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Endocrinology Vol. 144, No. 5 2075-2083
Copyright © 2003 by The Endocrine Society

Rat Thyroid Hyperplasia Induced by Gestational and Lactational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

N. Nishimura, J. Yonemoto, Y. Miyabara, M. Sato and C. Tohyama

Environmental Health Sciences Division (N.N., Y.M., M.S., C.T.), National Institute for Environmental Studies, Tsukuba 305-8506, Japan; Endocrine Disruptors and Dioxin Research Project (J.Y., Y.M., C.T.), National Institute for Environmental Studies, Tsukuba 305-8506, Japan; Core Research for Evolutional Science and Technology (J.Y., Y.M., C.T.), Japan Science and Technology, Kawaguchi 332-0012, Japan; and Institute of Clinical Medicine (M.S.), University of Tsukuba, Tsukuba 305-0006, Japan

Address all correspondence and requests for reprints to: Noriko Nishimura, Ph.D., Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba 305-8506, Japan. E-mail: nishimura.noriko{at}nies.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Effects of gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thyroid function of offspring were investigated in the rat. Pregnant Holtzman rats, TCDD-sensitive strain, were given a single oral dose of 200 ng or 800 ng TCDD/kg on gestational day 15. Parameters related to the thyroid functions were examined on postnatal days (PNDs) 21 and 49. Serum T4 levels in offspring decreased significantly on PND21 in the two TCDD-exposed groups but increased on PND 49 only in the high-dose group. A dose of 800 ng TCDD/kg exerted a more than 2-fold increase in serum TSH level in male offspring on PNDs 21 and 49. A significant induction of uridine diphosphate-glucuronosyltransferase-1 gene by TCDD was observed on PND 21 but returned to basal levels on PND 49. Gene expression of cytochrome P4501A1 was markedly induced in the liver treated with TCDD. Even a single oral perinatal exposure to 800 ng TCDD/kg resulted in hyperplasia of the thyroid gland of offspring on PND 49. Proliferating cell nuclear antigen immunocytochemistry also supported this finding. Thus, gestational and lactational exposure to TCDD was found to disrupt thyroid hormone homeostasis, which results in a sustained excessive secretion of TSH, followed by the hyperplasia of thyroid follicular cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DIOXIN AND DIOXIN-LIKE polychlorinated biphenyls (PCBs) are ubiquitously present in the environment, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent isomer among the large family of dioxin and related compounds. In addition to reproductive, immunological, neurobehavioral toxicities, teratogenicity, and carcinogenicity, the alteration of thyroid hormone metabolism and function by TCDD has been published. Thyroid hormone is required for brain development, neuronal maintenance, and metabolic function during fetal and early neonatal periods (1). In adult rodents, exposure to TCDD resulted in morphological and functional alterations in the thyroid (2, 3, 4, 5). Morphologically, TCDD exposure induced an increase in the volume of thyroid follicular cells, followed by a hyperplasia (6) and, eventually, follicular thyroid tumor in male Osborne Mendel rats (7). Exposure to TCDD also causes hypothyroxinemia by elevated excretion of T4 into bile after the induction of uridine diphosphate (UDP)-glucuronosyltransferase-1 (UGT1). This is accompanied by an increase in TSH-positive cells in the anterior pituitary, followed by a foamy change in the colloid of some follicles, an indication of accelerating the biosynthesis of T4 in the thyroid (8). Thus, a TCDD-caused induction of UGT1, an enzyme catalyzing T4-glucuronide formation, results in the enhanced biliary excretion of T4, which results in a reduced serum T4 level (9, 10). It is established that reduction of circulating T4 levels can accelerate TSH release by the negative feedback mechanism in the pituitary-thyroid axis, which, in turn, leads to compensatory production of T4 in the thyroid gland.

In human populations, dioxin, dioxin-like PCBs, and other chemicals (particularly, non-dioxin-like PCBs), all of which are ubiquitous in the environment, may affect thyroid metabolism as well as neurobehavioral development. A Dutch cohort study suggested that maternal exposure to dioxin and/or PCBs, as represented as concentrations in breast milk, was associated with an increased level of TSH and a decreased level of T4 in the serum of 105 children but not always for T3 (11, 12). Furthermore, the follow-up of the Dutch cohort study (42-month-old infants) found an association between decreased cognitive ability and PCB concentrations in the maternal blood at birth and that attention and activity in these preschool children might be impaired by prenatal, as well as postnatal, PCB exposure (Ref. 13 ; no dioxin concentrations in the infants were available because approximately 100 ml of blood specimens are needed for dioxin determinations). It is important to perform a mechanistic study to examine the possible effects of dioxin and PCBs on thyroid morphology and function.

