Download Mechanism of action of cathinone from khat

Mechanism of action of cathinone from khat (Catha edulis)
on behavioural and reproductive function in vervet
monkey (Chlorocebus aethiops).
A thesis submitted in fulfilment of requirements for Doctor of Philosophy
degree of University of Nairobi [Animal Physiology].
ALBERT WAFULA NYONGESA, BVM, MSc, (UON)
DEPARTMENT OF VETERINARY ANATOMY AND PHYSIOLOGY
UNIVERSITY OF NAIROBI
2013.
ii
DEDICATION
This work is dedicated to God the Almighty for guiding me through my education and to my
late father Daniel Kimungui for his incalculable contribution to my education. His words of
wisdom and financial support contributed immensely to my success. May Almighty God rest
his soul in eternal peace. I also dedicate this work to my mother and my beloved family for
their tireless support to my studies.
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ACKNOWLEDEGMENTS
Special thanks to the University of Nairobi for meeting my tution fee as well as the Dean’s
Committee Grant that enabled me do my research project. In the same regard, I wish to extend
my thanks to the Khat Research Program (KRP) under the Seed Grant for International
Students at the University of Minnesota, USA for funding the project. I also wish to express
my appreciation to my supervisors: Dr Jemimah Oduma of the University of Nairobi, Dr Pius
Adoyo of Institute of Primate Research, Karen and Prof Sanika Chirwa of Meharry Medical
College for their untiring efforts and willingness to guide me through the entire research
project. I also wish to express my appreciation to Prof Dominic Oduor-Okelo for his
assistance with technical knowledge on electron microscopy work and interpretation of
results. This work could not have succeeded without the co-operation and excellent technical
assistance of Gershon Deya, Eric Omolo and James Ndung’u and the entire Animal Resources
staff of Institute of Primate Research, Margaret Kagina, Robert Tsuma, Catherine Ngaywa,
Peter Irungu, Daniel Kwoba, Francis Okumu and Stanley Marete of Department of Veterinary
Anatomy and Physiology, University of Nairobi. Finally I wish to acknowledge Dr Charles
Kimwele, Dr Boniface Kavoi, Dr Philemon Towett, Dr Rodi Ojoo, Dr Allan Njogu, Dr
Johnson Nasimolo, Dr Catherine Kaluwa, Dr Dominic Ochwang’i, Jackson Mugweru and
Samuel Kamonde for encouraging me to pursue this work.
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PUBLICATIONS
1. Acute and sub-chronic effects of purified cathinone from khat (Catha edulis) on
behavioural profiles in vervet monkeys (Chlorocebus aethiops).
2. Dose-response inhibitory effects of purified cathinone from khat (Catha edulis) on
cortisol and prolactin release in vervet monkeys (Chlorocebus aethiops).
v
TABLE OF CONTENTS
DECLARATION ...................................................................................................................... ii
DEDICATION .........................................................................................................................iii
ACKNOWLEDEGMENTS .................................................................................................... iv
PUBLICATIONS ...................................................................................................................... v
LIST OF TABLES .................................................................................................................. xii
LIST OF FIGURES ...............................................................................................................xiii
ABSTRACT .......................................................................................................................... xvii
CHAPTER 1 .............................................................................................................................. 1
1.0 GENERAL INTRODUCTION AND LITERATURE REVIEW ................................... 1
1.2 RESEARCH OBJECTIVES ............................................................................................. 4
1.2.1 OVERALL OBJECTIVE .......................................................................................... 4
1.2.2 SPECIFIC OBJECTIVES ......................................................................................... 4
1.3 JUSTIFICATION OF STUDY ......................................................................................... 4
1.4 LITERATURE REVIEW ................................................................................................. 7
1.4.1 GENERAL OVERVIEW ON KHAT USE ............................................................... 7
1.4.2 CHEMICAL COMPOSITION .................................................................................. 9
1.4.3 PHARMACOKINETICS OF KHAT ...................................................................... 14
1.4.4 DOPAMINERGIC SYSTEM .................................................................................. 15
1.4.5 SEROTONERGIC SYSTEM .................................................................................. 17
1.4.6 NORADRENALINE ............................................................................................... 19
1.4.7 EFFECTS OF CATHINONE ON THE REWARD CIRCUIT ............................... 23
1.4.8 EFFECTS OF KHAT ON CARDIOVASCULAR SYSTEM ................................ 25
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1.4.9 CORRELATION OF BEHAVIOUR . .................................................................... 27
1.5.0 EFFECTS OF KHAT PITUITARY-ADRENAL AXIS ........................................ 28
1.5.1 HYPOTHALAMO-HYPOPHYSEAL-GONADAL AXIS .................................... 30
1.5.2 KHAT AND CATHINONE AND GONADAL AXIS ........................................... 32
1.5.3 SEX STEROID HORMONE METABOLISM ....................................................... 34
1.5.4 VERVET MONKEY BIOLOGY AND USE IN RESEARCH .............................. 36
1.5.4.1 Reproduction .................................................................................................... 36
1.5.4.2 Use in neuro-behavioural and reproductive health research ............................ 37
Vervet as an alternative to rhesus: ............................................................................ 37
Investigation of neurobehavioural phenotypes: ........................................................ 37
Investigation of the immune system and infectious disease: .................................... 38
CHAPTER 2............................................................................................................................ 39
2.0 GENERAL MATERIALS AND METHODS ................................................................ 39
2.1 ANIMALS AND HOUSING ......................................................................................... 39
2.2 CHEMICALS ................................................................................................................. 39
2.3 EXTRACTION OF CATHINONE FROM KHAT ........................................................ 40
2.4 EXPERIMENTAL DESIGN .......................................................................................... 41
2.4.1 IN VIVO STUDIES .................................................................................................. 42
2.4.1.1 Pre-treatment phase (40 days) .......................................................................... 42
2.4.1.2 Treatment phase (4 months) ............................................................................... 42
CHAPTER 3............................................................................................................................ 44
ACUTE AND SUB-CHRONIC EFFECTS ON BEHAVIOUR ........................................ 44
3.0 INTRODUCTION ............................................................................................................ 44
3.1 MATERIALS AND METHODS ................................................................................... 47
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3.1.1 ANIMALS AND HOUSING .................................................................................. 47
3.1.2 ANIMAL HANDLING AND EXPERIMENTAL DESIGN .................................. 47
3.1.2.1 Pre-treatment phase............................................................................................ 47
Environmental (cage) enrichment during pre-treatment phase ................................... 47
3.1.2.2 Treatment phase ................................................................................................. 48
i). Environmental (cage) enrichment during treatment phase ..................................... 49
ii). Behavioural studies .............................................................................................. 49
3.1.3 STATISTICS ............................................................................................................ 54
3.2 RESULTS ....................................................................................................................... 55
3.2.1 BEHAVIOURAL OBSERVATIONS ..................................................................... 55
3.2.1.1 Behavioural scores of aggression ....................................................................... 55
3.2.1.2 Behavioural scores of anxiety ............................................................................ 62
3.2.1.3 Abnormal behaviour .......................................................................................... 66
3.2.1.4 Appetitive behaviour.......................................................................................... 70
3.2.1.5 Withdrawal behavioural score ............................................................................ 72
3.3 DISCUSSION ................................................................................................................. 74
3.3.1 EFFECTS ON BEHAVIOUR ................................................................................. 75
3.3.1.1 Aggression ......................................................................................................... 75
3.3.1.2 Anxiety .............................................................................................................. 76
3.3.1.3 Abnormal behaviour .......................................................................................... 77
3.3.1.4 Withdrawal/Isolation and appetitive behaviour .................................................. 78
CHAPTER 4............................................................................................................................ 83
SERUM HORMONAL PROFILES IN VERVET MONKEYS ........................................ 83
4.0 INTRODUCTION ............................................................................................................ 83
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4.1 MATERIALS AND METHODS..................................................................................... 87
4.1.1 ANIMALS AND HOUSING .................................................................................. 87
4.1.2 EXPERIMENTAL DESIGN ................................................................................... 87
4.1.2.1 Pre-treatment phase............................................................................................ 87
4.1.2.2 Treatment phase ................................................................................................. 87
i). Drug administration............................................................................................... 87
ii) Blood sampling .................................................................................................... 88
iii) Hormonal analysis ............................................................................................... 88
LH bioassay .............................................................................................................. 89
4.1.3 STATISTICS ........................................................................................................... 90
4.2 RESULTS ....................................................................................................................... 91
4.2.1 Effect of (-)-cathinone on serum cortisol ................................................................ 91
4.2.2 Effect of (-)-cathinone on serum prolactin .............................................................. 92
4.2.3 Effect of (-)-cathinone on serum Lutenizing hormone ............................................ 92
4.2.4 Effect of (-)-cathinone on serum testosterone ......................................................... 93
4.2.5 Effect of (-)-cathinone on serum progesterone ........................................................ 93
4.3 DISCUSSION ............................................................................................................... 112
4.3.1 Effect of (-)-cathinone on cortisol and prolactin. .................................................. 112
4.3.2 Effect of (-)-cathinone on Luteinizing hormone, progesterone. ............................ 118
CHAPTER 5.......................................................................................................................... 122
IMMUNOHISTOCHEMICAL LOCALIZATION OF PITUITARY ............................ 122
5.0 INTRODUCTION .......................................................................................................... 122
5.1 MATERIALS AND METHODS ................................................................................. 124
5.1.1 ANIMALS AND HOUSING ................................................................................ 124
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5.1.2 EXPERIMENTAL DESIGN ................................................................................. 124
5.1.3 IMMUNOHISTOCHEMISTRY ........................................................................... 124
5.1.4 HISTOLOGICAL EVALUATION ....................................................................... 126
5.1.5 MORPHOMETRIC ANALYSIS .......................................................................... 126
5.2 RESULTS ..................................................................................................................... 128
5.3 DISCUSSION ............................................................................................................... 142
CHAPTER 6.......................................................................................................................... 146
ACUTE AND SUB-CHRONIC EFFECTS ON STEROID HORMONE ....................... 146
6.0 INTRODUCTION .......................................................................................................... 146
6.1 MATERIALS AND METHODS ................................................................................. 151
6.1.1 ANIMALS AND HOUSING ................................................................................ 151
6.1.2 EXPERIMENTAL DESIGN ................................................................................. 151
6.1.2.1 Animal grouping .............................................................................................. 151
6.1.2.2 Leydig cell function test ................................................................................... 152
6.1.2.3. Expression of steroidogenic enzymes ............................................................. 153
i) Total RNA extraction ........................................................................................... 153
ii) RNA characterization, purity and integrity .......................................................... 153
iii) First strand cDNA synthesis ............................................................................... 154
iv) Semi-quantitative PCR analysis for 3 β-HSD and 17 β-HSD type I . .................. 154
v) Real-time quantitative PCR analysis of 3 β-HSD and 17 β-HSD type I. .............. 155
6.1.2.4 Enzyme immunoassay for intracellular progesterone ....................................... 156
6.1.3 STATISTICS ......................................................................................................... 157
6.2 RESULTS ..................................................................................................................... 158
6.3 DISCUSSION ............................................................................................................... 169
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CHAPTER 7.......................................................................................................................... 173
MORPHOFUNCTIONAL EFFECTS OF CATHINONE ON TESTES ....................... 173
7.0 INTRODUCTION ............................................................................................................ 173
7.1 MATERIALS AND METHODS................................................................................... 177
7.1.1 ANIMALS AND HOUSING ................................................................................ 177
7.1.2 EXPERIMENTAL DESIGN ................................................................................. 177
7.1.3 TISSUE PROCESSING FOR ELECTRON MICROSCOPY .............................. 177
7.1.4 STATISTICAL ANALYSIS ................................................................................. 182
7.2 RESULTS ..................................................................................................................... 183
7.3 DISCUSSION ............................................................................................................... 196
CHAPTER 8.......................................................................................................................... 201
8.1 GENERAL DISCUSSION ............................................................................................ 201
8.2 CONCLUSIONS ........................................................................................................... 206
8.3 FUTURE GOALS ......................................................................................................... 207
REFERENCES ..................................................................................................................... 209
APPENDIX 1 ........................................................................................................................ 250
APPENDIX 2 ......................................................................................................................... 251
APPENDIX 3 ......................................................................................................................... 252
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LIST OF TABLES
Table 3-1. Animal grouping by number, sex, age and treatment dose .................................... 51
Table 3-2. Summary of composite behaviour definitions. ....................................................... 52
Table 5-1. Immunohistochemistry summary table.. .............................................................. 131
Table 5-2. Mean number of cells at high power magnification.. ........................................... 132
Table 6-1. Table showing hydroxysteroid dehydrogenases ................................................... 149
Table 6-2. Comparisons between samples based on “cycle threshold” (CT).. ...................... 165
Table 7-1. Mean volume densities of various organelles in Leydig cells .. ........................... 189
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LIST OF FIGURES
Figure 1-1. Chemical structures of S-(-)-cathinone. ................................................................ 10
Figure 1-2. Structure of K-19 (1) . ........................................................................................... 12
Figure 1-3. Summary of different classes of alkaloids in khat leaves ..................................... 13
Figure 1-4. Schematic representation of serotonin–dopamine interaction.. ............................ 18
Figure 1-5. Mid-saggital view of rat brain with serotonin-immunoreactive cell bodies.. ....... 19
Figure 1-6. The distribution of noradrenergic neurons in the brain. ....................................... 20
Figure 1-7. Brain areas receiving a prominent noradrenergic innervation. ............................. 21
Figure 1-9. Biosynthesis of steroid hormones in adrenal cortex and gonads.. ........................ 35
Figure 1-9. Biosynthesis of steroid hormones in adrenal cortex and gonads.. ........................ 35
Figure 3-1. Cage enrichment with a coloured toy ................................................................... 48
Figure 3-2. Intra-gastric administration of (-)-cathinone to the vervet monkey. ..................... 53
Figure 3-3 a. A significant increase in ‘yawn’ . ...................................................................... 56
Figure 3-3 b. Individual scores for ‘engage in a stare’ . ......................................................... 57
Figure 3-3 c. Behavioural scores for ‘jaw thrust’.. .................................................................. 58
Figure 3-3 d. A significant increase in ‘bouncing off cage walls’.. ........................................ 59
Figure 3-3 e. A significant increase in ‘vigorous shaking of cage walls’ ............................... 60
Figure 3-3 f. Increase in ‘head jerk’.. ...................................................................................... 61
Figure 3-4 a. A dose-dependent increase in ‘pacing’ .. .......................................................... 63
Figure 3-4 b. A significant increase in ‘scratch’. .................................................................... 64
Figure 3-4 c. A dose-dependent increase in ‘self directed’ behaviour. ................................... 65
Figure 3-5 a. A dose-dependent increase in ‘response independent of stimuli’...................... 67
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Figure 3-5 b. Behavioural scores for ‘fine motor stereotypy’. ................................................ 68
Figure 3-5 c. A significant increase in ‘whole body stereotypy’. ............................................ 69
Figure 3-6. A significant increase in latency of response to food . ........................................ 71
Figure 3-7. A significant increase in time spent by focal subjects withdrawn.. ...................... 73
Figure 4-1. Changes in cortisol levels across groups... ........................................................... 95
Figure 4-2. Changes in cortisol levels across study weeks. ..................................................... 96
Figure 4-3. Pattern of cortisol release over sampling periods. . .............................................. 97
Figure 4-4. Group differences in patterns of changes in serum cortisol.. ................................ 98
Figure 4-5. Group differences in cortisol levels across sampling periods.. ............................. 99
Figure 4-6. Changes in serum prolactin levels across treatment groups. .............................. 100
Figure 4-7. Pattern of serum prolactin levels during pre-treatment and treatment .. ............. 101
Figure 4-8. Changes in prolactin levels over sampling period.. ............................................ 102
Figure 4-9. Group differences of changes in serum prolactin across weeks.. ....................... 103
Figure 4-10. Group differences in serum prolactin levels across sampling periods.. ............ 104
Figure 4-11. Changes in serum LH levels across groups. .. .................................................. 105
Figure 4-12. Changes in serum LH levels across study weeks. ............................................ 106
Figure 4-13. Group differences in serum LH levels across sampling periods....................... 107
Figure 4-14. Group differences in testosterone across study weeks.. .................................... 108
Figure 4-15. Group differences in serum testosterone levels. ............................................... 109
Figure 4-16. Group differences in serum progesterone across study weeks. ........................ 110
Figure 4-17. Correlations between serum cortisol and prolactin .. ........................................ 111
Figure 5-1. Hematoxylin and eosin staining of adenohypophysis.. . ..................................... 133
Figure 5-2. Dose-dependent effect of (-)-cathinone on corticotrophs .................................. 134
xiv
Figure 5-3. Mean labelling index (LI) indicating number of corticotrophs. ......................... 135
Figure 5-4. The figure shows gonadotrophs .......................................................................... 136
Figure 5-5. Mean LI expressing number of gonadotrophs.. .................................................. 137
Figure 5-6. Immunostaining for lactotrophs .......................................................................... 138
Figure 5-7. Mean LI expressing number of lactotrophs ........................................................ 139
Figure 5-8. Immunostaining of adenohypophysis of vervet monkey with S100. .................. 140
Figure 5-9. Figure showing a skin tissue with melanoma positive for S100. ........................ 141
Figure 6-1. Diagramatic presentation of steroid hormone metabolism ................................. 148
Figure 6-2. Vervet testicular tissues in incubation media ..................................................... 156
Figure 6-3. Total RNA expression of steroidogenic enzymes in testis.. ............................... 161
Figure 6-4. mRNA expression of steroidogenic enzymes in vervet testicular tissues. . ........ 161
Figure 6-5 a and b. mRNA expression of steroidogenic enzymes.. ..................................... 162
Figure 6-6. Relative expression of 3- HSD type I in testis of vervet monkeys. .................. 163
Figure 6-7. Relative expression of 17- HSD type I in testis of vervet monkeys ................. 164
Figure 6-8. Local steroidogenesis following addition of LH and (-)-cathinone.. .................. 166
Figure 6-9. LH increased while LH and (-)-cathinone decreased androstenedione. ............. 167
Figure 6-10. LH increased while LH and (-)-cathinone decreased testosterone . . ............... 168
Figure 7-1. Asceptic surgery of testes of the vervet. ............................................................. 180
Figure 7-2. Weight reading of testis ...................................................................................... 180
Figure 7-3. Weight reading after immersion of testis ............................................................ 181
Figure 7-4. Testicular tissue trimming for immersion in fixative ......................................... 182
Figure 7-5. Vervet monkey Leydig cell ultrastructure. ......................................................... 186
Figure 7-6. A graph showing mean volume densities . . ...................................................... 188
Figure 7-7. Vervet monkey Sertoli cell. ................................................................................ 190
xv
Figure 7-8. Spermatocytes of vervet monkeys .. ................................................................... 192
Figure 7-9. Spermatocytes of vervet monkeys.. .................................................................... 193
Figure 7-10. Round spermatids of vervet monkeys in acrosomal phase. .............................. 194
Figure 7-11. Elongate spermatids of vervet monkeys. .......................................................... 195
xvi
ABSTRACT
Biological effects of cathinone on behavioural and reproductive functions are inadequately
investigated, and available reports in literature are conflicting. A number of these studies have
indicated varying degree of information on effect of khat and cathinone on hypothalamohypophyseo-adrenocortical axis, mesolimbic system as well as neuro-endocrine disorders and
histopathological lesions in testes of humans and experimental animals. This study
investigated the mechanism of action of cathinone on behavioural changes in both sexes of
vervet monkeys as well as neuro-endocrine alterations that influence reproductive function in
males of the same species. The significance of this study was to determine whether cathinone
use boosts or is detrimental to reproductive and mental health under varying degree of use.
Such information would be vital to social and health care professionals and service providers,
as well as policy makers, community-based health groups and international organizations.
Fourteen adult vervet monkeys were divided into tests (12 animals) and controls (2 animals),
and treated with escalating doses of cathinone at alternate days of each week for 4 months.
Controls were administered normal saline in the same pattern as tests. Administration was
done via oral gavage. One month of pre-treatment served to establish baseline values. Group
II and IV animals were administered GnRH agonist alongside (-)-cathinone two weeks after
treatment phase of 4 months. Composite behavioural scores of aggression, anxiety, abnormal
responses, withdrawal and appetite loss were done alongside analysis of serum prolactin,
cortisol, luteinizing hormone, progesterone and testosterone. Stereology of testicular subcellular organelles of Leydig cells was also done following electron microscopy of testicular
tissue of the animals. For ex vivo studies, isolated Leydig cells from testis of the animals were
co-incubated with luteinizing hormone and subjected to two doses of cathinone (low and
xvii
high) overnight at 37 0C. Androstenedione, progesterone and testosterone were analyzed and
compared with those in control cells that were treated with incubation media alone. Gene
expression of mRNA of hydroxysteroid dehydrogenase enzymes (3 beta- and 17 beta
hydroxysteroid dehydrogenase type 1) was done for comparison of results with those of
intracellular hormones of testicular cells. Behavioural and hormonal data were analysed for
within- and between-subjects effects using Greenhouse-Geisser correction. Separate
ANOVAs were performed for each behavioural and hormonal variables with Bonferroni post
hoc multiple comparisons using SPSS version 14. Results indicate a dose-dependent effect of
(-)-cathinone on behavioural and hormonal profiles. Composite scores of aggression, anxiety,
abnormal responses, withdrawal and appetite loss increased in a dose-dependent manner.
Serum prolactin, cortisol and progesterone decreased with escalating doses of (-)-cathinone.
Serum LH increased with increasing doses while testosterone increased with lower doses and
peaked with medium dose then decreased with high dose. The morphological picture showed
disruption in smooth and rough endoplasmic reticulum, lipid droplets, mitochondria and
Golgi apparatus of Leydig cells, which were either morphologically altered or reduced in
number. Morphometric data showed reduced volume densities of these steroidogenic
structures. Sertoli cells of treated animals were unaffected while spermatogonia,
spermatocytes, spermatids and spermatozoa were variously affected by (-)-cathinone
treatment. The effects observed ranged from disruption of cell membranes of spermatogonia,
condensation of chromatin material in spermatocytes accompanied by intracytoplasmic
vacuolation, disruption of formation acrosome granules in acrosome vesicles and failure of
flagella formation at high dose of (-)-cathinone. The mRNA expression of 3 beta- and 17 beta
hydroxysteroid dehydrogenase type 1 enzymes increased with low and medium doses but
decreased at high dose of (-)-cathinone. The findings demonstrate that sub-chronic exposure
xviii
to (-)-cathinone at high dose causes behavioural and hormonal alterations probably via
changes
in
presynaptic
striatal
dopamine
system
and
hypothalamo-hypophyseal-
adrenocortical and gonadal axis integrity. The processes of spermatogenesis and
steroidogenesis appear to be affected at high dose of cathinone as evidenced by morphological
alterations and suppression of steroidogenic enzyme expression of vervet monkey testis.
xix
CHAPTER 1
1.0 GENERAL INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
Khat is both socially and economically one of the most important plants to inhabitants of
areas where it is grown, not only to countries of Eastern Africa but also of the Middle East. It
is chewed as a social custom and a stimulant. Fresh leaves and shoots are chewed for their
euphoriant and stimulatory effects (Al-Bekairi et al., 1991). Cathinone is the principal
ingredient of khat, and is found naturally as (-)- enantiomer; the (+)- enantiomer is not found
(Kalix and Braenden, 1985). The general analogy between the effects of (-)-cathinone and
those of amphetamine, as well as their chemical similarity suggest that the two substances
might have the same mechanism of action (Graziani et al., 2008; Houghton, 2004; Cox and
Rampes, 2003) with similar potential for abuse (Kalix, 1984 a).
Its impact in the body of the consumer ranges from mental, respiratory, digestive to
reproductive system effects. There is a growing interest in trying to understand the underlying
neural mechanisms and biological functions following use and abuse of khat among
consumers. Research findings in humans and experimental animals have reported changes in
sleep patterns, mood, attention, aggression, anxiety, locomotor activity, and affiliative
behaviours (Kalix, 1994; Pantelis et al., 1989), learning and memory (Kimani and Nyongesa,
2008) and sexual behaviour (Tariq et al., 1990; Qureshi et al., 1988) following khat use.
These behaviours are partly influenced by disorders in the hypothalamic dopaminergic system
1
(Ishikawa et al., 2007) and partly due to dopaminergic activity in the mesolimbic system
(Eisch and Harburg, 2006; Jones and Bonci, 2005; Rang, 2003).
Studies on neuro-behavioural functions following khat use in humans and experimental
animals are scarce. Most of the findings have not given clarity to the corresponding
magnitude of psychological behavioural changes reported perhaps due to the paucity of
objective and, therefore, relevant human data. The framework for postulating khat/(-)cathinone associated neuro-behavioural changes in sleep patterns, mood, attention,
aggression, anxiety, locomotor activity, and affiliative behaviour has been extrapolated from
results of khat studies predominantly in mice (Kimani and Nyongesa, 2008), rats (Banjaw et
al., 2005; Kalix et al., 1995; Islam et al., 1990) and monkeys (Schuster and Johanson, 1979).
Behavioural studies on khat and (-)-cathinone in primates are scarce. The available literature
highlights on subjective effects of khat in humans such as induction of mood changes,
irritability, anorexia and insomnia following long-term khat use (Al-Motarreb et al., 2002;
Nencini et al., 1989). There is scanty literature on effects of khat and (-)-cathinone on
consummatory behaviour in humans and non-human primates. Most available literature is on
appetitive behaviour in rats (Islam et al., 1990; Eisenberg et al., 1987; Foltin et al., 1983;
Zelger and Carlini, 1980; Knoll, 1979), guinea pigs (Jansson et al., 1988) and humans
(Murray et al., 2008; Kalix and Braenden, 1985; Halbach, 1972). Appetitive behaviour
basically concerns ability or failure to seek food while consummatory behaviour refers to
eating according to the Pavlovian homeostatic model.
Mammalian reproduction is systematically controlled by nervous and endocrine systems.
Steroid hormones are primarily synthesized by the ovaries and placenta (females) and testes
2
(males) and to a lesser extent by adrenal glands in both sexes. In adult male mammals,
testicular tissue constitutes seminiferous tubules and interstitium. The seminiferous tubules
are composed of Sertoli cells, various forms of spermatogenic cells and cells of the basal
compartment while the interstitium is comprised of Leydig cells, fibroblasts, macrophages,
blood and lymphatic vessels. Toxic agents impact each of these structures differently through
diverse cellular and endocrine pathways. Contradictory findings have been reported on effects
of khat and (-)-cathinone on reproductive sex hormones and semen parameters in roosters
(Hammouda, 1978), testicular tissue morphology in rats (Islam et al., 1990) and humans (ElShoura et al., 1995), steroid hormones in mice (Nyongesa et al., 2007) and rabbits (Nyongesa
et al., 2008). Quite recently, studies on khat extract and (-)-cathinone in rats showed a
biphasic effect on sexual motivation and potency, with high doses diminishing sexual
motivation and potency while low doses enhanced motivation (Mohammed and Engidawork,
2011). On the other hand, studies in baboons demonstrated increased plasma testosterone and
decreased cortisol and prolactin following khat administration (Mwenda et al., 2006). The
measure on cortisol is in concordance with studies in rats (Mohammed and Engidawork,
2011). The discrepancy in results has left more questions than answers. Do khat and (-)cathinone interfere with neuroendocrine function at the meso-striatal regions of the brain,
hypothalamo-hypophyseal level with gonadotropin releasing factors or with enzymatic
activity in sex steroid metabolism or structural alteration at gonadal level? Does species
difference or dose schedule play role towards this variance in effects? These questions led to
the objectives of the present study.
3
1.2 RESEARCH OBJECTIVES
1.2.1 OVERALL OBJECTIVE

To investigate the possible mechanism of action of (-)-cathinone on behavioural and
reproductive function.
1.2.2 SPECIFIC OBJECTIVES

To determine effect of (-)-cathinone on behavioural changes in vervet monkeys

To determine plasma levels of serum cortisol, prolactin, testosterone and progesterone in
(-)-cathinone-treated animals.

To determine serum levels of luteinizing hormone (LH) in response to gonadotropin
releasing hormone (GnRH) agonist administration in (-)-cathinone-treated male vervet
monkeys.

To immunolocalize anterior pituitary cell types secreting above hormones and identify
potential adenohypophyseal cell apoptosis using S100 as biochemical marker of
degeneration.

To determine the suppressive effects of (-)-cathinone on Leydig cell and germ cell
function.

To determine the effect of (-)-cathinone on the enzymatic activity in sex steroid
metabolism
1.3 JUSTIFICATION OF STUDY
The mechanism of action of khat on hypothalamo-hypophyseal-adrenocortical axis influencing
neural behaviour and biological function in khat consumers remains to be elucidated. Studies
were advanced that increased adrenal phosphorylase activity, serum free fatty acids and urinary
4
17α- hydroxycorticosteroids could be attributed to stimulatory effect of khat on adrenal function
(Connock et al., 2007). Furthermore, studies showed that khat increases adrenocorticotrophic
hormone in humans (Nencini et al., 1984) and rabbits (Nyongesa et al., 2008). High
concentrations of corticotrophin releasing factor were shown to activate the hypothalamohypophyseal-adrenocortical axis and other stress systems in the amygdala resulting in dysregulated emotional state of drug addiction (Goeders and Guerin, 1996 b). Persistent
alterations in these systems may lead to pathophysiology that translates into psychiatric
disorders such as disturbances in sleep patterns, anxiety, mood, social isolation and sexual
behaviour changes manifested in psychotropic drug ‘addicts’.
The mechanism of action of khat on reproductive and neuro-endocrine functions also remains to
be elucidated. Quantitative analysis of the sub-cellular architecture revealed by electron
microscopy is a logical step in the investigation of the normal functioning of cells and their
pathological alterations. Enzymes involved in testosterone synthesis are located in both SER
and mitochondria of Leydig cells (Tamaoki, 1973). Testicular ultra-structural changes
interfere with morphological expression of testosterone by Leydig cells. Previously,
stereological techniques of volume of cells, nuclei, mitochondria, SER and surface area
densities of SER and mitochondrial cristae were used to establish the functional activity of
Leydig cells in rats (Mazzocchi et al., 1982).
The present study is designed to test the integrity of the pituitary and the central mechanism
involved in the regulation of LH release in (-)-cathinone-treated male vervets. The effect on
pituitary gland will be assessed by observation of plasma LH release in response to GnRH
agonist in (-)-cathinone-treated vervets. Testosterone metabolism was determined by
5
measurement of the expression of mRNA 3 β- HSD type 1 and 17 β- HSD type 1. The
isoforms of 3 β- HSD involved in mammalian testicular testosterone synthesis are type 1 and
3 (Penning, 1997) while the main izymes of 17 β- HSD in testosterone formation in mammals
is considered to be 17 β-HSD type 1 and 3, which catalyzes the reduction of androstenedione
to testosterone with high efficiency and are almost exclusively expressed in testis (Geissler et
al., 1994). The study quantified testicular Leydig cell hypofunction by assessing the
steroidogenic organelles of these cells while germ cell hypofunction was done by assessment
of ultrastructural alterations following (-)-cathinone treatment.
Khat use is exclusively a human habit. However, controlled and invasive studies in humans
are limited by ethical constraints. Monkeys have been used extensively as models for research
in human reproduction, developmental anatomy, nutritional disorders and effects of drugs
(Amaral, 2002) as well as in understanding the pathogenesis of AIDS (Ambrose et al., 2001).
In the present study, the vervet monkey was chosen for use as a model animal due to its
recognized utility as an old world monkey alternative to the rhesus macaque, of which there is
now a critical shortage for biomedical research. Vervets are currently the most populous
African non-human primates. They are similar to rhesus monkeys in behaviour and body
physiology (Disotell, 2000) and are closely related to humans (Raaum et al., 2005). They are
less expensive and may be accompanied by fewer health and safety risks (Gordon et al., 2005;
Baulu et al., 2002). Recently, research findings established paradigms that demonstrate the
use of vervet monkeys as models (other than humans) for elucidating cognitive function such
as working memory and response inhibition that are relevant to human psychiatric disorders
such as schizophrenia (James et al., 2007).
6
The present study was designed to test the integrity of the hypothalamo-hypophysealadrenocortical axis and the mesolimbic system involved in regulation of adrenocorticotrophic
hormone release in (-)-cathinone-treated vervet monkeys. The effect on the hypothalamohypophyseal-adrenocortical and gonadal axes as well as mesolimbic systems were observed
by measurement of serum cortisol, prolactin, progesterone, testosterone and LH levels as well
as accompanying immunohistochemical localization of corticotropes, lactotropes and
gonadotropes in the hypophysis. Potential hypophyseal cellular degeneration was done by
use of S100. The study was also designed to test the integrity of hypothalamo-hypophyseoadrenocortical and gonadal axes and sex steroid metabolism.
1.4 LITERATURE REVIEW
1.4.1 GENERAL OVERVIEW ON KHAT USE
Khat chewing is predominantly a male habit, but women also practice it. A general moral
battle is on-going over khat consumption in East Africa and the diaspora (Klein and
Beckerleg, 2007). Khat use has been associated with breakdown of marriages, prostitution and
a host of other social evils (Beckerleg, 2008) while male khat consumption is usually
highlighted as damaging and irresponsible. Thus, a study on consumption in Djibouti found
that khat consumption, mostly by men, is associated with higher inequality in distribution of
resources within the family (Borelli and Perali, 2004). In Somalia, women, particularly those
in the diaspora present equal discourse in their campaign against khat consumption (Klein and
Beckerleg, 2007).
7
The pattern of khat consumption and its socio-economic impact in Kenya is not any different.
According to reports of a survey carried out during the month of December, 2008 in over one
hundred homesteads in Maua area of Meru District, Kenya, married couples experience
marital problems associated with heavy and long-term use of khat (Nyongesa et al.,
unpublished data). Majority of the women, in particular, complain of their husbands spending
much of their time and money on khat consumption while neglecting family duties. Of those
interviewed, 60% claimed that their husbands were not sexually active especially after heavy
consumption of khat. About 70% of male khat addicts that were interviewed also reported
spermatorrhea following consumption of large quantities of khat while 30% of them reported
a boost on sexuality following use. This array of mixed reports parallels what has been
reported on khat by various investigators. Most of the research findings both in humans and
experimental animals confirm such reports (Nyongesa et al., 2008; El Shoura et al., 1995;
Islam et al., 1990) but other studies report differently (Mwenda et al., 2006; Adeoya-Osiguwa
and Fraser, 2005; Al-Mamary et al., 2002). Some studies have shown a biphasic effect on
testosterone in mice (Nyongesa et al., 2007) and rats (Mohammed and Engidawork, 2011).
These reports have been discussed extensively in later chapters of this thesis. These findings
indicate that khat contains some components that may have contraceptive efficacy either on
their own or in combination with other therapeutics. Such findings, however, remain to be
elucidated. In the past two decades, khat use has followed immigrants from the traditional use
regions around the Horn of Africa and the Middle East to western countries. Khat can now be
regarded as a psychoactive plant that is being targeted for illegalization in most western
countries such as the United States and most parts of Europe including United Kingdom,
Sweden and the Netherlands among others (Grayson, 2008; Carrier, 2007). The fact that
khat’s appearance in western countries has, nevertheless, created moral and political panic in
8
some circles (Carrier, 2007) and growing anxiety in the source countries because of the fear
of illegalization, is not surprising because most of such economies will lose on revenue
collections from khat exports. This is particularly so in countries where khat use is associated
with a lifestyle and its cultivation a strategy for national development. A proper approach into
investigations on khat and human health is a critical consideration whereupon vital
information is provided to social and health care professionals, health service providers,
community-based health groups as well as policy makers and international organizations.
1.4.2 CHEMICAL COMPOSITION
Catha edulis contains many biologically active alkaloids. Cathinone also referred to as S-(-)cathinone or [S- (-) - α-aminopropiophenone] is the most potent ingredient of khat (Kalix and
Braenden, 1985). It is fairly unstable, being metabolized relatively quickly to (+)norpseudoephedrine (cathine) and (-)-norephedrine, which are more stable and less potent
molecules as a result of a chemical reduction once the plant is harvested (Brenneisen and
Geisshüsler, 1985). A diastereomeric pair of phenylpropanolamines is produced from this
reaction, specifically (+)-norpseudoephedrine [1S, 2S-2-amino-1-phenyl-1-propanol] and (-)norephedrine [1R, 2S-2-amino-1-phenyl-1-propanol]. Cathine and (-)-cathinone are
phenylisopropylamine derivatives, which resemble amphetamine in chemical structure and
biological activity (Schorno and Steinegger, 1979).
The substance (+)-norpseudoephedrine was the first to be isolated and identified from the khat
plant (Wolfes, 1930) and historically labelled as cathine. Other constituents of khat include:
(+)-merucathinone, (+) – merucathine and (-)-pseudomerucathinone, which occur only in khat
found in the Meru area of Kenya (Kalix, 1988). There is a rapid stereo-selective metabolism
9
of
S-(-)-cathinone
to
(-)-norephedrine
and
(+)-norpseudoephedrine
following
its
administration in humans (Brenneisen et al., 1990; Nencini and Ahmed, 1989; Geisshüsler
and Brenneisen, 1987). Metabolism of S-(-)-cathinone to (+)-norpseudoephedrine involves
reduction of a keto group to an alcohol, a fairly common metabolic pathway in humans,
catalyzed by liver microsomal enzymes (Guantai and Maitai, 1983) (Fig. 1-1).
Figure 1-1. Chemical structures of S-(-)-cathinone, S,S-(+)-norpseudoephedrine (cathine) and
R,S-(-)-norephedrine (MW=151.2). Cathinone is transformed mainly into cathine in khat
10
leaves and mainly into norephedrine by human metabolism. (Adapted from Feyissa and Kelly,
2008).
(-)-Cathinone can also undergo other reactions such as dimerization and auto-oxidation to
form 3,6 dimethyl-2,5-diphenylpyrazine (Berrang et al., 1982; Szendrei, 1980). Enolization of
the keto-amine segment also facilitates racemization at the C2 site while S-(-)-cathinone is in
solution (Fig. 1-2). Racemization can also occur during drying process after the plant is
harvested (LeBelle et al., 1993).
11
Figure 1-2. Structure of K-19 (1), one of the most highly elaborated members of the
cathedulin family with its polyhydroxylated sesquiterpenoid euonyminol core. (Adapted from
White, 1994).
Studies have shown that khat contains many other chemical substances including:

Alkaloids, terpenoids, flavonoids, sterols, glycosydes, tannins (7 - 14% by weight) and
more than 10 amino acids including tryptophan, glutamic acid, glycine, alanine and
threonine (Szendrei, 1980; Luqman and Danowski, 1976; Halbach, 1972; Geisshüsler
and Brennesien, 1987; Elmi, 1983; Crombie, 1980).
12

Trace quantities of vitamins including vitamin C (15 mg/100 mg), thiamine, niacin,
riboflavin and carotene (Cox and Rampes, 2003; Nencini et al., 1989; Kalix and
Braenden, 1985; Luqman and Danowski, 1976).

