National Institute of Toxicological Research, Korea Food and Drug Administration, 5 Nokbun-dong Eunpyung-gu, Seoul 122704, Korea
Received July 19, 2001; accepted December 12, 2001
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ABSTRACT |
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Key Words: endocrine disrupters; female pubertal assay; vaginal opening; DES; tamoxifen; flutamide; testosterone; estrous cyclicity; hormone analysis.
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INTRODUCTION |
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The U.S. Environmental Protection Agency (EPA) established the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) in 1996. The EDSTAC has recommended the screening strategy to detect endocrine-active compounds that are agonist/antagonist to the estrogen/androgen receptors, steroid biosynthesis inhibitors, or altering thyroid hormone function (EDSTAC, 1998). The rodent 20-day pubertal female assay is one of the Tier I screening assay recommended by EDSTAC, and uses vaginal opening (VO) as an indicator of pubertal onset. The determination of the onset of puberty in the female rat is an endpoint that has been included in several standard toxicology test protocols. Moreover, several studies have demonstrated that the onset of puberty in female rat is associated with VO and first estrus (Ramaley, 1981
; Ramirez and Sawyer, 1965
). VO usually occurs at around 3037 days after birth in female rats, and the onset of the first estrus occurs at about 5 weeks, although variations occur between strains or between different colonies of the same strain (Clark, 1999
; Goldman et al., 2000
; Rivest, 1991
). Estrogens are potent regulatory factors of physiological responses and play an important role in puberty. Precocious VO can occur in response to estrogens or estrogen-like chemicals (Allen and Doisy, 1924
; Ashby et al., 1997
; Laws et al., 2000
; Nass et al., 1984
; Odum et al., 1997
). Conversely, inhibition of aromatase or steroid synthesis can delay the age at VO (Marty et al., 1999
). Ultimately, natural or synthetic estrogens may have serious consequences on the reproductive cycle in humans and animals (Korach, 1993
; Kuhnz and Putz, 1989
). Furthermore, the pubertal female assay is designed to detect chemicals that alter steroidogenesis or alter the hypothalamic-pituitary control of ovarian function. This assay also examines the endpoints associated with the development of female sex organs and secondary sexual characteristics including reproductive organs weights, hormonal status, and estrous cyclicity (EDSTAC, 1998
; Goldman et al., 2000
; Laws et al., 2000
).
The U.S. EPA is in the process of standardizing the operational protocol for the rodent pubertal female assay. Actually, relatively few studies have been conducted to detect estrogenic/antiestrogenic compounds under the current protocol (Laws et al., 2000; Marty et al., 1999
). Gray and coworkers (1989) demonstrated that the estrogenic or antiandrogenic pesticide methoxychlor significantly accelerated the onset of VO and the first estrus in Long-Evans rats using a similar protocol. The VO was advanced by 7 days and the length of the estrous cycles were increased following exposure to higher doses of methoxychlor (50 and 100 mg/kg, po). In addition, a number of other environmental compounds have been reported to affect the VO and estrous cycle. For example, the alkylphenol compounds, 4-tert-octylphenol and p-nonylphenol, were found to increase uterine weight in prepubertal rats and to advance VO (Gray and Ostby, 1998
; Laws et al., 2000
; Odum et al., 1997
).
Although it is well known that estrogenic compounds affect puberty, few studies have used this protocol to investigate female pubertal onset with compounds having androgenic/antiandrogenic activity. Therefore, we undertook this study to evaluate whether this rodent pubertal female assay can detect EDs with a variety of endocrine activities. Accordingly, we used this protocol to examine 4 positive EDs with diverse activities. The test compounds included an estrogen receptor agonist, diethylstilbestrol (DES), an estrogen receptor antagonist (tamoxifen), an androgen receptor agonist (testosterone), and an androgen receptor antagonist (flutamide).
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MATERIALS AND METHODS |
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Animals.
