ToxSci Advance Access originally published online on March 22, 2007
Toxicological Sciences 2007 97(2):308-317; doi:10.1093/toxsci/kfm063
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Low-Dose Effects of Ammonium Perchlorate on the Hypothalamic-Pituitary-Thyroid Axis of Adult Male Rats Pretreated with PCB126
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* Interdisciplinary Toxicology Program, University of Georgia, Athens, Georgia 30602
Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602
Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Air Force Research Laboratory, Human Effectiveness Directorate, Biosciences and Protection Division, Applied Biotechnology Branch, Wright-Patterson Air Force Base, Ohio 45433
¶ Section of Endocrinology, Diabetes, and Nutrition Center, Boston Medical Center, Boston, Massachusetts 02118
|| Statistics and Modeling Supporting Informed Decisions, Athens, Georgia 30606
||| Division of Toxicology, Agency for Toxic Substances and Disease Registry, Atlanta, Georgia 30333
1 To whom correspondence should be addressed at 206 Environmental Health Sciences Department, University of Georgia, Athens, GA 30602-2102, USA. Fax: 706-542-7472. E-mail: evad{at}uga.edu.
Received January 18, 2007; accepted March 16, 2007
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ABSTRACT |
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The objective of this research was to characterize the disturbances in the hypothalamic-pituitary-thyroid (HPT) axis resulting from exposure to a binary mixture, 3,3',4,4',5-pentachlorobiphenyl (PCB126) and perchlorate (ClO
), known to cause hypothyroidism by different modes of action. Two studies were conducted to determine the HPT axis effects of ClO on adult male Sprague-Dawley rats pretreated with PCB126. In dosing study I, rats were administered a single oral dose of PCB126 (0, 7.5, or 75 µg/kg) on day 0 and 9 days later ClO (0, 0.01, 0.1, or 1 mg/kg day) was added to the drinking water until euthanasia on day 22. Significant dose-dependent trends were found for all thyroid function indices measured following ClO in drinking water for 14 days. Seventy-five micrograms PCB126/kg resulted in a significant increase in hepatic T4-glucuronide formation, causing a decline in serum thyroxine and fT4, and resulting in increased serum thyroid-stimulating hormone (TSH). Serum TSH was also increased in animals that received 7.5 µg PCB126/kg; no other HPT axis alterations were found in these animals. When pretreated with PCB126, the ClO dose trends disappeared, suggesting a less than additive effect on the HPT axis. In dosing study II, animals were given lower doses of PCB126 (0, 0.075, 0.75, or 7.5 µg/kg) on day 0, and followed with ClO (0 or 0.01 mg/kg day) in drinking water beginning on day 1 and continuing for several days to explore transient HPT axis effects. No statistical effects were seen for PCB126 or ClO alone, and no perturbations were found when administered sequentially in dosing study II. In conclusion, these studies demonstrate that HPT axis disturbances following exposure to ClO are less than additive when pretreated with relatively high doses of PCB126. At relatively low doses, at or near the no-observed-effect-level for PCB126 and ClO, no interactions between the chemicals occur. Key Words: PCB126; perchlorate; rat; T4; thyroid; TSH; UDPGT.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Several important scientific challenges exist to improve chemical risk assessment practices, particularly with the reliance of human health risk assessments on laboratory animal toxicology studies. For example, most toxicology studies are conducted with a single chemical; however, humans are exposed to complex mixtures of chemicals. Environmental exposures to chemicals are typically much lower than the doses administered to laboratory animals. For toxicants that indirectly disturb endocrine system homeostasis, the interpretation of laboratory animal findings is confounded by significant species differences in endocrine physiology, such as the hypothalamic-pituitary-thyroid (HPT) axis (Capen, 1996
).