Elements including calcium, iron (Hattab and Angmar-Mansson, 2000; Halbach,
1972), manganese (Halbach, 1972), copper, zinc, and toxic metals like lead and
cadmium (Matloob, 2003) and a negligible amount of fluoride (Hattab and AngmarMansson, 2000).
Composition of alkaloids in khat leaves are diagrammatically presented (Fig. 1-3):
Figure 1-3. Summary of different classes of alkaloids in khat leaves
The concentrations of (-)-cathinone vary in different parts of the plant, but abundant in young
fresh leaves and shoots although it is reduced by three days after leaves have been removed
13
from khat tree (Balint and Balint, 1994). The potency of khat also depends on the variety of
khat. In Kenya, there are different sub-species of khat namely: Ghiza, kangeta and kata. Ghiza
has short stems and is said to be more potent. This is the variety that was used in the study.
In this thesis the effect of (-)-cathinone covers: the reward centers of the brain, effect on
hypothalamo-hypophyseal-adrenocortical and gonadal axes as well as effect on testosterone
biosynthesis ex vivo. Literature on these systems has been highlighted for purposes of
bringing the present study into the context.
1.4.3 PHARMACOKINETICS OF KHAT
During khat chewing session, leaves and barks of shoots are chewed slowly over several
hours, usually 2 to 10 h at which point between 100 and 500 g of khat is chewed by a
particular individual (Al-Hebshi and Skaug, 2005; Matloob, 2003; Nencini and Ahmed,
1989). The juice of masticated leaves is swallowed while residues are spat (Toennes et al.,
2003). Earlier studies on excretion of khat alkaloids in humans showed that both cathine and
norephedrine are slowly absorbed and metabolised (Maitai and Mugera, 1975). In another
study, four healthy non-drug using volunteers chewed four portions of 0.6 g khat leaves per
kilogram body weight for 1 hr and plasma concentration-time data analysed using a twocompartment model with two-segment absorption (Toennes et al., 2003). It was found that the
mucosa of the oral cavity is the first absorption segment where the major proportion of the
alkaloids is absorbed. According to the findings of World Health Organization (1985), khat is
rapidly absorbed after mastication, metabolized in the liver with only a small fraction
appearing in urine (Kalix and Braenden, 1985). The more rapid and intense action of
14
cathinone compared to cathine, is explained by its higher lipid solubility, facilitating quicker
access into the central nervous system (Zelger et al., 1980).
The pharmacokinetic parameters of cathinone and other constituents of khat leaves have been
determined over 8 h, with peak plasma levels attained after 1 – 3.5 h (Halket et al., 1995;
Brenneisen et al., 1990). Similarly, maximal plasma concentration (tmax) of cathinone, cathine
and norephedrine are reported to be reached at 2.3, 2.6 and 2.8 h, respectively (Toennes et al.,
2003). After ingestion of 0.8 mg/kg cathinone, it was reported that total amount of cathinone
absorbed in the body after 9 h was 25 ± 13 µg min ml-1 and terminal elimination half-life was
260 ± 102 min (Widler et al., 1994). There is rapid metabolism of S (-)-cathinone to
norephedrine and cathine following its administration to humans (Brenneisen et al., 1990;
Nencini and Ahmed, 1989). In four human subjects who chewed khat leaves for 1 h, then spat
out the residues and subsequently had their urine samples tested for the presence of cathinone
and its metabolites, cathinone was detected for up to approximately 26 h, while cathine and
norephedrine were detected for at least 80 h (Toennes and Kauert, 2002). Only 7% or less of
absorbed (-)-cathinone is excreted in the form of norephedrine and cathine (Toennes and
Kauert, 2002; Kalix et al., 1990; Brenneisen and Geisshusler, 1987). The amount of
norephedrine excreted in urine is much higher than that ingested suggesting that (-)-cathinone
is also metabolized to R, S (-)-norephedrine (Toennes and Kauert, 2002).
1.4.4 DOPAMINERGIC SYSTEM
Neurons of the ventral mesencephalon also referred to as A8, A9 and A10 cell groups, have
been collectively designated as the mesotelencephalic dopamine (DA) system (Dahlström and
15
Fuxe, 1964). These investigators actually identified twelve groups of catecholamine cells
(designated A1- A12) distributed from the medulla oblongata to the hypothalamus. Initially,
the mesolimbic DA system was thought to originate from the A10 cells of the ventral
tegmental area (VTA) that then project to structures closely associated with the limbic system.
This system was considered separate from the nigrostriatal DA system originating from the
more lateral substantia nigra (SN; A9 cell group) (White, 1996; Kalivas, 1993; Roth et al.,
1987; Bannon and Roth, 1983). Five additional cell groups A13-A17 are located in the
diencephalon. Olfactory bulb and retine as well as 3 adrenaline-containing cell groups C1 –
C3 were later added (Hökfelt et al., 1984). The DA neurons (A12) in the arcuate nucleus of
the hypothalamus have been shown to co-localize with various neuropeptides including
growth hormone releasing hormone, neurotensin, galanin, enkephalin and dynorphin,
suggesting a broader neuroendocrine role of these neuron subtypes (Lookingland and Moore,
2005).
The mesolimbic and mesocortical DA systems are important in modulation of functions such
as motivation, control of emotions and cognition sub-served by prefrontal cortex and limbic
regions (LeMoal and Simon, 1991). Substantial evidence indicates that the mesolimbic
pathway, particularly in DA cells innervating nucleus accumbens, is implicated in the
pleasurable reward following psycho-stimulation by use of natural or drug enforcers (Koob,
1992; Di Chiara and Imperato, 1988). Lesion to DA terminals in nucleus accumbens induces
hypo-exploration, enhanced latency in initiation of motor responses, disturbances in
organizing complex behaviours and inability to switch from one behavioural activity to
another (LeMoal and Simon, 1991). Therefore, the mesolimbic DA system is deemed
16
important for acquisition and regulation of goal-directed behaviours, established and
maintained by natural or drug reinforcers (Kiyatkin, 1995; LeMoal and Simon, 1991).
The nigro-striatal DA system originates from the substantia nigra (SN) (A9 cell group). It is
important due to its involvement in the pathogenesis of Parkinson’s disease (PD) (Grace and
Bunney, 1985). In mammals, the SN is a heterogeneous structure that includes two distinct
compartments: the substantia nigra pars compacta (SNc) and the substantia nigra pars
reticulata (SNr). The SNc represents the major source of striatal DA and, as already
mentioned, its degeneration causes PD. On the contrary, the SNr mainly contains g-amino-nbutyric acid (GABA)-ergic neurons which constitute one of the major efferences of the basal
ganglia (Grace and Bunney, 1985).
1.4.5 SEROTONERGIC SYSTEM
Serotonin is a neuromodulator whose detailed properties are as yet much more mysterious
than dopamine, but which is implicated in a wealth of important phenomena, ranging from
analgesia (LeBars, 1988; Sawynok, 1988), hallucinations (Aghajanian and Marek, 1999) to a
variety of mood disorders such as anxiety and depression (Westenberg et al., 1996). Virtually
all parts of the central nervous system (CNS) receive innervation from serotonergic fibres
arising from cell bodies located in two trunks of the midbrain serotonergic nuclei: the dorsal
raphe (DR) and the median raphe (MR) (Moukhles et al., 1997; Van Bockstaele et al., 1994).
Serotonin-containing bodies of the raphe nuclei project to dopaminergic cells in the VTA and
SN, as well as nucleus accumbens, prefrontal cortex and striatum (Moukhles et al., 1997; Van
Bockstaele et al., 1994; Hervé et al., 1987) (See Fig. 1-4). There are also serotonergic
17
projections from the raphe to the periacqueductal gray, which is involved in the control of
defensive and aversively motivated behaviours (see Graeff, 2003, for a review).
Figure
1-4.
Schematic
representation
of
serotonin–dopamine
interaction
in
the
mesocorticolimbic and nigrostriatal DA-ergic system. Serotonin-containing cell bodies of the
raphe nuclei send projections to dopaminergic cells in both the ventral tegmental area (VTA,
A10) and the substantia nigra (SN, A9), and to their terminal fields in the nucleus accumbens,
prefrontal cortex and striatum. [Adapted from Di Giovanni et al. (2008)].
At electron microscopy there is presence of synaptic contacts of 3H5-HT-labelled terminals
with both dopaminergic and non-dopaminergic dendrites in all sub-nuclei of the VTA, and in
the SNc and SNr (Moukhles et al., 1997; Kalivas, 1993; Hervé et al., 1987). There is
18
differential distribution of 5-HT receptor subtypes within the dopaminergic systems (Hoyer et
al., 1994; Barnes and Sharp, 1999) that have led to the insight of dopamine-serotonin systems
interaction in the brain (Fig. 1-5)
Figure 1-5. Mid-saggital view of the rat brain with serotonin-immunoreactive cell bodies.
The blue and red ovals encompass the two major subdivisions of the brain serotonergic
system. The major functions of 5-HT and the brain areas where they occur are also shown.
Abbreviations: DRN, dorsal raphe nucleus; MRN, medial raphe nucleus; NRM; nucleus raphe
magnus; NRO, nucleus raphe obscurus; SNc, substantia nigra pars compacta; VTA, ventral
tegmental area. [Adapted from Di Giovanni et al. (2008)].
1.4.6 NORADRENALINE
Noradrenaline,
3,
4-dihydroxyphenylethanolamine,
is
released
from
terminals
of
noradrenergic neurons in the brain from most postganglionic sympathetic neurons and from
19
chromaffin cells in the adrenal medulla. The cell bodies of central noradrenergic neurons are
all clustered within two bilateral groups of nuclei (A1 –A7) in the brain stem (Fig. 1-6). These
comprise the locus coeruleus (LC) complex and the lateral tegmental nuclei. The locus
coeruleus proper (nucleus A6) accounts for approximately 45% of all the noradrenergic
neurons in the brain (Stanford, 2001). The activity of noradrenergic neurons within locus
coeruleus is governed by GABAergic (inhibitory) projection from nucleus prepositus
hyperglossi and glutamatergic (excitatory) input from the nucleus paragingantocellularis
(Aston-Jones et al., 1991).
Figure 1-6. The distribution of noradrenergic neurons in the brain. The cell bodies are
clustered in nuclei (A1 – A7) in the pons/medulla regions of the brainstem and their axons
project both rostrally and caudally to most regions of the neuraxis. The major nucleus is the
locus coeruleus (A6). (Adapted from Stanford, 2001)
Many brain areas are innervated by neurons from LC and lateral tegmental area (LTA). The
frontal cortex, hippocampus and olfactory bulb appear to be innervated by neurons originating
20
from LC whereas most of hypothalamic nuclei are almost exclusively innervated by neurons
from LTA (Fig. 1-7).
Figure 1-7. Brain areas receiving a prominent noradrenergic innervation. Most brain regions
are innervated by neurons projecting from both the locus coeruleus and tegmental system.
(Adapted from Stanford, 2001).
Synthesis of dopamine, adrenaline and noradrenaline share a common pathway. The amino
acid l-tyrosine (precursor substrate) undergoes hydroxylation in the presence of tyrosine
hydroxylase (TH) to form L-dihydroxyphenylalanine (L-DOPA) followed by decarboxylation
by DOPA decarboxylase to form dopamine (Fig. 1-8). This occurs in the cytoplasm of
catecholamine-releasing neurons. Dopamine is transported to the storage vesicles where
dopamine -hydroxylase converts it to noradrenaline.
21
Figure 1-8. The synthetic pathway for noradrenaline
Noradrenaline neurons influence arousal behaviours such as sleep/wakefulness, depression
and anxiety (Stanford, 2001). The precise features of environmental stimuli that provoke
increased noradrenergic transmission are unclear. Previous studies showed neither ‘novelty’
nor ‘aversiveness’ of the stimulus alone is responsible (McQuade and Stanford, 2000).
Increased noradrenergic transmission in the brain mediates changes in selective attention.
Another concept is that noradrenergic transmission influence emotional impact of a given
stimulus. It is possible that the role and consequences of central noradrenergic transmission
depends on type or severity of stimulus or individual differences in neurobiological coding
22
behaviour. Overall, it is extremely unlikely that noradrenergic transmission is the sole factor
determining behavioural response to even simple emotional stimuli.
1.4.7 EFFECTS OF CATHINONE ON THE REWARD CIRCUIT OF THE BRAIN
Research on behavioural and cognitive function in human khat users has been focussed
mostly on observational and single-case studies, but little information has been posted in
literature on interventional studies (Hoffman and Al’Absi, 2010). The general understanding
on these findings is that the effects observed following khat consumption are generally of
central stimulation and include euphoria, excitation, anorexia, increased respiration,
hyperthermia, logorrhoea, analgesia and increased sensory stimulation (Nencini and Ahmed,
1989). Khat chewers believe that they reason more clearly and are more alert, although their
concentration and judgement of issues are objectively impaired (Pantelis et al., 1989). In view
of its potency and high lipid solubility (Hassan et al., 2007; Kalix and Braenden, 1985),
facilitating access into the central nervous system (Zelger et al., 1980), it can be assumed that
khat-induced psychostimulation is predominantly due to cathinone content of the leaves
(Kalix, 1990). This is substantiated by the brief stimulation after khat chewing (Kalix et al.,
1990), which is in agreement with the finding that cathinone is metabolized rapidly in the
body (Brenneisen and Geisshüsler, 1987). Cathine and norephedrine possess weaker central
stimulant properties since they are less lipophilic (Nencini and Ahmed, 1989).
There is enough evidence in existing literature suggesting that khat and cathinone induce
psychostimulation via meso-striato-corticolimbic dopaminergic pathway (Kalix, 1990). The
dependence-producing potential, analgesia and anorexic effects of khat and cathinone are
23
believed to be partly mediated via this pathway (Gosnell et al., 1996). Hypothalamohypophyseo-adrenocortical axis has also been demonstrated to be susceptible to drug abuse
via dopaminergic transmission (Steckler and Holsboer, 1999; Sillaber et al., 1988). Activation
of the hypothalamo-hypophyseo-adrenocortical axis and ascending catecholaminergic neurons
play a critical role in metabolic and behavioural adaptation to various forms of stress
(Ishikawa et al., 2007). High levels of glucocorticoids have been shown to contribute to
development, maintenance and outcome of substance abuse disorders (Karlsgodt et al., 2003;
King et al., 2003). In another related study, it was shown that psychostimulants increase
corticosterone levels (Mello and Mendelson, 1997). On the other hand, suppression of
glucocorticoids by adrenalectomy reduces extracellular concentrations of dopamine in nucleus
accumbens in response to psychostimulants (Barrot et al., 2000; Piazza et al., 1996).
Together, these findings indicate a relationship between pleasurable effects of the drug (that
influences neural behaviours in drug ‘addicts’) with activation of stress system in the body.
In general, khat consumers show a range of experiences and manifestations from minor
reactions to development of psychoses. Some of these include: logorrhoea, hyperactivity,
irritability, loss of concentration, anxiety, dizziness, agitation and aggression. These reactions
occur after low to moderate exposure to khat (Cox and Rampes, 2003; Griffiths et al., 1997).
A number of studies have reported on psychiatric disorders with features of manic-like
psychosis following khat use (Gough and Cookson, 1984; Giannini and Castellani, 1982),
schizophreniform psychosis (Yousef et al., 1995; McLaren, 1987; Luqman and Danowski,
1976), paranoid psychosis (Nielen et al., 2004; Alem and Shibre, 1997; Critchlow and Seifert,
1987) and depression (Pantelis et al., 1989).
24
1.4.8 EFFECTS OF KHAT, CATHINONE AND OTHER PSYCHOSTIMULANTS ON
CARDIOVASCULAR SYSTEM AND BODY TEMPERATURE
Several studies on khat effects in humans have shown elevated blood pressure and increase in
heart rate (Hassan et al., 2005; Hassan et al., 2000; Gugelmann et al., 1985). It was postulated
that this effect was mediated by beta adrenergic receptors (Hassan et al., 2005). Increase in
blood pressure, heart rate and cardiac contractile force in anaesthetized dogs (Kohli and
Goldberg, 1982) and positive ionotropic and chronotropic actions in isolated atria
(Gugelmann et al., 1985) were reported after the administration of the active ingredient, (-)cathinone. Incidences of acute myocardial infarction among regular khat chewers in Yemen
have recently been reported (Al-Motarreb et al., 2005). Findings on khat-induced coronary
spasm (Al-Motarreb et al., 2004) were supported by the amphetamine, cocaine and 3, 4methylenedioxymethamphetamine (MDMA), which is structurally and functionally similar to
cathinone and both are believed to increase risk of acute coronary syndrome (Ragland et al.,
1993).
Khat has been shown to cause central nervous system stimulation, presumably as a
consequence of the sympathomimetic effects of (-)-cathinone (Balint and Balint, 1994).
Central thermoregulatory disturbances from sympathomimetics may arise from complex
interactions between dopamine and 5-HT in the brain stem and hypothalamus (Callaway and
Clark, 1994; Yamawaki et al., 1983; Ricuarte et al., 1980). There are several reports linking
khat to increase in body temperature (Kalix, 1991; Luqman and Danowski, 1976). Previous
studies on rats have shown that a non-selective dopamine receptor agonist, such as
apomorphine, when given in the presence of a D2 dopamine receptor antagonist, such as
haloperidol, can elicit an effect on 5-HT receptors resulting in hyperthermia (Yamawaki et al.,
25
1983). Hyperthermia also commonly follows increased heat production from physical
activity. It has been shown by earlier investigators that khat at high doses causes, among other
effects, hyperactivity (Kalix, 1991; Pantelis et al., 1989), which may cause changes in core
body temperature. Both lack of central dopaminergic activity and serotonergic hyperstimulation have been associated with altered hypothalamic thermoregulatory control as well
as abnormal central sympathetic and motor activity that may influence peripheral body heat
production and dissipation (Parada et al., 1995; Schwartz et al., 1995; Nimmo et al., 1993;
Lee et al., 1985). Dopamine injection into the preoptic nuclei of anterior hypothalamus
decreases core body temperature in animal models, suggesting that decreased dopaminergic
activity at this level may precipitate hyperthermia (Yamawaki et al., 1983). An earlier study
investigating the role of (-)-cathinone in the brown fat tissue thermogenesis (uncoupling
oxidative phosphorylation in brown fat tissue cells) obtained evidence suggesting that betaadrenergic receptors might be involved in the responses obtained (Tariq et al., 1989).
Another proposed mechanism of sympathomimetic-induced hyperthermia suggests that these
agents may initiate neuronal damage and increase hypothalamic concentrations of interleukin1 beta commonly known as the cytokine of fever (Albers and Sonsall, 1995). Peripherally,
sympathomimetics such as 3, 4-methylenedioxyamphetamine have been shown to raise
metabolism by up to 118% while elevating core body temperature (Gordon et al., 1991).
Seizures, hyperkinetic muscle action and motor excitability may also contribute to a rise in
core body temperature.
Khat contains sympathomimetic compounds that cause vasoconstriction of peripheral vessels
in the rabbit (Kalix, 1991) and cardiac effects (Balint and Balint, 1994) thereby impairing heat
26
dissipation. Although the exact mechanism remains unknown, clinical data and animal studies
indicate that ambient temperature, motor activity, metabolic regulation, autonomic vascular
changes and central disturbances in thermoregulation all contribute to profound elevations in
core body temperature.
1.4.9 CORRELATION OF BEHAVIOUR AND PERIPHERAL BIOCHEMICAL
MARKERS OF MONOAMINERGIC ACTIVITY.
Biological correlates of human behaviour that rely heavily on measurements of various
parameters in peripheral tissues have been used in biomedical research to reflect some aspect
of central nervous system function. Since sympathomimetic drugs produce their effects by
increasing synaptic levels of biogenic amines: dopamine, noradrenaline and serotonin,
through multiple mechanisms (Fischman and Madras, 2005), the concentrations of these
amines can be used to correlate specific behaviours manifested by an experimental animal
model or in controlled clinical trials. Some of the peripheral variables that have been
suggested to measure aspects of central serotonergic function are blood serotonin (Rao et al.,
1998), plasma free tryptophan (Møller et al., 1980), and total plasma tryptophan (Fernstrom et
al., 1976). Plasma free and total plasma tryptophan have been shown to correlate positively
with some neural behaviours (approach, heterogroom and eat, and inversely with ‘avoid’ and
‘be solitary’) in studies done in the vervet monkeys. Whole blood serotonin correlated
inversely with “avoid” and “be solitary” (Raleigh et al., 1981). In these findings, it was
suggested that in normal, drug naive monkeys, plasma free and total plasma tryptophan are
better correlates of the central serotonergic activity influencing behaviour than is whole blood
serotonin.
27
Studies have shown that the biological correlates of central noradrenergic system activity
measured in cerebrospinal fluid/blood increased in relation to enhanced pharmacological
activity of Locus coeruleus (LC)- a noradrenergic locus (Stanford, 2001). These measures
included activity of tyrosine hydroxylase in the LC and noradrenaline release in LC projection
areas. An earlier study conducted in patients with psychiatric disorders demonstrated that an
increase in peripheral norepinephrine results from activation of sympathetic nervous system
and reflects, rather than causes anxiety (Starkman et al., 1990). Further investigations on
amphetamine have shown that plasma levels of glucocorticoids and adrenocorticotrophic
hormone (ACTH) are increased by its acute administration in rodents and humans (Smith et
al., 2004; Swerdlow et al., 1993; Jacobs et al., 1989; Halbreich et al., 1981).
1.5.0 EFFECTS OF KHAT, CATHINONE AND OTHER PSYCHOSTIMULANTS ON
HYPOTHALAMO-HYPOPHYSEAL-ADRENOCORTICAL AXIS
Hypothalamo-hypophyseo-adrenocortical axis and pre-striatal dopamine (mesolimbic) system
may be susceptible to drug abuse, since most of these drugs have an affinity for dopaminergic
transmission (Steckler and Holsboer, 1999). Activation of the hypothalamo-hypophyseoadrenocortical axis and ascending catecholaminergic neurons play a critical role in metabolic
and behavioural adaptation to various forms of stress (Ishikawa et al., 2007). Discrepancies
on
findings
regarding
effects
of
psychostimulants
on
hypothalamo-hypophyseal-
adrenocortical axis have been observed. Preliminary studies showed that psychostimulants
increase corticosterone levels (Mello and Mendelson, 1997) and these increased
glucocorticoids contribute to substance abuse disorders (Karlsgodt et al., 2003; King et al.,
2003). On the other hand, suppression of glucocorticoids reduces extracellular concentrations
of dopamine in nucleus accumbens in response to psychostimulants (Barrot et al., 2000;
28
Piazza et al., 1996). Studies on khat have demonstrated increases in adrenocorticotrophic
hormone in humans (Nencini et al., 1984) and plasma cortisol in rabbits (Nyongesa et al., 2008).
However, in baboons, khat decreased plasma cortisol and prolactin (Mwenda et al., 2006).
Glucocorticoids are important mediators of the relationship between stress and drug-seeking
behaviour in rodents (Marinelli and Piazza, 2002; Piazza and Le Moal, 1998). High levels of
glucocorticoids have also been shown to increase rates of response to psychostimulant drugs
(Goeders and Guerin, 1996 b; Piazza et al., 1991) though mechanism modulating the rise in
plasma cortisol remains speculative. This is supported by the finding that adrenolectomy
attenuate psychostimulant self-administration (Deroche et al., 1997; Goeders and Guerin,
1996 a) and reduction in the amount of alcohol intake in alcohol-preferring rats (Fahlke et al.,
1994).
In several other investigations designed to examine the relationship between cortisol levels,
mesolimbic dopamine release and subjective drug response to amphetamine in healthy
volunteers, increase in cortisol levels was associated with greater amphetamine-induced
dopamine release in left ventral striatum (LVS), left dorsal putamen (LDP) and right dorsal
putamen (RDP) (Oswald et al., 2005). These findings paralleled those implicating interaction
of stress with alteration of mesocorticolimbic dopamine neurotransmission (Marinelli and
Piazza, 2002). Glucocorticoids are not only activated by stress but their secretion also
precedes many goal-seeking behaviours such as food-seeking (Bassareo and Di Chiara, 1999;
Taber and Fibiger, 1997; Wilson et al., 1995; Salamone et al., 1994; Westerink et al., 1994)
and drug-seeking behaviour (Cadoni et al., 2003; Marinelli and Piazza, 2002).
29
There is also growing evidence demonstrating interaction of stress with serotonergic systems.
Functional interactions of corticotrophin-releasing hormone (CRH) with serotonergic neurons
in the midline raphe have been shown in experimental animals (Linthorst et al., 1997).
Findings have also demonstrated a precise overlap of processes containing CRH and serotonin
in the dorso-lateral sub-nucleus (Ruggiero et al., 1999). Corticotropin-releasing hormone
subtype 2 receptors were expressed in the brainstem raphe indicating a neuro-peptidergic
interaction with serotonin (Chalmers et al., 1995). An identical locus is enriched in CRH
receptors in the macaque (Millan et al., 1986) and interconnects with limbic and visceral
reflex centres (Fulwiler and Saper, 1984).
1.5.1 HYPOTHALAMO-HYPOPHYSEAL-GONADAL AXIS IN THE MALE
Of the three components of the male reproductive system, the hypothalamus and the anterior
pituitary gland have solely regulatory functions, which are mediated by the hormones secreted
from these two organs. The third component, the testes, also produces key hormones
controlling male sexual characteristics and behaviours, the most important of which is
testosterone. In addition, the testes are responsible for sperm production.
The hypothalamus, which is located at the base of the brain, is often called the master control
unit of the reproductive systems of both men and women. Among other hormones, the
hypothalamus produces gonadotropin releasing hormone (GnRH) which is secreted in pulses
into a system of blood vessels that connect the hypothalamus to the anterior pituitary gland
located just beneath the hypothalamus.
30
In response to the GnRH stimulus, the anterior pituitary gland produces two hormones that
control reproductive functions: luteinizing hormone (LH) and follicle-stimulating hormone
(FSH) and releases them into the general circulation. These two hormones have different
functions in males and females. In the male, LH stimulates the testes to produce testosterone,
whereas FSH plays an important role in sperm maturation. In addition, the anterior pituitary
gland produces prolactin. In the male, elevated prolactin levels play a role in reproduction by
indirectly suppressing testosterone levels in the body.
The testes consist primarily of the seminiferous tubules, the site of sperm cell formation and
maturation. Interspersed among those tubules are interstitial cells, including Leydig cells,
which produce testosterone. In addition to its function in reproduction, testosterone helps
regulate many diverse body functions, including bone and muscle development, red blood cell
turnover, and development and maintenance of male secondary sexual characteristics, such as
sexual drive, growth of facial and body hair, and deepening of the voice during puberty in
boys. Consequently, an insult to the hormonal system controlling testosterone production can
result not only in infertility but also in other deleterious consequences, such as accelerated
bone loss, decreased muscle function, and lower than normal numbers of red blood cells.
Another important testicular cell type is the Sertoli cell. These cells play a critical role in
sperm development by supporting and nourishing the germ cells during their development.
The three components of the male reproductive system: the hypothalamus, anterior pituitary,
and testes form a finely tuned system called the hypothalamo-pituitary-gonadal (HPG) axis,
which is controlled through a classic negative feedback mechanism. As testosterone levels in
the blood rise, the anterior pituitary becomes less responsive to stimulation by GnRH,
31
resulting in reduced LH and FSH secretion. Because LH induces testosterone production,
reduced LH secretion results in lowered testosterone levels. Conversely, if testosterone levels
in the blood decline, such as during injury to the testes, the anterior pituitary’s responsiveness
to GnRH increases and more LH and FSH are secreted, thereby promoting testosterone
production by Leydig cells. Following a classical negative feedback loop high testosterone
levels inhibit further secretion of GnRH.
In addition to this feedback loop, several hormones produced within and outside the testes
regulate testosterone production. One such hormone is β-endorphin, a molecule similar to
morphine produced within the testis. It suppresses testicular testosterone production and/or
release. Furthermore, β-endorphin released in the hypothalamus results in decreased GnRH
levels. Lower GnRH levels, in turn, lead to reduced LH and FSH secretion from the anterior
pituitary and reduced testosterone production by the Leydig cells. (More information on the
hypothalamo-pituitary-gondal axis function is found in article by Hiller-Sturmhöfel and
Bartke, 1998 pp. 153–164).
1.5.2 KHAT AND CATHINONE AND THE HYPOTHALAMO-HYPOPHYSEALGONADAL AXIS
Numerous and contradictory findings have been reported about khat and (-)-cathinone on
reproductive function. These reports are all convincingly supported by enough evidence,
leading to a dilemma as to whether or not khat is a boon or bane to humanity. Studies reported
khat as an aphrodisiac (for review see Feyissa and Kelly, 2008; Bentur et al., 2008), with its
cathine and norephedrine alkaloids being shown to stimulate the final stages of sperm
maturation and inhibition of acrosomal loss (Adeoya-Osiguwa and Fraser, 2005). Similar
32
findings showed that rabbits fed on freeze-dried leaves of khat for three months had an
increased rate of spermatogenesis (Al Mamary et al., 2002). Furthermore, cathine and
caffeine were shown to increase sexual motivation when given separately or in combination
(Taha et al., 1995). Similarly, studies in olive baboons showed enhanced plasma testosterone
and decreased prolactin and cortisol levels following khat treatment (Mwenda et al., 2006).
In a World Health Organization bulletin, reports of khat on impairment of sexuality, inability
to sustain erection, loss of libido and spermatorrhoea (WHO, 1980), consistent with those of
Pantelis et al. (1989) have been highlighted. Other studies reported a significant increase in
number of abnormal sperm cells following (-)-cathinone exposure in mice (Qureshi et al.,
1988), rats (Islam et al., 1990) and accompanying degenerative changes of testicular tissue
with decrease in plasma testosterone levels in rats (Islam et al., 1990). Mutagenicity of germ
cells in albino mice has also been demonstrated following (-)-cathinone treatment (Tariq et
al., 1990). Studies in humans showed that long-term khat consumption caused deleterious
effects on sperm parameters and steroidogenesis (El-Shoura et al., 1995). The biggest
question now is; which way forward? On the whole, however, a greater percentage of the
studies show solid proof that khat impairs rather than boosts sexuality.
There are about four possible mechanisms through which khat and (-)-cathinone can influence
plasma testosterone levels. Through:

Inhibition of release of luteinizing hormone-releasing hormone from the hypothalamus
resulting in a series of secondary effects on the pituitary and testes.

Blockage of the biosynthesis of the sex steroid hormones at the level of the testes.
33

Suppression of the synthesis and/or release of LH from the pituitary and thereby
inhibit LH-dependent testicular steroidogenesis.

