MICROSCOPY RESEARCH AND TECHNIQUE 00:00–00 (2015)
Aged Garlic Extract Ameliorates the Oxidative Stress, Histomorphological, and Ultrastructural Changes of Cisplatin-Induced Nephrotoxicity in Adult Male Rats ASHRAF YOUSSEF NASR* AND AMAL AL SHAHAT IBRAHIM Anatomy Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
KEY WORDS
cisplatin; aged garlic extract; kidney; rats; ultrastructure; biochemical; enzymes
ABSTRACT Aim: Aged garlic extract (AGE) is a natural dietary substance having different antioxidant free-radical-scavenger compounds that ameliorates the toxicity of the oxidative stress. This study aimed to investigate the effect of AGE on cisplatin (CP)-induced nephrotoxicity in rats. Materials and methods: Twenty-four, adult male Wistar albino rats were randomly divided into four groups namely control, AGE-treated (a single oral dose of 250 mg/kg/day for 21 days), CPtreated (a single intraperitoneal dose of 7.5 mg/kg on Day 16), and AGE 1 CP-treated (AGE at a dose of 250 mg/kg/once daily for 21 days and a single dose of CP of 7.5 mg/kg intraperitoneally on Day 16). Body weight and absolute and relative kidney weights of each rat were calculated. Serum creatinine, uric acid, and urea levels were determined. Level of malondialdehyde and reduced glutathione and activity of superoxide dismutase and catalase of renal tissues were measured. Renal specimens from each rat were prepared for both light and electron microscopic examinations. Results: Interstitial cell infiltration, hemorrhage, glomerular atrophy, necrosis, and tubular degeneration were observed after CP treatment. Superoxide dismutase and catalase activities and glutathione level were significantly decreased and malondialdehyde level was significantly increased in CP-treated rats compared with AGE 1 CP-treated animals. A remarkable improvement in the histopathological and ultrastructural changes induced by CP in renal tissues was observed in AGE 1 CP-treated rats. Conclusion: AGE exhibited antioxidant effect that could ameliorate the nephrotoxic effects of CP. Microsc. Res. Tech. 00:000–000, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Cisplatin (CP) is a highly efficient antineoplastic drug commonly used as a first-line therapy for many solid cancers, such as stomach cancer, ovarian cancer, lung cancer, bladder cancer, and germ cell tumors (Bergs et al., 2007). The anticancer effect of CP is mediated by apoptosis and DNA-crosslinks with subsequent cytotoxic lesions in malignant cells (Benedetti et al., 2013). However, the clinical use of CP is associated with dose- and duration-dependent nephrotoxic side effect (Saad et al., 2009). The exact mechanism of CP-induced nephrotoxicity is not well understood. Thus, several mechanisms have been suggested to explain such effect. Generation of reactive oxygen species (ROS), impaired glutathione (GSH) metabolism, alterations in the antioxidant enzymes, and increased lipid peroxidation are the most plausible mechanisms of CP-induced toxicity (Ajith et al., 2007; Razo-Rodrıguez et al., 2008). The main site of CP accumulation is the mitochondria of the tubular epithelial cell, namely the S3 segment of the proximal tubules (Hanigan and Devarajan, 2003). ROS are continuously scavenged by the action of mitochondrial antioxidant enzymes, such as superoxide dismutase (SOD), reduced GSH, GSH C V
2015 WILEY PERIODICALS, INC.
peroxidase, and catalase (CAT) (Mansour et al., 2006). CAT and SOD are enzymes of the intrinsic antioxidant defense system, which are responsible for the dissemination of free radicals such as superoxide radicals (Ajith et al., 2007). CP induces ROS in renal epithelial cells and reduces the activity of antioxidant enzyme through decreasing the concentration of intracellular GSH (Yao et al., 2007). Medicinal plants and natural herbal products are often administered in combination with chemotherapeutic drugs and conventional therapy to increase their potential antioxidant activity and provide better protection against their nephrotoxic effect (Adejuwon et al., 2014; Arhoghro et al., 2012; Razo-Rodrıguez et al., 2008). Recently, to increase the clinical usefulness of CP, various natural antioxidant free-radical scavengers have been used to prevent its nephrotoxic *Correspondence to: Ashraf Youssef Nasr, Anatomy Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt. E-mail: ashrafnaeem2013@gmail. com or [email protected] Received 1 August 2014; accepted in revised form 1 March 2015 REVIEW EDITOR: Prof. Alberto Diaspro DOI 10.1002/jemt.22494 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
2
A.Y. NASR AND A.A.S. IBRAHIM
effect (Abdelmaguid et al., 2010; Ali et al., in press; Arhoghro et al., 2012; Saad et al., 2009). Garlic is a commonly used food, and its medical properties have been well recognized for centuries (Bongiorno et al., 2008; Colin-Gonzalez et al., 2012). Garlic and its compounds have been reported to have diverse biological activities, such as regulating plasma lipid levels, anticarcinogenic, antithrombotic, antiartherosclerotic, antidiabetic, renoprotective, antioxidant and immune modulation, antibacterial, antihypertensive, and various other biological actions (Capasso, 2013; Razo-Rodrıguez et al., 2008). Aged garlic extract (AGE) contains various antioxidant organosulfur compounds, mainly S-allylcysteine and allicin (Bongiorno et al., 2008; Capasso, 2013; Colin-Gonzalez et al., 2012). This study was designed to investigate the effect of AGE on the oxidative stress and the changes of renal structure induced by CP in adult male Wistar albino rats. MATERIALS AND METHODS Animals and Experimental Design Twenty-four adult male Wistar albino rats (aged 12– 14 weeks) were obtained from the animal house, Faculty of Medicine, Zagazig University, Zagazig, Egypt. The rats were kept under appropriate conditions of temperature (25 C 6 2 C), humidity (60%–70%), and light (12-h dark–light cycles), with a free access to a commercial balanced diet and tap water. After 1 week of acclimatization, the animals were randomly divided into four equal groups in separate plastic cages, six rats each. Group I (control): Animals did not receive any medications. Group II (AGE-treated): Animals received AGE at a dose of 250 mg/kg once orally for 21 days (Alkreathy et al., 2010) and a single dose of normal saline (0.5 mL/rat, intraperitoneally (i.p.)) on Day 16. Group III (CP-treated): Animals received CP at a dose of 7.5 mg/kg once i.p. on Day 16 (Mansour et al., 2006; Nasr, 2013). Group IV (combined AGE 1 CPtreated): Animals received AGE (250 mg/kg once orally for 21 days) 1 a single dose of CP (7.5 mg/kg i.p.) on Day 16. The rats of all groups were frequently weighed on Days 1, 3, 7, 10, 13, 16, 19, and 22. Drugs and Chemicals CP was obtained in the form of commercial Egyptian Unistin Vial (Egyptian International medical Company (EIMC) United Pharmaceuticals, Cairo, Egypt). AGE (kyolic) was obtained from Wakunaga of America (Mission Viejo, CA). AGE used in this study contained 28.6% extracted solids (286 mg/mL), and S-allyl cysteine, the most abundant water-soluble compound in AGE, was present at a concentration of 1.47 mg/mL. Samples Collection and Biochemical Assays On Day 22 (6 days after CP injection), the rats were anesthetized by ether inhalation. Blood samples were collected through a direct intracardiac puncture from each rat in sterile labeled heparinized test tubes. The sera were separated and stored at 220 C for subsequent assessment of kidney functions. The kidneys of each rat were excised, cleaned, washed with normal saline, blotted with filter paper, and weighed. Relative kidney weight/body weight ratio was calculated accord-
ing to the following formula: organ weight/body weight ratio (%) 5 organ weight 3 100/body weight (Arhoghro et al., 2012). Assessment of Kidney Functions Serum creatinine, urea, and uric acid were measured (Dickey et al., 2005) using standard laboratory techniques and using the commercially available diagnostic kits by an auto analyzer (BT 300, Japan). Determination of Lipid Peroxidation and Antioxidant Enzymes A portion from each rat’s kidneys was minced and homogenized in ice-cold phosphate buffer saline (0.05 M, pH 7.4). A part of the homogenate was mixed with an equal volume of 10% trichloroacetic acid and centrifuged at 3,000 rpm for 15 min at 4 C. The supernatant was used to determine the content of the reduced GSH and malondialdehyde (MDA) enzyme (total lipid peroxidase). The remaining part of the homogenate was centrifuged at 17,000g for 60 min at 4 C, and the supernatant was used for estimation of the activity of both SOD and CAT enzymes. Lipid peroxidation was determined by estimating the level of thiobarbituric acid reactive substances that was used to measure the extent of MDA formed as a result of membrane lipid peroxidation, according to the method of Ohkawa et al. (1979), using commercial kits (Biodiagnostic, Cairo, Egypt). Antioxidant markers GSH reductase, SOD, and CAT were measured by a colorimetric method using commercial kits (Biodiagnostic) according to the methods of Carlberg and Mannervik (1985), Sun et al. (1988), and Aebi (1984), respectively. Tissue Processing Light Microscopy. Renal specimens were fixed in 10% neutral-buffered formalin solution for 48 h, dehydrated in ascending grades of ethyl alcohol, cleared in xylol, and embedded in paraffin blocks. Serial sections (3–5 mm) were cut using microtome. The sections were washed in a water bath, left in the oven for dewaxing, and were stained with hematoxylin and eosin, periodic acid Schiff (PAS), and Masson’s trichrome stains (Bancroft and Gamble, 2002). The stained tissue slides were mounted with DPX and covered with cover slips to examine by a light microscope. Electron Microscopy. . Specimens of 1 mm3 size from each kidney were immediately immersed in 2.5% glutaraldehyde buffered with 0.1 M phosphate buffer for 24–48 h. Then, the specimens were washed in phosphate buffer (pH 7.2–7.4) three to four times for 20 min every time and postfixed in a buffered solution of 1% osmium tetroxide for 2 h, and then washed in the same buffer four times for 20 min each. After fixation, the specimens were dehydrated in ascending grades of ethyl alcohol. Then, they were cleared in propylene oxide, embedded in a mixture of 1:1 of Epon resin– araldite for 1 h. Polymerization was performed in the oven at 65 C for 24 h (Hayat, 2000). Semi-thin sections (1 mm thick) were cut using a glass knife on LKB2000S ultramicrotome, mounted on glass slides, and stained with buffered toluidine blue. The appropriate areas were selected with the light microscope. The resin blocks were trimmed to get rid of the undesired Microscopy Research and Technique
GARLIC EXTRACT AND CISPLATIN NEPHROTOXICITY
3
TABLE 1. Effect of cisplatin (CP) and/or aged garlic extract (AGE) on body and kidney weights of rats Body weight Animal group
Initial BW
Control AGE Cisplatin AGE 1 CP
Kidney weight
Final BW
Changes
a,b
267.2 6 2.4 268 6 2.24 302.5 6 3.9 290.7 6 2.14
140.7 6 1.93 138.3 6 1.05 217.3 6 1.43 24.3 6 0.33
307.8 6 2.7 306.3 6 2.3a,b 284.8 6 4.5b,c 286.3 6 2.3c,d
Absolute
Relative (3100) a
2.38 6 0.026 2.38 6 0.032a 2.43 6 0.018c 2.38 6 0.018a
0.774 6 0.001a 0.771 6 0.0001a 0.853 6 0.001c 0.831 6 0.009a
Data are expressed as mean 6 SEM (n 5 6). a Significant difference vs. CP-treated group at P < 0.005. b Significant difference between initial and final body weights (BW) of same group at P < 0.001. c Significant difference vs. control group at P < 0.01. d No significant difference between initial and final BW (P 5 0.197).
