Introduction

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Fatty acid esters of estradiol (conjugated at the 17-hydroxyl group) are extremely lipophilic metabolites that are formed by enzymatic esterification of estradiol in the presence of either saturated or unsaturated fatty acyl-CoAs. Fatty acyl-CoA:estradiol acyltransferase, which catalyzes the esterification of estradiol, is present in liver as well as in extrahepatic tissues (Schatz and Hochberg, 1981; Mellon-Nussbaum et al., 1982; Adams et al., 1986; Martyn et al., 1987, 1988; Paris and Rao, 1989). Estradiol fatty acid esters have been found in human blood (Janocko and Hochberg, 1983) and in human ovarian follicular fluid (Larner et al., 1993), indicating that these esters are naturally occurring metabolites of estradiol that are present in humans. The physiological role of the esterified metabolites of estradiol, however, is not known.

Fatty acid esters of estradiol possess little or no estrogen receptor binding affinity (Janocko et al., 1984), and their hormonal activity comes from the release of the parent estradiol by esterase (Katz et al., 1987, 1991). Because of their high lipophilicity, these endogenous metabolites are present at very low concentrations in the blood, and at higher concentrations in fat (Larner et al., 1992). Estradiol fatty acid esters have very long half-lives in vivo (Hershcopf et al., 1985; Larner and Hochberg, 1985), and they may function as a reservoir, particularly in fat-rich tissues, for the prolonged release of hormonally active estradiol. Earlier studies indicated that estradiol fatty acid esters have prolonged estrogenic activity (Larner et al., 1985; Vazquez-Alcantara et al., 1985, 1989; Hochberg et al., 1990; Zielinski et al., 1991). It is expected that altering the metabolic formation of estradiol fatty acid esters in vivo may alter the hormonal activity of estradiol.

Clofibrate and gemfibrozil (classical peroxisome proliferators) are commonly prescribed hypolipidemic drugs (Hess et al., 1965; Reddy and Lalwani, 1983). Clinical studies indicated that men chronically receiving clofibrate manifested side effects related to disturbed sex hormone function such as decreased libido and breast tenderness or enlargement (The Coronary Drug Project Research Group, 1975). Recent studies from our laboratory indicated that treatment of rats with clofibrate or gemfibrozil caused a manyfold increase in the liver microsomal formation of estradiol fatty acid esters (Xu et al., 1997, 2001). These results suggest that clofibrate and gemfibrozil may prolong or enhance the hormonal action of endogenous estradiol, particularly in the fat-rich mammary gland.

In the present study, we evaluated the effect of pretreatment of ovariectomized rats with clofibrate on estradiol-induced increases in uterine weight, the number of lobules in the mammary gland, and the incorporation of bromodeoxyuridine into lobular and ductal cells in the mammary gland.

	Materials and Methods

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Chemicals. [2,4,6,7,16,17-³H(N)]Estradiol (110-170 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, MA). Estradiol, clofibrate, oleoyl-CoA, and bromodeoxyuridine were purchased from the Sigma Chemical Co. (St. Louis, MO). Solvents for extraction of metabolites and for HPLC assays were of HPLC grade, and they were purchased from Fisher Scientific (Pittsburgh, PA).

Animals. Five-week-old Sprague-Dawley rats (ovariectomized at 4 weeks) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). The animals were kept on a 12-h light/dark cycle, and allowed free access to water throughout the experiment. For studies on the assay of liver enzymes, the rats were fed different doses of clofibrate (0.15, 0.30, 0.45, or 0.60% by weight) in AIN-76A diet (Research Diets, New Brunswick, NJ) for 4 weeks. The animals were sacrificed, and liver samples were removed for the preparation of microsomes as previously described (Thomas et al., 1983). The protein concentration in microsomal preparations was determined by using a Bio-Rad protein assay (bovine serum albumin as a standard) according to the instructions of the provider.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Protocol for studies on the effect of feeding rats 0.6% clofibrate in AIN-76A diet or control AIN-76A diet alone on the action of estradiol.

