Immunohistochemical localization of anterior pituitary cell types of vervet monkey (Chlorocebus aethiops) following sub-chronic cathinone exposure

Author’s Accepted Manuscript Immunohistochemical localization of anterior pituitary cell types of vervet monkey (Chlorocebus aethiops) following sub-chronic cathinone exposure Albert Nyongesa, Jemimah Oduma, Mustafa al’Absi, Sanika Chirwa www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(15)30074-X http://dx.doi.org/10.1016/j.jep.2015.08.007 JEP9672

To appear in: Journal of Ethnopharmacology Received date: 14 April 2015 Revised date: 3 August 2015 Accepted date: 6 August 2015 Cite this article as: Albert Nyongesa, Jemimah Oduma, Mustafa al’Absi and Sanika Chirwa, Immunohistochemical localization of anterior pituitary cell types of vervet monkey (Chlorocebus aethiops) following sub-chronic cathinone e x p o s u r e , Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.08.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Immunohistochemical localization of anterior pituitary cell types of vervet monkey (Chlorocebus aethiops) following sub-chronic cathinone exposure

Albert Nyongesaa,*, Jemimah Odumaa, Mustafa al’Absib, Sanika Chirwac.

a

Department of Veterinary Anatomy and Physiology, University of Nairobi, P.O Box 3019700100, Nairobi, Kenya, bDuluth Medical Research Institute, University of Minnesota, Duluth MN, 55812, USA, cDepartment of Neuroscience and Pharmacology, Meharry Medical College, Nashville, USA.

* Corresponding author. Tel.: +254 20 4446764. Email: [email protected]. (A.Nyongesa)

ABSTRACT Ethnopharmacological Relevance: Khat (Catha edulis) contains cathinone, an active principal that is customarily 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 specific antibodies. Material and methods: Eleven vervet monkeys (6 males and 5 females) divided into tests (n = 9) and controls (n = 2) 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 insignificant. Subsequent GnRH agonist co-treatment with low and high cathinone doses enhanced 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 influencing 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 findings 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. Key Words: HPA axis; Immunohistochemistry; Monkey; Pituitary. Introduction

Khat is both socially and economically one of the most important plants to inhabitants of areas where it is grown in countries 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 (Krizevski et al., 2007; Al-Obaid et al., 1998; Kalix, 1990), a natural compound that is similar to (+) amphetamine in chemical structure and biological activity (Kelly, 2011; Feyissa and Kelly, 2008; Kalix, 1992). Psychostimulants increase adrenocorticotrophic hormone (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 degeneration (Zwain and Amato, 2000). Earlier studies in humans showed that long-term exposure to amphetamine and methamphetamine caused increase in clusterin-positive cells and gonadotrophs but a decrease in corticotrophs and lactotrophs (Ishikawa et al., 2007). These observations suggest that drug abuse influences 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. Whether 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 revenue from foreign exports. Following the recent ban of khat export by the governments of United Kingdom and the Netherlands, there has emerged socio-economic and political panic in source countries due to loss of revenue collection from khat exports. The affected communities are calling upon the government to intervene and lift the ban for the sake of economies of scale in these regions. To these people, the socio-economic benefits from khat outweigh the potential health risks accompanying its long-term and un-regulated use. Conversely, khat use has been associated with breakdown of marriages, prostitution and a host of other social evils (Beckerleg, 2008). The delicate balance between health risks and socio-economic value of the crop has degenerated into lack of clear laws in regulating khat use even with the full understanding of the dynamics of health risk factors posed to khat chewers. To this end, a proper approach into investigations of khat and human health is a critical consideration whereupon vital information is provided to social and health care professionals, policy makers and international organizations with sole aim of sensitizing affected populations. To further explore the interaction between cathinone effects on endocrine hormones, we carried out studies by immuno-localization of hormone-producing cells of the anterior pituitary gland. In particular, we have focussed on corticotrophs, lactotrophs and gonadotrophs. Neuropharmacological studies together with histochemical and immunofluorescence techniques have established that biogenic amines and other neurotransmitters play a crucial role in the modulation of anterior pituitary hormone secretion through action on hypothalamic- hypophysiotrophic neurons (Fuxe and Hökfelt, 1969). Various endocrine and reproductive effects of khat and cathinone in humans and experimental animals have been reported although some of the reports appear contradictory. On the one hand, increase in adrenocorticotrophic hormone in humans (Nencini et al., 1984) and plasma cortisol in rabbits (Nyongesa et al., 2008) following khat extract exposure were reported. On the other hand, decreased plasma cortisol and prolactin were observed in baboons following sub-chronic khat exposure (Mwenda et al., 2006). Other studies

