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ANDROGEN METABOLISM IN THYMUS OF FETAL AND ADULT RATS

Fraunhofer Institute of Toxicology and Experimental Medicine, Center for Drug Research and Medical Biotechnology, Hannover, Germany

(Received December 1, 2003; accepted March 16, 2004)

	Abstract

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Cytochrome P450 (P450) monooxygenases play a role in target tissue metabolic activation of xenobiotics and/or endogenous compounds, such as vasoactive molecules or hormones. Indeed, tissue-specific metabolism of steroids is important in a variety of organs, including thymus, and may alter tissue-specific functions. Steroids have been shown to regulate thymus growth and function, but surprisingly little is known about expression of the responsible enzyme systems in thymus tissue, nor is the thymus-specific biotransformation of testosterone known. We therefore investigated gene and protein expression, total protein content, and enzyme activity of major P450 isoforms and other key steroid-metabolizing enzymes in thymus tissue of adult and fetal rats. We detected 6ß-hydroxytestosterone (HT), 7-HT, 16-HT, 2-HT, and androstenedione to be major testosterone metabolites in the adult thymus. The high production of 7-HT and 16-HT correlated well with the gene and protein expression of CYP2A1/2 and CYP2B1/2 in thymus of adult animals. When compared with fetal thymic tissue, CYP2A1/2, 17ß-hydroxysteroid dehydrogenase isoform 1 (17ß-HSDH1) and the androgen receptor were 8-, 3-, and 3-fold more highly expressed in adult rats, whereas 17ß-HSDH2, 17ß-HSDH3, and 5-reductase were reduced to 12%, 0%, and 32% of those in fetal thymus. In conclusion, we demonstrated that rat thymus expresses a variety of cytochrome P450 monooxygenases and other steroid-metabolizing enzymes, and it successfully metabolizes testosterone. Changes of the underlying steroid-metabolizing enzyme systems may aid in understanding the role of androgens in altering biological functions of the thymus.

Because of the scarce information available and in view of its essential role in thymus biology, we investigated thymus-specific androgen metabolism and hormone receptor expression levels. We therefore studied gene and protein expression and enzyme activity of major P450 isoforms and key steroid-metabolizing enzymes, i.e., 17ß- and 3ß-hydroxysteroid dehydrogenases (HSDHs) and 5-steroid-reductase, in thymus tissue of fetal and adult male rats. We also correlate gene and protein expression level and enzyme activity of steroid-metabolizing enzymes with expression of the androgen and estrogen receptor .

Overall, our study aimed to investigate steroid metabolism in thymus for a better understanding of these messengers in organ function.

	Materials and Methods

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Animals. All animal procedures described in this article were approved by the ethical committee of the local government of Hannover, Germany. Male Sprague-Dawley or pregnant Sprague-Dawley rats weighing 200 to 250 g were obtained from Charles River (Sulzfeld, Germany). Food and water were given ad libitum. Rats were anesthetized with ketamine (anesthetic) and xylazine hydrochloride (muscle relaxant), with 0.1 ml of ketamine per 100 g body weight and 0.05 ml of xylazine hydrochloride per 100 g body weight. In addition, 2000 international units of heparin were given intraperitoneally before surgery. Thymus tissue was explanted under a magnifying glass to exclude contamination with nonthymic tissue. Then, all tissue samples were shock-frozen in liquid nitrogen and stored at -80°C to await further analysis.

Gene Expression Studies. RNA was isolated from thymus of fetal (gestation day 19; n = 5) and adult (9–12 months old; n = 3) male rats using the SV total RNA Isolation System (Macherey-Nagel, Düren, Germany) according to the manufacturer's recommendation. Quality of isolated RNA was checked using a 1.0% agarose gel. Total RNA (2 µg) from each sample was used for reverse transcription. RNA and random-primer (Roche, Mannheim, Germany) were preheated for 10 min at 70°C. Then, 5x reverse transcriptase-avian myeloblastosis virus buffer, deoxynucleoside-5'-triphosphates (1 mM; Roche), 40 U RNase inhibitor (Stratagene, Amsterdam, Netherlands), and 20 U avian myeloblastosis virus-reverse transcriptase (Promega, Mannheim, Germany) were added and volumes were adjusted to 20 µl with diethyl pyrocarbonate (Sigma Chemie, Deisenhofen, Germany)-treated water. Then, reverse transcription was carried out for 60 min at 42°C and was stopped by heating to 95°C for 5 min. The resulting cDNA was frozen at -20°C until further experimentation.

