Academic literature on the topic 'Other biomedical and clinical sciences not elsewhere classified'

Abstract Introduction Multiple Myeloma (MM) is considered a malignancy of post germinal center long-lived plasma cells. Nevertheless T-cell independent antigen stimulation before the exposure of the B-cell to the germinal center can happen and results to IgM secreting short lived plasma cells and lymphoplasmacytes representing thus a potential alternative normal counterpart for IgM plasma cell dyscrasias. IgM myeloma is an infrequent subtytpe of MM with an estimated prevalence of 0.5%. Due to its rarity little is known about its characteristics and prognosis in comparison with Waldestrom’s macroglobulinemia (WM) and the other MM subtypes. Purpose To identify the characteristics and the prognosis of IgM MM, and compare it predominantly with WM and subsequently with the rest of the MM subtypes. Methods We interogatted our Multiple Myeloma Data Base for cases of IgM MM and their respective Overall Survival (OS), Progression Free Survival (PFS), bone disease as defined by x-Rays, PET-CT and MRI, Gene Expression Profile (GEP), and common disease characteristics (anemia,calcium, creatinine) and compare it to the prognosis of WM and non-IgM MM. Diagnosis was based on the morphological and immunophenotypical findings of pathologically examined biopsy specimens along with the presence or not of typical clinical characteristics of MM (lytic bone lesions, hypercalcemia, renal failure) or typical clinical characteristics of WM (organomegaly, lymphadenopathy). Results There were 22 confirmed IgM MM cases. 14 of them presented at MIRT at initial diagnosis while 8 had previously been treated elsewhere. Osteolytic bone lesions and/or pathological fractures by x-ray and CT examination were evident in 16 cases. For the remaining 6 cases active bone focal lesions by either MRI or PET were identified in three. There was no organomegaly evident in cases with an available PET/CT at baseline, while only one had evidence of hilar and mediastinal lymphadenopathy along with calcified lung nodules. Elevated creatinine levels (>2.0 mg/dl) were evident in 4 cases at initial diagnosis. Their disease characteristics are depicted in the table 1. Median OS for IgM MM was 4.9 years while PFS could not be accurately estimated due to lack of data on patients treated elsewhere. Median OS for a historical control of 158 WM cases in MIRT was 9.2 years (Clin Lymphoma Myeloma Leuk. 11(1):139-42). Median OS of the WM group remained largely unaffected, even when the subgroup of the WM cases requiring treatment was analyzed (9.0 years).To further clarify if the IgM MM differs in terms of OS from the other isotypes of MM, we compared the IgM group to a group of 61 non-IgM MM cases which were matched by important prognostic clinical factors (age, creatinine> 2mg/dl, LDH>190u/L, b-2M >5.5mg/dl and Albumin<3.5gr/dl). No statistical difference was found for OS (p=0.846). Out of 22 cases, 14 of them had available GEP data on initial diagnosis. In 6 of these cases the cyclin D1 gene expression was high enough to be consistent with a t(11;14) translocation at FISH analysis, one case was consistent with a t(14;16) translocation, one with a t(4;14) translocation and two more were classified as belonging to the hyperdiploid subgroup. A comparative genomic analysis was performed on the IgM MM, the non-IgM MM and WM cases with available GEP data at initial diagnosis (14, 61 and 42 cases respectively). 1155 probesets that had expression level significantly different between WM and non IgM MM (FDR<3E-06) were identified. Then, the expression values of these 1155 probesets in all GEP samples, including WM, non IgM MM, and IgM MM, were used to build a clustering tree. We found that IgM MM mainly clustered with non IgM MM, supporting the findings of the clinical data. Conclusion IgM MM is a discrete clinical entity that should be distinguished from WM. Bone disease is evident in the majority of the cases, especially when specialized radiological techniques are incorporated at the initial work up. It holds a distinct prognosis from WM, while when balanced for prognostic factors that hold importance in MM it does not differ from the other MM isotypes. Finally analysis of the genetic data further supports the resemblance between IgM MM and the non IgM MM, and the difference with WM. Disclosures: Zhang: University of Arkansas for Medical Sciences: Co-inventor of the DNA probes for FISH of IGHC/IGHV (14q32), MMSET/FGFR3 (4p16), CCND3 (6p21), CCND1 (11q13), MAF (16q23), and MAFB (20q12) loci, sub. to the US Patent & Trademark Office as Prov. App# 61/726,327: Methods of Detecting 14q32 Translocations, Co-inventor of the DNA probes for FISH of IGHC/IGHV (14q32), MMSET/FGFR3 (4p16), CCND3 (6p21), CCND1 (11q13), MAF (16q23), and MAFB (20q12) loci, sub. to the US Patent & Trademark Office as Prov. App# 61/726,327: Methods of Detecting 14q32 Translocations Patents & Royalties.

Abstract Objectives Effective wound remodeling (WR) is a significant challenge associated with chronic wounds such as diabetic ulcers. Bioactive compounds such as polyphenols have been documented to influence wound healing. Previously, our lab recorded that a phenolic acid extract (PE) from Wild Blueberry (WB) promotes wound closure by promoting endothelial cell migration and angiogenesis. Still, the in vivo effect on WR for clinical translation is unknown. Thus, the objectives of this study are to examine the pre-clinical effect of phenolic extract (PE) from wild blueberries (WB) on histological and molecular pathways of tissue remodeling on rats' wounds. Methods Phenolics were extracted from WB through the Folin-Ciocalteu method. The extract was then incorporated into a gel and a cream base. Fifty-six Sprague-Dawley rats were classified into seven groups as follows: Group 1, Control (no treatment), Group 2, the cream carrier without the PE, Group 3, gel carrier, without PE, Group 4, gel with 500 ug/ml of the PE, Group 5, cream with 500 ug/ml of PE, Group 6, gel with 1000 ug/ml of PE, and Group 7, cream with 1000 ug/ml of PE. Dorsal wounds were created on all the rats and treated according to the above groups for six days. Skin tissues were excised and fixed for molecular analysis and Masson trichrome staining. Stained tissues were visualized and quantified under the light microscope for the pattern of collagen deposition as wound remodeling. Results Histology analysis showed a significant increase in the organized pattern of collagen deposition with the gel-PE 500 ug/ml treatment compared with the other groups. Currently, we are analyzing results for genes and proteins associated with the observed collagen deposition. Conclusions Our innovative research will elucidate the potential of PE to modulate collagen-mediated wound remodeling. Results from this project may benefit patients with chronic wounds through the clinical development of PE in gel carriers as a wound-promoting treatment. Funding Sources USDA National Institute of Food and Agriculture, The Wild Blueberry Association of North America, The Maine Technology Institute, The University of Maine Medicine Seed Grant Program, The Maine Innovation Research Technology Accelerator, i-CORPS NSF, The Graduate School of Biomedical Sciences and Engineering.

