Steroid hormones modulate expression of cytochrome P450 enzymes in male hamster reproductive tract and leiomyosarcomas
Chad E. Hudson,
Bradley A. Schulte1,,
Thomas R. Sutter2, and
James S. Norris3,
Department of Microbiology and Immunology,
1 Departments of Pathology and Laboratory Medicine and Otolaryngology and Communicative Sciences, Medical University of South Carolina, PO Box 250504, 173 Ashley Avenue, Charleston, SC 29425-2230 and
2 W.Harry Feinstone Center for Genomic Research, University of Memphis, Life Sciences Bldg, College of Arts & Sciences, 38152, USA
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Abstract
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Syrian hamsters treated with estrogen and androgen for 8 months develop leiomyosarcomas in the vas deferens. Metabolism of estrogen by cytochrome P450s (CYPs) produces catechols and reactive oxygen species, and may contribute to tumor formation. To examine this issue, male hamsters were treated with 17 ß-estradiol (E2), testosterone propionate (TP) or both hormones. Reproductive tract tissues from control and treated animals were immunostained with antibodies specific for four CYP enzymes (1A1, 1A2, 1B1 and 3A1/2). Immunoreactive CYP1A1 was not found in the reproductive tract of control or treated animals. In untreated hamsters, CYP1A2 was detected only in principal cells of the caput epididymis. TP alone had no effect, but treatment with E2 induced expression of CYP1A2 in columnar epithelial cells throughout the epididymis and lining of the vas deferens. Treatment with E2 + TP blocked the induction of CYP1A2 seen in surface epithelial cells treated with E2 alone, but not the constitutive expression of this enzyme. Instead, simultaneous exposure to both hormones induced CYP1A2 in basal cells of the epididymis and vas deferens. CYP3A1/2 was not detected in the reproductive tract of control or TP-treated males, but immunostaining was induced in the inner layer of vas deferens smooth muscle by E2, and in all smooth muscle layers by dual hormone treatment. In controls, CYP1B1 was present in smooth muscle lining the epididymis and surrounding the vas deferens and dual hormone treatment increased staining intensity for CYP1B1 in these cells. Immunoreactive CYP1A2 was not detectable in leiomyosarcomas but the enzyme was present in both columnar and basal cells of the vas deferens epithelium adjacent to the tumors. In contrast, tumor cells showed heterogeneous expression of both CYP1B1 and CYP3A1/2. The relationships between hormone treatment, differential CYP expression and tumor formation strengthen our hypothesis that metabolism of estrogen is an important element in this model of hormonal carcinogenesis.
Abbreviations: CYPs, cytochrome P450s; E2, 17 ß-estradiol; GST, glutathione S-transferase; LDL, low density lipoprotein; NGS, normal goat serum; NRS, normal rabbit serum; ROS, reactive oxygen species; TP, testosterone propionate.
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Introduction
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Treatment of Syrian hamsters for 8 months or longer with 17 ß-estradiol (E2) in combination with testosterone propionate (TP) causes leiomyosarcomas (smooth muscle tumors) in the vas deferens or uterus at a frequency of 100% (1). Withdrawal of either androgen or estrogen retards tumor growth, and withdrawal of both hormones results in tumor regression (1). Treatment with androgen alone causes mild smooth muscle hyperplasia in the vas deferens (2), while treatment with estrogen alone results in atrophy of the entire male reproductive tract, within 4 weeks (3). Treatment with estrogen alone also induces renal neoplasms in 100% of males, but not females (4).
The molecular mechanisms involved in the induction of leiomyosarcomas by androgen and estrogen have not been elucidated. We recently described steroid-induced changes in the expression of three isoforms of the phase II detoxification enzyme glutathione S-transferase (GST) in the male hamster reproductive tract (3). Treatment with E2 or E2 + TP for at least 4 weeks blocked the normal expression of GST
and GSTµ, but not that of GST
in the epithelial lining of the vas deferens (3). The decreased GST levels in vas deferens epithelium may expose underlying smooth muscle to endogenous carcinogens normally eliminated by these enzymes. However, it is also possible that the metabolism of E2 itself may contribute to tumor formation (3).
