Universidade Estadual Paulista “Júlio de Mesquita Filho” Gabriela Missassi EFFECTS OF SUBCHRONIC EXPOSURE TO SIBUTRAMINE ON REPRODUCTIVE PARAMETERS AND FERTILITY OF WISTAR ADULT MALE RATS EFEITOS DA EXPOSIÇÃO SUBCRÔNICA À SIBUTRAMINA SOBRE PARÂMETROS REPRODUTIVOS E FERTILIDADE DE RATOS MACHOS WISTAR ADULTOS Botucatu - SP 2016 Gabriela Missassi EFFECTS OF SUBCHRONIC EXPOSURE TO SIBUTRAMINE ON REPRODUCTIVE PARAMETERS AND FERTILITY OF WISTAR ADULT MALE RATS EFEITOS DA EXPOSIÇÃO SUBCRÔNICA À SIBUTRAMINA SOBRE PARÂMETROS REPRODUTIVOS E FERTILIDADE DE RATOS MACHOS WISTAR ADULTOS Relatório final, apresentado a Universidade Estadual Paulista ”Júlio de Mesquita Filho”, como parte das exigências para a obtenção do título de Bacharel em Ciências Biológicas. Orientadora: Profa. Dra. Wilma De Grava Kempinas Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu 2016 Palavras-chave: Epidídimo; Qualidade espermática; Sibutramina. Missassi, Gabriela. Efeitos da exposição subcrônica á sibutramina sobre parametros reprodutivos e fertilidade de ratos machos Wistar adultos / Gabriela Missassi. - Botucatu, 2016 Trabalho de conclusão de curso (bacharelado - Ciências Biológicas) - Universidade Estadual Paulista "Júlio de Mesquita Filho", Instituto de Biociências de Botucatu Orientador: Wilma De Grava Kempinas Capes: 20600003 1. Testes de toxicidade subcrônica. 2. Epidídimo. 3. Sibutramina. 4. Drogas - Efeito fisiológico. 5. Sêmen. 6. Rato como animal de laboratório. DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CÂMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSEMEIRE APARECIDA VICENTE-CRB 8/5651 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉC. AQUIS. TRATAMENTO DA INFORM. 1 EFFECTS OF SUBCHRONIC EXPOSURE TO SIBUTRAMINE ON REPRODUCTIVE PARAMETERS AND FERTILITY OF WISTAR ADULT MALE RATS Gabriela Missassi1*, Cibele S. Borges1, Raquel F. Silva1, Janete Anselmo-Franci2, Wilma G. Kempinas1. 1Laboratory of Biology and Toxicology of Reproduction and Development, Department of Morphology, Institute of Biosciences, UNESP–Universidade Estadual Paulista, Botucatu, São Paulo, Brazil 2Department of Morphology, Stomatology and Physiology, School of Dentistry, USP– Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil Running Title: Sibutramine administration in the dark phase impairs rat sperm quality *Corresponding author: Gabriela Missassi Laboratory of Reproductive and Developmental Biology and Toxicology Department of Morphology, Institute of Biosciences of Botucatu UNESP – Univ Estadual Paulista, 18618-970, Botucatu, SP, Brazil Phone: +55 14 98167-4847, e-mail: gabby_missassi@hotmail.com. 2 ABSTRACT 1 2 Sibutramine is a non-selective serotonin-norepinephrine reuptake inhibitor largely used for 3 weight loss. In a previous work, we showed the pharmacological mechanisms involved in the 4 reduction on sperm quality and fertility of rats exposed to this anorexigen in the light phase of 5 light-dark (l/d) cycle. Considering that rodents are nightlife animals, with higher metabolic 6 activity during the dark phase, the present work aimed to further investigate whether the 7 alterations found in rat sperm quality would be maintained after treatment in this period. For 8 this, adult male Wistar rats were treated with sibutramine (10 mg/kg/day) or vehicle for 15 days 9 in the dark phase of the light-dark cycle. Sibutramine decreased final body and reproductive 10 organ weights, as well as the testosterone levels and number of sperm with progressive motility. 11 These animals also had an acceleration in the sperm transit time in the epididymis and a 12 decrease in sperm concentrations throughout this organ (histopathological analysis). In 13 conclusion, the impairment of reproductive parameters were enhanced when the exposure 14 occurred during the dark phase of the l/d cycle, which corresponds to more active period of 15 these animals. 