Abstract
A liberal amount of acrylamide (AA) is produced as a result of frying or baking foods in high temperatures, and individuals take certain amounts of AA everyday by consuming these food items. Pregnant women are also exposed to AA originating from food during pregnancy and their fetus are probably affected. The rats were divided into five different groups: control (C), corn oil (CO), vitamin E (Vit E), AA, and Vit E + AA, with eight pregnant rats in each group. On the 20th day of pregnancy, fetuses were removed and brain tissues of fetuses were examined for biochemical and histological changes. AA caused degeneration in neuron structures in fetal brain tissue and caused hemorrhagic damages; dramatically decreased brain-derived neurotrophic factor levels; increased malondialdehyde, total oxidant capacity levels; and decreased reduced glutathione and total antioxidant capacity levels (p < 0.05). On the other hand, it was determined that the Vit E, a neuroprotectant and a powerful antioxidant, suppressed the effects of AA on fetal development and fetal brain tissue damage for the above-mentioned parameters (p < 0.05). It is recommended to consume food containing Vit E as a protection to minimize the toxic effects of food-oriented AA on fetus development due to the widespread nature of fast-food culture in today’s life and the impossibility of protection from AA toxicity.
Introduction
Acrylamide (AA) is a rather toxic amide that could be synthesized chemically, containing unsaturated double bond and widely used in various industrial fields such as textiles, paper, cosmetics manufacturing, and treatment of drinking water. 1,2 AA is formed naturally as a result of Maillard reaction in food that contains carbohydrates and especially asparagine, which contains AA, under temperatures higher than 120°C. 3,4 Studies conducted with humans and experimental animals demonstrated that AA causes neurotoxicity. 5 –7 Daily average AA intake dose was reported as 0.5 μg/kg/day; however, the intake dose varies based on factors such as lifestyle, nutritional habits, age, and gender. 8,9
AA is a highly water-soluble chemical that could permeate to infants via breast milk during breast feeding period. 10 Especially in intrauterine life and infancy, the level of exposure to AA could be higher than adults. This is due to smaller body size of fetuses and infants when compared to adults. 9,11 This could result in permanent AA-induced damages that could last a lifetime in fetuses and infants. Under normal physiologic conditions, there is a balance between antioxidant systems and free radicals, and due to this balance, free radicals are rendered harmless. Destabilizing this balance in favor of oxidants causes oxidative stress, which in turn results in oxidative tissue damage.
Vitamin E (Vit E) is a significant fat-soluble antioxidant that could be found in four tocopherol forms of α, β, γ, and δ. It prevents the oxidative stress formed by free radicals in the fat phase of the cell, thus protecting the cells against oxidative stress. It creates that effect by transforming free radicals into less reactive compounds. 12 –14 Vit E could easily pass through the placenta. 15 Vit E, which is a powerful antioxidant for several tissues, permeates the blood–brain barrier and shows a strong neuroprotective effect. 16,17
This study would attempt to determine the effects of AA applied to rats during pregnancy on fetal brain development and brain-derived neurotropic factor (BDNF) synthesis, hence to identify the neurotoxic mechanism of action of AA, and would test the protective effects of Vit E, which is a powerful antioxidant and neuroprotective agent, against the possible damage that AA would cause in the fetal brain.
Materials and methods
Animals
Forty young female Wistar albino rats weighing 250 ± 20 g and bred in İnönü University Faculty of Medicine, Experimental Animals Breeding and Research Center were used in the study. Rats were moved into special cages on 17:00 h in groups of two females and one male. They were kept in these cages until 08:00 h next morning. At the end of this period, males were separated from the females. Vaginal smear taken from female rats was examined under microscope and females whose smear exhibited the existence of sperm were accepted as half-day pregnant. Females who were not identified as pregnant + with the smear test were excluded from the study. Pregnant rats were kept in 12-h daylight, 12-h darkness chambers, which were continuously ventilated and kept under 21 ± 2°C, for 20 days (during pregnancy). Rats were fed ad libitum during the tests.
