African Journal of Pharmacy and Pharmacology Vol. 5(11), pp. 1389-1397, 22 September, 2011
Available online http://www.academicjournals.org/ajpp
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Impairment of active avoidance learning and sensory
motor reflexes in mice offspring induced by perinatal
acute toxic exposure to selenium
Gada Al Basher1, Hossam Ebaid1,2, Jamaan Ajarem1 and Gasem Abu-Taweel3
1Department of Zoology, College of Science, King Saud University, Saudi Arabia, P.O.Box 2455, Riyadh – 11451.
2Department of Zoology, Faculty of Science, El-Minia University, Egypt.
3Department of Biology, College of Education, Dammam University, P.O. 2375, Dammam - 31451, Saudi Arabia.
Accepted 7 June, 2011
Selenium is an essential element with a narrow margin between beneficial and toxic effects. The
learning and sensory motor reflexes-changes were studied after the perinatal exposure of mice to acute
toxic doses of sodium selenite. Atomic absorption as well as the behavioral observations were
employed. Adult pregnant mice was assigned into three groups: the first group was remained as a
control group; the second and the third groups were orally administrated sodium selenite at doses of 1
mg/Kg (1 ppm) and 4 mg/kg (4 ppm) of the diet, respectively started from the 7th day of gestation to the
15th day of birth. Results revealed that body weight gain came significantly lower in pups born to
treated mothers than those of the control pups. The appearance of body hair and opening of eyes of the
pups from treated mothers were delayed in a dose-dependent manner. Selenite also inhibited the
sensory motor reflexes in all elements in a dose dependent manner. The active avoidance test indicated
that selenite exposure was associated with learning impairment. Acetylcholine recorded a significant
decrease in treated pups. Significant high concentrations of selenium in the brain, liver and kidney was
detected, indicating active transfer of selenium from mothers during pregnancy and lactation periods.
Key words: Perinatal, sodium selenite, atomic absorption, acetylcholine, active avoidance test, sensory motor
Environmental toxicity of selenium in humans is
associated with symptoms such as anaemia, leucopoenia
and many other health problems (Sunde, 2000; Mataix
and Lippies, 2002; Tinggi, 2003). Attention has been paid
recently to residual selenium returned to the soil (Oldfield,
1997). Although, the need for selenium in nutrition is well
recognized, the question concerning the high doses of
selenium for supplemental use is still being debated.
Despite, sodium selenite is widely used as a source of Se
in animal feed, it is not a natural nutritional form of
selenium and it can create ecological problems (Kumar et
al., 2010). Receiving selenium as selenomethionine, the
chief natural nutritional form present in grain crops is better
*Corresponding author. E-mail: email@example.com.
than sodium selenite (Schrauzer and Surai, 2009). The
general population should be warned against the
employment of selenium supplements for prevention of
diseases. The benefits of selenium supplementation are
still uncertain, and their indiscriminate use could generate
an increased risk of toxicity (Miguel and Carmen, 2008).
Mechanisms lying behind detrimental effects of selenium
are poorly understood yet. They can involve DNA
damage induction and consequently, DNA damage
response and repair pathways may play a crucial role in
cellular response to selenium (Manikova et al., 2009).
Toxicity of selenium, however, depends on many factors
like the selenium species, amount ingested, age, physio-
logical status, and dietary interaction with other nutrients
(Mataix and Liopis, 2002). Data reported the memory and
the other cognitive and behavioral problems induced by
selenite–high doses are poorly available. This paper
1390 Afr. J. Pharm. Pharmacol.
describes an attempt to determine the high dose-selenite
induced neurobehavioral changes in an experimental
MATERIALS AND METHODS
Male and female Swiss-Webster strain mice (8 weeks old) were
housed in opaque plastic cages. Animals were kept under reversed
lighting 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 7th day of pregnancy to the 15th day of birth (PD15). The
common dose of selenium in animal feed is 0.3 mg/kg of diet of
sodium selenite (Leeson et al., 2008; Calamari et al., 2009). Offsprings
were subjected to the developmental, neurobehavioral and biochemical
studies from the day of birth (PD1) until the day 40 (PD40).
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 PD21. During the
weaning period, three pups of each litter were color marked from
the others without any consideration to its sex, and were subjected
to various behavioral tests (described below) under dim lighting. All
observations were recorded on PD1 and repeated every other day
until PD21. These observations were used to measure the early
development of sensory motor coordination reflexes together with
morphological development in the pups.
Sensory motor coordination reflexes
Body weight: Weight is a useful indicator of development. 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
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
Pups were placed on the 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. 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.
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
morphological indicators of
preceded an electric shock (1 mA for 5 s) by 5 s called the
unconditioned stimulus. If the animal avoided the unconditioned
stimulus by running 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 between the
chambers was recorded (intertrial crossing). The automated shuttle
box recorded this parameter during the whole experimental period
of 30 trials (Hacioglu et al., 2003).
Estimation of acetylcholinestrase
Brain of the tested animals was removed and gently rinsed in
physiological saline (0.9% NaCl), and then blotted in Whatman filter
paper. The organ fresh weights were recorded and frozen until use.
