Journal of Cell and Molecular Biology 11(1&2):47-58, 2013
Haliç University, Printed in Turkey.
http://jcmb.halic.edu.tr
Research Article 47
Effects of gestational exposure to lead acetate on implantation
and neonatal mice
Ragini SHARMA and Sheetal MOGRA*
Environmental and Developmental Toxicology Research Laboratory, Department of Zoology,
University College of Science, Mohanlal Sukhadia University, Udaipur- 313001 (Raj.) India.
(* author for correspondence; [email protected])
Received: 07 January 2012, Accepted: 30 November 2013
Abstract
Exposure to heavy metal lead acetate during pregnancy has been associated with adverse health
consequence on both the mother and the offspring. The aim of the study was to evaluate the
gestational and lactational exposure of lead acetate on implantation and on some neonatal parameters
in mice. Forty eight Swiss albino mice divided into 8 groups of 6 mice each served as subjects for this
study. Mice in group 1 were dosed with distilled water (control), while those in groups 2, 3 and 4 were
exposed to lead at a dose of 266 mg/kg BW, 532 mg/kg BW and 1064 mg/kg BW, respectively. Mice
in group 5 were dosed with vitamin C (166 mg/kg BW) while those in groups 6,7 and 8 were exposed
to lead with vitamin C at a dose of 266 mg/kg BW +166 mg/kg BW, 532 mg/kg BW +166 mg/kg BW
and 1064 mg/kg BW+ 166 mg/kg BW, respectively. All the pregnant females were dosed between
gestation day (GD) 10th - till parturition, gestation day 10th postnatal day (PND) 21 and monitored for
signs of toxicity and gestation length. At birth, the litter size and weight, and crown to rump length of
the pups were measured. The pups were evaluated for physical characteristics and death. The dams
were sacrificed at postnatal day 22, and the uterine horns evaluated for number of implantation sites.
The results showed a significant decrease in the weight and body size in pups exposed to lead acetate
in utero compared to the control. In addition, all the pups prenatally exposed to lead acetate were born
weak and shows poor health status, show delay in physical development. A dose-dependent increase
in percentage of postimplantation loss was observed in mice dosed with lead acetate. The
administration of vitamin C along with lead not reverse the adverse effect of lead and its coadministration produce more adverse effects then lead. In conclusion, exposure of pregnant mice to
lead caused postimplantation loss, decreased litter weight, survivability and body size.
Keywords: lead acetate, implantation, gestation, crown to rump length, litter size.
Özet
Kurşun asetata gestasyonel maruz kalımın gestasyona ve neonatal farelere etkisi
Hamilelik sırasında bir ağır metal olan kurşun asetata maruz kalmak hem anne, hem de doğacak
nesilde sağlık sorunları ile ilişkilendirilmiştir. Bu çalışmanın amacı, farelerde laktasyonel gestasyonel
olarak kurşun asetata maruz kalımın hamile kalım ve çeşitli neonatal parametreler üzerine etkilerinin
değerlendirilmesidir. Çalışmada her biri 6 fareden oluşan 8 gruba dahil edilmiş toplam 48 fare
kullanılmıştır. 1. gruptaki farelere distile su verilirken, grup 2, 3 ve 4‘teki fareler sırasıyla vücut
ağırlığına oranları 266 mg/kg, 532 mg/kg ve 1064 mg/kg olacak şekilde kurşuna maruz bırakılmıştır.
5. gruptaki farelere vitamin C verilirken (166mg/kg), grup 6,7 ve 8’teki farelere ise hem vitamin C,
hem de kurşun, sırasıyla şu dozlarda verilmiştir; 266 mg/kg – 166 mg/kg, 532 mg/kg – 166mg/kg,
1064mg/kg – 166 mg/kg. Hamile fareler bu kimyasallara 10. gestasyon gününden doğuma ve 10.
gestasyon gününden 21. postnatal güne kadar maruz bırakılmış ve toksisite ve gestasyon süresi
48 Ragini SHARMA and Sheetal MOGRA
açısından incelenmiştir. Doğum sonrasında batın büyüklüğü ve yavruların ağırlığı ve baştan sağrıya
kadar uzunlukları ölçülmüştür. Yavrular fiziksel özellikleri ve ölüm açısından incelenmiştir. Hamile
dişiler doğumdan sonraki 22. günde sakrifiye edilmiş ve uterusları implantasyon bölgelerinin sayısı
açısından incelenmiştir. Sonuçlar, in utero olarak kurşuna maruz bırakılmış yavruların kontrol
grubuna nazaran daha hafif ve kısa olduğunu göstermiştir. Ayrıca doğum öncesi kurşuna maruz kalan
bütün fareler zayıflık, genel sağlık problemleri ve gelişimde gecikme göstermiştir. Kurşun asetat
verilen farelerde dozla orantılı emplantasyon sonrası düşük oranlarında artış görülmüştür. Kurşun ile
beraber vitamin C verilmesi kurşunun etkilerini düzeltmemiş ve tek başına kurşuna maruz kalımdan
daha fazla olumsuz etki oluşmasına yol açmıştır. Sonuçlara göre hamile farelerin kurşuna maruz
bırakılması, postimplantasyon sonrası düşüğe, azalmış batın ağırlığına, hayatta kalıma ve azalmış
vücut boyuna sebep olmaktadır.
