Digital Comprehensive Summaries of Uppsala Dissertations

from the Faculty of Pharmacy 315

Anabolic Androgenic Steroids

Neurobiological Effects of Nandrolone, Testosterone,
Trenbolone, and Stanozolol

SOFIA ZELLEROTH

ACTA
UNIVERSITATIS
UPSALIENSIS ISSN 1651-6192
ISBN 978-91-513-1560-7
UPPSALA URN urn:nbn:se:uu:diva-480857
2022
Dissertation presented at Uppsala University to be publicly examined in B42, Biomedical
center, Husargatan 3, Uppsala, Friday, 23 September 2022 at 09:15 for the degree of Doctor
of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Faculty
examiner: PhD Astrid Bjørnebekk (Department of Mental Health and Addiction, Oslo
University Hospital).

Abstract
Zelleroth, S. 2022. Anabolic Androgenic Steroids. Neurobiological Effects of Nandrolone,
Testosterone, Trenbolone, and Stanozolol. Digital Comprehensive Summaries of Uppsala
Dissertations from the Faculty of Pharmacy 315. 87 pp. Uppsala: Acta Universitatis
Upsaliensis. ISBN 978-91-513-1560-7.

The use of anabolic androgenic steroids (AAS) for recreational purposes is a health concern,
as long-term AAS-use in supraphysiological doses is associated with severe physical and
psychological adverse effects. Several behavioral and cognitive problems are reported after
long-term AAS-use, and alterations in brain morphology as well as neurotransmitter systems
have been reported. The AAS-induced negative impact on the brain may depend on the type of
AAS used, but the rationale behind the adverse effects observed is still not clear.
The aim of the present thesis was to investigate the neurobiological impact of
supraphysiological doses of structurally diverse AAS; testosterone, nandrolone, stanozolol,
and trenbolone, as well as of the prodrugs nandrolone decanoate, testosterone undecanoate,
testosterone decanoate, and trenbolone decanoate. Wistar rats were used to study the influence
on behavior, effects on the brain, and additional somatic effects. Furthermore, in vitro models
of immature and mature primary rat cortical cell cultures were used to examine the potential
toxic properties of the AAS administered. In the in vitro studies, nandrolone and trenbolone
were identified as the most toxic of the AAS investigated, due to their adverse impact on
mitochondrial function, membrane integrity, apoptosis, and neurite outgrowth. In vivo, the AAS
demonstrated diverse somatic outcomes affecting body weight development, and organ weights
to different degrees. In addition, nandrolone decanoate caused a reduced general activity, an
effect possibly induced by increased stress vulnerability and alterations in the oxytocinergic
system. Furthermore, nandrolone decanoate induced impaired memory in the novel object
recognition test. Overall, nandrolone decanoate was identified as the most harmful steroid
investigated due to its prominent impact on body weight development, affecting multiple organs,
and being the only AAS causing impaired cognitive function.
In conclusion, the structurally different AAS exerted diverse effects on cell viability, neurite
development as well as regarding physical impairments and impact on behavior, suggesting that
harmful physiological, neurological, and psychological outcomes may be expected after AAS-
use. These findings highlight that the severity and type of adverse effects depend on the type of
AAS used, which is valuable information to consider in order to provide good healthcare and
treatment options to AAS-users.

Keywords: Anabolic androgenic steroids, testosterone, nandrolone, trenbolone, stanozolol,

central nervous system, primary cortical cell cultures, rats, multivariate concentric square
field, novel object recognition.

Sofia Zelleroth, Department of Pharmaceutical Biosciences, Box 591, Uppsala University,

SE-75124 Uppsala, Sweden.

© Sofia Zelleroth 2022

ISSN 1651-6192
ISBN 978-91-513-1560-7
URN urn:nbn:se:uu:diva-480857 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-480857)
In loving memory of my grandmother
“Det här har jag aldrig provat tidigare,
så det klarar jag helt säkert”

- Pippi Långstrump
List of Papers

This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.

I. Zelleroth, S., Nylander, E., Nyberg, F., Grönbladh, A., Hallberg,

M. (2019). Toxic Impact of Anabolic Androgenic Steroids in Pri-
mary Rat Cortical Cell Cultures. Neuroscience. 397, 172-183.
II. Zelleroth, S., Nylander, E., Örtenblad, A., Stam, F., Nyberg, F.,
Grönbladh, A., Hallberg, M. (2021). Structurally different ana-
bolic androgenic steroids reduce neurite outgrowth and neuronal
viability in primary rat cortical cell cultures. J Steroid Biochem
Mol Biol. 210, 105863.
III. Zelleroth, S., Nylander, E., Kjellgren, E., Grönbladh, A., Hall-
berg, M. (2022). Nandrolone decanoate and testosterone un-
decanoate differently affect stress hormones, neurotransmitter
systems, and general activity in the male rat. Behavioral Brain
Research. 432. 113971.
IV. Zelleroth, S., Stam, F., Nylander, E., Kjellgren, E., Gising, J.,
Larhed, M., Grönbladh, A., Hallberg, M. The decanoate-esters of
nandrolone, testosterone, and trenbolone induce steroid specific
memory impairment and somatic effects in the male rat. Manu-
script.

Reprints were made with permission from the respective publishers.

List of additional papers

Diwakarla, S., Nylander, E., Grönbladh, A., Vanga, SR., Khan, YS., Gutiér-
rez-de-Terán, H., Sävmarker, J., Ng, L., Pham, V., Lundbäck, T., Jenmalm-
Jensen, A., Svensson, R., Artursson, P., Zelleroth, S., Engen, K., Rosenström,
U., Larhed, M., Åqvist, J., Chai, SY., Hallberg, M. (2016). Aryl Sulfonamide
Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in
Primary Hippocampal Neuron Cultures. ACS Chem Neurosci, 7, 1383-1392.
Nylander, E., Grönbladh, A., Zelleroth, S., Diwakarla, S., Nyberg, F., Hall-
berg, M. (2016). Growth hormone is protective against acute methadone-in-
duced toxicity by modulating the NMDA receptor complex. Neuroscience.
339, 538-547.
Brolin, E., Johansson, J., Zelleroth, S., Diwakarla, S., Nyberg, F., Grönbladh,
A., Hallberg, M. (2017). The mRNA expression of insulin-like growth factor-
1 (Igf1) is decreased in the rat frontal cortex following gamma-hydroxybutyr-
ate (GHB) administration. Neuroscience Letters. 646, 15-20.
Brolin, E., Zelleroth, S., Jonsson, A., Hallberg, M., Grönbladh, A., Nyberg,
F. (2018). Chronic administration of morphine using mini-osmotic pumps af-
fects spatial memory in the male rat. Pharmacology, Biochemistry and Behav-
ior. 167, 1-8.
Nylander, E., Zelleroth, S., Nyberg, F., Grönbladh, A., Hallberg, M. (2018).
The protective and restorative effects of growth hormone and insulin-like
growth factor-1 on methadone-induced toxicity in vitro. International Journal
of Molecular Science. 19, 3627.
Nylander, E., Zelleroth, S., Stam, F., Nyberg, F., Grönbladh, A., Hallberg, M.
(2020). Growth hormone increases dendritic spine density in primary hippo-
campal cell cultures. Growth Hormone & IGF Research. 50, 42-47.
Balgoma, D., Zelleroth, S., Grönbladh, A., Hallberg, M., Pettersson, C.,
Hedeland., M. (2020). Anabolic androgenic steroids exert a selective remod-
eling of the plasma lipidome that mirrors the decrease of the de novo lipogen-
esis in the liver. Metabolomics. 16, 12.
Grönbladh, A., Nylander, E., Zelleroth, S., Hallberg, M. (2021). Assessing
Cell Viability Effects of Opioids in Primary Cortical Cells from Rat. Methods
in Molecular Biology. 2201, 171-180.
Nylander, E., Zelleroth, S., Nyberg, F., Grönbladh, A., Hallberg, M. (2021).
The effects of morphine, methadone, and fentanyl on mitochondria: A live cell
imaging study. Brain Research Bulletin. 171, 126-134.
Contents

Introduction ................................................................................................... 15
Testosterone.............................................................................................. 15
Biosynthesis ......................................................................................... 16
Signaling .............................................................................................. 17
AAS misuse .............................................................................................. 19
Prevalence ............................................................................................ 20
Common AAS ..................................................................................... 20
Administration patterns ....................................................................... 21
Pharmacology ...................................................................................... 22
Dependence and reinforcing effects .................................................... 23
Neurotoxicity ....................................................................................... 24
Adverse effects .................................................................................... 25
Cognitive deficits ................................................................................. 26
Stress ........................................................................................................ 27
Oxytocin’s role in stress ...................................................................... 28
Background to methodology ......................................................................... 29
Primary cell culturing ............................................................................... 29
Cell viability ........................................................................................ 29
Neurite outgrowth ................................................................................ 31
The rat as a research model ...................................................................... 31
Behavior ............................................................................................... 31
The multivariate concentric square fieldÔ test ................................... 32
The novel object recognition test ......................................................... 33
Multivariate data analysis ......................................................................... 34
Enzyme-linked immunosorbent assay ...................................................... 35
Quantitative polymerase chain reaction ................................................... 36
Aim................................................................................................................ 37
Materials and methods .................................................................................. 38
General procedures ................................................................................... 38
Ethical statement ...................................................................................... 39
Animals .................................................................................................... 39
Primary cell cultures ................................................................................. 39
Cell viability assays .................................................................................. 40
Immunocytochemistry .............................................................................. 40
 Neurite outgrowth analysis ....................................................................... 41
Gene expression analysis.......................................................................... 41
Isolation of RNA and cDNA synthesis ................................................ 41
Quantitative polymerase chain reaction............................................... 42
Animal drug treatment.............................................................................. 42
Behavioral testing ..................................................................................... 43
The novel object recognition test ......................................................... 43
The multivariate concentric square fieldÔ test ................................... 43
Trend analysis ...................................................................................... 44
Tissue collection ....................................................................................... 45
Enzyme-linked immunosorbent assay ...................................................... 45
Statistical analysis .................................................................................... 45
Conventional statistics ......................................................................... 45
Multivariate data analysis .................................................................... 46
Results and discussion .................................................................................. 48
AAS-specific impact on cell viability and neurite development .............. 48
AAS-induced impact on body weight development ................................. 53
AAS-induced effects on plasma stress hormone levels ............................ 54
AAS-specific alterations on organ weights .............................................. 55
Altered gene expression in the hypothalamus .......................................... 56
AAS-induced impact on the behavioral profile ........................................ 58
Nandrolone decanoate impair recognition memory ................................. 63
General discussion .................................................................................... 64
Future perspectives ................................................................................... 66
Conclusion .................................................................................................... 67
Populärvetenskaplig sammanfattning ........................................................... 69
Acknowledgement ........................................................................................ 71
References ..................................................................................................... 73
Abbreviations

1 DIV One day in vitro

7 DIV Seven days in vitro
AAS Anabolic androgenic steroids
ACTB Actin beta
ACTH Adrenocorticotropic hormone
ADHD Attention deficit hyperactivity disorder
Akt Protein kinase B
ANOVA Analysis of variance
AR Androgen receptor
ATP Adenosine triphosphate
CNS Central nervous system
COMT Catechol-O-methyltransferase
CPP Conditioned place preference
CRH Corticotropin-releasing hormone
D Duration
DAPI 4′ ,6-diamidino-2- phenylindole, dihydrochloride
DHT Dihydrotestosterone
DSM Diagnostic and Statistical Manual of mental disorders
ELISA Enzyme-linked immunosorbent assay
ER Estrogen receptor
F Frequency
FITC Fluorescein isothiocyanate
FSH Follicle stimulating hormone
GnRH Gonadotropin releasing hormone
GR Glucocorticoid receptor
HPA Hypothalamic-pituitary-adrenal
HPG Hypothalamic-pituitary-gonadal
i.c.v Intracerebroventricular
L Latency
LDH Lactate dehydrogenase
LH Luteinizing hormone
MAO A Monoamine oxidase A
MAO B Monoamine oxidase B
MAPK Mitogen-activated protein kinase
MCSF Multivariate concentric square field
MR Mineralocorticoid receptor
MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide
NMDA N-methyl-D-aspartate
NOR Novel object recognition
NPY Neuropeptide Y
NPY1R Neuropeptide Y1 receptor
NPY5R Neuropeptide Y5 receptor
OD Optical density
OXTR oxytocin receptor
PCA Principal component analysis
PLS-DA Partial least square-discriminant analysis
PR Progesterone receptor
qPCR Quantitative polymerase chain reaction
RPL19 Ribosomal protein 19
RPLP0 Ribosomal protein large
SD Standard deviation
SEM Standard error of mean
SRE Steroid response element
Tubb3 Tubulin beta 3
VIP Variable Importance for the Projection
Introduction

Anabolic androgenic steroids (AAS) are synthetic derivatives of the male sex
hormone, testosterone. In the 1950s, AAS-use started among elite athletes and
body builders (Kanayama and Pope, 2018). The performance enhancing and
muscle building effects made the steroids popular and the use rapidly in-
creased throughout the 1970s. In the 1980s the AAS-use for recreational pur-
poses also emerged into the general population. By the end of the 1980s the
typical steroid user was no longer an elite athlete, but rather a young adult or
adolescent male who used steroids to build muscles and strength in order to
improve physical appearance (Kanayama et al., 2010b, Kanayama and Pope,
2018). Long-term steroid use is associated with severe adverse effects, affect-
ing both physical and mental health (Albano et al., 2021). However, several
of the AAS-induced adverse effects rarely appear before middle-age, and as
most of the steroid users are still under the age of 50, relatively few steroid
users seek medical attention. As the steroid-using population is getting older,
it is likely that a higher proportion of this population will need medical help,
including for cognitive decline. Thus, it is important that physicians that will
meet these patients have information about AAS misuse and AAS-induced
adverse effects in order to be able to provide proper healthcare. Therefore,
increased knowledge, especially regarding cognitive and mental disabilities,
are warranted within this field.

Testosterone
Testosterone, the primary sex hormone in men, is well known to cause both
androgenic (male characteristics) and anabolic (muscle building) effects (Lee
and Chang, 2003, Freeman et al., 2001). Testosterone was first isolated and
described in 1935 (David et al., 1935, Wettstein, 1935), and in 1939 the re-
searchers, Butenandt and Ruzicka, were awarded the Nobel Prize in Chemis-
try for the synthesis of testosterone (NobelPrize.org, 1939). Rapidly thereaf-
ter, synthetic derivatives of the testosterone molecule with improved anabolic
properties and less androgenic effects, i.e., anabolic androgenic steroids
(AAS), were designed and by the mid 50s more than 200 AAS had been de-
scribed (Nieschlag and Nieschlag, 2014). Most AAS were developed with the
intention to treat different medical conditions such as certain types of anemia,

15
hypogonadism, osteoporosis, and severe burns. Many of these AAS are still
clinically used today.

