ToxSci Advance Access originally published online on December 9, 2005
Toxicological Sciences 2006 90(1):87-95; doi:10.1093/toxsci/kfj069
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Effect of PCB 126 on Hepatic Metabolism of Thyroxine and Perturbations in the Hypothalamic-Pituitary-Thyroid Axis in the Rat
* University of Georgia, Interdisciplinary Toxicology Program, Athens, Georgia 30602; Agency for Toxic Substances and Disease Registry (ATSDR), NCEH/ATSDR/DT, Chamblee, GA 30341; University of Georgia, College of Veterinary Medicine, Department of Pathology, Athens, GA 30602; USEPA/ORD/NHEERL, Neurotoxicology Division, Research Triangle Park, NC 27711; ¶ U.S. Environmental Protection Agency (USEPA/ORD/NHEERL) Neurotoxicology Division; and || Visiting Scientist, Department of Environmental Science, College of Natural Sciences, Kangwon, National University, Churcheon, Gangwon Do 200-701 South Korea
1 To whom correspondence should be addressed at University of Georgia, 206 Environmental Health Sciences Department, Athens, GA 30602–2102. Fax: (706) 542-7472. E-mail: jwfisher{at}uga.edu.
Received August 27, 2005; accepted November 23, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The objective of this research was to examine the time- and dose- dependent disturbances in the hypothalamic-pituitary-thyroid (HPT) axis of adult male rats administered a potent coplanar (non-ortho) PCB, 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Adult male Sprague-Dawley rats were administered a single oral bolus dose of 0, 7.5, 75, or 275 µg PCB 126/kg bw dissolved in corn oil. The rats were sacrificed periodically over 22 days. The 7.5-µg/kg dose induced hepatic ethoxyresorufin-O-deethylation EROD activity, but no changes were observed in hepatic uridine diphosphate glucuronyl transferases (UDPGTs) activity or serum TSH, T4, or fT4 concentrations. The two highest doses caused a modest decline in weight gain, induced hepatic EROD and UDPGT activities, increased serum TSH concentrations, and decreased serum T4 and fT4 concentrations. The amount of thyroxine glucuronide formed daily (pM/mg protein) increased linearly with the area-under-the-concentration-curve (AUCC) for PCB 126 in liver (µg/kg/day) and then slowed at the 275-µg/kg PCB 126 dose. Perturbations in the HPT axis were nonlinear with respect to PCB 126 dosing. As expected, an inverse relationship between the AUCC for serum T4 (µg/dl/day) and the AUCC for serum TSH (ng/dl/day) was observed; however, the relationship was highly nonlinear. These data support a mode of action for PCB 126 involving induction of hepatic UDPGTs by the aryl hydrocarbon receptor AhR. However, the dose-response characteristics of the HPT axis are nonlinear and complex, requiring sophisticated tools, such as PBPK models, to characterize dose response.
Key Words: PCB 126; thyroid hormones; P450, EROD; rat; TSH; UDPGT.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polychlorinated biphenyls (PCBs) were produced industrially from the years 1929 to 1977 for their unique chemical properties such as thermal stability, resistance to acids and bases, and low water solubility (ATSDR, 2000
). Production was halted due to increasing awareness of health effects associated with PCB exposure. PCBs are very lipophilic and persistent in the environment. PCB congeners or mixtures (Aroclors) have been associated with various toxic responses including cancers, hepatotoxicity, gastrointestinal and respiratory disturbances, neurotoxicity, immunotoxicity, reproductive and developmental effects, and endocrine disruption including thyroid toxicity (ATSDR, 2000; Li and Hansen, 1997).
