Chemosphere xxx (2014) xxx–xxx

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Protective effects of boron on cyclophosphamide induced lipid peroxidation and genotoxicity in rats Sinan Ince a,⇑, Ismail Kucukkurt b, Hasan Huseyin Demirel c, Damla Arslan Acaroz b, Erten Akbel d, Ibrahim Hakki Cigerci e a

Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, 03030 Afyonkarahisar, Turkey Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Biochemistry, 03030 Afyonkarahisar, Turkey Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Pathology, 03030 Afyonkarahisar, Turkey d Usak University, Usak Health Training School, 64100 Usak, Turkey e Afyon Kocatepe University, Faculty of Science and Arts, Department of Biology, 03030 Afyonkarahisar, Turkey b c

h i g h l i g h t s  B reduces cyclophosphamide (CYC) induced toxicity.  B inhibits lipid peroxidation and DNA damage in rats.  B regenerates CYC-induced histopathological changes in rat tissues.  B attenuates NO and restores SOD, CAT, and GSH in rats.

a r t i c l e

i n f o

Article history: Received 19 August 2013 Received in revised form 5 December 2013 Accepted 5 January 2014 Available online xxxx Keywords: Boron Cyclophosphamide Genotoxicity Lipid peroxidation Rat

a b s t r a c t The aim of the present study was to evaluate the possible protective effect of boron (B) on cyclophosphamide (CYC) induced oxidative stress in rats. Totally, thirty Wistar albino male rats were fed standard rodent diet and divided into 5 equal groups: physiological saline was given intraperitoneally (i.p.) to the control group (vehicle treated), to the second group only 75 mg kg1 CYC was given i.p. on the 14th d, and boron was administered (5, 10, and 20 mg kg1, i.p.) to the other groups for 14 d and CYC (75 mg kg1, i.p.) on the 14th d. CYC caused increase of malondialdehyde and decrease of glutathione levels, decrease of superoxide dismutase activities in erythrocyte and tissues, decrease of erythrocyte, heart, lung, and brain catalase, and plasma antioxidant activities. Also, CYC treatment caused to DNA damage in mononuclear leukocytes. Moreover, B exhibited protective action against the CYC-induced histopathological changes in tissues. However, treatment of B decreased severity of CYC-induced lipid peroxidation and genotoxicity on tissues. In conclusion, B has ameliorative effects against CYC-induced lipid peroxidation and genotoxicity by enhancing antioxidant defence mechanism in rat. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Cyclophosphamide (N,N-Bis(2-chloroethyl)tetrahydro-2H1,3,2-oxazaphosphorin-2-amine 2-oxide; CYC), is a cytotoxic bifunctional alkylating agent, belongs to the class of nitrogen mustards (Dollery, 1999; Tripathi and Jena, 2008). CYC is widely used at high dose for chemotherapy of various forms of cancer (bronchial, breast and ovarian cancer, lymphomas, leukemias, etc.) and at low dose for treatment of autoimmune diseases (rheumatoid arthritis), and also used as immunosuppressant after organ ⇑ Corresponding author. Tel.: +90 2722281312/142; fax: +90 2722281349. E-mail address: [email protected] (S. Ince).

transplantations (bone marrow transplantations) (Jolivet et al., 1983; Kuo et al., 2003; Buerge et al., 2006). In clinical application, it is important to prevent DNA damage of normal cells which is induced by CYC (Tripathi and Jena, 2009). Recent studies suggest that CYC generates reactive oxygen species like superoxide anion, hydroxyl radical and hydrogen peroxide (H2O2) during its oxidative metabolism, and depresses the antioxidant defense mechanisms in the liver (Bhattacharya et al., 2003; Stankiewicz et al., 2002). These free radicals may attack soluble cell compounds as well as membranes, eventually leading to the impairment of cell functions and cytolysis (Bergendi et al., 1999). Boron (B) is a mineral substance found in nature. A side from its traditional use in the health care system, B is widely used in

http://dx.doi.org/10.1016/j.chemosphere.2014.01.038 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Ince, S., et al. Protective effects of boron on cyclophosphamide induced lipid peroxidation and genotoxicity in rats. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.038

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S. Ince et al. / Chemosphere xxx (2014) xxx–xxx

