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Increased estrogen-16α-hydroxylase activity in women with breast and endometrial cancer
J. sreroidBio&em. Vol. 20, No. 4B, pp. 10’77-1081, 1984 Printedin Great Britain.AU rights reserved
~22~731~84 $3.00 + 0.00 Copyright Q 1984 PergamonPress Lid
INCREASED ESTROGEN-I6a-HYDROXYLASE ACTIVITY IN WOVEN WITH BREAST AND ENDO~ETRIAL CANCER
JACK FISHMAN, JILL SCHNEIDER,
RICHARDJ. HERSHCOPFand H. LJZQNBRADLOW The Rockefeller University, 1230 York Avenue, New York, New York 10021, U.S.A.
Sudan-We
have measured the three principal oxidative transfo~ations of estradiol by means of a radiometric procedure in women with breast or endometrial cancer and in age matched controls. No difference between the 17/?-01 oxidation or 2-hydroxylation of the hormone was observed between the study groups. In contrast, 16u-hydroxylation was strikingly elevated in the women with breast and endometrial cancer relative to the age matched controls. Evidence is presented that this increased activity precedes the clinical evidence of the disease and that it represents a significant risk factor for these estrogen dependent tumors. This risk may be mediated by one of the products of lo-hydroxyiation, lo-hydroxyestrone, which exhibits unique biological properties.
INTRODUCrION A wealth of animal and human data have provided evidence that estrogens play a role in the initiation and/or maintenance of mammary tumors. Because of this relationship, extensive studies have been carried out comparing the metabolism and excretion of estrogens in patients with breast cancer and control groups [l-3]. Despite a great deal of effort no consensus on any consistent differences between patients and control populations has been reached [4,5]. In retrospect, the absence of significant differences in the estrogenic patterns in breast cancer patients is perhaps not surprising. The development of breast cancer is a slow process with the initiation of the disease apparently taking place many years before overt symptoms appear. In the so called “window” hypothesis of breast cancer initiation [6] there are specific periods of increased vulnerability to such initiation in the early post pubertal years and again in the perimenopausal period. For example, studies of survivors of the Hiroshima bomb show that those exposed to the radiation during their teens show the greatest incidence of breast cancer 20-30 years later. The protective effect of a first pregnancy prior to age 25 also indicates that hormonal changes in this early age period [7] influence the appearance of the disease in postmenopaus~ years. Similarly, oophorectomy is protective of later development of breast cancer only if carried out prior to 30 years of age [8]. The many studies of estrogen secretion and metabolism in postmenopausal breast cancer patients, therefore, may not provide information about the endocrine milieu at the time when the disease was initiated, and hence may not offer insights into the role of estrogen metabolism as a risk factor for breast cancer. The metabolism of estradiol, which is primarily oxidative, consists of an initial oxidation of the
17/?-hydroxy group to yield estrone [9]. This steroid is subsequently metabolized mainly through either of two alternate hydroxylative pathways; hydroxylation at the C-2 or the 16a position. These hydroxylations are of particular interest in that they constitute competing reactions whose products are themselves active compounds characterized by markedly different biological properties. The 16~ -hydroxyestrogens--es&i01 and 16~-hydroxyestronedemonstrate uterotropic activity comparable to that of the parent hormone, estradiol [lo, 1I]. On the other hand, the principal 2-hydroxyestrogens2-hydroxyestrone and 2-methoxyestrone-exhibit virtually no peripheral estrogenic effects but play a role in neur~nd~~ne regulatory mediated events [12]. Two principal methods can be employed to evaluate the metabolic transformation of estrogens in the human. The classical approach involves the intravenous administration of a radiolabeled estrogen and the analysis of the radiolabeled metabolites in urine and blood samples obtained after the injection. The procedure is compiex, requiring enzyme and acid hydrolysis and fractionation by gradient partition, or thin layer chromatography, followed by reverse isotope dilution and recrystallization to constant specific activity. There are significant manipulative losses but most importantly urinary excretion is variable and accounts for only 50-70% of the administered radioactivity with no information being provided about the missing elements. Studies of hormonal changes in cancer patients can also be affected by non-specific illness effects which can significantly alter enterohepatic circulation and urinary metabolic patterns. The alternative radiomet~c procedure which we have devised [I 31, involves the administration of a precursor, labelled with tritium at a metabolically reactive site, from which the tritium is transferred to body water upon oxidation or hydroxylation at that posi-
1077
IO78
JACK FBHMAN
tion. The extent of reaction is followed by measuring the tritium specific activity of blood and urine samples collected at intervals over 24-48 h. This technique has the advantage that the total extent of a particular reaction can be obtained without the need to isolate or characterize any metabolites and without concern for further transformations or excretory routes. Its disadvantage resides in that the method provides no information regarding conjugative or metabolic steps. Since 2and further 16a-hydroxylation are largely mutually exclusive, they can be ascertained individually with little concern for the existence of dual transformations in the same molecule. In order to obtain information about the hormonal state existing at the time of tumor initiation, one should examine a metabolic transformation which is conserved with aging and which, when measured at the time of disease diagnosis will mirror the status at the time when the disease was initiated. We have, therefore, first examined the pattern of oxidative transformation of estradiol in pre- and postmenopausal women to determine which of the oxidative transformations is most conserved with age. We then repeated these studies in a series of women with breast and endometrial cancer and compared their patterns with those of matched normal controls. Some of these results have been reported in a preliminary communication [13].