The oxidative metabolism of E2 by cytochrome P450s (CYPs) results in production of catechol estrogens and reactive oxygen species (ROS) (5). Catechol estrogens are potential carcinogens, especially 4-hydroxy catechols, which form DNA adducts leading to depurination and mutations (6). These compounds also act as intermediates in the production of semiquinones and quinones, which covalently bind DNA. The rapid cycling from quinone to semiquinone results in formation of superoxide anion (5). The ROS generated by this redox cycling have been shown to cause DNA damage (7) and promote carcinogenesis (8). Exposure of rats, for example, to ethinylestradiol resulted in increased levels of 8-hydroxydeoxyguanosine and ultimately led to hepatocellular carcinoma (8). Simultaneous administration of antioxidants prevented both the formation of 8-hydroxydeoxyguanosine and the induction of carcinoma.
Estrogen also can act as a non-genotoxic carcinogen (9). Administration of diethylstilbesterol causes neoplastic transformation in Syrian hamster embryo cells in the absence of demonstrable genetic mutations (10). Another non-genotoxic effect of estrogen in neoplasia is the covalent binding of ß-tubulin by quinoid estrogens and subsequent chromosomal changes such as aneuploidy or non-disjunction caused by the disruption of microtubules (11). The induction of renal cell carcinoma in the Syrian hamster by estrogens is characterized by trisomies and tetrasomies of nine different chromosomes, including chromosome 6, which results in amplification of the c-myc protooncogene (12). This suggests that estrogen induces genomic instability and it has been hypothesized that the resulting inappropriate copy number of gene(s) regulating cell growth and/or division is part of a multi-step process leading to tumorigenesis (12). It remains unclear whether the carcinogenicity of estrogens is due to these non-genotoxic effects, direct damage of DNA or a combination of both. It is important to note in this context, that even the non-genotoxic effects are promoted by a quinoid estrogen (11). The CYP-mediated formation of estrogenic metabolites and/or metabolic by-products, therefore, is an essential step in both the genotoxic and non-genotoxic pathways to carcinogenesis.
The CYP superfamily includes over 700 enzymes, classified by protein sequence homology into families (>40% similarity) and subfamilies (>55% similarity) (13). These enzymes are not related to the cytochrome oxidases in the mitochondria, and in mammalian cells are most often located on smooth endoplasmic reticulum (13). CYP1A1, CYP1A2, or members of the CYP3A family form predominately the 2-hydroxy catechol estrogens (5,6,14), while the newly identified CYP1B1 forms predominately the 4-hydroxy catechols (1519). The specific activities of these enzymes have been investigated in Syrian hamster liver and kidney (20). Members of the CYP3A family are primarily responsible for 2- and 4-hydroxylation of E2 in the liver, while a CYP1A enzyme catalyzes 2-hydroxylation in hamster kidney (20). A small part of the 4-hydroxylation in the kidney may be catalyzed by CYP3A enzymes, but there is an additional, unidentified kidney enzyme responsible for the majority of this activity (20). This 4-hydroxylation activity was induced by ß-napthaflavone (20), a response that has been attributed to CYP1B1 in other systems (21) suggesting strongly that this unidentified hamster enzyme could be CYP1B1 (20). The importance of CYP-mediated metabolism in both genotoxic and non-genotoxic mechanisms of estrogen-induced carcinogenesis supports the theory that metabolism of exogenously administered E2 is involved in the hormonal induction of leiomyosarcomas (3). To address this question, we treated male Syrian hamsters with either E2, TP or both and performed immunohistochemical analysis of reproductive tract organs to determine CYP expression patterns in normal tissues and changes affected by hormone treatment.
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Materials and methods
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Animal treatment and tissue processing for immunohistochemistry
All Syrian hamsters were housed and handled under a protocol approved by the Medical University of South Carolina's Animal Care and Use Committee. Thirty 40 day-old males were used for immunohistochemical studies. Animals were implanted subcutaneously with pellets containing 20 mg TP, E2 or both hormones (Hormone Pellet Press, University of Kansas Medical Center, Kansas City, KS) at ~50 days after birth and were killed at 2, 4 and 6 weeks post-implantation. Two animals were used for each treatment protocol and each time point, along with one age-matched untreated animal for each time point. To analyze the effects of long term treatment with E2 + TP, another group of males was implanted with E2 + TP and killed after 6, 8 and 9 months of treatment. Two animals were used for each time point, along with one age-matched untreated control.
Animals were euthanized with 30 mg pentobarbital, and reproductive tract tissues (testis; caput, corpus and cauda epididymis; epididymisvas deferens junction; distal vas deferens) along with other systemic organs were harvested rapidly, sliced into appropriately sized pieces and immersed for 30 min in a solution of 10% formalin containing 0.5% zinc dichromate with the pH adjusted to 5.0 immediately before use. Tissues were dehydrated in a graded series of ethanols (70%, 2 h; 80%, 2 h; 95%, 2 h and 100%, 3x1 h), cleared in Histoclear (2x1 h) (National Diagnostics, Atlanta, GA) and embedded in paraffin (Paraplast Plus, 2x1 h at 58°C) (Curtin Matheson, Atlanta, GA). Serial sections at 4 µm thickness were mounted on glass slides. Every twenty-fifth section was stained with hematoxylin and eosin and selected sections were immunostained following the protocol outlined below.