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. 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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. 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Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).Key word: Salt Lake, Bacteria, biosurfactant, Chott, halophiles, hydrolases, 16S rRNAINTRODUCTIONSaline lakes cover approximately 10% of the Earth’s surface area. The microbial populations of many hypersaline environments have already been studied in different geographical regions such as Great Salt Lake (USA), Dead Sea (Israel), Wadi Natrun Lake (Egypt), Lake Magadi (Kenya), Soda Lake (Antarctica) and Big Soda Lake and Mono Lake (California). Hypersaline regions differ from each other in terms of geographical location, salt concentration and chemical composition, which determine the nature of inhabitant microorganisms (Gupta et al., 2015). Then low taxonomic diversity is common to all these saline environments (Oren et al., 1993). Halophiles are found in nearly all major microbial clades, including prokaryotic (Bacteria and Archaea) and eukaryotic forms (DasSarma and Arora, 2001). They are classified as slight halophiles when they grow optimally at 0.2–0.85 M (2–5%) NaCl, as moderate halophiles when they grow at 0.85–3.4 M (5–20%) NaCl, and as extreme halophiles when they grow at 3.4–5.1 M (20–30%) NaCl. Hyper saline environments are inhabited by extremely halophilic and halotolerant microorganisms such as Halobacillus sp, Halobacterium sp., Haloarcula sp., Salinibacter ruber , Haloferax sp and Bacillus spp. (Solomon and Viswalingam, 2013). There is a tremendous demand for halophilic bacteria due to their biotechnological importance as sources of halophilic enzymes. Enzymes derived from halophiles are endowed with unique structural features and catalytic power to sustain the metabolic and physiological processes under high salt conditions. Some of these enzymes have been reported to be active and stable under more than one extreme condition (Karan and Khare, 2010). Applications are being considered in a range of industries such as food processing, washing, biosynthetic processes and environmental bioremediation. Halophilic proteases are widely used in the detergent and food industries (DasSarma and Arora, 2001). However, esterases and lipases have also been useful in laundry detergents for the removal of oil stains and are widely used as biocatalysts because of their ability to produce pure compounds. Likewise, amylases are used industrially in the first step of the production of high fructose corn syrup (hydrolysis of corn starch). They are also used in the textile industry in the de-sizing process and added to laundry detergents. Furthermore, for the environmental applications, the use of halophiles for bioremediation and biodegradation of various materials from industrial effluents to soil contaminants and accidental spills are being widely explored. In addition to enzymes, halophilic / halotolerants microorganisms living in saline environments, offer another potential applications in various fields of biotechnology like the production of biosurfactant. Biosurfactants are amphiphilic compounds synthesized from plants and microorganisms. They reduce surface tension and interfacial tension between individual molecules at the surface and interface respectively (Akbari et al., 2018). Comparing to the chemical surfactant, biosurfactant are promising alternative molecules due to their low toxicity, high biodegradability, environmental capability, mild production conditions, lower critical micelle concentration, higher selectivity, availability of resources and ability to function in wide ranges of pH, temperature and salinity (Rocha et al., 1992). They are used in various industries which include pharmaceuticals, petroleum, food, detergents, cosmetics, paints, paper products and water treatment (Akbari et al., 2018). The search for biosurfactants in extremophiles is particularly promising since these biomolecules can adapt and be stable in the harsh environments in which they are to be applied in biotechnology.OBJECTIVESEastern Algeria features numerous ecosystems including hypersaline environments, which are an important source of salt for food. The microbial diversity in Chott Tinsilt, a shallow Salt Lake with more than 200g/L salt concentration and a superficies of 2.154 Ha, has never yet been studied. The purpose of this research was to chemically analyse water samples collected from the Chott, isolate novel extremely or moderate halophilic Bacteria, and examine their phenotypic and phylogenetic characteristics with a view to screening for biosurfactants and enzymes of industrial interest.MATERIALS AND METHODSStudy area: The area is at 5 km of the Commune of Souk-Naâmane and 17 km in the South of the town of Aïn-Melila. This area skirts the trunk road 3 serving Constantine and Batna and the railway Constantine-Biskra. It is part the administrative jurisdiction of the Wilaya of Oum El Bouaghi. The Chott belongs to the wetlands of the High Plains of Constantine with a depth varying rather regularly without never exceeding 0.5 meter. Its length extends on 4 km with a width of 2.5 km (figure 1).Water samples and physico-chemical analysis: In February 2013, water samples were collected from various places at the Chott Tinsilt using Global Positioning System (GPS) coordinates of 35°53’14” N lat. and 06°28’44”E long. Samples were collected randomly in sterile polythene bags and transported immediately to the laboratory for isolation of halophilic microorganisms. All samples were treated within 24 h after collection. Temperature, pH and salinity were measured in situ using a multi-parameter probe (Hanna Instruments, Smithfield, RI, USA). The analytical methods used in this study to measure ions concentration (Ca2+, Mg2+, Fe2+, Na+, K+, Cl−, HCO3−, SO42−) were based on 4500-S-2 F standard methods described elsewhere (Association et al., 1920).Isolation of halophilic bacteria from water sample: The media (M1) used in the present study contain (g/L): 2.0 g of KCl, 100.0/200.0 g of NaCl, 1.0 g of MgSO4.7HO2, 3.0 g of Sodium Citrate, 0.36 g of MnCl2, 10.0 g of yeast extract and 15.0 g agar. The pH was adjusted to 8.0. Different dilutions of water samples were added to the above medium and incubated at 30°C during 2–7 days or more depending on growth. Appearance and growth of halophilic bacteria were monitored regularly. The growth was diluted 10 times and plated on complete medium agar (g/L): glucose 10.0; peptone 5.0; yeast extract 5.0; KH2PO4 5.0; agar 30.0; and NaCl 100.0/200.0. Resultant colonies were purified by repeated streaking on complete media agar. The pure cultures were preserved in 20% glycerol vials and stored at −80°C for long-term preservation.Biochemical characterisation of halophilic bacterial isolates: Bacterial