http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, 2014; 36(7): 1095–1103 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2014.918812
LABORATORY STUDY
Protective effect of administration of Withania somifera against bromobenzene induced nephrotoxicity and mitochondrial oxidative stress in rats Mahima Vedi, Mahaboobkhan Rasool, and Evan Prince Sabina
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School of Bio Sciences and Technology, VIT University, Vellore, India
Abstract
Keywords
Background: The present study was conducted to elucidate the protective role of Withania somnifera against bromobenzene induced nephrotoxicity and mitochondrial dysfunction in rats. Methods: In this study, Wistar albino rats of either sex were divided into six groups consisting of six animals each. The first one was control, those in group II received bromobenzene (10 mmol/kg, intragastric intubation) once, but group III and IV animals received W. somnifera (250 and 500 mg/kg, orally), respectively for 8 days followed by bromobenzene once on the 8th day and silymarin (100 mg/kg, orally) was given for 8 days to group V animals and then bromobenzene on the last day. Group VI animals received only W. somnifera (500 mg/kg) for 8 days. Results: The levels of renal lipid peroxidation, lysosomal enzymes and glycoprotein were increased significantly (p50.05) in the bromobenzene alone treated rats and antioxidant status and mitochondrial enzymes were found to be decreased, when compared to the control group. The levels of kidney functional markers (urea, uric acid and creatinine) were also found to be abnormal in serum of bromobenzene alone treated rats. On the other hand, administration of W. somnifera (250 and 500 mg/kg) along with bromobenzene offered a significant dose-dependent protection to the biochemical alterations as observed in the bromobenzene alone treated rats, which was also evidenced by histopathology. Conclusion: Thus, the oral administration of W. somnifera significantly protected against the bromobenzene induced nephrotoxicity and renal mitochondrial dysfunction in rats.
Antioxidant, glutathione, lipid peroxidation, nephroprotective
Introduction Bromobenzene (BB) is an aryl halide (C6H5Br) that has profound application in industries. It represents a good model for toxicity studies as it causes necrosis in liver and kidney1 and the characteristics of the response induced by bromobenzene could also be helpful in understanding the toxicity induced by a variety of xenobiotics. Bromobenzene gets biotransformed in the liver to form reactive intermediates, which initially can be detoxified by conjugation with glutathione, thereby depleting it and eventually binds to cellular macromolecules. The secondary metabolites of bromobenzene such as bromophenoisomers, 4-bromocatechol, 2-bromohydroquinone and benzoquinone that are formed in the hepatic phase II reactions2 contribute to oxidative stress and nephrotoxicity. Most importantly, there is increased formation of reactive oxygen inside the mitochondria due to extensive depletion of cytosolic and mitochondrial glutathione levels during the
Address correspondence to Evan Prince Sabina, School of Bio Sciences and Technology, VIT University, Vellore 632014, India. Tel: +914162202324, +919080494445; E-mail:
[email protected] History Received 26 December 2013 Revised 6 March 2014 Accepted 24 April 2014 Published online 19 May 2014
bromobenzene metabolism.3 One possible protective strategy would be to enrich tissue mitochondria with antioxidants thereby limiting mitochondrial oxidative damage which has been proven by several studies.4–6 Withania somnifera or Ashwagandha or Indian Ginseng (dunal Solanaceae) is mainly cultivated in drier parts of India and all over the world. It has been found to exhibit antioxidant,7 anti-arthritic,8 anti-cancer,9 anti-inflammatory10 and anti-ageing properties.7 Furthermore, it has curative effects against carbendazim-induced histopathological changes in liver and kidney of rats11 and preventive effects against gentamicin induced nephrotoxicity.12 These properties may be due to the presence of various constituents such as alkaloids (isopellertierine, anserine), steroidal lactones (withanolides, withaferins), saponins, withanoloides and iron.13 The roots of W. somnifera are proven to be most therapeutically active among all the other parts of this plant and exhibit free radical scavenging and antioxidant properties.8 From the above evidences of antioxidant and protective effects of W. somnifera, the present study was carried out to investigate possible protective effect of W. somnifera on bromobenzeneinduced renal damage in rats.
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Materials and methods Drugs and chemicals Commercially available Ashwagandha (W. somnifera) root powder was obtained from Indian Medical Practitioner’s Co-operative Stores and Society, Chennai, Tamilnadu, India. Silymarin was obtained from Natural Remedies Private Limited, Bangalore, India. All other reagents used were standard laboratory reagents of analytical grade. The optimum dosage of W. somnifera,10 silymarin14 and bromobenzene15 was selected on the basis of previous studies.
