ToxSci Advance Access originally published online on January 4, 2006
Toxicological Sciences 2006 90(2):337-348; doi:10.1093/toxsci/kfj083
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Evaluation of Histological and Molecular Endpoints for Enhanced Detection of Thyroid System Disruption in Xenopus laevis Tadpoles
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* Department of Inland Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, D-12587 Berlin, Germany; Aquatic Ecology and Toxicology, Department of Zoology, University of Heidelberg, D-69120 Heidelberg, Germany; and Department of Endocrinology, Institute of Biology, Humboldt University Berlin, D-10115 Berlin, Germany
1 To whom correspondence should be addressed at Department of Inland Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Mueggelseedamm 310, D-12587 Berlin, Germany. Fax: +49 30 64181 799. E-mail: werner.kloas{at}igb-berlin.de.
Received August 24, 2005; accepted December 20, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Amphibian metamorphosis represents a promising model for the identification of thyroid system–disrupting chemicals due to the pivotal role played by thyroid hormones for the initiation and regulation of metamorphosis. An important aspect of bioassay development is the identification and evaluation of sensitive and diagnostic endpoints. In this study, several morphological, histological, and molecular endpoints were evaluated for their utility to detect alterations in thyroid system function after exposure of stage 51 Xenopus laevis tadpoles to various concentrations (1.0, 2.5, 10, 25, and 50 mg/l) of the anti-thyroidal compound ethylenethiourea (ETU). Analysis of developmental stages on exposure day 20 and monitoring of time to fore limb emergence (FLE) revealed retardation and complete arrest of tadpole development at 25 mg/l and 50 mg/l ETU, respectively. Development was not affected by 1.0, 2.5, and 10 mg/l ETU. Histological alterations in the thyroid gland were observed in FLE-displaying tadpoles after exposure to 2.5, 10, and 25 mg/l ETU, as well as in developmentally arrested tadpoles exposed to 50 mg/l ETU. Prevalence and severity of histological changes increased in a concentration-dependent manner. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) showed increased mRNA expression of the
- and ß-subunits of thyroid-stimulating hormone (TSH, TSHß) in pituitary tissue of tadpoles exposed to 25 and 50 mg/l ETU. Results demonstrate the successful detection of anti-thyroidal effects of ETU in Xenopus laevis tadpoles using various endpoints and highlight the particular sensitivity of thyroid gland histology to detect thyroid system disruption in tadpoles. Key Words: Xenopus laevis; amphibian metamorphosis; endocrine disruption; thyroid system; histology; RT-PCR.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies on the effects of endocrine-disrupting chemicals (EDC) have so far been predominantly concerned with alterations in sex steroid action and the consequences for reproduction and development in vertebrates (Colborn et al., 1993
; McLachlan, 2001). However, alterations of thyroid system function caused by EDC are also of concern because of the vital role played by thyroid hormones (TH) in the regulation of growth, development, metabolism, and reproduction (Brown et al., 2004; Brucker-Davis, 1998; Kloas, 2002). The complexity of the thyroid system provides multiple target sites for EDC to affect thyroid homeostasis and TH action (Capen, 1997; Zoeller, 2003). Mammalian studies have provided evidence that EDC-induced alterations of thyroid homeostasis can result from inhibition of thyroidal iodide uptake (Siglin et al., 2000), suppression of thyroid peroxidase activity (Yamasaki et al., 2002), displacement of TH from plasma transport proteins (Meerts et al., 2000), and upregulation of hepatic TH transformation/elimination (Hood et al., 2003). In addition, more recent data suggest that EDC interference with TH signaling events in peripheral tissues may represent another relevant pathway of thyroid disruption (Bogazzi et al., 2003; Yamada-Okabe et al., 2004). Accordingly, comprehensive testing approaches for detection of thyroid-disrupting chemicals should consider both changes in thyroid homeostasis and modulation of peripheral TH action.
The process of anuran postembryonic development (metamorphosis) provides a particularly promising biological model for the development of a bioassay to detect various modes of thyroid disruption (Kloas, 2002; Opitz et al., 2002). In anurans, the metamorphic development of tadpoles to juvenile froglets is dependent on TH (Shi, 1999). Treatment of tadpoles with antithyroidal compounds (e.g., perchlorate, methimazole) or synthetic thyroid hormone receptor antagonists (e.g., NH-3) leads to retardation or even complete blockade of metamorphic development (Goleman et al., 2002; Lim et al., 2002; Tietge et al., 2005). In turn, exposure of premetamorphic tadpoles to low nanomolar concentrations of thyroxine (T4), 3,5,3'-triiodothyronine (T3), or synthetic thyroid hormone receptor agonists (e.g., GC-1) causes precocious induction of metamorphosis (Furlow et al., 2004; Opitz et al., 2005c; Shi, 1999). Using tadpoles of the South African clawed frog Xenopus laevis as test organisms, we recently developed the Xenopus Metamorphosis Assay (XEMA) to detect thyroid-disrupting activities of EDC (Opitz et al., 2005a). During the initial evaluation of the XEMA test, monitoring of developmental rates was used as a surrogate measurement to assess the thyroidal status of the differentially treated tadpoles. In accordance with their known effect patterns on the thyroid system, T4 was found to accelerate tadpole development whereas mammalian anti-thyroidal compounds such as propylthiouracil (PTU) and ethylenethiourea (ETU) caused developmental retardation of X. laevis tadpoles (Opitz et al., 2005a). Although apical morphological endpoints such as developmental stage are easy to determine and provide an integrated measure of EDC effects on metamorphic development, the identification and evaluation of further endpoints that are more specific for changes in thyroid axis functions are required in order to confirm thyroid disruption in anuran tadpoles.
