P. A. Lefevre

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

A range of chemicals with various levels of activity as actual or potential endocrine disrupters have been evaluated for activity in the peripubertal male rat assay. The chemicals studied included anti‐androgens (vinclozolin), cyproterone acetate, flutamide, 2,2‐bis(4‐chlorophenyl)‐1,1‐dichloroethylene (DDE), metabolic modulators (anastrazole, finasteride, ketoconazole) and oestrogens (butyl benzyl phthalate (BBP), methoxychlor, bisphenol A (BPA), diethylstilboestrol (DES)), the suspected anti‐androgen dibutyl phthalate (DBP) and the non‐oestrogen fenitrothion. Dosing extended over postnatal days (pnd) 22–35, 36–50, 36–55 and 22–35, with recovery to pnd 55 or 22–55. The endpoints studied were changes in the weights of testes, epididymides, seminal vesicles and prostate. Changes in body weight and the weights of the liver and kidney were also monitored. In some experiments changes in the day of prepuce separation (PPS) were also determined. Only BBP and BPA were inactive in all the assays conducted. Changes in the weight of reproductive tissues provided a sensitive indicator of activity for the remaining chemicals with the exception of DDE, for which higher dose levels could have been used. However, none of the curtailed periods of exposure were able to detect all of the agents. Diethyl stilboestrol, and to a lesser extent DBP and DDE, delayed PPS when exposure occurred over the period pnd 22–55. A complex dependence of the day of PPS on the period of exposure and the body weight of the test animals was observed, and caution is recommended when assessing this endpoint in the presence of reductions in body weight. It is concluded that reproductive tissue weight changes in the peripubertal male have shown sensitivity to a range of biochemical modulators, oestrogens and anti‐androgens, and that as such the assay warrants further evaluation. Measurement of delays in PPS may be of value in cases of large delays, but delays of 1–2 days will be difficult to interpret with confidence. The present results are discussed within the context of the sexually mature male rat assay described by O’Connor and the castrated male rat assay described by Hershberger, both of which are the subject of current international study. It is concluded that a decision on the usefulness of the peripubertal male rat assay must await the generation of further data on each of these three assays. There is an urgent need for international agreement on a list of reference endocrine disrupters and their active dose levels, with which to validate individual endocrine disruption assays and batteries of assays. Copyright

Partial and weak oestrogenicity of the red wine constituent resveratrol: consideration of its superagonist activity in MCF-7 cells and its suggested cardiovascular protective effects

It was recently reported that the red wine phytoestrogen resveratrol (RES) acts as a superagonist to oestrogen‐responsive MCF‐7 cells. This activity of RES was speculated to be relevant to the ‘French paradox’ in which moderate red wine consumption is reported to yield cardiovascular health benefits to humans. We report here that RES binds to oestrogen receptors (ER) isolated from rat uterus with an affinity 5 orders of magnitude lower than does either the reference synthetic oestrogen diethylstilboestrol (DES) or oestradiol (E2). In comparison with E2 or DES, RES is only a weak and partial agonist in a yeast hER‐α transcription assay and in cos‐1 cell assays employing transient transfections of ER‐α or ER‐β associated with two different ER‐response elements. Resveratrol was also concluded to be inactive in immature rat uterotrophic assays conducted using three daily administrations of 0.03–120 mg kg−1/day−1 RES (administered by either oral gavage or subcutaneous injection). These data weaken the suggestion that the oestrogenicity of RES may account for the reported cardiovascular protective effects of red wine consumption, and they raise questions regarding the extent to which oestrogenicity data derived for a chemical using MCF‐7 cells (or any other single in vitro assay) can be used to predict the hormonal effects likely to occur in animals or humans. Copyright

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.

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.