R.C. Bonney

THE RELATIONSHIP BETWEEN 17β‐HYDROXYSTEROID DEHYDROGENASE ACTIVITY AND OESTROGEN CONCENTRATIONS IN HUMAN BREAST TUMOURS AND IN NORMAL BREAST TISSUE

The activity of 17β‐hydroxysteroid dehydrogenase (17βHSD) was measured in human breast tumours and in normal breast tissue from premenopausal, perimenopausal and postmenopausal women. Enzyme activity was higher in tumour tissue than in normal tissue from the same breast and under the conditions of the assay the oxidation of oestradiol was higher than the reduction of oestrone. The physiological status of the women in the study did not relate to the activity of the enzyme in either normal or tumour tissue although fibroadenomas had less activity than adenocarcinomas.

Endometrial phospholipase A2 enzymes and their regulation by steroid hormones

The presence of two phospholipase A2 (PLA2) enzymes, designated PLA2(i) and PLA2(ii), has been demonstrated in human endometrium. These enzymes differ with respect to pH and calcium requirements, location within the tissue and regulation by steroid hormones. Phospholipase A2(i) is calcium dependent, optimally active at pH 7.5-9.0 and present mainly in the glandular component of the endometrium. Changes in activity occur during the menstrual cycle which are indicative of regulation by ovarian steroids. Conversely, PLA2(ii) is calcium independent, optimally active at pH 7.0 and located predominantly in the stromal layer. Wide variation in PLA2(ii) activity was found between individual subjects and there was no relationship with the stage of the menstrual cycle. Activity was, however, much higher in pathological endometrium and in endometrium from subjects with severe dysmenorrhoea. Triton X-100 activated PLA2(i) but not PLA2(ii). In cultured explants of endometrium, both enzymes were inhibited by progesterone whereas oestradiol and dexamethasone had no effect. However, progesterone priming followed by treatment with oestradiol caused a 2-fold stimulation of PLA2(i) but not PLA2(ii). Phospholipase A2 is favoured as the rate-limiting step in the generation of arachidonic acid for prostaglandin synthesis. However, our studies so far do not support a direct relationship between PLA2 and endometrial concentrations of prostaglandins, which implies that other important regulatory steps are involved. Other enzymes which are potentially capable of mobilizing arachidonic acid should also be investigated.

Adrenal androgen concentrations in breast tumours and in normal breast tissue. The relationship to oestradiol metabolism

Concentrations of ADIOL, DHA and DHAS were measured in human breast tumours and normal tissue from the same breast and related to 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) activity in these tissues. ADIOL and DHA were significantly higher in tumour tissue compared to normal tissue from the same breast (paired t-test: P less than 0.05 and P less than 0.01 respectively) whereas the difference between concentrations of DHAS in normal tissue and tumour tissue was not significant. There was a positive correlation between ADIOL and DHA in both tissues (P less than 0.001) but for DHAS the relationship was only significant in normal tissue (ADIOL:DHAS, P less than 0.001; DHA:DHAS, P less than 0.002). An increase in 17 beta-HSD activity was associated with an increase in DHAS concentrations in both normal and tumour tissue (P less than 0.01 and P less than 0.001 respectively) and with an increase in DHA concentrations in normal tissue (P less than 0.05). These results might be explained by an impairment in the balance between sulphatase and sulphotransferase activity in breast tumours.

The role of tissue steroids in regulating aromatase and oestradiol 17β-hydroxysteroid dehydrogenase activities in breast and endometrial cancer

We have observed that oestradiol concentrations in breast and endometrial tumours are relatively higher than oestrone, in contrast to peripheral tissues. Infusion of radiolabelled oestrogen also suggested there was a difference in metabolism between normal and tumour tissue. We have therefore looked for factors which could modulate tissue steroid metabolism and conclude that progesterone may influence aromatase, and that the adrenal androgens can inhibit oestradiol dehydrogenase activity. The latter mechanism, in particular, may be important in increasing tissue exposure to oestradiol.

