Thyroid Follicular Cell Proliferation as a Function of Age

Beitr. Path. Bd. I48, I 52-I64 (I973)

Institute of Pathology, University of Freiburg (Director: Prof. Dr. W. SANDRITTER)

Thyroid Follicular Cell Proliferation as a Function of Age Proliferation der Follikelzellen der Schilddriise unter Beriicksichtigung des Alters K.

CHRISTOV*,

R.

BOLLMANN

and C.

THOMAS**

With 6 Figures' Received August 24, 1972 . Accepted October 3, 1972

Summary Thyroid follicular cell proliferation in normal and MTU-treated male newborn, 10, 60 and 34o-day-old Wistar rats was studied. In methylthiouracil(MTU)-treated newborn animals the thyroid weight growth curve consists of 2 phases: an almost exponential phase for approximately 8 days and a plateau phase between the roth and 24th day. In 10, 60 and 340-day-old animals the growth pattern of the thyroid weight is presented by a lag phase of 2 days, an exponential phase for about 13 days and a plateau phase until the end of the experimental period (24 days). By means of autoradiography it was found that during the first days after birth between 7 and 8 per cent of all follicular cells are labeled. The labeling index curve showed a sharp decrease after the second week and then reached an unchanged level in 60-day-old rats. About 0.2 per cent of all follicular cells in 60 and 34o-day-old animals were labeled. After MTU-treatment in all experimental groups the curves indicated a sharp increase of labeled follicular cells between the 2nd and 3rd day with a peak at the 8th day. Between the 8th and 24th day a considerable decrease of labeled cells was observed. The highest value for labeled follicular cells was 19 per cent in the newborn group, I4.5 per cent in lo-day-old animals, 8.4 per cent in 60-day-old animals and 4.3 per cent in the 34o-day-old group. The rate of follicular cell labeling after MTU-treatment is 3, 2,40 and 20 times larger in newborn, ro, 60 and 340day-old animals, respectively. From the labeled mitosis curve some cell cycle parameters of the follicular cells in 10 and 34o-day-old animals were established. Thus, in the 10day-old group the duration of the DNA-synthesis period (Ts) was 7.5 hrs, of the DNS-

* This work was carried out during a Research Fellowship of the International Agency for Research of Cancer, Lyon, France, while on leave from the Cancer Research Institute Sofia 56, Bulgaria. ** Supported by the Deutsche Forschungsgemeinschaft.

Thyroid Follicular Cell Proliferation .

I

53

post synthetic (pre mitotic) period (G 2) 3 hrs and of the mitosis (TM)-period 4.5 hrs. The same phases of the cell cycle in the 34o-day-old group were as follows: TS-IO hrs, T G2-3 hrs, T M-7.5 hrs. Factors affecting the thyroid cell proliferation during postnatal development are discussed.

Interest in thyroid tumors has grown considerably in recent years, owing to the high incidence of carcinomas among people who have been irradiated during early childhood in the cervical region for thymic enlargement, lymphadenitis or certain skin diseases (WINSHIP and ROSVOLL, 1961; H EMPELMAN, 1968; H ARPER and P AVOYAN, 1968). A high percentage of thyroid tumors was also found in people exposed to 131, 132, 133, and 135 I irradiation from the atomic explosions over Hiroshima, Nagasaki and the Marshal Islands (SOKOLOV, 1963; CONARD, 1970). It was also established that the younger the patient at the time Qf exposure, the greater the likelihood of his developing carcinoma of the thyroid (HEMPELMAN et ai., 1967; CONARD et ai., 1969). These data indicate that the thyroid of young and old people responds differently to irradiation. The high incidence of thyroid tumors after irradiation was related to the higher radiation doses received by the infant thyroid (CAROLL et ai., 1964; PIEFER et ai., 1968; WINSHIP and ROSVOLL, 1969) or, in young animals, to greater physiological cell proliferation (SHELINE, 1969). It was established that in proliferating cell systems many agents induce a higher incidence of tumors in a short time than in non-proliferating cell systems (PREI and H ARSONO, 1967; POUND, 1968). Other investigations also indicated that during cell division some phases of the cell cycle are more susceptible to the carcinogenic effect of different agents (WARWICK, 1971). Since in newborn and suckling animals increased cell proliferation is a physiological pheniomenon it was postulated and proven that many carcinogens have a considerably greater oncogenic effect when they are applied during embryonic development or in the first days after birth (THOMAS and BOLLMANN, 1968; D ELLA-PORTA and TERRACHINI, 1969). In order to trace this phenomenon in the thyroid gland of rats DONIACH, in 1969, injected 4-day-old rats intraperitoneally wirh 1. 1, 2.0 and 5.5 [LCi 131 I without finding any difference in the number of tumors between suckling and adult rats, which where treated in the same manner with 30 [Lei 131 1. However, there is still a lack of systematic research to indicate the carcinogenic effect of X-rays on the thyroid gland in suckling animals and to point out the relationship between cell proliferation and tumor induction after irradiation. This study was undertaken to determine: I. The thyroid cell proliferation in rats as a function of age. 2. The age-related difference in response to irradiation. 3. The DNA, RNA and histone synthesis during thyroid carcinogenesis.

