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The Protective Effect of Boric Acid on Aluminum-Induced Hepatotoxicity

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The Protective Effect of Boric Acid on Aluminum-Induced Hepatotoxicity H. TÜRKEZ, F. GEYİKOĞLU, S. ÇOLAK Turk J Biol 35 (2011) 293-301 © TÜBİTAK doi:10.3906/biy-0902-11 Th e protective eff ect of boric acid on aluminum-induced hepatotoxicity and genotoxicity in rats Hasan TÜRKEZ, Fatime GEYİKOĞLU, Suat ÇOLAK Department of Biology, Faculty of Science, Atatürk University, 25240, Erzurum - TURKEY Received: 05.02.2009 Abstract: Th e effi cacy of boric acid (BA) was examined on liver marker enzymes in aluminum (Al)-treated rats. Also, a liver micronucleus assay was performed to evaluate the genotoxicity in hepatocytes. With these aims, Sprague- Dawley rats were randomly separated into 10 groups of 5 animals. Aluminum chloride (5 mg/kg body weight (b.w.) AlCl3) and BA (3.25, 13, 36 and 58.5 mg/kg b.w.) alone were administered with injections to the experimental animals. Furthermore, the animals were also treated with Al for 4 consecutive days followed by BA exposure for 10 days. Th e rats were anesthetized aft er Al and BA injections and the levels of serum enzymes were determined. Hepatocytes were isolated for counting the number of micronucleated hepatocytes (MNHEPs). Aft er exposure to Al, the enzymatic activities of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) signifi cantly increased. Furthermore, this metal caused a signifi cant increase in MNHEPs’ incidence. In contrast, the applications of BA doses did not cause any adverse eff ect on the above parameters. Moreover, pretreatments with BA signifi cantly modulated the toxic eff ects of Al. Key words: Aluminum, boric acid, liver, micronucleus assay, rat, serum enzymes Sıçanda alüminyum ile uyarılmış hepatotoksisite ve genotoksisite üzerine borik asidin koruyucu etkisi Özet: Bu çalışmada, alüminyum ile muamele edilmiş sıçanlarda borik asitin (BA) karaciğer markör enzimleri üzerine etkisi incelendi. Ayrıca hepatositlerde genotoksiste değerlendirmesi için karaciğer mikronukleus testi uygulandı. Bu amaçlarla, Sprague-Dawley sıçanları rastgele on gruba ayrıldı ve her bir grupta beş hayvan yeraldı. Alüminyum klorid (5 mg/kg vücut ağırlığı (v.a.) AlCl3) ve borik asit (3,25, 13, 36 and 58,5 mg/kg v.a.) tek başlarına injeksiyonlar ile deney hayvanlarına verildi. Bunun yanı sıra, on günlük BA maruz kalmayı takiben dört gün boyunca da hayvanlar alüminyum ile muamele edildi. Alüminyum ve BA enjeksiyonundan sonra bayıltılmış sıçanlardan alınan kanlarda serum enzim düzeyleri tespit edildi. Mikronukleuslu hepatositlerin (MNHEPs) sayısını hesaplamak için de hepatositler izole edildi. Alüminyuma maruz kalmayı takiben, alkalin fosfataz (ALP), aspartat aminotransferaz (AST), alanin aminotransferaz (ALT) ve laktat dehidrojenaz (LDH) enzim aktiviteleri belirgin olarak yükseldi. Ayrıca, bu metal MNHEPs oranında belirgin bir yükselmeye neden oldu. Aksine, BA dozlarının uygulanması yukardaki parametreler üzerinde herhangi bir olumsuz etkiye sebep olmadı. Üstelik BA ile ön muameleler alüminyumun toksik etkilerini düşürdü. Anahtar sözcükler: Alüminyum, borik asit, karaciğer, mikronukleus testi, sıçan, serum enzimleri 293 Th e protective eff ect of boric acid on aluminum-induced hepatotoxicity and genotoxicity in rats Introduction the frequency of SCEs in Chinese hamster ovary Aluminum (Al) is the third most common (CHO) cells (18). BA did not induce chromosomal element on the Earth’s crust. Th e almost ubiquitous or mitotic spindle abnormalities in bone marrow presence of Al has so heavily contaminated our erythrocytes in the MN assay in Swiss-Webster environment that exposure is virtually inescapable mice (22). Besides these fi ndings, BA did not alter (1). Th e extensive use of this metal (food and the incidence of SCEs over control cells (23,24) but occupational exposure) was questioned due to its induced structural and total CAs (24). It was also toxic eff ects (2). Moreover, Al toxicity occurred as determined that boron mines did not have genotoxic a result of aluminum containing pharmaceutical and carcinogenic eff ects on boron miners and did not products such as aluminum based phosphate increase chromosomal anomalies (25). Th erefore, BA binders or antacid intake (3). Al has pro-oxidative remains a very interesting research topic due to its eff ects in vitro and in vivo (4). Th us, Al provoked equivocal and relatively unknown useful action, role cardiotoxicity, nephrotoxicity, and neurotoxicity in the treatment of various diseases, and interactions (3,5). Furthermore, Al induced hepatic dysfunctions, with other elements. DNA cross-linking in rat ascites hepatoma cells (6), Th e liver is a critical organ that contains most of MN and sister chromatid exchange (SCE) formations the accumulated metals and where toxic eff ects can in human peripheral blood lymphocytes (7-10). be expected ( 26,27). Al accumulated higher in the Boron is a trace mineral for plants, animals, and liver than in the brain, muscle, heart, or