In studies to determine whether Saccharomyces cerevisiae produced estrogens, the organism was grown in culture media prepared using distilled water autoclaved in polycarbonate flasks. The yeast-conditioned media showed the presence of a substance that competed with [3H]estradiol for binding to estrogen receptors (ER) from rat uterus. However, it soon became clear that the estrogenic substance in the conditioned media was not a product of the yeast grown in culture, but was leached out of the polycarbonate flasks during the autoclaving procedure. [3H]Estradiol displacement activity was monitored by ER RRA, and the active substance was purified from autoclaved medium using a series of HPLC steps. The final purified product was identified as bisphenol-A (BPA) by nuclear magnetic resonance spectroscopy and mass spectrometry. BPA could also be identified in distilled water autoclaved in polycarbonate flasks without the requirement of either the organism or the constituents of the culture medium. Authentic BPA was active in competitive RRAs, demonstrating an affinity approximately 1:2000 that of estradiol for ER. In functional assays, BPA (10-25 nM) induced progesterone receptors in cultured human mammary cancer cells (MCF-7) at a potency of approximately 1:5000 compared to that of estradiol. The BPA effect on PR induction was blocked by tamoxifen. In addition, BPA (25 nM) increased the rate of proliferation of MCF-7 cells assessed by [3H]thymidine incorporation. Thus, BPA exhibited estrogenic activity by both RRA and two functional bioresponse assays. Finally, MCF-7 cells grown in media prepared with water autoclaved in polycarbonate exhibited higher progesterone receptor levels than cells.grown in media prepared with water autoclaved in glass, suggesting an estrogenic effect of the water autoclaved in polycarbonate. Our findings raise the possibility that unsuspected estrogenic activity in the form of BPA may have an impact on experiments employing media autoclaved in polycarbonate flasks. It remains to be determined whether BPA derived from consumer products manufactured from polycarbonate could significantly contribute to the pool of estrogenic substances in the environment.

1. Bile salts of Petromyzon marinus L. ammocoetes appeared to consist solely or chiefly of a crystalline substance, whose chromatographic and i.r.-spectral characteristics suggested that it was a monosulphate ester of a bile alcohol having the 3alpha,7alpha,12alpha-trihydroxy pattern of substitution in a 5alpha-steroid nucleus. 2. This substance on cleavage with dioxan-trichloroacetic acid gave petromyzonol, n.m.r. and mass-spectral examination of which suggested the structure 5alpha-cholane-3alpha,7alpha,12alpha,24-tetrol. 3. 3alpha,7alpha,12alpha-Trihydroxy-5alpha-cholanoic acid (allocholic acid) from the lizards Anolis lineatopus lineatopus Gray and Cyclura carinata Harlan (family Iguanidae) was esterified with propan-1-ol and reduced by lithium aluminium hydride to 5alpha-cholane-3alpha,7alpha,12alpha,24-tetrol, identical with petromyzonol. 4. Chromic acid oxidation of petromyzonol sulphate from lamprey bile, followed by acid hydrolysis, gave 24-hydroxy-5alpha-cholane-3,7,12-trione; hence the sulphate ester group is at C-24. 5. Petromyzonol sulphate is both primitive and unique: a study of its biogenesis might improve our understanding of evolution at the molecular level.

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1. Methods have been developed for the isolation and identification of small amounts of bile salts and of bile acids and alcohols obtained by solvolysis. These methods involve preparative and analytical t.l.c., purification on columns of protonated Al(2)O(3) and Sephadex LH-20 and also g.l.c.-mass spectroscopy of solvolysis products. 2. Application to 29 species of frogs and toads has confirmed the constancy of bile salt patterns in a single species, including colour phases in two instances, and has revealed great variations between different species in some genera (e.g. Rana, Ptychadena) and little difference between widely distributed species in others (e.g. Bufo). 3. Taxonomic deductions should be made with caution and with regard to the physiological significance of the biochemical character considered. The molecular differences found might be interpreted as indicating variations in the rate of evolution.

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