Determination of estrogen 3-sulfates in biological fluids of mammary tumor-bearing rats by radioimmunoassay

O&383-2897/93 $6.00+ 0.00 Pergamon Press Ltd

Nucl. Med. Eiol. Vol. 20, No. 4, pp. 493-501, 1993 Printed in Great Britain

Determination of Estrogen 3-Sulfates in Biological Fluids of Mammary Tumor-bearing Rats by Radioimmunoassay NORIKO

SHIMURA*,

Faculty of Pharmaceutical

YUKIMICHI KOMORI, AKIKO KUBODERA

TOUICHI

TANAKA

and

Sciences, Science University of Tokyo, 12 Ichigaya Funagawara-machi, Shinjuku-ku, Tokyo 162, Japan (Received 13 October 1992)

Determination of estradiol3-sulfate (E2 3-S) and estriol 3-sulfate (E, 3-S) in plasma, urine or tumor tissues of mammary tumor-bearing rats were performed using the superior radioimmunoassay (RIA) system. The plasma E, 3-S, average tissues,

level of E, 3-S after tumorigenesis was found to be about one-third of the normal level. As to the levels in urine were significantly high both before and after tumorigenesis. Before that, the level was about 1.5 times, and after that, about 2.5 times as high as the normal level. In tumor an extremely high level of E, 3-S (399.6 f 113.9 pg/g tissue) was determined.

Introduction Estrogen plays an important role in growth of mammary tumors. For example, mammary tumor which was induced by a chemical carcinogen, 7,12-

dimethylbenz(a)anthracine (DMBA) (Huggins and Yang, 1962) in rats was hormone-dependent (Leung, 1982), so ovariectomy and adrenalectomy were effective in completely diminishing tumor growth. But the diminished tumors had grown again when estrogen was administered to them (Strental et al.,

1963). The biological levels of estrogen were significantly different between normal women and mammary cancer patients. In mammary cancer patients, the plasma levels of estrone (E,) or estradiol (E,) were found to be higher than those of normal women (Henderson et al., 1975). As for E,, it was reported that E, also had an ability to promote mammary tumor growth as well as E, and Ez (Rudali et al., 1975; Lippman et al., 1977). Moreover, E, was reported to promote cell division and growth of mammary gland (Lee, 1980). As mentioned above, not only E, and E, but also E,, which was known to have just a weak estrogen effect, has been closely related to mammary tumors. While many reports have indicated that plasma levels of estrogen 3-sulfates, major metabolites of estrogens are extremely high in patients with breast cancers (Noel et al., 1981; RCmy-Martin et al., 1983; Samojlik et al., 1982). But most assays for estrogen *Author for correspondence.

sulfates need a hydrolytic step to release the hydrophobic steroid moiety (Samojlik et al., 1982; RkmyMartin et al., 1983), solvent extraction (Wright et al., 1978) or chromatography (Loriaux et al., 1971), so reproducibility was not sufficient. For the above reasons, the relationship between mammary tumor and estrogen 3-sulfates has not become clear. Recently, we reported direct RIA systems of estrogen 3-sulfates without hydrolysis (Tanaka et al., 1984, 1985a,b,c). This time, we have tried to determine estrogen 3-sulfates in plasma, urine or tumor tissues of mammary tumor-bearing rats using RIA systems based on the above method in order to investigate the relationship between the levels of estrogen 3-sulfates and mammary tumor.

Materials and Methods Reagents 7,12_Dimethylbenz(a)anthracine (DMBA) was purchased from Kanto Chemical Ltd (Tokyo, Japan). Bovine gamma globulin and BSA fraction V were supplied by Sigma Chemical Co. (St Louis, MO). Sep-Pak C,, cartridge column was purchased from Waters Associates (Millford, MA). [6,7-3H]Estradiol (1.48 TBq/mmol), [6,7-3H]estriol (2.09 TBq/mmol) and [6,7-3Hlestrone sulfate (2.18 TBq/mmol) were bought from New England Nuclear (Boston, MA). The labeled compounds were purified by thin-layer chromatography on Silica Gel G prior to use. All other general reagents were from Nacarai Tesque Inc. (Kyoto, Japan).

