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). Th ese are experimental error, and stored at –20 °C. Th e following parameters the maximum diff erence between treatments that is were measured by an automated biochemical desired to detect, a signifi cance level, and power. With analyzer (Olympus AU 2700) with Bayer testing kits this information, the tables provided by Bowman and (Bioclinica): ALP, AST, ALT, and LDH. Kastenbaum were used to determine the diff erences Liver MN assay in the incidence of MNHEPs. Results presented as Th e liver MN assay was done by using the method mean ±SD values and the level of 0.05 was regarded of Suzuki et al. (31). Isolated hepatocytes from as statistically signifi cant. rats were prepared by the collagenase perfusion technique (36). Th e liver was perfused through the hepatic portal vein with calcium-free Hanks Results and discussion balanced salt solution to remove blood for about Activity levels of serum marker enzymes were

10 min at a fl ow rate of 2.5 mL/min. As soon as the found elevated markedly in rats treated with AlCl3 liver became grayish brown in color, a second buff er (Tables 1 and 2). No such changes were observed in solution containing collagenase (Hank’s balanced salt control rat samples. As is evident from the tables, BA supplemented with 4 mM calcium chloride and 0.5 doses alone (3.25, 13, 36, and 58.5 mg/kg b.w.) did not mg collagenase/mL) was perfused at the same rate change the activity levels of ALP, AST, ALT, and LDH. until the liver appeared to have broken up. Th en the Th e pre-treatment with BA could bring a signifi cant liver minced into 3- to 4-mm pieces with a sterile decrease in the activity levels of these enzymes when scalpel. Following mechanical dissociation, the cells compared to AlCl3 group. Moreover, the levels of all were fi ltered through gauze and centrifuged at 1350 ALT and AST enzyme samples showed similarity rpm for 5 min. Th e hepatocyte pellets were suspended with control values aft er treatments together with BA in 10% neutral buff ered formalin and stored under and AlCl3. Th e ALP (3.25 and 13 mg/kg b.w. BA) and

295 Th e protective eff ect of boric acid on aluminum-induced hepatotoxicity and genotoxicity in rats

Table 1. Th e changes in ALP and AST levels in the serum of rats treated with BA and AlCl3. Each value represents mean ± SD of 5 animals, means in the same column followed by the diff erent letters present signifi cant diff erences at the P < 0.05 level.

Treatments ALP (U/L) AST (U/L) Controls 379.4 ± 41.5a 127. 4 ± 13.9a

b b AlCl3 (5 mg/kg b.w.) 610.5 ± 57.9 278.6 ± 22.4 BA (3.25 mg/kg b.w.) 368.2 ± 46.5a 145.6 ± 16.2a BA (13 mg/kg b.w.) 371.7 ± 34.6a 118.3 ± 14.8a BA (36 mg/kg b.w.) 396.5 ± 42.8a 128.4 ± 12.1a BA (58.5 mg/kg b.w.) 372.2 ± 32.7a 122.5 ± 14.8a

a a AlCl3 + BA (3.25 mg/kg b.w.) 405.2 ± 39.1 114.7 ± 15.6

a a AlCl3 + BA (13 mg/kg b.w.) 408.7 ± 37.6 127.0 ± 12.7

b a AlCl3 + BA (36 mg/kg b.w.) 502.9 ± 61.7 123.5 ± 16.1

b a AlCl3 + BA (58.5 mg/kg b.w.) 515.4 ± 51.6 130.8 ± 16.4

Table 2. Th e changes in ALT and LDH levels in the serum of rats treated with BA and AlCl3. Abbreviations are as in Table 1.

Treatments ALT (U/L) LDH (U/L) Controls 45.2 ± 5.7a 932.8 ± 127.5a

b b AlCl3 (5 mg/kg b.w.) 75.5 ± 8.2 1350.7 ± 214.6 BA (3.25 mg/kg b.w.) 49.4 ± 5.8a 932.2 ± 125.6a BA (13 mg/kg b.w.) 45.0 ± 6.1a 1015.5 ± 142.7a BA (36 mg/kg b.w.) 39.7 ± 4.8a 1026.4 ± 155.8a BA (58.5 mg/kg b.w.) 44.4 ± 5.1a 1030.3 ± 148.6a

a a AlCl3 + BA (3.25 mg/kg b.w.) 39.3 ± 4.5 980.1 ± 133.0

a b AlCl3 + BA (13 mg/kg b.w.) 37.1 ± 4.6 1190.8 ± 172.8

a b AlCl3 + BA (36 mg/kg b.w.) 42.3 ± 5.5 1225.6 ± 189.8

a b AlCl3 + BA (58.5 mg/kg b.w.) 45.8 ± 6.0 1264.6 ± 211.4

LDH (3.25 mg/kg b.w. BA) enzyme activities were by AlCl3 toxicity. In the past, no proof was available normalized by low doses of BA exposure, Our results to support these data. It is known that the swelling clearly revealed that BA presented useful eff ects on and necrosis of hepatocytes results in the release of the activities of enzymes as directly related to the liver enzymes into the circulating blood (38). Th is dose for LDH and ALP and without depending on was associated with massive centrilobular necrosis, dose for ALT and AST against AlCl3-induced liver ballooning degeneration, and cellular infi ltration damages. of the liver (39). Th e chemicals as Al induce these Th e conclusions of the present study reveal that fi ndings (40,41) and overall perturbations observed BA inhibits the leakage of liver marker enzymes into in the metabolic profi le of serum demonstrate the circulation and, therefore, limits the damages caused impairment in the liver metabolism (42). Rahman

