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SUMMARY

Human skin from forehead, cheek and axilla was incubated in vitro with [7α-³H]dehydroepiandrosterone (DHA), [7α-³H]DHA sulphate, [7α-³H]-androstenedione and [7α-³H]testosterone. The following enzyme activities were detected: 3β-hydroxysteroid dehydrogenase Δ^4-5 isomerase, 17β-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, 5α-reductase, 5β-reductase, sulphotransferase, sulphatase, steroid hydroxylase. 5α-Reduced steroids were the major metabolites. All four substrates were converted to 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol. In axillary skin, conversion of 17-oxosteroids to 17β-hydroxysteroids was favoured, 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol being major metabolites. In facial skin, formation of 17-oxosteroids predominated with little accumulation of 5α-dihydrotestosterone or 5α-androstane-3α,17β-diol. 5α-Androstane-3β,17β-diol was a metabolite of DHA, androstenedione and testosterone but was found in lower amounts than 5α-androstane-3α,17β-diol. Similarly conversions to epiandrosterone were much lower than to androsterone in all the skin specimens.

It was concluded that the differences in accumulation of 5α-dihydrotestosterone were determined by the differences in 17β-oxidoreduction rather than differences in 5α-reductase, the activity of which was high in all skin specimens.

SUMMARY

The metabolism of oestrone, dehydroepiandrosterone (DHA) and testosterone by normal mammary gland tissue from pregnant rats and by dimethylbenzanthracene (DMBA)-induced mammary tumours has been investigated.

Oestradiol was the only compound regularly formed from oestrone. DHA was mainly converted to androst-5-ene-3β,17β-diol (Δ⁵-androstenediol), but another compound with the characteristics of an hydroxylated derivative of DHA was also formed. These tissues also had Δ⁴-5α-hydrogenase, 3-oxosteroid reductase and 17β-hydroxysteroid oxidoreductase activities when testosterone was used as substrate. Hormone-dependent and -independent tumours had higher enzyme activities towards oestrone and testosterone than had normal mammary tissue from pregnant rats. This difference cannot be accounted for solely by the difference in the cellularity of these tissues. The production of oestradiol by the hormone-dependent tumours was related to the growth rate of the tumour. No similar relationship was found for the metabolism of testosterone. Evidence is presented that hormone-independent tumours might be able to metabolize more oestrone than dependent tumours of similar growth rates.

SUMMARY

The metabolism of [7α-³H]pregnenolone and [4-¹⁴C]dehydroepiandrosterone (DHA) by a Sertoli cell tumour of the testis from a dog with signs of feminization has been studied in vitro and compared with the metabolism of the normal canine testis.

The tumour formed oestrone and oestradiol-17β from DHA thus providing direct evidence for the synthesis of oestrogen by this type of neoplasm.

Relative or complete inactivity of several enzyme systems involved in the synthesis of testosterone was found in the tumour tissue, and the conversion of either precursor to testosterone was considerably less than in the normal testis.

Suggestive evidence is presented for the occurrence of steroid-specific 17α-hydroxylase and 3β-hydroxysteroid dehydrogenase-isomerase systems in canine testicular tissue.

The formation of sulphates of pregnenolone and DHA was shown both in normal and in neoplastic tissues and, in addition, the tumour either formed the sulphate of 17α-hydroxypregnenolone or caused the 17α-hydroxylation of pregnenolone sulphate.