16 17 3 INTRODUCTION 1 2 Sibutramine is an anorexigen that acts as a non-selective inhibitor of norepinephrine 3 reuptake (Nisoli and Carruba 2000; Choi et al. 2011; Binsaleh 2012). Its efficacy as a weight 4 loss agent has been proven, since it reduced the incidence of mortality and morbidity related to 5 obesity (Yen and Ewald 2012; Suplicy et al. 2014). However, because of severe side effects, 6 such as increased blood pressure, several countries prohibited its use (James et al. 2010; Barétic 7 2013; Seimon et al. 2014; Krentz et al. 2016). 8 Currently, some studies in these countries reported the presence of sibutramine in mixed 9 teas and natural compounds to accelerate weight loss (Zhang et al. 2013; Kim et al. 2014; 10 Mathona et al. 2014). Hachem et al (2016) analyzed the composition of 100% natural food 11 supplements used for weight loss and observed that almost 50% had an active pharmaceutical 12 ingredient, among them, sibutramine. 13 After sibutramine intake, the drug is metabolized in the liver and forms two metabolites: 14 primary (M1) and secondary amines (M2). These compounds mediate sibutramine activity and 15 reach peak plasma concentrations 3-4 hours, respectively (Bae et al. 2009; Bae et al. 2011). 16 Studies have shown that between 2-8 hours after administration of sibutramine, the animals 17 showed a marked reduction in caloric intake due to the action that amines exert on α1-18 adrenoceptor, which is important to increase energy expenditure and thermogenesis (Jackson 19 et al. 1997). 20 Moreover, because male genital system receives innervation from sympathetic 21 (adrenergic fibers) and parasympathetic fibers (cholinergic fibers), which exert distinct control 22 functions mediated by neurotransmitters, such as norepinephrine (Purves et al. 2001), some 23 studies also investigate the effects of this compound in the reproductive system. In a previous 24 work, it was demonstrated that the increased availability of norepinephrine caused by 25 4 sibutramine administration impaired sperm quality and fertility in adult male rats (Bellentani 1 et al. 2011; Borges et al. 2013). 2 Considering these informations and the fact that rodents, unlike humans, are nocturnal 3 animals (Sthefan 1972; Bravo et al. 2014) and have higher metabolic activity during this period, 4 the administration of drugs at night possibly exerts more effect in their system. Thus, the 5 present study aimed to evaluate the effects of sibutramine on sperm quality of adult male rats 6 treated for 15 days in the dark phase of light/dark (l/d) cycle. 7 8 5 MATERIAL AND METHODS 1 Ethics Statement 2 The experimental procedures were approved by the local Ethics Committee for the Use 3 of Experimental Animals of the University of São Paulo State (protocol number 248-CEEA) 4 and were in accordance with the Guide for the Care and Use of Laboratory Animals (National 5 Institutes of Health). The euthanasia was performed by decapitation, all efforts were made to 6 minimize suffering. 7 8 Animals 9 Male (110 days old/ 490-540 g) Wistar rats were obtained from the Central Biotherium 10 of UNESP – Univ Estadual Paulista, and maintained under controlled conditions (25°C, 30% 11 air humidity, 12/12-h light/dark cycle) with food and water available ad libitum. 12 The male rats were randomly allocated into two groups: control, in which rats were 13 treated by gavage with the vehicle solution (33.3% dimethylsulfoxide and 66.7% saline) in 0.5 14 ml/kg, and treated, in which rats were trated with sibutramine (10 mg/kg/day) in vehicle 15 solution by gavage for 15 days. 16 17 Drugs and Solutions 18 Drugs were obtained from the following sources: sibutramine hydrochloride 19 monohydrate from Deg (Jiangyin Eas, China) and dimethylsulphoxide (DMSO) from Sigma 20 (St. Louis, MO, USA). 21 Sibutramine was diluted in 33.3% dimethylsulfoxide and 66.7% saline and 22 administered once a day in 0.5ml/kg. This dose was chosen as the minimum anorectic dose in 23 this experimental model. 