296 H. TÜRKEZ, F. GEYİKOĞLU, S. ÇOLAK

et al. (43) reported that the increase in the activities oxidative stress and hepatic damage may accelerate of diff erent enzymes in blood might be due to the the progression of chronic hepatodegenerative necrosis of liver, this showing the stress condition disorders, including enzymes increase induced by Al of the treated animals. At this point, our study (10,42). In contrast, increasing antioxidant capacity clearly supports that liver damage is induced by Al plays an important role as a hepatoprotective (54). administration. As a matter of fact, the elevation in Th erefore, there is great interest in the clinical roles transaminase is encountered in conditions causing of BA. It is noteworthy that even in small amounts hepatocellular damage, loss of functional integrity BA helps protect nucleic acids from damage by of the cell membrane, and necrosis such as in free radicals and reactive oxygen species that can chemically induced liver injury and elevation in be generated during normal metabolism as well as enzymes (44,45). Th us, it will be relevant to briefl y through exposure to toxins and pollutants (23,55). mention the importance of enzymic changes as However, it should be noted that boron itself is not manifestations of tissue toxicity. Elevations are seen an antioxidant, but is reported to strengthen the in the liver and myocardial injury, and a rise in serum antioxidant defense system of the tissues (56,57). AST and ALT is more specifi c and predominant in Al has high affi nity for proteins, carbohydrates, the liver and myocardial injury, respectively. Th e and polynucleotides and may exist as a reversible modulations in transaminase are also infl uenced by macromolecular complex of cation/polyanion. the degree of hepatic decompensation of cell necrosis If the cation/polyanion ratio exceeds a threshold (46). A signifi cant increase in ALP could occur in value, an irreversible complex can be formed, giving parenchymal liver disorders such as hepatitis and rise to inhibitory eff ect of Al. Once in the liver, Al cirrhosis, and striking elevation is encountered with increases cells’ iron uptake and oxidative status (58). extrahepatic biliary tract (mechanical) obstruction or Th e liver is the chief site of iron storage, containing with intrahepatic (functional cholestasis) (47). Again, 98% of total iron and a large abundance of transferrin the LDH in serum as a biological marker for liver receptors (TfRs). Th us, it produces toxic eff ects by damage increases (48). Cell necrosis leads to a rise in modifying iron homeostasis and interfering with the concentration of the LDH enzyme in serum and iron regulatory proteins (59). BA does not aff ect the tissue. Th e LDH released into the medium provides parameters of carbohydrate and protein metabolism an index of cell death and membrane permeability to (60). Besides, BA can protect biomembranes (56). LDH, and an increase in LDH activity in the medium On the other hand, Al-induced polynuclear ions occurs as a result of cell membrane disintegration can also produce membrane changes and aggregate and enzyme leakage (49-51). Th e present study also biomolecules (61). Again, the chemicals such as Al demonstrates that AlCl3 signifi cantly aff ects LDH induce the process of lipid peroxidation in the cell level in serum. Th e evidence reveals that the activities membrane (62), a principal cause of hepatotoxicity of LDH are used as a marker of Al toxicity (52). (63). Lipid peroxidation is linked with the excess Th us, it is obvious that the increase in degenerative generation of reactive oxygen species, which may eff ects of AlCl3 becomes more prominent with the be contributed to by exogenous or the endogenous increase in the LDH level. Results from previous sources. Boron (as borax) can help to maintain the investigations indicate that BA does not aff ect liver oxidant/antioxidant balance of the liver as a result of weight and histology (53). Our study also reveals that the decreased generation of lipid peroxidation (12). hepatocytes retain their capacity to normal function Additionally, Al intake infl icts membrane associated aft er BA alone exposures. Moreover, BA against Al proteins (Na-K ATPase, and PKC) (64). In contrast, presents useful eff ects on the activities of enzymes it has been proposed that boron contributes to living as directly related to the dose (LDH and ALP) or systems by acting indirectly as a proton donor and without depending on dose (ALT and AST). that it exerts an infl uence on cell membrane structure No single mechanism emerges to explain all the and function (65). Although the absolute essentiality systemic eff ects of Al. One of the mechanisms involves of boron for plants is well documented, studies to free radical-induced oxidative cell injury in Al date have not shown it to be unequivocally essential toxicity (52). As a matter of fact, interactions between for either animals or humans.

