A Mixture of Ammonium Perchlorate and Sodium Chlorate Enhances Alterations of the Pitutary-Thyroid Axis Caused by the Individual Chemicals in Adult Male F344 Rats

Abstract

Ammonium perchlorate (AP) and sodium chlorate (SC) have been detected in public drinking water supplies in many parts of the United States. These chemicals cause perturbations in pituitary-thyroid homeostasis in animals by competitively inhibiting iodide uptake, thus hindering the synthesis of thyroglobulin and reducing circulating T₄ (thyroxine). Little is known about the short-term exposure effects of mixtures of perchlorate and chlorate. The present study investigated the potential for the response to a mixture of these chemicals on the pituitary-thyroid axis in rats to be greater than that induced by the individual chemicals. Adult male F-344 rats were exposed, via their drinking water, to the nominal concentrations of 0.1, 1.0, 10 mg/L AP or 10, 100, 1000 mg/L SC and their mixtures for 7 days. Serum T₄ levels were significantly (p < 0.05) reduced in rats following exposure to the mixtures, but not after exposure to the individual chemicals. Serum T₃ (triiodothyronine) was not altered by treatment and TSH (thyroid stimulating hormone) was only increased after the high-dose chlorate treatment. Histological examination of the thyroid gland showed colloid depletion and hypertrophy of follicular epithelial cells in high-dose single chemical and all mixture-treated rats, while hyperplasia was observed only in some of the rats treated with mixtures (AP 10 + SC 100, AP 0.1 + SC 1000, and AP 10 + SC 1000 mg/L). These data suggest that short-term exposure to the mixture of AP and SC enhances the effect of either chemical alone on the pituitary-thyroid axis in rats.

Introduction

Humans and wildlife are routinely exposed to mixtures of chemicals present in the environment. Chemicals, such as ammonium perchlorate (AP) and sodium chlorate (SC) are of concern because their past and present use and improper disposal have resulted in widespread contamination of surface water and groundwater sources (Motzer, 2001; Greer et al., 2002; Urbansky, 2002). Studies have shown that subchronic single chemical exposure to AP or SC cause perturbations in pituitary-thyroid homeostasis in developing and adult mammals, amphibians, and fish (Brechner et al., 2000; Hooth et al., 2001; York et al., 2001, 2004; Goleman et al., 2002a; Patino et al., 2003). However, information is scanty about the short-term exposure effects of the biologically important mixture of AP and SC on the pituitary-thyroid axis in animals.

Ammonium perchlorate is widely used as an oxidizer in solid rocket propellants, munitions, fireworks, fertilizer, and air bag inflators (Fisher et al., 2000; Lawrence et al., 2000). It is highly soluble in water and the perchlorate ion dissociates from its cationic ligands and remains stable for long periods under normal environmental conditions (Urbansky, 1998). In the United States alone, surface water and groundwater contamination with AP has been reported in 14 states (Fisher et al., 2000; Smith et al., 2001; Merrill et al., 2003). Surface water and groundwater concentration of AP as high as 630,000 to 3,700,000 ppb was documented near Las Vegas, Nevada. In other states of the United States, most detections were found at or below 20 ppb (Motzer, 2001; Soldin, 2001; Greer et al., 2002). Perchlorate was detected at concentrations of 555–5,557,000 ppb (vegetation), 811–2038 ppb (aquatic insects), and up to 207, 580, and 2328 ppb in fish, frogs, and mammals, respectively, near an ammunition plant in Texas (Smith et al., 2001).

Subchronic exposure to AP causes perturbations in thyroid homeostasis in mice over the range of 1.0 nM–1.0 mM (Thuett et al., 2002), rabbits from 10–100 mg/kg (York et al., 2001a), rats from 1.0–30 mg/kg (Siglin et al., 2000; York et al., 2001b; Yu et al., 2002), Xenopus laevis from the LC50 of 510 ± 36 mg/kg (Goleman et al., 2002b), and in zebra fish from 18–667 mg/L (Patino et al., 2003). In humans, perchlorate is known to affect thyroid function at pharmacologic doses (Stanbury and Wyngaarden, 1952; Wolff, 1998). A recent study correlated abnormal thyroid function in newborn humans and consumption of drinking water from AP contaminated Colorado River water (Brechner et al., 2000).

