U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environment Effect Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Boulevard, Duluth, Minnesota 55804
1 To whom correspondence should be addressed at US EPA, Mid-Continent Ecology Division, Duluth, MN 55804-2595. Fax: (218) 529-5000. E-mail: degitz.sigmund{at}epa.gov.
Received March 29, 2005; accepted June 15, 2005
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ABSTRACT |
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Key Words: thyroid; metamorphosis; amphibian.
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INTRODUCTION |
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Anuran metamorphosis is a complex process by which increasing levels of thyroid hormones (TH) promote the remodeling of the aquatic larvae into an adult tetrapod. Virtually every tissue in the tadpole is a target of TH (Atkinson et al., 1996; Shi, 1996
). The dramatic structural and functional changes of larval tissues during metamorphosis have been studied extensively (Shi, 2000
). Nieuwkoop and Faber (1994)
have developed a detailed description of the major developmental events for X. laevis and provided a detailed guide for staging this species. Briefly, post-embryonic development and metamorphosis can be divided into three phases: premetamorphosis, prometamorphosis, and metamorphic climax. Premetamorphosis is the period of post-embryonic development that precedes thyroid gland function and is mainly a period of growth. Development during this interval is not mediated by T4 (Dodd and Dodd, 1976
). Prometamorphosis begins at Nieuwkoop and Faber (NF), stage 54 and is characterized by the onset of thyroid gland function, rising levels of T4, and the process of T4-dependent morphogenesis (Dodd and Dodd, 1976
). Prometamorphosis continues through approximately NF stage 60 and several tissues reach their respective metamorphic fates during this phase (e.g., limbs). At NF stage 60, metamorphic climax begins which is characterized by a rapid increase in T4 synthesis and the dramatic morphogenetic events, including remodeling of structures such as the craniofacial region and gut, differentiation of the liver, and resorption of the gill and tail (Dodd and Dodd, 1976
). A large body of literature exists regarding thyroid hormone control of amphibian metamorphosis and recent work has begun to define tissue-specific T4-dependent programs of gene activation and repression in the brain, tail, and limb (Brown et al., 1996
; Buckbinder and Brown, 1992
; Denver et al., 1997
; Shi, 1996
; Wang and Brown, 1993
).
In nearly all vertebrates synthesis and release of T4 is under the control of the hypothalamohypohysial-thyroid axis (HPT). Thyrotropin releasing hormone (TRH) is released from cells in the hypothalamus and is transported via portal circulation to the pituitary, where it stimulates a sub-population of secretory cells, thyrotropes, to synthesize and release thyroid stimulating hormone (TSH). Thyroid stimulating hormone travels via systemic circulation to the thyroid gland and stimulates thyroid cells to synthesize and release thyroid hormones into systemic circulation. The HPT in adult anurans follows this general vertebrate pattern (Dodd and Dodd, 1976). However, there are differences in the larval life stage with regard to stimulation of the thyrotropes by the hypothalamus. Current interpretations suggest that corticotropin releasing factor (CRF), rather than TRH, may be the primary hypothalamic signal which initiates TSH synthesis and release (Denver, 1996
). T4 (and Triiodothyronine [T3]) associate with serum proteins and are carried throughout the body to target tissues, where they cross the cell membranes and are subject to 5' and 5 deiodinase activity. In tissues programmed to do so, the relatively-inactive T4 is converted by 5' deiodination to the active form of the hormone, T3, which binds to nuclear receptors and initiates gene transcription (Norris, 1997
). 5 Deiodinase, deiodinates the tyrosyl ring of T4 and T3 to effect the production of rT3 and di-iodothyronines, respectively, inactive forms of T4. This can result in reductions in local T3 concentration and is thought to prevent inappropriate T4 stimulation throughout the metamorphic process (Becker et al., 1997
; Huang et al., 1999
; Kawahara et al., 1999
; Marsh-Armstrong et al., 1999
). In mammals the hormones are subject to conjugation and ultimately elimination, via sulfation and glucuronidation, and catabolism of the tyrosine residue via decarboxylation and deamination (Brucker-Davis, 1998
). However, the roles of these processes in anuran metamorphosis are poorly understood.
