ToxSci Advance Access originally published online on July 13, 2006
Toxicological Sciences 2006 93(2):278-285; doi:10.1093/toxsci/kfl063
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The Effects of Methimazole on Development of the Fathead Minnow, Pimephales promelas, from Embryo to Adult
* School of Biosciences, Hatherly Laboratories, Prince of Wales Road, University of Exeter, Exeter EX4 4PS, United Kingdom; AstraZeneca, Global Safety, Health and Environment, Brixham Environmental Laboratory, Freshwater Quarry, Brixham, Devon TQ5 8BA, United Kingdom; and Institute for the Environment, Brunel University, Uxbridge, Middlesex UB8 3PH, United Kingdom
1 To whom correspondence should be addressed at Environment Agency, Chemical Assessment Section, EA Science Group—Environment and Human Health, Red Kite House, Howbery Park, Wallingford, Oxfordshire OX10 8BD, United Kingdom. Fax: +44 01491-828427. E-mail: helen.jordinson{at}environment-agency.gov.uk.
Received April 26, 2006; accepted June 26, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The importance of thyroid hormones in regulating early developmental processes of many amphibian and fish species is well known, but the impacts of exposure to disrupters of thyroid homeostasis during the embryo-larval-juvenile transitions are unclear. To investigate these impacts, fathead minnows, Pimephales promelas, were exposed to a model thyroid axis disrupter, methimazole, an inhibitor of thyroid hormone synthesis, at control (0), 32, 100, and 320 µg/l, starting at <24-h postfertilization, for 28, 56, and 83/84 days postfertilization (dpf). Thyroid disruption was evident at 28 dpf, when survival was significantly reduced by 32 or 100 µg/l methimazole concomitant with a reduced thyroxine (T4) content. However, the T3 content of these fish was similar to that of control fish, and body mass was unaffected (as in all groups), suggesting compensatory mechanisms overcame reduced T4 synthesis. At the highest concentration of methimazole (320 µg/l), activation of feedback mechanisms on the hypothalamic-pituitary-thyroid axis was suggested by the normal T4 content after 28 dpf exposure to methimazole, although triiodothyronine (T3) content of these fish was significantly reduced. The generally less pronounced disruption of thyroid hormone homeostasis after 56 days exposure to methimazole also suggests compensatory mechanisms in juvenile/adult fish that may regulate T4 content, despite exposure to methimazole at 32 or 100 µg/l (in fish held in 320 µg/l methimazole, the T4 content was significantly higher than in controls). Whole body T3 content at 56 dpf was significantly depressed only in fish held in 100 µg/l methimazole. By 83/84 dpf, length, body mass, and thyroid hormone concentrations were similar in all experimental groups and controls, indicating that adult fish may achieve regulation of their thyroid axis despite prolonged exposures to thyroid disruptors throughout early development.
Key Words: thyroid; thyroxine; triiodothyronine; development; methimazole; fathead minnow.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies in mammals, birds, and amphibians have indicated that a range of contaminants, including flame retardants (polybrominated diphenyl ethers), polychlorinated biphenyl (PCB) congeners, dioxins, and bisphenol A, can interfere with thyroid hormone homeostasis and thyroid hormone binding to receptors, both in vivo and in vitro (Brouwer et al., 1998
; Fowles et al., 1994; Fritsche et al., 2005; Iwamuro et al., 2003; Kitamura et al., 2005; Kohn, 2000; Kohn et al., 1996; Yamada-Okabe et al., 2005). This has led to questions regarding the impact of thyroid disruptor chemicals on thyroid homeostasis of fish (Brown et al., 2004a; Organisation for Economic Co-operation and Development, 2003), particularly as thyroid hormones have known actions on teleost development, growth, osmoregulation, and metabolism (Boeuf et al., 1989; Liu and Chan, 2002).