Sodium chlorate is used as an oxidizing agent in the dye, explosives, matches, and leather industries. It is also found as a drinking water disinfection by-product after chlorine dioxide disinfection (Gallagher et al., 1994; ILSI, 1999). According to estimates, 10–30% of the chlorine dioxide in disinfected drinking water is converted to chlorate (Couri et al., 1982; Bolyard and Fair, 1993; McCauley et al., 1995). Chlorate has been detected in drinking water at concentrations as high as 2.0 mg/L (ILSI, 1999). Sodium chlorate exposure can result in potentially detrimental effects on thyroid homeostasis in rats. McCauley et al. (1995) reported significant reductions in serum T₃ and T₄ levels following exposure to 12–48 mM (approx 1400–5000 mg/L) of SC for 90 days. In a recent study, exposure to SC at 1.0–2.0 g/L for 4–21 days caused histological changes in the rat thyroid gland including depletion of colloid and hypertrophy and hyperplasia of follicular epithelial cells (Hooth et al., 2001).

Mechanistic studies suggest that AP and SC affect pituitary-thyroid homeostasis by competitively inhibiting iodide uptake at the sodium iodide symporter (NIS) of the thyroid gland, which results in decreased synthesis of thyroglobulin and reduced circulating thyroid hormone levels (Carrasco, 1993; Wolff, 1998; York et al., 2001a). Thyroid hormones are essential for normal body functions including metabolism, growth, maturation, and reproduction in developing and adult animals (Capen et al., 1991). Long-term exposure to such chemicals may disrupt many vital body functions. As a consequence, there may be persistent stimulation of the pituitary-thyroid axis, which may lead to hypertrophy and hyperplasia of thyroid follicular cells that may lead to tumor formation (Capen et al., 1991).

Despite different sources of origin, AP and SC may contaminate the drinking water simultaneously; thus, humans and wildlife may be exposed to mixtures of these chemicals. A default drinking water consumption rate of 2 L/day in humans could result in the potential consumption of 36 μg of perchlorate and 4 mg of chlorate per day (Gibbs et al., 1998). Most of the information documenting the adverse effects of AP or SC on thyroid homeostasis comes from single chemical, long-term, exposure studies (Siglin et al., 2000; York et al., 2001a; Goleman et al., 2002b; Greer et al., 2002). Information is not available regarding the interim toxicodynamics and risk from exposure to mixtures of these chemicals.

The present studies were conducted to characterize the effects of exposure to mixtures of AP and SC on the pituitary-thyroid axis in rats. The objectives were to describe and compare the short-term exposure effects of AP or SC on thyroid homeostasis, and to examine the potential for an enhancement of these effects by the mixtures of these chemicals.

Methods

Animals and Dosing

The present study was conducted in accordance with the EPA’s approved Laboratory Animal Project Review (LAPR) and Institutional Animal Care and Use Committee (IACUC) guidelines.

Six-week-old (42-day) male Fischer-344 rats were purchased from Charles River Laboratory, Raleigh, NC. They were allowed to acclimate in an EPA designated animal facility for 5 days prior to exposure under standard conditions (temp 72–74°F, humidity 42–46% and a 12-hr dark and light cycle). All animals had free access to a commercial rodent chow (Purina 5001, St. Louis, MO; Iodine content 0.8 ppm) and drinking water. At 7 weeks of age, the rats were divided into 16 different treatment groups (n = 10/dose group) including control (Table 1). Animals were housed in hanging plastic cages with ALPHA-dri paper bedding with 2 rats per cage. Individual rats were identified by an ear tag. Rats in different dose groups received their respective dose solutions for 7 consecutive days through the drinking water. The water bottles were examined daily for any leakage or obstruction throughout the study. The rats were weighed at the beginning of exposure and again at necropsy. Animals were examined daily in their cages for signs of toxicity or illness. Two rats from dose group AE did not drink water, lost weight, and appeared dehydrated. They were culled from the study following NHEERL/EPA guidelines.

