Thyroid Toxicity Due to Subchronic Exposure to a Complex Mixture of 16 Organochlorines, Lead, and Cadmium

Michael G. Wade*,1, Sophie Parent*, Kenneth W. Finnson{dagger}, Warren Foster{ddagger}, Edward Younglai{ddagger}, Avril McMahon*, Daniel G. Cyr{dagger} and Claude Hughes§

* Growth and Development Section, Environmental and Occupational Toxicology Section, Bureau of Chemical Hazards, Health Canada, Environmental Health Centre, Tunney's Pasture, Ottawa, Ontario, Canada; {dagger} INRS, Institute Armand Frappier, 245 Boulevard Hymus, Pointe-Claire, Québec, Canada; {ddagger} Department of Obstetrics and Gynecology, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada; and § Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina

Received October 26, 2001; accepted February 4, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human population in the industrialized world is ubiquitously exposed to complex mixtures of persistent pollutants that contaminate food, water, and air. A large number of these contaminants have been shown to cause significant toxicity to the hypothalamic-pituitary-thyroid (HPT) axis in laboratory animal studies, through a variety of mechanisms, although these effects occur at levels of exposure greatly in excess of common human exposure. While many of the mechanisms of thyroid toxicity of these substances are potentially complementary, little is known of the degree of interaction of common persistent contaminants on responses of the HPT axis. To investigate the potential effects of a complex, environmentally relevant mixture on the HPT axis, sexually mature male rats were administered a mixture of 16 common organochlorines (dichlorodiphenoxytrichloroethane [DDT], p,p`-dichlorodiphenoxydichloroethylene [p,p'-DDE], hexachlorobenzene [HCB], tetrachlorodibenzo-p-dioxin [TCDD], polychlorinated biphenyls [PCBs], methoxychlor, endosulfan, heptachlor, hexachlorocyclohexane, dieldrin, aldrin, mirex, and several chlorinated benzenes, and metal contaminants [lead, cadmium]). The doses of the mixture that were administered were related to minimum risk levels or tolerable daily intakes of these substances, as derived by risk assessment with the 1x, 10x, 100x, and 1000x groups receiving mixture components at doses equivalent to 1x, 10x, 100x, or 1000x the minimum risk level (or tolerable daily intake, reference dose), respectively. After 70 daily treatments by gavage, endpoints related to circulating thyroid hormone (serum thyroxine [T4], triiodothyronine [T3], thyroid stimulating hormone [TSH], and serum T3 uptake [T3-up]), thyroid gland histomorphology (thyroid follicle cross sectional area, epithelial height, follicle roundness or aspect ratio, colloid/epithelial ratio) and hepatic metabolism of thyroid hormone (UDP-glucuronyl transferase [UGT] and outer-ring deiodinase [ORD]) were assessed. All examined endpoints were significantly altered by the mixture albeit with great variability between endpoints in the sensitivity. While most endpoints examined did not show significant changes at mixture doses below 1000x, 2 endpoints, TSH and hepatic outer ring deiodinase activity, were significantly increased and decreased, respectively, by 1x dose and showed dose-related increases in severity with increasing dose. Median thyroid follicle cross sectional area was also increased by the lowest dose of the mixture but decreased with subsequent increases in dose until, at the highest dose, this parameter was significantly reduced relative to control. The relative sensitivity of endpoints of thyroid function in detecting toxicity of the mixture was TSH = ORD = median follicle area >> T3 > all other endpoints. These results demonstrate that low doses of ubiquitous environmental contaminants can alter HPT physiology in sexually mature males.

Key Words: organochlorines; dioxin; PCB; thyroxine; thyroid; outer ring deiodinase; UDP glucuronyltransferase.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Humans interact with their environments on a daily basis and, as a consequence, are exposed to a broad spectrum of synthesized chemicals present in the food they eat, the air they breathe, and the water they drink. A variety of synthetic halogenated organic compounds, such as dichlorodiphenoxytrichloroethane (DDT) and its persistent metabolite p,p`-dichlorodiphenoxydichloroethylene (p,p'-DDE), hexachlorobenzene (HCB), dioxins, furans, polychlorinated biphenyls (PCBs), other organochlorine pesticides, and metals, such as lead and cadmium, have been released into the environment through human activities and are routinely found as contaminants in tissues collected from the human population in Canada and in other nations throughout the world (Davies and Mes, 1987Go; Frank et al., 1988Go; Mes et al., 1990Go; Mes, 1992Go; Newsome et al., 1995Go; Szymczynski and Waliszewski, 1981aGo,bGo). While the levels of exposure for the vast majority of the population are below doses shown to cause adverse effects in laboratory studies examining the effects of single chemicals, the consequences of low level exposure to a wide variety of substances are not known and are the subject of considerable debate (Germolec et al., 1989Go; Pohl et al., 1997Go; Porter et al., 1999Go).