Thyroid hormones are critical for normal body metabolism, growth, development, and reproduction, and numerous toxicology studies with PCBs have documented the disruption of normal thyroid function in laboratory animals (Brouwer et al., 1998). Dioxins and coplanar PCBs disrupt the hypothalamic-pituitary-thyroid (HPT) axis in laboratory animals (Beetstra et al., 1991; Craft et al., 2002; Desaulniers et al., 1999; Martin, 2002; Van Birgelen et al., 1994a,b) by binding to the aryl hydrocarbon receptor (AhR) and increasing transcription of several hepatic proteins including uridine diphosphate glucuronyl transferases (UDPGTs) (Auyeung et al., 2003; Craft et al., 2002; Schuur et al., 1997). The induction of UDPGTs leads to increased glucuronidation of thyroxine (T4) and subsequent excretion in bile as T4-glucuronide (T4-G). The increased loss of T4 in bile and ultimately in feces is thought to result in disruption of the HPT axis (Hood and Klaassen, 2000). Nonplanar, ortho substituted PCB congeners do not bind to the AhR receptor, but do cause disturbances in the HPT axis, as evidenced by a decline in serum T4 concentrations (e.g., Craft et al., 2002; Desaulniers et al., 1999; Van Birgelen et al., 1994b). The decline in serum T4 concentrations results from increased metabolism of thyroxine to T4-G via upregulation of UDPGTs, but the induction of UDPGTs is controlled by the constitutive androstane receptor (CAR) and pregnane X receptors (PXR) (Kretschmer and Baldwin, 2005; Schuetz et al., 1998). Other mechanisms for disruption of the HPT axis have been proposed for PCBs, including direct effects on TSH release in the pituitary (Khan et al., 2003) and hydroxylated metabolites of PCBs displacing T4 from serum binding proteins (transthyretin), potentially resulting in the accelerated systemic clearance of T4 (Chauhan et al., 2000; Cheek et al., 1999; Lans et al., 1993).
The ultimate objective of our research is to develop physiological models that describe the HPT axis in the rat to predict dose-dependent perturbations in the HPT axis by chemicals. In this paper we present a portion of the experimental findings for adult male rats administered a potent coplanar (non-ortho) PCB, 3,3',4,4',5-pentachlorobiphenyl (PCB 126). PCB 126 has been studied in the rat. In a 30-day rat kinetic study with PCB 126, Yoshimura et al. (1985) estimated that 70% of the PCB 126 dose was still present in the liver 5 days after dosing, and the serum PCB 126 half-life was approximately 17 days. The sequestration of PCB 126 in the liver represents substantial binding of PCB 126 to P450 cytochrome (CYP) 1A2 (Chen et al., 2003). A primary concern of PCB 126 toxicity is its "dioxin-like" characteristics to cause cancer via the AhR receptor–mediated signaling pathways (Toyoshiba et al., 2004). The National Toxicology Program has conducted a 2-year cancer bioassay on PCB 126 (NTP, 2005), including an evaluation of the thyroid.
Several acute research studies have examined the effects of PCB 126 on the HPT axis. PCB 126 causes consistent dose-related declines in serum T4 concentrations and increases in hepatic CYP 1A1 and UDPGT activities (Craft et al., 2002; Desaulniers et al., 1999; Martin, 2002) with daily repeated doses of 2 to 7 days and dose rates ranging from 0.03 to 400 µg/kg/day. Serum T3 concentrations were either unchanged (Martin, 2002) by PCB 126 administration or decreased (Desaulniers et al., 1999). No consistent changes in TSH serum concentrations were found (Craft et al., 2002; Desaulniers et al., 1999).
There is a no time-course data published on the pharmacokinetics of PCB 126 in the rat, and there is no data published on the time- and dose-dependent changes of the HPT axis caused by PCB 126 in the rat. In the present study male adult rats were administered a single dose of 7.5, 75, or 275 µg/kg of PCB 126 by oral bolus intubation, and biological samples collected at various times after dosing. The goal was to collect temporal information on the kinetics of PCB 126 in the liver, the upregulation of hepatic enzymes, specifically UDPGT, which is responsible for the disruption of the HPT axis, and several biological indicators of thyroid status.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Study design.
Rats (n = 104) were administered a single oral bolus of PCB 126 in corn oil (0.0, 7.5, 75, or 275 µg/kg). The dosing volume was adjusted to give 1.0 ml/kg body weight. Eight rats were killed prior to dosing to obtain control information, and then eight rats per dose group (32 rats per sacrifice day) were killed on Days 5, 9, and 22. To embellish the PCB 126 pharmacokinetic studies, an additional 48 rats were dosed (n = 4 per dose group, excluding the control group) to obtain PCB 126 tissue concentrations of Days 1, 3, and 15 post dosing. Hepatic enzymes were measured on Day 1 in these rats.
Chemicals and reagents.
