E. Perel

The relationship between aromatase activity and body fat distribution

The metabolism of androstenedione (A) to estrone (E1) and 5 alpha-reduced androgens was studied in stromal cells derived from human adipose tissue from different body sites. The tissue was obtained from non-obese patients undergoing cosmetic liposuction or at the time of surgery for reduction mammoplasty. The conversion of A to E1 per 1x 10(6) cells was between 6- and 30-fold greater in the upper thigh, buttock, and flank than in the abdomen. These differences were present in primary culture and persisted to at least the third subculture. Estrogen formation in breast adipose tissue was similar to that found in cells from abdominal fat. The formation of 5 alpha-reduced metabolites (5 alpha-androstenedione, androsterone, and dihydrotestosterone) varied from patient to patient but was similar in cells from different body sites. These studies show that the regional distribution of fat may influence the metabolism of androgens in adipose tissue, with upper body fat tending to form a lower ratio of estrogens to 5 alpha-reduced androgens than lower body fat.

Steroid modulation of aromatase activity in human cultured breast carcinoma cells

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.

Influence of Adipose Tissue Distribution on the Biological Activity of Androgens

To establish whether the conversion of androstenedione (A) to estrogens and 5 alpha-reduced metabolites in human adipose tissue was determined by the site of origin of the tissue, studies were carried out on adipose stromal cells from different body sites. Adipose tissue was obtained from the breast, omentum, abdomen, lower thigh, upper thigh, buttock, and flank from patients undergoing liposuction for cosmetic reasons or at surgery. Stromal cells were isolated after incubation of the adipose tissue with collagenase and were grown in culture using alpha-minimal essential medium (MEM) + 15% fetal calf serum. Studies of A metabolism were carried out when the cells were between days 4 and 12 in culture. After an 8-hour incubation with (3H)-A as substrate, estrone (E1), testosterone (T), 5 alpha-androstanedione (5 alpha-A-dione), androsterone (AND), and dihydrotestosterone (DHT) were isolated using thin layer and paper chromatography. The conversion per 1 x 10(6) cells of A of E1 was more than 10-fold greater in the upper thigh, buttock, and flank than in the breast, lower thigh, abdomen, or omentum (0.13-3.0 vs 0.01-0.09%). The formation of 5 alpha-reduced androgens varied from 0.86-10% and was similar in tissue from different body sites. Cortisol (10(-7) M) stimulated E1 formation 3- to 10-fold in cells from all sites, whereas 5 alpha-reductase activity was either unchanged or increased moderately (up to twofold). In cells from the abdomen, omentum, and lower thigh, the formation of 5 alpha-reduced androgens was more than 10-fold greater than the formation of E1. In cells from the upper thigh, buttock, and flank, E1 formation was comparable to 5 alpha-reduced androgen formation. These studies show marked differences in the relative conversion of A to estrogens and 5 alpha-reduced androgens in adipose stromal cells depending on their site of origin, and they suggest that the distribution of body fat may be a major factor in determining the biologic effects of secreted androgens.

Aromatase activity in the breast and other peripheral tissues and its therapeutic regulation

Studies using [3H]androstenedione (A) demonstrated that this substrate can be aromatized to estrone (E1) in homogenates of breast carcinoma tissue and breast adipose tissue, in breast carcinoma and breast adipose stromal cells in culture, and in cultured adipose stromal cells from sites remote from the tumor. Using cultured breast carcinoma cells, it was shown that estrogen formation was stimulated by cortisol (10(-6) M) and inhibited by endogenous 5 alpha-reduced androgens: 5 alpha-androstene-dione greater than androsterone greater than dihydrotestosterone greater than epiandrosterone greater than 3 alpha- and 3 beta- androstanediol. It was also shown that 19-nortestosterone and 19-norandrostenedione (10(-6) M) inhibited E1 formation by 80%. Progesterone (10(-6) M) had no effect on aromatase activity, while the progestational agent R5020 (10(-6) M) caused a 70% inhibition. These studies emphasize that a variety of compounds can influence aromatase activity and that drugs which are used as aromatase inhibitors in patients with breast carcinoma may have multiple sites of action.

