Download Title: Embryotoxicity of Orally Administered Misoprostol to Pregnant

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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(Received 12 July, 2010, revised 1 October 2010)
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