In mammalian studies, measurements of plasma concentrations of T4, T3, and thyroid-stimulating hormone (TSH), combined with histopathological and morphometric analyses of the thyroid gland, represent a common approach to detect anti-thyroidal effects of EDC (O'Connor et al., 1999; Yamasaki et al., 2002). Blood samples are difficult to obtain from the small tadpoles of X. laevis, thus limiting the ability to detect treatment-related changes in plasma levels of T4, T3, and TSH. Therefore, analysis of TH-dependent gene expression in peripheral tissues might provide an alternative means of assessing thyroidal status indirectly in X. laevis tadpoles, because expression of thyroid hormone receptor ß (TRß) mRNA, for example, is closely correlated with changes in circulating TH (Krain and Denver, 2004; Opitz et al., 2005c). In addition, determination of mRNA expression of the - and ß-subunits of TSH (TSH, TSHß) in pituitary tissue should provide a valuable endpoint from which to characterize pituitary compensatory activities involved in the maintenance of overall thyroid homeostasis (Huang et al., 2001). Thyroid gland histopathology is another classical approach to detecting anti-thyroidal activities of chemicals (Capen, 1997), and previous studies involving exposure of anuran tadpoles to anti-thyroidal compounds reported histopathological changes in the thyroid gland similar to those seen in mammals (Goos et al., 1968; Miranda et al., 1996, Tietge et al., 2005). However, studies in amphibians often used only a single high concentration treatment to produce hypothyroidism in tadpoles (Goos et al., 1968; Miranda et al., 1996), and very little is known about concentration-dependent changes in thyroid gland histology of anuran tadpoles.
In the present study we investigated the utility of various morphological, histological, and molecular endpoints to detect thyroid system disruption in X. laevis tadpoles by the model anti-thyroidal compound ETU (Doerge and Takazawa, 1990; Tsuda, 1983). We showed that metamorphic retardation caused by ETU is associated with concentration-dependent histological changes in the thyroid gland and increased mRNA expression levels of TSHß in the pituitary.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and husbandry.
All experiments were carried out with X. laevis tadpoles reared from the animal stock of the Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany. Spawning of adult X. laevis was induced by injection of human chorionic gonadotropin (Sigma, Deisenhofen, Germany) into the dorsal lymph sac as described by Kloas et al. (1999)
. Eggs and tadpoles were reared in synthetic culture medium at 22° ± 1°C and pH 7.0 ± 0.5 during all phases of the experiments described below. The synthetic medium, formulated by adding 2.5 g of the commercial salt mixture "Tropic Marin Meersalz" (Tagis, Dreieich, Germany) to 10 liters of deionized water, was also used as dilution water to prepare the test solutions in the exposure experiments with ETU. All rearing tanks were continuously aerated by airstones. The light:dark cycle was 12:12 h. Starting at day 5 postfertilization, tadpoles were fed daily ad libitum with Sera Micron (Sera, Heinsberg, Germany). Developmental stages of tadpoles were determined according to the Normal Table of X. laevis (Nieuwkoop and Faber, 1994). All experimental aspects were conducted in compliance with the institutional guidelines for the care and use of animals.
Exposure Study with ETU.
The exposure experiment involved aqueous exposure of premetamorphic tadpoles for up to 90 days to five concentrations of ETU and a solvent control in a static renewal exposure regime. ETU and dimethylsulfoxide (DMSO) were obtained from Sigma. Stock solutions of ETU were prepared in 1% DMSO before each renewal of the test solutions. The exposure experiment was initiated with stage 51 tadpoles at day 14 post-fertilization. Twenty stage 51 tadpoles were placed in each of three replicate tanks containing nominal concentrations of 1.0, 2.5, 10, 25, and 50 mg/l ETU, as well as 0.005% DMSO (solvent control, SC). To reduce the total number of test animals, we omitted the use of a solvent-free control in the experimental design, because data from several preliminary experiments indicated that DMSO, at the low concentration used in this study, does not affect developmental rates, growth, survival, or any of the histological and molecular biological endpoints examined in X. laevis tadpoles (Kloas et al., 2003; Opitz et al., 2005b and unpublished data).
Exposures were conducted in 11-liter glass aquaria containing 10 liters of test solution; the larval density at test initiation was two tadpoles per liter. All test tanks were placed in a thermoregulated waterbath maintained at 22° ± 1°C. Test solutions were replaced completely three times a week (Monday, Wednesday, Friday) according to the procedures described for the XEMA ring-test (Opitz et al., 2005a). During exposure, tadpoles were fed daily a specified amount of Sera Micron which was added to the tanks as dry food. Initially, the total daily food ration was 200 mg/tank, but the daily food ration was increased during the course of the exposure experiment to account for tadpole growth.
