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Clinical Hemorheology and Microcirculation 46 (2010) 1–8 DOI 10.3233/CH-2010-1364 IOS Press

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Effects of oral acrylamide intake on blood viscosity parameters in rats

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Okan Arıhan∗ , Nurten B. Seringeç, Esin ˙Ileri Gürel and Neslihan H. Dikmeno˘glu

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Faculty of Medicine, Department of Physiology, Hacettepe University, Ankara, Turkey

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Received: 26 July 2010 Accepted: 13 August 2010

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1. Introduction

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Acrylamide (CH2 =CHCONH2 ) is a monomer used in polymer manufacture industry. Polyacrylamides are formed by polymerization of acrylamide monomers and have various applications such as, paper strengthening, waste water treatment, production of biomedical and scientific research materials like electrophoresis gels and personal care products such as lotions, cosmetics and deodorants [9]. Previously, exposure of humans to acrylamide was thought to originate only from industrial, occupational or accidental sources, but later when acrylamide was detected as a hemoglobin (Hb) adduct from the blood samples of humans unexposed to such sources, investigations were started to clarify this situation [37]. These investigations revealed that the source was heated starch-rich foods [39, 40]. Acrylamides are formed via a reaction between carbohydrates and proteins in the food. This reaction is known as Maillard reaction and takes place if such foods are processed at high temperatures (such as during frying and baking) [38]. Reducing sugars and asparagine are the main substrates in this reaction [13]. Acrylamide content of the food varies depending on several factors but it is widely accepted that acrylamide

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Abstract. Acrylamide which is formed via reaction of reducing sugars with amino acids during food processing at high temperatures is not only neurotoxic and carcinogenic, but it also damages erythrocyte membrane and generates micronucleated erythrocytes. In the present study, effects of chronic administration of acrylamide at a dose which does not induce neurotoxicity were evaluated on blood viscosity parameters (hematocrit, erythrocyte deformability, erythrocyte aggregation and plasma viscosity). Twenty adult male Sprague-Dawley rats were divided into control and acrylamide groups. The acrylamide group received 10 mg/kg/day acrylamide, whereas the control group received saline (vehicle), both in 10 ml/kg/day volume via gastric gavage. Erythrocyte aggregation and deformability were measured with LORCA and plasma viscosity with cone-plate viscometer. Erythrocyte deformability was measured before, and at the end of the 3rd and the 5th weeks of acrylamide administration. Hematocrit, erythrocyte aggregation and plasma viscosity were measured only at the end of the 5th week. Acrylamide caused a significant decrease in the deformability index of erythrocytes (at the end of the 3rd week, control: 0.606 ± 0.003, acrylamide: 0.595 ± 0.003, p < 0.05) (at the end of the 5th week, control: 0.606 ± 0.002, acrylamide: 0.588 ± 0.002, p < 0.01). Aggregation tendency and plasma viscosity were slightly higher in the acrylamide group, however the difference was not statistically significant. These results imply that acrylamide which does not cause neurotoxicity in rats may alter blood viscosity if chronically taken. Keywords: Acrylamide, erythrocyte deformability, hemorheology, blood viscosity

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∗ Corresponding author: Okan Arıhan, Faculty of Medicine, Department of Physiology, Hacettepe University, Ankara, Turkey. Tel.: +90 312 3051567; Fax: +90 312 3052186; E-mail: [email protected].

1386-0291/10/$27.50 © 2010 – IOS Press and the authors. All rights reserved

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O. Arıhan et al. / Effects of oral acrylamide intake on blood viscosity parameters in rats