To begin to understand some of the scientific challenges for mixtures toxicology and endocrine-disrupting chemicals, we designed studies to evaluate HPT disturbances in rats administered low to moderate binary doses of two thyroid-active chemicals that induce hypothyroidism by dissimilar mechanisms. Perchlorate (ClO) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) were selected because of their widespread distribution in the environment (ATSDR, 2000; NRC, 2005; NTP, 2006), detection in human tissues (Blount et al., 2006; CDC, 2005), and their well characterized mode of action on the HPT axis in rats (Craft et al., 2002; Fisher et al., 2006; NRC, 2005; and Yu et al., 2002). PCB126 (3,3',4,4',5-pentachlorobiphenyl) is a potent coplanar (non-ortho) dioxin-like PCB congener with a toxic equivalency factor of 0.1 (Safe, 1994) and generally exists with mixtures of multiple PCB congeners in the environment. PCBs are no longer used by industry, but are ubiquitous in the environment with detectable concentrations found across all media, including air, soil, water, sediment, and biota (NTP, 2006). The primary mode of action for PCB126-mediated disruption of the HPT axis is through increased phase II metabolism of the thyroid hormone, thyroxine (T4). PCB126 binds to and activates hepatic aryl hydrocarbon receptors (AhRs). AhR activation results in the upregulation of several hepatic enzymes, including uridine diphosphate glucuronyl transferases (UDPGTs). An increase in phase II conjugation of T4 (formation of T4-glucuronide [T4-G]) results in increased biliary excretion of T4-G (Craft et al., 2002) and decreased circulating T4, leading to hypothyroidism. Dose-response characteristics for PCB126 and HPT disturbances in the rat were recently characterized in our laboratory (Fisher et al., 2006).
Perchlorate has been the subject of several toxicology studies targeting the HPT axis in wildlife, laboratory animals and humans because of its presence in water and food supplies (NRC, 2005). The ammonium perchlorate (AP) salt is used as an oxidizer in pyrotechnics, solid rocket fuels, and air bags. AP is highly water soluble and dissociates in water forming the perchlorate anion (Motzer, 2001). Apparently, perchlorate is also formed naturally (Dasgupta et al., 2005). Perchlorate acts on the HPT axis by competing for thyroidal uptake of dietary iodide (I–), resulting in a decline in available iodide for synthesis of the thyroid hormones (Wolff, 1998 and Yu et al., 2002) and the onset of hypothyroidism. A specialized transporter protein, referred to as the sodium/iodide symporter (NIS), located on the basolateral side of the follicular cell actively transports iodide and possibly perchlorate from the blood supply into the thyroid gland. Competitive inhibition of thyroidal uptake of radiolabeled iodide by perchlorate in the adult rat has been carefully characterized, along with the subsequent perturbations in serum thyroid hormones and thyroid-stimulating hormone (TSH) (Yu et al., 2002).
Very few studies with mixtures of either PCBs or anions have been conducted to ascertain mixture composition contributions to disruption of the HPT axis in the rat. In one study, Crofton et al. (2005) evaluated serum T4 levels after four consecutive days of oral gavage dosing with a mixture of 18 polyhalogenated aromatic hydrocarbons, including PCB126. There was no deviation from additivity at the lowest mixture dose, but a greater-than-additive decrease in serum T4 was observed at the three highest mixture doses. Khan et al. (2005) reported that synergistic interactions occurred when rats ingested binary combinations of perchlorate and chlorate in drinking water for 7 days, as evidenced by greater decreases in serum T4 levels. Interestingly, Khan et al. (2005) also noted that male Fischer rats appear less sensitive than male Sprague-Dawley (SD) rats to ClO-induced alterations in the HPT axis. In vitro competitive inhibition studies using FTRL-5 and COS NIS-6 cells have been undertaken with several anions to estimate the affinity of anions for the NIS protein (Van Sande et al., 2003).
In the present study, we evaluated the combined effects of two chemicals, both of which induce hypothyroidism by different mechanisms in the rat. Rats were pretreated with a single oral bolus dose of a potent and persistent thyroid-active chemical (PCB126) that is cleared slowly from the body. Dose- and time-dependent perturbations of the HPT axis are well characterized for PCB126. At a specified time after dosing, the PCB126-pretreated rats were given drinking water containing a second thyroid-active chemical, ClO, for different periods of time. Dose- and time-dependent perturbations of ClO on the HPT axis have also been characterized. Our working hypothesis was that for rats in which serum TSH was elevated by pretreatment with PCB126, the blocking effects of ClO on thyroidal uptake of iodide would be diminished, resulting in less than additive perturbations in the HPT axis. TSH stimulates the production of the NIS protein, which results in increased thyroidal uptake of iodide (Eng et al., 2001) and the formation and secretion of thyroid hormones. If increases in TSH associated with PCB126 leads to increases in the NIS protein, perchlorate may be less effective at blocking the thyroidal uptake of iodide.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Dose Selection and Design
To study the interactions of PCB126 and ClO
, doses of each chemical were selected that are known to cause moderate disturbances in the HPT axis and those that are thought to cause minimal or no disturbances in the HPT axis in the adult male rat. PCB126 dose selection (single oral bolus administration of 7.5 and 75 µg/kg) was based on previous research in our laboratory with single oral bolus doses of PCB126 and resulting perturbations in the HPT axis of the male SD rat (Fisher et al., 2006). Rats were given ClO in drinking water for 2 weeks at dose rates of 0.01, 0.1, and 1.0 mg/kg day; the two highest doses (0.1 and 1.0 mg/kg day) were previously reported to cause moderate disturbances of the HPT axis (Yu et al., 2002).
