The effect of treatment with 4-hydroxyandrostenedione or medroxyprogesterone acetate on human breast tumour regression.

We have assessed cell deletion in breast tumours removed surgically from postmenopausal women by measuring the distribution ratio of acid phosphatase activity between the bound (in the lysosome) and free (in the phagosome) states. In benign breast tumours the acid phosphatase distribution ratio was much higher than that found in untreated malignant breast tumours. The acid phosphatase distribution ratio was also measured before and after treatment in malignant breast tumours removed from eight patients treated with 4-hydroxyandrostenedione (4-OH A) and in six patients treated with medroxyprogesterone acetate (MPA). Elevated acid phosphatase distribution ratios in benign compared with malignant tumours, and in malignant tumours after treatment from four of eight patients treated with 4-OH A and from three of six patients treated with MPA, may suggest higher cell deletion in these patients.

Control of aromatase activity in breast cancer cells: the role of cytokines and growth factors.

The aromatase complex has a key role in regulating oestrogen formation in normal and malignant breast tissues. Using dexamethasone-treated fibroblasts, derived from breast tumours, breast tumour cytosol and breast tumour-derived conditioned medium (CM) markedly stimulate aromatase activity. The cytokine, interleukin-6 (IL-6) has been identified as a factor present in CM which is capable of stimulating aromatase activity. To examine whether IL-6 may have a role in vivo in regulating breast tissue aromatase activity, IL-6 production and aromatase activity in breast tumour and adipose tissue from breast quadrants were examined. In 5/6 breasts examined so far, aromatase activity was highest in adipose tissue in the breast quadrant containing the tumour or on which the tumour impinged. There was a significant correlation (P < 0.05, Kendall's rank correlation) between IL-6 production and aromatase activity in these breast tissues. It is concluded that IL-6 may have an important role in regulating aromatase activity in breast tissues.

Inhibition of oestrogen synthesis in postmenopausal women with breast cancer.

The effectiveness in reducing oestrogen exposure, of an aromatase inhibitor, and a sulphatase inhibitor, as measured by in vivo studies in breast cancer patients, has been investigated. 4-Hydroxyandrostenedione (4HA) was shown to diminish plasma oestrogen levels, to inhibit peripheral and local aromatization and to cause a concomitant decrease in the activity of DNA-polymerase-alpha, measured as an indicator of cellular proliferation. The source of oestrone sulphate in breast tissues was examined, and it was shown that the tissue content of this conjugate derived from circulating oestrone, but no evidence could be found for the direct accumulation of conjugate from the plasma. Administration of Danazol was found to cause a fall in plasma oestrone levels, and to diminish the conversion ratio of oestrone sulphate to oestrone in some patients. It also inhibited tissue sulphatase activity. Although it is concluded that this drug is only a weak sulphatase inhibitor, these observations indicate the potential value of developing more efficient sulphatase inhibitors. Enzyme inhibition is now a proven effective treatment for breast cancer and the development of more efficient inhibitors is an important objective.

Interleukin-1 and interleukin-6 in breast cyst fluid: their role in regulating aromatase activity in breast cancer cells.

Gross cystic breast disease is a common benign disease which may be associated with an increased risk for breast cancer. Breast cyst fluid (BCF) contains many steroids, peptide growth factors and proteins. We have now identified interleukin-1 (IL-1) and IL-6 in BCF by specific radioimmunoassays. Concentrations of IL-1 were similar in BCF with low or high Na+/K+ ratios (ratio less than 3 vs greater than 3; 357 +/- 72 pg/ml vs 308 +/- 126 pg/ml). In contrast, IL-6 concentrations were significantly higher (P less than 0.01) in BCF with a Na+/K+ ratio greater than 3 (2.75 +/- 2.34 ng/ml) compared with BCF with a low electrolyte ratio (0.21 +/- 0.09 ng/ml). BCF (10%, v/v) stimulated aromatase activity when added to dexamethasone stimulated breast tumour-derived fibroblasts and there was a significant correlation between the stimulation of aromatase activity and BCF Na+/K+ ratio (r = 0.95, P less than 0.001). A significant correlation was also found between stimulation of aromatase activity and concentration of IL-6 in BCF (r = 0.80, P less than 0.01) but not IL-1 concentration (r = -0.39, not significant). Addition of IL-1 or IL-6 (50 ng/ml) to fibroblasts stimulated aromatase activity but was associated with a small (20%) decrease in cell growth. It is concluded that IL-6 may have an important role in regulating aromatase activity in breast cancer cells.

Adrenal steroid endocrinology--some unsolved problems.

