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Betaine supplementation mitigates cisplatin-induced nephrotoxicity by abrogation of oxidative/nitrosative stress and suppression of inflammation and apoptosis in rats Hanan Hagar a,d,∗ , Azza El Medany a , Reem Salam b,e , Gamila El Medany c , Omina A. Nayal a a

Pharmacology unit, Physiology Department, College of Medicine and King Khalid University Hospital, King Saud University, Saudi Arabia Clinical Chemistry Unit, Pathology Department, College of Medicine and King Khalid University Hospital, King Saud University, Saudi Arabia c Anatomy Department, College of Medicine and King Khalid University Hospital, King Saud University, Saudi Arabia d Pharmacology& Toxicology Department, Pharmacy College, Zagazig University, Egypt e Biochemistry Department, Medical College, Ain Shams University, Egypt b

a r t i c l e

i n f o

Article history: Received 7 September 2014 Accepted 5 November 2014 Keywords: Cisplatin Acute renal injury Betaine Oxidative stress Caspase-3 Nuclear factor-kappa.

a b s t r a c t Cisplatin is one of the most potent chemotherapeutic antitumor drugs used in the treatment of a wide range of solid tumors. Its primary dose-limiting side effect is nephrotoxicity. This study aims to investigate the effect of betaine supplementation on cisplatin-induced nephrotoxicity. A single intraperitoneal injection of cisplatin (5 mg/kg) deteriorated the kidney functions as reflected by elevated blood urea nitrogen and serum creatinine levels. Oxidative/nitrosative stress was evident in cisplatin group by increased renal thiobarbituric acid-reactive substances (TBARS), an indicator of lipid peroxidation, reduced renal total antioxidant status and increased renal nitrite concentration. Cisplatin resulted in a decline in the concentrations of reduced glutathione, glutathione peroxidase, catalase, and superoxide dismutase in renal tissues. Renal tumor necrosis factor-␣ (TNF-␣) was also elevated. Expressions of nuclear factor-kappa B (NF-␬B) and caspase-3 were up-regulated in renal tissues as indicated by immunohistochemical analysis. Histopathological changes were observed in cisplatin group. Betaine supplementation (250 mg/kg/day) orally via gavage for 21 days prior to cisplatin injection was able to protect against deterioration in kidney function, abrogate the decline in antioxidants enzymes and suppressed the increase in TBARS, nitrite and TNF-␣ concentrations. Moreover, betaine inhibited NF-␬B and caspase-3 activation and improved the histological changes induced by cisplatin. Thus, the present study demonstrated the renoprotective nature of betaine by attenuating the pro-inflammatory and apoptotic mediators and improving antioxidant competence in kidney tissues of cisplatin treated rats. Betaine could be a beneficial dietary supplement to attenuate cisplatin nephrotoxicity. © 2014 Published by Elsevier GmbH.

1. Introduction Betaine (trimethylglycine) is a natural component that is widely found in animals, plants, and microorganisms and rich dietary sources include seafood, especially marine invertebrates; wheat germ or bran; and spinach (Craig, 2004). It is rapidly absorbed and utilized as an osmolyte and a methyl donor due to the presence of three methyl groups in its structure and thereby helps to maintain

∗ Corresponding author at: Pharmacology Unit (31), College of Medicine & King Khalid University Hospital, King Saud University, P.O. BOX 2925, Riyadh 11461, Saudi Arabia. Tel.: +966 1 467 9289; fax: +966 1 467 9289. E-mail address: [email protected] (H. Hagar).

liver, heart, and kidney health. Betaine insufficiency is associated with lipid metabolism disorder, diabetes, metabolic syndrome, and vascular diseases in patients (Lever and Slow, 2010). Betaine can reduce the risk for inflammation-related diseases via inhibiting the expression of IL-6 and TNF-␣ (Go et al., 2007; Lv et al., 2009). Betaine is an endogenous metabolite of choline that is needed for the structural integrity and signaling functions of cell membranes; it directly affects cholinergic neurotransmission, transmembrane signaling, and lipid transport/metabolism (Zeisel and Blusztajn, 1994). It plays an important role in homocysteine metabolism (Barak et al., 1996). Betaine supplementation is effective for reducing plasma homocysteine levels in humans (Steenge et al., 2003) and in homocystinuria patients with MTHFR deficiency (Smolin et al., 1981). Betaine generally appears to be safe at a daily intake of 9–15 g (average of

http://dx.doi.org/10.1016/j.etp.2014.11.001 0940-2993/© 2014 Published by Elsevier GmbH.