Theoretically, normal T4 homeostasis and action can be disrupted at several sites in the pathway, including: interference of the negative feed back loop, T4 synthesis, T4 transport, metabolic conversion of T4 to active and inactive forms, receptor-mediated effects, and nonspecific effects. However, xenobiotic chemicals which are known to interfere with normal T4 homeostasis and action have been shown to act primarily by producing hypothyroidism via (1) inhibition of iodide uptake, (2) inhibition of T4 synthesis, (3) inhibition and/or up-regulation of deiodinases, or (4) up-regulation of catabolism of T4 (Brucker-Davis, 1998; DeVito et al., 1999
). Much of our current understanding of xenobiotic action on the thyroid axis comes for work which primarily has been conducted in mammalian species (Brucker-Davis, 1998
). Before an amphibian metamorphosis screen can be implemented, an understanding of the response to known antagonists must be developed in order to demonstrate that mechanisms which are operative in higher vertebrates will be picked up by the amphibian screen.
Until very recently there has been very little evidence that xenobiotic chemicals act by agonism or antagonism of the thyroid hormone receptors (TR) (DeVito et al., 1999). However, Kitamura et al. (2002)
have recently shown that the flame retardants, tetrabromobisphenol A and tetrachlorobisphenol A, have agonistic activity of the TR. Further Schapira et al. (2003)
demonstrated TR antagonism by groups of compounds with diverse chemical structures. Given this recent finding it is imperative that an amphibian thyroid screen also be capable of detecting xenobiotic interactions with the receptor.
In an effort to provide information for improving the EDSTAC recommended protocol we have conducted studies aimed at defining the organismal and thyroidal response of X. laevis to model chemicals which induced hypothyroidism. Towards this end we have conducted experiments with the classical vertebrate thyroid hormone synthesis inhibitors methimazole and PTU. These chemicals were selected because of their well-defined mechanisms of action in vertebrates and their demonstrated effectiveness at disrupting the amphibian HPT (Goos, 1968; Goos et al., 1968
; Iwasawa, 1958
). Further, we have examined the organismal response to the TR agonist T4. We have focused on defining the organismal response as a function of when in the metamorphic process the exposure is initiated and length of the exposure period. The studies described here are part of a much larger effort aimed at making the necessary improvement to the original screening protocol and to develop the necessary data to establish confidence in the amphibian assay as a vertebrate screen.
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MATERIALS AND METHODS |
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Experimental Design
Three main experimental objectives were to examine the effects of different test chemicals on thyroid histology and developmental rate, determine the differential sensitivity of stage 51 and 54 larvae and to establish the necessary duration of exposure. The primary endpoints were developmental stage and thyroid histology, though observations of mortality and growth were also made. To achieve these objectives, we employed two experimental approaches.
Experimental approach 1 (methimazole and PTU experiment 1).
Two concurrent studies were conducted with individuals from the same cohort of organisms for each chemical. One was initiated with stage 51 larvae and the other with stage 54 larvae. Both exposures were conducted for 14 days. Methimazole and PTU were tested in two independent experiments using animals from different breeding pairs. Eighteen days post-fertilization, larvae were anesthetized using 100 mg/l of MS-222 buffered with 200 mg/l of sodium bicarbonate, sorted by stage (Nieuwkoop and Faber, 1994). After recovery from the anesthesia, 240 stage 51 tadpoles were randomly placed into 12 tanks (20 tadpoles/tank). Remaining tadpoles (NF stages 52 and 53) were placed back in clean water. After four days organisms were anesthetized, confirmed to be at stage 54, and used to start (using similar methods described for the stage 51 tadpoles) the stage 54 portion of each study. Stage 51 and 54 larvae were exposed to five concentrations of methimazole (6.25, 12.5, 25, 50, and 100 mg/l) or PTU (1.25, 2.5, 5, 10, and 20 mg/l) and a control. Each test concentration and control were replicated with 20 organisms per replicate.
