A comparative study of the effect of the dose and exposure duration of anabolic androgenic steroids on behavior, cholinergic regulation, and oxidative stress in rats

Abstract

The aim of this study was to assess if the dose and exposure duration of the anabolic androgenic steroids (AAS) boldenone (BOL) and stanazolol (ST) affected memory, anxiety, and social interaction, as well as acetylcholinesterase (AChE) activity and oxidative stress in the cerebral cortex (CC) and hippocampus (HC). Male Wistar rats (90 animals) were randomly assigned to three treatment protocols: (I) 5 mg/kg BOL or ST, once a week for 4 weeks; (II) 2.5 mg/kg BOL or ST, once a week for 8 weeks; and (III) 1.25 mg/kg BOL or ST, once a week for 12 weeks. Each treatment protocol included a control group that received an olive oil injection (vehicle control) and AAS were administered intramuscularly (a total volume of 0.2 ml) once a week in all three treatment protocols. In the BOL and ST groups, a higher anxiety level was observed only for Protocol I. BOL and ST significantly affected social interaction in all protocols. Memory deficits and increased AChE activity in the CC and HC were found in the BOL groups treated according to Protocol III only. In addition, BOL and ST significantly increased oxidative stress in both the CC and HC in the groups treated according to Protocol I and III. In conclusion, our findings show that the impact of BOL and ST on memory, anxiety, and social interaction depends on the dose and exposure duration of these AAS.

Fig 1

Experimental Protocols: Protocol I: Intramuscular injection of vehicle (olive oil, 0.2 ml), stanazolol (ST, 5 mg/kg), or boldenone (BOL, 5 mg/kg) once a week for 4 weeks. Protocol II: Intramuscular injection of vehicle (olive oil, 0.2 ml), stanazolol (ST, 2.5 mg/kg), or boldenone (BOL, 2.5 mg/kg) once a week for 8 weeks. Protocol III: Intramuscular injection of vehicle (olive oil, 0.2 ml), stanazolol (ST, 1.25 mg/kg) or boldenone (BOL, 1.25 mg/kg) once a week for 12 weeks. Day 1: elevated plus maze task (EPM). Day 2: object recognition task, habituation. Day 3: object recognition task, training. Day 4: object recognition task, testing. Day 5: agonist behavior (resident–intruder paradigm), (n = 10).

Fig 2

Anxiogenic-like behavior of rats (n = 10) treated intramuscularly with vehicle (olive oil, 0.2 ml), boldenone (BOL), or stanazolol (ST) once a week according to Protocol I (4 weeks, 5 mg/kg), Protocol II (8 weeks, 2.5 mg/kg), or Protocol III (12 weeks, 1.25 mg/kg) as assessed in the elevated plus maze task. Time spent in the open-arms (A, B, and C), entry to open-arms (D, E, and F), time spent in closed-arms (G, H, and I), and crossing numbers (J; P1, P2 and P3). *Denotes significant difference from the vehicle group. # Denotes significant difference between BOL and ST groups.

Fig 3

Dominant behavior over the intruder of rats (n = 10) reated intramuscularly with vehicle (olive oil, 0.2 ml), boldenone (BOL), or stanazolol (ST) once a week according to Protocol I (4 weeks, 5 mg/kg), Protocol II (8 weeks, 2.5 mg/kg), or Protocol III (12 weeks, 1.25 mg/kg). Dominant behavior time for Protocol I (A), II (B), and III (C) and dominant behavior events for Protocol I (D), II (E), and III (F). *Denotes significant difference from the vehicle group. # Denotes significant difference between BOL and ST groups.

Fig 4

Dominant behavior over territory in rats treated intramuscularly with vehicle (olive oil, 0.2 ml), boldenone (BOL), or stanazolol (ST) once a week according to Protocol I (4 weeks, 5 mg/kg), Protocol II (8 weeks, 2.5 mg/kg), or Protocol III (12 weeks, 1.25 mg/kg). Time spent marking territory for Protocol I (A), II (B), and III (C) and territory-marking events for Protocol I (D), II (E), and III (F). *Denotes significant difference from the vehicle group. # Denotes significant difference between BOL and ST groups.

Fig 5

Submissive behavior of rats (n = 10) treated intramuscularly with vehicle (olive oil, 0.2 ml), boldenone (BOL), or stanazolol (ST) once a week according to Protocol I (4 weeks, 5 mg/kg), Protocol II (8 weeks, 2.5 mg/kg), or Protocol III (12 weeks, 1.25 mg/kg). Submissive behavior time for Protocol I (A), II (B), and III (C) and submissive behavior events for Protocol I (D), II (E), and III (F). *Denotes significant difference from the vehicle group. # Denotes significant difference between BOL and ST groups.

Fig 6

Aggressive behavior of rats (n = 10) treated intramuscularly with vehicle (olive oil, 0.2 ml), boldenone (BOL), or stanazolol (ST) once a week according to Protocol I (4 weeks, 5 mg/kg, graph A), Protocol II (8 weeks, 2.5 mg/kg, graph B), or Protocol III (12 weeks, 1.25 mg/kg, graph C). *Denotes significant difference from the vehicle group. # Denotes significant difference between BOL and ST groups.

Fig 7

Object recognition task index and acetylcholinesterase (AChE) activity in the brain of rats (n = 10) treated intramuscularly with vehicle (olive oil, 0.2 ml), boldenone (BOL), or stanazolol (ST) once a week (intramuscular) according to Protocol I (4 weeks, 5 mg/kg, graph A), Protocol II (8 weeks, 2.5 mg/kg, graph B), or Protocol III (12 weeks, 1.25 mg/kg, graph C). Object recognition task index for Protocol I (A), II (B), and III (C). AChE activity in the cerebral cortex for Protocol I (D), II (E), and III (F) and hippocampus for Protocol I (G), II (H), and III (I). *Denotes significant difference from the vehicle group. # Denotes significant difference between BOL and ST groups.

Fig 8

Parameters of oxidative stress in the cerebral cortex of rats (n = 10) treated intramuscularly with vehicle (olive oil, 0.2 ml), boldenone (BOL), or stanazolol (ST) once a week according to Protocol I (4 weeks, 5 mg/kg, graph A), Protocol II (8 weeks, 2.5 mg/kg, graph B), or Protocol III (12 weeks, 1.25 mg/kg, graph C). Reactive oxygen species (ROS) production for Protocol I (A), II (B), and II (C); malondialdehyde (MDA) levels for Protocol I (D), II (E), and III (F); reduced glutathione (GSH) levels for Protocol I (G), II (H), and III (I); non-protein thiol (NPSH) levels for Protocol I (J), II (K), and III (L). *Denotes significant difference from the vehicle group. # Denotes significant difference between BOL and ST groups.

Fig 9

Parameters of oxidative stress in the hippocampus of rats (n = 10) treated intramuscularly with vehicle (olive oil, 0.2 ml), boldenone (BOL), or stanazolol (ST) once a week according to Protocol I (4 weeks, 5 mg/kg, graph A), Protocol II (8 weeks, 2.5 mg/kg, graph B), or Protocol III (12 weeks, 1.25 mg/kg, graph C). Reactive oxygen species (ROS) production for Protocol I (A), II (B), and II (C); malondialdehyde (MDA) levels for Protocol I (D), II (E), and III (F); reduced glutathione (GSH) levels for Protocol I (G), II (H), and III (I); non-protein thiol (NPSH) levels for Protocol I (J), II (K), and III (L). *Denotes significant difference from the vehicle group. # Denotes significant difference between BOL and ST groups.