industrial, agricultural, and cosmetic applications. It is absorbed rapidly after its administration and rapidly distributed throughout the body via passive diffusion. Following administration of B, the concentration of it in the blood and tissue is reported to be in the ratio of 1:1 in rats and humans (Murray, 1998). B is primarily essential for plants and some animals, and beneficial for humans in nutritional amounts, and thus found in animal and human tissues at low concentrations (Hunt, 1996). B deficiency may occur in animals when their diet contains B at 0.3 mg kg1; the maximum tolerable level of B is 150 mg kg1 diet (Nielsen et al., 1987). B has effects on the metabolism of calcium and potassium (Meacham et al., 1994), vitamin D (Hunt, 1996), aldehyde dehydrogenase, xanthine oxidase, cytochrome B5 reductase (Hunt, 1996; Devirian and Volpe, 2003), insulin, oestrogen, testosterone, T3, T4 (Nielsen et al., 1987; Armstrong et al., 2001), triglycerides, glucose (Hunt, 1996), and reactive oxygen species (Turkez et al., 2007; Ince et al., 2010). The most important application of B is neutron capture therapy; it has been used extensively in the context of various cancers (Martin et al., 1989; Gregoire et al., 1993; Primus et al., 1996). The present study was designed to investigate the effect of B on lipid peroxidation (LPO), antioxidant status, DNA damage, and histopathological changes caused by CYC in Wistar albino rats. 2. Materials and methods 2.1. Materials Boric acid as a source of boron and CYC purchased from Sigma–Aldrich (Interlab, Turkey), and Baxter (Halle, Germany), respectively. All the other chemicals and reagents were of analytical reagent grade purchased from commercial sources. 2.2. Experimental protocol Healthy male Wistar albino rats, 60 d of age and weighing 250– 300 g, were purchased from The Animal Breeding Laboratories of the Experimental Animal Research and Application Center (Afyon, Turkey). The animals were kept at room temperature (25 °C) and relative humidity (50–55%) in a 12 h light/dark cycle with ad libitum access to standard rodent diet and water. Rats were allowed to acclimatise to the animal facility for at least 7 d before experiment started. Prior to the experiments, rats were fed with standard rodent diet for one week in order to adapt to the laboratory conditions. In this study, totally 30 male rats were randomly allocated into 5 groups, 6 rats in each group. All animals were fasted overnight before the experiment. Physiological saline was given intraperitoneally (i.p.) to the control group (vehicle treated), 75 mg kg1 CYC was given i.p. alone (Oboh et al., 2011) to the second group on the 14th d, and B (5, 10, and 20 mg kg1, i.p.) was given to the other groups for 14 d and they received CYC (75 mg kg1, i.p.) on the 14th day. The experimental protocols were also approved by the Animal Care and Use Committee at Afyon Kocatepe University and were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. 2.3. Blood collection and preparation of erythrocytes and homogenate Blood samples from each group were collected by cardiac puncture into heparinised and non-heparinised tubes under light ether anaesthesia at the end of 14 d. Within 30 min of blood collection, the erythrocytes were precipitated by centrifugation at 3500 rpm for 15 min at 4 °C, afterwards plasma and serum were removed. The erythrocytes were washed three times with isotonic saline

and the puffy coat was discarded. Then, same volume of isotonic saline and erythrocyte were added into vials and stored at 20 °C in the deep freeze. Erythrocyte suspension was destroyed by osmotic pressure, use five times of cold deionised water. The erythrocyte lysate was stored at 4 °C until measurements within 3 d (Winterbourn et al., 1975). Animals were sacrificed by cervical dislocation and their liver, lung, kidney, heart, and brain tissues were washed immediately with ice cold 0.9% NaCl. Each tissue was trimmed free of extraneous tissue, rinsed in chilled 0.15 M Tris–HCl buffer (pH 7.4). These tissues were blotted dry, and homogenised in 0.15 M Tris–HCl buffer (pH 7.4) to yield a 10% (w/v) homogenate. Then, they were centrifuged at 3500 rpm for 10 min at 4 °C. The pellets represented the nuclear fraction and the supernatants were subjected to centrifugation at 20 000 rpm for 20 min at 4 °C. The resultant pellets and the supernatants represented the mitochondrial fraction and the cytosolic (including microsomal fraction) fraction, respectively. Reactive oxygen species generation was observed in these fractions as well as whole homogenate (Kucukkurt et al., 2008). 2.4. Preparation of tissues for histopathological analysis At the end of experimental period, 30 male rats were sacrificed. Then, they were dissected and liver, lung, kidney, heart, and brain tissues from each animal were collected and fixed into 10% formalin solution for 48 h and then dehydrated through graded alcohol series (70–100%), cleared in xylene and embedded in paraffin. 5–6 lm thick paraffin sections were cut and stained with haematoxylin-eosin (H&E) and analyzed under a light microscope (Olympus Bx51 model, Tokyo, Japan) equipped with camera (Olympus DP20, Tokyo, Japan). 2.5. Measurement of LPO and reduced glutathione (GSH) in whole blood and tissue homogenates Malondialdehyde (MDA), as a marker for LPO, was determined according to the methods of Draper and Hardley (1990) in whole blood and in tissue homogenates (Ohkawa et al., 1979). The principle of the methods is based on spectrophotometric measurement of the colour production during the reaction of thiobarbituric acid (TBA) with MDA and its absorbance was measured spectrophotometrically at 532 nm. The concentration of MDA was calculated by the absorbance coefficient of MDA-TBA complex and expressed as lM in blood and nM in wet tissue. GSH concentration was measured using the method described by Beutler et al. (1993) in whole blood and tissue homogenates. Briefly, 0.2 mL sample was added to 1.8 mL distilled water. 3 mL of precipitating solution (1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 mL distilled water) was mixed with sample. The mixture was allowed to stand for approximately 5 min and then filtered (Whatman No. 42). 2 mL of filtrate was taken then it was added into another tube and 8 mL of the phosphate solution (0.3 M disodium hydrogen phosphate) and 1 mL 5,50 -Dithio-bis(2nitrobenzoic acid) (DTNB) were added. A blank was prepared with 8 mL of phosphate solution; 2 mL diluted precipitating solution and 1 mL DTNB reagent. A standard solution of the GSH was prepared (40 mg/100 mL). The optical density was measured at 412 nm on the spectrophotometer. Results were expressed as lM in blood and nM in wet tissue. 2.6. Measurement of superoxide dismutase (SOD) and catalase (CAT) activities in erythrocyte lysate and tissue homogenates The antioxidant enzyme activity of SOD in erythrocyte lysate and tissue homogenate was measured according to the method of Sun et al. (1988). The measurement of SOD is based on the