Study design
Forty female Wistar rats were used in the study. Before the experiments, propagated rats with positive vaginal smear results were randomly separated into five groups of eight rats each: Group 1: C group—No application was performed during pregnancy. Group 2: CO group—1 mL/kg corn oil was applied during pregnancy. Group 3: AA group—AA (Sigma A8887, St Louis, Missouri, USA) was dissolved in drinking water; 5 mg/kg body weight AA was applied during pregnancy. Group 4: Vit E group—α-Tocopherol (Sigma T3251) was dissolved in corn oil; 100 mg/kg body weight Vit E was applied during pregnancy. Group 5: AA + Vit E group—5 mg/kg AA and 100 mg/kg Vit E were applied during pregnancy.
Applications were performed at the same day and time via gavage. After the fetuses were delivered by caesarian section on the 20th day of pregnancy, the fetal brain tissues were used in histologic and biochemical analyses.
Histologic analysis method
Fetal brain tissues were fixed with 10% formaldehyde solution. Eight micrometers thick cross sections were obtained from paraffin blocks for histologic analysis. Obtained sections were deparaffinized and hematoxylin–eosin (H–E) staining method was applied. Stained sections were examined using Carl Zeiss Axiocam (Germany) ERc5 digital camera attached microscope and histopathologic analysis was conducted. Histopathologic analysis was conducted using images of neurons in cerebral cortex region taken by different lenses.
Biochemical analyses
Malondialdehyde (MDA), reduced glutathione (GSH), total antioxidant status (TAS), total oxidant status (TOS), and BDNF levels were measured on fetal brain tissues that were taken into liquid nitrogen tanks at the end of the study and then stored in −80°C deep freeze.
Preparation of the tissues for biochemical analyses
Fetal brain tissues preserved in deep freeze were removed and weighed on the day of the study. Phosphate buffer was added to produce a 10% homogenate and they were homogenized in ice for 1–2 min at 12,000 r/min (IKA, Germany). Supernatants were obtained by centrifuging tissue homogenates for 30 min at 5000 r/min and +4 degrees.
Measurement of reduced GSH levels
GSH analysis was conducted by the method described by Ellman. 18 GSH concentration was determined by the reaction of GSH located in the analysis cartridge with 5,5′-dithiobis (2-nitrobenzoic acid), producing a green-like color, and by reading the light intensity of this color using a 410-nm wavelength Spectrophotometer (LKB Biochrom Ultrospec Plus 4054 UV/visible).
Measurement of MDA levels
The method by Uchiyama et al. 19 was used in MDA analysis. MDA concentration was determined by the measurement of the supernatant extracted from n-butanol phase of the pink colored product, which was produced by the reaction of the MDA in supernatant with thiobarbituric acid under 95°C, using a spectrophotometer (LKB Biochrom Ultrospec Plus 4054 UV/visible) at 535 nm.
TOS measurement
Commercial TOS measurement kit (RelAssay Diagnostic, Turkey) was used for the determination of TOS. The principal of this method is based on the oxidation of ferrous ion–chelator complex into ferric ions by the oxidants in the sample and creation of a color by formed ferric ions with the chromogenous substance in acidic environment. 20 As detailed in the procedure for the kit, for TOS measurement, ELISA (enzyme-linked immunosorbent assay), (Basic Radim Immunoassay Operator (BRIO); Pomezia, Italy) was set at 25°C, 500 μL reactive 1 (measurement buffer) and 75 μL serum were mixed and its absorbance was measured at 530 nm. Twenty-five microliters reactive 2 (pro-chromogenous solution) was added to the mixture and it was incubated for 10 min and then absorbance was measured again at 530 nm to determine the TOS levels.
TAS measurement
Commercial TAS measurement kit (RelAssay Diagnostic) was used. Working principal of the method is based on the reduction of the dark blue–green 2,2′-azino-bis 3 ethylbenzothiazoline-6-sulphonic acid (ABTS) radical into colorless ABTS form by the antioxidants in the sample (23). As detailed in the procedure for the kit, for TAS measurement, ELISA (BRIO; Pomezia) was set at 25°C, 500 μL reactive 1 (measurement buffer) and 30 μL serum were mixed and its absorbance was measured at 660 nm. Seventy-five microliters reactive 2 (colored ABTS solution) was added to the mixture and it was incubated for 10 min and then absorbance was measured again at 660 nm to determine the TAS levels.
