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Pakistan J. Zool., vol. 43(3), pp. 511-521, 2011. Embryotoxicity of Orally Administered Misoprostol to Pregnant Mice Khadija Naeem*, Naveed Ahmad and Asmatullah Department of Zoology, University of the Punjab, Lahore, Pakistan Abstract.- Misoprostol (Prostaglandin E1) is an antiulcer drug. This study evaluated the embryotoxic effects of this medication in mice during organogenesis. The dose groups used were 0.02, 0.04, 0.06, 0.08 and 0.1 µg/g BW. The pregnant mice were exposed to these dose groups on days 8, 10 and 12 of gestation. Fetuses were recovered on day 18 of gestation and were subjected to morphological, morphometric, histological and skeletal studies. Morphological studies revealed defects like imbalanced axis, hydrocephaly, microphthalmia, cryptophthalmia, open eyelids, meromelia, forelimb micromelia, clinodactyly, syndactyly, subcutaneous hemorrhages and kinky tail. Moreover there was dose dependant reduction in litter size and increase in percentage of fetal resorptions. Histological deformities observed during this study included herniated lateral and fourth ventricle, underdeveloped retina, escaped ectoneural stem cells in fourth ventricle, poorly formed cochlea, nasal septum with atrophied inferior concha and hypoplasia in ventricular wall of heart. Study of skeletal parameter showed reduction in ossification in skull, ribs and limb region. Key words: Misoprostol, mice development, embryotoxicity. INTRODUCTION The incidence of pyrosis markedly increases during pregnancy (Castro, 1967; Marrero et al., 1992). The literature on peptic ulcer during pregnancy suffers from a preponderance of case reports and uncontrolled or poorly controlled studies. The incidence of peptic ulcers during pregnancy is estimated from case reports and retrospective clinical series. These estimates are unreliable (Cappell, 2003). Peptic ulcer disease is a chronic, recurrent problem with remission and exacerbation (Truelove, 1960). Peptic ulcers during pregnancy may be underdiagnosed because patients often treat themselves with nonprescription medications and do not seek medical attention for mild to moderate gastrointestinal symptoms. Physicians may attribute symptoms of abdominal pain, pyrosis or nausea and vomiting to gastroesophageal reflux disease or nausea and vomiting of pregnancy, more common condition during pregnancy (Singer and Brandt, 1991). Risk factors for peptic ulcer during pregnancy as well as in the general population include cigarette smoking, advanced age, NSAID use, alcoholism, genetic predisposition, gastritis and Halobacter pylori infection (Borum, 1998; Kurata, 1991). ______________________________ * Corresponding author: [email protected] 0030-9923/2011/0003-0511 $ 8.00/0 Copyright 2011 Zoological Society of Pakistan. The symptoms of peptic ulcers in pregnant women are similar to those in the general population and include epigastric pain, anorexia, postprandial nausea and vomiting, abdominal distention and eructation. Pain from a duodenal ulcer occurs several hours post prandially during the day, occurs nocturnally and is relived by eating or ingesting alkaline food (De Vore, 1980). In a study, eleven of twenty five (44%) women with chronic peptic ulcer disease experienced sudden remission of severe ulcer symptoms with the onset of pregnancy (Sandweiss et al., 1939). A study which was done with experimentally induced obstructive jaundice in Wistar albino rats concluded that administration of PGE1 provides protection against obstructive jaundice induced atrophy and damage of intestinal mucosa and thereby prevent translocation of enteric bacteria to underlying tissues (Gurleyik et al., 2006). Widespread use of nonsteroidal aintiinflammatory drugs can lead to a variety of symptoms of gastrointestinal injury. These drugs can cause damage to the gastrointestinal mucosa starting from esophagus to stomach, small bowel and large bowel gastrotoxicity is most common clinical manifestation of adverse effects of these drugs (Khokhar and Gill, 2003). Nonsteroidal antiinflammatory drugs block COX-I enzyme from forming beneficial prostaglandins such as PGE1. PGE1 plays a vital role in protecting the stomach and duodenum. Administration of synthetic prostaglandin E1 (Misoprostol) when metabolized 512 K. NAEEM ET AL. acts systemically to stimulate mucous production. A dose of 200µg of misoprostol also reduces gastric acid secretion (Lanas and Sopena, 2009). The optimal dose of misoprostol is 200µg two or three times a day for prevention of NSAID