Reprint From:

Aeg *-tLu

y& y iti.....iw j)2 Ill;'E? fgSV

Published byThe Faculty Of Medicine,Suez Canal University.

Ismailia , Egypt

ISSN 1110 - 6999

Vol. 12, No. 1 , March, 201035 -42

Suez Canal Univ Med J

Study of the Effect of Acrylamide on Purkinje Cells of the Cerebellum in Albino RatsAbdulmonem A. Al-Hciyani, Raid M. Hamdy and Hesham N. Abdel-RaheemDepartment of Anatomy, Faculty of Medicine, King Abdulaziz University, Jeddah, KSA

AbstractObjectives: Acrylamide has several toxic and carcinogenic effects. The current research aimed to study theharmful effects of acrylamide on the structure of the Purkinje cells of the cerebellum in the albino rats, inan 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 orallyor intraperitoneally.Conclusion: Exposure to acrylamide produced degenerative changes in the Purkinje cells of the cerebellumwhich were more prominent with the longer period of exposure.Keywords: Acrylamide, Cerebellum, Purkinje cells, Toxic Effects, Histological Structure, Neurotoxicity,Albino Rats, Fast Food.

IntroductionAcrylamide is a white crystalline odorless

compound, 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 crystallineform as flake-like crystals. It was found also thatacrylamide readily polymerizes on reaching meltingpoint or exposure to UV light. Solid acrylamide isstable at room temperature, but may polymerizeviolently when melted or exposed to oxidizingagents.

It was reported that acrylamide was generatedfrom food components during heat treatment asa result of the Maillard reaction between aminoacids asparagine in potatoes and cereals andreducing sugars such as glucose. Swedish FoodAdministration recently reported the presence ofacrylamide in heat-treated food products. Theformation of acrylamide is associated with high-temperature (higher than 200C) in cooking processat certain carbohydrate-rich foods, especially whenasparagines react with sugar.

Average daily adult intake of acrylamide in mostpopulations was estimated to be approximately 0.5pg/kg body weight. However, intake may varywidely from 0.3 - 2 pg/kg BW/day or may reacheven 5 pg/kg BW/day. The concluding estimate ofaverage daily human intake was 1 pg/kg BW/dayand 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, 120C 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, andcoffee 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 asprobably 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 biotransformation 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 gaitdisturbances 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 MethodsThis 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 SigmaAldrich Chemical Co. (St Louis, MO, USA); 99%purity, and freshly prepared solutions were prepared bydissolving in saline to obtain the required dosages0'.

Forty adult male albino rats weighing (250-300g) were used in the present study. The rats werehoused individually and maintained under a controlledenvironment with average temperature (20-27C)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 whilegroup 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 andwere 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 generalanesthesia, where their cerebella were extracted and fixedin 10% buffered neutral formalin, processed to obtainparaffin blocks. Serial sagittal sections (5 pm thick) weresliced 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 andan 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 thePurkinje 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 stainingshowed deeply stained argyrophilic dendritesarborizing into the basal part of the outer molecularlayer. In addition, the Purkinje cells manifested ahigh 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 intothe outer molecular layer. In addition, the Purkinjecell somata acquired a very high affinity to silver sothat they appeared more deeply stained between theouter molecular and inner granular layers (Figure7).

, .. V... t V

Sk* :r

Figure (1): A photomicrograph of sagittal section of thecerebellum of rat from control group showing the3 layers of the cerebellar cortex; outer molecularlayer with relatively few cells (ML), inner extensivecellular granular cell layer (GL) and Purkinje layerwhich is formed of largely spaced flask-shapedPurkinjecells (PC) with apically arranged dendritesarborizing into the molecular layer (H & E>

Al-Hayani et al.,38

ah ,ML

5 P6;'. OiV

o Figure (4): A photomicrograph of a sagittal section of the > .cerebellum of rat from group II (receiving 50 mg/kg Sintraperitoneally) 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*jsrmm,Figure (5): A photomicrograph of a sagittal section of the cerebellum of rat from

group II (receiving 50 mg/kg intraperitoneally) showing deeply stainedargyrophilic dendrites (arrows) arborizing into the basal part of the outermolecular layer (ML). Note that the Purkinje cells (PC) manifest a lesseraffinity 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 withdiminished arborization of their dendrites into !, , JLthe outer molecular layer. Note the 3 layers of * p the cerebellar cortex; outer molecular (ML), jPurkinje cell layer (PC) and inner granular(GL) layers (H & E * 400). JIBEEI. "

- * jMt* "* * -- VA*** * ' a if%t * .* ** r~

%$

r.