Chemicals, Dose Solution Preparation, and Chemical Confirmation Analysis

Ammonium perchlorate (NH₄ClO₄) CAS# 7790-98-9 and Sodium chlorate (NaClO₃) CAS# 7775-09-9, purity >99% were purchased from Sigma-Aldrich Chemical Co. Milwaukee, WI. Single chemical dose solutions were prepared by dissolving the individual chemicals in deionized water (d. water) to appropriate concentrations of AP (0.1, 1.0, and 10.0 mg/L) and SC (10, 100, and 1000 mg/L). Mixture solutions were prepared by using a full factorial design to provide all the possible combinations of the selected single chemical doses (Table 1). These doses were chosen based upon information available in the literature considering that these chemicals would not affect thyroid homeostasis at low levels, but would alter serum hormone levels at high doses without producing overt toxicity.

Dose solutions were prepared by weighing the chemicals individually, thoroughly dissolving in 1.0 liter of deionized water (Dracor, Kensington MD), and bringing the final volume to 4 liters in clean, separate and appropriately labeled carboys. All solutions were stored at room temperature in a dry cool place away from light. The rats received their respective treatments via clean brown glass drinking water bottles that were fitted with steel sippers and placed on cage tops. Water samples from each dose solution were collected for analysis in clean scintillation vials at the beginning, at water bottle change (middle), and at the end of the study.

The chemical analysis was accomplished by using high performance liquid chromatography (HPLC) (Waters, Milford, MA) with conductivity detector (CD 20). The HPLC system was a Waters (Milford, Ma) 600E pump with a 717 autosampler and column heater. The column was a 100 × 4.6 mm Allsep Anion column manufactured by Alltech Inc. (Deerfield, Ill). The detector was a CD20 conductivity detector manufactured by Dionex (Sunnyvale, Ca) with an ARS Ultra 4 mm suppressor equipped for either recycle or chemical suppression mode. Ammonium Perchlorate was analyzed by injecting 1mL of sample with a 2.8 mmole sodium bicarbonate, 2.2 mmole sodium carbonate, and 8 mmole p-cyanophenol aqueous isocratic mobile phase at 2 mL/min. The ARS Ultra 4 mm suppressor was in chemical suppression mode using 50 mmoler of sulfuric acid at a flow rate of 5 mL/min. Sodium chlorate was analyzed by injecting 100 μL of sample with a 2.8 mmole sodium bicarbonate and 2.2 mmole sodium carbonate aqueous isocratic mobile phase at 2 mL/min. The ARS Ultra 4 mm suppressor was in recycle mode.

The actual dose received by rats in each dose group (Table 1) was calculated by multiplying water chemical concentration with average water consumption by 2 rats (per cage) for 7 days and dividing this value by the average body weight of the 2 rats in that particular cage.

Necropsy and Tissue Collection

A fixed time schedule and necropsy order was followed to maintain consistency throughout the studies. All rats were euthanized by CO₂ asphyxiation followed by exsanguinations, then necropsied between 8:00 AM and 12 noon on each day of necropsy. Cardiac blood was aspirated and serum was collected by centrifuging blood samples at 5000 rpm for 20 min. Serum samples were stored at −80°C until hormone analysis. Tissues taken at necropsy (hypothalamus, pituitary, thyroid, and liver) were examined for gross lesions before fixation. Selected tissues from 4 rats in each dose group were quick-frozen in liquid nitrogen and stored at −80°C for further analysis. The thyroid from 6 rats in each dose group were fixed in 10% neutral buffered formalin, processed by routine methods, sectioned at 5 μm, and stained with hematoxylin and eosin for histological examination.