Dosing study I.
The average weight on day 0 for 128 SD rats used in this study was 216 ± 11 g. This study was divided into two groups (Table 1) with group 1 dosed 1 day prior to group 2. On day 0, a portion of the rats were administered single oral bolus doses of PCB126 dissolved in corn oil (7.5 or 75 µg/kg) or corn oil alone (controls), while others remained on house-supplied water. On day 9 after dosing with PCB126, rats were administered ClO (0, 0.01, 0.10, or 1.00 mg/kg day) in their drinking water for an additional 14 days. Rats were euthanized on day 22 between 7 and 10 A.M. and tissues collected for analysis (see "Tissue collection and preparation" section).
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Dosing study II.
The purpose of this study was to examine the interactions of lower doses of PCB126 with perchlorate. The time on treatment for dosing study II was shortened to 5 days or less after administration of a single oral gavage, lower dose of PCB126 (0, 0.075, 0.75, or 7.5 µg PCB126/kg) on day 0 to capture the transient perturbations in the HPT axis. The average weight on day 0 for 192 adult male SD rats used in this study was 250 ± 16 g. One day following PCB126 dosing, rats were administered ClO
in drinking water to obtain doses of 0 or 0.01 mg/kg day. A portion of the rats received only PCB126 and were euthanized and tissues collected 12 h, 1 day, 2 days, and 5 days after dosing. In addition to PCB126, a subset of rats also received ClO in drinking water that began 1 day after PCB126 dose, and these animals were euthanized 2 and 5 days after PCB126 dosing, respectively (Table 2).
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Chemicals and Reagents
PCB126 (100 µg/ml in isooctane) was obtained from Accustandard Corporation (New Haven, CT). Dosing solutions (target doses 0.0, 0.075, 0.75, 7.5, and 75 µg/kg) were prepared as detailed in Fisher et al. (2006)
having final concentrations of 0, 0.02, 0.2, 1.2, and 12.0 µg PCB126/ml corn oil, respectively. AP salt (99.8% pure) was obtained from Aldrich (Milwaukee, WI). Final ClO drinking water concentrations for target doses of 0, 0.01, 0.1, and 1.0 mg/kg day were 0, 0.09, 0.9, and 9.0 mg/l.
Animals
Adult male SD rats were obtained from Charles River Laboratories (Wilmington, MA) weighing 161–180 g on arrival. Rats were housed individually in a "shoebox" style cage at an accredited American Association for Accreditation of Laboratory Animal Care facility with humidity/climate control and a 12-h light/dark cycle. Rats were fed Purina PMI Certified Rodent Chow #5001 and provided water (with or without perchlorate) ad libitum. Rats were allowed to acclimate for 1 week prior to dosing. The studies were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. Body weights and food and water consumption were determined at the time of dosing, every 3 days during the study, and at euthanasia.
Tissue Collection and Preparation
Rats were euthanized by CO2 asphyxiation and exsanguinated from the inferior vena cava as described in Fisher et al. (2006). Whole blood was collected in serum separator tubes, allowed to clot, centrifuged, and the serum removed. Serum aliquots were stored at – 80°C until analysis of TSH and thyroid hormones. Livers were excised, weighed, and divided for analysis. Five grams of the liver were used to make microsomes for UDPGT activity (Fisher et al., 2006). Liver microsomes were also stored at – 80°C until analysis. Thyroids were excised free of fat and connective tissue and weights recorded. Both thyroid lobes from dosing study I animals and one lobe from animals in dosing study II were placed in 10% formalin for histomorphometric determination of the colloid/follicular volume ratio. The other lobe in dosing study II was frozen at – 80°C until analysis of iodide content.
