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Uptake of Tritiated Stilbestrol Diphosphate by the Prostate Gland
Vol. 104, July Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright © 1970 by The Williams & Wilkins Co.
UPTAKE OF TRITIATED STILBESTROL DIPHOSPHATE BY THE PROSTATE GLAND CAROL PROMISLOW, JOHN CONNOLLY
AND
ALBERT CLARKE
From the Departments of Urology and Biochemistry, Queen's University, Kingston, Ontario
The classical experiments of Huggins and Hodges led to the use of estrogen and/or castration therapy for prostatic carcinoma. 1 This approach continues to be the basis for nonoperative treatment of this neoplasm and is invariably used at some stage of the disease. While the hormone dependence of the tumor has been established and approximately 80 per cent respond to treatment, relapses are common.1- 3 The reason for the large number of eventual relapses is unknown. Testicular androgens are necessary to maintain growth and function of the prostate gland while exogenous estrogens have an opposite effect. Estrogens may produce this inhibition by acting at the cellular level or indirectly by inhibiting gonadotropin secretion. 2 • 4 • 5 In vitro studies have shown that estrogens inhibit testosterone production by the testes and, following estrogen administration, the binding of testosterone to serum proteins is also increased. 6 This may be of Accepted for publication June 25, 1969. Read at annual meeting of American Urological Association, San Francisco, California, May 11-15, 1969.
This study was supported by funds from The National Cancer Institute and from The Ontario Cancer Treatment and Research Foundation, grant No. 154. 1 Huggins, C. and Hodges, C. V.: Studies on prostatic cancer. I. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res., 1: 293, 1941. 2 Huggins, C. and Clark, P. J.: Quantitative studies of prostatic secretion. II. The effect of castration and of estrogen injection on the normal and on the hyperplastic prostate glands of dogs. J. Exp. Med., 72: 747, 1940. 3 Franks, L. M.: Estrogen-treated prostatic cancer: the variation in responsiveness of tumor cells. Cancer, 13: 490, 1960. 4 Goodwin, D. A., Rasmussen-Taxdal, D. S., Ferreira, A. A. and Scott W. W.: Estrogen inhibition of androgen-maintained prostatic secretion in the hypophysectomized dog. J. Urol., 86: 134, 1961.
6 Huggins, C.: Control of cancers of man by endocrinologic methods. Cancer Res., 17: 467,
1957.
6 Slaunwhite, W.R., Jr., Sandberg, A. A., Jackson, J. E. and Staubitz, W. J.: Effects of estrogen and HCG on androgen synthesis by human testes. J. Clin. Endocrinol. Metabolism, 22: 992, 1962.
considerable significance since protein-bound testosterone is biologically inactive. The mechanisms by which estrogen administration and/or androgen deprivation influence the growth of prostatic tumors are unknown. The malignant cells which have responded to treatment show pyknosis of the nuclei, vacuolization of the cytoplasm, rupture of the cell membrane and eventual replacement by fibrosis. Stilbestrol, a synthetic preparation with estrogen-like properties, is commonly used to treat carcinoma of the prostate. Because the prostate gland is rich in acid phosphatase the diphosphate form of stilbestrol has been recommended for the treatment of this disease. This compound, an inactive or transport form of stilbestrol, is activated when the phosphate groups are hydrolyzed by acid phosphatase. While in vitro dephosphorylation may be efficient there has been no conclusive in vivo demonstration of this. Therefore, we designed a series of experiments to determine whether quantities of the dephosphorylated compound are deposited in the prostate gland following the administration of tritiated diethylstilbestrol diphosphate (sodium salt). Because we have considerable evidence that this hormone depresses the activity of the phosphatase (necessary for its conversion into an active hormone), change in radioactive stilbestrol-diphosphate was compared with time against the changes in acid phosphatase in gland and serum. MATERIALS AND METHODS
We used an inbred strain of mature, male rabbits averaging 8 pounds in weight. Tritiumlabeled diethylstilbestrol diphosphate (50 to 100 mc./mm.) was given intravenously at 3 µc. per pound for a total dose of 21 µc. This dose was contained in 0.3 mg. stilbestrol diphosphate. Thirty rabbits were sacrificed at O (controls), 15 and 30 minutes; 1, U'2, 2}'2, 3, 4}'2, 6, 12 and 24 hours and on days 2, 4, 9 and 19. Whole blood was drawn by cardiac puncture and the rabbits were sacrificed. Samples of prostate, liver, bladder 146
UPTAKE OF TRITIATED STILBESTROL DIPHOSPHATE BY PROSTATE GLAND
