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The impact of nandrolone decanoate and growth hormone on biosynthesis of steroids in rats
Steroids 78 (2013) 1192–1199
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Steroids journal homepage: www.elsevier.com/locate/steroids
The impact of nandrolone decanoate and growth hormone on biosynthesis of steroids in rats Alfhild Grönbladh a,⇑, Jenny Johansson a, Mark M. Kushnir b, Jonas Bergquist c, Mathias Hallberg a a
Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, P.O. Box 591, SE-751 24 Uppsala, Sweden ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108-1221, USA c Department of Chemistry, BMC, Analytical Chemistry and SciLife Lab, Uppsala University, P.O. Box 599, SE-751 24 Uppsala, Sweden b
a r t i c l e
i n f o
Article history: Received 28 June 2013 Received in revised form 10 August 2013 Accepted 19 August 2013 Available online 5 September 2013 Keywords: Growth hormone Anabolic androgenic steroids Estrone Testosterone Androstenedione Thymus
a b s t r a c t Growth hormone (GH) and anabolic androgenic steroids (AAS) are commonly used in sports communities. Several studies have suggested an association between GH and AAS. We have investigated the impact of GH in rats treated with nandrolone decanoate (ND). Male Wistar rats received ND (15 mg/ kg) every third day during three weeks and were subsequently treated with recombinant human GH (1.0 IU/kg) for ten consecutive days. Plasma samples were collected and peripheral organs (i.e. heart, liver, testis and thymus) were dissected and weighed. Concentration of thirteen endogenous steroids was measured in the rat plasma samples using high specificity LC–MS/MS methods. Seven steroids were detected and quantified, and concentrations of estrone, testosterone, and androstenedione were significantly different among the groups, while concentrations of pregnenolone, DHEA, 17-hydroxyprogesterone and corticosterone were not altered. Administration of rhGH alone altered the plasma steroid distribution, and the results demonstrated significantly increased concentrations of plasma estrone as well as decreased concentrations of testosterone and androstenedione in the ND-treated rats. Administration of rhGH to ND-pretreated rats did not reverse the alteration of the steroid distribution induced by ND. Administration of ND decreased the weight of the thymus, and addition of rhGH did not reverse this reduction. However, rhGH administration induced an enlargement of thymus. Taken together, the plasma steroid profile differed in the four groups, i.e. control, AAS, rhGH and the combination of AAS and rhGH treatment. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Growth hormone (GH) is an endocrine polypeptide with a wide variety of biological functions e.g. in processes associated with metabolism and growth. In addition, GH plays an important role in the function of the central nervous system (CNS). GH and insulin-like growth factor 1 (IGF1), a peptide mediating many of the effects of GH, have been shown to increase neurogenesis, be involved in neuroprotection and counteract opioid-induced apoptosis in cells from mouse hippocampus [1,2]. In addition, it was shown that GH is involved in cognition and memory functions [3]. Studies suggest that GH affects function of the hypothalamic–pituitary–adrenal (HPA) axis [4] and influence the adrenal androgen secretion [5]. GH signaling is also believed to affect the hypothalamic–pituitary– gonadal (HPG) axis (for review see [6]). However, to the best of our ⇑ Corresponding author. Tel.: +46 18 4714117. E-mail addresses: [email protected] (A. Grönbladh), [email protected] (J. Johansson), [email protected] (M.M. Kushnir), [email protected] (J. Bergquist), [email protected] (M. Hallberg). 0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2013.08.012
knowledge, the influence of GH on concentrations of plasma steroids in intact rodents has not been examined. GH is known to be abused in sports communities, and is recognized as an anabolic agent. Another group of substances, which are abused for their anabolic attributes are the anabolic androgenic steroids (AAS), among which one of the most frequently abused is nandrolone decanoate (ND). ND has a structure very similar to testosterone, the main endogenous androgen biosynthesized in the testis (Fig. 1). Today the AAS abuse has spread beyond the world of sports and is commonly used by adolescents and young adults [7]. AAS effect many functions in the body, including the CNS, and are known to cause a range of adverse effects, including gynecomastia, increased risk of cardiovascular and hepatic disease, aggression, depression, and impaired cognitive functions [8,9]. Users of AAS often combine the steroid intake with other drugs of abuse and various pharmaceuticals, and combination with GH is common [10]. It is known that GH and IGF1 effects biosynthesis of sex steroids [6,11] and that gonadal steroids to some extent regulate GH secretion [12]. A recent study demonstrated that treatment
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Fig. 1. Pathway of steroid biosynthesis. The most common pathway of steroidogenesis in rodents is believed to be the D4-pathway, involving progesterone, 17hydroxyprogesterone and androstenedione [57], whereas the D5-pathway is more common in humans [58]. (1) Cholesterol side chain cleavage enzyme (CYP11A), (2) Cytochrome P450 17a-hydroxylase, 17,20 lyase (CYP17A1), (3) 3b-hydroxysteroid dehydrogenase 1 (3b-HSD1), (4) 21a-hydroxylase (CYP21), (5) 11b-hydroxylase 1 (CYP11B1), (6) 11b-hydroxysteroid dehydrogenase 2 (11b-HSD2), (7) 11b-hydroxysteroid dehydrogenase 1 (11b-HSD1), (8) 17b-hydroxysteroid dehydrogenase 3 (17bHSD3), (9) 5a-reductase, (10) aromatase (CYP19), (11) 17b-hydroxysteroid dehydrogenase 1 (17b-HSD1), (12) 17b-hydroxysteroid dehydrogenase 2 (17b-HSD2). Steroids in bold were measured in the present study.
with ND lead to decreased plasma concentrations of IGF1 [13], however, the mechanism behind the interaction between the two systems is not known. It is well known that AAS in humans induce a negative feedback function on the HPG-axis causing a decrease of concentrations of endogenous androgens [14–16]. GH is less studied in this context, but was shown to be present in the testis and play an important role for steroidogenesis and gametogenesis [17,18]. Kanzaki et al., showed that GH stimulates steroidogenesis in rat Leydig cells [19]. Notably, there have been no reports on combined effect of administration of ND and GH on the plasma steroid profile in intact animals. As mentioned above, both substances target functions of several peripheral organs, the effect of concurrent administration of ND and GH on organ weight have however not previously been studied in rats. The aims of this study were to examine the effects of GH and AAS on biosynthesis of endogenous steroids, and to investigate the effect of a supraphysiologic three-week long intake of ND, and a subsequent ten day long treatment with rhGH in intact rats. Furthermore, the impact of AAS and GH treatment on weight of liver, thymus, testis and heart was investigated. Concentration of thirteen steroids was measured in rat plasma using validated liquid chromatography tandem mass spectrometry (LC–MS/MS) methods.
