DEPLETION OF VITAMIN-C AND GLUTATHIONE BY
ACRYLAMIDE CAUSES DAMAGE TO HIPPOCAMPUS REGION
OF BRAIN IN CHICK EMBRYO
M. Venkataswamy1, K V Subbaiah2, B. Suman1, M. Meenabai1, K J Rao2 and K.
Thyaga Raju1*
1
Department of Biochemistry (DST FIST and UGC BSR) and 2Department of Zoology,
Sri Venkateswara University, Tirupati-517502, A. P., INDIA
*E-mail: thyagarajuk_2002@rediffmail.com
ABSTRACT
The present study deals with the effects on depletion of brain system based on
vitamin-c, glutathione and modification of histological analysis by the treatment of
acrylamide to chick embryo. The in vivo studies carried out with exposure of different
concentrations of Acrylamide (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6mg) on developing brain
tissue of various batches of experimental Bobcock strain chick embryo at different time
of intervals (24, 48 and 72h) showed a significant reduction in the contents of vitamin-c
and glutathione. Also the acrylamide effect caused mild structural damages at 0.3 and
0.4mg treatment and further mild hemorrhages, necrotic damages and formation of
vacuoles were observed at 0.5 and 0.6mg acrylamide treatments in hippocampus region
of chick embryo brain tissue under microscopic examination. Finally our experimental
results conclude that because of reduction of glutathione and vitamin-c the free radical
scavenger´s role was suppressed and histological analysis indicated propagation of
toxicity to hippocampus and selective hippocampus impairment in recognition, working
memory for nonspatial stimuli and inhibition of the nerve impulse transmission by
acrylamide.
KEY WORDS:
hippocampus.
Acrylamide,
chick
embryonic
brain,
glutathione,
vitamin-c,
INTRODUCTION
Acrylamide (CH2 CH CONH2), an industrially produced α, β-unsaturated
(conjugated) reactive molecule, is used worldwide to synthesize polyacrylamide.
Acrylamide has been identified in at least 3 of the 1,699 hazardous waste sites that have
been proposed for inclusion on the Environment protection agency (EPA) National
Priorities List (NPL). The National Toxicology Program (NTP) and the International
Agency for Research on Cancer consider acrylamide to be a “probable human
carcinogen,” based on studies in laboratory animals given acrylamide in drinking water.
Fig. 1: Structure of Acrylamide
Acrylamide and polyacrylamide are used primarily as flocculants for classifying
drinking, and treating municipal and industrial effluents. They are also used in the
production of dyes and organic chemicals, contact lenses, cosmetics and toiletries,
permanent-press fabrics, textiles, pulp and paper products; in ore processing, sugar
refining, and as a chemical grouting agent, and soil stabilizer for the construction of
tunnels, in sewers, wells, and reservoirs. Because of their use as mentioned above they
may be released to the environment and causes pollution to water. Residual monomer in
the polyacrylamide is the main source of drinking water contamination. Further it can be
released to soil and land from plastics, dye industries and from acrylamide-containing
grouting agents used in reservoirs and wells.
Acrylamide was identified in food samples cooked at high temperatures.
Concentrations of acrylamide in food vary with the type of food, cooking method,
temperature, water content, food thickness, and length of heating. Carbohydrate-rich
foods typically contain the highest levels of acrylamide. The chemical nature of
acrylamide is a white or colorless, and odorless crystalline solid. Acrylamide is stable at
room temperature but can violently polymerize at its melting point or under UV light1, 2, 3.
Acrid fumes, as well as NOx, may be released when it is heated to decomposition1.
Acrylamide is soluble in water, alcohol, and acetone, and is insoluble in benzene and
heptanes2, 4.
Neurological Effects of Acrylamide
Ataxia and skeletal muscle weakness are hallmark symptoms of acrylamide
induced central-peripheral neuropathy and symptoms were primarily associated with
occupational exposure potential via both inhalation and dermal contact5, 6. Available
information in animals primarily involves oral exposure. Clinical signs can be elicited
following single oral dosing in the range of 100–200 mg/kg; and lower dose levels
require repeated dosing to elicit clinical signs. Information in humans is available from
numerous case reports in which acrylamide exposure has been associated with signs of
impaired neurological performance in central and peripheral nervous systems that include
impaired motor function and muscle weakness. Evidence of degenerative lesions in
peripheral nerve fibers, as observed by light and electron microscopy, have been detected
at oral doses lower than those eliciting clinical signs and other overt indications of
functional deficit.
