induction of t4 udp-gt activity, serum thyroid stimulating hormone, and thyroid follicular cell...

Induction of T4 UDP-GT activity, serum thyroid stimulating hormone,and thyroid follicular cell proliferation in mice treated with

microsomal enzyme inducers

Alan Hood, Marcia L. Allen, YaPing Liu, Jie Liu, and Curtis D. Klaassen*Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160-7140, USA

Received 22 August 2002; accepted 12 December 2002

Abstract

The microsomal enzyme inducers phenobarbital (PB), pregnenolone-16�-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor1254 (PCB) are known to induce thyroxine (T4) glucuronidation and reduce serum T4 concentrations in rats. Also, microsomal enzymeinducers that increase serum TSH (i.e., PB and PCN) also increase thyroid follicular cell proliferation in rats. Little is known about theeffects of these microsomal enzyme inducers on T4 glucuronidation, serum thyroid hormone concentrations, serum TSH, and thyroid glandgrowth in mice. Therefore, we tested the hypothesis that microsomal enzyme inducers induce T4 UDP-GT activity, resulting in reducedserum T4 concentrations, as well as increased serum TSH and thyroid follicular cell proliferation in mice. B6C3F male mice were fed acontrol diet or a diet containing PB (600, 1200, 1800, or 2400 ppm), PCN (250, 500, 1000, or 2000 ppm), 3MC (62.5, 125, 250, or 500ppm), or PCB (10, 30, 100, or 300 ppm) for 21 days. All four inducers increased liver weight and hepatic microsomal UDP-GT activitytoward chloramphenicol, �-naphthol, and T4. PB and PCB decreased serum total T4, but PCN and 3MC did not. Serum thyroid stimulatinghormone was markedly increased by PCN and 3MC treatments, and slightly increased by PB and PCB treatments. All four microsomalenzyme inducers dramatically increased thyroid follicular cell proliferation in mice. The findings suggest that PB, PCN, 3MC, and PCBdisrupt thyroid hormone homeostasis in mice.© 2003 Elsevier Science (USA). All rights reserved.

Introduction

Phenobarbital (PB) is a known thyroid tumor promoter inrats (Hiasa et al., 1982; McClain et al., 1988). The mecha-nism of PB mediated thyroid tumor promotion has beenproposed to include the following events: (1) induction ofthyroxine (T4) glucuronidation, (2) reduction of serum T4

concentrations, (3) a compensatory increase in serum thy-roid stimulating hormone (TSH), and (4) TSH-dependentstimulation of thyroid gland function and growth (McClain,1992; McClain et al., 1988). This mechanism was proposedbecause the predominant pathway for T4 biotransformationin rats is conjugation with glucuronic acid, catalyzed by theenzyme uridine-5-diphosphoglucuronosyltransferase (UDP-GT) (Rutgers et al., 1989b). UDP-GT enzymes are found

mostly in the endoplasmic reticulum of the liver. The addi-tion of glucuronic acid to T4 is a deactivation reaction, andthe conjugate is excreted in bile. Induction of T4 UDP-GTactivity is associated with reduced serum T4 concentrationsin rats (Hood et al., 1999a; Liu et al., 1995).

Thyroid hormone concentrations are controlled by afeedback mechanism that involves the hypothalamus, ante-rior pituitary, and the thyroid gland (Larsen and Silva, 1983;Rapoport and Spaulding, 1996; Scanlon and Toft, 1996).Serum T4 and triiodothyronine (T3) concentrations are mon-itored by the hypothalamus and the anterior pituitary gland.A decrease in serum thyroid hormone concentrations stim-ulates the hypothalamus to secrete thyrotropin releasinghormone (TRH), which then stimulates the release of TSHfrom the anterior pituitary (Scanlon and Toft, 1996). Inaddition to TRH, the secretion of TSH from the anteriorpituitary is also regulated by circulating thyroid hormoneconcentrations (Larsen and Silva, 1983). Increased produc-

* Corresponding author. Fax: �1-913-588-7501.E-mail address: [email protected] (C.D. Klaassen).