In a study on Long-Evans rats, approximately 0.05% of the administered dose to dams was transferred to a fetus (14). Administration of a mixture of PCBs, such as Aroclor 1254, to pregnant Long-Evans rats affected thyroid hormone levels of offspring, and postnatal T4 replacement was shown to attenuate hearing loss and motor deficit (15). Investigations on toxicological effects of gestational and lactational exposure to TCDD on thyroid of offspring are limited, however. By administering TCDD at a daily dose of 0.1 µg/kg to Sprague Dawley rats during gestational days (GDs) 10–16, Seo et al. (16) demonstrated a slight decrease in plasma T4 concentration but not in T3 and TSH concentrations on postnatal day (PND) 21; they concluded that the degree of T4 suppression observed was too small to be clinically significant. Regarding carcinogenicity of TCDD, oral administration of TCDD, twice a week for 2 yr, resulted in follicular cell adenomas in female B6CF1 mice and male and female Osborne-Mendel rats in a dose-related fashion (17).

Because not many other studies showed carcinogenicity, particularly in the thyroid, there seems a remarkable difference in susceptibility to TCDD exposure among strains and/or species. Regarding lethality, the guinea pig is the most sensitive, the rat and mouse have intermediate sensitivity, whereas the hamster is relatively tolerant (18). The effective dose to induce changes in thyroid hormone levels in hamsters is higher than that in rats (19). Weber et al. (20) examined differences in sensitivity to TCDD toxicity using the TCDD-susceptible (C57Bl/6J) and the TCDD-less sensitive (DBA/2) mouse strains, and showed that doses of TCDD required to affect thyroid hormone levels were about 1000 times lower in C57Bl/6J mice than in DBA/2 mice. In the rat, a marked difference in responsiveness to TCDD has been shown by using CTP1A1 as a marker gene, with the Sprague Dawley rats being the least sensitive strain, in contrast to the higher sensitivity of the Holtzman rat strain (21).

In the present study, we evaluated the gestational and lactational effects of TCDD on offspring, by administering a single oral dose of TCDD to pregnant Holtzman rats on GD 15, and examined, on PNDs 21 and 49, a variety of parameters related to thyroid functions as well as morphology. Unexpectedly, we have found that this dosing regimen produced hyperplasia in the thyroid of the TCDD-exposed male and female offspring.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and exposure
Male and female Holtzman rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and bred at the National Institute for Environmental Studies (NIES, Tsukuba, Japan). They were maintained in controlled room at 23 ± 1 C and humidity at 50 ± 10% on a 12-h light, 12-h dark cycle. The animals received food and distilled water ad libitum and were handled with humane care under the guidelines of the NIES. Ten-week-old female rats in proestrus were mated 1:1 with males overnight, and females that had a vaginal plug the following morning were designated as d 0 of gestation. Pregnant rats (six per group) were dosed by gavage with corn oil (vehicle) and 200 or 800 ng TCDD/kg on GD 15. On PND 2, litters were randomly culled to eight offspring, with five males and three females, when possible. On PNDs 21 or 49, tissues (including liver, pituitary glands, and thyroid glands with the trachea) were excised from offspring anesthetized lightly with diethyl ether. Tissues were fixed in Zanboni’s solution (22) for 24 h at 4 C and processed for immunohistological examinations. For biochemical examination, tissues were snap-frozen in liquid nitrogen and stored at -80 C until analysis.

