Mechanism of Thiazopyr-Induced Effects on Thyroid Hormone Homeostasis in Male Sprague–Dawley Rats

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

142, 133–142 (1997)

TO968032

Mechanism of Thiazopyr-Induced Effects on Thyroid Hormone Homeostasis in Male Sprague–Dawley Rats KATHY J. HOTZ,* ALAN G. E. WILSON,*,1 DARYL C. THAKE,* MARSTON V. ROLOFF,* CHARLES C. CAPEN,† JOEL M. KRONENBERG,‡ AND DAVID W. BREWSTER§ *Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167; †Ohio State University, Department of Veterinary BioSciences, 1925 Coffey Road, Columbus, Ohio 43210; ‡AgrEvo USA Company, Regulatory Affairs, 2711 Centerville Road, Wilmington, Delaware 19808; and §Hoechst-Roussel Pharmaceuticals, P.O. Box 2100, Somerville, New Jersey 08876 Received July 19, 1996; accepted September 23, 1996

Mechanism of Thiazopyr-Induced Effects on Thyroid Hormone Homeostasis in Male Sprague-Dawley Rats. HOTZ, K. J., WILSON, A. G. E., THAKE, D. C., ROLOFF, M. V., CAPEN, C. C., KRONENBERG, J. M., AND BREWSTER, D. W. (1997). Toxicol. Appl. Pharmacol. 142, 133–142. Chronic administration of thiazopyr in the diet at dose levels of 1000 and 3000 ppm, but not 100 ppm, has demonstrated an increase in thyroid follicular cell tumors in male Sprague–Dawley rats. In the studies reported here we have evaluated the mechanism of thiazopyrinduced thyroid tumors by studying the effect of thiazopyr on a number of endpoints that indicate hypothalamic–pituitary–thyroid homeostasis. At a dose level of 3000 ppm, thiazopyr caused a marked depression in circulating levels of T4 as soon as 7 days after commencement of treatment. Concurrent with this decrease in T4 was an increase in TSH levels, an increase in thyroid and liver weights, a three- to sixfold increase in hepatic T4-uridine diphosphate glucuronosyl transferase (UDPGT) activity, and increases in thyroid follicular cell hypertrophy and hyperplasia. Dose-related changes associated with thiazopyr treatment were significant increases in liver weight, thyroid weight, and hepatic T4-UDPGT activity at high doses. Increased levels of serum TSH, T3 , and rT3 , decreased levels of T4 , and an increased incidence of thyroid follicular cell hypertrophy and hyperplasia were observed 56 days after the initiation of 3000 ppm thiazopyr. All the changes, except thyroid weight, were partially or completely reversible upon removal of thiazopyr from the diet. Increased thyroid T4 elimination, primarily via increased hepatic conjugation by T4-UDPGT, resulting in decreased serum T4 , appeared to be responsible for the increased TSH levels. The sustained increase in TSH by thiazopyr appears responsible for the stimulation of the thyroid follicular cells resulting in follicular cell hypertrophy, hyperplasia, and ultimately neoplasia. In summary, evidence is presented for a hormonally mediated, threshold-dependent process for the development of thyroid follicular cell tumors from high-dose thiazopyr administration in male rats. This mechanism is not considered to be relevant to humans, since the thyroid of humans is much less sensitive to this pathogenic phenomenon than rodents. q 1997 Academic Press

1

To whom correspondence should be addressed.

Thiazopyr (Fig. 1) was developed by Monsanto Company as a herbicide candidate for the preemergent control of grass and broadleaf weeds in soybeans, alfalfa, cotton, peanuts, and other crops. Oncogenicity studies have demonstrated that administration of thiazopyr for 24 months in the diet at dose levels of 1000 and 3000 ppm resulted in an increase in thyroid follicular cell adenomas (13 and 20%, respectively) in male rats (Naylor and McDonald, 1992). The incidence of carcinomas in the animals dosed with 3000 ppm thiazopyr was not significantly different from that in the control animals. No oncogenic or thyrotoxic effects were observed in female rats. Exposure of the rat thyroid gland to sustained elevated levels of TSH can result in the formation of thyroid gland hyperplasia and neoplasia (Furth, 1968). Increases in TSH levels can result from a reduction in levels of circulating thyroid hormones (T3 and T4) through feedback control of the hypothalamus and pituitary gland (Sawin, 1969; Lissitsky, 1976). Depressed levels of T4 may be caused by an inhibition of thyroid hormone synthesis or an increase in hormone clearance. Induction of hepatic microsomal enzymes plays an important role in the metabolism of these hormones, as well as other xenobiotics (McClain et al., 1989; McClain, 1989). The studies reported here delineate the effect of thiazopyr on a number of parameters which are critical to hypothalamic–pituitary–thyroid homeostasis (Brewster and Hotz, 1992; Hotz et al., 1993a,b). In these studies, liver and thyroid weights, thyroid morphology, serum hormone levels, and hepatic UDPGT activity were determined. The temporal and dose-related changes in these parameters have been determined as well as the reversibility of these parameters upon removal of thiazopyr from the diet. Blood T4 half-life, T4 biliary elimination, thyroidal iodine uptake and organification, and hepatic enzymatic activity (i.e., deiodinase activity) were also evaluated to determine the mechanism(s) involved in the thyroid changes induced by thiazopyr. The results of these studies have provided a basis for demonstrating the mode of action for the development of thyroid tumors which occur in male rats following chronic administration of thia-

