Gotowa bibliografia na temat „Animo acids – metabolism”

Chronic diseases are characterised by altered autophagy and protein metabolism disarrangement, resulting in sarcopenia, hypoalbuminemia and hypo-haemoglobinaemia. Hypo-haemoglobinaemia is linked to a worse prognosis independent of the target organ affected by the disease. Currently, the cornerstone of the therapy of anaemia is iron supplementation, with or without erythropoietin for the stimulation of haematopoiesis. However, treatment strategies should incorporate the promotion of the synthesis of heme, the principal constituent of haemoglobin (Hb) and of many other fundamental enzymes for human metabolism. Heme synthesis is controlled by a complex biochemical pathway. The limiting step of heme synthesis is D-amino-levulinic acid (D-ALA), whose availability and synthesis require glycine and succinil-coenzyme A (CoA) as precursor substrates. Consequently, the treatment of anaemia should not be based only on the sufficiency of iron but, also, on the availability of all precursor molecules fundamental for heme synthesis. Therefore, an adequate clinical therapeutic strategy should integrate a standard iron infusion and a supply of essential amino acids and vitamins involved in heme synthesis. We reported preliminary data in a select population of aged anaemic patients affected by congestive heart failure (CHF) and catabolic disarrangement, who, in addition to the standard iron therapy, were treated by reinforced therapeutic schedules also providing essential animo acids (AAs) and vitamins involved in the maintenance of heme. Notably, such individualised therapy resulted in a significantly faster increase in the blood concentration of haemoglobin after 30 days of treatment when compared to the nonsupplemented standard iron therapy.

To investigate changes in muscle metabolism during lactation, serial biopsy of the triceps brachii was conducted in first-parity sows subjected to three degrees of selective protein mobilization through restriction of dietary protein intake (see Clowes EJ, Aherne FX, Foxcroft GR, and Baracos VE. J Anim Sci 81: 753–764, 2003). Muscle biopsies were taken 7 days before parturition and at 12 and 23 days of lactation. The following changes occurred after parturition, were progressive, and were significantly magnified in animals under the greatest degree of dietary protein restriction and hence of protein mobilization. Decreased RNA-to-DNA ratio (capacity for protein synthesis) was observed. The presence of increased expression of several elements of the ubiquitin proteasome proteolytic pathway suggested a robust catabolic response. However, as lactation progressed, and especially under conditions of increased dietary protein restriction, protein mobilization increased, muscle RNA-to-DNA ratio fell further, protease gene expression continued to rise, tissue free glutamine levels rose dramatically, and essential amino acid levels, especially branched-chain amino acids and threonine, fell to below prepartum levels.

Narvaez, N., Alazzeh, A. Y., Wang, Y. and McAllister, T. A. 2014. Effect of Propionibacterium acidipropionici P169 on growth performance and rumen metabolism of beef cattle fed a corn- and corn dried distillers’ grains with solubles-based finishing diet. Can. J. Anim. Sci. 94: 363–369. A growth and metabolism experiment was conducted to evaluate the effect of Propionibacterium acidipropionici P169 on feedlot steers fed a corn- and corn dried distillers' grains with soluble (DDGS)-based finishing diet. Steers (40 non-cannulated and 8 ruminally cannulated) were divided into two groups and administered 10 g head−1 d−1 of maltodextrin containing 1×1011 colony-forming units (CFU) of P169 or the same amount of carrier (Control), top-dressed once daily upon feeding. Feed intake, growth rate and feed efficiency were determined over 115 d. For cannulated steers, ruminal pH was monitored continuously for 5 d during the second week of each month over the entire feeding period with rumen samples collected 3 h after feeding on days 1 and 5. Molar proportions of butyrate, branched-chain volatile fatty acids (VFA) and NH3-N concentration increased (P<0.01) with P169, whereas total VFA, molar proportions of propionate, the acetate:propionate ratio, and lactate concentration did not differ (P>0.05) between treatments. P169 had a limited effect on ruminal pH as duration and area under the curve both at pH 5.5 and 5.2 as well as frequency of acute ruminal acidosis bouts were similar (P>0.05) for both groups. Compared with control steers, steers fed P169 had more (P<0.05) bouts of subacute ruminal acidosis (SARA). All steers had similar (P>0.05) feed intake, growth rate, feed efficiency and carcass characteristics, except for longissiumus muscle area, being less (P<0.05) for P169 steers than controls. Supplementing P169 to beef cattle fed a corn- and corn DDGS-based finishing diet had no effect on growth performance of beef cattle or ruminal pH and increased the bouts of SARA.

Meadus, W. J., Duff, P., Rolland, D., Aalhus, J. L., Uttaro, B., and Dugan, M. E. R. 2011. Feeding docosahexaenoic acid to pigs reduces triglycerides in blood and induces gene expression for fat oxidation in liver and adipose but not in muscle. Can. J. Anim. Sci. 91: 601–612. The essential fatty acids required in diets of humans are linoleic acid (18:2n-6:LA) and α-linolenic acid (18:3n-3: ALA), and these can be elongated and desaturated to form long-chain omega-6 or omega-3, respectively. Even though not considered essential, consumption of long-chain omega-3 fatty acid is recommended for health benefits, including protection against cardiovascular disease. The omega-3 fatty acid, docosahexanoic acid (DHA), was supplemented in pig diets as a dried biomass of the microalgae Schizochytrium to see if there are unique physiological changes associated with DHA feeding. Pigs were fed a diet with 330 mg (low), 3600 mg (medium) or 9400 mg (high) DHA per day for the last 25 d before slaughter at market weight (∼110 kg). Blood triglycerides (TG) were assayed colormetrically and tissue samples were analyzed for gene expression patterns of RNA by quantitative real-time polymerase chain reaction. Fatty acids were analyzed by gas chromatography. Animal performance appeared to increase with DHA, as shown by a 14% improved feed:gain ratio of 2.74±0.27 (P<0.05). Blood triglycerides were reduced significantly from 0.40±0.23 mM to 0.20±0.09 mM. Pigs accumulated 14 times more DHA in their subcutaneous fat (SQ) (10.67 mg g−1) on the high diet compared with the control diet (0.75 mg g−1). Gene analysis showed that the expression of the fat oxidation biomarkers acyl CoA oxidase 1 (ACOX1), peroxisome proliferator-activated receptors alpha (PPARα) and gamma (PPARγ) were stimulated in the SQ and liver. The delta-6 desaturase (D6D) and elongase (Elov5), which are genes involved in the endogenous synthesis of DHA, were unchanged. Fatty acid synthase (FASN) was stimulated in the liver and muscle of pigs on the high DHA diet. Analysis of gene transcription activity suggested fat metabolism was stimulated in the liver and SQ fat, but the genes involved in the endogenous production of DHA remained unchanged.

Ramirez-Bribiesca, J. E., Wang, Y., Jin, L., Canam, T., Town, J. R., Tsang, A., Dumonceaux, T. J. and McAllister, T. A. 2011. Chemical characterization and in vitro fermentation of Brassica straw treated with the aerobic fungus, Trametes versicolor . Can. J. Anim. Sci. 91: 695–702. Brassica napus straw (BNS) was either not treated or was treated with two strains of Trametes versicolor; 52J (wild type) or m4D (a cellobiose dehydrogenase-deficient mutant) with four treatments: (i) untreated control (C-BNS), (ii) 52J (B-52J), (iii) m4D (B-m4D) or (iv) m4D+glucose (B-m4Dg). Glucose was provided to encourage growth of the mutant strain. All treatments with T. versicolor decreased (P<0.05) neutral-detergent fibre and increased (P<0.05) protein and the concentration of lignin degradation products in straw. Ergosterol was highest (P<0.05) in straw treated with B-52J, suggesting it generated the most fungal biomass. Insoluble lignin was reduced (P<0.05) in straw treated with B-52J and B-m4D, but not with B-m4Dg. Mannose and xylose concentration were generally higher (P<0.05) in straw treated with fungi, whereas glucose and galactose were lower as compared with C-BNS. The four treatments above were subsequently assessed in rumen in vitro fermentations, along with BNS treated with 2 mL g−1of 5 N NaOH. Concentrations of total volatile fatty acids after 24 and 48h were lower (P<0.05) in incubations that contained BNS treated with T. versicolor as compared with C-BNSor NaOH-treated BNS. Compared with C-BNS, in vitrodry matter disappearance and gas production were increased (P<0.05) by NaOH, but not by treatment with either strain of T. versicolor. Although treatment with T. versicolor did release more lignin degradation products, it did not appear to provide more degradable carbohydrate to in vitro rumen microbial populations, even when a mutant strain with compromised carbohydrate metabolism was utilized. Production of secondary compounds by the aerobic fungi may inhibit rumen microbial fermentation.

