Evidence of Chemical Stimulation of Hepatic Metabolism by an Experimental Acetanilide (FOE 5043) Indirectly Mediating Reductions in Circulating Thyroid Hormone Levels in the Male Rat

FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.

29, 251–259 (1996)

0029

Evidence of Chemical Stimulation of Hepatic Metabolism by an Experimental Acetanilide (FOE 5043) Indirectly Mediating Reductions in Circulating Thyroid Hormone Levels in the Male Rat1 W. R. CHRISTENSON,2 B. D. BECKER, B. S. WAHLE, K. D. MOORE, P. D. DASS, S. G. LAKE, D. L. VAN GOETHEM, B. P. STUART, G. K. SANGHA, AND J. H. THYSSEN Agriculture Division, Toxicology, Bayer Corporation., 17745 South Metcalf, Stilwell, Kansas 66085-9104 Received March 20, 1995; accepted July 20, 1995

Evidence of Chemical Stimulation of Hepatic Metabolism by an Experimental Acetanilide (FOE 5043) Indirectly Mediating Reductions in Circulating Thyroid Hormone Levels in the Male Rat. CHRISTENSON, W. R., BECKER, B. D., WAHLE, B. S., MOORE, K. D., DASS, P. D., LAKE, S. G., VAN GOETHEM, D. L., STUART, B. P., SANGHA, G. K., AND THYSSEN, J. T. (1996). Fundam. Appl. Toxicol. 29, 251–259. N-(4-Fluorophenyl)-N-(1-methylethyl)-2-[[5-(trifluoromethyl)1,3,4-thiadiazol-2-yl]oxy]acetamide (FOE 5043) is a new acetanilide-type herbicide undergoing regulatory testing. Previous work in this laboratory suggested that FOE 5043-induced reductions in serum thyroxine (T4) levels were mediated via an extrathyroidal site of action. The possibility that the alterations in circulating T4 levels were due to chemical induction of hepatic thyroid hormone metabolism was investigated. Treatment with FOE 5043 at a rate of 1000 ppm as a dietary admixture was found to significantly increase the clearance of [125I]T4 from the serum, suggesting an enhanced excretion of the hormone. In the liver, the activity of hepatic uridine glucuronosyl transferase, a major pathway of thyroid hormone biotransformation in the rat, increased in a statistically significant and dose-dependent manner; conversely, hepatic 5*-monodeiodinase activity trended downward with dose. Bile flow as well as the hepatic uptake and biliary excretion of [125I]T4 were increased following exposure to FOE 5043. Thyroidal function, as measured by the discharge of iodide ion in response to perchlorate, and pituitary function, as measured by the capacity of the pituitary to secrete thyrotropin in response to an exogenous challenge by hypothalamic thyrotropin releasing hormone, were both unchanged from the controlled response. These data suggest that the functional status of the thyroid and pituitary glands has not been altered by treatment with FOE 5043 and that reductions in circulating levels of T4 are being mediated indirectly through an increase in the biotransformation and excretion of thyroid hormone in the liver. q 1996 Society of Toxicology

N - ( 4 - Fluorophenyl ) - N - ( 1 - methylethyl ) - 2 - [ [ 5 - ( tri fluoromethyl)-1,3,4-thiadiazol-2-yl]oxy]acetamide (FOE 5043) is a prospective new chemical pesticide with herbicidal properties currently undergoing toxicological testing in support of regulatory approval. The general profile of FOE 5043-induced toxicity, following 3, 6, or 13 weeks of continuous dietary exposure at concentrations of 0, 25, 400, 1000, 1600, or 3000 ppm, was dominated by morphological and functional alterations in thyroid- and liver-related endpoints (Christenson et al., 1995). In the blood, thyroid changes were characterized by consistent and dose-dependent declines in circulating levels of T4 (400, 1600, and 3000 ppm at 3, 6, and 13 weeks; 1000 ppm at 3 weeks). Specifically, declines of 50–60%, relative to the untreated control, were measured in 1000- to 3000-ppm animals at 3 weeks. Alterations in serum levels of triiodothyronine (T3),3 reverse T3, and TSH were suggested at various time intervals at doses ¢1000 ppm; however, the changes, unlike those in T4, were generally inconsistent and transient and thus difficult to unequivocally attribute to the chemical. Pathologically, the thyroidal changes were characterized by increases in thyroid organ weight (1600 and 3000 ppm at 3, 6, and 13 weeks; 1000 ppm at 3 weeks) and hypertrophy of the follicular cells (3000 ppm at 3, 6, and 13 weeks). The response of the liver to FOE 5043 was suggestive of microsomal enzyme induction and included increases in organ weight (400, 1600, and 3000 ppm at 3, 6, and 13 weeks; 1000 ppm at 3 weeks) and cytochrome P450 content (400, 1600, and 3000 ppm at 3, 6, and 13 weeks), proliferation of the endoplasmic reticulum (3000 ppm at 13 weeks), and hepatocellular hypertrophy (400, 1600, and 3000 ppm at 3, 6, and 13 weeks; 1000 ppm at 3 weeks). Finally, no evidence Abbreviations used: 5*-DI, 5*-deiodinase; T4, thyroxine (3,5,3*,5*-tetraiodo-L-thyronine); T3, triiodothyronine (3,5,3*-triiodothyronine); TRH, thyrotropin releasing hormone; TSH, thyrotropin stimulating hormone; UDPGT, uridine diphosphoglucuronosyl transferase; PTU, propylthiouracil; wt, weight. 3

