ToxSci Advance Access originally published online on June 28, 2006
Toxicological Sciences 2006 93(2):268-277; doi:10.1093/toxsci/kfl053
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The Colloidal Thyroxine (T4) Ring as a Novel Biomarker of Perchlorate Exposure in the African Clawed Frog Xenopus laevis
* Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-3131; Department of Range, Wildlife and Fisheries Management and Texas Cooperative Fish & Wildlife Research Unit, Texas Tech University, Lubbock, Texas 79409-2120; Department of Environmental Toxicology and Texas Cooperative Fish and Wildlife Research Unit, Texas Tech University, Lubbock, Texas 79409-1160; and U.S. Geological Survey Texas Cooperative Fish and Wildlife Research Unit and Department of Range, Wildlife and Fisheries Management, Texas Tech University, Lubbock, Texas 79409-2120
1 To whom correspondence should be addressed. Fax: (806) 742-2963. E-mail: james.carr{at}ttu.edu.
Received April 26, 2006; accepted June 21, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The purpose of this study was to determine if changes in colloidal thyroxine (T4) immunoreactivity can be used as a biomarker of perchlorate exposure in amphibian thyroid tissue. Larval African clawed frogs (Xenopus laevis) were exposed to 0, 1, 8, 93, and 1131 µg perchlorate/l for 38 and 69 days to cover the normal period of larval development and metamorphosis. The results of this study confirmed the presence of an immunoreactive colloidal T4 ring in thyroid follicles of X. laevis and demonstrated that the intensity of this ring is reduced in a concentration-dependent manner by perchlorate exposure. The smallest effective concentration of perchlorate capable of significantly reducing colloidal T4 ring intensity was 8 µg perchlorate/l. The intensity of the immunoreactive colloidal T4 ring is a more sensitive biomarker of perchlorate exposure than changes in hind limb length, forelimb emergence, tail resorption, thyrocyte hypertrophy, or colloid depletion. We conclude that the colloidal T4 ring can be used as a sensitive biomarker of perchlorate-induced thyroid disruption in amphibians.
Key Words: perchlorate; biomarker; thyroid; amphibian; endocrine; disruption.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Amphibian metamorphosis depends on the timely and adequate production and systemic release of thyroid hormones (THs): thyroxine (T4) and triiodothyronine (T3) (Shi, 2000
). Thus, aquatic contaminants that impair thyroid function are likely to have major impacts on the recruitment of larval amphibians into adult populations. Perchlorate is a well-known goitrogen that inhibits TH synthesis and it is widely used in the manufacture of missiles, rockets, ammunitions, and fireworks (Logan, 2001). The widespread contamination of ground-, surface, and drinking water by perchlorate is of increasing concern. In the United States, perchlorate has been detected in groundwater (3 µg/l–3.7 mg/l), surface water (3–120 µg/l), and drinking water (1–811 µg/l) (USEPA, 2004). By competing with the sodium/iodide symporter responsible for transporting iodide into thyroid follicle cells, perchlorate interferes with TH synthesis and secretion and lowers circulating levels of THs in the blood (Siglin et al., 2000; York et al., 2001a,b). The adverse effects of perchlorate on thyroid function have been studied in mammals (Capen, 1997; Carr et al., in press; Siglin et al., 2000; York et al., 2001a,b), birds (McNabb et al., 2004, in press), fish (Crane et al., 2005; Mukhi et al., 2005; Patiño et al., 2003; Theodorakis et al., 2006), and amphibians (Carr and Theodorakis, in press; Carr et al., 2003; Goleman and Carr, 2006; Goleman et al., 2002a,b; Michael and Aziz, 1976; Sparling et al., 2003; Theodorakis et al., 2006; Tietge et al., 2005).
