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THYROID PROLIFERATIVE POTENTIAL AS A FUNCTION OF AGE

THYROID PROLIFERATIVE POTENTIAL AS A FUNCTION OF AGE Laboratory of Rudiobiology, Uiiiaersity of California Medical Center, San Francisco, Califoriiia, U.S.A. (Receiivd 28 October 1968; rerision receii>ed December 1968) 30 ABSTRACT The effect of a goitrogenic stimulus on thyroid weight and thyroid cell 3HTdR labeling of Sprague-Dawley rats varying from 2 to 40 weeks of age was determined. Propylthiouracil ad libitum in drinking water produced a spurt in follicle cell labeling index and thyroid weight evident after 24 hr for all age groups. The increase in labeling index reached a peak at 5-7 days and then decreased to a level a few times greater than that of the normal unstimulated thyroid. The tritiated thymidine labeling index for thyroid follicle cells and the effect of PTU thereon was determined for August male rats 01’3 days to 12 weeks of age. In the older rats, the follicle cell labeling index rose to 5-6% after 4-5 days of PTU treatment and then slowly fell to about 1 %, For the unstimulated control rat of comparable age, the labeling index was about 0.1 %. At all ages the thyroid showed a rapid response to PTU. Examination of the time sequence of mitotic labeling showed that the DNA synthesis period was 7.5 hr for normal 2-week-old rats and for 10-12-week-old rats that had received PTU for 4 days. There was no second wave of labeled mitoses in either group during the 48-hr interval studied. From the curve of thyroid weight DS time on PTU and from the labeled mitoses curve, inferences regarding the minimum fraction of proliferating follicle cells in the stimulated ‘adult’ rat thyroid were made. INTRODUCTION There is evidence suggesting that the thyroid in young and in old individuals responds differently to irradiation, insofar as neoplasia is concerned. However, the basis for this is not clear. Studies have shown a higher incidence of neoplasms in thyroids exposed to irradiation in infancy than would be expected in a non-irradiated population (Pincus, Reichlin & Hemplemann, 1967; Hemplemann et al., 1967). The high incidence of neoplasia found in the accidentally I3’I exposed Marshallese children may be related to the higher radiation doses received by the infant thyroid as compared with the adult thyroid (Robbins, Rall & Conard, 1967). Such a differential dose effect, however, does not explain the observaCorrespondence: Professor Glenn E. Sheline, Department of Radiology, University of California Medical Center, San Francisco, California 94122, U.S.A. Glenn E. Sheline tions of Sheline e t a ] . (1962). In the latter study of radioiodine-treated patients, each patient received approximately the same dose of l3lI per unit weight of thyroid gland, but an inverse relationship between age at time of treatment and incidence of neoplasia was found. DeLawter & Winship (1963), in a study of the effects of external X-ray therapy, found that the younger the patient at time of exposure, the greater the likelihood of developing carcinoma of the thyroid. Doniach (1957) failed to find a difference between the radiation sensitivity of the weanling rat and the adult rat thyroid as indicated by l 3 I I uptake and thyroid response to goitrogen. To explain the discrepancy between this and observations on the apparent radiosensitivity of infant human thyroid tissue, he suggested that small doses of irradiation to the infant thyroid may initiate a carcinogenic process and that tumor formation is promoted by subsequent cellular proliferation. Others have shown that although the adult thyroid can respond to stimulation by increase in weight, mitotic index, and number of follicular and stromal cells (Doniach & Logothetopoulos, 1955;Santler, 1957), it normally undergoes little cell replication (Leblond & Walker, 1956; Leblond, Messier & Kopriwa, 1959). Leblond & Walker (1956) compared the thyroid mitotic index with change in body weight and concluded that the thyroid of the 200-250 g rat does not undergo cell renewal. The low mitotic rate in the adult human thyroid, an organ of essentially constant weight, suggests a similar lack of cell renewal. Thus, in the adult, radiation-induced changes might find no opportunity for expression. There are no data to indicate whether the decrease in a fraction of cells undergoing mitosis in the adult thyroid is due to a lengthening of the cell cycle or to a decrease in the fraction of cells in the proliferative compartment. The present report is a study of age-related differences in thyroid follicle cell replication. It is the first step in an investigation of age-related differences in response to irradiation. The first part of this paper deals with changes in thyroid weight, follicle cell incorporation of tritiated thymidine (3HTdR), and response to stimulation by a goitrogenic substance as a function of age of the rat. The second part of the study compares 3HTdR uptake by thyroid follicle cell nuclei and the passage of such cells through mitosis in young normal glands and adult goitrogenically-stimulated glands. Because of the low labeling index of adult thyroid cells, a direct comparison of young and adult normal cells was not made. MATERIALS A N D METHODS Part Z Male Sprague-Dawley rats of 2-40 weeks of age were used. They were caged in groups of two to six depending upon size with free access to a diet of Purina mouse breeder chow and drinking water. Propylthiouracil (PTU) was administered to some groups in 0.1 % concentration in the drinking water; initially, to encourage drinking, 1 % sucrose was also added. The sucrose, however, appeared to have no influence on water intake and its use was discontinued. In the first phase of the study, rats of 2,4, 12 and 36 weeks of age were randomized into three