Unconjugated and Glucuronide Steroid Levels in Human Breast Cyst Fluid A. BELANGER AND F. LABRIE MRC Group in Molecular Endocrinology Lava1 University Medical Center Ste-Foy, Quibec, GI V 4G2 Canada
A. ANGEL1 Patologica Medica Universita di Torino Ospedale San Luigi Gonzaga Regime Gonzaga, 10 10043 Orbassano Turin, Italy
INTRODUCTION Since gross cystic disease of the breast is associated with a two- to four-fold increased risk of the development of breast cancer, the concentration of a wide variety of hormones, enzymes, tumor-associated antigens, and ions in breast cyst fluid has been measured in order to better understand the relationship between the hormonal environment and breast cancer.'" While the concentration of some steroids, namely progesterone (Prog ), androsterone (ADT), and dehydroepiandrosterone (DHEA), and its sulfate, was reported to be high in breast cyst fluid (BCF), this research area has attracted only a limited interest despite the well-known potential influence of steroids on tumor growth: In addition, the presence in BCF of steroid-bindingproteins (namely, progesterone-binding protein, albumin, corticosteroid-binding globulin [CBG], and testosterone-estradiol-bindingglobulin [TeBG], which all favor an accumulation of steroids in this fluid), contributes to the interest in this field.' The present report deals with the concentrations of C-21, C-19 and C-18 steroids as well as steroid glucuronides in BCF. The steroid glucuronides include androstane3a,17P-diol glucuronide (3A-DIOL-G), androsterone glucuronide (ADT-G), estradiol glucuronide (E2-G), and estrone glucuronide (El-G). Several recent studies on steroid g1ucuronides7-'' have associated the plasma levels of 3A-DIOL-G and ADTG as markers of the transformation of testosterone (TESTO) into dihydrotestosterone (DHT) as reflecting the total pool of androgens. In addition to its presence in the liver, the enzyme glucuronyl transferase has been shown to be present in human peripheral tissues, such as the skin and the prostate. Such a wide distribution of 93
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glucuronyl transferase could explain the large amount of Sa-reduced steroid glucuronides in plasma." Since the local accumulation of steroid glucuronides may reflect androgen conversion in breast tissue, and since androgens have been suggested to play a role in normal and cancer cell f ~ n c t i o n , ' ~it. 'was ~ of interest to determine the levels of these 5a-reduced steroid conjugates in BCF. Moreover, we have examined the levels of E2-G and El-G in BCF since these metabolites could also aid in estimation of estradiol transformation in this tissue. The concentration of the gross breast cystic disease protein 15 (GCDFP-15), one of the major protein constituents of human breast cyst fluid,15was also measured and its value was correlated with concentrations of unconjugated steroids as well as steroid conjugates. The interest of such study is strengthened by our recent finding of a potent inhibitory effect of estrogens on GCDFP15 mRNA levels and release in the human breast cancer cell line ZR-75-l.l6
SUBJECTS, MATERIALS AND METHOD The subjects included 75 women who had needle aspiration of breast cysts. After aspiration, the gross cystic disease fluid was stored at -20°C until assayed. The samples were analyzed by procedures originally devised for the analysis of steroid in p l a ~ m a . ~ *Intra-assay *~~'' variation for each steroid was less than 16% and to avoid inter-assay variation, all breast cyst samples were analyzed simultaneously. Assay of the unconjugated steroids employed extraction and column chromatography, while that for the steroid glucuronides involved an extraction followed by treatment with P-glucuronidase and column chr~matography.~ GCDFP- 15 was measured by radioimmunoassay using a modification of the method previously described by Haagensen et al. "." All RIA results are shown as the means 2 SEM of triplicate determinations on individual samples. Radioimmunoassays were calculated according to the method of Rodbard and Lewald.''
