e d a l e s s e r amount o f t h e i n j e c t e d d o s e o f 131 I ^ i n t h e t h y r o i d , t h a n t h e u n t r e a t e d c o n t r o l s . The 1. The te r m " i n h i b i t e d " i s u s e d h e r e t o mean t h e c o m p l e t e s u p p r e s s i o n o f t h e s y n t h e s i s o f t h y r o x i n e and t r i o d o t h y r o n i n e . 16 reduction was even more severe when f i s h were k i l l e d at 120 hours instead of the usual 96 hours. One group of f i s h held for 12 days i n 0.005$ thiourea were k i l l e d 120 hours after injection (Figure 4). The percent dose was 0.18$, while groups held for the same period and k i l l e d at 96 hours had percent dosages ranging.from 0.44 - 0.65$. It would appear that unless the thyroid gland synthesizes thyroxine, i t can not retain iodide ions to the same extent as under normal conditions. There was a tendency for groups of f i s h held for 32 days or longer to retain more of the injected dose than groups held for 10 - 11 days (Figures 2 - 5 ) . This may be explained on the basis of increased secretion of the thyroid stimulating hormone (T.S.H.) from the pituitary. It i s l i k e l y that by 32 days the blood level of the thyroid hormones had become depleted, and the inhibiting effect of the thyroid hormones on the pituitary had ceased.' Increased secretion of the T.S.H. hormone would then occur, and as a result, the thyroid gland would be more efficient i n trapping iodide. The control group for f i s h k i l l e d after 32 days of treat-ment had exceptionally high uptakes (Figures 2 - 4 ) . This may be explained on the basis of re-entry of the injected dose after i t was excreted into the water. Due to circumstances, i t was necessary to hold the control group i n a smaller aquarium containing 40 l i t r e s of water, which was not changed following 131 injection. The excreted I re-entered the animals again, and thus was available for trapping by the thyroid. Hickman (1958) found re-entry could affect thyroid uptake 24 hours after injection. 17 S i n c e t h e f i s h were h e l d f o r 96 h o u r s i t i s q u i t e l i k e l y t h a t t h e h i g h u p t a k e was due t o t h i s phenomenon. I n e x p e r i m e n t 25 ( s e e a p p e n d i x , T a b l e XV), 2 c o n t r o l f i s h were i n j e c t e d 4 - 5 m i c r o c u r i e s o f r a d i o i o d i n e as o p p o s e d t o t h e u s u a l 20 o r 25 m i c r o c u r i e s . When c a l c u l a t e d on t h e b a s i s o f a 20 m i c r o c u r i e s t a n d a r d , t h e p e r c e n t d ose i n t h e t h y r o i d (when i n j e c t e d w i t h 4 - 5 m i c r o c u r i e s ) was f o u n d to be 0.49%. The t h y r o i d s were s e p a r a t e l y d i g e s t e d , e x t r a c t e d and r a d i o c h r o m a t o g r a p h e d . I n b o t h c a s e s , d i i o d o t y r o s i n e and t h y r o x i n e were d e t e c t e d . The s i g n i f i c a n t p o i n t o f t h e above p r o c e d u r e i s t h a t t h e t h y r o i d s o f f i s h t r e a t e d w i t h 0.0025% and h i g h e r c o n c e n t r a t i o n s o f t h i o u r e a , h a d a p p r o x i m a t e l y t h e same amount o f r a d i o a c t i v i t y i n the t h y r o i d ( o f t e n more) a s n o r m a l f l o u n d e r i n j e c t e d w i t h 4 - 5 m i c r o c u r i e s . When i n d i v i d u a l l y a s s a y e d , no t h y r o x i n e c o u l d be d e t e c t e d i n f i s h e x p o s e d t o 0.0025% t h i o u r e a o r h i g h e r c o n c e n t r a t i o n s , , b u t i n n o r m a l u n t r e a t e d f i s h w i t h an e q u i v a l e n t amount o f r a d i o a c t i v i t y i n t h e t h y r o i d , t h y r o x i n e was r e a d i l y d e t e c t e d . F u r t h e r m o r e , i t s h o u l d be b o r n i n mind t h a t t h e t h y r o i d s o f a g r o u p o f t r e a t e d f i s h were a l w a y s p o o l e d and t h e n a s s a y e d f o r hormones. T h i s p r o c e d u r e i n c r e a s e d t h e r a d i o a c t i v i t y 5 - 1 3 f o l d ( d e p e n d i n g on t h e number o f f i s h ) , and i n e v e r y c a s e t h y r o x i n e was n o t d e t e c t e d . T hus t h e s y n t h e s i s o f t h y r o x i n e was v i r t u a l l y n o n - e x i s t e n t when f l o u n d e r were e x p o s e d t o 0.0025% t h i o u r e a o r h i g h e r . C. SERUM RESULTS Serum was e x t r a c t e d and r a d i o c h r o m a t o g r a p h e d f o r t h y r o i d 18 hormones i n those groups of f i s h exposed to 0.005%, 0.01$, 0.02$ thiourea for 32 days, and for groups exposed to the same concentrations for 69 days. No thyroid hormones were detected, although thyroxine was present i n sera from the untreated controls. 