The Hypothalamo-Pituitary-Thyroid (HPT) Axis: A Target of Nonpersistent ortho-Substituted PCB Congeners

Moazzam A. Khan*, Carol A. Lichtensteiger{dagger}, Obaid Faroon{ddagger}, Moiz Mumtaz{ddagger}, David J. Schaeffer* and Larry G. Hansen*,1

* Department of Veterinary Biosciences, College of Veterinary Medicine, 2001 S. Lincoln Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802; {dagger} Department of Veterinary Pathobiology, College of Veterinary Medicine, 2001 S. Lincoln Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802; and {ddagger} Division of Toxicology, Agency for Toxic Substances and Disease Registry (ATSDR), 1600 Clifton Road, Atlanta, Georgia 30333

Received August 14, 2001; accepted October 9, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Coplanar polychlorinated biphenyls (PCBs) cause adverse effects in developing and adult animals. Less is known about the effects of nonplanar ortho-substituted PCBs. We investigated the effects of 2 nonplanar PCB congeners, 95 (2,3,6-2',5'-penta CB) or 101 (2,4,5-2',5'-penta CB), and estradiol on selected endocrine parameters. In Study 1, weanling female Sprague-Dawley (S-D) rats were given a single dose of PCB 95 ip at 4, 8, 16, and 32 mg/kg/day for 2 consecutive days and killed 24 h after the last dose. PCB 95 exposure caused a dose-dependent (p < 0.001) decrease in serum thyroxine (T4) levels. Serum thyroid stimulating hormone (TSH) concentrations did not change, but prolactin (PRL) levels increased in a nonlinear (with dose) manner. No significant changes were seen in thyroid gland morphology and pituitary lactotroph number. In Study 2, progression or regression of effects was assessed by lengthening the time and a second congener was tested. Weanling female S-D rats received a single dose of PCB 95 or PCB 101 ip at 16 and 32 mg/kg/day for 2 days and were killed 48 h after the last dose. PCB 95 and PCB 101 both decreased serum T4 (p < 0.001) and hypothalamic dopamine (DA; p < 0.05) levels. No changes were seen in serum triiodothyronine (T3), TSH, and PRL concentrations. Morphological analysis of the thyroid gland showed a decrease (p < 0.05) in colloid area in rats treated with PCB 95 or 101. However, the epithelial cell height increased only in PCB 95 treated rats. Thyroid epithelial cell proliferation increased (p < 0.05) following exposure to estradiol and PCB 95. The results suggest that the HPT axis appears to be a target of ortho-substituted PCBs. PCB 95 was more effective than PCB 101 in causing these changes.

Key Words: episodic; PCBs; thyroid; thyroxine (T4); triiodithyronine (T3); pituitary; thyroid stimulating hormone (TSH); prolactin; hypothalamus; dopamine.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCBs) are widespread global contaminants. These compounds are highly lipophilic and persistent and, therefore, are present in the global ecosystem including air, water, fish, wildlife, as well as in human blood, adipose tissue, and milk (ATSDR, 2000Go; Hansen, 1998Go, 1999Go; Safe, 1994Go). Human and animal diseases that may result from exposure to PCBs include hepatotoxicity, immunotoxicity, and neurotoxicity. PCBs cause adverse effects on reproduction, development, and endocrine function including thyroid hormone homeostasis and estrogen-responsive tissues (Gray et al., 1999Go; Hansen, 1998Go; Morse et al., 1996Go; Safe, 1994Go).

Thyroid hormones and steroids are essential for normal body metabolism, growth, and development including reproduction, maturation, and aging (Capen et al., 1991Go). Both influence and are influenced by the hypothalamo-pituitary-thyroid (HPT) axis. Exposure to PCBs and related compounds causes reduction thyroid hormones in developing animals (Goldey et al., 1995Go; Li and Hansen, 1996Go; Morse et al., 1996Go; Ness et al., 1993Go) and adult animals (Kodavanti et al., 1998Go; Porterfield, 1994Go).

PCBs also influence estrogenic function. Several PCB congeners and mixtures can exhibit estrogenic activities whereas others show antiestrogenic activities (Jansen et al., 1993Go; Krishnan and Safe, 1993Go; Li and Hansen, 1996Go); ortho-substituted PCBs have shown generally weak estrogenic properties, but their precise mechanism of action is not well characterized.

The toxic effects of PCBs depend on their degree of chlorination and pattern of chlorine substitution. Coplanar PCBs and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause reductions in thyroid hormones mainly by binding to the aryl hydrocarbon receptor (AhR) and inducing hepatic UDP-glucuronosyl transferases (UDPGTs) that result in increased thyroxine (T4) biliary excretion (Kohn et al., 1996Go). PCB metabolites with hydroxyl groups on meta or para positions have structural resemblance to T4 and affinity for transthyretin (TTR), a thyroid hormone plasma transport protein. Thus, T4 is competitively displaced by PCB metabolites, rendering it vulnerable to metabolism and elimination (Chuhan et al., 2000; Lans et al., 1993Go). Nonplanar, i.e., ortho-substituted PCBs (with multiple chlorines at ortho positions) do not bind to the AhR and thus produce AhR-independent toxic effects. This group of PCBs induces CYP 2B and 3A enzymes, but their mechanisms of action are unclear (Hansen, 1998Go; Safe, 1994Go).

