Biologia 66/2: 357—364, 2011
Neurobehavioral changes in mice offspring induced
by prenatal exposure to acute toxicity of sodium selenite
Jamaan S. Ajarem1, Gada I. Al Basher1& Hossam Ebaid1,2*
1Department of Zoology, College of Science, King Saud University, Saudi Arabia, P.O.Box 2455, 11451 Riyadh, Saudi
Arabia; e-mail: email@example.com
2Department of Zoology, Faculty of Science, El-Minia University, Egypt
Abstract: Selenium is an essential element with a narrow margin between beneficial and toxic effects. This study was
aimed to determine the neurobehavioral changes resulted from the prenatal exposure of mice to high doses of sodium
selenite during fetal and early postnatal development. Atomic absorption for monitoring the placental transfer of selenium
to offspring was employed. The developmental observations as well as the behavioral tests, such as sensory motor reflexes,
and learning and memory test in automatic reflex conditioner (shuttle box) (active avoidance responses) were applied.
Adult mice was assigned into three groups: the first group was remained as a control group given phosphate buffered saline;
the second and the third groups were orally administrated sodium selenite at doses of 1 mg/kg and 4 mg/kg of the diet,
respectively started from the 7thday to the end of the gestation period. Appearance of body hair and opening of eyes of
the pups from treated mothers were delayed in a dose-dependent manner. The body weight gain came significantly lower
than those of the control especially at the higher dose. Selenite also inhibited the sensory motor reflexes in all elements
of acts and postures in a dose dependent manner. The active avoidance training-test indicated that selenite exposure was
associated with learning impairment. Acetylcholine recorded a significant decrease in almost all the period of this study. By
using atomic absorption, we found a significant high concentration of selenium in the brain, liver and kidney until the 40th
postnatal day, indicating active transfer of selenium from mothers to embryos.
Key words: selenium; atomic absorption; acetylcholine; learning; sensory motor reflexes
Environmental toxicity of selenium in humans is asso-
ciated with symptoms such as hypochromic anaemia,
leucopoenia, damaged nails, etc in long-term workers.
The high accidental ingestion of selenium has been re-
lated with vomiting, diarrhoea, mottling of the teeth as
well as neurological disturbances like acroparesthesias,
weakness, convulsion, etc. (Sunde 2000; Mataix Verdu
& Llopis, 2002; Tinggi 2003). Attention has been paid
recently to residual selenium returned to the soil (Old-
field 1997). Selenium may be given directly to livestock,
or applied as a fertilizer amendment to increase sele-
nium content of their feeds (Oldfield 1997). Although,
the need for selenium in human and animal nutrition is
well recognized, the question concerning the high doses
of selenium for supplemental use is still being debated.
Although, sodium selenite is widely used as a
source of Se in animal feed, it is not a natural nu-
tritional form of selenium and it can create ecologi-
cal problems (Kumar et al. 2010). Receiving selenium
as selenomethionine, the chief natural nutritional form
present in grain crops is better than sodium selenite
as a source (Schrauzer & Surai 2009). Selenomethion-
ine and sodium selenite have risks of toxicity if intake is
too high (Krittaphol et al. 2010). Therefore, the general
population should be warned against the employment of
selenium supplements for prevention of hepatopathies,
cardiovascular or cancer diseases. The benefits of se-
lenium supplementation are still uncertain, and their
indiscriminate use could generate an increased risk of
toxicity (Miguel & Carmen 2008). Although the gut
microbiota contributes to the excretion of excess sele-
nium through the production of methylated selenium
compounds and elemental selenium (Krittaphol et al.
2010), the high doses of this element are highly toxic.
Mechanisms lying behind detrimental effects of se-
lenium are poorly understood yet. They can involve
DNA damage induction and consequently, DNA dam-
age response and repair pathways may play a crucial
role in cellular response to selenium (Mániková et al.
