Developmental toxicity in rat fetuses exposed to the benzimidazole netobimin

Reproductive Toxicology 13 (1999) 295–302

Developmental toxicity in rat fetuses exposed to the benzimidazole netobimin Marc Navarroa, Lourdes Canutc, Ana Carreteroa, Carles Cristofolb, Fco–Javier Pe´rez–Aparicioa, Margarida Arboixb, Jesu´s Rubertea,* a

Dept. of Anatomy and Embryology, Veterinary Faculty, Autonomous University of Barcelona, 08193 Bellaterra, Barcelona, Spain b Dept. of Pharmacology and Toxicology, Veterinary Faculty, Autonomous University of Barcelona, 08193 Bellaterra, Spain c Laboratory of Teratology of CIDA (Centro de Investigacion y Desarrollo Aplicado. S.A.L.), 08130-Santa Perpetua de la Mogoda, Barcelona, Spain Received 22 October 1998; received in revised form 5 March 1999; accepted 12 March 1999

Abstract Netobimin (NTB) is a prodrug of albendazole (ABZ) and is used as a broad-spectrum anthelmintic both in human and veterinary medicine. Pregnant Sprague-Dawley rats were treated po with 50, 59.5 and 70.7 mg/kg of NTB on Gestational Day (GD) 10. The results, observed on GD 20, demonstrated that NTB induced a significant increase of resorptions. Moreover, decreased fetal body weight and an increase in skeletal malformations were observed in treated groups. We report the first study in which vascular malformations are described in rats after the administration of a benzimidazole compound. An interesting relationship between intercostal vessel and rib malformations was found. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Benzimidazoles; Pathogenesis of malformations; Corrosion casting; Scanning electron microscopy; Rat fetus; Teratology; Dysmorphogenesis; Embryolethality

1. Introduction Netobimin (NTB) is a prodrug of a benzimidazole compound and mainly is used as a broad-spectrum anthelmintic in veterinary medicine. Netobimin is cycled by gastrointestinal bacteria and transformed into albendazole (ABZ) [1]. Albendazole is the molecule that has the anthelmintic effect [2] and has been widely available in many countries, even in human medicine [3], for the treatment of gastrointestinal helminthiases. In humans, it has shown evidence of good efficacy against hydatid cysts [4]. We already have demonstrated the developmental toxicity of NTB in sheep embryos [5], the commonest species in which NTB is used as a broad-spectrum anthelmintic. Several studies suggest that the capacity of benzimidazole drugs to bind with cellular microtubules is responsible for the toxic effects on the embryo [6]. Once ABZ is absorbed, it is metabolized sequentially to the sulphoxide (ABZSO) and sulphone (ABZSO2) of ABZ [7]. Both ABZ and ABZSO exhibit toxic

* Corresponding author. Tel.: ⫹93-581-20-06; fax: ⫹93-581-18-46. E-mail address: [email protected] (J. Ruberte)

effects [8,9]. However, the significant correlation found in the rat embryo between the rate of developmental toxicity and metabolite concentration demonstrate that the ABZSO concentration could be the main factor accounting for toxicity [10]. Furthermore, the transplacental movement of ABZ metabolites after the administration of NTB in ewes supports that these metabolites may be responsible for the developmental toxicity of NTB [11]. Developmental toxicity has been shown in rats for benzimidazole compounds such as albendazole, parbendazole or flubendazole [9,12–14], demonstrating that the rat is equally sensitive to the teratogenic effects of Benzimidazoles. However, only a few investigations of the metabolism and pharmacology of NTB in rats can be found, and the description of the developmental toxicity of NTB in this species is rather scant [1,10]. The aim of this work was to study the developmental toxicity produced by NTB in rat fetuses. The study was designed to characterize the malformations induced by this compound, and to reproduce in an experimental species the teratogenic effects induced by NTB in sheep [5]. Resorption rate, fetal weight and malformations were studied. The investigation was focused on skeletal and

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vascular anomalies, as well as the possible correlation between them.

