Toxicological Sciences 55, 78-84 (2000)
Copyright © 2000 by the Society of Toxicology
Endocrine Toxicology |
Differential Effects of Microsomal Enzyme Inducers on in Vitro Thyroxine (T4) and Triiodothyronine (T3) Glucuronidation
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160-7140
Received August 4, 1999; accepted January 6, 2000
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Microsomal enzyme inducers that increase UDP-glucuronosyltransferase (UDP-GT) activity are suspected to affect the thyroid gland by increasing the glucuronidation of T4, which reduces serum thyroxine (T4). In response to reduced serum T4, serum thyroid-stimulating hormone (TSH) increases. However, not all microsomal enzyme inducers that reduce serum T4 produce an increase in serum TSH. We have shown that serum TSH is increased the most in rats treated with the microsomal enzyme inducers phenobarbital (PB) or pregnenolone-16
-carbonitrile (PCN), whereas TSH is affected less in rats treated with 3-methylcholanthrene (3MC) and Aroclor 1254 (PCB). It is unclear why serum TSH is differentially affected by various microsomal enzyme inducers. We propose that the glucuronidation of T3 might be the reason serum TSH is increased by some microsomal enzyme inducers but not by others. Male Sprague-Dawley rats were fed either a basal diet or a diet containing PB (at 300, 600, 1200, or 2400 ppm), PCN (at 200, 400, 800, or 1600 ppm), 3MC (at 50, 100, 200, or 400 ppm), or PCB (at 25, 50, 100, or 200 ppm) for 7 days; and T4 and T3 UDP-GT activities were then determined. T4 UDP-GT activity was increased in rats treated with PB (120%), PCN (250 to 400%), 3MC (400 to 600%), or PCB (300 to 430%). In contrast, T3 UDP-GT activity was increased in rats treated with PB (90%) or PCN (120 to 200%), whereas 3MC and PCB treatments did not have an appreciable effect. In conclusion, differential effects on T3 glucuronosyltransferase activity were found in rats treated with microsomal enzyme inducers.
Key Words: UDP-glucuronosyltransferase (UDP-GT) activity; thyroid stimulating hormone (TSH); phenobarbital (PB); pregnenolone-16-carbonitrile; Arochlor 1254 (PCB); 3-methylcholanthrine; glucuronidation.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Several microsomal enzyme inducers, such as phenobarbital (PB) and pregnenolone-16
-carbonitrile (PCN), have been shown to increase the glucuronidation of T4, reduce serum T4 concentration, increase serum thyroid-stimulating hormone (TSH) concentration, and increase thyroid gland 131I uptake, thyroid follicular-cell proliferation, and thyroid gland weight (Hood et al., 1999
; Liu et al., 1995). Increases in serum TSH by microsomal enzyme inducers has been proposed to mediate the thyroid tumor promoting effect of PB (McClain, 1992). Phenobarbital (PB) and PB-like compounds have been shown to promote thyroid tumors (Diwan et al., 1988; Hiasa et al., 1982), however other microsomal enzyme inducers, such as PCN, 3MC, and PCB, have not been shown to promote thyroid tumors. Until these other microsomal enzyme inducers are tested for thyroid tumor promotion, assessment of whether other microsomal enzyme inducers promote thyroid tumors is enhanced by understanding their effects on serum TSH concentrations, because TSH is thought to mediate the thyroid tumor promotion effect.
Several studies have investigated the effects of microsomal enzyme inducers on serum T4 and TSH concentrations. The microsomal enzyme inducers PB, PCN, 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) have been shown to reduce serum T4 concentration (Barter and Klaassen, 1994; De Sandro et al., 1991; Khanduja et al., 1987; Liu et al., 1995; McClain et al., 1989; Saito et al., 1991). The mechanism by which PB, PCN, 3MC, and PCB reduce serum T4 concentration is extrathyroidal (Barter and Klaassen, 1992b). This extrathyroidal mechanism is thought to be increased T4 glucuronidation in the liver, because PB and PCN increase the glucuronidation of T4 in vivo, i.e., biliary excretion of T4-glucuronide (Japundzic et al., 1976; McClain et al., 1989; Oppenheimer et al., 1968) and in vitro, i.e., T4 UDP-GT activity (Barter and Klaassen, 1992a; De Sandro et al., 1991; Liu et al., 1995). 3-Methylcholanthrene and PCB also increase the glucuronidation of T4 in vitro (Barter and Klaassen, 1992a; Liu et al., 1995). Therefore, microsomal enzyme inducer-mediated increases in T4 glucuronidation are associated with reductions in serum T4 concentration.