TABLE 2. Effects of cisplatin (CP) and/or aged garlic extract (AGE) treatment on kidney biomarkers of rats Control Creatinine (mg/dL) Uric acid (mg/dL) Urea (mg/dL)
AGE a
0.51 6 0.003 3.68 6 0.23a 25.7 6 1.23a
0.62 6 0.005 3.66 6 0.22a 26.5 6 0.99a
CP a,b
AGE-CP b
2.3 6 0.005 7.9 6 0.42b 66.8 6 2.26b
1.42 6 0.008a,b 5.7 6 0.2a,b* 43.2 6 1.56a,b
Data are expressed as mean 6 SEM (n 5 6). a Significant difference vs. CP-treated group at P < 0.001. b Significant difference vs. control group at P < 0.001. (a*: P < 0.005) AGE: aged garlic extract; CP: cisplatin.
tissue. Ultrathin sections (60–90 nm thick) were cut with a glass knife on a LKB ultramicrotome, mounted on copper grids, and double stained with uranyl acetate and lead citrate. The grids were examined and photographed using a transmission electron microscope (JEOL TEM-1200 EX, Tokyo, Japan) operated at 60–70 kV (Electron Microscope Unit, Faculty of Science, Ain Shams University, Egypt). Statistical Analyses Results were expressed as mean 6 SEM. Comparison of means was done by the Student’s t-test, one-way analysis of variance, and Mann–Whitney U-test. Values of P < 0.05 were considered statistically significant. Statistical evaluation was conducted with SPSS version 16.0 (SPSS, Chicago, IL). RESULTS Body Weight The final body weight of CP-treated rats revealed a significant reduction compared with both control and AGEtreated groups. In addition, the final body weight of control group exhibited a significant difference compared with those of CP-treated and combined AGE 1 CPtreated groups. However, insignificant change in the final body weight of the combined AGE 1 CP-treated rats was observed compared with that of CP-treated rats (Table 1). Kidney Weight Both absolute and relative kidney’s weights of CPtreated rats showed a significant difference compared with those of the other three groups, whereas the absolute and relative kidney weights of the control rats showed a significant difference compared with those of CP-treated rats only (Table 1). Biochemical Results Kidney Biomarkers. A significant increase in the serum level of kidney biomarkers was observed in CPtreated rats compared with those of the other three Microscopy Research and Technique
groups. However, the serum level of such biomarkers of control rats revealed no significant difference compared with those of AGE-treated rats. In addition, a significant reduction in the serum level of such biomarkers was observed in the control rats compared with those of CPtreated and combined AGE 1 CP-treated rats (Table 2). Lipid Peroxidation and Antioxidant Enzymes. MDA level in renal tissues of CP-treated rats displayed a significant increase compared with control, AGE-treated, and combined AGE 1 CP-treated rats. However, GSH level and SOD and CAT activities revealed a significant reduction in renal tissues of CPtreated rats compared with the other three groups. Moreover, a significant increase in GSH level and CAT and SOD activities accompanied with significant reduction in MDA level in the renal tissues of rats pretreated with AGE compared with that of control and CP-treated groups. However, the renal tissue of AGEtreated rats revealed no significant difference in MDA and GSH levels and the activity of SOD and CAT compared with that of control rats (Table 3). Histopathological Results Light microscopic examination of renal parenchyma revealed normal corpuscular and tubular structures in control (Fig. 1A) and AGE-treated (Fig. 1B) animals. However, in CP-treated rats, glomerular congestion and atrophy, degeneration, necrosis, desquamation with shedding of the apical microvilli of the proximal tubular epithelial lining cells, luminal dilation with excessive accumulation of homogenous casts in both proximal convoluted tubules (PCT) and distal convoluted tubules (DCT), intertubular hemorrhage, and mononuclear cells infiltration were observed (Fig. 1C). In the AGE-pretreated rats, degenerated few proximal tubules, dilated tubular lumen with accumulation of hyaline cast, and mononuclear cells infiltration were observed in the renal parenchyma (Fig. 1D). In PAS-stained renal sections, strong-positive reaction was observed in the brush border of PCT, basement
4
A.Y. NASR AND A.A.S. IBRAHIM
TABLE 3. Effects of aged garlic extract (AGE) administration on level of malondialdehyde (MDA), activity of superoxide dismutase (SOD), level of reduced glutathione (GSH), and activity of catalase (CAT) enzymes in renal tissues of rats treated with cisplatin (CP) Animal group (n 5 6 rats) Control AGE CP AGE 1 CP
MDA (nmol/mg protein) a
4.03 6 0.12 4.02 6 0.11a 9.17 6 0.19b 5.9 6 0.14a,b
SOD (U/mg protein) a
49.7 6 2.11 48.2 6 1.9a 21 6 1.63b 36 6 1.93a,b
CAT (U/mg protein) a
41.7 6 1.9 41.5 6 1.7a 29.5 6 1.3b 36.2 6 1.23*, **
GSH (U/mg protein) 15.5 6 1.3a 15.3 6 1.1a 7.9 6 0.84b 12.2 6 0.71, 11
Values are expressed as mean 6 SEM (n 5 6). a Significant difference vs. control group at P < 0.001. b Significant difference vs. CP-treated group at P < 0.001. *P 5 0.036; **P 5 0.0038; 1P 5 0.049; 11P 5 0.003. No significant difference was recorded between control and AGE-treated groups in all parameters (P 5 0.61–0.99).