"Biomarkers" are defined as xenobiotically induced alterations in behavior or in whole-body, cellular, or biochemical components or processes, structures or functions in a biological system or sample (Kendall et al., 2001). A biomarker approach to determine the effects of contaminants on organisms is currently at the core of environmental toxicity studies. Several thyroid biomarkers of thyroid disruption have been developed. Changes in thyroid histopathology, such as follicle cell hypertrophy, hyperplasia, and colloid depletion, and in related hormone levels, such as thyroid-stimulating hormone (TSH) and TH in the circulation, are widely used as biomarkers to investigate disorders of the thyroid gland. In amphibians, morphological changes during development and metamorphosis, such as forelimb emergence (FLE), hind limb growth, and tail resorption, have also been used to assess thyroid disruption (Degitz et al., 2005; Goleman et al., 2002a,b; Opitz et al., 2006; Tietge et al., 2005). However, the relative sensitivity of these biomarkers to low-concentration goitrogen exposures is still uncertain. For example, in mammals, birds, amphibians, and fishes, changes in circulating or whole-body THs are relatively insensitive biomarkers of perchlorate exposure, whereas direct measurements of thyroid function (follicle cell height and thyroid gland TH content) tend to be more sensitive to this goitrogen (Carr et al., in press). Measurements of the follicle cell hypertrophy and colloid depletion (Goleman et al., 2002a; Tietge et al., 2005) have been used previously in amphibians to gauge the direct effects of perchlorate on thyroid function, but the degree to which either of these measurement reflects changes in thyroid TH content is unclear. Moreover, direct measurement of thyroid TH content in larval amphibians is difficult due to their small body size. Recently, an immunocytochemistry-based novel biomarker of perchlorate exposure, the colloidal T4 ring, was reported in zebra fish (Mukhi et al., 2005). The ring is an immunoreactive accumulation of T4 along the periphery of colloid, at the interphase between the apical surface of the follicular epithelium and the lumen (colloid). Changes in colloid T4 ring intensity had greater sensitivity and longevity to perchlorate exposure than measurements of follicle cell hypertrophy or alterations of whole-body T4 levels (Mukhi et al., 2005). However, the existence of this ring and its sensitivity as a biomarker of thyroid disruption have not been examined in any amphibian species.
The objective of this study was to assess the utility of the colloidal T4 ring as a biomarker of perchlorate exposure in amphibians. The African clawed frog Xenopus laevis was used as the animal model. The specific goals were to validate the presence of colloidal T4 ring in amphibian thyroid follicles, to validate change of the intensity of the T4 ring upon goitrogen exposure, and to compare the sensitivity of T4 ring intensity with other biomarkers of thyroid disruption in frogs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Test materials.
Sodium perchlorate (minimum purity, 98%) was purchased from Sigma-Aldrich Chemical (St Louis, MO). Rabbit anti-T4 antiserum was purchased from ICN Biomedical (Costa Mesa, CA).
Animals.
Sexually mature male and female X. laevis were purchased from Xenopus Express (Homosassa, FL). Adults were maintained in 160-l flow-through aquaria (Aquatic Habitats, Apopka, FL) containing dechlorinated water on a 12:12 h light:dark regimen at 20 ± 2°C. Frog brittle (Nasco, Ft Atkinson, WI) was fed to the frogs three times weekly (four to six brittle nuggets per frog). The Texas Tech Animal Care and Use Committee (Lubbock, TX) approved all animal procedures.
To induce breeding, adult frogs were allowed to acclimate in 40-l glass aquaria containing 18 l Frog Embryo Teratogenesis Assay—Xenopus (FETAX) medium for 7 days. FETAX medium was prepared as outlined by Dawson and Bantle (1987): NaCl, 10.7mM; NaHCO3, 1.14mM; KCl, 0.4mM; CaCl2, 0.14mM; CaSO4, 0.35mM; and MgSO4, 0.62mM. The FETAX salts were dissolved in deionized water that had been passed through a 1.2-cu ft carbon filter immediately before use. A 50% static renewal of FETAX medium was performed daily. Spawning was induced by injecting adult X. laevis via the dorsal lymph sac with human chorionic gonadotropin (hCG, Sigma Chemical) dissolved in 0.9% NaCl as described by Goleman et al. (2002a). Immediately following hCG injection, breeding pairs were transferred to 21-l glass breeding tanks equipped with false bottoms made of silicone-coated 0.5-in. hardware mesh and left to breed overnight.
