Effect of Microsomal Enzyme Inducers on the Biliary Excretion of Triiodothyronine (T3) and Its Metabolites

Nichole R. Vansell and Curtis D. Klaassen,1

Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., 2019 Breidenthal, Kansas City, Kansas 66160–7417

Received June 26, 2001; accepted October 25, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been postulated that inducers of UDP-glucuronosyltransferase (UGT) decrease circulating thyroid hormone concentrations by increasing their biliary excretion. The inducers pregnenolone-16{alpha}-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) are each effective at reducing serum thyroxine concentrations. However, only PCN treatment produces a marked increase in serum levels of thyroid-stimulating hormone (TSH), whereas 3MC and PCB cause little to no increase in TSH. Excessive TSH elevation is considered the primary stimulus for thyroid tumor development in rats, yet the mechanism by which enzyme induction leads to TSH elevation is not fully understood. Whereas PCN, 3MC, and PCB all increase microsomal UGT activity toward T4, only PCN causes an increase in T3-UGT activity in vitro. The purpose of this study was to determine whether PCN, which increases serum TSH, causes an increase in the glucuronidation and biliary excretion of T3 in vivo. Male rats were fed control diet or diet containing PCN (1000 ppm), 3MC (250 ppm), or PCB (100 ppm) for 7 days. Animals were then given [125I]-T3, iv, and bile was collected for 2 h. Radiolabeled metabolites in bile were analyzed by reverse-phase HPLC with {gamma}-detection. The biliary excretion of total radioactivity was increased up to 75% by PCN, but not by 3MC or PCB. Of the T3 excreted into bile, approximately 75% was recovered as T3-glucuronide, with remaining amounts represented as T3-sulfate, T2-sulfate, T3, and T2. Biliary excretion of T3-glucuronide was increased up to 66% by PCN, while neither 3MC nor PCB altered T3-glucuronide excretion. These findings indicate that PCN increases the glucuronidation and biliary excretion of T3 in vivo, and suggest that enhanced elimination of T3 may be the mechanism responsible for the increases in serum TSH caused by PCN.

Key Words: triiodothyronine (T3); pregnenolone-16{alpha}-carbonitrile (PCN); 3-methylchloranthrene (3MC); polychlorinated biphenyl (PCB); glucuronidation; bile; rat; thyroid.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thyroid hormones are substrates of the hepatic microsomal enzyme family, UDP-glucuronosyltransferase (UGT). Their increased glucuronide conjugation in response to microsomal enzyme induction is of concern due to the corresponding functional adaptation of the thyroid gland. Thyroxine (T4) and triiodothyronine (T3)-glucuronides are the predominate metabolites of thyroid hormones and are excreted in bile (Visser, 1990Go). The glucuronidation of T4 in rats is increased by the anticonvulsants phenobarbital (McClain et al., 1989Go), phenytoin (Mendoza et al., 1966Go), carbamezapine (Connell et al., 1984Go), as well as the antibiotic imidazole (Comer et al., 1985Go), the synthetic steroids spironolactone and pregnenolone-16{alpha}-carbonitrile (Liu et al., 1995Go; Semler et al., 1989Go), the polycyclic aromatic hydrocarbons 3-methylcholanthrene and TCDD (Bastomsky, 1977Go; Bastomsky and Papapetrou, 1973Go), and polyhalogenated hydrocarbons (Bastomsky et al., 1976Go; Wilson et al., 1996Go), among others. All of these compounds decrease serum T4 concentrations. Among these, phenobarbital, pregnenolone-16{alpha}-carbonitrile (PCN), and spironolactone are associated with increases in circulating concentrations of thyroid stimulating hormone (TSH), which regulates thyroid hormone synthesis (Liu et al., 1995Go; Semler et al., 1989Go). Increased serum TSH concentrations are known to induce growth of the thyroid gland, and sustained increases in TSH have been associated with chemicals such as propylthiouracil and potassium perchlorate that induce thyroid cancer in lab animals (Hood et al., 1999bGo; Kanno et al., 1990Go). Phenobarbital and PCN increase thyroid cell proliferation, spironolactone produces thyroid gland follicular cell hypertrophy (Hood et al., 1999aGo; Jones and Clarke, 1993Go; Semler et al., 1989Go), and, thus far, phenobarbital is known to promote the incidence of thyroid tumors in rats (McClain et al., 1988Go). These observations led to the proposal that the effects of microsomal enzyme inducers on the thyroid are mediated by increased TSH, which results from decreases in serum T4 concentrations due to induction of T4 biotransformation by UGT (McClain et al., 1988Go; Semler et al., 1989Go).