Fig. 1. Light micrographs of rat kidney showing normal structure in control (A) and AGE-treated (B) groups. Glomerular atrophy (G), wide Bowman’s capsular space (C), degeneration (*) and exfoliation (arrow head) of the epithelial cells lining most of the proximal tubules (P), accumulation of hyaline cast (arrow) within the wide lumina (L) of both proximal (P) and distal (D) convoluted tubules, and extensive
inflammatory cell infiltrations (I) are noticed in CP-treated rats (C). Degenerative epithelial changes (*) and wide lumina (L) with accumulation of homogenous hyaline casts (arrow) are observed in a few of proximal tubules (P) in combined AGE and CP-treated rats (D). Hematoxylin and eosin stain was used. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
membrane of both renal tubules, and outer layer of glomerular capsule in control (Fig. 2A) and AGE-treated (Fig. 2B) rats. However, weak-positive PAS reaction was observed in the remaining brush border of the nonaffected PCT and the basement membrane of both proximal and distal renal tubules in CP-treated rats (Fig. 2C), whereas positive PAS reaction was noticed in the regenerated brush border of many PCT and the basement membrane of renal tubules and outer capsular membrane in the combined AGE 1 CP-treated rats (Fig. 2D). In Masson’s trichrome-stained renal sections, excessive connective tissues were observed around the blood
vessels, and wide vascular spaces were noticed in CPtreated rats (Fig. 3C), whereas little amount of connective tissues were observed around the vascular space and blood vessels within the cortical structure of the kidney in control (Fig. 3A), AGE-treated (Fig. 3B), and combined AGE 1 CP-treated (Fig. 3D) rats. Electron Microscopic Observations The glomerular and capsular structures of the renal corpuscles revealed normal ultrastructure in control (Fig. 4A) and AGE-treated (Fig. 4B) rats, whereas fused foot processes, dilated congested capillary loops Microscopy Research and Technique
Fig. 2. Light micrographs of renal cortex of rat showing a strongpositive PAS reaction in the brush border (arrow), basement membrane of renal tubules (arrow head), and outer layer of Bowman’s capsule (*) in control (A) and AGE-treated (B) rats. Weak-positive PAS reaction is seen in the remaining brush border (arrow) of a few proximal convoluted tubules (P) and in the basement membrane of renal tubules (arrow head) in cisplatin-treated rats (C). A strong-positive
PAS reaction in the brush border (arrow) of regenerated proximal tubules (P), basement membrane of renal tubules (arrow head), and outer layer of Bowman’s capsule (*) in combined AGE 1 cisplatintreated rats (D). C: Bowman’s capsule; G: glomerulus; P: proximal convoluted tubules; D: distal convoluted tubules. PAS stain was used. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 3. Light micrographs of renal cortex of rat showing excessive collagen fibers (R) around the blood vessels (A) and vascular spaces (VS) within the renal parenchyma of cisplatin-treated rats (C), whereas a little amount of collagen fibers (R) are seen within the renal parenchyma around the vascular spaces (VS) and blood vessels
(A) of control (A), AGE-treated (B), and combined AGE 1 cisplatintreated (D) rats. Masson’s trichrome stain was used. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
6
A.Y. NASR AND A.A.S. IBRAHIM
Fig. 4. Electron micrographs of the renal corpuscles of rat kidney showing normal outer parietal cells (Pe) and inner podocytes (Pc) with primary (P1) and secondary (P2) foot processes, glomerular capillaries (C) having thin regular basement membrane (m), and endothelial cells lining (E) with mesangial cells (MC) in-between in control (A) and AGE-treated (B) rats. Fusion (*) of the secondary foot
processes (P2), mesangial hyperplasia (MC), and thick irregular basement membrane (m) of the glomerular capillary (C) are seen in cisplatin-treated rats (C). Congested glomerular capillaries (C) are seen in combined AGE and cisplatin-treated rats (D). F: fenestrations between the foot processes. B: blood cells. Uranyl acetate and lead citrate stains were used.
with irregular thick basement membrane, and mesangial cell hyperplasia with excessive deposition of mesangial matrix were observed in the renal corpuscles of CP-treated rats (Fig. 4C). However, dilated congested glomerular capillaries with uniform thickness of their basal lamina were observed in the AGE-pretreated rats (Fig. 4D). The epithelial cells lining the PCT exhibited normal ultrastructure in control (Fig. 5A) and AGE-treated (Fig. 5B) rats. However, thick irregular basement membrane, loss of basal infoldings, fragmented apical microvilli, degenerated cell organelles, presence of many lysosomes, and apoptotic nuclear changes were observed in the PCT of CP-treated rats (Fig. 5C). However, in the AGE-pretreated rats, normal nuclei, long apical microvilli, few lysosomes, and cytoplasmic vacuoles were seen in the proximal tubular epithelial cells (Fig. 5D).
The DCT showed normal ultrastructure in control rats (Fig. 6A) and AGE-treated rats (Fig. 6B). However, the DCT of CP-treated rats showed a few small sized round-shape mitochondria, presence of many lysosomes, and basal cytoplasmic vacuoles (Fig. 6C), and the DCT of the combined AGE and CP-treated rats revealed regular basement membrane with numerous basal infoldings, regular apical nuclei, intact intercellular tight junctions, basal oblong mitochondria, a few lysosomes, and apical cytoplasmic vesicles (Fig. 6D). DISCUSSION CP is a potent antineoplastic drug. Its clinical use, however, revealed nephrotoxic side effect (Ajith et al., 2007). In this study, a significant reduction in the body weight and the absolute kidney weight with increases reno-somatic index was observed in the rats, 6 days Microscopy Research and Technique
GARLIC EXTRACT AND CISPLATIN NEPHROTOXICITY
7
Fig. 5. Electron micrographs of proximal tubular epithelial cells of rat kidney showing normal ultrastructure in control (A) and AGEtreated (B) rats. Marked destruction (*) of the apical microvilli (mv), many lysosomes (Ly) and cytoplasmic vacuoles (V), thick irregular basement membrane (BM), numerous small-sized electron-dense degenerated mitochondria (M), and irregular nucleus (N) with
peripheral condensation of its heterochromatin (h) on the nuclear inner aspect of the envelop (ne) are seen in cisplatin-treated rats (C). Regular nucleus (N), numerous apical microvilli (mv), many round mitochondria (M), and few lysosomes (Ly) are seen in combined AGE and cisplatin-treated rats (D). Uranyl acetate-lead citrate Stains
after a single intraperitoneal injection of CP. Abdelmaguid et al. (2010), Anusuya et al. (2013), Arhoghro et al. (2012), Chirino et al. (2004), and Nasr (2013) showed a significant reduction in the body and absolute kidney weights, 3–6 days after a single (5–7.5 mg/kg) intraperitoneal injection of CP. The reduction in the body weight in CP-treated rats might be due to the direct toxic effect of CP on renal tubules, with subsequent polyuria and dehydration (Ali and Al Moundhri, 2006; Azu et al., 2010; Yao et al., 2007), or due to gastrointestinal toxicity with subsequent decrease in appetite, ingestion, and assimilation of food (Arhoghro et al., 2012; Yao et al., 2007). The reduction in the absolute kidney weight in CP-treated rats could be due to the structural damage and/or reduction in functions of renal tissues (Park et al., 2009). However, the increase in reno-somatic index might be due to the
edema of renal parenchyma because CP is known to cause renal inflammation (Adejuwon et al., 2014; Anusuya et al., 2013; Mansour et al., 2006). On the other hand, an improvement in CP-induced reduction of the body weight was observed in the rats pretreated with AGE. Such effect indicates that AGE might exhibit a protective effect against the toxicity of CP on renal and intestinal tissues. The protective effect of AGE could be attributed to its anti-inflammatory effect that decreased the renal edema induced by CP (Anusuya et al., 2013). Serum creatinine, uric acid, and urea levels were considered the main parameters that determine the glomerular filtration rate (Azu et al., 2010). In this study, a significant elevation of renal biomarkers was recorded in CP-treated rats. Similar findings were previously reported in different studies (Arhogro et al.,
Microscopy Research and Technique
8
A.Y. NASR AND A.A.S. IBRAHIM
Fig. 6. Electron micrographs of the distal tubular epithelial cells of rat kidney showing regular basement membrane (BM), nuclei (N) with regular outline, oblonged basal mitochondria (M) between the basal infoldings (f), and few lysosomes (Ly) in control (A) and AGEtreated (B) rats. Thick basement membrane (BM), irregular nucleus (N), degenertaed mitochondria (M), and numerous lysosomal
vacuoles (V) are seen within the cytoplasm are seen in cisplatintreated rats (C). Regular basement membrane (BM) with many basal infoldings (f), regular oval nucleus (N), many round basal mitochondria (M), few apical lysosomes (Ly), and apical tight junctions (J) are seen in combined AGE and cisplatin-treated rats (D). L: tubular lumen. Uranyl acetate and lead citrate stains were used.
2012; Azu et al., 2010; Mansour et al., 2006; RazoRodrıguez et al., 2008). The elevation in the serum levels of renal biomarkers might be in part due to impaired renal function (Anusuya et al., 2013), tubular obstruction with back-leakage of the renal tubules (Azu et al., 2010), inhibition of protein synthesis in the tubular cells (Adejuwon et al., 2014), or the direct toxic effect of CP on the glomerular and tubular structures through the generation of ROS and enhancement of lipid peroxidation in renal tubules (Ajith et al., 2007; Mansour et al., 2006; Saad et al., 2009; Yao et al., 2007). In the animals pretreated with AGE, a remarkable improvement in the serum level of the renal biomarkers was recorded. These results confirm the ability of AGE to improve the renal functions through its
antioxidant and free radical scavenger activities (Anusuya et al., 2013). In agreement with our results, Abd El-Halim and Mohamed (2012), Alkreathy et al. (2010), Anusuya et al. (2013), and Razo-Rodrıguez et al. (2008) reported that feeding of AGE provided a protective effect in rats. In this study, significant increase in lipid peroxidation accompanied with marked reduction in level and activity of the antioxidant enzymes in the CP-treated animals, whereas pretreatment with AGE produced a remarkable improvement in level and activity of renal antioxidant enzymes, with a reduction in lipid peroxidation. These findings provide evidence about the antioxidant effect of AGE against CP-induced oxidative stress and lipid peroxidation. The results of our study were supported by the findings that were reported by Ajith Microscopy Research and Technique
GARLIC EXTRACT AND CISPLATIN NEPHROTOXICITY
et al. (2007), Mansour et al. (2006), and Razo-Rodrıguez et al. (2008), who stated that reduction in the activity of antioxidant enzymes (CAT and SOD) increased lipid peroxidation (MDA), and depletion of GSH in renal tissues was implicated in the pathogenesis of CP toxicity. Many studies have demonstrated that AGE exhibited a protective effect against different toxic agents through its powerful antioxidant and free-radical scavengers (Abd El-Halim and Mohamed, 2012; Alkreathy et al., 2010; Anusuya et al., 2013; Capasso, 2013). Consistent with the findings of this study, shedding of the apical microvilli, exfoliation and degeneration of the epithelial cells lining the PCT, and accumulation of homogenous cast were observed in rats exposed to different doses of CP (Abdelmaguid et al., 2010; Aleisa et al., 2007; Ali and Al Moundhri, 2006; Ali et al., in press; Chirino et al., 2004; Mansour et al., 2006; Nasr, 2013; Tufekci et al., 2009). Thus, the histopathological manifestations confirmed the biochemical changes. Moreover, in agreement with our ultrastructural findings, Abdelmaguid et al. (2010), Ali and Al Moundhri (2006), Chirino et al. (2004), and Nasr (2013) found fusion of foot processes of podocytes, mesangial cells proliferation with increase mesangial matrix, degeneration of the proximal tubular cells with distinct nuclear apoptotic changes, irregularity of tubular basement membrane, and loss of basal infoldings in renal tissues of CP-treated animals. In addition, Pratibha et al. (2010) stated that partial or complete shedding of the apical microvilli induced impairment in amino acids and glucose absorption. The authors added that presence of many mitochondria of variable length with dense matrix along basolateral folding might be due to a mitochondrial response to the direct stress of CP on renal tissues. Concomitant with the results of this study, the ultrastructure of renal tissues in AGE-pretreated rats revealed normal glomeruli, preserved apical microvilli, and restoration of nuclear structure of the proximal tubular epithelial cells (Benedetti et al., 2013; Tufekci et al., 2009). Also, no evidence of inflammatory cells infiltration was observed in AGE-pretreated animals (Tufekci et al., 2009). The author added that absence of inflammatory cells infiltration in renal tissues of the animal pretreated with AGE could be considered a proof of the anti-inflammatory effect of AGE. Thus, the inflammation could be considered as a mechanism in CP-induced nephrotoxicity (Benedetti et al., 2013). Apoptosis is an important way of cell death in normal and pathological processes. In this study, different nuclear apoptotic changes, in the form of membrane budding, cell shrinkage, and marginal condensation of chromatin were observed in CP-treated renal tissue. Disappearance of such morphological changes in the renal tissues of AGE-pretreated rats gives a clue evidence of antiapoptotic property of AGE.