showed a dose-dependent decrease in cortisol following cathinone treatment in rats (Mohammed and Engidawork, 2011) and decrease in cortisol and prolactin in vervet monkeys (Nyongesa et al., 2014 b). These findings though contradictory, are significant since they point to the responsiveness of the anterior pituitary cell types. It is not clear whether the observed discrepancies could be due to species difference or merely the responsiveness of pituitary cell types to hormone synthesis and secretion. It is noteworthy that majority of studies show effect on hormonal release but the precise effect on cells secreting these hormones mediating differences in endocrine responsiveness to cathinone remains unclear. No experiments have assessed immunohistochemical localization or antigen expression in anterior pituitary cell types and cellular morphology following drug use in the same experimental subjects. To this end, we used antibody specific markers to immuno-localize the cell types secreting hormones as well as examination of cellular morphology by histologic staining. In the present study we hypothesized that cathinone variously affects hormonal synthesis by gonadotrophs, corticotrophs and lactotrophs or release of hypophyseotrophic factors from hypothalamus. We were able to examine relationship between endocrine function and cellular morphology. Further, we used GnRH agonist, goserelin acetate (ZOLADEX) to study the effect of cathinone on the responsiveness of anterior pituitary gonadotrophs to hypothalamic GnRH. The study used the vervet monkey as an animal model. Vervet monkeys have long been the most important non-human primate model for biomedical research other than rhesus macaque (Baulu et al., 2002). They have been used extensively for research in human reproduction (Amaral 2002) and cognitive function relevant to human psychiatric disorders (James et al., 2007). Materials and methods 2.1. Animals and housing Eleven adult vervet monkeys were captured in the wild and put under quarantine at the Institute of Primate Research (IPR) for three months and housed at the same institution. Housing was done in individual standard monkey cages (0.6 m x 0.66 m x 0.8 m) for the duration of the study. The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the European Community guidelines (EEC Directive of 1986; 86/609/EEC). The research work was approved by Institute of Primate Research 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 (< 5mm) and dissolved in 250 ml methanol in a conical flask for extraction. The quantity of khat used followed the contention that a bundle of 100 g of fresh khat leaves contain a cathinone content of about 200 mg (EMCDDA, 2011). 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 re-extracted 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 (< 1 ml) 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 followed by chloroform extraction to remove neutral organic compounds as well as 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 and solution vacuum-dried at 337 millibar in a Rotar Vump for 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 Hewlett-Packard Model 5965B Infrared Detector. A total of about 1000 mg cathinone was extracted from the leaves and it was approximately 97% pure. 2.3. Experimental design, dose preparation and treatment The animals were randomly assigned to four groups. Group I were controls and comprised of two animals (1 male and 1 female) while group II, III and IV were tests and comprised of three animals (2 males and 1 female) each. The sample size of animals used in the study was chosen on ethical considerations. Three concentrations of cathinone (1.6 mg/ml, 6.4 mg/ml and 12.8 mg/ml) were prepared by reconstituting cathinone extract paste using normal saline and administered at three different doses (0.8 mg/kg, 3.2 mg/kg and 6.4 mg/kg body weight) respectively. In each case, the working volume (2 ml) of the stock solution, obtained by factoring in the body weight of the animal ( in this case 4 kg), was adjusted to an appropriate final volume (5 ml) using the same saline and administered via intra-gastric tube at alternate days of each week for 4 months. The doses were chosen based on previous studies in humans (Kalix, 1984) and rats (Mohammed and Engidawork, 2011) which indicated optimum effect of cathinone on various body parameters to be within this dose range. Controls were administered normal saline at the same volume and regimen as test animals. At the end of the 4- month cathinone alone treatment, animals on low and high doses (0.8 and 6.4 mg/kg) were administered ZOLADEX at 50 μg/kg body weight single subcutaneous injection while continuing with cathinone treatment for a further 2 weeks. Due to small sample size that was limited by ethical concerns only animals at low and high doses of cathinone were co-treated with ZOLADEX to establish antagonistic or synergistic effects of cathinone in presence of the GnRH agonist. The general health status of the animals was routinely monitored throughout the entire experimental period to ensure only health animals were used. Behavioural observations were also done and data has been published elsewhere (Nyongesa et al., 2014 a). At the end of treatment period, all animals were euthanized with high dose of ketamine and pituitary gland harvested for immunohistochemical evaluation. 2.4. Immunohistochemistry The adenohypophysis of all animals were harvested and fixed with 4% formaldehyde in phosphate-buffered saline (pH 7.2) for 1 h and, thereafter, 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 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 manufacturer’s 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 before insertion into Autostainer. Thereafter, slides were placed on a Dako autostainer containing respective antibodies. Primary antibodies were monoclonal 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) and polyclonal rabbit anti-human S100 (Ready-to-use, 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. After staining, slides were removed from autostainer, dehydrated, cleared and counterstained with hematoxylin. 2.5. Histological evaluation The sections were examined and fields of interest 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 based 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 histological evaluation. 2.6. 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 were 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 using one-way ANOVA at 5% significance level. In order to calculate the LI, the following formula was used: LI = (PiC+/ PiCT) x 100 Where, PiCT = total number of cells for each stain (ACTH, prolactin, LH) that was counted in a given field (positive and negative cells for adenohypophysis), PiC+ = number of immunostained cells (positive cells) for a given stain counted on the same area, LI = labelling index, usually expressed as a percentage Results 3.1. Cathinone effects on pituitary cell histology and ACTH immunostaining There were behavioural changes on vervet monkeys in the course of study as we reported in a recent publication (Nyongesa et al., 2014a). There was a dose and time-dependent increase in aggression, anxiety, abnormal responses, withdrawal and appetite loss. Most of these behaviours