Experimental design.
Naturally fertilized eggs were obtained from five pairs of adults. Within 24 h after fertilization, 60–63 X. laevis embryos at Nieuwkoop and Faber (NF) stage 1–10 (Nieuwkoop and Faber, 1994) were transferred to individual 20-l glass aquaria containing 8 l of FETAX medium with 0, 1, 10, 100, and 1000 µg/l of perchlorate (nominal concentration). Each treatment consisted of four replicates. All treatments were maintained at 22 ± 2°C on a 12:12 h light:dark regimen and aerated continuously throughout the exposure. Larvae were fed 0.4 g of powdered frog brittle (Nasco) mixed in 2 ml of FETAX solution per tank daily beginning on posthatch day 5. Tanks were checked daily for depletion of food, and additional food was provided as needed. Half the volume of medium in each tank was replaced every 3 days. Beginning on the day of hatch, daily records were kept on the percentage of frogs showing FLE (both forelimbs visible), and percentage of metamorphosed animals (complete tail resorption; NF stage 66). Dead animals were removed and preserved in 10% neutral buffered formalin.
Ten tadpoles were randomly collected from each tank (total of 40 per treatment) at day 38 for analysis. Postmetamorphic animals (NF stage 66) were removed from the tanks between days 38 and 69 as they completed metamorphosis. All exposures ended at day 69, at which time the remaining animals were collected for meristic measurements and a total of five postmetamorphic frogs per treatment were processed for histological analysis. At sampling, animals were removed from tanks, euthanized by immersion in 3-aminobenzoic acid ethyl ester (MS-222, Sigma) at 1 g/l, rinsed in distilled water, weighed, and preserved in Bouin's fixative (EMD Chemicals Inc, Gibbstown, NJ) for 48 h followed by a 24-h flow-through tap water rinse and stored in 70% ethanol. All animals were identified per NF stage and measured for snout-vent length (SVL), hind limb length (HLL), tail length, and body weight.
Water quality.
Unionized ammonia, pH, dissolved oxygen, salinity, and conductance were monitored every week for each tank. Water temperature was monitored daily. An YSI model 85 meter (Yellow Springs, OH) was used to monitor water temperature, dissolved oxygen, salinity, and conductance, and pH was determined with an Oakton pH meter (Gresham, OR). Mean water temperature and pH were 21.1 (range 20.2–21.9) and 7.4 (range 6.9–7.9), respectively, while mean specific conductivity and dissolved oxygen were 1633.2 µS/cm (range 1522 to 1707 µS/cm) and 37.3% (range 24.7–52.5%), respectively. Total ammonia nitrogen was measured in tank water using an U.S. Environmental Protection Agency–approved Nesslerization method with a Hach spectrophotometer model DR/2010 (Loveland, CO). Ammonia was calculated from total ammonia nitrogen based upon temperature and pH (Alleman, 1998; Emerson et al., 1975). Unionized ammonia ranged from 0 to 0.22 mg/l in FETAX control medium over the 69-day exposure.
Samples of diluted stock solutions as well as aliquots of control and test tank media were collected on the day of water exchange throughout the experiment. Perchlorate concentration was verified by ion chromatography (Tian et al., 2003).
Histopathology and image analysis.
Histological procedures followed the description of Carr et al. (2003). The lower jaw was used to prepare blocks of paraffin for thyroid histopathology. Sections were cut at 5-µm thickness, processed, and stained with hematoxylin and eosin. Histological sections through the middle of thyroid glands were chosen for analysis, and at least three to seven follicles from one lobe of the gland were analyzed. Digital images of the thyroid follicles were taken with an Olympus digital camera (DP70; Tokyo, Japan) attached to a compound microscope. Measurements were conducted digitally using ImagePro Express Software (Media Cybernetics, Silver Spring, MD). Thyroid follicle cell height and semiquantitative measurements of colloid depletion were conducted following the procedures of Mukhi et al. (2005). Briefly, four follicular epithelial cells at predetermined positions (12:00, 3:00, 6:00, and 9:00 A.M.) were measured in each of three to seven follicles per frog. Mean cell height was calculated for each follicle, and the mean of all measured follicles was determined for each sample. Colloid depletion was measured using a semiquantitative method by assigning a score for each of the three to seven follicles as previously reported (Mukhi et al., 2005): 0, no colloid depletion; 1, up to 1/3 of colloid depleted; 2, up to 2/3 of colloid depleted; and 3, full colloid depletion. The mean score of all measured follicles was used for each sample.