Please cite this article in press as: Ince, S., et al. Protective effects of boron on cyclophosphamide induced lipid peroxidation and genotoxicity in rats. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.038

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principle in which xanthine reacts with xanthine oxidase as a source of substrate (superoxide) and reduces nitroblue tetrazolium (NBT) as an indicator of superoxide. In this method, xantine–xantine oxidase was utilised to generate a superoxide flux. The absorbance obtained from NBT reduction to blue formazon by superoxide was determined at 560 nm spectrophotometrically. SOD activity was expressed in U mgHb1 erythrocyte and U lg1 protein tissue. CAT activities in erythrocyte lysate and tissue homogenate were determined according to the method of Luck (1955) and Aebi (1974), respectively. These methods are based on the decomposition of H2O2 by CAT. The reaction mixture was composed of 50 mM phosphate buffer of pH 7.0; 10 mM H2O2 and sample. The reduction rate of H2O2 was followed at 240 nm for 45 s. at room temperature. One unit of CAT is the amount of CAT decomposes 1.0 lmol of H2O2 per min at pH 4.5 at 25 °C, and the CAT activity (k; nmol min1) is expressed in k mgHb1 erythrocyte and k lg1 protein tissue. 2.7. Measurement of nitric oxide (NO) in plasma and tissue homogenate Decomposition of NO occurs rapidly in aerated solutions to form stable nitrite/nitrate products. Plasma nitrite/nitrate concentration was measured by a modified method of Griess assay, described by Miranda et al. (2001). The principle of this assay is reduction of nitrate by vanadium combined with detection by the acidic Griess reaction. Briefly, samples were deproteinized prior to assay. The plasma and homogenate were added to 96% cold ethanol at 1:2 (v/v) and then vortexed for 5 min. After incubating for 30 min at 4 °C, the mixture was centrifuged at 3500 rpm for 5 min and the supernatants were used for the Griess assay. Analysis was done in a microtiter plate. One hundred microliters of filtrated samples were mixed with 100 lL of VCl3 and was rapidly followed by the addition of the Griess reagents, which contain sulphanilamide 50 lL and N-(1-naphthyl)ethylenediamine dihydrochloride 50 lL. The determination was performed at 37 °C for 30 min. The absorbance was measured by a microplate reader (Multiskan Spectrum, Thermo Labsystems, Finland) at 540 nm. Nitrite/nitrate concentration was calculated using a NaNO2 standard curve and expressed as lM. 2.8. Measurement of antioxidant activity (AOA) in plasma The assay measured the AOA of the plasma to inhibit the production of thiobarbituric acid reactive substances from sodium benzoate under the influence of the free oxygen radicals derived from Fenton’s reaction. A solution of 1 mM uric acid was used as standard. This reaction can be measured by spectrophotometer at 532 nm and the inhibition of colour development illustrated as the AOA (Koracevic et al., 2001).

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Then, leukocytes were mixed with 100 lL of 0.5% low-melting agarose in PBS at 37 °C. Subsequently, 80 lL of this mixture was layered onto a slide pre-coated with a thin layer of 1% normal melting point agarose, covered immediately with a coverslip and stored for 5 min at 4 °C to allow the agarose to solidify. After removing the cover-slips, the slides were immersed in freshly prepared cold (4 °C) lysing solution (2.5 M NaCl, 100 mM EDTA-Na2; 1% Na-sarcosine, 10 mM Tris–HCl, pH 10–10.5; 1% Triton X-100 with 10% DMSO being added just before use) for at least 1 h. The slides were then electrophoresed (25 V/300 mA, 25 min) after they were immersed in freshly prepared alkaline electrophoresis buffer (0.3 M NaOH and 1 mM EDTA-Na2, pH > 13) at 4 °C for unwinding (40 min). All steps were carried out under minimal illumination. After electrophoresis, the slides were neutralized (0.4 M Tris–HCl, pH 7.5) for 5 min. The dried microscope slides were stained with 2 lg mL1 ethidium bromide (70 lL/slide), covered with a coverslip each and analyzed using a fluorescence microscope. The images of 100 randomly chosen nuclei were analyzed visually. Observations were made at a magnification of 400 using a fluorescent microscope (Olympus, Japan). Each image was classified according to the intensity of the fluorescence in the comet tail by being given a value of 0, 1, 2, 3 or 4 (from undamaged class 0 to maximally damaged class 4), and therefore the total score of the slides ranged from 0 to 400 arbitrary units (AU). Damage was detected by a tail of fragmented DNA that migrated from nuclei, causing a ‘comet’ pattern, whereas whole nuclei, without a comet, were not considered damaged. 2.10. Measurement of hemoglobin (Hb) and protein concentrations The Hb was determined colorimetric cyanomethemoglobin method according to Drabkin and Austin (1935), and tissue protein content was assayed according to the colorimetric method of Lowry et al. (1951). 2.11. Spectrophotometric measurements The spectrophotometric measurements were performed by using a Shimadzu 1601 UV–VIS spectrophotometer (Tokyo, Japan). 2.12. Statistical analyses Data obtained from experimental animals were expressed as means and standard deviation of means (±SD) and analysed using one-way analysis of variance (ANOVA), followed by Duncan post-hoc tests on the SPSS (11.5) software computer program. A difference in the mean values of p < 0.05 was considered to be significant. 3. Results 3.1. Effect on LPO and GSH