A few studies have examined the effects of candidate thyroid disruptors, for example, organochlorine pesticides and coplanar PCBs in adult fish (Brouwer et al., 1998; Brown et al., 2004b). However, compensation for perturbations of the thyroid status of adult fish can occur at multiple sites in the hypothalamic-pituitary-thyroid axis, including the hypothalamus, where somatostatin has an inhibitory action (Xiao and Lin, 2003), the pituitary which releases thyroid stimulating hormone (TSH) (Garcia Ayala et al., 2003; Hansen and Hansen, 1998), and at the thyroid gland, where thyroid peroxidase (TPO) activity can be modulated (Ebbesson et al., 1998). Additional regulation of hepatic and renal outer ring monodeiodinases, which convert thyroxine (T4) to triiodothyronine (T3), and inner ring monodeiodinase, which degrades T3, will determine circulating levels of T4 and T3 (Eales and Brown, 1993; Van der Geyton et al., 2001, 2005). These regulatory processes may limit the impacts of thyroid disrupting chemicals. For example, although the PCB congener 126 temporarily disturbed thyroid function in adult lake trout, a return to a balanced (euthyroid) state occurred during continued exposure (Brown et al., 2004b). Feedback loops that can potentially maintain a stable euthyroid state in the presence of thyroid disrupting chemicals may not, however, be fully developed in larval and juvenile stages of fish (Power et al., 2001; Yamano and Miwa, 1998), so early life stages are likely to be more sensitive to thyroid disruption. Such sensitivity could be of particular importance as in the early life stages, peaks in thyroid hormones (Crane et al., 2004; Deane and Woo, 2003; Szisch et al., 2005) play important roles in regulating the growth and development of many fish species (Boeuf et al., 1989; Liu and Chan, 2002; Reddy and Lam, 1992; Trijuno et al., 2002).
In order to gain insights into the possible effects of inhibitors of thyroid hormone synthesis in freshwater fish, and investigate the hypothesized differences in sensitivity of different life stages, we exposed fathead minnows (Pimephales promelas) as embryos (<24-h postfertilization) for 12 weeks to methimazole (1-methyl-2-mercaptoimidazole; CAS number 60-56-0). Methimazole is a well-characterized inhibitor of TPO, inhibiting the oxidation of iodide by follicular cells that leads to coupling of iodine to tyrosine within the thyroglobulin in the follicular lumen to form mono- and diiodotyrosines (MIT and DIT) (Engler et al., 1982), and the subsequent coupling of MIT and DIT that results in thyroid hormones (Capen, 1997). In the amphibian, Xenopus laevis, methimazole (25 µg/l) has been reported to inhibit tail resorption during metamorphosis (Fort and Stover, 1997). Recent studies indicate a dose-dependent delay in development of X. laevis during exposure of pre- and prometamorphic larvae to methimazole at 6.25–100 mg/l (Degitz et al., 2005). Amongst fish, methimazole (172 µg/l) delayed hatching of zebrafish larvae (Elsalini and Rohr, 2003), while exposure of zebrafish as embryos for 5 days to 114 µg/l methimazole caused severely stunted growth and retarded head cartilage development, but did not impact on levels of thyroid receptor 
and ß messenger RNA (Liu and Chan, 2002). These studies did not include measurement of circulating or tissue thyroid hormones in order to identify perturbations in the thyroid axis. In our study of fathead minnows, in order to determine whether effects of thyroid disruption persist from the early life stages through to adults, we exposed embryos to 32, 100, and 320 µg/l methimazole continuously from less than 24 h after fertilization for 83/84 days. We assessed hatching success, and survival and growth (as wet mass and body length), alongside measurements of thyroid hormone content (at 28 and 56 days postfertilization [dpf]) and plasma concentrations of thyroid hormones at 83/84 dpf.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flow-through exposure system.
In an 84-day study, fathead minnows were exposed to methimazole (nominal concentrations 0, 32, 100, and 320 µg/l) throughout development, from <24-h (postfertilization) through to adults, in a flow-through system (see below for details of test solution analyses). Concentrations of methimazole were selected to include the range (25–172 µg/l) that has been shown to inhibit hatching, growth, development, and metamorphosis of amphibian and fish embryos/larvae (Fort and Stover, 1997
; Liu and Chan, 2002). Dechlorinated water (25 ± 1°C) was delivered from a header tank and flowed through glass capillary tubing into four glass mixing vessels (2 l maximum volume) at 800 ± 80 ml/min. From each mixing vessel, water flowed directly into a glass flow-splitting chamber. Each flow-splitting chamber had one waste overflow and six glass capillary tube outlets allowing water to flow to six glass tanks. From each of four of these outlets, water flowed to glass tanks (12 l maximum volume, 9 l working volume) at 80 ± 8.0 ml/min to give four replicate tanks for each exposure group. These tanks were used for the first stage of the exposure until fish reached 33 dpf. The remaining two outlets from the flow-splitting chamber allowed water to flow at 225 ± 22.5 ml/min to 54-l tanks (45 l working volume) used from 33 dpf to the end of the study (84 dpf) forming the two replicates for each exposure group.
