Catechol estrogen formation in liver microsomes from female ACI and SpragueDawley rats: comparison of 2- and 4-hydroxylation revisited
Sonia Mesia-Vela1,
Rosa I. Sanchez1,
Jonathan J. Li3,
Sara Antonia Li3,
Allan H. Conney2 and
Frederick C. Kauffman1,4
1 Laboratory of Cellular and Biochemical Toxicology, Department of Pharmacology and Toxicology, and
2 Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854 and
3 Department of Pharmacology, Toxicology and Experimental Therapeutics, Hormonal Carcinogenesis Laboratory, Kansas Cancer Institute, The University of Kansas Medical Center, Kansas City, KS 66160-7158, USA
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Abstract
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Estradiol (E2)-hydroxylation was studied in liver microsomes from ACI and SpragueDawley female rats, which differ markedly in their susceptibility to E2-induced formation of mammary tumors. NADPH-dependent oxidation of E2 by liver microsomes from ACI and SpragueDawley rats produced several metabolites of which 2-hydroxyestradiol (2-OH-E2), estrone (E1), and 2-hydroxyestrone (2-OH-E1) were predominant. Incubations with either low (9 nM) or high (50 µM) concentrations of radiolabeled E2 and with varying amounts of microsomal protein indicated the formation of only small amounts of 4-hydroxyestradiol (4-OH-E2). The ratio of 2-OH-E2 to 4-OH-E2 formed with the low concentration of E2 was about 10:1 regardless of the amount of microsomal protein used, and about 20:1 using a high concentration of E2. Thus, oxidation of E2 by liver microsomes from female ACI and SpragueDawley rats occurs primarily via 2-hydroxylation, and 4-hydroxylation is only a minor pathway. These results are in disagreement with a recent report indicating substantial 4-hydroxylation of E2 by liver microsomes from female ACI rats.
Abbreviations: E2, estradiol; E1, estrone; 2-OH-E2, 2-hydroxyestradiol; 2-OH-E1, 2-hydroxyestrone; 4-OH-E2, 4-hydroxyestradiol
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Introduction
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The female ACI rat has attracted considerable attention recently as an animal model for studies on the initiation and progression of estradiol (E2)-induced and E2-dependent mammary tumors (13). The rapid induction of mammary tumorigenesis by subcutaneous implantation of a silastic tube or pellet containing E2 in the ACI rat (80100% incidence in 1620 weeks) renders this animal model useful for studies on mechanisms of estrogen-induced mammary carcinogenesis (13). A recent report by Wilson and Reed (4) indicated that the NADPH-dependent oxidation of E2 by liver microsomes from the estrogen-sensitive female ACI rat differed from that of the less sensitive SpragueDawley rat (5). Liver microsomes from ACI rats were reported to produce much higher amounts of 4-hydroxyestradiol (4-OH-E2) than 2-hydroxyestradiol (2-OH-E2) (4) which is the major metabolite produced by liver microsomes of the less sensitive SpragueDawley rat (6). It was suggested that the formation of a greater amount of hepatic 4-OH-E2 relative to that of 2-OH-E2 is related to the higher susceptibility of the ACI rat to E2-induced mammary tumors as 4-OH-E2 but not 2-OH-E2 is carcinogenic in the Syrian hamster kidney (79), and 4-OH-E2 is more carcinogenic towards the uterus of CD-1 mice than is 2-OH-E2 (10). Elevated 4-hydroxylation of E2 is observed in the Syrian hamster kidney (11,12) and in the CD-1 mouse uterus (13) and pituitary (14), all of which organs are susceptible to E2 carcinogenesis. Moreover, when compared with normal biopsies of breast tissues, elevated ratios of 4-OH-E2 to 2-OH-E2 formation have been reported in human breast tumor samples (15,16).
In the present report, we describe and compare the patterns of E2 hydroxylation in liver microsomes from the highly estrogen-sensitive female ACI rat with patterns in microsomes from the less estrogen-sensitive female SpragueDawley rat strain. Because of the potential importance of the high 4-hydroxylation of E2 in a rat strain that is highly sensitive to E2-induced mammary carcinogenesis, we have made an attempt to confirm and extend the studies of Wilson and Reed (4). The data presented herein, however, indicate that liver microsomal preparations from female ACI rats hydroxylate E2 predominantly in the 2-position, and only a very small amount of 4-OH-E2 is formed.