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SHIMURA

Animal treatment and samples

Mammary tumors were induced in 7-week-old Sprague-Dawley female rats (14&160 g) (Sankyo Labo Service Co. Inc., Tokyo, Japan) with a chemical carcinogen, by the method of Huggins and Yang (1962). 10 mg of DMBA dissolved in 1 mL of sesame oil was administered orally to the rats, and 1 week later, the same dose was fed again. Blood samples were collected by cutting the tails at intervals of 7 days each, before and after the tumorigenesis was checked by digital examination, and centrifuged. Urine samples were collected within 24 h of the appointed day. Both plasma and urine samples were stored at -20°C.

et

al.

stand at room temperature for 15 min and then centrifuged at 2500 rpm for 10 min. A 0.25 mL aliquot of supernatant was transferred into a counting vial containing Bray’s scintillator (10 mL) (Bray et al., 1960), and radioactivity was counted in an Aloka LSC-673 liquid scintillation spectrometer (Aloka, Tokyo, Japan). Standards were prepared by adding E, 3-S (2Cr5000 pg) to normal male rat plasma treated with charcoal, instead of mammary tumor-bearing rat plasma. E, 3-S in plasma was determined by the same procedure with [6,7-3H]E3 3-S as labeled antigen and anti-E, 3-S antiserum.

Preparation of antibody

(2) -&, E,

Anti-E, 3-S antiserum was raised in a male guinea pig against 6-oxoestradiol 3-sulfate-(O-carboxymethyl)oxime-bovine serum albumin (BSA) conjugate according to the method previously reported (Tanaka et al., 1984). Anti-E, 3-S antiserum was raised in the same way against 6-oxoestriol 3-sulfate-(O-carboxymethyl)oxime-BSA conjugate (Tanaka et al., 1985a). Antisera to E, or E, were raised in a male New Zealand white rabbit against 6-oxoestradiol(0-carboxymethyl)oxime-BSA conjugate or 6-0xoestriol-(0-carboxymethyl)oxime-BSA conjugate, according to the method of Wright et al. (1978).

RIA was performed by the slightly modified method of Wright et al. (1973). An aliquot (0.55 mL) of diluted plasma was extracted with ether (2.0mL) and washed with water (1 .OmL). [6,7-3H]E, (7000 dpm) in methanol (0.1 mL) was added to this extract and organic solvent was dried under nitrogen gas. To this residue was added the diluted anti-E, antiserum, and the procedure described above was followed. The level of E3 in plasma was determined by the same procedure with [6,7-3H]E3 as labeled antigen and anti-E, antiserum.

Preparation of radioligands [6,7-3 H]Estradiol

3-sulfate (2.18 TBq/mmol) was prepared by the reduction of [6,7-3H]estrone sulfate with sodium borohydride (Tanaka et al., 1984). [6,7-3H]Estriol 3-sulfate (1.67 TBq/mmol) was prepared by the sulfotransferase from a male guinea pig liver (Tanaka et al., 1985a). The labeled compounds (7 x lO’dpm/mL) were stored in methanol containing a drop of NH,OH at 4°C for the routine use. RIA in plasma of mammary tumor-bearing rats

RIA in urine of mammary tumor-bearing rats

[6,7-3H]E2 3-S (7000 dpm) in methanol (0.1 mL) was added to tubes and organic solvent was dried under nitrogen gas. To these tubes, urine samples of mammary tumor-bearing rats (0.05 mL) were added and then the diluted anti-E, 3-S antiserum (0.20 mL) was mixed. The following procedure was described above. For standards, Ez 3-S (20-5000 pg) was spiked into water (0.05 mL) instead of urine, and used. E, 3-S E, or E, in urine were determined by the same procedure with each labeled antigen and each antiserum.

(1) E2 3-S, E, 3-S

RIA in mammary tumor tissues

RIA was performed by the slightly modified method of Tanaka et al. (1984, 1985~). Plasma sample of mammary tumor-bearing rat (0.05 mL) was diluted with 0.07 M phosphate buffer (pH 7.5; 10 mL) and passed through a Sep-Pak C,, cartridge column. After washing with water, steroids in plasma were eluted with methanol (3.0mL). To this elute, [6,7-3H]E, 3-S (7000dpm) in methanol (0.1 mL) was added and organic solvent was evaporated in vacua. The anti-E, 3-S antiserum diluted with 0.06 M borate buffer (pH 8.0, containing 0.05% BGG and 0.06% BSA) (0.25 mL) was added to tubes and the mixture was incubated at room temperature for 1 h. After addition of 50 (w/v)% (NH4)*S04 solution (0.25 mL), the resulting suspension was allowed to

