ToxSci Advance Access originally published online on August 28, 2007
Toxicological Sciences 2007 100(1):194-202; doi:10.1093/toxsci/kfm219
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Published by Oxford University Press 2007.
Effects of Altered Food Intake during Pubertal Development in Male and Female Wistar Rats
Endocrinology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
1 To whom correspondence should be addresses at MD-72, NHEERL, U.S. EPA, Alexander Drive, Research Triangle Park, NC 27711. Fax: (919) 541-5138. E-mail: laws.susan{at}epa.gov.
Received May 14, 2007; accepted August 14, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The U.S. Environmental Protection Agency is currently validating assays that will be used in a Tier I Screening Battery to detect endocrine disrupting chemicals. A primary concern with the Protocols for the Assessment of Pubertal Development and Thyroid Function in Juvenile Male and Female Rats is that a nonspecific reduction in body weight (BWT) during the exposure period may potentially confound the interpretation of effects on the endocrine endpoints. Wistar rats were underfed 10, 20, 30, or 40% less than the ad libitum food consumed by controls from postnatal days (PNDs) 22 to 42 (females) or PNDs 23 to 53 (males). Terminal BWT of females and males were 2, 4, 12, and 19% and 2, 6, 9, and 19% lower than controls, respectively. In the females, neither the age of pubertal onset nor any of the thyroid hormone endpoints were affected by food restriction (FR) that led to a 12% decrease in BWT. Similarly, none of the male reproductive endpoints examined were altered by FR that led to a 9% BWT decrease. However, decreased triiodothyronine and thyroxin was observed in FR males with a 9% reduced BWT. While these data support the use of the maximum tolerated dose for BWT (10%) for the female protocol, effects on the male thyroid endpoints indicate that a slightly lower limit (
6% BWT loss) may be appropriate for the male pubertal protocol, and in cases where the BWT loss approaches 9–10%, additional studies and/or a weight of evidence approach should be used when interpreting the data for the thyroid endpoints. Key Words: pubertal development; food restriction; endocrine disruptors.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As mandated under the Food Quality Protection Act (1996), the U.S. Environmental Protection Agency (U.S. EPA) is implementing an endocrine disruptor screening program (http://www.epa.gov/scipoly/oscpendo/edspoverview/index.htm) to detect chemicals that alter the estrogen, androgen, and thyroid hormone systems in humans, fish, and wildlife. This program, based largely upon recommendations by the Endocrine Disruptor Screening and Testing Advisory Committee (U.S. EPA, 1998), is working toward (1) establishing procedural rules and policy for program implementation; (2) developing a strategy for prioritizing chemicals currently under the purview of the U.S. EPA for screening and testing; and (3) developing and validating protocols to conduct specific assays designed to detect endocrine disrupting chemicals.
The validation process for each assay is designed to demonstrate the biological relevance, as well as the ability of the assay to produce comparable data when conducted in multiple laboratories (e.g., assay reliability). Two protocols for the Assessment of Pubertal Development and Thyroid Function in Juvenile Rats (one for the male and one for the female) are currently undergoing validation for inclusion in the screening battery. These assays are designed to evaluate the effects of test chemicals on pubertal development and thyroid function in the rat through alterations in the estrogen, androgen, or thyroid hormone systems as well as to identify any impact of chemicals on the central nervous system regulation of pubertal development and thyroid hormone regulation. Goldman et al. (2000)
and Stoker et al. (2000b) have reviewed the scientific basis of the protocols and the influence of pharmaceutical and environmental toxicants on pubertal development and thyroid function in male and female rats. In addition, several studies have been published demonstrating the utility of these protocols to identify a wide variety of endocrine disrupting chemicals including the steroid biosynthesis/receptor modulators (Marty et al., 1999, 2001a), thyroid toxicants (Marty et al., 2001b; Stoker et al., 2004, 2006), and chemicals that affect hypothalamic function (Ashby et al., 2002; Laws et al., 2000, 2003; Stoker et al., 2000a, 2002).