Sprague-Dawley Crl:CD female rats (Charles River Laboratories) were obtained from the KFDA Laboratory Animal Resources (Seoul, Korea) under specific pathogen-free (SPF) conditions. Dams with litters of 18-day-old female rats were received and housed in clear polycarbonate cages for 3 days prior to the start of dosing. All animals were maintained under a 12 h light-dark cycle. Ambient air temperature was controlled at 23 ± 2°C and relative humidity was maintained at 50 ± 10%. Prior to the experiment, all animals were checked for overt signs of illness and only healthy animals were selected for the study. All animals were maintained on Certified Rodent LabDiet (#5057, Purina, USA), and filtered and chlorinated tap water was supplied using glass bottles. Food and water were available ad libitum.
Study groups.
All test compounds were dissolved in a minimal amount of 95% ethanol and diluted to a final working concentration with corn oil (final concentration of ethanol was less than 1.0%). The study design called for 10 animals per group. At weaning (21 days of age), the animals were randomized into the various treatment groups based on the body weight. The body weight variation per group was within the mean ± 5 g. The following treatment groups were used: vehicle control (oral gavage), DES (0.2, 1.0, and 5.0 µg/kg/day, oral gavage), tamoxifen (10, 50, and 200 µg/kg/day, oral gavage), testosterone (0.05, 0.2, and 1.0 mg/kg/day, sc), and flutamide (1, 5, and 25 mg/kg/day, oral gavage). According to the EDSTAC Final Report (1998), the recommended exposure route for this protocol is by oral gavage. In the present study, a preliminary experiment was performed to investigate the effects of testosterone (5 mg/kg/day) by oral gavage using this assay protocol. However, no significant differences between testosterone and the control were observed. Generally, testosterone has low bioavailability when given by the oral route, owing to gastrointestinal and hepatic inactivation. Thus, testosterone was administered by sc injection to maximize the sensitivity of this assay. In terms of dosage level selection, EDSTAC (1998) recommended that only one high dose of a test compound (at or just below the maximum tolerated dose or MTD) is required when conducting this assay. Generally, the MTD is broadly defined as the dose that a test animal can tolerate without any adverse physical effects. Most studies have used reductions in body weight (generally a body weight gain that is 10% lower than the control) as a single criterion. However, it is not clear in the present guidelines. Thus, a high dose of each compound was selected, as used by previously described studies. All test compounds were administered from 21 days of age for 20 days. The total amount of injection volume per rat was 3 ml/kg/day.
Clinical signs and body weights.
Throughout the study period, each animal was observed at least once daily for clinical signs of toxicity related to chemical treatment. On working days, all cages were checked in the morning and afternoon for dead or moribund animals. The body weight of each rat was recorded daily to the nearest 0.1 g, measured just prior to treatment.
Measurement of organ weights.
Twenty-four hours after the last treatment, each rat was anesthetized with CO2 in the same sequence as the test substance was administered. The uterus and ovary were dissected, and carefully trimmed free of fat, to avoid loss of lumenal contents. The body of the uterus was cut just above its junction with the cervix and at the junction of the uterine horns with the ovaries. The vagina was removed from the uterus at the level of the uterine cervix and the ovarian and vaginal weights were measured. The uterus was weighed with the lumenal contents. The liver, heart, kidney, thyroid, thymus, pituitary glands, and adrenal glands were carefully dissected and weighed.
Vaginal opening and estrous cyclicity.
Each animal was examined daily for VO beginning on postnatal day (PND) 21. On the day that VO was first detected, the age and body weights were recorded. Daily vaginal lavage was collected from the day following VO and then daily until the end of the study, by repeated pipetting of 0.9% saline into the vagina. The lavage fluid was applied to a clean glass slide and the smear was viewed immediately under low magnification (x100) with microscope. Cytology was evaluated and the stage of the estrous cycle was determined using the method of Everett (1989).
Hormonal measurements.
