Environmental Health Science Laboratory, Sumitomo Chemical Company, Ltd., 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-8558, Japan
Received October 7, 2003; accepted January 28, 2004
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ABSTRACT |
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Key Words: endocrine disruptor; Hershberger assay; thyroid hormone; screening.
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INTRODUCTION |
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The endocrine system is highly complex and includes many components, such as the hypothalamus, pituitary, testis, ovary, thyroid, adrenal, and pancreas. EDSTAC, an advisory committee to United States Environmental Protection Agency (U.S. EPA), has recommended that the potential to interact with either the sex steroids (estrogen and androgen) or with thyroid hormone function should primarily be evaluated (EDSTAC, 1998; Gray et al., 2002
).
EDSTAC has developed a screening and testing strategy to identify endocrine disrupters, in which the screening battery includes three in vitro assays and five in vivo assays for Tier 1 screening (T1S). The in vitro assays are an estrogen receptor binding or transcriptional activation assay, an androgen receptor binding or transcriptional activation assay, and a steroidogenesis assay using minced testis. The five recommended in vivo screens are a rodent three-day uterotrophic assay, a rodent 20-day pubertal female assay, a male rodent 57-day Hershberger assay, a frog metamorphosis assay for thyroid effects, and a fish gonadal recrudescence assay. In addition, the EDSTAC also proposed two alternative batteries deemed worthy of further evaluation: a 14-day intact adult male assay with thyroid and a rodent 30-day thyroid/pubertal male assay. Positive chemicals in the T1S would move into a second level (T2T) where more defined toxicological responses would be characterized in two-generation mammalian reproductive studies, for example.
At the same time, an OECD Task Force on Endocrine Disrupter Testing and Assessment (EDTA) has been established to provide a focal point within the OECD to identify and recommend priorities for the development and validation of methods for identifying endocrine active chemicals. The EDTA has selected the rodent Hershberger assay, the rodent uterotrophic assay, and the enhanced OECD 407 guideline study (28-day treatment study) to start international cooperative work (Gray et al., 2002).
The Hershberger assay is one in vivo assay proposed by both OECD and EDSTAC to test for chemicals that have the potential to act as androgens or anti-androgens, and indeed, it has long been widely used by the pharmaceutical industry for this purpose with drugs for potential therapeutic use (Dorfman, 1969a,b
; Hershberger et al., 1953
). Accessory sex glands/tissues require androgen stimulation to gain and maintain their weights during and after puberty. If male rodents are castrated (i.e., the endogenous testicular source of androgen is removed), exogenous androgen is necessary, if involution is to be avoided. In the castrated male rodent, therefore, effects on these tissues are likely to be direct and not a result of pituitary or gonadal secretion. For assessment of androgenicity, test chemicals are administered to castrated males, while for anti-androgenicity, test chemicals are given to castrated, testosterone-treated animals.
Since there are many variations in the protocol used for the Hershberger assay, development of an internationally recognized standard for the assay is in progress, which is currently being validated for specificity, sensitivity, and reproducibility within the OECD framework (Gray et al., 2002). The proposed OECD protocol for the Hershberger assay recommends 10 days of treatment to peripubertal male castrated rats, and includes weighing individual sex accessory tissues, since little is known about the response of individual sex accessory tissues to exogenous chemicals that may have androgenic effects. The mandatory organs are the ventral prostate, seminal vesicles together with coagulating glands, glans penis, Cowper's glands, and levator ani and bulbocavernosus muscles. In addition, determination of liver weight is highly recommended as some test substances appear to exert anti-androgenic effects by increasing metabolism of TP by hepatocytes. Furthermore, the weights of the adrenal glands and the kidneys and levels of serum luteinizing hormone and testosterone are included as optional endpoints (Gray et al., 2002
).
Recently, many articles concerning the Hershberger assay from the standpoint of endocrine disruptors have been published. All except for Vinggaard's study (Vinggaard et al., 2002) were focused solely on androgenicity and/or anti-androgenicity (Andrade et al., 2002
; Ashby and Lefevre, 2000b
; Ashby et al., 2001
; Date et al., 2002
; Kim et al., 2002
; Kitamura et al., 2003
; Kunimatsu et al., 2002
; Lambright et al., 2000
; Nellemann et al., 2001
; O'Connor et al., 1999a; Stroheker et al., 2003
; Sunami et al., 2000
; Wilson et al., 2002
; Yamada et al., 2000
, 2001
, 2003
; Yamasaki et al., 2001
, 2002a, 2003
). Vinggaard and coworkers determined effects of prochloraz, an imidazole fungicide, on levels of thyroxine (T4) and thyroid-stimulating hormone (TSH) as well as follicle-stimulating hormone (FSH) and testosterone in castrated rats treated with TP. However, there was no discussion about the reliability of the Hershberger assay for assessing thyroid hormone modulators (Vinggaard et al., 2002
).
