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

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

*Growth and Development Section, Environmental and Occupational Toxicology Section, Bureau of Chemical Hazards, Health Canada, EnvironmentalHealth Centre, Tunney’s Pasture, Ottawa, Ontario, Canada;†INRS, Institute Armand Frappier, 245 Boulevard Hymus, Pointe-Claire, Que´bec, Canada;

‡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

The human population in the industrialized world is ubiqui-tously exposed to complex mixtures of persistent pollutants thatcontaminate food, water, and air. A large number of these con-taminants have been shown to cause significant toxicity to thehypothalamic-pituitary-thyroid (HPT) axis in laboratory animalstudies, through a variety of mechanisms, although these effectsoccur at levels of exposure greatly in excess of common humanexposure. While many of the mechanisms of thyroid toxicity ofthese substances are potentially complementary, little is known ofthe degree of interaction of common persistent contaminants onresponses of the HPT axis. To investigate the potential effects of acomplex, environmentally relevant mixture on the HPT axis, sex-ually mature male rats were administered a mixture of 16 commonorganochlorines (dichlorodiphenoxytrichloroethane [DDT], p,p*-dichlorodiphenoxydichloroethylene [p,p’-DDE], hexachloroben-zene [HCB], tetrachlorodibenzo-p-dioxin [TCDD], polychlori-nated biphenyls [PCBs], methoxychlor, endosulfan, heptachlor,hexachlorocyclohexane, dieldrin, aldrin, mirex, and several chlo-rinated benzenes, and metal contaminants [lead, cadmium]). Thedoses of the mixture that were administered were related to min-imum risk levels or tolerable daily intakes of these substances, asderived by risk assessment with the 13, 103, 1003, and 10003groups receiving mixture components at doses equivalent to 13,103, 1003, or 10003 the minimum risk level (or tolerable dailyintake, reference dose), respectively. After 70 daily treatments bygavage, endpoints related to circulating thyroid hormone (serumthyroxine [T4], triiodothyronine [T3], thyroid stimulating hormone[TSH], and serum T3 uptake [T3-up]), thyroid gland histomor-phology (thyroid follicle cross sectional area, epithelial height,follicle roundness or aspect ratio, colloid/epithelial ratio) and he-patic metabolism of thyroid hormone (UDP-glucuronyl trans-ferase [UGT] and outer-ring deiodinase [ORD]) were assessed. Allexamined endpoints were significantly altered by the mixturealbeit with great variability between endpoints in the sensitivity.While most endpoints examined did not show significant changesat mixture doses below 10003, 2 endpoints, TSH and hepatic

outer ring deiodinase activity, were significantly increased anddecreased, respectively, by 13 dose and showed dose-related in-creases in severity with increasing dose. Median thyroid folliclecross sectional area was also increased by the lowest dose of themixture but decreased with subsequent increases in dose until, atthe highest dose, this parameter was significantly reduced relativeto control. The relative sensitivity of endpoints of thyroid functionin detecting toxicity of the mixture was TSH 5 ORD 5 medianfollicle area @ T3 > all other endpoints. These results demonstratethat low doses of ubiquitous environmental contaminants can alterHPT physiology in sexually mature males.

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

Humans interact with their environments on a daily basisand, as a consequence, are exposed to a broad spectrum ofsynthesized chemicals present in the food they eat, the air theybreathe, and the water they drink. A variety of synthetic halo-genated organic compounds, such as dichlorodiphenoxytri-chloroethane (DDT) and its persistent metabolitep,p9-dichlo-rodiphenoxydichloroethylene (p,p’-DDE), hexachlorobenzene(HCB), dioxins, furans, polychlorinated biphenyls (PCBs),other organochlorine pesticides, and metals, such as lead andcadmium, have been released into the environment throughhuman activities and are routinely found as contaminants intissues collected from the human population in Canada and inother nations throughout the world (Davies and Mes, 1987;Franket al., 1988; Meset al., 1990; Mes, 1992; Newsomeetal., 1995; Szymczynski and Waliszewski, 1981a,b). While thelevels of exposure for the vast majority of the population arebelow doses shown to cause adverse effects in laboratorystudies examining the effects of single chemicals, the conse-quences of low level exposure to a wide variety of substancesare not known and are the subject of considerable debate(Germolecet al., 1989; Pohlet al., 1997; Porteret al., 1999).

For noncancer risk assessments, estimates of safe exposure(minimum risk level, acceptable or tolerable daily intake, or

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

TOXICOLOGICAL SCIENCES67, 207–218 (2002)Copyright © 2002 by the Society of Toxicology

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reference dose) for a given substance are routinely derived, bya variety of public health organizations, from detailed exami-nation of evidence of toxicity from exposure to that substance(Barnes and Dourson, 1988). Ideally, this process involves theidentification of a no observable adverse effect level(NOAEL), the highest dose of exposure that is without toxiceffect on the most sensitive endpoint examined, which is thendivided by a safety factor to estimate a daily rate of exposurethat is predicted not to cause adverse effects. While this pro-cess may be appropriate for estimating risks of exposure toindividual compounds it does not attempt to estimate alter-ations in potency due to coexposure to other substances. Veryfew studies have attempted to assess the degree of hazardposed by simultaneous exposures to reference doses of multi-ple chemicals (Itoet al., 1995a,b).