2. Experimental 2.1. Animals In this study, 48 male Wistar rats obtained from Taconic Farms (Ejeby, Denmark) were allowed to adapt to the laboratory environment for approximately two weeks. The rats were ten weeks old, with a body weight of 316.1 ± 2.5 g, at the start of the experiment. All rats were group-housed (three in each cage) with free access to water and food and kept in an air-conditioned room with controlled temperature (22–24 °C) and humidity (50–60%). The rats were kept under a reversed 12-h dark/light cycle with lights off at 7 a.m. The animal experiments were performed under a protocol approved by the Uppsala Animal Ethical Committee. Data from these rats are also included in an earlier publication on effects of GH and ND on spatial memory, for further details see Ref. [13]. 2.2. Treatment Subcutaneous (s.c.) injections with ND (Deca-Durabol, Organon, Oss, Netherlands), 15 mg/kg, or peanut oil (Apoteket AB, Umeå, Sweden) was performed every third day during three weeks, starting from day 1 to 21 of the experiment. During days 22–31, the rats
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Table 1 Description of the four different treatment groups. The AAS (nandrolone decanoate) or peanut oil was administered every third day with subcutaneous (s.c.) injections and the rhGH or saline was given daily (s.c. injections).
Days 1–21 Days 22–31
Controls
GH
ND
ND + GH
Peanut oil Saline
Peanut oil Growth hormone 1.0 IU/kg
Nandrolone decanoate 15 mg/kg Saline
Nandrolone decanoate 15 mg/kg Growth hormone 1.0 IU/kg
were injected (s.c.) with rhGH (Amersham, Uppsala, Sweden) 1.0 IU/kg or saline for ten consecutive days (see Table 1). On the last day of the experiment the rats were decapitated and peripheral organs (e.g. thymus, testis, heart, and liver) were weighed and dissected and trunk blood was collected. 2.3. Plasma samples Trunk blood was collected in 0.1% EDTA, put on ice, and then centrifuged at 3000 r.p.m. at 4 °C, for 10 min. Plasma was collected and stored in 80 °C until further analysis. Samples were transported between the participating centers on dry ice. 2.4. Reagents and standards Testosterone, estrone, estradiol, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, cortisone, hydroxylamine, formic acid, trifluoroacetic acid, dansyl chloride, and sodium carbonate were purchased from Sigma Chemical Company (St. Louis, US). Androstenedione, dehydroepiandrosterone (DHEA), progesterone, and corticosterone were purchased from Steraloids Inc. (Newport, RI). The internal standards were deuterium labeled analogs of the steroids d3-testosterone, d3-pregnenolone, d2-11-deoxycortisol, d9-17-hydroxyprogesterone, d317-hydroxypregnenolone, d4-cortisone, d3-cortisol (purchased from Cambridge Isotope Laboratories, Andover, MA), and d4-estrone and d3-estradiol (purchased from CDN Isotopes, Toronto, ON). All other chemicals were of the highest purity commercially available.
steroid. Two mass transitions were monitored for each steroid and its internal standard. Quantitative data analysis was performed using Analyst™ 1.5.2 software. Calibration curves were generated with every set of samples using six calibrators, and three quality control samples were included with every set of samples. Specificity of the analysis for each steroid in every sample was evaluated by comparing concentrations determined using the primary and the secondary mass transitions of each steroid and its internal standard [25]. 2.6. Statistical analysis Statistical analysis was performed using the software Prism 6 (GraphPad Software, Inc. La Jolla, USA). The normality of the data distribution was examined using the Shapiro–Wilks W-test. The steroid profile data was analyzed using the Kruskal–Wallis test and Dunn’s post hoc test since these data followed a non-Gaussian distribution. For the purpose of statistical analysis of steroids for which concentrations were below the detection limit, the values were set to the concentration corresponding to the respective limit of quantification of the methods. Statistical analyses of the weight measurements were performed using one-way ANOVA and Tukey’s post hoc tests. Correlation analysis was performed with the nonparametric Spearman correlation test. Concentrations of plasma steroids are expressed as median, minimal and maximal values; weight measurements are presented as mean ± SEM, p-values less than 0.05 were considered significant. 3. Results
2.5. Liquid chromatography tandem mass spectrometry (LC–MS/MS)
3.1. Steroid analysis
Plasma samples were analyzed for pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, cortisone, corticosterone, DHEA, androstenedione, testosterone, progesterone, estrone and estradiol using LC–MS/MS as previously described [20–24]. Briefly, steroids were extracted from samples, corticosterone, DHEA, androstenedione, testosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, and 11-deoxycortisol were derivatized with hydroxylamine to form oxime derivatives; estrone, and estradiol was derivatized with dansyl chloride to form oxime derivatives [23]. Cortisol and cortisone were analyzed underivatized [24]. Limit of quantification (LOQ) was 0.05 ng/mL for pregnenolone, 17hydroxyprogesterone, and 11-deoxycortisol, 0.25 ng/mL for 17hydroxypregnenolone, 1 ng/mL for progesterone, 0.5 ng/mL for corticosterone, 0.01 ng/mL for testosterone and androstenedione, 0.05 ng/ml for DHEA, 1 ng/mL for cortisol and cortisone, and 1 pg/ml for estrone and estradiol. The intra-assay and inter-assay CV were <8% and 11%, respectively [23]. All steroids were analyzed in positive ion mode using an electrospray ion source on a triple quadruple mass spectrometer (API5500; AB Sciex, Foster City, CA). The HPLC system consisted of series 1260 HPLC pumps (Agilent Technologies), and an HTC PAL autosampler (LEAP Technologies, NC) equipped with a fast wash station. The quadrupoles Q1 and Q3 were tuned to unit resolution and the mass spectrometer conditions were optimized for maximum signal intensity of each
