Edited by: Chao Deng, University of Wollongong, Australia
Reviewed by: Agata Copani, University of Catania, Italy; Aram Megighian, University of Padua, Italy
*Correspondence: Patrizia Proia, Department of Sports Science (DISMOT), University of Palermo, Via Eleonora Duse 2, Palermo 90146, Italy. e-mail:
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Anabolic-androgenic steroids (AAS) are lipophilic hormones often taken in excessive quantities by athletes and bodybuilders to enhance performance and increase muscle mass. AAS exert well known toxic effects on specific cell and tissue types and organ systems. The attention that androgen abuse has received lately should be used as an opportunity to educate both athletes and the general population regarding their adverse effects. Among numerous commercially available steroid hormones, very few have been specifically tested for direct neurotoxicity. We evaluated the effects of supraphysiological doses of methandienone and 17-α-methyltestosterone on sympathetic-like neuron cells. Vitality and apoptotic effects were analyzed, and immunofluorescence staining and western blot performed. In this study, we demonstrate that exposure of supraphysiological doses of methandienone and 17-α-methyltestosterone are toxic to the neuron-like differentiated pheochromocytoma cell line PC12, as confirmed by toxicity on neurite networks responding to nerve growth factor and the modulation of the survival and apoptosis-related proteins ERK, caspase-3, poly (ADP-ribose) polymerase and heat-shock protein 90. We observe, in contrast to some previous reports but in accordance with others, expression of the androgen receptor (AR) in neuron-like cells, which when inhibited mitigated the toxic effects of AAS tested, suggesting that the AR could be binding these steroid hormones to induce genomic effects. We also note elevated transcription of neuritin in treated cells, a neurotropic factor likely expressed in an attempt to resist neurotoxicity. Taken together, these results demonstrate that supraphysiological exposure to the AAS methandienone and 17-α-methyltestosterone exert neurotoxic effects by an increase in the activity of the intrinsic apoptotic pathway and alterations in neurite networks.
Anabolic-androgenic steroids (AAS) are lipophilic hormones, derived from cholesterol, that include in the same family the natural male hormone testosterone, along with the related molecules methandienone, 17-α-methyltestosterone, nandrolone, and androsterone (Orlando et al.,
AAS exert their effects in many parts of the body, including the reproductive and endocrine tissues, muscle, bone, hair follicles in the skin, the liver and kidneys, and the hematopoietic, immune and central nervous systems (CNS) (Mooradian et al.,
Inappropriate activation of apoptosis in neurons has been associated with several neurological illnesses, including Huntington disease and Alzheimer disease (AD) (Varshney and Ehrlich,
Given that AAS abuse poses a significant public health problem and based upon the previously published data, we investigated the morphological, biochemical and molecular mechanisms leading to changes in neuronal physiology, in particular neuronal cell death, for supraphysiological concentrations of methandienone and 17-α-methyltestosterone, two AAS commonly found for sale on the internet and used for gain muscle mass but less studied than other hormones such as nandrolone and androsterone. Derivates of methandienone and 17-α-methyltestosterone also resist metabolism in the liver and contain modifications that are associated with significant hepatic toxicities (Kuhn,
There are a number of well characterized
Here we demonstrate that rat neurons and PC12 cells express the androgen receptor (AR). We describe a reduction in neurite networks and loss of survival signaling and enhanced apoptosis, as evidenced by a decrease in phospho-ERK and an increase in the levels of the active fragment of caspase 3 and cleaved poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP), as well as upregulation and cleavage of heat shock protein (Hsp) 90, occurring in a dose-dependent manner in androgen treated PC12 differentiated in NGF. Many of these observations were noted after long exposures of PC12 to AAS, suggesting a genomic effect, and through the use of hydroxyflutamide we demonstrate that AAS toxicity proceeds directly through the AR, likely altering gene transcription to affect cell survival (Heinlein and Chang,