Immunohistochemistry
T4 and proliferating cell nuclear antigen (PCNA) in the thyroid and TSH in the pituitary gland were stained in tissue sections by an indirect immunohistochemical (IHC) technique (23, 24). Briefly, the deparaffinized and rehydrated sections were pretreated in 0.01 M sodium citrate buffer, pH 6.0, with microwave heating and washed in PBS. To quench endogenous peroxidase activity, sections were covered with 0.3% H2O2 dissolved in 100% methanol. Rabbit antibody against T4 (RP 039; Diagnostic BioSystems, Pleasanton, CA) was diluted 1:200, and monoclonal mouse anti-PCNA (Clone PC 10, Code No. M 0879; DAKO Corp., Glostrup, Denmark) was diluted 1:300 or rabbit antibody against TSH (AB976; Chemicon International, Inc., Temecula, CA) was diluted 1:1000 in PBS. The primary antibodies were incubated over the sections in a humidified chamber for either 60 min at 25 C for T4 staining or 30 min at 37 C for TSH and PCNA immunostaining. Sections were subsequently washed with PBS, followed by incubation for 1 h with biotinylated goat antirabbit IgG (BA-1000; Vector Laboratories, Inc., Burlingame, CA) or antimouse IgG (BA-9200) that were diluted 1:200 in PBS. They were incubated with an avidin-biotinylated peroxidase complex (PK-4000; Vector Laboratories, Inc.). Immunoreactions were performed by using hydrogen-peroxide-activated 3,3'-diaminobenzidine-tetrahydrochloride (Sigma, St. Louis, MO). Sections were counterstained for 10 sec in Mayer’s hematoxylin. Negative controls, in which the primary antibody was replaced with normal rabbit IgG, did not show nonspecific staining. PCNA-positive cells were quantified by counting the number of cells per low-power field for three fields, by the use of an Image Processing and Analysis System (Version 2.2A; Leica Corp., Qwin, Cambridge, UK). The degree of thyroid alteration was also quantified by applying the system to hematoxylin and eosin-stained thyroid tissue sections. The follicular lumenal, parenchymal, and total areas were quantified, and the values reported for each animal represent means of three random fields per each tissue slice.

Thyroid hormone analyses
Serum total T4 and total T3 levels were determined with Amerlex RIA kits (Amersham International, Buckinghamshire, UK), according to the manufacturer’s instructions (6). Serum TSH levels were determined with a rat TSH enzyme immunoassay kit (Amersham International).

RNA extraction and RT-PCR
Total hepatic RNA was extracted by using Isogen (Nippon Gene, Tokyo, Japan). Enzymes and cofactors for reverse transcription (RT) and PCR were purchased from Takara (Otsu, Japan). All the primers used in the present study were purchased from Amersham Pharmacia Biotechnology (Piscataway, NJ). Sequences of PCR primers for amplification of cytochrome P4501A1, UGT1 (the common region of UGT1 isoforms), and ß-actin were the same as reported earlier (25). RT of RNA was performed in a final vol of 20 µl solution containing 5 mM MgCl2, 1 mM deoxynucleotide triphosphate, 0.25 U/µl avian myeloblastoma virus reverse transcriptase, 0.125 µM oligo dT-adaptor primer, 1 U/µl ribonuclease inhibitor, and 1 µg total RNA, using the RNA LA PCR kit (Takara). The RT samples were incubated at 42 C for 15 min and then at 99 C for 5 min for inactivation of reverse transcriptase. PCR was subsequently performed as follows: reaction mixture was incubated at 94 C for 4 min and then amplified at 94 C for 30 sec, 56 C for 30 sec, and 72 C for 1 min. Reactions were repeated for 21 cycles for UGT1 and 22 cycles for ß actin and cytochrome P4501A1 (CYP1A1). The PCR mixture (10 µl) contained 2.5 mM MgCl2, 0.25 U Takara LATaq, 0.2 µM each forward and reverse primer, and 2 µl RT products. PCR products were detected as a single band on 1.5% agarose gel in 1x Tris-borate EDTA containing 2 µg/ml ethidium bromide. Band intensity was quantified by the EDAS120 system (version 2.02; Eastman Kodak Co., Rochester, NY).

TCDD analysis
TCDD in tissue specimens (serum, liver, and adipose tissues) was determined by essentially the same method as described earlier (23). In brief, samples collected and pooled from five rats were weighed and spiked with 13C-2,3,7,8-TCDD (Wellington, Ontario, Canada) as the internal standard. The tissue was digested and washed, and TCDD was extracted with n-hexane. The n-hexane layer was cleaned by concentrated sulfuric acid, rinsed with water, and dried. The solution was concentrated and cleaned further by silica gel (Kieselgel 60; Merck KGaA, Darmstadt, Germany) and activated carbon-silica gel (active carbon impregnated-silica gel; Wako Pure Chemical Industries Ltd., Osaka, Japan) column chromatography.

