* Department of Biology, Virginia Tech, Blacksburg Virginia 24061-0406; Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Virginia Tech, Blacksburg VA 24061-0443
Received May 27, 2004; accepted August 4, 2004
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ABSTRACT |
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Key Words: perchlorate; thyroid; quail chicks; exposure time.
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INTRODUCTION |
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Recently we have investigated the effects of drinking AP solutions, ranging from 50 µg/l to 4,000 mg/l, on thyroid function of bobwhite quail chicks (McNabb et al., 2004). These studies showed that thyroid gland thyroxine content (thyroidal T4), which accounts for >95% of the gland stores of TH, was the most sensitive indicator of altered thyroid function. After 2 weeks of exposure to AP, thyroidal T4 was decreased in relation to increasing AP concentration in the drinking water for the range from 50 µg/l to 4000 mg/l. Thyroidal T4 decreased from 85% of that in controls at .05 mg/l AP, to 54% at 50 mg/l, to <12% at 5004000 mg/l. The decrease in thyroidal T4 was apparent at much lower AP concentrations than was the case for alterations in thyroid weight or plasma T4 concentrations. This study also included one 8-week experiment with concentrations of 2504000 mg/l AP in drinking water. Comparison of the 8-week study with the 2-week studies suggested some degree of adaptation in thyroid function with sustained exposure to AP.
Our objectives in this study were to describe further the effects of different times of AP exposure on thyroid function in bobwhite quail chicks. We used bobwhite quail (Colinus virginianus) which are a United States Environmental Protection Agency-approved model for studies of avian toxicology. As ground-dwelling birds, quail are dependent on local water supplies, so they experience the full impact of highly contaminated local water sources and of any perchlorate present in plants growing in the same region. Birds such as quail typically obtain most of their water by drinking (80%), with preformed water in their food and metabolic water contributing much smaller proportions of their water needs (
4% and 16%, respectively) when they are eating a seed diet (McNabb 1969
). These proportions may change somewhat if birds include insects in their diets at certain times of year, or if drinking water becomes limiting and they shift to a higher proportion of succulent dietary plants (for a general review of water balance in birds, see Bartholomew and Cade 1963
).
As in our previous study of AP effects in bobwhite chicks, we evaluated organismal thyroid status (plasma T4 and triiodothyronine [T3]), activation of the hypothalamic-pituitary-thyroid (HPT) axis (thyroid weight) and TH stores (thyroidal T4 and T3 content) over a wide range of AP concentrations (12.5 µg/l4000 mg/l) in drinking water and for three exposure times (2, 4, and 8 weeks). Our results are discussed in relation to two hypotheses: (1) that AP exposure will decrease plasma T4 concentrations, increase thyroid gland weight, and decrease thyroidal T4 content in quail and (2) that quail will show some degree of adaptation (compensation) in their thyroid function in response to sustained AP exposure.
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MATERIALS AND METHODS |
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Ammonium perchlorate was administered in distilled water. We did not observe any differences in drinking rates or any taste aversion to any perchlorate concentration used in these studies. Drinking rates were not measured. Quail chicks were exposed to AP concentrations from 12.5 µg/l to 4000 mg/l in drinking water (see Experimental Design, below). We estimate that doses ranged from 0.96 µg AP/kg per day (at 12.5 µg/l AP) to 308 mg AP/kg per day (4000 mg/l AP), assuming an ad libitum drinking rate of 7.7% of body weight/day as measured for bobwhite quail under thermoneutral conditions (McNabb, 1969). Exposures are expressed as mg/l of NH4ClO4 for comparison with common expressions in the literature; perchlorate ion represents 84.6% of the weight of AP. Ammonium perchlorate concentrations in the water were not measured. Fresh AP solutions were prepared at 23-day intervals and drinking devices were cleaned and replenished with AP solutions daily.
