Predominant 4-hydroxylation of estradiol by constitutive cytochrome P450s in the female ACI rat liver
Angela M. Wilson and
Gregory A. Reed1,
Department of Pharmacology, Toxicology and Therapeutics and Kansas Cancer Institute, University of Kansas Medical Center, Kansas City, KS 66160-7417, USA
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Abstract
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The ACI rat is extremely sensitive to estrogens as mammary carcinogens, whereas the SpragueDawley strain is relatively resistant. Comparison of the disposition and effects of estrogens in these two strains should provide insights into the mechanisms of estrogen carcinogenicity. We have begun this investigation by comparing the metabolism of [3H]17ß-estradiol (E2) by liver microsomes prepared from female rats from each strain. Both strains produce estrone (E1) as the major product at E2 concentrations >1 µM, with smaller amounts of 2-hydroxy-E2 formed. As the E2 concentration is decreased, however, aromatic hydroxylation becomes a more dominant pathway for both strains. At starting E2 concentrations as low as 3 nM, SpragueDawley liver microsomes produced comparable yields of 2-hydroxy-E2 and E1. In contrast, ACI liver microsomes yielded a profound shift to aromatic hydroxylation as the dominant pathway as E2 concentrations dropped below 1 µM, and this shift reflected the production of 4-hydroxy-E2 as the predominant product. The apparent Km for 4-hydroxylation of E2 is <0.8 µM, as opposed to ~4 µM for 2-hydroxylation, suggesting that different cytochrome P450s (CYPs) are responsible. Western immunoblotting of the liver microsomal preparations from ACI and SpragueDawley rats for CYPs known to catalyze 2- and 4-hydroxylation of E2 revealed that both strains contained comparable amounts of CYP 2B1/2 and 3A1/2, but no detectable amounts of CYP 1B1, the proposed E2 4-hydroxylase. Although this enzyme is not a constitutive CYP in SpragueDawley rat liver, its presence in ACI liver could provide a ready explanation for the predominance of 4-hydroxy-E2 as a product. The identity of the estradiol 4-hydroxylase in ACI rat liver and the role of this unique reaction in the heightened sensitivity to E2 carcinogenicity remain to be elucidated.
Abbreviations: CE1, 2/4-hydroxyestrone; COMT, catechol O-methyltransferase; CYP, cytochrome P450; DMBA, 7,12-dimethylbenz[a]anthracene; E1 estrone; E2, 17ß-estradiol; E3, estriol; 17ß-HSD, 17ß-hydroxysteroid dehydrogenase; 2-OH-E1, 2-hydroxyestrone; 4-OH-E1, 4-hydroxyestrone; 2-OH-E2, 2-hydroxyestradiol; 4-OH-E2, 4-hydroxyestradiol; 6
-OH-E2, 6
-hydroxyestradiol; RLM, rat liver microsomes.
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Introduction
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Estrogens are risk factors for breast cancer in humans. Epidemiological evidence indicates that an increase in level or duration of exposure to 17ß-estradiol (E2) and estrone (E1) is associated with an increased incidence of breast cancer (1,2). Early menarche and late menopause are risk factors which increase duration of exposure to endogenous estrogens (3). Interruption of normal estrogen levels during pregnancy decreases estrogen exposure, thus late first full-term pregnancy and nulliparity increase a woman's chance of developing breast cancer (2,47). In addition, long-term use of hormone replacement therapies increases breast cancer risk due to an increase in the level of exposure and the duration of exposure to estrogens (1,7).
Animal studies provide more direct and quantifiable evidence for a causal role of estrogens in tumorigenesis (reviewed in refs 8,9). E1, E2 and the stilbene estrogen diethylstilbestrol induce mammary gland tumors in both rats and mice and estriol (E3) is also a mammary carcinogen in mice. The synthetic steroids norethynodrel and mestranol are mammary carcinogens in rats and dogs, respectively. In addition to these effects in mammary gland, estrogens are also carcinogenic in the liver, kidney, pituitary and various organs of the genitourinary tract of several species (1012). Induction of tumors in multiple tissues and species strongly supports the IARC classification of estrogens as human carcinogens (13).