Enhancement of metabolism of testosterone by the liver
The present study considered the first three hypotheses: that is, determination of the integrity
of the hypothalamus, reproductive hormone profiles and examination of the sex steroid
hormone biosynthetic pathway and testicular ultrastructure.
1.5.3 SEX STEROID HORMONE METABOLISM
Steroid hormones are derived from acetate with cholesterol as an intermediate product. It is
well known that smooth endoplasmic reticulum (SER) is involved in endogenous synthesis of
cholesterol from acetate and glucose (Christensen, 1975) and that cholesterol is the main
product stored in lipid droplets (Moses et al., 1969). Cholesterol is metabolized into
pregnenolone, which in the presence of 17 α- hydroxylase and 3 β- hydroxysteroid
dehydrogenase (3 β-HSD) can be converted into 17 α- hydroxypregnenolone and
progesterone, respectively. The entire biosynthetic pathway gives rise to four major classes of
steroid hormones: the progestagens, estrogens, androgens and corticosteroids. Progestagens
lead to formation of androstenedione, which is converted into testosterone in the presence of
17 β –hydroxysteroid dehydrogenase (17 β-HSD). In the presence of 17, 20 desmolase, 17 αhydroxypregnenolone is converted into dehydroepiandrosterone, which is in turn converted
into androstenediol in the presence of 17 β-HSD. Androstenediol is then converted into
testosterone under the influence of 3 β-HSD. Steroid hormones are synthesized by gonads,
adrenal gland, ovaries and placenta. The figure below shows the role of different enzymes in
the synthesis of steroid hormones in gonads and adrenal cortex (Fig. 1-9).
34
Figure 1-9. Biosynthesis of steroid hormones in adrenal cortex and gonads. Individual
enzymes are highlighted and showing different isoforms of cytochrome P450s (CYP) and
hydroxysteroid dehydrogenases (HSD). (Adapted from Paynes and Hales, 2004).
Hydroxysteroid dehydrogenases (HSDs) play an important role in the biosynthesis and
inactivation of all steroid hormones. They exist in multiple isoforms, which show tissue
specificity in expression and this, coupled with properties of each isoform (reductase or
dehydrogenase), can determine the role of enzyme in steroid hormone action. In testis, 3βHSD and17 β-HSD play an essential role in testicular steroidogensis (Andersson et al., 1995).
Several isozymes of 17 β-HSD have been identified in human steroidogenic tissues. The
isozymes type I, II and III of 17 β-HSD (Andersson et al., 1995) and type IV (Adamski et al.,
1995) are found in microsomes of testis where they reduce androstenedione to testosterone.
35
1.5.4 VERVET MONKEY BIOLOGY AND USE IN BIOMEDICAL RESEARCH
1.5.4.1 Reproduction
Vervets are seasonal breeders. Among captive vervets in Kenya, the mating season lasts from
July to September and births are grouped between November and January (Eley et al., 1986).
In wild African populations, the breeding season lasts from April to June and the majority of
births are concentrated from October to December (Baldellou and Adan, 1997). In the West
Indies, births occur throughout the year but most are concentrated from April to July
(Horrocks, 1986). Seasonal breeding in vervet monkeys evolved due to the need to reproduce
during times of food abundance. After the rainy season, food resources are much more
plentiful compared to other times of the year. Females nursing their young ones, together with
their young ones are not as likely to be nutritionally stressed during this period, thereby
increasing their chances of survival (Baldellou and Adan, 1997).
In the wild, females attain sexual maturity at about four years of age and give birth at around
five years old (Cheney et al., 1988). However, in captivity vervets are not affected by
seasonal pattern of breeding but rather readily reproduce throughout the year (Seier, 2005).
Here, females mature fairly fast such that they can give birth at the age of two years
(Fairbanks and McGuire, 1985). Female vervets do not exhibit external signs of oestrus
(Cheney and Seyfarth, 1990; Eley et al., 1989). In captivity the ovarian cycle lasts about 32.5
days, and is characterized by menstruation. Peak sexual receptivity occurs around the 13th day
of the cycle (Eley et al., 1989; Else et al., 1986). Female vervets undergo ovarian cycles
throughout the year, but the cycles are irregular during non-breeding seasons (Else et al.,
1986). Males reach sexual maturity at around five years of age, but do not achieve full adult
weight until after six years of age. This seems to limit the opportunity for mating until then.
36
The gestation period in vervets is between 163 to 165 days (Andelman, 1987; Eley et al.,
1986) while the inter-birth interval varies from 1 to 2 years, depending on presence or absence
of a surviving offspring of the previous breeding season. In the West Indies, the inter-birth
interval is slightly less than 1 year (Horrocks, 1986).
1.5.4.2 Use in neuro-behavioural and reproductive health research
The vervet monkey (Chlorocebus aethiops, formerly Cercopithecus aethiops) has long been
among the most important non-human primate (NHP) models for biomedical research, other
than the rhesus macaque and much greater than that of any other NHP (Baulu et al., 2002).
The dramatic recent growth in the use of vervet as a model derives, in part, from its
recognized utility as an old world monkey alternative to the rhesus macaque, of which there is
now a critical shortage for biomedical research. In addition, Human Immunodeficiency Virus
(HIV) research also has an increasing focus on vervets because, in contrast to macaques, they
are readily infected with simian immunodeficiency virus (SIV) but do not progress to disease.
Vervet as an alternative to rhesus:
The vervet is similar to rhesus in behaviour and physiology (Disotell, 2000; Ziegler, 1990)
and is about equally closely related to humans (Raaum et al., 2005; Page, 2001). At the same
time it is more readily accessible, less expensive, and may be accompanied by fewer health
and safety risks (Gordon et al., 2005).
Investigation of neurobehavioural phenotypes:
Neuro-behavioural phenotypes form a central focus of vervet monkey investigation.
Behavioural observations over several decades have identified heritable traits relating to
aggression, maternal-infant interactions, anxiety, and novelty seeking and impulsivity
37
(Fairbanks et al., 2004; Fairbanks et al., 2001b; Fairbanks, 2001a; Fairbanks et al., 1999).
More recently, paradigms have been established that demonstrate the enormous value of
NHPs as models intermediate between humans and rodents for elucidating cognitive
processes such as working memory and response inhibition that are relevant to human
diseases such as schizophrenia and attention deficit disorder respectively (James et al., 2007).
Investigation of the immune system and infectious disease:
The vervet is growing in importance as a model species for acquired immunodeficient
syndrome (AIDS) research. African populations of the vervet are natural carriers of SIV and
unlike Asian macaques, do not show signs of illness when infected. Caribbean-derived
vervets are free of SIV and thus represent an excellent model for experimental study of the
immune response on initial infection. Several laboratories are using this model to understand
how the immune system prevents disease progression, with potential implications for the care
and prevention of HIV-related diseases in humans (Goldstein et al., 2006).
38
CHAPTER 2
2.0 GENERAL MATERIALS AND METHODS
2.1 ANIMALS AND HOUSING
Fourteen adult vervet monkeys were captured in the wild and put under quarantine at the
Institute of Primate Research (IPR) for three months during which time they were tuberculintested. The animals were housed in individual standard monkey cages (0.6 m x 0.66 m x 0.8 m)
for the duration of the study. Daily supply of monkey chow, vegetables, fruits and commercial
multivitamins was given around 0900 h, while water was provided ad libitum. Lighting
conditions with approximately 12 h: 12 h (light: dark cycle) and an average room temperature
of 23 0C with a relative humidity of approximately 60% was provided in the animal house.
Cage cleaning and regular change of beddings was also observed. Complete animal health care
and supervision was provided to the animals throughout course of study and only healthy
animals were selected for investigation. All study procedures followed accepted veterinary
protocols for analgesia and anaesthesia that were approved by the Institutional Review
Committee (IRC) at IPR. These protocols were guided by International Guiding Principles for
Biomedical Research involving animals, and developed by the Council for International
Organizations of Medical Sciences.
2.2 CHEMICALS
Goserelin acetate (Zoladex)- a Luteinizing Hormone Releasing Hormone (Sigma Ltd St. Louis,
MO, USA), immunohistochemistry antibodies of LH, prolactin and Adrenocorticotrophic
39
hormone (DakoCytomation, Denmark), Luteinizing hormone, androstenedione, progesterone,
cortisol, prolactin and testosterone enzyme immunoassay kits. Polymerase chain reaction
reagents included: ZR Tissue & Insect RNA Microprep Kit, RevertAid Premium First Strand
cDNA Synthetic Kit, DreamTaq DNA Polymerase, dNTP Mix, TopVision LE GQ Agarose, GR
Green Nucleic Acid Stain, Maxima Probe/ROX qPCR Master Mix ((Nova Tec
Immundiagnostica GMBH, Germany). Khat samples were obtained from Meru district in
Kenya.
2.3 EXTRACTION OF CATHINONE FROM KHAT
Fresh leaves and shoots of khat (ghiza variety) were weighed (500 g) and crushed with mortar
and pestle into very small pieces (< 5 mm) and dissolved in 250 ml methanol in a conical
flask. The extraction protocol followed that previously described by Lee (1995). In brief, the
mixture was shielded from light and sonicated at room temperature for 15 min with
intermittent shaking and stirring followed by filtration through cotton gauze and then grade 1
Whatman filter paper to get rid of fine particles. The non-filtered plant material was reextracted in 250 ml fresh methanol and sonicated for 24 h. The mixture was then filtered and
admixed with initial methanol-extracted khat material and condensed to near dryness (< 1ml)
using a stream of air. A very dilute solution of sulphuric acid (approximately 0.02 N) was
used to re-suspend and acidify the residue. This was followed by chloroform extraction to
remove neutral organic compounds as well as the remaining plant solids. A small amount of
saturated sodium bicarbonate solution was added to the aqueous solution to basify the extracts
followed by methylene chloride to extract cathinone and cathine. A stream of air was again
used to reduce the extract to a minimal amount. The resultant solution was then vacuum-dried
40
at 337 millibar in a Rotar Vump for 4 to 5 hours into an oily paste. The dry weight of the
extracts was determined and thin layer chromatography used to confirm the presence of
alkaloids. The plant extracts were spotted directly onto a pre-coated 5 by 10 cm silica gel 60
plate. The plate was then developed in ethyl acetate: methanol: aqueous ammonia (85:10:5),
and viewed under an ultraviolet lamp. The spots were visualized using a 0.5% ninhydrin
solution, and the plate heated using a heat gun. Colour development was used to localize the
alkaloids (purple for cathine and burnt orange for cathinone) among the moving spots. The Rf
values for cathinone and cathine were also calculated. The vapour phase infrared spectra was
obtained on a Hewlett-Packard Model 5890 Series II Gas Chromatograph, using a 12 m by
0.32 mm HP-5 (0.52 µm loading) capillary column and a temperature program of 70 0C for 1
min, 15 0C/min to 270 0C, with a final temperature hold for 5 min, equipped with a HewlettPackard Model 5965B Infrared Detector. About 300 g of total cathinone was extracted and it
was approximately 97% pure.
2.4 EXPERIMENTAL DESIGN
The fourteen adult vervet monkeys (eight males and six females) were randomly assigned to
five groups. Group I animals were controls and comprised of two animals while group II, III,
IV and V were test animals and comprised of three animals each. The animal identification by
group and sex was as follows: GI (2148_1 [male], 2149_1 [female], GII (2107_1, 2107_2
[males], 2107_3 [female]), GIII (2169_1, [male], 2169_3, 2169_4 [females]), GIV (2158_1,
2158_2 [males], 2158_3 [female]) and GV (2155_1, 2155_2 [males], 2155_3 [female]).
Group III had one male and two females while control group had one male and one female.
Group II, III, IV and V animals were administered (-)-cathinone at 0.8 mg/kg, 1.6 mg/kg, 3.2
41
mg/kg and 6.4 mg/kg body weight, respectively via intragastric tube. Controls were
administered normal saline. The study was carried out in two phases: Pre-treatment and
treatment phases.
2.4.1 IN VIVO STUDIES
2.4.1.1 Pre-treatment phase (40 days)
All animals were habituated to presence of observers and handling for 4 weeks before
commencement of experiments constituting pre-treatment phase (1 month). Caution was taken to
avoid stress to the animals. A detailed description of protocol on cage enrichment and animal
acclimatization to presence of observer is given in chapter 3 of this thesis.
The femoral vein of each animal was cannulated using a 22 G blood vessel cannula after
shaving and swabbing the area with 70% alcohol. Heparinised saline was introduced into the
cannula to prevent blood clotting inside the lumen. The adhesive tape was used to anchor the
cannula in situ to prevent it from dropping. Blood samples (3 ml) were then collected from the
femoral vein at 5-days interval for cortisol, prolactin, luteinizing hormone, progesterone and
testosterone analysis.
2.4.1.2 Treatment phase (4 months)
Control animals were administered 10 ml normal saline (0.9% sodium chloride) via oral gavage
three times a week (Mondays, Wednesdays and Fridays) for 4 months (Fig. 3-1). Control and test
animals were anaesthetized with ketamine hydrochloride intramuscularly (initial dose of 10
42
mg/kg body weight followed by 2.5 mg/kg body weight at 30 min intervals) at 0930 h before
administration of normal saline and (-)-cathinone at 1000 h, respectively. The dose of ketamine
used was not enough to induce narcosis but only sufficient to immobilize the animals. Test
animals, grouped into four groups were treated with respective doses (0.8, 1.6, 3.2 and 6.4 mg/kg
body weight) of (-)-cathinone via oral gavage three times a week (Monday, Wednesday and
Friday) for 4 months. The doses were chosen based on previous studies in humans (Kalix, 1984
b) and rats (Mohammed and Engidawork, 2011) which indicated optimum effect of (-)-cathinone
on various body parameters to be within this dose range. Specific experimental protocols
following treatment are discussed in the following chapters.
43
CHAPTER 3
ACUTE AND SUB-CHRONIC EFFECTS OF PURIFIED
CATHINONE
FROM
KHAT
BEHAVIOUR
PROFILES
IN
(CATHA
EDULIS)
VERVET
ON
MONKEYS
(CHLOROCEBUS AETHIOPS)
3.0 INTRODUCTION
Khat is customarily consumed by millions of inhabitants in the south-west part of Arabian
Peninsula and East African countries between Sudan and Madagascar namely: Djibouti,
Ethiopia, Somalia, Kenya, Tanzania and Uganda. Fresh leaves and shoots are chewed for their
euphoriant and stimulatory effects (Al-Bekairi et al., 1991). Cathinone is the principal
ingredient of khat, and is found naturally as (-)- enantiomer. The (+)- enantiomer is not found
(Kalix and Braenden, 1985). The (-)-cathinone resembles (+)-amphetamine in chemical
structure and biological activity (Zelger et al., 1980).
(-)-Cathinone is a lipophilic alkaloid and readily crosses the blood-brain barrier to reach the
primary sites of action in the central nervous system (Cox and Rampes, 2003). Although
behavioural consequences associated with khat and cathinone exposure in humans and
experimental animals have been extensively documented, the corresponding magnitude of
these psychological behavioural changes remain unclear due to the paucity of objective and,
44
therefore, relevant human data. The framework for postulating khat/cathinone associated
behavioural changes in sleep patterns, mood, attention, aggression, anxiety, locomotor
activity, and affiliative behaviour has been extrapolated from results of khat studies
predominantly in mice (Kimani and Nyongesa, 2008), rats (Banjaw et al., 2005; Kalix et al.,
1995; Islam et al., 1994), monkeys (Schuster and Johanson, 1979) and humans (Pantelis et al.,
1989). For example, long-term khat chewing has been shown to induce a depressive mood,
irritability, anorexia and insomnia (Al-Motarreb et al., 2002; Nencini et al., 1989). Reports on
studies on khat and cathinone in non-human primates are scarce. Earlier studies lack clarity on
behavioural observation over an extended period of time. There is also scanty literature on
effects of khat and cathinone on appetite. Most available literature is on rodents where khat
reduced food consumption, maternal weight gain and lowered food efficiency index in
pregnant rats (Islam et al., 1994).
In this study, we administered (-)-cathinone to adult male and female vervet monkeys housed
in single cages. Individual caging was considered since it allowed for individual behavioural
assessment independent of influence from other mates. Such measurements are highly
relevant for behavioural models of human psychological conditions and since the study was
designed to determine the effect of (-)-cathinone doses in presence/absence of cage
enrichment, the primary consideration was individual observations and whether or not cage
enrichment had any effect on specific behaviours. Cage enrichment is regarded as a reward
since animals are conditioned to differentiate between a reward and a no reward situation. In
the present study, cage enrichment aimed at determining whether behavioural changes
observed could be altered by a reward or was due to a reward. Behavioural studies on khat
and (-)-cathinone in primates is scarce. The available literature highlights on subjective effects
45
of khat in humans such as induction of mood changes, irritability, anorexia and insomnia
following long-term khat use (Al-Motarreb et al., 2002; Nencini et al., 1989). Similarly,
earlier studies do not indicate clarity on experimental design and behavioural observations
over an extended period of time. This has brought about generalization of characterizations
for interpreting (-)-cathinone effects in human khat consumers hence conflicting reports.
There is also scanty literature on effects of khat and (-)-cathinone on consummatory
behaviour in humans and non-human primates. Most available literature is on appetitive
behaviour in rats (Eisenberg et al., 1987; Foltin et al., 1983), guinea pigs (Jansson et al.,
1988) and humans (Murray et al., 2008; Kalix and Braenden, 1985). Appetitive behaviour
basically concerns ability or failure to seek food while consummatory behaviour refers to
eating according to the Pavlovian homeostatic model.
Khat use is a human habit; it is not consumed by other animals. However, controlled and
invasive studies in humans are limited by ethical constraints. This study aimed at determining
effects of acute and sub-chronic exposure to (-)-cathinone on specific types of behaviour in
presence and absence of cage enrichment. It was hypothesized that long-term administration
of (-)-cathinone, and cage enrichment influence behavioural alterations regardless of (-)cathinone dose. Vervet monkeys were used since they are a good model for investigating
neuro-behavioural phenotypes relating to aggression, maternal-infant interactions, novelty
seeking and impulsivity (Fairbanks et al., 2004; Fairbanks et al., 2001 b; Fairbanks et al.,
2001 a; Fairbanks et al., 1999) as well as elucidation of cognitive process (James et al., 2007).
46
3.1 MATERIALS AND METHODS
3.1.1 ANIMALS AND HOUSING
This is the same as was described previously in section 2.1 of general materials and methods.
3.1.2 ANIMAL HANDLING AND EXPERIMENTAL DESIGN
This is same as was described previously in section 2.4 of general materials and methods.
Briefly, a total of fourteen adult vervet monkeys (eight males and six females) were randomly
divided into four groups of 3 test animals each and a fifth group with two animals (one male and
one female) serving as controls. Group III had one male and two females while other test groups
had two males and one female. The summary is indicated in Table 3-1 below. The study was
carried out in two phases:
3.1.2.1 Pre-treatment phase
Pre-treatment period comprised of 2 months. However, the first month served to habituate the
animals to the presence of the observer as well as handling while during the second month of
pre-treatment phase, blood sampling together with individual behavioural observations were
done. Caution was taken to avoid stress to the animals.
Environmental (cage) enrichment during pre-treatment phase
Experimental animals did not have prior experience with the plastic toys used for cage
enrichment (Fig. 3-1). Cage enrichment was achieved with plastic toys that were hollow and
bright- coloured according to the protocol of Bayne (1989). Briefly, focal subjects were
observed on 3 days of each week: Monday, Wednesday and Friday. Day 1: no toys (control)
47
observed for 1 h; Day 2: first presentation of toy and observed for 1 h; Day 3: presentation of
same toy for 20 min followed by filling with bananas or carrots and removal of toy without
any fooe enrichment. Three observers standing at strategic positions in full view of focal
subjects made the behavioural scoring. This was done during acclimatization for 4 weeks
followed by 1 month of pre-treatment phase. The aim of behavioural observations during pretreatment phase was to establish baseline values of specific behaviours of interest.
Figure 3-1. Cage enrichment with a coloured toy
3.1.2.2 Treatment phase
Treatment given to the animals is as was described in general materials and methods under
section 2.4.1.2.
48
i). Environmental (cage) enrichment during treatment phase
The protocol was similar as that employed during pre-treatment phase. Similar toys were used
but with holes made on the sides that could be filled with food on some days. Focal subjects
were observed on 3 days of each week: Monday, Wednesday and Friday as for pre-treatment.
ii). Behavioural studies
Behavioural definitions
Fourteen individual behavioural categories were condensed into 5 composite behaviour
scores: aggression, anxiety, abnormal behaviour, withdrawal and appetitive/consummatory
behaviour using a one-zero sampling technique (Melega et al., 2008; Martin and Bateson,
1993). The aggression composite score was defined by yawn, engage in stare, jaw thrust,
bouncing off cages, shaking cage walls and head jerk (Melega et al., 2008). The anxiety
composite score combined 3 (pacing, scratch and self-directed) behaviours that have been
validated as indicators of anxiety in non-human primates (Melega et al., 2008; Castles et al.,
1999; Aureli, 1997; Maestripieri et al., 1992). The description of abnormal behaviour
combined measures of “responses independent of appropriate stimuli”, “fine motor” and
“whole body stereotypy” (Castner and Goldman-Rakic, 1999). Withdrawal behaviour was
defined by duration of time taken by a focal subject on the platform or at the corner of the
cage while appetitive/consummatory behaviour was defined by the latency of time to respond
to food introduced in the cage (Rapkin et al., 1995). Table 3-2 gives a summary of
behavioural definitions that were followed.
Behavioural scoring
49
All behaviours were observed from 1100 h to 1300 h of each observation day. Animals were
administered (-)-cathinone via intra-gastric tube (Fig. 3-2) at 1000 h three times a week
(Monday, Wednesday and Friday) as described under general materials and methods and cage
enrichment followed the protocol described by Bayne (1989). Focal subjects were observed
for their interest with empty toys and toys with food. A video camera (Type-Image video
camera CAM) connected to a video camera recorder (VCP-C10, Toshiba PTE Ltd.,
Singapore) was placed at a strategic position within the animal house and three different
observers who had been habituated to the animals scored individual behavioural scores. Interrator reliability, which was obtained by dividing the number of times behaviour was scored by
the observers by the number of times each of the observers scored the behaviour, was 90%.
Behavioural observations were collected on all focal subjects. Behaviour was scored if it
occurred one or more times during a 3- min session. Three x -3 minute sessions were done for
each of the frequency behaviours (aggression, anxiety and abnormal behaviour) per
experimental day. For durational behaviours of withdrawal and appetitive/consummatory
behaviour, three x 8- min sessions were done per each observation day.
50
Table 3-1. Animal grouping by number, sex, age and treatment dose
Animal group No. of animals Sex
(-)-Cathinone dose (mg/kg) Age
I
2
1M, 1F 0
A
II
3
2M, 1F 0.8
A
III
3
1M, 2F 1.6
A
IV
3
2M, 1F 3.2
A
V
3
2M, 1F 6.4
A
Total
14
8M, 6F
51
Table 3-2. Summary of composite behaviour definitions. [Adapted from Melega et al.
(2008)].
COMPOSITE
BEHAVIOR
Frequency
Behaviours
Aggression
Anxiety
Abnormal
behavior
Durational
behaviors
Withdrawal
Eat
BEHAVIOURAL
CATEGORY
DESCRIPTION
Yawn
Engage in a stare
Jaw thrust
Bouncing off cage
walls
Vigorous shaking of
cage walls
Head jerk
Pacing
Yawning with teeth showing
Open mouth, ears back and eyelids up
Grinding teeth
Focal subjects jumping off cage walls within
cages
Withdrawn/isolated
Focal subject withdraws to a corner or platform
within the cage.
Time taken for the focal subject to place food in
the mouth, chew, forages through food or carries
food in mouth or drinks water. (Rapkin et al.,
1995).
Rapid sideways jerking of the head
Any repetitive, stereotypic locomotor behaviour
at least 3 times in succession. (Melega et al.,
2008).
Scratch
Focal subjects scratching themselves
Self-directed
Engaged in a brief touch to face or body, hand or
foot rubbing, licking hand, body, hair picking,
masturbation, shrug. (Melega et al., 2008).
Responses
Engaged in abnormal attention or orienting
independent of
responses made in absence of apparent stimulus
stimuli
e.g grasp at air, stare off into space (Castner and
Goldman-Rakic, 1999).
Fine motor stereotypy Engaged in abnormal focussed repetitive motor
responses directed towards objects or selfdirected e.g repetitive grasping at face, and
repetitive movements of hand or foot. (Melega et
al., 2008).
Whole body
Engaged in abnormal repetitive whole body
stereotypy
movements, including circling, side-to-side,
somersaulting- other than pacing. (Melega et al.,
2008).
Latency to food
response
52
Figure 3-2. Intra-gastric administration of (-)-cathinone to the vervet monkey.
53
3.1.3 STATISTICS
Behavioural data for pre-treatment and treatment phases were analysed jointly. The mean
scores for each dependent variable were calculated for each subject across each of the
independent variables when it was deemed applicable. The mean scores were then used as raw
data for all ANOVAs. All behavioural observations were done during morning hours of each
observational day. For each dependent behavioural measure (aggression, anxiety, abnormal
behaviour, withdrawal and appetite), a 5 treatment group (cathinone dose: 0.8, 1.6, 3.2 and 6.4
mg/kg body weight, and control) x 25 observational days univariate repeated measures ANOVA
was performed, using treatment group as a between-subject factor and observational day as
within-subject factor. The Huynh-Feldt correction was applied for those tests and Bonferroni
adjustments used for post hoc multiple comparisons. Statistical significance was set at 5%
level.
54
3.2 RESULTS
3.2.1 BEHAVIOURAL OBSERVATIONS
3.2.1.1 Behavioural scores of aggression
The results show a significant effect of (-)-cathinone on ‘yawn’, ‘bouncing off cage walls’ and
‘head jerk’ as indicators of aggression. The individual scores for ‘yawn’ were more
observable during absence of cage enrichment as indicated by a significant main effect of
days (F(16, 144) = 26.6, P < 0.001). Groups with (-)-cathinone treatment showed greater scores
than the control group as indicated by a significant main effect of group (F(4, 9) = 12.9, P <
0.01) on this measure. There was a significant group by day interaction ([F(64, 144) = 2.45, P <
0.001]; Fig. 3-3 a) for this score. The individual behavioural scores of ‘engage in a stare’ (F(16,
144)
= 10.2, P < 0.001; Fig. 3-3 b) and ‘jaw thrust’ (F(10,
94)
= 10.2, P < 0.001; Fig. 3-3 c)
differed over days. Changes in scores for ‘bouncing off cage walls’ differed by group as
indicated by a significant group by day interaction (F(68,
154)
= 1.74, P < 0.01; Fig. 3-3 d).
Changes in scores on ‘vigorous shaking of cage walls’ were moderated by (-)-cathinone
groups as indicated by a significant group by day effect (F(44, 101) = 1.58, P < 0.05; Fig. 3-3 e).
(-)-Cathinone treatment group showed greater ‘head jerk’ than the control animals (Group
effect: F(4, 9) = 48.0, P < 0.001), and patterns of changes in this score differed among groups
(group by day effect: F(42,
94)
= 2.18, P < 0.01; Fig. 3-3 f). This increase of individual
behavioural scores was more pronounced in absence of cage enrichment. During pretreatment period individual scores for ‘engage in a stare’ were observable in both presence
and absence of cage enrichment. However, there was a fixed order of observations during
cage enrichment, which could be regarded as a limitation of the study.
55
9
Aggression - yawn
8
Group x day: p <.001
Group: p < .01 (con<0.8=1.6=3.2=6.4)
day: p<.001
7
mean frequency
6
5
Control
0.8
4
1.6
3
3.2
6.4
2
1
0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-3 a. A significant increase in ‘yawn’ for group by day interactions (P < 0.001),
between subject groups and over days (P < 0.01) in (-)-cathinone subjects compared to
controls (n = 14). Note that the increase was more pronounced in absence of cage enrichment.
56
3.0
Aggression - engage in a stare
Group x day: not significant
Group: not significant
day: p<.001
2.5
mean frequency
2.0
Control
1.5
0.8
1.6
1.0
3.2
6.4
0.5
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-3 b. Individual scores for ‘engage in a stare’ in (-)-cathinone subjects did not differ
significantly from controls for group by day interactions and between subject groups but
significant (P < 0.001) over experimental period (n = 14).
57
5.0
Aggression - jaw thrust
4.5
Group x day: not significant
Group: not significant
day: p<.001
4.0
mean frequency
3.5
3.0
Control
2.5
0.8
2.0
1.6
1.5
3.2
6.4
1.0
0.5
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-3 c. Behavioural scores for ‘jaw thrust’. There was no significant difference for
group by day and between subject groups on this measure. However, the increase was
significant over experimental period (P < 0.001). Note that the effect was more pronounced in
absence of cage enrichment among (-)-cathinone subjects compared to controls (n = 14).
58
6.0
Aggression - bouncing off cage walls
Group x day: p<.01
Group: p=.07
day: p<.001
5.0
Control
mean frequency
4.0
0.8
1.6
3.2
3.0
6.4
Control
2.0
0.8
1.6
1.0
3.2
6.4
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-3 d. A significant increase in ‘bouncing off cage walls’ for group by day (P < 0.01),
between (-)-cathinone subject groups (P = 0.07) and over days (P < 0.001). The increase was
more pronounced during absence of cage enrichment in (-)-cathinone subjects compared to
controls (n = 14).
59
6.0
Aggression - vigorous shaking of cage walls
Group x day: p<.05
Group: p=.07
day: p<.001
5.0
mean frequency
4.0
Control
3.0
0.8
1.6
2.0
3.2
6.4
1.0
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-3 e. A significant increase in ‘vigorous shaking of cage walls’ for group by day (P <
0.05), group (P = 0.07) and days (P < 0.001) in (-)-cathinone subjects compared to controls (n
= 14). The behaviours were more observable during absence of cage enrichment.
60
5.0
x day: p<.01
Aggression - head jerk Group
Group: p<.001 (con<0.8=1.6=3.2=6.4)
4.5
day: p<.001
4.0
mean frequency
3.5
3.0
Control
2.5
0.8
2.0
1.6
1.5
3.2
6.4
1.0
0.5
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-3 f. Increase in ‘head jerk’ for group by day (P<0.01), group (P<0.001) and days
(P<0.001) in (-)-cathinone-treated animals. The increase was more observable during absence
of cage enrichment in (-)-cathinone subjects compared to controls (n = 14).
61
3.2.1.2 Behavioural scores of anxiety
Scores for ‘pacing’ changed over days (F(19, 174) = 14.0 , P < 0.001), and were greater among ()-cathinone groups relative to controls (F(4,9) = 11.7, P < 0.01). However, these findings were
qualified by a group by day interaction (F(77,
174)
= 1.78, P < 0.01; Fig. 3-4 a). Scores for
‘scratch’ differed by days (F(12, 115) = 33.9 , P < 0.001), and by group with greater number of
observations in (-)-cathinone groups as compared with the control group (F(4, 9) = 26.4, P <
0.001), and there was a group by day interaction (F(51, 115) = 3.44, P < 0.001; Fig. 3-4 b). There
was a group (F(4, 9) = 44.9, P < 0.001) and group by day interaction (F(90, 203) = 4.67 , P < 0.001
interaction on ‘self-directed’ behavioural scores (Fig. 3-4 c). The effect of (-)-cathinone on
these measures of anxiety was dose-dependent.
62
16.0
Group x day: p<.01
Group: p<.01 (con<0.8=1.6=3.2=6.4)
day: p<.001
Anxiety - pacing
14.0
mean frequency
12.0
10.0
Control
8.0
0.8
1.6
6.0
3.2
4.0
6.4
2.0
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-4 a. A dose-dependent increase in ‘pacing’ for group by day (P < 0.01), group (P <
0.01) and day (P < 0.001) in (-)-cathinone subjects compared to controls (n = 14). There was
no significant difference on this measure both in presence and in absence of cage enrichment.
63
25.0
Group x day: p<.001
Group: p<.001 (con<0.8=1.6=3.2=6.4)
day: p<.001
Anxiety - scratch
mean frequency
20.0
15.0
Control
0.8
10.0
1.6
3.2
6.4
5.0
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-4 b. A significant increase in ‘scratch’ for group by day, between groups and over
days (P < 0.001) in (-)-cathinone subjects compared to controls (n = 14).
64
14.0
x day: p<.001
Anxiety - self-directed Group
Group: p<.001 (con<others; 0.8<6.4)
day: p<.001
12.0
mean frequency
10.0
8.0
Control
0.8
6.0
1.6
3.2
4.0
6.4
2.0
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-4 c. A dose-dependent increase in ‘self directed’ behaviour for group by day,
between subject groups and over days (P<0.05) in (-)-cathinone-treated animals compared to
controls. The increment was more elaborate during cage enrichment. (n = 14).
65
3.2.1.3 Abnormal behaviour
Results show a dose-dependent increase in stereotypical responses in (-)-cathinone subjects
compared to controls. Although there was a general increase in ‘response independent of
stimuli’ among all subjects over experimental period (F(22,
204)
= 69.8 , P < 0.001)) these
changes tended to be pronounced with increase in (-)-cathinone dose (group by day
interaction: (F(90,
204)
= 6.91 , P < 0.001; Fig. 3-5 a). Individual scores for ‘fine motor
stereotypy’ showed a significant increase across observational days (F(18,
166)
= 87.8 , P <
0.001), and there was also a group by day interaction (F(73, 166) = 8.13 , P < 0.001; Fig. 3-5 b).
Similarly, there were group versus observational day interaction (F(76, 171) = 8.06 , P < 0.001),
as well as group (F(4,9) = 74.5, P < 0.001) and days effects (F(19, 171) = 94.7, P < 0.001) in
‘whole body stereotypy’ (Fig. 3-5 c). The increase on these measures was more pronounced
for dose 6.4 mg/kg body weight compared to other doses (0.8, 1.6 and 3.2 mg/kg body
weight) of (-)-cathinone. Overall, (-)-cathinone subjects, at all doses, showed high levels of
abnormal behaviour compared to controls.
66
25.0
Abnormal - response independent of stimuli
Group x day: p<.001
Group: p<.001 (con<others; 6.4>0.8, 1.6)
day: p<.001
mean frequency
20.0
15.0
Control
0.8
10.0
1.6
3.2
6.4
5.0
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-5 a. A dose-dependent increase in ‘response independent of stimuli’ for group by
day, group and over days (P < 0.001) in (-)-cathinone subjects compared to controls (n = 14).
67
25.0
Abnormal - fine motor stereotypy
Group x day: p<.001
Group: p<.001 (con<others); 0.8<3.2)
day: p<.001
mean frequency
20.0
15.0
Control
0.8
10.0
1.6
3.2
6.4
5.0
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-5 b. Behavioural scores for ‘fine motor stereotypy’ showing a significant increase
for group by day interactions, between subject groups and over days (P < 0.001) in (-)cathinone subjects compared to controls (n = 14).
68
20.0
Abnormal - whole body stereotypy
18.0
Group x day: p<.001
Group: p<.001 (con<others; 0.8<6.4; 1.6<6.4)
day: p<.001
16.0
mean frequency
14.0
12.0
Control
10.0
0.8
8.0
1.6
6.0
3.2
6.4
4.0
2.0
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-5 c. A significant increase in ‘whole body stereotypy’ for group by day interactions,
between subject groups and over experimental period (P < 0.001). The increase was more
significant for dose 6.4 mg/kg bwt of (-)-cathinone compared to other doses and controls (n =
14).
69
3.2.1.4 Appetitive behaviour
The results show a significant increase in latency of response to introduction of food over
observational period (F(15, 139) = 1261 , P < 0.001), and on average the 6.4 mg/kg had greater
latency than other groups (F(4,9) = 2248 , P < 0.001). These findings were qualified by a
significant group by observational day interactions (F(62, 139) = 80 , P < 0.001) which indicate
dose-dependent relationship between (-)-cathinone dose and latency of response to food
across experimental period (Fig. 3-6). The increase was more elaborate among (-)-cathinone
subjects during cage enrichment compared to absence of cage enrichment. From these results
it is possible that cage enrichment, to some extent, affected the focus of focal subjects to the
introduction of food leading to a fixed order in the observations. This is regarded as a
limitation of the study methodology on environmental enrichment.
70
160.0
140.0
Appetite
Group x day: p<.001
Group: p<.001 (con<0.8<1.6<3.2<6.4)
day: p<.001
latency (seconds)
120.0
100.0
Control
80.0
0.8
1.6
60.0
3.2
40.0
6.4
20.0
0.0
1
3
6
1
3
6
9
1
3
Pretreat_Iso Pretreat_Rich
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-6. A significant increase (P < 0.001) in latency of response to food among (-)cathinone subjects compared to controls (n = 14). Note a more elaborate dose-dependent
increase during cage enrichment.
71
3.2.1.5 Withdrawal behavioural score
Duration of time spent by focal subjects in withdrawal to the corner of the cage increased over
observational period (F(11, 102) = 2006 , P < 0.001; Fig. 3-7) and (-)-cathinone groups showed
longer duration than the control group (F(4,9) = 1052 , P < 0.001). These findings were
qualified by a group by observational day interactions (F(45, 102) = 245 , P < 0.001) suggesting
dose-dependent increase in withdrawal score. This increase in withdrawal period of (-)cathinone subjects was significant following cage enrichment compared to period in absence
of cage enrichment. During pre-treatment phase there was no observable withdrawal of focal
subjects among all subject groups indicating that (-)-cathinone contributed greatly to this
behavioural observation.
72
450.0
400.0
duration (seconds)
350.0
Withdrawn to the corner of cage
Group x day: p<.001
Group: p<.001 (con<0.8<1.6<3.2<6.4)
day: p<.001
300.0
250.0
Control
0.8
200.0
1.6
150.0
3.2
6.4
100.0
50.0
0.0
1
3
6
1
3
6
9
Pretreat_Iso Pretreat_Rich
1
3
6
9 12 15 18 21 24 1
Treatment_Iso
day
3
6
9 12 15 18 21 24
Treatment_Rich
Figure 3-7. A significant increase (P < 0.001) in time spent by focal subjects withdrawn to
corner of cage for group by day, between groups and over days. Note a dose-dependent
significant increase during cage enrichment compared to absence of cage enrichment (n = 14).
73
3.3 DISCUSSION
The aim of this study was to establish, in single-caged vervet monkeys, a profile of
behavioural alterations resulting from acute- and sub-chronic exposure to (-)-cathinone in
presence and/or absence of cage enrichment over a period of 5 months. However, the first one
month was pre-treatment period, where animals were observed in order to establish baseline
levels on behaviour patterns under investigation. (-)-Cathinone was administered on alternate
days as described in earlier chapter. This took into consideration the plasma half-life of (-)cathinone in the body of vervets. Studies in humans who chewed khat for 1 h showed that (-)cathinone was detected in urine for up to approximately 26 h while cathine and norephedrine
were detected for at least 80 h (Toennes and Kauert, 2002). The regimen also took into
account the likely development of tolerance to (-)-cathinone by the animals. It is possible that
after multiple repeated exposures to khat and (-)-cathinone, tolerance develops. Tolerance and
cross-tolerance between (-)-cathinone and cathine have been reported in animal studies (Foltin
et al., 1983).
The results have shown a dose-dependent increase in frequencies of individual behavioural
scores that measure aggression, anxiety and abnormal-related behaviours. Durational
behaviours of withdrawal/isolation and eat/appetite also showed a dose-dependent increase
over observational period of time. The frequency of behavioural scores on aggression,
anxiety, abnormal responses, withdrawal and appetitive behaviours were not affected by cage
enrichment.
74
3.3.1 EFFECTS ON BEHAVIOUR
3.3.1.1 Aggression
During pre-treatment phase neither presence nor absence of cage enrichment influenced
behavioural patterns. However, following (-)-cathinone treatment behavioural changes
became apparent with increasing dose levels and over time period. All individual behavioural
categories defining aggression increased in both presence and absence of cage enrichment but
significantly so in absence of cage enrichment. The significant effect during absence of cage
enrichment might have been due to the novelty effect of (-)-cathinone. There were low mean
frequencies of ‘yawn’, ‘engage in a stare’, ‘jaw thrust’, ‘bouncing off cage walls’, ‘vigorous
shaking of cage walls’ and ‘head jerk’ during cage enrichment compared to absence of cage
enrichment. It is also apparent that mean frequency scores of ‘engage in a stare’ and ‘jaw
thrust’ did not differ significantly for group and group by day effects. This is unexpected and
may be attributed to limitation in methodology used. However, since ‘yawn’ was the only
behaviour observed during pre-treatment period while other observational categories defining
aggression were absent, it is likely that (-)-cathinone primarily influenced aggressive
behaviour in these animals.
Earlier studies on khat extracts and (-)-cathinone reported stereotyped behaviour, selfadministration and anorectic effects in animal species (Gordon et al., 1993, Calcagnetti and
Schechter, 1992) similar to that evoked by [S-(+)-amphetamine] (Goudie, 1985).
Furthermore, both khat extract and (-)-cathinone have been shown to enhance baseline
aggressive behaviour in isolated rats (Banjaw et al., 2005). These behaviours are partly
influenced by disorders in the hypothalamic dopaminergic system (Ishikawa et al., 2007) and
75
partly due to dopaminergic and serotonergic activity in the mesolimbic system (Eisch and
Harburg, 2006, Jones and Bonci, 2005). Further evidence has shown involvement of both khat
extract and (-)-cathinone in depletion of serotonin and its metabolite 5-hydroxyindole-acetic
acid in both anterior and posterior striatum (Banjaw et al., 2005). The present study simulated
the use of (-)-cathinone in vervet monkey model as it appears in human khat ‘addicts’. The
results show a dose- dependent increase in aggressive behaviour which is in agreement with
earlier findings where chronicity of use or multiple acute high doses of (-)-cathinone exposure
in humans showed a wide range of dose-dependent behavioural changes including aggression,
hyperactivity, various forms of psychotic illness, anxiety, paranoia and social withdrawal
(Berman et al., 2009).
3.3.1.2 Anxiety
The result of the present study showed that anxiety scores for ‘pacing’, ‘scratch’ and ‘selfdirected’ steadily increased in a dose-dependent manner in absence and presence of cage
enrichment and over observational period of time. (-)-Cathinone subjects exhibited more
anxiety levels during cage enrichment. Stereotypical behaviours have been demonstrated in
vervet monkeys following maternal separation and these measures are reminiscent of anxiety
symptoms in humans (Marais et al., 2006).
Studies on (-)-cathinone and amphetamine in animals have shown their sympathomimetic
effects on dopaminergic (Pehek et al., 1990) and serotonergic (Kalix, 1984 b) synapses as
well as peripherally via noradrenergic storage sites (Kalix, 1983 b). Centrally acting
neurotransmitters such as dopamine and serotonin have been shown to have modulatory
effects on specific behaviours (Spoont, 1992). The receptors for both dopamine and serotonin
76
appear overlapping in the limbic and cortical regions of the brain (Goldman-Rakic et al.,
1990), and this explains the inhibitory effect of serotonin on facilitatory effects of dopamine.
Noradrenaline has also been shown to influence specific behaviours when interacting with
other biogenic amines in the brain of different animal models and in human case studies. For
example, reduced levels of serotonin are found in patients with anxiety disorders (Charney et
al., 1990) and reduction in both serotonin and noradrenaline is associated with major
depression (Sulser, 1989; Meltzer and Lowy, 1987). Recent studies showed that anxiolytic
mechanism may include some interaction of noradrenaline and serotonin, rather than
increasing synaptic levels of serotonin (Marais et al., 2006). Studies on (-)-cathinone have
demonstrated maintenance of drug-seeking behaviour in rats habituated to amphetamine and
monkeys trained to lever-press for cocaine injection (Yanagita, 1979). Similar studies in
humans by use of amphetamine showed drug-related effects of insomnia, irritability, anxiety,
sadness and nightmares (Efron et al., 1997). (-)-Cathinone causes these effects via its effect
on central dopaminergic (Pehek et al., 1990) and serotonergic (Kalix, 1984 b) and
peripherally at the noradrenergic storage sites (Kalix, 1983 b).
3.3.1.3 Abnormal behaviour
Abnormal behavioural scores observed in the present study showed a steady increase in (-)cathinone subjects in a dose-dependent manner and over experimental period. These
behaviours were more apparent following cage enrichment. A study by Zelger et al. (1980)
reported stereotypical behaviour and hyper-locomotion in rats treated with (-)-cathinone, and
these paradigms characterize abnormal behaviour (Melega et al., 2008). The results of the
present study on scores for ‘fine motor’ and whole body stereotypy’ confirm these earlier
77
findings in other animals and, probably, a similar mechanism of action of these drugs is
involved in influencing these behaviours.
3.3.1.4 Withdrawal/Isolation and appetitive behaviour
The durational behaviours of withdrawal and appetite loss in (-)-cathinone–treated animals
showed an increase in a dose-dependent manner over the experimental period. This increase
was again more pronounced following cage enrichment compared to observations in absence
of cage enrichment but the difference in both set-ups was not significant. Cage enrichment
may have potentiated the effect on these measures. However, similar studies in humans and
experimental animals have reported changes in affiliative and anorectic behaviours following
khat use (Gordon et al., 1993, Pantelis et al., 1989). Studies have shown social withdrawal as
a common side effect of [S-(+)-amphetamine] in children with attention deficiency disorder
(Clinical Practice Guideline, 2001). On the other hand, anorectic behaviour is characterized
by development of tolerance (Zelger and Carlini, 1980). It is, therefore, possible that while
cage enrichment appeared to have influenced the observed behaviours, the effect may have
been primarily due to (-)-cathinone exposure as it showed a dose-dependent response.
Methamphetamine is a derivative of amphetamine and, like amphetamine, has been shown to
cause alterations in the human striatal dopamine system effects (Johanson et al., 2006, Wang
et al., 2004). Studies on effects of methamphetamine on the dopaminergic system showed
dopamine receptor density alterations following multiple exposures (Melega et al., 2008).
Although not tested, the findings of the present study are a possible pointer to the association
between behavioural changes and receptor density alterations or levels of catecholamines at
the periphery. This is explained by the fact that amphetamines and (-)-cathinone influence
78
release of catecholamines via a similar mechanism (Kalix, 1996). For example, studies have
shown that (-)-cathinone increases levels of dopamine, serotonin and noradrenaline in the
brain via catecholaminergic synapses (Calcagnetti and Schechter, 1993; Kalix, 1991).
Serotonin (5-Hydroxytryptamine [5HT]) is a short-acting widespread neurotransmitter which
acts on a number of receptor subtypes found at high density in the limbic system and raphe
nuclei as well as in the hypothalamus (Blundell, 1984). Agonists at the 5-HT2c receptor show
the most consistent inhibition of food intake and the 5-HT2c-knockout mouse is hyperphagic
and obese (Tecott et al., 1995). Another study investigating involvement of 5-HT2C receptors
in the regulation of food intake in the Siberian hamsters treated with fenfluramine
demonstrated that fenfluramine exerted a potent hypophagic effect (Schuhler et al., 2005).
Because fenfluramine is known to increase 5-HT release and inhibit its reuptake, these
observations demonstrated the involvement of the serotonergic system in the regulation of
food intake in the hamster. In general, agonists at the 5-HT receptors and drugs that inhibit the
uptake of serotonin reduce feeding. Additionally, 5-HT stimulates noradrenaline release and
modifies behaviour and mood (Neary et al., 2004). However, at high doses, the massive
release of 5 –HT not only gives rise to acute psychotic symptoms but also causes chemical
damage to the cells that release it (Kalant, 2001).
The observed effects of (-)-cathinone on appetitive/consummatory behaviour in the present
study are consistent with findings of Nyongesa et al. (unpublished data) which showed a
dose-dependent decrease in food intake and body weight gain of rabbits following exposure to
khat extract. The arcuate nucleus of the hypothalamus is involved in central appetite
regulation
by use of Agouti-related protein (AgRP) that increases food intake through
antagonism of melanocortin MC3 and MC4 receptors and thus blockade of inhibition of the
79
anorexigenic agonist α-melanocyte stimulating hormone (α-MSH) (Neary et al., 2004).
Virtually all AgRP neurones co-secrete neuropeptide Y (NPY), (Goldstone et al., 2002),
which have Y2R - a presynaptic inhibitory autoreceptor. The arcuate nucleus also contain
galanin-like peptide (GALP)-containing neurones, which plays a critical role in the regulation
of mammalian energy balance and reproduction (Gottsch et al., 2004), and several discrete
populations of neurones in this nucleus are targets for the regulatory hormone leptin (McMinn
et al., 2000). In rodents and primates, arcuate nucleus contains mRNA of neuropeptides
involved in feeding and/or reproduction including neuropeptide Y (NPY), α-melanocyte
stimulating hormone (α-MSH), agouti-related protein (AgRP), and galanin (Cone et al.,
2001). GALP infusions have been shown to stimulate luteinizing hormone (LH) release in a
dose-related manner in male rats, mice and macaques (Cunningham et al., 2004; Krasnow et
al., 2004) and this LH release is likely mediated via GnRH-1-dependent pathways
(Cunningham et al., 2004; Matsumoto et al., 2001). Previous studies, however, showed that
khat suppresses appetite independent of ghrelin and peptide YY (PYY) secretion (Murray et
al., 2008). From these observations, it is possible that alterations in consummatory behaviour
of the vervet monkeys following (-)-cathinone treatment may have been due to its effect on
the arcuate nucleus or as a result of increased levels of 5-HT in the brain. It is, therefore,
possible that (-)-cathinone caused dopamine and serotonin receptor density alterations that
influenced changes in feeding behaviour.
The study had its own limitations. The methodology was aimed at modelling an experimental
animal to emulate the situation in human khat chewers. Khat is chewed over a long period of
time while the juice is extracted. Toennes et al. (2003) reported a major role buccal mucosa
plays in the absorption of (-)-cathinonoe, cathine and norephedrine alkaloids extracted into
80
saliva following chewing. In the present study, it was not possible to achieve this route of
administration in monkeys, instead (-)-cathinone was given as a bolus via gastric gavage.
Khat chewing is a daily habit in addicts and so the observed effects in khat chewers may be as
a result of these multiple exposures. This regimen was hampered by the mode of
administration where animals were anaesthetized during each time of (-)-cathinone
administration due to difficulty in achieving intubation in awake monkeys. One possibility is
to identify sweetener that can be mixed with khat leaves so that experimental animals are able
to chew on them rather than administration via other routes. It was also unethical to keep
animals under anaesthesia daily for purposes of drug administration. Moreover, since
anaesthesia eliminates cardiovascular measures that are indicative of (-)-cathinone effects, this
is regarded as a limitation of the study. On the other hand, it is well understood that vervet
monkeys live in relatively stable multi-male, multi-female social groups (Isbell et al., 1991).
Isolation into individual cages done in this study may have partly influenced their behavioural
manifestations even though they were adequately habituated. There was also some individual
variation in behavioural parameters of vervet monkeys suggesting that larger sample size is
required when studying behaviour in this species. These are gaps for future consideration.
Conclusion
The findings of the present study have shown that, indeed, (-)-cathinone exposure caused
behavioural alterations in a dose-dependent manner. The effects were pronounced following