Immunoperoxidase staining
Deparaffinized and rehydrated sections were treated with 0.3% H2O2 to block endogenous peroxidase activity, and equilibrated in 0.1 M PBS, pH 7.2, containing 1% normal goat serum (NGS) or 1% normal rabbit serum (NRS), for subsequent incubation with primary antisera generated in rabbits or sheep, respectively. Sections were then incubated at 4°C overnight with one of the following antisera: rabbit anti-rat CYP1A1 (Chemicon International, Temecula, CA), diluted 1:1000 in PBS/NGS; sheep anti-rat CYP1A2 (Chemicon International), diluted 1:1000 in PBS/NRS; or rabbit anti-rat CYP3A1/2 (Xenotech LLC, Kansas City, KS), diluted 1:1000 in PBS/NGS.
Sections used for staining with rabbit anti-human CYP1B1, a rabbit polyclonal directed against human CYP1B1, were processed for antigen retrieval (22), with minor modifications. Following treatment with 0.3% H2O2, the sections were flooded with 10 mM citric acid monohydrate and heated for 20 min in an enclosed steamer (Rival Automatic Steamer) for 20 min. After cooling slowly to room temperature, the sections were equilibrated in PBS/NGS, and incubated overnight at 4°C with anti-CYP1B1, diluted to a final IgG concentration of 2 µg/ml in PBS/NGS.
Following exposure to primary antiserum, the sections were rinsed with PBS and incubated for 30 min with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) rinsed again with PBS, and flooded with an avidinbiotinhorseradish peroxidase complex (Vectastain ABC; Vector Laboratories) for 30 min. Sites of bound primary antibody were visualized by a 10 min development in 3,3'-diaminobenzidineH2O2 peroxidase substrate medium (Sigma Fast; Sigma, St Louis, MO). Sections from all treatment groups and all time points were stained in the same protocols to minimize method variability and provide side by side comparison of relative differences in immunostaining intensity.
Animal treatment and tissue processing for western blot analysis
Fifty male Syrian hamsters were used for western blot analysis with anti-CYP1B1. Two treatment protocols were employed. The first included five 40 day-old males treated with ß-NF exactly as described by Bhattacharyya et al. (21), and five age-matched controls. The second included forty 40 day-old males treated for 4 weeks with E2, TP or both hormones simultaneously, as described above. Five males received no hormone treatment and were used as age-matched controls, five males were treated with TP, 10 with E2 + TP and 20 with E2 (due to E2-associated atrophy of the male reproductive tract, more animals were required from the E2 and E2 + TP treatment groups to recover the same amount of microsomal protein). Animals were killed by barbiturate overdose and tissue samples were harvested immediately, pooled together by treatment group, frozen in liquid N2 and stored at 80°C until use. For microsomal preparations, samples were thawed slowly on ice and then homogenized in a buffer previously described by Otto et al. (23), using a Tissumizer (Tekmar, Cincinnati, OH) at 40% power with 35 intervals of 5 s each, allowing the samples to cool on ice between each interval. The samples were then centrifuged at 600 g for 10 min, 15 000 g for 20 min and 105000 g for 90min, saving the supernatant each time for use in the subsequent centrifugation. The resulting pellet was re-suspended using a Potter-Elvehjam homogenizer in a sodium pyrophosphate buffer described by Otto et al. (23) and recentrifuged at 105 000 g for 60 min. The microsomal pellets were then re-suspended in a Potter-Elvehjam tube with resuspension buffer (10 mM KPO4 pH 7.4, 2 mM MgCl2 and 2 mM dithiothreiotol), then aliquoted and stored at 80°C.
