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Effect of ivermectin on male fertility and its interaction with P-glycoprotein inhibitor (verapamil) in rats
Environmental Toxicology and Pharmacology 26 (2008) 206–211
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Effect of ivermectin on male fertility and its interaction with P-glycoprotein inhibitor (verapamil) in rats Abeer F. El-Nahas ∗ , Ibrahim M. El-Ashmawy ∗ Departments of Genetics and Pharmacology, Faculty of Veterinary Medicine, Alexandria University, Edfina, Behera, P.O. 22758, Egypt
a r t i c l e
i n f o
Article history: Received 19 December 2007 Received in revised form 20 March 2008 Accepted 24 March 2008 Available online 29 March 2008 Keywords: Ivermectin Verapamil P-glycoprotein Male fertility Meiosis
a b s t r a c t Administration of permeability-glycoprotein (Pgp) inhibitors can modify the pharmacological properties or induce toxic effects of Pgp substrates. The effects of administration of ivermectin (anthelmentic drug, Pgp substrate), either alone or simultaneously with verapamil (Pgp inhibitor) on male fertility were studied by determining mounting behavior, epididymal spermatozoal analysis, weight and histopathological examination of male reproductive organs and cytogenetic evaluation of meiotic chromosome. The results revealed that administration of ivermectin once weekly for 8 weeks induced slight fertility disturbances. While, pre-treatment with verapamil disturbed male fertility through altering different sperm parameters and histological structure of reproductive organs. Cytogenetic study revealed partial effect of ivermectin on meiosis. Meanwhile, the combined treatment of ivermectin and verapamil induced stronger effects on germ cells, increased frequency of meiotic structural chromosomal aberrations and increased X–Y chromosomal dissociation, raising the attention to the genetic quality of mature sperm. We concluded that ivermectin has slight effects on male fertility, but when taken with verapamil induced adverse effects on meiosis and fertility. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Ivermectin, an acaricide and anthelmentic drug of the family of avermectins, produced by Streptomyces avermitilis cultures, is a well-tolerated drug with no side effects in mammals at pharmacological doses (Fisher and Mrozik, 1992). Ivermectin is an agonist of ␥-aminobutyric acid (GABA) receptors and of glutamate-gated Cl− channels, the later restricted to invertebrates (Bloom, 1996). Some clinicians suggested that ivermectin may interfere with the gastrointestinal function of target parasites, resulting in starvation of the parasite (Renukaprasad et al., 1989). The low ivermectin toxicity has been attributed to its restricted access to some organs and brain tissues, especially for being a substrate of Pgp (Schinkel et al., 1994). Pgp is a member of ATP-binding cassette superfamily of transmembrane transporters and mediates the membrane transport of many hydrophobic compounds, including hormones, sterols, lipids, phospholipids, cytokines, and anticancer drugs (Bellamy, 1996). Pgp is located in many tissues and in the capillary endothelial cells of the testis and blood–brain barrier (Cordon-Cardo et al., 1990), where it functions as an efflux transporter of xenobiotics (Chen et al., 2004; Lin, 2003). Interactions with substances that inhibit Pgp are
∗ Corresponding authors. Fax: +20 45 2960450. E-mail addresses: [email protected] (A.F. El-Nahas), [email protected] (I.M. El-Ashmawy). 1382-6689/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2008.03.011
of great interest, as they can potentially enhance the absorption of important medicines that are generally poorly absorbed, such as chemotherapeutic medicines. Alternately, Pgp inhibition may theoretically increase the incidence of side effects or toxicity of some medicines, producing unwanted effects. Many compounds are known to modulate the Pgp by reducing the efflux activity of the pump, e.g. verapamil, cyclosporine A, erythromycin and their analogs (Ford and Hait, 1990). Therefore, the modulation of Pgp function by Pgp inhibitors, such as verapamil, can be an important factor in modifying the pharmacological actions of certain drugs. Inspite of the approval for use of ivermectin in all dogs. It can, however, cause neurotoxicity at very low doses to genetically sensitive canine breeds collies. Increased ivermectin levels in the brains of sensitive collies appear to be due to ineffective brain-to-blood efflux caused by Pgp transporter (Dowling, 2006). The chromosome abnormalities associated with infertility are of two types. Karyotype alterations affecting cells of both somatic and germ cell lines and mitotic abnormalities. Both types can produce infertility either by spermatogenesis arrest or formation of chromosomally unbalanced gametes leading to spontaneous abortion and/or offspring with mental deficiency and malformations (Navarro et al., 1987). It is also reported that these abnormal gametes are produced as a result of altered intra-testicular environment that affect negatively the mechanisms controlling chromosome segregation during cell division (De Palma et al., 2005). Many authors recorded the undesirable effects of iver-
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mectin on fertility (Tanyildizi and Bozkurt, 2002; Schroder et al., 1986). To our knowledge, this design is the first one concerning the drug–drug interaction between P-glycoprotein inhibitor and ivermectin on male fertility. In this study, the effect of ivermectin on male fertility was studied in rats and its interaction with verapamil with respect to different sperm parameters, weights and histopathological examination of the reproductive organs and cytogenetic examination of germ cells. 2. Materials and methods 2.1. Animals Adult male Wister rats (weighing 170–190 g, 10–12 weeks age) were used. The animals were obtained from a closed random bred colony at Faculty of Veterinary Medicine, Alexandria University, Egypt. The rats were maintained on food and water ad libitum and housed in groups of six in isolated cages. The animals were acclimatized for 2 weeks prior to usage. The investigation confirms to the guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH publication no. 83-23, revised 1996). The local ethical committee approved the study. 2.2. Experimental protocol Male rats were divided into four groups (12 rats each). Group 1 received once weekly physiological saline (2 ml/kg body weight) i.p. and after 1 h received propylene glycol (used as a vehicle for the drugs used in this study) (2 ml/kg body weight) i.p. Group 2 received once weekly saline and after 1 h received ivermectin, Moramectin® 1%, obtained from Arabcomed, Egypt (300 g/kg body weight) i.p. (Alvinerie et al., 1999). Group 3 received once weekly verapamil, Isoptin® from Knoll, Istanbul, Turkey (3 mg/kg body weight) i.p. (Schroder et al., 1986) and after 1 h received propylene glycol as in group 1. Group 4 received verapamil (3 mg/kg body weight) i.p. once weekly and after 1 h received ivermectin (300 g/kg body weight) i.p. After 8 weeks from the beginning of treatment, each group was divided equally; the first six male rats were chosen from each group and used for mounting behavior, reproductive organs weight (testes, epididymis and accessory sex organs), histopathological changes in these organs and semen analysis. 2.3. Mounting behavior at the end of treatment (8 weeks) To observe the lipido-oriented mounting behavior, non-oestrus female rats were paired with treated male rats. The male assuming the copulatory position over the female but failing to achieve intromission was considered as a mount (Subramonian et al., 1997). Male rats from each group were randomly chosen and suitably marked. The rats were placed in a clear aquarium and allowed to acclimatize for 15 min. After that a non-oestrus female was introduced into the arena. The number of mounts was recorded for 15 min 2.4. Reproductive organ weights All male rats were weighed and sacrificed. The testes, epididymides and accessory sex organs (seminal vesicles and prostate glands) were dissected out, grossly examined and weighed. The index weight (I.W.) of the organ was calculated by I.W. = organ weight (g)/100 × body weight (g). 2.5. Epididymal sperm count Epididymal spermatozoa were counted by a modified method of Yokoi et al. (2003). Briefly, the epididymis was minced in 5 ml of saline, placed in a rocker for 10 min and incubated at room temperature for 2 min. The supernatant fluid was diluted 1:100 with a solution containing 5 g NaHCO3 , 1 ml formalin (35%) and 25 mg eosin per 100 ml distilled water. About 10 l of the diluted semen was transferred to each counting chamber of the improved Neubaur haemocytometer (Deep 1/10 mm, LABART, Munich, Germany) and was allowed to stand for 5 min for counting under a light microscope at ×200 magnification. 2.6. Sperm motility Sperm-progressive motility was evaluated microscopically within 2–4 min of their isolation from the cauda epididymis as described by Sonmez et al. (2005). Fluid was obtained from the cauda epididymis with a pipette and diluted to 2 ml with tris buffer solution. The percentage of motility was evaluated at ×400 magnification.
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2.8. Histopathological studies Sections were taken from testis, epididymis, seminal vesicles and prostate glands immediately after the rats were killed. The tissues were fixed in 10% neutral formalin for a period of at least 24 h, dehydrated in graded (50–100%) alcohol and embedded in paraffin, cut into 4–5 m thick sections and stained with haematoxylin and eosin for photomicroscopic examination (Bancroft and Stevens, 1990). The second six rats from each group were used for: 2.9. Cytogenetic study Meiotic chromosomes were prepared according to Imai et al. (1981). The testes were dissected out and were cut into three pieces with a sharp knife. The testicular tissue was put in hypotonic solution (1% trisodium citrate solution with 0.005% colchicine). The mass of seminiferous tubules were fixed in 1:1 ethyl alcohol:glacial acetic acid for 30 min, followed by another fixation in 60% alcohol (1:1 fixative diluted with distilled water). The cells were resuspended in 1:1 fixative and two drops were added into clean slide, dried and stained with 3% Giemsa for 10 min at room temperature. The slides were examined by light microscope. Proportion of cells at spermatogonial metaphase (SPM), metaphase I (MI) and metaphase II (MII) of meiosis were calculated by examining 300 metaphases at ×400. Fifty well-spread spermatogonial metaphase and MII were examined at ×1000 for detection of chromosome structural and numerical aberrations. Fifty cells at MI were examined and the numbers of cells with autosomal and/or X–Y dissociation were calculated at ×1000. 2.10. Statistical analysis Data were submitted to analysis of variance (ANOVA), P ≤ 0.05, to compare the treated groups with the control group. When significant difference was found, means were compared used Duncan’s multiple range test (Duncan, 1955). Calculations were done using SAS system (SAS, 1987).
3. Results 3.1. Organ weights The weights of testis, epididymis and accessory sex glands were slightly decreased (P 0.05) in ivermectin-treated group versus control, while the decrease in weights of these organs was more in rats treated with ivermectin plus verapamil. Verapamil administration insignificantly changes the reproductive organs weight (Table 1). 3.2. Mounting behavior Administration of ivermectin once weekly for 8 weeks insignificantly decreased the mounting behavior compared to the control. While, prior administration of verapamil to ivermectin significantly (P ≤ 0.05) decreased the mounting behavior. Verapamil administration not significantly changed the mounting behavior (Table 2). 3.3. Sperm characteristics Epididymal sperm concentration, sperm motility and abnormal sperm rates are shown in Table 2 for ivermectin and/or verapamil treatments. Verapamil administration potentiates the ivermectininduced reduction in sperm concentration. Ivermectin alone insignificantly lowered sperm motility compared to the control group, but verapamil administration prior to ivermectin significantly reduced sperm motility (Table 2). When total sperm abnormalities (head and tail) were analyzed, only the verapamil and ivermectin-treated group had significant higher levels of abnormalities (Table 2). Verapamil insignificantly changed sperm motility and abnormalities (Table 2). 3.4. Histopathological findings
2.7. Sperm abnormality The method by Evans and Maxwell (1987) was used for determination of the percentage of morphologically abnormal spermatozoa. A total of 300 sperm cells were counted on each slide under light microscope at ×400 magnification.
The microscopic examination for the testes of the control rats revealed normal histologic structure and spermatogenesis in the seminiferous tubules (Fig. 1a). In case of treatment with ivermectin,
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Table 1 Effect of ivermectin and/or verapamil on the organs weight in male rats Groups Control Verapamil (V) Ivermectin (I) I+V
Initial body wt. (g) 184.3 181.0 180.5 191.2
± ± ± ±
10.0 7.4 7.5 8.0
Final body wt. (g) 231.4 235.0 231.0 242.0
± ± ± ±
I.W. of testis
7.1 8.6 6.9 4.7
1.40 1.29 1.10 1.08
I.W. of epididymis
± 0.05 ± 0.03a ± ± 0.05b ± 0.01b a
0.54 0.49 0.41 0.40
± ± ± ±
I.W. of accessory sex organ
a
0.01 0.02a 0.02b 0.01b
1.07 1.12 0.95 0.86
± ± ± ±
0.06a 0.03a 0.02b 0.06b
All values are expressed as mean ± S.E. Number of rats in each group is six. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test). wt. = weight, I.W. = organ weight (g)/100 × body weight (g). Table 2 Effect of ivermectin and/or verapamil on sperm characteristics, and mounting behavior in male rats Groups
Sperm count (×106 /ml)
Control Verapamil (V) Ivermectin (I) I+V
392.3 391.6 343.3 211.0
± ± ± ±
12.5a 16.2a 30.7a 21.4b
Sperm motility (%) 80.8 77.5 74.1 62.5
± ± ± ±
1.5a 1.1a 5.0a 5.5b
Sperm abnormality (%) 8.8 9.4 9.2 14.0
± ± ± ±
Mounting behavior/15 min
0.37b 0.9b 0.6b 0.7a
8.8 8.5 7.3 6.5
± ± ± ±
0.2a 0.4a 0.3a 0.3b
All values are expressed as mean ± S.E. Number of rats in each group is six. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test).
Fig. 1. Photomicrographs of rat testis, (a) control rats revealed normal histologic structure. (b) Treated with ivermectin, some of the spermatogonial cells in the seminiferous tubules were seen to be affected with vacuolar degeneration (arrows), in seminiferous tubules (st) H and E ×400, (c and d) treated with ivermectin + verapamil where, (c) showing degeneration in some tubules with presence of coagulated luminal contents (st) H and E ×250, (d) showing one degenerated seminiferous tubule (arrow) and other completely necrotic tubule (st) H and E ×250.
some of the spermatogonial cells in the seminiferous tubules were seen to be affected with vacuolar degeneration (Fig. 1b). No obvious changes could be seen in the testes of rats treated with only verapamil, while in case of treatment with (ivermectin + verapamil) some destructive changes were seen in the form degeneration in some tubules with presence of coagulated luminal contents in some seminiferous tubules, in addition to complete coagulative necrosis of some other tubules (Fig. 1c and d). Also, many tubules contain uncompleted spermatogenesis (Fig. 1c). At the same direction, the histopathological changes observed in the epididymis, prostate and seminal vesicles were pronounced in rats treated with verapamil plus ivermectin than ivermectin alone (data not shown).
of the control. The combination of both drugs caused significant increase in the proportion of spermatogonial metaphase in the 300 cells. Furthermore, neither ivermectin nor verapamil or its combination has effect on the proportion of the cells at MI. However, the number of the cells in MII significantly decreased in animals treated with ivermectin alone and when taken with verapamil as shown Table 3 Effect of ivermectin and/or verapamil on the distribution of spermatogonial metaphase, MI, and MII in 300 Metaphases Groups
SPM
3.5. Cytogenetic analysis
Control Verapamil (V) Ivermectin (I) I+V
159.0 144.2 165.4 183.8
MI
Cell distribution counts determined on cytogenetic preparation showed the proportion of the cells in spermatogonial metaphase in animals treated with ivermectin or verapamil to be similar to that
All values are expressed as mean ± S.E. Number of rats in each group is 6. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test). SPM: spermatogonial metaphase, MI: metaphase I, MII: metaphase II.
± ± ± ±
15.0b 12.8b 9.3b 6.1a
65.0 74.2 72.4 74.2
MII ± ± ± ±
3.6 2.3 5.3 6.4
73.6 79.8 57.2 50.8
MII/MI ratio ± ± ± ±
12.9a 11.9a 6.4b 8.3b
1.14 1.1 0.8 0.8
± ± ± ±
0.2 1.1 0.1 0.3
A.F. El-Nahas, I.M. El-Ashmawy / Environmental Toxicology and Pharmacology 26 (2008) 206–211 Table 4 Number of abnormal spermatogonial metaphase, MI, and MII in 50 cells induced by ivermectin and/or verapamil in male rats Groups Control Verapamil (V) Ivermectin (I) I+V
No. of cells
SPM
50 50 50 50
6.8 5.4 8.0 16.2
MI ± ± ± ±
b
0.7 0.5b 1.2b 2.1a
ivermectin alone. It has been reported that, co-administration of verapamil with some drugs as amidarane potentiate toxicity symptoms which is dose-dependent (Molento et al., 2004). The decrease in these reproductive organs weight could be due to the decrease in testosterone level as reported by our previous and other studies (Zaied, 2004; Srikhanth et al., 1999; El-Ashmawy and Mandour, 1996) which may be due to the direct effect of ivermectin on the central nervous system and gonadal tissues or its effects on hypothalamus–pituitary–testis axis. The mounting behavior was correlated with our previous findings (El-Ashmawy and Mandour, 1996). Pre-treatment with verapamil significantly potentiated the undesirable effect of ivermectin on the mounting behavior. Ivermectin administration alone did not significantly change sperm characteristics. But giving verapamil plus ivermectin significantly induced hazardous effects on sperm count, motility and abnormality. The reduced sperm content implies an adverse effect on spermatogenesis in rats that received ivermectin plus verapamil. Impaired sperm motility in these rats is indicative of a defect in the acquisition or maintenance of motility. This combination may alter the epididymal secretory products or has a direct action on sperm motility and morphology (Ballent et al., 2007; Zaied, 2004). The increase in sperm abnormalities such as sperm without head, hookless, coiled or abnormal tail were also observed in rats treated with verapamil plus ivermectin. Furthermore, these sperm abnormalities were also associated with histopathological abnormalities in rats that received both drugs. Cell distribution counts determined on cytogenetic preparation showed that distribution of SPM and MI is the same in all groups indicating that neither ivermectin nor verapamil or its combination has any adverse effect on mitotically divided cells (Spermatogonial, spermatocyte) and also these drugs has no effect on the progression of SPM spermatocyte to MI. This result is supported by histopathological findings, which showed incomplete suppression of spermatogenesis. The increased number of SPM metaphase in ivermectin plus verapamil treated group may reflect the decreased number of cell in MII in the 300 counted metaphases. The insignificant decrease of MII/MI ratio (0.8) in animals treated with verapamil or ivermectin plus verapamil may indicate some effect on the progress of spermatogenesis but not complete suppression of spermatogenesis. Carrara et al. (2004) found that in man with MII/MI ratio of 0.8 most cells entering MI meiosis I progressed to meiosis II and those with MII/MI > 0.5 showed complete suppression of spermatogenesis and this is not the case in our study. Chandley et al. (1976) reported a correlation between decreased number of cells reaching metaphase II and increased percentage of cells with unpaired sex chromosome, especially when this over 40% but in our study the frequency of X–Y chromosome dissociation is 21.6% (10.8/50) (Table 5) which also indicate incomplete arrest of spermatogenesis. Our study showed that combined treatment of ivermectin and verapamil significantly increased number of aberrant SPM, MI and MII cells compared to the other groups. Furthermore, this combined
MII
2.0 4.4 4.2 7.8
± ± ± ±
b
0.5 0.9b 1.2b 1.1a
4.0 2.6 3.4 7.6
± ± ± ±
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0.6b 0.6b 0.6b 1.5a
All values are expressed as mean ± S.E. Number of rats in each group is six. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test).
in Table 3. Insignificant decrease in MII/MI ratio was observed in groups that received ivermectin and ivermectin plus verapamil. No significant increase in the number of abnormal cells in spermatogonial metaphase, MI, or MII induced by ivermectin or verapamil (Table 4). However, the combination of both drugs caused significant increase in the abnormal spermatogonial metaphase, MI and MII cells. Administration of ivermectin and/or verapamil did not induce an increase in the number of polyploidy spermatogonial metaphase (Fig. 2a) or diploid MII (Fig. 2f) (numerical chromosomal aberrations) (Table 5). The animals that received verapamil plus ivermectin showed significant increase in the number of structural chromosomal aberrations (increase fragmented spermatogonial metaphase (Fig. 2b) and fragment in MII cells (Fig. 2e and Table 5). The frequency of X–Y dissociation significantly increased in animals that received verapamil plus ivermectin (Fig. 2c). Meanwhile there is no significant change in the number of autosomal dissociation (Fig. 2d) induced by ivermectin, verapamil or its combination (Table 5). 4. Discussion A pharmacological interaction is said to occur when the response to one medicine varies from what is usually predicted because another substance has altered the response or induced toxicity. It is generally accepted, however, that there are two major interaction mechanisms namely pharmacodynamic and pharmacokinetic interaction. Recently, different pharmacological approaches have been used in an attempt to increase the systemic availability of chemotherapeutic profiles of ivermectin (Ballent et al., 2007; Molento et al., 2004). Many authors recorded the undesirable effects of ivermectin on male fertility, as reduced semen concentration and sperm motility (Tanyildizi and Bozkurt, 2002; Schroder et al., 1986). At the present study, ivermectin administration once weekly for 8 weeks induced a decrease in weights of the testis, epididymis and accessory sex organs. These results are in accordance with that obtained by Zaied (2004); El-Ashmawy and Mandour (1996), but not correlated with those reported by Daurio et al. (1987). These differences in response may be due to species variety. By administrations of verapamil prior to ivermectin, the decrease in weights of these organs were more pronounced than
Table 5 Types of abnormalities observed in spermatogonial metaphase, MI, and MII in 50 cells induced by ivermectin and/or verapamil in male rats Groups
SPM
MI
Polyploidy Control Verapamil (V) Ivermectin (I) I+V
2.8 3.2 3.8 5.4
± ± ± ±
0.9 1.1 0.5 1.1
Fragment 5.0 3.6 4.0 10.8
± ± ± ±
MII
X–Y b
1.8 0.7b 1.1b 1.4a
0.4 1.0 0.6 2.8
Autosomal dissociation ± ± ± ±
b
0.2 0.6b 0.4b 0.7a
1.6 3.4 4.2 4.2
± ± ± ±
0.4 0.6 1.5 0.6
Fragment 3.8 2.4 3.2 7.2
± ± ± ±
b
0.7 0.6b 0.7b 1.3a
Diploid cells 0.2 0.4 0.4 0.8
± ± ± ±
0.2 0.2 0.7 0.6
All values are expressed as mean ± S.E. Number of rats in each group is six. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test).
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Fig. 2. Photomicrographs showing different chromosomal abnormalities in rat spermatogenesis treated with ivermectin and/or verapamil (a) polyploidy spermatogonial metaphase, (b) fragment in spermatogonial metaphase, (c) X–Y univalent at MI, (d) autosomal univalent at MI, (e) fragment in MII, (f) diploid MII.
treatment significantly increased structural chromosomal aberrations, and also increased X–Y chromosome dissociation. Verapamil showed co-clastogeneic effect on mitotic chromosome of mice when administered with acrylamid, cyclophosphamide and dioxidine (Nesterova et al., 1999). Furthermore Calogero et al. (2003) reported that abnormal meiotic chromosome complement is associated with sperm abnormalities. Furthermore, elevated frequency of X–Y dissociation in mice correlates with significantly lower testis weight and lower yield of spermatogenesis (Krzanowska, 1989). It was also reported that partial deletion of Y-chromosome (structural aberrations) affects efficiency of spermatogenesis, morphology of spermatozoa and their epididymal maturation and their capacity to reach the ampulla and fertilize eggs (Styra et al., 2002). All these studies explain decreased sperm count, motility and increased its abnormalities and also reduced reproductive organs weight associated with combined treatment with ivermectin and verapamil. However, concern exists regarding the genetic quality of mature gametes from animals that received ivermectin plus verapamil.
Kahraman et al. (2002) observed elevated sperm abnormalities associated with a greater rate of pregnancy failure. Cytogenetic study revealed partial effect of ivermectin on meiosis through reducing number of spermatocyte at MII. Meanwhile, the combined treatment of ivermectin plus verapamil induced stronger effect on germ cells, as they reduce number of spermatocyte at MII. Increased number of the structural chromosomal aberrations at SPM and MII and increased frequency of X–Y chromosome dissociation at MI, raise the attention to the genetic quality of mature sperm which need further investigation. The low ivermectin toxicity has attributed to its restricted access to some tissues, especially for being a substrate of Pgp (Griffin et al., 2005; Xu et al., 1998). Pgp is linked to the integrity of blood–tissue barriers, such as the blood–brain barrier, testis or placenta and a partial blockade of Pgp could be responsible for a new drug distribution in the organism with possible increases of drug rates in organs behind these barriers (Griffin et al., 2005). Therefore, concomitant administration of substrates and Pgp inhibitors would modify drug pharmacokinetics by increased bioavailability and organ uptake,
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leading to increased efficacy or more adverse reactions and toxicities. In the present study, verapamil is used to block Pgp (Ford and Hait, 1990). Many authors discussed the role of P-glycoprotein inhibitors, especially verapamil, on changing the pharmacological properties of ivermectin (Molento et al., 2004). Accordingly, verapamil administration may facilitate more passage of ivermectin through the blood–testis barriers, increasing its intracellular accumulation and retention. This suggestion may in part explain the pronounced toxic effects of ivermectin in animals pre-treated with verapamil compared with ivermectin alone. We concluded that ivermectin has slight effects on male fertility, but when taken with verapamil induced adverse effects on meiosis and fertility. References Alvinerie, M., Dupuy, J., Eckhoutte, C., Sutra, J.F., 1999. Enhanced absorption of pour-on ivermectin formulation in rats by co-administration of the multidrugresistance-reversing agent verapamil. Parasitol. Res. 85, 920. Ballent, M., Lifschitz, A., Virkel, G., Sallovitz, J., Lanusse, C., 2007. Involvement of P-glycoprotein on ivermectin kinetic behavior in sheep: itraconazole-mediated changes on gastrointestinal disposition. J. Vet. Pharmacol. Ther. 30, 242. Bancroft, J.D., Stevens, A., 1990. Theory and Practice of Histological Technique, third ed. Churchill-Livingstone, New York, NY. Bellamy, W.T., 1996. P-glycoprotein and multidrug resistance. Annu. Rev., Pharmacol. Toxicol. 36, 161. Bloom, F.E., 1996. Neurotransmission and the central nervous system. In: Goodman, Gilman (Eds.), The Pharmacological Basis of Therapeutics, ninth ed. McGrawHill, New York, NY, p. 267. Calogero, A.E., Burrello, N., Depalma, A., Barone, N., D’Agata, R., Vicari, E., 2003. Reprod. Biomed. 6, 310. Carrara, R.C.V., Yamasaki, R., Mazucatto, L.F., Valudo, M.A.L., Sartorato, F.I., Pina-Neto, R., 2004. Somatic and germ cells cytogenetic studies and AZF microdeletion screening in infertile man. Genet. Mol. Biol. 27, 122. Chandley, A.C., Fletcher, J., Watson, G.S., 1976. Cytogenetics and infertility in man. II. Testicular histology and meiosis. Ann. Hum. Genet. 40, 165. Chen, L.M., Liang, Y.J., Ruan, J.W., Dind, Y., 2004. Reversal of P-gp mediated multidrug resistance in-vitro and in-vivo by FG020318. J. Pharm. Pharmacol. 56, 1061. Cordon-Cardo, C., O’Brein, J.P., Boccia, J., Casals, J., Bertino, J.R., Melamed, M.R., 1990. Expression of the multi drug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J. Histochem. Cytochem. 38, 1277. Daurio, C.P., Gilman, M.R., Pullian, J.D., 1987. Reproductive evaluation of male beagles and the safety of ivermectin. Am. J. Vet. Res. 48, 1755. De Palma, A., Burrello, N., Barone, N., D’Agata, R., Vicari, E., Calogero, A.E., 2005. Patients with abnormal sperm parameters have an increased sex chromosome aneuploidy rate in peripheral leukocytes. Hum. Reprod. 20, 2153. Dowling, P., 2006. Pharmacogenetics: it’s not just about ivermectin in collies. Can. Vet. J. 47, 1165. Duncan, D.B., 1955. Multiple range and multiple F test. Biometrics 11, 1. El-Ashmawy, I.M., Mandour, A.A., 1996. Studies on the influence of ivermectin on reproductive organs and liver function in male rabbits. In: Third Vet. Med. Conf. Zagazig, Egypt, October 8–10. Evans, G., Maxwell, W.M.C., 1987. Handling and examination of semen. In: Maxwell, W.M.C. (Ed.), Salamon’s Artificial Insemination of Sheep and Goats. Butterworths, Sydney, Australia, p. 93.
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Fisher, M.H., Mrozik, H., 1992. The chemistry and pharmacology of avermectins. Annu. Rev., Pharmacol. Toxicol. 32, 537. Ford, J.M., Hait, W.N., 1990. Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol. Rev. 42, 155. Griffin, J., Fletcher, N., Clemence, R., Blanchflower, S., Brayden, D.J., 2005. Selametin is a potent substrate and inhibitor of human and canine P-glycoprotein. J. Vet. Pharmacol. Ther. 3, 257. Imai, H.T., Matsuda, Y., Shiroshi, T., Moriwaki, K., 1981. High frequency of X-Y chromosome dissociation in primary spermatocytes of F1 hybrids between Japanese wild mice (Mus musculus molossinus) and inbred laboratory mice. Cytogenet. Cell Genet. 29, 166. Kahraman, S., Kumtepe, Y., Sertyel, S., Donmez, E., Denkhalifa, M., 2002. Pronuclear morphology scoring and chromosomal status of embryos in severe male infertility. Hum. Reprod. 17, 3193. Krzanowska, H., 1989. X-Y chromosome dissociation in mouse strain differing in efficiency of spermatogenesis elevated frequency of univalents in pubertal males. Gamate Res. 23, 365. Lin, J.H., 2003. Drug–drug interaction mediated by inhibition and induction of Pglycoprotein. Adv. Drug. Deliv. Res. 55, 53. Molento, M.B., Lifschitz, A., Sallovitz, J., Lanusse, C., Prichard, R., 2004. Influence of verapamil on the pharmacokinetics of the antiparasitic drugs ivermectin and moxedectin in sheep. Parasitol. Res. 92, 121. Navarro, J., Bonet, J., Garcia, M., Vidal, L., Freixa, L., Templado, C., 1987. Citogenetica de la inferilidad Y La esterilidad. Medicine 100, 4223. Nesterova, E.V., Burnev, A.D., Seredenin, S.B., 1999. Verapamil contributes to the clastogenic effects of acrylamide, cyclophosphamide, and dioxidine of somatic cells of BALB/C and C57BL mice. Mutat. Res. 440, 171. Renukaprasad, C.M., Ramaswamy, M., Kumar, M., Gapal, M., Keshavamurthy, B.S., 1989. Therapeutic effect of ivermectin on rabbit mange. Indian Vet. J. 66, 1055. SAS, 1987. Statistical Analysis System. Users guide. Statistics SAS Institute Cary, North Carolina, USA. Schinkel, A.H., Smit, J.J.M., van Tellinger, O., Beijneun, J.H., 1994. Disruption of the mouse mdr la P-glycoprotein gene leads to a deficiency in the blood–brain barrier and to an increased sensitivity to drugs. Cell 77, 491. Schroder, J.G., Swan, E., Barrick, R.A., 1986. Effects of ivermectin on the reproductive potential of breeding rams. J. S. Afr. Vet. Assoc. 57, 211. Sonmez, M., Turk, G., Yuce, A., 2005. The effect of ascorbic acid supplementation on sperm quality in rats. Theriogenology 63, 2063. Srikhanth, V., Malini, M.T., Arunakaran, J., Govindarajulu, P., Balasubramanian, K., 1999. Effects of ethanol on epididymal secretory products and sperm maturation in albino rats. J. Pharmacol. Exp. Ther. 288, 509. Styra, J., Bilinska, B., Krzanowska, H., 2002. Effect of partial Y-chromosome deletion in Bio-BR-Ydel mice on testis morphology sperm quality and efficiency of fertilization. Reprod. Fertil. Dev. 14 (1–2), 101. Subramonian, A., Madhavachandran, R., Rajasekkaran, S., Pushpangadan, P., 1997. Aphrodisiac property of Trichopus zeylancus extract in male mice. J. Ethnopharmacol. 57, 21. Tanyildizi, S., Bozkurt, T., 2002. An investigation of the effects of Ivermectin on blood serum, semen hyaluronidase activities and spermatological characteristics in sheep. Turk. J. Vet. Anim. Sci. 26, 353. Xu, M., Molento, M., Blackhall, W., Ribeiro, P., Beech, R., Prichard, R., 1998. Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog. Mol. Biochem. Parasitol. 91, 327. Yokoi, K., Uthus, E.O., Nielsen, F.H., 2003. Nickel deficiency diminishes sperm quantity and movement in rats. Biol. Trace Elem. Res. 93, 141. Zaied, G.M., 2004. Some pharmacodynamic properties of ivermectin in comparison with doramectin in male rats. Ph.D. thesis presented to Pharmacology Department, Faculty of Veterinary Medicine Alexandria University, Egypt.
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Effect of ivermectin on male fertility and its interaction with P-glycoprotein inhibitor (verapamil) in rats Abeer F. El-Nahas ∗ , Ibrahim M. El-Ashmawy ∗ Departments of Genetics and Pharmacology, Faculty of Veterinary Medicine, Alexandria University, Edfina, Behera, P.O. 22758, Egypt
a r t i c l e
i n f o
Article history: Received 19 December 2007 Received in revised form 20 March 2008 Accepted 24 March 2008 Available online 29 March 2008 Keywords: Ivermectin Verapamil P-glycoprotein Male fertility Meiosis
a b s t r a c t Administration of permeability-glycoprotein (Pgp) inhibitors can modify the pharmacological properties or induce toxic effects of Pgp substrates. The effects of administration of ivermectin (anthelmentic drug, Pgp substrate), either alone or simultaneously with verapamil (Pgp inhibitor) on male fertility were studied by determining mounting behavior, epididymal spermatozoal analysis, weight and histopathological examination of male reproductive organs and cytogenetic evaluation of meiotic chromosome. The results revealed that administration of ivermectin once weekly for 8 weeks induced slight fertility disturbances. While, pre-treatment with verapamil disturbed male fertility through altering different sperm parameters and histological structure of reproductive organs. Cytogenetic study revealed partial effect of ivermectin on meiosis. Meanwhile, the combined treatment of ivermectin and verapamil induced stronger effects on germ cells, increased frequency of meiotic structural chromosomal aberrations and increased X–Y chromosomal dissociation, raising the attention to the genetic quality of mature sperm. We concluded that ivermectin has slight effects on male fertility, but when taken with verapamil induced adverse effects on meiosis and fertility. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Ivermectin, an acaricide and anthelmentic drug of the family of avermectins, produced by Streptomyces avermitilis cultures, is a well-tolerated drug with no side effects in mammals at pharmacological doses (Fisher and Mrozik, 1992). Ivermectin is an agonist of ␥-aminobutyric acid (GABA) receptors and of glutamate-gated Cl− channels, the later restricted to invertebrates (Bloom, 1996). Some clinicians suggested that ivermectin may interfere with the gastrointestinal function of target parasites, resulting in starvation of the parasite (Renukaprasad et al., 1989). The low ivermectin toxicity has been attributed to its restricted access to some organs and brain tissues, especially for being a substrate of Pgp (Schinkel et al., 1994). Pgp is a member of ATP-binding cassette superfamily of transmembrane transporters and mediates the membrane transport of many hydrophobic compounds, including hormones, sterols, lipids, phospholipids, cytokines, and anticancer drugs (Bellamy, 1996). Pgp is located in many tissues and in the capillary endothelial cells of the testis and blood–brain barrier (Cordon-Cardo et al., 1990), where it functions as an efflux transporter of xenobiotics (Chen et al., 2004; Lin, 2003). Interactions with substances that inhibit Pgp are
∗ Corresponding authors. Fax: +20 45 2960450. E-mail addresses: [email protected] (A.F. El-Nahas), [email protected] (I.M. El-Ashmawy). 1382-6689/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2008.03.011
of great interest, as they can potentially enhance the absorption of important medicines that are generally poorly absorbed, such as chemotherapeutic medicines. Alternately, Pgp inhibition may theoretically increase the incidence of side effects or toxicity of some medicines, producing unwanted effects. Many compounds are known to modulate the Pgp by reducing the efflux activity of the pump, e.g. verapamil, cyclosporine A, erythromycin and their analogs (Ford and Hait, 1990). Therefore, the modulation of Pgp function by Pgp inhibitors, such as verapamil, can be an important factor in modifying the pharmacological actions of certain drugs. Inspite of the approval for use of ivermectin in all dogs. It can, however, cause neurotoxicity at very low doses to genetically sensitive canine breeds collies. Increased ivermectin levels in the brains of sensitive collies appear to be due to ineffective brain-to-blood efflux caused by Pgp transporter (Dowling, 2006). The chromosome abnormalities associated with infertility are of two types. Karyotype alterations affecting cells of both somatic and germ cell lines and mitotic abnormalities. Both types can produce infertility either by spermatogenesis arrest or formation of chromosomally unbalanced gametes leading to spontaneous abortion and/or offspring with mental deficiency and malformations (Navarro et al., 1987). It is also reported that these abnormal gametes are produced as a result of altered intra-testicular environment that affect negatively the mechanisms controlling chromosome segregation during cell division (De Palma et al., 2005). Many authors recorded the undesirable effects of iver-
A.F. El-Nahas, I.M. El-Ashmawy / Environmental Toxicology and Pharmacology 26 (2008) 206–211
mectin on fertility (Tanyildizi and Bozkurt, 2002; Schroder et al., 1986). To our knowledge, this design is the first one concerning the drug–drug interaction between P-glycoprotein inhibitor and ivermectin on male fertility. In this study, the effect of ivermectin on male fertility was studied in rats and its interaction with verapamil with respect to different sperm parameters, weights and histopathological examination of the reproductive organs and cytogenetic examination of germ cells. 2. Materials and methods 2.1. Animals Adult male Wister rats (weighing 170–190 g, 10–12 weeks age) were used. The animals were obtained from a closed random bred colony at Faculty of Veterinary Medicine, Alexandria University, Egypt. The rats were maintained on food and water ad libitum and housed in groups of six in isolated cages. The animals were acclimatized for 2 weeks prior to usage. The investigation confirms to the guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH publication no. 83-23, revised 1996). The local ethical committee approved the study. 2.2. Experimental protocol Male rats were divided into four groups (12 rats each). Group 1 received once weekly physiological saline (2 ml/kg body weight) i.p. and after 1 h received propylene glycol (used as a vehicle for the drugs used in this study) (2 ml/kg body weight) i.p. Group 2 received once weekly saline and after 1 h received ivermectin, Moramectin® 1%, obtained from Arabcomed, Egypt (300 g/kg body weight) i.p. (Alvinerie et al., 1999). Group 3 received once weekly verapamil, Isoptin® from Knoll, Istanbul, Turkey (3 mg/kg body weight) i.p. (Schroder et al., 1986) and after 1 h received propylene glycol as in group 1. Group 4 received verapamil (3 mg/kg body weight) i.p. once weekly and after 1 h received ivermectin (300 g/kg body weight) i.p. After 8 weeks from the beginning of treatment, each group was divided equally; the first six male rats were chosen from each group and used for mounting behavior, reproductive organs weight (testes, epididymis and accessory sex organs), histopathological changes in these organs and semen analysis. 2.3. Mounting behavior at the end of treatment (8 weeks) To observe the lipido-oriented mounting behavior, non-oestrus female rats were paired with treated male rats. The male assuming the copulatory position over the female but failing to achieve intromission was considered as a mount (Subramonian et al., 1997). Male rats from each group were randomly chosen and suitably marked. The rats were placed in a clear aquarium and allowed to acclimatize for 15 min. After that a non-oestrus female was introduced into the arena. The number of mounts was recorded for 15 min 2.4. Reproductive organ weights All male rats were weighed and sacrificed. The testes, epididymides and accessory sex organs (seminal vesicles and prostate glands) were dissected out, grossly examined and weighed. The index weight (I.W.) of the organ was calculated by I.W. = organ weight (g)/100 × body weight (g). 2.5. Epididymal sperm count Epididymal spermatozoa were counted by a modified method of Yokoi et al. (2003). Briefly, the epididymis was minced in 5 ml of saline, placed in a rocker for 10 min and incubated at room temperature for 2 min. The supernatant fluid was diluted 1:100 with a solution containing 5 g NaHCO3 , 1 ml formalin (35%) and 25 mg eosin per 100 ml distilled water. About 10 l of the diluted semen was transferred to each counting chamber of the improved Neubaur haemocytometer (Deep 1/10 mm, LABART, Munich, Germany) and was allowed to stand for 5 min for counting under a light microscope at ×200 magnification. 2.6. Sperm motility Sperm-progressive motility was evaluated microscopically within 2–4 min of their isolation from the cauda epididymis as described by Sonmez et al. (2005). Fluid was obtained from the cauda epididymis with a pipette and diluted to 2 ml with tris buffer solution. The percentage of motility was evaluated at ×400 magnification.
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2.8. Histopathological studies Sections were taken from testis, epididymis, seminal vesicles and prostate glands immediately after the rats were killed. The tissues were fixed in 10% neutral formalin for a period of at least 24 h, dehydrated in graded (50–100%) alcohol and embedded in paraffin, cut into 4–5 m thick sections and stained with haematoxylin and eosin for photomicroscopic examination (Bancroft and Stevens, 1990). The second six rats from each group were used for: 2.9. Cytogenetic study Meiotic chromosomes were prepared according to Imai et al. (1981). The testes were dissected out and were cut into three pieces with a sharp knife. The testicular tissue was put in hypotonic solution (1% trisodium citrate solution with 0.005% colchicine). The mass of seminiferous tubules were fixed in 1:1 ethyl alcohol:glacial acetic acid for 30 min, followed by another fixation in 60% alcohol (1:1 fixative diluted with distilled water). The cells were resuspended in 1:1 fixative and two drops were added into clean slide, dried and stained with 3% Giemsa for 10 min at room temperature. The slides were examined by light microscope. Proportion of cells at spermatogonial metaphase (SPM), metaphase I (MI) and metaphase II (MII) of meiosis were calculated by examining 300 metaphases at ×400. Fifty well-spread spermatogonial metaphase and MII were examined at ×1000 for detection of chromosome structural and numerical aberrations. Fifty cells at MI were examined and the numbers of cells with autosomal and/or X–Y dissociation were calculated at ×1000. 2.10. Statistical analysis Data were submitted to analysis of variance (ANOVA), P ≤ 0.05, to compare the treated groups with the control group. When significant difference was found, means were compared used Duncan’s multiple range test (Duncan, 1955). Calculations were done using SAS system (SAS, 1987).
3. Results 3.1. Organ weights The weights of testis, epididymis and accessory sex glands were slightly decreased (P 0.05) in ivermectin-treated group versus control, while the decrease in weights of these organs was more in rats treated with ivermectin plus verapamil. Verapamil administration insignificantly changes the reproductive organs weight (Table 1). 3.2. Mounting behavior Administration of ivermectin once weekly for 8 weeks insignificantly decreased the mounting behavior compared to the control. While, prior administration of verapamil to ivermectin significantly (P ≤ 0.05) decreased the mounting behavior. Verapamil administration not significantly changed the mounting behavior (Table 2). 3.3. Sperm characteristics Epididymal sperm concentration, sperm motility and abnormal sperm rates are shown in Table 2 for ivermectin and/or verapamil treatments. Verapamil administration potentiates the ivermectininduced reduction in sperm concentration. Ivermectin alone insignificantly lowered sperm motility compared to the control group, but verapamil administration prior to ivermectin significantly reduced sperm motility (Table 2). When total sperm abnormalities (head and tail) were analyzed, only the verapamil and ivermectin-treated group had significant higher levels of abnormalities (Table 2). Verapamil insignificantly changed sperm motility and abnormalities (Table 2). 3.4. Histopathological findings
2.7. Sperm abnormality The method by Evans and Maxwell (1987) was used for determination of the percentage of morphologically abnormal spermatozoa. A total of 300 sperm cells were counted on each slide under light microscope at ×400 magnification.
The microscopic examination for the testes of the control rats revealed normal histologic structure and spermatogenesis in the seminiferous tubules (Fig. 1a). In case of treatment with ivermectin,
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Table 1 Effect of ivermectin and/or verapamil on the organs weight in male rats Groups Control Verapamil (V) Ivermectin (I) I+V
Initial body wt. (g) 184.3 181.0 180.5 191.2
± ± ± ±
10.0 7.4 7.5 8.0
Final body wt. (g) 231.4 235.0 231.0 242.0
± ± ± ±
I.W. of testis
7.1 8.6 6.9 4.7
1.40 1.29 1.10 1.08
I.W. of epididymis
± 0.05 ± 0.03a ± ± 0.05b ± 0.01b a
0.54 0.49 0.41 0.40
± ± ± ±
I.W. of accessory sex organ
a
0.01 0.02a 0.02b 0.01b
1.07 1.12 0.95 0.86
± ± ± ±
0.06a 0.03a 0.02b 0.06b
All values are expressed as mean ± S.E. Number of rats in each group is six. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test). wt. = weight, I.W. = organ weight (g)/100 × body weight (g). Table 2 Effect of ivermectin and/or verapamil on sperm characteristics, and mounting behavior in male rats Groups
Sperm count (×106 /ml)
Control Verapamil (V) Ivermectin (I) I+V
392.3 391.6 343.3 211.0
± ± ± ±
12.5a 16.2a 30.7a 21.4b
Sperm motility (%) 80.8 77.5 74.1 62.5
± ± ± ±
1.5a 1.1a 5.0a 5.5b
Sperm abnormality (%) 8.8 9.4 9.2 14.0
± ± ± ±
Mounting behavior/15 min
0.37b 0.9b 0.6b 0.7a
8.8 8.5 7.3 6.5
± ± ± ±
0.2a 0.4a 0.3a 0.3b
All values are expressed as mean ± S.E. Number of rats in each group is six. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test).
Fig. 1. Photomicrographs of rat testis, (a) control rats revealed normal histologic structure. (b) Treated with ivermectin, some of the spermatogonial cells in the seminiferous tubules were seen to be affected with vacuolar degeneration (arrows), in seminiferous tubules (st) H and E ×400, (c and d) treated with ivermectin + verapamil where, (c) showing degeneration in some tubules with presence of coagulated luminal contents (st) H and E ×250, (d) showing one degenerated seminiferous tubule (arrow) and other completely necrotic tubule (st) H and E ×250.
some of the spermatogonial cells in the seminiferous tubules were seen to be affected with vacuolar degeneration (Fig. 1b). No obvious changes could be seen in the testes of rats treated with only verapamil, while in case of treatment with (ivermectin + verapamil) some destructive changes were seen in the form degeneration in some tubules with presence of coagulated luminal contents in some seminiferous tubules, in addition to complete coagulative necrosis of some other tubules (Fig. 1c and d). Also, many tubules contain uncompleted spermatogenesis (Fig. 1c). At the same direction, the histopathological changes observed in the epididymis, prostate and seminal vesicles were pronounced in rats treated with verapamil plus ivermectin than ivermectin alone (data not shown).
of the control. The combination of both drugs caused significant increase in the proportion of spermatogonial metaphase in the 300 cells. Furthermore, neither ivermectin nor verapamil or its combination has effect on the proportion of the cells at MI. However, the number of the cells in MII significantly decreased in animals treated with ivermectin alone and when taken with verapamil as shown Table 3 Effect of ivermectin and/or verapamil on the distribution of spermatogonial metaphase, MI, and MII in 300 Metaphases Groups
SPM
3.5. Cytogenetic analysis
Control Verapamil (V) Ivermectin (I) I+V
159.0 144.2 165.4 183.8
MI
Cell distribution counts determined on cytogenetic preparation showed the proportion of the cells in spermatogonial metaphase in animals treated with ivermectin or verapamil to be similar to that
All values are expressed as mean ± S.E. Number of rats in each group is 6. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test). SPM: spermatogonial metaphase, MI: metaphase I, MII: metaphase II.
± ± ± ±
15.0b 12.8b 9.3b 6.1a
65.0 74.2 72.4 74.2
MII ± ± ± ±
3.6 2.3 5.3 6.4
73.6 79.8 57.2 50.8
MII/MI ratio ± ± ± ±
12.9a 11.9a 6.4b 8.3b
1.14 1.1 0.8 0.8
± ± ± ±
0.2 1.1 0.1 0.3
A.F. El-Nahas, I.M. El-Ashmawy / Environmental Toxicology and Pharmacology 26 (2008) 206–211 Table 4 Number of abnormal spermatogonial metaphase, MI, and MII in 50 cells induced by ivermectin and/or verapamil in male rats Groups Control Verapamil (V) Ivermectin (I) I+V
No. of cells
SPM
50 50 50 50
6.8 5.4 8.0 16.2
MI ± ± ± ±
b
0.7 0.5b 1.2b 2.1a
ivermectin alone. It has been reported that, co-administration of verapamil with some drugs as amidarane potentiate toxicity symptoms which is dose-dependent (Molento et al., 2004). The decrease in these reproductive organs weight could be due to the decrease in testosterone level as reported by our previous and other studies (Zaied, 2004; Srikhanth et al., 1999; El-Ashmawy and Mandour, 1996) which may be due to the direct effect of ivermectin on the central nervous system and gonadal tissues or its effects on hypothalamus–pituitary–testis axis. The mounting behavior was correlated with our previous findings (El-Ashmawy and Mandour, 1996). Pre-treatment with verapamil significantly potentiated the undesirable effect of ivermectin on the mounting behavior. Ivermectin administration alone did not significantly change sperm characteristics. But giving verapamil plus ivermectin significantly induced hazardous effects on sperm count, motility and abnormality. The reduced sperm content implies an adverse effect on spermatogenesis in rats that received ivermectin plus verapamil. Impaired sperm motility in these rats is indicative of a defect in the acquisition or maintenance of motility. This combination may alter the epididymal secretory products or has a direct action on sperm motility and morphology (Ballent et al., 2007; Zaied, 2004). The increase in sperm abnormalities such as sperm without head, hookless, coiled or abnormal tail were also observed in rats treated with verapamil plus ivermectin. Furthermore, these sperm abnormalities were also associated with histopathological abnormalities in rats that received both drugs. Cell distribution counts determined on cytogenetic preparation showed that distribution of SPM and MI is the same in all groups indicating that neither ivermectin nor verapamil or its combination has any adverse effect on mitotically divided cells (Spermatogonial, spermatocyte) and also these drugs has no effect on the progression of SPM spermatocyte to MI. This result is supported by histopathological findings, which showed incomplete suppression of spermatogenesis. The increased number of SPM metaphase in ivermectin plus verapamil treated group may reflect the decreased number of cell in MII in the 300 counted metaphases. The insignificant decrease of MII/MI ratio (0.8) in animals treated with verapamil or ivermectin plus verapamil may indicate some effect on the progress of spermatogenesis but not complete suppression of spermatogenesis. Carrara et al. (2004) found that in man with MII/MI ratio of 0.8 most cells entering MI meiosis I progressed to meiosis II and those with MII/MI > 0.5 showed complete suppression of spermatogenesis and this is not the case in our study. Chandley et al. (1976) reported a correlation between decreased number of cells reaching metaphase II and increased percentage of cells with unpaired sex chromosome, especially when this over 40% but in our study the frequency of X–Y chromosome dissociation is 21.6% (10.8/50) (Table 5) which also indicate incomplete arrest of spermatogenesis. Our study showed that combined treatment of ivermectin and verapamil significantly increased number of aberrant SPM, MI and MII cells compared to the other groups. Furthermore, this combined
MII
2.0 4.4 4.2 7.8
± ± ± ±
b
0.5 0.9b 1.2b 1.1a
4.0 2.6 3.4 7.6
± ± ± ±
209
0.6b 0.6b 0.6b 1.5a
All values are expressed as mean ± S.E. Number of rats in each group is six. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test).
in Table 3. Insignificant decrease in MII/MI ratio was observed in groups that received ivermectin and ivermectin plus verapamil. No significant increase in the number of abnormal cells in spermatogonial metaphase, MI, or MII induced by ivermectin or verapamil (Table 4). However, the combination of both drugs caused significant increase in the abnormal spermatogonial metaphase, MI and MII cells. Administration of ivermectin and/or verapamil did not induce an increase in the number of polyploidy spermatogonial metaphase (Fig. 2a) or diploid MII (Fig. 2f) (numerical chromosomal aberrations) (Table 5). The animals that received verapamil plus ivermectin showed significant increase in the number of structural chromosomal aberrations (increase fragmented spermatogonial metaphase (Fig. 2b) and fragment in MII cells (Fig. 2e and Table 5). The frequency of X–Y dissociation significantly increased in animals that received verapamil plus ivermectin (Fig. 2c). Meanwhile there is no significant change in the number of autosomal dissociation (Fig. 2d) induced by ivermectin, verapamil or its combination (Table 5). 4. Discussion A pharmacological interaction is said to occur when the response to one medicine varies from what is usually predicted because another substance has altered the response or induced toxicity. It is generally accepted, however, that there are two major interaction mechanisms namely pharmacodynamic and pharmacokinetic interaction. Recently, different pharmacological approaches have been used in an attempt to increase the systemic availability of chemotherapeutic profiles of ivermectin (Ballent et al., 2007; Molento et al., 2004). Many authors recorded the undesirable effects of ivermectin on male fertility, as reduced semen concentration and sperm motility (Tanyildizi and Bozkurt, 2002; Schroder et al., 1986). At the present study, ivermectin administration once weekly for 8 weeks induced a decrease in weights of the testis, epididymis and accessory sex organs. These results are in accordance with that obtained by Zaied (2004); El-Ashmawy and Mandour (1996), but not correlated with those reported by Daurio et al. (1987). These differences in response may be due to species variety. By administrations of verapamil prior to ivermectin, the decrease in weights of these organs were more pronounced than
Table 5 Types of abnormalities observed in spermatogonial metaphase, MI, and MII in 50 cells induced by ivermectin and/or verapamil in male rats Groups
SPM
MI
Polyploidy Control Verapamil (V) Ivermectin (I) I+V
2.8 3.2 3.8 5.4
± ± ± ±
0.9 1.1 0.5 1.1
Fragment 5.0 3.6 4.0 10.8
± ± ± ±
MII
X–Y b
1.8 0.7b 1.1b 1.4a
0.4 1.0 0.6 2.8
Autosomal dissociation ± ± ± ±
b
0.2 0.6b 0.4b 0.7a
1.6 3.4 4.2 4.2
± ± ± ±
0.4 0.6 1.5 0.6
Fragment 3.8 2.4 3.2 7.2
± ± ± ±
b
0.7 0.6b 0.7b 1.3a
Diploid cells 0.2 0.4 0.4 0.8
± ± ± ±
0.2 0.2 0.7 0.6
All values are expressed as mean ± S.E. Number of rats in each group is six. Values with different letters at the same column are significantly different at P ≤ 0.05 (ANOVA with Duncan’s multiple range test).
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Fig. 2. Photomicrographs showing different chromosomal abnormalities in rat spermatogenesis treated with ivermectin and/or verapamil (a) polyploidy spermatogonial metaphase, (b) fragment in spermatogonial metaphase, (c) X–Y univalent at MI, (d) autosomal univalent at MI, (e) fragment in MII, (f) diploid MII.
treatment significantly increased structural chromosomal aberrations, and also increased X–Y chromosome dissociation. Verapamil showed co-clastogeneic effect on mitotic chromosome of mice when administered with acrylamid, cyclophosphamide and dioxidine (Nesterova et al., 1999). Furthermore Calogero et al. (2003) reported that abnormal meiotic chromosome complement is associated with sperm abnormalities. Furthermore, elevated frequency of X–Y dissociation in mice correlates with significantly lower testis weight and lower yield of spermatogenesis (Krzanowska, 1989). It was also reported that partial deletion of Y-chromosome (structural aberrations) affects efficiency of spermatogenesis, morphology of spermatozoa and their epididymal maturation and their capacity to reach the ampulla and fertilize eggs (Styra et al., 2002). All these studies explain decreased sperm count, motility and increased its abnormalities and also reduced reproductive organs weight associated with combined treatment with ivermectin and verapamil. However, concern exists regarding the genetic quality of mature gametes from animals that received ivermectin plus verapamil.
Kahraman et al. (2002) observed elevated sperm abnormalities associated with a greater rate of pregnancy failure. Cytogenetic study revealed partial effect of ivermectin on meiosis through reducing number of spermatocyte at MII. Meanwhile, the combined treatment of ivermectin plus verapamil induced stronger effect on germ cells, as they reduce number of spermatocyte at MII. Increased number of the structural chromosomal aberrations at SPM and MII and increased frequency of X–Y chromosome dissociation at MI, raise the attention to the genetic quality of mature sperm which need further investigation. The low ivermectin toxicity has attributed to its restricted access to some tissues, especially for being a substrate of Pgp (Griffin et al., 2005; Xu et al., 1998). Pgp is linked to the integrity of blood–tissue barriers, such as the blood–brain barrier, testis or placenta and a partial blockade of Pgp could be responsible for a new drug distribution in the organism with possible increases of drug rates in organs behind these barriers (Griffin et al., 2005). Therefore, concomitant administration of substrates and Pgp inhibitors would modify drug pharmacokinetics by increased bioavailability and organ uptake,
A.F. El-Nahas, I.M. El-Ashmawy / Environmental Toxicology and Pharmacology 26 (2008) 206–211
leading to increased efficacy or more adverse reactions and toxicities. In the present study, verapamil is used to block Pgp (Ford and Hait, 1990). Many authors discussed the role of P-glycoprotein inhibitors, especially verapamil, on changing the pharmacological properties of ivermectin (Molento et al., 2004). Accordingly, verapamil administration may facilitate more passage of ivermectin through the blood–testis barriers, increasing its intracellular accumulation and retention. This suggestion may in part explain the pronounced toxic effects of ivermectin in animals pre-treated with verapamil compared with ivermectin alone. We concluded that ivermectin has slight effects on male fertility, but when taken with verapamil induced adverse effects on meiosis and fertility. References Alvinerie, M., Dupuy, J., Eckhoutte, C., Sutra, J.F., 1999. Enhanced absorption of pour-on ivermectin formulation in rats by co-administration of the multidrugresistance-reversing agent verapamil. Parasitol. Res. 85, 920. Ballent, M., Lifschitz, A., Virkel, G., Sallovitz, J., Lanusse, C., 2007. Involvement of P-glycoprotein on ivermectin kinetic behavior in sheep: itraconazole-mediated changes on gastrointestinal disposition. J. Vet. Pharmacol. Ther. 30, 242. Bancroft, J.D., Stevens, A., 1990. Theory and Practice of Histological Technique, third ed. Churchill-Livingstone, New York, NY. Bellamy, W.T., 1996. P-glycoprotein and multidrug resistance. Annu. Rev., Pharmacol. Toxicol. 36, 161. Bloom, F.E., 1996. Neurotransmission and the central nervous system. In: Goodman, Gilman (Eds.), The Pharmacological Basis of Therapeutics, ninth ed. McGrawHill, New York, NY, p. 267. Calogero, A.E., Burrello, N., Depalma, A., Barone, N., D’Agata, R., Vicari, E., 2003. Reprod. Biomed. 6, 310. Carrara, R.C.V., Yamasaki, R., Mazucatto, L.F., Valudo, M.A.L., Sartorato, F.I., Pina-Neto, R., 2004. Somatic and germ cells cytogenetic studies and AZF microdeletion screening in infertile man. Genet. Mol. Biol. 27, 122. Chandley, A.C., Fletcher, J., Watson, G.S., 1976. Cytogenetics and infertility in man. II. Testicular histology and meiosis. Ann. Hum. Genet. 40, 165. Chen, L.M., Liang, Y.J., Ruan, J.W., Dind, Y., 2004. Reversal of P-gp mediated multidrug resistance in-vitro and in-vivo by FG020318. J. Pharm. Pharmacol. 56, 1061. Cordon-Cardo, C., O’Brein, J.P., Boccia, J., Casals, J., Bertino, J.R., Melamed, M.R., 1990. Expression of the multi drug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J. Histochem. Cytochem. 38, 1277. Daurio, C.P., Gilman, M.R., Pullian, J.D., 1987. Reproductive evaluation of male beagles and the safety of ivermectin. Am. J. Vet. Res. 48, 1755. De Palma, A., Burrello, N., Barone, N., D’Agata, R., Vicari, E., Calogero, A.E., 2005. Patients with abnormal sperm parameters have an increased sex chromosome aneuploidy rate in peripheral leukocytes. Hum. Reprod. 20, 2153. Dowling, P., 2006. Pharmacogenetics: it’s not just about ivermectin in collies. Can. Vet. J. 47, 1165. Duncan, D.B., 1955. Multiple range and multiple F test. Biometrics 11, 1. El-Ashmawy, I.M., Mandour, A.A., 1996. Studies on the influence of ivermectin on reproductive organs and liver function in male rabbits. In: Third Vet. Med. Conf. Zagazig, Egypt, October 8–10. Evans, G., Maxwell, W.M.C., 1987. Handling and examination of semen. In: Maxwell, W.M.C. (Ed.), Salamon’s Artificial Insemination of Sheep and Goats. Butterworths, Sydney, Australia, p. 93.
211
Fisher, M.H., Mrozik, H., 1992. The chemistry and pharmacology of avermectins. Annu. Rev., Pharmacol. Toxicol. 32, 537. Ford, J.M., Hait, W.N., 1990. Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol. Rev. 42, 155. Griffin, J., Fletcher, N., Clemence, R., Blanchflower, S., Brayden, D.J., 2005. Selametin is a potent substrate and inhibitor of human and canine P-glycoprotein. J. Vet. Pharmacol. Ther. 3, 257. Imai, H.T., Matsuda, Y., Shiroshi, T., Moriwaki, K., 1981. High frequency of X-Y chromosome dissociation in primary spermatocytes of F1 hybrids between Japanese wild mice (Mus musculus molossinus) and inbred laboratory mice. Cytogenet. Cell Genet. 29, 166. Kahraman, S., Kumtepe, Y., Sertyel, S., Donmez, E., Denkhalifa, M., 2002. Pronuclear morphology scoring and chromosomal status of embryos in severe male infertility. Hum. Reprod. 17, 3193. Krzanowska, H., 1989. X-Y chromosome dissociation in mouse strain differing in efficiency of spermatogenesis elevated frequency of univalents in pubertal males. Gamate Res. 23, 365. Lin, J.H., 2003. Drug–drug interaction mediated by inhibition and induction of Pglycoprotein. Adv. Drug. Deliv. Res. 55, 53. Molento, M.B., Lifschitz, A., Sallovitz, J., Lanusse, C., Prichard, R., 2004. Influence of verapamil on the pharmacokinetics of the antiparasitic drugs ivermectin and moxedectin in sheep. Parasitol. Res. 92, 121. Navarro, J., Bonet, J., Garcia, M., Vidal, L., Freixa, L., Templado, C., 1987. Citogenetica de la inferilidad Y La esterilidad. Medicine 100, 4223. Nesterova, E.V., Burnev, A.D., Seredenin, S.B., 1999. Verapamil contributes to the clastogenic effects of acrylamide, cyclophosphamide, and dioxidine of somatic cells of BALB/C and C57BL mice. Mutat. Res. 440, 171. Renukaprasad, C.M., Ramaswamy, M., Kumar, M., Gapal, M., Keshavamurthy, B.S., 1989. Therapeutic effect of ivermectin on rabbit mange. Indian Vet. J. 66, 1055. SAS, 1987. Statistical Analysis System. Users guide. Statistics SAS Institute Cary, North Carolina, USA. Schinkel, A.H., Smit, J.J.M., van Tellinger, O., Beijneun, J.H., 1994. Disruption of the mouse mdr la P-glycoprotein gene leads to a deficiency in the blood–brain barrier and to an increased sensitivity to drugs. Cell 77, 491. Schroder, J.G., Swan, E., Barrick, R.A., 1986. Effects of ivermectin on the reproductive potential of breeding rams. J. S. Afr. Vet. Assoc. 57, 211. Sonmez, M., Turk, G., Yuce, A., 2005. The effect of ascorbic acid supplementation on sperm quality in rats. Theriogenology 63, 2063. Srikhanth, V., Malini, M.T., Arunakaran, J., Govindarajulu, P., Balasubramanian, K., 1999. Effects of ethanol on epididymal secretory products and sperm maturation in albino rats. J. Pharmacol. Exp. Ther. 288, 509. Styra, J., Bilinska, B., Krzanowska, H., 2002. Effect of partial Y-chromosome deletion in Bio-BR-Ydel mice on testis morphology sperm quality and efficiency of fertilization. Reprod. Fertil. Dev. 14 (1–2), 101. Subramonian, A., Madhavachandran, R., Rajasekkaran, S., Pushpangadan, P., 1997. Aphrodisiac property of Trichopus zeylancus extract in male mice. J. Ethnopharmacol. 57, 21. Tanyildizi, S., Bozkurt, T., 2002. An investigation of the effects of Ivermectin on blood serum, semen hyaluronidase activities and spermatological characteristics in sheep. Turk. J. Vet. Anim. Sci. 26, 353. Xu, M., Molento, M., Blackhall, W., Ribeiro, P., Beech, R., Prichard, R., 1998. Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog. Mol. Biochem. Parasitol. 91, 327. Yokoi, K., Uthus, E.O., Nielsen, F.H., 2003. Nickel deficiency diminishes sperm quantity and movement in rats. Biol. Trace Elem. Res. 93, 141. Zaied, G.M., 2004. Some pharmacodynamic properties of ivermectin in comparison with doramectin in male rats. Ph.D. thesis presented to Pharmacology Department, Faculty of Veterinary Medicine Alexandria University, Egypt.