long-term use. There was continual increase in some scores of aggression in a dose-dependent
manner in (-)-cathinone-treated animals while the same scores decreased in controls over
time. Time-dependent changes in frequency behaviours of aggression, anxiety and abnormal
responses as well as durational behaviours of appetite and withdrawal demonstrated how
81
long-term drug use influences different behavioural profiles. Cage enrichment did not have an
effect on measures of aggression and anxiety while those measures defining abnormal
behaviour, withdrawal and appetite/consummatory behaviour showed a significant effect on
following (-)-cathinone exposure. However, the study did not consider counter-balancing
between cage enrichment and its absence on (-)-cathinone effects to different behaviours
leading to a fixed order in observations of different behaviours following (-)-cathinone
administration.
82
CHAPTER 4
SERUM HORMONAL PROFILES FOLLOWING ACUTE AND
SUB-CHRONIC
CATHINONE
EXPOSURE
IN
VERVET
MONKEYS (CHLOROCEBUS AETHIOPS)
4.0 INTRODUCTION
Khat has been used as a culturally sanctioned stimulant not only in many countries of eastern
Africa but also in the Middle East. (-)-Cathinone [S-(-)-alpha aminopropiophenone] is the
primary psychoactive alkaloid of khat (Kalix and Braenden, 1985; Kalix, 1984 c). The general
semblance between biochemical effects of (-)-cathinone and those of amphetamine as well as
their chemical structure points to the fact that both substances have similar mechanism of
action (Graziani et al., 2008; Houghton, 2004; Cox and Rampes, 2003), with equal potential
for abuse (Kalix, 1984 a). Acute and chronic exposure of khat and (-) cathinone in consumers
have been shown to cause a wide range of effects from mental, respiratory, digestive to
reproductive dysfunction. Research findings in humans and experimental animals have
reported changes in sleep patterns, mood, attention, aggression, anxiety, locomotor activity,
and affiliative behaviours (Pantelis et al., 1989; Kalix, 1994), learning and memory (Kimani
and Nyongesa, 2008) and sexual behaviour (Tariq et al., 1990). There is growing evidence in
literature indicating that khat and (-)-cathinone induce psychostimulation primarily via mesostriato-corticolimbic dopaminergic pathway (Kalix, 1990), although there may be other
systems involved. The addiction potential, analgesia and anorexic effects of khat and (-)83
cathinone are believed to be partly mediated via this pathway (Gosnell et al., 1996). This is
consistent with studies demonstrating the involvement of hypothalamo-hypophysealadrenocortical axis in psychostimulation via dopaminergic transmission (Steckler and
Holsboer, 1999). Preliminary studies showed increased corticosterone levels following use of
psychostimulants (Mello and Mendelson, 1997) and these high levels were shown to
contribute to substance abuse disorders (King et al., 2003). Studies on khat showed increase
in adrenocorticotrophic hormone in humans (Nencini et al., 1984), consistent with cortisol
measurement in rabbits following khat extract exposure (Nyongesa et al., 2008) but different
from findings in baboons where decreased plasma cortisol was observed (Mwenda et al.,
2006). These conflicting findings especially with respect to cortisol may be explained partly by
the time of exposure to khat/(-)-cathinone and possibly species difference. The aforementioned
investigations on khat covered an acute phase of exposure, in which elevated cortisol
measurements, independent of treatment, are expected as a result of lack of adequate habituation
of animals to handling and manipulations. Similarly, acute exposure is not quite a reflection of
effects in a regular khat chewer. Species difference may also have contributed to the variance in
results. The present study considered both acute and sub-chronic exposure of (-)-cathinone to the
vervet monkeys.
Prolonged elevations of glucocorticoid concentrations have been shown to reduce the
response of luteinizing hormone (LH) to gonadotropin releasing hormone (GnRH) (Wang,
1986). The concentrations of circulating gonadotropins in turn depend on the frequency and
amplitude of GnRH pulses discharged into the pituitary portal vessels, which in turn depend
on endocrine status (Dierschke et al., 1970). Previous studies have shown that (-)-cathinone is
an inhibitor of several anterior pituitary hormones (Wagner et al., 1982). This was indeed the
84
findings in rabbits where LH levels were suppressed in a dose-dependent manner following
crude khat extract administration (Nyongesa et al., 2008).
Several investigations have similarly shown that khat and (-)-cathinone have effects on
circulating levels of testosterone although these reports are contradictory. For example, (-)cathinone has been shown to have cytotoxic effect on gonads leading to androgenic deficiency
in mice (Tariq et al., 1987). Khat and (-)-cathinone have also been reported to interefer with
mitosis in body cells (Al-Meshal, 1987) leading to a decrease in plasma testosterone in
humans (El-Shoura et al., 1995; Balint et al., 1991; Kalix and Braenden, 1985) and rats (Islam
et al., 1990). On the contrary, reports have cited khat as an aphrodisiac (Krikorian, 1984;
Margetts, 1967), a medicament for premature ejaculation (Pantelis et al., 1989; Luqman and
Danowski, 1976) and a libido booster that increases sexual desire (Elmi, 1983) and sperm
power booster (Adeoya-Osiguwa and Fraser, 2005). These findings are similar to those in
baboons (Mwenda et al., 2006) which showed that khat causes an increase in plasma
testosterone levels. This may explain the increase in aggressive behaviour presented in
chapter 3 of this thesis where increasing doses of (-)-cathinone caused aggression in a doseand time-dependent manner. Khat and (-)-cathinone have also been shown to have biphasic
effect on reproductive function. For instance, in vitro studies on effect of khat extract on
isolated mouse interstitial cells showed that high concentrations (30 mg/ml and 60 mg/ml) of
khat extract adversely affected cell viability by about 50% while low concentrations (0.06
mg/ml, 0.6 mg/ml and 6 mg/ml) caused minimal effect (Nyongesa et al., 2007). These
findings showed a similar pattern to those of 6-hydroxydopamine on mouse Leydig cells that
showed increasing doses being stimulatory then inhibitory to testosterone production (Wango
et al., 1995).
85
Previous studies focused on acute effects of (-)-cathinone; however, no study has
systematically examined sub-chronic effects of (-)-cathinone treatment. The present study was
designed to investigate the effects of (-)-cathinone on hypothalamo-hypophysealadrenocortical and gonadal axes. We hypothesized that sub-chronic to chronic administration
of (-)-cathinone would alter neurobiological functions in the hypothalamo-hypophysealadrenocortical and gonadal systems, leading to changes in production of cortisol, prolactin,
LH, testosterone and progesterone hormones. Progesterone measurements were done for
comparison with those in a parallel ex vivo study designed to determine effects of (-)cathinone on testosterone biosynthetic pathway described in chapter 6 of this thesis.
86
4.1 MATERIALS AND METHODS
4.1.1 ANIMALS AND HOUSING
The same animals described under general materials and methods were used for this study.
4.1.2 EXPERIMENTAL DESIGN
Animal identification and grouping is same as was described previously in section 2.4 of
general materials and methods.
4.1.2.1 Pre-treatment phase
This was done according to the previous description under section 3.1.2.1 of chapter 3. The
femoral vein of each animal was cannulated using a 22 G blood vessel cannula after shaving
and swabbing the area with 70% alcohol. Heparinised saline was introduced into the cannula
to prevent blood clotting inside the lumen. The adhesive tape was used to anchor the cannula
in situ to prevent it from dropping off and blood collection done. Blood samples (2 ml) were
then collected from femoral vein at 5-days interval for cortisol, prolactin, LH, progesterone and
testosterone analysis.
4.1.2.2 Treatment phase
i). Drug administration
(-)-Cathinone administration followed the previous description under section 3.1.2.2 of
treatment phase. At the end of this 1st treatment phase that lasted 4 months, group II and V
animals were co- treated with GnRH agonist goserelin acetate (ZOLADEX) in the presence of
87
low and high dose (0.8 and 6.4 mg/kg body weight) of (-)-cathinone for additional 2 weeks
during which blood samples were collected at 5- day intervals and processed for hormone
analysis. ZOLADEX (3.6 mg) was prepared in 10% DMSO in PBS and and an equivalent of 50
μg/kg body weight/injection was administered subcutaneously.
ii) Blood sampling
Food was restricted prior to use of anaesthetics. Blood samples were collected from femoral
vein of animals following anaesthesia with 10 mg/kg body weight of ketamine hydrochloride
intramuscular. Femoral vein of each animal was cannulated as described under 4.1.2.1 above
and, thereafter, 2 ml blood samples collected into heparinized LP3 tubes at 20 min- interval
for 2.5 h starting at 10th min following (-)-cathinone administration. Sampling was done at
1000 h on Mondays and Wednesdays of every week. Serum was stored at -200C until assayed
for cortisol, prolactin, LH, progesterone and testosterone. Dopamine concentrations were
estimated from serum prolactin based on inverse proportionality. This was based on findings
that spontaneous secretion of prolactin is inhibited by dopamine (Birge et al., 1970; MacLeod
et al., 1970) and that dopamine receptors are found on lactotroph membranes (Goldsmith et
al., 1979). Other studies showed that dopamine is found in hypophysial stalk plasma (Plotsky
et al., 1978; Ben-Jonathan et al., 1977) in amounts sufficient to inhibit prolactin release
(Gibbs and Neill, 1978).
iii) Hormonal analysis
Hormonal assays for serum cortisol, prolactin, progesterone, LH and testosterone were done
by use of enzyme immunoassay technique using the kits from Nova Tec Immundiagnostica
GMBH, Germany. The technique uses the principle of competition of hormone in sample
with enzyme conjugated hormone for limited binding sites on the specific antibody.
88
Validation of the assay method for use in the vervet monkey followed that of Eley et al.,
(1989). In all hormone assay procedures, assays were done in triplicate. The optical density of
specimen was measured using HumaReader HS (Gessellschaft für Biochem und Diagnostica,
mbH, Germany).
Cortisol enzyme immunoassay
The assay procedure of the protocol supplied in the kit was followed
Prolactin enzyme immunoassay
This followed the assay procedure stipulated in the protocol supplied with the kit.
Progesterone enzyme immunoassay
The assay procedure within the protocol of the kit was followed
Testosterone enzyme immunoassay
The assay procedure within the protocol of the kit was followed
LH bioassay
Male animals were anaesthetised with 10 mg/kg ketamine hydrochloride intramuscularly and
both testes harvested. The animals were later sacrificed and samples collected for other
experiments. The testes were decapsulated and fat trimmed off, then rinsed in ice-cold 10 ml
Modified Eagle Medium (MEM) in plastic Petri dish. The media was aerated with medical
gas (95% O2: 5% CO2) in ice-cold water before use. Testes were minced with a pair of
scissors and cells dispersed mechanically using a Fin pipette by sucking in and out several
89
times. The minced tissue was transferred to a 50 ml conical flask containing 100 ml media,
and mixed gently for 15 min at 4 C. The suspension was then filtered through fine cotton
gauze to obtain interstitial cells which were transferred into 100 ml conical flask and
incubated for 15 min in 5% carbon-dioxide water bath at 4 0C. The cell suspension was then
aliquoted into 6 plastic test tubes and subjected to percoll density gradient centrifugation three
times and the pellets pooled into a conical flask. The pooled pellets were re-suspended in
medium and Leydig cells isolated by sieving the suspension through cotton gauze, diluted
with the incubation media and purified by 3 beta hydroxysteroid dehydrogenase (3β-HSD)
staining method. The purified Leydig cells were then counted using a haemocytometer to
obtain a working concentration of approximately 2 x 105 cells/ml. Luteinizing hormone levels
were determined by assaying testosterone levels in the suspension medium following the
enzyme immunoassay (EIA) protocol.
4.1.3 STATISTICS
Results are expressed as mean ± SEM. Hormonal data for pre-treatment and treatment phases
for cortisol, prolactin, progesterone, testosterone and LH were analysed jointly. For each
hormonal measure, a 5 treatment group (0, 0.8, 1.6, 3.2 and 6.4 mg/kg body weight) x 20
experimental weeks (4 weeks of pre-treatment phase and 16 weeks of treatment phase) x 7
sampling times (at 20, 40, 60, 80, 100, 120 and 140 min period) repeated measures ANCOVA
was conducted. Treatment group was used as a between-subject factor, week and sampling
time as within-subject factors and sex as a covariate. Huynh-Feldt correction was applied for
the analysis and Bonferroni adjustments performed for post hoc multiple comparison tests.
Statistical significance was set at 5% level.
90
4.2 RESULTS
The results on effect of GnRH agonist on LH are not reported here due to the small sample
size used for group II and V animals that were involved. There were no controls for
comparison. However, effect of GnRH agonist on immunolocalization of gonadotrophs is
reported in chapter 5 of this thesis. The results of all hormones reported below showed no
significant effect of cage enrichment on these parameters over the experimental period,
suggesting that the effects observed were primarily due to (-)- cathinone treatment.
4.2.1 Effect of (-)-cathinone on serum cortisol
The results show a significant group effect [F
(4, 8)
= 218, P < 0.001] indicating dose-
dependent decrease in cortisol levels (Fig. 4-1). Cortisol also changed across the 20-week
period [F (18, 142) = 21.7, P < 0.001] with levels during the treatment phase (from week 5 to
week 20) being generally lower than those during the 4 week pre-treatment phase (Fig. 4-2).
There was also variation in cortisol levels over sampling time [F (6, 48) = 7.98, P < 0.001] (Fig.
4-3). Multiple comparison tests indicated that levels at the first sampling time of the pretreatment phase was greater than the last two sampling times (P < 0.05) suggesting
habituation. There was also a significant group x week interaction [F
(71, 142)
= 4.86, P <
0.001], which indicated that reduction in cortisol levels over study weeks was pronounced as
(-)-cathinone dose increased (Fig. 4-4). Decrease in cotisol levels over sampling period tended
to be pronounced during the treatment phase relative to pre-treatment phase as indexed by
group x time interaction [F (24, 48) =2.06, P < 0.05] (Fig. 4-5).
91
4.2.2 Effect of (-)-cathinone on serum prolactin
Higher (-)-cathinone- induced decrease in overall prolactin concentrations as indicated by a
significant group effect [F
(4, 8)
= 267, P < 0.001] (Fig. 4-6) was observed. Prolactin levels
observed during treatment phase were lower than pre-treatment phase showing main effect of
week: [F (15, 118) = 129, P < 0.001] on this measure (Fig. 4-7). Prolactin changed over sampling
period [F
(6, 48)
= 19.5, P ≤ 0.001] with lower levels in the last four sampling periods relative
to the first sampling period (P ≤ 0.001) (Fig. 4-8). In addition, a significant group x week
interaction [F
(59, 118)
= 13.03, P < 0.001] indicated dose- and time-dependent effects of (-)-
cathinone on serum prolactin levels (Fig. 4-9). Decrease in prolactin across sampling period
tended to be pronounced during the treatment phase as indicated by group x time interaction
[F (24, 48) = 3.65, P < 0.001] (Fig. 4-10).
4.2.3 Effect of (-)-cathinone on serum Lutenizing hormone
The result also show that high doses of (-)-cathinone increased LH concentrations as indicated
by a significant group effect [F (4, 8) = 14612.4, P < 0.001] (Fig. 4-11). There was a significant
group by week interaction with the two high dose F (4, 76) = 333.8, P < 0.001] indicating doseand time-dependent effects of (-)-cathinone on LH secretion (Fig. 4-12). Luteinizing hormone
levels observed during treatment phase were higher than pre-treatment phase showing main
effect of week: [F (1, 19) = 1185, P < 0.001; multiple comparisons: P ≤ 0.001] on this measure.
There was significant effect of (-)-cathinone on LH levels over sampling period [F
(4, 24)
=
344.5, P < 0.001] indicating a group by sampling period interaction (Fig. 4-13). Overall, the
results show that (-)-cathinone had a strong effect on hypothalamo-hypophyseal axis as a
function of dose, sampling period and experimental time as shown by week x sampling period
x group interaction [F (4, 24) = 7.4, P < 0.001].
92
4.2.4 Effect of (-)-cathinone on serum testosterone
The changes in serum testosterone in (-)-cathinone subjects and controls are presented in
Figures 4-14 and 4-15. The results show a significant group effect [F (4, 8) = 28.85, P < 0.001]
based on linearly independent pair-wise comparisons among estimated marginal means. There
was also a significant difference in means across 20-week period [F (1, 19) = 10.25, P < 0.001]
at highest dose (6.4 mg/kg) compared to moderate doses and controls with levels during the
treatment phase (5 - 20 weeks) being generally lower than those during the 4 week pretreatment phase. The results show a biphasic effect of (-)-cathinone on this parameter. At dose
3.2 mg/kg body weight, serum testosterone increased then decreased as shown by group x
week interaction [F
(4, 76)
= 7.14, P < 0.001] (Fig. 4-14). Low and medium doses generally
increased serum levels of testosterone while highest dose significantly decreased testosterone
levels as indicated by group by period interaction [F (4, 24) = 3.19, P < 0.001] (Fig. 4-15).
4.2.5 Effect of (-)-cathinone on serum progesterone
The results show a general pattern of decrease in serum progesterone in dose-and timedependent manner as shown by group and week interaction [F (4, 76) = 3.72, P < 0.001] (Fig. 416). There was also a significant group effect [F
(4, 8)
= 114.53, P < 0.001] based on linearly
independent pair-wise comparisons among estimated marginal means. Serum progesterone
levels observed during treatment phase were lower than pre-treatment phase as shown by
effect of week: [F (1, 19) = 38.66, P < 0.001 as well as for controls.
Correlational analysis was conducted to examine associations between cortisol and prolactin.
There were no correlation between the two hormones during the pre-treatment phase except
one inverse relationship (r = -0.57, P < 0.05) observed at 40 min of sampling period in the
93
third week. In contrast, there were many positive correlations between the two hormones
during the treatment phase. For example, during the fifth week of treatment, there were
positive correlations at all sampling periods (r > 0.53, Ps < 0.05). Similar patterns of positive
relationships were found during week 6, 7 and 8. In the latter half of the treatment phase (i.e.,
from the eleventh to twentieth week), positive correlations of cortisol and prolactin were
found mostly from 100 min, 120 min and 140 min of sampling period (P < 0.05) (Fig. 4-17).
94
195
*
**
Cortisol (ng/ml)
190
**
185
***
180
175
170
Control
0.8
1.6
3.2
6.4
Group (mg/kg)
Figure 4-1. Changes in cortisol levels across groups. Entries show mean and standard error of
the mean. There was a significant dose-dependent decrease in serum cortisol in treatment
groups compared to controls. * P < 0.05, ** P < 0.01, *** P < 0.001. * means statistically
significant. (n = 14).
95
195
cortisol (ng/ml)
190
185
180
175
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Week (1-4: Pre-treatment; 5-20: Treatment)
Figure 4-2. Changes in cortisol levels across study weeks. Entries show mean and standard
error of the mean. Serum cortisol levels during the treatment phase were lower than during
pre-treatment phase. (n = 14).
96
Cortisol (ng/ml)
188
*
**
120
140
186
184
182
20
40
60
80
100
Time (minutes)
Figure 4-3. Pattern of cortisol release over sampling periods. Note that cortisol levels at the
last two periods were significantly lower than the cortisol levels at 20th to 80th minute of
sampling indicating maximum plasma concentration of (-)-cathinone at these periods. *
means statistically significant at P < 0.05, ** means statistically significant at P < 0.01, (n =
14).
97
Control
0.8
1.6
3.2
6.4
205
Cortisol (ng/ml)
195
185
175
165
155
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Week (1-4: Pre-treatment; 5-20: Treatment)
Figure 4-4. Group differences in patterns of changes in serum cortisol across experimental
period. There was a significant (P < 0.001) group by week interaction among treatment
groups compared to controls. (n = 14).
98
Control
0.8
1.6
3.2
6.4
40
60
80
100
120
Cortisol (ng/ml)
198
188
178
168
20
140
Time (minutes)
Figure 4-5. Group differences in patterns of change in serum cortisol levels across sampling
periods. Note a group x sampling period interaction with dose group 3.2 and 6.4 mg/kg body
weight of (-)-cathinone showing a significant (P<0.05) decrease compared to low dose groups
and controls. (n = 14).
99
30
***
Prolactin (ng/ml)
**
25
**
20
***
15
10
Control
0.8
1.6
3.2
6.4
Group (mg/kg)
Figure 4-6. Changes in serum prolactin levels across treatment groups. Entries show mean
and standard error of the mean. Note a significant decrease in serum prolactin at (-)-cathinone
dose 6.4 mg/kg body weight compared to other treatment groups and controls. ** P < 0.01,
*** P < 0.001. * - statistically significant. (n = 14).
100
Prolactin (ng/ml)
31
27
23
19
15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Week (1-4: Pre-treatment; 5-20: Treatment)
Figure 4-7. Pattern of serum prolactin levels during pre-treatment and treatment weeks.
Entries show mean and standard error of the mean. Levels during the treatment phase were
lower than the levels during the pre-treatment phase. (n = 14).
101
Prolactin (ng/ml)
22
***
***
**
***
80
100
120
140
21
20
19
18
20
40
60
Time (minutes)
Figure 4-8. Changes in prolactin levels over sampling period. Entries show mean and
standard error of the mean. Levels at the last four periods were lower than the levels at the
first period. ** P < 0.01, *** P < 0.001. * - statistically significant. (n = 14).
102
Control
0.8
1.6
3.2
6.4
35
Prolactin (ng/ml)
30
25
20
15
10
5
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
Week (1-4: Pre-treatment; 5-20: Treatment)
Figure 4-9. Group differences in patterns of changes in serum prolactin across study weeks
showing a strong group by week interaction. There was a significant group by week
interaction with treatment group 3.2 and 6.4 mg/kg body weight of (-)-cathinone compared to
that of (-)-cathinone dose 0.8 mg/kg and 1.6 mg/kg body weight. (n = 14).
103
Control
0.8
1.6
3.2
6.4
32
28
Prolactin (ng/ml)
24
20
16
12
8
20
40
60
80
100
120
140
Time (minutes)
Figure 4-10. Group differences in patterns of change in serum prolactin levels across
sampling periods. There was a significant group by sampling period interaction compared to
controls. (n = 14).
104
160
140
*
LH conc (mUI/ml)
120
100
80
60
40
***
**
**
20
0
0
0.8
1.6
3.2
6.4
Treatment group (mg/kg)
Figure 4-11. Changes in serum LH levels across groups. Entries show mean ± standard error
of the mean. Note a significant increase (P<0.001) in LH levels for dose 3.2 and 6.4 mg/kg
body weight of (-)-cathinone compared to low doses and controls (n = 14).
105
0 mg/kg
0.8 mg/kg
1.6 mg/kg
3.2 mg/kg
6.4 mg/kg
200
180
160
LH conc (mIU/ml)
140
120
100
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
Week (1-4: pre-treatment; 5-20: treatment)
Figure 4-12. Changes in serum LH levels across study weeks. Entries show mean ± standard
error of the mean. At dose 3.2 mg/kg and 6.4 mg/kg body weight of (-)-cathinone from week
5 to week 20 LH levels were significantly higher (P<0.001) than at dose 0.8 mg/kg and 1.6
mg/kg body weight of (-)-cathinone as well as controls.
106
0 mg/kg
0.8 mg/kg
3.2 mg/kg
6.4 mg/kg
40
80
1.6 mg/kg
180
160
LH conc (mIU/ml)
140
120
100
80
60
40
20
0
0
20
60
100
120
140
160
Time (min)
Figure 4-13. Group differences in patterns of change in serum LH levels across sampling
periods. Note a significant (P<0.001) group x sampling period interaction with (-)-cathinonetreated animals at dose 3.2 and 6.4 mg/kg body weight showing a steady increase compared to
dose 0.8 mg/kg and 1.6 mg/kg body weight and controls (n = 14).
107
0 mg/kg
0.8 mg/kg
1.6 mg/kg
3.2 mg/kg
6.4 mg/kg
5
4.5
Testosterone conc (ng/ml)
4
3.5
3
2.5
2
1.5
1
0.5
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
Week (1-4: pretreatment; 5-20: treatment)
Figure 4-14. Group differences in patterns of changes in testosterone across study weeks.
Note a significant increase in testosterone at dose 3.2 mg/kg and a significant decrease at dose
6.4 mg/kg body weight of (-)-cathinone compared to controls. (n=14).
108
0 mg/kg
0.8 mg/kg
1.6 mg/kg
3.2 mg/kg
6.4 mg/kg
4.5
Testosterone conc (ng/ml)
4
3.5
3
2.5
2
1.5
1
0.5
0
0
20
40
60
80
100
120
140
160
Time (min)
Figure 4-15. Group differences in patterns of change in serum testosterone levels across
sampling periods (a significant group x sampling period interaction). Note the biphasic effect
with two high doses (3.2 mg/kg increased while 6.4 mg/kg decreased testosterone levels over
time) of (-)-cathinone, (n=14).
109
0 mg/kg
0.8 mg/kg
1.6 mg/kg
3.2 mg/kg
6.4 mg/kg
3
Progesterone conc (ng/ml)
2.5
2
1.5
1
0.5
0
0
1
2
3
4
5 6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
Week (1-4: preatreatment; 5-20: treatment)
Figure 4-16. Group differences in patterns of changes in serum progesterone across study
weeks. There was a dose- and time-dependent decrease in serum progesterone over study
period in treatment groups compared to controls, (n=14).
110
Prolactin (ng/ml)
35
20
y = 0.4984x - 72.227
R2 = 0.5289
5
160
185
210
Cortisol (ng/ml)
Figure 4-17. Correlations between serum cortisol and prolactin during week 1 of treatment
phase. Note a strong positive correlation r2 = 0.53 between serum prolactin and cortisol.
111
4.3 DISCUSSION
4.3.1 Effect of (-)-cathinone on cortisol and prolactin.
The results of the present study show that administration of (-)-cathinone produced a doseand time-dependent decrease in serum cortisol and prolactin. The effects are suggestive of
pleasurable responses from reward centres in the brain that necessitate psychic dependence on
khat use (Halbach, 1972). Recent findings showed a decrease in serum cortisol levels in
Sprague Dawley rats treated with 5 mg/kg body weight of (-)-cathinone (Mohammed and
Engidawork, 2011). These results are consistent with those of other findings in baboons
(Mwenda et al., 2006) indicating that khat causes a decrease in plasma cortisol and prolactin
levels.
Although the present study indicated a decrease in serum cortisol and prolactin, most previous
studies concur with findings on prolactin but not cortisol measurements. This is supported by
earlier reports implicating (-)-cathinone in inhibition of release of several anterior pituitary
hormones (Wagner et al., 1982). Previous studies have shown that (-)-cathinone causes an
increase in levels of extracellular dopamine but decreases dihydroxyphenyl acetic acid
(DOPAC) and homovanillic acid (HVA) in vivo (Pehek et al., 1990). Findings on earlier in
vitro studies and of tissue content (reviewed in Kalix and Braenden, 1985) indicating that (-)cathinone, like amphetamine, causes release of dopamine and inhibits uptake of dopamine in
vivo points to a similar mechanism. It appears that the dose-dependent inhibitory effect of (-)cathinone on prolactin in the present study shared a similar mechanism of action. Studies on
behavioural effects of khat and (-)-cathinone have shown strong evidence to suggest
involvement of dopaminergic system (Kalix and Braenden, 1985; Zelger et al., 1980). It is
112
very likely, therefore, that (-)-cathinone influenced behaviours reported in chapter 3 of this
thesis via observed low serum prolactin levels in treated animals.
Previous studies reported varying findings on use of psychostimulants and their effects on the
hypothalamo-hypophyseal-adrenocortical system. For example, high levels of glucocorticoids
were shown to contribute to development, maintenance and outcome of substance abuse
disorders (King et al., 2003). Other studies on khat have reported increase in plasma cortisol
in rabbits (Nyongesa et al., 2008) and adrenocorticotrophic hormone in humans (Nencini et al.,
1984). These findings are at variance with those in baboons (Mwenda et al., 2006), rats
(Mohammed and Engidawork, 2011) and those of the present study. Studies have shown that
stress stimulates adrenocorticotrophic hormone production and subsequent adrenal steroid
secretion (Czeisler et al., 1976), and this response in adrenocortical secretion correlates
positively with the intensity of stressful stimuli (Murton et al., 1998). From the
aforementioned, animal handling and manipulations are likely to exaggerate cortisol
measurements in response to a given drug exposure. This has been explicitly explained by
Dettmer et al. (1996) in a study on behavioural and cortisol responses to repeated capture and
venipuncture in Cebus apella. In this study, it was shown that animals that were behaviourally
habituated to handling and blood collection procedures had low cortisol levels compared to
non-habituated naive subjects. In the present study, all animals were habituated to handling
for four weeks before experimentation and during blood sampling both controls and test
animals were handled the same way. Furthermore, serial blood sampling was done at constant
time (1000 h) of every sampling day of the week to avoid influence of time of day on cortisol
levels as glucocorticoid hormone release is characterized by a circadian cycle (Akana et al.,
1996). The observed pattern of cortisol hormone release in the present study is likely a
113
function of (-)-cathinone treatment. The (-)-cathinone doses used in the present study were
based on knowledge of previous studies in humans and experimental animals (Mohammed
and Engidawork, 2011; Widler et al., 1994; Pehek et al., 1990; Brenneisen et al., 1990). Most
of these studies did interventional studies with similar protocol as that employed in the
present study. From the results of the present study, it is apparent that behavioural scores
reported in chapter 3 were not influenced by cortisol secretion. Earlier studies reported
locomotor responses induced by injection of psychostimulants in nucleus accumbens (Delfes
et al., 1990) and decrease in locomotor responses occur by suppression of corticosterone and
re-established by restoring basal levels of the hormone (Marinelli et al., 1994). In another
separate study glucocorticoid secretion was shown to precede many goal-seeking behaviours
such as food-seeking (Bassareo and Di Chiara, 1999; Wilson et al., 1995) and drug-seeking
behaviour (Cadoni et al., 2003). In the present study, however, the appetitive/consummatory
behaviour was impaired in (-)-cathinone subjects indicating little role of cortisol on the
observed behaviours.
The findings of the present study also show a strong positive correlation between cortisol and
prolactin during treatment phase suggesting that the mode of action of (-)-cathinone on the
two systems producing these hormones may be similar and appear to influence each other.
Studies have shown that activation of the hypothalamo-hypophyseo-adrenocortical axis and
ascending catecholaminergic neurons play an important role in metabolic and behavioural
adaptation to stress (Ishikawa et al., 2007). Similarly, a precise overlap of processes
containing corticotrophin-releasing hormone (CRH) and serotonin in the dorso-lateral subnucleus have been demonstrated (Ruggiero et al., 1999). Since cortisol is synthesized in
specific cells of the adrenal glands, released in the peripheral blood flow and its production
114
controlled centrally by classical negative feedback system of the hypothalamo-hypophysealadrenocortical axis (McEwen, 2000), its low levels observed in the present study may partly
be explained by reduced synthesis of CRH and/or release at the hypothalamus or reduced
responsiveness of its receptors at the pituitary and adrenal gland as a function of (-)- cathinone
exposure.
Another hypothesis that attempts to explain the observed low serum cortisol levels in the
present study is the possible involvement of the extra-hypothalamic CRH systems that have
been implicated in behavioural arousal (Landgraf, 2005) and depression (Mitchell, 1998).
Studies in humans showed an association between CRH innervations and serotonergic
neurons in the midline raphe (Ruggiero et al., 1999), consistent with the findings in rats that
demonstrated localization of the CRH neuropeptide and its receptor binding sites (De Souza,
1995) as well as mRNA expression of subtype 2 to the midline raphe (Chalmers et al., 1995).
Serotonin-containing bodies of the raphe nuclei project to dopaminergic cells in the ventral
tegmental area, substantia nigra, nucleus accumbens, prefrontal cortex and striatum
(Moukhles et al., 1997; Van Bockstaele et al., 1994). This association may explain, in part,
the behavioural output following a given stimulus to the hypophyseo-adrenocortical axis.
Studies in humans, for example, showed that pregnant mothers undergoing depression
produced decreased salivary cortisol and increased serotonin and dopamine (Field et al.,
2005), consistent with a similar study on postpartum depressive mothers that showed
behavioural and stress hormone changes including decrease in anxiety levels and salivary
cortisol following massage therapy (Field et al., 1996). It appears that the rewarding system in
the brain of humans including non human primates consists of a network of connections
between serotonergic and dopaminergic projections in the meso-corticolimbic system and
115
hypothalamic CRH projections, and that these interconnections may be unique in these
species different from other animals. The present study did not consider investigation in this
direction. Further research may prove essential to the understanding of pharmacokinetics of
psychostimulants to the reward centres of the brain. However, the serotonergic system has
been reported to be involved in cathinone-induced stereotyped movement, aggression and
sexual arousal (Abdulwaheb et al., 2007; Banjaw et al., 2006; Connor et al., 2002).
Aggression and stereotypy that characterize abnormal behaviour have been reported in
chapter 3 of this thesis. It is possible (-)-cathinone caused the observed behaviours via its
action on the serotonergic system.
The similarity in results on hormonal profiles in non-human primates as seen in the present
study and that of Mwenda et al. (2006) but different from humans (Nencini et al., 1984),
raises important unanswered questions: Is there a threshold of (-)-cathinone exposure above
which persistent hormonal changes in hypophyseal-adrenocortical system are induced? What
factors influence differences in vulnerability to persistent neuro-endocrine changes following
cathinone exposure? Does the cumulative exposure as a result of daily khat use produce
persistent dopaminergic and hormonal changes associated with behavioural manifestations
observed in khat ‘addicts’? Is there a species difference in the reward centres of the brain
connecting the hypothalamo-hypophyseo-adrenocortical axis? Does the choice of dose of
drug matter when dealing with different species of animals under experimentation? These
questions underscore the need to determine which animal species, dose range and/or
experimental paradigms best simulate daily human khat consumption. The mechanisms
underlying changes in hypothalamo-hypophyseal-adrenocortical axis and dopaminergic
system following khat use remain speculative. However, some evidence demonstrate marked
116
species differences in vulnerability to simulate induced hormonal changes in rabbits
(Nyongesa et al., 2008), baboons (Mwenda et al., 2006) and rats (Mohammed and
Engidawork, 2011). It is also possible that after multiple repeated exposures to khat, tolerance
develops and this may have contributed to variability in effects. Tolerance and cross-tolerance
between (-)-cathinone and cathine have been reported in animal studies (Foltin et al., 1983)
and these effects are probably mediated by pre-synaptic dopamine release (Schechter, 1990).
Similar reports indicate that after chronic exposure to amphetamines, animals exhibit either
tolerance or sensitization during subsequent drug administration, indicating adaptations in the
neurobiological substrates of these behaviours (Berman et al., 2009).
In the present study, a dose- and time-dependent effect of (-)-cathinone on cortisol and
prolactin hormonal profiles in vervet monkeys are presented. The pattern of dose and time as
functions of khat/(-)-cathinone stimulatory effects has been demonstrated in several studies.
Neuropharmacological evaluation of effects of khat in rats showed that different
concentrations caused psycho-stimulation followed by depression, characterized by a
stimulatory electro-encephalogram (EEG) pattern with lower doses (50 – 100 mg/kg) while
higher doses (400 mg/kg) caused initial activation followed by EEG depression (Saleh et al.,
1988). These EEG patterns were observed to resemble the progression of psychostimulation
and excitation to that of sedation, anxiety and depression with continued khat chewing
(Hassan et al., 2002), indicating a dose and time effect to neurobiological manifestations in
the khat chewer. Similarly, acute and sub-chronic administration of Catha edulis leaves or S
(-)-cathinone showed increased locomotor activity in rats (Banjaw and Schmidt, 2006).
Similarities between (-)-cathinone and amphetamine have also been demonstrated with regard
to induction of stereotyped behaviour in rats (Banjaw and Schmidt, 2005) and mice (Al-
117
Meshal et al., 1991) at high doses. Earlier reports showed that cathinone induces tremor at
low doses and seizure at high doses (Berardelli et al., 1980). Recently, studies showed a doseand time-dependent effect of khat extract on spatial learning and memory in mice (Kimani
and Nyongesa, 2008). In this study it was observed that low doses of khat extract had no
significant effect on learning but impaired memory while high doses impaired learning but
improved memory. Increase in aggression, anxiety and abnormal behaviours as well as
withdrawal and impaired consummatory/appetitive behaviour following (-)-cathinone
administration to vervet monkeys have been described in chapter 3 of this thesis. These
differences on neuro-behavioural manifestations indicate the effect of dose of khat/(-)cathinone and duration of exposure on different neurobiological systems of the brain.
4.3.2 Effect of (-)-cathinone on Luteinizing hormone, progesterone and testosterone.
The results of the present study show a dose- and time-dependent increase in LH with the
significant increase at high doses of (-)-cathinone. Conversely, serum progesterone levels
decreased in a dose- and time-dependent manner while the effect of (-)-cathinone on
testosterone were biphasic, with low and moderate doses increasing while high dose
decreasing its production. From the results, it appears (-)-cathinone played a stimulatory role
either at the level of the hypothalamus or directly on pituitary gland to cause the observed
increase in LH. There is prevailing evidence that gonadotrophic releasing hormone (GnRH)
produced by the hypothalamic neurosecretory cells is involved in regulation of both LH and
Follicular stimulating hormone (FSH) secretion (Hadley, 1988; Bardin and Paulsen, 1981).
The increase in testosterone following increasing LH in the present study is interesting.
Following the classical negative feedback loop, it is expected that increasing levels of
testosterone could have inhibited further production of LH although this depends on whether
118
or not elevated testosterone levels persisted for long. However, earlier studies showed
evidence of a differential control of LH and FSH secretion (Burger, 1989; Hall et al., 1988).
Norepinephrine has been shown to play a principal role in promoting LH release via GnRH
while serotonin inhibits LH release (Knol, 1991). Since khat and (-)-cathinone are
sympathomimetic in that they produce their effects by increasing synaptic levels of biogenic
amines: dopamine, noradrenaline and serotonin, through multiple mechanisms (Fischman and
Madras, 2005), it is possible the observed effects in the present study were due to levels of
these neurotransmitters in circulation.
The biphasic effects of khat and (-)-cathinone have been reported by several investigators. For
example, previous studies showed that low dose of khat was stimulatory while high dose was
inhibitory to sexual motivation and performance in male rats (Abdulwaheb et al., 2007),
consistent with testosterone measurement in mice isolated interstitial cells in vitro (Nyongesa
et al., 2007). Quite recently, a study in rats demonstrated that mild doses of khat improved
sexual motivation but did not interfere with performance while high doses impaired both
motivation and performance (Mohammed and Engidawork, 2011). It was suggested that
alteration of both dopamine (at low dose) and serotonin (at high dose) levels in the central
nervous system could explain the biphasic sexual behaviour of rats after khat administration
(Abdulwaheb et al., 2007; Taha et al., 1995). However, the role of testosterone on this
measure can not be ignored. The effect of (-)-cathinone on stereodogenic structures,
particularly smooth endoplasmic reticulum and mitochondria cristae, as well as
dehydroxysteroid dehydrogenase enzymes (3- HSD and 17- HSD) have been reported in
chapter 5 and 6 of this thesis, all of which point to the observed testosterone measurements.
The increase in testosterone following (-)-cathinone treatment may in part explain increases in
119
indicators of aggression (yawn, engage in a stare, bouncing off cages walls, vigorous shaking
of cages and head jerk) reported in chapter 3 of this thesis. In primates, the correlation
between aggression and increase in circulating testosterone levels has been reported (Alberts
et al., 1992; Bernstein et al., 1979).
The decrease in serum progesterone in (-)-cathinone subjects may be explained independently
or in relation to the observed testosterone secretion pattern. Firstly, progesterone is one of the
intermediate hormones that are produced in the steroid biosynthetic pathway that ultimately
ends up with testosterone and estrogen production (Christensen, 1975). The decreasing levels
of progesterone may, therefore, be explained by its enzymatic breakdown by hydroxysteroid
dehydrogenases into formation of androstenedione and subsequently testosterone (Andersson
et al., 1995). Secondly, high levels of LH and testosterone in the present study may explain
decreasing levels of progesterone. Earlier studies have shown that endogenous corticosteroid
and progesterone are negatively correlated to LH and testosterone (Welsh et al., 1981). The
observed increase in LH and testosterone in the present study may have influenced the
secretion of progesterone hormones.
It is worth noting that vervet monkeys are social animals that live under social complex
interactions and so studies on sexual function under captive conditions must be interpreted
with caution. However, previous studies have shown that vervet monkeys readily adapt to
captivity and reproduce throughout the year (Seier, 2005) and so the observed hormone
profiles may not be due to seasonality in reproduction. Owing to the necessity of obtaining
blood samples at regular intervals as well as repeated (-)-cathinone administration as per
stringent schedule, it would be a stressful undertaking for group-housed vervets.
120
Conclusively, the findings of the present study have shown that (-)-cathinone exposure caused
hormonal alterations in a dose- and time- dependent manner. These results confirm and
extend previous findings, in demonstrating effects of (-)-cathinone on hypothalamohypophyseal-adrenocortical and gonadal axes as well meso-corticolimbic systems.
121
CHAPTER 5
IMMUNOHISTOCHEMICAL
LOCALIZATION
OF
ANTERIOR
PITUITARY CELL TYPES OF VERVET MONKEYS (CHLOROCEBUS
AETHIOPS) FOLLOWING SUB-CHRONIC CATHINONE EXPOSURE
5.0 INTRODUCTION
The cells within the pituitary gland respond to continuously changing central nervous system
as well as adrenocortical and gonadal signals and provide appropriate responses. As a result,
changes in pituitary cell function serve to integrate a number of complex mechanisms
associated with response to various stressful stimuli or successful reproduction. It would,
therefore, be expected that natural products that are routinely consumed or used by human
population may affect aspects of reproductive capacity or elicit stressful responses via
alteration of pituitary function. However, studies on use of psychotropic drugs often ignore
measures of hypothalamo-pituitary or pituitary function in response to various degrees of
exposure to these drugs.
Many drugs of abuse exert an inhibitory or stimulatory action on pituitary function by
modifying hypothalamic control of pituitary hormone secretion (Steckler and Holsboer, 1999;
Sillaber et al., 1988). They do so by altering neurotransmitter and neuropeptide activity
(Cooper et al., 1986). Previous studies have shown that amphetamine, which resembles (-)cathinone in chemical structure and function (Graziani et al., 2008; Houghton, 2004; Cox and
122
Rampes, 2003) and with similar potential for abuse (Kalix, 1984 a), causes increasse in
gonadotrophic cells following long-term use (Tsai et al., 1996). In a similar study long-term
use of amphetamine caused an increase in mixed cell follicles in the adenohypophysis and
reduction in number of lactotrophs in the immunohistochemical sections (Ishikawa et al.,
2007).
Previous studies on khat and (-)-cathinone have shown varying effects on the pituitary gland.
For example, earlier studies showed that (-)-cathinone is an inhibitor of several anterior
pituitary hormones (Wagner et al., 1982). Similar studies on effects of escalating doses of
khat extracts suppressed plasma LH levels in rabbits (Nyongesa et al., 2008). In other studies
there was increase in adrenocorticotrophic hormone in humans (Nencini et al., 1984),
consistent with cortisol measurement in rabbits following khat extract exposure (Nyongesa et
al., 2008) but different from findings in baboons where decreased plasma cortisol was
observed (Mwenda et al., 2006). The levels of these pituitary hormones correspond to the
activity of the specific cell types that synthesize them that is: gonadotrophs, corticotrophs and
lactotrophs. It is this scanty and contradictory information that led to the hypothesis of the
present study. It was hypothesized that cathinone variously affects the expression of
gonadotrophs, corticotrophs and lactotrophs and consequently their hormonal secretions or
release of releasing factors from hypothalamus. This study investigated effect of (-)-cathinone
on hypothalamo-pituitary- adrenocortical and gonadal axes. The hypothalamic function was
tested
by
use
of
GnRH
agonist
goserelin
acetate
(ZOLADEX)
followed
by
immunolocalization of gonadotrophs on anterior pituitary sections while pituitary function
was evaluated by direct immunolocalization of lactotrophs and corticotrophs.
123
5.1 MATERIALS AND METHODS
5.1.1 ANIMALS AND HOUSING
See section 2.1 of general materials and methods.
5.1.2 EXPERIMENTAL DESIGN
The experimental design for this study is the same as was described previously in section 2.4
of general materials and methods. At the end of the 4 month (-)-cathinone alone treatment,
animals on low and high doses (0.8 and 6.4 mg/kg body weight) of (-)-cathinone were
administered ZOLADEX at 50 μg/kg body weight single subcutaneous injection for a further 2
weeks while continuing with (-)-cathinone treatment. ZOLADEX was prepared at 3.6 mg in
10% DMSO in PBS. At the end of treatment period, all animals were euthanized with a high
dose of ketamine and anterior pituitary gland harvested for immunohistochemical evaluation.
5.1.3 IMMUNOHISTOCHEMISTRY
The anterior pituitary glands of all the animals were harvested and fixed with 4%
formaldehyde in phosphate-buffered saline (pH 7.2) for 1 h then embedded in paraffin wax.
Serial sections (5 μ thick) were carefully cut and labelled with animal number and the stain to
be used. Immunostaining was done by avidin-biotin complex method and colour development
done using 3, 3’ diaminobenzidine. The slides were placed on Dako Autostainer Slide Racks
(code S3704) and inserted in an oven at 100 0C for 15 minutes and allowed to cool before
proceeding with antigen retrieval. Appropriate labels of slides were generated from a
computer connected to Dako Link 48 instrument, fixed carefully on slides to avoid damage to
the barcode. Thereafter, slides were inserted into the Dako target retrieval solution in PT Link
124
tanks (code PT100/PT101), which incorporated pre-heat temperature, antigen retrieval
temperature and time as well as cool down settings at 970C for 20 minutes. The Dako target
retrieval solution was prepared (3:1) according to specifications and used for both
deparaffinization and antigen retrieval. Each autostainer slide rack was removed from PT
Link tanks and immediately dipped into PT Link rinse solution (code PT109) containing
diluted room temperature Dako Wash buffer (10x) (code S3006) and left for 1 – 5 minutes.
The slides were then removed and washed gently with diluted room temperature wash buffer
to remove any debris on slides before insertion into Autostainer instrument. Thereafter, slides
were placed on a Dako autostainer instrument containing respective antibodies. Primary
antibodies were monoclonal mouse anti-human adrenocorticotrophic hormone, monoclonal
mouse anti-human luteinizing hormone, polyclonal rabbit anti-human prolactin and
polyclonal rabbit anti-human S100 (Dako). Detection was performed using anti-mouse or
anti-rabbit Dako Envision system (biotinylated secondary antibodies for mouse and rabbit) as
appropriate. Peroxidase activity was revealed by diamino 3, 3’-benzidine Substrate
Chromogen System (Dako) for LH, prolactin, ACTH and S100. Hematoxylin and eosin were
used for assessing normal morphology of cells of the pituitary gland.
A summary of type of antigen, antibody, species, source as well as dilution of the antibodies
for staining are given (Table 5-1).
The Autostainer machine runs at an automated duration of time, which appears on the screen
during the entire staining period. After completion of staining, the slides were carefully
removed from the autostainer and drained of excess distilled water and arranged in a staining
rack followed by dehydration, clearing and counterstaining with hematoxylin and cover
slipped for microscopic examination.
125
5.1.4 HISTOLOGICAL EVALUATION
The sections were examined and desired fields photographed using a Zeiss Axioskop 40 plus
microscope (Carl Zeiss MicroImaging GmbH, Germany) and a ProgRes CT5 USB C plus
digital camera (Jena, Germany) with ProgRes Capture Pro 2.8 software (JENOPTIK/Optical
Systems). Immunohistochemistry results for antibodies of interest were classified on presence
or absence of staining as well as average number of immunostained cells per three randomly
selected high magnification fields (x 40 objective). Slides that were stained with hematoxylin
and eosin were used for standard histology.
5.1.5 MORPHOMETRIC ANALYSIS
Quantification of immunostained cells for ACTH, prolactin and LH on anterior pituitary
sections was done by direct manual counting technique previously applied in measuring the
mitotic rates in the endometrium of primates (Brenner et al., 2003). Immunostained cells were
observed under light microscopy and three non-overlapping fields at high power
magnification (x 400) randomly selected with the help of an ocular grid and counted using a
mechanical tabulator. The labelling index (LI) was used to express the number of stained cells
as a percentage of the entire population of cells on a section of the slide. The mean values of
LI were then used for comparison among different treatment groups. In order to calculate the
LI, the following formula was used:
LI = (PiC+/ PiCT) x 100
Where,
126
PiCT = total number of cells for each stain (ACTH, prolactin, LH) that was counted in
a given field (positive and negative cells for anterior pituitary),
PiC+ = number of immunostained cells (positive cells) for a given stain counted on the
same area,
LI = labelling index, usually expressed as a percentage
127
5.2 RESULTS
Pituitary sections of all the animals (n = 14) from all the four (-)-cathinone dose groups were
generally histologically similar compared to controls. The sections were characterized by
clusters and islands of cells separated by thin sinusoidal spaces and slightly larger vascular
profiles. The anterior pituitary parenchyma consisted of predominantly acidophils with rare
numbers of basophils and chromophobes. Acidophils appeared uniform in size, stained deeply
eosinophilic, and laden with cytoplasmic granules. Some of the acidophils exhibited
degranulation. Basophils stained slightly basophilic and chromophobes showed pale
cytoplasm. The sinusoids were lined by single layer of endothelium and appeared slightly
widened in some areas. Fairly dilated sinusoids were distinguished as an effect of fixation and
processing but not of any morphological significance. Some eosinophilic cells with lobulated
nuclei were also observed to be a common occurrence in the vervet monkey since these
features were seen in sections of controls and treated groups. There were no other significant
morphological changes observed. Histology of the sections showed physiologically active
glands (Fig. 5-1).
Since immunochemical staining with all antibodies did not show significant difference in
pituitary sections of animals treated with 0.8 mg/kg and 1.6 mg/kg body weight of (-)cathinone, the immunohistochemical pictures in the latter case are not presented here but the
former are presented for data comparison since it is the lowest (-)-cathinone dose used in the
study. Immunohistochemistry for staining of basophils showed no obvious difference in the
intensity of cytoplasmic staining across treatment groups and controls. In sections stained
with mouse anti-human ACTH antibody, two cell populations were identified with the