Esterification of Estradiol by Rat Liver Microsomes. Enzyme assays for the esterification of estradiol by rat liver microsomes were carried out as described previously (Xu et al., 2001). Incubation mixtures consisted of 50 µM ³H-labeled estradiol (~1-2 µCi), 100 µM oleoyl-CoA, together with 5 mM magnesium chloride in 0.1 M sodium acetate buffer (pH 5.0). For the preparation of the incubation mixture, radioactive estradiol in ethanol was added first, dried under nitrogen, and then the remaining components of the incubation mixture (including nonradioactive estradiol in 5 µl of ethanol) were added. This procedure resulted in uniform distribution of radioactive and nonradioactive estradiol throughout the incubation mixture. The reaction was initiated by the addition of hepatic microsomes (1 mg of protein/ml for microsomes from control rats or 0.5 mg of protein/ml for microsomes from induced rats). After incubation at 37°C for 30 min, the reaction was arrested by placing the tubes on ice, followed by addition of 0.5 ml of ice-cold sodium acetate buffer (pH 5.5) and 5 ml of ethyl acetate. The samples were vortexed immediately and then centrifuged for 10 min at 3000g. The organic phase was removed and the extraction was repeated a second time. The organic solvent extracts were combined and evaporated to dryness under a stream of nitrogen. The resulting residues were dissolved in 100 µl of methanol and were analyzed by HPLC with a radioactivity detector as described earlier (Xu et al., 2001). It is important to note that high-purity ethyl acetate (99.9% pure) must be used, since the use of ethyl acetate of lower purity results in the formation of artifact peaks on the HPLC chromatography.

Deesterification of the Oleoyl Ester of Estradiol by Fat Microsomes and Cytosol (Esterase Assay). Abdominal fat pads from 10 female rats (8 weeks old) treated with 0.5% clofibrate in AIN-76A diet for 2 weeks or fat pads from 10 rats treated with AIN-76A diet alone were removed. Two batches of five fat pads from each group were pooled to provide two pooled fat samples from control rats and two pooled fat samples from clofibrate-treated rats. The fat was ground in liquid nitrogen to obtain small particles. The fat particles were then homogenized in 2.5 volumes of homogenizing buffer [0.05 M Tris-HCl (pH 7.4) in 1.15% KCl] at 4°C with a Polytron homogenizer and centrifuged at 9000g for 30 min. The supernatant (minus the upper fat layer) was centrifuged at 105,000g for 90 min. The upper supernatant cytosol fraction was filtered through a Kimwipe to obtain a clear cytosolic supernatant fraction. The microsomal pellet was resuspended in 0.25 M sucrose.

BrdU Labeling. Two hours before sacrifice, animals from experiments 1 and 2 were injected i.p. with 50 µg of BrdU/g of body weight (dissolved in ~0.5 ml of sterile saline). The abdominal pair of mammary glands was removed, fixed in Carnoy's fixative for 3 h, transferred to 80% ethanol, embedded in paraffin blocks, cut into 5-µm sections, and placed on Superfrost microscope slides (Fisher Scientific). The sections were placed in xylene to remove paraffin and rehydrated using a gradient of ethanol with decreasing concentrations. The slides were placed in 4.0 N HCl for 20 min for denaturing, and then placed in a solution of 3% H₂O₂ (in methanol) to quench endogenous peroxidase activity. Sections were blocked using 1% normal horse serum. The sections were incubated with anti-BrdU primary antibody (Novocastra, Newcastle, UK) at a 1:100 dilution for 60 min, followed by a 1:100 dilution of a biotinylated horse anti-mouse secondary antibody (rat absorbed) (Vector Laboratories, Burlingame, CA) for 45 min at room temperature. Detection was performed using ABC Elite reagent (Vector Laboratories), and color was developed using diaminobenzidine (Sigma Chemical Co.) for 5 min. Harris hematoxylin (Sigma Chemical Co.) was used as a counterstain, and the slides were preserved using Permount (Fisher Scientific). The number of lobules per microscopic field and the labeling index (number of cells that incorporated BrdU/total number of cells counted × 100) in the ducts and lobules of the mammary gland were determined using a light microscope. For determination of the number of lobules, the magnification was 100-fold (10× objective, 10× ocular). For studies on BrdU labeling, the magnification was 400-fold (10× objective, 40× ocular).

Statistical Analysis. Data are presented as the mean ± S.E.M. Differences between means were assessed by using the two-way ANOVA test. Since there were interactions between the two factors studied (clofibrate and estradiol), we compared the means between the control group and clofibrate-treated group at each dose of estradiol infusion (Kendall, 1993). P values <0.05 were considered significant.