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12 g). Subacute and subchronic rat studies determined that betaine is nontoxic at all doses studied (0–5% of the diet) (Hayes et al., 2003). Cisplatin (cis-diamminedichloroplatinum II, CDDP), is the most important platinum anticancer drug widely used in the treatment of head, neck, ovarian, and testicular cancers. Nevertheless, its full clinical utility is limited because of toxicity including renal and hepatic toxicity (Arany and Safirstein, 2003; O’Dwyer et al., 1999). 25–35% of patients experience a significant decline in renal function after the administration of a single dose of cisplatin (Ries and Klastersky, 1986). Nephrotoxicity is one of the most important sideeffects of cisplatin therapy, affecting primarily the S3 segment of the proximal tubules (Townsend et al., 2003; Miller et al., 2010). The kidney accumulates cisplatin to a greater degree than other organs and is the major route for its excretion. Cisplatin selectively damage proximal tubular epithelial cells (Camano et al., 2010). Its concentration in proximal tubular cells is about five times the serum concentration (Kuhlmann et al., 1997), this disproportionate accumulation of cisplatin in renal tissue contributes to cisplatininduced nephrotoxicity (Arany and Safirstein, 2003). The mechanisms of cisplatin nephrotoxicity are complex and involve numerous processes as inflammation (Faubel et al., 2007), production of reactive oxygen, (Davis et al., 2001) and nitrogen species (Chirino and Pedraza-Chaverri, 2009), as well as cell apoptosis (Camano et al., 2010). Although the production of reactive oxygen species (ROS) by the cisplatin has been implicated in the pathogenesis of cisplatin-induced renal injury (Matsushima et al., 1998), the precise mechanisms underlying the disorders remain to be unknown and still a matter of debate. Moreover, efficient pharmacotherapies to attenuate this devastating complication of cisplatin chemotherapy are not available. Targeting and modulating the internal antioxidant mechanisms by chemopreventive agents has become a part of many therapeutic strategies. A large number of natural products and dietary components have been evaluated as potential chemoprotective agents. Nothing is known about the role of betaine in cisplatin-induced nephrotoxicity. The aim of the present work is to investigate the effect of betaine supplementation on acute renal injury induced by cisplatin in rats hoping to accentuate the progression of renal injury as a side effect of cisplatin treatment. Efficacy of betaine supplementation against the nephrotoxicity was evaluated in terms of biochemical estimation of oxidative/nitrosative stress markers, antioxidant enzyme activities, inflammatory and apoptotic markers and histopathological changes.

2.2. Animals Adult male Wistar rats, weighing 220–250 g, were used in this study. They were obtained from the Animal Care Centre, College of Pharmacy, King Saud University. All the animals were fed a standard rat chow and water ad libitum and kept in a temperature-controlled environment (20–22 ◦ C) with an alternating cycle of 12-h light and dark. The animals used in this study were handled and treated in accordance with the strict guiding principles of the National Institution of Health for experimental care and use of animals. 2.3. Experimental design The animals were divided into four groups of 10 rats each. The details of groups are: Group 1: saline control group which received the saline vehicle alone; group II: betaine group in which betaine (250 mg/kg/day) was given orally via gavage for 21 days; group III: cisplatin group in which rats were injected with a single intraperitoneal injection of cisplatin (5 mg/kg) and group IV: cisplatin + betaine group in which betaine (250 mg/kg/day) was given orally via gavage for 21 days before the single cisplatin injection and daily for 5 days after cisplatin. The doses of cisplatin and betaine were chosen depending upon the literature (Behling et al., 2006; Ganesan et al., 2010). 2.4. Estimation of renal function Serum levels of blood urea nitrogen (BUN) and creatinine were estimated spectrophotometrically using commercial diagnostic kits (Biomérieux Inc., France). 2.5. Sample preparation for biochemical studies At the end of experiments, the animals were sacrificed; blood and kidneys were collected. Blood samples were collected and centrifuged for 10 min at 3000 rpm to obtain clear sera which were stored at −20 ◦ C for subsequent measurement of renal functions. Kidneys were quickly excised, washed immediately with ice-cold physiological saline, blotted dry, and weighed. Portions were taken for histopathological and immunohistochemical studies and the remaining parts of kidneys were homogenized in ice-cold saline to produce 10% (w/v) homogenates, which were centrifuged at 1000g for 10 min at 4 ◦ C. The supernatants were divided into aliquots and were kept at −80 ◦ C until assayed for biochemical studies. 2.6. Biochemical assays for oxidative stress markers

2. Materials and methods 2.1. Chemicals Betaine, cis-platin, Thiobarbituric acid, 5, 5-dithiobis-(2nitrobenzoic acid) were purchased from Sigma St. Louis (Mo, USA). Blood urea nitrogen and creatinine were measured using kits from Biomérieux Inc., (France). Total Antioxidant Status (TAS), glutathione peoxidase, and superoxide dismutase were measured using diagnostic kits provided by Randox Chemical Co. (Antrim, United Kingdom). Chemicals used for measuring catalase and reduced glutathione were obtained from Sigma St. Louis (Mo, USA). Antibodies used for nuclear factor kappa-␤ and caspase-3 immunohistochemical studies were purchased from R& D System (MN, USA). Caspase-3 activity was measured using caspase-3 colorimetric assay (catalog number BF 3100) provided by R&D Company (MN, USA). TNF-␣ and nitrite/nitrate concentration were measured using enzyme-linked immunosorbent assay kits from R&D Systems (MN, USA).