predominant proportion of mostly acidophils that appeared negative for ACTH stain.
128
Positively staining cells, interpreted as corticotrophs, revealed uniform intracytoplasmic
staining and appeared arranged individually and occasionally in clusters of 2 - 3 cells (Fig. 52 a-d). A random selection of three fields at x 40 objective showed a dose-dependent decrease
in LI of cells. Mean LI of corticotrophs were: 48 ± 4, 36 ± 5, 35 ± 5.6, 22 ± 6 and 14 ± 3.5 for
doses 0, 0.8, 1.6, 3.2 and 6.4 mg/kg body weight of (-)-cathinone (Fig. 5-3).
The sections stained with mouse anti-human Luteinizing Hormone showed a similar pattern
obtained with ACTH staining, with two cell populations under light microscopy. The larger
percentage of cells was composed mostly of acidophils that were negative for LH stain. The
staining pattern was cytoplasmic and positive cells, interpreted as gonadotrophs, were mostly
scattered throughout the field and occasionally in groups of 2 – 3 cells. These cells maintained
a dispersed pattern and intensity of staining was similar for within- and between- treatment
groups. However, the number of stained cells when taken at three randomly selected high
power fields (x 40 objective) was significantly different across treatment subjects as shown by
their LI (Fig. 5-4). The mean LI for positive cells for low dose of (-)-cathinone co-treated with
GnRH agonist was higher (46 ± 8) compared to doses 1.6 and 3.2 mg/kg body weight as well
as controls. At doses 1.6 and 3.2 mg/kg body weight, mean values of 28 ± 5.4 and 43 ± 10,
respectively, were counted indicating that GnRH agonist indeed boosted the expression of
positive staining cells. At the highest (6.4 mg/kg body weight) dose of (-)-cathinone, there
was a significantly higher (P < 0.05) number of positive cells (78 ± 14.8) compared to other
doses and controls (20 ± 10). The results show a dose-dependent effect of (-)-cathinone on
positive cell count and the synergistic effect of GnRH agonist (Fig. 5-5).
129
In sections stained with polyclonal rabbit anti-human prolactin, two cell populations were
observed at light microscopy, most of which were negative for prolactin. Positive cells
(lactotrophs) were scattered in the field occasionally in clusters of 2-4 cells. These positive
cells maintained a dispersed pattern, and intensity of staining was similar for within- and
between- treatment groups. However, the LI of stained cells when taken at three randomly
selected high power fields (x 40 objective) was significantly different across treatment
subjects (Fig. 5-6). Results show a dose-dependent decrease in number of positive cells
counted in three randomly selected x 40 fields. At doses (0.8, 1.6, 3.2 and 6.4 mg/kg body
weight) of (-)-cathinone, 61 ± 3, 58 ± 5, 36 ± 6.2 and 18 ± 5, respectively compared to
controls (63 ± 4.5) were counted (Fig. 5-7).
Immunostaining of cells with polyclonal rabbit anti-human S100, a multifunctional marker of
cellular degeneration, showed no apparent staining in both pituitary sections of (-)-cathinone
subjects and controls (Fig. 5-8). In order to validate the specificity of the assay, positive
control for S100 (skin melanoma known to be positive for this stain) was performed in
parallel with test samples (Fig. 5-9). This means that the observed difference in labelling
index of positive cells with different antibodies was due to the effect of (-)-cathinone on
expression of the hormones in the positive cells and not due to cellular degeneration. The
absence of staining with S100 in samples and staining for positive control shows that the
assay worked but cellular degeneration was absent.
A summary of effect of escalating doses of (-)-cathinone on anterior pituitary cell types
considered in this study are shown (Table 5-2).
130
Table 5-1. Immunohistochemistry summary table. B stands for antigen retrieval for 20
minutes at 970C in Dako target retrieval solution.
Antigen
Antibody
Species
Source Dilution
code
ACTH
M3501
Antigen
retrieval
Monoclonal mouse anti- Dako
1:50
B
1:50
B
1:200
B
Polyclonal rabbit anti- Dako
Ready-to-
B
human
use
human
LH
M3502
Monoclonal mouse anti- Dako
human
Prolactin A0569
Polyclonal rabbit anti- Dako
human
S100
IR504
131
Table 5-2. Mean number of cells at high power magnification for each (-)-cathinone dose. LI
means labelling index; a* in presence of GnRH agonist and significantly different (P<0.05); *
- significantly different at (P<0.05). bwt means body weight.
LI (%) of positive cells
(-)-Cathinone dose (mg/kg bwt)
0
LI
for
ACTH
at
x
400 48 ± 4
0.8
1.6
3.2
6.4
36 ± 5
35±5.6 22 ±6*
14±3.5*
46±8a*
28±5.4 43±10
78±
magnification)
LI for LH at x 400 magnification)
LI
for
PRL
at
x
20±10
+
14.8a*
agonist
+ agonist
400 63±4.5 61±3
magnification)
132
58±5
36±6.2* 18±5*
Figure 5-1. Hematoxylin and eosin staining of adenohypophysis of vervet monkey. Similar
morphological features between treatment groups and controls were observed. The gland
shows presence of acidophils (a), basophils (b) and sinusoids (s) lined by epithelial cells
(filled up arrow). Open arrow shows degranulation into sinusoids. Original magnification x
400.
133
Figure 5-2. Dose-dependent effect of (-)-cathinone on corticotrophs in anterior pituitary
gland. The pictures show positive staining for corticotrophs ( full arrows) in saline-treated
controls (A), (-)-cathinone-treated animals at 0.8 mg/kg (B), 3.2 mg/kg (C) and 6.4 mg/kg
(D). Original magnification x 400
134
Mean Labelling index for corticotrophs (%)
60
50
40
**
30
***
20
10
0
0
0.8
1.6
3.2
6.4
Cathinone dose (mg/kg body weight)
Figure 5-3. Mean labelling index (LI) indicating number of corticotrophs as a percentage of
total cells in sections of control animals and (-)-cathinone-treated groups. Results show a
dose-dependent decrease in mean LI of cells counted in three random x 40 fields with a
significant decrease (P < 0.05) at dose 6.4 mg/kg body weight of (-)-cathinone, (n=14). **
means statistically significant at P <0.05, *** means statistically significant at P <0.01.
135
Figure 5-4. The figure shows gonadotrophs ( open arrows) in anterior pituitary gland of
controls vervet monkeys (A) and vervet monkeys exposed to (-)-cathinone doses of 0.8 mg/kg
plus GnRH agonist (B), 3.2 mg/kg (C) and 6.4 mg/kg plus GnRH agonist (D). (-)-Cathinone
increased expression of gonadotrophs in a dose-dependent manner. Original magnification x
400.
136
Mean Labelling index for gonadotrophs (%)
***
90
80
70
**
60
**
50
40
30
20
10
0
0
0.8 +GnRH
agonist
1.6
3.2
6.4 +GnRH
agonist
Cathinone dose (mg/kg body weight)
Figure 5-5. Mean LI expressing number of gonadotrophs as a percentage of total pituitary
cells of control and (-)-cathinone-treated animals. Note that animals on low and high doses of
(-)-cathinone and co-treated with GnRH agonist expressed significantly higher (P<0.05)
number of cells compared to (-)-cathinone alone and controls (n =14) when counted on three
random fields at x 400 magnification. ** means statistically significant at P < 0.05, *** means
statistically significant at P < 0.01.
137
Figure 5-6. Immunostaining for lactotrophs (arrows) in anterior pituitary gland of controls
vervet monkeys (A) and vervet monkeys treated with (-)-cathinone doses of 0.8 mg/kg (B),
3.2 mg/kg (C) and 6.4 mg/kg (D). Original magnification x 400
138
Mean Labelling index for lactotrophs (%)
80
70
60
50
*
40
**
30
20
10
0
0
0.8
1.6
3.2
6.4
Cathinone dose (mg/kg body weight)
Figure 5-7. Mean LI expressing number of lactotrophs in controls and (-)-cathinone-treated
groups. Results showed a dose-dependent decrease in number of cells counted in three
random x 40 fields. The high doses (3.2 and 6.4 mg/kg body weight) of (-)-cathinone caused a
significant decrease (P < 0.05) compared to other doses and controls (n =14). * means
statistically significant at P<0.05, ** means statistically significant at P<0.01.
139
Figure 5-8. Immunostaining of adenohypophysis of vervet monkey with S100. The cells were
negative for the stain. Original magnification x 400.
140
Figure 5-9. Figure showing a skin tissue with melanoma positive for S100. Note the melona
around the hair follicle (bigger full arrow). Arrow head shows nuclei of muscle cells. Original
magnification x 400
141
5.3 DISCUSSION
Psychotropic drugs cause varying effects in the body of the user based on type and degree of
use. Most of these drugs influence the central nervous system function via disregulation of the
hypothalamic dopaminergic system (Quan et al., 2005). Drugs such as methamphetamine
have been shown to reduce long-term potentiation while increasing synaptic transmission in
the hippocampus of mouse (Swant et al., 2010). It is on the understanding of these varying
effects of psychotropic drugs on the central nervous system that the hypothesis of the present
study was designed.
The present study provides insight into the immunolocalization of anterior pituitary gland
cells specifically: corticotrophs, gonadotrophs and lactotrophs of vervet monkeys following
exposure to escalating doses of (-)-cathinone. The results show that increasing doses of (-)cathinone suppressed the labelling index of corticotrophs and lactorophs in a dose-dependent
manner. The significant effects were observed with high doses (3.2 and 6.4 mg/kg body
weight) of (-)-cathinone. These results can explain the dose-dependent decrease in serum
cortisol and prolactin levels reported in chapter 4 of this thesis. On the other hand, cell density
of gonadotrophs increased in a dose-dependent manner. However, in the presence of GnRH
agonist, low (0.8 mg/kg body weight) and high dose (6.4 mg/kg) of (-)-cathinone significantly
increased the immunolocalization of gonadotrophs. Luteinizing hormone levels in (-)cathinone subjects co-treated with GnRH agonist are not reported. Due to small sample size of
animals there were no controls for this intervention. However, results on immunolocalization
of pituitary cells in animals co-treated with GnRH agonist are reported here. This finding is
significant in that it points to the hypothalamic and pituitary integrity following (-)-cathinone
142
exposure. However, previous studies reported the inhibition of gonadotropin release by
dopamine binding to the receptors on basophils thereby activating a membrane G protein
(Yen, 1991). Previously, studies in rabbits reported a decrease in luteinizing hormone
production following khat extract administration (Nyongesa et al., 2008). Hormonal results of
the present study in chapter 4 show that (-)-cathinone increased and then decreased serum
testosterone levels in vervets. High dose of (-)-cathinone, however, increased LH as well as
labelling index of gonadotrophs but decreased serum testosterone levels. It appears that (-)cathinone at high dose acted through different mechanism of action leading to variability on
these measures. One possible mechanism is the cytotoxic effect of (-)-cathinone on Leydig
cells (Tariq et al., 1987). Earlier studies showed that luteinizing hormone binds to its
receptors on Leydig cells (Bardin and Paulsen, 1981) and stimulates synthesis of testosterone
(Castro et al., 2002). From the foregoing, it is hypothesized that effect of (-)-cathinone on
hypothalamo-hypophyseal axis may be species specific and that it influences hypothalamohypophyseo-gonadal axis via different mechanisms. Studies on receptor binding are required
to confirm the responsiveness of the anterior pituitary cells to hypophyseotrophic hormones
from the hypothalamus.
In this thesis, it is reported that (-)-cathinone caused a dose-dependent decrease in number of
lactotrophs, consistent within dose-dependent decrease in serum prolacin levels reported in
chapter 4. This is expected, given the understanding that (-)-cathinone induces release of
dopamine from several brain regions including striatum (Pehek et al., 1990; Kalix and
Braenden, 1985; Kalix, 1983 a). Since dopamine is a well known inhibitory factor to prolactin
release, its increase leads to a proportionate decrease in prolactin levels. This finding is
consistent with another study that showed decrease in prolactin levels following
143
administration of khat extract to baboons (Mwenda et al., 2006). The findings explain
behavioural manifestations reported in chapter 3 of this thesis. On the contrary, previous
findings on long-term use of amphetamine in humans showed hyperprolactemia due to
hyperfunction of prolactin cells (Ishikawa et al., 2007). This is unexpected since amphetamine
and (-)-cathinone have been shown to have same mechanism of action (Graziani et al., 2008;
Houghton, 2004; Cox and Rampes, 2003) at central dopaminergic (Kalix and Braenden,
1985) as well as other peripheral sites (Kalix and Braenden, 1985; Nencini et al., 1984; Knoll,
1979).
Results also showed a dose-dependent decrease in corticotrophs in anterior pituitary sections
as shown by the labelling index. Corticotrophs synthesize adrenocorticotrophic hormone
(ACTH) that in turn stimulates adrenal cortex to produce glucocorticoids. Cortisol is one such
glucocorticoid. The results on decreased immunolocalization of corticotrophs reflect dosedependent decrease in cortisol levels reported in chapter 4 of this thesis. This finding is
similar to that in baboons where khat extract caused a decrease in plasma cortisol (Mwenda et
al., 2006) but different from studies in rabbits (Nyongesa et al., 2008). The difference in
findings may be explained partly by the time of exposure to khat/(-)-cathinone, possibly species
difference and animal handling. Glucocorticoid hormone release is characterized by a circadian
cycle (Akana et al., 1996). In the present study, animals were habituated to handling and
presence of observer for one month before blood collection, and control animals were handled in
the same way as treatment groups.
This study investigated the possibility of cellular degeneration that led to variation in the
labelling index of various pituitary cell types following exposure to escalating doses of (-)-
144
cathinone. (-)-Cathinone has been reported to be cytotoxic (Tariq et al., 1987) and
mitodepressive (Al-Meshal et al., 1989; Al-Meshal, 1987; De Hondt et al., 1984). Earlier studies
using S100 (a multifunctional marker of cell degeneration) demonstrated presence of clusterincontaining mixed follicles in long-term amphetamine abusers which corresponded to cellular
degeneration (Ishikawa et al., 2007). In the present study, S100 was used in parallel with a
known positive control (skin melanoma) to detect any possible cellular degeneration in the
anterior pituitary gland. All cells of pituitary sections of (-)-cathinone subjects and negative
controls were negative for the stain when compared to the positive control. The variation in
labelling index in various sections, therefore, is not suggestive of cellular degeneration but rather
the reflection in variation of respective antigens in the anterior pituitary cell types.
Overall, the results demonstrated immunolocalization of anterior pituitary cell types following ()-cathinone exposure and that these numbers correspond to the concentration of hormones in
circulation. The results indicate that (-)-cathinone at different degree of exposure, indeed, has an
effect on the hypothalamo-pituitary- adrenocortical and gonadal axes. It is interesting to note that
the effect on gonadotrophs is, however, opposite to that of other anterior pituitary cell types. The
significance of this lies in the selective action of (-)-cathinone on different functional systems of
the body.
145
CHAPTER 6
ACUTE AND SUB-CHRONIC EFFECTS OF CATHINONE ON SEX
STEROID HORMONE METABOLISM IN ISOLATED INTERSTITIAL
TESTICULAR CELLS OF VERVET MONKEYS (CHLOROCEBUS
AETHIOPS)
6.0 INTRODUCTION
Sex steroids regulate numerous physiological processes by exerting their effects on target
organs including reproductive, cardiovascular and musculoskeletal systems as well as liver
and brain (Ihemelandu, 1981). These hormones are synthesized by smooth endoplasmic
reticulum (SER) of Leydig cells of testis, cells of inner layer of adrenal cortex, granulosa and
luteal cells of the ovaries as well as placenta from cholesterol as the precursor molecule
(Christensen, 1975). Cholesterol is stored mainly in lipid droplets (Moses et al., 1969) and is
metabolized into pregnenolone. In the presence of hydroxysteroid dehydrogenases, a series of
chemical reactions take place that result into conversion of 5- 3 hydroxysteroids,
pregnenolone, 17 hydroxypregnenolone and dehydroepiandrosterone (DHEA) to the 4- 3ketosteroids, progesterone, 17- hydroxyprogesterone and androstenedione, respectively
(Payne and Hales, 2004). These hydroxysteroid dehydrogenases are of two types: 3 beta
hydroxysteroid dehydrogenase (3-HSD) and 17 beta hydroxysteroid dehydrogenase (17HSD) and both belong to the same phylogenetic protein family (Penning, 1997). They are
146
involved in reduction and oxidation of steroid hormones requiring NAD+/NADP+ as acceptors
and their reduced forms as donors of reducing equivalents.
The human steroidogenic tissue contains several isoforms for 3-HSD (Payne and Hales,
2004) and isozymes of the 17-HSDs (Adamski and Jakob, 2001; Adamski et al., 1995), and
each is a product of a distinct gene. The number of these isoforms or isozymes varies in
different species, tissue distribution, catalytic activity, substrate and co-factor specificity, subcellular localization and mechanism of regulation (Payne and Hales, 2004). Studies in the
mouse showed classification of 3-HSD into several isoforms: I, III and VI as classic
dehydrogenases/isomerases while IV and V as 3- keto reductases and, therefore, not involved
in active steroidogenesis (Abbaszade et al., 1995; Clarke et al., 1993). In humans two distinct
isoforms of 3-HSD (type I and II) have been identified (Payne and Hales, 2004). 3-HSD I
is expressed in placenta, skin, and breast while 3-HSD II is expressed in adrenal cortex,
ovary and testis (Simard et al., 1996; Rheaume et al., 1991). Mouse 3-HSD I is expressed in
testis, ovary and adrenal gland while 3-HSD VI is expressed in placenta, skin and testis
(Abbaszade et al., 1997). It is not known if this scenario in expression is similar in vervet
monkey testis. On the other hand, 17-HSDs are not involved in biosynthesis of adrenal
steroids. In humans nine different isozymes have been identified (Adamski and Jakob, 2001),
with types I, III, and VII participating in gonadal steroidogenesis (Payne and Hales, 2004).
The hydroxysteroid dehydrogenase enzymes involved in sex steroid synthesis in human and
mouse are highlighted below (Table 6-1) while substrates involved in testosterone
biosynthesis are shown (Fig. 6-1).
147
Acetate
Cholesterol
Pregnenolone
17 αHydroxypregnenolone
Progesterone
Pregnanediol
17αHydroxyprogesterone
Androstenedione
11-deoxycortisol
Cortisol
Dehydroepiandrosterone
Androstenediol
Testosterone
17β-Oestradiol
Figure 6-1. Diagramatic presentation of steroid hormone metabolism
148
5α-dihydrotestosterone
Table 6-1. Table showing hydroxysteroid dehydrogenases involved in active steroidogenesis
Gene
Chromosomal
Protein
location
name
Synonyms
Tissue
specific
expression
(molecular
mass)
Human
A 17
Mouse
Human
Mouse
17q11-
11
17HSD1
17- HSD1, estradiol-
Ovary, placenta,
q21
(60.25cM)
(35 kDa)
17
mammary gland
hydroxysteroid
dehydrogenase
HSD17B1
Hsd11b2
dehydrogenase,
17-oxidoreductase
HSD17B3
HSD17B7
B HSD3B1
Hsd11b3
Hsd17b7
Hsd3b6
9q22
1q23
1p 13.1
13
1
3
(49.1
cM)
17HSD3
17- HSD3, testicular
Testis
(34.5 kDa)
17-HSD
(microsomal)
17HSD
17-HSD/17-keto
Corpus
(37.3 kDa)
steroid reductase 7
(microsomal)
3
3-HSD
Placenta,
HSD1
type
(type
luteum
mouse
human
IV), 3-HSD/5- 4
giant trophoblast
3 HSDVI
isomerase,
cells,
mouse (42
hydroxy- 5 steroid
(microsomal and
k Da)
dehydrogenase, 3 -
mitochondria)
3-
skin
hydroxy-5-ene steroid
dehydrogenase
HSD3B2
Hsd3b1
1p 13.1
3
cM)
(49.1
3-HSDII
human
See above
Ovary,
Leydig
cells,
adrenal
cortex
(microsomal and
149
mitochondria)
3-HSDI
See above
mouse
Testosterone is synthesized through metabolism of pregenenolone and DHEA by 3-HSD and
17-HSD and subsequently aromatized in the presence of P450 aromatase cytochrome into
oestrogen (Aizawa et al., 2007). To date, it is not clear whether steroidogenesis-related
enzymes are affected by psychostimulants such as (-)-cathinone. In the present study, it was
hypothesized that vervet monkey testes contain steroidogenic enzymes of different isoforms
or isozymes of hydroxysteroid dehydrogenases and that their biosynthetic functions are
variably affected by (-)-cathinone exposure. In this study, gene (mRNA) expression of these
enzymes from testicular tissue of controls and (-)-cathinone-treated vervet monkeys was
determined and their concentrations quantified by real-time polymerase chain reaction (PCR).
150
6.1 MATERIALS AND METHODS
6.1.1 ANIMALS AND HOUSING
The animals and housing procedures are same as was described previously under section 2.1
of general materials and methods.
6.1.2 EXPERIMENTAL DESIGN
6.1.2.1 Animal grouping
The same pattern of animal grouping used here has been described under general materials
and methods in chapter 2, section 2.4 of this thesis. Briefly 14 animals were divided into 5
groups (I, II, III, IV and V) and labelled as GI for controls while GII, III, IV and V as
treatment groups with (-)-cathinone at 0.8, 1.6, 3.2 and 6.4 mg/kg body weight, respectively.
Controls were treated with normal saline. However, group III animals are not discussed in this
chapter since results of these animals in the pilot study were not significantly different from
group II animals. Only male animals were used in this study for investigation of expression of
steroidogenic enzymes by use of semi-quantitative and real-time PCR. However, animal
2169_1 was not used since its testicular samples were not properly preserved for this study
but is described in chapter 7 of this thesis. Samples of animal 2148_1 were duplicated into
2148_2. These same animals were used for ex vivo studies for measurement of intracellular
progesterone, androstenedione and testosterone levels following testicular harvest and culture
of interstitial cells. The harvested cells were incubated in culture medium in test tubes and
treated as described below.
151
6.1.2.2 Leydig cell function test
To assess for regulation of 3 β- hydroxysteroid dehydrogenase (HSD) type I and 17 β- HSD
type I, both in vivo and ex vivo studies were required. This is because long-term primary
culture of Leydig cells was necessary and these Leydig cells could not withstand long-term
culture period. Similarly, during drug metabolism, there are possible inherent differences in
the sensitivity of in vivo and ex vivo preparations. Therefore, both in vivo and ex vivo studies
for assessment of enzymatic regulation of steroidogenesis were done for data comparison.
The harvested testes were decapsulated, fat trimmed off and rinsed in ice-cold 10 ml Minimal
Essential Medium Eagle (modified) in plastic Petri dish to remove excess blood followed by
homogenization (Fig. 6-2). Isolated Leydig cells from testes of all animals were cultured
in100 μl of incubation media in test tubes as follows: controls (2 tubes) and test cells (10
tubes). Test cells were grouped and treated as follows: 2 tubes (50 μl of LH alone), 2 tubes
(50 μl of LH + 100 μl of 0.8 mg/kg of (-)-cathinone [low dose]), 2 tubes (50 μl of LH+ 100 μl
of 6.4 mg/kg of (-)-cathinone [high dose]), 2 tubes (low dose alone) and 2 tubes (high dose
alone). The cells were incubated for 1 hr 15 min at 37 0C. The control cells contained killed
cells (by boiling in water for 5 min according to Oduma et al. (2006). At the end of the
incubation period, the cell suspensions were centrifuged at 1500 x g, the supernatant decanted
and analysed for progesterone, androstenedione and testosterone by enzyme immunoassay
while pellets collected and stored at -80 0C for mRNA expression of steroidogenic enzymes (3
β-HSD type I and 17 β-HSD type I).
152
6.1.2.3. Expression of steroidogenic enzymes in isolated Leydig cells co-incubated with (-)cathinone
The cells collected above were stored at -800C in RNALater for stabilization and protection of
RNA from degradation and later analyzed for mRNA expression of 3 β-HSD type I and 17 βHSD type I by semi-quantitative RT-PCR and real-time quantitative PCR analysis.
Reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA expression of 3
β-HSD type I isoform and 17 β-HSD type I isozyme of hydroxysteroid dehydrogenase.
i) Total RNA extraction
RNA was extracted from tissues using TRIzol® Reagent (Invitrogen, Burlington, Ontario,
Canada), a monophasic mixture of phenol and guanidine isothiocyanate. The reagent is capable of
maintaining the integrity of the RNA due to highly effective inhibition of RNase activity while
disrupting cells and dissolving cell components during sample homogenization. After
homogenizing the sample with TRIzol® Reagent, chloroform was added, and the homogenate
allowed to separate into a clear upper aqueous layer (containing RNA), an interphase and a red
lower organic layer (containing the DNA and proteins, respectively). RNA was precipitated from
the aqueous layer using isopropanol, followed by a subsequent wash and re-suspension into
TRIzol® Reagent for use as a template for first strand cDNA synthesis. Complete protocol for
RNA extraction is found at:
http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf
ii) RNA characterization, purity and integrity
RNA yield was quantified using a NanoDrop® ND-1000 spectrophotometer (Inqaba Biotech,
South Africa) based on the convention that one absorbance unit at 260 nm equals 40 μg/ml
RNA (Sambrook et al., 1989). Purity of the RNA was judged on the basis of A260: A280
153
ratio and its integrity and overall quality judged by inspection of the 28S and 18S ribosomal
RNA (rRNA) bands on agarose gel.
iii) First strand cDNA synthesis
First strand cDNA was synthesized from total RNA of each sample (5µg) using the
SuperScript™ III System for RT-PCR (Invitrogen, Burlington, Ontario, Canada).
SuperScript® III Reverse Transcriptase is widely used because of its ability to reduce RNase
H activity and provide increased thermal stability (Gerard et al., 1986; Kotewicz, 1985).
Moreover, the enzyme is not significantly inhibited by ribosomal and transfer RNA and can
be used to synthesize cDNA from total RNA. A complete protocol is found at:
http://tools.invitrogen.com/content/sfs/manuals/superscriptIIIfirststrand_pps.pdf
iv) Semi-quantitative PCR analysis for mRNA expression of 3 β-HSD type I isoform and
17 β-HSD type I isozyme.
Semi-quantitative PCR was performed according to the method described by Iemitsu et al.
(2000). Each PCR reaction contained 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2,
dNTP at 200 mM each, and gene-specific primers:
HSD3B-F (5′-GGGAGGAATTTTCCAAGCTC-3′),
HSD3B-R (5′-CCAGAGGCTCTTCTTCATGG-3′),
HSD17B-F (5′-TGGGATCCCTAGAGACGTTG -3′),
HSD17B-R (5′-GACTCTCGCATAAGCCTTCG- 3′) at 0.5 μM each, and 0.025 U/μl ExTq
polymerase.
The
3
-hydroxysteroid
dehydrogenase
type
I
(Accession
number:
XM_002801725.1) and 17 -hydroxysteroid dehydrogenase type I (Accession number:
NM_001047132.1) used for designing primers were from a closely related species, Macaca
mulata, selected from the regions that were seen to be highly conserved in this species. The
154
vervet monkey genome has not been sequenced. The genes were identified from NCBI and
gene-specific primers designed using primer 3 software (http://primer3.wi.mit.edu/). The
designed primers were able to detect a specific isoform or isozyme found in the testes (type I).
The PCR procedure was carried out using a PCR thermal cycler (GeneAmp PCR System
9700, ABI). The cycle profile included denaturation for 30 seconds at 94 0C, annealing for 1
min at 58 0C and extension for 1 min at 72 0C. These steps were repeated 40 times.
Amplification products were separated on 1 % agarose gels stained with ethidium bromide
and photographed by ScionImage UVP under UV light. Samples without reverse transcriptase
were used as internal controls while samples without reverse-transcribed cDNA or without
Taq gene were used as negative controls.
v) Real-time quantitative PCR analysis of mRNA expression of 3 β-HSD type I isoform and
17 β-HSD type I isozyme.
a) Optimizing the PCR Reaction for Real-Time Analysis
Before running the sample on the real-time thermal cycler, it was worthwhile to verify the
specificity of the reaction by optimization in a regular thermal cycler without real-time
analysis capabilities and product analysis by gel electrophoresis using 3 µl of amplicon (PCR
product).
b) Real-time detection of PCR products
Real-time PCR incorporates traditional PCR thermal cycler technology with integrated
fluorimeters and detectors that provide the ability to both excite a fluorochrome and detect the
emitted light. In the current implementation, the excitation and detection channels took
advantage of SYBR Green whose fluorescence increases 100- to 200-fold when bound to the
155
minor groove of double stranded DNA. The analysis was used to measure relative mRNA
expression.
Each PCR amplification was performed in triplicate by 1 cycle of 95 0C for 10 minutes and 40
cycles of 94 0C for 15 sec and 60 0C for 1 min. The expression of β-actin mRNA was used as
an internal control. The quantitative values of 3 β-HSD type I and 17 β-HSD type I were
normalized by that of β-actin mRNA expression.
6.1.2.4 Enzyme immunoassay for intracellular progesterone, androstenedione and
testosterone
Enzyme immunoassay technique was employed to assay for progesterone, androstenedione
and testosterone using ELISA kits from Nova Tec Immundiagnostica GMBH, Germany and
protocols of the manufacturer were followed.
Figure 6-2. Vervet testicular tissues in incubation media (Minimal Essential Media).
156
6.1.3 STATISTICS
Values are expressed as mean ± SEM. Hormonal analysis was done using one-way ANOVA
followed by Tukey’s post hoc multiple comparisons test. Statistical significance was set at 5%
level.
157
6.2 RESULTS
The present study examined the existence of steroidogenic enzymes (3- HSD type I and 17HSD type I) in vervet testis. Consistent with the hypothesis, mRNA expression of 3- HSD
type I and 17- HSD type I was detected in testis of vervet monkeys. The RNA that was
extracted was of good yield based on the convention that one absorbance unit at 260 nm
equals 40 μg/ml RNA (Sambrook et al., 1989). The purity of RNA for all samples was good
following A260: A280 ratio and found to be in the range of 1.5 - 2 while the integrity and
overall quality was judged by inspection of the 28S and 18S rRNA bands on agarose gel (Fig.
6-3). In this picture the 28S and 18S RNA bands appear prominent in animals 2148_1 and
2148_2 (controls). Animals at low dose (0.8 mg/kg body weight) of (-)-cathinone: 2107_1
showed a strong 28S and weak 18S while for animal 2107_2 both 28S and 18S RNA bands
appeared strong. RNA of animal 2158_1 shows only 18S band while 2158_2 showed strong
28S and weak 18S. Animals treated with high dose (6.4 mg/kg) of (-)-cathinone (2155_1 and
2155_2) showed that both 28S and 18S RNA bands appeared strong (Fig 6-3). It is not very
clear the cause of variance in RNA bands but it may be attributed to storage. Tissues of each
animal were stored in separate vials in RNALater.
Optimization in a regular thermal cycler and product analysis by gel electrophoresis using 3
µl of amplicon showed good specificity of the reaction before performing real-time PCR (Fig.
6-4). Quantification of mRNA levels was done by real-time PCR. In order to detect the
amplicon, the level of fluorescence signal was set sufficiently above background, that is, at
cycle 16. This was informed by consideration of the detection limits of the equipment and
background fluorescence due to the fluorescent chemistry used. The cycle at which the
158
threshold was met or exceeded, called the “cycle threshold” (CT), was used for making
comparisons between samples. Standard curve analysis performed on the BioRad iQ5 realtime PCR instrument is shown (Fig. 6-5 a and b)
The results show that medium and high doses (3.2 and 6.4mg/kg body weight) of (-)cathinone administered to animals (2158_1 and 2158_2; 2155_1 and 2155_2, respectively)
significantly lowered (P < 0.05) while low dose (0.8 mg/kg body weight) of (-)-cathinone
administered to animals (2107_1 and 2107_2) increased (P<0.05) mRNA levels of 3- HSD
type I enzyme expression (Fig. 6-6). Similarly, high doses of (-)-cathinone significantly
suppressed (P < 0.05) mRNA expression of 17- HSD type I while the effect of low dose of ()-cathinone did not differ significantly from controls (Fig. 6-7). A clear comparison of cycle
threshold of the expression of the two enzymes among different treatment groups and controls
is shown (Table 6-2).
Finally, the study also sought to determine whether escalating doses of (-)-cathinone could
affect hormone synthesis along the testosterone biosynthetic pathway. This was done by
measuring levels of intracellular progesterone, androstenedione and testosterone in cultured
interstitial cells of testes. Cultured interstitial cells exposed to luteinizing hormone (LH)
standard showed increase in progesterone concentration while exposure to low dose (LD) and
high dose (HD) of (-)-cathinone at 100 µl in the presence of LH showed a decrease in
progesterone levels. This decrease in progesterone levels was significant with high dose of ()-cathinone alone (Fig. 6-8). Animals exposed to low dose of (-)-cathinone in vivo showed
increasing concentration of progesterone both in presence and absence of LH. Results also
show a similar pattern for androstenedione release by cultured Leydig cells in presence of LH
159
alone, LH and LD and HD of (-)-cathinone and (-)-cathinone alone (Fig. 6-9). High doses of
(-)-cathinone significantly suppressed (P<0.05) synthesis of testosterone in animals exposed
to medium (2158_1) and high (2155_1) doses of (-)-cathinone of in vivo studies. However,
2107_1 that was exposed to low dose of (-)-cathinone in vivo showed increased testosterone
levels following exposure to (-)-cathinone alone ex vivo (Fig. 6- 10).
160
Figure 6-3. Total RNA expression of steroidogenic enzymes in testis of vervet monkeys.
Images on ethidium bromide-stained denaturing agarose gel showing integrity and quality of
RNA extracted.
Figure 6-4. mRNA expression of steroidogenic enzymes in vervet testicular tissues.
Representative images showing expression of 3- HDS type I and 17- HSD type I as
revealed by RT-PCR. -actin was used as an internal control.
161
Figure 6-5 a and b. mRNA expression of steroidogenic enzymes. Relative expression of 3HSD type I and 17- HSD type I in vervet monkey testis (n = 8) as revealed by real-time
quantitative PCR. Animals administered high doses of (-)-cathinone showed high cycle
threshold for expression of genes for both enzymes compared to low dose of (-)-cathinone and
controls. –actin mRNA was used as an internal control for normalization.
162
50
***
***
Mean cycle threshold
45
**
40
**
35
30
25
3- Beta HSD
20
15
10
5
0
2148_1 2148_2 2107_1 2107_2 2158_1 2158_2 2155_1 2155_2
control (0mg/kg)
Low dose
(0.8mg/kg)
Medium dose
(3.2mg/kg)
High dose
(6.4mg/kg)
Figure 6-6. mRNA expression of steroidogenic enzymes. Relative expression of 3- HSD
type I in testis of vervet monkeys (n = 8) as revealed by real-time quantitative PCR. -actin
mRNA was used as an internal control for normalization. Note a significant increase in mean
cycle threshold in mRNA expression of 3-HSD for medium and high dose (-)-cathinone
subjects compared to controls and low dose (-)-cathinone subjects. (n = 8). ** means
statistically significant at P<0.01, *** means statistically significant at P<0.001.
163
50
**
45
Mean cycle threshold
40
*
**
**
**
*
35
30
25
17-Beta HSD
20
15
10
5
0
2148_1
2148_2
Control (0mg/kg)
2107_1
2107_2
Low dose
(0.8mg/kg)
2158_1
2158_2
Medium dose
(3.2mg/kg)
2155_1
2155_2
High dose
(6.4mg/kg)
Figure 6-7. mRNA expression of steroidogenic enzymes. Relative expression of 17- HSD
type I in testis of vervet monkeys (n = 8) as revealed by real-time quantitative PCR. -actin
mRNA was used as an internal control for normalization. There was a significant suppression
(P<0.05) of mRNA expression of 17- HSD type I with medium and high dose of (-)cathinone as shown by increase in mean cycle threshold on expression of this enzyme with the
two doses. (n = 8). * means statistically significant at P<0.05, ** means statistically
significant at P<0.01.
164
Table 6-2. Comparisons between samples based on “cycle threshold” (CT).  means samples
with low CT, X means samples with high CT. Note that most samples expressing 17- HSD
showed high CT compared to those of 3- HSD indicating that mRNA expression of 17HSD was more suppressed by (-)-cathinone than was that of 3- HSD.
Sample
3B HSD
17BHSD
2158_1
40
X
27
√
40
X
28
√
26
√
25
√
27
√
30
√
40
X
40
X
40
X
40
X
36
X
32
√
31
√
32
√
2158_2
2155_1
2155_2
2148_1
2148_2
2107_1
2107_2
165
2
1.8
Progesterone conc. (ng/ml)
1.6
1.4
1.2
2148_1 (Control)
1
2107_1 (0.8mg/kg)
2158_1 (3.2mg/kg)
0.8
2155_1 (6.4mg/kg)
0.6
0.4
0.2
0
Basal
levels
LH alone LH+LD
100ul
LH+HD LD 100ul
100ul
HD
100ul
Leydig cell exposure to LH and cathinone
Figure 6-8. Local steroidogenesis following addition of LH and (-)-cathinone to cultured
interstitial cells. Results show that LH alone increased while LH and low or high dose of (-)cathinone decreased progesterone secretion. The decrease was significant in presence of high
dose of (-)-cathinone alone when compared to controls (n = 12). Control cells were incubated
in media alone. Note that cells of animal exposed on low dose of (-)-cathinone (2107_1)
showed relative increase in progesterone levels.
166
0.8
0.7
Androstenedione conc. (ng/ml)
0.6
0.5
2148_1 (Control)
0.4
2107_1 (0.8mg/kg)
2158_1 (3.2mg/kg)
2155_1 (6.4mg/kg)
0.3
0.2
0.1
0
-0.1
Basal
levels
LH
alone
LH+LD
100ul
LH+HD
100ul
LD
100ul
HD
100ul
Leyd cell exposure to LH and cathinone
Figure 6-9. Local steroidogenesis following addition of LH and (-)-cathinone to cultured
interstitial cells. Results show that LH alone increased while LH and low or high dose of (-)cathinone decreased androstenedione secretion. The decrease was significant in presence of
high dose of (-)-cathinone alone compared to controls (n = 12). Control cells were incubated
in media alone. Note that cells of animal exposed to low dose of (-)-cathinone (2107_1)
showed relative increase in androstenedione levels compared to high dose of (-)-cathinone.
167
1.6
Testosterone conc. (ng/ml)
1.4
1.2
1
2148_1 (Control)
2107_1 (0.8mg/kg)
2158_1 (3.2mg/kg)
0.8
2155_1 (6.4mg/kg)
0.6
0.4
0.2
0
Basal
levels
LH alone
LH+LD
100ul
LH+HD
100ul
LD 100ul HD 100ul
Leydig cell exposure to LH and cathinone
Figure 6-10. Local steroidogenesis when LH and (-)-cathinone were added in cultured
interstitial cells. Results show that LH alone increased while LH and low or high dose of (-)cathinone decreased testosterone secretion. The decrease was significant in presence of high
dose of (-)-cathinone alone compared to controls (n = 12). Control cells were treated with
incubation medium alone. Note that cells of animal exposed on low dose of (-)-cathinone
(2107_1) showed increase in testosterone levels in presence of LD and HD of (-)-cathinone.
168
6.3 DISCUSSION
The present study demonstrates, for the first time, the existence of 3- HSD I and 17-HSD I
in vervet monkey testis and effect on their expression following (-)-cathinone exposure.
Several previous studies have demonstrated expression of steroidogenic enzymes in various
tissues. For example, 3- HSD has been expressed in testis, ovary, adrenal gland and brain
tissues (Zwain and Yen, 1999; Abbaszade et al., 1997) and 17-HSD abundantly expressed in
testis, ovary and brain (Payne and Hales, 2004; Zwain and Yen, 1999). Other studies also
demonstrated existence of 3-HSD, 17-HSD and P450 aromatase cytochrome in rat skeletal
muscle (Aizawa et al., 2007).
Results show that moderate and high doses of (-)-cathinone in animals (2158_1, 2158_2,
2155_1 and 2155_2) significantly suppressed (P<0.05) expression of mRNA of 3-HSD type
I in vervet testis while low doses of (-)-cathinone administered to animals (2107_1 and
2107_2) increased its expression. Previous studies showed that human 3-HSD type II and
mouse 3-HSD type I and VI isoforms participate in active sex steroid biosynthesis in gonads
(Abbaszade et al., 1997; Simard et al., 1996; Rheaume et al., 1991). These isoforms of 3HSD
catalyze
conversion
hydroxypregnenolone
and
of
DHEA
5to
3-hydroxysteroids,
the
4-
3-ketosteroids,
pregnenolone,
progesterone,
1717-
hydroxyprogesterone and androstenedione respectively, (Payne and Hales, 2004).
Steroidogenic enzymes are characterized under multiple isoforms or isozymes and are
expressed in cell and tissue-specific manner (Payne and Hales, 2004). The 3-HSDs are
membrane-bound enzymes distributed on mitochondria and microsomal membranes. From
169
the results of the present study it appears that 3-HSD type I is expressed in the testis of the
vervet monkeys and is homologous in amino acid sequence to that in humans (3- HSD II)
and mouse (3- HSD I) and that all catalyze steroidogenesis. The results also showed that
high dose of (-)-cathinone impair steroid biosynthesis and, therefore, by extension,
reproductive capacity of an individual.
The results also showed expression of mRNA of 17-HSD I in testicular tissue of the vervet
monkeys and that the expression of this enzyme was suppressed by high dose of (-)-cathinone
but not low dose. Previous studies in humans showed the exclusive involvement of 17-HSD
I, III and VII in the conversion of androstenedione to testosterone in gonads and not in adrenal
glands (Payne and Hales, 2004; Andersson et al., 1995). These isozymes, however, differ in
homology, tissue distribution, catalytic preferences, substrate specificity, subcellular
localization and mechanism of regulation. From the foregoing, it appears the isozymes of
17-HSD involved in human and vervet monkeys are similar. The 17-HSDs convert inactive
17- ketosteroids into their active 17- hydroxy forms. The finding on impairment of
expression of mRNA of both 3-HSD I and 17-HSD I by high dose of (-)-cathinone is
supported by the observed significant decrease in serum testosterone discussed in chapter 4 of
this thesis even though testosterone hormone results did not show any disturbance in
hypothalamo-pituitary integrity. This finding is supported by results on immunolocalization
of pituitary trophs in chapter 5 of this thesis, where there was increase in labelling index of
gonadotrophs with increasing doses of (-)-cathinone. To this end, interefence along the
hypothalamo-hypophyseal-gonadal axis following (-)-cathinone exposure seems to be
localized at the gonadal level.
170
Escalating doses of (-)-cathinone in presence or absence of luteinizing hormone affected
concentration of intracellular progesterone, androstenedione and testosterone differently. For
instance, progesterone, androstenedione and testosterone levels were suppressed by high dose
of (-)-cathinone while low dose of (-)-cathinone increased testosterone levels. Along the
steroid biosynthetic pathway, pregnenolone synthesized from cholesterol is converted to
progesterone in the presence of 3-HSD. Similarly, dehydroepiandrosterone is converted to
androstenedione in the presence of the same enzyme. Although not a subject of consideration
in the present study, earlier studies by Gordon et al. (1980) provided evidence that alcohol
inhibits 5- 3-HSD via depletion of NAD+ during metabolism. In a similar study, alcohol
and acetaldehyde significantly affected conversion of androstenedione to testosterone (Cicero
and Bell, 1980). Quantification of 3-HSD by real-time PCR performed in the present study
indicate the suppression of this enzyme by high dose of (-)-cathinone and, therefore, confirms
the observed decrease in progesterone and androstenedione measurements ex vivo. From the
results intracellular testosterone levels were also affected by (-)-cathinone in cultured cells
with low dose increasing while high dose suppressing its production. Overall, the
quantification of the steroidogenic enzymes by real-time PCR correlated very well with data
on hormone measurements ex vivo. In chapter 4 of this thesis it is shown that high dose of (-)cathinone (6.4 mg/kg body weight) suppressed serum testosterone while low doses (0.8 and
1.6mg/kg body weight) and medium dose (3.2 mg/kg body weight) of (-)-cathinone increased
its secretion consistent with the increase and then decrease in expression of 3-HSD I as well
as intracellular testosterone following low and high dose of (-)-cathinone. This biphasic effect
of khat and (-)-cathinone on testosterone production was demonstrated in earlier studies in
mice (Nyongesa et al., 2007) and rats (Mohammed and Engidawork, 2011). In another related
study, Abdulwaheb et al. (2007) reported that low doses of khat extract (200 mg/kg/day)
171
enhanced sexual motivation while high doses of the extract (400 mg/kg/day) suppressed
sexual motivation and performance in male rats. It was suggested that alteration of both
dopamine (at low dose) and serotonin (at high dose) levels in central nervous system could
explain the biphasic sexual behaviour of rats after khat administration (Abdulwaheb et al.,
2007; Taha et al., 1995). From the foregoing, it appears (-)-cathinone directly affects
conversion of pregnenolene to progesterone and androstenedione to testosterone ex vivo and
this effect is compounded in vivo by the same mechanism.
Conclusively, the results of the present study strongly validate the assumption that changes in
endocrine state are integrally involved in (-)-cathinone’s acute and chronic effects in many
organ systems including reproductive system. The results have also shown that most of the
reproductive complications reported in regular and long-term khat consumers are attributed to
a greater extend to its localized effect on testicular function itself. These findings are
significant since they are informative to the reproductive health implications in the chronic
consumers of khat.
172
CHAPTER 7
MORPHOFUNCTIONAL EFFECTS OF CATHINONE ON VERVET
MONKEY (CHLOROCEBUS AETHIOPS) TESTES FOLLOWING SUBCHRONIC EXPOSURE
7.0 INTRODUCTION
The testicular parenchyma is broadly divided into numerous coiled seminiferous tubules and
interstitium. The tubular epithelium consists of germ cells at various stages of development
interspersed among Sertoli cells, and both cell populations depend on each other for the
success of spermatogenesis. The testicular interstitium of primates, generally, comprise of
wide areas of areolar connective tissue drained by large lymphatic vessels (Fawcett et al.,
1973). The cellular elements of the interstitial tissue are many and varied; the most important
and frequently encountered being the Leydig cell. Leydig cells of most species of animals
studied are polygonal in shape with eccentric nuclei containing one or several nucleoli and
numerous smooth and rough endoplasmic reticulum, abundant mitochondria, Golgi bodies
and lipid droplets (Christensen, 1975). The ultrastructure and steroidogenic function of the
Leydig cell have been described in several reviews (Ewing and Brown, 1977; Christensen,
1975; Hall, 1970; Christensen and Gillim, 1969). The steroidogenic enzymes responsible for
converting cholesterol to testosterone are sequestered in mitochondria and smooth
endoplasmic reticulum membranes of the Leydig cell (Zirkin et al., 1980). Any noxious
173
agents that interfere with these structures may impair the process of steroidogenesis and
reproductive capacity of an animal or individual as a whole.
The process of spermatogenesis occurs within the seminiferous tubules of the testis. The
germinal epithelia within these structures proliferate, differentiate and mature among Sertoli
cells in a pattern that characterize the seminiferous cycle (Onyango et al., 2001). Premature
germ cells undergo active mitotic division and exhibit the greatest dynamic transformation of
chromatin. Within these forms of germ cells are abundant histones that are highly susceptible
to noxious agents while maturation stages of sperm formation have highly condensed
chromatin due to displacement of lysine-rich histones by small argenine and cysteine-rich
protamines (Carlson, 1999). Insulting agents may, therefore, impair spermatogenesis by
interfering with mitotic process of germ cells.
Khat and (-)-cathinone have been shown to have varying effects on the reproductive function
in humans and experimental animals studied. Khat consumption in humans is most prevalent
in males (Al Motarreb et al., 2002). It is due to this gender prevalence or bias of khat use that
has necessitated most studies in males compared to females. Numerous reports on khat use
show contradictory findings on male reproductive function. Impairment of sexuality (WHO,
1980; Halbach, 1972), inability to sustain erection (Elmi, 1983), loss of libido (Krikorian,
1984) and spermatorrhoea (Pantelis et al., 1989; Granek et al., 1988) have been reported.
Khat extract and (-)-cathinone have also been shown to depress cell proliferation and inhibit
RNA, DNA and protein synthesis in dividing cells leading to reduced spermatogenesis (AlMeshal et al., 1989; De Hondt et al., 1984; Hammouda, 1971). In another separate study,
feeding rabbits for 3 months with food containing different amounts of dried, ground khat
174
leaves stimulated spermatogenesis yet cauda epididymides and Leydig cells were unaffected
when compared to controls (Al Mamary et al., 2002). This finding is similar to those in
baboons where Leydig cells were unaffected by khat extract (Mwenda et al., 2006) but at
variance with what was reported in the rat (Islam et al., 1990) where (-)-cathinone caused
atrophy of Leydig and Sertoli cells as well as degeneration of spermatocytes and spermatids.
Khat has also been reported to lower plasma testosterone levels in rabbits (Nyongesa et al.,
2008). Similar studies by Nyongesa et al. (unpublished data) on effects of khat extract on
rabbit testicular tissue revealed vacuolation in spermatogonia and zygotene spermatocytes
while pachytene spermatocytes, spermatids and spermatozoa were unaffected. Vacuolation in
seminiferous epithelium were reported in rats treated with heptachlor (Wango et al., 1997)
and goats treated with ethane dimethanesulphonate (Onyango et al., 2001). From the
foregoing, it appears khat and (-)-cathinone may have effects on testicular structure and
function that affect reproductive performance. In the present study, it was hypothesized that ()-cathinone causes structural alterations in the testis that influence changes in sexual
responses and these variations may be species specific.
Species difference in testosterone secretion is attributed to some qualitative differences in
Leydig cell structure and/or function (Ewing et al., 1979). The same investigators speculated
that there might be a strong correlation between testosterone secretion and amount of
membranous structures present in either the mitochondria and/or smooth endoplasmic
reticulum (SER) of Leydig cells in different species. This view was confirmed by Zirkin et al.
(1980) who reported a positive linear correlation between testosterone secretion and the
volume density of SER in five mammalian species, including the rabbit. The present study
was designed to investigate the effects of (-)-cathinone on volume density of membranous
175
structures present in mitochondria, SER, Golgi apparatus and lipid droplets as well as
potential morphological changes in germinal epithelium affecting spermatogenesis.
Understanding the function of testicular tissue requires reliable quantitative data describing
the surface area and volume densities of cellular constituents of testis. Such data are needed
for exact characterization of testicular function. Studies on effects of khat and (-)-cathinone
on testicular ultrastructure and spermatogenesis in the vervet monkeys are scarce. This study
was designed to elucidate through morphologic and morphometric means the hypothesis that
seminiferous tubular epithelium and interstitial tissue of testis of vervet monkeys undergo
transient changes following sub-chronic (-)-cathinone exposure thereby interfering with
steroidogenesis and spermatogenesis.
176
7.1 MATERIALS AND METHODS
7.1.1 ANIMALS AND HOUSING
Eight males out of fourteen animals described under general materials and methods were used
for this study. A detailed description is given in section 2.1 of general materials and methods.
7.1.2 EXPERIMENTAL DESIGN
This is same as was described previously in section 2.4 of general materials and methods. At
the end of treatment phase 8 males were anaesthetized with 10 mg/kg body weight ketamine
hydrochloride and asceptically castrated. One testis of each animal was used for this study
(Fig. 7-1). The other testis was used for ex vivo studies discussed in the previous chapter 6.
7.1.3 TISSUE PROCESSING FOR TRANSMISSION ELECTRON MICROSCOPY
A little of 2.5% glutaraldehyde was injected beneath the tunica albuginea. The tissues were
then immersed in sample bottles containing 2.5% glutaraldehyde in 0.1 M cacodylate buffer
(pH 7.2) for electron microscopy. The testicular volumes were calculated using Scherle
method (Scherle, 1970), also known as Water Immersion Volumetry. The whole testis of each
animal was immersed in a jar containing phosphate buffer of known weight placed on a
calibrated weighing balance (Fig. 7-2). The change in weight reading on the balance was
taken to be the volume of the immersed testis (Fig. 7-3). This was based on the Archimede’s
Principle that the buoyant force on a submerged object is equal to the weight of the fluid that
is displaced by the object. The mean score on volume was taken after three successive
readings of the same tissue. Testicular tissues were then trimmed into sufficiently small size
(1 mm3) to permit proper fixation and processing (Fig. 4).
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Standard techniques for electron microscopy processing were followed. Briefly, trimmed and
fixed testicular samples were post-fixed with 4% Osmium tetroxide for about 2 h. Tissues
were then rinsed in phosphate buffer for 15 min followed by dehydration in increasing
concentrations of ethanol (20, 40, 50, 60, 70, 80, 90 and 100 [absolute]) three times every 15
min. Tissue blocks were then cleared using propylene oxide twice every 15 min followed by
infiltration with epoxy resin mixture (EMS Sciences, UK) (13 ml Epon-812, 7 ml MNA, 8 ml
DDSA and 16 drops of DMP-30) in the following proportions:

