Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland, Michigan 48674
Received July 21, 2000; accepted December 6, 2000
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ABSTRACT |
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Key Words: endocrine disruption; endocrine modulation; EDSTAC; puberty; pubertal onset; preputial separation; thyroid; prolactin; dopamine.
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INTRODUCTION |
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The centerpiece of the alternate battery 2 is the male pubertal onset assay. In this assay, immature male rats are dosed with test material daily by gavage for approximately 34 weeks, then the age and weight at which these rats attain puberty is measured. Puberty is noted externally as the completion of balano-preputial separation (PPS). Because growth rate can impact puberty onset, body weights are monitored throughout the dosing period. Once dosing is complete, rats are euthanized, and the weights of the testes, epididymides, levator ani plus bulbocavernosus muscle, ventral prostate, and seminal vesicles plus coagulating glands are recorded. At necropsy, a serum sample is collected for the analysis of serum hormone levels, including thyroid-stimulating hormone (TSH) and thyroxine (T4). Analyses of other hormone levels, i.e., testosterone, estradiol, leutinizing hormone (LH), prolactin (PRL), and triiodothyronine (T3), are optional. Histological evaluations of the testes, epididymides, and thyroid gland are required according to the EDSTAC protocol, whereas weights and histology from the liver, kidneys, adrenals, and pituitary are optional. If further mechanistic data are desired, EDSTAC suggested that measurements of ex vivo testis and pituitary hormone production and hypothalamic neurotransmitter levels may be conducted. Since its original design in 1998, alternative approaches to this assay have been proposed (Ashby and Lefevre, 2000; Stoker et al., 2000b
).
The primary biomarker of pubertal onset in this assay is PPS, which is known to be androgen dependent. PPS normally occurs at approximately 43.6 ± 1.0 days of age in Sprague-Dawley rats (mean ± SD) (Clark, 1999). Agents operating through a variety of different mechanisms may alter pubertal assay end points, including agents that are estrogenic (Ashby and Lefevre, 2000
; Gray et al., 1989
) or antiandrogenic (Ashby and Lefevre, 1997
, 2000
; Monosson et al., 1999
), or compounds that modulate gonadotropins (Kennedy et al., 1985
), PRL (Maric et al., 1982
), growth hormone (Ramaley and Phares, 1983
), or hypothalamic or thyroid function (Huhtaniemi et al., 1986
; Valle et al., 1985
).
Despite the use of PPS as an apical measure of endocrine function for many years (Korenbrot et al., 1977), a standard protocol for the male pubertal assay has yet to be firmly established or validated. This study was designed as an initial evaluation of this assay's ability to detect known endocrine-active compounds that alter PRL or thyroid homeostasis. The endocrine-active compounds used in this study encompassed different modes of action and included a dopamine antagonist that increases PRL levels (haloperidol; HALO), a dopamine agonist that decreases PRL levels (bromocryptine; BRC), a weak antithyroid agent (phenobarbital; PB), and a strong goitrogen (propylthiouracil; PTU). These compounds were administered at dose levels reported to cause endocrine-mediated effects by the oral route in previous studies. The dopamine antagonist HALO was administered at doses equal to or greater than those reported to increase serum PRL levels (Lotz and Krause, 1978
) and produce behavioral effects with longer-term dosing (e.g., oral dyskinesias) (Gao et al., 1997
; Kaneda et al., 1992
). Administration of HALO at these dose levels reportedly produces rat plasma drug levels equivalent to those achieved in humans when using HALO therapeutically as an antipsychotic (Gao et al., 1997
; Kaneda et al., 1992
). Dose levels of the dopamine agonist BRC were selected from a study by Tozawa (1993), wherein female rats receiving oral BRC exhibited delayed puberty. Note that compounds that affect PRL levels are not a primary focus of EDSTAC screening, but use of these compounds establishes whether mechanisms distinct from estrogens, androgen, or thyroid-mediated alterations can affect pubertal onset. Furthermore, compounds that alter PRL secretion were included to facilitate comparison of this assay with another alternate Tier I assay, the 14-day intact male assay (O'Connor et al., 2000a
). The doses of the weak (PB) and strong (PTU) thyroid agents were selected from studies in which these chemicals were used to induce hypothyroidism (Bunick et al., 1994
; McClain et al., 1989
; de Sandro et al., 1991
; Sandrini et al., 1991
). These agents operate by distinct mechanisms to produce hypothyroidism; PB operates by inducing hepatic microsomal enzymes to enhance thyroid hormone clearance (McClain et al., 1989
), whereas PTU decreases thyroid hormone synthesis via direct effects on the thyroid gland (Capen, 1997
; Shiroozu et al., 1983
).