The gas chromatograph/mass spectrometer (GC/MS) analysis was performed in the selected ion mode on a JMS700 high-performance double-focusing mass spectrometer (JEOL Inc., Tokyo, Japan) coupled to an HP 6890 gas chromatograph (Hewlett-Packard Co., Wilmington, DE). Sample solution was introduced into an HP 6890 equipped with a CP-SIL 8CB/MS column (Chrompack, EA Middelburg, The Netherlands; 30 m x 0.25 mm inside diameter, film thickness 0.25 µm). Identification was based on the correct isotope ratio of M+ to (M + 2)+ (±15%), recoveries (50–120%), and retention time (±4.0 sec) of the GC separation. The area of mass profile peaks of the quantification ions was used for the quantitative analysis of TCDD. Quantified values were calculated by the internal standard methods.

Statistical analysis
StatView for Windows (version 5.0; SAS Institute, Inc., Cary, NC) was used for statistical analysis. Data are expressed as mean ± SEM. Differences in means among the three groups were analyzed by one-way ANOVA followed by Scheffé’s test as post hoc comparison. Comparisons of mean values between males and females were performed by Student’s t test. P values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reproductive and developmental outcomes
No signs of toxicity were observed in dams treated with TCDD at a dose of 200 ng/kg (low-dose group), whereas a significant decrease in litter size was observed in the 800-ng TCDD/kg (high-dose group; Table 1Go). TCDD treatment did not cause statistically significant changes in sex ratios and birth weights of offspring. Body weight, brain weight, and thyroid (right lobe) weight of the offspring at the time of weaning were not affected by the treatment (data not shown).


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  		Table 1. Reproductive outcomes of dams exposed to TCDD on GD15

 
Thyroid hormone concentrations
Administration of TCDD to dams on GD 15 decreased (in a dose-dependent manner) total T4 concentrations in the serum from both male and female offspring on PND 21 (Fig. 1AGo). In particular, the T4 concentrations were significantly lower in male offspring in both the low- and high-dose groups than those in vehicle-treated offspring. Conversely, on PND 49, the serum T4 concentration of male offspring in the high-dose group was significantly increased (Fig. 1AGo). A similar trend was observed in the female rats in the high-dose group, although the increase was not statistically significant. Serum T3 concentration was significantly increased on PND 21 in female offspring in the high-dose group, compared with the vehicle-treated control, but no statistical difference was observed on PND 49 (Fig. 1BGo).



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  		Figure 1. Effects of gestational and lactational exposure to TCDD on serum levels of T4 (A) and T3 (B) on PNDs 21 and 49. Values are mean ± SEM (n = 4–6). *, Significantly different from vehicle-treated controls (P < 0.05).

 
This elevated level of biosynthesis of T4 in response to TCDD was confirmed immunohistochemically in the offspring thyroid gland. On PND 49, the marked induction of T4 was clearly shown in the male thyroid glands of the high-dose group. The staining intensity for T4 was stronger in the colloid lumina of follicles in male and female TCDD-treated offspring, compared with the vehicle-treated controls (Fig. 2Go, A–D). The cytoplasm of most of the follicular cells was negative at this time. However, no significant change in the localization pattern for T4 was found in male and female thyroid glands on PND 21 (data not shown). In these offspring, T4 was positively stained in the cytoplasm of follicle epithelial cells and weakly and homogeneously in the colloid lumina of follicles. Because the localization of T4 is very similar to that of thyroglobulin (22), the detection of T4 immunoreactivity found in the present study is thought to reflect the localization of both non-Tg-bound and Tg-bound T4.



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  		Figure 2. IHC detection of T4 in thyroid glands of male offspring, on PND 49, after gestational and lactational exposure to TCDD. A, Thyroid gland from a vehicle-treated offspring. The follicles with or without immunostaining for T4 were dispersed in the thyroid gland. B, High magnification of A. T4 localized in the follicular colloids but not in the follicular epithelial cells. C, Thyroid gland from offspring after gestational and lactational exposure to 800 ng TCDD/kg. An increased intensity of immunostaining in the thyroid follicles was noted. Intensely stained thyroid follicles were dispersed in thyroid gland, which was characterized by smaller size and an increase in total number of follicles, compared with the vehicle-treated control. D, High magnification of C. Strong T4 immunostaining was observed mainly in the follicular colloid. Bar, 50 µm.