Once in the test system mortality observations were made daily and any dead larvae were removed. Water samples were taken at least three times throughout each test and measured for the respective test chemicals. The measured water concentrations were very near nominal concentrations for all experiments and so the effects data are presented below as nominal (Table 1). On exposure day 8, three organisms per replicate (six organisms per treatment) were randomly selected, anesthetized in MS-222, evaluated for developmental stage (investigator was blind to treatment group), weighed, and preserved in Bouin's solution for histological analysis of the thyroid glands. On exposure day 14, all remaining organisms in the study were similarly treated, with five organisms per replicate (ten per treatment) preserved for histological analysis. Other procedures or methods (dissolved oxygen, hardness, pH) not specified followed those recommended by the American Society for Testing and Materials (ASTM, 1995).
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Once in the test system mortality observations were made daily and any dead larvae were removed. Water samples were taken at least three times throughout each test and measured for the respective test chemicals. Chemical concentrations were measured at multiple times throughout the exposure period in both the A and B replicates (Table 1). The measured water concentrations were very near nominal concentrations for all experiments. Further, chemical concentrations were very consistent throughout the exposure period as indicated by the small standard deviation and showed very good agreement between replicate tanks. As a result the effects data are presented below as nominal. At the end of the exposure period organisms where anesthetized in MS-222, evaluated for developmental stage (investigator was blind to treatment group), weighed, and preserved in Bouin's solution for histological analysis of the thyroid glands. Other procedures or methods (dissolved oxygen, hardness, ph) not specified followed those recommended by the American Society for Testing and Materials (ASTM, 1995).
Toxicant Solutions
Test chemicals (T4, methimazole, and PTU) were obtained from Sigma Chemical Co. (St. Louis, MO). Stock solutions for methimazole were made in a 19 L glass carboy by dissolving the chemical in Lake Superior water using a stir plate and a magnetic stir bar. Stock solutions for PTU were made in a 19 L glass carboy using a high speed top stirrer to dissolve the chemical in Lake Superior water that had been previously heated to 40°C. T4 stock solutions (0.0256 mg/l) were prepared by dissolving 4.91 mg T4 in 15 ml NaOH (50 mm) and diluting to 196 in Lake Superior water.
Exposure System
A computerized exposure system was used for all studies. This system, whose components are glass, stainless steel, and teflon, generated five duplicated exposure concentrations for each chemical with a dilution factor of 0.5 for methimazole and PTU, as well as duplicate controls. Exposure tanks were glass aquaria (22.5 x 14.0 x 16.5 cm deep) equipped with 13 cm standpipes, which resulted in an actual tank volume of 4.0 l. The flow rate to each tank was 25 ml/min. Fluorescent lamps provided a photoperiod of 12 h light:12 h dark at an intensity that ranged from 61 to 139 lumens at the water surface.
Water Characteristics
Lake Superior water used for all tests was filtered through sand, a 5-micron filter, a 0.45-micron filter, sterilized with ultraviolet light and heated to the appropriate test tank temperature of 20.9 ± 0.2°C (n = 588). Exposure tanks were immersed in a water bath system (water bath temperatures were continuously monitored) to maintain temperature uniformity between tanks. Dissolved oxygen (DO) was measured weekly during all tests using a dissolved oxygen meter (calibrated prior to use by the air saturation method) on a minimum of twelve exposure tanks. The mean, SD, and range of DO measurements were 7.04 ± 0.64 mg/l (5.847.88 mg/l) n = 72. All other water characteristics were measured using methods described by the APHA (1992). The mean, SD, and range of pH readings (conducted weekly on a minimum of twelve tanks during all tests) was 7.7 ± 0.1 (7.577.92) n = 72. Hardness and alkalinity determinations were made on a minimum of three tanks (one control, one intermediate, and one high concentration tank) once during each study. The mean and range for total hardness was 47.3 (46.547.5) mg/l as CaCO3 (n = 4) for all analyses. The mean and range for all alkalinity measurements was 40.8 (40.541.0) mg/l as CaCO3 (n = 4).
