might be due to a direct action on the thyroid, like that of the thiouracil

Transcription

1 288 J. Physiol. (1953) I20, COMPARISON OF THE EFFECTS OF THIOURACIL, THY- ROXINE AND CORTISONE ON THE THYROID FUNCTION OF RABBITS BY N. B. MYANT* From the Department of Clinical Research, University College Hospital Medical School, London (Received 21 August 1952) It is now well established that cortisone reduces the uptake of radio-iodine by the thyroid (Money, Kirschner, Kraintz, Merrill & Rawson, 1950; Frederickson, 1951), but the mechanism of this inhibition is not understood. The inhibition might be due to a direct action on the thyroid, like that of the thiouracil group of substances; or it might be caused indirectly through an action like that of thyroxine on the pituitary stimulus to the thyroid. When thiouracil is given continuously to an animal, the inhibition of thyroxine synthesis causes a reduction in the blood concentration of thyroid hormone which, in turn, leads to an increased output of thyrotrophin by the pituitary (Astwood, 1945). It is known that thyrotrophin increases the rate at which thyroid hormone, already stored in the thyroid, is secreted into the circulation (Keating, Rawson, Peacock & Evans, 1945). If, therefore, cortisone has a direct action on the thyroid, partial inhibition by cortisone continued for long enough to reduce the blood thyroxine concentration should lead to an abnormally rapid passage through the thyroid of any radio-iodine that is taken up by it. If, on the other hand, cortisone acts on the pituitary stimulus to the thyroid, it should lead to an abnormally slow passage of radio-iodine through the thyroid. The effects of continued doses of thiouracil, thyroxine and cortisone on the uptake and discharge of radio-iodine by the thyroid are compared in the experiments described below. METHODS Adult male rabbits were used as test animals. They were given M.R.C. diet no. 18 without fresh vegetables. The proportion of a dose of radio-iodine taken up in the thyroid at 24 hr and the rate of loss of radio-iodine from the thyroid were measured in living animals by a method previously described (Myant, 1953). * Work undertaken on behalf of the Medical Research Council.

2 EFFECT OF CORTISONE ON THYROID Methyl thiouracil was given by mixing 0-2 g with every 100 g of food. This quantity of methyl thiouracil was the maximum that the rabbits would eat, but was not enough to cause adequate changes in thyroid function. An additional quantity was given by daily subcutaneous injections, each of 10 mg of methyl thiouracil in 5 ml. of water. Sodium DL-thyroxine was given intramuscularly as a 0.001% solution in m/50-sodium carbonate, each injection containing 50,tg of thyroxine. A fresh solution of thyroxine was used for each set of injections. The radio-iodine was given intravenously to eliminate variations in the rate of absorption from the injection site. Cortisone, as an aqueous suspension, was given daily by intramuscular injections each of 15 mg, the control rabbits receiving the same volume of the suspending solution. The amounts of thiouracil, thyroxine and cortisone used in these experiments were found by preliminary tests to be sufficient, when given continuously for 2 weeks, to reduce thyroid uptake of radio-iodine to about half the normal value. RESULTS The effect of thiouracil One pair of litter-mates was used. Before the thiouracil was started both rabbits were given a test dose of 12,uc of radio-iodine. The radio-iodine content of the thyroid was then determined at 24 hr and the loss of radioiodine from the thyroid was measured during the next week. Then, during Test (6 weeks, 45 s 44 ~~~~-, ~~~~~~~~~~ ~~~~~~~afte r *f < 7 4 S 75th test) 289 Weeks Fig. 1. Arrangement of radio-iodine tests during thiouracil and control periods without. Heavy black line indicates a period of. Arrow with horizontal line indicates an uptake test in both rabbits, followed by measurement of the rate of fall of radio-iodine in the thyroid. consecutive periods of 3*weeks the rabbits alternately received thiouracil, as in Fig. 1. The uptake of radio-iodine by the thyroid was tested in both rabbits at the beginning of the 3rd week of each period, and the loss of radio-iodine was followed in both until the end of the period (Fig. 1). Finally, both rabbits were tested 6 weeks after the 5th test. At each pair of tests made during the period, the treated animal was given 24,uc of radio-iodine ancd the untreated one was given 12,uc. The larger dose was given to the treated animal in order to make the total radiation dosage delivered to the thyroids of the two animals as nearly equal as possible. At each pair of tests during the period, the thyroid of the treated animal took up a smaller proportion of the dose than the untreated animal's thyroid and the treated animal's thyroid showed the more rapid rate of loss (Table 1). Moreover, in the treated animal the uptake of radio-iodine was less, PH. Cxx. 19