Hepatic Enzyme Analysis
T4-G formation rates catalyzed by UDPGTs and glucuronic acid were determined for all doses and dose combinations based on the method of Visser et al. (1993) as modified by Zhou et al. (2001). The calculated activity of hepatic UDPGTs was reported as picomoles of T4-G formed per milligram of protein per minute. The minimal UDPGT activity detection using T4 as the substrate was 0.05 pmol T4-G per milligram of protein per minute. Hepatic microsomal protein was determined using the Folin phenol reagent method published by Lowry et al. (1951).
Serum Hormone and TSH Analyses
Serum aliquots of 0.5 ml were stored at – 80°C until analysis, which occurred less than 4 months after collection. A previously unfrozen serum aliquot was used for each assay. Serum-free T4 (fT4) concentrations were measured by equilibrium dialysis using a radioisotopic assay kit (No. 40-2210, Nichols Institute Diagnostics, San Juan Capistrano, CA). Serum total T4 was determined by radioimmunoassay as in Fisher et al. (2006) using T4-15 antisera obtained from Endocrine Sciences (Calabasas, CA). Serum TSH was measured using a purchased (commercially available) rat TSH radioimmunoassay kit (MP Biomedicals #07C-90102, Orangeburg, NY).
The intra-assay coefficients of variation were 4.8, 9.4, and 6.0% for dosing study I measurements of fT4, T4, and TSH, respectively. For dosing study II, the intra-assay coefficients of variation were 19.4, 7.7, and 14.6%.
Thyroid Histopathology and Iodide Content
Thyroid glands were collected and prepared for histomorphometric analysis with a hematoxylin and eosin stain (Fisher et al., 2006). Two sections, for each lobe of the thyroid when available, were examined microscopically and two photographs of each section were taken for digital analysis of area ratios. Images with average area of 0.55 mm2 were analyzed using the computer software Image-Pro Plus (MediaCybernetics, Silver Spring, MD). Follicular epithelial cell area was contrasted with colloid area (black vs. white, respectively) and the total black versus white area was computationally determined. These values were then used to determine the colloid volume to epithelial follicular cell volume (C/EFC) ratios. An average of four ratios (four images) was determined for each animal.
In dosing study I, both lobes of the thyroid from each animal were used for histomorphometric analysis; however, in dosing study II, one lobe from each thyroid, alternating right and left, was reserved for iodide analysis. Total thyroidal endogenous iodide content (127I) was determined using the method of Benotti et al. (1965) for a portion of dosing study II animals.
Statistical Analyses
All statistical analyses were performed using the statistical software package Statistical Analysis System (SAS) v8.2 (SAS Institute Inc., Cary, NC). Dosing study I data were transformed by taking the square root prior to analysis. ANOVA was used to determine if there were differences between the measurements taken on the 2 days of collection for controls or animals treated with only perchlorate. No statistical differences (p 
0.05) were determined between days of euthanasia for the control or ClO only dose groups. Therefore, results from these rats were grouped together, which resulted in a total of 16 rats in the control and ClO only dose groups for dosing study I.
Subsequently, the transformed data were evaluated by ANOVA to determine treatment-related effects (PCB126 or ClO) and followed by Tukey's multiple comparison (MC) test (p 0.05) to compare treatment means (individual compounds and mixtures) to control means. In addition, Tukey MC test was used for comparison of the mixture means to the responses of the individual chemicals. ANOVA followed by Tukey MC test was also utilized for analysis of dosing study II data. Data presented are expressed as percent of control (100%) ± SEM.
The Tukey–Ciminera–Heyse (TCH) trend test (Tukey et al., 1985) was used, in addition to ANOVA and Tukey's MC test, in order to detect nonzero trends in response to the test compounds. The TCH test was conducted sequentially, using contrast coefficients calculated using SAS v8.2 and equations and methods described by Antonello et al. (1993), to determine the no-statistical-significance-of-trend (NOSTASOT) dose. For dosing study I, contrast coefficients were calculated for ClO doses (0, 0.01, 0.10, and 1.0 mg/kg day), and the TCH test was used to determine ClO trends at different dose concentrations of PCB126 (0, 7.5, and 75 µg/kg) pretreatment for each variable. The contrast coefficients for dosing study II were calculated for PCB126 doses (0, 0.075, 0.75, and 7.5 µg/kg) and used to determine PCB126 trends at each time point, with (2 and 5 days) and without ClO (0.5, 1, 2, and 5 days). A significant nonzero trend was identified when p 0.05. The NOSTASOT dose was determined when p > 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Dosing Study I
Body and organ weights.