Protein concentration was determined using the Bradford method (Bio-Rad Protein Assay, Bio-Rad, Hercules, CA) with BSA as a standard. To characterize anti-CYP1B1 specificity, western blot analysis of the first treatment protocol group was performed using 10 µg total microsomal protein from the adrenal, testis, epididymis or vas deferens of control or ß-NF treated males. Semi-quantitative western blot analysis of the second treatment protocol group was then used to determine the effects of hormone treatment on levels of immunodetectable CYP1B1 in the male hamster reproductive tract. An 8% Trisglycine gel was loaded with 2.5, 5, 10 and 20 µg microsomal protein from one sample to verify the linear relationship of the assay. Microsomal protein (10 µg) from the epididymis of males treated with E2, TP or E2 + TP for 4 weeks was also incubated in the same gel. For all western blots, microsomal protein samples were electrophoresed using a denaturing 8% Trisglycine gel until the 7.5 kDa molecular weight standard reached the bottom of the gel, followed by transfer at 250 mA for 2 h onto a nitrocellulose membrane, with a transfer buffer containing 25 mM Tris, 190 mM glycine and 20% methanol. The membrane was blocked overnight at room temperature with 3% non-fat milk in TBS (10 mM TrisHCl, 0.15 mM NaCl, pH 6.5), and then incubated with anti-CYP1B1 diluted to a final IgG concentration of 5 µg/ml in incubation buffer (TBS with 0.3% Tween 20, and 1% non-fat milk). The membrane was washed with TBST (TBS with 0.1% Tween 20) twice, for 10 min each, and once with TBS for 10 min, and then incubated with HRP-conjugated goat anti-rabbit IgG (Pierce, Rockford, IL) diluted 1:75 000 in incubation buffer. Following three washes for 10 min each with TBST and two washes for 10 min each with TBS, the membrane was incubated with Super Signal Ultra Chemiluminescent Substrate (Pierce, Rockford, IL) for 5 min, and then exposed to X-ray film (CL-Xposure film; Pierce) and/or digitally photographed (ChemiImager 4400 Low Light Imaging System; Alpha Innotech, San Leonardo, CA) for densitometric analysis (1D-Multi Line Densitometry, AlphaEase software package; Alpha Innotech).
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Results
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Antibody reactivity and specificity
The three commercially available antibodies used for immunohistochemistry have been well characterized as reactive and specific in other species. Western blot analysis with anti-rat CYP1A1 (Chemicon International) demonstrates specific binding to CYP1A1 and not CYP1A2 in hepatic microsomes from rats treated with 3-methylcholanthrene. Furthermore, anti-CYP1A1 showed no immunoreactivity in hepatic microsomes from untreated rats (which contain CYP1A2 only), or to hepatic microsomes from rats treated with phenobarbital, pregnenolone, 16-
-carbonitrile or clofibrate, which induces CYP2B, CYP2C, CYP3A and CYP4A, respectively (personal communication, Nadia Mohammad, Chemicon International) Anti-ratCYP1A2 (Chemicon International), binds specifically to CYP1A2 in rat, human and mouse hepatic microsomes, and shows no cross-reactivity with other CYPs, including CYP1A1 (2427). In western blot analysis with rat hepatic microsomes, anti-rat CYP3A1/2 (Xenotech LLC), binds specifically to CYP3A1 and CYP3A2, and shows no cross-reactivity with other CYPs (2830). Furthermore, anti-CYP3A1/2 cross-reacts with CYP3A1 and CYP3A2 in dog, rhesus, CD1 and B6C3F1 mice (personal communication, Kammie Settle, Xenotech LLC).
Reactivity and specificity of these three commercially available antibodies in hamster tissue were confirmed by immunohistochemical staining in serial sections, processed and stained in the same protocol to allow a direct comparison of immunoreactivity (Table I
). In the large intestine, anti-CYP1A1 showed immunoreactivity in the cytoplasm, but not the nucleus, of epithelial cells, in contrast to the consistent nuclear staining seen in these epithelial cells with anti-CYP1A2 (unpublished observations). Anti-CYP3A1/2 stained the lamina propria, but not the epithelium, of the large intestine. In the liver, anti-CYP1A1 showed strong nuclear staining in some, but not all hepatocytes, while anti-CYP1A2 showed weaker nuclear immunoreactivity, in a different subset of hepatocytes than those stained with anti-CYP1A1. In contrast to the anti-CYP1A family members, anti-CYP3A1/2 showed no immunoreactivity in the liver. In the kidney, anti-CYP3A1/2 reacted strongly with the interstitial cells of the renal medulla, where anti-CYP1A1 and anti-CYP1A2 showed only weak staining limited to the epithelium of medullary collecting ducts (unpublished observations). The well characterized reactivity and specificity of these antibodies in other species and the specific staining patterns seen in serial sections from hamster tissues indicate that there is no cross-reactivity with non-specific hamster CYP antigens.
CYP1A1
No immunoreactive CYP1A1 was detected in the reproductive tract of untreated control males, or males treated with E2, TP or both hormones simultaneously for any of the treatment durations analyzed. Reactivity and specificity of this antiserum with hamster antigen were confirmed as described above.