Throughout the 90-day exposure, all tanks were inspected daily for dead tadpoles or tadpoles showing abnormal behavior (e.g., uncoordinated swimming, hyperventilation, atypical quiescence). Dead tadpoles were removed from the tanks as soon as they were observed. In addition, all tanks were monitored daily for tadpoles showing FLE, which characterizes the transition of tadpoles from developmental stage 57 to stage 58 (Nieuwkoop and Faber, 1994). On the day of the first observation of FLE (exposure day 20 in this study), developmental stage and whole body length (WBL) were determined for all tadpoles in all tanks. Developmental stage (Nieuwkoop and Faber, 1994) was assessed by visual inspection under a binocular dissection microscope, and WBL was measured from the tip of the snout to the tail end according to the standard operating procedures of the XEMA test (Opitz et al., 2005a). From exposure day 20 onward, individual tadpoles displaying FLE were removed from the tanks and anesthetized by chilling on ice, after which WBL was measured. Tadpoles were then killed by decapitation and dissected under a binocular microscope to assess gross morphology of the thyroid gland and to collect tissues for histological and molecular analyses.
Subsets of three to four FLE-displaying tadpoles per replicate tank were randomly selected for histological analyses of the thyroid glands and for collection of brain and pituitary tissues for gene expression analysis. In the 50 mg/l ETU treatment group, tadpoles were arrested in premetamorphic development, and stage 53/54-arrested animals (three animals per replicate tank) were sampled on exposure days 28 and 90 for thyroid histological and gene expression analyses. For thyroid gland histology, the lower jaw was completely removed and fixed in Bouin's fixative (Sigma) for 24 h, followed by two 6 h rinsing steps in 70% ethanol (Roth, Karlsruhe, Germany). Fixed tissues were finally stored in 70% ethanol at 4°C until further processing. For gene expression analysis, the whole brain including the pituitary was removed. Subsequently, the pituitary gland, together with a defined portion of surrounding brain tissue (pituitary/brain tissue volume ratio of approximately 1:5), was microdissected to obtain a sufficient amount of tissue for RNA extraction. For this dissection method, we took advantage of the fact that, with the exception of the developmentally arrested tadpoles in the 50 mg/l ETU group, the sampled organisms were at the same developmental stage and thus displayed the same landmarks of brain gross morphology that were used for orientation during dissection. This approach to pituitary tissue sampling was used to obviate the need for pooling pituitary tissue samples from several tadpoles. The remaining brain tissue was collected separately, and brain and pituitary tissue samples were immediately frozen in liquid nitrogen and stored at –80°C until RNA isolation.
Thyroid gland histology and morphometry.
Lower jaw tissue samples containing the paired thyroid gland were dehydrated in a graded series of alcohol, embedded in paraffin (Paraplast, Roth, Karlsruhe, Germany), and sectioned in a transverse plane from dorsal to ventral at 5 µm thickness. Serial sections were mounted on glass slides, stained with hematoxylin and eosin, and embedded with glass coverslips. For each tadpole, five sections of the middle part of the right thyroid lobe were qualitatively analyzed for exposure-related changes under a Zeiss Axiovert 200 microscope (Zeiss, Jena, Germany) equipped with a Show View II digital camera (Olympus, Hamburg, Germany). Image analysis was performed with AnalySIS software (Soft Imaging Systems, Münster, Germany). For determination of epithelial cell heights, serial sectioning throughout the whole right lobe of the thyroid gland was performed to trace individual follicles throughout the lobe and to identify a total of five different sections containing different follicles. For each of the preselected sections, the histological integrity of all follicles present was assessed. Three different follicles were then randomly selected, and the height of four epithelial cells per follicle was determined, resulting in a total of 60 measurements per individual. For each animal, a single mean value for epithelial cell height was calculated and at least five tadpoles were analyzed for each treatment group.
Semiquantitative RT-PCR.
Total RNA was extracted from brain and pituitary tissues of individual tadpoles using RNeasy Micro Kit according to the manufacturer's protocol (Qiagen, Hilden, Germany). RNA was eluted from the RNeasy Micro spin columns with 16 µl RNase-free water and UV-absorbance of RNA solutions was measured at 260 and 280 nm using a Spectrafluor Plus microplate reader (Tecan, Crailsheim, Germany). Prior to reverse transcription-polymerase chain reaction (RT-PCR), all RNA samples were diluted to a concentration of 1 µg total RNA per 8 µl RNase-free water. cDNA was reverse transcribed from 1 µg total RNA using Avian Myeloblastosis Virus reverse transcriptase (AMV-RT; Biometra, Göttingen, Germany). RNA was first incubated with 7.5 pmol oligo(dT) primer (sequence: 5'-CCTGAATTCTAGAGCTCA(T)17–3'; Biometra) in a 20 µl reaction at 70°C for 3 min followed by cooling on ice. After addition of 15 nmol of each dNTP (Biometra), 10 units AMV-RT and the reaction buffer (25 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl2) supplied with the enzyme, the resultant 30-µl reaction was incubated at 37°C for 60 min, heated to 94°C for 2 min, and then cooled on ice. The cDNA was diluted 10-fold before use in PCR.