Immunocytochemistry and image analysis.
Alternate slides prepared from the same animals used for histopathology were used for immunocytochemistry analysis. Immunocytochemistry was performed using the VectaStain Elite ABC kit (Vector Laboratories, Burlingame, CA) and followed the same procedures as previously reported for zebra fish (Mukhi et al., 2005). Briefly, the rabbit anti-T4 antiserum was diluted (1:4000) in phosphate-buffered saline (pH 7.4) containing Tween 20 (0.3%). This dilution factor was determined based on pilot trials using rabbit anti-T4 dilutions of 1:1000, 1:2000, 1:4000, and 1:8000 (data not shown). Digital images of three to seven follicles per frog were used to measure the optical density of the colloidal T4 ring at eight predetermined positions in each follicle (12:00, 1:50, 3:00, 4:50, 6:00, 7:50, 9:00, and 10:50 A.M.). Background staining was also measured in the same manner immediately outside of the thyroid gland. Background intensity was subtracted from the value of ring intensity. An average ring intensity value was obtained from all follicles measured and assigned to each frog. The specificity of the rabbit anti-T4 antiserum has been reported previously (Mukhi et al., 2005). The antiserum cross-reacts 100% with both the enantiomers of T4 but shows low cross-reactivity to T3 (1.2%) and does not cross-react with monoiodo-L-tyrosine or diiodo-L-tyrosine.
Statistical analysis.
For continuous data, such as body weight, tail length, HLL, and SVL, differences among tanks within a treatment were tested by one-way analysis of variance (ANOVA) using InStat software (GraphPad, San Diego, CA). If there were no significant differences among tank replicates, data from each tank within a treatment were pooled for further analysis. Incidence data were analyzed as percentages per tank. HLL, follicle cell height, and colloidal T4 ring optical density of all treatments were analyzed by two-way ANOVA conducted by SPSS (SPSS for Windows 11.0, SPSS Inc., Chicago, IL) using developmental stage and perchlorate concentration (treatments) as independent factors. If significant treatment or interaction effects were determined, one-way ANOVA was performed for treatment at each developmental stage. Homogeneity of variances was assessed using Bartlett's test, and normality of the data was tested using the Kolmogorov and Smirnov method. If the assumptions of parametric statistics were met, then one-way ANOVA followed by the Tukey-Kramer multiple comparison test were used. If the assumptions of parametric tests were not met, then the Kruskal-Wallis (KW) ANOVA by ranks followed by Dunn's multiple comparisons test were used. Differences in colloid depletion were tested using KW ANOVA by ranks followed by Dunn's multiple comparisons test. Spearman correlation and linear regression analysis were performed to determine the correlation between developmental stage (a nonmetric variable) and the test biomarkers. An 
value of 0.05 was used to assess significant differences. Data are reported as mean ± SEM.
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RESULTS |
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Perchlorate Analysis
Perchlorate concentrations in test solutions (n = 16) collected through the exposure were 0 ± 0.00, 0.87 ± 0.09, 8.18 ± 0.38, 93.24 ± 7.45, and 1130.71 ± 29.83 µg perchlorate/l, respectively, for the 0, 1, 10, 100, and 1000 µg/l nominal concentrations. Chi-square tests (
2 = 17.89, df = 4, p < 0.01) indicated that there were significant differences between nominal and measured perchlorate concentrations; thus, rounded mean actual concentrations of 0, 1, 8, 93, and 1131 µg perchlorate/l are reported.