Acosta, D. A. V., Pfeifer, L. F. M., Schmitt, E., Schneider, A., Silveira, P. A. S., Jacometo, C. B., Brauner, C. C., Rabassa, V. R., Corrêa, M. N. and Del Pino, F. A. B. 2013. Effect of prepartum somatotropin injection in late pregnant Holstein heifers with high body condition score on metabolic parameters, resumption of ovulation and milk production. Can. J. Anim. Sci. 93: 287–292. In the early post-partum period of dairy cows the duration and intensity of negative energy balance, the level of body condition score (BCS) loss and the milk yield are strongly associated with the timing of the first ovulation. The aim of this study was to determine the effect of pre-partum injections of somatotropin in dairy heifers with high BCS on the metabolism, resumption of ovarian activity and milk production. Holstein heifers (n=20) with high BCS, were divided randomly into two groups: somatotropin (n=10), which received two doses of somatotropin (500 mg) at −28 and −14 d from calving and Control (n=10), which received placebo. Blood samples were collected for evaluation of β-hydroxybutyrate (BHBA) and non-esterified fatty acids (NEFA) concentrations. Follicular development was also monitored via ultrasound. Somatotropin had no effect on plasma NEFA (P=0.35 and P=0.46) or BHBA (P=0.20 and P=0.44,) concentrations in the pre-partum and post-partum period, respectively. Milk production was not different between control (17.53±0.66 kg cow−1 d−1) and somatotropin groups (16.13±0.67 kg cow−1 d−1) (P=0.14). Pre-partum somatotropin administration did not affect (P=0.28) the time of the first post-partum ovulation. The proportion of cows ovulating the first post-partum follicular wave was not different between groups (P=0.49). In conclusion, pre-partum somatotropin treatment in dairy heifers with high body condition score seems not to have any effect on markers of energy balance, milk production or development of the first follicular wave in the early post-partum period.

Low molecular weight chemical compounds or intermediate products of a metabolism or other cellular processes are called metabolites. The objective of the present study was to investigate the variations in the metabolite concentration in the milk from crossbred water Buffalo (B), Holstein cross (H), Indigenous cattle (I) and Red Chittagong Cattle (R) and the relationships among those metabolites. The concentration of citric acid, ? -keto glutaric acid (?-keto GA), orotic acid, pyruvic acid, succinic acid, lactic acid, formic acid, uric acid and propionic acid were measured by using High Performance Liquid Chromatography. Pooled milk samples of nine B, fifteen I and twenty R cows were collected from the research farm of Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh. The pooled milk samples of H were collected from twenty five cows of Central Cattle Breeding Station and Dairy Farm, Savar, Dhaka, Bangladesh. All the sampling was done from the morning milking. The samples were preserved by adding ‘bronopol tablet’ and frozen and stored at -20 °C. Except propionic acid, all the metabolites varied significantly (p<0.01) among the milk types and B milk had lowest concentration of them. The highest concentration of citric acid (ppm) was 2245.50±39.90 in I milk followed by 2156.60±26.60 in R, 1858.30±8.70 in H and 1366.70±33.70 in B milk. The H, I and R milk were found similar in their ?-keto GA content. Similarities were also found for succinic acid between H and R milk and so for H and I milk in uric acid content. Highest formic acid was found in H milk (392.78±2.23 ppm) and the lowest was 308.91±3.75 ppm in R milk while B and I milk was found similar. The lowest concentration of uric acid was found 0.44±0.26 ppm in B milk and R milk had the highest content (11.38±1.10 ppm). Pyruvic, formic and propionic acids showed no significant relationship among them and with others as well. The most highly significant (p<0.01) correlation was found between orotic and ?-keto GA (r=0.915) and between uric and succinic acid (r=0.914). So, the metabolites showed a considerable variation in their concentration in different types of milk and also some of the parameters showed significant relationship among them. DOI: http://dx.doi.org/10.3329/bjas.v42i2.18504 Bang. J. Anim. Sci. 2013. 42 (2): 152-157

Introduction Methods Resuts Discussion Conclusions Acknowledgments Authors' contributions Competing interests Ethical approval References Effect of Methotrexate and Omega-3 Combination on Cytogenetic Changes of Bone Marrow and Some Enzymatic Antioxidants: An Experimental Study Inaam N. Ali1, Muthana M. Awad2, Alaa S. Mahmood2,* 1 Water and Environment Directorate, Ministry of Sciences and Technology, Baghdad, Iraq 2 Department of Biology, College of Science, University of Anbar, Anbar, Iraq * Corresponding author: A. S. Mahmood (alaashm91@gmail.com) Abstract: Objective: To assess the effect of methotrexate and omega-3 combination on cytogenetic changes of bone marrow and activities of some enzymatic antioxidants. Methods: Fifty-six mature male Wistar rats were divided into two experimental groups and a control group. The first experimental group was sub-divided into three sub-groups depending on the concentration of methotrexate (MTX): X1 (0.05 mg/kg MTX), X2 (0.125 mg/kg MTX) and X3 (0.250 mg/kg MTX), which were given intraperitoneally on a weekly basis for eight weeks. The second experimental group (MTX and omega-3 group) was also sub-divided into three sub-groups (Y1, Y2 and Y3), which were injected intraperitoneally with 0.05, 0.125 and 0.25 mg/kg MTX, respectively, weekly for eight weeks accompanied by the oral administration of 300 mg/kg omega-3. The rats of the control group were given distilled water. The enzymatic activity of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) were measured in the sera of rats. In addition, the mitotic index (MI) and chromosomal aberrations of bone marrow were also studied. Results: MTX resulted in a significant decrease in the activities of CAT, SOD and GR compared to the controls. It also increased the MI and chromosomal aberrations of rat bone marrows. On the other hand, omega-3 significantly increased the activities of the investigated enzymatic antioxidants and reduced the MI and chromosomal aberrations in treated mice when given in combination with MTX. Conclusions: MTX has a genotoxic effect on the bone marrow by increasing the MI and all types of chromosomal aberrations and decreasing the enzymatic activity of CAT, SOD and GR. The addition of omega-3 can lead to a protective effect by reducing the toxic and mutagenic effects of MTX. Keywords: Methotrexate, Omega-3, Antioxidant, Wistar rat, Chromosomal aberration, Mitotic index 1. Introduction Methotrexate (MTX) is a folic acid antagonist because of their chemical similarity [1]. Vezmar et al. [2] showed that MTX affects the synthesis of nucleic acids deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by interfering with the biosynthesis of thymine and purines. It also directly affects the rapidly dividing and intact cells, especially those in the mucous membranes of the mouth, intestine and bone marrow [3]. Omega-3 