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POSSIBLE ALDOSTERONISM IN STILBŒSTROL-TREATED GUINEAPIGS
1192 With this in mind we have analysed various drinks for their sodium, potassium, and chloride concentration. These are summarised in table 11, and of them the pure fruit-juices contain the most potassium with least sodium. We have given up to a litre a day of pure fruit-juices to several patients and been able thereby substantially to reduce the daily amounts of potassium given as salts. The drinks did not interfere with the patient’s appetite, and no side-effects were observed. means.
Schemm, F. R., Cámara,
Preliminary
(1954) Circulation, 10,
17-OXOSTEROIDS
(mg.
Urinary 17-oxosteroid per pig per 24 hr. ±S.E.)
Group
10 animals and 15-22 estimations per group.
for 17-ketosteroids), which has been measured Dr. R. D. Bulbrook and Dr. F. C. Greenwood, who have kindly allowed us to use some of their data here. Table i gives figures for the output of such steroids (which do not include aldosterone) in guineapigs which have been castrated or have been treated with large doses of stilboestrol (implants of 15-mg. tablets renewed every three to four months) for more than a year. The output of 17-oxosteroids is increased equally in intact and castrated guineapigs treated with stilbcestrol, and there is other evidence (particularly from the increase produced by adrenocorticotrophin) that the 17-oxosteroid output is one index of adrenal-cortical activity. The excretion of 17-oxogenic steroids is also increased in parallel fashion. (2) The evidence for loss of potassium in treated guineapigs is given in table 11, which records the over. new name
REFERENCE A. A.
TABLE I—DAILY OUTPUT OF URINARY
by
430.
Communications
POSSIBLE ALDOSTERONISM IN
STILBŒSTROL-TREATED GUINEAPIGS THE evidence is so far indirect ; but the similarities between the condition of primary aldosteronism or Conn’s syndrome in man and the effects of large doses of stilbopstrol in male guineapigs seem suggestive enough to report. In the human disease excessive secretion of aldosterone by the adrenal cortex is associated with loss of potassium.l2 There are also changes in the kidneys of the patients (vacuolation of the epithelia] cells of the renal tubules 3) like those produced in the early stages of potassium deficiency in rats.4 Later the kidneys of
TABLE
II—NIGHTLY
OUTPUT
URINARY
OF
SODIUM
AND
POTASSIUM
whether normal potassium intake is restored or not, show a different lesion—cystic It is this late result of dilatation of the tubules.4 5 potassium deficiency that we believe is reproduced in the treated guineapigs, and our suggestion that the cause may be excessive secretion of aldosterone is based on evidence that the stilbœstro] treatment causes adrenal overactivity and loss of potassium. A further similarity between human patients and the treated guineapigs is that both have polydipsia and polyuria.
potassium-deficient rats,
Nightly output (m.eq. per pig ±S.E.) Group Sodium
Potassium
excretion of sodium and potassium mea. four nights weekly for five weeks in 4 normal guineapigs and 4 guineapigs which had been treated with stilboestrol 05-20 mg. by mouth daily for three months. Despite the large variation in the nightly excretion of sodium the mean figure is virtually the same in the two groups. There was less variation in the potassium output which was significantly greater (almost doubled) in the treated animals than in the controls. The mean bodyweight of the two groups did not differ by more than 5% during the course of the urine collections. The food intake was not measured but had borne a constant relation to body-weight in other experiments. (3) Both intact and castrated guinea pigs in which stilbœstrol is implanted die, during the second year of treatment, from renal failure with a raised blood-
night urinary
sured
EVIDENCE
(1) The evidence for adrenal overactivity depends on urinary excretion of 17-oxosteroids (the chemists’
the
1. Conn, J. W. J. Lab. clin. Med. 1955, 45, 3. 2. Chalmers, T. M., FitzGerald, M. G., James, A. H., Scarborough, H. Lancet, 1956, i, 127. 3. Conn, J. W. J. Lab. clin. Med. 1955, 45, 661. 4. Follis, R. H. jun., Orent-Keiles, E., McCollum, E. V. Amer. J. Path. 1942, 18, 29. 5. Fourman, P., McCance, R. A., Parker, R. A. Brit. J. exp. Path. 1956, 37, 40.
on
urea level, voluminous pericardia! effusion, and abnormal kidneys.