Mammary tumor tissue (0.5 g) was homogenized in 0.07 M phosphate buffer (pH 7.5; 2mL). This tissue homogenate was treated with methanol (3mL) in order to remove proteins and centrifuged at 2500 rpm for 10min. To this supernatant, [6,7-3H]E, 3-S (3000 dpm) in methanol (0.1 mL) was added and concentrated by evaporation in vacua. Then, the lipids in this mixture were removed with ether (3 mL). The aqueous phase was diluted with 0.07 M phosphate buffer (pH 7.5; 10 mL) and passed through Sep-Pak Cl8 cartridge column. Absorbed substances to Sep-Pak Cl8 column were eluted with methanol (3 mL) and methanol was evaporated to dryness in vacua. To this residue, 0.07 M phosphate buffer (pH 7.5; 0.1 mL) was added. Ether extraction and

Estrogen 3-sulfates in biological fluids Table I. Accuracy of the assay by determination of the recovery of E, 3-S added to normal rat plasma Estradiol Added 100 200

IO00

3-sulfate

(pg/mL) ~ Found + SD

Recovery (%)*

117.x & 10.4 187.8 t 12.5 982.3 k 41.1

117.8 93.9 98.2

Table

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3. The intra-assay and inter-assay variations of the radioimmunoassay of E, 3-S in female rat plasma Intra-assay

Inter-assay

Subject No.

Found + SD

CV (%)*

Found f SD

CV (%)*

I 2 3

149.9 * 19.9 485.1 i41.3 I I I I .9 i 95.4

13.3 x.5 X.6

131.9+ 15.6 463.5 & 25.0 1159.5 I 60.3

II 8 54 5.2

*?I = 5.

*n = 5.

passing through the column were repeated twice to remove lipids and proteins. The resulting methanol solution (0.3 mL) was divided into three fractions (0.1 mL each), and one was used for calculation of recovery ratio, the others were used for RIA. [6,7-‘H]Ez 3-S (5000dpm) was added to these two fractions. The following procedure was described above. E, 3-S in plasma was determined by the same procedure with [6,7-3H]E3 3-S as labeled antigen and anti-E, 3-S antiserum. In order to know the normal level of each estrogen, rats which were not administered with DMBA, were prepared and each estrogen level was determined similarly by RIA.

Results Mammary tumors were found 2 or 3 months after the initial injection of DMBA. E1 3-S or E3 3-S can be determined in the range of 50-5000 pg/tube. Accuracy of the assay was assessed by estimating recoveries of known amounts of E, 3-S added to steroid free rat plasma. As for E, 3-S the same procedure was performed. Recovery data estimated for three levels were satisfactory as shown in Tables 1 and 2. The intra-assay and inter-assay variations are shown in Tables 3 and 4. The intraassay variations in the RIA were measured with three different samples at points on the calibration curve. The coefficient of variation (CV) values were less than 13.3% at each point in the measurement of E2 3-S. While the values of CV in the measurement of E, 3-S were less than 4.2% at each point. The inter-assay variations were measured with analysis of three different samples. The CV values were found to be less than 11.8% in the measurement of E, 3-S and for E, 3-S less than 8.5% at each point. The recovery ratios of E, 3-S and E, 3-S in plasma were 93.9-l 17.8 and 95.2-104.3%, respectively.

Table 2. Accuracy of the assay by determination of the recovery of E, 3-S added to normal rat plasma Estriol Added 100 500 2500 *II = 5.

3-sulfate (pg/mL) ~ Found + SD 95.2 & 4.8 493.2 k 20. I 2607.7 f 44.8

Recovery (%)* 95.2 98.6 104.3

Figures show the concentrations of E, 3-S E, 3-S Ez or E, measured by RIA system we developed. These levels varied with the days before and after tumorigenesis. The normal range of each estrogen in rats, which were not administered with DMBA was shown as the band enclosed by dotted lines. E2 and E_, 3-S in plasma Figure 1 shows the plasma levels of Ez and EZ 3-S in rats. Ez levels were higher (2725 + 529.2 pgjmL. mean 1 SE) than the average of normal levels (280 & 56.4 pg/mL) after tumorigenesis significantly (P < 0.01). On the contrary to El, as the day of tumorigenesis was approaching, E, 3-S levels gradually decreased and after tumorigenesis, its levels became lower (105.1 k 27.6 pg/mL, mean f SE) than the average of normal levels (392 k 68.9 pg/mL) significantly (P < 0.01) in every week. E2 and E, 3-S in urine E, and E, 3-S levels in the urine are shown in Fig. 2. E, levels (62.3 +4.5 ng/day) were almost the same as the average of normal levels (64.7 + 9.0 ng/day) in every week. While E, 3-S levels before tumorigenesis (116 +_ 19.3 ng/day) were almost the same as the average of normal levels (81.6 k 11.9 ng/day), but at 3-5 weeks after tumorigenesis, the levels increased significantly (277 k 62.5 ngjday) (P < 0.05). E., and E., 3-S in plasma Plasma levels of E, in rats (1752.1 k 288.5 pg/mL) were almost the same as the average of normal levels (1118 f 478.5 pg/mL) (Fig. 3). While, the concentrations of E, 3-S in plasma (792.6 f 182.5 pg/mL) were almost the same as the average of normal levels (632 f 101 pg/mL) before tumorigenesis, but 34 weeks after that, they showed a tendency to increase (Fig. 3).