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0041-008X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Chemical name and structure of thiazopyr.

zopyr at relatively high dose levels. The results show that thiazopyr perturbs thyroid hormone homeostasis as a consequence of hepatic UDPGT induction, which increases biliary excretion of T4 and results in a compensatory increase in TSH secretion which predisposes the rat thyroid to develop follicular cell tumors in chronic studies. MATERIALS AND METHODS Test material and chemicals. The test material for this study was thiazopyr (purity 96.4%) which was supplied by Monsanto Company. All other chemicals were of the highest quality available and were purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific Products (Pittsburgh, PA) unless otherwise specified. Analysis of the test material. Dietary concentrations of thiazopyr, homogeneity of the diet mixtures, and stability of thiazopyr in the diet were determined by gas chromatographic analysis using an electron capture detector. The test material was determined to be stable under study conditions. Animals and treatment. Male Sprague–Dawley rats, Crl:CD(SD)BR, were purchased from Charles River Laboratories, Inc. (Portage, MI). The rats were 11–12 weeks old at the start of the studies. However, the animals in the time–course study were 11–23 weeks old so that all animals were approximately the same age at termination. Animals were identified by eartags and bar-coded cage cards and were acclimated for 7 to 30 days before being randomly assigned to study groups. The animals were housed individually in stainless steel suspension cages in an environmentally controlled animal room (temperature 64.4–78.87F and humidity 40–70%) on a 12-hr light/dark cycle. Purina Certified Rodent Chow (No. 5002; Purina Mills, St. Louis, MO) and tap water were provided ad libitum. Food consumption was determined weekly and animal body weights and clinical observations were recorded every other week. Animals were not fasted prior to euthanization. Five groups of animals with 40 animals per group (20 control and 20 treated) were fed control or 3000 ppm thiazopyr-amended diet for 7, 14, 28, 56, and 90 days. Commencement of thiazopyr-amended diet administration was staggered so that all animals were approximately the same age at termination. In another study, seven groups of animals, with 20 animals/ group, were fed untreated diet or thiazopyr-treated diet (0, 10, 30, 100, 300, 1000, and 3000 ppm) for 56 days (an optimal time point obtained from the time–course study). Four additional groups were used to assess the reversibility of effects induced by thiazopyr. Control animals were fed