2.9. Comet assay in rat mononuclear leukocytes Mononuclear leukocytes were separated in order to use in Comet assay. For this, the method of Kocyigit et al. (2005) was followed. In this method; heparinezed blood samples were leaked into histopaque 1077 on the test tubes and after forming a tiny layer the test tubes were centrifuged at 2100 rpm for 30 min (25 °C). After that, the middle layer (contains mononuclear leukocytes) was transferred into 1 mL of salinated phosphate buffer (PBS) (pH 7.4) and mixed with it. Then, this mixture was again centrifuged at 1600 rpm for 10 min (25 °C). After discharging the supernatant, the pellet was diluted as including 106 in mm3 by PBS (pH 7.4).

MDA level is widely used as a marker of free-radical mediated LPO. A highly significant elevation was observed in whole blood and liver (p < 0.001), kidney and brain (p < 0.01), and heart and lung (p < 0.05) MDA levels of CYC administered rats compared to control rats. In contrast, supplementation of B in a dose-dependent manner showed significantly decreased in whole blood and all tissue (p < 0.05) than the CYC group (Table 1). GSH is a nonenzymatic antioxidant in the detoxification pathway that reduces the toxic metabolites of pesticides. GSH levels of whole blood (p < 0.05) kidney, and heart (p < 0.001) of CYC groups were found to be lower than the control group. In contrast, administration of B in a dose-dependent manner showed higher GSH levels of blood,

Please cite this article in press as: Ince, S., et al. Protective effects of boron on cyclophosphamide induced lipid peroxidation and genotoxicity in rats. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.038

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Table 1 Effects of cyclophosphamide (CYC) at dose 75 mg kg1 and CYC + boron (B) at dose 5 (B5), 10 (B10), and 20 (B20) mg kg1 on malondialdehyde levels in blood, kidney, heart, lung, liver, and brain of rats. Treatment design

Blood (lM)

Kidney (nM)

Heart (nM)

Lung (nM)

Liver (nM)

Brain (nM)

Control CYC B5 + CYC B10 + CYC B20 + CYC

2.61 ± 0.39c 5.64 ± 0.70a 4.34 ± 0.15b 4.53 ± 0.34b 4.37 ± 0.49b

0.20 ± 0.09b 0.83 ± 0.41a 0.47 ± 0.26b 0.46 ± 0.20b 0.27 ± 0.19b

0.03 ± 0.01b 0.14 ± 0.06a 0.11 ± 0.09a 0.08 ± 0.05ab 0.07 ± 0.02ab

0.05 ± 0.03b 0.25 ± 0.17a 0.18 ± 0.09ab 0.15 ± 0.09ab 0.09 ± 0.04b

0.06 ± 0.03c 0.15 ± 0.03a 0.13 ± 0.02ab 0.09 ± 0.01bc 0.07 ± 0.04c

0.16 ± 0.02b 0.30 ± 0.10a 0.23 ± 0.05ab 0.21 ± 0.05b 0.17 ± 0.02b

Values are mean ± standard deviations; n = 6. a,b,c In the same column values with different letters show statistically significant differences in blood, liver (p < 0.001), kidney, brain (p < 0.01), heart and lung (p < 0.05).

kidney and heart than the CYC group (Table 2). In addition, there were no changes in lung, liver, and brain GSH levels of rats.

kidney and liver tissues compared to control group. In contrast, administration of B in a dose dependent manner was reversed CYC-induced alteration of SOD and CAT activities.