Table 4. The intra-assay and inter-assay variations of the radioimmunoassay of E, 3-S in female rat plasma Intra-assay

Inter-assay

Subject No.

Found f SD

CV (%)*

Found + SD

CV (%)*

I 2 3

93.7 f 2.9 563.9 f 23.8 2648.9 + 93.9

3.1 4.2 3.5

90.3 * 2.1 537.1 k 16.2 2618.4 f 223.8

2.3 3.0 8.5

*n =s.

NORIKOSHIMURAet al.

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Fig. 1. Changes of estradiol (E,)(A) and estradiol 3-sulfate (E2 3-S)(B) concentrations in plasma of rats administered with DMBA, before and after tumorigenesis. Each point represents the mean rf: SE (n = 4 for E,,_. n = 7 for E, 3-S). The bold and dotted lines represent the mean concentration and the range of SE of each substance in normal rat plasma (n = S), respectively.

E, and E, 3-S in urine In urine, E, levels were 54.6 _+4.1 ng/day before tumorigenesis and 84.1 + 7.6 ng/day after tumorigenesis. These values were higher than the average of normal levels (30.9 f 2.9 ng/day) significantly (P < 0.01). Especially, after tumorigenesis, the level was about 2.7 times as high as the average of normal levels (Fig. 4). Similarly, E, 3-S levels were significantly higher than the average of normal levels both before and

after tumorigenesis (P < 0.01). Before tumorigenesis, its level was 333.7 _+26.4ng/day and this value was 1.5 times as high as the average of normal levels (204 f 35.8 ng/day). After tumorigenesis, the level was 530.7 f 54.2 ngjday and this was 2.5 times as high as the average of normal levels (Fig. 4). Ez 3-S and EJ 3-S in mammary

tumor tissues

In mammary tumor tissues, E, 3-S level was lower than lOpg/g tissue. On the other hand, E, 3-S level

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Fig. 2. Changes of E,(A) and E, 3-S(B) concentrations in urine of rats administered with DMBA, before and after tumorigenesis. Each point represents the mean + SE (n = 8 for E,, n = 6 for E, 3-S). The bold and dotted lines represent the mean concentration and the range of SE of each substance in normal rat plasma (n = 5), respectively.

was found to be extremely higher (399.6 f 113.9pg/g tissue) than that of E2 3-S. The extraction ratios of E, 3-S and E, 3-S during the extraction process were 78.7 f 5.4 (mean + SD)% and 70.2 f 6.2%, respectively.

Discussion Previous studies demonstrated that E, concentrations in plasma and tumor tissues of mammary cancer patients were higher than those of normal

women, and that was also demonstrated for the animals (Henderson et al., 1975; Bonney et al., 1984; Santen et al., 1984). DMBA-induced mammary tumor-bearing rats had also high plasma levels of E2 both before and after tumorigenesis in our study. E, 3-S which is one of the metabolites of E,, however, showed an entirely different pattern from E,. The levels of E, 3-S in plasma were gradually decreased as the week of tumorigenesis was approaching, and after tumorigenesis, more decreased.

NORIKOSHIMURAet al.

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Fig. 3. Changes of estriol (E,)(A) and estriol 3-sulfate (E3 3-S)(B) concentrations in plasma of rats administered with DMBA, before and after tumorigenesis. Each point represents the mean & SE (n = 10 for E,, n = 8 for E, 3-S). The bold and dotted lines represent the mean concentration and the range of SE of each substance in normal rat plasma (n = 5), respectively.