Methimazole test solutions.
The exposure concentrations were randomly assigned to the four tank clusters. Methimazole (Sigma, U.K.) stock solutions, at concentrations 10,000 times greater than the exposure concentrations (320 mg/l, 1 g/l, and 3.2 g/l), were made at 15-day intervals. Stock concentrates were delivered to the relevant mixing vessels at 0.08 ml/min using a multiple channel peristaltic pump with polyvinyl chloride pump tubing. Stocks were thus delivered to the mixing vessels at a rate 10,000 times lower than the delivery of dechlorinated water allowing dilution of the methimazole to the required concentrations. Stock solutions and the mixing vessels were constantly stirred.
Methimazole was weighed out in a fume cupboard in accordance with safety assessment of handling procedures and the flow-through studies were carried out in the open laboratory.
Analysis of methimazole test solution.
Test solution samples were taken weekly from each tank for analysis by high-performance liquid chromatography (HPLC). Where samples were not analyzed immediately, they were stored at – 80°C. At the same time as taking test solution samples, solutions were prepared, from each stock concentrate, at the same nominal concentrations as the test solutions, to act as analytical "controls" to determine loss during freezing, and were held frozen, along with test samples, until HPLC alongside test solutions. Measured values were adjusted to account for the small losses during storage (<35%).
Embryo selection, maintenance, and exposure.
All animal procedures were carried out according to U.K. Home Office regulations (License number PCD 30 8301, Environmental Safety Testing of Chemicals) and with local ethical approval. On day 0 of the study, fathead minnow embryos (800), at the blastula and morula developmental stages, were gathered from mating tiles in the fish husbandry unit at AstraZeneca's Brixham Environmental Laboratory (BEL), Devon, U.K. Healthy embryos were selected, five at a time, and randomly assigned to incubation cups, until each contained 25 embryos. Incubation cups were hung on oscillating incubation units over the experimental tanks (two egg cups per tank), maintaining the embryos constantly moving up and down through the test water at two cycles per minute. Embryos were inspected on a daily basis and any dead ones removed. On 4 and 5 dpf, all surviving embryos hatched into their experimental tanks.
Feeding of exposed fish.
Larvae were inspected daily and mortalities recorded. From 1 to 7 days posthatch (dph) larvae were fed a suspension of rotifers, Brachionus plicatilis, (3 ml of 3000 rotifers/ml, three times per day). At 7 dph, fry were also fed cultured Artemia nauplii three times. From 8 to 15 dph, larvae were fed Artemia alone three times daily (twice at weekends), and from 16 to 28 dph, fish were fed Artemia twice daily and ground pellet food (Ecostart 17, Biomar, Denmark) once daily. Fish were always fed to satiation. From 29 to 63 dph, fish were fed frozen brine shrimp twice daily and pellet food once daily (one feed of each type per day at weekends). For the remainder of the study, fish were given food pellets twice daily and frozen brine shrimp three times daily. To ensure satiation, the quantity of pellet and frozen brine shrimp was increased as the fish developed.
Sampling regime.
At 28 and 56 dpf, fathead minnows were sampled as whole bodies; at 83 and 84 dpf, plasma samples were collected. Fish were sacrificed between 6:00 A.M. and 10:00 A.M. by immersion in neutral buffered ethyl 3-aminobenzoate methanesulfonate (MS222) (750 mg/l). Each fish was weighed (wet mass) and measured using digital calipers to determine standard length. For whole body analysis, fish were immediately snap frozen on dry ice (20 fish per experimental regime). At 83 and 84 dpf, blood was sampled by cardiac puncture (10 fish per experimental regime) using 0.5 ml syringes flushed with ammonium heparin solution (approximately 5200 units/ml). Blood samples were kept on ice prior to plasma separation by centrifugation, and plasma volumes determined prior to freezing on dry ice. Fish sexes were determined by examination of internal sex organs. Plasma samples were held at – 80°C until extraction of T4 and T3.
Sample analysis.