Undifferentiated pheochromocytoma 12 cells (PC12, ATCC, Manassas, VA) were cultured in RPMI-1640 medium containing 10% horse serum and 5% fetal bovine serum (Sigma-Aldrich, St. Louis, MO). For induction of differentiation, cells were grown in 12-well plates on poly-D-lysine (Sigma-Aldrich) at an initial concentration of 1 × 105 cells per square centimeter in a medium supplemented with 200 ng/ml of nerve growth factor (NGF, Promega Corporation, Madison, WI) for 5 days. During this time, cells attached to the substratum and produced a network of neurites. Differentiated PC12 cultures were treated with vehicle (dimethyl ether, DME) or the steroid hormones androsterone, nandrolone, methandienone and 17-α-methyltestosterone (Cerilliant Corporation, Round Rock, Texas) at a concentration of 50–75 μM for times defined as short term (24 h), and long term (48 h) (Duranti et al.,
Immunohistochemistry on rat brain slices was performed following the protocol described previously (Basile et al.,
Control or methandienone and 17-α-methyltestosterone treated PC12 cells were fixed with 4% paraformaldehyde in PBS for 10 min. on ice and subsequently permeabilized in cold methanol for an additional 5 min. After three washes with PBS, cells were blocked with 5.5% FBS in PBS with 0.1% Triton X-100 for 45 min. at room temperature. The cells were then incubated with AR antibody (Santa Cruz, CA) in 3% BSA/ PBS overnight at 4°C. The next day, cells were washed three times in 0.1% Triton/ PBS and once in PBS and incubated with biotinylated secondary antibody (Dako North America, Carpinteria, CA) for 45 min. Cells were then washed three times in 0.1% Triton/ PBS and then once again in PBS, followed by an incubation in 0.6% H2O2 for 30 min. at room temperature to quench endogenous peroxidase. Cells were then incubated in strep ABC complex (Dako North America) at room temperature for 30 min, washed and incubated with DAB peroxidase substrate (Vector Laboratories, Youngstown, OH) following manufacturer's instructions. Counterstain was performed in dilute Harris hematoxylin (Sigma-Aldrich). Image acquisition was performed as described for immunohistochemistry (see above).
1 × 105 PC12 cells per square centimeter growing on poly-D-lysine coated cover slips were differentiated for 5 days in 200 ng/ml of NGF (Promega), followed by treatment with 75 μM of methandienone or 17-α-methyltestosterone (Cerilliant) or equal amounts of DME (as the carrier control). Cells were then fixed with 96% ice-cold ethanol for 10 min. and permeabilized for 5 min. with 0.1% Triton X-100 in PBS. Cells were blocked in 3% fetal bovine serum for 30 min., followed by 1 h incubation in a humidity chamber at room temperature with rabbit polyclonal anti-NF antibody (Cell Signaling Technology, Beverly, MA). The secondary antibody was anti-rabbit IgG conjugated to fluorescein (Sigma Aldrich). The samples were mounted with Vectashield mounting medium containing 4-6-diamino-2-phenyl-indole (DAPI, Vector Laboratories). Morphological analysis and quantification of neurite bearing cells were carried out using an Aperio Scanscope (Aperio Technologies, Vista, CA). Ten randomly separated microscopic fields were observed and the proportion of cells with neurites equal to or greater than the length of one cell body were scored positive for neurite outgrowth, with the final result expressed as a percentage of the total number of cells counted. Neurite extension length was also measured for all identified positive neurite bearing cells per field by tracing the longest length neurite using the Neuron J module of Image J software (NIH, Bethesda, MD, version 1.46c). The value of neurite length in pixels (average maximal neurite length per neurite-bearing cell in 10 fields) was calculated and designated as one experiment. All experiments were repeated at least three times on separate days and data are expressed as mean ± SD.
Cell death was evaluated by staining PC12 cells treated for 48 h with 75 μM methandienone, 17-α-methyltestosterone or DME with acridine orange/ethidium bromide mixture (Sigma-Aldrich), each at a concentration of 100 μg/ml in PBS as previously described (Schiera et al.,
After the indicated treatment, cells were collected, washed with PBS, and homogenized in lysis buffer (Cell Signaling Technology) supplemented with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 μ l/ml aprotinin and leupeptin, Sigma-Aldrich) and phosphatase inhibitors (2 mM NaF and 0.5 mM sodium orthovanadate, Sigma-Aldrich). After centrifugation, protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad Hercules, CA). 15 μg of protein was loaded onto each lane of 12% acrylamide-SDS denaturing gels. After electrophoretic separation, samples were electroblotted onto a PVDF membrane (Immobilon P, Millipore Corp., Billerica, MA). The blotting membrane was blocked with 5% milk and then immunostained with one of the following antibodies: anti-phospho-ERK (Cell Signaling Technology); rabbit polyclonal anti-Hsp90 (Cell Signaling Technology); rabbit polyclonal anti-cleaved caspase 3 (Cell Signaling Technology); rabbit polyclonal anti-PARP (Cell Signaling Technology); anti-GAPDH (Sigma-Aldrich).
