Environmental Health Science Laboratory, Sumitomo Chemical Company, Ltd., 3-1-98, Kasugade-naka, Konohana-ku, Osaka 554-8558, Japan
Received May 17, 1999; accepted September 2, 1999
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ABSTRACT |
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Key Words: endocrine disruptor; screening; Hershberger assay; pubertal male assay; flutamide; antiandrogen; rat.
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INTRODUCTION |
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Chemicals have been evaluated for adverse effects by toxicity testing guidelines such as the multigeneration reproduction study, the chronic toxicity study, and the carcinogenicity study, etc. However, it has been argued that these studies were not specifically designed to be used as screening tools to detect endocrine-disrupting compounds. In the U.S.A., the Food Quality Protection Act (FQPA) of 1996 and the Safe Drinking Water Act (SDWA) of 1996 require the United States Environmental Protection Agency (U.S. EPA) to develop a screening/testing strategy for identification of endocrine-disrupting compounds. Subsequently, the U.S. EPA formed a committee composed of multiple and varied stakeholders, the Endocrine-Disruptor Screening and Testing Advisory Committee (EDSTAC). The endocrine-disrupting effects of many xenobiotics may result from interference with the hormonal regulation of the normal development and function of the male and female reproductive tracts (Danzo, 1998; Kelce and Wilson, 1997
; Kelce et al., 1998
). EDSTAC developed a screening and testing strategy to identify compounds that are androgenic/antiandrogenic, estrogenic/antiestrogenic, or steroid biosynthesis inhibitors, as well as compounds that alter thyroid hormone function (U.S. EPA, 1998
). The Organization for Economic Co-operation and Development (OECD) has also published a review paper: appraising test methods for these chemicals (OECD, 1997). For the purpose of identifying environmental compounds that potentially disrupt endocrine function, several different screening assays for detecting these endocrine-active compounds (EACs) have been proposed (U.S. EPA, 1998
; Gray et al., 1997
; OECD, 1997). Validation of the usefulness of these assays will be conducted in many laboratories.
A rodent 57 day Hershberger assay is one of the assays in the proposed Tier 1 Screening Battery by EDSTAC (U.S. EPA, 1998). OECD has also selected the Hershberger assay for priority validation. The purpose of this assay is to identify potential androgenic and antiandrogenic compounds based on the response of androgen-dependent tissues in the castrated, or castrated and testosterone (T)-treated animals (Gray et al., 1997
; Hershberger et al., 1953
; OECD, 1997; U.S. EPA, 1998
). In the EDSTAC proposal, a rodent 20-day thyroid/pubertal male assay is one of the candidates to replace the Hershberger assay (U.S. EPA, 1998
). The evaluation of a pubertal male assay is also in progress (Ashby and Lefevre, 1997
; Kelce and Wilson, 1997
).
As a part of our research program to develop short-term in vivo screening methods to detect EACs, we initiated a validation of the Hershberger assay. In the current study, we compared the activity of the 5-day Hershberger assay utilizing mature male rats and the pubertal male assay for detecting antiandrogenic agents. Flutamide was used as a standard androgen receptor antagonist in this early phase validation (Kelce et al., 1997; Koch, 1984
).
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MATERIALS AND METHODS |
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Animals and Housing
All experiments were performed under The Guide for Animal Care and Use of Sumitomo Chemical Co. Ltd. Male Crj:CD(SD)IGS rats were purchased from Charles River Japan, Inc. (Shiga, Japan). They were 9 weeks or 1415 days of age (the day of birth of the animal was counted as day 0) upon arrival, and were acclimatized to the laboratory environment for 7 days before use. Immature rats were received at our laboratory with their dams or foster dams and acclimatized with them. During the quarantine period of 7 days, all animals were examined for clinical signs and weighed, and animals judged to be in good health, based on these findings, were selected for the study. Rats were housed 2 per cage in stainless steel wire mesh cages under controlled environmental conditions, including a temperature of 24 ± 2°C, a relative humidity of 55 ± 10%, a frequency of ventilation of more than 10 air exchanges/h, and a 12-h light/dark cycle (light on, 08002000 h). Drinking water and pellet rodent diet were available ad libitum. Prior to operation (Experiments 1 and 2) or treatment (Experiment 3), rats were assigned to each group by the stratified randomization method using a computer program based upon body weight and body weight gain during a quarantine period, so no significant difference in mean body weight was observed among the groups.