Biosynthesis
Testosterone plays an important role in the development of the reproductive
system and sex characteristics. Testosterone is derived from cholesterol and
mainly produced in the Leydig cells of the testes in men, and in low amounts
in the ovaries in females. In addition, the hormone is synthesized both in the
adrenal glands and in the brain of both sexes (Kicman, 2008, Tobiansky et al.,
2018). The steroid biosynthesis consists of a series of enzymatic steps that
converts cholesterol into other steroid hormones. The rate-limiting step of the
steroid biosynthesis is the transport of cholesterol into the mitochondria,
which is conducted by the steroidogenic acute regulatory protein (Lin et al.,
1995). The secretion of testosterone is regulated by the hypothalamus-pitui-
tary-gonadal axis (HPG-axis), illustrated in figure 1. Gonadotropin releasing
hormone (GnRH) is secreted from the hypothalamus and controls the release
of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from the
anterior pituitary, which in turn stimulates the Leydig cells to secrete testos-
terone. Increasing levels of testosterone subsequently inhibit the release of
GnRH and LH by acting on the hypothalamus and pituitary through a negative
feedback loop (Tyagi et al., 2017). In addition, the pituitary secretes adreno-
corticotropic hormone (ACTH) which stimulates the adrenal glands to release
glucocorticoids, and to some extent androgens (Handa et al., 1994). The se-
cretion of ACTH is regulated by the hypothalamic hormone, corticotropin-
releasing hormone (CRH), and the interaction between these hormones, the
hypothalamus, pituitary, and the adrenal gland is known as the hypothalamic-
pituitary-adrenal axis (HPA-axis) (Handa et al., 1994), illustrated in figure 1.

16
Figure 1. Illustration of the hypothalamic-pituitary-adrenal axis (HPA-axis) and hy-
pothalamic-pituitary-gonadal axis (HPA-axis). Illustration by Sofia Zelleroth.

Signaling
Testosterone reaches the target tissues through the blood stream and mediates
its effect by activation of the intracellular androgen receptor (AR), which has
wide-spread expression in various tissues and organs throughout the body, in-
cluding the central nervous system (CNS) (Simerly et al., 1990). The AR is a
member of the steroid and nuclear receptor superfamily that also include min-
eralocorticoid (MR), glucocorticoid (GR), estrogen (ER), and progesterone
receptors (PR) (Heinlein and Chang, 2002, Lu et al., 2006). In the absence of
testosterone, AR exists in its inactive form, forming complexes together with
different chaperone molecules, such as the heat shock protein (HSP). Testos-
terone passively diffuses into the cell, and there directly binds and activates
the AR. Testosterone may also be modulated by metabolism in the cells, which
is dependent of the locally present steroid-converting enzymes in the target
tissue. For example, in androgen tissues, such as the reproductive organs, tes-
tosterone is reduced by 5a-reductase to the more potent metabolite dihydro-
testosterone (DHT). DHT binds the AR with greater affinity compared to tes-
tosterone, and because of this testosterone can be considered as a prohormone
in these tissues. Binding of testosterone or DHT to the AR causes dissociation
of HSP, which results in AR activation. Active AR form dimers, which are
translocated to the cell nucleus, where they interact with the steroid response
element (SRE) and generate the formation of a transcription complex. The

17
transcription complex is a cluster of coregulators that activates certain genes,
resulting in gene transcription, and production of new proteins, which affects
cell function, growth, and differentiation (Bennett et al., 2010). This is referred
to as the genomic pathway or the classical signaling pathway, illustrated in
figure 2. In addition, activated AR may also potentiate non-genomic signaling
pathways, where the response appears within seconds to minutes after expo-
sure. Activated AR induce these effects by interaction with signaling mole-
cules at the plasma membrane, such as the tyrosine-kinase Scr, resulting in the
release of intracellular calcium and activation of the mitogen-activated protein
kinase (MAPK) pathway. This triggers activation of signaling cascades and
phosphorylation of co-regulators which in turn regulates other cytoplasmic
signaling events, as well as gene transcription (Foradori et al., 2008). Further-
more, testosterone is suggested to mediate non-genomic effects through a
membrane bound AR (Heinlein and Chang, 2002). Activation of this receptor
is suggested to act on a G-protein resulting in activation of the
RAS/MEK/ERK pathway in the cell (Foradori et al., 2008). Moreover, other
receptors may be involved in the actions of steroids. For example, AAS have
been demonstrated to directly modulate the function of GABAA receptors
(Bitran et al., 1993, Yang et al., 2005), where AAS binding resulted in altered
neuronal activity (Lambert et al., 1995). In addition, AAS affect the actions of
neurosteroids at the sigma-1-receptor (Elfverson et al., 2011), and high levels
of testosterone have been demonstrated to cause antagonistic effects on GRs,
leading to inhibition of glucose synthesis and protein catabolism (Mayer and
Rosen, 1975). Also, certain AAS demonstrate relatively high binding affinity
to the PR (Bauer et al., 2000), and possibly mediates effects through PR-acti-
vation. Furthermore, regarding the enzymatic conversion of testosterone the
enzyme, aromatase, plays an important role. In tissues expressing aromatase,
testosterone may be converted to estrogen which mediates its effects by acti-
vation of estrogen receptors (ER). Thus, both 5a-reductase and aromatase are
present in the brain and are important for the mechanism of action of steroids
in the brain (Kicman, 2008).

18
Figure 2. Illustration of testosterone signaling. A) Testosterone (T) diffuses through
the cell membrane and is reduced to dihydrotestosterone (DHT) in the presence of 5a-
reductase. T or DHT binds to the intracellular androgen receptor (AR) resulting in the
release of heat shock proteins (hsp) and activation of the receptor. Activated AR forms
dimers, which are translocated to the cell nucleus, where they bind to specific DNA
sequences, called steroid receptor elements (SRE). This allows recruitment of co-ac-
tivators and results in gene transcription. B) Activated AR may also interact with Scr
resulting in activation of the MAPK-pathway and phosphorylation of co-regulators,
and thereby affecting other cytoplasmatic events and gene transcription. C) Non-ge-
nomic effects of T may also be mediated through a membrane bound AR (mAR).
Activation of this receptor is suggested to act on a G-protein resulting in activation of
the RAS/MEK/ERK pathway. Illustration by Sofia Zelleroth.

AAS misuse
As previously mentioned, the large majority of the AAS-users today are not
elite athletes, but instead young adults and adolescents using steroids in order
to improve their physical appearance rather than increase their ability in sports
(Kindlundh et al., 1998, Cohen et al., 2007). Both men and women are known
to use AAS, although to a much lesser extent in women (Havnes et al., 2021,
Kindlundh et al., 1999, Kanayama et al., 2007). Even if the use among women

19
is rare, women generally report more severe side-effects compared to men
(Havnes et al., 2021). In addition, adolescent-users might be subjected to
higher risks compared to adult users as their brains are still under develop-
ment.

Prevalence
The exact prevalence of AAS-use in the society is difficult to estimate (Pope
et al., 2014a), but problems have been reported by several countries in the
recent years (Sagoe et al., 2014, Hearne et al., 2021, Andreasson and Henning,
2019). Furthermore, AAS-use is particularly high in certain gym communi-
ties, where the prevalence has been reported to be as high as 10 % (Althobiti
et al., 2018). This may however be an underestimation based on the amount
of AAS that exist on the illegal market. For example, during 2020-2021 the
Swedish Customs made over 1400 confiscations of performance-enhancing
drugs (Tullverket.se, 2021) and earlier this year (January 2022) the Swedish
police made one of the largest AAS confiscations, consisting of at least one
million units (SVT.se, 2022).

Common AAS
There are hundreds of different AAS that have been synthesized over the
years. World-wide there are approximately 10-15 steroids that occur more fre-
quently, and these also occur as different prodrugs where the ester side chains
may differ in length. In the studies included in this thesis, we have chosen to
investigate testosterone, nandrolone, trenbolone, and stanozolol, i.e., four of
the most frequently used AAS for recreational purposes, illustrated in figure
3a-d (Börjesson et al., 2020a). Thus, these four AAS will be focused on in the
present doctoral thesis, together with the prodrugs nandrolone decanoate, tes-
tosterone undecanoate, testosterone decanoate, and trenbolone decanoate (il-
lustrated in figure 3e-h).

20
Figure 3. Chemical structure of AAS included in the thesis. A) Testosterone, B) nan-
drolone, C) stanozolol, D) trenbolone, E) nandrolone decanoate, F) testosterone un-
decanoate, G) testosterone decanoate, and H) trenbolone decanoate.

Administration patterns
Most AAS-users administer steroids in cycles of approximately 8-16 weeks,
with two or three cycles per year, using doses ranging up to 100 times higher
than the recommended therapeutic dose of testosterone (Kanayama et al.,
2009b). To avoid some of the adverse effects associated with AAS, the ad-
ministration pattern usually involves a gradual increase in the beginning of the
cycle, which thereafter is decreased, and followed by a “clean” period without
AAS prior the start of next cycle (Trenton and Currier, 2005, Kanayama et al.,
2009a). To receive a lean and muscular body the users often combine several
pharmacologically different AAS, referred to as “stacking”, with the belief to
receive a synergistic effect (Wilson, 1988). It is common that depot steroids,
such as nandrolone decanoate, are combined with orally administered AAS,
for example stanozolol, in order to receive optimal results (Trenton and
Currier, 2005). In addition, users may also administer other performance-en-
hancing drugs (e.g., growth hormone and insulin), as well as substances to
counteract certain side-effects, such as aromatase inhibitors, estrogen antago-
nists, and chorion gonadotropin releasing hormones (Kanayama et al., 2020).
The majority of the substances used for recreational purposes are illegal with-
out a prescription in most countries, but are available through local under-
ground drug dealers or via numerous Internet sites. Furthermore, it is not un-
usual that these compounds are illegally produced which results in additional

21
health risks, a fact that seems to be of little concern as many AAS-users often
perceive themselves as being invulnerable (Denham, 2019).

Pharmacology
As previously mentioned, the aim of synthesizing new AAS have historically
been to increase the anabolic potency and to reduce the androgenic side-ef-
fects. In order to develop such AAS, structural changes have been made, pri-
marily by modifications of carbon atoms in the positions 2, 9, 11, or 19 of the
testosterone molecule, see figure 3a. Other common modifications are in-
tended to optimize the oral bioavailability of AAS as well as to generate more
long-acting steroids (Fragkaki et al., 2009). However, despite these structural
modifications all available AAS to date still cause androgenic effects when
used in high doses and for a long period of time.
Some of the most commonly used parenteral preparations of AAS include
esterified forms of testosterone, nandrolone, and trenbolone, where testos-
terone enanthate, nandrolone decanoate, and trenbolone acetate seem to be of
particular popularity among steroid users. The length of the ester at the 17b-
position is used for the parenteral preparation to modulate the rate of absorp-
tion from the vehicle. Once in the circulation, hydrolysis occurs to yield the
active compound. The duration of action of the esters depends on the absorp-
tion rate, which is dependent on the length of the ester. In general, the longer
ester-chain length, the more slowly the preparation is released into the circu-
lation (Kicman, 2008). In previous study the effects of different esters were
investigated and demonstrated that shorter esters caused higher and earlier
peak concentrations and had a shorter mean residence time after injection
compared to longer esters (Minto et al., 1997).
The AAS included in the thesis, do not only differ in structure and rout of
administration, they also display pharmacological differences. In comparison
with testosterone, nandrolone has a higher binding affinity to the AR, but is
reduced by 5a-reductase to 5a-dihydro-19-nortestosterone, which has lower
binding affinity than nandrolone itself. Also, nandrolone is a poor substrate to
aromatase and therefore cause minor estrogenic effects (Kicman, 2008).
Trenbolone on the other hand has the highest binding affinity to the AR when
compared to nandrolone and testosterone. However, trenbolone is neither re-
duced by 5a-reductase nor converted to estradiol by aromatase (Scippo et al.,
2002, Yarrow et al., 2010). Stanozolol has the weakest affinity to the AR of
the four AAS. However, stanozolol is still a potent activator of the AR, an
effect thought to depend on the specific ligand-receptor confirmation which
determines the interaction with co-regulators and transcription factors
(Saartok et al., 1984, Feldkoren and Andersson, 2005), which is suggested to
be AAS-specific (Holterhus et al., 2002). In addition to the AR, trenbolone
and nandrolone bind the PR as well (Bauer et al., 2000), suggesting an

22
alternative mechanism of action apart from AR activation. The pharmacolog-
ical differences between the four AAS affect their anabolic and androgenic
properties and will probably induce individual steroid specific effects on the
brain and body.

Dependence and reinforcing effects

In the late 1980 the first reports regarding AAS dependence were published
(Tennant et al., 1988, Brower et al., 1989). Since then, the prevalence of indi-
viduals developing AAS dependence syndrome has increased (Kanayama et
al., 2009a, Ip et al., 2012, Hildebrandt et al., 2011, Kanayama et al., 2010a,
Kanayama et al., 2009c, Pope et al., 2014c, Wood, 2008). Several studies have
demonstrated long-term AAS-use in supraphysiological doses to cause de-
pendence, as steroid users continue administration of AAS despite the
knowledge of severe adverse effects (Brower et al., 1991, Brower et al., 1990,
Hays et al., 1990). Various withdrawal symptoms have also been reported, and
in fact approximately 30% of the AAS-users have been reported to become
dependent (Kanayama et al., 2009b). This is described in a number of inves-
tigations where AAS dependence have been diagnosed using the Diagnostic
and Statistical Manual of mental disorders (DSM-III or DSM-IV criteria), re-
viewed by Kanayama and colleagues (Kanayama et al., 2010b). The literature
also suggest that men with AAS-dependence display specific features com-
pared to non-dependent AAS-users (Kanayama et al., 2009c). For example,
steroid dependence is associated with impaired emotion recognition (Hauger
et al., 2019a), executive dysfunction (Hauger et al., 2020), and in fact depend-
ent AAS-users have been reported to display characteristic brain abnormali-
ties, with morphological changes in pre-frontal cortical areas (Hauger et al.,
2019b).
Three possible pathways contributing to the progression of AAS depend-
ence have been suggested (Kanayama and Pope, 2018). Firstly, AAS-users
might become addicted to the muscle growing effect, and continue AAS-use
in order to maintain their muscles and appearance, a condition recognized and
referred to as “muscle dysmorphia”. This condition was first described as “re-
verse anorexia nervosa” (Pope et al., 1993), where an individual sees himself
as small and weak, even though he has a very muscular physique. Muscle dys-
morphia has been recognized as a risk factor to AAS-use in young men, thus
potentiating AAS dependence syndromes (Kanayama and Pope, 2018). Sec-
ondly, AAS inhibit the HPG/HPA-axis resulting in low concentrations of en-
dogenous testosterone. Once the AAS are discontinued the users enters a state
of hypogonadism which is associated with decreased energy, loss of libido,
erectile dysfunction, and depression. Due to these distressed feelings, many
individuals may continue their steroid use in order to feel better (Kashkin and
Kleber, 1989). Thirdly, individuals may develop AAS dependence due to the
compounds hedonic and rewarding properties. AAS do not induce an acute

23
rewarding effect in the same manner as other drugs of abuse, e.g., alcohol,
cocaine, or opiates. Still, it is well known that AAS induce satisfying feelings,
boosting self-esteem, and self-confidence (Pope et al., 2014b). It is however
difficult to determine whether these reinforcing effects seen in humans are
direct effects caused by the AAS, or an indirect result from the increased
strength and muscle size. Interestingly, animal studies have demonstrated re-
inforcing properties of testosterone in both hamsters and rats (Triemstra and
Wood, 2004, Wood, 2004, Wood et al., 2004). Hamsters self-administer tes-
tosterone using intracerebroventricular (i.c.v) injections to the point of death
(Peters and Wood, 2005). In addition, nandrolone and drostanolon are demon-
strated to be more reinforcing compared to stanozolol and oxymetholone in
hamsters self-administrating (i.c.v) these compounds (Ballard and Wood,
2005). Furthermore, testosterone has been demonstrated to have rewarding
effects when using the conditioned place preference (CPP) in both mice and
rats (Packard et al., 1998, Schroeder and Packard, 2000, Arnedo et al., 1999,
Frye et al., 2002). The CPP is a test that reveals the rewarding effects of a drug
by comparing the time spent in a compartment associated with the drug, with
the time spent in a compartment associated with control substance. The induc-
tion of CPP has been shown to depend on the type of AAS used, and the re-
warding effect seems to be dose-dependent (Prus et al., 2009). In addition to
these behavioral studies, AAS have also been shown to affect neurotransmitter
signaling systems e.g., the dopaminergic system (Birgner et al., 2008,
Kindlundh et al., 2002, Kindlundh et al., 2001, Kindlundh et al., 2004), en-
dogenous opioid system (Johansson et al., 2000c, Johansson et al., 1997,
Magnusson et al., 2009, Nyberg and Hallberg, 2012), and serotonergic system
(Rainer et al., 2014, Ambar and Chiavegatto, 2009), within brain regions as-
sociated with reward, which probably contribute to the hedonic and rewarding
properties of these compounds.
These findings indicate several differences between structurally diverse
AAS, which may affect their rewarding properties, and probably affect their
dependence potential. Therefore, most likely, other AAS-induced effects
could also be steroid-specific. The differences in steroid-induced effects, to-
gether with the differences seen between dependent and non-dependent AAS-
users, are important knowledge for steroid users and could have important im-
plications for treatment strategies.

