DEVELOPMENTAL TOXICITY OF IBUPROFEN TREATED MICE

Abstract

The effect of ibuprofen – an anti-inflammatory, analgesic drug was tested on pregnant females at different developmental phases. The fetus was collected on 12th, 15th and 18th gestation days (gd). Embryos on the day of parturition and 5 day old new born were considered for experimental study. Ovary and uterus of the mother was also taken into account for histopathological examination. The adverse effects on pregnancy outcome included a significant reduction in the number of implantation and significant increase in percentage of post implantation loss. The body weight as well as physical characteristics varied due to the treatment at different phases of gestation. The concentrations of brain DNA and RNA of the fetus and the pups were also lower compared to controls. A major change in the histological architecture was seen in the sections of ovary and uterus of the mother. These findings suggest the susceptibility of the drug to the mother as well as to the embryo.

Full text

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Research article
DEVELOPMENTAL TOXICITY OF IBUPROFEN TREATED MICE
C.R. SAHU
Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India
Received- 03 March 09, Revised and Accepted- 28 March 09

ABSTRACT
The effect of ibuprofen – an anti-inflammatory, analgesic drug was tested on pregnant
females at different developmental phases. The fetus was collected on 12th, 15th and 18th
gestation days (gd). Embryos on the day of parturition and 5 day old new born were
considered for experimental study. Ovary and uterus of the mother was also taken into
account for histopathological examination. The adverse effects on pregnancy outcome
included a significant reduction in the number of implantation and significant increase in
percentage of post implantation loss. The body weight as well as physical characteristics
varied due to the treatment at different phases of gestation. The concentrations of brain
DNA and RNA of the fetus and the pups were also lower compared to controls. A major
change in the histological architecture was seen in the sections of ovary and uterus of the
mother. These findings suggest the susceptibility of the drug to the mother as well as to
the embryo.
Key Words: Ibuprofen, Fetus, Histopathology, DNA and RNA
INTRODUCTION
A number of reports suggest that non-
steroidal anti-inflammatory drugs
(NSAID) have been used primarily for
the treatment of rheumatic disease,
musculoskeletal disorders, dysmenorrhoea,
inflammation and for post-operative
pain1-3. Besides being anti-inflammatory
these drugs are analgesic and anti-
pyretic. More than 100 million NSAIDs
are prescribed throughout the world4.
The chemical class of 2-arylpropionate
to which ibuprofen belongs is now being
widely used as largest single group of
NSAID. Nevertheless, ibuprofen has
also been associated with gastric lesions
and bleeding, which is due to the
inhibition of COX, the enzyme which is
responsible for synthesis of prostaglandin5,6.
More recently another group of NSAID,
a non-specific COX inhibitor, have gained
attention as an effective therapy for
tumor patient, against the incidence and
mortality of colorectal cancer and
reported to potentates the anti-cancer
effects of cisplatin on human invasive
bladder cancer7. Prior toxicological
studies on ibuprofen were limited to rats

International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 1, Issue 1, July-Sep. 2009

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and dogs with repeated dose toxicity and
the side effects of the drug thus showed
gastrointestinal lesions. Drug developmental
effects after triple daily doses
administration of ibuprofen showed
developmental anomalies in rats8.
Though the drugs enter the fetal
circulation, it does not show any
teratogenic activity but influence to
change in organ weight in rats. The
present studies were conducted to
examine the foetotoxic potential of
ibuprofen in terms of the estimates of
the dimension of each developmental
period as well as measurements of the
content of nucleic acid in the brain of
mice. Microscopic examination of the
reproductive tissues particularly the
ovary and the uterus were used to
characterize the effects of the drug on
the reproductive parts.
MATERIALS AND METHODS
Animals
Sexually matured laboratory mice (Mus
musculus) were housed in cages and
received food and water ad libitum. The
animal rooms were maintained at 22ºC
to 25ºC and a relative humidity of 50 to
60 % with 12 h light and dark cycles.
Female mice were allowed to mate to
adult males of proven fertility overnight
and were separated the following
morning. Insemination was determined
by the presence of sperm in the vaginal
smears taken at the time of separation.
The day on which sperm were found
designated as day-0, and the female was
placed in an individual cage at that time.
The experiments have been conducted
with due permission from Institutional
Animal Ethics Committee (IAEC)
formulated through CPCSEA.
Experimental designs
As per therapeutic doses pregnant
females were fed 100 mg of ibuprofen
(Boots India Ltd.)/ Kg of body weight,
at different developmental phases
separately according to the following
schedule.
Phase
Pre-implantation (PI)
Gestation
1-4
Early Placental (EP) 5-10
Placental (PL) 11-19
The control set of 21 to 24 mice was
similarly administered distilled water.
The animals were sacrificed on 12th, 15th
and 18th day of gestation to collect fetus.
The embryos on the day of parturition
and 5 day old new born were also taken
into consideration.
Maternal and fetal observation
Animals were observed daily for clinical
sign of toxicity. On specific gd. mice
were killed by cervical dislocation.
Uterine contents (i.e. the number of
implantation sites, resorptions, dead and
live fetuses) were evaluated.