induced ulcers (Raskin et al., 1995). Misoprostol is widely used antiulcer agent in patients who are at high risk for nonsteroidal anti-inflammatory drugs (NSAID) mediated gastritis and ulcer disease (Numo, 1992). Misoprostol was also shown to induce uterine contractions when administered to pregnant women and causes abortion or preterm labor (Ngai et al., 1995; Wing et al., 1995). Misoprostol is used for a range of obstetric and gynecologic applications including first and second trimester abortion, treatment of miscarriage, induction of labor and prevention and management of postpartum hemorrhage (Blanchard et al., 2000; Broekhuizen, 2000; Goldberg et al., 2001). Misoprostol tablets are well absorbed after oral, sublingual, vaginal and rectal administration and are excreted in the colostrums in low concentration (Abdel-Aleem et al., 2003; Tang et al., 2002; Khan and El-Rafaey, 2003). In view of the above mentioned literature, the present study was designed and carried out in mice embryos because of their possible extrapolation in human applications. MATERIALS AND METHODS Sexually mature albino mice Mus musculus were used during this study. Females in proestrus stage were caged overnight with males of the same stock. The females were examined next morning. Presence of vaginal plug was taken as day 0 of gestation. These pregnant females were separated. A total of 60 pregnant females were used during this study. They were divided into a group of 10 for control and 10 for each of the five dose groups. Misoprostol Searle Pharmaceuticals Inc. was used because it is stable at room temperature (Ginath and Zakut, 2001). The drug is soluble in water. The doses were prepared by diluting a tablet of 200µg in distilled water in such a way that each 0.1ml of the solution contained desired dose. Five doses used during this experiment were 0.02, 0.04, 0.06, 0.08 and 0.1µg/g BW. The doses were applied orally on days 8, 10 and 12 of gestation. A vehicle treated control group was also maintained alongside which was treated with 0.1ml of distilled water. On day 18 of gestation, the treated dams were weighed and anaesthetized with anesthetic Ether. The dams were given midline incision in the abdomen and the uteri were exposed. The number of implantations and resorptions were recorded. The fetuses were removed from the uteri. These fetuses were wiped with tissue paper and weighed then were fixed in Bouin’s fluid for 48 hours. After 48 hours, fetuses were shifted to 70% ethanol. The morphological studies were done to record anomalies of craniofacial region, trunk, limbs, tail and axis. The fetuses were macrophotographed. The morphometric studies involved recording of fetal weight, crown rump length, head circumference, eye circumference, length of fore and hind limbs and tail length. The head and eye circumference values (p=mm2) were calculated for each fetus with the help of a computer based program the “Ellipse Circumference Calculator” downloaded from CSG Network (CSGN, 2006). The morphometric data were subjected to ANOVA by using SPSS software. The data collected for maternal and toxicity was also analyzed for ANOVA on SPSS software. The analysis was done on 95% confidence interval error of mean and levels of significance p<0.05, p<0.01 and p<0.001 For histological preparations selected fetuses from each dose groups were processed for paraffin sections which were stained with hematoxylin and eosin. For fetal skeletal preparation, the fetuses were preserved in 95% ethanol. These fetuses were eviscerated through a small abdominal incision and all thoraco-abdominal organs were removed. These eviscerated fetuses were placed in 2% KOH solution unless the flesh was completely decolorized and bones were clear. These were then placed in Alizarin Red S (1% aqueous solution made alkaline b 2-3 drops of 1% KOH) for 30 minutes. The deeply stained fetuses were placed in 1% KOH until the skeleton was clearly visible through surrounding tissue. These stained skeletons were further cleared in 20% glycerinated 1% KOH. The stained specimens were preserved in 50% ethanolic glycerol MISOPROSTOL TERATOGENICITY for microscopic studies and macrophotography (Kawamura et al., 1990). RESULTS During present study, maternal toxicity showed an increase in maternal weights of vehicle control and dose group 0.02µg/g but with a difference of (p<0.05) (Table I). However there was a reduction in maternal weights of dose groups 0.04 and 0.06 (p<0.01) and 0.08 and 0.1µg/g BW (p<0.001) (Table I). The study of fetal toxicity showed a significant reduction (p<0.001) in fetal litter size between vehicle control and the entire dose groups (Table II). Similarly a significant increase in (p<0.001) percentage of malformed fetuses was observed between vehicle control and all the dose groups (Table II). However, a significant increase (p<0.001) in percentage of resorptions was observed between vehicle control and dose groups 0.06, 0.08 and 0.1µg/g BW (Table II). Table I.