Assessment of DE-71, a Commercial Polybrominated Diphenyl Ether (PBDE) Mixture, in the EDSP Male and Female Pubertal Protocols

Tammy E. Stoker*,1,2, Susan C. Laws*,1, Kevin M. Crofton{dagger}, Joan M. Hedge{dagger}, Janet M. Ferrell* and Ralph L. Cooper*

* Reproductive Toxicology Division, {dagger} Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

2 To whom correspondence should be addressed at Reproductive Toxicology Division, MD-72, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-5138. E-mail: stoker.tammy{at}epa.gov.

Received August 4, 2003; accepted October 29, 2003

ABSTRACT

DE-71, a commercial mixture, was used to test the sensitivity of the female and male pubertal protocol to detect thyroid active chemicals. These protocols are being evaluated for the U.S. EPA's Endocrine Disruptor Screening Program as part of a Tier I Screening Battery. To examine the ability of these protocols to screen for chemicals that induce the clearance of thyroid hormone, we examined male and female Wistar rats following DE-71 exposure. Rats were gavaged daily with 0, 3, 30, or 60 mg/kg DE in corn oil from postnatal day (PND) 23-53 in the male or PND 22-41 in the female. The temporal effects of DE-71 on liver enzymes and thyroid hormones were measured in another group of males and females following only 5 days of dosing (PND 21 to 26 in females and PND 23 to 28 in males). Serum T4 was significantly decreased at 30 and 60 mg/kg following the 5-day exposures and in the 21-day exposed females. Doses of 3, 30, and 60 mg/kg decreased T4 in 31-day exposed males. Serum T3 was decreased and TSH elevated by 30 and 60 mg/kg in the 31-day exposed males only. Decreased colloid area and increased follicular cell heights (indicative of the hypothyroid state) were observed in thyroids of the 60 mg/kg groups of 20- and 31-day exposed female and males. Increased liver-to-body weight ratios coincided with a significant induction of uridinediphosphate-glucuronosyltransferase (UDGPT; two to four-fold), and ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD) at the two highest doses in all exposures. Of the androgen dependent tissues in the 31-day exposed males, seminal vesicle (SV) and ventral prostate (VP) weights were reduced at 60 mg/kg, while testes and epididymal weights were not affected. Preputial separation (PPS) was also significantly delayed by doses of 30 and 60 mg/kg. In the female, the 60 mg/kg dose also caused a significant delay in the age of vaginal opening. Based upon the thyroid hormone response data, this study provides evidence that the 31-day alternative Tier 1 male protocol is a more sensitive test protocol than the 5-day or female pubertal protocol for thyrotoxic agents that act via up-regulation of hepatic metabolism. This apparent greater sensitivity may be due a greater body burden attained following the longer dosing regimen as compared with that of the female protocol, or to gender specific differences in thyroid hormone metabolism. Also, the delay in PPS and reduction in SV and VP weights may indicate a modification or inhibition of endogenous androgenic stimulation directly by DE-71 or a secondary effect that occurs in response to a DE-induced change in thyroid hormones.

Key Words: polybrominated diphenyl ethers; EDSP Tier I screen; pubertal development; thyroid hormones; hepatic enzyme activity.

The amendments to the Food Quality Protection Act of 1996 (FQPA) and the Safe Drinking Water Act of 1996 (SDWA) mandate that the EPA develop a screening and testing program to determine whether pesticides and drinking water contaminants "may have an effect in humans that is similar to an effect produced by a naturally-occurring estrogen, or other such endocrine effect as the Administrator may designate" (U.S. EPA EDSP Web site). EPA's Endocrine Disruptor Screening and Testing Advisory Committee (U.S. EPA, EDSTAC final report; www.epa.gov/scipoly/oscpendo/) determined that there was both a strong scientific basis and feasibility, considering time and resource constraints, to expand the scope of the screening program to include the androgen- and thyroid-hormone systems. Currently, the U.S. EPA is implementing the first phase of their Endocrine Disruptor Screening Program (EDSP), which includes the development and validation of in vitro and in vivo assays for use in the Tier 1 screening battery (T1S) for detecting chemicals with endocrine actions. Two protocols for the evaluation of pubertal development and thyroid function are currently being considered for inclusion in the T1S, the male and female pubertal protocols (Goldman et al., 2000Go; Stoker et al., 2000Go, www.epa.gov/scipoly/oscpendo/). These pubertal protocols were designed to detect alterations in pubertal development, thyroid function, and hypothalamic-pituitary-gonadal (HPG) system peripubertal maturation (see Fig. 1). Currently, relatively little is known about the efficacy of these protocols as screens to detect thyrotoxicants.



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FIG. 1. The timeline for the male and female pubertal protocols currently used in the U.S. EPA EDSP Tier 1 Screening Battery.