Experiments. To investigate the effects of sustained AP exposure on thyroid function in quail, after different lengths of time, we exposed quail chicks to 0, 0.05, 0.5, 50, and 250 mg/l AP in drinking water and measured thyroid variables at 1, 2, 4, and 8 weeks of exposure (Experiment 1). This experiment was used to explore the time course of perchlorate effects. Subsequently, because neither facilities nor sampling constraints allowed testing of all the AP concentrations or times that needed to be investigated within a single experiment, we combined the data from Experiment 1 and a series of additional experiments (Experiments 26; Table 1) employing overlapping ranges of AP in drinking water (from 0.0125 mg/l to 4000 mg/l). All studies were initiated at 34 days posthatch. A previous article reported our studies of 2-week exposures and one 8-week exposure (McNabb et al. 2004). This article reports on additional studies of 4-week and 8-week exposures and includes the data previously reported to examine the effects of AP for different durations. The combined experiments are shown in Table 1. The high AP exposures were used to establish the nature of perchlorate effects on thyroid function in birds, and as a frame of reference for investigations of lower AP exposures and more sustained exposure times. Each experiment included control birds (drinking distilled water) and one or more AP concentrations that showed thyroid effects in a previous study. The purpose of this overlap was to evaluate the consistency of effects in multiple studies.
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Organismal thyroid status was evaluated by measuring T4 and T3 concentrations in plasma by radioimmunoassay. In brief, plasma T4 and T3 concentrations were determined by a double antibody radioimmunoassay on duplicate aliquots (12.5 µl for T4; 25 µl for T3) for each plasma sample using the methods of Wilson and McNabb (1997). Primary antibodies were purchased from Esoterix, Endocrinology (Calabasas Hills, CA), 125I-labeled hormones (high specific activity; 1200 µCi/µg) were from PerkinElmer Life Sciences (Boston, MA). Two levels of Lyphochek Immunoassay Plus Control Serum from BioRad (Irvine, CA) were included with each assay to evaluate assay performance. We validated the radioimmunoassays, which use standards prepared in hormone-stripped chicken plasma, for hormone measurements on bobwhite quail plasma by demonstrating parallelism between the standard curve and diluted and hormone-spiked samples of bobwhite plasma. Precision tests on a pooled plasma sample indicated that ± 2 SE was 3.1% of the mean for T4 (n = 6) and 2.6% of the mean for T3 (n = 6). The lower limit of sensitivity of the T4 assay was 1.25 ng/ml, for T3 it was 0.125 ng/ml.
Hypothalamic-pituitary-thyroid axis activation was evaluated by weighing thyroid glands. This indirect measurement of HPT axis activation assesses feedback stimulation of pituitary thyrotropin release and was used because assays for avian thyrotropin are not available and radioimmunoassays that use heterologous antibodies (directed against thyrotropin from other vertebrate classes) do not effectively measure avian plasma thyrotropin (unpublished studies in our laboratory).
Thyroid gland stores of THs were measured by digesting glands with a protease to release T4 and T3 from within thyroglobulin, then extracting the THs in ethanol and measuring them in the extract by radioimmunoassay (RIA; method of McNabb and Cheng 1985). Thyroid tissue (5 mg or less) was digested in 350 µl of digestion medium containing 25 mg of Pronase (Sigma-Aldrich Chemical, St. Louis, MO) at 37°C in a shaking water bath for 24 h. Following the digestion, 1.0 ml of absolute ethanol was added, the tubes were vortexed, and the THs were extracted for 24 h at 20°C, then centrifuged at 13,500 x g for 5 min. The supernatant was removed and stored at 20°C until analysis. Dilutions of the supernatants, in 75% ethanol, were analyzed for T4 and T3 by RIA as described above, except that the standards were prepared in 75% ethanol.
Statistical analyses. Doseresponse data within individual experiments were analyzed using linear regression of either raw data or log-transformed data, if the relationship was exponential. When differences between the thyroid responses for exposure to different AP concentrations was indicated by analysis of variance (ANOVA), Fisher's Protected Least Significant Difference test was used to determine which treatments differed from control values. Before combining experiments, we determined that the thyroid responses, compared to controls, were the same for AP concentrations used across different studies; then the combined studies were analyzed as described above for each exposure time (2, 4, and 8 weeks). Assumptions of the methods used were checked and they hold. Probabilities of p < 0.05 were considered indicative of statistically significant relationships between thyroid variables and AP concentrations, and between treatments analyzed by analysis of variance (ANOVA). Because these statistical analyses were done after a series of experiments, and because there were missing "cells," the data were not suitable for analysis by two-way ANOVA with time as a variable.