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MATERIALS AND METHODS |
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Chemicals.
HALO (2 or 4 mg/kg/day), BRC (10 or 50 mg/kg/day), PTU (240 mg/kg/day), and PB (50 or 100 mg/kg/day) were purchased from Sigma Chemical Company (St. Louis, MO). The estimated purity of each compound was determined to be 98% by the chemical supplier.
Experimental design.
In the first series of experiments, 10 litters, each containing 12 pups and one lactating dam, were used. Each litter comprised six male and six female pups, so that both the male and female pubertal onset assays could be conducted simultaneously. Results of the female pubertal assay were presented previously (Marty et al., 1999) and additional results for the male pubertal assay are presented separately (Marty et al., 2000
). In the first experiment, 10 males/treatment group received vehicle, steroid biosynthesis inhibitors (presented separately), or PTU. In the second series of experiments, 11 litters containing 10 male pups per litter were used. In the second experiment, 10 rats/group were treated with steroid biosynthesis inhibitors (presented separately), HALO, BRC, or PB. In accordance with our study design, pups were weighed at weaning (21 days of age) and randomized into treatment groups such that each group had approximately equal body weight mean and variance. Furthermore, pups were blocked by litter to ensure that littermates were not assigned to the same treatment group. Because control males from the first series of experiments were similar to control males from the second series of experiments (mean weanling weight 51.7 ± 2.7 vs. 52.7 ± 4.6 g; body weight at PPS 237.1 ± 18.6 vs. 237.3 ± 21.1 g; age at PPS 43.8 ± 1.5 vs. 45.0 ± 2.3 days of age; body weight at study termination 259.6 ± 20.2 vs. 251.1 ± 23.1 g), results from the controls were combined prior to analysis.
In accordance with EDSTAC recommendations for oral exposure, males were administered test compounds daily (7:309:30 A.M.) by gavage. The vehicle was 0.5% METHOCEL® A4M and the dose volume was 3 ml/kg. As mentioned previously, doses of test compounds were selected on the basis of literature reports citing endocrine changes with each compound treatment.
The present study differed from the protocol proposed by EDSTAC in several ways. First, obtaining pups at 14 days of age differs from the EDSTAC recommended protocol, which requires that laboratories used time-mated females. According to this approach, pregnant females would deliver their litters "in-house," allowing the litters to be culled to eight to ten pups per litter on pnd 3 or 4. Presumably, the rationale for the EDSTAC approach is to ensure equal growth rates for pups across litters. In the current study, weanling pup weights varied from 82.3 to 111.9% of the group mean body weights. The two pups with the greatest variance from the group mean body weight (9.3 g below and 6.2 g above their respective group means) were 44 and 42 days of age at the time of PPS, values which are at or below the mean age at PPS in control animals (44.4 days of age). Furthermore, receiving litters of male pups at 14 days of age prevented the needless euthanasia of female offspring that are not required for this assay. It also reduced the time required to conduct this screening assay by 3 weeks compared with receiving time-mated females. Aside from the age of the animals, there is uncertainty as to whether a 20-day dosing period beginning at weaning is sufficient (Ashby and Lefevre, 2000); therefore, animals were dosed for 30 days beginning at 21 days of age (weaning). Additional changes included the following:
The optional measurements of serum testosterone, DHT, and liver weights were included in this study. In our initial work with this assay, we focused on puberty onset, the reproductive organs and the thyroid; hence, accessory sex organ and liver weights were not collected for compounds run in the first series of experiments, which included PTU.
Observations.
All animals were examined at least once per day throughout the study for clinical signs of toxicity. Body weights were recorded daily and used to adjust dose volume. Body weights were compared statistically at 21, 27, 34, 41, and 50 days of age, and body weight gains were analyzed during the intervals of 2127, 2134, 2141, and 2150 days of age.
Males were examined for PPS beginning at 35 days of age. On the day that PPS was achieved, age and body weight of the affected animals were recorded. Males were examined daily for PPS until this end point was achieved or until 50 days of age, the day prior to scheduled necropsy. If an animal had not achieved PPS by 50 days of age, that animal was arbitrarily assigned a value of 51 days of age. This artificial value was applied to one animal in the high-dose PB group, to two animals in the high-dose BRC groups, and to all of the animals in the PTU treatment group.
Pathology.