24 25 6 Body and reproductive organ weights 1 The animals were weighed during the treatment and euthanized by decapitation at the 2 end of it. The right testis, epididymis, ventral prostate, and seminal vesicle (without the 3 coagulating gland) were removed and their weights recorded. 4 5 Hormonal measurements 6 Blood samples were allowed to coagulated and the serum obtained by centrifugation 7 (2400 rpm) for 20 min at 4°C and stored at -20°C. Serum testosterone, FSH and LH levels were 8 determined by radioimmunoassay. Testosterone levels were measured using the Coat-A-9 Count® assay (Diagnostics Products Corporation, Los Angeles, USA) while LH and FSH were 10 measured using specific kits supplied by the National Institute of Arthritis, Diabetes and 11 Kidney Diseases (NIADDK). All samples were measured within the same assay to avoid the 12 inter-assay errors. Intra-assay variabilities were 3.4% for LH, 2.8% for FSH, and 4% for 13 testosterone. 14 15 Sperm counts, daily sperm production, and sperm transit time through the epididymis 16 Homogenization-resistant testicular spermatids (stage 19 of spermiogenesis) in the 17 testis were counted as described previously, with adaptations adopted by Fernandez et al. 18 (2007). Briefly, the testis, was decapsulated, weighed, and soon after the collection, was 19 homogenized in 5 ml of NaCl 0.9% containing Triton X 100 0.5%, followed by sonication for 20 30 s. After a 10-fold dilution, one sample was transferred to Neubauer chambers (4 fields per 21 animal), and mature spermatids were counted. To calculate the daily sperm production (DSP), 22 the number of spermatids at stage 19 was divided by 6.1, which is the number of days of the 23 seminiferous cycle during which these spermatids are present in the seminiferous epithelium. 24 In the same manner, caput/corpus and cauda epididymidis were cut into small fragments with 25 7 scissors and homogenized, and sperm counted as described for the testis. The sperm transit 1 time through the epididymis was determined by dividing the number of sperm in each portion 2 by DSP. 3 4 Sperm motility 5 The male rats were used for sperm isolation as described elsewhere. Briefly, the sperm 6 were released from the left proximal epididymal cauda, first site where fertile sperm is 7 encountered in the rat, by pinching the duct and collecting the sperm in 2 mL of modified 8 human tubular fluid (HTF) medium (Irvine Scientific). Sperm motility was evaluated using an 9 aliquot of 10μL of sperm suspensions was immediately transferred to a Makler chamber 10 maintained at 34oC. Using a phase-contrast microscope (400 x magnification), 100 sperm were 11 counted and classified as Type A (mobile with progressive movement) Type B (mobile without 12 progressive movement) and Type C (immobile). 13 14 Histological Analysis of Testis and Epididymis 15 The left testis and epididymis were fixed in Bouin solution for 24 h. The pieces were 16 dehydrated in a graded ethanol series and routinely processed for embedding in paraffin, 17 sectioned at 5 m, and subsequently stained with hematoxylin and eosin (H&E). Testis and 18 epididymis sections were examined by light microscopy following specific guidelines for 19 toxicological studies (Foley, 2001). 20 21 Statistical analysis 22 Data are presented as mean ± standard error of mean (SEM) or median and interquartil 23 range. Student’s t-test or Mann-Whitney test were used for comparison of parametric or 24 8 nonparametric variables respectively. Differences were considered significant when p < 0.05. 1 The statistical analyses were performed by GraphPad InStat (version 5). 2 3 9 RESULTS 1 2 During treatment, the weight of the animals was monitored and from the twelfth day, 3 until the last day of treatment the sibutramine group had a significant reduction (p <0.05) in 4 weight gain compared with the control group (Figure 1). 