MATERIALS
AND METHODS
Subjects
Thirty-three patients with either primary or metastatic breast cancer (mean age 58.5 years; range 43-74 years) were studied. None were receiving hormonal treatment or chemotherapy at the time of the study. Two women had been treated by oophorectomy more than 2 years prior to the studies, but none had undergone adrenalectomy or other ablative therapy. Estrogen receptor status was positive in tumor tissue obtained from 27 of these patients. Nine of the 33 patients were perimenopausal, having had their last menstrual period 6-12 months previously. The remaining 24 patients were postmenopausal and had no menses for at least 2 years. Ten patients, a11postmenopausal, with primary endometrial cancer were included in the study. The individuals studied had no history of hver, kidney, or endocrine dysfunction, and none had used oral contraceptives within the previous 2 years. Fourteen of the subjects were receiving analgesics or mild antihypertensive medications, but these were discontinued several days prior to the estrogen metabolism studies. The results obtained in these individuals did not differ from those of other patients. None of the subjects had taken drugs known to alter steroid metabolism within a 6 month interval prior to the studies.
et ut.
Ten normal women (mean age 59.8 years: range 48-70 years) were studied as controls. These subjects were postmenopausal by at least Zyears. had previously had regular menses, were in good health with no history of liver, kidney, or endocrine dysfunction and were taking no medications. The racial and geographical backgrounds of the normal subjects and patients were similar and all participants were within 90-1357;) of ideal body weight according to the Metropolitan Life Insurance Company tables. The studies directed to assess the influence of age on the transformation utilized a different group of normal women of various ages who were also screened to exclude individuals with a history of (or evidence for) kidney or liver disease and who were free from any major drug intake for a period of at least 3 months and from oral contraceptives for at least 2 years. Obesity, amenorrhea and dysthyroidism were also criteria for exclusion from the study. Preparation qf [2-‘HJestrudiol, [I&-.‘H]estrudiol [1 7u3H]estradiol tracers
und
The [2-3H]-, [16~-~H]- and [ 17n-3H]estradiol tracers were prepared and the specificity of the labels at the desired positions was confnmed as described [I 31. For radiotracer, substrate the [ 16~-3H]estradiol homogeneity exceeded 96%, with 81% of the “H being situated in the 16a and 15% in the 16f3 orientation. Hydroxylation of estradiol at both the C-2 and 16 positions proceeds without an isotope effect [ 141,and a “National Institutes of Health” shift does not occur in the case of the former [14]. In the case of the [16cr-3H]estradiol tracer, a small contribution to the amount of -‘H,O formed may be anticipated from the release of 3H from the 168 position as a consequence of the formation of two minor estrogen metabolites--16~-hydroxyestrone and 16~,17~-triol. However, in view of the preeminence of the 16ff-hydroxylated D-ring metabolites of estradiol in humans relative to the 168 metabolites, the extent of 3H release from this tracer is likely to closely approximate the extent of 16a-hydroxylation. Procedure
Labeled estradiol tracers were dissolved in sterile propylene glycol and single dose aliquots were then stored in sealed ampoules. A measured weight of each tracer was administered as an intravenous bolus injection and serial blood samples were drawn as described [13]. For the [2-3H]estradiol and [ 16ct-3H]estradiol studies, blood samples (10 ml for the former study and 15 ml for the latter) were obtained before and 1, 2, 4, 8, 12, 24, 36 and 48 h after isotope administration. In this report the extent of 16aJH release in both populations was determined by using only those studies conducted for 48 h after the administration of [16a-3H]estradiol because it has now been established that this time interval is required to adequately measure the maximal extent of
1079
Increased estrogen-16a-hydroxylase activity Table 1. 2- and 16a-hydroxylation
of estradiol in normal women
y0 Dose hydroxylated, 2-Hydroxylation
Subjects Premenopausal women Postmenooausal women
Table 2. Maximal
mean f SEM 16a-Hydroxylation
38.9 f 1.2 (6) 32.6 + 1.3 (7)
percentage
oxidation
10.7 + 2.7 (7) 8.5 + 1.4(9)
of [2-‘Hlestradiol,
[16a-‘H]estradiol
and
[17a-‘H]estradiol in viva * Estradiol metabolism,t Subjects
2-Hydroxylation
Breast cancer patients Normal women P
32.1 + 2.7 (24) 31.1 i4.0(10) NS
% oxidation
16a -Hydroxylation
17-Oxidation 73.0 + 4.2 (13) 76.9 f 5.3 (13) NS
14.9 * 1.5 (15) 9.3 + 0.8 (10) 0.01
NS, not significant. *In breast cancer patients and normal controls. tMean f SEM; the number of subjects studied is shown in parentheses
this reaction. Blood (10 ml) was obtained before and 0.33, 0.67, 1, 2, 4, 8, 12 and 24 h after administration of [ 17~ -3H]estradiol. When multiple tracer studies were performed [16a-3H]estradiol, [Z3H]estradiol and [ 17~-3H]estradiol were administered sequentially over a 5 day interval. The three tracer studies were repeated in seven normal subjects to establish the reproducibility of the method. Analysis of the data by the paired Student t-test revealed no significant differences between duplicate studies. Data analysis
Blood specimens were stored at -20°C for up to 96 h, lyophilized and the 3Hz0 thereby obtained was assayed in a Packard 2650 scintillation counter for a time sufficient for a counting accuracy of f5%. Total body water was measured in each individual by a 2H20 dilution procedure: 75 ml of 99.5% 2H,0 (Merck) was administered orally to each subject and a blood sample was obtained 3 h later, at which time equilibration with total body water is achieved [15]. The atom% excess of 2H in the body water was determined by mass spectrometry and these results were used to calculate total body water. The percentage of oxidation at each time point after the administration of [2-3H]-, [16c(-)H]- or [ 17cr-3H]estradiol was then determined as the product of the plasma-water specific activity (dpm/l) and total body water volume (1) divided by the dose of 3H-labeled steroid (dpm) administered, the fraction multiplied by 100.