A 10% (w/v) homogenate of each frozen tissue was prepared and
the supernatant was applied
acetylcholinestrase activity in the homogenized brain tissue was
estimated by using acetyl chloride as the substrate. The specific
activity of acetylcholinestrase was expressed as micromoles
acetylcholine chloride hydrolysed per gram wet tissue weight per
hour at 37±1° C.
Total selenium measurement
This method was performed according to the previously described
technique by Lechner-Doll et al. (1990). Total selenium in brain,
kidney and liver tissues was measured 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,
Varian Instruments, Walnut Creek, CA). Tissues were homogenized
in 0.25% triton X-100 (10 ml diluent/1g tissue weight). Standard
curves ranging from 40 to 800 ng/ml were performed. Tissue
homogenates were diluted 1:5 in a diluent consists of 0.2% nitric
acid, 0.1% Triton™ X-100, 1% Pd(NO3)2 and 0.1% Mg(NO3)2 and
then 20 ?l was injected. Two concentrations (600 and 100 ng/ml)
from the standard curves prepared from tissue homogenate were
processed in duplicate and used as quality control samples for
tissue samples. Other quality control samples spiked with selenite
or L-selenomethionine at two different concentrations (one at 500
ng/ml and one at 150 ng/ml)) 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 variation greater than established acceptable values
(that is,>15% for high-QCs; by >20% for low-QCs).
The data of body weight, morphological developments and sensory
motor reflexes were compared within the experimental groups by
the analysis of variance (ANOVA) and were subsequently analyzed
by Student’s t-test (Yamane, 1973).
Tissue concentration of
The concentration of selenium was analyzed by atomic
absorption in order to confirm the placental transfer of
selenium to the tested organs. Significant high concen-
tration of selenium was detected in the brain, kidney and
for enzyme assay. The
selenium and the
Basher et al. 1391
Figure 1. Total selenium measurement (mg/g tissue) in the brain (A), kidney (B) and
hepatic (C) tissues; Selenium was measured by electrothermal atomic absorption
spectrophotometry. Selenium was detected in the developed pups-tissues at PD20 and
PD40. Results are expressed as mean and standard errors (M±SE). The result was
considered significant * when P < 0.05 and highly significant ** when P < 0.01.
liver tissues when tested on PD20 (Figure 1). On PD40, it
was found also significant high concentrations of
selenium in the same tested tissues. Acetylcholine is a
clear biomarker to the behavioral changes. It recorded a
significant decrease until PD 14 whenever, it started to
elevate (Figure 2).
1392 Afr. J. Pharm. Pharmacol.
Figure 2. 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 mean and standard errors (M±SE). The result was considered significant * when P
< 0.05 and highly significant ** when P < 0.01.
Figure 3. The morphological developments, namely, the body weight (upper), eye opening and body
hair appearance (lower) of the developed control pups and those born to mothers consuming sodium
selenite. All observations were recorded on PD1 and repeated every other day until PD21. Results are
expressed as mean and standard errors (M±SE). The result was considered significant * when P <
0.05 and highly significant ** when P < 0.01.
Selenium delayed the hair appearance and the eye
Postnatal developments are crucial indicators for the
selenite prenatal exposure-stress. From PD1 to PD21,
the body weight gain, the hair appearance and the
opening of eyes were daily observed. The body weight of
pups born to mothers consuming sodium selenite lagged
behind controls from the day of birth and remained so
almost throughout their weaning period until PD21 in a
dose-dependent manner. Eye opening and body hair
appearance were significantly (p<0.05) delayed after
those of the controls (Figure 3).
Early development of sensory motor reflexes were
inhibited by selenium
Neural tissues during embryogenic developments attract
selenium. It was, therefore, expected that a direct change
Basher et al. 1393
Figure 4. The early development of the different sensory motor reflexes in the 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
comparing to the controls. All observations were recorded on PD1 and repeated every other
day until PD21. Results are expressed as mean and standard errors (M±SE). The result was
considered significant * when P < 0.05 and highly significant ** when P < 0.01.
in behavioral elements must be occurred. Both the
sensory motor reflexes and the ability of the tested
animals for learning were addressed. Perinatal exposure
of mice to selenite had a significant and dose dependent
inhibitory effect on the early development of all sensory
motor reflexes in the pups. During the first three weeks of
the postnatal development, selenite had a dose-
dependent and significant inhibitory effect on the righting
reflex, the rotating reflex and the cliff avoidance activity
especially at the higher dose (Figure 4).
1394 Afr. J. Pharm. Pharmacol.
Figure 5. Learning and memory test in automatic reflex conditioner (shuttle box) showing the latency to avoid shock
in seconds (upper right), the number of intertrial crossing between the two compartments (upper left), the number of
reinforced crossing (lower right) and the number of stimulating crossings in seconds (lower left). 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 seconds. The results were expressed as the mean percentage of avoidance responses for each daily
shuttle box session. The result was considered significant * when P < 0.05 and highly significant ** when P < 0.01.