In this study thirteen steroids were measured in rat plasma, of these steroids six were not detected. The LC–MS/MS methods used in this study were developed and validated for analysis of human serum and plasma samples and were shown to perform well for analysis of the rat plasma samples. The present study demonstrated that ND treated animals had very low plasma concentrations of testosterone and androstenedione (below the detection limit in all the rats of the group) (Fig. 2). Plasma concentration of estradiol was significantly elevated in the ND and the ND + GHtreated rats compared to controls, no alteration of estradiol was seen in the rhGH-treated rats compared to controls. The median plasma concentrations in the control group of estrone, testosterone, and androstenedione were 2.30 pg/ml, 0.80 ng/ml, and 0.07 ng/ml, respectively. The corresponding median concentrations in the ND treated group were 3.50 pg/ml, 0.01 ng/ml, and 0.01 ng/ ml, respectively. The median concentrations in the group treated with rhGH were 2.80 pg/ml, 0.80 ng/ml, and 0.04 ng/ml, respectively. Corresponding concentrations in the group treated with both ND and GH were 3.30 pg/ml, 0.01 ng/ml, and 0.01 ng/ml, respectively. No significant difference in plasma concentration of pregnenolone was observed among the treatment groups. The corticosterone concentration varied between the rats. A few animals (5 out of 12 rats) in the rhGH treated group had highly elevated concentrations. Although the result overall was not statistical significant when all the rats in the groups were included in
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Fig. 2. Box plots with distribution of plasma concentrations of endogenous steroids in rats after treatment of ND and GH. Values are presented as median, minimum and maximum values, n = 11–12/group. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
the comparison (p = 0.34). The concentration of 17-hydroxyprogesterone was elevated in the GH treated animals compared to the ND treated (p = 0.027). A positive correlation between corticosterone and 17-hydroxyprogesterone concentrations was found in all treatment groups (controls r2 = 0.94, p < 0.0001, GH r2 = 0.49, p = 0.01, ND r2 = 0.52, p = 0.0077, ND + GH r2 = 0.98, p < 0.0001). Concentrations of DHEA did not differ significantly between the treatment groups, however, in all groups some of the DHEA measurements were under the detection limit suggesting low concentrations of DHEA in rat plasma. Concentrations of cortisol, cortisone, 11-deoxycortisol, progesterone, estradiol, and 11-hydroxypregnenolone were below the detection limit of the methods, irrespective of the type of treatment. Considering the difference in distribution of the concentrations of pregnenolone, 17-hydroxyprogesterone, DHEA, corticosterone, androstenedione, testosterone, and estradiol on a pie diagram (Fig. 3), both rhGH and ND induced a change in the plasma steroid profile. 3.2. Weight of peripheral organs The weight of heart, liver, thymus, and testis in the sacrificed rats was measured and a summary of the data is shown in Fig. 4. Due to the differences in body weight between the animals in different treatment groups (Table 2), the weights of the various organs are expressed as normalized to the body weight (shown as mg tissue of 100 g of the body weight). The results demonstrated (a) decreased liver weight in the animals receiving ND + GH compared to controls and rhGH-treated animals and (b) a significant decrease of the weight of the thymus in the rats receiving ND and ND + GH (c) an increase in the weight of the thymus gland of
the GH rats as compared to the controls (d) the weight of the testis differed between the treatment groups: rats receiving only ND had a significantly larger testis compared to all other groups, including the ND + GH group. The results did not demonstrate any significant alteration of weight of the heart among the treatment groups.
4. Discussion In this study, the impact of AAS administration alone or in combination with GH, on the biosynthesis of steroids in male rats was investigated. Seven steroids were successfully measured and the results demonstrated an altered steroid profile from rhGH and ND administration. Administration of ND decreased the concentration of testosterone and androstenedione, and elevated the estradiol concentration; addition of rhGH did not effect these alterations. Herein a LC–MS/MS method was applied for the analysis and determination of concentrations of central steroids in rat plasma. The methodology has previously successfully been used for analyses of steroids in plasma from humans [23], as well of steroids in various other biological fluids [26–29]. The small sample volumes needed, high specificity, high sensitivity and use for the methods for routine measurement of steroids in clinical diagnostic settings represent advantages with the method. The divergence concentrations of steroids we observe in comparison with outcomes from other studies where alternative analytical techniques were used is reflecting e.g. different detection ranges and lower specificity of analysis [30–32]. The administration of ND decreased plasma concentrations of testosterone. This was an expected observation since AAS are well
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Fig. 3. Pie charts with relative concentration of seven steroids measured in the rat plasma, plotted as percent of total plasma steroid concentrations, after treatment with ND and GH (n = 11–12/group).
Fig. 4. The impact of ND and GH on weight of liver, testis, heart and thymus. Values are presented as mean ± SEM, n = 11–12/group. ⁄p < 0.05,
known to activate the negative feedback loop of the HPA/HPG-axis and decrease concentrations of LH and FSH [14–16]. We observed a significant decrease of androstenedione, the precursor of
⁄⁄
p < 0.01,
⁄⁄⁄
p < 0.001.
testosterone, caused by the ND administration, which could also be explained by the negative feedback of the HPA/HPG-axis. Our results did not demonstrate a significant difference in the
A. Grönbladh et al. / Steroids 78 (2013) 1192–1199 Table 2 Body weight (g) on the last day of the experiment. ⁄p < 0.05 compared to GH and ND groups, ⁄⁄⁄p < 0.001 compared to control and GH groups. Values are presented as mean ± SEM.