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untreated diet for 112 or 168 days. The treated animals were administered 3000 ppm of thiazopyr via the diet for 56 days followed by untreated diet for 56 or 112 days. In a third study, four groups of animals consisting of 10 to 20 animals were fed untreated diet or thiazopyr-treated diet (3000 ppm) for approximately 56 days to determine T4 half-life, biliary elimination, thyroid iodine uptake, and hepatic enzymatic activity. Euthanization procedures. Animal euthanizations took place between 7 AM and 12 noon and alternated between control and all groups of treated animals to minimize the diurnal variation of TSH (Singh et al., 1967; Fukuda et al., 1975). Every effort was taken to maintain quiet in the animal room since excess noise, cage movement, and stress on the animals are known to influence serum hormone levels (Dohler et al., 1979). The rats were anesthetized with CO2 :O2 (80:20) and then maintained with CO2 :O2 (60:40) while blood was collected via cardiac puncture or from the posterior vena cava. Serum was separated by centrifugation and stored frozen at 0707C. Serum samples were shipped on dry ice to Ani Lytics, Inc. (Gaithersburg, MD) for the determination of T3 , rT3 , T4 , and TSH concentrations. Levels of T3 , rT3 , and T4 were determined using commercially available radioimmunoassay (RIA) kits supplied by Diagnostic Products (Los Angeles, CA). Reagents obtained from the National Pituitary Hormones and Antisera Reference Center (University of California, Los Angeles, CA) were used in determining TSH concentrations (personal communication). At termination, a section of the trachea with the thyroid glands attached was removed and fixed in 10% neutral buffered formalin for 48 to 72 hr. After fixation, the thyroid glands along with the thyroid isthmus were isolated, cleaned of fat and connective tissue, blotted, and weighed. After processing, the thyroids were embedded in paraffin, sectioned at approximately 5 mm, and stained with hematoxylin and eosin for histological evaluation. At termination, the liver was also removed, rinsed in saline, blotted, and weighed. Livers were stored frozen at 0707C for enzyme analysis. T4-Uridine diphosphate glucuronosyl transferase (UDPGT) activity. Hepatic microsomes from frozen livers were prepared according to a modified method of Dent et al. (1976). Briefly, a homogenate of liver with cold 20 mM Tris/1.15% KCl buffer (1/4) was prepared using a Teflon – glass homogenizer. The homogenate was centrifuged at 10,000g for 20 min at 47C. The resulting supernatant was centrifuged at 47C for 1 hr at 100,000g. The microsomal pellet was resuspended in 0.25 M sucrose – 5.4 mM EDTA – 20 mM Tris buffer (pH 7.4) to a final volume of 1 g of liver/ml of buffer. Protein concentration was determined using the dye-binding method of Bradford (1976). Hepatic UDPGT activity was determined using 125I-T4 (Amersham, Arlington Heights, IL) as the substrate according to McClain et al. (1989) with slight modifications; microsomes (0.2 mg microsomal protein/ml) were incubated for 60 min at 377C in 66 mM Tris-HCl buffer (pH 7.8) along with 10 mM MgCl2 , 2.5 mM UDPNAG (uridine 5*-diphospho-N-acetylglucosamine), 1 mM thyroxine, and 125I-thyroxine (5 ml/ml buffer-substrate) in a volume of 175 ml. The reaction was started by the addition of 25 ml UDPGA (from a 40 mM stock). Blanks were prepared by adding UDPGA to the reaction mixtures after the reaction was terminated by the addition of cold methanol. Activity was expressed as pmol/mg protein/min and as pmol/min/total liver. Blood T4 half-life determination. After approximately 56 days on diet, 10 control and 10 thiazopyr-treated animals were administered approximately 10 mCi of [125I]T4 (New England Nuclear, Wilmington, DE) by intravenous (iv) injection into the lateral tail vein. Blood was collected by retro-orbital bleeding at 15, 30, and 45 min after [125I]T4 administration, then at 1, 2, 4, 6, 8, 24, 48, and 72 hr. The amount of radiolabel appearing in the blood was measured using a Micro-Medic ME Plus automatic gamma counter (Systems, Inc., Huntsville, AL). The half-life of T4 in the blood was determined by nonlinear regression analysis performed by an exponential fit computer program, which is a modified Gauss–Newton method based on the method of residuals and consists of sequentially feathering, or peeling off, successive exponential terms (Gibaldi and Perrier, 1982; Tuey, 1980).

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TABLE 1 Temporal Effects of Dietary Administration of Thiazopyr on Thyroid Function in Male Ratsa Treatment 7-day Control Treated 14-Day Control Treated 28-Day Control Treated 56-Day Control Treated 90-Day Control Treated

Body wt (g)

Liver wt (g)

Thyroid wt (mg)

T4 (mg/dl)

T3 (ng/dl)

rT3 (ng/dl)

641 { 13 645 { 13

24.2 { 0.7 33.2 { 0.9*

24.7 { 0.9 30.9 { 1.1*

3.7 { 0.2 2.5 { 0.1*

74 { 3 73 { 3

— —

2.6 { 0.2 4.2 { 0.5*

632 { 10 621 { 12

23.6 { 0.6 34.5 { 0.9*

25.3 { 0.7 31.7 { 0.8*

2.8 { 0.1 1.9 { 0.1*

66 { 2 66 { 3

— —

3.3 { 0.3 4.4 { 0.4

662 { 15 639 { 12

25.5 { 0.9 41.9 { 0.9*

25.7 { 1.1 35.8 { 1.1*

2.5 { 0.1 1.9 { 0.1*

61 { 4 92 { 3*

3.1 { 0.3 4.6 { 0.3*

4.2 { 0.4 5.7 { 0.5*

641 { 9 627 { 10

25.4 { 0.8 43.5 { 1.1*

24.5 { 0.8 36.3 { 1.7*

2.3 { 0.1 1.0 { 0.1*

63 { 4 67 { 4

— —

2.5 { 0.2 5.0 { 0.4*

657 { 10 624 { 11

25.2 { 0.7 46.9 { 1.3*

26.4 { 1.1 39.9 { 1.3*

3.6 { 0.2 1.7 { 0.1*

67 { 3 76 { 4

— —

2.9 { 0.2 5.2 { 0.4*

TSH (ng/ml)

a Rats were treated with 0 or 3000 ppm of thiazopyr in the diet for 7, 14, 28, 56, or 90 days. Data represent the mean { standard error of the mean for 19 or 20 rats per group. * Significantly different from control with Tukey’s test for unconfounded comparisons after Random Factorial ANOVA or with Student’s t test (p £ 0.05).