The subtle responses and potential mechanism(s) of action of many nonplanar environmental PCBs are poorly defined and almost half of them, including some that are very prevalent, have not even been investigated (Hansen, 1998Go). These nonplanar PCBs are environmentally threatening, based on their frequency of occurrence in environmental samples, relative abundance in animal tissue, and potential neuroendocrine actions (Hansen, 1998Go, 1999Go; Seegal et al., 1990Go; Wong and Pessah, 1996Go). Frequencies of detection of less persistent congeners are lower in human samples because of "episodic" exposures (Hansen, 2001Go); thus, they are often unreported and mostly ignored in toxicological studies (Hansen, 1998Go, 1999Go, 2001Go). Only a few episodic PCB congeners have been evaluated for endocrine system effects in vivo. Structure-activity relationships (SARs) for single endpoints such as ryanodine receptors (RyR; Wong and Pessah, 1996Go) are emerging, but integration of complex actions in vivo is rare.

The present studies were conducted with the objectives (1) to investigate the acute endocrine disruption potential of broad-acting, nonpersistent ortho-substituted PCB congeners and (2) to evaluate in vivo tissue parameters, screening for more sensitive indicators of endocrine effects. We focused on endpoints that may eventually help identify changes in the HPT axis following acute exposure to PCBs and related compounds. PCB 95 is weakly estrogenic (Rogers and Dennison, 2000; Sajid 1996Go). Our assay was not optimized for uterotropic effects, but estradiol was included because of known effects on the HPT axis, especially T4 (Dohler et al., 1979Go) and RyRs (Sundaresan et al., 1997Go). Comparisons of effects on the HPT axis thus may suggest estrogen-like activities of the PCBs.

Two structurally similar, but chemically distinct congeners, PCBs 95 and 101, were compared to estradiol. These congeners are present in the environment and food chain, but generally do not bioconcentrate in birds and mammals due to the labile 2,3,6 and 2,5 substitutions (Hansen, 1999Go, 2001Go). The multiple ortho chlorines, however, make these compounds volatile (enriched in air) and they also tend to accumulate in plants and fish (Hansen, 1999Go).

Acute exposure has been used (e.g., Jansen et al., 1993Go; Li and Hansen, 1996Go) because it is more relevant to common episodic exposure (Hansen, 2001Go) that may be significant in developing animals. Episodic PCB exposure could result in disruption of vital systems due to parent compound and/or metabolites. This disruption would be more biologically significant in developing animals than in healthy adults. Immature or developing animals are of greatest concern because the insult to the endocrine system at this age may cause irreversible damage and a transient insult could cause altered development. Weanling rats are used because they are still subject to developmental effects, but most major systems including the HPT axis, are fully functional (Dohler et al., 1979Go). Hepatic cytochrome P450 (CYP) enzymes are still in flux, but this stage is more relevant to developing animals.

Except for CYP induction (3 doses), PCB effects are generally assessed following 4–21 days of dosing. Since multiple mechanisms and cascading responses are almost certainly involved in the HPT axis, the short-term in vivo studies were conducted to identify initial targets. Longer duration permit some recovery/repair as well as progression of secondary responses. Modest (< 50 mg/kg) doses were used to avoid gross toxicity. Endpoints were assessed 24 and 48 h following exposure to determine whether initial effects were transient or progressive.

Study 1 focused on pituitary-thyroid morphology (colloid area, cell height, lactotrophs), function (TSH, PRL), effect (T4), and estrogenic effects 24 h following exposure to PCB 95. Study 2 was performed with more parameters (hypothalamic dopamine, PCNA, T3), longer duration (48 h) and a second ortho-PCB (PCB 101), which structurally resembles PCB 95, to gain some insight into mechanisms and structural requirements. Both congeners are episodic, but previous studies have suggested that the more labile 2,3,6-chlorination (e.g., PCB 95) is more thyrotoxic than the more stable 2,4,5-chlorination (e.g., PCB 101) (Li et al., 1998Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and animal husbandry.
Sixteen-day-old female Sprague-Dawley pups along with dam were obtained from Harlan (Indianapolis, IN). The rats were housed in separate plastic cages as 10 pups + dam per cage in the Office of Lab Animal Care (OLAC) facility at the College of Veterinary Medicine, University of Illinois at Urbana-Champaign. Standard rearing conditions (temperature 22–24°C, humidity 34–37% and 12 h light/dark cycle) were maintained throughout the studies. Water and feed (American Institute of Nutrition [AIN] diet 93G supplemented to 2 ppm iodine [Harlan Teklad, Madison, WI]) were available ad libitum to pups and dams. The pups were acclimatized by weighing and handling individually daily for 5 days prior to dosing. At 21 days of age, the pups were weaned and randomly divided into different dose groups. Rats in each dose group were identified by a tail tatoo and were kept 2 pups/cage.

