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Immunohistochemical localization of anterior pituitary cell types of vervet monkey (Chlorocebus aethiops) following sub-chronic cathinone exposure Albert Nyongesa a,n , Jemimah Oduma a , Mustafa alAbsi b , Sanika Chirwa c a Department of Veterinary Anatomy and Physiology, University of Nairobi, P.O. Box 30197, 00100 Nairobi, Kenya b Duluth Medical Research Institute, University of Minnesota, Duluth, MN 55812, USA c Department of Neuroscience and Pharmacology, Meharry Medical College, Nashville, USA article info Article history: Received 14 April 2015 Received in revised form 3 August 2015 Accepted 6 August 2015 Available online 12 August 2015 Keywords: HPA axis Immunohistochemistry Monkey Pituitary abstract Ethnopharmacological relevance: Khat (Catha edulis) contains cathinone, an active principal that is cus- tomarily used as a psychostimulant that wards off fatigue and to some extent used as an aphrodisiac. Aim of study: To investigate effects of escalating doses of cathinone on hormone expression by different anterior pituitary cell types using specic antibodies. Material and methods: Eleven vervet monkeys (6 males and 5 females) divided into tests (n ¼9) and controls (n ¼2) were used. Animals were allocated as group I (saline controls), group II (0.8 mg/kg), group III (3.2 mg/kg) and group IV (6.4 mg/kg) of cathinone. All treatments were via oral route at alternate days of each week. At the end of 4-month treatment phase, GnRH agonist (ZOLADEX) was administered to group II (low dose) and group IV (high dose) alongside cathinone for 2 additional weeks. Results: High cathinone dose at long-term exposure caused proliferation of gonadotrophs but decrease in lactotrophs and corticotrophs in anterior pituitary sections of animals while effect of low dose on these cells was insignicant. Subsequent GnRH agonist co-treatment with low and high cathinone doses en- hanced gonadotroph proliferation but no change on decline of lactotrophs and corticotrophs. Conclusion: We believe that there was a possible potentiation of cathinone on pituitary hormone synthesis thereby inuencing reproductive function. Suppression of corticotrophic and lactotrophic functions suggest lowering of stress levels and modulation of reproductive function based on dose level and chronicity of exposure. The ndings are consistent with the hypothesis that cathinone interferes with pituitary cell integrity and consequently target organs, but further studies are required to address the precise mechanism underlying this phenomenon. & 2015 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Khat is both socially and economically one of the most im- portant plants to inhabitants of areas where it is grown in coun- tries of Eastern Africa and the Middle East. It is chewed as a social custom and a stimulant by an estimated over 20 million people (Al-Motarreb et al., 2002). Fresh leaves contain cathinone (Kri- zevski et al., 2007; Al-Obaid et al., 1998; Kalix, 1990), a natural compound that is similar to ( þ ) amphetamine in chemical struc- ture and biological activity (Kelly, 2011; Feyissa and Kelly, 2008; Kalix, 1992). Psychostimulants increase adrenocorticotrophic hor- mone (ACTH) (Ishikawa et al., 2007) and corticosterone levels (Mello and Mendelson, 1997). Anterior pituitary gland of drug abusers has been shown to have mixed cell follicles containing clusterin, a multifunctional glycoprotein, related to cellular de- generation (Zwain and Amato, 2000). Earlier studies in humans showed that long-term exposure to amphetamine and metham- phetamine caused increase in clusterin-positive cells and gona- dotrophs but a decrease in corticotrophs and lactotrophs (Ishikawa et al., 2007). These observations suggest that drug abuse inu- ences expression of clusterin in mixed cell follicles and endocrine function in anterior pituitary cells in response to chronic activation of hypothalamic-pituitary-adrenocortical and gonadal axes. Whe- ther long-term use of cathinone causes similar effects in the user remains speculative. Given the widespread use of cathinone, it seems prudent to evaluate its mechanism of action on functional systems in the body of the user. In Kenya, khat consumption is associated with a lifestyle and plays role in marriage negotiations among communities where it is grown. Its cultivation also contributes to the government Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology http://dx.doi.org/10.1016/j.jep.2015.08.007 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved. n Corresponding author. E-mail address: [email protected] (A. Nyongesa). Journal of Ethnopharmacology 174 (2015) 168177

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  • Journal of Ethnopharmacology 174 (2015) 168–177

    Contents lists available at ScienceDirect

    Journal of Ethnopharmacology

    http://d0378-87

    n CorrE-m

    journal homepage: www.elsevier.com/locate/jep

    Immunohistochemical localization of anterior pituitary cell types ofvervet monkey (Chlorocebus aethiops) following sub-chronic cathinoneexposure

    Albert Nyongesa a,n, Jemimah Oduma a, Mustafa al’Absi b, Sanika Chirwa c

    a Department of Veterinary Anatomy and Physiology, University of Nairobi, P.O. Box 30197, 00100 Nairobi, Kenyab Duluth Medical Research Institute, University of Minnesota, Duluth, MN 55812, USAc Department of Neuroscience and Pharmacology, Meharry Medical College, Nashville, USA

    a r t i c l e i n f o

    Article history:Received 14 April 2015Received in revised form3 August 2015Accepted 6 August 2015Available online 12 August 2015

