(Received 27 November 1953)

Transcription

1 51 J. Physiol. (I954) I25, 5I-55 CHANGES IN THE COLLAGEN CONTENT OF THE THYROID IN RATS TREATED WITH THIOURACIL BY MARGARET L. R. HARKNESS, R. D. HARKNESS AND JOYCE E. SANTLER From the Departments of Physiology and of Anatomy and Embryology, University College, London (Received 27 November 1953) A number of previous investigations have been made on the relation of growth of cellular tissue to growth of intercellular supporting tissue as judged by chemical estimation of collagen. In nerve and regenerating liver (Abercrombie & Johnson, 1946, 1947; Harkness, 1952; Harkness & Harkness, 1953 a) the process of collagen formation is slow compared with the other changes in the tissue, whereas in the pregnant uterus (Harkness & Harkness, 1953 b) it is relatively rapid and follows a course similar to the change in total weight. It seemed of interest to extend these observations to other tissues, and we have accordingly investigated the change in the collagen content of the thyroid gland of the rat treated with thiouracil. Collagen formation was found to be variable but generally slower than increase in the weight of the thyroid. A considerable amount of the collagen formed under the influence of thiouracil treatment disappeared when the treatment was discontinued. METHODS The animals used were adult male albinos of the local strain. They were kept at a temperature of approximately 210 C in cages of six. They were given at all times free access to water and food, M.R.C. diet no. 31 (Parkes, 1946). Thiouracil (005 %, w/v) was given in the drinking water. For the removal of the thyroid the animals were anaesthetized with ether. In some cases the whole thyroid was used for collagen estimation, in others only half, the other half being used for a histological investigation the results of which will be published separately. Collagen was estimated by the method of Neuman & Logan (1950). Tyrosine was estimated on some samples by the method of Medes (1932) and found to be present in such small amounts as to require negligible correction of the hydroxyproline values. No such correction has been made. RESULTS Two main experiments were done. In the first, rats were killed 3, 6, 12, 24 and 50 days after the commencement of thiouracil treatment. Two separate groups of rats were used (A and B, Table 1). Treatment was started at different times 4-2

2 52 MARGARET L. R. HARKNESS AND OTHERS so that all rats in a given experiment (A or B) were killed on the same day. The results are given in Table 1 and Fig. 1. Thiouracil treatment for 24 days increased the weight of the thyroid to about 4 times the control value in both TABLE 1. Effect of thiouracil on weight and collagen content of the thyroid Duration of thiouracil treatment (days) Control r A rats Expt. A Body weight at ±9 243± Wet weightof 16-5± ±2-1 31*9± ± ±8-5 Total collagen of 0*350±0*030 0*338+0* ± Expt. B Body weight at Wet weight of ± Total collagen of 0-369± ' The estimate of variation is the standard error of the mean. There were four rats in each group for each experiment (A and B), except for the 12-day group of Expt. A which contained three rats. 400 A.5V v 2000 U)C 0 O II Duration of treatment with thiouracil (days) Fig. 1. Collagen content and weight of the thyroid gland after varying duration of treatment with thiouracil. The letters A and B refer to the separate experiments of Table 1. groups A and B. The effect on the collagen content of the thyroid was more variable than was the increase in total weight of the gland, but in both groups there was a significant increase in collagen by the end of the experiment, though less increase than in gland weight. In both groups the weight of the

3 COLLAGEN IN THYROID 53 gland was first significantly above the control value at 6 days (t test, P < 0.05, Fisher, 1946). The collagen content of the gland was not significantly above the control value till the 24th day in group A, and the 12th day in group B. In the second main experiment rats were treated for 30 days with thiouracil. A sample of the treated rats was then killed and the treatment of the remainder stopped. Half of this remainder was killed 7 days, and half 30 days after the end of the treatment. Two groups of control untreated rats were used, one killed at 30 days and the other at the end of the experimental period. The results are given in Table 2. Increases in weight and collagen content of the TABLE 2. Reabsorption of collagen from the thyroid after cessation of thiouracil treatment Days from start of experiment Control, Control,, - 30-day 60-day Body weight at 286±8 331±11 248±11 264±8 326±7 Wet weight of 15-2± ±0-6 44*8±2t8 28*1± ±1-8 Total collagen of 0-482± ± ± ±0-060 Thiouracil treatment was stopped 30 days from the start of the experiment. The estimate of variation is the standard error of the mean. There were eight rats in each group of control and experimental animals. thyroid were similar to those found in the first experiment. When the thiouracil treatment was stopped the weight of the thyroid fell and the new collagen disappeared. The disappearance seems to have been rapid at first, but subsequently, between the 37th and 60th day, slower. It appears to have been incomplete, though in fact the 60th day figure is not significantly above either of the control values (P > 0-05, t test loc. cit.). We can offer no explanation for the anomalously high 30-day control value (cf. Table 1). DISCUSSION It is generally agreed that the action of thiouracil on the thyroid gland is both direct and indirect. A direct inhibition of thyroxine production results in an increase in output of thyrotropic hormone by the anterior lobe of the pituitary gland, which in turn causes the thyroid gland to increase in size. We may consider that the changes which we have investigated are mainly the result of the action of thyrotropic hormone on the thyroid. We have observed an increase in the total quantity of collagen present in the thyroid glands of animals treated with thiouracil. The increase, however, differed considerably in degree in different experiments. By contrast the effect of thiouracil on the increase in weight of the gland varied less. So long as we have no information to enable us to break down this added weight into individual components, there is an element of uncertainty in its use as an index of