 
Thyroid hormone homeostasis is controlled by a sensitive feedback mechanism within the hypothalamic-pituitary-thyroid axis (Capen, 1997Go). Thyroid simulating hormone (TSH) induces the thyroid to synthesize thyroxine (T4), which is then 5'-mono-deiodinated to the more biologically active triiodothyronine (T3). The rate of TSH release is controlled by the amount of thyrotropin-releasing hormone (TRH) released by the hypothalamus, as well as by the circulating concentrations of T3 and T4. Thus, alterations in thyroid hormone homeostasis may result from disturbances in thyroid hormone synthesis, secretion, or catabolism (Capen, 1997Go). For example, disturbances may occur as a result of inhibition of iodine uptake or via increased thyroid hormone clearance. An alteration in thyroid hormone homeostasis can also occur as a result of induced hepatic microsomal enzymes or by an inhibition of 5'-deiodinase, the intracellular enzyme responsible for converting T4 to T3 (Capen, 1997Go; McClain et al., 1989Go; Masaki et al., 1984Go).

Previous research has suggested the utility of a short-term (4-day) in vivo exposure to weanling rats for detecting alterations in T4 following exposure to a number of polyhalogenated aromatic hydrocarbons (Craft, 2002Go; Zhou et al., 2001Go). Using this 4-day dose regimen, Zhou et al. (2001)Go recently showed that exposure to DE-71, a commercial mixture of polybrominated diphenyl ethers (PBDEs), induced hypothyroxinemia by increasing hepatic microsomal phase II enzyme uridine diphospho-glucuronosyl transferase (UDPGT) activity in Long-Evans female rats. T4 is conjugated with glucuronic acid in a reaction catalyzed by UDPGT before being excreted into the bile (Capen, 1997).

PBDE mixtures are synthesized in large quantities as flame retardants for commercial products such as electronic equipment and textiles (IPCS, 1994Go). Over 50,000 tons are produced globally each year (Sellstrom and Jansson, 1995Go). PBDEs have recently been detected in wildlife species and human milk and plasma samples (Andersson and Blomkvist, 1981Go; de Boer et al., 1998Go; Ohta et al., 2002Go; Sjodin et al., 1999Go) and appear to be increasing in concentrations (Meironyté et al., 1999Go; Sellstrom et al., 1993Go; She et al., 2002Go; Stern and Ikonomou, 2000Go), due to their persistance and bioaccumulation (de Wit, 2002Go). For these reasons, the potential health effects of such exposures are currently being investigated (Darnerud et al., 2001Go; Hooper and McDonald, 2000Go; McDonald, 2001Go). The predominant PBDEs found in wildlife and human samples are 2,2',4,4'-tetraBDE (IUPAC: BDE-47) and 2,2',4,4',5-pentaBDE (BDE-99; Kierkegaard et al., 1999Go; Lindstrom et al., 1999Go; Meironyté et al., 1999Go; Sellstrom et al., 1993Go; Sjodin, 2000Go; Sjodin et al., 1999Go; Strandman et al., 1999Go).

DE-71 is a commercial mixture which contains mostly tetra and penta congeners (Sjodin, 2000Go) and about 8000 tons are produced in the U.S. each year. Short-term exposure to DE-71 has been shown to induce hypothyroxinemia in both rats and mice (Darnerud and Sinjari, 1996Go; Fowles et al., 1994Go; IPCS, 1994Go; Zhou et al., 2001Go). A reduction in plasma thyroxine (T4) has been reported in young rodents (both rats and mice) following a 4- and 14-day exposure to this commercial PBDE. However, there was no effect on plasma TSH concentration (Fowles et al., 1994Go; Darnerud and Sinjari, 1996Go; Hallgren and Darnerud, 2002Go; Zhou et al., 2001Go). DE-71 induces both phase I (ethoxyresorufin-O-deethylase [EROD] and pentoxyresorufin-O-deethylase [PROD]) and phase II (uridinediphosphate-glucuronosyltransferase [UDPGT]) metabolic enzyme activities (Carlson, 1980aGo,bGo; Fowles et al., 1994Go; Hallgren and Darnerud, 2002Go; Zhou et al., 2001Go). Increased T4-glucuronidation by phase II UDPGT enzymes in liver (for review see Mackenzie et al., 1997Go) has been suggested as one of the primary mechanisms by which PBDEs deplete T4 and interfere with thyroid hormone homeostasis (Brouwer et al., 1998Go; Hallgren and Darnerud, 2002Go; Zhou et al., 2001Go).

The present study was conducted to determine the effectiveness of the EDSP Tier 1 male and female pubertal protocols as screens for thyroid active chemicals that act via the induction of hepatic microsomal enzymes, using DE-71 as the test chemical. In addition, we compared the dose-response relationships of the short-term exposure in the weanling rat (Zhou et al., 2001Go) to a longer (20- or 31-day) exposure to this thyrotoxicant.