Neurotoxicity
The AAS-induced effects in the brain are not fully understood, but several in
vitro studies suggest that supraphysiological concentrations (micromolar) of
various AAS have neurotoxic effects, causing cell death by induction of apop-
tosis (Basile et al., 2013, Estrada et al., 2006). AAS may also cause enhanced
cellular vulnerability to other toxic insults (Orlando et al., 2007, Caraci et al.,
2011). The results regarding testosterone are however contradictive, as both

24
neurotoxic and neuroprotective (Hammond et al., 2001) effects have been re-
ported. A possible explanation is that low concentrations of testosterone have
neuroprotective effects, while high concentrations are neurotoxic (Orlando et
al., 2007). It is also suggested that the protective effects of testosterone are
mediated by estrogens, as no protective effects could be found to be induced
by low concentration of the non-aromatizable DHT (Azcoitia et al., 2001). In
fact, estrogens have previously been demonstrated to have neuroprotective ac-
tions, reviewed by Arevalo et al., (Arevalo et al., 2015). Thus, differences in
neurotoxicity between structurally diverse AAS could be expected, due to
their pattern of metabolism.
The neurotoxic effects of AAS are also suggested to be mediated by the
membrane bound AR, whereas the protective effects are suggested to be
caused by activation of the intracellular AR (Gatson et al., 2006, Gatson and
Singh, 2007). Gatson and colleagues (Gatson et al., 2006, Gatson and Singh,
2007) proposed that activation of the membrane bound AR in C6 glial cells,
by the impermeable DHT-BSA, sensitizes the cells to toxic insults resulting
in enhanced cell death. The underlying mechanism to this enhanced vulnera-
bility may be reduced phosphorylation and expression of Akt (protein kinase
B). On the contrary, other researchers have demonstrated AAS to induce apop-
tosis through the genomic pathway by activation of classical intracellular AR
(Cunningham et al., 2009, Basile et al., 2013). The contradictive findings re-
ported in the literature highlights the complexity of the mechanisms of AAS
induced neurotoxicity. However, regardless of the mechanism responsible for
the toxic outcomes, the AAS-induced neurotoxicity observed in humans using
AAS in high doses and for long period of time is a potential risk factor for
developing neurodegenerative diseases, including cognitive deficits.

Adverse effects
As briefly described above, using AAS in supraphysiological doses for a long
period of time is not without risk. AAS-use is associated with several severe
adverse effects, affecting both physical and mental health. AAS affect the ma-
jority of the organs and cells in the human body e.g., the liver, cardiovascular
system, reproductive system, musculoskeletal system, urinary system, and he-
matological system, for reviews see (Albano et al., 2021, Goldman and
Basaria, 2018). Withdrawal from AAS may cause prolonged, and sometimes
irreversible, hypogonadism in men (Kanayama et al., 2015). In addition, AAS
users experiencing psychological adverse effects often report mood disturb-
ances as withdrawal symptoms (Malone et al., 1995, Kanayama et al., 2018).
Regarding the mental health, behavioral problems associated with heavy
AAS-use have been observed as a consequence of long-term use in both adults
and adolescents (Kanayama et al., 2018, Kanayama et al., 2008). Both human
and experimental animal studies have documented steroid-use to be associated
with increased aggression, irritability, and also violent behavior (Breuer et al.,

25
2001, Morrison et al., 2015, Pope et al., 2000, Ricci et al., 2013, Steensland et
al., 2005). Increased psychopathic traits in AAS-users have been suggested to
be one of the underlying mechanism to anger problems seen in these patients
(Nelson et al., 2022). Furthermore, AAS-users suffer from higher risk of de-
veloping mood disorders, such as anxiety and depression, but also mania com-
pared to non-users (Lindqvist Bagge et al., 2017, Joksimovic et al., 2019,
Rosic et al., 2014, Pope et al., 2000, Franey and Espiridion, 2018, Kanayama
et al., 2008, Kanayama et al., 2009c, Ip et al., 2012). This is also observed in
experimental animal studies where AAS have been shown to cause both de-
pressive-like behavior (Tucci et al., 2012, Zotti et al., 2014) and anxiogenic
symptoms in rodents (Rocha et al., 2007, Rosic et al., 2014, Minkin et al.,
1993). As previously mentioned, the AAS-induced effects on mood are pri-
marily reported as a withdrawal symptom, as the majority of these symptoms
occur directly after AAS discontinuation (Malone et al., 1995, Kanayama et
al., 2018). The underlying cause for the higher prevalence of psychiatric
symptoms among AAS-users compared to non-users (Pope and Katz, 1994,
Malone et al., 1995) is not fully established, but it is suggested that several
additional factors, apart from steroid-use, such as concomitant drug use, pre-
existing psychiatric disorders, and family background may be involved and
enhance the effects described in humans (Börjesson et al., 2020b). Even
though the psychological adverse effects associated with AAS-use seem to be
idiosyncratic, only affecting a minority of AAS-users (Kanayama et al., 2008)
it is still important with an increased awareness and further studies on this
subject. Interestingly, a recent study report AAS-use among weightlifters to
be associated with attention deficit hyperactivity disorder (ADHD) symptoms
and lower cognitive performance (Kildal et al., 2022). Furthermore, the liter-
ature suggests certain predisposed individuals to express anti-social behaviors
more frequently (Hallgren et al., 2015), and to be involved in violent crimes
and murders (Pope and Katz, 1990, Aknouche et al., 2021), as well as commit
suicide attempts to higher degrees (Thiblin et al., 1999). Thus, the wide-range
of psychiatric symptoms indicates long-term AAS-use to be a serious health
problem.

Cognitive deficits
Cognition, or information processing, involves higher brain functions such as
perception, attention, decision making, learning, and memory. As previously
mentioned, cognitive impairments are suggested as a consequence of long-
term AAS-use (Kanayama et al., 2013). This raises concern regarding the pos-
sibility that these individuals may be at risk of developing neurodegenerative
diseases. As most of the AAS users still are middle-aged or younger, and the
cognitive effects will appear later in life, it is tempting to speculate that these
effects are just in the beginning of being explored.

26
 So far, high-dose methyltestosterone has been demonstrated to cause for-
getfulness and confusion in humans (Su et al., 1993), and nandrolone decano-
ate has been demonstrated to impair spatial learning and memory when stud-
ied in the Morris water maze task (Magnusson et al., 2009, Pieretti et al., 2013,
Tanehkar et al., 2013), as well as causing impaired social memory in rodents
(Kouvelas et al., 2008). In addition, deficits in visuospatial memory have also
been detected in humans using AAS, an effect that correlated with the lifetime
AAS dose (Kanayama et al., 2013). A poor memory function and morpholog-
ical changes in prefrontal cortical areas have also been observed in male
weightlifting steroid-users (Bjørnebekk et al., 2019). Furthermore, the amyg-
dala has been demonstrated to be enlarged in long-term steroid-users, and an
altered functional connectivity between amygdala and areas associated with
cognitive control have been demonstrated (Kaufman et al., 2015, Westlye et
al., 2017). In a recent study, dependent AAS-users were especially affected in
regard to alterations in cognitive function, as these subjects displayed poorer
mental flexibility, problem solving, and executive function, including working
memory (Hauger et al., 2020).
There are also contradictive reports regarding AAS-induced effects on cog-
nition. For example, previous study demonstrated methandrostenolone and a
steroid ‘cocktail’ of testosterone cypionate, boldenone undecylenate, and nan-
drolone decanoate not to affect performance in the Morris water maze task
(Clark et al., 1995). In addition, certain experimental animal studies have not
been able to detect testosterone-induced learning or memory impairments
(Smith et al., 1996, Wood and Serpa, 2020). In fact, testosterone is instead
suggested to impair cognitive flexibility, as it alters decision-making in the
operant discounting tasks (Wallin et al., 2015).
The various findings on the relation between AAS-use and cognition fur-
ther highlights both the importance of dose and plasma concentration of the
steroids, as well as the possibility of steroid-specific adverse effects. Thus, the
type of AAS used, dose, and duration of use, as well as age of onset, may be
important factors contributing to the degree and type of cognitive impairment
reported.

Stress
Stress is the physiological response to a stressor e.g., a chemical or biological
agent, environmental condition, or external stimulus the individual considers
demanding, challenging, or threatening. The HPA-axis (illustrated in figure 1)
together with the autonomic nervous system are the two major systems that
responds to stressors, where glucocorticoids and catecholamines are the re-
spective mediators. The autonomic nervous system activates the fight-or-
flight response, while the HPA-axis influence several metabolic, psychologi-
cal, and immunological functions. In response to a stressor the hypothalamus

27
releases CRH, which act on the pituitary to secrete ACTH into the circulation.
ACTH stimulate the release of glucocorticoids (cortisol in humans and corti-
costerone in rodents) from the adrenal gland. Therefore, stress may alter many
body functions, including both physiological and psychological functions, in-
creasing the risk of medical problems, including mental illness (Yaribeygi et
al., 2017).

Oxytocin’s role in stress

The oxytocin-system is primarily known for its involvement in reproductive
processes and behaviors (Veening et al., 2015), but it is also a neuromodulator
contributing to the regulation of other behaviors (Misrani et al., 2017), and is
for example suggested to be involved in several neuropsychiatric conditions
(Marazziti and Catena Dell’ Osso, 2008). Oxytocin is produced in the hypo-
thalamus, secreted from the pituitary, and mediates its effects through the ox-
ytocin receptor. Several stressors are shown to induce the release of oxytocin
within the brain (Veenema and Neumann, 2008), and oxytocin is suggested to
be able to modulate the stress response by inhibiting the secretion of ACTH
and thereby decreasing the production and release of glucocorticoids (Li et al.,
2019, Onaka and Takayanagi, 2019, Onaka et al., 2012). Furthermore, oxyto-
cin has been demonstrated to be mutually regulated with the HPA-axis, thus
mutual dysregulation of these systems, as a consequence of stress, could pos-
sibly contribute to increased vulnerability to the psychopathologic effects of
stress (Dabrowska et al., 2011, Li et al., 2019).

28
Background to methodology

Primary cell culturing

Primary cell cultures from rodents are useful in vitro models to study drug-
induced effects on cell viability, and other cellular functions, as well as inves-
tigating the underlying mechanisms of these effects. The advantage with pri-
mary cell cultures compared to immortalized cell lines is that they are more
similar to the in vivo state, as they exhibit several physiological and biochem-
ical features. The primary cortical cell cultures from rat used in paper I and II
contained mixed glial and neuronal cells to mimic a physiological environ-
ment. The cells started to form neurites already after one day in vitro (1 DIV),
and after seven days in vitro (7 DIV) they showed prominent networks of neu-
rites and were considered mature.

Cell viability
Cell viability functions such as, mitochondrial activity, membrane integrity,
and apoptotic activity, can be assessed in primary cortical cells and are useful
markers to determine the impact of different chemicals and drugs on cell via-
bility.
Mitochondria are responsible for the majority of the adenosine triphosphate
(ATP) production in the cell through oxidative phosphorylation. Mitochondria
have several functions, and are involved in numerous cellular processes to
maintain cellular function. For example, mitochondria are known to be in-
volved in the regulation of apoptosis and cellular death. Therefore, mitochon-
drial dysfunction has been implicated in several diseases (Nunnari and
Suomalainen, 2012). In paper I and II, mitochondrial activity was assessed in
primary cortical cells exposed to AAS, as a measure of cell viability, using the
colometric 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide (MTT) assay (Sigma Aldrich). In functional mitochondria the MTT is
metabolized to a purple formazan product by mitochondrial dehydrogenase,
as illustrated in figure 4a. The colorimetric alteration is measured in the MTT-
assay, and reflects active mitochondria.
Lactate dehydrogenase (LDH) is an enzyme naturally occurring in the cell
cytoplasm of almost all tissues. It is an important enzyme of the anaerobic
metabolic pathway, where it catalyzes the conversion of lactate to pyruvate
with the reduction of NAD+ to NADH. LDH has clinical significance as it

29
serves as an indicator of acute and chronic diseases (Khan et al., 2020). When
membrane integrity is compromised due to cellular damage, LDH leaks out of
the cell into the surrounding environment. Quantification of LDH in cellular
media following drug-induced damage can therefore effectively be used as a
marker for cytotoxicity. In paper I and II, the AAS-induced damage to the cell
membrane was determined by measuring the amount of LDH in the cell me-
dium. The LDH cytotoxicity detection kit (Roche Life Science) was used to
quantify the LDH present in the cell medium. The LDH kit contains a tetrazo-
lium salt which is reduced to a red formazan product reflecting the amount of
LDH and damaged cells, as illustrated in figure 4b.
Apoptosis is a regulated form of cell death, characterized by specific mor-
phological and biochemical features. Apoptosis occurs in all cells, where it
plays an important role maintaining cell homeostasis and function. The initi-
ation of apoptosis is regulated by certain caspases, where caspase 3 and 7 are
the executioner enzymes. Therefore, detection of active caspases can be used
as a marker of apoptosis. Abnormal initiation of apoptosis, e.g., drug-induced
apoptosis, may cause cellular dysfunction and contribute to several diseases
(Agostini et al., 2011). In paper I, the activity of caspase 3 and 7 in cortical
cells was measured following AAS treatment using the Caspase 3/7 GLO kit
(Promega). The luminance kit contains a caspase 3/7 substrate which is
cleaved to form luciferin by active caspase 3/7. Thereafter, luciferin reacts
with the supplied luciferase that in turn produces a luminance signal, as illus-
trated in figure 4c.

Figure 4. Cell viability assays. A) the MTT-assay, B) the LDH-assay, and C) the
caspase 3/7-assay. Illustration by Sofia Zelleroth.