series with subgroups of four to six animals each. One series served as controls. In the other two, the rats were placed on PTU and sacrificed after either 1 week or 4 weeks. Thus, the animals ranged from 3-40 weeks of age at sacrifice. Thirty minutes prior to sacrifice, each rat received an intraperitoneal injection of 1 microcurie (1 pCi) of 3HTdR/g body weight. Immediately after death the thyroid was dissected from the trachea, weighed, and Thyroid proliferative potential as a function o age f placed in neutral buffered formalin. A dissection microscope was used for the smaller glands. The thyroids were subsequently imbedded in paroplast and cut into 3 p thick sections. Standard hematoxylin and eosin slides were made. Radioautographs were prepared with Kodak AR 10 stripping film. After exposure, up to 8 weeks as necessary, the films were developed and the sections stained with hematoxylin and eosin. The number of individual grains over the labeled nuclei of 1000 follicular cells was tabulated for each thyroid. Nuclei with three or more grains were assumed to be labeled by 3HTdR. Preliminary examination of the radioautographs suggested that there was little or no difference between the labeling index of follicular cells near the periphery and those in the central portion of the thyroid. An effort was made, howcver, to include various areas in the counting of each thyroid section. I t appeared, but was not quantitated, that stromal cells were labeled with a frequency roughly similar to that of the follicular cells. In the second phase of the study, randomized groups of six animals each were placed on PTU at various ages and sxrificed at such intervals thereafter that they were either 7 or 40 weeks of age. The time o n PTU ranged from I to 28 days. The thyroids were studied as described above. Part I1 The rats were newborn to 3-month-old August males bred in the colony of the Biophysics Department, Institute of Cancer Research, Surrey, England. They received M.R.C. diet 418 and water ad libitirm. The drinking water for some of the animals contained 0.025% PTU. Animals to be sacrificed within 24 hr of starting PTU also received ml of the PTU solution by stomach tube at time zero. Except for studies of labeled mitoses, each animal received 0.5 pCi/g body weight of 3HTdR intraperitoneally 1 hr before sacrifice. Rats used for labeled mitosis studies were given 1 pCi/g and sacrificed a t intervals up to 48 hr thereafter. After killing with ether, the thyroid glands were dissected, weighed, and placed in neutral formalin. In rats less than 3 weeks of age the trachea and attached thyroid were dissected and placed in neutral formalin as a unit without effort to identify the gland. For radioautographs 4 p paraffin sections were mounted on slides and dipped in a 50% solution of llford K-5 nuclear emulsion. The slides were dried and stored at 4°C for periods of 2-6 weeks. The emulsion was then developed in Kodak D 19 B. Sections for counting labeled mitoses were stained with Feulgen and the others with hematoxylin and eosin. Fifty oil immersion fields were examined and the number of labeled follicle cell nuclei counted. In every fifth field the total number of follicle cell nuclei was determined; this usually ranged from 150 to 225 per field. The follicle cell labeling index (FCLI) was then calculated. Individual animals of the same age and treatment occasionally showed a 2- to 3-fold variation of FCLI. This variation appears to represent differences between animals since in ten rats where duplicate slides were counted the difference between duplicates did not exceed 25 %. To determine the follicle cell labeled mitosis index (FCLMI), oil immersion fields were examined until twenty-five follicle cell nuclei in metaphase or anaphase were found. RESULTS AND DISCUSSION In Fig. 1, rat body weight is shown as a function of age and time treated with PTU. These curves have two obvious components. Initially. growth was comparatively rapid. After about Glenn E. Sheline 10-12 weeks there was an abrupt change to a lower growth rate which continued for the duration of the study, namely 40 weeks. In comparison to the controls, animals treated with PTU for 1 or 4 weeks exhibited a lower body weight, the difference being greater for the longer treatment period. However, PTU treatment did not seem to affect the shape of the growth curves. How much of the effect on body weight of the PTU treated animals was due to hypothyroidism, to the lowered fluid intake, or to some other influence of PTU is not known. It is known that hypothyroidism reduces growth rate. Furthermore, Lewis, Cheever & VanderLaan (1965) have shown that 0.03% PTU in Purina chow results in a disappearance of growth hormone from the rat pituitary. While the reluctance to drink was particularly Body weight ( 9 ) Thyroid weight (mg) Age ( w e e k s ) FIG.1. Body weight and thyroid weight of rats of differing ages. One group on PTU for 1 week (a) another for 4 weeks ( 0 ) ; A, control rats. and evident during the first 24 hr, it continued to a lesser extent for the full 4 weeks. The low fluid intake also meant a decreased consumption of sucrose. However, in later experiments when no sucrose was added to the water, there was a similar difference in weight between treated and control animals. Furthermore, groups of rats, not otherwise included in this report, in which the PTU concentration was reduced to 0.025% failed to grow normally. Dobyns & Didtschenko (1961) have also described an impairment of body growth in rats on 0.1 % thiouracil. Weight of the thyroid gland as related to the age of rat and time on PTU is also presented in Fig. 1. As with body weight, the thyroid weight of treated and control rats increased rapidly up to about 10-12 weeks of age and thereafter increased at a much lower