R

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tion and secretion of TSH leads to increased serum TSHconcentrations, followed by stimulation of the thyroid glandto synthesize T4 and T3 that restores the deficit of thyroidhormones in serum (Rapoport and Spaulding, 1996).

It has been proposed that chemicals that induce UDP-GTs can increase the glucuronidation and biliary excretionof T4, resulting in reduced serum T4 concentration, in-creased serum TSH, and ultimately promote thyroid tumors(McClain, 1992). The role of TSH in mediating the thyroidtumor promoting effects of microsomal enzyme inducers,such as PB, is plausible because TSH is known to stimulatethyroid gland growth (i.e., hypertrophy and hyperplasia)(Capen, 1996). Studies in rats have shown that PB inducesT4 UDP-GT activity, reduces serum T4 concentrations, in-creases serum TSH, and promotes thyroid tumors (De San-dro et al., 1991; Hiasa et al., 1982; Liu et al., 1995; McClainet al., 1988). Although PB, and PB-like compounds, is theonly microsomal enzyme inducer shown to promote thyroidtumors in rats (Diwan et al., 1985; Hiasa et al., 1982;McClain et al., 1988), the microsomal enzyme inducer preg-nenolone-16�-carbonitrile (PCN) has also been shown toinduce T4 UDP-GT activity, reduce serum T4 concentration,and increase serum TSH (Hood et al., 1999a; Liu et al.,1995). Although the microsomal enzyme inducers 3-meth-ylcholanthrene (3MC) and Aroclor 1254 (PCB) induce T4

UDP-GT activity and decrease serum T4 concentrations,serum TSH is unaffected in these rats (Hood et al., 1999a;Liu et al., 1995). The thyroid tumor promoting effects ofPCN, 3MC, and PCB have not been reported.

The effects of microsomal enzyme inducers on T4 glu-curonidation, serum T4 concentration, serum TSH, thyroidgland growth, and the promotion of thyroid tumors has beenstudied exclusively in rats. However, in addition to rats,mice are extensively used in carcinogenic bioassays. There-fore, information on the effects of microsomal enzyme in-ducers on thyroid hormone homeostasis and on thyroidfunction in mice is important to thoroughly understandwhether the mechanism of thyroid hormone disruption bymicrosomal enzyme inducers is species-specific, or whetherthe mechanism may someday be extrapolated to humans.Therefore, this study tested the hypothesis that microsomalenzyme inducers produce similar thyroid hormone disrupt-ing effects (i.e., increased T4 UDP-GT activity, reducedserum T4 concentration, increased serum TSH concentra-tions, and stimulation of thyroid gland growth) in mice.

Materials and methods

Materials. UDP-glucuronic acid, Brij-58, saccharic acid-1-4-lactone, 3MC, protease, and contrast green were pur-chased from Sigma Chemical Co. (St. Louis, MO). Total T4

and total T3 radioimmunoassay kits were obtained fromDiagnostic Products Corp. (Los Angeles, CA). PB wasobtained from Spectrum Chemical Mfg. Corp. (Gardena,CA). Polychlorinated biphenyl (Aroclor 1254) was pro-

vided by Dr. Steve Aust (Utah State University, Logan,UT). PCN was synthesized from 16- dehydropregnenolone,obtained from Pfaltz and Bauer, Inc. (Waterbury, CT), asdescribed by Sonderfan and Parkinson (1988). The mono-clonal antibody to BrdU was obtained from Becton–Dick-inson (Mountain View, CA). Enhanced black buffer solu-tion, DAB solution (3,3-diaminobenzidine), and peroxidesolution were purchased from Kirkegaard and Perry Labo-ratories, Inc. (Gaithersburg, MD). Serum TSH levels wereassayed by materials provided by the National Hormone andPituitary Program of NIDDK (Torrance, CA). Normal horseserum, biotinylated anti-mouse IgG (rat absorbed antibody),and Avidin-biotin-peroxidase complex (ABC) was pur-chased from Vector Laboratories (Burlingame, CA). Adsor-bosil-Plus TLC plates were purchased from Alltech Asso-ciates, Inc. (Deerfield, IL). Thyroxine-L-[125I] was obtainedfrom NEN DuPont (Boston, MA).