As described above, thyroid hormone, as well as estrogen and androgen, is a high priority hormone to be evaluated. Thyroid dysfunction leads to abnormal development, altered growth patterns, and a variety of physiological perturbations in mammals (EDSTAC, 1998). As for tests to screen thyroid hormone modulators, the T1S battery recommended by EDSTAC includes a rodent 20-day pubertal female assay, the 14-day intact adult male assay and the rodent 30-day pubertal male assay, and the OECD has proposed the enhanced OECD 407 guideline (Gray et al., 2002
). Apart from thyroid hormone modulators, these assays are also designed to detest agents that affect steroid biosynthesis. Concerning thyroid hormone modulators, measurement of circulating levels of T4, tri-iodothyronine, (T3), and TSH, and thyroid histopathology are included in these tests. Additionally, measurement of the thyroid gland weight is required in the current draft protocol of the enhanced OECD 407 guideline study. Concerning dosing period, many in the literature discuss two weeks as the soonest they see microscopic changes (Biegel et al., 1995
; Hood et al., 1999a
; Jones and Clarke, 1993
; McClain et al., 1989
; O'Connor et al., 1999b
). Thus there is a possibility that a 10-day Hershberger assay would be able to detect thyroid modulators. Although the original Hershberger assay is mainly applied to assess (anti-)androgenic activities, if it were also reliable for assessing thyroid hormone modulating activity, it will become more comprehensive screening. In the present preliminary study, therefore we evaluated the reliability of the enhanced Hershberger assay to detect thyroid modulating activity, while concentrating attention on possible influence on confounding evaluation of (anti-)androgenic activity. As thyroid modulators, a thyroperoxidase inhibitor, propylthiouracil (PTU), and two hepatic enzyme inducers that enhance the clearance of thyroid hormones, phenobarbital (PB) and 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), were employed.
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MATERIALS AND METHODS |
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Animals and housing
All experiments were performed in accordance with 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) at the age of five weeks and acclimated to the laboratory environment for sevendays before use. During the experiment, the rats were housed two per cage in suspended aluminum cages with a stainless steel wire-mesh front and floor 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 (lighting period, 08002000 h). Drinking water and pellet rodent diet were available ad libitum. After the quarantine period, animals in good health (based on clinical signs and body weights) were selected for operations. Male rats at six weeks of age were castrated via the scrotum under ether anesthesia, and chemical treatment was not initiated until seven days later to allow for complete recovery from surgical stress.
Prior to administration of chemicals, rats were assigned to groups by a stratified randomization method using a computer program based on body weight and body weight gain during the quarantine and recovery periods to avoid body weight variation among the groups. At the commencement of treatment, it was confirmed that no significant differences existed in mean body weights among the groups, since such variation may be the source of variation in tissue weight (Ashby and Lefevre, 2000a; Monosson et al., 1999
; Sunami et al., 2000
). The initial body weight variation among the animals was within ± 20% of the mean body weight, and the body weight variance approximately equal across all groups in an experiment at the start of the treatment (the age of rats was seven weeks).
Study design
PTU, PB, or p,p'-DDE were administered for 10 days by oral gavage to castrated male rats (Experiment 1) or 0.2 mg/kg/day TP-treated (sc injections) castrated male rats (Experiment 2). Data for p,p'-DDE with TP-treated castrated rats (10-day treatment) were obtained from an experiment performed previously (Experiment 3), documented in an earlier publication (Yamada et al., 2001). Test substances and TP were dissolved in vehicle (corn oil). As the point of departure, based on the previous studies, dose levels expected to give positive responses, but not exceed maximum tolerated dose (MTD) levels, were selected for each chemical: 2.5 mg/kg/day of PTU (dose levels used in the previous studies: 240 mg/kg/day by po gavage, Marty et al., 2001
; 0.02510 mg/kg/day by ip injection, O'Connor et al., 1999b
; 0.120 mg/kg/day by po gavage, O'Connor et al., 2002b
; and 0.01 and 1 mg/kg/day by po gavage, Yamasaki et al., 2002b); 125 mg/kg/day of PB (dose levels used in the previous studies: 50 and 100 mg/kg/day by oral gavage, Marty et al., 2001
; and 5100 mg/kg/day by ip injection and po gavage, O'Connor et al., 1999b
and 2002b
, respectively); and 100 mg/kg/day of p,p'-DDE (100300mg/kg/day by ip injection and po gavage, O'Connor et al., 1999a
; 50300mg/kg/day by po gavage, O'Connor et al., 2002a
). The daily amounts for administration were 5 ml/kg BW for po gavage and 0.5 ml/kg BW for sc injections. The amount administered for each animal was calculated based on the body weight on that day. Each group consisted of six animals. The study conditions described above are consistent with those in a current draft protocol proposed by the OECD.
Endpoints
Daily cage-side observation was performed to detect moribund or dead rats. For all experiments, body weights, food consumption, and weights of liver and kidneys were assessed as indices of systemic toxicity. Food consumption for each cage was measured over two consecutive days. The daily consumption per animal was calculated and presented in Tables 1-3.
