OBJECTIVE:The Organisation for Economic Co-operation and Development (OECD) has completed phase 2 of an international program to validate the rodent Hershberger bioassay. DESIGN:The Hershberger bioassay is designed to identify suspected androgens and antiandrogens based on changes in the weights of five androgen-responsive tissues (ventral prostate, paired seminal vesicles and coagulating glands, the levator ani and bulbocavernosus muscles, the glans penis, and paired Cowper's or bulbourethral glands). Protocol sensitivity and reproducibility were tested using two androgen agonists (17alpha-methyl testosterone and 17beta-trenbolone), four antagonists [procymi-done, vinclozolin, linuron, and 1,1-dichoro-2,2-bis-(p-chlorophenyl)ethylene (p,p'-DDE)], and a 5alpha-reductase inhibitor (finasteride). Sixteen laboratories from seven countries participated in phase 2. RESULTS:In 40 of 41 studies, the laboratories successfully detected substance-related weight changes in one or more tissues. The one exception was with the weakest antiandrogen, linuron, in a laboratory with reduced sensitivity because of high coefficients of variation in all tissue weights. The protocols performed well under different experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). There was good agreement and reproducibility among laboratories with regard to the lowest dose inducing significant effects on tissue weights. CONCLUSIONS:The results show that the OECD Hershberger bioassay protocol is reproducible and transferable across laboratories with androgen agonists, weak androgen antagonists, and a 5alpha-reductase inhibitor. The next validation phase will employ coded test substances, including positive substances and negative substances having no androgenic or antiandrogenic activity.

We have previously used genome-wide transcript profiling to investigate the relationships between changes in gene expression and physiological alterations during the response of the immature mouse uterus to estrogens. Here we describe the identification of a functionally inter-related group of estrogen-responsive genes associated with iron homeostasis, including the iron-binding protein lactotransferrin, the ferroxidase ceruloplasmin, the iron delivery protein lipocalin 2 and the iron-exporter ferroportin. Quantitative real-time PCR revealed that the expression of these genes increases with time during the uterotrophic response, reaching maximal levels in the post-proliferative phase (between 48 and 72 h). In contrast, the heme biosynthesis genes aminolevulinic acid synthase 1 and 2 were maximally induced by estrogen at 2 and 4 h, respectively, prior to increased cell proliferation. Together, these data reveal that estrogen induces the temporally coordinated expression of iron homeostasis genes in the mouse uterus, and suggest an important role for iron metabolism during sex steroid hormone-induced uterine cell growth and differentiation.

The Organisation for Economic Cooperation and Development (OECD) has completed phase 1 of the Hershberger validation intended to identify in vivo activity of suspected androgens and anti-androgens. Seventeen laboratories from 7 countries participated in phase 1, and results were collated and evaluated by the OECD with the support of an international committee of experts. Five androgen-responsive tissues (ventral prostate, paired seminal vesicles and coagulating glands, levator ani and bulbocavernosus muscles, glans penis, and paired Cowper's or bulbourethral glands) were evaluated. The standardized protocols used selected doses of a reference androgen, testosterone propionate (TP), and an antiandrogen, flutamide (FLU). All laboratories successfully detected TP-stimulated increases in androgen-responsive tissue weight and decreases in TP-stimulated tissue weights when FLU was co-administered. The standardized protocols performed well under a variety of conditions (e.g., strain, diet, housing protocol, bedding). There was good agreement among laboratories with regard to the TP doses inducing significant increases in tissue weights and the FLU doses decreasing TP-stimulated tissue weights. Several additional procedures (e.g., weighing of the dorsolateral prostate and fixation of tissues before weighing) and serum component measurements (e.g., luteinizing hormone) were also included by some laboratories to assess their potential utility. The results indicated that the OECD Hershberger protocol was robust, reproducible, and transferable across laboratories. Based on this phase 1 validation study, the protocols have been refined, and the next phase of the OECD validation program will test the protocol with selected doses of weak androgen agonists, androgen antagonists, a 5α-reductase inhibitor, and chemicals having no androgenic activity.