30
Neurite outgrowth
Neurites are projections from the cell body of neurons that form neuronal net-
works allowing neuronal connectivity and communication between neurons.
Development of neuronal networks includes several mechanisms, both intrin-
sic and extrinsic factors, to control the modulation and regulation of neurite
length and shape. These processes are essential to obtain functional axons and
dendrites which in turn are critical for neuronal connectivity (Radio and
Mundy, 2008). Deficits in neurite development may alter brain function, and
possibly affect cognitive function, cause psychiatric disorders, and overall af-
fect behavior (Huang and Song, 2019). A convenient method to model neurite
development in vitro, is the use of immunocytochemistry in order to measure
neurite length in cultured neurons. The obtained results are able to generate
useful toxicological and mechanistic cell data which may be used to predict
the toxicity of various compounds and chemicals, such as AAS.

The rat as a research model

The Norway rat (Rattus norvegicus) was the first animal species to be domes-
ticated for scientific purposes and has been used in experimental studies for
more than a century (Modlinska and Pisula, 2020). Today, the laboratory rat
is essential in biomedical research, as it offers a characterized mammalian
model system with unique advantages for modelling human diseases, studying
the effects of various compounds, and pharmaceutical drug development.

Behavior
Ethology is the scientific and objective study of animal behavior. Konrad Lo-
renz, Nikolaas Tinbergen, and Karl von Frish are considered the founders of
this field. In 1973, they were awarded the Nobel Prize in Physiology or Med-
icine for their work in developing ethology (NobelPrize.org, 1973). Behavior
is what an animal does, including muscle activity, vocal expressions, and glan-
dular activity, which is initiated or affected by internal or external stimuli.
Also, behavior can be divided into innate and acquired behavior, where innate
behavior is evolutionary established and specific to one or several species,
resulting in less individual variation. On the other hand, acquired behavior is
learned and can be improved by practice and it is specific for the individual,
resulting in large individual variations. Unfortunately, there is no perfect test
to study behavior and the most suitable one has to be chosen based on the
research question and the objectives with the experiment. In order to conduct
good scientific research, it is of importance to understand the biological basis
for production of behavior for the species of interest. Not only in order to
know what to measure and how to interpret the results when performing

31
behavioral studies, but also in order to provide high welfare and fulfill the
needs of the laboratory animals (APA, 2022).
In this thesis, two different behavioral tests have been used; the multivariate
concentric square fieldÔ (MCSF) test and the novel object recognition (NOR)
test. The MCSF-test, used in paper III, was chosen in order to investigate
whether the AAS induced an overall effect on the behavior, causing a specific
behavioral profile that affected the personality of the rat. The test was also
used in paper IV, where a negative reinforcement, aversive air-puff, was in-
cluded in the test in order to increase the risk-taking aspect of the test. In paper
IV, the NOR-test was used in order to investigate whether the AAS treatments
cause memory impairment. Both tests are described in detail below.

The multivariate concentric square fieldÔ test

The MCSF-test was developed by Meyerson and colleagues in 2006
(Meyerson et al., 2006), as a consequence to fulfill the need of an appropriate
behavioral test that provides the opportunity of multiple measures, and thereby
generates a behavioral profile rather than single parameters. The MCSF arena
provides an environment where different types of functional behaviors (i.e.,
general activity, exploratory activity, shelter seeking, risk assessment, and
risk-taking) can be examined in the same apparatus and trial session. The rat
has a free choice to visit and explore areas with different qualities, i.e., open,
elevated, different lightening, areas of exploration, and sheltered areas, which
creates an experimental setting closer to the natural milieu of the rat (as illus-
trated in figure 5). This is considered an advantage compared to the batteries
of different tests often used to study different behavioral variables, as the
MCSF is not predisposed to a specified behavior, but instead creates an op-
portunity of studying a more diverse behavioral profile. Also, with the MCSF-
test the risk of carry-over effects seen with batteries of behavioral tests is
avoided (Meyerson et al., 2006). The MCSF-test is based on “forced explora-
tion”, meaning that the animal is released directly in the arena. The explora-
tory strategi of the rat is influenced by its risk/benefit assessment, as well as
its emotional and mental state. This indicates the complexity and difficulty of
understanding the mechanisms underlying various behaviors, and emphasizes
the need of tests where the rat is allowed to express a wide-ranging behavioral
repertoire. In addition, Meyerson and colleagues also provides a statistical
procedure, called “trend-analysis”, to measure and evaluate variation in be-
havioral traits (Meyerson et al., 2013). The trend analysis, in combination with
multivariate data analysis, provides an opportunity to extract information and
to illustrate the results obtained in the MCSF-test. The MCSF-test can be used
to study a wide range of behaviors and questions. The test has over the years,
for example been used to study the impact on behavioral profiles in psychiatric
and neurological diseases (Ekmark-Lewen et al., 2016, Magara et al., 2015),

32
brain trauma (Ekmark-Lewen et al., 2016), age (Lundberg et al., 2019), ma-
ternal separation (Lundberg et al., 2017, Lundberg et al., 2020), selective
breeding programs (Roman et al., 2012), and voluntary alcohol intake
(Karlsson and Roman, 2016, Momeni et al., 2014).

Figure 5. Illustration of the multivariate concentric square fieldÔ (MCSF) arena. The
arena is divided into zones by walls or imagined boundaries (dashed lines). Zones in
the arena: central circle, center, corridors, dark corner room, hurdle, slope, bridge en-
trance, and bridge. Illustration by Sofia Zelleroth.

The novel object recognition test

The NOR-test was first described by Ennaceur and Delacour (Ennaceur and
Delacour, 1988) as a test used for neurobiological and pharmacological stud-
ies of memory in rats. The test is considered a “pure” working-memory test as
it does not include any reference-memory component, nor positive or negative
reinforcements. The test is based on the spontaneous exploratory behavior of
the rat towards different objects. First, the rat is exposed to two identical ob-
jects, which it can explore freely. In a second trial, one of the familiar objects
is replaced by a novel object. Because of the rat’s preference of novelty, the
rat will spend increased time exploring the novel object compared to the fa-
miliar object. When summarizing the data, the object-recognition can be
measured as the difference in exploration time of the novel and familiar object.
The main index of recognition used is the discrimination of novel and familiar
objects, often presented as discrimination ratio. This is measured in the second
trial as the time spent exploring the novel object divided by the time exploring
both objects. The recognition data is influenced by the time the rat is allowed
to explore the two identical objects in the first trial, as well as the time between

33
the first and second trial. In paper IV, the experiment was designed in a way
that control rats should form a memory of the objects in the first trial, called
training (15 min), and remember that object in the second trial, called testing
(24 hours later), as illustrated in figure 6. With this approach, possible AAS-
induced impairments on recognition memory could be identified.

Figure 6. Illustration of the novel object recognition test (NOR-test). The rats were
allowed 20 min habituation to the arena, following a 24-hour delay the rats were pre-
sented with two identical objects which they were allowed to explore for 15 min (train-
ing). Following another 24-hour delay, one of the familiar objects was replaced with
a novel object, and the rats were tested for discrimination during a 5 min test session.
Illustration by Sofia Zelleroth.

Multivariate data analysis

The MCSF-test generates complex information with several dependent varia-
bles and creates data tables that are “wide and short”, i.e., multivariate data.
Therefore, Meyerson and colleagues suggest that multivariate data analyses
can be used as a complement to the conventional statistics, based on the fact
that multivariate data analysis allows for extraction of additional information,
as patterns and relationships may be defined (Meyerson et al., 2006, Meyerson
et al., 2013).
In this thesis, two different multivariate data analyses were used, the prin-
cipal component analysis (PCA), and partial least square-discriminant analy-
sis (PLS-DA). The PCA is an unsupervised method which examines the rela-
tionship between several X:s (parameters), whereas the PLS-DA which is a
regression extension of the PCA and a supervised method examining the rela-
tionship between several X:s (parameters) and a class-Y variable (group).
Both PCA and PLS-DA are used for dimension reduction of the data. In PCA
the largest difference between the samples is shown, which is useful in order
to detect possible relationships between the individuals, and enables interpre-
tation of the relatedness between individuals and identification of groups and
outliers. However, the PCA does not take group-information into considera-
tion, and focus on highlighting the difference between the individuals and not
the groups. With the PLS-DA, the model knows which group each sample
belongs to from the beginning, and focus on finding the largest difference be-
tween the groups. Therefore, the PLS-DA provides information of which

34
parameters that are most likely to be responsible for discrimination between
the groups.
These methods project the data into a score-plot, which provides a good
summary of the data. The coefficients of the projections, i.e., how the param-
eters are combined to create the relationships shown in the score plot, are
called loadings or weights. These are shown in the loading plot, which pro-
vides information about which parameters that are of most importance for the
model, and how the parameters relate to each other.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) is a well-established technique
to detect antigens in biological samples. There are four general types of
ELISA; direct ELISA, indirect ELISA, sandwich ELISA, and competitive
ELISA, reviewed in (Aydin, 2015). A sandwich and competitive ELISA were
used in paper III and IV in this thesis. The sandwich ELISA setup includes
two antibodies specific for different epitopes on the same antigen. One of the
antibodies is coated on the multi-well plate enabling immobilization of the
antigen, and is therefore referred to as capture-antibody. The other antibody is
conjugated and facilitates detection of the antigen. The competitive ELISA
setup is based on the principle that the sample antigen competes with a labeled
reference antigen of binding to a primary antibody. The primary antibody is
coated on the multi-well plate and incubated with labeled reference antigen
and sample antigen. Lower amount of antigen in the sample results in stronger
signal due to more labeled reference antigen in the well.

Figure 7. Illustration of different ELISA setups. A) Sandwich ELISA and B) compet-

itive ELISA. Illustration by Sofia Zelleroth.

35
Quantitative polymerase chain reaction
Quantitative polymerase chain reaction (qPCR), or real-time PCR, is widely
used in research and diagnostics to determine and quantify nucleic acid in var-
ious biological samples. In research, qPCR is a widely used tool for the quan-
tification of changes in gene expression by measuring changes in cellular
mRNA levels e.g., up- or down-regulation of the expression of specific genes
after pharmacological treatment (Bustin et al., 2009). For that purpose, qPCR
is combined with reverse-transcription PCR, where RNA obtained from the
biological sample is reverse transcribed to cDNA, and the cDNA is used as a
template in the qPCR amplification. In qPCR, the amplified DNA product is
measured as the reaction progress, and quantified after each cycle. Detection
of the amplified DNA product is enabled by inclusion of fluorescent reporter
molecules in each reaction, where the fluorescent signal increases as the
amount of DNA product is increased. The measured fluorescence is propor-
tional to the total amount of amplified DNA.
In paper II and III, qPCR was used to quantify possible changes in expres-
sion of certain genes following AAS treatment. SyBr Green was used for the
detection of cDNA, together with the cDNA template, specific primers of the
target genes of interest, dNTPs, and DNA polymerase. In the qPCR reaction,
cDNA is denatured by increased temperature, allowing the single stranded
primers to bind to the specific target sequence. DNA polymerase attaches to
the primed template and starts to incorporate complementary nucleotides.
DNA polymerase extends the sequence by incorporating additional nucleo-
tides that are complementary to the cDNA template. SYBR green binds to the
newly synthetized double stranded DNA and fluoresces. After repeating the
cycles approximately 35-40 times, the starting amounts of cDNA can be de-
termined.

36
Aim

The overall aim for the present doctoral thesis was to evaluate and compare
the adverse impact of structurally diverse anabolic androgenic steroids, focus-
ing on the effects in the central nervous system.

The specific aims were:

• to investigate the potential toxic effects of different doses and exposure
times of four commonly used AAS; testosterone, nandrolone, stanozo-
lol, and trenbolone, using an in vitro model of mature primary cortical
cell cultures.
• to investigate the impact of testosterone, nandrolone, stanozolol, and
trenbolone on neurite development and cell viability, using an in vitro
model of immature primary cortical cell cultures.
• to compare and examine the impact of nandrolone decanoate and tes-
tosterone undecanoate with regard to their effects on stress hormones,
selected neurotransmitter systems, behavioral profiles, and body
weight gain in the male rat.
• to investigate differences between nandrolone decanoate, testosterone
decanoate, and trenbolone decanoate with regard to their effects on
recognition memory, behavioral profile, body weight development, or-
gan weights, and plasma corticosterone levels in the male rat.

37
Materials and methods

General procedures
In order to investigate the neurobiological impact of structurally diverse AAS,
in vitro models of primary rat cortical cell cultures, and in vivo models of male
Wistar rats were used. The AAS included in the thesis; testosterone, nandro-
lone, trenbolone, and stanozolol, were chosen as they are frequently used for
recreational purposes. The primary rat cortical cells cultures were used in pa-
per I and II in order to study the toxic properties of the AAS. Male Wistar rats
were used in paper III and IV in order to study the behavioral impact, effects
on the brain, and additional somatic effects induced by the AAS. Figure 8
illustrates the experimental outline of the studies included in the thesis.

Figure 8. Experimental outline of A) paper I, B) paper II, C) paper III, and D) paper
IV. Illustration by Sofia Zelleroth.

38
Ethical statement
All animal experimental procedures included in paper I, II, III, and IV were
approved by the local animal ethics committee at Uppsala University (C14/15;
5.8.18-18550/2018; 5.8.18-02249/2017), and were conducted in accordance
to Swedish guidelines regarding animal experiments (Animal Welfare Act
SFS 1998:56) and the European Union Directive on the Protection of Animals
Used for Scientific Purposes (Directive 2010/63/EU).

Animals
Pregnant Wistar or Sprauge-Dawly dams (paper I and II) were purchased from
Charles River (Germany or Italy). The male Wistar rats (paper III and IV)
were purchased from Envigo (Netherlands). At arrival to the animal facility
the male rats were seven (paper III) or five (paper IV) weeks of age. All rats
used for behavioral experiments (paper III and IV) were allowed two weeks
of acclimatization prior the start of any experimental procedures in order to
adapt to the facility and the reversed light/dark cycle, which was separated by
a dusk/dawn shift. Also, the main experimenter handled all animals to allow
the rats to habituate to different situations in order to minimize stress during
the experiments. All rats (paper I-IV) were housed in the animal facility in
transparent type IV cages (59 × 38 × 20 cm) with raised cage lids and bedding
consisting of wood-chip, paper sheets, and a wooden house as enrichment un-
der standardized housing conditions (i.e., 20-24 °C and 45-65 % humidity).
The animals were monitored daily and had free access to food (Type R36,
Lantmännen, Kimstad, Sweden) and water ad libitum.

Primary cell cultures

On embryonic day 17 the dams were euthanized with increasing levels of car-
bon dioxide, thereafter the embryos were removed and their brains were dis-
sected. Cortical tissues (frontal cortex) were carefully dissected prior to enzy-
matical and mechanical dissociation in order to obtain a cell suspension. The
cells were seeded in tissue culture plates and grown in cell medium favoring
neuronal growth supplemented with fetal bovine serum, B27, GlutaMaxÔ,
penicillin, and streptomycin. The cells were stored in a humified incubator at
37 °C and 5 % CO2. The AAS used in paper I and II (testosterone, nandrolone,
stanozolol, and trenbolone), and the AR antagonist (flutamide) were pur-
chased from Sigma-Aldrich. In order to examine the effects of AAS on pri-
mary cortical cell cultures, the cells were administered with 10 μM, 30 μM, or
100 μM of the AAS. In paper I, cortical cells were exposed to AAS after 7
DIV for 24 hours (DIV 7-8) or repeatedly every 24 hours over three days (DIV

39
7-10). In paper II, immature cortical cells (1 DIV) were exposed to the AAS
repeatedly every 24 hours over three days (DIV 1-4) in order to study the im-
pact on neurite development. In addition, to determine whether the effect is
mediated by the AR, the cells were co-administered with the AAS together
with 10 μM of flutamide.