    Keywords:HPA axisImmunohistochemistryMonkeyPituitary

    x.doi.org/10.1016/j.jep.2015.08.00741/& 2015 Elsevier Ireland Ltd. All rights rese

    esponding author.ail address: [email protected] (A. Ny

    a b s t r a c t

    Ethnopharmacological relevance: Khat (Catha edulis) contains cathinone, an active principal that is cus-tomarily used as a psychostimulant that wards off fatigue and to some extent used as an aphrodisiac.Aim of study: To investigate effects of escalating doses of cathinone on hormone expression by differentanterior pituitary cell types using specific antibodies.Material and methods: Eleven vervet monkeys (6 males and 5 females) divided into tests (n¼9) andcontrols (n¼2) were used. Animals were allocated as group I (saline controls), group II (0.8 mg/kg), groupIII (3.2 mg/kg) and group IV (6.4 mg/kg) of cathinone. All treatments were via oral route at alternate daysof each week. At the end of 4-month treatment phase, GnRH agonist (ZOLADEX) was administered togroup II (low dose) and group IV (high dose) alongside cathinone for 2 additional weeks.Results: High cathinone dose at long-term exposure caused proliferation of gonadotrophs but decrease inlactotrophs and corticotrophs in anterior pituitary sections of animals while effect of low dose on thesecells was insignificant. Subsequent GnRH agonist co-treatment with low and high cathinone doses en-hanced gonadotroph proliferation but no change on decline of lactotrophs and corticotrophs.Conclusion: We believe that there was a possible potentiation of cathinone on pituitary hormonesynthesis thereby influencing reproductive function. Suppression of corticotrophic and lactotrophicfunctions suggest lowering of stress levels and modulation of reproductive function based on dose leveland chronicity of exposure. The findings are consistent with the hypothesis that cathinone interfereswith pituitary cell integrity and consequently target organs, but further studies are required to addressthe precise mechanism underlying this phenomenon.

    & 2015 Elsevier Ireland Ltd. All rights reserved.

    1. Introduction

    Khat is both socially and economically one of the most im-portant plants to inhabitants of areas where it is grown in coun-tries of Eastern Africa and the Middle East. It is chewed as a socialcustom and a stimulant by an estimated over 20 million people(Al-Motarreb et al., 2002). Fresh leaves contain cathinone (Kri-zevski et al., 2007; Al-Obaid et al., 1998; Kalix, 1990), a naturalcompound that is similar to (þ) amphetamine in chemical struc-ture and biological activity (Kelly, 2011; Feyissa and Kelly, 2008;Kalix, 1992). Psychostimulants increase adrenocorticotrophic hor-mone (ACTH) (Ishikawa et al., 2007) and corticosterone levels(Mello and Mendelson, 1997). Anterior pituitary gland of drug

    rved.

    ongesa).

    abusers has been shown to have mixed cell follicles containingclusterin, a multifunctional glycoprotein, related to cellular de-generation (Zwain and Amato, 2000). Earlier studies in humansshowed that long-term exposure to amphetamine and metham-phetamine caused increase in clusterin-positive cells and gona-dotrophs but a decrease in corticotrophs and lactotrophs (Ishikawaet al., 2007). These observations suggest that drug abuse influ-ences expression of clusterin in mixed cell follicles and endocrinefunction in anterior pituitary cells in response to chronic activationof hypothalamic-pituitary-adrenocortical and gonadal axes. Whe-ther long-term use of cathinone causes similar effects in the userremains speculative. Given the widespread use of cathinone, itseems prudent to evaluate its mechanism of action on functionalsystems in the body of the user.

    In Kenya, khat consumption is associated with a lifestyle andplays role in marriage negotiations among communities where itis grown. Its cultivation also contributes to the government

    www.elsevier.com/locate/jephttp://dx.doi.org/10.1016/j.jep.2015.08.007http://dx.doi.org/10.1016/j.jep.2015.08.007http://dx.doi.org/10.1016/j.jep.2015.08.007http://crossmark.crossref.org/dialog/?doi=10.1016/j.jep.2015.08.007&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.jep.2015.08.007&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.jep.2015.08.007&domain=pdfmailto:[email protected]://dx.doi.org/10.1016/j.jep.2015.08.007

  • A. Nyongesa et al. / Journal of Ethnopharmacology 174 (2015) 168–177 169

    revenue from foreign exports. Following the recent ban of khatexport by the governments of United Kingdom and the Nether-lands, there has emerged socio-economic and political panic insource countries due to loss of revenue collection from khat ex-ports. The affected communities are calling upon the governmentto intervene and lift the ban for the sake of economies of scale inthese regions. To these people, the socio-economic benefits fromkhat outweigh the potential health risks accompanying its long-term and un-regulated use. Conversely, khat use has been asso-ciated with breakdown of marriages, prostitution and a host ofother social evils (Beckerleg, 2008). The delicate balance betweenhealth risks and socio-economic value of the crop has degeneratedinto lack of clear laws in regulating khat use even with the fullunderstanding of the dynamics of health risk factors posed to khatchewers. To this end, a proper approach into investigations of khatand human health is a critical consideration whereupon vital in-formation is provided to social and health care professionals,policy makers and international organizations with sole aim ofsensitizing affected populations.