PCB 126 was obtained from Accustandard Corporation (New Haven, CT) at purity greater than 99%. Stock solutions were prepared by dissolving PCB 126 in n-hexane, and the concentrations were checked by gas chromatography using a micro-electron capture detector (GC/µECD). The n-hexane and PCB 126 stock solutions were then mixed with corn oil (MazolaTM, Best Foods, Englewood Cliffs, NJ), and then the n-hexane was removed by evaporation under a mild stream of nitrogen gas (adapted from DeVito et al., 1993). All solutions were prepared by dilution of equal amounts of hexane or hexane/PCB 126 with corn oil. Four dosing solutions were prepared (0.0, 7.5, 75, 275 µg/kg) with final concentrations of 0.0, 0.002, 0.020, and 0.0599 mg/ml, respectively.
Animals.
Male Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA) weighing 151–175 grams. Rats were housed two per "shoe-box" style cage and allowed to acclimate for 5 days before use. Animals had unrestricted access to PMI #5001 rodent chow (PMI feeds, St. Louis, MO) and water. Rats were kept in a humidity/climate-controlled facility with a 12-h light/dark cycle which is accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). The study was conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Rats were weighed upon dosing and on Days 5, 9, 15, and 22. The number of rats weighted per dose group per day diminished to an n = 8 by Day 22 post dosing. Rats were euthanatized by CO2 asphyxiation.
Tissue collection and preparation.
Blood was collected from the inferior vena cava using an 18
-gauge needle (Becton Dickinson) and 10-cc syringe (Becton Dickinson). Blood was placed into serum separator tubes, allowed to clot, and centrifuged for 15 min at room temperature. Serum aliquots were stored at –80°C until analysis of TSH and thyroid hormones. Blood and fat were also collect for analysis of PCB 126 (not reported). Livers were excised, and portions of the liver divided for analysis of cytochrome P450 (CYP) 1A1 activity, UDPGT activity, and Type I 5'-deiodinase activity (not reported). Hepatic protein content was measured using the method of Lowry et al. (1951). Another portion of the liver was frozen for later analysis of PCB 126.
Liver microsomes were prepared by taking 5 g of liver sample from each animal, homogenizing in 25 ml of 0.02 M Tris–HCl containing 0.15 M KCl (pH 7.4), and centrifuging at 10,000 x g for 30 min at 4°C. The pellet was then washed, resuspended in 0.25 M sucrose containing 10 mM EDTA and 1.15% KCl (pH 7.4), and stored at –80°C. Liver homogenates for Type I 5'-deiodinase activity were prepared by homogenizing 1 g of liver in 5 ml of a buffer containing 100 mM potassium phosphate, 1 mM ethylenediamine tetraacetate (EDTA), and 1 mM dithiothreitol (DTT) (pH 7.0) and stored at –80°C. Thyroids were carefully dissected free of fat and connective tissue, weighed, and placed in 10% formalin for histopathology to determine thyroid colloid volume/follicle epithelium volume ratios.
Analysis of PCB 126 in tissue.
PCB 126 was extracted from liver by placing 0.3 g of tissue into a 20 ml vial containing 2 µl of an internal standard (4,4'-dibromobiphenyl) solution and 0.3 g of powered anhydrous sodium sulfate. Methanol (0.3 ml) and 5 ml of hexane were added, and the mixture homogenized (TISSUE TEAROR, Biospec Products, Inc.) for 1 min. The supernatant organic phase was transferred to a test tube and centrifuged at 3,500 rpm for 10 min, and 1.5 ml of the organic phase (hexane) was loaded onto a preconditioned (1 ml of hexane without vacuum) Bond Elut PCB cartridge (Varian, Inc.). One ml of hexane was used to elute the organic phase under light vacuum for 3 min. This step was repeated three additional times for an accumulated elution volume of 4 ml. The eluate was then concentrated to dryness under a mild stream of nitrogen and redissolved in 0.3 ml of hexane. Two µl of the sample was injected into a gas chromatograph (GC) equipped with a micro-electron capture detector (µECD) (Agilent 6890) and an Agilent autosampler 7683.
The oven condition was initially set to 100°C for 3 min, increased at a rate of 7°C/min to a temperature of 220°C, increased at a rate 3°C/min to a temperature of 260°C, and finally increased at a rate of 15°C/min to a temperature of 280°C. The run time was about 45 min for each sample. The inlet was 250°C with a 20:1 split. A HP-5 capillary column (15 m x 0.53 mm x 1.5 µM) was used with an initial carrier flow rate of 2.2 ml/min. The µECD detector was heated to 300°C. Under the conditions described, the limit of quantification for PCB 126 in liver was 2.7 µg/kg. The average extraction efficiency was over 92% for liver.