Castration and Testosterone Capsule Implantation
Rats were anesthetized with ether and then castrated. In addition, testosterone (T)-containing capsules were inserted subcutaneously through an incision made posterior to the scapulae. The capsule preparation was similar to the previous reports (Bookstaff et al., 1990a,b
; Kelce et al., 1997
; Smith et al., 1977
). Briefly, capsules were made by tamping crystalline testosterone (approximately 30 mg/capsule) into silicone rubber tubing (1.6 mm id, 3.2 mm od, 25 mm length). Each end of the capsule was sealed by silicone, and wiped clean with ethanol.
Study Design
Experiment 1 (optimization for conditions of capsule implantation).
Male rats (10 weeks of age, 6 rats/dose) were castrated and immediately implanted with 2 empty capsules, or with 1 or 2 capsules containing T. This was performed to determine a suitable value of T implanted into castrated rats that indicates accessory organ weights equivalent to those of intact rats. Control rats were sham-operated without the castration and capsule implantation. Based on body weight and food consumption, the animals were considered recovered from the operation stress within 1 week. Six days after the operation, rats were transferred to a room adjacent to the necropsy room in the morning. All rats were kept quietly at least one h prior to blood sampling (to avoid the effects of transfer stress on hormone levels). All rats were euthanized by blood withdrawal from the abdominal aorta under light ether anesthesia. The light ether anesthesia was previously confirmed for not affecting unduly circulating T and LH levels. Blood sampling from the abdominal aorta was selected since it is able to provide enough volume for analyses. Collected blood was used for determination of serum T and LH levels. After careful trimming to remove fat and other contiguous tissue in a uniform manner, wet weights of ventral prostate and seminal vesicles (with coagulating glands) were examined (seminal vesicles were weighed after fixation overnight in a 10% neutral-buffered formalin). Ventral prostate and seminal vesicles (with fluid) were weighed separately since these organs respond to different androgens (Gray et al., 1997; Prahalada et al., 1998
).
Experiment 2 (activity evaluation of the 5-day Hershberger assay for flutamide).
The operated male rats (11 weeks of age, 6 rats/dose) were treated by gavage with flutamide (0, 0.15, 0.6, 2.5, or 10 mg/kg/day). This was performed to determine whether the castrated, T-implanted rats respond to the antiandorogenic activity of flutamide at lower doses. In the current experiment, 2 capsules containing T were implanted in each rat based on results in Experiment 1. Seven days after the operation, rats were dosed for 5 days, then euthanized on the morning of test day +6, following the same procedure as Experiment 1. The highest dose (10 mg/kg/day) was selected based on the previous study: the antiandrogenic effect of flutamide was clearly observed (Cook et al., 1997; Ladics et al., 1998
; O'Connor et al., 1998
). In the current experiment (together with the following Experiment 3), carboxymethylcellulose (0.5%) was used as the vehicle, and the dose volume was 5.0 ml/kg body weight. Organ weights (ventral prostate, seminal vesicles with coagulating glands, and levator ani muscle plus bulbocavernosus muscle), and serum hormone levels (T and LH) were determined together with the indices of toxicity such as body weight, food consumption, hematology, and blood biochemistry. Histopathology of the ventral prostate, seminal vesicles, and levator ani plus bulbocavernosus muscle followed standard procedure.
Experiment 3 (comparison of an activity with other assays).