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Test animals The study was performed using Wistar albino rats of either sex, having a mean weight of 150 g, procured from VIT animal house, VIT University, Vellore, Tamil Nadu, India. The rats were fed commercial pelleted feed from Hindustan Lever Ltd. (Mumbai, India) and water was provided ad libitum. The animals were well treated and cared for in accordance of the guidelines recommended by the as per the guidelines of the Guide for the Care and Use of Laboratory Animals (National Institute of Health Publication). The experimental procedure was approved by the ethical committee (VIT/IAEC/VIIth/17) of VIT University, Vellore, India. Experimental design Animals were allocated randomly in six groups of six animals each and all experimental rats except group I and group VI received bromobenzene dosage (intragastric tube) only once. Aqueous suspensions of each of silymarin (100 mg/kg b.w.) and W. somnifera (250 and 500 mg/kg b.w.) root powder were made in double distilled water for administration to rats. Rats were made to fast for 24 h before and 19 h after the last dosage before the sacrifice. Group I. Control, which received 0.1 mL of coconut oil by intragastric intubation for 8 days. Group II. Received bromobenzene once only (10 mmol/kg in 0.1 mL coconut oil) by intragastric intubation. Group III. Received W. somnifera (250 mg/kg, orally) for 8 days and then bromobenzene once only (10 mmol/kg body weight in 0.1 mL coconut oil) by intragastric intubation on the 8th day. Group IV. Received W. somnifera (500 mg/kg respectively, orally) for 8 days and then bromobenzene once only (10 mmol/kg in 0.1 mL coconut oil) by intragastric intubation on the 8th day. Group V. Received silymarin (100 mg/kg, orally) for 8 days and bromobenzene once only (10 mmol/kg body weight, in 0.1 mL coconut oil) by intragastric intubation on the 8th day Group VI. Received W. somnifera (500 mg/kg, orally) for 8 days At the end of the experimental period, all the animals were decapitated 19 h after the last dosage under ether anesthesia; the trunk blood was collected and allowed to coagulate at ambient temperature for 30 min. Serum was separated by centrifugation at 175 g for 10 min. Kidneys were immediately removed and washed in ice-cold saline to
Ren Fail, 2014; 36(7): 1095–1103
remove the blood. The tissues were sliced and homogenized in 0.1 M Tris–HCl buffer (pH 7.0). The homogenates were centrifuged at 48 g for 10 min and supernatant was used for further analysis. Biochemical analysis Levels of cholesterol and triglycerides were determined in the serum and levels of urea, uric acid and creatinine were determined in the serum and 24 hour urine samples of control and experimental rats using respective kits (Autospan diagnostics, Bangalore, India) according to manufacturer’s protocol. Evaluation of oxidative stress Lipid peroxidation in the kidney tissue homogenate was determined by the method of Ohkawa et al.16 using malondialdehyde (MDA), which is the end product of lipid peroxidation and thus used as indicator for evaluation of oxidative stress. Superoxide dismutase (SOD) was assayed by the method of Marklund and Marklund.17 The activity of the enzyme was measured by using 50% inhibition of pyrogallol auto-oxidation. Catalase (CAT) was assayed according to the method of Sinha18 and chromic acetate produced by the action of catalase on dichromate acetic reagent was measured spectrophotometrically at 610 nm. Glutathione peroxidase (GPx) was assayed by the method of Rotruk et al.19 based on the reaction between glutathione remaining after the action of GPx 5,50 -dithiobis-(2-nitrobenzoic acid) to form a complex that has maximum absorption at 412 nm. Glutathione-s-transferase or GST20 was measured using 1-Chloro-2, 4-dinitrobenzene as substrate and reduced glutathione was evaluated by the method of Moron et al.21 Total protein was estimated using the method of Lowry’s et al.22 using bovine serum albumin as standard. Assay of lysosomal enzymes and glycoproteins N-acetyl glucosaminidase activity was assessed by the method of Maruhn23 and the activity of b-galactosidase was