    To further explore the interaction between cathinone effects onendocrine hormones, we carried out studies by immuno-locali-zation of hormone-producing cells of the anterior pituitary gland.In particular, we have focussed on corticotrophs, lactotrophs andgonadotrophs. Neuro-pharmacological studies together with his-tochemical and immunofluorescence techniques have establishedthat biogenic amines and other neurotransmitters play a crucialrole in the modulation of anterior pituitary hormone secretionthrough action on hypothalamic-hypophysiotrophic neurons (Fuxeand Hökfelt, 1969). Various endocrine and reproductive effects ofkhat and cathinone in humans and experimental animals havebeen reported although some of the reports appear contradictory.On the one hand, increase in adrenocorticotrophic hormone inhumans (Nencini et al., 1984) and plasma cortisol in rabbits(Nyongesa et al., 2008) following khat extract exposure were re-ported. On the other hand, decreased plasma cortisol and prolactinwere observed in baboons following sub-chronic khat exposure(Mwenda et al., 2006). Other studies showed a dose-dependentdecrease in cortisol following cathinone treatment in rats (Mo-hammed and Engidawork, 2011) and decrease in cortisol andprolactin in vervet monkeys (Nyongesa et al., 2014b). These find-ings though contradictory, are significant since they point to theresponsiveness of the anterior pituitary cell types. It is not clearwhether the observed discrepancies could be due to species dif-ference or merely the responsiveness of pituitary cell types tohormone synthesis and secretion. It is noteworthy that majority ofstudies show effect on hormonal release but the precise effect oncells secreting these hormones mediating differences in endocrineresponsiveness to cathinone remains unclear. No experimentshave assessed immunohistochemical localization or antigen ex-pression in anterior pituitary cell types and cellular morphologyfollowing drug use in the same experimental subjects. To this end,we used antibody specific markers to immuno-localize the celltypes secreting hormones as well as examination of cellularmorphology by histologic staining. In the present study we hy-pothesized that cathinone variously affects hormonal synthesis bygonadotrophs, corticotrophs and lactotrophs or release of hypo-physeotrophic factors from hypothalamus. We were able to ex-amine relationship between endocrine function and cellularmorphology. Further, we used GnRH agonist, goserelin acetate(ZOLADEX) to study the effect of cathinone on the responsivenessof anterior pituitary gonadotrophs to hypothalamic GnRH. Thestudy used the vervet monkey as an animal model. Vervet mon-keys have long been the most important non-human primatemodel for biomedical research other than rhesus macaque (Bauluet al., 2002). They have been used extensively for research in hu-man reproduction (Amaral, 2002) and cognitive function relevant

    to human psychiatric disorders (James et al., 2007).

    2. Materials and methods

    2.1. Animals and housing

    Eleven adult vervet monkeys were captured in the wild and putunder quarantine at the Institute of Primate Research (IPR) forthree months and housed at the same institution. Housing wasdone in individual standard monkey cages(0.6 m�0.66 m�0.8 m) for the duration of the study. The re-search was conducted in accordance with the internationally ac-cepted principles for laboratory animal use and care as found inthe European Community guidelines (EEC Directive of 1986; 86/609/EEC). The research work was approved by Institute of PrimateResearch Review Committee.

    2.2. 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(o5 mm) and dissolved in 250 ml methanol in a conical flask forextraction. The quantity of khat used followed the contention thata bundle of 100 g of fresh khat leaves contain a cathinone contentof about 200 mg (European Monitoring Centre for Drugs and DrugAddiction, 2011). The extraction protocol followed that previouslydescribed by Lee (1995). In brief, the mixture was shielded fromlight and sonicated at room temperature for 15 min with inter-mittent shaking and stirring followed by filtration through cottongauze and then grade 1 Whatman filter paper to get rid of fineparticles. The non-filtered plant material was re-extracted in250 ml fresh methanol and sonicated for 24 h. The mixture wasthen filtered and admixed with initial methanol-extracted khatmaterial and condensed to near dryness (o1 ml) using a stream ofair. A very dilute solution of sulfuric acid (approximately 0.02 N)was used to re-suspend and acidify the residue followed bychloroform extraction to remove neutral organic compounds aswell as plant solids. A small amount of saturated sodium bi-carbonate solution was added to the aqueous solution to basify theextracts followed by methylene chloride to extract cathinone andcathine. A stream of air was again used to reduce the extract to aminimal amount and solution vacuum-dried at 337 mbar in aRotar Vump for 5 h into an oily paste. The dry weight of the ex-tracts was determined and thin layer chromatography used toconfirm the presence of alkaloids. The plant extracts were spotteddirectly onto a pre-coated 5 by 10 cm silica gel 60 plate. The platewas then developed in ethyl acetate: methanol: aqueous ammonia(85:10:5), and viewed under an ultraviolet lamp. The spots werevisualized using a 0.5% ninhydrin solution, and the plate heatedusing a heat gun. Color development was used to localize the al-kaloids (purple for cathine and burnt orange for cathinone) amongthe moving spots. The Rf values for cathinone and cathine werealso calculated. The vapour phase infrared spectra was obtained ona Hewlett-Packard Model 5890 Series II Gas Chromatograph, usinga 12 m by 0.32 mm HP-5 (0.52 mm loading) capillary column and atemperature program of 70 °C for 1 min, 15 °C/min to 270 °C, witha final temperature hold for 5 min, equipped with a Hewlett-Packard Model 5965B Infrared Detector. A total of about 1000 mgcathinone was extracted from the leaves and it was approximately97% pure.

    2.3. Experimental design, dose preparation and treatment

    The animals were randomly assigned to four groups. Group Iwere controls and comprised of two animals (1 male and 1 female)

  • A. Nyongesa et al. / Journal of Ethnopharmacology 174 (2015) 168–177170

    while groups II, III and IV were tests and comprised of three ani-mals (2 males and 1 female) each. The sample size of animals usedin the study was chosen on ethical considerations. Three con-centrations of cathinone (1.6 mg/ml, 6.4 mg/ml and 12.8 mg/ml)were prepared by reconstituting cathinone extract paste usingnormal saline and administered at three different doses (0.8 mg/kg, 3.2 mg/kg and 6.4 mg/kg body weight) respectively. In eachcase, the working volume (2 ml) of the stock solution, obtained byfactoring in the body weight of the animal ( in this case 4 kg), wasadjusted to an appropriate final volume (5 ml) using the samesaline and administered via intra-gastric tube at alternate days ofeach week for 4 months. The doses were chosen based on previousstudies in humans (Kalix, 1984) and rats (Mohammed and En-gidawork, 2011) which indicated optimum effect of cathinone onvarious body parameters to be within this dose range. Controlswere administered normal saline at the same volume and regimenas test animals. At the end of the 4-month cathinone alonetreatment, animals on low and high doses (0.8 and 6.4 mg/kg)were administered ZOLADEX at 50 μg/kg body weight singlesubcutaneous injection while continuing with cathinone treat-ment for a further 2 weeks. Due to small sample size that waslimited by ethical concerns only animals at low and high doses ofcathinone were co-treated with ZOLADEX to establish antagonisticor synergistic effects of cathinone in presence of the GnRH agonist.The general health status of the animals was routinely monitoredthroughout the entire experimental period to ensure only healthanimals were used. Behavioural observations were also done anddata has been published elsewhere (Nyongesa et al., 2014a). At theend of treatment period, all animals were euthanized with highdose of ketamine and pituitary gland harvested for im-munohistochemical evaluation.

