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Chem. Res. Toxicol. 2000, 13, 1208-1213
Quantitation of Catechol Estrogens and Their N-Acetylcysteine Conjugates in Urine of Rats and Hamsters Madoka Nakagomi*,† and Emako Suzuki† Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan Received August 21, 2000
A method for the analysis of N-acetylcysteine conjugates of catechol estrogens [catechol estrogen mercapturates (CE SRs)], which are likely to be urinary markers of estrogen-induced tumors, was established in this study. The characteristics of the method that was established were (1) cleanup of urine using the immunoaffinity column of CE SRs, (2) detection of catechol estrogens (CEs) and CE SRs by electrochemical detection, which provided the high specificity, and (3) stability of CE SRs through the cleanup. Using this method, the simultaneous quantitation of 2-hydroxy-17β-estradiol (2-OHE2), 4-hydroxy-17β-estradiol (4-OHE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestrone 1-N-acetylcysteine thioether (2-OHE1 1SR), 2-hydroxyestrone 4-N-acetylcysteine thioether (2-OHE1 4SR), and 4-hydroxyestrone 2-N-acetylcysteine thioether (4-OHE1 2SR) in the range of 1-15 ng was performed. We first demonstrated the presence of CE SRs, 2-OHE1 1SR and 2-OHE1 4SR, in urine from rats treated intraperitoneally with 17β-estradiol (E2) at a dose of 5 mg/kg. In female rats, the amount of 2-OHE1 1SR was several-fold greater than that of 2-OHE1 4SR, while the presence of 4-OHE1 2SR was not confirmed. The level of CEs and CE SRs in male rats was approximately 1/2-1/20 of that in female rats. The excretion rate following administration of 2-OHE1 at 2 mg/kg and that following the administration of 4-OHE1 at 2 mg/kg were different in female rats. In addition, 4-OHE1 2SR was present in the urine of male Syrian hamsters treated intraperitoneally with E2, whereas it was absent in rats.
Introduction Although it is generally accepted that estrogens potentiate the development of cancer by a receptor-mediated process, recent studies suggested that estrogens play other important roles and concluded that estrogens were also complete carcinogens capable of tumor initiation by mutation potentially in critical genes (1-3). Catechol estrogens (CEs),1 2- and 4-hydroxyestrogens, which are the main metabolites of estradiol (E2), are converted to o-quinone in the body by cytochrome P450 (P450) or peroxidase (Figure 1). Recently, o-quinone has been highlighted as an active intermediate in the development of cancer (4-9), since the presence of the o-quinone-DNA complex has been confirmed (5-7). It has been demonstrated that the o-quinone of CEs reacts with glutathione (10) and that 2-hydroxyestradiol (2-OHE2) reacts with glutathione, producing N-acetylcysteine conjugates (SRs) in an in vitro system using rat liver (11) (Figure 1). In addition, it has been suggested that the urinary levels * To whom correspondence should be addressed. Telephone: +81265-73-8611. Fax: +81-265-73-8612. E-mail:
[email protected]. † Present address: Analysis Services Department, Ina Research Inc., 2148 Nishiminowa, Ina, Nagano 399-4501, Japan. 1 Abbreviations: E , 17β-estradiol; 2-OHE , 2-hydroxy-17β-estradiol; 2 2 4-OHE2, 4-hydroxy-17β-estradiol; 2-OHE1, 2-hydroxyestrone; 4-OHE1, 4-hydroxyestrone; 2-OHE1 1SR, 2-hydroxyestrone 1-N-acetylcysteine thioether; 2-OHE1 4SR, 2-hydroxyestrone 4-N-acetylcysteine thioether; 4-OHE1 2SR, 4-hydroxyestrone 2-N-acetylcysteine thioether; CEs, catechol estrogens; CE SRs, catechol estrogen N-acetylcysteine thioethers (catechol estrogen mercapturates); SG, glutathione thioether; P450, cytochrome P450; EIA, enzyme immunoassay.
of mercapturate can be used as a biomarker for exposure to active nucleophilic materials such as benzene and N-nitrosodibutylamine (12-14). Thus, it might be meaningful to determine the urinary levels of CE SRs as markers of estrogen-induced tumorigenesis. However, the presence of these conjugates in urine has not been confirmed as such. This is because no highly specific and sensitive methods of analysis of CE SRs in body fluid containing many materials with similar structures have been established, and also CE SRs are unstable metabolites. We therefore established a method for quantitation of CEs and CE SRs in urine using an immunoaffinity column, and subsequently determined the levels of CEs and CE SRs in urine of rats or hamsters treated with E2 or CEs using this method.