Although estrogens are known mammary carcinogens in animals and humans, the mechanisms of carcinogenesis are not clear. One proposed mechanism is based on the mitogenic action of estrogens in hormone-specific tissues (i.e. uterus and breast) mediated via estrogen receptors (2,8,14). With longer periods of enhanced proliferation there is an increased chance of mutations. Another mechanism has been proposed in which estrogens act as direct genotoxic carcinogens (15). Estrogen metabolites are able to covalently bind to DNA and the resulting modified sites in DNA are proposed to be pre-mutagenic lesions (11,16). The third proposed mechanism of E2 carcinogenicity is that estrogens or their metabolites are indirectly genotoxic. Reactive oxygen species, generated by redox cycling of estrogen metabolites, such as the catechols and hydroquinones, are suggested to be DNA damaging. This is thought to occur by oxidation or hydroxylation of the DNA to produce pre-mutagenic lesions which can lead to mutations (17,18). Finally, an epigenetic mechanism has been proposed based on the induction of aneuploidy which is concurrent with the emergence of cell transformation (1921).
Determination of the primary mechanism of estrogen carcinogenicity will require an appropriate model system. The rat provides an excellent model for mammary carcinogenesis based on a low spontaneous tumor incidence and efficient induction of tumors by chemical agents (8,9). In addition, chemically induced rat mammary tumors clearly resemble human breast tumors based on their similar morphology and on the high degree of estrogen dependence seen in both rat and human tumors (reviewed in ref. 9). What is even more striking is the pronounced strain difference in rat sensitivities to mammary carcinogenicity. ACI rats are extremely sensitive to estrogens as mammary carcinogens, developing palpable tumors after 3 months treatment with E2 and 100% mammary tumor incidence after 6 months treatment (10,22). SpragueDawley rats, however, are much less sensitive to estrogens, exhibiting no mammary tumors after 7 months treatment (23,24). This strain difference in susceptibilities to mammary carcinogenicity is apparently specific for estrogens as causative agents. ACI rats are less sensitive to induction of mammary tumors by 7,12-dimethylbenz[a]anthracene (DMBA), a known mammary carcinogen, than are SpragueDawley rats. In fact, 100% of SpragueDawley rats treated with DMBA develop mammary tumors within a few weeks (25), whereas, only 30% of ACI rats develop mammary tumors after 8.5 months treatment. ACI rats are relatively insensitive to other genotoxic mammary carcinogens as well (26,27). This unique strain difference, in which the ACI rat is specifically more sensitive to estrogens as mammary carcinogens, presents an invaluable tool for determining the key mechanistic steps of E2 carcinogenicity.
SpragueDawley rats and ACI rats differ markedly in their responses to E2 with regard to mammary tumorigenesis. These differences could be due to differences in E2 pharmacodynamics, pharmacokinetics or both. By careful comparison of the interactions between E2 and the cells and tissues from these two strains we may dissect out critical processes which underlie the pronounced difference in susceptibility to tumor induction. These critical differences may also illuminate the major mechanism involved in estrogen carcinogenicity in the mammary gland. We have begun by comparing the metabolism of E2 by liver microsomes from the two strains. Although hepatic metabolism may not play as central a role in the mammary effects of E2 as target tissue metabolism, we nonetheless consider the characterization of hepatic E2 metabolism to be a logical first step in comparing these strains and their overall response to E2. We have observed quantitative and qualitative differences in E2 metabolism in ACI and SpragueDawley liver microsomes. The differences in hepatic metabolism of E2 between the two strains of rats may be the first step in elucidating the key differences affecting susceptibility to mammary carcinogenesis.