Pentachloronitrobenzene (PCNB) has been shown to inhibit foci-formation for MCF-7 cells in vitro (Zou, E., Hatakeyama, M., Matsumra, F., 2002. Foci-formation of MCF-7 cells as an in vitro screening method for estrogenic chemicals. Environ. Toxicol. Pharmacol. 11, 71) This effect was referred to as representing an anti-estrogenic property of PCNB. However, we have found no evidence that PCNB acts as either an estrogen or an anti-estrogen, either in vitro or in vivo. The assays conducted were binding to human and rat estrogen receptors (ER), a hER yeast trans-activation assay, the immature rat uterotrophic assay and a pubertal female rat assay. Nonetheless, when PCNB was evaluated as a possible anti-estrogen against estradiol in the immature rat uterotrophic assay, it enhanced, rather than reduced the activity of estradiol. Absence of an effect by PCNB on the uterotrophic activity of diethylstilbestrol suggests that the effect with estradiol was related to alteration of its metabolism. However, PCNB was not hepatotoxic and failed to inhibit cytochrome P450 or estradiol sulphotransferase. Pentachlorophenol, a major metabolite of PCNB, was inactive as an estrogen and failed to enhance the uterotrophic activity of estradiol.

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.

Laboratory animal diets for studies to determine the endocrine-disrupting potential of chemicals are under scrutiny because they can affect both assay control values and assay sensitivity. Although phytoestrogen content is important, we have previously shown that a phytoestrogen-rich diet and a phytoestrogen-free diet were equally uterotrophic to rats and advanced vaginal opening (VO) when compared with the standard diet RM1. Abolition of the effects by the gonadotrophin-releasing hormone antagonist Antarelix indicated that these effects were mediated through the hypothalamus-pituitary-reproductive organ axis. In the present study, we investigated the relationship between cumulative energy intake and sexual maturation in female rats. Infant formula (IF) at different concentrations and synthetic diets, with a wide range of metabolizable energy (ME) values, were used to modulate energy intake. Increasing energy intake was associated with an increase in uterine weight (absolute and adjusted for body weight) for both IF and the synthetic diets. In both cases, the increased uterine weight was directly proportional to energy intake. Body weight was unaffected by IF consumption but, in the case of the diets, was increased proportionally with energy consumption. Antarelix abolished the uterine weight increases with both formula and the diets, whereas body weight was unaffected. The mean day of VO was also advanced by high-ME diets and IF, whereas body weight at VO was unaffected. VO occurred at an energy intake of approximately 2,300 kJ/rat determined by measuring total food intake from weaning to VO, indicating that this cumulative energy intake was the trigger for puberty. ME is therefore a critical factor in the choice of diets for endocrine disruption studies.

2-Acetylaminofluorene (2AAF) is shown to be reproducibly active in inducing UDS in cultured hepatocytes from a B6C3F1 mouse liver.

A major challenge in the emerging field of toxicogenomics is to define the relationships between chemically induced changes in gene expression and alterations in conventional toxicologic parameters such as clinical chemistry and histopathology. We have explored these relationships in detail using the rodent uterotrophic assay as a model system. Gene expression levels, uterine weights, and histologic parameters were analyzed 1, 2, 4, 8, 24, 48, and 72 hr after exposure to the reference physiologic estrogen 17 beta-estradiol (E2). A multistep analysis method, involving unsupervised hierarchical clustering followed by supervised gene ontology-driven clustering, was used to define the transcriptional program associated with E2-induced uterine growth and to identify groups of genes that may drive specific histologic changes in the uterus. This revealed that uterine growth and maturation are preceded and accompanied by a complex, multistage molecular program. The program begins with the induction of genes involved in transcriptional regulation and signal transduction and is followed, sequentially, by the regulation of genes involved in protein biosynthesis, cell proliferation, and epithelial cell differentiation. Furthermore, we have identified genes with common molecular functions that may drive fluid uptake, coordinated cell division, and remodeling of luminal epithelial cells. These data define the mechanism by which an estrogen induces organ growth and tissue maturation, and demonstrate that comparison of temporal changes in gene expression and conventional toxicology end points can facilitate the phenotypic anchoring of toxicogenomic data.

