74 THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII-1 Feb.

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1 74 THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII-1 Feb EFFECTS OF GENTAMICIN ON THE THYROID FUNCTION OF RATS YOSHIE KAJIMOTO, MASAAKI KURAMOTO, YASUKO ISHIMURA, TSUNEO KATORI and SHIUNG-YI LEE Department of Pharmacology, School of Medicine, Tokushima University, Tokushima (Director: Prof YOSHIE KAJIMOTO) (Received November 21, 1974) Not only the thyroid, hypothalamus, pituitary gland, pituitary-adrenal system, pancreas and insulin receptors but also all of the other endocrine organs may participate endocrinically in the mechanism of stimulant action on growth MAGSOOD1) attempted to establish the relationship between thyroid function and growth of rats and reported that the amount of thyroid hormones to be secreted from the organ affects greatly the growth of rats and the secretion of the hormones decreases with advancing years On the other hand, TEIR et al2) reported that the body weight of adult female rats is not affected by administration of thyroid tissue suspension TUSQUES3) emphasized that the presence of the thyroid gland is essential to the process from the newborn to maturity According to SCOW4), BOOD5) and LEE6), removal of rat thyroid can result in growth inhibition of the whole body WILLIAMS7), HUGHES8), BARKER9) and THYSSEN10) demonstrated that growth of experimental animals is inhibited with thiouracil In Japan, KAMEI11) described the relationship among the process of rat growth, actions of thyroid preparations and effects of antithyroid agents The results of all investigations mentioned above are suggestive of evidences that the thyroid plays an important role in stimulation of growth of experimental animals In a previous communication") it was shown by us that the body weight of rats is increased after administration of gentamicin (GM) in a small dosage We have tried recently to investigate changes in the thyroid gland after dosing of GM This paper is concerned with the result of the study Experimental Methods 1 Determination of131i-uptake Wistar male rats weighing about 300g at 11 weeks of age were employed in determination of 181I-uptake (%) The animals were grouped of 6 each At a dose level of 10mg/kg body weight, GM was given once or continuously for 4 weeks After 2 `3 hours of intramuscular injection of GM at a single dose, Na131I in a dose of 02ƒÊc was given subcutaneously to the back of rats, and the thyroid gland was extirpated 4 hours later to determine the radioactivities in 05ml of KREBS-RINGER'S solution by a well-type scintillation counter Rats in other groups received GM intramuscularly or orally for 4 consecutive weeks Because the constituents of amino acids (described later) were determined in the same rats, the thyroid was removed 24 hours after subcutaneous injection of 10ƒÊc of Na131I to the back of rats The organ was homogenized in 1 ml of KREBS-RINGER'S solution under ice-cooling, and a portion of the homogenate was diluted for counting the radioactivity

