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Toxicon 51 (2008) 316319 www.elsevier.com/locate/toxicon

Short communication

Teratogenicity of Mimosa tenuiora seeds to pregnant rats$

R.M.T. Medeiros, A.P.M. de Figueiredo, T.M.A. Ben cio, F.P.M. Dantas, F. Riet-Correa
rio, CSTR, Universidade Federal de Campina Grande, Campus de Patos, Patos, PB, Paraba, Brazil Hospital Veterina Received 2 April 2007; received in revised form 16 June 2007; accepted 18 June 2007 Available online 26 June 2007

Abstract Bone and other malformations caused by the ingestion of Mimosa tenuiora are common in ruminants in the Brazilian semiarid. The aim of this research was to study the teratogenic effects of M. tenuiora in Wistar rats (Rattus novergicus). The experimental group had 15 females and was fed, from the 6th to the 21st day of pregnancy, with a ration containing 10% of M. tenuiora seeds. The control group, with 10 females, was fed with the same ration without seeds. There were no differences in weight gains, and food and water consumption between treated and control rats. Ninety bone malformations were observed in 40 of the 101 fetuses born in the experimental group, and four malformations were observed in three of the 58 fetuses born in the control group (po0.05). The weight and number of ossication centers of the fetuses from the experimental group were higher than those from the control group (po0.05). It is concluded that the seeds of M. tenuiora are teratogenic to rat fetuses. r 2007 Published by Elsevier Ltd.
Keywords: Rats; Malformations; Teratogenic plants; Mimosa tenuiora

Bone and other malformations are very common in the Brazilian semiarid in goats (Riet-Correa et al., 2004) and sheep (Nobrega et al., 2005). The main defects are permanent exure of the forelimbs, craniofacial anomalies including cleft palate, brachygnathia, agnathia, cleft lip due to unilateral or bilateral aplasia or hypoplasia of the incisive bones, and eye malformations including microphtalmia,

$ Ethical statement: The experimental procedures used in the experiments reported in this paper were performed following the legal and ethical national requirements of the Federal University of Campina Grande. Corresponding author. Tel.: +558334233409; fax: +558334214659. E-mail address: rmtmed@uol.com.br (R.M.T. Medeiros).

ocular dermoids, and corneal opacity. Other malformations are kyphosis, scoliosis, lordosis, cranioschisis, segmental stenosis of the colon, and meningocele. Many animals had multiple malformations, mainly in the head and legs. Malformations are also observed in cattle, but are less frequent than in small ruminants (Riet-Correa et al., 2006). Some of those malformations were induced experimentally in pregnant goats ingesting Mimosa tenuiora, suggesting that this plant is the cause of such anomalies (Pimentel et al., 2007). The aim of this paper was to study the effects of seeds of M. tenuiora in pregnant rats (Rattus novergicus) and to verify whether this species can be used as an experimental model to study the embryotoxicity and fetotoxicity of the plant and its active principle.

0041-0101/$ - see front matter r 2007 Published by Elsevier Ltd. doi:10.1016/j.toxicon.2007.06.012

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Twenty-ve Wistar pregnant female rats were divided into two groups. The experimental group with 15 females, was fed with a ration containing 10% of M. tenuiora seeds. The control group, with 10 females, was fed with the same food without seeds of M. tenuiora. The rats were housed in 40 50 20 cm plastic cages, under controlled temperature (2226 1C), in a natural cycle of light. After the conrmation of pregnancy, the females were maintained alone in the cage until the 21st day of pregnancy. The seeds of M. tenuiora were collected in the municipality of Patos, state of Para ba. The ration for the experimental group was prepared with 80% commercial pelleted rat food (LABINAs), which was ground and mixed with 10% ground M. tenuiora seeds and 10% corn starch in hot water. After mixing the seeds and ration with the starch, pellets were made by hand and dried in the shade. The food for the control group was prepared in the same way, but with 90% rat commercial food and 10% corn starch. For mating, one male and two females were allotted in a cage during 1214 h, from evening to the following morning. Females with evidence of mating (vaginal plug or vaginal smear with sperm cells) were assigned to one of the two groups. Females that failed to become pregnant by two consecutive times were rejected. Food consumption, water ingestion, and body weight were veried on days 6, 10, 14, and 21. On the 21st day of pregnancy, the rats were anesthetized by ether inhalation and the ovaries and uteri were removed by cesarean section. The number of corpora lutea in each ovary was recorded and the gravid uterus was weighed. The fetuses were removed from the uteri, dried of amniotic uid, weighed and examined for conformation of the eyes, mouth, head, limbs, tail, and ears, and the presence of the anal perforation, with the aim of verifying external abnormalities and malformations. The placentas and the live fetuses were weighed. The number of implantation sites and resorptions was recorded in both uterine horns. After being weighed, the fetuses were euthanatized with ether, xed in acetone for 24 h, examined for cleft palate, and eviscerated. For examination of the skeleton, the fetuses were submersed in a solution of 0.8% potassium hydroxide with alizarin-red S, which was changed daily during 34 days (Staples and Schenell, 1964). Then the fetuses were cleared in a solution of 40% ethylic alcohol, 40% glycerin, and