Two parts of propylene oxide in 1 part of epoxy-resin mixture for 30 min

One part of propylene oxide in 2 parts of epoxy-resin mixture for 30 min

Infiltration in pure epoxy-resin mixture without accelerator (DPM-30) overnight.

Finally, embedded in resin mixture as previously prepared, comprising of 100 ml resin
mixture and 1.8 ml DMP-30 in plastic capsules and incubated in the oven at 600C
overnight for curing.
Gold sections (70 nm) were done using Sorvall ultra microtome, placed on copper grids and
left to dry. Grids were stained using lead citrate and uranyl acetate (EMS Sciences, UK) and
viewed by means of transmission electron microscope. Selected areas were photographed and
micrographs printed.
The volume densities (volume fraction) of mitochondria, smooth endoplasmic reticulum,
Golgi apparatus and lipid droplets of Leydig cells were estimated from micrographs of central
parts of testis photographed at x 6000 magnification. The volume density occupied by
mitochondria, lipid droplets, Golgi apparatus and ‘membrane space’ occupied by smooth and
rough endoplasmic reticulum membranes was evaluated by differential point counting as
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earlier described (Kavoi et al., 2012; Weibel, 1979). Briefly, a transparent test grid with a
square lattice of points was overlaid with random positioning on testicular electron
micrographs projected on a computer monitor. The number of points hitting the structures of
interest (mitochondria, smooth endoplasmic reticulum, lipid droplets and Golgi apparatus)
and those falling on the projected field of testicular interstitial tissue were counted. Here, the
volume density of component of interest [Vv (l)] was calculated as a percentage using the
following formula:
Vv (l) = (NPl/NPi) x 100,
Where, Vv (l) is volume density of cell component, NPl is the number of test points hitting
the image of the evaluated component of interest, and NPi is the number of all points falling
on the cell image (interstitium).
By substituting in the equation above, volume densities of mitochondria, smooth endoplasmic
reticulum, lipid droplets and Golgi apparatus used the following formulae, respectively:
Vv (m) = (NPm/NPi) x 100
Vv (ser) = (NPser/NPi) x 100
Vv (l) = (NPl/NPi) x 100
Vv (ga) = (NPga/NPi) x 100
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Figure 7-1. Asceptic surgery of testes of the vervet.
Figure 7-2. Weight reading on the weighing balance before immersion of testis
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Figure 7-3. Weight reading after immersion of testis
181
Figure 7-4. Testicular tissue trimming for immersion in fixative
7.1.4 STATISTICAL ANALYSIS
Morphometric data from each animal was averaged per experimental group and standard error
of means determined. Morphometric data was analysed using one way ANOVA and
comparisons in means among groups performed using Tukey’s multiple comparisons post hoc
test. Statistical significance was set at P < 0.05.
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7.2 RESULTS
The interstitial tissue of testis comprised of Leydig cells, generally pleomorphic in shape but
mostly polyhedral or polyangular in outline, among which were other cells including
macrophages, fibroblasts and a few lymphatic vessels. In control animals, Leydig cell
cytoplasm showed abundant sub-cellular organelles including numerous smooth and rough
endoplasmic reticulum, spherical or elongate mitochondria, Golgi apparatus and a few lipid
droplets (Fig. 7-5 A). Experimental animals on low dose (-)-cathinone (0.8 mg/kg and 1.6
mg/kg) presented similar findings with an increase in number of lipid droplets (Fig. 7-5 B). In
experimental animals on medium (3.2mg/kg body weight) (Fig. 7-5 D) and high dose
(6.4mg/kg body weight) of (-)-cathinone (Fig. 7-5 E and F), there were variations in these
organelles. There was ballooning of mitochondria with evidence of disrupted mitochondria
cristae and in some cases signs of degeneration of mitochondria were observed. Some
mitochondria also appeared to be engulfing some material as shown by asterisk in Figure 7-5
F. Rough endoplasmic reticulum appeared disorganized as opposed to a regular pattern of
arrangement in controls. At high dose of (-)-cathinone smooth endoplasmic reticulum and
lipid droplets were rare. Table 7-1 shows a summary of the morphometric data on volume
densities of various sub-cellular organelles that were evaluated in Leydig cells. These are
mean values obtained from data scoring at three different fields on the same testicular section.
The stereologic data showed that medium and high dose of (-)-cathinone significantly
(P<0.05) altered the morphology of organelles involved in steroidogenesis, primarily: SER,
RER, Golgi apparatus, mitochondria and lipid droplets (Fig. 7-6).
The seminiferous epithelium showed two populations of cells: Sertoli cells and germ cells at
different stages of development. Spermatogonia were found along the basal lamina of
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seminiferous tubules next to Sertoli cells (Fig. 7-7 A-D). (-)-Cathinone affected these cells
differently. For instance, at high dose the nuclear membrane integrity of spermatogonia was
disrupted (Fig. 7-7 D) compared to controls (Fig. 7-7 C). The chromatin material of
spermatogonia at high (-)-cathinone dose appeared condensed, indicative of degeneration.
Primary spermatocytes that were characterized by condensed chromatin in spherical nucleus
were also affected by (-)-cathinone (Fig. 7-8 A-D). The results showed vacuolation in
cytoplasmic organelles of spermatocytes at low (Fig. 7-8 B), medium (Fig. 7-8 C) and high
(Fig. 7-8 D) dose of (-)-cathinone. The intensity of vacuolation was highest with high dose.
Similarly, there was also evidence of decreased cytoplasmic mass in primary spermatocytes in
(-)-cathinone- treated animals compared to controls. At high dose of (-)-cathinone the nuclear
material appeared more condensed or disorganized. Similar vacuolations were observed in
pachytene spermatocytes (Fig. 7-9). At low (Fig. 7-9 B), medium (Fig. 7-9 C) and high (Fig.
7-9 D) doses of (-)-cathinone, there were numerous vacuolations accompanied by
displacement of the nucleus to the periphery of the cells. At the high dose most of the
cytoplasmic organelles were displaced by an amorphous material and the nuclei shrunk. The
cell membrane appeared irregular (Fig. 7-9 D).
The findings of this study also showed a marked effect on the process of spermiogenesis itself
as shown (Fig. 7-10 A-D). The figures showed acrosomal phase spermatids characterized by
presence of acrosome granule within the acrosome vesicle on one end of the nucleus of round
spermatids similar to controls (Fig. 7-10 A). (-)-Cathinone interfered with spermiogenesis as
evidenced by appearance of acrosome granule outside acrosome vesicle at low (-)-cathinone
dose (Fig. 7-10 B), loss of the acrosome granule at medium dose (Fig. 7-10 C) and regression
of the nucleus and disorganization of acrosome vesicle without granule at high (-)-cathinone
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dose (Fig. 7-10 D). At these doses the cytoplasmic organelles appear disorganized and few
while the general shape of the cells bear irregular outline. This is indicative of cellular
degeneration. Finally, elongate spermatids appear to have been affected in a similar pattern
like round spermatids. In controls (Fig. 7-11 A), elongate spermatids had a spear- shaped
nucleus covered by the acrosomal membrane with flagella lined by a sheet of mitochondria.
At low dose of (-)-cathinone (Fig. 7-11 B) similar features were observed. However, moderate
(Fig. 7-11 C) and high dose (Fig. 7-11 D) of (-)-cathinone interfered with spermiogenesis.
This was evidenced by oblong shapes of elongate spermatids with no signs of flagella
formation. The spermatids appeared clumped together as a sign of possible phagocytosis by
Sertoli cells.
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Figure 7-5. Vervet monkey Leydig cell ultrastructure of control (A), (-)-cathinone-treated
animals at 0.8mg/kg (B), 1.6mg/kg (C and D), 3.2mg/kg (E), 6.4 mg/kg (F). Note abundance
of mitochondria (m), smooth endoplasmic reticulum (Se) and well arranged rough
endoplasmic reticulum (arrow) in controls. At low dose (B), rough endoplasmic reticulum
were few and disorganized. Lipid droplets (Ld), Golgi apparatus (arrow head) and membrane
Whorls (w) were observed at medium dose (C and D). Mitochondria appeared to be losing
integrity of inner cristae and in some areas degenerating (star) and engulfed by other
mitochondria (D and E). Rough endoplasmic reticulum were few and appeared scattered. (n =
5). Bar = 0.5 µm in A-F.
186
187
16
14
Volume density (%)
12
10
Mitochondria
LD
8
SER
6
RER
4
GA
2
0
-2
0
0.8
1.6
3.2
6.4
Cathinone dose (mg/kg)
Figure 7-6. A graph showing mean volume densities (%) of mitochondria, lipid droplets
(LD), Golgi apparatus (GA), smooth (SER) and rough endoplasmic reticulum (RER) in
Leydig cell of control and (-)-cathinone-treated vervet monkeys. Note a significant (P<0.05)
decrease in volume densities of these organelles at dose 3.2 and 6.4 mg/kg body weight (n =
5).
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Table 7-1. Mean volume densities of various organelles in Leydig cells of vervet monkey
testis. SER - smooth endoplasmic reticulum, RER - rough endoplasmic reticulum, * significant difference at 5% level, (n = 5).
Mean volume densities (%) (-)-Cathinone dose (mg/kg body weight)
Controls
0.8
Mitochondria
11.96±1
12.46±0.5 12.76±1.1 7.56±0.8* 6.61±0.6*
Lipid droplets
4.12±0.4 6±1.4
6.32±1
0.87±1.1* 0.8±0.9*
SER
8±1.2
8.76±0.9
3.72±1*
2.97±1.2*
RER
9.47±0.7 9.54±0.8
9.29±1.4
5.08±1.1
4.58±1*
Golgi apparatus
5.49±1.3 5.25±1.2
5.45±1
2.4±0.7*
1.55±0.9*
8.4±1
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1.6
3.2
6.4
Figure 7-7. Vervet monkey Sertoli cell of controls (A) and test animals (B-D). There were no
morphological differences in Sertoli cell among treatment groups compared to controls.
However, (-)-cathinone caused morphological changes in early spermatocytes (Sp) at dose 3.2
mg/kg (C) and 6.4 mg/kg (D). Infiltration of material in cytoplasm of spermatocyte (arrow)
interfered with integrity of nuclear membrane (n). Spermatogonia type A (Sa) are present. (n
= 5). Bar = 0.5 µm in A-D
190
191
Figure 7-8. Spermatocytes of vervet monkeys showing controls (A) and (-)-cathinone-treated
groups (B - D). Note cytoplasmic vacuolations (v) in treated animals. Some spermatocytes
appear to lose most of the cytoplasmic content (C and D) at dose 3.2 and 6.4 mg/kg body
weight, respectively. Chromatin material in the nucleus (n) of spermatocytes in C and D
appear disorganized. (n = 5). Bar = 0.5 µm in A-D.
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Figure 7-9. Spermatocytes of vervet monkeys showing controls (A) and (-)-cathinone-treated
groups (B - D). Numerous cytoplasmic vacuolations (v) accompanied by disorganization of
nuclear membrane in treated groups was observed. At high dose (6.4mg/kg) (D) nucleus (n)
appear shrunken and cytoplasm is devoid of most of organelles with disrupted cell membrane.
Chromatin material in the nucleus appeared more condensed indicative of degenerative
changes. (n = 5). Bar = 0.5 µm in A-D.
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Figure 7-10. Round spermatids of vervet monkeys in acrosomal phase of controls (A) and
treated animals (B – D). (-)-Cathinone inteferred with acrosome formation with low dose (B)
showing acrosome granule (arrow) outside acrosome vesicle (V) while at medium (C) and
high dose (D), there was complete granule loss and shape of acrosome vesicle appear
distorted. The nucleus (n) appeared shrunken at high dose (D). (n = 5). Bar represents 0.5 µm
in A - D.
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Figure 7-11. Elongate spermatids of vervet monkeys. Controls (A) and low dose (0.8 mg/kg)
of (-)-cathinone (B) shows spermatids with intact acrosome (arrow) and developing flagella
with emerging mitochondrial sheet (m). At dose 3.2 mg/kg (C) and 6.4 mg/kg (D), normal
sperm development is impaired. Note aggregation of spindle-shaped spermatids with few
mitochondria next to disorganized round spermatid (Rs) in C. At medium and high doses of ()-cathinone spermatids appear tailless with no signs of flagella formation. (n = 5). Bar = 0.5
µm in A-D.
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7.3 DISCUSSION
The results of the present study show that high doses of (-)-cathinone at sub-chronic exposure
to vervet monkeys resulted in alteration of sub-cellular organelles in Leydig cells implicated
in steroidogenesis. This is principally due to decrease in SER, mitochondrial cristae, lipid
droplets and Golgi apparatus. The decrease in SER may be a result of inhibition of formation
of new SER membranes at high doses of (-)-cathinone following breakdown of old ones.
Gonadotropins have been shown to play a crucial role in maintenance of protein synthesis
(Rommerts and Brinkman, 1981). These findings are supported by those on hormonal studies
reported in chapter 4 and immunohistochemical localization of pituitary cells reported in
chapter 5 of this thesis. Hormonal results showed a dose- and time-dependent upregulation of
LH while serum testosterone showed a biphasic pattern of secretion following (-)-cathinone
exposure with low dose increasing while high dose decreased its production.
Immunolocalization studies of pituitary cell types reported in chapter 5 showed an increase in
gonadotrophs following administration of high dose of (-)-cathinone. The increase was
significant in animals co-treated with GnRH agonist indicating that (-)-cathinone did not
impair the integrity of hypothalamus or pituitary gland on this measure. In chapter 6 of this
thesis, it is reported that at high dose of (-)-cathinone 3- HSD I and 17- HSD I were
impaired. Since these enzymes play a crucial role in steroidogenesis (Payne and Hales, 2004),
interference in their gene expression is indicative of impaired steroidogenesis. Further tests
done on ex vivo measurement of intracellular testosterone of cultured interstitial cells showed
decrease in testosterone levels. From these findings, it seems likely that (-)-cathinone
interfered with steroidogenesis at the gonadal level. The stereological data concerning SER
and mitochondria fit well with the results in chapter 6 which indicate adverse effects of high
196
dose and sub-chronic exposure of cathinone on steroidogenic enzymes. These enzymes are
located on membranes of SER and mitochondrial cristae (Mazzocchi et al., 1982; Nussdorfer
et al., 1980; Tamaoki, 1973). Therefore, decrease in number of SER and mitochondria
coupled with ballooning effect of these mitochondria leading to disorganization of
mitochondrial cristae may have contributed to the observed decrease in testosterone
production at high dose of (-)-cathinone.
The results of the present study also showed decrease in lipid droplets following sub-chronic
exposure to (-)-cathinone. This finding also points towards effect of (-)-cathinone on
steroidogenesis. Cholesterol, a product stored in lipid droplets (Moses et al., 1969) is
precursor of steroid hormones (Mazzocchi et al., 1982). On the same note, it is possible that
the observed reduced serum testosterone levels in chapter 4 of this thesis was partly due to
depletion of lipid droplets in Leydig cells following high dose (-)-cathinone exposure. The
SER are involved in synthesis of cholesterol from acetate and glucose (Christensen, 1975).
Similarly, reduction in the amount of Golgi apparatus in the present study is also a pointer
towards impairment of steroidogenesis. A large body of evidence suggest that smooth
endoplasmic reticulum and Golgi apparatus are integral in steroidogenesis (Mazzocchi et al.,
1982; Christensen and Gillim, 1969). Overall, the findings of the present study on Leydig cell
morphology are consistent with those in rats (Islam et al., 1990). However, it is difficult to
make a logical comparison between findings of the present study and those in baboons
(Mwenda et al., 2006) since in the latter case Leydig cell ultrastructure was not considered.
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From the foregoing, it is reasonable to conclude that (-)-cathinone at high dose resulted in
structural alterations of steroidogenic organelles that interfered with their function primarily at
the enzyme level. These structural alterations of steroidogenic structures represent decreased
steroidogenic enzyme expression and, therefore, testosterone synthesis at high dose of (-)cathinone and following long-term exposure. The findings are consistent with those in earlier
studies that reported cytotoxic effects of (-)-cathinone in relation to androgenic deficiency in
mice (Tariq et al., 1987).
In the present study sub-chronic exposure to high doses of (-)-cathinone had adverse effects
on developing germ cells in the seminiferous tubules of vervet monkeys, pointing towards
impairment of spermatogenesis. The results show interference of germ cell proliferation,
differentiation and maturation. The disorganized shape of spermatogonia as shown by cell
membrane outline as well as vacuolation in primary and secondary spermatocytes, all point to
degeneration in these cells. Previous studies by Nyongesa et al. (unpublished data) showed
vacuolation in premature germ cells of rabbit testis following administration of 40.5 g/kg
body weight of khat extract. It is argued that these premature germ cells are highly susceptible
to noxious agents due to the abundance of histones in the chromatin material compared to
mature forms that contain highly condensed chromatin due to argentine and cysteine-rich
protamines (Carlson, 1999). Vacuoles in seminiferous epithelium are frequently encountered
following impaired spermatogenesis. Similar vacuolations were reported in rats treated with
heptachlor (Wango et al., 1997) and goats treated with ethane dimethanesulphonate (Onyango
et al., 2001). There is also evidence of impairment in nuclear function as shown by irregular
outline of nuclear membrane in primary and secondary spermatocytes. The volume of
cytoplasm in these cells is generally reduced. Round spermatids also show peripheral
198
margination of chromatin material. All these occurrences are suggestive of impairment of cell
division and development (Onyango et al., 2001). The findings of the present study compare
favourably with those of previous investigators which showed depressed cell proliferation and
inhibition of RNA, DNA and protein synthesis in actively dividing cells (Al-Meshal et al.,
1989; De Hondt et al., 1984; Hammouda, 1971). Other studies reported effects of khat on
impairment of spermatogenesis in roosters (Hammouda, 1978), mice (Qureshi et al., 1988),
rat (Islam et al., 1990), and humans (El- Shoura et al., 1995).
The acrosomal phase of spermatids also showed defective development. Some (-)-cathinone
subjects displayed displacement of acrosome granule from acrosome vesicles while in others
the acrosome granule was absent altogether. At high dose the nucleus of these round
spermatids was markedly reduced in size. This finding is supported by reports in rats where ()-cathinone enantiomers caused degeneration of spermatocytes and spermatids (Islam et al.,
1990). Results also showed that elongate spermatids of control animals had spear-shaped
condensed nucleus and a flagellum with a mitochondrial sheet along mid and principal piece
indicative of normal process of spermiogenesis (Johnson et al., 1970). Elongate spermatids of
moderate and high (-)-cathinone dose groups appeared oblong in shape and showed tailless
heads with no centrioles. The elongate spermatids appeared clumped together within sleevelike pockets of Sertoli cells, possibly for subsequent phagocytosis.
The results of the present study show that the Sertoli cell ultrastructure was unaffected by (-)cathinone administration to vervet monkeys. The ultrasructure of Sertoli cells revealed large
multi-lobed (4 – 5) nuclei with prominent nucleoli in the basal region of the cytoplasm and
dispersed heterochromatin and eurochromatin consistent with findings of Lebelo and Van der
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Horst (2010). This is at variance with the findings of Islam et al. (1990) where (-)-cathinone
caused degeneration in Sertoli and Leydig cells of rats. It is not clear whether the variance in
findings is attributed to species differences or research protocol employed. Sertoli cells play a
very crucial role in the process of spermatogenesis including: phagocytosis, spermiogenesis,
formation of blood-testis barrier, spermiation and production of some regulatory proteins
(Russell and Griswold, 1993; Johnson, 1991; Tindall et al., 1985). From the results of this
study, the impaired spermatogenesis was a direct effect of (-)-cathinone on developing germ
cells themselves and not as a secondary effect of Sertoli cells.
In conclusion, the findings of this study have shown that (-)-cathinone, at high dose and longterm exposure, has adverse effects on testicular function with particular reference to
spermatogenesis and steroidogenesis. It appears from these findings that regular and longterm consumption of khat interferes with the reproductive capacity of an individual through
direct effect on both processes, which seem to influence each other. It is, however, not clear
whether these effects of (-)-cathinone on gonadal function are irreversible. Further research
need to consider investigations on reversal effects of (-)-cathinone upon withdrawal from
long-term exposure as the direction for future studies.
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CHAPTER 8
8.1 GENERAL DISCUSSION
This thesis highlights the possible mechanism of action of (-)-cathinone on behavioural and
reproductive function in vervet monkeys. Behavioural studies were undertaken by direct
scoring of specific indicators of those behaviours captured by video camera and related to
effects on endocrine function by measurement of hormonal profiles. Reproductive function
studies considered both hormonal and morphological interplay of structures along the
hypothalamo-pituitary-adrenocortical and gonadal axes influencing these measures. The
results showed a clear connection between the nervous and endocrine systems and their
relationship with target organs. These findings provide an indirect in-sight on the impact of
long-term khat use by human ‘khat addicts’ on the behavioural and reproductive health
aspects.
Behavioural findings indicated that (-)-cathinone caused an increase in aggressive and
abnormal behaviour and anxiety in a dose-dependent manner both in presence and absence of
cage enrichment. Durational behaviours of latency to eat and withdrawal as indexed by
jumping on to the platform within the cage or towards a corner similarly increased with higher
doses of (-)-cathinone. Since the characteristic property of khat consumption is
psychostimulation and attainment of euphoria, the behavioural pharmacology of khat has
attracted attention of many investigators. Earlier studies reported stereotyped behaviour, selfadministration and anorectic effects in animal species (Gordon et al., 1993, Calcagnetti and
Schechter, 1992) following khat or (-)-cathinone exposure. These findings were similar to
those evoked by [S-(+)-amphetamine] (Goudie, 1985). In related studies, both khat extract
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and (-)-cathinone showed enhanced baseline aggressive behaviour in isolated rats (Banjaw et
al., 2005). Investigators have linked these behaviours to disorders in the hypothalamic
dopaminergic system (Ishikawa et al., 2007) as well as dopaminergic and serotonergic
activity in the mesolimbic system (Eisch and Harburg, 2006, Jones and Bonci, 2005).
Biogenic amines such as dopamine and serotonin have been shown to have modulatory effects
on specific behaviours (Spoont, 1992). For example, khat and (-)-cathinone induced
stereotyped movement, aggression and sexual arousal (Abdulwaheb et al., 2007; Banjaw et
al., 2006; Connor et al., 2002). The findings of the present study indicate a possible
involvement of serotonergic system on these measures.
The increased latency in food intake observed in the present study is best characterised as
consummatory rather than appetitive behaviour. Two models have been proposed to explain
reduction in food intake following exposure to psychostimulants (Wolgin and Munoz, 2006).
Here, appetitive behaviour is defined as the suppression of food intake as evidenced by loss of
ability to seek food while consummatory behaviour is failure to eat available food. In the
present set up, animals were supplied with food but responded differently to its presence.
However, most available findings in literature highlight on anorectic effects of khat and (-)cathinone. For example, previous studies showed induction of anorexia in rhesus monkeys
(Foltin and Schuster, 1983), rats (Eisenberg et al., 1987; Foltin et al., 1983), pregnant guinea
pigs (Jansson et al., 1988) and humans (Murray et al., 1998) following treatment with (-)cathinone and cathine. Noradrenergic, dopaminergic and opioid systems have been implicated
in khat/(-)-cathinone-induced analgesia and anorexia (Connor et al., 2000; Della Bella et al.,
1985; Nencini et al., 1984). The findings of the present study may, in part, be explained by
these anorectic effects of khat and (-)-cathinone that led to the observed consummatory
202
behaviours. It is possible (-)-cathinone caused these effects via involvement of central
dopaminergic and serotonergic as well as peripheral noradrenergic systems.
In the present study, (-)-cathinone had pronounced effects on hypothalamo-hypophysealadrenocortical and gonadal axes as evidenced by alterations in hormonal profiles. These
alterations of hormonal concentrations are explained by varied immunolocalization of anterior
pituitary cell types secreting these hormones, testicular morphological changes and
stereodogenic function that have been discussed in this thesis. There was a dose-dependent
increase in LH and decrease in serum cortisol, prolactin and progesterone levels. Serum
testosterone showed a biphasic pattern with low doses of (-)-cathinone increasing while high
dose decreasing its production. The increase in LH at high dose and decrease of testosterone
at the same dose points towards susceptibility of testicular steroid forming structures to (-)cathinone. Earlier studies showed gonadotropin-independent decrease in plasma testosterone
levels via exogenous cortisol and dexamethasone administration (Welsh et al., 1981). In
humans, a rise in plasma testosterone with no corresponding increase in LH was observed
during exercise (Murray et al., 1998). Gonadal effects of (-)-cathinone in the present study are
clearly demonstrated. Results showed that high doses of (-)-cathinone suppressed expression
of hydroxysteroid dehydrogenases involved in testosterone biosynthesis. Mitochondrial
cristae and smooth and rough endoplasmic reticulum were structurally altered by high dose of
(-)-cathinone as indexed by their volume densities. Ex vivo measurements of intracellular
hormones found along testosterone synthesis pathway showed decreased androstenedione,
progesterone and testosterone in a dose-dependent manner confirming effect of (-)-cathinone
on 3 HSD and 17 HSD.
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In this thesis, results on correlation of hormones are presented. There was a strong positive
correlation between cortisol and prolactin during treatment phase suggesting that the mode of
action of (-)-cathinone to the two systems producing these hormones may be similar and
appear to influence each other. Studies have shown that activation of the hypothalamohypophyseal-adrenocortical axis and ascending catecholaminergic neurons play an important
role in metabolic and behavioural adaptation to stress (Ishikawa et al., 2007). The decreased
cortisol levels following (-)-cathinone administration in the present study may reflect the
situation in humans, who chew khat to achieve a state of pleasure, euphoria and elation
(Berman et al., 2009). Some of the observed behaviours in the present study may be
correlated to hormonal alterations. For example, increasing levels of serum testosterone with
(-)-cathinone following low and medium dosing may explain the aggressive behaviour
observed. In primates, the correlation between circulating testosterone and aggressive
behaviour is tenuous (Alberts et al., 1992; Bernstein et al., 1979).
Results also show disrupted spermatogenesis and steroidogenesis following (-)-cathinone
exposure. (-)-Cathinone affected all stages of germinal epithelial development indicating its
cytotic effects (Tariq et al., 1987). On the other hand, Sertoli cells were unaffected implying
that interference of spermatogenesis was due to (-)-cathinone effects on germ cells but not as
a result of failure of the supportive role of Sertoli cells. Since testosterone is involved in
spermatogenesis, its low levels at high (-)-cathinone dose in the present study may have also
contributed to disruption of spermatogenesis. From the aforementioned, it appears the effects
of (-)-cathinone on sexual function are compound and involve the hypothalamo-hypophysealadrenocortical and gonadal axes and not as simple as it is literally thought.
204
The study encountered some limitations that may have possibly influenced the results. The
methodology was aimed at modelling an experimental animal to emulate the situation in
human khat chewers. The present study used a purified (-)-cathinone instead of use of whole
fresh leaves and twigs as is normal the case with human khat chewers. The observed effects in
khat chewers are likely a combination of all compounds of khat and multiple exposures. It is,
therefore, possible that the effect of cathine and norephedrine and other alkaloids extracted
into saliva following chewing were not considered. In the present study, it was not possible
for monkeys to chew on the leaves but instead (-)-cathinone was given as a bolus via gastric
gavage. In this study, it was not possible to obtain serial blood collection intravenously from
awake animals and so animals were anaesthetized each time of (-)-cathinone administration
and blood sampling. It is not certain whether ketamine influenced the observed changes in
hormonal profiles. However, animals were maintained on ketamine at 2.5 mg/kg body weight,
a dose that is sufficient to immobilize the animals without causing narcosis (Rizvi et al.,
2000).
The insignificant effect on hormone profiles over time and dose by time interaction as well as
some individual variation in behavioural parameters of vervet monkeys suggest that larger
sample size is required in such studies. The small sample size per group used in the present
study did not allow for a clear follow up on statistics of dose by time interaction on hormone
profiles and morphological evaluation by treatment groups. Finally, the choice of (-)cathinone doses was informed by what has been used in humans. However, this did not
consider the metabolic rate of the vervet monkeys as a factor to the pharmacokinetics of the
drug in the body and this may have led to the discrepancy in the results observed.
205
8.2 CONCLUSIONS
The thesis presents results of (-)-cathinone on behavioural and reproductive function in vervet
monkeys. These findings are summarized as follows:

Results on behavioural and endocrine function showed the involvement of
hypothalamo-hypophyseal-adrenocrotical and gonadal axes. The effects of (-)cathinone on hormones seem to influence, to a great extent, different behaviours
suggesting that there is an interplay between endocrine and meso-corticolimbic
systems.