 
The serum TSH concentrations in male and female offspring were significantly increased in the high-dose group, in comparison with the vehicle-treated control, and remained elevated until PND 49 (Fig. 3Go). The IHC staining for TSH confirmed the induced biosynthesis of TSH by TCDD in the anterior pituitary gland of male and female offspring. The staining intensity and the number of positive cells for TSH were increased in the anterior pituitary of TCDD-exposed offspring on PND 21 (Fig. 4BGo) and PND 49 (Fig. 4DGo).



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  		Figure 3. Effects of gestational and lactational exposure to TCDD on serum levels of TSH on PNDs 21 and 49. Values are mean ± SEM (n = 4–6). *, Significantly different from vehicle-treated controls (P < 0.05); a, significantly different from female rat (Student’s t test, P < 0.05).

 


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  		Figure 4. IHC detection of TSH in the pituitary gland of male offspring after gestational and lactational exposure to 800 ng TCDD/kg. Note an increased intensity of immunostaining and the number of positive cells for TSH in the anterior pituitary of the TCDD-treated rat, compared with the vehicle-treated control. A, Pituitary gland from a vehicle-treated offspring on PND 21. B, Pituitary gland from a TCDD-treated offspring on PND 21. C, Pituitary gland from a vehicle-treated offspring on PND 49. D, Pituitary gland from a TCDD-treated offspring on PND 49. Bar, 50 µm.

 
UGT1 and CYP1A1 mRNA in the liver
UGT1 and CYP1A1 mRNA levels in the liver of offspring were quantified by RT-PCR (Fig. 5Go). On PND 21, a significant increase in the UGT1 mRNA level was observed in male and female offspring in the low- and high-dose groups (P < 0.05), but differences were not longer apparent on PND 49 (Fig. 5CGo). However, a marked induction of CYP1A1, attributable to TCDD, was found in the liver of male and female offspring on both PNDs 21 and 49 (Fig. 5BGo).



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  		Figure 5. Dose-response of CYP1A1 (A and B) and UGT1 (A and C) mRNA expression in livers of male and female offspring, on PNDs 21 and 49, after perinatal exposure to TCDD, by RT-PCR analysis. ß-actin (A, bottom panel) was used to check for equal loading of mRNA. Values are mean ± SEM (n = 4–6). *, Significantly different from vehicle-treated controls (P < 0.05).

 
TCDD concentrations in the tissues
TCDD concentrations in male offspring tissues are summarized in Table 2Go. We analyzed TCDD concentrations in a single specimen for each dose group and for each time point by combining tissue specimens from six rats, because dioxin determination by high-resolution GC-MS was extremely laborious and costly. Consistent with an earlier study (16), large amounts of TCDD were found in the offspring liver and adipose tissue on PND 21. The tissue TCDD concentration had decreased markedly by PND 49, for example, to almost 2% of that of the liver on PND 21. The TCDD concentration was reversed between the liver and adipose tissue, particularly in the high-dose group, on PND 49.


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  		Table 2. Distribution of TCDD in liver, adipose, and serum of offspring, on PNDs 21 and 49, after gestational and lactational exposure to TCDD

 
Thyroid histology
From hematoxylin and eosin staining, maternal exposure to 800 ng TCDD/kg (high-dose group) resulted in the diffuse hyperplasia of follicular cells in the male rat thyroid on PND49 (Fig. 6CGo), in contrast to no apparent changes in vehicle-treated rat thyroid (Fig. 6Go, A and B). A high-power view of thyroid tissues (Fig. 6DGo) demonstrated that the TCDD-exposed thyroid exhibited hyperplasia, characterized by small follicles and tall columnar epithelium having a slightly larger nucleus. In addition to hyperplasia in the follicular epithelium, TCDD exposure produced an increase in the number of abnormally shaped follicles, as well as fibroplastic lesions in the interstitial connective tissue.



