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In previous studies from our laboratory of the metabolism of androstenedione (A) and testosterone (T) in breast adipose and breast carcinoma tissue, the aromatization of these compounds, and their interconversion were demonstrated. The present study describes the conversion of androstenedione and testosterone to C19 metabolites in homogenates of normal breast tissue and breast carcinoma tissue and examines the C19 metabolites in homogenates of benign prostatic hypertrophy (BPH) tissue under similar conditions. Tissue homogenates were incubated for 90 min in Krebs-Ringer bicarbonate buffer (pH 7.4) with ATP (3 mM) and NADPH (2.4 mM) as co-factors. Following extraction with ethyl acetate, the metabolites were separated into neutral and phenolic fractions. Identification of specific products was made by thin layer and paper chromatography and recrystallization procedures. The formation of C19 metabolites was 10-fold greater in prostate than in breast tissue. Androsterone was the major product of androstenedione in both breast and prostate. The other 5 alpha-metabolites of androstenedione identified were dihydrotestosterone (DHT) and epiandrosterone (EPI). The 5 beta-metabolite, etiocholanolone (ET10), was identified in both breast and prostate following incubation with androstenedione. Using testosterone as substrate, dihydrotestosterone was the major product in normal breast and BPH. Etiocholanolone was detected in breast, but not in prostate following incubation with testosterone. Aromatization was demonstrated in all incubations with breast tissue, but not in prostate. The results indicate that androstenedione is actively metabolized by both breast and prostate to 5 alpha reduced metabolites. They also identify the formation of etiocholanolone in both tissues. Aromatization was demonstrated only in breast tissue.

Cortisol and steroids with progestational or androgenic activity were studied to determine the effects of these steroids on the conversion of androstenedione (A) to estrone (E1) in human cultured breast carcinoma cells. Cortisol (10(-6) M) stimulated aromatase activity in two estrogen unresponsive cell lines (MD, DM) and in an estrogen responsive cell line (MCF7) with the maximum stimulation occurring during confluence. Cortisol inhibited the replication of MCF7 cells but not MD and DM. Dihydrotestosterone, androsterone and 5 alpha-androstanedione (10(-6) M) inhibited the conversion of A to E1 by greater than 90% under basal and cortisol stimulated conditions. Progesterone (10(-6) M) had no effect on aromatase activity while the progestational agent R5020 (10(-6) M) produced a 30% inhibition. The anabolic steroids 19-nortestosterone and 19-norandrostenedione which also have progestational activity inhibited the conversion of A to E1 in a dose dependent manner with 90% inhibition at 10(-6) M. Danazol (10(-6) M) a drug with both androgenic and progestational activity inhibited E1 formation by 30%. Under the same conditions, the known inhibitor of aromatase, 4-hydroxyandrostenedione (10(-6) M) decreased E1 formation by more than 90% and aminoglutethimide (10(-6) M) caused only 25% inhibition. These studies demonstrate that endogenous and exogenous steroids may have significant effects in modulating the local formation of estrogens from androgen precursors in cultured breast carcinoma cells. This effect on estrogen formation may be a factor in the biological response of breast tissue.

We have used 5 alpha-dihydrotestosterone (DHT) and two synthetic, non-metabolizable androgens, methyltrienolone (MT) and mibolerone (MB), to study intact genital skin fibroblasts from four subjects with familial incomplete androgen resistance. In each, the free androgen receptor has normal binding capacity at 37 degrees C and normal half-lives at 37-43 degrees C. In three the mutant receptor misbehaves in a pattern that is ligand-specific and temperature-dependent. At 37 degrees C the equilibrium (Kd) and non-equilibrium (k) dissociation constants, and the ability to augment binding activity during prolonged exposure to androgen, are impaired with DHT, but not with MT; with MB, only the k is abnormal. Mutant MT-receptor complexes dissociate normally even at 42 degrees C; yet, in cells post-incubated at 42 degrees C with cycloheximide and a saturating concentration of ligand, their pool size decays in the rank order, MT greater than MB greater than normal. This measure of lability is nonlinear as a semilogarithmic function of time; it varies directly with temperature and the concentration of cycloheximide, but inversely with that of ligand. Thus, MT and MB evoke distinct forms of thermal dysfunction from the androgen receptor in ligand-sensitive androgen resistance. This observation will help to elucidate the combinatorial properties of normal androgen-receptor complexes that enable them to regulate gene transcription differentially in various androgen target tissues.