Reprint From:

A

eg *-tLu

y& y it

i.....iw ««j)2 Ill

;'E?

fgS

V

Published by

The Faculty Of Medicine,

Suez Canal University.

Ismailia , Egypt

ISSN 1110 - 6999

Vol. 12, No. 1 , March, 2010

35 -42Suez 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 AbdulazizUniversity, Jeddah, Saudi Arabia through the years 2008-2010. A daily dose of 50 mg/kg body weight ofacrylamide was administrated to adult male albino rats orally and intraperitoneally. Their cerebella wereobtained after two and four weeks of acrylamide administration, where serial sagittal sections were stainedwith H & E, and silver stains and examined microscopically.

Results: Rats treated with acrylamide 50 mg/kg body weight for two weeks showed mild degenerativechanges in the form of diminished dendrites with less arborization of the Purkinje cells, while rats treatedwith the same dose/or four weeks showed severe degenerative changes of Purkinje cells in tire form ofdisintegrated dendrites and ill-defined arborization into the outer molecular layer. Moreover, Purkinje cellsbodies showed marked irregularity in cell boundary. Silver staining showed deeply stained argyrophilicdendrites arborizing into the basal part of the outer molecular layer. In addition, the Purkinje cells manifesteda 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 odorlesscompound, which is soluble in water, alcohol, andother organic solvents(l). The chemical compoundacrylamide (acrylamideylic-amide) has thechemical formula C3H5NO and its IUPAC name(International Union of Pure and Applied Chemistry)is 2-propenamide. Acrylamide is incompatible withacids, bases, oxidizing agents, iron and iron salts. Itdecomposes non-thermally to form dimethylamineand thermal decomposition produces carbonmonoxide, carbon dioxide and oxides of nitrogen®.

Acrylamide exists in two forms; a monomer(severely toxic) and a polymer (nontoxic), themonomer occurs in a white flowing crystalline

form as flake-like crystals®. It was found also thatacrylamide readily polymerizes on reaching melting

point or exposure to UV light. Solid acrylamide is

stable at room temperature, but may polymerizeviolently 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

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, furthermorefoods prepared or purchased in restaurants hadconcentrations up to almost 4 mg/kg (in one sampleof potato crisps)00'. The early findings tended tofocus on starch-rich foods such as fried potatoes(hash browns), French fries, potato crisps and crisp-bread, all of which showed relatively high levelsof acrylamide. The parallel finding that fried meat(pork, chicken, beef, cod, sausages, and hamburger)contained only low amounts ofacrylamide suggestedthat carbohydrate-rich but not protein-rich foodsprovided the precursors of acrylamide formation.Bread (especially bread crust), cereals, coffee, and

coffee surrogates were found to contain significantlevels of acrylamide. Besides potatoes, particularcereals, coffee, and crisp-bread were considered asrelevant sources of human exposure, since they areconsumed on a regular basis by a broad group ofconsumers00.

Acrylamide was evaluated by the InternationalAgency for Research on Cancer in 1994 as“probably carcinogenic to humans02'. Based on thepositive bioassay results in mice and rats, supportedby evidence that acrylamide is biotransformedin mammalian tissues to a chemically reactivegenotoxic metabolite. This process of biotrans¬formation is possible in humans and can bedemonstrated to occur efficiently in both human androdent tissues03'. Severe exposure to acrylamidemight produce CNS symptoms as confusion andhallucinations. Drowsiness, loss of concentrationand ataxia were also seen. Cerebellar signs such asdysarthria, tremors, positive Romberg sign and gait

disturbances were most common. Visual changes(reduction of red and green discrimination), ahypertensive retinopathy were associated04'. Onthe other hand, it was reported that long-termacrylamide exposure produced a motor and sensorypolyneuropathy that was insidious and distal inonset; the presence of ataxia, dysarthria and tremorsuggested central midbrain involvement. Signs andsymptoms included weakness, parasthesias, fatigue,lethargy,decreasedpinpricksensation,vibratory loss,

decreased reflexes, positive Romberg sign. Severitywas worse in distal portions of the extremities.

Desquamation of the palms, soles, sweating andperipheral vasoconstriction were more prominentin acrylamide peripheral neuropathy compared withother industrial neuropathies05'. Although the toxiceffects of the acrylamide were studies extensively,

its effect on the cerebellar structure was not studiedin details. Therefore, the aim of the present workwas to study the harmful effects of acrylamide onthe structure of the Purkinje cells of the cerebellumin the albino rat, in an attempt to clarify its potentialrisk on the human health.