The mechanism by which these microsomal enzyme inducers affect serum TSH is much less understood. For instance, in recent studies that we have performed, PB and PCN treatments increased serum TSH, whereas 3MC and PCB treatments did not have an appreciable effect on serum-TSH concentration (Hood et al., 1999; Liu et al., 1995). This is surprising because 3MC and PCB treatments are as effective, if not more, at increasing T4 UDP-GT activity and reducing serum T4 concentration as PB and PCN treatments. The different effects of PB, PCN, 3MC, and PCB on serum TSH concentration is not explained by reduced serum T3 concentrations, because serum T3 is not appreciably affected by these microsomal enzyme inducers (Hood et al., 1999; Liu et al., 1995). This could be due to the body`s ability to maintain serum T3 concentrations (Pazos Moura et al., 1991). Other studies have also been unable to quantitatively correlate reduced serum levels of thyroid hormone to compensatory increases in serum TSH of microsomal enzyme inducer-treated rats (Barter and Klaassen, 1994; Saito et al., 1991).
In the present study, we investigated whether the reason why some microsomal enzyme inducers increase serum TSH, and others do not, is due to a difference in their ability to increase the glucuronidation of T3. We were first led to this hypothesis because a few studies have investigated the effect of microsomal enzyme inducer treatment on T3 UDP-GT activity (Saito et al., 1991; Visser et al., 1993b). Second, microsomal enzyme inducers have been shown to induce different UDP-GT isozymes (Barter and Klaassen, 1992a). Third, T4 and T3 appear to be glucuronidated by different glucuronosyltransferases (Beetstra et al., 1991; Visser et al., 1993a). Therefore, we reasoned that the effect microsomal enzyme inducers have on T3 UDP-GT activity may differ from their effect on T4 UDP-GT activity.
In the present study, we hypothesized that microsomal enzyme inducers have differential effects on T3 UDP-GT activity. If true, then these results may have important implications for why some microsomal enzyme inducers increase serum TSH (PB and PCN), whereas other microsomal enzyme inducers (3MC and PCB) do not. Although the effects of these microsomal enzyme inducers on T4 UDP-GT activity have been reported previously (Liu et al., 1995), the effect of microsomal enzyme inducers on T4 UDP-GT activity was re-examined to determine whether T3 and T4 UDP-GT activities are differentially affected by microsomal enzyme inducer treatment.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Materials.
Phenobarbital (PB), 16-dehydropregnenolone, 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) were obtained from Spectrum Chemical, Mfg., Corp. (Gardena, CA), Pfaltz and Bauer, Inc. (Waterbury, CT), Sigma Chemical Co. (St. Louis, MO), and Chem Service Corp. (West Chester, PA), respectively. 16-Dehydropregnenolone was used to synthesize pregnenolone-16
-carbonitrile (PCN) as described by Sonderfan and Parkinson (1988). Thyroxine (T4), triiodothyronine (T3), propylthiouracil (PTU), UDP-glucuronic acid (UDP-GA), magnesium chloride (MgCl2), LH-20 resin, Tris-HCl, sucrose, and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma Chemical Co. (St. Louis, MO). 125I-T4 and 125I-T3 (labeled on the outer ring, 3' or 5') were obtained from the Dupont Company NEN Research Products (Boston, MA). Radioimmunoassay (RIA) kits for total and free T4 and T3 were obtained from Diagnostic Products Corp. (Los Angeles, CA). Rat TSH (for radioiodinations), anti-rat TSH serum (rabbit) and reference preparation rat TSH were kindly provided by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (Baltimore, MD). All other reagents were obtained from Fisher Chemical Company.
Animals and treatments.
Male Sprague-Dawley rats, 225–250 g, were housed in polypropylene cages containing corn-cob bedding and maintained at approximately 70°F on a 12-h light/dark cycle. Phenobarbital was dissolved in methanol, whereas PCN, 3MC, and PCB were dissolved in acetone. Each treatment was added to 1 kg of Purina Rodent Laboratory Chow 5001 (iodine content of 0.7 ppm), mixed thoroughly, and allowed to dry. The rats were divided into groups (5–6 rats each) and fed PB (at 300, 600, 1200, or 2400 ppm), PCN (at 200, 400, 800 or 1600 ppm), 3MC (at 50, 100, 200, or 400 ppm), or PCB (at 25, 50, 100, or 200 ppm). Control group received non-treated rodent chow. All rats received feed and water, ad libitum, for 7 days. Rats were monitored every 2 days by recording body weights and feed consumption.
Sampling.
On experimental day 7, rats were weighed and then lightly anesthetized with diethyl ether. Approximately 1.5 ml of blood was sampled from the aorta. Serum was collected by allowing the blood to clot at 4°C for 2 h, centrifuged, and supernatant stored at –80°C. Liver was removed, weighed, frozen in liquid nitrogen, and stored at –80°C.
Microsome preparations.