Blood was collected from the abdominal aorta approximately 24 h after the last treatment of test compounds. Serum was prepared immediately and stored at -75°C until analyzed for serum hormone concentrations. Commercially available radioimmunoassay (RIA) kits were used to determine serum concentrations of E2 and T4 (Amersham Corp., Arlington Height, IL).
Statistical analysis.
All values are expressed as mean ± SE (n = 10 animals). Data for mean initial or necropsy body weights, the mean ages and body weights at VO, organ weights, and hormone levels were analysed statistically for homogeneity of variance using Bartlett's test. When samples were proven to be homogeneous, nonparametric analysis of variance was applied. Absolute organ weights were analysed using analysis of covariance (ANCOVA) with the body weight at necropsy as a covariate. When a significant treatment effect was present, Dunnett's test (control vs. treatment group) was used to compare treatment groups. If significant heterogeneity of variance was apparent, some data transformations were performed prior to statistical analyses. In addition, hormone data were log transformed for statistical analysis. Where significant differences between the groups were detected, the Dunnett's or Bonferroni's tests were used. The level of statistical significance was set a priori at = 0.05.
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RESULTS |
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Vaginal Opening
In the present study, the mean age and body weight at VO were 32.3 ± 0.5 days (range: 3035 days) and 117.4 ± 7.2 g in control animals, respectively. DES significantly advanced VO to 24 days of age for animals treated with 5.0 µg/kg DES (Fig. 2). The mean body weight at the time of VO was also significantly reduced in 5.0 µg/kg doses of DES (64.7 ± 1.7 g; Fig. 3
). VO was first detected at 24 days of age in all animals treated with 5.0 µg/kg DES (Fig. 4
). Tamoxifen significantly accelerated VO to 27.8 ± 0.5 and 25.1 ± 0.1 days of age in animals treated with 50 and 200 µg/kg of DES, respectively (Fig. 2
). Mean body weights at VO were also significantly reduced by tamoxifen at 50 µg/kg (85.0 ± 2.2 g) and 200 µg/kg (67.9 ± 0.9 g; Fig. 3
).
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Neither 1 mg/kg nor 5 mg/kg flutamide affected the age at VO. However, the highest dose (25 mg/kg) of flutamide significantly accelerated VO (26.1 ± 0.1 days; Fig. 2). Mean body weight at VO was also significantly decreased at 25 mg/kg of flutamide (73.8 ± 2.1 g) compared to the control (117.4 ± 7.2 g).
Estrous Cyclicity
The estrous cycles of individual animals were observed from the day after VO until the end of the study. Schematic diagrams of the estrus cyclicity of individual animals are shown in Figure 4. The number of days in each stage of the estrous cycle and cycle length were documented. In the case of DES treatment, the number of days in estrus was increased at the high dose of DES treatment (5.0 µg/kg). The estrous cycle of female rats treated with tamoxifen was also significantly affected in a dose-dependent manner, and at 50 µg/kg, the number of days in diestrus increased (data not shown). Tamoxifen treatment had profound effects at 200 µg/kg, and tamoxifen treatment at this level produced a persistent diestrus smear throughout the entire observation period. Moreover, none of the rats had a normal estrous cycle during the study period (Fig. 4
). Testosterone also had a profound effect on the estrous cycle at all doses examined, and the number of days in proestrus was not observed at high testosterone dose (1.0 mg/kg; Fig. 4
). The estrous cycles of rats treated with 1.0 and 5.0 mg/kg flutamide were unaffected. However, the higher dose of flutamide (25 mg/kg) showed a similar pattern to tamoxifen; the number of days in diestrus was increased (Fig. 4
).