However, more recent studies suggest that the glucuronidation of T4 may not mediate increased TSH in rats following treatment with phenobarbital and PCN, since other microsomal enzyme inducers that decrease serum T4, such as 3-methylcholanthrene (3MC) and the polychlorinated biphenyl mixture, Aroclor 1254 (PCB), do not alter TSH (Hood et al., 1999aGo; Liu et al., 1995Go). Moreover, when the effects of these 4 inducers on UGT activity toward T3 in liver microsomes were determined, only phenobarbital and PCN produced an increase in T3-UGT activity; 3MC and PCB did not (Hood and Klaassen, 2000aGo). This differential effect on T3 glucuronidation mirrors the differential effect of these inducers on TSH, suggesting that T3 glucuronidation may mediate increases in serum TSH produced by certain microsomal enzyme inducers. It was hypothesized, then, that induction of T3 glucuronidation, rather than T4, results in increased biliary excretion of T3, thereby increasing turnover of T3 and altering one or more factors that regulate TSH.

In the present study, the significance of increased T3-UGT activity demonstrated in liver microsomes was assessed by determining whether an increase in the biliary excretion of T3 occurs in vivo, and whether T3 biliary excretion relates to serum TSH. It is known that different members of the UGT family are inducible by unique classes of microsomal enzyme inducers, and that treatment with various classes of inducers results in the increased glucuronidation of specific substrates (Arand et al., 1991Go; Emi et al., 1995Go; Fournel et al., 1987Go; Lilienblum et al., 1982Go; Ullrich and Bock, 1984Go; Watkins et al., 1982Go). It is thought that T4 and T3 are glucuronidated by different UGT enzymes: T4 by bilirubin (UGT1A1) and phenol (UGT1A6) UGTs, and T3 by androsterone UGT (2B2) (Beetstra et al., 1991Go; van Raaij et al., 1993Go; Visser et al., 1993aGo,bGo). The inducibility of T4 biliary excretion and fecal clearance by chemicals that also induce phenol and bilirubin glucuronidation has been examined (Bastomsky, 1977Go; Bastomsky and Papapetrou, 1973Go; Beetstra et al., 1991Go; van Raaij et al., 1993Go; Visser et al., 1991Go), but the induction of T3 glucuronidation and biliary excretion has not been well characterized. Thus, in this study, rats were treated with PCN, a chemical that increases UGT activity toward T4 and T3, as well as increasing serum TSH. Rats were also treated with 3MC and PCB, prototypical chemicals that effectively reduce serum T4, but that increase only T4-UGT activity and do not increase serum TSH. The biliary excretion of radiolabeled T3 was examined, and the radioactive components of bile were determined by HPLC. It is hypothesized that because PCN treatment induces microsomal T3-UGT activity, the same treatment will increase the biliary excretion of T3-glucuronide, while 3MC and PCB treatments will not. This study further addresses the hypothesis that increased T3 glucuronidation may mediate the increases in serum TSH produced via microsomal enzyme inducers by determining whether induction of T3 glucuronidation results in the increased biliary excretion of T3, and whether this increase coincides with elevated TSH.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents.
16-Dehydropregnenolone, 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) were obtained from Steraloids, Inc. (Newport, RI), Sigma Chemical Co. (St. Louis, MO), and Chem Service, Inc. (West Chester, PA), respectively. Pregnenolone-16{alpha}-carbonitrile (PCN) was synthesized from 16-dehydropregnenolone as previously described (Sonderfan and Parkinson, 1988Go). [125I]-T3 was obtained from NEN Research Products (Boston, MA). Heparin was supplied by Elkins-Sinn, Inc. (Cherry Hill, NJ). ß-Glucuronidase type IX-A was obtained from Sigma Chemical Co. (St. Louis, MO). Ultima Flo-M Scintillant was purchased from Packard Instrument Co, Inc. (Meridian, CT). All other reagents were obtained from Fisher Scientific (Pittsburgh, PA).