Kidney tissues were homogenized in phosphate buffer (pH = 7) in a ratio of 1/10 (w/v) and centrifuged at 3000 × g for 5 min and the supernatant was used for biochemical assays. 2.6.1. Determination of total antioxidant status Total Antioxidant Status (TAS) was determined using kit from Randox Company. The principle of the method is based on the generation of the ABTS radical cation (ABTS•+) from the interaction between metmyoglobin, 2,2 -azinobis-(3-ethyl-benzothiazoline6-sulphonic acid) (ABTS) and a stabilized form of hydrogen peroxide. The TAS assay was performed using a 20 ␮l sample and assay read time of 3 min. Absorbance was measured at 600 nm. The results were expressed as mmol/min/mg protein. 2.6.2. Assessment of thiobarbituric acid-reactive substances concentration The amount of renal thiobarbituric acid-reactive substances (TBARS) was measured by the thiobarbituric acid assay (TBA) as

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previously described by Buege and Aust (1978). Briefly, 0.5 ml of homogenates was added to 2 ml of TBA reagent containing 0.375% TBA, 15% trichloroacetic acid and 0.25 N HCl. Samples were boiled for 15 min, cooled and centrifuged. Absorbances of the supernatants were spectrophotometrically measured at 532 nm. TBARS concentrations were calculated by the use of 1,1,3,3 tetraethoxypropane as a standard. The results were expressed as nmol/g wet tissue weight. All assays were done using samples in duplicate from each animal. 2.6.3. Determination of superoxide dismutase activity Superoxide dismutase (SOD) activity was determined using RANSOD kit from Randox Company and according to the method of (Sun et al., 1988). The principle of the method is based on the inhibition of NBT reduction by the xanthine–xanthine oxidase system as a superoxide generator. SOD activity is measured by the degree of inhibition of this reaction. One unit of SOD is that which causes 50% inhibition in the NBT reduction rate. SOD activity was expressed as units per milligram protein. 2.6.4. Measurement of catalase activity Catalase (CAT) activity was measured according to the method described by Higgins et al. (1978). The decomposition of hydrogen peroxide (H2 O2 ) was followed spectrophotometrically at 240 nm in 50 mM potassium phosphate buffer (pH 7.05) with 19 mM H2 O2 . Twenty microliter of supernatants was used for 1 ml of reaction mixture. The specific activity of catalase was expressed as the number of ␮mol H2 O2 decomposed/min/g renal tissue weight. 2.6.5. Determination of reduced glutathione Reduced glutathione (GSH) was determined as non-protein and total sulphydryl contents in rat kidneys using the method by Ellman (1959) and modified by Nagi et al. (1992). Briefly, tissues were homogenized in 5.0 ml of cold KCl (1.15%), and the samples were precipitated with trichloroacetic acid. The reaction mixture containing 0.5 ml supernatant, 2.0 ml Tris-EDTA buffer (pH 8.9), and 0.1 ml 5, 5 -dithio-bis-2-nitrobenzoic acid (DTNB). The solution was read at 412 nm on a spectrophotometer (Genesys, Spectronic Instruments). The results were expressed as ␮mol/g wet tissue weight. 2.6.6. Determination of glutathione peroxidase activity Renal glutathione peroxidase (GSH-Px) was estimated according to the method described by Paglia and Valentine (1967) using RANSEL kit. GPH-Px activity was defined as the number of ␮mol NADPH oxidized/min/g wet tissue weight. 2.7. Estimation of renal nitrite concentration Renal nitrite/nitrate concentration was measured using a specific enzyme-linked immunosorbent assay kit (R&D Systems) according to the manufacturer’s directions. The nitrite levels were expressed as ␮mol/g wet tissue. 2.8. Determination of tumor necrosis factor-alpha Tumor necrosis factor-␣ (TNF-␣) level in renal homogenates was determined by enzyme-linked immunosorbent assay (ELISA) using rat TNF-␣ immunoassay kit according to the recommendations of the manufacturer (R&D Systems, USA). 2.9. Assessment of renal caspase-3 activity Caspase-3 activity, as a marker of apoptosis, was estimated in 96-well plates according to the manufacturer’s protocol by