19 Results TABLE I Results from groups of Starry Flounder exposed to 0.03% Thiourea. Number Duration Thyroxine Synthesis of of of Synthesis Lower Fish Treatment Analogues 6 74 days Inhibited None detected 5 63 days Inhibited Trace-compound unidentified TABLE II Results from groups of Starry Flounder exposed to 0.02$ Thiourea. Number Duration Thyroxine Synthesis of of of Synthesis Lower Fish Treatment Analogues 11 32 days Inhibited Moniodotyrosine detected Diiodotyrosine a trace 9 61 days Inhibited None detected 7 69 days Inhibited None detected 20 TABLE III Results from two groups of Starry Flounder exposed to 0.01% Thiourea. Number of Fish Duration of Treatment Thyroxine Synthesis Synthesis of Lower Analogues 11 32 days Inhibited Moniodotyrosine detected 8 69 days Inhibited None detected TABLE IV Results from groups of Starry Flounder exposed to 0.005% Thiourea. Number of Fish Duration of Treatment Thyroxine Synthesis Synthesis of Lower Analogues 7 11 days Inhibited None detected 11 12 days Inhibited None detected 12 32 days Inhibited Moniodotyrosine and Diiodotyrosine ; detected 10 69 days Inhibited None detected , 21 TABLE V Results from a group of Starry Flounder exposed to 0.0025$ Thiourea. Number of Fish Duration of Treatment Thyroxine Synthesis Synthesis of Lower Analogues 13 11 days Inhibited None detected T&BLE VI Results from a group of Starry Flounder exposed to 0.001$ Thiourea. Number of Fish Duration of Treatment Thyroxine Synthesis Synthesis of Lower Analogues 8 10 days Not Detected Moniodo and Diiodotyrosine detected 22 TABLE V I I R e s u l t s f r o m a g r o u p o f S t a r r y F l o u n d e r e x p o s e d t o 0.0005% T h i o u r e a . Number o f F i s h D u r a t i o n o f T r e a t m e n t T h y r o x i n e S y n t h e s i s S y n t h e s i s o f Lower A n a l o g u e s 10 10 d a y s Not I n h i b i t e d Not I n h i b i t e d TABLE V I I I R e s u l t s f r o m a g r o u p o f S t a r r y F l o u n d e r e x p o s e d t o 0.0001% T h i o u r e a . Number o f F i s h D u r a t i o n o f T r e a t m e n t T h y r o x i n e S y n t h e s i s S y n t h e s i s o f Lower A n a l o g u e s 10 10 d a y s Not I n h i b i t e d Not [ I n h i b i t e d 23 Figure 1. Average percent dose i n the thyroid approximately 96 hours after injection of 25 microcuries of 131 I ^ of groups of starry flounder exposed to 0.03$ thiourea. Open bar, untreated controls, shaded bar, treated f i s h . 63 DAYS 24 Figure 2. Average percent dose i n the thyroid approximately 96 hours after injection of I of groups of starry flounder exposed to 0.02% thiourea. The 32 and 69 day groups were injected with 20 microcuries - 61 day group was injected with 25 microcuries. Open bar untreated controls, shaded bar treated f i s h . 25 r, F i g u r e 3» A v e r a g e p e r c e n t d ose i n t h y r o i d a p p r o x i m a t e l y 96 131 h o u r s a f t e r i n j e c t i o n o f 20 m i c r o c u r i e s o f I ^ o f g r o u p s o f s t a r r y f l o u n d e r e x p o s e d t o 0.01% t h i o u r e a . Open b a r , u n t r e a t e d c o n t r o l s , s h a d e d b a r t r e a t e d f i s h . 7 DAYS 26 Figure 4. Average percent dose i n the thyroid approximately 131 96 hours after injection of I ^ of groups of starry flounder exposed to 0.005$ thiourea. 11, 52, and 69 day groups injected with 20 microcuries; 12 day group injected with 25 microcuries. Percent dose i n 12 day group was measured 120 hours after injection. Open bar, untreated controls, shaded bars, treated f i s h . 69 27 Figure 5. Average percent dose i n the thyroid 96 hours after injection of 20 microcuries of I 1 3 1 . 11 day group exposed to 0.0025% thiourea, 10 day group exposed to 0.001%. Open bar untreated control; shaded bar treated f i s h . 7 -i d CO O Z i d U DC I d CL 5 -II DAYS 28 Figure 6. Average percent dose i n the thyroid approx-imately 96 hours after an injection of 20 microcuries of I 1 3 1 of a group of flounder exposed to 0.0005% thiourea, open bar un-treated controls; shaded bar, treated f i s h . Average percent dose i n the thyroid approximately 96 hours after injection of 131 20 microcuries of I ' of a group of starry flounder exposed to 0.0001% thiourea, open bar, untreated controls; shaded bar, treated f i s h . 28a i F i g u r e 7. An a u t o r a d i o g r a m o f t h y r o i d e x t r a c t s f r o m a g r o u p o f s t a r r y f l o u n d e r e x p o s e d t o 0 . 