Weight (g)
Controls
GH
ND
ND + GH
392 ± 7
407 ± 7
340 ± 6⁄⁄⁄
367 ± 8⁄
concentration of 17-hydroxyprogesterone, which could indicate a down-regulation of the conversion of 17-hydroxyprogesterone to androstenedione. It has been postulated that rat adrenals lack the 17alpha-hydroxylase/17,20 lyase (CYP17) enzyme [33,34] although, there are reports supporting adrenal testosterone production in rats [35,36]. The 17-hydroxyprogesterone is in rat testes mainly formed from progesterone by CYP17 hydroxylase activity, and then converted to androstenedione by CYP17 lyase enzyme, i.e. the C–C bond in the 17-position is cleaved. In humans, it has been suggested that the lyase and hydroxylase function of CYP17 are regulated separately [37], so an explanation for the reduction of concentrations of androstenedione and not 17-hydroxyprogesterone could be an alteration of the CYP17 phosphorylation state, because CYP17 has only low or no lyase activity when under-phosphorylated [38]. However, if this were true, we should have detected increased 17-hydroxyprogesterone concentrations due to its accumulation [39]. Alternatively, the increased expression of CYP19 (aromatase) that occurs after ND administration, which has been shown to be induced by an androgen receptor mediated system [40,41] could account for the lower concentrations of testosterone and its precursor androstenedione. However, although the estradiol concentrations were found to be increased, the concentrations of estradiol are still very low compared to concentrations of androstenedione and testosterone. It is therefore not likely that the significant reduction in concentrations of androstenedione and testosterone is primarily attributed to the up-regulation of aromatase causing a conversion of androstenedione to estrone. One should bear in mind the fact that estrone is in equilibrium with estradiol. Normally, estrogens can be synthesized by either conversion of testosterone to estradiol, or by conversion of androstenedione to estrone, both reactions involving the enzyme CYP19 (aromatase). Estrone can then be further converted to estradiol by the enzyme 17b-hydroxysteroid dehydrogenase (17bHSD2) and vice versa (see Fig. 1). However, since nandrolone is known to be a substrate to aromatase we suggest that the major part of the estrone probably originates from the ND administration. Supporting this are early studies demonstrating aromatization of nandrolone to estrone [42,43] and ND administration to male patients also increased estrone concentrations [44]. Although, studies have suggested that nandrolone is not aromatized to the same extent as testosterone [45]. The treatment with rhGH was found to increase 17-hydroxyprogesterone concentrations. This has also been demonstrated in patients treated with rhGH during one year [46]. Another study could not detect any alteration of 17-hydroxyprogesterone concentrations from GH treatment [47], thus, the exact impact of the hormone remains uncertain. In some of the GH-treated rats, we found the corticosterone concentration to be highly elevated, indicating that GH stimulates secretion of this glucocorticoid, however, the among-groups comparison did not show statistically significant differences. Earlier studies have demonstrated increases of plasma corticosterone concentrations after GH treatment [48–50] and it has been proposed that this effect could be mediated through a stimulation of ACTH release via the hypothalamus [48]. Previous studies on the effect of AAS on corticosterone concentrations have been inconsistent [51–54], implying that length of steroid administration, time of the blood collection, and the administration route can have an
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impact on the corticosterone concentration. Long-term AAS intake in humans decreased ACTH concentrations [55]. In rats, ND induced an increase of ACTH and corticosterone concentrations one hour, but not 24 h after the last injection [52] suggesting a biphasic effect of AAS on the HPA-axis. Corticosterone is the main glucocorticoid in rats and only traces of cortisol have been detected in rats [56]. Furthermore, earlier studies suggest that the D4 (4-ene) pathway of the steroid synthesis is the predominant pathway in rats [57], whereas the D5 (5-ene) pathway is predominant in humans [58] (see Fig. 1). Thus, in this study, the low concentrations or absence of some of the steroids characteristic of the pathway of steroids biosynthesis in humans (D5) were expected [34,56]. Although an influence of GH on the HPG-axis has been established, the administration of rhGH did not induce any alteration of the testosterone concentrations in this study. This observation is consistent with the data from earlier studies, GH supplementation in humans did not affect concentrations of testosterone [59,60]. In an animal model, overexpression of the GH releasing hormone (GHRH) was shown to increase concentration of GH, which resulted in a decrease in concentration of testosterone [61]. The rhGH treatment had no influence on the inhibition of biosynthesis of endogenous testosterone, and the combined effects of rhGH and ND did not have a major impact on the steroid concentrations measured herein. A recent study examined the effect of a combined treatment with GH and testosterone in men undergoing chronic glucocorticoid treatment and detected an additive effect on skeletal muscle mass compared to testosterone or GH alone [62]. Since ND is known to have a half-life of approximately six days [63], the rats were expected to have high plasma concentration of ND on the last day of the experiment. The ND treatment was discontinued on day 21 of the experiment and these results demonstrated that the endogenous androgen production was decreased ten days after the last ND injection. Another indicator of the effect of ND was a large decrease of thymus observed in the animals receiving ND, this observation is in agreement with earlier studies, where a significant effect of AAS on thymus also was observed [53,64]. Indeed, it is hypothesized that AAS produce thymolytic effects by stimulating the secretion of molecular entities by the thymic epithelial cells that may induce regression of the thymus gland [65]. Furthermore, an increase of thymus was seen from the rhGH administration, indicating thymus as a target for GH actions (for a review, see [66]). For example, in AIDS patients receiving longterm treatment with GH, a significant increase of thymus has been demonstrated [67]. In humans excessive levels of exogenous androgens have been reported to decrease spermatogenesis and induce testicular atrophy [68,69]. In addition, administration of ND for 14 weeks reduced the testis volume in rats [70]. In the present study, the rats receiving ND exhibited a slight increase of the normalized testis weight (mg tissue/100 g body weight). The non-normalized testis weights (g) did however not differ significantly between the treatment groups (data not shown). Interestingly, the testis weight was previously demonstrated to be unaffected by two weeks of ND administration in male Sprague Dawley rats, suggesting the effect of AAS on testis atrophy may require a long-term treatment [53]. In conclusion, repeated administration of a high dose of ND to male rats resulted in significantly elevated concentrations of estrone in plasma, and reduced concentrations of testosterone and androstenedione. Treatment of the rats with rhGH altered the steroid profile but administration of rhGH to the AAS treated rats did not reverse the ND-induced changes in the steroid profile. Furthermore, administration of rhGH to rats induced an enlargement of thymus, however rhGH did not reverse the reduction of thymus caused by ND. This study further highlights the major impact of nandrolone decanoate on biological and endocrine functions.
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Acknowledgements This study was supported by grants from the Swedish Research Council (Grant 9459, 621-2011-4423) and ARUP Institute for Clinical & Experimental Pathology, Salt Lake City, USA.