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measuring enzymatic cleavage of the substrate Ac-DEVD-AMC (R&D System). Renal tissues were placed in 0.15 M KCl and homogenized in a polytron homogenizer for 10 strokes. The resulting homogenates were centrifuged at 3000 rpm for 15 min, and the supernatant fractions were used for caspase-3 activity measurement. Protein content in supernatants was determined and concentration was adjusted for activity measurements (1–4 ␮g/␮l). Fifty microliter of homogenate (200 ␮g of total protein) was added to 50 ␮l of the assay buffer followed by 5 ␮L of caspase-3 colorimetric substrate (DEVD-pNA) and incubated at 30 ◦ C for 2 h. Absorbance was measured at 405 nm using a microplate reader (Bmg Lab Technologies). Experiments were performed in duplicate. The caspase-3 activity was expressed as unit/mg tissue wt. Unit is defined as the amount of enzyme needed to convert one picomole of substrate per minute at 30 ◦ C.

2.10. Measurement of protein content Total protein concentration was estimated according to the method of Lowry et al. (1951) with bovine albumin as a standard.

2.11. Histological studies Animals were sacrificed; sections of the kidney were fixed in 10% formalin, embedded in paraffin wax and cut at 5-␮m thickness. Kidney sections were then processed and stained with hematoxylin and eosin dye (H&E) for histological evaluation of renal injury, according to standard protocols. The slides were coded to prevent observer bias during evaluation. All tissue sections were examined in an Olympus BH-2 microscope for characterization of histopathological changes at 400× magnification. The following parameters were chosen to indicate the severity of the morphological damage to the kidney after cisplatin injection: brush border loss, tubule dilatation, tubule degeneration, tubule necrosis, and tubular cast formation. These parameters were evaluated on a scale of 0–4, which ranged from absent (0), mild (1), moderate (2), severe (3), to very severe (4) (Megyesi et al., 1998). Each parameter was determined in six different rats.

2.12. Immunohistochemical studies Four micrometer thick sections were prepared from different animal groups and immunohistochemistry was performed. Sections were deparaffinised, rehydrated, and endogenous peroxidase activity was blocked with H2 O2 in methanol. Sections were pre-treated in citrate buffer (pH 6.0) in a microwave. Sections were incubated at room temperature with monoclonal anti-nuclear factor-␬B (NF-␬B), and anti-caspase-3 antibodies (1:200). Sections were incubated with biotinylated goat anti-polyvalent, then with streptavidin peroxidase and finally with DAB plus chromogen. Slides were counterstained with hematoxylin. The slides were visualized under light microscope and the extent of cell immunopositivity was assessed.

2.13. Statistical analysis The results were expressed as the mean ± S.E. in each group. Statistical analysis were performed using analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons test. The data were analyzed with Graph prism statistical software and a statistical probability of P < 0.05 is considered to be significant.

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3.2. Effect of betaine on renal total antioxidant status in cisplatin-treated rats Renal total antioxidant activity in cisplatin group was significantly (P < 0.05) reduced indicating enhanced oxidative stress compared to normal control group. Betaine supplementation was able to attenuate the reduction in total antioxidant status in cisplatin-treated rats as compared to cisplatin group alone (Table 1). Betaine alone had no effect on total antioxidant status. 3.3. Effect of betaine on renal lipid peroxides in cisplatin-treated rats Thiobarbituric acid reactive substances (TBARS) concentration was used as a measure of lipid peroxidation. TBARS concentrations were similar in control and betaine groups (Table 1). Renal TBARS concentration increased up to 221.3% in cisplatin-treated group compared to normal control rats. Treatment with betaine inhibited cisplatin-induced lipid peroxidation and resulted in a significant decrease in renal TBARS level as compared to cisplatin group alone (Table 1). 3.4. Effect of betaine on renal SOD activity in cisplatin-treated rats Cisplatin administration caused a significant (P < 0.05) inhibition in renal SOD activity by 47.15% as compared to normal control rats (Table 1). Treatment with betaine provided protection against the decline in SOD activity induced by cisplatin in renal tissue (P < 0.05) (Table 1). 3.5. Effect of betaine on renal GSH concentrations in cisplatin-treated rats Cisplatin produced a 50% reduction in renal GSH content as compared to normal control group. Betaine was able to abrogate the decline in GSH content in cisplatin-treated rats (Table 1). Betaine alone produced no significant change in renal GSH content. 3.6. Effect of betaine on renal GSH-Px activity in cisplatin-treated rats Renal GSH-Px activity was reduced in renal tissues in cisplatintreated rats by 44% as compared to normal control rats (Table 1). Treatment with betaine ameliorated the decline in renal GSH-Px activity (P < 0.05) as compared to cisplatin group alone (Table 1). Betaine per se did not alter GSH-Px activity. Fig. 1. Effect of betaine (250 mg/kg/d, orally via gavage) for 21 days on kidney functions in cisplatin treated rats. (A) Serum creatinine level; (B) blood urea nitrogen. Values are expressed as mean ± SEM, n = 10. * P < 0.05 compared with control group. # P < 0.05 compared with cisplatin-treated group.