Replicate (pups) consisted of 1 littermate assigned to each dose group. Consistency was maintained throughout both studies by following a fixed time schedule for handling, weighing, dosing, and killing the rats. To minimize variations in animal response to circadian rhythms, all doses were administered between 0900 and 1100 h; single rats from different dose groups were killed in forward-reverse order, (group 1, 2, 3, 4, 5, 5, 4, 3, 2, 1, 1, 2, 3...etc.).

Dosing.
PCB 95 (2,3,6–2',5'), purity 100% and PCB 101 (2,4,5–2',5'), purity 99.7%, were purchased from AccuStandard Inc. (New Haven, CT). Estradiol 17ß (approx. 98%) was purchased from Sigma Chemical Co. (St. Louis, MO). Dose solutions were prepared by adding corn oil (Mazola) purchased from a local grocery. Dosage regimen was selected to establish a dose range that would not be lethal, but cause serum hormone changes and allow identification of other possible target organ effects. In order to compare the test compounds, the 2 highest doses were selected for Study 2.

Study 1.
Twenty-one-day-old pups (n = 6–9/dose group) were dosed with PCB 95 ip in 0.1 ml of corn oil at 4, 8, 16, and 32 mg/kg/day for 2 days. An estradiol positive control group received 100 ng ip estradiol/rat/day. Negative controls received 0.1 ml of corn oil only. Twenty-four h after the last dose, the pups were decapitated by guillotine and tissue samples were collected. Necropsies were performed on individual rats immediately following decapitation.

Study 2.
Twenty-one-day-old pups (n = 9/dose group) received PCB 95 or 101 ip at 16 and 32 mg/kg/day for 2 days and were killed by decapitation 48 h after the last dose. Controls received corn oil only. The estradiol positive group was administered 1.0 µg/rat/day dose. In this study, the estradiol dose was increased to examine uterine complement C3 expression (Howe et al., 1990Go) to allow for the longer duration between the last dose and euthanasia. The duration, however, was too long to detect changes in C3 expression.

Blood collection.
Blood was collected from the decapitated neck region in 1.5 ml microcentrifuge tubes and held on ice until blood samples were collected from all rats. The blood sample was centrifuged (5000 rpm for 10 min), serum was collected, and stored at –20°C until hormone analysis.

Tissue collection.
Thyroid and adrenal glands (right and left) were removed and fixed in 10% neutral buffered formalin for 24 h. Livers were removed, quick frozen by using liquid nitrogen, and stored at –70°C. Uteri were carefully trimmed free of surrounding fat and connective tissue, excised at the cervical os, removed, weighed, and stored in –70°C. Livers, adrenals, and uteri were weighed before fixation. Relative organ weight (%) was = organ weight/terminal body weight x 100. Following decapitation, the hypothalamus was dissected out, weighed on an electronic balance, quick frozen in liquid nitrogen, and stored in a –70°C freezer until further analysis. Pituitaries and ovaries were removed, fixed in Bouin's solution for 4 h followed by 2 washes in 50% ethanol and left in 70% ethanol for 24 h. After routine tissue processing and embedding, the pituitaries and ovaries were sectioned and mounted on glass slides.

For morphological and histopathological examination, thyroids were processed and embedded in paraffin, sectioned to a thickness of 4 microns, and stained with hematoxylin and eosin. In addition, proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed on thyroid sections in Study 2 in order to evaluate proliferative changes.

Serum total T4, T3, TSH, and PRL analysis.
Serum total thyroxine (T4), total triiodothyronine (T3), rat thyroid stimulating hormone (TSH), and prolactin (PRL) analysis were performed as described previously (Khan et al., 1999Go) by using commercially available radioimmunoassay (RIA) kits.

For T4 and T3 analyses, total T4 and total T3 RIA kits (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA) were used. Kit manufacturer's instructions were followed using duplicate calibrator, control, and test samples. Rat thyroid stimulating hormone (rTSH) [125I] and rat prolactin (rPRL) [125I] assay systems (Amersham Life Science, Buckinghamshire, England) were used for the analysis of serum TSH and PRL according to the supplier instructions. The minimum detection limits for T4, T3, TSH, and PRL assays were 0.5 µg/dl, 7.0 ng/dl, 0.2 ng/tube and 0.2 ng/tube respectively. The intra-assay coefficient of variance (CV%) was 1.4, 3.1, 4.0, and 6.7 respectively.