were not observed in controls. Time-dependent changes demonstrated effect of long-term exposure to cathinone on behavioural profiles. However, there were no significant sex differences in immunohistochemical results and data presentation is grouped together. Histological picture of the sections of treated and control animals showed physiologically active glands with no significant morphological changes observed (Fig. 1). Each antibody staining was done on treatment subjects pituitary sections alongside that of control that was treated with normal saline through 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. Positively staining cells, interpreted as corticotrophs, revealed uniform intra-cytoplasmic staining and appeared arranged individually and occasionally in clusters of 2 - 3 cells (Fig. 2a A-D). A random selection of three fields at x 40 objective showed a dose-dependent decrease in LI of corticotrophs for 0.8 mg/kg (F3, 7 = 36; P < 0.05), 3.2 mg/kg (F3, 7 = 22; P < 0.5) and 6.4 mg/kg (F3, 7 = 14; P < 0.05) body weight of cathinone compared to controls (F3, 7 = 48; P < 0.05) (Fig. 2b). 3.2. Cathinone effects on LH immunostaining in presence and absence of GnRH agonist The sections stained with mouse anti-human LH showed a similar pattern obtained with ACTH staining, with two cell populations under light microscopy. The larger percentages of cells were 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 was significantly different across treatment subjects as shown 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-treated with 50 µg/kg GnRH agonist were higher (F3, 7 = 46; P < 0.05 and F3, 7 = 78; P < 0.05, respectively ) compared to dose 3.2 mg/kg (F3, 7 = 43; P < 0.05) and controls (F3, 7 = 20; P < 0.05) indicating that GnRH agonist boosted the antigen expression of positive staining cells. The results show a dose-dependent effect of cathinone on positive cell count and the synergistic effect of GnRH agonist (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 negative for prolactin. Positive cells (lactotrophs) were scattered in the field occasionally in clusters of 2-4 cells and maintained a dispersed pattern with a similar intensity of staining for within- and between- treatment groups. However, the LI of stained cells when taken at three randomly selected high power fields was significantly different across treatment subjects (Fig. 4a). Results show a dose-dependent decrease in number of lactotrophs (F3, 7 = 61; P < 0.05, F3, 7 = 36; P < 0.05 and F3, 7 = 18; P < 0.05) at doses (0.8, 3.2 and 6.4 mg/kg body weight) of cathinone, respectively compared to controls (F3, 7 = 63; P < 0.05) (Fig. 4b). 3.4. Cathinone effects on S100 protein immunostaining Immunostaining of cells with polyclonal rabbit anti-human S100 showed no apparent staining in pituitary sections of treatment groups and controls indicating absence of S-100-positive cells

