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Argan oil reduces oxidative stress, genetic damage and emperipolesis in rats treated with acrylamide
Biomedicine & Pharmacotherapy 94 (2017) 873–879
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ScienceDirect www.sciencedirect.com
Original article
Argan oil reduces oxidative stress, genetic damage and emperipolesis in rats treated with acrylamide lua,* , Birsen Aydınb , Vedat Şekerog lua Zülal Atlı Şekerog a b
Department of Molecular Biology and Genetics, Faculty of Science, Ordu University, 52200-Ordu, Turkey Department of Biology, Faculty of Science, Amasya University, 05100-Amasya, Turkey
A R T I C L E I N F O
Article history: Received 1 June 2017 Received in revised form 4 August 2017 Accepted 7 August 2017 Keywords: Acrylamide Argan oil Oxidative stress DNA damage Megakaryocytic emperipolesis
A B S T R A C T
Acrylamide (AA), a well-known toxicant, is present in high-temperature-processed foods in heated foods. Argan oil (AO), a natural vegetable oil, is receiving increasing attention due to its powerful biological properties. However, limited information is available about its effects in lymphoid organs and bone marrow. The aim of this study is to investigate the effects of AO on hematological parameters, 8hydroxydeoxyguanosine (8-OHdG), thiobarbituric acid reactive substances (TBARs), protein carbonyl (PCO), glutathione (GSH), myeloperoxidase (MPO) levels, the formation of micronucleus (MN) and megakaryocytic emperipolesis (ME) against AA-induced toxicity in rats. The animals were treated with AA (50 mg/kg/day), AO (6 ml/kg/day per day) and AA + AO (50 mg + 6 ml/kg/day) for 30 days. Treatment of rats with AA significantly decreased the hematological parameters, GSH and MPO activity and PCEs ratio while it increased TBARs, PCOs and 8-OHdG levels and formation of MN and ME. No significant differences were observed in the animals received the AO alone. Co-treatment with AA + AO ameliorated almost all of the alterations caused by AA and exhibited protective effect in rats. Based on the obtained results, we suggest that integration of AO in diet or using its supplements may be a good strategy for improving tissue injury in many diseases. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Acrylamide (AA) is an odorless, colorless, highly water-soluble, rapidly polymerizable, crystalline substance. Polymers of AA are widely used in industry and biological research [1]. In foods, AA is generated from some food components during heat treatment at high temperatures (above 120 C) [2]. In addition to the occupational routes of exposure, AA may be absorbed from mainstream cigarette smoke [1]. Therefore, high temperatureprocessed foods and smoking are considered as important sources of human exposure to AA [3]. Certain foods such as french fries, potato crisps, bread, cookies, and coffee exert the highest contribution to dietary exposure of AA [4]. Children seem to be under more threat than adults in terms of exposure of AA because of their higher caloric intake relative to body weight and their higher consumption of certain AA-rich foods [4,5]. AA, a well-known toxicant, has been classified as a probable carcinogen to human (Group 2A). Therefore, AA has raised significant concerns about the possible exposure and health risks
* Corresponding author. lu). E-mail addresses: [email protected], [email protected] (Z.A. Şekerog http://dx.doi.org/10.1016/j.biopha.2017.08.034 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
for the general population [3]. Some in vitro and in vivo studies have demonstrated that AA can induce oxidative stress, neurotoxicity, developmental and reproductive toxicity, immunotoxicity, genotoxicity, and carcinogenicity [1,2,6–8]. In recent years, researchers have focused on some of natural antioxidants that may be effective on the toxicity of AA [7,9–11]. Argan oil (AO), a natural vegetable oil, is obtained from the fruits of the argan tree (Argania spinosa L.) which is an endemic tree growing in the south-western region of Morocco [12–14]. It is used in folk medicine and in cosmetics to repair various skin conditions and to prevent hair loss [15]. Chemical analysis of this oil showed that it is rich in polyunsaturated fatty acids (80%) like oleic and linoleic acids. It also contains minor compounds such as tocopherols, sterols (schottenol and spinasterol), phenols (ferulic, syringic and vanillic acid), triterpene alcohols, carotenoids, xanthophyls and squalene. Because of these compounds, AO is an important and powerful antioxidant source [12–15]. In addition, it protects against oxidation because it is an important source of vitamin E [13]. It has been stated that AO possess choleretic, hepatoprotective, cardiovascular-protective, chemopreventive, antioxidant, anti-inflammatory, anti-malarial, anti-diabetic, antiatherosclerotic, anti-hypercholesterolemic, anti-hypertensive, antiproliferative and anticarcinogenic properties [12,14–16].
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Because of these properties, AO may play an important role in treatment of certain diseases such as hypertension, diabetes mellitus, hypercholesterolemia and cancer. It can be also used as a balanced dietary supply without marked adverse effects on immune cell function [17]. AO can prevent mutations induced by urethane and methyl methanesulfonate in D. melanogaster [16]. It has been stated that AO may play a significant role in the protection of genetic materials against some environmental mutagens thanks to its antioxidants and antimutagenic constituents. However, there are few data about its protective potential against AA-induced toxicity. Moreover, although the antioxidant effects of AO were studied in various tissues, its antioxidant effects in lymphoid organs and antigenotoxic effects in bone marrow have also not been investigated yet. Therefore, the purpose of this study was to represent the effects of AO on the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs), the formation of micronucleus (MN) and megakaryocytic emperipolesis (ME) in bone marrow, 8-hydroxydeoxyguanosine (8-OHdG) levels in urine, thiobarbituric acid reactive substances (TBARS), protein carbonyl (PCO) and glutathione (GSH) concentrations in spleen, thymus and bone marrow and, myeloperoxidase (MPO) activity in polymorphonuclear (PMN) leukocytes against AA-induced toxicity in rats. 2. Materials and methods 2.1. Chemicals AA was purchased from Sigma-Aldrich (St. Louis, MO, USA, CAS Number 79-06-1). Organic Moroccan AO was purchased from Alassala Ltd. (Nottingham, UK). According to the certificate information of the product, the content of organic Moroccan argan oil is shown in Table 1. All other chemicals and reagents used in this study were of analytical grade. 2.2. Animals and experimental design Twenty healthy adult (12–14 weeks of age) female SpragueDawley rats weighing 225–275 g were obtained from the Experimental Animal Center of University of Ondokuz Mayıs (Samsun, Turkey). The study was approved by the Committee of Ethics in Research with animals of the same university (HADYEK 2014/24). All procedures were performed with approval by the Committee and in accordance with the guidelines provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animals were housed in cages under laboratory conditions (12 h day/12 h night cycle, air temperature of 21 1 C, standard rations and tap water ad libitum) during the experiment.