Thyroid hormones were extracted from whole bodies and entire plasma samples (4–30 µl) using a method based on that used by Greenblatt et al. (1989) and fully described in Crane et al. (2004). During the extractions, the presence of anilino naphthalene sulfonic acid ensured that thyroid hormones were not bound to the thyroid hormone–binding proteins present in plasma (Prasad and Hollander, 1979). Where fish had a wet weight of 300 mg or greater, they were cut into at least two portions, extracted separately and extracts recombined prior to analysis of T3 and T4 by radioimmunoassays (RIAs) validated for use in fathead minnows (Crane et al., 2004). Values were adjusted for the extraction efficiencies (Crane et al., 2004). The RIAs were validated by checking parallelism with serial dilutions of tissue sample extracts against standard curves. The minimum detectable levels were 2.04 pg per tube (T3) and 8.16 pg per tube (T4); intraassay variability was 9.68% for T3 and 5.33% for T4; interassay variability was 11.66% for T3 and 20.37% for T4. As a protease digestion step was not included in the extraction procedure, the stored thyroid hormones bound to thyroglobulin within the thyroid follicles were not released and, therefore, measured whole body thyroid hormone concentrations comprised total thyroid hormones in circulation and associated with nonthyroidal tissues. Thyroid hormones were also extracted from pellet food (Ecostart 17), fed to fish during the latter part of the study, to determine the T4 content by RIA. T3 was not measured. T4 content of the diet was 9.1 ng/g.
Statistical analysis.
All statistics were performed using Sigmastat (Version 2.0, SPSS, Inc., Chicago, USA) and significance was accepted at p < 0.05. All values shown are mean ± standard error (SE). Statistical analysis excluded the female control group at 83 or 84 dpf, the size of which could not be predetermined, and that only included two fish.
The effects of methimazole on hatching and survival, body mass and length, T4 and T3 content or concentration, and hormone ratios were each identified using one-way analyses of variance (ANOVAs) with post hoc multiple comparisons by Tukey honestly significantly different test. Hormone ratios at 28, 83, and 84 dpf, T4 at 56 dpf, and 83 or 84 dpf (male only), and T3 in male fish at 83/84 dpf were log10 transformed to meet requirements of normality and homogeneity of variance prior to ANOVA. Thyroid hormone ratio data could not be normalized at 56 dpf and therefore Kruskall-Wallis ANOVA on ranks with post hoc Dunn's test was used. Percentage hatch and percentage survival data were arcsine square root transformed prior to analysis, with adjustment of the values 0 or 1 (U.S. Environmental Protection Agency, 1994). At 83/84 dpf, comparison of T4 and T3 concentrations in male and female fish (except in controls, where n = 2 females precluded analysis) was made using t-tests, except for T3 concentrations of fish exposed to 320 µg/l, where a Mann-Whitney U-test was used as data could not be normalized; T4 concentrations in fish exposed to 100 µg/l were log10 transformed before applying a t-test.
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RESULTS |
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Analytical Chemistry
The measured concentrations of methimazole over the duration of the study were 30.8 ± 1.1 µg/l (96% of nominal 32 µg/l; n = 36), 113.6 ± 3.4 µg/l (114% of nominal 100 µg/l; n = 36), and 343.9 ± 3.6 µg/l (107% of nominal 320 µg/l; n = 36). Test concentrations in the subsequent discussion refer to nominal concentrations, according to standard ecotoxicology procedures (Organization for Economic Cooperation and Development, 1992
).
Hatchability and 28-Day Survival
All groups showed at least 88% hatch with most larvae hatching at 4 or 5 dpf (Table 1). However, the rate of hatching was significantly increased in fish exposed to 32 µg/l methimazole compared to all other groups (Table 1; 76.4% of larvae exposed to 32 µg/l had hatched by day 4, whereas 23.5–30.8% of larvae had hatched in all other groups).
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The highest percentage survival to 28 dpf was that of the controls. Fish exposed to 32 and 100 µg/l methimazole showed significantly reduced survival in comparison to controls (Table 1), although survival still exceeded 93%. After 28 dpf, there were only a total of three mortalities during the remainder of the study.