1

Portions of this work were presented at the annual meeting of the Society of Toxicology, Dallas, TX, March 1994. 2 To whom correspondence should be addressed. Fax: (913)897-9125.

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0272-0590/96 $12.00 Copyright q 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.

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of FOE 5043-induced variation in any of the thyroidal or hepatic parameters examined above was observed through 3 weeks of exposure at the 25-ppm level. In line with these observations, a clear precedent for interaction between the thyroid and the liver following chemical exposure is well-documented in the literature as various investigators have reported reductions in serum levels of thyroid hormones in animals exposed to hepatic microsomal enzyme inducers (Bock et al., 1973, 1979; Lucier et al., 1975; Bastomsky et al., 1976; Bastomsky, 1977; Wishart, 1978a, b; Batt et al., 1981; Gorski and Rozman, 1987; McClain et al., 1989). Moreover, noting that T4 itself is principally metabolized in the rat by the hepatic microsomal enzyme uridine diphosphate glucuronosyltransferase (UDPGT) (Taurog et al., 1952; Galton, 1968), it has been postulated that the changes observed in serum thyroid hormone levels following exposure to microsomal enzyme inducers are due, at least in part, to an increased T4 glucuronidation and secretion into the bile as a result of chemical induction of UDP-GT (Comer et al., 1985; Henry and Gasiewicz, 1987; Semler et al., 1989; Saito et al., 1991; de Sandro et al., 1991; Barter and Klaassen, 1992; Johnson et al., 1993). Because investigative studies conducted at this laboratory to characterize the origin or nature of FOE 5043-induced thyroidal changes also provided supportive evidence that a non-thyroidally-driven action was involved in the changes in thyroidal homeostasis observed, the focus of the studies described here was to examine the hypothesis that chemically induced stimulation of the metabolizing capacity of the liver was the initiating or primary event leading to hepatically driven and secondarily mediated alterations in circulating thyroid hormone levels which were observed following exposure of the male rat to the microsomal enzyme inducer FOE 5043. To test this hypothesis experimentally, the structural and/ or functional integrity of the hypothalamic–pituitary–thyroid–hepatic axis was systematically examined for evidence of chemical interference with respect to the synthesis, metabolism, or regulation of thyroid hormone to provide evidence to support the most probable target site or sites consistent with the FOE 5043-induced thyroidal changes observed. Two primary considerations in arriving at the dosing approach which was selected to conduct these studies were (1) the consistency of the pattern of change of FOE 5043-induced declines in T4 blood levels observed in 400- to 3000ppm animals with respect to magnitude, time, and dose described above and (2) the desire to remain as consistent as possible with the dietary exposure regimen of 25, 400, and 800 ppm, which was used in the lifetime bioassay with FOE 5043 in the rat. These factors played a significant role in the rationale behind the decision to use 2- to 3-week exposures at a low dose of 25 and a top dose of either 1000 or 3000 ppm FOE 5043 admixed in the diet to conduct the mechanistic investigations described in this report.