154' K.-CHRlSTOV, R. BOLLMANN and C. THOMAS

4. The ultramicroscopic changes in some cell structures after irradiation

and hyperstimulation. 5. The latent period, structure and growth pattern of the induced thyroid tumors. The present report deals with changes in thyroid epithelial cell proliferation of rats as a function of age in normal conditions and after hyperstimulation. H yperstimulation by thyroidstimulating hormone (TSH) was provided by the administration of methylthiouracil (MTU) which by blocking the thyroid hormone synthesis causes an increase in the output of TSH from the anterior lobe of the pituitary (PURVES, 1964).

Material and Methods Rats. Male Wistar rats (Fa. Ivariovas, Kisslegg, Germany) between I and 365 days of age were used (Table l). In order w obtain suckling rats male and female 60-day-old rats were kept together and checked for litters every 12 hrs during the last days of pregnancy. The mothers were kept with their newborn offspring in a single cage under standard conditions and fed ad libitum with an "Altromin R" diet (Altromin GmbH Lage, Germany). The adult (60 and Ho-day-old) rats were caged in groups of 3 with free access to food and drinking water. Methylthoiuracil (MTU). o. I per cent 4-Methyl-2-thiouracil (Fluka AG., Chemische Fabrik Buchs, Switzerland) was given in the drinking water for a period of 24 days. The MTU-treated newborn ~nd lo-day-old animals were daily injected intraperitoneally with 0.2 cc and 0.5 cc, respectively, of an o. I per cent solution of MTU. The mothers of these suckling animals were given the same solution of MTU as the adult animals. Thymidine-(Methyl-)3 H. (Radiochemical Centre, Amersham, England, specific activity 6.7 Cijmmole). Forty-five minutes before being killed 4 rats from each group received an intraperitoneal injection of I fJ.Ci of3H-thymidine/g body weight. Ten and Ho-day-old

Table

1.

Age at sacrifice (days)

Experimental groups Newborn Control MTU

0

6 8

6 6 6

2 4

6 6

6

6

6

8

6

6

10 15 20 24 Total

lo-day-old Control MTU

5 6 52

5I

6 6 6

44

60-day-old Control MTU

Ho-day-old Control MTU

6

6

5 6

5 6

6 6 6 6

6 6

46

6 6

5 6

6 6

5 6 40

43

42

45

Thyroid Follicular Cell Proliferation· 15 5 rats were used for labeled mitosis studies. They were killed at varying intervals up to the 58th hour after thymidine injection. After killing the animals with ether the thyroid gland was dissected, was weighed on an analytical balance and then was placed in Bouin's solution. A dissection microscope was used for the smaller glands. After dehydration the thyroid glands were subsequently embedded in paraplast and cut into 5 fJ. thick sections. For autoradiography the slides were dipped in 50 per cent solution of Ilford K 5. Some slides were covered with Kodak AR 10 stripping film. After exposure (up to 6 weeks as necessary) the films or emulsion were developed in Kodak D 19B developer. The sections were stained with hematoxylin and eosin. The number of labeled nuclei per 5,000 nuclei was determined. Only nuclei with 3 or more grains were assumed to the labeled. A labeled mitosis index of follicular cell was determined in meta-or anaphayse nuclei. In order to obtain a sufficient number of labeled nuclei the newborn thyroids were cut into a number of parallel sections and every 5th was examined. For histological study the slides were stained with hematoxylin and eosin as well as by the PAS reaction.