Measurement of BDNF levels
Fresh brain tissues were rapidly weighed and transferred into liquid nitrogen tank and stored in −80°C until the day of the experiment. At that time, fetal brain samples were thawed and phosphate buffer (pH: 7.5, 0.05 molar, including protease inhibitor cocktail) by four times the volume of the tissue weight was added and they were homogenized in ice at 12,000 r/min (IKA), and then supernatants were obtained by centrifuging the samples for 10 min at 10,000 g. In accordance with the kit procedure, the samples were measured in an enzyme-linked immunosorbent assay (ELISA) (BRIO; Pomezia) equipment at 450 nm to determine BDNF levels.
Statistical analyses
Statistical analyses were conducted with SPSS 21.0 for Windows software package. Normal distribution of the data was analyzed using Shapiro–Wilk test. Since the data did not exhibit normal distribution, it was summarized with median (minimum to maximum). Comparison of the groups was conducted with Kruskal–Wallis test. Paired comparisons following the Kruskal–Wallis test were conducted with Conover method. In all analyses, significance level was set at 0.05 (p ≤ 0.05).
Results
Histopathological findings
Fetal brain tissue cross sections of experimental groups were analyzed in optical microscope. Histopathological examination was conducted on structures and numerical density of neurons in the cerebral cortex region. It was observed that numerical density of cerebral cortex neurons was decreased in AA group fetal brains compared to the control and other study groups. Furthermore, the neuron layer thickness was found to be decreased in the region when compared to other groups. It was found that both numerical density and the layer thickness were increased in Vit E and AA + Vit E applied groups compared to AA applied group. However, when compared to the control group, there were no significant differences between the groups except the AA applied group (Figure 1).
(a) C group: Structures and numerical density of cerebral cortex neurons are normal. H–E: ×40. (b) CO group: Histological picture is similar to the C group. H–E: 40. (c) AA group: Numerical density of the neurons located at cerebral cortex of fetus brains was reduced compared to control and other groups. Neuron layer in the region depicted with an asterisk (*) was reduced when compared to other groups. H–E: ×40. (d) Degeneration (thin arrow) and edematous neurons (thick arrow) were identified in the neurons located at the cerebral cortex of fetuses in the AA group. H–E: ×100. (e) Vit E group: No anomalies were observed in regions and neurons in the cerebral cortex. H–E: ×40. (f) AA + Vit E group: Histological image was almost similar to the C group. H–E: ×40. C: control; CO: corn oil; AA: acrylamide; Vit E: vitamin E; H–E: hematoxylin–eosin.
Biochemical findings
When compared to the C group, fetal brain tissue MDA and TOS levels statistically significantly increased in the AA group (p < 0.05), while Vit E application statistically significantly reduced (p < 0.05) MDA and TOS levels up to that of the C group.
When compared to the control group, fetal brain tissue GSH and TAS levels statistically significantly decreased in the AA group (p < 0.05), while Vit E application statistically significantly increased (p < 0.05) GSH and TAS levels up to that of the C group.
When compared to the control group, fetal brain tissue BDNF levels statistically significantly decreased in the AA group (p < 0.05), while Vit E application statistically significantly increased (p < 0.05) BDNF levels up to that of the control group. Histopathologic findings of the present study were consistent with the biochemical findings. MDA, TOS, GSH, TAS, and BDNF median results are presented in Table 1.
Fetus brain tissue oxidant–antioxidant parameters of all groups.a
C: control; CO: corn oil; AA: acrylamide; Vit E: vitamin E; AA + Vit E: acrylamide + vitamin E; gwt: gram wet tissue.
aData are expressed median (Min–Max) of eight animals. Different letters in columns are significant p < 0.05.
Discussion
Water-soluble AA permeates placenta directly in experimental animals, reaching fetal tissue and causing developmental disorders and tissue damage depending on the daily intake rate. 10 It was determined that AA has neurotoxic effect on neural axons of the peripheral and central nervous systems and causes membrane division and tubulovascular changes along the nerve terminals, however the mechanism of the neurotoxic effect is yet to be discovered. 21 It was suggested that these tissue damages were due to AA-induced oxidative stress. 22 –24
Vit E, which is a powerful antioxidant for several tissues, could penetrate the blood–brain barrier and displays powerful neuroprotective effects. 16,17 Vit E easily permeates the placenta during intrauterine period and is substantially absorbed by the fetal brain tissue. 15 Brain is especially sensitive to oxidative stress due to its low antioxidant capacity and requires the protective effect of powerful antioxidants such as Vit E. 25
Alam et al. 22 applied 10 mg/kg/day oral AA from the 7th day of the pregnancy until delivery (prenatal) and until the postdelivery 28th day (perinatal) to pregnant rats. The researchers observed that AA delayed the reproduction in granular layer histologically and concurrently caused Purkinje cell loss. Histopathological examination of Purkinje cells of the perinatal group demonstrated micro space formations and cell loss. Alam et al. demonstrated with the above-mentioned study that prenatal and perinatal AA or its metabolites prevented biochemical mechanism, caused oxidative stress, and resulted in structural changes in the developing rat cerebellum.