Hepatic enzyme analysis.
Ethoxyresorufin-O-deethylation (EROD) was measured as indicator of activity of CYP 1A1 on complete time-course data sets. The fluorimetric technique described by Lubet et al. (1985) was used to quantify EROD activity (nM/min/mg protein). This provided the rate of CYP 1A1–mediated deethylation of 7-ethoxyresorufin to resorufin. The Day 9 275-µg/kg liver samples for EROD analysis were not used because of experimental error.
Hepatic microsomal T4-G (thyroxine glucuronide) formation rates catalyzed by uridine diphosphate glucuronyl transferases (UDPGTs) and glucuronic acid were assayed for the complete time-course data sets based on the method of Visser et al. (1993) as modified by Zhou et al. (2001). The calculated UDPGT activity was reported as pM T4-G formed/mg protein/min. The limit of detection for UDPGT activity with T4 as the substrate was 0.05 pM T4-G/mg protein.
Serum hormone and TSH analyses.
Serum fT4 concentrations were measured on samples from Days 5 and 22 in all dose groups. Serum total T4 (T4), total T3 (T3), and reverse T3 (rT3) concentrations were collected on Days 5, 9, and 22 in all dose groups. Serum TSH was measured for all doses on days 5, 9, and 22.
Serum free thyroxine (fT4) analysis by direct dialysis was carried out at first thaw of the serum aliquots to avoid altered serum binding due to repeated freeze-thaw cycles. Nondialysis fT4 immunoassays often used in rat serum samples are designed for human serum samples, which have much higher concentrations of thyroxine binding globulin than animal sera (Refetoff et al., 1970). Systematic underestimation of fT4 has been documented to occur in animal and human sera (Nelson et al., 2005; Rabin et al., 2004; and Schachter et al., 2004) when nondialysis fT4 assays were employed. The fT4 direct dialysis assay kit (Nichols Institute Diagnostics, Inc., San Clemente, CA) used in these studies involved the overnight dialysis of 200 µl of serum against 2.4 ml of buffer at 37°C prior to the sensitive radioimmunoassay of the dialysate for T4. All samples were performed in the same assay. The immunoassay intra-assay variation of the high standard provided by the manufacturer was 5.9%, and the low standard was 10.0%.
Serum total T4 and T3 (not reported) were measured by radioimmunoassay (Chopra et al., 1980; Eltom et al., 1992) using the T4-15 and T3-38 antisera obtained from Endocrine Sciences (Calabasas, CA). Serum rT3 concentrations (not reported) were measured in the same assay by radioimmunoassay as described by Ferguson and Peterson (1992) for dog serum, but modified for rat serum. All immunoassays included iodothyronine-free serum prepared by activated charcoal extraction of endogenous hormone as previously described (Eltom et al., 1992). Radiolabeled T4 (125I-thyroxine, S.A. >1500 µCi/µg) was obtained from Amersham Biosciences, and radiolabelled T3 (125I-3,5,3'-triiodothyronine, S.A 1080–1320 µCi/µg) and rT3 ([125I] 3,3',5'-triiodothyronine, S.A 1080–1320 µCi/µg) were obtained from Perkin Elmer (Boston, MA). The intra-assay coefficients of variation for the T4, T3, and rT3 assays in this study were, respectively: 8.8%, 7.7%, and 29.7%. We would note that the rT3 concentrations measured were quite low. The mean and standard deviation serum concentration of the chosen pool was 5.6 ± 1.7 ng/dl for an assay with a detection limit of 2 ng/dl.
Serum rat TSH concentrations were measured using a radioimmunoassay kit (ICN Diagnostics #07C-90102, Orangeburg, NY). The intra-assay coefficient of variation was 9.5%.
Thyroid histomorphometry.
Thyroid glands were collected on Day 22 for all PCB 126 dose groups. Both thyroid glands from each rat were fixed in neutral buffered 10% formalin solution, embedded in paraffin, sectioned sagittally at 3 µm, and stained with hematoxylin and eosin. All sections were examined microscopically. To obtain a quantitative measure of the relative amount of colloid and follicular epithelial cell volume for each rat, a stereological procedure was performed. The volume fractions of follicular epithelial cells and of colloid were measured by a stereological technique using light microscopy at 200x and an eyepiece reticle (Russ, 1986). A grid with 121 equidistant points was placed over two midline sections of each thyroid gland, and volume fractions (V%) for colloid and follicular epithelium were calculated using the formula:
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). All four measurements for each of the rats were averaged to obtain mean volume fractions. The colloid volume fraction (CV) to follicular volume fraction (FV) ratio for each rat was calculated using the formula:
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Statistical analysis.