PCRs were carried out in a thermal cycler (T3; Biometra, Göttingen, Germany) using the melting, annealing, and extension cycling conditions, as shown in Table 1. DNA contamination was checked for by direct amplification of RNA extracts before conversion to cDNA, and any contamination of RNA extracts with genomic DNA could be excluded. For PCR amplification, a 20-µl reaction mixture was prepared containing 10 µl of HotStar TaqDNA Polymerase Mastermix (QIAGEN GmbH, Hilden, Germany), a 400 nM concentration of the 3'- and 5'-specific primer (see Table 1), and 1 µl of cDNA. PCRs were done within the linear range of amplification; amplification products were separated using a 1.5% agarose gel and stained with ethidium bromide. Gels were photographed on a transilluminator (Kodak Image Station 440; Eastman Kodak, Rochester, NY; see Fig. 1).

View larger version (36K): [in this window] [in a new window]   FIG. 1. Gene expression of P450 monooxygenases, 17ß-HSD1–4, 3ß-HSD1, 5-reductase, estrogen receptor (ER), and the androgen receptor (AR) in thymus tissue of adult and fetal rats. a, ethidium bromide-stained agarose gels (reverse images of the gel are shown). b, results are presented as ratios of the gene of interest/cyclophilin in percentage of adult animals. Data represent mean ± S.D. of n = 3 (adult rats) and n = 5 (fetal rats) different animals. , p < 0.05. M, marker; A, adult animal; F, fetal animal.

Western Immunoblotting. Microsomal extracts from thymus (100 µg) of adult rats were immediately denaturated at 95°C for 5min, followed by SDS-polyacrylamide gel electrophoresis on 12% polyacrylamide gels, and blotted onto a polyvinylidene difluoride membrane (PerkinElmer Life and Analytical Sciences, Rodgau-Jügesheim, Germany) at 350 mA for 2 h in a buffer containing 400 mM glycine, 50 mM Tris (pH 8.3). We used baculovirus-insect cell-expressed Supersomes (50 µg; NatuTec, Frankfurt, Germany) containing CYP1A1, CYP2A, CYP2B, or CYP2E1 as positive controls. Nonspecific binding sites were blocked with Rotiblock (Roth, Karlsruhe, Germany) in 1x TBS buffer. After electroblotting of proteins, membranes were incubated with polyclonal antibodies (dilution 1:100 to 1:400) for CYP1A1 (Rubitec Gmbh, Bochum, Germany; catalog number Mab 1A3-03), CYP2A (Santa Cruz Biotechnology, Heidelberg, Germany; catalog number sc-9896), CYP2B (Chemicon International, Hofheim, Germany; catalog number AB-1268), and CYP2E1 (Chemicon International; catalog number AB-1274) for 1 h at room temperature and washed three times with 1x TBS buffer containing 0.1% Tween 20 (Roth). Subsequently, the membranes were incubated with a 1:10,000 diluted anti--sheep antibody (Chemicon International; CYP2A, CYP2B, CYP2E1) or a 1:5000 diluted anti--mouse antibody (Chemicon International; CYP1A1) for 1 h at room temperature followed by three successive washes with 1x TBS buffer containing 0.1% Tween 20 (Roth). Immunoreactive proteins were visualized with a chemiluminescence reagent kit (PerkinElmer Life and Analytical Sciences) according to the manufacturer's instructions, and bands were scanned with the Kodak Image Station CF 440 and analyzed using the Kodak 1D 3.5 imaging software.

Preparation of Microsomes and Measurement of Total P450 Content. Microsomes from thymus tissue were prepared as described previously (Thum and Borlak, 2002). Briefly, tissue pieces from adult rat thymus were cut into small pieces and homogenized with an Ultra-Turrax homogenizer (IKA Labortecnik, Staufen, Germany) in KCl buffer (0.15 M, pH 7.4). After centrifugation for 30 min at 11,000g and 4°C, the supernatant was centrifuged at 170,000g and 4°C for 60 min. The pellet was resuspended in KCl buffer (0.15 M, pH 7.4) and recentrifuged for 40 min at 200,000g and 4°C, and the microsomal fraction was transferred into Tris-sucrose buffer (0.25 M sucrose, 20 mM Tris buffer, 5 mM EDTA). Microsomal solutions were shock-frozen in liquid nitrogen and stored at -80°C until further use. All experimental results were obtained from at least n = 3 individual animals. Microsomal protein concentrations were determined according to the method of Smith et al. (1985) using the BCA test, a modification of the Lowry test (Lowry et al., 1951).

Protein content was adjusted to approximately 1 mg of protein/ml. Microsomal samples were diluted in 0.1 M Tris buffer (pH 7.4) to approximately 1 mg of protein/ml, and the diluted sample was added to both sample and reference cuvettes to record the baseline between 400 and 500 nm. Sodium dithionite was added to both cuvettes and the sample cuvette was gently bubbled with carbon monoxide for approximately 1 min. The spectrum was then scanned from 400 to 500 nm, and the absorbance difference between 450 and 490 nm was calculated. Using Beer's Law, the cytochrome P450 concentration was measured by the following equation: Abs.Diff · Path Length/Ext.Coeff = P450 (nmol/ml) (extinction coefficient of 91 for the absorbance difference between 450 and 490 nM; see Omura and Sato, 1964).