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Assay procedure
Nucleic acid extraction was done
following the method of Geel &
Timirus9. DNA was assayed
colorimetrically according to Burton
Diphenyl procedure10 using calf thymus
DNA (Sigma) for the standard. RNA
was assayed colorimetrically according
to Ceriotti’s orcinol procedure11 using
yeast RNA (Sigma) for the standard.
Histopathological examination
The ovary and uterus were fixed for
histological examination. Tissues were
trimmed, embedded in paraffin,
sectioned and stained with Hematoxylene
and Eosin. Representative cross sections
were then examined for evidence of
pathological changes.
Statistical evaluation
Statistical evaluation of quantitative data
relative to fetus and pups (body weight,
nucleic acid concentration) were performed
using ANOVA and the means were
compared using Student’s ‘t’ test.
Probability test (∝ = 0.05) was used for
pair-wise comparison between each ibuprofen
treated group and the control group.
RESULTS
General reproductive performance
No maternal death or distinctive clinical
sign were observed during the period
under study. The results of the general
reproductive performance are summarized
in Table 1. Treatment related adverse
effects on pregnancy outcome included
a significant reduction in the number of
implantation sites for litter both in PI
and EP phase (Table 1).
Table 1 : Table shows developmental toxicity in mice following maternal exposure
to ibuprofen
Days
18
91.3
21/23
13.77±0.40
Control
12
87.5
21/24
13.20±0.10
15
95.6
22/23
14.40±0.50
0 5
% pregnant
(No. preg. / No. mated )
Average No. of
implantation sites/dam
%post-implantation
loss/dam
Average No. of life
fetuses/dam
91.7
22/24
13.83±0.40
92.9
24/26
14.01±1.10
0.30±0.02 0.32± 0.02 0.30±0.03 0.31±0.01 0.31±0.04
12.9±1.2 11.9±1.5 11.3±1.3 11.7±1.2 12.0±1.4
Pre-implantation
94.7
18/19
5.87±0.02
% pregnant (No.preg./
No. mated
Average No. of
implantation sites/dam
Average No. of life
fetuses/dam
% post-implantation
loss/dam
88.2
15/17
5.47±0.02
90.0
18/20
5.58±0.02
94.4
17/18
5.85±0.02
95.0
19/20
5.89±0.02
NA NA NA NA NA
99.99 98.99 98.99 99.00 99.00

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Early placental
95.0
19/20
6.01±0.13
% pregnant
(No.preg./No. mated
Average No. of
implantation sites/dam
% post-implantation
loss/dam
Average No. of live
fetus/dam
89.47
17/19
6.00±0.13
94.4
17/18
5.98±0.13
90.0
18/20
5.70±0.13
95.4
21/22
6.54±0.13
11.10±0.23 13.04±0.27 18.18±0.19 18.50±0.23 23.07±0.l2
5.32±0.28 5.19±0.32 4.90±0.35 4.89±0.49 4.61±0.51
Placental
% pregnant
(No.preg./No. mated
Average No. of
implantation sites/dam
% post-implantation
loss/dam
Average No. of live
fetus/dam
NA= Not available. Statistically significant as compared to control p<0.05

