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Oral erdosteine administration attenuates cisplatin-induced renal tubular damage in rats
Pharmacological Research 47 (2003) 149–156
Oral erdosteine administration attenuates cisplatin-induced renal tubular damage in rats Zeki Yildirim a,∗ , Sadik Sogut b , Ersan Odaci c , Mustafa Iraz d , Huseyin Ozyurt b , Mahir Kotuk a , Omer Akyol b a
Department of Pulmonary, Turgut Ozal Medical Center, Inonu University School of Medicine, 44069 Malatya, Turkey b Department of Biochemistry, Inonu University School of Medicine, Malatya, Turkey c Department of Histology, Ondokuz Mayis University School of Medicine, Samsun, Turkey d Department of Pharmacology, Inonu University School of Medicine, Malatya, Turkey Accepted 14 October 2002
Abstract The effect of oral erdosteine on tissue malondialdehyde (MDA) and nitric oxide (NO) levels, and catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities are investigated in the cisplatin model of acute renal failure in rats. A single dose of cisplatin caused kidney damage manifested by kidney histology as well as increases in plasma creatinine and blood urea nitrogen (BUN) levels. Treatment with free radical scavenger erdosteine attenuated increases in plasma creatinine and BUN, and tissue MDA and NO levels, and provided a histologically-proven protection against cisplatin-induced acute renal failure. Erdosteine also reduced depletion in the tissue CAT, GSH-Px, and SOD activities. These results show that erdosteine may be a promising drug for protection against cisplatin-induced nephrotoxicity. However, further studies with different doses of erdosteine are warranted for clarifying the issue. © 2002 Published by Elsevier Science Ltd. Keywords: Erdosteine; Cisplatin nephrotoxicity; Nitric oxide; Free radicals
1. Introduction Erdosteine [N-(carboxymethylthioacetyl)-homosysteine thiolactone], as a thiol derivate, contains two blocked sulfhydryl groups which become free only after hepatic metabolisation. The reducing potential of these sulfhydryl groups accounts for free radical scavenging and antioxidant activity of erdosteine [1,2]. Experimental and clinical studies have demonstrated the free radical scavenging properties of erdosteine. Biagi et al. showed that orally administered erdosteine may prevent loss of the elastase inhibitory capacity of ␣,1-antitrypsin induced by exposure to cigarette smoke in rats [3]. It has been documented that the S isomer of erdosteine at oral doses of 200 and 400 mg kg−1 is effective in protecting mice against lethal doses of paraquat which was able to form free radicals when administered by i.p. route [4]. A trial in 24 healthy smokers treated with erdosteine for 4 weeks in a double-blind fashion revealed an increase in the level of functional ␣1 -antitrypsin in the ∗ Corresponding author. Tel.: +90-422-3410785; fax: +90-422-3410728. E-mail address: [email protected] (Z. Yildirim).
1043-6618/02/$ – see front matter © 2002 Published by Elsevier Science Ltd. PII: S 1 0 4 3 - 6 6 1 8 ( 0 2 ) 0 0 2 8 2 - 7
bronchoalveolar lavage fluid [5]. In a study by Ciaccia et al. 16 smokers with chronic bronchitis pre-treated with 300 mg erdosteine three times daily for 2 weeks were studied and an incomplete reduction of smoke-induced decline in responsiveness of polymorphonuclear cells to chemotactic agent casein 1 g l−1 and formyl-methionin–leucine–phenylalanine 1 mmol l−1 was observed. Responsiveness to casein increased 49% after erdosteine administration compared with a 13% decline after placebo. Similar findings were observed by formyl-methionin–leucine–phenylalanine [6]. Cisplatin is a widely used anti-neoplastic agent in the treatment of solid tumors including lung cancer and nephrotoxicity is its major dose limiting side effect. Recently, evidences have been accumulated that this side effect is closely related to reactive oxygen metabolites [7,8]. Several agents such as amifostine have been proposed to reduce this side effect. Although amifostine introduced into the clinical usage, when it supplemented with cisplatin, their prices are approximately 10-fold higher than cisplatin alone and 3-fold higher than carboplatin, a second-generation analogue of cisplatin with less nephrotoxicity which has replaced cisplatin in many chemotherapeutic regimens. Thus, it is warranted a more cost saving drug for nephroprotection. Based on the
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potent free radical scavenging effect and the lower price of erdosteine, we suggest that it may be useful to protect renal tubular damage. The major purpose of the current study is to examine the role of erdosteine in the protection of cisplatin-induced nephrotoxicity in rats besides the effect of erdosteine on some antioxidant enzymes, malondialdehyde (MDA) and NO in the kidney tissue.