Hormone Measurements
Serum hormone concentrations were evaluated individually in all female rats regardless of estrous cycle stage. Serum estradiol (E2) concentrations were not different from the control group by DES, testosterone, and flutamide treatment. However, tamoxifen (50 µg/kg) administration caused a statistically significant increase in serum E2 levels to 158% of the control level of 15.81 ± 1.74 pg/ml (Table 3). Serum TSH levels were significantly increased by tamoxifen (10 and 50 µg/kg), testosterone (0.2 mg/kg), and flutamide (1.0 and 25 mg/kg) compared with the control animals (0.65 ± 0.17 ng/ml). Serum T4 concentrations were similar to the control after DES, tamoxifen, and flutamide treatment. However, serum T4 levels were significantly reduced after testosterone treatment at 0.05, 0.2, and 1.0 mg/kg to 55, 60, and 64% of the control levels (3.27 ± 0.23 µg/dl). Serum T3 levels were significantly increased after DES, tamoxifen (10 and 50 µg/kg), testosterone (1.0 mg/kg), and flutamide (1.0 and 5 mg/kg) treatment (Table 3
).
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DISCUSSION |
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Changes of VO day, the body weight at VO, and estrous cyclicity in prepubertal Sprague-Dawley rats following daily exposure to estrogenic/antiestrogenic or androgenic/antiandrogenic compounds from PND 21 to PND 40 days were examined. The summary results of the pubertal female assay are shown in Table 4. Control rats had a mean VO age of 32.3 ± 0.5 days. DES (5.0 µg/kg), tamoxifen (50 and 200 µg/kg), and flutamide (25 mg/kg) significantly advanced VO day, whereas treatment with testosterone (1.0 mg/kg, sc) significantly delayed this process. Several studies have reported that VO in rodents usually occurs around 3342 days after birth. In the present study, the mean range of VO was 3035 days in Sprague-Dawley female rats, which is consistent with those of previously reported data (Clark, 1999
; Cooper et al., 1993
; Goldman et al., 2000
).
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In order to evaluate the ability of this assay to detect estrogenic compounds, weaning rats were exposed to DES (0.2, 1.0, and 5.0 µg/kg) and were examined for VO, body weight at VO, and estrous cytology. As expected, DES (5.0 µg/kg) significantly accelerated VO to 8.4 days earlier than the control (Fig. 2). VO was first detected at 24 days of age in all rats treated with 5.0 µg/kg DES, which also significantly decreased body weights at the time of VO by more than 45% compared to the control. These results are agreement with previous data that DES (silastic implants) advanced VO days followed by 1217 days of persistent diestrus before regular estrus cycles (Nass et al., 1984
). Several studies have demonstrated that estrogenic chemicals, such as nonylphenol and octylphenol affect VO in rodents (Gray and Ostby, 1998
; Laws et al., 2000
). In addition, methoxychlor (25 mg/kg) administered by oral gavage starting at weaning was found to advance VO (average 6 days advanced), the appearance of the first estrus, and the onset of estrous cycles (Gray et al., 1989
).
DES (1.0 µg/kg) also significantly reduced ovarian weights, whereas no significant differences in necropsy body weights or uterine weights were observed in any treatment group. In our preliminary study, higher doses of DES (> 5.0 µg/kg) significantly decreased body weight as compared with the controls (data not shown). Thus, such high doses were not selected for this assay validation, because pubertal development can be influenced by body weight differences (Glass and Swerdloff, 1980). This was particularly important when evaluating results of the female pubertal assay, since it is known that lower body weight during the prepubertal period will delay the onset of puberty (Goldman et al., 2000
). A statistically significant decrease (87% of the control) in absolute kidney weight was observed at 1.0 µg/kg of DES. The reason for this effect is not known.
In order to detect antiestrogenic compounds, weaning rats were exposed to tamoxifen (10, 50, and 200 µg/kg). A statistically significant acceleration of VO (an average advance of 5 and 7 days) was observed in rats treated with tamoxifen (50 and 200 µg/kg). The ranges of age at VO were 2630 days and 2526 days in rats treated with 50 µg/kg and 200 µg/kg of tamoxifen, respectively. Wakeling and Bowler (1988) found that tamoxifen significantly accelerated VO in neonate rats treated with 25 µg/rat/day for 46 days. Our data are consistent with those of a previous report, which found that tamoxifen produces estrogen-like responses in the rodent pubertal female assay (Wakeling and Bowler, 1988). Tamoxifen treatment in ovariectomized CD1 mice has been found to increase both the lumenal epithelial thickness and the BrdU labeling index in the endometrial stroma of the uterus. (Carthew et al., 1999
). In contrast, tamoxifen was found to exert an antiestrogenic effect on the uterotrophic response to estradiol benzoate when the 2 chemicals were administered concurrently (Wakeling, 1989
).