    2.4. Immunohistochemistry

    The adenohypophysis of all animals were harvested and fixedwith 4% formaldehyde in phosphate-buffered saline (pH 7.2) for1 h and, thereafter, embedded in paraffin wax. Serial sections (5 μthick) were carefully cut and labeled with animal number and thestain to be used. Immunostaining was done by avidin-biotincomplex method and color development done using 3, 3′ diami-nobenzidine. The slides were placed on Dako Autostainer SlideRacks (code S3704) and inserted in an oven at 100 °C for 15 minand allowed to cool before proceeding with antigen retrieval.Appropriate labels of slides were generated from a computerconnected to Dako Link 48 instrument, fixed carefully on slides toavoid damage to the barcode. Thereafter, slides were inserted intothe Dako target retrieval solution in PT Link tanks (code PT100/PT101), which incorporated pre-heat temperature, antigen re-trieval temperature and time as well as cool down settings at 97 °Cfor 20 min. The Dako target retrieval solution was prepared (3:1)according to manufacturer’s specifications and used for both de-paraffinization and antigen retrieval. Each autostainer slide rackwas removed from PT Link tanks and immediately dipped into PTLink rinse solution (code PT109) containing diluted room tem-perature Dako Wash buffer (10� ) (code S3006) and left for 1–5 min. The slides were then removed and washed gently withdiluted room temperature wash buffer before insertion into Au-tostainer. Thereafter, slides were placed on a Dako autostainercontaining respective antibodies. Primary antibodies were mono-clonal mouse anti-human adrenocorticotrophic hormone (1:50,Dako), monoclonal mouse anti-human luteinizing hormone (1:50,Dako), polyclonal rabbit anti-human prolactin (1:200, Dako) andpolyclonal rabbit anti-human S100 (Ready-to-use, Dako). Detec-tion was performed using anti-mouse or anti-rabbit Dako Envisionsystem (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 assessingnormal morphology of cells of the pituitary gland. After staining,slides were removed from autostainer, dehydrated, cleared andcounterstained with hematoxylin.

    2.5. Histological evaluation

    The sections were examined and fields of interest photo-graphed using a Zeiss Axioskop 40 plus microscope (Carl ZeissMicroImaging GmbH, Germany) and a ProgRes CT5 USB C plusdigital camera (Jena, Germany) with ProgRes Capture Pro 2.8 soft-ware (JENOPTIK/Optical Systems). Immunohistochemistry resultsfor antibodies of interest were classified based on presence orabsence of staining as well as average number of immunostainedcells per three randomly selected high magnification fields (�40objective). Slides that were stained with hematoxylin and eosinwere used for standard histological evaluation.

    2.6. Morphometric analysis

    Quantification of immunostained cells for ACTH, prolactin andLH on anterior pituitary sections was done by direct manualcounting technique previously applied in measuring the mitoticrates in the endometrium of primates (Brenner et al., 2003). Im-munostained cells were observed under light microscopy andthree non-overlapping fields at high power magnification wererandomly selected with the help of an ocular grid and countedusing a mechanical tabulator. The labelling index (LI) was used toexpress the number of stained cells as a percentage of the entirepopulation of cells on a section of the slide. The mean values of LIwere then used for comparison among different treatment groupsusing one-way ANOVA at 5% significance level. In order to calcu-late the LI, the following formula was used:

    LI PiC /PiCT x100= ( + )

    where,PiCT¼total number of cells for each stain (ACTH, prolactin, LH)

    that was counted in a given field (positive and negative cells foradenohypophysis),

    PiCþ¼number of immunostained cells (positive cells) for agiven stain counted on the same area,

    LI¼ labelling index, usually expressed as a percentage.

    3. Results

    3.1. Cathinone effects on pituitary cell histology and ACTHimmunostaining

    There were behavioural changes on vervet monkeys in thecourse of study as we reported in a recent publication (Nyongesaet al., 2014a). There was a dose and time-dependent increase inaggression, anxiety, abnormal responses, withdrawal and appetiteloss. Most of these behaviours were not observed in controls.Time-dependent changes demonstrated effect of long-term ex-posure to cathinone on behavioural profiles. However, there wereno significant sex differences in immunohistochemical results anddata presentation is grouped together. Histological picture of thesections of treated and control animals showed physiologicallyactive glands with no significant morphological changes observed(Fig. 1). Each antibody staining was done on treatment subjectspituitary sections alongside that of control that was treated withnormal saline through study. Immunohistochemistry for stainingof basophils showed no obvious difference in the intensity of

  • Fig. 1. Hematoxylin and eosin staining of anterior pituitary gland of vervet monkey.Animals received intra-gastric administration of 6.4 mg/kg at alternate days of eachweek for 4 months and anterior pituitary glandular cell immunostaining done asdescribed under Section 2. The morphological features in this group of animals(n¼3) were similar to those of controls (n¼2) that receive normal saline hence itwas taken as a control slide. The slide shows presence of acidophils (a), basophils(b) and sinusoids (s) lined by epithelial cells (filled up arrow). White arrow showsdegranulation into sinusoids indicating a physiologically active gland. (Originalmagnification �400).