0 3 $ t h i o u r e a f o r 63 d a y s . C o n t r o l g r o u p a l s o r e p r e s e n t e d , S o l v e n t , N. B u t a n o l , d i o x i n e a n d 2 N. NH.0H. / l ^ b . (So*, ^ ^ t u t * A t i ^ t n . Thyroxine Thyroxine Inorganic Iodide Diiodo-tyro sine 0 n g x n 'Z Control Pish Treated. Pish Tagged Synthetic Thyroxine 29 DISCUSSION A. EFFECTS OF INHIBITING CONCENTRATIONS OF THIOUREA ON PERCENT DOSE IN THE THYROID With the exception of certain anions (see introduction), there i s general agreement that the iodide concentrating mechanism of the thyroid i s not affected by thiourea and other antithyroid compounds (Taurog e_b a l . , 1947). This discovery was based on mammalian material , and subsequently, i t was revealed that f i s h responded i n a similar manner (Leloup, 1952). In the normal animal, iodide i s accumulated by the gland, most of i t i s incorporated into an organic form (i . e . the hormones), and then i s stored within the f o l l i c l e s linked to a protein. In this state, the iodine i s non-diffusable and only returns slowly to the blood as the hormone after the enzymatic hydrolysis of the protein. In the inhibited animal, iodide i s accumulated at the same rate, but owing to the failure of organic synthesis, i t remains as the iodide ion -an osmotically active particle which i s subject to the forces of diffusion. It rapidly returns to the blood becoming liable to elimination by the kidney. 2. Nadler and Leblond (1955) estimate that i n the rat, 45% of the accumulated iodide which enters the gland is discharged as such, the remainder ut i l i z e d for synthesis of the hormones. 5. Under conditions of prolonged inhibition, the hormonal blood level f a l l s , allowing the increased secretion of T.S.H. by the pituitary which accelerates the trapping of iodide by the thyroid. 30 Taurog et, a l - (1947) have shown that the a b i l i t y of the 4 thyroid gland to trap iodide ions i s limited by the level of iodide i n "the blood. Since l i t t l e of the accumulated iodide i s retained for lack of organic synthesis i n the inhibited animal, and plus the fact that the injected dose has been subject to excretion, the level of radioactivity i n the blood rapidly.falls. In the inhibited flounder 96 hours after injection, we have a situation where trapping i s continuing, but owing to the decreased level of the injected dose i n the blood, the thyroid i s unable to concentrate the same amount of ions as a normal animal. The significant point of the above discussion i s that flounder held i n 0.0025$ thiourea and higher concentrations had a lower percent dose i n the thyroid than untreated control f i s h . Concomitant with the lower percent dose was the fact that no thyroxine was detected by radiochromatography i n treated f i s h exposed to the above concentrations (see results, Tables I - V). The low percent dose values support the conclusions based on radi©chromatographic evidence that the thyroids were inhibited. There were some exceptions to the rule that a low percent dose i n the thyroid signifies inhibition. The exceptions occurred in the cases of long term exposure to thiourea. In experiment 26 (see appendix, Tables XVI - XIX) where flounder 4. Iodide ions at pharmacological levels behave as interfering ions inhibiting the trapping mechanism of the thyroid. 31 were h e l d f o r 69 d a y s , 6 f i s h had p e r c e n t d o s a g e s e q u a l t o t h e c o n t r o l s o r e v e n h i g h e r . The t h y r o i d s i n t h e c a s e o f t h e s e h i g h u p t a k e f i s h were i n d i v i d u a l l y a n a l y z e d f o r hormones, b u t none were d e t e c t e d - t h e r a d i o a c t i v i t y b e i n g i n o r g a n i c . To e x p l a i n t h e above e x c e p t i o n s i s d i f f i c u l t and no c o n c l u s i o n s can be made. One m i g h t a r g u e t h a t an e x c e s s i v e s e c r e t i o n o f T.S.H. i s m a k i n g t h e i o d i d e t r a p more e f f i c i e n t . I t i s p o s s i b l e t h a t e x c e s s i v e T.S.H. s e c r e t i o n had p r o d u c e d a h y p e r e m i c c o n d i t i o n i n t h e t h y r o i d r e s u l t i n g i n a b l o o d p o o l -t h e b l o o d i o d i d e c o n t r i b u t i n g t o t h e r a d i o a c t i v e c o u n t . An a l t e r n a t i v e e x p l a n a t i o n m i g h t a t t r i b u t e t h e h i g h u p t a k e s t o a f a i l u r e o f t h e f i s h t o e x c r e t e t h e d o s e a t t h e n o r m a l r a t e . A s u s t a i n e d h i g h - b l o o d l e v e l o f r a d i o a c t i v i t y w o u l d a l l o w the t h y r o i d t o m a i n t a i n a s t e e p g r a d i e n t between t h e b l o o d and t h e g l a n d , d e s p i t e t h e f a c t t h a t t h e t h y r o i d l o s e s i o d i d e f o r l a c k o f o r g a n i c s y n t h e s i s . O t h e r l o n g t e r m e x p o s u r e s t o t h i o u r e a had no e x c e p t i o n s ( s e e a p p e n d i x , T a b l e s V I - V I I ) . A c h e c k on r e n a l c l e a r a n c e , o r t h e o v e r a l l r a