There is currently a great deal of scientific and public inter-est in the possibility that many of these persistent environmen-tal contaminants and other anthropogenic substances may becausing alterations in the function of endocrine systems,thereby disrupting developmental and physiological functionin animals and humans. Most of this attention has been directedto investigating effects related to sex steroid physiology al-though there is a growing body of evidence showing that thereare many common, persistent substances that are potent dis-rupters of thyroid homeostasis (Brouweret al., 1998). Whilethese have been reported to cause reductions of circulatingthyroid hormones or histopathological lesions to the thyroidgland, the doses required to induce these effects in experimen-tal animals are well above doses to which the human popula-tion are generally exposed. However, the mechanisms of actionby which these substances interfere with thyroid physiologyare diverse and potentially complementary. Coexposure tomultiple substances, which have been shown to have differentmechanisms of action in inducing thyroid disruption, is areality in the human populations throughout the industrializedworld. As such, the possibility that the cumulative effects ofthe mixture could have greater impact in inducing thyroiddysfunction than would occur from exposure to comparabledoses of the individual components alone has significant publichealth implications. The current study examines the effects ofsubchronic daily exposure to a complex mixture of ubiquitouspersistent contaminants on thyroid hormone physiology andmetabolism in adult male rats.

MATERIALS AND METHODS

Mixture formulation. The formulation of the mixture and selection ofdose levels is described elsewhere (Wadeet al., 2002). Briefly, the contami-nant mixture was formulated to include some of the persistent organic andinorganic contaminants to which the human population of Canada couldconceivably be exposed. The dose levels of each component in the mixturereflect the currently promulgated safe levels of exposure as published by theATSDR (minimum risk level; MRL), Canadian Environmental Protection ActChemical Assessments (tolerable daily intake; TDI), U.S. EPA (referencedose; RfD), or at the lowest NOEL available in the scientific literature (Table

1). The dosing solutions provided a daily dose of each component equivalentto 13, 103, 1003, or 10003 their daily safe or no effect levels of exposure.

Animals. Sexually mature, male Sprague Dawley rats (49 days of age)were purchased from Charles River (St. Constant, Que´bec, Canada) andacclimated to holding facilities for 1 week prior to commencement of dosing.Animals were caged in pairs in clear plastic cages containing wood chips forbedding and maintained under controlled temperature (24°C), humidity (30–70%), and light (12:12 light:dark). All animals were provided standard labo-ratory rat chow (Labdiet 5001 rodent chow, Ralston Purina, St. Louis, MO)and waterad libitum. The protocol for animal experimentation detailing allanimal manipulations was scrutinized and approved by the McMaster Univer-sity Institutional Animal Care Committee prior to initiation of the study. Allanimal care and handling were in accordance with Canadian Council forAnimal Care guidelines.

Rats were randomly assigned to control (n 5 9), 13(10), 103(10),1003(10), or 10003(10) treatment groups. Rats received corn oil (vehicle) orappropriate treatment in a volume of 1ml/g body weight daily by gavage basedon the most recent body weight, which was recorded on Mondays, Wednes-days, or Fridays of every week. As reproductive endpoints were also examinedin this study, as described elsewhere (Wadeet al.,in press), rats were dosed for70 consecutive days, which is equivalent to 1 complete cycle of spermatogen-esis. On the day following the final dose, rats were anesthetized with isoflu-orane and, after recording terminal body weight, were sacrificed by exsangui-nation via cardiac puncture followed by decapitation. Blood was collected inSST Vacutainer tubes (Becton-Dickinson), held for no more than 4 h beforeserum was collected by centrifugation (30003 g for 15 min), aliquoted andfrozen at –80°C until analysis. Serum that was visibly red was not analyzedfurther as this indicated extensive haemolysis after blood collection. Commer-cially available RIA kits were used to assay serum content of thyroid stimu-lating hormone (TSH; rat-specific, Cat. No. RPA5541, Amersham Pharmacia,Piscataway, NJ), thyroxine, triiodothyronine, and serum triiodothyronine up-take (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, werefrozen 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 freeof connective tissue and fixed in PBS containing 4% paraformaldehyde for 3to 4 days until all samples were simultaneously embedding in paraffin using anautomated histoprocessor.

Thyroid gland histomorphology. Sections of thyroid gland, 5mm thick,were cut parallel to the longitudinal axis of the trachea and stained withPeriodic Acid Schiff. Areas photographed for analysis were from areas show-ing as few artifacts as possible, had little nonfollicle tissue, were clearly notadjacent to the preiphery of the gland, and were immediately adjacent to theparathyroid gland. The latter 2 criteria are to avoid including measurementstaken from follicles on the surface of the gland as these tend to be much lessactive than those within the gland (Low, 1982). Histomorphological parame-ters were quantified for follicle cross sections in 2 digital images, eachequivalent to an area of 6253 410 mm, from 2 separate histological sectionsper animal for 5 animals per treatment using ImagePro software (release 4.0,Media Cybernetics, Silver Spring, MD). All images were captured and allmeasures of thyroid gland morphology were collected from 2 digital imagesfrom each animal by a single investigator (S. P.), who was unaware ofspecimen treatment.

Agents that disrupt thyroid physiology cause an increase in the thickness ofthyroid follicle epithelium and reduce the volume of follicle colloid and overallfollicle diameter indicating toxicity to thyroid gland (Capen, 1999). Theseeffects were estimated using 5 separate parameters calculated from severalbasic measures of follicle morphology graphically depicted in Figure 1. Peri-odic Acid Schiff stains the noncellular central colloid of thyroid follicles anintense purple stain that can be identified and selected using a color-recogni-tion feature of the digital image analysis software. Once selected, the crosssectional area of follicular colloid and length of the longest and shortest axes(Lc and Wc, respectively, in Fig. 1) are automatically calculated for every

208 WADE ET AL.

complete follicle cross section visible in a digital image. As hyperactivefollicles tend to collapse as the intrafollicular colloid is exhausted, the round-ness and aspect ratio were calculated as [(colloid perimeter)2/4p(colloid area)]and [Lc/Wc], respectively. The mean roundness and aspect ratio attempted toestimate the degree to which the follicle cross section deviated from thenormal, round shape. For example, a perfect circle would receive a value of 1with either parameter, and the value would increase as the perimeter becameless circular and more convoluted. As height of thyroid epithelial cells isinfluenced in animals that are challenged with agents that disrupt thyroidhormone homoeostasis, the height and relative proportion of total follicle crosssectional area of follicle epithelium were estimated. Total follicle area wasidentified for the 5 largest follicles that had no evidence of any artifactualdistortion in each image by manually tracing the basement membrane. Epi-thelial height was estimated as [(Lf – Lc) 1 (Wf – Wc)]/4. In an attempt tocapture the relative changes in epithelial height and volume of the colloid in asingle 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 avoidintroducing variability, as this parameter was highly dependent on the size offollicular cross section.