Acrylamide is a carcinogen with the potential to cause nervous system damage7
Ingestion of foodstuffs containing acrylamide appears to be one of the most common
methods of exposure for the general public. Average estimated intake of acrylamide from
food sources ranged from 0.8 to 6.0 μg/kg bw/day for short-term exposure and 0.3 to 0.8
μg/kg bodyweight/day for long-term exposure8, 9. Children may be susceptible to foodborne exposure 2–3 times that of adults on a body weight basis8, 9. Based on evidence of
early structural and functional damage10, suggested that nerve terminals were of the
primary site to acrylamide action and that synaptic dysfunction and subsequent
degeneration by acrylamide neurotoxicity.
Developmental effects of acrylamide
Three developmental toxicity studies are reported in rats, including one study of
developmental neurotoxicity. No significant treatment-related effects on embryo/fetal
viability, growth, or development or dose-related increases in fetal malformations were
found in off-spring of female rats administered up to 15 mg/kg/day on gestational days 6
to 20 or of female mice administered up to 15 mg/kg/day on gestational days 6 to 17. In
the off-spring of female rats treated from gestational day 6 to lactational day 10, there
were no clinical signs of toxicity and pup survival was not affected at birth or up to 21
days at maternal doses of 10 mg/kg/day or lower11. In this study, pup body weight
(preweaning) at a maternal dose of 15 mg/kg/day and reduced pup survival at birth and
for days 1 to 3 at a maternal dose of 20mg/kg/day were observed; however, maternal
toxicity and signs of neurotoxicity were significant at these doses . Developmental
neurotoxicity has been assessed in rats. At doses equal to or less than 10 mg/kg/day
during gestation, no effects in behavioral tests (open-field motor activity, auditory startle
habituation, or passive avoidance) were noted in offspring when tested either within 21
days post-natal or as adults. Decreases in catecholamine (dopamine) levels in cerebellum,
pons medulla, and mid-brain but not the hypothalamus or basal ganglia were noted and
were more pronounced in younger rats (12, 15, or 21 days old) than older rats (60 days)
when exposed directly to acrylamide (25 mg/kg/day for 5 days) at various ages (12, 15,
21, or 60 days post-partum).
Histopathological effects of acrylamide
Histopathological evidence of acrylamide-induced peripheral neuropathy has
been observed in rats receiving oral doses as low as 1 mg/kg/day for 3 months; the
observed degenerative effects in peripheral nerve fibers at such dose levels have been
shown to be completely reversible within a few months following the cessation of
exposure. Considering all the facts experiments were conducted on chick embryos to
study the effect of acrylamide on developing brain.
MATERIALS AND METHODS
Acrylamide (99.9%), a monomer form, purchased from Bio-Rad laboratories
(Richmond, USA), and other chemicals purchased indigenously were of pure and used
for the analysis of various samples of our research.
Source of fertilized eggs and incubation conditions
Freshly laid Bobcock strain zero day old fertilized eggs were purchased from Sri
Venkateswara Veterinary University, Tirupati and Sri Balaji hatcheries, Chittoor, Andhra
Pradesh. The eggs were incubated horizontally at 37.5±0.5˚C with a relative humidity of
65% in an egg incubator, we consider day1 (d1) as an incubation period of 24h. The
humidity of the incubator was maintained by keeping the trough full of water inside. The
water was replaced for every alternate day and the water level was maintained to keep the
same percentage of humidity throughout the incubation. Eggs were rotated manually four
times a day and were examined through the Candler every day for the proper growth and
viability. The dead eggs were removed immediately from the incubator. During all
experiments, the embryos were maintained at 37±0.5˚C except for brief intervals (60-120
seconds) required during the different treatment conditions. During this interval embryos
experienced ambient room temperature (29-30˚C).
Treatment
Acrylamide treatment and collection of brain tissue
A group of six eggs (n=6) were maintained for each time point and dose, 0.1, 0.2,
0.3, 0.4, 0.5 and 0.6 mg, of acrylamide in saline was administered as single dose
separately to fertilized chick embryos on day8 (d8),day9 (d9) and day10 (d10) of
incubation. On 11th day the embryo’s brain was collected and stored for further use at 20°c.