epithelium and adipose tissue were stored at minus IOC until used. Whole blood was allowed to clot for 20 minutes and the serum fraction was collected and frozen. Because steroid hormones are bound to serum albumin the radioactivity is concentrated in the serum fraction. 7 Therefore, serum rather than whole blood was measured. In addition, the pigment in whole blood and other deeply colored tissues causes quenching. Quenching occurs when the 13-particles and light energy are absorbed by the colored solution and are not picked up by the liquid scintillation counter. Preparation of tissue counting. 1) Hyamine hydroxide-toluene phosphor system. Between 100 and 250 mg. tissue were prepared for liquid scintillation counting, using a hyamine hydroxidetoluene phosphor system. 8 The results were expressed as the total counts per minute per gm. tissue. A 0.2 ml. sample of serum was used because with larger quantities the phases separate during counting. To minimize quenching, 3 drops of hydrogen peroxide were added to the deeply colored counting solutions (e.g. liver). 2) Ether extraction-toluene phosphor system. This method was used to determine the distribution of the ether-soluble, radioactive material (stilbestrol) in the various tissue samples. A 10 JJer cent homogenate of bladder and prostate prepared with distilled water was placed in a centrifuge tube. The homogenate was extracted 3 times with wccessive 5 ml. portions of ether. After each addition of ether the tubes were shaken vigorously and centrifuged for 15 minutes at 2,000 revolutions per minute. The ether layer was removed with a Pasteur pipette, placed in a counting vial and evaporated under nitrogen. Ten ml. of toluene-phosphor was then added to each vial and the sample was counted. All counting wa~ done using a liquid scintillation counter. To correct the variable quenching effect of the various tissues when the hyamine hydroxide-toluene phosphor system was used, each sample wa.~ counted before and after an internal standard of known radioactivity was added. To obtain the percentage of counts in the 7 Eik-Nes, K., Schellman, J. A., Lumry, H.. and Samuels, L. T.: The binding of steroids to protein. I. Solubility determinations. J. Biol. Chem., 206: 411, 1954. 8 Herberg, R. J.: Determination of carbon-14 and tritium in blood and other whole tissues. Anal. Chem., 32: 42, 1960.
147
ether-soluble fraction we corrected for differences in counting efficiencies between the 2 systems. The regular toluene-phosphor system was 4.48 times more efficient than the hyamine hydroxidetoluene phosphor system. Determination of acid phosphatase in serum and tissue. Using Manning's technique a 1 per cent homogenate of prostate and bladder was prepared. 9 Acid phosphatase levels in both serum and homogenates were determined at the various times using the Bodansky method. 10 The acid and alkaline phosphatase activities of the serum and of a 1 per cent homogenate of bladder and prostate obtained from the 2 control animals were measured 1rning non-radioactive stilbestrol diphosphate as the substrate.11 RESULTS
After a single, intravenous dose of 0.3 mg. tritiated stilhestrol diphosphate, we detected no apparent preferential uptake by prostatic tissue. In various tissues examined the maximum concentration of radioactivity occurred within the first half hour (fig. 1). After this peak the declined rapidly. After 6 hours only trace amounts of radioactivity remained. The fluctuations in tissue activity paralleled those of the serum. The greatest concentration of activity appears in the serum (fig. 1). The uptake of stilbestrol di phosphate by the prostate gland was surprisingly low. At all time intervals examined the total activity of the prostate is less than that of the liver, bladder and serum. The high activity of liver is partly due to its role in detoxification and conjugation of these compounds. A tissue is hormone-responsive when it incor]Jorates and retains a specific hormone over long periods. The incorporation pattern of a non.responsive tissue parallels that of blood. 12 Evidence obtained with tagged stilbestrol diphos9 Manning, J.P., Babson, A. L., Butler, M. C. and Priester, S. F.: Determination of acid phosphatase activity in tissue homogenates. Canad. J. Biochem., 44: 755, 1966. 10 Bodansky, A.: Phosphatase studies. II. Determination of serum phosphatase. Factors influencing the accuracy of the determination. J. Biol. Chem., 101: 93, 1933. 11 Johnson, W., Jasmin, R. and Corte, G.: Enzymic hydrolysis of stilbestrol diphosphate in vitro and in vivo. Proc. Soc. Exp. Biol. Med., 106: 327, 1961. 12 Jensen, E. V. and Jacobson, H. I.: Basic guides to the mechanism of estrogen action. Recent Progr. Hormone Res., 18: 387, 1962.