	Results

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Stimulatory Effect of Clofibrate Administration on Liver Microsomal Fatty Acyl-CoA:Estradiol Acyltransferase in Ovariectomized Rats

Administration of 0.15, 0.30, 0.45, or 0.60% clofibrate in AIN-76A diet to ovariectomized rats for 4 weeks stimulated liver microsomal fatty acyl-CoA:estradiol acyltransferase activity by 6-, 14-, 16-, and 21-fold, respectively (Fig. 2). The liver/body weight ratios were increased 13, 29, 55, and 99%, respectively (data not shown). These results are similar to what we observed in female intact rats in an earlier study (Xu et al., 2001). In an additional study, we examined the time course for the effect of clofibrate administration on the induction of fatty acyl-CoA:estradiol acyltransferase. Female rats were fed 0.6% clofibrate for 4, 8, 14, and 21 days, and maximum induction was observed after 8 days of treatment (data not shown).

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Stimulatory effect of dietary clofibrate administration on the formation of estradiol-oleoyl ester by rat liver microsomes. Ovariectomized rats were fed AIN-76A diet or 0.15 to 0.60% clofibrate in AIN-76A diet for 4 weeks. Liver was removed for the preparation of microsomes. The incubation mixture consisted of 1 mg of microsomal protein per milliliter from control rats or 0.5 mg of microsomal protein per milliliter from clofibrate-treated rats, 50 µM 3H-labeled estradiol, and 100 µM oleoyl-CoA. Formation of estradiol oleoyl ester was measured as described under Materials and Methods. Each value is the mean ± S.E.M. obtained from liver microsomes from three rats.

Stimulatory Effect of Clofibrate Administration on the Deesterification of Estradiol-Oleoyl Ester by Fat Microsomes and Cytosol

Administration of 0.5% clofibrate in AIN-76A diet for 2 weeks stimulated fat microsomal and fat cytosolic esterase activity by 125 and 90%, respectively (10 rats/group). The mean and individual esterase activity values (pmol of estradiol formed/mg of protein/h) for two pooled fat pad samples (each obtained from five rats) from control animals and two pooled fat pad samples from clofibrate-treated animals (each obtained from five rats) were 33.9 (40.1, 27.6) pmol of estradiol formed/mg of protein/h for fat microsomes from control rats, 76.2 (77.7, 74.7) pmol of estradiol formed/mg of protein/h for fat microsomes from clofibrate-treated rats, 33.6 (40.2, 27.1) pmol of estradiol formed/mg of protein/h for fat cytosol from control rats, and 63.8 (68.2, 59.4) pmol of estradiol formed/mg of protein/h for fat cytosol from clofibrate-treated rats.