MATERIALS AND METHODS

Animals.
Twenty timed-pregnant female Wistar rats were purchased from Charles River Laboratories (Raleigh, NC) and shipped to arrive on gestation day 13. Upon arrival the rats were housed one per cage in an AAALAC accredited facility maintained at 22°C and on a 14 h:10 h light:dark cycle (on 0800 h, off 2200 h). Food (Purina laboratory rat chow 5001) and water were provided ad libitum. The day of delivery was designated postnatal day (PND) 0. An overview of the male and female pubertal protocols and the 5-day exposures are shown on Figure 1. On PND 3, the pups were culled to 8 to 10 per litter to maximize uniformity in growth rates. On PND 21, all male and female pups were weaned and weighed to the nearest 0.1 g and weight ranked. Pups were then assigned so that treatment groups had similar body weight means and variances. Littermates were also equally distributed among the treatment groups, with only one pup from a given litter in each treatment group. After assignment, similarly treated males and females were housed two per cage and weighed daily throughout treatment. The female dosing started on PND 22 and the males on PND 23. A subset of the animals were killed for the 5-day exposures on PND 26 in females and PND 27 in males. The females and males for the pubertal protocols were killed on PND 41 (females) or PND 53 (males) for the protocol endpoint measures.

Treatment.
All treatments were administered daily by oral gavage for either 5 days in the short-term exposures or 20 or 31 consecutive days (from PND 23 to PND 53 in the male or PND 22 to 41 in the female) until the day of necropsy (see Fig. 1 for the necropsy timepoints) at a volume of 0.5 ml per 100 g body weight between 0800 and 0900 h. Body weights were recorded daily and the dose administered each day was adjusted for body weight. DE-71 (lot 7550OK20A) was generously supplied by the Great Lakes Chemical Corporation (West Lafayette, IN). The composition of the DE-71 lot contained 58.1% penta-BDE and 24.6% tetra-BDE (Sjodin, 2000Go). The dosing solution was prepared by mixing the compound with corn oil, sonicating for 30 min at 40°C, and diluting in series with corn oil to the desired concentrations of 3, 30, or 60 mg/kg. Doses were selected on the basis of the earlier weanling study (Zhou et al., 2001Go), which examined alterations of thyroid hormones. Control animals received corn oil only.

Pubertal Development
Preputial separation.
The separation of the foreskin of the penis from the glans penis, preputial separation (PPS), is an early reliable marker of the progression of puberty in male rats (Korenbrot et al., 1997Go). In the present study, PPS was monitored beginning on PND 33, until all males showed separation. All males were examined once daily at approximately the same time each day. A partial separation with a thread of cartilage remaining was recorded as "partial," but only the day of complete separation was used in the data analyses.

Vaginal opening.
Vaginal patency is estrogen dependent and generally indicative of the occurance of the first ovulation and the onset of estrous cyclicity in the rat. Throughout the dosing period, animals were examined at the same time daily for vaginal opening. The age at complete vaginal opening was recorded. Beginning on the day of vaginal opening, daily vaginal smears were collected and observed under a low-power light microscope for the presence of leukocytes, nucleated epithelial cells, or cornified epithelial cells, to determine the age of the first complete estrous cycle after vaginal opening. The vaginal smears were classified as diestrus (presence of luekocytes), proestrus (nucleated epithelial cells), or estrus (cornified epithelial cells) as characterized by Everett (1989)Go. Daily vaginal cytology was recorded for each female until the day of sacrifice.

Necropsy
The day before necropsy (PND 25 or 40 for females and PND 26 or 52 for males), the rats were placed in a holding room adjacent to the necropsy room. This room was maintained under the same lighting conditions as the animal room but the location allowed for each animal to be decapitated immediately (routinely less than 15 s after removal from their home cage) to minimize stress-induced changes in hormones. The time of necropsy was counterbalanced by dose groups.

Five-day exposures.
Following the 5-day exposures, approximately 2 h after the last dose of DE-71 (1000 h), the males and females were decapitated as described above. The blood was collected and the liver was removed and weighed. A portion of the liver was weighed and immediately frozen in liquid nitrogen and stored at -80°C until enzyme analysis.

Thirty-one-day exposure in males.
Following the male 31-day exposure, the males were decapitated approximately 2 h after the last dose of DE-71 (1000 h) and blood was collected. The pituitary, liver, kidneys, adrenals, testes, ventral and lateral prostates, epididymides, seminal vesicles with coagulating gland (with fluid) were removed and weighed. The epididymides, left testis, and thyroid (with the bracketing trachea) were removed, fixed in 10% neutral phosphate-buffered formalin for 24 h before transferring to 70% ethanol until later processing in paraffin for histology (hematoxylin and eosin or H&E stain) and pathology. Immediately following sacrifice, thyroid glands from these males were removed and fixed and stored in ethanol as described above until histological evaluation of transverse sections.

Twenty-day exposure in females.
Animals were killed by decapitation approximately 2 h after the last dose on PND 41. Tissue weights for each animal were recorded for liver, kidney, adrenals, ovaries, uterus, and pituitary. Immediately following sacrifice, the uteri, ovaries, and thyroid glands were removed, fixed and stained for histology as described above.