CONCLUSION The findings of this study revealed that AGE has protective effects against CP-induced nephrotoxicity mostly through its antioxidant, anti-inflammatory, and antiapoptotic properties. However, the effect of AGE on Microscopy Research and Technique
9
antitumor activity of CP was not elaborated in this study. So, this point can be investigated in another study. ACKNOWLEDGMENT The authors thank the staff members of Electron Microscopic Centre, Faculty of Science, Ain Shams University, Egypt, for their technical support. REFERENCES Abd El-Halim SS, Mohamed MM. 2012. Garlic powder attenuates acrylamide-induced oxidative damage in multiple organs in rat. J Appl Sci Res 8:168–173. Abdelmaguid EN, Hania NC, Noura SA. 2010. Protective effect of silymarin on cisplatin-induced nephrotoxicity in rats. Pak J Nutr 9:624–636. Adejuwon AS, Femi-Akinlosotu OM, Omirinde JO, Owolabi OR, Afodun AM. 2014. Launaea taraxacifolia ameliorates cisplatininduced hepato-renal injury. Eur J Med Plant 4:528–541. Aebi H. 1984. Catalase in vitro. Methods Enzymol 105:121–126. Ajith TA, Usha S, Nivitha V. 2007. Ascorbic acid and alpha tocopherol protect anticancer drug cisplatin induced nephrotoxicity in mice: A comparative study. Clin Chim Acta 375:82–86. Aleisa AM, Al-Majed AA, Al-Yahya AA, Al-Rejaie SS, Bakheet SA, AlShabanah OA, Sayed-Ahmed MM. 2007. Reversal of cisplatininduced carnitine deficiency and energy starvation by propionyl-lcarnitine in rat kidney tissues. Clin Exp Pharmacol Physiol 34: 1252–1259. Ali BH, Al Moundhri MS. 2006. Agents ameliorating or augmenting the nephrotoxicity of cisplatin and other platinum compounds: A review of some recent research. Food Chem Toxicol 44:1173–1183. Ali DA, Abdeen AM, Ismail MF, Mai AM. 2013. Histological, ultrastructural and immunohistochemical studies on the protective effect of ginger extract against cisplatin-induced nephrotoxicity in male rats. Toxicol Ind Health. Alkreathy H, Zoheir AD, Nessar A, Mark S, Soad SA, Osman AM. 2010. Aged garlic extract protects against doxorubicin-induced cardiotoxicity in rats. Food Chem Toxicol 48:951–956. Anusuya N, Durgadevi P, Dhinek A, Mythily S. 2013. Nephroprotective effect of ethanolic extract of garlic (Allium sativum) on cisplatin induced nephrotoxicity in male Wistar rats. Asian J Pharm Clin Res 6:97–100. Arhoghro EM, Kpomah DE, Uwakwe AA. 2012. Ocimum gratissimum aqueous extract enhances recovery in cisplatin-induced nephrotoxicity in albino Wistar rats. Indian J Drugs Dis 1:129–142. Azu OO, Duru FIO, Osinubi AA, Noronha CC, Elesha SO, Abayomi OO. 2010. Protective agent, Kigelia africana fruit extract, against cisplatin induced kidney oxidant injury in Sprague-Dawley rats. Asian J Pharm Clin Res 3:54–58. Bancroft JD, Gamble M. 2002. Theory and practice of histological techniques, 5th ed. Edinburgh: Churchill Livingstone. pp. 125– 138, 172–175, 184–193, 593–620. Benedetti G, Fredriksson L, Herpers B, Meerman J, van de Water B, de Graauw M. 2013. TNF-a-mediated NF-jB survival signaling impairment by cisplatin enhances JNK activation allowing synergistic apoptosis of renal proximal tubular cells. Biochem Pharmacol 85:274–286. Bergs JW, Franken NA, Haveman J, Geijsen ED, Crezee J, Van Bree C. 2007. Hyperthermia, cisplatin and radiation trimodality treatment: A promising cancer treatment? A review from preclinical studies to clinical application. Int J Hyperthermia 23:329–341. Bongiorno PB, Fratellone PM, Pina L. 2008. Potential health benefits of garlic (Allium sativum): A narrative review. J Complement Integr Med 5:1–26. Capasso A. 2013. Antioxidant action and therapeutic efficacy of Allium sativum L. Molecules 18:690–700. Carlberg I, Mannervik B. 1985. Glutathione reductase. Methods Enzymol 113:484–490. Chirino YI, Hernandez-Pando R, Pedraza-Chaverri J. 2004. Peroxynitrite decomposition catalyst ameliorates renal damage and protein nitration in cisplatin-induced nephrotoxicity in rats. BMC Pharmacol 4:20. Colin-Gonzalez AL, Santana RA, Silva-Islas CA, Chanez-Cardenas ME, Santamarıa A, Perla DM. 2012. The antioxidant mechanisms underlying the aged garlic extract- and S-allylcysteine-induced protection. Oxid Med Cell Longev 2012:article ID 907162. Dickey DT, Wu YJ, Muldoon LL, Neuwelt EA: Protection against cisplatin-induced toxicities by N-acetylcysteine and sodium thiosulfate as assessed at the molecular, cellular, and in vivo levels. J Pharmacol Exp Ther 2005, 314:1052–1058.
10
A.Y. NASR AND A.A.S. IBRAHIM
Hanigan MH, Devarajan P. 2003. Cisplatin nephrotoxicity: Molecular mechanisms. Cancer Ther 1:47–61. Hayat MA. 2000. Chemical fixation. In: Hayat MA, editor. Principles and techniques of electron microscopy: Biological applications, 4th ed. New York: Cambridge University Press. pp. 4–85. Mansour HH, Hafezm HF, Fahmy NM. 2006. Silymarin modulates cisplatin 10 mg kg1 induced oxidative stress and hepatotoxicity in rats. J Biochem Mol Biol 39:656–661. Nasr AY. 2013. Effect of misoprostol on ultrastructural changes of renal tissues in cisplatin- treated adult rats. J Cytol Histol 4:3. Ohkawa H, Ohishim N, Yagim K. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358. Park HR, Ju EJ, Jo SK, Jung U, Kim SH, Yee ST. 2009. Enhanced antitumor efficacy of cisplatin in combination with HemoHIM in tumor-bearing mice. BMC Cancer 9:85. Pratibha R, Dayanand A, Bhiwgade, Sameer K, Padmanabh V, Rataboli PV, Chitra YD. 2010. Cisplatin induced histological changes in renal tissue of rat. J Cell Anim Biol 4:108–111.
Razo-Rodrıguez AC, Chirino YI, S anchez-Gonz alez DJ, MartınezMartınez CM, Cruz C, Pedraza-Chaverri J. 2008. Garlic powder ameliorates cisplatin-induced nephrotoxicity and oxidative stress. J Med Food 11:583–586. Saad AA, Youssef MI, El-Shennawy LK. 2009. Cisplatin induced damage in kidney genomic DNA and nephrotoxicity in male rats: The protective effect of grape seed proanthocyanidin extract. Food Chem Toxicol 47:1499–1506. Sun Y, Oberley LW, Li Y. 1988. A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500. Tufekci O, Gunes D, Ozo gul C, Kolatan E, Altun Z, Yilmaz O, Aktas¸ S, Erbayraktar Z, Kirkim G, Mutafo glu K, Soylu A, Serbetc¸io glu B, G€ uneri EA, Olgun N. 2009. Evaluation of the effect of acetyl l-carnitine on experimental cisplatin nephrotoxicity. Chemotherapy 55:451–459. Yao X, Panichpisal K, Kurtzman N, Nugenmt K. 2007. Cisplatin nephrotoxicity: A review. Am J Med Sci 334:115–124.