assessed by the method of Rosenblit et al.24 Cathepsin D activity was calculated by the method of Biber et al.25 using hemoglobin as the substrate and the activity expressed as mmol of tyrosine liberated/h/mg protein. The kidney tissue samples were defatted and weighed amount of defatted tissue was mixed in 3 mL of 2 M HCl and heated at 90 C for 4 h, which was then cooled and 3 mL of 2 M NaOH was added. This was used to obtain aliquots for the estimation of sialic acid and hexosamine contents. Sialic acid was measured using the method of Aminoff26 with Niebes27 modifications. Exactly 0.25 M periodate (in 0.1 N H2SO4) was added to a fraction of tissue homogenate, and the reaction was stopped after 30 min using arsenite solution. Then the contents were heated after addition of thiobarbituric acid and the pink color that developed due to the reaction was measured at 540 nm. For the estimation of hexosamine, acetyl acetone reagent consisting of trisodium phosphate and potassium tetraborate with acetyl acetone was added to tissue homogenate and the mixture was boiled. After cooling, Ehrlich’s reagent was added and the pink color development was measured at 540 nm.28
DOI: 10.3109/0886022X.2014.918812
Protective effect of W. somnifera against nephrotoxicity
Isolation of mitochondria
of oxaloacetate and 0.2 mL mitochondrial suspension was prepared. The control tubes contained all reagents except NADH. The change in OD at 340 nm was measured for 2 min at intervals of 15 sec in a UV spectrophotometer. The activity of the enzyme was expressed as nmol of NADH oxidized/min/mg protein.
For the isolation of kidney mitochondria (Johnson and Lardy),29 a 20% (w/v) homogenate was prepared in 0.25 M sucrose containing 0.05 M Tris–HCl buffer and 5.0 mM EDTA, which was centrifuged at 600 g for 10 min. The supernatant fraction was separated and then centrifuged at 10,000 g for 5 min at 4 C to bring down the mitochondrial pellet, which was re-suspended in KCl and used for the estimation of various parameters. Evaluation of mitochondrial enzymes
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Isocitrate dehydrogenase activity Exactly 0.4 mL of Tris–HCl, 0.2 mL of trisodium isocitrate, 0.2 mL of NADP and the required amount of enzyme was incubated for 60 min at 37 C, and then 1.0 mL of coloring reagent was added followed by 0.5 mL of EDTA. After 20 min 10 mL of NaOH was added and the color developed was read at 420 nm after 10 min. A calibration curve was established with a-ketoglutarate as standard. The isocitrate dehydrogenase activity was expressed as nmol of a-ketoglutarate formed/h/mg protein.30 -Ketoglutarate dehydrogenase activity The activity of a-ketoglutarate dehydrogenase was assayed by the method of Reed and Mukherjee.31 Exactly 0.15 mL of phosphate buffer and 0.1 mL each of thiamine pyrophosphate, magnesium sulfate, a-ketoglutarate and potassium ferricyanide were mixed. After making up the total volume to 1.2 mL with distilled water, 0.2 mL of mitochondrial suspension was added and incubated at 30 C for 30 min and the reaction was terminated by the addition of 1 mL of tricarboxylic acid (TCA). The tubes were centrifuged and 0.1 mL of potassium ferricyanide, 1 mL of duponol and 0.5 mL of ferric ammonium sulfate–duponol reagent were added to 1 mL of supernatant and then incubated at room temperature for 30 min. The color developed was measured at 540 nm. A standard containing potassium ferrocyanide was assayed simultaneously. The activity of a-ketoglutarate dehydrogenase was expressed as nmol of ferrocyanide formed/h/mg protein. Succinate dehydrogenase activity The activity of succinate dehydrogenase was assayed according to the method of Slater and Bonner.32 Exactly 1.0 mL of Phosphate buffer, 0.1 mL of EDTA, 0.1 mL of BSA, 0.3 mL of sodium succinate and 0.2 mL of potassium ferricyanide were mixed and made up to 2.8 mL with double-distilled water. Exactly 0.2 mL of mitochondrial suspension was added and the change in optical density (OD) was recorded at 15 sec intervals for 5 min at 420 nm. The succinate dehydrogenase activity was expressed as nmol of succinate oxidized/min/mg protein. Malate dehydrogenase activity The activity of malate dehydrogenase was assayed by the method of Mehler et al.33 Reaction mixture containing 0.75 mL of phosphate buffer, 0.15 mL of NADH, 0.75 mL