Anahtar sözcükler: kurşun asetat, implantasyon, gestasyon, baştan sağrıya mesafe, batın büyüklüğü.
Introduction
Lead is of public health concern due to their toxic
effects on vulnerable fetuses, persistence in
pregnant and breastfeeding mother and widespread
occurrence in the environment (Dorea, 2004).
Theoretically, pregnant women can no longer be
exposed to occupational sources with the
application of public health regulations but other
sources including water contamination, wall paint,
industrial wastes and automobile exhaust fumes can
not be ignored (Klein et al., 1994). Significant
quantities of lead were transferred in to the fetus;
however, the placenta appeared to greatly limit the
passage of lead since large maternal-fetal
concentration gradient existed (Mc Clain, 1975). As
lead is a persistent metal, thus it is still present in
the environment. Persistent substance (lead)
accumulates in the body of the mother, may
redistribute, thus leading exposure to the unborn
child. Pregnancy and lactation are time of
physiologic stress during which bone turnover is
accelerated (Gulson et al., 2004). Mobilization of
maternal bone lead stores has been clearly
identified as a major source of fetal lead exposure
(Gulson et al., 2003; Hernandez Avila, 2002).
The mammalian embryo or fetus is surrounded
by and dependent on the maternal system. Except
for physical agents, all exposure passes through the
maternal blood stream and placenta before they
reach the developing embryo or fetus. T he
metabolism, storage and excretion could
significantly alter a chemical agents delivery to the
embryo or fetus. Even after birth the newborn may
continue to depend on maternal nutrition if it is
breast fed and may be exposed to toxic agents
(Hood, 1997). Breastfeeding is essential to
complete infant development (Dorea and
and Donangelo, 2006). Lead also affects the
postnatal development. In the postnatal
development the effect of lead on the fetus
are mainly caused by lactation (O’Halloran,
1992-93). Many substances, especially
lipophilic ones to which the lactating mother
is exposed or has been exposed at an earlier
period in her life, are secreted into the breast
milk and some substance may even
concentrate in breast milk (EPA, 1997).
Pregnancy and breastfeeding can cause a
state of physiologic stress that increase bone
turnover of lead. Pregnancy related
hormonal
changes,
affect
calcium
metabolism and also cause lead to leave the
bone and enter the blood. Wherever
maternal blood lead levels become elevated,
it is easily available to the fetus and has
negative impact on fetal development
(Keller et al., 1980). There is a strong
correlation between maternal and umbilical
cord blood lead levels, including the transfer
of lead from mother to fetus ( Gardella,
2001).In addition to transfer of lead
prenatally , lead levels in breast milk also
increase with the lead level in maternal
blood, posing an additional risk to the
neonate (Li et al., 2000).
Transfer of lead via placenta and human
milk was shown by higher lead levels in
milk and blood of infant (Xuezhi, 1992). A
great deal of research demonstrates that
lactational exposure represents a substantial
aspect of lead transmission to neonates
(Beach and Henning, 1998). It suggests that
maternal transfer and /or neonatal absorption
Gestational exposure to lead acetate 49
appears to be much more efficient than direct
exposure of adults. The unborn child as well as
infants is considered potentially more susceptible to
adverse effects of lead than adults. Because of its
widespread uses, the present study is aimed at
evaluating the effect of lead exposure to pregnant
mice on the pregnancy status (post-implantation
loss and gestation length) and the effect of
gestational and lactational exposure on some
developmental parameters (litter size and weight,
survivability and body size ) in neonates. The
present study also investigates the role of vitamin C
in lead toxicity during the period of gestation and
lactation.
Materials and Methods
Test Chemicals
Following test chemicals were used for the present
investigation:
Lead acetate: Laboratory reagent lead acetate,
manufactured by ‘S.D. Fine Chem. Ltd.’, Mumbai
was used for the experiments.
Vitamin C: ‘Limcee’ capsule of vitamin C
containing 500 mg of ascorbic acid, manufactured
by ‘Sarabhai Chemicals’, Vadodara were used for
the investigation.
All other chemicals used were of analytical grade.