Chemical Analysis
Methimazole and PTU.
Water samples (900 µl) collected from the exposure chambers were placed into vials containing methanol (100 µl), mixed and immediately analyzed for methimazole or PTU. Analyses were conducted using a Hewlett-Packard 1050 HPLC equipped with a diode-array detector ( 258 nm for methimazole,
276 nm for PTU). An aliquot of sample (25 µl) was injected directly into a Lichrosorb RP-18 column and the column was eluted with a 0.025 M phosphate buffer (pH 3) and methanol gradient program at a flow rate of 1 ml/min. The run time per injection was 30 min. The retention times for methimazole and PTU were 9.2 and 14.8 min, respectively.
T4.
Water samples collected from the exposure chambers were immediately analyzed for T4 by high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). For initial analyte separation, an Agilent 1100 series HPLC system (Wilmington, DE) was used. An aliquot of sample (75 µl) was injected directly into a Synergi Hydro RP, 4 µ (50 x 3.0 mm) column (Phenomenex, Torrance, CA) that was maintained at 23°C. The column was eluted with a phosphate buffer (10 mM, pH 3.0) and methanol gradient program at 0.45 ml/min. The column effluent was connected to a high pressure micro-splitter valve where it was mixed with a post column reagent (10% nitric acid:90% water) delivered from an isocratic pump at 0.55 ml/min. The post column reagent was used to reduce the methanol concentration introduced into the ICP-MS (Michalke et al., 2000). The combined HPLC effluent and post column reagent (combined flow of 1.0 ml/min) was connected directly to the nebulizer of a Varian UltraMass ICP-MS system (Mulgrave, Victoria, Australia) for subsequent iodine detection.
The ICP-MS was equipped with a Sturman-Masters spray chamber and a demountable torch with a small bore (0.8 mm i.d.) injector tube. The plasma, auxiliary and nebulizer argon gas flow rates were 15.5 l/min, 1.25 l/min, and 0.83 l/min, respectively. The ICP-MS was operated in the time-resolved mode and iodine was determined at m/z of 127 with a scan time of 100 msec. The time resolved data was imported into GRAMS/32 AI (version 6.00) chromatography software (Thermo Galactic, Salem, NH) for chromatogram generation and peak smoothing and integration. Microsoft Excel 97 (Redmond, WA) was then used to determine iodine concentrations (i.e., ng I/ml) for T4 using the external standard method of quantification with a 5 point linear calibration curve. Iodine concentrations were converted and reported as concentrations of ngT4/ml. Recovery of T4 in the spiked water samples was 101%. The agreement of duplicate samples was 100%.
Histological Procedures
Following the anesthetization of the organisms sampled for thyroid histology, the organisms were decapitated caudal to the eyes and were fixed in Bouin's fluid for approximately 48 h, then dehydrated and embedded in paraffin using standard histological procedures. The cut face of the tissues were oriented toward the leading edge of the paraffin block and sectioned at 58 µm and stained with hematoxylin and eosin. Histological slides were blindly evaluated for the presence follicular cell hypertrophy, follicular cell hyperplasia, morphology of the follicle, and colloid depletion criteria described by Hooth et al. (2001). Presence of a given lesion within an individual tadpole resulted in assignment of a value of 1 such that if the gland contained all four lesions the gland received a score 4. Individual scores were used to generate a mean severity score for each treatment group. The frequencies of the given lesions within a treatment are also presented.
Statistics
Developmental stage was analyzed based on the distribution of stages at 8 and 14 days of exposure for both tests conducted with stage 51 and 54 larvae. The non-parametric Kruskal-Wallis one way analysis of variance was conducted on the entire data set for each time and initial stage. When this analysis was significant (p 0.05), pairwise comparisons between treated and control treatments were conducted using the Dunn's method (1-tailed test), with a significance level of p
0.05. In the case of all three chemicals tested exposure resulted in a shift in developmental stage relative to the controls at one or more of the test concentrations. As a result the controls and treated animals were at different developmental stages and in some cases there were no common stages observed. Given that organism weight is related to the developmental stage we could not develop a rational statistical strategy for making weight comparisons. Instead the weight data are presented simply as treatment means.