Although we now have a good understanding of some of the mechanisms which control pituitary-adrenal activity in human subjects, there are several important problems which still require a solution. The mechanism which controls the diurnal rhythm of aldosterone secretion is not yet identified, and although ACTH is clearly an important factor in the control of adrenocortical activity, it does not account for the pattern of these changes, or for the changes which occur in adrenal androgen secretion. Corticosteroids are well known to have suppressive effects on the release of ACTH, but the retention of pituitary-adrenal responsiveness in patients receiving ACTH therapy, and the prolonged suppression of the system caused by cortisol-secreting tumours, are not well explained by the model currently used, which needs further refinement.

Primary hyperaldosteronism in England and Wales: a review of the use of a Supraregional Assay Service laboratory for the measurement of aldosterone and plasma renin activity.

To ascertain the use being made of a Supraregional Assay Service laboratory in the diagnosis of primary hyperaldosteronism, follow-up data were obtained on 60 patients in whom the diagnosis was suggested by the biochemical results. In 36 patients an adrenal adenoma had been removed; 14 patients had evidence of an adenoma on CT scan; 10 patients were presumed to have bilateral adrenal hyperplasia. The data used to make the diagnosis of primary hyperaldosteronism and to assess the likelihood of the presence of an adenoma indicate that patients studied at 40 centres in the UK show results very similar to those of North American series where large numbers of patients have been described but all have been studied in the same centre. The majority of patients in our series were treated at the hospital at which the diagnosis was made, thus avoiding referral to a centre distant from the patient's home and indicating that the service was being used as originally intended when the Supraregional Assay Service was set up.

Investigation of the salivary 18-hydroxycorticosterone:aldosterone ratio in man using a direct assay.

A method for the direct determination of 18-hydroxycorticosterone (18OHB) in human saliva has been developed and validated. Saliva was collected at 30 min and 1 h intervals between 0600 and 2200 h from healthy men and women for the determination of 18OHB (SHB), aldosterone (SA) and glucocorticoids (SGC = cortisol + cortisone). SHB was highly correlated with SA (r = 0.75; P less than 0.001) but even more highly with SGC (r = 0.89; P greater than 0.001). Multiple regression analysis confirmed that SGC was a more important determinant of SHB than was SA. Though the concentrations of 18OHB and aldosterone were highly correlated there was considerable variation in the 18OHB:aldosterone ratio during the period of saliva collection. This ratio tended to be highest in the morning and lowest in the evening and was weakly correlated with SGC level (r = 0.62; P less than 0.01). The 18OHB:aldosterone ratio in saliva approximates to, and is highly correlated with, that in plasma. We suggest that the fluctuations in SHB:SA ratio correspond to the relative rates of secretion of 18OHB and aldosterone and that this ratio is modulated either by ACTH or by cortisol. Whether this indicates that 18OHB is a by-product of glucocorticoid as well as aldosterone metabolism, or whether this implies a separate physiological role for the steroid remains to be clarified.

Endothelin-like immunoreactivity in human breast cyst fluid.

Immunoreactive endothelin has been detected in 21 of 43 samples of breast cyst fluid (21 cases; 3.5 +/- 0.6 pmol/l, mean +/- SEM. Other 22 cases; not detectable, less than 0.5 pmol/l). Fast protein liquid chromatographic analysis of the immunoreactive endothelin of pooled breast cyst fluid showed two immunoreactive peaks; one in the void volume and the other in the position of endothelin-1. It is probable that endothelin-1 is produced by the epithelial cells lining breast cysts, but significance of the presence of endothelin-1 in breast cyst fluid remains to be elucidated.

Mitogenic peptides in breast cyst fluid: relationship with intracystic electrolyte ratios.

Women with palpable breast cysts which are lined with apocrine epithelium may be at higher risk of developing breast cancer than women with breast cysts which are lined with flattened epithelium, the former group being characterized by intracystic sodium to potassium ratios below 3, while the latter group has intracystic sodium to potassium ratios above 3. In this study the distribution of intracystic concentrations of the mitogenic peptides, epidermal growth factor, endothelin and gastrin-releasing peptide in the 2 groups of breast cysts were compared to see whether differences in concentrations between the 2 cyst groups might provide an explanation for the higher risk of breast cancer observed in women with "apocrine" breast cysts. The concentrations of epidermal growth factor and gastrin-releasing peptide were significantly higher in the low electrolyte ratio group (p less than 0.001). There was no difference in endothelin concentrations between the 2 groups. Negative correlations were found between epidermal growth factor concentrations and Na+/K+ and between gastrin-releasing peptide concentrations and Na+/K+ (p less than 0.001). A positive correlation was found between gastrin-releasing peptide and epidermal growth factor concentrations in breast cyst fluid (p less than 0.001). The significantly higher intracystic concentrations of both epidermal growth factor and gastrin-releasing peptide in the low-electrolyte-ratio group may provide an explanation for the higher risk of breast cancer which has been observed in women with "apocrine" breast cysts.