Metabolism Experiments with Testosterone. One milligram of microsomal protein was incubated with 100 µM testosterone and 1 mg/ml NADPH in 1 ml of Tris buffer (20 mM; Sigma Chemie) for 4 h at 37°C in a shaking water bath. Samples were shock-frozen in liquid nitrogen and were stored at -80°C until further analysis.

Testosterone and its metabolites were analyzed by high-performance liquid chromatography, according to the method of Arlotto et al. (1991) with slight modifications as described in Thum and Borlak (2002). 11-Hydroxyprogesterone was used as an internal standard for the quantitative determination of testosterone metabolites.

Statistical Analysis. Data represent mean ± standard deviation. The Student's t test was used and differences were considered significant at p < 0.05.

	Results

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Gene Expression. Cytochrome P450 monooxygenases. We detected transcript copies of CYP1A1, CYP2A1/2, CYP2B1/2, CYP2E1, and CYP2J3 in thymus tissue of adult or fetal rats, whereas expression of CYP1A2, CYP2C11, CYP3A1, CYP3A2, or CYP4A1 was below the limit of detection. In strong contrast, all of the P450 isoforms investigated are expressed in rat liver or cultured rat hepatocytes, as reported recently (Borlak and Thum, 2001). Transcripts of CYP2A1/2 were significantly (p < 0.05) higher in thymus tissue of adult rats, whereas CYP2J3 mRNA levels were significantly (p < 0.05) more abundantly expressed in thymus tissue of fetal rats (see Figs. 1 and 2a). Furthermore, CYP2E1 was 2-fold more highly expressed in fetal thymic tissue, but no statistical significance was reached.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Western immunoblotting of CYP1A1, CYP2A, CYP2B, and CYP2E1 in Supersomes (positive control) and microsomal extracts (100 µg) of adult thymus tissue. M, marker; C, positive control; T1–3, adult rat thymus tissue.

Hydroxysteroid dehydrogenases, 5-steroid reductase estrogen, and androgen receptor. When compared with thymus tissue of fetal rats, the 17ß-hydroxysteroid dehydrogenase isoform 1 (17ß-HSDH1) and the androgen receptor were expressed >3-fold higher in adult rats, whereas expression of 17ß-HSDH2, 17ß-HSD3, and 5-reductase were 12%, 0%, and 32% higher, respectively. No significant change in expression levels of 17ß-HSDH4, 3ß-HSDH1, or the estrogen receptor was observed when fetal and adult rat thymus were compared (see Figs. 1 and 2, a and b).

Protein Expression. We detected CYP2A and CYP2B expression in thymus tissue of adult rats. CYP2E1 protein expression was low, and in the case of CYP1A1, levels were below the limit of detection (see Fig. 3). Protein expression of P450 monooxygenases could not be determined in fetal thymus because of the small amounts of tissue available.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Total P450 content in microsomal extracts (1 mg) of liver and thymus of adult rats (n = 3) (a) and production of testosterone metabolites with microsomal suspensions of adult rat thymus tissue (b). a, data represent mean ± S.D.; , p < 0.05. b, data represent mean ± S.D.

Androgen Metabolism in the Thymus. Total P450 content and enzyme activity could only be measured in thymus and liver of adult animals, since the tissue weight of fetal thymic tissue was too small to enable microsomal enzyme assays.

We used a considerably long incubation time to gain maximal metabolic production; therefore, the enzymatic activities were determined under nonlinear conditions. In thymus, total P450 content was about 8% of liver (see Fig. 3a), and testosterone was metabolized to 6-HT, 7-HT, 16-HT, 2-HT, and androstenedione as major metabolites. The production of 16-HT and 7-HT correlated well with the gene expression of CYP2B1/2 and CYP2A1/2, respectively. When compared with microsomal membranes of liver, production of 16-HT and 7-HT was 3% and 5%, respectively, in thymus tissue (see Table 2 and Fig. 3b).

	Discussion

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We show rat thymus to express several cytochrome P450 and other steroid-metabolizing enzymes and demonstrate tissue-specific metabolism of testosterone. We also demonstrate an age-dependent transcript expression of isoforms of P450 and 17ß-HSDH, as well as the androgen receptor, in thymus tissue of fetal and adult rats. We thus extend earlier findings of a time-dependent P450 monooxygenase gene expression during liver organogenesis to the thymus (Borlakoglu et al., 1993; Yang et al., 1995; Juchau et al., 1998).