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  		Figure 6. Histological comparison of offspring thyroid glands, on PND 49, after gestational and lactational exposure to TCDD. A, Thyroid gland from a male vehicle-treated offspring. B, High magnification of A. C, Thyroid gland from a male offspring after gestational and lactational exposure to 800 ng TCDD/kg. Note the increase in the number of small follicles, compared with A. D, High magnification of C, showing small follicles and abnormally shaped follicles as well as fibroplastic lesion in interstitial connective tissue. E, Thyroid gland from a female vehicle-treated offspring. F, Thyroid gland from a female TCDD-treated offspring. Note the many small follicles in thyroid glands, but hyperplastic capability was not as prominent in females as in males. Sections were stained with hematoxylin and eosin. Bar, 100 µm for A, C, E, and F; 50 µm for B and D. G, Quantification of hyperplastic changes in thyroid gland by measuring the ratio of parenchymal area to thyroid lumenal area. Values are mean ± SEM (n = 4–6). *, Significantly different from vehicle-treated controls (P < 0.05); a, significantly different from female rat (Student’s t test, P < 0.05).

 
The thyroid of 49-d-old female offspring responded to TCDD in a slightly different manner than that in male offspring (Fig. 6Go, E and F). The hyperplasia in the female offspring was not as prominent (Fig. 6Go, E and F), although many small follicles are similarly observed. Hyperplastic areas, quantitatively analyzed in terms of the ratio of parenchymal area to follicular area, showed that the ratio was significantly increased in both male and female offspring in the high-dose group (Fig. 6GGo). In addition, the area ratio (parenchymal/luminal) of thyroid was significantly higher in male offspring than female offspring.

PCNA immunocytochemistry was performed to assess the proliferative action of TCDD in the thyroid gland of 49-d-old male offspring. Follicular cells that had PCNA-labeled nuclei were found in vehicle-treated control rat thyroid (Fig. 7AGo), whereas a drastic increase in number of PCNA-positive cells was found in the offspring thyroid from the high-dose group (Fig. 7BGo), indicating an ability of TCDD to induce cell proliferation. When the ratio of PCNA-positive cells to total number of follicular cells was calculated and compared between TCDD-treated offspring and vehicle-treated control offspring on PND 49, we found a significant increase in the ratio in the thyroid from both male and female offspring in the high-dose group (Fig. 7CGo). There was a tendency for a much higher frequency of PCNA-labeled nuclei to be observed in males than in females (P < 0.05).



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  		Figure 7. PCNA staining in thyroid glands from male offspring, on PND 49, after gestational and lactational exposure to 800 ng TCDD/kg. A, Thyroid gland from a vehicle-treated offspring. B, Thyroid gland from a TCDD-treated offspring. Note the dramatic increase in the number of PCNA-positive cells in the thyroid gland. C, The ratio of PCNA-positive cells to total number of thyroid follicular cells. *, Significantly different from vehicle-treated controls (P < 0.05); a, significantly different from female rat (Student’s t test, P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The most remarkable, but unexpected, finding in the present study is that even a single oral administration of TCDD to pregnant rats was sufficient to produce a hyperplastic alteration in the thyroid of both male and female offspring on PND49. The hyperplasia, which was confirmed by morphometric analysis (Fig. 6GGo), was characterized by small follicles with cubical and columnar epithelium with an elevated number of PCNAs (Fig. 7Go), which was also observed in adult animals exposed to TCDD and many goitrogenic xenobiotics. For example, Sewall et al. (6) found similar morphological changes in the adult rat thyroid after a 30-wk continuous treatment with TCDD; they suggested that prolonged stimulation of the thyroid by TSH is responsible for these changes. Although no thyroid neoplasia or nodules were observed after TCDD treatment in the present study, the diffuse thyroid hyperplasia induced by continued stimulation by TSH might progress to nodules and eventually neoplasia (see review in Ref. 26). These data, as well as the present observations, support the notion that long-term perturbations of the pituitary-thyroid axis by xenobiotics or physiological alterations are likely to predispose rats to a higher incidence of proliferative lesions (27).