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After sampling of blood, the sex accessory glands/tissues (the ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, LABC muscles, glans penis, and Cowper's glands) as well as the thyroid, pituitary, liver, and kidneys were dissected. Although the weights of dorso-lateral prostate and pituitary are not mandatory endpoints in the current draft OECD protocol of the Hershberger assay, they were also determined in this study for better understanding. Liver, kidneys, LABC muscles, and glans penis were weighed fresh (without fixation). The ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, Cowper's glands, thyroid, and pituitary were carefully trimmed to remove fat and other contiguous tissue in a uniform manner after fixation overnight in a 10% neutral-buffered formalin, and then weighed wet. This procedure has been confirmed to be reliable (Yamada et al., 2001). It was performed for the rats from the various dosage groups in a randomized fashion. The combined values of ventral and dorso-lateral prostate were also evaluated. In the experiment assessing androgenicity, many animals failed to achieve preputial separation (PPS) so glans penis weights were not collected on these animals. Both absolute weight and relative weights adjusted to body weight were evaluated in the present study. However, body weights were not affected by the treatment, and statistical significance in relative weights was also observed as well as those in absolute weights. Therefore, data of absolute weights were only presented in the tables. For the thyroid, paraffin-embedded tissues were cut at about 5 µm in thickness and stained with hematoxylin and eosin for microscopic examination.
Statistical analysis
Data for body weights, food consumption, organ weights, and serum hormones in each chemical group were analyzed with the F-test for homogeneity of variance (Gad and Weil, 1982). If homogeneous, the data was analyzed by the Student's t-test, and if not homogeneous, the data were analyzed by the Aspin-Welch's t-test (Aspin, 1949
; Welch, 1938
). Serum hormone data were analyzed by the Mann-Whitney U-test if values were included that were below the limit of quantification. In fact, in the PTU-treated group, seven out of seven animals in T4 of both castrated and TP-treated castrated rats, two out of seven animals in T3 of castrated rats, and one out of seven animals in T3 of TP-treated castrated rats showed below the limit of quantification. Incidences of quantitative histopathological findings were analyzed by the Mann-Whitney's U-test. The significance of differences from the control group was estimated at probability levels of 1 and 5%.
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RESULTS |
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In TP-treated castrated rats (Table 2), thyroid weights were significantly increased by PTU (to 266% of the control value). Liver weights were significantly decreased in rats treated with PTU (to 90% of the control value), whereas PB increased liver weights (to 132% of the control value). Kidney weights were significantly decreased in rats treated with PTU. Some marginal changes were observed in androgen-related organs (except LABC muscles) of rats treated with PTU and PB; however, they were not statistically significant and within historical control values in our laboratory. Weights of LABC muscles were significantly decreased in rats treated with PB (to 86% of the control value). Data for p,p'-DDE are summarized in Table 3. p,p'-DDE significantly decreased weights of androgen-related organs and significantly increased liver weight; however, it did not significantly affect on thyroid weight.
Serum Concentrations of Thyroid Hormones and TSH
Data for serum levels of thyroid hormones and TSH in castrated or TP-treated castrated rats treated with PTU, PB, and p,p'-DDE are shown in Table 4. PTU markedly decreased serum T4 and T3 concentrations and increased serum TSH concentrations, irrespective of TP-treatment. PB and p,p'-DDE decreased serum T4 levels with statistical significance but not T3 and TSH. Some marginal changes were observed in T3 and TSH, and that may suggest some physiological responses to weak thyroid modulators but also a possibility as natural variance in hormone measurements given the small sample size. p,p'-DDE significantly increased serum T3 concentrations in TP-treated castrated rats. However, this alteration was considered spurious since it was not reproduced in other studies, including Experiment 1 and another study conducted in our laboratory.
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DISCUSSION |
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Reliability of Assessing for Thyroid Hormone Modulating Activities
In order to evaluate the sensitivity of the enhanced Hershberger assay for detecting thyroid hormone modulators, we used three known chemicals with thyroid hormone modulating activity: PTU, PB, and p,p'-DDE as a point of departure. PTU is a potent inhibitor of thyroid hormone synthesis (Capen, 1997; Kohrle, 1990
), and PB and p,p'-DDE are hepatic enzyme inducers that enhance the clearance of thyroid hormones (Barter and Klaassen, 1994
; Capen, 1997
; McClain et al., 1989).
Three endpoints, which are routinely used for identifying chemicals that alter thyroid function, are (1) peripheral levels of thyroid hormone and TSH, (2) thyroid weight, and (3) histopathology of thyroid gland. Since rodents do not have thyroxine binding globulin, total T3, total T4, and TSH are routinely used for evaluating thyroid hormone homeostasis (Biegel et al., 1995; Capen, 1997
; Marty et al., 2001
; O'Connor et al., 1999a
,b
, 2000
, 2002a
,b
; Yamasaki et al., 2002). The typical pattern of findings induced by thyroid hormone modulators is as follows: increased TSH and decreased T3 and/or T4, coupled with increased thyroid gland weight and/or histopathologic changes (follicular cell hypertrophy/hyperplasia, colloid depletion and/or pale staining) (Biegel et al., 1995; Capen, 1997; McClain, 1995; O'Connor et al., 1999b). Many factors can affect thyroid hormone levels including diet, stress, age, and circadian rhythm (Capen, 1997; DAdohler et al., 1979). Therefore precautions minimizing confounding factors were taken in this study: (1) consistency of strain, age, housing condition, diet, etc. in all experiments, (2) necropsy was conducted at similar time of the day (08301100 h) and by identical method in all experiments, (3) the order in which the animals were necropsied was designed such that one animal from each of the group is necropsied in a random fashion before necropsy of the second animal from each group, and (4) in order to minimize stress, based on our previous study in which effect of cage transport on serum TSH and prolactin (which is considered as indicator of stress) levels were determined, time point for blood collection was set as at least 1 h of quiet time following cage transport. According to Döhler et al. (1979)
, serum TSH levels increased significantly within 5 min and continued to rise for 60 min after cage transport. The reason for this discrepancy between Döhler's and our findings is unknown.
