ToxSci Advance Access originally published online on January 6, 2007
Toxicological Sciences 2007 96(2):246-254; doi:10.1093/toxsci/kfm001
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Published by Oxford University Press 2007.
Effects of Prolonged Exposure to Perchlorate on Thyroid and Reproductive Function in Zebrafish
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* Department of Environmental Toxicology, Texas Tech University, Lubbock, Texas 79409-1160
U.S. Geological Survey Texas Cooperative Fish and Wildlife Research Unit
Department of Range Wildlife and Fisheries Management
Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-2120
2 To whom correspondence should be addressed. Fax: (806) 742-2946. E-mail: reynaldo.patino{at}ttu.edu.
Received October 27, 2006; accepted January 3, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The objectives of this study were to determine the effects of prolonged exposure to perchlorate on (1) thyroid status and reproductive performance of adult zebrafish (Danio rerio) and (2) F1 embryo survival and early larval development. Using a static-renewal procedure, mixed sex populations of adult zebrafish were exposed to 0, 10, and 100 mg/l nominal concentrations of waterborne perchlorate for 10 weeks. Thyroid histology was qualitatively assessed, and females and males were separated and further exposed to their respective treatments for six additional weeks. Eight females in each tank replicate (n = 3) were paired weekly with four males from the same respective treatment, and packed-egg (spawn) volume (PEV) was measured each of the last five weeks. At least once during weeks 14–16 of exposure, other end points measured included fertilization rate, fertilized egg diameter, hatching rate, standard length, and craniofacial development of 4-day–postfertilization larvae and thyroid hormone content of 3.5-h embryos and of exposed mothers. At 10 weeks of exposure, perchlorate at both concentrations caused thyroidal hypertrophy and colloid depletion. A marked reduction in PEV was observed toward the end of the 6-week spawning period, but fertilization and embryo hatching rates were unaffected. Fertilized egg diameter and larval length were increased by parental exposure to perchlorate. Larval head depth was unaffected but the forward protrusion of the lower jaw–associated cartilage complexes, Meckel's and ceratohyal, was decreased. Exposure to both concentrations of perchlorate inhibited whole-body thyroxine content in mothers and embryos, but triiodothyronine content was unchanged. In conclusion, prolonged exposure of adult zebrafish to perchlorate not only disrupts their thyroid endocrine system but also impairs reproduction and influences early F1 development.
Key Words: perchlorate; thyroid; reproduction; embryo; larvae; development; fish.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Perchlorate has been used for many years in the manufacture of ammunitions, as an oxidant in fuel for missiles and rockets, and in industries such as leather processing, electroplating, aluminum refining, and rubber manufacturing (Logan, 2001
). Salts of perchlorate are highly soluble in water, and perchlorate is often detected in surface and ground waters near sites of manufacture, use, or disposal (Tipton et al., 2003; Urbansky, 2002). Perchlorate has a similar partial specific volume to iodide, with which it competes for the sodium-iodide symporter (Van Sande et al., 2003). Perchlorate-dependent iodide deficiency can result in decreased thyroid hormone (TH) synthesis and eventually may lead to hypothyroidism (Capen, 2001). The toxicological effects of perchlorate on vertebrate thyroid gland/follicles have been reported by various authors (e.g., Bradford et al., 2005; Crane et al., 2005; Goleman et al., 2002a,b; Hu et al., 2006; Liu et al., 2006; McNabb et al., 2004; Mukhi et al., 2005; Patiño et al., 2003; Theodorakis et al., 2006; Thuett et al., 2002; Tietge et al., 2005; York et al., 2001).
