Developmental toxicity in rat fetuses exposed to the benzimidazole netobimin

Developmental toxicity in rat fetuses exposed to the benzimidazolenetobimin

Marc Navarroa, Lourdes Canutc, Ana Carreteroa, Carles Cristofolb,Fco–Javier Pe´rez–Aparicioa, Margarida Arboixb, Jesu´s Rubertea,*

aDept. of Anatomy and Embryology, Veterinary Faculty, Autonomous University of Barcelona, 08193 Bellaterra, Barcelona, SpainbDept. of Pharmacology and Toxicology, Veterinary Faculty, Autonomous University of Barcelona, 08193 Bellaterra, Spain

cLaboratory 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 veterinarymedicine. 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 anincrease in skeletal malformations were observed in treated groups. We report the first study in which vascular malformations are describedin rats after the administration of a benzimidazole compound. An interesting relationship between intercostal vessel and rib malformationswas 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 com-pound and mainly is used as a broad-spectrum anthelminticin veterinary medicine. Netobimin is cycled by gastrointes-tinal 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 inhuman medicine [3], for the treatment of gastrointestinalhelminthiases. In humans, it has shown evidence of goodefficacy against hydatid cysts [4]. We already have demon-strated the developmental toxicity of NTB in sheep embryos[5], the commonest species in which NTB is used as abroad-spectrum anthelmintic. Several studies suggest thatthe capacity of benzimidazole drugs to bind with cellularmicrotubules is responsible for the toxic effects on theembryo [6]. Once ABZ is absorbed, it is metabolized se-quentially to the sulphoxide (ABZSO) and sulphone(ABZSO2) of ABZ [7]. Both ABZ and ABZSO exhibit toxic

effects [8,9]. However, the significant correlation found inthe rat embryo between the rate of developmental toxicityand metabolite concentration demonstrate that the ABZSOconcentration could be the main factor accounting for tox-icity [10]. Furthermore, the transplacental movement ofABZ metabolites after the administration of NTB in ewessupports that these metabolites may be responsible for thedevelopmental toxicity of NTB [11].

Developmental toxicity has been shown in rats for benz-imidazole compounds such as albendazole, parbendazole orflubendazole [9,12–14], demonstrating that the rat is equallysensitive to the teratogenic effects of Benzimidazoles. How-ever, only a few investigations of the metabolism and phar-macology of NTB in rats can be found, and the descriptionof the developmental toxicity of NTB in this species israther scant [1,10].

The aim of this work was to study the developmentaltoxicity produced by NTB in rat fetuses. The study wasdesigned to characterize the malformations induced bythis compound, and to reproduce in an experimentalspecies the teratogenic effects induced by NTB in sheep[5]. Resorption rate, fetal weight and malformations werestudied. The investigation was focused on skeletal and

* Corresponding author. Tel.:193-581-20-06; fax:193-581-18-46.E-mail address:[email protected] (J. Ruberte)

Reproductive Toxicology 13 (1999) 295–302

0890-6238/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.PII: S0890-6238(99)00013-1

vascular anomalies, as well as the possible correlationbetween 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 proestruswere caged overnight with males of the same strain. Thepresence of sperm in a vaginal smear the following morningwas considered Gestation Day (GD) 0.

Dams were individually housed (Makrolon, 373 21.4318 cm with hardwood chip bedding) in the CIDA laboratoryin a room maintained at approximately 19–25°C, 40–60%relative humidity, and a 12 h light/dark cycle. Air changewas carried out 6–8 times per hour. Animals were fed acommercial diet (UAR A04C; Usine d’Alimentation Ratio-nelle, Villemoisson sur Orge, France) and had free access tofood and to tap water. Manipulations and experimentalprocedures were performed according to the OECD Princi-ples 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 (6or 7 rats/group). Three groups received NTB doses of 50,59.5, and 70.7 mg/kg body weight (Hapasil® suspension5%; Schering–Plough), in a volume of 5 mL/kg (accordingto the individual body weight of each dam), orally (gastricintubation) on GD 10. The control group was administeredthe same volume of vehicle (0.2% sodium carboxymethylcellulose in 1% Tween 80 in double distilled water). GD 10in rats is equivalent to the embryonic stage in ewes (17thday of pregnancy) that gives rise to developmental toxicityafter oral treatment with NTB [5]. Netobimin doses wereselected from previous studies [1], that showed fetal mal-formations when pregnant rats were treated with NTB dosesof 44.2 mg/kg or higher. We selected 50 mg/kg as the firstdose and the higher doses were obtained by a geometricprogression, multiplying by the square root of two.

