Sensitivity of Thyroid Gland Growth to Thyroid Stimulating Hormone ...

49, 263–271 (1999) Copyright © 1999 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Sensitivity of Thyroid Gland Growth to Thyroid Stimulating Hormone (TSH) in Rats Treated with Antithyroid Drugs Alan Hood,* Ya Ping Liu,* Vincent H. Gattone II,† and Curtis D. Klaassen* ,1 *Department of Pharmacology, Toxicology, and Therapeutics, and †Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 –7140 Received October 13, 1998; accepted January 22, 1999

Antithyroid drugs and phenobarbital (PB) have been shown to promote thyroid tumors in rats. It has been proposed that increased thyroid-stimulating hormone (TSH) mediates the thyroid tumor-promoting effect of antithyroid drugs and PB, and is increased because of decreased thyroxine (T 4) concentration. However, PB is much less effective than antithyroid drugs at increasing TSH. It has been proposed that small increases in serum TSH produced by PB treatment is sufficient to promote thyroid tumors. However, the level to which TSH must be increased to stimulate the thyroid gland has not been reported. Therefore, we have examined the effect of increasing serum TSH concentration on thyroid growth by measuring thyroid gland weight and thyroid follicular cell proliferation. Serum TSH concentrations were increased by feeding rats various concentrations of propylthiouracil (PTU) or methimazole (MMI) for 21 days. Serum total T 4, free T 4, total T 3 (triiodothyronine), free T 3, and TSH concentrations were measured by radioimmunoassay. Thyroid follicular cell proliferation was measured by autoradiography and expressed as a labeling index (LI). PTU and MMI treatments reduced total and free T 4 more than 95% by day 21, whereas total and free T 3 were reduced 60%. TSH, thyroid follicular cell proliferation and thyroid weight were increased 560%, 1400%, and 200%, respectively, by day 21. TSH was significantly correlated with thyroid weight and LI. Moderate increases in serum TSH of between 10 and 20 ng/ml increased the number of proliferating thyroid follicular cells, but had no effect on thyroid weight. These results support that small increases in serum TSH can be sufficient to stimulate thyroid follicular cell proliferation. Furthermore, thyroid follicular cell proliferation may be more useful than thyroid weight alone for assessing alterations in thyroid growth in rats treated with chemicals that produce only small to moderate increases in serum TSH. Key words: thyroid stimulating hormone (TSH); thyroxine (T 4); triiodothyronine (T 3); propylthiouracil (PTU); methimazole (MMI); phenobarbitol (PB); thyroid follicular cell proliferation; rat.

Thyroid stimulating hormone (TSH) has been proposed to mediate the thyroid tumor-promoting effects of antithyroid drugs and the microsomal enzyme-inducer, phenobarbital (PB) 1

To whom correspondence should be addressed. Fax: (913) 588-7501. E-mail: [email protected].

(McClain et al., 1988). This postulate was based on observations that showed antithyroid chemicals and PB reduce serum thyroxine (T 4) concentration, which reduces the negative feedback on TSH synthesis and secretion and stimulates thyroid gland growth. In support of this mechanism, thyroid hormone replacement therapy decreases serum TSH concentrations, and decreases the incidence of thyroid tumors in thyroid tumorpromotion studies using antithyroid drugs or PB (Jemec, 1980; McClain et al., 1988). Decreasing serum TSH reduces the thyroid tumor-promoting effect of PB (Hiasa et al., 1987; McClain et al., 1988), suggesting that TSH plays an important role in mediating the effects of PB on the thyroid gland. Although TSH is thought to be the principal mediator in thyroid-tumor promotion by antithyroid drugs and PB, these chemicals affect tumor promotion through differing pathways. Antithyroid drugs inhibit thyroid hormone synthesis (Engler et al., 1983; Nakashima et al., 1978; Taurog, 1976), whereas PB increases hepatic degradation (glucuronidation) of T 4 (Barter and Klaassen, 1992; Japundzic et al., 1976; McClain et al., 1989). Because the mechanisms of action are different, antithyroid drugs decrease serum T 4 and T 3, and increase serum TSH concentrations more than does PB (De Sandro et al., 1991). Phenobarbital-elicited increases in serum TSH are much less when compared to antithyroid drugs, and are not sustained (McClain et al., 1989). Several antithyroid drugs, such as aminotriazole, propylthiouracil (PTU), methimazole (MMI), and sulfamethazine, promote thyroid tumors in rats (Hiasa et al., 1982b; Jemec, 1980; Kitahori et al., 1984; McClain, 1992), whereas PB is the only microsomal enzyme inducer known to increase T 4 glucuronidation and promote thyroid tumors in rats (Hiasa et al., 1982a). Other microsomal enzyme inducers increase T 4 glucuronidation in vitro, such as pregnenolone-16acarbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) (Barter and Klaassen, 1994; Liu et al., 1995), but have not been evaluated for their ability to promote thyroid tumors in rats. It is not known whether other microsomal enzyme inducers that increase the glucuronidation of T 4 (i.e., PCN, 3MC, and PCB) promote thyroid tumors in rats. Until thyroid tumorpromotion studies are performed using microsomal enzyme