Animals and treatments. B6C3F male mice (7 weeks) wereobtained from Charles River Breeding Laboratories, Inc.(Portage, MI). Mice were housed in polypropylene cagescontaining corn-cob bedding and maintained at 21–22°C ona 12-h light/dark cycle. Food and tap water were providedad libitum. Mice were divided into 17 groups of six to eightmice. PB was dissolved in methanol, whereas PCN, 3MC,and PCB were dissolved in acetone. Each treatment wasadded to 1 kg of Purina Rodent Laboratory Chow 5001(iodine content of 0.7 ppm), mixed thoroughly, and allowedto dry. Control mice were fed a normal rodent chow, whilethe remaining 16 groups were fed rodent chow mixed withPB (600, 1200, 1800, or 2400 ppm), PCN (at 250, 500,1000, or 2000 ppm), 3MC (at 62.5, 125, 250, or 500 ppm),or PCB (at 10, 30, 100, or 300 ppm) for 21 days. Thesedoses were based on our previous studies of feeding thesemicrosomal enzyme inducers to rats. Mice were monitoredby recording body weights and feed consumption.

Thyroid hormone and TSH assays. After 21 days on thevarious diets, mice were lightly anesthetized with ether and0.8 to 1.0 ml of blood was drawn from the orbital sinuscavity. After clotting at 4°C, serum was separated by cen-trifugation and stored at �80°C prior to total T4 and total T3

radioimmunoassays. Serum TSH levels were determined aspreviously described (Hood et al., 1999c).

Assessment of thyroid follicular cell proliferation. After themice had been on the various diets for 21 days, each mousereceived an ip injection of BrdU (100 mg/kg) dissolved insaline. Two hours later, thyroid glands were removed, fixedin 10% buffered formalin, and embedded in paraffin. Quan-tification of thyroid follicular cell proliferation was deter-mined as described previously (Hood et al., 1999a).

Hepatic microsome preparation. UDP-GT activity was de-termined in liver microsomes. Liver microsomes were pre-pared by homogenizing liver tissue in 4 vol of buffer con-

7A. Hood et al. / Toxicology and Applied Pharmacology 188 (2003) 6–13

taining 50 mM Tris–HCl and 150 mM potassium chloride.Homogenates were then centrifuged at 10,000g for 20 min.The supernatant was decanted into ultracentrifuge tubes andcentrifuged at 100,000g for 60 min. The cytosol was re-moved and 1 ml wash buffer (10 mM EDTA and 150 mMpotassium chloride) was added to the microsomal pellet.The microsomal pellet was resuspended in wash buffer andhomogenized. The homogenate was centrifuged again at100,000g for 60 min. The supernatant was removed and0.25 M sucrose was added to the microsomal pellet, ho-mogenized, and stored at �80°C. Protein in liver micro-some samples was determined using the bicinchoninic acidmethod (Smith et al., 1985).

Hepatic UDP-GT activity. T4 UDP-GT activity was deter-mined as described by Barter and Klaassen (1992). T4-Glucuronide was separated from free T4 by thin-layer chro-matography as described by Henry and Gasiewicz (1987)and quantified by gamma scintillation spectrometry.UDP-GT activities toward [14C]chloramphenicol (Youngand Lietman, 1978) and �[14C]naphthol (Bock et al., 1978)were determined as described.