is a type of unsaturated fats, which are classified as essential fatty acids that cannot be manufactured by the body and should be taken with food [4]. Sources of omega-3 include fish oils, such as salmon, sardines and tuna, as well as soybeans, walnuts, raisins and linseed, almonds and olive oils [5]. Omega-3 is used in the prevention of a number of diseases such as rheumatoid arthritis, ulcerative colitis, asthma, atherosclerosis, cancer, and cardiovascular diseases [6]. A large amount of evidence indicates that omega-3 fatty acids have significant health benefits, including anti-inflammatory and antioxidant properties besides their effect on blood cholesterol levels [7]. Antioxidants retard the oxidation process by different mechanisms such as the removal of free radicals [8]. Enzymatic antioxidants include catalase (CAT), which is the first line of defense in the cell that removes hydrogen peroxide formed during biological processes by converting it into an aldehyde, and superoxide dismutase (SOD). There are three major families of SOD enzymes: manganese SOD (Mn-SOD) in the mitochondria and peroxisomes, iron SOD (Fe-SOD) in prokaryote cells and copper/zinc SOD (Cu-Zn SOD) in the cytoplasm of eukaryote cells [9]. Therefore, changes in the metal co-factors (manganese, iron, copper and zinc) can alter the effectiveness of SOD and may lead to diseases as a result of oxidative stress [10]. Glutathione reductase (GR) is also an enzymatic antioxidant that converts the oxidized glutathione to the reduced glutathione in the presence of NADPH, which is oxidized to NADP [11]. Therefore, the aim of the present study was to assess the effects of MTX and omega-3 on the cytogenetic changes of bone marrow as well as the activities of CAT, SOD and GR enzymatic antioxidants in male rats. 2. Method 2.1. Laboratory animals and experimental design Fifty-six mature male Wistar rats (Rattus norvegicus), aged 10–12 weeks old and weighing 250–300 gm, were used in the present study. The rats were kept in separate cages, with natural 13- hour light and 11-hour dark periods in a contamination-free environment with a controlled temperature (28.0 ± 1.0°C). In addition, rats were maintained on a standard diet and tap water ad libitum. The rats were randomly allocated to two experimental groups and a control group. The first experimental group (MTX group) included 24 rats injected intraperitoneally with different MTX dilutions with distilled water [12]. It was sub-divided into three sub-groups (eight rats per sub-group) according to MTX concentration as follows: X1 (0.05 mg/kg MTX), X2 (0.125mg/kg MTX) and X3 (0.25 mg/kg MTX). All rats were given a single dose of the specified MTX concentration weekly for eight weeks. The second experimental group (MTX and omega-3 group) included 24 rats allocated to three sub-groups (Y1, Y2 and Y3), which were injected intraperitoneally with 0.05, 0.125 and 0.25 mg/kg MTX, respectively, weekly for eight weeks accompanied by the oral administration of 300 mg/kg omega-3. The control group included eight rats that were intraperitoneally injected with distilled water and given a single dose of distilled water orally weekly for eight weeks. 2.2. Blood collection and processing After the end of the dosing period, 5 ml of blood were withdrawn from the heart (by cardiac puncture) using a 5 cc disposable syringe. The collected blood was immediately poured into a clean sterile screw-capped tube (plain tube) and left for coagulation in a water bath at 37°C for 15 minutes. After coagulation of blood, the plain tube was centrifuged for 5 minutes at 1500 rpm. Then the samples were stored at -20°C for subsequent analysis. 2.3. Measurement of the activity of antioxidant enzymes The antioxidant activities of CAT, SOD and GR were measured using enzyme-linked immunosorbent assay kits purchased from Kamiya Biomedical Company (Seattle, WA, US), according to the manufacturer's instructions. 2.4. Cytogenetic study of bone marrow Rats were killed by cervical dislocation, and their hip bones were cleaned from surrounding muscles and then dissected by cutting both ends of the bone. Five milliliters of physiological buffered saline were injected inside the bone to withdraw bone marrow into a test tube. Tubes were centrifuged at 2000 rpm/10 minutes. The supernatant was then removed, and 10 ml of KCL solution (0.075 M) were added to the sediment. The mixture was then incubated at 37 °C in a water bath for 30 minutes, with shaking from time to time. The tubes were then centrifuged at 2000rpm/10 minutes to remove the supernatant. However, 5 ml of a freshly prepared fixative solution (methanol: glacial acetic acid 1:3) were added gradually in the form of droplets into the inner wall of the tube with constant mixing. After that, the tubes were placed at 4 °C for half an hour to fix the cells. This process was repeated for three times, and the cells were then suspended in 2 ml of the fixative solution. The tubes were centrifuged at 2000 rpm for 5 minutes, and the supernatant was then removed while the cells were re-suspended in 1-2 ml of cold fixative solution. After shaking the tubes, 4–5 drops were then taken from each tube onto a clean slide from a height of about three feet to provide an opportunity for the cells and nuclei to spread well. The slides were stained with acridine orange solution (0.01%) for 4–5 minutes, incubated in Sorensen’s buffer (0.06M, pH 6.5) for a minute. and then examined using a fluorescence microscope Olympus BX 51 America at a wavelength of 450–500 nm [13, 14]. A total of 1000 cells were examined, and both dividing and non-dividing cells were calculated [13]. Mitotic index (MI) was calculated according to the following formula [13]: MI= No. of dividing cells / 1000 × 100 2.5. Analysis of chromosomal aberrations of bone marrow cells A total of 1000 dividing cells were examined on the stained slides under a fluorescence microscope at a wavelength of 45–500 nm. The examined cells were at the first metaphase of the mitotic division, where chromosomal aberrations are clear and can be easily seen [13]. 2.6. Statistical analysis Data were analyzed using the Statistical Analysis System (SAS®) software, version 9.1 (Cary, NC, USA) [15]. Effects were expressed as mean ± standard error (SE) and statistically compared using a completely randomized design analysis of variance and least significant differences. Differences at P values <5 were considered statistically significant. 3. Results 3.1. Effects of MTX and MTX-omega-3 combination on antioxidant enzymatic activities Table (1) shows significantly lower SOD activities among rats treated with MTX or MTX-omega-3 compared to controls. Moreover, sera of rats receiving relatively high doses of MTX (sub-groups X2 and X3) showed the lowest enzymatic activities of 4.29 ± 0.01 IU and 3.93 ± 0.11 IU, respectively. On the other hand, CAT activity differed significantly between treated and control rats as well as among treated rats themselves, In this respect, the controls showed the highest activity of 39.38 ±0.02 IU, while those receiving the highest MTX concentration, either alone or in combination with omega-3 (sub-groups X3 and Y3), showed the lowest activities of 30.97 ± 0.03 IU and 32.12± 0.06 IU, respectively. Regarding GR activity, control rats showed a higher activity of 53.09± 0.05 IU compared to treated ones; however, the differences in GR activities in rats given low doses of MTX, either alone or in combination with omega-3 (sub-groups X1 and Y1), were not statistically significant. On the other hand, rats in sub-groups X3 and Y3 showed the lowest GR activities of 34.59 ± 0.63 IU and 37.15 ±0.01, respectively, with statistically significant differences from other sub-groups. 