The animals were randomly divided into four groups of five rats. Animals in control (C) group were orally gavaged with a constant volume of 1 ml/kg bw of 0.9% NaCl solution. AA was administered intraperitoneally at a dose of 50 mg/kg/day three times per week for 30 days. AO was administered by oral gavage at a dose of 6 ml/ kg per day for 30 days. The remaining animals were treated with together AA (50 mg/kg/day three times per week) and AO (6 ml/kg/ day) for 30 days until they were euthanized. At the end of time, all animals were sacrificed by cervical dislocation. 2.3. Preparations of blood samples Blood samples were collected by cardiac puncture with a heparinized disposable syringe. The blood samples were used to determine some hematological parameters such as red blood cell count (RBC), hemoglobin (Hb), mean corpuscular Hb concentration (MCHC), hematocrit (HCT), lymphocytes, monocytes, eosinophils and white blood cell count (WBC) and to separate of polymorphonuclear leukocytes (PMNs). These hematological parameters were analyzed using auto-analyzer (Cobas Integra 800; Roche Diagnostics GmbH; Mannheim, Germany). 2.4. Determination of antioxidant parameters in lymphoid organs The spleen and thymus were placed in ice-cold 0.15 M NaCI solution. They were perfused with the same solution to remove red blood cells and then blotted on filter paper. The thymus and spleen indices were calculated according to the following formula: Thymus or spleen index = weight of thymus or spleen/body weight 100. These samples were also stored at 80 C until use. The tissues were centrifuged 4000g for 10 min and then supernatants were used for biochemical assays. Bone marrows were collected from femur by flushing with 1 ml of ice-cold Tris-HCI buffer (0.1 M, pH7.4). The material was centrifuged at 1000 rpm for 3 min and pellet containing marrow cells was stored at 80 C until use. The cells were homogenized using liquid nitrogen and then 1000 g supernatants were used for biochemical analyses. The protein content of tissue homogenates was determined by following the method described by Lowry et al. [18] using bovine serum albumin as a standard. GSH levels in the lymphoid organs were determined by the method of Moron et al. [19]. This method is based on the reaction of GSH with 5, 50-dithiobis (2nitrobenzoic acid). It produces 5-thio-2-nitrobenzoic acid which is a yellow product measured at 412 nm. The extent of lipid peroxidation in terms of TBARS formation was measured according to the method of Esterbauer and Cheeseman [20]. Malondialdehyde (MDA) as a part of TBARS it is commonly used as an indicator of lipid peroxidation. Considering 99% of TBARS is MDA, TBARS concentrations of the samples were calculated by using the
Table 1 The composition of organic Moroccan argan oil. Fatty acids Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid
myristic (C 14: 0) pentadecanoïc (C 15: 0) palmitic (C 16: 0) palmitoleic (C 16: 1) heptadecanoïc (C 17: 0) Stearic (C 18: 0) oleic (C 18: 1) Linoleic (C 18: 2) Linoleic(C18:3) arachidic (C 20: 0) gadoleic (C 20: 1) Hellenic (C 22: 0)
%
Sterols
%
Tocopherols
%
Phenolic compounds (3262 mg/kg oil)
%
0,1 0,1 12 0,1 0,1 5,6 46,1 34 0,1 0,4 0,4 0,1
Schottenol Spinasterol A 7- avenasterol Stigmasta-8,22-dièn-3p-ol. Campesterol Cholesterol
44 34 4–7 3,2 5,7 0,4 0,4
Alpha-tocopherol Beta-tocopherol Gamma-tocopherol Delta-tocopherol
6–9 0,1–0,3 80–91 5–10,2
Ferulic acid Vanilic acid Syringic acid Tyrosol
96,47 2,05 1,13 0,36
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extinction coefficient of MDA, which is 1.56 10 5M 1cm 1 [21]. Levels of PCOs, a marker of protein oxidation, in the lymphoid organs and PMNs were determined by the method of Levine et al. [22]. 2.5. Evaluation of myeloperoxidase (MPO) activity in PMNs PMNs were isolated according to Godfrey et al. [23]. Briefly, the blood was centrifuged at 2500 rpm for 5 min. The leukocyte-rich “buffy coat” was removed and subjected to gelatin sedimentation by mixing with equal volume of 2% gelatin in 0.9% NaCl and then incubation at 37 C for 40 min. After centrifugation at 1000 rpm for 10 min, the cell pellet contained the PMNs was resuspended in cold erythrocyte lysing solution (155 mM NH4Cl, 2 mM NaHCO3, 0.1 mM EDTA). The cell suspension was centrifuged at 275g for 5 min. After the supernatant was discarded, the pellet was washed tree times with Hank’s balanced solution. The PMNs suspension was homogenized using liquid nitrogen and then supernatant obtained from PMNs by mild centrifugation at 250g for 20 min was used for biochemical analyses. MPO activity was evaluated according to Bradley et al. [24]. An aliquot of PMNs was homogenized in 0.5% hexadecyltrimethylammonium bromide (HTAB) to solubilize membrane-bound MPO. The mixture was cycled three times through freezing and thawing, and the supernatant obtained from the PMNs homogenate by centrifugation at 10,000g for 30 min at 4 C was used for MPO activity. 2.6. Measurement of urinary 8-OHdG Formation of 8-OHdG was measured using OxiselectTM Oxidative DNA Damage ELISA kit (Cell Biolabs, Inc., San Diego, CA). Urinary samples were collected from all rats and stored at 80 C until analysis. The samples were centrifuged at 3000g for 10 min prior to use and used for the 8-OHdG ELISA assay, following the manufacturer's instructions. In brief, the unknown 8-OHdG samples or 8-OHdG standards are first added to an 8-OHdG/BSA conjugate preabsorbed EIA plate. After a brief incubation, an anti8-OHdG monoclonal antibody is added, followed by an HRP conjugated secondary antibody. Absorbance of each microwell was read on a microplate reader using 450 nm as the primary wave length. The quantity of 8-OHdG in the specimens were determined by comparing its absorbance with known 8-OHdG standard curve. 2.7. Sampling Preparation for ME and MN assay The MN test was performed as described by Schmid [25] and the frequency of ME was evaluated according to Yener and Dikmenli [26] with minor modifications. Briefly, bone marrow was flushed out from both femurs using 2 ml of fetal bovine serum and
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centrifuged at 500g for 10 min and the supernatant was discarded. The pellet was resuspended in a small amount of fetal calf serum and spread on clean slides. The smears were stained with MayGrünwald-Giemsa method. The frequency of ME was analyzed from one hundred randomly selected and morphologically distinguishable megakaryocytes per animal. ME index was also calculated for per animal using the following formula: ME index = frequency of ME X number of entering cells per megakaryocyte/100. To calculate the MN frequencies, 2000 PCEs were scored per animal. To determine the ratio of PCEs to normochromatic erythrocytes (NCEs), two hundred immature and mature erythrocytes were scored per animal. 2.8. Statistical analysis All statistical comparisons were performed by using one-way analysis of variance (ANOVA) using the SPSS software package for Windows. The Tukey post hoc testing was performed for intergroup comparisons. Differences with p < 0.05 were considered to be statistically significant. All results were expressed as mean standard deviation (SD). 3. Results and discussion 3.1. Hematological parameters and indexes of spleen and thymus Effects of AA and/or AO on the levels of some hematological parameters and the index of the spleen and thymus of rats are shown in Table 2. AA decreased all the hematological parameters and these decreases were found statistically significant (p < 0.05) except for mean corpuscular Hb concentration. No significant alterations were observed in any of these parameters in the animals received the AO alone when compared to control group. Decreased levels of hematological parameters caused by the effects of AA were found significantly increased after co-treatment with AA + AO (p < 0.05) except for Hb concentration. In other words, AO normalized the altered values in rats. No significant differences in the spleen and thymus indexes were observed between the control group and treatment groups (p < 0.05). Previous studies have demonstrated that AA can change some hematological parameters. AA covalently binds to the cysteine residues and forms adducts with sulphydryl group on Hb resulting in the reduction of the amount of Hb in blood [27]. 36 mg/kg AA administration during 30 days decreased WBC, lymphocytes, monocytes and granulocyte in female BALB/c mice [8]. Significantly changed hematological parameters were detected in rats exposed to 5, 10 or 50 mg/kg AA for 10 weeks [28]. AA treatment resulted in the decrease of Hb level and RBC count in mice [11]. Consistent with previous findings, data from the present study indicate that AA significantly decreased the hematological
Table 2 Effects of acrylamide (AA) and/or argan oil (AO) on hematological parameters, spleen index and thymus index in rats. .