Immunohistochemical staining of pituitary lactotrophs.
To investigate a correlation between pituitary lactotroph number and serum PRL concentration, immunohistochemical staining of pituitary lactotrophs was performed. The Avidin-Biotin method (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA) was used to perform immunostaining (protocol and antibodies provided by Dr. Paul Vancutsem, Department of Veterinary Pathobiology, University of Illinois). Frawley's antirat prolactin rabbit polyclonal antibodies were used as the primary antibody to detect the lactotrophs. Immunostaining and lactotroph count was performed as described by Poulet et al. (1997). A total of 400 lactotroph cells were identified and scored from each pituitary slide in randomly selected microscope fields at 40X magnification. The results were expressed as PRL labeling index (LI), computed as the number of immunopositive cells divided by total number of cells counted x 100 (Poulet et al., 1997Go).

Evaluation of morphological changes in the thyroid gland.
Morphometry data were collected by using a fully integrated software-controlled computerized microscopy system, Stereo Investigator version 2.1 (MicroBrightField, Inc., Colchester, VT). Stereo Investigator works in conjunction with a microscope and associated microscope instrumentation that performs image overlays, precisely controls the lateral movement of a motorized stage, and precisely controls the focus of the microscope. Once optimized and calibrated, the computer controlled motorized stage will allow only random examination of the tissue section placed on glass slide.

Images (from thyroid tissue slides) were captured by using a high resolution Chromachip-II camera (Javelin Electronics) mounted on an Olympus BH-2 microscope (40X magnification) attached to an IBM Pentium-II computer system. Subsequently, the images were analyzed by measuring colloid area and epithelial cell height and by counting PCNA positive cells using Fractionator and Nucleator probes. Twenty random follicles from each animal were measured for colloid area. Epithelial cell height was measured from 60 cells as described previously (Khan et al., 1999Go).

Measurement of hypothalamic dopamine.
The hypothalamic dopamine (DA) concentration was measured as described by (Kuhananthan et al., 1991Go). For DA analysis, the frozen hypothalamic tissue was placed in a microcentrifuge tube containing 500 µl of filtered 0.1M perchloric acid. Following sonication, the homogenate was centrifuged at 4°C at 10,000 rpm for 20 min. The supernatant was filtered using a 22 µM filter. Twenty µl of filtered samples were subjected to high-performance liquid chromatography-electrochemical detection (HPLC-ECD; Bioanalytical Systems). Freshly prepared standards were used and DA concentration was measured as pg/mg of tissue. All samples were kept on ice during the entire process of preparation.

Assessment of cell proliferation activity in the thyroid gland (PCNA staining).
Immunostaining of the thyroid tissue was performed using antibodies against proliferating nuclear cell antigen (PCNA). PCNA is a 36 kDa acidic nonhistone nuclear protein that functions as an auxiliary protein for DNA polymerase and is an absolute requirement for DNA synthesis (Kurki et al., 1986Go; Velicky et al., 1997Go). PCNA is commonly accepted as a reliable indicator of increase in cell proliferation activity (Velicky et al., 1997Go) and may help in evaluating thyroid tumor-promoting potential of xenobiotics.

The thyroid tissue from control and treated rats was routinely fixed in 10% neutral buffered formalin and embedded in paraffin wax. Four-micron-thick sections were mounted on poly-L-lysine coated glass slide. Antibodies targeting PCNA, diaminobenzidine (DAB) substrate, Optimax buffer, biotinylated antimouse antibody, and avidin-biotin were purchased from BioGenex, San Ramon, CA. To minimize variation, all slides were processed and stained as a single batch. For PCNA staining, slides were deparaffinized using xylene and graded alcohol. The sections were then washed with water and incubated with normal goat serum to reduce nonspecific antibody binding. The positive slides were applied with primary PCNA antibody 1:10,000 dilution and negative control slides with negative control serum. Slides were rinsed with Optimax buffer and incubated with avidin and biotin for 15 min to block endogenous biotin. A secondary antibody, biotinylated antimouse immunoglobulin was applied for 20 min and slides were rinsed in Optimax buffer. Peroxidase conjugated streptavidin was applied for 20 min, followed by Diaminobenzidine (DAB) substrate for 5 min and counterstaining with hematoxylin for 1 minute. Finally, slides were rinsed in water, dehydrated, cleared, and mounted in permanent mounting medium.

Cell proliferation activity was determined by following guidelines described by Kurki et al. (1986). Cells in G0 phase were characterized by no immunostaining. Cells with minimal nuclear staining were regarded in G1 stage cycle. Nuclei with intense dark brown staining were characterized as S phase (DNA duplication stage) nuclei or PCNA positive cells. Cells were counted randomly by using a computerized system, Stereo Investigator, until a total of 500 labeled and unlabeled nuclei were assessed. The labeling index (LI) was calculated as the number of PCNA positive nuclei divided by the total number of nuclei x 100 (Velicky et al., 1997Go).