Results Body and thyroid weight changes during the first 24 days after birth are given in Table 2. The physiological increase of the thyroid weight in the controls is more intense during the first 4 days after birth. MTU-administration causes the rat thyroid to undergo an additional increase in weight (the goitrogenic response). From Figure I it can be seen that in MTUtreated animals the growth curve consists of 2 phases: an almost exponential phase for approximately 8 days and a plateau phase between the loth and 24th day. In the Io-day-old group the thyroid weight growth is not as marked as it is in the newborn control group. The changes of the thyroid weight after MTU-treatment in this group are characterized by a lag phase

Table 2. Newborn Animals Age at sacrifice (days) 0 2 4 6 8 10 15 20 24

Body weight (g) Controls MTU 8.0± 2.1 9·3 ± 2·4 1O.4± 1.1 12.2±2·3 14·3 ± 1.7 I2·9± 3. 2 24·5 ± 3·7 3503 ± 6·3 41.6± 503

7· 2 ± 1.4 9·5 ± I.7 11.7± 4.2 9.5 ± 2.1 12.1±2.2 1Q±1.9 14·3 ± 2·5 17·3 ± 2·4 22·4± 5.7

Thyroid weight (mg) Controls MTU

Labeling index (%) Controls MTU

1.2 + 0.2 1.4± 0.15 2.2 ± 0.3 2.6± 0.9 3.1 ± 0.8 3· 0 ±0·5 5· 0 ± 0.3 8.1±0·5 1O.2± 0.9

7. 1 ± 0·3 8.6 ± 0.2 7· 6 ± 0·4 8·4± 0·7 7·4± 0·4 7.0± 0.2 6.0± 0.2 4. 0 ± 0·3 3. 2 ± 0·5

1.2± 0.15 1.43 ± 0·4 4·17 ± 0·5 10.5 6 ± 0·7 17.20± 1.1 19.34± 2.2 23·4 ± 1.7 24. 1 ± 1.2 22.1 ± 1.1

7· 1 ±0·3 7· 6 ±0.2 15.0 ± 1. I 17.4± 1.6 19.0± 1.0 17.8 ± 0.6 13.0± 2.0 10.2 ± 0.8 8.8 ± 1.1

1 56 . K.

CHRISTOV,

R. BOLL MANN and C.

T HOMAS

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Day s on MTU Newborn Rots.o - o Con t ro!s.~_ )(. MTU · treated

Fig . I. Log mean follicular cell labeling index and thyroid weight in control and MTUt reated Wistar rats during the first 24 days after birth .

r

0

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0 N

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Days on MTU

Fi g. 2

lI)-Day- ol d Rats, 0 - 0 Con t rols ."- " MTU - treated

Fig. 2. Log mean follicular cell labeling index and thyroid weight in Wistar rats treated with MTU for 24 days starting on the loth day of age.

Thyroid Follicular Cell Proliferation .

I

57

10 ' en dO

10'-

10°

r~

o~'" dO

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Days on MTU 60-Day-old Rat~ a-aC ontrols, x-.MTU - treated

Fig. 3. Log mean follicular cell labeling index and thyroid weight in Wistar rats treated with MTU for 24 days starting on the 60th day of age.

of 2 days, an exponential growth for about 13 days and a plateau phase until the end of the experimental period (24 days-Fig. 2). Such 3-phase curves were also found in 60 and 340-day-old animals (Fig. 3 and 4). A retardation of the body growth after MTU-treatment in newborn and Io-day-old animals was also observed. Similar changes of the body weight in the adult animal groups were not established. Labeling index. If we assume that all labeled cells are dividing cells, then the proliferative capacity of the follicular cells shows considerable variation in the different age groups (Fig. 5). Between 7 and 8 per cent of all follicular cells in the newborn animals were labeled. No noteworthy changes were found in the labeling index until the loth day. The labeling index decreases rapidly after the second week and did not reach a constant level until about 8 weeks later. 'This level remained unchanged until the end of the first year. During the first 2 days after birth, a small increase of the labeled follicular cells was noted in the MTU-injected rats (Fig. I). The curve showed sharp increases between the 2nd and 4th days. 'The curve then reaches a peak at 8 days after starting MTU. Between the 8th and 24th day on MTU a

158 . K.

