RESUMO
Partially purified lipid extracts of Saccharomyces cerevisiae contain a substance that displaces tritiated estradiol from rat uterine cytosol estrogen receptors. The yeast product induces estrogenic bioresponses in mammalian systems as measured by induction of progesterone receptors in cultured MCF-7 human breast cancer cells and by a uterotrophic response and progesterone receptor induction after administration to ovariectomized mice. The findings raise the possibility that bakers' yeast may be a source of environmental estrogens.
Assuntos
Estrogênios/biossíntese , Saccharomyces cerevisiae/metabolismo , Animais , Bioensaio , Neoplasias da Mama/metabolismo , Estradiol/metabolismo , Estrogênios/farmacologia , Feminino , Humanos , Camundongos , Receptores de Progesterona/metabolismo , Útero/efeitos dos fármacosRESUMO
We have previously shown the presence of 17 beta-estradiol in extracts of commercially prepared Saccharomyces cerevisiae ss well as the production of estradiol by yeast grown in the laboratory. In our current study, yeast grown in a chemically defined medium synthesized estradiol in only small amounts, (less than 500 pg/liter). We have analyzed a variety of media commonly used for growing yeast and found that substantial estradiol production (greater than 5 ng/liter) was obtained when yeast were grown in medium supplemented with Bacto-peptone. The peptone was shown to contain significant amounts of estrone, and the results of the experiments establish a precursor-product relationship where estrone from the medium is metabolized to estradiol by S. cerevisiae. Studies with added [3H]estrone demonstrated rapid conversion into [3H]estradiol and a 3H-labeled nonpolar estrogen derivative. The commercially obtained yeast used previously had been grown in a molasses medium. We demonstrate here that the molasses medium contains substantial amounts of estrone and estradiol. We conclude that the conversion of estrone in a culture medium to estradiol in laboratory grown yeast and estrone and estradiol present in the commercially grown yeast medium account for the majority of estradiol found in yeast.
Assuntos
Estrogênios/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Estradiol/análise , Estrona/análise , Melaço , Peptonas , Radioimunoensaio , Fatores de TempoRESUMO
This paper further characterizes the estrogen-binding protein we have described in the cytosol of the yeast Saccharomyces cerevisiae. [3H]Estradiol was used as the radioprobe, and specific binding of cytosol fractions was measured by chromatography on Sephadex minicolumns. Other 3H-steroids did not exhibit specific binding. [3H]Estradiol binding was destroyed by treatment with trypsin, but not RNase, DNase, or phospholipase; N-ethylmaleimide substantially decreased the binding. The yeast did not metabolize estradiol added to the medium, and extraction and chromatography of the bound moiety showed it to be unmetabolized estradiol. Scatchard analysis of cytosol from both a and alpha mating types as well as the a/alpha diploid cell revealed similar binding properties: an apparent dissociation constant or Kd(25 degrees) for [3H]estradiol of 1.6-1.8 nM and a maximal binding capacity or Nmax of approximately 2000-2800 fmol/mg of cytosol protein. Gel exclusion chromatography on Sephacryl S-200 and high performance liquid chromatography suggested a Stokes radius of approximately 30 A. Sucrose gradient centrifugation showed a sedimentation coefficient of approximately 5 S, and the complex did not exhibit ionic dependent aggregation. The estrogen binder in S. cerevisiae differed in its steroidal specificities from classical mammalian estrogen receptors in rat uterus. 17 beta-Estradiol was the best competitor, 17 alpha-estradiol had about 5% the activity, and diethylstilbestrol exhibited negligible binding affinity as did tamoxifen, nafoxidine, and the zearalenones. In summary, a high affinity, stereospecific, steroid-selective binding protein has been demonstrated in the cytosol of the simple yeast S. cerevisiae. We speculate that this molecule may represent a primitive hormone receptor system, possibly for an estrogen-like message molecule.
Assuntos
Estradiol/metabolismo , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Citosol/metabolismo , Estabilidade de Medicamentos , Cinética , Peso Molecular , Receptores de Estrogênio/isolamento & purificaçãoRESUMO
Saccharomyces cerevisiae possesses a high-affinity estrogen binding protein and an endogenous ligand that displaces [3H]estradiol from both the yeast binding protein and mammalian estrogen receptors. Semipurified preparations of this ligand have been shown to exhibit estrogenic activity in mammalian systems. We now describe the purification procedure and ultimate identification of the estrogenic substance in extracts of S. cerevisiae as 17 beta-estradiol. Organic solvent extracts of commercially obtained dried yeast were sequentially chromatographed on silica gel columns and then subjected to a series of reversed phase HPLC fractionations. Active ligand was monitored by [3H]estradiol displacement in a rat uterine cytosol assay. After seven chromatography steps, the purified and highly active ligand exhibited a single peak with retention times identical to those of 17 beta-estradiol on both HPLC and GC. The yeast material was identified as 17 beta-estradiol by its UV absorbance and mass spectrometric fragmentation pattern. In addition, radioimmunoassay confirmed the presence of approximately the same mass of 17 beta-estradiol (approximately equal to 800 ng/1.5 kg of yeast) as estimated both by a competitive binding assay with estrogen receptor and by mass spectrometry. Extraneous contamination by estradiol was excluded by repeat experiments with different batches of starting material and demonstration of estradiol by RIA in conditioned medium and cell pellets of laboratory-grown S. cerevisiae whereas non-conditioned medium did not possess the steroid. We conclude that 17 beta-estradiol is a yeast product.
Assuntos
Estradiol/isolamento & purificação , Saccharomyces cerevisiae/análise , Estradiol/imunologia , RadioimunoensaioRESUMO
It has been shown by us and others that progesterone inhibits the growth of Trichophyton mentagrophytes and that the organism escapes from this inhibition over time. We report here studies which show that escape from growth inhibition is related to the enzymatic transformation of progesterone to polar metabolites. Isolation and identification of the progesterone metabolites confirm the production of 15 alpha-hydroxyprogesterone. In addition, three other metabolites were isolated. Two of these were determined to be 1-dehydroprogesterone and 11 alpha-hydroxyprogesterone. The third metabolite was a 1-dehydro-hydroxyprogesterone, but the location of the hydroxyl group could not be determined unequivocally. Studies using authentic 15 alpha-hydroxyprogesterone, 1-dehydroprogesterone, and 11 alpha-hydroxyprogesterone reveal that these derivatives are significantly less inhibitory to the growth of T. mentagrophytes than progesterone. Pretreatment of organisms with progesterone augments the rate of metabolism and enhances escape. We have described previously a progesterone-binding protein (PBP) in cytoplasmic extracts of T. mentagrophytes and hypothesized that progesterone mediates growth inhibition by binding to the PBP of this organism. The relative binding affinity that progesterone and its metabolites display for PBP correlates with the relative growth inhibitory potency of these compounds. These results suggest that metabolism of progesterone to more polar and less inhibitory compounds, which exhibit lower affinity for PBP, is the mechanism of escape from progesterone-mediated inhibition of growth in this organism.