t e of. d i s a p p e a r -a nce f r o m t h e body o f t h e i n j e c t e d t r a c e r d ose would s e t t l e t h i s q u e s t i o n . The f a c t t h a t a low p e r c e n t d o s e i n t h e t h y r o i d s i g n i f i e s i n h i b i t i o n o f t h e t h y r o i d c o u l d p r o v e t o be a u s e f u l and c o n -v e n i e n t method f o r d e t e r m i n i n g w hether o r n o t t h e g l a n d i s i n h i b i t e d . B. COMPLETENESS OP INHIBITION C o n c e n t r a t i o n s o f 0.0025$ t h i o u r e a and h i g h e r b l o c k e d t h y r o x i n e s y n t h e s i s c o m p l e t e l y . However, m o n i o d o t y r o s i n e and 32 diiodotyrosine were detected i n groups of f i s h k i l l e d after 32 days exposure to thiourea (see Results, Tables II - IV). There was no correlation between strength of concentration and the presence of the lower analogues. As to why these precursors of thyroxine should appear after 32 days treatment, and not before or after,is puzzling. No explanation i s available. At the time of Astwood's (1955) review, i t was generally believed that antithyroid compounds affected the iodination of tyrosine only, and that i t was unlikely the coupling reaction was inhibited. Richards and Ingbar (1959) present evidence that the coupling of 2 diiodotyrosine molecules i s affected by propylthiouracil i n the rat. Results from this investigation support this view. Diiodotyrosine was synthesized, but no thyroxine was produced in the case of f i s h treated for 32 days by thiourea. Of interest i s the demonstration by Slingerland et a l . (1959),that the second iodination reaction of tyrosine to form diiodotyrosine i s more sensitive to propylthiouracil than the formation of moniodotyrosine. Similar results were evident in this study as the moniodotyrosine spot on the autoradiogram was darker, indicating that a greater amount of radioactivity was present. Prom the comparative physiological view point,the possibility that thiourea i s acting in a similar manner as propylthiouracil i n the rat i s interesting. However, more work i s necessary to confirm this po s s i b i l i t y . 33 The fact that the synthesis of moniodotyrosine and t diiodotyrosine was not always inhibited by the concentrations of thiourea used i n this study, does not necessarily detract from the conclusions that thyroid activity was inhibited. Barker (1955) points out that moniodotyrosine and diiodotyrosine never appear i n the blood except in cases of pathological disturbances i.e. cancer, radiation injury and the effects of surgical trauma. According to Roche, Michel, and Michel (1952), there i s i n the thyroid an enzyme desiodase, which deiodinates moniodotyrosine and diiodotyrosine liberating iodide which would return to the iodide pool. Although this study i s based on mammals, i t i s conceivable that a similar enzyme exists in f i s h . Since no thyroxine was formed, deiodination would presumably occur. In those instances where serum was extracted for labelled thyroid hormones, i n no case was moniodotyrosine or diiodotyrosine detected in the sera of either normal or treated f i s h . Thus, one can be assured that although some organic synthesis does occur, i t would be of no physiological consequence. C. ESCAPEMENT As described earlier (see Introduction), there are numerous cases i n the literature where there i s good evidence that the thyroid sometimes escapes the inhibiting effects of an antithyroid compound. Of interest i s the case reported by Preiders (1949), who observed a resumption of growth and a return to normal 34 5 thyroid histology after 3 weeks of treatment by Trichomaster  tricopterus, a small fresh water aquarium f i s h exposed to 0.0025% phenyl thiourea. Three other species exposed to the same conditions remained inhibited throughout the 8 week experiment. Starry flounder held for over 9 weeks in concentrations of thiourea ranging f r om 0.005% - 0.03% failed to "escape". Undoubtedly, as Freiders has demonstrated, a species difference can exist. In addition, different antithyroid drugs may act in a different manner (Astwood, 1955). The possibility of escapement s t i l l may exist, since 0.0025% thiourea, the lowest inhibiting concentration of thiourea tested, was followed for only an 11 day period. Experiments of longer duration at lower concentrations may demonstrate "escapement" of the thyroid from thiourea i n the case of the starry flounder. D. DOSAGE MP TOXICITY Perhaps the most significant point revealed by this study has been the demonstration of the potency of thiourea. A concentration of 0.0025% in sea water was the lowest effective dose tested. The next lowest dose, 0.001%, did not abolish thyroid activity, although uptake i n some f i s h in this concen-tration was reduced significantly (see Appendix, Table XXIII) 5. Under excessive T.S.H. secretion, the thyroid responds morphologically by an increase in c e l l height (hypertrophy), an increase i n the number of f o l l i c l e s (hyperplasia), an increase in the blood supply (hyperemia), and changes in the nature of the colloid. These histological changes of the thyroid have been widely used as c r i t e r i a for determining inhibition. 