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Cytochrome c oxidase activity The activity of cytochrome c oxidase was assayed according to the method of Pearl et al.34 One milliliter of phosphate buffer, 0.2 mL of 0.2% N-phenyl-p phenylene diamine, 0.1 mL of 0.01% cytochrome c, 0.5 mL of distilled water were mixed and incubated at 25 C for 5 min. Mitochondrial suspension of 0.2 mL was added and the change in absorbance was recorded at 550 nm for 5 min at intervals of 15 sec. The enzyme activity was expressed as change in absorbance/min/mg protein. NADH dehydrogenase activity The enzyme activity of NADH dehydrogenase was assayed by the method of Minakami et al.35 The reaction mixture was prepared containing 1.0 mL of phosphate buffer, 0.1 mL of potassium ferricyanide, 0.1 mL of NADH and 0.2 mL of mitochondrial suspension. The total volume was made up to 3.0 mL with water. NADH was added just before the addition of the enzyme source (mitochondrial suspension). The change in absorbance was recorded at 420 nm for 3 min at 15 sec intervals. The activity of NADH dehydrogenase was expressed as nmol of NADH oxidized/min/mg protein. Histopathological examinations Immediately after sacrifice, a portion of the kidney was fixed in 10% formalin, then washed, dehydrated in descending grades of isopropanol and finally rinsed with xylene. The tissues were then embedded in molten paraffin wax. Sections were cut into 5 mm thickness, stained with hematoxylin and eosin and observed microscopically for histopathological changes. Statistical analysis All results were expressed as mean ± SD compared to normal control rats. The intergroup variation between various groups was measured by one way analysis of variance (ANOVA) using the Graph Pad Prism, version 5.0 (La Jolla, CA) and the comparisons between two groups were conducted by Student Newman-Keul’s test. Results were considered statistically significant when p50.05.
Results Effects of W. somnifera on biochemical parameters The protective effects of W. somnifera against bromobenzene induced nephrotoxicity were monitored by estimating the levels of creatinine, urea and uric acid in the serum and 24 hour urine samples of control and experimental rats. In the bromobenzene induced group, the levels of serum creatinine, urea and uric acid significantly increased (p50.05) whereas, they were found to decrease in the 24 urine samples when compared with the control group (Table 1). Pre-treatment
M. Vedi et al.
Table 4 shows the activity of TCA cycle enzymes (Isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase) and respiratory enzymes (NADH dehydrogenase and cytochrome c oxidase). The activities of these enzymes were found to be significantly reduced (p50.05) in the only bromobenzene treated group as compared to the control group. The W. somnifera (250 and 500 mg/kg) or silymarin administration enhanced the activities of mitochondrial enzymes to near normal levels.
20 ± 3.63b* 328.05 ± 13.90b* 44.16 ± 2.48b* 14.16 ± 1.16a*b* 294.32 ± 5.76a*b* 35.09 ± 5.27b* 21.66 ± 1.63b* 320.66 ± 8.35b* 38.83 ± 5.27b* 7.10 ± 1.70a* 171.16 ± 8.97a* 20.5 ± 3.39a* 20.83 ± 3.43 331.50 ± 13.33 42 ± 5.09
15.01 ± 1.89a*b* 253.83 ± 18.78a*b* 34.16 ± 2.85b*
0.54 ± 0.03b* 6.57 ± 0.15b* 12.68 ± 0.22b* 137.84 ± 2.44b* 73.07 ± 3.54b* 0.50 ± 0.05b* 7.84 ± 0.16a*b* 13.28 ± 0.22b* 151.02 ± 1.74b* 83.69 ± 10.22a*b* 0.41 ± 0.01b* 6.58 ± 0.28b* 12.87 ± .04b* 132.73 ± 2.35a*b* 79.40 ± .72b* 0.69 ± 0.07b* 8.19 ± 0.16a*b* 14.09 ± 0.56b* 182.14 ± 1.28a*b* 82.81 ± 1.06a*b*
Group VI (WS-500 mg/kg) Group V (Silymarin-100 mg/kg +BB) Group IV (WS-500 mg/kg) +BB)
Notes: Each value represents the mean ± SD of six rats. Comparisons were made as follows: a – group I versus groups II, III, IV, V, VI; b – group II versus group III, IV, V, VI. The symbols represent statistical significance at *p50.05. Statistical analysis was calculated by one way analysis of variance (ANOVA) using the Graph Pad Prism followed by the Student Newman–Keul’s test.