Androgen metabolism in male and female breast tissue

Incubation studies have been carried out using normal breast tissue and breast tissue from patients with gynecomastia, mammary dysplasia and breast carcinoma to determine the pattern of androstenedione metabolism. All tissues formed estrone (E1) and testosterone (T) in all incubations. Estradiol (E2) was isolated in incubations of tissue from 1 of 6 patients with mammary dysplasia, 5 of 6 patients with gynecomastia and in all incubations with normal and carcinoma tissue. Estrone formation was lowest in mammary dysplasia and gynecomastia, and higher in apparently normal breast tissue. The greatest E1 formation was found in incubations with breast carcinoma tissue, although there was considerable variation within this tissue group. Estradiol formation was low in all tissues, with the highest conversion rates in carcinoma tissue. Testosterone formation in carcinoma tissue was greater than in mammary dysplasia or gynecomastia, but similar to apparently normal tissue. These results indicate that breast tissue from different pathological states varies in its capacity to aromatize androstenedione (A) to estrogenic products and to convert it to other androgens. They have also shown that the pattern of metabolism is distinctive for the nature of the pathological abnormality.

The relationship between growth and androstenedione metabolism in four cell lines of human breast carcinoma cells in culture

The conversion of androstenedione (A) to estrogens, testosterone (T) and 5 alpha-reduced metabolites was studied in different phases of cell growth in 4 lines of cultured human breast carcinoma cells. Aromatase activity was 10-fold greater in MD and DM than in MCF7 cells and was undetectable in ZR75 cells. Estrogen formation in MD and DM lines increased during the phase of exponential growth and decreased to 20% of maximum during confluence. 5 alpha-Reductase activity was determined by the formation of 5 alpha-androstane-3,17-dione (5 alpha-A-dione) and androsterone (AND), and was 5-fold greater in ZR75 cells than MD cells and 2-fold greater than in MCF7 cells. This activity was relatively constant during exponential growth and decreased during confluence. T accumulation was inversely related to 5 alpha-reductase activity. The MCF7 and ZR75 cells which contain estrogen receptors had the highest levels of 5 alpha-reductase activity while the MD line which lacks estrogen receptors had the lowest 5 alpha-reductase activity. The assessment of aromatase and 5 alpha-reductase activity in addition to estrogen and progesterone receptors may be helpful in predicting hormone sensitivity in human breast tumours.

Effects of 4-hydroxy-4-androstene-3, 17-dione and 10-propargylestr-4-ene-3, 17-dione on the metabolism of androstenedione in human breast carcinoma and breast adipose tissues.

The effects of 4-hydroxy-4-androstene-3, 17-dione (4-OH-A) and 10-propargylestr-4-ene-3, 17-dione (PED) on the aromatization of androstenedione (A) and the conversion of A to testosterone (T) were studied in incubations with breast carcinoma and breast adipose tissues. Parallel studies were carried out to determine the effects of 4-OH-A and PED on A metabolism in tissue from 5 patients with breast carcinoma. At 11 micro M, both compounds fully inhibited aromatization, whereas the conversion of A to T was decreased in only 2 incubations. Studies with varying concentrations of 4-OH-A and PED demonstrated that both compounds inhibited estrone (E1) formation by 80% at a concentration of 0.085 micro M, with maximum effect at 0.34 micro M. 90% inhibition of estradiol (E2) formation was observed at inhibitor concentrations of 0.17 micro M or greater. T formation was slightly affected at 0.67 microM, but was progressively inhibited with increasing 4-OH-A or PED concentrations, reaching 70% at 11 micro M. Similar experiments with 4-OH-A in breast adipose tissue homogenates showed that a concentration of 0.1 micro M was sufficient to inhibit aromatization while T inhibition required 11 micro M. 4-OH-A and PED are selective inhibitors of aromatization in human breast tissues and may provide a mechanism for controlling estrogen responsive processes.

Androstenedione metabolism in epithelial cells derived from early-lactation human milk

Epithelial cells derived from duct epithelium were cultured from early lactation human milk in medium supplemented with 15% fetal calf serum, insulin (0.3 u/ml), cortisol 21-sodium succinate (6 micrograms/ml) and amikacin (50 micrograms/ml). The capacity of these cells to metabolize androstenedione to estrone, estradiol and C19 metabolites was studied during continuous culture. After extraction of the medium, the products were subjected to phenolic partition and separated by thin-layer and paper chromatography, followed by recrystallization to constant specific activity. The study demonstrated a progressive increase in the formation of estrone and testosterone over the first 24 h in culture, while estradiol formation showed an initial 2-4 h lag, then increased slowly. The C19 compounds identified were androsterone, 5 alpha-androstanedione, epiandrosterone, dihydrotestosterone and etiocholanolone. 5 alpha-Androstanedione and androsterone were the major 5 alpha-reduced metabolites. Since these cells are derived from normal duct epithelium, their metabolic characteristics may be more representative of normal breast tissue than those of tissue removed from patients with pathological breast disorders.