Primers for X. laevis TRßA, TSH, and TSHß were designed based on published cDNA sequences (Buckbinder and Brown, 1993; Yaoita et al., 1990) and were synthesized by TIB Molbiol (Berlin, Germany). Elongation factor 1 (EF) was used for normalization of target gene data as described by Opitz et al. (2005c). Polymerase chain reaction was performed in a 25 µl reaction using 3 µl of cDNA solution as template. The PCR reactions contained 2 nmol of each dNTP (Biometra), 10 pmol of each gene-specific PCR primer, 1 unit of Taq DNA polymerase (Invitrogen, Karlsruhe, Germany), and the buffer (20 mM Tris-HCl, pH 8.4, 1.5 mM MgCl2) supplied with the enzyme. The following gene-specific primers were used in the PCR: TRßA (forward) 5'-GTC GCT TCA AAA AGT GCA TCG-3'; TRßA (reverse) 5'-ACC CTC GGG CGC ATT AAC TAT-3'; TSH (forward) 5'-ACG GGT CAC AAG TGA TGG-3'; TSH (reverse) 5'-GGG ATC ACA TCA TGC AGA TGA-3'; TSHß (forward) 5'- AGA GTG CGC TTA CTG CCT TG-3'; TSHß (reverse) 5'-GGT AGG AAA AGA GCG GGT TC-3'; EF (forward) 5'-TGC CAA TTG TTG ACA TGA TCC C-3'; EF (reverse) 5'-TAC TAT TAA ACT CTG ATG GCC-3'. The PCR amplification was carried out in a thermal cycler (Biometra) according to the following protocols: for TSH, 94°C for 3 min followed by 21 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 30 s, followed by extension at 72°C for 10 min; for TSHß, 94°C for 3 min followed by 24 cycles of 94°C for 30 s, 62°C for 30 s, 72°C for 30 s, followed by extension at 72°C for 10 min; for TRßA, 94°C for 3 min, 29 cycles of 94°C for 30 s, 67°C for 30 s, 72°C for 30 s, followed by extension at 72°C for 10 min; for EF, 94°C for 3 min, 16 cycles of 94°C for 30 s, 59°C for 30 s, 72°C for 30 s, followed by extension at 72°C for 10 min. For each of the genes analyzed, PCR cycle numbers were empirically determined to ensure detection within the linear range of the PCR reaction. Amplified PCR products were subjected to electrophoresis on a 1.8% agarose gel and stained with ethidium bromide (GIBCO/BRL, Eggenstein, Germany). Images of ethidium bromide–stained gels were taken using the gel documentation system GelDoc 2000 (Bio-Rad, Munich, Germany) and Quantity One software (Bio-Rad). After a background subtraction, intensities of separated bands were quantified using the band analysis quick guide tool provided by the Quantity One software. Densitometric values for EF were used to normalize TSH, TSHß, and TRßA values. Results from triplicate analyses of individual RNA samples for specific gene expression were averaged, yielding a single value for each RNA sample to be used in statistical analysis. The PCR products were extracted from agarose gels using QIAquick kit (Qiagen), and the identity of the PCR products was confirmed by sequence analysis (Sequence Laboratories, Göttingen, Germany). To verify that RNA preparations were not contaminated by residual genomic DNA, RT-PCR was conducted for RNA samples without the addition of AMV-RT. In addition, primers for TRßA-specific PCR were designed targeting sequences in different exons (exon 2 and exon 4) of the TRßA gene. Using these primers, PCR amplification of genomic DNA of X. laevis yielded a single PCR product of >1.5 kb, whereas RT-PCR amplification of purified RNA of X. laevis yielded a single PCR product of 298 bp (data not shown). Therefore, monitoring of agarose gels for the presence of a second PCR product of >1.5 kb in TRßA-specific RT-PCR assays provided an additional means to control for residual genomic DNA.
Data analysis.
Whole body length was analyzed by Dunnett's test for differences between the solvent control group and individual ETU treatments. The non-parametric Kruskal-Wallis test was used to determine differences in developmental stages on day 20 and Dunn's multiple comparison test was used for pairwise comparisons with the control group. Individual tadpoles were used as the experimental units in all analyses. Comparisons between tanks within treatments did not identify significant tank effects, and data from all tanks within a given treatment were pooled for final analyses of treatment effects. Differences in time to FLE were analyzed by the Kruskal-Wallis test followed by Dunn's multiple comparison test to compare each ETU treatment with the control group. Epithelial cell heights and densitometric data from RT-PCR assays were log-transformed to satisfy criteria of normality and homogeneity of variance. In initial analyses, analysis of covariance (ANCOVA) models were used with time to FLE as covariate, but the covariate was not significant and was removed from the final model. One-way analysis of variance (ANOVA) was used for the final analyses, followed by Dunnett's test to compare control data to all other treatment groups or by the Tukey-Kramer multiple comparison test for pairwise comparisons to identify significant differences among treatment groups. Statistical analysis was performed using the software package Sigma Stat 2.0 (SPSS-Jandel Scientific, Erkrath, Germany). Differences were considered significant at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of ETU on Survival, Growth, and Development
No signs of systemic toxicity were observed during the 90-day exposure of X. laevis tadpoles to ETU. No gross morphological or behavioral abnormalities were detected in any treatment group, and only one tadpole died in one replicate tank of the 50 mg/l ETU treatment group on day 71. Growth of tadpoles, when assessed by means of WBL measurements on exposure day 20, was also not affected by ETU treatment (Table 1).