A comparison was made with the activity of the 5-day Hershberger assay utilizing mature male rats versus 2 other assays: a 5-day assay utilizing mature intact male rats and a 20-day assay utilizing immature intact male rats. The 20-day treatment period was selected since a rodent 20-day thyroid/pubertal male assay is a candidate to replace the Hershberger assay (U.S. EPA, 1998). Age-matched intact male rats (11 weeks of age, 6 rats/dose) were treated by gavage with flutamide (0, or 10 mg/kg/day) for 5 days. Immature male rats (2223 days of age, 10 rats/dose) were treated by gavage with flutamide (0, 0.15, 0.6, 2.5, or 10 mg/kg/day) for 20 days. One day after the last dosing, all animals were euthanized as described for Experiment 1. Endpoints described in Experiment 2 were examined. For the pubertal male assay, statistical analysis was performed on data from all 10 animals per dose, or on data from the first or last 6/10 animals examined per dose. These analyses were performed to identify the sensitivity and power of large numbers (Tables 2 and 3
).
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Hormone measurement.
Serum was prepared from the collected blood and stored at 80° until analysis of serum hormone concentrations. Serum T and LH were measured using commercially available RIA kits. All samples were measured in duplicate in the same assay, the intra-assay coefficients of variations being 89% for testosterone, and 915% for LH, respectively.
Statistical analyses.
All data were analyzed by one-way analysis of variance (ANOVA) followed by a least significant difference (LSD) test. The hormone data were analyzed by the Kruskal-Wallis test, followed by Mann-Whitney U-test, if values were below a limit of quantitation. The significant difference from the control group was estimated at a probability level of 1 and 5%.
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RESULTS |
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Activity Evaluation of the 5-Day Hershberger Assay for Flutamide
Castrated rats implanted with two capsules containing T (6 rats/dose) were treated by gavage for 5 days with vehicle or flutamide (0.15, 0.6, 2.5, or 10 mg/kg/day). Based on data from body weights, food consumption, hematology, blood biochemistry, and organ weights apart from accessory sex organs, no serious systemic toxicity occurred following the administration of flutamide (data not shown). Serum T and LH levels and weights of ventral prostate and seminal vesicles in controls were similar to those of the castrated rats implanted with 2 capsules containing T in Experiment 1. Therefore, the procedure exhibits high reproducibility (Table 1 and Figs. 1, 2, and 3
). Although significant increases in serum LH levels were observed at doses of 2.5 and 10 mg/kg/day flutamide (217 and 254% of control, respectively), serum T levels in all groups were similar to that of control (Table 1
). With respect to weights of the designated organs, flutamide decreased in a dose-dependent manner: statistical significance was observed in seminal vesicle and levator ani and bulbocavernosus muscles (
0.6 mg/kg/day) and ventral prostate (
2.5 mg/kg/day). For the values exhibiting statistical significance, percentage values compared to control were 86, 67, and 43% for the seminal vesicles; 89, 86, and 72% for levator ani muscle plus bulbocavernosus muscle; and 72 and 54 % for ventral prostate, respectively (Fig. 4
). Histopathological analysis revealed flutamide induced atrophy as follows: 4/6 rats dosed at 2.5 mg/kg/day and 5/6 rats dosed at 10 mg/kg/day showed ventral prostate atrophy; 2/6 rats dosed at 2.5 mg/kg/day and 6/6 rats dosed at 10 mg/kg/day showed seminal vesicle atrophy; and 1/6 rat dosed at 10 mg/kg/day showed atrophy of levator ani plus bulbocavernosus muscles.
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Antiandrogenic effects of flutamide were found in age-matched intact male rats after treatment with 10 mg/kg/day for 5 days, however, the responses were less than those found in the 5-day Hershberger assay (Fig. 5). For the 10-mg/kg/day treatment group, atrophy of the ventral prostate was observed in 3/6 rats; and 1/6 rats showed seminal vesicle atrophy. As shown in Table 1
, a dose of 10-mg/kg/day flutamide increased T levels (5-fold from the controls), concomitant with a slight increase in serum LH levels (127% of the controls). There were no changes in the testis or epididymis weights; however, a significant increase in the pituitary weight (118% of control) at 10 mg/kg/day flutamide was noted (data not shown).