In the present study, as expected, the potent thyroid toxicant, PTU markedly increased thyroid weight, and caused changes in thyroid hormones (decreased serum T3 and T4, and increased serum TSH). Furthermore, PTU caused marked microscopic alteration of the thyroid gland. No increase was observed in liver weight with PTU treatment, consistent with previous studies (O'Connor et al., 1999b, 2002b). On the other hand, PB and p,p'-DDE also revealed microscopic alterations of the thyroid gland, concomitant with increased liver weights. Regarding thyroid hormones, PB and p,p'-DDE significantly decreased serum T4 but not T3 or TSH. The effects of PB and p,p'-DDE on T3 and TSH levels may appear similar as PTU; however, the possibility that these effects are not toxicologically relevant but rather reflect the natural variance in hormone measurements given the small sample size, can not be ruled out. Statistically significant changes were not observed in thyroid weights of rats treated with PB or p,p'-DDE. These findings demonstrated higher thyrotrophic potency for PTU as compared to PB and p,p'-DDE, reflecting of their mechanism(s) of action. Overall, the endpoint profiles obtained for PTU, PB, and p,p'-DDE were similar to the effects observed in previous studies (Biegel et al., 1995
; Capen, 1997
; Marty et al., 2001
; McClain et al., 1989
; O'Connor et al., 1999a
,b
, 2002a
,b
; Saito et al., 1991
; Yamasaki et al., 2002). The findings of the present study demonstrate that these test substances were identified as endocrine-active substances, consistent with their known/proposed mechanism(s) of action, suggesting that the enhanced Hershberger assay is reliable for screening of thyroid hormone modulators.
At a workshop focused on screening for chemicals that alter thyroid hormone function and homeostasis, determination of serum thyroid hormone concentrations following chemical exposure in rodents was thought to be reasonable as an initial step, with concurrent histological evaluation of the thyroid to strengthen the reliability (DeVito et al., 1997, 1999
). Hood and coworkers (1999b) demonstrated that thyroid follicular cell proliferation may be more useful than thyroid weight alone for assessing alteration in thyroids of rats treated with chemicals that produce only small to moderate increases in serum TSH. Our findings also indicated that thyroid gland histopathology and serum T4 are the most reliable parameters for identifying chemicals with thyroid hormone modulating activity, consistent with the literature (DeVito et al., 1997
, 1999
; Hood et al., 1999b).
Concerning dosing period, many previous studies found that a two-week treatment is sufficient for some chemical-induced changes in the thyroid gland (Biegel et al., 1995; Hood et al., 1999a; Jones and Clarke, 1993; McClain et al., 1989; O'Connor et al., 1999b). On the other hand, the workshop on Screening Methods for Chemicals that Alter Thyroid Hormone Action, Function and Homeostasis (DeVito et al., 1997, 1999
) concluded that at least 26 weeks of dosing is required to observe consistent thyroid responses. In fact, there are some cases in which longer exposure may be more sensitive. In order for a screen to be useful it must be comprehensive, but it must also be cost-effective and of short duration (O'Connor et al., 1999b). Based on the present study, at least, the effects of PTU 2.5 mg/kg, PB 125 mg/kg, and p,p'-DDE 100 mg/kg (these dose levels do not exceed the MTD) on the thyroid could be detected by the 10-day dosing. To confirm that 10-day dosing is sufficient to detect thyroid modulators, dose-response study using lower dose levels should be performed.
The thyroid gland is one of the nonclassical target organs for sex steroids and the presence of androgen receptor in the thyroid glands of mammalian species is well documented (Banu et al., 2002; Pelletier, 2000
). Testosterone administration has been found to cause a statistically significant decrease in serum T3 and T4 concentrations, but not histopathological changes in the thyroid gland, in a 15-day intact adult male assay (O'Connor et al., 2000
). Stimulatory effects of testosterone on the expression of TSH mRNA in the pituitary of normal Wistar rats are also documented (Ross, 1990
). Furthermore, androgen may enhance thyroid tumor growth indirectly by increasing TSH production through altered hypothalamic or pituitary function (Hofmann et al., 1986; Paloyan et al., 1982). Administration of the testosterone biosynthesis inhibitor, ketoconazole decreased serum T3 and T4 in the 15-day intact adult male assay (O'Connor et al., 2002b) and Banu and coworkers (2001)
have demonstrated that gonadectomy significantly decreases serum TSH and TSH-receptor concentrations in the thyroid in vivo. These findings suggest that presence/absence of androgens may affect thyroid hormone homeostasis. In the present study, there were no obvious differences in responses of peripheral levels of thyroid hormones and TSH, and thyroid weight between castrated and TP-treated castrated rats, whereas histopathological alteration appeared to be more robust in TP-treated castrated rats. This may allow use of only one condition (namely, TP-treated castrated) for determination of whether a test chemical has thyroid hormone modulating activity. Further studies are necessary for confirmation.