It has been reported that mammary tumor tissue has estrogen sulfotransferase activity and can sulfurylate steroids (Adams, 1979), whereas it has also estrogen sulfatase activity and can convert steroids into physiologically active metabolites (Adams, 1964; Adams and Wong, 1968). But although mammary tumor tissues have these enzyme activities, we can not think that these varied concentrations only resulted from the change be-

tween E, and E, 3-S. For instance, it is well known that estrone sulfate (E,-S) is a quantitatively abundant estrogen in plasma with mammary carcinoma (Franz et al., 1979; Noel et al., 1981; R&my-Martin et al., 1983; Roberts et al., 1980) and the transformation of E,-S into E2 in mammary carcinoma has also been reported by Vignon et al. (1980), and Wilking et al. (1980). Therefore the increased levels of E, in plasma may be influenced by E,-S levels.

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Fig. 4. Changes of E, (A) and E, 3-S (B) concentrations in urine of rats administered with DMBA, before and after tumorigenesis. Each point represents the mean k SE (n = 12 each). The bold and dotted lines represent the mean concentration and the range of SE of each substance in normal rat plasma (n = 5), respectively.

While it is well known that free estrogen has an ability to grow mammary tumor (Rudali, 1975; Simpson-Herren et al., 1970). Gelly et al. (1986) demonstrated the fact that mammary tumor tissues could reversibly transform a biologically active estrogen into its inactive sulfate suggested that this transformation could be important in the control of estrogen action. Both estrogen sulfatase and estrogen sulfotransferase may play a role in regulating the estrogen action by shifting the intracellular equilibrium be-

tween free estrogen and estrogen sulfates (Tseng et al., 1982). That is, sulfates can reduce the action of estrogen, such as growing mammary tumor (Fishman et al., 1983; Gross0 et al., 1984). In this report, Ez 3-S levels in plasma were decreased when rats were administered with DMBA. E, 3-S may be an intermediate substance which will be transformed into other inactive conjugate, or may be a precursor of active estrogen (E,, etc.). Only quantitative change could hardly show the reason.

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NORIKO SHIMURAet al.

In tumor tissues, Er 3-S level was below lops/g tissue, so it has been found that E2 3-S could not been remained in tumor without being excreted from tissues to extracellular fluid. As seen in Fig. 2, E, levels in urine was normal. Urine levels of E, 3-S were around normal levels before the week of tumorigenesis, but 3-5 weeks after that, the levels became higher (Fig. 2). Taking into consideration that steroid sulfates are excreted mainly in urine (Levitz et al., 1960) it is reasonable that urine levels of E, 3-S were increased according to the levels of E, in plasma. As to E, in plasma, the levels were about normal range (Fig. 3). E, is known to have just a weak biological action of estrogen, compared with E, or E2, and it is also one of the metabolites of these estrogens. But it has been reported that E, also has an ability to promote the mammary tumor’s growth (Rudali et al., 1975; Lippman et al., 1977). Therefore, E, can be sulfated to be reduced estrogen action. Plasma levels of E, 3-S were not different from the normal level significantly (Fig. 3). In urine, E, levels were always higher than the normal level, but it showed a tendency to decrease 5-6 weeks after tumorigenesis (Fig. 4). E, 3-S levels in urine were clearly higher than the normal level, especially after tumorigenesis, extremely high concentrations were detected (Fig. 4). While, in mammary tumor tissues, high level of E3 3-S was determined being different from E, 3-S. From these findings, it has been suggested that a large amount of E, 3-S existed in tumor tissues and excreted in urine of DMBA-induced mammary tumor-bearing rats. It may also be suggested that estrogens can be sulfated to be reduced estrogen action and mostly excreted as a form of E, 3-S. DMBA-induced mammary tumor is hormonedependent and tumor cells produce hormone (Leung, 1982). In fact, our study shows that the E3 3-S level in tumor tissues was about 4 times higher than that in plasma. It is suggested that this is not due to the accumulation from plasma but the production of the cancer cells. From the result, E, 3-S will be expected as a new in vivo imaging agent. In this report, we tried to apply RIA system, which we developed previously (1984, 1985a,b,c), for determination of estrogen sulfates in rats with mammary tumor. So quantitative observation of E, 3-S or E, 3-S, which had not been cleared about the distribution in the body, has become enabled. This study is thought to be significant as the first step in order to investigate about the enzyme activities, the metabolic pathways of estrogens, or the diagnosis based on quantitative change of estrogens. References Adams J. B. (1964) Enzymic synthesis of steroid sulfates. II. Presence of steroid sulphokinase in human mammary

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