postmenopausal women but the change in the former reaction was so small as to suggest that this enzymatic activity is largely conserved with age. The results of the cancer studies using [2-‘Hlestradiol, [ 16a-3H]estradiol and [ 17a-3H]estradiol are shown in Table 2. The extent of oxidative metabolism at positions 17c(, C-2 and 16a in the normal postmenopausal females were 76.9 f 5.3, 3 1.1 f 4.0 and 9.3 + 0.8x, respectively. Corresponding values obtained for the patients with breast cancer were 73.0 + 4.2, 32.7 f 2.7 and 14.9 + 1.5x, respectively. There was a statistically significant difference (P < 0.01) between these two populations for the biotransformation of estradiol at the C-16ahydroxylation. Furthermore, only one of the breast cancer patients exhibited 16-hydroxylation lower than 11% and only one of the normal controls was higher than 11%. The results obtained in the endometrial cancer patients are provided in Table 3. The average value for 16a-hydroxylation was 15% which compares to the breast cancer patients and is significantly greater than the normal controls. Again only one of the endometrial cancer patients exhibited a value lower than 11%. In contrast no differences were noted in the 2-hydroxylation in these patients compared to normal controls. Table 3. Results of radiometric studies on C-2 and C-16 hydroxylation in patients with endometrial cancer 0/OOxidation
Statistical analysis
The results are expressed as mean f SEM values. statistical analysis was performed by using Student’s two tailed t-test; P values of x0.05 were considered to be significant. RESULTS AND DISCUSSION
The degree of 2- and 16~ -hydroxylation in pre- and postmenopausal women is shown in Table 1. There was a decrease in 16a- and 2-hydroxylation in the
Postmenopausal
1 2 3 4 5 6 7 8 9 10 AVG
c-2
C-16
30.6 21.9 34.0 10.6 43.3 41.3 28.6 34.3 43.2 30.9 31.9
12.1 13.5 28.6 10.6 12.6 15.0 12.5 15.0
1080
JACK FISHMAN rt ul.
The findings of increased 16r-hydroxylation in breast cancer patients, as determined by radiometry, conform to our previous observations obtained by the classical procedures of an increased metabolism toward 16a-hydroxylated metabolites in both men [ 161and women with breast cancer [I 77. It should be noted that when the urinary estriol content measured in patients with breast cancer was compared with the level in normals it was variously reported to be elevated or reduced [IS, 191. These results can be rationalized since increased 16~-hydroxyiation need not be reflected in increased excretion of estriol, but in an excess of the initial product of the reaction, 16cr-hydroxyestrone (160HE,). Indeed in SLE the one other disease in which 16~-hydroxylat~on is increased greater formation of 16a-hydroxyestrone rather is observed 1201. Thus than estriol I6cc-hydroxylation needs to be considered both in terms of its initial product, 16or-OHE,, which is a potent and possibly unique estrogen agonist [21] and its further metabolite, estriol, which appears to behave as a conventional estrogen agonist under conditions. presence of physiological The 16a-hydroxyestrone, apart from conjugation, is then controlled by two reactions: its formation via 16a-hydroxylation and its reduction to estriol. In view of the biologic properties of the ketol and its potential relation to oncogenesis, both of these enzymatic transformations assume significant importance. That an increased formation of 16c(-OHE, is biologically significant is indicated by an examination of its properties. It appears to be fully equivalent to estradiol as an estrogen agonist in several test systems and in addition has full biological availability in the human because it does not bind to SHBG, the principal estrogen binding protein in plasma. Moreover, it has the unique property of forming covalent biological with amino groups on bonds macromolecules [2 I]. Because both the breast and the endometrium are estrogen target sites it is not unreasonable to expect that estrogen induced tumor initiation may have similar mechanisms in both tissues. The distinctive difference between the sites resides in the temporal nature of such initiation since any transformed cells in the endometrium would be periodically discarded. An essential question that needs to be addressed is whether the elevated enzymatic activity in the cancer patients is the result of the disease or preceded it. Some suggestion that the latter is the case can be supplied by the results of a study in which the endogenous urinary and plasma hormone concentrations in a population of women who were at risk for familial breast cancer, because of one or more first or second degree relatives with the disease, were measured and compared with a closely matched low risk control group [22]. No differences were detected in the wide variety of plasma and urinary hormones and metabolites measured daily over an entire menstrual cycle. The exception was a highly significant