In the present study, tamoxifen, like DES, significantly decreased ovarian weights. Moreover, tamoxifen (25 mg/kg) significantly decreased liver, heart, and kidney weights compared to the control, and this decrease in organ weights was accompanied by a significant decrease in final body weight. In addition, tamoxifen significantly decreased the pituitary and adrenal gland weights in a dose-dependent manner. It is considered that these results were caused by the effect of negative feedback inhibition of the cortical hormones, rather then being caused by the direct organ toxicity of tamoxifen, because the biosynthesis and secretion of adrenal cortical hormones is under the direct control of the trophic hormone, adrenocorticotrophic hormone (ACTH). Moreover, the secretion of ACTH is controlled by the negative feedback mechanism modulated by the circulating cortical hormones.
In order to evaluate the ability of this assay to detect androgenic compounds, weaning rats were exposed to testosterone (0.05, 0.2, and 1.0 mg/kg, sc). Testosterone significantly delayed VO and increased the body weight at VO. In general, serum testosterone concentrations increased during the onset of puberty in female rats (Hutter and Gibson, 1988; Lephart et al., 1989
). Mathews and coworkers (1987) reported that serum testosterone levels begin to rise during early proestrus and reach a 9-fold increase at the time of the first LH surge. Testosterone implanted at physiological concentrations (2 or 6 mg/ml in oil) into 28 days of age juvenile rats induced precocious VO and early proestrus, but not the first ovulation. They suggest that these results were closely associated with a direct effect on vaginal epithelium probably mediated by the local aromatization of testosterone. Bloch et al. (1995) have also reported that exposure to testosterone (silastic implants) during days 15 to 30 in female rats advanced VO.
Zarrow et al. (1969) observed that a single dose of 0.1 mg of testosterone on day 21 failed to alter the age of pubertal onset, while the same dose exposure during days 21 to 23 advanced both VO and the first estrus. In addition, a 10-day exposure (PND 21 to 30) advanced VO, but extended the time to the first estrus. They suggest that local estrogen produced by aromatization may mediate changes in the timing of VO by acting directly on the vaginal epithelium (Lephart et al., 1989). Although aromatase activity increased in vaginal epithelium of immature rats prior to the onset of puberty, serum estradiol concentrations were not increased in immature female rats treated with physiological levels of testosterone (Mathews et al., 1987
). These results are inconsistent with our data demonstrating that testosterone dose-dependently delayed VO, and that the highest dose (1.0 mg/kg) of testosterone significantly delayed by an average 5 days as compared with the control (Fig. 2
). The mechanism of action of this testosterone-induced delay on VO is unclear, but our data suggest that the high dose of testosterone delays VO by acting directly on the vaginal epithelium, and that this occurs despite the fact that physiological levels of testosterone can induce precocious VO.
In the present study, all doses of testosterone significantly reduced ovarian weight and the high dose of testosterone also significantly reduced thyroid and uterine weights, but necropsy body weights were significantly increased in high dose group (1.0 mg/kg). A statistically significant decrease in pituitary weights was observed at 0.2 and 1.0 mg/kg of testosterone. Although the decreased pituitary weight may be due to, in part, a direct effect of testosterone on the pituitary, the mechanisms are unclear.