Effects of Perchlorate on Growth, Development, and Metamorphosis
Although there were no significant treatment-related effects of perchlorate on body weight and SVL, there were clear effects on development and completion of metamorphosis. The maximum developmental stage of tadpoles collected after 38 days of exposure was NF stage 62, the minimum stage was 49, and the mean developmental stage was dependent upon the concentrations of perchlorate. There were significant reductions in HLL (one-way ANOVA, p < 0.001) in tadpoles exposed to 93 and 1131 µg perchlorate/l and collected on day 38. Hind limb growth measured in stage 66 animals throughout the exposure period and in unmetamorphosed tadpoles collected on day 69 was significantly reduced by 93 and 1131 µg perchlorate/l. The cumulative percentage of tadpoles exhibiting FLE or completing tail resorption, recorded daily throughout the exposure, was significantly decreased (one-way ANOVA, p < 0.001) by 93 and 1131 µg perchlorate/l (Fig. 1). None of the tadpoles exposed to 1131 µg perchlorate/l completed metamorphosis during the 69-day exposure.
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Effects of Perchlorate on Thyroid Histopathology and Colloidal T4 Ring Intensity
Thyroid follicles in frogs from all treatments were lined with a single layer of epithelial cells. At day 38, these epithelial cells were cuboidal in control or low–perchlorate concentration treatments (1 and 8 µg perchlorate/l) and columnar or high columnar in the greater perchlorate exposure concentrations (93 and 1131 µg perchlorate/l). The follicle lumens were filled with colloid in tadpoles exposed to the FETAX control medium, 1 and 8 µg perchlorate/l. Pinocytotic vesicles of colloid were formed around the lumen in all treatment groups (Fig. 2A). Colloid formation could be observed by NF stage 49 in all treatments; however, little or no colloid was qualitatively observed in the follicle lumens of later-stage tadpoles exposed to 93 and 1131 µg perchlorate/l (Fig. 2B).
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Significant increases in follicle cell height were observed in 38-day tadpoles exposed to 93 and 1131 µg perchlorate/l relative to control tadpoles (one-way ANOVA, p < 0.001) (Fig. 3A). There was a trend for increased follicle cell height in the 8-µg perchlorate/l treatment, but this trend was not statistically different from the controls (one-way ANOVA, p > 0.05). Follicle cell height was positively correlated with developmental stage in tadpoles from the control group (Spearman correlation, two tailed, p < 0.001) (Fig. 4A) as well as from the other treatments. Perchlorate-dependent effects on colloid depletion were also observed (nonparametric ANOVA, p < 0.001), with significant depletion in tadpoles exposed to 93 and 1131 µg perchlorate/l compared to controls (Fig. 3B). The degree of colloid depletion was also correlated with development stage in 38-day tadpoles from the control as well as all other treatment groups (Spearman correlation, two tailed, p < 0.05).
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Immunoperoxidase staining revealed an intense colloidal T4 ring in 38-day tadpoles exposed to 0, 1, and 8 µg perchlorate/l (Figs. 5A and 5B), but this ring was not present in tadpoles exposed to 93 and 1131 µg perchlorate/l. Two-way ANOVA indicated that there was a significant interaction between developmental stage and perchlorate concentration as independent factors, so all treatment-related intensity data were analyzed by one-way ANOVA for each stage. Within a given treatment, there were no differences in ring intensity among tadpoles of different developmental stages (one-way ANOVA, p > 0.05), except for tadpoles exposed to 8 µg perchlorate/l. In these tadpoles, mean T4 ring intensity at stage 56 and 57 (0.160 ± 0.023 optical absorbance units, n = 19) was significantly different from the values at stage 60–62 (0.3995 ± 0.074, n = 4; p < 0.05); and mean T4 ring intensity at stage 57 (0.155 ± 0.021, n = 13) was significantly lower in the 8-µg perchlorate/l treatments compared to controls (0.373 ± 0.032, n = 9; p < 0.05). Exposure to perchlorate for 38 days caused a significant decrease (one-way ANOVA, p < 0.001) in the mean T4 ring intensity of tadpoles exposed to 8, 93, and 1131 µg perchlorate/l relative to the controls (Fig. 3C). Correlation analysis indicated that there was no significant association (Spearman correlation, two tailed, p > 0.05) between developmental stage and T4 ring intensity in the control, 1-, or 8-µg perchlorate/l treatments (Fig. 4B).