Statistics. Differences between control and treated animalswere determined using ANOVA analysis followed by theDuncan’s multiple range post-hoc test. Significant differ-ences between treated and control groups (p � 0.05) areindicated by asterisks in the figures. Statistical analyseswere performed using STATISTICA 4.5, Statsoft Inc.(Tulsa, OK).

Results

Effect of microsomal enzyme inducers on liver weights

Liver weights were increased up to (i.e., maximum) 77,42, and 75% in mice treated with PB (2400 ppm), PCN(2000 ppm), or PCB (300 ppm), respectively (Fig. 1). Micetreated with 3MC (62.5 and 250 ppm) resulted in a slightincrease (10%) in liver weight. PB and 3MC treatments didnot produce clear dose-dependent increases in liver weight.

Effect of microsomal enzyme inducers onchloramphenicol, �-naphthol, and T4 UDP-GT activities

All four microsomal enzyme inducers increased chlor-amphenicol and �-naphthol UDP-GT activities (Fig. 2).Chloramphenicol UDP-GT activity (top) was increased upto 50, 45, 50, and 40% in mice treated with PB (600 and2400 ppm), PCN (2000 ppm), 3MC (125 ppm), or PCB (300ppm), respectively. �-Naphthol UDP-GT activity (bottom)was increased up to 130, 80, 75, and 120% in mice treatedwith PB (2400 ppm), PCN (2000 ppm), 3MC (125 ppm), orPCB (300 ppm), respectively. 3MC treatment did not pro-duce a clear dose-dependent increase in chloramphicol or�-naphthol UDP-GT activities.

All four microsomal enzyme inducer treatments in-creased T4 UDP-GT activity (Fig. 3). T4 UDP-GT activitywas increased up to 114, 86, 107, and 105% in mice treatedwith PB (2400 ppm), PCN (1000 ppm), 3MC (500 ppm),and PCB (100 ppm), respectively.

Fig. 1. Effect of microsomal enzyme inducers on liver weight. Liver weight (g/kg body wt) was determined in mice fed either a basal diet or a diet containingPB (600, 1200, 1800, or 2400 ppm), PCN (250, 500, 1000, or 2000 ppm), 3MC (62.5, 125, 250, or 500 ppm), or PCB (10, 30, 100, or 300 ppm) for 21 days.The hashed bar represents the mean (�SE) liver weight in control mice. Each point represents the mean � SE of 6–8 mice. *Significantly different fromcontrol (p � 0.05).

8 A. Hood et al. / Toxicology and Applied Pharmacology 188 (2003) 6–13

Effect of microsomal enzyme inducers on serum totalT4 and T3

Serum total T4 (TT4) concentrations were reduced inmice treated with PB or PCB. PCB (300 ppm) treatment hadthe greater effect, reducing serum T4 concentrations asmuch as 62%, as compared to the 49% reduction in PB(1800 ppm)-treated mice (Fig. 4, top). In contrast to PB andPCB, serum TT4 concentrations were increased in PCN-(41% at 500 ppm) and 3MC (36% at 125 ppm)-treated mice.

Serum total T3 (TT3) concentrations were not affected byPB treatment, and only one dose (250 ppm) of PCN pro-duced a slight reduction (17%; Fig. 4, bottom). Both the 30and 100 ppm doses of PCB reduced serum TT3 concentra-tions approximately 25%, but the highest dose (300 ppm) ofPCB had no effect. 3MC treatment reduced serum TT3

concentrations, with the highest dose (500 ppm) having thegreatest effect (34%).

Effect of microsomal enzyme inducers on serum TSH andthyroid follicular cell proliferation

All four microsomal enzyme inducer treatments in-creased serum TSH above control mice (Fig. 5, top). PB

(1800 ppm) and PCB (100 ppm) treatments increased serumTSH approximately 25%. Compared to PB and PCB, PCNand 3MC treatments were more effective at increasing se-rum TSH concentrations. Serum TSH concentrations wereincreased up to 110 and 100% in mice treated with PCN(2000 ppm) or 3MC (62.5 ppm), respectively. The lowestdose of 3MC (62.5 ppm) produced the largest increase inserum TSH, which dose-dependently decreased.