(-)-Cathinone has a selective action on different pituitary cell types. In this thesis a
dose-dependent increase in immunolocalization of gonadotrophs whose expression
was enhanced by co-treatment with GnRH agonist goserelin acetate has been reported.
The results have demonstrated, for the first time, that the synthesis and secretion of
GnRH as well as the responsiveness of the gonadotrophs to its presence is not affected
by (-)-cathinone. However, the population of lactotrophs and corticotrophs reduced
with increasing doses of (-)-cathinone.

The results have shown that (-)-cathinone enhances LH production but suppresses
prolactin, progesterone and cortisol production in a dose-dependent manner. There
was a biphasic effect of (-)-cathinone on testosterone synthesis with low and medium
doses enhancing while high dose suppressing its production.

The study has also demonstrated, for the first time, the expression of 3- HSD I and
17- HSD I in vervet monkey testis. The results showed that (-)-cathinone has a direct
effect on gonadal function with low and medium doses enhancing while high dose
interfering with expression of 3- HSD I and 17- HSD I. Similarly, low and medium
206
doses of (-)-cathinone did not have significant effect on morphology of steroidogenic
tissue while high dose led to morphological alterations in smooth endoplasmic
reticulum, lipid droplets, mitochondria cristae and Golgi apparatus of Leydig cells
leading to disruption of steroidogenesis. This explains the biphasic effect of (-)cathinone on testosterone production reported in this thesis.