Effect of Dietary Clofibrate on the Action of Estradiol in Ovariectomized Rats

Experiment 1. Ovariectomized rats were pretreated with 0.6% clofibrate in AIN-76A diet for 8 days. An Alzet pump (model 2002) that delivered an estradiol solution at a constant rate (0.5 µl/h; 0.1 or 1 µg of estradiol per day) was then implanted subcutaneously, and dietary clofibrate was continued. An earlier study showed that estradiol implantation that provided a dose of 1 µg/day produced a stable estradiol plasma level comparable to the plasma estradiol concentration at diestrus during the estrous cycle in rats (Butcher et al., 1978).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Lack of effect of clofibrate administration on the uterotropic response to estradiol (E2). In experiment 1, ovariectomized rats were fed AIN-76A () diet or 0.6% clofibrate in AIN-76A diet () for 18 days. On the 8th day of the experiment, an Alzet pump that delivered 0.1 or 1 µg of estradiol per day was implanted subcutaneously. After 10 days of estradiol infusion, the animals were sacrificed and uterine wet weights were measured. Each value is the mean ± S.E.M. obtained from five rats. In experiment 2, ovariectomized rats were fed AIN-76A diet () or 0.6% clofibrate in AIN-76A diet () for 24 days. On the 8th day of the experiment, an Alzet pump that delivered 0.2 or 1 µg of estradiol per day was implanted subcutaneously. After estradiol infusion for 10 days, the pump was removed. Animals were sacrificed before removal of the estradiol pump and on the 3rd and 6th day after removal of the pump. Each value is the mean ± S.E.M. obtained from 9 to 10 rats.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Stimulatory effect of clofibrate administration on the number of lobules per microscopic field in the mammary gland. In experiment 1, ovariectomized rats were fed AIN-76A () or 0.6% clofibrate in AIN-76A diet () for 18 days. On the 8th day of the experiment, an Alzet pump that delivered 0.1 or 1 µg of estradiol per day was implanted subcutaneously. After 10 days of estradiol infusion, the animals were sacrificed and the pair of abdominal mammary glands was removed for staining and histological examination. The number of lobules in 10 microscopic fields (100-fold magnification) was determined in mammary glands from each rat. Each value is the mean ± S.E.M. obtained from three to five rats. In experiment 2, ovariectomized rats were fed AIN-76A diet () or 0.6% clofibrate in AIN-76A diet () for 24 days. On the 8th day of the experiment, an Alzet pump that delivered 0.2 or 1 µg of estradiol per day was implanted subcutaneously. After estradiol infusion for 10 days, the pump was removed. Animals were sacrificed immediately before removal of the estradiol pump and on the 3rd and 6th day after removal of the pump. The number of lobules per microscopic field in mammary glands from each rat was determined as described in experiment 1. Each value is the mean ± S.E.M. obtained from 6 to 10 rats. *P < 0.05 compared with control diet group implanted with estradiol (1 µg/day). **P < 0.001 compared with control diet group implanted with estradiol (1 µg/day).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Effect of clofibrate administration on BrdU labeling in lobular and ductal cells in the mammary gland. In experiment 1, ovariectomized rats were fed AIN-76A or 0.6% clofibrate in AIN-76A diet for 18 days. On the 8th day of the experiment, an Alzet pump that delivered 0.1 or 1 µg of estradiol (E2) per day was implanted subcutaneously. After 10 days of estradiol infusion, the animals were injected i.p. with bromodeoxyuridine (BrdU; 50 µg/g of body weight) 2 h before sacrifice. The labeling index (number of cells that incorporated BrdU/total number cells counted × 100) was determined in ducts and lobules of the mammary gland using 400-fold magnification (10× objective, 40× ocular). Each value is the mean ± S.E.M. obtained from three to five rats. In experiment 2, ovariectomized rats were fed AIN-76A or 0.6% clofibrate in AIN-76A diet for 24 days. On the 8th day of the experiment, an Alzet pump that delivered 0.2 or 1 µg of estradiol per day was implanted subcutaneously. After estradiol infusion for 10 days, BrdU was injected as described above, and the animals were sacrificed 2 h later. Additional animals were sacrificed on the 3rd and 6th day after removal of the pump. BrdU labeling index in ductal and lobular cells was determined as described above. Each value is the mean ± S.E.M. obtained from 6 to 10 rats. *P < 0.05 compared with the control diet group implanted with estradiol (1 µg/day).

Experiment 2. In experiment 1, little or no estrogenic effect on the mammary gland was observed in rats infused with a low dose of estradiol (0.1 µg/day) for 10 days. In experiment 2, we selected a higher dose of estradiol (0.2 µg/day) and repeated the 1-µg/day dose. After 10 days of estradiol infusion, the estradiol pump was removed and the estrogenic effects on the uterus and mammary gland were determined just before discontinuation of estradiol infusion and at 3 and 6 days after discontinuation of the estradiol infusion.

View larger version (150K): [in this window] [in a new window]   Fig. 6.   Histological appearance of the mammary gland from rats treated with estradiol or clofibrate and estradiol. Ovariectomized rats were fed AIN-76A diet or 0.6% clofibrate in AIN-76A diet for 24 days. On the 8th day, an Alzet pump that delivered 1 µg of estradiol per day was implanted subcutaneously. After estradiol infusion for 10 days, the animals were injected i.p. with BrdU (50 µg/g of body weight) and the animals were killed 2 h later. Tissues were processed as described under Materials and Methods. Pictures were taken using 100-fold magnification (10× objective, 10× ocular). Photographs (3.5 × 5 inches) were prepared. The brown-staining nuclei indicate BrdU incorporation into cellular DNA. The arrows indicate lobules. Control group (A), clofibrate-treated group (B), estradiol-infused (1 µg/day) group (C), clofibrate-treated + estradiol-infused (1 µg/day) group (D).