CHRISTOV,

R.

I

BOLLMANN

and C.

THOMAS

b

1 -J o

8 12 16 20

8

12 16 20 2

Days on MTU Fig.4 340 -Day- old Rats, o-oControls, x-.~o1TU - treated

Fig. 4. Mean follicular cell labeling index and thyroid weight in Wi star rats treated with MTU for 24 days starting on the Hoth day of age.

o

6 F i9. 5

10

12

'

48

51

Age/weeks

Fig. 5. Follicular cell labeling index in Wistar rats during the first year after birth.

Thyroid Follicular Cell Proliferation·

100

159

o .... q

/./1'\\ / 0\,

01 I

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15 Fig.6

0_0

30

45

60

Hours after 3H -Thymidine administration

10 -Day-old

x--x340-Day-old

Fig. 6. Percentage of labeled mitoses of follicular cells in 10 and 340-day-old Wistar rats.

constant decrease in the number of labeled cells was observed. In lo-dayold animals the labeled follicular cells curve after MTU-treatment was very similar to that of the newborn animals; a small increase in the first 2 days, a sharp rise between the 2nd and 8th day and a decrease after the loth day (Fig. 2). The changes in the labeled nuclei curve in the 60 and Ho-day-old groups were very similar to those of the lo-day-old group (Fig. 2, 3 and 4). The highest value for labeled follicular cells was as follows: 19 per cent in the newborn group, and 14.5, 8-4 and 4.3 per cent for the 10, 60 and Ho-day-old group, respectively. Eight days on MTU produces an increase of the labeled follicular cell nuclei in the newborn-group of about 3 times, in the Io-day-old rats about 2 times, in the 6o-day-old group about 40 times and in 340-day-old group about 20 times. In the newborn and lo-day-old rats the peaks were not as sharp as in the 60 and 340-day-old groups, but the peaks were more round and smooth like a plateau. For determining some phases of the cell cycle in 10 and Ho-day-old animals the fraction of the labeled mitosis after a single injection of 3Hthymidine was used (Fig. 6). Every point of the curve represents the mean value of the labeled mitosis (meta- and anaphases in 4 animals). Neither curve reaches the 100 per cent value. In lo-day-old animals the highest level (96.4 and 97.5 per cent) of labeled mitoses was found at the 8th and loth hrs, respectively, after 3H-thymidine injection. The mitotic labeled curve for 340-day-old animals is wider at the 50 per cent level and reaches its peak (94.3 per cent labeled mitoses) about 3 hrs later. The gap

160 . K.

CHRISTOV,

R.

BOLLMANN

and C.

THOMAS

of both curves was similar with an exception between the 20th and 24th hr where a plateau phase of the 340-day-old rats curve was observed. The time interval between the addition of 3H-thymidine and the appearance of the first labeled mitosis corresponds to the minimal time for a cell to pass from the end of the S-period to division (minimal postsynthetic periodG 2)-3 hrs in both groups. The interval between the injection of 3Hthymidine and the 50 per cent value of the ascending limb of the labeled mitosis curve gives the mean post-synthetic + ~ mitotic time (TG2+1/,M)4.5 hrs in lo-day-old animals and 6 hrs in the Ho-day-old group. The time between the appearance of the first labeled mitosis and their 100 per cent point explains the duration of the mitosis (T M)-4. 5 hrs in the lo-day-old group and 7.5 hrs in 340-day-old animals. The DNA-synthesis period (Ts)-7.5 and 10 hrs, respectively, for the 10 and 340-day-old animals is the time between the 50 per cent points on the ascending and descending limbs of the curves. Until 58 hrs no second wave for labeled mitoses was observed.

