J. Poortman

Plasma concentration gradient of steroid hormones across human mammary tumours in vivo.

Abstract The paraendocrine behaviour of human breast tumours was studied in vivo. From 10 women with an established mammary tumour, blood samples were taken during mastectomy from the main internal artery of the mammary gland and from its corresponding vein. In these blood samples 10 steroid hormones (oestrogens and androgens) were estimated. All tumours investigated show a positive or negative gradient for one or more steroid hormones. The results indicate that also in vivo human breast tumours are able to modify their own endocrine environment by a wide variety of metabolic conversions. With the exception of Adione we cannot conclude whether these metabolic conversions are of significance for the development of mammary tumours because of the inconsistent pattern in the changes of hormone levels between arterial and venous blood.

Androgens in postmenopausal breast cancer: Excretion, production and interaction with estrogens

Abstract The role of androgens and estrogens has been investigated in postmenopausal breast cancer patients. No differences were found in the urinary excretion of estrone and of estriol between 41 primary mammary cancer patients and 48 normal postmenopausal women, representative of the normal population. A significantly lower excretion of androgen metabolites (11-DOKS) was found in the patients. Because 11-DOKS in postmenopausal women arise mainly from three secretory products and estrogens are mainly derived from peripheral conversion of androstenedione to estrone, production rates of DHEA, DHEAS and androstenedione and the conversion of androstenedione to estrone were estimated. No significant difference was found between the two groups for the blood production rate of androstenedione, neither for its conversion to estrone. The urinary production rate of DHEAS was definitely lower in the selected breast cancer patients compared to normal controls. The DHEA production rate was also lower but statistical significance was not achieved. On account of these results the hypothesis was tested that DHEAS, DHEA or one of their metabolites might interfere with the binding of estradiol to its specific receptor, an essential step in its mechanism of action. From the results of an in vitro incubation study of receptors from human myometrial and mammary tumour tissue with several steroids, evidence was obtained that the estradiol binding was inhibited, in a molar concentration ratio not far beyond the physiological range, by 5-androstene-3β,17β-diol, a steroid closely related to DHEA. If these in vitro findings may be applied to in vivo conditions, it is conceivable that androstenediol is a regulating agent of estrogenic action at the cellular level.

The endogenous concentration of estradiol and estrone in normal human postmenopausal endometrium

The endogenous estrone (E1) and estradiol (E2) levels (pg/g tissue) were measured in 54 postmenopausal, atrophic endometria and compared with the E1 and E2 levels in plasma (pg/ml). The results from the tissue levels of both steroids showed large variations and there was no significant correlation with their plasma levels. The mean E2 concentration in tissue was 420 pg/g, 50 times higher than in plasma and the E1 concentration of 270 pg/g was 9 times higher. The E2/E1 ratio in tissue of 1.6, was higher than the corresponding E2/E1 ratio in plasma, being 0.3. We conclude that normal postmenopausal atrophic endometria contain relatively high concentrations of estradiol and somewhat lower estrone levels. These tissue levels do not lead to histological effects.

Uptake and Concentration of Steroid Hormones in Mammary Tissues

In order to exert their biological effects, steroid hormones must enter the cells of target tissues and after binding to specific receptor molecules must remain for a prolonged period of time in the nucleus. Therefore the endogenous levels and the subcellular distribution of estradiol, estrone, DHEAS, DHEA ad 5-Adiol were measured in normal breast tissues and in malignant and nonmalignant breast tumors from pre- and postmenopausal women. For estradiol the highest tissue levels were found in the malignant samples. No differences were seen in these levels between pre- and postmenopausal women despite the largely different peripheral blood levels. For estrone no differences were found between the tissues studied. Although the estradiol concentration was higher in the estradiol-receptor-positive than in the receptor-negative tumors, no correlation was calculated between the estradiol and the receptor consent. Striking differences were seen between the breast and uterine tissues for the total tissue concentration of estradiol, the ratio between estradiol and estrone, and the subcellular distribution of both estrogens. At similar receptor concentrations in the tissues these differences cannot easily be explained. Regarding the androgens, the tissue/plasma gradient was higher for DHEA than for 5-Adiol, and for DHEAS there was very probably a much lower tissue gradient. The highly significant correlation between the androgens suggests an intracellular metabolism of DHEAS to DHEA and 5-Adiol. Lower concentrations of DHEAS and DHEA were observed in the malignant tissues compared with the normal ones and the benign lesions. For 5-Adiol no differences were found and therefore these data do not support our original hypothesis on the role of this androgen in the etiology of breast abnormalities. Hence the way in which adrenal androgens express their influence on the breast cells remains unclear.

MEASUREMENT OF ENDOGENOUS SUBCELLULAR CONCENTRATION OF STEROIDS IN TISSUE

A reliable method for the extraction of steroid hormones from human uterine tissue and the subsequent measurement of these hormones in the subcellular compartments by radioimmunoassay is described. Extraction of radioactive steroid hormones from in vivo labelled human uterine tissue by different methods reveals that an almost quantitative extraction of steroid hormones from the nuclear fraction is obtained by sonication in ethanol-acetone. Extraction of steroid hormones with diethylether from a high speed cytosol is incomplete. Using a more potent denaturating agent prior to extraction with diethyl ether leads to complete extraction of unconjugated steroids.

Subcellular distribution of androgens and oestrogens in target tissue.