The results of a number of studies have indicated that TH may regulate reproduction in some teleost species (Cyr and Eales, 1988; Cyr et al., 1988; Van der Geyten et al., 2001; Weber et al., 1992). For example, in study with rainbow trout, experimental inhibition of the transformation of thyroxine (T4) into triiodothyronine (T3; the active TH) caused hypothyroidism in females and depressed their gonadosomatic index (GSI), whereas moderate hyperthyroidism induced by exogenous T3 treatment led to higher GSI relative to control values (Cyr and Eales, 1988). Conversely, the results of other studies seem to be inconsistent with a positive regulatory effect of TH on teleost reproduction. For example, in a earlier study with zebrafish, Patiño et al. (2003) found that exposure to perchlorate (18 mg/l) did not negatively affect the volume of eggs spawned although the numbers of eggs per spawn volume was increased and thyroid histology was also greatly affected after an exposure period of 8 weeks. Further, a recent study with zebrafish reported that a 3-week exposure to the thyroid-disrupting chemical, propylthiouracil, induced a near doubling in the numbers of eggs produced despite the development of hypothyroid conditions (Van der Ven et al., 2006). In some other vertebrates previously examined, exposure to perchlorate did not seem to cause major impairments in reproductive performance (Gentles et al., 2005; York et al., 2001, 2005).
The inconsistent observations concerning the reproductive effects of perchlorate at environmentally relevant concentrations—and thus also the involvement of the thyroid endocrine system as transducer of these effects—may be at least partly due to differences in the concentrations of perchlorate used and, perhaps more importantly, the length of the treatment application. In zebrafish, for example, T4 concentration (whole-body content) was not affected even after a 12-week exposure to perchlorate at concentrations up to 11 mg/l (Mukhi et al., 2005). The resiliency of the T4 content of the animal despite exposures to perchlorate at concentrations that affect thyroid follicle function is likely due to the large reserves of T4 stored in follicular colloid (e.g., Brown et al., 2004). This situation could explain why zebrafish reproduction, even if it were a TH-regulated activity, did not seem to be negatively impacted in an earlier study using a relatively short period of treatment with perchlorate (Patiño et al., 2003). Thus, the first objective of the present study was to determine the effects of prolonged exposure to perchlorate on the reproductive performance of zebrafish. The length of the exposure period in the present study was not predetermined, but based on the real-time appearance (if any) of clear reproductive effects.
THs are maternally transferred into teleost eggs, and it has been suggested that maternal TH is necessary for embryonic development (e.g., Power et al., 2001). The second objective of this study was to determine the consequences of parental exposure to perchlorate on parameters of gamete quality and early F1 development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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General animal husbandry.
Four-month-old, wild-type zebrafish were obtained from a local vendor (Lubbock, TX) and allowed 1 week to acclimatize to our laboratory conditions prior to the onset of exposure. Animal husbandry procedures for this study were as described by Mukhi et al. (2005)
. Briefly, each aquarium was filled with 30 l of zebrafish water (25 g of R/O right/100 l of reverse osmosis water) and fitted with two hand-made internal (glass) biofilters. Thirty-five fish from a mixed sex population were randomly assigned to each of nine aquaria. The aquaria were treated with Stresszyme (Aquatic Pharmaceuticals, Pittsburgh, PA) weekly to facilitate mineralization of nitrogenous waste. The range of observed water quality parameters were pH 6.0–8.0 (this range includes extreme values rarely observed; typically, values were maintained between 6.5 and 7.5), temperature 27–29°C, and 12 h/12 h light/dark cycle. Fish were fed twice daily to satiation with adult frozen Artemia and/or Tetramin flakes (Tetra Sales, Blacksburg, VA). Leftover food and fecal material were removed by siphoning every evening. Temperature and pH were measured daily and dissolved oxygen, specific conductivity, salinity, unionized ammonia, and nitrate were measured at least once weekly. Half of the water volume (15 l) was removed and replaced with clean system water twice weekly. The protocol for the use of animals in this study was reviewed and approved by the Texas Tech University Animal Care and Use Committee (Lubbock, TX).
Perchlorate exposure prior to spawning.
Sodium perchlorate (CAS # 07601-89-0) was used as the source of perchlorate for this study and was added directly to system water. A static-renewal exposure procedure was applied as previously described (Mukhi et al., 2005; Patiño et al., 2003). The nominal perchlorate concentrations were 0 (control), 10, and 100 mg/l, and each treatment was conducted in triplicated aquaria. Fish maintenance procedures were as already described. The length of this phase of the exposure was 10 weeks, at the end of which three females per replicate were sampled for thyroid histological analysis. For this purpose, the sampled fish were euthanized in 1 mg/l tricaine methanesulfonate (MS-222; Argent Chemical Laboratories, Inc., Redmond, WA), fixed in Bouin's solution at 4°C for 48 h, rinsed in tap water, and stored in 70% ethanol until processed. Histological procedures and thyroid follicle analyses were as described by Patiño et al. (2003).
