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

2 Suez Canal Univ Med J Vol. 12, No. 1, March, 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 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 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, 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 of Pure and Applied 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. 35 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 hightemperature (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 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.

3 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 crispbread, all of which showed relatively high levels of acrylamide. The parallel finding that fried meat (pork, chicken, beef, cod, sausages, and hamburger) contained only low amounts of acrylamide suggested 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,vibratory loss, 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, Al-Hayani et al., 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 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 ( 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 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 folia of the cerebellar cortex consisting of outer 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 Effect 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/kg orally 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 arborization into theoutermolecular layer.moreover, 37 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 (Figure 6). Silver staining showed deeply stained argyrophilic dendrites 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.,.. V... t V Sk* :r ;ÿ fiv. 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). Sll mwm llgpi X 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 Purkinje cells (PC) with apically arranged dendrites arborizing into the molecular layer (H & E><400).. -* -.., A * i*l' V * Vi 4 v fell M WB. I agm i i 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 stain x 400).

5 38 Al-Hayani et al., V 5 Figure (4): A photomicrograph of a sagittal section of the >. P6;' o». Oi 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). mm, ML ah, sm WMf +m*jsr 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 * 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!,, the outer molecular layer. Note the 3 layers of the cerebellar cortex; outer molecular (ML), Purkinje cell layer (PC) and inner granular > - f «* jmt* "* * - JL - VA*** * ' a if * * * p»» (GL) layers (H & E * 400). JIBEEI. " t $ aw r. * s. % * * r~ j %. F;i< ' yip Figure(7): Aphotomicrograph of a sagittal 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). mm a aggr

6 Effect of Acrylamide on the Purkinje cells 39 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 highdose; 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 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 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 term inal degeneration 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.

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 forthe aforementioned 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, 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. Financial support: This work was supported financially by grant No. 428/009 of the Deanship of Scientific Research, King Abdulaziz University, Saudi Arabia. References 1. Giese J. Acrylamide in Foods. Food Technology; 2002, 56(l0): Raloff J. Launches Acrylamide Investigations. Science News; 2002, 162: Tyl R, Crump K. Acrylamide in Food. Food Standards Agency; 2003,5: Hagmar L, Tornqvist M. Inconclusive results from an epi demiological study on dietaiy acrylamide and cancer. Br J Cancer; 2003, 89: 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: 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: 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: 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: Parzefall W. Minireview on the toxicity of dietary acryl amide. Food Chem Toxicol; 2008, 46(4): Sharp D. Acrylamide in food. Lancet; 2003, 361(9355): 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: 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: 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; :385ÿ 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: Fernandez S, Kurppa L, Hyvonen L. Content of acrylamide decreased in potato chips with addition of a proprietary flavonoid spice mix in flying. Innovations in Food Technol ogy; 2003, 56: Drury R and Wallington E. Carlton s Histological Tech niques. Oxford University Press. New York 5th ed.; 1980, pp. 237.!

8 Effect 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: Lehning E, Balaban C, Ross J, LoPachin M. Acrylamide neuropathy: III. Spatiotemporal characteristics of nerve cell damage in rat forebrain. NeuroToxicology; 2002, 23: 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: LoPachin M, Lehning E, Jortner S. Rate of neurotoxicant exposure determines morphologic manifestations of distal axonopathy. Toxicol Appl Pharmacol; 2000, 167: LoPachin M, Lehning E, Ross F. Nerve terminals as the primary site of acrylamide action: a hypothesis. NeurbToxicology; 2002, 23: LoPachin M, Balaban C, Ross F. Acrylamide axonopathy revisited. Toxicol Appl Pharmacol; 2003, 188(3): Correspondence to Raid M Hamdy, MD Department of Anatomy, Faculty of Medicine, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia raidhamdy@hotmail.com

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