263

264

HOOD ET AL.

inducers other than PB, evaluation of the thyroid tumor-promoting potential of other microsomal enzyme inducers, such as PCN, 3MC, or PCB, will be dependent upon understanding the key event(s) that mediate thyroid-tumor promotion. McClain (1992) has proposed that key events in thyroid-tumor promotion by antithyroid drugs and PB are a reduction in serum T 4 concentration that results in an increase in serum TSH concentration, because of the negative feedback effect of thyroid hormones on TSH synthesis and secretion (Larsen and Silva, 1983). Decreases in serum T 4 concentration and increases in serum TSH have been reported to be dose-dependent (McClain, 1995). Furthermore, McClain (1992) has proposed that small increases in serum TSH produced by PB is sufficient to promote thyroid tumors in rats. However, detailed studies of the relationship between serum TSH and thyroid tumors has not been reported. Also, it is currently unknown how much TSH needs to be increased to stimulate the thyroid gland. Therefore, in the present study, we attempted to determine how sensitive the thyroid gland is to TSH stimulation. Because TSH is known to stimulate thyroid gland growth, and uncontrolled growth plays a role in the tumorigenic process, increase in thyroid gland growth rate was used to assess the degree of its stimulation by TSH. Thyroid gland growth was assessed by measuring changes in (a) weight of the gland and (b) the number of proliferating thyroid follicular cells. Rats were treated with various doses of two antithyroid drugs, either propylthiouracil (PTU) or methimazole (MMI). Propylthiouracil and MMI were used in the present study because these agents are more effective at decreasing serum T 4 and T 3 concentrations (as well as increasing serum TSH concentration) than PB and other microsomal enzyme-inducing agents (Cooper et al., 1983; Delia and Thompson, 1988; De Sandro et al., 1991; Mannisto et al., 1979; Rondeel et al., 1992; Stringer et al., 1981). We tested the hypothesis that small increases in serum TSH results in stimulation of thyroid gland growth. MATERIALS AND METHODS Materials. Propylthiouracil (PTU), methimazole (MMI), lactoperoxidase, and glucose oxidase VII (168,200 units/gm solid) were obtained from Sigma Chemical Co. (St. Louis, MO). Radioimmunoassay (RIA) kits for total and free T 4 and free T 3 were obtained from Diagnostic Products Corp. (Los Angeles, CA). Na 125I was obtained from NEN Research Products (Boston, MA). Rat TSH (for radioiodinations) and anti-rat-TSH antibody were kindly provided by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (Baltimore, MD). 3H-Thymidine (2 Ci/mmol) was obtained from Amersham Corporation (Arlington Heights, IL). Animals and treatments. Male Sprague-Dawley rats, 225–250 g, were housed in polypropylene cages containing corn-cob bedding and maintained at approximately 21°C on a 12-h light cycle. Propylthiouracil and MMI were dissolved in methanol, then added to 1 kg of Purina Rodent Laboratory Chow 5001 (iodine content of 0.7 ppm), mixed thoroughly, and allowed to dry. The rats were divided into groups and fed various dietary concentrations of PTU (at 1, 3, 10, 30, 100, or 300 ppm) or MMI (at 3, 10, 30, 100, 300, or 1000 ppm). Control groups received nontreated rodent chow. All rats were allowed free access to feed and water for 21 days. During the 21-day treatment period, rats