rate. Histologic examination of slides from the control group showed that some of the increase in thyroid weight of the older animals was probably due to colloid storage. Since the thyroid cells became smaller with increased age, the apparent correlation between thyroid weight Thyroid prolqeratiue potential as a function of age gain and increase of colloid storage does not rule out the possibility of acontinuing increase in number of thyroid follicle cells. One week on propylthiouracil produced an increase in thyroid weight of 50-75 % irrespective of age. Three more weeks of PTU treatment caused an additional doubling of thyroid weight. Since response to PTU, especially in the older rats, involved a histologically evident increase in thyroid cell volume and a decrease in colloid storage, changes in thyroid weight cannot be directly related to numbers of cells present. The per cent of follicular cells which were labeled with 3HTdR is given in Fig. 2. In untreated control animals of 2-4 weeks of age, about 3 % were labeled. The labeling index G - cn -6 : 2 l ? 4- decreased rapidly to about 0.3 % at 6-8 weeks and remained at this level for the duration of the study. It is not evident whether continuation of labeling in older animals represents a replacement of follicle cells or a slowly expanding cell population. One week of treatment with PTU produced about a 5-fold increase in labeling in rats of 2-3 weeks of age, and a 20to 25-fold increase in the 36 -40-week-old group. After 4 weeks on PTU, the labeling index was still above the levels for control animals but considerably lower than in rats after only 1 week of PTU treatment. Regardless of age when started on PTU, these thyroids showed 1.1-1.4% of cells labeled after 4 weeks on the drug. Figs. 3 and 4 present body weights, thyroid weights, and labeling indices of rats on PTU for varying lengths of time, and sacrificed at 7 and 40 weeks of age. These ages were chosen so that one group would fall in the rapid and one in the slow growth periods. The results in these two age groups were similar in many respects. The maximum effect of PTU on thyroid growth rate occurred within the first few days after starting PTU. The labeling index began to rise after the first 24 hr and reached a sharp peak at 5 days in the older rats and at 7 days in the younger rats. The labeling index then rapidly decreased, and after 4 weeks on PTU it was between 1 and 2 % for both age groups. These observations are contrary to the suggestion of Crooks eta/.(1964) that, during the first 2 weeks of a goitrogenic challenge, thyroid growth Glenn E. Sheline Time on PTU (days) FIG.3 . Sprague-Dawley rats 7 weeks of age at sacrifice. Shows body weight (A), thyroid weight (0) and labeling index (0) various periods of time on PTU. for 15 20 25 Time on PTU (days) FIG.4. Sprague-Dawley rats 40 weeks of age at sacrifice. Shows body weight (A), thyroid and for weight (0) labeling index (0) various periods of time on PTU. Thyroid proliferative potential as a-function of age is mainly due to hypertrophy and that it is during the third and fourth weeks that the growth spurt is due to cellular division. Other authors have described an initial response to goitrogenic stimulus which was not maintained even though the goitrogen was continued. Doniach & Logothetopoulos (1955) administered 0.06 % propylthiouracil ad lihituriz in drinking water to 3-4-month-old rats and found the mitotic index to increase up to about 8 days, and then decrease to nearly zero by 24 days. Santler (1957) using adult male rats and 0.05 < methylthiouracil found an increase ; in mitotic index and in total number of mitoses per lobe for both follicular and stromal cells with peak values at about 6 days. The mitotic indices dropped thereafter. According to Santler, the total number of follicular and of stromal cells per lobe showed no increase between 24 and 50 days although mitoses were still present at the later time. Time on PTU (days) FIG.5. August male rats of 10-12 weeks of age. Thyroid weight, i standard deviation, shown l as a function of time on I’TU. If there is no change in the DNA synthesis period and in the fraction of proliferating cells, it can be inferred from the declining labeling index that there is a decrease in the proliferation rate of the follicle cells even though PTU treatment continues. It may be that the decrease in thyroid cell proliferation is due to hypertrophy and hyperplasia sufficient to raise the hormone output of the thyroid to such a level that the thyrotrophic hormone output of the pituitary is depressed. If the fall in labeling index with continuing PTU stimulation were due to a limited number of permissible cell divisions, one might expect the decrease in labeling of the younger thyroid to appear only after it had reached the size at which the adult thyroid exhibited this effect. Whatever the mechanism responsible for the decline, the labeling index remains above normal for at least 4 weeks. Figs. 5-8 present data obtained with the August male rats. Thyroid weight versus time on PTU for two separate experiments (four to five animals per point) is shown in Fig. 5. These rats were 10-12 weeks of age and weighed 175-225 g; they were past the phase of high FCLI, and presumably of rapid thyroid growth, and were considered as young adults. The initial Glenn E. Sheline Time on P T U (days) FIG. 6. August male rats of 10-12 weeks of age. Follicle cell labeling index, f l standard deviation, plotted against time on PTU. slope of the curve is rather flat; after 2 days on PTU, thyroid weight begins to increase rapidly. After about 7 days the slope again decreases. The slope of the curve at 4 days suggests a thyroid weight doubling time of about 5 days, whereas between 7 and 21 days the slope is consistent with a 16-day doubling time. Hyperplasia