Ogawa et al. 26 studies the effects of 0, 4, 20, and 100 ppm dose AA administered between the 10th and 21st days of pregnancy on the fetuses of pregnant rats. In 20 and 100 ppm AA administered groups, it was determined that neurons in the hippocampus region of neonatal rat brain tissue were affected adversely. Researchers stated that anomalies that progress by the reduction of immature granular cells due to the effects on hippocampal neurogenesis that targets the reproduction of type-3 precursor cells and results in neurotoxicity and developmental disorders could occur.
El-Sayad et al. 21 fed study group rats with a diet that included 30 mg/kg body weight AA and 30% fried potato chips (FPCs) during pregnancy from the 6th day of pregnancy and provided a standard diet to the control group rats and compared the babies’ brain tissues histologically. A serious decrease in the number of Purkinje cells and serious thinning of internal granular layers were observed during the optical microscope examinations of the cerebral cortex of AA and FPC applied animals. The existence of significant necrosis was observed in the brain Purkinje cells and neurons of the babies born from AA and FPC applied mothers. Furthermore, histopathologic examination of Purkinje cells also demonstrated changes in endoplasmic reticulum, degeneration in normal polyribosome structure, bulging mitochondria with abnormally differentiated crista and an abnormal Golgi organization.
Among the results of the present study, degeneration in neuroglia nuclei, decrease in numerical density of neurons, degenerated neuron extensions, edema in neurons, neuron losses, increase in dead cell count, and chromatolysis were striking findings. Contrary to the AA group, decrease in regions with neuron loss and degenerated neurons and an increase in healthy neuron count were determined in the AA + Vit E group. Considering the above-mentioned histopathological findings, the results of the current study are consistent with the findings of the previous similar studies in the field.
During regular physiologic metabolism, certain levels of free oxygen radicals (FORs) are formed in all eukaryotic cells. Primary FORs are superoxide anion radical (O2 •), hydrogen peroxide (H2O2), hydroxyl radical (•OH), singlet oxygen (1O2), and peroxyl radicals (LOO•). While free radicals continuously form in the body under physiological conditions, these harmful radicals are detoxified by inhibitive antioxidant mechanisms. These antioxidant structures are antioxidant enzymes (superoxide dismutase (SOD), catalase, glutathione-S-transferase, glutathione peroxidase, vitamins (A, E, and C)) and other organic and inorganic molecules (GSH, melatonin, and selenium).
Under normal physiological conditions, there is an equilibrium between the antioxidant systems and free radicals and due to this equilibrium, free radicals are rendered harmless. An imbalance favoring the oxidants would result in oxidative stress, which would in turn cause oxidative tissue damage.
In the study that Alam et al. 22 applied 10 mg/kg/day oral AA from the 7th day of the pregnancy until delivery (prenatal) and until the postdelivery 28th day (perinatal) to pregnant rats, it was found that MDA levels in the brain tissues of 28-days-old baby rats seriously increased and GSH levels significantly decreased. Yonguç et al. 27 applied 100 mg/kg body weight Vit E orally to diabetes model induced male rats and observed that TAS levels in rat hippocampus tissues increased significantly when compared to control and diabetes groups and at the same time TOS levels decreased significantly.
Alzoubi et al. 28 studied the protective effect of Vit E application on cognitive deficiency induced by chronical sleep withdrawal and the possible molecular targets of Vit E on cognitive deficiency induced by chronical sleep withdrawal. Sleep withdrawal was initiated with modified multiplatform model on rats and 100 mg/kg Vit E was added to the rats’ diet. Behavior measurement study was conducted to test the spatial learning and memory using radial arm maze test. Furthermore, nonenzymatic and enzymatic antioxidant levels such as GSH, GSSG, and GSH/GSSG; GPX; catalase; and SOD were measured. Study results demonstrated that chronic sleep withdrawal deteriorated both (short-term and long-term) memories, however Vit E application compensated for this deterioration. In addition, Vit E returned the decrease in SOD, catalase, and GPX activity levels in the hippocampus and the decrease in GSH/GSSG ratio to normal levels.