were monitored by recording body weights and feed consumption every 2 to 3 days. Feed was replenished as needed throughout the 21-day treatment period. This time period was chosen because previous studies have shown serum thyroid hormone concentrations to be maximally reduced in rats treated with antithyroid drugs (Cooper et al., 1983; Wynford-Thomas et al., 1982b). Although thyroid gland growth increases during the 21-day treatment period, thyroid follicular cell proliferation has been shown to peak 7 days after treatment with an antithyroid agent, and then returned to control levels (Wynford-Thomas et al., 1982b). Sampling. On experimental days 23, 13, 17, 114, and 121 rats were lightly anesthetized with ether, and blood was sampled from the orbital sinus. Because thyroid hormones and TSH exhibit a circadian rhythm, blood was obtained between 9:00 and 11:00 a.m. in a randomized fashion. Analysis of serum thyroid hormones. Approximately 1.5 ml of blood was collected. The blood was centrifuged, serum removed, and the serum was stored at 280°C. Total and free T 4 and total and free T 3 were determined with RIA kits. Limits of detection for these kits were: 0.25 mg/dl, total T 4; 0.01 ng/dl, free T 4; 7 ng/dl, total T 3; and 0.2 pg/ml, free T 3. Analysis of serum TSH. Rat TSH, obtained from the National Hormone and Pituitary Program, was iodinated by the glucose oxidase/lactoperoxidase method (Tower et al., 1977). TSH bound to anti-rat-TSH antibody was separated from unbound TSH by immunoprecipitation with a goat anti-rabbit secondary antibody. The amount of bound TSH was determined by gamma spectrometry. Analysis of thyroid gland growth. Stimulation of thyroid gland growth was determined by measuring thyroid gland weight and thyroid follicular cell proliferation. On experimental day 21, thyroid glands were removed and placed in 10% buffered formalin, at room temperature, for approximately two h. Excess tissue was removed from each thyroid gland, weighed, and formalinfixed for 24 h. Thyroid follicular cell proliferation was measured by autoradiography. Two h before removing the thyroid glands, rats were injected with 3 H-thymidine (33 microcuries per 100 g body weight). Thyroid glands were removed, fixed, embedded in paraffin, and sliced into 5 mm sections. Autoradiography was performed using routine techniques (Christov, 1985). Slides were incubated for 4 weeks at 4°C, then developed with D-19 developer (at 8°C) and Kodak fixer. The slides were then lightly stained with hematoxylin and eosin. Assessment of thyroid follicular cell proliferation. The autoradiographic labeling index (LI) was determined by counting the number of nuclei labeled with black silver grains. The criterion for a labeled thyroid follicular cell nucleus was that it had to have 10 or more black silver grains. One thousand nuclei (labeled plus unlabeled) of each thyroid gland were counted. To reduce bias, slides were randomized, viewed under a microscope (203), and tissue sections (8 per slide) were randomly selected. An area within a tissue section was also selected randomly, and thyroid follicular cell nuclei were counted (labeled and unlabeled). Steps 3 and 4 were repeated until a total of 1000 nuclei had been counted. Statistics. Differences between control and treated animals were determined using a one-way ANOVA. Significant differences between groups were determined using the Duncan’s multiple range post-hoc test (p , 0.05), and indicated by an asterisk. Correlational analysis (r) was used to determine the strength of the relationship between serum TSH concentrations and increases in thyroid growth. Correlational analysis was performed because serum TSH concentrations (x variable) is not fixed, assumptions for correlational analysis was not violated, and nonlinear models resulted in a worse fit. Statistical analyses were performed using STATISTICA 4.5, Statsoft Inc. (Tulsa, Oklahoma) and Prophet 5, BBN Technologies (Cambridge, Massachusetts).

RESULTS

Effect of PTU and MMI on body weight and feed consumption. Although the data is not shown, body weights among rats were similar prior to administering PTU and MMI. By

SENSITIVITY OF THYROID GROWTH TO TSH

265

PTU or MMI, which remained reduced throughout the 21-day treatment period. Control rats had total serum T 4 concentrations of approximately 5 mg/dl throughout the 21-day period (Fig. 2). Serum total T 4 concentration was either not affected or reduced 35% on treatment day 14 in rats treated with 3 ppm or 10 ppm of PTU or MMI, respectively. Serum total T 4 concentrations were reduced more than 95% by day 7 in rats treated with higher dietary concentrations of PTU or MMI, and these remained diminished for the remainder of the 21-day treatment period. Serum TSH concentration in control rats was approximately 6.3 ng/ml throughout the 21-day period (Fig. 3). Treatment

FIG. 1. Effects of PTU or MMI on serum total T 3. Serum total T 3 concentration was measured on treatment days –3, 3, 7, 14, and 21. Symbols indicate the different feed concentrations of PTU [control (F); 1 ppm (E); 3 ppm (■); 10 ppm (h); 30 ppm (Œ); 100 ppm (‚); and 300 ppm (), respectively] or MMI [control (F); 3 ppm (E); 10 ppm (■); 30 ppm (h); 100 ppm (Œ); 300 ppm (‚); and 1,000 ppm (), respectively]. Each value represents the mean 6 SE of 4 or 5 rats. The lowest dose producing a significant difference (p , 0.05) from controls is indicated with an asterisk (*); doses producing greater reductions are also significantly different from controls.

treatment day 21, rats treated with high dietary concentrations of PTU (greater than 10 ppm) and MMI (greater than 100 ppm), body weight was reduced approximately 17%. Feed consumption was significantly increased in rats treated with 300 ppm of PTU, but not affected by MMI treatment. Effects of PTU and MMI on serum total T 3, total T 4, and TSH. Serum total T 3 concentration from control rats was between 75 and 90 ng/dl throughout the 21-day period (Fig. 1). Treatment with low dietary concentrations of PTU (3 ppm) or MMI (10 ppm) decreased serum total T 3 by 30%, which tended to return to control levels by day 21. Larger reductions in serum total T 3, as much as 65% by the 3rd treatment day, occurred in rats treated with higher dietary concentrations of