Animals and treatments.
Male Sprague-Dawley rats (Sasco, Wilmington, MA) weighing 150–200 g were randomly assigned to 1 of 4 groups of 12 animals each. Following a 1-week acclimation period, animals were placed on either control diet or diet containing one of the following inducers: PCN (1000 ppm), 3MC (250 ppm), PCB (100 ppm). Dosages selected are those previously demonstrated to have significantly reduced serum T4, increased T4 glucuronidation, and in the case of PCN, increased serum TSH (Liu et al., 1995Go). Rats were allowed ad libitum food and water. At the end of 7 days, animals were anesthetized with sodium pentobarbital (50 mg/ml) combined 1:1 with potassium iodide (1 mg/ml) at 2 ml/kg to prevent uptake of 125I into the thyroid. The femoral vein and artery were cannulated (PE 50) and primed with either saline or heparinized saline (33 units/ml), respectively. Approximately 1 ml of blood was sampled from the artery at this time, from which serum was collected and stored at –80°C for further assay. The bile duct was cannulated (PE 10), and the animal warmed under a heat lamp to 37°C body temperature. After 10 min, the animal was given 1 ml of [125I]-T3, iv, (10 µCi/ml in 10 mM NaOH saline plus 1% normal rat serum), and bile was collected on ice for 2 h at 30-min intervals. Fifteen min following the start of bile collection and 3 more times at intervals of 30 min, approximately 300 µl blood aliquots were collected from the femoral artery. At the end of the 2-h collection period, the urinary bladder was exposed and urine collected by puncture with a syringe and needle. The liver was removed, weighed, snap-frozen, and stored at –80°C. Bile and urine volumes were determined gravimetrically. Blood samples were centrifuged for 5 min to collect serum. Two aliquots (10 µl each) were taken from the bile samples, and 2 aliquots (15 µl each) from urine and serum samples for gamma spectroscopy. Following the addition of methanol (1:2) and storage at –20°C for 1 h to precipitate protein, bile was centrifuged at 12,000 x g (4°C) for 10 min and the supernatant collected for analysis by HPLC.

HPLC analysis.
Beckman System Gold equipment and software (version 8.1), consisting of an Autosampler 507, Programmable Solvent Module 126, Radioisotope Detector 171, and 110B Solvent Delivery Module for pumping scintillation cocktail, were utilized for HPLC. Reverse-phase HPLC was performed on a 10 x 0.3-cm ChromSpher C18 column in combination with both a ChromSep 10 x 2-mm reverse-phase guard column (Chrompack, Inc., Raritan, NJ) and a 7.5 x 4.6-mm Adsorbosphere C18 reverse-phase guard column (Alltech Associates, Inc., Deerfield, IL) with a 16–40% gradient of acetonitrile run against 0.02-M ammonium acetate, pH 4 (de Herder et al., 1988Go). The step-wise linear gradient was as follows: 6 to 18 min, 16 to 27% acetonitrile; 22 to 27 min, 27 to 40% acetonitrile; 37–47 min, 40 to 16% acetonitrile. The sample volume injected was 20 µl. To identify iodothyronine glucuronides, 100 µl bile aliquots were incubated for 4 h at 37°C with 250 units (168 µg) ß-glucuronidase in 100 µl of 100 mM phosphate buffer (pH 6.8). The reaction was stopped by the addition of 50-µl methanol and cooling on ice. Samples were then concentrated to ~100 µl by vacuum centrifugation and analyzed by HPLC for the absence of glucuronide metabolites. Acid labile conjugates were identified after treatment of 100 µl bile for one h at 80°C in 600 µl 1M HCl as previously described (Rutgers et al., 1989Go), again followed by cooling on ice and concentration by vacuum centrifugation.

Determination of serum T3, T4, and TSH.
The concentrations of total (free and protein-bound) serum T3 and T4 at Day 7, as well as TSH, prior to the collection of bile, were determined by radioimmunoassay (RIA) kits (total T3 and T4: Diagnostic Products Corp., Los Angeles, CA; rat TSH: Amersham Life Science, Inc., Arlington Heights, IL). Limits of detection for these kits were 7 ng/dl, 0.25 µg/dl, and 0.50 ng/ml, respectively.