and hypertrophy of follicular and stromal cells and variations in colloid content are all reflected in thyroid weight. It is, therefore, difficult to translate measurement of weight into numbers of follicular cells. Santler (1957) described experiments in which adult male rats were given 0.05 % methylthiouracil in drinking water and measurements made of thyroid weight and of total number of follicular and stromal cells per lobe. If Santler’s data are averaged and an interpolation made for a treatment time of 4 days, one finds a 38 % increase in thyroid weight accompanied by an increase of only 19 % in number of follicle cells. If it is pertinent to apply these data to the presently observed 5-day doubling time for thyroid weight, the follicle cell doubling time in the present adult rat after 4 days on PTU would be about 10 days. Age at socrifice (weeks) FIG.7. August male rats. Follicle cell labeling index, $1 standard deviation, plotted against age at sacrifice for control animals (A) and for rats on PTU 4 days ( ) 0 . Thyroid proliferative potential as a function of age The per cent of labeled follicle cells for rats used in Fig. 5 was averaged for each PTU treatment time. The results are given in Fig. 6 . The FCLI was 0.1 % for control animals. After a 1-2 day lag, the curve rises sharply to an apparent peak at 4-5 days and then falls to a much lower value, but one still well above that of the control animals. The FCLI was 5.2% at 4 days and 1.2% at 21 days. The changes in FCLI corresponded in time with the changes in slope of the thyroid weight curve and are similar to the observations in SpragueDawley rats (Figs. 3 and 4). Fig. 7 shows the FCLI for untreated control animals and for animals receiving PTU for 4 days. These rats ranged from 3 days to about 12 weeks of age at time of sacrifice. The FCLI is about the same after 5 weeks of age in the PTU-treated rats as in the I-2-week-old rats without treatment. On the basis of these data, it was decided to use 2-week-old untreated and 10-12-week-old PTU-treated rats for the study of labeled mitoses. This would contrast ‘young’ thyroids with ‘older’ thyroids and each group should have a FCLl of about 5%. Non-stimulated older thyroids show very infrequent labeled mitoses and a comparison of ‘young’ and ‘old’ non-stimulated thyroids was not attempted. Time after pulse label ( h r ) FIG. 8. Follicle cell labeled mitosis index for 2-week-old untreated August male rats (A) and for 10-12-week-old treated August male rats (0). The treated group received PTU for 4 days prior to injection of 3HTdR. Fig. 8 presents the per cent of labeled mitoses (FCLMJ) found in thyroid follicle cells up to 48 hr after a single injection of 3HTdR. The labeled mitosis curves for 2-week-old and for 10-12-week-old rats are similar except that the curve for the older group is about 2 hr delayed. In the older group. PTU was started 4 days prior to ’HTdR administration and continued until sacrifice. Using the 50% point on the curves to delineate the various parts of the generation cycle, G2 i 3 mitosis time is 4.7 hr for the younger and 7.0 hr for the older rats. Synthesis time (S) is approximately 7.5 hr for each group. Histologically, it was noted that when the fraction of labeled mitoses begins to decrease, adjacent pairs of labeled nuclei occur with great frequency. Throughout the 48 hr, there is no indication of a second wave of labeled mitoses. Because of desynchronization and the rapidly changing growth kinetics, as shown in Figs. 2 and 3, it did not seem worthwhile to extend the curves for a longer time. For example. the mitotic activity in thyroids from 4-day PTU-treated rats would be quite different after 3 or 4 days than it was only a few hours after thymidine injection; hence, a second wave of labeled mitoses would give little information regarding the generation time applicable at the beginning of the experiment. Since the FCLI curves change less rapidly in Glenn E. Sheline rats of 1 week or less of age and in the older group on PTU for 2 weeks or more (Figs. 6 and 7), interpretation might have been simpler if such groups of animals had been used for the labeled mitosis study. This combination was ruled out because of the relatively small number of follicle cells available in the thyroid of rats of only a few days of age and because long periods of PTU treatment seemed to have adverse effects on the rats. In Fig. 8, it appears that the follicle cell generation time is at least 45 hr. Assuming that in the PTU-treated 10-1 2-week-old rats the follicle cell population doubling time, as derived utilizing Santler’s (1957) data, is 10 days, the per cent of follicle cells in the proliferation cycle is at least 20%. Jf the fraction undergoing proliferation were smaller, maintenance of this doubling rate would require a shorter generation time and a second wave of labeled mitoses would have appeared. Because of lack of data on young thyroid weights, a similar calculation cannot be made for the 2-week-old untreated rats. ACKNOWLEDGMENTS 1 would like to thank Professor Leonard F. Lamerton, Dr Gordon Steel and Professor Harvey Patt for many helpful suggestions and discussions, Dr Claire Shellabarger for some of the data of Figs. 5 and 6 , and Mrs Margaret Miller for her technical assistance. I also wish to thank Professor Lamerton for the opportunity to conduct Part 11 of this study in his laboratory at the Institute of Cancer Research, Surrey, England. In addition, I should like to express my appreciation to the Commonwealth Fund for financial assistance during my stay in England. This work was performed under the auspices of the U.S. Atomic Energy Commission. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Proliferation Wiley

THYROID PROLIFERATIVE POTENTIAL AS A FUNCTION OF AGE

S heline , G lenn E.