FIG. 2. Effects of PTU and MMI on serum sotal T 4. Serum total T 4 concentration was measured on treatment days –3, 3, 7, 14, and 21. Symbols indicate the different feed concentrations of PTU: control (F); 1 ppm (E); 3 ppm (■); 10 ppm (h); 30 ppm (Œ); 100 ppm (‚); and 300 ppm (), respectively] or MMI [control (F); 3 ppm (E); 10 ppm (■); 30 ppm (h); 100 ppm (Œ); 300 ppm (D); and 1,000 ppm (), respectively]. Each value represents the mean 6 SE of 4 or 5 rats. The lowest dose producing a significant difference (p , 0.05) from controls is indicated with an asterisk (*), doses producing greater reductions are also significantly different from controls.

266

HOOD ET AL.

60, 40, and 50%, respectively, in rats treated with 10 ppm or greater of PTU. Dietary concentrations higher than 30 ppm of PTU reduced serum total and free T 4 93 and 96%, respectively, and reduced serum total and free T 3 64 and 73%, respectively. Figure 5 also illustrates that reductions of serum total and free T 4 and T 3 were dependent on the dietary concentration of MMI. Serum total T 4 and free T 3 were increased 42 and 27%, respectively, in rats treated with the lowest dietary concentration (3 ppm) of MMI. Serum total T 4, free T 4, and free T 3 were decreased 67, 78, and 47%, respectively, in rats treated with 30 ppm or more of MMI. Dietary concentrations greater than 300 ppm of MMI decreased serum total and free T 4 by 96%, and decreased serum total and free T 3 by 61 and 73%, respectively. Effects of PTU and MMI on serum TSH, thyroid follicular cell proliferation, and thyroid weight on treatment day 21.

FIG. 3. Effects of PTU and MMI on serum TSH. Serum TSH concentration was measured on treatment days –3, 3, 7, 14, and 21. Symbols indicate the different feed concentrations of PTU: control (F); 1 ppm (E); 3 ppm (■); 10 ppm (h); 30 ppm (Œ); 100 ppm (‚); and 300 ppm (), respectively; or MMI: control (F); 3 ppm (E); 10 ppm (■); 30 ppm (h); 100 ppm (Œ); 300 ppm (‚); and 1,000 ppm (), respectively. Each value represents the mean 6 SE of 4 or 5 rats. The lowest dose producing a significant difference (p , 0.05) from controls is indicated with an asterisk (*); doses producing greater increases are also significantly different from controls.

with PTU (3 ppm) or MMI (10 ppm) increased serum TSH concentrations 170% on day 14 and 110% on day 3, respectively, but the increase was transient, as serum TSH was not increased by the end of the 21-day treatment period. Treatment with higher dietary concentrations of PTU or MMI resulted in significant increases in serum TSH concentration that had not reached a plateau during the 21-day treatment period. Effects of PTU and MMI on serum total and free thyroid hormones on treatment day 21. Figure 4 illustrates that reductions in serum total and free T 4 and serum total and free T 3 on day 21 were dependent on the dietary concentration of PTU. Serum total T 4, free T 4, total T 3, and free T 3 were reduced 80,

FIG. 4. Dietary concentration-dependent effects of PTU on serum thyroid hormone concentrations. Total T 4 (mg/dl), free T 4 (ng/dl), total T 3 (ng/dl) and free T 3 (pg/ml) were measured on treatment day 21.*Significantly different (p , 0.05) from controls.


267

shaped thyroid follicular cells, large colloid spaces, and few labeled nuclei. Tissue sections from PTU and MMI treated rats contained cuboidal to columnar shaped thyroid follicular cells, small irregularly shaped colloid spaces, and numerous labeled nuclei. Sensitivity of thyroid gland growth to TSH. Figure 9 shows two scatter graphs of serum TSH concentrations versus labeling index (top panel) and serum TSH concentration versus thyroid weight (bottom panel). The dotted-lined box shows baseline values of TSH, labeling indices, and thyroid weights of control rats. Increases in serum TSH concentration were significantly correlated with increases in labeling index and thyroid weight (r 5 0.72* and 0.81*, respectively) at an alpha level of p 5 0.0001.

FIG. 5. Dietary concentration-dependent effects of MMI on serum thyroid hormone concentrations. Total T 4 (mg/dl), free T 4 (ng/dl), total T 3 (ng/dl) and free T 3 (pg/ml) were measured on treatment day 21. *Significantly different (p , 0.05) from controls.