Fish Weight and Length
The wet weight of control fish and fish exposed to methimazole was not significantly different at any sampling point (Table 2); however, significant differences in overall length were apparent (Fig. 1). By 28 dpf, fish exposed to 32 µg/l methimazole had significantly greater standard lengths than control fish, and this effect persisted at 56 dpf (Fig. 1) when fish exposed to 320 µg/l also had significantly greater standard lengths than control fish. At 83/84 dpf, fish could be sexed and data separated for male and female fish. As only two control female fish were available, statistical analysis was not feasible although data are included in Figure 1; there were no longer any significant differences in the standard lengths of control and experimental male fish (Fig. 1).
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Thyroid Hormone Levels
At 28 dpf, fish exposed to 32 and 100 µg/l methimazole had significantly lower whole body T4 than control fish (Fig. 2, Panel A). However, whole body T3 showed no significant differences between fish exposed to 32 or 100 µg/l and control fish, while fish exposed to 320 µg/l had significantly lower whole body T3 content compared to all other treatment groups (Fig. 2, Panel B). In fish exposed to 100 µg/l, unchanged whole body T3 content (compared to control fish) coupled with reduced whole body T4 resulted in a significant elevation in T3/T4 ratio (Fig. 2, Panel C).
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By 56 dpf, whole body T4 content was significantly increased in fish exposed to the highest levels of methimazole (320 µg/l) compared to that in all other treatment groups (Fig. 2, Panel A). The T3 content showed no significant difference between control fish and fish exposed to 32 or 320 µg/l, but fish exposed to 100 µg/l had significantly depressed whole body T3 content compared to all other groups (Fig. 2, Panel B). The T3:T4 ratios in fish exposed to methimazole concentrations for 56 days showed no significant deviations from those of control fish (Fig. 2, Panel C).
At 83 dpf, it was possible to collect sufficient blood for analysis of plasma T3 and T4 and to sex these fish. As sexes could not be predetermined and were unequal (Table 3), with only two control females, within-sex statistical analysis of the effects of methimazole on female fish was not feasible. After exposure to 320 µg/l methimazole, female fish had significantly higher plasma T3 concentrations than male fish, and when exposed to 32 µg/l methimazole, females had statistically higher plasma T4 concentrations, and therefore significantly lower T3/T4 ratios than male fish (Table 3). In male fish exposed to methimazole for 83/84 days, there were no significant differences in plasma concentrations of T4 or T3 (or thyroid hormone ratios) from those of control male fish (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our studies have investigated the effects of the thyroid synthesis inhibitor, methimazole, on thyroid homeostasis in an established fish model, the fathead minnow, throughout embryo and larval development. This has allowed assessment of thyroid homeostasis in larval, juvenile, and adult fish to establish whether any effects persist to the adult stage. Methimazole belongs to the group of thionamides (methimazole, propylthiouracil, thiouracil, and thiourea) that pharmacologically inhibit TPO, the enzyme that leads to the formation of thyroid hormones in the follicles (Capen, 1997
; Engler et al., 1982). In fish, the thyroid gland synthesizes principally T4, and T3 is largely formed by peripheral deiodination by the liver and kidney (Brown et al., 2004a; Eales and Brown, 1993), so exposure to methimazole can be predicted to inhibit T4 secretion. However, effects of thyroid toxicants on T3, the predominantly active thyroid hormone, are less easily predictable. Our measurements of whole body thyroid hormones in larval and juvenile fish excluded a protease digestion step for extraction of thyroid hormones coupled to thyroglobulin within the thyroid follicles, and therefore provide an index of circulating and tissue levels. At 28 dpf, T4 content was significantly reduced in fish exposed to 32 or 100 µg/l methimazole, presumably as the result of the known inhibitory action on TPO activity and reduced formation and release of T4. However, this effect was not apparent at 28 dpf in fish exposed to 320 µg/l methimazole, at 56 dpf (at any concentration of methimazole), or in adult fish, suggesting that the larval stage is the most sensitive to endocrine disruption.