Impairment of active avoidance learning and memory
in shuttle box
Results of learning showed that high concentration of
selenite has impaired the avoidance conditioning in the
shuttle box. Impairment of active avoidance learning 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 compartment during
the unconditioned stimulus on almost all days of the
experiment and recorded high success rates of
avoidance response (Figure 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. The
number of the intertrial crossings between the two
compartments, the number of stimulating crossings and
the number of the reinforced crossing between the two
compartments (Figure 5) were significantly decreased.
Since it belongs to nutrients that are essential for health,
selenium is recently applied to many attempts in different
therapeutic approaches on animal models (Bartel et al.,
2010; Hu et al., 2010; Venkitaraman et al., 2010; Jung
and Seo, 2010; Williams et al., 2010; Serdar et al., 2009).
The developmental toxicity of selenium is a nutritional,
environmental and medicinal concern, with sodium
selenite being the most compound of choice. We studied
the perinatal exposure of gestated mice females 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 exposure to high
doses of selenium is extremely harmful to the developing
pups indicating an active transfer of selenium from
mother to embryos. This was proved in the present study
by the atomic absorption analysis that revealed high
levels of selenium in all investigated tissues (brain, liver
and kidney) of the offspring. The placental 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.
Results strongly revealed a statistically significant
decline in body weight at selenite high-concentration as
was found by Ferm et al. (1990). They noted fetal body
weights and lengths were reduced in a dose-dependent
manner. Selenium caused anorexia (Satoh et al., 1981)
in which food and water uptake of mothers decreased to
the minimum. Exposure to selenite also causes a decline
in the ability of pups to lactation (Satoh, 2007), blocks the
intestinal absorption of different food derivatives (Kasik
and Rice, 1995) and decreases the level of thyroxine
which affects the body weight (Kaprara and Krassas,
2006). Beside the possible oxidation and inhibition of
protein synthesis in the body of the developed pups,
selenite might induce oxidative stress in the secretary
cells of the mammary glands in mothers. Therefore, this
quantitatively decreased the feeding rate of their offspring
and its subsequent body weight.
Recent studies have reported the toxicity and related
oxidative stress of selenium (El-Demerdash, 2001;
Fujimoto et al., 2009; Isai et al., 2009). There is a large
body of evidence implicating oxidative stress and reactive
oxygen species (ROS) in the mechanism of selenium
toxicity (Isai et al., 2009; Misra and 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;
Manikova 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 redox (Misra and
Niyogi, 2009). It is more likely that in this study, liver
greatly affected, since atomic absorption analysis proved the
presence of high concentration of this element within the
Basher et al. 1395
hepatic tissues. This explains the effects resulted within
the cells during the early developmental 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 embryotoxicity
quantitative changes in proteins, growth inhibition and
morphological abnormalities of cultured embryos.
Selenium affects the metabolic processes via changing
either the structure and function of mitochondria or the
mechanism of action of endocrine hormones which
controls metabolism (Kohrle et al., 2005). Additionally, the
delay in hair appearance might be due to the delay 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 (Georgieff, 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 neurons of the optic nerve is
directly affected by selenium (Satoh et al., 1981). The
possible DNA damage by selenite during pregnancy
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 rodents. The
high uptake of selenium in the embryo was in the neuro-
epithelium (Danielsson et al., 1990; Ferm et al., 1990;
Usami and Ohno, 1996). Thus, significant abnormalities
are involved the nervous system during developmental
stages after exposure to selenite. Atomic absorption
analysis proved the presence of high concentration of
selenium within the brain tissues of the developed pups.
The active avoidance training-test indicated that selenite
exposure was associated with learning impairment.
Similar results for aluminum were obtained by Sun et al.
(1999), Miu et al. (2003) and Abu et al. (2010). Selenium
impaired 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
impairments as discussed above (Kucukatay et al., 2007;
Yamada et al., 1995). The increased production of
oxidants 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
system) (Geyer et al., 1997). Acetylcholine can be a
marker in teratological studies (Ajarem and 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
1396 Afr. J. Pharm. Pharmacol.
the acetylcholine at PD7 and at PD14. Thus, delayed
sensory motor reflexes are also explained by the
significant decrease of acetylcholine, the most important
neurotransmitter. The direct result of this decrease is the
disturbance of neuromuscular signals and therefore, the
inhibitory effects in all elements of acts and postures in
pups born to treated mothers. It has been reported that
alteration in the brain enzymes are among the factors
responsible for disturbances in behavioral activities of the
affected animals (Ajarem and Ahmad, 1998). Taken
together, these could explain why sensory motor reflexes
This study, by atomic absorption analysis, confirmed
the placental transfer of selenium to the tissue of
embryos from treated mothers in a prenatal exposure.
Selenium which was detected in the brain, liver and
kidney of the pups, strongly affected the learning,
sensory motor reflexes 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 and Carmen, 2008; Ajarem et
al., 2011), 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
gestation, selenium must be eventually monitored.
The Authors extend their appreciation to the Deanship of
Scientific Research at king Saud University for funding
the work through the research group project No. RGP-
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