Discussion The growth curves for the thyroid weight in 10, 60 and 340-day-old rats under a goitrogenic stimulus are similar to those described by PHILP et al. (1969). The existence of a lag phase lasting 2 days, an exponential phase lasting about 10 days and a plateau phase which persists for as long as the administration of MTU (24 days) was well confirmed. The biphasic growth curve observed in our newborn animals is most probably the result of a higher growth potential of the thyroid epithelial and stromal cells during the first days after birth (Fig. I). Because of little colloid storage in these animals, no loss of thyroid weight due to resorption of colloid as in the adult groups could be expected. The increase of follicular cell size and cell proliferation compensates for the removal of the colloid and results in an increase of the thyroid weight during the exponential phase. The findings of CROOKS et al. (1964) that follicular cell hypertrophy procedes cell hyperplasia after MTU-treatment were not confirmed in our studies. Our results demonstrate that during the lag phase cell division increases. This means that the thyroid weight growth could not be divided into two separate phases of hypertrophy and hyperplasia. Morever, during cell division there are epithelial and mesenchymal cells which are in G 2 phase with double the cell volume in comparison to the cells in the G 1 phase. A moderate cell volume had to be expected during the S period. Considering all these facts, it appears that there are 2 types of follicular cell hypertrophy; first hypertrophy dependent upon cell division and second independent or

Thyroid Follicular Cell Proliferation .

161

"true hypertrophy" which was observed in the nondividing cells (PHILP et aI., 1969). In long-term experiments some other factors modify the thyroid weight growth. After 150 days on MTU there is an increase in growth potential of the thyroid weight which is the result of colloid accumulation as well as nodular hyperplasia and the development of tumors (CHRISTOV and RAICHEV, 1972). Since the increase in body weight in the newborn and in the lo-day-old groups is more prominent than in the other groups a reduction of the body weight growth after MTU-treatment was observed only in the first 2 groups. In the adult animals there is also retardation of body-growth potential, but it was necessary to follow a large group of animals for a long period of time (CHRISTOV, 1968). The reduction of the body growth rate in MTU-treated animals is probably the result of hypothyroidism, since MTU inhibits thyroid hormone synthesis. In such animals there is also a disappearance of growth hormone because growth hormone production requires the presence of thyroid hormones (PURVES, 1964). Another effect of body growth retardation may be diminished fluid intake and a toxic effect of MTU on some other cell system, such as liver or the hemopoetic system (WILLIS, 1962). Our results demonstrate a great variance in the proliferative capacity of thyroid epithelial cells between newborn and adult animals. The higher per cent of labeled follicular cells in suckling animals than in the adults (Fig. 5) correlates with a higher level of cell proliferation in other cell systems (U:iBLECKE et aI., 1969; STOCKER and HEINE, 1971). In the control group during the first 6 days after birth, there is a tendency towards an increase in the labeled follicular cells which may be a physiological reaction to the increased TSH-stimulation. The 35-fold drop in the per cent of labeled cells between the loth and the 60th day corresponds to the results of SHELINE, 1969. which were obtained in propylthiouracil (PTU)-treated Fischer rats and could explain to some extent the retardation of thyroid weight growth potential during postnatal development. The involution of thyroid cell proliferation after birth is either the result of functional specialization of most of the dividing cells (they enter Go) or only a prolongation of the cell cycle time, since there is no morphological evidence for cell loss. MTU treatment causes a rapid increase in labeled follicular and mesenchymal cells in all experimental groups with a peak at the 7th to 8th day for follicular cells and at the 4th to the 5th day for the mesenchymal cells (unpublished data). In suckling rats (newborn and 10 days of age) the highest value for labeled epithelial cells was about 3 times greater than in the control groups, whereas in 60 and Ho-day-old animals an

162 . K.

CHRISTOV,

R.

BOLLMANN

and C.