Red blood cell count (1012/L) Hemoglobin (g/L) Mean corpuscular Hb concentration (g/L) Hematocrit (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) White blood cell count (109/L) Spleen index Thymus index a b
Control
AA
AO
AA + AO
7,52 0,46 141,50 5,45 315,40 9,48 45,04 1,96 67,86 1,94 3,82 0,24 1,82 0,15 7,14 0,45 0,40 0,06 0,16 0,01
6,38 0,36a 115,40 7,44a 309,60 11,50 39,09 0,78a 56,18 1,76a 3,35 0,31a 1,51 0,15a 5,72 0,33a 0,37 0,03 0,18 0,02
7,48 0,44b 137,40 5,98b 322,80 9,65 44,30 1,45b 66,00 1,58b 4,06 0,19b 1,92 0,13b 6,94 0,54b 0,39 0,03 0,16 0,02
6,92 0,34 128,20 5,89a 311,00 10,25 45,04 1,96b 62,94 3,24b 3,89 0,23b 1,64 0,07 6,37 0,37 0,37 0,02 0,17 0,01
Compared with control group difference is statistically significant. Compared with AA difference is statistically significant (p < 0.05).
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parameters. In spite of lymphocytes rates (increased in females and decreased in males) and eosinophiles levels (increased in males), no significant changes in other hematological parameters (WBC, monocytes, basophiles, RBC and platelets) have been detected in AO supplemented rats for 13 weeks [29]. Similar to these results, our results indicate that no significant differences were observed on hematological parameters in rats treated with AO alone. Furthermore, co-treatment with AO + AA significantly increased and improved the hematological parameters when compared to AA treated rats. Abnormal standard deviation values have not been observed in hematological or other parameters in female animals. This results may indicate that the cycle periods of the animals probably do not effect on these parameters. Although no significant differences in the spleen and thymus indexes were observed in our study, weight losses in spleen have been observed in rats treated with AA [6,28]. Increased oxidative damage in the spleen caused by the effect of AA can cause the reduction in RBCs [6]. 3.2. Antioxidant parameters in lymphoid organs TBARs, PCOs and GSH levels in spleen, thymus and bone marrow in AA and/or AO treated rats are shown in Table 3. The intraperitoneal administration of AA caused oxidative damage in all the lymphoid organs as reflected by significant increases in TBARs and PCOs levels and decreases in GSH levels (p < 0.05). No significant alterations, except for TBARs and GSH levels in the thymus, were observed in any of these parameters in the animals received the AO alone when compared to control group. AO decreased the lipid peroxidation and protein carbonyl levels and the increased GSH levels in almost all the lymphoid organs when compared to AA treated group. According to TBARs levels, we can say that AA increased the lipid peroxidation levels in spleen, thymus and bone marrow, respectively. These increases were significant in spleen and thymus when compared to control group (p < 0.05). Co-treatment with AA + AO significantly reduced the lipid peroxidation in the spleen and thymus when compared to treatment with AA alone (p < 0.05). When compared with control group, PCOs levels in AA treated rats significantly increased in spleen, thymus and bone marrow, respectively (p < 0.05). Although co-treatment with AA + AO decreased the PCOs levels in all the lymphoid organs, statistically significant decrease of PCOs levels was detected only in the spleen (p < 0.05). When compared to control group, significant decreases GSH levels in animals treated with AA were found in spleen, thymus and bone marrow, respectively (p < 0.05). Cotreatment with AA + AO significantly increased and normalized the GSH levels in all the lymphoid organs (p < 0.05). Some in vivo studies have shown that AA causes oxidative stress in plasma, liver, testis, brain and kidney of animals [30,31]. While the administration of AA reduced antioxidant enzyme levels such as catalase (CAT), superoxide dismutase (SOD), peroxidase, GSH peroxidase (GSH-Px) and GSH, it increased the ROS and MDA levels in different organs of rats [4,10,11,30,32–34]. AA also increased the
levels of lipid peroxidative product, PCO content, hydroxyl radical and hydroperoxide in rat brain [9]. The obtained results in the present study are agreement with the previous findings because of significant decreases in the GSH levels and significant increases in lipid peroxidation and PCOs levels in all lymphoid tissues of AA treated rats. The protective effects of AO against oxidative stress are probably due to its high contents of powerful antioxidants [13]. The activities of cytosolic CAT were significantly higher in rats treated with AO (5 ml/kg/day for 8 weeks) [35]. Lower plasma lipoperoxide and LDL cholesterol levels and higher a-tocopherol concentrations were observed in healthy subjects were regularly consuming AO (mean daily 15 g) [36]. The phenolic fraction of AO has been shown to inhibit low-density lipoprotein oxidation in macrophages [37] and to have an antiproliferative effect on human prostate cancer cell lines [38]. After a 3-week consumption of AO in dyslipidemic patients, platelet lipid peroxidation levels were about 34% lower and platelet GPx activity was about 22% higher when compared to the control group in which AO was replaced by butter [12]. AO (5 ml/kg b.w by gavage for 3 weeks) significantly improved oxidative stress parameters against mercuric chloride-induced toxicity by increasing GSH levels, GSH-Px, glutathione-S-transferase (GST) and CAT activities and decreasing lipid peroxidation level in rats [13]. AO significantly decreased plasma markers of lipid peroxidation in rats compared to rats fed high-fat diet. In addition, plasma vitamin E concentrations, CAT and SOD activities were significantly increased in AO-treated rats [14]. Administration of AO restored both the cytosolic and mitochondrial oxidative stress by normalizing nicotinamide adenine dinucleotide phosphate generating enzymes in acrylamide treated rat brain [39]. Few studies have investigated the direct effect of AO on the immune system cells. Benzaria et al. [17] reported that AO improved proliferation and phospholipase D activity of rat thymocytes. The obtained results in the present study are agreement with the previous findings because AO administration together with AA significantly decreased oxidative stress in lymphoid tissues. 3.3. MPO activity in PMNs Fig. 1 shows the effects of AA and/or AO on MPO activity in PMNs of rats. AA significantly decreased the MPO activity in PMNs when compared to control group (p< 0.05). No significant alteration was observed in the activity in the animals received the AO alone when compared to control group (p < 0.05). Cotreatment with AA + AO significantly ameliorated and completely normalized the MPO activity against AA-induced toxicity (p< 0.05). Increased MPO activity is an important local mediator of inflammation and subsequent organ damage in tissues. Lower MPO activities in blood leukocytes were observed in multiple sclerosis, diabetes and autoimmune lupus nephritis patients [40]. MPO also plays an important role in the various functions of neutrophils in innate and adaptive immunity in PMNs lymphocytes [41]. Increased MPO activity was found in different tissues of
Table 3 TBARs, PCOs and GSH levels in spleen, thymus and bone marrow in rats treated with acrylamide (AA) and/or argan oil (AO). Treatment groups
Control AA AO AA + AO a b
TBARs (nmol malondialdehyde/mg protein)
PCOs (nmol carbonyl/mg protein)
GSH (nmol GSH/mg protein)
Spleen
Thymus
Bone marrow
Spleen
Thymus
Bone marrow
Spleen
Thymus
Bone marrow
10,53 0,66 14,96 2,43a 11,49 0,94b 12,57 1,46
16,02 0,57 22,02 1,42a 13,00 0,74a,b 17,51 1,26b
3,18 0,38 3,92 0,93 3,39 0,70 3,50 0,83
18,05 2,66 22,97 1,53a 19,92 2,41 18,62 1,20b
11,72 1,80 16,79 0,81a 12,19 0,89b 14,73 1,01a
14,01 1,63 18,64 2,28a 15,09 1,75 17,80 2,07a
1,54 0,18 1,03 0,25a 1,75 0,30b 1,46 0,35
1,13 0,04 0,96 0,06a 1,48 0,10a,b 1,20 0,09b
0,32 0,05 0,24 0,02a 0,37 0,03b 0,33 0,02b
Compared with control group difference is statistically significant. Compared with AA difference is statistically significant (p < 0.05).
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Fig. 1. The effects of acrylamide (AA) and/or argan oil (AO) on myeloperoxidase (MPO) activity in polymorphonuclear leukocytes (PMNs) in rats. Experiments were repeated three times. a: Compared with control group difference is statistically significant, b: Compared with AA difference is statistically significant (p < 0.05).