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RESULTS |
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Thyroidal T4 per gland pair was systematically decreased with increasing AP concentrations in the drinking water at 1, 2, 4, and 8 weeks of exposure (Fig. 1). The decreases were greatest at 2 weeks with chicks on all treatments 0.5 mg/l AP having significantly lower thyroidal T4 than controls. By 4 and 8 weeks, chicks on the three lowest AP concentrations showed restoration of thyroidal T4 content to levels equal to those in control birds, and only chicks drinking 50 and 250 mg/l AP had significantly less thyroidal T4 than controls. At the highest AP concentration used in this study (250 mg/l), chicks had marked, significant thyroidal T4 depletion at all sampling weeks. The magnitude of the AP effect on thyroidal T4 increased up to 4 weeks of exposure, then there was some adaptation to sustained AP, suggested by the partial restoration of thyroidal T4 between weeks 4 and 8 (week 1, 44.8% of controls; week 2, 33.7%; week 4, 21.9%; week 8, 50.4%). At the second highest AP concentration used, 50 mg/l AP, thyroidal T4 was significantly depleted compared to controls at 2 and 4 weeks, but by 8 weeks the thyroidal T4 content had increased and did not differ from controls. Weight-specific thyroidal T4 content as well as thyroidal T3 (both weight specific and per pair of thyroids) showed an essentially identical pattern to that of thyroidal T4 content per gland pair (data not shown).
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Thyroid gland weight increases were significantly related to increased drinking water AP concentrations. ANOVA indicated that gland weights were significantly greater than those of controls at 500 mg/l at 2 weeks and at
1000 mg/l AP at 8 weeks; AP concentrations in these ranges were not included in any of the studies with sampling at 4 weeks (Fig. 3).
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DISCUSSION |
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Plasma T4 concentrations, which reflect organismal thyroid status, might be expected to be the most useful measures for evaluating thyroid disruption. However, in all our experiments, plasma T4 concentrations were highly variable. We speculated previously that this variability occurs because of the cycling of HPT axis responses to plasma THs (McNabb et al., 2004). Thus, if circulating THs decrease as a result of exposure to a chemical such as perchlorate, feedback to the HPT axis results in increased thyrotropin release, which in turn stimulates all aspects of thyroid gland function including the release of stored THs. When this release of stored hormone results in euthyroid (or even elevated) levels of circulating THs, the axis response will decrease. This intermittency of feedback signals and HPT axis responses may explain why thyroid hypertrophy (which requires sustained thyrotropin release), like plasma THs, is not a sensitive indicator of developing hypothyroidism. Cycling of HPT responses is known to occur in mammals with iodine deficiency (Delange and Ermans, 1996
) and appears to occur in laboratory mammals exposed to perchlorate (York et al., 2001
). This cycling in circulating TH patterns could be verified by daily plasma sampling using chronically implanted catheters and a larger avian model. This technique has been employed for studies of pulsatile patterns of hormone release in the growth hormone axis in domestic chickens and turkeys, which are large enough that sampling at short regular intervals is not harmful (see, for example, Bacon et al. 1989
; Vasilatos-Younken et al. 1990
).
In addition to considering T4, the predominant TH, we also evaluated T3, which appears to be the most metabolically active TH in birds (for a review, see McNabb, 2000), as it is in mammals (Engler and Berger, 1984). Patterns of T3 both in the thyroid gland and in the circulation gave essentially identical indications of AP effects to those for T4 in bobwhite quail chicks. The pattern of increases in thyroidal T3/T4 ratios with increasing concentration of AP and increasing exposure times further emphasizes the effects of the AP-associated iodine deficiency in the thyroid. This effect also is seen in mammals under iodine-deficient conditions (Taurog, 1996
). Overall, measurements of T3 give similar information, or slightly less sensitive information compared to T4.
Our first hypothesis, that plasma T4 concentrations would be decreased, thyroid gland weights would be increased, and thyroidal T4 content would be decreased was supported by the exposures of quail chicks to very high AP concentrations (>10004000 mg/l for 2 and 8 weeks; see combined studies in Figures 2, 3, and 4 and note that the studies at 4 weeks did not include this range of AP concentrations). However, as discussed above, thyroid function is altered at much lower AP concentrations (down to 0.05 mg/l), as indicated by depleted thyroidal T4 content. Thus, thyroidal T4 stores play an important role in homeostatic responses that maintain organismal euthyroidism, for at least some time, at many of the AP concentrations used in this study.
Our second hypothesis, that young quail show some degree of adaptation in their thyroid function in response to sustained AP exposure, also was supported by this study. Our preliminary study (Experiment 1), with 0.05250 mg/l AP in the drinking water and sampling at 1, 2, 4, and 8 weeks, provided some evidence that bobwhite chicks could partially compensate over time for the initial effects of AP exposure (Fig. 1). In this study, thyroidal T4 content was depleted in relation to AP exposure during the first 2 weeks; then, partial or complete restoration of thyroidal T4 was seen by 8 weeks of AP exposure, depending on the specific AP concentration. It should be noted that this compensation is against the background of developmental increases in thyroidal hormone content that are occurring (thyroidal T4 increased 6.1 times between weeks 1 and 8 of this study).