We have evaluated whether mixtures of estrogens, present in the mix at doses that are individually inactive in the immature rat uterotrophic assay, can give a uterotrophic response. Seven chemicals were evaluated: nonylphenol, bisphenol A (BPA), methoxychlor, genistein (GEN), estradiol, diethylstilbestrol, and ethinyl estradiol. Dose responses in the uterotrophic assay were constructed for each chemical. The first series of experiments involved evaluating binary mixtures of BPA and GEN at dose levels that gave moderate uterotrophic responses when tested individually. The mixtures generally showed an intermediate or reduced uterotrophic effect compared with when the components of the mixture were tested alone at the dose used in the mixture. The next series of experiments used a multicomponent (complex) mixture of all seven chemicals evaluated at doses that gave either weakly positive or inactive uterotrophic responses when tested individually in the assay. Doses that were nominally equi-uterotrophic ranged over approximately six orders of magnitude for the seven chemicals. Doses of agents that gave a weak uterotrophic response when tested individually gave a marginally enhanced positive response in the assay when tested combined as a mixture. Doses of agents that gave a negative uterotrophic response when tested individually gave a positive response when tested as a mixture. These data indicate that a variety of different estrogen receptor (ER) agonists, present individually at subeffective doses, can act simultaneously to evoke an ER-regulated response. However, translating these findings into the process of environmental hazard assessment will be difficult. The simple addition of the observed, or predicted, activities for the components of a mixture is confirmed here to be inappropriate and to overestimate the actual effect induced by the mixture. Equally, isobole analysis is only suitable for two- or three-component mixtures, and concentration addition requires access to dose-response data and EC50 values (concentration giving 50% of the maximum response) for the individual components of the mixture--requirements that will rarely be fulfilled for complex environmental samples. Given these uncertainties, we conclude that it may be most expedient to select and bioassay whole environmental mixtures of potential concern.

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.

While defining the no effect level for the 5 alpha-reductase inhibitor finasteride in the Hershberger assay, we encountered an inverted-U low-dose trophic effect on the prostate gland of the rat. Two attempts to confirm this observation were unsuccessful, and we concluded that the positive effect initially observed was associated with normal biologic variability. During the same period we attempted, unsuccessfully, to repeat our own observation of weak uterotrophic activity in the rat for the sunscreen 3-(4-methylbenzylidene)camphor (4MBC). Further evaluation led us to conclude that 4MBC is uterotrophic only when the control uterine weights are at the low end of their normally encountered range. This led us to reevaluate our earlier mouse uterotrophic assay data for bisphenol A (BPA). Originally we had concluded that BPA gave irreproducible evidence of weak uterotrophic activity, but upon ordering the eight experiments we had conducted, according to decreasing control uterine weight, we confirmed reproducible weak uterotrophic activity for BPA when the control uteri were at the low end of their normal range. In this article, we describe these observations, together with a reanalysis of the data associated with several reported instances of weak or low-dose endocrine effects that have proven difficult to confirm in independent laboratories. These include the activity of BPA on the CF1 mouse prostate; the activities of BPA, octylphenol, and nonylphenol on the rat testis; and the effect of polycarbonate caging on control mouse uterine weight. In all of these cases, variability among controls provides a major obstacle to data interpretation and confirmation. Our recommendations on experimental design are also presented, with a view to ending the current impasse on the reality, or otherwise, of low-dose or weak endocrine toxicities.

Our attempts to confirm reports of low-dose/hormetic effects in rodent endocrine toxicity studies are reviewed. It is concluded that our present failure to confirm any such effects is due, in large part, to a general lack of understanding of confounding influences and the failure of most investigators to confirm their findings before publication. The major potential confounding factor is suggested to be variability of the parameters under study within control groups, a factor that assumes increased importance when attempting to demonstrate weak low-dose effects. This is illustrated by our studies with bisphenol A in the mouse uterotrophic assay and of finasteride in the Hershberger antiandrogenicity assay. In both of these cases our ability to demonstrate a low-dose effect is dependent on whether concurrent or recent control values are used.

M. Sakaue et al. (2001,J. Occup. Health vol. 43, pp. 185-190) have described how oral exposure of sexually mature male rats to bisphenol A (BPA) between postnatal days (PND) 91-97 led to a reduction in daily sperm production (DSP) 5 weeks later (18 weeks of age). Activity was observed over the dose range 20 microgram/kg-200 mg/kg BPA, with an absence of activity over the dose range 2 ng/kg-2 microgram/kg BPA. There was no evidence of a dose response relationship over the active dose range (five orders of magnitude range). The observation of endocrine disruption (ED) effects for BPA at such low doses, and in sexually mature animals, was unexpected. It was therefore decided to mount an independent repeat of their study. A total of four independent studies were conducted according to the protocol used by Sakaue et al. Doses of 20 microgram/kg, 2 mg/kg, or 200 mg/kg BPA were administered to adult Sprague-Dawley (SD) rats over PND 91-97, and the studies were terminated when the rats reached the age of 18 weeks. Three different rodent diets were employed (RM3, Purina 5002, and CE2), the last of which had been used by Sakaue et al. BPA failed to give any evidence of ED activities, including the changes in DSP reported by Sakaue et al. 2001. During the course of these studies, the test protocol was adapted to coincide more precisely with that used by Sakaue et al.; this included restricting the number of animals per cage, removing bedding from the cages, and changing to the use of glass water bottles in the cages. The only thing of interest to emerge from our studies was the observation of a significant difference in DSP between the control groups of our first and second study. As the change in diet from RM3 to Purina 5002 was the major difference between those two studies, we conducted a repeat of the second study, but we were unable to confirm the differences seen between the first and second study. The probability that those differences arose either by chance, or as the result of intrinsic study-to-study variability, was strengthened by the absence of significant differences in the sperm parameters in a final (fifth) study where the sperm parameters for control animals maintained on the three different diets were compared under the conditions of the main experiments. No explanation for our failure to replicate the effects reported by Sakaue et al. is evident. A review of DSP values reported in the recent literature is provided and discussed, and it is concluded that use of the term DSP/g testis rather than DSP/testis could increase the sensitivity of DSP assessments.