The first visible -change in the is in the glomeruli, where the intercapillary cells proliferate and the intercapillary substance increases relatively early-after about three months. Later, about a year after the start of treatment, more widespiead changes appear, whose main feature is dilatation, often cystic, of some of the tubules throughout the renal cortex. with atrophy of other tubules and interstitial fibrosis (see
kidneys
Kidney of guineapig, aged 22
mos.,
castrated and treated with and eosin. x 31/2.)
(Haematoxylin
stilboestrol for -
17
months.
6.
Trevan, D. Lancet, July 7, 1956. p. 22.
1193
figure). The early glomerular change may be similar to glomerular changes seen in oestrogen-treated hamsters7 deoxycortone-treated rats,8 but without comparison of the actual sections we cannot be sure
direct about this. The later tubular dilatation is also rather difficult to assess ; some dilatation occurs in deoxycortone-treated rats 8 but it is not so widespread or characteristic. The lesion most closely resembling that in the treated guineapigs that we have found described is that in rats after the acute phase of potassium deficiency.5Fourman et al., in describing this lesion, are careful to state that it may not be specific ; they have found similar tubular dilatation in old rats, as we have done in old guineapigs. Nevertheless, when the evidence of potassium loss in our animals is taken into account, the similarity in appearance seems more than fortuitous. (4) In treated guineapigs the intake of water and the output of urine are increased sixfold or sevenfold.
and in
COMMENTS
It remains to
suggest why we have not detected the renal signs of potassium deficiency, and why stilboestrol treatment has not given rise to such chronic renal lesions in other species. The failure to detect the acute lesions is not surprising, because we have examined very few kidneys from animals killed during the early stages of treatment. The lack of reports of similar renal lesions in other species treated with oestrogen may be due to species differences in adrenal response to oestrogen treatment. Direct evidence is not available for many species, but what evidence there is suggests that adrenal function under oestrogen treatment is unchanged or reduced in man,9—11 reduced in rats,12 but increased in
early
guineapigs.133 The complexities of the action of adrenal-cortical secretions on electrolyte metabolism are well known. We do not suggest that excess aldosterone is the only explanation of our findings : it only seems to us the most likely one. One obvious objection is that our guineapigs have increased secretion of 17-oxosteroids which the human may excrete abnormally steroids 14 or of corticosterone.15 This difference does not necessarily contradict our hypothesis, because we have evidence that the fluid balance and electrolyte changes are not correlated with the excretion of such steroids, and some differences may be expected in comparing a pathological human condition with what we assume to be an exaggerated physiological condition in the treated animals. Until the excretion of aldosterone by our guineapigs has been studied, the evidence summarised above is no more than suggestive ; but it seems sufficient, in combination with that from another species presented by two of us in the accompanying communication,16 to deserve consideration.
patients do not, though they large amounts of 17-oxogenic
We some
are very grateful to Dr. Paul Fourman for of his rat material for study.
sending
us
F. C. CHESTEKMAN M.B. Lond.
L. M. FRANKS M.B. Durh.
E. T. KNUDSEN* *
7. 8. 9.
Imperial Cancer Research Fund, London, N.W.7. Courtauld Institute of Biochemistry, Middlesex Hospital, London, W.1
M.B. Lond.
P. C. WILLIAMS B.Sc. Lond.
Horning, E. S. Z. Krebsforsch. 1956, 61, 1. Herlant, M., Timiras, P. S. Acta anat. 1951, 12, 229. Dean, A. L., Woodard, H. Q., Twombley, G. H. Surgery, 1944,
16, 169. 10. Birke, G., Franksson, C., Plantin, L. O. Acta endocr., Copen11.