Spawning procedures and sample collections.
After the initial 10 weeks of exposure, females and males were separated into new aquaria. Three female tanks per treatment were set up; each female tank contained eight fish derived from respective (single) original aquaria (females were not pooled among treatment replicates during this transition). Only one male tank per treatment was maintained; each male tank contained four fish from each of the three original aquaria per treatment.
For spawning purposes, four randomly chosen males were paired with each replicate of eight females from the same treatment. Spawning was conducted once weekly, and the exposure was continued until clear effects on reproductive output were noted (see "Results" section). The combined wet weight of the eight female fish from each tank was taken in the evening before spawning by placing them in a prezeroed, 1-l beaker (with water) immediately before their transfer to a spawning chamber within each female tank. The spawning chambers contained false bottoms to collect eggs as described earlier (Patiño et al., 2003). The following morning at 2.5 h after lights on (artificial dawn), fish were removed from the spawning chambers and returned to their tanks. The spawn was collected from each chamber and fecal matter and other debris were removed by rinsing 4 times with fresh zebrafish water and using glass pipettes. Packed-egg volume (PEV) was determined by volume displacement in a graduated 5-ml glass cylinder. The first spawning was conducted at week 11 of exposure to synchronize breeding, but no data was collected from this spawning event. The fish were then spawned once weekly for five additional weeks. Spawning was discontinued at this time because of the large treatment effects observed on PEV (see "Results" section).
Additional measurements were taken from week 14 to week 16. Because of limited PEV in the perchlorate-treated groups especially toward the end of the exposure period, not all measurements could be taken at each spawning event. Fertilization and hatching rates were measured at week 14; embryonic T3 and T4 contents were determined at weeks 14 and 15, respectively (samples were snap frozen in liquid nitrogen and stored at – 80°C until analyzed); standard length and craniofacial development of 4 days postfertilization (dpf) larvae were determined at week 15 (for this purpose, larvae were fixed in 4% paraformaldehyde at 4°C overnight and stored in 70% ethanol until further processed); egg diameters were measured at week 16; and whole-body T4 and T3 content in the exposed females were determined at week 16 (fish were snap frozen in liquid nitrogen and stored at – 80°C until further processing). Larvae and adults were euthanized in a lethal concentration of MS-222 (1 g/l) prior to processing. Embryo and larval rearing were conducted in untreated zebrafish water (28°C).
TH extraction and analysis.
THs, T3 and T4, were extracted from two pooled females per tank replicate; T3 was extracted from 0.7 to 2.8 g of PEV per tank replicate; and T4 was extracted from 0.4 to 2.2 g of PEV per tank replicate. Extraction procedures were as previously described for whole body of zebrafish (Mukhi et al., 2005). Samples were homogenized using a Polytron homogenizer (Glen Mills Inc., Clifton, NJ) in ice-cold methanol (volume in ml = tissue weight (g) x 4] containing 1mM propylthiouracyl and then sonicated. Approximately 1000 counts per minute of [125I]-T3 or [125I]-T4 in 50 µl of methanol (containing 1mM propylthiouracyl) were then added to each sample to monitor the recovery of endogenous hormone. Following a 30-min incubation, homogenates were centrifuged and the supernatants were removed, mixed with two volumes of chloroform, and back extracted into an aqueous phase with 2N ammonium hydroxide. The back extraction was repeated two more times. The aqueous fractions were pooled and dried in a Jouan centrifugal evaporator overnight at 30°C. The dried samples were reconstituted in 300 µl of barbital buffer (barbital 15.47 g/l, ethylenediaminetetraacetic acid 0.5 g/l, bovine gamma globulin 1 g/l, and thimerosal 1 g/l; pH 8.6) prior to the assay, and the recovery of radiotracer was determined in an aliquot using a Cobra 5005 gamma counter (Packard, Downers Grove, IL). Under these conditions, the average extraction recovery efficiencies for T3 and T4 were 64% and 67%, respectively, for the embryos and 71% and 62%, respectively, for whole-body homogenates.