Serum TSH and thyroid weight were increased 300 and 95%, respectively, in rats treated with 10 ppm or more of PTU (Fig. 6). Treatment with 30 ppm or more of PTU increased serum TSH 490%, thyroid follicular cell proliferation 1,400%, and thyroid weight 200%. Serum TSH, thyroid follicular cell proliferation, and thyroid weight were increased 300%, 1,000%, and 64%, respectively, in rats treated with 30 ppm MMI (Fig. 7). Treatment with 100 ppm or more of MMI increased serum TSH 480%, thyroid follicular cell proliferation 1,300%, and thyroid weight 200%. Thyroid gland morphology. Figure 8 is a photomicrograph of representative sections of thyroid glands from a control (top panel) rat and rats treated with either 300 ppm of PTU (middle panel) or 1000 ppm of MMI (bottom panel) for 21 days. Tissue sections from control rats contained squamous to cuboidal

FIG. 6. Dietary concentration-dependent effects of PTU on serum TSH, thyroid weight, and thyroid follicular cell proliferation. Serum TSH (ng/ml), thyroid weight (mg/kg), and thyroid follicular cell proliferation, expressed as a labeling index, were determined on treatment day 21. *Significantly different (p , 0.05) from controls.

268

HOOD ET AL.

gland to stimulation by TSH. Stimulation of the thyroid gland was assessed by determining the number of proliferating thyroid follicular cells (expressed as a labeling index) and by recording thyroid gland weights. Because increases in serum TSH concentration of PTU- and

FIG. 7. Dietary concentration-dependent effects of MMI on serum TSH, thyroid weight, and thyroid follicular cell proliferation, expressed as a labeling index, were determined on treatment day 21. *Significantly different (p , 0.05) from controls.

DISCUSSION

Increased serum TSH concentration has been proposed to mediate the thyroid tumor-promoting effect of antithyroid drugs and phenobarbital (PB) (McClain et al., 1988). However, serum TSH is only modestly increased, when compared to antithyroid drugs, and the increase in TSH of PB-treated rats is not sustained; that is it returns to control levels after 12 weeks of treatment (McClain et al., 1989). This is an important finding, as 12 weeks is the time required for the development of thyroid tumors in PB-mediated thyroid tumor-promotion studies (Hiasa et al., 1983). It is unclear whether the early increase in serum TSH concentration of PB-treated rats could be expected to stimulate the thyroid gland, because the increase in serum TSH of PB-treated rats is very small, compared to other chemicals, such as antithyroid drugs. The goal of the present study was to determine the sensitivity of the thyroid

FIG. 8. Thyroid gland morphology. Representative photomicrographs (173 magnification) of thyroid glands from a control (top panel) rat and rats treated with either 300 ppm of PTU (middle panel) or 1000 ppm of MMI (bottom panel) for 21 days. Thyroid follicular cell nuclei labeled with 3Hthymidine appear black.


FIG. 9. Relationship between serum TSH and thyroid weight and follicular cell proliferation in rats treated with PTU or MMI for 21 days. Correlational coefficient (r) values are given and show the strength of the linear relationship between serum TSH concentrations and increases in thyroid growth. The r 2 values show the proportion of the variability in the y-values that are explained by the variation associated with the x-values. *Indicates statistical significance (p , 0.05; n, 35). Note that the data for rats treated with PTU and MMI were pooled for this analysis.

MMI-treated rats is thought to be a result of reduced serum T 4 and T 3 concentrations (Scanlon and Toft, 1996), it was important that treatment with PTU and MMI was of sufficient length to result in maximum reductions in serum T 4 and T 3 concentrations. Maximum reductions in serum T 3 and T 4 (Figs. 1 and 2, respectively) were observed after one week of treatment with high dietary concentrations of PTU or MMI. This is consistent with findings of previous studies (Delia and Thompson, 1988; Rondeel et al., 1992). Furthermore, the reductions in serum T 3 and T 4 were dependent upon the dietary concentration of PTU or MMI administered (Figs. 4 and 5, respectively). PTU and MMI were more effective at reducing serum T 4 concentration than serum T 3, which suggests mechanisms that maintain serum T 3 concentration were activated in rats treated with PTU or MMI. Mechanisms that may play such a role include: extrathyroidal conversion of T 4 to T 3 catalyzed by deiodinases (Silva et al., 1984), recovery of conjugated T 3-sulfate (T 3-S) by sulfatases (Visser, 1990), and/or enterohepatic circulation (Visser, 1990). These mechanisms may also explain the small increase in serum T 4 and T 3 observed in rats treated with the lowest dose of MMI (Fig. 5, top and bottom paanels). These results suggest that the mechanisms that maintain serum thyroid hormone concentrations in antithyroid drug-