Animals and Treatment
Healthy adult female Swiss albino mice 8-10
weeks of age and average body weight (BW) of 30
g were used in this study. After reaching an average
weight of 30g, six females were mated with two
males. Female mice were examined every day in
the morning and female showing vaginal plug were
isolated and duration of their gestation period were
recorded. Presence of spermatozoa in the vagina the
following morning was called day 1 of gestation.
Confirmed pregnant female mice were maintained
on sterilized rice husk bedding in polypropylene
cages and kept at a temperature of about 23±3 C
with 12±1 h L:D cycle. Animals were fed on
standard pellet diet (Pranav Agro, Baroda). Food
and water were given ad libitum.
Experimental protocol was approved by the
Institutional Animal Ethics Committee. Handling of
animals was according to the guidelines of
committee for the purpose of control and
supervision of experiments on Animals (CPCSEA),
Ministry of Environment and forest.
174 mg/kg was reported by Material SafetyData
Sheets (MSDS) provided by manufactures
(2007) and Sadykov et al. (2009) for lead
acetate; considering this aspect dose level
which may show adverse effect on
developmental parameter were selected as
the dose for the present study.
Dose for antioxidant was calculated in
the present study with the same previous
doses of vitamin that is 166 mg/kg BW for
vitamin C which was already tested in adult
animals by Banu and Sharma (2005) during
the period of gestation and lactation.
Dose of lead acetate and vitamin C were
prepared in distilled water. Experimental
protocol-The animals were dosed with test
chemicals starting from 10th day of gestation
to 17th day of gestation and from 10th day of
gestation (dg) till parturition and lactation
(PND 21) using the following protocol.
Mice in group 1 control (treated with
distilled water) and groups 2, 3 and 4 were
dosed with lead acetate at a dose 266 mg/kg
BW(8mg/animal/day),
532
mg/kg
BW(16mg/animal/day) and 1064 mg/kg BW
(32mg/animal/day) respectively. Mice in
group 5,6,7 and 8 were dosed with vitamin
C at a dose 166 mg/kg BW, 266 mg/kg
BW+ 166 mg/kg BW ,532 mg/kg BW + 166
mg /Kg BW and 1064 mg/kg BW +166
mg/kg BW respectively . The pilot
experiments were performed at different
dose tolerance study. As there were no
significant alterations in the developmental
land mark at different dose levels, the
optimum doses were used for the
experimentation.
Toxic signs and body weight-After
administration of lead acetate alone and lead
with vitamin C to 8 groups; mice were
operated separately for experimental period.
All the control and treated mice were
observed carefully for appearance of any
toxic signs. At birth, the litter size and
weight of pups, physical status and
survivability were evaluated. The crown to
rump length (tip of nose to rump) of each
pup was measured on postnatal day (PND)
1. The dams were allowed to raise the pups
up to weaning at PND 20. At PND 21, the
dams were anesthetized with the help of
sodium pentathiol, and the uterine horns
The
50 Ragini SHARMA and Sheetal MOGRA
were dissected, rinsed and immersed directly in 2%
sodium hydroxide (NaOH), potassium hydroxide
(KOH) solution or one of several other alkaline
solutions for one hour, the uteri were cleared. The
number of implantation sites was determined by the
best and most convenient solution for clearing the
rat uterus and for staining implantation sites was
2% NaOH or KOH as described by Yamada et al.,
(1988).Thereafter, the implantation sites appearing
as dark rings were counted. The number of
implantation sites in each dam was then correlated
with the litter size to determine if there had been
post implantation loss, using the following formula
(US EPA, 1996).
% Post-implantation loss= No. of implantation sites — No. of litters × 100
No. of implantation sites
Body weight of the control and treated mice fetus
and pups were taken at the end of 17th day of
gestation, PND 1 and PND 21.
Statistical Evaluation-Data obtained in
the present study were analyzed statistically
using one way ANOVA and the values are
expressed as mean +Standard deviation. A
probability value of P<0.05, 0.01 was
considered as statically significant.
Results
Effects of lead on gestation length
The gestation length of mice in all the
treated groups was not significantly different
from the control, although they were slightly
shorter. The administration of lead along
with vitamin C did not produce any
significant changes in the length of gestation
period when compare to the lead treated as
well as Vitamin C treated group (Table 1).
Table 1. Effects of in utero exposure to lead acetate on gestation length
Treatment of Groups
Gestation length
Control
20.1667± 0.7528
8mg
20.0000 ± 0.6325 aN.S.
16 mg
19.6667± 0.8165 aN.S.
32 mg
19.6667± 1.2111 aN.S.
Vitamin C
19.3333 ± 0.5164 aN.S.
8mg + vitamin C
19.8333 ± 0.4082 bN.S., eN.S.
16mg + vitamin C
19.8333 ± 0.4082 aN.S., cN.S., e N.S.