Hepatic UDP-glucuronyltransferase activity. A portion of the liver wasthawed on ice and homogenized in 2.5 volume of cold 0.2M Tris containing1.15% KCl (pH 7.4) with a teflon-glass homogenizer, to prepare a 40% (w/v)homogenate. Homogenates were centrifuged at 10,0003 g, 4°C for 20 minand the resulting postmitochondrial supernatant, also called S9 liver homog-enate preparation, was recovered and used as UDP-glucuronyltransferase(UDP-GT) enzymatic source. The S9 homogenates were aliquoted and storedat –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 ofeither of 2 substrates;o-amino phenol (OAP; ICN Biomedicals, Aurora, OH)and 125I-T4; NEN (Life Science Products, Boston, MA) independently. Todetermine glucuronyltransferase activity towards T4, 2.5 mM L-T 4 (SigmaChemical, St. Louis, MO) and125I-T4 (nominal specific activity of 1mCi/nmol), were added to 33 mM Tris-HCl buffer (pH 7.4) containing 10 mMMgCl2, 0.05% Brij-58 (Sigma), 1.4 mM saccharic acid-1,4-lactone (Sigma), 1mM 6-n-propyl-2-thiouracil (Sigma), and 2 mg/ml of S-9 protein sample(adapted from Barter and Klaassen, 1992). All samples were incubated induplicate in a final reaction volume of 0.2 ml in parallel with blank containingS-9 sample but no UDPGA. After a 5-min preincubation at 37°C, the reactionwas 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 waspelleted by centrifugation and an aliquot (40ml) of supernatant was applied toWhatman TLC silica-gel G plates (Fisher Scientific, Fair Lawn, NJ) anddeveloped in ethyl acetate:methyl ethyl ketone:formic acid:water mixture(50:30:10:10) as previously described (McClainet al., 1989). The developedplates were air dried and analyzed by densitometric analysis, using Image-Quant software version 5.0, of image generated using a Storm 840 Phos-phorimager (Molecular Dynamics, Sunnyvale, CA) after overnight exposure.The fraction of the total radioactivity in the form of T4-glucuronide wasdetermined by comparison to the total radioactivity of125I-T4 standards alsospotted on the plates.

UDP-GT activity towards OAP was evaluated using a similar reactionmixture except that T4 (cold and labeled), saccharic acid-1,4-lactone and6-n-propyl-2-thiouracil were excluded from the mixture and 0.14 mM OAP

TABLE 1Composition of Dosing Mixtures

Chemical Supplier Purity (%) 13 MRL/RfD/TDI a

Aldrin Accustandard 98.6 30 ngb

p,p’-DDT Sigma 98 30 ngb,c

p,p’-DDE Sigma 99 570 ngb,c

Dieldrin Sigma 97 50 ngb

Endosulfan Accustandard 99.6 50 ngb

Heptachlor Polysciences Corp. Technical 0.5mgd

Hexachlorobenzene Aldrich 991 0.3mgb

Hexachlorocyclohexane Polysciences Corp. Technical 0.3mgb

Mirex Gift e 98 0.8mgb

Methoxychlor Aldrich 95 2mgb

1,2,3-Trichlorobenzene Aldrich 97 0.77mgf

1,2,4-Trichlorobenzene Aldrich 99 2.3mgf

1,2,3,4-Tetrachlorobenzene Aldrich 98 0.2mgf

Pentachlorobenzene Aldrich 98 0.5mgf

TCDD Accustandard 99 1 ngg

PCB (as Arochlor 1254) Accustandard Technical 1mgh

Cadmium chloride Fisher 991 0.7mgb

Lead chloride Fisher 991 0.1 ngd

a13 dose is equivalent to the estimate dose, in mass/kg body weight/day, of daily exposure that will not cause adverse health effects in humans or experimentalNOAEL. Where MRL for chronic exposure differed from MRL for intermediate exposure (as for HCB) the intermediate value was used.

bMinimum risk level (ATSDR).cMRL for DDT and DDE includes all isomers and metabolites of DDT: Relative levels of DDT and DDE in mixture reflect relative concentrations in serum

of Canadian women (Fosteret al., 1996).dRfD 5 reference dose (EPA).eGift from Dr. L. S. Kaminsky, Dept. of Health, Albany, NY to Dr. I. Chu, Health Canada.fTDI 5 tolerable daily intake (CEPA).gNOAEL 5 no observable adverse effect level (Feeley and Grant, 1993).hPTDI 5 provisional tolerable daily intake (Health Canada; Grant, 1983).

209THYROID EFFECTS OF PERSISTENT POLLUTANT MIXTURE

was added. The reaction was carried out, in triplicate, with 1 mg of S-9 proteinin a final volume of 0.5 ml. The reaction was initiated by the addition of 1 mMUDPGA after a 5 min preincubation at 37°C and was terminated by theaddition of ice cold precipitation mixture containing 1M trichloroacetic acid in1M phosphate buffer (pH 2.1). After termination and centrifugation OAPglucuronide levels analyzed using a colorimetric method (Dutton and Story,1962) and were quantitated by comparison to OAP glucuronide standards thatwere prepared from frozen stock solutions for each determination.