12. 13. 14. 15. 16.
hagen, 1954, 15, 1. Halkerston, I. D. K., Hillman, J., Palmer, D., Rundle, A. J. Endocrin. 1956, 13, 433. Vogt, M. J. Physiol. 1955, 130, 601. Zondek, B., Burstein, S. Endocrinology, 1952, 50, 419. Foye, L. V. jun., Feichtmeir, T. V. Amer. J. Med. 1955, 19, 966. Mader, I. J., Iseri, L. T. Ibid, p. 976. Franks, L. M., Chesterman, F. C. Lancet, 1956, ii, 1193.
EXPERIMENTAL PRODUCTION OF MORPHOLOGICAL CHANGES RESEMBLING CONN’S SYNDROME Conn’s syndrome1 is characterised by a disturbance of electrolyte balance, considerable loss of potassium, characteristic lesions in the heart-muscle and kidneys, and either hyperplasia or tumour formation in the adrenal glands. In a study of the effects of long-continued stilboestrol treatment of golden hamsters we have seen similar morphological changes.. The animals used were stock hamsters, and the changes described were seen in both sexes treated with subcutaneous implants of stilbcestrol 15 mg. (renewed usually at three-month intervals) alone or together with an initial application of methylcholanthrene (39 hamsters). There were no similar in 36 untreated hamsters or in 19 hamsters changes treated with methylcholanthrene alone or together with other hormones. In the adrenal there was usually a diffuse hyperplasia, but tumour nodules were occasionally found. The initial change appeared to begin in the middle zone of the zona fasciculata. This change was similar to that described by Peczenikbut ultimately more severe. After a month isolated pale-staining cells, with finely vacuolated cytoplasm and rather darkly staining nuclei, could be seen in the zona fasciculata. After three months these cells were much more numerous, and large cells with abundant cytoplasm and large nuclei lay between the cords of the zona fasciculata. In addition a zone in the middle of the fasciculate layer showed nuclear irregularity with variation in shape, size, and intensity of staining. The cytoplasm was often eosinophil. By six months the changes were well developed. The normal trabecular arrangement of the adrenal cells was lost, the cords being replaced by irregular masses of large cells with eosinophil, sometimes coarsely granular, cytoplasm and large irregular nuclei. There were many bizarre and irregular cells. In some the cytoplasm was foamy and finely vacuolated ; in others the vacuoles were large. The nuclei were sometimes pyknotic and fragmented. This change at first involved only the middle zone of the fasciculata but later affected most of the gland, although as a rule the zona glomerulosa remained intact. These changes were found in all the stilboestrol-treated hamsters, of either sex, except for 4 of 6 males segregated and treated at the age of 3 months for periods of five, six, ten and twelve months. The 2 other hamsters, treated for eight and twelve months, showed slight changes only. After six months’ treatment degenerative changes appeared in these presumably hyperplastic cells. The cells underwent an eosinophil coagulative necrosis which at first affected only small groups in the middle of the zona fasciculata but later involved the whole zone and ultimately the whole gland except the zona glomerulosa. Haemorrhage with rupture of sinusoid walls occurred in these necrotic areas, and there seems to be little doubt that this had followed the necrosis. The latter change in some, but not all, cases took place about ten to fourteen days after reimplantation with stilboestrol and appeared after seven to twelve months’ total treatment. In several animals there was an ill-defmed nodular hyperplasia of the cells of the inner zones of the adrenal cortex, and in 3 there were definite tumours. 2 were obviously derived from the hyperplastic fasciculate cells (figs. 1 and 2), but the 3rd appeared to have arisen from the zona glomerulosa and resembled small glomerulosa-cell tumours sometimes seen in untreated old animals (Chesterman, unpublished
observations). In some hamsters with pronounced adrenal changes lesions associated with severe potassium deficiency 3—6 In the myocardium were focal areas were also found. of muscle degeneration, often subendocardial, associated Conn, J. W. J. Lab. clin. Med. 1955, 45, 3, 661. Peczenik, O. Proc. roy. Soc. Edinb., B 1944, 62, 59. Follis, R. H. jun., Orent-Keiles, E., McCollum, E. V. Amer. J. Path. 1942, 18, 29. 4. Perkins, J. G., Petersen, A. B., Riley, J. A. Amer. J. Med. 1950, 8, 115. 5. Keye, J. D. jun. Circulation, 1952, 5, 766. 6. Fourman, P., McCance, R. A., Parker, R. A. Brit. J. exp. Path. 1956, 37, 40. 1. 2. 3.