Data analysis.
Data obtained were analyzed by using Systat version 8.0 computerized program. Differences between treatment groups were analyzed using one-way ANOVA, followed by Tukey's HSD multiple comparison. The Kruskal-Wallis test was used on occasion due to inhomogenity of variance. Regression analysis was carried out to assess the dose-dependent effects. Differences were considered significant if p < 0.05. Quantitative results are expressed as mean ± SEM.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study 1 (24 h after 2 Consecutive Daily Doses)
Terminal body weight and relative organ weights.
Exposure to the selected doses of PCB 95 did not cause overt signs of toxicity. No changes were seen in body weights of treated and control rats. Relative liver and uterus weights did not differ among PCB 95 treated animals. The relative adrenal weight was smaller (p > 0.05) in the 32 mg/kg group than in the 16 mg/kg group (Table 1Go). Regression analysis indicated no linear trend, but suggested (p = 0.056) a quadratic (convex) trend in relative adrenal weights 24 h after 2 consecutive daily doses of PCB 95. This trend was absent after 48 h (Table 2Go). Relative uterine weight (p < 0.001) increased significantly in estradiol (100 ng/rat/day) positive control rats (Table 1Go).


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TABLE 1 Terminal Body Weight and Relative Organ Weight in Immature Female Rats 24 h following Acute Exposure to PCB 95
 

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TABLE 2 Terminal Body Weight and Relative Organ Weight in Immature Female Rats 48 h following Acute Exposure to PCB 95 or 101 PCB 101
 
Serum total T4, TSH, and PRL analysis.
Serum total T4 levels decreased following exposure to PCB 95 (Fig. 1Go). Linear regression analysis revealed that serum T4 levels decreased in a dose-dependent manner (p < 0.001) by 39, 47, and 53% in the 8, 16, and 32 mg/kg groups respectively, when compared to control. However, serum T4 concentration increased (p < 0.001) following estradiol (100 ng) exposure as compared to control. No changes were seen in serum TSH levels (Fig. 2Go). Serum PRL levels increased significantly (p = 0.02) in estradiol-treated rats as compared to control (Fig. 3Go). Polynomial regression analysis showed a nonlinear (p < 0.001) relationship between serum PRL concentration and the PCB 95 dose administered (Fig. 3Go).



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FIG. 1. Serum total T4 levels in weanling female rats that received corn oil (control), estradiol (100 ng/day), and PCB 95 at 4, 8, 16, and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 24 h following the last dose. Data are presented as mean ± SEM µg/dl. *Significant difference (p < 0.001) from control.

 


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FIG. 2. Serum TSH levels in weanling female rats that received corn oil (control), estradiol (100 ng/day), and PCB 95 at 4, 8, 16, and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 24 h following the last dose. Data are presented as mean ± SEM ng/ml.

 


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FIG. 3. Serum PRL levels in weanling female rats that received corn oil (control), estradiol (100 ng/day), and PCB 95 at 4, 8, 16, and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 24 h following the last dose. Data are presented as mean ± SEM ng/ml. *Significant difference (p < 0.001) from control.

 
Pituitary lactotroph LI.
Despite alterations in the serum PRL concentration, no changes were seen in the number of lactotrophs in the anterior pituitary gland of rats exposed to either PCB 95 or estradiol (Fig. 4Go).



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FIG. 4. Pituitary lactotroph labeling index (%) in weanling female rats that received corn oil (control), estradiol (100 ng/day), and PCB 95 at 4, 8, 16, and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 24 h following the last dose. Labeling index calculated as prolactin positive cells/a total of 400 cells x 100.

 
Thyroid gland morphometry.
In spite of the distinct decreases in serum T4 concentration, changes in thyroid gland colloid area and follicular epithelial cell height were subtle (Fig. 5Go).



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FIG. 5. Mean ± SEM measurements of thyroid colloid area (µm2) of 20 follicles and cell height (µm) of 60 follicular epithelial cells from weanling female rats that received corn oil (control), estradiol (100 ng/day), and PCB 95 at 4, 8, 16, and 32 mg/kg/day for 2 consecutive days.

 
Study 2 (48 h after 2 Consecutive Daily Doses)
Final body and organ weights.
No clinical signs of toxicity or moribundity occurred among animal groups. PCB 95 and 101 exposure did not cause changes in body weight or relative organ weight (%) of treated animals. An increase (p < 0.001) was seen in relative uterine wet weight in the estradiol (1.0 µg/rat/day) positive group when compared to control (Table 2Go).

Serum total T4, total T3, TSH, and PRL analysis.
Serum total T4 concentration decreased (p < 0.001) following exposure to PCB 95 or 101 (Fig. 6Go). While PCB 95 exposure at 16 and 32 mg/kg caused 59 and 60% reductions in T4 levels, exposure to PCB 101 at 16 and 32 mg/kg resulted in smaller 43 and 46% decreases in T4 levels, respectively when compared to control (Fig. 6Go). Serum T3 levels appeared slightly depressed in rats treated with PCB 95 as compared to control and PCB 101 group, but these changes were statistically nonsignificant (Fig. 7Go). Serum TSH levels rose slightly only in PCB 95 treated rats (Fig. 8Go) and serum PRL concentration decreased in rats treated with either PCB 95 or PCB 101 (Fig. 9Go). Alterations in serum TSH and PRL were not different when compared to control (Figs. 8 and 9GoGo). No changes were seen in serum T4, T3, TSH, and PRL concentration in estradiol-treated rats (Figs. 6, 7, 8, and 9GoGoGoGo).