2.3. Observations

On GD 20, pregnant rats were euthanatized with halo-thane, laparotomized and, after a necropsy to study thepossible maternal effects resulting from the treatment, thenumber of resorptions and dead or live fetuses were re-corded. All live fetuses were individually removed, sexed,weighed, euthanatized with halothane, and examined forgross external abnormalities.

Approximately two-thirds of the live fetuses were ran-domly selected for skeletal examination using a clearingtechnique consisting of maceration in 1% KOH and staining

with alizarin red S [15]. To determine vascular anomalies,the remaining fetuses were injected through the umbilicalartery to obtain casts of their vascular system. The umbilicalartery was dissected and cannulated with an angulated Pas-teur’s pipette. The pipette was fire polished to a gaugesimilar to the artery to be injected, and sealed to the vesselwith a drop of cyanocrylate (Loctite®) [16]. The resins usedfor corrosion casting were Araldite CY 223, hardener HY2967 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% methylmethacrylatemonomer [17].

After the polymerization process, fetuses were maceratedin 5% KOH. The casts thus obtained were dissected undera stereomicroscope, mounted on stubs, sputtered with gold,and observed in a Hitachi S-570 scanning electron micro-scope at an accelerating voltage of 8 to 10 KV (see 18 fordetails on the technique). All the cast were compared withthe normal arterial pattern of the GD 20 rat fetus [18].

The litter was considered the experimental unit for pur-poses of statistical evaluation. Differences in fetal weight,because it is a continuous variable, were analyzed withANOVA and incidence data such as resorptions rate andnumber of malformations, were analyzed with the Kruskal–Wallis test. If a significant difference was detected, theBonferroni or the Dunn’s tests were respectively used tocompare the control group with each treated group. Thesignificance level accepted for differences between doseswasP , 0.05 for all tests.

Although we have used some of the terminology con-tained in the work of Wise et al. [19], most of the termi-nology used in the description of developmental abnormal-ities conforms to the Nomina Anatomica and NominaEmbryologica Veterinaria [20] because we think it gives abetter understanding of the mechanisms involved in theembryogenesis of the malformations.

3. Results

No maternal effects resulting from the treatment werenoticed.

3.1. Fetal mortality and fetal body weight

In the group treated with 70.7 mg/kg of NTB the rate ofresorptions exceeded 50% and was significantly higher thanin all the other groups (P , 0.02) (Table 1). Fetal bodyweight was significantly (P , 0.003) decreased in all treatedgroups compared with controls (Table 1).

3.2. External malformations

No control fetuses were found with external defects.External malformations only appeared when the rats weretreated with 59.5 mg/kg of NTB or more (Table 1) and were

296 M. Navarro et al. / Reproductive Toxicology 13 (1999) 295–302

mostly tail anomalies and anal atresia. Tail anomalies con-sisted 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 centersin the sternum and incomplete ossification of the skull andmetacarpals were commonly seen at all doses, even in theunexposed group, and were classified as variations.

Fourteen percent of the control fetuses were observed tohave vertebral and rib anomalies. In contrast, the percentageof these malformations in treated groups was four or moretimes that of the control fetuses (P , 0.02). Although theincidence of affected fetuses did not increase dose depen-dently, the number of malformations per fetus tended to risein response to the treatment (Table 2).