Further Analyses
Blood was allowed to clot for 1 h and then centrifuged at 1260 x g for 30 min; the serum collected and stored frozen at -80°C for subsequent hormone assays. In the adult males, anterior pituitaries were frozen on dry ice and stored at -80°C for subsequent hormonal analyses.

Radioimmunoassays.
Serum thyroxine (T4), tri-iodothyronine (T3), and thyroid stimulating hormone (TSH) radioimmunoassays were performed on the serum collected. Total T3 and T4 were measured using Coat-a-Count radioimmunoassay kits obtained from Diagnostic Products Corporation (Los Angeles, CA). In the Total T4 assay, some of the treated (30 and 60 mg/kg DE-71) samples were below the lowest standard of 1.0 µg/dl. For those samples, a lowest detectable limit of 1.0 µg/dl was used as the concentration used for analysis. In the adult males, serum testosterone (DPC, Los Ageles, CA) and serum and pituitary luteinizing hormone (LH) and prolactin (PRL) were measured by radioimmunoassay. The pituitary peptide RIAs were performed using the following materials supplied by the National Hormone and Pituitary Agency for LH, PRL, and TSH, respectively: iodination preparation I-9, I-6, I-9; reference preparation RP-3, RP-3, RP-3; and antisera S-11, S-9, S-6. Iodination material was radiolabeled with 125I (Dupont/New England Nuclear) by a modification of the chloramine-T method of Greenwood et al. (1963)Go. Labeled antigen was separated from unreacted iodide by gel filtration chromatography as described previously (Goldman et al., 1986). Sample serum and pituitary homogenate were pipetted with appropriate dilutions to a final assay volume of 500 µl with 100 mM phosphate buffer containing 1% bovine serum albumin (BSA). Standard reference preparations were serially diluted for the standard curves. Two hundred milliliters of primary antisera in 100 mM potassium phosphate, 76.8 mM EDTA, 1% BSA, and 3% normal rabbit serum (pH 7.4) were pipetted into each assay tube, vortexed, and incubated at 5°C for 24 h. One hundred milliliters of the iodinated hormone were then added to each tube, and the tube was vortexed and incubated for 24 h. A second antibody (Goat Anti-Rabbit Gamma Globulin at a dilution of 1 unit /100 µl; Calbiochem,) was then added, vortexed, and incubated for 24 h. The samples were centrifuged at 1260 x g for 30 min and the supernate aspirated and the sample tube, with pellet, was counted on a gamma counter. Intra assay coefficients of variation for the LH, PRL, and TSH assays were 1.1, 0.9, and 2.2%, respectively.

Hepatic enzyme activities assay.
All chemicals used in enzyme assays were purchased from Sigma Chemical Co. (St. Louis, MO) and were of the highest grade commercially available. Liver microsomal fractions were prepared as described previously (DeVito et al., 1993Go). Microsomal protein concentrations were determined using a protein assay kit (Bio-Rad, Richmond, CA) with bovine serum albumin as the standard. Hepatic microsomal EROD (a marker for CYP1A1 activity) and PROD (a marker of CYP2B activity) activities were assayed using the method of DeVito et al. (1993)Go. All substrate concentrations were 1.5 nM. Both EROD and PROD values were calculated as pmol resorufin per mg protein per min and graphically represented as a percentage of control activity.

Hepatic microsomal T4-UDPGT activity was assayed based on the method of Visser et al. (1993)Go as modified by Zhou et al. (2001)Go. 125I-labeled T4 was purchased from NEN Life Science Products Inc. (Boston, MA). Aliquots of liver microsomes were diluted to 2 mg protein per ml with 100 mM Tris-HCl (pH = 7.8) containing 5 mM MgCl2. Then, 100 µl of diluted microsomes were incubated without detergent at 37°C, with 50 µl of a purified 4 µM T4 stock containing 1 mM 125I-labeled T4 (around 50,000 dpm) as substrate, 1 mM unlabeled T4,100 mM 6-n-propyl-2-thiouracil (for preventing deiodination), and 20 mM UDPGA (as cofactor, or no UDPGA for blanks) over a 30-min period. The reaction was stopped by the addition of 200 µl ice-cold methanol, followed by centrifugation at 4°C at 1278 x g for 5 min. A 200-µl aliquot of supernatant was transferred to a microtube and mixed with 750 µl 0.1N HCl. A 50 µl aliquot from each microtube was collected for use as total counts for the assay and counted for 1 min on the gamma-counter. The remaining T4-glucuronyl product (T4-G) was then separated by chromatography on Supelco Filtration Columns filled with 2 ml of lipophilic sephadex LH-20 in gel suspension. This column was conditioned with 3 ml 0.1N HCl, which was then eluted with an additional 2 ml of 0.1N HCl which was collected and discarded. The column was further eluted with 8 ml of deionized water, and 1 ml of these collected fractions were counted for radioactivity for 1 min on the gamma-counter. The calculated UDPGT activity was expressed as pmol T4-G per mg protein per min and graphically represented as percentage of control group mean activity. The limit of detection for UDPGT was 0.05 pmol T4-G/mg protein.