3.2. Effects of MTX and MTX-omega-3 combination on mitotic index of bone marrow cells Figure (1) shows a significant decrease in the MI in all treated groups compared to control. In addition, there was a reverse association between MTX concentration and MI, where rats treated with the highest dose of MTX (sub-group X3) showed a significant decrease in MI compared to all other treated rat sub-groups. In addition, rats in sub-groups treated with MTX and omega-3 (sub-groups Y1, Y2 and Y3) showed a significant increase in MI compared to their counterpart rats receiving MTX only. Table 1. Activity of antioxidant enzymes in rats treated with MTX and MTX-omega-3 Group Enzymatic activity (mean± SE) SOD (IU) CAT (IU) GR (µmol) Control 6.41±0.02 a 39.38±0.02 a 53.09±0.05 a X1 (0.05 mg MTX/ kg) 5.33±0.01 b 37.81±0.01 c 51.12±0.06 a Y1 (0.05 mg MTX + 300 mg omega-3/ kg) 6.08±0.04 a 38.40±0.02 b 51.97±0.03 a X2 (0.125 mg MTX/ kg) 4.29±0.01 cd 33.13±0.01 e 42.34±0.03 b Y2 (0.125 mg MTX + 300 mg omega-3/ kg) 4.99±0.40 b 36.68±0.02 d 43.02±3.04 b X3 (0.25 mg MTX/ kg) 3.93±0.11 d 30.97±0.03 g 34.59±0.63 c Y3 (0.25 mg MTX + 300 mg omega-3/ kg) 4.47±0.02 c 32.12±0.06 f 37.15±0.01 c SE, Standard error; IU, international unit; SOD, superoxide dismutase; CAT, catalase; GR, glutathione reductase; *statistically significant at P < 0.05; **statistically significant at P < 0.01. Means with different letters within the same column showed a statistically significant difference. 3.3. Effects of MTX and MTX-omega-3 combination on chromosomal aberrations of bone marrow cells Rats receiving higher concentrations of MTX (sub-group X3) showed a significant increase in all types of chromosomal aberrations, i.e., chromatid gaps, chromosome gaps, chromatid breaks, chromosome breaks, deletions and simple fragments (Figure 2 and Table 2) than those of the control group or other treated sub-groups. All rats treated with MTX-omega-3 combination showed a significant decrease in almost all types of chromosomal aberrations compared to their counterpart rats receiving MTX alone (Table 2). Figure 1. Effect of MTX and MTX-omega-3 on the MI of bone marrow cells of treated rats compared to the controls. The groups X1 (0.05 MTX), X2 (0.125 MTX) and X3 (0.250 MTX) were compared to the control group, while the groups Y1 (0.05 MTX+ omega-3), Y2 (0.125 MTX+ omega-3) and Y3 (0.25 MTX+ omega-3) were compared to X1, X2 and X3, respectively. Figure 2. Effect of MTX and MTX-omega-3 on chromosomal aberration as seen under fluorescence microscope after staining with acridine orange: (1) a simple fragment; (2) a chromatid gap; (3) a chromosomal gap (A) and a chromosomal break (B). 4. Discussion The present experiment reveals that the addition of omega-3 to MTX alleviates its effects on the activities of the antioxidant enzymes CAT, SOD and GR, and decreases the MI as well as all types of chromosomal aberrations in the bone marrow cells. Daham et al. [16] showed that the decline in antioxidants associated with chemotherapy is attributed to the increase in lipid peroxidation caused by these kinds of drugs, which increase the level of free radicals. In addition, Weijl et al. [17] showed that some chemotherapeutic drugs have a negative effect on the antioxidant levels such as GR, whose activity decreases as a result of its involvement in many cellular processes such as cell defenses against the toxicity of some compounds. Al-Dalawy et al. [18] found that the decrease in the level of SOD is an evidence of its increased activity due to the increased release of free radicals. MTX causes an increase in the release of free radicals, including the OH radical that causes direct damage to DNA [16]. Al-Helaly [19] showed that the amount of food taken has an effect on antioxidants, where nutritional deficiency decreases the antioxidant levels, thus increasing free radicals that cause damage to DNA. Table 2. Chromosomal aberrations of bone marrow cells in rats treated with MTX and MTX-omega-3 Group Type of chromosomal aberration(mean ± SE) Chromatid gap Chromosome Gap Chromatid breaks Chromosome breaks Deletion Simple Fragments Chromosomal aberration (%) Control 1.33±0.33 e 0.00±0.00 e 1.67±0.33 c 0.33±0.15 c 0.00±0.00 0.67±0.33 cd 0.04±0.005 f X1 2.75±0.47 cd 1.50±0.28 cd 2.50±0.64 bc 1.00±0.41 bc 0.50±0.28 bc 0.75±0.25 bcd 0.09±0.02 de Y1 1.75±0.47 de 0.75±0.25 de 1.50±0.28 c 1.00±0.00 bc 0.75±0.25 abc 0.75±0.25 abc 0.065±0.005 ef X2 4.67±0.33 b 2.67±0.33 ab 2.67±0.33 bc 1.67±0.33 ab 0.67±0.33 abc 1.67±0.33 ab 0.14±0.006 bc Y2 3.00±0.00 c 2.00±0.00 bc 3.00±0.057 bc 1.33±0.33 b 0.67±0.33 abc 0.33±0.15 d 0.106±0.003 cd X3 6.80±0.37 a 3.00±0.31 a 4.60±0.74 a 2.40±0.24 a 1.40±0.24 a 1.80±0.37 a 0.20±0.017 a Y3 5.60±0.40 ab 2.40±0.24 ab 3.60±0.24 ab 1.80±0.20 ab 1.20±0.20 ab 1.40±0.24 abc 0.16±0.003 b LSD 1.231** 0.814** 0.602** 0.841** 0.774* 0.941** 3.499* SE, Standard error; * statistically significant at P < 0.05; ** statistically significant at P < 0.01. Means with different letters within the same column showed a statistically significant difference. X1 (0.05 mg MTX/ kg); X2 (0.125 mg MTX/ kg); X3 (0.25 mg MTX/ kg); Y1 (0.05 mg MTX + 300 mg omega-3/ kg); Y2 (0.125 mg MTX + 300 mg omega-3/ kg); Y3 (0.25 mg MTX + 300 mg omega-3/ kg). In the present study, the intraperitoneal administration of MTX to rats also caused a decrease in the MI of bone marrow and a significant increase in the rate of abnormal chromosomal aberration compared to the control rats. This finding is consistent with those reported previously [20], [21]. The effect of MTX can be attributed to its ability to interfere with the genetic material, leading to the appearance of toxic and mutagenic consequences. Rushworth et al. [22] reported that MTX leads to a lack of dihydrofolate reductase, which is the key to the growth and cell division processes. This, in turn, leads to a reduction of the nucleotides involved in the building of DNA and, therefore, to a stop or obstruction of the repair mechanisms of the damaged DNA. In addition, Wong and Choi [23] concluded that MTX inhibits the action of enzymes controlling the purine metabolism, which leads to the accumulation of adenosine in addition to the damage of the molecule itself and to the occurrence of chromosomal aberrations. Jafer et al. [24] reported the ability of MTX to induce chromosomal aberration in humans or animals by preventing the repair of DNA and affecting the proteins found in chromosomes. These findings were also confirmed by Hussain et al. [25], who found that MTX causes an increase in chromosomal aberrations. In the present study, the MI showed a significant increase in rat sub-groups treated with MTX-omega-3 combination, but there was a decrease in the rate of chromosomal aberration, which confirms the role of omega-3 unsaturated fatty acids in protecting the cell from the impact of free radicals [26], [27]. Attia and Nasr [28] reported the antioxidant effect of omega-3, which was attributed to the reduction in lipid peroxidation and the increase in SOD and CAT or the stimulation of GR. It is noteworthy that GR leads to the synthesis of reduced glutathione, which is important in the defense of the cell against toxic substances and the prevention of the occurrence of mutations [29]. 