Serum T₄, T₃, and TSH Analysis

Serum total thyroxine (T₄) and total triiodothyronine (T₃) analyses were performed by using commercially available Total T₄and T₃ RIA kits (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA) following manufacturer’s instructions and using duplicate calibrator, control, and test samples (Khan et al., 1999). The assay sensitivity range for total T₄ was 1.0 to 24.4 μg/dL and that of the T₃ assay was 0.5 to 5.6 ng/ml. Serum TSH concentration was measured by using materials supplied by the National Hormone and Pituitary Program, Torrance, CA (Iodination preparation NIDDK-rTSH-I-9, reference preparation NIDDK-rTSH-RP-3, and the antiserum NIDDK-antirat TSH-RIA-6). Iodination material was radiolabeled with ¹²⁵I (Perkin Elmer/New England Nuclear, Boston, MA) by a modification of the chloramine-T method (Greenwood et al., 1963). Labeled TSH was separated from untreated iodide by gel filtration chromatography (Sowers et al., 2003). The assay was conducted in duplicate, as described in (Thibodeaux et al., 2003) and assay sensitivity was 0.9 to 16.8 ng/ml.

Histological Examination

The thyroid, pituitary, and liver were examined for histological changes without knowledge of treatment. The thyroid tissues were graded for colloid depletion, hypertrophy, or hyperplasia of follicular epithelial cells by following criteria described in Hooth et al. (2001). Briefly, colloid depletion was identified when there was either reduction or absence of colloid in the follicular lumen or when there was pale, lacy, and/or granular material in the follicular lumen. Follicular cell hypertrophy was considered present when thyroid follicles were uniformly lined by tall cuboidal to columnar epithelium, and there was an increase in cytoplasm to nuclear ratio along with an increase in cell width and height. Thyroid hyperplasia was evaluated using a combined assessment of amount of hyperplasia within follicles, in addition to the area of thyroid affected. Pituitary tissue slides were graded in accordance with the increase or decrease in the number of acidophilic or basophilic cells in the anterior pituitary gland. Since TSH cells in the anterior pituitary are basophilic, it was anticipated that an increase in the number of basophils would be reflected by the increased intensity of blue (basophilic) color. Liver slides were also examined for histological changes.

Data Analysis

Data obtained were analyzed using SigmaStat Version 3.0 software (SPSS, Inc). Differences between treatment groups were analyzed using 1 way analysis of variance (ANOVA), followed by Dunnett’s 2 sided test for comparison of means of treated vs. control groups. Differences were considered significant if p < 0.05. Results are expressed as mean ± standard error of the mean (SEM). Pairwise comparisons of serum hormone levels were made using 1 way ANOVA followed by Tukey’s method and log-linear model was used to examine the association between chemical exposure and hypertrophy or hyperplasia of thyroid tissue.

Results

Chemical Confirmation Analysis

The actual concentration of chlorate and perchlorate in the dosing solutions is presented in Tables 1, 2, and 3. Unexpectedly, chlorate was detected at a mean concentration of 0.5 ± 0.04 mg/L in deionized water that was used as a control and vehicle to prepare the dose solutions. The source of SC contamination in the deionized water supply was not determined. While it is possible that there was a contaminant in the samples that eluted at the same time in the ion chromatogram as chlorate, it is unlikely. Identification is made by retention time and visual inspection of peak shape. The retention time was appropriate for chlorate, and the peak shape did not indicate a coeluting contaminant. The water analysis was conducted after completion of the in-life portion of the study at which time it was discovered that the nominal concentrations desired were not achieved in all exposure groups.

Animals and Dosing

No clinical signs of toxicity or toxicity-related deaths occurred among rats in any treatment group in the present studies. Also, no biologically significant differences were noted in mean body weight and water consumption between rats in the control and other treatment groups. Some of the rats in group CD had a problem with the water bottles, which impacted their weight gain unrelated to treatment. The mean ± SEM body weight, water consumption, rate, and actual dose of chemicals received by rats in different treatment groups are presented in Table 1.