Treatment-related effects at each time point were first evaluated with an analysis of variance followed by Dunnett's test (p < 0.05) for comparison of treatment means to control means. All analyses were performed using SAS V 8.2 (Cary, NC). Some of the data are presented as percent or fold changes from control values. Statistical analyses were carried as mentioned above, and an asterisk signifies a statistically significant difference from control (p < 0.05).
Berkley Madonna (Berkeley Madonna, Inc) software was used to perform linear interpolations and calculate dose-dependent time-integrated measures of mean hepatic PCB 126 concentrations, T4-G production rates, and EROD activity, and serum TSH, and T4 concentrations. Integrations were carried out for 22 days and then averaged on a daily basis. The integration method for stiff systems (Rosenbrock) was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Kinetics of PCB 126
Single oral bolus PCB 126 doses slowed mean body weight gain by Day 22 post dosing in the 75- and 275-µg/kg dose groups by 13 and 17%, respectively (Fig. 1a). Peak hepatic PCB 126 concentrations for the 7.5-, 75-, and 275-µg/kg dose groups were observed rapidly by Days 1, 3, and 1 post dosing, respectively. Hepatic clearance of PCB 126 was slow (Fig. 2a). The time-integrated liver exposure to PCB 126 or area-under-the-concentration-curve (AUCC) (µg/kg/day) was modestly curvilinear as a function of administered dose (Fig. 2b).
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), 75.0 (
), or 275 (
) µg/kg of PCB 126. Statistically significant deceases in weight gain are denoted with an asterisk (*).
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PCB 126 Dose- and Time-Dependent Perturbations in Hepatic Enzymes and the HPT Axis
Liver.
Dose- and time-dependent increases in the hepatic rate of formation of T4-G were observed for the two highest doses of PCB 126 (Fig. 3a). Peak induction of T4-G formation rates occurred on Day 5 after dosing and was elevated 3.4- and 6.6-fold for the 75- and 275-µg/kg dose groups, respectively. The 7.5-µg/kg dose of PCB 126 did not induce significant increases in T4-G production. The amount (pM) of T4-G produced in the liver/mg of protein/day slowed at the 275-µg/kg dose and was not linearly related with the time-integrated (AUCC) hepatic concentration of PCB 126 (Fig. 3b).
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EROD activity was significantly increased for the study period in the two highest groups and only on Day 1 in the lowest dose group (Fig. 4a). Peak hepatic induction of CYP 1A1 activity (EROD) on Day 5 was similar for both the 75- and 275-µg/kg dose groups (about 30-fold), while the peak hepatic CYP 1A1 activity for the 7.5-µg/kg dose group was about 12-fold on Day 1. Interestingly, the daily EROD activity was linearly related with the time-integrated hepatic PCB 126 concentration (Fig. 4b) above
100 µM/mg protein/day or the lowest administered dose.
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Serum.
Serum T4 and fT4 concentrations were not altered by the lowest dose of PCB 126, while the two higher doses of PCB 126 caused a decline in serum T4 and fT4 concentrations (Figs. 5a and 5b) over the study period. Serum T4 concentrations reached their nadir in the 75- and 275-µg/kg dose groups on Day 9. Serum fT4 concentrations were only measured on Day 5 and 22. No dose-related changes were observed in serum T3 or rT3 concentrations, except for transient decreases in T3 on Day 22 and rT3 on Day 5 (data not shown).
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The two highest PCB 126 doses caused increases in serum TSH concentrations (Fig. 6) over the study period. Surprisingly, the mean serum TSH concentrations for the 75- and 275-µg/kg dose groups were similar. The reason for this is unknown. Serum TSH concentrations were not significantly elevated in the 7.5-µg/kg dose group.
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Thyroid.