Effect of W. somnifera on mitochondrial enzymes
3.35 ± 0.32a* 23.57 ± 0.14a* 66.38 ± 1.56a* 263.06 ± 12.06a* 109.64 ± 3.07a*
Figure 1 depicts that the glycoproteins levels (hexosamine and sialic acid) were found to be significantly higher in bromobenzene alone treated rats than the control rats. On the other hand, the rats treated with W. somnifera in addition to bromobenzene, the glycoprotein levels (hexosamine and sialic acid) were found to be significantly restored to near normal level similar to that of silymarin treatment.
0.61 ± 0.05 6.42 ± 0.27 12.64 ± 0.17 144.97 ± 3.62 72.07 ± 2.36
Effect of W. somnifera on glycoproteins
Serum Creatinine (mg/dl) Uric acid (mg/dl) Urea (mg/dl) Triglycerides (mg/dl) Cholesterol (mg/dL) Urine Creatinine (mg/day) Uric acid (mg/day) Urea (mg/day)
From Table 3 the protective effect of W. somnifera on the activities of lysosomal enzymes in control and experimental group of rats can be seen. The activity of the lysosomal enzymes such as N-acetyl glucosaminidase, b-galactosidase and cathepsin D were found to be significantly elevated in the kidney of bromobenzene alone treated group as compared to the control rats, whereas in rats pre-treated with W. somnifera in addition to bromobenzene administration, the enzyme levels were found to be significantly decreased in a dose dependent manner.
Group III (WS-250 mg/kg +BB)
Effect of W. somnifera on lysosomal enzymes
Group II (BB-10 mmol/kg)
As shown in Table 2 levels of catalase and superoxide dismutase were decreased significantly (p50.05) due to generation of oxidative stress by the action of bromobenzene in the kidney tissue. It was compensated and brought near to normal levels by the pre-treatment of W. somnifera (250 and 500 mg/kg) as evident in this study. It has been observed that in kidney tissues bromobenzene metabolites bind with the glutathione and thus depleting its levels. This can be observed in group II that levels of reduced glutathione, glutathione peroxidase and glutathione-s-transferase were found to decrease due to administration of bromobenzene. Pre-administration of W. somnifera (250 and 500 mg/kg) was able to restore the levels of antioxidants (Table 2).
Group I (Control)
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Effect of W. somnifera on antioxidant status
Parameters
with W. somnifera in the fourth group (W. somnifera 500 mg/ kg + bromobenzene) maintained the levels of creatinine, urea and uric acid in serum and 24 urine samples of bromobenzene induced animals near to the normal level as the control group. Also, the standard reference drug silymarin showed similar results. Serum levels of cholesterol and triglycerides were found to be significantly elevated in the bromobenzene alone treated group of rats as seen in Table 1. These were brought near to normal levels by pre-treatment of W. somnifera (250 and 500 mg/kg) and silymarin.
Ren Fail, 2014; 36(7): 1095–1103
Table 1. Effect of administration of bromobenzene (BB) with or without the prior administration of W. somnifera (WS) or silymarin on serum and urinary parameters in rats.