35 i suggesting some degree of inhibition had occurred. The s i g n i f i -cance of the results becomes apparent when one reviews the literature i n an attempt to compare dosages used i n other investigations. A review by Pickford and Atz (1957) reveals that a widely used concentration of thiourea has been 0.03 - 0.033$ (about 12 times higher than 0.0025$). Concentrations of 0.05$ (20 times) and 0.1$ (40 times) have been popular, but to a lesser extent. A wide variety of species have been exposed to these concentrations, and this fact immediately gives rise to the question of whether or not the popular dosages of thiourea (0.03 - 0.1$) have been unnecessarily high. Lever e_t a l . (1949) treated Lebistes reticulatus with 0.01$ thiourea and noted hypertrophy and hyperplasia of the thyroid, which suggests inhibition.(from Pickford and Atz, 1957). Buser-Lahaye (1953) used 0.02$ on Gambusia af f i n i s and noted a similar response (from Pickford and Atz, 1957). The above concentrations were effective on the starry flounder. Whether or not lower con-centrations of thiourea would be effective for most species of teleosts remains to be demonstrated and one can only speculate at this point. It i s entirely possible that some species might require high concentrations of thiourea to cause thyroid inactivation. This necessity could well be due to differences in kidney function. Thiourea in the elasmobranch Squalus acanthias i s fi l t e r e d through glomeruli with l i t t l e reabsorption by the kidney tubule (Clark and Smith, 1932). As a result, assuming 36 t h a t most o t h e r f i s h e x c r e t e t h i o u r e a i n a s i m i l a r manner, a f r e s h w a t e r f i s h w i t h i t s c o p i o u s u r i n e o u t p u t w o u l d c l e a r 1 t h i o u r e a f r o m t h e "blood much more r a p i d l y t h a n a m a r i n e t e l e o s t . I t i s p o s s i b l e t h a t r e n a l f u n c t i o n c o u l d c a u s e a g r a d i e n t t o e x i s t between t h e t h i o u r e a i n t h e s e a w a t e r and t h e b l o o d t o s u c h a n e x t e n t / t h a t i n h i b i t i n g t h r e s h o l d c o n c e n t r a t i o n s o f t h i o u r e a would d i f f e r c o n s i d e r a b l y between d i f f e r e n t s p e c i e s . S u c h a p o s s i b i l i t y c o u l d e a s i l y be i n v e s t i g a t e d u s i n g an e u r y h a l i n e s p e c i e s i n d i f f e r e n t s a l i n i t i e s . ' 37 . CONCLUSIONS 1. T h i o u r e a i n c o n c e n t r a t i o n s o f 0.0025$, 0.005$, 0.01$, 0.02$ and 0.03$ p r o v e d e f f e c t i v e i n p r e v e n t i n g t h e s y n t h e s i s o f t h y r o x i n e "by t h e t h y r o i d g l a n d o f t h e s t a r r y f l o u n d e r . C o n c e n t r a t i o n s o f 0.001$, 0.0005$ and 0.0001$ t h i o u r e a proved], t o be o n l y p a r i t a l l y e f f e c t i v e . 2. The t h y r o i d s o f f l o u n d e r h e l d i n c o n c e n t r a t i o n s o f 0.005$ -0.03$ t h i o u r e a f o r o v e r 9 weeks r e m a i n e d i n h i b i t e d . No escapement by t h e t h y r o i d f r o m t h e i n h i b i t i n g e f f e c t s o f t h i o u r e a o c c u r r e d d u r i n g t h e 9 week p e r i o d . 3. F l o u n d e r h e l d i n i n h i b i t i n g c o n c e n t r a t i o n s o f t h i o u r e a l ^ l c o n s i s t e n t l y had l o w u p t a k e s o f I by t h e t h y r o i d when compared t o c o n t r o l s . 4. I t i s s u g g e s t e d t h a t t h e measurement o f t h y r o i d u p t a k e c o u l d p r o v e to be a u s e f u l and c o n v e n i e n t method f o r a s s e s s i n g i n h i b i t i o n . 5. A l t h o u g h t h e s y n t h e s i s o f t h y r o x i n e was b l o c k e d by t h e i n h i b i t i n g c o n c e n t r a t i o n s o f t h i o u r e a , some s y n t h e s i s o f m o n i o d o t y r o s i n e and d i i o d o t y r o s i n e o c c u r r e d . APPENDIX 38 TABLE I Experiment 19 - Starry Flounder exposed to 0.03% Thiourea for 74 days. Fish Weight (gms.) % dose i n Thyroid 89 hours after injection 1 151 0.5% 2 185 l.'88% * 3 156 0.5% 4 174 3.3% * 5 139 0.93% 6 387 1.88% Ave. - 1.49% Moniodotyrosine - not detected Diiodotyrosine - not detected Thyroxine - not detected * Chromatographed separately - no thyroid hormones detected. 