UDP-glucuronosyltransferase (UDP-GT) activity was determined in liver microsomes. Liver microsomes were prepared by homogenizing liver tissue in 4 volumes of buffer containing 50 mM Tris–HCl and 150 mM potassium chloride. Homogenates were then centrifuged at 10,000 g for 20 min. The supernatant was decanted into ultracentrifuge tubes and centrifuged at 100,000 x g for 60 min. The cytosol was removed and 1 ml of wash buffer (10 mM EDTA and 150 mM potassium chloride) was added to the microsomal pellet. The microsomal pellet was resuspended in wash buffer and homogenized. The homogenate was centrifuged again at 100,000 x g for 60 min. The supernatant was removed and 0.25 M sucrose was added to the microsomal pellet, homogenized, and stored at –80°C. Protein in liver microsome samples was determined using the bicinchoninic-acid method (Smith et al., 1985). Enzyme activities in these microsomal preparations was determined in either duplicate or triplicate (see figure legends for details).
Purification of 125I-rT3.
Free 125I was removed from stock 125I-T4 or 125I-T3 immediately prior to determining UDP-GT activity (Leonard and Rosenberg, 1980). 125I-T4 and 125I-T3 were purified by applying stock 125I-T4 or 125I-T3 to an LH-20 column (0.5 ml bed volume), equilibrated with 0.1 N HCl. Residual 125I was removed from the column by rinsing with 4 ml of 0.1 N HCl. The LH-20 column was rinsed with 4 ml of deionized water. 125I-T4 or 125I-T3 was eluted by rinsing the column with 1.5 ml of 98% ethanol 2% ammonium hydroxide. The volume of 125I-T4 or 125I-T3 eluate was reduced to 
200 µl using a gentle stream of nitrogen gas.
UDP-GT activities toward T4 and T3.
UDP-GT activity toward T4 and T3 was determined by quantifying the amount of 125I-T4-glucuronide or 125I-T3-glucuronide produced (Beetstra et al., 1991). Unless indicated otherwise, all reactions contained 150 µl of reaction mixture (pH 7.8) containing: 75 mM Tris–HCl, 7.5 mM MgCl2, 30 mM UDP-glucuronic acid, 1 µM of T4 or T3 (100,000 cpm), and 0.1 mM PTU to inhibit outer-ring deiodinase activity. Reactions were started by adding 50 µl of protein (final concentration of 250 µg/ml for T4 and 81 µg/ml for T3) and incubated for 60 min at 37°C in a water bath. The reactions were stopped by adding 200 µl of ice-cold 100% methanol, and placing each sample on ice. The samples were centrifuged for 45 min at 10,000 x g (5°C). Two hundred µl of supernatant was added to a Sephadex LH-20 column (1 ml bed volume) that had been equilibrated with 6 ml of 0.1 N HCl. Free 125I was eluted first by rinsing the LH-20 columns with 2 ml of 0.1 N HCl. The columns were rinsed with 2 ml of deionized water. 125I-T4-glucuronide or 125I-T3-glucuronide was eluted from the columns by rinsing with 3 ml of deionized water. To ensure that all of the 125I-T4-glucuronide or 125I-T3-glucuronide was eluted, the columns were rinsed with an additional 3 ml of deionized water. 125I-T4 and 125I-T3 were then eluted from the columns by rinsing with 3 ml of ethanol-0.1 N NaOH (1:1 vol/vol). The amount of 125I-T4 or 125I-T3 in the eluates was quantified using a gamma counting instrument (Beckman Instruments, Palo Alto, CA).
Statistics.
Differences between control and treated animals were determined using the ANOVA analysis followed by the Duncan's multiple range post hoc test. Significant differences between treated and control groups (p < 0.05) are indicated by asterisks in the figures. Statistical analyses were performed using STATISTICA 4.5, Statsoft Inc. (Tulsa, Oklahoma). Kinetic parameters (Km and Vmax) were determined using hyperbolic regression analysis, the Hyper.exe (J.B. Easterly, Liverpool, UK).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Characterization of T4 and T3 UDP-GT Activities
Effect of various T4 or T3 concentrations on UDP-GT activity.
T4 UDP-GT activity was linear up to about 50 µM T4 and reached a plateau at concentrations above 100 µM T4 (Fig. 1
). T3 UDP-GT activity was linear over the range of T3 concentrations used in this study (Fig. 1). Some of the T4 and T3 did not appear to remain in solution at concentrations above 50 µM and 100 µM, respectively. However, the estimated apparent Km value for T4 was 53 µM. Because saturating concentrations of T3 was unobtainable in the present study, accurate estimation of Km for T3 was not possible.
|
Effect of various UDP-glucuronic acid concentrations on UDP-GT activity toward T4 and T3.
T4 UDP-GT activity was linear up to 10 mM UDP-glucuronic acid, and reached a plateau at concentrations above 25 mM UDP-glucuronic acid (Fig. 2
, top).
|
T3 UDP-GT activity was linear up to 1 mM UDP-glucuronic acid (Fig. 2
, bottom). T3 UDP-GT activity reached a plateau at concentrations above 5 mM UDP-glucuronic acid; however, the activity was higher at the 60-mM concentration (Fig. 2, bottom).