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MATERIALS AND METHODS Chemicals. All experiments were conducted with technical grade FOE 5043 (FOE 5043), which was synthesized and acquired from Bayer AG of Leverkusen, Germany. The chemical name of the active ingredient (AI) of FOE 5043 is N-(4-fluorophenyl)-N-(1-methylethyl)-2-[[5-( trifluoromethyl)-1,3,4-thiadiazol-2-yl]oxy] acetamide (Chemical Abstracts Services Registry No. 142459-58-3). Thyroxine (T4), triiodothyronine (T3), thyrotropin releasing hormone (TRH), and propylthiouracil (PTU) were obtained from Sigma Chemical Co. (St. Louis, MO). [125I]T4 (sp act 1080– 1620 mCi/mg) and Na125I (sp act 17.4 mCi/mg) were obtained from New England Nuclear Du Pont (N. Billerica, MA). All other chemicals used were at least reagent grade and commonly available. Storage and analysis of the test batch of FOE 5043. FOE 5043 was stored continuously under freezer conditions (approx 0237C) and analyzed periodically to confirm the stability of the AI. For the four analyses conducted prior to, during, and following conclusion of the entire series of 2to 3-week studies described in this report, the mean purity { SD of the FOE 5043 test batch was determined to be 97.2 { 1.6% (analyzed 8/91, 2/92, 9/92, and 3/93). These data confirm that the AI of FOE 5043 remained stable in the freezer during these investigations. Analysis of FOE 5043 as a dietary admixture. The homogeneity, stability, and concentration of FOE 5043 in its dietary matrix were analytically verified using methodology described by Moore and Shelton (1991). Briefly, homogeneity was confirmed by a comparative analysis of the concentration of nine samples taken from three distinct sections (three samples/section) of the mixing bowl. The stability of the AI of FOE 5043, when mixed in the diet and stored at room and freezer (approx 0237C) temperatures, was confirmed for 14 and 28 days, respectively. Additionally, the concentration of AI in the various test diets was periodically confirmed during the course of each experiment as well. Animal husbandry. Male rats (CDF[F-344]/BR), ranging in weight from 180 to 250 g when placed on study, were procured either from SASCO, Inc. (Madison, WI), or from Hilltop Lab Animals, Inc. (Scottdale, PA). The animals were maintained and exposed to the FOE 5043 test material at an American Association for Accreditation of Laboratory Animal Careaccredited facility. Upon receipt from the vendor, animals were examined and subsequently killed (CO2 asphyxiation) if deviations in general appearance and/or behavior were observed. Those animals considered acceptable were individually housed and acclimated to their ambient laboratory conditions (room temperature 18 to 267C, relative humidity 40 to 70%, and a daily photoperiod of 12 hr of light (7:00 AM to 7:00 PM) alternating with 12 hr darkness) for approximately 1 week (with the exception of the bile duct-cannulated rats, which were subject to a 3-day acclimation period) prior to their release for study. While on study, animals were individually housed in either suspended stainless steel wire-mesh cages or polycarbonate shoebox-type enclosures (bile duct-cannulated rats) containing Alpha-dri bedding (Shepards Specialty Papers, Kalamazoo, MI). Food (Purina Mills rodent lab chow 50014 in ‘‘etts’’ form, St. Louis, MO) and tap water (municipal water supply of the city of Kansas City, MO) were provided continuously for ad libitum consumption. Animal treatment and preparation of the test diets. Prior to treatment, animals were distributed to dose groups using weight stratification-based computer programs, obtained from either INSTEM Computer Systems (Stone, Staffordshire, UK) or SAS Institute, Inc. (Cary, NC). In these studies, animals were administered FOE 5043, relative to the analytically determined percentage of purity of the chemical, for periods of time ranging from 2 to 3 weeks at constant concentrations in the feed of 0, 25, 1000, or 3000 ppm. Up until the time an animal was killed, its particular test diet remained continuously available for ad libitum consumption. An acetone/ corn oil mixture was used to dissolve the FOE 5043 prior to mixing in the diet with a Hobart Model A200T or D300T mixer (Troy, OH). The control