Hepatic outer-ring deiodinase activity. Outer ring deiodinase activity wasassayed in microsomes prepared from liver S9 diluted 1:10 with 0.2 MTris-1.15 % KCl (pH5 7.4) homogenization buffer. Diluted S9 homogenateswere centrifuged (Beckman L8-80 Ultracentrifuge; Ti-80 rotor, BeckmanCoulter, Fullerton, CA) at 25,2003 g for 20 min to obtain the crude mito-chondrial/lysosomsal pellet. The post mitochondrial/lysosomal supernatantwas centrifuged at 110,0003 g for 67 min to obtain the microsomal pellet. Themicrosomal pellets were resuspended in 1 ml of homogenization buffer andstored at –20°C prior to deiodinase assay.

The deiodinase assay was based on a previously published procedure (Sand-erset al.,1997). Deiodination was measured by the quantitation of radioiodide(125I) released by outer-ring deiodination (ORD) of [39,59-125I]-T 4 (NEN LifeScience Products, Boston, MA). Microsomal pellets were diluted 1:10 with 0.1M sodium phosphate buffer (pH5 7.2) containing 2mM EDTA and 10 mMdithiothreitol to obtain a final microsomal protein concentration of roughly 0.1mg/ml. Aliquots of 500ml of the diluted microsomal fraction were preincu-bated at 37°C for 20 min. Control tubes received 500ml of sodium phosphatebuffer alone. The reaction was started by adding 20ml of 125I-T4 (100,000 cpm;

final concentration 10 nM) in 0.1 N NaOH. After 60 min, the reaction wasstopped by the addition of 250ml of 5% BSA followed by 1.25ml of 10%trichloroacetic acid (TCA) to precipitate [125I]-labeled iodothyronines. Sampleswere vortexed and centrifuged at 3000 rpm for 5–10 min. A 250-ml aliquot ofthe supernatant was added to Sephadex LH-20 minicolumns containing 750mlof 1.0 N HCl (Finnson and Eales, 1999). Columns were swirled, drained, andfree radioiodide eluted with 3 ml of 0.1 N HCl. The radioiodide fraction wascounted 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 experi-mental tubes (reaction mixture containing sample microsomes). Deiodinationactivity was expressed as fmol of T4 deiodinated.min–1.mg protein–1. Proteinconcentration was determined as described above.

Statistical analyses. Animals were killed between 0900 and 1300 h oneither of 2 consecutive days with the number of animals per treatment groupbalanced between days. Data that were expressed as proportions (e.g., percentof thyroid follicles with greater than 1000mm2 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 withmixture dosage and day of necropsy being the 2 factors tested. Specifictreatment effects were identified using Student Neuman Keuls post hoc test formultiple comparisons. All data sets were tested for homogeneity of varianceand normal distribution and, where homoscedasticity or normality not indi-cated, data were retested after log transformation or, where either homosce-dasticity or normality tests were not satisfied by transformation, reanalyzedusing Krustal Wallace test of ranks followed by Dunn’s test for multiplecomparisons to test the effect of treatment only. The accepted level of signif-icance was set atp # 0.05.

RESULTS

The effect of sacrificing animals on 2 days did introducesome variability to the data as there were statistically signifi-cant differences between days for several endpoints. However,these differences were generally small with questionable bio-logical significance and, for the data reported in here, there wasno interaction between day and treatment for any measure. Theeffects of treatment on animal body weight, major organweights, and various immune system, reproductive, and otherendpoints are reported elsewhere (Wadeet al., 2002).

Circulating Hormones

Circulating T4 levels were significantly reduced only inresponse to 10003 treatment while T3 levels were increasedin 1003 and 10003 animals (Table 2). Stress to the hypo-thalamic-pituitary-thyroid (HPT) axis at lower doses was indi-cated by a dose-related increase in circulating TSH with sig-nificantly elevated levels seen in response to 13 mixture.Serum T3 uptake was also diminished by treatment with themixture but this was significant only in 10003 animals. Otherindices of thyroid hormone status, T4/TSH, free thyroid index(FTI; serum T4 3 T3 uptake) and T3/T4 ratio were calculatedfrom these data and, while these measures were influenced bythe mixture, none were as sensitive an indicator of mixtureeffects as serum TSH. After TSH, the order of sensitivity wasT4/TSH (LOEL 5 103) . T3 (1003) . T4 5 T3 uptake5%T3/T4 5 FTI (all 10003).

FIG. 1. Diagram demonstrating normal thyroid follicle structure with anindication of the morphological measurements taken from each thyroid follicleto calculate indices of thyroid function. Follicle dimensions were measuredautomatically from colloid cross section, identified automatically by imageanalysis software or from total follicle cross section based on a manual tracingaround 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.

210 WADE ET AL.

Thyroid Gland Histomorphology

Treatment with the highest dose of the mixture led to strik-ing changes in thyroid gland morphology characterized byreduced amount of colloid within follicle cross sections, in-

creased size and vacuolization of follicular epithelium, and achange in cross sectional shape from oval to highly convoluted(Fig. 2). The total number of follicles identified on the 2 digitalimages examined for each animal varied greatly between ani-mals (median 96, range 49–164) due, at least in part, to the size

TABLE 2Serum Hormone Levels at Necropsy for Male Rats Exposed to Complex Mixture

of Persistent Environmental Contaminants for 70 Days

Hormone measurement Control 13 103 1003 10003 ANOVA p

TSH (ng/ml) 8.416 1.55a 12.136 6.20b 11.946 2.81b 16.796 2.77c 21.126 8.00c , 0.001Total T4 (mg/dl) 3.986 0.28b,c 4.306 0.64b,c 3.776 0.41b 4.476 0.39c 3.016 0.32a , 0.001Total T3 (ng/dl) 61.126 11.31a 63.286 9.22a 65.546 8.81a,b 77.906 11.94b 97.466 12.28c , 0.001T4/TSH 0.4896 0.103c 0.4136 0.182b,c 0.3426 0.132b 0.2726 0.047b 0.1706 0.081a , 0.001%T3/T4 0.1556 0.035a 0.1356 0.055a 0.1756 0.026a 0.1756 0.027a 0.3256 0.040b , 0.001Serum T3 uptake (%) 55.496 2.18b 55.456 2.68b 54.576 4.46b 53.256 4.00b 48.846 3.35a 0.004FTI (mg/dl) 2.216 0.20b,c 2.386 0.38c 2.066 0.26b 2.386 0.24c 1.476 0.21a , 0.001

