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K. GRIFFITHS

SUMMARY

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.

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K. GRIFFITHS

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

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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 2.7.7.6) 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

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T. JONES and K. GRIFFITHS

SUMMARY

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.

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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

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K. GRIFFITHS and D. GLICK

SUMMARY

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.

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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. article image

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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

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G. V. GROOM and K. GRIFFITHS

SUMMARY

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.

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A. H. BAILLIE and K. GRIFFITHS

SUMMARY

One hundred and thirty-two male Swiss white mice were killed in batches of twelve, between birth and the end of the 10th week of postnatal life inclusive, a total of eleven groups. Sections of testis from every animal were incubated with three steroid substrates to demonstrate 3β-hydroxysteroid dehydrogenase histochemically. The substrates were (1) 3β: 17α-dihydroxypregn-5-en-20-one (17α-hydroxypregnenolone), (2) 3β-hydroxypregn-5-en-20-one (pregnenolone) and (3) 3β-hydroxyandrost-5-en-17-one (DHA). When 17α-hydroxypregnenolone was used as a substrate no 3β-hydroxysteroid dehydrogenase activity was demonstrable in the testis until the end of the 10th week of postnatal life. With pregnenolone as a substrate 3β-hydroxysteroid dehydrogenase activity was demonstrable throughout the age groups studied. It was present at birth and increased progressively until the end of the 6th week of postnatal life. Thereafter the activity decreased progressively during the ensuing 4 weeks. With DHA as substrate activity was again demonstrable in all age groups studied and increased progressively from birth until the end of the 7th week of postnatal life after which a relatively constant high level was maintained. On the basis of these findings the existence of more than one 3β-hydroxysteroid dehydrogenase enzyme is postulated, each enzyme being substrate specific.

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A. H. BAILLIE and K. GRIFFITHS

SUMMARY

One hundred and ten male Swiss white mice were killed in batches of ten weekly between birth and the end of the 10th week of postnatal life. To demonstrate 3β-hydroxysteroid dehydrogenase activity histochemically, sections of testis from every animal were incubated with the following steroid substrates: (1) sodium 3β-sulphoxypregn-5-en-20-one (pregnenolone sulphate), (2) sodium 3β-sulphoxy-17α-hydroxypregn-5-en-20-one (17α-hydroxypregnenolone sulphate), (3) sodium 3β-sulphoxyandrost-5-en-17-one (DHA sulphate), (4) 3β,16α-dihydroxypregn-5-en-20-one (16α-hydroxypregnenolone), (5) pregn-5-ene-3β,20β-diol (pregnenediol), (6) androst-5-ene-3β, 17β-diol (androstenediol).

Pregnenolone sulphate was rapidly used by the entire interstitium at all ages. 17α-Hydroxypregnenolone sulphate was metabolized by some Leydig cells of all age groups. DHA sulphate was not utilized histochemically by the Leydig cells of the various age groups, but formazan deposition occurred in the mature seminiferous epithelium. This is the only steroid so far investigated to give an histochemical reaction with the germinal epithelium, and 3β-hydroxysteroid dehydrogenase activity has not previously been described in the seminiferous tubules. The utilization of steroid sulphates differently from the free steroids in the histochemical demonstration of 3β-hydroxysteroid dehydrogenase activity suggests that the presence of a sulphate group may affect enzyme-substrate binding.

With 16α-hydroxypregnenolone and pregnenediol as substrates, 3β-hydroxysteroid dehydrogenase activity was demonstrable at birth, increased progressively until the 6th week of postnatal life, and subsequently decreased during the ensuing 4 weeks. This growth curve closely resembles the growth curve obtained with pregnenolone. Androstenediol gave a histochemical reaction with the Leydig cells of all age groups studied, and the sigmoid growth curve resembles that obtained with 3β-hydroxyandrost-5-en-17-one (DHA). These differing growth curves are regarded as further evidence of substrate-specific 3β-hydroxysteroid dehydrogenases.