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Exposure of tadpoles to ETU resulted in a concentration-dependent inhibition of metamorphic development. Determination of developmental stage of all test organisms on exposure day 20 revealed that 50 mg/l ETU caused a complete inhibition of metamorphic development at late premetamorphic stages 53/54 (Table 1). Significant developmental retardation was also detected for 25 mg/l ETU; however, metamorphosis was not completely inhibited, but if compared to the SC group proceeded much slower as evident from the observation of earlier developmental stages on exposure day 20. Treatment of tadpoles with 1.0, 2.5, and 10 mg/l ETU did not cause distinct differences in day 20 developmental stages.
A similar effect pattern was observed when time to FLE was used as endpoint to assess ETU treatment-related alterations in metamorphic development (Fig. 1). The first tadpoles displaying FLE were observed on exposure day 20 in the control group (7 animals) and in the 1.0 mg/l (8 animals) and 2.5 mg/l ETU (2 animals) treatment groups. In the 10 mg/l ETU treatment group, the onset of FLE was slightly delayed, as the first tadpoles showing FLE were observed on day 22 (4 animals). However, compared to the SC group, median time to FLE was not significantly different for the 1.0, 2.5, and 10 mg/l ETU treatment groups, and all tadpoles exposed to ETU concentrations 
10 mg/l ETU reached the FLE stage within 38 days of exposure. Monitoring of tadpoles for FLE thus confirmed the strong inhibition of metamorphosis by 25 mg/l ETU and 50 mg/l ETU. In the 25 mg/l ETU treatment group, the first tadpole showing FLE was observed on day 23, but at test termination after 90 days of exposure, only 83% of the tadpoles in this group exhibited FLE. None of the tadpoles exposed to 50 mg/l ETU showed FLE, and their development was still arrested at stages 53/54 on exposure day 90.
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Effects on the Thyroid Gland
Although not quantified in the present study, visual examination of thyroid gland gross morphology of ETU-exposed specimens revealed increases in glandular size at 10, 25, and 50 mg/l ETU (Fig. 2). Hematoxylin-eosin stained paraffin sections of the thyroid gland were used to evaluate the histological appearance of thyroid glands collected from FLE-displaying tadpoles. Because tadpoles exposed to 50 mg/l ETU did not show development to stage 58, developmentally arrested tadpoles were sampled for thyroid gland histology on exposure days 28 and 90, respectively. Histologically, thyroid glands from FLE-displaying tadpoles in the SC group were characterized by fairly uniform follicles lined by a single layer of cuboidal follicular epithelial cells (Fig. 3). The colloid showed homogeneous tinctoral properties, and peripheral vacuolation of the colloid was minimal. Histological examination of ETU-treated tadpoles revealed a variety of histological alterations, which are summarized in Table 2. Histological changes observed in thyroid glands from the 1.0 and 2.5 mg/l ETU treatment group included mild increases in peripheral vacuolation of the colloid (1.0 and 2.5 mg/l ETU) and mild follicle distension (2.5 mg/l ETU). The prevalence of peripheral colloid vacuolation was increased at 2.5 mg/l ETU (Fig. 3C). At 10 mg/l ETU, many thyroid glands showed a marked increase in follicle size, accompanied by partial colloid depletion (Fig. 3D). In some tadpoles, treatment with 10 mg/l ETU resulted in a foamy appearance and pale staining of the colloid. The follicular epithelium still consisted of a single layer of follicular cells. No signs of follicular cell hypertrophy were evident in thyroid glands from tadpoles exposed to
10 mg/l ETU. Interestingly, in the 10 mg/l ETU treatment group, epithelial cells appeared flattened compared to the control group. Thyroid glands of tadpoles exposed to 25 and 50 mg/l ETU showed a high prevalence of follicular cell hypertrophy and hyperplasia (Fig. 3E, 3F). Colloid depletion was generally enhanced in thyroid glands from these treatment groups as was evident from a foamy and faintly-staining colloid. Follicle size was markedly increased at 25 and 50 mg/l ETU, leading to pronounced diffuse enlargement of the glands. However, follicular architecture displayed a considerable heterogeneity within individual glands. As depicted in Figure 3E and 3F, some follicles were greatly increased in size, contained a foamy and faintly staining colloidal mass, and showed only a moderate increase in the height of the epithelial cell layer. Another type of follicle could be characterized as collapsed follicles, almost devoid of colloid and lined by highly hypertrophic epithelial cells.
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Follicular cell height was determined in a subset of thyroid glands to evaluate quantitatively the treatment-related changes in follicular cell morphology. This analysis included glands from FLE-displaying tadpoles of the different treatments and glands sampled on day 28 from stage 53/54-arrested tadpoles of the 50 mg/l ETU treatment. Results from epithelial cell height measurements revealed a biphasic response pattern to ETU exposure (Fig. 4). Compared to the SC group, statistically significant increases in mean epithelial cell heights were observed in thyroid glands from tadpoles exposed to 25 and 50 mg/l ETU. In contrast, mean values for epithelial cell height in thyroid glands from tadpoles treated with 1.0, 2.5, and 10 mg/l ETU were lower than in control tadpoles. However, only the decrease in epithelial cell height observed at 10 mg/l ETU was statistically significant.