Data analysis.
All results are expressed as the mean ± SE. For analysis of individual T3 metabolites, the percent total peak area of each biliary metabolite as determined by HPLC was multiplied by the total biliary radioactivity to determine the amount for each metabolite. Means were compared by one-way ANOVA followed by Dunnett's test. Statistical significance is reported at the p < 0.05 level.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After 7 days of treatment, each of the 3 inducers produced an increase in liver to body weight ratio (Fig. 1Go). PCN and PCB produced statistically significant increases of 48% and 27%, respectively. 3MC increased liver to body weight ratio by 15% greater than control, but was not statistically significant.



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FIG. 1. Effect of the microsomal enzyme inducers pregnenolone-16{alpha}-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) on liver weight, expressed as g/kg body weight. Values are the means ± SE. *Significantly different from control (p < 0.05).

 
Concentrations of serum thyroid hormones after 7 days of treatment with each of the inducers are shown in Figure 2Go. Serum T4 concentrations were decreased 45% by PCN treatment, 49% by 3MC treatment, and 67% by PCB treatment (upper panel). Serum concentrations of T3 were not decreased by 3MC, but were reduced to a similar extent by PCN and PCB, 23% and 21%, respectively (middle panel). Serum TSH concentrations were increased by 90% above control, only by PCN treatment.



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FIG. 2. Effect of pregnenolone-16{alpha}-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) on serum concentrations of total T4, T3, and TSH after 7 days of inducer treatment. Values are means ± SE. *Significantly different from control (p < 0.05).

 
Following surgical implantation of the biliary cannula, animals were administered [125I]-T3, iv, and during the collection of bile, the disappearance of [125I]-T3 from the serum was measured (Fig. 3Go). 3MC treatment had no effect on serum concentrations of [125I]-T3. At 15 min following the injection of [125I]-T3, serum concentrations in PCN-treated animals were reduced by 22%, although this was not statistically significant. Serum [125I]-T3 concentrations in PCB-treated animals were significantly lower than in control rats at both 15 and 45 min after administration (by 36% and 33%, respectively).



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FIG. 3. CON, control. Effect of pregnenolone-16{alpha}-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) on the clearance of T3 from serum. The concentration of total radioactivity was sampled from serum at 30-min intervals following iv administration of [125I]-T3. Values are means ± SE. *Significantly different from control (p < 0.05).

 
The rate of biliary excretion of total [125I]-T3 (T3 and metabolites) is depicted in the upper panel of Figure 4Go. Neither 3MC nor PCB treatments produced an increase in the total biliary excretion of T3 and its metabolites. However, PCN treatment increased the biliary excretion of T3 and its metabolites at all time points after the first 30 min, with the peak rate of excretion occurring at 60 min (58% above control). At 90 min, biliary excretion rates in PCN-treated animals increased to 63% above control rates, and reached double that of control rats at 120 min. The cumulative biliary excretion of total [125I]-T3 over the 2-h collection period is shown in the lower panel. Again, PCN treatment increased the cumulative amount of T3 and metabolites excreted into bile by 56, 75, and 67%. Neither 3MC nor PCB affected the excretion of total [125I]-T3 in bile.



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FIG. 4. CON, control. Effect of pregnenolone-16{alpha}-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) on the biliary excretion of T3. Total radioactivity excreted was measured in bile collected at 30-min intervals following iv administration of [125I]-T3. Excretion rate of bile radioactivity is given in fmol/min/kg body weight; cumulative amount of radioactivity excreted in bile is given in fmol/kg body weight over 2 h. Values are mean ± SE. *Significantly different from control (p < 0.05).

 
The chemical form of [125I]-T3 excreted into bile of control rats was primarily in the form of T3-glucuronide (74%), followed by T3 sulfate (16%), non-conjugated T2 (6%), T2 sulfate (3%), and nonconjugated T3 (2%). Each metabolite was quantified by HPLC, and its cumulative excretion in control rats and rats treated with the 3 microsomal enzyme inducers is depicted in Figure 5Go. PCN treatment produced 66% and 55% increases in the cumulative excretion of T3-glucuronide at 90 and 120 min, respectively (top panel), and 112, 164, and 138% increases in the cumulative excretion of T2 sulfate at 60, 90, and 120 min, respectively (Fig 5Go, second from bottom). PCN had no effect on the biliary excretion of T3 sulfate, T3, or T2.