    A. Nyongesa et al. / Journal of Ethnopharmacology 174 (2015) 168–177 171

    cytoplasmic staining across treatment groups and controls. Insections stained with mouse anti-human ACTH antibody, two cellpopulations were identified with the predominant proportion ofmostly acidophils that appeared negative for ACTH stain. Positivelystaining cells, interpreted as corticotrophs, revealed uniform intra-cytoplasmic staining and appeared arranged individually and oc-casionally in clusters of 2–3 cells (Fig. 2a A–D). A random selectionof three fields at �40 objective showed a dose-dependent de-crease in LI of corticotrophs for 0.8 mg/kg (F3, 7¼36; Po0.05),3.2 mg/kg (F3, 7¼22; Po0.5) and 6.4 mg/kg (F3, 7¼14; Po0.05)body weight of cathinone compared to controls (F3, 7¼48;Po0.05) (Fig. 2b).

    3.2. Cathinone effects on LH immunostaining in presence and ab-sence of GnRH agonist

    The sections stained with mouse anti-human LH showed a si-milar pattern obtained with ACTH staining, with two cell popu-lations under light microscopy. The larger percentages of cellswere composed mostly of acidophils that were negative for LHstain. The staining pattern was cytoplasmic and positive cells, in-terpreted as gonadotrophs, were mostly scattered throughout thefield and occasionally in groups of 2–3 cells. These cells main-tained a dispersed pattern, and intensity of staining was similar forwithin- and between-treatment groups. However, the number ofstained cells when taken at three randomly selected high powerfields was significantly different across treatment subjects asshown by LI (Fig. 3a). The mean LI for positive cells for low dose(0.8 mg/kg) and high dose (6.4 mg/kg) of cathinone co-treatedwith 50 mg/kg GnRH agonist were higher (F3, 7¼46; Po0.05 andF3, 7¼78; Po0.05, respectively ) compared to dose 3.2 mg/kg(F3, 7¼43; Po0.05) and controls (F3, 7¼20; Po0.05) indicatingthat GnRH agonist boosted the antigen expression of positivestaining cells. The results show a dose-dependent effect of cath-inone on positive cell count and the synergistic effect of GnRHagonist (Fig. 3b).

    3.3. Cathinone effects on lactotroph immunostaining

    In sections stained with polyclonal rabbit anti-human prolactin,

    two cell populations were observed, most of which were negativefor prolactin. Positive cells (lactotrophs) were scattered in the fieldoccasionally in clusters of 2–4 cells and maintained a dispersedpattern with a similar intensity of staining for within- and be-tween- treatment groups. However, the LI of stained cells whentaken at three randomly selected high power fields was sig-nificantly different across treatment subjects (Fig. 4a). Resultsshow a dose-dependent decrease in number of lactotrophs(F3, 7¼61; Po0.05, F3, 7¼36; Po0.05 and F3, 7¼18; Po0.05) atdoses (0.8, 3.2 and 6.4 mg/kg body weight) of cathinone, respec-tively compared to controls (F3, 7¼63; Po0.05) (Fig. 4b).

    3.4. Cathinone effects on S100 protein immunostaining

    Immunostaining of cells with polyclonal rabbit anti-humanS100 showed no apparent staining in pituitary sections of treat-ment groups and controls indicating absence of S-100-positivecells (Fig. 5a). In order to validate the specificity of the assay, po-sitive control for S100 (skin melanoma known to be positive forthis stain) was performed in parallel with test samples (Fig. 5b). Asummary of effect of escalating doses of cathinone on anteriorpituitary cell types considered in this study is shown (Table 1).

    4. Discussion

    A major aim of this study was to determine whether the effectsof cathinone on endocrine function along hypothalamic–pituitary–adrenocortical and gonadal axes are related to potential morpho-logical changes of anterior pituitary cells. Here, we assessed thesub-chronic in vivo effects of cathinone on anterior pituitary cellfunction, specifically corticotrophs, gonadotrophs and lactotrophsby immunohistochemical localization. Further, we explored theinfluence of GnRH agonist on gonadotrophic function followingcathinone exposure. The data presented here provides a me-chanistic link that supports published data suggesting alterationsin hormonal profiles following cathinone (Nyongesa et al., 2014b,Mohammed and Engidawork, 2011, Islam et al., 1990) and khat(Nyongesa et al., 2008, 2007; Mwenda et al., 2006) exposure tohumans and experimental animals. The results of the presentstudy show that cathinone at medium (3.2 mg/kg) and high(6.4 mg/kg) dose and at sub-chronic exposure suppressed corti-cotrophic and lactotrophic antigen expression. In agreement withour recently published data (Nyongesa et al., 2014 b), decrease inLI of corticotrophs and lactotrophs corresponded with suppressedcortisol and prolactin hormone levels. On the other hand, celldensity of gonadotrophs increased in a dose-dependent manner.This was compounded by co-treatment with GnRH agonist for lowand high dose of cathinone compared to cathinone-alone mediumdose. The histological evaluation of these pituitary cell typesshowed no significant changes between treatment groups andcontrols. The results of our study, however, are correlative and donot directly address causative relationship between hormonemeasurement and cellular morphological integrity.

    4.1. Effect on corticotrophs

    The decrease in LI of corticotrophs in the present study may beexplained by disturbances in cleavage of propiomelanocortin(POMC) into adrenocorticotrophic hormone (ACTH), β-endorphinand β-lipotropin following cathinone treatment. Studies on re-peated administration of amphetamine twice daily for 14 daysmoderately increased POMC mRNA in pituitary gland with no al-teration following adrenolectomy (Jaworska et al., 1994). Sinceglucocorticoids suppress POMC synthesis and causes reduction inACTH stores in secretory granules (Gann et al., 1981), a rise in

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    Fig. 2. (a) Dose-dependent effect of cathinone on corticotrophs (full arrows) in controls (n¼2) (A), cathinone-treated animals (n¼3) at 0.8 mg/kg plus 50 mg/kg GnRHagonist (B), 3.2 mg/kg body weight of cathinone alone (n¼3) (C) and 6.4 mg/kg body weight of cathinone plus 50 mg/kg GnRH agonist (n¼3) (D). Note that GnRH agonist didnot have significant effect on expression of ACTH-positive cells (Original magnification �400). (b) Mean LI showing number of corticotrophs as percentage of total cells insections of control and cathinone-treated groups. Results show a dose-dependent decrease in mean LI of cells counted in three random �40 fields with a significantdecrease (Po0.05) at dose 6.4 mg/kg body weight of cathinone. ** Means statistically significant at Po0.05 compared with all other groups, *** means statistically significantat Po0.01 compared with all other groups.