In a separate empirical study, when Ghorbel et al. 29 applied 20 mg/kg body weight AA to female rats for 21 days using oral gavage, a significant increase in brain tissue MDA levels and a significant decrease in GSH levels were observed compared to the control group. In the study that Alam et al. 22 applied 10 mg/kg/day oral AA from the 7th day of the pregnancy until delivery (prenatal) and until the postdelivery 28th day (perinatal) to pregnant rats, it was found that MDA levels in the brain tissues of 28-days-old baby rats seriously increased and GSH levels significantly decreased. In an empirical study by Krishna and Muralidhara 30 conducted by adding 50, 100, and 200 ppm AA to the drinking water of pregnant rats between the 6th and 19th days of pregnancy, it was reported that 200 ppm AA application significantly increased the MDA levels in fetal brain tissue.
In the present study conducted by applying 5 mg/kg/day AA between the 1st and 20th days of the pregnancy, 100 mg/kg/day Vit E was given as a protective against the possible neurotoxic effects of AA. MDA, GSH, TAS, and TOS levels were measured in the brain tissue of fetuses aborted on the 20th day of the pregnancy by caesarian section. As a result, it was determined that AA application significantly increased the MDA and TOS levels and significantly decreased GSH and TAS levels in rat fetal brain tissue; however, Vit E application in addition to AA significantly reduced MDA and TOS levels and significantly increased GSH and TAS levels in rat fetal brain tissue.
These results demonstrated that AA application caused oxidative stress in fetal brain tissues and the protective antioxidant effects of Vit E against AA neurotoxicity were fully realized. These findings of the current study are consistent with the results of other similar studies.
One of neurotrophic factors, BDNF is an extremely important brain development factor that maintains neuron growth, synaptic functions, and neural plasticity in these stages of brain development. The most important functional properties are protection of neurons and providing their survival. BDNF supports the development of noradrenergic and serotonergic neurons and protects them from toxic damages. It regulates neuronal continuity and plasticity with its positive effect on dendrite growth. Studies showed that oxidative stress reduced the BDNF levels in the brain. 31
Sakr et al. 32 applied 100 mg/kg Vit E as a protective to 4 weeks old rats exposed to exercise for 2 months. In this study, BDNF gene expression levels in young rat brain tissue were analyzed. Study results showed that BDNF gene expression levels significantly decreased in the group exposed to exercise, however BDNF gene expression levels significantly increased as a result of Vit E application.
Madhyastha et al. 33 set a stress model in rats between the 1st and 10th days of pregnancy and applied 10 mg/kg/day resveratrol orally for protection during pregnancy. The study examined the changes in BDNF levels due to stress and resveratrol in fetal hippocampal tissue between the 1st and 10th days of pregnancy. Study results demonstrated that stress substantially decreased BDNF levels, while resveratrol application increased BDNF levels. Chakrabarty et al. 34 created a hypothyroidism model by applying propyl-thiouracile (PTU) to pregnant rats and examined the changes in BDNF levels during perinatal 3–7th days in the brain tissues of young rats. The study reported that PTU application substantially decreased the BNDF levels in young rat hippocampi.
Conclusion
The oxidant and antioxidant parameters obtained from the AA applied groups in the present study demonstrated that AA caused oxidative stress in fetal brain tissue by permeating through the placenta. Probably, AA-induced oxidative stress and consequent oxidative tissue damage reduced fetal brain tissue BDNF levels. On the other hand, we consider that Vit E applied concomitantly with AA removed AA-induced free radicals with its sweeper effect, suppressed oxidative stress as a result, and normalized BDNF levels.
This study is the first of its kind that demonstrated that AA seriously decreased BDNF levels in fetal brain tissue. Thus, for the complete discovery of the BDNF reduction mechanisms of AA in fetal brains, further studies are required to scrutinize BDNF gene transcription phase and BDNF mRNA translation phase.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by a grant from the Scientific Research Fund of Inonu University (Grant number: İNU-BAP 2015/92).