decrease in the excretion of estradiol and estrone glucuronides in the high risk populations and an increase in their urinary estrone sulfate excretion [23]. These differences were concentrated in the periovulatory period when estrogen secretion is acutely increased suggesting a possible effect of loading on the capacity of the metabolic system. The former and the present studies, the first prospective and the second retrospective, employed totally different procedures and measured quite different end points. Nevertheless, the results obtained in the two studies may reflect the same underlying phenomenon. In the high risk group, the conjugative pattern of estrogens was apparently shifted from glucuronidation toward sulfation relative to the low risk group. If, as is the case in the guinea-pig[24], the sulfates are better substrates for 16a-hydroxyestrone formation relative to the free estrogens, then the increased sulfation in the high risk subjects would be consistent with the increased 16c(-hydroxylation observed in the cancer patients. The high risk group showed normal urinary estriol excretion but the excessive sulfation could be reflected solely in increased lbcr-OHE, formation, since presence of the sulfate group increases 16cr-hydroxylation but retards subsequent reduction to estriol. Similarly, the increased 16cc-hydroxylation deduced for the cancer patients by the radiometric procedure could have, as its principal consequence, increased 16cc-hydroxyestrone formation. If the case is borne out that the increased 16a-hydroxylation precedes breast and endometrial cancer and constitutes a risk for these diseases, it would be possible to identify at risk individuals. More importantly it would provide an important insight into the mechanism of estrogen induced oncogenesis. Acknowledgements-This
work was supported by grant CA
22795 from the National Cancer Institute. We wish to thank Dr John Lewis, Dr Virginia Pierce and Dr David Kinne for their essential and valuable patient studies. REFERENCES 1. Zumoff B., Fishman J., Bradlow H. L. and Hellman L.:
2.
3.
4. 5.
6. 7.
Hormone profiles in hormone dependent cancers. Guncer Res. 35 (1975) 3365-3373. Cole P., Cramer D., Yen S., Paffenbarger R., MacMahon B. and Brown J.: Estrogen profiles of premenopausal women with breast cancer. Cancer Res. 38 (1978) 745-748. Jones M. K., Ramsey I. D., Collins W. P. and Dyer G. I.: The relationship of plasma prolactin to 17a-ostradiol in women with tumors of the-breast. Eur. J. Cancer 13 (1977) 1109-I II2 Miller A. B.: A n overview of ho~on~sso~iated cancers. Cancer Res. 38 (1978) 3985-3990. Lemon H. M., Wotiz H. H., Parsons L. and Mozdan P. J.: Reduced estriol excretion in patients with breast cancer prior to endocrine therapy. JAMA 1% (1966) 1128-1136. Korenman S. G.: Oestrogen window hypothesis of the aetiology of breast cancer. Lancet i (1980) 700-701. MacMahon B., Cole P. and Brown J.:’ Etiology of human breast cancer: a review. J. natn Cancer inst. 50 (1973) 21-42.
Increased estrogen-16a -hydroxylase activity
8. Kelsey J. L.: A review of the ~idemiolo~
of human breast cancer. Epics. Rev. 1 (1979) 74-109. 9. Fishman J., Bradlow H. L. and Gallagher T. F. Oxidative metabohsm of estradiol. J. biol. Chem. 235 (1960) 3104-3107. 10. Martucci C. and Fishman J.: Direction of e&radio1 metabolism as a control of its hormonal actionuterotropic activity of estradiol metabohtes. Endocrinology 101 (1977) 1709-1715. 11. Clark J. H., Paszko Z. and Peck E. J.: Nuclear binding and retention of the receptor estrogen complex: relation to the agonistic and antagonistic properties of estriol. Endocrinology 100 (1977) 9 l-96. 12. Fishman J. and Martucci C.: Biological properties of 16u-hydroxyestrone: implications m estrogen physiology and pathophysiolo~y. J. c/in. Endocr. Metab. 51 (1980) 611-615. 13. Fishman J., Bradlow H. L., Schneider J., Anderson K. E. and Kappas A.: Radiometric analysis of oxidation in man: sex differences in estradiol metabolism. Proc. nafn. Acad. Sci, U.S.A. 77 (1980) 49.57-4960.