For noncancer risk assessments, estimates of safe exposure (minimum risk level, acceptable or tolerable daily intake, or reference dose) for a given substance are routinely derived, by a variety of public health organizations, from detailed examination of evidence of toxicity from exposure to that substance (Barnes and Dourson, 1988Go). Ideally, this process involves the identification of a no observable adverse effect level (NOAEL), the highest dose of exposure that is without toxic effect on the most sensitive endpoint examined, which is then divided by a safety factor to estimate a daily rate of exposure that is predicted not to cause adverse effects. While this process may be appropriate for estimating risks of exposure to individual compounds it does not attempt to estimate alterations in potency due to coexposure to other substances. Very few studies have attempted to assess the degree of hazard posed by simultaneous exposures to reference doses of multiple chemicals (Ito et al., 1995aGo,bGo).

There is currently a great deal of scientific and public interest in the possibility that many of these persistent environmental contaminants and other anthropogenic substances may be causing alterations in the function of endocrine systems, thereby disrupting developmental and physiological function in animals and humans. Most of this attention has been directed to investigating effects related to sex steroid physiology although there is a growing body of evidence showing that there are many common, persistent substances that are potent disrupters of thyroid homeostasis (Brouwer et al., 1998Go). While these have been reported to cause reductions of circulating thyroid hormones or histopathological lesions to the thyroid gland, the doses required to induce these effects in experimental animals are well above doses to which the human population are generally exposed. However, the mechanisms of action by which these substances interfere with thyroid physiology are diverse and potentially complementary. Coexposure to multiple substances, which have been shown to have different mechanisms of action in inducing thyroid disruption, is a reality in the human populations throughout the industrialized world. As such, the possibility that the cumulative effects of the mixture could have greater impact in inducing thyroid dysfunction than would occur from exposure to comparable doses of the individual components alone has significant public health implications. The current study examines the effects of subchronic daily exposure to a complex mixture of ubiquitous persistent contaminants on thyroid hormone physiology and metabolism in adult male rats.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mixture formulation.
The formulation of the mixture and selection of dose levels is described elsewhere (Wade et al., 2002Go). Briefly, the contaminant mixture was formulated to include some of the persistent organic and inorganic contaminants to which the human population of Canada could conceivably be exposed. The dose levels of each component in the mixture reflect the currently promulgated safe levels of exposure as published by the ATSDR (minimum risk level; MRL), Canadian Environmental Protection Act Chemical Assessments (tolerable daily intake; TDI), U.S. EPA (reference dose; RfD), or at the lowest NOEL available in the scientific literature (Table 1Go). The dosing solutions provided a daily dose of each component equivalent to 1x, 10x, 100x, or 1000x their daily safe or no effect levels of exposure.


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TABLE 1 Composition of Dosing Mixtures
 
Animals.
Sexually mature, male Sprague Dawley rats (49 days of age) were purchased from Charles River (St. Constant, Québec, Canada) and acclimated to holding facilities for 1 week prior to commencement of dosing. Animals were caged in pairs in clear plastic cages containing wood chips for bedding and maintained under controlled temperature (24°C), humidity (30–70%), and light (12:12 light:dark). All animals were provided standard laboratory rat chow (Labdiet 5001 rodent chow, Ralston Purina, St. Louis, MO) and water ad libitum. The protocol for animal experimentation detailing all animal manipulations was scrutinized and approved by the McMaster University Institutional Animal Care Committee prior to initiation of the study. All animal care and handling were in accordance with Canadian Council for Animal Care guidelines.

Rats were randomly assigned to control (n = 9), 1x(10), 10x(10), 100x(10), or 1000x(10) treatment groups. Rats received corn oil (vehicle) or appropriate treatment in a volume of 1 µl/g body weight daily by gavage based on the most recent body weight, which was recorded on Mondays, Wednesdays, or Fridays of every week. As reproductive endpoints were also examined in this study, as described elsewhere (Wade et al., in press), rats were dosed for 70 consecutive days, which is equivalent to 1 complete cycle of spermatogenesis. On the day following the final dose, rats were anesthetized with isofluorane and, after recording terminal body weight, were sacrificed by exsanguination via cardiac puncture followed by decapitation. Blood was collected in SST Vacutainer tubes (Becton-Dickinson), held for no more than 4 h before serum was collected by centrifugation (3000 x g for 15 min), aliquoted and frozen at –80°C until analysis. Serum that was visibly red was not analyzed further as this indicated extensive haemolysis after blood collection. Commercially available RIA kits were used to assay serum content of thyroid stimulating hormone (TSH; rat-specific, Cat. No. RPA5541, Amersham Pharmacia, Piscataway, NJ), thyroxine, triiodothyronine, and serum triiodothyronine uptake (T4, T3, and T3-UP, respectively; Cat. Nos. 06B-254029, -256447, and -237124, respectively; ICN Biomedicals, Aurora, OH).

The liver was dissected out and 4 pieces of the liver, roughly 1 g each, were frozen in liquid nitrogen and stored at –80°C prior to hepatic enzyme analysis. A portion of the trachea, with intact thyroid gland attached, was dissected free of connective tissue and fixed in PBS containing 4% paraformaldehyde for 3 to 4 days until all samples were simultaneously embedding in paraffin using an automated histoprocessor.