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Generally, little or no colloid depletion was observed in 69-day frogs that finished metamorphosis (NF stage 66) from the control, 1-, 8-, and 93-µg perchlorate/l treatments (a total of five postmetamorphic frogs from each of these four treatments were used in this analysis; metamorphosis was not completed in any of the individuals exposed to 1131 µg perchlorate/l). However, moderate (scores 1–2) to severe (score 3) colloid depletion was observed in one control and two 1-µg perchlorate/l samples. Although colloid depletion was negligible in NF stage 66 frogs exposed to 93 µg perchlorate/l, their thyroid glands were large and contained many microfollicles. No treatment-related differences in follicle cell height or in colloid depletion were observed in 69 day, NF stage 66 frogs collected at the end of the exposure (Figs. 6A and 6B).
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Due to the presence of pinocytotic vesicles around the periphery of the colloid, the appearance of the colloidal T4 rings in X. laevis tadpoles (Figs. 5A and 5B) was not like the typical ring observed in fish thyroid follicles (Mukhi et al., 2005
). However, the rings became similar to those of fish as the frogs completed metamorphosis (NF stage 66; Figs. 5C and 5D). At 69 days, NF stage 66 frogs exposed to 8 and 93 µg perchlorate/l had depressed mean colloidal T4 ring intensities relative to that of control frogs (Fig. 6C). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Larval anurans require normal TH secretion in order to complete metamorphosis. Our finding that perchlorate exposure inhibited metamorphosis in a concentration-dependent manner is consistent with previous studies showing that environmentally relevant concentrations of perchlorate retard metamorphosis in X. laevis (Goleman and Carr, 2006
; Goleman et al., 2002a,b; Tietge et al., 2005) and Hyla versicolor (Sparling et al., 2003). In the present study, exposure to 93 and 1131 µg perchlorate/l significantly inhibited metamorphosis in X. laevis, as determined by reduced HLL and failure to observe FLE and tail resorption in larvae. The signs of reduced HLL were shown as early as 38 days after the start of the exposures. Although exposure to 93 µg perchlorate/l did not completely prevent metamorphosis, this concentration of perchlorate still significantly affected morphological end points relative to control values. The lower perchlorate concentrations (1 and 8 µg perchlorate/l) did not significantly reduce the hind limb growth and the rate of metamorphosis, although there were trends for reduced HLL in tadpoles exposed to 8 µg perchlorate/l for 38 days. Previous work has also indicated that exposure to 5 or 18 µg/l ammonium perchlorate reduced, but did not prevent, FLE and HLL in larvae exposed for 70 day (Goleman et al., 2002b). However, ammonium perchlorate may be more effective at lower concentrations than sodium perchlorate at producing outward signs of thyroid disruption in X. laevis, such as reduction in HLL and FLE (Goleman and Carr, 2006).
Histopathological changes in the thyroid gland, including follicle cell hypertrophy and colloid depletion, have been previously used to assess perchlorate effects on thyroid function in frogs (Carr et al., 2003; Goleman et al., 2002a,b; Tietge et al., 2005), fishes (Crane et al., 2005; Mukhi et al., 2005; Patiño et al., 2003), and mammals (Siglin et al., 2000; York et al., 2001a,b). In the present study, cell height was measured quantitatively, and relative colloid depletion was assessed semiquantitatively in tadpole thyroids after 38 days of exposure. Our data indicate that both end points were significantly altered in tadpoles exposed to 93 and 1131 µg perchlorate/l, and these findings are consistent with the observed effects of perchlorate on indices of development (hind limb growth) and metamorphosis (FLE and tail resorption) after 38 days of exposure. These results are similar to those of previous studies with larval amphibians, where exposure to waterborne ammonium perchlorate or sodium perchlorate during development causes follicle cell hypertrophy (Goleman et al., 2002a,b; Tietge et al., 2005), with a reported Lowest Observed Effects Concentration (LOEC) of 16 µg perchlorate/l (Tietge et al., 2005). Also, as previously reported for other anuran species (Pseudacris triseriata, Rana catesbeiana, and Rana palustris [Etkin, 1936] and Rana pipiens [D'Angelo and Charipper, 1939]) and urodeles (Taricha torosa [Uhlenhuth, 1934; Uhlenhuth et al., 1945], Ambystoma maculatum and Ambystoma opacum [Uhlenhuth, 1925]; as reviewed by Norman et al., 1987), follicle cell height in the present study was positively correlated with tadpole developmental stage. All observations of the present and previous studies of perchlorate and metamorphosis are consistent with knowledge of the endocrine regulation of metamorphosis in frogs and of the mechanisms of action of perchlorate. Namely, frog metamorphosis is under the control of hypothalamus-pituitary-thyroid axis, and THs are responsible for initiation and completion of metamorphosis (Norris, 1997). The reduced TH synthesis caused by perchlorate exposure not only impairs the initiation and completion of metamorphosis but also disrupts the negative feedback mechanism of THs on the pituitary. The disruption of feedback mechanisms causes an increased production of pituitary TSH, which in turn causes follicle cell hypertrophy and colloid depletion (Capen, 2001).