The pattern shown by combining data from Experiments 27 provides additional evidence that some adaptation in thyroid function occurs in quail chicks with sustained AP exposure. The degree of this adaptation in relation to AP exposure time is concentration dependent and this is illustrated in Figure 6, in which the AP concentrations we used are separated into three concentration categories to show the different responses to AP exposure with time. Thus at the lowest range of AP concentrations tested (0.0135 mg/l) at 2 weeks of exposure there were decreases in TG-T4 of as much as 50%, as shown in Figure 6a). At 4 weeks, the TG-T4 content was partially restored (Fig. 6b), and at 8 weeks (Fig. 6c) there was an overshoot with TG-T4 content averaging about 25% above that of controls for this range of AP exposures. These data suggest that increased thyrotropin in AP-exposed birds (from HPT axis responses that are too subtle to be reflected by thyroid hypertrophy), stimulated thyroid gland functional capacity resulting in greater TH synthesis and storage, thereby compensating for the effects of AP in this concentration range. Thyrotropin is known to upregulate the thyroidal sodium-iodide symporter in rats (Ohmori et al., 1998), and it seems likely that the same effect occurs in birds. In addition, increases in iodide trapping are known to occur in association with iodine deficiency in both laboratory mammals and in humans under clinical conditions (Delange and Ermans, 1996
). An increase in iodide symporters should increase the absolute amount of iodide uptake, and that could, in turn, enhance TH synthesis and storage. In addition, increases in iodide trapping in association with iodine deficiency are known to occur in both laboratory mammals and human clinical conditions (Delange and Ermans, 1996
). This explanation of apparent thyroid adaptation is compatible with our data in chicks exposed to AP concentrations up to 5 mg/l in this study.
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The adaptation to sustained exposure to AP that we observed in growing chicks suggests developmental plasticity of the thyroid system during a period when thyroid capacity for hormone synthesis is increasing (McNichols and McNabb, 1988). One of the key factors involved in the adaptive responses may be increasing iodide trapping capability of the thyroid during development. For example, 1-day-old Japanese quail have about 3x the thyroidal iodine content of 14-day embryos (Stallard and McNabb, 1990
). Thus, as thyroidal iodide trapping increases with age, the resulting increases in thyroid iodide content may play a role in adaptation to sustained AP exposure. Differences in iodide availability are known to interact with perchlorate effects in mammals (Wolff 1998
), but there have been no studies of these interactions in birds.
Our observation that quail chicks can adapt to some perchlorate exposures raises the question of whether this adaptation occurs at all life stages or whether it is present only in developing birds. We have investigated this in adult bobwhite quail and found no evidence of adaptation in thyroid function for up to 8 weeks of AP exposure. A preliminary report of these findings has been presented (McNabb and Queral-Kirkpatrick, 2003). This comparison suggests there may be developmental plasticity of thyroid function in young quail that is not present in adult birds.
It should be noted that perchlorate concentrations like those that caused sustained organismal hypothyroidism in our studies have been measured in local water sources at highly contaminated sites (Urbansky, 1998) and would cause severe alterations in thyroid function in ground dwelling birds using these water sources. At some lower AP concentrations, this study suggests that young birds may, over time, adapt to sustained perchlorate exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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Parts of this work were presented orally at SETAC meetings: McNabb, F. M. A., G. A. Fox, and K. A. Grasman. 2002. Comparison of variables for evaluating pollutant effects on thyroid function in birds. (Presentation at SETAC, Abstr. 588, p. 128); McNabb, F. M. A., L. T. Queral-Kirkpatrick. 2003. Perchlorate exposure level, exposure time and perchlorate-associated cation effects in birds. (Presentation at SETAC, Abstr. 250, p. 57).
This work has been submitted to Toxicological Sciences for possible publication. Exclusive license to publish may be granted without notice, after which this version may no longer be accessible.
1 To whom correspondence should be addressed at Department. of Biology, 2119 Derring Hall, Virginia Tech, Blacksburg, VA 24061-0406. Fax: (540) 231-9307. E-mail: happy{at}vt.edu
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