148
PROMISLOW, CONNOLLY AND CLARKE 19,500
16,500
13,500
cpm/g I0,500
TISSUE
4,500
1,500 3 4 5 6 12 24 4 B 12 16 20 0 -------HOURS---------DAYS--TIME AFTER INJECTION
Fm. 1. Radioactivity in rabbit tissues after single intravenous injection of tritiated diethylstilbestrol diphosphate (3 µc./pound). phate and counting techniques suggests that the prostate behaves as a non-responsive gland. The uptake pattern we observed (fig. 1) resembles that obtained by Jensen and Jacobson in tissues shown to be unresponsive to estradiol. 12 Free stilbestrol is ether-soluble and stilbestrol diphosphate is not. After stilbestrol diphosphate is given the ether-soluble fraction should indicate how much hydrolysis has occurred. To determine whether the prostate acid phosphatase was successfully dephosphorylating the stilbestrol diphosphate we measured the amount of free stilbestrol by homogenizing prostatic tissue in water, extracting with ether and counting. Since the total activity in the bladder was high the ether-soluble components of this tissue were also extracted and counted. Within the first U-z hours between 55.0 and 69.5 per cent of the radioactivity in the prostate gland was in the ether-soluble fraction (table 1). This figure was 24.2 per cent at the end of 24 hours. The activity in the ether fraction of the bladder ranged from 12.0 to 18.8 per cent over the first 3 hours, then decreased sharply at 6 hours. We found few ether-soluble components in serum; only 7.1 per cent of the activity was ether soluble at 15 minutes. The concentration of radioactivity in the ether-soluble fraction of prostatic tissue was actually higher than that of bladder or serum, although the total activity (water plus ether-soluble components) in the prostate (fig. 1) was lower than any of the other tissues examined except fat. In a parallel study, using the Bodansky method, we determined the acid phosphatase activity in
TABLE
l. Percentage of radioactivity in ether fraction
Time Interval 15 mins. 30 mins. 1 hr. H"2 hrs. 3 hrs. 6 hrs. 12 hrs. 24 hrs.
Serum
Bladder
Prostate
7.1 5.9 4.4 2.4 1.6 0.4 0.1 0.3
14. 9 13.0 12.0 12.9 18.8 6.3 4. 6 1.6
69.5 66.0 55.8 55.0 19.8 20.0 24.2
2. Acid phosphatase activity in rabbit serum and 1 per cent homogenate after intravenous injection of tritiated stilbestrol diphosphate (3 µc./pound
TABLE
(Bodansky mg.%)
Prostate (Bodansky units/gm.)
Control Control
0.85 0.75
3.8 4.3
15 mins. 30 mins.
1.7 1.8 1.5 1.4 1.4 1.4 0.6 0.4 0.5 0.4 0.1 0
3.0 3.3 3.1 2.6 1.8 2.8 2.8 2.4 2.2 2.9 2.8 2.9
Serum
1 hr. 1¼ hrs. 3 hrs. 6 hrs. 12 hrs. 24 hrs. 48 hrs. 4 days 9 days 19days
serum and 1 per cent homogenate of bladder and prostate. 10 After we gave tritiated stilbestrol diphosphate, the serum acid phosphatase levels showed that the enzymatic activity increased
UPTAKE OF TRITIATED STILBESTROL DIPHOSPHATE BY PROSTATE GLAND
149
19,500
1,500 5 +-------1HOURS--------TIME AFTER INJECTION
Fm. 2. Radioactivity in rabbit tissues after single intravenous injection of tritiated diethylstilbestrol diphosphate (3 µc./pound). over the first 6 hours (table 2). The levels then decreased to those of the control animals reaching zero at 19 days. The fluctuations in the serum acid phosphatase closely matched those of serum radioactivity. However, the acid phosphatase content of prostatic homogenates showed little change after stilbestrol diphosphate was injected (table 2). In addition we did in vitro studies on 1 per cent homogenates prepared from the bladder and prostate of control animals to determine their ability to hydrolyze a buffered substrate prepared from non-radioactive stilbestrol diphosphate. The prostatic homogenate rapidly and almost completely dephosphorylated the stilbestrol diphosphate (fig. 2). Within 2 hours 97 per cent of this substrate was hydrolyzed (pH 5.0). Bladder homogenates (pH 5.0) hydrolyzed 31 per cent of the substrate at 2 hours. The enzymic activity of both prostate and bladder (at pH 9.0) was low.
While the effects of estrogens on the prostatic epithelium are known, the exact mechanism of their action remains unexplained. After the injection of tritiated stilbestrol diphosphate the prostate gland does not take up appreciable quantities of the compound. The maximum prostatic uptake of the labeled compound occurred 30 minutes after injection (fig. 1). This finding agrees with those of Fergusson13 and
Ghaleb14 who studied the uptake of C14-labeled stilbestrol diphosphate in a small number of patients with cancer of the prostate. It is difficult to explain the high concentration of radioactive compound in the bladder. However, since the bladder contains both acid and alkaline phosphatase, some of the ester may be dephosphorylated (fig. 2). Serum also contains measurable quantities of the enzyme but the radioactivity in its ether-soluble fraction was extremely low (table 1). The total radioactivity in the bladder after the injection of stilbestrol diphosphate was high. However, the percentage of activity found in the ether-soluble fraction averaged 14.3 per cent over the first 3 hours (table 1). This amount of free stilbestrol in the bladder is not due to the free stilbestrol in the serum since this was low (table 1). Therefore, there was hydrolysis of stilbestrol diphosphate in the bladder. This is probably due to the measurable quantities of acid phosphatase found in the rabbit bladder (fig. 2). Because the quantity of free stilbestrol does not build up in the bladder with time, we concluded that the rate of dephosphorylation does not keep up with clearance from the bladder. Conversely, the prostate gland appears to efficiently hydrolyze the ester. At 15 minutes 69.5 per cent of the total radioactivity appeared in the ether-soluble fraction, a value that fell to 24.2 per cent by 24 hours. The rate of dephos-
13 Fergusson, J. D.: Tracer experiments showing the distribution and fate of injected phosphorylated estrogens in cancer of the prostate. Brit. J. Urol., 33: 442, 1961.
