The compound 2-methyl-1,2-bis-(3-pyridyl)-1-propanone (SU4885) described by Chart, Sheppard, Allen, Bencze & Gaunt (1958) has been considered a relatively specific inhibitor of 11β-hydroxylation of steroids (Liddle, Island, Lance & Harris, 1958) both in vivo in man and in vitro in the ox. Grant (1960) showed that SU4885 caused a rapid fall in the cortisol content of adrenal venous blood when administered to patients during bilateral adrenalectomy. Homogenates of adrenals removed from these patients had a decreased 11β-'hydroxylase' activity, but the 17- and 21-hydroxylating enzymes were unaffected. As both the 11β-'hydroxylase' (Sweat, 1951) and the 19-'hydroxylase' (Hayano & Dorfman, 1955) enzyme systems are mitochondrial in the adrenal gland, it was decided to ascertain whether the 19-'hydroxylase' was also inhibited by SU4885. Earlier studies (Griffiths, 1963) had shown that the adrenal of the golden hamster possessed active steroid 11β- and 19-hydroxylating enzymic systems. Six male golden hamsters weighing approximately 115 g. received 8·3
The metabolism of 11-deoxycorticosterone (DOC) in vitro by homogenates of adrenal glands from various species has been studied.
Homogenates of 'non-fatty' adrenal glands, especially those prepared from golden hamster adrenals, were shown to possess an active 19-hydroxylating enzyme system. In the case of the golden hamster adrenal, approximately 30% of the added DOC was converted to 19-hydroxy-11-deoxycorticosterone (19-OH-DOC). Negligible transformation was observed using homogenates prepared from 'fatty' adrenal glands, similar to the type found in man. 19-OH-DOC was, however, formed from DOC by a homogenate of a human foetal adrenal gland.
No relationship was observed between the 11β-hydroxylating activity of the adrenal homogenate, and the reported cortisol/corticosterone ratio found in the adrenal venous blood of various animals.
C. GILES and K. GRIFFITHS
It has been shown recently (Griffiths, 1963) that administration of 2-methyl-1: 2-bis-(3-pyridyl)-1-propanone (SU 4885) to golden hamsters decreases the steroid '19-hydroxylase' activity of the adrenal gland as well as that of the ' 11β-hydroxylase' on which SU 4885 was considered to have a specific inhibitory action (Liddle, Island, Lance & Harris, 1958). SU 4885 has also been shown by Kahnt & Neher (1962) to inhibit 19-hydroxylation in beef adrenal cortex homogenates. Since hydroxylation of the angular C-19 methyl group appears to be an essential stage in the biosynthesis of oestrogens (Meyer, 1955; Ryan, 1959; Longchampt, Gual, Ehrenstein & Dorfman, 1960), it was decided to ascertain whether SU 4885 has any effect on the steroid aromatizing enzyme system of human placental tissue.
Fresh placentae were dissected free of foetal membranes and processed at 4°. Saline-washed tissue was minced, and 200 g. were homogenized in 65 ml. buffer containing 0·05 m-phosphate, (pH 7), 0·25 m-sucrose
T. JONES and K. GRIFFITHS
Although dehydroepiandrosterone sulphate has been recognized as one of the major secretory products of the human adrenal gland neither its biosynthetic pathway nor its site of formation within the cortex have been extensively studied. Ultramicrochemical techniques, which relate enzymic activity to well-defined groups of cells, have been used in the work now described to study the sulphation of dehydroepiandrosterone in the zones of the guinea-pig adrenal cortex. It has been shown that dehydroepiandrosterone sulphokinase activity resides only in the compact cell of the zona reticularis.
P. DAVIES and K. GRIFFITHS
Various metabolites of testosterone, compounds such as 5α-dihydrotestosterone and the 5α-androstanediols, appear to have specific roles in eliciting certain of the androgenic responses (Baulieu, Lasnitzki & Robel, 1968 a, b). Furthermore, some of these compounds can stimulate in vitro the activity of DNA-dependent RNA polymerase (nucleoside triphosphate-RNA nucleotidyltransferase, EC 220.127.116.11) of nuclei prepared from rat and dog prostatic tissue (Davies, Fahmy, Pierrepoint & Griffiths, 1972). To investigate this matter further, the effects of a series of testosterone metabolites on the RNA polymerase activity isolated from specimens of tissue from patients with benign prostatic hypertrophy (bph) were studied, and the inhibitory effects of various stilboestrol derivatives were also investigated.