Histology.
Following paraffin embedding, central 4–6 µm sections of the ovaries, uteri, testes, and epididymides were stained with hematoxylin and eosin (H&E) for pathological evaluation, which was performed by Veritas Laboritories (Burlington, NC). In addition, each thyroid gland (bracketing the trachea) was paraffin embedded and cut into 4–6 µm transverse sections. The central thyroid sections were stained with H&E and pathologically evaluated by Experimental Pathology Laboratories, Inc. (Reseach Triangle Park, NC). The follicular epithelial height and colloid areas were scored subjectively using a five-point grading scale (1 = shortest/smallest; 5 = tallest/largest, respectively). Any lesions were also noted in this thyroid evaluation.

Statistics.
All data were analyzed for age and treatment effects by ANOVA using the General Linear Model (GLM) procedures (SAS, version 8.1, SAS Institute, Inc., Cary, NC), and for homogeneity of variance using Bartlett's test (GraphPad InStat; GraphPad Software, San Diego, CA). When significant treatment effects (p < 0.05) were indicated by GLM, the Dunnett's and Duncan's t-tests were used to compare each treatment group with the control.

Benchmark doses.
The U.S. Environmental Protection Agency (USEPA, 1995Go) now uses benchmark doses (BMD; Barnes et al., 1995Go; Crump, 1984Go) in addition to the no-observed-adverse-effect-level (NOAEL) approach for non-cancer risk assessment. Therefore, in this study, we used Benchmark Dose Software (USEPA, 2000Go) to calculate the BMD5 and BMDL5 values for T4 concentrations following exposure to DE-71. The latter was defined as the lower 95% confidence limit of the administered dose predicted to cause a 5% increase in response (Allen et al., 1994Go). The percentage change in serum T4 concentrations was the only endpoint used to calculate the BMDs, which allowed for comparisons to be made between the male and female and the two exposure scenarios. Selection of a specific curve-fitting model for the BMD determination was based on the Akaike's Information Criterion (AIC) value. The AIC is -2L + 2p, where L is the log-likelihood at the maximum likelihood estimates for the parameters, and p is the number of model parameters estimated. The model with the lowest AIC value is presumed to be the most appropriate.

RESULTS
Weight and General Toxicity
No visible signs of toxicity were observed in any of the treated animals in this study. Liver-to-body weight ratios were increased following the 5-, 20-, and 31-day exposures to 30 and 60 mg/kg DE-71 in males and females (Table 1). However, no treatment-related changes in growth (data not shown) or necropsy body weight were observed in males or females (Tables 2 and 3).


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TABLE 1 Liver Weights following Exposure to DE-71

 

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TABLE 2 Male Pubertal Endpoints following a 31-Day Exposure to DE-71 in the Male

 

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TABLE 3 Female Reproductive Endpoints on PND 41 following a 20-Day Exposure to DE-71

 
Liver Enzymes
Five- and 31-day exposed males.
Increased hepatic enzymatic activities were observed following each exposure period to DE-71 in this study (Fig. 2). There was a significant UDGPT (uridinediphosphate-glucuronosyltransferase) induction following the 5-day exposure (349% of control) and 31-day exposure (208% of control) of 60 mg/kg/day (Fig. 2a). EROD (ethoxy-resorufin-O-deethylase) activity following the 5- and 31-day exposures was also significantly increased at both 30 and 60 mg/kg/day, with an increase of 1429 and 1442% of control following 5 days and 1027 and 1654% following 31 days (Fig. 2b). Similarly, PROD (pentoxy-resorufin-O-deethylase) activity was significantly increased at the 30 and 60 mg/kg/day exposure with an increase of 1436 and 2823% over controls following the 5-day exposure and 19,183 and 20,291% of control following the 31-day exposure (Fig. 2c).



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FIG. 2. The effect of 5- and 31-day exposures to DE-71 on liver microsomal enzyme activity in the male rats, represented as a percentage of control activity ± SEM. (a) UDGPT activity, (b) EROD activity, (c) PROD activity. *p < 0.05.

 
Five- and 20-day exposed females.
There was a significant UDGPT induction of 209 and 267% of control following the 5-day exposure and 254 and 290% following the 20-day exposure of both 30 and 60 mg/kg/day, respectively (Fig. 3a). Similarly, EROD activity following the 5- and 20-day exposures was also significantly increased at 30 and 60 mg/kg/day, with an increase of 488 and 1347% of control following the 5-day exposure and 1222 and 1624% of control following the 20-day exposure to both 30 and 60 mg/kg/day (Fig. 3b). PROD activity was also induced by the 30 and 60 mg/kg/day exposures to DE-71 at both time points (Fig. 3c). There was a 1033 and 1036% increase following the 5-day exposure and a 21,899 and 22,602% following the 20-day exposure.