Most of the vertebral abnormalities were located at thethoracic vertebrae. No vertebral defects were noted in thecervical region. The most common vertebral defect in allgroups was the bilobulate vertebral body, being the onlyvertebral defect that appeared in the control group (Table 2).This anomaly is characterized by the incomplete fusion ofthe two chondrification centres of the vertebral body, eithersymmetrically (Fig. 1A) or asymmetrically (Fig. 1B). Whenthere was not fusion between the two chondrification cen-tres, and depending on their sizes, the vertebral malforma-tion was classified as symmetric (Fig. 1C) or asymmetric(Fig. 1D) bilateral hemivertebra. If one of the chondrifica-tion centres of the vertebral body was not formed, theanomaly was called unilateral hemivertebra (Fig. 1E). Thelack of chondrification centres was called vertebral bodyagenesis (Fig. 1F).

Fused vertebrae were less common (Table 2) and af-fected either the body or the arch of the vertebrae. Withrespect to the vertebral body, we observed different typesof fusion such as fused unilateral hemivertebrae (Fig.1G), two vertebral bodies partially fused (Fig. 1H), orcomplete fusion of several vertebral bodies (Fig. 1I). At

least in two 59.5 and one 70.7 mg/kg treated fetuses, allfrom different litters, fusion of the vertebral arches (Fig.1J) was accompanied by hypoplasia of these arches, thusproducing spina bifida oculta in the thoracolumbar re-gion.

The short supernumerary ribs showed a pattern ofappearance quite similar to that of the bilobulate verte-bral bodies. Other kinds of rib malformations were onlyfound in treated fetuses (Table 2). The main rib malfor-mations were fusion and agenesis. The fused ribs wereclassified into different types (Table 2): dorsal (Fig. 2A)or dorsocentral (Figs. 2B,3C); central (Fig. 2C), that wasthe most common fusion; centroventral (Figs. 2D,3D) orventral (Fig. 3E); complete (Fig. 2E), that was the less

Table 1Effects of NTB on fetal development in rats

Observations

Dose (mg NTB/kg)

0 50 59.5 70.7

No. litters 6 6 7 6No. implantations/litter 11.5 10.5 14.1 13.2No. resorptions/littera 3.8 2.4 13.4 58.1b

No. dead fetuses/litter 0 0.2 0 0No. live fetuses (per litter) 66 (11.0) 60 (10.0) 84 (12.0) 38 (6.3)Fetal weight (g6 SE) 5.576 0.05 5.066 0.05c 4.896 0.05c 4.866 0.08c

% of fetuses with externalmalformations

0 0 13.1 13.1

% of fetuses with skeletal malformations 14.2 52.9c 71.4c 68.0c

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

Table 2Percentage of fetuses with vertebral and rib malformations produced byNTB

Observations

Dose (mg NTB/kg)

0 50 59.5 70.7

Vertebral malformationsSupernumerary thoracic vertebrae 0.0 26.4 21.4 16.0Bilobulate vertebral body 7.1 29.4 55.3 48.0Bilateral hemivertebra 0.0 26.4 48.2 20.0Unilateral hemivertebra 0.0 11.7 19.6 20.0Vertebral body agenesis 0.0 0.0 7.4 8.4Fused vertebral bodies 0.0 2.9 8.9 8.0Fused vertebral arches 0.0 2.9 16.0 12.0

Rib malformationsShort supernumerary ribs 9.5 50.0 37.5 24.0Short rib (no. 13) 0.0 5.8 8.9 12.0Fused ribs 0.0 11.7 33.9 20.0Rib agenesis 0.0 0.0 14.2 8.0

Types of fused ribsa

Dorsal or dorsocentral 0.0 5.4 14.5 0.0Central 0.0 1.8 30.9 10.9Centroventral or ventral 0.0 1.8 5.4 5.4Complete 0.0 0.0 1.8 5.4Multiple 0.0 1.8 9.0 5.4

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

297M. Navarro et al. / Reproductive Toxicology 13 (1999) 295–302

frequent fusion; and multiple fusion of more than tworibs (Fig. 2F). The rib agenesis could be partial (inter-rupted ossification) (Fig. 2B) or total (absent rib). Thelatter mainly affected the 13th rib and, similar to thevertebral body agenesis, only appeared in the groupstreated with 59.5 mg/kg or more.