2010). Toxicity of selenium, however, depends on many
factors like the selenium species, amount ingested, age,
physiological status, and dietary interaction with other
nutrients (Mataix Verdu & Llopis 2002). Data reported
the memory and the other cognitive and behavioral
problems induced by selenite–high doses are poorly
available. This paper describes an attempt to determine
* Corresponding author
c ?2011 Institute of Zoology, Slovak Academy of Sciences
J.S. Ajarem et al.
the high dose-selenite induced neurobehavioral changes
in an experimental model.
Material and methods
Male and female Swiss-Webster strain mice (8 weeks old)
were housed in opaque plastic cages (three females to one
male in each cage). Animals were kept under reversed light-
ing conditions with white lights. The ambient temperature
was regulated between 18 and 22◦C. Food and water were
available ad libitum, unless otherwise indicated. The males
were removed from the cages after pregnancy (appearance
of vaginal plug was considered as day one of pregnancy),
and the females were subjected to experimental treatments.
Pregnant females was assigned into three groups: 1) the
first group was remained as a control group given phosphate
buffered saline via oral route, 2) the second and the third
groups were orally administrated sodium selenite in the diet
at doses of 1 mg/kg and 4 mg/kg, respectively started from
the 7thday to the end of the pregnancy period. Offspring
were subjected to the developmental, neurobehavioral and
biochemical studies from the day of birth (PD1) until the
day 21 (PD21).
Behavioral and developmental motor reflexes
The pups of each experimental group were culled to only
eight per dam on PD0 and were left with their mothers
until PD22. During the weaning period, three pups of each
litter were color marked from the others without any consid-
eration to its sex, and were subjected to various behavioral
tests (described below) under dim lighting. In all, 21 pups
belonging to seven litters from each treatment category were
considered. All observations were recorded on PD1 and re-
peated every other day until PD21 in the same three color
marked pups of each litter. These observations were used
to measure the early development of sensory motor coor-
dination reflexes together with morphological development
in the pups. For statistical analysis, the mean of all three
color marked pups per litter was considered as a single score.
Thus, seven replicates from each treatment category were
considered in these observations.
Body weight. Weight is a useful indicator of develop-
ment. Thus, the pups were weighed every alternate day from
PD1 until PD21.
Eye opening and hair appearance. The day at which
the body hair fuzz appeared, and the eyes opened were also
recorded. These two parameters are also useful morpholog-
ical indicators of development.
Righting reflex. The time taken by a pup placed on its
back to turn over and place all four paws on the substrate
was recorded. An upper limit of 2 min is set for this test.
Rotating reflex. The surface used to measure the ro-
tating reflex was the same as that used for righting reflex,
except that it was inclined at an angle of 30◦. The pups were
placed on this surface with their heads pointed downwards.
The time elapsed until the pup rotate its body through 180◦
geonegatively and face its head upwards was recorded as the
rotating time. The upper limit of this test was also set at 2
Cliff avoidance. Pups were placed on the edge of a table
top with the forepaws and face over the edge. The time
taken by the pup to back away and turn from the “cliff”
was recorded. Again an upper limit of 2 min was chosen. A
latency of 2 min was attributed when the animal fell from
Estimation of acetylcholinestrase
Brain of the tested animals was removed and gently rinsed in
physiological saline (0.9% NaCl), and then blotted in What-
man filter paper. The organ fresh weights were recorded and
frozen until use. A 10% (w/v) homogenate of each frozen tis-
sue was prepared and the supernatant was applied for en-
zyme assay. The acetylcholinestrase activity in the homog-
enized brain tissue was estimated by using acetyl chloride
as the substrate. The specific activity of acetylcholinestrase
was expressed as micromoles acetylcholine chloride hydrol-
ysed per gram wet tissue weight per hour at 37 ± 1◦C.
Learning and memory test in automatic reflex conditioner
Animals of PD35 were tested using an automated shuttle-
box (Ugo basile, Italy). The shuttle-box was divided into
two chambers of equal size with a gate providing access to
the adjacent compartment. A light of 60 W for 5 s was
switched alternately in the two compartments and used as a
conditioned stimulus (CS) which preceded an electric shock
(1 mA for 5 s) by 5 s called the unconditioned stimulus. If
the animal avoided the the unconditioned stimulus by run-
ning into the dark compartment within 5 s after the onset
of the CS, the shuttle box recorded an avoidance response.