Microscopy Research and Technique
Aged Garlic Extract Ameliorates the Oxidative Stress, Histomorphological, and Ultrastructural Changes of Cisplatin-Induced Nephrotoxicity in Adult Male Rats ASHRAF YOUSSEF NASR* AND AMAL AL SHAHAT IBRAHIM Anatomy Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
KEY WORDS
cisplatin; aged garlic extract; kidney; rats; ultrastructure; biochemical; enzymes
ABSTRACT Aim: Aged garlic extract (AGE) is a natural dietary substance having different antioxidant free-radical-scavenger compounds that ameliorates the toxicity of the oxidative stress. This study aimed to investigate the effect of AGE on cisplatin (CP)-induced nephrotoxicity in rats. Materials and methods: Twenty-four, adult male Wistar albino rats were randomly divided into four groups namely control, AGE-treated (a single oral dose of 250 mg/kg/day for 21 days), CPtreated (a single intraperitoneal dose of 7.5 mg/kg on Day 16), and AGE 1 CP-treated (AGE at a dose of 250 mg/kg/once daily for 21 days and a single dose of CP of 7.5 mg/kg intraperitoneally on Day 16). Body weight and absolute and relative kidney weights of each rat were calculated. Serum creatinine, uric acid, and urea levels were determined. Level of malondialdehyde and reduced glutathione and activity of superoxide dismutase and catalase of renal tissues were measured. Renal specimens from each rat were prepared for both light and electron microscopic examinations. Results: Interstitial cell infiltration, hemorrhage, glomerular atrophy, necrosis, and tubular degeneration were observed after CP treatment. Superoxide dismutase and catalase activities and glutathione level were significantly decreased and malondialdehyde level was significantly increased in CP-treated rats compared with AGE 1 CP-treated animals. A remarkable improvement in the histopathological and ultrastructural changes induced by CP in renal tissues was observed in AGE 1 CP-treated rats. Conclusion: AGE exhibited antioxidant effect that could ameliorate the nephrotoxic effects of CP. Microsc. Res. Tech. 00:000–000, 2015. V 2015 Wiley Periodicals, Inc. C
INTRODUCTION Cisplatin (CP) is a highly efficient antineoplastic drug commonly used as a first-line therapy for many solid cancers, such as stomach cancer, ovarian cancer, lung cancer, bladder cancer, and germ cell tumors (Bergs et al., 2007). The anticancer effect of CP is mediated by apoptosis and DNA-crosslinks with subsequent cytotoxic lesions in malignant cells (Benedetti et al., 2013). However, the clinical use of CP is associated with dose- and duration-dependent nephrotoxic side effect (Saad et al., 2009). The exact mechanism of CP-induced nephrotoxicity is not well understood. Thus, several mechanisms have been suggested to explain such effect. Generation of reactive oxygen species (ROS), impaired glutathione (GSH) metabolism, alterations in the antioxidant enzymes, and increased lipid peroxidation are the most plausible mechanisms of CP-induced toxicity (Ajith et al., 2007; Razo-Rodrıguez et al., 2008). The main site of CP accumulation is the mitochondria of the tubular epithelial cell, namely the S3 segment of the proximal tubules (Hanigan and Devarajan, 2003). ROS are continuously scavenged by the action of mitochondrial antioxidant enzymes, such as superoxide dismutase (SOD), reduced GSH, GSH C V
2015 WILEY PERIODICALS, INC.
peroxidase, and catalase (CAT) (Mansour et al., 2006). CAT and SOD are enzymes of the intrinsic antioxidant defense system, which are responsible for the dissemination of free radicals such as superoxide radicals (Ajith et al., 2007). CP induces ROS in renal epithelial cells and reduces the activity of antioxidant enzyme through decreasing the concentration of intracellular GSH (Yao et al., 2007). Medicinal plants and natural herbal products are often administered in combination with chemotherapeutic drugs and conventional therapy to increase their potential antioxidant activity and provide better protection against their nephrotoxic effect (Adejuwon et al., 2014; Arhoghro et al., 2012; Razo-Rodrıguez et al., 2008). Recently, to increase the clinical usefulness of CP, various natural antioxidant free-radical scavengers have been used to prevent its nephrotoxic *Correspondence to: Ashraf Youssef Nasr, Anatomy Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt. E-mail: ashrafnaeem2013@gmail. com or [email protected] Received 1 August 2014; accepted in revised form 1 March 2015 REVIEW EDITOR: Prof. Alberto Diaspro DOI 10.1002/jemt.22494 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
2
A.Y. NASR AND A.A.S. IBRAHIM
effect (Abdelmaguid et al., 2010; Ali et al., in press; Arhoghro et al., 2012; Saad et al., 2009). Garlic is a commonly used food, and its medical properties have been well recognized for centuries (Bongiorno et al., 2008; Colin-Gonzalez et al., 2012). Garlic and its compounds have been reported to have diverse biological activities, such as regulating plasma lipid levels, anticarcinogenic, antithrombotic, antiartherosclerotic, antidiabetic, renoprotective, antioxidant and immune modulation, antibacterial, antihypertensive, and various other biological actions (Capasso, 2013; Razo-Rodrıguez et al., 2008). Aged garlic extract (AGE) contains various antioxidant organosulfur compounds, mainly S-allylcysteine and allicin (Bongiorno et al., 2008; Capasso, 2013; Colin-Gonzalez et al., 2012). This study was designed to investigate the effect of AGE on the oxidative stress and the changes of renal structure induced by CP in adult male Wistar albino rats. MATERIALS AND METHODS Animals and Experimental Design Twenty-four adult male Wistar albino rats (aged 12– 14 weeks) were obtained from the animal house, Faculty of Medicine, Zagazig University, Zagazig, Egypt. The rats were kept under appropriate conditions of temperature (25 C 6 2 C), humidity (60%–70%), and light (12-h dark–light cycles), with a free access to a commercial balanced diet and tap water. After 1 week of acclimatization, the animals were randomly divided into four equal groups in separate plastic cages, six rats each. Group I (control): Animals did not receive any medications. Group II (AGE-treated): Animals received AGE at a dose of 250 mg/kg once orally for 21 days (Alkreathy et al., 2010) and a single dose of normal saline (0.5 mL/rat, intraperitoneally (i.p.)) on Day 16. Group III (CP-treated): Animals received CP at a dose of 7.5 mg/kg once i.p. on Day 16 (Mansour et al., 2006; Nasr, 2013). Group IV (combined AGE 1 CPtreated): Animals received AGE (250 mg/kg once orally for 21 days) 1 a single dose of CP (7.5 mg/kg i.p.) on Day 16. The rats of all groups were frequently weighed on Days 1, 3, 7, 10, 13, 16, 19, and 22. Drugs and Chemicals CP was obtained in the form of commercial Egyptian Unistin Vial (Egyptian International medical Company (EIMC) United Pharmaceuticals, Cairo, Egypt). AGE (kyolic) was obtained from Wakunaga of America (Mission Viejo, CA). AGE used in this study contained 28.6% extracted solids (286 mg/mL), and S-allyl cysteine, the most abundant water-soluble compound in AGE, was present at a concentration of 1.47 mg/mL. Samples Collection and Biochemical Assays On Day 22 (6 days after CP injection), the rats were anesthetized by ether inhalation. Blood samples were collected through a direct intracardiac puncture from each rat in sterile labeled heparinized test tubes. The sera were separated and stored at 220 C for subsequent assessment of kidney functions. The kidneys of each rat were excised, cleaned, washed with normal saline, blotted with filter paper, and weighed. Relative kidney weight/body weight ratio was calculated accord-
ing to the following formula: organ weight/body weight ratio (%) 5 organ weight 3 100/body weight (Arhoghro et al., 2012). Assessment of Kidney Functions Serum creatinine, urea, and uric acid were measured (Dickey et al., 2005) using standard laboratory techniques and using the commercially available diagnostic kits by an auto analyzer (BT 300, Japan). Determination of Lipid Peroxidation and Antioxidant Enzymes A portion from each rat’s kidneys was minced and homogenized in ice-cold phosphate buffer saline (0.05 M, pH 7.4). A part of the homogenate was mixed with an equal volume of 10% trichloroacetic acid and centrifuged at 3,000 rpm for 15 min at 4 C. The supernatant was used to determine the content of the reduced GSH and malondialdehyde (MDA) enzyme (total lipid peroxidase). The remaining part of the homogenate was centrifuged at 17,000g for 60 min at 4 C, and the supernatant was used for estimation of the activity of both SOD and CAT enzymes. Lipid peroxidation was determined by estimating the level of thiobarbituric acid reactive substances that was used to measure the extent of MDA formed as a result of membrane lipid peroxidation, according to the method of Ohkawa et al. (1979), using commercial kits (Biodiagnostic, Cairo, Egypt). Antioxidant markers GSH reductase, SOD, and CAT were measured by a colorimetric method using commercial kits (Biodiagnostic) according to the methods of Carlberg and Mannervik (1985), Sun et al. (1988), and Aebi (1984), respectively. Tissue Processing Light Microscopy. Renal specimens were fixed in 10% neutral-buffered formalin solution for 48 h, dehydrated in ascending grades of ethyl alcohol, cleared in xylol, and embedded in paraffin blocks. Serial sections (3–5 mm) were cut using microtome. The sections were washed in a water bath, left in the oven for dewaxing, and were stained with hematoxylin and eosin, periodic acid Schiff (PAS), and Masson’s trichrome stains (Bancroft and Gamble, 2002). The stained tissue slides were mounted with DPX and covered with cover slips to examine by a light microscope. Electron Microscopy. . Specimens of 1 mm3 size from each kidney were immediately immersed in 2.5% glutaraldehyde buffered with 0.1 M phosphate buffer for 24–48 h. Then, the specimens were washed in phosphate buffer (pH 7.2–7.4) three to four times for 20 min every time and postfixed in a buffered solution of 1% osmium tetroxide for 2 h, and then washed in the same buffer four times for 20 min each. After fixation, the specimens were dehydrated in ascending grades of ethyl alcohol. Then, they were cleared in propylene oxide, embedded in a mixture of 1:1 of Epon resin– araldite for 1 h. Polymerization was performed in the oven at 65 C for 24 h (Hayat, 2000). Semi-thin sections (1 mm thick) were cut using a glass knife on LKB2000S ultramicrotome, mounted on glass slides, and stained with buffered toluidine blue. The appropriate areas were selected with the light microscope. The resin blocks were trimmed to get rid of the undesired Microscopy Research and Technique
GARLIC EXTRACT AND CISPLATIN NEPHROTOXICITY
3
TABLE 1. Effect of cisplatin (CP) and/or aged garlic extract (AGE) on body and kidney weights of rats Body weight Animal group
Initial BW
Control AGE Cisplatin AGE 1 CP
Kidney weight
Final BW
Changes
a,b
267.2 6 2.4 268 6 2.24 302.5 6 3.9 290.7 6 2.14
140.7 6 1.93 138.3 6 1.05 217.3 6 1.43 24.3 6 0.33
307.8 6 2.7 306.3 6 2.3a,b 284.8 6 4.5b,c 286.3 6 2.3c,d
Absolute
Relative (3100) a
2.38 6 0.026 2.38 6 0.032a 2.43 6 0.018c 2.38 6 0.018a
0.774 6 0.001a 0.771 6 0.0001a 0.853 6 0.001c 0.831 6 0.009a
Data are expressed as mean 6 SEM (n 5 6). a Significant difference vs. CP-treated group at P < 0.005. b Significant difference between initial and final body weights (BW) of same group at P < 0.001. c Significant difference vs. control group at P < 0.01. d No significant difference between initial and final BW (P 5 0.197).