3.2. Effect on antioxidant enzymes 3.3. Effect on NO and AOA Antioxidant enzymes, SOD and CAT activities were determined in erythrocyte, kidney, heart, liver, and brain tissue of rats and they are shown in Tables 3 and 4, respectively. In the CYC group, SOD activities were found to be lower in erythrocyte, liver, kidney (p < 0.01) heart, and brain (p < 0.001) tissues compared to control group. Nonetheless, CAT activities were decreased in erythrocyte, heart (p < 0.001) and brain tissues (p < 0.01) compared to control group. In addition these levels were not found to be different in

Levels of NO were determined in whole blood, kidney, heart, lung, liver, and brain tissue of rats and shown in Table 5. In the CYC group, NO levels were found to be higher in erythrocyte, heart, lung (p < 0.001), and kidney (p < 0.05) tissues compared to control group whereas they were not found in liver and brain tissues. Nonetheless, AOA levels were determined in plasma of rats and shown in Fig. 1A. AOA levels were decreased in plasma (p < 0.05)

Table 2 Effects of cyclophosphamide (CYC) at dose 75 mg kg1 and CYC + boron (B) at dose 5 (B5), 10 (B10), and 20 (B20) mg kg1 on glutathione levels in blood, kidney, heart, lung, liver, and brain of rats. Treatment design

Blood (lM)

Kidney (nM)

Heart (nM)

Lung (nM)

Liver (nM)

Brain (nM)

Control CYC B5 + CYC B10 + CYC B20 + CYC

14.22 ± 0.39a 12.48 ± 0.70b 12.51 ± 0.34b 14.00 ± 0.15a 14.75 ± 0.49a

6.94 ± 1.64bc 6.27 ± 1.48c 7.08 ± 1.29bc 8.08 ± 0.77ab 8.74 ± 0.84a

8.93 ± 2.42a 4.28 ± 0.26c 5.20 ± 0.71bc 6.08 ± 2.70bc 6.91 ± 1.23ab

6.94 ± 1.62 5.94 ± 0.65 6.44 ± 1.00 6.50 ± 1.83 6.50 ± 1.93

7.32 ± 0.62 6.00 ± 3.68 6.33 ± 0.73 6.66 ± 1.02 7.10 ± 0.98

6.86 ± 2.29 5.78 ± 0.41 5.97 ± 1.17 6.06 ± 0.41 6.33 ± 0.53

Values are mean ± standard deviations; n = 6. In the same column values with different letters show statistically significant differences in heart (p = 0.001), blood (p < 0.01), and kidney (p < 0.05).

a,b,c

Table 3 Effects of cyclophosphamide (CYC) at dose 75 mg kg1 and CYC + boron (B) at dose 5 (B5), 10 (B10), and 20 (B20) mg kg1 on superoxide dismutase activities in erythrocyte, kidney, heart, lung, liver, and brain of rats. Treatment design

Erythrocyte (U mgHb1)

Kidney (U lg1 protein)

Heart (U lg1 protein)

Lung (U lg1 protein)

Liver (U lg1 protein)

Brain (U lg 1 protein)

Control CYC B5 + CYC B10 + CYC B20 + CYC

35.38 ± 8.06b 19.05 ± 6.04c 41.07 ± 5.66b 42.84 ± 9.55b 66.97 ± 6.81a

0.26 ± 0.06bc 0.20 ± 0.03c 0.27 ± 0.04ab 0.31 ± 0.06ab 0.34 ± 0.04a

0.23 ± 0.10c 0.12 ± 0.03d 0.28 ± 0.05bc 0.38 ± 0.06a 0.36 ± 0.06ab

0.65 ± 0.18 0.66 ± 0.10 0.64 ± 0.15 0.69 ± 0.17 0.66 ± 0.25

0.31 ± 0.03b 0.28 ± 0.04c 0.38 ± 0.04ab 0.40 ± 0.08a 0.41 ± 0.08a

0.41 ± 0.08c 0.43 ± 0.05c 0.58 ± 0.15c 0.76 ± 0.05b 1.53 ± 0.24a

Values are mean ± standard deviations; n = 6. a,b,c,d In the same column values with different letters show statistically significant differences in heart, brain (p < 0.001), erythrocyte, kidney and liver (p < 0.01).

Table 4 Effects of cyclophosphamide (CYC) at dose 75 mg kg1 and CYC + boron (B) at dose 5 (B5), 10 (B10), and 20 (B20) mg kg1 on catalase activities in erythrocyte, kidney, heart, lung, liver, and brain of rats. Treatment design

Erythrocyte (k mgHb1)

Kidney (k lg1 protein)

Heart (k lg1 protein)

Lung (k lg1 protein)

Liver (k lg1 protein)

Brain (k lg1 protein)

Control CYC B5 + CYC B10 + CYC B20 + CYC

2097.20 ± 582.7c 1240.62 ± 235.68c 3652.33 ± 1081.23b 4476.87 ± 1015.32b 5832.28 ± 1007.31a

1.59 ± 0.36 1.57 ± 0.49 1.54 ± 0.53 1.64 ± 0.22 1.73 ± 0.32

0.24 ± 0.05d 0.16 ± 0.02d 0.35 ± 0.10c 0.48 ± 0.07b 0.67 ± 0.13a

0.65 ± 0.18b 0.56 ± 0.10b 0.64 ± 0.15b 0.69 ± 0.17b 1.26 ± 0.25a

3.44 ± 1.04 3.29 ± 0.63 4.48 ± 1.25 3.89 ± 1.01 4.06 ± 1.31

0.07 ± 0.01d 0.05 ± 0.02d 0.15 ± 0.03c 0.23 ± 0.05b 0.37 ± 0.05a

Values are mean ± Standard deviations; n = 6. a,b,c,d In the same column values with different letters show statistically significant differences in erythrocyte, heart, lung (p < 0.001), and brain (p < 0.01).