THs were measured in duplicate 50-µl aliquots of extract following procedures of MacKenzie et al. (1978). Authentic T3 or T4 standards were used for assay calibration and run in duplicate for each concentration. The hormone content of the samples was determined using a four parameter logistic transformation of [125I]-T3 or [125I]-T4 displacement by the authentic standards. The sample values obtained were corrected for the estimated recovery of each sample. This assay procedure was validated for both embryo and whole-body homogenates by confirming the direct proportionality of hormone dilution in serially diluted extracts (Pearson r = 0.97–0.99; p < 0.05) and by confirming the recovery of known concentrations of exogenously added authentic T3 and T4 into the extracts (all within 86–93% of expected values).
F1 embryo-larval measurements.
Fertilization rates were estimated at approximately 9 h after lights on as the number (percent) of viable embryos relative to the total number of eggs in a subsample of approximately 100 eggs per replicate; and hatching rates were determined as the number (percent) of the viable embryos that hatched by 3 dpf (72 h). Egg diameters were measured in 40–50 eggs per tank replicate using an ocular micrometer attached to a stereomicroscope and calibrated against an external (stage) micrometer. As the zebrafish egg is spherical, the diameter was measured randomly on any part of the egg.
Standard length and craniofacial development were examined in 15 larvae per replicate reared through hatching until 4 dpf, at which time they were sampled for analysis. Gross developmental deformities were also recorded. Craniofacial development was assessed only in embryos derived from the control and 100-mg/l perchlorate groups. Parameters of craniofacial development examined included head depth at the middle of the eye and lengths of the Meckel's and ceratohyal cartilage complexes (see below). These measurements were also made with an ocular micrometer. Four dpf was chosen for larval analysis because a previous study reported that craniofacial development in zebrafish is sensitive to TH during the embryo-to-larva transition period (3–5 dpf; Liu and Chan, 2002).
Whole-mount cartilage staining with Alcian Blue was conducted to facilitate cartilage analysis according to procedures described by Liu and Chan (2002). Briefly, the larvae preserved in 70% ethanol were dehydrated with series of graded concentrations of ethanol and stained overnight with a 0.1% solution of Alcian Blue dissolved in 80% ethanol/20% glacial acetic acid. The samples were washed with acidic ethanol (80% ethanol/20% glacial acetic acid and then 95% ethanol/5% glacial acetic acid), rehydrated to phosphate buffer saline (PBS), and rinsed twice with PBS. The samples were then consecutively treated with 50 mg trypsin per milliliter of a saturated sodium tetraborate solution at 4°C overnight and with 3% H2O2 for 5 min. The stained samples were washed several times with PBS and preserved in 50% glycerol (in PBS). The Meckel's and ceratohyal cartilage complexes each form a U- or V-shaped structure that can be approximated to an isosceles triangle (Fig. 1). The length (l) of each structure was estimated by measuring the side (s) and the base (b) of the triangle under a stereoscope and applying the Pythagorean formula, l = square root (s2 – b2/4). Because of the anteroventral direction of the ceratohyal complex in whole mounts, the "length" of this cartilage as measured in the present study is likely an underestimate and should be considered more as an estimate of the forward protrusion of the cartilage.
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Data analysis.
All statistical analyses were done with the Statistica Data Miner software package (StatSoft, Tulsa, OK) at the level of significance of
= 0.05. Parameters such as wet weight of females, PEV, TH content of embryo and whole-body (pool) extracts, hatching rate, and fertilization rate are measures of tank performance. Thus, in all these cases, sample size per treatment for statistical analyses is the number of tank replicates (n = 3). PEV was measured and analyzed as weekly PEV (two-way ANOVA, treatment x spawning event; followed by one-way ANOVA for treatment effects within each spawning event) or as cumulative PEV (repeated-measure ANOVA). Wet weight of females, TH content in mothers and embryos, hatching rates, and fertilization rates were analyzed by one- or two-way ANOVA as appropriate. Measurements taken from individuals (eggs or fish) such as egg diameter, larval length, and cartilage complex length were analyzed by one-way nested ANOVA (tanks nested into treatment). Separation of means was done using Tukey's honestly significant differences (HSD) test. All percent data were arcsine transformed and egg diameter, larval length, cartilage lengths, and T4 and T3 data were log transformed prior to analysis to achieve homogeneity of variances. The results are shown as untransformed mean ± SE. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of Perchlorate on Maternal Thyroid Histology
A qualitative assessment of the thyroid follicles in female zebrafish at the time of separation of the fish into spawning tanks (week 10) indicated a mild colloid depletion in fish exposed to perchlorate at 10 mg/l (Fig. 2B) and mild to severe depletion at 100 mg/l (Fig. 2C). Females from both perchlorate treatment groups exhibited hypertrophic thyroid follicle cells. In addition, some females exposed to perchlorate at 100 mg/l contained follicles that had completely collapsed or lost their follicular structure (Fig. 2D). Control fish had thyroid follicles full of colloid that contained nonhypertrophic epithelial cells (Fig. 2A).