TABLE 2. Effects of cisplatin (CP) and/or aged garlic extract (AGE) treatment on kidney biomarkers of rats Control Creatinine (mg/dL) Uric acid (mg/dL) Urea (mg/dL)
AGE a
0.51 6 0.003 3.68 6 0.23a 25.7 6 1.23a
0.62 6 0.005 3.66 6 0.22a 26.5 6 0.99a
CP a,b
AGE-CP b
2.3 6 0.005 7.9 6 0.42b 66.8 6 2.26b
1.42 6 0.008a,b 5.7 6 0.2a,b* 43.2 6 1.56a,b
Data are expressed as mean 6 SEM (n 5 6). a Significant difference vs. CP-treated group at P < 0.001. b Significant difference vs. control group at P < 0.001. (a*: P < 0.005) AGE: aged garlic extract; CP: cisplatin.
tissue. Ultrathin sections (60–90 nm thick) were cut with a glass knife on a LKB ultramicrotome, mounted on copper grids, and double stained with uranyl acetate and lead citrate. The grids were examined and photographed using a transmission electron microscope (JEOL TEM-1200 EX, Tokyo, Japan) operated at 60–70 kV (Electron Microscope Unit, Faculty of Science, Ain Shams University, Egypt). Statistical Analyses Results were expressed as mean 6 SEM. Comparison of means was done by the Student’s t-test, one-way analysis of variance, and Mann–Whitney U-test. Values of P < 0.05 were considered statistically significant. Statistical evaluation was conducted with SPSS version 16.0 (SPSS, Chicago, IL). RESULTS Body Weight The final body weight of CP-treated rats revealed a significant reduction compared with both control and AGEtreated groups. In addition, the final body weight of control group exhibited a significant difference compared with those of CP-treated and combined AGE 1 CPtreated groups. However, insignificant change in the final body weight of the combined AGE 1 CP-treated rats was observed compared with that of CP-treated rats (Table 1). Kidney Weight Both absolute and relative kidney’s weights of CPtreated rats showed a significant difference compared with those of the other three groups, whereas the absolute and relative kidney weights of the control rats showed a significant difference compared with those of CP-treated rats only (Table 1). Biochemical Results Kidney Biomarkers. A significant increase in the serum level of kidney biomarkers was observed in CPtreated rats compared with those of the other three Microscopy Research and Technique
groups. However, the serum level of such biomarkers of control rats revealed no significant difference compared with those of AGE-treated rats. In addition, a significant reduction in the serum level of such biomarkers was observed in the control rats compared with those of CPtreated and combined AGE 1 CP-treated rats (Table 2). Lipid Peroxidation and Antioxidant Enzymes. MDA level in renal tissues of CP-treated rats displayed a significant increase compared with control, AGE-treated, and combined AGE 1 CP-treated rats. However, GSH level and SOD and CAT activities revealed a significant reduction in renal tissues of CPtreated rats compared with the other three groups. Moreover, a significant increase in GSH level and CAT and SOD activities accompanied with significant reduction in MDA level in the renal tissues of rats pretreated with AGE compared with that of control and CP-treated groups. However, the renal tissue of AGEtreated rats revealed no significant difference in MDA and GSH levels and the activity of SOD and CAT compared with that of control rats (Table 3). Histopathological Results Light microscopic examination of renal parenchyma revealed normal corpuscular and tubular structures in control (Fig. 1A) and AGE-treated (Fig. 1B) animals. However, in CP-treated rats, glomerular congestion and atrophy, degeneration, necrosis, desquamation with shedding of the apical microvilli of the proximal tubular epithelial lining cells, luminal dilation with excessive accumulation of homogenous casts in both proximal convoluted tubules (PCT) and distal convoluted tubules (DCT), intertubular hemorrhage, and mononuclear cells infiltration were observed (Fig. 1C). In the AGE-pretreated rats, degenerated few proximal tubules, dilated tubular lumen with accumulation of hyaline cast, and mononuclear cells infiltration were observed in the renal parenchyma (Fig. 1D). In PAS-stained renal sections, strong-positive reaction was observed in the brush border of PCT, basement
4
A.Y. NASR AND A.A.S. IBRAHIM
TABLE 3. Effects of aged garlic extract (AGE) administration on level of malondialdehyde (MDA), activity of superoxide dismutase (SOD), level of reduced glutathione (GSH), and activity of catalase (CAT) enzymes in renal tissues of rats treated with cisplatin (CP) Animal group (n 5 6 rats) Control AGE CP AGE 1 CP
MDA (nmol/mg protein) a
4.03 6 0.12 4.02 6 0.11a 9.17 6 0.19b 5.9 6 0.14a,b
SOD (U/mg protein) a
49.7 6 2.11 48.2 6 1.9a 21 6 1.63b 36 6 1.93a,b
CAT (U/mg protein) a
41.7 6 1.9 41.5 6 1.7a 29.5 6 1.3b 36.2 6 1.23*, **
GSH (U/mg protein) 15.5 6 1.3a 15.3 6 1.1a 7.9 6 0.84b 12.2 6 0.71, 11
Values are expressed as mean 6 SEM (n 5 6). a Significant difference vs. control group at P < 0.001. b Significant difference vs. CP-treated group at P < 0.001. *P 5 0.036; **P 5 0.0038; 1P 5 0.049; 11P 5 0.003. No significant difference was recorded between control and AGE-treated groups in all parameters (P 5 0.61–0.99).