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Materials and methods
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Animals and chemicals
Sexually immature (67-week-old) and sexually mature (1214-week-old) female SpragueDawley and ACI rats were purchased from Harlan Laboratories (Indianapolis, IN). [2,4,6,7-3H]E2 (72 Ci/mmol) and [4-14C]E2 (54.1 mCi/mmol) were products of New England Nuclear (Boston, MA). E2, E1, 2-hydroxyestradiol (2-OH-E2), 4-hydroxyestradiol (4-OH-E2), E3, 2-hydroxyestrone (2-OH-E1), 4-hydroxyestrone (4-OH-E1), 6
-hydroxyestradiol (6
-OH-E2), clotrimazole and all other substrates, enzymes and cofactors were from Sigma Chemical Co. (St Louis, MO). All solvents and reagent chemicals were of HPLC grade and were purchased from Fisher Scientific (St Louis, MO). Scintillant fluid was Ultima Flo M from Packard Instrument Co. (Meriden, CT). NuPage gels and reagents used in western immunoblot analysis were purchased from Novex (San Diego, CA). The primary antibodies for CYPs 2B1/2 and 3A1/2 were provided by Xenotech LLC (Kansas City, KS), whereas rabbit anti-CYP 1B1 (28,29) was generously supplied by Thomas Sutter (Johns Hopkins University, Baltimore, MD). The secondary antibody utilized for visualization of CYPs 2B1/2 and 3A1/2 was alkaline phosphatase-conjugated anti-rabbit IgG and was purchased from Southern Biotechnology Associates Inc. (Birmingham, AL). The BCIP/NBT phosphatase substrate was obtained from Kirkegaard Perry Laboratories (Gaithersburg, MD) and the PVDF membrane was from Millipore (Bedford, MA). Bound antibody against CYP 1B1 was detected with a horseradish peroxidase-conjugated donkey anti-rabbit IgG using the enhanced chemiluminescence method (Amersham Corp., Arlington Heights, IL).
Microsomal preparation
Where indicated, rats were treated with a s.c. cholesterol pellet with or without E2 (20 mg/pellet) for 1 week. Rats were anesthetized with CO2, then killed by decapitation. The livers from 48 rats were pooled and homogenized in TrisKCl buffer, pH 7.4, at 4°C. The homogenate was fractionated by differential centrifugation and the microsomal pellets were resuspended in phosphate/MgCl2 buffer, pH 7.4, and stored at 80°C. Protein concentrations of the resulting rat liver microsomes (RLM) were determined using the Bradford reagent with bovine serum albumin as the standard.
Microsomal metabolism and analysis
[3H]E2 (0.1 µCi/incubation) or [14C]E2 (0.05 µCi/incubation) was mixed with unlabeled E2 and incubated with RLM in 50 mM TrisHCl buffer, pH 7.4, containing 5 mM MgCl2, 5 mM glucose 6-phosphate and 1 mM ascorbate at 37°C in a shaking water bath. E2 was added as a stock solution in DMSO, with a constant final concentration of 2% DMSO (v/v). The NADPH generating system, where used, included 1 U/ml glucose 6-phosphate dehydrogenase. Reactions were initiated by the addition of 1 mM NADP. Reactions with NADP and NAD as cofactors were initiated by addition of these compounds, to a final concentration of 1 mM, in the absence of glucose 6-phosphate dehydrogenase. Incubations were continued for 20 or 60 min as indicated, depending on substrate concentration. The reaction was stopped by extraction with 2x3 vol ethyl acetate. Recoveries of labeled compounds by extraction were routinely >99%. The combined extracts were evaporated under vacuum and the residue dissolved in the initial acetonitrile/methanol/water/acetic acid mix for HPLC elution. Unlabeled estrogen metabolite standards were added to verify retention times. All incubations were performed in triplicate and data are presented as means ± SD of three replicates.
HPLC analysis
Analysis of E2 metabolites was performed on a Supelcosil C18 column (5 µm, 4.6 mmx25 cm) at 30°C (Supelco, Bellefonte, PA). The elution conditions were based on a system developed by Robert Breuggemeier (personal communication). A binary solvent gradient consisting of acetonitrile/methanol/water/acetic acid was used for elution of the compounds. Specifically, solvent A was 21% acetonitrile/22% methanol/57% water/0.1% acetic acid and solvent B was 40% acetonitrile/60% water/0.33% acetic acid. The gradient was as follows: 015 min 100% solvent A; a linear increase from 1525 min to 19% solvent B; a linear increase from 2535 min to 20% solvent B; a linear increase from 3153 min to 100% solvent B; the column was returned to initial conditions over 20 min. Retention times for unlabeled metabolite standards, as detected by monitoring absorbance at 280 nm, were as follows: 6
-OH-E2, 6.00 min; E3, 7.00 min; 4-OH-E2, 13.50 min; 2-OH-E2, 15.50 min; 2-OH-E1 and 4-OH-E1 co-eluted at 17.75 min; E2, 24.00 min; E1, 29.50 min. Radiolabeled metabolites were detected and quantified using a Packard Flo-One Beta detector with Ultima Flo M liquid scintillant. The limit of detection for individual metabolite peaks was ~0.3% of total substrate. Tabulated data represent means ± SD from analysis of triplicate incubations.