To study the endogenous concentration of androgens and oestrogens in target tissue, three different methods have been used: (a) measurement of concentration gradient across mammary tumours, (b) long-term infusion of subphysiological amounts of labelled oestrogens and measurement of tissue-plasma gradients in human uterine tissue and (c) measurement of endogenous concentrations in mammary and uterine tissues. In addition to the tissue-plasma gradient, the subcellular distribution was also measured. An improved method is presented for processing the tissue and for the quantitative extraction of steroid hormones from cytosol and nuclear fraction. The data obtained show clearly that steroid hormones have a tissue-plasma gradient that varies from tissue to tissue and from one hormone to another. As steroid hormones exert their influence intracellularly at the site of receptor binding, our findings may have major consequences for the study of hormone-related cancers of the breast and of the uterus.

The endogenous concentration and subcellular distribution of androgens in normal human premenopausal endometrium, myometrium and vagina

The endogenous concentrations of testosterone (Testo), androstenedione (Adion), 5-androstene-3 beta, 17 beta-diol (Adiol), dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) have been measured in endometrium, myometrium and vagina from 23 premenopausal women at different stages of the cycle. The tissue to plasma gradient was positive for all androgens except DHEAS; a significant correlation existed between plasma and tissue levels of Adiol and DHEAS. In all tissues significant correlations were calculated between Adiol and DHEA, Adion and Testo, and Adiol and DHEAS. Except for Testo, androgen concentrations were different in the tissues with a significant correlation between the tissues, each tissue seeming to be able to create its own specific intratissue androgen pattern. During the cycle Testo was highest in myometrium and vagina and Adion in myometrium and endometrium during the secretory phase. Except for DHEA, androgens were preferentially localized in the cytosol fraction of the cells. From this localization and the known inhibitory effects of androgens on the activity of estrogen metabolizing enzymes, it is postulated that androgens play a role in enzymatic rather than receptor-mediated processes in uterine cells.

Uptake and metabolism of oestriol in human target tissues

The uptake, metabolism and subcellular distribution of oestradiol and oestriol in endometrial, myometrial and vaginal tissue of postmenopausal women under physiological conditions were studied by giving 3H-labelled oestradiol or oestriol in subphysiological doses by continuous infusion lasting 12 h before hysterectomy. The three tissues obtained from each woman were separated into three fractions: two cytosol fractions (free oestrogens and specifically bound) and one nuclear fraction. The results show an accumulation of both oestrogens in the target tissues, we found an approximately 33 times higher [3H]E2 concentration in endometrium (dpm per g) than in plasma (dpm/ml), 20 times in myometrium and 10 times in vaginal tissue. After the E3 infusions the tissue/plasma gradient was 37 for endometrium, 19 for myometrium and 11 for vagina. In plasma and tissues a metabolite of E3 could tentatively be identified as 16 alpha-hydroxyoestrone. The subcellular distribution showed that 60-80% of E2 and E3 is accumulated in the nuclear fraction of all tissues studied, no nuclear bound oestrone could be detected. From these results the conclusion was drawn that oestradiol still is the major tissue oestrogen in postmenopausal women and that it is mainly nuclear bound. Endometrium of postmenopausal women accumulates higher concentrations of E2 and E3 than vaginal tissue from the same individual, no preferential uptake of oestriol occurs under physiological conditions.

Metabolism of estradiol-17β, 5-androstene-3β,17β-diol and testosterone in human breast cancer cells in long-term culture

The human breast cancer cell line MCF-7 is able to metabolize steroids, which are added in order to study the growth rate of these cells. The following steroids: estradiol-17β, 5-androstene-3β,17β-diol and testosterone were incubated with these cells for 48 h under identical conditions used for growth-rate studies in our laboratory. The metabolites formed were identified. The conversion of estradiol-17β to estrone was 7% and no estriol was formed. The conversion of 5-androstene-3β-17β-diol to dehydroepiandrosterone was 8.1%, to testosterone 1.0% and to 5α-androstane-3β,17β-diol 3.2%. The conversion of testosterone to androstenedione was 6.5%, to etiocholanolone 0.4%, to 5α-dihydrotestosterone 0.7%, to 5-androstene-3β,17β-diol 0.5% and to androstenediol 0.2%. Some of these metabolites, due to their relative binding affinity, may have a biological effect on these cells that contain androgen and estrogen receptors.

Metabolic fate of intraperitoneally administered 5-androstene-3β,17β-DIOL, estradiol-17β and their combination in the immature female rat

Abstract Previous experiments in our laboratory have shown that the intraperitoneal administration to immature female rats of 1000 μg 5-androstene-3β,17β-diol (ADIOL) leads to effects, which are almost identical to those of 2.5 μg estradiol-17β (E 2 ) in several estrogen target organs. Moreover, the combination of these two hormones displayed strongly enhanced effects, which were shown to be related to a biphasic nuclear translocation of the estrogen receptor. The present investigation was undertaken to establish whether these effects of ADIOL could be due to its aromatization and to assess the contribution of the two steroids to the biphasic nuclear translocation of the estrogen receptor, which occurred with the combination of the two hormones. The distribution of E 2 and ADIOL over the plasma and the subcellular compartments of the uterus (cytosol and nucleus) was studied in experiments in which the steroids were administered in the same way as in our previous experiments. The conversion of ADIOL to E 2 in the plasma was found to be maximally 0.02%. No increase in the levels of E 2 in the cytosol and nuclear fraction was found. The addition of ADIOL to E 2 caused a protracted elevation of plasma and nuclear E 2 levels relative to those found with E 2 alone. Most probably this was caused by inhibition of the metabolic degradation of E 2 . This protracted elevation of E 2 levels, possibly together with the high levels of ADIOL, explain the occurrence of the second wave of nuclear translocation of the estrogen receptor observed in our earlier experiments. Unexpectedly we found that the immature female rat has a relatively high basal plasma level of estrone (E 1 ) as compared to the E 2 level. After injection of E 2 there was a large conversion of E 2 to E 1 .