- Effects of misoprostol on maternal weight after exposure on days 8, 10 and 12 of gestation. Dose groups Vehicle control (96)a 0.02µg/g BW (80)a 0.04µg/g BW (70)a 0.06µg/g BW (64)a 0.08µg/g BW (48)a 0.1µg/g BW (40)a Maternal weights Percentage Percentage increase decrease 513 present in 0.08µg/g BW, moreover microophthalmia was observed in 0.1µg/g BW. In dose groups 0.02 and 0.08µg/g BW, micrognathia was observed while agnathia was observed in 0.02µg/g BW. Forelimb micromelia was shown in dose groups 0.02 and 0.04µg/g BW, while low set limbs were observed in 0.02 and 0.06µg/g BW. Micromelia was manifested only in highest dose group 0.1µg/g BW, while clinodactyly was observed in 0.04µg/g BW and syndactyly was found in 0.08µg/g BW. Similarly, subcutaneous skin hemorrhages were found in 0.06 and 0.08µg/g BW dose groups. Kinky tail was found in 0.02 and 0.04µg/g BW dose groups (Table III). Table II.- Dose groups Vehicle control 0.02µg/g BW 0.04µg/g BW 0.06µg/g BW 0.08µg/g BW 0.1µg/g BW Fetal toxicity as a result of exposure to misoprostol on days 8, 10 and 12 of gestation. Litter size Percentage of malformed fetuses Resorption (%age) 96 0.00 0.00 80*** 12.52*** 70*** 64*** 48*** 40*** 33.33*** 47.22*** 64.28*** 72.22*** 0.00 0.00 4.74*** 18.54*** 47.22*** p<0.001*** 29.02 3.95* - 1.33** 6.97** 10.35*** 13.67*** a= No. of fetuses recovered p<0.05*, p<0.01**, p<0.001*** Morphological analysis revealed that the fetuses recovered from vehicle control group had well developed body organs like brain, eyes, snout, fore and hind limbs and tail (Fig. 1A). But among dose treated groups, imbalanced axis was observed in dose groups 0.02 and 0.08µg/g BW (Fig. 1B) (Table III). Moreover, hydrocephaly was observed in 0.08 µg/g BW (Fig. 1F) (Table III). Similarly, cryptophthalmia was observed in dose groups 0.02, 0.06 and 0.1 µg/g BW, while open eyelids were The morphometric observations during this study sowed a significant reduction (p<0.001) in weight, CR length, brain and eye circumferences and fore and hindlimb lengths as compared to vehicle treated control fetuses (Figs. 2-5). Histological studies of fetuses recovered from vehicle control group showed well developed brain, eye and heart. Within the brain, diencephalon, lateral, third and fourth ventricles pinna were found well develop (Fig. 6A). Moreover, well developed medulla oblongata, pinna and nasal cavity were also observed in vehicle control fetuses. Eyes were also well developed with well formed lens, cornea and retina. The eyelids were closed at this stage of development (Fig. 6B). The serial sections of the fetuses recovered from 0.1µg/g BW dose group showed herniated lateral and fourth ventricles. In eyes, the retina was poorly developed (Fig. 6C). 514 K. NAEEM ET AL. Fig. 1. Eighteen days old mice fetuses recovered from mothers exposed to different doses of Misoprostol on days 8, 10 and 12 of gestation. A, vehicle control; B, 0.02 µg/g BW; C and D, 0.04 µg/g BW; E, 0.06µg/g BW; F and G, 0.08µg/g BW; H, 0.1µg/g BW; B, well developed brain; E, well formed eyes with closed eye lids; F, well developed forelimbs; H, well developed hindlimb; u, cryptophthalmia, black arrow; forelimb micromelia, yellow arrow; kinky tail, green arrow; low set forelimb, grey arrow; hydrocephaly; sn, syndactyly; ru, resorbed uterus; mo, microophthalmia, purple arrow; micromelia. MISOPROSTOL TERATOGENICITY Table III.Parameter/ Dose groups 515 Morphological abnormalities induced in 18 days old fetuses recovered from pregnant mice treated with different dose (µg/g body weight) of Cytotec on days 8, 10 and 12 of gestation. Axis (%) Brain (%) Eyes (%) Snout (%) Limbs (%) Claws (%) Skin (%) Vehicle control (96)a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02µg/g (80)a Imbalanced axis (4.6) 0.00 Cryptophthalmia (2.5) Micrognathia (2.3) Agnathia (2.3) Fore limb micromelia (1.2), Low set forelimb (2.5) 0.00 0.00 Kinky tail (1.2) 0.04µg/g (70)a 0.00 0.00 0.00 0.00 Fore limb micromelia (1.4) Clinodactyly (7.1) 0.00 Kinky Tail (1.4) 0.06µg/g (64)a 0.00 0.00 Cryptophathalmia (13.04) 0.00 Low set fore limb (3.12) 0.00 Subcutaneous hemorrhage (13.4) 0.00 0.08µg/g (48)a Imbalanced axis (4.3) Hydrocephaly (4.17) Open Eyelids (4.17) Micrognathia (25) 0.00 Syndactyly (3.2) Subcutaneous hemorrhage (14.3) 0.00 0..1µg/g (40)a 0.00 0.00 Cryptophthalmia (11.1) Microophthamia (5) 0.00 Micromelia (5) 0.00 0.00 0.00 of fetuses recovered. Escaped ectoneual stem cells in the fourth ventricle were also observed. Along with these poorly formed cochlea and nasal septum with atrophied inferior concha were also taken into observation. 