Original article

M. KOPANSKA1, J. CZECH1, P. ZAGATA2, L. DOBREK3, P. THOR1, G. FORMICKI2

EFFECT OF THE DIFFERENT DOSES OF ACRYLAMIDE ON ACETYLOCHOLINOESTERASE ACTIVITY, THIOL GROUPS, MALONDIALDEHYDE CONCENTRATIONS IN HYPOTHALAMUS AND SELECTED MUSCLES OF MICE

1Department of Human Physiology, Faculty of Medicine, University of Rzeszow, Rzeszow, Poland; 2Department of Animal Physiology and Toxicology, Pedagogical University in Cracow, Cracow, Poland; 3Department of Pharmacology, Faculty of Medicine and Health Sciences, Andrzej Frycz Modrzewski Cracow University, Cracow, Poland
Acrylamide is a chemical compound that typically forms in starchy food products during high-temperature cooking, including frying, baking and roasting. Acrylamide is a known lethal neurotoxin. Its discovery in some cooked starchy foods in 2002 prompted concerns about the carcinogenicity of those foods. Little is known about acrylamide’s influence on the peripheral nerves. In our research we measured acrylamide’s influence on the acetylcholinesterase activity in hypothalamus, heart muscle, skeletal muscles of the thigh and smooth muscle of the small intestine (males, Swiss strain) in relation to the thiol groups and malondialdehyde concentration. Acrylamide was injected intraperitoneally (20 and 40 mg/kg, i.e. 0.52 and 1.04 mg per animal). The hypothalamus and muscles were taken 24, 48, and 192 h after the injection. Acetylcholinesterase activity was significantly lower (P < 0.001 to P < 0.05) in all structures. It was accompanied by the statistically significant (P < 0.001 to P < 0.05) increase in malondialdehyde concentrations in most of the studied structures time periods and ACR doses. –SH groups concentrations were significantly depleted in selected structures (P < 0.001 to P < 0.05). The AChE activity evaluation in mice muscles and hypothalamus was very important because there are many evidences that acrylamide affects directly on the peripheral nerves. Thus, it causes structural damages and physiological changes. The results obtained in the present study provide evidence for the occurrence of oxidative stress after intraperitoneal injection of acrylamide to hypothalamus, heart muscle, skeletal muscles of the thigh and smooth muscle of the small intestine.
Key words:
acrylamide, oxidative stress, hypothalamus, muscles, mice

INTRODUCTION

Acrylamide is a low-molecular-weight organic compound composed of carbon atoms (50.69%), hydrogen (7.09%), nitrogen (19.71%) and oxygen (22.51%) with a molecular weight of 71.08 g. It is highly reactive due to the occurrence of a conjugated double bond, an amide fragment in its structure and the presence of multiple bonds with electrophilic properties. The double bond is an electrophilic center, which easily binds to amino groups (–NH2) or sulfhydryl (–SH) of amino acids, peptides and proteins. Moreover, the acrylamide can form hydrogen bonds due to the presence of an amide group (1). Acrylamide is formed during thermal processing of food products (above 120°C) and it is related to the formation of acrolein, which is produced by the thermal decomposition of glycerol followed by the oxidation of acrolein to acrylic acid. Acrylic acid as well as acrolein can be formed from fatty triglycerides released during frying of food products (2, 3). Furthermore, acrylamide has been classified by the International Agency for Research on Cancer (4) as a carcinogen assigned to the 2A group (5, 6). The European Union also ranked it as a category of carcinogenic and mutagenic substances (7). In 2002, the Swedish Food Administration (SFA) paid particular attention to the very high content of acrylamide in many food products (2, 8). Based on the results of experiments conducted on mice and rats, it has been shown that acrylamide, which enters the body primarily through the digestive and respiratory system and also through the skin is rapidly metabolized and then, metabolites are excreted in the urine. The biotransformation and elimination of acrylamide occurs in the liver. With the involvement of cytochrome P450 2E1, acrylamide is converted to an epoxy derivative - glycidamide (9, 10). Cytochrome P450 (CYP) belongs to the hemoprotein superfamily. They play a very important role in the bio-activation and detoxification of many harmful substances. It has been assumed that CYP1A and CYP2E mainly metabolize carcinogenic substances, whereas CYP3A, CYP2D and CYP2C are responsible for drug metabolism (11). Both acrylamide and glycidamide undergo conjugation reactions with glutathion. Both substances are neurotoxic (12). Acrylamide exhibits toxic effects after oral, inhalational or intraperitoneal application. The first toxic signs are limb numbness, tingling and ataxia (10, 13). Long-term exposure to acrylamide may damage the nervous system, resulting in the inhibition of axonal and direct neurotransmission (14). Based on studies conducted on animals, it is observed that acrylamide damages cells both in the nervous system and in the reproductive system. It also contributes to the occurrence of tumors in some hormone-dependent tissues.

