Gene expression changes in the immature rat uterus: effects of uterotrophic and sub-uterotrophic doses of bisphenol A.

J. C. Gould et al., 1998, Mol. Cell Endocrinol. 142, 203-214, have reported that administration of 5-150 mg/kg/day BPA to immature rats leads to increases in uterine peroxidase activity and progesterone receptor (PR) protein levels in the absence of a uterotrophic response. These observations are of interest given current concerns regarding the adequacy of the uterotrophic assay to act as a sentinel for the estrogenic activity of chemicals in vivo. Therefore, the uterotrophic activity of BPA to the immature rat has been re-evaluated over the dose range 2 microg/kg-800 mg/kg/day. Expression levels of three estrogen responsive uterine genes were determined using real-time RT-PCR--namely, complement component 3, lipocalin 2, and PR. 18S rRNA and RNA polymerase II large subunit acted as control genes. Observations of gene expression were made 4 h and 72 h after the first of three daily po administrations of BPA. Increases in gene expression were observed over the uterotrophic dose range (approximately 200-800 mg/kg BPA). Over the dose range 2 microg/kg-20 mg/kg BPA there was no uterotrophic response and no increase in gene expression. We conclude that BPA does not produce reproducible changes in gene expression in the uterus of immature rats at dose levels that are not also uterotrophic. Therefore, in the present study, the no effect level for uterotrophic activity for BPA coincided with the no transcriptional effect level for uterine genes.

Spontaneous regression of hypercalcemia in a patient with primary hyperparathyroidism and prolactinoma.

We describe a unique case of spontaneous resolution of hyperparathyroidism in a lady with combined parathyroid adenoma and prolactinoma, raising the possibility of underlying multiple endocrine neoplasia (MEN) 1 syndrome. We also discuss the mechanism and natural history of such spontaneous remission.

Testosterone-stimulated weanlings as an alternative to castrated male rats in the Hershberger anti-androgen assay.

We showed previously that stimulation of weanling male rats with the synthetic androgen 17-methyltestosterone (17MT) caused premature growth of the sex accessory tissues such that the activity of the two anti-androgens flutamide and DDE could be demonstrated (Regul. Toxicol. Pharmacol. 35 (2002) 280). We suggested that that protocol should be evaluated as an alternative to the castrated male rat Hershberger assay. In the present paper we justify changing the assay protocol to use testosterone propionate (TP), in place of 17MT, as the stimulating androgen. This change enables biochemical formation of dihydrotestosterone from testosterone, a conversion not possible when using 17MT. This change in the protocol enables detection of the testosterone-5-reductase inhibitor finasteride. The modified TP-stimulated weanling male rat assay is shown to have similar sensitivity to that of the castrated male rat Hershberger assay in detection of the anti-androgens flutamide, procymidone, vinclozolin, and DDE, and of the biochemical inhibitor finasteride. The anti-androgen linuron and the anabolic steroid trenbolone were also detected as positive by the TP-stimulated weanling male assay. It is suggested that this modified assay for anti-androgens should be validated as an alternative to the Hershberger assay, thereby reducing animal stress by obviating the need for surgical castration.

Concept evaluation: an assay for receptor-mediated and biochemical antiestrogens using pubertal rats.

At present, assessment of chemicals for receptor-mediated antiestrogenic activity involves inhibition of uterine growth stimulated by coadministration of a reference estrogen in either ovariectomized or immature rodents. In the present paper, we describe an alternative assay for both receptor-mediated and biochemical antiestrogens. The assay involves treatment of immature rats from postnatal (pnd) 25 or 26 for either 7 or 14 days and monitors two benchmarks of puberty, the mean day of vaginal opening and the weight of the uterus, that require estrogen activity. The receptor-mediated antiestrogens ZM 189,154 and Faslodex (ICI 182,780), the aromatase inhibitor Arimidex (Anastrozole), and the GnRH inhibitor Antarelix were each effective in preventing uterine growth and in delaying vaginal opening for the course of the experiments. The 5alpha-reductase inhibitor Finasteride was inactive in the assay indicating assay specificity for antiestrogens. Delays in uterine growth were clearly evident in the 7-day experiments, but assessment of vaginal opening required the 14-day protocol. No significant changes in body weight were observed in any of the experiments. It is concluded that the assay holds promise as a simple method of detecting antiestrogens and that it is worthy of further study.