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FIG. 5. CON, control. Enhanced biliary excretion of T3 metabolites following pregnenolone-16{alpha}-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) treatment. Biliary excretion of radioactive T3 metabolites was determined by HPLC at 30-min intervals following iv administration of [125I]-T3. Data points represent the mean cumulative excretion of each metabolite in fmol/kg body weight over 2 h ± SE. The excretion of five metabolites is depicted from top to bottom: T3-glucuronide; T3-sulfate; T3; T2-sulfate; T2. *Significantly different from control (p < 0.05).

 
Treatment with 3MC had no effect on biliary excretion of T3-glucuronide, but did increase significantly the amount of T3 sulfate in bile by 68, 64, 60, and 51% at 30, 60, 90, and 120 min, respectively (Fig 5Go, second from top). 3MC treatment had no effect on the biliary excretion of any other T3 metabolite. PCB treatment did not affect the biliary excretion of any T3 metabolite.

The amount of total [125I]-T3 in urine was determined as well, but individual metabolites were not quantified because of their low urinary excretion. Figure 6Go compares the cumulative biliary excretion (top panel) of [125I]-T3 after 2 h to that in urine (bottom panel). In control animals, the radioactivity in urine was only 5% that in bile. The total [125I]-T3 in bile was increased 67% by PCN, and although PCN tended to increase total [125I]-T3 in urine, this was not statistically significant. PCB treatment tended to decrease the cumulative urinary excretion of T3 and its metabolites, but it was not statistically significant either.



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FIG. 6. Effect of pregnenolone-16{alpha}-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) on the cumulative excretion of T3 in bile versus urine. The amount of radioactivity excreted in bile and urine was measured over 2 h after iv administration of [125I]-T3. Values represent the total amount of radioactivity present in bile or urine in fmol/kg body weight (mean ± SE).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, the effects of microsomal enzyme inducers on T3 glucuronidation in vivo are consistent with those previously demonstrated using in vitro liver microsomal preparations (Hood and Klaassen, 2000aGo). Of the inducers tested, only PCN increased the biliary excretion of T3, whereas 3MC and PCB did not (Fig. 4Go). When bile from control and inducer-treated rats was analyzed by HPLC, the primary component was T3-glucuronide, as previously reported (de Herder et al., 1988Go), and the excretion of T3-glucuronide in bile was increased solely by PCN treatment (Fig. 5Go). This finding is consistent with the increase in T3-UGT activity shown in microsomal preparations from PCN-treated rats. In the present study, urinary excretion was minor in comparison to biliary excretion of T3 and its metabolites in both control and inducer-treated animals (Fig. 6Go).

Total T4 serum concentrations were reduced after 7 days by all 3 inducer treatments (Fig. 2Go), findings that are consistent with previous studies (Hood et al., 1999aGo; Liu et al., 1995Go). It has been shown that PCN, 3MC, and PCB reduce serum T4 by an extrathyroidal mechanism. This mechanism is believed to be the increased hepatic elimination of T4 (Barter and Klaassen, 1992Go). The increases in liver weight produced by inducer treatment (Fig. 1Go) also support an increase in liver enzyme activity. In the present study, PCN and PCB treatments reduced total T3 serum concentrations as well. Previous reports of microsomal enzyme-inducer treatments reducing serum T3 are variable (Bastomsky, 1977Go; Gorski and Rozman, 1987Go; Liu et al., 1995Go; Masubuchi et al., 1997Go; Semler et al., 1989Go). Though serum T3 concentrations in this study appear to be maintained compared to serum T4, both PCN and PCB treatments significantly reduced serum total T3. It was recently reported that activity of outer-ring deiodinase (ORD), which catalyzes the conversion of T4 to T3, was reduced by microsomal inducer treatment (Hood and Klaassen, 2000bGo); therefore, it is possible that the reduced deiodination may contribute to reduced serum T3. However, the effects on ORD activity were similar for PCN, 3MC, and PCB, not differential, as are the effects on serum T3. PCB and PCN treatments do appear to increase the clearance of T3 from serum (Fig. 3Go), suggesting an increase in the tissue uptake of serum T3. Future studies are required to further elucidate this mechanism. Despite reductions in serum T4, 3MC had no effect on serum T3, consistent with findings in previous studies that have examined effects of 3MC on serum thyroid hormones for up to 20 days duration (Barter and Klaassen, 1994Go).