Cell viability assays

To investigate the effect of the AAS on the viability of the cortical cells the
mitochondrial activity, the membrane integrity (paper I and II), and apoptotic
activity (paper I) were assessed.
To measure mitochondrial activity, the MTT-assay (Sigma-Aldrich) was
used. Cortical cells were seeded and grown in 96-well tissue culture plates and
treated with the specific compounds. Following drug treatment, the MTT was
added and the plates were incubated for 30 min to allow the metabolism to
occur. Thereafter, the cells were lysed using dimethyl sulfoxide, and the ab-
sorbance was measured using a plate-reader (FLUOstar Omega, BMG Lab-
tech) at 570 nm.
To evaluate membrane integrity, the LDH cytotoxicity detection kit
(Roche Life Science) was used. The cell medium from the same cells used in
the MTT-assay was collected and the amount of LDH was quantified. The
LDH reaction kit was added to the wells and the plates were kept in the dark
for 30 min, prior to measuring the absorbance at 492 nm using a plate-reader.
To determine whether the AAS initiated apoptosis, the activity of caspase
3 and caspase 7 was analyzed using the Caspase 3/7 GLO kit (Promega). For
the caspase-assay, the cortical cells were cultured in 96-well tissue culture
plates. Following drug treatments, the caspase 3/7-GLO reagent buffer was
added and the plates were protected from light. After 30 min incubation the
contents were carefully transferred to an empty white-walled 96-well plate
and luminescence was analyzed using a plate-reader.

Immunocytochemistry
Immunofluorescent staining was used to investigate the expression of the AR
in both mature (paper I) and immature (paper II) primary cortical cell cultures.
Also, in paper II immunocytochemistry was performed in order to detect neu-
rites. Cells were fixed with 4% paraformaldehyde, permeabilized with triton-
X-100, and blocked with normal donkey serum before primary antibody of
interest was added. An anti-androgen receptor antibody (Santa Cruz Biotech-
nology) was used to detect the AR, and an anti-beta-III tubulin antibody
(Sigma Aldrich) was used for the detection of neurites. The primary antibodies
were added and incubated for 1 hour before appropriate fluorescent-

40
conjugated secondary antibody was added. Lastly, the nuclei marker 4′ ,6-di-
amidino-2- phenylindole, dihydrochloride (DAPI; Sigma-Aldrich) was added
to visualize cell nuclei. Images were acquired using ImageXpress (Molecular
Devices).

Neurite outgrowth analysis

The analysis of neurite outgrowth by measuring the neurite length and number
of neurons was assessed with a high-throughput screening image-based ap-
proach using the high content screening system, ImageXpress. Nine images
per well and channel were acquired with a × 20 objective, resulting in a total
of 324 images per treatment and concentration, as the experiments were re-
peated six times (n=6). Automated images analysis in Image J (version 1.52
h) with a macro written by the authors of paper II were used to analyze the
images. Briefly, the DAPI channel was used to count number of cells and the
fluorescein isothiocyanate (FITC) channel was used to determine the neurite
length. Only cells positive for anti-beta-III-tubulin were characterized as neu-
rons. In image J, the algorithm “Isodata” was used to threshold the 16-bit im-
ages and the function “skeletonize” was used to skeletonize the binary images.
The skeletonized FITC images were then used to determine neurite length by
measuring the total area (in pixels), which corresponds to the total length of
neurites. To ensure the functionality of the macro, certain parameters were
monitored in each site, such as the nuclei area, intensity, and circularity.

Gene expression analysis

Isolation of RNA and cDNA synthesis
RNA was extracted from the primary cortical cells after 4 DIV (paper II) and
from the brain areas hypothalamus, frontal cortex, amygdala, striatum, and
hippocampus (paper III) using Qiagen RNeasy Mini kit (Qiagen). Prior isola-
tion of RNA, cells and tissues were lysed and homogenized, and 70 % ethanol
was added for optimal binding conditions to the RNeasy membrane. The cells
were lysed using the RPL buffer provided with the kit, and the brain areas
were lysed using QIAzol lysis reagent (Qiagen) and chloroform. The RNeasy
spin columns were used to obtain high-quality RNA by eluting the samples in
RNase free water. The RNA concentrations were quantified using a NanoDrop
ND-1000 (NanoDrop Technologies) and diluted to 100 ng/μL. To examine the
quality of the RNA the Experion RNA analysis kit (Bio-rad instruments) was
used. Samples having a RNA quality indicator between 7–10, and demonstrat-
ing clear 18S and 28S ribosomal RNA peaks were used for the cDNA synthe-
sis. To obtain cDNA, the iScript cDNA synthesis kit (Bio-rad instruments)

41
was used and all reactions included 250 ng RNA, 5 × iScript reaction mix,
iScript reverse transcriptase, and RNase free H2O in a total volume of 20 μL.
The reaction was performed using following cycling protocol: 5 min at 25 °C,
30 min at 42 °C, and 5 min at 85 °C.

Quantitative polymerase chain reaction

To measure the mRNA expression of selected genes, qPCR was used in paper
II and III. The genes of interest were the Tubb3 (tubulin beta 3), OXTR (oxy-
tocin receptor), AR, NPY (neuropeptide Y), NPY1R (neuropeptide Y1 recep-
tor), NPY5R (neuropeptide Y5 receptor), COMT (Catechol-O-methyltrans-
ferase), MAO-A (monoamine oxidase A), and MAO-B (monoamine oxidase
B). The primer sequences were designed utilizing the Primer-BLAST tool
(NCBI), and all primers were validated in silico using the UCSC genome
browser. The qPCR reactions were performed in 96-well plates (Bio-rad)
where each reaction contained 5 ng cDNA, 20 μM of the forward and reverse
primers (ThermoFisher Scientific), iQ SyBr Green Supermix (Bio-rad), and
RNase free H2O in a total volume of 25 μL. Negative controls were included
and all reactions were performed in duplicates. The following cycling protocol
was used on the CFX96 real-time PCR detection system, version 3.1 (Bio-
Rad); 95 °C for 3 min, followed by 40 cycles of 95 °C for 15s, 60 °C for 20s,
72 °C for 10s. To ensure specific amplification product a melt curve was in-
corporated after each run. The mean amplification efficiency for each primer
set was calculated utilizing the LinRegPCR software and Cq-values were ob-
tained using the CFX managing software. To obtain normalized gene expres-
sion, the qBASE software, version 3.2 (Biogazelle), was used together with
three reference genes, ribosomal protein 19 (RPL19), actin beta (ACTB), and
ribosomal protein large (RPLP0), which were selected after evaluation with
GeNorm (a part of qBASEplus).

Animal drug treatment

The AAS used in paper III, nandrolone decanoate (Deca-Durabol®) and tes-
tosterone undecanoate (Nebido®), and in paper IV, nandrolone decanoate, tes-
tosterone decanoate, and trenbolone decanoate were dissolved and/or diluted
with peanut oil (50 mg/mL). The rats received subcutaneous injections (15
mg/kg) of the selected AAS every third day throughout the experiments in a
maximum injection volume of 100 µL. The control group received subcuta-
neous injections of peanut oil, following the same administration schedule as
the AAS groups.

42
Behavioral testing
The novel object recognition test
The NOR-test used in paper IV was performed as described by Feltmann et
al., (Feltmann et al., 2015) with minor modifications. Briefly, the NOR-test
was performed over three consecutive days, starting with habituation (day 1),
followed by training (day 2), and ended with the testing (day 3) when the rats
were approximately 9 weeks of age. The NOR arena consisted of a rectangular
box (80×30×50 cm) with solid black plastic walls, visual cues of different
black-and-white patterns on the short sides, and an open roof. The floor of the
arena was primed with bedding material from the cages of all rats included in
the experiment. The objects used for discrimination, a Lego brick cube and
ceramic cup, were placed on the short sides, and the illumination in the arena
was 9 lux. During habituation the rats were allowed to spend 20 min in the
NOR arena to familiarize with the new environment. The training session was
performed following 24-hours, and then two identical objects were placed in
the NOR arena for the animals to explore for 15 min. Following another 24-
hours, the test session was performed. The object the rat spent the least amount
of time exploring during the training session was then replaced by a novel
object. The rats were allowed to explore the familiar and the novel object for
five minutes. Each test session was recorded and the time of active exploration
(during training and testing), together with midline crossings (during training)
was manually measured using the EthoVision system version 15 (Noldus In-
formation). The discrimination ratio (time exploring the novel object divided
with the sum of time exploring both objects) was calculated as a measure of
remembrance.

The multivariate concentric square fieldÔ test

In paper III and IV, the MCSF-test was used as described by Meyerson et al.,
(Meyerson et al., 2006), with minor modifications. The MCSF arena consist
of an outer (100 ´ 100 cm) squared field with black solid walls on three sides
and a transparent wall on the fourth. An inner (70 ´ 70 cm) square field gives
rise to an open area in the middle, called center, three corridors which are to
be reached through openings in the internal walls, and the elevated brightly
illuminated (600 lux) bridge. The open area (approximately 25 cm in diame-
ter) in the middle of the center is called central circle. In one of the corners a
dark sheltered area is located, called the dark corner room. In opposite corner,
a hole-board device called the hurdle is found. The hurdle is elevated 10 cm
from the floor and consists of a platform with two openings and a photocell to
register nose-pokes. In the third corner a slope leads up to the bridge. The
slope and the bridge are constructed of a stainless-steel wire-mesh. These dif-
ferent areas in the arena are associated with a specific type of behavior, i.e.,

43
general activity, shelter seeking, risk assessment, risk taking, and exploratory
behavior. The rats are transported to the MCSF arena in a transportation
bucket and placed in the center facing the walls without openings. The rat is
allowed to freely move around and explore the arena for 20 min. After each
trial session fecal boli and number of urinations were noted together with num-
ber of nose-pokes in the hurdle. Before the start of next trial, the arena was
cleaned with 10% ethanol (v/v). In paper IV, the rats were subjected to an
aversive air-puff when entering the bridge. Each test session was recorded and
monitored from an adjacent room. The observer manually performed the air-
puff when the rats entered the bridge, and scored rearing, grooming and
stretched attend posture by direct observation. The EthoVision system version
13 (paper III) and 15 (paper IV) was used to score distance moved (total, cm)
and velocity (mean, cm/s), as well as the zone measures; latency (L, s) to first
visiting a zone, frequency (F) of visits in specific zone, and duration (D, s) of
total time spent in specific zone. In addition, following descriptive parameters
were obtained latency to leave center (L leave), the total activity (sum of all
frequencies), the mean duration per visit in specific zone (D/F), total corridors
(sum of frequencies, duration and D/F, respectively, for the corridors), the
slope/bridge interval ((L slope-L bridge)/L slope), and frequency and duration
of the risk/shelter indices ((F bridge-F dark corner room)/(F bridge+F dark
corner room) and (D bridge-D dark corner room)/(D bridge+ D dark corner
room)). The rats were tested in the MCSF at approximately 12 weeks of age
on experimental day 17 (paper III) and at approximately 10 weeks of age at
experimental day 23 (paper IV).

Trend analysis
The ranked order procedure, called trend analysis, is performed in order to
measure and evaluate behavioral traits. In the MCSF-test, certain descriptive
parameters correlate and form a functional category, i.e., general activity,
shelter seeking, risk assessment, risk taking, and exploratory behavior. In pa-
per III, following descriptive parameters were selected for the functional cat-
egories; general activity (total activity, distance arena, F center, F and D/F
total corridors), exploratory activity (D total corridors, D center, D hurdle,
rearing, nose pokes), risk assessment (D, F and D/F for slope and bridge en-
trance, stretched attend posture in center and slope), risk-taking (D, F and D/F
for bridge and central circle), and shelter seeking (D, F and D/F dark corner
room). In paper IV, following descriptive parameters were selected for the
functional categories; general activity (total activity, distance arena, F center,
F and F/D total corridors), exploratory activity (D total corridors, D center, D
hurdle, rearing, and nose pokes), risk assessment (D, F and D/F slope), risk-
taking (D, F and D/F for bridge and central circle), and shelter seeking (D, F
and D/F dark corner room). For each descriptive parameter the rats are ranked
against each other, where the animal with the highest score is given the highest

44
rank value and the animal with the lowest score is given the lowest rank value.
The individual rank values are then summed for each functional category re-
sulting in group-wise comparison based on the relative position of the animal
within the entire population. The parameters that are negatively related to the
functional category (D center, D and D/F total corridors) were inversely
ranked before the rank values were summed.

Tissue collection
The day after the MCSF-test were performed, experimental day 18 (paper III)
and experimental day 24 (paper IV), the animals were euthanized by decapi-
tation. In paper III, trunk blood together with following brain areas were ob-
tained; hypothalamus, amygdala, frontal cortex, hippocampus, and striatum.
Brain areas were dissected using a brain matrix. In paper IV, trunk blood was
collected together with following peripheral organs, heart, liver, testis, kidney,
and thymus which were weighed directly after dissection. The brain areas and
peripheral organs were immediately frozen on dry ice. To obtain blood
plasma, the blood samples were collected in lithium/heparin tubes (Sarstedt)
and centrifuged at 3000 rpm for 10 min at 4 °C. All plasma samples and tissues
were stored in -80° C until further biochemical analysis.

Enzyme-linked immunosorbent assay

The blood plasma samples obtained in paper III and IV were used to measure
the concentration of stress hormone levels utilizing ELISA. Concentrations of
corticosterone and ACTH were analyzed in paper III, while the concentration
of corticosterone was analyzed in paper IV. The commercial kits, corti-
costerone ELISA kit (ab108821) and mouse/rat ACTH ELISA kit (ab263880)
purchased from Abcam, were used for the analyses in accordance with the
instructions from the manufacturer. The controls, standards, and samples were
performed in duplicates. In the corticosterone ELISA kit the density of color-
ation was detected by measuring the mean absorbance at 450 nm and 570 nm,
and in the ACTH ELISA kit the OD was recorded at 450 nm using the FLU-
Ostar Omega spectrophotometer (BMG Labtech).

Statistical analysis
Conventional statistics
The statistical analyses were performed using the GraphPad software (Prism
version 7-9). In paper I and II the cells obtained from embryos from one

45
individual rat were considered as one individual cell culture (n=1), and all in
vitro experiments were repeated at least three times (n=3). In paper I, raw data
was used for the statistical analyses. The two-way analysis of variance (two-
way ANOVA) was used with treatment and culture ID as factors to account
for differences. When an overall effect was found on treatment further com-
parisons were performed using Dunnett’s post hoc test. In paper II, the data
was normalized to percent of control or fold of control, and all statistical dif-
ferences were indicated with one-way analysis of variance (one-way
ANOVA), followed by Dunnett’s post hoc test when appropriate. In paper III
and IV, the behavioral measurements did not follow a normal distribution ac-
cording to Saphiro-Wilks W normality test. Therefore, non-parametric anal-
yses were performed. Measurements obtained from the MCSF-test and the
NOR-test were analyzed using the Mann-Whitney test or Kruskal-Wallis test
when appropriate. When an overall effect was identified further group-wise
comparison was made by Dunn’s or Dunnett’s multiple comparison test. Body
weight measurements were statistically analyzed using two-way ANOVA
with time and treatment as factors to account for differences, followed by mul-
tiple comparison with Tukey´s or Dunnet’s post hoc test. In paper III, the
plasma stress hormone levels did not follow normal distribution, therefore, the
Kruskal-Wallis test was used for the overall comparison between the groups,
followed by Dunnett’s post hoc test when appropriate. Data from the qPCR
analysis followed a gaussian distribution and was statistically analyzed using
the one-way ANOVA, followed by Tukey’s multiple comparison test when
appropriate. Correlation analysis was performed using Pearson correlation. In
paper IV, the organ weight measurements and corticosterone levels followed
a gaussian distribution and were analyzed with one-way ANOVA, and further
group-wise comparison was made with Dunnett’s post hoc test. The results
from the trend analysis performed in paper III and IV were statistically ana-
lyzed using one-way ANOVA followed by Tukey´s or Dunnett’s post hoc test.
MCSF measurements obtained at different time-points (paper IV) were statis-
tically compared using two-way ANOVA, followed by Sidak’s multiple com-
parison test. Occurrences obtained from the MCSF-test were analyzed with
the Chi-square test.