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FIG. 3. The effect of 5- and 20-day exposures to DE-71 on liver microsomal enzyme activity in the female rats, represented as a percentage of control activity ± SEM. (a) UDGPT actitiy, (b) EROD activity, (c) PROD activity. *p < 0.05.

 
Pubertal Development and Reproductive Endpoints
   Females.
There was a significant delay (1.8 days) in the age of vaginal opening in the 60 mg/kg group in the females exposed to DE-71 from PND 22 to 41 (Fig. 4). Because there were no changes in body weight from the exposure, no evaluation of vaginal opening with body weight as a covariant was performed. Following vaginal opening, the females in all treatment groups showed regular estrous cyclicity until the necropsy day. There were no significant changes in pituitary, adrenal, kidney, ovary or uterine (wet or dry) weights in any of the treatment groups following the 20-day dosing period (Table 3).



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FIG. 4. The mean age of vaginal opening following post-weaning exposure to DE-71 in the female Wistar rat. *p < 0.05 as compared to control mean.

 
The two highest doses of DE-71, 30 and 60 mg/kg, significantly decreased serum T4 levels following both the 5- and 20-day exposure periods (Fig. 5). However, the serum T3 concentration was not different from controls at either time point (Fig. 6). Likewise, although there was a nonsignificant increase in the mean serum TSH in the highest treatment group, it was not significantly different from the control mean in this study (Fig. 7). As the females were sacrificed on different days of the estrous cycle (shown on Table 3), which would greatly increase the variability within treatment groups, no additional reproductive hormones were measured in the female.



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FIG. 5. The effect of 5- and 20-day exposures to DE-71 on mean total serum thyroxine (T4) concentrations in the female rat. *p < 0.05 as compared to control mean.

 


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FIG. 6. The effect of 5- and 20-day exposures to DE-71 on mean total serum tri-iodothyronine (T3) concentrations in the female rat. *p < 0.05 as compared to control mean.

 


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FIG. 7. The effect of 5- and 20-day exposures to DE-71 on mean serum TSH concentrations in the female rat. *p < 0.05 as compared to control mean.

 
   Males.
DE-71 exposure from PND 23 to 53 significantly delayed age of onset of preputial separation by 1.7 and 2.1 days in the 30 and 60 mg/kg groups, respectively, compared to the 31-day control males (Fig. 8). Lateral prostate, epididymal, and testicular weights were not different from the control in any of the 31-day DE-71 rats killed on PND 53 (Table 2). However, ventral prostate and seminal vesicle weights were significantly decreased following the 31-day exposure to 60 mg/kg DE-71 (Table 2). There was also a significant increase in the anterior pituitary weight in the 30 mg/kg group following 31 days of exposure, but this effect was not dose dependent.



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FIG. 8. The mean age of preputial separation following postweaning exposure to DE-71 in the male Wistar rat. *p < 0.05 as compared to control mean. **p < 0.01 as compared to control mean.

 
Serum T4 was decreased in the 30 and 60 mg/kg groups after 5 days of exposure (Fig. 9), but the 3 mg/kg T4 values were not significantly different than controls. After 31 days, T4 was significantly decreased in all three treatment groups. Serum T3 concentrations were similar to controls in all three dose groups following 5 days of exposure to DE-71 (Fig. 10). In contrast, the 31-day exposure resulted in a decrease in serum T3 at 30 and 60 mg/kg. Similarly, the 5-day exposure did not affect serum TSH levels at any dose of DE-71, while the 31-day exposure significantly increased serum TSH in a dose-related manner in the 30 and 60 mg/kg dose groups, with no effect at 3 mg/kg (Fig. 11). Finally, there was a two-fold increase in serum prolactin following the 31-day exposure in the highest dose groups, but no changes in serum LH, pituitary LH (pLH), or pituitary prolactin (pPRL) following the 31-day exposure in the males (Table 4).



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FIG. 9. The effect of 5- and 31-day exposures to DE-71 on mean total serum thyroxine (T4) concentrations in the male rat. *p < 0.05 as compared to control mean.

 


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FIG. 10. The effect of 5- and 31-day exposures to DE-71 on mean total serum tri-iodothyronine (T3) concentrations in the male rat. *p < 0.05 as compared to control mean.

 


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FIG. 11. The effect of 5- and 31-day exposures to DE-71 on mean serum TSH concentrations in the male rat. *p < 0.05 as compared to control mean.