Microscopically, all thyroid glands appeared normal, i.e., there was no evidence of gross thyroid hyperplasia. Follicular size and staining intensity of colloid varied considerably within individual thyroid glands. Qualitatively, a modest increase in the size and height of the follicular epithelium and decrease in size of follicular lumens was noted in rats receiving the 75-and 275-µg/kg dose on Day 22 post dosing. Quantitatively, the ratio of colloid volume fraction/follicular epithelial volume fraction was significantly lower (Table 1) in only the 275-µg/kg dose group on Day 22. A lower CV/FV is a morphologic indicator consistent with the bioactivity of increased serum TSH concentrations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The objective of these experiments was to gain insights into the time- and dose-dependent nature of PCB 126 induction of hepatic UDPGT which results in increased phase II metabolism and systemic clearance of thyroxine (T4). As a consequence of increased metabolism of T4 to thyroxine glucuronide (T4-G), the hypothalamus/pituitary responds to lowered serum levels of T4 by releasing increased amounts of TSH (negative feedback) into the blood supply. TSH then binds to receptors on the thyroid gland and upregulates the production of thyroid hormones. In our study we found that hepatic metabolism of T4 was not induced 1 day post dosing, but by 5 days post dosing the hepatic metabolism was induced in the 75- and 275-µg/dose groups. Serum T4 concentrations were also less than controls by Day 5 post dosing in these dose groups. Serum TSH was not elevated by Day 5, suggesting that sustained low serum T4 concentrations were not yet sufficient (negative feedback loop) to upregulate production of TSH. However by Day 9 post dosing, serum TSH concentrations were elevated in these dose groups. The partial recovery of serum T4 and fT4 by 22 days post dosing is because hepatic UDPGT activity was slowly returning to a control level, and serum TSH continued to stimulate the thyroid gland. Morphometric analysis of the thyroid gland on Day 22 indicated that the gland was still stimulated by TSH by the 275-µg/kg dose. In the low-dose PCB 126 rats, a consistent negative response was found for hepatic UDPGT activity and serum TSH and thyroid hormones; only hepatic EROD activity was increased.
Using pharmacokinetic tools to analyze the data, we examined the relationships between internal dose of PCB 126 (liver) and perturbations in the HPT axis. A clear inverse relationship was observed between the daily amount of T4 metabolized to T4-G and the AUCC values for T4 in serum (Fig. 7). This provides strong kinetic support for induction of UDPGTs as the primary mechanism of action for PCB 126 on the HPT axis. At the 275-µg/kg dose of PCB 126, compensatory mechanisms may have maintained the time-integrated serum T4 concentrations despite a continued rise in induction of hepatic UDPGT activity. This resulted in less than expected decrease in serum AUCC T4 values (Fig. 7) or nonlinear kinetic behavior. When evaluating the inverse relationship between the time-integrated concentrations (AUCC) of TSH and serum T4, the AUCC TSH values for the 75-µg/kg PCB 126 dose were apparently similar or greater than the 275-µg/kg dose (Fig. 8). This suggests that that sustained TSH production may have been compromised in the 275-µg/kg dose group. Fig. 8 demonstrates the nonlinear relationship between serum T4 and TSH systemic availability, suggesting possible saturation of regulatory elements in the HPT axis. Taken together, these observations demonstrate that the HPT axis is a highly nonlinear system when disturbed by PCB 126 and will require the use of PBPK models to describe this endocrine system.
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Other mechanisms that may disturb the HPT axis include hydroxyl metabolites of PCB 126 (Lans et al., 1993), which may displace thyroid hormones bound to serum proteins, although PCB 126 is cleared from the body very slowly by fecal excretion and metabolism, suggesting the role of metabolism may be minimal. The inclusion of induction of hepatic EROD (CYP1A1) activity is needed for the HPT axis to help understand how PCB 126 apportions itself between the AhR and CYP1A2 binding sites, and CYP1A1 induction may play a role in metabolism of PCB 126. The importance of CYP1A1 will become more apparent when a PBPK model for PCB 126 is developed.
In this study, T4-G formation rates were increased to a maximum of 3.4- and 6.6-fold 5 days after dosing in the 75- and 275-µg/kg PCB 126 dose groups, respectively. Craft et al. (2002) reported approximately 3.5- and 7.4-fold inductions of T4-G in rats 1 day after daily dosing for 4 days with 10 and 40 µg/kg of PCB 126. Kohn et al. (1996) developed a PBPK model for dioxin and its disturbances of the HPT axis and surmised that the excretion of T4-G from the body was the primary mode of action. There was very little kinetic data in this study to validate these conclusions.