3 290 N. B. MYANT TABLE 1. Percentage uptake and rate of loss of radio-iodine with and without thiouracil Test occasion Rabbit, I_ no Uptake as % of dose Half-period of loss (in days) Normal type: test without ; heavy type: test with x E X X.50 I I I I 0 ~ ~ ~ ~ ~ ~ ~ ~~~ ~~~I -1 '~~~ 345 Days ~ ~ ~ ~ I _ I Days It Weeks Fig. 2. Output of radio-iodine from the thyroid of rabbit no. 44 (tests 2 and 3) showing the effect of thiouracil on the rate of loss of 1311 from the thyroid. Thick black line: thiouracil ; arrow: test dose of 131L. Log. scale. and the rate of loss was more rapid, than in the tests on the same animal made before and after the period. A typical pair of consecutive tests is illustrated in Fig. 2. The effect of the thiouracil was to reduce the 24 hr uptake from an average of 22-7 % ± 1-43 in the eight tests maae without to 11-1 % ± 3-0, in the four tests made at the second week of, and to shorten the half period of fall from 4-12 days to 2-25 days It may be argued that tests 45 (3) and (5) and 44 (4) and (6) should be excluded from the estimate of the average value in the normal state because at 2 weeks after the end of the previous course of the thyroid might not have returned to normal. However, if these values are excluded, the averages would not be appreciably changed (22-8%+1-2 and 3-76 days ± 0-64). If, therefore, cortisone inhibition is due to a thiouracil-like action, an amount of cortisone sufficient to halve the 24-hr uptake, when given over a similar length of time, should almost double the rate of fall of radio-iodine in the thyroid.

4 EFFECT OF CORTISONE ON THYROID Tahe effect of thyroxine Four litter-mates were used, two animals receiving the while the other two acted as controls. The arrangement of the periods and radio-iodine tests was the same as that used in the thiouracil experiments. As in the previous experiment, the treated animals were given 24,uc of 1311, and the untreated animals were given 12,uc. Thyroxine is known to be broken down by the body with the release of its iodine atoms as iodide. The rate of breakdown is not known exactly, but experiments with thyroxine labelled by radio-iodine suggest that the mean life of thyroxine molecules in the body tissues may be about 2 or 3 days in humans (Myant & Pochin, 1950). Therefore, in any experiment in which thyroxine is given continuously for more than a few days, the amount of iodide in the animal's body is likely to be increased during part of the time when the observations are made. For this reason, the control animals in the thyroxine experiments were given daily injections of 40,ug of potassium iodide, containing an amount of iodine about equal to the amount of iodine in the 50,tg of thyroxine given to the treated animals. None of the animals received KI at the first test, made 1 week before the beginning of the first period, and none was given KI after the end of the 5th test period. At each set of tests, both the treated animals took up less radio-iodine in their thyroids than the control pair, and the radio-iodine was lost more slowly from the thyroids of the treated animals (Table 2). The uptake of radio-iodine TABLE 2. Percentage uptake and rate of loss of radio-iodine with and without thyroxine Test occasion Rabbit, A no Uptake as % of dose * * * Half-period of loss (in days) i * Normal type: test without (test occasions 1 and 6) or with KI only; heavy type: test with thyroxine. 291 by the control pair given KI was consistently less than the uptake in the two sets of tests without KI (tests 1 and 6), suggesting that part of the action of thyroxine, in the quantities used in this experiment, was due to iodide resulting from its breakdown in the body. If this is so, then the amount of inhibition caused directly by the thyroxine can best be estimated by a comparison between the values in the tests with thyroxine and those with KI. 19-2