2. Materials and methods 2.1. Animals Female Wistar albino rats weighing 200–250 g were purchased from experimental research center, University of Erciyes (Kayseri, Turkey) and housed in animal laboratory of our university. The animals were fed with a standard diet and kept on a physiological day–night rhythm. Rats were divided into three groups: rats given cisplatin as a cisplatin-induced acute renal failure model, rats given cisplatin plus erdosteine, and rats given isotonic saline solution alone as a control group. 2.2. Cisplatin-induced acute renal failure Cisplatin model of acute renal failure was induced in rats as described by Zhang et al. [9]. The animals received an intraperitoneal injection of cisplatin (Cisplatinum Ebewe, 0.5 mg ml−1 ) at a dose of 7 mg kg−1 body weight and were sacrificed 5 days after cisplatin injection. Blood and kidneys were obtained for the various measurements. Renal impairment was assessed by blood urea nitrogen (BUN) and plasma creatinine levels as well as kidney histology. BUN and creatinine were determined by the use of Sigma diagnostic kits. Kidney histology was performed as described in the following section. 2.3. Effect of erdosteine on cisplatin model of acute renal failure The erdosteine was obtained from a drug company (Ilsan, Turkey), dissolved in distilled water and administered orally once a day at a dose of 10 mg kg−1 body weight via plastic disposable syringes. First dose of erdosteine was given 24 h prior to cisplatin injection and continued until sacrifice. 2.4. Control rats Isotonic saline solution (an equal volume of cisplatin) was administered by intraperitoneal injection. In addition, NaHCO3 dissolved with distilled water was given orally (as equal volume of erdosteine) 24 h prior to the saline injection followed by once a day at the same dose until sacrifice.
2.5. Kidney histology The kidney were sectioned and fixed in 10% formalin, dehydrated and embedded in paraffin. Tissues were then sectioned at 3 m, stained with hematoxylin and eosin (HE), and examined for tubular necrosis and dilatation. 2.6. Biochemical measurements All tissues were washed two times with cold saline solution, placed into glass bottles, labeled, and stored in a deep freeze (−30 ◦ C) until processing (maximum 10 h). Tissues were homogenized in a four volumes of ice-cold Tris–HCl buffer (50 mM, pH 7.4) using a glass Teflon homogenizer (Tempest Virtishear, Model 278069; The Virtis Company, Gardiner, NY) after cutting of the kidney into small pieces with a scissors (for 2 min at 5000 rpm). Nitric oxide and MDA measurements were made at this stage. The homogenate was then centrifuged at 5000 × g for 60 min to remove debris. Clear upper supernatant fluid was taken and CAT and GSH-Px activities and protein concentration were carried out in this stage. The supernatant solution was extracted with an equal volume of ethanol/chloroform mixture (5/3, volume per volume [v/v]). After centrifugation at 5000 × g for 30 min, the clear upper layer (the ethanol phase) was taken and used in the SOD activity and protein assays. All preparation procedures were performed at +4 ◦ C. 2.7. MDA determination Kidney MDA levels were determined by Wasowicz’s method [10] based on the reaction of MDA with thiobarbituric acid at 95–100 ◦ C. Fluorescence intensity was measured in the upper n-butanol phase by a fluorescence spectrophotometer (Hitachi, Model F-4010) adjusted exitation at 525 nm and emission at 547 nm. Arbitrary values obtained were compared with a series of standard solutions (1,1,3,3-tetramethoxypropane). Results were expressed as nanomole per gram tissue protein of kidney (nmol g−1 protein tissue). 2.8. NO determination Since NO measurement is very difficult in biological specimens, tissue nitrite (NO2 − ) and nitrate (NO3 − ) were estimated as an index of NO production. The method for kidney nitrite and nitrate levels was based on the Griess reaction [11]. Samples were initially deproteinized with Somogyi reagent. Total nitrite (nitrite + nitrate) was measured after conversion of nitrate to nitrite by copporized cadmium granules by a spectrophotometer at 545 nm (Ultraspec Plus, Pharmacia LKB Biochrom Ltd., UK). A standard curve was established with a set of serial dilutions (10−8 –10−3 mol l−1 ) of sodium nitrite. Linear regression was done by using the peak area from nitrite standard. The resulting equation was
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then used to calculate the unknown sample concentrations. Results were expressed as nanomole per gram tissue protein.
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groups. All analyses were made using the SPSS statistical software package. A P-value <0.05 was considered as statistically significant.