Effect of various protein concentrations and incubation times on UDP-GT activity toward T4 and T3.
The effect of increasing protein concentration and time on T4 and T3 UDP-GT activities (data not shown) was also determined in the present study and was consistent with previous studies (Barter and Klaassen, 1992a). Briefly, T4 UDP-GT activity was linear up to 0.25 mg/ml of microsomal protein and reached a plateau at concentrations above 0.5 mg/ml. T3 UDP-GT activity was linear up to 0.125 mg/ml of protein and reached a plateau at concentrations above 0.5 mg/ml. UDP-glucuronosyltransferase activity toward T4 and T3 were linear over a 4-hour incubation period.
Effect of detergent on UDP-GT activity toward T4 and T3.
We investigated the effects of detergent on UDP-GT activity toward T4 and T3, because these substances are widely used in UDP-GT activity assays. Figure 3 shows the effect of 0.025% Brij-56, Brij-58, and CHAPS on UDP-GT activity toward T4 and T3 in control, PB (2400 ppm), PCN (1600 ppm), 3MC (400 ppm), and PCB (200 ppm)-treated rats. Brij-56, Brij-58, or CHAPS did not appreciably activate T4 UDP-GT activity in control, PB-, or PCN-treated rats compared to those incubations that did not include detergent (Fig. 3, top). In contrast to rats treated with PB and PCN, Brij-56 and Brij-58 reduced (70%) T4 UDP-GT activity in rats treated with 3MC or PCB (Fig. 3, top). CHAPS had minimal effects on UDP-GT activity toward T4 (Fig. 3, top).
|
Brij-56 and Brij-58 had an even greater inhibitory effect on the glucuronidation of T3 than that on T4, because Brij-56 and Brij-58 reduced T3 UDP-GT activity in control (
50%), PB (50%)-, and PCN (78%)-, as well as 3MC (70%)- and PCB (75%)-treated rats, compared to reaction mixtures that did not contain detergent (Fig. 3, bottom). Again, CHAPS had minimal effects on UDP-GT activity toward T3 (Fig. 3, bottom).
Optimal conditions for determining T4 and T3 UDP-GT activity.
Based on the results from the studies above, reaction conditions for T4 and T3 UDP-GT activities included 75 mM Tris–HCl, 7.5 mM MgCl2, 50 mM (T4) or 30 mM (T3) UDP-glucuronic acid, 0.1 mM PTU, 250 µg/ml (T4) or 81 µg/ml (T3) protein, and 1 µM T4 or T3, and 100,000 CPM of 125I-T4 or 125I-T3.
Effect of microsomal enzyme inducers on UDP-GT activity toward T4.
T4 UDP-GT activity was increased the most in rats treated with PCN, 3MC, or PCB, and less in rats treated with PB (Fig. 4). For instance, T4 UDP-GT activity was increased up to 270, 250, and 400% in PCN-, 510, 480, and 600% in 3MC-, and 300, 310, and 430% in PCB-treated rats when the activity was expressed per mg protein/min (Fig. 4, top), per g tissue/min (Fig. 4, middle), or per whole organ/min (Fig. 4, bottom), respectively. Phenobarbital treatment did not increase T4 UDP-GT activity when the activity was expressed per mg protein/min or per g tissue/min. However, PB treatment did increase T4 UDP-GT activity 120% when it was expressed as per whole organ/min. The enhanced induction of T4 UDP-GT activity, when expressed as per whole organ/min, is probably due to increases in liver weight of PB (38%), PCN (43%), 3MC (23%), or PCB (31%) treatments (data not shown).
|
Effect of microsomal enzyme inducers on UDP-GT activity toward T3.
T3 UDP-GT activity was increased the most in rats treated with PCN, less in rats treated with PB, and not increased in rats treated with 3MC or PCB (Fig. 5
). For instance, T3 UDP-GT activity was increased 140, 120, and 200% in PCN-treated rats when the activity was expressed per mg protein/min, per g tissue/min, or per whole organ/min, respectively (Fig. 5). Although PB treatment did not significantly affect T3 UDP-GT activity when the activity was expressed per mg protein/min (Fig. 5, top) or per g tissue/min (Fig. 5, middle), it was increased 90% when expressed per whole organ/min (Fig. 5, bottom). UDP-GT activity toward T3 was not affected in rats treated with 3MC or PCB, regardless of how it was expressed (Fig. 5).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the present study, we tested the hypothesis that those microsomal enzyme inducers that increase serum TSH concentration, i.e., PB and PCN, increase T3 UDP-glucuronosyltransferase (UDP-GT) activity. However, those microsomal enzyme inducers that do not increase serum TSH concentration, i.e., 3MC and PCB, do not increase UDP-GT activity toward T3. Before we tested this hypothesis it was important to first characterize the in vitro conditions for determining T4 and T3 UDP-GT activities, because (1) the optimal UDP-glucuronic acid concentration has not been shown for determining UDP-GT activity toward T4 or T3, (2) the use of Brij-56 has been shown to reduce UDP-GT activity toward T4 in Wistar rats treated with 3MC-type microsomal enzyme inducers (Visser et al., 1993b
), and (3) to ensure that we use optimal in vitro assay conditions for determining T4 and T3 UDP-GT activities. Some of the results from these preliminary studies, which are presented in this publication and briefly discussed below, were surprising.