Human pituitary LH (1200 i.u.) was infused for 4 h (from 10.00 to 14.00 h) into six women with anorexia nervosa and into five women with polycystic ovarian disease (PCO). Plasma dehydroepiandrosterone sulphate (DHAS), androstenediol sulphate, dehydroepiandrosterone (DHA), androstenediol and testosterone were estimated by gas–liquid chromatography in blood samples taken every 2 h from 10.00 to 20.00 h. The values were compared with those obtained at the same times on the previous control day. There were no significant changes in the plasma levels of DHAS and androstenediol sulphate in response to LH at any of the sampling times in either the anorexia nervosa or the PCO patients. In the anorexia nervosa women, plasma DHA levels were significantly increased at 16.00 (P<0·001), 18.00 (P<0·001) and 20.00 h (P<0·05) after LH infusion. In the PCO women, DHA levels increased significantly at 14.00 (P<0·01), 16.00 (P<0·001), 18.00 (P<0·001) and 20.00 h (P<0·001) in response to LH infusion. Plasma androstenediol levels increased significantly in the anorexia nervosa patients at 12.00 (P<0·001), 14.00 (P<0·01) and 16.00 h (P<0·01) in response to LH. Similar increases were also found in the PCO patients at 12.00 (P<0·01), 14.00 (P<0·001) and 16.00 h (P<0·01). Plasma testosterone decreased progressively in the anorexic women in response to LH, becoming significant at 16.00 (P<0·05), 18.00 (P<0·05) and 20.00 h (P<0·01). A similar progressive decrease in plasma testosterone was seen in the PCO women, the levels being significantly lower than controls at 16.00 (P<0·05), 18.00 (P<0·05) and 20.00 h (P<0·05).

The results represent the first experimental evidence for a direct regulatory role for LH on androgen secretion in women. In addition, the data have a significant bearing on the pathogenesis of the PCO syndrome and the development of hirsutism which may be directly related to the high androgen levels in PCO women in whom the levels of LH are normally raised. The data may also offer an explanation for the mechanisms responsible for the low androgen levels in anorexia nervosa patients in whom there is a gonadotrophin deficiency.

SUMMARY

A granulosa cell multilocular cystadenoma was removed from a 7-yr.-old girl who showed breast growth and had a history of almost continuous vaginal bleeding for 9 days. Tissue, which microscopically was shown to consist almost exclusively of granulosa cells, was incubated with [4-¹⁴C]-DHA and [7-³H]pregnenolone in Krebs-Ringer bicarbonate solution. The ability of this tissue to synthesize oestrogens from these precursors was investigated. These results and those of pre- and post-operative urinary steroid estimations are discussed in relation to current theories of steroid biosynthesis.

Yolk sac and endometrial tissue were obtained from tammar wallabies between 11 and 25 days after the removal of pouch young. Tissues were examined histologically and steroid-metabolizing enzymes were identified by incubation for 3 h at 37 °C in Medium 199 containing labelled steroid precursors. Yolk sac membrane (YSM) incubated with labelled pregnenolone produced a small amount of progesterone and pregnanediols; 80·5 ± 8·4 (s.e.m.) % of the original substrate remained unmetabolized. Labelled androstenedione was metabolized to 5α-androstane-3,17-dione and androsterone, and only 5·8 ± 3·8% of the original substrate remained at the end of incubation. Incorporation of androstenedione or dehydroepiandrosterone (DHA) into phenolic compounds was low (0·5 ± 0·1%). There was no evidence for the enzymes, arylsulphatase or sulphotransferase, in YSM. Endometrial tissue from the same animals metabolized pregnenolone, DHA and androstenedione, converted progesterone to androstenedione, and produced aqueous-soluble steroid conjugates. The results demonstrated that YSM contains enzymes associated predominantly with steroid catabolism and with incipient progesterone synthesis. The findings are discussed in relation to the histological appearance of the tissues and compared with placental steroid synthesis in eutherian mammals.

SUMMARY

Treatment of male guinea-pigs daily with an oral dose of 2 mg dehydroepiandrosterone (DHA) sulphate/100 g body weight for 2 weeks significantly reduced the glucose-6-phosphate dehydrogenase (G-6-PDH) activity of erythrocytes, liver, kidney and testis. Lactate dehydrogenase activity in plasma also decreased, but l-aspartate: 2-oxoglutarate aminotransferase (GOT) and l-alanine: 2-oxoglutarate aminotransferase (GPT) activity in plasma remained unaffected. In liver and kidney, however, a significant rise in GOT and GPT was observed.