Note.Values are mean6 SD of measurements fromn samples. For control group,n 5 9; for all other groups,n 5 10. FTI was calculated as the product oftotal T4 and serum T3 uptake.

a,b,cMeans with the same superscript are not significantly different (p . 0.05).

FIG. 2. Photomicrographs of representative sections from a representative (A) control animal and (B) 10003 animal demonstrating the effects of subchronicmixture treatment. Control follicles are oval shaped containing colloid of uniform color surrounded by orderly cuboidal epithelium surrounded in turn by abasement membrane. Follicles in high dose animals are irregular in shape and enclosed by hypertrophied columnar epithelial cells containing many vacuoles intheir cytoplasm. Colloid in the follicular lumen is denoted by “c.” The large arrow indicates the follicle basement membrane and the small arrow indicates thethyroid epithelium. Bar5 20 mm.

211THYROID EFFECTS OF PERSISTENT POLLUTANT MIXTURE

of follicles. The effect of mixture on mean number of folliclesper 2 images per was not statistically significant (p 5 0.09) butshowed a tendency towards increased follicle number in thehighest dose group (Table 3). Other indices sensitive to folliclevolume showed significant effect of mixture dose althoughsensitivity of these parameters varied considerably. The meanproportion of follicles whose individual cross-sectional areaexceeded 1000mm2 was significantly reduced in the highest dosegroup but not at lower doses (Table 3). Although the mean of thisparameter for the 13 group was 57% larger than that of controlanimals, this difference was not statistically significant.

As follicle cross sectional area varied greatly between fol-licles and was not normally distributed within animals (the vastmajority of follicles being much less than 500mm2) a subset ofthe largest 23 follicles from each image, 46 follicles total peranimal, was selected for parametric measures of follicle mor-phology. Analysis of median follicle cross sectional area iden-tified a biphasic effect of mixture treatment with a significantincrease in median area at the lowest dose and a subsequentdecline with increasing dose such that median area was signif-icantly lower than control in response to the highest dose. Bothmean follicle roundness and aspect ratio were significantlyincreased in animals exposed to the 10003 dose, but did notdiffer from control in animals exposed to lower doses of themixture (Table 3).

To evaluate the effects of the mixture on follicle epithelialcells both the height of the epithelial layer and the proportionof total follicle cross sectional area occupied by epithelium wasdetermined for the 5 largest follicles from each image (10 peranimal). The 5 largest were selected instead of 5 randomlychosen follicles to reduce the variability in the data that wouldbe introduced by selecting follicles of variable size as epithelialto colloid ratio is very dependent on follicle size. Only thehighest dose of the mixture caused a significant increase inboth of these parameters relative to control.

Hepatic T4 Metabolism

To determine if the mixture influenced hepatic metabolismof thyroid hormones, the activities of hepatic T4 ORD, theenzyme involved in the conversion of T4 to the more physio-logically active form, T3, and of UDP-GT, the enzyme systemresponsible for conjugation of T4 to the hormonally inactiveT4-glucuronide. The activity of UDP-GT was determined usingeither OAP or radiolabeled T4 as the enzyme substrates. Ac-tivity towards OAP was evaluated to determine if the assayusing this substrate, which is far less technically challengingthan the assay using125I-T4, is able to predict activity towardsT4 as has been previously suggested (Masubuchiet al.,1997).UDP-GT activity towards OAP was significantly elevated inanimals exposed to the 1003 dose (184% of control) andfurther increased in the 10003 animals to a maximum level588% of control activity (Fig. 3A). The glucuronidation of125I-T4 by liver S-9 was much less efficient than glucuronida-tion of OAP with the rate of UDP-GT activity towards OAPper mg protein per min being greater than 11- and 26-foldhigher than T4 glucuronidation activity in control and highdose groups, respectively (Fig. 3B). Although UDP-GT activ-ity towards 125I-T4 was numerically increased in the 1003group (120% of control), this difference did not reach statisti-cal significance.

To determine if the mixture influenced hepatic conversion ofT4 to the more active form of thyroid hormone, T3, the activityof ORD on T4 was assayed in liver microsomes. Treatmentwith the mixture caused a dose-related reduction in ORDactivity with the lowest dose causing a significant reductionrelative to control (Fig. 4). ORD activity was further reducedby increasing dose with activity in microsomes from animalsexposed to the highest dose being less than 28% of activity incontrol microsomes.