Western immunoblot
Liver microsomes (50 µg) were resolved by SDSPAGE on 412% gradient NuPage gels in MOPS buffer. They were then transferred to a PVDF membrane (Immobilon-P; Millipore). Non-specific binding was blocked using Tris-buffered saline (10 mM TrisHCl, pH 7.4, 150 mM NaCl, containing 10% non-fat milk) for 1 h at room temperature. The membranes were probed for 2 h at room temperature with polyclonal antibodies for CYP 1B1, 2B1/2 and 3A1/2. Antibody binding for CYPs 2B1/2 and 3A1/2 was detected using alkaline phosphatase-conjugated anti-rabbit IgG, then visualized using BCIP/NBT phosphatase substrate. Anti-CYP 1B1 antibody was detected by incubation for 1 h with a horseradish peroxidase-linked donkey anti-rabbit IgG using the enhanced chemiluminescence method.
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Results
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Initial studies of the protein and time dependence of E2 metabolism by RLM from female ACI and SpragueDawley rats demonstrated a linear increase from 0.3 to 3.0 mg protein/ml and similar increases from 20 to 60 min incubation (data not shown). Based on these results, the experimental conditions were standardized to 1.0 mg protein/ml and 20 min incubation. Next, microsomes from ACI and SpragueDawley rats were incubated with a range of concentrations of E2 from 9 nM to 30 µM, to characterize the dependence on substrate concentration. Striking quantitative and qualitative changes in metabolic profiles were observed as the E2 concentration was decreased. These changes are demonstrated by the data in Figure 1
, showing representative HPLC profiles resulting from E2 metabolism at the high and low ends of this concentration range.

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Fig. 1. Estradiol metabolite profiles as a function of strain and estradiol concentration. [3H]E2 was incubated with rat liver microsomes (1 mg microsomal protein/ml) for 20 min with a NADPH generating system. Ethyl acetate extracts were processed and analyzed by reverse phase HPLC as described in Materials and methods. These data were generated using RLM from mature rats. Similar results were obtained with immature animals.
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With an initial concentration of 30 µM E2 the major metabolite produced by RLM from both strains was E1, with minor amounts of 2-OH-E2 as the only other detectable product (Figure 1A and C
). In the SpragueDawley preparation containing 9 nM E2 the major metabolite formed was still E1, but there was a nearly equal contribution of aromatic hydroxylation, specifically formation of 2-OH-E2 and 2/4-hydroxyestrone (CE1) (Figure 1B
). A more dramatic change in metabolism was seen in the ACI RLM at nanomolar E2 concentrations (Figure 1D
). At this E2 concentration E1 was no longer the major metabolite produced and instead aromatic hydroxylation was the major pathway. Moreover, the predominant catechol estrogen formed was 4-OH-E2, with lesser amounts of 2-OH-E2 and CE1 found. Examination of the kinetics of individual product formation at times from 5 to 20 min incubation showed that 2-OH-E2, 4-OH-E2 and E1 yields increased steadily with incubation time, whereas CE1 formation was only detectable after the 5 min incubation time (data not shown). This is consistent with their formation as primary and secondary metabolites of E2. Parallel incubations were performed with 3H- and 14C-labeled E2 at initial substrate concentrations of 0.3, 1 and 10 µM, which produced identical product distributions, demonstrating that loss of tritium was not a significant factor in our studies (data not shown).