(Fig. 5a). 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. 5b). A summary of effect of escalating doses of cathinone on anterior pituitary cell types considered in this study is shown (Table 1). Discussion A major aim of this study was to determine whether the effects of cathinone on endocrine function along hypothalamic-pituitary-adrenocortical and gonadal axes are related to potential morphological changes of anterior pituitary cells. Here, we assessed the sub-chronic in vivo effects of cathinone on anterior pituitary cell function, specifically corticotrophs, gonadotrophs and lactotrophs by immunohistochemical localization. Further, we explored the influence of GnRH agonist on gonadotrophic function following cathinone exposure. The data presented here provides a mechanistic link that supports published data suggesting alterations in 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 to humans and experimental animals. The results of the present study show that cathinone at medium (3.2 mg/kg) and high (6.4 mg/kg) dose and at sub-chronic exposure suppressed corticotrophic and lactotrophic antigen expression. In agreement with our recently published data (Nyongesa et al., 2014 b), decrease in LI of corticotrophs and lactotrophs corresponded with suppressed cortisol and prolactin hormone levels. On the other hand, cell density of gonadotrophs increased in a dose-dependent manner. This was compounded by co-treatment with GnRH agonist for low and high dose of cathinone compared to cathinone-alone medium dose. The histological evaluation of these pituitary cell types showed no significant changes between treatment groups and controls. The results of our study, however, are correlative and do not directly address causative relationship between hormone measurement and cellular morphological integrity. 4.1. Effect on corticotrophs The decrease in LI of corticotrophs in the present study may be explained by disturbances in cleavage of propiomelanocortin (POMC) into adrenocorticotrophic hormone (ACTH), βendorphin and β-lipotropin following cathinone treatment. Studies on repeated administration of amphetamine twice daily for 14 days moderately increased POMC mRNA in pituitary gland with no alteration following adrenolectomy (Jaworska et al., 1994). Since glucocorticoids suppress POMC synthesis and causes reduction in ACTH stores in secretory granules (Gann et al., 1981), a rise in POMC mRNA may imply impairment of ACTH synthesis hence glucocorticoids. The expression of POMC, secretion of corticotrophic releasing hormone and its function on corticotrophs and adrenal cortex can be regulated by cytokines such as interleukin 1β and 6, tumor necrosis factor α and interferons α and γ (Marx et al., 1998). Sympathomimetics have been shown to induce hyperthermia via neuronal damage and increase in hypothalamic concentrations of interleukin 1 β (Albers and Sonsall, 1995). It is noteworthy that since cathinone is a sympathomimetic alkaloid that causes peripheral vasoconstriction (Kalix, 1991) and cardiac effects (Balint and Balint, 1994), the argument that cathinone may have interfered with corticotrophic function via effect on immune regulation of hypothalamic-pituitary-adrenal axis may be relevant. Recently, we published data indicating dose-dependent decrease in cortisol levels in vervet monkeys following sub-chronic cathinone exposure (Nyongesa et al., 2014 b). Studies in rats also showed that high levels of cathinone suppressed corticosterone levels (Mohammed and Engidawork, 2011). These findings parallel those in baboons where khat extracts suppressed serum cortisol levels (Mwenda et al., 2006) but different from studies in

