Supraphysiological doses of performance enhancing anabolic-androgenic steroids exert direct toxic effects on neuron-like cells

Abstract

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.

Keywords: PC12; anabolic-androgenic steroids; apoptosis; neuritin; neurotoxicity.

Figure 1

Immunohistochemistry in Purkinje cells of the rat cerebellum demonstrates expression of AR. Original magnification 200 X (A). AR expression in neurons of the rat brain stem. Original magnification 200 X (B). Immunocytochemistry for AR on PC12 in culture. Details of receptor expression are shown in the inset. Original magnification 200 X (C). AR expression in PC12 cells. Original magnification 400 X (D).

Figure 2

Immunofluorescence for neurofilament to demonstrate neurite outgrowth in PC12 cells differentiated in NGF in tissue culture, either control treated (left panel), treated with 75 μM methandienone (center panel) or treated with 75 μM of 17-α-methyltestosterone (right panel) (A). Quantification of neurite outgrowth in the three conditions shown in (A), as determined by cells exhibiting neurites equal to or greater than the length of one cell body, as a percentage of the total number of cells counted (Y-axis). Experiments were repeated at least three times and data expressed as mean ± SD (^**P ≤ 0.01). These AAS demonstrate toxic effects on formed neurite networks in differentiated PC12 (B). Average of the sums of neurite length for identified neurite bearing cells in 10 high-power fields (Y-axis). Experiments were repeated at least three times and data expressed as mean ± SD (^**P ≤ 0.01) (C).

Figure 3

PC12 cells were control treated (left panel) or treated with 75 μM methandienone (center panel) or 75 μM 17-α-methyltestosterone (right panel) for 48 h and a vitality assay was performed. Green cells are vital; cells in early cell death are yellow; orange indicates late cell death (A). Vitality status expressed as percentage of total live cells counted in 10 high power fields (Y-axis) reveals that methandienone and 17-α-methyltestosterone induce cell death in PC12. Experiments were repeated at least three times and data expressed as mean ± SD (^*P ≤ 0.05) (B).

Figure 4

Immunoblots demonstrate decreasing levels of phospho-ERK (top panel) in PC12 treated for 48 h with 75 μM androsterone, nandrolone, methandienone, and 17-α-methyltestosterone, compared to control treated cells (A). Immunoblots for caspase 3 (top panel), PARP (second panel), and Hsp90 (third panel) in PC12, control treated or treated 48 h with 75 μM androsterone, nandrolone, methandienone, and 17-α-methyltestosterone demonstrate increasing levels of the activated fragment of caspase 3 and cleavage of PARP and Hsp90, indications of apoptosis and cell death (B). PC12 were control treated or treated for 48 h with 50 or 75 μM methandienone (left column) or 17-α-methyltestosterone (right column). Increasing concentrations of these AAS resulted in appearance of the activated fragment of caspase 3 and cleavage of PARP and Hsp90 in a dose-dependent manner (C). GAPDH was used as the loading control for all blots (bottom panels).

Figure 5

Immunoblots for caspase 3 (top panel), PARP (second panel) (A) and Hsp90 (B) in PC12, control treated or treated for 48 h with 75 μ M methandienone and 17-α-methyltestosterone alone or with 10 μM hydroxyflutamide. Indicators of apoptosis are decreased in the presence of hydroxyflutamide. GAPDH was used as the loading control for all blots (bottom panels).

Figure 6

Levels of neuritin mRNA (ΔC_T, Y-axis) in PC12 treated for 24 h in the indicated concentrations of AAS, as determined by RT-qPCR normalized to GAPDH, reveal a dose-dependent increase in transcription. Experiments were repeated three times and data expressed as mean ± SD (^*P ≤ 0.05; ^**P ≤ 0.01).