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Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
The impact of nandrolone decanoate and growth hormone on biosynthesis of steroids in rats Alfhild Grönbladh a,⇑, Jenny Johansson a, Mark M. Kushnir b, Jonas Bergquist c, Mathias Hallberg a a
Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, P.O. Box 591, SE-751 24 Uppsala, Sweden ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108-1221, USA c Department of Chemistry, BMC, Analytical Chemistry and SciLife Lab, Uppsala University, P.O. Box 599, SE-751 24 Uppsala, Sweden b
a r t i c l e
i n f o
Article history: Received 28 June 2013 Received in revised form 10 August 2013 Accepted 19 August 2013 Available online 5 September 2013 Keywords: Growth hormone Anabolic androgenic steroids Estrone Testosterone Androstenedione Thymus
a b s t r a c t Growth hormone (GH) and anabolic androgenic steroids (AAS) are commonly used in sports communities. Several studies have suggested an association between GH and AAS. We have investigated the impact of GH in rats treated with nandrolone decanoate (ND). Male Wistar rats received ND (15 mg/ kg) every third day during three weeks and were subsequently treated with recombinant human GH (1.0 IU/kg) for ten consecutive days. Plasma samples were collected and peripheral organs (i.e. heart, liver, testis and thymus) were dissected and weighed. Concentration of thirteen endogenous steroids was measured in the rat plasma samples using high specificity LC–MS/MS methods. Seven steroids were detected and quantified, and concentrations of estrone, testosterone, and androstenedione were significantly different among the groups, while concentrations of pregnenolone, DHEA, 17-hydroxyprogesterone and corticosterone were not altered. Administration of rhGH alone altered the plasma steroid distribution, and the results demonstrated significantly increased concentrations of plasma estrone as well as decreased concentrations of testosterone and androstenedione in the ND-treated rats. Administration of rhGH to ND-pretreated rats did not reverse the alteration of the steroid distribution induced by ND. Administration of ND decreased the weight of the thymus, and addition of rhGH did not reverse this reduction. However, rhGH administration induced an enlargement of thymus. Taken together, the plasma steroid profile differed in the four groups, i.e. control, AAS, rhGH and the combination of AAS and rhGH treatment. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Growth hormone (GH) is an endocrine polypeptide with a wide variety of biological functions e.g. in processes associated with metabolism and growth. In addition, GH plays an important role in the function of the central nervous system (CNS). GH and insulin-like growth factor 1 (IGF1), a peptide mediating many of the effects of GH, have been shown to increase neurogenesis, be involved in neuroprotection and counteract opioid-induced apoptosis in cells from mouse hippocampus [1,2]. In addition, it was shown that GH is involved in cognition and memory functions [3]. Studies suggest that GH affects function of the hypothalamic–pituitary–adrenal (HPA) axis [4] and influence the adrenal androgen secretion [5]. GH signaling is also believed to affect the hypothalamic–pituitary– gonadal (HPG) axis (for review see [6]). However, to the best of our ⇑ Corresponding author. Tel.: +46 18 4714117. E-mail addresses: [email protected] (A. Grönbladh), [email protected] (J. Johansson), [email protected] (M.M. Kushnir), [email protected] (J. Bergquist), [email protected] (M. Hallberg). 0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2013.08.012
knowledge, the influence of GH on concentrations of plasma steroids in intact rodents has not been examined. GH is known to be abused in sports communities, and is recognized as an anabolic agent. Another group of substances, which are abused for their anabolic attributes are the anabolic androgenic steroids (AAS), among which one of the most frequently abused is nandrolone decanoate (ND). ND has a structure very similar to testosterone, the main endogenous androgen biosynthesized in the testis (Fig. 1). Today the AAS abuse has spread beyond the world of sports and is commonly used by adolescents and young adults [7]. AAS effect many functions in the body, including the CNS, and are known to cause a range of adverse effects, including gynecomastia, increased risk of cardiovascular and hepatic disease, aggression, depression, and impaired cognitive functions [8,9]. Users of AAS often combine the steroid intake with other drugs of abuse and various pharmaceuticals, and combination with GH is common [10]. It is known that GH and IGF1 effects biosynthesis of sex steroids [6,11] and that gonadal steroids to some extent regulate GH secretion [12]. A recent study demonstrated that treatment
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Fig. 1. Pathway of steroid biosynthesis. The most common pathway of steroidogenesis in rodents is believed to be the D4-pathway, involving progesterone, 17hydroxyprogesterone and androstenedione [57], whereas the D5-pathway is more common in humans [58]. (1) Cholesterol side chain cleavage enzyme (CYP11A), (2) Cytochrome P450 17a-hydroxylase, 17,20 lyase (CYP17A1), (3) 3b-hydroxysteroid dehydrogenase 1 (3b-HSD1), (4) 21a-hydroxylase (CYP21), (5) 11b-hydroxylase 1 (CYP11B1), (6) 11b-hydroxysteroid dehydrogenase 2 (11b-HSD2), (7) 11b-hydroxysteroid dehydrogenase 1 (11b-HSD1), (8) 17b-hydroxysteroid dehydrogenase 3 (17bHSD3), (9) 5a-reductase, (10) aromatase (CYP19), (11) 17b-hydroxysteroid dehydrogenase 1 (17b-HSD1), (12) 17b-hydroxysteroid dehydrogenase 2 (17b-HSD2). Steroids in bold were measured in the present study.
with ND lead to decreased plasma concentrations of IGF1 [13], however, the mechanism behind the interaction between the two systems is not known. It is well known that AAS in humans induce a negative feedback function on the HPG-axis causing a decrease of concentrations of endogenous androgens [14–16]. GH is less studied in this context, but was shown to be present in the testis and play an important role for steroidogenesis and gametogenesis [17,18]. Kanzaki et al., showed that GH stimulates steroidogenesis in rat Leydig cells [19]. Notably, there have been no reports on combined effect of administration of ND and GH on the plasma steroid profile in intact animals. As mentioned above, both substances target functions of several peripheral organs, the effect of concurrent administration of ND and GH on organ weight have however not previously been studied in rats. The aims of this study were to examine the effects of GH and AAS on biosynthesis of endogenous steroids, and to investigate the effect of a supraphysiologic three-week long intake of ND, and a subsequent ten day long treatment with rhGH in intact rats. Furthermore, the impact of AAS and GH treatment on weight of liver, thymus, testis and heart was investigated. Concentration of thirteen steroids was measured in rat plasma using validated liquid chromatography tandem mass spectrometry (LC–MS/MS) methods.