Cell Proliferation , Volume 2 (2) – Apr 1, 1969
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Publisher
Wiley
Copyright
1969 Blackwell Science Limited
ISSN
0960-7722
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1365-2184
DOI
10.1111/j.1365-2184.1969.tb00998.x
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Abstract

Laboratory of Rudiobiology, Uiiiaersity of California Medical Center, San Francisco, Califoriiia, U.S.A. (Receiivd 28 October 1968; rerision receii>ed December 1968) 30 ABSTRACT The effect of a goitrogenic stimulus on thyroid weight and thyroid cell 3HTdR labeling of Sprague-Dawley rats varying from 2 to 40 weeks of age was determined. Propylthiouracil ad libitum in drinking water produced a spurt in follicle cell labeling index and thyroid weight evident after 24 hr for all age groups. The increase in labeling index reached a peak at 5-7 days and then decreased to a level a few times greater than that of the normal unstimulated thyroid. The tritiated thymidine labeling index for thyroid follicle cells and the effect of PTU thereon was determined for August male rats 01’3 days to 12 weeks of age. In the older rats, the follicle cell labeling index rose to 5-6% after 4-5 days of PTU treatment and then slowly fell to about 1 %, For the unstimulated control rat of comparable age, the labeling index was about 0.1 %. At all ages the thyroid showed a rapid response to PTU. Examination of the time sequence of mitotic labeling showed that the DNA synthesis period was 7.5 hr for normal 2-week-old rats and for 10-12-week-old rats that had received PTU for 4 days. There was no second wave of labeled mitoses in either group during the 48-hr interval studied. From the curve of thyroid weight DS time on PTU and from the labeled mitoses curve, inferences regarding the minimum fraction of proliferating follicle cells in the stimulated ‘adult’ rat thyroid were made. INTRODUCTION There is evidence suggesting that the thyroid in young and in old individuals responds differently to irradiation, insofar as neoplasia is concerned. However, the basis for this is not clear. Studies have shown a higher incidence of neoplasms in thyroids exposed to irradiation in infancy than would be expected in a non-irradiated population (Pincus, Reichlin & Hemplemann, 1967; Hemplemann et al., 1967). The high incidence of neoplasia found in the accidentally I3’I exposed Marshallese children may be related to the higher radiation doses received by the infant thyroid as compared with the adult thyroid (Robbins, Rall & Conard, 1967). Such a differential dose effect, however, does not explain the observaCorrespondence: Professor Glenn E. Sheline, Department of Radiology, University of California Medical Center, San Francisco, California 94122, U.S.A. Glenn E. Sheline tions of Sheline e t a ] . (1962). In the latter study of radioiodine-treated patients, each patient received approximately the same dose of l3lI per unit weight of thyroid gland, but an inverse relationship between age at time of treatment and incidence of neoplasia was found. DeLawter & Winship (1963), in a study of the effects of external X-ray therapy, found that the younger the patient at time of exposure, the greater the likelihood of developing carcinoma of the thyroid. Doniach (1957) failed to find a difference between the radiation sensitivity of the weanling rat and the adult rat thyroid as indicated by l 3 I I uptake and thyroid response to goitrogen. To explain the discrepancy between this and observations on the apparent radiosensitivity of infant human thyroid tissue, he suggested that small doses of irradiation to the infant thyroid may initiate a carcinogenic process and that tumor formation is promoted by subsequent cellular proliferation. Others have shown that although the adult thyroid can respond to stimulation by increase in weight, mitotic index, and number of follicular and stromal cells (Doniach & Logothetopoulos, 1955;Santler, 1957), it normally undergoes little cell replication (Leblond & Walker, 1956; Leblond, Messier & Kopriwa, 1959). Leblond & Walker (1956) compared the thyroid mitotic index with change in body weight and concluded that the thyroid of the 200-250 g rat does not undergo cell renewal. The low mitotic rate in the adult human thyroid, an organ of essentially constant weight, suggests a similar lack of cell renewal. Thus, in the adult, radiation-induced changes might find no opportunity for expression. There are no data to indicate whether the decrease in a fraction of cells undergoing mitosis in the adult thyroid is due to a lengthening of the cell cycle or to a decrease in the fraction of cells in the proliferative compartment. The present report is a study of age-related differences in thyroid follicle cell replication. It is the first step in an investigation of age-related differences in response to irradiation. The first part of this paper deals with changes in thyroid weight, follicle cell incorporation of tritiated thymidine (3HTdR), and response to stimulation by a goitrogenic substance as a function of age of the rat. The second part of the study compares 3HTdR uptake by thyroid follicle cell nuclei and the passage of such cells through mitosis in young normal glands and adult goitrogenically-stimulated glands. Because of the low labeling index of adult thyroid cells, a direct comparison of young and adult normal cells was not made. MATERIALS A N D METHODS Part Z Male Sprague-Dawley rats of 2-40 weeks of age were used. They were caged in groups of two to six depending upon size with free access to a diet of Purina mouse breeder chow and drinking water. Propylthiouracil (PTU) was administered to some groups in 0.1 % concentration in the drinking water; initially, to encourage drinking, 1 % sucrose was also added. The sucrose, however, appeared to have no influence on