The pure androgen receptor antagonist flutamide has been fully characterized in the female pubertal onset assay. In the present study, the effect of flutamide in the pubertal female assay was found to be similar to that of DES. Flutamide (25 mg/kg) significantly accelerated VO (7 days earlier than the control) and increased the body weight at VO. Generally, flutamide binds to the androgen receptor and effectively blocks androgen recognitions, and thus stimulation of gonadotropin release from the anterior pituitary caused an increase in the serum testosterone concentration (Lephart et al., 1989). Thus, our data suggest that the acceleration of VO is probably due to an increase in the estrogenic stimulus within the uterus caused by the attenuation of androgenic stimulus in immature rats. Therefore, a high dose of AR antagonist can alter female pubertal onset, which could play an important role in the development of the female reproductive function.
Flutamide at 25 mg/kg significantly decreased the liver weight. It is hypothesized that the increased liver weight induced by flutamide is associated with an induction of hepatic enzyme activity. Similar to tamoxifen, flutamide also significantly reduced the weights of the pituitary (1.0 and 25 mg/kg) and adrenal glands (1.0 mg/kg). However, the mechanisms of these changes in organ weights caused by flutamide treatment are unclear.
Vaginal smears are widely used to identify the phase of the estrous cycle and can indicate circulating estrogen (Cooper et al., 1993; Everett, 1989
; Ojeda and Urbanski, 1994
). Generally, a regular estrous cycle (diestrus, proestrus, and estrus) is defined as being 4 to 6 days and containing 1 to 2 days of estrus. Although we did not extend the study period to characterize the regular estrous cycles, this was partially successful in all treatment groups, except for the testosterone group. Nass et al. (1984) reported that regular estrous cyclicity is disrupted during adulthood following exposure to estrogen, DES, or testosterone from day 12 through to VO, which is substantially supported by our present data. In particular, high doses of tamoxifen (200 µg/kg) and flutamide (25 mg/kg) increased the number of days of diestrus. Although we did not investigate the histopathology of the ovaries, these changes may have been due to an alteration in the timing of the LH surge rather than being caused by the estrogen-like induction of a pseudopregnancy.
Serum hormone level may be useful for identifying compounds that alter hormone function. However, such data require cautious interpretation because many factors can affect hormone levels, e.g., diet, stress, and age (Donohoe et al., 1984). In addition, the measurement of steroid hormones, especially E2, has limited value because serum hormone levels are markedly influenced by the estrous cycle. In the present study, serum hormone concentrations were measured individually in all female rats regardless of estrous cycle stage, because the hormonal measurements were performed to determine whether compound-induced alterations in serum hormone concentrations could be detected, not to describe overall hormonal homeostasis. Serum E2 concentrations were found to be unaffected by DES, testosterone, and flutamide treatment, whereas tamoxifen administration caused a significant increase in serum E2 levels. In the case of hormones associated with thyroid function, serum T4 concentrations were unaffected by DES, flutamide, and tamoxifen treatment. However, testosterone significantly decreased serum T4 levels, and this change was correlated with thyroid weights. Several biochemical steps in the biosynthesis of thyroid hormones are vulnerable to interference by exogenous agents. In general, thyroid hormone (T4) concentrations may be affected by testosterone, but testosterone did not produce the hormonal pattern (increased TSH level and thyroid gland weight) characteristically induced by thyroid toxicants. Our data did not allow an exact interpretation of the correlation between the VO and the change in hormone levels observed in the female pubertal assay. A great number of animals per treatment group would be needed in any future study to clarify the effects of hormone balance on VO in female rats.
In summary, 4 potent active compounds were used to evaluate the female pubertal assay for the detection of EDs. DES and tamoxifen significantly advanced VO and decreased body weight at VO. Flutamide produced a pattern of responses characteristic of an ER agonist/antagonist in this assay, whereas testosterone dose-dependently delayed VO, and significantly decreased pituitary and adrenal weights. Our data suggest that the female pubertal assay may be useful for identifying chemicals that operate through a variety of mechanisms, although the compounds used in the present study were strong endocrine-active agents. Additional data is required on other compounds with weak endocrine disrupting activity to further characterize the sensitivity of the female pubertal assay.
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ACKNOWLEDGMENTS |
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NOTES |
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