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Effects of Perchlorate on Maternal and Embryonic TH Contents
Measurements made at week 16 indicated that whole-body T4 content, but not T3 content, was greatly reduced in perchlorate-treated females compared to control females (one-way ANOVA, Tukey's HSD, p < 0.05; Figs. 3A and 3B). Although there appeared to be a trend for whole-body T3 content to be lower in perchlorate-treated fish (Fig. 3A), this trend was not statistically significant (p > 0.05).
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Embryonic T3 and T4 contents were measured at weeks 14 and 15, respectively. Embryonic T3 content was not affected by maternal exposure to perchlorate (one-way ANOVA, p > 0.05; Fig. 4A). However, embryonic T4 content was decreased in both the 10- and 100-mg/l groups compared to the control treatment (one-way ANOVA, Tukey's HSD, p < 0.05; Fig. 4B).
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Effect of Perchlorate on Maternal Wet Weight prior to Spawning
Two-way ANOVA (treatment x length of exposure) indicated that both treatment and length of exposure affected the wet weight of females (p < 0.05). At week 12 of exposure, the mean weight of females prior to spawning was significantly lower in the 100-mg/l group compared to the control and 10-mg/l groups (one-way ANOVA, Tukey's HSD, p < 0.05). From week 13 to week 16, the mean weight of females in the 10- and 100-mg/l treatment groups were significantly lower than control values (one-way ANOVA, p < 0.05), but there was no significance difference between the 10- and 100-mg/l treatment groups (p > 0.05; Fig. 5).
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Effect of Perchlorate on PEV Production
Two-way ANOVA (treatment x length of exposure) indicated that treatment, length of exposure, and their interaction all had significant effects on PEV (p < 0.05). One-way ANOVA conducted at each spawning date indicated that treatment effects were significant (p < 0.05) at 12, 13, 15, and 16 weeks of exposure. The inhibitory effect of perchlorate on PEV was evident earlier in the 10-mg/l exposure group (weeks 12, 13, 15, and 16) than in 100-mg/l exposure group (weeks 15 and 16) (one-way ANOVA, Tukey's HSD, p < 0.05; Fig. 6A). The cumulative PEV in the 10- and 100-mg/l treatment groups were significantly lower than the control cumulative PEV (repeated-measure ANOVA, Tukey's HSD, p < 0.05; Fig. 6B).
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Effects of Parental Exposure to Perchlorate on F1 Embryo-Larval Development
Measurements of fertilization and hatching rates at week 14 indicated that these parameters of gamete and embryo quality were not affected by parental exposure to perchlorate at 10 or 100 mg/l (one-way ANOVA, p > 0.05; Table 1). However, mean egg diameter at week 16 was increased in spawns from both perchlorate treatments compared to control values (nested ANOVA, Tukey's HSD, p < 0.05; Table 1).