Data from individual animals (or the group means) were fitted to the general equation: C Å Ae0at / Be0bt, where C is the concentration of thiazopyr at time t (hr), and A and B are the intercept values extrapolated from alpha (a) and beta (b), the first-order elimination rate constants. The appropriate number of exponential terms required to fit the data were determined visually, and also by examination of the goodness of fit as evaluated by the values for the residuals. Functions Ae0at and Be0bt were designated components 1 and 2, respectively. The half-life values (t12) for the a and b phases were calculated using the relationship: Half-life (t12) Å ln 2 (0.693)/elimination rate constant. Total area under the curve (AUC), calculated as the sum of A/a and B/b, was expressed as the appropriate concentration unit 1 appropriate time unit/ml (i.e., % dose 1 min/ml). Clearance was calculated as the dose/AUC and was expressed as ml/min. Biliary elimination. Approximately 56 days after commencement of the study, 10 control and 10 thiazopyr-treated animals were anesthetized with 50

mg of pentobarbital/kg body wt (intraperitoneal). This was followed by an oral dose of 90 mg pentobarbital/kg which was diluted with isotonic saline to a final volume of 3 ml/kg body wt. The bile duct was ligated and cannulated. Animals were administered approximately 10 mCi of [125I]T4 (New England Nuclear) by tail vein injection and bile was collected at 30-min intervals over a 4-hr period. The bile samples were weighed and the radioactivity appearing in the bile was determined using a Micro-Medic ME Plus automatic gamma counter. Results were expressed as cumulative % of the administered dose in the bile and as the average group rate of excretion of T4 . Iodine uptake and organification. Approximately 56 days after commencement of the study, five control and five thiazopyr-fed animals were administered approximately 10 mCi of [125I]iodine (New England Nuclear) plus 10 mg of potassium iodide carrier in Ç0.25 ml of isotonic saline by tail vein injection. Approximately 24 hr later, the animals were euthanized by CO2 asphyxiation. Blood was collected from the posterior vena cava, and the liver and thyroid glands were removed and weighed. The thyroid glands were homogenized in 5 ml of cold 0.15 M NaCl containing 1 mM potassium iodide according to Comer et al. (1985). The amount of radioac-

TABLE 2 Dose-Related Effects of Dietary Administration of Thiazopyr on Thyroid Function in Male Ratsa Treatment Control 10 ppm 30 ppm 100 ppm 300 ppm 1000 ppm 3000 ppm

Body wt (g) 565 590 576 560 578 579 566

{ { { { { { {

9 9 12 10 12 11 9

Liver wt (g) 21.2 22.4 21.6 21.7 24.1 28.4 38.5

{ { { { { { {

0.8 0.5 0.7 0.6 0.8* 1.1* 0.1*

Thyroid wt (mg) 23.2 24.4 24.5 23.8 25.5 29.1 33.9

{ { { { { { {

0.7 0.7 0.8 0.6 0.7 0.8* 1.3*

T4 (mg/dl) 4.1 4.3 3.9 4.1 4.0 4.0 2.9

{ { { { { { {

0.2 0.3 0.2 0.2 0.2 0.2 0.1*

T3 (ng/dl)

rT3 (ng/dl)

84 { 3 82 { 4 68 { 2* 84 { 3 82 { 3 91 { 4 110 { 6*

0.047 { 0.004 — — — — — 0.071 { 0.006*

a

TSH (ng/ml) 2.7 3.5 2.7 3.1 2.9 3.1 4.3

{ { { { { { {

Rats were treated with thiazopyr in the diet for 56 days. Data represent the mean { standard error of the mean for 19 or 20 rats per group. * Significantly different from control with Dunnett’s test after ANOVA (p £ 0.05).