RESULTS FIGURE 1 shows the concentrations in cyst fluid of the C-21 and C-19 steroids, namely, Prog, pregnenolone (Preg), 17-hydroxy-progesterone ( 17-0H-Prog), 17-hydroxy-pregnenolone ( 17-0H-Preg), DHEA, androst-5-ene-3P,17P-diol ( 5-ene-Diol), androstenedione (4-ene-Dione) and TESTO. The concentrations of Prog and Preg exceed those of their respective 17-hydroxylated derivatives by almost 100-fold. Among the C- 19 steroids, 5-ene-Diol, 4-ene-Dione, and TESTO were found at approximately 5 nmol/L in BCF, whereas DHEA levels reached 31 2 4 nmol/L. FIGURE 2 shows that the ADT levels at 6.2 2 0.9 n m o l h were higher than those of DHT and 3A-DIOL. The estrogens estrone ( E l ) and estradiol (E2), on the other hand, were measured at concentrations of 0.67 2 0.03 and 0.12 2 0.01 nmolh, respectively. At concentrations of 125 2 24 nmolh, ADT-G is the major steroid glucuronide found in BCF and exceeds by more than 10- and 100-fold that of 3ADIOL-G and El-G or E2-G, respectively. The correlation between the concentrations of the various steroids in BCF was then examined. The statistical relationship between cyst fluid DHEA and 4-ene-Dione,
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TESTO, and 4-ene-Dione, TESTO and DHT, 3A-DIOL and DHT, 3A-DIOL and 3A-DIOL-G as well as 3A-DIOL-G and ADT-G levels can be expressed by the linear 3 and 4. A significant positive correlation was also regressions shown in FIGURES observed between 4-ene-Dione and El, 4-ene-Dione and El-G as well as El-G and E2-G (FIG. 5). The concentration of GCDFP-15 in BCF has been measured at 2745 2 234 p g l ml. While no correlation was observed between GCDFP-15 and C-21 or C-19 steroids, a significant negative relationship is seen between El-G and GCDFP-15 levels (FIG. 6). However, the correlations between the unconjugated estrogens and GCDFP- 15 as well as between E2-G and GCDFP-15, do not reach statistical significance.
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C19 SIemMs In Cyst Rui
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_I
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17-0H-P
A. ANGEL1 Patologica Medica Universita di Torino Ospedale San Luigi Gonzaga Regime Gonzaga, 10 10043 Orbassano Turin, Italy
INTRODUCTION Since gross cystic disease of the breast is associated with a two- to four-fold increased risk of the development of breast cancer, the concentration of a wide variety of hormones, enzymes, tumor-associated antigens, and ions in breast cyst fluid has been measured in order to better understand the relationship between the hormonal environment and breast cancer.'" While the concentration of some steroids, namely progesterone (Prog ), androsterone (ADT), and dehydroepiandrosterone (DHEA), and its sulfate, was reported to be high in breast cyst fluid (BCF), this research area has attracted only a limited interest despite the well-known potential influence of steroids on tumor growth: In addition, the presence in BCF of steroid-bindingproteins (namely, progesterone-binding protein, albumin, corticosteroid-binding globulin [CBG], and testosterone-estradiol-bindingglobulin [TeBG], which all favor an accumulation of steroids in this fluid), contributes to the interest in this field.' The present report deals with the concentrations of C-21, C-19 and C-18 steroids as well as steroid glucuronides in BCF. The steroid glucuronides include androstane3a,17P-diol glucuronide (3A-DIOL-G), androsterone glucuronide (ADT-G), estradiol glucuronide (E2-G), and estrone glucuronide (El-G). Several recent studies on steroid g1ucuronides7-'' have associated the plasma levels of 3A-DIOL-G and ADTG as markers of the transformation of testosterone (TESTO) into dihydrotestosterone (DHT) as reflecting the total pool of androgens. In addition to its presence in the liver, the enzyme glucuronyl transferase has been shown to be present in human peripheral tissues, such as the skin and the prostate. Such a wide distribution of 93
94