CYP1A2 (Table II
)
In untreated animals the principal cells (the epithelial cells lining the tubules of the epididymis) of the most proximal region of the caput epididymis were stained with anti-CYP1A2 (Figure 1A and B
). The enzyme's distribution was limited to a supranuclear cellular compartment, consistent with the localization of CYPs in smooth endoplasmic reticulum in other mammalian cells (13). Immunoreactivity with anti-CYP1A2 was characterized by intense staining limited to a distinct contingent group of tubules in the epididymis, with no immunoreactivity in adjacent tubules of the distal caput, corpus or cauda epididymis, or the vas deferens. The tubules stained with anti-CYP1A2 represent ~10% of the epididymal tubules, and the staining pattern was remarkable in that no apparent tissue demarcation or anatomic structure separated the immunoreactive tubules from the directly adjacent, non-staining tubules. This pattern was not affected by treatment with TP for 2, 4 or 6 weeks (data not shown). In contrast, treatment with E2 for 2, 4 or 6 weeks resulted in new immunoreactivity with anti-CYP1A2 in 100% of the principal cells throughout the epididymis (Figure 1C and D
). Treatment with E2 also resulted in immunostaining with anti-CYP1A2 in the columnar epithelial cells of the vas deferens (Figure 1E
). This staining was detectable only in the proximal vas deferens near its junction with the epididymis after 2 weeks of treatment, and extended into more distal regions of the vas deferens with increased duration of E2 treatment. The E2-induced immunoreactive CYP1A2 in epithelial cells of the epididymis and vas deferens again was limited to the supranuclear region.

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Fig. 1. Immunohistochemical localization of CYP1A2 in (A, B) epididymis from untreated controls; (C, D) epididymis or (E) vas deferens after treatment with E2 for 2 weeks; (F, G) epididymis after treatment with E2 and TP for 2 weeks and (H) a large nodular tumor in the vas deferens after treatment with E2 + TP for 8 months. Basal cells are labeled with arrows in (G) and (H). Abbreviations: n, nucleus; epi, epithelium; sm, smooth muscle. (A, C and H) Bar, 100 µm; (BG) bar, 50 µm.
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Simultaneous treatment with E2 + TP for 2, 4 or 6 weeks blocked the increase in immunostaining with anti-CYP1A2 in the epididymis and vas deferens induced by E2 alone, but had no effect on the immunostaining pattern in the most proximal region of the caput epididymis which remained similar to that seen in untreated animals (Figure 1F
). In contrast to treatment with E2 alone, simultaneous treatment with E2 + TP for 2, 4 or 6 weeks resulted in new immunoreactivity with anti-CYP1A2 in basal cells throughout the epididymis (Figure 1F and G
).
All animals treated for 8 and 9 months with both E2 + TP had large nodular tumors in the vas deferens. Prolonged treatment with both hormones resulted in increased staining for anti-CYP1A2 in the vas deferens, first detectable in both the columnar epithelial cells and basal cells of the proximal vas deferens near its junction with the epididymis after 6 months of treatment, and in more distal regions of the vas deferens at 8 and 9 months of treatment. This staining pattern was heterogeneous throughout the vas deferens including the epithelium overlying the fully developed tumors. There were regions in the vas deferens where either the basal cells or the columnar, or both or neither cell type was stained (Figure 1H
).
CYP3A1/2
Immunoreactive CYP3A1/2 was not detectable in the untreated reproductive tract (Figure 2A
). Reactivity and specificity of this antiserum with hamster antigen were confirmed as described above.

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Fig. 2. Expression of CYP3A1/2 in vas deferens from (A) untreated controls, (B) treatment with E2, (C) treatment with E2 + TP for 2 weeks and (D) a vas deferens tumor after treatment with E2 + TP for 8 months. Abbreviations: epi, epithelium; sm, smooth muscle. Bar, 50 µm.
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Treatment with TP failed to induce staining with anti-CYP3A1/2 in the male reproductive tract, but treatment with E2 for 2, 4 or 6 weeks resulted in intense staining in the inner-circular smooth muscle layer of the vas deferens (Figure 2B
). In contrast, treatment with E2 + TP induced anti-CYP3A1/2 staining throughout all smooth muscle layers in the vas deferens, but with a more disperse and heterogeneous pattern than that seen after treatment with E2 alone (Figure 2C
). Anti-CYP3A1/2 also stained a subset of tumor cells in the large nodular tumors in animals which had been treated with E2 + TP for 8 or 9 months (Figure 2D
).