Please cite this article in press as: Ince, S., et al. Protective effects of boron on cyclophosphamide induced lipid peroxidation and genotoxicity in rats. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.038

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Table 5 Effects of cyclophosphamide (CYC) at dose 75 mg kg1and CYC + boron (B) at dose 5 (B5), 10 (B10), and 20 (B20) mg kg1 on nitric oxide levels in plasma, kidney, heart, lung, liver, and brain of rats. Treatment design

Plasma (lM)

Kidney (lM)

Heart (lM)

Lung (lM)

Liver (lM)

Brain (lM)

Control CYC B5 + CYC B10 + CYC B20 + CYC

5.72 ± 0.42b 7.89 ± 0,68a 4.10 ± 2.06c 4.44 ± 0.73bc 4.42 ± 0.43bc

1.90 ± 0.26b 2.51 ± 0.34a 1.99 ± 0.36b 1.97 ± 0.37b 1.72 ± 0.40b

1.00 ± 0.18bc 1.27 ± 0.25ab 0.91 ± 0.15c 0.74 ± 0.37c 1.45 ± 0.23a

1.04 ± 0.24c 2.35 ± 0.65a 1.74 ± 0.55b 1.88 ± 0.38b 1.34 ± 0.26bc

1.95 ± 0.44 2.31 ± 0.56 2.28 ± 0.55 1.88 ± 0.30 1.83 ± 0.31

1.81 ± 0.45 2.17 ± 0.15 2.13 ± 0.33 1.77 ± 0.21 1.88 ± 0.28

Values are mean ± Standard deviations; n = 6. In the same column values with different letters show statistically significant differences in plasma, heart, lung (p < 0.001), and kidney (p < 0.05).

a,b,c

of CYC group (2.68 ± 0.65 mM) and no difference has been observed in B5 plus CYC (3.43 ± 0.33 mM), B10 plus CYC (3.48 ± 0.50 mM), and B20 plus CYC (3.75 ± 0.41 mM) groups compared to control group (3.78 ± 0.49 mM).

3.4. Effect on DNA damage DNA damage was determined in mononuclear leukocytes of rats and shown in Fig. 1B. In the CYC group, DNA damage levels were found to be high level (21.83 ± 4.36 AU) compared to control group (5.17 ± 1.17 AU) (p < 0.001). DNA damages were also found to be at 19.00 ± 2.58, 15.00 ± 3.16, and 14.25 ± 3.40 AU in B5, B10, and B20 plus CYC, respectively. This results suggested that administration of B could not alleviated enough the DNA damage compared to control (p < 0.01) but in a dose dependent manner it prevented the CYC-induced alteration of DNA damage in mononuclear leukocytes compared to CYC group (p < 0.05).

3.5. Histopathological examination Histopathological changes in organs of experimental groups were largely described and shown in Fig. 2. In CYC group, dilatation and degeneration of sinusoids and leukocyte infiltration have been observed in liver (Fig. 2B1) of rats. There was hyaline cylinder in the lumen of the kidney tubuli (Fig. 2B2) and hyaline degeneration was found in heart (Fig. 2B3) of rats. Focal gliosis and neuronal degeneration were found in brain (Fig. 2B4) and fibrin deposition was also found in lung (Fig. 2B5) of rats. In B groups especially B20, slight histopathological changes have been observed in liver, kidney, heart, lung, and brain tissues of rats (Fig 2C, D and E1–5, respectively). In the control group, no significant histopathological changes were observed in liver, kidney, heart, lung, and brain tissues of rats (Fig. 2A1–5, respectively).

4. Discussion Increased oxidative stress represents an imbalance between intracellular product of free radicals and the cellular defence mechanisms; notably, MDA is one of the most important markers of oxidative stress (Kucukkurt et al., 2010). Many researches demonstrated that CYC is a chemotherapeutic agent cause oxidative stress in a dose- and time-dependent manner (Manda and Bhatia, 2003; Tripathi and Jena, 2009), and increases levels of MDA, depletes GSH, and decreases activities of antioxidant enzymes such as SOD and CAT (Premkumar et al., 2001). These reports suggested that the generation of oxidative products are mainly related to the DNA damage caused by CYC. In addition, Zhang et al. (2008) reported that CYC at a dose of 100 and 200 mg kg1, i.p. significantly caused DNA damages in both mouse bone marrow cells and peripheral lymphocyte cells, and markedly inhibited the activities of glutathione peroxidase and SOD, and increased MDA contents in mouse blood. In our study, the enhanced production of blood and tissue lipid peroxides observed is an agreement with other studies. B administration in a dose dependent manner was found to produce significantly less lipid peroxides than CYC-treated rats. Consistent with our results, B acted as scavenger of superoxide, hydroxyl radical and singlet molecular oxygen (Ince et al., 2010, 2012a). Previous studies have shown that GSH plays a key role in the detoxification of the reactive toxic metabolites of CYC, and that cell death begins when GSH stores are markedly depleted (Manda and Bhatia, 2003). Also, Ince et al. (2010) have demonstrated that B supplementation induces reduced glutathione activity in rats. In this study, the observed increase in lipid peroxidation and concomitant depletion of GSH levels in CYC group suggests that the increased peroxidation could be a consequence of depleted GSH stores. Treatment of CYC-induced rats with B especially 10 and 20 mg kg1 yielded normal levels of GSH, which may have resulted from the B-mediated reduction of peroxidation activity among cells.