AA treated rats (40 mg/kg i.p.for 10 days) [32,33]. Contrary to previous results, we also observed that MPO activity significantly decreased in PMNs of AA treated rats. The immune system, especially PMNs cells are sensitive to oxidative stress [42]. So far, the effects of AO on MPO activity have not been investigated. Our results indicate that co-treatment with AO + AA improved MPO activity in PMNs. 3.4. Urinary 8-OHdG As shown in Fig. 2, AA significantly increased the urinary concentration of 8-OHdG when compared to control group (p < 0.05). No significant alterations were observed in 8-OHdG levels in the animals received the AO alone and co-treatment with AO in combination with AA when compared to control group. Our results show that AO significantly reduced and improved the increased urinary 8-OHdG levels induced by AA in rats. 8-OHdG, an indicator of oxidative DNA damage, is eliminated by excretion in the urine [1]. Numerous reports indicate that if it is not repaired, it can contribute to the induction of carcinogenesis and many other diseases [43]. Therefore, 8-OHdG levels can be used to predict risk of some diseases such as cancer, atherosclerosis, and diabetes [1,43]. Significantly increased 8-OHdG levels were
detected in AA treated rats (40 mg/kg i.p.for 10 days) [32,33]. The level of 8-OHdG in the liver increased significantly in the AAtreated mice [10]. Serum levels of 8-OHdG were significantly increased in AA treated rats [34]. Because AA increased the 8OHdG level in rat urine in our study, our findings support the idea that there is significant correlation between urinary 8-OHdG levels and AA consumption. Effects of AO on 8-OHdG formation have not been previously studied. Our results show that AO showed protective activity against to AA by decreasing the formation of 8-OHdG formation in rat urine. 3.5. ME and MN assay Table 4 represents the effects of AA and/or AO on the frequencies of MN and ME and the ratio of PCEs to NCEs in rat bone marrow. AA significantly increased the formation of MN and decreased the ratio of PCEs to NCEs in bone marrow (p < 0.05). No significant differences in MN and PCE/NCE values were observed in the animals received the AO compared to the control group. Cotreatment with AA and AO significantly increased the PCE/NCE ratio and improved the PCE/NCE value against AA-induced cytotoxicity (p < 0.05). Significant decreased MNPCE frequency was also found when rats were co-treated with AO and AA
Fig. 2. The effects of acrylamide (AA) and/or argan oil (AO) on urinary levels of 8-hydroxydeoxyguanosine (8-OHdG) in rats. Experiments were repeated three times. a: Compared with control group difference is statistically significant, b: Compared with AA difference is statistically significant (p < 0.05).
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Table 4 Effects of acrylamide (AA) and/or argan oil (AO) on megakaryocytic emperipolesis (ME), micronucleated polychromatic erythrocytes (MNPCE) frequency and the ratio of PCEs to NCEs in rat bone marrow. Treatment groups
ME%
ME index
MNPCE frequency%
PCE/NCE Ratio
Control AA AO AA + AO
2.17 0.39 3.60 0.69a 2.27 0.30b 2.83 0.31
2.54 1.03 6.75 2.15a 2.55 0.66b 4.09 0.94b
0.32 0.06 0.55 0.07a 0.33 0.04b 0.42 0.10b
1.71 0.28 1.20 0.09a 1.73 0.31b 1.59 0.12b
a b
Compared with control group difference is statistically significant. Compared with AA difference is statistically significant (p < 0.05).
compared to AA alone (p < 0.05). The results of the present study show that AO ameliorated the MN frequency and the ratio of PCEs to NCEs in rat bone marrow. As shown in Table 4, AA also significantly increased the frequencies of ME and ME index compared to the control group in bone marrow (p < 0.05). No significant differences in these values were observed in the animals received the AO compared to the control group. Co-treatment with AA + AO significantly decreased the ME and ME index values compared to those treated with AA alone (p < 0.05). The results of the present study show that AO significantly reduced and improved the ME and ME index values (p < 0.05). ME is enclosing or engulfing of diverse bone marrow cells by megakaryocytes. Although various hypotheses have been proposed to explain the physiological mechanism of the ME, it has not yet been clearly clarified. Nevertheless, it is known to increase in many disorders such as various cancer types and different tumours, hypoxia, anemia and blood loss. The hyperplasia of haematopoietic cells is also closely associated with ME [26]. Yener and Dikmenli [26] reported that administration of AA (60 mg/kg) to rats by gavage for five consecutive days significantly decreased the incidence of ME in rat bone marrow compared to control group. Contrary to the finding, the obtained results in the present study indicated that intraperitoneal administration of AA in a dose of 50 mg/kg/day at the same time every other day for 30 days significantly increased ME frequency in rat bone marrow. Observed differences in these studies may result from the differences in the preferred administration route and treatment time of AA to animals. Many studies have shown genotoxic, reproductive and carcinogenic effects of AA. According to these studies, AA can induce specific DNA adducts and breaks, gene mutation, chromosome aberrations, MN formation, sister chromatin exchanges, unscheduled DNA synthesis, aneuploidy, polyploidy, carcinogenesis and sperm malformation rate [2,11]. There are many scientific studies with negative and positive results about the effect of AA on MN formation in mice and rats. It has been stated that AA significantly induced the MN frequency in mice [11,44–49] and in rats [4,52], while others have found no effect [45,50,51]. As shown, the MN results are conflicting in mice and rats. We think the reasons for these observed inconsistent results may be different routes chosen for administration of AA to animals or some minor differences in methodological procedures in different laboratories. Significantly increased the frequencies of MNPCEs and decreased the ratio of PCE/NCE were found in bone marrow of rats at single oral doses of 125, 150 or 175 mg/kg for 48 h [52]. DNA strand break and MN formation were increased in rats treated with 5, 10 and 15 mg/kg/ day of AA for 4 or 12 weeks [4]. The obtained results in the present study are agreement with the previous findings because AA increased the MN frequency in rat bone marrow. There is no study examining effects of AO on ME and MN formation and on PCE/NCE ratio. We clearly observed that AO decreased the formation of genetic damage in parallel with the formation of ME in rat bone marrow against AA toxicity.