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Observations conducted at week 15 indicated that parental exposures to perchlorate did not cause gross developmental deformities in F1 progeny (data not shown). Larval length was slightly but significantly increased in progeny from the 100 mg/l–treated parents compared to progeny from the control parents (Table 2). Also, analysis of jaw morphometry indicated that the lengths of Meckel's and ceratohyal cartilage complexes were reduced compared to control larvae (Table 2). Cartilage measurements could not be normalized for larval length because these measurements were not recorded for the same individual fish (larvae from each spawn were processed in groups for cartilage staining). However, because mean standard length in the 100-mg/l larvae was longer than in control larvae, cartilage measurements normalized for larval length would only have amplified the difference observed. Parental exposure to perchlorate had no effect on larval head depth (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Perchlorate competitively inhibits iodide uptake into thyroid follicles and thus can impair TH production (Capen, 2001
; Wolff, 1998). Although the perchlorate concentrations used in this study are high (nominal 10 and 100 mg/l), they are within the range of values reported in surface and ground waters of highly contaminated sites in the United States (http://www.epa.gov/fedfac/pdf/detect0305.pdf). At environmentally relevant concentrations, perchlorate disrupts the histology of thyroid follicles in several aquatic vertebrates including the African clawed frog, Xenopus laevis (Goleman et al., 2002a; Hu et al., 2006), fathead minnow, Pimephales promelas (Crane et al., 2005), and zebrafish (Mukhi et al., 2005; Patiño et al., 2003; Van der Ven et al., 2006; present study). In zebrafish, perchlorate-induced changes in thyroid follicles include hypertrophy, hyperplasia, colloidal depletion, angiogenesis, and suppression of the immunocytochemistry-based colloidal T4 ring (Mukhi et al., 2005; Patiño et al., 2003). The intensity of colloidal T4 ring, which may represent the level of TH synthetic activity in thyroid follicles, was inhibited after exposure to perchlorate at levels as low as 11 ppb (Mukhi et al., 2005). Several of these histological conditions were evident in the perchlorate-treated fish of the present study prior to the onset of the spawning trials, and maternal T4 content was also affected toward the end of the trials. These observations confirm the effectiveness of perchlorate as disruptor of thyroid function in the adult zebrafish used for the present study.
A previous study with adult zebrafish found that exposure to perchlorate at concentrations up to 11 mg/l for 12 weeks had no effect on whole-body T4 concentration; however, the same study suggested that longer exposure periods may be necessary to alter TH content in this species (Mukhi et al., 2005). Indeed, the results of the present study showed that exposure to perchlorate at 10 or 100 mg/l for 16 weeks greatly suppressed whole-body T4 concentrations in adult fish. Other studies with various vertebrate species have yielded inconsistent results regarding the effects of perchlorate on T4 concentrations. For example, exposure to perchlorate caused slight declines in whole-body T4 concentration of African clawed frog (Goleman et al., 2002a) and mosquito fish, Gambusia holbrooki (Bradford et al., 2005) and in plasma T4 of rabbits (York et al., 2001), whereas perchlorate treatment increased T4 concentration in whole body of fathead minnow (Crane et al., 2005) and in plasma of deer mice, Peromyscus maniculatus (Thuett et al., 2002). The reason for these inconsistent observations with T4 is uncertain, although it could be related to compensation (i.e., increased TH production) or exhaustion (i.e., decreased TH production) of the thyroid system in relation to xenobiotic dose and length of exposure (e.g., Brown et al., 2004). It is notable that T3 concentrations seem to be more resistant to change than T4 concentrations during exposure to perchlorate. For example, exposure to perchlorate did not affect T3 levels in rabbits (plasma; York et al., 2001) and zebrafish (whole body; present study) despite a decline in T4 levels in both cases. (Although whole-body T3 concentrations in the present study appeared to show a trend to decrease in perchlorate-treated adult fish, this trend was not statistically significant.) The relative resilience of T3 homeostasis was also recently demonstrated in a study of rats (Rattus norvegicus) fed iodine-deficient diets, in which reductions in the T3 content of various tissue compartments occurred only after the corresponding T4 content had declined by 75–95% (Pedraza et al., 2006). In the present study, whole-body T4 content in female fish exposed for 16 weeks to 100 mg/ml perchlorate was reduced by 95% compared values recorded in control fish. Thus, zebrafish treated with perchlorate may have been near hypothyroid at the end of the 16-week exposure period, and it is possible that longer exposures could have led to clear reductions in T3 content.