Ideally, the optimal substrate concentration for the in vitro enzyme activity assay is the concentration that saturates enzyme activity. In the present study, we were unable to obtain a concentration of T3 that saturates T3 UDP-GT activity (Fig. 1, bottom). In addition, not all of the T4 and T3, at concentrations above 50 µM, was in solution, making it difficult to determine actual saturating concentrations. The most likely reason that T4 and T3 did not remain in solution at high substrate concentration was because of the low pH (7.8) of the reaction mixture. Although T4 and T3 are most readily dissolved in a basic solution (pH 11 or higher), a pH of 7.8 was selected because previous studies reported T4 UDP-GT activity to be optimal between pH of 7.4 to 7.8, and T3 UDP-GT activity to be optimal between pH of 7.8 to 8.2 (Beetstra et al., 1991). The insolubility of thyroid hormones at high concentration may explain why a saturating concentration of T3 was not obtained in the present study, and accurate estimation of Km was not possible. Although we report a Km of 53 µM for T4 UDP-GT activity, this estimate may not represent the true Km because of the insolubility of T4 at high substrate concentrations. Previous studies have used 1 µM of T4 and T3 for in vitro UDP-GT activity assays (Beetstra et al., 1991; Visser et al., 1993b). Although 1 µM is below saturating concentrations of T4/T3, this concentration appears to be adequate, because only 35 and 22% of T4 and T3, respectively, were consumed in reactions that had the highest activities: 400 ppm of 3MC for T4 (Fig. 4) and 1600 ppm PCN for T3 (Fig. 5).
The apparent Km value of 53 µM for T4 UDP-GT activity is lower than that reported in previous studies: 100 µM for T4 and T3 (Beetstra et al., 1991; Visser et al., 1993b). These previous studies did not report the solubility problem that was experienced in the present study. Because we found that T4 did not remain in solution at high substrate concentrations, we expected that our estimation of Km would be higher than the true Km, which doesn't appear to be the case. Our apparent Km estimate is lower than that reported in previous studies. It is possible that the lower Km values obtained in the present study were due to the higher concentration of UDP-glucuronic acid (50 mM for T4) used, compared to the 5 mM used in previous studies (Beetstra et al., 1991; Liu et al., 1995; McClain et al., 1989; Visser et al., 1993b). UDP-glucuronic acid concentrations of 50 mM and 30 mM for T4 and T3, respectively, were used in the present study because UDP-GT activity toward T4 and T3 was saturated at these concentrations (Fig. 2), which has not been reported previously.
Detergent is commonly used in UDP-GT activity assays. However, in the present study, we found Brij detergents (Brij-56 and Brij-58) to be more detrimental than beneficial for determining UDP-GT activity toward T4 or T3 in rats treated with microsomal enzyme inducers (Fig. 3). The effects of Brij-56 on UDP-GT activity toward thyroid hormones has been reported previously (Visser et al., 1993b); however, this is the first report to show that T4 UDP-GT activity is reduced by Brij detergents in rats treated with 3MC or PCB. In addition, T3 UDP-GT activity is reduced even more by Brij detergents in untreated rats, as well as in rats treated with PB, PCN, 3MC, or PCB. Overall, these results suggest that Brij detergents inhibit, rather than activate, some UDP-GT isozymes that glucuronidate T4 and T3.
In the present study, not all of the microsomal enzyme inducers that increased UDP-GT activity toward T4 increased its activity toward T3. Figure 4 (bottom panel) shows that PB, PCN, 3MC, and PCB increase T4 UDP-GT activity when expressed per whole organ/min, which is consistent with previous studies (Barter and Klaassen, 1994; Liu et al., 1995). T3 UDP-GT activity was increased in rats treated with PB (90%) or with PCN (200%), when the activity was expressed per whole organ/min (Fig. 5, bottom panel), but not in rats treated with 3MC or PCB. The differential effects of microsomal enzyme inducers on UDP-GT activity toward T4 and T3 is likely the result of induction of different UDP-GT isozymes by these microsomal enzyme inducers (Barter and Klaassen, 1992a). Also, T4 and T3 are thought to be glucuronidated by different glucuronosyltransferases (Beetstra et al., 1991; Visser et al., 1993a). Thyroxine is glucuronidated by phenol and bilirubin UDP-GTs, whereas T3 is glucuronidated by an androsterone UDP-GT (Visser et al., 1993a).