14 Ghaleb, H.: Studies on prostatic carcinoma. In: British Empire Cancer Campaign-ThirtySeventh Annual Report of the Grand Council, p. 145, London, 1959.
DISCUSSION
150
PROMISLOW, CONNOLLY AND CLARKE
phorylation is probably higher in man than in the rabbit (as observed in this experiment) because acid phosphatase concentration (units/ gram) of human prostate is approximately 300 times that of the rabbit. 15 Although dephosphorylation is efficient, little free stilbestrol is deposited in the rabbit prostate. If stilbestrol was accumulating in the gland, total activity and the activity of the ethersoluble fraction should increase with time. Since this does not happen, the prostate gland, by definition, is not estrogen-responsive. 12 Although the prostate incorporates less tritiated stilbestrol diphosphate, it concentrates more free stilbestrol (as measured by the ether-soluble fraction) than any of the other tissues tested. Measurement of acid phosphatase activity in serum taken during the first 6 hours after injection of tritiated stilbestrol diphosphate indicates increased enzyme levels (table 2). This increase parallels the radioactive uptake of the material. By 12 hours the serum enzyme levels have fallen to less than those of the control animals and by 19 hours are zero. If the levels of acid phosphatase in serum are to change, the relative rates of production, release into the circulation, inhibition and destruction must change. The rapid increase in enzyme levels, we showed, may be due to binding or inactivation of a natural acid phosphatase inhibitor by the injected hormone. Steroid hormones are known to bind to serum protein and it is possible that the inhibitor is more strongly bound than the enzyme. 7 After hormone injection the enzyme activity in the prostate gland deviates from that seen in the control animals but the difference between the levels at 15 minutes and at 19 days is significant. It is possible that we did not give enough stilbestrol diphosphate to suppress enzyme levels in the normal rabbit prostate. The dose used (0.3 mg./7 pound animal) is equivalent to 8 mg. per 180-pound man. However, the acid phosphatase concentration in the rabbit gland is low. 15 Woodard noted that after estrogen therapy in men, serum enzyme levels declined and the low levels in the cancerous gland were 15 Gutman, A. B.: The development of the acid phosphatase test for pros ta tic carcinoma. Bull. N.Y. Acad. Med., 44: 63, 1968.
even less after estrogen therapy. 16 If this is valid, after the first injection of stilbestrol diphosphate the amount of enzyme available to convert transport stilbestrol to free stilbestrol would be lower when the next injection was given. Arcadi observed that glycoproteins in the basement membrane of the prostatic acini became insoluble after estrogen treatment. 17 He suggested that the impermeability of the ground substance chiefly determines the resistance the ground substance offers to the growth and spread of tumor cells. Thus the formation of tough glycoprotein in the basement membrane might keep acid phosphatase from escaping from the gland and this woukl explain the near normal levels of this enzyme in the gland when the serum levels approach zero. SUMMARY
Tritium-labeled diethylstilbestrol diphosphate was administered to 30 inbred, male rabbits which were then sacrificed at various times. Samples of serum, liver, bladder, prostate and fat were analyzed for radioactivity. The total radioactivity in the prostate was less than that of liver, bladder and serum at all times. No preferential accumulation of activity was observed in the prostate. The behavior of this tissue resembled that of non-responsive tissue. However, the gland appears to hydrolyze tritiated stilbestrol diphosphate efficiently because 69.5 per cent of the radioactivity appeared in the ether-soluble fraction at 15 minutes. This conclusion was supported by in vitro studies. A 1 per cent homogenate of prostate from control animals dephosphorylated 97 per cent of a (non-radioactive) stilbestrol diphosphate substrate (pH 5.0) in 2 hours. In the rabbit the level of acid phosphatase in the serum was elevated after stilbestrol diphosphate was injected, decreased to normal at 6 hours and zero at 19 days. The concentration of acid phosphatase in the prostate was constant at all times. 16 Woodard, H. Q. and Dean, A. L.: Significance of phosphatase findings in carcinoma of the prostate. J. Urol., 67: 158, 1947. 17 Arcadi, J. A.: The influence of hormones upon some constituents of connective tissue in prostatic cancer. Texas Reports Biol. Med., 13: 591, 1955.