isolates were studied for Gram’s reaction, cell morphology and pigmentation. Enzymatic assays (catalase, oxidase, nitrate reductase and urease), and assays for fermentation of lactose and mannitol were done as described by Smibert (1994).Optimization of growth conditions: Temperature, pH, and salt concentration were optimized for the growth of halophilic bacterial isolates. These growth parameters were studied quantitatively by growing the bacterial isolates in M1 medium with shaking at 200 rpm and measuring the cell density at 600 nm after 8 days of incubation. To study the effect of NaCl on the growth, bacterial isolates were inoculated on M1 medium supplemented with different concentration of NaCl: 1%-35% (w/v). The effect of pH on the growth of halophilic bacterial strains was studied by inoculating isolates on above described growth media containing NaCl and adjusted to acidic pH of 5 and 6 by using 1N HCl and alkaline pH of 8, 9, 10, 11 and 12 using 5N NaOH. The effect of temperature was studied by culturing the bacterial isolates in M1 medium at different temperatures of incubation (4°C–55°C).Screening of halophilic bacteria for hydrolytic enzymes: Hydrolase producing bacteria among the isolates were screened by plate assay on starch, tributyrin, gelatin and DNA agar plates respectively for amylase, lipase, protease and DNAse activities. Amylolytic activity of the cultures was screened on starch nutrient agar plates containing g/L: starch 10.0; peptone 5.0; yeast extract 3.0; agar 30.0; NaCl 100.0/250.0. The pH was 7.0. After incubation at 30 ºC for 7 days, the zone of clearance was determined by flooding the plates with iodine solution. The potential amylase producers were selected based on ratio of zone of clearance diameter to colony diameter. Lipase activity of the cultures was screened on tributyrin nutrient agar plates containing 1% (v/v) of tributyrin. Isolates that showed clear zones of tributyrin hydrolysis were identified as lipase producing bacteria. Proteolytic activity of the isolates was similarly screened on gelatin nutrient agar plates containing 10.0 g/L of gelatin. The isolates showing zones of gelatin clearance upon treatment with acidic mercuric chloride were selected and designated as protease producing bacteria. The presence of DNAse activity on plates was determined on DNAse test agar (BBL) containing 10%-25% (w/v) total salt. After incubation for 7days, the plates were flooded with 1N HCl solution. Clear halos around the colonies indicated DNAse activity (Jeffries et al., 1957).Milk clotting activity (coagulase activity) of the isolates was also determined following the procedure described (Berridge, 1952). Skim milk powder was reconstituted in 10 mM aqueous CaCl2 (pH 6.5) to a final concentration of 0.12 kg/L. Enzyme extracts were added at a rate of 0.1 mL per mL of milk. The coagulation point was determined by manual rotating of the test tube periodically, at short time intervals, and checking for visible clot formation.Screening of halophilic bacteria for biosurfactant production. Oil spread Assay: The Petridis base was filled with 50 mL of distilled water. On the water surface, 20μL of diesel and 10μl of culture were added respectively. The culture was introduced at different spots on the diesel, which is coated on the water surface. The occurrence of a clear zone was an indicator of positive result (Morikawa et al., 2000). The diameter of the oil expelling circles was measured by slide caliber (with a degree of accuracy of 0.02 mm).Surface tension and emulsification index (E24): Isolates were cultivated at 30 °C for 7 days on the enrichment medium containing 10-25% NaCl and diesel oil as the sole carbon source. The medium was centrifuged (7000 rpm for 20 min) and the surface tension of the cell-free culture broth was measured with a TS90000 surface tensiometer (Nima, Coventry, England) as a qualitative indicator of biosurfactant production. The culture broth was collected with a Pasteur pipette to remove the non-emulsified hydrocarbons. The emulsifying capacity was evaluated by an emulsification index (E24). The E24 of culture samples was determined by adding 2 mL of diesel oil to the same amount of culture, mixed for 2 min with a vortex, and allowed to stand for 24 h. E24 index is defined as the percentage of height of emulsified layer (mm) divided by the total height of the liquid column (mm).Biosurfactant stability studies : After growth on diesel oil as sole source of carbone, cultures supernatant obtained after centrifugation at 6,000 rpm for 15 min were considered as the source of crude biosurfactant. Its stability was determined by subjecting the culture supernatant to various temperature ranges (30, 40, 50, 60, 70, 80 and 100 °C) for 30 min then cooled to room temperature. Similarly, the effect of different pH (2–11) on the activity of the biosurfactant was tested. The activity of the biosurfactant was investigated by measuring the emulsification index (El-Sersy, 2012).Molecular identification of potential strains. DNA extraction and PCR amplification of 16S rDNA: Total cellular DNA was extracted from strains and purified as described by Sambrook et al. (1989). DNA was purified using Geneclean® Turbo (Q-BIO gene, Carlsbad, CA, USA) before use as a template in polymerase chain reaction (PCR) amplification. For the 16S rDNA gene sequence, the purified DNA was amplified using a universal primer set, forward primer (27f; 5′-AGA GTT TGA TCM TGG CTC AG) and a reverse primer (1492r; 5′-TAC GGY TAC CTT GTT ACG ACT T) (Lane, 1991). Agarose gel electrophoresis confirmed the amplification product as a 1400-bp DNA fragment.16S rDNA sequencing and Phylogenic analysis: Amplicons generated using primer pair 27f-1492r was sequenced using an automatic sequencer system at Macrogene Company (Seoul, Korea). The sequences were compared with those of the NCBI BLAST GenBank nucleotide sequence databases. Phylogenetic trees were constructed by the neighbor-joining method using MEGA version 5.05 software (Tamura et al., 2011). Bootstrap resembling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies.Nucleotide sequence accession numbers: The nucleotide sequences reported in this work have been deposited in the EMBL Nucleotide Sequence Database. The accession numbers are represented in table 5.Statistics: All experiments were conducted in triplicates. Results were evaluated for statistical significance using ANOVA.RESULTSPhysico-chemical parameters of the collected water samples: The physicochemical properties of the collected water samples are reported in table 1. At the time of sampling, the temperature was 10.6°C and pH 7.89. The salinity of the sample, as determined in situ, was 224.70 g/L (22,47% (w/v)). Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions (table 1). SO4-2 and Mg+2 was present in much smaller amounts compared to Na +and Cl- concentration. Low levels of calcium, potassium and bicarbonate were also detected, often at less than 1 g/L.Characterization of isolates. Morphological and biochemical characteristic feature of halophilic bacterial isolates: Among 52 strains isolated from water of Chott Tinsilt, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for further characterization (table 2). The colour of the isolates varied from beige, pale yellow, yellowish and orange. The bacterial isolates A1, A2, A4, B1 and B5 were rod shaped and gram negative (except B5), whereas A3 and B4 were cocci and gram positive. All strains were oxidase and catalase positive except for B1. Nitrate reductase and urease activities were observed in all the bacterial isolates, except B4. All the bacterial isolates were negative for H2S formation. B5 was the only strain positive for mannitol fermentation (table 2).We isolated halophilic bacteria on growth medium with NaCl supplementation at pH 7 and temperature of 30°C. We studied the effect of NaCl, temperature and pH on the growth of bacterial isolates. All the isolates exhibited growth only in the presence of NaCl indicating that these strains are halophilic. The optimum growth of isolates A3 and B1 was observed in the presence of 10% NaCl, whereas it was 15% NaCl for A1, A2 and B5. A4 and B4 showed optimum growth in the presence of 20% and 25% NaCl respectively. A4, B4 and B5 strains can tolerate up to 35% NaCl.The isolate B1 showed growth in medium supplemented with 10% NaCl and pH range of 7–10. The optimum pH for the growth B1 was 9 and they did not show any detectable growth at or below pH 6 (table 2), which indicates the alkaliphilic nature of B1 isolate. The bacterial isolates A1, A2 and A4 exhibited growth in the range of pH 6–10, while A3 and B4 did not show any growth at pH greater than 8. The optimum pH for growth of all strains (except B1) was pH 7.0 (table 2). These results indicate that A1, A2, A3, A4, B4 and B5 are neutrophilic in nature. All the bacterial isolates exhibited optimal growth at 30°C and no detectable growth at 55°C. Also, detectable growth of isolates A1, A2 and A4 was observed at 4°C. However, none of the bacterial strains could grow below 4°C and above 50°C (table 2).Screening of the halophilic enzymes: To characterize the diversity of halophiles able to produce hydrolytic enzymes among the population of microorganisms inhabiting the hypersaline habitats of East Algeria (Chott Tinsilt), a screening was performed. As described in Materials and Methods, samples were plated on solid media containing 10%-25% (w/v) of total salts and different substrates for the detection of amylase, protease, lipase and DNAse activities. However, coagulase activity was determined in liquid medium using milk as substrate (figure 3). Distributions of hydrolytic activity among the isolates are summarized in table 4.From the seven bacterial isolates, four strains A1, A2, A4 and B5 showed combined hydrolytic activities. They were positive for gelatinase, lipase and coagulase. A3 strain showed gelatinase and lipase activities. DNAse activities were detected with A1, A4, B1 and B5 isolates. B4 presented lipase and coagulase activity. Surprisingly, no amylase activity was detected among all the isolates.Screening for biosurfactant producing isolates: Oil spread assay: The results showed that all the strains could produce notable (>4 cm diameter) oil expelling circles (ranging from 4.11 cm to 4.67 cm). The average diameter for strain B5 was 4.67 cm, significantly (P < 0.05) higher than for the other strains.Surface tension and emulsification index (E24): The assimilation of hydrocarbons as the sole sources of carbon by the isolate strains led to the production of biosurfactants indicated by the emulsification index and the lowering of the surface tension of cell-free supernatant. Based on rapid growth on media containing diesel oil as sole carbon source, the seven isolates were tested for biosurfactant production and emulsification activity. The obtained values of the surface tension measurements as well as the emulsification index (E24) are shown in table 3. The highest reduction of surface tension was achieved with B5 and A3 isolates with values of 25.3 mN m−1 and 28.1 mN m−1 respectively. The emulsifying capacity evaluated by the E24 emulsification index was highest in the culture of isolate B4 (78%), B5 (77%) and A3 (76%) as shown in table 3 and figure 2. These emulsions were stable even after 4 months. The bacteria with emulsification indices higher than 50 % and/or reduction in the surface tension (under 30 mN/m) have been defined as potential biosurfactant producers. Based on surface tension and the E24 index results, isolates B5, B4, A3 and A4 are the best candidates for biosurfactant production. It is important to note that, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was choosen for futher analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4.biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was chosen for further analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4. The biosurfactant produced by this strain was shown to be thermostable giving an E-24 Index value greater than 78% (figure 4A). Heating of the biosurfactant to 100 °C caused no significant effect on the biosurfactant performance. Therefore, the surface activity of the crude biosurfactant supernatant remained relatively stable to pH changes between pH 6 and 11. At pH 11, the value of E24 showed almost 76% activity, whereas below pH 6 the activity was decreased up to 40% (figure 4A). The decreases of the emulsification activity by decreasing the pH value from basic to an acidic region; may be due to partial precipitation of the biosurfactant. This result indicated that biosurfactant produced by strain B4 show higher stability at alkaline than in acidic conditions.Molecular identification and phylogenies of potential isolates: To identify halophilic bacterial isolates, the 16S rDNA gene was amplified using gene-specific primers. A PCR product of ≈ 1.3 kb was detected in all the seven isolates. The 16S rDNA amplicons of each bacterial isolate was sequenced on both strands using 27F and 1492R primers. The complete nucleotide sequence of 1336,1374, 1377,1313, 1305,1308 and 1273 bp sequences were obtained from A1, A2, A3, A4, B1, B4 and B5 isolates respectively, and subjected to BLAST analysis. The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus as shown in table 5. The halophilic isolates A2 and A4 showed 97% similarity with the Halomonas variabilis strain GSP3 (accession no. AY505527) and the Halomonas sp. M59 (accession no. AM229319), respectively. As for A1, it showed 96% similarity with the Halomonas venusta strain GSP24 (accession no. AY553074). B1 and B4 showed for their part 96% similarity with the Salinivibrio costicola subsp. alcaliphilus strain 18AG DSM4743 (accession no. NR_042255) and the Planococcus citreus (accession no. JX122551), respectively. The bacterial isolate B5 showed 98% sequence similarity with the Halobacillus trueperi (accession no. HG931926), As for A3, it showed only 95% similarity with the Staphylococcus arlettae (accession no. KR047785). The 16S rDNA nucleotide sequences of all the seven halophilic bacterial strains have been submitted to the NCBI GenBank database under the accession number presented in table 5. The phylogenetic association of the isolates is shown in figure 5.DICUSSIONThe physicochemical properties of the collected water samples indicated that this water was relatively neutral (pH 7.89) similar to the Dead Sea and the Great Salt Lake (USA) and in contrast to the more basic lakes such as Lake Wadi Natrun (Egypt) (pH 11) and El Golea Salt Lake (Algeria) (pH 9). The salinity of the sample was 224.70 g/L (22,47% (w/v). This range of salinity (20-30%) for Chott Tinsilt is comparable to a number of well characterized hypersaline ecosystems including both natural and man-made habitats, such as the Great Salt Lake (USA) and solar salterns of Puerto Rico. Thus, Chott Tinsilt is a hypersaline environment, i.e. environments with salt concentrations well above that of seawater. Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions, as in most hypersaline ecosystems (with some exceptions such as the Dead Sea). These chemical water characteristics were consistent with the previously reported data in other hypersaline ecosystems (DasSarma and Arora, 2001; Oren, 2002; Hacěne et al., 2004). Among 52 strains isolated from this Chott, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for phenotypique, genotypique and phylogenetique characterization.The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus. Genera obtained in the present study are commonly occurring in various saline habitats across the globe. Staphylococci have the ability to grow in a wide range of salt concentrations (Graham and Wilkinson, 1992; Morikawa et al., 2009; Roohi et al., 2014). For example, in Pakistan, Staphylococcus strains were isolated from various salt samples during the study conducted by Roohi et al. (2014) and these results agreed with previous reports. Halomonas, halophilic and/or halotolerant Gram-negative bacteria are typically found in saline environments (Kim et al., 2013). The presence of Planococcus and Halobacillus has been reported in studies about hypersaline lakes; like La Sal del Rey (USA) (Phillips et al., 2012) and Great Salt Lake (Spring et al., 1996), respectively. The Salinivibrio costicola was a representative model for studies on osmoregulatory and other physiological mechanisms of moderately halophilic bacteria (Oren, 2006).However, it is interesting to note that all strains shared less than 98.7% identity (the usual species cut-off proposed by Yarza et al. (2014) with their closest phylogenetic relative, suggesting that they could be considered as new species. Phenotypic, genetic and phylogenetic analyses have been suggested for the complete identification of these strains. Theses bacterial strains were tested for the production of industrially important enzymes (Amylase, protease, lipase, DNAse and coagulase). These isolates are good candidates as sources of novel enzymes with biotechnological potential as they can be used in different industrial processes at high salt concentration (up to 25% NaCl for B4). Prominent amylase, lipase, protease and DNAase activities have been reported from different hypersaline environments across the globe; e.g., Spain (Sánchez‐Porro et al., 2003), Iran (Rohban et al., 2009), Tunisia (Baati et al., 2010) and India (Gupta et al., 2016). However, to the best of our knowledge, the coagulase activity has never been detected in extreme halophilic bacteria. Isolation and characterization of crude enzymes (especially coagulase) to investigate their properties and stability are in progress.The finding of novel enzymes with optimal activities at various ranges of salt concentrations is of great importance. Besides being intrinsically stable and active at high salt concentrations, halophilic and halotolerant enzymes offer great opportunities in biotechnological applications, such as environmental bioremediation (marine, oilfiel) and food processing. The bacterial isolates were also characterized for production of biosurfactants by oil-spread assay, measurement of surface tension and emulsification index (E24). There are few reports on biosurfactant producers in hypersaline environments and in recent years, there has been a greater increase in interest and importance in halophilic bacteria for biomolecules (Donio et al., 2013; Sarafin et al., 2014). Halophiles, which have a unique lipid composition, may have an important role to play as surface-active agents. The archae bacterial ether-linked phytanyl membrane lipid of the extremely halophilic bacteria has been shown to have surfactant properties (Post and Collins, 1982). Yakimov et al. (1995) reported the production of biosurfactant by a halotolerant Bacillus licheniformis strain BAS 50 which was able to produce a lipopeptide surfactant when cultured at salinities up to 13% NaCl. From solar salt, Halomonas sp. BS4 and Kocuria marina BS-15 were found to be able to produce biosurfactant when cultured at salinities of 8% and 10% NaCl respectively (Donio et al., 2013; Sarafin et al., 2014). In the present work, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% NaCl. To our knowledge, this is the first report on biosurfactant production by bacteria under such salt concentration. Biosurfactants have a wide variety of industrial and environmental applications (Akbari et al., 2018) but their applicability depends on their stability at different environmental conditions. The strain B4 which can produce biosurfactant at 25% NaCl showed good stability in alkaline pH and at a temperature range of 30°C-100°C. Due to the enormous utilization of biosurfactant in detergent manufacture the choice of alkaline biosurfactant is researched (Elazzazy et al., 2015). On the other hand, the interesting finding was the thermostability of the produced biosurfactant even after heat treatment (100°C for 30 min) which suggests the use of this biosurfactant in industries where heating is of a paramount importance (Khopade et al., 2012). To date, more attention has been focused on biosurfactant producing bacteria under extreme conditions for industrial and commercial usefulness. In fact, the biosurfactant produce by strain B4 have promising usefulness in pharmaceutical, cosmetics and food industries and for bioremediation in marine environment and Microbial enhanced oil recovery (MEOR) where the salinity, temperature and pH are high.CONCLUSIONThis is the first study on the culturable halophilic bacteria community inhabiting Chott Tinsilt in Eastern Algeria. Different genera of halotolerant bacteria with different phylogeneticaly characteristics have been isolated from this Chott. Culturing of bacteria and their molecular analysis provides an opportunity to have a wide range of cultured microorganisms from extreme habitats like hypersaline environments. Enzymes produced by halophilic bacteria show interesting properties like their ability to remain functional in extreme conditions, such as high temperatures, wide range of pH, and high salt concentrations. These enzymes have great economical potential in industrial, agricultural, chemical, pharmaceutical, and biotechnological applications. Thus, the halophiles isolated from Chott Tinsilt offer an important potential for application in microbial and enzyme biotechnology. In addition, these halo bacterial biosurfactants producers isolated from this Chott will help to develop more valuable eco-friendly products to the pharmacological and food industries and will be usefulness for bioremediation in marine environment and petroleum industry.ACKNOWLEDGMENTSOur thanks to Professor Abdelhamid Zoubir for proofreading the English composition of the present paper.CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.Akbari, S., N. H. Abdurahman, R. M. Yunus, F. Fayaz and O. R. Alara, 2018. Biosurfactants—a new frontier for social and environmental safety: A mini review. Biotechnology research innovation, 2(1): 81-90.Association, A. P. H., A. W. W. Association, W. P. C. 