water intake and its use was discontinued. In the first phase of the study, rats of 2,4, 12 and 36 weeks of age were randomized into three series with subgroups of four to six animals each. One series served as controls. In the other two, the rats were placed on PTU and sacrificed after either 1 week or 4 weeks. Thus, the animals ranged from 3-40 weeks of age at sacrifice. Thirty minutes prior to sacrifice, each rat received an intraperitoneal injection of 1 microcurie (1 pCi) of 3HTdR/g body weight. Immediately after death the thyroid was dissected from the trachea, weighed, and Thyroid proliferative potential as a function o age f placed in neutral buffered formalin. A dissection microscope was used for the smaller glands. The thyroids were subsequently imbedded in paroplast and cut into 3 p thick sections. Standard hematoxylin and eosin slides were made. Radioautographs were prepared with Kodak AR 10 stripping film. After exposure, up to 8 weeks as necessary, the films were developed and the sections stained with hematoxylin and eosin. The number of individual grains over the labeled nuclei of 1000 follicular cells was tabulated for each thyroid. Nuclei with three or more grains were assumed to be labeled by 3HTdR. Preliminary examination of the radioautographs suggested that there was little or no difference between the labeling index of follicular cells near the periphery and those in the central portion of the thyroid. An effort was made, howcver, to include various areas in the counting of each thyroid section. I t appeared, but was not quantitated, that stromal cells were labeled with a frequency roughly similar to that of the follicular cells. In the second phase of the study, randomized groups of six animals each were placed on PTU at various ages and sxrificed at such intervals thereafter that they were either 7 or 40 weeks of age. The time o n PTU ranged from I to 28 days. The thyroids were studied as described above. Part I1 The rats were newborn to 3-month-old August males bred in the colony of the Biophysics Department, Institute of Cancer Research, Surrey, England. They received M.R.C. diet 418 and water ad libitirm. The drinking water for some of the animals contained 0.025% PTU. Animals to be sacrificed within 24 hr of starting PTU also received ml of the PTU solution by stomach tube at time zero. Except for studies of labeled mitoses, each animal received 0.5 pCi/g body weight of 3HTdR intraperitoneally 1 hr before sacrifice. Rats used for labeled mitosis studies were given 1 pCi/g and sacrificed a t intervals up to 48 hr thereafter. After killing with ether, the thyroid glands were dissected, weighed, and placed in neutral formalin. In rats less than 3 weeks of age the trachea and attached thyroid were dissected and placed in neutral formalin as a unit without effort to identify the gland. For radioautographs 4 p paraffin sections were mounted on slides and dipped in a 50% solution of llford K-5 nuclear emulsion. The slides were dried and stored at 4°C for periods of 2-6 weeks. The emulsion was then developed in Kodak D 19 B. Sections for counting labeled mitoses were stained with Feulgen and the others with hematoxylin and eosin. Fifty oil immersion fields were examined and the number of labeled follicle cell nuclei counted. In every fifth field the total number of follicle cell nuclei was determined; this usually ranged from 150 to 225 per field. The follicle cell labeling index (FCLI) was then calculated. Individual animals of the same age and treatment occasionally showed a 2- to 3-fold variation of FCLI. This variation appears to represent differences between animals since in ten rats where duplicate slides were counted the difference between duplicates did not exceed 25 %. To determine the follicle cell labeled mitosis index (FCLMI), oil immersion fields were examined until twenty-five follicle cell nuclei in metaphase or anaphase were found. RESULTS AND DISCUSSION In Fig. 1, rat body weight is shown as a function of age and time treated with PTU. These curves have two obvious components. Initially. growth was comparatively rapid. After about Glenn E. Sheline 10-12 weeks there was an abrupt change to a lower growth rate which continued for the duration of the study, namely 40 weeks. In comparison to the controls, animals treated with PTU for 1 or 4 weeks exhibited a lower body weight, the difference being greater for the longer treatment period. However, PTU treatment did not seem to affect the shape of the growth curves. How much of the effect on body weight of the PTU treated animals was due to hypothyroidism, to the lowered fluid intake, or to some other influence of PTU is not known. It is known that hypothyroidism reduces growth rate. Furthermore, Lewis, Cheever & VanderLaan (1965) have shown that 0.03% PTU in Purina chow results in a disappearance of growth hormone from the rat pituitary. While the reluctance to drink was particularly Body weight ( 9 ) Thyroid weight (mg) Age ( w e e k s ) FIG.1. Body weight and thyroid weight of rats of differing ages. One group on PTU for 1 week (a) another for 4 weeks ( 0 ) ; A, control rats. and evident during the first 24 hr, it continued to a lesser extent for the full 4 weeks. The low fluid intake also meant a decreased consumption of sucrose. However, in later experiments when no sucrose was added to the water, there was a similar difference in weight between treated and control animals. Furthermore, groups of rats, not otherwise included in this report, in which the PTU concentration was reduced to 0.025% failed to grow normally. Dobyns & Didtschenko (1961) have also described an impairment of body growth in rats on 0.1 % thiouracil. Weight of the thyroid gland as related to the age of rat and time on PTU is also presented in Fig. 1. As with body weight, the thyroid weight of treated and control