39 TABLE II Experiment 19 - Untreated Control Pish Eish Weight (gms.) % dose i n Thyroid 89 hours after injection 1 49 3.3% 2 86 5.25% 3 61 4.-3% 4 35 3.4% 5 81 2.35% Ave. - 3.72% Moniodotyrosine - detected Diiodotyrosine - detected Thyroxine - detected 40 TABLE III * Experiment 20 - Starry Flounder exposed to 0.?01% Thiourea for 10 days. , •• Fish Weight (gms.) % dose i n Thyroid 94 hours after injection 1 269 0.62% 2 115 0.53% 3 63 0.38% 4 150 0.78% 5 81 0:45% 6 100 0.68% 7 124 0.'85% 8 99 0;62% 9 85 0.98% 10 85 1.46% Ave. - 0.73% * Chromatographic analysis for Thyroid hormones not attempted. 41 TABLE I V * Experiment 20 - Untreated Control Fish Fish Weight (gms.) $ dose in Thyroid 94 hours after injection 1 143 2.9$ 2 114 1^56$ 3 75 2.81$ 4 - 2.84$ 5 - 2.56$ Ave. - 2.53$ 42 TABLE V Experiment 21 - Starry Flounder exposed to 0.005% Thiourea for 12 days. Pish Weight (gms.) % dose i n Thyroid 120 hours after injection 1 156 0.18% 2 43 0.08% 3 69 0.08% 4 45 0.27% 5 34 0.03% 6 58 0.01% 7 55 0.22% 8 - 0.22% 9 29 0.28% 10 88 0.31% 11 94 0.39% Ave. - 0.18% Moniodotyrosine - not detected Diiodotyrosine - not detected Thyroxine - not detected 4 3 TABLE VI Experiment 22- Starry Flounder exposed to 0.02% Thiourea for 61 days. J Fish Weight (gms.) % dose i n Thyroid 94 hours after injection 1 73 l.:05% 2 156 0.81% 3 157 0.73% 4 152 0.'18% 5 77 0.35% 6 84 0.74% 7 25 0.16% 8 320 1.80% 9 250 0.87% Ave. - 0.73% Moniodotyrosine - not detected Diiodotyrosine - not detected Thyroxine - not detected 44 TABLE V I I Experiment 23 - Starry Flounder exposed to 0.03$ Thiourea for 63 days. Fish Weight (gms;) $ dose i n Thyroid 94 hours after injection 1 98 0.14$ 2 99J 0.:81% 3 96 1.14$ 4 68 1.8$ 5 230 0.78$ Ave. - 0.93$ Moniodotyrosine - trace-compound Diiodotyrosine - unidentified Thyroxine - not detected 45 TABLE VIII Experiment 22-23 - Untreated Control Pish Pish Weight % dose i n Thyroid 96 hours after injection I 1 45 3.60% 2 97 4.20% 3 85 3.00% 4 47 0.96% 5 36 2.70% Ave. - 2.90% Moniodotyrosine - detected Diiodotyrosine - detected Thyroxine - detected 46 T A B L E IX Experiment 24 - Starry Flounder exposed to 0.005$ Thiourea for 32 days. Fish Weight (gms.) % dose i n Thyroid > 90 hours after injection 1 98 0.54$ 2 61 2.54$ 3 64 1.60$ 4 190 l.'20$ 5 54 ll. J50$ * 6 55 0.8$ 7 95 1.45$ 8 64 1.70$ 9 52 0.55$ 10 60 1.03$ 11 77 1.15$ 12 76 0.55$ Ave. - 1.17$ Moniodotyrosine - detected Diiodotyrosine - detected-trace Thyroxine - not detected * Not included in average. 47 TABLE X Experiment 24 - Starry Flounder exposed to 0.01% Thiourea for 32 days. Fish Weight (gms•) % dose i n Thyroid 94 hours after injection 13 194 1.25% 14 62 0.32% 15 84 1.25% 16 95 1.35% 17 51 0.41% 18 93 1.45% 19 46 0.39% 20 61 1.15% 21 64 0.95% 22 49 0.82% 23 30 0.87% AveJ - 0.93% Moniodotyrosine - detected Diiodotyrosine - not detected Thyroxine - not detected 48 TABLE X I Experiment 24 - Starry Flounder exposed to 0.02% Thiourea for 32 days. Fish Weight (gms.) % dose in Thyroid j 97 hours after injection 24 145 4.J30% * 25 61 0.87% 26 75 0.56% 27 53 0.40% 28 73 2.40% 29 44 0.40% 30 45 0.85% 31 38 0.49% 32 59 0.07% 33 77 0.143% 34 83 1.07% 35 83 0.60% Ave. - 0.74% Moniodot' yrosine - detected Diiodotyrosine - detected-a trace Thyroxine - not detected * Not included i n average. 49 TABLE X I I Experiment 24 - Untreated Control Fish Fish Weight (gms.) % dose i n Thyroid 98 hours after injection 36 42 8.0% 37 47 6.25% 38 45 8.35% 39 40 6.45% 40 38 4.34% 41 35 9.10% 42 25 9.50% Ave. - 7.42% Moniodotyrosine - detected Diiodotyrosine - detected Thyroxine - detected 50 TABLE XIII Experiment 25 - Starry Flounder exposed to 0.0025$ Thiourea for 11 days. Fish 1 Weight (gms.) $ dose i n Thyroid ^ 94 hours after injection 19 106 0.68$ 20 75 0.86$ 22 46 0,'30$ 23 133 0.93$ 24 39 0.36$ 25 34 0.56$ 26 59 0.45$ 27 137 1.07$ 28 65 0.45$ 30 73 0.58$ 31 60 0.29$ 32 254 0.68$ 33 238 1.20$ Ave. - 0.65$ Moniodotyrosine - not conclusively detected Diiodotyrosine - not conclusively detected Thyroxine - not detected 51 TABLE XIV Experiment 25 - Starry Flounder exposed to 0.005% Thiourea for 11 days. Pish Weight (gms.) % dose i n Thyroid 90 hours after injection 1 2 3 4 5 6 7 318 274 37 42 46 41 34 0.81% 0.59% 0.42% 0.24% 0.33% 0.29% 0.43% Ave. - 0.44% Moniodotyrosine Diiodotyrosine Thyroxine - not detected - not detected - not detected 52 TABLE XV Experiment 25 - Untreated Control Pish Pish Weight (gms.) % dose i n Thyroid 92 hours after •injection 14 68 3.6% 15 54 0.49% * + 16 55 3.5% 17 36 0.49% * + Ave. - 3.6% Moniodotyrosine - detected Diiodotyrosine - detected Thyroxine - detected * Not included in average. +• Injected with only 4-5 microcuries of radioiodine. Digested and radiochromatographed separately -Thyroxine and Diiodotyrosine detected. 