20% benzylic alcohol. The degree of fetal bone development was evaluated by counting the ossication centers in some fetal bones (phalanges of the fore limbs, metacarpus, metatarsus, sternebrae, and caudal vertebrae). The kidney, lung, and liver of the fetuses were weighed. The rats were euthanized and necropsied. Lung, liver, heart, and kidneys were weighed and samples of these tissues were xed in 10% buffered formalin, embedded in parafn, sectioned at 5 mm, and stained by hematoxylin and eosin for histological examination. The software GraphPad Instat V2.01 (Graphpad, 1993) was used for the statistical analysis. Food consumption, water ingestion, body weight gains, organ weight, and date of the offspring were analyzed by the Student t test. Frequency of skeletal abnormalities and malformations were evaluated by Fishers exact test. The percentages of pre- and post-implantation losses and the degree of ossication were submitted to the Mann Whitney U test. The intake of experimental ration containing M. tenuiora seeds was well tolerated. No signs of sickness or abnormal behavior were observed. None of the females aborted, delivered prematurely, or died during the experiment. Maternal food and water consumption during gestation, and mean weight gains were similar in the treated and control groups (Table 1). Liver and heart weights of the dam from the experimental group were signicantly lower (po0.05) than those of the rats from the control group (Table 2). No gross or histologic lesions were observed in the liver, kidney, heart, and lung of rats from both groups. No differences were found between liver, heart, and kidney weights of the fetuses from both groups. Reproductive data are summarized in Table 3. No dead fetuses were observed. No statistical differences were observed in the number of corpora lutea, resorptions and implants, and placental weight. Gravid uterus weight and mean fetal weight
Table 1 Food and water consumption, and weight gains (mean7SD), from the 6th to the 21st day of gestation, of rats from the M. tenuiora group and control group Control group Food consumption (g) Water consumption (g) Weight gains (g) 251.54723.96 486.507118.8 63.074715.89 M. tenuiora group 268.17751.72 476.687101.10 60.03719.33

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318 R.M.T. Medeiros et al. / Toxicon 51 (2008) 316319 Table 4 Number and percentage of skeletal malformations in 101 fetuses from rats of the M. tenuiora group and 58 fetuses of the control group Malformation Lung Heart Liver Right kidney Left kidney 1.1170.19 0.7270.08 8.8770.76 0.5770.05 0.5070.06 0.9970.17 0.6370.09** 7.7071.19** 0.5270.12 0.5370.07 Fetuses affected in the control group Fetuses affected in the M. tenuiora group 33 (32.67%)* 9 (8.9%)* 1 (0.99%) 3 (2.97%) 0 (0%) 1 (0.99%) 8 (7.92%)* 2 (1.98%) 1 (0.99%) 1 (0.99%) 14 (13.86%)* Table 2 Weight (mean in grams7SD) of the lung, heart, liver, and kidney of rats from the M. tenuiora group and control group Organ Control group M. tenuiora group

Signicantly different (**P40.001) from the control group.

Table 3 Reproductive performance in rats from the M. tenuiora group and control group Control group (mean7SD) M. tenuiora group (mean7SD) 48.60710.59* 9.3372.25 4.3470.79** 0.3970.05 11.7672.35 0.6670.47 12.61723.32 7.98713.97

Gravid uterus weight (g) Number of fetuses Weight of the fetuses (g) Weight of the placenta (g) Number of corpora lutea Number of resorptions Embryo losses preimplantation Embryo losses postimplantation

47.37719.32 8.8073.55 3.7070.58 0.4170.09 12.7071.49 0.6070.96 21.61726.05 7.94715.49

Cleft palate Scoliosis Bid sternum Aplasia of one sternebra Aplasia of two sternebrae Aplasia of three sternebrae Hypoplastic sternebrae Disarranged sternebrae Hypoplasia of the ischium Hypoplasia of the femur Concavity of the parietal, interparietal and supraoccipital bones Hypoplasia of the orbit (microphtalmia) Hypoplasia of the nasal bone Deformed occipital bone Number of fetuses with malformations

0 0 0 1 (1.72%) 1 (1.72%) 0 0 1 (1.72%) 0 0 0

0 0 1 (1.72%) 4

3 (2.97%) 8 (7.92%)* 0 (0%) 90

Signicantly different (*Po0.05) from the control group.

Signicantly different (*P40.01) and (**P40.001) from the control group.

