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Differential effects of amitriptyline treatment

Differential effects of amitriptyline treatment International Journal of Integrative Biology
A journal for biology beyond borders
Differential effects of amitriptyline treatment
on testicular and liver functions
Afify M 1,*, Abd Elmaksoud M D 1, Mosa T 1, Elshaer M 2, Kotb N 3
1 Biochemistry Department, National Research Centre, Dokki, Egypt 2 Pathology Department, National Research Centre, Dokki, Egypt 3 National Organization for Drug Control and Research (NODCAR), Egypt Submitted: 30 Sep. 2009; Revised: 13 Dec. 2009; Accepted: 15 Dec. 2009 Abstract
The aim of our study was to investigate the influence of exposure to amitriptyline treatment with different doses
on the activity of liver and testicular indices; and examined these organs histopatholgically to confirm the effect
of amitriptyline. This study conducted on 80 adult male rats. The 40 rats were divided into two groups according
to received dose of amitriptyline (low and high doses) and the third group (20 rats) received toxic dose of
cyclophosphamide to serve as a positive control, beside 20 healthy rats as a control group. After 30 days of
treatment, the animals were sacrificed and blood and tissue samples were collected. The results showed that
there was no significant difference in rats treated by low dose of amitriptyline as regards liver enzymes (ALT,
AST and γGT), testicular functions (testosterone and prolactin levels, and spermatic count) and
histopatholgically changes in the tissue of these organs. While the high dose showed significant difference in the
liver and testicular functions proved by, the changes occurred in the liver and testicular tissue that is like the
toxic effect of cyclophosphamide. In conclusion, high dose of amitriptyline has toxic effects on the metabolic
functions of the liver and reduction in the productive functions of the testis beside the toxic histopathological
changes in the tissue of these organs.
Keywords: amitriptyline, liver, testis and cyclophosphamide.

INTRODUCTION
serotonin reuptake and of norepinephrine (NE) reuptake (Diaz et al., 2008). Tricyclic may also possess It well known that treatment-emergent sexual an affinity for muscarinic and histamine H1 receptors dysfunctions occur with many antidepressive to varying degrees. Although the pharmacologic effect compounds. Antidepressants are widely prescribed for occurs immediately, often the patient's symptoms do the chronic treatment of several anxiety disorders not respond for 2 to 4 weeks (Thase et al., 2005). (Feighner, 1999; Zohar and Westenberg, 2000). Although norepinephrine and dopamine are generally Amitriptyline the older tricyclic antidepressant used in considered stimulatory neurotransmitters, tricyclic the treatment of anxiety disorders (Feighner, 1999). antidepressants also increase the effects H1 histamine, and thus most have sedative effects (Landen et al., Amitriptyline hydrochlorate is a tricyclic antidepressant 2005; Forbes et al., 1996). with sedative and analgesic properties (Bryson and Wilde, 1996). The mechanism of the sedation induced The antidepressants are among those drugs, which by amitriptyline is related to its antihistaminic actions cause toxic effects on much of organ system especially while the analgesic mechanism is not fully understood, male reproductive system. About 15% of these drugs although γ2A–adrenoreceptors appear to have have adverse effects on hormonal levels and target significant role (O'zdogan et al., 2004). This drug is organ like testes, which secrete hormones and produces approximately equally active as an inhibitor of male germ cells during spermatogenesis. Studies showed that the effects of antidepressant on sexual dysfunction are more than 60%. Effects of drugs on Corresponding author:
Mie Afify Mohammed, Ph.D. sexual dysfunction and spermatogenesis appear to be Biochemistry Department, due to changes in hormones level such as testosterone, Genetic Engineering and Biotechnology, Research Division LH, FSH, prolactin and estrogen (Clayton and Montejo, National Research Centre, Tahrir Street, Dokki, Giza, 12311, Egypt Email: [email protected] International Journal of Integrative Biology
IJIB, 2009, Vol. 8, No. 1, 50 IJIB, All rights reserved Differential effects of amitriptyline treatment The gonads and adrenals secrete several male sex • Group I- included 20 rats were treated daily with low hormones, called androgens. All are steroid hormones - dose of amitriptyline 0.4 mg/dl orally taken for 1 that is, derived from cholesterol and containing a basic skeleton of four fused carbon rings. Testosterone is the most potent and abundant androgen. Gonadotropin- • Group II- included 20 rats were treated daily with releasing hormone (GnRH) from the hypothalamus high dose of amitriptyline 0.4 mg/dl orally taken for promotes anterior pituitary release of luteinizing 10 days, then 0.8 mg/dl for 10 days, then 1.6 mg/dl for hormone (LH) and follicle-stimulating hormone (FSH). another 10 days. LH stimulates the interstitial cells of Leydig in the testes to synthesize and secrete testosterone. • Group III- 20 rats were treated by 25 mg/kg body Testosterone secretion occurs in pulsatile bursts, about weight of cyclophosphamide intraperitoneally which six per day, with a morning peak and an early evening is a toxic dose for the rats to serve as a positive control trough, and is regulated through a negative feedback on the hypothalamus and pituitary (Freeman et al., 2001). Beside 20 normal healthy rats (untreated) served as Amitriptyline hydrochloride is a prescription control group. After 30 days of treatment, the animals medication that is used for the treatment of depression. were sacrificed and the blood and tissue samples were As with any medicine, there are possible side effects with amitriptyline. Some of these side effects can affect a person's sexual well-being. In the case of amitriptyline, there have been a few sexual side effects reported, a decreased sex drive (libido). The effects of Blood samples