2.9. SOD activity determination Total (Cu–Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al. [12] with a slight modification by Durak et al. [13]. The principle of the method is based on the inhibition of NBT reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the supernatant after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. SOD activity was also expressed as units per milligram protein. 2.10. Catalase activity determination CAT activity was determined according to Aebi’s method [14]. The principle of the method was based on the determination of the rate constant (s−1 , k) of the H2 O2 decomposition rate at 240 nm. Results were expressed as k (rate constant) per gram protein. 2.11. Glutathione peroxidase activity determination GSH-Px activity was measured by the method of Pagia et al. [15]. The enzymatic reaction was initiated in a tube, which is containing following items: NADPH, reduced glutathione (GSH), sodium azide, and glutathione reductase by addition of H2 O2 and the change in absorbance at 340 nm was monitored by a spectrophotometer. Activity was given in units per gram protein. All samples were assayed in duplicate. 2.12. Protein determinations Protein assays were made by the method of Lowry et al. [16]. 2.13. Data analysis Data are expressed as means ± standard deviation. The one-way ANOVA analysis of variance and post hoc multiple comparison tests (LSD) were performed on the data of biochemical variables to examine the differences among
3. Results Cisplatin administration at a dose of 7 mg kg−1 body weight resulted in acute renal failure similar to a previous study [9]. As shown in Table 1, significant elevations in the mean plasma creatinine and BUN levels were seen after treatment with cisplatin alone as compared to the control (P < 0.002). Erdosteine attenuated increases in serum creatinine and BUN levels but did not reach a statistically significant level (P > 0.05). Similarly, NO and MDA levels in the kidney tissue were increased by the cisplatin management and erdosteine prevented these increments at a statistically significant level (P < 0.0001 for NO and P = 0.023 for MDA) (Fig. 1). In the control group, the mean renal tissue concentrations of SOD, GSH-Px and CAT activities were 0.048 ± 0.0008 U mg−1 protein, 3.33 ± 0.42 U mg−1 protein, and 0.82 ± 0.11 kg−1 protein, respectively (Fig. 2). In the cisplatin group, these enzyme activities significantly decreased in the kidney tissues and were measured to be 0.068 ± 0.00047 U mg−1 protein, 1.89 ± 0.21 U g−1 protein and 0.55 ± 0.0073 kg−1 protein, respectively. In the erdosteine plus cisplatin administered rats, mean GSH-Px (2.70 ± 0.33 U g−1 protein) and CAT activities (0.66 ± 0.11 U mg−1 protein) (P = 0.02), but not SOD activity (0.0041±0.00046 U mg−1 protein), were significantly higher than the cisplatin administered rats (P < 0.0001 for GSH-Px and P = 0.02 for CAT). However, as shown in Fig. 1b, one rat in erdosteine pre-treated group has higher CAT activity than other rats. When excluding this value, it was seen that the difference in CAT activity was not statistically significant (P > 0.05). As shown in Fig. 3, control rats showed no abnormality for the kidney histology. Cisplatin administration to the rats revealed a remarkable proximal tubular necrosis with extensive epithelial vacuolization, swelling, and tubular dilatation as compared to the controls and erdosteine-treated rats. The wall height (between two opposite direction of arrow heads) of proximal tubules in control kidneys is higher than both of cisplatin and erdosteine group. The glomeruli appeared normal in all groups.
Table 1 Plasma creatinine and urea levels in control, cisplatin-treated and erdosteine pre-treated plus cisplatin-treated rats
BUN (mg dl−1 ) Creatinine (mg dl−1 ) a b
Control (n = 6)
Cisplatina (n = 9)
Cisplatin + erdosteineb (n = 8)
19.50 ± 2.90 0.40 ± 0
78.77 ± 42.70 1.33 ± 1.3
69.75 ± 25.50 1.15 ± 0.8
P = 0.002 and 0.012 for BUN and creatinine, respectively, between control and cisplatin groups. P = 0.810 and 0.846 for BUN and creatinine, respectively, between cisplatin and cisplatin plus erdosteine groups.
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Fig. 1. The scatter plot graph of MDA and NO in control, cisplatin-treated and erdosteine plus cisplatin-treated rats. A single dose intraperitoneal administration of cisplatin treatment resulted in a limited increase in renal tissue MDA level while a significant increase in the renal tissue NO level when compared to the control rats. Prophylactic erdosteine treatment significantly prevented the increase in both MDA and NO levels in the damaged renal tissue.
4. Discussion Our study shows that BUN and creatinine levels are markedly increased in the blood of rats treated with cisplatin. We indicate that prophylactic oral administration of the erdosteine in rats may have protective potential on cisplatin-induced acute renal failure as measured by BUN and creatinine level. Although the increment of mean BUN and creatinine concentration cannot reach the statistically significant level, it is less prominent in erdosteine
pre-treated rats than cisplatin alone. Kidney histology provided a marked reduction in the extent of tubular damage in those animals treated with erdosteine (Fig. 3). In addition, our study indicates that erdosteine significantly prevents the depletion of the renal tissue CAT and GSH-Px activities by scavenging free radicals produced by cisplatin (Fig. 2). But further statistical analysis of the data showed that the difference in CAT activity between cisplatin and cisplatin plus erdosteine-treated rats was due to the higher CAT activity of a rat in erdosteine pre-treated group as shown in Fig. 1b.
Fig. 2. The scatter plot graph of renal tissue CAT, GSH-Px and SOD activities in control, cisplatin and erdosteine plus cisplatin-treated rats. A single dose intraperitoneal administration of cisplatin treatment resulted in significant decrease in renal tissue activity of these antioxidant enzyme activities as compared to the control rats. Prophylactic erdosteine treatment significantly prevented the depletion of damaged renal tissue GSH-Px activity. However, erdosteine has a limited protective effect on the reduction of tissue CAT and SOD activity caused by cisplatin administration.
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Fig. 3. Three micrographs that have been taken from the cortex of kidney in each group, which are control (A), cisplatin (B) and erdosteine plus cisplatin (C). Control rat showing no abnormality. The extensive epithelial cell vacuolization, swelling, desquamation and necrosis are clearly observed in the kidney of the cisplatin-treated rat. The larger tubular lumens in cisplatin-treated rats than those of the control and the erdosteine groups are showing extensive necrosis (B). The wall height (between two opposite direction of arrow heads) of proximal tubules (P) in the control kidney is higher than both of cisplatin and erdosteine groups. The free radical scavenger erdosteine shows a marked reduction in the extent of tubular damage (C) (scale bar 10 m).