Animals were moved to the necropsy room on the day prior to scheduled necropsy. In the morning (8 A.M.noon), approximately 24 h after the final dose, animals were weighed and anesthetized with methoxyflurane, and a terminal serum sample was collected by orbital sinus puncture. While still anesthetized, animals were decapitated and given a limited gross necropsy with a focus on reproductive organs. The testes, epididymides, seminal vesicles, prostate, and liver were removed and weighed. Seminal vesicle weights included seminal fluid. Thyroid glands were removed, fixed in neutral, phosphate-buffered 10% formalin, weighed, and examined histologically. Terminal serum samples were collected for analysis of testosterone, DHT, TSH, T4, and PRL, depending on the test material. Hormone assays were conducted by Anilytics, Inc. (Gaithersburg, MD).
Statistical analysis.
Throughout this report, data are presented as mean ± standard deviation for all parameters. Variables (age at PPS, body weight at PPS, body weights, serum hormone data, and absolute and relative organ weights) were evaluated by Bartlett's test for equality of variances (Winer, 1971). Based on the results of Bartlett's test, some data transformations were required prior to analyses. Transformed data included the inverse of age at PPS for the PB treatment group, log transformation of thyroid weights for PTU-treated animals, serum PRL data for the HALO treatment group, and relative thyroid weight for the PTU treatment group. Serum PTU TSH data were transformed by square root. After Bartlett's test, age at PPS, body weight at PPS, serum hormone data, body weights, and absolute organ weights were evaluated by an analysis of covariance (ANCOVA) with body weight at weaning as the covariate (U.S. EPA, 1998
). Relative organ weights were evaluated by analysis of variance (ANOVA). In accordance with the recommendations in the EDSTAC final report (U.S. EPA, 1998
), significant differences (p < 0.05) were examined using least square means (LSM) to compare the vehicle to treatment groups during post hoc comparisons. In most cases, analyses were grouped based on test compound such that each analysis contained results from control animals compared with results from animals treated with one test compound. The level of statistical significance for all analyses was set a priori at p = 0.05.
To examine the requirement to control for weaning weight during statistical analyses, data from these experiments also were analyzed by analysis of variance (ANOVA) and Dunnett's test, and compared with the results using ANCOVA and LSM analyses. Differences in the analyses included an ANOVA-identified statistically significant decrease in absolute epididymal weight at high-dose PB (p = 0.054 with ANCOVA analysis), a finding that seems plausible given that this concentration of PB also reduced absolute testes, prostate, and seminal vesicle weights. However, ANOVA failed to identify a statistically significant difference in T4 levels at 4.0 HALO (p = 0.054), a finding that was detected with ANCOVA. Both ANCOVA and ANOVA detected a significant difference in this parameter at 2.0 HALO. Furthermore, although relative thyroid weight was evaluated by ANOVA in both instances, a post hoc comparison using LSM detected a significant increase in relative thyroid weight at 50 PB, which was not identified with Dunnett's test. Given these minimal differences, ANOVA and ANCOVA methods of statistical analysis are approximately equivalent in cases where weanling weight is adequately controlled at the time of randomization into treatment groups.
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RESULTS |
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Treatment effects on mean body weight gain are shown in Figures 1A and 1B. Males treated with HALO, BRC, high-dose PB, and PTU exhibited reduced body weight gains at each time interval evaluated. Low-dose PB-treated animals had significantly decreased body weight gains during the latter half of the study, the day 2141 and day 2150 time intervals. These reduced body weight gains contributed to a significantly lower mean body weight in all treatment groups at study termination (Fig. 2
). Animals treated with PTU exhibited extreme delays in growth, having body weights that were 5, 23, 37, and 51% below controls at 27, 34, 41, and 50 days of age, respectively.
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In the evaluation of thyroid inhibitors, neither dose of PB significantly changed the mean age at PPS; however, PB at 50 and 100 mg/kg/day decreased the mean body weight at PPS in a dose-dependent manner to 228.2 ± 22.2 and 215.6 ± 24.3 g, respectively (p = 0.054 in ANCOVA analysis). PTU (240 mg/kg/day) significantly delayed puberty such that none of the PTU-treated animals had achieved PPS at 50 days of age, the last time point measured. Consequently, all animals in this dose group were artificially assigned the value of 51 days of age for puberty onset. Similarly, the body weight at PPS (Fig. 3B) was artificially set to the terminal body weight of these animals and was significantly lower than controls.