Assay of non-enzymatic antioxidants
Reduced Glutathione (GSH)
Reduced Glutathione content was determined according to the method of Ellman
and Boyne12. 1 g of tissue was homogenized in10 ml of phosphate buffer. From this 0.5
ml was pipette out and precipitated with 2.0 ml of 5% TCA. 1.0 ml of the supernatant
was taken after centrifugation and added to it 0.5 ml of Ellman’s reagent followed by 3.0
ml of phosphate buffer. The yellow color developed was read at 412 nm. A series of
standards were treated in a similar manner along with a blank containing 3.5 ml of buffer.
Ascorbic acid (Vit-C)
Ascorbic acid levels were determined according to the method of Omaye13. 0.5 ml
of tissue homogenate was mixed thoroughly with 1.5 ml of 6%TCA and centrifuged for
20 minutes at 3500 x g. To 0.5 ml of the supernatant, 0.5 ml of DNPH reagent was added
and mixed well. The tubes after 3 hours of incubation at room temperature placed in icecold water and added 2.5 ml of 85% sulphuric acid and further allowed to stand for 30
minutes. A set of standards containing 10-50 mg of ascorbic acid were taken and
processed similarly along with a blank, containing 0.5 ml of 4% TCA. The color
developed was read at 530 nm.
Preparation of slides for histopathology
The developing brains tissues of d11 chick control and treated were dissected and
they were gently rinsed with physiological saline to remove blood and debris adhering to
them. They were fixed in Bouin’s solution until processing. The tissues were washed
with running tap water, overnight to remove Bouin’s solution. After dehydrating through
a graded series of alcohols, the tissues were cleared in methyl benzoate and embedded in
paraffin wax. Sections were cut at 6μ thickness and stained with haematoxylin14 and
counter stained with Eosin dissolved in 95% alcohol. After dehydration and cleaning,
sections were mounted in Canada balsam. Histological examinations of the tissues were
followed according to Humason15 and the specimens were observed under the light
microscope.
Statistical analysis:
Results were expressed as the means ± standard deviation (SD). Differences
between groups were evaluated by using one-way ANOVA, followed by Duncan’s test.
All statistical analyses were performed using the statistical software SPSS 11.0 (SPSS
Ltd., Surrey, UK). The P value of less than 0.05 was considered as statistically
significant.
RESULTS AND DISCUSSION
Glutathione (GSH)
The brain Reduced glutathione (GSH) levels of d(11) developing chick embryos
control and AC-treated 24, 48 and 72h time of interval groups were presented in Table 1.
The glutathione (GSH) levels at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg AC-treated 24h time of
interval groups were 5.716, 5.597, 4.583, 4.010, 3.666 and 2.978, respectively, and there
was a 1.012, 1.034, 1.263, 1.443, 1.579 and 1.943 fold decrease was found in treated than
in the control and 1.037, 1.120, 1.365, 1.474, 1.694, 2.014, 1.045, 1.205, 1.407, 1.579,
1.781 and 2.269 fold decrease in reduced glutathione levels were observed in the brain of
all six AC-treated 48 and 72h time of interval groups when compared to control.
Table-1. Effect of acrylamide on glutathione and ascorbic acid levels in the brain of
11th day old chick embryo.
S. No
Treatment
(mg)
Glutathione
Ascorbic acid
24h
48h
72h
24h
48h
72h
1
Control
5.789 ± 0.15a
5.789 ± 0.15a
5.789 ± 0.15a
1.32 ± 0.04a
1.32 ± 0.04a
1.32 ± 0.04a
2
0.1
5.716 ± 0.16e
5.581 ± 0.13f
5.537 ± 0.08f
1.24 ± 0.04e
1.17 ± 0.04e
1.09 ± 0.04e
3
0.2
5.597 ± 0.11e
5.166 ± 0.21e
4.801 ± 0.17e
1.09 ± 0.06d
1.05 ± 0.03d
0.93 ± 0.04d
4
0.3
4.583 ± 0.15d
4.239 ± 0.21d
4.114 ± 0.20d
0.94 ± 0.06c
0.84 ± 0.04c
0.77 ± 0.04c
5
0.4
4.011 ± 0.18c
3.927 ± 0.12c
3.666 ± 0.10c
0.85 ± 0.04
0.78 ± 0.06c
0.74 ± 0.04c
6
0.5
3.666 ± 0.19b
3.416 ± 0.15b
3.249 ± 0.20b
0.66 ± 0.04a
0.62 ± 0.03b
0.58 ± 0.04b
7
0.6
2.978 ± 0.22a
2.874 ± 0.16a
2.551 ± 0.13a
0.61 ± 0.04a
0.53 ± 0.04a
0.49 ± 0.05a
8
F-Value
230.79
212.36
272.13
141.71
194.00
181.44
Units: mg/g tissue. Each value represents the mean ± SD (n=6). Different letters are
significantly different at the level of p < 0.05 according to the Duncan’s test.