Human bph specimens, obtained immediately after prostatectomy, were homogenized using a Latapie press in combination with a Potter—Elvehjem homogenizer. Nuclei were purified from homogenates by centrifugation through discontinuous gradients of hypertonic sucrose (0·8–·8–2·1 mol/l) and were judged free of cytoplasmic fragments by
K. GRIFFITHS and D. GLICK
Recent biochemical studies on the functional zonation of the human adrenal cortex suggest that one of the earlier principal effects of corticotrophin (ACTH) on steroid biosynthesis occurs at the fasciculo-reticular border. This report deals with procedures for the study of the 11β-hydroxylase activity in microgram amounts of adrenal tissue and with the distribution of this activity throughout the adrenal cortex of the rat. ACTH administration is shown to stimulate the 11β-hydroxylase activity at the fasciculo-reticular border.
K. GRIFFITHS and C. A. GILES
Earlier studies (Griffiths, 1963) have shown that the adrenal gland of the golden hamster possesses an active 19-hydroxylating enzyme system. This was demonstrated by measuring the conversion of 11-deoxycorticosterone (DOC) to 19-hydroxy DOC. The ability of this enzyme system to hydroxylate C19-steroids has now been investigated.
Adrenal glands (300 mg.) from twelve male hamsters were homogenized in 500 μl. 0·25 m-sucrose containing 0·12 m-nicotinamide, as described earlier (Griffiths, 1963). The homogenate (450 μl.) was incubated with 3 μc [4-14C]testosterone (sp.ac. 8·8 μc/μmole) for 60 min. at 37° in 900 μl. medium containing 677 μmoles tris buffer, pH 7·4, 675 μmoles potassium chloride, 135 μmoles potassium fumarate, 2·94 μmoles NADP, 49·7 μmoles MgSO4, 14·7 μmoles ATP, 22·5 μmoles glucose-6-phosphate (G6P) and 1·0 Kornberg unit of G6P-dehydrogenase. Procedures for the isolation of the neutral steroid fraction, paper chromatography systems and steroid estimation and identification were those described
G. V. GROOM and K. GRIFFITHS
Plasma levels of LH, FSH, prolactin, oestradiol and progesterone were determined daily during two consecutive menstrual cycles in six women volunteers. During the first (control) cycle no treatment was given and normal secretion of these hormones was observed. Oral administration of tamoxifen (20 mg/day), for either 5 or 10 days of the follicular phase of the second cycle, caused no change in either the overall length of the cycle or the time of occurrence of the mid-cycle gonadotrophin surge. There was little difference in the secretion of LH, FSH and progesterone during the control and test cycles.
A two- to eight-fold increase in oestradiol levels was observed during the test cycle which was most pronounced at the times of mid-cycle and mid-luteal hormone peaks. There was a significant decrease in plasma prolactin levels at mid-cycle but no real difference could be seen during the remainder of the cycle.
The data suggest that tamoxifen may act directly on the ovary to stimulate oestradiol release without intermediary gonadotrophin stimulation. As the drug apparently inhibited prolactin secretion even in the presence of high oestradiol levels, an alternative explanation may be that the reduced prolactin concentration permits augmented ovarian stimulation by normal concentrations of gonadotrophins.
E. H. D. CAMERON and K. GRIFFITHS
The removal of a large clear cell adenoma (67 g.) from a patient in the Cardiff Royal Infirmary presented an opportunity to investigate the relative importance of alternative metabolic pathways to cortisol in adrenal tissue consisting primarily of a single cell type. Experiments were performed in which chopped tumour tissue was incubated simultaneously with [4-14C]pregnenolone and [7α-3H]progesterone for periods of time ranging from 5 to 120 min.
The conversion of these precursors to a variety of corticosteroids and intermediates was determined by radioisotope dilution techniques. From the results obtained, it was concluded that progesterone played only a minor role in the transformation of pregnenolone to cortisol in this tissue. It appeared that a large proportion of any progesterone formed from pregnenolone is rapidly metabolized to the 17-deoxycorticosteroids.
T. C. HAMILTON, P. DAVIES and K. GRIFFITHS
The specific retention of androgens and oestrogens by cytoplasmic components of human ovarian tumours was investigated. High-affinity, low-capacity binding of androgens was observed in 88% of tumour specimens and oestrogen binding in 32%. Retention of oestrogens did not occur in the absence of androgen binding. The androgen-binding component, of sedimentation coefficient 7·5–8·5S, showed specificity for 5α-dihydrotestosterone and 17β-hydroxy-17α-methyl-estra-4,9,11-trien-3-one (R1881). In some instances, competition for R1881-binding sites indicated the presence of progesterone receptor-like binding. The data presented suggest strongly the existence of androgen and oestrogen receptors in some ovarian tumours and may be relevant to histopathological classification and therapeutic rationales.