Fig. 1. Light micrographs of rat kidney showing normal structure in control (A) and AGE-treated (B) groups. Glomerular atrophy (G), wide Bowman’s capsular space (C), degeneration (*) and exfoliation (arrow head) of the epithelial cells lining most of the proximal tubules (P), accumulation of hyaline cast (arrow) within the wide lumina (L) of both proximal (P) and distal (D) convoluted tubules, and extensive
inflammatory cell infiltrations (I) are noticed in CP-treated rats (C). Degenerative epithelial changes (*) and wide lumina (L) with accumulation of homogenous hyaline casts (arrow) are observed in a few of proximal tubules (P) in combined AGE and CP-treated rats (D). Hematoxylin and eosin stain was used. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
membrane of both renal tubules, and outer layer of glomerular capsule in control (Fig. 2A) and AGE-treated (Fig. 2B) rats. However, weak-positive PAS reaction was observed in the remaining brush border of the nonaffected PCT and the basement membrane of both proximal and distal renal tubules in CP-treated rats (Fig. 2C), whereas positive PAS reaction was noticed in the regenerated brush border of many PCT and the basement membrane of renal tubules and outer capsular membrane in the combined AGE 1 CP-treated rats (Fig. 2D). In Masson’s trichrome-stained renal sections, excessive connective tissues were observed around the blood
vessels, and wide vascular spaces were noticed in CPtreated rats (Fig. 3C), whereas little amount of connective tissues were observed around the vascular space and blood vessels within the cortical structure of the kidney in control (Fig. 3A), AGE-treated (Fig. 3B), and combined AGE 1 CP-treated (Fig. 3D) rats. Electron Microscopic Observations The glomerular and capsular structures of the renal corpuscles revealed normal ultrastructure in control (Fig. 4A) and AGE-treated (Fig. 4B) rats, whereas fused foot processes, dilated congested capillary loops Microscopy Research and Technique
Fig. 2. Light micrographs of renal cortex of rat showing a strongpositive PAS reaction in the brush border (arrow), basement membrane of renal tubules (arrow head), and outer layer of Bowman’s capsule (*) in control (A) and AGE-treated (B) rats. Weak-positive PAS reaction is seen in the remaining brush border (arrow) of a few proximal convoluted tubules (P) and in the basement membrane of renal tubules (arrow head) in cisplatin-treated rats (C). A strong-positive
PAS reaction in the brush border (arrow) of regenerated proximal tubules (P), basement membrane of renal tubules (arrow head), and outer layer of Bowman’s capsule (*) in combined AGE 1 cisplatintreated rats (D). C: Bowman’s capsule; G: glomerulus; P: proximal convoluted tubules; D: distal convoluted tubules. PAS stain was used. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 3. Light micrographs of renal cortex of rat showing excessive collagen fibers (R) around the blood vessels (A) and vascular spaces (VS) within the renal parenchyma of cisplatin-treated rats (C), whereas a little amount of collagen fibers (R) are seen within the renal parenchyma around the vascular spaces (VS) and blood vessels
(A) of control (A), AGE-treated (B), and combined AGE 1 cisplatintreated (D) rats. Masson’s trichrome stain was used. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
6
A.Y. NASR AND A.A.S. IBRAHIM
Fig. 4. Electron micrographs of the renal corpuscles of rat kidney showing normal outer parietal cells (Pe) and inner podocytes (Pc) with primary (P1) and secondary (P2) foot processes, glomerular capillaries (C) having thin regular basement membrane (m), and endothelial cells lining (E) with mesangial cells (MC) in-between in control (A) and AGE-treated (B) rats. Fusion (*) of the secondary foot
processes (P2), mesangial hyperplasia (MC), and thick irregular basement membrane (m) of the glomerular capillary (C) are seen in cisplatin-treated rats (C). Congested glomerular capillaries (C) are seen in combined AGE and cisplatin-treated rats (D). F: fenestrations between the foot processes. B: blood cells. Uranyl acetate and lead citrate stains were used.
with irregular thick basement membrane, and mesangial cell hyperplasia with excessive deposition of mesangial matrix were observed in the renal corpuscles of CP-treated rats (Fig. 4C). However, dilated congested glomerular capillaries with uniform thickness of their basal lamina were observed in the AGE-pretreated rats (Fig. 4D). The epithelial cells lining the PCT exhibited normal ultrastructure in control (Fig. 5A) and AGE-treated (Fig. 5B) rats. However, thick irregular basement membrane, loss of basal infoldings, fragmented apical microvilli, degenerated cell organelles, presence of many lysosomes, and apoptotic nuclear changes were observed in the PCT of CP-treated rats (Fig. 5C). However, in the AGE-pretreated rats, normal nuclei, long apical microvilli, few lysosomes, and cytoplasmic vacuoles were seen in the proximal tubular epithelial cells (Fig. 5D).
The DCT showed normal ultrastructure in control rats (Fig. 6A) and AGE-treated rats (Fig. 6B). However, the DCT of CP-treated rats showed a few small sized round-shape mitochondria, presence of many lysosomes, and basal cytoplasmic vacuoles (Fig. 6C), and the DCT of the combined AGE and CP-treated rats revealed regular basement membrane with numerous basal infoldings, regular apical nuclei, intact intercellular tight junctions, basal oblong mitochondria, a few lysosomes, and apical cytoplasmic vesicles (Fig. 6D). DISCUSSION CP is a potent antineoplastic drug. Its clinical use, however, revealed nephrotoxic side effect (Ajith et al., 2007). In this study, a significant reduction in the body weight and the absolute kidney weight with increases reno-somatic index was observed in the rats, 6 days Microscopy Research and Technique
GARLIC EXTRACT AND CISPLATIN NEPHROTOXICITY
7
Fig. 5. Electron micrographs of proximal tubular epithelial cells of rat kidney showing normal ultrastructure in control (A) and AGEtreated (B) rats. Marked destruction (*) of the apical microvilli (mv), many lysosomes (Ly) and cytoplasmic vacuoles (V), thick irregular basement membrane (BM), numerous small-sized electron-dense degenerated mitochondria (M), and irregular nucleus (N) with
peripheral condensation of its heterochromatin (h) on the nuclear inner aspect of the envelop (ne) are seen in cisplatin-treated rats (C). Regular nucleus (N), numerous apical microvilli (mv), many round mitochondria (M), and few lysosomes (Ly) are seen in combined AGE and cisplatin-treated rats (D). Uranyl acetate-lead citrate Stains
after a single intraperitoneal injection of CP. Abdelmaguid et al. (2010), Anusuya et al. (2013), Arhoghro et al. (2012), Chirino et al. (2004), and Nasr (2013) showed a significant reduction in the body and absolute kidney weights, 3–6 days after a single (5–7.5 mg/kg) intraperitoneal injection of CP. The reduction in the body weight in CP-treated rats might be due to the direct toxic effect of CP on renal tubules, with subsequent polyuria and dehydration (Ali and Al Moundhri, 2006; Azu et al., 2010; Yao et al., 2007), or due to gastrointestinal toxicity with subsequent decrease in appetite, ingestion, and assimilation of food (Arhoghro et al., 2012; Yao et al., 2007). The reduction in the absolute kidney weight in CP-treated rats could be due to the structural damage and/or reduction in functions of renal tissues (Park et al., 2009). However, the increase in reno-somatic index might be due to the
edema of renal parenchyma because CP is known to cause renal inflammation (Adejuwon et al., 2014; Anusuya et al., 2013; Mansour et al., 2006). On the other hand, an improvement in CP-induced reduction of the body weight was observed in the rats pretreated with AGE. Such effect indicates that AGE might exhibit a protective effect against the toxicity of CP on renal and intestinal tissues. The protective effect of AGE could be attributed to its anti-inflammatory effect that decreased the renal edema induced by CP (Anusuya et al., 2013). Serum creatinine, uric acid, and urea levels were considered the main parameters that determine the glomerular filtration rate (Azu et al., 2010). In this study, a significant elevation of renal biomarkers was recorded in CP-treated rats. Similar findings were previously reported in different studies (Arhogro et al.,
Microscopy Research and Technique
8
A.Y. NASR AND A.A.S. IBRAHIM
Fig. 6. Electron micrographs of the distal tubular epithelial cells of rat kidney showing regular basement membrane (BM), nuclei (N) with regular outline, oblonged basal mitochondria (M) between the basal infoldings (f), and few lysosomes (Ly) in control (A) and AGEtreated (B) rats. Thick basement membrane (BM), irregular nucleus (N), degenertaed mitochondria (M), and numerous lysosomal
vacuoles (V) are seen within the cytoplasm are seen in cisplatintreated rats (C). Regular basement membrane (BM) with many basal infoldings (f), regular oval nucleus (N), many round basal mitochondria (M), few apical lysosomes (Ly), and apical tight junctions (J) are seen in combined AGE and cisplatin-treated rats (D). L: tubular lumen. Uranyl acetate and lead citrate stains were used.
2012; Azu et al., 2010; Mansour et al., 2006; RazoRodrıguez et al., 2008). The elevation in the serum levels of renal biomarkers might be in part due to impaired renal function (Anusuya et al., 2013), tubular obstruction with back-leakage of the renal tubules (Azu et al., 2010), inhibition of protein synthesis in the tubular cells (Adejuwon et al., 2014), or the direct toxic effect of CP on the glomerular and tubular structures through the generation of ROS and enhancement of lipid peroxidation in renal tubules (Ajith et al., 2007; Mansour et al., 2006; Saad et al., 2009; Yao et al., 2007). In the animals pretreated with AGE, a remarkable improvement in the serum level of the renal biomarkers was recorded. These results confirm the ability of AGE to improve the renal functions through its
antioxidant and free radical scavenger activities (Anusuya et al., 2013). In agreement with our results, Abd El-Halim and Mohamed (2012), Alkreathy et al. (2010), Anusuya et al. (2013), and Razo-Rodrıguez et al. (2008) reported that feeding of AGE provided a protective effect in rats. In this study, significant increase in lipid peroxidation accompanied with marked reduction in level and activity of the antioxidant enzymes in the CP-treated animals, whereas pretreatment with AGE produced a remarkable improvement in level and activity of renal antioxidant enzymes, with a reduction in lipid peroxidation. These findings provide evidence about the antioxidant effect of AGE against CP-induced oxidative stress and lipid peroxidation. The results of our study were supported by the findings that were reported by Ajith Microscopy Research and Technique
GARLIC EXTRACT AND CISPLATIN NEPHROTOXICITY
et al. (2007), Mansour et al. (2006), and Razo-Rodrıguez et al. (2008), who stated that reduction in the activity of antioxidant enzymes (CAT and SOD) increased lipid peroxidation (MDA), and depletion of GSH in renal tissues was implicated in the pathogenesis of CP toxicity. Many studies have demonstrated that AGE exhibited a protective effect against different toxic agents through its powerful antioxidant and free-radical scavengers (Abd El-Halim and Mohamed, 2012; Alkreathy et al., 2010; Anusuya et al., 2013; Capasso, 2013). Consistent with the findings of this study, shedding of the apical microvilli, exfoliation and degeneration of the epithelial cells lining the PCT, and accumulation of homogenous cast were observed in rats exposed to different doses of CP (Abdelmaguid et al., 2010; Aleisa et al., 2007; Ali and Al Moundhri, 2006; Ali et al., in press; Chirino et al., 2004; Mansour et al., 2006; Nasr, 2013; Tufekci et al., 2009). Thus, the histopathological manifestations confirmed the biochemical changes. Moreover, in agreement with our ultrastructural findings, Abdelmaguid et al. (2010), Ali and Al Moundhri (2006), Chirino et al. (2004), and Nasr (2013) found fusion of foot processes of podocytes, mesangial cells proliferation with increase mesangial matrix, degeneration of the proximal tubular cells with distinct nuclear apoptotic changes, irregularity of tubular basement membrane, and loss of basal infoldings in renal tissues of CP-treated animals. In addition, Pratibha et al. (2010) stated that partial or complete shedding of the apical microvilli induced impairment in amino acids and glucose absorption. The authors added that presence of many mitochondria of variable length with dense matrix along basolateral folding might be due to a mitochondrial response to the direct stress of CP on renal tissues. Concomitant with the results of this study, the ultrastructure of renal tissues in AGE-pretreated rats revealed normal glomeruli, preserved apical microvilli, and restoration of nuclear structure of the proximal tubular epithelial cells (Benedetti et al., 2013; Tufekci et al., 2009). Also, no evidence of inflammatory cells infiltration was observed in AGE-pretreated animals (Tufekci et al., 2009). The author added that absence of inflammatory cells infiltration in renal tissues of the animal pretreated with AGE could be considered a proof of the anti-inflammatory effect of AGE. Thus, the inflammation could be considered as a mechanism in CP-induced nephrotoxicity (Benedetti et al., 2013). Apoptosis is an important way of cell death in normal and pathological processes. In this study, different nuclear apoptotic changes, in the form of membrane budding, cell shrinkage, and marginal condensation of chromatin were observed in CP-treated renal tissue. Disappearance of such morphological changes in the renal tissues of AGE-pretreated rats gives a clue evidence of antiapoptotic property of AGE.