There were no significant differences in cumulative mean body weight gain over the 22-day study period after either a single oral bolus dose of PCB126 (7.5 or 75 µg/kg), 14-day exposure to ClO
in drinking water (0.01, 0.10, or 1.00 mg/kg day), or a combination of the two after a 9-day pretreatment period with PCB126. The average weight gain for all animals was 120 ± 24 g. In addition, food and water consumption were not altered during the course of the study with daily intakes averaging 24.9 ± 1.0 g and 38.9 ± 2.2 ml, respectively. Rats administered 75 µg PCB126/kg had a significant increase of 18% in mean liver to body weight ratio (Table 3). No treatment-related differences in thyroid weights were found (data not shown).
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Individual chemical treatment.
Administration of 7.5 and 75 µg PCB126/kg resulted in a dose-dependent increase in the rate of hepatic T4-G formation, although only the 75-µg PCB126/kg dose group was significantly elevated by 166% above control (Fig. 1). Seventy-five but not 7.5 µg PCB126/kg resulted in a significant decrease in serum T4 (49%) and fT4 (51%) concentrations compared to controls at 22 days after treatment. Elevated serum TSH concentrations were detected for both PCB126 dose groups (Fig. 1). However, the 7.5-µg/kg dose group TSH concentrations were slightly higher than the 75-µg/kg dose group concentrations, although not statistically different from one another.
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When the TCH trend test was employed to evaluate trends in the data across the three ClO
dose groups, dose-dependent trends were detected for all thyroid function indices measured. A dose-dependent increase in mean serum TSH concentrations was observed in rats that received 0.01, 0.1, or 1.0 mg/kg day of ClO in drinking water for 14 days (Fig. 1). The NOSTASOT dose, or lowest dose of ClO that did not cause a statistical alteration in serum TSH, was 0.01 mg/kg day. An increasing trend was also determined for rate of T4-G formation, while a decreasing trend was seen in serum T4 and fT4 concentrations with a NOSTASOT dose of 0.10 mg ClO/kg day for these indices (Fig. 1). Histopathology analysis of thyroids from animals that received either PCB126 or ClO did not result in any statistical differences from control. The mean colloid:epithelial follicular cell (C/EFC) ratio for vehicle control animals that received no test compounds was 1.19 ± 0.25 (see supplementary data).
Binary mixture treatment.
No dose-dependent trends were detected for the effect of ClO on the rate of T4-G formation when animals were pretreated with 7.5 or 75 µg PCB126/kg. Thus, PCB126 masked the effect of exposure to ClO for 14 days in drinking water on the rate of T4-G formation (Fig. 1). Results from Tukey's MC test also support this finding since no statistical differences between the coexposed animals and animals that received only PCB126 were seen.
PCB126 also masked the effect of ClO on serum fT4, T4, and TSH measured after a 9-day pretreatment period with either 7.5 or 75 µg PCB126/kg and followed with exposure to ClO in drinking water for 14 days (0.01, 0.1, or 1.0 mg/kg day). As previously stated, ClO dose-dependent trends were observed for changes in serum T4, fT4, and TSH concentrations when ClO was administered alone for 14 days in drinking water; however, no ClO trends were found when it was administered to rats that were pretreated with PCB126.
No changes in the volume of colloid or follicular cells (C/EFC ratio) were seen in animals that received both PCB126 and ClO; similar C/EFC ratios were determined for animals that received only one test compound (see supplementary data).
In summary, the binary mixture of 7.5 or 75 µg PCB126/kg and 0.01, 0.1, or 1.0 mg ClO/kg day resulted in the disappearance of the ClO dose-dependent HPT axis effects (Fig. 1), indicating a less than additive response for the binary mixture.
Dosing Study II
Dosing study II provided information on binary mixtures for lower doses of PCB126 determined in this study to be at or near NOSTASOT doses for PCB126, combined with a NOSTASOT ClO dose of 0.01 mg/kg day determined in dosing study I. To carry out these studies in the same fashion as dosing study I, shorter treatment periods were selected as described in the "Materials and Methods section."
Body and organ weights.