	Discussion

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In an earlier study, we found that treatment of rats with clofibrate for several days stimulated the liver microsomal metabolism of estradiol to fatty acid esters by fatty acyl-CoA:estradiol acyltransferase (Xu et al., 2001). This study, which was the first demonstration of an effect of an environmental agent on the esterification of an estrogen, provided an opportunity to explore the physiological significance of changes in estradiol esterification. Fatty acid esters of estradiol that are formed by fatty acyl-CoA:estradiol acyltransferase are extremely lipophilic, and have prolonged hormonal activity because they are slowly metabolized (Hershcopf et al., 1985; Larner and Hochberg, 1985). Large amounts of fatty acid esters of estradiol have been found in fat (Larner et al., 1992). We hypothesized that treatment of rats with clofibrate would enhance the hepatic metabolism of estradiol to lipophilic fatty acid esters that would concentrate in fat and in fat-rich tissues such as the mammary gland in vivo. The sequestered fatty acid esters of estradiol would be expected to undergo hydrolysis by esterases to biologically active estradiol in the mammary gland (Katz et al., 1987), thereby providing a source of estradiol and a strong estrogenic stimulus to the mammary gland.

Consistent with the above-described hypothesis, we observed in the present study that administration of clofibrate to ovariectomized rats that were infused with estradiol had a selective stimulatory effect on the action of estradiol in the mammary gland but not in the uterus. In both experiments 1 and 2, we observed that in ovariectomized rats infused with estradiol (1 µg/day) for 10 days, clofibrate administration enhanced the potency of estradiol toward the mammary gland, which is indicated by an increase in the BrdU labeling index in lobules and an increase in the number of lobules in the mammary gland (Figs. 4 and 5). We further determined whether the increased formation of estradiol fatty acid esters by clofibrate administration could prolong the duration of estrogenic activity after the estradiol implantation pump was removed (experiment 2). The results showed that on the 3rd day after estradiol administration was discontinued, clofibrate-treated rats maintained a somewhat higher level of cell proliferation in the mammary gland lobules than in control rats (Fig. 5). On the 6th day after the discontinuation of estradiol administration, there was no difference in the low level of proliferation of lobular and ductal cells in the mammary gland between control and clofibrate-treated animals. A possible reason for the lack of a prolonged estrogenic response in clofibrate-treated animals after the removal of the estradiol pump could be due to rapid hydrolysis of estradiol fatty acid esters in fatty tissue. In the present study, we found that esterase activity in fat tissue was increased about 100% in clofibrate-treated animals. Therefore, estradiol fatty acid esters sequestered in the mammary gland and in other fatty tissues of clofibrate-treated rats might be hydrolyzed to estradiol very quickly to exert a strong but rather short-lived estrogenic effect.

A stimulatory effect of clofibrate treatment on the estrogenic actions of an infusion of 1 µg of estradiol per day on the mammary gland was observed, but this effect was not seen with a lower dose of 0.1 or 0.2 µg of estradiol per day. Although administration of these low-dose levels of estradiol had a strong stimulatory effect on uterine growth (Fig. 3), these dosage regimens did not increase the number of lobules (Fig. 4) and had no or only a small stimulatory effect on BrdU incorporation into lobular and ductal DNA in the mammary gland (Fig. 5). The 1-µg/day dose of estradiol used in our study appears to be a physiologically relevant dose. An earlier study by Butcher et al. (1978) indicated that implantation of an estradiol Alzet osmotic pump at a dose level of 1 µg/day to ovariectomized rats produced a physiological plasma level of estradiol equivalent to that which occurs during diestrus in intact rats. It would be of interest to explore the effect of clofibrate administration on the hormonal actions of endogenous estradiol in intact rats.

The biological importance of enhanced estradiol esterification by clofibrate administration is not clear, and additional studies are needed to determine the effect of clofibrate administration on the in vivo levels of estradiol and estradiol fatty acid esters. It has been reported that some men treated chronically with clofibrate have breast tenderness or enlargement (The Coronary Drug Project Research Group, 1975). The possibility that these side effects of clofibrate are related to clofibrate-induced formation of estradiol fatty acid esters requires further investigation. Epidemiological studies are also needed to determine whether long-term treatment of patients with clofibrate and related hypolipidemic drugs alters the risk of breast cancer, endometrial cancer, osteoporosis, or other diseases that are influenced by estrogen.