Ascorbic acid (Vit-C)
The brain ascorbic acid (Vit-C) levels of d(11) developing chick embryos control
and AC-treated 24, 48 and 72h time of interval groups was presented in Table 1. The
level of ascorbic acid (Vit-C) at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg AC-treated 24h time of
interval groups were 1.24, 1.09, 0.94, 0.85, 0.66 and 0.61, respectively, and there was a
1.064, 1.211, 1.404, 1.552, 2.00 and 2.163 fold decrease in treated than in the control,
and 1.128, 1.257, 1.571, 1.692, 2.129, 2.490, 1.211, 1.419, 1.714, 1.783, 2.275 and 2.693
fold decreased ascorbic acid (Vit-C) levels were observed in the brain of all six ACtreated 48 and 72h time of interval groups and compared to control.
Histopathological studies
The results of control and treated chick embryonic hippocampus region of brain
histology studies were presented in the Fig. 2-8. In these results the control and 0.1mg
AC treated (24, 48 and 72h) hippocampus of brain showed normal cytoarchitectural Fig.2
and 3. The 0.2, 0.3mg AC treated showed mild degenerative changes in hippocampus of
brain tissue Fig. 4-5, 0.4mg AC treated showed mild and structural damages Fig. 6. The
chick embryonic brain treated with 0.5mg AC at 24, 48h shows structural damages and
72h shows mild hemorrhages (lesion), structural damage shown in Fig. 7. The 0.6mg AC
treated showed structural damages at 24, 48h and 72h showing necrotic damage,
formation of vacuole shown in Fig. 8.
Fig. 2: Histological analysis of Hippocampus region of brain section (11 day old
chick embryo) under the light Microscope (10X) showing normal cytoarchitectural
(Control).
Note: Arrows indicate modification of tissue at an appropriate site in the slide
Fig. 3: 0.1 AC treated 11 day old chick embryonic brain hippocampus region. A)
24h, B) 48h, C) 72h showing no damage
A
B
C
Fig. 4: 0.2 AC treated 11 day old chick embryonic brain hippocampus region, A)
24h, B) 48h showing no damage C) 72h showing mild damage
A
B
C
Fig. 5: 0.3 AC treated 11 day old chick embryonic brain hippocampus region, A)
24h, B) 48h, C) 72h showing mild damages
A
B
C
Fig. 6: 0.4 AC treated 11 day old chick embryonic brain hippocampus region., A)
24h, B) 48h showing mild damages C) 72h showing structural damage
A
B
Fig. 7: 0.5 AC treated 11 day old chick embryonic brain hippocampus region., A)
24h, B) 48h structural damages C) 72h showing mild hemorrhages (L), structural
damage
A
B
C
L
Fig. 8: 0.6 AC treated 11 day old chick embryonic brain hippocampus region., A)
24h, B) 48h structural damages C)72h showing necrotic damage (L), formation of
vacuole (L1)
L
L1
The acrylamide, a synthetic monomer with a wide scope of industrial
applications, is a known industrial toxic chemical that produce neurotoxicity
characterized by progressive neuronal degeneration. Acrylamide may be released to the
environment during production and use of polyacrylamides. Acrylamide is not considered
highly persistent in the environment. It is expected to be highly mobile in soil and water
and is highly susceptible to biodegradation in both media. The International Agency for
Research on Cancer (IARC) has classified acrylamide as “probably carcinogenic to
humans”16.
Acrylamide reacts rapidly with SH groups of biomolecules. Extensive reaction
occurs with proteins too, both on their SH groups and with amino groups. The most
important reaction of acrylamide with proteins is the adduction to hemoglobin (Hb), SH
groups of cysteine and glutathione, and on the NH2 groups of the N-terminal valines.
Further it also forms adducts with cytoskeletal proteins and protamines17. Acrylamide
may pose more than a neurotoxic health hazard to exposed ones which shall have signs of
impaired neurological performance in central and peripheral nervous systems that include
impaired motor function and muscle weakness. Acrylamide and its principal metabolite,
glycidamide (epoxide), react with various biologically significant targets. AC enhances
the production of reactive oxygen species and potentially affects brain.