32mg + vitamin C
19.0000 ± 1.0954 aN.S., dN.S., eN.S.
Values are expressed as mean ± S.D. for six animals in each group, P value > 0.05 non significant (N.S). < 0.05 significant (*).
< 0.01 highly significant (**).
a= when compare to control, b= when compare to 8 mg, c= when compare to 16 mg, d= when compare to 32 mg, e= when
compare to Vitamin C.
th
Effect of lead exposure on litter weight at 17 day
of gestation
There was no significant change in the litter weight
of fetus between the control and in lower dose of
lead treated group but at higher dose level (16, 32
mg) there were significant (p<0.05) decreases in the
litter weight of fetus from the control. In vitamin C
group there was a significant (p<0.05) increase in
litter weight. The groups treated with Vitamin C
along with lead acetate showed significant decrease
(p<0.05) in litter weight. However, in groups
treated with 8 mg + vitamin C showed decrease but
the decrease was non-significant from control. The
combined treatment of Vitamin C + lead showed
significant decrease when compared with
only lead treated and Vitamin C treated
group (Table 2).
Effects of lead exposure on neonates body
weight at PND 1
There was no significant change in the
neonate weight between the control and in
lower dose of lead treated group (p>0.06)
but at higher dose level (16, 32mg) there
was significant decrease (p<0.05) in the
litter weight of neonate from the control. In
Vitamin C treated group there was a
significant increase (p<0.05) in litter weight.
The groups treated with Vitamin C along
Gestational exposure to lead acetate 51
with lead acetate showed significant decrease (p<
0.05) in litter weight of pups from control.
However, in group treated with 8mg + vitamin C
showed decrease in weight but the decrease was
non significant from control. The combined
treatment of vitamin C + lead show non significant
decrease in all the groups as compared to lead
treated groups. The administration of vitamin C
along with lead showed significant decrease (p<
0.05) in body weight when compare with vitamin C
(Table 2).
the litter weight of pups from the control. In
Vitamin C treated group there was a
significant increase (p< 0.05) in litter
weight. The groups treated with vitamin C
along with lead acetate showed significant
decrease while in 16mg + vitamin C treated
group showed significant increase in litter
weight of pups. However, in group treated
with 8mg + vitamin C showed non
significant increase from control. The
combined treatment of vitamin C + lead
show significant increase in 16mg + vitamin
C (p< 0.05) and 32mg + vitamin C (p< 0.05)
from lead treated groups. The administration
of vitamin C along with lead showed
significant decreased (p<0.05) when
compare with vitamin C treated group
(Table 2).
Effects of lead exposure on neonatal body weight
at PND 21
There was no significant change in the neonate
weight between the control and in lower dose of
lead treated group but at higher dose level (16,
32mg) there were significant decreases (p< 0.05) in
Table 2. Effects of lead on body weight of fetus and neonate of treated mothers on 17th day of
gestation, on PND 1 and PND 21
Treatment of Groups
Control
8mg
16 mg
32 mg
Vitamin C
8mg + vitamin C
16mg + vitamin C
32mg + vitamin C
Age of animals
17 dg
0.7067± 0.0592
0.6867± 0.0356
PND 1
1.3750 ± 0.0660
1.3067 ± 0.0647
aN.S.
aN.S.
0.6167 ± 0.0378
1.1917 ± 0.2759
PND 21
6.1933 ± 0.1777
6.1067 ± 0.1226
aN.S
.
5.5750 ± 0.3631
a*
a*
a**
0.5950 ± 0.0517
0.9767 ± 0.1669
4.7650 ± 0.1048
a**
a**
a**
0.7900± 0.0494
1.6717 ± 0.0880
9.6100 ± 0.3888
a*
a**
a*
0.6417± 0.0426
1.2867 ± 0.739
6.2367 ± 0.1372
aN.S., bN.S., e **
aN.S., bN.S., e**
aN.S., bN.S., e**
0.5233± 0.0635
1.1733 ± 0.0750
6.8333 ± 0.6237
a*, c*, E***
a*, c N.S., e**
a**, c**, e**
0.3883± 0.0293
0.9217± 0.0763
5.2067 ± 0.1155
a**, d**, e**
a**, dN.S., e**
a**, d*, e**
Values are express as mean ± S.D. for six animals in each group, • P value >0.05 non significant (N.S.), <0.05 significant (*),
<0.01 highly significant (**). a= when compare to control, b= when compare to 8 mg, c= when compare to 16 mg, d= when
compare to 32 mg, e= when compare to Vitamin C.
Effects of lead on pup’s survival and morphology
All the pups delivered by mice in groups 32mg
lead, 16mg + vitamin C and 32mg + vitamin C
were born weak. The pups of 32mg + vitamin C
were weaker and most of the pups died 2 - 3 days
post delivery. On the other hand, pups from mice in the
control group, only vitamin C treated groups were
stronger and they all survived.