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Also, erdosteine prevented the increase of NO and MDA concentrations in the renal tissue of rat at a statistically significant level (Fig. 1). Cisplatin acts mostly on the proximal renal tubule of the kidney. Proximal tubular epithelial cells take up cisplatin actively and the concentration of the drug in these cells exceeds plasma concentration by a factor of five. Cisplatin toxicity in proximal tubular cells is morphologically characterized by tubular necrosis [17]. Despite the underlying mechanism of cisplatin-induced nephrotoxicity is still not well known, many recent in vitro and in vivo studies indicate an important role of reactive oxygen metabolites in the pathogenesis of this nephrotoxicity [18]. Cisplatin induces free radical production causing oxidative renal damage. Antioxidants have been shown to be protective in cisplatin nephrotoxicity [19]. Various free radical scavengers have been shown to be effective in protection from cisplatin-induced nephrotoxicity and treatment with such agents provided significant protection against cisplatin-induced acute renal failure [20–22]. In the present study, lipid peroxidation was monitored by measuring of MDA which results from free radical damage to membrane components of the cells. We observed a moderate increase in the MDA concentration in the kidney tissue of rats treated with cisplatin alone. Erdosteine significantly attenuated the increase of MDA concentration in renal tissue. This is probably due to its elimination capacity for free oxygen radicals. The protection of renal failure by erdosteine may also due to the reduction of lipid peroxidation of renal tubular cells in rat kidney. Also, preventive effect of erdosteine in the depletion of CAT and GSH-Px activities may be due to its free radical scavenging and antioxidant activity. Recently, it has been discussed the role of NO in the pathophysiology of acute renal failure [23]. Increasing evidences suggest that NO has an important role in modulating oxidant stress and tissue damage. Peresleni et al. demonstrated that oxidant stress to the epithelial cells caused an increase in immunodetectable inducible NO synthase (iNOS), which results in an elevation in NO release, nitrite production, and decreased cell viability [24]. The mechanism mediating induction of iNOS due to the free radical exposure remains unknown. It has recently hypothesized that cytotoxic effect of NO production depends on redox state of the cell and its ability to generate peroxynitrite (ONOO− ) anion. Peroxynitrite, a highly reactive oxidant formed during the interaction between NO and O2 − , can attack a wide variety of biological targets. Despite extensive researches evaluated the role of NO in cyclosporine A-induced acute renal failure [25], studies in cisplatin-induced acute renal failure are scant and inconclusive. The presented study indicated the marked elevation in NO level in damaged kidney tissue of the cisplatin-treated rats and erdosteine significantly attenuated this increment (P < 0.0001). This increment of NO generation in the renal tissue of cisplatin administrated rats support above mentioned mechanism relating genera-
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tion of NO caused by free radicals under oxidative stress [25]. Based on this evidence of the important role of NO in cisplatin-induced acute renal failure in rat, it appears that the inhibition of NO during cisplatin treatment may offer the protection against cisplatin nephrotoxicity. In conclusion, although the preventive effect of erdosteine on the plasma BUN and creatinine levels can not reach the statistically significant levels, we think that erdosteine may be a promising drug against cisplatin-induced renal failure and further studies are warranted to define optimum dosage of this drug. Also, additional studies are warranted to determine whether erdosteine can prevent nephrotoxicity in humans as well as whether erdosteine therapy affects the anti-tumor activity of cisplatin. In addition, these data indicate that NO may play a role in the pathogenesis of cisplatin nephrotoxicity. The effect of NO inhibition on cisplatin-nephrotoxicity should also be further investigated.
References [1] Dechant KL, Noble S. Erdosteine. Drugs 1996;52:875–81. [2] Braga PC, Dal Sasso M, Zuccotti T. Assessment of the antioxidant activity of the SH metabolite I of erdosteine on human neutrophil oxidative bursts. Arzneimittelforschung 2000;50:739–46. [3] Biagi GL, Fregnan GB, Gazzani G, Vandoni G. Erdosteine protection from cigarette smoke-induced loss of alpha 1-antitrypsin activity in rat lungs. Int J Clin Pharmacol Ther Toxicol 1989;27:235–7. [4] Inglesi M, Nicola M, Fregnan GB, et al. Synthesis and free radical scavenging properties of the enantiomers of erdosteine. Farmaco 1994;40:703–8. [5] Vagliasindi M, Fregman GB. Erdosteine protection against cigarette smoking-induced functional antiprotease deficiency in human bronchiolo-alveolar structures. Int J Clin Farmacol Toxicol 1989;27:238– 41. [6] Ciaccia A, Papi A, Tschirky B, Fregnan B. Protection of erdosteine on smoke-induced peripheral neutrophil dysfunction both in healthy and in bronchitic smokers. Fundam Clin Pharmacol 1992;6:375–82. [7] Matsushima H, Yonemura K, Ohishi K, Hishida A. The role of oxygen free radicals in cisplatin-induced acute renal failure in rats. J Lab Clin Med 1998;131:518–26. [8] Baliga R, Zhang Z, Baliga M, et al. In vitro and in vivo evidence suggesting a role for iron in cisplatin-induced nephrotoxicity. Kidney Int 1998;53:394–400. [9] Zhang JG, Viale M, Esposito M, Lindup WE. Tiopronin protects against the nephrotoxicity of cisplatin in the rat. Hum Exp Toxicol 1999;18:713–7. [10] Wasowicz W, Neve J, Peretz A. Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage. Clin Chem 1993;39:2522–6. [11] Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36:1440–3. [12] Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500. [13] Durak I, Yurtarslani Z, Canbolat O, Akyol O. A methodological approach to superoxide dismutase (SOD) activity assay based on inhibition of nitroblue tetrazolium (NBT) reduction. Clin Chim Acta 1993;214:103–4. [14] Aebi H. Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysis. New York and London: Academic Press; 1974. p. 673–7.