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Background: Reperfusion following ischemia can lead to more injuries than ischemia itself especially in diabetic patients. The aim of this study was to evaluate the effect of dexmedetomidine on ischemia-reperfusion injury (IRI) in rats with have hepatic IRI and diabetes mellitus. Methodology: Twenty-eight Wistar Albino rats were randomised into four groups as control (C), diabetic (DC), diabetic with hepatic ischemia-reperfusion injury (DIR), and diabetic but administered dexmedetomidine followed by hepatic IRI (DIRD) groups. Hepatic tissue samples were evaluated histopathologically by semiquantitative methods. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathion s-transpherase (GST), and catalase (CAT) enzyme levels were investigated in liver and kidney tissues as oxidative state parameters. Results: In Group DIR; hepatocyte degeneration, sinusoidal dilatation, pycnotic nucleus, and necrotic cells were found to be more in rat hepatic tissue; while mononuclear cell infiltration was higher in the parenchyme. MDA levels were significantly lower; but SOD levels were significantly higher in Group DIRD with regard to Group DIR. In the IRI induced diabetic rats’ hepatic and nephrotic tissues MDA levels, showing oxidative injury, were found to be lower. SOD levels, showing early antioxidant activity, were higher. Conclusion: The enzymatic findings of our study together with the hepatic histopathology indicate that dexmedetomidine has a potential role to decrease IRI. Key words: Hepatic ischemia reperfusion injury; Diabetes mellitus; Dexmedetomidine; Rat; MDA; SOD Citation: Sezen SC, Işık B, Bilge M, Arslan M, Çomu FM, Öztürk L, Kesimci E, Kavutçu M. Effect of dexmedetomidine on ischemia-reperfusion injury of liver and kidney tissues in experimental diabetes and hepatic ischemia-reperfusion injury induced rats. Anaesth Pain & Intensive Care 2016;20(2):143-149 Received: 21 November 2015; Reviewed: 10, 24 December 2015, 9, 10 June 2016; Corrected: 12 December 2015; Accepted: 10 June 2016 INTRODUCTİON Perioperative acute tissue injury induced by ischemia-reperfusion is a comman clinical event caused by reduced blood supply to the tissue being compromised during major surgery. Ischemia leads to cellular injury by depleting cellular energy deposits and resulting in accumulation of toxic metabolites. The reperfusion of tissues that have remained in ischemic conditions causes even more damage.1 Furthermore hepatic ischemia-reperfusion injury (IRI) demonstrates a strong relationship with peri-operative acute kidney injury.2 The etiology of diabetic complications is strongly associated with increased oxidative stress (OS). Diabetic patients are known to have a high risk of developing OS or IRI which results with tissue failure.3 The most important role in ischemia and reperfusion is played by free oxygen radicals.1 In diabetes, characterized by hyperglycemia, even more free oxygen radicals are produced due to oxidation of glucose and glycosylation of proteins.3 The structures which are most sensitive to free oxygen radicals in the cells are membrane lipids, proteins, nucleic acids and deoxyribonucleic acids.1 It has been reported that endogenous antioxidant enzymes [superoxide dismutase (SOD), glutathion s-transpherase (GST), catalase (CAT)] play an important role to alleviate IRI.4-8 Also some pharmacological agents have certain effects on IRI.1 The anesthetic agents influence endogenous antioxidant systems and free oxygen radical formation.9-12 Dexmedetomidine is a selective α-2 adrenoceptor agonist agent. It has been described as a useful and safe adjunct in many clinical applications. It has been found that it may increase urine output by considerably redistributing cardiac output, inhibiting vasopressin secretion and maintaining renal blood flow and glomerular filtration. Previous studies demonstrated that dexmedetomidine provides protection against renal, focal cerebral, cardiac, testicular, and tourniquet-induced IRI.13-18 Arslan et al observed that dexmedetomidine protected against lipid peroxidation and cellular membrane alterations in hepatic IRI, when given before induction of ischemia.17 Si et al18 demonstrated that dexmedetomidine treatment results in a partial but significant attenuation of renal demage induced by IRI through the inactivation of JAK/STAT signaling pathway in an in vivo model. The efficacy of the dexmedetomidine for IRI in diabetic patient is not resarched yet. The purpose of this experimental study was to evaluate the biochemical and histological effects of dexmedetomidine on hepatic IRI in diabetic rat’s hepatic and renal tissue. METHODOLOGY Animals and Experimental Protocol: This study was conducted in the Physiology Laboratory of Kirikkale University upon the consent of the Experimental Animals Ethics Committee of Kirikkale University. All of the procedures were performed according to the accepted standards of the Guide for the Care and Use of Laboratory Animals. In the study, 28 male Wistar Albino rats, weighing between 250 and 300 g, raised under the same environmental conditions, were used. The rats were kept under 20-21 oC at cycles of 12-hour daylight and 12-hour darkness and had free access to food until 2 hours before the anesthesia procedure. The animals were randomly separated into four groups, each containing 7 rats. Diabetes was induced by a single intraperitoneal injection of streptozotocin (Sigma Chemical, St. Louis, MO, USA) at a dose of 65 mg/kg body weight. The blood glucose levels were measured at 72 hrs following this injection. Rats were classified as diabetic if their fasting blood glucose (FBG) levels exceeded 250 mg/dl, and only animals with FBGs of > 250 mg/dl were included in the diabetic groups (dia­betes only, diabetes plus ischemia-reperfusion and diabetes plus dexmedetomidine-ischemia-reperfusion). The rats were kept alive 4 weeks after streptozotocin injection to allow development of chronic dia­betes before they were exposed to ischemia-reperfusion.