Comparison of the developmental and reproductive toxicity of diethylstilbestrol administered to rats in utero, lactationally, preweaning, or postweaning.

The objective of the study was to determine which period of exposure produces the most marked effects on the reproductive capacity and sexual development of the rat, with particular emphasis on the relative sensitivity of in utero and postnatal exposures. The endocrine active chemical, diethylstilbestrol (DES) was used as an agent known to affect many of the endpoints examined. Hitherto, such comparisons have been made between studies, rather than within a study. Our data will be helpful in the interpretation of future multigenerational assay data. In preliminary studies, DES was shown to be active in the immature rat uterotrophic assay with a lowest detected dose of 0.05 mg DES/kg body weight by sc injection and 10 mg DES/l (1.6 mg DES/kg body weight) by administration in drinking water. A dose of 60 microg DES/l drinking water ( approximately 6.5mg DES/kg body weight/day) was selected for the main study since this represented the midpoint of the drinking water uterotrophic dose response and produced no overt maternal toxicity. The study used 10 groups of concomitantly pregnant animals, including 2 control groups. The first comparison was between the effects of exposure to DES in utero, and exposure from conception to weaning. Another group of animals was exposed to DES in utero and cross-fostered to untreated pregnant females to prevent lactational transfer of DES to pups. Two groups were exposed to DES neonatally, either from birth to postnatal day (PND) 10 (pups thus having only lactational exposure), or from birth until weaning (PND 21; pups thus having both lactational exposure and self-exposure via drinking water). In addition, a dose response study to DES was conducted on animals exposed from weaning to PND 100, when the first phase of the study was terminated. Pups exposed to DES in utero and pups exposed from weaning to PND 100 were bred to assess fertility of the F1 animals and the sexual development of F2 offspring. This last comparison was to determine the extent to which weanling rats could be used in endocrine toxicity studies to assess their potential to show activity in utero. The most sensitive period of exposure for inducing developmental effects in F1 animals was from weaning onwards. The neonatal to weaning period (PND 1-21) was the next most sensitive. Essentially no effects were induced in F1 animals exposed in utero. No effects of any kind were observed in animals only exposed over the early neonatal period of PND 1-10. The mean day of vaginal opening, testes descent, and prepuce separation was only altered in groups where postnatal exposure to DES continued beyond PND 10, or was started at weaning. No changes were observed in anogenital distance or caudal sperm counts. Some changes in organ weights were observed, but the interpretation of these was often confused by concomitant changes in body weight. In general, histopathological examination of tissues yielded no additional information. In breeding studies with animals exposed to DES in utero, or from weaning, reduced litter sizes and marginal advances in the day of vaginal opening were observed in the offspring, together with changes in organ weights. However, no unique sensitivity was noted for exposure in utero. Evaluation of the several exposure periods and the many markers monitored in this study may have individual strengths in individual cases, but when rigorously compared using the reference estrogen DES, many preconceptions regarding their absolute or relative value were not upheld. Further, each of these markers is subject to natural variability, as demonstrated by comparisons made among the 5 separate control groups available in parts of the present study. This variability increases the chance that small changes observed in endocrine toxicity studies employing small group sizes and a single control group, or no concomitant control group, may be artifactual. The most marked effects observed in this study were on the developmental landmarks in the F1 animals induced by exposures after PND 10. Some effects on developmental landmarks and organ weights were observed in F2 animals following exposure either in utero or postweaning. This study therefore does not establish a unique role for exposures in utero or during the early neonatal period.

Detection of aromatase inhibitors in vitro using rat ovary microsomes.

Inhibition of aromatase activity in vitro is one of the Tier 1 screening assays proposed by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) for the detection of potential endocrine disrupters. In this report a rat ovarian aromatase inhibition assay has been evaluated using the reference aromatase inhibitors anastrozole, fadrozole, letrozole and CGS 18320B. Rat ovary microsomes were used as the enzyme source, as endocrine disruption studies are most commonly carried out in this species. Aromatase activity was inhibited in vitro by anastrozole, fadrozole, letrozole and CGS 18320B with IC(50)s of 25, 7, 7 and 5 nM, respectively. This assay, therefore, appears to have good sensitivity to aromatase inhibitors and may be useful as a general screening assay and in mechanistic studies.