No significant difference in body weight gain was observed over the 5-day period after a single oral gavage dose of 0.075, 0.75, or 7.5 µg PCB126/kg. Also, no significant changes were found in liver or thyroid weights (data not shown). Food consumption was not monitored in this study because no differences were found at the higher doses in dosing study I. Water consumption was monitored to calculate ClO intake. The average daily intake of water was 41.5 ± 6.6 ml.
Individual chemical treatment.
A dose-dependent increase in rate of T4-G formation was observed for animals treated with PCB126 at 2 and 5 days after dosing (Fig. 2). The rate of T4-G formation peaked at day 2 and began to return to control values by day 5 (see supplementary data). The NOSTASOT dose for increase in rate of T4-G formation was found to be the lowest dose of PCB126 (0.075 µg/kg) administered.
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Interestingly, these low doses of PCB126 resulted in a dose-dependent decrease in serum fT4 at 12 and 24 h after dosing with NOSTASOT doses of <0.075 and 0.75, respectively (see supplementary data). No trend or statistically significant differences from controls were detected at later time points of 2 or 5 days after dosing (Fig. 2). No PCB126 dose-dependent trends were determined for serum T4 (Fig. 2). An increasing trend in serum TSH due to PCB126 exposure (NOSTASOT dose of 0.75 µg PCB126/kg) was found at 12 h after dosing (see supplementary data), but no trends were evident at later time points.
There were no statistical differences (Tukey's MC test) in the rate of T4-G formation or serum T4, fT4, or TSH concentrations in rats administered 0.01 mg ClO/kg day for 1 or 4 days (Fig. 2).
Binary mixture treatment.
For the binary mixture in dosing study II, animals were pretreated with PCB126 (0.075, 0.75, or 7.5 µg/kg) for 1 day and exposed to 0.01 mg ClO/kg day in drinking water for 1 or 4 days. The TCH trend test failed to find any significant PCB126 dose trends in the coexposure data for dosing study II (Fig. 2). The serum TSH concentrations were not statistically different from control values (Tukey's MC test) and the TCH test did not detect a trend across PCB126 doses.
No statistical significant differences in thyroid morphology were observed in these animals (see supplementary data). Total thyroidal iodide content was not significantly altered by treatment. Stable iodide (127I) content ranged from 10 to 15 µg per thyroid gland (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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The objective of these experiments was to characterize the low-dose interactions between two thyroid-active compounds, PCB126 and perchlorate (ClO
), which act via different modes of action to disturb the HPT axis. PCB126 is thought to act primarily by binding to the AhR to induce hepatic UDPGT enzymes which increase the metabolism of T4; ClO acts by inhibiting iodide uptake into the thyroid gland, resulting in decreased thyroid hormone production. The studies were designed to evaluate HPT axis disturbances caused by low doses of ClO on rats with modest preexisting disturbances in the HPT axis as a result of PCB126. The serum half-life (t1/2) of PCB126 in rats is approximately 17 days (Yoshimura et al., 1985), while the plasma t1/2 of iv administered 36ClO is 7.3 h (Yu et al., 2002). The long t1/2 of PCB126 allowed for administration of a single dose of PCB126 several days before treatment with ClO was initiated. This experimental design may mimic human exposures to these chemicals. PCB126 human exposure occurs from contaminated diet and its t1/2 is approximately 4.5 years (Ogura, 2004), while ClO exposure occurs primarily from ingestion of water and food with a t1/2 of 6–8 h (NRC, 2005).
Generally speaking, the dose-response characteristics of PCB126 on the HPT axis were similar to those obtained previously at higher doses (Fisher et al., 2006). That is, serum TSH concentrations were elevated, serum thyroid hormones were either unchanged or decreased and hepatic T4-G production rates were increased. Interestingly, in this study, serum TSH concentrations in animals dosed with 7.5 µg/kg were similar to the 75-µg/kg dose group. Fisher et al. (2006) reported that 75 µg/kg of PCB126 resulted in elevated serum TSH concentrations greater than the 275-µg/kg dose group. Other PCB126 studies report variable findings for treatment-related changes in serum TSH concentrations (Martin, 2002; NTP, 2006), suggesting that PCB126 may be disturbing the HPT axis by more than one mechanism of action. Further evidence for this suggestion comes from the present study. Serum fT4 concentrations declined by 12 h after dosing with PCB126, but a corresponding increase in hepatic T4-G formation was not observed (see supplementary data). Similar findings have been reported for other PCBs in which declines in serum T4 were not accompanied by UDPGT induction (Hansen, 1998; Li and Hansen, 1996). This may suggest that PCB126 disturbs the thyroid axis by another mechanism that has yet to be elucidated.