14. Fishman J., Guzik H.‘and Hellman L.: Aromatic ring hydroxylation of estradiol in man. Biochemistry 9 (1970) 1593-1598.
15. Schloerb P. R., Friis-Hansen B. J., Edelman I. S., Solomon A. K. and Moore F. D.: The measurement of total body water in the human subject by deuterium oxide dilution. J. c[in. inuest. 29 (1950) 1296-1310. 16. Hellman L.. Zumoff B., Fishmau J. and Gallagher T. F.: Peripheral metabolism of [5Njestradiol and the excretion of endogenous es&one and estriol glucosiduronate in women with breast cancer. J. clin. Endow. ~etab. 33 (1971) 138-144. 17. Gallagher T. F., Fishman J., Zumoff B., Cassouto J.
and Hellman L.: Studies of production and metabolism
of estrogens I~uencing
1081
in humans.
Host
In
E~dogenoas
Factors
Tumor 3aimce
(Edited by R. W. Wissier, T. L. Dao and S. Wood). University of Chicago Press, Illinois (1967) pp. 127-135. 18. ZumofT B., F&man J., Bradlow H. L. and Hellmau L.: Hormone profiles in hormone-dependent cancers. Cuncer Res. 35 (1975) 3365-3370. 19. Marmoston J., Crowley L. G., Myers S. M., Stern E. and Hopkins C. E.: II. Urinary excretion of estrone, estradiol and estriol by patients with breast cancer and benign breast disease. Am. J. Obstet. Gynec. 92 (1963) AM-A67 .__ .-..
20. Labita R. G., Bradlow H. L., Kunkel H. G. and Fishman J.: Increased 16a-hydroxylation of estradiol in systemic lupus erythematosus. J. clin. Endocr. Metab. 53 (1981) 174-178.
21. Bucala R., Fisman J. and Cerami A.: Formation of cortisol between and adducts covalent 16u-hydroxylation and protein-possible role in the pathogenesis of cortisol toxicity and systemic lupus erythematosus. 22. Fishman J., Fukushima D. K., O’Connor J. Lynch H. T.: Low urinary estrogen glucuronides in women at risk for familial breast cancer. Science 204 (1979) 1089-1091. 33 Fishman J., Bradlow H. L., Fukushima D. K., -“’ O’Connor J., Rosenfeld R. S., Graepel 0, Elston R. and Lynch H. T.: Abnormal estrogen conjugation in women at risk for familial breast cancer at the periovulatory stage of the menstrual cycle. Cancer Res. 43 (1983) 1884-1890. 24. Hobkirk R., Nilsen M. and Jennings B.: Evidence for 16a-hydroxylation of estrone 3-sulfate by guinea-pig liver shces. Can. J. Bioehem. 53 (1975) 1133-1134.
~22~731~84 $3.00 + 0.00 Copyright Q 1984 PergamonPress Lid
INCREASED ESTROGEN-I6a-HYDROXYLASE ACTIVITY IN WOVEN WITH BREAST AND ENDO~ETRIAL CANCER
JACK FISHMAN, JILL SCHNEIDER,
RICHARDJ. HERSHCOPFand H. LJZQNBRADLOW The Rockefeller University, 1230 York Avenue, New York, New York 10021, U.S.A.
Sudan-We
have measured the three principal oxidative transfo~ations of estradiol by means of a radiometric procedure in women with breast or endometrial cancer and in age matched controls. No difference between the 17/?-01 oxidation or 2-hydroxylation of the hormone was observed between the study groups. In contrast, 16u-hydroxylation was strikingly elevated in the women with breast and endometrial cancer relative to the age matched controls. Evidence is presented that this increased activity precedes the clinical evidence of the disease and that it represents a significant risk factor for these estrogen dependent tumors. This risk may be mediated by one of the products of lo-hydroxyiation, lo-hydroxyestrone, which exhibits unique biological properties.