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0.2 0.4 0.1 0.4 0.3 0.2 0.4*

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FIG. 2. Effect of removal of thiazopyr from the diet on liver and thyroid weights in rats fed thiazopyr for 56 days followed by control diet for 56 or 112 days. a56 days on thiazopyr (3000 ppm) diet. b56 days on thiazopyr (3000 ppm) diet followed by 56 days on control diet. c56 days on thiazopyr (3000 ppm) diet followed by 112 days on control diet. *Significantly different from respective control with Dunnett’s test, p £ 0.05.

tivity in an aliquot of the thyroid homogenate was determined using a gamma counter. Protein was precipitated from the thyroid homogenate with 4 ml of 10% trichloroacetic acid (TCA) and collected by centrifugation at 1300g for 10 min. The resulting protein pellet was resuspended in 4 ml of 5% TCA and centrifuged. The pellet was resuspended in 2 ml of isotonic saline and the amount of radioactivity was measured. Protein was determined using the dye-binding assay of Bradford (1976). Hepatic deiodinase. Approximately 56 days after commencement of diet, 10 control and 10 thiazopyr-fed animals were terminated by CO2 asphyxiation and the livers were removed, weighed, and stored frozen at approximately 0707C. Hepatic deiodinase activity was measured in 5 control and 5 thiazopyrfed animals according to the method of Scammell and Fregly (1981). Homogenates of liver were prepared in 3 vol of phosphate buffer (0.15 M, pH 7.4, 47C) which were then centrifuged at 2000g for 20 min at 47C. The supernatant (0.2 ml) was incubated with 6.4 mM nonradioactive T4 and 2.8 mM dithiothreitol in a total volume of 1 ml for 30 min at 377C. The reaction was stopped by the addition of 2 ml of 95% ethanol and the samples were mixed and immediately placed on ice. After centrifugation (2000g, 20 min) the supernatant was decanted and stored in the freezer (Ç 0107C) until assayed by RIA (Diagnostic Products). The T3 in the blank samples (samples containing T4 , but not incubated) was subtracted from the T3 measured in the test samples to account for the amount of T3 already present in the homogenates and commercial T4 . Protein concentration in the 2000g supernatant was determined using the method of Bradford (1976) and activity was calculated as ng T3 generated/mg protein/30 min or as mg T3 generated/total liver weight/30 min. Statistical analysis. Results were calculated as the means { standard error of the means (SEM) for the number of animals indicated. Comparisons between control and treated animals were made with Tukey’s test, Student’s t test or Dunnett’s test (p £ 0.05 or p £ 0.01, one- or two-sided) (Snedecor and Cochran, 1978; Steel and Torrie, 1980) unless indicated otherwise. Fisher’s exact test (p £ 0.05 or p £ 0.01) was used to compare microscopic findings of the thyroid glands (Fisher, 1950). Outliers were eliminated using Grubbs’ test (Grubbs, 1969; Grubbs and Beck, 1972).

RESULTS

Clinical Observations, Terminal Body and Organ Weights No overt signs of toxicity were observed in any animal on study and all animals survived the entire treatment period.

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Tables 1 and 2 summarize the terminal body, liver, and thyroid weights. There were no significant differences in final body weight between control and treated animals at any of the time points or dose levels examined; however, cumulative body weight gain was decreased at 3000 ppm at 90 days which is consistent with previous toxicity studies establishing 3000 ppm as the MTD (data not shown). Thiazopyr, at a dose level of 3000 ppm, produced a significant time-dependent increase in absolute liver weights as soon as 7 days after administration (Table 1). Dose-related increases in liver weights were observed at 300, 1000, and 3000 ppm after 56 days on thiazopyr-treated diet (Table 2). Liver weights were 114, 134, and 182% of control animals, respectively. Thyroid weights were increased significantly at all time points with the greatest increase at 90 days (Table 1). Thyroid weights were also increased significantly in animals that received 1000 and 3000 ppm (not 300 ppm or less) of thiazopyr for 56 days (125 and 146% of controls, respectively) (Table 2). When animals administered diets containing 3000 ppm thiazopyr for 56 days were removed from thiazopyr-treated diets and placed on untreated diets for an additional 56 or 112 days, absolute liver weights returned to control levels and thyroid weights returned to near control levels (i.e., only 15–17% increase above controls) (Fig. 2). Thyroid Hormone Concentrations Serum TSH, T4 , T3 , and rT3 concentrations after 7, 14, 28, 56, and 90 days of thiazopyr treatment at 3000 ppm are shown in Table 1. T4 levels were significantly less than controls at all time points. TSH levels were increased at all time points; however, the increase at 14 days was not statistically significant. T3 levels were significantly increased

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FIG. 3. Effect of removal of thiazopyr from the diet on serum TSH, T4 , T3 , and rT3 in rats fed thiazopyr for 56 days followed by control diet for 56 or 112 days. a56 days on thiazopyr (3000 ppm) diet. b56 days on thiazopyr (3000 ppm) diet followed by 56 days on control diet. c56 days on thiazopyr (3000 ppm) diet followed by 112 days on control diet. *Significantly different from control with Dunnett’s test, p £ 0.05.