tricyclic antidepressants on the endocrine system can result in sexual dysfunction including libido decrease, Peripheral fasting venous blood samples (3 ml) were impotence, testicular swelling, ejaculation dysfunction, drawn from each rat. One ml blood put in a tube breast enlargement, and galactorrhea in females or containing EDTA to separate the plasma after gynecomastia in males. The syndrome of inappropriate centrifuging for 10 minutes. The other two ml blood secretion of antidiuretic hormone (SIADH) has been was left to clot at room temperature to separate sera reported (Taylor, 2006). after centrifuging for 10 minutes at 3000 r.p.m. Sera and plasma were separated, divided into several Therefore, with the point of view that it might be aliquots and stored at – 70°C until assay. interesting and possibly fruitful to study the influence of exposure to amitriptyline treatment with Tissue samples
pharmacological and toxic doses on the activity of liver Liver and testicular tissue samples were obtained by and testicular indices in adult male rats. We also taking biopsies of the fresh specimen, testis and the examined the hepatic and testicular tissues liver were dissected, weighted and microscopically histopatholgically to confirm the toxic effect of large doses of amitriptyline on these organs. All animals were subjected to the following
MATERIALS AND METHODS
1. Determination of serum aspartate transaminase (AST), serum alanine transaminase (ALT) and Experimental design
serum gamma glutamyle transferase (γGT) by using the method recommended by the Committee After approval of the ethical committee of the National on Enzymes of the Scandinavian Society for Research Center, this study was conducted on 80 adult Chemistry and Clinical Physiology Wistar male rats (body weight, 250–315 gm.), and (1974), the test was performed using already standard laboratory conditions were done during commercially available kit from Boehringer- experiment. We used amitriptyline tablet (25mg) and Mannhiem Company, Germany. dissolved in 125 ml distilled water, for oral 2. Detection of prolactin hormone (PL) level by administration of the drug, we used a 1-ml syringe enzyme immunoassay (EIA) using (without the needle) directly introducing 0.4 ml of already commercially available Prolactin ELISA solution into the animal's mouth. kit in a Rat/ Rat plasma which is used for the quantitative measurement of prolactin from The rats were divided according to the treatment the Calbiotech, Inc. (CBI), (Catalog No.: PR063F- 100. The CBI prolactin kit is based on a solid phase sandwich ELISA method (Duhau et al., 1991). International Journal of Integrative Biology
IJIB, 2009, Vol. 8, No. 1, 51 IJIB, All rights reserved Differential effects of amitriptyline treatment 3. Detection of testosterone hormone level by enzyme immunoassay using already commercially available kit DRG® Testosterone ELISA (EIA-1559), the DRG® ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle of competitive binding (Tietz, 1986). 4. Evaluation of spermatic count from testicular biopsy. The epididymides were extracted and the sperms were sampled, some drops of the sperm suspension were put on haemocytometer to count the sperm. 5. Histopathological analysis: The whole testis and liver biopsy, fixed in 10% formaldehyde dehydrated in ethanol and embedded in paraffin wax and sectioned on 5μm thin section and they were Figure 1: Histopathology of hepatic tissue of normal adult rat, exposed to at low dose,
high dose of amitriptyline, and toxic dose of cyclophosphamide as +ve control. (A)
stained with haematoxylin and eosin Normal liver with central vein and surrounding hepatocytes, sinusoids lined with Kupffer for light microscopic evaluation. For cells. (B) Group I, showed minimal changes with interlobular inflammatory cellular
all tissues studied, a blinded infiltrations and hepatocytic vacuolation. (C) Group II, showed marked obliteration of the
blood sinusoids, inflammatory cellular infiltration around the hepatic vein and hepatocytic microscopic evaluation of vacuolation. (D) Group III showed marked obliteration of the blood sinusoids, marked
the sections was made. cytoplasmic degeneration of hepatocytes, focal permeation by lymphoplasmic cells, focal pyknotic, nectrotic and apoptic nuclei were remarkable Statistical analysis
as normal testosterone and prolactin with means values Statistical analysis was performed using the SPSS (6.6 and 13.5 ng/ml respectively). software package for Windows (SPSS (UK) Ltd., Surrey, United Kingdom). ANOVA was used to Group I: rats treated by low dose of amitriptyline, the
determine the difference between the means of the serum levels of liver function tests (AST, ALT and GT) groups. Further analysis was carried out using a with a mean values of (19.8, 19.2 and 15.8 U/L nonparametric test for two independent samples respectively) and the testicular function (testosterone (Mann-Whitney U test), whereas t-test was used for and prolactin hormones) with a mean values of (6.3, continuous variables. P value considered significant 12.9 ng/ml respectively) compared to the control group. when it was < 0.05.