Athough serum concentrations of T4, T3, and TSH were measured in the present study, these data were not included in this report because they were reported in a previous publication (Hood et al., 1999). Table 1 summarizes the effects of PB, PCN, 3MC, and PCB on T4/T3 UDP-GT activity and serum concentrations of T4, T3, and TSH. Increasing the glucuronidation of T4 has been proposed as resulting in reduced serum T4 concentrations, which in turn leads to increases in serum TSH in rats treated with PB and possibly other microsomal enzyme inducers (McClain, 1992; McClain et al., 1989). In the present study, PB, PCN, 3MC, and PCB reduced serum free T4 concentration 45, 50, 53, and 95%, respectively (Hood et al., 1999), as well as increasing T4 UDP-GT activity (Table 1). These results support the hypothesis that reductions in serum T4 concentration of microsomal enzyme inducer-treated rats is the result of increases in T4 glucuronidation. This finding is consistent with previous studies that have found T4 UDP-GT activity to be correlated with reductions in serum T4 concentrations (Barter and Klaassen, 1994; Saito et al., 1991). However, only PB and PCN increased serum TSH, 65 and 90%, respectively, (Hood et al., 1999), whereas 3MC and PCB did not affect serum TSH. It is unclear why 3MC and PCB did not increase serum TSH in these rats.
|
In the present study, those microsomal enzyme inducers that increased T3 UDP-glucuronosyltransferase activity (PB and PCN) increased serum TSH (Table 1
). In contrast, those microsomal enzyme inducers that did not increase T3 UDP-glucuronosyltransferase activity did not alter serum TSH concentration (Table 1). This finding suggests that glucuronidation of T3 might be important in mediating increases in serum TSH of rats treated with microsomal enzyme inducers. The role of T3 glucuronidation in mediating increases in serum TSH of microsomal enzyme inducer-treated rats needs further investigation. There are other possible explanations for why some microsomal enzyme inducers (3MC and PCB) do not increase serum TSH. For instance, 3MC and PCB may directly affect the pituitary that compromises its ability to synthesize and secrete TSH. In a previous study, we ruled out the possibility that differences in pituitary deiodinase activity could explain the differential effects of these microsomal enzyme inducers on serum TSH (Hood and Klaassen, 2000). Preliminary studies also did not support that 3MC and PCB directly affect pituitary function (data not shown). However, other factors, such as thyrotropin releasing hormone (TRH), are involved in TSH synthesis and secretion, which may be adversely affected by 3MC or PCB treatments.
In conclusion, unlike the effect on T4 glucuronosyltransferase activity, microsomal enzyme inducers have differential effects on T3 glucuronosyltransferase activity. Furthermore, only those microsomal enzyme inducers that increase T3 UDP-glucuronosyltransferase activity also increased serum TSH in our study (Table 1). This finding suggests that induction of T3 glucuronidation may play a role in mediating increases in serum TSH of microsomal enzyme inducer-treated rats. The mechanism by which induction of T3 glucuronidation mediates increases in serum TSH is unclear. We hypothesize that induction of T3 glucuronidation results in increased biliary excretion of T3, resulting in increased turnover rates of T3, which alters one or more factors that regulate TSH synthesis and secretion. Therefore, factors known to regulate TSH synthesis and secretion, such as TRH and pituitary levels of T3, need to be investigated. Serum T3 concentrations are unaffected in microsomal enzyme inducer-treated rats because of physiological mechanisms that maintain serum T3 concentrations (Pazos Moura et al., 1991).
|
ACKNOWLEDGMENTS |
|---|
This work was supported by NIH Grant ES08156 and EPA Grant R826297. A.H. was supported by NIH Training Grant ES07079.
|
NOTES |
|---|
1 To whom correspondence should be addressed. Fax: (913) 588-7501. E-mail: cklaasse{at}kumc.edu.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Barter, R. A., and Klaassen, C. D. (1992a). Rat liver microsomal UDP-glucuronosyltransferase activity toward thyroxine: Characterization, induction, and form specificity. Toxicol. Appl. Pharmacol. 115, 261–267.[Web of Science][Medline]
Barter, R. A., and Klaassen, C. D. (1992b). UDP-glucuronosyltransferase inducers reduce thyroid hormone levels in rats by an extrathyroidal mechanism. Toxicol. Appl. Pharmacol. 113, 36–42.[Web of Science][Medline]
Barter, R. A., and Klaassen, C. D. (1994). Reduction of thyroid hormone levels and alteration of thyroid function by four representative UDP-glucuronosyltransferase inducers in rats. Toxicol. Appl. Pharmacol. 128, 9–17.[Web of Science][Medline]
Beetstra, J. B., van Engelen, J. G., Karels, P., van der Hoek, H. J., de Jong, M., Docter, R., Krenning, E. P., Hennemann, G., Brouwer, A., and Visser, T. J. (1991). Thyroxine and 3,3',5-triiodothyronine are glucuronidated in rat liver by different uridine diphosphate-glucuronyltransferases. Endocrinology 128, 741–746.