INTRODUCrION A wealth of animal and human data have provided evidence that estrogens play a role in the initiation and/or maintenance of mammary tumors. Because of this relationship, extensive studies have been carried out comparing the metabolism and excretion of estrogens in patients with breast cancer and control groups [l-3]. Despite a great deal of effort no consensus on any consistent differences between patients and control populations has been reached [4,5]. In retrospect, the absence of significant differences in the estrogenic patterns in breast cancer patients is perhaps not surprising. The development of breast cancer is a slow process with the initiation of the disease apparently taking place many years before overt symptoms appear. In the so called “window” hypothesis of breast cancer initiation [6] there are specific periods of increased vulnerability to such initiation in the early post pubertal years and again in the perimenopausal period. For example, studies of survivors of the Hiroshima bomb show that those exposed to the radiation during their teens show the greatest incidence of breast cancer 20-30 years later. The protective effect of a first pregnancy prior to age 25 also indicates that hormonal changes in this early age period [7] influence the appearance of the disease in postmenopaus~ years. Similarly, oophorectomy is protective of later development of breast cancer only if carried out prior to 30 years of age [8]. The many studies of estrogen secretion and metabolism in postmenopausal breast cancer patients, therefore, may not provide information about the endocrine milieu at the time when the disease was initiated, and hence may not offer insights into the role of estrogen metabolism as a risk factor for breast cancer. The metabolism of estradiol, which is primarily oxidative, consists of an initial oxidation of the
17/?-hydroxy group to yield estrone [9]. This steroid is subsequently metabolized mainly through either of two alternate hydroxylative pathways; hydroxylation at the C-2 or the 16a position. These hydroxylations are of particular interest in that they constitute competing reactions whose products are themselves active compounds characterized by markedly different biological properties. The 16~ -hydroxyestrogens--es&i01 and 16~-hydroxyestronedemonstrate uterotropic activity comparable to that of the parent hormone, estradiol [lo, 1I]. On the other hand, the principal 2-hydroxyestrogens2-hydroxyestrone and 2-methoxyestrone-exhibit virtually no peripheral estrogenic effects but play a role in neur~nd~~ne regulatory mediated events [12]. Two principal methods can be employed to evaluate the metabolic transformation of estrogens in the human. The classical approach involves the intravenous administration of a radiolabeled estrogen and the analysis of the radiolabeled metabolites in urine and blood samples obtained after the injection. The procedure is compiex, requiring enzyme and acid hydrolysis and fractionation by gradient partition, or thin layer chromatography, followed by reverse isotope dilution and recrystallization to constant specific activity. There are significant manipulative losses but most importantly urinary excretion is variable and accounts for only 50-70% of the administered radioactivity with no information being provided about the missing elements. Studies of hormonal changes in cancer patients can also be affected by non-specific illness effects which can significantly alter enterohepatic circulation and urinary metabolic patterns. The alternative radiomet~c procedure which we have devised [I 31, involves the administration of a precursor, labelled with tritium at a metabolically reactive site, from which the tritium is transferred to body water upon oxidation or hydroxylation at that posi-
1077
IO78
JACK FBHMAN
tion. The extent of reaction is followed by measuring the tritium specific activity of blood and urine samples collected at intervals over 24-48 h. This technique has the advantage that the total extent of a particular reaction can be obtained without the need to isolate or characterize any metabolites and without concern for further transformations or excretory routes. Its disadvantage resides in that the method provides no information regarding conjugative or metabolic steps. Since 2and further 16a-hydroxylation are largely mutually exclusive, they can be ascertained individually with little concern for the existence of dual transformations in the same molecule. In order to obtain information about the hormonal state existing at the time of tumor initiation, one should examine a metabolic transformation which is conserved with aging and which, when measured at the time of disease diagnosis will mirror the status at the time when the disease was initiated. We have, therefore, first examined the pattern of oxidative transformation of estradiol in pre- and postmenopausal women to determine which of the oxidative transformations is most conserved with age. We then repeated these studies in a series of women with breast and endometrial cancer and compared their patterns with those of matched normal controls. Some of these results have been reported in a preliminary communication [13].
MATERIALS
AND METHODS
Subjects
Thirty-three patients with either primary or metastatic breast cancer (mean age 58.5 years; range 43-74 years) were studied. None were receiving hormonal treatment or chemotherapy at the time of the study. Two women had been treated by oophorectomy more than 2 years prior to the studies, but none had undergone adrenalectomy or other ablative therapy. Estrogen receptor status was positive in tumor tissue obtained from 27 of these patients. Nine of the 33 patients were perimenopausal, having had their last menstrual period 6-12 months previously. The remaining 24 patients were postmenopausal and had no menses for at least 2 years. Ten patients, a11postmenopausal, with primary endometrial cancer were included in the study. The individuals studied had no history of hver, kidney, or endocrine dysfunction, and none had used oral contraceptives within the previous 2 years. Fourteen of the subjects were receiving analgesics or mild antihypertensive medications, but these were discontinued several days prior to the estrogen metabolism studies. The results obtained in these individuals did not differ from those of other patients. None of the subjects had taken drugs known to alter steroid metabolism within a 6 month interval prior to the studies.
et ut.