A 2- to 3·7-fold increase of C₁₉-steroids was observed in plasma, liver and kidney. In extracts of liver and kidney more than 60% of steroids were isolated from the sulphatide fraction. Only minor changes were detected in the metabolic pattern of C₁₉-steroids, 17-hydroxysteroids prevailing in the free and sulphatide fractions, while 17-oxosteroids predominated in the sulphate and glucuronide fractions.

A slight rise of cyclic AMP concentrations in liver and kidney tissue was attributed to the inhibition of phosphodiesterase by the DHA/G-6-PDH system.

3α,16α-Dihydroxyandrost-5-en-17-one was first isolated by YoungLai & Solomon (1967) as a major radioactive urinary metabolite after administration of [7-³H]16α-hydroxydehydroepiandrosterone (16α-hydroxy DHA) to two human subjects. We have isolated this metabolite after glucuronidase hydrolysis and chloroform extraction of a urine sample from a patient who had an inoperable asymptomatic adrenal carcinoma and who excreted about 5 mg of each of this compound and 16α-hydroxy DHA/24 h. The isolated steroid gave a purple colour with blue tetrazolium salt, characteristic of an α-ketol group. On a silica gel plate approximately 3 μg of this steroid could be visualized with picric acid reagent (Eberlein, 1965) specific for 5-ene-3-hydroxysteroids. The free and acetylated steroid could not be distinguished from authentic standards by gas—liquid chromatography (g.l.c.) using SE-30 and QF-1 columns. Mass spectra obtained by g.l.c. linked to mass spectrometry of the trimethylsilyl ether of authentic and isolated steroid confirmed its identity. The spectrum

Recent studies in which [7α-³H]dehydroepiandrosterone sulphate (DHA-S) was shown to be converted to oestrogen by minced corpora lutea (Fahmy, Griffiths & Turnbull, 1968a), also showed the formation of 19-hydroxyandrostenedione (19-OH-A) but not 19-hydroxytestosterone (19-OH-T). Little is known concerning the role of testosterone as a direct precursor of oestradiol-17β synthesis in the ovary, and of 19-OH-T as an obligatory intermediate. Even in the placenta the role of 19-OH-T remains equivocal. Perfusion studies of Bolté, Mancuso, Dray, Baulieu & Diczfalusy (1964) suggested that testosterone was directly converted to oestradiol-17β, whereas incubation studies have produced contradictory results (Baulieu, Wallace & Lieberman, 1963; Menini & Engel, 1967). It was therefore decided to investigate further the precursor role of 19-OH-A and 19-OH-T in human luteal tissue.

Radioactive 19-OH-A and 19-OH-T were prepared by incubating either [4-¹⁴C]- or [7α-³H]androstenedione with golden hamster adrenal homogenates (Griffiths & Giles, 1965). Incubations

SUMMARY

As part of a continuing study of adrenal steroids in relation to breast cancer, various experiments were performed in order to study relationships between androgen and corticosteroid biosynthesis. Chopped tumour tissue from a 'mixed cell' adrenal adenoma (7·4 g.) removed from a patient in Cardiff Royal Infirmary was incubated with [4-¹⁴C]pregnenolone and [7α³H]17α-hydroxypregnenolone for periods of time ranging from 30 to 120 min. The results of this work suggest that 17α-hydroxyprogesterone may not be an obligatory intermediate in androgen or cortisol synthesis. Evidence from further experiments with 'normal' human adrenal tissue removed from breast cancer patients using previously established ultramicrochemical techniques indicates that dehydroepiandrosterone (DHA) sulphokinase enzyme system is confined to the zona reticularis of the gland. The conversion of [7α-³H]DHA sulphate, [7α-³H]androstenedione and [7α-³H]testosterone to oestrogens and their conjugates by adrenal homogenates was also investigated. Conversions were extremely low from all precursors.