Thyroid gland histomorphology.
Sections of thyroid gland, 5 µm thick, were cut parallel to the longitudinal axis of the trachea and stained with Periodic Acid Schiff. Areas photographed for analysis were from areas showing as few artifacts as possible, had little nonfollicle tissue, were clearly not adjacent to the preiphery of the gland, and were immediately adjacent to the parathyroid gland. The latter 2 criteria are to avoid including measurements taken from follicles on the surface of the gland as these tend to be much less active than those within the gland (Low, 1982Go). Histomorphological parameters were quantified for follicle cross sections in 2 digital images, each equivalent to an area of 625 x 410 µm, from 2 separate histological sections per animal for 5 animals per treatment using ImagePro software (release 4.0, Media Cybernetics, Silver Spring, MD). All images were captured and all measures of thyroid gland morphology were collected from 2 digital images from each animal by a single investigator (S. P.), who was unaware of specimen treatment.

Agents that disrupt thyroid physiology cause an increase in the thickness of thyroid follicle epithelium and reduce the volume of follicle colloid and overall follicle diameter indicating toxicity to thyroid gland (Capen, 1999Go). These effects were estimated using 5 separate parameters calculated from several basic measures of follicle morphology graphically depicted in Figure 1Go. Periodic Acid Schiff stains the noncellular central colloid of thyroid follicles an intense purple stain that can be identified and selected using a color-recognition feature of the digital image analysis software. Once selected, the cross sectional area of follicular colloid and length of the longest and shortest axes (Lc and Wc, respectively, in Fig. 1Go) are automatically calculated for every complete follicle cross section visible in a digital image. As hyperactive follicles tend to collapse as the intrafollicular colloid is exhausted, the roundness and aspect ratio were calculated as [(colloid perimeter)2/4{pi}(colloid area)] and [Lc/Wc], respectively. The mean roundness and aspect ratio attempted to estimate the degree to which the follicle cross section deviated from the normal, round shape. For example, a perfect circle would receive a value of 1 with either parameter, and the value would increase as the perimeter became less circular and more convoluted. As height of thyroid epithelial cells is influenced in animals that are challenged with agents that disrupt thyroid hormone homoeostasis, the height and relative proportion of total follicle cross sectional area of follicle epithelium were estimated. Total follicle area was identified for the 5 largest follicles that had no evidence of any artifactual distortion in each image by manually tracing the basement membrane. Epithelial height was estimated as [(Lf – Lc) + (Wf – Wc)]/4. In an attempt to capture the relative changes in epithelial height and volume of the colloid in a single parameter, the ratio of the area of the epithelium (Total Area – Colloid) to colloid area was calculated for the 5 largest follicles from each digital image. The 5 largest follicles from each image were arbitrarily chosen to avoid introducing variability, as this parameter was highly dependent on the size of follicular cross section.



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FIG. 1. Diagram demonstrating normal thyroid follicle structure with an indication of the morphological measurements taken from each thyroid follicle to calculate indices of thyroid function. Follicle dimensions were measured automatically from colloid cross section, identified automatically by image analysis software or from total follicle cross section based on a manual tracing around the basement membrane. The methods used for measuring Lf, Lc, Wf, and Wc and for the calculation of specific indices are described in the text.

 
Hepatic UDP-glucuronyltransferase activity.
A portion of the liver was thawed on ice and homogenized in 2.5 volume of cold 0.2M Tris containing 1.15% KCl (pH 7.4) with a teflon-glass homogenizer, to prepare a 40% (w/v) homogenate. Homogenates were centrifuged at 10,000 x g, 4°C for 20 min and the resulting postmitochondrial supernatant, also called S9 liver homogenate preparation, was recovered and used as UDP-glucuronyltransferase (UDP-GT) enzymatic source. The S9 homogenates were aliquoted and stored at –80°C until assayed. Protein concentration was determined by Bio-Rad Bradford protein assay (Bio-Rad, Hercules, CA) using bovine serum albumin (Fraction V, Sigma) as standard.

UDP-GT activity in liver S-9 was assayed by examining glucuronidation of either of 2 substrates; o-amino phenol (OAP; ICN Biomedicals, Aurora, OH) and 125I-T4; NEN (Life Science Products, Boston, MA) independently. To determine glucuronyltransferase activity towards T4, 2.5 µM L-T4 (Sigma Chemical, St. Louis, MO) and 125I-T4 (nominal specific activity of 1 µCi/nmol), were added to 33 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgCl2, 0.05% Brij-58 (Sigma), 1.4 mM saccharic acid-1,4-lactone (Sigma), 1 mM 6-n-propyl-2-thiouracil (Sigma), and 2 mg/ml of S-9 protein sample (adapted from Barter and Klaassen, 1992Go). All samples were incubated in duplicate in a final reaction volume of 0.2 ml in parallel with blank containing S-9 sample but no UDPGA. After a 5-min preincubation at 37°C, the reaction was initiated by addition of 3 mM UDPGA, allowed to progress for 30 min, and terminated by the addition of 0.2 ml of ice-cold methanol. Protein was pelleted by centrifugation and an aliquot (40 µl) of supernatant was applied to Whatman TLC silica-gel G plates (Fisher Scientific, Fair Lawn, NJ) and developed in ethyl acetate:methyl ethyl ketone:formic acid:water mixture (50:30:10:10) as previously described (McClain et al., 1989Go). The developed plates were air dried and analyzed by densitometric analysis, using ImageQuant software version 5.0, of image generated using a Storm 840 Phosphorimager (Molecular Dynamics, Sunnyvale, CA) after overnight exposure. The fraction of the total radioactivity in the form of T4-glucuronide was determined by comparison to the total radioactivity of 125I-T4 standards also spotted on the plates.