It is important to identify possible mechanisms of acrylamide neurotoxicity, since such actions have been observed not only in animals but also in humans exposed to acrylamide. According to many studies (15-17), acrylamide is likely to cause abnormalities in redox biology, especially oxidative stress. Lipid peroxidation, one of the oxidative stress effects, is the process in which reactive oxygen species (ROS) result in the oxidative deterioration of lipids. Malondialdehyde (MDA) is a major lipid peroxidation product. The increase in MDA concentration is found in an elevated production of ROS in the body. Animal studies have also shown that acrylamide already using very low doses causes oxidative stress in various organs (18). To prevent ROS-mediated damage, the body has a defense system of antioxidants. An example of such endogenous compounds is reduced glutathione (GSH) which disables ROS protecting reactive protein groups from irreversible inactivation (19). GSH can be conjugated to toxic compounds, such as acrylamide. It causes a decrease in acrylamide concentration in the cell. Reduced GSH levels in the brain and muscles can also be a result of its use in free-radical reactions produced in excess after acrylamide’s administration. In consequence, oxidative balance in cells can be seriously disturbed (20). The antioxidant capacity of the body also depends on the content and activity of the antioxidant enzymes. The antioxidants prevent the process of producing new ROS and lipid peroxidation. This function is performed e.g. by albumins which have the capacity to bind different transition metal ions such as copper and iron that contain unpaired electrons, which provides protection against free radical reactions (20). The mechanism of many neurotoxins action is mainly related to their influence on neurotransmission (21). Even minor changes in the neurotransmitter system may have a significant effect on brain bioelectrical activity and behavior. It is associated with interactions between individual neurotransmitters and neurons. Acetylcholine is a major neurotransmiter in the cholinergic system. Acrylamide as a neurotoxin can cause a severe dysfunction in synaptic transmission in cholinergic neurons in both the central and peripheral nervous systems (17). It is also known, that the inhibition of AChE activity may cause excessive stimulation of postsynaptic membranes by acetylcholine, leading to neurotransmission impairment and muscle paralysis (22). In this way many neurotoxins interact with the animal organs (23).

In the present study, we hypothesized that acrylamide can disturb synaptic transmission in cholinergic neurons of the central and peripheral nervous system by affecting the activity of AChE. This study is a continuation and extension of the research presented previously by Kopanska et al. (24). AChE activity has also been investigated in the hypothalamus and heart muscle, transverse thigh muscle and smooth muscle of the small intestine of the SWISS mice.