*aw s

yip Figure (7):Aphotomicrograph ofasagittal section of the cerebellum of rat from group(receiving 50 mg/kg orally) showing deeply stained argyrophilic Purkinjecell layer (PC). Note deeply stained argyrophilic dendrites arborizing into theouter 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 longerduration exposure resulted in affection of Purkinjecell bodies as well.

Based on results from previous investigations,the possibility existed that at higher dose ofacrylamide exposure, axonopathy was expressedin CNS. Therefore, to determine degeneratingneuronal somata, dendrites, terminals and axonsin nervous tissues of acrylamide-intoxicated rats,silver stain techniques were used. Results from thepresent study showed that intoxication of rats at 50mg/kg/day produced nerve terminal degeneration indifferent layers of the cerebellum. This effect wasspecific for terminals since argyrophilic changesin axons or other nerve cell components (i.e. cellbody or dendrite) were evident at any time duringintoxication at the higher dose-rate(l9).

It was reported that the argyrophilic terminalsappearing in nervous tissues might be in the formof dying-back effects characterizing acrylamidestarting from the dendrites and axons then to tiresoma(20). As intoxication at the higher acrylamidedose-rate continued, the intensity and scope of nerveterminal damage in cerebrum nuclei progressed.Maximal neurological effects (severe) on fourthweek of the acrylamide dosing paradigm coincidedwith moderate-to-heavy nerve terminaldegenerationin numerous brain areas. The shorter exposureperiods of acrylamide also produced selective nerveterminal degeneration, although the correspondingdamage was less pervasive than that produced bythe longer periods of exposure. Thus, irrespectiveof acrylamide dosing conditions, nerve terminaldegeneration was the sole neuropathologic effect inrat cerebrum, cerebellum and peripheral nervesl:!0,.Moreover, several lines of evidence suggested that,regardless of dose-rate, nerve terminal damage wasan early consequence of acrylamide intoxication inboth central and peripheral nervous systems(l0). Thepresent study suggests that the acrylamide damageis related to cumulative effects i.e. the problem is inthe time factor rather than the dose given.

Discussion

In the present study, different stages of Purkinjecell degeneration were observed in the cerebellumunder the influence of different periods of exposureto high dose of acrylamide. Hematoxylin and eosinstaining revealed that, when the acrylamide wasgiven for two weeks, depletion of dendrites in themolecular layer was observed. Moreover, increasingthe duration of exposure to acrylamide up to fourweeks resulted in a severe damage in the form ofdisintegration and ill-defined arborization of thedendrites, together with marked irregularity in theoutline of Purkinje cell bodies.

In a previous study*l7), the effects of high-doseacrylamide treatment of up to 50 mg/kg/day for 4-10 days in comparison to the low-dose subchronicexposure, up to 12 mg/kg/day for 90 days wasstudied. The investigators found that in the high-dose; Purkinje cells, long ascending tracts of thespinal cord, optic tract terminal, preterminal regionsin superior colliculus, sensory ganglion cells anddistal large-caliber peripheral axons were severelyaffected; Purkinje cells and fasciculus gracilischanges were the earliest lesions. On the other hand,in the low-dose, the dominant lesion was confined tothe distal peripheral axon with only minor changesoccurring in spinal cord and medulla; paranodalswellings with the characteristic appearance ofneurofilament aggregations were seen.

Silver staining confirmed Purkinje celldegeneration by showing a prominent increase inthe argyrophilia. Such increase in argyrophilia waspositively correlated with the duration of exposureto acrylamide so that, with the two weeks exposure,the dendrites and axons showed increased affinityto silver while the soma of Purkinje cells werefaintly stained. After four weeks of exposure, thebodies, dendrites and axons of Purkinje cells allshowed dense argyrophilia. These observationswere consistent with the work of Lehning et a!.

40 Al-Hayani et al.,

Regarding the mechanism of acrylamideneurotoxicity, LoPachin et al.(2l) has shown thatacrylamide inhibits K+-evoked neurotransmitterrelease from brainstem and cerebrocorticalsynaptosomes, which could provide an explanationfortheaforementioned electrophysiologicalfindings.Moreover, reports of increased neurotransmitter(i.e. dopamine, serotonin) receptor binding instriatum and other forebrain areas of intoxicatedrats were consistent with compensatory responsesto acrylamide-induced synaptic dysfunction. Basedon these considerations, acrylamide neurotoxicitywas represented by nerve terminal dysfunction incentral and peripheral nervous systems*22*.

In conclusion, the present study expandedthe available information concerning the hazardscarried by the consumption of acrylamide on thecerebellum. Although the doses of acrylamideutilized in the present investigation were higherthan the average dietaiy daily intake in humans,0.4-5 pg/kg body weight/day, yet the cumulativeeffects of such toxicant on human health still awaitto be fully identified*9*. Further studies focusing onthe influence of acrylamide on different organs insmaller doses for prolonged periods could aid inthe full understanding of hazards implicated by thissubstance.