THE JOURNAL OF UROLOGY
Copyright © 1970 by The Williams & Wilkins Co.
UPTAKE OF TRITIATED STILBESTROL DIPHOSPHATE BY THE PROSTATE GLAND CAROL PROMISLOW, JOHN CONNOLLY
AND
ALBERT CLARKE
From the Departments of Urology and Biochemistry, Queen's University, Kingston, Ontario
The classical experiments of Huggins and Hodges led to the use of estrogen and/or castration therapy for prostatic carcinoma. 1 This approach continues to be the basis for nonoperative treatment of this neoplasm and is invariably used at some stage of the disease. While the hormone dependence of the tumor has been established and approximately 80 per cent respond to treatment, relapses are common.1- 3 The reason for the large number of eventual relapses is unknown. Testicular androgens are necessary to maintain growth and function of the prostate gland while exogenous estrogens have an opposite effect. Estrogens may produce this inhibition by acting at the cellular level or indirectly by inhibiting gonadotropin secretion. 2 • 4 • 5 In vitro studies have shown that estrogens inhibit testosterone production by the testes and, following estrogen administration, the binding of testosterone to serum proteins is also increased. 6 This may be of Accepted for publication June 25, 1969. Read at annual meeting of American Urological Association, San Francisco, California, May 11-15, 1969.
This study was supported by funds from The National Cancer Institute and from The Ontario Cancer Treatment and Research Foundation, grant No. 154. 1 Huggins, C. and Hodges, C. V.: Studies on prostatic cancer. I. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Res., 1: 293, 1941. 2 Huggins, C. and Clark, P. J.: Quantitative studies of prostatic secretion. II. The effect of castration and of estrogen injection on the normal and on the hyperplastic prostate glands of dogs. J. Exp. Med., 72: 747, 1940. 3 Franks, L. M.: Estrogen-treated prostatic cancer: the variation in responsiveness of tumor cells. Cancer, 13: 490, 1960. 4 Goodwin, D. A., Rasmussen-Taxdal, D. S., Ferreira, A. A. and Scott W. W.: Estrogen inhibition of androgen-maintained prostatic secretion in the hypophysectomized dog. J. Urol., 86: 134, 1961.
6 Huggins, C.: Control of cancers of man by endocrinologic methods. Cancer Res., 17: 467,
1957.
6 Slaunwhite, W.R., Jr., Sandberg, A. A., Jackson, J. E. and Staubitz, W. J.: Effects of estrogen and HCG on androgen synthesis by human testes. J. Clin. Endocrinol. Metabolism, 22: 992, 1962.
considerable significance since protein-bound testosterone is biologically inactive. The mechanisms by which estrogen administration and/or androgen deprivation influence the growth of prostatic tumors are unknown. The malignant cells which have responded to treatment show pyknosis of the nuclei, vacuolization of the cytoplasm, rupture of the cell membrane and eventual replacement by fibrosis. Stilbestrol, a synthetic preparation with estrogen-like properties, is commonly used to treat carcinoma of the prostate. Because the prostate gland is rich in acid phosphatase the diphosphate form of stilbestrol has been recommended for the treatment of this disease. This compound, an inactive or transport form of stilbestrol, is activated when the phosphate groups are hydrolyzed by acid phosphatase. While in vitro dephosphorylation may be efficient there has been no conclusive in vivo demonstration of this. Therefore, we designed a series of experiments to determine whether quantities of the dephosphorylated compound are deposited in the prostate gland following the administration of tritiated diethylstilbestrol diphosphate (sodium salt). Because we have considerable evidence that this hormone depresses the activity of the phosphatase (necessary for its conversion into an active hormone), change in radioactive stilbestrol-diphosphate was compared with time against the changes in acid phosphatase in gland and serum. MATERIALS AND METHODS
We used an inbred strain of mature, male rabbits averaging 8 pounds in weight. Tritiumlabeled diethylstilbestrol diphosphate (50 to 100 mc./mm.) was given intravenously at 3 µc. per pound for a total dose of 21 µc. This dose was contained in 0.3 mg. stilbestrol diphosphate. Thirty rabbits were sacrificed at O (controls), 15 and 30 minutes; 1, U'2, 2}'2, 3, 4}'2, 6, 12 and 24 hours and on days 2, 4, 9 and 19. Whole blood was drawn by cardiac puncture and the rabbits were sacrificed. Samples of prostate, liver, bladder 146
UPTAKE OF TRITIATED STILBESTROL DIPHOSPHATE BY PROSTATE GLAND