The thyroid labeling index (LI) for control mice givenBrdU was 2.0 � 0.31 (Fig. 5, bottom). The LI was increasedup to 450 and 550% by PB (1800 and 2400 ppm) and PCN(1000 ppm) treatments, respectively. In mice treated with3MC, 250 ppm increased LI 450% and was unchanged atthe other doses. Compared to the other three treatments,PCB treatment produced the largest increase (900% at 300ppm) in the LI.

Discussion

The effects of PB, PCN, 3MC, and PCB on thyroidhormone homeostasis has been extensively studied in rats.Previous studies have shown that all four of these microso-mal enzyme inducers increase T4 UDP-GT activity, as wellas reduce serum T4 concentrations in rats (Hood and Klaas-sen, 2000; Liu et al., 1995). Furthermore, two microsomalenzyme inducers (PB and PCN) increase serum TSH con-centrations and stimulate thyroid gland growth in rats (Hoodand Klaassen, 2000). In comparison to rats, only a fewstudies have reported the effects of microsomal enzymeinducers on thyroid hormone homeostasis in mice (Book-staff et al., 1996; Viollon-Abadie et al., 1999). Bookstaff etal. (1996) reported reductions in serum T4 concentrationsand increases in serum TSH in mice treated with PB, but didnot examine T4 UDP-GT activity or alterations in thyroid

Fig. 3. Effect of microsomal enzyme inducers on T4 UDP-GT activity.UDP-GT activity toward T4 is expressed as picomoles T4-G/mg protein/min (top). Each value represents the mean � SE of 6–8 mice. *Signifi-cantly different from control (p � 0.05).

Fig. 2. Effect of microsomal enzyme inducers on chloramphenicol and �-naphthol UDP-GT activities. Chloramphenicol (top) and �-naphthol (bot-tom) UDP-GT activities are expressed as nmol/mg protein/min. Each valuerepresents the mean � SE of 6–8 mice. *Significantly different fromcontrol (p � 0.05).


gland function or growth. Viollon-Abadie et al. (1999) re-ported that microsomal enzyme inducer treatment did notaffect T4 UDP-GT activity, serum TSH, or thyroid glandfunction in mice. Therefore, additional studies investigatingthe effects of microsomal enzyme inducers on thyroid hor-mone homeostasis, and thyroid gland growth in mice waswarranted. In the present study, the hypothesis was testedthat microsomal enzyme inducers disrupt thyroid hormonehomeostasis by inducing T4 UDP-GT activity, resulting inreduced serum T4 concentrations, increased serum TSH,and stimulation of thyroid gland growth in mice.

In the present study, PB, PCN, 3MC, and PCB affectedthe liver consistently with enzyme induction. PB, PCN, andPCB treatments increased liver weight (Fig. 1). Although3MC treatment was the least effective at increasing liverweight, this microsomal enzyme inducer was effective atinducing UDP-GT activity. Chloramphenicol and �- naph-thol UDP-GT activities were induced in the present study byall four microsomal enzyme inducer treatments (Fig. 2).Chloramphenicol and �-naphthol UDP-GT activities were

measured in the present study because it has been shownthat PB and PCN preferentially induces chloramphenicolUDP-GT activity, whereas 3MC and PCB preferentiallyinduces �-naphthol UDP-GT activity in rats (Barter andKlaassen, 1994). Because all four microsomal enzyme in-ducers increased chloramphenicol and �-naphthol UDP-GTactivities in mice, the results from the present study suggestthat these microsomal enzyme inducers do not induce spe-cific UDP-GT isozymes in mice.