Given the depressed T4 content of 28 dpf fish exposed to the two lower methimazole concentrations, it is likely that fish exposed to the higher concentration (320 µg/l) had also initially suffered similar effects of methimazole on TPO activity, but that feedback mechanisms were subsequently activated. Increased release of T4 from follicular stores could occur, but would be unlikely to have a lasting benefit. Activation of the well-known mechanism of feedback regulation of the thyroid axis in fish, and increased release of TSH from the pituitary (Brown et al., 2004a; Eales and Brown, 1993; Leiner and MacKenzie, 2003; Pradete-Balade et al., 1997) is therefore likely to have occurred. However, since methimazole inhibits TPO activity (Capen, 1997; Engler et al., 1982), the effectiveness of any compensatory drive to raise T4 production ought to be impaired. Nevertheless, the significant increase in whole body T4 of 56 dpf fish held in 320 µg/l suggests that in this group the possibility of persistent overstimulation by pituitary TSH, with circulating levels of TSH sufficiently elevated to overcome the level of inhibition induced by methimazole. In order to corroborate this theory, measurements of TSH would be needed, and as yet there is no available assay for fathead minnows. Alternatively, the increased T4 may be the result of altered thyroid hormone metabolism. Thyroid hormones are conjugated in the liver by sulpho-transferases and glucuronosyl-transferases in order to inactivate and excrete them (Finnson and Eales, 1999). A decrease in conjugation rates could also result in increased circulating T4. There are no reports of methimazole influencing the metabolism of thyroid hormones; however, this might be a regulatory response in order to maintain circulating T4.
Although T4 content of 28 dpf larval fish exposed to 32 or 100 µg/l methimazole was reduced, these fish showed no change in whole body T3 content. T3 levels are determined both by the available pool of T4 and its conversion by 5'-monodeiodinase to form T3, together with the deiodination of T3 to di- and monoiodothyronines (Eales and Brown, 1993; Eales et al., 1993; Mol et al., 1997) and conjugation by sulpho-transferases and glucuronosyl-transferases to inactivate and excrete the hormones (Distefano et al., 1998; Eales and Brown, 1993; Finnson and Eales, 1999). Significantly higher T3/T4 ratios in fish held in 100 µg/l methimazole suggest an increased conversion of T4 to T3 or reduced degradation and conjugation during continued exposure to methimazole.
At the highest level of methimazole (320 µg/l), however, even though T4 content was maintained at 28 dpf, T3 content was less than that of both control fish and fish exposed to the two lower concentrations of methimazole. To our knowledge there are no previous reports of any direct actions of methimazole on 5'-monodeiodinase or T3 degradation, and with the pool of T4 unaltered it is difficult to fully explain the low T3 content of these fish. A similar though less pronounced depression in T3, when again T4 was unaffected, occurred at 56 dpf in fish exposed to 100 µg/l methimazole. A lag time before released T4 is converted to T3, or a nonspecific action of methimazole, may explain the reduced T3 in these fish. Whatever the explanation, an impaired regulation of T3 occurred both at 28 dpf in the presence of 320 µg/l methimazole and, to a lesser extent, at 56 dpf when exposed to 100 µg/l methimazole. These results indicate a lower sensitivity of these fish to the lower exposure concentrations of methimazole. As T3 is the main physiologically active hormone in fish (Leatherland, 1993) depressed T3 levels in these early stages of development could have damaging effects on physiological processes such as metabolism, growth, and development.
At the final sampling point (83/84 dpf), plasma thyroid hormone concentrations of methimazole-exposed and control male fish were not significantly different, indicating an ability to recover and ultimately maintain physiological levels of thyroid hormones in adult fish that had been exposed to methimazole from prior to hatch. In the late stages of development, fish were fed increasing amounts of pelleted food containing 9.1 ng T4/g food. The T3 content of the pellets was not measured in these studies, but is likely to be less than the T4 content, given the absence of thyroid storage. From 34 to 68 dpf, pellet food accounted for around a third of food intake and thereafter, pelleted food was increased to around 40% of the food intake. Assuming a food intake of 2% body mass per day, dietary T4 can be estimated as 0.03–0.15 ng/day and T3 at somewhat less than this amount. To assess the possible amelioration of methimazole effects due to the dietary intake of thyroid hormones, the estimated intake needs to be compared to T4 and T3 production rates but these are unknown for most fish species, including fathead minnows. Juvenile salmonids have been reported to secrete 1.5–4.5 ng T4/h/100 g body mass (Specker et al., 1984) and if similar secretion rates occur in fathead minnows then dietary T4 would represent only 5–20% of T4 secretion rates. Furthermore, absorption of T4 by the fish gut is extremely low (MacKenzie et al., 1993; Moon et al., 1994; Sweeting and Eales, 1992), so T4 acquisition from the diet seems likely to play only a minimal role in the achievement of the stable T4 concentrations that were found in adult fathead minnows exposed to methimazole. Gut absorption of dietary T3 does, however, occur in fish (Moon et al., 1994; Van der Geyton et al., 2005), although dietary acquisition is unlikely to represent more than a small proportion (<20%) of normal T3 production, and may be much less. In tilapia held at 24°C, the T3 appearance rate was reported as 36 ng/h/100 g body mass (DiStefano et al., 1998). If similar values apply in fathead minnows, then dietary acquisition of T3 would be insignificant (<1% of normal production rates). Even if T3 production rates are lower, akin to those reported for salmonids, it is improbable that dietary intake of T3 accounts for the stable plasma T3 concentration that was found in adult fathead minnows exposed to methimazole. Instead, it is more likely that negative feedback control of circulating thyroid hormone levels occurs at the hypothalamic and/or pituitary levels (Han et al., 2004; Sukumar et al., 1997), although further studies are required to substantiate this hypothesis, as well as assessment of the possible role of monodeiodinases.