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MECHANISM OF FOE 5043-INDUCED DECLINES IN RAT SERUM THYROXINE diet (including the acetone/corn oil mixture) was prepared in the same manner as the treated diet, only excluding the FOE 5043. Replacement admixtures for a particular dose group were generally prepared on a weekly basis and stored under freezer conditions until presented to the animals the following week. Serum thyroid hormone determinations. Blood was consistently drawn for analysis between the hours of 0800 and 0930. Depending upon the requirements of the experiment, the sample was obtained from either the retroorbital sinus or the inferior vena cava. Once clotted, serum was isolated by centrifugation and stored at 0207C pending assay. With the exception of TSH, all circulating thyroid hormone concentrations were measured by using commercially available kits, characterized for use with rat serum. The specific hormones measured, the intra-assay coefficient of variation, and the minimum detectable concentration, respectively, for each kit were as follows: total T4, 8%, 0.32 ng/ml (ICN Diagnostics, Irvine, CA); free T4, 6%, 0.05 ng/dl (Incstar Corp., Stillwater, MN); total T3, 7%, 0.25 ng/ ml (Abbott Diagnostics, Chicago, IL); and free T3, 16%, 0.05 pg/ml (Incstar Corp.). Serum TSH concentrations were determined using rat TSH radioimmunoassay immunoreagents, supplied by Pituitary Hormones and Antisera Center, Harbor–UCLA Medical Center (Torrance, CA). Determination of hepatic UDP-GT and 5*-deiodinase activities. In general for these procedures liver tissue was perfused with ice-cold saline, frozen in liquid nitrogen, and stored at 0807C pending assay. The activity of UDP-GT toward p-nitrophenol was determined as described previously (Bock et al., 1973; Comer et al., 1985; Semler et al., 1989). UDP-GT activity was determined by measuring the disappearance of p-nitrophenol and expressed as nanomoles of p-nitrophenyl glucuronide formed per minute per unit of liver or liver protein. The estimation of 5*-deiodinase (5*DI) activity was based on the procedure described by Jones et al. (1988). Briefly, liver tissue was homogenized and centrifuged, and an aliquot of the supernatant was incubated at 377C for 10 min in the presence of phosphate buffer, dithiothreitol, and T4. The level of hepatic deiodinase activity measured was expressed as picograms of T3 formed per minute per unit of liver or liver protein. The protein content of the hepatic microsomal preparations was estimated by the method of Bradford (1976). Perchlorate discharge test. This test, carried out as described by Atterwill et al. (1987), is designed to assess the general competency of the thyroid gland by simultaneously detecting changes in the capacity of the gland to both trap and concentrate iodide (I0) and then to organify the I0 into thyroid hormones. The test is based on the principle that if free unreacted I0 has accumulated in the thyroid, such as through chemical interference with the two critical thyrogenesis steps described above, then administration of perchlorate (ClO04 ), a competitive inhibitor of thyroidal iodide transport/ trapping (Halmi et al., 1956), would be expected to cause a diffusional discharge of excess I0 from the gland. Animals (six/dose group) received either 0 or 1000 ppm FOE 5043 as a dietary admixture for 21 days. On Day 21 (Day 0, initiation of dosing) and between the hours of 0800 and 0930, all animals received an ip injection of approximately 33 mCi/kg body wt (0.4 ml/kg body wt) of carrier-free Na125I. Six hours after injection of the tracer, KClO4 was administered (10 mg/kg body wt; ip); approximately 2.5 min after injection of the ClO04 , the animal was asphyxiated in a CO2 chamber and terminated by exsanguination. Thyroids were excised and weighed, and the radioactive content in the blood and thyroid was determined with a gamma counter (Abbott Ansr gamma counter Model 7157). Background counts were subtracted and data calculated and expressed as a percentage of total 125I administered. To provide positive control data for comparative purposes, a group of rats was treated, as adapted from Atterwill and Hillier (1991), with the antithyroid agent PTU (200 mg/kg body wt/day; po) for 4 days prior to administration of ClO04 , approximately 24 hr after the final dose of PTU. Thyrotropin releasing hormone challenge test. This test, carried out as described previously (Lifschitz et al., 1978; Brown et al., 1987), is

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designed to assess the functional integrity of the pituitary gland. Animals (six/dose group) received either 0 or 1000 ppm FOE 5043 as a dietary admixture for 21 days. On Day 21 (Day 0, initiation of dosing) and between the hours of 0800 and 1030, blood was drawn via the retroorbital sinus for determination of circulating levels of thyrotropin. Samples were collected at 10 { 5 min before and then again at 15 { 2 and 60 { 5 min after a tail vein injection of approximately 5 mg TRH/kg body wt. Clearance of [125I]T4 from the circulation. Animals (five/dose group) received either 0 or 1000 ppm of FOE 5043 as a dietary admixture for 21 days. On Day 21 (Day 0, initiation of dosing) and between the hours of 0800 and 0930, all animals received an iv injection of approximately 8 mCi/kg body wt [125I]T4 into a tail vein. Prior to injection, [125I]T4 (sp act approximately 1250 mCi/mg T4) was diluted with 0.9% NaCl to give a solution of approximately 20 mCi/ml. Blood was drawn via the retroorbital sinus at 4, 8, 24, 48, 72, and 96 hr after administration of the tracer. Blood samples were centrifuged, and the radioactive content of an aliquot (°0.1 ml) of serum was determined with a gamma counter (Abbott Ansr gamma counter Model 7157). Background counts were subtracted and data for each time point calculated and expressed as a percentage of the total [125I]T4 administered per milliliter of serum. The clearance of [125I]T4 was estimated for each rat using the total area under the plasma concentration vs time curve (AUC), as calculated by the trapezoidal rule and applying the equation clearance (Cl) Å iv dose/AUC (Renwick, 1994). Biliary excretion of [125I]T4. Bile duct-cannulated animals were obtained from Hilltop Lab Animals, Inc. Animals (12–13/dose group) received either 0 or 1000 ppm of FOE 5043 as a dietary admixture for 14 days. On Day 14 (Day 0, initiation of dosing) and between the hours of 0800 and 1030, all animals were administered an iv injection of approximately 9 mCi/ kg body wt of [125I]T4 into a lateral tail vein. Prior to injection, [125I]T4 (sp act approximately 1250 mCi/mg T4) was diluted with 0.9% NaCl to give a solution of approximately 25 mCi/ml. Animals were anesthetized with a 3:3:1 mixture of ketamine, xylazine, and acepromazine, respectively, prior to administration of the tracer. During the bile collection process, the rats were placed in a Bollman restraining cage. In addition to bile, liver and blood samples were collected and their radioactive content was determined as described previously (McClain et al., 1989). Statistics. Data were analyzed for statistically significant differences by either Student’s t test (unpaired) or a one-way analysis of variance (Snedecor and Chochran, 1967) followed by Dunnett’s test (Dunnett, 1955, 1964). Differences with p values £0.05 were considered statistically significant. All statistical evaluations were performed using software from INSTEM Computer Systems, SAS Institute Inc., or Jandel Scientific (Corte Madera, CA).