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RESULTS |
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Significant inhibition in development was observed in stage 51 at 10 mg/l and above and in stage 54 larvae at 20 mg/l (Tables 5 and 6). In the stage 51 exposure, controls developed to stages 5557 with the majority (91%) reaching stage 57. At the highest treatment larvae developed to stages 55 and 56 with the majority (91%) being stage 55. At intermediate concentrations organisms spanned four developmental stage, 5457. In stage 54 larvae, the controls spanned four developmental stages (stages 5760) with stage 58 and 59 being nearly equally represented at 38 and 44%, respectively. At the highest concentration, larvae progressed to developmental stages 56, 57, and 58 with the majority (65%) at stage 56. The span of developmental stage at intermediate concentrations fell between the controls and the highest treatment.
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Effects of T4 Treatment (Experimental Approach 2)
Growth and mortality.
Growth and mortality were calculated when the test was terminated. T4 treatment only resulted in an increase in mortality relative to controls at 4 µg/l (15%) when exposure was initiated at stage 51. Weight was also measured at test termination and T4 treatment resulted in a decrease in mean body weight (Table 7). T4 accelerated development and the organisms at the higher concentrations were at more advanced stages of development. The observed weight loss is consistent with the decrease in weight that is observed in organisms proceeding through metamorphic climax.
Stage 51 developmental effects.
The range of developmental stages achieved in controls after 21 day was quite variable with organisms at 6 different stages 5762, with the majority (37.5%) at stage 59 (Table 9). Variability was consistent across the lower treatments with organism spanning 6 developmental stages. There was a decrease in the number of developmental stages represented with increasing TH concentrations (Table 9). At the highest test concentration (4 µg/l) larvae developed to stages 6365 with the majority (44.2%) being at stage 64. Significant acceleration in development was observed at 2 and 4 µg/l T4.
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DISCUSSION |
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Methimazole and PTU were used to produce depletion in T4 levels. Methmizaole and PTU are well characterized inhibitors of T4 synthesis in a number of species and have been shown to retard the metamorphic process in amphibians (Callery and Elinson, 2000; Goos et al., 1968
; Iwasawa, 1958
). It is well established that both methimazole and PTU have the ability to inhibit TH synthesis by blocking thyroid peroxidase coupling of iodine to the tyrosine precursor contained within thyroglobulin (Davidson et al., 1978
; Engler et al., 1982a
,b
; Nagasaka and Hidaka, 1976
). In addition to this activity, PTU also has been shown to be an inhibitor of deiodinase activity in a number of species (Kohrle et al., 1987
).
Table 10 compares the test chemical concentrations at which significant effects on developmental rate were observed. In the case of methimazole and T4, the stage of initiation had no impact on the concentration at which significant effects on developmental rate were observed. Results with PTU, the chemical for which we have the most data, indicate that if the exposure is held to 14 days for both life stages, stage 51 is slightly more sensitive. However, if the test duration is extended to 21 days for stage 51 and compared to test started with stage 54 larvae exposed for 14 day, stage 51 is slightly less sensitive. In addition to the experiments described here we have conducted experiments with the model iodide uptake inhibitor perchlorate in which we examined the impact of extending exposure period to include metamorphic climax (Tietge et al., 2005). Surprisingly, this did not result in dramatic increase in sensitivity for detecting HPT inhibition. Previous work with amphibians indicates that later stage prometamorphic tadpoles are less sensitive to the T4 synthesis inhibitors thiourea (Iwasawa, 1956
) and methimazole (Callery and Elinson, 2000
). By stage 60, X. laevis tadpoles become almost completely insensitive to classic vertebrate T4 synthesis inhibitors, such as methimazole and perchlorate, (Denver et al., 1997
; Goleman et al., 2002
). The combination of our data and this published literature leads us to conclude that there is little to be gained from examining the sensitivity of tadpoles between stage 54 and 60. Further, we have shown that increasing the duration of the exposure does not greatly increase sensitivity. In fact, the data suggest that lengthening the exposure may reduce sensitivity due to increased variability in tadpole development.