2 Feb THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII Determination of 131I-triiodothyronine resine sponge uptake (Triosorb test or 131I-T3 RSU) and determination of 125I-thyroxine resine sponge uptake (Tetrasorb test or 125I-T4 RSU) Wistar male rats weighing about 180g at 6 weeks of age were divided into groups of 6 each GM in a dose of 10mg/kg/day was given intramuscularly and orally for 4 continuous weeks except for Sunday The blood was collected from the heart 4 weeks later, and both 1811_7-8 and 125I-T 4 RSU in the serum were determined In this experiment were used Dainabbott Determination Kit, Triosorb and Tetrasorb 3 Determination of synthetic ability of hormone in thyroid In order to examine the process of hormone synthesis in the thyroid, paper partition chromatography was conducted for the hydrolyzate solution of the thyroid tissue homogenate in accordance with the methods of SHIMODA and GREER13) and OTAKI14) At the dose level of 10ƒÊc, Na131I was injected subcutaneously to the back of the control rats and GM-treated rats After the lapse of 24 hours, their thyroid glands were extirpated The wet organ was weighed, and subsequently homogenized in 05ml of KREBS-RINGER'S solution along with methiocil to make 1 ml in total To the solution, 10 mg of pancreatin was added as an proteolytic enzyme Moreover, 1 drop of toluene, 100ƒÊg each of 3-monoiodo-l-tyrosine, 3, 5-diiodo-l-tyrosine, 3, 3, 5-triiodo-l-thyronine and l-thyroxine and 500ƒÊg of KI as carriers were added to the solution The solution was incubated at 37 Ž for 48 hours The hydrolyzate thus obtained was tested by paper partition chromatography Toyo Filter Paper No 51 was used as the filter paper The test was carried out in a dark room as possible as we could Using two solvent systems e, BAW system (n-butanol-acetic acid-water, 4:1:5) and BEA system (n-butanol-absolute alcohol- 05 N ammonia, 5:1:2), the sample was developed for 17 hours The filter paper was dried and iodinated amino acids were colored by spraying PAULY'S diazo reagent and a 2% CuSO4 solution of inorganic iodine The radioactivities of each colored spots were determined by a gas flow counter The ratio of each iodinated amino acid was calculated by a planimeter,i, Results 1 Determination of 131I-Uptake The percent 131I-uptake by the thyroid gland of the control group and the GM-treated group (at a single dose level of 10mg/kg, im) was 71 }13 (%) and 79 }04 (%) respectively, whereas the values in the control, intramuscularly treated group (for 4 weeks) and orally treated group (for 4 weeks) were 63 }12, 70 }19 and 81 }31 (%) respectively Though the GMtreated groups indicated a slightly higher tendency to "'I-uptake than the control group, the values were not significantly different between both groups (Fig 1) 2 Determination of 131I-T8 RSU (%) (Triosorb Test) 181I-T 3 RSU values after im injection and p o Fig 1 I-Uptake (%) after a single or continuous administration of GM for 4 weeks Each value is the mean±sd (P<005 n=6 `10) administration of GM (10mg/kg/day) for 4 successive weeks were 512 }32% and 501 }36%, respectively, while the value in the control group was 585 }19% The value in the GM-treated groups was decreased significantly as compared with that in the control group (P<001) (Fig 2) 3 Determination of 125I-T4 RSU (Tetrasorb Test) 125I-T4 RSU values in groups treated with intramuscular and oral GM (10mg/kg/day for 4 con-

3 76 THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII-1 Feb Fig 2 131I-T3, RSU after continuous administration of GM for 4 weeks Each value is the mean }SD (P<005 n=5 `6) **: value differs from control (P<001) Fig 3 125I-T4RSU after continuous administration of GM for 4 weeks Each value is the mean }SD (P<005 n=6 `8) * or **: value differs from control (P<005 or P<001) secutive weeks) were 53 }05ƒÊg/dl and 45 }10ƒÊg/dl respectively, whereas the value in the control group was 68 }04ƒÊg/dl The values in GM-treated groups were significantly lower than those in the control group (Fig 3) 4 Assessment of Thyroid Function to Synthesize the Hormones Ten pc of Nell was injected subcutaneously to the back of rats that received 10mg/kg/ day of GM intramuscularly and orally for 4 continuous weeks like Triosorb and Tetrasorb tests Thus, the fractions of iodinated amino acids were examined 24 hours after injection of the radioactive compound Table 1 represents the fractions of iodinated amino acids which can be developed by BEA system of solvents The change in monoiodotyrosine (MIT) after treatment with GM was slight, but diiodotyrosine (DIT) was increased Both T3 and T4 values were significantly lower in GM-treated groups than in the control group (P<005) The fractions of iodinated amino acids which were developed by BAW system of solvents are given in Table 2 There existed a decreasing tendency of T3+T4 values in the GM-treated group Fig 4 depicts the wet weight of the thyroid and adrenal gland Atrophic tendency of the Table 1 Distribution of 1311-labeled amino acids of hydrolyzates of thyroid after continuous administration of GM for 4 weeks Solvent: n-butanol-absolute alchohol -05 N ammonia (5:1:2) Each value is the mean }SD P<005 n=5 `6 *: Value differs from control P<0 05