2. Experimental 2.1. Animals In this study, 48 male Wistar rats obtained from Taconic Farms (Ejeby, Denmark) were allowed to adapt to the laboratory environment for approximately two weeks. The rats were ten weeks old, with a body weight of 316.1 ± 2.5 g, at the start of the experiment. All rats were group-housed (three in each cage) with free access to water and food and kept in an air-conditioned room with controlled temperature (22–24 °C) and humidity (50–60%). The rats were kept under a reversed 12-h dark/light cycle with lights off at 7 a.m. The animal experiments were performed under a protocol approved by the Uppsala Animal Ethical Committee. Data from these rats are also included in an earlier publication on effects of GH and ND on spatial memory, for further details see Ref. [13]. 2.2. Treatment Subcutaneous (s.c.) injections with ND (Deca-Durabol, Organon, Oss, Netherlands), 15 mg/kg, or peanut oil (Apoteket AB, Umeå, Sweden) was performed every third day during three weeks, starting from day 1 to 21 of the experiment. During days 22–31, the rats
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Table 1 Description of the four different treatment groups. The AAS (nandrolone decanoate) or peanut oil was administered every third day with subcutaneous (s.c.) injections and the rhGH or saline was given daily (s.c. injections).
Days 1–21 Days 22–31
Controls
GH
ND
ND + GH
Peanut oil Saline
Peanut oil Growth hormone 1.0 IU/kg
Nandrolone decanoate 15 mg/kg Saline
Nandrolone decanoate 15 mg/kg Growth hormone 1.0 IU/kg
were injected (s.c.) with rhGH (Amersham, Uppsala, Sweden) 1.0 IU/kg or saline for ten consecutive days (see Table 1). On the last day of the experiment the rats were decapitated and peripheral organs (e.g. thymus, testis, heart, and liver) were weighed and dissected and trunk blood was collected. 2.3. Plasma samples Trunk blood was collected in 0.1% EDTA, put on ice, and then centrifuged at 3000 r.p.m. at 4 °C, for 10 min. Plasma was collected and stored in 80 °C until further analysis. Samples were transported between the participating centers on dry ice. 2.4. Reagents and standards Testosterone, estrone, estradiol, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, cortisone, hydroxylamine, formic acid, trifluoroacetic acid, dansyl chloride, and sodium carbonate were purchased from Sigma Chemical Company (St. Louis, US). Androstenedione, dehydroepiandrosterone (DHEA), progesterone, and corticosterone were purchased from Steraloids Inc. (Newport, RI). The internal standards were deuterium labeled analogs of the steroids d3-testosterone, d3-pregnenolone, d2-11-deoxycortisol, d9-17-hydroxyprogesterone, d317-hydroxypregnenolone, d4-cortisone, d3-cortisol (purchased from Cambridge Isotope Laboratories, Andover, MA), and d4-estrone and d3-estradiol (purchased from CDN Isotopes, Toronto, ON). All other chemicals were of the highest purity commercially available.
steroid. Two mass transitions were monitored for each steroid and its internal standard. Quantitative data analysis was performed using Analyst™ 1.5.2 software. Calibration curves were generated with every set of samples using six calibrators, and three quality control samples were included with every set of samples. Specificity of the analysis for each steroid in every sample was evaluated by comparing concentrations determined using the primary and the secondary mass transitions of each steroid and its internal standard [25]. 2.6. Statistical analysis Statistical analysis was performed using the software Prism 6 (GraphPad Software, Inc. La Jolla, USA). The normality of the data distribution was examined using the Shapiro–Wilks W-test. The steroid profile data was analyzed using the Kruskal–Wallis test and Dunn’s post hoc test since these data followed a non-Gaussian distribution. For the purpose of statistical analysis of steroids for which concentrations were below the detection limit, the values were set to the concentration corresponding to the respective limit of quantification of the methods. Statistical analyses of the weight measurements were performed using one-way ANOVA and Tukey’s post hoc tests. Correlation analysis was performed with the nonparametric Spearman correlation test. Concentrations of plasma steroids are expressed as median, minimal and maximal values; weight measurements are presented as mean ± SEM, p-values less than 0.05 were considered significant. 3. Results
2.5. Liquid chromatography tandem mass spectrometry (LC–MS/MS)
3.1. Steroid analysis
Plasma samples were analyzed for pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, cortisone, corticosterone, DHEA, androstenedione, testosterone, progesterone, estrone and estradiol using LC–MS/MS as previously described [20–24]. Briefly, steroids were extracted from samples, corticosterone, DHEA, androstenedione, testosterone, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, progesterone, and 11-deoxycortisol were derivatized with hydroxylamine to form oxime derivatives; estrone, and estradiol was derivatized with dansyl chloride to form oxime derivatives [23]. Cortisol and cortisone were analyzed underivatized [24]. Limit of quantification (LOQ) was 0.05 ng/mL for pregnenolone, 17hydroxyprogesterone, and 11-deoxycortisol, 0.25 ng/mL for 17hydroxypregnenolone, 1 ng/mL for progesterone, 0.5 ng/mL for corticosterone, 0.01 ng/mL for testosterone and androstenedione, 0.05 ng/ml for DHEA, 1 ng/mL for cortisol and cortisone, and 1 pg/ml for estrone and estradiol. The intra-assay and inter-assay CV were <8% and 11%, respectively [23]. All steroids were analyzed in positive ion mode using an electrospray ion source on a triple quadruple mass spectrometer (API5500; AB Sciex, Foster City, CA). The HPLC system consisted of series 1260 HPLC pumps (Agilent Technologies), and an HTC PAL autosampler (LEAP Technologies, NC) equipped with a fast wash station. The quadrupoles Q1 and Q3 were tuned to unit resolution and the mass spectrometer conditions were optimized for maximum signal intensity of each