Liver Weights
Absolute and relative liver weights, optional end points in the male pubertal assay, are illustrated in Figure 4. Both dose levels of HALO significantly decreased absolute liver weights compared with liver weights in control animals (Fig. 4A
); however, these decreases were secondary to decreased body weights, because mean relative liver weights were unchanged in these animals (Fig. 4B
). In contrast, both absolute and relative liver weights were increased at both dose levels of PB, a finding that has been reported previously with repeated PB exposure (McClain et al., 1989
; de Sandro et al., 1991
). Note that liver weights were not recorded in our initial experiments with the male pubertal onset assay; therefore, there are no values for liver weights in animals treated with PTU.
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Accessory Sex Gland Weights
Recent data from Monosson et al. (1999) and Ashby and Lefevre (2000) suggest that comparison of absolute weights is the most critical for accessory sex glands, because these organs exhibit nonlinear weight increases in contrast to linear body weight increases during the peripubertal period. For completeness, both absolute and relative accessory sex gland weights will be presented here, with a subsequent focus on altered absolute organ weights. HALO had no effect on either absolute or relative prostate and seminal vesicle weights (Figs. 6A6D). High-dose BRC reduced absolute prostate weight (p = 0.011, Fig. 6A
), whereas relative prostate weights were not altered with this treatment (Fig. 6B
). Absolute and relative seminal vesicle weights (fluid included) also were reduced with high-dose BRC (p
0.003 in both cases). Similar to BRC, high-dose PB decreased absolute prostate weight (p = 0.009), and absolute and relative seminal vesicle weights (p
0.008). Accessory sex gland weights were not recorded for the PTU dose group.
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Because HALO, PB, and PTU significantly affected relative thyroid weights, TSH and T4 were measured in serum samples from animals in these dose groups. As shown in Figure 9A, PTU significantly increased serum TSH to 19.25 ± 4.81 ng/ml (p = 0.0001; control TSH = 2.02 ± 0.92 ng/ml), a finding that was consistent with thyroid histology for this group. Conversely, T4 was decreased significantly in PTU-treated animals to 0.35 ± 1.12 ng/ml compared with 6.72 ± 1.00 ng/ml in control animals (p = 0.0001; Fig. 9B
). Although TSH was not detectably altered, serum T4 levels were significantly reduced at both concentrations of PB and HALO. PB decreased T4 to 5.83 ± 0.96 and 4.06 ± 0.86 ng/ml at 50 and 100 mg/kg/day, respectively (p = 0.029 and p = 0.0001), whereas HALO reduced serum T4 to 5.45 ± 0.97 ng/ml at 2.0 mg/kg/day and 5.84 ± 0.99 ng/ml at 4.0 mg/kg/day. Although HALO produced clinical signs in treated animals, it had minimal effects on reproductive end points; therefore, serum PRL was evaluated in HALO-treated animals to determine whether this treatment was efficacious. At 24 h after the last treatment, HALO increased mean serum PRL, although these increases were not dose related nor were they statistically identified. Serum PRL values were 200.47 ± 240.10 and 103.63 ± 181.25 ng/ml with 2.0 and 4.0 mg HALO/kg/day, respectively, compared with 55.60 ± 74.72 ng/ml in control animals (p = 0.288). Serum PRL was not measured for BRC-treated animals, because BRC altered a variety of end points, including increased age at PPS and decreased absolute prostate and seminal vesicle weights. Given these endocrine-mediated effects, it was presumed that serum prolactin levels had been sufficiently altered with BRC treatment.
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DISCUSSION |
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One factor of critical importance in the pubertal male screening assay is the effect of body weight on assay end points. Because the treatments in this study affected body weight gains, these data can be used to examine complex interactions between body weight and pubertal onset. For example, at 44 days of age, which is the mean age at PPS in the control group, high-dose PB-treated animals had a 15% lower mean body weight than the control animals. Despite this body weight differential, the delay in the age at which PPS was achieved was not statistically significant (46.4 ± 3.8 compared with 44.4 ± 2.0 days of age in control animals). Conversely, PTU-treated animals had a marked delay in puberty (> 6.6 days), which was statistically identified. These animals weighed 42% less than the control animals at 44 days of age. Overall, these data suggest that the mean age at PPS is not readily altered by moderate decreases in body weight gains, but can be dramatically altered by large changes in body weight gain. This concept has been substantiated by previous studies (Aguilar et al., 1984; Carney et al., 1998
). In a study by Aguilar et al. (1984), age at preputial separation was not altered by rearing male rats in litters with 8 vs. 12 offspring, despite an approximate 20% differential in body weight at the time of preputial separation (Aguilar et al., 1984
). Furthermore, in animals given 30% less feed than controls, PPS was delayed by only 1 day, despite an 11% decrease in body weight at PPS. With a 50% dietary restriction, which resulted in a 23% decrease in body weight at PPS, puberty was delayed by 6.8 days (Carney et al., 1998
).