5. Conclusions MTX significantly decreases the activity of enzymatic antioxidants, reduce the MI and increase the chromosomal aberrations of all types in bone marrow. This gives further evidence on the genotoxic effects of MTX on the bone marrow. On the other hand, omega-3 shows a protective effect by reducing the toxic and mutagenic effects of MTX. Acknowledgments The authors thank the staff of the Water and Environment Directorate, Ministry of Science and Technology, Baghdad, Iraq for their cooperation. They also thank Dr. Jasim Al-Niami for his technical and scientific guidance. Authors' contributions INA, MMA and ASM contributed to the study design and analyzed data. All authors contributed to the manuscript drafting and revising and approved the final submission. Competing interests The authors declare that they have no competing interests associated with this article. Ethical approval The ethical clearance of this study was obtained from the Ethics Committee of the College of Science, University of Anbar (Reference No. A. D. 51 in 30/8/2015). References Yuen CW, Winter ME. Methotrexate (MTX). In: Basic clinical pharmacokinetics, Winter ME, editor. Philadelphia, USA: Lippincott Williams & Wilkins; 2010. p.p. 304–25. Google Scholar Vezmar S, Becker A, Bode U, Jaehde U. Biochemical and clinical aspects of methotrexate neurotoxicity. Chemotherapy 2003; 49: 92–104. DOI PubMed - Google Scholar Tian H, Cronstein BN. Understanding the mechanisms of action of methotrexate implications for the treatment of rheumatoid arthritis. Bull NYU Hosp Jt Dis 2007; 65: 168–73. PubMed - Google Scholar El-Khayat Z, Rasheed WI, Elias T, Hussein J, Oraby F, Badawi M, et al. Protective effect of either dietary or pharmaceutical n-3 fatty acids on bone loss in ovariectomized rats. Maced J Med Sci 2010; 3: 9–16. DOI - Google Scholar Kris-Etherton PM, Harris WS, Appel LJ; Nutrition Committee. Fish consumption, fish oil, omega-3 fatty acids and cardiovascular disease. Arterioscler Thromb Vasc Biol 2003; 23: e20–30. DOI - PubMed - Google Scholar Calder PC. Polyunsaturated fatty acids and inflammation. Prostaglandins Leukot Essent Fatty Acids 2006; 75: 197–202. DOI - PubMed - Google Scholar Begin ME, Ells G, Das UN, Horrobin DF. Differential killing of human carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids. J Natl Cancer Inst 1986; 77: 1053–62. DOI - PubMed - Google Scholar Shan B, Cai YZ, Sun M, Corke H. Antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents. J Agric Food Chem. 2005; 53: 7749–59. DOI - PubMed - Google Scholar Matiax J, Quiles JL, Huertas JR, Battino M. Tissue specific interactions of exercise, dietary fatty acids, and vitamin E in lipid peroxidation. Free Radic Biol Med 1998; 24 : 511–21. DOI - PubMed - Google Scholar Dean RT, Fu S, Stocker R, Davies MJ. Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 1997; 324: 1–8. PubMed - Google Scholar Bashir A, Perham RN, Scrutton NS, Berry A. Altering Kinetic mechanism and enzyme stability by mutagenesis of the dimmer interface of glutathione reductase. Biochem J 1995; 312: 527–33. PubMed - Google Scholar Perret-Gentil MI. Rat Biomethodology. Laboratory Animal Resources Center. The University of Texas at San Antonio. [Cited 1 Feb. 2015]. Available from: https://www.utdallas.edu/research/docs/rat_biomethodology/ Allen JW, Shuler CF, Menders RW, Olatt SA. A simplified technique for in vivo analysis of sister chromatid exchange using 50 bromodeoxyuridine tablets. Cytogenet Cell Genet 1977; 18: 231–7. DOI PubMed - Google Scholar Forsum U, Hallén A. Acridine orange staining of urethral and cervical smears for the diagnosis of gonorrhea. Acta Derm Venereol 1979; 59: 281–2. PubMed - Google Scholar Statistical Analysis System user's guide. Version 9.1. Cary, NC, USA: SAS Institute Inc.; 2012. Daham HH, Rahim SM, Al-Hmesh MJ. The effect of radiotherapy and chemotherapy in several physiological and biochemical parameters in cancer patients. Tikrit J Pure Sci 2012; 17: 83–91. Weijl N, Elseendoorm TJ, Lentjes EG, Hopman CD, Wipkink-Bakker A, Zwinderman AH, et al. Supplementation with antioxidant micronutrients and chemotherapy-induced toxicity in cancer patients treated with cisplatin-based chemotherapy: a randomised, double-blind placebo-controlled study. Eur J Cancer 2004; 40: 1713–23. DOI - PubMed - Google Scholar Al-Dalawy SS, Al-Salehy FK, Al-Sanafi AI. Efficient enzymatic antioxidants for oxidative stress syndrome in patients with hypertension. J Dhi Qar Sci 2008; 2: 32–3. Al-Helaly LA. Some antioxidant enzymes in workers exposed to pollutants. Raf J Sci 2011; 22: 29–38. Google Scholar Othman GO. Protective effects of linseed oil against methotrexate induced genotoxicity in bone marrow cells of albino mice Mus musculus. ZJPAS. 2016; 28: 49–53. Google Scholar Ashoka CH, Vijayalaxmi KK. Cytogenetic effects of methotrexate in bone marrow cells of Swiss albino mice. Int J Sci Res Edu 2016; 4: 4828–34. DOI - Google Scholar Rushworth D, Mathews A, Alpert A, Cooper Dihydrofolate reductase and thymidylate synthase transgenes resistant to methotrexate interact to permit novel transgene regulation. J Biol Chem 2015; 290: 22970–9. DOI - PubMed - Google Scholar Wong PT, Choi SK. Mechanisms and implications of dual-acting methotrexate in folate-targeted nanotherapeutic delivery. Int J Mol Sci 2015; 16: 1772–90. DOI - PubMed - Google Scholar Jafer ZMT, Shubber EK, Amash HS. Cytogenetic analysis of Chinese hamster lung fibroblasts spontaneously resistant to methotrexate. Nucleus 2001; 44: 28–35. Google Scholar Hussain ZK, AL-Mhdawi F, AL-Bakri N. Effect of methotrexate drug on some parameters of kidney in newborn mice. Iraqi J Sci 2014; 55: 968–73. Google Scholar Ghazi-Khansari M, Mohammadi-Bardbori A. Captopril ameliorates toxicity induced by paraquat in mitochondria isolated from the rat liver. Toxicol in Vitro 2007; 21: 403–7. DOI - PubMed - Google Scholar Dinic-olivira RJ, Sousa C, Remiao F, Durte JA, Navarro SA, Bastos L, et al. Full survival of paraquat-exposed rats after treatment with sodium salicylate. Free Radic Biol Med 2007; 42: 1017–28. DOI - PubMed - Google Scholar Attia AM, Nasr HM. Dimethoate-induced changes in biochemical parameters of experimental rat serum and its neutralization by black seed (Nigella sativa) oil. Slovak J Anim Sci 2009; 42: 87–94. Google Scholar Al-Rubaie AH.M. Effect of natural honey and mitomycin C on the effectiveness of the enzyme glutathione reductase in mice Mus musculus. Babylon Uni J 2008; 15: 1385–91.