297 Th e protective eff ect of boric acid on aluminum-induced hepatotoxicity and genotoxicity in rats

Table 3 shows the results of the liver MN assay (III) bone marrow is not a target organ for some in Sprague-Dawley rats. AlCl3 induced a statistically classes ofcarcinogens (21). Th us, in the present study, signifi cant increase in formations of MNHEPs we assessed the liver MN assay. Th e results of in vitro although BA did not cause any alterations dependent studies in mammalian and bacterial systems have not upon the number of doses administered. Moreover, revealed mutagenic eff ects of aluminum compounds BA (3.25 and 13 mg/kg b.w.) modulated the increased (67,68) although some genotoxicity studies have

MNHEPs rates by AlCl3 (Figure 1A, B, and C). shown signifi cant increases in MN formations Th e bone marrow and liver MN assays were and structural chromosome aberrations in human recommended for evaluating systemic genetic lymphocytes by Al (7,9,69). Manna and Das (70) toxicity of chemicals (66). Using the liver instead reported the genotoxic eff ects of intraperitoneally of the bone marrow is important for the following injected aluminum chloride in mice. Agency reasons: (I) many chemicals are metabolized in the for Toxic Substances and Disease Registry (71) liver but not in the bone marrow, (II) short-lived also suggested that further genotoxicity studies, metabolites generated in the liver (or other tissue) particularly in vivo exposures of Al, would be useful may not allow effi cient exposure in bone marrow, for verifying the results of the Manna and Das (70)

Table 3. Results of the liver MN assay in rats treated with AlCl3 and BA. HEP, hepatocyte; means in the same column followed by the diff erent letters present signifi cant diff erences at the P < 0.05 level

MNHEP (%)/2000 HEP, mean ± standard Treatments No. of animals deviation Controls 5 0.38 ± 0.08a c AlCl3 (5 mg/kg b.w.) 5 0.94 ± 0.51 BA (3.25 mg/kg b.w.) 5 0.30 ± 0.17a BA (13 mg/kg b.w.) 5 0.35 ± 0.20a BA (36 mg/kg b.w.) 5 0.53 ± 0.28ab BA (58.5 mg/kg b.w.) 5 0.49 ± 0.22ab a AlCl3 + BA (3.25 mg/kg b.w.) 5 0.43 ± 0.19 ab AlCl3 + BA (13 mg/kg b.w.) 5 0.51 ± 0.24 bc AlCl3 + BA (36 mg/kg b.w.) 5 0.82 ± 0.37 c AlCl3 + BA (58.5 mg/kg b.w.) 5 1.04 ± 0.58

Figure 1. Sample photomicrographs of hepatocytes treated with (A) BA (13 mg/kg b.w.), (B) AlCl3 (5 mg/kg b.w.) and (C) AlCl3 + BA (58.5 mg/kg b.w.). Arrows show micronuclei. Th e cells were stained with AO-DAPI and viewed with epiluminescent fl uorescent lighting; ×1000.

298 H. TÜRKEZ, F. GEYİKOĞLU, S. ÇOLAK

study and for evaluating other potential end points It is noteworthy that the toxic eff ect of Al on of genotoxicity. At this point, the fi ndings of our human health cannot be ruled out either, and thus study support the limited in vivo reports. As a matter exposure to Al should be monitored and limited. of fact, it is established that a signifi cant increase Foremost, the detrimental eff ects of Al as a risk factor in the rates of MNHEPs occurs aft er in vivo Al for developing tissue damages should be decreased as exposure. It is also determined that that BA exhibits far as possible. In the present study, the boron-related antigenotoxic properties against AlCl3. Existing data remedy was especially important against aluminum- suggest that genotoxicity is not an area of concern induced hepatic dysfunction and genetic damage. following exposure to boron compounds in humans Th us, BA can be fi rstly proposed to prevent Al and animals (72). Likewise, negative results in a large toxicity as a nutritional supplement or a functional number of in vitro and in vivo mutagenicity assays food component. indicate that boron compounds, especially boric acid and borax, are not genotoxic (23,24,25,55). Furthermore, recent fi ndings show that these Acknowledgements compounds exhibit anti-oxidant and anti-genotoxic Th e authors are grateful to Gökhan Yüksel for properties against toxic chemicals including titanium the linguistic support in preparing the article. Th is and thioacetamide (12,55). Very little is known about investigation was supported by Atatürk University the biological eff ects of boron exposure in animal (Grant Number: 2007-161). cells (73). Although the physiological function of the boron in hepatocytes is unknown, a hypothesis is that Al-induced genetic damage can be prevented by Corresponding author: inductive eff ects of boron compounds on antioxidant Hasan TÜRKEZ capacity, because the toxic eff ects of Al appear to be Department of Biology, Faculty of Sciences, mediated, at least in part, by free radicals (74). As known, genetic damage mainly develops related to Atatürk University, 25240, Erzurum - TURKEY oxidative stress. E-mail: [email protected]

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