Multivariate data analysis

Multivariate data analysis was used in paper III and IV and all analyses were
performed using SIMCA 15 (Umetrics AB) utilizing the autofit option to cre-
ate the models resulting in the maximum number of significant components.
In paper III, the PLS-DA was used with the intention to get an overview
and summary of all the data collected. With the PLS-DA it is possible to iden-
tify relationships between the study groups and the parameters obtained from
the study, which suggest which parameters that are of most importance for
discrimination between the studied groups. The VIP (Variable Importance for

46
the Projection) plot was derived from the model, which summarizes the im-
portance of the parameters to explain the model. The results from the PLS-DA
were therefore used to identify key findings of the study. All experimental
data obtained from the study, except latencies, risk/shelter indices,
slope/bridge interval, and rank values for the functional categories, were in-
cluded in the analysis
The PCA was used to examine the relationship of the descriptive parameters
obtained in the MCSF-test. The PCA allows for identification of groups and
outliers, and which descriptive parameters that are of importance for creating
these relationships. The test also allows for interpretation of how the descrip-
tive parameters correlates to each other. All descriptive parameters obtained
from the MCSF-test, except latencies, were included in the analysis.

47
Results and discussion

Studying and evaluating the impact of AAS misuse on the brain and behavior
is a difficult task, and trying to distinguish between AAS-specific effects in
human users is almost impossible due to individual differences and polydrug
use (Sagoe et al., 2015). The majority of preclinical studies evaluating the
central effects of AAS, and their impact on the brain and behavior, often in-
vestigates either a single compound or a cocktail of several steroids. It is not
until recently comparison of different types of AAS, and their respective ef-
fects, have gained increased interest and attention. Because of the pharmaco-
logical differences between structurally diverse AAS, it is of interest to eluci-
date and compare if these compounds also cause different impact on the brain
and behavior. The results presented in the present thesis further highlights the
importance of AAS type used, the concentration used, and the exposure-time.
All these factors seem to have an impact on the biological, as well as the be-
havioral outcome detected after steroid-use.

AAS-specific impact on cell viability and neurite

development
The investigated AAS, testosterone, nandrolone, stanozolol and trenbolone,
affected the cell viability to different degrees in primary cortical cells. The
reduced viability measured as reduced mitochondrial function, impaired mem-
brane integrity, induced apoptotic cell death, and effect on neurite length de-
pends on the type of AAS.
In paper I, AAS exposure to mature primary cortical cells reduced cell via-
bility and increased cell cytotoxicity in a dose-dependent and time-dependent
manner. With respect to the parameters assessed, nandrolone was identified
as the most toxic AAS as it significantly caused an overall reduction in mito-
chondrial activity as indicated by ANOVA, as well as disrupted membrane
integrity and activated caspase 3/7 after acute exposure (figure 9). Further-
more, nandrolone demonstrated prominent impact on the viability of the cells
after repeated exposure (figure 10). Flutamide was able to prevent all altera-
tions caused by acute AAS exposure (data not shown), which strongly sup-
ports the effects to be mediated by the AR.

48
Figure 9. Mature primary cortical cells treated with a single dose of 10, 30, and 100
µM of testosterone, nandrolone, stanozolol, and trenbolone. Cell viability was as-
sessed 24 hours after exposure by measuring A) mitochondrial activity, B) LDH re-
lease, and C) caspase 3/7 activity. Data is presented as mean values and standard error
of mean (mean ± SEM), n=3-4. Overall differences were calculated with two-way
ANOVA, followed by Dunnet’s post hoc test in comparison with control when appro-
priate. Probability values of p < 0.05 were considered statistically significant,
*p < 0.05, **p < 0.01.

Trenbolone, testosterone and stanozolol demonstrated similar effects on cell

viability as nandrolone, but to a lesser extent. Given the increase of the exe-
cutionary caspases 3/7, it is likely that the toxic effects mediated from nandro-
lone, trenbolone and testosterone are caused by apoptosis (figure 10c), which
is in accordance with previous findings (Estrada et al., 2006, Basile et al.,
2013). Interestingly, stanozolol did not activate the caspases 3/7, indicating
stanozolol to possibly induce necrosis (figure 10c). Dysregulated cell death
has been associated with neurodegenerative diseases (Agostini et al., 2011),
and therefore the findings on AAS-induced apoptosis further supports the pos-
sibility of increased risk of developing neurodegenerative disorders among
long-term AAS-users. Flutamide inhibited the caspase activation after re-
peated exposure of the AAS (data not shown), suggesting the induction of the
apoptotic cascade to be dependent on AR-activation. Furthermore, flutamide
was able to prevent the effects caused by repeated exposure of testosterone
and stanozolol, while the impact of nandrolone and trenbolone were de-
creased, but not to control levels (data not shown). This suggests the toxic

49
effects induced by nandrolone and trenbolone, at least in part, to be mediated
by an AR independent pathway.

Figure 10. Mature primary cortical cells treated with 10, 30, and 100 µM of testos-
terone, nandrolone, stanozolol, and trenbolone repeatedly every 24 hour for three
days. A) AAS effect on mitochondrial activity. B) AAS effect on membrane integrity.
C) AAS effect on caspase 3/7 activity. Data is presented as mean values and standard
error of mean (mean ± SEM), n=3-4. Overall differences were calculated with two-
way ANOVA, followed by Dunnet’s post hoc test in comparison with control when
appropriate. Probability values of p < 0.05 were considered statistically significant,
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

In paper II, the AAS induced effect on neurite outgrowth was assessed in im-
mature primary cortical cells. Trenbolone was identified to cause most serious
harm, as neurite length and number of neurons were critically affected. Figure
11 demonstrates representative images of primary cortical cells that were used
for the measurement of the neurite length. The reduction in number of cells
together with the reduced mitochondrial activity indicates trenbolone to de-
crease the viability of the cells (data not shown). However, the reduction in
neurite length was not interpreted as a direct result of increased neuronal death
as the surviving neurons in the treated cell cultures displayed a reduced aver-
age neurite length per neuron (figure 12a). In addition, the mRNA expression
of the Tubb3 gene, encoding the protein beta-III tubulin, was reduced in cells
treated with 100 µM of trenbolone (figure 12b), indicating the trenbolone-

50
induced impact on neurite outgrowth to be mediated by genomic effects.
Moreover, nandrolone and testosterone impaired the development of neurites
as well, indicated as reduced average neurite length per neuron (figure 12a).
However, nandrolone or trenbolone did not seem to affect neither mitochon-
drial activity, membrane integrity, or Tubb3 expression. Interestingly,
stanozolol did not have an impact on any of the assays included in paper II.

Figure 11. Representative images of immature primary cortical cells treated with 10,
30, and 100 µM of A) testosterone, B) nandrolone, C) stanozolol, and D) trenbolone
repeatedly every 24 hour for three days.

51
Figure 12. The AAS effect on immature primary cortical cells, A) neurite length per
neuron, and B) Tubb3 mRNA expression, after repeated treatment (10-100 µM) of
testosterone, nandrolone, stanozolol, and trenbolone alone or in combination with
flutamide (10 µM). Data is presented as percent of control or fold of control (mean ±
SEM), n=3-6. Overall differences were calculated with one-way ANOVA, followed
by Dunnet’s post hoc test in comparison with control when appropriate. Probability
values of p < 0.05 were considered statistically significant, *p < 0.05, **p < 0.01,
****p < 0.0001.

Regarding the mechanism underlying the observed toxic effects, paper I indi-
cated that AAS activation of the AR is primarily responsible for the toxic out-
comes observed, as the AR antagonist flutamide inhibited some toxic effects.
On the contrary, flutamide was not able to inhibit the effects on neurite out-
growth or Tubb3 expression demonstrated in paper II, suggesting another
mechanism of action, apart from AR activation to be responsible for the effect.
Both nandrolone and trenbolone also bind to the progesterone receptor (Bauer
et al., 2000), enabling an alternative mechanism of action. Furthermore, the
highest AAS concentrations used in paper I and II (100 µM), together with the
repeated treatment regime, increase the probability of affecting other receptor-
signaling systems in the cells. The concentrations used for the in vitro experi-
ments (paper I and II) were supposed to mimic heavy steroid use, and were
chosen based on previous published findings (Basile et al., 2013, Estrada et
al., 2006, Cunningham et al., 2009, Caraci et al., 2011, Ma and Liu, 2015),
where micromolar concentrations of various AAS have been suggested as neu-
rotoxic. However, the concentrations used (10 µM - 100 µM) cannot be inter-
preted as the actual concentration the brain of long-term steroid users is ex-
posed to. Furthermore, the differences in AAS-induced toxicity detected in
paper I and II might depend on the possibility that the expression and func-
tionality of the AR in developing neurons is different compared to cells with
full-grown neuronal networks. Additionally, the cellular mechanisms in-
volved in the regulation of neurite outgrowth and cell viability might be de-
pendent on the developing state of the cell. Moreover, it is possible that certain
AAS-induced effects are AR-mediated, while other effects are caused by

52
direct or indirect modulation of other receptor-signaling systems, which is fur-
ther indicated by previous findings that demonstrate nandrolone decanoate to
alter several neurotransmitter systems within the rat brain i.e., the glutama-
tergic system (Le Greves et al., 1997, Le Grevès et al., 2002), the peptidergic
system (Johansson et al., 2000a, Johansson et al., 2000c, Johansson et al.,
1997, Hallberg et al., 2000, Hallberg et al., 2005), the monoaminergic system
(Lindqvist et al., 2002, Kindlundh et al., 2001, Kindlundh et al., 2004), as well
as the GABAergic system (Gronbladh et al., 2014).

AAS-induced impact on body weight development

Steroid use in combination with weight-lifting exercise is well known to cause
muscle building effects resulting in increased lean body mass. The results in
paper III and IV demonstrated nandrolone decanoate to have a prominent im-
pact on weight development, where the steroid caused a reduction in body
weight gain in comparison to the control, as well as to other AAS (figure 13).
Treatment with testosterone undecanoate did not have an impact on body
weight gain, however in paper IV where the treatment with testosterone dec-
anoate was prolonged, a reduction in body weight gain was detected at exper-
imental day 24. The difference in esters used (decanoate compared to un-
decanoate) could possibly affect the outcome. However, with the treatment
regime used (15 mg/kg every third day) high systemic AAS concentrations
were expected regardless of the ester used.
The demonstrated effect of nandrolone decanoate on body weight gain is in
accordance with previous published findings (Lindblom et al., 2003,
Gronbladh et al., 2013). Lindblom and colleagues (Lindblom et al., 2003) also
suggest the effect to be a result of reduced food intake. Further supporting this
suggestion, the expression of genes associated with food intake and satiety
(NPY5R and OXTR) within the hypothalamus (presented below) were in pa-
per III found to be affected by the AAS treatment. Decreased food intake and
loss of body weight are signs of reduced well-being in rodents. Therefore,
these findings together with the additional somatic effects described i.e., al-
terations in organ weights and plasma stress hormone levels (presented be-
low), indicate the AAS, and especially nandrolone decanoate, to affect the
well-being of the rats.

53
Figure 13. Body weight development of rats treated with A) nandrolone decanoate
and testosterone undecanoate, and B) nandrolone decanoate, testosterone decanoate,
and trenbolone decanoate 15 mg/kg every third day. Data is presented as body weight
gain in percent from initial body weight (mean ± SEM), n=12 rats per group. Statisti-
cal testing was performed using a repeated measurement two-way ANOVA, followed
by Tukey’s multiple comparison test or Dunnett´s post hoc test. A p-value less than
0.05 was considered significant, * indicates difference in comparison with the control
(peanut oil), and # indicates difference between the nandrolone decanoate group and
the testosterone undecanoate group, *p < 0.05, **p < 0.01, ****p < 0.0001,
####
p < 0.0001.

AAS-induced effects on plasma stress hormone levels

Different types of stressors are known to affect the well-being of rats. A quan-
titative measure of stress is to measure stress hormone levels in blood plasma.
In paper III, concentrations of ACTH and corticosterone were analyzed, and
the result demonstrated nandrolone decanoate to reduce ACTH levels in com-
parison to control and to increase the levels of corticosterone in comparison
to testosterone undecanoate treated rats (figure 14a-b). These results indicate
an active stress response in nandrolone decanoate treated animals. This could
possibly contribute to some of the detected effects on the reduced well-being
and behavioral alterations seen in paper III following the nandrolone decano-
ate treatment. However, in paper IV there was no change in concentrations of
corticosterone caused by nandrolone decanoate (figure 14c). The difference in
results between the two studies could indicate nandrolone decanoate to cause
an increased stress vulnerability, making the rats more vulnerable to outer
stressors, rather than causing a direct effect on the HPA-axis. With that aspect
in mind, the differences in results obtained in paper III and IV could be ex-
plained by the fact that the experimental procedures in the two studies differ
in several aspects, including possible stressors.

54
Figure 14. Stress hormone levels in plasma. A) ACTH levels and B) corticosterone
levels obtained from rats treated with nandrolone decanoate (ND) and testosterone
undecanoate (TU) 15 mg/kg every third day for 18 days. C) Corticosterone levels
obtained from rats treated with nandrolone decanoate (ND), testosterone decanoate
(TD), and trenbolone decanoate (TrD) 15 mg/kg every third day for 24 days. Data is
presented as percent of control with mean and SEM, n=10-12 samples per group. Sig-
nificance is indicated by Kruskal-Wallis test, followed by Dunn’s multiple compari-
son test. A p-value less than 0.05 was considered significant, *p < 0.05, **p < 0.01.

AAS-specific alterations on organ weights

In paper IV, organ weights were obtained during the dissection. The three
AAS investigated, nandrolone decanoate, testosterone decanoate, and
trenbolone decanoate, significantly reduced the thymus gland (figure 15a).
The thymus gland is well known to be sensitive to steroids, and nandrolone
has previously been reported to induce thymus atrophy (Johansson et al.,
2000b, Grönbladh et al., 2013, Hince et al., 2008). Therefore, this observed
effect confirms that the AAS-treatments caused a systemic effect. In addition,
AAS-specific effects were found on kidney, liver, and testis weights (figure
15b-d). Nandrolone decanoate and trenbolone decanoate significantly in-
creased the weight of the kidney. Hypertrophy of the kidneys has previously
been reported following exposure to nandrolone decanoate (Brasil et al.,
2015). In addition, nandrolone decanoate reduced the weight of the liver. Alt-
hough hepatic alterations are primarily associated with 17a-alkylated AAS
(Goldman and Basaria, 2018), liver morphology, enzymes, and proteins are
reported to be affected by supraphysiological doses of nandrolone decanoate
(Vieira et al., 2008). Interestingly, testosterone decanoate was the only AAS
reducing the weight of the testicle. In humans, hypogonadism is a well-known
problem among AAS-using males, often reported as a withdrawal symptom
and known to be caused by activation of the negative feedback loop of the
HPG/HPA-axis resulting in decreased levels of endogenous testosterone
(Christou et al., 2017, Duca et al., 2019). However, the lacking effect of nan-
drolone is surprising, as the HPG-axis is downregulated in human AAS users.