Introduction Methods Resuts Discussion Conclusions Acknowledgments Authors' contributions Competing interests Ethical approval References Effect of Methotrexate and Omega-3 Combination on Cytogenetic Changes of Bone Marrow and Some Enzymatic Antioxidants: An Experimental Study Inaam N. Ali1, Muthana M. Awad2, Alaa S. Mahmood2,* 1 Water and Environment Directorate, Ministry of Sciences and Technology, Baghdad, Iraq 2 Department of Biology, College of Science, University of Anbar, Anbar, Iraq * Corresponding author: A. S. Mahmood (alaashm91@gmail.com) Abstract: Objective: To assess the effect of methotrexate and omega-3 combination on cytogenetic changes of bone marrow and activities of some enzymatic antioxidants. Methods: Fifty-six mature male Wistar rats were divided into two experimental groups and a control group. The first experimental group was sub-divided into three sub-groups depending on the concentration of methotrexate (MTX): X1 (0.05 mg/kg MTX), X2 (0.125 mg/kg MTX) and X3 (0.250 mg/kg MTX), which were given intraperitoneally on a weekly basis for eight weeks. The second experimental group (MTX and omega-3 group) was also sub-divided into three sub-groups (Y1, Y2 and Y3), which were injected intraperitoneally with 0.05, 0.125 and 0.25 mg/kg MTX, respectively, weekly for eight weeks accompanied by the oral administration of 300 mg/kg omega-3. The rats of the control group were given distilled water. The enzymatic activity of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) were measured in the sera of rats. In addition, the mitotic index (MI) and chromosomal aberrations of bone marrow were also studied. Results: MTX resulted in a significant decrease in the activities of CAT, SOD and GR compared to the controls. It also increased the MI and chromosomal aberrations of rat bone marrows. On the other hand, omega-3 significantly increased the activities of the investigated enzymatic antioxidants and reduced the MI and chromosomal aberrations in treated mice when given in combination with MTX. Conclusions: MTX has a genotoxic effect on the bone marrow by increasing the MI and all types of chromosomal aberrations and decreasing the enzymatic activity of CAT, SOD and GR. The addition of omega-3 can lead to a protective effect by reducing the toxic and mutagenic effects of MTX. Keywords: Methotrexate, Omega-3, Antioxidant, Wistar rat, Chromosomal aberration, Mitotic index 1. Introduction Methotrexate (MTX) is a folic acid antagonist because of their chemical similarity [1]. Vezmar et al. [2] showed that MTX affects the synthesis of nucleic acids deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by interfering with the biosynthesis of thymine and purines. It also directly affects the rapidly dividing and intact cells, especially those in the mucous membranes of the mouth, intestine and bone marrow [3]. Omega-3 is a type of unsaturated fats, which are classified as essential fatty acids that cannot be manufactured by the body and should be taken with food [4]. Sources of omega-3 include fish oils, such as salmon, sardines and tuna, as well as soybeans, walnuts, raisins and linseed, almonds and olive oils [5]. Omega-3 is used in the prevention of a number of diseases such as rheumatoid arthritis, ulcerative colitis, asthma, atherosclerosis, cancer, and cardiovascular diseases [6]. A large amount of evidence indicates that omega-3 fatty acids have significant health benefits, including anti-inflammatory and antioxidant properties besides their effect on blood cholesterol levels [7]. Antioxidants retard the oxidation process by different mechanisms such as the removal of free radicals [8]. Enzymatic antioxidants include catalase (CAT), which is the first line of defense in the cell that removes hydrogen peroxide formed during biological processes by converting it into an aldehyde, and superoxide dismutase (SOD). There are three major families of SOD enzymes: manganese SOD (Mn-SOD) in the mitochondria and peroxisomes, iron SOD (Fe-SOD) in prokaryote cells and copper/zinc SOD (Cu-Zn SOD) in the cytoplasm of eukaryote cells [9]. Therefore, changes in the metal co-factors (manganese, iron, copper and zinc) can alter the effectiveness of SOD and may lead to diseases as a result of oxidative stress [10]. Glutathione reductase (GR) is also an enzymatic antioxidant that converts the oxidized glutathione to the reduced glutathione in the presence of NADPH, which is oxidized to NADP [11]. Therefore, the aim of the present study was to assess the effects of MTX and omega-3 on the cytogenetic changes of bone marrow as well as the activities of CAT, SOD and GR enzymatic antioxidants in male rats. 2. Method 2.1. Laboratory animals and experimental design Fifty-six mature male Wistar rats (Rattus norvegicus), aged 10–12 weeks old and weighing 250–300 gm, were used in the present study. The rats were kept in separate cages, with natural 13- hour light and 11-hour dark periods in a contamination-free environment with a controlled temperature (28.0 ± 1.0°C). In addition, rats were maintained on a standard diet and tap water ad libitum. The rats were randomly allocated to two experimental groups and a control group. The first experimental group (MTX group) included 24 rats injected intraperitoneally with different MTX dilutions with distilled water [12]. It was sub-divided into three sub-groups (eight rats per sub-group) according to MTX concentration as follows: X1 (0.05 mg/kg MTX), X2 (0.125mg/kg MTX) and X3 (0.25 mg/kg MTX). All rats were given a single dose of the specified MTX concentration weekly for eight weeks. The second experimental group (MTX and omega-3 group) included 24 rats allocated to three sub-groups (Y1, Y2 and Y3), which were injected intraperitoneally with 0.05, 0.125 and 0.25 mg/kg MTX, respectively, weekly for eight weeks accompanied by the oral administration of 300 mg/kg omega-3. The control group included eight rats that were intraperitoneally injected with distilled water and given a single dose of distilled water orally weekly for eight weeks. 2.2. Blood collection and processing After the end of the dosing period, 5 ml of blood were withdrawn from the heart (by cardiac puncture) using a 5 cc disposable syringe. The collected blood was immediately poured into a clean sterile screw-capped tube (plain tube) and left for coagulation in a water bath at 37°C for 15 minutes. After coagulation of blood, the plain tube was centrifuged for 5 minutes at 1500 rpm. Then the samples were stored at -20°C for subsequent analysis. 2.3. Measurement of the activity of antioxidant enzymes The antioxidant activities of CAT, SOD and GR were measured using enzyme-linked immunosorbent assay kits purchased from Kamiya Biomedical Company (Seattle, WA, US), according to the manufacturer's instructions. 2.4. Cytogenetic study of bone marrow Rats were killed by cervical dislocation, and their hip bones were cleaned from surrounding muscles and then dissected by cutting both ends of the bone. Five milliliters of physiological buffered saline were injected inside the bone to withdraw bone marrow into a test tube. Tubes were centrifuged at 2000 rpm/10 minutes. The supernatant was then removed, and 10 ml of KCL solution (0.075 M) were added to the sediment. The mixture was then incubated at 37 °C in a water bath for 30 minutes, with shaking from time to time. The tubes were then centrifuged at 2000rpm/10 minutes to remove the supernatant. However, 5 ml of a freshly prepared fixative solution (methanol: glacial acetic acid 1:3) were added gradually in the form of droplets into the inner wall of the tube with constant mixing. After that, the tubes were placed at 4 °C for half an hour to fix the cells. This process was repeated for three times, and the cells were then suspended in 2 ml of the fixative solution. The tubes were centrifuged at 2000 rpm for 5 minutes, and the supernatant was then removed while the cells were re-suspended in 1-2 ml of cold fixative solution. After shaking the tubes, 4–5 drops were then taken from each tube onto a clean slide from a height of about three feet to provide an opportunity for the cells and nuclei to spread well. The slides were stained with acridine orange solution (0.01%) for 4–5 minutes, incubated in Sorensen’s buffer (0.06M, pH 6.5) for a minute. and then examined using a fluorescence microscope Olympus BX 51 America at a wavelength of 450–500 nm [13, 14]. A total of 1000 cells were examined, and both dividing and non-dividing cells were calculated [13]. Mitotic index (MI) was calculated according to the following formula [13]: MI= No. of dividing cells / 1000 × 100 2.5. Analysis of chromosomal aberrations of bone marrow cells A total of 1000 dividing cells were examined on the stained slides under a fluorescence microscope at a wavelength of 45–500 nm. The examined cells were at the first metaphase of the mitotic division, where chromosomal aberrations are clear and can be easily seen [13]. 