In this study thirteen steroids were measured in rat plasma, of these steroids six were not detected. The LC–MS/MS methods used in this study were developed and validated for analysis of human serum and plasma samples and were shown to perform well for analysis of the rat plasma samples. The present study demonstrated that ND treated animals had very low plasma concentrations of testosterone and androstenedione (below the detection limit in all the rats of the group) (Fig. 2). Plasma concentration of estradiol was significantly elevated in the ND and the ND + GHtreated rats compared to controls, no alteration of estradiol was seen in the rhGH-treated rats compared to controls. The median plasma concentrations in the control group of estrone, testosterone, and androstenedione were 2.30 pg/ml, 0.80 ng/ml, and 0.07 ng/ml, respectively. The corresponding median concentrations in the ND treated group were 3.50 pg/ml, 0.01 ng/ml, and 0.01 ng/ ml, respectively. The median concentrations in the group treated with rhGH were 2.80 pg/ml, 0.80 ng/ml, and 0.04 ng/ml, respectively. Corresponding concentrations in the group treated with both ND and GH were 3.30 pg/ml, 0.01 ng/ml, and 0.01 ng/ml, respectively. No significant difference in plasma concentration of pregnenolone was observed among the treatment groups. The corticosterone concentration varied between the rats. A few animals (5 out of 12 rats) in the rhGH treated group had highly elevated concentrations. Although the result overall was not statistical significant when all the rats in the groups were included in
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Fig. 2. Box plots with distribution of plasma concentrations of endogenous steroids in rats after treatment of ND and GH. Values are presented as median, minimum and maximum values, n = 11–12/group. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
the comparison (p = 0.34). The concentration of 17-hydroxyprogesterone was elevated in the GH treated animals compared to the ND treated (p = 0.027). A positive correlation between corticosterone and 17-hydroxyprogesterone concentrations was found in all treatment groups (controls r2 = 0.94, p < 0.0001, GH r2 = 0.49, p = 0.01, ND r2 = 0.52, p = 0.0077, ND + GH r2 = 0.98, p < 0.0001). Concentrations of DHEA did not differ significantly between the treatment groups, however, in all groups some of the DHEA measurements were under the detection limit suggesting low concentrations of DHEA in rat plasma. Concentrations of cortisol, cortisone, 11-deoxycortisol, progesterone, estradiol, and 11-hydroxypregnenolone were below the detection limit of the methods, irrespective of the type of treatment. Considering the difference in distribution of the concentrations of pregnenolone, 17-hydroxyprogesterone, DHEA, corticosterone, androstenedione, testosterone, and estradiol on a pie diagram (Fig. 3), both rhGH and ND induced a change in the plasma steroid profile. 3.2. Weight of peripheral organs The weight of heart, liver, thymus, and testis in the sacrificed rats was measured and a summary of the data is shown in Fig. 4. Due to the differences in body weight between the animals in different treatment groups (Table 2), the weights of the various organs are expressed as normalized to the body weight (shown as mg tissue of 100 g of the body weight). The results demonstrated (a) decreased liver weight in the animals receiving ND + GH compared to controls and rhGH-treated animals and (b) a significant decrease of the weight of the thymus in the rats receiving ND and ND + GH (c) an increase in the weight of the thymus gland of
the GH rats as compared to the controls (d) the weight of the testis differed between the treatment groups: rats receiving only ND had a significantly larger testis compared to all other groups, including the ND + GH group. The results did not demonstrate any significant alteration of weight of the heart among the treatment groups.
4. Discussion In this study, the impact of AAS administration alone or in combination with GH, on the biosynthesis of steroids in male rats was investigated. Seven steroids were successfully measured and the results demonstrated an altered steroid profile from rhGH and ND administration. Administration of ND decreased the concentration of testosterone and androstenedione, and elevated the estradiol concentration; addition of rhGH did not effect these alterations. Herein a LC–MS/MS method was applied for the analysis and determination of concentrations of central steroids in rat plasma. The methodology has previously successfully been used for analyses of steroids in plasma from humans [23], as well of steroids in various other biological fluids [26–29]. The small sample volumes needed, high specificity, high sensitivity and use for the methods for routine measurement of steroids in clinical diagnostic settings represent advantages with the method. The divergence concentrations of steroids we observe in comparison with outcomes from other studies where alternative analytical techniques were used is reflecting e.g. different detection ranges and lower specificity of analysis [30–32]. The administration of ND decreased plasma concentrations of testosterone. This was an expected observation since AAS are well
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Fig. 3. Pie charts with relative concentration of seven steroids measured in the rat plasma, plotted as percent of total plasma steroid concentrations, after treatment with ND and GH (n = 11–12/group).
Fig. 4. The impact of ND and GH on weight of liver, testis, heart and thymus. Values are presented as mean ± SEM, n = 11–12/group. ⁄p < 0.05,
known to activate the negative feedback loop of the HPA/HPG-axis and decrease concentrations of LH and FSH [14–16]. We observed a significant decrease of androstenedione, the precursor of
⁄⁄
p < 0.01,
⁄⁄⁄
p < 0.001.
testosterone, caused by the ND administration, which could also be explained by the negative feedback of the HPA/HPG-axis. Our results did not demonstrate a significant difference in the
A. Grönbladh et al. / Steroids 78 (2013) 1192–1199 Table 2 Body weight (g) on the last day of the experiment. ⁄p < 0.05 compared to GH and ND groups, ⁄⁄⁄p < 0.001 compared to control and GH groups. Values are presented as mean ± SEM.