CONCLUSION The findings of this study revealed that AGE has protective effects against CP-induced nephrotoxicity mostly through its antioxidant, anti-inflammatory, and antiapoptotic properties. However, the effect of AGE on Microscopy Research and Technique
9
antitumor activity of CP was not elaborated in this study. So, this point can be investigated in another study. ACKNOWLEDGMENT The authors thank the staff members of Electron Microscopic Centre, Faculty of Science, Ain Shams University, Egypt, for their technical support. REFERENCES Abd El-Halim SS, Mohamed MM. 2012. Garlic powder attenuates acrylamide-induced oxidative damage in multiple organs in rat. J Appl Sci Res 8:168–173. Abdelmaguid EN, Hania NC, Noura SA. 2010. Protective effect of silymarin on cisplatin-induced nephrotoxicity in rats. Pak J Nutr 9:624–636. Adejuwon AS, Femi-Akinlosotu OM, Omirinde JO, Owolabi OR, Afodun AM. 2014. Launaea taraxacifolia ameliorates cisplatininduced hepato-renal injury. Eur J Med Plant 4:528–541. Aebi H. 1984. Catalase in vitro. Methods Enzymol 105:121–126. Ajith TA, Usha S, Nivitha V. 2007. Ascorbic acid and alpha tocopherol protect anticancer drug cisplatin induced nephrotoxicity in mice: A comparative study. Clin Chim Acta 375:82–86. Aleisa AM, Al-Majed AA, Al-Yahya AA, Al-Rejaie SS, Bakheet SA, AlShabanah OA, Sayed-Ahmed MM. 2007. Reversal of cisplatininduced carnitine deficiency and energy starvation by propionyl-lcarnitine in rat kidney tissues. Clin Exp Pharmacol Physiol 34: 1252–1259. Ali BH, Al Moundhri MS. 2006. Agents ameliorating or augmenting the nephrotoxicity of cisplatin and other platinum compounds: A review of some recent research. Food Chem Toxicol 44:1173–1183. Ali DA, Abdeen AM, Ismail MF, Mai AM. 2013. Histological, ultrastructural and immunohistochemical studies on the protective effect of ginger extract against cisplatin-induced nephrotoxicity in male rats. Toxicol Ind Health. Alkreathy H, Zoheir AD, Nessar A, Mark S, Soad SA, Osman AM. 2010. Aged garlic extract protects against doxorubicin-induced cardiotoxicity in rats. Food Chem Toxicol 48:951–956. Anusuya N, Durgadevi P, Dhinek A, Mythily S. 2013. Nephroprotective effect of ethanolic extract of garlic (Allium sativum) on cisplatin induced nephrotoxicity in male Wistar rats. Asian J Pharm Clin Res 6:97–100. Arhoghro EM, Kpomah DE, Uwakwe AA. 2012. Ocimum gratissimum aqueous extract enhances recovery in cisplatin-induced nephrotoxicity in albino Wistar rats. Indian J Drugs Dis 1:129–142. Azu OO, Duru FIO, Osinubi AA, Noronha CC, Elesha SO, Abayomi OO. 2010. Protective agent, Kigelia africana fruit extract, against cisplatin induced kidney oxidant injury in Sprague-Dawley rats. Asian J Pharm Clin Res 3:54–58. Bancroft JD, Gamble M. 2002. Theory and practice of histological techniques, 5th ed. Edinburgh: Churchill Livingstone. pp. 125– 138, 172–175, 184–193, 593–620. Benedetti G, Fredriksson L, Herpers B, Meerman J, van de Water B, de Graauw M. 2013. TNF-a-mediated NF-jB survival signaling impairment by cisplatin enhances JNK activation allowing synergistic apoptosis of renal proximal tubular cells. Biochem Pharmacol 85:274–286. Bergs JW, Franken NA, Haveman J, Geijsen ED, Crezee J, Van Bree C. 2007. Hyperthermia, cisplatin and radiation trimodality treatment: A promising cancer treatment? A review from preclinical studies to clinical application. Int J Hyperthermia 23:329–341. Bongiorno PB, Fratellone PM, Pina L. 2008. Potential health benefits of garlic (Allium sativum): A narrative review. J Complement Integr Med 5:1–26. Capasso A. 2013. Antioxidant action and therapeutic efficacy of Allium sativum L. Molecules 18:690–700. Carlberg I, Mannervik B. 1985. Glutathione reductase. Methods Enzymol 113:484–490. Chirino YI, Hernandez-Pando R, Pedraza-Chaverri J. 2004. Peroxynitrite decomposition catalyst ameliorates renal damage and protein nitration in cisplatin-induced nephrotoxicity in rats. BMC Pharmacol 4:20. Colin-Gonzalez AL, Santana RA, Silva-Islas CA, Chanez-Cardenas ME, Santamarıa A, Perla DM. 2012. The antioxidant mechanisms underlying the aged garlic extract- and S-allylcysteine-induced protection. Oxid Med Cell Longev 2012:article ID 907162. Dickey DT, Wu YJ, Muldoon LL, Neuwelt EA: Protection against cisplatin-induced toxicities by N-acetylcysteine and sodium thiosulfate as assessed at the molecular, cellular, and in vivo levels. J Pharmacol Exp Ther 2005, 314:1052–1058.
10
A.Y. NASR AND A.A.S. IBRAHIM
Hanigan MH, Devarajan P. 2003. Cisplatin nephrotoxicity: Molecular mechanisms. Cancer Ther 1:47–61. Hayat MA. 2000. Chemical fixation. In: Hayat MA, editor. Principles and techniques of electron microscopy: Biological applications, 4th ed. New York: Cambridge University Press. pp. 4–85. Mansour HH, Hafezm HF, Fahmy NM. 2006. Silymarin modulates cisplatin 10 mg kg1 induced oxidative stress and hepatotoxicity in rats. J Biochem Mol Biol 39:656–661. Nasr AY. 2013. Effect of misoprostol on ultrastructural changes of renal tissues in cisplatin- treated adult rats. J Cytol Histol 4:3. Ohkawa H, Ohishim N, Yagim K. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358. Park HR, Ju EJ, Jo SK, Jung U, Kim SH, Yee ST. 2009. Enhanced antitumor efficacy of cisplatin in combination with HemoHIM in tumor-bearing mice. BMC Cancer 9:85. Pratibha R, Dayanand A, Bhiwgade, Sameer K, Padmanabh V, Rataboli PV, Chitra YD. 2010. Cisplatin induced histological changes in renal tissue of rat. J Cell Anim Biol 4:108–111.
Razo-Rodrıguez AC, Chirino YI, S anchez-Gonz alez DJ, MartınezMartınez CM, Cruz C, Pedraza-Chaverri J. 2008. Garlic powder ameliorates cisplatin-induced nephrotoxicity and oxidative stress. J Med Food 11:583–586. Saad AA, Youssef MI, El-Shennawy LK. 2009. Cisplatin induced damage in kidney genomic DNA and nephrotoxicity in male rats: The protective effect of grape seed proanthocyanidin extract. Food Chem Toxicol 47:1499–1506. Sun Y, Oberley LW, Li Y. 1988. A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500. Tufekci O, Gunes D, Ozo gul C, Kolatan E, Altun Z, Yilmaz O, Aktas¸ S, Erbayraktar Z, Kirkim G, Mutafo glu K, Soylu A, Serbetc¸io glu B, G€ uneri EA, Olgun N. 2009. Evaluation of the effect of acetyl l-carnitine on experimental cisplatin nephrotoxicity. Chemotherapy 55:451–459. Yao X, Panichpisal K, Kurtzman N, Nugenmt K. 2007. Cisplatin nephrotoxicity: A review. Am J Med Sci 334:115–124.
Microscopy Research and Technique