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[15] Pagia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–70. [16] Lowry O, Rosenbraugh N, Farr L, Randall R. Protein measurement with the Folin-phenol reagent. J Biol Chem 1951;183:265–75. [17] Anand AJ, Bashey B. Newer insights into cisplatin nephrotoxicity. Ann Pharmacother 1993;27:1519–25. [18] Baliga R, Zhang Z, Baliga M, Ueda N, Shah S. Role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity. Kidney Int 1998;54:1562–9. [19] Baliga R, Ueda N, Walker PD, Shah SV. Oxidant mechanisms in toxic acute renal failure. Drug Metab Rev 1999;3:971–97. [20] Babu E, Gopalakrishnan VK, Sriganth IN, Gopalakrishnan R, Sakthisekaran D. Cisplatin induced nephrotoxicity and the modulating effect of glutathione ester. Mol Cell Biochem 1995;144:7–11.
[21] Sheikh-Hamad D, Timmins K, Jalali Z. Cisplatin-induced renal toxicity: possible reversal by N-acetylcysteine treatment. J Am Soc Nephrol 1997;8:1640–4. [22] Weichert-Jacobsen KJ, Bannowski A, Kuppers F, Loch T, Stöckle M. Direct amifostine effect on renal tubule cells in rats. Cancer Res 1999;59:3451–3. [23] Goligorsky MS, Brodsky AV, Noiri E. Nitric oxide in acute renal failure: NOS versus NOS. Kidney Int 2002;61:855–61. [24] Peresleni T, Noiri E, Bahou WF, Goligorsky MS. Antisense oligodeoxynucleotides to inducible NO synthase rescue epithelial cells from oxidative stress injury. Am J Physiol 1996;270(6 Pt 2):F971–7. [25] Gossmann J, Radounikli A, Bernemann A, Schellinski O, Raab HP, Bickeboller R, et al. Pathophysiology of cyclosporine-induced nephrotoxicity in humans: a role for nitric oxide? Kidney Blood Press Res 2001;24:111–5.
Oral erdosteine administration attenuates cisplatin-induced renal tubular damage in rats Zeki Yildirim a,∗ , Sadik Sogut b , Ersan Odaci c , Mustafa Iraz d , Huseyin Ozyurt b , Mahir Kotuk a , Omer Akyol b a
Department of Pulmonary, Turgut Ozal Medical Center, Inonu University School of Medicine, 44069 Malatya, Turkey b Department of Biochemistry, Inonu University School of Medicine, Malatya, Turkey c Department of Histology, Ondokuz Mayis University School of Medicine, Samsun, Turkey d Department of Pharmacology, Inonu University School of Medicine, Malatya, Turkey Accepted 14 October 2002
Abstract The effect of oral erdosteine on tissue malondialdehyde (MDA) and nitric oxide (NO) levels, and catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities are investigated in the cisplatin model of acute renal failure in rats. A single dose of cisplatin caused kidney damage manifested by kidney histology as well as increases in plasma creatinine and blood urea nitrogen (BUN) levels. Treatment with free radical scavenger erdosteine attenuated increases in plasma creatinine and BUN, and tissue MDA and NO levels, and provided a histologically-proven protection against cisplatin-induced acute renal failure. Erdosteine also reduced depletion in the tissue CAT, GSH-Px, and SOD activities. These results show that erdosteine may be a promising drug for protection against cisplatin-induced nephrotoxicity. However, further studies with different doses of erdosteine are warranted for clarifying the issue. © 2002 Published by Elsevier Science Ltd. Keywords: Erdosteine; Cisplatin nephrotoxicity; Nitric oxide; Free radicals
1. Introduction Erdosteine [N-(carboxymethylthioacetyl)-homosysteine thiolactone], as a thiol derivate, contains two blocked sulfhydryl groups which become free only after hepatic metabolisation. The reducing potential of these sulfhydryl groups accounts for free radical scavenging and antioxidant activity of erdosteine [1,2]. Experimental and clinical studies have demonstrated the free radical scavenging properties of erdosteine. Biagi et al. showed that orally administered erdosteine may prevent loss of the elastase inhibitory capacity of ␣,1-antitrypsin induced by exposure to cigarette smoke in rats [3]. It has been documented that the S isomer of erdosteine at oral doses of 200 and 400 mg kg−1 is effective in protecting mice against lethal doses of paraquat which was able to form free radicals when administered by i.p. route [4]. A trial in 24 healthy smokers treated with erdosteine for 4 weeks in a double-blind fashion revealed an increase in the level of functional ␣1 -antitrypsin in the ∗ Corresponding author. Tel.: +90-422-3410785; fax: +90-422-3410728. E-mail address: [email protected] (Z. Yildirim).