Effects of Alteration of Thyroid Hormone Homeostasis on Reliability of Assessment of (Anti-)androgenic Activities
Although androgens play a predominant role in the growth and maintenance of the size of androgen dependent tissues, several other factors can influence tissue weights (Luke et al., 1994), suggesting that thyroid modulators may affect the reliability of the Hershberger assay for screening for (anti-)androgenicity. We therefore examined whether our test compounds affect the capacity of the Hershberger assay to assess (anti-)androgenic effects.
p,p'-DDE is thought to have weak anti-androgenic activity in vitro and in vivo (Kelce et al., 1995). In utero and lactational exposure to 100 mg/kg/day of p,p'-DDE induced antiandrogenic responses such as reduction in male anogenital distance, increase in retention of male thoracic nipples, and alteration in expression of AR (Kelce et al., 1995; You et al., 1998). In our previous studies conducted under identical conditions on the present study, 100 mg/kg/day of p,p'-DDE showed the expected anti-androgenic responses in the target tissues of castrated TP-treated rats: decreased weights of the ventral prostate, seminal vesicles together with coagulating glands, glans penis, Cowper's glands, and LABC muscles (Yamada et al., 2001, 2003
). Therefore, these findings indicate the expected anti-androgenic responses of p,p'-DDE can be detected in this assay protocol. On the other hand, PTU and PB clearly demonstrated thyroid modulating influence, whereas they had no effects on weights of any androgen related tissues except LABC muscles in this assay, which is consistent with previous findings (O'Connor et al., 1999b; they did not examine the weight of LABC muscles). The findings thus indicate that reliability of the Hershberger assay for assessing (anti-)androgenicity is not confounded by alteration of thyroid hormone homeostasis. PB treatment reduced LABC muscle weight in TP-treated castrated rats. We also know that PB-treatment reduced the LABC muscle weight in male pubertal assay (data not published). Though the mechanism of this alteration by PB-treatment is unknown, the fact that the muscle complex growth, unlike the other sex accessory tissues, is regulated by testosterone and not DHT (Blohm et al., 1986) might be related.
In conclusion, this preliminary study suggests that the enhanced Hershberger assay, with evaluation of thyroid histopathology and weights, and hormone levels, appears to be reliable for screening for not only (anti-)androgenic chemicals but also thyroid modulators. If so, the Hershberger assay will be more comprehensive screening. As the Hershberger assay using castrated rats is currently designed, it can be used to detect androgens and anti-androgens. Because this assay is performed in castrated animals that do not produce endogenous sex hormones, other assays such as pubertal assay and enhanced OECD 407 are needed to detect agents that affect steroid hormone synthesis. On the other hand, the current OECD protocol of Hershberger assay includes measurement of adrenal weights as option. So far, there is no data indicating that weight measurement and/or histopathological examination of adrenals in the Hershberger assay is reliable for assessing steroid hormone inhibitors (to our knowledge, nobody has examined it). Recently, the Hershberger assay using immature noncastrated rats is proposed to validate in the OECD program. Thus, if the assay can detect agents that affect steroid hormone synthesis, and additionally detect thyroid modulators, it might be possible to reduce the number of animals, and overall time and cost by extending the parameters evaluated. In order to evaluate whether the sensitivity and specificity of such a thyroid assay is great enough for routine screening purposes, future experiments including dose-response studies using lower dose levels have to be performed.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: +81-66466-5443. E-mail: yamadat8{at}sc.sumitomo-chem.co.jp
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ankley, G. T., Johnson, R. D., Detenbeck, N. E., and Bradbury, S. P. (1997). Development of a research strategy for assessing the ecological risk of endocrine disruptors. Rev. Toxicol. 1, 71106.
Ashby, J., and Lefevre, P. A. (2000a). The peripubertal male rat assay as an alternative to the Hershberger castrated male rat assay for the detection of anti-androgens, oestrogens and metabolic modulators. J. Appl. Toxicol. 20, 3547.[ISI][Medline]
Ashby, J., and Lefevre, P.A. (2000b). Preliminary evaluation of the major protocol variables for the Hershberger castrated male rat assay for the detection of androgens, anti-androgens and metabolic modulators. Regul. Toxicol. Pharmacol. 31, 92105.[CrossRef][ISI]
Ashby, J., Lefevre, P. A., Deghenghi, R., and Wallis, N. (2001). Replacement of surgical castration by GnRH-inhibition or Leydig cell ablation in the male rat Hershberger antiandrogen assay. Regul. Toxicol. Pharmacol. 34, 188203.[CrossRef][ISI][Medline]
Aspin, A. A. (1949). Table for use in comparisons whose accuracy involves two variances, separately estimated. Biometrika 36, 290292.[ISI]