rabbits (Nyongesa et al., 2008). However, the difference in findings may be explained partly by duration of exposure to khat/cathinone, possibly species difference and animal handling. Furthermore, it is worth noting that recent clinical observations in khat users showed disrupted adrenocortical functions (al’Absi et al., 2013) suggesting a long-term effect of khat use on cortisol, possibly mediated by central effects at the pituitary and hypothalamic levels. In the present study, animals were habituated to handling and presence of observer for 1 month before blood collection, and control animals were handled in the same way as treatment groups. Effect on lactotrophs In this study, we have reported a dose-dependent decrease in LI of lactotrophs in cathinonetreated animals. The results are consistent with dose-dependent decrease in serum prolactin levels in cathinone-treated vervet monkeys (Nyongesa et al., 2014b) and khat extract-treated baboons (Mwenda et al., 2006). The precise mechanism that might be responsible for suppressed lactotroph function is not known, but data showing that cathinone induces release of dopamine from several brain regions including striatum (Pehek et al., 1990) could be relevant. There is evidence suggesting that dopamine may act directly as the primary prolactin inhibiting factor (Ben-Jonathan et al., 2001) and that dopamine D2 receptors are expressed on plasma membrane of lactotrophs (Goldsmith et al., 1979). To this end, it is instructive to compare long-term endocrine effects of cathinone in vervet monkeys with those of amphetamine in humans for clinical interpretation. Studies on long-term use of amphetamines in humans reported increase in prolactin-secreting cells and gonadotrophs with a decrease in somatotrophs with no change in corticotrophs and thyrotrophs as well as increase in clusterin-containing follicles (Ishikawa et al., 2007). The authors associated increase in lactotrophs to dysfunction of hypothalamic dopaminergic neurons in chronic drug abusers. Related studies in rodents have shown that acute to chronic exposure to methamphetamine or amphetamine both lead to striatal dopamine depletion and physical destruction of dopamine terminals (Fumagalli et al., 1998) as well as decreased levels of presynaptic markers of dopamine function such as tyrosine hydroxylase (Schmidt and Gibb, 1985), dopamine transporter (Wagner et al., 1982) and vesicular monoamine transporter (Frey et al., 1997). The observed decrease in LI of lactotrophs in the present study is indicative of modulation leading to down-regulation of particular functional proteins rather than dysfunction of hypothalamic dopaminergic neurons. However, dopamine measurement, its transporters or metabolites were not considered in this study. Effect on gonadotrophs The cell density of gonadotrophs in pituitary sections of cathinone-treated animals increased in a dose-dependent manner. This was compounded by co-treatment with GnRH agonist for low and high dose of cathinone compared to cathinone-alone medium dose treatment, suggesting a possible enhancement of responsiveness of gonadotrophs to GnRH following cathinone exposure. This significant finding indicates a possible up-regulation of gonadotroph function or receptor responsiveness to GnRH in presence of cathinone. Studies utilizing a bioactive GnRH analogue coupled to ferritin (Hopkins and Gregory, 1977), a bioactive rhodamine derivative of GnRH (Naor et al., 1981) and [biotinyl-D Lys6] GnRH (Childs et al., 1983) as receptor probes indicated that GnRH receptors are initially distributed evenly on the cell surface and then form clusters which subsequently become internalized. It has been shown that receptor-bound GnRH agonist is internalized via coated pits and is subsequently routed either to lysosomes or to secretory granules (Hazum and Keinan, 1983) where endosomes and Golgi complex are involved