De Sandro, V., Chevrier, M., Boddaert, A., Melcion, C., Cordier, A., and Richert, L. (1991). Comparison of the effects of propylthiouracil, amiodarone, diphenylhydantoin, phenobarbital, and 3-methylcholanthrene on hepatic and renal T4 metabolism and thyroid gland function in rats. Toxicol. Appl. Pharmacol. 111, 263–278.[Web of Science][Medline]
Diwan, B. A., Rice, J. M., Nims, R. W., Lubet, R. A., Hu, H., and Ward, J. M. (1988). P-450 enzyme induction by 5-ethyl-5-phenylhydantoin and 5,5-diethylhydantoin, analogues of barbiturate-tumor promoters phenobarbital and barbital, and promotion of liver and thyroid carcinogenesis initiated by N-nitrosodiethylamine in rats. Cancer Res. 48, 2492–2497.
Hiasa, Y., Kitahori, Y., Ohshima, M., Fujita, T., Yuasa, T., Konishi, N., and Miyashiro, A. (1982). Promoting effects of phenobarbital and barbital on development of thyroid tumors in rats treated with N-bis(2-hydroxypropyl)nitrosamine. Carcinogenesis 3, 1187–1190.
Hood, A., Hashmi, R., and Klaassen, C. D. (1999). Effects of microsomal enzyme inducers on thyroid-follicular cell proliferation, hyperplasia, and hypertrophy. Toxicol. Appl. Pharmacol. 160, 163–170.[Web of Science][Medline]
Hood, A., and Klaassen, C. D. (2000). Effects of microsomal enzyme inducers on outer-ring deiodinase activity toward thyroid hormones in various rat tissues. Toxicol. Appl. Pharmacol. 163, 240–248.[Web of Science][Medline]
Japundzic, M. M., Bastomsky, C. H., and Japundzic, I. P. (1976). Enhanced biliary thyroxine excretion in rats treated with pregnenolone-16-carbonitrile. Acta Endocrinol. Copenh. 81, 110–119.
Khanduja, K. L., Gupta, M. P., Pathak, C. M., Vaidyanathan, S., Koul, A., Koul, I. B., and Sharma, R. R. (1987). Effect of phenobarbital and 3-methylcholanthrene treatment on thyroid hormones in rat. Indian J. Med. Res. 86, 375–381.[Web of Science][Medline]
Leonard, J. L., and Rosenberg, I. N. (1980). Iodothyronine 5'-deiodinase from rat kidney: Substrate specificity and the 5'-deiodination of reverse triiodothyronine. Endocrinology 107, 1376–1383.
Liu, J., Liu, Y., Barter, R. A., and Klaassen, C. D. (1995). Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: A dose-response study. J. Pharmacol. Exp. Ther. 273, 977–985.
McClain, R. M. (1992). Thyroid gland neoplasia: non-genotoxic mechanisms. Toxicol. Lett. 64–65, 397–408.
McClain, R. M., Levin, A. A., Posch, R., and Downing, J. C. (1989). The effect of phenobarbital on the metabolism and excretion of thyroxine in rats. Toxicol. Appl. Pharmacol. 99, 216–228.[Web of Science][Medline]
Oppenheimer, J. H., Bernstein, G., and Surks, M. I. (1968). Increased thyroxine turnover and thyroidal function after stimulation of hepatocellular binding of thyroxine by phenobarbital. J. Clin. Invest. 47, 1399–1406.
Pazos-Moura, C. C., Moura, E. G., Dorris, M. L., Rehnmark, S., Melendez, L., Silva, J. E., and Taurog, A. (1991). Effect of iodine deficiency and cold exposure on thyroxine 5'-deiodinase activity in various rat tissues. Am. J. Physiol. 260, E175–182.