MATERIAL AND METHODS

Study design

The research was conducted on Swiss male mice 12 weeks old, weighing 26 g. The animals were segregated into six experimental and three control groups. Each group consisted of 6 animals. The total number of animals was 108. Animals were fed with standard diet and grown in 12/12 light photoperiods. Granules for rodents (Agropol, Motycz, Poland) used for feeding contained proteins (16%), fat and oils (3.30%), phosphorus (0.65%), calcium (1.30%), sodium (up to 0.08%), lysine (0.77%), methionine (0.33%), cellulose (5.00%), ash (7.50%), and additives (vitamins A, D3, E). Animals from experimental groups were injected intraperitoneally with ACR doses of 20 mg/kg body weight (b.w.) and 40 mg/kg b.w., i.e., 0.52 mg and 1.04 mg per animal, respectively. ACR injected intraperitoneally is efficiently absorbed by the liver and undergoes biotransformation, which is essential for the ACR toxicity. Moreover, Sumner et al. (25) found that there were no significant differences in ACR metabolism and ACR metabolite excretion in the urine between rats exposed orally and intraperitoneally to ACR. In our experiments, the animals were injected one, two, or three times, which did not influence their condition. The doses of ACR were calculated on the basis of the experiment conducted by Zhu et al. (17). They indicated symptoms of ataxia in rats exposed to ACR doses of 60 mg/kg b.w. for 3 weeks (three ACR doses per week). In our experiments, we used the ACR dose of 40 mg/kg b.w. and lower one (20 mg/kg b.w.), and the animals were exposed one, two, or three times to ACR during 8 days. Thus, the doses used in our studies may be considered as subacute for rodents. They were still much higher than the mean ACR intake in daily diet of a human; so the results presented in our work must be interpreted carefully.

The experiment was performed in three series. Animals of the first series were injected on the first day of the experiment and sacrificed after 24 hours. The second series of animals was injected with ACR on the first day of the experiment and after 24 hours of the experiment (two doses of ACR). They were sacrificed 24 hours after the last injection, i.e. after 48 hours of the experiment. Animals of the third series were injected with ACR three times, i.e. on the first day of the experiment and next after 24 and 168 hours of the experiment. They were sacrificed 24 hours after the last injection, i.e. 192 hours (8 days) after the beginning of the experiment.

ACR was purchased from Sigma-Aldrich (Saint Louis, MO). ACR was dissolved in physiological saline and injected in the volume of 0.3 mL per animal. Control animals were injected with physiological saline (Polfa, Krakow, Poland) exclusively in the volume of 0.3 mL. All the injections were done at 7 am.

The hypothalamus, heart muscle, skeletal muscles of the thigh and smooth muscle of the small intestine were dissected and kept frozen at –80°C until the analyses. All the applied procedures were accepted by the First Local Ethic Committee on Experiments on Animals in Krakow (resolution number: 175/2012).

Acetylcholinesterase activity

Acetylcholinesterase activity was measured by preparing homogenates of tissue, as described below. Hypothalamus, 100 mg skeletal muscels, cardiac muscles and small intestinal were washed in phosphate-buffered saline (PBS) immediately after weighting. Next, the tissues were homogenized for 2 min in ice-cooled (4°C) 0.1 M sodium phosphate buffer (pH = 8). The proportion of 5 ml sodium phosphate buffer per 100 mg of the tissue was used to prepare the homogenates. Then, the homogenates were centrifuged at 15 000 rpm for 15 min (4°C). The supernatant obtained after centrifugation of tissue homogenate was used to measure acetylcholinesterase activity (AChE, EC 3.1.1.7) by the Ellman’s et al. colorimetric procedures. AChE catalyze the hydrolysis of acetylthiocholine (AcSCh) that is sulfur analogs of their respective natural substrate, acetylcholine. Upon hydrolysis, this substrate analog produce acetate and thiocholine. Thiocholine in the presence of the highly reactive dithiobis-2-nitro-benzonic acid (DTNB) reacts to generate the yellow of 5-thio-2-nitrobenzoate product. The assay is based on measurement of the change in color intensity at λ = 412 nm that is directly proportional to the activity of AChE. The results are presented as µM of acetylthiocholine iodide per g protein × h–1. AChE activity detection was performed with a MARCEL S330 spectrophotometer (Marcel, Poland).