Each mouse was given 30 trials daily with a fixed intertribal
interval of 20 s. Without shock, the number of crossings be-
tween the chambers was recorded (intertrial crossing). The
automated shuttle box recorded this parameter during the
whole experimental period of 50 trials (Hacioglu et al. 2003).
Total selenium measurement
This method was performed according to the previously
described technique by Lechner-Doll et al. (1990). To-
tal selenium in brain, kidney and liver tissues was mea-
sured using atomic absorption spectrophotometry, using a
PC-based ZL4100 Atomic Absorption Spectrophotometer
(Perkin-Elmer) equipped with auto-sampler or automated
Zeeman-effect background correction (Spectra AA-600, Var-
ian Instruments, Walnut Creek, CA). Tissues were homog-
enized in 0.25% triton X-100 (10 ml diluent/1 g tissue
weight). Standard curves ranging from 40–800 ng ml−1were
performed. Tissue homogenates were diluted 1 : 5 in a dilu-
ent consists of 0.2% nitric acid, 0.1% TritonTMX-100, 1%
Pd(NO3)2 and 0.1 % Mg(NO3)2 and then 20 µl was in-
jected. Two concentrations (600 ng ml−1and 100 ng ml−1)
from the standard curves prepared from tissue homogenate
were processed in duplicate and used as quality control sam-
ples for tissue samples. Other quality control samples spiked
with selenite or L-selenomethionine at two different concen-
trations (one at 500 ng ml−1and one at 150 ng ml−1) were
run with the tested samples. The standard procedure called
for repeated analysis of samples for which the corresponding
quality control (QC) sample showed a coefficient of varia-
tion greater than established acceptable values (i.e., > 15%
for high-QCs; by > 20% for low-QCs).
The data of body weight, morphological developments and
sensory motor reflexes were compared within the experimen-
tal groups by the analysis of variance (ANOVA) and were
subsequently analyzed by Student’s t-test (Yamane 1973).
Results and discussion
The common dose of selenium in animal feed is 0.3
mg/kg of diet of sodium selenite (Leeson et al. 2008;
Prenatal effects of sodium selenite on mice offspring
Fig. 1. Morphological development of mice: body weight (upper),
eye opening and body hair appearance (lower) of control pups
and those born to mothers consuming sodium selenite. All obser-
vations were recorded on PD1 and repeated every other day until
PD21. Results are expressed as means and standard errors (M ±
SE). * P < 0.05, ** P < 0.01.
Calamari et al. 2009). In this study, doses of 1mg/kg
and 4mg/kg of sodium selenite of the diet were applied.
Postnatal developments are crucial indicators for the se-
lenite prenatal exposure-stress. From PD1 to PD21 the
hair appearance, opening of eyes and the body weight
gain were observed. The present results clearly show
that the body weight of the pups born to mothers con-
suming sodium selenite during prenatal period lagged
behind their controls from the day of their birth (PD1)
and remained so almost throughout their weaning pe-
riod until PD21 in a dose-dependent manner. The de-
cline in body weight at lower and higher doses was sta-
tistically insignificant throughout the weaning period
(Fig. 1). All tested morphological developments, like
eye opening and body hair appearance took place after
those of the control. Both of these morphological fea-
tures were significantly (P < 0.05) declined, at both the
lower and the higher dose of the selenite (Fig. 1).
It is known that neural tissues during embryonic
Fig. 2. Early development of different sensory motor reflexes in
pups born to both control and treated mothers. A: The mean
righting reflex in seconds; B: The rotating reflex; C: The cliff
avoidance reflex in seconds. Pups born to treated mothers were
inactive compared to the controls. All observations were recorded
on PD1 and repeated every other day until PD21. Results are
expressed as means and standard errors (M ± SE). * P < 0.05,
** P < 0.01.
development attract selenium. It was, therefore, ex-
pected that a direct change in behavioral elements must
be occurred. Both the sensory motor reflexes and the
ability of the tested animals for learning were addressed.