Serum T₄, T₃, and TSH Analysis

Exposure to mixtures of chlorate and perchlorate resulted in a significant (p < 0.05) reduction in serum T₄ as compared to control (Table 2). Despite reductions in T₄, no changes were seen in serum T₃ or TSH levels in any treatment group except the SC high-dose group where TSH levels were significantly greater than control (Table 2). Pairwise analysis of serum hormone levels following exposure to AP or SC and their mixtures are described in Table 4. Briefly, serum T₄ concentration decreased significantly in mixture treated animals as compared to the single chemical exposure group (Table 4). Serum TSH levels, however, did not show consistent trends, but showed significant differences among SC or mixture-treated rats.

Histological Examination

Histological examination of the thyroid gland showed depletion of colloid accompanied by hyperplasia of follicular epithelial cells in rats that received high doses of AP or SC and in all rats exposed to the mixtures of these chemicals (Table 3; Figure 1). Hypertrophy of thyroid follicular epithelial cells was present following single chemical exposure to SC and the high dose of AP as well as the mixtures of the 2 chemicals. An increase in the incidence and severity of follicular cell hyperplasia was present in rats that received mixture solutions containing medium and high concentration of AP and SC (Table 3). The log linear model analysis of the data showed an association (p < 0.0001) between hypertrophy and SC exposure. Hyperplasia was associated with both, AP (p < 0.02) and SC (p < 0.002) exposure. Examination of the anterior pituitary gland revealed an apparent increase in the number of basophils (basophilia) only in rats that received high doses of AP or SC and their mixtures. These are the same groups that also had colloid depletion. No treatment-related lesions were observed following gross and microscopic examination of the liver (Table 3).

Discussion

Short-term exposure to mixtures of AP and SC caused significant reductions in serum T₄ levels in contrast to the lack of change in serum hormone levels following treatment with a single chemical. These observations are different from those reported following long-term exposure to AP or SC, i.e., reductions in serum T₃ and T₄ levels followed by a compensatory increase in serum TSH concentration (Siglin et al., 2000; York et al., 2001a, 2001b). However, it is important to note that the current investigation is unique from earlier studies because first, the animals were exposed to AP or SC for only 7 days and second, this study examined the mixture effects of the AP or SC that have not been studied previously. Also, the changes seen in serum hormone levels following exposure to mixtures of AP or SC resemble those seen following exposure to various polychlorinated biphenyls (PCBs) congeners (Khan et al., 2002; Khan and Hansen, 2003). Results of the present studies suggest that 7-days of exposure to the mixtures of AP and SC enhances the effect on the rat pituitary-thyroid axis and results in hypothyroxinemia, a condition in which serum levels of total or free T₄ decrease but serum T₃ and TSH remain within normal range (Lavado-Autric et al., 2003). In the past, hypothyroxinemia was generally regarded as harmless, but there is increasing evidence of potentially adverse outcomes from studies of populations of pregnant women who are hypothyroxinemic from dietary iodine deficiency or exposure to environmental endocrine disruptors (Khan et al., 2002; Lavado-Autric et al., 2003). Recent evidence suggests that maternal hypothyroxinemia can be harmful to offspring especially during early gestation in animals, including humans (Calvo et al., 1992, 1997; Morreale de Escobar et al., 2000; Lavado-Autric et al., 2003).