rats increased rapidly up to about 10-12 weeks of age and thereafter increased at a much lower rate. Histologic examination of slides from the control group showed that some of the increase in thyroid weight of the older animals was probably due to colloid storage. Since the thyroid cells became smaller with increased age, the apparent correlation between thyroid weight Thyroid prolqeratiue potential as a function of age gain and increase of colloid storage does not rule out the possibility of acontinuing increase in number of thyroid follicle cells. One week on propylthiouracil produced an increase in thyroid weight of 50-75 % irrespective of age. Three more weeks of PTU treatment caused an additional doubling of thyroid weight. Since response to PTU, especially in the older rats, involved a histologically evident increase in thyroid cell volume and a decrease in colloid storage, changes in thyroid weight cannot be directly related to numbers of cells present. The per cent of follicular cells which were labeled with 3HTdR is given in Fig. 2. In untreated control animals of 2-4 weeks of age, about 3 % were labeled. The labeling index G - cn -6 : 2 l ? 4- decreased rapidly to about 0.3 % at 6-8 weeks and remained at this level for the duration of the study. It is not evident whether continuation of labeling in older animals represents a replacement of follicle cells or a slowly expanding cell population. One week of treatment with PTU produced about a 5-fold increase in labeling in rats of 2-3 weeks of age, and a 20to 25-fold increase in the 36 -40-week-old group. After 4 weeks on PTU, the labeling index was still above the levels for control animals but considerably lower than in rats after only 1 week of PTU treatment. Regardless of age when started on PTU, these thyroids showed 1.1-1.4% of cells labeled after 4 weeks on the drug. Figs. 3 and 4 present body weights, thyroid weights, and labeling indices of rats on PTU for varying lengths of time, and sacrificed at 7 and 40 weeks of age. These ages were chosen so that one group would fall in the rapid and one in the slow growth periods. The results in these two age groups were similar in many respects. The maximum effect of PTU on thyroid growth rate occurred within the first few days after starting PTU. The labeling index began to rise after the first 24 hr and reached a sharp peak at 5 days in the older rats and at 7 days in the younger rats. The labeling index then rapidly decreased, and after 4 weeks on PTU it was between 1 and 2 % for both age groups. These observations are contrary to the suggestion of Crooks eta/.(1964) that, during the first 2 weeks of a goitrogenic challenge, thyroid growth Glenn E. Sheline Time on PTU (days) FIG.3 . Sprague-Dawley rats 7 weeks of age at sacrifice. Shows body weight (A), thyroid weight (0) and labeling index (0) various periods of time on PTU. for 15 20 25 Time on PTU (days) FIG.4. Sprague-Dawley rats 40 weeks of age at sacrifice. Shows body weight (A), thyroid and for weight (0) labeling index (0) various periods of time on PTU. Thyroid proliferative potential as a-function of age is mainly due to hypertrophy and that it is during the third and fourth weeks that the growth spurt is due to cellular division. Other authors have described an initial response to goitrogenic stimulus which was not maintained even though the goitrogen was continued. Doniach & Logothetopoulos (1955) administered 0.06 % propylthiouracil ad lihituriz in drinking water to 3-4-month-old rats and found the mitotic index to increase up to about 8 days, and then decrease to nearly zero by 24 days. Santler (1957) using adult male rats and 0.05 < methylthiouracil found an increase ; in mitotic index and in total number of mitoses per lobe for both follicular and stromal cells with peak values at about 6 days. The mitotic indices dropped thereafter. According to Santler, the total number of follicular and of stromal cells per lobe showed no increase between 24 and 50 days although mitoses were still present at the later time. Time on PTU (days) FIG.5. August male rats of 10-12 weeks of age. Thyroid weight, i standard deviation, shown l as a function of time on I’TU. If there is no change in the DNA synthesis period and in the fraction of proliferating cells, it can be inferred from the declining labeling index that there is a decrease in the proliferation rate of the follicle cells even though PTU treatment continues. It may be that the decrease in thyroid cell proliferation is due to hypertrophy and hyperplasia sufficient to raise the hormone output of the thyroid to such a level that the thyrotrophic hormone output of the pituitary is depressed. If the fall in labeling index with continuing PTU stimulation were due to a limited number of permissible cell divisions, one might expect the decrease in labeling of the younger thyroid to appear only after it had reached the size at which the adult thyroid exhibited this effect. Whatever the mechanism responsible for the decline, the labeling index remains above normal for at least 4 weeks. Figs. 5-8 present data obtained with the August male rats. Thyroid weight versus time on PTU for two separate experiments (four to five animals per point) is shown in Fig. 5. These rats were 10-12 weeks of age and weighed 175-225 g; they were past the phase of high FCLI, and presumably of rapid thyroid growth, and were considered as young adults. The initial Glenn E. Sheline Time on P T U (days) FIG. 6. August male rats of 10-12 weeks of age. Follicle cell labeling index, f l standard deviation, plotted against time on PTU. slope of the curve is rather flat; after 2 days on PTU, thyroid weight begins to increase rapidly. After about 7 days the slope again decreases. The slope of the curve at 4 days suggests a thyroid weight doubling time of about 5 days, whereas between 7 