TABLE 3The Effects of Mixture Exposure on Thyroid Gland Histomorphological Parameters

Endpoint Control 13 103 1003 10003 ANOVA p

Mean no. of follicles 95.46 14.9 84.46 31.5 76.46 11.4 88.66 18.5 116.26 27.8 0.09% Follicles. 1000mm 22.66 9.5b 35.66 19.6b 28.36 9.2b 23.36 4.4b 6.06 5.6a , 0.0001Median area (mm2) 14226 287b 22236 549c 14436 342b 14836 112b 6206 236a , 0.0001Roundness3 1.296 0.12a 1.296 0.10a 1.226 0.05a 1.326 0.07a 1.816 0.31b 0.013Aspect ratio3 1.606 0.17a 1.496 0.09a 1.546 0.06a 1.566 0.18a 1.966 0.25b 0.0015Epithelium/colloid4 1.316 0.29a,b 1.026 0.08a 1.436 0.47a,b 1.716 0.38b 3.866 0.86c , 0.0001Epithelial height4 10.26 1.3a,b 9.56 1.0a 10.86 1.6a,b 12.26 1.0b 14.86 0.4c , 0.0001

Note.Mean number of follicles, per 2 images; % Follicles. 1000mm, determined for the measurements of all complete thyroid follicles in 2 images peranimal. Value reported is the mean6 SD of 5 animals per treatment group. Median area (mm2), values reported are mean6 SD of the median follicle size forthe largest 26 follicles from 2 images per animal (52 follicles per animal) of 5 animals per treatment group. Roundness and aspect ratio, values reported representthe mean6 SD of mean roundness estimated from 5 animals per treatment group. Mean values for individual animals were calculated from measurements ofthe largest 26 follicles from 2 images per animal. Epithelium/colloid and epithelial height, values reported represent the mean6 SD of the mean measures fromfollicle cross sections from 5 animals per treatment group. Mean values for individual animals were calculated for the 5 largest follicles per image from 2 imagesper animal.

a,b,cMeans with the same superscript are not significantly different (p . 0.05).

212 WADE ET AL.

DISCUSSION

The current study demonstrates that subchronic exposure toa complex mixture of persistent contaminants has adverseeffects on thyroid hormone physiology in sexually mature malerats. Effects of the mixture were seen on all endpoints exam-ined related to (1) thyroid gland morphology, (2) serum hor-mone levels, and (3) hepatic thyroid hormone metabolism. Asindicated by 3 separate endpoints, thyroid homeostasis wassignificantly altered at the lowest dose of the mixture tested.This dose is based on promulgated estimates of levels of safeexposure for each individual component and is, therefore,below the lowest dose at which each substance has been shownto cause significant adverse effectsin vivo. It should be noted,however, that few of the substances in the mixture have beentested for effects on any of the endpoints examined in thecurrent study.

The human populations of the industrialized world are ex-posed to all or most of the components of the mixture (Ander-son et al., 1998; Conacher and Mes, 1993; Davies and Mes,1987; Fosteret al., 1996; Franket al., 1988, 1993; Mes andMalcolm, 1992; Newsomeet al.,1995; Van Hove Holdrinetet

al., 1977), primarily through contamination of food (Conacherand Mes, 1993; Davies, 1988; Davies and Mes, 1987). Thedaily rates of exposure of the vast majority of the population,for most components, are well below the doses used in thecurrent study although little is known about the rates of humanexposures to some of these (e.g. trichlorobenzenes; CEPA,1993) as there has been little effort to document levels of thesein human populations.

Although all endpoints of thyroid function were altered byexposure to some dose of the mixture, there were large differ-ences in the sensitivity to insults on thyroid physiology. The 2most sensitive endpoints, both being significantly altered at adose of mixture 3 orders of magnitude below the dose causinga significant reduction in circulating T4 (13 vs. 10003), wereserum TSH and hepatic ORD activity. These are also the only2 endpoints measured that have been shown to be directlyresponsive to thyroid hormone stimulation (Berryet al.,1990,1991; Miyashitaet al., 1995; Shupniket al., 1986).

Hepatic ORD activity, ascribed to Type I 59-deiodinase, hasbeen shown to be directly regulated by thyroid hormone.Hypothyroid rats exhibit much lower hepatic ORD activity andlower Type I deiodinase gene expression while both enzymeactivity and gene expression were greatly elevated in rats madehyperthyroid by daily injections of T3 or T4 (Berryet al.,1990,1991; Miyashitaet al., 1995). In addition, both ORD activityand Type I deiodinase mRNA are increased in parallel in ratliver cells in response to T3 treatmentin vitro (Davieset al.,1996). Treatment of rats with some substances shown to reducecirculating T4 levels results in reduced hepatic ORD activity(Hood and Klaassen, 2000; Raasmajaet al., 1996). As such,

FIG. 4. The effects of subchronic mixture administration on T4 ORDactivity determined in hepatic microsomes. The data represent means6 SDsof 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).

FIG. 3. The effects of subchronic mixture administration on UDP-glucu-ronyl transferase activity directed towards either (A) T4 or (B) o-aminophenolas determined in hepatic S9 preparation as described in the text. The datarepresent means6 SDs of enzyme activity determined for 9 (control) or 10 (allother treatments) animals per group. Means bearing the same letter are notsignificantly different (p , 0.05).

213THYROID EFFECTS OF PERSISTENT POLLUTANT MIXTURE

the reduction in hepatic ORD activity in response to mixturetreatment may be the result of the disruption of T3 signaling inhepatocytes by mixture components through some currentlyunknown mechanism.