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DISCUSSION |
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In intact animals, homeostatic mechanisms attempt to maintain steady-state conditions against external stresses. For males, the gonadal-hypothalamic-pituitary axis maintains peripheral androgen levels with some physiological variations. Testicular Leydig cells produce and secrete androgens, i.e., testosterone, in response to LH released from the anterior pituitary, which is stimulated by luteinizing hormone-releasing hormone (LHRH) from the median eminence in the hypothalamus. The elevated T level suppresses the release of LHRH and/or LH, which is subject to feedback inhibition by androgens. Consequently, peripheral androgen levels are maintained with some physiological variations. When the screening of EACs is performed by an in vivo assay using intact animals, although hormone levels are thought to be more sensitive in an intact animal (as discussed by Cook et al., 1997 and O'Connor et al., 1998), the possible endocrine activity of the test compounds in target organ levels may be modulated by homeostatic mechanisms. In addition, if the goal of screening is to specifically identify androgen receptor-mediated toxicants, intact animals may decrease a specificity of screening (Gray et al., 1997). The Hershberger assay is designed to detect androgenic and antiandrogenic effects of the test compounds in critical conditions, with a consistent level of serum androgens (Hershberger et al., 1953
). In this assay, the weights of the ventral prostate and seminal vesicles, preferably levator ani and bulbocavernosus muscles, are measured in castrated, or castrated, T-treated male rats after 47 days of treatment with the test compounds. A 5-day treatment period was selected for the current study since a shorter term would suffice for routine screening. The advantage of this assay, compared to other in vivo procedures, is that it is a fairly simple, short term, and relatively specific screening method for direct androgenic/antiandrogenic effects (Gray et al., 1997
).
The sensitivity of the end points described above was enhanced through the use of castrated rather than intact male rats (Fig. 5), which is consistent with the previous findings (Gray et al., 1997
; Hershberger et al., 1953
). For castrated male rats, under our study conditions, serum LH levels were increased by flutamide at doses of 2.5 mg/kg/day and more; however, serum T levels were similar to the control levels. On the other hand, in intact mature rats, serum T levels were increased by flutamide, attributed to the increased steroidogenic capacity from increased serum LH levels (O'Connor et al., 1998
). Flutamide also increases T production in the ex vivo testis explant assay (Powlin et al., 1998
). These changes appear to compensate for the antiandrogenic effects of flutamide at the accessory sex-organ levels. However, in castrated male rats, the observed effects of flutamide treatment on accessory sex-organ weights were considered to arise from the net antiandrogenic effects of flutamide, since serum T levels are relatively consistent.
For the Hershberger assay, there are 2 stages used to detect endocrine activity of the test chemicals. Castrated animals are used to screen for androgenicity, and castrated, T-treated animals are utilized for antiandrogenic responses. Regarding systemic T treatment for antiandrogenicity, injection or implantation is used (reviewed by Smith et al., 1977) because of the lipid affinity of steroid hormones; T can be dissolved or suspended in oil and administered subcutaneously or intramuscularly. This method prolongs hormone effectiveness, but because of the short half-life of hormonal steroids in the circulation, either multiple injections or very large doses must be used to maintain effective blood levels throughout most of the day. However, a large volume of oil treatment may have an effect on steroid metabolism in the liver, since oil alters the metabolism of cholesterol (precursor of steroids), possibly due to phytosterols in the oil (Howell et al., 1998). The implantation procedure has been used in many previous studies (Bookstaff et al., 1990a
,b
; Danzo, 1995
; Kelce et al., 1997
; Shirai et al., 1991
; Smith et al., 1977
). For injection procedures, a time lag of treatment between testosterone and test compound may alter the sensitivity of the screening. In contrast, the implantation procedure can leave the time lag out of consideration. Because of this, we selected implantation for the current study. As shown in Figures 1 and 2