Schemm, F. R., Cámara,
Preliminary
(1954) Circulation, 10,
17-OXOSTEROIDS
(mg.
Urinary 17-oxosteroid per pig per 24 hr. ±S.E.)
Group
10 animals and 15-22 estimations per group.
for 17-ketosteroids), which has been measured Dr. R. D. Bulbrook and Dr. F. C. Greenwood, who have kindly allowed us to use some of their data here. Table i gives figures for the output of such steroids (which do not include aldosterone) in guineapigs which have been castrated or have been treated with large doses of stilboestrol (implants of 15-mg. tablets renewed every three to four months) for more than a year. The output of 17-oxosteroids is increased equally in intact and castrated guineapigs treated with stilbcestrol, and there is other evidence (particularly from the increase produced by adrenocorticotrophin) that the 17-oxosteroid output is one index of adrenal-cortical activity. The excretion of 17-oxogenic steroids is also increased in parallel fashion. (2) The evidence for loss of potassium in treated guineapigs is given in table 11, which records the over. new name
REFERENCE A. A.
TABLE I—DAILY OUTPUT OF URINARY
by
430.
Communications
POSSIBLE ALDOSTERONISM IN
STILBŒSTROL-TREATED GUINEAPIGS THE evidence is so far indirect ; but the similarities between the condition of primary aldosteronism or Conn’s syndrome in man and the effects of large doses of stilbopstrol in male guineapigs seem suggestive enough to report. In the human disease excessive secretion of aldosterone by the adrenal cortex is associated with loss of potassium.l2 There are also changes in the kidneys of the patients (vacuolation of the epithelia] cells of the renal tubules 3) like those produced in the early stages of potassium deficiency in rats.4 Later the kidneys of
TABLE
II—NIGHTLY
OUTPUT
URINARY
OF
SODIUM
AND
POTASSIUM
whether normal potassium intake is restored or not, show a different lesion—cystic It is this late result of dilatation of the tubules.4 5 potassium deficiency that we believe is reproduced in the treated guineapigs, and our suggestion that the cause may be excessive secretion of aldosterone is based on evidence that the stilbœstro] treatment causes adrenal overactivity and loss of potassium. A further similarity between human patients and the treated guineapigs is that both have polydipsia and polyuria.
potassium-deficient rats,
Nightly output (m.eq. per pig ±S.E.) Group Sodium
Potassium
excretion of sodium and potassium mea. four nights weekly for five weeks in 4 normal guineapigs and 4 guineapigs which had been treated with stilboestrol 05-20 mg. by mouth daily for three months. Despite the large variation in the nightly excretion of sodium the mean figure is virtually the same in the two groups. There was less variation in the potassium output which was significantly greater (almost doubled) in the treated animals than in the controls. The mean bodyweight of the two groups did not differ by more than 5% during the course of the urine collections. The food intake was not measured but had borne a constant relation to body-weight in other experiments. (3) Both intact and castrated guinea pigs in which stilbœstrol is implanted die, during the second year of treatment, from renal failure with a raised blood-
night urinary
sured
EVIDENCE
(1) The evidence for adrenal overactivity depends on urinary excretion of 17-oxosteroids (the chemists’
the
1. Conn, J. W. J. Lab. clin. Med. 1955, 45, 3. 2. Chalmers, T. M., FitzGerald, M. G., James, A. H., Scarborough, H. Lancet, 1956, i, 127. 3. Conn, J. W. J. Lab. clin. Med. 1955, 45, 661. 4. Follis, R. H. jun., Orent-Keiles, E., McCollum, E. V. Amer. J. Path. 1942, 18, 29. 5. Fourman, P., McCance, R. A., Parker, R. A. Brit. J. exp. Path. 1956, 37, 40.
on
urea level, voluminous pericardia! effusion, and abnormal kidneys.