CYP1B1
Reactivity and specificity of anti-CYP1B1 with hamster antigen were confirmed by western blot analysis of microsomal samples pooled from five control males or five males treated with ß-NF as described in Materials and methods, or Figure 3
. Anti-CYP1B1 recognized a single band approximating the molecular weight of the 56 kDa control CYP1B1 from human lymphoblasts (Gentest, Woburn, MA). This protein was detected in the untreated epididymis, and was further induced by treatment with ß-NF. It was detected in the testis and vas deferens only after induction by ß-NF, similar to results from rat uterus (21). Although high levels of CYP1B1 are present in rat adrenal gland (21), no CYP1B1 was detected in microsomal fractions of adrenal glands from control or ß-NF treated hamsters.

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Fig. 3. Western blot demonstrating reactivity and specificity of anti-CYP1B1 with hamster antigen in microsomal samples pooled from five control males or five males treated with ß-NF as described in Materials and methods. Individual lanes contained commercially available CYP1B1 from human lymphoblasts (lane 1), adrenal gland from untreated (lane 2) or ß-NF treated males (lane 3); testis from untreated (lane 4) or ß-NF treated hamsters (lane 5); epididymis from untreated (lane 6) or ß-NF treated hamsters (lane 7) and vas deferens from untreated (lane 8) or ß-NF treated hamsters (lane 9). The relative location of molecular weight standards (kDa) is indicated on the left.
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Immunohistochemical analysis of untreated tissues showed strong staining with anti-CYP1B1 of smooth muscle lining the tubules in all regions of the epididymis (Figure 4A and B
), and weaker staining of smooth muscle lining the vas deferens (Figure 4C
). There was no change in the staining pattern with anti-CYP1B1 in the reproductive tract after treatment with E2 alone or TP alone for 2, 4 or 6 weeks, compared with untreated controls (data not shown). Treatment with E2 + TP, however, resulted in increased peri-nuclear staining in smooth muscle cells surrounding the vas deferens (Figure 4D
). The increased peri-nuclear staining was present after 2 weeks of treatment and remained through 9 months of treatment, when intense immunoreactivity was seen within the fully developed tumors (Figure 4E and F
). Combined treatment with E2 + TP also resulted in a slight increase in the intensity of anti-CYP1B1 staining in epididymal smooth muscle. The increase in CYP1B1 expression after treatment with E2 + TP was evaluated by semi-quantitative western blot analysis. Small yields from microsomal preparations from vas deferens, especially after treatment with E2 or E2 + TP, make western blot analysis with these tissues impractical. Instead, the epididymis from control and hormone treated males was used for this purpose. A denaturing gel was loaded with 2.5, 5, 10 and 20 µg microsomal protein from one sample for verifying the linear relationship of the assay. Microsomal protein (10 µg) from animals treated with E2, TP or E2 + TP for 4 weeks was incubated in the same gel. After transfer to nitrocellulose and incubation with anti-CYP1B1, the chemiluminescent signal used for detection was exposed to X-ray film (Figure 5A
) or collected using a digital camera, and analyzed to measure the signal intensity. Linear regression analysis of total protein loaded and detection signal intensity produced a slope-intercept formula with a correlation coefficient of 0.975. The signal intensity for the E2 + TP treated sample was 2.4 times greater than that for the control sample (Table III
and Figure 5A
). The nitrocellulose filter stained with 0.1% amido black (Figure 5B
) showed similar amounts of total protein in each lane, confirming that the difference in signal intensity was not due to differences in total protein loaded for each sample.

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Fig. 4. Immunohistochemical analysis of reproductive organs with anti-CYP1B1. Serial sections of the epididymis from untreated controls were stained with (A) hematoxylin and eosin (H&E), or (B) anti-CYP1B1. The thin layer of smooth muscle cells labeled with arrows in (A) is intensely stained in (B); immunostaining for CYP1B1 in the vas deferens of (C) an untreated control and (D) treatment with E2 + TP for 2 weeks. Serial sections of a large nodular tumor in the vas deferens after treatment with E2 + TP for 8 months were stained with (E) H&E and (F) anti-CYP1B1. Abbreviations: epi, epithelium; sm, smooth muscle. Bar, 50 µm.
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Fig. 5. (A) Microsomal fractions of epididymis probed with anti-CYP1B1. Lane 1, no treatment; lane 2, 4 weeks E2 alone; lane 3, 4 weeks TP alone; lane 4, 4 weeks E2 + TP. (See Table III for densitometric results.) (B) Nitrocellulose filter used for western blot analysis stained with 0.1% amido black. The lane containing molecular weight standards is labeled `STD'.