, implantation of two 25-mm capsules maintains the serum T levels within a physiological range, as well as serum LH levels and the weights of accessory sex organs. Kelce et al. (1997) found that implantation of two 25-mm capsules proved more effective. The reason for this difference is unknown; however, it may be due to a difference in capsule components. Sensitivity of this assay is affected by serum T levels in T-treated rats: levels below or above the suitable levels of serum T might either reduce a range of weight differences and decrease sensitivity or may make the response resistant to androgen antagonists and less sensitive. In fact, the magnitude of effects by antiandrogens are relatively higher in rats with physiological serum T levels than in rats with higher serum T levels (Kelce et al., 1997
). Therefore, we were more concerned with achieving physiological serum T levels than with the actual value of the T dose. Under the hormone conditions of our experiments, antiandrogenic effects of flutamide were detected in a dose-related manner, as shown in Figures 3 and 4
. In comparison, with a 14-day intact mature male assay (Cook et al., 1997
; O'Connor et al., 1998
), the magnitude of response was greater or equivalent. Furthermore, although the preliminary data remains to be confirmed, our system was able to detect an antiandrogenic effect of a weaker antiandrogen, p,pprime;-DDE, at a dose level of 100 mg/kg/day: the weights of seminal vesicles and ventral prostate were approximately 75% of control. These findings suggest that our study conditions are suitable for screening of antiandrogens.
According to the EDSTAC, the Rodent 20-day thyroid/pubertal male assay is one of the candidates to replace the Hershberger assay. The usefulness of a weanling male rat assay for antiandrogens has been described (Ashby and Lefevre, 1997; Kelce and Wilson, 1997
). If a single assay can detect compounds that alter thyroid hormone function and androgenic/antiandrogenic compounds, its use would be very cost effective. Therefore, in the current study, we comparatively evaluated the activity of the pubertal male assay for detecting antiandrogenic effects of flutamide. Results indicate that the pubertal male assay (if 10 animals per dose were used) was able to detect antiandrogenicity. However, in the case of 6 animals per dose, statistically significant changes were observed in the seminal vesicles and levator ani plus bulbocavernosus muscles but not in the ventral prostate. This may be due to the large variation of individual data because of the small size of the organs. Therefore, the reliability and relevance of the assay utilizing immature intact rats remains to be determined, although it appears to be a useful screening tool.
Based on our findings, the seminal vesicles, and possibly the levator ani plus bulbocavernosus muscles, appear to be more sensitive to flutamide than the ventral prostate. This is consistent with previous findings (Kelce et al., 1997; O'Connor et al., 1998
). According to the O'Connor data, the magnitude of the T increase by flutamide was marginally higher than that of dihydrotestosterone (DHT), as follows: dosages were 1, 5, and 20 mg/kg/day; 175, 264 and 464% of control for T; and 155, 184, and 343% of control for DHT. Prostate growth is dependent upon the enzymatic reduction of T to DHT (Prahalada et al., 1998
), whereas those of the seminal vesicle and the levator ani plus bulbocavernosus muscles are T-dependent (Gray et al., 1997
). We did not determine the DHT levels; however, the differences of sensitivity among organs may be due to differences of the growth regulation in each organ. Anyway, the weights of seminal vesicle and ventral prostate, and possibly levator ani and bulbocavernosus muscles, are considered to be suitable endpoints for screening of antiandrogenic agents.
In conclusion, the 5-day Hershberger assay utilizing mature male rats was more sensitive than the 5-day assay utilizing mature intact male rats or the pubertal male assay. For detection of the antiandrogenic activity of flutamide, the implantation procedure utilized in this study contributed to high sensitivity of the 5-day Hershberger assay when compared with other assays, suggesting that our study conditions are relevant. Consequently, our findings support that the rat 5-day Hershberger assay utilizing mature male rats is a sensitive and short-term screening method for the detection of antiandrogenic compounds in vivo. On the other hand, if 10 animals per dose are used, the pubertal male assay also appears to be useful; however, further studies are required to investigate the reliability and relevance of this assay.
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ACKNOWLEDGMENTS |
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NOTES |
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