Neonates of lead treated group show
morphological changes as compared to control
group animals. Neonates of control group shows
normal appearance, soft smooth hair and active
state of animals (Plate). Lead treated animals show
rough body surface, dispersed hair with patchy
52 Ragini SHARMA and Sheetal MOGRA
skin. Neonates of lead + vitamin C treated group also
shows morphological changes at lesser degree as
compared to lead treated group.
vitamin C treated group showed same results
as obtained in control group.
The administration of vitamin C along with
lead (32mg + vitamin C) showed significant
decrease (p<0.05) and non-significant (p>
There was a comparative decrease in the crown to
0.05) decrease in 16mg + vitamin C group
rump length of fetus in all the groups except in
when compared with lead treated groups.
vitamin C treated groups as compared to control
However in group 8mg + vitamin C showed
group. The crown to rump length of fetus in the
non-significant increase (p>0.05) in the
control group was significantly different from those
crown to rump length. The groups treated
obtained in group vitamin C and lead + vitamin C
with vitamin C along with lead acetate
treated groups. All the lead treated groups showed
showed significantly decrease (p<0.05) in
non-significant decrease (p>0.05) while in 8mg +
the crown to rump length of groups when
compared to vitamin C (Table 3).
Table 3. Effects of in utero exposure to lead acetate on crown to rump length
Treatment of Groups
Body size (17th dg)
Control
1.6167 ± 0.0753
Effects of gestational exposure to lead on crown to
rump length
8mg
1.5667 ± 0.0516aN.S.
16 mg
1.5000 ± 0.0894aN.S.
32 mg
1.7000 ± 0.0894aN.S.
Vitamin C
1.7833 ± 0.1602a*
8mg + vitamin C
1.6167 ± 0.1169 bN.S., e*
16mg + vitamin C
1.4333 ± 0.1506a**, cN.S., e **
32mg + vitamin C
1.3167 ± 0.1169a**, d**, e **
Values are express as mean ± S.D. for six animals in each group, • P value >0.05 non significant (N.S), <0.05 significant (*),
<0.01 highly significant (**). a= when compare to control, b= when compare to 8 mg, c= when compare to 16 mg, d= when
compare to 32 mg, e= when compare to Vitamin C.
Table 4. Effects of gestational lead exposure on post implantation loss on 17 th (dg) and PND 1
Groups
Control
8 mg
16 mg
32 mg
vit C
8+C
16 + C
Postimplantation
losses at 17th dg
Arc sign mean ± SD
44.9819±0.0000
44.9819±0.0000
53.4146±2.3629 a*
67.2729±15.9791 a**
44.9819±0.0000
44.9819±0.0000
51.7773±1.2932 aN.S.,
Retransform
ed
value
0
0
14.5
35.11
0
0
Postimplantation
losses at PND 1
Arc sign mean ± SD
44.9819± 0.0000
44.9819±0.0000
50.9021±1.1131
64.7583±3.0747 a**
44.9819±0.0000
44.9819±0.0000
52.2684±1.7349 aN.S., cN.S.,
Retransforme
d value
0
0
10.26
31.85
0
0
cN.S., eN.S.
11.75
eN.S.
12.59
63.1114±11.4944 a**, dN.S.,
32 + C
53.3382±3.2943 a*, d**, e*
14.38
e**
29.58
Values are express as mean ± S.D. for six animals in each group, • P value >0.05 non significant (N.S), <0.05 significant (*),
<0.01 highly significant (**).
a= when compare to control, b= when compare to 8 mg, c= when compare to 16 mg, d= when compare to 32 mg, e= when
compare to Vitamin C.
Gestational exposure to lead acetate 53
Table 5. Effect of gestation lead exposure on implantation sites and litter size (17th dg)
Groups
Control
8 mg
16 mg
32 mg
vit C
8+C
16 + C
32 + C
Number of implantation site at 17th
day of gestation
11.25 ± 0.9574
5.500 ± 0.5774 a**
7.250 ± 1.7078 a**
7.750 ± 1.7078 a**
11.50 ± 0.5774 aN.S.
9.750 ± 0.9574 aN.S., b**., e*
8.750 ± 1.7078 a**.,cN.S., e**
8.750 ± 0.9574 a**., dN.S., e**
No. of litter size at 17th day of
gestation
11.250 0.9574
5.500 ± 0.5774 a**
6.250 ± 1.7078 a**
5.250 ± 0.9574 a**
11.500 ± 0.5774 aN.S.
