Study of the effect of acrylamide on purkinje cells of the cerebellum in albino rats

1. Reprint From:
A
eg *-tLu
y& y it
i.....i
w ««
j)2 Ill
;'E?
fg
S
V
Published by
The Faculty Of Medicine,
Suez Canal University.
Ismailia , Egypt
ISSN 1110 -6999

2. Vol. 12, No. 1 , March, 2010
35 -42
Suez Canal Univ Med J
Study of the Effect of Acrylamide on Purkinje Cells of the Cerebellum in Albino Rats
Abdulmonem A. Al-Hciyani, Raid M. Hamdy and Hesham N. Abdel-Raheem
Department of Anatomy, Faculty of Medicine, King Abdulaziz University, Jeddah, KSA
Abstract
Objectives: Acrylamide has several toxic and carcinogenic effects. The current research aimed to study the
harmful effects of acrylamide on the structure of the Purkinje cells of the cerebellum in the albino rats, in
an attempt to clarify its potential risk on the human health.
Methods: The study was performed at the Department of Anatomy, Faculty of Medicine, King Abdulaziz
University, Jeddah, Saudi Arabia through the years 2008-2010. A daily dose of 50 mg/kg body weight of
acrylamide was administrated to adult male albino rats orally and intraperitoneally. Their cerebella were
obtained after two and four weeks of acrylamide administration, where serial sagittal sections were stained
with H & E, and silver stains and examined microscopically.
Results: Rats treated with acrylamide 50 mg/kg body weight for two weeks showed mild degenerative
changes in the form of diminished dendrites with less arborization of the Purkinje cells, while rats treated
with the same dose/or four weeks showed severe degenerative changes of Purkinje cells in tire form of
disintegrated dendrites and ill-defined arborization into the outer molecular layer. Moreover, Purkinje cells
bodies showed marked irregularity in cell boundary. Silver staining showed deeply stained argyrophilic
dendrites arborizing intothe basal part ofthe outer molecularlayer. Inaddition,the Purkinjecellsmanifested
a high affinity to silver so that they appeared brown in color, whether acrylamide was administered orally
or intraperitoneally.
Conclusion: Exposure to acrylamide produced degenerative changes in the Purkinje cells of the cerebellum
which were more prominent with the longer period of exposure.
Keywords: Acrylamide, Cerebellum, Purkinje cells, Toxic Effects, Histological Structure, Neurotoxicity,
Albino Rats, Fast Food.
Introduction
Acrylamide is a white crystalline odorless
compound, which is soluble in water, alcohol, and
other organic solvents(l). The chemical compound
acrylamide (acrylamideylic-amide) has the
chemical formula C3H5NO and its IUPAC name
(International Union ofPureandApplied Chemistry)
is 2-propenamide. Acrylamide is incompatible with
acids, bases, oxidizing agents, iron and iron salts. It
decomposes non-thermally to form dimethylamine
and thermal decomposition produces carbon
monoxide, carbon dioxide and oxides of nitrogen®.
Acrylamide exists in two forms; a monomer
(severely toxic) and a polymer (nontoxic), the
monomer occurs in a white flowing crystalline
form as flake-like crystals®. It was found also that
acrylamide readily polymerizes on reaching melting
point or exposure to UV light. Solid acrylamide is
stable at room temperature, but may polymerize
violently when melted or exposed to oxidizing
agents®.
It was reported that acrylamide was generated
from food components during heat treatment as
a result of the Maillard reaction between amino
acids asparagine in potatoes and cereals and
reducing sugars such as glucose®. Swedish Food
Administration recently reported the presence of
acrylamide in heat-treated food products®. The
formation of acrylamide is associated with high-
temperature (higher than 200°C) in cooking process
at certain carbohydrate-rich foods, especially when
asparagines react with sugar®.
Average daily adult intake of acrylamide in most
populations was estimated to be approximately 0.5
pg/kg body weight®. However, intake may vary
widely from 0.3 - 2 pg/kg BW/day or may reach
even 5 pg/kg BW/day. The concluding estimate of
average daily human intake was 1 pg/kg BW/day
and for high consumers it was estimated to be 4 pg/
kg BW/day®.
