Lipid peroxidation and apoptotic response in rat brain areas induced by long-term administration of nandrolone: the mutual crosstalk between ROS and NF-kB

Abstract

The aim of this study was to evaluate the played by oxidative stress in the apoptotic response in different brain areas of rats chronically treated with supra-physiological doses of nandrolone decanoate (ND). Immunohistochemical study and Western blot analysis were performed to evaluate cells' apoptosis and to measure the effects of expression of specific mediators, such as NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), Bcl-2 (B-cell lymphoma 2), SMAC/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low PI) and VMAT2 (vesicular monoamine transporter 2) on apoptosis. The results of the present study indicate that a long-term administration of ND promotes oxidative injury in rat brain specific areas. A link between oxidative stress and NF-κB signalling pathways is supported by our results. In addition to high levels of oxidative stress, we consistently observed a strong immunopositivity to NF-κB. It has been argued that one of the pathways leading to the activation of NF-κB could be under reactive oxygen species (ROS)-mediated control. In fact, growing evidence suggests that although in limited doses, endogenous ROS may play an activating role in NF-κB signalling, while above a certain threshold, they may negatively impact upon this signalling. However, a mutual crosstalk between ROS and NF-κB exists and recent studies have shown that ROS activity is subject to negative feedback regulation by NF-κB, and that this negative regulation of ROS is the means through which NF-κB counters programmed cells.

Keywords: apoptosis; nandrolone decanoate; neurotoxicity; oxidative stress.

Figure 1

MDA (nmol/mg tissue) in rat brain areas after administration of nandrolone decanoate 1.875 mg/kg twice per week by intramuscular injection for 8 weeks (each value is the mean ± S.D. of three animals). Cont: Control; PFC: frontal cotex; Str: striatum; Hipp: hippocampus; Cer: cerebellum.

Figure 2

Strong and uniform NF‐kB neuronal positivity was found in the frontal cortex (A), striatum and hippocampus (B), of the nandrolone group. Western blot analysis detects the chemiluminescent blots of NF‐kB (C). (D) control group with negative results.

Figure 3

Confocal laser scanning microscopy showed markedly Bcl‐2 positive cytoplasmic reaction (in brown) on the striatum (A) and hippocampus (in brown) (B) in rats after nandrolone treatment. (C) Western blot analysis detects the chemiluminescent blots of Bcl‐2 in the treated group. (D) control group with negative results.

Figure 4

A strong SMAC/DIABLO localization on the dendrites and neuronal cell body positivity located in the frontal cortex (A), striatum (B) and hippocampus areas was revealed for treated rats. (C) Chemiluminescent blots of SMAC/DIABLO in the treated group. (D) Control group with negative results.

Figure 5

VMAT‐2 weaker reactions on the dendrites and neuronal cell bodies of treated rats compared to controls (D). In particular the most significant difference was found in striatum (A) and hippocampus (B) samples. (C) Western blot analysis detects the chemiluminescent blots of VMAT2 in the treated group.

Figure 6

The neuronal nuclei labelled by TUNEL assay showed an intense, widespread, positive reaction in the treated group, especially in the frontal cortex: confocal laser scanning microscopy shows a marked positive nuclear reaction (in green apoptotic bodies) (A), and hippocampus (B) samples. Spotted positive nuclei were observed in cerebellum samples (in bleu) (C) and in the control group (arrow) (D).

Figure 7

Mechanisms of the neuropathological effects of AASs: evidence has shown the recurrence of increased neuronal susceptibility to apoptotic stimuli as a source of the neurodegenerative and neurotoxic potential of these compounds. ROSs represent a serious hazard for cells, because they are powerful oxidizing molecules able to damage proteins, lipids and DNA. ROSs act as second messengers in various biological responses, among which the induction of programmed cell death is of paramount importance in our understanding of many common diseases and degenerative conditions. Growing evidence suggests that endogenous ROS may play an activating role in NF‐kB signalling, and above a certain threshold, they may negatively impact upon this signalling. ROS are thought to have an inhibitory effect on NF‐kB activity.