9.750 ± 0.9574 aN.S., b**., e*
7.750 ± 1.7078 a**., cN.S., e**
7.500 ± 1.0000 a**., d*., e**
Values are express as mean ± S.D. for six animals in each group, • P value >0.05 non significant (N.S), <0.05 significant (*),
<0.01 highly significant (**). a= when compare to control, b= when compare to 8 mg, c= when compare to 16 mg, d= when
compare to 32 mg, e= when compare to Vitamin C.
Table 6. Effect of gestation lead exposure on implantation sites and litter size (PND 1)
Groups
Control
8 mg
16 mg
32 mg
vit C
8+C
16 + C
32 + C
Number of implantation site
PND 1 day
8.0000 ± 0.8165
6.0000 ± 0.8165 a*
10.0000 ± 1.8257 a*
9.5000 ± 2.6458 aN.S.
7.7500 ± 0.9574 a*
5.5000 ± 0.5774 a*., bN.S., e*
7.5000 ± 1.2910 aN.S., c*., eN.S.
9.7500 ± 0.9574 aN.S., d*., e*
at
No. of litter size at PND 1 day
8.0000 ± 0.8165
6.0000 ± 0.8165 a*
9.0000 ± 1.8257 aN.S.
6.5000 ± 1.9149 aN.S.
7.7500 ± 0.9574 aN.S.
5.5000 ± 0.5774 a**., b**.,e*
6.5000 ± 1.2910 aN.S., cN.S., eN.S.
7.0000 ± 1.4142 aN.S., d*., eN.S.
Values are express as mean ± S.D. for six animals in each group, • P value >0.05 non significant (N.S), <0.05 significant (*),
<0.01 highly significant (**). a= when compare to control, b= when compare to 8 mg, c= when compare to 16 mg, d= when
compare to 32 mg, e= when compare to Vitamin C.
Effects of gestational lead exposure on post
implantation loss on 17th dg
The numbers of implantation sites observed in the
uterus of mice were significantly higher (p< 0.01)
in the control group as compared to those in lead
treated groups and lead + vitamin C groups. There
was no significant difference in the number of
implantation sites in the control group compared to
those in vitamin C groups. There was no significant
change (p> 0.05) in the number of implantation
sites and litter size in the control and 8mg, Vitamin
C, 8mg + vitamin C groups, though in the latter
group (16 mg + vitamin C), there was a marginal
but non significant reduction in the litter size as
compared to number of implantation sites.
However, a significant reduction (P <0.05)
in litter size as compared to the number of
implantation sites were observed in group
16mg, 32mg, 32mg + vitamin C groups.
Correlation of the implantation site with the
litter size as shown in Table 5, indicated that
no post implantation loss was recorded in
the control group, 8mg, vitamin C, 8mg +
vitamin C while in other groups post
implantation losses were recorded . In
comparison to control group there was
significant increase (p<0.05) in post
implantation losses in 16mg, 32 mg, and
32mg + vitamin C groups.
The administration of lead along with
Vitamin C reduced the post implantation
losses as compared to lead treated groups.
54 Ragini SHARMA and Sheetal MOGRA
The reduction was significant (p< 0.05) in 32mg +
vitamin C while in 16mg + vitamin C the reduction
was non-significant (p> 0.05). However in 8mg +
vitamin C treated group showed no post
implantation loss as found in only 8 mg treated
group.
The percentage of post implantation losses were
significantly (p< 0.05) increased in 32mg + vitamin
C treated group while non-significant (p > 0.05)
increase in 16mg + vitamin C group when they
compared with vitamin C.
Effects of gestational lead exposure on post
implantation loss on PND 1
There was no significant (p> 0.05) change in the
number of implantation sites and litter size in the
control and 8mg, Vitamin C, 8mg + vitamin C
group, though in the latter group (16mg + vitamin
C and 16 mg), there was a marginal but non
significant reduction in the litter size as compared
to number of implantation sites. However, a
significant reduction (p < 0.05) in litter size
as compared to the number of implantation
sites was observed in mice in group 32mg,
32mg + vitamin C groups. Correlation of the
implantation site with the litter size as
shown in Table 6 indicated that no post
implantation loss was recorded from mice in
the control group, 8mg, vitamin C, 8mg +
vitamin C while in other groups post
implantation losses were recorded.
In comparison to control group there was
significant increase in post implantation
losses in 32mg, 32mg + vitamin C groups.
The administration of lead along with
vitamin C did not show any clear cut
changes. In 32 + C group there was decrease
while in 16mg + vitamin C there was
increase but both the results were not
significant. However, 8mg + vitamin C
treated group showed no post implantation
loss as found in only 8 mg.
Figure 1. A: Normal morphological appearance, soft and smooth fur and active state of neonate
(control group). B: No observable morphological change (266 mg/kg BW lead). C: Shows hair loss in
head region (asteriks). (532 mg/kg BW lead). D: Shows rough body surface dispersed hair, severe hair
loss (asteriks) in head and abdomen. (1064 mg/kg) BW lead). E: Normal morphological appearance.