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FIG. 6. Serum total T4 levels in weanling female rats that received corn oil (control), estradiol (1.0 µg/day), and PCB 95 or PCB 101 at 16 and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 48 h following the last dose. Data are presented as mean ± SEM µg/dl. *Significant difference (p < 0.001) from control.

 


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FIG. 7. Serum total T3 levels in weanling female rats that received corn oil (control), estradiol (1.0 µg/day), and PCB 95 or PCB 101 at 16 and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 48 h following the last dose. Data are presented as mean ± SEM ng/ml.

 


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FIG. 8. Serum TSH levels in weanling female rats that received corn oil (control), estradiol (1.0 µg/day), and PCB 95 or PCB 101 at 16 and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 48 h following the last dose. Data are presented as mean ± SEM ng/ml.

 


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FIG. 9. Serum PRL levels in weanling female rats that received corn oil (control), estradiol (1.0 µg/day), and PCB 95 or PCB 101 at 16 and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 48 h following the last dose. Data are presented as mean ± SEM ng/ml.

 
Hypothalamic dopamine concentration.
PCB 95 or 101 exposure at 16 and 32 mg/kg doses caused reductions in hypothalamic dopamine concentration (Fig. 10Go). Dopamine concentration decreased by 45.3 and 38.7% in PCB 95-exposed rats and 35.1 and 31.9% in PCB 101 treated animals when compared to control, but a maximum response was approached since the response did not differ between the 2 doses. PCB 95 was more effective (p < 0.001) in decreasing hypothalamic DA levels as compared to PCB 101 (p < 0.05). An apparent decrease (p = 0.09) was seen in hypothalamic DA levels in animals in the estradiol positive group when compared to control (Fig. 10Go).



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FIG. 10. Hypothalamic dopamine concentration in weanling female rats that received corn oil (control), estradiol (1.0 µg/day), and PCB 95 or PCB 101 at 16 and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 48 h following the last dose. Data are presented as mean ± SEM pg/mg. Significant difference from control, *p < 0.05, **p < 0.001.

 
Evaluation of morphological changes in the thyroid gland.
Mean ± SEM changes in thyroid colloid area (follicle size) and epithelial cell height are illustrated in Fig. 11Go. Exposure to PCB 95 or 101 (at both doses) decreased (p < 0.05) thyroid colloid area when compared to control. The PCB treated groups were not different from each other. The epithelial cell height increased (p < 0.05) in PCB 95 treated rats (low and high doses) when compared to control, but did not increase in PCB 101 treated rats.



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FIG. 11. Mean ± SEM measurements of thyroid colloid area (µm2) of 20 follicles and cell height (µm) of 60 follicular epithelial cells from weanling female rats that received corn oil (control), estradiol (1.0 µg/day), and PCB 95 or PCB 101 at 16 and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 48 h following the last dose.

 
Evaluation of thyroid epithelial cell proliferation (PCNA staining).
Thyroid epithelial cell proliferation (PCNA labeling index) was found increased (p < 0.05) in rats treated with estradiol and PCB 95 (both doses), but exposure to PCB 101 did not cause significant changes in the PCNA labeling index (Fig. 12Go).



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FIG. 12. Thyroid epithelial cell proliferation labeling index (%) in weanling female rats that received corn oil (control), estradiol (1.0 µg/day), and PCB 95 or PCB 101 2 x 16 and 32 mg/kg/day for 2 consecutive days. Rats were sacrificed 48 h following the last dose. Labeling index calculated as number of PCNA positive cell/a total of 500 cells x 100. *Significant difference (p < 0.05) from control.

 
Histopathology.
Histopathologic examination of thyroid, adrenal, and pituitary gland did not show any changes of pathologic significance in either study.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The current study investigated acute endocrine effects of 2 labile PCBs in weanling female Sprague-Dawley rats following 2 consecutive daily doses. A summary of significant observations is included in Tables 3 and 4GoGo. Two daily doses up to 32 mg/kg/day did not produce any clinical signs of toxicity nor did this treatment and duration cause any changes of pathological significance in the tissues examined. At 2 x 64 or 2 x 96 mg/kg, PCB 95 did not affect pup body weight, but increased liver weight (Sajid, 1996Go). The highest dose administered in the present studies (32 mg/kg) did not affect the liver weight (Tables 1 and 2GoGo).