This study showed that a colloidal T4-immunoreactive ring is present in thyroid follicles of larval and postmetamorphic X. laevis. The presence of T4 immunoreactivity concentrated in a ring of colloid adjacent to follicle cells was first reported in rainbow trout, medaka (Raine et al., 2001), larval zebra fish (Wendl et al., 2002), and adult zebra fish (Mukhi et al., 2005), and a similar radioactive iodide ring was observed in rat (Wollman and Etholm, 1981), but this is the first observation of such a ring in an amphibian species. Our results also indicate that the intensity of the colloidal T4 ring is reduced in a concentration-dependent manner by perchlorate exposure. The smallest concentration at which we observed perchlorate-dependent reduction in colloidal T4 ring intensity was 8 µg/l. This concentration is more than 10-fold smaller than the smallest concentration of perchlorate (93 µg/l) capable of affecting HLL, FLE, tail resorption, thyroid follicle cell hypertrophy, and colloid depletion in the present study. Moreover, compared to measurements of follicle cell height, which presumably reflect changes in circulating TSH concentrations, measurements of colloidal T4 ring intensity provide an indirect measurement of the changes in thyroid T4 content and are, thus, more likely to reflect changes in the amount of T4 available to the organism. Although the perchlorate concentration range tested in this study might be considered too large to allow for precise calculation of LOEC values, our data nonetheless illustrate that measurements of colloid T4 ring intensity were more sensitive to sodium perchlorate exposure than other morphological and histopathological end points. Goleman et al. (2002b), using a different perchlorate salt (ammonium perchlorate), previously reported that perchlorate concentrations as low as 5 and 18 µg/l slightly inhibited FLE and HLL, respectively. The LOEC (5 µg/l) for partial inhibition of FLE by perchlorate reported by Goleman et al. (2002b) is comparable to the apparent LOEC for depletion of colloidal T4 ring intensity (8 µg/l) in the present study; however, the precise LOEC for perchlorate-induced changes in colloidal ring intensity presumably resides between 8 and 1 µg/l as other concentrations within this range were not tested and 1 µg perchlorate/l had no effect on colloid ring intensity.