Experimental Section Chemicals. Bovine serum albumin (BSA) was supplied by Sigma Chemical Co. (St. Louis, MO). Complete Freund’s adjuvant was obtained from Difco Laboratory (Detroit, MI). E2 was purchased from Rousell Uclaf. 2-OHE2, 4-hydroxyestradiol (4OHE2), 2-hydroxyestrone (2-OHE1), and 4-hydroxyestrone (4OHE1) were prepared in our laboratory according to the method of Stubenrauch et al. (15). 2-Hydroxyestrone 1-N-acetylcysteine thioether (2-OHE1 1SR), 2-hydroxyestrone 4-N-acetylcysteine thioether (2-OHE1 4SR), and 4-hydroxyestrone 2-N-acetylcysteine thioether (4-OHE1 2SR) (Figure 1) were synthesized by the method described previously (16). All other chemicals were reagent grade commercial products. Preparation of the Immunogen. To the solution of 2-OHE1 1SR (39.1 mg) in dehydrated DMSO (390 µL) were added
10.1021/tx000182a CCC: $19.00 © 2000 American Chemical Society Published on Web 11/14/2000
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Figure 1. Metabolic activation and deactivation of estrogens. N-hydroxysuccinimide (16.5 mg) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide HCl (27.3 mg), and the mixture was stirred at room temperature for 3 h under nitrogen. The resulting solution was added dropwise to BSA (113 mg) in 0.05 M phosphate buffer (pH 7.4, 1170 µL) with stirring, and the mixture was stirred for a further 30 min at room temperature under nitrogen. The resulting solution was subjected to gel filtration (PD-10 columns; Pharmacia Biotech, Uppsala, Sweden), and lyophilization gave the hapten-BSA conjugate (94.1 mg) as a fluffy powder. Preparation of the Antiserum. The hapten-BSA conjugate (2 mg) dissolved in sterile isotonic saline (0.5 mL) was emulsified with complete Freund’s adjuvant (0.5 mL), and the emulsion was immediately injected intradermally into a domestic male albino rabbit (Kbl:JW; Kitayama Labes Co., Nagano, Japan) at multiple sites along the back. This procedure was repeated once per 2 week interval for the first 2 months and once a month thereafter. After 5 months, whole blood collected from the heart of the rabbit was centrifugated at 2050g for 10 min to obtain the antiserum. The antiserum was stored at 4 °C with 0.1% (w/v) NaN3. The titer and the cross reactivity of the antiserum were checked by the comparable enzyme immunoassay (EIA) procedure as described in our previous report (17). In the first trial of immunization, the 2-OHE1 1SR-, 2-OHE1 4SR-, or 4-OHE1 2SR-BSA conjugate at 1 mg/animal was used as an immunogen, and only low-titer antiserum was obtained. When the immunogens were applied at a dose of 2 mg/animal, high-titer antiserum was obtained in one of three rabbits immunized with the 2-OHE1 1SR-BSA conjugate. Immobilization of the Antibody. The antiserum (20 mL) was purified using Protein A Sepharose 4 Fast Flow (Pharmacia Biotech). The eluate from the column was subjected to dialysis against distilled water and lyophilized to obtain Ig G (397 mg) as a fluffy powder. The Ig G (300 mg) was dissolved in 5 mL of 50 mM phosphate buffer (pH 7.5). Affi-Gel 10 (9 mL, Bio-Rad Laboratories, Hercules, CA) was washed with 10 mM cold acetate buffer (pH 4.5), followed by cold deionized water, and
subsequently mixed with the Ig G solution. After the mixture had been shaked gently for 4 h at 4 °C, 1 M ethanolamine (0.6 mL, pH 8.0) was added to the mixture and agitation was continued for 1 h at ambient temperature. Finally, the gel coupled with Ig G was washed with deionized water, added to a polypropylene column, and stored at 4 °C in 50 mM phosphate buffer (pH 7.3) containing 0.2% NaN3. Cleanup of Urine Samples for Determination of CE and CE SR Levels. The urine (0.75 mL for female and 1.5 mL for male) was filtered with disposable disk filters (HLC-DISK 25, pore size of 0.45 µm; Kanto Chemical Co., Inc., Tokyo, Japan) and diluted with 4 mL of 50 mM phosphate buffer (pH 7.3) to bring the pH to neutral. The diluted urine was applied to the column (gel volume of 0.9 mL; see Immobilization of the Antibody), which was equilibrated with 50 mM phosphate buffer (pH 7.3). After the column had been washed with 50 mM phosphate buffer (pH 7.3) containing 0.001% ascorbic acid (4 mL), the desired fraction was eluted with 95% methanol containing 0.001% ascorbic acid (3.5 mL), and the eluate that was collected was evaporated under reduced pressure at 35 °C. The residue was redissolved in methanol (0.1 mL) and injected into the HPLC column (40 µL). HPLC Analysis. The HPLC system linked to an electrochemical detector (ECD, Coulochem 2 Model 5011, ESA, Inc.) set at 0.15 V was used for the detection of CEs and CE SRs. Inertsil ODS-3 [4.6 mm (i.d.) × 150 mm; GL Science Co., Inc., Tokyo, Japan] was used as the HPLC column at ambient temperature. The solvent systems [70:26:8 and 70:30:0 (v/v) 0.5% NH4H2PO4 (pH 3.5)/CH3CN/CH3OH] were used for the analysis of rat and hamster urine, respectively. To separate 4-OHE1 2SR from unknown metabolites, the 70:27:5 (v/v) 0.5% NH4H2PO4 (pH 3.5)/CH3CN/CH3OH system was used. The flow rate was 1.0 mL/min. Each standard curve was prepared over the range of 1-15 ng; the known amounts of 2-OHE1 1SR, 2-OHE1 4SR, 4-OHE1 2SR, 2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1 (2.5, 5, 12.5, 25, and 37.5 ng in each estrogen) were spiked into 50 mM phosphate buffer (pH 7.3), and cleanup of