UDP-GT activity towards OAP was evaluated using a similar reaction mixture except that T4 (cold and labeled), saccharic acid-1,4-lactone and 6-n-propyl-2-thiouracil were excluded from the mixture and 0.14 mM OAP was added. The reaction was carried out, in triplicate, with 1 mg of S-9 protein in a final volume of 0.5 ml. The reaction was initiated by the addition of 1 mM UDPGA after a 5 min preincubation at 37°C and was terminated by the addition of ice cold precipitation mixture containing 1M trichloroacetic acid in 1M phosphate buffer (pH 2.1). After termination and centrifugation OAP glucuronide levels analyzed using a colorimetric method (Dutton and Story, 1962Go) and were quantitated by comparison to OAP glucuronide standards that were prepared from frozen stock solutions for each determination.

Hepatic outer-ring deiodinase activity.
Outer ring deiodinase activity was assayed in microsomes prepared from liver S9 diluted 1:10 with 0.2 M Tris-1.15 % KCl (pH = 7.4) homogenization buffer. Diluted S9 homogenates were centrifuged (Beckman L8-80 Ultracentrifuge; Ti-80 rotor, Beckman Coulter, Fullerton, CA) at 25,200 x g for 20 min to obtain the crude mitochondrial/lysosomsal pellet. The post mitochondrial/lysosomal supernatant was centrifuged at 110,000 x g for 67 min to obtain the microsomal pellet. The microsomal pellets were resuspended in 1 ml of homogenization buffer and stored at –20°C prior to deiodinase assay.

The deiodinase assay was based on a previously published procedure (Sanders et al., 1997Go). Deiodination was measured by the quantitation of radioiodide (125I) released by outer-ring deiodination (ORD) of [3`,5`-125I]-T4 (NEN Life Science Products, Boston, MA). Microsomal pellets were diluted 1:10 with 0.1 M sodium phosphate buffer (pH = 7.2) containing 2mM EDTA and 10 mM dithiothreitol to obtain a final microsomal protein concentration of roughly 0.1 mg/ml. Aliquots of 500 µl of the diluted microsomal fraction were preincubated at 37°C for 20 min. Control tubes received 500 µl of sodium phosphate buffer alone. The reaction was started by adding 20 µl of 125I-T4 (100,000 cpm; final concentration 10 nM) in 0.1 N NaOH. After 60 min, the reaction was stopped by the addition of 250 µl of 5% BSA followed by 1.25 µl of 10% trichloroacetic acid (TCA) to precipitate [125I]-labeled iodothyronines. Samples were vortexed and centrifuged at 3000 rpm for 5–10 min. A 250-µl aliquot of the supernatant was added to Sephadex LH-20 minicolumns containing 750 µl of 1.0 N HCl (Finnson and Eales, 1999Go). Columns were swirled, drained, and free radioiodide eluted with 3 ml of 0.1 N HCl. The radioiodide fraction was counted in a gamma counter (Packard System, Mississauga, Ontario, Canada). Deiodination rate was determined by subtracting the cpm of control tubes (containing total reaction mixture without microsomes) from cpm of experimental tubes (reaction mixture containing sample microsomes). Deiodination activity was expressed as fmol of T4 deiodinated.min-1.mg protein-1. Protein concentration was determined as described above.

Statistical analyses.
Animals were killed between 0900 and 1300 h on either of 2 consecutive days with the number of animals per treatment group balanced between days. Data that were expressed as proportions (e.g., percent of thyroid follicles with greater than 1000 µm2 cross sectional colloid area) were normalized by arcsine transformation prior to analysis by ANOVA. Unless otherwise indicated, all data were analyzed by two-way ANOVA with mixture dosage and day of necropsy being the 2 factors tested. Specific treatment effects were identified using Student Neuman Keuls post hoc test for multiple comparisons. All data sets were tested for homogeneity of variance and normal distribution and, where homoscedasticity or normality not indicated, data were retested after log transformation or, where either homoscedasticity or normality tests were not satisfied by transformation, reanalyzed using Krustal Wallace test of ranks followed by Dunn's test for multiple comparisons to test the effect of treatment only. The accepted level of significance was set at p <= 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effect of sacrificing animals on 2 days did introduce some variability to the data as there were statistically significant differences between days for several endpoints. However, these differences were generally small with questionable biological significance and, for the data reported in here, there was no interaction between day and treatment for any measure. The effects of treatment on animal body weight, major organ weights, and various immune system, reproductive, and other endpoints are reported elsewhere (Wade et al., 2002Go).

Circulating Hormones
Circulating T4 levels were significantly reduced only in response to 1000x treatment while T3 levels were increased in 100x and 1000x animals (Table 2Go). Stress to the hypothalamic-pituitary-thyroid (HPT) axis at lower doses was indicated by a dose-related increase in circulating TSH with significantly elevated levels seen in response to 1x mixture. Serum T3 uptake was also diminished by treatment with the mixture but this was significant only in 1000x animals. Other indices of thyroid hormone status, T4/TSH, free thyroid index (FTI; serum T4 x T3 uptake) and T3/T4 ratio were calculated from these data and, while these measures were influenced by the mixture, none were as sensitive an indicator of mixture effects as serum TSH. After TSH, the order of sensitivity was T4/TSH (LOEL = 10x) > T3 (100x) > T4 = T3 uptake = %T3/T4 = FTI (all 1000x).