Inhibition of 17β-hydroxysteroid dehydrogenase activity in human endometrium by adrenal androgens

The effect of dehydroepiandrosterone sulphate (DHA-S) and its metabolites dehydroepiandrosterone (DHA) and 5-androstene-3 beta, 17 beta-diol (ADIOL) on the activity of 17 beta-hydroxysteroid dehydrogenase in human endometrial tissue was investigated by an isotope ratio technique. The apparent KM for oestradiol was 1.59 X 10(-6) M. All three androgens inhibited the metabolism of oestradiol and the apparent Ki values were: ADIOL, 2.05 X 10(-6) M; DHA-S and DHA, 1.59 X 10(-6) M. However, ADIOL acted by direct competition with oestradiol for the active enzyme site whereas inhibition by DHA and its sulphate was non-competitive. DHA-S and DHA were more potent inhibitors of oestradiol metabolism than was ADIOL. These results support the hypothesis that adrenal androgens could be involved in the development of endometrial hyperplasia and adenocarcinoma. Inhibition of oestradiol metabolism could increase the concentration of oestradiol in endometrial tissue and if unopposed by progesterone, e.g. after the menopause or in subjects with ovulatory defects, could stimulate abnormal endometrial growth.

The interrelationship between plasma 5-ene adrenal androgens in normal women

Plasma concentrations of 5-androstene-3 beta,17 beta-diol (ADIOL) dehydroepiandrosterone (DHA) dehydroepiandrosterone sulphate (DHAS) and cortisol were measured by radioimmunoassay in a group of women aged between 27 and 88 years of age. There was a significant negative correlation with increased age for all three adrenal androgens but not for cortisol. The decrease in adrenal androgens was not related to an excessive divergence from ideal body weight. There was a highly significantly positive correlation between plasma concentrations of all three adrenal androgens which supports a metabolic interrelationship.

The relationship between oestradiol metabolism and adrenal steroids in the endometrium of postmenopausal women with and without endometrial cancer

The aim of the present study was to investigate the hypothesis that adrenal androgens are contributory to the development of endometrial cancer either by the oestrogenic action of 5-androstene-3 beta, 17 beta-diol (androstenediol) or through the inhibition of oestradiol metabolism. Concentrations of androstenediol, dehydroepiandrosterone (DHA), DHA sulphate (DHAS), oestrone and oestradiol were measured in plasma and endometrium from postmenopausal women with and without endometrial cancer. There was no difference between normal postmenopausal women and endometrial cancer patients with respect to either tissue or plasma adrenal androgens although there was a tendency for plasma DHAS levels to be increased in cancer patients (normal women: 640 +/- 156 ng/ml; cancer patients: 808 +/- 159 ng/ml). There was a positive correlation between endometrial tissue concentrations of androstenediol and DHA in both normal women (P less than 0.05) and cancer patients (P less than 0.01) but for DHAS the relationship was only significant for non-malignant tissue (androstenediol: DHAS, P less than 0.05; DHA: DHAS, P less than 0.02). A significant positive correlation was found between all three plasma adrenal androgens for both groups. In cancer patients there was a trend towards an inverse correlation between endometrial tissue concentrations of DHAS and the enzyme 17 beta-hydroxysteroid dehydrogenase (17OHSD) although the relationship was not significant (r = 0.49). In endometrium, oestradiol was present in significantly higher concentrations than oestrone whereas in plasma the reverse was the case. There was also a tendency for plasma oestradiol levels to be elevated in the cancer subjects. These data do not support a substantial role for adrenal androgens in endometrial cancer but suggest that a relationship may exist between DHAS and 17OHSD and that an imbalance between sulphatase and sulphotransferase activities may be involved.