Alternatively, mixture-related reduction in hepatic ORD ac-tivity could be due to specific inhibition of this enzyme bymixture components. Methoxychlor, the most prominent com-ponent, by weight, of the mixture, has been shown to bemetabolized by hepatic microsomes into an intermediate thatbinds covalently to a single liver protein identified as Type Ideiodinase (Zhouet al., 1995). Further, these investigatorsdemonstrated that male rats exposed orally for 4 consecutivedays to levels of methoxychlor several orders of magnitudehigher than the dose received by 13 rats in our study, showeda moderate reduction in hepatic ORD activity. One couldspeculate that the reduction in ORD activity in the 13 animalscould possibly be due to the much longer period of exposure ofrats in the current study. In addition, coexposure with othermixture components could influence the efficiency of conver-sion of methoxychlor to its active metabolite as has beensuggested (Bulgeret al., 1983). Other mixture componentshave also been implicated in inhibition of hepatic ORD, in-cluding several PCB congeners or their hydroxyl metabolites(as cited in Brouweret al.,1998) and lead or cadmium (Chaur-asia et al., 1996; Guptaet al., 1997; Gupta and Kar, 1997,1998). In the current study, only ORD enzyme activity, notgene expression, was measured. Consequently, it is unclearwhether the change in activity was due to reduced ORD ex-pression, due to a reduction in the thyroid hormone signalperceived by the liver cells, through some unknown mecha-nism, or due to mixture components directly impairing thefunction of ORD protein. As ORD is responsible for localconversion of the major circulating form of thyroid hormone(T4) into the more bioactive T3, the consequences of directinhibition of this enzyme in the absence of any change incirculating T4 (as seen in response to the 1, 10, and 1003mixtures) would be the reduction of bioactive thyroid hormoneavailable to hepatocytes. However, serum T3 level was in-creased at the 2 highest doses of mixture that may mean thatany shortfall in T3 to hepatic tissue due to reduced localconversion from T4 may be at least partially alleviated. Thefact that circulating T3 was increased in spite of a reductionhepatic ORD supports the observation that hepatic outer ringdeiodination is less important than the thyroid gland as a sourceof serum T3 (Chanoineet al., 1993). One could speculate thatincreased serum T3 may be a consequence of increased circu-lating TSH as Type I deiodinase has been shown to be inducedin the thyroid gland by TSH (Wuet al.,1985). The activity ofORD in tissues other than liver was not determined in thisstudy, making mixture effects on ORD in nonhepatic tissuesentirely speculative. However, if the reduction in hepatic ORDactivity is due to covalent binding of a methoxychlor metabo-lite to ORD, as previously suggested (Bulgeret al.,1983; Zhouet al.,1995), it is unlikely that the mixture would cause similar

reductions in activity and, hence, local T3 availability, in othertissues as the levels of cytochrome P450-mediated xenobioticbiotransforming activity is dramatically lower or nonexistent intissues other than liver.

It has been well established that TSH secretion by thepituitary is inversely related to thyroid hormone levels thatserve as the basis for feedback control of circulating thyroidhormone. The synthesis of TSH protein subunits and TSHsecretion are strongly suppressed by T3 but are upregulated inthe absence of T3 (Shupniket al., 1986). As such, increasedlevels of circulating TSH are likely due to reduced thyroidhormone signal perceived by the pituitary gland. It is puzzling,therefore, that TSH levels are elevated at doses of mixture wellbelow those causing significant reduction in circulating T4. Asneither circulating T4 or T3 levels were significantly altered in13 animals it is tempting to speculate that the mechanismunderlying the increase in circulating TSH relates either toreduced conversion of T4 to T3 in the pituitary or hypothalamicnuclei controlling pituitary thyrotrophes, or to an inhibition ofsignaling through the pituitary thyroid hormone receptor. Cur-rently, there are no xenobiotics known to inhibit thyroid hor-mone receptor molecules (Cheeket al., 1999; DeVitoet al.,1999) although not all components of the mixture have beentested for this activity. It is also possible that the mixtureinhibited local conversion of T4 to T3 in the pituitary andhypothalamus, thereby reducing the local amount of availableT3 and subsequently increasing TSH secretion. The enzymeresponsible for this conversion in pituitary and brain, Type IIdeiodinase, is distinct from the hepatic isoform in that it isencoded by a distinct gene whose expression and enzymeactivity are upregulated in response to hypothyroidism (Esco-bar-Morrealeet al.,1997) and developmental exposure to sometoxicants (Morseet al., 1996). The effects of mixture compo-nents on the activity or expression of this enzyme are entirelyunstudied, so any suggestions that increased TSH productionby the pituitary is secondary to reduced local conversion of T3

to T4 are speculative. It is also possible that the thyrotoxicmode of activity of the mixture at low doses did involvereduced circulating T4 but that the apparent euthyroid levels ofT4 are an artifact of compensatory recovery after long termexposure, as has been shown for other thyroid toxicants (e.g.,Hoothet al.,2001). We can only conclude that the lowest doseof the mixture caused increased circulating TSH by some as yetunknown mechanism.

The lowest dose of mixture also caused a significant increasein median thyroid follicle colloid cross sectional area. The factthat the mixture had a biphasic effect on this parameter, ascross sectional area in the highest dose animals being signifi-cantly reduced compared to control, is interesting although thesignificance of this finding is unclear. This increase in lumenarea at the low dose is associated with a roughly 50% increasein circulating TSH, which could be expected to increase thesecretory activity of thyroid epithelium. This involves bothsecretion of colloid material into the follicle lumen and the

214 WADE ET AL.

removal of colloid material from the lumen to liberate thyroidhormone. The increase in follicle lumen area at the low dosemay suggest that the initial increase may be due to increasedstimulation of secretory activity by the low dose while increas-ing mixture exposure shifts the balance of these processes tofavor colloid removal. However, as these increases in lumenarea are not associated with any increase in circulating T4 orwith any increase in hepatic T4 UDP-GT activity, which wouldindicate an increase in T4 clearance, the biological significanceof the increase and subsequent decrease in colloid area is notclear.