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Materials and methods
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Chemicals
[2,4,6,7,16,17-3H(N)]Estradiol (110170 Ci/mmol) or [4-14C]estradiol (4560 mCi) were purchased from NEN Life Science Products (Boston, MA). Estradiol, ascorbic acid, NADPH, reference compounds estriol, estrone, 2-hydroxyestradiol and 4-hydroxyestradiol were purchased from Sigma Chemical (St Louis, MO). 2-Hydroxyestrone and 4-hydroxyestrone were purchased from Steraloids (Newport, RI). AIN-76A diet was purchased from Dyets (Bethlehem, PA) and Teklad rodent diet 8604 was from Ralston-Purina (Teklad, Madison, WI). All other chemicals used were of the highest grade from standard sources.
Animals
Seven-week-old, intact, female, outbred ACI rats, and 6-week-old, intact, female, outbred SpragueDawley rats were supplied by Harlan SpragueDawley (Indianapolis, IN). The animals were housed individually in an AALAC accredited barrier facility under controlled temperature, humidity and lighting conditions. The animal studies were carried out in adherence to guidelines established in the Guide for the Care and the Use of Laboratory Animals (US Department of Health and Human Resources, NIH, 1985). The rats were fed either AIN-76A diet for 4 weeks, or Teklad Rodent Diet 8604 for 6 months. All animals received tap water ad libitum.
Preparation of liver microsomes
Animals were killed by decapitation. The livers were quickly removed, and microsomal fractions were prepared by differential centrifugation as described previously (17). Microsomal fractions were suspended in 0.25 M sucrose and stored at 80°C. Protein was determined using a Bio Rad protein assay kit (Pierce Chem. Co., Rockport, IL) with BSA as a standard.
NADPH-dependent microsomal metabolism of estradiol
Liver microsomal fractions were incubated with 5 mM ascorbic acid, 3 mM magnesium chloride, 50 µM sodium-phosphate buffer (pH 7.4), and 9 nM or 50 µM [3H]E2 (0.5 µCi) in a total volume of 500 µl, at 37°C, with gentle agitation, for 20 min. The reactions were initiated by the addition of 2 mM NADPH. The reactions were terminated by the addition of 5 ml of ethyl acetate. The precipitated protein was sedimented at 2000 g, for 8 min, and extracted twice. The combined organic solvent extracts were evaporated to dryness under nitrogen. The resulting residue was dissolved in 110 µl of methanol, and 90 µl aliquots were analyzed immediately for HPLC separation of estrogen metabolites.
HPLC analysis
Either [3H] or [14C]E2 metabolites were separated and quantified using an HPLC system coupled with in-line UV and radioactivity detection as described previously (18). The HPLC system consisted of a Waters 600E separation module (Milford, MA), a Waters Lambda-Max model 481 UV detector (set at 280 nm), and a solid cell radioactive flow detector (ß-ram from IN/US, Fairfield, NJ). Estrogen metabolites were separated on a 5 uODS Ultracarb 30 column (150x4.6 mm) (Phenomenex, Torrance, CA). All separations were performed at room temperature, at a flow rate of 1.2 ml/min. The solvent system used for the separation of estradiol metabolites consisted of: acetonitrile (solvent A)/0.1% acetic acid in water (solvent B) and 0.1% acetic acid in methanol (solvent C). The solvent gradient (solvent A/solvent B/solvent C) used for eluting estradiol metabolites was as follows: 8 min of isocratic at 16/68/16, 7 min of a concave gradient (curve number 9) to 18/64/18, 13 min of a concave gradient (curve number 8) to 20/59/21, 10 min of a convex gradient (curve number 2) to 22/57/21, 13 min of a concave gradient (curve number 8) to 58/21/21, followed by a 0.1 min step to 92/5/3 and 5 min isocratic at 92/5/3. The gradient was then returned to the initial condition (16/68/16) and held for 15 min before analysis of the next sample. The calculation of the amount of each E2 metabolite formed was based on the amount of radioactivity detected for each corresponding metabolite peak. Data were converted to pmol/20 min incubation based on the specific activity of E2 used in the incubation mixture. The relative retention times of the standard compounds given in relation to E2 are: 0.34 for E3 (16 -OH-E2), 0.79 for 4-OH-E2, 0.84 for 2-OH-E2, 0.92 for 2-hydroxyestrone (2-OH-E1), 0.94 for 2-OH-E2 and 1.04 for E1 (estrone). The data are representative of at least three separations. The retention times of the standards did not vary by >5% between runs.