RNA was extracted from treated and untreated PC12 cells using the TRIZOL reagent (Life Technologies, Grand Island, NY) according to the protocol provided by the manufacturer. After the last step of the protocol, the RNA was air-dried and then dissolved in RNAase free water for quantification by spectrophotometer. 1 μg of RNA was used for reverse transcription to cDNA using the AMV reverse-transcriptional system (Promega) in the presence of random hexamers (Invitrogen, Life Technologies). The cDNA was used for quantitative real-time PCR (RT-qPCR) with specific gene primers as follows: Neuritin sense: 5′-gcatctggtgaataatcgctcacg-3′, anti-sense: 5′-actgaaggaggcgacgacaatagc-3′; GAPDH sense: 5′-atcccatcaccatcttccag-3′, anti-sense: 5′-cctgcttcaccaccttcttg-3′. The CT method was used for data analysis of neuritin mRNA expression, estimated in triplicate samples and normalized to GAPDH expression levels.
Student's paired
The AR, a member of the nuclear receptor superfamily of transcription factors, is capable of binding the principal steroidal androgens testosterone and its metabolite 5α-dihydrotestosterone, as well as other AAS, and mediating their effects within the cell (Lee and Chang,
Neuron cell cultures are a useful system to study potential deleterious effects of different compounds. In this model, alterations in sprout formation and neurite length are used as a determinant of neurotoxicity (Radio and Mundy,
To further determine toxicity, PC12 were grown in 75 μM of methandienone and 17-α-methyltestosterone and examined for cell death in a vitality assay. In this system, cells are analyzed by immunofluorescence to detect membrane integrity based upon the uptake or exclusion of a dye from the cell. Ethidium bromide (EB) fluoresces red and is only able to pass through the membrane of a dead or dying cell, while acridine orange (AO), which fluoresces green, is a membrane-permeable dye that will stain all cells in the sample. Cells fluorescing yellow are taking up both EB and AO and represent an early stage of cell death, with a more orange color indicating a later stage in the process. We observed that most control treated, differentiated PC12 remained vital (Figure
To confirm that the cell death we observed was apoptosis, and to compare with other AAS to determine if this could be a general mechanism of toxicity for a variety of androgens, we examined by immunoblot for levels of active, phosphorylated ERK, an indicator of cell survival. The MAPK cascade, and in particular ERK, has been shown to be protective in neuronal cell types, allowing them to survive exposure to pro-apoptotic compounds (Karmarkar et al.,
Examining methandienone and 17-α-methyltestosterone in more detail, we noted upregulation of the active fragment of caspase 3 occurring in a dose dependent manner following treatment of both of these AAS (Figure
To determine if apoptosis proceeds through the AR, we treated cells with methandienone and 17-α-methyltestosterone but this time in the presence of the anti-androgen hydroxyflutamide (Nguyen et al.,
Neuritin is a neurotrophic factor that plays an important role in neurite growth and survival. It is known to be upregulated in damaged, stressed or ischemic neurons as they attempt to re-establish connectivity following injury (Ujike et al.,
There are great challenges in attempting to characterize the potential risks of neurotoxicity for environmental chemicals and pharmacological agents such as AAS. Indeed, among the innumerable commercial compounds available, a relative few have been adequately characterized for their potential effects on human health in general, and fewer still specifically tested for neurotoxicity. It is important to note that the number of studies with rigorous scientific methodology that have derived significant conclusions is small, whereas the intensity of the underground marketing and promotion of most AAS is intense, far exceeding the data supporting their use. At the same time it is important to note that non-hormonal supplements, such as vitamins, amino acids, caffeine and ephedrine often contain anabolic steroids that are not declared on the labels of the products. The most abundant steroids found in these supplements are methandienone and 17-α-methyltestosterone.