The implantation site did not show any
95.0
19/20
6.30±0.07
100
20/20
7.01±0.03


7.69±0.01 7.80±0.03

5.83±0.01 6.91±0.01
further differentiation in PI phase
whereas animals of EP phase treated
group did show some pups lesser in
number in comparison to controls
suggesting implantation with further
growth and differentiation. Similarly,
the outcome of PL treated groups was
more in terms of the implantation site
and hence formation of pups than EP,
but was lesser than control. No
statistically significant differences
between the groups EP and PL as to
litter size and to ibuprofen feed were
seen. Further adverse effect on
pregnancy outcome included a
significant increase in the percentage of
post-implantation loss (resorptions and
dead fetuses) for litter above control in
EP phase and also the gradual increased
percentage of loss with the advancement
of age. Few abnormalities like shorten
of one of the uterine horns (Fig.1) and
stunted growth were noted. Negligible
amount of post-implantation loss were
also observed in PL phase. In PL phase
the increased percentage of post
implantation loss also evident during
postnatal development. However this
variation was not significant. A
significant treatment related decrease in
live litter size was seen in both EP and
PL treated group of animals during
different phases of development. The
body weight (Table 2) of fetuses of the
EP phase oriented groups of animals
gradually increased in different gestation
days except on 18th gd. With the
advancement of age of embryo the body
weight of the postnatal embryo

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increased compared to the controls.
Then length of the embryo (Table 2)
increased greatly between gd 12 to 5-
day postnatal embryo resulting in
significant change in length with time in
Table 2 : Table shows physical characteristics and brain nucleic acid content of
embryo of mice treated orally with ibuprofen.
control embryo. Likewise the width of
the embryo (Table 2) increased
reflecting the elongation of the embryo
during embryogenesis in the control
series.
Age of
embryo
12C
12EP
15C
15EP
18C
18EP
0-day C
0-day EP 4.555±0.122 0.899±0.038 1.905±0.094 0.163±0.021
0-day PL 4.002±0.161 0.961±0.094 1.672±0.063 0.690±0.057
5-day C
6.144±0.106 1.216±0.028 2.490±0.156 0.606±0.103
5-day EP 7.033±0.132 1.255±0.078 3.305±0.155 0.140±0.010
5-day PL 6.700±0.159 1.000±0.035 2.710±0.105 0.492±0.035
Values are expressed as mean ± SE, P value (C-T) significant at p < 0.01, C= Control, EP= Early Placental
PL= Placental

A significant increase in the body
Embryonic
length (cm)
0.977±0.064 0.065±0.044 0.031±0.001 3.006±0.352
1.066±0.179 0.305±0.201 0.031±0.007 1.898±0.292
1.377±0.213 0.716±0.030 0.048±0.005 2.624±0.301
1.700±0.105 0.416±0.024 0.032±0.002 0.163±0.013
3.200±0.207 0.800±0.024 1.163±0.109 2.103±0.264
3.788±0.139 0.538±0.024 1.075±0.034 0.170±0.008
3.800±0.156 1.033±0.033 1.324±0.123 1.518±0.169
Embryonic
width (cm)
Body
weight (gm)
DNA
(mg/100mg)
RNA
(µ µ µ µg/100mg)
228.48±16.43
257.10±10.24
205.94±10.57
173.86± 8.22
174.45±22.89
166.29±11.31
142.85± 5.46
111.59± 4.73
128.13± 7.29
132.70±14.26
90.82± 4.14
106.38± 6.41
weight for embryo and pups in both EP
and PL treated animals was noted during
embryogenesis. With the 6-fold increase
in length between 12 gd and 5 postnatal
embryos the width of the embryo
increased 4 fold. Whereas, the length of
the embryo showed an increase in body
length compared to controls in EP
treated animals, the width of the embryo
did not increase, resulted an effect of the
treatment. Similar results in increase of
body length and decrease of body width
were observed in embryo and pups of
PL treated animals.
Brain nucleic acid concentration
Brain RNA content of the fetus and the
pups of EP phase treatment animal
showed both increase and decrease
reflecting an infliction in the change in
drug. However the PL phase treated
animals showed a reduction in RNA
content in their pups. When both doses
were compared, significant reduction
(p< 0.01) in RNA content was observed.
Likewise brain DNA content of the