This concentration-dependent shift in the spectrum of metabolites formed by RLM from each strain is shown by the tabulated product yields (Table I
) and by the normalized product distributions (Figure 2
). The E2 concentrations chosen for this experiment were 30 µM, a concentration where E1 formation predominates, and 100 nM, where both strains produced significant yields of both dehydrogenation and hydroxylation products. At 30 µM E2 the major metabolite produced for both strains and both age groups was E1, accounting for 5595% of metabolites (Figure 2A
). 2-OH-E2 is the next most abundant metabolite, ranging from 8% in immature ACI rats to >40% in mature SpragueDawley rats. The only group that produced detectable 4-OH-E2 at this E2 concentration was the mature ACI rats and this was <10% of total metabolites. At 100 nM E2, E1 remained the major product from SpragueDawley microsomes, but fell to 45% of total metabolites (Figure 2B
). 2-OH-E2 production remained the same in each strain for each age when the E2 concentration was decreased. Also, at 100 nM E2 there was production of CE1 by all groups but immature ACI rats. Formation of the catechol estrones requires two oxidative steps, presumably the conversion of E2 to estrone followed by aromatic hydroxylation to the catechol. Our HPLC procedure could not resolve the catechol estrones 2-OH-E1 and 4-OH-E1, but it could distinguish the catechol estrones from the catechol estradiols 2-OH-E2 and 4-OH-E2. Strikingly, at 100 nM E2 RLM from both age groups of ACI rats produced more 4-OH-E2 than any other metabolite, accounting for 35% of metabolites in mature ACI rats and 70% of metabolites formed in immature ACI rats. It is key to note that the clear qualitative differences in metabolite profile for E2 oxidation are between strains and that only minor quantitative differences are observed between immature and mature female rats of the same strain.
The ACI preparations were incubated with various cofactors and inhibitors to determine what enzymes or families of enzymes were responsible for E2 metabolism (Figure 3
). We chose 1 µM E2 as the initial substrate concentration to ensure significant production of E1 and of both aromatic hydroxylation products. Incubations containing the standard NADPH generating system were used as a control. The major metabolite produced by ACI liver microsomes was 4-OH-E2, with lesser amounts of 2-OH-E2 and E1 being formed. Incubations of 1 µM E2 with ACI RLM with the NADPH generating system and 10 µM clotrimazole, a broad CYP inhibitor, showed a 75% decrease in production of 4-OH-E2 and 2-OH-E2, indicating that these are products of CYP-dependent reactions. Also, there was an increase in E1 production, a 17ß-hydroxysteroid dehydrogenase (17ß-HSD)-dependent reaction. When the cofactor NADP was added without glucose 6-phosphate dehydrogenase there was a 70% decrease in production of 4-OH-E2 and 2-OH-E2, with a corresponding increase in E1 production. This is consistent with the ability of NADP to serve as a cofactor for 17ß-HSD (30,31) but not for CYP-dependent hydroxylation. There was some production of 4-OH-E2 and 2-OH-E2, which could be attributed to reduction of the NADP to NADPH by 17ß-HSD or other microsomal dehydrogenases, which could then serve as a cofactor for CYP-dependent metabolism of E2 to the catechols. When NAD, a cofactor that cannot be reduced to NADPH, was added there was complete abolition of formation of the catechol estrogens.

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Fig. 3. Modulation of estradiol oxidation by ACI rat liver microsomes. RLM (1 mg/ml) were incubated for 20 min with 1 µM [3H]E2 with either a NADPH generating system or initiation by addition of 1 mM NAD or NADP. Clotrimazole was added 10 min prior to the initiation of reactions. Extraction and analysis were as described under Materials and methods and for Figure 1 . Data represent the means ± standard deviations from the analysis of three replicate incubations. Comparison of the rate of individual metabolite formation to the rate observed with the NADPH generating system was performed by one-way ANOVA followed by Dunnett's test. *, Significant difference from NADPH generating system, P < 0.05.
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The differences in hydroxylation products formed in the two strains of rats suggests that there are different CYPs present in liver microsomes from these two strains. Different enzymes might be expected to exhibit different kinetic constants. SpragueDawley and ACI RLM were incubated for 20 min with E2 concentrations of 0.003, 0.01, 0.03, 0.10, 0.30, 1, 3, 10, 30 and 60 µM. Using graphical analysis of initial rate data, kinetic constants were obtained for the three metabolites of interest for ACI and SpragueDawley rats (Table II
). Catechol estrones, which result from two sequential enzymatic reactions, were not included in the calculation of initial rate, either as distinct products or as contributors to either aromatic hydroxylation of dehydrogenation. We found that the Vmax and Km for E1 in both rat strains are significantly higher than for aromatic hydroxylation. This is consistent with the known properties of 17ß-HSD, a high capacity and relatively high Km enzyme for E2 oxidation. Aromatic hydroxylation, however, is catalyzed by CYPs. Second, there is no significant difference between the E2 Km value for 2-hydroxylation in SpragueDawley and ACI rats. In contrast, the E2 Km value for 4-hydroxylation in ACI rats is significantly lower than the Km for 2-hydroxylation in ACI rats. These data suggest that the same CYP may catalyze 2-hydroxylation of E2 in both strains, but that the 4-hydroxylation seen with ACI RLM is catalyzed by a different enzyme.