35

3. Al-Hayani et al.,36
It was found that the foodstuffs heated above
, 120°C yielded acrylamide concentrations up to 1
mg/kg in carbohydrate-rich foodstuffs, furthermore
foods prepared or purchased in restaurants had
concentrations up to almost 4 mg/kg (in one sample
of potato crisps)00'. The early findings tended to
focus on starch-rich foods such as fried potatoes
(hash browns), French fries, potato crisps and crisp-
bread, all of which showed relatively high levels
of acrylamide. The parallel finding that fried meat
(pork, chicken, beef, cod, sausages, and hamburger)
contained only lowamountsofacrylamidesuggested
that carbohydrate-rich but not protein-rich foods
provided the precursors of acrylamide formation.
Bread (especially bread crust), cereals, coffee, and
coffee surrogates were found to contain significant
levels of acrylamide. Besides potatoes, particular
cereals, coffee, and crisp-bread were considered as
relevant sources of human exposure, since they are
consumed on a regular basis by a broad group of
consumers00.
Acrylamide was evaluated by the International
Agency for Research on Cancer in 1994 as
“probably carcinogenic to humans02'. Based on the
positive bioassay results in mice and rats, supported
by evidence that acrylamide is biotransformed
in mammalian tissues to a chemically reactive
genotoxic metabolite. This process of biotrans¬
formation is possible in humans and can be
demonstrated to occur efficiently in both human and
rodent tissues03'. Severe exposure to acrylamide
might produce CNS symptoms as confusion and
hallucinations. Drowsiness, loss of concentration
and ataxia were also seen. Cerebellar signs such as
dysarthria, tremors, positive Romberg sign and gait
disturbances were most common. Visual changes
(reduction of red and green discrimination), a
hypertensive retinopathy were associated04'. On
the other hand, it was reported that long-term
acrylamide exposure produced a motor and sensory
polyneuropathy that was insidious and distal in
onset; the presence of ataxia, dysarthria and tremor
suggested central midbrain involvement. Signs and
symptoms included weakness, parasthesias, fatigue,
lethargy,decreasedpinpricksensation,vibratoryloss,
decreased reflexes, positive Romberg sign. Severity
was worse in distal portions of the extremities.
Desquamation of the palms, soles, sweating and
peripheral vasoconstriction were more prominent
in acrylamide peripheral neuropathy compared with
other industrial neuropathies05'. Although the toxic
effects of the acrylamide were studies extensively,
its effect on the cerebellar structure was not studied
in details. Therefore, the aim of the present work
was to study the harmful effects of acrylamide on
the structure of the Purkinje cells of the cerebellum
in the albino rat, in an attempt to clarify its potential
risk on the human health.
Material and Methods
This study was performed at the Department
of Anatomy, Faculty of Medicine, King Abdulaziz
University, Jeddah, Saudi Arabia through the years 2008-
2010 after approval of the Faculty Ethical Committee.
Acrylamide powder was obtained from Sigma—
Aldrich Chemical Co. (St Louis, MO, USA); 99%
purity, and freshly prepared solutions were prepared by
dissolving in saline to obtain the required dosages0'.
Forty adult male albino rats weighing (250-300
g) were used in the present study. The rats were
housed individually and maintained under a controlled
environment with average temperature (20-27°C)
throughout the experimental period, water and food
availability and standard light-dark cycle at the animal
house. After one week of acclimatization, the animals
were divided into three main groups; (I, II and III).
Group I rats (16 rats) received a daily dose of 50 mg/
kg body weight of acrylamide for two weeks while
group II animals (16 rats) received the same dose of
acrylamide for four weeks. Group III rats (8 rats)received
equivalent amounts of saline for the same periods and
were considered as controls. Each of groups I and II
animals were subdivided into two subgroups, each of
them consisted of 8 rats; the first subgroup was given
acrylamide via intraperitoneal injections while the second
subgroup was given acrylamide orally via endogastric
tube respectively. The rats were sacrificed under general
anesthesia, where their cerebellawereextracted and fixed
in 10% buffered neutral formalin, processed to obtain
paraffin blocks. Serial sagittal sections (5 pm thick) were
sliced and stained with Hematoxylin and eosin and silver
(modified Glees)06’.