RESULTS

FOE 5043 Dietary Exposure Rates For the experimental results described below, the mean daily intake of the AI of FOE 5043 (mg/kg body wt/day { SE), calculated from feed consumption, body weight, and diet analysis data, for animals administered the chemical over approximately 3 weeks at nominal dietary concentrations of 25, 1000, or 3000 ppm was 1.7 { 0.1, 70.5 { 4.1, and 224.2 { 35.0, respectively. FOE 5043 was not detected in control feed. Effect of FOE 5043 on Body Weight and Food Consumption Over 21 days of continuous and uninterrupted dietary exposure to FOE 5043, declines in body weight and increases

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FIG. 1. Effect of FOE 5043 on body weight and food consumption in the male rat. Animals were treated for 21 days with 0, 25, 1000, or 3000 ppm FOE 5043 as a dietary admixture to the feed. (Inset) Corresponding profile of food consumption over the same time period. Each data point represents the xV { SE for groups of five animals. An asterisk indicates statistical significance relative to control.

in food consumption were noted at a concentration of 3000 ppm (Fig. 1). However, neither body weight nor food consumption was affected at exposure levels up to and including 1000 ppm in the feed.

release of negative feedback inhibition accompanying a fall in serum T4 levels, with an increased TSH secretion from the pituitary, presumably to drive the thyroid to restore circulating T4 levels to normal (DeGroot, 1979). In this procedure the functional status of the FOE 5043-exposed pituitary gland was assessed in terms of its response (release of TSH into the circulation) to an exogenous challenge with the hypothalamic and TSH-regulating tripeptide TRH. Circulating TSH levels in the serum approximately 10 min before and approximately 15 and 60 min after administration of TRH are shown in Fig. 3. Despite the apparent lack of a compensating elevation in serum TSH levels in the face of a marked decline in FOE 5043-induced circulating T4 concentrations, the data from this experiment provide no indication of pituitary impairment as a result of treatment with FOE 5043. As illustrated in Fig. 3, the responses of the pituitary gland of the control and treated animals at the two time points evaluated following the TRH challenge were unequivocally comparable. In addition, histopathologic analyses conducted previously (Christenson et al., 1995) on the pituitary as well as the hypothalamus of rats administered up to 3000 ppm FOE 5043 in the diet for 13 weeks provided no morphological evidence of a chemically mediated interference with the homeostasis of these glands. Effect of FOE 5043 and PTU on Perchlorate-Induced Discharge of Radioiodide from the Thyroid Gland The effect on control, FOE 5043-, and PTU-treated animals of a pulse dose of ClO40 is shown in Table 1. As depicted in the table, no significant difference in the response

Effect of FOE 5043 on Serum Thyroid Hormone Concentrations The effects of FOE 5043 on circulating levels of total and free T4, total T3, and TSH are shown in Fig. 2. Statistically significant declines relative to controls of 50 and 30%, respectively, in circulating levels of total and free T4 were measured in 21-day 1000-ppm FOE 5043-treated animals. However, no effect on serum levels of either T3 or TSH over the same exposure interval was indicated. Most notably, the lack of change in TSH is curious, as it suggests the possibility of an FOE 5043-induced interference with the regulation of thyroid hormone as the result of a compromised pituitary gland that is unable to respond appropriately to a depressed serum T4 concentration. Effect of FOE 5043 on the Response of the Pituitary Gland to Thyrotropin Releasing Hormone In the face of a marked decline in serum T4 concentration (as noted above), a functionally competent pituitary gland would be expected to respond, as a result of the apparent

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FIG. 2. Effect of FOE 5043 on circulating concentrations of total and free thyroxine (T4), triiodothyronine (T3), and thyrotropin (TSH) in the male rat. Animals were treated for 21 days with 0 or 1000 ppm FOE 5043 as a dietary admixture to the feed. Each bar represents the xV { SE for groups of four or five animals. An asterisk indicates statistical significance relative to control by Student’s t test (unpaired); p £ 0.05.