We found that perinatal exposure to TCDD caused a marked decrease in the circulating T4 level at weaning (PND 21), in a dose-dependent manner (Fig. 1AGo), and serum TSH level was increased significantly on PNDs 21 and 49 in the high-dose group (Fig. 3Go). We also observed a significant correlation between induction of UGT1 gene expression and suppression in serum T4 levels. Although the elevated level of UGT1 mRNA did not last until PND 49 (Fig. 5Go), it is possible that a drastic decline of hepatic TCDD concentration, approximately 2% of the value in the rat liver on PND 21 (Table 2Go), is responsible for this phenomenon. This perturbation of thyroid hormone balance in the TCDD-exposed offspring is thought to be caused by the enhanced excretion of T4-glucuronide via induction of UGT1 in response to TCDD, as has been reported for TCDD-exposed adult animals (5). Because TCDD is reported to induce UGT1A6 (28), it is reasonable to speculate that UGT1A6 is primarily responsible for T4 glucuronidation in offspring after maternal TCDD exposure in the present study. Vansell et al. (29) also suggested that PCB (Aroclor 1254) enhanced T4 UGT activity by increased expression of UGT1A6. Recently, using an antibody against UGT1A6- or UGT1A1-specific peptide, we observed an induction of UGT1A6, but not UGT1A1, in the centrilobular region of the liver of TCDD-exposed rats (30). Among nine isoforms of UGT1A family, TCDD-induced UGT1A6 is thought to glucuronidate T4. In our recent study, we have found an elevation of UGT1A7 mRNA in the liver of rats perinatally exposed to TCDD (30), which may suggest that UGT1A7 might be responsible for glucuronidation of T4.

Circulating T3 concentrations were observed to be elevated in female offspring in the high-dose group on PND 21 but not on PND 49. Because a single dose of TCDD was reported to decrease in type I deiodinase activity in a dose-dependent manner in adult rats (31), it is not likely that type I deiodinase is associated with the above-mentioned increase in the serum T3 in the present study. Because sulfation is responsible for the elimination of T3 from liver to the bile (26), it is worth studying whether and how TCDD might affect this metabolic pathway.

Next, we addressed questions of whether or not this perturbation in the hypothalamus-pituitary-thyroid axis is mediated by an aryl hydrocarbon receptor (AhR)-dependent mechanism and which period of exposure (gestational or lactational) is more responsible for this perturbation. In a previous study (32), we administered a single oral dose of TCDD to pregnant AhR knock-out mice on GD 12. No induction of UGT1 gene expression by TCDD administration in offspring (PND 21) was observed, indicating that this induction was dependent on AhR. To clarify the critical exposure period in terms of perturbation of the thyroid homeostasis by TCDD, a cross-fostering study might answer the question. Earlier studies suggested that there may be a different critical window period for each endpoint (33, 34). Recently, we have found that lactational, rather than gestational, exposure to TCDD plays a crucial role in disrupting the thyroid hormone balance (30).

It has been established that circulating thyroid hormone levels, T3 and T4, are regulated by the negative feedback mechanism in the hypothalamus, which produces TRH, and in the pituitary, which secretes TSH, leading to the control of circulating T3 and T4 concentrations. Some explanations may be drawn from epidemiological and laboratory studies. Serum T3 and T4 levels were reported to be significantly higher in Yusho patients who had accidentally ingested rice oil that contained PCBs, polychlorinated dibenzofurans, and polychlorinated quaterphenyls; and several Yusho patients had elevated serum TSH levels (35). In a Dutch epidemiologic study (36), 11-wk-old infants who were breast-fed with a relatively higher background level of dioxin and PCBs in their mothers’ milk were found to have higher levels of serum total T4, T4/T4-binding globulin ratio, and TSH, which led to the conclusion that these compounds modulate the hypothalamic-pituitary-thyroid regulatory system in the newborns. The coincidentally induced levels of T4 and TSH in the rat, on TCDD exposure (Figs. 1Go and 3Go), were supported by the enhanced biosynthesis of TSH and T4 by TCDD in the target organs, pituitary and thyroid, respectively (Figs. 2Go and 4Go), and may be relevant to the above-mentioned observations in humans. An alternative explanation for coincidental increases in T4 and TSH levels would be that TCDD disrupts the negative feedback mechanism in the hypothalamic-pituitary-thyroid axis, which results in secretion of pituitary TSH independently of serum levels of thyroid hormones.