After 14 days of ClO treatment in drinking water, serum thyroid hormone concentrations were similar to control values across the dose groups that received only ClO, suggesting that the modest thyroid upregulation by TSH provided adequate compensation for thyroidal iodide uptake and thyroid hormone synthesis. The possible induction of hepatic T4-G formation by ClO deserves further study since rate of T4-G formation has not been reported previously for ClO-treated animals. In the present study, the NOSTASOT dose, also considered to be the no-observed-effect-level (NOEL), for alterations in serum TSH was 0.01 mg ClO/kg day. The alterations in serum T4, fT4, and TSH for rats exposed to ClO alone in dosing study I agree with previously published data for the 0.10 and 1.00 mg/kg day exposures (Yu et al., 2002). The determination of 0.01 mg ClO/kg day as a NOSTASOT dose based on serum TSH extended the dose-response curve for ClO established in Yu et al. (2002) into the low-dose region.
The binary mixtures data collected from dosing study I support our hypothesis that ClO is less effective as a thyroid axis disruptor in rats pretreated with PCB126. In the data from animals coexposed with the high PCB126 (75 µg/kg) dose from dosing study I, it is evident that PCB126 dominated the HPT axis responses in these coexposed animals. Serum total and free T4 in animals coadministered 75 µg PCB126/kg and 1.0 mg ClO/kg day were significantly below control values by about 40%, which corresponded to the decrease seen in PCB126 (75 µg/kg) only animals (50%). At this highest dose of ClO (1.0 mg/kg day) administered in dosing study I, a dose-dependent decrease in serum total and free T4 (8 and 13%, respectively) and subsequent increase in TSH of 100% was found. This suggests that the upregulation and stimulation of the thyroid by TSH at this dose of ClO was not sufficient to maintain normal thyroidal iodide levels for hormone production. However, in animals pretreated with 75 µg PCB126/kg prior to administration of the high dose of ClO (1.0 mg/kg day), ClO was unable to exacerbate the hypothyroid condition further. That is, there was no further decrease in serum total or free T4, and no ClO dose-related statistical trend in serum TSH was found in animals pretreated with PCB126.
Additionally, it is speculated that due to prior exposure to PCB126, the degree to which ClO is able to block thyroidal iodide uptake (and subsequently disturb the HPT axis) is diminished in the presence of elevated TSH, which is known to stimulate NIS protein expression and activity (Dohan et al., 2003). PCB126 appeared to mask the effect of ClO in these animals, which is supported by the lack of ClO dose-response trends in the binary mixture studies conducted in dosing study I. Since ClO dose-response trends were found when the compound was administered alone, the disappearance of these trends in PCB126 and ClO coexposed animals suggests that the effect is less than additive at the dose combinations tested in dosing study I. This is also supported by evaluating expected additive responses based on the absolute mean percent change from control (supplementary data). The response as percent of control for hepatic rate of T4-G formation averaged 39% less than additive. On average for the binary mixture combinations tested in dosing study I, TSH was 35% less than expected under the response additivity assumption for chemicals of dissimilar modes of action, and total T4 averaged 5% less than additive. The free T4 deviation from additivity was different for each dose of PCB126. At the low dose of PCB126 (7.5 µg/kg), animals coexposed had mean serum-free T4 levels on average 17% greater than expected under the additivity assumption; however, animals coexposed with 75 µg PCB126/kg had free T4 levels 13% less than the predicted additive response. The reason for the difference at these two PCB126 doses is not known but may be related to displacement of the hormone from carrier proteins in the blood. PCBs and their hydroxyl metabolites have been shown to displace T4 from the serum-binding protein transthyretin (TTR) in rats (Brower and van den Berg, 1986; Chauhan et al., 2000; and Cheek et al., 1999); at low concentrations, the M-1 metabolite of PCB126 (Koga, 1990) may play a similar role to PCB metabolites already identified to have this behavior.
In animals that are hypothyroid, as indicated by elevated serum TSH and decreased serum T4 concentrations prior to ClO exposure, the apparent dose-response curve for ClO inhibition of iodide uptake is shifted to the right. The ClO dose-response curve shift to the right in hypothyroid, or TSH-stimulated animals, suggests that a higher dose of ClO is needed to result in the same degree of inhibition of iodide uptake at the NIS that is seen in TSH normal, euthyroid rats.