The first visible -change in the is in the glomeruli, where the intercapillary cells proliferate and the intercapillary substance increases relatively early-after about three months. Later, about a year after the start of treatment, more widespiead changes appear, whose main feature is dilatation, often cystic, of some of the tubules throughout the renal cortex. with atrophy of other tubules and interstitial fibrosis (see
kidneys
Kidney of guineapig, aged 22
mos.,
castrated and treated with and eosin. x 31/2.)
(Haematoxylin
stilboestrol for -
17
months.
6.
Trevan, D. Lancet, July 7, 1956. p. 22.
1193
figure). The early glomerular change may be similar to glomerular changes seen in oestrogen-treated hamsters7 deoxycortone-treated rats,8 but without comparison of the actual sections we cannot be sure
direct about this. The later tubular dilatation is also rather difficult to assess ; some dilatation occurs in deoxycortone-treated rats 8 but it is not so widespread or characteristic. The lesion most closely resembling that in the treated guineapigs that we have found described is that in rats after the acute phase of potassium deficiency.5Fourman et al., in describing this lesion, are careful to state that it may not be specific ; they have found similar tubular dilatation in old rats, as we have done in old guineapigs. Nevertheless, when the evidence of potassium loss in our animals is taken into account, the similarity in appearance seems more than fortuitous. (4) In treated guineapigs the intake of water and the output of urine are increased sixfold or sevenfold.
and in
COMMENTS
It remains to
suggest why we have not detected the renal signs of potassium deficiency, and why stilboestrol treatment has not given rise to such chronic renal lesions in other species. The failure to detect the acute lesions is not surprising, because we have examined very few kidneys from animals killed during the early stages of treatment. The lack of reports of similar renal lesions in other species treated with oestrogen may be due to species differences in adrenal response to oestrogen treatment. Direct evidence is not available for many species, but what evidence there is suggests that adrenal function under oestrogen treatment is unchanged or reduced in man,9—11 reduced in rats,12 but increased in
early
guineapigs.133 The complexities of the action of adrenal-cortical secretions on electrolyte metabolism are well known. We do not suggest that excess aldosterone is the only explanation of our findings : it only seems to us the most likely one. One obvious objection is that our guineapigs have increased secretion of 17-oxosteroids which the human may excrete abnormally steroids 14 or of corticosterone.15 This difference does not necessarily contradict our hypothesis, because we have evidence that the fluid balance and electrolyte changes are not correlated with the excretion of such steroids, and some differences may be expected in comparing a pathological human condition with what we assume to be an exaggerated physiological condition in the treated animals. Until the excretion of aldosterone by our guineapigs has been studied, the evidence summarised above is no more than suggestive ; but it seems sufficient, in combination with that from another species presented by two of us in the accompanying communication,16 to deserve consideration.
patients do not, though they large amounts of 17-oxogenic
We some
are very grateful to Dr. Paul Fourman for of his rat material for study.
sending
us
F. C. CHESTEKMAN M.B. Lond.
L. M. FRANKS M.B. Durh.
E. T. KNUDSEN* *
7. 8. 9.
Imperial Cancer Research Fund, London, N.W.7. Courtauld Institute of Biochemistry, Middlesex Hospital, London, W.1
M.B. Lond.
P. C. WILLIAMS B.Sc. Lond.
Horning, E. S. Z. Krebsforsch. 1956, 61, 1. Herlant, M., Timiras, P. S. Acta anat. 1951, 12, 229. Dean, A. L., Woodard, H. Q., Twombley, G. H. Surgery, 1944,
16, 169. 10. Birke, G., Franksson, C., Plantin, L. O. Acta endocr., Copen11.
12. 13. 14. 15. 16.
hagen, 1954, 15, 1. Halkerston, I. D. K., Hillman, J., Palmer, D., Rundle, A. J. Endocrin. 1956, 13, 433. Vogt, M. J. Physiol. 1955, 130, 601. Zondek, B., Burstein, S. Endocrinology, 1952, 50, 419. Foye, L. V. jun., Feichtmeir, T. V. Amer. J. Med. 1955, 19, 966. Mader, I. J., Iseri, L. T. Ibid, p. 976. Franks, L. M., Chesterman, F. C. Lancet, 1956, ii, 1193.