Effect of rodent diets on the sexual development of the rat.

Five rodent diets have been evaluated for their possible effect on the sexual development of the rat. Groups of 12 pregnant Alpk rats were fed one of the following combinations of diets during pregnancy and postnatally: RM3/RM1, AIN-76A/AIN-76A, RM3/AIN-76A, Teklad Global 2016 (Global)/Global and Purina 5001/Purina 5001. AIN-76A is phytoestrogen-free while the other diets contained varying amounts of phytoestrogens. The phytoestrogens genistein and daidzein were determined in the diets studied, and the concentrations found agreed with earlier estimates. RM3/RM1 was selected as the control group, as this has been used routinely in this laboratory for the past decade. Determinations were made in offspring of the times of vaginal opening and first estrus among the females, and of prepuce separation and testes descent among the males. At postnatal day (PND) 26 the females from 6 of the 12 litters were terminated and tissue weights measured. Males from 6 of the 12 litters were similarly studied at PND 68. Animals from the remaining litters were transferred to RM1 diet at PND 70. Termination of the study was at PND 128 (males) and PND 140 (females) when body weights and tissue weights were determined. Marked differences in body weight, sexual development, and reproductive tissue weights were observed for rats maintained on AIN-76A or Purina 5001, with only minimal effects among rats maintained on the Global diet. These comparisons were against RM3/RM1 as the reference diet. However, using Purina 5001 as the reference diet reversed the direction of the differences seen when using RM3/RM1 as the reference diet. The differences observed when using RM3/RM1 as reference diet occurred mainly postnatally. In addition, the fact that similar differences were seen for the phytoestrogen-free diet, AIN-76A, and the phytoestrogen-rich diet, Purina 5001, indicate that these effects are more likely to be caused by nutritional differences between the diets that then have centrally mediated effects on rodent sexual development, rather than individual dietary components affecting peripheral estrogen receptors (ER). This proposal is supported by abolition of the uterotrophic activity of AIN-76A and Purina 5001 (relative to RM3/RM1) in the immature rat by coadministration of the gonadotrophin-releasing hormone (GnRH) antagonist ANTARELIX: The present data indicate that choice of diet may influence the timing of sexual development in the rat, and consequently, that when evaluating the potential endocrine toxicity of chemicals, the components of rodent diets used should be known, and as far as is possible, controlled.

The uterotrophic activity of nonylphenol in the rat is not mediated by aromatase enzyme induction.

p-Nonylphenol (NP) is weakly estrogenic to rodents and to some species of fish. All evidence to date has indicated that the estrogenic effects of NP are due to the interaction of NP with the estrogen receptor. Recent findings of increased plasma estradiol in fish exposed to NP have, however, led to the proposal of an alternative mechanism for NP-induced estrogenicity in this species, possibly via induction of aromatase enzymes. In the present studies, this hypothesis was investigated in rats using the aromatase inhibitor anastrozole. The results indicated that the uterotrophic action of NP, as with estradiol used as a positive control, is mediated directly by its interaction with uterine ER, rather than an indirect effect via aromatase enzyme induction. Circulating levels of estradiol were unchanged after NP treatment and the aromatase inhibitor anastrozole failed to inhibit NP-induced uterine growth. These results are consistent with previous published data on NP in rodents.

Obstacles to the prediction of estrogenicity from chemical structure: assay-mediated metabolic transformation and the apparent promiscuous nature of the estrogen receptor.

Information on structure-activity relationships (SAR) and pathways of metabolic activation would facilitate the preliminary screening of chemicals for estrogenic potential. Published crystallographic studies of the estrogen receptor (ER) imply an essential role of the two hydroxyl groups on estradiol (17beta-E(2)) for its binding to ER. The influence of these hydroxyl groups on ER binding and estrogenicity was evaluated by the study of 17beta-E(2) with one or both of these hydroxyl groups removed (17beta-desoxyestradiol and 3, 17beta-bisdesoxyestradiol, respectively). 6-Hydroxytetralin (17beta-E(2) with its C- and D-rings removed) and other synthetic estrogens were also studied. The estrogenicity assays comprised a yeast ER-mediated transcription assay, mammalian cell transcription assays incorporating either ER alpha or ER beta, and the immature rat uterotrophic assay. With the exception of 6-hydroxytetralin in the uterotrophic assay, all the chemicals were active in all the assays. Hydroxylation of the two desoxy compounds to estradiol was shown to occur in immature female rats, but metabolism was not implicated in the responses observed in the ER-binding and yeast systems. It is concluded that the 3-hydroxyl and 17beta-hydroxyl groups of 17beta-E(2) are not absolute requirements for estrogenicity. It would therefore be of value to the derivation of SAR for estrogenicity were the crystal structure of the bisdesoxy-E(2)/ER complex to be evaluated.