269

treated and microsomal enzyme inducer-treated rats should be studied further. Serum TSH concentrations steadily increased over the 21day treatment period and did not appear to reach a plateau (Fig. 3). This result is consistent with a previous study, which showed that a 21-day treatment period with an antithyroid drug is insufficient to increase serum TSH concentrations maximally (Wynford-Thomas et al., 1982b). Nevertheless, 30 ppm or more of PTU and 300 ppm or more of MMI produced maximum increases in serum TSH (Figs. 6 and 7, respectively). Furthermore, thyroid gland weight and thyroid follicular cell proliferation were also maximally increased by the higher dietary concentrations of PTU and MMI (Figs. 6 and 7, respectively). The large increase in the number of proliferating thyroid follicular cells is consistent with the morphological changes seen in rats treated with PTU and MMI (Fig. 8). Both treatments resulted in classical morphological signs of TSH stimulation. For instance, morphological evidence of thyroid follicular cell hyperplasia (increased cell number) and hypertrophy (increased cell size), as well as reduced size and irregularly-shaped colloid spaces, was apparent (Fig. 8). Because these morphological changes were not quantified using morphometric techniques, the degree that these signs of TSH stimulation changed in these rats is unknown. Because thyroid follicular cell proliferation was increased more than serum TSH in the present study, which has also been shown to occur in rats treated with aminotriazole (Wynford-Thomas et al., 1982b), this finding suggests that thyroid gland growth may be highly sensitive to TSH stimulation. The sensitivity of thyroid gland growth is further supported by the data illustrated in Figure 9, which shows two scatter graphs of serum TSH and thyroid gland growth. Because TSH is considered to be the major factor regulating thyroid growth (Rapoport and Spaulding, 1996), it is not surprising to find that serum TSH concentrations is correlated with thyroid gland growth. However, the correlation was not strong. The data suggested that 34 to 50% (1 – r 2 5 the proportion of unexplained variability) of the variance in thyroid growth is due to something other than TSH. Growth factors, such as insulin-like growth factor (IGF), have been shown to play a role in regulating thyroid follicular cell proliferation. However, the contribution of these other growth factors working independently of TSH is unclear. For example, IGF and TSH have been found to work together to potentiate the proliferation response of thyroid follicular cells (Smith et al., 1986; Tramontano et al., 1986). Besides other growth factors, the high variability in the number of proliferating thyroid follicular cells could also be due to the desensitization of thyroid follicular cells to TSH. Thyroid follicular cells are known to become desensitized to TSH stimulation, resulting in a burst of proliferating thyroid follicular cells within the first week of treatment with an antithyroid drug, followed by a decline in the number of proliferating thyroid follicular cells (Smith et al., 1987; Wynford-Thomas et al., 1982a). It should be mentioned that previ-

270

HOOD ET AL.

ous studies also report similar amounts of variation in thyroid follicular cell proliferation indices (Wynford-Thomas et al., 1982a,b). We found it interesting that the high variability in thyroid growth appeared to be restricted to rats that had high (greater than 25 ng/ml) serum TSH concentrations (Fig. 9). Rats with serum TSH concentrations between 10 and 20 ng/ml, representing a 60 –200% increase, had a 150 –250% increase in the number of proliferating thyroid follicular cells that was much less variable. This can also be seen in the dose-response curves of rats treated with PTU or MMI (Figs. 6 and 7, respectively). In these same rats, thyroid weight was not much different from baseline thyroid-weight levels. These results further illustrate the sensitive nature of thyroid gland growth to TSH stimulation. These findings support the concept that small to moderate increases in serum TSH is sufficient to stimulate thyroid gland growth. Also, thyroid follicular cell proliferation indices may be a more useful thyroid growth parameter than recording thyroid gland weights. However, one must keep in mind that others have shown that large increases in thyroid follicular cell proliferation is a transient response, although the transient nature of thyroid follicular cell proliferation has not been demonstrated in microsomal enzyme inducer-treated rats. In conclusion, these findings support the hypothesis that thyroid gland growth is sensitive to TSH stimulation. Furthermore, thyroid follicular cell proliferation indices may be a very useful thyroid growth parameter, because (a) the increase in the number of proliferating thyroid follicular cells was much greater than the increase in thyroid gland weight, and (b) moderate increases in serum TSH resulted in increased number of proliferating thyroid follicular cells, whereas thyroid gland weight was not increased in these rats. The sensitivity of the rat thyroid gland to TSH stimulation may explain why some chemicals, such as PB, that produce small increases in serum TSH can adversely affect the thyroid, such as promoting thyroid tumors. The mechanism by which PB is thought to promote thyroid tumors is by increasing the glucuronidation of thyroid hormones and resulting in moderate increases in serum TSH. If this is correct, then other microsomal enzyme inducers that increase the glucuronidation of thyroid hormones and produce similar moderate increases in serum TSH should also promote thyroid tumors. Because the microsomal enzyme inducer, pregnenolone-16a-carbonitrile (PCN), increases serum TSH more than does PB (Liu et al., 1995), PCN treatment should be more effective at promoting thyroid tumors than PB in rats, and this needs to be demonstrated in future studies.