It has been suggested that tests based on a variety of
The development of the CNS involves coordinated gene expression and an ordered initiation of specific cellular events regulating proliferation, differentiation, cell migration, neurite outgrowth, synapse formation, myelination, and programmed cell death. Theoretically, chemically mediated disruption of one or more of these events could potentially impair CNS development or function (Barone et al.,
The concentrations of steroids used in our experiments are comparable to that used in other studies examining different compounds considered to be performance-enhancing. For example, in Duranti's study, rat L6C5 and mouse C2C12 skeletal muscle cells were treated with up to 20 μM of the β2-adrenergic receptor agonist salmeterol at a concentration of 1 × 104 cells per square centimeter, conditions comparable to our AAS experiments (Duranti et al.,
AAS that previously had been shown to damage neurite networks in PC12 cells have also been shown to activate the apoptotic pathway. Having detected evidence of toxicity in the neurite outgrowth assay, we then wanted to determine if the continued exposure of differentiated PC12 to AAS might induce cell death. Therefore, we examined PC12 cells in a vitality assay and observed evidence of permeability to an acridine orange/ethidium bromide mixture, and hence loss of membrane integrity and cell death, following exposure to methandienone and 17-α-methyltestosterone, with the latter showing slightly more cell death than the former. To determine the nature of cell death, we investigated phospho-ERK, a marker of ell vitality and survival, and several components of the apoptotic pathway. Phosphorylation of ERK was reduced upon exposure to androsterone, nandrolone, methandienone and 17-α-methyltestosterone. Caspase 3 is one of the key executioners of apoptosis, engaging in the proteolytic cleavage of many key proteins such as the nuclear enzyme PARP. Indeed, the finding that caspase 3 is expressed in PC12 suggests a role for this protease in PC12 cell death (Haviv et al.,
We noted that the effects on cells following AAS treatment were delayed, suggesting that these hormones might exert their effects by acting on AR-mediated genomic pathways. Though previous efforts have failed to detect the AR receptor in PC12 cells by RT-PCR (Nguyen et al.,
Hsp90 is a molecular chaperone responsible for controlling numerous signaling pathways in the cell (Bishop et al.,
We also observed a dose-dependent increase in neuritin mRNA levels in PC12 cells treated with methandienone and 17-α-methyltestosterone, which to our knowledge is the first
Here we show that at high concentrations, methandienone and 17-α-methyltestosterone exert detrimental effects on differentiated PC12 cells expressing AR, including inhibition of neurite network maintenance, induction of cell death through apoptosis and cleavage of the protective chaperone protein Hsp90. Between these two compounds we noted greater cell death and higher neuritin transcription in PC12 in response to 17-α-methyltestosterone treatment, supporting the belief that this AAS is the more toxic to neuron-like cells of the two compounds tested. These findings will be pursued in future investigations but currently suggest another potentially harmful physiological effect in the abuse of steroids, that of CNS toxicity.
John R. Basile provided protocols and support for immunohistochemistry and immunoblot experiments and helped draft the manuscript. Nada O. Binmadi assisted in immunohistochemistry, immunocytochemistry and neurite outgrowth experiments. Hua Zhou and Ying-Hua Yang performed the RT-PCR for neuritin. Antonio Paoli helped plan out experiments and draft the manuscript. Patrizia Proia conceived and designed the study, performed the vitality assays and helped draft the manuscript. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank Dr. Dong Wei of the Department of Neural and Pain Sciences at the University of Maryland Dental School for providing the rat brains for AR immunohistochemistry and Dr. Lorena Souza of the National Institutes of Health for assistance with immunofluorescence imaging. Special thanks to Dr. Alessia Gallo and Dr. Gabriella Schiera for their support and critical comments. John R. Basile is supported by the National Cancer Institute, NIH (R01 CA133162).
anabolic-androgenic steroids
central nervous system
androgen receptor
poly (adenosine diphosphate [ADP]-ribose) polymerase
heat shock protein
nerve growth factor
pheochromocytoma 12 cells
Ethidium bromide
Acridine orange.