Results
Thecerebellum of the control groupshowed folia
of thecerebellar cortexconsisting ofouter molecular
layer, Purkinje cell layer, inner granular layer and
an underlying central core of white matter. Purkinje
cells were characterized by a large flask shaped
cell body with apical arrangement of dendrites,
that were arborizing into the overlying molecular
layer. Detailed examination of Purkinje cell bodies
revealed that their nuclei were pale stained and
contained deeply stained nucleolus (Figure 1).

4. 37Effect of Acrylamide on the Purkinje cells
The cerebellum of the rats receiving 50 mg/kg
intraperitoneally for two weeks showed that the
Purkinje cells manifested degenerative changes
in the form of diminished dendrites with less
arborization into the outer molecular layer (Figure
2). While examination of the cerebellar sections of
rats receiving 50 mg/kgorally for 2 weeks displayed
Purkinje cells with similar findings. Silver staining
of the same group showed that Purkinje cells
acquired more affinity for staining. Strikingly,
an increased density of argyrophilic arborizing
dendrites extending into the outer molecular layer
was observed (Figure 3).
The cerebellum of the rats receiving 50 mg/kg
for four weeks intraperitoneally showed severe
degenerative changes affecting Purkinje cells in
the form of disintegrated dendrites and ill-defined
arborizationintotheoutermolecularlayer.Moreover,
Purkinje cell bodies showed marked irregularity in
cell boundary (Figure 4). Moreover, silver staining
showed deeply stained argyrophilic dendrites
arborizing into the basal part of the outer molecular
layer. In addition, the Purkinje cells manifested a
high affinity to silver so that they appeared brown
in color (Figure 5).
The cerebellum of the rats receiving 50 mg/kg
for four weeks orally showed more irregularity in
the shape of the Purkinje cells and degeneration of
their dendritic tree(Figure6).Silverstainingshowed
deeply stained argyrophilicdendrites arborizing into
the outer molecular layer. In addition, the Purkinje
cell somata acquired a very high affinity to silver so
that they appeared more deeply stained between the
outer molecular and inner granular layers (Figure
7).
, .. V..‘. t V •
Sk* :r
Figure (1): A photomicrograph of sagittal section of the
cerebellum of rat from control group showing the
3 layers of the cerebellar cortex; outer molecular
layer with relativelyfewcells(ML), innerextensive
cellular granular cell layer (GL) and Purkinje layer
which is formed of largely spaced flask-shaped
Purkinjecells(PC) withapically arranged dendrites
arborizing into the molecular layer (H & E><400).
# -
;ÿ
fiv.X
V.
.
”
•
-* -..,
• A * i*L' V v
• *Vi •4
Figure (2): A photomicrograph of a sagittal section of the cerebellum of rat
from group I (receiving 50 mg/kg intraperitoneally) showing Purkinje
cells (PC)) with depleted arborization of their dendrites into the outer
molecular layer. Note the outer molecular (ML) and inner granular (GL)
layers (H & E x 400).
llgpi®
Sll
mwm Figure (3): A photomicrograph of a sagittal section of the cerebellum of rat from
group I (receiving 50 mg/kg intraperitoneally) showing that Purkinje cells (PC)
acquired more affinity for staining. Note the increased density of argyrophilic
dendrites (arrows) running in different directions in the basal part of the outer
molecular layer (ML) close to Purkinje cells. Note Granular layer (GL) (Silver
stainx 400).
fell
Iagm
®i™iMWB.

5. Al-Hayani et al.,38
ah ,
ML
5
P6;'
. Oi
V
o» „ „
Figure (4): A photomicrograph of a sagittal section of the > .
cerebellum of rat from group II (receiving 50 mg/kg S
intraperitoneally) showing Purkinje cells (PC) with JM
severely depleted arborization of their dendrites into g
the outer molecular layer. Note the outer molecular
(ML) and inner granular (GL) layers (H & E x 400).