53 TABLE XVI Experiment 26 - Starry Flounder exposed to 0.005% Thiourea for 69 days. Pish Weight (gms.) % dose i n Thyroid 94 hours after injection 1 58 2.'31% 2 64 1.17% 3 29 0..44% 4 41 0.59% 5 56 1.45% 6 43 0.70% 7 80 9.91% * + 8 50 0.76% 9 90 l.;65% 10 34 0.'89% Ave. - 1.10% Moniodotyrosine - not detected Diiodotyrosine - not detected Thyroxine - not detected * Not included i n average. ••«+• Digested and radio chromatographed separately - Moniodotyrosine Diiodotyrosine and Thyroxine not detected. 54 TABLE XVII Experiment 26 - Starry Flounder exposed to 0.01% Thiourea for 69 days.' Fish Weight (gms.) % dose i n Thyroid 98 hours after injection 21 38 0.55% 22 49 1.03% 25 72 6.5% * + 24 60 1.25% 25 41 0.47% 26 51 3.76% 27 111 2.24% 28 65 1:^63% 29 64 1^21% 50 70 5.0% * + Ave. - 1..51% Moniodotyrosine - not detected Diiodotyrosine - not detected Thyroxine - not detected * Not included i n average. + Digested and radiochromatographed separately - Thyroxine Moniodotyrosine Diiodotyrosine - not detected. 55 TABLE XVIII Experiment 26 - Starry Flounder exposed to 0.02% Thiourea for 69 days. Fish Weight (gms.) %. dose i n Thyroid '96 hours after injection 131 60 11.6% 12 81 1.82% 13 65 1.46% 14 74 1.25% 15 123 1.528% 16 139 4.5% * + 17 68 2 a 4% 18 89 4.7% * X 19 75 4.17% * X 20 44 0.675% Ave. - 1.-40% Moniodotyrosine - not detected Diiodotyrosine - not detected Thyroxine - not detected •Not included i n average Digested and radiochromatographed separately - no thyroid hormones detected. X Pooled, digested, and autoradiographed together - no thyroid hormones detected 56 TABLE XIX Experiment 26 - Starry Flounder Untreated Controls Fish Weight (gms.) $ dose i n Thyroid 99 hours after I injection j 31 114 3.61$ 32 128 3.61$ 33 132 1.86$ 34 133 4.78$ 35 140 3.44$ 36 125 2.42$ 37 154 3.38$ 38 172 4.50$ + 39 210 3.38$ 40 103 3.66$ 41 53 3.82$ Ave. - 3-50$ Moniodotyrosine - detected Diiodotyrosine - detected Thyroxine - detected + Digested, autoradiochromatographed separately - Moniodotyrosine, Diiodotyrosine and Thyroxine detected. 57 TABLE XX Experiment 30 - Starry Flounder - Untreated Control Fish Fish r — | Weight (gms.) % dose i n Thyroid 92 hours after injection 31 58 6.3% 32 61 6.75% 33 76 5.55% 34 51 5.10% 35 52 4.97% 36 45 A. 00% 37 67 5.55% 38 65 7.70% 39 66 7.00% 40 33 5.10% Ave. - 5.80$ Moniodotyrosine - detected Diiodotyrosine - detected Thyroxine - detected 58 TABLE XXI Experiment 30 - Starry Flounder exposed to 0.0005% Thiourea for 10 days. Pish Weight (gms.) % dose i n Thyroid 90 hours after injection 11 79 8.82% 12 71 9.47% 13 39 3.15% 14 70 4.66% 15 84 4.71% 16 44 2.18% 17 116 4.02% 18 50 4.04% 19 71 2.77% 20 60 5.03% Ave; - 4.-88% Moniodotyrosine - detected Diiodotyrosine - detected Thyroxine - detected 59 TABLE X X I I Experiment 30 - Starry Flounder exposed to 0.0001% Thiourea for 10 days. Eish Weight (gms.) % dose i n Thyroid 89 hours after injection 1 59 3.10% 2 90 4.18% 3 46 8.61% 4 97 6.73% 5 44 10.80% 6 113 6.21% 7 66 4.84% 8 68 4.71% 9 90 4.'5 4% 10 43 4.90% Ave. - 5.86% Moniodotyrosine - detected Diiodotyrosine - detected Thyroxine - detected 60 TABLE XXIII Experiment 30 - Starry Flounder exposed to 0.001$ Thiourea for 10 days. Fish Weight (gms.) $ dose in Thyroid 91 hours after injection 22 55 11.5$ 23 75 5.08$ 24 72 1.94$ 25 132 2.30$ 27 67 2.94$ 28 66 6.10$ 29 57 2.56$ 30 43 1.40$ Ave. - 4.:22$ Moniodotyrosine - detected Diiodotyrosine - detected Thyroxine - detected 61 LITERATURE CITED Astwood, E.B. 1955. Mechanism of action of antithyroid compounds. Brookhaven Symp. Biol., 7: 61-73. Barker, S.B. 1955. The circulating thyroid hormone. Brookhaven Symp. Biol., 7: 74-88. Buser-Lahaye, J. 1953. Etude experimentale du determinisme de l a regeneration des nageoires chez les poissons teleosteens. Annee Biol., 29: 23-29. Chambers, H.A. 1953. Toxic effects of thiourea on the liv e r of the adult male k i l l f i s h , Fundulus heteroclitus (Linn.). Bull. Bingham Oceanogr. Coll., I j H ^ ) : 69-93. Charipper, H.A. and A.S. Gordon. 1947. The biology of antithyroid agents. Vitam.' and Horm., 5: 273-316. Chester Jones, I. 1957. "The Adrenal Cortex". Cambridge University Press.' Clark, R.W. and H.W.' Smith. 1932. Absorption and excretion of water and salts by elasmobranch fishes. I l l , The use of xylose as a measure of the glomerular f i l t r a t e i n Squalus acanthias. J. Cellular Comp. Physiol. 