epithelium and adipose tissue were stored at minus IOC until used. Whole blood was allowed to clot for 20 minutes and the serum fraction was collected and frozen. Because steroid hormones are bound to serum albumin the radioactivity is concentrated in the serum fraction. 7 Therefore, serum rather than whole blood was measured. In addition, the pigment in whole blood and other deeply colored tissues causes quenching. Quenching occurs when the 13-particles and light energy are absorbed by the colored solution and are not picked up by the liquid scintillation counter. Preparation of tissue counting. 1) Hyamine hydroxide-toluene phosphor system. Between 100 and 250 mg. tissue were prepared for liquid scintillation counting, using a hyamine hydroxidetoluene phosphor system. 8 The results were expressed as the total counts per minute per gm. tissue. A 0.2 ml. sample of serum was used because with larger quantities the phases separate during counting. To minimize quenching, 3 drops of hydrogen peroxide were added to the deeply colored counting solutions (e.g. liver). 2) Ether extraction-toluene phosphor system. This method was used to determine the distribution of the ether-soluble, radioactive material (stilbestrol) in the various tissue samples. A 10 JJer cent homogenate of bladder and prostate prepared with distilled water was placed in a centrifuge tube. The homogenate was extracted 3 times with wccessive 5 ml. portions of ether. After each addition of ether the tubes were shaken vigorously and centrifuged for 15 minutes at 2,000 revolutions per minute. The ether layer was removed with a Pasteur pipette, placed in a counting vial and evaporated under nitrogen. Ten ml. of toluene-phosphor was then added to each vial and the sample was counted. All counting wa~ done using a liquid scintillation counter. To correct the variable quenching effect of the various tissues when the hyamine hydroxide-toluene phosphor system was used, each sample wa.~ counted before and after an internal standard of known radioactivity was added. To obtain the percentage of counts in the 7 Eik-Nes, K., Schellman, J. A., Lumry, H.. and Samuels, L. T.: The binding of steroids to protein. I. Solubility determinations. J. Biol. Chem., 206: 411, 1954. 8 Herberg, R. J.: Determination of carbon-14 and tritium in blood and other whole tissues. Anal. Chem., 32: 42, 1960.
147
ether-soluble fraction we corrected for differences in counting efficiencies between the 2 systems. The regular toluene-phosphor system was 4.48 times more efficient than the hyamine hydroxidetoluene phosphor system. Determination of acid phosphatase in serum and tissue. Using Manning's technique a 1 per cent homogenate of prostate and bladder was prepared. 9 Acid phosphatase levels in both serum and homogenates were determined at the various times using the Bodansky method. 10 The acid and alkaline phosphatase activities of the serum and of a 1 per cent homogenate of bladder and prostate obtained from the 2 control animals were measured 1rning non-radioactive stilbestrol diphosphate as the substrate.11 RESULTS
After a single, intravenous dose of 0.3 mg. tritiated stilhestrol diphosphate, we detected no apparent preferential uptake by prostatic tissue. In various tissues examined the maximum concentration of radioactivity occurred within the first half hour (fig. 1). After this peak the declined rapidly. After 6 hours only trace amounts of radioactivity remained. The fluctuations in tissue activity paralleled those of the serum. The greatest concentration of activity appears in the serum (fig. 1). The uptake of stilbestrol di phosphate by the prostate gland was surprisingly low. At all time intervals examined the total activity of the prostate is less than that of the liver, bladder and serum. The high activity of liver is partly due to its role in detoxification and conjugation of these compounds. A tissue is hormone-responsive when it incor]Jorates and retains a specific hormone over long periods. The incorporation pattern of a non.responsive tissue parallels that of blood. 12 Evidence obtained with tagged stilbestrol diphos9 Manning, J.P., Babson, A. L., Butler, M. C. and Priester, S. F.: Determination of acid phosphatase activity in tissue homogenates. Canad. J. Biochem., 44: 755, 1966. 10 Bodansky, A.: Phosphatase studies. II. Determination of serum phosphatase. Factors influencing the accuracy of the determination. J. Biol. Chem., 101: 93, 1933. 11 Johnson, W., Jasmin, R. and Corte, G.: Enzymic hydrolysis of stilbestrol diphosphate in vitro and in vivo. Proc. Soc. Exp. Biol. Med., 106: 327, 1961. 12 Jensen, E. V. and Jacobson, H. I.: Basic guides to the mechanism of estrogen action. Recent Progr. Hormone Res., 18: 387, 1962.