4 Feb THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII-1 77 Table 2 Distribution of 131I-labeled amino acid of hydrolyzates of thyroid after continuous administration of GM for 4 weeks Solvent: n-butanol-acetic acid-water (4:1:5) Each value is the mean }SD P<005 n=5 `6 Fig 4 Weight of thyroid and adrenal gland Weight of thyroid Weight of adrenal gland mg/rat mg/rat Each value is the mean }SD (P<005 n=6 `8) thyroid was noticed in the GM-treated groups, but the statistical test disclosed no significant differences between the GMtreated group and control group No change was seen in the wet weight of the adrenal gland after GM dosing Table 3 summarizes the overall results The drug-treated groups indicated a slightly higher tendency to 131I-uptake which represents a test of the thyroid function per se than the control group Table 3 Effects of GM on thyroid function in rats did The former showed a slightly atrophic tendency to the same organ in terms of wet weight as compared with the latter However, there were no significant differences on 181I-uptake and wet weight of the thyroid between treated and untreated groups As the thyroid function, the thyroid hormone level in the blood was determined by triosorb test The blood TB level was apparently lower in the GM-treated groups than in the control group Equally, the serum T4 level of the GM-treated group, determined by tetrasorb test, was significantly lower than that of the control group The result obtained by assessment of

78 THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII-1 Feb the thyroid function for hormone synthesis 24 hours after injection of the radioactive iodine compound revealed an elevation of DIT value and

5 78 THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII-1 Feb the thyroid function for hormone synthesis 24 hours after injection of the radioactive iodine compound revealed an elevation of DIT value and decreases of both T3 and T4 values in the GM-treated groups in comparison with the values in the control group, which led to the decrease in the value of T3+T4/MIT+DIT Therefore, it seems likely that the thyroid function for synthesis of the hormone is affected by GM in one way or another, viz, the biosynthesis of T3 and T4 from DIT are retarded or inhibited by GM Discussion The stimulation to the rat growth after administration of GM was investigated from endocrinic standpoints In particular, the metabolic change in the thyroid having a close relation to the stimulation to the rat growth was examined in conjunction with the action of GM The essential constituents of the thyroid hormone are both amino acids and iodine, and therefore, the production of the thyroid hormone may be disturbed by the deficiency of any of these substances The decrease in synthesis of thyroid hormones due to metabolic abnormality of amino acids has never been reported up to date On the other hand, abnormality on the production of thyroid hormones due to deficit in iodine is well documented for a long time In the meanwhile, because abnormality on thyroid hormone production in man develops in those who live in such area where deficiency of iodine ingestion cannot be believed, a considerable amount of research has been reported on this problem, ie, it has been elucidated that goiter manifests by ingestion of salt, soybean, cabagge, etc15) A variety of inference has been therefore proposed for the etiology of abnormal production of thyroid hormones If the provocative causes of abnormal hormone production which are introduced into body along with food ingestion are assumed as extrinsic factors, congenital abnormality of the thyroid which was clarified by STANBURY et al16) as the resultant in abnormality of enzyme system being participant in synthesis of thyroid hormones is considered as one of the intrinsic factors However, it was proved biochemically that the abnormalities are encountered in the whole process of synthesis and release of the thyroid hormone The provocative causes are now accepted as follows: (1) Defect in iodine ingestion (Trapping defect) (2) Defect in oxidation of ingested iodine (Inability of oxidation from I to I2) (3) Defect in synthetic process from oxidized iodine to iodotyrosine (Inability to halogenated tyrosine in the presence of elemental iodine) (4) Defect in coupling reaction of two DIT residues (Inability to couple iodinated tyrosines) (5) Abnormality of hydrolase for thyroglobulin (Inability to hydrolyze thyroglobulin) (6) Defect in dehalogenase (Inability to deiodinate "free" iodotyrosine) (7) Abnormal thyroglobulin (Abnormal formation or release of thyroglobulin or "thyroglobulinlike" substance) Assuming the abnormality on thyroidal hormones after GM treatment to be extrinsic cause, the drug was examined biochemically The iodine uptake in the GM-treated group tended to be increased, as compared with that in the control group However, the increase was not significantly different between both groups In oxidation process of ingested iodine, the element is oxidized enzymically to convert from I to I2 followed by formation of iodotyrosine When there are defects in this process, neither MIT nor DIT are synthesized Though the DIT level remained unchanged in the control group, the level was evidently increased after administration of GM Hence, it is reasonable to presume that oxidation of iodine and biosynthesis of iodotyrosine undergo smoothly even in the presence of GM STANBURY et al reported that when there exists a defect in the coupling reaction of two DIT residues, both T3 and T4 cannot be detected in the extirpated thyroid T3 and T4 levels were decreased markedly, while DIT value was increased remarkably after the administration of GM This fact suggests that GM affects the process of coupling of two DIT residues After thyroxine has been synthesized from the ingested iodine, the element is liberated from thyro-