55
Possibly, the nandrolone-induced effect varies due to treatment regime. Also,
the effect on testes is contradictive as testicular weight has been reported to
both increase (Grönbladh et al., 2013) and decrease (Sretenovic et al., 2021)
in rats after exposure to nandrolone decanoate.

Figure 15. Organ weights of male rats, A) thymus, B) liver, C) kidney, and D) testis,
treated with 15 mg/kg of nandrolone decanoate (ND), testosterone decanoate (TD),
trenbolone decanoate (TrD) or control (peanut oil). Data is presented as percent of
body weight with mean ± SEM, n=11-12 samples per group. Statistical differences
were obtained using one-way ANOVA, followed by Dunnett’s post hoc test when
appropriate. Probability values of p < 0.05 were considered statistically significant,
**p < 0.01, ***p < 0.001, ****p < 0.0001.

Altered gene expression in the hypothalamus

In paper III, nandrolone decanoate and testosterone undecanoate were demon-
strated to induce AAS-specific changes in mRNA expression of specific genes
in certain areas of the brain, as demonstrated in figure 16.

56
Figure 16. Hypothalamic mRNA expression of A) OXTR, B) MAOA, C) MAOB, D)
COMT, E) NPY, F) NPY1R, G) NPY5R, and H) AR of rats treated with 15 mg/kg of
nandrolone decanoate (ND), testosterone undecanoate (TU), and control (peanut oil)
every third day for 18 days. Statistical analysis was performed using the one-way
ANOVA, followed by Tukey’s multiple comparison test when appropriate. Data is
presented as mean ± SD, and a p-value less than 0.05 was considered significant,
*p < 0.05, **p < 0.01, ****p < 0.0001.

In the hypothalamus, the expression of the oxytocin receptor was significantly

increased by both AAS. In addition, nandrolone decanoate caused an elevation
in the expression of the NPY5R and COMT genes, while testosterone un-
decanoate increased the expression of the COMT, as well as the AR gene.
The multivariate data analysis revealed the altered OXTR expression to be
of certain importance and identified as one of the key findings of the study. In
addition to the involvement in reproductive processes and behaviors (Veening
et al., 2015), the oxytocin-system contributes to the regulation of food intake,
by reducing appetite and stimulate satiety (Sabatier et al., 2013, Onaka and
Takayanagi, 2019). Another signaling-system well-known to regulate feeding
behavior and energy homeostasis is the NPY-system, where the neuropeptide
NPY stimulates hunger by activation of NPY-receptors within the hypothala-
mus (Mercer et al., 2011, Levine and Morley, 1984). Alterations in the oxyto-
cin-system, as well as the NPY-system, such as changes in the expression of
the receptors (i.e., OXTR and NPY5R), could possibly affect the appetite reg-
ulation and feeding behavior of the rats, resulting in altered body weight gain.
This is further supported by the PLS-DA, where the loading plot indicated a
negative relationship between the OXTR expression in the hypothalamus and
body weight gain, as these parameters load in opposite to each other (data not
shown). This relationship was confirmed by a correlation analysis indicating
significant negative correlation between OXTR mRNA expression in hypo-
thalamus and body weight gain (figure 17). The nandrolone-induced decrease
in food-intake and body weight is previously suggested to be a result of alter-
ations in the melanocortin system, as mRNA levels of pro-opiomelanocortin

57
are demonstrated to be decreased in the arcuate nucleus following exposure to
nandrolone decanoate (Lindblom et al., 2003).
In paper III the mRNA levels and not the protein levels were investigated,
which is important to have in mind as changed gene expression not always
result in changes of the final protein. Nevertheless, the alterations in brain
gene expression found indicate the AAS to induce an AAS-specific impact on
the brain, which possibly also contributes to behavioral changes.

Figure 17. Relationship between body weight gain (experimental day 18) and mRNA
expression of OXTR in the hypothalamus of male rats. Data is presented as percent
of initial body weight and percent of control. The relationship was obtained using the
Pearson correlation, n=36 and p<0.05 was considered statistically significant.

AAS-induced impact on the behavioral profile

The MCSF-test was used in paper III and paper IV in order to investigate ster-
oid-specific impact on the behavioral profile of the rats. In paper III, nandro-
lone decanoate was demonstrated to cause reduced general activity in male
rats, as indicated by the trend-analysis (figure 18a). Additionally, the follow-
ing descriptive parameters were significantly affected by the treatment with
nandrolone decanoate; total activity, velocity and distance moved in the arena
as well the center, number of visits and time spent in the center and corridors,
together with time spent per visit in the center (data not shown). The PCA
gives an overview of the descriptive parameters obtained from the MCSF-test
in paper III (figure 19). The majority of the control rats load in the upper right
quadrant, associated with high activity in descriptive parameters of general
activity. The majority of rats treated with AAS tend to load in opposite to the
control rats, somewhat spread in the two lower quadrants of the score plot,
indicating negative relation with parameters associated with general activity,
which further supports the conventional statistics and the trend analysis. The
AAS-induced effect on activity is however contradictive, as the literature de-
scribe both reduced and increased activity to be caused by AAS in rats
(Salvador et al., 1999, Van Zyl et al., 1995).

58
Figure 18. Trend analyses obtained from the MCSF-test. A) Behavioral profiling of
rats treated with nandrolone decanoate (ND) and testosterone undecanoate (TU) 15
mg/kg every third day and tested in the MCSF on experimental day 17. B) Behavioral
profiling of rats treated with nandrolone decanoate (ND), testosterone decanoate
(TD), and trenbolone decanoate (TrD) 15 mg/kg every third day and tested in the
MCSF on experimental day 23. Data is presented as mean with SEM, n=12 rats per
group. Statistical analyses were performed using one-way ANOVA, followed by
Tukey’s multiple comparison test or Dunnett’s post hoc test when appropriate and p
< 0.05 was considered statistically significant, *p < 0.05.

Interestingly, in paper IV general activity was not affected by the nandrolone

decanoate treatment, and to our surprise none of the AAS investigated in paper
IV (nandrolone decanoate, testosterone decanoate, and trenbolone decanoate)
affected the behavioral profile of the rats, as no significant effects were seen
in the trend analysis (figure 18b), as well as no clear separation was detected
in the PCA (figure 20). In paper III, the nandrolone decanoate-induced effect
on general activity was suggested as a consequence of increased stress vulner-
ability. The absence of nandrolone-induced effect on general activity in paper
IV further supports this theory, as no alteration in stress hormone levels could
be detected in these rats.

59
Figure 19. Behavioral profiling in the MCSF-test of rats included in paper III. A)
Individual scores, and B) parameter loadings from the PCA, n=36, R2X[1]=0.345;
R2X[2]=0.111.

60
Figure 20. Behavioral profiling in the MCSF-test of rats included in paper IV. A)
Individual scores, and B) parameter loadings from the PCA, n=46, R2X[1]=0.245;
R2X[2]=0.114.

AAS-use is associated with increased risk-taking behavior, especially among

adolescent users. It was therefore hypothesized that the AAS would affect the
risk-taking behavior in the MCSF-test. Surprisingly, risk-taking behavior, as
well as risk assessment, were unaffected by the AAS treatments, both in paper
III and IV, as indicated by the trend analysis. However, in paper III there was
a trend towards difference for a specific descriptive parameter (time spent per
visit on the bridge) associated with risk-taking behavior, as well as the PCA
indicated descriptive parameters associated with risk-taking and exploratory
behavior to be of importance for the loading of steroid treated rats, as these
descriptive parameters load in the lower right quadrant of the loading plot. As
there seemed to be a relationship between these indicators, the MCSF-test
used in paper IV was designed with an aversive stimulus (air-puff) located in
the entrance of the bridge, with the intention to increase the risk associated
with the bridge. Rats exposed to the air-puff on the bridge have previously
been demonstrated to avoid the bridge up to two weeks after exposure to the

61
aversive stimulus (Karlsson et al., 2009). Surprisingly, the AAS did not alter
the functional category, risk-taking behavior, in the trend analysis. The behav-
ior on the bridge was however further investigated in order to evaluate the
effect of the air-puff as a threatening stimulus and whether the reaction to the
air-puff was affected by the AAS-treatments. The majority of the AAS-treated
rats returned to the bridge after exposure to the air-puff (figure 21) i.e., ap-
proximately 70% of nandrolone decanoate treated rats, 90% of testosterone
treated rats, and 70% of the trenbolone treated rats, which could imply an im-
paired association of the aversive stimulus and the bridge. However, these
numbers were not statistically significant in comparison to the control group,
where approximately 60% of the rats went back to the bridge. The fact that
many of the control rats returned to the bridge was surprising, as previous
findings indicate that more than 70% of naïve rats exposed to an air-puff avoid
the bridge up to two weeks after exposure (Karlsson et al., 2009).

Figure 21. Behavior on the bridge in the MCSF-test in paper IV. A) Number of rats
visiting the bridge during the 20 min MCSF trial. B) Number of rats returning to the
bridge after being subjected to the aversive air-puff. C) Number of visits on the bridge
during the first 5 min period and the last 5 min period of the MCSF trial. Occurrences
(visited bridge and returned to bridge) were analyzed using the Chi-square test. Num-
ber of visits on the bridge measured over time was analyzed with two-way ANOVA,
with time and treatment as factors to account for differences, followed by Sidak’s
multiple comparison test when appropriate. Number of visits on the bridge over time
is presented as mean ± SEM. A p-value less than 0.05 was considered statistically
significant, *p < 0.05.

To further investigate the behavior associated with the air-puff in the MCSF-
test the descriptive parameters duration bridge and frequency bridge were as-
sessed over time by dividing the 20-min MCSF trial into five-min periods.
Interestingly, nandrolone decanoate treated rats visited the bridge significantly
more times in the beginning of the trial compared to the end of the trial, a
numerical trend which could be seen in the other AAS-treated groups as well,
but not in the control group (figure 21). This further supports the speculation
regarding an AAS-induced altered reaction towards aversive stimuli, which is
in accordance with previous findings demonstrating AAS to cause increased
impulsivity (Garcia-Argibay, 2019, Hildebrandt et al., 2014, Scarth et al.,

62
2022) and poor decision making (Wallin et al., 2015, Wallin-Miller et al.,
2018, Wood and Serpa, 2020) which possibly could contribute to the increased
violent behavior seen among AAS-users (Klotz et al., 2007, Lundholm et al.,
2010, Skårberg et al., 2010, Thiblin et al., 1999, Hauger et al., 2021).

Nandrolone decanoate impair recognition memory

Cognitive deficits are associated with long-term AAS-use in humans where
speed of processing, working memory, and problem solving seem to be par-
ticularly affected (Bjørnebekk et al., 2019, Kanayama et al., 2013, Kaufman
et al., 2015, Kildal et al., 2022). The impact on memory function is also known
to depend on the period of time of AAS-use (Bjørnebekk et al., 2019). In paper
IV, nandrolone decanoate was identified as the only AAS causing impaired
recognition memory in the NOR-test, indicated by the fact that the rats spend
equal amount of time exploring the novel and familiar object during the test
session (figure 22). However, because of the experimental design and treat-
ment regime used in paper IV it is not possible to determine whether the im-
paired recognition memory displayed is a cause of altered memory acquisi-
tion, consolidation, or recall. Nevertheless, the effect on memory does not
seem to be caused by altered exploratory behavior or motor function, as no
effects were found in regard to preference of objects, preference of side, loco-
motor activity, or exploration time. The results obtained are in line with pre-
vious published findings, where nandrolone decanoate has been described to
impair memory in other types of memory tests. For example, both spatial
memory function in the Morris water maze task (Magnusson et al., 2009,
Pieretti et al., 2013, Tanehkar et al., 2013), as well as olfactory social memory
(Kouvelas et al., 2008) are reported to be altered following nandrolone deca-
noate treatment. Testosterone, on the other hand, has previously been de-
scribed not to affect memory (Clark et al., 1995, Wood and Serpa, 2020),
which also is in accordance with the results from paper IV. Surprisingly,
trenbolone decanoate did not induce an impairment in the NOR-test.
Trenbolone display several pharmacological similarities with nandrolone dec-
anoate, including the ability to cause neurodegeneration (Ma and Liu, 2015).
Furthermore, trenbolone has been described by AAS-users to cause particular
harm (Scarth and Bjørnebekk, 2021). In our studies, trenbolone however did
not have an impact on cognitive function.

63
Figure 22. Novel object recognition. Discrimination ratio (time exploring the novel
object)/(time exploring both objects) during the test session of the NOR-test. Rats
were exposed to 15 mg/kg of nandrolone decanoate (ND), testosterone decanoate
(TD), and trenbolone decanoate (TrD) every third day. Statistical analysis was made
using the Kruskal-Wallis test and when an overall effect was found, further group-
wise analysis was made using Dunn’s multiple comparison test. Data is presented as
median with interquartile range, n=12 rats per group. A p-value <0.05 was considered
statistically significant, *p < 0.05.