2.6. Statistical analysis Data were analyzed using the Statistical Analysis System (SAS®) software, version 9.1 (Cary, NC, USA) [15]. Effects were expressed as mean ± standard error (SE) and statistically compared using a completely randomized design analysis of variance and least significant differences. Differences at P values <5 were considered statistically significant. 3. Results 3.1. Effects of MTX and MTX-omega-3 combination on antioxidant enzymatic activities Table (1) shows significantly lower SOD activities among rats treated with MTX or MTX-omega-3 compared to controls. Moreover, sera of rats receiving relatively high doses of MTX (sub-groups X2 and X3) showed the lowest enzymatic activities of 4.29 ± 0.01 IU and 3.93 ± 0.11 IU, respectively. On the other hand, CAT activity differed significantly between treated and control rats as well as among treated rats themselves, In this respect, the controls showed the highest activity of 39.38 ±0.02 IU, while those receiving the highest MTX concentration, either alone or in combination with omega-3 (sub-groups X3 and Y3), showed the lowest activities of 30.97 ± 0.03 IU and 32.12± 0.06 IU, respectively. Regarding GR activity, control rats showed a higher activity of 53.09± 0.05 IU compared to treated ones; however, the differences in GR activities in rats given low doses of MTX, either alone or in combination with omega-3 (sub-groups X1 and Y1), were not statistically significant. On the other hand, rats in sub-groups X3 and Y3 showed the lowest GR activities of 34.59 ± 0.63 IU and 37.15 ±0.01, respectively, with statistically significant differences from other sub-groups. 3.2. Effects of MTX and MTX-omega-3 combination on mitotic index of bone marrow cells Figure (1) shows a significant decrease in the MI in all treated groups compared to control. In addition, there was a reverse association between MTX concentration and MI, where rats treated with the highest dose of MTX (sub-group X3) showed a significant decrease in MI compared to all other treated rat sub-groups. In addition, rats in sub-groups treated with MTX and omega-3 (sub-groups Y1, Y2 and Y3) showed a significant increase in MI compared to their counterpart rats receiving MTX only. Table 1. Activity of antioxidant enzymes in rats treated with MTX and MTX-omega-3 Group Enzymatic activity (mean± SE) SOD (IU) CAT (IU) GR (µmol) Control 6.41±0.02 a 39.38±0.02 a 53.09±0.05 a X1 (0.05 mg MTX/ kg) 5.33±0.01 b 37.81±0.01 c 51.12±0.06 a Y1 (0.05 mg MTX + 300 mg omega-3/ kg) 6.08±0.04 a 38.40±0.02 b 51.97±0.03 a X2 (0.125 mg MTX/ kg) 4.29±0.01 cd 33.13±0.01 e 42.34±0.03 b Y2 (0.125 mg MTX + 300 mg omega-3/ kg) 4.99±0.40 b 36.68±0.02 d 43.02±3.04 b X3 (0.25 mg MTX/ kg) 3.93±0.11 d 30.97±0.03 g 34.59±0.63 c Y3 (0.25 mg MTX + 300 mg omega-3/ kg) 4.47±0.02 c 32.12±0.06 f 37.15±0.01 c SE, Standard error; IU, international unit; SOD, superoxide dismutase; CAT, catalase; GR, glutathione reductase; *statistically significant at P < 0.05; **statistically significant at P < 0.01. Means with different letters within the same column showed a statistically significant difference. 3.3. Effects of MTX and MTX-omega-3 combination on chromosomal aberrations of bone marrow cells Rats receiving higher concentrations of MTX (sub-group X3) showed a significant increase in all types of chromosomal aberrations, i.e., chromatid gaps, chromosome gaps, chromatid breaks, chromosome breaks, deletions and simple fragments (Figure 2 and Table 2) than those of the control group or other treated sub-groups. All rats treated with MTX-omega-3 combination showed a significant decrease in almost all types of chromosomal aberrations compared to their counterpart rats receiving MTX alone (Table 2). Figure 1. Effect of MTX and MTX-omega-3 on the MI of bone marrow cells of treated rats compared to the controls. The groups X1 (0.05 MTX), X2 (0.125 MTX) and X3 (0.250 MTX) were compared to the control group, while the groups Y1 (0.05 MTX+ omega-3), Y2 (0.125 MTX+ omega-3) and Y3 (0.25 MTX+ omega-3) were compared to X1, X2 and X3, respectively. Figure 2. Effect of MTX and MTX-omega-3 on chromosomal aberration as seen under fluorescence microscope after staining with acridine orange: (1) a simple fragment; (2) a chromatid gap; (3) a chromosomal gap (A) and a chromosomal break (B). 4. Discussion The present experiment reveals that the addition of omega-3 to MTX alleviates its effects on the activities of the antioxidant enzymes CAT, SOD and GR, and decreases the MI as well as all types of chromosomal aberrations in the bone marrow cells. Daham et al. [16] showed that the decline in antioxidants associated with chemotherapy is attributed to the increase in lipid peroxidation caused by these kinds of drugs, which increase the level of free radicals. In addition, Weijl et al. [17] showed that some chemotherapeutic drugs have a negative effect on the antioxidant levels such as GR, whose activity decreases as a result of its involvement in many cellular processes such as cell defenses against the toxicity of some compounds. Al-Dalawy et al. [18] found that the decrease in the level of SOD is an evidence of its increased activity due to the increased release of free radicals. MTX causes an increase in the release of free radicals, including the OH radical that causes direct damage to DNA [16]. Al-Helaly [19] showed that the amount of food taken has an effect on antioxidants, where nutritional deficiency decreases the antioxidant levels, thus increasing free radicals that cause damage to DNA. Table 2. Chromosomal aberrations of bone marrow cells in rats treated with MTX and MTX-omega-3 Group Type of chromosomal aberration(mean ± SE) Chromatid gap Chromosome Gap Chromatid breaks Chromosome breaks Deletion Simple Fragments Chromosomal aberration (%) Control 1.33±0.33 e 0.00±0.00 e 1.67±0.33 c 0.33±0.15 c 0.00±0.00 0.67±0.33 cd 0.04±0.005 f X1 2.75±0.47 cd 1.50±0.28 cd 2.50±0.64 bc 1.00±0.41 bc 0.50±0.28 bc 0.75±0.25 bcd 0.09±0.02 de Y1 1.75±0.47 de 0.75±0.25 de 1.50±0.28 c 1.00±0.00 bc 0.75±0.25 abc 0.75±0.25 abc 0.065±0.005 ef X2 4.67±0.33 b 2.67±0.33 ab 2.67±0.33 bc 1.67±0.33 ab 0.67±0.33 abc 1.67±0.33 ab 0.14±0.006 bc Y2 3.00±0.00 c 2.00±0.00 bc 3.00±0.057 bc 1.33±0.33 b 0.67±0.33 abc 0.33±0.15 d 0.106±0.003 cd X3 6.80±0.37 a 3.00±0.31 a 4.60±0.74 a 2.40±0.24 a 1.40±0.24 a 1.80±0.37 a 0.20±0.017 a Y3 5.60±0.40 ab 2.40±0.24 ab 3.60±0.24 ab 1.80±0.20 ab 1.20±0.20 ab 1.40±0.24 abc 0.16±0.003 b LSD 1.231** 0.814** 0.602** 0.841** 0.774* 0.941** 3.499* SE, Standard error; * statistically significant at P < 0.05; ** statistically significant at P < 0.01. Means with different letters within the same column showed a statistically significant difference. X1 (0.05 mg MTX/ kg); X2 (0.125 mg MTX/ kg); X3 (0.25 mg MTX/ kg); Y1 (0.05 mg MTX + 300 mg omega-3/ kg); Y2 (0.125 mg MTX + 300 mg omega-3/ kg); Y3 (0.25 mg MTX + 300 mg omega-3/ kg). In the present study, the intraperitoneal administration of MTX to rats also caused a decrease in the MI of bone marrow and a significant increase in the rate of abnormal chromosomal aberration compared to the control rats. This finding is consistent with those reported previously [20], [21]. The effect of MTX can be attributed to its ability to interfere with the genetic