and 21 days the slope is consistent with a 16-day doubling time. Hyperplasia and hypertrophy of follicular and stromal cells and variations in colloid content are all reflected in thyroid weight. It is, therefore, difficult to translate measurement of weight into numbers of follicular cells. Santler (1957) described experiments in which adult male rats were given 0.05 % methylthiouracil in drinking water and measurements made of thyroid weight and of total number of follicular and stromal cells per lobe. If Santler’s data are averaged and an interpolation made for a treatment time of 4 days, one finds a 38 % increase in thyroid weight accompanied by an increase of only 19 % in number of follicle cells. If it is pertinent to apply these data to the presently observed 5-day doubling time for thyroid weight, the follicle cell doubling time in the present adult rat after 4 days on PTU would be about 10 days. Age at socrifice (weeks) FIG.7. August male rats. Follicle cell labeling index, $1 standard deviation, plotted against age at sacrifice for control animals (A) and for rats on PTU 4 days ( ) 0 . Thyroid proliferative potential as a function of age The per cent of labeled follicle cells for rats used in Fig. 5 was averaged for each PTU treatment time. The results are given in Fig. 6 . The FCLI was 0.1 % for control animals. After a 1-2 day lag, the curve rises sharply to an apparent peak at 4-5 days and then falls to a much lower value, but one still well above that of the control animals. The FCLI was 5.2% at 4 days and 1.2% at 21 days. The changes in FCLI corresponded in time with the changes in slope of the thyroid weight curve and are similar to the observations in SpragueDawley rats (Figs. 3 and 4). Fig. 7 shows the FCLI for untreated control animals and for animals receiving PTU for 4 days. These rats ranged from 3 days to about 12 weeks of age at time of sacrifice. The FCLI is about the same after 5 weeks of age in the PTU-treated rats as in the I-2-week-old rats without treatment. On the basis of these data, it was decided to use 2-week-old untreated and 10-12-week-old PTU-treated rats for the study of labeled mitoses. This would contrast ‘young’ thyroids with ‘older’ thyroids and each group should have a FCLl of about 5%. Non-stimulated older thyroids show very infrequent labeled mitoses and a comparison of ‘young’ and ‘old’ non-stimulated thyroids was not attempted. Time after pulse label ( h r ) FIG. 8. Follicle cell labeled mitosis index for 2-week-old untreated August male rats (A) and for 10-12-week-old treated August male rats (0). The treated group received PTU for 4 days prior to injection of 3HTdR. Fig. 8 presents the per cent of labeled mitoses (FCLMJ) found in thyroid follicle cells up to 48 hr after a single injection of 3HTdR. The labeled mitosis curves for 2-week-old and for 10-12-week-old rats are similar except that the curve for the older group is about 2 hr delayed. In the older group. PTU was started 4 days prior to ’HTdR administration and continued until sacrifice. Using the 50% point on the curves to delineate the various parts of the generation cycle, G2 i 3 mitosis time is 4.7 hr for the younger and 7.0 hr for the older rats. Synthesis time (S) is approximately 7.5 hr for each group. Histologically, it was noted that when the fraction of labeled mitoses begins to decrease, adjacent pairs of labeled nuclei occur with great frequency. Throughout the 48 hr, there is no indication of a second wave of labeled mitoses. Because of desynchronization and the rapidly changing growth kinetics, as shown in Figs. 2 and 3, it did not seem worthwhile to extend the curves for a longer time. For example. the mitotic activity in thyroids from 4-day PTU-treated rats would be quite different after 3 or 4 days than it was only a few hours after thymidine injection; hence, a second wave of labeled mitoses would give little information regarding the generation time applicable at the beginning of the experiment. Since the FCLI curves change less rapidly in Glenn E. Sheline rats of 1 week or less of age and in the older group on PTU for 2 weeks or more (Figs. 6 and 7), interpretation might have been simpler if such groups of animals had been used for the labeled mitosis study. This combination was ruled out because of the relatively small number of follicle cells available in the thyroid of rats of only a few days of age and because long periods of PTU treatment seemed to have adverse effects on the rats. In Fig. 8, it appears that the follicle cell generation time is at least 45 hr. Assuming that in the PTU-treated 10-1 2-week-old rats the follicle cell population doubling time, as derived utilizing Santler’s (1957) data, is 10 days, the per cent of follicle cells in the proliferation cycle is at least 20%. Jf the fraction undergoing proliferation were smaller, maintenance of this doubling rate would require a shorter generation time and a second wave of labeled mitoses would have appeared. Because of lack of data on young thyroid weights, a similar calculation cannot be made for the 2-week-old untreated rats. ACKNOWLEDGMENTS 1 would like to thank Professor Leonard F. Lamerton, Dr Gordon Steel and Professor Harvey Patt for many helpful suggestions and discussions, Dr Claire Shellabarger for some of the data of Figs. 5 and 6 , and Mrs Margaret Miller for her technical assistance. I also wish to thank Professor Lamerton for the opportunity to conduct Part 11 of this study in his laboratory at the Institute of Cancer Research, Surrey, England. In addition, I should like to express my appreciation to the Commonwealth Fund for financial assistance during my stay in England. This work was performed under the auspices of the U.S. Atomic Energy Commission.

Journal

Cell Proliferation – Wiley

Published: Apr 1, 1969

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