(166 mg/kg BW vit C)
importance of early development and the
detrimental effects on development due to
Discussion
lead toxicity. Our results illustrate the
greater risk to adverse health outcomes
This paper address the importance of the first 21
among the children due to lead intoxication.
days of life in the developing pups, examines the
Gestational exposure to lead acetate 55
During the first 21 days of life; pup development is
dynamic and involves the maturation of interrelated functioning such as physical and mental
development. It is a period marked by rapid
physical and neurological development. From our
result it is clear that short-term gestational exposure
of lead can also negatively affect the fetal growth.
Susan et al. 2001 reported that the response
from exposure to a toxic agent during development
may vary depending on the dose, time of exposure
and the mode of action. As we found in our
previous study that early gestational exposure
inhibit the developmental process while in case of
exposure from 10th day gestation and there after
fetal toxicity sharply declined.
The embryo toxicity and teratogenicity of lead
(Pb) in animals have been reported by McClain and
Becker (1975).Kimmel et al. (1980) and Beaudoin
and Fisler (1981) also reported embryo growth
retardation and malformations in their studies.
Papanikolaou et al. (2005) reported that lead
crosses the placenta during pregnancy and has been
associated with intrauterine death, prematurity and
low birth weight.
Our experimental result shows that newly born
pups of treated mother displayed slight perinatal
growth retardation and that many of the pups had
reduced body weights; although the litter size and
postpartum pup death to weaning were unaffected
by lead exposure. At higher dose level
postimplantation losses and general retardation of
development were also encountered. The
postimplantation losses observed in groups exposed
to lead were apparently due to foetal death and
resorption. Pups derived from lead exposed females
were examined grossly and some morphological
alteration was evident. Birth weight predicts
infant’s survival, growth and development. A
change in offspring body weight is a sensitive
indicator of developmental toxicity, in part because
it is a continuous variable. So we can say that the
late fetal stages of mice prenatal development
appeared uniquely sensitive to inorganic lead
exposure.
Studies of Kennedy et al. (1975) were parallel
with our results. They administered lead (up to
714 mg) or tetraethyl lead (10 mg/kg) by gavage in
mice during the period of organogenesis (day 5-15).
At the higher dose levels maternal toxicity, fetal
resorption and general retardation of development
were encountered. Foetus derived from lead
exposed females were examined grossly for
external changes and for internal structural
and skeletal development but no teratogenic
response was evident , even at dose levels at
which frank signs of maternal toxicity were
observed.
Carole et al. (1980), reported that
exposure of lead acetate (at 0.0.5,5,25,50,or
250 ppm) did not affect the ability to
conceive, to carry a normal litter to term or
to deliver the young. They also reported that
the percentage of malformed fetuses,
resorption and postpartum pup deaths to
weaning were unaffected by lead exposure
but our result are not in conformity with
these findings. They also reported that at
250ppm exposure body length of female
offspring were significantly shorter than
those of controls, and there was a tendency
for all young in this group to be smaller.
The results of postnatal experimentation
illustrate that maternal transfer of lead both
gestational and lactational are very efficient.
It is also apparent that lead uptake is greater
during the fetal/neonatal period. Gestation
and lactation exposure adversely affect the
postnatal development of pups. It reduces
the pup body weight, length and delays their
physical development. At higher dose level
stillbirth were also observed. At cessation
of exposure PND 21, morphological
abnormalities were observed in pups i.e. the
exoskeleton of was not properly developed.
The coat colour was also different from
controls. It was quite scanty and dirty cream
in colour in contrast to the full smooth and
show white fur of controls. The presences
of patchy skin, altered coat colour and
texure change in fur indicates that
development of fur may be affected. The
snout region also showed structural
deformity. Postnatal effects, especially on
body weight and behaviors of the pups may
be induced via effect on the mother. For
example, lactation or maternal care may be
affected and potentially any alteration in
maternal physiology and behaviors may in
turn influence the offspring.
Reiter et al. (1975) also observed
developmental delays in rat offspring which
were exposed to lead (50ppm) throughout
56 Ragini SHARMA and Sheetal MOGRA
gestation and lactation. Kurtais et al. (1994) also
reported that prenatal lead exposure also appears to
be associated with reduced birth weight.
In contrast to our result Schroeder and
Mitchener (1971) reported that lead exposed mice
produced fewer litters and experienced early death
but they agree with our statement that many of the
offspring had reduced body weight. Kimmel et al.