The search for this different CYP-dependent enzyme was carried out by western analysis of RLM. Immunoblot analysis was performed on the microsomal fractions of each rat strain and each age group to determine the expression level of CYPs known to be involved in E2 metabolism. Under the conditions studied there is expression of CYPs 2B1/2 and 3A1/2 in both strains of rat (Figure 4
). There are no major differences in the expression of CYP 2B1/2 or 3A1/2 between the two age groups of ACI rats. Both immature and mature SpragueDawley rats express comparable levels of CYP 2B1/2, but immature SpragueDawley rats have diminished 3A1/2 expression relative to mature rats. In this study we also examined the effect of chronic E2 administration on hepatic CYP expression. No effects were detected. This was mirrored by a lack of detectable effect of E2 treatment on the ability of RLM to metabolize E2 (data not shown). ACI and SpragueDawley RLM were analyzed for expression of CYP 1B1 as well. There was no detectable expression of CYP 1B1, a known estradiol 4-hydroxylase, in RLM from either strain of rat (data not shown).

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Fig. 4. Western analysis of cytochrome P450 expression in rat liver microsomes. Rat liver microsomes (50 µg) were resolved by SDSPAGE on 412% gradient gels, transferred to a PVDF membrane and probed with polyclonal antibodies for CYPs 2B1/2 and 3A1/2 for 2 h at room temperature. Antibody reactions were detected using alkaline phosphatase-conjugated anti-rabbit IgG, then visualized using BCIP/NBT phosphatase substrate. SD, SpragueDawley; C, control; E2, 17ß-estradiol-treated; 6 wk, sexually immature animals; 12 wk, sexually mature animals.
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Discussion
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We have demonstrated that liver microsomes from ACI and SpragueDawley rats both efficiently metabolize E2 over a wide range of initial substrate concentrations. These liver preparations catalyze only aromatic hydroxylation and the 17ß-dehydrogenation of E2. This is in marked contrast to male SpragueDawley liver microsomes, which yield a far more complex mixture of oxidized metabolites (G.A.Reed, unpublished observation; 32). Similar metabolite profiles are produced by RLM from females of the two strains at high initial E2 concentrations (i.e.
10 µM), but this changes dramatically when the substrate concentration is lowered into the nanomolar range. SpragueDawley rats yield more 2-OH-E2 relative to E1 as the initial substrate concentration is decreased, but E1 remains the major product. In ACI rats, however, aromatic hydroxylation dominates and 4-OH-E2 is produced as the predominant metabolite at lower E2 concentrations.
Previous reports on the metabolism of E2 by microsomal preparations and by purified CYPs have employed extremely high substrate concentrations, ranging from 20 to 200 µM (3234; reviewed in ref. 35). Our qualitative findings at high E2 concentrations, i.e. that E1 is the major metabolite formed followed by 2-OH-E2, are consistent with other laboratories studying E2 metabolism by RLM from female SpragueDawley rats and from other strains as well (33,34). With microsomes from the ACI rat, however, this qualitative result changes dramatically, but only when the initial E2 concentration is lowered toward more realistic levels. We have shown that rat liver microsomes oxidize E2 at low concentrations, nearing actual physiological concentrations. These lower, more physiologically relevant concentrations, which have not been examined before, were required in order to observe the dramatic differences in metabolite profiles between these two rat strains.
In rats hepatic hydroxylation of E2 to the catechols 2-OH-E2 and 4-OH-E2 is catalyzed by CYPs 1A2, 2B1/2, 2C6, 2C11, the 2D family and the 3A family (reviewed in 11,35). Production of 2-OH-E2 by these CYPs greatly exceeds formation of 4-OH-E2, with 8090% production of 2-OH-E2 and only 1020% production of 4-OH-E2. This greater production of 2-OH-E2 reflects the orientation of E2 in the active site of the enzyme. Thus, formation of more 2-OH-E2 than 4-OH-E2 is a consequence of the binding interaction between substrate and CYP and should not be dependent on the concentration of E2. In contrast, human CYP 1B1 is unique in that it is reported to be a specific 4-hydroxylase of E2 (36). The presence of CYP 1B1 as a constitutive enzyme in female ACI rat liver would be consistent with our observed metabolite profile. By comparing the Km values determined for ACI RLM, it is apparent that 4-hydroxylation of E2 is catalyzed by a different enzyme with a lower Km than that for 2-hydroxylation. The Km determined for 4-hydroxylation of E2 in ACI RLM, substantially lower than that determined for 2-hydroxylation of E2 in the same incubations, matches the reported E2 Km value for 4-hydroxylation by recombinant human CYP 1B1 (36), providing an additional suggestion that this CYP may be responsible for the observed biotransformation of E2.