Results also showed that (-)-cathinone interferes with seminiferous germ cell
development at all stages of growth and development thereby leading to impairement
of spermatogenesis.
Overall, the results of the present study point towards the possible mechanism of action
through which (-)-cathinone influences behavioural manifestations and impairs reproductive
function. The observed increase in aggression could have been the function of testosterone
which peaked at medium dose of (-)-cathinone. The decreased population of lactotrophs and
corticotrophs explains observed reduced prolatin and cortisol hormones respectively, and
these may have influenced anxiety and abnormal behaviour to some extent. The reduced
expression of 3- HSD I and 17- HSD I as well as morphological alterations in the
steroidogenic tissue at high dose of (-)-cathinone demonstrates intereference of catalytic
activity of these enzymes leading to low testosterone production. This possibly contributed to
the observed impaired spermatogenesis although (-)-cathinone also appeared to have a direct
effect on the germinal epithelia itself.
8.3 FUTURE GOALS
The future direction on khat research is to determine whether the observed effects on gonadal
function particularly on the processes of spermatogenesis and steroidogenesis are reversible
207
by considering the withdrawal period following long-term exposure. There is also need to
focus on the possible cause of addiction following concomitant use of khat and other drugs of
abuse such as cannabis and tobacco and whether or not the observed behaviours in the chronic
user are a product of a combination of khat and other substance abuse or that these other drugs
boost the effects of khat. Quite frequently, khat addicts concurrently use other substances
especially tobacco. There is also need to model and animal to simulate the actual chewing
process of khat as it occurs in humans. One possibility is to identify sweetener that can be
mixed with khat leaves so that experimental animals are able to chew on them rather than
administration via other routes. There was also need to consider mating as a significant
behaviour in the study. There is also need to measure the levels of cathinone directly in blood
as an indicator to the effects observed in the animal model. These are gaps for future
consideration.
208
REFERENCES
Abbaszade, I.G., Clarke, T.R., Park, C.H. and Payne, A.H. (1995): The mouse 3hydroxysteroid dehydrogenase multigene family includes two functionally distinct
groups of proteins. Molecular Endocrinology 9: 1214 – 1222.
Abbaszade, I.G., Arensburg, J., Park, C.H, Kasa-Vubu, J.Z., Orly, J. and Payne, A.H. (1997):
Isolation of a new mouse 3- hydroxysteroid dehydrogenase isoform, 3HSD VI,
expressed during early pregnancy. Endocrinology 138: 1392 – 1399.
Abdulwaheb, M., Makonnen, E., Debela, A. and Abeba, D. (2007): Effect of Catha edulis
Forsk (khat) extracts on male rat sexual behaviour. Journal of Ethnopharmacology
110: 250 – 256.
Adamski, J., Normand, T., Leenders, F., Monte, D., Begue, A., Stehelin, D., Jungblut, P.W.
and du Launoit, Y. (1995): Molecular cloning of a novel widely expressed human 80
kDa 17 β hydroxysteroid dehydrogenase IV. Biochemistry Journal 311: 437 – 443.
Adamski, J. and Jakob, F.J. (2001): A guide to 17- hydroxysteroid dehydrogenase.
Molecular Cell Endocrinology 171: 1 – 4.
Adeoya-Osiguwa, S.A. and Fraser, L.R. (2005): Cathine and norephedrine, both
phenylpropanolamines, accelerate capacitation and then inhibit spontaneous acrosome
loss. Human Reproduction 20: 198 - 207.
Aghajanian,
G.K.
and
Marek,
G.J.
(1999):
Serotonin
and
hallucinogens.
Neuropsychopharmacology 21: 165 – 235.
Aizawa, K., Iemitsu, M., Maeda, S., Jesmin, S., Otsuki, T., Mowa, C.N., Miyaachi, T. and
Mesaki, N. (2007): Expression of stereoidogenic enzymes and synthesis of sex steroid
209
hormones from DHEA in skeletal muscle of rats. American Journal of Physiology,
Endocrinology and Metabolism 292: E577 – E584.
Akana, S.F., Casio, C.S., Du, J.Z., Levin, N. and Dallman, M.F. (1996): Reset of feedback in
the adrenocortical system: an apparent shift in sensitivity of adrenocorticotrophin to
inhibition by corticosterone between morning and evening. Endocrinology 119: 2325
– 2332
Albers, D.S. and Sonsall, P.K. (1995): Methamphetamine-induced hyperthermia and
dopaminergic neurotoxicity in mice: Pharmacological profile of protective and nonprotective agents. Journal of Pharmacology and Experimental Therapeutics 275:
1104.
Alberts, S.C., Salposky, R.M. and Altman, J. (1992): Behavioural, endocrine and
immunological correlates of immigration by an aggressive male into a natural primate
group. Hormones and Behaviour 26: 167 – 178.
Alem, A. and Shibre, T. (1997): Khat induced psychosis and its medico-legal implication: a
case report. Ethiopian Medical Journal 35: 137 – 139.
Al-Bekairi, A.M., Abulaban, F.S., Qureshi, S. and Shah, A.H. (1991): The toxicity of Catha
edulis (Khat). A review. Fitoterapia LXII: 291– 300.
Al-Hebshi, N.N. and Skaug, N. (2005): Khat (Catha edulis) - an updated review.
Addiction Biology 10: 299 – 307.
Al Mamary, M., Al Habori, M., Al Aghbari, A.M. and Baker M.M. (2002): Investigation into
the toxicological effects of Catha edulis leaves: a short term study in animals.
Phytotherapy Research 16: 127-132.
Al-Meshal, I.A. (1987): Mitodepressive effects of (-)-cathinone from khat (Catha edulis) on
the meristematic region of Allium cepa roots tips. Toxicon 25: 451 - 454.
210
Al- Meshal, I.A., Qureshi, S., El- Feraly, F.S. and Tariq, M. (1989): The toxicity of (-)cathinone from Catha edulis on prolonged treatment in Swiss mice. Proceedings of the
35th International Congress on Alcoholism and Drug Dependence: Oslo, Norway 1:
64.
Al-Meshal, I.A., Qureshi, S., Ageel, A.M. and Tariq M. (1991): The toxicity of Catha edulis
(khat) in mice. Journal of Substance Abuse 3: 107 – 115.
Al-Motarreb, A., Baker, K. and Broadley, K.J. (2002): Khat: Pharmacological and medical
aspects and its social use in Yemen. Phytotherapy Research 16: 403 - 413.
Al-Motarreb, A. and Broadley, K.J. (2004): Coronary and aortic vasoconstriction by
cathinone, the active constituent of khat. Autonomic and Autocoid Pharmacology 23:
319 – 326.
Al-Motarreb, A., Briancon, S., Al-Jaber, N., Al-Adhi, B., Al-Jailani, F., Salek, M.S. and
Broadley, K.J. (2005): Khat chewing is a risk actor for acute myocardial infarction: a
case-control study. British Journal of Clinical Pharmacology 59: 574 - 581.
Amaral, D.G. (2002): The primate amygdala and the neurobiology of social behaviours for
understanding social anxiety. Biology and Psychiatry 51: 11 - 17.
Ambrose, Z., Larsen, K., Thompson, J., Stevens, Y., Finn, E., Hu, S.I. and Bosch, M.L.
(2001): Evidence of early local viral replication and local production of antiviral
immunity upon mucosal simian-human immunodeficiency virus SHIV infection in
Macaca nemestrina. Journal of Virology 75: 8589 – 8596.
Andelman, S.J. (1987): Evolution of concealed ovulation in vervet monkeys (Cercopithecus
aethiops). The American Naturalist 129: 785 – 799.
211
Andersson, S., Geissler, W.M., Patel, S. and Wu, L. (1995): The molecular biology of
androgenic 17 β hydroxysteroid dehydrogenases. Journal of Steroid Biochemistry and
Molecular Biology 53: 37 – 39.
Aston-Jones, G., Shipley, M.T., Chouvet et al. (1991): Afferent regulation of locus coeruleus
neurons: Anatomy, Physiology and Pharmacology. Progress in Brain Research 88: 47
– 73.
Aureli, F. (1997): Post-conflict anxiety in non-human primates: the mediating role of emotion
in conflict resolution. Aggressive Behaviour 23: 315 – 328.
Baldellou, M. and Adan, A. (1997): Time, gender and seasonality in vervet activity: A
chronobiological approach. Primates 38: 31 – 43.
Balint, G.A., Ghebrekidan, H. and Balint, E. (1991): Catha edulis: an international sociomedical problem with considerable pharmacological implications. East African
Medical Journal 68: 555 - 561.
Balint, G.A. and Balint, E.E. (1994): On the medico-social aspects of khat (Catha edulis)
chewing habit. Human Psychopharmacology 9: 125 - 128.
Banjaw, M.Y. and Schmidt, W.J. (2005): Behavioural sensitization following repeated
intermittent oral administration of Catha edulis in rats. Behavioural Brain Research
156: 181 – 189.
Banjaw, M.Y. and Schmidt, W.J. (2006): Catha edulis extract and its active principle
cathinone induce ipsilateral rotation in unilaterally lesioned rats. Behavioural
Pharmacology 17: 615 – 620.
Banjaw, M.Y., Miczek, K. and Schmidt, W.J. (2006): Repeated Catha edulis oral
administration enhances the baseline aggressive behaviour in isolated rats. Journal of
Neural Transmission 113: 543 - 556.
212
Bannon, M.J. and Roth, R.H. (1983). Pharmacology of mesocortical dopamine neurons.
Pharmacological Reviews 35: 53 – 68.
Barnes, N.M. and Sharp, T. (1999): A review of central 5-HT receptors and their function.
Neuropharmacology 38: 1083 – 1152.
Barrot, M., Marinelli, M., Abrous, D.N., Rouge-Pont, F., Le Moal, M. and Piazza, P.V.
(2000): The dopaminergic hyper-responsiveness of the shell of the nucleus accumbens
is hormone-dependent. European Journal of Neuroscience 12: 973 – 979.
Bassareo, V. and Di Chiara, G. (1999): Differential responsiveness of dopamine transmission
to food stimuli in nucleus accumbens shell/core compartments. Neuroscience 89: 637
– 641.
Bardin, C.W. and Paulsen, C.A. (1981): The Testis. In: Williams, R.H. (Ed.). Textbook of
Endocrinology (6th edn.). Philadelphia: Saunders.
Baulu, J., Evans, G. and Sutton, C. (2002): Pathogenic agents found in Barbados Chlorocebus
aethiops sabaeus and in Old World Monkeys commonly used in Biomedical Research.
Laboratory Primate Newsletter 41: 4 – 6.
Bayne, K.A. (1989). Nylon balls revisted. Laboratory Primate Newsletter 28: 5 – 6.
Beckerleg, S. (2008): Khat in East Africa: taking women into or out of sex work. Substance
Use and Misuse 43: 1170 – 1185.
Ben-Jonathan, N., Oliver, C., Weiner, H.J., Micah, R.S. and Porter, J.C. (1977): Dopamine
in hypophysial portal plasma of the rat during estrous cycle and throughout pregnancy.
Endocrinology 100: 452 – 458.
Bentur, Y., Bloom-Krasik, A. and Raikhlin-Eisenkraft, B. (2008): Illicit cathinone (“Hagigat”) poisoning. Clinical Toxicology 46: 206 – 210.
213
Berardelli, A., Capocaccia, L., Pacitti, C., Tancredi, V., Quinter, F. and Elmi A.S. (1980):
Behavioural and EEG effects induced by an amphetamine-like substance (cathinone)
in rats. Pharmacological Research Communications 12: 959 – 964.
Berman, S.M., Kuczenski, R., McCracken, J.T. and London, E.D. (2009): Potential adverse
effects of amphetamine treatment on brain and behaviour: A review. Molecular
Psychiatry 14: 123 – 142.
Bernstein, I.S., Rose, R.M., Gordon, T.P. and Grady, C.L. (1979): Agonistic rank,
aggression, social context and testosterone in male pigtail monkeys. Aggressive
Behaviour 5: 329 – 339.
Berrang, B.D., Lewin, A.H. and Carrol, F.I. (1982): Enantiomeric -aminopropiophenones
(Cathinone): preparation and investigation. Journal of Organic Chemistry 47: 2643 –
2647.
Birge, C.A., Jacobs, L.S., Hammer, C.T. and Daughaday, W.H. (1970): Catecholamine
inhibition of prolactin secretion by isolated adenohypophyses. Endocrinology 86: 120
– 130.
Blundell, J.E. (1984): Serotonin and appetite. Neuropharmacology 23: 1537 -1551.
Borelli, S. and Perali, F. (2004): Drug consumption and intra-household distribution of
resources. In: Dagum, C., Ferrari, G. (Eds.). Household Behaviour, Equivalence
scales, Welfare and Poverty. Physica-Verlag, Heidelberg.
Burger, H.G. (1989): Regulation of gonadotropin secretion. A 1987 perspective. In: Wass,
J.A. and Scanlon, M.F. (Eds). Neuroendocrine perspectives. New York: SpringerVerlag. Vol 6.
Brenneisen, R. and Geisshüsler, S. (1985): Psychotropic drugs III: analytical and chemical
aspects of Catha edulis Forsk. Pharm. Acta Helv 60: 290 – 301.
214
Brenneisen, R. and Geisshüsler, S. (1987): Phenylpentenylamines from Catha edulis. Journal
of Natural Products 50: 1188 – 1189.
Brenneisen, R., Fisc, H.U., Koelbing, U., Geisshüsler, S. and Kalix, P. (1990):
Amphetamine-like effects in humans of the khat alkaloid cathinone. British Journal of
Clinical Pharmacology 30: 825 - 828.
Brenner, R.M., Slayden, O.D., Rodgers, W.H., Critchley, H.O., Caroll, R., Nie X.J. and Mah,
K. (2003): Immunohistochemical assessment of mitotic activity with an antibody to
phosphorylated histone H3 in the macaque and human endometrium. Human
Reproduction 18: 1185 – 1193.
Cadoni, C., Solinas, M., Valentini, V. and Di Chiara, G. (2003): Selective psychostimulant
sensitization by food restriction: differential changes in accumbens shell and core
dopamine. European Journal of Neuroscience 18: 2326 - 2334.
Calcagnetti, D.J. and Schechter, M.D. (1992): Increases in the locomotor activity of rats after
intracerebral administration of cathinone. Brain Research Bulletin 29: 843 – 846.
Calcagnetti, D.J. and Schechter, M.D. (1993): Place preference for the psychostimulantcathinone is blocked by pretreatment with a dopamine release inhibitor. Progress in
Neuro-psychopharmacology and Biological Psychiatry 17: 637 - 649.
Callaway, C.W. and Clarke, R.F. (1994): Hyperthermia and psychostimulant overdose.
Annals of Emergency Medicine 24: 68.
Carlson, B.M. (1999): Human embryology and developmental biology, (2nd edn.). Mosby,
Chicago, New York, London, Toronto, Sydney, Milan, Tokyo.
Carrier, N. (2007): A strange drug in a strange land. In: Vanderbrock, A.P. (Ed.). Travelling
cultures and plants. The ethnology and Ethnopharmacology of Human Migrations.
Berghahn Books: New York and Oxford, pp 186 – 203.
215
Castles, D.L., Whiten, A. and Aureli, F. (1999): Social anxiety, relationships and selfdirected behaviour among wild female olive baboons. Animal Behaviour 58: 1207 –
1215.
Castner, S.A. and Goldman-Rakic, P.S. (1999): Long-lasting psychotomimetic consequences
of repeated low-dose amphetamine exposure in rhesus monkeys.
Neuropsychopharmacology 20: 10 - 28.
Castro, A.C., Berndtson, W.E. and Cardoso, F.M. (2002): Plasma and testicular testosterone
levels, volume density and number of Leydig cells and spermatogenic efficiency of
rabbits. Brazilian Journal of Medical and Biological Research 35: 493 - 498.
Chalmers, D.T., Lovenberg, T.W. and De Souza, E.B. (1995): Localization of novel
corticotrophin-releasing factor receptor (CRF2) mRNA expression to specific
subcortical nuclei in rat brain: comparison with CRF1 receptor mRNA expression.
Journal of Neuroscience 15: 6340 – 6350.
Charney, D.S., Drystal, J.J., Southwick, S.M., Nagy, L.M., Woods, S.W. and Heninger, G.R.
(1990): Serotonin function in panic and generalized anxiety disorders. Psychiatry
Annals 20: 593 – 604.
Cheney, D.L., Seyfarth, R.M., Andelman, S.J. and Lee, P.C. (1988): Reproductive success in
vervet monkeys. In: Reproductive success. Glutton-Brock, T.H. (Ed.). Studies of
individual variation in contrasting breeding systems. University of Chicago Press:
Chicago, pp 384 – 402.
Cheney, D. and Seyfarth, R. (1990): Attending to behaviour versus attending to knowledge:
examining monkeys’ attribution of mental states. Animal Behaviour 40: 742 – 753.
216
Christensen, A.K. and Gillim, S.W. (1969): The correlation of fine structure and function in
steroid-secreting cells with emphasis on those of the gonads. In: The Gonads.
McKerns, K.W. (Ed.). Appleton-Century-Crofts: New York.
Christensen, A.K. (1975): Leydig cells. In: Greep, R.O. and Astwood, E.B. (Eds.). Handbook
of Physiology. American physiological Society: Washington DC 5 Sect 7: 57.
Cicero, T.J. and Bell, R.D. (1980): Effects of ethanol and acetaldehyde on the biosynthesis of
testosterone
in
the
rodent
testes.
Biochemical
and
Biophysical
Research
Communications 94: 814 – 819.
Clarke, T.R., Bain, P.A., Greco, T.L. and Payne, A.H. (1993): A novel mouse kidney 3hydroxysteroid dehydrogenase complementary DNA encodes a 3- ketosteroid
reductase instead of a 3- hydroxysteroid dehydrogenase/5- 4-isomerase.
Molecular Endocrinology 7: 1569 – 1578.
Clinical Practice Guideline (2001): Treatment of the school-aged child with attention
deficit/hyperactivity disorder. Pediatrics 108: 1033 – 1044.
Cone, R.D., Cowley, M.A., Butler, A.A., Fan, W., Marks, D.L. and Low, M.J. (2001): The
arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis.
Internal Journal of Obesity and Related Metabolic Disorders 25: 563 – 567.
Connock, M., Juarez-Garcia, A. and Jowett, S. (2007): Methadone and buprenorphine for the
management of opioid dependence: a systematic review and economic evaluation.
Health Technology and Assessment 11: 1 – 171.
Connor, J., Makonnen, E. and Rostom, A. (2000): Comparison of analgesic effects of khat
(Catha edulis Forsk) extract, D-amphetamine and ibuprofen in mice. Journal of
Pharmacy and Pharmacology 52: 107 – 110.
217
Connor, J., Rostom, A. and Makonnen, E. (2002): Comparison of effects of khat extract and
amphetamine on motor behaviour in mice. Journal of Ethnopharmacology 81: 65 –
71.
Cox, G. and Rampes, H. (2003): Adverse effects of khat. A review: Advances in Psychiatry
Treatment 9: 456 – 463.
Cooper, R.L., Goldman, J.M. and Rehnberg, G.L. (1986): Pituitary function following
treatment with reproductive toxins. Environmental Health Perspectives 70: 177 – 184.
Critchlow, S. and Seifert, R. (1987): Khat-induced paranoid psychosis. British Journal of
Psychiatry 150: 247 – 249.
Crombie, L. (1980): The cathedulin alkaloids. Bulletin of Narcotics 32: 37 – 50.
Cunningham, M.J., Shahab, M., Grove, K.L., Scarlett, J.M., Plant, T.M., Cameron, J.L.,
Smith, M.S., Clifton, D.K. and Steiner, R.A. (2004): Galanin-like peptide as a possible
link between metabolism and reproduction in the macaque. Journal of Clinical
Endocrinology and Metabolism 89: 1760 – 1766.
Czeisler, C.A., Ede, M.C., Regenstein, Q.R., Kisch, E.S., Fang, V.S. and Ehrlich, E.N.
(1976): Episodic 24-hour cortisol secretory patterns in patients awaiting elective
cardiac surgery. Journal of Clinical Endocrinology and Metabolism 42: 273 – 284.
Dahlström, A. and Fuxe, K. (1964): Evidence for the existence of monoamine-containing
neurons in the central nervous system. I. Demonstration of monoamines in the cell
bodies of brain stem neurons. Acta Physiologica Scandinavica 62: 1 – 55.
De Hondt, H.A., Fahmy, A.M. and Abdelbaset, S.A. (1984): Chromosomal and biochemical
studies on the effect of khat extract on laboratory rats. Environmental Mutagenesis 6:
851 - 860.
218
Delfes, J.M., Schreiber, L. and Kelly, A.E. (1990): Microinjection of cocaine into the nucleus
accumbens elicits locomotor activation in the rat. Journal of Neuroscience 10: 303 –
310.
Della Bella, D., Carenzi, A., Frigeni, V., Reggiani, A. and Zamboni, A. (1985): Involvement
of monoaminergic and peptidergic components in cathinone-induced analgesis.
European Journal of Pharmacology 114: 231 – 234.
Deroche, V., Marinelli, M., Le Moal., M. and Piazza, P.V. (1997): Glucocorticoids and
behavioural effects of psychostimulants II. Cocaine intravenous self-administration
and reinstatement depend on glucocorticoid levels. Journal of Pharmacology and
Experimental Therapeutics 281: 1401 - 1407.
De Souza, E.B. (1995) Corticotropin-releasing factor receptors: Physiology, Pharmacology,
Biochemistry and role in central nervous system and immune disorders.
Psychoneuroendocrinology 20: 789 – 819.
Dettmer, E.L., Phillips, K.A., Rager, D.W., Bernstein, I.S. and Fragaszy, D.M. (1996):
Behavioural and cortisol responses to repeated capture and venipuncture in Cebus
paella. American Journal of Primatology 38: 357 – 362.
Di Chiara, G. and Imperato, A. (1988): Drugs abused by humans preferentially increase
dopamine concentrations in the mesolimbic system of freely moving rats. Proceedings
of the National Academy of Sciences, USA 85: 5274 – 5278.
Di Giovanni, G., Di Matteo, V., Pierucci, M. and Esposito, E. (2008): Serotonin-dopamine
interaction: electrophysiological evidence. In: Di Giovannni, G., Di Matteo, V. and
Esposito, E. (Eds.). Progress in Brain Research 172: 45 – 71.
219
Dierschke, D.J., Bhattacharya, A.N., Atkinson, L.E. and Knobil, E. (1970): Circhoral
oscillations of plasma LH levels in the ovariectomized monkey. Endocrinology 87:
850 - 853.
Disotell, T. R. (2000): Molecular systematics of the Cercopithecidae. In: Whitehead, P.F. and
Jolly, C.J. (Eds.). Old World Monkeys. Cambridge: Cambridge University Press. pp.
29-56.
Efron, D., Jarman, F. and Barker, M. (1997): Side effects of methylphenidate and
dexamphetamine in children with attention deficit disorder: a double-blind,
Cross-over trial. Pediatrics 100: 662 – 666.
Eisch, A.J. and Harburg, G.C. (2006): Opiates, psychostimulants, and adult hippocampal
neurogenesis: Insights for addiction and Stem Cell Biology.
Hippocampus 16: 271 –286.
Eisenberg, M.S., Maher, T.J. and Silverman, H.I. (1987): A comparison of the effects of
phenylpropanolamine, d-amphetamine and d-norpseudoephedrine on open-field
locomotion and food intake in the rat. Appetite 9: 31 – 37.
Eley, R.M., Else, J.G., Gulamhusein, N. and Lequin, R.M. (1986): Reproduction in the vervet
monkey (Cercopithecus aethiops): I. Testicular volume, testosterone, and seasonality.
American Journal Primatology 10: 229 – 235.
Eley, R.M., Tarara, R.P., Worthman, C.M. and Else, J.G. (1989): Reproduction in the vervet
monkey (Cercopithecus aethiops): III. The menstrual cycle. American Journal of
Primatology 17: 1 – 10.
Elmi, A.S. (1983): The chewing of khat in Somalia. Journal of Ethnopharmacology 8: 163 176.
220
El-Shoura, S.M., Abdel Aziz, M. and Elmalik, E.M. (1995): Deleterious effects of khat
addiction on semen parameters and sperm ultrastructure. Human Reproduction 9:
2295 - 2300.
Else, J.G., Eley, R.M., Wangula, C. and Lequin, R.M. (1986): Reproduction in the vervet
monkey (Cercopithecus aethiops): II. Annual menstrual patterns and seasonality.
American Journal of Primatology 11: 333 – 342.
Ewing, L.L. and Brown, B.L. (1977): Testicular steroidogenesis. In: The Testis. Johnson, A.J.
and Gomes, W.R. (Eds.). Academic Press: New York, 4: 239.
Ewing, L.L., Zirkin, B.R., Cohran, R.C., Kromann, N., Peters, C. and Ruiz-Bravo, N. (1979):
Testosterone secretion by rat, rabbit, guinea pig, dog and hamster testes perfused in
vitro: correlation with Leydig cell mass. Endocrinology 105: 1135 -1142.
Fahlke, C., Engel, J.A, Eriksson, C.J., Hard, E. and Soderpalm, B. (1994): Involvement of
corticosterone in the modulation of ethanol consumption in the rat. Alcohol 11: 195 202.
Fairbanks, L.A. and McGuire, M.T. (1985): Relationships of vervet mothers with sons and
daughters from one through three years of age. Animal Behaviour 33: 40 – 50.
Fairbanks, L.A., Fontenot, M.B., Phillips-Conroy, J.E., Jolly, C.J., Kaplan, J.R. and
Mann, J.J. (1999): CSF monoamines, age and impulsivity in grivet monkeys
(Cercopithecus aethiops aethiops). Brain, Behavaviour and Evolution 53: 305 – 312.
Fairbanks, L.A. (2001a): Individual differences in response to a stranger: social
impulsivity as a dimension of temperament in vervet monkeys. Journal of
Comparative Psychology 115: 22 – 28.
221
Fairbanks, L.A., Melega, W.P., Jorgensen, M.J., Kaplan, J.R. and McGuire, M.T. (2001b):
Social impulsivity inversely associated with CSF 5-HIAA and fluoxetine exposure
invervet monkeys. Neuropsychopharmacology 24: 370 – 378.
Fairbanks, L.A., Jorgensen, M.J., Huff, A., Blau, K., Hung, Y.Y. and Mann J.J. (2004):
Adolescent impulsivity predicts adult dominance attainment for male vervet monkeys.
American Journal of Primatology 64: 1 – 17.
Fawcett, D.W., Neaves, W.B. and Flores, M.N. (1973): Comparative observations on
intertubular lymphatics and the organization of the interstitial tissue of the mammalian
testis. Biology of Reproduction 9: 500 – 532.
Fernstrom, J.D., Hirsch, M.J. and Fllaer, D.V. (1976): Tryptophan concentration in rat brain.
Failure to correlate with free serum tryptophan or its ratio to the sum of other serum
neural amino acids. Biochemical Journal 160: 589 – 596.
Feyissa, A.M. and Kelly, J.P. (2008): A review of the neuropharmacological properties of
khat. Progress in Neuropharmacology and Biological Psychiatry 32: 1147 – 1166.
Field, T., Grizzle, N., Scafidi, F. and Schanberg, S. (1996): Massage and relaxation therapies’
effects on depressed adolescent mothers. Adolescence 31: 903 – 911.
Field, T., Hernandez-Reif, M. and Diego, M. (2005): Cortisol decreases and serotonin and
dopamine increases following massage therapy. International Journal Neuroscience
115: 1397 – 1413.
Fischman,
A.J.
and
Madras,
B.K.
(2005):
The
Neurobiology
of
Attention
Deficit/Hyperactivity Disorder. Biological Psychiatry 57: 1374 – 1376.
Foltin, R.W. and Schuster, C.R. (1983): Interaction between the effects of intragastric meals
and drugs on feeding in rhesus monkeys. Journal of Pharamcology and Experimental
Therapeutics 226: 405 – 410.
222
Foltin, R.W, Woolverton, W.L. and Schuster, C.R. (1983): Effects of psychomotor stimulants
alone and in pairs, on milk drinking in the rat after intraperitoneal and intragastric
administration. Journal of Pharmacology and Experimental Therapeutics 226: 411 –
418.
Fulwiler, C.E. and Saper, C.B. (1984): Subnuclear organization of the efferent connections of
the parabrachial nucleus in the rat. Brain Research. Brain Research Reviews 7: 229 –
259.
Geisshüsler, S. and Brenneisen, R. (1987): The content of psychoactive phenylpropyl and
phenylpentenyl khatamines in Catha edulis Forsk of different origin. Journal of
Ethnopharmacology 19: 269 - 277.
Geissler, W.M., Davis, D.L., WuL, Bradshaw, K.D., Patel, S., Mendonca, B.B., Elliston,
K.O.,
Wilson,
J.D.,
Russell,
D.W.
and
Andersson,
S.
(1994):
Male
pseudohermaphroditism caused by mutations of testicular 17-hydroxysteroid
dehydrogenase 3. Natural Genetics 7: 34–39.
Gerard, R.D., Chien, K.R. and Meidell, R.S. (1986): Molecular biology of tissue
plasminogen activator and endogenous inhibitors. Molecular Biology and Medicine 3:
446 – 457.
Giannini, A. and Castellani, S.A. (1982): A maniac-like psychosis due to khat (Catha edulis).
Journal of Toxicology 19: 455 - 459.
Gibbs, D.M. and Neill, J.D. (1978): Dopamine levels in hypophysial stalk blood in the rat are
sufficient to inhibit prolactin secretion in vivo. Endocrinology 102: 1895 – 1900.
Goeders, N.E. and Guerin, G.F. (1996a): Effects of surgical and pharmacological
adrenolectomy on the initiation and maintenance of intravenous cocaine selfadministration in rats. Brain Research 722: 145 - 152.
223
Goeders, N.E. and Guerin, G.F. (1996b): Role of corticosterone in intravenous administration
in rats. Neuroendocrinology 64: 337 - 348.
Goldman-Rakic, P.S, Lidow, M.S. and Gallager, D.W. (1990): Overlap of dopaminergic
adrenergic and serotonergic receptors and complementarity of
their subtypes in
primate prefrontal cortex. Journal of Neuroscience 10: 2125 – 2138.
Goldsmith, P.C., Cronin, M. and Weiner, R.I. (1979): Dopamine receptor sites in the anterior
pituitary. Journal of Histochemistry and Cytochemistry 27: 1205 – 1207.
Goldstein, S., Brown, C.R., Ourmanov, I., Pandre, I., Buckler-White, A., Erb, C., Nandi, J.S.,
Foster, G.J., Autissier, P., Schmitz, J.E. and Hirsch, V.M. (2006): Comparison of
simian immunodeficiency virus SIVagmVer replication and CD4+ T-cell dynamics in
vervets and sabaeus African Green Monkeys. Journal of Virology 80: 4868 – 4877.
Goldstone, A.P., Unmehopa, U.A., Bloom, S.R. and Swab, D.F. (2002): Hypothalamic NPY
and agouti-related protein are increased in human illness but not in Prader- Will
Syndrome and other obese subjects. Journal of Clinical Endocrinolgy and Metabolism
87: 927 - 937.
Gordon, G.G., Vit tek, J., Southren, A.L., Munnangi, P. and Lieber, C.S. (1980): Effect of
chronic alcohol ingestion on the biosynthesis of steroids in rat testicular homogenate
in vitro. Endocrinology 106: 1880 – 1885.
Gordon, C.I., Watkinson, W.P., O’Callaghan, J.P. and Miller, D.B. (1991): Effects of 3, 4
methylenedioxymethamphetamine on autonomic thermoregulatory responses of the
rat. Pharmacology Biochemistry and Behaviour 38: 339.
Gordon, T.L., Meehan, S.M. and Schechter, M.D. (1993): Differential effects of nicotine but
not cathinone on motor activity of P and NP rats. Pharmacology, Biochemistry and
Behaviour 44: 657 – 659.
224
Gordon, S., Pandrea, I., Dunham, R., Apetrei, C. and Silvestri, G. (2005): The Call of The
World: What can be learned from studies of SIV Infection of Natural Hosts? HIV
Sequence Pp 2 – 29.
Gosnell, B.A., Yracheta, J.M., Bell, S.M. and Lane, K.E. (1996): Intravenous self
administration of cathinone by rats. Behavioural Pharmacology 7: 526 – 531.
Gottsch, M.L., Clifton, D.K. and Steiner, R.A. (2004): Galanin-like peptides as a link in the
integration of metabolism and reproduction. Trends in Endocrinology and Metabolism
15: 215 – 221.
Goudie, A.J. (1985): Comparative effects of cathinone and amphetamine on fixed interval
operant responding: a rate-dependency analysis. Pharmacology, Biochemistry and
Behavior 23: 355 – 365.
Gough, S. and Cookson, I.B. (1984): Khat-induced schizophreniform psychosis in the United
Kingdom. Lancet 1: 455.
Gugelmann, R., Von Allmen, M., Brenneisen, R. and Portzig, H. (1985): Quantitative
differences in the pharmacological effect of (+) and (-)-cathinone. Experientia 41:
1568 – 1571.
Grace, A. and Bunney, B. (1985): Dopamine. In: Rogawski, M.A. and Barker, J.L. (Eds.).
Neurotransmitter Action in the Vertebrate Nervous System. Plenum Press: New York,
pp 285 – 319.
Graeff, F.G. (2003): On serotonin and experimental anxiety. Psychopharmacology 163: 467
– 476.
Granek, M., Shalev, A. and Weingarten, A.M. (1988): Khat-induced hypnagogic
hallucinations. Acta Psychiatrica Scandinavica 78: 458 – 461.
225
Graziani, M., Michele, S. and Paolo, N. (2008): Khat chewing from the pharmacological
point of view: an update. Substance Use and Misuse 43: 762 – 783.
Grayson, K. (2008): Chasing Dragons: Security, Identity, and Illicit Drugs in Canada,
University of Toronto Press, Toronto.
Griffiths, P., Gossop, M., Wickenden, S., Dunworth J., Harris, K. and Lloyd, C (1997): A
trans-cultural pattern of drug use: qat (khat) in the UK. British Journal of Psychiatry
170: 281 – 284.
Guantai, A.N. and Maitai, C.K. (1983): Metabolism of cathinone to d-norpseudoephedrine in
humans. Journal of Pharmaceutical Sciences 72: 1217 – 1218.
Hadley, M.E. (1988): Endocrinology (2nd edn.). Englewood Cliffs: Prentice Hall.
Halbach, H. (1972): Medical aspects of the chewing of khat leaves. Bulletin of World Health
Organization 46: 21.
Halbreich, U., Sachar, E.J., Asnis, G.M., Nathan, R.S. and Halpern, F.S. (1981): Diurnal
cortisol
responses
to
dextroamphetamine
in
normal
subjects.
Psychoneuroendocrinology 6: 223 - 229.
Halket, J.M., Karasu, Z. and Murray-Lyon, I.M. (1995): Plasma cathinone levels following
chewing khat leaves (Catha edulis Forsk). Journal of Ethnopharmacology 9: 111 –
113.
Hall, P.F. (1970). Endocrinology of the testis. In: The Testis. Johnson, A.J., Gomes W.R. and
Van Demark, N.L. (Eds). Academic Press: New York, 2: 1.
Hall, J.E., Brodie, T.D., Badger, T.M., Rivier, J., Vale, W., Conn, M., Schoenfeld, D. and
Crowley, W.F. (1988): Evidence of differential control of FSH and LH secretion by
gonadotropin-releasing hormone (GnRH) from the use of a GnRH antagonist. Journal
of Clinical Endocrinology and Metabolism 67: 524 – 531.
226
Hammouda, E.S. (1971): Effect of khat extract, D-cycloserine and thiadomide on RNA of
the neurons of 7 and 19 days of chick embryo. Bulletin of Sciences 64.
Hammouda, E.S. (1978): Effects of dietary khat extract on the testes of the White Leghorn
(Gallus domesticus). Bulletin of Sciences 2: 7 - 22.
Hassan, N.A., Gunaid, A.A. and Abdo-Rabbo, A.A. (2000): The effect of khat chewing on
blood pressure and heart rate in healthy volunteers. Tropical Doctor 30: 107 - 108.
Hassan, N.A., Gunaid, A.A., El-Khally, F.M. and Murray-Lyon, I.M. (2002): The subjective
effects of chewing qat leaves in human volunteers. Saudi Medical Journal 23: 850 –
853.
Hassan, N.A., Gunaid, A.A., Khally, F.M., Al-Naomi, M.Y. and Murray-Lyon, I.M. (2005):
Khat chewing and arterial blood pressure: a randomized controlled clinical trial of
alpha-1 and selective beta-adrenoceptor blockade. Saudi Medical Journal 26: 537 –
541.
Hassan, N.A., Gunaid, A.A. and Murray-Lyon, I.A. (2007): Khat (Catha edulis) health
aspects of khat chewing. East Mediterrenean Health Journal 13: 706 – 718.
Hattab, F.N. and Angmar-Mânsson, B. (2000): Fluoride content in khat (Catha edulis)
chewing leaves. Archives of Oral Biology 45: 253 – 255.
Hervé, D., Pickel, V.M., Joh, T.H. and Beaudet, A. (1987): Serotonin axon terminals in the
ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic
neurons. Brain Research 435: 71 – 83.
Hiller-Sturmhöfel, S. and Bartke, A. (1998): The endocrine system: an overview. Alcohol
Health and Research World 22: 153 – 164.
Hoffman, R. and Al’Absi, M. (2010): Khat use and neurobehavioural functions: Suggestions
for future studies. Journal of Ethnopharmacology 132: 554 – 563.
227
Hökfelt, T., Mårtensson, R., Björklund, A., Kleinau, S. and Goldstein, M. (1984):
Distribution maps of tyrosine hydroxylase-immunoreactive neurons in the rat brain.
In: Handbook of chemical neuroanatomy. Björklund, A. and Hökfelt, T. (Eds.).
Classical transmitters in the CNS (1st edn.). Elsevier, pp 277 – 379.
Horrocks, J.A. (1986). Life-history characteristics of a wild population of vervets
(Cercopithecus aethiops sabaeus) in Barbados, West Indies. International Journal of
Primatology 7: 31- 45.
Houghton, P. (2004): Khat- a growing concern in UK. The Pharmaceutical Journal 272: 163.
Hoyer, D., Clarke, D.E., Fozard, J.R., Harting, P.R., Martin, G.R., Mylecgarane, E.J., Saxena,
P.R. and Humphrey, P.P. (1994): VII. International Union of Pharmacology
Classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacological
Reviews 46: 157 – 203.
Iemitsu, M., Miyauchi, T., Maeda, S., Yuki, K., Kobayashi, T., Shimojo, N., Yamaguchi, I.
and Matsuda, M. (2000): Intense exercise causes decrease in expression of both
endothelial NO synthetase and tissue NOx level in hearts. American Journal of
Physiology- Regulatory, Integrative and Comparative Physiology 279: R951–R959.
Ihemelandu, E.C. (1981): Comparison of effect of oestrogen on muscle development of male
and female mice. Acta Anatomica 110: 311 – 317.
Isbell, L.A., Cheney, D.L. and Seyfarth, R.M. (1991): Group fusions and minimum group
sizes in vervet monkeys (Cercopithecus aethiops). American Journal of Primatology 25:
57 – 65.
Ishikawa, T., Li Zhu, B., Miyashi, S., Ishizu, H. and Maeda, H. (2007): Increase in clusterincontaining follicles in the adenohypophysis of drug abusers. International Journal of
Legal Medicine 121: 395 – 402.
228
Islam, M.W., Tariq, M., Ageel, A.M., El-Feraly, F.S., Al-Meshal, I.A. and Ashaf, I. (1990):
An evaluation of the male reproductive toxicity of cathinone. Toxicology 603: 223 234.
Islam, M.W., Al-Shabanah, O.A., Al-Harbi, M.M. and Al-Gharaby, N.M. (1994): Evaluation
of teratogenic potential of khat (Catha edulis Forsk) in rats. Drug Chemical
Toxicology 17: 51 - 68.
Jacobs, D., Silverstone, T. and Rees, L. (1989): The neuroendocrine response to oral
dextroamphetamine in normal subjects. International Clinical Psychopharmacology 4:
135 - 147.
James, A.S., Groman, S.M., Seu, E., Jorgensen, M., Fairbanks, L.A. and Jentsch, J.D. (2007):
Dimensions of impulsivity are associated with poor spatial working memory
performance in monkeys. Journal of Neuroscience 27: 14358 - 14364.
Jansson, T., Kristiansson, B. and Qirbi, A. (1988): Effect of khat on maternal food intake,
maternal weight gain and fetal growth in the late-pregnant guinea pig. Journal of
Ethnopharmacology 23: 11 - 17.
Johanson, C.E., Frey, K.A., Lundahl, L.H., Keenan, P., Lockhart, N., Roll, J., Galloway,
G.P., Koeppe, R.A., Kilbourn, M.R., Robbins, T. and Schuster, C.R. (2006): Cognitive
function and nigrostriatal markers in abstinent methamphetamine abusers.
Psychopharmacology (Berl) 185: 327 – 338.
Johnson, A.D., Gomes, W.R. and Van Demark, N.L. (1970): Development, Anatomy and
Physiology. In: The Testis. Johnson, A.D., Gomes, W.R. and Van Demark, N.L.
(Eds.). Academic Press: New York and London, Vol 1
Johnson, L. (1991): Spermatogenesis. In Cupps, P.T (Ed). Reproduction in domestic animals
(4th edn.). Academic Press Inc., San Diego: California, pp 173 – 219.
229
Jones, S. and Bonci, A. (2005): Synaptic plasticity and drug addiction. Current Opinion in
Pharmacology 5: 20 – 25.
Kalant, H. (2001): The pharmacology and toxicology of “ecstacy” (MDMA) and related
drugs. Canadian Medical Association Journal 165: 917 – 928.
Kalivas, P. (1993): Neurotransmitter regulation of dopamine neurons in the ventral tegmental
area. Brain Research. Brain Reviews 18: 75 – 113.
Kalix, P. (1983 a): Mechanism of action of (-) cathinone, a new alkaloid from khat leaves.
Alcohol 18: 301 - 303.
Kalix, P. (1983 b): The pharmacology of khat and of the khat alkaloid cathinone. In:
International Conference on Khat. The health and socioeconomic aspects of khat use.
International Council on Alcohol and Addictions: Madagascar., January 17 – 21, pp
140 – 143.
Kalix, P. (1984 a): Effect of the alkaloid (-) cathinone on the release of radioactivity from rat
striatal tissue pre-labeled with 3H-serotonin. Neuropsychobiology 12: 127 – 129.
Kalix, P. (1984 b): The pharmacology of khat. General Pharmacology 15: 179 – 187.
Kalix, P. (1984 c): Recent advances in khat research. Alcohol and Alcoholism 19: 319 -323.
Kalix, P. and Braenden, O. (1985): Pharmacological aspects of the chewing of khat leaves.
Pharmacological Reviews 37: 149 - 164.
Kalix, P. (1988): Khat: a plant with amphetamine effects. Journal of Substance Abuse
Treatment 5: 163 - 169.
Kalix, P. (1990): Pharmacological properties of the stimulant khat. Pharmacology and
Therapeutics 48: 397 – 416.
230
Kalix, P., Geisshusler, S., Brenneisen, R., Koelbing, U. and Fisch, H.U. (1990): Cathinone, a
phenylapropylamine alkaloid from khat leaves that has amphetamine effects in
humans. NIDA Research Monograph 105: 289 – 290.
Kalix, P. (1991): The pharmacology of psychoactive alkaloids from ephedra and catha.
Journal of Ethnopharmacology 32: 201 - 208.
Kalix, P. (1992): Cathinone: a natural amphetamine. Pharmacological Toxicology 70: 77 86.
Kalix, P. (1994): Khat: an amphetaminhe-like stimulant. Journal of Psychoactive Drugs 26:
69 - 74.
Kalix, P, Taha, S.A., Ageel, A.M., Islam, M.W. and Ginawi, O.T. (1995): Effect of (-)cathinone, a psychoactive alkaloid from khat (Catha edulis Forsk) and caffeine on
sexual behaviour in rats. Pharmacology Research 31: 299 - 303.
Kalix, P. (1996): Catha edulis, a plant that has amphetamine effects. Pharmacy World and
Science 18: 69 – 73.
Karlsgodt, K.H., Lukas, S. and Elman, I. (2003): Psychosocial stress and the duration of
cocaine dependence. American Journal of Drug Alcohol Abuse 29: 539 - 551.
Kavoi, B.M., Makanya, A.N., Plendl, J. and Kiama, S.G (2012): Morphofunctional
adaptations of the olfactory mucosa in postnatally developing rabbits. The Anatomical
Record 295: 1352 – 1363.
Kimani, S.T. and Nyongesa, A.W. (2008): Effects of single daily khat (Catha edulis) extract
on spatial learning and memory in CBA mice. Behavioural Brain Research 195: 192 –
197.
231
King, A.C., Bernardy, N.C. and Hauner, K. (2003): Stressful events, personality and mood
disturbance: gender differences in alcoholics and problem drinkers. Addiction and
Behaviour 28: 171 - 187.
Kiyatkin, E.A. (1995): Functional significance of mesolimbic dopamine. Neuroscience and
Biobehavioral Reviews 19: 573 – 598.
Klein, A. and Beckerleg, S. (2007): Building castles of spit- the role of khat in ritual, leisure
and work. In: Goodman, J., Lovejoy, P. and Sherrat, A. (Eds.). Consuming Habits:
Global and Historical Perspectives on How Cultures Define Drugs (New Edition).
Routledge: Abingdon, pp 238 – 254.
Knoll, J. (1979): Studies on the central effects of (-) cathinone. Problems of Drug
Dependence. Proceedings of the 41st Annual Scientific Meeting. National Institute of
Drug Abuse, U.S Government Printing Office: Washington D.C. Research Monograph
27: 322 - 323.
Knol, B.W. (1991): Stress and the endocrine hypothalamus-pituitary-testis syste: a review.
Veterinary Quaterly 13: 104 – 114.
Kohli, J. and Goldberg, L. (1982): Cardiovascular effect of (-)-cathinone in the anaesthetized
dog: comparison with (+) - amphetamine. Journal of Pharmacy and Pharmacology
34: 338 - 340.
Koob, G.F. (1992): Drugs of abuse: anatomy, pharmacology and function of reward
pathways. Trends in Pharamcological Sciences 13: 177 – 184.
Kotewicz, M.L., D’Alessio, Driftmier, K.M. and Blodgett, K.P. (1985): Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in E. coli. Gene
35: 249 – 258.
232
Krasnow, S.M., Hohmann, J.G., Gragerov, A., Clifton, D.K. and Steiner, R.A. (2004):
Analysis of the contribution of galanin receptors 1 and 2 to the central actions of
galanin-like peptide. Neuroendocrinology 79: 268 – 277.
Krikorian, A.D. (1984): Khat and its use: a historical perspective. Journal of
Ethnopharmacology 12: 115.
Landgraf, R. (2005): Neuropeptides in anxiety modulation. Handbook of Experimental
Pharmacology 169: 335 – 369.
LeBars, D. (1988): Serotonin and pain. In: Osborne, N.N. and Hamon, M. (Eds.). Neuronal
serotonin, pp 171 – 226.
Lebelo, S.L. and Van der Horst, G. (2010): The ultrastructure of the Sertoli cell of the vervet
monkey, Chlorocebus aethiops. Tissue and Cell 42: 348 – 354.
LeBelle, M.J., Lauriault, G. and Lavoie, A. (1993): Gas chromatographic- mass spectrometric
identification of chiral derivatives of the alkaloids of khat. Forensic Science
International 61: 53 – 64.
Lee, T.F., Mora, F. and Myers, R.D. (1985): Dopamine and thermoregulation: An evaluation
with specific reference to dopaminergic pathways. Neuroscience and Biobehavioural
Reviews 9: 589.
Lee, M.M. (1995): The identification of cathinone in khat (Catha edulis). A time study.
Journal of Forensic Science 40: 116 – 121.
Le Moal, M. and Simon, H. (1991): Mesocorticolimbic dopaminergic network: functional
and regulatory roles. Physiological Reviews 71: 155 – 234.
Linthorst, A.C., Flachskamm, C., Hopkins, S.J., Hoadley, M.E., Labeur, M.S., Holsboer, F.
and Reul, J.M. (1997): Long-term intracerebroventricular infusion of corticotrophinreleasing hormone alters neuroendocrine, neurochemical, autonomic, behavioural and
233
cytokine responses to a systemic inflammatory challenge. Journal of Neuroscience 17:
4448 – 4460.
Lookingland, K.J. and Moore, K.E. (2005): Functional neuroanatomy of hypothalamic
dopaminergic neuroendocrine systems. In: Handbook of chemical neuroanatomy
Dunnet, S.B., Bentivoglio, M., Björklund, A. and Hökfelt, T. (Eds.). Dopamine 21:
435 – 523.
Luqman, W. and Danowski, T.S. (1976): The use of khat (Catha edulis) in Yemen: Social
and Medical observations. Annals of Internal Medicine 85: 246 - 249.
MacLeod, R.M., Fontham, E.H. and Lehmey, J.E. (1970): Prolactin and growth hormone
production are influenced by catecholamines. Neuroendocrinology 6: 283 – 294.
Maestripieri, D., Schino, G., Aureli, F. and Troisi, A. (1992): A modest proposal:
Displacement activities as an indicator of emotions in primates. Animal Behaviour 44:
967 - 979.
Maitai, C. and Mugera, G. (1975): Excretion of the active principle of Catha edulis (miraa) in
human urine. Journal of Pharmaceutical Sciences 64: 702 - 703.
Marais, L., Daniels, D., Brand, L., Viljoen, F., Hugo, C. and Stein, D.J. (2006):
Psychopharmacology of maternal separation anxiety in vervet monkeys. Metabolism
and Brain Disorders 21: 201 – 210.
Margetts, E. (1967): Miraa and myrrh in East Africa: clinical notes about Catha edulis.
Economic Botany 21: 358 - 362.
Marinelli, M., Piazza, P.V., Deroche, V., Maccari, S., Le Moal, M. and Simon, H. (1994):
Corticosterone circadian secretion differentially facilitates dopamine-mediated
psychomotor effect of cocaine and morphine. Journal of Neuroscience 14: 2724 2731.
234
Marinelli, M. and Piazza, P.V. (2002): Interaction between glucocorticoid hormones, stress
and psychostimulant drugs. European Journal of Neuroscience 16: 387 - 394.
Martin, P. and Bateson, P. (1993): Measuring behaviour: An introductory Guide. Cambridge
University Press: Cambridge.
Matloob, M.H. (2003): Determination of cadmium, lead, copper and zinc in Yemen khat by
anodic stripping voltammetry. East Mediterrenean Health Journal 9: 28 - 36.
Matsumoto, H., Noguchi, J., Takastu, Y., Horikoshi, Y., Kumano, S., Ohtaki, T., Kitada, C.,
Itoh, T., Onda, H., Nishimura, O. and Fujino, M. (2001): Stimulation effect of galaninlike peptide on LHRH-mediated LH secretion in male rats. Endocrinology 142: 3693 –
3696.
Mazzocchi, G., Robba, C., Rebuffat, P., Gottardo, G. and Nussdorfer, G.G. (1982): Effects of
a chronic treatment with testosterone on the morphology of the interstitial cells of rat
testis: an ultrastructural stereologi study. International Journal of Andrology 5: 130 –
136.
McEwen, B.S: (2000): The neurobiology of stress. From serendipity to clinical relevance.
Brain Research 886: 172 – 189.
McLaren, P. (1987): Khat psychosis. British Journal of Psychiatry 150: 712.
McMinn, J.E., Baskin, D.G. and Schwartz, M.W. (2000): Neuroendocrine mechanisms
regulating food intake and body weight. Obesity Reviews 1: 37 – 46.
McQuade, R. and Stanford, S.C. (2000): A microdialysis study of the noradrenergic response
in rat frontal cortex and hypothalamus to a conditioned cue for aversive, naturalistic
environmental stimuli. Psychopharmacology 148: 201 – 208.
Melega, W.P., Jorgensen, M.J., Lacan, G., Way, B.M., Pham, J., Morton, G., Cho, A.K. and
Fairbanks, L.A. (2008): Long-term methamphetamine administration in the vervet
235
monkey models aspects of a human exposure: Brain neurotoxicity and behavioural
profiles. Neuropsychopharmacology 33: 1441 – 1452.
Mello, N.K. and Mendelson, J.H. (1997): Cocaine’s effects on neuroendocrine systems:
clinical and preclinical studies. Pharmacology, Biochemistry and Behavior 57: 571 –
599.
Meltzer, H.Y. and Lowy, M.T. (1987): The serotonin hypothesis of depression. In:
Psychopharmacology: The Third Generation of Progress. Meltzer, H.Y (Ed). Raven
Press, New York, pp 513 – 526.
Millan, M.A., Jacobowitz, D.W., Hauger, R.L., Catt, K.J. and Aguilera, G. (1986):
Distribution of corticotrophin-releasing factor receptors in primate brain. Proceedings
of the National Academy of Sciences, USA 83: 1921 – 1925.
Mitchell, A.J. (1998): The role of corticotrophin releasing factor in depressive illness: A
critical review. Neuroscience and Biobehavioural Reviews 22: 635 – 651.
Mohammed, A. and Engidawork, E. (2011): Reproductive parameters are differentially
altered following subchronic administration of Catha edulis Forsk (Khat) extract and
cathinone in male rats. Journal of Ethnopharmacology 134: 977 – 983.
Moses, H.L., Davis, W.W., Rosenthal, A.S. and Garren, L.D. (1969): Adrenal cholesterol:
Localization by electron microscope audiordiography. Science 163: 1203.
Moukhles, H., Bosler, O., Bolam, J.P., Valleé, A., Umbriacco, D., Geffard, M. and Doucet,
G. (1997): Quantitative and morphometric data indicate precise cellular interactions
between serotonin terminals and post-synaptic targets in rat substantia nigra.
Neuroscience 76: 1159 – 1171.
236
Møller, S.E., Kirk, L. and Honoré, P. (1980): Relationship between plasma ratio of
tryptophan to competing amino acids and the response to L-tryptophan treatment in
endogenously depressed patients. Journal of Affective Disorders 2: 47 – 59.
Murray, F.T., Cameron, D.F., Vogel, R.B., Thomas, R.G., Wijss, H.U. and Zauner, C.W.
(1998): The pituitary-testicular axis at rest and during moderate exercise in males with
diabetes mellitus and normal sexual sexual function. Journal of Andrology 9: 197 –
206.
Murray, C.D., Le Roux, C.W., Emmanuel, A.V., Halket, J.M., Pryborowska, A.M., Kamm,
M.A. and Murray-Lyon, I.M. (2008): The effect of khat (Catha edulis) as an appetite
suppressant is independent of ghrelin and PYY secretion. Appetite 51: 747 – 750.
Murton SA, Tan ST, Pricket TC, Frampton C. and Donald, R.A. (1998): Hormone responses
to stress in patients with major burns. British Journal of Plastic Surgery 51: 388 –
392.
Mwenda, J.M., Owuor, R.A., Kyama, C.M., Wango, E.O., Arimi, M.M. and Langat, D.K.
(2006): Khat (Catha edulis) up-regulates testosterone and decreases prolactin and
cortisol levels in the baboon. Journal of Ethnopharmacology 103: 379 - 384.
Neary, N.M., Goldstone, A.P. and Bloom, S.R. (2004): Appetite regulation: From the gut to
the hypothalamus. Clinical Endocrinology 60: 153 - 160.
Nencini, P., Ahmed, A., Amicon, G. and Elmi, A. (1984): Tolerance develops to sympathetic
effects of khat in humans. Pharmacology 28: 150 - 154.
Nencini, P. and Ahmed, A.M. (1989): Khat consumption: a pharmacological review. Drug
and Alcohol Dependence 23: 19 – 29.
237
Nencini, P., Grassi, M.C., Botan, A.A., Asseyr, A.F. and Paroli, E. (1989): Khat-chewing
spread to the Somali community in Rome. Drug and Alcohol Dependence 23: 255 –
258.
Nielen, R.J., van der Heijden, F.M., Tuiner, S. and Verhoeven, W.M. (2004): Khat and
mushrooms associated with psychosis. World Journal of Biological Psychiatry 5: 49 –
53.
Nimmo, S.M., Kennedy, B.W. and Tullett, W.M. (1993): Drug-induced hyperthermia.
Anaesthesia 48: 892.
Nussdorfer, G.G., Robba, C., Mazzochi, G. and Rebuffat, P. (1980): Effects of human
chorionic gonadotropins on interstitial cells of the rat testis: a morphometric and
radioimmunological study. International Journal of Andrology 3: 319.
Nyongesa, A.W., Patel, N.B., Onyango, D.W., Odongo, H.O. and Wango, E.O. (2008): Khat
(Catha edulis) lowers plasma luteinizing hormone (LH) and testosterone secretion, but
increases cortisol levels in male rabbits. Journal of Ethnopharmacology 116: 245 250.
Nyongesa, A.W., Patel, N.B., Onyango, D.W., Wango, E.O. and Odongo, H.O. (2007): In
vitro study of the effects of khat (Catha edulis Forsk) extract on isolated mouse
interstitial cells. Journal of Ethnopharmacology 110: 401 - 405.
Oduma, J.A., Oduor Okelo, D., Odongo, H. and Makawiti, D.W. (2006): The pesticide
heptachlor affects steroid hormone secretion in isolated follicular and luteal cells of
rat. Comparative Biochemistry and Physiology, Part C 144: 76 – 84.
Onyango, D.W., Wango, E.O., Oduor-Okelo., D. and Werner, G. (2001): Early testicular
response to intraperitoneal administration of ethane dimethanesulphonate (EDS) in the
238
goat (Capra hircus). Journal of Submicroscopic Cytology and Pathology 33: 117 –
124.
Oswald, L.M., Wong, D.F., McCaull, M., Zhou, Y., Kuwabara, H., Choi, L., Brasic, J. and
Wand, G.S. (2005): Relationships among ventral striatal dopamine release, cortisol
secretion and subjective response to amphetamine. Neuropsychopharmacology 2665:
1 - 12.
Page, S.L. and Goodman, M. (2001): Catarrhine Phylogeny: Non-coding DNA evidence for a
diphylectic origin of the Mangabeys and for human- chimpanzee clade. Molecular and
Phylogenetic Evolution 18: 14 – 25.
Pantelis, C., Hindler, C.G. and Taylor, J.C. (1989): Use and abuse of khat distribution,
pharmacology, side effects and description of psychosis attributed to khat chewing.
Psychological Medicine 19: 657 - 668.
Parada, M.A, de Parada, M. P., Rada, P. and Hernandez, L. (1995): Supride increases and
dopamine decreases intracranial temperature in rats when injected in the lateral
hypothalamus: An animal model for the neuroleptic malignant syndrome. Brain
Research 674: 117.
Payne, A.H. and Hales, D.B. (2004): Overview of steroidogenic enzymes in the pathway
from cholesterol to active steroid hormones. Endocrine Reviews 25: 947 – 970.
Pehek, E.A., Schechter, M.D. and Yamamoto, B.K. (1990): Effects of cathinone and
amphetamine on the neurochemistry of dopamine in vivo. Neuropharmacology 29:
1171 – 1176.
Penning, T.M. (1997): Molecular endocrinology of hydroxysteroid dehydrogenases.
Endocrine Reviews 18: 281 – 305.
239
Piazza, P.V., Maccari, S., Deminiere, J.M., Le Moal., Mormede, P. and Simon, H (1991):
Corticosterone levels determine individual vulnerability to amphetamine selfadministration. Proceedings of the National Academy of Sciences, USA 88: 2088 2092.
Piazza, P.V., Barrot, M., Rouge-Pont, F., Marinelli, M., Maccari, S., Abrous, D.N., Simon, H.
and Le Moal, M. (1996): Suppression of glucocorticoid secretion and antipsychotic
drugs has similar effects on the mesolimbic dopaminergic transmission. Proceedings
of the National Academy of Sciences, USA 93: 15445 – 15450.
Piazza, P.V. and Le Moal, M. (1998): The role of stress in drug self-administration. Trends in
Pharmacological Sciences 19: 67 - 74.
Plotsky, P.M., Gibbs, D.M. and Neill, J.D (1978): Liquid chromatographic-electrochemical
measurement of dopamine in hypophysial stalk blood of rats. Endocrinology 102:
1887 – 1894.
Quan, L., Ishikawa, T., Michiue, T et al. (2005). Ubiquitin-immunoreactive structures in the
midbrain of methamphetamine abusers. Legal Medicine (Tokyo) 7: 144 – 150.
Qureshi, S., Tariq, M., Pamar, N.S. and Al- Meshal, I.A. (1988): Cytological effects of khat
(Catha edulis) in somatic and male germ cells of mice. Drug Chemistry and
Toxicology 11: 151 - 165.
Raaum, R.L., Sterner, K.N., Noviello, C.M., Stewart, C.B. and Disotell, T.R. (2005):
Catarrhine primate divergence dates estimated from complete mitochondrial genomes:
concordance with fossil and nuclear DNA evidence. Journal of Human Evolution 48:
237 - 57.
Ragland, A.S., Ismail, T. and Arsura, E.L. (1993): Myocardial infarction after amphetamine
use. American Heart Journal 125: 247 – 249.
240
Raleigh, M.J., Yuwiler, A., Brammer, G.L., McGuire, M.T. and Flannery, J.W. (1981):
Peripheral correlates of serotonergically-influenced behaviors in vervet monkeys
(Cercopithecus aethiops sabaeus). Psychopharmacology 72: 241 – 246.
Rang, H.P. (2003): Pharmacology. Edinburgh: Churchill Livingstone, Pp 596.
Rao, M.L., Barbar, H., Papassotiropoulos, A., Diester, A. and Frahnert, C. (1998): Upregulation of the platelet serotonin 2A receptor and low blood serotonin in suicidal
psychiatric patients. Neuropsychobiology 38: 84 – 89.
Rapkin, A.J., Pollack, D.B., Raleigh, M.J., Stone, B. and McGuire, M.T. (1995): Menstrual
cycle and social behaviour in vervet monkeys. Psychoneuroendocrinology 20: 289 –
297.
Rheaume, E., Lachance, Y., Zhao, H.F., Breton, N., Dumont, M., de Launoit, Y., Trudel, C.,
Luu-The, V., Simard, J. and Labrie, F. (1991): Structure and expression of a new
complementary
DNA
encoding
the
almost
exclusive
3-
hydroxysteroid
dehydrogenase/5- 4 – Isomerase in human adrenals and gonads. Molecular
Endocrinology 5: 1147 – 1157.
Ricuarte, G.A., Schuster, C.R. and Seider, L.S. (1980): Long term effects of repeated
methamphetamine administration at dopamine and serotonin neurons in the rat brain.
A regional study. Brain Research 193: 153.
Rizvi, S.R., Weinbauer, G.F., Arslan, M., Partch, C-J. and Nieschlag, E. (2000): Testosterone
modulates growth hormone secretion at the hypothalamic but not at the hypophyseal
level in the adult male rhesus monkey. Journal of Endocrinology 165: 337 – 344.
Rommerts, F.F and Brinkman, A.O (1981): Modulation of steroidogenic activities in testis
Leydig cells. Molecular and Cell Endocrinology 21: 15.
241
Roth, R.H., Wolf, M.E. and Deutch, A.Y. (1987): Neurochemistry of midbrain dopamine
systems. In: Psychopharamcology. The Third Generation of Progress. Meltzer, H.Y.
(Edn.). Raven Press: New York, pp 81 – 94.
Ruggiero, D.A., Underwood, S.M., Rice, P.M., Mann, J,J. and Arango, V. (1999):
Corticotropin-releasing hormone and serotonin interact in the human brain stem:
Behavioural implications. Neuroscience 91: 1343 – 1354.
Russell, L.D. and Griswold, M.D. (1993): The Sertoli cell. Cache River Press: Clear Water
Florida, USA.
Salamone, J.D., Cousins, M.S., McCullough, L.D., Carriero, D.L. and Berkowitz, R.J.
(1994): Nucleus accumbens dopamine release increases during instrumental level
pressing fir food but not free food consumption. Pharmacology Biochemistry and
Behavior 49: 25 – 31.
Saleh, M., Mekkawy, H. and el-Komy, F. (1988): Acute effects of a khat extract on the rat
electro-encephalogram. Journal of Ethnopharmacology 23: 291 – 298.
Sambrook, J., Fritsch, E. and Maniatus, T. (1989): A Laboratory Manual (3rd Edn.). Cold
Spring Harbor Press.
Sawynok, J. (1988): The role of ascending and descending noradrenergic and serotonergic
pathways in opioid and non-opioid anti-nociception as revealed by lesion studies.
Canadian Journal of Physiology and Pharmacology 67: 975 – 988.
Schechter, M.D. (1990): Rats become acutely tolerant to cathine after amphetamine or
cathinone administration. Psychopharmacology (Berl) 101: 126 – 131.
Scherle, W. (1970): A simple method for volumetry of organs in quantitative stereology.
Mikroscopie 26: 57 – 60.
242
Schorno, H.X. and Steinegger, E. (1979): CNS-active phenylpropylamine of Catha edulis of
Kenyan origin. Experientia 35: 572.
Schuhler, S., Clark, A., Joseph, W., Patel, A., Lehnen, K., Stratford, E., Horan, T.L., Fone,
K.C. and Ebling, F.J. (2005): Involvement of 5-HT2C receptors in the regulation of
food intake in Siberian hamsters. Journal of Neuroendocrinology 17: 276 – 285.
Schuster, C. and Johanson, R. (1979): Behavioural studies of cathinone in monkeys and rats.
In: Problems of Drug Dependence. National Institute of Drug Abuse, U.S Government
Printing Office, Washington D.C. Research Monogaph 27: 324 - 325.
Schwartz, P.J., Wehr, T.A. and Rosenthal, N.E. (1995): Serotonin and thermoregulation:
Physiologic and pharmacologic aspects of control revealed by intravenous M-CPP in
normal human subjects. Neuropsychopharmacology 13: 105.
Seier, J.V. (2005): Vervet monkey breeding. In: The Laboratory primate. Wolfe-Coote, S.
(Ed.). New York, Elsevier. Pp 175 – 178.
Sillaber, I., Montkowski, A. and Landgraf, R. (1988): Enhanced morphine-induced
behavioural effects and dopamine release in the nucleus accumbens in a transgenic
mouse model of impaired glucocorticoid (type II) receptor function: influence of longterm treatment with the antidepressant moclobemide. Neuroscience 85: 415–425.
Simard, J., Durocher, F., Mebarki, F., Turgeon, C., Sanchez, R., Labrie, Y., Couet, J., Trudel,
C., Rheaume, E., Morel, Y., Luu-The, V. and Labrie, F. (1996): Molecular biology
and genetics of the 3 hydroxysteroid dehydrogenase/5- 4- isomerise gene family.
Journal of Endocrinology 150 (suppl): S189 – S207.
Smith, S.M., Vaughan, J.M., Donaldson, C.J., Rivier, J., Li, C. and Chen, A. (2004): Cocaine
and amphetamine-regulated transcript activates the hypothalamic-pituitary-adrenal
243
axis through a corticotrophin-releasing factor receptor-dependent mechanism.
Endocrinology 145: 5202 - 5209.
Spoont, M.R. (1992): The modulatory role of serotonin in neural information processing:
implications for human psychopathology. Psychology Bulletin 112: 330 – 350.
Stanford, S.C. (2001). Noradrenaline. In: Neurotransmitters, drugs and brain function.
Webster, R.A (Ed.). John Willey and Sons Ltd. Baffins Lane, Chichester, West
Sussex, UK, pp 163 – 185.
Starkman, M.N., Oliver, M.S., Cameron, O.G., Nesse, R.M. and Zelnik, T. (1990):
Peripheral catecholamine levels and the symptoms of anxiety: Studies in patients with
and without pheochromocytoma. Psychosomatic Medicine 52: 129 – 142.
Steckler, T. and Holsboer, F. (1999): Enhanced conditioned approach responses in transgenic
mice with impaired glucocorticoid receptor function. Behavioural Brain Research
102: 151 – 163.
Sulser, F. (1989): New perspectives on the molecular pharmacology of affective disorder.
European Archives of Psychiatry and Neurological Sciences 238: 231 – 239.
Swant, J., Chirwa, S., Stanwood, G. and Khoshbouei. (2010): Methamphetamine reduces
LTP and increases baseline synaptic transmission in the CA1 region of mouse
hippocampus. PloS ONE 5: e1 1382.
Swerdlow, N.R., Koob, G.F., Cador, M., Lorang, M. and Hauger, R.L. (1993): Pituitaryadrenal axis responses to acute amphetamine in the rat. Pharmacology Biochemistry
and Behavior 45: 629 - 637.
Szendrei, K. (1980): The chemistry of khat. Bulletin of Narcotics 32: 5 – 36.
Taber, M.T. and Fibiger, H.C. (1997): Feeding-evoked dopamine release in the nucleus
accumbens: regulation by glutamatergic mechanisms. Neuroscience 76: 1105 – 1112.
244
Taha, S., Ageel, A., Islam, M. and Ginawi, O (1995): Effect of (-)-cathinone, a psychoactive
alkaloid from khat (Catha edulis Forsk) and caffeine on sexual behaviour in rats.
Pharmacological Research 31: 299 – 303.
Tamaoki, B.I. (1973): Steroidogenesis and cell structure. Biochemical pursuit of sites of
steroid synthesis. Journal of Steroid Biochemistry 4: 89.
Tariq, M., Parmar, S., Qureshi, S., El-Feraly, F.S. and Al-Meshal, I.A. (1987): Clastogenic
evaluation of cathinone and amphetamine in somatic cells of mice. Mutation
Research/ Reviews in Mutation Research 190: 153.
Tariq, M., Islam, M.W., Al- Meshal, I.A., El- Feraly, F.S. and Ageel, A.M. (1989):
Comparative study of cathinone and amphetamine on brown adipose thermogenesis.
Life Sciences 44: 95.
Tariq, M., Qureshi, S., Ageel, A.M. and Al-Meshal, I.A. (1990): The induction of dominant
lethal mutations upon chronic administration of qat (Catha edulis) in albino mice.
Toxicology Letters 50: 349 - 353.
Tecott, L.H., Sun, L.M Akana, S.F., Strack, A.M, Lowestein, D.H., Dallman, M.F. and Julius,
D. (1995): Eating disorder and epilepsy in mice lacking 5-HT2c serotonin receptors.
Nature 374: 542 - 546.
Tindall, D.J., Rowley, D.R., Murthy, L., Lipshultz, L.I. and Chang, C.H. (1985): Structure
and biochemistry of the Sertoli cell. International Review of Cytology 94: 127 – 149.
Toennes, S.M. and Kauert, G.F. (2002): Excretion and detection of cathinone, cathine and
phenylpropanolamine in urine after khat chewing. Clinical Chemistry 48: 1715 - 1719.
Toennes, S.W., Harder, S., Schramm, M., Neiss. and Kauert, G. (2003): Pharmacokinetics of
cathinone, cathine and norephedrine after the chewing of khat leaves. British Journal
of Clinical Pharmacology 56: 125 - 130.
245
Tsai, S.C., Chiao, Y.C. and Lu, C.C. (1996): Inhibition by amphetamine of testosterone
secretion through a mechanism involving an increase of cyclic AMP production in rat
testes. British Journal of Pharmacology 118: 984 – 988.
Van Bockstaele, E.J., Cestari, D.M. and Pickel, V.M. (1994): Synaptic structure and
connectivity of serotonin terminals in the ventral tegmental area: Potential sites for
modulation of mesolimbic dopamine neurons. Brain Research 647: 307 – 322.
Wagner, G., Preston, K., Ricuarte, G., Schuster, C and Seiden, L. (1982): Neurochemical
similarities between d- 1- cathinone and d- amphetamine. Drug and Alcohol
Dependence 9: 279 - 284.
Wang, L. (1986): Effect of cortisol on luteinizing hormone release during sexual behaviour in
the boar. Canadian Journal of Veterinary Research 50: 540 – 542.
Wang, G.J., Volkow, N.D., Chang, L., Miller, E., Sedler, M., Hitzemann, R., Zhu, W., Logan,
J., Ma, Y. and Fowler, J.S. (2004): Partial recovery of brain metabolism in
methamphetamine abusers after protracted abstinence. American Journal of Psychiatry
161: 242 – 248.
Wango, E.O., Odongo, H.O., Oduma, J. and Oduor-Okelo, D. (1995): Effects of 6hydroxydopamine on testosterone production by mouse Leydig cells in vitro. Acta
Biologica Hungarica 46: 75 - 85.
Wango, E.O., Onyango, D.W., Odongo, H., Okindo, E. and Mugweru, J. (1997): In vitro
production of testosterone and plasma levels of luteinising hormone, testosterone and
cortisol in male rats treated with heptachlor. Comparative Biochemistry and
Physiology 118: 381 – 386.
246
Welsh, T.H., Randel, R.D. and Johnson, B.H. (1981): Interrelationships of serum
corticosteroids, LH and testosterone in male bovine. Archives of Andrology 6: 141 –
150.
Weibel, E.R. (1979): Stereological methods: Practical methods for biological morphometry.
London: American press.
Westerink, B.H Teisman, A. and de Vries, J.B. (1994): Increase in dopamine release from the
nucleus accumbens in response to feeding: a model to study interactions between
drugs and naturally activated dopaminergic neurons in the rat brain. Naunyn
Schmiedelbergs Archives of Pharmacology 349: 230 – 235.
Westernberg, H.G., den Boer, J.A., Murphy, D.L (Eds.). (1996): Advances in the
Neurobiology of Anxiety Disorders. Wiley: New York.
White, J.D. (1994): The chemistry of khat. An approach to the synthesis of euonyminol. Pure
and Applied Chemistry 66: 2183 – 2188.
White, F.J. (1996): Synaptic regulation of mesocorticolimbic dopamine neurons. Annual
Review of Neuroscience 19: 405 – 436.
Widler, P., Mathys, K., Brenneisen, R., Kalix, P. and Fisch, H.U. (1994): Pharmacodynamics
and pharmacokinetics of khat: a controlled study. Clinical Pharmacology and
Therapeutics 55: 269 - 277.
Wilson, C., Nomikos, G.G., Collu, M. and Fibiger, H.C. (1995): Dopaminergic correlates of
motivated behaviour: importance of drive. Journal of Neuroscience 15: 5169 – 5178.
Wolfes, O. (1930): Uber das Vorkommen von D-nor- iso ephedrine in Catha edulis. Archives
of Pharmacology 268: 81 - 83.
Wolgin, D.L. and Munoz, J.R. (2006): Role of instrumental learning in tolerance to cathinone
hypophagia. Behavioural Neuroscience 120: 362 – 370.
247
World Health Organization Advisory Group (1980): Review on pharmacology of khat.
Bulletin on Narcotics 3: 83.
World Health Organization (1985): Expert Committee on addiction-producing drugs.
Cathinone. WHO Technical Report Series 729: 8 - 9.
Yamawaki, S., Lai, H. and Horita, A. (1983): Dopaminergic and serotonergic mechanisms of
thermoregulation: mediation of thermal effects of apomorphine and dopamine.
Journal of Pharmacology and Experimental Therapeutics 227: 383.
Yanagita, T. (1979): Studies on cathinone: cardiovascular and behavioral effects in rats and
self-administration experiments in monkeys. In: Problems of Drug Dependence.
National Institute of Drug Abuse, U.S Government Printing Office, Washington D.C.
Research Monograph 2: 326 - 327.
Yen, S.S. (1991): The hypothalamic control of pituitary hormone secretion. In: Reproductive
endocrinology.Yen, S.S. and Jaffe, R.B. (Eds.). Saunder, W.B. Company: Harcourt
Brace Jovanorich, Inc., 65 – 104.
Yousef, G., Huq, Z. and Lambert, T. (1995): Khat chewing as a cause of psychosis. British
Journal of Hospital Medicine 54: 322 – 326.
Zelger, J. and Carlini, E. (1980): Anorexigenic effects of two amines obtained from
Catha edulis Forsk (khat) in rats. Pharmacology, Biochemistry abd Behavavior 12:
701 – 705.
Zelger, J., Schorno, H. and Carlin, E. (1980): Behavioural effects of cathinone: an
amphetamine obtained from Catha edulis: comparisons with amphetamine,
norpseudoephedrine, apomorphine and nomifensine. Bulletin on Narcotics 32: 67 - 81.
Ziegler, T.E. and Bercovitch, F.B. (Eds.). (1990): Socio-endocrinology of Primate
Reproduction, New York: Wiley-Liss.
248
Zirkin, B.R., Ewing, L.L., Kromann, N. and Cohran, R.C. (1980): Testosterone secretion by
rat, rabbit, guinea pig, dog and hamster testes perfused in vitro: correlation with
Leydig cell ultrastructure. Endocrinology 107: 1867 - 1874.
Zwain, I.H. and Yen, S.S. (1999): Neurosteroidogenesis in astrocytes, oligodendrocytes, and
neurons of cerebral cortex of rat brain. Endocrinology 140: 3843 – 3852.
249
APPENDIX 1
1.0UNITS
µl
Microlitre
µ
Micron
µg
Microgram
µm
Micrometer
ml
Milliter
IU
International Units
g
Gram
Kg
Kilogram
%
Per cent
0
Degrees Celcius
C
pH
The negative log of H+
mM
Milmole
nmol/l Nanomole per liter
mm
Milmeter
mg
Milgram
cm
Centimeter
mm3
Cubic milmeter
250
APPENDIX 2
2.0 ABBREVIATIONS
H
Hour
Min
Minute
Sec
Second
SD
Standard deviation
SEM Standard error of mean
cDNA Complementary deoxyribonucleic acid
RNA Ribonucleic acid
UK
United Kingdom
Ltd
Limited
O2
Oxygen
CO2
Carbon dioxide
N
Normality
G
Gauge
P
P value
Rf
Refractory Index

Beta

Delta
DMSO Dimethyl sulphoxide
ANOVA Analysis of Variance
251
APPENDIX 3
3.0 PREPARATION OF REAGENTS
3.1 0.1 M PHOSPHATE BUFFER SALINE, pH 7.2
A stock solution of 0.1 M phosphate buffer was prepared by dissolving 17.6 g Na2HPO4.H2O,
MW 178.05 in 500 ml double distilled water to make first stock solution. A second stock
solution was prepared by dissolving 13.8 g Na2HPO4.H2O, MW 138.01 in 500 ml double
distilled water. The final stock solution of 0.1M was made by mixing 360 ml of the first solution
with 140 ml of second solution and diluting it to 1 litre with double distilled water to give 0.1M
phosphate buffer saline pH 7.2.
3.2 4% OSMIUM TETROXIDE (OsO4)
Into a flask containing 25 ml double distilled water, 1 g of Osmium tetroxide was added, shaken
vigorously and flask wrapped with aluminium foil and stored at 40C until required for use.
3.3 Modified Minimum Essential Media (MEM)
Add 2 g Sodium bicarbonate in a flask containing 1 liter of double distilled water. Ajust the
pH to between 7.2 and 7.4 and incubate for 5 days at 370C in 3-5% CO2 to check for sterility.
The media is then filtered and store at -40C for use.
252