Weight (g)
Controls
GH
ND
ND + GH
392 ± 7
407 ± 7
340 ± 6⁄⁄⁄
367 ± 8⁄
concentration of 17-hydroxyprogesterone, which could indicate a down-regulation of the conversion of 17-hydroxyprogesterone to androstenedione. It has been postulated that rat adrenals lack the 17alpha-hydroxylase/17,20 lyase (CYP17) enzyme [33,34] although, there are reports supporting adrenal testosterone production in rats [35,36]. The 17-hydroxyprogesterone is in rat testes mainly formed from progesterone by CYP17 hydroxylase activity, and then converted to androstenedione by CYP17 lyase enzyme, i.e. the C–C bond in the 17-position is cleaved. In humans, it has been suggested that the lyase and hydroxylase function of CYP17 are regulated separately [37], so an explanation for the reduction of concentrations of androstenedione and not 17-hydroxyprogesterone could be an alteration of the CYP17 phosphorylation state, because CYP17 has only low or no lyase activity when under-phosphorylated [38]. However, if this were true, we should have detected increased 17-hydroxyprogesterone concentrations due to its accumulation [39]. Alternatively, the increased expression of CYP19 (aromatase) that occurs after ND administration, which has been shown to be induced by an androgen receptor mediated system [40,41] could account for the lower concentrations of testosterone and its precursor androstenedione. However, although the estradiol concentrations were found to be increased, the concentrations of estradiol are still very low compared to concentrations of androstenedione and testosterone. It is therefore not likely that the significant reduction in concentrations of androstenedione and testosterone is primarily attributed to the up-regulation of aromatase causing a conversion of androstenedione to estrone. One should bear in mind the fact that estrone is in equilibrium with estradiol. Normally, estrogens can be synthesized by either conversion of testosterone to estradiol, or by conversion of androstenedione to estrone, both reactions involving the enzyme CYP19 (aromatase). Estrone can then be further converted to estradiol by the enzyme 17b-hydroxysteroid dehydrogenase (17bHSD2) and vice versa (see Fig. 1). However, since nandrolone is known to be a substrate to aromatase we suggest that the major part of the estrone probably originates from the ND administration. Supporting this are early studies demonstrating aromatization of nandrolone to estrone [42,43] and ND administration to male patients also increased estrone concentrations [44]. Although, studies have suggested that nandrolone is not aromatized to the same extent as testosterone [45]. The treatment with rhGH was found to increase 17-hydroxyprogesterone concentrations. This has also been demonstrated in patients treated with rhGH during one year [46]. Another study could not detect any alteration of 17-hydroxyprogesterone concentrations from GH treatment [47], thus, the exact impact of the hormone remains uncertain. In some of the GH-treated rats, we found the corticosterone concentration to be highly elevated, indicating that GH stimulates secretion of this glucocorticoid, however, the among-groups comparison did not show statistically significant differences. Earlier studies have demonstrated increases of plasma corticosterone concentrations after GH treatment [48–50] and it has been proposed that this effect could be mediated through a stimulation of ACTH release via the hypothalamus [48]. Previous studies on the effect of AAS on corticosterone concentrations have been inconsistent [51–54], implying that length of steroid administration, time of the blood collection, and the administration route can have an
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impact on the corticosterone concentration. Long-term AAS intake in humans decreased ACTH concentrations [55]. In rats, ND induced an increase of ACTH and corticosterone concentrations one hour, but not 24 h after the last injection [52] suggesting a biphasic effect of AAS on the HPA-axis. Corticosterone is the main glucocorticoid in rats and only traces of cortisol have been detected in rats [56]. Furthermore, earlier studies suggest that the D4 (4-ene) pathway of the steroid synthesis is the predominant pathway in rats [57], whereas the D5 (5-ene) pathway is predominant in humans [58] (see Fig. 1). Thus, in this study, the low concentrations or absence of some of the steroids characteristic of the pathway of steroids biosynthesis in humans (D5) were expected [34,56]. Although an influence of GH on the HPG-axis has been established, the administration of rhGH did not induce any alteration of the testosterone concentrations in this study. This observation is consistent with the data from earlier studies, GH supplementation in humans did not affect concentrations of testosterone [59,60]. In an animal model, overexpression of the GH releasing hormone (GHRH) was shown to increase concentration of GH, which resulted in a decrease in concentration of testosterone [61]. The rhGH treatment had no influence on the inhibition of biosynthesis of endogenous testosterone, and the combined effects of rhGH and ND did not have a major impact on the steroid concentrations measured herein. A recent study examined the effect of a combined treatment with GH and testosterone in men undergoing chronic glucocorticoid treatment and detected an additive effect on skeletal muscle mass compared to testosterone or GH alone [62]. Since ND is known to have a half-life of approximately six days [63], the rats were expected to have high plasma concentration of ND on the last day of the experiment. The ND treatment was discontinued on day 21 of the experiment and these results demonstrated that the endogenous androgen production was decreased ten days after the last ND injection. Another indicator of the effect of ND was a large decrease of thymus observed in the animals receiving ND, this observation is in agreement with earlier studies, where a significant effect of AAS on thymus also was observed [53,64]. Indeed, it is hypothesized that AAS produce thymolytic effects by stimulating the secretion of molecular entities by the thymic epithelial cells that may induce regression of the thymus gland [65]. Furthermore, an increase of thymus was seen from the rhGH administration, indicating thymus as a target for GH actions (for a review, see [66]). For example, in AIDS patients receiving longterm treatment with GH, a significant increase of thymus has been demonstrated [67]. In humans excessive levels of exogenous androgens have been reported to decrease spermatogenesis and induce testicular atrophy [68,69]. In addition, administration of ND for 14 weeks reduced the testis volume in rats [70]. In the present study, the rats receiving ND exhibited a slight increase of the normalized testis weight (mg tissue/100 g body weight). The non-normalized testis weights (g) did however not differ significantly between the treatment groups (data not shown). Interestingly, the testis weight was previously demonstrated to be unaffected by two weeks of ND administration in male Sprague Dawley rats, suggesting the effect of AAS on testis atrophy may require a long-term treatment [53]. In conclusion, repeated administration of a high dose of ND to male rats resulted in significantly elevated concentrations of estrone in plasma, and reduced concentrations of testosterone and androstenedione. Treatment of the rats with rhGH altered the steroid profile but administration of rhGH to the AAS treated rats did not reverse the ND-induced changes in the steroid profile. Furthermore, administration of rhGH to rats induced an enlargement of thymus, however rhGH did not reverse the reduction of thymus caused by ND. This study further highlights the major impact of nandrolone decanoate on biological and endocrine functions.
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Acknowledgements This study was supported by grants from the Swedish Research Council (Grant 9459, 621-2011-4423) and ARUP Institute for Clinical & Experimental Pathology, Salt Lake City, USA.
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