Malondialdehyde concentrations (MDA)

Twenty five mg of tissue was homogenized on ice with homogenization solution containing 10 ml of RIPA buffer and 10 µl of protease inhibitor. The tissue was homogenized with the homogenization solution in a proportion of 250 µl solution per 25 mg of the tissue. Next, the homogenate was centrifuged at 4 500 rpm for 10 min (4°C). After centrifugation, 100 µl of supernatant was taken and transferred into a high temperature resistant tube. Then, SDS and a mixture of TCA and TBA were added to the sample. Then, the whole was stirred and boiled at 100°C for 1 hour. After this, the sample was cooled on ice for 10 min to stop the reaction and centrifuged at 3,500 rpm for 10 min (4°C). 150 µl of supernatant was placed on microplates and the absorbance was measured at λ = 540 nm using TECAN microplate reader. MDA concentration is presented as µM/mg protein.

Reduced glutathione concentrations

Tissues were homogenized in 0.1 M phosphate buffer with pH of 7.4 containing 1 mM EDTA. Homogenates were centrifuged at 4°C and 15,000 rpm. The supernatants were denatured by blending of 100 µl of supernatant, 100 µl of TCA and 100 µl of EDTA. The mixture was cooled in a refrigerator for 10 min and then centrifuged for 5 min with 6,300 rpm. The GSH was determined according to the Ellman colorimetric method (1959). A solution was prepared on a microplate. A blank test was also prepared. The prepared samples were stirred and cooled for 10 min (4°C). Extinctions were measured using a microplate reader (TECAN SUNRISE Infinite® F50) at wavelength λ = 412 nm. The concentration of reduced glutathione (µmol/g of a protein) was determined using the calibration curve. The calibration curve was determined using pure reduced glutathione (Sigma Aldrich).

General protein concentration

General protein concentration was determined using the Bradford method (26). The maximum of absorption spectrum for dye in acidic solution is at 465 nm. When the protein is bound the maximum moves towards longer wavelengths and occurs at 595 nm. The calibration curve was determined based on the BSA protein measurements (bovine albumin). Linear dependence of absorbance on concentration was determined in the protein concentration range of 0.1 – 1.4 mg/mL. To carry out measurements 4 µl of the test sample and 200 µl of Bradford reagent were placed on microplates. The measurement was performer using microplate reader at wavelength λ = 595nm.

Statistical analysis

The results were statistically analyzed using STATISTICA 10.0 Stat-Soft. Distribution of dependent variables was tested using the Shapiro-Wilk test. The homogeneity of variance was checked by Levene’s ANOVA. Differences between control and treatments were examined by a one-way analysis of variance (ANOVA) followed by a post hoc Dunnett’s test. Differences were considered as statistically significant at P < 0.05.

RESULTS

Acetylcholonesterase activity in hypothalamus

AChE activity in the hypothalamus exhibits a significant decrease exposed to acrylamide. In all periods of time, it was statistically significant. The greatest decrease was 75.09% in comparison to the control sample and it was noted after 24 of hours after ACR application (40 mg/kg ACR) (Fig. 1).

Figure 1
Fig. 1. AChE activity in mouse hypothalamus after 24, 48, and 192 hours of experiment. Differences statistically significant compared to control at **P < 0.01; ***P < 0.001.

Acetylcholonesterase activity in heart muscle

The greatest decrease was 52.20% in comparison to the control sample and it was determined after 192 hours of ACR application (40 mg/kg/b.w.) (Fig. 2).

Figure 2
Fig. 2. AChE activity in the mouse myocardium after 24, 48 and 192 hours of experiment. Differences statistically significant compared to the control samples at ***P < 0.001.

Acetylcholonesterase activity in the smooth muscle of the small intestine of mice

The greatest decrease in the activity of the enzyme was 56.84% and it was measured after 192 hours of intraperitoneal injection of acrylamide (40 mg/kg) (Fig. 3).

Figure 3
Fig. 3. AChE activity in the smooth muscle of the small intestinal of mice after 24, 48, and 192 hours of experiment. Differences statistically significant compared to control at **P < 0.01; *** P < 0.001.

Acetylcholonesterase activity in skeletal muscle of the thigh

With regard to the control sample, the greatest decrease in AChE activity of 68.51% was observed after 192 hours of ACR application (40 mg/kg). A comparable decrease in AChE activity (67.27%) with respect to controls was also reported after 48 hours of ACR application (40 mg/kg) (Fig. 4).