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TABLE 2 Serum Hormone Levels at Necropsy for Male Rats Exposed to Complex Mixture of Persistent Environmental Contaminants for 70 Days
 
Thyroid Gland Histomorphology
Treatment with the highest dose of the mixture led to striking changes in thyroid gland morphology characterized by reduced amount of colloid within follicle cross sections, increased size and vacuolization of follicular epithelium, and a change in cross sectional shape from oval to highly convoluted (Fig. 2Go). The total number of follicles identified on the 2 digital images examined for each animal varied greatly between animals (median 96, range 49–164) due, at least in part, to the size of follicles. The effect of mixture on mean number of follicles per 2 images per was not statistically significant (p = 0.09) but showed a tendency towards increased follicle number in the highest dose group (Table 3Go). Other indices sensitive to follicle volume showed significant effect of mixture dose although sensitivity of these parameters varied considerably. The mean proportion of follicles whose individual cross-sectional area exceeded 1000 µm2 was significantly reduced in the highest dose group but not at lower doses (Table 3Go). Although the mean of this parameter for the 1x group was 57% larger than that of control animals, this difference was not statistically significant.



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FIG. 2. Photomicrographs of representative sections from a representative (A) control animal and (B) 1000x animal demonstrating the effects of subchronic mixture treatment. Control follicles are oval shaped containing colloid of uniform color surrounded by orderly cuboidal epithelium surrounded in turn by a basement membrane. Follicles in high dose animals are irregular in shape and enclosed by hypertrophied columnar epithelial cells containing many vacuoles in their cytoplasm. Colloid in the follicular lumen is denoted by "c." The large arrow indicates the follicle basement membrane and the small arrow indicates the thyroid epithelium. Bar = 20 µm.

 

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TABLE 3 The Effects of Mixture Exposure on Thyroid Gland Histomorphological Parameters
 
As follicle cross sectional area varied greatly between follicles and was not normally distributed within animals (the vast majority of follicles being much less than 500 µm2) a subset of the largest 23 follicles from each image, 46 follicles total per animal, was selected for parametric measures of follicle morphology. Analysis of median follicle cross sectional area identified a biphasic effect of mixture treatment with a significant increase in median area at the lowest dose and a subsequent decline with increasing dose such that median area was significantly lower than control in response to the highest dose. Both mean follicle roundness and aspect ratio were significantly increased in animals exposed to the 1000x dose, but did not differ from control in animals exposed to lower doses of the mixture (Table 3Go).

To evaluate the effects of the mixture on follicle epithelial cells both the height of the epithelial layer and the proportion of total follicle cross sectional area occupied by epithelium was determined for the 5 largest follicles from each image (10 per animal). The 5 largest were selected instead of 5 randomly chosen follicles to reduce the variability in the data that would be introduced by selecting follicles of variable size as epithelial to colloid ratio is very dependent on follicle size. Only the highest dose of the mixture caused a significant increase in both of these parameters relative to control.

Hepatic T4 Metabolism
To determine if the mixture influenced hepatic metabolism of thyroid hormones, the activities of hepatic T4 ORD, the enzyme involved in the conversion of T4 to the more physiologically active form, T3, and of UDP-GT, the enzyme system responsible for conjugation of T4 to the hormonally inactive T4-glucuronide. The activity of UDP-GT was determined using either OAP or radiolabeled T4 as the enzyme substrates. Activity towards OAP was evaluated to determine if the assay using this substrate, which is far less technically challenging than the assay using 125I-T4, is able to predict activity towards T4 as has been previously suggested (Masubuchi et al., 1997Go). UDP-GT activity towards OAP was significantly elevated in animals exposed to the 100x dose (184% of control) and further increased in the 1000x animals to a maximum level 588% of control activity (Fig. 3AGo). The glucuronidation of 125I-T4 by liver S-9 was much less efficient than glucuronidation of OAP with the rate of UDP-GT activity towards OAP per mg protein per min being greater than 11- and 26-fold higher than T4 glucuronidation activity in control and high dose groups, respectively (Fig. 3BGo). Although UDP-GT activity towards 125I-T4 was numerically increased in the 100x group (120% of control), this difference did not reach statistical significance.



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FIG. 3. The effects of subchronic mixture administration on UDP-glucuronyl transferase activity directed towards either (A) T4 or (B) o-aminophenol as determined in hepatic S9 preparation as described in the text. The data represent means ± SDs of enzyme activity determined for 9 (control) or 10 (all other treatments) animals per group. Means bearing the same letter are not significantly different (p < 0.05).

 
To determine if the mixture influenced hepatic conversion of T4 to the more active form of thyroid hormone, T3, the activity of ORD on T4 was assayed in liver microsomes. Treatment with the mixture caused a dose-related reduction in ORD activity with the lowest dose causing a significant reduction relative to control (Fig. 4Go). ORD activity was further reduced by increasing dose with activity in microsomes from animals exposed to the highest dose being less than 28% of activity in control microsomes.