4. Conclusions The results of the present study demonstrate that AO have a protective effect against AA-induced toxicity in rats as evidenced by levels of GSH, PCO, TBARs, MPO and 8-OHdG, concentrations of hematological parameters, the ratio of PCEs and frequencies of ME and MN. Therefore, the ameliorating effects of AO may focus attention on the beneficial effect of this substance to protect against oxidative stress-related diseases. Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this article. All the authors reviewed the paper and approved the final version. Acknowledgements This study was supported by the Scientific Research Funding of Amasya University (Turkey) [Grant number FBM-BAP-15-0982015]. References [1] C.Y. Lin, H.L. Lee, Y.C. Chen, G.W. Lien, L.Y. Lin, L.L. Wen, C.C. Liao, K.L. Chien, F.C. Sung, P.C. Chen, T.C. Su, Positive association between urinary levels of 8hydroxydeoxyguanosine and the acrylamide metabolite N-acetyl-S(propionamide)-cysteine in adolescents and young adults, J. Hazard. Mater. 261 (2013) 372–377. [2] Y. Ishii, K. Matsushita, K. Kuroda, Y. Yokoo, A. Kijima, S. Takasu, Y. Kodama, A. Nishikawa, T. Umemura, Acrylamide induces specific DNA adduct formation and gene mutations in a carcinogenic target site the mouse lung, Mutagenesis 30 (2015) 227–235. [3] K. Ji, S. Kang, G. Lee, S. Lee, A. Jo, K. Kwak, D. Kim, D. Kho, S. Lee, S. Kim, S. Kim, Y. F. Hiuang, K.Y. Wu, K. Choi, Urinary levels of N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), an acrylamide metabolite, in Korean children and their association with food consumption, Sci. Total Environ. 456–457 (2013) 17–23. [4] W.K.B. Khalil, H.H. Ahmed, H.F. Aly, M.G. Eshak, Toxicological effects of acrylamide on testicular function and ımmune genes expression profile in rats, Int. J. Pharm. Sci. Rev. Res. 24 (2014) 143–151. [5] R.R. Maronpot, R.J.M.M. Thoolen, B. Hansen, Two-year carcinogenicity study of acrylamide in Wistar Han rats with in utero exposure, Exp. Toxicol. Pathol. 67 (2015) 189–195. [6] S.I. Zaidi, S. Raisuddin, K.P. Singh, A. Jafri, R. Husain, M.M. Husain, S.A. Mall, P.K. Seth, P.K. Ray, Acrylamide induced immunosuppression in rats and its modulation by 6-MFA: an interferon inducer, Immunopharmacol. Immunotoxicol. 16 (1994) 247–260. [7] W. Chen, Y. Shen, H. Su, X. Zheng, Hispidin derived from Phellinus linteus affords protection against acrylamide-induced oxidative stress in Caco-2 cells, Chem. Biol. Interact. 219 (2014) 83–89. [8] J. Fang, C.L. Liang, X.D. Jia, N. Li, Immunotoxicity of acrylamide in female BALB/ cmice, Biomed. Environ. Sci. 27 (2014) 401–409. [9] D. Lakshmi, K. Gopinath, G. Jayanthy, S. Anjum, D. Prakash, G. Sudhandiran, Ameliorating effect of fish oil on acrylamide induced oxidative stress and neuronal apoptosis in cerebral cortex, Neurochem. Res. 37 (2012) 1859–1867. [10] L. Zhang, H. Zhang, Y. Miao, S. Wu, H. Ye, Y. Yuan, Protective effect of allicin against acrylamide-induced hepatocyte damage in vitro and in vivo, Food Chem. Toxicol. 50 (2012) 3306–3312. [11] M. Zhao, X. Liu, Y. Luo, H. Guo, X. Hu, F. Chen, Evaluation of protective effect of freeze-dried strawberry grape, and blueberry powder on acrylamide toxicity in mice, J. Food Sci. 80 (2015) 869–874. [12] A. Haimeur, H. Messaouri, L. Ulmann, V. Mimouni, A. Masrar, A. Chraibi, G. Tremblin, N. Meskini, Argan oil prevents prothrombotic complicationsby
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Original article
Argan oil reduces oxidative stress, genetic damage and emperipolesis in rats treated with acrylamide lua,* , Birsen Aydınb , Vedat Şekerog lua Zülal Atlı Şekerog a b
Department of Molecular Biology and Genetics, Faculty of Science, Ordu University, 52200-Ordu, Turkey Department of Biology, Faculty of Science, Amasya University, 05100-Amasya, Turkey
A R T I C L E I N F O
Article history: Received 1 June 2017 Received in revised form 4 August 2017 Accepted 7 August 2017 Keywords: Acrylamide Argan oil Oxidative stress DNA damage Megakaryocytic emperipolesis
A B S T R A C T
Acrylamide (AA), a well-known toxicant, is present in high-temperature-processed foods in heated foods. Argan oil (AO), a natural vegetable oil, is receiving increasing attention due to its powerful biological properties. However, limited information is available about its effects in lymphoid organs and bone marrow. The aim of this study is to investigate the effects of AO on hematological parameters, 8hydroxydeoxyguanosine (8-OHdG), thiobarbituric acid reactive substances (TBARs), protein carbonyl (PCO), glutathione (GSH), myeloperoxidase (MPO) levels, the formation of micronucleus (MN) and megakaryocytic emperipolesis (ME) against AA-induced toxicity in rats. The animals were treated with AA (50 mg/kg/day), AO (6 ml/kg/day per day) and AA + AO (50 mg + 6 ml/kg/day) for 30 days. Treatment of rats with AA significantly decreased the hematological parameters, GSH and MPO activity and PCEs ratio while it increased TBARs, PCOs and 8-OHdG levels and formation of MN and ME. No significant differences were observed in the animals received the AO alone. Co-treatment with AA + AO ameliorated almost all of the alterations caused by AA and exhibited protective effect in rats. Based on the obtained results, we suggest that integration of AO in diet or using its supplements may be a good strategy for improving tissue injury in many diseases. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Acrylamide (AA) is an odorless, colorless, highly water-soluble, rapidly polymerizable, crystalline substance. Polymers of AA are widely used in industry and biological research [1]. In foods, AA is generated from some food components during heat treatment at high temperatures (above 120 C) [2]. In addition to the occupational routes of exposure, AA may be absorbed from mainstream cigarette smoke [1]. Therefore, high temperatureprocessed foods and smoking are considered as important sources of human exposure to AA [3]. Certain foods such as french fries, potato crisps, bread, cookies, and coffee exert the highest contribution to dietary exposure of AA [4]. Children seem to be under more threat than adults in terms of exposure of AA because of their higher caloric intake relative to body weight and their higher consumption of certain AA-rich foods [4,5]. AA, a well-known toxicant, has been classified as a probable carcinogen to human (Group 2A). Therefore, AA has raised significant concerns about the possible exposure and health risks
* Corresponding author. lu). E-mail addresses: [email protected], [email protected] (Z.A. Şekerog http://dx.doi.org/10.1016/j.biopha.2017.08.034 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
for the general population [3]. Some in vitro and in vivo studies have demonstrated that AA can induce oxidative stress, neurotoxicity, developmental and reproductive toxicity, immunotoxicity, genotoxicity, and carcinogenicity [1,2,6–8]. In recent years, researchers have focused on some of natural antioxidants that may be effective on the toxicity of AA [7,9–11]. Argan oil (AO), a natural vegetable oil, is obtained from the fruits of the argan tree (Argania spinosa L.) which is an endemic tree growing in the south-western region of Morocco [12–14]. It is used in folk medicine and in cosmetics to repair various skin conditions and to prevent hair loss [15]. Chemical analysis of this oil showed that it is rich in polyunsaturated fatty acids (80%) like oleic and linoleic acids. It also contains minor compounds such as tocopherols, sterols (schottenol and spinasterol), phenols (ferulic, syringic and vanillic acid), triterpene alcohols, carotenoids, xanthophyls and squalene. Because of these compounds, AO is an important and powerful antioxidant source [12–15]. In addition, it protects against oxidation because it is an important source of vitamin E [13]. It has been stated that AO possess choleretic, hepatoprotective, cardiovascular-protective, chemopreventive, antioxidant, anti-inflammatory, anti-malarial, anti-diabetic, antiatherosclerotic, anti-hypercholesterolemic, anti-hypertensive, antiproliferative and anticarcinogenic properties [12,14–16].
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Because of these properties, AO may play an important role in treatment of certain diseases such as hypertension, diabetes mellitus, hypercholesterolemia and cancer. It can be also used as a balanced dietary supply without marked adverse effects on immune cell function [17]. AO can prevent mutations induced by urethane and methyl methanesulfonate in D. melanogaster [16]. It has been stated that AO may play a significant role in the protection of genetic materials against some environmental mutagens thanks to its antioxidants and antimutagenic constituents. However, there are few data about its protective potential against AA-induced toxicity. Moreover, although the antioxidant effects of AO were studied in various tissues, its antioxidant effects in lymphoid organs and antigenotoxic effects in bone marrow have also not been investigated yet. Therefore, the purpose of this study was to represent the effects of AO on the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs), the formation of micronucleus (MN) and megakaryocytic emperipolesis (ME) in bone marrow, 8-hydroxydeoxyguanosine (8-OHdG) levels in urine, thiobarbituric acid reactive substances (TBARS), protein carbonyl (PCO) and glutathione (GSH) concentrations in spleen, thymus and bone marrow and, myeloperoxidase (MPO) activity in polymorphonuclear (PMN) leukocytes against AA-induced toxicity in rats. 2. Materials and methods 2.1. Chemicals AA was purchased from Sigma-Aldrich (St. Louis, MO, USA, CAS Number 79-06-1). Organic Moroccan AO was purchased from Alassala Ltd. (Nottingham, UK). According to the certificate information of the product, the content of organic Moroccan argan oil is shown in Table 1. All other chemicals and reagents used in this study were of analytical grade. 2.2. Animals and experimental design Twenty healthy adult (12–14 weeks of age) female SpragueDawley rats weighing 225–275 g were obtained from the Experimental Animal Center of University of Ondokuz Mayıs (Samsun, Turkey). The study was approved by the Committee of Ethics in Research with animals of the same university (HADYEK 2014/24). All procedures were performed with approval by the Committee and in accordance with the guidelines provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animals were housed in cages under laboratory conditions (12 h day/12 h night cycle, air temperature of 21 1 C, standard rations and tap water ad libitum) during the experiment.