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TABLE 3 Summary of Endocrine Changes in Female Weanling Rats 24 h following Acute Exposure to PCB 95 (Study 1)
 

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TABLE 4 Summary of Endocrine Changes in Female Weanling Rats 48 h following Acute Exposure to PCBs 95 or 101 (Study 2)
 
In Study 1, PCB 95 caused a dose-dependent reduction (p < 0.001) in serum T4 levels (Fig. 1Go). Serum T4 was decreased 53% below that of corn oil controls 24 h following 2 x 32 mg/kg PCB 95 (Fig. 1Go). In Study 2, serum T4 was decreased 60% from a higher control 48 h following the same dose (Fig. 6Go). The higher serum T4 (Fig. 6Go) reflects pups one day older during a dynamic phase of development; T4 levels are generally higher on days 24 and 25 than on days 21 and 22 (Dohler et al., 1979Go). An obvious increase in T4 levels in estradiol-treated rats (Fig. 1Go) was not seen in the older rats, but the PCB effect was intensified. PCB 101 was slightly less effective, but also caused (p < 0.001) reduction (46%) in serum T4 from control, 48 h after 2 x 16 and 2 x 32 mg/kg exposure (Fig. 6Go).

AhR agonists amplify biliary excretion of T4 due to induction of UDP-glucuronosyltransferase (UDPGT) enzymes (Barter and Klassen, 1994; Hansen, 1998Go; Kohn et al., 1996Go) and/or increased T4 sequestration by the liver (Capen et al., 1991Go; Hansen, 1998Go; Martin et al., 2001Go). AhR agonists are more potent, while noncoplanar PCBs act through multiple mechanisms and are more efficacious (Ness et al., 1993Go; Seo et al., 1995Go).

In normal thyroid physiology, reduction of circulating T4 levels is compensated for by release of TSH by the pituitary that stimulates the thyroid gland to synthesize more thyroid hormones and produce thyroid gland hypertrophy and hyperplasia (Capen et al., 1991Go). PCB mixtures and related compounds caused prolonged reductions in circulating T4 levels, but there may or may not be a compensatory increase in plasma TSH levels in adult and neonate rats (Barter and Klassen, 1994; Capen et al., 1991Go; Morse et al., 1996Go). In our studies, exposure to ortho-PCB congeners 95 or 101 reduced T4 levels, but did not reduce T3 or increase TSH levels suggesting interruption of the feedback mechanism(s) of the HPT axis. Several lines of evidence support a distinctive hypothesis regarding thyroid hormone mimics and/or TSH release.

It is possible that the ortho-substituted PCB congeners tested in the present studies (or their metabolites) may have bound to thyroid hormone receptors so that the hypothalamo-pituitary axis could not perceive any reductions in thyroid hormone levels and, therefore, no increase was seen in TSH levels. SAR studies have shown that PCBs with ortho-substitution and hydroxylated PCB metabolites have significant affinity for binding to a T4 binding protein, transthyretin (TTR) (Chuhan et al., 2000; Lans et al., 1993Go) that may competitively inhibit T4 binding to TTR. Molecular modeling studies have revealed that the structure of thyroid hormones closely resemble PCBs, including those with chlorines at ortho positions. These PCBs may interact with thyroid hormone receptors (McKinney and Waller, 1994Go) and produce thyroid hormone-like effects without causing changes in TSH concentration. This aspect is also supported by evidence that a PCB mixture (Aroclor 1254) produced thyromimetic effects on the developing rat brain. Zoeller et al. (2000) examined the expression of 2 thyroid hormone-responsive genes, RC3/Neurogranin and the myelin basic protein (MBP), in the developing brain that are regulated by thyroid receptor. Aroclor 1254 treatment reduced serum total T4 levels, but the expression of RC3 was increased at day 15, suggesting thyromimetic effects of PCBs. The PCB congeners tested in our present studies may resemble thyroid hormones enough to interact with the thyroid receptor causing thyromimetic effects and deceiving the body from perceiving thyroxinemia.

An alternate or additional mechanism may involve TSH release from the pituitary. Intracellular Ca2+ is known to regulate a large number of cellular functions including hormone secretion. PCB 95 (with tri-ortho chlorine substitution) was found to have direct and potent activity towards the ryanodine-sensitive Ca2+ release channels (RyRs) in the skeletal and smooth muscles (Wong and Pessah, 1996Go) suggesting ortho-substituted PCB 95 perturbs Ca2+ homeostasis by targeting one of the RyR accessory protein complexes, FKBP12/RyR (Wong and Pessah, 1997Go). Influx of Ca2+ ions is necessary for the contraction of microfilaments and discharge of TSH-containing granules from the anterior pituitary gland (Capen et al., 1991Go). RyR types 2 and 3 are expressed and hormonally regulated in rat pituitaries (Sundaresan et al., 1997Go). Pituitary function is compromised by ruthenium red (a RyR antagonist), which caused a 40% decrease in the spike phase and 35% decrease in plateau phase of GnRH-induced leutinizing hormone (LH) release from the rat pituitary gland (Sundaresan et al., 1997Go). It is possible that, in our studies, there were alterations in pituitary intracellular Ca2+ homeostasis following exposure to PCB 95 (tri-ortho) and 101 (di-ortho) which may have resulted in blockage of TSH secretion. Other indications of pituitary dysfunction would add some support to this hypothesis.