One concern with these two protocols as potential Tier I screens is that many of the endpoints included may be sensitive to alterations in body weight (BWT) per se, and thus changes in BWT associated with exposure to the test chemical may confound the interpretation of the data. There is little doubt that rigorous food restriction (FR) regimens resulting in decreased BWTs of greater than 50% (vs. control) prior to- and/or during pubertal development will produce moderate to severe reproductive alterations in organ weights, fertility, and reproductive development (Bronson and Heideman, 1990; Hamilton and Bronson, 1986; Kennedy and Mitra, 1963; Merry and Holehan, 1979, 1985; Widdowson and McCance, 1960). These assays are designed to evaluate the effects of tests chemicals on pubertal development and thyroid function in the rat through alterations in the estrogen, androgen, or thyroid hormone systems. In addition, the assays can detect chemicals that disrupt pubertal development through changes in the regulation of the central nervous system. In this regard, several studies have shown that minor reductions in daily food consumption (e.g., 10–30% of pair-fed controls) and associated weight loss of 10% do not alter the endpoints that are included in the proposed pubertal assays (Auguilar et al., 1984; Chapin et al., 1993; Laws et al., 2000; O'Connor et al., 2000; Ronnekleiv et al., 1978). These studies support the practice of using BWT as a part of the dose-setting process. Thus, depending on the amount of information available on the toxicity of the test compound, the guidelines for these protocols will likely require that a maximum tolerated dose (MTD) based on terminal BWT be used. The MTD for BWT is defined as the dose that produces a 10% reduction in terminal BWT as compared with the appropriate control group (Hodgson, 1987; IUPAC, 1997). This approach assumes that a 10% reduction in terminal BWT alone would not alter the endpoints in the pubertal assays. However, this assumption has not been fully examined. Thus, we conducted the following experiments to define the extent to which the endpoints examined in the pubertal assay are affected by FR regimens that approach the MTD for BWT at the time of necropsy.
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MATERIALS AND METHODS |
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Animals.
Wistar rats (14-day timed pregnant) were obtained from Charles Rivers Laboratories, Raleigh, NC, and were maintained under controlled temperature (20°C–24°C), humidity (40–50%), and light (14-h light/10-h dark) conditions with Purina Laboratory Rat Chow (5001) and water available ad libitum in an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility. Pregnant dams (n = 18) were allowed to deliver their pups naturally; 3 days postpartum (postnatal day [PND] 3, PND 0 = the morning of birth) all litters were standardized to 10 pups, maintaining an equal number of males and females per litter when possible. Females were weaned on PND 21, ranked by BWT, and placed into treatment groups such that the mean BWT ± SD for all groups were similar. Males were weaned on PND 22, ranked by BWT, and placed into treatment groups such that the mean BWTs and SD were similar. In addition, litter mates were equally distributed among the treatment groups with no more than one male or female from a single litter assigned to each treatment group.
Experimental design.
The experimental design followed the Protocols for the Assessment of Pubertal Development and Thyroid Function in the male (Stoker et al., 2000b) and female rats (Goldman et al., 2000). To evaluate the effect of FR and the resulting reduction in BWT during pubertal development, groups of animals were studied during a 20-day period in the females (PNDs 22–41) and a 30-day period in the males (PNDs 23–53). This time period encompasses the age of the onset of puberty in both sexes. For any given FR treatment group, each animal within that group was paired with a control rat of similar initial BWT. Food consumption of the control animals was measured daily beginning at PND 21. Each day during the treatment period, animals in the FR groups were fed 10, 20, 30, or 40% less food than that consumed by its respective control animal the previous day. BWT was recorded daily as were indications of vaginal opening in the female or preputial separation (PPS) in the male. Females were killed by decapitation on PNDs 41 and 42, and liver, kidney, adrenal, ovary, uterus, and pituitary weights were recorded. Males were killed by decapitation on PNDs 53 and 54, and liver, kidney, adrenal, pituitary, left testis, ventral prostate, left epididymis, seminal vesicles with coagulating gland (with fluid) were removed and weights recorded. Thyroid and reproductive tissues (ovary, uterus, right testis, and epididymis) were removed and prepared for histological evaluation. Serum was stored at – 80°C until assayed for triiodothyronine (T3), thyroxin (T4), thyroid-stimulating hormone (TSH), glucose, and leptin. Pituitaries (males only) were stored at – 80°C for prolactin and luteinizing hormone (LH) assays.