(19) The rats were weighed before the study. Rats were anesthetized with intraperitoneal ketamine 100 mg/kg. The chest and abdomen were shaved and each animal was fixed in a supine position on the operating table. The abdomen was cleaned with 1% polyvinyl iodine and when dry, the operating field was covered with a sterile drape and median laparotomy was performed. There were four experimental groups (Group C (sham-control; n = 7), (Group DC (diabetes-sham-control; n = 7), Group DIR (diabetes-ischemia-reperfusion; n = 7), and Group DIRD (diabetes-ischemia-reperfusion-dexmedetomidine; n = 7). Sham operation was performed on the rats in Group C and Group DC. The sham operation consisted of mobilization of the hepatic pedicle only. The rats in this group were sacrificed 90 min after the procedure. Hepatic I/R injury was induced in Groups DIR and DIRD respectively with hepatic pedicle clamping using a vascular clamp as in the previous study of Arslan et al.(17) After an ischemic period of 45 min, the vascular clamp was removed. A reperfusion period was maintained for 45 min. In Group DIRD, dexmedetomidine hydrochloride 100 μg/kg, (Precedex 100 μg/2 ml, Abbott®, Abbott Laboratory, North Chicago, Illinois, USA) was administrated via intraperitoneal route 30 minutes before surgery. All the rats were given ketamine 100 mg/kg intraperitoneally and intracardiac blood samples were obtained. Preserving the tissue integrity by avoiding trauma, liver and renal biopsy samples were obtained. Biochemical Analysis: The liver and renal tissues were first washed with cold deionized water to discard blood contamination and then homogenized in a homogenizer. Measurements on cell contest require an initial preparation of the tissues. The preparation procedure may involve grinding of the tissue in a ground glass tissue blender using a rotor driven by a simple electric motor. The homogenizer as a tissue blender similar to the typical kitchen blender is used to emulsify and pulverize the tissue (Heidolph Instruments GMBH & CO KGDiax 900 Germany®) at 1000 U for about 3 min. After centrifugation at 10,000 g for about 60 min, the upper clear layer was taken. MDA levels were determined using the method of Van Ye et al,(20) based on the reaction of MDA with thiobarbituric acid (TBA). In the TBA test reaction, MDA and TBA react in acid pH to form a pink pigment with an absorption maximum at 532 nm. Arbitrary values obtained were compared with a series of standard solutions (1,1,3,3-tetraethoxypropane). Results were expressed as nmol/mg.protein. Part of the homogenate was extracted in ethanol/chloroform mixture (5/3 v/v) to discard the lipid fraction, which caused interferences in the activity measurements of T-SOD, CAT and GST activities. After centrifugation at 10.000 x g for 60 min, the upper clear layer was removed and used for the T-SOD, CAT, GST enzyme activity measurement by methods as described by Durak et al21, Aebi22 and Habig et al23 respectively. One unit of SOD activity was defined as the enzyme protein amount causing 50% inhibition in NBTH2 reduction rate and result were expressed in U/mg protein. The CAT activity method is based on the measurement of absorbance decrease due to H2O2 consumption at 240 nm. The GST activity method is based on the measurement of absorbance changes at 340 nm due to formation of GSH-CDNB complex. Histological determinations: Semiquantitative evaluation technique used by Abdel-Wahhab et al(24) was applied for interpreting the structural changes investigated in hepatic tissues of control and research groups. According to this, (-) (negative point) represents no structural change, while (+) (one positive point) represents mild, (++) (two positive points) medium and (+++) (three positive points) represents severe structural changes. Statistical analysis: The Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA) 20.0 softwre was used for the statistical analysis. Variations in oxidative state parameters, and histopathological examination between study groups were assessed using the Kruskal-Wallis test. The Bonferroni-adjusted Mann-Whitney U-test was used after significant Kruskal-Wallis to determine which groups differed from the others. Results were expressed as mean ± standard deviation (Mean ± SD). Statistical significance was set at a p value < 0.05 for all analyses. RESULTS There was statistically significant difference observed between the groups with respect to findings from the histological changes in the rat liver tissue (hepatocyte degeneration, sinüsoidal dilatation, pycnotic nucleus, prenecrotic cell) determined by light microscopy according to semiquantitative evaluation techniques (p < 0.0001). In Group DIR, hepatocyte degeneration was significantly high compared to Group C, Group DC and Group DIRD (p < 0.0001, p < 0.0001, p = 0.002, respectively), (Table 1, Figure 1-4). Similarly, sinüsoidal dilatation was significantly higher in Group DIR (p < 0.0001, p = 0.004, p = 0.015, respectively). Although, pcynotic nucleus was decreased in Group DIRD, it did not make a significant difference in comparison to Group DIR (p = 0.053), (Table 1, Figure 1-4). The prenecrotic cells were significantly increased in Group DIR, with respect to Group C, Group DC and Group DIRD (p < 0.0001, p = 0.004, p < 0.0001, respectively), (Table 1, Figure 1-4). Table 1. The comparison of histological changes in rat hepatic tissue [Mean ± SD)] p**: Statistical significance was set at a p value < 0.05 for Kruskal-Wallis test *p < 0.05: When compared with Group DIR Figure 1: Light microscopic view of hepatic tissue of Group C (control). VC: vena centralis, *: sinusoids. ®: hepatocytes, k: Kupffer cells, G: glycogen granules, mc: minimal cellular changes, Hematoxilen & Eosin x 40 Figure 2: Light-microscopic view of hepatic tissue of Group DC (diabetes mellitus control) (G: Glycogen granules increased in number, (VC: vena centralis, *:sinusoids. ®:hepatocytes, k:Kupffer cells, G: glycogen granules, mc: minimal cellular changes; Hematoxylin & Eosin x 40) Figure 3: Light-microscopic view of hepatic tissue of Group DIR (Diabetes Mellitus and ischemia-reperfusion) (VC: vena centralis, (H) degenerative and hydrophic hepatocytes, (dej) vena centralis degeneration (centrolobar injury) (*): sinusoid dilatation. (←) pycnotic and hyperchromatic nuclei, MNL: mononuclear cell infiltration, (¯) congestion, K: Kupffer cell hyperplasia, (­) vacuolar degeneration (Hematoxylin & Eosin x 40) Figure 4: Light-microscopic view of hepatic tissue of Group DIRD (Diabetes Mellitus and ischemia-reperfusion together with dexmedetomidine applied group) (VC: vena centralis, (MNL) mononuclear cell infiltration, (dej) hydrophilic degeneration in hepatocytes around vena centralis, (conj) congestion, G: glycogen granules, (←) pycnotic and hyperchromatic nuclei, sinusoid dilatation (*) (Hematoxylin & Eosin x 40) Besides, in liver tissue parenchyma, MN cellular infiltration was a light microscopic finding; and showed significant changes among the groups (p < 0.0001). This was significantly higher in Group DIR, compared to Group C, DC, and DIRD (p < 0.0001, p=0.007, p = 0.007, respectively), (Table 1, Figure 1-4). The enzymatic activity of MDA, SOD and GST in hepatic tissues showed significant differences among the groups [(p = 0.019), (p = 0.034). (p = 0.008) respectively]. MDA enzyme activity was