ANNALS NEW YORK ACADEMY OF SCIENCES
glucuronyl transferase could explain the large amount of Sa-reduced steroid glucuronides in plasma." Since the local accumulation of steroid glucuronides may reflect androgen conversion in breast tissue, and since androgens have been suggested to play a role in normal and cancer cell f ~ n c t i o n , ' ~it. 'was ~ of interest to determine the levels of these 5a-reduced steroid conjugates in BCF. Moreover, we have examined the levels of E2-G and El-G in BCF since these metabolites could also aid in estimation of estradiol transformation in this tissue. The concentration of the gross breast cystic disease protein 15 (GCDFP-15), one of the major protein constituents of human breast cyst fluid,15was also measured and its value was correlated with concentrations of unconjugated steroids as well as steroid conjugates. The interest of such study is strengthened by our recent finding of a potent inhibitory effect of estrogens on GCDFP15 mRNA levels and release in the human breast cancer cell line ZR-75-l.l6
SUBJECTS, MATERIALS AND METHOD The subjects included 75 women who had needle aspiration of breast cysts. After aspiration, the gross cystic disease fluid was stored at -20°C until assayed. The samples were analyzed by procedures originally devised for the analysis of steroid in p l a ~ m a . ~ *Intra-assay *~~'' variation for each steroid was less than 16% and to avoid inter-assay variation, all breast cyst samples were analyzed simultaneously. Assay of the unconjugated steroids employed extraction and column chromatography, while that for the steroid glucuronides involved an extraction followed by treatment with P-glucuronidase and column chr~matography.~ GCDFP- 15 was measured by radioimmunoassay using a modification of the method previously described by Haagensen et al. "." All RIA results are shown as the means 2 SEM of triplicate determinations on individual samples. Radioimmunoassays were calculated according to the method of Rodbard and Lewald.''
RESULTS FIGURE 1 shows the concentrations in cyst fluid of the C-21 and C-19 steroids, namely, Prog, pregnenolone (Preg), 17-hydroxy-progesterone ( 17-0H-Prog), 17-hydroxy-pregnenolone ( 17-0H-Preg), DHEA, androst-5-ene-3P,17P-diol ( 5-ene-Diol), androstenedione (4-ene-Dione) and TESTO. The concentrations of Prog and Preg exceed those of their respective 17-hydroxylated derivatives by almost 100-fold. Among the C- 19 steroids, 5-ene-Diol, 4-ene-Dione, and TESTO were found at approximately 5 nmol/L in BCF, whereas DHEA levels reached 31 2 4 nmol/L. FIGURE 2 shows that the ADT levels at 6.2 2 0.9 n m o l h were higher than those of DHT and 3A-DIOL. The estrogens estrone ( E l ) and estradiol (E2), on the other hand, were measured at concentrations of 0.67 2 0.03 and 0.12 2 0.01 nmolh, respectively. At concentrations of 125 2 24 nmolh, ADT-G is the major steroid glucuronide found in BCF and exceeds by more than 10- and 100-fold that of 3ADIOL-G and El-G or E2-G, respectively. The correlation between the concentrations of the various steroids in BCF was then examined. The statistical relationship between cyst fluid DHEA and 4-ene-Dione,
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TESTO, and 4-ene-Dione, TESTO and DHT, 3A-DIOL and DHT, 3A-DIOL and 3A-DIOL-G as well as 3A-DIOL-G and ADT-G levels can be expressed by the linear 3 and 4. A significant positive correlation was also regressions shown in FIGURES observed between 4-ene-Dione and El, 4-ene-Dione and El-G as well as El-G and E2-G (FIG. 5). The concentration of GCDFP-15 in BCF has been measured at 2745 2 234 p g l ml. While no correlation was observed between GCDFP-15 and C-21 or C-19 steroids, a significant negative relationship is seen between El-G and GCDFP-15 levels (FIG. 6). However, the correlations between the unconjugated estrogens and GCDFP- 15 as well as between E2-G and GCDFP-15, do not reach statistical significance.
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