Neonatal exposure of male rats to nonylphenol has no effect on the reproductive tract.

P. C. Lee (1998, ENDOCRINE: 9, 105-111) has reported that neonatal exposure of SD rats to nonylphenol (NP; 8 mg/kg/day) by daily intraperitoneal (ip) injection in DMSO results in decreased ventral prostate and epididymides weights, and delayed testes descent, at post natal day (pnd) 31. These effects were surprising given that similar effects were not reported in an earlier multi-generation study of NP. We have repeated the central experiment described by Lee and were unable to confirm the effects reported. Alpk (Wistar derived) rats were exposed to NP (8mg/kg/day by ip injection in either arachis oil or DMSO) from pnd 1-10 and assessed on pnd 34-36. No significant effects on animal body weights were observed. The weights of the epididymides, seminal vesicles, testes, and ventral prostate were unaffected using either vehicle. Testes descent proceeded normally, with both test and control testes fully descended by pnd 29. Possible reasons for this divergence in findings for NP are discussed.

Re-evaluation of the first synthetic estrogen, 1-keto-1,2,3, 4-tetrahydrophenanthrene, and bisphenol A, using both the ovariectomised rat model used in 1933 and additional assays.

1-Keto-1,2,3,4-tetrahydrophenanthrene (THP-1) was reported by Cook et al. in 1933 as the first synthetic estrogen. Estrogenic activity was assessed by the induction of vaginal cornification in ovariectomised rats. The corresponding 4-isomer (THP-4) was shown to be inactive. Both chemicals have been re-synthesised and assessed for hormonal activity. Each chemical bound weakly and to the same extent to isolated estrogen receptors, but only at high concentrations. However, they each lacked estrogenic or anti-estrogenic activity when evaluated in vitro using a yeast hER assay, and both failed to induce vaginal cornification or uterotrophic effects in ovariectomised rats. THP-1, and to a lesser extent THP-4, were shown to possess weak androgenic and anti-androgenic activity in vitro when evaluated using an hAR yeast assay. Estrogenic activity for bisphenol A (BPA) was subsequently demonstrated by [Dodds and Lawson, Synthetic, oestrogenic agents without the phenanthrene nucleus, Nature 137, (1936)] using the same ovariectomised rat protocol, and this activity has been confirmed and supplemented by positive uterotrophic effects for BPA in the same bioassays. The present results illustrate the complexity of deriving conclusions regarding the hormonal activities of chemicals. First, some activities observed in isolated hormonal receptor binding assays may not be expressed in functional hormonal assays. This indicates the need for functional hormonal assays in any screening programme. Second, that activities observed for a chemical in one hormonal assay may not be reflected in related hormonal assays. This indicates the need to define assay protocols with some precision when incorporating them into screening batteries. Finally, that some literature reports of hormonal activity for chemicals may not be capable of independent confirmation under apparently identical conditions of test. This illustrates the need to use lists of hormonally active chemicals with care.

Effects of p-nonylphenol (NP) and diethylstilboestrol (DES) on the Alderley Park (Alpk) rat: comparison of mammary gland and uterus sensitivity following oral gavage or implanted mini-pumps.