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Discussion
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Data from other species and the one report from Syrian hamster tissues (20) suggest that the CYPs most involved in E2 metabolism are members of the CYP1A and CYP3A family, and CYP1B1. Our findings here demonstrate that the male Syrian hamster reproductive tract constitutively expresses at least some CYPs necessary for the metabolism of E2. Moreover treatment with E2 alone altered the basal expression pattern, promoting increases in both CYP1A2 and CYP3A1/2.
In normal tissues, CYP1A2 was detected only in the most proximal region of the epididymis, whereas treatment with E2 for as little as 2 weeks induced expression throughout the epididymis and in the proximal vas deferens. The progressive induction of this enzyme in more distal regions of the male reproductive tract points to some form of communication between the epithelial cells lining the proximal and more distal regions of the epididymis and vas deferens. Such a response could be driven either by direct cell to cell communication possibly via gap junctions or mediated in a paracrine fashion via secretion by the testis or epididymis of an unidentified E2-responsive factor. Lengthening the duration of exposure to E2 would allow the signal to travel further distally through the reproductive tract, and would result in the time-dependent increase in CYP1A2 expression observed in more distal regions of the vas deferens. This time-dependent increase may also have occurred in the epididymis, but on a much more rapid time scale. Confirming this would require examining E2's effects on CYP1A2 expression at time points earlier than 2 weeks of treatment. There was no evidence for a similar temporal response in the induction of CYP3A1/2 in smooth muscle cells of the vas deferens. It is remarkable, however, that expression of CYP3A1/2 was induced by E2 in a cell type (i.e. smooth muscle) which expresses no detectable enzyme under normal conditions.
The E2-induced effects on CYP expression were altered markedly by combined treatment with E2 + TP. In the epididymis, TP treatment blocked the E2-dependent induction of CYP1A2, but not its normal constitutive expression, as demonstrated by the continued positive staining in the most proximal region of the epididymis in males treated with TP or E2 + TP. Inhibition of the E2-dependent induction of CYP1A2 by TP may be related to the anti-estrogenic potential of androgens. Experiments using the MCF-7 breast cancer cell line indicate that the E2-induced synthesis of specific proteins is blocked by testosterone without affecting the `formation, activation, nuclear binding or nuclear processing of estrogenreceptor complexes (31).' In vivo studies in immature rat uteri also have shown that testosterone decreases E2-induced RNA transcription in general, as measured by [3H]UTP incorporation, without changing the binding affinity or the concentration of estrogen receptor in either the nucleus or the cytoplasm (32). Similar results were seen in studies of the E2-dependent induction of the low density lipoprotein (LDL) receptor in the human hepatocellular carcinoma cell line, HepG2. Compounds with androgen receptor agonist activity, like the synthetic androgen R1881, decrease the ability of E2 to induce the LDL receptor in a manner independent of E2 concentration (33). While it is likely that interactions between steroid hormones and their receptors may vary in different species and experimental models, there appears to be a common mechanism in which androgens do not interfere with estrogen-receptor binding or activity, but block the effects of estrogens by altering the transcription or translation of estrogen-responsive targets. In the Syrian hamster model, TP may act to prevent the transcription or translation of the hypothetical E2-responsive factor which induces CYP1A2.
Conversely, simultaneous treatment with E2 and TP appeared to have a cell-type specific and time-dependent synergistic effect on the expression of CYPs. This was demonstrated by the induction of CYP1A2 expression in the basal cells of the epididymis after only 2 weeks, and with longer term treatment, the induction of heterogeneous expression of CYP1A2 in basal and columnar epithelial cells of the vas deferens, including the epithelium adjacent to large nodular tumors. Moreover, treatment with E2 + TP induced CYP3A1/2 expression throughout the vas deferens smooth muscle, in contrast to the limited expression seen only in the inner-most layer of smooth muscle cells after treatment with E2 alone. This heterogeneous expression of CYP3A1/2 in cells dispersed throughout the smooth muscle surrounding the vas deferens was also observed in a subset of tumor cells after long term treatment with E2 + TP. Combined treatment with E2 + TP also promoted an increase in CYP1B1 expression. Semiquantitative western blot results indicated that the relative level of CYP1B1 in epididymal microsomes from animals treated with E2 + TP was more than twice that measured in untreated controls. This approximate doubling in protein expression was difficult to discern in immunohistochemical preparations. However, a significant increase in peri-nuclear staining with anti-CYP1B1 in the smooth muscle cells of the vas deferens after treatment with E2 + TP and in the tumor itself was readily apparent with immunohistochemical analysis. Although it was not practical to perform western blot analysis on vas deferens tissues, the immunostaining results suggest that the increase in CYP1B1 after treatment with E2 + TP in the vas deferens was much greater than the 2.4-fold increase measured in the epididymis.