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FIG. 4. The effects of subchronic mixture administration on T4 ORD activity determined in hepatic microsomes. The data represent means ± SDs of enzyme activity determined for 9 (control) or 10 (all other treatments) animals per group. Means bearing the same letter are not significantly different (p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The current study demonstrates that subchronic exposure to a complex mixture of persistent contaminants has adverse effects on thyroid hormone physiology in sexually mature male rats. Effects of the mixture were seen on all endpoints examined related to (1) thyroid gland morphology, (2) serum hormone levels, and (3) hepatic thyroid hormone metabolism. As indicated by 3 separate endpoints, thyroid homeostasis was significantly altered at the lowest dose of the mixture tested. This dose is based on promulgated estimates of levels of safe exposure for each individual component and is, therefore, below the lowest dose at which each substance has been shown to cause significant adverse effects in vivo. It should be noted, however, that few of the substances in the mixture have been tested for effects on any of the endpoints examined in the current study.

The human populations of the industrialized world are exposed to all or most of the components of the mixture (Anderson et al., 1998Go; Conacher and Mes, 1993Go; Davies and Mes, 1987Go; Foster et al., 1996Go; Frank et al., 1988Go, 1993Go; Mes and Malcolm, 1992Go; Newsome et al., 1995Go; Van Hove Holdrinet et al., 1977Go), primarily through contamination of food (Conacher and Mes, 1993Go; Davies, 1988Go; Davies and Mes, 1987Go). The daily rates of exposure of the vast majority of the population, for most components, are well below the doses used in the current study although little is known about the rates of human exposures to some of these (e.g. trichlorobenzenes; CEPA, 1993Go) as there has been little effort to document levels of these in human populations.

Although all endpoints of thyroid function were altered by exposure to some dose of the mixture, there were large differences in the sensitivity to insults on thyroid physiology. The 2 most sensitive endpoints, both being significantly altered at a dose of mixture 3 orders of magnitude below the dose causing a significant reduction in circulating T4 (1x vs. 1000x), were serum TSH and hepatic ORD activity. These are also the only 2 endpoints measured that have been shown to be directly responsive to thyroid hormone stimulation (Berry et al., 1990Go, 1991Go; Miyashita et al., 1995Go; Shupnik et al., 1986Go).

Hepatic ORD activity, ascribed to Type I 5`-deiodinase, has been shown to be directly regulated by thyroid hormone. Hypothyroid rats exhibit much lower hepatic ORD activity and lower Type I deiodinase gene expression while both enzyme activity and gene expression were greatly elevated in rats made hyperthyroid by daily injections of T3 or T4 (Berry et al., 1990Go, 1991Go; Miyashita et al., 1995Go). In addition, both ORD activity and Type I deiodinase mRNA are increased in parallel in rat liver cells in response to T3 treatment in vitro (Davies et al., 1996Go). Treatment of rats with some substances shown to reduce circulating T4 levels results in reduced hepatic ORD activity (Hood and Klaassen, 2000Go; Raasmaja et al., 1996Go). As such, the reduction in hepatic ORD activity in response to mixture treatment may be the result of the disruption of T3 signaling in hepatocytes by mixture components through some currently unknown mechanism.

Alternatively, mixture-related reduction in hepatic ORD activity could be due to specific inhibition of this enzyme by mixture components. Methoxychlor, the most prominent component, by weight, of the mixture, has been shown to be metabolized by hepatic microsomes into an intermediate that binds covalently to a single liver protein identified as Type I deiodinase (Zhou et al., 1995Go). Further, these investigators demonstrated that male rats exposed orally for 4 consecutive days to levels of methoxychlor several orders of magnitude higher than the dose received by 1x rats in our study, showed a moderate reduction in hepatic ORD activity. One could speculate that the reduction in ORD activity in the 1x animals could possibly be due to the much longer period of exposure of rats in the current study. In addition, coexposure with other mixture components could influence the efficiency of conversion of methoxychlor to its active metabolite as has been suggested (Bulger et al., 1983Go). Other mixture components have also been implicated in inhibition of hepatic ORD, including several PCB congeners or their hydroxyl metabolites (as cited in Brouwer et al., 1998Go) and lead or cadmium (Chaurasia et al., 1996Go; Gupta et al., 1997Go; Gupta and Kar, 1997Go, 1998Go). In the current study, only ORD enzyme activity, not gene expression, was measured. Consequently, it is unclear whether the change in activity was due to reduced ORD expression, due to a reduction in the thyroid hormone signal perceived by the liver cells, through some unknown mechanism, or due to mixture components directly impairing the function of ORD protein. As ORD is responsible for local conversion of the major circulating form of thyroid hormone (T4) into the more bioactive T3, the consequences of direct inhibition of this enzyme in the absence of any change in circulating T4 (as seen in response to the 1, 10, and 100x mixtures) would be the reduction of bioactive thyroid hormone available to hepatocytes. However, serum T3 level was increased at the 2 highest doses of mixture that may mean that any shortfall in T3 to hepatic tissue due to reduced local conversion from T4 may be at least partially alleviated. The fact that circulating T3 was increased in spite of a reduction hepatic ORD supports the observation that hepatic outer ring deiodination is less important than the thyroid gland as a source of serum T3 (Chanoine et al., 1993Go). One could speculate that increased serum T3 may be a consequence of increased circulating TSH as Type I deiodinase has been shown to be induced in the thyroid gland by TSH (Wu et al., 1985Go). The activity of ORD in tissues other than liver was not determined in this study, making mixture effects on ORD in nonhepatic tissues entirely speculative. However, if the reduction in hepatic ORD activity is due to covalent binding of a methoxychlor metabolite to ORD, as previously suggested (Bulger et al., 1983Go; Zhou et al., 1995Go), it is unlikely that the mixture would cause similar reductions in activity and, hence, local T3 availability, in other tissues as the levels of cytochrome P450-mediated xenobiotic biotransforming activity is dramatically lower or nonexistent in tissues other than liver.