5 292 N. B. MYANT The uptake in the eight tests with thyroxine averaged 5-7 ± 0-80 of the dose at 24 hr and the half period of fall averaged 12-8 days + 1F94, compared with / and 4*8 days in the eight tests with KI. Thus, if cortisone inhibition is due to a thyroxine-like action, a continued dose of cortisone sufficient to reduce the uptake of radio-iodine to 40% of the normal value should reduce the rate of output to about a third of the control rate. The effect of cortisone Three pairs of litter-mates were used. The arrangement of the tests and periods of are shown for the first pair in Fig. 3, the groups of tests being related to the periods in four different ways. (1) Four tests before any had been given (38 (1); 40 (1), (2) and (3)). (2) Four tests during the 3rd week of a course of. (3) Three tests during the 3rd week after the end of a course of (38 (3) and (6); 40 (5)). (4) Three tests during the 6th week after the end of a course of (38 (4) and (7); 40 (6)). Test I I I I I I I I I I 1 I I I I I ' I Weeks Fig. 3. Arrangement of tests with and without cortisone. Symbols as in Fig. 1. The same arrangement was used for the other two pairs of rabbits. As in the experiments with thiouracil and thyroxine, the treated animals were given 24,uc of radio-iodine and the untreated animals were given 12,uc. In the twelve pairs of tests made during a period of (i.e. in the test occasions (2), (4), (5) and (7)) the percentage uptake by the treated animals averaged / compared with 25-2% in the untreated animals (Table 3), representing an average inhibition to about 40% below the control value. This estimate of the degree of inhibition is justified only if thyroid function was at the normal level in those animals which were used as controls, but which had already had one or more courses of. The true amount of inhibition might, for instance, be overestimated if there were a rebound increase in thyroid activity after a period of inhibition or it rfuight be underestimated if thyroid function did not recover fully for several weeks. In the whole set of control values, the error due to either of these factors is unlikely

6 EFFECT OF CORTISONE ON THYROID 293 TABLE 3. Percentage uptake of a dose of radio-iodine with and without cortisone Test occasion Rabbit no Mean Normal type: test without ; heavy type: test with. to be large, because there was not a significant difference between the averages of the values before and after (see next section). In the same twelve pairs of tests the half period of loss averaged 4-5 days , compared with 2-51 days in the untreated animals from the same test pairs (Table 4). Thus, the effect of a course of cortisone sufficient to TABLE 4. Half period of loss (days) of radio-iodine in the thyroid with and without cortisone Test occasion A Rabbit, no Mean Normal type: test without ; heavy type: test with. TABLE 5. Comparison of effects of thiouracil thyroxine and cortisone on 24 hr uptake and rate of loss 24 hr uptake Rate of loss* A,A Treatment Control With % change Control With % change Thiouracil Thyroxine Cortisone * ExpresEed as reciprocal of half period of loss. inhibit the thyroid uptake of radio-iodine to 40 % below the control value is to decrease the rate of loss to about 55 % of the control rate. The effects of thiouracil, thyroxine and cortisone are compared in Table 5. After-effects of cortisone inhibition. Some of the percentage uptake values in the later control period were much higher than those in the tests made before