The first 28-day period of this study represents the most critical phase of rapid development in fathead minnows when thyroid hormones peak (Crane et al., 2004) presumably linked to developmental processes, as in other species (de Jesus, 1994; de Jesus et al., 1993, 1998; Schreiber and Specker, 1998). We have previously used fathead minnows as a model species to study the effects of ammonium perchlorate on the thyroid axis of fish (Crane et al., 2005). These studies showed pronounced changes in weight of fish exposed to perchlorate, with delayed scale and pigment development at the larval to juvenile transition. The results of this study show the more subtle effects of methimazole, which produced only minor effects on development. Survival was significantly reduced in fish exposed to 32 and 100 µg/l methimazole, but mortality rates were very low, and the small differences observed seem unlikely to be biologically significant. Despite the effects of low levels of methimazole on the T4 content of 28 dpf fish, and at the highest level of methimazole, the reduced T3 content in 28 dpf fish, assessment of body mass and standard length did not reveal any major effects of methimazole on growth and development. Although there were statistically significant changes in length on days 28 and 56, the changes are not thought to be of biological significance. These observations contrast with reported effects of methimazole and other thionamides on other fish species and on amphibians. For example, methimazole (25 µg/l) inhibited metamorphosis of X. laevis (Fort and Stover, 1997). In fish, this compound also delayed metamorphosis (evident as slowed resorption of dorsal and pelvic fin spines and acquisition of pigmentation) in association with depressed whole body T4 content that was reported in coral trout grouper (Plectropomus leopardus) exposed to 30 mg/l thiourea (Trijuno et al., 2002). The lack of noticeable effects on the development of fathead minnows in this study may reflect an early development of self-regulatory mechanisms and the regulation of T3 content that is achieved in the two lowest concentrations of methimazole by 28 dpf. Despite this, assessment of thyroid status at 28, 56, and 83/84 dpf during continuous methimazole exposure has shown disruption of the thyroid axis, although the results indicate a complex pattern of responses related to both the length of the exposure and the concentration of methimazole, with several potential self-regulatory points.
Our data suggest a high susceptibility of larval fish to the effects of methimazole on the thyroid axis, indicating that the period of early development in fathead minnows is a good stage for assessing thyroid disruption. We have also shown the ability of the thyroid axis to overcome continuous assault by thyroid disrupting chemicals during the transition from larva through to adult. It remains to be seen whether higher concentrations of methimazole might cause more persistent perturbations in thyroid hormones that are manifest in adult fish. It is possible that more substantial alterations in thyroid physiology might be demonstrated prior to day 28, and future studies should address thyroid disruption in earlier stages, when the thyroid axis feedback mechanisms are less well developed. At this early stage (prior to 28 dpf), perchlorate has been seen to significantly affect thyroid function alongside a significant inhibition of development (Crane et al., 2005), which indicates the potential for development of early life stage tests of thyroid disruption for comparison with the currently favored amphibian model used in chemical risk assessments (Degitz et al., 2005; Organisation for Economic Co-operation and Development, 2004).
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ACKNOWLEDGMENTS |
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This work was supported by AstraZeneca and funded by a Natural Environment Research Council studentship (NER/S/C/1999/0453) awarded to H.C. We thank Anita Young and Ingrid Maas for carrying out the HPLC analysis; Mike Field, Martin Canty, and Lisa Bickley from BEL for help with fish husbandry and sampling; and Sue Frankling from the University of Exeter for technical assistance with RIAs.
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