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74.23 ± 2.01 92.15 ± 4.14 19.26 ± 1.03 5.56 ± 0.27 24.8 ± 0.18 0.32 ± 0.31
Group I (Control) 35.04 ± 2.73a* 26.60 ± 0.17a* 9.08 ± 0.21a* 3.96 ± 0.70a* 18.00 ± 0.22a* 0.94 ± 0.10a*
Group II (BB-10 mmol/kg) 51.66 ± 1.82b* 65.75 ± 4.65a*b* 18.8 ± 0.05a*b* 7.85 ± 0.26a*b* 22.63 ± 0.13a*b* 0.42 ± 0.02b*
Group III (WS-250 mg/kg + BB) 68.83 ± 0.63a*b* 80.75 ± 4.51a*b* 18.89 ± 0.20a*b* 8.46 ± 0.22a*b* 24.27 ± 0.08a*b* 0.45 ± 0.05b*
Group IV (WS-500 mg/kg) + BB) 63.96 ± 5.39a*b* 69.15 ± 5.61a*b* 18.03 ± 0.40a*b* 9.22 ± 0.19a*b* 24.22 ± 0.17a*b* 0.38 ± 0.09b*
Group V (Silymarin 100 mg/kg +BB)
74.10 ± 4.49b* 84.95 ± 1.57a* 18.60 ± 0.14a*b* 7.80 ± 0.93a*b* 24.32 ± 0.14a*b* 0.45 ± 0.11b*
Group VI (WS-500 mg/kg)
35.41 ± 0.93 8.11 ± 0.44 47.39 ± 1.49
Group I (Control) 71.01 ± 1.81a* 18.37 ± 0.83a* 70.64 ± 1.96a*
Group II (BB-10 mmol/kg)
51.13 ± 0.84a*b* 10.03 ± 0.67a*b* 56.83 ± 1.72a*b*
Group III (WS-250 mg/kg +BB)
51.64 ± 0.85a*b* 9.92 ± 0.62a*b* 56.06 ± 0.91a*b*
Group IV (WS-500 mg/kg) +BB)
50.34a ± 0.59a*b* 10.98 ± 0.71a*b* 51.99 ± 1.01a*b*
Group V (Silymarin 100 mg/kg +BB)
36.32 ± 0.72b* 8.17 ± 0.47b* 45.14 ± 1.64b*
Group VI (WS-500 mg/kg)
Notes: Each value represents the mean ± SD of six rats. Comparisons were made as follows: a – group I versus groups II, III, IV, V, VI; b – group II versus group III, IV, V, VI. The symbols represent statistical significance at *p50.05. Statistical analysis was calculated by one way analysis of variance (ANOVA) using the Graph Pad Prism followed by the Student Newman–Keul’s test.
b-galactosidase (mmol p-nitrophenol formed/h/mg protein) Cathepsin-D (mmol of tyrosine liberated/h/mg of protein) N-acetyl glucosaminidase (mmol p-nitrophenol formed/h/mg protein)
Parameters
Table 3. Effect of administration of bromobenzene (BB) with or without the prior administration of W. somnifera (WS) or silymarin on lysosomal enzymes in kidney of control and experimental rats.
Notes: Each value represents the mean ± SD of six rats. Comparisons were made as follows: a – group I versus groups II, III, IV, V, VI; b – group II versus group III, IV, V, VI. The symbols represent statistical significance at *p50.05. Statistical analysis was calculated by one way analysis of variance (ANOVA) using the Graph Pad Prism followed by the Student Newman–Keul’s test.
Catalase (Units/min/mg protein) SOD (U/mg protein) GST (nmol/min mg protein) Reduced glutathione (mmol/mg protein) Glutathione peroxidase (nmol/min mg protein) Lipid peroxidation (nmol/mg protein)
Parameters
Table 2. Effect of administration of bromobenzene (BB) with or without the prior administration of W. somnifera (WS) or silymarin on antioxidant status in rats.
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DOI: 10.3109/0886022X.2014.918812
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Figure 1. Effect of administration of bromobenzene (BB) with or without the prior administration of W. somnifera (WS) or silymarin on glycoproteins in kidney of control and experimental rats.
Renal histopathology The representative sections of the kidney clearly illustrate the protective effects of W. somnifera on bromobenzene induced nephrotoxicity (Figure 2). In group I or control rats there are no visible changes and kidney tubular structure can be seen intact (Figure 2a). In the bromobenzene treated rats (Figure 2b), the presence of damaged renal tubules, including peritubular inflammatory cell infiltrates can be observed which has been attenuated by the pre-treatment of W. somnifera (Figures 2c and d). In silymarin treated group, few inflammatory cell infiltrates were observed indicating mild tubular damage (Figure 2).