0.02 0.04 0.06 0.08 0.1 20 0.06 0.08 0.1 600 *** *** *** 15 *** 800 Fetal CR Length (mm S.E.) *** *** *** 1000 *** Fetal Weight (mg±S.E.) 1200 0.04 25 1600 1400 0.02 *** Control Control *** aNo. Tail (%) 10 400 5 200 0 Dose Groups (µg/g BW) 0 Dose Groups(µg/g BW) Fig 2. Effect of different doses of Cytotec on fetal weight of 18 days old mice fetuses recovered from the mothers treated on different days of gestation. Significant difference *** p<0.001 Fig. 3. Effect of different doses of Cytotec on fetal CR length of 18 days old mice fetuses recovered from the mothers treated on different days of gestation. Significant difference *** p<0.001 516 K. NAEEM ET AL. Control 0.02 0.04 0.06 0.08 0.1 *** *** *** *** 20 *** 15 *** *** *** 10 *** 5 *** Fetal Brain amd eye Circumference (mm±S.E.) 25 0 Dose Groups(µg/g BW) Fig. 4. Effect of different Cytotec on fetal brain circumference of 18 days old mice fetuses recovered from the mothers treated on different days of gestation. Significant difference *** p<0.001 Control 0.02 0.04 0.06 0.08 DISCUSSION 0.1 10 *** *** *** *** *** *** *** 7 *** 6 5 *** 4 *** Fetal Fore and hindlimb size (mm±S.E.) 9 8 development, three layers of heart walls i.e. epicardium, myocardium and endocardium were well developed. A well developed interventricular septum was also observed (Fig. 8A). The histological preparation of the fetus treated with 0.1µg/g BW dose showed ventricular thrombus in walls of heart. The study of skeletal parameter in the vehicle control fetuses showed well ossified axial and appendicular skeleton. Well ossified skull, vertebral column, ribs, limbs and digits were observed in skeletal formation of vehicle control fetuses. On the other hand skeletal preparation of the fetus recovered from 0.02µg/g BW dose administered group showed reduced ossification in skull and forelimb regions. Similarly, reduced ossification was observed in the skull and rib regions of the fetuses recovered from 0.04µg/g BW dose group (Fig. 8). 3 2 1 0 Dose Groups (µg/g BW) Fig. 5. Effects of Cytotec on Fetal forelimb size of 18 days old fetuses recovered from the mothers exposed with different doses (i.e. single, double, triple and chronic). Significant difference *** p<0.001 The observation of cardiac region in the serial section of the fetus from vehicle control group showed well developed chambers of heart i.e. two atria and two ventricles. At this stage of mouse Misoprostol is primarily used to prevent or treat gastrointestinal injury or blood loss related to nonsteroidal anti-inflammatory drugs (NSAIDs) ingestion. It is particularly helpful in patients with arthritis who have to continue taking NSAIDs despite suffering from peptic ulcer that refuses to respond to histamine blockers. The drug promotes healing of these ulcers and may also be effective in individuals who continue to smoke (Wilson, 1987, 1988). Misoprostol was the first synthetic prostaglandin analogue to be made available for the treatment of peptic ulcer. It has been licensed for this condition in over eighty countries (Clark et al., 2002). The recommended dosage for treatment of patients with benign gastric or duodenal ulcers is 200µg four times daily for at least four weeks (Monk and Clissold, 1987; Watkinson et al., 1988). In a study, 13 and 14 day rat embryos treated with misoprostol were maintained in culture medium. It was seen that crown rump of fetuses reduced from 9 to 7mm. Some embryos developed grossly visible hemorrhages in the ventral region of rhombencephalon and/ or rhomboncephalic ventricle. Histologic studies confirmed these ventricle hemorrhages (Shepard and Lemire, 1996). MISOPROSTOL TERATOGENICITY Fig. 6. Histological sections of 18 days old mouse fetuses from cranial and optic regions. A and B, transverse section from vehicle control; C and D, transverse sections from drug treated fetuses. Pinna (p); diencephalon (d); lateral ventricle (lv); choroids plexus within third ventricle (cp); medulla oblongata (mb); third ventricle (tv); cerebral hemisphere (ch); cochlea (c); lens (l); nasal cavity (nc); Herniated fourth ventricle ( pink arrow); poorly