    A. Nyongesa et al. / Journal of Ethnopharmacology 174 (2015) 168–177172

    POMC mRNA may imply impairment of ACTH synthesis henceglucocorticoids. The expression of POMC, secretion of cortico-trophic releasing hormone and its function on corticotrophs andadrenal cortex can be regulated by cytokines such as interleukin1β and 6, tumor necrosis factor α and interferons α and γ (Marxet al., 1998). Sympathomimetics have been shown to induce hy-perthermia via neuronal damage and increase in hypothalamicconcentrations of interleukin 1β (Albers and Sonsall, 1995). It isnoteworthy that since cathinone is a sympathomimetic alkaloidthat causes peripheral vasoconstriction (Kalix, 1991) and cardiaceffects (Balint and Balint, 1994), the argument that cathinone mayhave interfered with corticotrophic function via effect on immuneregulation of hypothalamic–pituitary–adrenal axis may be re-levant. Recently, we published data indicating dose-dependent

    decrease in cortisol levels in vervet monkeys following sub-chronic cathinone exposure (Nyongesa et al., 2014b). Studies inrats also showed that high levels of cathinone suppressed corti-costerone levels (Mohammed and Engidawork, 2011). These find-ings parallel those in baboons where khat extracts suppressedserum cortisol levels (Mwenda et al., 2006) but different fromstudies in rabbits (Nyongesa et al., 2008). However, the differencein findings may be explained partly by duration of exposure tokhat/cathinone, possibly species difference and animal handling.Furthermore, it is worth noting that recent clinical observations inkhat users showed disrupted adrenocortical functions (al’Absiet al., 2013) suggesting a long-term effect of khat use on cortisol,possibly mediated by central effects at the pituitary and hy-pothalamic levels. In the present study, animals were habituated

  • 0

    10

    20

    30

    40

    50

    60

    70

    80

    90

    100

    0 0.8 + GnRH agonist 3.2 6.4 + GnRH agonist

    Cathinone dose (mg/kg body weight)

    Mea

    n La

    belli

    ng in

    dex

    for g

    onad

    otro

    phs

    (%)

    ****

    ***

    Fig. 3. (a). The figure shows gonadotrophs (open arrows) in anterior pituitary gland of control vervet monkeys (n¼2) (A) and vervet monkeys (n¼3) exposed to cathinonedoses at 0.8 mg/kg plus 50 mg/kg GnRH agonist (B), 3.2 mg/kg body weight of cathinone alone (n¼3) (C) and 6.4 mg/kg body weight of cathinone plus 50 mg/kg GnRH agonist(n¼3) (D) at alternate days of the week for 4 months as described under Section 2. Cathinone increased the LI for gonadotrophs in a dose-dependent manner. (Originalmagnification �400). (b) Mean LI expressing number of gonadotrophs as percentage of total anterior pituitary gland cells of control (n¼2) and cathinone-treated animals(n¼3 vervet monkeys/group). Note that animals at low and high doses of cathinone and co-treated with GnRH agonist expressed significantly higher (Po0.05; n¼6)number of cells compared to those exposed to cathinone alone as well as controls when counted on three fields at �400 magnification. ** Means statistically significant atPo0.05 compared to other groups, *** means statistically significant at Po0.01 compared to other groups.

    A. Nyongesa et al. / Journal of Ethnopharmacology 174 (2015) 168–177 173

    to handling and presence of observer for 1 month before bloodcollection, and control animals were handled in the same way astreatment groups.

    4.2. Effect on lactotrophs

    In this study, we have reported a dose-dependent decrease in LIof lactotrophs in cathinone-treated animals. The results are con-sistent with dose-dependent decrease in serum prolactin levels incathinone-treated vervet monkeys (Nyongesa et al., 2014b) andkhat extract-treated baboons (Mwenda et al., 2006). The precisemechanism that might be responsible for suppressed lactotroph

    function is not known, but data showing that cathinone inducesrelease of dopamine from several brain regions including striatum(Pehek et al., 1990) could be relevant. There is evidence suggestingthat dopamine may act directly as the primary prolactin inhibitingfactor (Ben-Jonathan and Hnasko, 2001) and that dopamine D2receptors are expressed on plasma membrane of lactotrophs(Goldsmith et al., 1979). To this end, it is instructive to comparelong-term endocrine effects of cathinone in vervet monkeys withthose of amphetamine in humans for clinical interpretation. Stu-dies on long-term use of amphetamines in humans reported in-crease in prolactin-secreting cells and gonadotrophs with a de-crease in somatotrophs with no change in corticotrophs and

  • 0

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    0 mg/kg 0.8 mg/kg 3.2 mg/kg 6.4 mg/kg

    Cathinone dose

    Mea

    n La

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    ng in

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    for l

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    s (%

    )

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    Fig. 4. (a). Immunostaining for lactotrophs (arrows) in anterior pituitary gland of controls vervet monkeys (A) and vervet monkeys treated with cathinone doses at 0.8 mg/kgbody weight of cathinone plus 50 mg/kg GnRH agonist (B), 3.2 mg/kg body weight of cathinone alone (C) and 6.4 mg/kg body weight of cathinone plus 50 mg/kg GnRH agonist(D) at alternate days of the week for 4 months. Note that GnRH agonist did not have significant effect on expression of lactotrophs (Original magnification �400). (b) Mean LIexpressing number of lactotrophs in controls (n¼2) and cathinone-treated groups (n¼3 vervet monkeys/group). Results showed a dose-dependent decrease in number ofcells counted in three random �40 fields. Dose 3.2 and 6.4 mg/kg body weight of cathinone caused a significant decrease (Po0.05) compared to 0.8 mg/kg and controls. *Means statistically significant at Po0.05 compared with all other groups, ** means statistically significant at Po0.01 compared with all other groups.