The serum TSH and hepatic ORD data lead to speculationthat antithyroid effects of the mixture are due to effects onhormone conversion in peripheral tissues or to interferencewith thyroid hormone action. However, most of the compo-nents of the mixture have previously been shown, when ad-ministered at much higher doses, to adversely affect thyroidhormone physiology in male rats by a variety of alternativemechanisms. These include the induction of hepatic T4-di-rected UDP-GT activity by TCDD, HCB, and PCBs (van Raaijet al., 1993; Visseret al., 1993), the displacement of T4 fromserum carrier proteins by PCB, trichloro-, tetrachloro-, or hexa-chlorobenzene metabolites (Brouweret al., 1990; van denBerg, 1990; van den Berget al.,1991; Den Bestenet al.,1991;van Raaijet al.,1993), inhibition of iodothyronine sulfotrans-ferases by HCB, PCB, and dioxin metabolites (Schuuret al.,1998a,b), and effects on the thyroid gland epithelium structureand function by various organochlorines (Brouweret al.,1998;Li and Hansen, 1997). Some literature does demonstrate inhi-bition of ORD by some mixture components including me-thoxychlor (Zhouet al.,1995), several PCB congeners or theirhydroxyl metabolites (as cited in Brouweret al., 1998), andlead or cadmium (Chaurasiaet al., 1996; Guptaet al., 1997;Gupta and Kar, 1997, 1998), as discussed above, supportingthe contention that this may be the mechanism leading toreduced ORD activity and alteration of thyroid physiology.The activity of UGT on T4 is clearly elevated by the mixturebut this effect is only significant in animals exposed to 10003dose. Induction of this activity and the subsequent increasedmetabolic clearance of circulating T4 has been suggested asbeing the most significant mechanism by which PCBs, dioxins,and other xenobiotics induce toxicity to thyroid physiology(Barter and Klaassen, 1994; Visseret al., 1993). The vastmajority of thyroid physiology-related endpoints assessed inthe current study, including circulating T4, were significantlyaltered only in animals with significantly elevated hepaticUGT, which may indicate that the stress due to increasedremoval of T4 from circulation stressed the HPT axis beyondits capacity to compensate. Regardless of the etiology of thy-roid toxicity, it is clear that frank pathology, as indicated by thesevere hypertrophy of the thyroid epithelium and reduction incirculating T4, is only apparent in animals exposed to the10003 dose.

It is interesting to note that hepatic microsomal UGT-T4

activity has been observed to double in response to subchronicexposure to daily doses of TCDD (26 ng/kg/day) alone (VanBirgelen et al., 1995) that are almost 2 orders of magnitudebelow the daily TCDD exposure contained in the mixtureshown to significantly increase this parameter in the currentstudy. The reason for this discrepancy between this finding andthe results of the current study is not clear, although possibleexplanations could be that mixture components may haveblocked TCDD action on this parameter in the current study orthat differences in tissue preparation (homogenate supernatantin the current study vs. microsomal preparation) may haveresulted in a difference in activity. This latter explanation isunlikely as the ratio of UGT-OAP activity in high dose versuscontrol in the current study was similar if assayed using ho-mogenate supernatant or microsomes (data not shown).

The significance of thyroid toxicity for intact adult male ratsis not clear. While the hypertrophy of the thyroid gland seen inhigh dose animals is likely to be precancerous lesions (Capen,1992, 1999), the slight elevation in circulating TSH levels andreduction of liver ORD activity seen in the low dose animalsare of questionable significance to the long term health ofsexually mature male animals. As thyroid hormones have apermissive influence on the regulation of anabolic metabolism,cellular differentiation, growth and development of organ sys-tems, toxicity to the HPT axis would have the greatest conse-quences for fetal or juvenile animals in which cellular differ-entiation is occurring in many tissues through the body. Assuch processes do occur in a limited number of tissues inmature animals there is the possibility that, in cases of severetoxicity, some processes in adult male rats may be impaired.For example, severe thyroid disruption in mature animals hasbeen shown to impair recovery from toxicant-induced Leydigcell depletion as differentiation of Leydig cells from interstitialstem cells is impaired (Ariyaratneet al., 2000). In addition,thyroidectomy or severe toxicant-induced hypothyroidism im-pairs the ability of adult rats to thermoregulate (Potteret al.,1986; Zaninovichet al., 2000) and synthesize fatty acids(Freakeet al.,1989). However, thyroid toxicity would have farmore significant consequences for fetal or neonatal rats due tothe critical role thyroid hormone plays in permitting the devel-opment of the central nervous system (Koibuchi and Chin,2000; Porterfield, 1994; Porterfield and Hendrich, 1993) andreproductive tract (Cookeet al., 1991, 1992). Experimentsexamining the effects of prenatal exposure of pregnant rats toa very similar mixture on gestational days 9 through 15 re-sulted in a significant increase in the circulating T4 levels inadult male, but not female pups (Wadeet al., unpublished)suggesting effects of the mixture on the development of theHPT axis and that the effects on TSH and ORD seen in thecurrent study may be predictive of effects ofin uteroexposure.

The results from the current study imply that MRLs or noeffect levels, the criteria used in selecting doses for eachcomponent of the mixture, may not be sufficiently stringent toprotect against subtle toxicant effects on the HPT axis. How-

215THYROID EFFECTS OF PERSISTENT POLLUTANT MIXTURE

ever, it is important to note that promulgated MRLs are notstatic and are periodically altered to reflect advances in under-standing of the toxicity of substances. Further, the effectsobserved in 13 animals, unlike the frank toxicity evident byhistopathological changes in the thyroid gland in 10003 ani-mals, could be described as adaptive responses to toxicanttreatment. Consequently, it is not clear that any of these mea-sures would be identified as a critical effect for risk assessment.As the highest dose (10003) also caused significant systemictoxicity (Wade et al., 2002), altered thyroid status may notbe the effect that has the greatest significance for health.However, it will be important to examine the degree to whichchanges in these sensitive parameters in adult male rats canpredict thyroid related developmental effects in order to deter-mine the significance of thyroid toxicity of this mixture. As thecomponents of the mixture were selected based on the knowncontaminants in human tissues, the relationship between thelevels of exposure used in the current study and the levels towhich the general human population are exposed is relevant topublic health.

ACKNOWLEDGMENTS

This study was supported in part by a grant from the Canadian ChemicalProducers association through the Canadian Chlorine Coordinating Committeeand by the Great Lakes Research Program of Health Canada. The authorsgratefully acknowledge the technical assistance of Lorraine Casavant.

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