1043-6618/02/$ – see front matter © 2002 Published by Elsevier Science Ltd. PII: S 1 0 4 3 - 6 6 1 8 ( 0 2 ) 0 0 2 8 2 - 7
bronchoalveolar lavage fluid [5]. In a study by Ciaccia et al. 16 smokers with chronic bronchitis pre-treated with 300 mg erdosteine three times daily for 2 weeks were studied and an incomplete reduction of smoke-induced decline in responsiveness of polymorphonuclear cells to chemotactic agent casein 1 g l−1 and formyl-methionin–leucine–phenylalanine 1 mmol l−1 was observed. Responsiveness to casein increased 49% after erdosteine administration compared with a 13% decline after placebo. Similar findings were observed by formyl-methionin–leucine–phenylalanine [6]. Cisplatin is a widely used anti-neoplastic agent in the treatment of solid tumors including lung cancer and nephrotoxicity is its major dose limiting side effect. Recently, evidences have been accumulated that this side effect is closely related to reactive oxygen metabolites [7,8]. Several agents such as amifostine have been proposed to reduce this side effect. Although amifostine introduced into the clinical usage, when it supplemented with cisplatin, their prices are approximately 10-fold higher than cisplatin alone and 3-fold higher than carboplatin, a second-generation analogue of cisplatin with less nephrotoxicity which has replaced cisplatin in many chemotherapeutic regimens. Thus, it is warranted a more cost saving drug for nephroprotection. Based on the
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potent free radical scavenging effect and the lower price of erdosteine, we suggest that it may be useful to protect renal tubular damage. The major purpose of the current study is to examine the role of erdosteine in the protection of cisplatin-induced nephrotoxicity in rats besides the effect of erdosteine on some antioxidant enzymes, malondialdehyde (MDA) and NO in the kidney tissue.
2. Materials and methods 2.1. Animals Female Wistar albino rats weighing 200–250 g were purchased from experimental research center, University of Erciyes (Kayseri, Turkey) and housed in animal laboratory of our university. The animals were fed with a standard diet and kept on a physiological day–night rhythm. Rats were divided into three groups: rats given cisplatin as a cisplatin-induced acute renal failure model, rats given cisplatin plus erdosteine, and rats given isotonic saline solution alone as a control group. 2.2. Cisplatin-induced acute renal failure Cisplatin model of acute renal failure was induced in rats as described by Zhang et al. [9]. The animals received an intraperitoneal injection of cisplatin (Cisplatinum Ebewe, 0.5 mg ml−1 ) at a dose of 7 mg kg−1 body weight and were sacrificed 5 days after cisplatin injection. Blood and kidneys were obtained for the various measurements. Renal impairment was assessed by blood urea nitrogen (BUN) and plasma creatinine levels as well as kidney histology. BUN and creatinine were determined by the use of Sigma diagnostic kits. Kidney histology was performed as described in the following section. 2.3. Effect of erdosteine on cisplatin model of acute renal failure The erdosteine was obtained from a drug company (Ilsan, Turkey), dissolved in distilled water and administered orally once a day at a dose of 10 mg kg−1 body weight via plastic disposable syringes. First dose of erdosteine was given 24 h prior to cisplatin injection and continued until sacrifice. 2.4. Control rats Isotonic saline solution (an equal volume of cisplatin) was administered by intraperitoneal injection. In addition, NaHCO3 dissolved with distilled water was given orally (as equal volume of erdosteine) 24 h prior to the saline injection followed by once a day at the same dose until sacrifice.
2.5. Kidney histology The kidney were sectioned and fixed in 10% formalin, dehydrated and embedded in paraffin. Tissues were then sectioned at 3 m, stained with hematoxylin and eosin (HE), and examined for tubular necrosis and dilatation. 2.6. Biochemical measurements All tissues were washed two times with cold saline solution, placed into glass bottles, labeled, and stored in a deep freeze (−30 ◦ C) until processing (maximum 10 h). Tissues were homogenized in a four volumes of ice-cold Tris–HCl buffer (50 mM, pH 7.4) using a glass Teflon homogenizer (Tempest Virtishear, Model 278069; The Virtis Company, Gardiner, NY) after cutting of the kidney into small pieces with a scissors (for 2 min at 5000 rpm). Nitric oxide and MDA measurements were made at this stage. The homogenate was then centrifuged at 5000 × g for 60 min to remove debris. Clear upper supernatant fluid was taken and CAT and GSH-Px activities and protein concentration were carried out in this stage. The supernatant solution was extracted with an equal volume of ethanol/chloroform mixture (5/3, volume per volume [v/v]). After centrifugation at 5000 × g for 30 min, the clear upper layer (the ethanol phase) was taken and used in the SOD activity and protein assays. All preparation procedures were performed at +4 ◦ C. 2.7. MDA determination Kidney MDA levels were determined by Wasowicz’s method [10] based on the reaction of MDA with thiobarbituric acid at 95–100 ◦ C. Fluorescence intensity was measured in the upper n-butanol phase by a fluorescence spectrophotometer (Hitachi, Model F-4010) adjusted exitation at 525 nm and emission at 547 nm. Arbitrary values obtained were compared with a series of standard solutions (1,1,3,3-tetramethoxypropane). Results were expressed as nanomole per gram tissue protein of kidney (nmol g−1 protein tissue). 2.8. NO determination Since NO measurement is very difficult in biological specimens, tissue nitrite (NO2 − ) and nitrate (NO3 − ) were estimated as an index of NO production. The method for kidney nitrite and nitrate levels was based on the Griess reaction [11]. Samples were initially deproteinized with Somogyi reagent. Total nitrite (nitrite + nitrate) was measured after conversion of nitrate to nitrite by copporized cadmium granules by a spectrophotometer at 545 nm (Ultraspec Plus, Pharmacia LKB Biochrom Ltd., UK). A standard curve was established with a set of serial dilutions (10−8 –10−3 mol l−1 ) of sodium nitrite. Linear regression was done by using the peak area from nitrite standard. The resulting equation was
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then used to calculate the unknown sample concentrations. Results were expressed as nanomole per gram tissue protein.