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the mixtures was performed according to the method described above. Animals and Treatment. Female and male Sprague-Dawley rats (6 weeks old) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Male Syrian hamsters (6 weeks old) in the body weight range of 70-110 g were purchased from SLC Japan Co. Ltd. (Shizuoka, Japan). The animals were acclimated for at least 1 week prior to use, were kept under a 12 h light/ dark cycle, and had free access to food (CE-2, Clea, Tokyo, Japan) and water. Rats were pretreated intraperitoneally (ip) with phenobarbital (induced group) or saline (noninduced group) for four consecutive days (30 mg/kg for one day and 60 mg/kg for three days). E2, 2-OHE1, or 4-OHE1 was suspended in corn oil by sonication and administered ip. Immediately after administration of these estrogens, rats or hamsters were housed individually in metabolic cages and their urine was collected for 24 h on three consecutive days into polypropylene tubes containing ascorbic acid (200 mg per tube per day in rats and 50 mg per tube per day in hamsters). Urine samples obtained each day were stored at -80 °C until they were assessed. The stabilities of 2-OHE1 1SR, 2-OHE1 4SR, 4-OHE1 2SR, 2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1 in urine for 1 month were found to be 64.6, 97.9, 78.7, 88.4, 87.7, 93.4, and 87.9% under -80 °C, respectively.
Results Cross Reactivity. The specificity of anti-2-OHE1 1SR antiserum, which was used for preparation of the immunoaffinity column, was confirmed by analysis of cross reactivity to 10 related compounds. The values are presented as percentages of the amount of 2-OHE1 1SR that reduced half the enzyme activity to the amount of each compound that reduced half the enzyme activity. The cross reactivities of anti-2-OHE1 1SR antiserum were 30.0% for E1, 5.2% for E2, 112.5% for 2-OHE1, 12.9% for 2-OHE2, 17.5% for 4-OHE1, 4.2% for 4-OHE2, 100.0% for 2-OHE1 4SR, 21.0% for 4-OHE1 2SR, 21.0% for 2-OHE1 2-methyl ether, and 85.3% for 2-OHE1 3-methyl ether. Reliability of the Analytical Method. Each standard curve of 2-OHE1 1SR, 2-OHE1 4SR, 4-OHE1 2SR, 2-OHE2, 4-OHE2, 2-OHE1, or 4-OHE1 exhibited good linearity (R > 0.999) over the range of 1-15 ng. The accuracy and precision of the analytical method with cleanup using the immunoaffinity column were checked by analyzing the percentage recovery and the coefficients of variation of 2-OHE1 1SR, 2-OHE1 4SR, 4-OHE1 2SR, 2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1; known amounts of 2-OHE1 1SR, 2-OHE1 4SR, 4-OHE1 2SR, 2-OHE2, 4-OHE2, 2-OHE1, and 4-OHE1 (2.5, 5, 12.5, 25, and 37.5 ng in each estrogen) spiked into 50 mM phosphate buffer (pH 7.3) were determined by HPLC after the cleanup procedure described above had been carried out and were calculated from each standard curve. The total accuracy and precision of the values for seven kinds of CEs and CE SRs at four or five concentrations (five samples per concentration of each compound) were 84.1-117% and e9.93%, respectively. A typical standard HPLC chromatogram is shown in Figure 2. Standard peaks of seven kinds of CEs and CE SRs could be distinguished from each other, suggesting that this method would allow their simultaneous quantitation. Urinary Excretion of CE and CE SR in Rats Treated with E2. The levels of CEs and CE SRs in urine of rats treated ip with E2 at 5 mg/kg and typical HPLC results are shown in Table 1 and Figure 3, respectively. The values are presented as total urinary amount of each rat for 3 days per kilogram of body weight. Only 2-hy-
Figure 2. Typical HPLC chromatogram of catechol estrogens and their mercapturates obtained from a standard sample: (1) 2-OHE1 1SR, (2) 2-OHE1 4SR, (3) 4-OHE1 2SR, (4) 4-OHE2, (5) 2-OHE2, (6) 2-OHE1, and (7) 4-OHE1. Table 1. Urinary Excretion of Catechol Estrogens and Their Mercapturates in Rats Treated with E2 urinary excretion (µg/kg) estrogen metabolite
female
male
2-OHE1 1SR 2-OHE1 4SR 2-OHE2 2-OHE1
2.75 ( 1.14a 0.60 ( 0.22 1.15 ( 0.85 14.24 ( 4.41
0.12 ( 0.05b -c 0.45 ( 0.16 1.51 ( 0.63
a Values represent the means ( SD (N ) 6 for females, N ) 4 for males). b Calculated using the data below the limits of quantification (