Saito, K., Kaneko, H., Sato, K., Yoshitake, A., and Yamada, H. (1991). Hepatic UDP-glucuronyltransferase(s) activity toward thyroid hormones in rats: Induction and effects on serum thyroid hormone levels following treatment with various enzyme inducers. Toxicol. Appl. Pharmacol. 111, 99–106.[Web of Science][Medline]
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85.[Web of Science][Medline]
Sonderfan, A. J., and Parkinson, A. (1988). Inhibition of steroid 5-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450. Arch. Biochem. Biophys. 265, 208–218.[Web of Science][Medline]
Visser, T. J., Kaptein, E., van Raaij, J. A., Joe, C. T., Ebner, T., and Burchell, B. (1993a). Multiple UDP-glucuronyltransferases for the glucuronidation of thyroid hormone with preference for 3,3',5'-triiodothyronine (reverse T3). FEBS Lett. 315, 65–68.[Web of Science][Medline]
Visser, T. J., Kaptein, E., van Toor, H., van Raaij, J. A., van den Berg, K. J., Joe, C. T., van Engelen, J. G., and Brouwer, A. (1993b). Glucuronidation of thyroid hormone in rat liver: Effects of in vivo treatment with microsomal enzyme inducers and in vitro assay conditions. Endocrinology 133, 2177–2186.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. L. Flippin, J. M. Hedge, M. J. DeVito, G. A. LeBlanc, and K. M. Crofton Predictive Modeling of a Mixture of Thyroid Hormone Disrupting Chemicals That Affect Production and Clearance of Thyroxine International Journal of Toxicology, September 1, 2009; 28(5): 368 - 381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Hedge, M. J. DeVito, and K. M. Crofton In Vivo Acute Exposure to Polychlorinated Biphenyls: Effects on Free and Total Thyroxine in Rats International Journal of Toxicology, September 1, 2009; 28(5): 382 - 391. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Zorrilla, E. K. Gibson, S. C. Jeffay, K. M. Crofton, W. R. Setzer, R. L. Cooper, and T. E. Stoker The Effects of Triclosan on Puberty and Thyroid Hormones in Male Wistar Rats Toxicol. Sci., January 1, 2009; 107(1): 56 - 64. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Emi, S.-i. Ikushiro, and Y. Kato Thyroxine-Metabolizing Rat Uridine Diphosphate-Glucuronosyltransferase 1A7 Is Regulated by Thyroid Hormone Receptor Endocrinology, December 1, 2007; 148(12): 6124 - 6133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L A Burns-Naas, M Zorbas, B Jessen, W Evering, G Stevens, J L Ivett, T E Ryan, J C Cook, C C Capen, M Chen, et al. Increase in thyroid follicular cell tumors in nelfinavir-treated rats observed in a 2-year carcinogenicity study is consistent with a rat-specific mechanism of thyroid neoplasia Human and Experimental Toxicology, December 1, 2005; 24(12): 643 - 654. [Abstract] [PDF]
|
|
|
|
|
|
A. M. Zavacki and P. R. Larsen CARs and Drugs: A Risky Combination Endocrinology, March 1, 2005; 146(3): 992 - 994. [Full Text] [PDF]
|
|
|
|
|
|
N. R. Vansell, J. R. Muppidi, S. M. Habeebu, and C. D. Klaassen Promotion of Thyroid Tumors in Rats by Pregnenolone-16{alpha}-Carbonitrile (PCN) and Polychlorinated Biphenyl (PCB) Toxicol. Sci., September 1, 2004; 81(1): 50 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Chen, J. L. Staudinger, and C. D. Klaassen NUCLEAR RECEPTOR, PREGNANE X RECEPTOR, IS REQUIRED FOR INDUCTION OF UDP-GLUCURONOSYLTRANSFERASES IN MOUSE LIVER BY PREGNENOLONE-16{alpha}-CARBONITRILE Drug Metab. Dispos., July 1, 2003; 31(7): 908 - 915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Dalton, R. T. Miller, and S. A. Meyer The Herbicide Metolachlor Induces Liver Cytochrome P450s 2B1/2 and 3A1/2, but Not Thyroxine-Uridine Dinucleotide Phosphate Glucuronosyltransferase and Associated Thyroid Gland Activity International Journal of Toxicology, July 1, 2003; 22(4): 287 - 295. [Abstract] [PDF]
|
|
|
|
|
|
E. S. Craft, M. J. DeVito, and K. M. Crofton Comparative Responsiveness of Hypothyroxinemia and Hepatic Enzyme Induction in Long-Evans Rats Versus C57BL/6J Mice Exposed to TCDD-like and Phenobarbital-like Polychlorinated Biphenyl Congeners Toxicol. Sci., August 1, 2002; 68(2): 372 - 380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. R. Vansell and C. D. Klaassen Increase in Rat Liver UDP-Glucuronosyltransferase mRNA by Microsomal Enzyme Inducers that Enhance Thyroid Hormone Glucuronidation Drug Metab. Dispos., March 1, 2002; 30(3): 240 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Zhou, M. M. Taylor, M. J. DeVito, and K. M. Crofton Developmental Exposure to Brominated Diphenyl Ethers Results in Thyroid Hormone Disruption Toxicol. Sci., March 1, 2002; 66(1): 105 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. R. Vansell and C. D. Klaassen Effect of Microsomal Enzyme Inducers on the Biliary Excretion of Triiodothyronine (T3) and Its Metabolites Toxicol. Sci., February 1, 2002; 65(2): 184 - 191. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. Capen Overview of Structural and Functional Lesions in Endocrine Organs of Animals Toxicol Pathol, January 1, 2001; 29(1): 8 - 33. [Abstract] [PDF]
|
|
|
|
|
|
C. D. Klaassen and A. M. Hood Effects of Microsomal Enzyme Inducers on Thyroid Follicular Cell Proliferation and Thyroid Hormone Metabolism Toxicol Pathol, January 1, 2001; 29(1): 34 - 40. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||