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Gene Expression Analysis
Expression of TSH
and TSHß mRNA in pituitary tissue was analyzed by semiquantitative RT-PCR. When determined in FLE-displaying tadpoles, no significant differences to the SC group were detected in mRNA expression levels of TSH and TSHß in response to 1.0, 2.5, and 10 mg/l ETU (Fig. 5). However, mRNA expression of both TSH subunits was significantly increased in FLE-displaying tadpoles exposed to 25 mg/l ETU and in developmentally arrested tadpoles of the 50 mg/l ETU treatment group. For stage 53/54-arrested tadpoles from the 50 mg/l ETU treatment group, pituitary gene expression was analyzed separately for tissue samples collected on exposure days 28 and 90, respectively. At both time points, mRNA expression levels of TSH and TSHß were lower than in FLE-displaying tadpoles from the 25 mg/l ETU treatment group. The difference in TSH expression between the two highest ETU concentrations was statistically significant.
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When comparing TRßA mRNA expression levels in brain tissue of FLE-displaying tadpoles of the different treatment groups, no significant differences were detectable between the SC group and any of the ETU treatment groups (Fig. 6). Significantly lower expression levels of TRßA mRNA were only detected in brain tissue of stage 53/54-arrested tadpoles of the 50 mg/l ETU treatment group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development of sensitive and diagnostic bioassays for detection of thyroid system-disrupting chemicals has been recognized as an important research need (Colborn, 2002
; Yamasaki et al., 2002; Zoeller, 2003). We recently reported the development and initial evaluation of a Xenopus Metamorphosis Assay (XEMA), which exploits the TH-dependent processes of metamorphic development in X. laevis tadpoles as a model to detect disruption of thyroid system function (Opitz et al., 2005a). In the previous study, apical morphological endpoints (e.g., developmental stage, whole body length) were successfully used to identify alterations in X. laevis metamorphic development (Opitz et al., 2005a), but additional measurements of thyroid system-related endpoints are needed to enhance the sensitivity and specificity of the XEMA testing protocol for detection of thyroid system disruption. In the present study we therefore examined the utility of various morphological, histological, and molecular biological endpoints to characterize chemically induced changes in thyroid system function and metamorphic development of X. laevis tadpoles. Ethylenethiourea was used as a model compound because of its well-known anti-thyroidal activity in mammalian model systems (Tsuda, 1983; Graham and Hansen, 1972) and its potent inhibitory effects on X. laevis metamorphosis (Opitz et al., 2005a). In the thyroid gland, ETU inhibits the activity of thyroid peroxidase resulting in a suppression of TH synthesis (Doerge and Takazawa, 1990; Marinovich et al., 1997).
For the assessment of ETU effects on metamorphic development, two different approaches were used in the present study. Both determination of the developmental stage of all test animals after a defined exposure period (exposure day 20 in this study) and monitoring of tadpoles for the time required until FLE consistently revealed a strong retardation of metamorphosis at concentrations 
25 mg/l ETU. No sensitivity difference was observed between these two approaches. Determination of the time required until FLE (Ankley et al., 1998) or until completion of metamorphosis (Allran and Karasow, 2000) are commonly used approaches to assess chemical effects on metamorphic development in anuran amphibians. However, a disadvantage of time to FLE as an endpoint is that a test protocol based on this endpoint will be time-consuming and unpredictable in its duration. Thus, determination of developmental stages after distinct exposure periods should be the preferred endpoint measurement to assess developmental effects in a standardized test protocol for thyroid disruption (Opitz et al., 2005a; Tietge et al., 2005).
The failure of tadpoles in the 50 mg/l ETU treatment group to develop to prometamorphic stages (which requires a low level of circulating TH) during 90 days of exposure suggests an almost complete inhibition of TH synthesis at 50 mg/l ETU. Developmental data for the 25 mg/l ETU treatment group indicate a marked suppression of the thyroid system, although the extent of the resulting developmental retardation was highly variable among individual tadpoles. Overall, the developmental effects observed in the present study for concentrations 25 mg/l ETU are consistent with previous results obtained using the standardized 28-day XEMA test protocol (Opitz et al., 2005a).
A major goal of the present study was the examination of further endpoints that may bear the potential to increase the sensitivity and diagnostic power of the amphibian test model for detection of thyroid system disruption. Despite acting via different modes of action, the primary effect of many compounds influencing thyroid system function is a reduction in circulating levels of TH (Brucker-Davis, 1998). The present model of pituitary-thyroid axis function holds that reduced TH concentrations in serum will trigger increases in plasma TSH levels via reduced negative feedback of TH on the biosynthetic and secretory activity of thyrotropic cells in the pituitary (O'Connor et al., 1999). Increased release of TSH by the pituitary, in turn, leads to a stimulation of TH synthesis and secretion by the thyroid gland, and, as a consequence of the mitogenic activity of TSH, may result in follicular cell proliferation and thyroid gland enlargement (Capen, 1997). There is evidence for the existence of similar feedback signaling pathways in tadpoles undergoing metamorphic development (Buckbinder and Brown, 1993; Manzon and Denver, 2004). Thus, particular attention was given to the evaluation of endpoints that provide information about mechanisms of compensation along the pituitary–thyroid axis (Capen, 1997). Considering the methodological limitations in characterizing the thyroidal status of the differentially treated tadpoles by means of analysis of serum T4, T3, and TSH concentrations, we examined whether RT-PCR analysis of TSHß mRNA expression in pituitary tissue in combination with histological examination of thyroid gland tissue might provide alternative measurements to detect anti-thyroidal effects in X. laevis tadpoles.