Feb THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII-1 79 globulin by hydrolysis and transported into the blood flow in a form of T4 to act as a hormone However, insufficient hydrolysis of thyroglobulin

6 Feb THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII-1 79 globulin by hydrolysis and transported into the blood flow in a form of T4 to act as a hormone However, insufficient hydrolysis of thyroglobulin may induce hypothyrodism HAMILTON et al") investigated 2 patients with goitrous cretinism and found that T4 levels in their thyroid glands are nearly normal Although T3 and T4 concentrations in the thyroid gland homogenate and in the serum were lower in GM-treated groups than in the control group, both were clearly detected In this sense, GM does not affect hydrolase which acts on thyroglobulin T4 is liberated from the protein of thyroglobulin by hydrolysis and transported as a hormone in the blood flow A considerable amount of MIT and DIT are simultaneously liberated by the hydrolysis of thyroglobulin, these compounds are rarely transported in the blood under normal conditions because both are subject to deiodination by a potentially active deiodinase which acts specifically on free MIT and DIT in the thyroid gland and in turn the liberated free iodine is then available for reutilization in the synthesis of T4 Hence, in the absence of normal functioning of deiodinase system, MIT and DIT appear in the blood without receiving deiodination and are eventually excreted This can result in excessive loss of precursors of thyroid hormones from body, which leads to hypofunction of hormone synthesis KUSAKABE18) reported a variety of abnormalities on deiodinase, viz, enzymic abnormalities are mostly attributed to disturbance of DIT-deiodinase, while MIT-deiodinase activity is hardly disturbed In the paper partition chromatographic study on the thyroid homogenate obtained from GM-treated rats, DIT level was found to be high This fact indicates that massive loss of DIT in the blood due to abnormality of deiodinase by GM cannot be suspected The decreased production of thyroidal hormones resulting from abnormalities on the iodinemetabolism related enzymic system has been discussed above Abnormalities on thyroglobulin or release of abnormal thyroglobulin into the blood may also elicit metabolic abnormalities of thyroid hormones, which lead to development of goiter or hypothyroidism When abnormal thyroglobulin appears in the thyroid gland, it is left at the originally spotted place in paper partition chromatography because abnormal thyroglobulin is insoluble in butanol It is known that the presence of abnormal thyroglobulin in the thyroid gland causes abnormally high tetrasorb value in many cases The present paper partition chromatographic study on the thyroid tissue homogenate of GM-treated rats revealed the absence of characteristic iodide substances at the original spot and a low tetrasorb value These facts substantiate an evidence that GM never induces abnormality of thyroglobulin As mentioned above, it was examined whether or not and how extrinsic factor in terms of administration of GM participates in enzymic abnormalities on synthesis and release of thyroid hormones in this study It was found that GM never results in abnormalities on iodine uptake, iodine oxidation, synthesis of iodinated tyrosine, deiodination process and thyroglobulin It was presumed that GM may provoke abnormalities on only some processes of coupling reaction from DIT to T3 and T4 because GM induces an increase in DIT value and decreases in T3 and T4 values Patients with goitrous cretinism are often observed in practice However, NIKI19) reported that goitrous cretinism does not manifest in patients with KOMURO'S goiter Based upon the facts that BASEDOW'S disease being one of the hyperthyroidism causes emaciation and that the secretion of the thyroid hormone decreases with advancing years, it seems much more likely that the abnormality on thyroid hormones due to administration of GM plays in any way a role as a factor in stimulation to growth of rats Summary The effect of gentamicin (GM) on the thyroid function was investigated in relation with its stimulating action to the growth of rats at a very small dose level The results may be summarized as follows : (1) Changes in 131I-uptake of the thyroid after administration of GM were unremarkable The change in wet weight of the thyroid was also unremarkable Therefore, GM may not