Because T4 is the predominant TH synthesized and stored in thyroid follicles of teleosts, it is not surprising that exposure to compounds that inhibit T4 synthesis (e.g., perchlorate) take relatively long periods of time to significantly suppress T4 levels. T3 is biologically more active than T4, and in teleosts it is synthesized by peripheral tissues via outer ring deiodination of T4 (Brown et al., 2004). Treatment of tilapia (Oreochromis mossambicus) with methimazole, also a TH synthesis inhibitor, induces the activities of type I and type II deiodinases (which enhance T3 production from T4) and suppresses the activity of type III deiodinase (which metabolizes T3 and T4 to inactive forms) in the liver (Van der Geyten et al., 2001). These changes in deiodinase activities following disruption of TH synthesis in tilapia (Van der Geyten et al., 2001) and other teleosts (Brown et al., 2004) are believed to help maintain T3 homeostasis under conditions of reduced but still adequate T4 supply. Thus, increased conversion rates of T4 to T3 under near-hypothyroid conditions may explain why T3 levels are even more resistant to change than T4 levels following exposure to goitrogens. It is also important to note that whole-body measurements of TH—as in the present study—could mask changes that occur in individual tissues. For example, iodine deficiency may have different or even opposite effects on the T3 content of different target tissues (Pedraza et al., 2006). Thus, the possibility cannot be ruled out that, in the present study, perchlorate exposure caused hypothyroid conditions in some tissues.
Coupled with the decline in whole-body maternal T4 content, PVE was also clearly suppressed by perchlorate after an exposure period of 12–16 weeks. The inhibitory effects of perchlorate were consistent for both concentrations (10 and 100 mg/l) at weeks 15 and 16, although early during the spawning period it appeared that perchlorate was more effective at 10 mg/l than 100 mg/l. The reason for the earlier reduction in egg volume at 10 mg/l perchlorate than 100 mg/l is not clear. A previous study with zebrafish did not find an effect of perchlorate (18 mg/l) on PEV following an 8-week exposure (Patiño et al., 2003), but unfortunately TH content was not measured in that study. A subsequent study suggested that T4 levels in zebrafish exposed to similar concentrations of perchlorate take longer than 12 weeks to change (Mukhi et al., 2005). Taken together, these observations with zebrafish (Mukhi et al., 2005; Patiño et al., 2003; present study) suggest that perchlorate impairs egg production in this species only if the exposure conditions are such that overall T4 homeostasis is also disrupted. These observations are also consistent with the view that endogenous TH plays a role in the regulation of zebrafish reproduction. In support of this view, there is correlative as well as experimental evidence suggesting that TH is necessary for gonadal recrudescence in various teleost species (Cyr and Eales, 1988, 1996; Cyr et al., 1988; Van der Geyten et al., 2001; Weber et al., 1992). Although GSI was not measured in the present study, the lower body weight of perchlorate-treated female fish immediately prior to spawning is consistent with the view that their GSI were also suppressed. However, the results of the present study cannot rule out the possibility of a direct effect of perchlorate on the reproductive system, especially since a clear hypothyroid condition (T3 deficiency) was not achieved.
Not all studies of teleost reproduction have reported negative consequences of goitrogen exposure. An earlier study with zebrafish suggested the possibility of a slight stimulatory effect of perchlorate on the number of eggs produced (Patiño et al., 2003) under conditions where whole-body TH content may not have been affected (Mukhi et al., 2005). In eastern mosquito fish (G. holbrooki), conditions of perchlorate exposure that yield only small declines in T4 content (Bradford et al., 2005) also seem to lead to a small stimulatory effect on the fecundity of female fish (Park et al., 2006). In marked contrast with the results of the present study, it was recently reported that exposure of zebrafish to goitrogen caused a considerable increase in egg production (egg numbers) concomitant with clear declines in (plasma) T4 as well as T3 levels (Van der Ven et al., 2006). In this recent study, zebrafish were exposed to propylthiouracil (like perchlorate, an inhibitor of TH synthesis) for 3 weeks and were spawned twice weekly over the exposure period. Each unit of replication (n = 3) in this recent study consisted of two females and one male, and control fish spawned an average of 2.3 times out of 6 spawning attempts (Van der Ven et al., 2006). Thus, the low numbers of animals per spawning unit coupled with a spawning failure of over 50% in the control fish raise the possibility of biased results due to random differences in the pretreatment condition of the experimental fish. In the present study, each unit of replication (n = 3) consisted of eight females and four males, and reproductive cycles were synchronized by a trial spawn prior to the initiation of data collection. The results of the present study clearly indicated that zebrafish fecundity (spawn volume and spawned egg numbers) is suppressed after prolonged exposure to perchlorate. However, the possibility of short-term increases in the number of eggs or embryos produced following goitrogen exposure (Park et al., 2006; Patiño et al., 2003; Van der Ven et al., 2006) deserves further examination.