General discussion
The underlying mechanism for the AAS-induced effects on the brain resulting
in behavioral alterations, and cognitive impairments is not fully understood.
As previously mentioned, several researchers suggest neurotoxicity, and ei-
ther direct induction of apoptosis or enhanced vulnerability to other insults to
be involved (Basile et al., 2013, Estrada et al., 2006). The results from paper
I and II demonstrating increased cell death and deficits in neurite development
after AAS-exposure further supports these theories and could possibly con-
tribute to the smaller cortical volume and thinner cortex seen among AAS-
using weightlifters (Bjornebekk et al., 2017). Well-functioning neurite devel-
opment is an essential mechanism, both for CNS development and neuronal
regeneration (Schaefer et al., 2017), and deficits in neurite outgrowth may al-
ter brain development, as well as neuronal networking and connectivity, and
will possibly result in neurobiological and psychological disabilities.
The differences in AAS-induced effects presented in this thesis possibly
depend on the pharmacological differences between the structurally diverse
AAS of investigation. In all studies, nandrolone and/or trenbolone were iden-
tified to cause more harm in comparison to testosterone and stanozolol. Both
nandrolone and trenbolone display a high binding affinity for the AR (Scippo
et al., 2002, Death et al., 2004), as well as relatively high affinity for the PR
(Bauer et al., 2000), indicating two possible receptor pathways. In addition,
nandrolone and trenbolone are poor substrates for aromatase and therefore not
converted to estrogen (Ryan, 1959). The lower toxicity and behavioral effects
caused by testosterone could possibly be explained by the fact that

64
testosterone is converted to estrogen, which seem to display neuroprotective
effects (Arevalo et al., 2015). Furthermore, stanozolol has relatively low bind-
ing affinity to the AR compared to the other AAS (Saartok et al., 1984), which
may be a possible explanation for the low cellular toxicity demonstrated by
stanozolol.
Regardless of whether the androgen, progesterone, or estrogen receptors
are involved in the mechanism of action for AAS in the CNS, several receptor
systems are affected by AAS, in most cases probably as a secondary effect.
For example, nandrolone decanoate is suggested to impair cognitive function
via an increase in GABAB receptor functionality (Gronbladh et al., 2014), as
well as dynorphinergic actions, by elevating mRNA levels of prodynorphin
within the hippocampus, an area associated with cognitive function
(Magnusson et al., 2009). Also, the endogenous opioid-system is implied to
be altered in other areas of the brain, regulating emotions, dependence, defen-
sive reactions, and aggression (Johansson et al., 2000b). Furthermore, the im-
pulsive and aggressive behavior seen in AAS-users is suggested as a cause of
imbalanced MAO activities in the brain, as nandrolone decanoate is demon-
strated to affect mRNA levels of both MAO A and MAO B in several brain
areas (Birgner et al., 2008). In addition, AAS-induced serotonergic effects are
suggested to contribute to increased aggression, as nandrolone decanoate
treated rats display lower levels of serotonin in various areas of the brain
(Lindqvist et al., 2002). Furthermore, nandrolone decanoate alter the mRNA
expression of N-methyl-D-aspartate (NMDA) receptor subunits in hippocam-
pus and hypothalamus, an effect also suggested to contribute to the aggressive
behavior (Le Greves et al., 1997).
The nandrolone-induced decrease in general activity demonstrated in paper
III was, as previously mentioned, suggested as a result of increased stress vul-
nerability, which may be mediated by alteration in the oxytocin-system in the
hypothalamus. Oxytocin is mutually regulated with the HPA-axis (Dabrowska
et al., 2011, Li et al., 2019) and known to increase after stressful stimuli
(Veenema and Neumann, 2008). Thus, oxytocin seem to have a role in mod-
ulating the stress response (Onaka and Takayanagi, 2019, Onaka et al., 2012).
Interestingly, oxytocin is also previously reported to reduce locomotor activity
in rats (Uvnäs-Moberg et al., 1994).
Long-term AAS-use is associated with increased risk of developing mood
disorders, such as depression (Piacentino et al., 2015, Kanayama et al., 2008,
Ip et al., 2012), and well-known risk factors for depression are, among others,
dysregulation of the HPA-axis and chronic stress (Khan and Khan, 2017). In-
terestingly, the oxytocin-system has been implicated to be involved in the eti-
ology of depression as well (Meynen et al., 2007, Parker et al., 2010, Purba et
al., 1996). It is suggested that a negative environment, such as stress, causes
imbalance in the oxytocin-system resulting in psychopathological behavior
i.e., aggression, anxiety, and cognitive disorders (Lesse et al., 2017,
Murgatroyd et al., 2015, Neumann and Landgraf, 2012, Wulsin et al., 2016).

65
In addition, neuroadaptations of the oxytocinergic receptor system in brain
areas associated with stress, emotional regulation, and social bonding have
been demonstrated to be induced by other drugs of abuse (Zanos et al., 2015,
Zanos et al., 2014a, Zanos et al., 2014b). Therefore, the nandrolone-induced
effects demonstrated in this thesis i.e., cellular toxicity, impaired body weight
gain, increased stress response, reduced general activity, and impaired cogni-
tive function, at least in part, could be explained by a dysregulation of the
oxytocin-system.

Future perspectives
The present thesis highlights differences in adverse effects and possible mech-
anisms of action between four structurally diverse AAS. However, further in-
vestigations including additional AAS are needed to determine other possible
differences among these compounds in regards to adverse effects. In addition,
the exact underlying mechanism or mechanisms of action resulting in neuro-
toxic effects and altered behavior is not fully understood, and need to be fur-
ther elucidated. Based on the fact that the psychological adverse effects asso-
ciated with AAS-use seem to be idiosyncratic, it is possible that AAS-users
respond differently to AAS based on their personality and individual vulnera-
bility. Also, it is possible that the typical AAS-user has a certain type of per-
sonality, especially in regards to risk-taking behavior. Therefore, it would be
interesting to characterize the behavioral profile of the rats before the start of
AAS-treatment, for example into high and low risk-takers, in order to investi-
gate the effects of AAS on a certain type of personality. Another important
question is gender differences. In the four studies presented in this thesis we
primarily studied male rats, and even though the majority of AAS-users are
men the steroid-use for recreational purposes occurs among women as well.
Women report more severe adverse effects compared to men, and there are
few studies conducted including females. It would therefore be interesting to
address gender differences in future studies. Moreover, as the literature, in-
cluding this thesis, demonstrate AAS-use to cause neurotoxicity, which may
correspond with behavioral and cognitive deficits seen among steroid-users,
there is a need of possible treatment strategies to revert AAS-induced damage.
This highlights another research area that needs further attention in the future.

66
Conclusion

The overall conclusion of this doctoral thesis is that the examined structurally
diverse AAS exert adverse effects in regard to cell viability, neurite develop-
ment, physical impairments, and behavior, suggesting that harmful physiolog-
ical, neurological, and psychological outcomes might be expected after AAS-
use. These findings are of relevance in order to highlight that the severity and
types of adverse effects seen after long-term AAS-use differ dependent on the
type of AAS used.

• Paper I: the study revealed dose-dependent, time-dependent, as well

as AAS specific negative impact on mitochondrial activity, membrane
integrity, and induction of apoptosis in mature cortical cells. The toxic
effects seemed to be primarily mediated by AR activation. Nandrolone
was identified as the most toxic AAS when compared to trenbolone,
testosterone and stanozolol.
• Paper II: the study demonstrated trenbolone to cause the greatest im-
pairment on neurite outgrowth and cell viability in immature cortical
cells. Nandrolone and testosterone displayed similar effects, but with
less impact on neurite development, whereas stanozolol that did not
affect the cells in any of the assays performed.
• Paper III: the study revealed nandrolone decanoate treated rats to
have an impaired body weight development, decreased general activity
in the MCSF, and altered plasma levels of stress hormones. The AAS
caused brain region-dependent changes in mRNA expression, where
the hypothalamus was identified as the region most affected by the
AAS. The effects seem to involve a dysregulation in the oxytocinergic
system and increased stress vulnerability.
• Paper IV: the study revealed nandrolone decanoate to impair recogni-
tion memory in male rats, indicating altered cognitive function. In ad-
dition, various somatic effects were demonstrated by nandrolone dec-
anoate, testosterone decanoate, and trenbolone decanoate, as indicated
by alterations in body weight development and weight of thymus, liver,
kidney, and testis, possibly indicating reduced well-being. Especially
nandrolone decanoate was identified to cause a prominent impact on

67
 body weight development, affecting multiple organs, and being the
only AAS causing impaired cognitive function. The behavioral profile
and plasma stress hormone levels were not affected by the AAS ad-
ministration.

68
Populärvetenskaplig sammanfattning

Anabola androgena steroider (AAS) är syntetiska varianter av det manliga

könshormonet testosteron och har på grund av sina prestationshöjande och
muskelfrämjande egenskaper använts i dopingsyfte av elitidrottare och mus-
kelbyggare sedan 50-talet. Under sent 80-tal fick steroiderna en bredare sprid-
ning i samhället och sedan dess är den typiska steroidanvändaren inte längre
elitidrottare, utan istället är det främst unga män som använder dessa preparat
för att bygga muskler och enligt dem själva, för att förbättra sitt utseende.
Steroidanvändningen sker ofta i så kallade cykler, eller kurer. Det innebär
att steroider administreras under ett visst antal veckor och sedan följer ett antal
veckor utan steroider. Doserna av steroider som används är väldigt höga, och
kan vara upp till hundra gånger högre än de kliniska doser som används inom
vården. I början av en cykel är det vanligt att lägre doser används, vilka sedan
successivt ökas, för att i slutet av cykeln trappas ner igen. Det är också vanligt
förekommande att flera olika typer av steroider administreras under en cykel,
både orala och de som injiceras. Denna administreringsstrategi är främst av-
sedd för att optimera de önskade effekterna och för att minimera bieffekter.
Användningen av steroider i detta syfte är nämligen inte utan risker. Det
förekommer många allvarliga bieffekter, både fysiska och psykiska effekter,
vilka framförallt uppkommer efter en lång tids användning. De psykiska bief-
fekterna påverkar steroidanvändarens mående, mentala hälsa och beteende.
Några av de mest kända bieffekterna är ökad aggressivitet, irritabilitet och så
kallade ”roid rage”. Dessa effekter har gjort att steroidanvändare i högre grad
är involverade i olika våldsbrott. Andra psykiska effekter, som kanske inte är
lika välkända, är de som påverkar steroidanvändarens psykiska mående. Ste-
roidanvändare som brukat steroider under lång tid kan nämligen ha en ökad
risk för att utveckla ångest, depression och andra psykiska sjukdomar.
Studier på människor och djur har visat att steroider förändrar strukturen
och storleken på olika delar av hjärnan, men även att hjärnans signalsystem
och olika celler är påverkade. Dessa förändringar i hjärnan kan bidra till ett
förändrat beteende, men ytterligare studier behövs för att klargöra de exakta
detaljerna om varför förändringarna uppstår.
I den här avhandlingen har vi studerat och försökt särskilja effekterna av
fyra vanligt förekommande steroider; testosteron, nandrolon, stanozolol och
trenbolon. Dessa har undersökts i avseende på deras påverkan på det centrala
nervsystemet och hur de bidrar till ett förändrat beteende. Vi har använt oss

69
av en cell-modell med hjärnceller från råtta för att undersöka steroidernas tox-
iska effekter, hur de påverkar cellernas funktion och livsduglighet, så kallad
viabilitet. Vi har i två studier påvisat att dessa fyra steroider, i olika grad och
sannolikt genom olika mekanismer, påverkar cellernas mitokondriefunktion,
cellmembran och utvecklingen av neuronala nätverk. De inducerar också olika
typ av celldöd. Vi har även använt oss av en djur-modell där vi undersökt hur
hanråttor påverkas av höga doser av steroider som de exponerats för var tredje
dag i ungefär tre veckor, ett doseringsschema som ska motsvara en så kallad
steroidkur. Därefter har steroidernas effekter på råttornas beteende och minne
studerats i olika relevanta beteendetest. Vi har också undersökt vissa fysiska
effekter, till exempel kroppsvikt och organvikter. Utöver detta har vi även ge-
nomfört biokemiska analyser där vi mätt plasmakoncentrationerna av olika
stresshormon och uttrycket av specifika gener i olika delar av hjärnan. I dessa
studier påvisas skillnader mellan de olika steroiderna i avseende på fysiska
effekter och förändringar i beteende. Nandrolondekanoat identifierades att
vara en särskilt skadlig steroid, då den inducerade ett förändrat beteende hos
råttorna i form av minskad aktivitet och försämrat minne. Nandrolondekanoat
orsakade också minskad viktuppgång hos råttorna i jämförelse med kontroll-
råttorna och även flera organs vikter var påverkade, bland andra lever och nju-
rar. Studierna påvisade även steroid-specifika förändringar i uttrycket av olika
gener i framförallt hjärnregionen hypotalamus. Flera av dessa gener är bland
annat involverade i regleringen av födointag och mättnadskänslor, samt stress-
reaktionen, vilket skulle kunna förklara övriga resultat till viss del.
Sammanfattningsvis belyser denna avhandling de allvarliga konsekvenser
som steroidanvändning kan ha på kroppen, hjärnan och beteende. De steroid-
specifika effekterna indikerar att typen av steroid som används är av betydelse
för vilka bieffekter som uppkommer och hur allvarliga dessa är. Utöver detta
har dosen och exponeringstiden betydelse för graden av bieffekter. Kunskap
om dessa preparat är av stor vikt för alla som i sin profession eller vardag är i
kontakt med steroidanvändare, framförallt för vården, då det ger förutsätt-
ningar för att kunna tillgodose varje patient bästa möjliga behandling och vård.

70
Acknowledgement

The studies included in this thesis were carried out at the department of Phar-
maceutical Biosciences, Division of Neuropharmacology and Addiction Re-
search, Faculty of Pharmacy, Uppsala University, Sweden. The Swedish Re-
search Council and the Kjell and Märta Beijer Foundation supported this
work. Special thanks to Apotekarsocieteten for providing me with scholar-
ships to travel to international conferences.
This thesis could not have been produced without the help I have received
from all wonderful colleagues at the Faculty of Pharmacy. I would like to ex-
press my sincere gratitude to all of you who have made my years as a PhD
student pleasant and enjoyable.
First, I would like to thank my main supervisor, Professor Mathias Hallberg.
I am so grateful for the opportunity to learn from you. Thank you for your
support and guidance regarding both research and teaching related matters,
and for always believing in me.
My co-supervisor, Alfhild Grönbladh, who taught me everything worth know-
ing in the lab! Thank you for your endless support, encouragement, and friend-
ship.
The senior Professor in our group, Fred Nyberg, thank you for introducing me
to the best research group and giving me the opportunity as an undergraduate
student in your lab, also for your enthusiasm and support over the years!
Erik Nylander, thank you for being a great roommate and friend! For all good
laughs and fun discussions regarding everything from the latest episode of
Game of Thrones to cell culturing. Thank you for all the fun at conferences,
and for all burgers and beers!
To my wonderful friend Frida Stam, thank you for always being there for me,
both in the lab and outside of work! I am grateful for all the hours together in
the gym, pushing each other to the limit and chatting about everything from
heaven to earth. You are the best “training-buddy” there is!
Past and present members of the research group, Erika Brolin, thank you for
taking care of me as an undergraduate student and during my first years as a
PhD student, for guiding me in the lab, and for teaching me your excellent
organizing skills. Jenny, Anna J, Shanti, Anna L, Elena, Olga, Sara, John,

71
Maria, Anne-Lie, Fredrik, Viktoria, Åsa, Joep, and Nikita, thank you for en-
couragement, guidance, and nice discussions, as well as the fun over lunch,
fika, and AW´s! Thanks to all of our master students for your hard work and
bringing joy to the lab-group.
To all FDR members for your excellent work and all interesting discussions!
Thanks to Erika Roman, Lova Segerström, Stina Lundberg, Nikita Tjernström,
and Åsa Konradsson Geuken for great advice regarding behavioral experi-
ments and valuable discussions.
To the personnel at the animal facility for taking care of my rats.
All past and present members of FarmBio for providing a pleasant and wel-
coming atmosphere, and for all the nice fika´s! Special thanks to all adminis-
trative personnel that made my life easier during these years.
To all my friends and loved ones outside of BMC. I could not have done this
without you!
My amazing Biomedz-girls, Elin, Karin, Anneli, and Sofia for your support
and friendship since my first day in Uppsala. You are simply the best!
My best friends, Mikaela, Cecilia, Lina, Beatrice L, Martina, Amelie, and Be-
atrice Å, thank you for always being there no matter what. Life would not be
the same without you!
I would also like to thank my family and relatives for all support and encour-
agement. Your help with practical things, with everything from baby-sitting
Cleo to preparing dinner, is much appreciated and have made our life so much
easier. I feel the happiest when I am with you!
Special thanks to my beloved family. My incredible little sisters, Ylva and Ikil,
I am so lucky to have you in my life! My parents, Eva and Magnus, the best
mom and dad in the world! Thank you for always believing in me, listening,
understanding, and helping me become the person I am today. Your encour-
agement, support, and endless love means everything to me.
To my little one, Cleo, no one is as precious to me as you! I will always be
there for you. I love you, always, forever!
Martin, you are the love of my life, my best friend, and the greatest father to
Cleo. You are my hidden strength! I am grateful for all the things you do for
us, making life beautiful. Thank you for loving me just the way I am!

With love,
Sofia Uppsala, August 2022

72
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Acta Universitatis Upsaliensis
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