The animals were randomly divided into four groups of five rats. Animals in control (C) group were orally gavaged with a constant volume of 1 ml/kg bw of 0.9% NaCl solution. AA was administered intraperitoneally at a dose of 50 mg/kg/day three times per week for 30 days. AO was administered by oral gavage at a dose of 6 ml/ kg per day for 30 days. The remaining animals were treated with together AA (50 mg/kg/day three times per week) and AO (6 ml/kg/ day) for 30 days until they were euthanized. At the end of time, all animals were sacrificed by cervical dislocation. 2.3. Preparations of blood samples Blood samples were collected by cardiac puncture with a heparinized disposable syringe. The blood samples were used to determine some hematological parameters such as red blood cell count (RBC), hemoglobin (Hb), mean corpuscular Hb concentration (MCHC), hematocrit (HCT), lymphocytes, monocytes, eosinophils and white blood cell count (WBC) and to separate of polymorphonuclear leukocytes (PMNs). These hematological parameters were analyzed using auto-analyzer (Cobas Integra 800; Roche Diagnostics GmbH; Mannheim, Germany). 2.4. Determination of antioxidant parameters in lymphoid organs The spleen and thymus were placed in ice-cold 0.15 M NaCI solution. They were perfused with the same solution to remove red blood cells and then blotted on filter paper. The thymus and spleen indices were calculated according to the following formula: Thymus or spleen index = weight of thymus or spleen/body weight 100. These samples were also stored at 80 C until use. The tissues were centrifuged 4000g for 10 min and then supernatants were used for biochemical assays. Bone marrows were collected from femur by flushing with 1 ml of ice-cold Tris-HCI buffer (0.1 M, pH7.4). The material was centrifuged at 1000 rpm for 3 min and pellet containing marrow cells was stored at 80 C until use. The cells were homogenized using liquid nitrogen and then 1000 g supernatants were used for biochemical analyses. The protein content of tissue homogenates was determined by following the method described by Lowry et al. [18] using bovine serum albumin as a standard. GSH levels in the lymphoid organs were determined by the method of Moron et al. [19]. This method is based on the reaction of GSH with 5, 50-dithiobis (2nitrobenzoic acid). It produces 5-thio-2-nitrobenzoic acid which is a yellow product measured at 412 nm. The extent of lipid peroxidation in terms of TBARS formation was measured according to the method of Esterbauer and Cheeseman [20]. Malondialdehyde (MDA) as a part of TBARS it is commonly used as an indicator of lipid peroxidation. Considering 99% of TBARS is MDA, TBARS concentrations of the samples were calculated by using the
Table 1 The composition of organic Moroccan argan oil. Fatty acids Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid
myristic (C 14: 0) pentadecanoïc (C 15: 0) palmitic (C 16: 0) palmitoleic (C 16: 1) heptadecanoïc (C 17: 0) Stearic (C 18: 0) oleic (C 18: 1) Linoleic (C 18: 2) Linoleic(C18:3) arachidic (C 20: 0) gadoleic (C 20: 1) Hellenic (C 22: 0)
%
Sterols
%
Tocopherols
%
Phenolic compounds (3262 mg/kg oil)
%
0,1 0,1 12 0,1 0,1 5,6 46,1 34 0,1 0,4 0,4 0,1
Schottenol Spinasterol A 7- avenasterol Stigmasta-8,22-dièn-3p-ol. Campesterol Cholesterol
44 34 4–7 3,2 5,7 0,4 0,4
Alpha-tocopherol Beta-tocopherol Gamma-tocopherol Delta-tocopherol
6–9 0,1–0,3 80–91 5–10,2
Ferulic acid Vanilic acid Syringic acid Tyrosol
96,47 2,05 1,13 0,36
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extinction coefficient of MDA, which is 1.56 10 5M 1cm 1 [21]. Levels of PCOs, a marker of protein oxidation, in the lymphoid organs and PMNs were determined by the method of Levine et al. [22]. 2.5. Evaluation of myeloperoxidase (MPO) activity in PMNs PMNs were isolated according to Godfrey et al. [23]. Briefly, the blood was centrifuged at 2500 rpm for 5 min. The leukocyte-rich “buffy coat” was removed and subjected to gelatin sedimentation by mixing with equal volume of 2% gelatin in 0.9% NaCl and then incubation at 37 C for 40 min. After centrifugation at 1000 rpm for 10 min, the cell pellet contained the PMNs was resuspended in cold erythrocyte lysing solution (155 mM NH4Cl, 2 mM NaHCO3, 0.1 mM EDTA). The cell suspension was centrifuged at 275g for 5 min. After the supernatant was discarded, the pellet was washed tree times with Hank’s balanced solution. The PMNs suspension was homogenized using liquid nitrogen and then supernatant obtained from PMNs by mild centrifugation at 250g for 20 min was used for biochemical analyses. MPO activity was evaluated according to Bradley et al. [24]. An aliquot of PMNs was homogenized in 0.5% hexadecyltrimethylammonium bromide (HTAB) to solubilize membrane-bound MPO. The mixture was cycled three times through freezing and thawing, and the supernatant obtained from the PMNs homogenate by centrifugation at 10,000g for 30 min at 4 C was used for MPO activity. 2.6. Measurement of urinary 8-OHdG Formation of 8-OHdG was measured using OxiselectTM Oxidative DNA Damage ELISA kit (Cell Biolabs, Inc., San Diego, CA). Urinary samples were collected from all rats and stored at 80 C until analysis. The samples were centrifuged at 3000g for 10 min prior to use and used for the 8-OHdG ELISA assay, following the manufacturer's instructions. In brief, the unknown 8-OHdG samples or 8-OHdG standards are first added to an 8-OHdG/BSA conjugate preabsorbed EIA plate. After a brief incubation, an anti8-OHdG monoclonal antibody is added, followed by an HRP conjugated secondary antibody. Absorbance of each microwell was read on a microplate reader using 450 nm as the primary wave length. The quantity of 8-OHdG in the specimens were determined by comparing its absorbance with known 8-OHdG standard curve. 2.7. Sampling Preparation for ME and MN assay The MN test was performed as described by Schmid [25] and the frequency of ME was evaluated according to Yener and Dikmenli [26] with minor modifications. Briefly, bone marrow was flushed out from both femurs using 2 ml of fetal bovine serum and
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centrifuged at 500g for 10 min and the supernatant was discarded. The pellet was resuspended in a small amount of fetal calf serum and spread on clean slides. The smears were stained with MayGrünwald-Giemsa method. The frequency of ME was analyzed from one hundred randomly selected and morphologically distinguishable megakaryocytes per animal. ME index was also calculated for per animal using the following formula: ME index = frequency of ME X number of entering cells per megakaryocyte/100. To calculate the MN frequencies, 2000 PCEs were scored per animal. To determine the ratio of PCEs to normochromatic erythrocytes (NCEs), two hundred immature and mature erythrocytes were scored per animal. 2.8. Statistical analysis All statistical comparisons were performed by using one-way analysis of variance (ANOVA) using the SPSS software package for Windows. The Tukey post hoc testing was performed for intergroup comparisons. Differences with p < 0.05 were considered to be statistically significant. All results were expressed as mean standard deviation (SD). 3. Results and discussion 3.1. Hematological parameters and indexes of spleen and thymus Effects of AA and/or AO on the levels of some hematological parameters and the index of the spleen and thymus of rats are shown in Table 2. AA decreased all the hematological parameters and these decreases were found statistically significant (p < 0.05) except for mean corpuscular Hb concentration. No significant alterations were observed in any of these parameters in the animals received the AO alone when compared to control group. Decreased levels of hematological parameters caused by the effects of AA were found significantly increased after co-treatment with AA + AO (p < 0.05) except for Hb concentration. In other words, AO normalized the altered values in rats. No significant differences in the spleen and thymus indexes were observed between the control group and treatment groups (p < 0.05). Previous studies have demonstrated that AA can change some hematological parameters. AA covalently binds to the cysteine residues and forms adducts with sulphydryl group on Hb resulting in the reduction of the amount of Hb in blood [27]. 36 mg/kg AA administration during 30 days decreased WBC, lymphocytes, monocytes and granulocyte in female BALB/c mice [8]. Significantly changed hematological parameters were detected in rats exposed to 5, 10 or 50 mg/kg AA for 10 weeks [28]. AA treatment resulted in the decrease of Hb level and RBC count in mice [11]. Consistent with previous findings, data from the present study indicate that AA significantly decreased the hematological
Table 2 Effects of acrylamide (AA) and/or argan oil (AO) on hematological parameters, spleen index and thymus index in rats. .