THOMAS

increase in the labeled cells of 40 and 20 times, respectively, was found. One week on PTU produced a 5-fold increase in labeled follicular cells in 2-3-week-old suckling animals. However, in the 36-40-week-old animals a 25-fold increase of labeled follicular cells was observed (SHELINE, 1969). In young Fisher rats whose age was not given a 50-fold increase of the labeling index was noted after 3 days of MTU-treatment (WOLLMAN et al., 1968). A multifold increase of the mitotic index after treating adult rats with goitrogens was also found in the studies of DONIACH and LOGOTHETOPOULUS (1955) and SANTLER (1957). The variety of these data regarding labeled cells and mitotic indices is obviously determined by the difference in the strains, the goitrogenic agents and the unequal starting value for dividing cells in suckling and adult animals. The higher proliferative potential of thyroid epithelial cells in the 60-day-old rats than in the 340day-old group (with an almost identical level of labeled cells) could be interpreted mainly as a result of a more susceptible period of the thyroid follicular cells that exists with increased TSH stimulation. Similar more sensitive phases for cell proliferation during postnatal development are well known in other cell systems, principally those which are hormonedependent (BANERJEe et al., 1971; EPIFANOVA, 1971). The drop of the labeling index after an early peak may be due to the fact that thyroid hypertrophy and hyperplasia are sufficient to raise the hormone output of the thyroid to a level sufficient to suppress the TSH output of the pituitary (SHELINE, 1969). It is also possible that the thyroid tissue becomes less responsive despite continued stimulation by TSH. That is the case, for instance, in some thyroid tumors (CHRISTOV, 1972). It may also be presumed that the TSH-level in the blood falls during prolonged goitrogenic stimulation because of a depletion of TSH-production (WOLLMAN and BREITMAN, 1971). The labeled mitosis curves point our that until 58 hrs after a single injection of 3H-thymidine no second wave of labeling exists. This means that the cell cycle time in 10 and Ho-day-old rats is longer than 58 hrs. If we extend the mitotic curve in suckling animals for a long time whole cell system is no longer in a steady state as the rate of dividing cells rapidly decreases with age. This means that the second wave of labeled mitoses in this group cannot give precise information about the generation time of the follicular cells. The observed differences in some phases of the cell cycle between young and adult animals correlate with the changes of the cell cycle time during postnatal development (MALAMUD. 1971). In order to estimate the cell cycle parameters and growth differences between normal and neoplastic thyroid cell systems several experiments are in progress.

Thyroid Follicular Cell Proliferation . 163

Z usammenfassung An neugeborenen, 10,60 und 340 Tage alten Wistar-Ratten wird die Proliferation der Follikelzellen der Schilddriise untersucht. Bei den mit Methylthiouracil (MTU) behandelten Ratten zeigt die Gewichtskurve der Schilddriise wah rend des Wachstums zunachst bis zum 8. Versuchstag eine exponentielle Zunahme und spater - zwischen dem 8. und 24. Versuchstag - ein Plateau. Bei den 10,60 und 340 Tage alten Tieren liegen ahnliche Verhaltnisse vor. Histoautoradiographische Untersuchungen mit 3H-Thymidin zeigen in den ersten Tagen nach der Geburt 7-8% markierte Follikelzellen. Die Markierungskurve weist nach der 2. Woche eine rasche Abnahme auf und bleibt bei den 60 Tage alten Ratten unverandert. Bei diesen und den 340 Tage alten Ratten nimmt nach MTU-Behandlung die Zahl an markierten Follikelzellen zwischen dem 2. und 4. Versuchstag zu, erreicht am 8. Tag einen Gipfel und nimmt dann wieder abo Die hbchsten Markierungswerte betragen bei neugeborenen Ratten 19%, im Alter von 10 Tagen 14,5%,60 Tagen 8,4% und 340 Tagen 4,3%. Die Zunahme an markierten Zellen ist bei neugeborenen Ratten 3mal grbBer als bei unbehandelten Tieren (10 Tage: 2mal, 60 Tage: 40mal und 340 Tage: 20mal). Es werden die Zellzykluszeiten und die verschiedenen Faktoren, die die Proliferation von Follikelzellen beeinflussen, diskutiert.