PCB 126 concentrations were much greater in liver than in fat (data not shown), indicating that binding of PCB 126 to hepatic-derived CYP 1A2 is substantial (Chen et al., 2003). These findings are consistent with other studies that have compared PCB 126 concentrations in liver and fat after administration of PCB 126 (Angelique et al., 1994; Chen et al., 2001; Lyche et al., 2004). Both CYP 1A1 and CYP 1A2 catalyze the metabolism of many xenobiotics and have broad ligand specificities. There is a much greater amount of CYP 1A2 in rat liver than CYP 1A1. The high-affinity binding of PCB 126 to CYP 1A2 results in competitive inhibition of CYP 1A2 (methoxyresorufin, MROD) activity, with an inhibition constant (Ki) equal to 0.026 µM (Chen et al., 2003). On the other hand, CYP 1A1 (EROD) activity was highly induced by PCB 126. Hepatic induction of EROD (CYP 1A1) by PCB 126 has been reported in several in vitro and in vivo studies, with some studies focused on the HPT axis (Craft et al., 2002; Desaulniers et al., 1999; Martin, 2002). CYP 1A1 has been reported to be induced with PCB 126 as great as 95-fold in rats (Toyoshiba et al., 2004) and up to 1000-fold induction has been reported in hepatic cells (CYP 1A1 mRNA) (Broccardo et al., 2004).
Eltom et al. (1992) showed the TCDD affected 5'-deiodianse activity (possibly directly) in thyroidectomized rats despite T3 replacement therapy. Usually the serum T3 and rT3 concentrations reflect the status of Type I 5'-deiodinase activity. T3 serum concentrations in this acute dosing study did not change appreciably, but chronic administration of PCB 126 causes an increase in serum T3 and TSH concentrations and a decrease in T4 concentrations (NTP, 2005). We speculate that, in the NTP study with PCB 126, adaptive responses occurred to maintain T3 concentrations, such as a decrease in metabolism of T3 or increased production of T3 by the thyroid (Schuur et al., 1997).
Our findings differ in some respects from previous acute dosing studies with PCB 126 in rats. TSH serum levels were increased in this study, while Desaulniers et al. (1999) and Martin (2002) reported no changes in serum TSH concentrations. Desaulniers et al. (1999) also reported reductions in total T3 serum concentrations, which was not the case in this study. On the other hand, in the present study a morphometric analysis of the thyroid gland revealed a typical pattern of decreased colloid, indicating TSH stimulation of the thyroid gland. Desaulniers et al. (1999) also analyzed the histology of the thyroid glands of PCB 126-treated animals and found that the colloid was less dense than in control animals, which is consistent with depletion by TSH stimulation.
To our knowledge, this is one of the first studies to apply the technique of fT4 measurements by equilibrium dialysis to studies of toxin effects on thyroid hormone metabolism. Several studies of PCBs and the thyroid axis in nonprimate species showed declines in serum fT4 concentrations using nondialysis methodology. Nondialysis procedures are generally not accurate in nonprimate species because of the lower amount of the high-affinity binding protein, thyroid binding globulin (TBG), in these species. This results in an underestimation of the fT4 concentrations in serum by analog procedures (Nelson, 2005; Schachter et al., 2004).
In conclusion, our pharmacokinetic study suggests that the primary mode of action of PCB 126 on the HPT axis in the adult male Sprague Dawley rat is increased phase II metabolism of thyroxine, presumably mediated by AhR. Recent studies with nuclear receptors indicate that nuclear receptors exhibit cross talk and coordinated cellular activities, suggesting that other receptors play important roles in thyroid hormone homeostasis (Bock and Köhle, 2004), particularly as it relates to chemical/drug-specific disturbances in the HPT axis. Our pharmacokinetic analysis suggests that the dose-response characteristics of PCB 126–induced perturbations in the HPT axis are complex and nonlinear. The design of experimental studies, such as this study, to examine both the temporal- and dose-dependent changes in the HPT axis is important for the development of dose-response curves for thyroid-active chemicals. The development of a PBPK model for the HPT axis will aid in understanding complex nonlinear dose-response data for thyroid active chemicals.
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ACKNOWLEDGMENTS |
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Primary support for this research was kindly provided by ATSDR grant U61/ATU472105-(02, 03, and 04). Additional support was provided by U.S. EPA, ORD/NHEERL and the University of Georgia, College of Agricultural and Environmental Sciences. Special thanks to Deirdre Mahle, John Swint, Wilson Everett, Andy Smith, and Reiko Perleberg for helping with the animal studies. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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