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2. Materials and methods

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2.1. Animals

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content increases with increasing temperature. When heated above 120◦ C, acrylamide concentration in carbohydrate-rich foods can reach up to 1 mg/kg [29]. The main dietary source of acrylamide differs with feeding habits, depending on the abundance of either potato or bread in the diet, or intake of coffee. Daily acrylamide intake varies with age and feeding habits but estimates are 0.43 ␮g/kg for adults and 1.6 ␮g/kg for 2 to 5 years old children in the United States [7]. Workplace exposure levels were found to be 1.4–18.6 ␮g/kg whereas intake from food consumption was approximately 0.4 [43] to 0.75 ␮g/kg [7]. A review by Shipp et al. [30] gives detailed information about the metabolism of acrylamide in rats, mice and humans. Briefly, acrylamide is converted into glycidamide via cytochrome P450 2E1 pathway. Conversion rate is around 60% in humans and 40% in rats [12]. Both acrylamide and glycidamide have oxidative, neurotoxic and carcinogenic effects. When human MCF7 cells are treated with glycidamide in vitro, genes related to oxidative stress – glutathione S-transferases (GSTs), and epoxide hydrolase are up-regulated [8]. According to carcinogenesis studies in mice and rats, acrylamide and glycidamide are classified as possible carcinogens by U.S. Environmental Protection Agency [42], EU [11] and National Toxicology Program [26]. However, since their binding is confined only in the sugar moiety, excluding the base-pairing region of DNA, the mutagenic effect can be considered only as “potential” [17]. Exposure of humans to acrylamide from mainly industrial and accidental origins was reported to cause neurotoxicity [31, 33, 35]. Although research has focused generally on its potential carcinogenic and neurotoxic properties, acrylamides also have adverse effects on erythrocytes. In human erythrocytes, acrylamide increases lipid peroxidation [6]. Acute acrylamide toxicity damages erythrocyte membrane and decreases its hemolytic resistance [41]. It was demonstrated that acrylamide administration caused formation of micronucleated erythrocytes in blood [48]. Acrylamide also binds to hemoglobin and forms stable reaction products called hemoglobin adducts [3]. Viscosity of blood is a measure of its inner resistance to flow and is an important factor in the cardiovascular system both at macro and microcirculation [4, 22, 24]. Parameters that determine blood viscosity are: Erythrocyte deformability, erythrocyte aggregation, plasma viscosity and hematocrit value. Erythrocyte deformability, the ability of erythrocytes to change shape as they move through the circulation, decreases the resistance of blood to flow and hence the viscosity of blood. This property is particularly important during passage through capillaries smaller than the diameter of erythrocytes. Membrane skeleton which is composed of peripheral and integral proteins forming a network next to the phospholipid membrane is the main structure responsible for deformability of erythrocytes [10]. Formation of extra bonds between the proteins within the membrane skeleton itself or between proteins of the skeleton and hemoglobin, due to external causes such as oxidative stress and various chemicals, impairs the deformability of these cells [34]. Therefore, acrylamide is expected to alter physiological properties of erythrocytes. This study aimed to evaluate the effects of chronic acrylamide administration on blood viscosity parameters in rats. We hypothesized that systemic administration of acrylamide may alter blood viscosity. For this purpose we selected a dose which does not cause apparent neurotoxicity and applied it for 5 weeks.

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Male Sprague-Dawley rats, weighing 270–300 grams in average were used in the experiments. Animals were kept in a room where ambient temperature and relative humidity were stable with a light-dark cycle


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2.2. Acrylamide application

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of 12 h/12 h. Water and food were available ad libitum. Two groups were formed, acrylamide and control groups which were composed of 10 animals each.

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2.3. Erythrocyte deformability

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Acrylamide (Merck) was dissolved in tap water since it is highly soluble in water (215.5 g/100 ml at 30◦ C) and given via gastric gavage. Acrylamide (10 mg/kg) was given to animals daily in a 10 ml/kg volume. Since higher doses are reported to cause neurotoxicity and poor survival, a lower dose with no apparent toxic effect was preferred [5]. The control animals were given saline 10 ml/kg. All procedures were performed in accordance with Helsinki Declaration, with the permission of Hacettepe University Ethical Committee for Animals (19.3.2009 and #2009/12).

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2.4. Aggregation

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Erythrocyte deformability was measured three times for each animal: At the beginning of the study before acrylamide administration, at the end of the 3rd week and at the end of the 5th week of acrylamide administration. Measurements were done by LORCA (Laser-Assisted Optical Rotational Cell Analyzer) (Mechatronics, Holland) at 30 Pa shear stress and 37◦ C [2]. For this purpose venous blood was drawn from lateral tail vein into a heparin (15 U/ml) coated insulin injector. 0.1 ml of blood was drawn from each animal and 25 ␮l of it was mixed with 5 ml PVP medium (polyvinylpyrrolidone, 360,000 MW, 300 mOsm/kg). One ml of this suspension was placed into LORCA, into the gap between two concentric glass cylinders. In this system the revolving outer cylinder creates a shear stress on the suspension causing the erythrocytes in it to change shape (deform) from biconcave to ellipsoid. A continuous laser beam traverses the suspension and the cell suspension causes diffraction of the beam the pattern of which also changes from circular into ellipsoid with the increasing deformation in erythrocytes. The elongation index (EI) which is a measure of erythrocyte deformability is calculated via a computerized ellipse-fitting program [14, 15]. A lower EI means attenuated deformability.