lung (28). humans (11). It probably strengthens the antioxidant Hence, in recent years, it has become especially defense mechanism by a yet unknown mechanism important to examine useful antidotes against the (12). Research fi ndings suggest that physiological toxic eff ects of Al. Th e supplementation with free amounts of supplemental dietary boron aff ect a wide radical scavengers (e.g. BA) may protect animals range of metabolic parameters in animals (13). It is from the harmful eff ect of Al. Th e role of BA in liver released in the form of BA that is water soluble and against aluminum-induced changes has not so far biologically available. BA is found in an array of been studied. Usually, the extent of hepatic damage is consumer goods including fi reproofi ng for fabrics assessed by the increased level of cytosolic enzymes and wood, insecticides, and in many cosmetics and such as ALT, AST, and ALP (29,30). Liver MN assays personal care products as well (14). It is absorbed are particularly useful for the detection of the in vivo from the gastrointestinal tract into the blood stream genotoxic eff ects of many chemicals by providing (15) and plays an important role in improving the necessary metabolic activation pathways (31). In arthritis, plasma lipid profi les, and brain function this study, our major goal was to elucidate the in vivo (11,16). Boronated agents with hypolipidemic, anti- potential protective role of BA against Al-induced infl ammatory, or anticancer properties are also hepatotoxicity and genotoxicity in rats. Th us, here we developed (16). Short-term mutagenicity studies focused on changes in serum enzymes as a marker clearly revealed that boron compounds are not associated with hepatic dysfunction and alterations genotoxic. BA was found negative for mutagenicity in in MN formations in hepatocytes as genotoxic the Escherichia coli Sd-4 and Salmonella typhimurium endpoint. With these aims, the present study was assays (17,18). Moreover, results in mammalian designed to determine levels of liver enzymes and genotoxicity test systems were all negative. BA was rate of MNHEPs formations in rats aft er exposure to negative in inducing unscheduled DNA synthesis 3.25, 13, 36, and 58.5 mg/kg b.w. concentrations of in cultured rat hepatocytes (19) and did not BA and 5 mg/kg b.w. AlCl3. induce forward mutations in mouse lymphoma cells (18,20). Similarly, borax and borax ores were Materials and methods found to be negative in assays for mutagenicity in Chinese hamster cells, mouse embryo fi broblasts, Animals and human foreskin fi broblasts (21). BA did not Th e experiment was carried out on male Sprague- induce chromosome aberrations (CAs) or increase Dawley rats, 8 weeks old, weighing 150-200 g. Th e 294 H. TÜRKEZ, F. GEYİKOĞLU, S. ÇOLAK animals were kept on a 12-h light-dark cycle and refrigeration. Immediately prior to evaluation, 10- allowed free access to food and water. All experiments 20 μL of hepatocyte suspension was mixed with were performed in accordance with the Guide for the an equal volume of acridine orange (AO)–DAPI Care and Use of Laboratory Animals (32). (4´, 6-diamidino-2-phenylindole dihydrochloride) Experimental design stain solution (AO, 0.5 mg/mL; DAPI, 10 μg/mL) for fl uorescent staining. Approximately 10-20 Th e animals were randomly divided into 10 μL of the mixture was dropped onto a glass slide groups: (1) controls, which were intact with no and covered with a cover glass. Samples of well- previous experimental history; (2) rats that received isolated hepatocytes were evaluated with the aid of 5 mg/kg b.w. AlCl (CAS No. 7784-13-6, Merck®) 3 a fl uorescence microscope counting the number injection intraperitoneally (ip) for 4 days; (3, 4, 5, of MNHEPs in 2000 hepatocytes for each animal. and 6) rats that received 3.25, 13, 36, and 58.5 mg/ MNHEPs were defi ned as hepatocytes with round kg b.w. BA (CAS No. 10043-35-3, Sigma®) ip for 10 or distinct MNs that stained like the nucleus, with days; (7, 8, 9, and 10) rats were treated with AlCl 3 a diameter 1/4 or less than that of the nucleus, and for 4 consecutive days followed by diff erent doses confi rmed by focusing up and down, taking into of BA exposure for 10 days. Th e doses were selected account hepatocyte thickness. according to the literature data (33-35). Th ere were 5 rats in each group. Aft er the following injections Statistical analysis of all chemicals, the animals were anesthetized with Biochemical and cytogenetic data were analyzed ether. using one-way analysis of variance (ANOVA) and Biochemical studies Fischer’s least signifi cant diff erence (LSD) and Duncan’s tests to determine whether any treatment Blood samples were collected into serum separator signifi cantly diff ered from the controls or each tubes (Microtainer; Becton Dickinson, Franklin other. On the other hand, the 4 data elements are Lakes, NJ, USA), allowed to stand (75-90 min), required for the application of the Kastenbaum and centrifuged (3000 rpm, 5 min), serum harvested, Bowman method (37).
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