2. Materials and methods 2.1. Animal husbandry Twenty-five 9 to 10 week old Sprague–Dawley rats (Crl: CDR) weighing 210 –300 g, were used. Rats in proestrus were caged overnight with males of the same strain. The presence of sperm in a vaginal smear the following morning was considered Gestation Day (GD) 0. Dams were individually housed (Makrolon, 37 ⫻ 21.4 ⫻ 18 cm with hardwood chip bedding) in the CIDA laboratory in a room maintained at approximately 19 –25°C, 40 – 60% relative humidity, and a 12 h light/dark cycle. Air change was carried out 6 – 8 times per hour. Animals were fed a commercial diet (UAR A04C; Usine d’Alimentation Rationelle, Villemoisson sur Orge, France) and had free access to food and to tap water. Manipulations and experimental procedures were performed according to the OECD Principles of Good Laboratory Practice, C(81)30, Paris, 12 May, 1981. Annex 2. 2.2. Experimental schedule Pregnant rats were randomly assigned to four groups (6 or 7 rats/group). Three groups received NTB doses of 50, 59.5, and 70.7 mg/kg body weight (Hapasil® suspension 5%; Schering–Plough), in a volume of 5 mL/kg (according to the individual body weight of each dam), orally (gastric intubation) on GD 10. The control group was administered the same volume of vehicle (0.2% sodium carboxymethyl cellulose in 1% Tween 80 in double distilled water). GD 10 in rats is equivalent to the embryonic stage in ewes (17th day of pregnancy) that gives rise to developmental toxicity after oral treatment with NTB [5]. Netobimin doses were selected from previous studies [1], that showed fetal malformations when pregnant rats were treated with NTB doses of 44.2 mg/kg or higher. We selected 50 mg/kg as the first dose and the higher doses were obtained by a geometric progression, multiplying by the square root of two.

with alizarin red S [15]. To determine vascular anomalies, the remaining fetuses were injected through the umbilical artery to obtain casts of their vascular system. The umbilical artery was dissected and cannulated with an angulated Pasteur’s pipette. The pipette was fire polished to a gauge similar to the artery to be injected, and sealed to the vessel with a drop of cyanocrylate (Loctite®) [16]. The resins used for corrosion casting were Araldite CY 223, hardener HY 2967 and red color DW (all from Ciba–Geigy), or Mercox® (Mercox–Jap. Vilene Co. supplied by Ladd Research Ind., Inc. Williston, VT) diluted with 25% methylmethacrylate monomer [17]. After the polymerization process, fetuses were macerated in 5% KOH. The casts thus obtained were dissected under a stereomicroscope, mounted on stubs, sputtered with gold, and observed in a Hitachi S-570 scanning electron microscope at an accelerating voltage of 8 to 10 KV (see 18 for details on the technique). All the cast were compared with the normal arterial pattern of the GD 20 rat fetus [18]. The litter was considered the experimental unit for purposes of statistical evaluation. Differences in fetal weight, because it is a continuous variable, were analyzed with ANOVA and incidence data such as resorptions rate and number of malformations, were analyzed with the Kruskal– Wallis test. If a significant difference was detected, the Bonferroni or the Dunn’s tests were respectively used to compare the control group with each treated group. The significance level accepted for differences between doses was P ⬍ 0.05 for all tests. Although we have used some of the terminology contained in the work of Wise et al. [19], most of the terminology used in the description of developmental abnormalities conforms to the Nomina Anatomica and Nomina Embryologica Veterinaria [20] because we think it gives a better understanding of the mechanisms involved in the embryogenesis of the malformations.

3. Results No maternal effects resulting from the treatment were noticed. 3.1. Fetal mortality and fetal body weight

2.3. Observations On GD 20, pregnant rats were euthanatized with halothane, laparotomized and, after a necropsy to study the possible maternal effects resulting from the treatment, the number of resorptions and dead or live fetuses were recorded. All live fetuses were individually removed, sexed, weighed, euthanatized with halothane, and examined for gross external abnormalities. Approximately two-thirds of the live fetuses were randomly selected for skeletal examination using a clearing technique consisting of maceration in 1% KOH and staining

In the group treated with 70.7 mg/kg of NTB the rate of resorptions exceeded 50% and was significantly higher than in all the other groups (P ⬍ 0.02) (Table 1). Fetal body weight was significantly (P ⬍ 0.003) decreased in all treated groups compared with controls (Table 1). 3.2. External malformations No control fetuses were found with external defects. External malformations only appeared when the rats were treated with 59.5 mg/kg of NTB or more (Table 1) and were

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297

Table 1 Effects of NTB on fetal development in rats Dose (mg NTB/kg) Observations

0

50

59.5

70.7

No. litters No. implantations/litter No. resorptions/littera No. dead fetuses/litter No. live fetuses (per litter) Fetal weight (g ⫾ SE) % of fetuses with external malformations % of fetuses with skeletal malformations