WMf
sm+m*jsr
mm,
Figure (5): A photomicrograph of a sagittal section of the cerebellum of rat from
group II (receiving 50 mg/kg intraperitoneally) showing deeply stained
argyrophilic dendrites (arrows) arborizing into the basal part of the outer
molecular layer (ML). Note that the Purkinje cells (PC) manifest a lesser
affinity to silver so that they appear brown in color. (Granular layer: GL)
(Silver stainx 400).
Slip
Sift® *
f>
Figure (6): A photomicrograph of a sagittal section
of the cerebellum of rat from group receiving »
50 mg/kg orally showing Purkinje cells with
diminished arborization of their dendrites into !, , ® JL
the outer molecular layer. Note the 3 layers of
* p »»
the cerebellar cortex; outer molecular (ML), j
Purkinje cell layer (PC) and inner granular
(GL) layers (H & E * 400). JIBEEI. "
-«* jMt*‘
"* * -
-VA*** * ' a if
%
t
•* •.* *
* r~
%
$
r.
*aw
s’
yip Figure(7):Aphotomicrographofasagittalsectionofthecerebellumofratfromgroup
(receiving 50 mg/kg orally) showing deeply stained argyrophilic Purkinje
cell layer (PC). Note deeply stained argyrophilic dendrites arborizing into the
outer molecular layer (ML) (granular layer: GL) (Silver stain* 400).
.
mmF;i< '
-
... -
a
aggr

6. Effect of Acrylamide on the Purkinje cells 39
restricted to the dendrites and axons while longer
duration exposure resulted in affection of Purkinje
cell bodies as well.
Based on results from previous investigations,
the possibility existed that at higher dose of
acrylamide exposure, axonopathy was expressed
in CNS. Therefore, to determine degenerating
neuronal somata, dendrites, terminals and axons
in nervous tissues of acrylamide-intoxicated rats,
silver stain techniques were used. Results from the
present study showed that intoxication of rats at 50
mg/kg/day produced nerve terminal degeneration in
different layers of the cerebellum. This effect was
specific for terminals since argyrophilic changes
in axons or other nerve cell components (i.e. cell
body or dendrite) were evident at any time during
intoxication at the higher dose-rate(l9).
It was reported that the argyrophilic terminals
appearing in nervous tissues might be in the form
of dying-back effects characterizing acrylamide
starting from the dendrites and axons then to tire
soma(20). As intoxication at the higher acrylamide
dose-rate continued, the intensity and scopeof nerve
terminal damage in cerebrum nuclei progressed.
Maximal neurological effects (severe) on fourth
week of the acrylamide dosing paradigm coincided
withmoderate-to-heavy nerveterminaldegeneration
in numerous brain areas. The shorter exposure
periods of acrylamide also produced selective nerve
terminal degeneration, although the corresponding
damage was less pervasive than that produced by
the longer periods of exposure. Thus, irrespective
of acrylamide dosing conditions, nerve terminal
degeneration was the sole neuropathologic effect in
rat cerebrum, cerebellum and peripheral nervesl:!0,.
Moreover, several lines of evidence suggested that,
regardless of dose-rate, nerve terminal damage was
an early consequence of acrylamide intoxication in
both central and peripheral nervous systems(l0). The
present study suggests that the acrylamide damage
is related to cumulative effects i.e. the problem is in
the time factor rather than the dose given.
Discussion
In the present study, different stages of Purkinje
cell degeneration were observed in the cerebellum
under the influence of different periods of exposure
to high dose of acrylamide. Hematoxylin and eosin
staining revealed that, when the acrylamide was
given for two weeks, depletion of dendrites in the
molecular layer was observed.Moreover, increasing
the duration of exposure to acrylamide up to four
weeks resulted in a severe damage in the form of
disintegration and ill-defined arborization of the
dendrites, together with marked irregularity in the
outline of Purkinje cell bodies.