Red blood cell count (1012/L) Hemoglobin (g/L) Mean corpuscular Hb concentration (g/L) Hematocrit (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) White blood cell count (109/L) Spleen index Thymus index a b
Control
AA
AO
AA + AO
7,52 0,46 141,50 5,45 315,40 9,48 45,04 1,96 67,86 1,94 3,82 0,24 1,82 0,15 7,14 0,45 0,40 0,06 0,16 0,01
6,38 0,36a 115,40 7,44a 309,60 11,50 39,09 0,78a 56,18 1,76a 3,35 0,31a 1,51 0,15a 5,72 0,33a 0,37 0,03 0,18 0,02
7,48 0,44b 137,40 5,98b 322,80 9,65 44,30 1,45b 66,00 1,58b 4,06 0,19b 1,92 0,13b 6,94 0,54b 0,39 0,03 0,16 0,02
6,92 0,34 128,20 5,89a 311,00 10,25 45,04 1,96b 62,94 3,24b 3,89 0,23b 1,64 0,07 6,37 0,37 0,37 0,02 0,17 0,01
Compared with control group difference is statistically significant. Compared with AA difference is statistically significant (p < 0.05).
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parameters. In spite of lymphocytes rates (increased in females and decreased in males) and eosinophiles levels (increased in males), no significant changes in other hematological parameters (WBC, monocytes, basophiles, RBC and platelets) have been detected in AO supplemented rats for 13 weeks [29]. Similar to these results, our results indicate that no significant differences were observed on hematological parameters in rats treated with AO alone. Furthermore, co-treatment with AO + AA significantly increased and improved the hematological parameters when compared to AA treated rats. Abnormal standard deviation values have not been observed in hematological or other parameters in female animals. This results may indicate that the cycle periods of the animals probably do not effect on these parameters. Although no significant differences in the spleen and thymus indexes were observed in our study, weight losses in spleen have been observed in rats treated with AA [6,28]. Increased oxidative damage in the spleen caused by the effect of AA can cause the reduction in RBCs [6]. 3.2. Antioxidant parameters in lymphoid organs TBARs, PCOs and GSH levels in spleen, thymus and bone marrow in AA and/or AO treated rats are shown in Table 3. The intraperitoneal administration of AA caused oxidative damage in all the lymphoid organs as reflected by significant increases in TBARs and PCOs levels and decreases in GSH levels (p < 0.05). No significant alterations, except for TBARs and GSH levels in the thymus, were observed in any of these parameters in the animals received the AO alone when compared to control group. AO decreased the lipid peroxidation and protein carbonyl levels and the increased GSH levels in almost all the lymphoid organs when compared to AA treated group. According to TBARs levels, we can say that AA increased the lipid peroxidation levels in spleen, thymus and bone marrow, respectively. These increases were significant in spleen and thymus when compared to control group (p < 0.05). Co-treatment with AA + AO significantly reduced the lipid peroxidation in the spleen and thymus when compared to treatment with AA alone (p < 0.05). When compared with control group, PCOs levels in AA treated rats significantly increased in spleen, thymus and bone marrow, respectively (p < 0.05). Although co-treatment with AA + AO decreased the PCOs levels in all the lymphoid organs, statistically significant decrease of PCOs levels was detected only in the spleen (p < 0.05). When compared to control group, significant decreases GSH levels in animals treated with AA were found in spleen, thymus and bone marrow, respectively (p < 0.05). Cotreatment with AA + AO significantly increased and normalized the GSH levels in all the lymphoid organs (p < 0.05). Some in vivo studies have shown that AA causes oxidative stress in plasma, liver, testis, brain and kidney of animals [30,31]. While the administration of AA reduced antioxidant enzyme levels such as catalase (CAT), superoxide dismutase (SOD), peroxidase, GSH peroxidase (GSH-Px) and GSH, it increased the ROS and MDA levels in different organs of rats [4,10,11,30,32–34]. AA also increased the
levels of lipid peroxidative product, PCO content, hydroxyl radical and hydroperoxide in rat brain [9]. The obtained results in the present study are agreement with the previous findings because of significant decreases in the GSH levels and significant increases in lipid peroxidation and PCOs levels in all lymphoid tissues of AA treated rats. The protective effects of AO against oxidative stress are probably due to its high contents of powerful antioxidants [13]. The activities of cytosolic CAT were significantly higher in rats treated with AO (5 ml/kg/day for 8 weeks) [35]. Lower plasma lipoperoxide and LDL cholesterol levels and higher a-tocopherol concentrations were observed in healthy subjects were regularly consuming AO (mean daily 15 g) [36]. The phenolic fraction of AO has been shown to inhibit low-density lipoprotein oxidation in macrophages [37] and to have an antiproliferative effect on human prostate cancer cell lines [38]. After a 3-week consumption of AO in dyslipidemic patients, platelet lipid peroxidation levels were about 34% lower and platelet GPx activity was about 22% higher when compared to the control group in which AO was replaced by butter [12]. AO (5 ml/kg b.w by gavage for 3 weeks) significantly improved oxidative stress parameters against mercuric chloride-induced toxicity by increasing GSH levels, GSH-Px, glutathione-S-transferase (GST) and CAT activities and decreasing lipid peroxidation level in rats [13]. AO significantly decreased plasma markers of lipid peroxidation in rats compared to rats fed high-fat diet. In addition, plasma vitamin E concentrations, CAT and SOD activities were significantly increased in AO-treated rats [14]. Administration of AO restored both the cytosolic and mitochondrial oxidative stress by normalizing nicotinamide adenine dinucleotide phosphate generating enzymes in acrylamide treated rat brain [39]. Few studies have investigated the direct effect of AO on the immune system cells. Benzaria et al. [17] reported that AO improved proliferation and phospholipase D activity of rat thymocytes. The obtained results in the present study are agreement with the previous findings because AO administration together with AA significantly decreased oxidative stress in lymphoid tissues. 3.3. MPO activity in PMNs Fig. 1 shows the effects of AA and/or AO on MPO activity in PMNs of rats. AA significantly decreased the MPO activity in PMNs when compared to control group (p< 0.05). No significant alteration was observed in the activity in the animals received the AO alone when compared to control group (p < 0.05). Cotreatment with AA + AO significantly ameliorated and completely normalized the MPO activity against AA-induced toxicity (p< 0.05). Increased MPO activity is an important local mediator of inflammation and subsequent organ damage in tissues. Lower MPO activities in blood leukocytes were observed in multiple sclerosis, diabetes and autoimmune lupus nephritis patients [40]. MPO also plays an important role in the various functions of neutrophils in innate and adaptive immunity in PMNs lymphocytes [41]. Increased MPO activity was found in different tissues of
Table 3 TBARs, PCOs and GSH levels in spleen, thymus and bone marrow in rats treated with acrylamide (AA) and/or argan oil (AO). Treatment groups
Control AA AO AA + AO a b
TBARs (nmol malondialdehyde/mg protein)
PCOs (nmol carbonyl/mg protein)
GSH (nmol GSH/mg protein)
Spleen
Thymus
Bone marrow
Spleen
Thymus
Bone marrow
Spleen
Thymus
Bone marrow
10,53 0,66 14,96 2,43a 11,49 0,94b 12,57 1,46
16,02 0,57 22,02 1,42a 13,00 0,74a,b 17,51 1,26b
3,18 0,38 3,92 0,93 3,39 0,70 3,50 0,83
18,05 2,66 22,97 1,53a 19,92 2,41 18,62 1,20b
11,72 1,80 16,79 0,81a 12,19 0,89b 14,73 1,01a
14,01 1,63 18,64 2,28a 15,09 1,75 17,80 2,07a
1,54 0,18 1,03 0,25a 1,75 0,30b 1,46 0,35
1,13 0,04 0,96 0,06a 1,48 0,10a,b 1,20 0,09b
0,32 0,05 0,24 0,02a 0,37 0,03b 0,33 0,02b
Compared with control group difference is statistically significant. Compared with AA difference is statistically significant (p < 0.05).