Gross morphological examination of thyroid glands from tadpoles exposed to 25 mg/l ETU showed a marked increase in glandular size, and diagnostic evidence for thyroid disruption by ETU was obtained from histological analysis of thyroid gland sections. Some of the changes observed, including diffuse enlargement of the thyroid gland, distended follicles, follicular cell hypertrophy, and hyperplasia, as well as colloid depletion, resemble histological alterations previously reported in mammalian studies with ETU (Graham and Hansen, 1972; Tsuda, 1983) and other TH synthesis inhibitors such as PTU (O'Connor et al., 1999) and amitrole (Strum and Karnovsky, 1971). The presence of hypertrophic/hyperplastic changes noted in thyroid gland sections from tadpoles exposed to 25 mg/l ETU is also consistent with findings of previous studies in which anuran tadpoles were treated with high concentrations of TH synthesis inhibitors (Goos et al., 1968; Miranda et al., 1996). In contrast, the light microscopic appearance of follicular architecture in glands from ETU-treated tadpoles in this study differs considerably from the histological changes observed following sodium perchlorate exposure of X. laevis tadpoles (Tietge et al., 2005; own unpublished data). Perchlorate represents a different type of TH synthesis inhibitor that acts via blocking of iodide uptake into follicular cells (Wolff, 1998). A prominent feature in the hypertrophic glands from perchlorate-exposed tadpoles is a concentration-dependent reduction in colloid content ultimately leading to a predominance of collapsed follicles depleted of colloid. Although some collapsed follicles containing little or no colloid were observed in glands from ETU-treated tadpoles in the present study, there was a remarkable heterogeneity in how individual follicles responded to ETU treatment. In contrast to perchlorate treatment, glands from ETU-treated tadpoles showed a high incidence of markedly distended follicles with large luminal compartments and only moderately hypertrophic epithelial cell layers. The mechanisms that generate the marked heterogeneity in follicle architecture in response to ETU treatment are unknown. However, the contrasting response patterns of the thyroid gland evoked by inhibitors of iodide uptake and thyroid peroxidase suggest that the diagnostic value of thyroid gland histology is not limited to the confirmation of thyroid system disruption but might also provide indications as to the character of the underlying mode of action. This aspect deserves further comparative histological studies using anti-thyroidal compounds that act via different modes of action.
In addition to a qualitative assessment, morphometric analyses of thyroid gland sections have been used in many studies to evaluate quantitatively the specific alterations in follicular architecture (Herrmann et al., 1989). Measurements of epithelial cell height, for example, represent a classical method for assessing follicular cell hypertrophy (Miranda et al., 1996, Goleman et al., 2002). The present study illustrates that quantitative epithelial cell height measurements can provide a useful endpoint to confirm follicular cell hypertrophy in thyroid glands of goitrogen-treated tadpoles. It should be noted, however, that in the case of ETU, other qualitative endpoints (e.g., follicle size and colloid depletion) proved to be more sensitive in detecting changes in thyroid gland histology.
Overall, type and severity of histological alterations in thyroid glands from tadpoles treated with 25 mg/l ETU indicated a permanent stimulation of the thyroid gland by TSH. This assumption was confirmed by RT-PCR analysis of pituitary gland gene expression. Increased expression of TSH and TSHß mRNA was detected in pituitary tissue of tadpoles exposed to the two highest ETU concentrations (25 and 50 mg/l). An interesting observation was that mRNA expression of both TSH subunits was more markedly increased at 25 mg/l ETU than at 50 mg/l ETU. Results from several studies indicate that negative feedback of circulating TH (Manzon and Denver, 2004), as well as hypothalamic factors, is involved in controlling pituitary thyrotrophic cell activity (Denver, 1996). Moreover, it has been shown that both the hypothalamus and the median eminence, a structure providing the neurovascular link between neurosecretory centers in the hypothalamic preoptic area and the pituitary, require TH for proper functional differentiation (Aronsson and Enemar, 1992; Goos et al., 1968). Thus, if hypothalamic hormones are involved in TSH upregulation in hypothyroid tadpoles, it appears reasonable to assume a less marked hypothalamic stimulation of pituitary TSH synthesis in stage 53/54-arrested tadpoles (50 mg/l ETU treatment group) due to reduced functional properties of the median eminence. However, more studies are needed to elucidate the precise contribution of hypothalamic factors to the regulation of TSH synthesis in hypothyroid tadpoles.
For the anuran metamorphosis model, we assumed that chemically induced perturbations of TH homeostasis may only become apparent as alterations in developmental rates, if the capacity of the hypothalamus–pituitary–thyroid axis to maintain sufficient TH levels is exhausted. In our study, no significant changes in developmental rates were detectable for ETU concentrations ranging from 1.0 to 10 mg/l, but the observation of distinct treatment-related changes in thyroid gland histology (follicle distension, partial depletion of colloid) strongly suggest that TH homeostasis had already been affected at these lower ETU concentrations. However, none of the lower ETU concentrations caused a detectable increase in TSH or TSHß mRNA expression, and no signs of thyroid follicular cell hypertrophy were observed upon histological examination of thyroid glands from tadpoles exposed to 10 mg/l ETU. The latter observation was quantitatively confirmed by morphometric measurements of epithelial cell heights showing rather reduced cell heights at 2.5 and 10 mg/l ETU.