148
PROMISLOW, CONNOLLY AND CLARKE 19,500
16,500
13,500
cpm/g I0,500
TISSUE
4,500
1,500 3 4 5 6 12 24 4 B 12 16 20 0 -------HOURS---------DAYS--TIME AFTER INJECTION
Fm. 1. Radioactivity in rabbit tissues after single intravenous injection of tritiated diethylstilbestrol diphosphate (3 µc./pound). phate and counting techniques suggests that the prostate behaves as a non-responsive gland. The uptake pattern we observed (fig. 1) resembles that obtained by Jensen and Jacobson in tissues shown to be unresponsive to estradiol. 12 Free stilbestrol is ether-soluble and stilbestrol diphosphate is not. After stilbestrol diphosphate is given the ether-soluble fraction should indicate how much hydrolysis has occurred. To determine whether the prostate acid phosphatase was successfully dephosphorylating the stilbestrol diphosphate we measured the amount of free stilbestrol by homogenizing prostatic tissue in water, extracting with ether and counting. Since the total activity in the bladder was high the ether-soluble components of this tissue were also extracted and counted. Within the first U-z hours between 55.0 and 69.5 per cent of the radioactivity in the prostate gland was in the ether-soluble fraction (table 1). This figure was 24.2 per cent at the end of 24 hours. The activity in the ether fraction of the bladder ranged from 12.0 to 18.8 per cent over the first 3 hours, then decreased sharply at 6 hours. We found few ether-soluble components in serum; only 7.1 per cent of the activity was ether soluble at 15 minutes. The concentration of radioactivity in the ether-soluble fraction of prostatic tissue was actually higher than that of bladder or serum, although the total activity (water plus ether-soluble components) in the prostate (fig. 1) was lower than any of the other tissues examined except fat. In a parallel study, using the Bodansky method, we determined the acid phosphatase activity in
TABLE
l. Percentage of radioactivity in ether fraction
Time Interval 15 mins. 30 mins. 1 hr. H"2 hrs. 3 hrs. 6 hrs. 12 hrs. 24 hrs.
Serum
Bladder
Prostate
7.1 5.9 4.4 2.4 1.6 0.4 0.1 0.3
14. 9 13.0 12.0 12.9 18.8 6.3 4. 6 1.6
69.5 66.0 55.8 55.0 19.8 20.0 24.2
2. Acid phosphatase activity in rabbit serum and 1 per cent homogenate after intravenous injection of tritiated stilbestrol diphosphate (3 µc./pound
TABLE
(Bodansky mg.%)
Prostate (Bodansky units/gm.)
Control Control
0.85 0.75
3.8 4.3
15 mins. 30 mins.
1.7 1.8 1.5 1.4 1.4 1.4 0.6 0.4 0.5 0.4 0.1 0
3.0 3.3 3.1 2.6 1.8 2.8 2.8 2.4 2.2 2.9 2.8 2.9
Serum
1 hr. 1¼ hrs. 3 hrs. 6 hrs. 12 hrs. 24 hrs. 48 hrs. 4 days 9 days 19days
serum and 1 per cent homogenate of bladder and prostate. 10 After we gave tritiated stilbestrol diphosphate, the serum acid phosphatase levels showed that the enzymatic activity increased
UPTAKE OF TRITIATED STILBESTROL DIPHOSPHATE BY PROSTATE GLAND
149
19,500
1,500 5 +-------1HOURS--------TIME AFTER INJECTION
Fm. 2. Radioactivity in rabbit tissues after single intravenous injection of tritiated diethylstilbestrol diphosphate (3 µc./pound). over the first 6 hours (table 2). The levels then decreased to those of the control animals reaching zero at 19 days. The fluctuations in the serum acid phosphatase closely matched those of serum radioactivity. However, the acid phosphatase content of prostatic homogenates showed little change after stilbestrol diphosphate was injected (table 2). In addition we did in vitro studies on 1 per cent homogenates prepared from the bladder and prostate of control animals to determine their ability to hydrolyze a buffered substrate prepared from non-radioactive stilbestrol diphosphate. The prostatic homogenate rapidly and almost completely dephosphorylated the stilbestrol diphosphate (fig. 2). Within 2 hours 97 per cent of this substrate was hydrolyzed (pH 5.0). Bladder homogenates (pH 5.0) hydrolyzed 31 per cent of the substrate at 2 hours. The enzymic activity of both prostate and bladder (at pH 9.0) was low.
While the effects of estrogens on the prostatic epithelium are known, the exact mechanism of their action remains unexplained. After the injection of tritiated stilbestrol diphosphate the prostate gland does not take up appreciable quantities of the compound. The maximum prostatic uptake of the labeled compound occurred 30 minutes after injection (fig. 1). This finding agrees with those of Fergusson13 and
Ghaleb14 who studied the uptake of C14-labeled stilbestrol diphosphate in a small number of patients with cancer of the prostate. It is difficult to explain the high concentration of radioactive compound in the bladder. However, since the bladder contains both acid and alkaline phosphatase, some of the ester may be dephosphorylated (fig. 2). Serum also contains measurable quantities of the enzyme but the radioactivity in its ether-soluble fraction was extremely low (table 1). The total radioactivity in the bladder after the injection of stilbestrol diphosphate was high. However, the percentage of activity found in the ether-soluble fraction averaged 14.3 per cent over the first 3 hours (table 1). This amount of free stilbestrol in the bladder is not due to the free stilbestrol in the serum since this was low (table 1). Therefore, there was hydrolysis of stilbestrol diphosphate in the bladder. This is probably due to the measurable quantities of acid phosphatase found in the rabbit bladder (fig. 2). Because the quantity of free stilbestrol does not build up in the bladder with time, we concluded that the rate of dephosphorylation does not keep up with clearance from the bladder. Conversely, the prostate gland appears to efficiently hydrolyze the ester. At 15 minutes 69.5 per cent of the total radioactivity appeared in the ether-soluble fraction, a value that fell to 24.2 per cent by 24 hours. The rate of dephos-
13 Fergusson, J. D.: Tracer experiments showing the distribution and fate of injected phosphorylated estrogens in cancer of the prostate. Brit. J. Urol., 33: 442, 1961.