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ble dose. Quantitatively, this difference was expressed in terms of an overall mean plasma clearance (xV { SE) rate for the entire sampling period of 1.98 { 0.09 ml/hr for the controls compared to a statistically significantly and approximately three-fold higher rate of 5.96 { 0.71 ml/hr for the FOE 5043-exposed animals. Effect of FOE 5043 on the Hepatocellular Binding of [125I]Thyroxine The distribution of radioactivity between the liver and the plasma (expressed as the percentage of administered dose per unit tissue) in control and FOE 5043-treated animals at 4 hr after administration of a radiolabeled dose of T4 is shown in Fig. 5. A statistically significant 66% increase in the liver/plasma ratio of the administered dose in treated animals relative to that in controls was measured. Effect of FOE 5043 on Hepatic UDP-GT and 5*DI Activities FIG. 3. Effect of FOE 5043 on circulating levels of thyrotropin (TSH) following an exogenous administration of thyrotropin releasing hormone (TRH) to the male rat. Animals were treated for 21 days with 0 or 1000 ppm of FOE 5043 as a dietary admixture to the feed. On Day 21 (Day 0, initiation of dosing), rats received an iv injection of TRH. Blood was drawn via the orbital sinus for determination of circulating levels of TSH at approximately 10 min before and then again at approximately 15 and 60 min after administration of TRH. Each bar represents the xV { SE for groups of six animals.

to perchlorate was observed between control and FOE 5043treated animals, suggesting that in this experiment exposure to FOE 5043 did not interfere with the capacity of the thyroid gland to take up and organify iodide ion during the process of hormonogenesis. In contrast, the response to ClO40 of the animals treated with the positive control PTU, a thioamide known to inhibit the production of T4 at both the organification (inhibition of peroxidase activity) and the coupling level (Grodsky, 1983), was characterized, relative to both FOE 5043 and control animals, by a significantly lower thyroid/ blood ratio of exogenously administered radiolabeled iodine. Effect of FOE 5043 on Clearance of [125I]Thyroxine from the Circulation Mean plasma [125I]T4 disappearance curves for control and FOE 5043-treated animals are shown in Fig. 4. The graph illustrates that treatment with FOE 5043 significantly increased the capacity of the rat to clear an exogenously administered dose of [125I]T4 from the plasma. By 4 hr after administration of the T4 tracer, blood levels of radiolabeled T4 (expressed as the mean percentage of administered dose per milliliter of plasma) in the FOE 5043-exposed animals were markedly lower than those of controls given a compara-

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The activities of the membrane-bound enzymes UDP-GT and 5*DI in the liver following 3 weeks of exposure to FOE 5043 are shown in Tables 2 and 3, respectively. Both enzymes have fundamental roles in the regulation and disposition of thyroid hormones, which are metabolized primarily in the liver (Hill et al., 1989). Insofar as chemical induction

TABLE 1 The Effect on the Process of Thyroidal Organification as Measured by the Perchlorate Discharge Test, Following Exposure of the Male Rat to Either FOE 5043 or Propylthiouracila Treatment Control (0 ppm) FOE 5043 (1000 ppm) Propylthiouracil

(%

Thyroid I dose/g)b

125

(%

Blood I dose/ml)

125

Thyroid/ blood ratio

1741 { 58c 1352 { 168

0.39 { 0.06 0.31 { 0.03

4906 { 562 4366 { 339

16.19 { 4.2d

0.35 { 0.05

47.6 { 6.6d

a Rats were treated for 21 days with 0 or 1000 ppm FOE 5043 as a dietary admix. On Day 21 (Day 0, initiation of dosing) all animals received an ip injection of Na 125I. Six hours after administration of the tracer, all animals were dosed with potassium perchlorate (10 mg/kg body wt; ip); approximately 2.5 min after injection of the perchlorate, the animal was sacrificed, and the radioactive content of the thyroid and the blood was determined in a gamma counter. To serve as a positive control, five rats were administered propylthiouracil, a thioamide capable of inhibiting the synthesis of thyroxine, at a dosage of 200 mg/kg body wt/day (po) for 4 days prior to administration of the perchlorate on Day 4. b Data are expressed in terms of the percentage of the total dose of Na 125 I administered per unit weight of thyroid tissue or per unit volume of blood. c Each value represents the mean { standard error for five or six rats per dose group. d Indicates statistically significant difference relative to control by Student’s t test (unpaired); p £ 0.05.