Table 5 Number of ossication centers (mean7SD) in fetuses from the M. tenuiora group and fetuses from the control group Bones Phalanges of the fore limbs Metacarpus Metatarsus Sternebrae Caudal vertebrae Control group 0.1670.54 (n 50) 3.0870.27 3.9270.56 5.9370.31 3.3470.84 (n 50) (n 50) (n 49) (n 50) Treated group 1.4571.50** (n 95) 3.8170.39* (n 95) 4.2670.44** (n 96) 5.9370.35 (n 95) 4.1470.90** (n 97)

of the group fed with M. tenuiora seeds were signicantly heavier than those from the control group (po0.05). Some fetuses of the treated group had evident bone malformations including scoliosis, lordosis, and a shorter head. Ninety bone malformations were observed in 40 of the 101 fetuses born in the experimental group, and four malformations were observed in three of the 58 fetuses born in the control group (po0.05). Results of bone anomalies in fetuses from the treated and control groups are presented in Table 4. Results in the number of ossication centers found in fetuses from both groups are presented in Table 5. The observation of different malformations in fetuses from rats fed with 10% of seeds of M. tenuiora during pregnancy demonstrated that the plant is teratogenic, suggesting that it caused the malformations observed in Northeaster Brazil in ruminants (Riet-Correa et al., 2004, 2006; Nobrega et al., 2005). The similar food and water consump-

n Number of fetuses examined; *P40.01; **P40.001.

tion and weight gains in the rats from the group fed with M. tenuiora and in rats from the control group suggest that the plant is not toxic for the dam. Despite the fact of causing malformation the plant did not affect the fetal development. By contrast, the fetal weight and the number of ossication centers were higher in the fetuses from rats fed with M. tenuiora seeds than in fetuses from the control group.

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M. tenuiora is a xerophilous tree very common in degraded areas in the semiarid region, where sometimes it is the only forage available. The teratogenic effect of this plant was also demonstrated in pregnant goats (Pimentel et al., 2007). M. tenuiora is particularly rich in tannins (Guimaraes-Beelen et al., 2006), and contains triterpenoidal and steroidal saponins (Jiang et al., 1991a, b) and indolic alkaloids (Meckes et al., 1990). The cortex of this plant is a popular remedy utilized in Mexico for the treatment of skin lesions, and the dried and powdered bark applied directly on the lesion is an effective remedy to treat skin burns and wounds (Rivera-Arce et al., 2007). The susceptibility of pregnant Wistar rats to the teratogenic effect of M. tenuiora suggests that this species can be used as an experimental model to study the embryotoxicity and fetotoxicity of the plant and its active principle. This work was nancially supported by Institutos do Milenio, National Council of Scientic and Technologic Development (CNPq), Grant No. 420012/2005-2 and by the Programa de Apoio a Nucleos de Excelencia (PRONEX), Grant No. 001/ 04, CNPq, FAPESQ, MCT. References
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and ruminal enzymatic activity in Saanen goats. Small Rum. Res. 61, 3544. GraphPad Software, 1993. GraphPad Instat V2.01. GraphPad Software, San Diego, (1 computer disk, 31in, IBM). 2 Jiang, Y., Massiot, G., Lavaud, C., Teulon, J.M., Guechot, C., Haag, M., Anton, R., 1991a. Triterpenoid glycosides from the bark of Mimosa tenuiora. Phytochemistry 30, 23572360. Jiang, Y., Massiot, G., Lavaud, C., Teulon, J.M., Guechot, C., Haag, M., Anton, R., 1991b. Structure of a new saponin from the bark of Mimosa tenuiora. J. Nat. Prod. 54, 12471253. Meckes, M., Lozoya, X., Gonzalez, L., Mart nez, M., 1990. Efecto producido por la fraccion de alcaloides de Mimosa tenuiora (tepescohuite) sobre el reejo peristaltico del leon del cobayo. Arch. Invest. Med. 21, 171174. Nobrega Jr., J.E., Riet-Correa, F., Nobrega, R.S., Medeiros, J.M., Vasconcelos, J.S., Simoes, S.V.D., Tabosa, I.M., 2005. Mortalidade perinatal de ovinos no semi-arido da Para ba. Pesq. Vet. Bras. 25, 171178. Pimentel, L.A., Riet-Correa, F., Gardner, D., Panter, K.E., Dantas, A.F.M., Medeiros, R.M.T., Mota, R.A., Araujo, J.A.S., 2007. Mimosa tenuiora as a cause of malformations in ruminants in the northeastern Brazilian semiarid rangelands. Vet. Pathol. 44, 928931. Riet-Correa, F., Medeiros, R.M., Neto, S.A., Tabosa, I.M., Pimentel, L.A., 2004. Malformacoes osseas em caprinos na - regiao semi-arida do nordeste do Brasil. Pesq. Vet. Bras. 24 (Supl), 4549. Riet-Correa, F., Medeiros, R.M.T., Dantas, A.F.M., 2006. Plantas Toxicas da Para ba. Centro de Saude e Tecnologia Rural, Patos, PB, SEBRAE/PB, 58p. Rivera-Arce, E., Chavez-Soto, M.A., Herrera-Arellano, A., Arzate, S., Aguero, J., Feria-Romero, I.A., Cruz-Guzman, A., Lozoya, X., 2007. Therapeutic effectiveness of a Mimosa tenuiora cortex extract in venous leg ulceration treatment. J. Ethnopharmacol. 109, 523528. Staples, R.E., Schenell, V.L., 1964. Renements in rapid clearing technique in the KOH-alizarin red S method for fetal bone. Stain Technol. 39, 6163.