Fig. 1. Effect of boron (B) on plasma antioxidant activity (AOA) levels (A) and DNA damage (B) in mononuclear leukocytes of rats treated intraperitoenally with 75 mg kg1 cyclophosphamide (CYC). Values are expressed as the mean ± SD of 6 rats per group. Statistical significance: ⁄⁄⁄ p < 0.001, ⁄⁄ p < 0.01, or ⁄ p < 0.05 versus control group.

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Fig. 2. The effect of boron (B) on cyclophosphamide (CYC) induced damage in liver (1), kidney (2), heart (3), brain (4) and lung (5) of rats. Representative figures were stained with H&E. The original magnification was 20 and the scale bars represent 100 lm. Dilatation and degeneration of sinusoids and leukocyte infiltration in liver (Fig. 2B1) hyaline cylinder in the lumen of the kidney tubules (Fig. 2B2) and hyaline degeneration in heart (Fig. 2B3) focal gliosis and neuronal degeneration in brain (Fig. 2B4) and fibrin deposition in lung (Fig. 2B5) of rats, respectively. (A) Control group, (B) animals treated intraperitoenally (i.p.) with 75 mg kg1 CYC on the 14th d, (C) animals treated with 5 mg kg1, i.p. B during 14 d and 75 mg kg1 CYC i.p. on the 14th d, (D) animals treated with 10 mg kg1 B i.p. during 14 d and 75 mg kg1 CYC i.p. on the 14th d and (E) animals treated with 20 mg kg1 B i.p. during 14 d and 75 mg kg1 CYC i.p. on the 14th d.

Antioxidants can inhibit free radical formation. Both SOD and CAT can degrade O2 and decompose H2O2, results in a decrease in oxidative stress, which is the effective way of cell protection from damage (Nagi and Almakki, 2009). These enzymes work together to eliminate active oxygen species, and small deviations in physiological concentrations may have a dramatic effect on the resistance of cellular lipids, proteins and DNA to oxidative damage (Ince et al., 2012b). Decreased activities of SOD and CAT in animals were also reported with many chemotherapeutic agents such as CYC, doxorubicin, vincristine, cisplatin and prednisolone (Singh et al., 2010; Popovic et al., 2007; Ognjanovic´ et al., 2012). CYC treatment on the 14th d decreased SOD and CAT activities which is consistent with these studies. In the CYC groups, low levels of SOD and CAT might be related to the consumption of these enzymes due to increased oxidative stress in the erythrocyte and tissue. SOD and CAT activities of rats increased significantly in the B groups as compared to the CYC groups, suggesting that B in a dose dependent manner has the ability to restore and maintain the activity of these enzymes. Administration of CYC caused NO metabolites (nitrite-nitrate) increase in rat urine and plasma (Souza-Filho et al., 1997) as well as in ferret (Alfieri and Gardner, 1997). In addition, Xu and Malavé (2001) suggested that CYC treatment at 150 mg kg1 produced NO metabolites in urine and plasma of rats. Similarly, in this study, CYC treatment increased plasma and tissue NO levels of rats. However, B treatment in a dose dependent manner reversed NO levels and it may play a role in cell-membrane functions and established thiols protect cells against CYC-induced damage (Nielsen, 1991).

High-dose chemotherapy has been shown to decrease plasma nutrient antioxidant concentrations, including concentrations of vitamin C, a-tocopherol, and b-carotene (Faure et al., 1996). Stankiewicz and Skrzydlewska (2003) and Stankiewicz et al. (2002) reported that 150 mg kg1 CYC caused a statistically significant decrease in total antioxidative status in kidney and lung of rats. Similarly, in this study, CYC treatment decreased plasma AOA levels whereas B enhance in rats because B compounds increased total antioxidant status in humans (Turkez et al., 2007). Erytrochytes could be more sensitive to chemotherapeutics (Franssen et al., 1990) and CYC or its metabolites can bind DNA, cause damage that may results in chromosome breaks, micronucleus formation and cell death (Morre et al., 1995; Murata et al., 2004). In this study, CYC led to DNA damage in mononuclear leukocytes. In this study, CYC induced a high extent of DNA damage compared to the control. Some researchers suggested that CYC generates active metabolites, like 4-hydroxycyclophosphamide, phosphoramide mustard and acrolein. These metabolites preferably alkylate N7 position of the guanine residue of DNA and this leads to inter- and intra-strand cross-links, DNA strand breaks, cessation of DNA synthesis, DNA-protein cross-links and DNA adduct formation (Maccubbin et al., 1991; Selvakumar et al., 2006). In contrast, B exhibited protective and anti-genotoxic effects on DNA damage in this study and this may be attributed to its antioxidant (Ince et al., 2010) and cytoprotective activity (Alsaif, 2004). In this study, CYC treatment at 75 mg kg1, i.p. produced apparent histopathological changes in liver, kidney, heart, lung, and brain of rats. Dilatation and degeneration of sinusoids and leukocyte infiltration have been observed in the liver of rats. Similarly,