The reduction of total T3 serum concentrations by both PCN and PCB seems to be inconsistent with the overall increase in T3 biliary excretion, which occurred only following PCN treatment. However, it is important to note that total T3 serum concentrations, not free T3 serum concentrations, were measured in the present study. In general, free T3 concentrations are maintained following microsomal enzyme inducer treatment by mechanisms that are not well understood (Hood and Klaassen, 2000bGo). Neither free nor total T3 serum concentrations appear to reflect the status of the pituitary-thyroid axis (i.e., thyroid activation by TSH), and exactly when and how the pituitary senses changes in serum T3 are not clearly defined. Changes in rodent total T4 and total T3 serum concentrations following treatment with PCN, 3MC, and PCB have been well characterized, and were compared in the present study at a single time point (day 7) to verify reproducibility against previous studies using these compounds.

Reductions in serum thyroid hormones produced by treatment with microsomal enzyme inducers are considered important because of the associated increases in serum TSH that follow, due to the decreased negative feedback effect at the hypothalamus and pituitary. It has been well documented that constant stimulation of the thyroid gland in rats with elevated levels of TSH causes goiter, thyroid hyperplasia, adenomas, and carcinomas (Curran and DeGroot, 1991Go). McClain et al. (1988) originally hypothesized that T4 glucuronidation mediates increases in serum TSH of microsomal enzyme inducer-treated rats. Many thyroid endocrine disruptors have been shown to reduce serum T4; however, their effect on TSH is, at best, variable. For example, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCB), and 3-methylcholanthrene (3MC) produce marked to dramatic reductions in serum T4 in rats, with no change in serum TSH and little stimulation of the thyroid gland (Henry and Gasiewicz, 1987Go; Hood et al., 1999aGo; Liu et al., 1995Go; Schuur et al., 1997Go). Recently, a poor correlation between the ability of microsomal enzyme inducers to increase T4 glucuronidation and increase serum TSH has been demonstrated (Hood and Klaassen, 2000aGo). In the aforementioned study, PCN increased T4-UGT activity and serum TSH, while 3MC and PCB increased T4-UGT activity but had no effect on serum TSH, despite reducing serum T4. PCN also increased T3-UGT activity whereas 3MC and PCB had no effect on T3-UGT activity. Because only microsomal enzyme inducers that induce T3-UGT activity increase serum TSH, the suggestion is that glucuronidation of T3, rather than T4, mediates the increase in serum TSH. This was the first association made between increases in serum TSH and induced T3 glucuronidation in rats treated with microsomal enzyme inducers. The present study has demonstrated that these differential effects of PCN, 3MC, and PCB are maintained when examining in vivo glucuronidaion of T3. The present data suggest that increased glucuronidation and biliary excretion of T3-glucuronide may be the mechanism by which PCN treatment increases serum TSH; 3MC and PCB, which do not increase serum TSH, did not increase the biliary excretion of T3 and its metabolites.

In addition to glucuronidation, iodothyronines may also be sulfated to facilitate their excretion, though this is a relatively minor pathway of T3 metabolism compared to glucuronidation. Treatment with both 3MC and PCN produced increases in the sulfated metabolites of T3, namely T3S, and T2S, respectively (Fig. 5Go). There is known to be an increase in the mRNA for the sulfotransferase enzyme SULT1B1 following treatment of rats with the same dose of PCN used in the present study (Dunn et al., 1999Go). SULT1B1 has been shown to conjugate thyroid hormones, including T2 (Fujita et al., 1999Go). Therefore, the increase in T2S biliary excretion following PCN treatment can likely be attributed to an increase in sulfotransferase activity.