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groups. All analyses were made using the SPSS statistical software package. A P-value <0.05 was considered as statistically significant.
2.9. SOD activity determination Total (Cu–Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al. [12] with a slight modification by Durak et al. [13]. The principle of the method is based on the inhibition of NBT reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the supernatant after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. SOD activity was also expressed as units per milligram protein. 2.10. Catalase activity determination CAT activity was determined according to Aebi’s method [14]. The principle of the method was based on the determination of the rate constant (s−1 , k) of the H2 O2 decomposition rate at 240 nm. Results were expressed as k (rate constant) per gram protein. 2.11. Glutathione peroxidase activity determination GSH-Px activity was measured by the method of Pagia et al. [15]. The enzymatic reaction was initiated in a tube, which is containing following items: NADPH, reduced glutathione (GSH), sodium azide, and glutathione reductase by addition of H2 O2 and the change in absorbance at 340 nm was monitored by a spectrophotometer. Activity was given in units per gram protein. All samples were assayed in duplicate. 2.12. Protein determinations Protein assays were made by the method of Lowry et al. [16]. 2.13. Data analysis Data are expressed as means ± standard deviation. The one-way ANOVA analysis of variance and post hoc multiple comparison tests (LSD) were performed on the data of biochemical variables to examine the differences among
3. Results Cisplatin administration at a dose of 7 mg kg−1 body weight resulted in acute renal failure similar to a previous study [9]. As shown in Table 1, significant elevations in the mean plasma creatinine and BUN levels were seen after treatment with cisplatin alone as compared to the control (P < 0.002). Erdosteine attenuated increases in serum creatinine and BUN levels but did not reach a statistically significant level (P > 0.05). Similarly, NO and MDA levels in the kidney tissue were increased by the cisplatin management and erdosteine prevented these increments at a statistically significant level (P < 0.0001 for NO and P = 0.023 for MDA) (Fig. 1). In the control group, the mean renal tissue concentrations of SOD, GSH-Px and CAT activities were 0.048 ± 0.0008 U mg−1 protein, 3.33 ± 0.42 U mg−1 protein, and 0.82 ± 0.11 kg−1 protein, respectively (Fig. 2). In the cisplatin group, these enzyme activities significantly decreased in the kidney tissues and were measured to be 0.068 ± 0.00047 U mg−1 protein, 1.89 ± 0.21 U g−1 protein and 0.55 ± 0.0073 kg−1 protein, respectively. In the erdosteine plus cisplatin administered rats, mean GSH-Px (2.70 ± 0.33 U g−1 protein) and CAT activities (0.66 ± 0.11 U mg−1 protein) (P = 0.02), but not SOD activity (0.0041±0.00046 U mg−1 protein), were significantly higher than the cisplatin administered rats (P < 0.0001 for GSH-Px and P = 0.02 for CAT). However, as shown in Fig. 1b, one rat in erdosteine pre-treated group has higher CAT activity than other rats. When excluding this value, it was seen that the difference in CAT activity was not statistically significant (P > 0.05). As shown in Fig. 3, control rats showed no abnormality for the kidney histology. Cisplatin administration to the rats revealed a remarkable proximal tubular necrosis with extensive epithelial vacuolization, swelling, and tubular dilatation as compared to the controls and erdosteine-treated rats. The wall height (between two opposite direction of arrow heads) of proximal tubules in control kidneys is higher than both of cisplatin and erdosteine group. The glomeruli appeared normal in all groups.
Table 1 Plasma creatinine and urea levels in control, cisplatin-treated and erdosteine pre-treated plus cisplatin-treated rats
BUN (mg dl−1 ) Creatinine (mg dl−1 ) a b
Control (n = 6)
Cisplatina (n = 9)
Cisplatin + erdosteineb (n = 8)
19.50 ± 2.90 0.40 ± 0
78.77 ± 42.70 1.33 ± 1.3
69.75 ± 25.50 1.15 ± 0.8
P = 0.002 and 0.012 for BUN and creatinine, respectively, between control and cisplatin groups. P = 0.810 and 0.846 for BUN and creatinine, respectively, between cisplatin and cisplatin plus erdosteine groups.
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Fig. 1. The scatter plot graph of MDA and NO in control, cisplatin-treated and erdosteine plus cisplatin-treated rats. A single dose intraperitoneal administration of cisplatin treatment resulted in a limited increase in renal tissue MDA level while a significant increase in the renal tissue NO level when compared to the control rats. Prophylactic erdosteine treatment significantly prevented the increase in both MDA and NO levels in the damaged renal tissue.