7 THE JAPANESE JOURNAL OF ANTIBIOTICS XXVIII-1 affect the thyroid itself (2) GM-treated rats clearly showed a decrease in 131I-triiodothyronine resine sponge uptake (triosorb test) as compared with the control animals, viz, GM-treated rats indicated hypofunction of the thyroid (3) Thyroxin (T4) levels in the serum, determined by 125I-T4 resine sponge uptake (tetrasorb test), were apparently lower in the GM-treated rats than in the control (4) The thyroid function test for synthesis of the hormones revealed an increase in diiodotyrosine (DIT) value and decreases in 3, 5, 3'-triiodothyronine (T3) and T4 values in the GMtreated groups as compared with the values in the control (5) In view of the above results it was obvious that GM does not cause abnormalities on iodine-uptake, iodine oxidation, organic conversion to iodinated tyrosines, deiodination and thyroglobulin In contrast, GM may induce abnormality on coupling reaction from DIT to T8 and T4 because the increase in DIT value and decreases in T, and T4 values are seen following administration of GM The whole of these facts seems to play an important role in stimulation to growth of rats after administration of the drug References 1) MAGSOOD, M: Influence of thyroid status on body growth Experientia 7(4): 150, ) TEIR, H; E CARPAN & I CARPAN: Effects of intraperitoneal injections of thyroid gland suspensions and oral methyl thiouracil administration in rats Acta Endocrinologica 22: , ) TUSQUES, J: Action de la thyroxine sur les rats blancs nouveau-nes Existence de zones sensibles (sensibles locales) C R Soc Biol 145(5): , ) Scow, R 0 & M E SIMPSON: Response to pituitary growth hormone of rats thyroidectomized on day of birth Anat Rec 91: , ) BOAS, N F & R O Scow: Apparent exophthalmos in the rat following cortisone treatment of thyroidectomy Endociinology 55(2): , Aug ) LEE, M O: Studies on the oestrous cycle in the rat The effect of thyroidectomy Endocrinology 9(5): , Sept/Oct ) WILLIAMS, R H ; A R WEINGLASS, G W BISSELL & J B PETERS: Anatomical effects of thiouracil Endocrinology 34: , ) HUGHES, A M: Cretinism in rats induced by thiouracil Endocrinology 34: 69-76, ) BARKER, S B: Influence of thiouracil on reproduction and growth in rat J Endocrinol 6: , ) THYSSEN, J: Thiouracil and thyrotoxicosis Acta Pharmacol Toxicol 3 (Suppl 2): 274, ) KAMEI, T: Effet de la thyroide sur la croissance chez les rats blancs J Pharm Soc Jap 55: , ) KAJIMOTO, Y & M KURAMOTO: Toxicity of gentamicin Chemotherapy 15(4): , July ) SHIMODA, S & M A GREER: Effect of graded doses of propylthiouracil on in vitro iodoamino acid synthesis in the rat thyroid gland Endocrinology 78: , ) STANBURY, J B; K OHELA & R PITT-RIVERS: The metabolism of iodine in 2 goitrous cretins compared with that in 2 patients receiving methimazole J Clin Endocrinol 15(1): 54-72, Jan ) HAMILTON, J G; M H SOLEY, W A REILLY & K B ELCHORN: Radio active iodine studies in childhood hypothyroidism Am J Dis Child 66(5): , Nov ) NIKI, M: Clinical studies on " Omuro goiter " 1 Environment and incidence of goiter in Omuro community, Kochi prefecture Shikoku Acta Med 26(4): , Aug 1970

Hypothalamo-Pituitary-Thyroid Axis

SMGr up Hypothalamo-Pituitary-Thyroid Axis Orluwene Chituru Godwill 1 * and Ohiri John U 1 1 Chemical Pathology Department, University of Port Harcourt Teaching Hospital, Nigeria *Corresponding author: