Teratogenic and cytogenetic effects of ivermectin and its interaction with P-glycoprotein inhibitor

Research in Veterinary Science 90 (2011) 116–123

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Teratogenic and cytogenetic effects of ivermectin and its interaction with P-glycoprotein inhibitor Ibrahim M. El-Ashmawy a,*, Abeer F. El-Nahas b,**, Aida E. Bayad c a

Department of Pharmacology, Faculty of Veterinary Medicine, Alexandria University, Egypt Department of Genetics, Faculty of Veterinary Medicine, Alexandria University, Egypt c Department of Physiology, Faculty of Veterinary Medicine, Alexandria University, Egypt b

a r t i c l e

i n f o

Article history: Received 30 July 2009 Accepted 14 May 2010

Keywords: Ivermectin Verapamil P-glycoprotein Feto-maternal Development Genotoxicity

a b s t r a c t Experiments in animals proved that P-glycoprotein (Pgp) forms a functional barrier between maternal and fetal blood circulation in the placenta, thus protecting the fetus from exposure to xenobiotics during pregnancy. In this study we aimed to demonstrate the effects of administration of ivermectin (anthelmentic drug, Pgp substrates), either alone or simultaneously with verapamil (Pgp inhibitor) in Wister rats on fetal development, maternal bone marrow for detection of micronuclei (MN), chromosomal aberrations and mitotic index (MI) and embryonic liver cells for cellular proliferation indicated by MI, and bleeding from umbilical vessels for detection of embryonic micronuclei (MN). The results revealed that administration of ivermectin or verapamil at 6th through 15th day of gestation did not significantly altered fetal development. While, co-administration of ivermectin and verapamil clearly disturbed fetal development as indicated from abnormal feto-maternal attachment and a significant decrease in fetal weights and numbers. Furthermore, co-administration of both drugs induced a significant increase in resorption sites, post-implantation loss and external, visceral and skeletal abnormalities. They also induced genotoxicity in both dam and embryonic cells indicated by reduced mitotic index, increased number of micronucleated erythrocytes in both, and increased different types of chromosomal aberrations in dam cells, while ivermectin alone show some genotoxic effect on somatic cells of dams and the embryos. Verapamil induced reduction of embryonic mitotic index. We concluded combined treatment of ivermectin and verapamil severely affect fetal genetic material and development and induced genotoxic effect in somatic cells of the dams. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Therapeutic compounds cross placenta depending on their lipid solubility, molecular size, degree of ionization and plasma protein binding. This function is facilitated by various transporters that are expressed differentially in the functional unit of placenta (Ganapathy et al., 2000). The drug-transporting P-glycoprotein (Pgp) originally discovered in multidrug resistance tumor cells (MDR) (Juliano and Ling, 1976), belongs to the superfamily of ATP binding cassette proteins. Pgp is expressed in a variety of normal tissues mostly of epithelial origin (Thiebaut et al., 1987; Croop et al., 1989; Song et al., 1995). Pgp (the product of mdr1a gene) was demonstrated in biologically important protective barriers, blood– brain barrier, blood testis barrier, maternal fetal barrier and intes-

* Correspondence to: Ibrahim M. El-Ashmawy, Departments of Pharmacology, Faculty of Veterinary Medicine, Alexandria University, P.O. Post 22758, Egypt. Fax: +20 45 2960450. ** Corresponding author. E-mail addresses: [email protected] (I.M. El-Ashmawy), abeerelnaha [email protected] (A.F. El-Nahas). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.05.020

tinal barrier (Croop et al., 1989; Schurr et al., 1989; Schinkel et al., 1994). One major physiological role of drug-transporting Pgps is the protection of an organism against potentially toxic compounds that can be encountered in the environment by limiting the passage of drugs and compounds into the fetus (Smit et al., 1999). Placental Pgp can be partially or completely blocked by administration of Pgp inhibitors resulting in greatly increased transplacental passage to the embryo. Furthermore, placental Pgp tends to increase during pregnancy (Croop et al., 1989). Effectively, progesterone is a potent inhibitor of Pgp activity and its level increase with pregnancy age. Thus Pgp activity is probably strongly down modulated in mature placentas. Pgp substrates are usually organic molecules ranging in size from 200 Da to almost 1900 Da. Most of them are uncharged or weekly basic in nature, but some acidic compounds can also be transported (Schinkel and Jonker, 2003). Drugs and xenobiotics that bear significant structural similarity to the physiological substrates have the potential to be recognized by the transporters expressed in the placenta (Ganapathy et al., 2000). Fetal tolerance to a certain level of maternal exposure to poor Pgp substrates will be lower than for equivalent amounts of drugs that are good Pgp

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substrates (Smit et al., 1999). Ivermectin (Pgp substrates), an acaricide and anthelmentic drug of the family avermectins, produced by Streptomyces avermitilis cultures, is a well tolerated drug and except for some genetically-modified animals (collie dog), no side effects in mammals at pharmacological doses were detected (Fisher and Mrozik, 1992; Mealey et al., 2001). 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). Interactions with substances that inhibit Pgp are of great interest, as they can potentially enhance the absorption of important medicines that are generally poorly absorbed, such as chemotherapeutic medicines. Additionally, Lankas et al. (1998) provided evidence that placental Pgp may also play an important role in the protection of the developing fetus. They also showed that the placental Mdr1a (Pgp gene product) is present in the fetus-derived epithelial cells that make up the exchange border between the fetal and maternal blood circulation, and that Pgp faces the maternal blood side. It was shown that absence of Mdr1a in naturally occurring Mdr1a mutant mice is associated with enhanced sensitivity of the fetus to isomer of the pesticide avermectin. Hence, functional Mdr1a Pgp may largely limit the fetal penetration of this compound (Lankas et al., 1997, 1998). They further showed that enhanced fetal drug penetration paralleled the increased avermectin sensitivity in Mdr1a Pgp mutant fetuses. In this study we aimed to demonstrate the effects of administration of ivermectin (anthelmentic drug, Pgp substrates), either alone or simultaneously with verapamil (Pgp inhibitor) on fetal development, maternal bone marrow for detection of MN, chromosomal aberrations and cellular proliferation and embryonic liver cells for cellular proliferation, and bleeding from umbilical vessels for detection of embryonic MN.

2. Materials and methods 2.1. Animals Adult female and 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, Egypt. The rats were maintained on food and water ad libitum and housed in groups of four 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.

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Group 3: received once daily verapamil, IsoptinÒ from Knoll, Istanbul, Turkey (3 mg/kg b.wt. i.p.) (Schroder et al., 1986) and after 1 h received propylene glycol as in group 1. Group 4: received once daily verapamil (3 mg/kg b.wt. i.p.) and after 1 h received ivermectin (300 lg/kg b.wt. i.p). It was recommended that the selected period of drug administration (6th day through 15th day of gestation) is considered the period of organogenesis in rats. This period is known to be the more sensitive period of gestation to any adverse insult, which can produce bioeffects and be easier to be detected (Christian, 2001). 2.3. Maternal observations, gestation and body weight All animals were observed twice daily for signs of treatmentrelated effects. Maternal body weights were recorded on GDO, daily until GD20. At cesarean sections (10 dams from each group) on GD20, a complete gross postmortem examination was performed. 2.4. Reproductive parameters The number of corpora lutae, number and position of implantations, resorptions, and live and dead fetuses were recorded. Uteri with no visible implantations were stained with ammonium sulfide 10% (Kopf and Salewski, 1964) and examined for evidence of early resorptions. 2.4.1. Fetal observations Each fetus was individually identified, weighed, sexed, and given a gross examination for external malformations/variations including observation for palatal defects. Approximately one-half of the fetuses in each litter were evaluated for visceral malformations/variations (Staples, 1974). The fetuses selected for visceral examination were injected abdominally with 0.2 ml Bouin’s solution and then placed in Bouin’s fixative for overnight then turned to formalin solution 10% and subsequently sectioned and examined (Wilson, 1965). Whereas, the other fetuses were eviscerated and processed; the ossified skeletal structures were stained with alizarin red S and the cartilagenus parts were stained with alcian blue stain (Dawson, 1976). Five dams from each group were used for the following study: 2.5. Cytogenetic analysis of the embryos and their dams The dams were sacrificed 1–2 h after injection of 4 mg/kg b.wt. colchicine.

2.2. Experimental protocol 2.6. Cytogenetic evaluation of the dams Female rats with regular oestrous cycle were obtained after repeated vaginal smear examination. Females in oestrus phase were paired overnight with well proven fertile males. At the next morning, vaginal smear were examined microscopically for the presence of spermatozoa, where zero day of pregnancy (GDO) was determined. Pregnant females were divided randomly into four equal groups (15 each) and given the following treatments from the day 6 to the day 15 of gestation. Group 1: received once daily physiological saline (2 ml/kg b.wt. i.p.) and after 1 h received propylene glycol (used as a vehicle for the drugs used in this study) 2 ml/kg b.wt. i.p. Group 2: received once daily saline and after 1 h received ivermectin, MoramectinÒ 1%, obtained from Arabcomed, Egypt (300 lg/kg b.wt. i.p. Alvinerie et al., 1999).

Bone marrow preparation from dam were made according to Giri et al. (1986). The cells were spread into clean slide, air dried stained with Gur Giemsa and 50 well spread metaphases per animal were selected for analysis of chromosomal aberrations including fragment, deletions, ring chromosome, and polyploidy. The mitotic indices (MI) were calculated from 1000 cells per animal. Micronucleus (MN) preparations. MN was prepared according to Schmid (1976) 1000 polychromatic erythrocytes were demonstrated for each animal. 2.7. Cytogenetic evaluation of embryo Micronucleus assay of red blood cells (MN) . Wright– Giemsa-stained blood smear were made from bleeding through umbilical vessels of the embryo (Chester et al., 1998). To avoid fetal

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blood clotting, a small drop of heparin (anticoagulant) was added on slide to which the fetal blood was added, for obtaining better embryonic blood smear. 500 erythrocytes were demonstrated for each embryo for detection of MN. Embryonic mitotic index. Embryonic liver cellular proliferation was detected by counting the mitotic indices from 1000 cells per embryo. The procedures of preparing the embryonic liver chromosome were the same as previously described in bone marrow chromosome according to Giri et al., 1986. The liver tissue were homogenized and maintained in hypotonic for 30 min. and the procedures were completed as in bone marrow. Enough metaphases were observed but were not spread enough to detect different types of chromosomal aberrations.

pre-implantation loss and number of fetuses (Table 2). Meanwhile, the post-implantation loss, and resorptions or dead fetuses per dam were significantly increased in dams treated with ivermectin plus verapamil compared with other groups (Table 2/ Fig. 1A). Additionally, there was a significant decrease in fetal body weight from dams also treated with ivermectin plus verapamil compared with other groups (Table 2). 3.3. Fetal observations

All females survived to scheduled study termination on GD20. There were no abnormal clinical signs in all groups. There were no significant differences in the maternal body weight gain in any of the treatment groups at any time during the study (Table 1). There were, however, significant reductions in the final absolute maternal body weight in dams treated with ivermectin plus verapamil compared to all other groups (Table 1).

Table 3 summarizes the fetal data collected during the study. There were some gross anomalies (Fig. 1). Stunted growth of the obtained fetuses from dams treated with ivermectin plus verapamil and clear abnormalities of their attachment with their dams were found (Fig. 1B). However, there was a low incidence of stunted fetuses or abnormal fetal attachment in other experimental groups either ivermectin or verapamil compared with the control (Fig. 1C). Subcutaneous hemorrhage was the most prominent lesion in the group treated with ivermectin plus verapamil (Table 3 and Fig. 1B). It was observed that these hemorrhages were pronounced at the group treated with ivermectin plus verapamil in comparison with other groups. Collectively, there was a significant increase in the incidence of visceral and skeletal variations in the group treated with verapamil plus ivermectin in comparison with other groups (Table 3 and Fig. 1D–F). Visceral malformations observed consisted of dilated cerebral ventricles, mal-positioned thymus, heart and lung hypoplasia (Table 3). The most prominent skeletal malformations observed consisted of delayed ossifications of skull, vertebrae and sternebrae and absence or fused ribs (Table 3). Other visceral and skeletal variations observed throughout the experimental groups were generally of low incidence and not significantly different between the control and treated groups.

3.2. Reproductive parameters

3.4. Cytogenetic study

There were no significant differences between any of the treatment groups in the number of corpora lutae, implantation sites,

Regarding to the cytogenetic effect, cellular proliferation indicated by mitotic index (MI) in the mother bone marrow cells

2.8. Statistical analysis All data were statistically analyzed by one-way analysis of variance (ANOVA). Multiple comparisons were performed by Duncan’s multiple range test. All values reported as means ± S.E. For all experimental data, the significance level was set at P 6 0.05, when appropriate. 3. Results 3.1. Maternal observations, gestation and body weight

Table 1 Body weights and weight gains in rats given ivermectin and/or verapamil in gestational days 6th through 15th. Body weight

Control Verapamil (V) Ivermectin (I) I+V

Weight gain

GD 0

GD 20

GD 0–6

GD 6–9

GD 9–12

GD 12–16

GD 6–20

178.8 ± 1.3 181.1 ± 1.9 181.1 ± 2.2 182.0 ± 1.4

263.9 ± 1.6a 265.8 ± 2.2a 261.4 ± 2.1a 248.6 ± 1.9b

12.2 ± 0.5 11.2 ± 0.4 11.3 ± 0.4 10.7 ± 0.4

5.5 ± 0.3 5.3 ± 0.21 5.4 ± 0.26 5.5 ± 0.3.

7.2 ± 0.3 7.0 ± 0.25 7.3 ± 0.3 7.0 ± 0.3

12.9 ± 0.3 12.2 ± 0.32 13.1 ± 0.23 12.2 ± 0.4

39.0 ± 0.5 39.5 ± 0.73 38.4 ± 0.47 38.0 ± 0.76

All values are expressed as mean ± S.E. Number of rats in each group 10. Values with different letters at the same column are significantly different at P 6 0.05 (ANOVA with Duncan’s multiple range test).

Table 2 Reproductive parameters in rats given ivermectin and/or verapamil in gestational days 6th through 15th.

Control Verapamil (V) Ivermectin (I) I+V

No. of corpora lutea

Implantation sites

Pre-implantation loss (%)*

No. of fetuses

Resorbed and dead fetuses

Post-implantation loss (%)**

Fetal body weight (g)

11.0 ± 0.6 11.5 ± 0.4 11.3 ± 0.4 11.0 ± 0.3

10.0 ± 0.5 10.7 ± 0.3 10.3 ± 0.3 10.2 ± 0.4

8.9 ± 2.2 6.9 ± 1.2 8.5 ± 2.2 7.5 ± 1.3

9.5 ± 0.4 8.6 ± 0.3 9.6 ± 0.3 8.0 ± 0.4

0.3 ± 0.2b 0.6 ± 0.2b 0.4 ± 0.2b 0.9 ± 0.3a

3.4 ± 1.7b 5.4 ± 2.0b 3.8 ± 1.6b 8.6 ± 2.5a

4.3 ± 0.1b 3.7 ± 0.1b 4.4 ± 0.1b 2.2 ± 0.1a

All values are expressed as mean ± S.E. Number of pregnant dams in each group 10. Values with different letters at the same column are significantly different at P 6 0.05 (ANOVA with Duncan’s multiple range test). * (Corpora lutae-implantation sites)/corpora lutea  100. ** (Implantation sites-live fetuses)/implantation sites  100.

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Fig. 1. A, uterus of female rat treated with ivermectin plus verapamil showing resorption at different embryonic stages with growth retardation. B, rat fetus treated as in C, showing subcutaneous hemorrhage and abnormal attachment with the placenta. C, rat fetus from dams treated with ivermectin alone showing normal attachment to the placenta. D, E and F, rat fetuses from dams treated with ivermectin plus verapamil showing incomplete ossifications of vertebrae, ribs and skull, respectively.

significantly lowered in the group treated with ivermectin alone or when taken with verapamil (Table 4). The combination of ivermectin and verapamil increased the rate of micronucleated erythrocyte compared with the other groups. (Table 4, Fig. 2A). The genotoxic effect of the combination of ivermectin and verapamil on dams was observed also in increased number of aberrant cells, and different types of chromosomal aberrations as fragment, deletion, ring chromosome and polyploidy (Table 5, Fig. 2C–F). Treatment with ivermectin also showed some genotoxic effect on somatic cell of the mother compared with the control. These genotoxic effects include increased number of aberrant cells and fragment. Regarding to the cytogenetic effect on the embryo, liver cellular proliferation significantly lowered in the group received ivermectin plus verapamil compared with the other groups. Meanwhile, the administration of each drug alone caused lowering in mitotic index compared with the control (Table 4).

The rate of MN in red blood cell from umbilical cord in embryos received ivermectin alone or with verapamil was higher than controls (Table 4, Fig. 2B).

4. Discussion Drugs may produce teratogenicity either by direct action on the embryo or through a drug metabolite, that interfere with biochemical processes by competitive inhibition of essential cellular components, and thereby produce birth defects. Additionally, teratogenic drugs may produce their effects by influencing the metabolism of proteins and nucleic acids or interfere with cell division and fetal malformations may be correlated with chromosomal anomalies (Sisodia, 1972). Furthermore, drugs induced teratogenesis through alteration of expression of Pgp gene which has been found in the maternal/fetal placental barrier. Pgp can significantly

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Table 3 External, visceral and skeletal abnormalities of rat fetuses obtained from ivermectin and/or verapamil treated dams at 6th to 15th days of gestation. Parameters

Control

No of fetuses (no. of litters) examined External alterations 90 (10) Visceral alterations 40 (10) Skeletal alterations 50 (10) No of fetuses (no. of litters) affected External observations Stunted (<3 g) 2 (1) Subcutaneous hemorrhage 0 Multiple facial anomalies 0 Visceral observations Dilated cerebral ventricles 0 Mal-positioned thymus 1 (1) Heart hypoplasia 0 Lung hypoplasia 0 Skeletal observations Skull Delayed ossifications 0 Vertebrae Delayed ossifications 0 Ribs Fused 1 (1) Sternebrae Delayed ossifications 1 (1)

Treatment I

V

I+V

80 (10) 40 (10) 40 (10)

80 (10) 40 (10) 40 (10)

80 (10) 40 (10) 40 (10)

4 (2) 0 0

2(1) 0 0

65 (10)* 10 (5)* 3 (3)*

1 2 1 1

1 (1) 1 (1) 0 0

4 6 4 2

1(1)

1(1)

4(4)*

1 (1)

1 (1)

4 (3)

1 (1)

0

4 (4)*

2 (2)

1 (1)

8 (6)*

(1) (1) (1) (1)

(3)* (3)* (3)* (2)*

*

Significantly different compared to all groups P 6 0.05 (ANOVA with Duncan’s multiple range test).

Table 4 Number of micronucleus and mitotic index in the embryo and their dams received ivermectin and/or verapamil in gestational days 6th through 15th.

Control Verapamil (V) Ivermectin (I) I+V

Embryo MN*

Embryo mitotic index***

Maternal MN**

Maternal mitotic index***

1.08 ± 0.3b 1.1 ± 0.3b

135.3 ± 9.4a 118.8 ± 9.7b

1.3 ± 1.3b 2.0 ± 0.2b

119.6 ± 8.7a 123.0 ± 15.6a

2.0 ± 0.4a

118.8 ± 15.0b

1.8 ± 0.3b

69.5 ± 2.0b

2.1 ± 0.3a

99.0 ± 13.8c

4.3 ± 0.9a

(I)67.0 ± 5.5b

All values are expressed as mean ± S.E. Number of rats in each group 5. Values with different letters at the same column are significantly different at P 6 0.05 (ANOVA with Duncan’s multiple range. * Embryonic MN was calculated in 500 RBS from embryonic umblical cord. ** Maternal MN was calculated in 1000 polychromatic erythrocyte from bone marrow. *** Mitotic index in both embryo and dam were calculated in 1000 cells.

reduce fetal exposure to chemicals potentially harmful to developing embryo. So drugs may reach to the fetuses through inhibition of Pgp expression (Croop et al., 1989; Lankas et al. (1998)). Our results demonstrated that there was a significant reduction in the final absolute maternal body weight in dams co-treated with ivermectin and verapamil compared to all other groups, with no abnormal clinical signs. These findings may be attributed to the significant decrease in their fetus weights and numbers which may be due to the observed abnormal attachment of these fetuses with their dams and the recorded subcutaneous hemorrhage may explain the stunted growth of the fetuses. Moreover, the postimplantation loss, resorptions and dead fetuses per dam were significantly increased in dams treated with ivermectin plus verapamil, compared to all other groups. Collectively, there was a significant increase in the incidence of external, visceral and skeletal abnormalities also, in the group co-treated with ivermectin and verapamil in accordance to all other groups.

It is well known that, the development of mammalian embryo is controlled by complex factors, maternal, placental and autogenous; these factors include hormones, protective mechanisms (immune system) and nutritional factors. Changes in these factors might be expected to lead to developmental abnormalities (Saxen, 1976). Accordingly, increased accumulation of ivermectin may alter any of the previously mentioned factors causing fetal abnormalities. Furthermore, many authors explain causes of the decrease in fetal weights and their viability. Tuchman-Duplessis (1975) attributed the decrease in the percent of viable fetuses and their weights to the accumulation of the drugs in the fetal body than the maternal body. Such accumulation could be enhanced by the very simplified structure of rats’ placenta which allowed the passage of drugs from their circulation and concentrated in fetal tissues or act as inhibitors of membrane enzymes involved in embryonic nutrition. It is a fact that the fetal body weights reflect the fetal development and neonatal mortality coupled with the concept that many chemicals may destroy cellular active DNA and so reduced biosynthesis of essential components, like protein and energy source (ATP and NAD/NADP) and consequently the fetal growth (Haschek and Rousseaux, 1993). Additionally, they provided attractive suggestion for fetal resorption, where they recorded that the critical point of intrauterine development, the first interferes with the implantation of the embryo or destroy their chromosomes. Furthermore, Collins and Collins (1979) reported that the mechanisms of action of most teratogens occurred through interference with nucleic acid replication/transcription, or RNA translation, deficiency of energy supply for metabolism of the organism by restricting the availability of substrates either directly or through the presence of analogs or antagonist of vitamins, essential amino acids and others. McFeely (1993) mentioned that it is well documented that chromosomal aberrations, ring and sticky chromosomes, centromer alterations, hypoploidy and polyploidy cause embryonic death which eventually lead to fetal resorption. Recently, different pharmacological approaches have been used in an attempt to increase the systemic availability of chemotherapeutic profiles of ivermectin (Molento et al., 2004; Ballent et al., 2007). Expression of the Mdr1a multidrug resistance efflux transporter Pgp in organs such as the brain capillary endothelium, and placenta play an important role in systemic, central nervous system, and fetal exposure to a variety of natural toxins and pharmaceuticals (Kwei et al., 1999). Many authors recorded the undesirable effects of avermectin and ivermectin on fetuses from normal and genetically-modified animals. Animals with wild-type (+/+) produce Pgp or deficient genotypes differ markedly in their sensitivity to the neurotoxicity and teratology induced by abamectin and ivermectin, two members of avermectin family of anthelmentics, attributed to differences in accumulation of these compounds in the brain and fetus (Lankas et al., 1997, 1998). The use of Pgp inhibitors as a means of enhancing systemic and tissue bioavailability of drugs has been demonstrated in vitro (Chervinsky et al., 1993) and in vivo (Didier and Loor, 1995; van Asperen et al., 1997; Alvinerie et al., 1999) and reversal of multidrug resistance with this strategy may be beneficial or harmful. The presence of these efflux transporters in maternofetal barriers also restricts the penetration of substrates in the fetuses. As many drugs and their metabolites are excellent substrates (e.g. ivermectin) for these transporters, they can have a dramatic impact on the toxicity of drugs (Seaman et al., 1987; Lankas et al., 1997). On the other hand, targeted inhibition of Pgp offers a tool for drug therapy, allowing drug penetration into the pharmacological sanctuaries behind the blood–tissue barrier such as the brain or the fetus (Lin, 2003). Regarding to the cytogenetic effect on dam bone marrow, ivermectin showed some genotoxic effects represented by decrease in

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Fig. 2. Photomicrographs showing different abnormalities in rat treated with ivermectin and/or verapamil. A, micronucleated erythrocyte (arrow) from dam bone marrow compared with normal erythrocyte (arrow head). B, embryonic micronucleated erythrocyte from bleeding of umbilical vessels (arrow) compared with normal (arrow head), C, D, E, F, maternal chromosome where, C, indicate fragment, D, deletion, E, ring chromosome and F, indicate polyploidy.

Table 5 Number of aberrant cells and different types of chromosomal aberrations in mice treated with ivermectin and/or verapamil in gestational days 6th through 15th. Group

No. of aberrant cells

Fragment

Deletion

Ring chromosome

Polyploidy cells

Control Verapamil (V) Ivermectin (I) I+V

5.0 ± 0.2c 6.2 ± 0.3c 9.0 ± 0.3b 11.5 ± 1.6a

5.4 ± 1.0b 6.6 ± 0.8b 8.2 ± 0.4a 9.8 ± 1.2a

0.4 ± 0.2c 1.4 ± 0.5b 1.6 ± 0.5b 2.6 ± 0.5a

0.2 ± 0.1b 0.4 ± 0.2b 0.6 ± 0.4b 1.4 ± 0.4a

0.6 ± 0.2b 0.8 ± 0.3b 1.0 ± 0.3b 3.0 ± 0.8a

All values are expressed as mean ± S.E. Number of rats in each group 5. Values with different letters at the same column are significantly different at P 6 0.05 (ANOVA with Duncan’s multiple range).

mitotic index and increased number of aberrant cells. Molinari et al. (2009) demonstrated the genotoxic effect of ivermectin and its commercial formulation ivomecÒ on Chinese hamster ovary cell, they induced DNA-strand breaks and cytotoxicity was observed at lower concentration indicated by lowering mitotic index

and the higher concentration, followed by a complete depression of mitotic activity. Furthermore, avermectin (avermectin B1a), induced single strand DNA breaks in rat hepatocytes from rats treated in vivo (Pesticide Fact Sheet, 1989). However, there is a lack of available literature on the genotoxicity of ivermectin in vivo.

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We also demonstrated that combined treatment with verapamil and ivermectin induced more genotoxic effect in dam, represented by decrease in mitotic index, increased number of micronucleated erythrocytes, number of aberrant cells and different types of chromosomal aberrations as fragment, deletion, ring chromosome and polyploidy cells. Nesterova et al. (1999) demonstrated that verapamil potentiate the clastogenic effect of acrylamide, cyclophosphamide and dioxidine in somatic cell of mice. Also, Grujicic et al. (2008) demonistrated that combined therapy with ritodrine, erythromycin and verapamil significantly increased the frequency of MN in peripheral blood lymphocytes of pregnant woman. Furthermore, Ferguson and Baguley (1988) proved that verapamil acts as a co-mutagen in the Salmonella/ mammalian microsome mutagenicity test. The co-mutagenic effect of verapamil is possible to explain in terms of the ‘accumulation theory’, according to which calcium antagonists-including verapamil inhibit the removal of cytotoxic agents from the cell, and the resulting accumulation of these agents leads to an increase in their mutagenicity (Scheid et al., 1991; Scheid and Traut, 1993). It is also known that verapamil increase the cytotoxic effects of some antitumor antibiotics (Chatterjee et al., 1999). This was explained as verapamil act as inhibitors of P-glycoprotein substrate of the cells which is either interferes with the passive diffusion of drugs into the cell (Eytan et al., 1996) or is involved in their active transport out of the cell thus reducing their intracellular concentration (Chuman et al., 1996). Therefore, concomitant administration of substrates and Pgp inhibitors would modify drug pharmacokinetics by increased bioavailability and organ uptake leading to increased efficacy or more adverse reactions and toxicities and our results agree with these studies. Regarding to the cytogenetic effect on the embryonic liver cell, administration of either ivermectin or verapamil alone caused lowering in mitotic index compared with the control. Meanwhile, the combination of both drugs caused more reduction in hepatic cell mitotic index. Furthermore, the rate of MN in red blood cell from umbilical cord in embryos received ivermectin alone or with verapamil was significantly high compared with the other groups. Grujicˇic´ et al. (2007) demonstrated a significant increase in MN frequency in umbilical cord blood lymphocyte of human neonates received verapamil and beta mimetic. Pgp is linked to the integrity of blood–tissues barriers, such placenta and a partial blockade of Pgp could be responsible for a new drug distribution in the organism with possible increases of drug rate in organs behind these barriers (Griffin et al., 2005). In the present study, verapamil is used to block Pgp (Ford and Hait, 1990). Many authors discussed the role of Pgp inhibitors, especially verapamil, on changing the pharmacological properties of ivermectin. Molento et al. (2004) reported that ivermectin plus verapamil treatment in sheep resulted in higher ivermectin plasma concentrations over the whole drug detection period of 12–15 days compared with ivermectin alone. Accordingly, verapamil administration may facilitate more passage of ivermectin through the blood–placental 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. It may not always be easy to predict to what extent a Pgp substrate drug will be affected by placental Pgp. Digoxin, is a very good Pgp substrate drug both in vitro and in vivo, it penetrated and entered fetal tissue easily and accumulation observed in fetuses, and the penetration of digoxin through the placenta may be due to physicochemical properties of the drug, or to other factors, including the presence of carrier proteins in the placenta that facilitate the transfer of digoxin (Schinkel et al., 1995; Mayer et al., 1996, 1997; Smit et al., 1999). This may explain the slight fetal genotoxicity induced by administration of ivermectin alone (Pgp substrate)

observed in this study which was not strong enough to affect fetal development. Nesterova et al. (1999) provided evidence that verapamil being nonclastogenic and this agree with our result in both somatic cell of dams and embryonic cells. However, verapamil showed a decrease in embryonic mitotic index. Schmidt et al. (1988) and Jian et al. (2007), observed the antiprolefrative effect of verapamil alone on tumor cell in vivo and in vitro. Accordingly, in veterinary medicine, Molento et al. (2004) reported that verapamil increased the efficacy of ivermectin and moxidectin when administered in combination with these compounds against a moxidectin-resistant strain of Haemonchus contortus in sheep. Moreover, the significant alteration in the plasma disposition of ivermectin in sheep induced by verapamil, possibly due to interference with a Pgp-mediated elimination mechanism, may have important impact on efficacy against resistance parasites and on the persistency of its antiparasitic activity. The differences in response to ivermectin toxicity in large animals may be due to species differences especially at placentas (Lankas et al., 1998). It can be assumed, that assessment of Pgp activity in the placenta will eventually affect drug of choice in pregnancy. For instance, usage of recognized Pgp substrates could result in limited hazard for the fetus. On the other hand, drugs that are not Pgp substrates might be preferred in cases where the fetus is the target of pharmacotherapy (Ceckova-Novotna et al., 2006). Pgp is found in the placentas of gravid rat and tends to increase during pregnancy (Croop et al., 1989). Effectively, progesterone is a potent inhibitor of Pgp activity and its levels increase with pregnancy age (Croop et al., 1989). We suggested that verapamil (Pgp inhibitor) with short half life may be potentiated by progesterone (a potent inhibitor of Pgp) in increasing the level of ivermectin to the fetus. We concluded that ivermectin at therapeutic doses has slight genotoxic effects on rat fetuses and their dams, but when taken with Pgp inhibitor (verapamil) induced more genotoxicity in dam somatic cell and fetus which induced adverse effects on fetal development. Hence, potential risk of Pgp mediated interactions should be carefully assessed when inhibitor and substrate of Pgp are administered concomitantly to the pregnant dams.

Conflict of interest statement None declared.

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