3. Results 3.1. Effect of betaine on kidney functions in cisplatin-treated rats Cisplatin-induced nephrotoxicity was indicated by the significant increase in serum creatinine level (Fig. 1A) and blood urea nitrogen (Fig. 1B). There was no significant difference in serum creatinine and BUN levels between normal control and betaine control groups (P > 0.05). Betaine improved renal functions in cisplatintreated rats as manifested by the significant (P < 0.05) reduction in serum creatinine level and blood urea nitrogen (Fig. 1A and B).

3.7. Effect of betaine on renal catalase activity in cisplatin treated rats Renal catalase activity decreased in cisplatin group down to 47.14% as compared to normal control value (Table 1). Rats which pre-treated with betaine before cisplatin administration had significantly higher catalase levels compared to cisplatin control group. Betaine alone was not able to change renal catalse activity. 3.8. Effect of betaine on renal nitrite level Renal nitrite level, which indicate the extent of nitric oxide (NO), was significantly (P < 0.05) elevated in the kidneys of the cisplatin control group (Table 2). Betaine pre-treatment before cisplatin administration significantly reduced renal NO level (Table 2). Betaine alone did not change renal nitrite level.

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Table 1 Effects of betaine (250 mg/kg/d, orally via gavage) for 21 days on renal total antioxidant status (TAS), lipid peroxides (TBARS), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), and catalase (CAT) concentrations in cisplatin treated rats.

TAS (mmol/mg protein) TBARS (nmole/g) SOD (U/mg) GSH (␮mole/g) GSH-Px (␮mol/min/g) CAT (␮mol/min/g)

Control

Betaine

Cisplatin

Cisplatin + betaine

0.15 ± 0.022 44.2 ± 3.6 19.3 ± 1.3 3.8 ± 0.12 8.9 ± 0.52 7 ± 0.50

0.13 ± 0.023 48.6 ± 4.1 20.1 ± 4.27 3.5 ± 0.05 8.2 ± 0.20 6 ± 0.24

0.05 ± 0.01* 97.8 ± 7.7* 9.1 ± 0.22* 1.9 ± 0.07* 5.2 ± 0.11* 3.3 ± 0.12*

0.085 ± 0.01** 32.1 ± 1.3** 14.16 ± 1.9** 3.3 ± 0.01** 7.4 ± 0.32** 6.5 ± 0.31**

All values are expressed as mean ± SEM, n = 10. The inter group variation between various groups were measured by one way analysis of variance (ANOVA) followed by followed by Tukey–Kramer multiple comparisons test. * P < 0.05 compared with normal control group. ** P < 0.05 compared with cisplatin-treated group. Table 2 Effects of betaine (250 mg/kg/d, orally via gavage) for 21 days on renal caspase-3 and TNF-␣ concentration in cisplatin treated rats.

Caspase-3 (␮mole/g tissue) TNF-␣ (pg/g tissue) Nitrite level (␮mol/g tissue)

Control

Betaine

Cisplatin

Cisplatin + betaine

1.5 ± 0.03 3.82 ± 0.01 12 ± 1.1

1.7 ± 0.05 5.95 ± 0.02 13 ± 1.2

3.1 ± 0.09* 77.1 ± 4.7* 42 ± 4.1*

1.9 ± 0.02** 34.5 ± 4.7** 15 ± 2.4**

All values are expressed as mean ± SEM, n = 10. The inter group variation between various groups were measured by one way analysis of variance (ANOVA) followed by followed by Tukey–Kramer multiple comparisons test. * P < 0.05 compared with normal control group. ** P < 0.05 compared with cisplatin-treated group. Table 3 Semiquantitative analysis of histology of kidney of rats.

in animals pre-treated with betaine (Fig. 2; Table 3). Treatment with betaine alone caused no significant morphologic alterations

Groups

Dilatation

Degeneration

Necrosis

Casts

Control Betaine Cisplatin Cisplatin + betaine

0 0 2 ± 0.07* 1.2 ± 0.08**

0 0 3.7 ± 0.1* 2.1 ± 0.09**

0 0 3.5 ± 0.2* 1.9 ± 0.06**

0 0 2.7 ± 0.1* 1.1 ± 0.02**

Rats were treated with cisplatin and betaine and histological grading was performed as described in Materials and methods section. Values are mean from six rats. * P < 0.05 compared with normal control group. ** P < 0.05 compared with cisplatin-treated group.