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Moroni et al. (1997) observed that severe cholestasis with minimal hepatocellular injury, a picture completely different from the patterns described in patients who developed hepatotoxicity caused by CYC. High dose of CYC (150 mg kg1) treatment to rats occurred diffuse marked swelling of hepatocytes and narrowing of sinusoidal spaces in liver. At the histopathological level, high doses of CYC caused kidney damage in the form of tubular necrosis and desquamation of lining epithelial cells with collection of eosinophilic granules within lumen of the kidney (Senthilkumar et al., 2006; Abraham et al., 2007; Sayed-Ahmed, 2010). These histopathological changes induced by CYC in kidney tissues are confirmed in this study, in which CYC caused hyaline cylinder in the lumen of kidney tubules. Viswanatha-Swamy et al. (2013) reported that high dose of CYC (200 mg kg1) produced massive change in the myocardium showed a varying degree of vacuolar changes in the cardiac muscle fibres mainly in the form of degeneration of myocardial tissue, vacuolization of the cardiomyocytes, infiltration of inflammatory cells and myofibrillar loss. Farrell et al. (1997) suggested that CYC (100 mg kg1) treatment has a differential effect on the infiltration of leukocyte subtypes and some abnormal behaviour in brain of mice. Sulkowska et al. (2002) revealed that CYC treatment to rats showed that intensified fibroplasias processes were found to occur in the interstitial of interalveolar septa of lung. The other histopathological study of lung revealed foci of oedema and congestion and alveolar septa thickening by lymphocytes and macrophage (Sulkowska et al., 2002). In this study, CYC at 75 mg kg1, i.p. resulted hyaline degeneration in the heart, focal gliosis and neuronal degeneration in the brain, and edema, congestion, and fibrin deposition in the lung of rats. In contrast, B in a dose dependent manner has protected liver, kidney, heart, lung, and brain tissues against CYC-induced cellular damage. In conclusion, the results of this study demonstrate that B was effective for the prevention of CYC-induced oxidative stress and DNA damage in rats. The results of this study show that the protective effects of B may be due to both an increase in the activity of the antioxidant defence system, as well as inhibit lipid peroxidation. Also, B protects cells and proliferation leads to enhanced regeneration after tissue damage in rats. References Abraham, P., Indirani, K., Sugumar, E., 2007. Effect of cyclophosphamide treatment on selected lysosomal enzymes in the kidney of rats. Exp. Toxicol. Pathol. 59, 143–149. Aebi, H., 1974. Catalase in vitro. In: Bergmeyer, U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York and London, pp. 673–677. Alfieri, A., Gardner, C., 1997. The NK1 antagonist GR 203040 inhibits cyclophosphamide-induced damage in the rat and ferret bladder. Gen. Pharmacol. 29, 245–250. Alsaif, M.A., 2004. Inhibition of gastric mucosal damage by boric acid pretreatment in rats. J. Med. Sci. 4, 102–109. Armstrong, T.A., Spears, J.W., Lloyd, K.E., 2001. Inflammatory response, growth, and thyroid hormone concentrations are affected by long-term boron supplementation in gilts. J. Anim. Sci. 79, 1549–1556. Bergendi, L., Benes, L., Durackova, Z., Ferencik, M., 1999. Chemistry, physiology and pathology of free radicals. Life Sci. 65, 1865–1874. Beutler, E., Duron, O., Kelly, B.M., 1993. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 61, 882–888. Bhattacharya, A., Lawrence, R.A., Krishnan, A., Zaman, K., Sun, D., Fernandes, G., 2003. Effect of dietary n-3 and n-6 oils with and without food restriction on activity of antioxidant enzymes and lipid peroxidation in livers of cyclophosphamide treated autoimmuneprone NZB/W female mice. J. Am. Coll. Nutr. 22, 388–399. Buerge, I.J., Buser, H.R., Poiger, T., Muller, M.D., 2006. Occurrence and fate of the cytostatic drugs cyclophosphamide and ifosfamide in wastewater and surface waters. Environ. Sci. Technol. 40, 7242–7250. Devirian, T.A., Volpe, S.L., 2003. The physiological effects of dietary boron. Crit. Rev. Food Sci. Nutr. 43, 219–231. Dollery, C., 1999. Therapeutic Drugs. Churchill Livingstone, Edinburgh. pp C349– C354. Drabkin, D.L., Austin, J.H., 1935. Spectrophotometric studies. II. Preparations from washed blood cells; nitric oxide hemoglobin and sulfhemoglobin. J. Biol. Chem. 112, 51–65.

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