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FIG. 4. Effect of FOE 5043 on the clearance of [125I]T4 from the circulation of the male rat as shown by mean [125I]T4 disappearance curves plotted semilogarithmically. Animals were treated for 21 days with 0 or 1000 ppm of FOE 5043 as a dietary admixture to the feed. On Day 21 (Day 0, initiation of dosing), rats received an iv injection of [125I]T4. At various times after administration of the tracer, blood was drawn from the orbital sinus, and the radioactive content of the plasma determined using a gamma counter. Radioactivity was expressed in terms of the mean percentage of the total dose of [125I]T4 administered per milliliter of plasma. Each data point represents the xV { SE for groups of six animals. An asterisk indicates statistical significance relative to control by Student’s t test (unpaired); p £ 0.05.

of one or both metabolizing enzymes could account for or contribute to the reduction in circulating T4 concentration, which was observed in the FOE 5043-exposed animals, the activity of both enzymes was assessed in the liver. Statistically significant and dose-related increases in UDP-GT activity were measured in the 1000- and 3000-ppm groups following 3 weeks of dietary dosing. These changes would be consistent with the postulate that FOE 5043 is indirectly mediating reductions in serum T4 levels through stimulation of the liver’s capacity for glucuronidation. However, FOE 5043 appeared to have the opposite effect on 5*DI as the activity of the enzyme appeared to trend downward with dose. Effect of FOE 5043 on Bile Flow and the Biliary Excretion of [125I]Thyroxine

FIG. 5. Effect of FOE 5043 on the hepatic uptake of [125I]T4 from the circulation of the male rat. Animals were treated for 21 days with 0 or 1000 ppm of FOE 5043 as a dietary admixture to the feed. On Day 21 (Day 0, initiation of dosing) rats received an iv injection of [125I]T4. Approximately 4 hr after administration of the tracer, animals were terminated by exsanguination, and the radioactive content of the liver and plasma was determined in a gamma counter. Radioactivity was expressed in terms of the mean percentage of the total dose of [125I]T4 administered per gram of liver and per milliliter of plasma. Each bar represents the xV { SE for groups of six animals. An asterisk indicates statistical significance relative to control by Student’s t test (unpaired); p £ 0.05.

most likely as a function of the modest increase in bile flow (Oppenheimer et al., 1968), by 45% with respect to control. DISCUSSION

Investigative work at this laboratory with the herbicidal agent FOE 5043 suggested that the effects of the chemical TABLE 2 Effect of FOE 5043 on Hepatic Uridine Diphosphate Glucuronosyltransferase Activity in the Male Rat a Activity (nmol/min)b Dose (ppm)

mg protein01

0 25 1000 3000

1.5 2.3 5.5 10.1

{ { { {

0.1c 0.4 0.6d 0.8d

a

As is typically seen with microsomal enzyme inducers with the ability to reduce circulating levels of thyroid hormone (Hill et al., 1989), both bile flow and the biliary excretion of exogenously supplied [125I]T4 (expressed as the percentage of the administered dose excreted per hour) were significantly increased in FOE 5043-treated animals (Fig. 6). For the 4 hr following administration of the T4 tracer, the cumulative biliary excretion of radioactivity was increased,

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g liver01 188.7 300.6 827.0 1401.2

{ { { {

20.1 59.7 88.9d 53.8d

Rats were treated for 21 days with 0, 25, 1000, or 3000 ppm FOE 5043 as a dietary admix. On Day 21 (Day 0, initiation of dosing) animals were sacrificed and the livers removed for estimation of uridine diphosphate glucuronosyltransferase activity. b Data are expressed as nanomoles p-nitrophenyl glucuronide formed per minute per milligram of microsomal protein or per gram liver. c Each value represents the mean { standard error for five rats per dose group. d Indicates statistically significant difference relative to control by oneway analysis of variance and Dunnett’s tests; p £ 0.05.

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MECHANISM OF FOE 5043-INDUCED DECLINES IN RAT SERUM THYROXINE

TABLE 3 Effect of FOE 5043 on Hepatic 5*-Deiodinase Activity in the Male Rata Activity (pg/min)b Dose (ppm)

mg protein01

0 25 1000 3000

6.8 6.1 4.3 3.9

{ { { {

0.8c 0.3 0.4d 0.3d

g liver01 862.6 837.3 649.3 580.0

{ { { {

84.6 50.8 40.5 24.8d

a

Rats were treated for 21 days with 0, 25, 1000, or 3000 ppm FOE 5043 as a dietary admix. On Day 21 (Day 0 Å initiation of dosing) animals were sacrificed and the livers removed for estimation of 5*-deiodinase activity. b Data are expressed as picograms triiodothyronine formed per minute per milligram of microsomal protein or per gram liver. c Each value represents the mean { SE for groups of five rats. d Indicates statistically significant difference relative to control by Student’s t test (unpaired); p £ 0.05.