Statistical analysis of data
Data are presented as the mean ± SD. Differences between means were assessed by using a two-way Students t test.
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Results
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Figure 1
depicts a typical HPLC profile for E2 metabolites in liver microsomal fractions from ACI rats fed AIN-76A diet for 4 weeks. Comparable profiles of metabolism were produced by microsomes from SpragueDawley rats (figure not shown).

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Fig. 1. Representative E2 metabolite profiles as a function of microsomal protein of ACI rats. 9 nM of [3H]E2 (0.5 µCi) was incubated with rat liver microsomes (1 and 2 mg protein) for 20 min at 37°C with an NADPH generating system and extracted with ethyl acetate as described in the Materials and methods. All the experiments were performed using liver microsomes from four animals per group. Quantification of metabolites formed is detailed in Table I .
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Six major metabolites were separated and identified. Oxidation of the 17-hydroxyl group to E1 and 2-hydroxylation of E2 to 2-OH-E2 and to 2-OH-E1 were the predominant metabolites found in microsomal fractions from both rat strains at E2 concentrations as low as 9 nM and as high as 50 µM (Table I
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Table 1. Estradiol hydroxylation by liver microsomes from ACI and SD rats using a low and high concentration of estradiol
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When the reaction was performed at a fixed low concentration of E2 (9 nM) and increasing amounts of protein, no significant relative changes were observed in the formation of 2- and 4-OH-E2 (Table IA
, Figure 1
). The ratio of 2-OH-E2 to 4-OH-E2 formation was 1012:1 regardless of the amount of microsomal protein used. Similar results were observed using the same conditions in liver microsomes from SpragueDawley rats. Moreover, when incubations of liver microsomal fractions from ACI or SpragueDawley rats were carried out at 50 µM E2, the ratio of 2-OH-E2 to 4-OH-E2 increased to 20:1 (Table IB
).
Similar assays of E2 hydroxylation were performed using microsomal fractions from ACI rats fed Teklad Rodent Diet 8604 for 6 months. These reactions were performed at 37°C for 20 min in 1 ml of 50 µM sodium-phosphate buffer (pH 7.4) containing 10 mM sucrose, 1 mM ascorbic acid, 3 mM magnesium chloride, 1 mM NADPH and different concentrations (5 nM to 5 µM) of [4-14C]E2 (0.51 µCi). Extraction and HPLC procedures were similar to those described above. Results from these studies showed a similar pattern of E2 hydroxylation by liver microsomes as those observed in Figure 1
(data not shown). The formation of 2-OH-E2 was always significantly greater than that observed for 4-OH-E2, regardless of the E2 concentration or the amount of protein present in the incubation. The ratio of 2-OH-E2 + 2-OH-E1 to 4-OH-E2 + 4-OH-E1 metabolite formation averaged 4.4, without significant changes within a range of from 5 nM to 5 µM E2.