6 11.5 3.8 0 66 (11.0) 5.57 ⫾ 0.05 0

6 10.5 2.4 0.2 60 (10.0) 5.06 ⫾ 0.05c 0

7 14.1 13.4 0 84 (12.0) 4.89 ⫾ 0.05c 13.1

6 13.2 58.1b 0 38 (6.3) 4.86 ⫾ 0.08c 13.1

14.2

52.9c

71.4c

68.0c

a

All resorptions were early. Significantly different from controls and from the 50 and 59.5 mg/kg doses. c Significantly different from controls. b

mostly tail anomalies and anal atresia. Tail anomalies consisted of tail agenesis and short and/or bent tail. Usually, anal atresia and tail agenesis were associated. 3.3. Skeletal malformations Fetuses with variation in shape of the ossification centers in the sternum and incomplete ossification of the skull and metacarpals were commonly seen at all doses, even in the unexposed group, and were classified as variations. Fourteen percent of the control fetuses were observed to have vertebral and rib anomalies. In contrast, the percentage of these malformations in treated groups was four or more times that of the control fetuses (P ⬍ 0.02). Although the incidence of affected fetuses did not increase dose dependently, the number of malformations per fetus tended to rise in response to the treatment (Table 2). Most of the vertebral abnormalities were located at the thoracic vertebrae. No vertebral defects were noted in the cervical region. The most common vertebral defect in all groups was the bilobulate vertebral body, being the only vertebral defect that appeared in the control group (Table 2). This anomaly is characterized by the incomplete fusion of the two chondrification centres of the vertebral body, either symmetrically (Fig. 1A) or asymmetrically (Fig. 1B). When there was not fusion between the two chondrification centres, and depending on their sizes, the vertebral malformation was classified as symmetric (Fig. 1C) or asymmetric (Fig. 1D) bilateral hemivertebra. If one of the chondrification centres of the vertebral body was not formed, the anomaly was called unilateral hemivertebra (Fig. 1E). The lack of chondrification centres was called vertebral body agenesis (Fig. 1F). Fused vertebrae were less common (Table 2) and affected either the body or the arch of the vertebrae. With respect to the vertebral body, we observed different types of fusion such as fused unilateral hemivertebrae (Fig. 1G), two vertebral bodies partially fused (Fig. 1H), or complete fusion of several vertebral bodies (Fig. 1I). At

least in two 59.5 and one 70.7 mg/kg treated fetuses, all from different litters, fusion of the vertebral arches (Fig. 1J) was accompanied by hypoplasia of these arches, thus producing spina bifida oculta in the thoracolumbar region. The short supernumerary ribs showed a pattern of appearance quite similar to that of the bilobulate vertebral bodies. Other kinds of rib malformations were only found in treated fetuses (Table 2). The main rib malformations were fusion and agenesis. The fused ribs were classified into different types (Table 2): dorsal (Fig. 2A) or dorsocentral (Figs. 2B,3C); central (Fig. 2C), that was the most common fusion; centroventral (Figs. 2D,3D) or ventral (Fig. 3E); complete (Fig. 2E), that was the less Table 2 Percentage of fetuses with vertebral and rib malformations produced by NTB Dose (mg NTB/kg) Observations Vertebral malformations Supernumerary thoracic vertebrae Bilobulate vertebral body Bilateral hemivertebra Unilateral hemivertebra Vertebral body agenesis Fused vertebral bodies Fused vertebral arches Rib malformations Short supernumerary ribs Short rib (no. 13) Fused ribs Rib agenesis Types of fused ribsa Dorsal or dorsocentral Central Centroventral or ventral Complete Multiple a

0

50

59.5

70.7

0.0 7.1 0.0 0.0 0.0 0.0 0.0

26.4 29.4 26.4 11.7 0.0 2.9 2.9

21.4 55.3 48.2 19.6 7.4 8.9 16.0

16.0 48.0 20.0 20.0 8.4 8.0 12.0

9.5 0.0 0.0 0.0

50.0 5.8 11.7 0.0

37.5 8.9 33.9 14.2

24.0 12.0 20.0 8.0

0.0 0.0 0.0 0.0 0.0

5.4 1.8 1.8 0.0 1.8

14.5 30.9 5.4 1.8 9.0

0.0 10.9 5.4 5.4 5.4

The percentage of the different types of fused ribs is expressed over the total number of fused ribs found.