Material and Methods

This study was performed at the Department

of Anatomy, Faculty of Medicine, King AbdulazizUniversity, 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 foodavailability and standard light-dark cycle at the animalhouse. After one week of acclimatization, the animalswere 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 ofacrylamide for four weeks. Group III rats (8 rats) receivedequivalent amounts of saline for the same periods and

were considered as controls. Each of groups I and IIanimals were subdivided into two subgroups, each ofthem consisted of 8 rats; the first subgroup was givenacrylamide via intraperitoneal injections while the secondsubgroup was given acrylamide orally via endogastrictube respectively. The rats were sacrificed under general

anesthesia, where their cerebella were extracted 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

The cerebellum of the control group showed foliaof the cerebellar cortex consisting of outer molecularlayer, Purkinje cell layer, inner granular layer and

an underlying central core of white matter. Purkinjecells were characterized by a large flask shapedcell body with apical arrangement of dendrites,

that were arborizing into the overlying molecularlayer. Detailed examination of Purkinje cell bodiesrevealed that their nuclei were pale stained andcontained deeply stained nucleolus (Figure 1).

37Effect of Acrylamide on the Purkinje cells

The cerebellum of the rats receiving 50 mg/kgintraperitoneally for two weeks showed that the

Purkinje cells manifested degenerative changesin the form of diminished dendrites with lessarborization into the outer molecular layer (Figure2). While examination of the cerebellar sections ofrats receiving 50 mg/kg orally for 2 weeks displayedPurkinje cells with similar findings. Silver stainingof the same group showed that Purkinje cellsacquired more affinity for staining. Strikingly,an increased density of argyrophilic arborizingdendrites extending into the outer molecular layerwas observed (Figure 3).

The cerebellum of the rats receiving 50 mg/kgfor four weeks intraperitoneally showed severedegenerative changes affecting Purkinje cells inthe form of disintegrated dendrites and ill-definedarborization into theoutermolecular layer.Moreover,

Purkinje cell bodies showed marked irregularity incell boundary (Figure 4). Moreover, silver staining

showed deeply stained argyrophilic dendritesarborizing into the basal part of the outer molecularlayer. In addition, the Purkinje cells manifested a

high affinity to silver so that they appeared brownin color (Figure 5).

The cerebellum of the rats receiving 50 mg/kgfor four weeks orally showed more irregularity inthe shape of the Purkinje cells and degeneration oftheir dendritic tree (Figure 6). Silver stainingshoweddeeply stained argyrophilic dendrites arborizing into

the outer molecular layer. In addition, the Purkinje

cell somata acquired a very high affinity to silver sothat they appeared more deeply stained between the

outer molecular and inner granular layers (Figure7).

, .. 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 relatively few cells (ML), inner extensive

cellular granular cell layer (GL) and Purkinje layer

which is formed of largely spaced flask-shaped

Purkinjecells (PC) with apically 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®Sllmwm

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.

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 JMseverely depleted arborization of their dendrites into gthe outer molecular layer. Note the outer molecular

(ML) and inner granular (GL) layers (H & E x 400).

WMfsm+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).SlipSift®

*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 !, , ® JLthe 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.

*aws’

yip Figure (7):Aphotomicrograph ofasagittal section of the cerebellum of rat from group

(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

Effect of Acrylamide on the Purkinje cells 39

restricted to the dendrites and axons while longer

duration exposure resulted in affection of Purkinjecell 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 tiresoma(20). As intoxication at the higher acrylamide

dose-rate continued, the intensity and scope of nerve

terminal damage in cerebrum nuclei progressed.

Maximal neurological effects (severe) on fourth

week of the acrylamide dosing paradigm coincided

with moderate-to-heavy nerve terminaldegeneration

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 was confined 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

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

fortheaforementioned electrophysiologicalfindings.

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.

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, 1 88(3): 1 35-53.18. 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.

k.fit

.

hi

milU

fcf

I

ti\i

hI

rfff

tfc%

ir

f;

fsi,

£>

ri

*t

*£

-CV

fcr

t£

fci

SIt

i,-

E*

r I

to

t 1 p £ T: L 1 Ift

V-fit fit

hll

Iit

cf-1

"*-•*

ru*

be

6f

eB

et

-Cs

-Cti

%Z

'G

If|If

fft

fi

Vi

itiU

zli-t

"It

llli£

| f11

iIlf

felt

ti--

i It

U|h

(4(t

•L

,I

U’:

Lft

£E*

<LISL

CL

V.

Elr

c1fi

£V%

c_c

£&*

&i

1&

k

&• !<L

I t l f

*£

£ 3 I f