It has been well established that TSH secretion by the pituitary is inversely related to thyroid hormone levels that serve as the basis for feedback control of circulating thyroid hormone. The synthesis of TSH protein subunits and TSH secretion are strongly suppressed by T3 but are upregulated in the absence of T3 (Shupnik et al., 1986Go). As such, increased levels of circulating TSH are likely due to reduced thyroid hormone signal perceived by the pituitary gland. It is puzzling, therefore, that TSH levels are elevated at doses of mixture well below those causing significant reduction in circulating T4. As neither circulating T4 or T3 levels were significantly altered in 1x animals it is tempting to speculate that the mechanism underlying the increase in circulating TSH relates either to reduced conversion of T4 to T3 in the pituitary or hypothalamic nuclei controlling pituitary thyrotrophes, or to an inhibition of signaling through the pituitary thyroid hormone receptor. Currently, there are no xenobiotics known to inhibit thyroid hormone receptor molecules (Cheek et al., 1999Go; DeVito et al., 1999Go) although not all components of the mixture have been tested for this activity. It is also possible that the mixture inhibited local conversion of T4 to T3 in the pituitary and hypothalamus, thereby reducing the local amount of available T3 and subsequently increasing TSH secretion. The enzyme responsible for this conversion in pituitary and brain, Type II deiodinase, is distinct from the hepatic isoform in that it is encoded by a distinct gene whose expression and enzyme activity are upregulated in response to hypothyroidism (Escobar-Morreale et al., 1997Go) and developmental exposure to some toxicants (Morse et al., 1996Go). The effects of mixture components on the activity or expression of this enzyme are entirely unstudied, so any suggestions that increased TSH production by the pituitary is secondary to reduced local conversion of T3 to T4 are speculative. It is also possible that the thyrotoxic mode of activity of the mixture at low doses did involve reduced circulating T4 but that the apparent euthyroid levels of T4 are an artifact of compensatory recovery after long term exposure, as has been shown for other thyroid toxicants (e.g., Hooth et al., 2001Go). We can only conclude that the lowest dose of the mixture caused increased circulating TSH by some as yet unknown mechanism.

The lowest dose of mixture also caused a significant increase in median thyroid follicle colloid cross sectional area. The fact that the mixture had a biphasic effect on this parameter, as cross sectional area in the highest dose animals being significantly reduced compared to control, is interesting although the significance of this finding is unclear. This increase in lumen area at the low dose is associated with a roughly 50% increase in circulating TSH, which could be expected to increase the secretory activity of thyroid epithelium. This involves both secretion of colloid material into the follicle lumen and the removal of colloid material from the lumen to liberate thyroid hormone. The increase in follicle lumen area at the low dose may suggest that the initial increase may be due to increased stimulation of secretory activity by the low dose while increasing mixture exposure shifts the balance of these processes to favor colloid removal. However, as these increases in lumen area are not associated with any increase in circulating T4 or with any increase in hepatic T4 UDP-GT activity, which would indicate an increase in T4 clearance, the biological significance of the increase and subsequent decrease in colloid area is not clear.

The serum TSH and hepatic ORD data lead to speculation that antithyroid effects of the mixture are due to effects on hormone conversion in peripheral tissues or to interference with thyroid hormone action. However, most of the components of the mixture have previously been shown, when administered at much higher doses, to adversely affect thyroid hormone physiology in male rats by a variety of alternative mechanisms. These include the induction of hepatic T4-directed UDP-GT activity by TCDD, HCB, and PCBs (van Raaij et al., 1993; Visser et al., 1993Go), the displacement of T4 from serum carrier proteins by PCB, trichloro-, tetrachloro-, or hexachlorobenzene metabolites (Brouwer et al., 1990Go; van den Berg, 1990Go; van den Berg et al., 1991Go; Den Besten et al., 1991Go; van Raaij et al., 1993), inhibition of iodothyronine sulfotransferases by HCB, PCB, and dioxin metabolites (Schuur et al., 1998aGo,bGo), and effects on the thyroid gland epithelium structure and function by various organochlorines (Brouwer et al., 1998Go; Li and Hansen, 1997Go). Some literature does demonstrate inhibition of ORD by some mixture components including methoxychlor (Zhou et al., 1995Go), several PCB congeners or their hydroxyl metabolites (as cited in Brouwer et al., 1998Go), and lead or cadmium (Chaurasia et al., 1996Go; Gupta et al., 1997Go; Gupta and Kar, 1997Go, 1998Go), as discussed above, supporting the contention that this may be the mechanism leading to reduced ORD activity and alteration of thyroid physiology. The activity of UGT on T4 is clearly elevated by the mixture but this effect is only significant in animals exposed to 1000x dose. Induction of this activity and the subsequent increased metabolic clearance of circulating T4 has been suggested as being the most significant mechanism by which PCBs, dioxins, and other xenobiotics induce toxicity to thyroid physiology (Barter and Klaassen, 1994Go; Visser et al., 1993Go). The vast majority of thyroid physiology-related endpoints assessed in the current study, including circulating T4, were significantly altered only in animals with significantly elevated hepatic UGT, which may indicate that the stress due to increased removal of T4 from circulation stressed the HPT axis beyond its capacity to compensate. Regardless of the etiology of thyroid toxicity, it is clear that frank pathology, as indicated by the severe hypertrophy of the thyroid epithelium and reduction in circulating T4, is only apparent in animals exposed to the 1000x dose.