material, leading to the appearance of toxic and mutagenic consequences. Rushworth et al. [22] reported that MTX leads to a lack of dihydrofolate reductase, which is the key to the growth and cell division processes. This, in turn, leads to a reduction of the nucleotides involved in the building of DNA and, therefore, to a stop or obstruction of the repair mechanisms of the damaged DNA. In addition, Wong and Choi [23] concluded that MTX inhibits the action of enzymes controlling the purine metabolism, which leads to the accumulation of adenosine in addition to the damage of the molecule itself and to the occurrence of chromosomal aberrations. Jafer et al. [24] reported the ability of MTX to induce chromosomal aberration in humans or animals by preventing the repair of DNA and affecting the proteins found in chromosomes. These findings were also confirmed by Hussain et al. [25], who found that MTX causes an increase in chromosomal aberrations. In the present study, the MI showed a significant increase in rat sub-groups treated with MTX-omega-3 combination, but there was a decrease in the rate of chromosomal aberration, which confirms the role of omega-3 unsaturated fatty acids in protecting the cell from the impact of free radicals [26], [27]. Attia and Nasr [28] reported the antioxidant effect of omega-3, which was attributed to the reduction in lipid peroxidation and the increase in SOD and CAT or the stimulation of GR. It is noteworthy that GR leads to the synthesis of reduced glutathione, which is important in the defense of the cell against toxic substances and the prevention of the occurrence of mutations [29]. 5. Conclusions MTX significantly decreases the activity of enzymatic antioxidants, reduce the MI and increase the chromosomal aberrations of all types in bone marrow. This gives further evidence on the genotoxic effects of MTX on the bone marrow. On the other hand, omega-3 shows a protective effect by reducing the toxic and mutagenic effects of MTX. Acknowledgments The authors thank the staff of the Water and Environment Directorate, Ministry of Science and Technology, Baghdad, Iraq for their cooperation. They also thank Dr. Jasim Al-Niami for his technical and scientific guidance. Authors' contributions INA, MMA and ASM contributed to the study design and analyzed data. All authors contributed to the manuscript drafting and revising and approved the final submission. Competing interests The authors declare that they have no competing interests associated with this article. Ethical approval The ethical clearance of this study was obtained from the Ethics Committee of the College of Science, University of Anbar (Reference No. A. D. 51 in 30/8/2015). References Yuen CW, Winter ME. Methotrexate (MTX). In: Basic clinical pharmacokinetics, Winter ME, editor. Philadelphia, USA: Lippincott Williams & Wilkins; 2010. p.p. 304–25. Google Scholar Vezmar S, Becker A, Bode U, Jaehde U. Biochemical and clinical aspects of methotrexate neurotoxicity. Chemotherapy 2003; 49: 92–104. DOI PubMed - Google Scholar Tian H, Cronstein BN. Understanding the mechanisms of action of methotrexate implications for the treatment of rheumatoid arthritis. Bull NYU Hosp Jt Dis 2007; 65: 168–73. PubMed - Google Scholar El-Khayat Z, Rasheed WI, Elias T, Hussein J, Oraby F, Badawi M, et al. Protective effect of either dietary or pharmaceutical n-3 fatty acids on bone loss in ovariectomized rats. Maced J Med Sci 2010; 3: 9–16. DOI - Google Scholar Kris-Etherton PM, Harris WS, Appel LJ; Nutrition Committee. Fish consumption, fish oil, omega-3 fatty acids and cardiovascular disease. Arterioscler Thromb Vasc Biol 2003; 23: e20–30. DOI - PubMed - Google Scholar Calder PC. Polyunsaturated fatty acids and inflammation. Prostaglandins Leukot Essent Fatty Acids 2006; 75: 197–202. DOI - PubMed - Google Scholar Begin ME, Ells G, Das UN, Horrobin DF. Differential killing of human carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids. J Natl Cancer Inst 1986; 77: 1053–62. DOI - PubMed - Google Scholar Shan B, Cai YZ, Sun M, Corke H. Antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents. J Agric Food Chem. 2005; 53: 7749–59. DOI - PubMed - Google Scholar Matiax J, Quiles JL, Huertas JR, Battino M. Tissue specific interactions of exercise, dietary fatty acids, and vitamin E in lipid peroxidation. Free Radic Biol Med 1998; 24 : 511–21. DOI - PubMed - Google Scholar Dean RT, Fu S, Stocker R, Davies MJ. Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 1997; 324: 1–8. PubMed - Google Scholar Bashir A, Perham RN, Scrutton NS, Berry A. Altering Kinetic mechanism and enzyme stability by mutagenesis of the dimmer interface of glutathione reductase. Biochem J 1995; 312: 527–33. PubMed - Google Scholar Perret-Gentil MI. Rat Biomethodology. Laboratory Animal Resources Center. The University of Texas at San Antonio. [Cited 1 Feb. 2015]. Available from: https://www.utdallas.edu/research/docs/rat_biomethodology/ Allen JW, Shuler CF, Menders RW, Olatt SA. A simplified technique for in vivo analysis of sister chromatid exchange using 50 bromodeoxyuridine tablets. Cytogenet Cell Genet 1977; 18: 231–7. DOI PubMed - Google Scholar Forsum U, Hallén A. Acridine orange staining of urethral and cervical smears for the diagnosis of gonorrhea. Acta Derm Venereol 1979; 59: 281–2. PubMed - Google Scholar Statistical Analysis System user's guide. Version 9.1. Cary, NC, USA: SAS Institute Inc.; 2012. Daham HH, Rahim SM, Al-Hmesh MJ. The effect of radiotherapy and chemotherapy in several physiological and biochemical parameters in cancer patients. Tikrit J Pure Sci 2012; 17: 83–91. Weijl N, Elseendoorm TJ, Lentjes EG, Hopman CD, Wipkink-Bakker A, Zwinderman AH, et al. Supplementation with antioxidant micronutrients and chemotherapy-induced toxicity in cancer patients treated with cisplatin-based chemotherapy: a randomised, double-blind placebo-controlled study. Eur J Cancer 2004; 40: 1713–23. DOI - PubMed - Google Scholar Al-Dalawy SS, Al-Salehy FK, Al-Sanafi AI. Efficient enzymatic antioxidants for oxidative stress syndrome in patients with hypertension. J Dhi Qar Sci 2008; 2: 32–3. Al-Helaly LA. Some antioxidant enzymes in workers exposed to pollutants. Raf J Sci 2011; 22: 29–38. Google Scholar Othman GO. Protective effects of linseed oil against methotrexate induced genotoxicity in bone marrow cells of albino mice Mus musculus. ZJPAS. 2016; 28: 49–53. Google Scholar Ashoka CH, Vijayalaxmi KK. Cytogenetic effects of methotrexate in bone marrow cells of Swiss albino mice. Int J Sci Res Edu 2016; 4: 4828–34. 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Abstract Objective Preoperative chronic stress (CS) is associated with postoperative brain injury in patients undergoing open heart cardiac surgery. This research is to explore the potential molecular biological mechanisms of brain damage following cardiac surgery in preoperative CS rats by the analyses combining proteomics and metabolomics. Methods We constructed the chronic unpredictable stress (CUS) and cardiac surgery models in adult rats. We proved the brain injury in CUS cardiac surgery rats by Hematoxylin–Eosin (H&E) staining, followed by separating the hippocampal tissue and investigating the potential mechanisms of brain injury by the methods of data-independent acquisition proteomics and untargeted metabolomics. Results The signaling pathways of glycoproteins and metabolism of amino acids were the main possible mechanisms of brain injury in CUS rats following cardiac surgery according to the proteomics and metabolomics. In addition, the pathways of animo acids metabolism such as the pathways of lysine degradation and β-alanine metabolism may be the main mechanism of cardiac surgery related brain injury in preoperative CUS rats. Conclusions The pathways of animo acids metabolism such as lysine degradation and β-alanine metabolism may be the potential mechanisms of brain injury in CUS rats following cardiac surgery. We should focus on the varieties of bioproteins and metabolites in these pathways, and related changes in other signaling pathways induced by the two pathways.