Prenatal exposure of mice to selenite had a significant
and dose dependent inhibitory effect on the early devel-
opment of all sensory motor reflexes in the pups. Pups
born to treated mothers were inactive compared to the
controls. During the first three weeks of the postnatal
development, selenite had a dose-dependent and signif-
icant inhibitory effect on the righting reflex (Fig. 2A),
the rotating reflex (Fig. 2B) and the cliff avoidance ac-
J.S. Ajarem et al.
Fig. 3. Learning and memory test in automatic reflex conditioner
(shuttle box) showing the latency to avoid shock in seconds (A)
and the number of intertrial crossing between the two compart-
ments (B). All observations were recorded on PD35 and repeated
for 3 days and each mouse was given 30 trials daily with a fixed
intertribal interval of 20 s. Results were expressed as the mean
percentage of avoidance responses for each daily shuttle box ses-
sion. * P < 0.05, ** P < 0.01.
tivity (Fig. 2C) especially at the higher dose. The lower
dose showed a significant inhibition of the cliff avoid-
ance activity (Fig. 2C) from the PD5 onwards until
PD21 as compared to the controls.
Results of learning showed that high concentration
of selenite has impaired the avoidance conditioning in
the shuttle box. Impairment of active avoidance learn-
ing in all tested groups was clearly observed. It was
observed that the control groups learned to avoid the
unconditioned stimulus by running into the other com-
partment during the unconditioned stimulus on almost
all days of the experiment and recorded high success
Fig. 4. Learning and memory test in automatic reflex conditioner
(shuttle box) showing the number of stimulating crossings in sec-
onds (A) and the number of the reinforced crossing between the
two compartments (B). All observations were recorded on PD35
and repeated for 3 days and each mouse was given 30 trials daily
with a fixed intertribal interval of 20 s. Results were expressed as
the mean percentage of avoidance responses for each daily shuttle
box session. * P < 0.05, ** P < 0.01.
rates of avoidance response (Figs 3, 4). The pups born
to selenite-treated mothers did not learn to avoid the
unconditioned stimulus and the number of crossings
during the conditioned stimulus decreased over time
during the experiment. They showed a dose-dependent
significant increase of the latency to avoid the shock
(Fig. 3A). The number of the intertrial crossings be-
tween the two compartments (Fig. 3B), the number
of stimulating crossings (Fig. 4A) and the number of
the reinforced crossing between the two compartments
(Fig. 4B) were significantly decreased.
Acetylcholine is a clear biomarker of behavioral
Prenatal effects of sodium selenite on mice offspring
Fig. 5. Acetyl cholinesterase estimation in the brain homogenates.
Offspring were subjected to biochemical studies from the day of
birth (PD1) until the day 21 (PD21). Results are expressed as
means and standard errors (M ± SE). * P < 0.05, ** P < 0.01.
changes. It significantly decreased until PD 14 when-
ever, it started to elevate as the concentration of selen-
ite gradually decreased in the brain and other tested
tissues (Fig. 5). The concentration of selenium was an-
alyzed by atomic absorption in order to confirm the
placental transfer of selenium to the tested organs. Sig-
nificant high concentration of selenium was detected
in the brain, kidneys and liver tissues when tested on
PD20 (Figs 6A–C). On PD40, it was found also signifi-
cant high concentrations of selenium in the same tested
Selenium has a major role in biological activities
as it is a part of the anti-oxidant system. Because it be-
longs to nutrients that are essential for health, selenium
is recently applied to many attempts in different thera-
peutic approaches on animal models (Serdar et al. 2009;
Bartel et al. 2010; Hu et al. 2010; Venkitaraman et al.