Studies have shown that subchronic single chemical exposure to AP or SC results in perturbations of pituitary-thyroid homeostasis in developing and adult animals. Significant changes were seen in serum thyroxine (T₄), triiodothyronine (T₃), and thyroid stimulating hormone (TSH) concentration in adult male and female Sprague–Dawley rats that were orally exposed to 0.2, 1.0, and 10 mg/kg/day of AP through drinking water for 14 days and at 0.01, 0.2, 1.0, and 10 mg/kg/day for 90 days. However, the histopathological effects were evident only after high-dose exposure of AP at 10 mg/kg/day for 14 or 90 days (Siglin et al., 2000). In a 2-generation study, oral administration of 0.3, 3.0, or 30 mg/kg of AP to rats resulted in alterations of serum thyroid hormones and thyroid gland histology in the parents (P) and their F1 and F2 male and female pups. In all animals, changes in the thyroid gland histology (hypertrophy and hyperplasia) occurred at lower doses (3.0 mg/kg) than those (30 mg/kg) that caused significant alterations in serum T₄, T₃, and TSH concentration (York et al., 2001b). A developmental study conducted in New Zealand White Rabbits showed dose-dependent reductions in maternal serum T₄levels and changes in thyroid histology after exposure to AP at 10, 30, and 100 mg/kg/day, but no effects were seen in offspring (York et al., 2001a), suggesting species-specific effects of AP. The thyroid disruption potential of AP has also been reported in fish and amphibians in addition to mammals (Goleman et al., 2002b; Patino et al., 2003; Tietge et al., 2005).

Information is limited, but studies have shown that SC also disrupts thyroid homeostasis in a manner similar to that of AP. Serum T₃ and T₄ decreased and TSH levels increased following 4 days of exposure to SC at 1.0 or 2.0 g/L and after 21 days of treatment with SC at 2.0 g/L in F-344 rats. An increase in thyroid follicular cell hyperplasia and significant colloid depletion was seen in rats exposed to 1.0 and 2.0 g/L of SC for 14 or 90 days (Hooth et al., 2001). Although the results of these studies may vary because of different study designs and species specificity, it is apparent that the pituitary-thyroid axis is a target of both AP and SC.

In the present studies, single chemical oral exposure to AP or SC (at selected doses) did not cause alteration in serum T₃, T₄, and except for the high dose SC, TSH in rats. This response was not unexpected because this study was not intended to expose the rats to overtly high doses of chemicals. It is possible that the changes in serum thyroid hormone levels occur at very early time points following exposure to these chemicals that normalized within 7 days. However, histological changes in the thyroid gland, especially at high doses of AP or SC, (Table 3) are consistent with other short-term studies where morphological changes in the thyroid gland occurred prior to obvious hormonal changes (Ness et al., 1993; Khan et al., 1999; York et al., 2001b). The exact reason for such a response is not clear, but a few possibilities can be suggested based upon the information available. The thyroid gland is highly innervated and can regulate itself without the influence of TSH (Thomas and Williams, 1992). Recently, it has been reported that iodine deficiency triggers an increase in the thyroid gland’s sympathetic nervous system (SNS) activity, which causes morphological changes in the thyroid (Young et al., 2005).

It is likely that, in the present studies, iodine deficiency caused by exposure to AP or SC and their mixtures activated the thyroid SNS activity which could result in histological changes independent of TSH stimulation. Another possibility for such a response could be due to the fact that the thyroid gland takes more time to return to its normal structure even after cessation of exposure to thyroid disruptors than it takes for serum hormone levels to equilibrate (Thomas and Williams, 1992). Why there is no increase in TSH levels in the face of significantly reduced T₄ levels is not clear. The present data also indicates that serum TSH were low in most of the animals exposed to mixtures of chemicals. Evidence suggests that the pituitary gland response may have been impaired such that would not secrete compensatory TSH following decreased T₄ levels. Such responses have been seen following exposure to some environmental PCB congeners (Khan and Hansen, 2003). Whether mixtures of AP and SC act through the described mechanism(s) is not clear. In the present study, morphological changes in the thyroid gland may have occurred early and we may be observing the histological changes in a gland that is involuting. Furthermore, thyroid gland growth and proliferation are highly sensitive to modest TSH stimulation (Fail et al., 1999; Hood et al., 1999). A mild increase in TSH levels could have resulted in the histological changes in the thyroid gland in the present study, which would take time to return to normal. The current data also suggests synergistic effects following exposure to the mixture of AP and SC (Table 2). Serum T4 levels reduced significantly in mixture treated rats (Table 4). It may have been that exposure to even low doses of a mixture of AP and SC resulted in an exhaustion of the pituitary-thyroid axis to an extent that could not respond further. Therefore, no added effects were noticed in high-dose groups. In this study, SC appeared to be more effective in causing changes in serum thyroid hormone levels than AP. An overall association between hypertrophy and hyperplasia of the thyroid gland after SC treatment also suggests this. It is possible that exposure to relatively high doses of SC may be responsible for these changes.