Many commercial laboratory diets have detectable levels of isoflavones (e.g., phytoestrogens such as genistein [GN]) that have weak estrogenic activity both in vitro and in vivo. During validation studies of the uterotrophic bioassay, diet samples from 20 participating laboratories were collected and analyzed for three major phytoestrogens: GN, daidzein (DN), and coumestrol (CM). Soy phytoestrogens GN and DN were found at total phytoestrogen levels from 100 to 540 microg/g laboratory diet; a forage phytoestrogen, CM, ranged from nondetectable to 4 microg/g laboratory diet. The phytoestrogen levels were compared with both baseline uterine weights of the control groups and with the relative uterine weight increase of groups administered two weak estrogen agonists: bisphenol A (BPA) and nonylphenol (NP). The comparison uses a working assumption of additivity among the phytoestrogens, despite several significant qualifications to this assumption, to estimate total genistein equivalents (TGE). Some evidence was found that phytoestrogen levels in the diet > 325-350 microg/g TGE could diminish the responsiveness of the uterotrophic bioassay to weak agonists. This was especially true for the case of the intact, immature female version of the uterotrophic bioassay, where higher food consumption relative to body weight leads to higher intakes of dietary phytoestrogens versus ovariectomized adults. This dietary level is sufficient in the immature female to approach a biological lowest observable effect level for GN of 40-50 mg/kg/day. These same data, however, show that low to moderate levels of dietary phytoestrogens do not substantially affect the responsiveness of the assay with weak estrogen receptor agonists such as NP and BPA. Therefore, laboratories conducting the uterotrophic bioassay for either research or regulatory purposes may routinely use diets containing levels of phytoestrogens < 325-350 microg/g TGE without impairing the responsiveness of the bioassay.

BACKGROUND The Hershberger assay for antiandrogens and modifiers of steroid biosynthesis uses surgically-castrated rats. We described an adaptation of the assay using the GnRH inhibitor Antarelix in place of surgical castration [Ashby J, Lefevre PA, Deghenghi R, Wallis N. Regulatory Toxicology and Pharmacology 34:188-203, 2001], and concomitantly described changes in expression of the androgen-dependent prostatic genes PBP C3, TRPM-2, and ODC as a possible complement to gravimetric analysis of the sex accessory tissues (SAT) [Nellemann C, Vinggaard AM, Dalgaard M, Hossaini A, Larsen J-J. Toxicology 163:29-38, 2001. METHODS The present study describes the results of combining these two modifications into a single assay. During the course of these experiments it was shown that SD rats gave similar results to AP rats and that the higher stimulatory dose of testosterone propionate (TP) used in our experiments gave stronger assay responses to FLU than the lower dose of TP used by some earlier investigators. The potent antiandrogen flutamide (FLU) and the weak antiandrogen DDE were used to evaluate this modified assay. RESULTS For all parameters studied (SAT weights and changes in expression of the 3 prostatic genes) FLU gave the expected positive results. The weak antiandrogen DDE gave variable and mainly non-reproducible responses. Use of DDE as a weak antiandrogen accelerated assessment of the new assay. CONCLUSIONS Possible reasons for this failure to detect DDE are discussed, and it is concluded that the modified assay is unsuitable for use in its present form. The use of gene expression analyses together with evaluation of SAT weights is a promising tool as an early and sensitive marker of antiandrogen action, but more work is needed on the choice of time frame as well as the selection of genes to monitor.