3.9. Effect of betaine on renal TNF-˛ concentration in cisplatin treated rats Renal TNF-␣ concentration increased in cisplatin group as compared to normal control value. Prior-administration with betaine significantly (P < 0.05) reduced the renal TNF-␣ concentration as compared to cisplatin group alone (Table 2). 3.10. Effect of betaine on renal caspase-3 concentration in cisplatin-treated rats Cisplatin significantly (P < 0.01) increased the executioner caspase-3 level in kidney tissues of cisplatin control group, indicating apoptosis of renal cells (Table 2). The activity of the executioner caspase-3 was significantly (P < 0.05) lowered in the combination group compared to the cisplatin group (Table 2). The differences in caspase-3 levels were insignificant (P > 0.05) between normal control and betaine control groups. 3.11. Effects of betaine on renal histological changes in cisplatin-treated rats The histological changes in the kidney tissues from various treatment groups are shown in (Fig. 2.) and the score for tissue damage is depicted in (Table 3). Cisplatin administration caused severe and widespread necrosis with dilatation, vacuolar degeneration, epithelial desquamation, and intraluminal cast formation in the proximal convoluted tubules. The histopathological changes and the severity of the damage induced by cisplatin were lessened

3.12. Effect of betaine on renal caspase-3 immunostaining in cisplatin-treated rats Caspase-3 was undetectable in normal control (Fig. 3A). On the other hand, kidneys obtained from rats treated with cisplatin alone demonstrated marked increase of caspase-3 in the cytoplasm of proximal tubular cells (Fig. 3C). Kidneys obtained from rats treated with betaine (250 mg/kg) demonstrated marked reduction in staining for caspase-3 (Fig. 3D). 3.13. Effect of betaine on renal NF-ˇ immunostaining in cisplatin-treated rats Nuclear factor kappa–␤ was undetectable in normal control, and −ve control (Fig. 4A and B). Imnmunohistochemical staining of rat kidney cortex obtained from rats treated with cisplatin demonstrated marked increase in nuclear factor kappa-␤ immune reactivity in all areas of kidney (Fig. 4C). Kidneys obtained from rats treated with betaine demonstrated markedly reduced staining for nuclear factor kappa-␤ (Fig. 4D). 4. Discussion Cisplatin is a key drug in the chemotherapy for cancers, including lung, gastrointestinal, and genitourinary cancer (Boulikas and Vougiouka, 2004). However, treatment with cisplatin should often discontinue due to its adverse reactions (Pinzani et al., 1994). Platinum analogs as carboplatin and oxaliplatin have been developed to date to overcome these cisplatin-related disadvantages but those regimens including cisplatin still constitute the standard treatment for lung cancer, stomach cancer and testicular cancer (Horwich et al., 1997). In recent years, there has been a global trend towards the use of natural substances existing in fruits, vegetables, and herbs as antioxidants and functional nutrients. Betaine is one of naturally occurring antioxidants which can be obtained from a variety of foods including wheat, shellfish, spinach, and sugar beets (Sakamoto et al., 2002).

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Fig. 2. Photomicrographs of rat kidney (H&E, 100×) from: (A) control group and (B) betaine alone treated group showing normal renal architecture;(C) cisplatin group showing extensive necrosis with dilatation, vacuolar degeneration, epithelial desquamation and intraluminal cast formation in the proximal tubules; (D) betaine + cisplatin group displaying marked improvement in the histological picture which is comparable to that of the control group.