2010; Jung & Seo 2010; Williams et al. 2010). Its biolog-
ical activity, however, mainly depends on its dose, with
a potential of selenium to induce detrimental effects at
high doses. The developmental toxicity of selenium is a
nutritional, environmental and medicinal concern, with
sodium selenite being the most compound of choice.
We studied the prenatal exposure of pregnant fe-
males to the widely applied, sodium selenite, on the
postnatal development of pups, with an inhibitory role
of selenium on the different investigated parameters.
Thus, the present results clearly suggest that prenatal
exposure to high doses of selenium is extremely harm-
ful to the developing pups. A strong correlation between
maternal selenium-exposure and the effects on various
aspects of the developed pups was found indicating pre-
natal transfer of selenium from mother to embryos. This
was proved in the present study by the atomic absorp-
tion analysis that revealed high levels of the selenium
in all investigated tissues (brain, liver and kidney) of
Fig. 6. Total selenium measurement analysis in brain (A), kid-
ney (B) and hepatic (C) tissues; Selenium was measured by elec-
trothermal atomic absorption spectrophotometry. Selenium was
detected in the developed pups-tissues at PD20 and PD40. Re-
sults are expressed as means and standard errors (M ± SE). * P
< 0.05, ** P < 0.01.
the offspring born to the treated mothers. The placen-
tal transfer of selenium in embryonic and fetal tissues
has been investigated in mice in early, mid, and late
pregnancy by Danielsson et al. (1990). They found that
the placental transfer of selenium was increased with
time after dosing and with progression from embryonic
through fetal stages suggesting active placental trans-
Results strongly revealed a statistically significant
decline in body weight at selenite high-concentration.
The significant decline in the body weight was in agree-
ment with the results obtained by Ferm et al. (1990).
They noted fetal body weights and lengths were re-
duced in a dose-dependent manner. Selenium derived
mothers to anorexia (Satoh et al. 1981) in which food
and water uptake decreased to the minimum. Prenatal
exposure to selenite also causes decline in the ability
of pups to lactation (Satoh 2007), blocks the intestinal
absorption of different food derivatives (Kasik & Rice
1995) and decreases the level of thyroxine which affects
J.S. Ajarem et al.
the body weight (Kaprara & Krassas 2006). Beside the
possible oxidation and inhibition of protein synthesis
in the body of the developed pups, selenite might in-
duce oxidative stress in the secretary cells of the mam-
mary glands in mothers. Therefore, this quantitatively
decreased the feeding rate of their offspring and its sub-
sequent body weight.
Recent studies have reported the toxicity and re-
lated oxidative stress of selenium (El-Demerdash 2001;
Isai et al. 2009; Fujimoto et al. 2009). There is a large
body of evidence implicating oxidative stress and re-
active oxygen species (ROS) in the mechanism of sele-
nium toxicity (Isai et al. 2009; Misra & Niyogi 2009).
In oxidative stress, the metabolism of oxygen leads to
generation of ROS which are able to oxidize almost all
classes of macromolecules, including proteins, lipids and
nucleic acids (Zachara et al. 2006; Fujimoto et al. 2009;
Mániková et al. 2010). It was proved that selenite ions
have the potential to induce oxidative DNA damage
in the liver cell culture through either an increase in
ROS formation (Fujimoto et al. 2009) or probably by
inducing the imbalance of intracellular glutathione re-
dox (Misra & Niyogi 2009). It is more likely that in
this study, liver greatly affected, since atomic absorp-
tion analysis proved presence of high concentration of
this element within the hepatic tissues. This explains
the effects resulted within the cells during the early de-
velopmental stages. Therefore, an important reason of
the decline in body weight gain might be that selenite
inhibited the protein synthesis and/or oxidize protein
macromolecules which were much more needed in the
developed body muscles, especially at the beginning of
life. Usami et al. (2008) investigated selenium embry-
otoxicity and found quantitative changes in proteins,
growth inhibition and morphological abnormalities of
Selenium affects the metabolic processes via chang-
ing either the structure and function of mitochondria or
the mechanism of action of endocrine hormones which
controls metabolism (K¨ ohrle et al. 2005). Additionally,
the delay in hair appearance might be due to the de-
lay in the collagen protein-synthesis by oxidation. The
retardation in the opening of eyes as it was attributed
to an abnormality in the neural developments (Georgi-
eff 2007), it could strongly explain what has happened
in the learning and memory, and in the sensory motor
reflexes as discussed below. Myelin sheath of the neu-
rons of the optic nerve is directly affected by selenium
(Satoh et al. 1981).
