Abstract
The abuse of anabolic androgenic steroids (AAS) has been considered a major public health problem during decades. Supraphysiological doses of AAS may lead to a variety of neuroendocrine problems. Precisely, the hypothalamic-pituitary-gonadal (HPG) axis is one of the body systems that is mainly influenced by steroidal hormones. Fluctuations of the hormonal milieu result in alterations of reproductive function, which are made through changes in hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH). In fact, previous studies have shown that AAS modulate the activity of these neurons through steroid-sensitive afferents. To increase knowledge about the cellular mechanisms induced by AAS in GnRH neurons, we performed proteomic analyses of the murine hypothalamic GT1-7 cell line after exposure to 17α-methyltestosterone (17α-meT; 1 μM). These cells represent a good model for studying regulatory processes because they exhibit the typical characteristics of GnRH neurons, and respond to compounds that modulate GnRH in vivo. Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry analyses identified a total of 17 different proteins that were significantly affected by supraphysiological levels of AAS. Furthermore, pathway analyses showed that modulated proteins were mainly associated to glucose metabolism, drug detoxification, stress response and cell cycle. Validation of many of these proteins, such as GSTM1, ERH, GAPDH, PEBP1 and PDIA6, were confirmed by western blotting. We further demonstrated that AAS exposure decreased expression of estrogen receptors and GnRH, while two important signaling pathway proteins p-ERK, and p-p38, were modulated. Our results suggest that steroids have the capacity to directly affect the neuroendocrine system by modulating key cellular processes for the control of reproductive function.
Highlights
The anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone, created to provide anabolic potency and low androgenic effect [1]
Basal testosterone levels of cerebrospinal fluid (CSF) ranged between 0.0001–0.001 μM [41,42], systemic exposure to high levels of methyltestosterone during 6 days (40–240 mg/day) led to concentrations in the CSF that ranged between 0.065–0.9 μM [43]. This dose is similar to the dose tested in our in vitro study. These results suggest that cell populations in the brain of AAS abusers can be under the direct influence of supraphysiological concentrations of the drug, and that anabolic steroids have the potential to cause negative consequences at the molecular, physiological and/or behavioral levels
By interrogating the proteome of the hypothalamic GT1-7 cell line as a model for studying AAS-induced regulatory processes of reproduction, we found that anabolic steroids modulate proteins associated to metabolism, cytoarchitecture, cellular homeostasis and hormonal regulation
Summary
The anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone, created to provide anabolic potency and low androgenic effect [1]. Under the influence of AAS, the hypothalamic-pituitary-gonadal (HPG) axis is one of the most affected body systems [4,5]. The neural control of this axis resides within the hypothalamus, which is characterized by the expression of high levels of androgen and estrogen receptors (AR and ER) [6]. GnRH neurons respond to fluctuations of gonadal steroids, which result in neuroplasticity changes leading to pulsatile secretion of this peptide. In vivo and in vitro studies have shown that exposure to steroids is associated with alterations on GnRH expression and secretion. Studies in rodents [8], and cells [9,10] showed that exposure to either androgens or estrogens reduced GnRH transcripts
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