References BANERJEE, M. R., WAGNER, J. E. and KINDER, D. L.: Life Sci. IO, 867 (1971) BASERGA, R. and WIEBEL, F.: Int. Rev. expo Path. 7, I (1969) CARROLL, R. E., HADDON, R. E., HANDY, F. H. and WIEBEN, E. E.: J. nat. Cancer Inst. }}, 277 (19 6 4) CHRlSTOV, K.: Thesis Candidate of Medical Sciencies, Sofia 228 (1968) CHRISTOV, K.: Endocrinologie 59, 382 (1972) CHRISTOV, K. and RAICHEV, R.: Curro Top. Path. 56, 79 (1972) CONARD, R. A., SUTOV, W. W., COLCOCK, B. P., DOBINS, B. M. and PAGIA, D. E.: In: Radiation induced cancer, p. 325. Int. Atomic Energy Agency, Wienna (1969) CONARD, R. A., DOBINs, B. M. and SUTOV, W. W.: J. Amer. med. Ass. 2I4, 316 (1970) CROOKS, J., GREIG, W. R., MACGREGOR, A. B. and MACINTOSH, 1. A. R.: Brit. J. Radiol. }7, 380 (19 64) DELLA-PORTA, G. and TARRACHINl, B.: Progr. expo Tumor Res. (Basel) 2, 334 (1969) DONIACH, 1.: In: Thyroid cancer, ed. CHR. HEDINGER, UICC monogr. 12, p. 174. Springer-Verlag, Berlin-Heidelberg-New York (1969) DONIACH,1. and LOGOTHETOPOLOUS, J. H.: Brit. J. Cancer 9,117 (1955) EpIFANOVA, O. J.: In: The cell cycle and cancer, ed. R. BASERGA, p. 142. Marcel Dekker New York (1971) FREl, J. V. and HARSONO, T.: Cancer Res. 27,1482 (1967) HAPPER, P. V. and PAVOJYAN, D.: Surg. Clin. N. Amer. 49, 57 (1968) HEMPELMANN, L. H., PIEFER, J. F., BURKE, G., TERRY, T. and AMES, W. R.: J. nat. Cancer Inst. }8, 317 (1967) HEMPELMANN, L. H.: Science I60, 1959 (1968) LOBLECKE, E. A., SCHULTZE, B. and MAURER, W.: Exp. Cell Res. JJ, 116 (1969) MALAMUD, D.: In: The cell cycle and cancer, ed. R. BASERGA, p. 125. Marcel Dekker, New York (1971) PHILP, J. R., CROOKS, J., MACGREGOR, A. G. and MACINTOSH, J. A. R.: Brit. J. Cancer 2}, 515 (1969)

164. K. CHRISTOV, R. BOLLMANN and C. THOMAS PIEFER, J. W., HEMPELMANN, L. H., DODDGES, H. and HODGES, F. ].: Amer. J. Roentgenol. IOJ, 13 (1968) POUND, A. W.: N. Z. med. ]. 67, Suppl. 88 (1968) PURVES, H. D.: In: The thyroid gland, ed. R. PITT-RIVERS and W. TROTTER, 2, I. Butterworth, London (1964) SANTLER,]. E.: J. Endocr. If, 15 1 (1957) SHELINE, G. E.: Cell Tiss. Kinet. 2,123 (1969) SOCOLOV, E. L., HASHIZUMA, A., NERISHI, S. and NATANI, N.: New Engl.]. Med. 268, 4 06 (19 6 3) STOCKER, E. und HEINE, W. D.: Verh. dtsch. Ges. Path. JJ, 483 (1971) THOMAS, C. und BOLLMAN, R.: Z. Krebsforsch. 71, 129 (1968) WARWICK, G. P.: Fed. Proc. JO, 1760 (1971) WILLIS, ].: ]. Path. Bact. 82,23 (1961) WINSHIP, T. and ROSVOLL, R. V.: In: Thyroid cancer, ed. CHR. HEDINGER, VICC monogr. 12, p. 75. Springer Verlag, Berlin-Heidelberg-New York (1969) WOLLMAN, S. H., ANDROS, G., CANNON, G. B. and AEGLETON, G. B.: In: Thyroid neoplasia, ed. S. YOUNG, D. INMAN, p. 20I. Academic Press, London-New York (1968) WOLLMAN, S. H. and BREITMAN, T. R.: Endocrinology 86, 322 (1970) Dr. K. CHRISTOV, Dr. R. BOLLMANN und Prof. Dr. C. THOMAS, Pathologisches Institut, D-78 Freiburg, AlbertstraBe 19