Importantly, steroid hormones are versatile signaling molecules and are metabolized via several enzyme systems, including P450 monooxygenases, hydroxysteroid dehydrogenases/reductases, and aromatases, and are usually bound reversibly to carrier proteins in the blood. Upon receptor-mediated endocytosis (Porto et al., 1995), they interact specifically with steroid-hormone-receptor proteins in the cytoplasm and nucleus (Becker et al., 1986). Binding of the hormone activates the receptor, enabling high affinity to specific DNA sequences to act as transcriptional enhancers. It is known that thymus size and weight are very sensitive to changes in the androgen status, and more recently, testosterone was shown to participate in apoptosis (Olsen et al., 1998). Indeed, androgen-resistant mice failed to trigger apoptosis in thymus upon androgen treatment, thus suggesting a requirement for a functional androgen receptor in thymus apoptosis (Olsen et al., 1998). Regulation of thymocyte apoptosis by androgens may mediate processes of thymocyte selection and might lead to changes in the peripheral T cell repertoire. We observed up to 3-fold higher expression of the androgen receptor in thymus mRNA extracts of adult animals. This might lead to enhanced androgen receptor-mediated thymocyte apoptosis in aged animals, as reported by Olsen et al. (1998).

The cellular targets of androgen action in the thymus are not completely known but may include lymphoid cells (thymocytes), and/or nonlymphoid epithelial cells. Androgen-resistant testicular feminization mice showed significant thymus enlargement (Olsen et al., 1998), which suggests the androgen receptor to be an important modulator in the control of thymus development.

17ß-HSDH3 is predominantly expressed in the testes and catalyzes the reaction from androstenedione to testosterone, whereas 17ß-HSD1 is highly expressed in the placenta, catalyzing the reactions from estradiol to estrone, testosterone to androstenedione, and androst-5-ene-3ß and 17ß-diol to dehydroepiandrosterone (Labrie et al., 2000). Notably, expression of 17ß-HSDH2, 3, and 4 was detected in fetal thymus tissue, whereas in adult thymus, expression was confined to the isoforms 17ß-HSDH1 and 4. This difference in the expression of 17ß-HSDHs between fetal and adult thymic tissue is likely to play an important role in the maintenance of tissue-specific steroid level and, by implication, in the onset and progression of hormone-sensitive thymus atrophy.

Interestingly, the lowest enzyme activities were associated with poorly transcribed genes in thymic tissue, whereas we found good correlation between CYP2A and CYP2B protein expression and production of the testosterone metabolites 16-HT and 7-HT. However, there is a need for in-depth experiments to establish enzyme kinetics of cytochromes P450 in thymic tissue. It is well known that the above-named cytochrome P450 monooxygenases play a major role in the metabolism of testosterone but are also competent in drug oxidation. In view of the expression of CYP2A and CYP2B, the thymus may have, additionally, tissue-specific drug metabolism capacity. For instance, CYP2A-catalyzed N-demethylation of cocaine leads to a hepatotoxic compound (Aoki et al., 2000) and is reported to have immunosuppressive and toxic effects on the thymus (Xu et al., 1998). It is reasonable to speculate that expression of CYP2A in the thymus is, at least in part, responsible for the production of a toxic metabolite to result in metabolically induced toxicity. Interestingly, expression of 5-reductase was >3-fold higher in fetal animals. This is suggestive for enhanced local production of dihydrotestosterone (DHT) to be higher in the fetal thymus. The effects of testosterone and DHT on thymus are controversial. For instance, Greenstein et al. (1988) reported testosterone to inhibit thymus growth in adult animals, whereas no effects were observed with DHT.

We used a long incubation time (4 h) to gain a maximum of testosterone metabolite production, especially from thymic tissue. We did not aim to perform a time line of metabolite production but, rather, focused on the type of metabolites that are produced in thymic tissue. It is noteworthy that thymic tissue consists of several cell types, including lymphoid thymocytes, nonlymphoid epithelial cells, endothelial cells, fibroblasts, macrophages, dendritic cells, and others. Further studies are therefore needed to understand in detail the role of each cell type and its metabolic competence for a better understanding of steroid metabolism and organ function. Moreover, metabolism experiments with testosterone were only done with microsomal extracts from adult thymus tissue, because the thymus tissue harvest from fetal animals was too low.

In conclusion, we demonstrate thymus tissue to be metabolically competent and to express several steroid-metabolizing enzymes. We speculate that the observed differences in steroid metabolism are important signals for thymus growth and function.

	Acknowledgments

 
We thank Dr. Regina Bökenkamp (Department of Pediatric Cardiology, University of Leyden, Netherlands) for preparation of fetal thymic tissue.

Address correspondence to: Prof. Dr. Jürgen Borlak, Fraunhofer Institute of Toxicology and Experimental Medicine, Center for Drug Research and Medical Biotechnology, Nikolai-Fuchs-Str. 1, D-30625 Hannover, Germany. E-mail: Borlak{at}item.fraunhofer.de

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