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Discussion
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Lacassagne (19) was the first to show that injection of estrogens, either at birth or at an early age caused mammary tumors to develop with the same frequency in male and female mice. Thereafter, numerous reports of estrogen-induced tumors appeared and many rodent tumor models have been introduced (20). The ACI rat differs from earlier animal models, and it is particularly valuable because estrogen-induced mammary tumorigenesis can be achieved within a relatively short period of time (1618 weeks) following chronic exposure to very low amounts of E2. Mammary tumors are induced consistently in female ACI rats given E2 in either silastic implants (3) or in cholesterol pellets (1). In our previous studies, chronic exposure of ACI rats to low E2 concentrations (pellet containing 3 mg E2, 17 mg cholesterol) led to serum levels that were
6.5-fold above basal values (data not shown)
Endogenous estrogens are hydroxylated at multiple positions by several hepatic and non-hepatic microsomal monooxygenase systems (reviewed in refs 21,22). In mammals, the liver contains high levels of cytochromes P450, which catalyze NADPH-dependent oxidation of estrogens to various hydroxylated or keto metabolites (21,22). 2-Hydroxylation is the major liver microsomal metabolic oxidation pathway of E2 in all mammalian species studied including humans (6,12,18,2329). Although E2 is known to be metabolized in target tissues (22), hepatic metabolism is a critical determinant in regulating systemic levels of circulating E2 and its metabolites. As it has been suggested that formation of 4-OH-E2 may be an essential pathway leading to E2-induced mammary carcinogenesis (11,12,15), the recent report indicating the formation of a large amount of the 4-OH-E2 by liver microsomes of female ACI rats (4), highly sensitive to E2-induced mammary carcinogenesis, has been considered supportive of this hypothesis.
Our results on the hydroxylation of E2 by liver microsomes from ACI and SpragueDawley rats indicate that 2-OH-E2 is the predominant metabolite produced from microsomes by both of these rodent strains. These results differ from those reported by Wilson and Reed (4) who indicated that liver microsomes from the sensitive ACI strain produced very high amounts of 4-OH-E2. Further, our results show that there is little or no difference in the pattern of hydroxylation between ACI and SpragueDawley rats. Most reports employing liver microsomes from different mammals including rodents and humans are in accord with our data showing a high ratio for the formation of 2-OH-E2 to the formation of 4-OH-E2 (6,12,18,2328).
Reasons for the disparity between our results and those of Reed and Wilson are unclear. It is unlikely that differences in age of the animals or diet underlie differences noted by their laboratory and ours. Animals used in the two studies were the same age and from the same source. Although Reed and Wilson, do not describe the diet fed to their animals, it is unlikely that diet differences can explain the differential metabolism observed because the same pattern of hydroxylation was obtained in microsomes from animals fed AIN76A diets at Rutgers University and those obtained in microsomes from animals maintained on Teckland Rodent diet 8604 at the University of Kansas. It is also unlikely that differences in the extraction of 2- and 4-hydroxylated metabolites differed as recoveries of these metabolites from incubation media are essentially the same, 85100% (18). Although, Wilson and Reed (4) described the retention times of 4-OH-E2 and 2-OH-E2 as not overlapping, the 4-OH-E2 peak shown is non-symmetrical and wide, approaching that for 2-OH-E2, thus suggesting that it may contain artifacts possibly due to the oxidation of E2 or its metabolites during the extraction procedure. During the past several years, we have occasionally observed the generation of artifactual radioactive peaks that occur after the extraction of radioactive E2 metabolites with impure batches of ethyl acetate. We routinely use the highest quality HPLC grade ethyl acetate for our extractions.
Although, our results clearly show that formation of 2-hydroxyestradiol metabolite is the major metabolite produced by rat liver microsomes from both strains, the possibility that other pathways exist in extra hepatic tissues cannot be ruled out. Therefore, it is particularly important to delineate pathways of estrogen hydroxylation in the mammary glands of ACI and SD rats, which differ markedly in their susceptibility to E2-induced tumors. It should be noted, however, that the total amount of E2 metabolism in mammary gland microsomes of female SD rats is
1% of that of liver microsomes derived from the same animal (30).
In summary, although liver E2 metabolism is distal to the site of mammary gland oncogenesis, the unique sensitivity of the female ACI rat to E2-induced neoplastic transformation in the mammary gland does not appear to be related to a higher capacity of this strain to form 4-hydroxyestradiol. It is evident from the results presented herein that liver microsomes from female ACI rats catalyze extensive formation of 2-hydroxyestradiol, but only minute amounts of 4-hydroxyestradiol.
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Notes
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4 To whom correspondence should be addressed Email: kauffma{at}rci.rutgers.edu 
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Acknowledgments
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This investigation was supported in part by grants CA58030, NIH (S.A.L.), ES05022, NIEHS Center and Midwest Research Institute/KUMC research funds.
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Received February 22, 2002;
revised April 3, 2002;
accepted April 26, 2002.