Ashby and Lefevre (2000) recently conducted experiments examining the effect of body weight on pubertal onset and final organ weight. These investigators found that age at puberty was most closely correlated to the initial body weight of these animals at 3536 days of age; that is, lighter animals were older at PPS, whereas heavier animals were younger at PPS. This paper nicely illustrates the relationship between body weight and age at PPS as a continuum. Interestingly, the control group in this study, which had median body weights between the lightest and heaviest groups, had the largest variability in the age at pubertal onset, exhibiting PPS at 44.8 ± 2.6 days of age. Furthermore, the lightest animals in this study, which weighed 17% less than controls at 3536 days of age, 8% less at the time of PPS, and 10% less at study termination, experienced a 1.4-day delay in PPS from the control animals. Given the group size in the present study, this 1.4-day delay would not achieve statistical significance; however, a body weight effect such as this could heighten the sensitivity of this group of animals to statistically detectable changes in PPS in response to a weak chemical treatment. Thus, the interpretation of delayed puberty in the presence of body weight changes is complex, in that the relative contribution of decreased body weight compared with the unknown endocrine activity of a given chemical can be difficult to discern. Still, data from this study suggests that although body weight influences pubertal onset, only the most dramatic effects on body weight would be expected to statistically impact age at PPS in the male pubertal assay if body weight is adequately controlled at the start of the study at weaning. Thus, despite the reduction in body weight with BRC treatment, it is likely that the delay in PPS (+ 3.2 days) is endocrine mediated, because animals exposed to high-dose PB had greater reductions in body weight without a significant effect on the age at PPS. To more accurately examine the impact of body weight on pubertal onset, this issue should be reexamined with 15 animals per treatment group to determine the sensitivity of age at preputial separation to body weight changes. Furthermore, the impact of reduced body weight on organ weight end points merits further investigation.
Aside from body weight, it appears that a number of other factors contribute to pubertal onset, factors that exhibit a large degree of interanimal variability and contribute to the large variability in age at pubertal onset in control animals. For example, the mean age for puberty onset in male control rats was 44.4 ± 2.0 days of age. This value is within the range reported by others for puberty onset in male rats (Clark, 1999). At the time these experiments were conducted, the U.S. EPA had not clearly defined the number of animals per treatment group. Since that time, a group size of 15 animals per treatment group has been proposed. Fifteen animals per group would improve the statistical power of the male pubertal assay such that the probability of detecting a 2.0-day change in the age at preputial separation would be 56%, whereas the probability of detecting a 2.0-day change is only 40% with ten animals per treatment group (Clark, 1999
). In these power calculations, Clark (1999) has assumed a standard deviation of 2.5 days. This standard deviation is greater than that observed in the present study and may be due to the examination of animals every other day for PPS in that data set. On the other hand, these values are based on the litter as the unit of measure; therefore, these probabilities would likely be lower with individual animal data (Clark, 1999
). This large interanimal variability was problematic with regard to hormone measurements as well. The large degree of variability encountered with these parameters was undoubtedly confounded by the 24-h time interval between administration of the final dose of test material and serum sample collection. Presumably, collecting serum samples within a smaller time interval after dosing will improve the consistency of these measures. This protocol, which recommends necropsy on the final day of treatment, is under consideration by the U.S. EPA within the context of its Endocrine Disrupter Screening Program. Aside from a more proximate necropsy time, an additional five animals per treatment group would have reduced the variability in the hormone values. Lastly, despite efforts to control stress (e.g., moving the animal cages to the necropsy room the day prior to necropsy), stress probably contributed to the increased variability in hormone measurements, particularly in the case of PRL, where basal serum levels were 5-fold higher than reported PRL levels in rats (O'Connor et al., 2000a
). However, the relative contribution of stress to the PRL increase is not known, because mean T4, TSH, and testosterone, all hormones subject to modulation by stress, were not notably different from other reported values in rats (Döhler et al., 1979
; Monosson et al., 1999
).
Although not required by EDSTAC, PRL was of interest in this assay, because prepubertal elevations in PRL via hypothalamic implants or dopamine receptor antagonism (sulpiride at 0.5 g/l in drinking water) from 22 or 23 days of age until puberty have been associated with precocious puberty in females (Advis and Ojeda, 1978; Advis et al., 1982
). To examine the role of PRL in puberty onset, males were treated with the dopamine antagonist HALO to increase circulating levels of PRL (Dickerman et al., 1972
; O'Connor et al., 2000a
), a phenomenon that was difficult to confirm by serum radioimmunoassay in the present study. To evaluate hypoprolactinemia, the dopamine agonist BRC was used (MacLeod and Lehmeyer, 1974
).