Histology.
Immediately following dissection at necropsy, thyroid, uterus, paired ovaries, right testis, and epididymis were placed in 10% neutral buffered formalin for 24 h. The tissues were rinsed and stored in 70% alcohol until embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissues were evaluated by Veritos Labs (Burlington, NC) for pathological and treatment-related effects.
Radioimmunoassays.
Serum TSH, serum and pituitary prolactin (PRL), and LH were measured by radioimmunoassay (RIA) using material supplied by the National Hormone and Peptide Program (Torrance, CA; http://www.humc.edu/hormones). The following materials were used for the LH, PRL, and TSH assays, respectively: iodination preparation I-9, I-6, and I-9; reference preparation RP-3, RP-3, and RP-3; and antisera S-11, S-9, and S-6. The iodination preparation was radiolabeled with 125I (PerkinElmer, Life and Analytical Sciences, Inc., Waltham, MA) using a modification of the chloramine-T methods (Greenwood et al., 1963). Labeled TSH was separated from free iodide by gel filtration chromatography, and the RIA was conducted as described by Goldman et al. (1986). Sample serum and pituitary homogenate were pipetted with appropriate dilutions to a final assay volume of 500 µl with 100mM phosphate buffer containing 1% bovine serum albumin (BSA). Standard reference preparations were serially diluted for the standard curves. Two hundred microliters of primary antisera in 100mM potassium phosphate, 76.8mM 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 microliters 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, Calbiochem, at a dilution of 1 unit/100 µl) was then added, vortexed, and incubated 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. Total serum T4, T3, and testosterone (T) were measured using coat-a-count RIA kits from Siemens Medical Solutions Diagnostics, (TKT4, TKT3, and TKTT, Los Angeles, CA). The limits of sensitivity for T3, T4, and TSH were 7.0 ng/dl, 0.25 µg/dl, and 0.04 ng/ml respectively.
Leptin and glucose assays.
Serum glucose (D-glucose) concentrations were determined using the Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189, Invitrogen Corporation, Carlsbad, CA). Standards were prepared according to the protocol at concentrations of 0–100µM. The level of sensitivity for glucose was 3µM. Leptin concentrations were determined using a Rat Leptin Elisa Kit (RL-83K, Linco Research, Inc., St Charles, Missouri). The standard curve ranged from 0.2 to 20 ng/ml with a level of sensitivity of 0.04 ng/µl.
Statistical analyses.
Data from the males and females were analyzed separately for treatment effects by ANOVA using the General Linear Model procedure (Statistical Analysis System [SAS], SAS Institute, Inc., Cary, NC). In cases where a significant treatment effect (p 0.05) was observed, the dose-response data were further evaluated by the Dunnett multiple comparisons test (control compared within each treatment group). Treatment means for each endpoint were tested for homogeneity of variance using the Bartlett Test (GraphPad InStat, GraphPad Software, San Diego, CA), and where heterogeneity was evident, the Welch t-test or Kruskal-Wallis nonparametric test with Dunn’s multiple comparison test were used. Relative organ weights were determined using necropsy BWT and compared to control relative weights by ANOVA and the Dunnett test as described above. All data are reported as the mean ± SEM (n).
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RESULTS |
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BWT/Growth
Figures 1 and 2 show the mean daily BWTs for each treatment group during pubertal development. Mean BWTs for all groups were equal on the initial day of the study (weaning) but gradually became distinct from the control at approximately PND 27 for the females (Fig. 1) and PND 30 for the males (Fig. 2). The mean age at vaginal opening in the control females was PND 33.0 ± 0.55. On PND 33, the mean BWTs of the 30 and 40% FR groups were significantly lower than controls, 7.7 and 14.4%, respectively (Table 1). At PND 41, the differences in BWT between control and FR females were 2, 4, 12, and 19% for the 10–40% FR groups with the 12 and 19% reductions being statistically significant. A similar pattern was observed in the males. In the controls, the mean age at PPS was PND 44.9 ± 0.5. On PND 44, the mean BWTs of the two groups with the greatest FR were significantly lower than the control with an 8.5 and 19% lower BWT in the 30 and 40% FR animals, respectively. At PND 54, the mean terminal BWTs of the FR animals were 2, 6, 9, and 19% lower than the controls with the BWTs of the animals in the two groups with the greatest amount of FR being significantly lower than the controls.