The glutathione plays a critical role in protecting cells from oxidative stress and
xenobiotics, in the central nervous system18 (CNS) with a particular emphasis on the
mechanism by which neuronal GSH synthesis is regulated. In brain GSH depletion leads
to an increased production of superoxide, hydroxyl and H2O2 radicals19. The intracellular
GSH pool is important for limiting oxidative stress–induced neuronal injury. GSH as
neuromodulator is required for cell proliferation and neuronal differentiation20. In fact,
brain GSH is reduced in some age-related neurodegenerative diseases. For example
Alzheimer’s disease (AD) is a leading age-related neurodegenerative disease, which
pathogenically involves oxidative stress21. The depletion of total glutathione is one of the
earliest biochemical change seen in the Parkinson’s disease (PD). The primary
neuropathological feature of PD is the loss of dopaminergic neurons of the substantia
nigra pars compacta (SN) that projects to striatum22.
The ascorbate content of developing rat brain doubles in late pregnancy, which
fits well with results of studies showing that ascorbate enhances the differentiation of
embryonicstem cells into neurons in a culture medium23. This is associated with increases
in expression of genes involved in neurogenesis, maturation, and neurotransmission24.
Ascorbate at 200µM concentration induces synaptic maturation of the neurons, based on
findings increased mumber of miniature excitatory post-synaptic currents in the cultured
neurons. The ascorbate effects were not mimicked by other antioxidants, such as GSH
and vitamin E, leading to the conclusion that they occurred by some other mechanism.
The ascorbate deficit in between 37 and 67 percents causes death to animals without any
scurvy disease. Standard hematoxylin and eosin staining of the brain and spinal cord
showed no abnormalities except in at Ischemia initial stages depletes intracellular GSH25
and ascorbate26 in brain.
Chick embryos have been used in the past for several years to investigate the
effect of environmental chemicals and radiations on developmental effects,
morphogenesis, etc. Avian embryonic tissues are characterized by the accumulation of
polyunsaturated lipids during the final week of in ovo development. During development
The brain differs from other tissues in being a highly aerobic and totally oxygendependent tissue and may be especially at risk from free radical attack, because this tissue
is characterized by a low content of natural antioxidants and generates a greater amount
of free radicals per gram of tissue than any other organ27.
The data obtained in our laboratory confirm the statistically significant
reduction in the levels of ascorbic acid and glutathione in brain of d (11) developing
chick embryos after AC-treatment (Table 1). AC-induced free radical production in
neurons has been suggested to be responsible for the oxidative brain damage. This fact
proves the intensification of lipid peroxidation in patients with central nervous system 28
as obtained similar results in patients operated on for a brain tumor glioblastoma
multiforme and meningioma. In the opinion of the authors the growth of a tumour may be
a cause of the disturbance of the equilibrium of redox reactions intensification. Louw29
showed an increase in the lipid peroxidation process in patients with astrocytoma. Some
reports Arora and Landolt30, 31 indicate that a higher level of oxygen-derived free radicals
may be responsible for the intensification of the proliferation of neoplasmatic cells,
which is followed by the development of a tumour. Arora30 showed that the addition of a
well-known free radical scavenger as lazaroids to brain tumour cell cultures leads to
inhibition of neoplasm growth. Also our (Fig. 3-8) results showed increase degenerative
changes, mild damage, hemorrhages, structural damages, necrotic damage, and formation
of vacuole shows damage in the hippocampus region of chick embryonic brain tissue.
CONCLUSION
On the basis of results obtained we conclude that acrylamide cause
disturbances in the oxidative status and the effect was pronounced with the high doses,
indicated enhanced brain damage risk during exposure to acrylamide. In chick embryo
brain GSH depletion leads to increased production of superoxide, hydroxyl radicals, and
also H2O2. The intracellular GSH pool is important for limiting oxidative stress–induced
neuronal injury and suppression of cell proliferation and neuronal differentiation.
Because of reduction of glutathione and vitamin-c the free radical scavenger´s role was
suppressed and histological analysis indicates acrylamide may probably involved in
propagation of toxicity to hippocampus region of chick embryonic brain, hippocampus
damage impairs memory for the order of a series and inhibits the nerve impulse
transmission.
ACKNOWLEDGMENTS
We are grateful to University Grants Commission (UGC) for providing financial
support under Rajiv Gandhi National Fellowship (RGNF) and DRDO to carry out the
research work. Prof. Thyaga Raju is recipient of UGC BSR OTG (2013).
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