In Study 1, a nonlinear relationship was seen between PCB 95 dose and serum PRL levels (Fig. 3Go). Estradiol (100 ng/rat/day) treatment increased (p < 0.001) serum PRL levels (Fig. 3Go), which was expected because serum PRL levels increase in adult female rats at the onset of vaginal opening when there is surge of endogenous estradiol, but decline within 1–3 days (Voogt et al., 1970Go). This remarkable decline in estradiol-induced serum PRL was obvious in Study 2 where PRL levels were decreased 71% from the 24 h value 48 h following a higher estradiol (1.0 µg/rat/day) dose (Fig. 9Go). The 48 h PRL level was only half as high as control values in estradiol-treated rats (Fig. 9Go). The trend toward higher control PRL in rats one day older may be due to rapid changes during development (Dohler et al., 1979Go; Voogt et al., 1970Go). No pattern was apparent for 48-h serum PRL in PCB-treated rats, suggesting a transient disruption. The uterine weight increase was also less 48 h after a high dose of estradiol (Table 2Go) than 24 h after a lower dose (Table 1Go).

As with changes in thyroid homeostasis, PCB 95 was more effective in reducing hypothalamic DA levels than PCB 101. The exact mechanism by which PCBs reduce the DA concentration is not clear. PCB 95 and 101 may cause changes in intracellular Ca2+ levels that may lead to increased DA secretion from neurons causing eventual reductions in hypothalamic DA levels. Our results support this hypothesis as PCB 95 (the most potent RyR ligand of the PCBs tested) was more effective than the less potent PCB 101 in causing alterations in serum T4 and PRL levels and hypothalamic DA concentration.

In Study 2, colloid area was decreased following exposure to PCB 95 or 101 (at both doses) suggest an early thyroid response (Fig. 11Go). Short-term xenobiotic exposure can produce similar thyroid responses (Khan et al., 1999Go; Ness et al., 1993Go; Saeed and Hansen, 1997Go). Mechanistic studies suggest that, in response to reductions in serum T4 concentration, serum TSH increases as a compensatory response causing thyroid gland hyperplasia and hypertrophy eventually leading to the development of thyroid tumors (Capen et al., 1991Go). PCB 95 or 101 exposure did not change TSH but did change thyroid morphology. It is proposed that T3 is the direct regulator of TSH secretion (Shupnik et al., 1985Go). We did not observe any changes in T3 levels. It can be speculated that PCB 95 or 101 is interfering with TSH production and release at the pituitary.

In Study 2, estradiol and PCB 95 exposure increased the thyroid epithelial cell proliferation (PCNA labeling index). PCB 95 has shown weak estrogenic activity (Rogers and Dennison, 2000; Sajid, 1996Go). The presence of estrogen receptor (ER{alpha}) in thyroid cells is known and it has been reported that estradiol increases the rate of cell proliferation in vitro in Fischer rat thyroid cell line (FRTL-5) in a time- and concentration-dependent manner independent of the TSH signaling pathway (Furlanetto et al., 1999Go). It is possible that this mechanism is responsible for the significant increase in PCNA labeling index in vivo as observed in the present study.

It is interesting to note that only PCB 95 exposure (both doses) increased the PCNA labeling index in our studies. It appears that PCB 95 and 101 act on the HPT axis through different mechanisms. Moreover, PCB 95 appears to be more effective in inducing mitosis as compared to PCB 101. Whether the PCNA labeling index can be used as an early and reliable indicator for in vivo carcinogenicity testing would require more detailed studies.

In summary, our results demonstrate that (1) ortho-substituted PCB 95 or 101 can significantly affect the endocrine system in immature animals even after acute episodic exposure; (2) the HPT axis appears to be a potential target of ortho-PCBs and related compounds; (3) circulating thyroid hormones and hypothalamic dopamine concentrations in the intact weanling female rats are sensitive endocrine indicators following exposure to these compounds; and (4) histological and morphological changes should be interpreted carefully, especially in acute exposure studies. As with estradiol, some responses to the weakly estrogenic PCB 95 (adrenal weight, serum PRL) are nonlinear with dose; therefore, careful attention must be paid to dose, to alleviating stress, and to the rapidly changing hormone levels in developing animals. The present studies have provided information regarding the potential targets for xenobiotics in the endocrine system. The elucidation of mechanism(s) by which ortho-PCBs alter the HPT axis is the focus of our ongoing studies.


    ACKNOWLEDGMENTS
 
The authors acknowledge and thank Dr. Victor Ramirez, Dr. Sue Woods, and David Kuehl for their contribution to these studies. We are also thankful to Charles Capen, Gabriele Ludewig, Daniel Ness, and Raphael Witorsch for reviewing this manuscript and providing thoughtful suggestions. Funding for these studies was provided, in part, by the Agency for Toxic Substances and Disease Registry (ATSDR), the University of Illinois Research Board, and the Hansen-Ducker Heritage Fund.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (217) 244-1625. E-mail: lhansen{at}cvm.uiuc.edu. Back


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