5 At the end of the experiment, the animals were weighed and killed. The absolute weight 6 of all reproductive organs and accessory glands collected from animals exposed to sibutramine 7 were reduced (Table 1). In addition, the hormone measurements demonstrated a significant 8 reduction in serum testosterone levels of these animals (Figure 2), although the levels of FSH 9 and LH were not altered. 10 Daily sperm production was similar in both groups (Figure 3A), however, despite the 11 short period of treatment, sibutramine group presented lower sperm reserves that was shown 12 in the histopathological analysis (Figure 4C - 4F) and sperm count (Figure 3B). Besides that, 13 this groups also had an acceleration in sperm transit time through epididymal caput/corpus and 14 cauda (Figure 3C). Regardless of these alterations, the histopathological analysis showed no 15 changes on the structure and dynamics of spermatogenesis in the testis (Figure 4A and 4B). 16 The analysis of sperm motility showed an increase in the percentage of immotile sperm 17 as well as a reduction of motile sperm in sibutramine-treated group (Figure 3D). 18 19 10 DISCUSSION 1 Pharmacotherapy designed to treat obese patients besides helping them to lose weight, 2 significantly reduces the risk of associated morbidities like type 2 diabetes and cardiovascular 3 disease. Despite the adverse effects associated with these medications, and the fact that many 4 countries prohibit its use (Krentz et al. 2016), it is common to find traces of these compounds 5 in natural food supplements and teas (Zhang et al. 2013; Kim et al. 2014; Mathona et al. 2014; 6 Hachem et al. 2016). 7 Sibutramine is considered the method of choice for weight loss due to suppression of 8 appetite through inhibition of monoaminergic neurotransmitters reuptake in the central nervous 9 system (Araujo and Martel 2012). Thus, from the twelfth day until the last, the group treated 10 with this drug had a significant reduction in weight gain compared to control, and this confirms 11 its efficacy as an anoxerigen. 12 The assessment of absolute and relative organ weights is an important parameter to 13 evaluate the risk of toxicity on male reproductive system (Clegg 2001). In a previous work, we 14 showed that sibutramine reduced the weight of epididymis, ventral prostate and seminal vesicle 15 (Borges et al. 2013). In the present study, the same results were observed but additionally, this 16 group also had a decrease in the weight of testis. It is known that testicular development and 17 the formation of secondary sexual characters are mediated by testosterone and for that reason, 18 its concentration is essential to spermatogenesis and sperm production. Despite the fact that 19 LH and FSH levels were unchanged, there was a significantly reduction in testosterone 20 concentrations that did not alter the morphology of the testis but is possibly the cause for low 21 testicular weight. 22 In the same manner, the reduced weights of prostate and seminal vesicle may have 23 resulted from low levels of testosterone combined with the sympathomimetic effect of the drug. 24 Studies demonstrated that sibutramine increased the contractile activity of ventral prostate 25 11 (Borges et al. 2013) and seminal vesicle (Nojimoto et al. 2009) leading to a more prominent 1 fluid elimination and consequently decreasing their weight. 2 Regarding the epididymis, the low sperm reserves resulted from the acceleration in 3 sperm transit time was possibly the cause for its weight reduction. This alteration was also 4 confirmed once the histopathological analysis showed less sperm present in this organ. 5 Considering the accelerated sperm transit time caused by sibutramine exposure, it might be 6 resulted from increased contractility of this organ in the presence of this drug (Borges et al. 7 2013) and from low levels of testosterone (Fernandez et al. 2007). 