ESTROGENIC ACTIVITY INVIVO AND INVITRO OF SOME DIETHYLSTILBESTROL METABOLITES AND ANALOGS

The diethylstilbestrol (DES) metabolite (β- dienestrol), which had been identified in mouse, rat, monkey, and human urine, and 2 proposed metabolic intermediates (diethylstilbestrol α,α'-epoxide and α,α'- dihydroxy DES) were synthesized and their estrogenic activities determined. In addition, 3 DES analogs, α-dienestrol, DES-dihydroxy diethyl phenanthrene (DES-phenanthrene), and 1-(α-ethyl, 4α-hydroxyphenyl)indanyl- 5-ol (indanyl-DES), were studied. Estrogenic activities of the compounds in vivo were determined by the immature mouse uterine weight bioassay; in vitro, their estradiol receptor binding activity (competitive equilibrium binding, sucrose gradient analysis, and association rate inhibition assays) was determined. Results of the mouse uterine weight bioassay gave the following order of estrogenicity: DES > α-dienestrol ≥ DES-epoxide > indanyl-DES > dihydroxy DES > β-dienestrol > DES-phenanthrene. Results of competitive equilibrium binding analyses of these compounds with estradiol- 17β for the mouse uterine cytosol receptor followed the same order seen for the bioassay, except for indanyl-DES. DES, indanyl-DES, and α-dienestrol had the greatest affinities (Ka values approximately 0.5-19.1 x 1010 M-1), while DES-phenanthrene had the lowest (Ka = 3.5 x 107 M-1 ± 1.2). Sucrose gradient analysis of the above competition preparations illustrated the displacement of [3H]estradiol from the receptor peak. This displacement was receptor specific and concentration dependent and correlated with the equilibrium binding concentrations. In addition, the most hormonally active substances demonstrated the greatest rate inhibition in the estradiol cytosol receptor association rate reaction (V0). The rank order of estrogenicity of the compounds determined in this study should be useful in evaluating alternative metabolic pathways of DES as well as distinguishing biologically active metabolites from relatively inactive ones.