In a previous study*l7), the effects of high-dose
acrylamide treatment of up to 50 mg/kg/day for 4-
10 days in comparison to the low-dose subchronic
exposure, up to 12 mg/kg/day for 90 days was
studied. The investigators found that in the high-
dose; Purkinje cells, long ascending tracts of the
spinal cord, optic tract terminal, preterminal regions
in superior colliculus, sensory ganglion cells and
distal large-caliber peripheral axons were severely
affected; Purkinje cells and fasciculus gracilis
changes were the earliest lesions. On the other hand,
in the low-dose, the dominant lesion wasconfined to
the distal peripheral axon with only minor changes
occurring in spinal cord and medulla; paranodal
swellings with the characteristic appearance of
neurofilament aggregations were seen.
Silver staining confirmed Purkinje cell
degeneration by showing a prominent increase in
the argyrophilia. Such increase in argyrophilia was
positively correlated with the duration of exposure
to acrylamide so that, with the two weeks exposure,
the dendrites and axons showed increased affinity
to silver while the soma of Purkinje cells were
faintly stained. After four weeks of exposure, the
bodies, dendrites and axons of Purkinje cells all
showed dense argyrophilia. These observations
were consistent with the work of Lehning et a!.<IS),
who noticed that with short period exposure time,
the degeneration affecting Purkinje cells was

7. 40 Al-Hayani et al.,
Regarding the mechanism of acrylamide
neurotoxicity, LoPachin et al.(2l) has shown that
acrylamide inhibits K+-evoked neurotransmitter
release from brainstem and cerebrocortical
synaptosomes, which could provide an explanation
fortheaforementionedelectrophysiologicalfindings.
Moreover, reports of increased neurotransmitter
(i.e. dopamine, serotonin) receptor binding in
striatum and other forebrain areas of intoxicated
rats were consistent with compensatory responses
to acrylamide-induced synaptic dysfunction. Based
on these considerations, acrylamide neurotoxicity
was represented by nerve terminal dysfunction in
central and peripheral nervous systems*22*.
In conclusion, the present study expanded
the available information concerning the hazards
carried by the consumption of acrylamide on the
cerebellum. Although the doses of acrylamide
utilized in the present investigation were higher
than the average dietaiy daily intake in humans,
0.4-5 pg/kg body weight/day, yet the cumulative
effects of such toxicant on human health still await
to be fully identified*9*. Further studies focusing on
the influence of acrylamide on different organs in
smaller doses for prolonged periods could aid in
the full understanding of hazards implicated by this
substance.
5. Donald M, Pellerone F, Adam B, Bouquet M, Thomas H,
i Dry B. Identification of resistance gene analogs linked to a
powdery mildew resistance locus in grapevine. Theor Appl
Genet; 2002, 104:610-8.
6. Konings E, Baars A, van Kiaveren D, Spanjer M, Rensen
P, Hiemstra M, van Kooij J, Peters P. Acrylamide exposure
from foods of the dutch population and an assessment of the
consequent risks. Food Chem Toxicol; 2003, 41:1569-79.
7. Rydberg P, Eriksson S, Tareke E, Karlsson P, Ehrenberg
L, Tornqvist M. Investigations of factors that influence
the acrylamide content of heated foodstuffs. J Agric Food
Chem; 2003,51:7012-8.
8. Amrein M, Bachmann S, Noti A. Potential of acrylamide
formation, sugars, and free asparagine in potatoes: a com¬
parison of cultivars and farming systems. J Agric Food
Chem; 2003, 51:5556-60.
9. Parzefall W. Minireview on the toxicity of dietary acryl¬
amide. Food Chem Toxicol; 2008, 46(4):1360-4.
10. Sharp D.Acrylamide in food. Lancet; 2003, 361(9355):361-
2.
11. Lingncrt H, Grivas S, Jagerslad M, Skog K, Tornqvist M,
Aman P.Acrylamide in food: Mechanisms of formation and
influencing factors during heating of foods. Scand J Nutri;
2002, 46:159-72.