3.4. Vascular malformations

All vascular anomalies were detected in the corrosioncasts of the treated rat fetuses. However, because not all

the vascular casts were complete, the number of fullcorrosion casts examined was too low to be statisticallyanalyzed (Table 3). The majority of the vascular malfor-mations (10 cases), were observed in those fetuses fromrats treated with NTB at 59.5 mg/kg. Only one cast fromthe group treated with the 50 mg/kg dose showed vascu-lar anomalies. No fetus from the highest dose group hadpresented malformations, although the number of fetusesof this dose that could be examined was very low due tothe high percentage of resorptions.

Some of these anomalies, such as ectopic origins (Fig.

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


3A) or duplications (Fig. 3F) were considered as varia-tions. The vascular anomalies found in the intercostalvessels associated with rib malformations were charac-terized as vascular malformations. For instance, theanomalous dorsal fusion of an intercostal artery (Fig. 3B)or vein (Fig. 3C) appeared in relation to dorsal or dor-socentral fused ribs. Furthermore, when distal fusion oftwo ribs occurred, we found just one intercostal arteryrelated to them (Figs. 3D,3E). Total (Fig. 3A) or partial(Figs. 3C,3E) agenesis of the intercostal vessels wasrelated to fused ribs (Figs. 3A,3E) or to partial agenesisof 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 previousfindings in sheep [5] and indicate that NTB administeredorally produces decreased fetal body weight and increasedfetal mortality and incidence of malformations. The rate ofresorptions was increased at the highest dose of NTB withrespect to all the other groups. Although, differences amonggroups treated with different doses of NTB were not signif-icant, an apparent dose dependently decrease of the fetalweight could be observed.

External malformations, such as tail defects and analatresia, were produced only at NTB doses of 59.5 mg/kg or

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-ventralfused ribs. E. Complete fused ribs. F. Multiple fused ribs.


more. Tail defects can be found as spontaneous variations inrats, although in a smaller percentage (0.4%) [21], and havebeen induced by treatment with albendazole [13] and otherbenzimidazoles, such as the flubendazole [14].

We have demonstrated in previous work that after oraladministration of NTB in rats, ABZ concentration and notthat of its metabolites significantly increases in plasma withdose [10]. Because ABZ is sequentially metabolized in theliver to ABZSO and ABZSO2 [1], the metabolic steps pro-duced in the hepatic microsomal system are probably satu-rable with high doses of NTB, that probably explains theabsence of dose-dependent differences.

At the time of treatment, the development of cervical

vertebrae was apparently too advanced to be influenced byNTB. The most common vertebral anomaly was the bilobu-late vertebral body, in accordance with other authors usingABZ and ABZ metabolites [9]. Together with this anomaly,supernumerary lumbar ribs were the only malformationsthat appeared in controls. The percentage of affected controlfetuses was however clearly lower than in the treatedgroups. Supernumerary ribs can occur spontaneously in rats[21] and are classified as variations in many teratologicworks. Nevertheless, the increased incidence of this ribdefect seen in exposed fetuses may be taken as an indicatorof possible teratogenic potential of the drug [22]. Moreover,in Sprague–Dawley rats, GD10 has been found to be the

Fig. 3. Vascular malformations observed in the fetuses treated with NTB. A. Ectopic origin (black arrows) and agenesis (white arrow) of the intercostalarteries. Aorta (A). Bar5 0.5 mm. B. Dorsal fused intercostal arteries (arrows). Aorta (A). Bar5 0.4 mm. C. Dorsal fused intercostal veins (arrows). Oneof them presents partial agenesis (black arrow) related to a pair of dorso-central fused ribs (*). Intercostal artery (a). Intercostal vein (v). Bar5 0.3 mm. D.Intercostal artery (a) related to a pair of centro-ventral fused ribs (*). Bar5 0.3 mm. E. Partial agenesis (arrow) of an intercostal artery related to a pair ofventral fused ribs (*). Bar5 0.5 mm. F. Duplication of the renal artery (stars). Bar5 0.6 mm.