    A. Nyongesa et al. / Journal of Ethnopharmacology 174 (2015) 168–177174

    thyrotrophs as well as increase in clusterin-containing follicles(Ishikawa et al., 2007). The authors associated increase in lacto-trophs to dysfunction of hypothalamic dopaminergic neurons inchronic drug abusers. Related studies in rodents have shown thatacute to chronic exposure to methamphetamine or amphetamineboth lead to striatal dopamine depletion and physical destructionof dopamine terminals (Fumagalli et al., 1998) as well as decreasedlevels of presynaptic markers of dopamine function such as tyr-osine hydroxylase (Schmidt and Gibb, 1985), dopamine transporter(Wagner et al., 1982) and vesicular monoamine transporter (Freyet al., 1997). The observed decrease in LI of lactotrophs in the

    present study is indicative of modulation leading to down-reg-ulation of particular functional proteins rather than dysfunction ofhypothalamic dopaminergic neurons. However, dopamine mea-surement, its transporters or metabolites were not considered inthis study.

    4.3. Effect on gonadotrophs

    The cell density of gonadotrophs in pituitary sections of cath-inone-treated animals increased in a dose-dependent manner.This was compounded by co-treatment with GnRH agonist for low

  • Fig. 5. (a) Immunostaining of anterior pituitary gland of vervet monkeys at sub-chronis exposure of cathinone at 6.4 mg/kg body weight with anti-human S100. The cellswere negative for the stain indicating absence of expression of S100 protein in pituitary sections of this treatment group (n¼3). (Original magnification �400). (b) A skintissue with melanoma, a known positive for S100 protein, comparing staining ability of the anti-human S-100 antibody. The antibody immunostaining worked well as shownby melanoma (full arrow) around the hair follicle (white arrow). Original magnification �400.

    Table 1Mean number of cells at high power magnification for 0.8 mg/kg, 3.2 mg/kg and6.4 mg/kg body weight of cathinone (n¼3 vervet monkeys/group) as well as con-trols (n¼2). The doses were administered via intra-gastric tube at alternate days ofthe week for 4 months as described under Section 2.

    LI (%) of positive cells Cathinone dose (mg/kg body weight)

    0 0.8 3.2 6.4

    LI for ACTH (at �400magnification)

    4874 3675 2276n 1473.5n

    LI for LH (at �400magnification)

    20710 4678an

    þagonist43710 787 14.8an

    þagonistLI for PRL (at �400magnification)

    6374.5 6173 3676.2n 1875n

    LI means labelling index.an means in presence of 50 mg/kg GnRH agonist and significantly different (Po0.05)compared with all other groups.

    n Means significantly different at (Po 0.05) compared with all other groups.

    A. Nyongesa et al. / Journal of Ethnopharmacology 174 (2015) 168–177 175

    and high dose of cathinone compared to cathinone-alone mediumdose treatment, suggesting a possible enhancement of respon-siveness of gonadotrophs to GnRH following cathinone exposure.This significant finding indicates a possible up-regulation of go-nadotroph function or receptor responsiveness to GnRH in pre-sence of cathinone. Studies utilizing a bioactive GnRH analoguecoupled to ferritin (Hopkins and Gregory, 1977), a bioactive rho-damine derivative of GnRH (Naor et al., 1981) and [biotinyl-D Lys6]GnRH (Childs et al., 1983) as receptor probes indicated that GnRHreceptors are initially distributed evenly on the cell surface andthen form clusters which subsequently become internalized. It hasbeen shown that receptor-bound GnRH agonist is internalized viacoated pits and is subsequently routed either to lysosomes or tosecretory granules (Hazum and Keinan, 1983) where endosomesand Golgi complex are involved in GnRH-receptor processing(Childs et al., 1986). The present study did not consider work onreceptor binding, which may be critical in confirming the re-sponsiveness of anterior pituitary cells to hypophyseotrophichormones from the hypothalamus. The other possible mechanismthat led to up-regulation of LI of gonadotroph compared to downregulation of lactotrophs and corticotrophs lies in the role of sec-ond messengers to responsiveness of secretory stimuli. The lit-erature on intracellular signaling and hormone secretion in go-nadotrophs is complex and contains evidence for changes of andactions for cyclic AMP, cyclic GMP, arachidonic acid and the

    lipoxygenase pathway, as well as diacylglycerols and lipid derivedfrom activation of phospholipase D (Dan-Cohen et al., 1992). Al-though we did not consider measurement of intracellular signalingin gonadotrophs in the present study, these earlier findings pro-vide insights in to the possible mechanism of action of cathinoneon gonadotroph function. Increase in gonadotrophic cell stainingsuggests that impairment of sexual function following cathinonetreatment (Islam et al., 1990; Mohammed and Engidawork, 2011)involve the gonads and not the pituitary gland. Some studies have,however, shown discrepancies to these findings. In one study,Wagner et al. (1982) showed that cathinone inhibit the release ofseveral anterior pituitary hormones. This finding parallels resultsof cortictotroph and lactotroph function but differ on gonadotrophfunction in the present study. Other studies in rabbits reported adecrease in LH following khat extract administration (Nyongesaet al., 2008). From these varying observations, some unansweredquestions arise: Is there a significant difference between gonado-tropins of males and females? To what degree are the GnRH re-sponses normally subject to modulation by steroids, neuro-transmitters, peptides or catecholamines of portal circulation?How do effects of psychotropic drugs on catecholamines andneurotransmitters in the brain affect pituitary function? Are therespecies differences in response to khat or cathinone on pituitaryfunction? A good part of these questions has not been fully ad-dressed. However, our study did not find any significant differencebetween males and females with respect to cathinone effects onthis measure.