In this study, the administration of a single dose of cisplatin (5 mg/kg, i.p.) resulted in impairment in kidney functions as reflected by increased serum levels of creatinine and urea nitrogen. Moreover, the kidneys of rats treated with cisplatin showed characteristic morphological changes. In the current study, cisplatin-treated rats showed an elevated concentration of renal TBARS, an index of lipid peroxidation, supporting the hypothesis that cisplatin nephrotoxicity is related to involvement of reactive oxygen species. Previous studies suggest that lipid peroxidation due to generation of reactive oxygen species is one of the prime factors in cisplatin-induced nephrotoxicity (Sahu et al., 2011; El-Naga, 2014). In our study, cisplatin not only increased lipid peroxides but also reduced the concentrations of TAS, GSH, GSH-Px, catalase, and SOD in renal tissues. This decline in antioxidants further aggravates the levels of reactive oxygen species and prevents kidney protection against cisplatin toxicity. Oxidative stress may have a causal role in the induction of renal injury by cisplatin (Valentovic et al., 2014). Oxidative stress is a condition where the reduction and oxidation (redox) balance between reactive species such as superoxide anion (· O2− ), hydrogen peroxide (H2 O2 ), peroxynitrite (ONOO− ), and hydroxyl radical (· OH) and antioxidant species such as glutathione (GSH) and glutathione peroxidase (GSH-Px) are disrupted. In the current study, cisplatin treated rats showed a decline in renal GSH concentration and these results corroborated the findings of Saad et al. (2004) and Shimeda et al. (2005) that cisplatin treatment depletes GSH in the kidneys of rats. Glutathione redox cycle is the most important intracellular antioxidant system which maintains cell integrity (Wu et al., 2004). By participating in the glutathione redox cycle, reduced GSH together with GSH-Px convert lipid peroxides to non-toxic products thus maintain the integrity of mitochondria and cell membranes. Depletion of renal GSH stores by cisplatin can account for the inhibition of renal GSH-Px activity obtained in our study. GSH peroxidase and SOD are important

enzymes involved in regulation of cellular oxidative stress. It is not known how cisplatin interferes with the activities of GSH-Px and SOD. It is reported that cisplatin induced depletion of these enzymes due to loss of copper and zinc, which are essential for SOD activity or selenium, which is required for GSH-Px (Badary et al., 2005). Another possibility is that cisplatin directly binds to sulfhydryl groups on cysteine in SOD and GSH peroxidase causing diminished activity (Meier et al., 2007). Betaine supplementation prevented cisplatin-induced lipid peroxidation and protected against the decline in TAS and severe depletion of antioxidant enzymes in cisplatin-treated rats. Furthermore, renal functional and morphological damages were significantly improved by betaine pre-treatment. Previous studies have demonstrated protective effects of betaine against renal injury induced by carbon tetrachloride in rats (Ozturk et al., 2003), high-fructose in rats (Fan et al., 2014) and hypertonicity in Madin Darley canine kidney cells (Horio et al., 2001). Betaine was also able to protect against oxidative stress mediated injury in other tissues as liver (Kim et al., 2005; Váli et al., 2007) and heart (Ganesan et al., 2010). In patients with chronic renal failure, betaine reduced post-methionine hyperhomocysteinemia (McGregor et al., 2002). Betaine is highly lipotropic and can readily pass across the membrane lipid bilayer and diffuses into intracellular compartments and this boosts the capabilities of the betaine to act as an antioxidant (Kanabak et al., 2001). Betaine contains an electrophilic methyl group that ameliorates pathologic states induced by reductive and oxidative stress (Ghyczy and Boros, 2001). Although betaine per se does not interact directly with oxidants, its antioxidant activity is most probably expressed via its effect on the metabolism of sulfur-containing substances in the trans-sulfuration pathway (Kim et al., 2009). Betaine is involved in the synthesis of methionine, which serves as a major supplier of cellular cysteine via the trans-sulfuration pathway for the synthesis of the antioxidant,

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Fig. 3. Immunohistochemical staining of caspase-3 in rat kidney (100×) from: (A) Control group and (B) Betaine alone treated group showing no expression of caspase 3; (C) Cisplatin group without betaine treatment showing a significant increase in caspase-3 immunoreactivity in the cytoplasm of proximal tubular cells; (D) betaine + cisplatin group demonstrating a significant reduction in caspase-3 immunostaining. Brown color indicates caspase-3 positivity.

reduced glutathione that protects the cell from reactive metabolites (Kim and Kim, 2002). This may accounts for its ability to replenish the thiol pool (Go et al., 2007). These results suggest that the preservation of antioxidant defense capacity may account for the nephroprotective activity of betaine. A gradual rise of oxidative stress due to disrupted redox regulation can influence the gene transcription and signal transduction pathways. The gene response to oxidative stress is seen due to the redox-sensitive transcription factors that modulate genes encoding inflammatory cytokines, adhesion molecules, and chemokines (Collins et al., 1995). NF-␬B is among the most important transcription factors shown to be exquisitely sensitive to oxidative stress and its activation is pivotal in the expression of pro-inflammatory cytokines like TNF-␣. In previous studies, cisplatin administration caused NF-␬B activation with subsequent inflammatory reactions responsible for renal injury (Sung et al., 2008; Kang et al., 2009). In the current investigation, NF-␬B activity was up-regulated in cisplatin treated rats and betaine suppresses NF-␬B activity probably via correction of redox imbalance by maintaining thiol homeostasis during cisplatin toxicity. These results are concord with other studies showing that betaine suppressed the redox-sensitive transcription factor, NF-␬B (Go et al., 2007; Fouad et al., 2010; Lee et al., 2013). Further, NF-␬B inhibitors have shown protection against cisplatin-induced nephrotoxicity (Camp et al., 2004; Francescato et al., 2007). NF-␬B signaling pathway activation can promote the transcription of TNF-␣ and iNOS genes (Morishima et al., 2009; ElNaga, 2014). In our study, TNF-␣ and nitrite levels were elevated in cisplatin treated rats suggesting implication of inflammation in mediating cisplatin-induced renal injury (Faubel et al., 2007; Zhang et al., 2007). TNF-␣ can be released by epithelial and mesangial cells leading to apoptosis of epithelial cells lining the tubular