sensitive period for lumbar rib induction [23] and, in fact,this malformation has been observed after treatment withsome benzimidazolic compounds [9,24]. No clear relationbetween supernumerary ribs and thoracic vertebrae wasfound, as occurs with other teratogenic compounds in mice[25]. However, an evident correlation was seen betweenvertebral body and rib agenesis. Although the incidence ofspina bifida was low and not significantly different from thatof other groups, the fact that it was seen in three fetusesfrom different treated litters may indicate that it could be atreatment effect, that would be in accordance with the re-sults obtained in sheep [6].

Congenital vascular anomalies commonly appear in therat. However, except for some heart anomalies described byMantovani et al. [13], no teratologic works with benzi-midazolic compounds have studied vascular anomalies inthis species. Although the number of vascular casts studieddid not allow a significant conclusion, the fact that vascularmalformations were only found in exposed groups suggestsan effect of the treatment, and supports our previous find-ings in sheep [5].

Most of the vascular malformations produced by NTBwere associated with skeletal malformations, as we havedemonstrated in sheep [5]. Moreover, the close relationshipbetween malformations of the ribs and their intercostalvessels is very striking. The present study demonstrates thatthe fusion between two ribs involves the agenesis of theintercostal vessels of one of them. We never found twointercostal vessels related to one fused or unfused rib.Therefore, the normal anatomic topography between the riband its intercostal vessel is preserved even during dysmor-phogenesis. There are no data available to support a mech-anistic conclusion and, therefore, any discussion would bepurely speculative. The intercostal arteries arise from thedorsal aorta and grow ventrally by angiogenesis, thus byvascular sprouts. Different authors have demonstrated thatangiogenesis is induced by several angiogenic factors, such

as the vascular endothelial growth factor (VEGF) [26,27].The striking relationship between rib and vascular malfor-mations raises the possibility that some of these vasculargrowth factors could be expressed by cellular osseous pop-ulations. In fact, the expression and secretion of VEGF inosteoblast-like cells and bone tissue has been documentedby several authors [28–31]. We think that, more specifi-cally, the osteoblasts of the caudal edge of the rib, that growventrally from the somites and are closely related to theendothelial cells of the intercostal vessels, could be impli-cated in the regulation of angiogenesis of these vessels.

In this process, cellular division and migration are veryimportant and a cytotoxic effect of ABZ and ABZSO [32,33] due to inhibition of the polymerization of the mamma-lian 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 archmalformations [34]. Our work was not designed as a mech-anistic study although, we think that the antimitotic andantimigrating effect induced by NTB could produce celldeath in the normal development of the ribs and intercostalvessels. We selected these malformations as the focus ofthis paper, although the other skeletal and external malfor-mations suggest more widespread cytotoxic effects.

The ectopic origin of the intercostal arteries, that we haveclassified as variations, could be due to the pressure of themalformed vertebral body against the artery [35].

Finally, we have demonstrated that the use of vascularcorrosion casts studied by scanning electron microscopy, aswe describe in a previous work [18], may be particularlyhelpful in observing the small arteries of rat fetuses and canbe useful for secondary stage characterization of a substanceknown or suspected of inducing teratogenic vascular ef-fects.

Acknowledgments

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

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Table 3Vascular malformations associated with NTB treatment during gestation

Observation

NTB dose (mg/kg/d)

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Successfully injected(% of total indose group)

14 (56) 12 (46) 16 (57) 6 (46)

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Fusion ofintercostal vessels

0 0 4 0

Agenesis ofintercostal vessel

0 0 4 0

Duplication ofrenal artery

0 0 1 0


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Developmental toxicity in rat fetuses exposed to the benzimidazole netobimin