only at 28 days. Reverse T3 (rT3) was measured only at the 28-day time point and was approximately 48% higher than control levels. Thiazopyr, at 3000 ppm in the diet for 56 days, caused a significant increase (160% of controls) in circulating levels of TSH and a significant decrease in T4 (30% lower than controls) (Table 2). Serum T3 and rT3 concentrations were increased at the 3000 ppm dose level (Table 2). A slight decrease in T3 was observed at 30 ppm; however, this effect appeared spurious since it was not evident at the lower or higher dose levels. The effects of removal of thiazopyr from the diet are shown in Fig. 3. TSH levels in treated animals were significantly greater than controls following 56 days of administration of diets containing 3000 ppm thiazopyr but returned to control levels after 56 days of untreated diets and were slightly lower than controls after 112 days of untreated diets.

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T4 levels began to increase to control levels after 56 and 112 days of untreated diet. Serum T3 and rT3 concentrations returned to control levels after 112 days of untreated diet. T4-Hepatic UDPGT Activity Specific and total estimated hepatic T4-UDPGT activities were shown to be significantly increased by 3000 ppm thiazopyr at all time points (Figs. 4A and 4C). Hepatic T4UDPGT was also significantly increased approximately 2to 2.5-fold of control levels in animals administered 1000 and 3000 ppm thiazopyr for 56 days (Fig. 4B). When expressed as estimated total activity/liver, there was an approximately sixfold increase in activity at 3000 ppm (Fig. 4D). The activity in animals treated with thiazopyr for 56 days and then maintained on control diet for 56 or 112 days was still slightly elevated but was not significantly different from controls (data not shown).

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HOTZ ET AL. Specific T4-UDPGT Activity:

FIG. 4. Temporal and dose-dependent effects of thiazopyr on specific and total thyroxine glucuronosyl transferase (T4-UDPGT) activity. (A) T4UDPGT activity (pmol/mg protein/min) in rats fed 3000 ppm thiazopyr in the diet for 7, 14, 28, 56, and 90 days. (B) T4-UDPGT activity (pmol/mg protein/min) in rats fed 3000 ppm thiazopyr in the diet for 56 days. (C) Total T4-UDPGT activity (adjusted for liver weight) in rats fed 3000 ppm thiazopyr in the diet for 7, 14, 28, 56, and 90 days. (D) Total T4-UDPGT activity (adjusted for liver weight) in rats fed 3000 ppm thiazopyr in the diet for 56 days. *Significantly different from respective control with Student’s t test or Dunnett’s test, p £ 0.05.

Thyroid Gland Histopathology

Blood T4 Half-Life

The combined diagnosis ‘‘hypertrophy/hyperplasia’’ was used to refer to diffuse changes in the thyroid glands. The use of this diagnosis indicated that while both of these changes appeared to occur in most affected glands, the relative degree to which each was present was variable and not quantitated. Thyroid follicular cell hypertrophy/hyperplasia was a prominent histopathological change induced by 3000 ppm thiazopyr at 14, 28, 56, and 90 days (Table 3). The maximal effect was observed at 56 days after administration of 3000 ppm thiazopyr (Table 3, Figure 5). Although a 35% incidence of follicular hypertrophy/hyperplasia was observed in the animals administered thiazopyr diet (3000 ppm) for 56 days, no hypertrophy/hyperplasia was observed in thiazopyrtreated animals which were returned to untreated diet for either 56 or 112 days (Table 3).

The level of T4 in the blood of the treated animals was less than that of the control animals. Analysis of the kinetics of elimination of T4 (analyzed as total radioactivity in the blood) demonstrated a significant difference in the pharmacokinetic parameters between control and thiazopyr-treated animals (Table 4). Total AUC was significantly less for thiazopyr-treated rats (Ç55% of control) and the clearance of T4 from the blood in 72 hr was twice that of control animals (Table 4). These changes indicated a faster rate of elimination of T4 from the blood of thiazopyr-treated rats relative to controls.