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Age at Vaginal Opening and PPS
The mean age at vaginal opening was PND 33.0 ± 0.55. Age at vaginal opening was not significantly different from controls for all FR groups (Table 1) with BWTs ranging from 97.6 to 123.5 g in the controls as compared with 90.1 to 131.4 g in the females in the highest FR group.
The age at PPS in the males was not significantly altered by FR (Table 2). The range of BWTs at the age of PPS for the controls (44.9 ± 0.54 days) was 180.4–259.4 g, as compared with the highest FR group which had BWTs ranging from 151.3 to 208.2 g.
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Organ Weights
Liver, kidney, adrenal, and pituitary weights at necropsy for males and females are shown in Table 3. Absolute weights of each of these tissues were significantly reduced in the two highest FR groups. In the males, the mean absolute liver weights were significantly decreased compared to controls in all FR groups. In addition, a comparison of the liver weight relative to terminal BWT revealed that the mean liver weights in the 20, 30, and 40% FR groups were significantly different from controls. A similar finding was observed for male kidney weight, with a significant decrease in the absolute mean in the 20, 30, and 40% groups as compared to controls but only in the 40% group when analyzed on a relative weight basis. The mean adrenal weights of the male were decreased after 10, 30, and 40% of FR, while the relative weights were not significantly affected. The mean pituitary weights in the male were significantly decreased in the 20–40% FR groups, but again, the mean relative weights were not different.
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In the females, there was a significant decrease in absolute liver weight in the 30 and 40% FR groups as compared to controls. Only the 40% FR group remained significantly smaller when analyzed relative to BWT. Also in the female, the mean kidney, adrenal, and pituitary weights were decreased in the 30 and 40% FR groups; however, none of these weights were different when analyzed relative to the BWT at necropsy.
Reproductive Tract Weights at Necropsy
In the female, the absolute ovarian weights were decreased in the 30 and 40% FR group, with no significance in the comparison of the relative weights (Table 4). The mean uterine weights were not different from control in either comparison in any of the FR groups. In the male, the absolute mean ventral prostate and seminal vesicle weights were significantly lower than the controls in the 40% FR, with no significance in relative weight at necropsy (Table 5). Similarly, the absolute epididymis weights were significantly decreased in the 40% FR group, but relative weight at necropsy was increased in this group. There were no differences in the absolute mean testis weight at necropsy, but the relative weight was increased in the 30 and 40% FR groups as compared to controls
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Histology
Histopathological analysis of the testes and epididymides revealed no significant alterations in testes structure or number of spermatids in the epididymides. No significant alterations were observed in thyroid histopathology in the males or females of any of the FR groups. In the female, no significant changes were observed in the ovaries or uteri of the FR rats as compared to the controls.
Hormones
As shown in Table 5, there were no significant differences in serum T4, T3, or TSH in any of the FR groups as compared to controls in the females when evaluated at necropsy (PNDs 41 and 42). In the males, serum T3 and T4 means were significantly lower than the controls (Table 4) in the 30 and 40% FR groups, and TSH was significantly decreased in the 40% restricted group at necropsy (PNDs 53 and 54). No differences in serum T, luteinizing hormone, or prolactin were noted in any of the males.