(1980) examined the effects of lead (Pb) on the
growth and development of embryos. Rat were
exposed to lead acetate for 6-7 weeks, then mated
and exposed continually through gestation and
lactation. Their offspring showed delayed vaginal
opening in the 50-and 250 ppm groups, and
significantly growth retardation 1 to 3 weeks after
exposure. McClain and Becker (1975) reported that
single dose of 25-70 mg/kg of lead nitrate
administered to pregnant rats on day 9 of gestation,
caused
a
urorectocaudal
syndrome
of
malformations and skeletal anomalies.
The administration of lead along with vitamin C
did not produced any significant changes in the
length of gestation period when compare to the lead
treated as well as vit C. The administration of lead
along with vit C reduced the post implantation
losses as compared to lead treated groups. The
percentage of post implantation losses were shows
significantly increased in 32 + vit C treated group
while non significant increase in 16 +C group when
they compared with vit C. In the present
investigation vit C does not cause any adverse
effect regarding development but the combine
treatment of antioxidant + lead (vit C + lead ) are
not as beneficial as alone antioxidant. There was an
increase in postimplantation losses in lead treated
group as compare to control. The group treated
with only vitamin C did not show any post
implantation loss. The administration of lead + vit
C decrease the postimplantation loss in comparison
to lead treated group while there was an increase
when compare with vit C.
Neonates appear to be unaffected or positively
affected by large oral doses of vitamin C as
determined by their body weight and overall
appearance during gestation and lactation, nor did
the excessive amounts of vitamin C seem to affect
resorption.
Litter size and the weight and survival of the
pups also did not show negative result by maternal
vitamin C administration. Neither did excessive
intake of vitamin C produce fetal malformations.
Several other observational studies also
showed positive correlations between
maternal vitamin C status and birth weight
(Rao et al., 2001; Mathews et al., 1999;
Doyle et al., 1990).
In contrast, decreased body weight was
noted by Barja et al. (1994) with both low
(33mg/kg) and very high (13,200 mg/kg)
levels of vitamin C intake. Nash et al.
(2007) also reported that maternal
administration of high dose vitamin C
(250mg) plus vitamin E (100mg) regimen
throughout gestation has limited efficacy
and potential adverse effect as a therapeutic
intervention for vitamin E neurobehavioral
teratogenicity. Poston et al. 2006 concluded
that vit C (1000 mg) does not prevent pre eclampsia in women at risk, but does
increase the rate of babies born with a low
birth weight. As such, use of high dose
antioxidant is not justified during pregnancy.
The unconducive intrauterine environment
resulting from alteration in hormonal levels,
increased oxidative stress and the direct
effect of lead on the foetus may have
contributed to the increased gestation length
and reduced litter size and weight observed
in lead-exposed animals in the present study.
In conclusion, the present study has shown
the ability of lead to increase the gestation
length and postimplantation losses, reduce
the pups body weight, survivability and
body size. Therefore, measures targeted
towards decreasing contact of pregnant
animals or humans with heavy metal lead
should be put in place in order to minimize
the risk of adverse effects associated with in
utero exposure to lead on neonates.
The post-implantation losses observed in
the groups administered with lead in the
present study were dose dependent. The
postimplantation losses observed in groups
exposed to lead were apparently due to
foetal death and resorption. The leadinduced post-implantation losses recorded in
mice exposed to lead in the present study
may have been due to in utero exposure of
the pups to the lead. Lead have been shown
to pass through the placenta and may have
also been found in amniotic fluid; indicating
direct contact with the foetus. It may have
Gestational exposure to lead acetate 57
also resulted in alteration of the maternal hormonal
levels either from the effect of lead on the CNS,
suppressing the brain release of gonadotropins.
This alteration may result in changes of the uterine
biochemical content, hence alteration in intrauterine
environment, leading to foetal death and resorption.
The pups exposed in utero to lead have been shown
by this study to have lower chances of survival as
all the pups were born weak and died as newborns.
This is in agreement with other earlier studies. The
low birth weight observed in the present study may
have been due to the induction of oxidative stress
by lead and alteration of the biochemical
environment of the foetus arising from changes in
the hormonal pattern of the dam.
The differing results of the various studies could
be due in part to differences in amount of
antioxidant that was use during pregnancy, because
the present study shows that it can influence the
period of gestation, lactation and effects of lead on
fetal and neonatal growth.
The mechanisms whereby Pb reduces newborn
size are unknown. According to Hernandez-Avila,
et al. (2002), one possible explanation would be a
reduction in fetal thyroid hormones caused by the
presence of Pb. However, the relationship between
thyroid function and prenatal exposure to Pb is still
controversial, and further studies are necessary to
confirm this hypothesis.
It is clear that both earlier and later stages of
development are vulnerable to toxic insult, results
in certain structural abnormalities that can be
classified appropriately as malformations. In
addition to structural malformations, behavioral
abnormalities as well as generalized growth
retardation, manifested as developmental delays are
within the realm of developmental toxicology.
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