An earlier report by Colerangle and Roy indicated that administration of p-nonylphenol (NP) to Noble rats, via subcutaneously implanted mini-pumps at estimated doses of 53.2 and 0.073 mg kg(-1) day(-1) for 11 days, led to proliferation of the mammary gland. Those results indicated a ca. 600-fold enhancement in assay sensitivity to NP over that of the standard 3-day rat uterotrophic assay. The potential importance of these observations led us to repeat the experiments in the Noble rat, as described earlier. Although our earlier results confirmed the reported effects of diethylstilboestrol (DES) on the mammary gland of Noble rats, we found no effects with NP. The present report extends our investigations of the effects of NP and DES on the mammary gland and uterus of other rat strains using both oral dosing and exposure via mini-pumps. The 3-day oral uterotrophic assay responses to NP were similar for immature Alderly Park (Alpk; Wistar-derived) and immature Sprague-Dawley rats. Likewise, oral administration of NP to ovariectomized Alpk rats for 11 days gave responses of a similar magnitude to those seen in the 3-day immature assays and in earlier 3-and 11-day oral assays conducted using Noble rats. Administration of NP via mini-pumps to ovariectomized Alpk rats, at the implant doses employed by Colerangle and Roy, gave a negative uterotrophic response. The highest achieved dose levels of NP in the implant experiment (27 mg kg(-1) day(-1)) were lower than in the above assays and the negative response was therefore consistent with the previously defined minimum detection level for NP in the uterotrophic assay of ca. 40 mg kg(-1) day(-1) day(-1). It is concluded that the uterotrophic activity of NP is independent of the strain of rat, the duration of dosing and the route of exposure. Two mammary gland studies were conducted on NP and DES in the Alpk rat. In the first study (a repeat of the techniques used in earlier studies with the Noble rat), NP was administered via mini-pumps (achieved doses of 0.052 and 37.4 mg kg(-1) day(-3) NP) and produced no effect on mammary gland development, whereas DES gave the expected trophic response. In the second mammary gland study, NP was administered orally to Alpk rats at 100 mg kg(-1) day(-1) for 11 days (a dose that produced a positive uterotrophic response in ovariectomized rats). In this experiment, DES, and to a lesser extent NP, increased mammary gland differentiation and cell proliferation. The present studies have demonstrated that the rat mammary gland responds predictably to oestrogenic stimulation but does not show increased sensitivity to oestrogens when compared to the rat uterus. It is also concluded that the minimum detection level for oestrogenic responses of NP in rodents, following oral, dietary and implant routes of exposure, is ca. 40 mg kg(-1) day(-1).

Comparative activities of p-nonylphenol and diethylstilbestrol in noble rat mammary gland and uterotrophic assays.

Colerangle and Roy (1996, Endocrine 4, 115-122) have described the apparent ability of both diethylstilbestrol (DES) and p-nonylphenol (NP) to cause extensive cell proliferation and lobular development in the mammary glands of young adult Noble rats. The chemicals were administered over 11 days via subcutaneously implanted minipumps. The dose level of DES used (0.076 mg/kg/day) was about 70 times higher than its minimum detection level in rodent uterotrophic and reproductive toxicology studies. In contrast, the lowest active dose level of NP (0.073 mg/kg/day) in the Noble rat mammary gland study was about 600 times lower than its minimum detection level in rat uterotrophic and multigeneration studies. The apparent enhanced sensitivity of the Noble rat mammary gland to the estrogenic activity of NP was considered worthy of further study. Ovariectomized Noble rat uterotrophic assays with NP (minimum detection level approximately 40 mg/kg/day, 3 or 11 days, oral gavage) revealed similar assay sensitivity to that observed for earlier immature and ovariectomized Alderley Park (AP) rat uterotrophic assays of this chemical. The response of the ovariectomized Noble rat uterotrophic assay to DES and estradiol was also as expected from earlier immature AP rat assays. It is concluded that the general sensitivity to estrogens of the Noble rat and the AP rat is similar. A repeat of the Noble rat mammary gland study with DES (11 x 0.076 mg/kg/day) and NP (11 x either 0.073 or 53.2 mg/kg/day), as originally reported by Colerangle and Roy (1996), revealed a strong positive response to DES and no response to NP. It is concluded that the minimum detection level of NP as a weakly estrogenic material in the rat should be based on the results of rat uterotrophic and multigeneration studies and therefore be set at approximately 40 mg/kg/day. It is also concluded that induced S-phase in the rodent mammary gland is best monitored using BRDU, as opposed to PCNA staining, and that use of subcutaneously implanted minipumps/pellets is inappropriate for risk/hazard assessment studies of chemicals already established as estrogenic in vitro and in vivo, as are NP and DES.