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 ApplGenet; 2002, 104:610-8.

6. Konings E, Baars A, van Kiaveren D, Spanjer M, RensenP, Hiemstra M, van Kooij J, Peters P. Acrylamide exposurefrom foods of the dutch population and an assessment of theconsequent risks. Food Chem Toxicol; 2003, 41:1569-79.

7. Rydberg P, Eriksson S, Tareke E, Karlsson P, EhrenbergL, Tornqvist M. Investigations of factors that influencethe acrylamide content of heated foodstuffs. J Agric FoodChem; 2003,51:7012-8.

8. Amrein M, Bachmann S, Noti A. Potential of acrylamideformation, sugars, and free asparagine in potatoes: a comparison of cultivars and farming systems. J Agric FoodChem; 2003, 51:5556-60.

9. Parzefall W. Minireview on the toxicity of dietary acrylamide. 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 andinfluencing factors during heating of foods. Scand J Nutri;2002, 46:159-72.

12. Fix S, Stitzel R, Ridder M. Switzer C. MK-801 neurotoxicity in cupric-silver-stained sections: Lesion reconstructionby three-dimentional computer image analysis. ToxicolPathol; 2000, 28:84-90.

!Financial support: This work was supported

financially by grant No. 428/009 of the Deanshipof Scientific Research, King Abdulaziz University,Saudi Arabia.

13. Tyl R, Friedman M, Losco P, Fisher L, Johnson K, StrotherD, Wolf C. Rat two-generation reproduction and dominantlethal study of acrylamide in drinking water. Reprod Toxicol; 2000. 14:385401.

References1. Giese J. Acrylamide in Foods. Food Technology; 2002,

56(l0):71-2.

2. Raloff J. Launches Acrylamide Investigations. ScienceNews; 2002, 162:15.

14. Biedermann M, Biedermann-Brem S, Noti A, Grob K,Mandli I-I. Two GC-MS methods for the analysis of acrylamide in foods. Mitt Lebensmittelunters Hyg;. 2002,93:638-52.

15. Fernandez S, Kurppa L, Hyvonen L. Content of acrylamidedecreased in potato chips with addition of a proprietary fla-vonoid spice mix in flying. Innovations in Food Technology; 2003, 56:170-7.

16. Drury R and Wallington E. Carltons Histological Techniques. Oxford University Press. New York 5th ed.; 1980,pp. 237.

3. Tyl R, Crump K. Acrylamide in Food. Food StandardsAgency; 2003,5:215-22.

4. Hagmar L, Tornqvist M. Inconclusive results from an epidemiological study on dietaiy acrylamide and cancer. Br JCancer; 2003, 89:774-5.

41Effect of Acrylamide on the Purkinje cells

17. Nemoto S, Takatsuki S, Sasaki K, Maitani T. Determinationof acrylamide in foods by GC/MS using 13C-labeled acrylamide as an internal standard. Shokuhin Eiseigaku Zasshi;2002, 43:371-6.

21. LoPachin M, Lehning E, Ross F. Nerve terminals as theprimary site of acrylamide action: a hypothesis. NeurbToxi-cology; 2002, 23:43-59.

22. LoPachin M, Balaban C, Ross F. Acrylamide axonopathyrevisited. 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 celldamage in rat forebrain. NeuroToxicology; 2002, 23:302-

Correspondence toRaid M Hamdy, MDDepartment of Anatomy,

Faculty of Medicine,

King Abdulaziz University,Jeddah, Kingdom of Saudi ArabiaEmail: raidhamdy@hotmail.com

56.

19. Lehning E, Balaban C, Ross J, LoPachin M. Acrylamideneuropathy: IT. Spatiotemporal characteristics of nerve celldamage in rat brainstem and spinal cord. NeuroToxicology;2002;23:417-31.

20. LoPachin M, Lehning E, Jortner S. Rate of neurotoxicantexposure determines morphologic manifestations of distalaxonopathy. Toxicol Appl Pharmacol; 2000, 167:75-86.

k.fi

t.

himi

lUfcf

Iti\

ihI

rfff

tfc%

ir

f;

fsi

,>

ri*

t *

-CV

fcr

t

fci

SIt

i,-

E*

r I

tot 1 p T: L 1 I

ft V-fit fit

hllI

itc f

-1"*-

*r

u*

be6

fe

Be

t-C

s-C

ti%

Z'G

If|If

fft

fi

Vi

itiU

zli-t

"Itl

lli|

f11

i Ilff

eltt

i --i

ItU

|h(4

(t

L,I

U:

Lft

E*