8 The smooth muscle present in the epididymis cauda receives sympathetic innervation 9 that is manly mediated by the activation of α1-adrenoreceptors (Ventura and Pennefather 1991; 10 Ventura and Pennefather 1994; Ricker 1998; Gerendai et al. 2001). Considering that, drugs that 11 act in the autonomic nervous system exerts influence in sperm transit time (Pholpramool et al. 12 1984; Billups et al. 1990; Kempinas et al. 1998). Once the passage through the epididymis has 13 a fundamental role in the post-testicular sperm maturation and current alterations in this 14 parameter may impair this process (Klinefelter 2002; Fernandez et al. 2007; Bellentani et al. 15 2011; Borges et al. 2013), the acceleration observed in the sibutramine group could be the cause 16 for the increased percentage of immotile sperm. 17 In conclusion, sibutramine administered in the dark phase of circadian rhythms of rats 18 promoted enhanced deleterious reproductive effects, as shown by reduced reproductive organ 19 weights, lower sperm quality and quantity and decreased serum testosterone levels. More 20 studies are needed to investigate the outcomes of longer periods of sibutramine exposure. 21 Considering that this drug is widely used as an anorexigen, the results of the present work may 22 have implications for human fertility. 23 12 CONFLICT OF INTEREST STATEMENT 1 The authors declare that there are no conflicts of interest. 2 3 13 ACKNOWLEDGEMENTS 1 The authors are grateful to Ruither de Oliveira Gomes Carolino of the Department of 2 Morphology, Stomatology and Physiology, Dental School of Ribeirão Preto, University of São 3 Paulo – USP, for the collaboration with the hormonal assays, to Dr. Adriana Lúcia Mendes 4 endocrinologist of the Department of Clinical Medicine, Medicine School of Botucatu, São 5 Paulo State University – UNESP, for the collaboration with the sibutramine prescription, and 6 to José Eduardo Bozano, assisting academic support, of the Department of Morphology, 7 Institute of Biosciences, São Paulo State University – UNESP, for the collaboration and 8 assistance throughout the project. 9 10 14 REFERENCES 1 Araujo, J.R. and Martel, F. 2012. 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J Med Toxicol. 8: 145-152. 24 25 19 Zhang, Y., Huang, X., Liu, W., Cheng, Z., Chen, C. and Yin, L. 2013. Analyses of drug 1 Illegally added into Chinese traditional patent medicine using surface-enhanced raman 2 scattering. Anal Sci. 29: 985-990. 3 4 20 FIGURE LEGENDS 1 Figure 1. Evolution of body weight gain during the treatment with sibutramine or vehicle. The 2 data are expressed as the mean + SEM. * p < 0.05 (Student’s t-test). 3 4 Figure 2. FSH (folicle-stimulating hormone), LH (Luteinizing hormone) and T (testosterone). 5 The data are expressed as the mean + SEM. * p < 0.05 (Student's t- test). 6 7 Figure 3. A: Sperm number in the testis of the control and sibutramine-treated groups. B: 8 Sperm reserves in caput/corpus and cauda of the control and sibutramine-treated groups. C: 9 Sperm transit time in caput/corpus and cauda of the control and sibutramine-treated groups. A-10 C: The data are expressed as the mean + SEM. * p < 0.05, ** p < 0.01 (Student's t- test). D: 11 Sperm motility with Type A (mobile with progression; p < 0.05) Type B (mobile without 12 progression) and Type C (immobile; p < 0.05). The data are expressed as median. * p < 0.05 13 (Mann- Whitney test). 14 15 Figure 4. Histological aspect of testis from control (a), and sibutramine group (b), the 16 epididymal caput and cauda from animals of control (c,e) and sibutramine (d,f) group (n=5/ 17 group). GC: germ cells; L: lumen; In: interstitial tissue; Spz: spermatozoa; Ep: epithelium. 18 Final magnification: 200x.19 21 Figure 1. 22 Figure 2. 23 Figure 3. 24 Figure 4. 25 Table 1.