Figure 4
Fig. 4. AChE activity in skeletal muscle of mouse thighs after 24, 48, and 192 hours of experiment. Differences statistically significant compared to the control samples at **P < 0.01; ***P < 0.001.

Malondialdehyde concentations

According to the obtained results, MDA concentrations in the hypothalamus increased at particular time periods in comparison to the control sample. The highest increase in MDA compared to control mice was observed after 48 hours (45.12%) and 192 hours (46.43%) of ACR exposure (40 mg/kg). Moreover, in the heart muscle, with regard to the control sample, the greatest increase in MDA concentration (58.84%) was observed after 192 hours of 40 mg/kg acrylamide exposure. Comparable increase was observed in other time periods (after 24 hours (57.01%) and 48 hours (56.31%) of ACR application (40 mg/kg). In the smooth muscle of the small intestine. The highest increase of MDA concentration (48.15%) was observed after 192 hours of ACR 40 mg/kg treatment compared to control group. A comparable growth (47.28%) was also observed after 48h in mice receiving 40 mg/kg of ACR. What is more, in the mouse skeletal muscle of the thigh, the highest increase of an index of lipid peroxidation was observed after 48 hours of ACR 40 mg/kg treatment compared to control group. It was 43.25%. (see Table 1).

Table 1. MDA Concentration (µM/g protein ± S.E.M.) in hypothalamus, heart muscle, skeletal muscles of the thigh and smooth muscle of the small intestine after acrylamide application.
Table 1

Reduced glutathione concentration

Administration of both ACR doses (20 mg/kg and 40 mg/kg) caused a decrease in the –SH groups level in the brains of the experimental animals as compared to their corresponding controls. In the hypothalamus, the highest decrease of GSH concentration (70.08% in comparison with the control group) was observed after 192 hours. The highest decrease of GSH concentration in heart muscle was observed after 24 hours of ACR 40 mg/kg treatment (89.47%). It is also noteworthy that GSH levels decrease for the remaining period of time; after 48 hours - 82.55% and after 192 hours - 80.38% of ACR 40mg/kg treatment. Moreover, in the smooth muscle of small intestine, the highest decrease of GSH concentrations (72.9%) was observed after 24 hours of ACR 40 mg/kg treatment compared to control group. In the skeletal muscle of the thigh, the highest decrease of GSH concentration (36.54%) was observed after 48 hours of ACR 40 mg/kg treatment (Table 2).

Table 2. GSH Concentration (µM/g protein ± S.E.M.) in hypothalamus, heart muscle, skeletal muscles of the thigh and smooth muscle of the small intestine after acrylamide application.
Table 2

DISCUSSION

In the present study, we hypothesized that the AChE activity, an enzyme associated with cholinergic transmission, can be an important determinant of the neurotoxic properties of acrylamide. Our study indicates decrease AChE activity in mice hypothalamus and selected muscles. Cholinergic neurons are involved in many functions in the brain including memory, emotion, stress response, body human motion control, and control of the basic life functions. Cholinergic synapses are distributed in autonomic fibers, also in parasympathetic fibers and in some sympathetic fibers (27). Kopanska et al. (24) have shown that acrylamide affects the activity of AChE in the selected brain structures, such as hemispheres, cerebellum and brainstem. Therefore, it seemed important to continue the study and evaluate the activity of AChE in the hypothalamus and the muscles of mice because there are several evidences that acrylamide acts directly on the peripheral nerves and causes structural damage and functional changes. Ultrastructural damage (e.g., cell membrane dysfunction) and functional changes (e.g., a decrease of neurotransmitter release) suggest that acrylamide disrupts the fusion of synaptic vesicles (13). The connection between the presynaptic membrane and the synaptic vesicle is the basis of neurophysiological processes and it is crucial for the construction of axon terminals and the release of neurotransmitters. Acrylamide disrupts the connections between the membranes and therefore it exerts toxic effect on the fusion of synaptic vesicles with the presynaptic membrane. Acrylamide also causes degeneration of the peripheral nerves by reducing neurotransmission (13).