Blood Glucose and Leptin
Serum glucose concentration in the males at PNDs 53 and 54 was not different from controls. However, the female rats in the 30 and 40% FR groups had significantly reduced serum glucose when compared to the controls on PNDs 41 and 42. Mean serum leptin concentrations were decreased in the 30 and 40% FR groups in the male but were only reduced in the 40% FR group in the female as compared to controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here we have evaluated the relationship between reduced BWT during pubertal development with the age of onset of puberty, organ weights at necropsy, and thyroid hormones. For these studies, the aim was to achieve a terminal BWT for the FR groups that was 10% lower than the controls as this is a well-established limit for setting the MTD for toxicology studies (Hodgson, 1987
; IUPAC, 1997). The results of this study demonstrate that neither the age of pubertal onset nor any of the thyroid hormone endpoints included in the female pubertal assay were affected by restrictions in food intake that led to decreases in BWT up to 12% in this strain of rats. Similarly, none of the male reproductive endpoints examined were altered by FR that led to a decrease in BWT up to 9%. However, we did observe a decrease in serum T3 and T4 in the males whose necropsy BWTs were 9% lower than the controls. Several of these endpoints in both sexes were altered if the animals were subjected to the greatest amount of FR which resulted in a 19% reduction in terminal BWT for both the females and males. Finally, the absolute weights, but not the relative weights, for pituitary, adrenals, liver, and kidney were significantly lower in the females with a 12% reduction in BWT. The same was true for the males in FR groups that led to a 6 and 9% lower BWT, with the exception of a significant change in relative liver weight in those animals with a 6, 9, and 19% BWT reduction. It is generally accepted that liver weight is dependent upon BWT, and when evaluated relative to BWT, any significant differences are no longer observed (Feron et al., 1973). However, in this study as well as FR studies reported by Marty et al. (2003) and O'Connor et al. (1999), both the absolute and relative liver weights were also significantly different from controls in males with reduced BWTs of 10 or 15% indicating that data for this endpoint should be carefully evaluated when BWTs approach this limit. Overall, these data support the use of the MTD for BWT of 10% for the female based on the lack of differences in the relative weights of the nonreproductive or reproductive organ weights or in the in-life measurements of age at vaginal opening in females with at least 12% loss in BWT. However, the effects observed for thyroid endpoints in the male indicate that a slightly lower limit ( 6% reduction in terminal BWT) may be appropriate for the male pubertal protocol, and in cases where the terminal BWT difference approaches 9–10%, additional studies and/or a weight of evidence approach should be used when interpreting the data for the thyroid endpoints.
As mentioned, several studies have evaluated the role of BWT and growth on reproduction and development by altering the food intake of rats using several different approaches. However, the overwhelming majority of these studies employed severe FR regimens (i.e., at least 50% restriction) or complete removal of the food for extended periods of time. Thus, these studies do not speak to the issue that is relevant to the dosing regimen employed in the pubertal protocols, namely to determine the extent to which less severe decreases in BWT may affect the required endpoints of the male and female pubertal protocols. By comparison, the relatively small changes in BWT that are permissible for the pubertal protocols (e.g., < 10%) that have been examined previously in our laboratory (Laws et al., 2000) and by others (Carney et al., 2004; Marty et al., 2003) have demonstrated no impact on the age of onset of puberty in the male or female. Other studies agree with data from this study in that no effects on the reproductive tract were observed following FR in the adult (Chapin et al., 1993, O'Connor et al., 1999).
In the current study, serum T was not significantly altered in the FR males, which would be consistent with the finding that PPS (which is androgen dependent) was also not affected by any of the FR regimens employed in our study. This observation is similar to our previous finding (Stoker et al., 2002) that serum T in male rats weighing 15% less following a similar FR regime was not different than control males. However, this result is in contrast to a report by Trentacoste et al. (2001). These investigators identified a significant reduction in serum T in males underfed from PNDs 22 to 47 and whose terminal BWT at PND 48 was 10% lower than controls. The lack of a difference in serum T observed in the present study would be consistent with the lack of difference in the age at PPS in both our studies and that reported by Marty et al. (2003). Trentacoste et al. (2001) did not measure PPS in their study and, although they reported a decrease in the absolute weight of the ventral prostate and seminal vesicle, there did not appear to be a difference in the relative weight of these organs. Regardless, the changes reported by Trentacoste and the effect on the reproductive tract endpoints in this study were only observed when the MTD of 10% or greater change in BWT was achieved. When the decrease in BWT was kept below this 10% limit, none of the reproductive endpoints were significantly different from controls.