In vivo treatment with 3MC did not have an effect on SULT1B1, and has even been reported to suppress mRNA for another sulfotransferase, SULT1A1 (Runge-Morris, 1998Go). SULT1C1 has also been reported to be important for sulfation of thyroid hormones in male rat liver, but again, this enzyme is not inducible (Fujita et al., 1999Go; Visser et al., 1998Go). In addition, examination of T3 sulfation in cytosolic fractions from inducer-treated rats has found it to be unaffected (Hood and Klaassen, 2000bGo). It does not appear then, that the increased biliary excretion of T3S following 3MC treatment is due to an increase in T3 sulfation. Rather, the accumulation of T3S in the bile of 3MC-treated rats is most likely due to an inhibition of inner-ring deiodinase (IRD) activity. T3 is the outer ring deiodination product of T4, while 3,3`-T2 is the inner ring deiodination (Visser, 1996Go). Sulfation is known to facilitate deiodination, and studies have demonstrated that T3S is rapidly deiodinated in rat liver (Moreno et al., 1994Go; Visser et al., 1983Go,1984Go). This activity is carried out in liver by Type I iodothyronine deiodinase (ID-I) and by Type III iodothyronine deiodinase (ID-III), mainly in rat brain and placenta (Visser, 1990Go; Visser and Schoenmakers, 1992Go). T3S is a substrate for ID-I in rat hepatocytes, and this activity is subject to physiological regulation and inhibition by propylthiouracil (Rooda et al., 1989Go). If IRD activity were inhibited, one would expect T3S to accumulate in bile and urine, as demonstrated in vivo following PTU inhibition of ID-I (de Herder et al., 1988Go). To date, no investigations into 3MC inhibition of IRD activity exist. The inhibition of Type I ORD activity following microsomal enzyme inducer treatment has been reported, but IRD activity was not examined (Hood and Klaassen, 2000bGo). It is important to recall that accumulation of T3S in bile following 3MC treatment was minor in the present study, and did not affect the total amount of T3 excreted in bile or urine (Fig. 6Go).

The present study demonstrates an increase in the biliary excretion of T3 produced by PCN, and proposes that this increase is due primarily to increased glucuronidation of T3. In order to further understand this mechanism, it is important to investigate which UDP-glucuronosyltransferase (UGT) is responsible for the glucuronidation of T3 and whether this enzyme is induced by PCN treatment. UGT2B2 is the UGT that has been suggested to glucuronidate T3, because (1) Beetstra et al. (1991) showed that LA Wistar rats have reduced ability to glucuronidate androsterone and T3, and (2) UGT2B2 glucuronidates androsterone (Haque et al., 1991Go). However, the presence of UGT2B2 mRNA and protein has not been directly examined for inducibility by PCN.

In the future, it will also be important to consider the role of other processes that are involved in the disposition of T3, including hepatic uptake and transport of T3G into bile. Herein, PCN tended to decrease the concentration of T3 in the serum within 15 min of administration (Fig. 3Go). This suggests enhanced disposition of T3 from serum to some tissue(s) following PCN treatment, which may be due to increased hepatic T3 uptake. Thyroid hormones contain a polar alanine side chain making their passage through membranes by diffusion a difficult process. Evidence now suggests that thyroid hormones are actively transported across plasma membranes (Hennemann et al., 1998Go; Hennemann and Visser, 1997Go; Hood and Klaassen, 2000aGo). However, the mechanism by which the liver takes up T3 and makes it available for glucuronidation, and the chemical inducibility of this process are yet unresolved. Recently, several proteins that transport chemicals into the liver have been identified and cloned from human and rat. Rat proteins that have been shown to transport thyroid hormones include oatp1, oatp2, and oatp3 (Abe et al., 1998Go; Docter et al., 1997Go; Eelkman Rooda et al., 1989Go; Rondeel et al., 1995Go), which have fairly similar affinities for T3. Currently, investigations into the chemical inducibility of these transporters are limited, but it has been reported that oatp1 is regulated by testosterone and estrogen in rat kidney (Lu et al., 1996Go). It has also been determined that oatp2 mRNA and protein are induced by PCN treatment in rat liver (Rausch-Derra et al., 2001Go). The increased uptake of T3 into liver by oatp2 may also, then, contribute to the increased biliary excretion of T3 following PCN treatment.

In conclusion, these data provide evidence that PCN treatment induces the glucuronidation and biliary excretion of T3, whereas 3MC and PCB treatments do not. This increased biliary excretion is accompanied by an increase in serum TSH, which does not occur following treatment of rats with 3MC and PCB. These in vivo findings are in agreement with those demonstrated in liver microsomes (Hood and Klaassen, 2000aGo), and support the mechanism for increased TSH following treatment with microsomal enzyme inducers to be the result of increases in T3 metabolism and excretion.


    ACKNOWLEDGMENTS
 
This research was supported by NIH Grant ES-08156 and NIH Training Grant ES-07079 (to N.R.V.).


    NOTES
 
1 To whom correspondence should be addressed. Fax: (913) 588-7501. E-mail: cklaasse{at}kumc.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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