7 gi'est roup 294 N. B. MYANT any had been given (compare, for example, test (1) with (4) and (6) in rabbit 38). This suggests the possibility of a rebound increase in thyroid uptake after a period of inhibition. However, analysis of the whole group of thirty control observations (Table 6, test groups (1), (3) and (4)) shows that there was not a significant difference between the average uptake in the twelve tests made before and the average uptake either at the 3rd or 6th week after the end of a course of. Similarly, there was no significant difference between the average rates of loss in tests made before and after with cortisone (Table 6). TABLE 6. Period of test 1 Before any 2 3rd week of 3 3rd week after 4 6th week after Average uptake and rate of lose of radio-iodine by the thyroid in groups of tests made before, during, and after cortisone Mean Significance of the half No. Mean difference between the means* period of uptake A t A of loss tests (%) Group 2 Group 3 Group 4 (days) ± Sig. Not sig. Not sig (P<0-01) (P>0.2) (P>0-1) Sig. (P <0-05) Sig. (P <001) 4-5± ± *8± 0*25 Significance of the difference between the means* Group 2 Group 3 Group 4 Sig. (P<0 01) Not sig. (P>0-1) Not sig. (P>0-1) - sig. (P <0-05) Sig. (P <0-01) * The values in these columns give the probability that the difference between the means could have occurred by chance. Calculations baesed on the test for differences between small samples of unequal size. The variations in the values from the control periods may, therefore, be explained by random errors of measurement; but we cannot exclude the possibility that cortisone inhibition tends to be followed by a rebound increase in some rabbits but not in others. Further tests were made on rabbit 38, in which this effect was most strongly suggested, but values as high as those in tests (4) and (6) were not observed again (see Table 3). DISCUSSION The mechanism of cortisone inhibition. In these experiments, cortisone given continuously in doses large enough to inhibit the thyroid uptake of radioiodine caused a diminution in the rate at which radio-iodine was lost from the thyroid. The low rate of loss in the treated animals was not an effect of the radiations from the test doses because the thyroids of the treated and control animals received about the same radiation dose. Moreover, the thyroids of the thiouracil-treated animals were irradiated to about the same extent, but in these there was an increased rate of loss of radio-iodine. The

8 EFFECT OF CORTISONE ON THYROID 295 increase in the rate of loss of radio-iodine brought about by thiouracil has been shown by others (Wolff, 1951; Perry, 1951). In experiments in which the thiouracil is continued after the initial uptake of radio-iodine, the effect is probably due to two causes. First, thiouracil blocks the re-entry into the thyroid of radio-iodine which has been released from the periphery by the breakdown of radio-active hormone; and, secondly, the output of thyrotrophin from the pituitary is increased and this increases the rate at which the thyroid discharges its hormone. The diminished rate of loss in the cortisone-treated animals cannot be due to an increase in the rate of re-entry of radio-iodine because the iodineaccumulating function of the thyroid must have remained at an abnormally low level throughout the 3rd week of the. It follows, therefore, that cortisone reduces the rate at which radio-iodine is released from the thyroid. In this respect, the inhibitory action of cortisone is qualitatively similar to that of thyroxine. Furthermore, a rough quantitative comparison between the effects of cortisone and thyroxine shows that for a given degree of inhibition of uptake by the thyroid the effect on the rate of loss of radio-iodine is about the same. These results suggest, therefore, that cortisone and thyroxine act at the same level in the chain of events controlling the formation and secretion of the thyroid hormone. The action of a substance which diminishes the rate of secretion of the thyroid hormone is most simply explained by supposing that the pituitary stimulus to the thyroid is reduced. There is ample experimental evidence that this is the way in which thyroxine inhibits the thyroid, but there is disagreement about the extent to which thyroxine inhibition is due to blocking the release of thyrotrophin from the pituitary (Griesbach & Purves, 1945), or to an action against thyrotrophin after it has reached the circulation (Cortell & Rawson, 1944). Either or both of these actions would explain the effects of cortisone observed here. Hill, Reiss, Forsham & Thorn (1950) have shown that cortisone inhibition of the thyroid can be reversed by thyrotrophin, and they have concluded that cortisone probably acts by inhibiting the pituitary. But their results could be explained equally well by a quantitative reaction between thyrotrophin and cortisone. Woodbury, Ghosh & Sayers (1951) have reported that cortisone reduces the thyroid-stimulating activity of thyrotrophin injected into the hypophysectomized rat. Since the amounts of cortisone they gave were comparable, on a weight for weight basis, with the amounts given to the rabbits in these experiments, the whole of the cortisone effect on the rabbit's thyroid could be explained by an action against thyrotrophin after it has been secreted. Observations, to be reported later, on the histological changes in the pituitaries of cortisone-treated rabbits support this explanation. After-effects of cortisone inhibition. It would be reasonable to expect that after a period of inhibition continued for long enough to reduce the concentra-