Effect of breast cyst fluid on oestrogen 17-oxidoreductase activity in MCF-7 breast cancer cells.

Breast cyst fluid (BCF) was found to stimulate oestrogen 17-oxidoreductase activity in the reductive direction, i.e., conversion of oestrone (E1) to oestradiol (E2), in MCF-7 breast cancer cells. Dialysis of BCF revealed that this property of BCF was present in both dialysed BCF and dialysate, implying that both high and low mol. wt. substances were responsible for stimulating E1 to E2 conversion. Gel filtration of dialysed BCF revealed that the high mol. wt. substances responsible for the stimulation of E1 to E2 conversion had mol. wts. of approximately 11 kD and 68 kD. This property of BCF would serve to increase the concentration of E2, a steroid which may play a role in mammary carcinogenesis.

In vitro metabolism or testosterone by breast microcysts.

Breast microcysts are considered to be a normal findings in the adult female breast without any increased risk of developing carcinomatous change. Breast cysts fluid contains steroid but not studies have been reported on the ability of breast microcysts to metabolise steroid hormones. It was, therefore, the aim of this study to identify the metabolites formed on incubation of radiolabelled testosterone with microcysts. In all instances dihydrotestosterone and androstenedione were formed. Oestrogens were not identified. Tis study, therefore, provides evidence for th presence of 5-alpha-reductase and 17-oxidoreductase enzyme systems in breast microcysts.

In vivo conversion of norethisterone to ethynyloestradiol in perimenopausal women.

The extent to which norethisterone is converted to ethynyloestradiol is controversial. To investigate the conversion of norethisterone to ethynyloestradiol we have used a double isotope infusion technique to measure the conversion in vivo. The use of acids or bases was precluded to prevent possible artefactual formation of phenolic metabolites of norethisterone. Transfer constants for the conversion of norethisterone to ethynyloestradiol in two perimenopausal women were 2.26 and 2.34% as measured in blood and 2.27 and 0.38% in urine. Results from this study show that a small but significant proportion of norethisterone is converted to ethynyloestradiol in vivo.

Analysis by DNA polymerase alpha activity of human breast tumour proliferation and the effect of endocrine therapy.

Cytosols of human breast tumours have been assayed for DNA dependent DNA polymerase alpha activity. DNA polymerase alpha activity in benign tumours was found to be significantly lower than in untreated malignant tumours. Biopsy samples removed surgically before and after endocrine therapy showed reduced DNA polymerase alpha activity in 6 out of 9 patients treated with 4-hydroxyandrostenedione, and in 6 out of 7 patients treated with MPA. DNA polymerase alpha activity in malignant breast tumours was higher in oestrogen receptor negative than oestrogen receptor positive tumours.

Time-course and relationship of the early changes in renal sodium excretion and aldosterone secretion during dietary sodium restriction in normal man.

1. The fall in renal sodium excretion after dietary sodium restriction is prompt and reproducible. The importance of increased aldosterone secretion during the early phase (within 48 h) of this response is unclear. Using two indirect measures of aldosterone secretion (in urine and saliva), we have tried to relate changes in excretion and concentration of this hormone to renal sodium excretion during the abrupt transition from a normal (approximately 150 mmol/day) or high (260 mmol/day) to a low (5-25 mmol/day) sodium intake in 11 and seven male volunteers, respectively. 2. All subjects showed reduced renal sodium excretion within 36 h of dietary restriction, but the times at which increases in renal aldosterone excretion, saliva aldosterone concentration and plasma renin activity became statistically significant varied widely (8-72 h, 2.5- greater than 62.5 h and less than 4- greater than 38 h for renal aldosterone secretion, saliva aldosterone concentration and plasma renin activity, respectively). Circadian fluctuations in saliva aldosterone concentration were apparent and increased in amplitude during sodium restriction. 3. Urine flow rate tended to increase on the first day of sodium restriction and this reached statistical significance in the group initially on a high sodium intake (64.0 +/- 8.8 to 84.3 +/- 11.2 ml/h, P less than 0.01); although the pattern of urine flow did correlate with plasma arginine vasopressin concentration (r = -0.49, P less than 0.01), there was no significant decrease in mean plasma arginine vasopressin concentration [1.15 (0.92-1.44) to 0.90 (0.72-1.12) pmol/l, P = 0.08; geometric mean and 95% confidence limits].(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor and oestradiol in human breast cyst fluid.