Group II: rats treated with high dose of amitriptyline,
the serum levels of (AST, ALT and GT) abruptly
increased significantly in all rats with means of (100.2, 64.47 and 57.53 U/L respectively). While the In this study, 80 mature male Wistar rats were used testosterone and prolactin levels were significantly and standard laboratory conditions were done during decreased (P<0.05) with mean values of (2.4 and 8.8 experiment. The 60 rats were divided in three groups, ng/ml respectively) compared to the controls and group two groups were treated with low (therapeutic) and high (toxic) of amitriptyline for 30 days orally and the third group was treated by high dose (toxic) of Group III: rats treated with toxic dose of
cyclophosphamide intraperitoneally to serve as a +ve cyclophosphamide to detect the toxic effect on the liver control group, and 20 rats remained untreated as functions acting as a +ve control group, the serum levels of (AST, ALT and GT) abruptly increased significantly in all rats with a means of (107.2, 69.9 Table 1 [Supplementary data] showed the effect of different and 68.53 U/L respectively). The levels of testosterone doses of amitriptyline on the serum levels of liver and and prolactin were significantly decreased (p<0.05) testicular functions. In the control group, all rats had with a means of (2.75 and 8.5 ng/ml respectively) normal serum levels of AST, ALT and γGT with mean compared to the control group. While no significant values of (17.8, 18.8 and 16.2 U/L respectively) as well changes (p>0.05) in the levels of these parameters between groups (II) and (III). This considered as a International Journal of Integrative Biology
IJIB, 2009, Vol. 8, No. 1, 52 IJIB, All rights reserved

Differential effects of amitriptyline treatment increment was non-
significant (p>0.05) with
a mean of 22.53x106/ml
as compared to control
group.
Group II rats treated
with high dose of
amitriptyline, the sperm
count was abruptly
decreased with a mean of
10.0x106/ml and these
decrement was
significant (p<0.05) as
compared to the control
and group (I).
Group III rats treated
with toxic dose of
cyclophosphamide to
detect its toxic effect on
the sperm counts acting
as a +ve control group,
the sperm count was
abruptly decreased with
a mean of 9.59x106/ml
and these decrement was
significant (p<0.05) as
compared to the control
and group (I). While no
significant changes
(p>0.05) in the sperm
counts between groups (II) and (III). This Figure 2: Histopathology of testicular tissue of normal adult rat, exposed to low dose, high dose of
amitriptyline and toxic dose of cyclophosphamide as +ve control (A) normal testis tissue, ST: Seminiferous
tubules; GS: Germ cells; SC: Sertoli cells; and LC: Leydig cells. (B) Group I showed minimal changes as
confirmatory test to compared to control slid (C) Group II showed showing focal pyknosis and damaged spermatogonia lining
detect the toxic effect of cells. Absent spermatids were remarkable in most of the seminiferous tubules. (D) Group III, (+ve control)
amitriptyline in high showed focal pyknotic and damaged spermatids, marked interstitial oedema, intratubular and intraluminal dose on the liver and testicular functions. confirmatory test to detect the toxic effect of amitriptyline in high dose on the liver and testicular Histopathological results
Histopathological study of testicular tissue and hepatic The gonadal weight in the different studied groups tissue of normal adult rat (control), exposed to low dose showed non-significant changes compared to the (group I), high dose of amitriptyline (group II) and control groups with means of (1.53 gm. for control, toxic dose of cyclophosphamide (+ve control) 1.54 gm. for group (I), 1.45 gm. for group (II) and 1.51 illustrated in Fig. 1 and Fig. 2. gm. for group (III). Table 2 [Supplementary data] showed the effect of DISCUSSION
different doses of amitriptyline on spermatic counts in the different studied groups. In the The antidepressants are among those drugs, which control group, all
rats had normal sperm count; the mean was cause toxic effects on much of organ system especially male reproductive system. About 15% of these drugs have adverse effects on hormonal levels and target organ like testes that secrete hormones and produces Group I rats treated with low dose of amitriptyline, the
sperm count showed slightly increased but these
male germ cells during spermatogenesis. Results showed that the effects of antidepressant on sexual International Journal of Integrative Biology