The observed effect pattern of mild changes in thyroid gland histology in the absence of increased pituitary TSHß mRNA expression is difficult to explain. One possible source of error in TSHß mRNA determination may be the pituitary tissue dissection approach used in the present study, which included dissection of a fixed portion of surrounding brain tissue. However, a recent study used RNA from individual pituitaries free of surrounding brain tissue for RT-PCR analysis of TSHß mRNA expression and confirmed the lack of increased TSHß mRNA expression at 10 mg/l ETU (Opitz et al., 2005b). Still another possibility is that mRNA expression of TSHß may not accurately reflect pituitary TSH synthesis and secretion. However, given that TSHß mRNA expression was only analyzed after tadpoles had been developed to FLE stage 58, the gene expression data obtained may merely represent a snapshot, providing information at a given developmental stage. In particular, it should be noted that the anti-thyroidal treatment in the present study covered a period during which the different components of hypothalamus–pituitary–thyroid gland axis still undergo profound developmental changes (Denver, 1996; Regard, 1978). It may thus be possible that the effect pattern observed in FLE-displaying tadpoles—enlarged thyroid follicles lined by flat epithelial cells with no alteration in TSHß mRNA expression—may represent a new steady state of pituitary–thyroid axis function that was established in response to modulating effects on thyroid gland activity and growth earlier in the exposure period. Interestingly, studies in rats and chickens have also shown that early stimulation of the developing thyroid gland by TSH could have long-lasting effects on functioning of thyrocytes and overall follicular architecture, leading to persistently enlarged follicles with increased luminal areas and decreased epithelial cell heights (Bakke et al., 1970; Leung and Marsh, 1976). At present, however, it is not known whether the tadpoles exposed to the lower ETU concentrations in our study experienced increased TSH stimulation during early time points of the exposure phase that were not covered by endpoint measurements.
Because the increased TSHß mRNA expression in FLE-displaying tadpoles exposed to 25 mg/l ETU was interpreted as an indication of reduced levels in circulating TH, we examined whether mRNA expression of TH-dependent genes in peripheral tissues is reduced in these tadpoles. A large body of information is available for the TH-dependent expression of TRßA mRNA (Shi, 1999), which increases in tadpole brain during prometamorphic and early climax stages (own unpublished data), closely following the developmental profile of circulating plasma TH levels (Leloup and Buscaglia, 1977). However, TRßA mRNA expression in brain of FLE-displaying tadpoles was not affected by ETU treatment, and reduced expression was only detected in the developmentally arrested tadpoles of the 50 mg/l ETU treatment group. Thus, although several other endpoints indicate hypothyroid conditions in ETU-treated tadpoles at the FLE stage, analysis of TRßA mRNA expression was insensitive to these conditions.
Assuming that ETU inhibits TH synthesis in X. laevis tadpoles in a manner similar to that reported for mammals, the effects profile observed for ETU indicates that impaired TH synthesis leads to retardation of development but does not impair tadpole growth. In response to lowered TH concentrations, pituitary TSH synthesis is increased at the gene transcription level and—although not determined in the present study—an accompanying increase in plasma TSH levels most likely accounts for the observed hyperstimulation of the thyroid gland. The histological finding of concentration-dependent increases in colloid depletion suggests a reduction in colloidal TH stores that are not refilled. Together, the present findings not only support the existence of pituitary–thyroid feedback signaling similar to mammalian species, they also suggest that assessment of endpoints related to the compensatory action of the pituitary–thyroid axis could provide diagnostic evidence for thyroid system disruption in anuran tadpoles.
In comparisons of the sensitivities of the different endpoints to detect changes in thyroid system function, histological examination of the thyroid gland provided the most sensitive measurement endpoints (follicle distension and colloid depletion), whereas analysis of TSH gene expression was as sensitive as developmental stage determination. It should be noted, however, that the observation of increased TSH gene expression provided strong diagnostic evidence for disruption of TH homeostasis by ETU in X. laevis tadpoles. In contrast, analysis of TRßA mRNA expression levels in brain was the least sensitive endpoint in the present study. Regarding the use of the endpoints examined in an enhanced XEMA testing protocol, it is concluded that both thyroid histopathology and RT-PCR analysis of TSH gene expression would markedly increase not only the sensitivity but also the diagnostic value of the XEMA test for detection of thyroid system disrupters. Histological analysis of the thyroid gland allowed detection of anti-thyroidal effects at 10-fold lower ETU concentrations compared to determination of apical morphological endpoints. Given this enhanced sensitivity of thyroid gland histology and the differential effect patterns caused by ETU (this study) and perchlorate treatment (Tietge et al., 2005), the development of a structured and standardized assessment scheme for histopathological changes in the thyroid is desirable to fully exploit the information provided by histological endpoints. In addition, time-course studies of the different endpoint responses are required to further increase understanding of the regulatory mechanisms induced in hypothyroid tadpoles.
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ACKNOWLEDGMENTS |
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This study was supported by the German Federal Environmental Agency under contract nos. 200 67 409, 203 67 450, and 204 67 454. The authors thank Marcel Simon for his help with performing the histological analyses.
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REFERENCES |
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