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Fig. 1. The effects of acrylamide (AA) and/or argan oil (AO) on myeloperoxidase (MPO) activity in polymorphonuclear leukocytes (PMNs) in rats. Experiments were repeated three times. a: Compared with control group difference is statistically significant, b: Compared with AA difference is statistically significant (p < 0.05).
AA treated rats (40 mg/kg i.p.for 10 days) [32,33]. Contrary to previous results, we also observed that MPO activity significantly decreased in PMNs of AA treated rats. The immune system, especially PMNs cells are sensitive to oxidative stress [42]. So far, the effects of AO on MPO activity have not been investigated. Our results indicate that co-treatment with AO + AA improved MPO activity in PMNs. 3.4. Urinary 8-OHdG As shown in Fig. 2, AA significantly increased the urinary concentration of 8-OHdG when compared to control group (p < 0.05). No significant alterations were observed in 8-OHdG levels in the animals received the AO alone and co-treatment with AO in combination with AA when compared to control group. Our results show that AO significantly reduced and improved the increased urinary 8-OHdG levels induced by AA in rats. 8-OHdG, an indicator of oxidative DNA damage, is eliminated by excretion in the urine [1]. Numerous reports indicate that if it is not repaired, it can contribute to the induction of carcinogenesis and many other diseases [43]. Therefore, 8-OHdG levels can be used to predict risk of some diseases such as cancer, atherosclerosis, and diabetes [1,43]. Significantly increased 8-OHdG levels were
detected in AA treated rats (40 mg/kg i.p.for 10 days) [32,33]. The level of 8-OHdG in the liver increased significantly in the AAtreated mice [10]. Serum levels of 8-OHdG were significantly increased in AA treated rats [34]. Because AA increased the 8OHdG level in rat urine in our study, our findings support the idea that there is significant correlation between urinary 8-OHdG levels and AA consumption. Effects of AO on 8-OHdG formation have not been previously studied. Our results show that AO showed protective activity against to AA by decreasing the formation of 8-OHdG formation in rat urine. 3.5. ME and MN assay Table 4 represents the effects of AA and/or AO on the frequencies of MN and ME and the ratio of PCEs to NCEs in rat bone marrow. AA significantly increased the formation of MN and decreased the ratio of PCEs to NCEs in bone marrow (p < 0.05). No significant differences in MN and PCE/NCE values were observed in the animals received the AO compared to the control group. Cotreatment with AA and AO significantly increased the PCE/NCE ratio and improved the PCE/NCE value against AA-induced cytotoxicity (p < 0.05). Significant decreased MNPCE frequency was also found when rats were co-treated with AO and AA
Fig. 2. The effects of acrylamide (AA) and/or argan oil (AO) on urinary levels of 8-hydroxydeoxyguanosine (8-OHdG) in rats. Experiments were repeated three times. a: Compared with control group difference is statistically significant, b: Compared with AA difference is statistically significant (p < 0.05).
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Table 4 Effects of acrylamide (AA) and/or argan oil (AO) on megakaryocytic emperipolesis (ME), micronucleated polychromatic erythrocytes (MNPCE) frequency and the ratio of PCEs to NCEs in rat bone marrow. Treatment groups
ME%
ME index
MNPCE frequency%
PCE/NCE Ratio
Control AA AO AA + AO
2.17 0.39 3.60 0.69a 2.27 0.30b 2.83 0.31
2.54 1.03 6.75 2.15a 2.55 0.66b 4.09 0.94b
0.32 0.06 0.55 0.07a 0.33 0.04b 0.42 0.10b
1.71 0.28 1.20 0.09a 1.73 0.31b 1.59 0.12b
a b
Compared with control group difference is statistically significant. Compared with AA difference is statistically significant (p < 0.05).
compared to AA alone (p < 0.05). The results of the present study show that AO ameliorated the MN frequency and the ratio of PCEs to NCEs in rat bone marrow. As shown in Table 4, AA also significantly increased the frequencies of ME and ME index compared to the control group in bone marrow (p < 0.05). No significant differences in these values were observed in the animals received the AO compared to the control group. Co-treatment with AA + AO significantly decreased the ME and ME index values compared to those treated with AA alone (p < 0.05). The results of the present study show that AO significantly reduced and improved the ME and ME index values (p < 0.05). ME is enclosing or engulfing of diverse bone marrow cells by megakaryocytes. Although various hypotheses have been proposed to explain the physiological mechanism of the ME, it has not yet been clearly clarified. Nevertheless, it is known to increase in many disorders such as various cancer types and different tumours, hypoxia, anemia and blood loss. The hyperplasia of haematopoietic cells is also closely associated with ME [26]. Yener and Dikmenli [26] reported that administration of AA (60 mg/kg) to rats by gavage for five consecutive days significantly decreased the incidence of ME in rat bone marrow compared to control group. Contrary to the finding, the obtained results in the present study indicated that intraperitoneal administration of AA in a dose of 50 mg/kg/day at the same time every other day for 30 days significantly increased ME frequency in rat bone marrow. Observed differences in these studies may result from the differences in the preferred administration route and treatment time of AA to animals. Many studies have shown genotoxic, reproductive and carcinogenic effects of AA. According to these studies, AA can induce specific DNA adducts and breaks, gene mutation, chromosome aberrations, MN formation, sister chromatin exchanges, unscheduled DNA synthesis, aneuploidy, polyploidy, carcinogenesis and sperm malformation rate [2,11]. There are many scientific studies with negative and positive results about the effect of AA on MN formation in mice and rats. It has been stated that AA significantly induced the MN frequency in mice [11,44–49] and in rats [4,52], while others have found no effect [45,50,51]. As shown, the MN results are conflicting in mice and rats. We think the reasons for these observed inconsistent results may be different routes chosen for administration of AA to animals or some minor differences in methodological procedures in different laboratories. Significantly increased the frequencies of MNPCEs and decreased the ratio of PCE/NCE were found in bone marrow of rats at single oral doses of 125, 150 or 175 mg/kg for 48 h [52]. DNA strand break and MN formation were increased in rats treated with 5, 10 and 15 mg/kg/ day of AA for 4 or 12 weeks [4]. The obtained results in the present study are agreement with the previous findings because AA increased the MN frequency in rat bone marrow. There is no study examining effects of AO on ME and MN formation and on PCE/NCE ratio. We clearly observed that AO decreased the formation of genetic damage in parallel with the formation of ME in rat bone marrow against AA toxicity.
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