IJIB, 2009, Vol. 8, No. 1, 53 IJIB, All rights reserved Differential effects of amitriptyline treatment dysfunction are more than 60%. Effects of drugs on matched with the changes that occurred due to the sexual dysfunction and spermatogenesis appear to be toxic dose of cyclophosphamide which act as +ve due to changes in hormones level such as testosterone, LH, FSH, prolactin and estrogen (Clayton and Montejo, These results were in accordance with Bahmanpour et al., who showed that the amitriptyline doses have Using three different experimental models, we different effects on hormonal levels. The higher dose searched for the side effect of antidepressant (25 mg/kg) decreased the testosterone and prolactin (amitriptyline) drug on the liver or testicular functions. levels but increased the FSH levels. Amitriptyline The obtained results indicated that amitriptyline in low changes the hormonal levels and disrupts the dose did not show any side effect on the liver activity testosterone and estrogens ratio. They concluded that as regards the liver enzyme levels (AST, ALT, ALP, the toxic effects of the amitriptyline caused the and γGT). Also there were no significant changes in disruption of sex hormone and can leads to sexual hepatic morphology between rats treated with low dose dysfunction and infertility (Bahmanpour et al., 2008). of amitriptyline and the control group. While the high dose of amitriptyline affect the liver activity in the A repeated oral treatment (twice daily, for 21 form of significant elevation of all liver enzymes consecutive days) with 10 mg/kg of antidepressants compared to controls. Also there were significant imipramine, amitriptyline, citalopram, affects the level morphological changes in the liver tissue including of testosterone and its metabolites (5 alpha- marked obliteration of the blood sinusoids, dihydrotestosterone and estradiol-17 beta) in the serum inflammatory cellular infiltration around the hepatic and brain structures (cerebral cortex, hypothalamus). vein and hepatocytic vacuolation; more confirmatory Citalopram and mianserin increased significantly the these changes were matched with the changes that serum testosterone concentration, while imipramine occurred due to the toxic dose of cyclophosphamide and amitriptyline reduced the concentration of 5 alpha- which act as +ve control. dihydrotestosterone (Przegalinski et al., 1987). On reviewing the literature through several medical On the other hand, a study done by Pardon and databases, we found few studies dealt with the effect of Nodarse in which evaluation of the effects of the tricyclic antidepressant (amitriptyline) on both hepatic antidepressive drug amitriptyline on the semen of 20 and testicular functions. These results were in infertile men with oligoasthenozoospermia was carried accordance of Davila et al., who used primary cell out. Quantitative assessment of semen in the whole cultures of neonatal hepatocytes to examine the group showed significantly higher sperm counts, an protective effect of flavonoids in the presence of increased proportion with normal sperm morphology hepatotoxins. The leakage of lactate dehydrogenase and an increased semen volume after treatment, with a (LDH), aspartate aminotransferase (AST) and alanine high positive correlation between sperm count before aminotransferase (ALT), as well as morphological and after treatment. Individual qualitative evaluation parameters, were used as indices of hepatotoxicity of showed an increased sperm count in 50% and amitriptyline (AT), and nortriptyline (NT) in large dose. increased motility in 35% of patients. They concluded These hepatotoxins caused significant LDH, AST, and that amitriptyline has a beneficial effect on semen in ALT leakage when compared to untreated control some of these patients (Pardon and Nodarse, 1980). groups. Changes in morphology were evident after 1 h of treatment with the toxicants, including vacuole We concluded from this study that high dose of formation, size deformation and cell necrosis. As the amitriptyline has toxic effects on the metabolic concentration of hepatotoxins was increased, the functions of the liver proved by elevation of the hepatic changes were more pronounced (Davila et al., 1989). enzymes and histopathological changes of hepatic tissue. Also, the use of high dose of amitriptyline led to As regards the effect of amitriptyline on the testicular reduction in the productive functions of the testis function, the results demonstrated that amitriptyline in proved by decreased both of hormonal levels low dose did not affect the testicular functions as there (testosterone and prolactin) and spermatic count with were non significant changes in the testosterone and histopathological changes of the testicular tissue. prolactin levels, spermatic counts, and morphologically as compared to the control group. While amitriptyline in high dose produced significant decreased in the References
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