Despite intensive studies of the neurotoxicity of acrylamide, there is little data available on its effect on AChE activity in cholinergic neurons. All the information above indicates that there was a disruption of oxidative balance in the hypothalamus and also in the cardiac muscle, skeletal muscle of the thigh and smooth muscle of the small intestine. Oxidative stress propagates the lipid peroxidation process. MDA is a major lipid peroxidation product. The increase in MDA concentration is found in an elevated production of ROS in the body. Lipid peroxidation is an avalanche process. Once it started it runs until the substrate is exhausted. Lipid structures of the cell membrane layers are affected leading to changes in their liquefaction and the membrane transport integrity is lost. Additionally, a receptor/ligand binding affinity is changed. In consequence, lysosomal enzymes are released what is a major mechanism of tissue damage (28, 29). MDA is an indicator of a lipid peroxidation. According to the obtained results, the significant increase of a lipid peroxidation was observed in studied brain and muscle structures of mice after acrylamide application.

Increasing in the production of reactive oxygen species causes disorders in cell membrane permeability, dissociation of oxidative phosphorylation in the mitochondria which may induce a cell apoptosis (30).

Malonic dialdehyde modifies the physical properties of cell membranes, causing disturbance of the lipid hydrophobicity and violation of the bilayer membrane structure (31). Thus, MDA influences the cellular structure, leading to disturbances in their normal function, and consequently to the brain and muscle dysfunction.

The results obtained in the present study indicate an increase in MDA levels in the hypothalamus as well as in selected muscles. Observed decrease in GSH concentration in the brain and selected muscles are consistent with Srivastava et al. (32). These authors have shown that the intensification of the lipid peroxidation process is linked to the decrease of the glutathione pool.

Zhu et al. (17) have revealed the presence of disorders in oxidative-reducing balance after ACR application in sciatic nerve, hippocampus, spinal cord, and cerebellar cortex.

A closer examination of certain biological processes and activities of pharmacological agents and metabolites often shows their newly documented pro-oxidative properties, and identifies molecular pathways linking their toxic oxidative effect with the protective biological anti-oxidative mechanisms. For example, as we can see from our results, oxidative stress caused by acrylamide can be very dangerous, but it can be controlled, to some extent, by the detoxifying, anti-oxidative activity of glutathione.

The well-investigated are the links between exercise, oxidative damage and resulting inflammation. The results of these studies usually show the dynamics of the anti-oxidative repair/defence mechanisms following the dynamics of the oxidative stress. Changes in lipid peroxidation measured as MDA, and in MPO activity in rats undergoing renovascular hypertension during exercise have been described (33). Sakr et al. showed a significant, exercise-related increase in expression of the brain-derived neurotrophic factor BDNF, what reveals that the oxidative stress resulting from hypoxia and/or exercise - here in rats - controls gene expression of this factor involved in the proliferation of neurons in the brain cortex (34).

The unique association between glutathione-related antioxidant defense system in endothelial pathophysiology of hypertension and endothelial injury has also been documented (35). These are only a few out of many examples of recent investigations pointing into a vast complexity of oxidative stress and anti-oxidative defence mechanisms in biological organisms.

The results of our study provide evidence for the occurrence of oxidative stress after intraperitoneal injection of ACR in hypothalamus, as well as in the myocardium, smooth muscle of the small intestine and skeletal muscle of the thigh.

Acknowledgments: Authors kindly thank Professor Zbigniew Dlugosz for his great support of this work. Special thanks to the staff of the Department of Animal Physiology, Slovak University of Agriculture. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflicts of interests: Non declared.

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R e c e i v e d : May 5, 2017
A c c e p t e d : August 25, 2017
Author’s address: Dr. Marta Kopanska, Department of Human Physiology, Faculty of Medicine, University of Rzeszow, 16C Rejtana Street, 35-959 Rzeszow, Poland. e-mail: martakopanska@poczta.onet.pl