Ten normal women (mean age 59.8 years: range 48-70 years) were studied as controls. These subjects were postmenopausal by at least Zyears. had previously had regular menses, were in good health with no history of liver, kidney, or endocrine dysfunction and were taking no medications. The racial and geographical backgrounds of the normal subjects and patients were similar and all participants were within 90-1357;) of ideal body weight according to the Metropolitan Life Insurance Company tables. The studies directed to assess the influence of age on the transformation utilized a different group of normal women of various ages who were also screened to exclude individuals with a history of (or evidence for) kidney or liver disease and who were free from any major drug intake for a period of at least 3 months and from oral contraceptives for at least 2 years. Obesity, amenorrhea and dysthyroidism were also criteria for exclusion from the study. Preparation qf [2-‘HJestrudiol, [I&-.‘H]estrudiol [1 7u3H]estradiol tracers
und
The [2-3H]-, [16~-~H]- and [ 17n-3H]estradiol tracers were prepared and the specificity of the labels at the desired positions was confnmed as described [I 31. For radiotracer, substrate the [ 16~-3H]estradiol homogeneity exceeded 96%, with 81% of the “H being situated in the 16a and 15% in the 16f3 orientation. Hydroxylation of estradiol at both the C-2 and 16 positions proceeds without an isotope effect [ 141,and a “National Institutes of Health” shift does not occur in the case of the former [14]. In the case of the [16cr-3H]estradiol tracer, a small contribution to the amount of -‘H,O formed may be anticipated from the release of 3H from the 168 position as a consequence of the formation of two minor estrogen metabolites--16~-hydroxyestrone and 16~,17~-triol. However, in view of the preeminence of the 16ff-hydroxylated D-ring metabolites of estradiol in humans relative to the 168 metabolites, the extent of 3H release from this tracer is likely to closely approximate the extent of 16a-hydroxylation. Procedure
Labeled estradiol tracers were dissolved in sterile propylene glycol and single dose aliquots were then stored in sealed ampoules. A measured weight of each tracer was administered as an intravenous bolus injection and serial blood samples were drawn as described [13]. For the [2-3H]estradiol and [ 16ct-3H]estradiol studies, blood samples (10 ml for the former study and 15 ml for the latter) were obtained before and 1, 2, 4, 8, 12, 24, 36 and 48 h after isotope administration. In this report the extent of 16aJH release in both populations was determined by using only those studies conducted for 48 h after the administration of [16a-3H]estradiol because it has now been established that this time interval is required to adequately measure the maximal extent of
1079
Increased estrogen-16a-hydroxylase activity Table 1. 2- and 16a-hydroxylation
of estradiol in normal women
y0 Dose hydroxylated, 2-Hydroxylation
Subjects Premenopausal women Postmenooausal women
Table 2. Maximal
mean f SEM 16a-Hydroxylation
38.9 f 1.2 (6) 32.6 + 1.3 (7)
percentage
oxidation
10.7 + 2.7 (7) 8.5 + 1.4(9)
of [2-‘Hlestradiol,
[16a-‘H]estradiol
and
[17a-‘H]estradiol in viva * Estradiol metabolism,t Subjects
2-Hydroxylation
Breast cancer patients Normal women P
32.1 + 2.7 (24) 31.1 i4.0(10) NS
% oxidation
16a -Hydroxylation
17-Oxidation 73.0 + 4.2 (13) 76.9 f 5.3 (13) NS
14.9 * 1.5 (15) 9.3 + 0.8 (10) 0.01
NS, not significant. *In breast cancer patients and normal controls. tMean f SEM; the number of subjects studied is shown in parentheses
this reaction. Blood (10 ml) was obtained before and 0.33, 0.67, 1, 2, 4, 8, 12 and 24 h after administration of [ 17~ -3H]estradiol. When multiple tracer studies were performed [16a-3H]estradiol, [Z3H]estradiol and [ 17~-3H]estradiol were administered sequentially over a 5 day interval. The three tracer studies were repeated in seven normal subjects to establish the reproducibility of the method. Analysis of the data by the paired Student t-test revealed no significant differences between duplicate studies. Data analysis
Blood specimens were stored at -20°C for up to 96 h, lyophilized and the 3Hz0 thereby obtained was assayed in a Packard 2650 scintillation counter for a time sufficient for a counting accuracy of f5%. Total body water was measured in each individual by a 2H20 dilution procedure: 75 ml of 99.5% 2H,0 (Merck) was administered orally to each subject and a blood sample was obtained 3 h later, at which time equilibration with total body water is achieved [15]. The atom% excess of 2H in the body water was determined by mass spectrometry and these results were used to calculate total body water. The percentage of oxidation at each time point after the administration of [2-3H]-, [16c(-)H]- or [ 17cr-3H]estradiol was then determined as the product of the plasma-water specific activity (dpm/l) and total body water volume (1) divided by the dose of 3H-labeled steroid (dpm) administered, the fraction multiplied by 100.
postmenopausal women but the change in the former reaction was so small as to suggest that this enzymatic activity is largely conserved with age. The results of the cancer studies using [2-‘Hlestradiol, [ 16a-3H]estradiol and [ 17a-3H]estradiol are shown in Table 2. The extent of oxidative metabolism at positions 17c(, C-2 and 16a in the normal postmenopausal females were 76.9 f 5.3, 3 1.1 f 4.0 and 9.3 + 0.8x, respectively. Corresponding values obtained for the patients with breast cancer were 73.0 + 4.2, 32.7 f 2.7 and 14.9 + 1.5x, respectively. There was a statistically significant difference (P < 0.01) between these two populations for the biotransformation of estradiol at the C-16ahydroxylation. Furthermore, only one of the breast cancer patients exhibited 16-hydroxylation lower than 11% and only one of the normal controls was higher than 11%. The results obtained in the endometrial cancer patients are provided in Table 3. The average value for 16a-hydroxylation was 15% which compares to the breast cancer patients and is significantly greater than the normal controls. Again only one of the endometrial cancer patients exhibited a value lower than 11%. In contrast no differences were noted in the 2-hydroxylation in these patients compared to normal controls. Table 3. Results of radiometric studies on C-2 and C-16 hydroxylation in patients with endometrial cancer 0/OOxidation
Statistical analysis
The results are expressed as mean f SEM values. statistical analysis was performed by using Student’s two tailed t-test; P values of x0.05 were considered to be significant. RESULTS AND DISCUSSION
The degree of 2- and 16~ -hydroxylation in pre- and postmenopausal women is shown in Table 1. There was a decrease in 16a- and 2-hydroxylation in the
Postmenopausal
1 2 3 4 5 6 7 8 9 10 AVG
c-2
C-16
30.6 21.9 34.0 10.6 43.3 41.3 28.6 34.3 43.2 30.9 31.9
12.1 13.5 28.6 10.6 12.6 15.0 12.5 15.0
1080
JACK FISHMAN rt ul.