REFERENCES Barter, R. A., and Klaassen, C. D. (1992). UDP-glucuronosyltransferase inducers reduce thyroid hormone levels in rats by an extrathyroidal mechanism. Toxicol. Appl. Pharmacol. 113, 36 – 42. Barter, R. A., and Klaassen, C. D. (1994). Reduction of thyroid hormone levels and alteration of thyroid function by four representative UDP-glucuronosyltransferase inducers in rats. Toxicol. Appl. Pharmacol. 128, 9 –17. Christov, K. (1985). Cell population kinetics and DNA content during thyroid carcinogenesis. Cell Tissue Kinet. 18, 119 –131. Cooper, D. S., Kieffer, J. D., Halpern, R., Saxe, V., Mover, H., Maloof, F., and Ridgway, E. C. (1983). Propylthiouracil (PTU) pharmacology in the rat. II. Effects of PTU on thyroid function. Endocrinology 113, 921–928. De Sandro, V., Chevrier, M., Boddaert, A., Melcion, C., Cordier, A., and Richert, L. (1991). Comparison of the effects of propylthiouracil, amiodarone, diphenylhydantoin, phenobarbital, and 3-methylcholanthrene on hepatic and renal T 4 metabolism and thyroid gland function in rats. Toxicol. Appl. Pharmacol. 111, 263–278. Delia, A. J., and Thompson, E. B. (1988). A time-course study of hypothyroidism-induced hypotension: its relation to food deprivation. Arch. Int. Pharmacodyn. Ther. 296, 210 –223. Engler, H., Taurog, A., Luthy, C., and Dorris, M. L. (1983). Reversible and irreversible inhibition of thyroid peroxidase-catalyzed iodination by thioureylene drugs. Endocrinology 112, 86 –95. Hiasa, Y., Kitahori, Y., Katoh, Y., Ohshima, M., Konishi, N., Shimoyama, T., Sakaguchi, Y., Hashimoto, H., Minami, S., and Murata, Y. (1987). Effects of castration before and after treatment with N-bis (2-hydroxypropyl)nitrosamine (DHPN) on the development of thyroid tumors in rats treated with DHPN followed by phenobarbital. Jpn. J. Cancer Res. 78, 1063–1067. Hiasa, Y., Kitahori, Y., Konishi, N., Enoki, N., and Fujita, T. (1983). Effect of varying the duration of exposure to phenobarbital on its enhancement of N-bis(2-hydroxypropyl)nitrosamine-induced thyroid tumorigenesis in male Wistar rats. Carcinogenesis 4, 935–937. Hiasa, Y., Kitahori, Y., Ohshima, M., Fujita, T., Yuasa, T., Konishi, N., and Miyashiro, A. (1982a). Promoting effects of phenobarbital and barbital on development of thyroid tumors in rats treated with N-bis(2-hydroxypropyl)nitrosamine. Carcinogenesis 3, 1187–1190. Hiasa, Y., Ohshima, M., Kitahori, Y., Yuasa, T., Fujita, T., and Iwata, C. (1982b). Promoting effects of 3-amino-1,2,4-triazole on the development of thyroid tumors in rats treated with N-bis(2-hydroxypropyl)nitrosamine. Carcinogenesis 3, 381–384. Japundzic, M. M., Bastomsky, C. H., and Japundzic, I. P. (1976). Enhanced biliary thyroxine excretion in rats treated with pregnenolone-16a-carbonitrile. Acta Endocrinol. Copenh. 81, 110 –119. Jemec, B. (1980). Studies of the goitrogenic and tumorigenic effect of two goitrogens in combination with hypophysectomy or thyroid hormone treatment. Cancer 45, 2138 –2148. Kitahori, Y., Hiasa, Y., Konishi, N., Enoki, N., Shimoyama, T., and Miyashiro, A. (1984). Effect of propylthiouracil on the thyroid tumorigenesis induced by N-bis(2-hydroxypropyl)nitrosamine in rats. Carcinogenesis 5, 657– 660. Larsen, P. R., and Silva, J. E. (1983). Intrapituitary mechanisms in the control of TSH secretion. In Molecular Basis of Thyroid Hormone Action (J. H. Oppenheimer, H. H. Samuels, J. W. Apriletti, Eds.), pp. 351–383. Academic Press, New York.

ACKNOWLEDGMENTS

Liu, J., Liu, Y., Barter, R. A., and Klaassen, C. D. (1995). Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study. J. Pharmacol. Exp. Ther. 273, 977–985.