14 Ghaleb, H.: Studies on prostatic carcinoma. In: British Empire Cancer Campaign-ThirtySeventh Annual Report of the Grand Council, p. 145, London, 1959.
DISCUSSION
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PROMISLOW, CONNOLLY AND CLARKE
phorylation is probably higher in man than in the rabbit (as observed in this experiment) because acid phosphatase concentration (units/ gram) of human prostate is approximately 300 times that of the rabbit. 15 Although dephosphorylation is efficient, little free stilbestrol is deposited in the rabbit prostate. If stilbestrol was accumulating in the gland, total activity and the activity of the ethersoluble fraction should increase with time. Since this does not happen, the prostate gland, by definition, is not estrogen-responsive. 12 Although the prostate incorporates less tritiated stilbestrol diphosphate, it concentrates more free stilbestrol (as measured by the ether-soluble fraction) than any of the other tissues tested. Measurement of acid phosphatase activity in serum taken during the first 6 hours after injection of tritiated stilbestrol diphosphate indicates increased enzyme levels (table 2). This increase parallels the radioactive uptake of the material. By 12 hours the serum enzyme levels have fallen to less than those of the control animals and by 19 hours are zero. If the levels of acid phosphatase in serum are to change, the relative rates of production, release into the circulation, inhibition and destruction must change. The rapid increase in enzyme levels, we showed, may be due to binding or inactivation of a natural acid phosphatase inhibitor by the injected hormone. Steroid hormones are known to bind to serum protein and it is possible that the inhibitor is more strongly bound than the enzyme. 7 After hormone injection the enzyme activity in the prostate gland deviates from that seen in the control animals but the difference between the levels at 15 minutes and at 19 days is significant. It is possible that we did not give enough stilbestrol diphosphate to suppress enzyme levels in the normal rabbit prostate. The dose used (0.3 mg./7 pound animal) is equivalent to 8 mg. per 180-pound man. However, the acid phosphatase concentration in the rabbit gland is low. 15 Woodard noted that after estrogen therapy in men, serum enzyme levels declined and the low levels in the cancerous gland were 15 Gutman, A. B.: The development of the acid phosphatase test for pros ta tic carcinoma. Bull. N.Y. Acad. Med., 44: 63, 1968.
even less after estrogen therapy. 16 If this is valid, after the first injection of stilbestrol diphosphate the amount of enzyme available to convert transport stilbestrol to free stilbestrol would be lower when the next injection was given. Arcadi observed that glycoproteins in the basement membrane of the prostatic acini became insoluble after estrogen treatment. 17 He suggested that the impermeability of the ground substance chiefly determines the resistance the ground substance offers to the growth and spread of tumor cells. Thus the formation of tough glycoprotein in the basement membrane might keep acid phosphatase from escaping from the gland and this woukl explain the near normal levels of this enzyme in the gland when the serum levels approach zero. SUMMARY
Tritium-labeled diethylstilbestrol diphosphate was administered to 30 inbred, male rabbits which were then sacrificed at various times. Samples of serum, liver, bladder, prostate and fat were analyzed for radioactivity. The total radioactivity in the prostate was less than that of liver, bladder and serum at all times. No preferential accumulation of activity was observed in the prostate. The behavior of this tissue resembled that of non-responsive tissue. However, the gland appears to hydrolyze tritiated stilbestrol diphosphate efficiently because 69.5 per cent of the radioactivity appeared in the ether-soluble fraction at 15 minutes. This conclusion was supported by in vitro studies. A 1 per cent homogenate of prostate from control animals dephosphorylated 97 per cent of a (non-radioactive) stilbestrol diphosphate substrate (pH 5.0) in 2 hours. In the rabbit the level of acid phosphatase in the serum was elevated after stilbestrol diphosphate was injected, decreased to normal at 6 hours and zero at 19 days. The concentration of acid phosphatase in the prostate was constant at all times. 16 Woodard, H. Q. and Dean, A. L.: Significance of phosphatase findings in carcinoma of the prostate. J. Urol., 67: 158, 1947. 17 Arcadi, J. A.: The influence of hormones upon some constituents of connective tissue in prostatic cancer. Texas Reports Biol. Med., 13: 591, 1955.