4. Discussion Our study shows that BUN and creatinine levels are markedly increased in the blood of rats treated with cisplatin. We indicate that prophylactic oral administration of the erdosteine in rats may have protective potential on cisplatin-induced acute renal failure as measured by BUN and creatinine level. Although the increment of mean BUN and creatinine concentration cannot reach the statistically significant level, it is less prominent in erdosteine
pre-treated rats than cisplatin alone. Kidney histology provided a marked reduction in the extent of tubular damage in those animals treated with erdosteine (Fig. 3). In addition, our study indicates that erdosteine significantly prevents the depletion of the renal tissue CAT and GSH-Px activities by scavenging free radicals produced by cisplatin (Fig. 2). But further statistical analysis of the data showed that the difference in CAT activity between cisplatin and cisplatin plus erdosteine-treated rats was due to the higher CAT activity of a rat in erdosteine pre-treated group as shown in Fig. 1b.
Fig. 2. The scatter plot graph of renal tissue CAT, GSH-Px and SOD activities in control, cisplatin and erdosteine plus cisplatin-treated rats. A single dose intraperitoneal administration of cisplatin treatment resulted in significant decrease in renal tissue activity of these antioxidant enzyme activities as compared to the control rats. Prophylactic erdosteine treatment significantly prevented the depletion of damaged renal tissue GSH-Px activity. However, erdosteine has a limited protective effect on the reduction of tissue CAT and SOD activity caused by cisplatin administration.
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Fig. 3. Three micrographs that have been taken from the cortex of kidney in each group, which are control (A), cisplatin (B) and erdosteine plus cisplatin (C). Control rat showing no abnormality. The extensive epithelial cell vacuolization, swelling, desquamation and necrosis are clearly observed in the kidney of the cisplatin-treated rat. The larger tubular lumens in cisplatin-treated rats than those of the control and the erdosteine groups are showing extensive necrosis (B). The wall height (between two opposite direction of arrow heads) of proximal tubules (P) in the control kidney is higher than both of cisplatin and erdosteine groups. The free radical scavenger erdosteine shows a marked reduction in the extent of tubular damage (C) (scale bar 10 m).
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Also, erdosteine prevented the increase of NO and MDA concentrations in the renal tissue of rat at a statistically significant level (Fig. 1). Cisplatin acts mostly on the proximal renal tubule of the kidney. Proximal tubular epithelial cells take up cisplatin actively and the concentration of the drug in these cells exceeds plasma concentration by a factor of five. Cisplatin toxicity in proximal tubular cells is morphologically characterized by tubular necrosis [17]. Despite the underlying mechanism of cisplatin-induced nephrotoxicity is still not well known, many recent in vitro and in vivo studies indicate an important role of reactive oxygen metabolites in the pathogenesis of this nephrotoxicity [18]. Cisplatin induces free radical production causing oxidative renal damage. Antioxidants have been shown to be protective in cisplatin nephrotoxicity [19]. Various free radical scavengers have been shown to be effective in protection from cisplatin-induced nephrotoxicity and treatment with such agents provided significant protection against cisplatin-induced acute renal failure [20–22]. In the present study, lipid peroxidation was monitored by measuring of MDA which results from free radical damage to membrane components of the cells. We observed a moderate increase in the MDA concentration in the kidney tissue of rats treated with cisplatin alone. Erdosteine significantly attenuated the increase of MDA concentration in renal tissue. This is probably due to its elimination capacity for free oxygen radicals. The protection of renal failure by erdosteine may also due to the reduction of lipid peroxidation of renal tubular cells in rat kidney. Also, preventive effect of erdosteine in the depletion of CAT and GSH-Px activities may be due to its free radical scavenging and antioxidant activity. Recently, it has been discussed the role of NO in the pathophysiology of acute renal failure [23]. Increasing evidences suggest that NO has an important role in modulating oxidant stress and tissue damage. Peresleni et al. demonstrated that oxidant stress to the epithelial cells caused an increase in immunodetectable inducible NO synthase (iNOS), which results in an elevation in NO release, nitrite production, and decreased cell viability [24]. The mechanism mediating induction of iNOS due to the free radical exposure remains unknown. It has recently hypothesized that cytotoxic effect of NO production depends on redox state of the cell and its ability to generate peroxynitrite (ONOO− ) anion. Peroxynitrite, a highly reactive oxidant formed during the interaction between NO and O2 − , can attack a wide variety of biological targets. Despite extensive researches evaluated the role of NO in cyclosporine A-induced acute renal failure [25], studies in cisplatin-induced acute renal failure are scant and inconclusive. The presented study indicated the marked elevation in NO level in damaged kidney tissue of the cisplatin-treated rats and erdosteine significantly attenuated this increment (P < 0.0001). This increment of NO generation in the renal tissue of cisplatin administrated rats support above mentioned mechanism relating genera-
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tion of NO caused by free radicals under oxidative stress [25]. Based on this evidence of the important role of NO in cisplatin-induced acute renal failure in rat, it appears that the inhibition of NO during cisplatin treatment may offer the protection against cisplatin nephrotoxicity. In conclusion, although the preventive effect of erdosteine on the plasma BUN and creatinine levels can not reach the statistically significant levels, we think that erdosteine may be a promising drug against cisplatin-induced renal failure and further studies are warranted to define optimum dosage of this drug. Also, additional studies are warranted to determine whether erdosteine can prevent nephrotoxicity in humans as well as whether erdosteine therapy affects the anti-tumor activity of cisplatin. In addition, these data indicate that NO may play a role in the pathogenesis of cisplatin nephrotoxicity. The effect of NO inhibition on cisplatin-nephrotoxicity should also be further investigated.
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