Colloidal T4 ring formation in fishes is believed to represent the accumulation of bound T4 at or near the apical border of thyrocytes (Mukhi et al., 2005). Perchlorate, by virtue of reducing T4 synthesis, presumably causes less T4 to accumulate in this area of the colloid. The present study indicated that, as in fishes, measurement of the intensity of the colloidal T4 ring is a sensitive biomarker of thyroid disruption in tadpoles and young postmetamorphic amphibians. Namely, perchlorate at a concentration of 8 µg/l caused a significant reduction in ring intensity after an exposure period of 38 days, whereas exposure to 93 µg perchlorate/l completely blocked the formation of the ring in tadpoles exposed for 38 days. The smallest concentration capable of reducing colloidal T4 ring intensity in this study in X. laevis is similar to the LOEC of 11 µg perchlorate/l for colloidal T4 ring in zebra fish after a 12-week exposure (Mukhi et al., 2005). Our data are also consistent with the observations of McNabb et al. (2004), who reported that direct measurement of changes in thyroid TH content was more sensitive to perchlorate exposure than other organism-level end points of thyroid function such as circulating plasma levels of THs. Surprisingly, the intensity of the colloidal T4 ring was not correlated with developmental stage, suggesting that the utility of this biomarker for detecting perchlorate exposure is not confounded by the developmental stage of the animal. The fact that colloidal T4 ring intensity does not change with plasma TH levels during metamorphosis may be explained by the fact that measurements of T4 immunoreactivity reflect a steady state between hormone synthesis, storage, and uptake across the apical plasma membrane of follicle cells. Thus, if TH synthesis increases to the same degree as apical T4 uptake during metamorphosis, we would not expect a change in steady-state levels of T4 in colloid. In fact, storing T4 during periods of low iodide availability, such as during metamorphic climax when the gastrointestinal (GI) tract is being reorganized and tadpoles may not eat, could be beneficial to the animal.
Examination of NF stage 66 (postmetamorphic) animals collected on the last day of exposure (69 days) revealed no perchlorate-associated effects on follicle cell hypertrophy or colloid depletion, although perchlorate did disrupt the organization of thyroid follicles. This observation may be due to the hypertrophy of follicle cells in control animals at 69 days relative to control tadpoles at 38 days of exposure. Follicle cell height was positively correlated with developmental stage in our study, and follicle cell hypertrophy and colloid depletion have been reported to accompany metamorphosis in other anuran species as reviewed by Norman et al. (1987). However, marked effects of perchlorate (at both 8 and 93 µg/l) on colloidal T4 ring intensity were still evident in postmetamorphic frogs after 69 days of exposure. This suggests that despite changes in the level of T4 stored in colloid, animals exposed to 8 µg/l had sufficient circulating T4 to complete metamorphosis, as none of the end points of metamorphosis (HLL, FLE, and tail resorption) were significantly different in these animals relative to controls. This same phenomenon occurred in a few of the animals exposed to 93 µg perchlorate/l, as a small percentage of these animals also completed metamorphosis during the 69-day exposure. It is possible that animals were able to complete metamorphosis despite reductions in colloid T4 by compensating in other ways, such as increasing T4 deiodination in target tissues or by altering levels of free (unbound) circulating T4 in plasma via changes in transthyretin affinity or capacity. It is also important to note that the colloidal T4 immunoreactivity was not completely eliminated in animals exposed to either 8 or 93 µg perchlorate/l, so that animals may simply have had T4 stored in amounts sufficient to carry them through metamorphosis.
In conclusion, the results of the present study indicate that the intensity of the colloidal T4 ring can be used a biomarker of thyroid dysfunction in tadpoles and young postmetamorphic amphibians. Furthermore, the intensity of colloidal T4 ring is a more sensitive biomarker of perchlorate exposure than HLL, FLE, tail resorption, and follicle cell hypertrophy or colloid depletion. The lack of correlation between colloidal T4 ring intensity and development stage suggests that the endogenous changes in thyroid activity that accompany metamorphosis (e.g., TH synthesis, storage, and release) do not alter the amount of T4 present in the colloidal ring, at least as gauged by immunocytochemistry. Finally, although additional characterization of this biomarker is necessary before its functional significance is understood, we suggest that it also has potential to provide useful information for assessing the thyroid endocrine status of natural amphibian populations.
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NOTES |
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Disclaimer: The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of any office of the U.S. Government.
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ACKNOWLEDGMENTS |
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We acknowledge T. Anderson and J. Cañas for technical advice and use of equipment in their laboratory. This research was supported by funding from the U.S. Geological Survey Amphibian Research and Monitoring Initiative program. The Texas Cooperative Fish and Wildlife Research Unit is jointly supported by the U.S. Geological Survey, Texas Tech University, Texas Parks and Wildlife, The Wildlife Management Institute, and the U.S. Fish and Wildlife Service.
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REFERENCES |
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