Discussion Once bromobenzene enters the kidney, its secondary phase metabolites combine with glutathione and produce various mono- and di-substituted derivatives which accumulate in the kidney and cause necrosis.36 Urea, uric acid and creatinine levels are the most important clinical parameters for evaluating abnormality in renal function. In fact creatinine is more reliable indicator of nephrotoxicity as its levels are increased in first phase of kidney disease.37 It may be assumed that renal tubular function is affected more by bromobenzene-induced toxicity than glomerular filtration.5 In the present study, intragastric intubation of bromobenzene (alone) once instigated renal injury that was evidenced by the elevation of serum urea, uric acid and creatinine and depletion in urinary levels of urea, uric acid and creatinine. From the results, it can be observed that pre-treatment of W. somnifera for 8 days to
bromobenzene treated rats was able to normalize the levels of serum urea, uric and uric acid thus restoring the renal function and this may be due to some of its active components like alkaloids, sitoinosides and steroidal lactones. Protection against reactive oxygen species is provided by antioxidant such as superoxide dismutase, catalase and glutathione. Superoxide dismutase converts the superoxide ion to hydrogen peroxide while catalase and glutathione peroxidase break down hydrogen peroxide to non-toxic forms and protects the tissue from highly reactive hydroxyl radicals. Reduced glutathione is a non-enzymatic antioxidant which reduces hydrogen peroxides and hydroperoxides by its redox and detoxification reaction outside and inside the cells.38 It is converted to its oxidized form by glutathione peroxidase and reverts back to reduced form by glutathione reductase. Glutathione-s-transferase is a phase II detoxifying enzyme for xenobiotic compounds which provides protection against toxic chemicals by catalyzing the formation of GSH-electrophile conjugates so, it is considered as toxicity marker in the vital organs. In the present study, the levels of antioxidants were found to be decreased in the kidney tissue after the administration of bromobenzene. This resulted in the decreased ability of the kidney to scavenge toxic free radicals such as hydrogen peroxide and lipid peroxides. Pre-administration of bromobenzene treated rats with W. somnifera was able to restore the levels of antioxidants and glutathione in a dose dependent manner. Lipid peroxidation is one of the important indicators of oxidative disturbance as it has been observed to be a major mediator in the toxicity of many xenobiotics by causing changes in substantial characteristics in living beings.39 There was an increase in lipid peroxidation
Protective effect of W. somnifera against nephrotoxicity Notes: Each value represents the mean ± SD of six rats. Comparisons were made as follows: a – group I versus groups II, III, IV, V, VI; b – group II versus group III, IV, V, VI. UA, nmol of a-ketoglutarate formed/ h; UB, nmol of ferrocyanide formed/h; UC, nmol of succinate oxidised/min; UD, nmol of NADH oxidised/min; UE, nmol of NADH oxidised/min; UF, change in OD 9 10–2/min. The symbols represent statistical significance at *p50.05. Statistical analysis was calculated by one way analysis of variance (ANOVA) using the Graph Pad Prism followed by the Student Newman–Keul’s test.
440 ± 9.47b* 102.59 ± 2.65a*b* 52.31 ± 2.84 b* 262.72 ± 8.54a*b* 32.81 ± 3.41a*b* 9.03 ± 0.95a*b* 414.12 ± 12.37a*b* 99.38 ± 11.25a*b* 39.61 ± 4.29a*b* 240.31 ± 6.69a*b* 28.69 ± 4.26a*b* 6.43 ± 0.84a*b* 431.14 ± 12.34a*b* 101.73 ± 9.85a*b* 41.05 ± 5.25a*b* 246.35 ± 5.61a*b* 34.21 ± 1.08a*b* 8.59 ± 0.97a*b* 423.03 ± 20.97a*b* 94.61 ± 2.69a*b* 36.61 ± 4.96a*b* 231.53 ± 11.25a*b* 25.92 ± 1.34a*b* 4.63 ± 2.01a*b* 303.94 ± 10.21a* 65.44 ± 4.77a* 20.52 ± 4.97a* 160.32 ± 9.35a* 17.86 ± 2.79a* 4.85 ± 1.08a* 445.01 ± 11.40 109.53 ± 9.50 53.22 ± 3.32 267.40 ± 14.70 36.81 ± 4.13 10.15 ± 1.18 Isocitrate dehydrogenase (UA/mg protein) a-Ketoglutarate dehydrogenase (UB/mg protein) Succinate dehydrogenase (UC/mg protein) Malate dehydrogenase (UD/mg protein) NADH dehydrogenase (UE/mg protein) Cytochrome-c-oxidase (UF/mg protein)
Group VI (WS-500 mg/kg) Group V (Silymarin 100 mg/kg + BB) Group IV (WS-500 mg/kg) + BB) Group III (WS-250 mg/kg + BB) Group II (BB-10 mmol/kg) Group I (Control) Parameters
Table 4. Effect of administration of bromobenzene (BB) with or without the prior administration of W. somnifera (WS) or silymarin on mitochondrial enzymes in kidney of control and experimental rats.