in GnRH-receptor processing (Childs et al., 1986). The present study did not consider work on receptor binding, which may be critical in confirming the responsiveness of anterior pituitary cells to hypophyseotrophic hormones from the hypothalamus. The other possible mechanism that led to up-regulation of LI of gonadotroph compared to down regulation of lactotrophs and corticotrophs lies in the role of second messengers to responsiveness of secretory stimuli. The literature on intracellular signalling and hormone secretion in gonadotrophs is complex and contains evidence for changes of and actions for cyclic AMP, cyclic GMP, arachidonic acid and the lipoxygenase pathway, as well as diacylglycerols and lipid derived from activation of phospholipase D (Dan-Cohen et al., 1992). Although we did not consider measurement of intracellular signalling in gonadotrophs in the present study, these earlier findings provide insights in to the possible mechanism of action of cathinone on gonadotroph function. Increase in gonadotrophic cell staining suggests that impairment of sexual function following cathinone treatment (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 of several anterior pituitary hormones. This finding parallels results of cortictotroph and lactotroph function but differ on gonadotroph function in the present study. Other studies in rabbits reported a decrease in LH following khat extract administration (Nyongesa et al., 2008). From these varying observations, some unanswered questions arise: Is there a significant difference between gonadotropins of males and females? To what degree are the GnRH responses normally subject to modulation by steroids, neurotransmitters, peptides or catecholamines of portal circulation? How do effects of psychotropic drugs on catecholamines and neurotransmitters in the brain affect pituitary function? Are there species differences in response to khat or cathinone on pituitary function? A good part of these questions has not been fully addressed. However, our study did not find any significant difference between males and females with respect to cathinone effects on this measure. Effect on S100 protein secreting cells In order to confirm whether other factors contributed to the observed increase in gonadotrophic and decrease in lactotrophic and corticotrophic immunostaining, we further immuno-labelled for S-100 protein in the same pituitary sections. S100-proteins are rarely observed in adenohypophysis of humans (Ishikawa et al., 2003) but in alcoholics with fatty liver or cirrhosis, S-100 protein increases alongside increase in number of somatotrophs and lactotrophs (Takanashi and Ishikawa, 1984). Other studies reported findings 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 presence of growth hormone in S-100 cells (Shirasawa et al., 1984), indicating that S-100 cells may belong to a class of stem cells or progenitor cells of growth hormone-producing cells. It is not clear whether S-100 protein plays any role in anterior pituitary cell function and whether non-human primate pituitaries also express it. The present study investigated the presence and possible involvement of S-100 protein in anterior pituitary cell function in vervet monkeys under sub-chronic cathinone exposure. The pituitary sections immunostained with anti-human S-100 were negative for the stain with accompaniment of decreased LI of lactotrophs and corticotrophs but increase in gonadotroph staining suggesting that anterior pituitary cell function was independent of S-100 protein in this animal species. In our study, S-100 antibody was used in parallel with a known positive control (skin melanoma) (Fig 5b). The histology of pituitary sections at all doses of cathinone showed an

actively performing gland with normal cellular morphological outline contrary to earlier findings of Ishikawa et al. (2007) where there was increase in clusterin in pituitary mixed cell follicles that was associated with cellular damage following various forms of stress induced by amphetamine. It is, therefore, likely that the observed effects were largely due to cathinone exposure. Overall, our immunohistochemical study is the first to demonstrate immuno-localization of anterior pituitary cells following sub-chronic in vivo cathinone exposure in vervet monkeys. The labelling indices of immuno-stained cells were correlative with serum hormonal concentrations reported in a separate publication (Nyongesa et al., 2014 b). The endocrine responses could not be explained as physiological circadian endocrine rhythms. For instance, prolactin is detected in plasma at all times during the day but in discrete pulses superimposed on basal secretion and exhibits a diurnal rhythm with peak values in the early morning hours (Veldman et al., 2001). Similarly, the hypothalamicpituitary-adrenal axis of cortisol secretion is typically maximal during the day and lower in the evening and night (Larsen, 2003). In the present study, blood for hormonal analysis was collected at constant times of each sampling day (1000 h). Our findings showed a steady decline in LI of lactotrophs and corticotrophs over experimental period. Gonadotroph immuno-labelling, on the other hand, increased with increase in cathinone dose and chronicity of exposure. The LI of gonadotrophs was further enhanced by GnRH agonist. The findings of our study were likely indicative of cathinone effects on endocrine function along hypothalamic-pituitary- adrenocortical and gonadal axes. It is likely cathinone exposure at sub-chronic exposure led to down-regulation of particular functional proteins within glandular cells rather than general destruction of cell types in pituitary gland. This conception complements data on methamphetamine where chronic use led to selective destruction of particular dopamine terminals or cell bodies (Wilson et al., 1996) and not whole cells. The absence of cell loss in our study may explain why human cathinone/khat abusers do not have many gross behavioural deficits that remain unrecoverable after withdrawal from use. It is noteworthy that the effect on gonadotrophs is, however, opposite to that of other anterior pituitary cell types studied. The significance of this lies in the possible selective action of cathinone on different functional systems of the body. The increase in LI of gonadotrophs following cathinone exposure and co-treatment with GnRH agonist may explain why in some instances khat/cathinone is viewed as a booster to sexual performance. Taken together with other published data, the present findings in vervet monkeys provide additional support for the hypothesis that endocrinological and immunohistological staining should be examined as possible diagnostic evaluations for stimulant addiction and subsequent management measures. Declaration of conflict of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding This study was funded by the Deans Committee # 500-655-635 of the University of Nairobi, Khat Research Program (KRP) and grant from the office of international programs at the University of Minnesota. Acknowledgements