structures and persuading damage of the kidney architecture, which is a hallmark of acute renal toxicity (Ramesh and Reeves, 2002; Zhang et al., 2007; El-Naga, 2014). In addition, increased renal NO production may enhance cellular injury. Excess NO reacts with superoxide anion to generate peroxynitrite radical, a potent prooxidant and cytotoxic intermediate that causes protein nitration and tissue injury. Also, excess NO depletes intracellular GSH thus increasing the susceptibility to oxidative stress (Jung et al., 2009). In the current study, the nephroprotective effect of betaine can be attributed to its ability to inhibit NF-␬B signaling pathway activation which promotes overproduction of TNF-␣ and nitric oxide. The anti-inflammatory effect of betaine has been demonstrated in other studies (Detopoulou et al., 2008). Several antioxidants and anti-inflammatory agents were proved effective in protecting the kidney against the deleterious effects of cisplatin (Kang et al., 2009; Khan et al., 2009). In previous studies, cisplatin induced DNA damage and cell apoptosis via activation of reactive oxygen species, pro-inflammatory cytokines that ultimately culminate in activation of caspase family of proteases (Kaushal et al., 2001; Yano et al., 2007). The present study examined the effect of betaine on caspase-3 activity, an executioner of cell apoptosis, to verify whether betaine can also modulate caspase-3 following cisplatin-induced oxidative stress. Caspase-3 level was increased by cisplatin in renal tissues, an action that was corrected by betaine administration. These results imply that the protective action of betaine is related not only to correction of redox status but also to abrogation of inflammation and apoptosis. It is possible that betaine diminished the signal generated either via integral membrane death receptor proteins such as Fas and TNFR1 or via mitochondrial cytochrome c release pathway, and finally reducing the expression of effector caspase-3,

Please cite this article in press as: Hagar H, et al. Betaine supplementation mitigates cisplatin-induced nephrotoxicity by abrogation of oxidative/nitrosative stress and suppression of inflammation and apoptosis in rats. Exp Toxicol Pathol (2014), http://dx.doi.org/10.1016/j.etp.2014.11.001

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Fig. 4. Immunohistochemical staining of nuclear factor-B (NF-B) in rat kidney (100×) from:(A) Normal control group and (B) betaine alone treated group showing no expression of NF-␬␤; (C) cisplatin group without betaine treatment showing a significant increase in NF-␬␤ immunoreactivity in the cytoplasm of proximal tubular cells; (D) betaine + cisplatin group demonstrating a significant reduction in NF-␬␤ immunostaining. Brown color indicates NF-␬␤ positivity.

thereby attenuating the apoptotic death and disruption of renal tubular cells caused by cisplatin. Moreover, betaine belongs to methylamine osmolytes, which have protein stabilizing effect on macromolecules (Horio et al., 2001) and this may contribute to the protection provided by betaine against cisplatin-induced DNA damage and apoptosis. Betaine inhibited the induction of caspase3, 8, and 9 activities after osmotic stress in Madin Darley canine kidney cells (Horio et al., 2001). Betaine supplementation significantly ameliorated bile acid-induced hepatocytes apoptosis in vivo and in vitro following bile duct ligation (Graf et al., 2002). In conclusion, betaine supplementation can attenuate cisplatininduced renal dysfunctions. Betaine was capable of restoring redox balance, thereby suppressing inflammation, NF-␬B activation and apoptosis during cisplatin toxicity. Betaine could be a beneficial dietary supplement in reducing the complications of nephrotoxicity induced by anticancer agents such as cisplatin in cancer chemotherapy. However, there is a need for further studies on this issue before clinical application can be recommended. Acknowledgments This work was funded by a grant from Deanship of Scientific Research, College of Medicine Research Center (CMRC), King Saud University, Saudi Arabia. References Arany I, Safirstein RL. Cisplatin nephrotoxicity. Semin Nephrol 2003;23(5):460–4. Badary OA, Abdel-Maksoud S, Ahmed WA, Owieda GH. Naringenin attenuates cisplatin nephrotoxicity in rats. Life Sci 2005;76(18):2125–35.

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