We found, however, that in female ACI rat liver there are no qualitative differences in the CYPs tested from what is expressed in the SpragueDawley female liver. There was expression of CYPs 2B1/2 and 3A1/2 but no detectable expression of CYP 1B1 in microsomes from either strain of rat. This suggests that 4-OH-E2 formation in ACI rats is due either to an additional CYP not probed for or a polymorphism in an existing CYP that alters the site of E2 hydroxylation. This latter proposal is particularly interesting in the light of a recent report from Shimada et al. (37). They characterized polymorphic human CYP 1B1 variants and found that these variants altered the ratio of 4-hydroxylation to 2-hydroxylation of E2 catalyzed by these enzymes. This subtle modulation of the orientation of E2 in the active site of a CYP and the resultant alteration in the site selectivity for E2 hydroxylation is precisely what we believe is responsible for the different metabolite distribution produced by ACI as opposed to SpragueDawley liver.
The proposed mechanisms for the carcinogenic action of estrogens depend on either estrogenicity or reactivity of the active species and both can be modulated by oxidative metabolism of estrogens. 2-OH-E2 has a lower affinity for the estrogen receptor (38,39) and has lower estrogenicity than the parent hormone E2 (40,41). Rapid O-methylation of 2-OH-E2 by catechol O-methyltransferase (COMT) results in a more rapid clearance in vivo and may also result in product inhibition of tumor cell proliferation (42,43). The resulting O-methylation of 2-OH-E2 may be the reason for its lack of carcinogenicity. 4-OH-E2, however, is a carcinogenic metabolite of E2 (44,45). It binds to the estrogen receptor with a similar affinity to E2 and it also activates the estrogen receptor (38). 4-OH-E2 takes longer to dissociate from the estrogen receptor compared with E2, therefore, there could be increased biological effects of 4-OH-E2 in comparison with E2 (46). COMT-catalyzed O-methylation is inhibited by 4-OH-E2. Regardless of the mechanism, 4-OH-E2 is important in the carcinogenicity of E2. It is not surprising that ACI rats, which are more sensitive to E2 as a mammary carcinogen, produce more of this detrimental metabolite 4-OH-E2 than SpragueDawley rats at near physiological levels of substrate.
Liver metabolism of E2 is an important first step in understanding the role of metabolism in mammary carcinogenesis. The liver plays a role in controlling the systemic levels of circulating metabolites. In addition to the liver, however, there is much interest in the metabolism of E2 in target tissues (35). Our next goal is to investigate the metabolism of E2 in the mammary gland to see whether there are differences in the target tissue that may be responsible for the differences in susceptibility of the two strains of rat. If there are differences in metabolism in the target tissue (the mammary gland) this would provide additional support for the role of metabolism of E2 in carcinogenicity.
In summary, these results reveal that there are dramatic differences in the hepatic metabolism of E2 in ACI and SpragueDawley rats and that these differences are only apparent as the conditions approach physiological concentrations of E2. Since 4-OH-E2 is thought to be the main carcinogenic metabolite of E2, it is even more interesting that ACI rat liver produces primarily this metabolite when challenged with near physiological levels of E2. The exclusive formation of this metabolite by the rat strain which is more sensitive to the carcinogenic effects of E2 suggests a role for this difference in E2 disposition in the different responses of the strains.
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Notes
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1 To whom correspondence should be addressed at: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160-7417, USA Email: greed{at}kumc.edu 
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Acknowledgments
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Western analysis for CYP 1B1 was performed by Nigel Walker (National Institute for Environmental Health Sciences, Research Triangle Park, NC). This work was supported by the US Army Breast Cancer Research Program (DAMD17-1-7155) and by NIH ES-07079.
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Received July 26, 2000;
revised September 28, 2000;
accepted October 3, 2000.