With regard to the male pubertal assay end points, HALO had no effect on age or body weight at pubertal onset, absolute epididymal weights, relative accessory sex gland weights, or relative liver weight. These data contradict previous reports by Maric et al. (1982) and Coert et al. (1985) that elevated PRL increased seminal vesicle and prostate weights in male rats. Maric et al. (1982) also observed decreased testicular growth and reduced or delayed androgen secretion with sustained hyperprolactinemia during the peripubertal period. Discrepancies between these studies and the current study may be due to the method used to produce hyperprolactinemia, which was via ectopic pituitary grafts in the former studies versus HALO in the present study. Furthermore, data from Maric et al. (1982) suggest that prolonged elevations of PRL prior to puberty may attenuate or delay large subsequent secretions of androgens, whereas short-term elevations do not produce these effects. Thus, it is possible that PRL elevations in the present study were not of sufficient magnitude or not maintained for a sufficient period to induce these long-term effects.
Interestingly, HALO significantly reduced thyroid weight and serum T4. Mean serum TSH was reduced slightly with HALO treatment, but this effect was not statistically different from control values. HALO had minimal effects on thyroid histology. Given that a majority of studies report an inhibitory effect of dopamine on TSH release (Burrow et al., 1977; Connell et al., 1985
; Krulich et al., 1977
; Ranta et al., 1977
; Tuomisto et al., 1975
) and that HALO stimulates TSH release via DA antagonism (Delitala et al., 1981
), these thyroid effects were unexpected. In contrast to these reports, a few studies describe a decrease in TSH release following DA antagonist treatment (Collu et al., 1975
; Mueller et al., 1976
; Nemeroff et al., 1978
). It is possible that HALO reduced TSH levels via weak noradrenergic inhibition of thyrotropin releasing hormone (TRH) release (Janssen et al., 1968
), because norepinephrine reportedly stimulates TRH release (Grimm and Reichlin, 1973
).
In the present study, PRL appeared to be elevated by HALO treatment, but this increase was not statistically identified, presumably due to the time lapse between our final dose and terminal blood collection and/or the use of single daily dosing with HALO. Researchers have reported that elevations in serum PRL are enhanced with multiple daily doses of HALO to sustain higher blood levels of test material (O'Connor et al., 1996; 2000a
). To further complicate detection of serum PRL changes, nonspecific factors such as stress can alter PRL levels (Cooke, 1995
; O'Connor et al., 2000a
). To some extent, stress contributed to the variability in PRL measures, because basal PRL levels are elevated above cited control values of
10 ng/ml (Stoker et al., 2000a
; O'Connor et al., 1998b
, 2000a
). It is possible that some of the elevation is due to the method of serum collection, because orbital sinus puncture has been associated with an approximately 70% increase in basal PRL values in anesthetized animals (Döhler et al., 1978
). Furthermore, these animals were euthanized in March, a peak month for serum PRL levels in Sprague-Dawley rats, despite maintenance under controlled laboratory conditions. This circannual variation could account for a 2-fold increase in serum PRL values (Wong et al., 1983
). Despite this elevation in basal serum PRL, PRL has a large dynamic range, and HALO has been reported to increase PRL to > 1000 ng/ml (Dickerman et al., 1972
); consequently, a change in serum PRL could still be detected in these treated animals, although sensitivity would be reduced. Although stress may have contributed to elevations in serum PRL, it had minimal impact on other serum hormones measured in this study. Testosterone, T4, and TSH, which are subject to modulation by stress, were not notably elevated above reported values (Döhler et al., 1979
; O'Connor et al., 1999a
; Monosson et al., 1999
). The variability of the serum PRL values may suggest that this end point is not well suited to EDSTAC screening studies.