Gross cystic breast disease is a common condition in women. Women with apocrine breast cysts (breast cyst fluid Na+/K+ less than 3) may be at higher risk of breast cancer than women who have cysts lined by flattened epithelium (Na+/K+ greater than or equal to 3). Breast cyst fluid concentrations of epidermal growth factor were significantly higher in the low electrolyte ratio group than in the high electrolyte ratio group (356.2 ng/ml vs 104.1 ng/ml, P less than 0.0003). A negative correlation was obtained between intracystic epidermal growth factor concentrations and Na+/K+ (rs = -0.666, P less than 0.001). No significant difference was found between the total oestradiol concentrations in the two cyst groups. However, the unbound oestradiol concentrations on a limited number of samples were significantly higher in the low electrolyte ratio group than in the high electrolyte ratio group (P less than 0.05). The higher concentrations of EGF in cyst fluid with Na+K+ less than 3 may explain why women with apocrine breast cysts may be at increased risk of developing breast cancer.

Effect of insulin-like growth factor-type I (IGF-I) and insulin on the secretion of sex hormone binding globulin and IGF-I binding protein (IBP-I) by human hepatoma cells.

Dietary factors are known to modulate concentrations of sex hormone-binding globulin (SHBG). In the present study we have investigated the possibility that insulin like growth factor-type I (IGF-I) may be an additional regulator of SHBG using cultured human hepatoma cells which secrete SHBG. The inhibitory effect of insulin on SHBG secretion by these cells was confirmed but, in addition, IGF-I was shown to inhibit SHBG secretion by about 40% at a concentration of 100 nmol/l. A similar degree of inhibition was achieved using insulin at a concentration of 10 mumol/l. Insulin, but not IGF-I, was also found to inhibit the secretion of a low molecular weight IGF-binding protein (IBP-I), which is also secreted by hepatoma cells. It is concluded that IGF-I is an additional regulator of SHBG secretion by these cells and that it may be involved in regulating SHBG secretion in vivo in response to dietary factors.

A possible mechanism for increased breast cell proliferation by progestins through increased reductive 17 beta-hydroxysteroid dehydrogenase activity.

We have investigated whether progestins may be able to regulate breast cell proliferation by altering the fraction of oestradiol relative to oestrone, using the human breast cancer cell line MCF-7. The ability of the two oestrogens, oestradiol and oestrone, to stimulate breast tumour cell proliferation was investigated. Oestradiol in concentration was of 10-fold greater proliferative potency than oestrone. The progestin MPA increased both reductive and oxidative 17 beta-hydroxysteroid oxidoreductase activity when the tissue culture media pH indicator phenol red was included in the media. When phenol red was excluded from the tissue culture media, MPA increased predominantly the reductive 17 beta-hydroxysteroid oxidoreductase activity, and to a far greater extent than in the presence of phenol red. Other progestins such as levonorgestrel, norethisterone and norethisterone acetate also increased predominantly reductive 17 beta-hydroxysteroid oxidoreductase activity in the absence of phenol red. The action of MPA on reductive 17 beta-hydroxysteroid oxidoreductase activity was increased by treatment with oestradiol to a small but significant extent. We propose that the progestational increase of reductive 17 beta-hydroxysteroid oxidoreductase activity is a possible mechanism by which progestins may increase breast cell proliferation in vivo.

Effect of treatment with 4-hydroxyandrostenedione on the peripheral conversion of androstenedione to estrone and in vitro tumor aromatase activity in postmenopausal women with breast cancer.

The effect of treatment with the aromatase inhibitor, 4-hydroxyandrostenedione (4-OHA) on the peripheral conversion of androstenedione to estrone has been examined in eight postmenopausal women with advanced breast cancer. Before treatment conversion of androstenedione to estrone ([p]AEIBB) ranged from 0.81 to 3.7% and was almost completely inhibited after treatment with 4-OHA (two doses of 500 mg i.m. with an interval of 12 days between doses). Transfer constants were also measured by the urinary method ([p]AEIBU) for some subjects and decreased from 2.3 +/- 0.52% to 0.24 +/- 0.11% after treatment, a mean reduction of 90%. Mean plasma concentration of estradiol (37.4 +/- 16.6 pmol/liter) and estrone (99.0 +/- 32.2 pmol/liter) decreased significantly (P less than 0.01) to 15.7 +/- 4.6 pmol/liter and 52.4 +/- 8.9 pmol/liter, respectively, after treatment. Aromatase and DNA polymerase alpha (a marker of cell proliferation) activities were measured in seven samples of breast tumor tissue obtained before and after treatment. For three samples there was a marked (67 +/- 17%) decrease in tumor aromatase activity after treatment, for two, little change occurred, while tumor aromatase activity in the other two samples appeared to be resistant to the effect of 4-OHA. The correlation between tumor aromatase and DNA polymerase alpha activities (r = 0.45) failed to reach a significant level.