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in rats treated with bromobenzene. Pre-administration of W. somnifera was able to cause dose dependent decrease in lipid peroxidation which shows reduction in oxidative disturbance. It has been suggested that W. somnifera contains sitoindosides and withaferin A, which have antioxidant activity and they enhance the free radical scavenging enzymes.40 Lysosomes are sub-cellular organelles responsible for catabolism of intracellular materials to generate smaller biomolecules for various biological processes.41 In this study, the activities of lysosomal enzymes were found to be increased significantly in the extracellular fluid, as a result of decreased lysosomal membrane stability and kidney damage. The elevated levels of lysosomal enzymes during bromobenzene induced toxicity eventually affect the metabolism of various connective tissue constituents, that is glycosaminoglycans, glycoproteins, collagen, resulting in irreversible tissue damage. The increment in levels of glycoprotein components of kidney tissue is due to the leakage of cell membrane glycoconjugates into the cytosol of kidney tissue. Pre-treatment of W. somnifera significantly decreased the lysosomal enzyme leakage and glycoprotein levels in bromobenzene alone treated rats proving the membrane stabilizing effects of W. somnifera. Since the pathogenesis of kidney injury promotes a variety of chemical reactions including oxidative stress with direct effect on organelles like mitochondria, we examined the effect of bromobenzene on mitochondrial enzymes and oxidative stress. In the present study, we observed the decreased activities of mitochondrial TCA cycle enzymes such as isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase due to the administration of bromobenzene to rats. Hence, the decline in isocitrate dehydrogenase activity may result in the perturbation of the balance between oxidants and antioxidants and subsequently lead to a pro-oxidant condition. We propose that bromobenzene causes mitochondrial dysfunction, followed by lipid peroxidation which provokes lethal cell injury.42 Intake of W. somnifera increased the activities of TCA cycle enzymes, probably by recovering the mitochondrial antioxidant defence system, and prevailed over the complications associated with the decreased TCA cycle operation. Further study is needed to clarify the relationship between lipid peroxidation and mitochondrial dysfunction and the mutual contribution to cell injury. Histological monograph of kidney shows normal architecture of tubules in control group of rats (Figure 2a). Bromobenzene treated group shows visible inflammatory infiltrate in the interstitium of the kidney tissue and congested glomerulus and tubular epithelial damage (Figure 2b). Preadministration of W. somnifera (250 mg/kg) for 8 days reduced the renal damage and congested blood vessels in glomerulus can be seen in Figure 2(c). However, W. somnifera (500 mg/kg) was able to restore the kidney histoarchitecture to normal levels as normal glomerulus and tubules can be observed (Figure 2d). In silymarin treated rats kidney with tubular damage containing inflammatory infiltrate is visible and in rats which were treated only with W. somnifera kidney appears to be normal. Thus it proves that W. somnifera (500 mg/kg) was able to provide significant protection against
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Figure 2. Kidney sections (stained with H&E) from rats (a) control showed normal tubules and glomerulus, (b) Bromobenzene (10 mmol) treated group showed peritubular inflammatory cell filtrates (diamond), (c) W. somnifera (250 mg/kg)+Bromobenzene treated group showed congested blood vessels in glomerulus (arrow), (d) W. somnifera (500 mg/kg)+Bromobenzene treated group showed near normal histoarchitecture of renal glomerulus and tubules, (e) Silymarin (100 mg/kg)+Bromobenzene treated group showed congestion in peritubular capillaries (arrow),dissolution of nucleus and tubular epithelial damage (dark arrow) and congestion of glomerular vessels (triangle) and (f) W. somnifera (500 mg/kg) showing normal glomerulus.
bromobenzene induced nephrotoxicity to rats. To the best of our knowledge, this is the first study to show the protective effect of W. somnifera against bromobenzene induced nephrotoxicity in rats and still further research is required for depicting the mechanism of action of W. somnifera in providing protecting against nephrotoxicity.
Declaration of interest The authors report no declaration of interest. The authors alone are responsible for the content and writing of the paper.
References Conclusion It may be hypothesized that the active components present in W. somnifera may scavenge the free radicals produced due to the abnormal metabolism and may enhance the production of non-enzymatic and enzymatic antioxidants, which may possibly lead to decreased damage due to the reactive oxygen species thereby reducing the oxidative stress and lipid peroxidation. Hence, our work opens a novel window to manage bromobenzene-induced nephrotoxicity. Further elucidation of the mechanisms may provide insights into nephrotoxicity resulting due to various xenobiotic compounds and may help in preventing it by the use of W. somnifera.
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