Given these results, the question remains as to whether the male pubertal assay would identify HALO for Tier II testing based upon thyroid effects. Note that thyroid weight is not a required end point in the current male pubertal assay protocol (U.S. EPA, 1998); thus, the probability of HALO triggering Tier II studies based on T4 changes alone must be contemplated. Furthermore, the question remains as to how to interpret reduced thyroid function. Typically, potential thyroid toxicants induce one or more of the following responses: elevations in TSH, decreases in T3/T4, increased thyroid weight and/or altered thyroid histology (O'Connor et al., 1998a
,b
; 1999a
,b
), whereas HALO produced decreased T4 and decreased thyroid weight. Under these circumstances, a consensus on interpretation of central nervous systemmediated reductions in thyroid size and function (reduced TSH and T4) has not been discussed. Because elevations in TSH are required to induce thyroid pathology (Hood et al., 1999
) and HALO did not elevate TSH nor produce significant tissue structural changes, this condition may warrant less concern. An alternate explanation to effects of HALO could be that serum TSH, T3, and T4 were decreased secondary to body weight effects (O'Connor et al., 1999a
). This explanation seems unlikely, however, because different response profiles exist for feed-restricted and HALO-treated animals. Specifically, relative liver weights were decreased and relative thyroid weights were unchanged with 15 days of feed restriction in male rats (O'Connor et al., 1999a
), whereas HALO treatment did not alter relative liver weight but decreased relative thyroid weight in the present study.
To compare results using HALO in the male pubertal assay with other Tier I in vivo assays, the 14-day intact male battery and the 5-day ovariectomized female battery relied upon altered serum PRL levels as the only end point for the detection of HALO (O'Connor et al., 2000a). These investigators used much lower doses of HALO (
1.0 mg/kg/day) administered by ip injection to adult intact male rats and ovariectomized female rats. In contrast to the present findings, O'Connor et al. (2000a) did not produce significant body weight changes, nor were any thyroid effects noted with HALO treatment, both of which may suggest that the dose of HALO was insufficient to alter these parameters. Alternatively, the age of the animals at the time of exposure may contribute to differential sensitivity to dopaminergic/PRL changes.
As mentioned previously, high-dose BRC significantly delayed the age at which puberty was achieved, an effect attributed to BRC-mediated endocrine effects rather than effects on body weight gain. Low-dose BRC animals, which also had a lower mean body weight than control animals at puberty, experienced no delay in the age at PPS. Previous studies have shown that BRC hypoprolactinemia delayed pubertal onset in immature female rats (Advis et al., 1981; Shaban and Terranova, 1986
) but was ineffective at altering pubertal onset in juvenile male rats (Hostetter and Piacsek, 1977
; Huhtaniemi and Catt, 1981
). Interestingly, the studies conducted in female rats noted decreased PRL secretion coupled with decreased LH release, a finding that does not correlate with the current study, because testosterone and DHT levels were not decreased. Huhtaniemi and Catt (1981) reported no changes in serum testosterone levels in juvenile male rats treated with BRC. Note that the route of administration in the previous male rat studies differed from the current study, so the inability to alter puberty may have been due to an inability to sustain a sufficient concentration of the hypoprolactinemic agent.
In a previous study in which male rats were treated with BRC, body weight and absolute testes weights were decreased (Pérez-Villamil et al., 1992). A reduction in testes weights corresponds with evidence that PRL, coupled with follicle stimulating hormone, contributes to testicular formation of LH and/or human chorionic gonadotropin receptors (Huhtaniemi and Catt, 1981
; Kolena and Seböková, 1983
). In the present study, absolute mean testicular weight was decreased by 12% with high-dose BRC; however, given the high interanimal variability and the lack of statistical significance, this reduction in testicular weight alone by BRC would be unlikely to trigger concern. However, this reduction, together with decreased prostate and seminal vesicle weights, would indicate endocrine activity. Due to the variety of effects produced by BRC, measurement of serum PRL was deemed unnecessary to verify the endocrine activity of this compound.
Therefore, the male pubertal assay detected BRC through delayed PPS, as well as a decrease in absolute prostate and seminal vesicle weights, but the detection of dopamine receptor agonists may be problematic in alternative Tier I screening assays. The 14-day intact male and 5-day ovariectomized female assays had difficulty detecting an alternate dopamine receptor agonist, apomorphine (O'Connor et al., 2000a). In these studies, apomorphine was not detected, although this compound should be retested at higher concentrations.