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Biliary Elimination of T4 The kinetics of elimination of 125I-T4 in the bile of thiazopyr-treated rats was increased 40% compared to controls

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TABLE 3 Thyroid Follicular Cell Hypertrophy/Hyperplasia in Male Rats Exposed to Thiazopyr in the Diet Number of animals with hypertrophy/hyperplasiaa Time–course

b

Exposure time (days) 7 14 28 56 90

1 7** 7** 15** 13**

iodine/g of thyroid tissue of the treated animals was not significantly different from the controls (data not shown). Hepatic Deiodinase Activity Thiazopyr had no effect on hepatic deiodination of T4 to T3 , measured as ng T3/mg protein/30 min. However, when expressed on a total liver basis there was a small increase (1.6-fold) in total hepatic deiodinase activity in animals which were administered 3000 ppm thiazopyr for 56 days. This resulted from the increase observed in liver weights (data not shown). DISCUSSION

Dose–responsec Dose level (ppm) 0 10 30 100 300 1000 3000

1 1 0 1 1 2 7* Reversibility

Reversal time (days) 56d 112e

0 0

a

20 animals/group at risk. 3000 ppm thiazopyr diet. c 56 days of dietary exposure. d 56 days on thiazopyr diet (3000 ppm)/56 days on control diet. e 56 days on thiazopyr diet (3000 ppm)/112 days on control diet. * Significantly different from control with Fisher’s exact test, p £ 0.05. ** Significantly different from control with Fisher’s exact test, p £ 0.01. b

(Table 5). This increased elimination is consistent with the increase seen in hepatic T4-UDPGT (Fig. 4). Comparison of T4 Elimination in the Blood and Bile As mentioned above, the rate of T4 accumulation in the bile of treated animals was increased 40% compared to controls in 4 hours (Table 5). For comparison, the blood data were reanalyzed for the first 4 hr of collection. In these 4 hr, the clearance of T4 from the blood of the controls was 0.006 ml/min versus 0.009 ml/min for the thiazopyr-treated animals (50% increase) (data not shown). Thyroidal Iodine Uptake There was a slight increase (38%) in the percentage of the administered [125I]iodine in the thyroid of treated animals compared to controls. The amount of protein-bound [125I]iodine/mg protein and the amount of protein-bound [125I]-

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Chronic administration of thiazopyr in the diet resulted in an increase in thyroid follicular cell tumors in male Sprague– Dawley rats. In this report the mode of action for this male rat specific increase in thyroid tumors has been discussed. The results of these studies have indicated that thiazopyr produces a time- and dose-dependent increase in TSH production which appears to be causally associated with the increase in thyroid tumors observed in chronic studies with thiazopyr. Current knowledge on the induction of thyroid follicular cell tumors in experimental animals supports the view that tumor formation is controlled by promotional mechanisms (Andrae and Greim, 1992). Rats are generally more susceptible to thyroid tumors than mice, and males typically are more sensitive than females. Hepatic microsomal enzymes play an important role in controlling thyroid hormone levels (McClain, 1989). Many xenobiotics which have been shown to induce hepatic enzymes also induce thyroid tumors in rats. Most of the hepatic microsomal enzyme inducers are not DNA reactive, are not mutagenic, and do not have intrinsic carcinogenic activity (Capen et al., 1991; Curran and DeGroot, 1991). Thiazopyr appears to act by a mechanism similar to that of many other chemicals which induce hepatic drug metabolizing enzymes (Curran and DeGroot, 1991). Consistent with this is the observation that thiazopyr is not mutagenic, induces hepatic UDPGT activity, and produces thyroid tumors only in the male rat at high doses in chronic studies. Furthermore, thiazopyr does not induce thyroid tumors in the female rat or male and female mice. It is well established that circulating thyroid hormone levels are controlled via the hypothalamic–pituitary–thyroid axis, which can be disrupted by xenobiotic chemicals that induce hepatic microsomal enzymes (i.e., T4-UDPGT) and increase the biliary excretion of T4 (Curran and DeGroot, 1991). As serum levels of T4 decline, the pituitary gland is stimulated to secrete TSH through either release of thyrotropin-releasing hormone from the hypothalamus or local decrease in conversion of T4 to T3 by monodeiodination. TSH

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FIG. 5. Thiazopyr-induced thyroid follicular cell hypertrophy/hyperplasia in male Sprague–Dawley rats. (A) Thyroid follicular cells from a control animal. (B) Thyroid follicular cells with hypertrophy/hyperplasia in a male rat treated with 3000 ppm thiazopyr in the diet for 56 days. H&E, 1200.