The findings of increased 16r-hydroxylation in breast cancer patients, as determined by radiometry, conform to our previous observations obtained by the classical procedures of an increased metabolism toward 16a-hydroxylated metabolites in both men [ 161and women with breast cancer [I 77. It should be noted that when the urinary estriol content measured in patients with breast cancer was compared with the level in normals it was variously reported to be elevated or reduced [IS, 191. These results can be rationalized since increased 16~-hydroxyiation need not be reflected in increased excretion of estriol, but in an excess of the initial product of the reaction, 16cr-hydroxyestrone (160HE,). Indeed in SLE the one other disease in which 16~-hydroxylat~on is increased greater formation of 16a-hydroxyestrone rather is observed 1201. Thus than estriol I6cc-hydroxylation needs to be considered both in terms of its initial product, 16or-OHE,, which is a potent and possibly unique estrogen agonist [21] and its further metabolite, estriol, which appears to behave as a conventional estrogen agonist under conditions. presence of physiological The 16a-hydroxyestrone, apart from conjugation, is then controlled by two reactions: its formation via 16a-hydroxylation and its reduction to estriol. In view of the biologic properties of the ketol and its potential relation to oncogenesis, both of these enzymatic transformations assume significant importance. That an increased formation of 16c(-OHE, is biologically significant is indicated by an examination of its properties. It appears to be fully equivalent to estradiol as an estrogen agonist in several test systems and in addition has full biological availability in the human because it does not bind to SHBG, the principal estrogen binding protein in plasma. Moreover, it has the unique property of forming covalent biological with amino groups on bonds macromolecules [2 I]. Because both the breast and the endometrium are estrogen target sites it is not unreasonable to expect that estrogen induced tumor initiation may have similar mechanisms in both tissues. The distinctive difference between the sites resides in the temporal nature of such initiation since any transformed cells in the endometrium would be periodically discarded. An essential question that needs to be addressed is whether the elevated enzymatic activity in the cancer patients is the result of the disease or preceded it. Some suggestion that the latter is the case can be supplied by the results of a study in which the endogenous urinary and plasma hormone concentrations in a population of women who were at risk for familial breast cancer, because of one or more first or second degree relatives with the disease, were measured and compared with a closely matched low risk control group [22]. No differences were detected in the wide variety of plasma and urinary hormones and metabolites measured daily over an entire menstrual cycle. The exception was a highly significant
decrease in the excretion of estradiol and estrone glucuronides in the high risk populations and an increase in their urinary estrone sulfate excretion [23]. These differences were concentrated in the periovulatory period when estrogen secretion is acutely increased suggesting a possible effect of loading on the capacity of the metabolic system. The former and the present studies, the first prospective and the second retrospective, employed totally different procedures and measured quite different end points. Nevertheless, the results obtained in the two studies may reflect the same underlying phenomenon. In the high risk group, the conjugative pattern of estrogens was apparently shifted from glucuronidation toward sulfation relative to the low risk group. If, as is the case in the guinea-pig[24], the sulfates are better substrates for 16a-hydroxyestrone formation relative to the free estrogens, then the increased sulfation in the high risk subjects would be consistent with the increased 16c(-hydroxylation observed in the cancer patients. The high risk group showed normal urinary estriol excretion but the excessive sulfation could be reflected solely in increased lbcr-OHE, formation, since presence of the sulfate group increases 16cr-hydroxylation but retards subsequent reduction to estriol. Similarly, the increased 16cc-hydroxylation deduced for the cancer patients by the radiometric procedure could have, as its principal consequence, increased 16cc-hydroxyestrone formation. If the case is borne out that the increased 16a-hydroxylation precedes breast and endometrial cancer and constitutes a risk for these diseases, it would be possible to identify at risk individuals. More importantly it would provide an important insight into the mechanism of estrogen induced oncogenesis. Acknowledgements-This
work was supported by grant CA
22795 from the National Cancer Institute. We wish to thank Dr John Lewis, Dr Virginia Pierce and Dr David Kinne for their essential and valuable patient studies. REFERENCES 1. Zumoff B., Fishman J., Bradlow H. L. and Hellman L.:
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