on the thyroidal economy of the male rat were secondary to chemical induction of the liver’s capacity to clear from the circulation, metabolize, and excrete thyroxine. To test this mechanistic hypothesis, the structural and/or functional integrity of each potential target site or sites comprising the hypothalamic–pituitary–thyroid–hepatic axis was examined following exposure to FOE 5043. Twenty-one days of treatment with FOE 5043 as a dietary admixture at a constant rate of 1000 ppm were found to significantly increase the clearance of [125I]T4 from the blood, suggesting that FOE 5043 is promoting, presumably via the liver, an enhanced excretion of the hormone. Correspondingly, in a dose–response study carried out over the same 21-day interval of time, but at dietary concentrations of 25, 1000, and 3000 ppm, the hepatic activity of the T4-glucuronidating enzyme UDP-GT was induced in a statistically significant and doserelated manner. Conversely under the same experimental conditions, the level of hepatic 5*DI activity, which if chemically induced, could also account for reductions in serum T4 levels, trended downward with dose. In the absence of corresponding changes in circulating T3 levels, the biological significance of this observation is unclear. Moreover, because 5*DI activity is known to decline in hypothyroid rats (Larsen et al., 1981; Berry et al., 1990; Berry and Larsen, 1992), it is possible that the reduction in hepatic 5*DI activity is not due to a direct action on the enzyme but rather represents a secondary response to the diminished level of thyroid hormone economy in the FOE 5043-exposed rat. This hypothesis, which is currently being examined experimentally, may also have implications with respect to the possibility of chemically mediated deiodinase-based toxicities which have a secondary rather than a primary origin. The critical role that the presence of adequate deiodinase activity plays in both the regulation of thyroid hormone and

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the subsequent expression of thyroid hormone-governing events in such tissues/organs as the brain, the pituitary, and the thyroid gland itself (Visser, 1990) cannot be overlooked in terms of its potential toxicological significance. Other metabolically significant FOE 5043-induced liver changes included an increased bile flow and an increased hepatocellular binding and biliary excretion of [125I]T4. Schwartz et al. (1969) have suggested that increases in hepatocellular binding of T4 are characteristic of those types of microsomal enzyme inducers, such as phenobarbital (Hill et al., 1989) and presumably FOE 5043, that have the capacity to promote proliferation of the endoplasmic reticulum, thus providing a greater surface area within the liver cell in which to interact, bind, and subsequently metabolize the hormone. In contrast to the significant hepatic influence on thyroidal economy induced by exposure to FOE 5043, thyroidal function, as measured by the perchlorate-induced discharge of iodide from the thyroid gland, and pituitary function, as measured by the capacity of the pituitary to secrete TSH in response to an exogenous challenge by hypothalamic TRH, were both unchanged from the controlled response.

FIG. 6. Effect of FOE 5043 on bile flow and the biliary excretion of [125I]T4 in the male rat. Bile duct-cannulated animals were treated for 14 days with 0 or 1000 ppm FOE 5043 as a dietary admixture to the feed. On Day 14 (Day 0, initiation of dosing), all animals received an iv injection of [125I]T4 and bile was collected at 1-hr intervals. The cumulative biliary excretion of the T4 tracer, expressed in terms of the percentage of the total dose of [125I]T4 administered, was determined over a 4-hr time period. Each bar represents xV { SE for groups of 12–13 animals. An asterisk indicates statistical significance relative to control by Student’s t test (unpaired); p £ 0.05.

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CHRISTENSON ET AL.

These functionally based thyroidal and pituitary assessments were in turn consistent with histopathological conclusions reached in earlier FOE 5043 studies (Christenson et al., 1995) in which no evidence of structural lesions in pituitary, thyroidal, or hypothalamic tissue was observed, following exposure to FOE 5043 over both a longer duration (13 weeks) and at a greater concentration (1600 ppm) than the 21-day 1000-ppm dosing regimen used in the functionality experiments described above. In summary, the results of these studies support the conclusion that FOE 5043-induced changes in total and free T4 serum concentrations are mediated through a mechanism separate and distinct from a direct action on the synthesizing and secreting functions of the pituitary, thyroid, or hypothalamus. Alternatively, the data suggest that the actions of FOE 5043 are characteristic of a phenobarbital-type microsomal enzyme inducer (increased liver size and weight with proliferation of the endoplasmic reticulum) (McClain et al., 1988; Hill et al., 1989; Oppenheimer et al., 1968, 1971) whose effects on thyroidal economy are secondary to a chemically induced stimulation of the capacity of the liver to clear from the circulation (increased hepatocellular binding), deactivate (increased UDP-GT activity), and excrete (increased bile flow and biliary clearance) thyroid hormone. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Margi Landes, Mary Mejia, Beth Longmoor, Terry Jensen, Hoang Hung, Bob Mueller, Vivian Clayton, Tammy Brown, Julie Ranjbar, Ron Jones, Joyce Christopher, Donna Voightj, Dale Largent, Bill Bopp, Martha Pypes, and Diana Cortner.

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