To examine the role of the thyroid gland in puberty onset and maturation, animals were treated with a strong thyroid agent, PTU, or a weak thyroid agent, PB. PTU operates by inhibiting thyroid hormone synthesis, whereas PB induces hepatic microsomal enzymes, thereby accelerating thyroxine metabolism and clearance (McClain et al., 1989). In contrast to previous reports investigating the thyroid effects of 100 mg PB/kg/day (McClain et al., 1989
; Hood et al., 1999
), rats in the present study exhibited significant decreases in body weight and body weight gains. This effect was due to the administration of PB as a bolus dose compared with dosing via the diet. However, in agreement with previous reports (Hood et al., 1999
; McClain et al. 1989
; de Sandro et al., 1991
), PB increased relative liver and thyroid weights and decreased serum T4 levels. Although Hood et al. (1999) detected a significant change in T4 and TSH when dosing rats with 100 mg PB/kg/day for 2 weeks, both T4 and TSH levels had recovered to control values by 12 weeks. Davies (1993) also reported difficulties in detecting TSH increases in response to PB treatment. Increased serum TSH was not observed in the present study at 30 days postdosing. Interestingly, decreases in absolute testes, prostate, and seminal vesicle weights were observed at the highest PB dose. These decreases in androgen-sensitive tissue weights may be due to decreased LH release following PB treatment (O'Connor et al., 1999a
). Alternatively, induction of hepatic microsomal enzymes by PB may accelerate testosterone metabolism and clearance (Levin et al., 1974
). This hypothesis is difficult to substantiate, however, because there were no reductions in serum testosterone or DHT with PB treatment. Once again, the decrease in serum androgens may have escaped detection due to sampling 24 h after the final dose of PB. In general, changes in organ weights may require greater scrutiny than hormonal changes, because organ weights represent a culmination of events over time, whereas serum hormone levels represent a single point in time.
PB-induced effects on thyroid function are believed to be compensatory to adjust for increased thyroid hormone clearance (McClain et al., 1989). Thus, with continued PB treatment, thyroid hormones were not as severely affected as after short-term PB treatment, indicating some compensation or recovery of thyroid function (Hood et al., 1999
; McClain et al., 1989
). Changes in thyroid histology with PB treatment, which included hypertrophy and hyperplasia of the follicular cells and a decreased amount of colloid, occurred secondary to increased T4 metabolism and elevated TSH secretion (Japundzic, 1969
). After 30 days of PB treatment, thyroid follicular cell proliferation was no longer enhanced, but relative thyroid weights remained significantly increased at this time point (Hood et al., 1999
), a finding consistent with the results of the present study. Several studies have reported difficulty in detecting thyroid histology changes with 14 weeks of dosing with weak-acting thyroid agents like PB (Hood et al., 1999
; O'Connor et al., 1999a
). One plausible explanation for the minimal microscopic changes seen with PB is that weak thyroid agents have a lesser ability to directly influence thyroid gland function (Capen, 1997
). Although not a required end point in the male pubertal assay, thyroid weight appears to be a valuable indicator of thyroid effect, particularly for mild to moderate goitrogenic compounds like PB (McClain et al., 1989
).
PTU, a potent goitrogen, significantly delayed maturation of juvenile male rats, a finding substantiated by numerous end points. PTU had significant effects on body weights, body weight gains, age at puberty, weight at puberty, and absolute testes and epididymal weights. In fact, terminal body weights were decreased 52% in PTU-treated animals. Serum testosterone and DHT were reduced. These results, delayed sexual maturation with reduced testes weights, have been reported previously with PTU treatment (Valle et al., 1985). Furthermore, the thyroid effects observed with PTU were similar to those observed by Wilen et al. (1981), including decreased T4 and striking increases in TSH and relative thyroid weight. Thyroid histology was markedly altered in a manner similar to that reported by Hood et al. (1999). It is well established that prolonged hypersecretion of TSH produces thyroid follicular cell proliferative lesions (Capen, 1997
; Davies, 1993
). PTU did not increase relative liver weight, a factor that helps differentiate direct acting thyroid agents from thyroid agents that operate through hepatic enzyme induction (O'Connor et al., 1999a
). Similar to the male pubertal onset assay, the 14-day intact male assay readily detected PTU (0.25 mg/kg/day) based on thyroid hormone measures and histology (O'Connor et al., 1999a
).
Overall, the male pubertal assay is capable of detecting chemicals that operate through a variety of mechanisms. BRC, PB, and PTU were deemed positive for endocrine activity in the current male pubertal assay study. Data from the dopaminergic agent HALO is more difficult to interpret. Although HALO produced thyroid effects, previous studies have focused on elevations in TSH and increased thyroid weights, so the interpretation for reduced thyroid activity is ill defined. Because this assay involves a number of end points, all of which are apical in nature, some consensus on interpretation of data generated with this assay will be needed before it can be applied to a broad range of chemicals of unknown activity. This will only be possible by examining results for positive and negative compounds in order to determine the activity profiles for various endocrine-active compounds and which end points are the most robust. Furthermore, an in-depth comparison of results generated with the male pubertal assay and other studies using the same compounds in alternate assays (e.g., Hershberger assay, 14-day intact male assay) would provide additional perspective on the utility of this assay.
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NOTES |
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2 Present address: Dow Corning Corporation, Midland, MI 48686.
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