In dosing study II, the objective was to dose rats with low doses of PCB126 and monitor transient changes in the HPT axis to determine a NOEL dose for PCB126. Also, binary experiments were conducted at low doses of both PCB126 and ClO to further characterize HPT axis responses. This study resulted only in a few statistically significant trends for PCB126 up to 1 day after dose and no statistical differences from control for animals treated with 0.01 mg ClO/kg day for 1 or 4 days. A NOEL for PCB126, based on its well-defined primary mode of action of PCB126 (phase II conjugation of T4), was found to be 0.075 µg/kg. Results from dosing study II demonstrate that doses of PCB126 and ClO, which do not cause alternations in the HPT axis when administered alone, will not result in HPT axis disturbances when administered sequentially. Thus, it appears no interaction (synergism or potentiation) occurs at relatively low doses between PCB126 and ClO for the thyroid axis indices measured in this study.
No statistically significant differences in thyroid morphology were determined for either study. Changes in thyroid gland have been seen in studies of the individual compounds. Fisher et al. (2006) found a statistically significant change in the ratio of colloid volume to epithelium volume 22 days after dose at the highest dose of PCB126 administered (275 µg/kg). In addition, female rats exposed to 30 mg ClO/kg day for 2 weeks prior to mating through lactation day 22 exhibited altered thyroid morphology, measured by colloid depletion, follicular hyperplasia and hypertrophy (York et al., 2005). To a much lesser extent, animals exposed to 0. 1 and 1.0 mg ClO/kg day for the same length of exposure exhibited some colloid depletion and follicular hyperplasia, while no colloid depletion was found in animals exposed to 0.01 mg ClO/kg day, and follicular hyperplasia was not different from controls (York et al., 2005). Thus, since no differences in thyroid colloid volume and follicular epithelial cell volume ratios were found for the PCB126 and ClO experiments presented in this article, either the treatment period was too short or the doses too low to result in structural changes within the thyroid gland itself.
One issue confronting toxicologists today is accurately extrapolating data from high-dose toxicology studies to low-dose exposures seen in the environment. In many cases, low-dose studies are needed to simulate more realistic human environmental exposures, and to provide information to minimize uncertainty in the low-dose area of the dose-response curve. However, challenges exist when implementing studies in the laboratory to explore endocrine effects in the low-dose region. Minor changes in hormone levels that result from low-dose exposures are difficult to discern because of hormone inter-individual and intra-assay variability between laboratories. Since the statistical power to detect differences in treated groups can be affected by this variability, future experiments, with a greater number of rats and more refined assays for hormone determination, could be conducted to support the conclusion that PCB126 masked effects of ClO at these low-dose rates examined.
In conclusion, these studies demonstrate that in animals treated with relatively high doses PCB126 prior to ClO exposure, HPT axis disturbances are less than additive and the ClO dose-response curve appears shifted to the right. In addition, when animals are coexposed with doses at or near the NOEL for each compound, no interaction between the compounds is observed for the thyroid indices measured.
The data from this study and previously published individual chemical studies will be utilized in the development of biologically based pharmacokinetic models for the adult male rat HPT axis. These models will be used to characterize and further the understanding of dose-response relationships for exposure to mixtures of thyroid-disrupting chemical mixtures. In addition, HPT axis mathematical models will help to interpret the nonlinear dose-response based on the primary well-defined modes of action.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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NOTES |
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2 Barry Harmon has passed away since submission of this article.
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ACKNOWLEDGMENTS |
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Primary funding for this research was kindly provided by ATSDR grant U61/ATU472105-(02, 03, 04, and 05). Additional research support was provided by the National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency and the U.S. Air Force Research Laboratory, Human Effectiveness Directorate, Biosciences and Protection Division, Applied Biotechnology Branch. E.D.M. supported through National Science Foundation Fellowship (DGE0229577) and currently EPA STAR Fellowship (FP-91679301-0). Special thanks to Dr W. Matthew Henderson, Debbie Ebalobo, and John Swint for helping with the animal studies. The views expressed in this article are those of the authors and do not represent official opinions of the Department of Defense, U.S. Environmental Protection Agency, or Agency for Toxic Substances and Disease Registry. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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