We found a sex difference in the degree of development of hyperplasia and an alteration of thyroid hormone status (Fig. 3Go). Male rats developed a higher degree of hyperplasia and a higher concentration of serum TSH than female rats in the high-dose group, suggesting that female offspring were not as sensitive to TCDD exposure as male offspring (Figs. 6Go and 7Go). A sex difference in thyroid tumorigenesis was reported by Hiasa et al. (37), in which promotion of thyroid tumors by phenobarbital was found to be more conspicuous in male rats than female rats. McClain et al. (38) reported that nitrosamine administration significantly induced thyroid tumors in male rats, compared with no tumors in female rats; they attributed this sex difference in tumor induction to the lower basal level of TSH in female rats, compared with male rats. A plausible explanation would be a difference in basal TSH concentrations between male and female rats in their study, but this explanation cannot be applied to the present study because no sex differences in the basal level of TSH were observed. It should be noted that, in a carcinogenicity study by the U.S. National Toxicology Program (17), Osborne-Mendel rats given gastric instillations of TCDD twice a week for 104 wk showed follicular-cell adenomas of the thyroid, both in males and females. Thus, no clear-cut explanation is possible for the sex difference in thyroid tumorigenesis observed in the present study.

The serum T4 concentrations after TCDD exposure in Holtzman rat offspring in the present study differed from the data from a previous report in which TCDD was orally administered to Sprague Dawley rats on GDs 10–16 (16), in which there was a slight decrease in plasma T4 concentrations in female offspring but not in male offspring. In addition, plasma T3 and TSH concentrations were not affected in any of the groups (16). A difference in dosing protocol, 0.025 or 0.10 µg/kg/d for 7 d, as well as a strain difference in sensitivity to TCDD, may (at least partly) explain this apparent inconsistency. The Holtzman strain is classified as a high responder, whereas the Sprague Dawley strain is considered to be a low responder, in terms of CYP1A1 induction (21, 39, 40).

The question arises as to whether and how the present experimental results are relevant to human findings. As mentioned earlier, our cross-fostering study showed that lactational (rather than gestational) exposure to TCDD played a crucial role in disrupting the thyroid hormone homeostasis in Holtzman rats (32). In the general population, breast-fed infants exposed to higher dioxin-TEQ levels had lower plasma T4 and higher TSH levels, at 2 wk after birth, in a Dutch cohort study (11); and dioxin-TEQ in breast milk was found negatively correlated with serum T4 level of infants in Japan (41). It has been reported that in industrialized countries, newborns were exposed to approximately 25-fold higher levels of dioxin-TEQ (for example, 112 vs. 4 pg TEQ/kg as tolerable daily intake) (42). Although the rat thyroid harbors transthyretin other than T4-binding globulin as a T4 carrier protein and is thought to be more susceptible than human thyroid in the development of hyperplasia caused by the shorter plasma half-life of T4 (43), it is still plausible to speculate that both human and rodent newborns are relatively at risk by being exposed to substantial amounts of dioxins via breast milk.

In conclusion, perinatal exposure to a low dose of TCDD was demonstrated to disrupt thyroid morphology and functions in offspring, leading to a hyperplasia of the thyroid gland, with a modulation of negative feedback mechanism through the hypothalamic-pituitary-thyroid axis. Whether this perturbation affects the development of brain functions warrants additional study.


   Acknowledgments
 
The authors thank Drs. S. Osako, H. Sone, and M. Ishizuka (NIES); Dr. H. Nishimura (Aichi Mizuho University); and Dr. V. E. Reeve (University of Sydney) for their useful discussions and comments. The authors are grateful to M. Ohmura and C. Yokoi for their excellent technical assistance.


   Footnotes
 
This work was supported, in part, by a Core Research for Evolutional Science and Technology grant from Japan Science and Technology Corporation (to C.T.), by the Science and Technology Agency (to N.N.), and by the Health and Labour Sciences Research Grant.

Abbreviations: AhR, Arly hydrocarbon receptor; CYP1A1, cytochrome P4501A1; GC/MS, gas chromatograph/mass spectrometer; GD, gestational day; IHC, immunohistochemical; NIES, National Institute for Environmental Studies; PCB, polychlorinated biphenyl; PCNA, proliferating cell nuclear antigen; PND, postnatal day; RT, reverse transcription; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; UDP, uridine diphoshate; UGT, UDP-glucuronosyltransferase-1.

Received July 22, 2002.

Accepted for publication January 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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