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2.5. Plasma viscosity

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Erythrocyte aggregation was measured at the end of the 5th week of acrylamide application. Aggregation measurements were performed by LORCA at 37◦ C. Before each measurement blood was oxygenated for 10–15 minutes, and then 1 ml of whole blood was placed into the gap between the cylinders. The decrease in the intensity of the back-scattered light as erythrocytes aggregate, is recorded as a syllectogram from which the amplitude (AMP, representing the total extent of aggregation), aggregation index (AI, a larger index represents greater and/or quicker aggregation), half time (t1/2, time that elapses until the peak intensity is reduced by half, reflects the kinetics of aggregation), ␥Isc max (threshold shear rate needed to prevent aggregation) are calculated by a computer program [2, 14, 15].

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Plasma viscosity was measured at the end of the 5th week. 0.5 ml plasma was used for each measurement. Measurements were performed by a cone-plate viscometer (Brookfield LVDV-II+PRO CP40) at 900 s−1 (120 rpm) shear rate and 37◦ C temperature.

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2.6. Hematocrit Hematocrit values were also recorded at the end of the 5th week using heparinized capillary tubes and a centrifuge specific for this purpose.

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2.7. Statistical analysis

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3. Results

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All statistical analyses were done using “SPSS 12.0 for Windows”. “Mann-Whitney U test” was used to compare the groups. Results were expressed as mean ± SEM. p < 0.05 was accepted to be statistically significant.

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3.1. Effects of acrylamide on erythrocyte deformability

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Animals in both of the groups were normal in their posture and general appearance during the study period. Diarrhea or other possible complications related with acrylamide or with gavage application were absent. Both of the groups gained weight within normal limits.

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3.2. Effects of acrylamide on erythrocyte aggregation, plasma viscosity and hematocrit

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Oral acrylamide administration slightly increased aggregation tendency and plasma viscosity, while slightly decreasing hematocrit. However these effects were not statistically significant (Table 1 ).

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Elongation indices of both groups at the beginning of the study prior to acrylamide or saline application were similar (Control: 0.602 ± 0.003; Acrylamide: 0.606 ± 0.001). At the end of the 3rd week elongation indices of the acrylamide group was significantly lower than that of the control group (Control: 0.602 ± 0.003; Acrylamide: 0.595 ± 0.003, p < 0.05). Erythrocyte deformability was further attenuated in the acrylamide group at the end of the 5th week (Control: 0.606 ± 0.002; Acrylamide: 0.588 ± 0.002, p < 0.01). Elongation indices are given in Fig. 1.

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Erythrocyte deformability (EI)

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Fig. 1. Effect of oral acrylamide intake on erythrocyte deformability (*, p < 0.05; **, p < 0.01).


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Table 1 Effect of oral acrylamide intake on erythrocyte aggregation and plasma viscosity

Control (saline) Acrylamide (10 mg/kg)

AMP (au)

AI (%)

t1/2 (sec)

γIsc max (sec−1 )

Pl.Viscosity (mPa.sec)

Hematocrit (%)