Our results establish potentially important relationships between hormone treatment and the regulation of CYP expression in the hormonally-induced formation of leiomyosarcomas. There is an absolute requirement for combined treatment with E2 + TP for tumor formation in this experimental model (1). The differential expression of CYPs in the reproductive tract induced by dual hormone treatment may be an important element in carcinogenesis. The elevated CYP1A2 levels in basal cells of the epithelium lining the epididymis and vas deferens induced by E2 + TP may generate metabolites and/or metabolic by-products which upon release could pass through the adjacent basal lamina and reach underlying smooth muscle cells. In contrast, under basal conditions or with E2 treatment alone, expression of CYP1A2 was limited to the apical compartment of columnar epithelial cells, a situation in which potentially harmful metabolites and/or metabolic by-products presumably would be eliminated through some apical transport mechanism and fail to reach underlying smooth muscle. A similar scenario could explain the differential upregulation of CYP3A1/2 and its possible role in tumor induction. After treatment with E2 alone, potentially carcinogenic end-products of CYP3A1/2 metabolism would be formed primarily in smooth muscle closest to the luminal epithelial lining of the vas deferens, and could be removed via transepithelial passage into the lumen. The specific transport mechanisms involved in this removal process may not be as readily able to eliminate the metabolic end-products generated by CYP3A1/2 expressed in the deeper layers of the vas deferens' smooth muscle lining after treatment with E2 + TP. The resulting accumulation of potential carcinogens could then lead to tumor formation.
An intriguing effect of combined treatment with E2 + TP was the induction of CYP1B1 in the actual tissue susceptible to transformation. It is possible that anti-CYP1B1 staining simply identifies a subset of cells more susceptible to carcinogenesis. However, this enzyme has been shown to catalyze the formation of 4-hydroxy catechol estrogens, which are more genotoxic and carcinogenic than the 2-hydroxy catechols (6,18,19), and the increased staining with anti-CYP1B1 may represent increased enzyme activity. Hammond et al. (20) correlated metabolite profiles with CYPs identified by western blot in microsomal samples, but did not conclusively prove the specific activities of these enzymes in hamster tissues. They also failed to positively identify CYP1B1 in hamster tissues. Although no definite conclusions can be made about the activities of these enzymes or the metabolites formed in these tissues, our results do suggest that CYP1B1-catalyzed metabolism may be an important step in the carcinogenic process. This conclusion is supported by the observation that the dramatic increase in CYP1B1 in vas deferens smooth muscle was seen only in response to dual treatment with E2 + TP, which is an absolute requirement for tumor induction. Furthermore, the original report of this tumor model noted that withdrawal of either hormone after large, nodular tumors had formed prevented further growth of the tumor (1). This phenomenon could also be related to increased CYP1B1 expression, which is seen only after treatment with both hormones simultaneously. This invokes the hypothesis that products of CYP1B1 metabolism may promote tumor growth.
The hormonal regulation of CYPs involved in the metabolism of E2 may be an important factor in estrogen-dependent carcinogenesis. We have shown here that the hormone treatment which leads to tumor formation in the hamster vas deferens also promotes dramatic changes in the expression pattern of CYP's which catalyze the oxidative metabolism of E2. These results were remarkable in that there were no inconsistencies seen among the individuals included in the different treatment groups. There was also a 100% frequency of tumor induction in the Syrian hamster vas deferens after treatment with E2 + TP for 8 months (1). Finally, one or more of the hypothesized CYP-mediated mechanisms discussed above may be involved in this model of tumor formation. The identification of the specific activities for these CYPs and the metabolites formed in these tissues is still required for a more complete explanation of their role in carcinogenesis.
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Notes
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3 To whom correspondence should be addressed E-mail: norrisjs{at}musc.edu 
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Acknowledgments
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The authors wish to thank Barbara Schmiedt for assistance with immunohistochemical techniques, Nancy Smythe for assistance with image analysis and Janie Nelson for assistance with manuscript preparation. This work was supported by NIH grants CA 49949-08, DC 00713-09 and ES 08148.
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Received October 30, 2000;
revised January 4, 2001;
accepted January 18, 2001.