This work was funded by NIH Grant ES-08156. AH was supported by NIH Training Grant ES-07079.

Mannisto, P. T., Ranta, T., and Leppaluoto, J. (1979). Effects of methylmercaptoimidazole (MMI), propylthiouracil (PTU), potassium perchlorate (KClO4) and potassium iodide (KI) on the serum concentrations of thyro-

SENSITIVITY OF THYROID GROWTH TO TSH trophin (TSH) and thyroid hormones in the rat. Acta Endocrinol. (Copenh.) 91, 271–281. McClain, R. M. (1992). Thyroid gland neoplasia: Non-genotoxic mechanisms. Toxicol. Lett. 64 – 65, 397– 408. McClain, R.M. (1995). The use of mechanistic data in cancer risk assessment: Case example—sulfonamides. In Low-Dose Extrapolation of Cancer Risks: Issues and Perspectives (S. Olin, W. Farland, C. Park, L. Rhomberg, R. Scheuplein, T. Starr, J. Wilson, Eds.), pp. 163–173. International Life Sciences Institute, Washington, DC. McClain, R. M., Levin, A. A., Posch, R., and Downing, J. C. (1989). The effect of phenobarbital on the metabolism and excretion of thyroxine in rats. Toxicol. Appl. Pharmacol. 99, 216 –228. McClain, R. M., Posch, R. C., Bosakowski, T., and Armstrong, J. M. (1988). Studies on the mode of action for thyroid gland tumor promotion in rats by phenobarbital. Toxicol. Appl. Pharmacol. 94, 254 –265. Nakashima, T., Taurog, A., and Riesco, G. (1978). Mechanism of action of thioureylene antithyroid drugs: factors affecting intrathyroidal metabolism of propylthiouracil and methimazole in rats. Endocrinology 103, 2187– 2197. Rapoport, B., and Spaulding, S. W. (1996). Mechanism of action of thyrotropin and other thyroid gland growth factors. In Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (L. E. Braverman, and R. D. Utiger, Eds.), pp. 207–219. Lippincott–Raven, Philadelphia. Rondeel, J. M., de Greef, W. J., Klootwijk, W., and Visser, T. J. (1992). Effects of hypothyroidism on hypothalamic release of thyrotropin-releasing hormone in rats. Endocrinology 130, 651– 656. Scanlon, M. F., and Toft, A. (1996). Regulation of thyrotropin secretion. In Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text (L. Braverman, and R.D. Utiger, Eds.), pp. 220 –240. J. B. Lippincott, New York. Silva, J. E., Gordon, M. B., Crantz, F. R., Leonard, J. L., and Larsen, P. R.

271

(1984). Qualitative and quantitative differences in the pathways of extrathyroidal triiodothyronine generation between euthyroid and hypothyroid rats. J. Clin. Invest. 73, 898 –907. Smith, P., Williams, E. D., and Wynford-Thomas, D. (1987). In vitro demonstration of a TSH-specific growth desensitising mechanism in rat thyroid epithelium. Mol. Cell Endocrinol. 51, 51–58. Smith, P., Wynford-Thomas, D., Stringer, B. M., and Williams, E. D. (1986). Growth factor control of rat thyroid follicular cell proliferation. Endocrinology 119, 1439 –1445. Stringer, B., Wynford-Thomas, D., Jasani, B., and Williams, E. D. (1981). Effect of goitrogen administration on the circadian rhythm of serum thyroid stimulating hormone in the rat. Acta Endocrinol. (Copenh.) 98, 396 – 401. Taurog, A. (1976). The mechanism of action of the thioureylene antithyroid drugs. Endocrinology 98, 1031–1046. Tower, B. B., Clark, B. R., and Rubin, R. T. (1977). Preparation of 125I polypeptide hormones for radioimmunoassay using glucose oxidase with lactoperoxidase. Life Sci. 21, 959 –966. Tramontano, D., Cushing, G. W., Moses, A. C., and Ingbar, S. H. (1986). Insulin-like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Graves’-IgG. Endocrinology 119, 940 –942. Visser, T. J. (1990). Importance of deiodination and conjugation in the hepatic metabolism of thyroid hormone. In The Thyroid Gland (M.A. Greer, Ed.), pp. 255–283. Raven Press, New York. Wynford-Thomas, D., Stringer, B. M., and Williams, E. D. (1982a). Desensitisation of rat thyroid to the growth-stimulating action of TSH during prolonged goitrogen administration. Persistence of refractoriness following withdrawal of stimulation. Acta Endocrinol. Copenh. 101, 562–569. Wynford-Thomas, D., Stringer, B. M., and Williams, E. D. (1982b). Dissociation of growth and function in the rat thyroid during prolonged goitrogen administration. Acta Endocrinol. (Copenh.) 101, 210 –216.