EXPERIMENTAL PRODUCTION OF MORPHOLOGICAL CHANGES RESEMBLING CONN’S SYNDROME Conn’s syndrome1 is characterised by a disturbance of electrolyte balance, considerable loss of potassium, characteristic lesions in the heart-muscle and kidneys, and either hyperplasia or tumour formation in the adrenal glands. In a study of the effects of long-continued stilboestrol treatment of golden hamsters we have seen similar morphological changes.. The animals used were stock hamsters, and the changes described were seen in both sexes treated with subcutaneous implants of stilbcestrol 15 mg. (renewed usually at three-month intervals) alone or together with an initial application of methylcholanthrene (39 hamsters). There were no similar in 36 untreated hamsters or in 19 hamsters changes treated with methylcholanthrene alone or together with other hormones. In the adrenal there was usually a diffuse hyperplasia, but tumour nodules were occasionally found. The initial change appeared to begin in the middle zone of the zona fasciculata. This change was similar to that described by Peczenikbut ultimately more severe. After a month isolated pale-staining cells, with finely vacuolated cytoplasm and rather darkly staining nuclei, could be seen in the zona fasciculata. After three months these cells were much more numerous, and large cells with abundant cytoplasm and large nuclei lay between the cords of the zona fasciculata. In addition a zone in the middle of the fasciculate layer showed nuclear irregularity with variation in shape, size, and intensity of staining. The cytoplasm was often eosinophil. By six months the changes were well developed. The normal trabecular arrangement of the adrenal cells was lost, the cords being replaced by irregular masses of large cells with eosinophil, sometimes coarsely granular, cytoplasm and large irregular nuclei. There were many bizarre and irregular cells. In some the cytoplasm was foamy and finely vacuolated ; in others the vacuoles were large. The nuclei were sometimes pyknotic and fragmented. This change at first involved only the middle zone of the fasciculata but later affected most of the gland, although as a rule the zona glomerulosa remained intact. These changes were found in all the stilboestrol-treated hamsters, of either sex, except for 4 of 6 males segregated and treated at the age of 3 months for periods of five, six, ten and twelve months. The 2 other hamsters, treated for eight and twelve months, showed slight changes only. After six months’ treatment degenerative changes appeared in these presumably hyperplastic cells. The cells underwent an eosinophil coagulative necrosis which at first affected only small groups in the middle of the zona fasciculata but later involved the whole zone and ultimately the whole gland except the zona glomerulosa. Haemorrhage with rupture of sinusoid walls occurred in these necrotic areas, and there seems to be little doubt that this had followed the necrosis. The latter change in some, but not all, cases took place about ten to fourteen days after reimplantation with stilboestrol and appeared after seven to twelve months’ total treatment. In several animals there was an ill-defmed nodular hyperplasia of the cells of the inner zones of the adrenal cortex, and in 3 there were definite tumours. 2 were obviously derived from the hyperplastic fasciculate cells (figs. 1 and 2), but the 3rd appeared to have arisen from the zona glomerulosa and resembled small glomerulosa-cell tumours sometimes seen in untreated old animals (Chesterman, unpublished
observations). In some hamsters with pronounced adrenal changes lesions associated with severe potassium deficiency 3—6 In the myocardium were focal areas were also found. of muscle degeneration, often subendocardial, associated Conn, J. W. J. Lab. clin. Med. 1955, 45, 3, 661. Peczenik, O. Proc. roy. Soc. Edinb., B 1944, 62, 59. Follis, R. H. jun., Orent-Keiles, E., McCollum, E. V. Amer. J. Path. 1942, 18, 29. 4. Perkins, J. G., Petersen, A. B., Riley, J. A. Amer. J. Med. 1950, 8, 115. 5. Keye, J. D. jun. Circulation, 1952, 5, 766. 6. Fourman, P., McCance, R. A., Parker, R. A. Brit. J. exp. Path. 1956, 37, 40. 1. 2. 3.