The possible DNA damage by selenite during preg-
nancy could affect the sensory motor reflexes of the
early developmental stages. This obviously reflected
on the different inhibited reflex activities. Gupta et
al. (2001) observed a suppression effect of selenium
on the sensory motor activities and imbalance in ro-
dents. Danielsson et al. (1990) reported that the high-
est uptake of selenium in the embryo was in the
neuro-epithelium. Therefore, sodium selenite caused
abnormalities such as a swelling of the rhomben-
cephalon in embryos (Usami & Ohno 1996). Also Ferm
et al. (1990) found malformations, mainly encephalo-
celes, with maternal selenium-toxicity manifested by
weight loss in pregnant hamsters treated with selenite
during embryogenesis. Thus, significant abnormalities
are involved the nervous system during developmen-
tal stages after exposure to selenite. Atomic absorp-
tion analysis proved the presence of high concentration
of selenium within the brain tissues of the developed
Acetylcholine can be a marker in teratological
studies (Ajarem & Ahmad 1991, 1998), since any
change in the behavior process due to any toxicant is
indicated by alteration in acetylcholine (Reddy et al.
2003). It was found that selenite suppressed the acetyl-
choline at PD7 and at PD14. Thus, delayed sensory
motor reflexes are also explained by the significant de-
crease of acetylcholine, the most important neurotrans-
mitter. The direct result of this decrease is the dis-
turbance of neuromuscular signals and therefore, the
inhibitory effects in all elements of acts and postures
in pups of treated mothers. It has been reported that
alteration in the brain enzymes are among the fac-
tors responsible for disturbances in behavioral activi-
ties of the affected animals (Ajarem & Ahmad 1998).
Taken together, these could explain why sensory mo-
tor reflexes were retarded during the postnatal devel-
The active avoidance training-test indicated that
selenite exposure was associated with learning impair-
ment. Similar results for aluminum were obtained by
Sun et al. (1999) and Miu et al. (2003). Selenium im-
paired the learning and memory centers in the brain
(Roig et al. 2006), especially when exposure taken place
during gestation (Lam et al. 2002). It also leads to the
degeneration of neurons in hippocampus which plays
an important role in learning process (Guo et al. 2010).
A growing body of evidence suggests that reactive ROS
play a crucial role in the development of these impair-
ments as discussed above (Yamada et al. 1995; Ku-
cukatay et al. 2007). The increased production of ox-
idants altered neurotransmitter release and increased
membrane permeability (Gupta et al. 1991). Besides,
free radicals may degrade nitric oxide (NO), which plays
an important role as a diffusible intercellular signaling
molecule (a neurotransmitter in the central nervous sys-
tem) (Geyer et al. 1997).
This study, by atomic absorption analysis, con-
firmed the placental transfer of selenium to the tis-
sue of embryos from treated mothers. Selenium which
was detected in the brain, liver and kidney of the
pups, strongly affected the learning, sensory motor re-
flexes and acetylcholine levels. Thus, selenium at high
doses is highly toxic to the tissue of embryos. Based
on this study and other previous studies (Goldhaber
2003; Miguel & Carmen 2008), we expect that chronic
supplementation of selenium can cause serious effects
especially abnormal functioning of the nervous system.
Therefore, when applied in the therapeutic approaches,
especially in case of pregnancy, selenium must be even-
Prenatal effects of sodium selenite on mice offspring
The authors are grateful to the department of Zoology, Col-
lege of Sciences, King Saud University for support.
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Received April 28, 2010
Accepted November 5, 2010