It is interesting to note that hepatic microsomal UGT-T4 activity has been observed to double in response to subchronic exposure to daily doses of TCDD (26 ng/kg/day) alone (Van Birgelen et al., 1995Go) that are almost 2 orders of magnitude below the daily TCDD exposure contained in the mixture shown to significantly increase this parameter in the current study. The reason for this discrepancy between this finding and the results of the current study is not clear, although possible explanations could be that mixture components may have blocked TCDD action on this parameter in the current study or that differences in tissue preparation (homogenate supernatant in the current study vs. microsomal preparation) may have resulted in a difference in activity. This latter explanation is unlikely as the ratio of UGT-OAP activity in high dose versus control in the current study was similar if assayed using homogenate supernatant or microsomes (data not shown).

The significance of thyroid toxicity for intact adult male rats is not clear. While the hypertrophy of the thyroid gland seen in high dose animals is likely to be precancerous lesions (Capen, 1992Go, 1999Go), the slight elevation in circulating TSH levels and reduction of liver ORD activity seen in the low dose animals are of questionable significance to the long term health of sexually mature male animals. As thyroid hormones have a permissive influence on the regulation of anabolic metabolism, cellular differentiation, growth and development of organ systems, toxicity to the HPT axis would have the greatest consequences for fetal or juvenile animals in which cellular differentiation is occurring in many tissues through the body. As such processes do occur in a limited number of tissues in mature animals there is the possibility that, in cases of severe toxicity, some processes in adult male rats may be impaired. For example, severe thyroid disruption in mature animals has been shown to impair recovery from toxicant-induced Leydig cell depletion as differentiation of Leydig cells from interstitial stem cells is impaired (Ariyaratne et al., 2000Go). In addition, thyroidectomy or severe toxicant-induced hypothyroidism impairs the ability of adult rats to thermoregulate (Potter et al., 1986Go; Zaninovich et al., 2000Go) and synthesize fatty acids (Freake et al., 1989Go). However, thyroid toxicity would have far more significant consequences for fetal or neonatal rats due to the critical role thyroid hormone plays in permitting the development of the central nervous system (Koibuchi and Chin, 2000Go; Porterfield, 1994Go; Porterfield and Hendrich, 1993Go) and reproductive tract (Cooke et al., 1991Go, 1992Go). Experiments examining the effects of prenatal exposure of pregnant rats to a very similar mixture on gestational days 9 through 15 resulted in a significant increase in the circulating T4 levels in adult male, but not female pups (Wade et al., unpublished) suggesting effects of the mixture on the development of the HPT axis and that the effects on TSH and ORD seen in the current study may be predictive of effects of in utero exposure.

The results from the current study imply that MRLs or no effect levels, the criteria used in selecting doses for each component of the mixture, may not be sufficiently stringent to protect against subtle toxicant effects on the HPT axis. However, it is important to note that promulgated MRLs are not static and are periodically altered to reflect advances in understanding of the toxicity of substances. Further, the effects observed in 1x animals, unlike the frank toxicity evident by histopathological changes in the thyroid gland in 1000x animals, could be described as adaptive responses to toxicant treatment. Consequently, it is not clear that any of these measures would be identified as a critical effect for risk assessment. As the highest dose (1000x) also caused significant systemic toxicity (Wade et al., 2002Go), altered thyroid status may not be the effect that has the greatest significance for health. However, it will be important to examine the degree to which changes in these sensitive parameters in adult male rats can predict thyroid related developmental effects in order to determine the significance of thyroid toxicity of this mixture. As the components of the mixture were selected based on the known contaminants in human tissues, the relationship between the levels of exposure used in the current study and the levels to which the general human population are exposed is relevant to public health.


    ACKNOWLEDGMENTS
 
This study was supported in part by a grant from the Canadian Chemical Producers association through the Canadian Chlorine Coordinating Committee and by the Great Lakes Research Program of Health Canada. The authors gratefully acknowledge the technical assistance of Lorraine Casavant.


    NOTES
 
1 To whom correspondence should be addressed at EHC-315, PL#0803D, Growth & Development Section, Environmental Health Directorate, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0L2. Fax: (613) 946-2600. E-mail: mike_wade{at}hc-sc.gc.ca. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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