The physiology and control of the pituitary-thyroid axis is very similar in humans and rats (Fukuda et al., 1975); however, data should be interpreted cautiously because of species-specific differences. Many studies highlight the thyroid-disruption potential of AP or SC in experimental animals (McCauley et al., 1995; Wolff, 1998; Siglin et al., 2000; Hooth et al., 2001), but information is inconsistent regarding the ability of these chemicals to disrupt thyroid homeostasis in humans. Brechner et al. (2000) reported significantly higher median TSH levels in newborn babies exposed to perchlorate-contaminated drinking water. In contrast, many studies have shown that environmentally relevant levels (16 μg/dl) of perchlorate lifetime occupational exposure (8000–88000 μg/kg) do not produce adverse effects in adult humans (Gibbs et al., 1998; Lamm et al., 1999; Lamm and Doemland, 1999; Lawrence et al., 2000; Greer et al., 2002). Also, perchlorate has been used at therapeutic doses of 600–1000 mg/kg/day to treat patients suffering from thyrotoxicosis and no adverse effects were observed even at 1500–2000 mg/kg (Crooks and Wayne, 1960; Soldin et al., 2001). Most of the available information on effects in human populations is after perchlorate exposure. There are very limited data regarding the thyroid disruption potential of SC in humans.

The adult human pituitary-thyroid axis may be less vulnerable to adverse effects of AP or SC and related chemicals because of certain chemical and biological factors. For example, AP has a half-life of 6–8 hours and about 95% is excreted within 72 hrs (Eichler, 1929). Also, in humans, the T₄ is bound to a high affinity thyroxine-binding globulin (TBG), which is absent in rodents and other animals. As a consequence, rodents have a higher thyroid turnover rate and shorter half-life (12–24 hours) of T₄as compared to adult humans (5–7 days) (Capen, 1991). Therefore, adult humans may be resistant to perturbation of the pituitary-thyroid axis, especially following short-term exposures to thyroid endocrine disruptors. However, the disruption of pituitary-thyroid axis in expectant mothers, the fetus, and infants occurs under entirely different circumstances because of the precise hormonal environment and the developing pituitary-thyroid axis. The human fetus does not have a fully functional thyroid system before 10–12 weeks of gestation and low maternal thyroxine levels during sensitive stages of development may cause irreversible damage to an immature fetus (Pop et al., 1999). Moreover, other environmental, physical, chemical, and nutritional factors may also contribute to perturbations of the thyroid system resulting in marginal or low circulating T₄ levels in pregnant women.

In conclusion, humans and wildlife are rarely exposed to single chemicals, and there is a need to explore the interactions between the biological systems and mixtures of chemicals present in our food and environment. The pregnant mother, developing fetus, and growing child are of greatest concern, because a short-term toxic insult during crucial periods of growth and development may cause irreparable damage. The present study suggests that mixtures of AP and SC enhance the effect of either chemical alone and can alter circulating thyroid hormone levels and produce associated histological changes even after short-tem exposure. However, these results should be interpreted carefully because the current study is a preliminary attempt to address the important issues regarding the effects of chemical mixtures present in drinking water. A thorough knowledge of the pharmacokinetics or toxicokinetics of chemical mixtures is needed for a comprehensive analysis.