9 N.BMYN N. B. MYANT tion of thyroxine in the circulation and thus to stimulate the pituitary, there might be an increase in thyroid activity above the normal level when the inhibitory influence is withdrawn. The experiments described here were designed with this possibility in mind. It is known that the pituitary response to thyroxine deficiency induced by thyroidectomy is well developed in rabbits by the 3rd or 4th week (Griesbach, 1952, personal communication). If cortisone inhibits the pituitary stimulus to the thyroid, any increase in pituitary activity that may be caused by the inhibition of thyroid function, would not begin to affect the thyroid until after the cortisone had been stopped. It seemed likely, therefore, that a post-inhibition rebound would be most readily detectable between the 3rd and 6th weeks after the end of the. Tests at these intervals did not provide definite evidence of a rebound increase, although the average values after were higher than those before. A small rebound effect at these intervals cannot, therefore, be excluded, and it is also possible that a significant effect might have been found if it had been looked for at earlier intervals after the end of the. SUMMARY 1. A comparison was made of the inhibitory actions of thiouracil, thyroxine and cortisone on the uptake and rate of loss of radio-iodine by the thyroids of living rabbits. 2. Cortisone, in doses sufficient to reduce the percentage uptake of a test dose of radio-iodine, reduces the rate at which radio-iodine is discharged from the thyroid. In this respect, cortisone resembles thyroxine but differs from thiouracil, which increases the rate of discharge of radio-iodine. 3. A rebound increase in thyroid uptake after cortisone inhibition may occur in some rabbits. 4. The possible ways in which cortisone may inhibit the thyroid are discussed. I am grateful to Mr W. West for making many of the measurements. REFERENCES ASTWOOD, E. B. (1945). Chemotherapy of hyperthyroidism. Harvey Lect. 40, CORTELL, R. & RAWSON, R. W. (1944). Effect of thyroxin on response of thyroid gland to thyrotropic hormone. Endocrinology, 35, FREDEICKSON, D. S. (1951). Effect of massive cortisone therapy on thyroid function. J. clin. Endocr. 11, 760. GRIMESBACH, W. E. & PIuRvEs, H. D. (1945). The significance of the basophil changes in the pituitary accompanying various forms of thyroxine deficiency. Brit. J. exp. Path. 26, HILL, S. R., REIss, R. S., FoRsH&M, P. H. & THORN, G. W. (1950). Effect of adreno-corticotropin and cortisone on thyroid function: thyroid-adrenocortical interrelationships. J. clin. Endocr. 10,

10 EFFECT OF CORTISONE ON THYROID 297 K&ATnG, F. R., RAWSON, R. W., PEACOCK, W. & EvANs, R. D. (1945). Collection and loss of radioactive iodine compared with anatomic changes induced in thyroid of chick by injection of thyrotropic hormone. Endocrinology, 36, MONEY, W. L., KiRscHNR, L., KRAITZ, L., MERRILL, P. & RAWSON, R. W. (1950). Effect of adrenal and gonadal products on weight and radio-iodine uptake of the thyroid gland in rat. J. clin. Endocr. 10, MYANT, N. B. (1953). The uptake of radio-iodine by the rabbit's thyroid measured in vivo. J. Phy8iol. 120, MYANT, N. B. & POCHIN, E. E. (1950). The metabolism of radiothyroxine in man. Clin. Sci. 9, PmRY, W. F. (1951). A method for measuring thyroid hormone secretion in the rat with its application to the bio-assay of thyroid extracts. Endocrinology, 48, WoLFF, J. (1951). Some factors that influence the release of iodine from the thyroid gland. Endocrinology, 48, WOODEBURY, D. M., GHOSH, B. N. & SAYERS, G. (1951). Modification of TSH action by ACTH and cortisone in hypophysectomized rats. J. clin. Endocr. 11, 761.