    4.4. Effect on S100 protein secreting cells

    In order to confirm whether other factors contributed to theobserved increase in gonadotrophic and decrease in lactotrophicand corticotrophic immunostaining, we further immuno-labeledfor S-100 protein in the same pituitary sections. S100-proteins arerarely observed in adenohypophysis of humans (Ishikawa et al.,2003) but in alcoholics with fatty liver or cirrhosis, S-100 proteinincreases alongside increase in number of somatotrophs and lac-totrophs (Takanashi and Ishikawa, 1984). Other studies reportedfindings suggesting that S-100 protein exerts effect on lactotroph,corticotroph, somatotroph and gonadotroph (Ishikawa et al., 2007)secretion. Studies in goat anterior pituitary gland reported pre-sence of growth hormone in S-100 cells (Shirasawa et al., 1984),indicating that S-100 cells may belong to a class of stem cells orprogenitor cells of growth hormone-producing cells. It is not clear

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    whether S-100 protein plays any role in anterior pituitary cellfunction and whether non-human primate pituitaries also expressit. The present study investigated the presence and possible in-volvement of S-100 protein in anterior pituitary cell function invervet monkeys under sub-chronic cathinone exposure. The pi-tuitary sections immunostained with anti-human S-100 were ne-gative for the stain with accompaniment of decreased LI of lacto-trophs and corticotrophs but increase in gonadotroph stainingsuggesting that anterior pituitary cell function was independent ofS-100 protein in this animal species. In our study, S-100 antibodywas used in parallel with a known positive control (skin melano-ma) (Fig. 5b). The histology of pituitary sections at all doses ofcathinone showed an actively performing gland with normal cel-lular morphological outline contrary to earlier findings of Ishikawaet al. (2007) where there was increase in clusterin in pituitarymixed cell follicles that was associated with cellular damage fol-lowing various forms of stress induced by amphetamine. It is,therefore, likely that the observed effects were largely due tocathinone exposure.

    Overall, our immunohistochemical study is the first to demon-strate immuno-localization of anterior pituitary cells following sub-chronic in vivo cathinone exposure in vervet monkeys. The labellingindices of immuno-stained cells were correlative with serum hor-monal concentrations reported in a separate publication (Nyongesaet al., 2014b). The endocrine responses could not be explained asphysiological circadian endocrine rhythms. For instance, prolactin isdetected in plasma at all times during the day but in discrete pulsessuperimposed on basal secretion and exhibits a diurnal rhythmwithpeak values in the early morning hours (Veldman et al., 2001). Si-milarly, the hypothalamic-pituitary-adrenal axis of cortisol secretionis typically maximal during the day and lower in the evening andnight (Larsen, 2003). In the present study, blood for hormonal ana-lysis was collected at constant times of each sampling day (1000 h).Our findings showed a steady decline in LI of lactotrophs and cor-ticotrophs over experimental period. Gonadotroph immuno-label-ling, on the other hand, increased with increase in cathinone doseand chronicity of exposure. The LI of gonadotrophs was further en-hanced by GnRH agonist. The findings of our study were likelyindicative of cathinone effects on endocrine function along hy-pothalamic-pituitary-adrenocortical and gonadal axes. It is likelycathinone exposure at sub-chronic exposure led to down-regulationof particular functional proteins within glandular cells rather thangeneral destruction of cell types in pituitary gland. This conceptioncomplements data on methamphetamine where chronic use led toselective destruction of particular dopamine terminals or cell bodies(Wilson et al., 1996) and not whole cells. The absence of cell loss inour study may explain why human cathinone/khat abusers do nothave many gross behavioural deficits that remain unrecoverable afterwithdrawal from use. It is noteworthy that the effect on gonado-trophs is, however, opposite to that of other anterior pituitary celltypes studied. The significance of this lies in the possible selectiveaction of cathinone on different functional systems of the body. Theincrease in LI of gonadotrophs following cathinone exposure and co-treatment with GnRH agonist may explain why in some instanceskhat/cathinone is viewed as a booster to sexual performance. Takentogether with other published data, the present findings in vervetmonkeys provide additional support for the hypothesis that en-docrinological and immunohistological staining should be examinedas possible diagnostic evaluations for stimulant addiction and sub-sequent management measures.

    Declaration of conflict of interest

    The authors declare that there is no conflict of interest thatcould be perceived as prejudicing the impartiality of the research

    reported.

    Funding

    This study was funded by the Deans Committee # 500-655-635of the University of Nairobi, Khat Research Program (KRP) andgrant from the office of international programs at the University ofMinnesota (Grant No. R21DA024626).

    Acknowledgments

    The authors are thankful to Agha Khan Teaching Hospital forgranting me laboratory space to carry out immunohistochemistrywork.

    Appendix A. Supplementary materials

    Supplementary data associated with this article can be found inthe online version at http://dx.10.1016/j.jep.2015.08.007.

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    Immunohistochemical localization of anterior pituitary cell types of vervet monkey (Chlorocebus aethiops) following...IntroductionMaterials and methodsAnimals and housingExtraction of cathinone from khatExperimental design, dose preparation and treatmentImmunohistochemistryHistological evaluationMorphometric analysis

    ResultsCathinone effects on pituitary cell histology and ACTH immunostainingCathinone effects on LH immunostaining in presence and absence of GnRH agonistCathinone effects on lactotroph immunostainingCathinone effects on S100 protein immunostaining

    Discussion4.1. Effect on corticotrophsEffect on lactotrophsEffect on gonadotrophsEffect on S100 protein secreting cells

    Declaration of conflict of interestFundingAcknowledgmentsSupplementary materialsReferences