Metabolism of [3H]oestradiol in vivo by normal breast and tumour tissue in postmenopausal women☆

Tumour and normal breast tissue was obtained from postmenopausal breast cancer patients following [3H]oestradiol infusion (50 mu Ci over a 12 h period). The fraction of radioactivity present as oestradiol or oestrone was measured and the results expressed both as the ratio of oestradiol-oestrone and as the percentage oestrogen present as oestrone, and the findings compared with in vitro measurements of 17 beta-hydroxysteroid dehydrogenase activity. Concentrations of 5-androstene-3 beta, 17 beta-diol, dehydroepiandrosterone and its sulphate and testosterone were measured and related to oestradiol metabolism. The study demonstrated that tumour tissue is less able to metabolise oestradiol to oestrone than is normal breast tissue and indicated that the ability of the tissue to detoxify oestradiol may be dependent on cofactor availability. The results also supported the possibility that increased tissue concentrations of adrenal androgens inhibit oestradiol and thus increase tissue exposure to oestradiol.

PHOSPHOLIPASE C ACTIVITY IN HUMAN ENDOMETRIUM: ITS SIGNIFICANCE IN ENDOMETRIAL PATHOLOGY

Phospholipase C activity was measured in human endometrium using an assay based on the release of total labelled water soluble products (inositol, inositol phosphates) from L‐3‐phosphatidyl‐[2‐3H] inositol. The enzyme was shown to be calcium dependent and to have an optimum pH of 5.5. There was no difference between proliferative phase and secretory phase endometrium with respect to phospholipase C activity either in women with normal menstrual blood loss (proliferative phase: 3.7 ± 0.7 (mean ± SD), secretory phase: 4.5 ± 2.0 nmol/mg protein/min) or in those complaining of severe menorrhagia (proliferative phase: 5.8 ± 2.8, secretory phase: 7.0 ± 2.8 nmol/mg protein/min). However, women complaining of severe menorrhagia had significantly higher endometrial phospholipase C activity than those in the normal group (P<0.01 and P<0.02 for proliferative and secretory phases respectively). Endometrial phospholipase C activity was also elevated in the presence of other gynaecological disorders, e.g. dysmenorrhoea, adenocarcinoma of the cervix and endometrial hyperplasia. The results indicate that phospholipase C activity in human endometrium is not related to the stage of the menstrual cycle but that in the presence of menorrhagia and other gynaecological disorders, activity is increased. Phospholipase C could be implicated in the generation of arachidonic acid for prostaglandin synthesis which may in turn be associated with these abnormalities.

Modulation of phospholipase A2 activity in human endometrium and amniotic membrane by steroid hormones

Phospholipase A2 (PLA2) activity was measured in endometrium and amnion by a double isotope ratio technique using 1-palmitoyl-2-oleoyl phosphatidylcholine as substrate in the presence and absence of a range of unconjugated steroids and steroid sulphates (0.2-6.4 X 10(-4) M). In the presence of 0.1% Triton, PLA2 activity was inhibited by the majority of steroids tested, pregnenolone sulphate being the most effective (12.9 +/- 3.0% control activity) while oestradiol sulphate, oestrone and testosterone had only a minimal or no effect (99.1 +/- 19.0, 85.4 +/- 4.4 and 104.2 +/- 16.3% control respectively). In the absence of Triton, the inhibitory effect of the free steroids was reduced or absent but oestradiol sulphate and testosterone sulphate stimulated activity by 2-13 and 1.5-3 times respectively. The effect was dose related, linear with time and independent of the stage of the menstrual cycle. Inhibition by pregnenolone sulphate, dehydroepiandrosterone (DHA sulphate and oestrone sulphate was maintained in the absence of Triton (24.9 +/- 3.8, 67.1 +/- 10.1 and 87.2 +/- 13.8% control respectively). In amnion all 5 steroid sulphates caused a marked stimulation of PLA2 activity in both the presence and absence of Triton. The effect was greatest without Triton and at 6.4 X 10(-4) M, testosterone, pregnenolone, oestrone, DHA and oestradiol sulphates increased PLA2 activity 20, 15, 12, 10 and 6-fold respectively. These findings indicate a direct action of steroid sulphates on PLA2 activity in endometrium and amnion.