Because T4 and T3 could not be both measured on the same batch of eggs collected at any given time (due to limited material), conclusions about the effects of perchlorate on embryonic T4 and T3 levels need to be made with caution. However, it seemed reasonably clear that exposure to perchlorate affected the TH content of early F1 embryos in a pattern similar to that observed in their mothers. Namely, T4 content was decreased and T3 content was unchanged in embryos derived from mothers exposed to perchlorate at 10 or 100 mg/l for 14–15 weeks. Maternally derived TH concentrations decline during development of the teleost embryo prior to the onset of endogenous hormone production (e.g., Power et al., 2001) and, in zebrafish, endogenous TH production does not begin until after hatching (Wendl et al., 2002). Because TH concentrations in whole embryos were determined shortly after fertilization (3.5 h), these concentrations are most likely a direct reflection of maternally inherited TH with little or no contribution from embryonic T4 or T3 metabolism. Thus, embryos of perchlorate-treated zebrafish had suppressed reserves of T4 at the beginning of their development and may have become hypothyroid (T3 deficient) prior to the completion of embryogenesis and hatching. The results of an earlier study with zebrafish indicated that lower jaw development and other aspects of craniofacial development are at least partly regulated by TH during the embryo-to-larva transition (Liu and Chan, 2002). In the present study, 4-dpf larvae derived from parents exposed to perchlorate (100 ppm) showed a reduced forward protrusion of the Meckel's cartilage (which forms the lower jaw) and the ceratohyal cartilage (which supports the lower jaw). This observation is consistent with the view that an overall reduction in maternally derived TH (due to maternal exposure to perchlorate) affects the craniofacial development of the F1 progeny during their embryo-to-larva transition. However, whereas larval head depth was also reduced in the earlier study (Liu and Chan, 2002), this parameter of craniofacial development was not affected in the present study; this difference in results between the two studies is perhaps due to differences in methodology (embryo-larval treatment versus maternal treatment, respectively).
Despite the negative effects on fecundity (see preceding discussion), exposure to perchlorate did not affect egg fertilization and hatching rates in the present study. These observations are consistent with those of a similar study with medaka, where fertilization and hatching of hypothyroid F1 eggs and embryos derived from thiourea-treated hypothyroid mothers did not differ from those of control individuals (Tagawa and Hirano, 1991). Other studies with zebrafish have also reported no effects of maternal goitrogen exposure on egg fertilization or hatching rates (Patiño et al., 2003; Van der Ven et al., 2006). Curiously, the diameter of fertilized eggs and the length of 4-dpf larvae were increased by maternal exposure to perchlorate in the present study. The reason for these phenomena is uncertain, but it could be related to a physiologically regulated trade-off between fecundity and egg size. In mosquito fish, maternal exposure to perchlorate also caused a slight increase in the mass of individual embryos (Park et al., 2006).
In conclusion, prolonged exposure of adult zebrafish to perchlorate impairs their thyroid endocrine system as well as their spawning success and also influences early F1 development. The concentrations of perchlorate used in this study are high in an environmental context. However, the results of this study make it clear that the severity of the reproductive effects of perchlorate greatly increases with time of exposure within the adult life span of an individual. Full life cycle or multigenerational studies will be needed to determine whether the reproductive effects of perchlorate are also of concern at the lower, more commonly reported environmental concentrations.
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NOTES |
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1 Present address: Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21218.
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ACKNOWLEDGMENTS |
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We acknowledge the help of Aaron Urbanczyk and James Dumbauld during experimental exposures. Drs James Carr and Duncan MacKenzie read a draft version of this manuscript and provided useful criticism. This research was supported by funding from the Texas Cooperative Fish and Wildlife Research Unit, which 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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