Banu, K. S., Govindarajulu, P., and Aruldhas, M. M. (2001). Testosterone and estradiol modulate TSH-binding in the thyrocytes of Wistar rats: Influence of age and sex. J. Steroid Biochem. Mol. Biol. 78, 329342.[CrossRef][ISI][Medline]
Banu, S. K., Govindarajulu, P., and Aruldhas, M. M. (2002). Testosterone and estradiol up-regulate androgen and estrogen receptors in immature and adult rat thyroid glands in vivo. Steroids 67, 10071014.[CrossRef][ISI][Medline]
Barter, R. A., and Klaassen, C. D. (1994). Reduction of thyroid hormone levels and alteration of thyroid function by four representative UDP-glucuronosyltransferase inducers in rats. Toxicol. Appl. Pharmacol. 128, 917.[CrossRef][ISI][Medline]
Blohm, T. R., Laughlin, M. E., Benson, H. D., Johnston, J. O., Wright, C. L., Schatzman, G. L., and Weintraub, P. M. (1986). Pharmacological induction of 5-reductase deficiency in the rat: separation of testosterone-mediated and 5
-dihydrotestosterone-mediated effects. Endocrinology 119, 959966.[Abstract]
Biegel, L. B., Cook, J. C., O'Connor, J. C., Aschiero, M., Arduengo, A. J., III, and Slone, T. W. (1995). Subchronic toxicity study in rats with 1-methyl-3-propylimidazole-2- thione (PTI): Effects on the thyroid. Fundam. Appl. Toxicol. 27, 185194.[CrossRef][ISI][Medline]
Capen, C. C. (1997). Mechanistic data and risk assessment of selected toxic end points of the thyroid gland. Toxicol. Pathol. 25, 3948.[ISI][Medline]
Colborn, T., vom Saal, F. S., and Soto, A. M. (1993). Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ. Health Perspect. 101, 378384.[ISI][Medline]
Date, K., Ohno, K., Azuma, Y., Hirano, S., Kobayashi, K., Sakurai, T., Nobuhara, Y., and Yamada, T. (2002). Endocrine-disrupting effects of styrene oligomers that migrated from polystyrene containers into food. Food Chem. Toxicol. 40, 6575.[CrossRef][ISI][Medline]
DeVito, M., Biegel, L., Brouwer, A., Brown, S., Brucker-Davis, F., Cheek, A. O., Christensen, R., Colborn, T., Cooke, P., Crissman, J., Crofton, K., Doerge, D., Gray, E., Hauser, P., Hurley, P., Kohn, M., Lazar, J., McMaster, S., McClain, M., McConnell, E., Meier, C., Miller, R., Tietge, J., and Tyl, R. (1999). Screening methods for thyroid hormone disruptors. Environ. Health Perspect. 107, 407415.[ISI][Medline]
DeVito, M., Crofton, K., and McMaster, S. (1997). Workshop report: Screening methods for chemicals that alter thyroid hormone action, function and homeostasis. Durham, NC, June 2325, 1997. United States Environmental Protection Agency. EPA/600/R-98/057.
Döhler, K.-D., Wong, C.C., and Von Zur Mühlen, A. (1979). The rat as model for the study of drug effects on thyroid function: Consideration of methodological problems. Pharmac. Ther. 5, 305318.[CrossRef][ISI]
Dorfman, R. I. (1969a). Androgens and anabolic agents. In Methods in Hormone Research Vol. II A. (R. I. Dorfman, Ed.), pp. 151220. Academic Press, San Diego.
Dorfman, R. I. (1969b). Antiandrogens. In Methods in Hormone Research Vol. II A. (R. I. Dorfman, Ed.), pp. 221249. Academic Press, San Diego.
EDSTAC. (1998). Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Final Report.
Gad, S. and Weil C. S. (1982). Statistics for toxicologist. In Principles and Methods of Toxicology (H. A. Wallance, Ed.), pp. 285286. Raven Press, New York.
Gray, L. E., Ostby, J., Wilson, V., Lambright, C., Bobseine, K., Hartig, P., Hotchkiss, A., Wolf, C., Furr, J., Price, M., Parks, L., Cooper, R. L., Stoker, T. E., Laws, S. C., Degitz, S. J., Jensen, K. M., Kahl, M. D., Korte, J. J., Makynen, E. A., Tietge, J. E., and Ankley, G. T. (2002). Xenoendocrine disrupters-tiered screening and testing. Filling key data gaps. Toxicology 181182, 371382.[CrossRef][ISI]
Hershberger, L. G., Shipley, E. G., and Meyer, R. K. (1953). Myotrophic activity of 19-nortestosterone and other steroids determined by modified levator ani muscle method. Proc. Soc. Exp. Biol. Med. 83, 175180.
Hofmann, C., Oslapas, R., Nayyar, R., McCall, A., and Paloyan, E. (1986). Testosterone enhancement of thyroid carcinoma in rats: the role of TSH. Surgery 100, 10781087.[ISI][Medline]
Hood, A., Liu, J., and Klaassen, C. D. (1999a). Effects of phenobarbital, pregnenolone-16a-carbonitrile and propylthiouracil on thyroid follicular cell proliferation. Toxicol. Sci. 50, 4553.[Abstract]
Hood, A., Liu, Y. P., Gattone, V. H., II, and Klaassen, C. D. (1999b). Sensitivity of thyroid gland growth to thyroid stimulating hormone (TSH) in rats treated with antithyroid drugs. Toxicol. Sci. 49, 263271.[Abstract]
Jones, H. B., and Clarke, N. A. (1993). Assessment of the influence of subacute phenobarbitone administration on multi-tissue cell proliferation in the rat using bromodeoxyuridine immunocytochemistry. Arch. Toxicol. 67, 622628.[ISI][Medline]
Kavlock, R. J. (1999). Overview of endocrine disruptor research activity in the United States. Chemosphere 39, 12271236.[CrossRef][ISI][Medline]
Kavlock, R. J., Daston, G. P., DeRosa, C., Fenner-Crisp, P., Gray, L. E., Kaattari, S., Lucier, G., Luster, M., Mac, M. J., Maczka, C., Miller, R., Moore, J., Rolland, R., Scott, G., Sheehan, D. M., Sinks, T., and Tilson, H. A. (1996). Research needs for the risk assessment of health and environmental effects of endocrine disruptors: A report of the U.S. EPA-sponsored workshop. Environ. Health Perspect. 104(Suppl. 4), 715740.
Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Kemppainen, J. A., and Wilson, E. M. (1995). Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist. Nature 375, 581585.[CrossRef][ISI][Medline]
Kim, H. S., Han, S. Y., Kim, T. S., Kwack, S. J., Lee, R. D., Kim, I. Y., Seok, J. H., Lee, B. M., Yoo, S. D., and Park, K. L. (2002). No androgenic/anti-androgenic effects of bisphenol-A in Hershberger assay using immature castrated rats. Toxicol. Lett. 135, 111123.[CrossRef][ISI][Medline]
Kitamura, S., Suzuki, T., Ohta, S., and Fujimoto, N. (2003). Antiandrogenic activity and metabolism of the organophosphorus pesticide fenthion and related compounds. Environ. Health Perspect. 111, 503508.[ISI][Medline]
Kohrle, J. (1990). Thyrotropin (TSH) action on thyroid hormone deiodination and secretion: One aspect of thyrotropin regulation of thyroid cell biology. Horm. Metab. Res. Suppl. 23, 1828.[Medline]
Kunimatsu, T., Yamada, T., Ose, K., Sunami, O., Kamita, Y., Okuno, Y., Seki, T., and Nakatsuka, I. (2002). Lack of (anti-) androgenic or estrogenic effects of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) in the Hershberger and uterotrophic assays. Regul. Toxicol. Pharmacol. 35, 227237.[CrossRef][ISI][Medline]
Lambright, C., Ostby, J., Bobseine, K., Wilson, V., Hotchkiss, A. K., Mann, P. C., and Gray, L. E., Jr. (2000). Cellular and molecular mechanisms of action of linuron: An antiandrogenic herbicide that produces reproductive malformations in male rats. Toxicol. Sci. 56, 389399.
Luke, M. C., and Coffey, D. S. (1994). The male sex accessory tissues. Structure, androgen action, and physiology. In The Physiology of Reproduction, 2nd ed. (E. Knobil and J. D. Neil, Eds.), pp. 14351487. Raven Press, New York.
Marty, M. S., Crissman, J. W., and Carney, E. W. (2001). Evaluation of the male pubertal assay's ability to detect thyroid inhibitors and dopaminergic agents. Toxicol. Sci. 60, 6376.
McClain, R. M. (1995). Mechanistic considerations for the relevance of animal data on thyroid neoplasia to human risk assessment. Mutat. Res. 333, 131142.[CrossRef][ISI][Medline]
McClain, R. M., Levin, A. A., Posch, R., and Downing, J. C. (1989). The effect of phenobarbital on the metabolism and excretion of thyroxine in rats. Toxicol. Appl. Pharmacol. 99, 216228.[ISI][Medline]
Monosson, E., Kelce, W. R., Lambright, C., Ostby, J., and Gray, L. E., Jr. (1999). Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat. Toxicol. Ind. Health 15, 6579.[ISI][Medline]
Nellemann, C., Vinggaard, A. M., Dalgaard, M., Hossaini, A., and Larsen, J. J. (2001). Quantification of antiandrogen effect determined by Lightcycler technology. Toxicology 163, 2938.[CrossRef][ISI][Medline]
O'Connor, J. C., Davis, L. G., Frame, S. R., and Cook, J. C. (2000). Evaluation of a Tier I screening battery for detecting endocrine-active compounds (EACs) using the positive controls testosterone, coumestrol, progesterone, and RU486. Toxicol. Sci. 54, 338354.
O'Connor, J. C., Frame, S. R., Davis, L. G., and Cook, J. C. (1999a). Detection of the environmental antiandrogen p,p'-DDE in CD and Long-Evans rats using a tier I screening battery and a Hershberger assay. Toxicol. Sci. 51, 4453.[Abstract]
O'Connor, J. C., Frame, S. R., Davis, L. G., and Cook, J. C. (1999b). Detection of thyroid toxicants in a tier I screening battery and alterations in thyroid endpoints over 28 days of exposure. Toxicol. Sci. 51, 5470.[Abstract]
O'Connor, J. C., Frame, S. R., and Ladics, G. S. (2002a). Evaluation of a 15-day screening assay using intact male rats for identifying antiandrogens. Toxicol. Sci. 69, 92108.
O'Connor, J. C., Frame, S. R., and Ladics, G. S. (2002b). Evaluation of a 15-day screening assay using intact male rats for identifying steroid biosynthesis inhibitors and thyroid modulators. Toxicol. Sci. 69, 7991.
OECD. (1997). Draft detailed review paper: Appraisal of test methods for sex-hormone disrupting chemicals. Environmental Health and Safety Publications, Paris.