The authors are thankful to Agha Khan Teaching Hospital for granting me laboratory space to carry out immunohistochemistry work.

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Table

Table 1 Mean number of cells at high power magnification for 0.8 mg/kg, 3.2 mg/kg and 6.4 mg/kg body weight of cathinone (n = 3 vervet monkeys/group) as well as controls (n = 2). The doses were administered via intra-gastric tube at alternate days of the week for 4 months as described under materials and methods. LI means labelling index; a* means in presence of 50 µg/kg GnRH agonist and significantly different (P < 0.05) compared with all other groups; * means significantly different at (P < 0.05) compared with all other groups.

Table

Table 1 LI (%) of positive cells

Cathinone dose (mg/kg body weight) 0

0.8

3.2

6.4

LI for ACTH (at x 400 magnification)

48 ± 4

36 ± 5

22 ±6*

14±3.5*

LI for LH (at x 400 magnification)

20±10

46±8a*

43±10

78± 14.8a*

+ agonist LI for PRL (at x 400 magnification)

63±4.5

61±3

+ agonist 36±6.2*

18±5*

Table

Table 1 LI (%) of positive cells

Cathinone dose (mg/kg body weight) 0

0.8

3.2

6.4

LI for ACTH (at x 400 magnification)

48 ± 4

36 ± 5

22 ±6*

14±3.5*

LI for LH (at x 400 magnification)

20±10

46±8a*

43±10

78± 14.8a*

+ agonist LI for PRL (at x 400 magnification)

63±4.5

61±3

+ agonist 36±6.2*

18±5*

Figure

Fig. 1.

Figure

Fig. 2 a.

Figure

Fig. 2 a). Dose-dependent effect of cathinone on corticotrophs (full arrows) in controls (n = 2) (A), cathinone-treated animals (n = 3) at 0.8 mg/kg plus 50 µg/kg GnRH agonist (B), 3.2 mg/kg body weight of cathinone alone (n = 3) (C) and 6.4 mg/kg body weight of cathinone plus 50 µg/kg GnRH agonist (n = 3) (D). Note that GnRH agonist did not have significant effect on expression of ACTH-positive cells (Original magnification x 400)

Figure

Mean Labelling index for corticotrophs (%)

60

50

40

**

30

*** 20

10

0 0 mg/kg

0.8 mg/kg

3.2 mg/kg

Cathinone dose

Fig. 2 b.

6.4 mg/kg

Figure

Fig. 2 b). Mean LI showing number of corticotrophs as percentage of total cells in sections of control 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. ** means statistically significant at P < 0.05 compared with all other groups, *** means statistically significant at P < 0.01 compared with all other groups.

Figure

Fig. 3 a.