12. Fix S, Stitzel R, Ridder M. Switzer C. MK-801 neurotoxic¬
ity in cupric-silver-stained sections: Lesion reconstruction
by three-dimentional computer image analysis. Toxicol
Pathol; 2000, 28:84-90.
!
Financial support: This work was supported
financially by grant No. 428/009 of the Deanship
of Scientific Research, King Abdulaziz University,
Saudi Arabia.
13. Tyl R, Friedman M, Losco P, Fisher L, Johnson K, Strother
D, Wolf C. Rat two-generation reproduction and dominant
lethal study of acrylamide in drinking water. Reprod Toxi¬
col; 2000. 14:385ÿ401.
References
1. Giese J. Acrylamide in Foods. Food Technology; 2002,
56(l0):71-2.
2. Raloff J. Launches Acrylamide Investigations. Science
News; 2002, 162:15.
14. Biedermann M, Biedermann-Brem S, Noti A, Grob K,
Mandli I-I. Two GC-MS methods for the analysis of acryl¬
amide in foods. Mitt Lebensmittelunters Hyg;. 2002,
93:638-52.
15. Fernandez S, Kurppa L, Hyvonen L. Content of acrylamide
decreased in potato chips with addition of a proprietary fla-
vonoid spice mix in flying. Innovations in Food Technol¬
ogy;2003, 56:170-7.
16. Drury R and Wallington E. Carlton’s Histological Tech¬
niques. Oxford University Press. New York 5th ed.; 1980,
pp. 237.
3. Tyl R, Crump K. Acrylamide in Food. Food Standards
Agency; 2003,5:215-22.
4. Hagmar L, Tornqvist M. Inconclusive results from an epi¬
demiological study on dietaiy acrylamide and cancer. Br J
Cancer; 2003, 89:774-5.

8. 41Effect of Acrylamide on the Purkinje cells
17. Nemoto S, Takatsuki S, Sasaki K, Maitani T. Determination
of acrylamide in foods by GC/MS using 13C-labeled acryl¬
amide as an internal standard. Shokuhin Eiseigaku Zasshi;
2002, 43:371-6.
21. LoPachin M, Lehning E, Ross F. Nerve terminals as the
primary site of acrylamide action: a hypothesis. NeurbToxi-
cology; 2002, 23:43-59.
22. LoPachin M, Balaban C, Ross F. Acrylamide axonopathy
revisited. Toxicol Appl Pharmacol; 2003, 188(3):135-53.18. Lehning E, Balaban C, Ross J, LoPachin M. Acrylamide
neuropathy: III. Spatiotemporal characteristics of nerve cell
damage in rat forebrain. NeuroToxicology; 2002, 23:302-
Correspondence to
Raid M Hamdy, MD
Department of Anatomy,
Faculty of Medicine,
King Abdulaziz University,
Jeddah, Kingdom of Saudi Arabia
Email: raidhamdy@hotmail.com
56.
19. Lehning E, Balaban C, Ross J, LoPachin M. Acrylamide
neuropathy: IT. Spatiotemporal characteristics of nerve cell
damage in rat brainstem and spinal cord. NeuroToxicology;
2002;23:417-31.
20. LoPachin M, Lehning E, Jortner S. Rate of neurotoxicant
exposure determines morphologic manifestations of distal
axonopathy. Toxicol Appl Pharmacol; 2000, 167:75-86.

9. k.fit.
himi
lUfcfI
tiihIrf
fftfc%irf;fsi,£>ri*t*£-CVfcr
t£fci
SIti,-
E*rI
to
t
1
p
£
T:
L
1
IftV-fitfit
hllIit
cf-1"*-•*r
u*be6feBet-Cs-Cti%Z'G
If|IffftfiV
iitiUzli-t
"Itllli£|f11
iIlffeltti--iIt
U|h(4(t•L,IU’:Lft
£E*<LISLCL
V.Elrc1fi£V%c_c
£&*&i1&k
&•
!<L
It
l
f
*£
£
3
I
f