Not only do PB, PCN, 3MC, and PCB induce chloram-phenicol and �-naphthol UDP- GT activities, but they alsoinduce T4 UDP-GT activity in mice (Fig. 3), suggesting thatthyroid hormone homeostasis may be disrupted in this spe-cies. This result is consistent with the thyroid hormonedisrupting effects of these microsomal enzyme inducers inthe rat (Hood and Klaassen, 2000; Liu et al., 1995). How-ever, unlike the rat, there is a poor correlation betweeninduction of T4 UDP-GT activity and reductions in serumT4 concentrations in mice, because serum T4 concentrationswere reduced in PB and PCB treated mice, but were in-

Fig. 4. Effect of microsomal enzyme inducers on serum total T4 and T3 concentrations. Serum total T4 (top) and total T3 (bottom) were determined in micefed either a basal diet or a diet containing PB (600, 1200, 1800, or 2400 ppm), PCN (250, 500, 1000, or 2000 ppm), 3MC (62.5, 125, 250, or 500 ppm),or PCB (10, 30, 100, or 300 ppm) for 21 days. Serum total T4 and total T3 concentrations are expressed as �g/dl and ng/dl, respectively. The hashed barrepresents the mean (�SE) serum total T4 and T3 concentrations in control mice. Each point represents the mean � SE of 6–8 mice. *Significantly differentfrom control (p � 0.05).


creased in PCN- and 3MC-treated rats (Fig. 4). A likelyexplanation for the increase in serum T4 concentrations inPCN- and 3MC-treated mice is stimulation of thyroidal T4

synthesis by TSH (Rapoport and Spaulding, 1996), becauseserum TSH concentration was increased in PCN- and 3MC-treated mice (Fig. 5, top). The increased TSH in PB- andPCB-treated mice (Fig. 5, top) probably was not largeenough to restore the deficit of serum T4 observed in thesemice. These findings suggest that the feedback mechanismis more efficient at restoring serum T4 concentrations inmice than in rats, because reductions in serum T4 are ob-served in rats despite increases in serum TSH. Alternatively,these microsomal enzyme inducers may directly impair thy-roid and/or pituitary function in rats, but not mice. OnlyPCB has been reported to affect thyroid morphology in rats(Collins et al., 1977). However, we have found that thedoses of 3MC or PCB used in our studies have no directadverse effect on thyroid gland growth or morphology inrats (Hood et al., 1999a). Therefore, it is unclear whetherthese microsomal enzyme inducers directly compromise

pituitary and/or thyroid function and warrant further inves-tigation.

In comparison to serum T4 concentrations, serum T3

concentrations were less affected by microsomal enzymeinducer treatment (Fig. 4, bottom), which is consistent withthe effects of these microsomal enzyme inducers on serumT3 concentrations in the rat (Hood et al., 1999a; Liu et al.,1995). This finding suggests that mice, in addition to rats,have a strong ability to maintain serum T3 concentrations.The reason that serum T3 concentrations were less affectedby microsomal enzyme inducer treatment than serum T4 inmice might be due to activation of homeostatic mechanismsthat maintain serum T3 concentrations, such as increasedsynthesis of T3 by the thyroid gland due to TSH stimulation,increased T3 synthesis in extrathyroidal tissues by outer-ring deiodination, recovery of T3 from T3-SO4 by sulfatases,and/or increased enterohepatic circulation (de Herder et al.,1989; Kohrle et al., 1991; Rutgers et al., 1989a; Visser,1988; Visser et al., 1988).