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Fig. 1. Types of vertebral malformations observed in the fetuses treated with NTB. A. Symmetric bilobulate vertebral body. B. Asymmetric bilobulate vertebral body. C. Symmetric bilateral hemivertebra. D. Asymmetric bilateral hemivertebra. E. Unilateral hemivertebra. F. Vertebral body agenesis. G. Fused unilateral hemivertebrae. H. Vertebral bodies partially fused. I. Fusion of several vertebral bodies. J. Fused vertebral arches.

frequent fusion; and multiple fusion of more than two ribs (Fig. 2F). The rib agenesis could be partial (interrupted ossification) (Fig. 2B) or total (absent rib). The latter mainly affected the 13th rib and, similar to the vertebral body agenesis, only appeared in the groups treated with 59.5 mg/kg or more. 3.4. Vascular malformations All vascular anomalies were detected in the corrosion casts of the treated rat fetuses. However, because not all

the vascular casts were complete, the number of full corrosion casts examined was too low to be statistically analyzed (Table 3). The majority of the vascular malformations (10 cases), were observed in those fetuses from rats treated with NTB at 59.5 mg/kg. Only one cast from the group treated with the 50 mg/kg dose showed vascular anomalies. No fetus from the highest dose group had presented malformations, although the number of fetuses of this dose that could be examined was very low due to the high percentage of resorptions. Some of these anomalies, such as ectopic origins (Fig.

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299

Fig. 2. Types of fused ribs observed in the fetuses treated with NTB. A. Dorsal fused ribs. B. Dorso-central fused ribs. C. Central fused ribs. D. Centro-ventral fused ribs. E. Complete fused ribs. F. Multiple fused ribs.

3A) or duplications (Fig. 3F) were considered as variations. The vascular anomalies found in the intercostal vessels associated with rib malformations were characterized as vascular malformations. For instance, the anomalous dorsal fusion of an intercostal artery (Fig. 3B) or vein (Fig. 3C) appeared in relation to dorsal or dorsocentral fused ribs. Furthermore, when distal fusion of two ribs occurred, we found just one intercostal artery related to them (Figs. 3D,3E). Total (Fig. 3A) or partial (Figs. 3C,3E) agenesis of the intercostal vessels was related to fused ribs (Figs. 3A,3E) or to partial agenesis of the rib (Fig. 3C). We never found one rib, fused or not, associated with two intercostal vessels.

4. Discussion The results of this investigation confirm our previous findings in sheep [5] and indicate that NTB administered orally produces decreased fetal body weight and increased fetal mortality and incidence of malformations. The rate of resorptions was increased at the highest dose of NTB with respect to all the other groups. Although, differences among groups treated with different doses of NTB were not significant, an apparent dose dependently decrease of the fetal weight could be observed. External malformations, such as tail defects and anal atresia, were produced only at NTB doses of 59.5 mg/kg or

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Fig. 3. Vascular malformations observed in the fetuses treated with NTB. A. Ectopic origin (black arrows) and agenesis (white arrow) of the intercostal arteries. Aorta (A). Bar ⫽ 0.5 mm. B. Dorsal fused intercostal arteries (arrows). Aorta (A). Bar ⫽ 0.4 mm. C. Dorsal fused intercostal veins (arrows). One of them presents partial agenesis (black arrow) related to a pair of dorso-central fused ribs (*). Intercostal artery (a). Intercostal vein (v). Bar ⫽ 0.3 mm. D. Intercostal artery (a) related to a pair of centro-ventral fused ribs (*). Bar ⫽ 0.3 mm. E. Partial agenesis (arrow) of an intercostal artery related to a pair of ventral fused ribs (*). Bar ⫽ 0.5 mm. F. Duplication of the renal artery (stars). Bar ⫽ 0.6 mm.

more. Tail defects can be found as spontaneous variations in rats, although in a smaller percentage (0.4%) [21], and have been induced by treatment with albendazole [13] and other benzimidazoles, such as the flubendazole [14]. We have demonstrated in previous work that after oral administration of NTB in rats, ABZ concentration and not that of its metabolites significantly increases in plasma with dose [10]. Because ABZ is sequentially metabolized in the liver to ABZSO and ABZSO2 [1], the metabolic steps produced in the hepatic microsomal system are probably saturable with high doses of NTB, that probably explains the absence of dose-dependent differences. At the time of treatment, the development of cervical

vertebrae was apparently too advanced to be influenced by NTB. The most common vertebral anomaly was the bilobulate vertebral body, in accordance with other authors using ABZ and ABZ metabolites [9]. Together with this anomaly, supernumerary lumbar ribs were the only malformations that appeared in controls. The percentage of affected control fetuses was however clearly lower than in the treated groups. Supernumerary ribs can occur spontaneously in rats [21] and are classified as variations in many teratologic works. Nevertheless, the increased incidence of this rib defect seen in exposed fetuses may be taken as an indicator of possible teratogenic potential of the drug [22]. Moreover, in Sprague–Dawley rats, GD10 has been found to be the