significantly incresed in Group DIR, according to Group C and Group DIRD (p = 0.011, p = 0.016, respectively), (Table 2). In Group DIR SOD enzyme activity was lower with respect to Group C and Group DIRD (p = 0.010, p = 0.038, respectively), (Table 2). The GST enzyme activity was significantly higher in Group DIR, when compared to Group C, DC and DIRD (p = 0.007, p = 0.038, p = 0.039, respectively), (Table 2). Table 2. Oxidative state parameters in rat hepatic tissue [Mean ± SD] p**: Statistical significance was set at a p value < 0.05 for Kruskal-Wallis test *p < 0.05: When compared with Group DIR The enzymatic activity of MDA, SOD in renal tissues, showed significant differences among the groups [(p < 0.0001), (p = 0.008) respectively ]. MDA enzyme activity was significantly incresed in Group DIR, according to Group C and Group DIRD (p < 0.0001, p < 0.0001, respectively). Also MDA enzyme activity level was significantly increased in Group DC, in comparison to Group C and Group DIRD (p = 0.003, p = 0.001, respectively), (Table 3). In Group DIR SOD enzyme activity was lower with respect to Group C and Group DIRD (p = 0.032, p = 0.013, respectively), (Table 3). The GST enzyme activity was significantly higher in Group DIR than the other three groups, however; CAT levels were similar among the groups (Table 3). Table 3: Oxidative state parameters in rat nephrotic tissue [Mean ± SD)] p**: Statistical significance was set at a p value < 0.05 for Kruskal-Wallis test *p < 0.05: When compared with Group DIR DISCUSSION In this study, we have reported the protective effect of dexmedetomidine in experimental hepatic and renal IRI model in the rat by investigating the MDA and SOD levels biochemically. Besides, hepatic histopathological findings also supported our report. Ischemic damage may occur with trauma, hemorrhagic shock, and some surgical interventions, mainly hepatic and renal resections. Reperfusion following ischemia results in even more injury than ischemia itself. IRI is an inflammatory response accompanied by free radical formation, leucocyte migration and activation, sinusoidal endothelial cellular damage, deteoriated microcirculation and coagulation and complement system activation.1 We also detected injury in hepatic and renal tissue caused by reperfusion following ischemia in liver. Experimental and clinical evidence indicates that OS is involved in both the pathogenesis and the complications of diabetes mellitus.25,26 Diabetes mellitus is a serious risk factor for the development of renal and cardiovascular disease. It is also related to fatty changes in the liver.27 Diabetes-related organ damage seems to be the result of multiple mechanisms. Diabetes has been associated with increased free radical reactions and oxidant tissue damage in STZ-induced diabetic rats and also in patients.26Oxidative stress has been implicated in the destruction of pancreatic β-cells28 and could largely contribute to the oxidant tissue damage associated with chronic hyperglycemia.29 A number of reports have shown that antioxidants can attenuate the complications of diabetes in patients30 and in experimental models.28,31 This study demonstrated that diabetes causes a tendency to increase the IRI. There is a lot of investigations related to the pharmacological agents or food supplements applied for decreasing OS and IRI. Antioxidant agents paly an important role in IRI by effecting antioxidant system or lessening the formation of ROS. It has been reported that anesthetic agents too, are effective in oxidative stress.1 During surgical interventions, it seems rational to get benefit from anesthetic agents in prevention of OS caused by IRI instead of using other agents. It has been declared that; dexmedetomidine; as an α-2 agonist with sedative, hypnotic properties; is important in prevention of renal, focal, cerebral, cardiac, testicular and tourniquet-induced IRI.13-18 On the other hand Bostankolu et al. concluded that dexmedetomidine did not have an additional protective role for tournique induced IRI during routine general anesthesia.32 In this study; we have shown that dexmedetomidine has a reducing effect in IRI in diabetic rats. Some biochemical tests and histopathological evaluations are applied for bringing up oxidative stress and IRI in the tissues. Reactive oxygen species (ROS) that appear with reperfusion injury damage cellular structures through the process of the lipid peroxidation of cellular membranes and yield toxic metabolites such as MDA.33 As an important intermidiate product in lipid peroxidation, MDA is used as a sensitive marker of IRI.34 ROS-induced tissue injury is triggered by various defense mechanisms.35 The first defence mechanisms include the antioxidant enzymes of SOD, CAT, and GPx. These endogenous antioxidants are the first lines of defence against oxidative stres and act by scavenging potentially damaging free radical moieties.36 There is a balance between ROS and the scavenging capacity of antioxidant enzymes.1-8 In this study, for evaluation of oxidative damage and antioxidant activity, MDS, SOD, GST and CAT levels were determined in liver and kidney tissues. MDA levels in hepatic and renal tissues were higher in Group DIR compared to Group C and Group DIRD. GST levels were higher in Group DIR compared to all the other three groups. When the groups were arranged from highest to lowest order, with respect to CAT levels, the order was; Group DIR, Group DIRD, Group DC and Group C. However, the difference was not significant. The acute phase reactant MDA, as a marker of OS, was found to be high in Group DIR and low in Group DIRD. This could be interpreted as the presence of protective effect of dexmedetomidine in IRI. IRI developing in splanchnic area causes injury also in the other organs.35 Leithead et al showed that clinically significant hepatic IRI demonstrates a strong relationship with peri-operative acute kidney injury.2 In our experimental research that showed correlation to that of research by Leithead et al. After hepatic IRI in diabetic rats renal OS marker MDA levels were significantly more in Group DIR than Group DIRD. In our study, we observed histopathological changes in the ischemic liver tissue and alterations in the level of MDA, SOD, GST and CAT levels which are OS markers. Histopathological changes of the liver tissues are hepatocyt degeneration, sinusoidal dilatation, nuclear picnosis, celluler necrosis, mononuclear cell infiltrationat paranchimal tissue. These histopathological injury scores were significantly lower in the Group DIRD than those in group DIR. LIMITATION Study limitation is there was no negative control group, as this type of surgical intervention is not possible in rats without anesthesia. CONCLUSION The enzymatic findings of our study together with the hepatic histopathology indicate that dexmedetomidine has a potential role to decrease ischemia-reperfusion injury. Conflict of interest and funding: The authors have not received any funding or benefits from industry or elsewhere to conduct this study. 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