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THIAZOPYR EFFECT ON THYROID FUNCTION

TABLE 4 T4 Blood Elimination Kinetics 72 Hr after an Intravenous Dose of (125I)T4 in Rats Fed Control or 3000 ppm Thiazopyr Diet for 56 days Control Half-life (min) a b Area under the curve (% dose 1 min/ml) a b Total Clearance (ml/min)

47.7 { 5.4 1,012.7 { 26.7

2,100 42,346 44,446 0.002

{ { { {

273 1457 1448 0.0001

Thiazopyr

83.7 { 16.8* 923.5 { 37.4*

2,805 21,670 24,475 0.004

{ { { {

538 1356** 1043** 0.0002*

* Significantly different from control with Student’s t test, p £ 0.05. ** Significantly different from control with Student’s t test, p £ 0.01.

in turn acts upon the thyroid gland to generate additional T4 and leads initially to hypertrophy and later hyperplasia of follicular cells (Sawin, 1969; Lissitsky, 1976; Capen et al., 1991). The long-term stimulation of the follicular cells by TSH in rats (especially males) increases the risk of developing thyroid follicular cell tumors (usually benign) in chronic carcinogenicity/toxicity studies (Bielschowsky, 1955; Furth, 1968; Comer et al., 1985; Sanders et al., 1988; Semler et al., 1989; Hill et al., 1989; McClain et al., 1988, 1989; Thomas and Williams, 1991). In this study, the induction of hepatic glucuronidation appeared to be the major factor accounting for the decrease in circulating T4 and the compensatory increase in TSH. The results suggest that the increase in serum TSH concentration developed secondary to the induction of hepatic T4-UDPGT rather than by alterations of the thyroid synthetic pathway. The elevation in serum T3 after administration of the high dose (3000 ppm) of thiazopyr appeared to be related to an increase in hepatic deiodinase activity and monodeiodination of T4 . The observed changes in serum hormone levels and liver weight were reversed upon removal of thiazopyr from the diet. Thyroid weight did not completely reverse, most likely due to the unique structure and development of colloid involution of follicles in response to the decrease in serum TSH. Reversal of thyroid weight begins shortly after removal of the TSH stimulus; however, if the stimulus has been persistent over a number of weeks (as in these studies) thyroid weight may remain elevated even though there is no residual evidence of hypertrophy/hyperplasia (Hill et al., 1989; Capen, 1991). The extent to which morphological progression in the thyroid can be reversed also depends on the severity and duration of the insult causing TSH stimulation (Hill et al., 1989). Other studies have shown varying degrees of reversibility after removal of the TSH stimulus (WynfordThomas et al., 1982; Arnold et al., 1983).

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Experimental evidence indicates that elevations in TSH in rats, induced by a wide variety of xenobiotic chemicals, predisposes the development of neoplasia of thyroid follicular cells in chronic studies (Capen, 1995). This hormone-mediated mechanism of carcinogenesis is considered to be a threshold phenomenon to which male rats are particularly susceptible. The findings in humans are markedly different from those in the rat, and there is strong evidence that prolonged TSH stimulation of the thyroid gland in humans does not lead to the development of thyroid cancer (Curran and DeGroot, 1991). For example, reduced iodide intake leads to thyroid tumors in rats, which is not the case in humans (Hill et al., 1996). The differences in sensitivity between rats and humans may be influenced by protein carriers of thyroid hormones in the blood. Rats lack a high-affinity binding protein, thyroxine-binding globulin, which binds T4 (and to some degree T3) in humans. In rats, more T4 is susceptible to being removed from the blood, metabolized, and excreted from the body since it is bound to low-affinity proteins. The half-life of T4 is significantly shorter in rats (õ1 day), compared to humans (5 – 9 days) (Hill et al., 1996). Rats have higher levels of serum TSH than humans and the turnover of thyroid hormones is greater. In summary, thiazopyr increased hepatic glucuronidation, deiodination, and biliary excretion of T4 which resulted in an increased rate of elimination of T4 from the blood. The result of the decreased circulating T4 was a sustained rise in circulating levels of TSH, an increase in thyroid weight, and an increase in the incidence of thyroid follicular cell hypertrophy/hyperplasia. The prolonged elevation in TSH predisposes the rodent thyroid gland to develop an increased incidence of follicular cell neoplasia in chronic studies. These results provide strong evidence that a hormonally mediated, threshold-dependent mechanism is operative in the production of thiazopyr-induced thyroid tumors in rats. Understanding the mechanism of action of a chemical on the rat thyroid therefore provides a more rational basis for extrapolating the findings from long-term rodent studies to the assessment of human risk. TABLE 5 Biliary Elimination Kinetics of (125I)T4 in Rats Fed Control or 3000 ppm Thiazopyr Diet for 56 Days

Bileb Control Treated

Cumulative % of dose

Rate of excretiona (% of dose/hr)

% Control

4.1 { 0.2 5.8 { 1.4

1.0 1.4*

140

a

Data compiled as the average rate for the individual collection periods. Bile collected for 4 hr. * Significantly different from control with Student’s t test, p £ 0.05.

b

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