Paloyan, E., Hofmann, C., Prinz, R. A., Oslapas, R., Shah, K. H., Ku, W. W., Ernst, K., Smith, M., and Lawrence, A. M. (1982). Castration induces a marked reduction in the incidence of thyroid cancers. Surgery 92, 839848.[ISI][Medline]
Pelletier, G. (2000). Localization of androgen and estrogen receptors in rat and primate tissues. Histol. Histopathol. 15, 12611270.[ISI][Medline]
Ross, D. S. (1990). Testosterone increases TSH-beta mRNA, and modulates alpha-subunit mRNA differentially in mouse thyrotropic tumor and castrate rat pituitary. Horm. Metab. Res. 22, 163169.[ISI][Medline]
Saito, K., Kaneko, H., Sato, K., Yoshitake, A., and Yamada, H. (1991). Hepatic UDP-glucuronyltransferase(s) activity toward thyroid hormones in rats: Induction and effects on serum thyroid hormone levels following treatment with various enzyme inducers. Toxicol. Appl. Pharmacol. 111, 99106.[ISI][Medline]
Stroheker, T., Cabaton, N., Berges, R., Lamothe, V., Lhuguenot, J. C., and Chagnon, M. C. (2003). Influence of dietary soy isoflavones on the accessory sex organs of the Wistar rat. Food Chem. Toxicol. 41, 11751183.[CrossRef][ISI][Medline]
Sunami, O., Kunimatsu, T., Yamada, T., Yabushita, S., Sukata, T., Miyata, K., Kamita, Y., Okuno, Y., Seki, T., Nakatsuka, I., and Matsuo, M. (2000). Evaluation of a 5-day Hershberger assay using young mature male rats: methyltestosterone and p,p'-DDE, but not fenitrothion, exhibited androgenic or antiandrogenic activity in vivo. Toxicol. Sci. 25, 403415.
Vinggaard, A. M., Nellemann, C., Dalgaard, M., Jorgensen, E. B., and Andersen, H. R. (2002). Antiandrogenic effects in vitro and in vivo of the fungicide prochloraz. Toxicol. Sci. 69, 344353.
Welch, B. L. (1938). The significance of the differences between two means when the population variances are unequal. Biometrika 29, 350362.
Wilson, V. S., Lambright, C., Ostby, J., and Gray, L. E., Jr. (2002). In vitro and in vivo effects of 17beta-trenbolone: a feedlot effluent contaminant. Toxicol. Sci. 70, 202211.
Yamada, T., Kunimatsu, T., Sako, H., Yabushita, S., Sukata, T., Okuno, Y., and Matsuo, M. (2000). Comparative evaluation of a 5-day Hershberger assay utilizing mature male rats and a pubertal male assay for detection of flutamide's antiandrogenic activity. Toxicol. Sci. 53, 289296.
Yamada, T., Sunami, O., Kunimatsu, T., Kamita, Y., Okuno, Y., Seki, T., Nakatsuka, I., and Matsuo, M. (2001). Dissection and weighing of accessory sex glands after formalin fixation, and a 5-day assay using young mature rats are reliable and feasible in the Hershberger assay. Toxicology 162, 103119.[CrossRef][ISI][Medline]
Yamada, T., Ueda, S., Yoshioka, K., Kawamura, S., Seki, T., Okuno, Y., and Mikami, N. (2003). Lack of estrogenic or (anti-)androgenic effects of d-phenothrin in the uterotrophic and Hershberger assays. Toxicology 186, 227239.[CrossRef][ISI][Medline]
Yamasaki, K., Sawaki, M., Noda, S., and Takatsuki, M. (2002a). Uterotrophic and Hershberger assays for n-butylbenzene in rats. Arch. Toxicol. 75, 703706.[CrossRef][ISI][Medline]
Yamasaki, K., Sawaki, M., and Takatsuki, M. (2001). Strain sensitivity differences in the Hershberger assay. Reprod. Toxicol. 15, 437440.[CrossRef][ISI][Medline]
Yamasaki, K., Tago, Y., Nagai, K., Sawaki, M., Noda, S., and Takatsuki, M. (2002b). Comparison of toxicity studies based on the draft protocol for the Enhanced OECD Test Guideline no. 407 and the research protocol of Pubertal Development and Thyroid Function in Immature Male Rats with 6-n-propyl-2-thiouracil. Arch. Toxicol. 76, 495501.[CrossRef][ISI][Medline]
Yamasaki, K., Takeyoshi, M., Sawaki, M., Imatanaka, N., Shinoda, K., and Takatsuki, M. (2003). Immature rat uterotrophic assay of 18 chemicals and Hershberger assay of 30 chemicals. Toxicology 183, 93115.[CrossRef][ISI][Medline]
You, L., Casanova, M., Archibeque-Engle, S., Sar, M., Fan, L. Q., and Heck, H. A. (1998). Impaired male sexual development in perinatal Sprague-Dawley and Long-Evans hooded rats exposed in utero and lactationally to p,p'-DDE. Toxicol. Sci. 45, 162173.[Abstract]
You, L., Sar, M., Bartolucci, E., Ploch, S., and Whitt, M. (2001). Induction of hepatic aromatase by p,p'-DDE in adult male rats. Mol. Cell Endocrinol. 178, 207214.[CrossRef][ISI][Medline]
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