developed retina (brown notched arrow); herniated lateral ventricle (grey arrow); undifferentiated ectoneural stem cells (yellow arrow); poorly formed cochlea (blue arrow); nasal septum with atrophied inferior concha (gold arrow); 517 518 K. NAEEM ET AL. Fig. 7. Eighteen days old mouse fetuses through cardiac region. A, section of vehicle control fetus; B, sections of Drug treated fetuses. sc, spinal cord; lg, lungs; dm, dorsal muscles; lv, lumen of ventricle; iv, interventricular septum, off white arrow; ventricular thrombus of heart, pink arrow tissue necrosis of liver. A B C Fig. 8. Fetal skeletal preparation of 18 days old mice fetuses. A, vehicle control skeleton showing well ossified skeleton; B, skeletons form 0.02µg/g BW misoprostol treated fetuses showing reduced ossification in the skull and foreli, b regions; C, skeletons form 0.04µg/g BW misoprostol treated fetuses showing reduced ossification in the skull and rib regions. MISOPROSTOL TERATOGENICITY A systematic review was performed to estimate the risk of congenital anomalies and other adverse events in children exposed to misoprostol during fetal life. It was found that prenatal exposure to misoprostol is associated with an increased risk of Mobius sequence and terminal transverse limb defects (Dal Pizzol et al., 2006). In another study, it was found that women exposed to misoprostol presented a risk of anomalies that was 2.6 times greater than non-exposed women (Dal Pizzol et al., 2005). Qazi (2006) reported birth of a girl after failed attempt to pregnancy termination. The offspring was with multiple anomalies like scalp defects, protrusion of dura mater, microcephaly and equinovarus deformities. During present study hydrocephaly, cryptophthalmia, microphthalmia and open eyelids were observed. Dal Pizzol et al. (2008) presented a study in which birth defects associated with misoprostol included meningomyelocoele, microcephaly, club feet, syndactyly and fingernail defects. During present study clinodactyly and syndactyly were observed. A number of papers described a group of children who had limb anomalies and whose mothers had taken misoprostol as an abortifacient (Coelho et al., 2000; Genest et al., 1999). A number of limb anomalies were observed during present study which included forelimb micromelia, low set forelimbs and micromelia. In a number of studies, misoprostol have been found associated with Mobius syndrome (Gonzalez et al., 1993, 1998). A study demonstrated that analogues of prostaglandin E1 rioprostil and misoprostol cause anomalies including gastroschisis, umbilical hernia, spina bifida and caudal vertebral defects in case of rioprostil and umbilical hernia, omphalocoele and/or gastroschisis in case of misoprostol. Based on the pharmacological profile a plausible mechanism responsible for teratogenicity is altered uterine tone, uterine or umbilical vasotonia (Clements et al., 1992; 1997). CONCLUSIONS Present study was conducted on teratogenic potential of an antiulcer drug Misoprostol. The 519 findings of this study suggest that this drug exhibits a wide variety of morphological, morphometric, histological and skeletal abnormalities at different dose groups. The morphological abnormalities include microphthalmia, cryptophthalmia, open eyelids, micrognathia, agnathia, micromelia, clinodactyly, syndactyly, subcutaneous hemorrhages and kinky tail. Histological derangements include herniated third and lateral ventricle, poorly developed retina, undifferentiated ectoneural stem cells, poorly formed cochlea, nasal septum with atrophied inferior concha, ventricular thrombus of heart and tissue necrosis of liver. Moreover reduced ossification was observed during the study of skeletal abnormalities. On the basis of above mentioned findings it is suggested that misoprostol should be used with extreme care and it should be avoided altogether during pregnancy. ACKNOWLEDGEMENTS We are highly grateful to Department of Zoology, for providing space and facilities to conduct this research work. We are also obliged to Higher Education Commission of Pakistan for providing funding for this research work. 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A): 2-8. WILSON, D.E., 1988. Misoprostol and gastroduodenal mucosal protection (cytoprotection). Postgrad. med. J., 64(S1): 7-11. WING, F.A., RAHALL, A., JONES, M.M., GOODWIN, T.M. AND PAUL, R.H., 1995. Misoprostol: an effective agent for cervical ripening and labor induction. Am. J. Obstet. Gynecol., 172: 1811-1816. (Received 12 July, 2010, revised 1 October 2010) 522 1 K. NAEEM ET AL.