 

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TABLE 4 Hormones in the Male on PND 53 following a 31-Day Exposure to DE-71

 
Histology
No significant differences or lesions were noted between the control and DE-71 treated PND 53 males in the sections of testes or epididymides following H&E staining. In addition, no differences were observed between the control and treated PND 41 female ovaries or uteri following H&E staining. However, in the microscopic evaluations of the H&E stained thyroid gland sections, there was an increase in follicular epithelial height and a decreased colloid area (colloid depletion) in the highest dose group of DE-71 in both the male and female rats following the 20- or 31-day exposures (Fig. 12). The follicular epithelial height score mean (1 = shortest; 5 = tallest) was significantly increased from 2.21 ± 0.19 in the control females to 3.73 ± 0.18 in the 60 mg/kg group scores (mean ± SEM, p < 0.01). For the male, the follicular height score mean was significantly increased from 2.43 ± 0.20 in the control males to 3.88 ± 0.29 in the 60 mg/kg group (p < 0.01). In the females, the colloid area score mean (1 = smallest; 5 = largest) was significantly decreased from 3.36 ± 0.29 in the control group to 2.60 ± 0.21 in the 60 mg/kg group (p < 0.05). Likewise, in the males, the colloid area was decreased from 3.57 ± 0.29 in the control group to 2.00 ± 0.33 in the highest dose group of DE-71. In addition, there was follicular degeneration in 13.3% of the female and 12.5% of the male thyroid sections from the 60 mg/kg groups. This degeneration was not observed in the control thyroid sections (Fig. 12).



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FIG. 12. Representative thyroid gland hematoxylin and eosin-stained sections from male rats dosed for 31 days with (a) control vehicle or (b) 60 mg/kg DE-71. Note the decreased colloid area and increase in follicular epithelial height in b as compared to (a). A thyroid section with a region of follicular degeneration is shown in (c) from a male dosed with 60 mg/kg DE-71. Each photo was taken with a 20x objective (total magnification 200x) and the bar denotes 100 µm.

 
BMD Results
NOELs and model estimates for BMD5s and BMDL5s for the serum T4 data are shown in Table 5. For the 31-day male exposure to DE-71, the dose of 3 mg/kg/day was determined to be the LOEL. Although in this study we did not reach a NOEL in the 31-day exposure in the male, the BMD5 estimate for T4 was 0.913 mg/kg/day. In the female 20-day exposure and in both 5-day exposures (i.e., male and female) to DE-71, the BMD5 estimate for T4 was lower than the 3 mg/kg NOEL for these three studies.


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TABLE 5 NOEL, BMD5, and BMDL5 Values for DE-71 Serum T4 Concentrations

 
DISCUSSION
In agreement with previous reports, the present study demonstrated that, in rats, pre- and peripubertal exposure to DE-71 will decrease thyroid hormones, increase hepatic CYP1A1 and CYP2B activity, and increase T4-UGT activity (Carlson, 1980aGo,bGo; Fowles et al., 1994Go; Hallgren and Darnerud, 1998; Zhou et al., 2001Go). More importantly, the results demonstrate that both the male and female pubertal protocols are able to detect environmental toxicants that act via this metabolic pathway. While the female pubertal protocol currently being evaluated for inclusion in the T1S Battery of the U.S. EPA EDSP requires 20 days of dosing, the male pubertal protocol, considered an alternative Tier 1 Test by EDSTAC requires 31 days of dosing. Although both of the pubertal protocols identified an effect of DE-71 on the thyroid hormone concentrations and thyroid histopathology, the dose required to bring about a significant change in these hormones in the male were clearly lower than that observed in the female suggesting that the male protocol may represent a more sensitive estimate of potential thyrotoxic agents. Whether this difference was the result of differences in the two sexes or duration of testing remains to be determined. It is clear from our current results that short-term (i.e., five days) exposure in the weanling male and female did not demonstrate a sex difference in response to DE-71 exposure in any of the thyroid hormones measured. The absence of a sex difference in these measures in the prepubertal animal is in agreement with previous reports (Craft et al., 2002Go; Fukuda et al., 1975Go; Kieffer et al., 1976Go). However, the male pubertal assay, employing a longer exposure period, did appear to enhance the ability of the assay to detect an effect at lower doses. Again, it was the male pubertal assay that appeared the most sensitive based upon the thyroid hormone data, with a LOEL of 3 mg/kg for serum T4 concentrations and the significant change in serum T3 and TSH concentrations following exposure to 30 and 60 mg/kg DE-71. The BMD5 calculations for T4 were a realistic estimate for the male results, but the 5-day and 20-day female estimates were below the NOELs for this study and did not agree with our results.

In addition to the longer dosing period used in the male pubertal assay, the male may be, in general, more sensitive to thyrotoxicant exposure. In other studies, using the same dosing duration, it has been proposed that the male rat is more sensitive than the female to perturbations of thyroid homeostasis, as indicated by an increased incidence of thyroid tumors following subchronic (Siglin et al., 2000Go) or chronic exposures to environmental chemicals (Capen, 1997). Siglin et al. (2000)Go also found that adult male rats exposed to perchlorate for two weeks showed a hypothyroid hormonal profile (decreased T4 and T3 with an increased TSH), while the adult female cohort altered T4, but not TSH or T3. The basis for this difference has not been elucidated. In general the serum T4 and TSH concentrations in the adult rat differ between the two sexes (male higher TSH and T4; Fukuda et al., 1975Go; Kieffer et al., 1976Go), although in the present study only the T4 levels were higher in the male controls. Thus, the initial difference in T4 levels between the two sexes may contribute to the differences observed in sensitivity to DE-71.