Figure

Fig. 3 a). The figure shows gonadotrophs (open arrows) in anterior pituitary gland of control vervet monkeys (n = 2) (A) and vervet monkeys (n = 3) exposed to cathinone doses at 0.8 mg/kg plus 50 µg/kg GnRH agonist (B), 3.2 mg/kg body weight of cathinone alone (n = 3) (C) and 6.4 mg/kg body weight of cathinone plus 50 µg/kg GnRH agonist (n = 3) (D) at alternate days of the week for 4 months as described under materials and methods. Cathinone increased the LI for gonadotrophs in a dose-dependent manner. (Original magnification x 400)

Figure

Mean Labelling index for gonadotrophs (%)

100

***

90 80 70

**

60

**

50 40 30 20 10 0 0

0.8 + GnRH agonist

3.2

Cathinone dose (mg/kg body weight)

Fig. 3 b.

6.4 + GnRH agonist

Figure

Fig. 3b). Mean LI expressing number of gonadotrophs as percentage of total anterior pituitary gland cells of control (n = 2) and cathinone-treated animals (n = 3 vervet monkeys/group). Note that animals at low and high doses of cathinone and co-treated with GnRH agonist expressed significantly higher (P < 0.05; n = 6) number of cells compared to those exposed to cathinone alone as well as controls when counted on three fields at x 400 magnification. ** means statistically significant at P < 0.05 compared to other groups, *** means statistically significant at P < 0.01 compared to other groups.

Figure

Fig. 4 a.

Figure

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/kg body weight of cathinone plus 50 µg/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 µg/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 x 400).

Figure

Mean Labelling index for lactotrophs (%)

80 70 60 50

* 40

**

30 20 10 0 0 mg/kg

0.8 mg/kg

3.2 mg/kg

Cathinone dose

Fig. 4 b.

6.4 mg/kg

Figure

Fig. 4b). Mean LI expressing number of lactotrophs in controls (n = 2) and cathinone-treated groups (n = 3 vervet monkeys/group). Results showed a dose-dependent decrease in number of cells counted in three random x 40 fields. Dose 3.2 and 6.4 mg/kg body weight of cathinone caused a significant decrease (P < 0.05) compared to 0.8 mg/kg and controls. * means statistically significant at P < 0.05 compared with all other groups, ** means statistically significant at P < 0.01 compared with all other groups.

Figure

Fig. 5.

Figure

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 each week for 4 months and anterior pituitary glandular cell immunostaining done as described under materials and methods. The morphological features in this group of animals (n = 3) were similar to those of controls (n = 2) that receive normal saline hence it was 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 shows degranulation into sinusoids indicating a physiologically active gland. (Original magnification x 400).

Figure

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 each week for 4 months and anterior pituitary glandular cell immunostaining done as described under materials and methods. The morphological features in this group of animals (n = 3) were similar to those of controls (n = 2) that receive normal saline hence it was 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 shows degranulation into sinusoids indicating a physiologically active gland. (Original magnification x 400).

Figure

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 cells were negative for the stain indicating absence of expression of S100 protein in pituitary sections of this treatment group (n = 3). (Original magnification x 400); b) A skin tissue with melanoma, a known positive for S100 protein, comparing staining ability of the anti-human S-100 antibody. The antibody immunostaining worked well as shown by melanoma (full arrow) around the hair follicle (white arrow). Original magnification x 400.

Figure

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 cells were negative for the stain indicating absence of expression of S100 protein in pituitary sections of this treatment group (n = 3). (Original magnification x 400); b) A skin tissue with melanoma, a known positive for S100 protein, comparing staining ability of the anti-human S-100 antibody. The antibody immunostaining worked well as shown by melanoma (full arrow) around the hair follicle (white arrow). Original magnification x 400.

*Graphical Abstract

Fresh khat leaves and reproductive health effects in males Release phenyl propylamines: cathinone, cathine, norephedrine Body functional systems

Accumulation of dopamine, serotonin, noradrenaline in neuronal synapses Central nervous system

Hypothamohypophyseal axis

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Gonadotrophic activity ↓Lactotrophic activity ↓Corticotrophic activity

Hypophyseal-gonadal axis

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Testosterone production at low khat dose and acute exposure ↓Testosterone production at high khat dose and chronic exposure

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Hypophyseal-adrenocortical axis

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↓Prolactin

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↓Glucorcoticoid production

Aphrodisiac-like effects at short-term and low khat use Impaired reproductive performance and fertility with addiction of khat use