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An apical endpoint is desirable because it integrates the effects of a chemical over the entire HPT and ultimately aids in predicting the hazard associated with a given compound. However, a change in the developmental rate alone is not a sufficient indicator of thyroid axis disruption. In fact, chemicals that cause a delay in development may impact processes unrelated to the thyroid axis (i.e., energetics and metabolism) yet retard development. The lack of diagnostic specificity in a test that solely relies on apical measures will invariably yield a high percentage of false positive results, thus limiting the utility of this assay as a screen designed to decrease further testing. This necessitates the development of additional measures which will aid in confirming the action of a chemical on the HPT. In the case of the HPT, the thyroid gland itself has considerable potential in this regard. The HPT responds to decreased circulating T4 by increasing the release of TSH which in turn acts on the gland to increase T4 synthesis (Norris, 1997). Prolonged depletion of blood T4 concentrations is accompanied by follicular cell/glandular hypertrophy, which is readily detectable using conventional histological procedures.
Using model inhibitors, we have begun to establish thyroid gland histology as a diagnostic indicator of HPT disruption in Xenopus. Both methimazole and PTU caused concentration-dependent histological changes in the thyroid tissue sampled after eight days of exposure. As was the case for both chemicals, thyroid histology was a slightly more sensitive indicator of HPT disruption than the apical measures (Tables 2 and 5). These results are consistent with those for perchlorate in which thyroid histology was shown to be a more sensitive indicator of thyroid axis inhibition (Tietge et al., 2005). Although methimazole and PTU caused similar effects on the follicular cells (hypertrophy/hyperplasia) at higher concentrations, qualitative differences of the thyroid follicles were observed. PTU exposure resulted in an increase in the size of the follicles. These findings with PTU are consistent with, although apparently less dramatic than, those reported by Goos et al. (1968)
, following exposure to 100 mg/l. Methimazole exposure, by contrast resulted in collapsed/larger follicles and a loss of colloid at higher concentrations. Iwasawa (1958)
has reported that treatment of Rhacophorus schlegelii larva with 125 mg/l methimazole resulted in severe glandular and follicular cell hypertrophy. However, no mention was made as to the morphology of the follicles or the colloid. In addition to the results presented here for PTU and methimazole we have reported elsewhere on the concentration dependent effects of perchlorate on thyroid gland histology (Tietge et al., 2005
) In contrast to PTU and methimazole, perchlorate exposure results in a concentration dependent increase in glandular hypertrophy associated with loss of colloid and collapse of follicles even at low test concentrations. These apparent differing responses are likely the result of a combination of factors, including the differing modes of action of the test chemicals, time at which the effective concentration is achieved at the site of action in the thyroid gland and impact of TSH on the thyroid tissue directly. In the case of both the T4 synthesis inhibitors, changes in thyroid histology are consistent with reduced circulating T4 which results in TSH elevations and stimulation of the thyroid gland in order to maintain circulating T4 concentrations.
In summary, we describe the initial phase of development of a X. laevis screening model for evaluating chemicals' abilities to disrupt the HPT. Development of this assay is by no means complete and there are additional aspects which must be considered before a screening protocol can be proposed. In the current studies we have made progress towards understanding stage sensitivity and an appropriate exposure duration for X. laevis. However, before a decision can be made as to the specifics of the screening protocol it must be demonstrated that the organismal and thyroidal responses reported here for PTU, methimazole and T4 are consistent with the response to chemicals which produce hyperthyroidism/hypothyroidism via other mechanism. Thus, experiments directed at investigating these additional plausible mechanisms are still needed.
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NOTES |
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ACKNOWLEDGMENTS |
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