A consequence of maintaining normal concentrations of

Fig. 5. Effect of microsomal enzyme inducers on serum TSH concentrations and thyroid follicular cell proliferation. Serum TSH (top) and thyroid follicularcell proliferation, reported as a labeling index (bottom), were determined in mice fed either a basal diet or a diet containing PB (600, 1200, 1800, or 2400ppm), PCN (250, 500, 1000, or 2000 ppm), 3MC (62.5, 125, 250, or 500 ppm), or PCB (10, 30, 100, or 300 ppm) for 21 days. Serum TSH concentrationand Ll are expressed as ng/ml and as the number of labeled nuclei per 1000 nuclei, respectively. The hashed bar represents the mean (�SE) serum TSHconcentration and mean (�SE) Ll in control mice. Each point represents the mean � SE of 6–8 mice. *Significantly different from control (p � 0.05).


serum thyroid hormones is the inability to use serum thyroidhormone concentrations as predictive measures of thyroidhormone disruption by microsomal enzyme inducer treat-ment. However, serum TSH was increased in mice treatedwith the four microsomal enzyme inducers (Fig. 5, top),suggesting that thyroid hormone homeostasis was disruptedin the present study. Therefore, the data from the presentstudy suggest that serum TSH concentrations, rather thanserum thyroid hormone concentrations; are a more usefulparameter for evaluating the thyroid hormone disruptingeffects of microsomal enzyme inducers. Furthermore, mi-crosomal enzyme inducers that increased serum TSH inmice also increased thyroid follicular cell proliferation (Fig.5, bottom), confirming that serum TSH concentrations wereincreased in the present study, and that thyroid gland growthwas affected by microsomal enzyme inducer treatment inmice. This finding is consistent with the effects of micro-somal enzyme inducers that increase serum TSH on thyroidgland growth in rats (Hood et al., 1999a).

Thyroid follicular cell proliferation is known to be sen-sitive to TSH stimulation (i.e., small increases in serumTSH results in large increases in proliferation) in rats treatedwith microsomal enzyme inducers or with antithyroid drugs(Hood et al., 1999a, 1999c; Wynford-Thomas et al., 1982b).Thyroid follicular cell proliferation also appears to be sen-sitive to TSH stimulation in mice, because thyroid follicularcell proliferation was increased many times greater (450 to900%) than serum TSH (25 to 110%; Fig. 5). Althoughthere does not appear to be a quantitative correlation be-tween serum TSH and thyroid follicular cell proliferation inmice treated with microsomal enzyme inducers for 21 days,the correlation between serum TSH and thyroid follicularcell proliferation is also poor in rats treated with these samemicrosomal enzyme inducers for the same treatment period(Hood et al., 1999b). The poor correlation between serumTSH and thyroid follicular cell proliferation may be due todesensitization of thyroid follicular cells to constant TSHstimulation, which has been shown to occur in rats (Wyn-ford-Thomas et al., 1982a). We have shown that thyroidfollicular cell proliferation peaks after 7 days of treatmentwith microsomal enzyme inducers that increase serum TSHin rats and then declines to control levels (Hood et al.,1999b). Similar time-course studies need to be done in miceto confirm whether desensitization of thyroid follicular cellsto TSH stimulation occurs in this species.

In conclusion, the microsomal enzyme inducers PB,PCN, 3MC, and PCB induce T4 UDP-GT activity, increaseserum TSH, and increase thyroid follicular cell proliferationin mice, suggesting that these microsomal enzyme inducersdisrupt thyroid hormone homeostasis in a manner similar torats. Also, serum thyroid hormone concentrations may notbe a reliable indicator of thyroid hormone disruption inmice. The findings that microsomal enzyme inducers in-crease serum TSH and thyroid gland growth in mice suggestthat microsomal enzyme inducers, which increase serumTSH, may promote thyroid tumors in mice, as well as in

rats. There is some evidence supporting the finding thatmicrosomal enzyme inducers promote thyroid tumors inmice, because PB has been reported to promote thyroidtumors in mice (Diwan et al., 1989); however, the thyroidtumor promoting effect of other microsomal enzyme induc-ers that increase serum TSH needs to be investigated.

Acknowledgments

This work was supported by NIH Grant ES08156. A.H.and M.L.A. were supported by NIH Training GrantES07079.

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