M. Navarro et al. / Reproductive Toxicology 13 (1999) 295–302 Table 3 Vascular malformations associated with NTB treatment during gestation NTB dose (mg/kg/d) Observation

0

50

59.5

70.7

Successfully injected (% of total in dose group) Fetuses with vascular malformations Ectopic origin of intercostal vessel Fusion of intercostal vessels Agenesis of intercostal vessel Duplication of renal artery

14 (56)

12 (46)

16 (57)

6 (46)

0

1

9

0

0

1

2

0

0

0

4

0

0

0

4

0

0

0

1

0

sensitive period for lumbar rib induction [23] and, in fact, this malformation has been observed after treatment with some benzimidazolic compounds [9,24]. No clear relation between supernumerary ribs and thoracic vertebrae was found, as occurs with other teratogenic compounds in mice [25]. However, an evident correlation was seen between vertebral body and rib agenesis. Although the incidence of spina bifida was low and not significantly different from that of other groups, the fact that it was seen in three fetuses from different treated litters may indicate that it could be a treatment effect, that would be in accordance with the results obtained in sheep [6]. Congenital vascular anomalies commonly appear in the rat. However, except for some heart anomalies described by Mantovani et al. [13], no teratologic works with benzimidazolic compounds have studied vascular anomalies in this species. Although the number of vascular casts studied did not allow a significant conclusion, the fact that vascular malformations were only found in exposed groups suggests an effect of the treatment, and supports our previous findings in sheep [5]. Most of the vascular malformations produced by NTB were associated with skeletal malformations, as we have demonstrated in sheep [5]. Moreover, the close relationship between malformations of the ribs and their intercostal vessels is very striking. The present study demonstrates that the fusion between two ribs involves the agenesis of the intercostal vessels of one of them. We never found two intercostal vessels related to one fused or unfused rib. Therefore, the normal anatomic topography between the rib and its intercostal vessel is preserved even during dysmorphogenesis. There are no data available to support a mechanistic conclusion and, therefore, any discussion would be purely speculative. The intercostal arteries arise from the dorsal aorta and grow ventrally by angiogenesis, thus by vascular sprouts. Different authors have demonstrated that angiogenesis is induced by several angiogenic factors, such

301

as the vascular endothelial growth factor (VEGF) [26,27]. The striking relationship between rib and vascular malformations raises the possibility that some of these vascular growth factors could be expressed by cellular osseous populations. In fact, the expression and secretion of VEGF in osteoblast-like cells and bone tissue has been documented by several authors [28 –31]. We think that, more specifically, the osteoblasts of the caudal edge of the rib, that grow ventrally from the somites and are closely related to the endothelial cells of the intercostal vessels, could be implicated in the regulation of angiogenesis of these vessels. In this process, cellular division and migration are very important and a cytotoxic effect of ABZ and ABZSO [32, 33] due to inhibition of the polymerization of the mammalian tubulin has been demonstrated [6]. Supporting this idea, experiments that block the migration of neural crest cells, that partially form the aortic arches, led to a variety of arch malformations [34]. Our work was not designed as a mechanistic study although, we think that the antimitotic and antimigrating effect induced by NTB could produce cell death in the normal development of the ribs and intercostal vessels. We selected these malformations as the focus of this paper, although the other skeletal and external malformations suggest more widespread cytotoxic effects. The ectopic origin of the intercostal arteries, that we have classified as variations, could be due to the pressure of the malformed vertebral body against the artery [35]. Finally, we have demonstrated that the use of vascular corrosion casts studied by scanning electron microscopy, as we describe in a previous work [18], may be particularly helpful in observing the small arteries of rat fetuses and can be useful for secondary stage characterization of a substance known or suspected of inducing teratogenic vascular effects.

Acknowledgments This work was performed with the support of a CICYT (SAF92– 0470.7) grant from the Spanish Government, and a grant (CI1-CT94 – 0113) from the European Community.

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