In the present study, in the 30 and 40% FR groups (or 9 and 19% BWT loss), T3 and T4 were both significantly decreased in the males. TSH was also decreased in the most severely FR (e.g., 19% reduction in BWT) group in the males. The serum thyroid hormones of the female rats were not altered in any group. No histopathological changes were observed in the thyroid of either sex. Thus, it appears that the male thyroid axis may be more sensitive to smaller changes in BWT than the female. Or, it is possible that these results reflect the extra 10-day exposure period in the male as opposed to the female protocol. This unexpected observation is in contrast to our earlier finding that neither T3 nor T4 were altered in male FR Wistar rats whose mean terminal BWT was 15% lower than controls (Stoker et al., 2002). O'Connor et al. (1999) reported effects on T3 and T4 in FR males with terminal BWT 15% lower than controls. However, a 10% reduction in BWT had no effect on these thyroid endpoints. Regardless, this finding underscores the fact that a weight of evidence decision must be used during which BWT, thyroid hormone concentrations, as well as thyroid histology are evaluated before concluding that the thyroid homeostasis has been disrupted by exposure to a test chemical.
Although serum glucose and leptin concentrations are not required endpoints for the pubertal protocols, they were evaluated in this study to determine the impact that any alteration in circulating levels may have on pubertal development. While there were no effects of FR on serum glucose concentrations in the male, there was a significant decrease in the females following 30 and 40% FR. During periods of protein and/or energy deficits, as with inadequate food intake, compensatory mechanisms serve to lessen the impact of the deficiencies. During acute FR, circulating glucose and glycogen are initially used as fuel sources (Senoo, 2000). Over time, certain tissues such as the heart and brain, which normally obtain energy from glucose oxidation, acquire the ability to use keto acids for fuel requirements, and the protein consumed is utilized more efficiently. Although the mean concentration of glucose was decreased in the more severe FR female groups (e.g., 12 and 19% lower BWT as compared with control), this did not alter the age of pubertal onset. Absence of any effect on glucose concentration in the male rat may be due to a longer adjustment period for the compensatory mechanisms of energy homeostasis or the male may have had higher stores of energy fuel sources available at weaning than the female. In any case, the energy sources appear to have been adequate for the maintenance of the developing reproductive tract and physiological events associated with the onset of puberty. Several studies have shown that the presence of leptin is necessary, but not sufficient, for reproductive development (Barash et al., 1996; Bronson, 2001; Cunningham et al., 1999; Mann and Plant, 2001). Our data support the idea that circulating leptin concentrations may act in a permissive role rather than as a trigger for the onset of puberty. Here, a reduction in circulating leptin concentrations at necropsy was observed in FR males (30 and 40%) and females (40%). However, reductions in leptin concentrations in these animals did not cause any change in the age of pubertal onset.
In summary, the results of the present experiments indicate that reductions in BWT up to 9% in the male and 12% in the female are without effect on the reproductive endpoints examined in the male and female pubertal protocols. The no effect level for the thyroid endpoints was 6% in the males and > 19% in the females. These observations support the concept that a 10% reduction in BWT is a reasonable basis for setting the maximum dose for the female pubertal protocol in conjunction with any other information concerning the toxicity of the test compound (e.g., clinical signs, other toxicity; Hodgson, 1987). However, the changes observed in the male thyroid hormones indicate that a slightly lower MTD ( 6% reduction in terminal BWT) may be appropriate for the male pubertal protocol.
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NOTES |
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2 Both authors contributed equally to this work.
Disclaimer: This manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendations for use.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge author Walden, Al Moore, Faye Poythress, Debbie Crawford, and Guillermo Orozco of New Year Tech for their technical support and assistance with animal care; Suzanne Hodge, Kate Bremser, and Keith McElroy for monitoring the animal body weight changes, preparing the daily aliquots of chow, and assisting with the animal necropsy and RIAs, and Drs. Jim Stevens and Charles Eldridge for their reviews and helpful comments on earlier drafts of the manuscript.
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