1: 131-143. Prieders, P. 1949. The effect of thiourea and phenylthiourea on growth and pigmentation of several species of f i s h . Master's dissertation, Catholic University, Washington, D.C. Gish, G. and A.J. Gatz. 1951. Effects of prolonged administra-tion of thiouracil on rat thyroids. Anat. Rec., 109:296. Goodman, L.S. and Gilman, A. 1956. "The Pharmacological Basis of Therapeutics". New York: The MacMillan Co. 1831 pp. Gudernatsch, J.P. 1911. The thyroid gland of teleosts. J. Morph., 21: 709-782. Hickman, CP. Jr. 1958. Doctoral dissertation. Univ. of Briti s h Columbia, Vancouver, B.C. Hoar, W.S. 1957. Endocrine organs, from "The Physiology of Pishes", Brown, M.E., Ed., Ch. VI, Vol. 1, pp. 245-285. New York: Academic Press Inc. Hoar, W.S. 1959. Endocrine factors i n the ecological adaptation of fishes, from "Comparative Endocrinology", Gorbman, A. Ed., pp. 1-23. New York: John Wiley and Sons. 62 L e v e r , J . , J . M i l t e n b u r g and G . J . v a n O o r d t . 1949. The e f f e c t o f a s h o r t t r e a t m e n t w i t h t h i o u r e a upon t h e f i s h t h y r o i d g l a n d . P r o c . Akad. S c i . , Amst., 52: 296-300. L e l o u p , J . 1952. A c t i o n s des a n t i t h y r o i d i e n s s u r l a f i x a t i o n de l ' i o d e r a d i o a e t i f e t l a s y n t h e s e de l a t h y r o x i n e dans l a t h y r o i d de deux t e l e o s t e e n s m a r i n s . C R . S o c . B i o l . , P a r i s , 146: 1017-1020. M a c k e n z i e , J . B . , C.G. M a c k e n z i e and E.V. M c C o l l u m . 1941. The e f f e c t o f S u l f a n i l y l g u a n i d i n e on t h e t h y r o i d o f t h e r a t . S c i e n c e , 94: 518-519. N a d l e r , N . J . and C P . L e b l o n d . 1955. The s i t e and r a t e o f f o r m a t i o n o f the t h y r o i d hormone. B r o o k h a v e n Symp. B i o l . , 7: 40-60. P i c k f o r d , G.E. and J.W. A t z . 1957. "The P h y s i o l o g y o f t h e P i t u i t a r y G l a n d o f P i s h e s " . New Y o r k : New Y o r k Z o o l . S o c . 613 p p . Rawson, R.W., J . E . R a i l and M. S o n e n b e r g . 1955. The c h e m i s t r y and p h y s i o l o g y o f t h e t h y r o i d , f r o m "The Hormones", P i n c u s , G. and K.V. Thimann, E d . pp.-433-519. New Y o r k , A c a d e m i c P r e s s . R i c h a r d s , J . B . and S.H. I n g b a r . 1959. The e f f e c t s o f p r o p y l t h i o u r a c i l and p e r c h l o r a t e on t h e b i o g e n e s i s o f t h e t h y r o i d hormone. E n d o c r i n o l o g y , 65.: 178-188. Roche, J . , S. L i s s i t z k y and R. M i c h e l . 1954. Methods o f b i o c h e m i c a l a n a l y s i s . I : 243. Roche, J . , and R. M i c h e l . 1955. N a t u r e , b i o s y n t h e s i s , and m e t a b o l i s m o f t h y r o i d hormones. P h y s i o l . Rev., 35: 583-610. Roche, J . , R. M i c h e l , 0. M i c h e l and S. L i s s i t z k y . 1952. S u r l a d e s h a l o g e n a t i o n e n z y m a t i q u e des i o d o t y r o s i n e s p a r l e c o r p s t h y r o i d e e t s u r s o n r o l e p h y s i o l o g i q u e . B i o c h e m i c a and B i o p h y s i c a A c t a . 9: 161-169. S a l t e r , W.T. 1950. The c o n t r o l o f t h y r o i d a c t i v i t y , f r o m "The Hormones". P i n c u s , G. and K.V. Thimann. E d . V o l . 2: pp. 301-349. New Y o r k , A c a d e m i c P r e s s . S m i t h , D . C , S.A. S l a d e k and A.W. K e l l n e r . 1953. The e f f e c t o f mammalian t h y r o i d e x t r a c t on t h e g r o w t h r a t e and s e x u a l d i f f e r e n t i a t i o n i n t h e f i s h , L e b i s t e s  r e t i c u l a t u s , t r e a t e d w i t h t h i o u r e a . P h y s i o l . Z o o l . . 26s 117-124. 63 S l i n g e r l a n d , D.W., D.E. Graham, R.K. J o s e p h s , P.Pi M u l v e y J r . , A.P. T r a k a s , and E . Y a m a z a k i . 1959. The e f f e c t o f p r o p y l t h i o u r a c i l on t h e c o n v e r s i o n o f m o n i o d o t y r o s i n e t o d i i o d o t y r o s i n e . E n d o c r i n o l o g y . 65_: 178-188. T a u r o g , A., I . L . C h a i k o f f and D.D. F e l l e r . 1947. The mechanism o f i o d i n e c o n c e n t r a t i o n by t h e t h y r o i d g l a n d : i t s n o n - o r g a n i c i o d i n e b i n d i n g c a p a c i t y i n t h e n o r m a l and p r o p y l t h i o u r a c i l - t r e a t e d r a t . J . B i o l . Chem. 171: 189-201. W i l s o n . 1955. Comments made d u r i n g d i s c u s s i o n o f E.B. Astwood's p a p e r . B r o o k h a v e n Symp. B i o l . 7: 61-73. Wollman, S.H. 1955. Comments made d u r i n g d i s c u s s i o n o f E.B. Astwood's p a p e r . B r o o k h a v e n Symp. B i o l . 7: 61-73. OMISSION G l e a s o n , G.I. 1955. Some n o t e s on t h e exch a n g e o f i o d i n e w i t h t h y r o x i n e h o m o l o g u e s . J . B i o l . Chem. 213: 837-841. 

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