4 54 MARGARET L. R. HARKNESS AND OTHERS growth. With this proviso we may say that it appears that the growth of collagen in the thyroid was generally slower than that of the tissue as a whole, and in this respect the changes resemble those found in the liver after partial hepatectomy, rather than those in the uterus during pregnancy (Harkness, 1952; Harkness & Harkness, 1953a, b). However, of more interest than the relation ofincrease in collagen and weight is the disappearance of collagen which takes place after the cessation of treatment with thiouracil. Disappearance of autologous collagen has been shown to take place from rat liver after withdrawal from the diet of carbon tetrachloride used to produce the cirrhosis (Morrione, 1949). It has also been recorded in atrophic limbs (Slack, 1953), and in the puerperal rat uterus (Harkness & Harkness, 1953 b). Such disappearance could represent a shift in dynamic equilibrium as does the disappearance of protein from the liver in fasting for example. If a substance is in such an equilibrium the quantity present can be reduced either by an increase in the rate of breakdown or by a reduction in the rate of synthesis. The latter mechanism would be the simpler and the easier to accept; and, if synthesis ceased, one would expect the substance concerned to disappear at a rate proportional to the turnover rate as measured by isotopic studies. However the turnover rate of collagen has been found to be very low in those tissues of the rat in which it has been investigated, viz. tendon, skin, liver, and bone (Neuberger, Perrone & Slack, 1951; Neuberger & Slack, 1953). Also, owing to the slowness of the observed turnover of liver collagen, a cessation of synthesis cannot readily account for the disappearance of collagen from the liver found by Morrione (1949); turnover rates similar to those in the above tissues would also be too low to account for the disappearance of collagen from uterus and thyroid. However, as both these latter tissues undergo considerably greater changes in size and structure in the course of normal life than do the tissues on which isotopic studies have been made, it would not be unexpected to find a more rapid rate of turnover of collagen in them; nevertheless, it seems unlikely that it would be rapid enough to account for the observed disappearance of collagen. It seems more likely that the removal of collagen is an active process and probably integrated with other vital processes. The low turnover rates observed in isotopic studies show that native collagen is not in itself chemically unstable in the body. Yet collagen from extraneous sources disappears fairly rapidly if introduced into the tissues of the body. The stability of collagen in tendon, etc., is thus not due to any inherent difficulty in removing it. It would appear that it must normally be stabilized in some way by the surrounding cells as might be done for example by the secretion of material preventing access of proteolytic enzymes to the fibres. Thus native collagen is more resistant to trypsin than collagen which has undergone treatment which might be expected to allow freer access to the

5 COLLAGEN IN THYROID 55 enzyme (Sizer, 1949). Similarly, collagen of tendon which has been treated with hyaluronidase to remove polysaccharide material becomes soluble to a greater extent in dilute acids (Jackson, 1953). SUMMARY 1. The effect of oral administration of thiouracil for various times up to 50 days on the collagen content (estimated chemically) of the rat's thyroid gland has been investigated. 2. During treatment with thiouracil both the weight and collagen content of the thyroid gland increased, the former more than the latter. 3. After cessation of treatment with thiouracil collagen was reabsorbed, both the weight and collagen content of the gland decreasing. We should like to express our thanks to the Nuffield Foundation whose financial assistance made this work possible, and to Shirley M. Fitch and Winifred Stidworthy for technical assistance. REFERENCES ABERCOMBIE, M. & JOHNSON, M. L. (1946). Collagen content of rabbit sciatic nerve during Wallerian degeneration. J. Neurol. 9, ABEtCROMBIE, M. & JoHNsow, M. L. (1947). The effect of reinnervation on collagen formation in degenerating sciatic nerves of rabbits. J. Neurol. 10, FISHER, R. A. (1946). Statistical Method8for Research Workers, 10th ed., 13dinburgh and London: Oliver and Boyd. HARKNESs, M. L. R. & HARNE8SS, R. D. (1953a). Further observations on collagen in the liver of the rat after partial hepatectomy. J. Physiol. 120, 6P. HARKNESS, M. L. R. & HARKNEss, R. D. (1953b). Collagen in the reproductive tract of the rat during pregnancy and lactation. J. Physiol. 120, 7P. HARKNESs, R. D. (1952). Collagen in regenerating liver of the rat. J. Physiol. 117, JACKSON, D. S. (1953). Chondroitin sulphuric acid as a factor in the stability of tendon. Biochem. J. 54, MEDEs, G. (1932). A new error of tyrosine metabolism: tyrosinosis. The intermediary metabolism of tyrosine and phenylalanine. Biochem. J. 26, MORIONE, T. G. (1949). Factors influencing collagen content in experimental cirrhosis. Amer. J. Path. 25, NEUBERGER, A., PERRONE, J. C. & SLACK, H. G. B. (1951). The relative metabolic inertia of tendon collagen in the rat. Biochem. J. 49, NEIuBERGER, A. & SLACK, H. G. B. (1953). The metabolism of collagen from liver, bone, skin and tendon in the normal rat. Biochem. J. 53, NEUMAN, R. E. & LOGAN, M. A. (1950). The determination of collagen and elastin in tissues. J. biol. Chem. 186, PARKES, A. S. (1946). Feeding and breeding of laboratory animals-rat and mouse cubes and cube containers. J. Hyg., Camb., 44, SIZER, I. W. (1949). The action of trypsin and other proteases on collagen. Enzymologia, 13, SLACK, H. G. B. (1953). Metabolism of collagen in the rat. In On the Nature and Structure of Collagen, ed. Jackson, S. F. & Randall, J. T. London: Butterworth.