It has also been suggested that gonadal hormones modulate thyroid gland function and regulation. For example, thyroperoxidase expression is higher in young males than in female rats (da Costa et al., 2001Go). Da Costa et al. (2001) demonstrated that as gonadal hormones decrease in the aging rat, there is a concommitant decrease in thyroid peroxidase. However, there is little evidence indicating that serum TSH and T4 concentrations fluctuate over the estrous cycle (Fukuda et al., 1975Go; Kieffer et al., 1976Go). Additionally, body weight was unaffected in either sex in this study and therefore this variable does not appear to be a confounder. Thus, one explanation for the observed difference between the sexes may be a gender specific control of the thyroid homeostasis in addition to the more extended period of exposure to the toxicant leading to a greater body burden of the compound.

However, the changes in the thyroid histopathology in both the male and the female rats were similar following DE-71 exposure. Both sexes demonstrated the decrease in colloid area and increase in follicular cell height in the highest dose group, with observations of follicular degeneration in a few thyroids. These data would indicate that such microscopic changes in the thyroid are relatively sensitive indicators of perturbations in thyroid homeostasis following a 20- or 31-day exposure, with the thyroid hormones and TSH being the most sensitive indicators. As thyroid weight is not a required endpoint in the male and female protocol at this time, we did not obtain thyroid weights. However, this endpoint has also been shown to be a very sensitive indicator of an alteration of thyroid homeostasis following a 2- or 4-week exposure to thyrotoxicants (Connor et al., 1999Go). Within the context of the U.S. EPA's efforts to validate the male and female protocols, it should be noted that thyroid weight is being evaluated to determine whether or not this endpoint should be included in the final required protocols.

In the male pubertal assay, DE-71 significantly delayed preputial separation in both the 30 and 60 mg/kg groups. In addition, androgen-dependent tissues, including the ventral prostate and seminal vesicle weights were decreased at the time of necropsy (PND 53). A delay in puberty and development of androgen-dependent tissues may be indicative of a decrease or interference with androgen function. Whether this effect is secondary to a decrease in thyroid function that subsequently influences steroidogenesis or a direct effect on androgen receptor binding and activation remains to be determined. There are conflicting reports regarding the effects of hypothyroidism on serum pituitary hormone levels, testosterone secretion, and the weight of the reproductive tissues. Testicular function does not appear to be affected if hypothyroidism is induced during fetal life or later during development or adulthood (Francavilla et al., 1991Go; Kalland et al., 1978Go; Maqsood, 1954Go). However, if hypothyroidism is induced in early neonatal through weaning (i.e., Francavilla et al., 1991Go; Valle et al., 1985Go, PND 1-30), there is a corresponding delay in sexual maturation, testicular atrophy, impaired accessory reproductive tract development, reduced gonadotrophins, and inhibition of steroidogenesis. Thus, the period of life at which hypothyroidism is present, relative to the maturity of the reproductive system, are key in explaining the differences of the reproductive system parameters measured in the pubertal male. In addition, our investigations with the male pubertal protocol using other toxicants that suppressed thyroid hormones (e.g., perchlorate, which impairs iodine uptake) did not effect pubertal development (Stoker et al., unpublished). Perchlorate reduced serum T4 to approximately 45% of control with no noticeable effect on the reproductive axis. DE-71 (30 and 60 mg/kg) reduced T4 to less than 20% of controls. Thus, whether the pubertal delay with corresponding decreased growth of androgen-dependent tissues observed in this study was due to the disruption of thyroid homeostasis or a more direct disruption of the reproductive axis remains to be determined.

The two-fold increase in serum prolactin observed in this study may correspond to the altered thyroid homeostasis. The reason for this appears to be the fact that decreased thyroid hormone feedback to the hypothalamus results in an increased release of TRH. In addition to stimulating TSH secretion from the pituitary, TRH also stimulates pituitary prolactin release (for review, Schally et al., 1973Go; Mueller et al., 1973Go; Vale et al., 1973Go).

In conclusion, the results demonstrate that both the male and female pubertal protocols will detect environmental thyroxicants that alter serum T4 and T3 concentration by upregulating their catabolism. Although the female pubertal protocol was able to detect the alteration of T4 and altered thyroid histology following exposure to DE-71 at the higher doses, the male pubertal protocol identified changes in T4 at a lower dose and additional changes (T3 and TSH) at the higher doses. Importantly, the results of the male pubertal assay also indicate that DE-71 may modify reproductive development by delaying preputial separation and suppressing the growth of the androgen dependent tissues. Whether this latter effect is mediated by decreased steroidogenesis or altered androgen receptor function remains to be determined. Thus, future work is planned to examine this PBDE mixture using both in vivo and in vitro androgen studies to determine the specific anti-androgen mechanisms.

NOTES

The information in this document has been funded wholly by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1 These authors contributed equally to this article. Back

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