18.50 ± 0.51 19.31 ± 0.52

51.74 ± 1.53 49.51 ± 1.44

3.67 ± 0.26 4.17 ± 0.31

49.00 ± 2.77 55.00 ± 5.63

1.27 ± 0.01 1.29 ± 0.01

43.3 ± 0.86 42.0 ± 0.63

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We found that chronic acrylamide administration in rats resulted in a decrease in erythrocyte deformability. By the end of the 3rd week, erythrocyte deformability indices of the acrylamide group showed a marked decrease. This trend continued for the acrylamide group and at the end of the 5th week erythrocyte deformability revealed a further decrease. The deterioration in erythrocyte deformability may be caused by various factors including peroxidation of membrane lipids and proteins, formation of hemoglobin adducts, binding of acrylamide with spectrin, formation of micronucleated erythrocytes [3, 6, 41]. All of these conditions have been reported following exposure to acrylamides in previous studies. Although oxidative stress parameters were not measured in this study, acrylamide has previously been reported to cause protein and lipid peroxidation in erythrocytes. Catalgol et al. [6] have reported that in vitro incubation of human erythrocytes with acrylamide increased MetHb formation representing oxidative damage to hemoglobin, increased MDA formation representing lipid peroxidation, changed the activities of SOD, GSH-Px, CAT and GSH levels representing an increased defense against oxidative stress. Tarkiksh [41] has reported that MDA level of the erythrocytes increased within 3 hours after intraperitoneal administration of acrylamide to rats. On the other hand, oxidative damage affects the lipids of erythrocyte membrane and proteins of erythrocyte membrane skeleton which is primarily responsible for the ability of erythrocytes to change shape [25, 34, 36]. MDA is a product of fatty acid peroxidation and it has been reported that incubation of human erythrocytes with MDA decreases erythrocyte deformability [16, 27]. Oxidation of oxyhemoglobin to methemoglobin causes release of superoxide anions and exposure of human erythrocytes to superoxide anions have been reported to result in a decrease in the deformability of erythrocytes [44]. Both acrylamide and glycidamide react with hemoglobin to form adducts, the blood levels of which are used to determine the level of exposure [3]. Acrylamide does not only bind to hemoglobin, but it also causes oxidative damage to the hemoglobin molecule. Although we did not determine the ratio of acrylamide binding and the ratio of methemoglobin conversion, it is shown that oxidatively damaged hemoglobin cross-links with spectrin to form globin-spectrin complexes which in turn decreases erythrocyte deformability [34]. Acrylamide itself binds skeletal proteins of the erythrocytes such as: Spectrin, ankyrin and protein 4.1. This binding reaction is used routinely in fluorescent quenching of these proteins in research about their physicochemical behavior [18]. Acrylamide and glycidamide increase micronucleated (MN) reticulocytes and micronucleated erythrocytes in blood [1, 46, 48]. In mice, acrylamide at doses greater than 6 mg/kg increased MN reticulocytes and at 4 mg/kg MN erythrocytes [48]. However, in rats due to their efficient removal by the spleen, the

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4. Discussion

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Values for erythrocyte aggregation, plasma viscosity and hematocrit. Differences between control and acrylamide groups were found to be statistically insignificant (p > 0.05).

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Acknowledgements

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level of MN reticulocytes is lower both in the control animals and in those treated with acrylamide [28]. Still, microscopy-based counting (though not the flow cytometric measurements) revealed a rise in the level of MN reticulocytes in rats in response to acrylamide treatment [47]. Taking into consideration that Witt et al. [47] have introduced acrylamide for 3 days, it is likely that MN cells have increased in our study as well. The effect of MN reticulocytes and erythrocytes on erythrocyte deformability is not known. However, it was demonstrated that a rise in the number of reticulocytes in blood results in a decrease in erythrocyte deformability [32]. Effect of acrylamide binding to DNA and its possible outcome of production of faulty skeletal proteins requires further investigation in the scope of erythrocyte membrane functioning. This study clearly demonstrated that even at doses which do not induce apparent neuro-toxicity in rats, acrylamide impairs erythrocyte deformability. Erythrocyte deformability is one of the main determinants of blood viscosity, and lower erythrocyte deformability increases the viscosity of blood. Increased blood viscosity means decreased fluidity of blood which increases occurrence of cardiovascular diseases [19–21, 23]. The dose applied in this study is higher than daily dietary intake of various countries. However, humans are exposed to lower levels, for longer periods of time. Although the protocol used in this study includes higher dose and shorter period of time, and also performed on a different species – a rat – it strongly implies that chronic exposure to acrylamide interferes with blood viscosity. Further human studies are needed to evaluate the effects of dietary acrylamide intake in long term. Although daily intake of acrylamide from dietary sources is relatively small when compared to industrial resources, it should be considered seriously. Since acrylamide cannot be detoxified and excreted from the body easily, the risk of accumulation of acrylamide and its active metabolites may cause serious health problems. Various dietary sources such as fried foods – mainly potato-, bread and coffee increase daily acrylamide intake in diets. Food prepared fresh or processed below 100◦ C should be preferred to frying and baking as a dietary choice, in order to reduce acrylamide intake from dietary sources. Last but not the least, acrylamide exposure from materials used daily, should also be considered. During polyacrylamide manufacture trace amount of monomers may remain in the final product (such as daily care products and cosmetics) and pose a small risk for the consumers compared to intake from food [45]. Although small, this is an additional risk and when intake from various resources is considered together with the difficult detoxification of acrylamide in human body and untoward effect of environmental toxins such as tobacco smoke the damage may cumulatively augment the effect of acrylamide on blood viscosity, mainly the erythrocyte deformability.

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Authors would like to thank Prof. Dr. Rüs¸tü Onur for critical reading of the article, Murat Do˘gan M.D. and Serkan Karaismailo˘glu M.Sc. for their support during experimental procedures.

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