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The effects of erdosteine on the activities of some metabolic enzymes during cisplatin-induced nephrotoxicity in rats
Pharmacological Research 50 (2004) 287–290
The effects of erdosteine on the activities of some metabolic enzymes during cisplatin-induced nephrotoxicity in rats H. Ramazan Yilmaz a,∗ , Mustafa Iraz b , Sadik Sogut c , Huseyin Ozyurt d , Zeki Yildirim e , Omer Akyol f , Serdar Gergerlioglu g a
Department of Medical Biology and Genetics, Faculty of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey b Department of Pharmacology, Faculty of Medicine, Inonu University, Malatya, Turkey c Department of Biochemistry, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey d Department of Biochemistry, Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey e Department of Pulmonary, Faculty of Medicine, Inonu University, Malatya, Turkey f Department of Biochemistry, Faculty of Medicine, Inonu University, Malatya, Turkey g Department of Physiology, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey Accepted 1 March 2004
Abstract Cisplatin is one of the widely used chemothrapeutic agents. One of the major side effects of the drug is renal toxicity. The aims of the presented study was (1) to investigate the effect of cisplatin on some renal metabolic enzyme activities such as hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) in an experimental model of acute renal failure and (2) to examine the protective role of erdosteine, an expectorant agent which has also antioxidant properties on cisplatin–induced nephrotoxicity and the enzyme activities mentioned above. Female Wistar albino rats were divided into three groups: sham operation group (n = 6), cisplatin group (n = 9), erdostein + cisplatin group (n = 8). All the chemicals used were applied intraperitoneally. Hexokinase, G6PD, LDH, and MDH activities were determined in the kidney supernatant at the end of the surgical procedures. Spectrophotometric methods were used to determine the activities of above-mentioned enzymes in the kidney tissue. Hexokinase and G6PD activities were found to be increased in cisplatin group compared to control group. G6PD activities were found to be decreased in erdosteine + cisplatin group compared to cisplatin group. There were minimal changes in LDH and MDH activities of the two study groups compared with the control group. The results obtained suggested that the glucose metabolizing metabolic pathways of renal tissue were partially affected from cisplatin toxicity and erdosteine have some protective effects on these enzyme activities. © 2004 Elsevier Ltd. All rights reserved. Keywords: Erdosteine; Cisplatin; Hexokinase; Glucose-6-phosphate dehydrogenase; Lactate dehydrogenase; Malate dehydrogenase; Kidney
1. Introduction Cisplatin (cis-dichlorodiammineplatinum(II), CDDP) is widely used in the treatment of solid tumors [1]. Nevertheless, its full clinical utility is limited due to some adverse side effects including renal toxicity [2]. It is known that cisplatin preferentially accumulates in cells of the proximal tubule [3]. Morphological correlates of tubulotoxicity are cellular necrosis, loss of microvilli, alterations in number and size of lysosomes, and mitochondrial vacuolization [2]. Although the production of reactive oxygen species (ROS) by the cisplatin has been implicated in the pathogenesis ∗ Corresponding author. Tel.: +90-246-211-3330; fax: +90-246-237-1165. E-mail address: [email protected] (H.R. Yilmaz).
1043-6618/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2004.03.003
of cisplatin-induced renal injury, the precise mechanisms underlying the disorders remain to be unknown. The free radicals frequently attack lipid component of cell membrane, resulting in lipid peroxidation. Proteins are also subject to free radical-mediated denaturation which may lead to structural or enzymatic deactivation [3], and free radical can also causes mitochondrial dysfunction [4]. Therefore, we hypothesized that the enzymatic deactivation in carbohydrate metabolism as well as mitochondrial dysfunction in the renal cells may contribute to the pathogenesis of cisplatin-mediated renal failure and that free radical scavengers may modify this metabolic pathway by scavenging free radical. Erdosteine (N-(caboxymethylthioacetyl)-homosysteine thiolactone), as a thiol derivate, contains two blocked sulphydryl groups which became free only after hepatic metab-
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olization. The reducing potential of these sulphydryl groups accounts for free radical scavenging and antioxidant activity of erdosteine [5,6]. Experimental and clinical studies demonstrate the free radical scavenging properties of erdosteine. In a previous study, authors showed that erdosteine attenuated cisplatin-induced acute renal failure in rats [7]. In the presented study, we further analyzed the damaged renal tissue used in the previous study to investigate the effects of erdosteine on the activities of hexokinase (HK; EC 2.7.1.1), glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49), malate dehydrogenase (MDH; EC 1.1.1.37), and lactate dehydrogenase (LDH; EC 1.1.1.27) in the damaged kidney tissue by cisplatin in rats and possible role of these enzymes in the pathogenesis of cisplatin nephrotoxicity [7].
2. Materials and methods Animal model of cisplatin-induced renal failure were described in the previous study [7,8]. Briefly, the animals received an intraperitoneal injection of cisplatin (cisplatinum ebewe, 0.5 mg ml−1 ) at a dose of 7 mg kg−1 BW and were sacrificed 5 days after cisplatin injection. Kidneys were obtained for the various biochemical measurements. Erdosteine (Ilsan, Turkey) was dissolved in distilled water with NaHCO3 and administered orally once a day at a dose of 10 mg kg−1 BW via plastic disposable syringes. First dose of erdosteine was given 24 h prior to cisplatin injection and continued until sacrifice. Isotonic saline solution (an equal volume of cisplatin) was injected intraperitoneally into the control rats. NaHCO3 served as control, dissolved with distilled water was given orally (as equal volume of erdosteine) 24 h prior to the saline solution injection, followed by once a day at the same dose until sacrifice. 2.1. Biochemical measurements After kidney tissues were obtained for biochemical analysis, the specimens were washed out from contaminating blood with ice-cold buffered saline. They were weighted and cut very thinly with a clean scalpel blade and then homogenized in 0.15 M ice-cold KCl for 3 min at 16,000 rpm with
a homogenizer (Ultra Turrax Type T-25-B; Labortechnic, Staufen, Germany). The homogenates were then centrifuged for 1 h at 4 ◦ C at 5000 × g. All measurements were done at the supernatant. 2.2. Enzyme activity determinations The activities of HK, G6PD, MDH, and LDH enzymes were determined spectrophotometrically [9], from the oxidation of NADH (for MDH and LDH) or the reduction of NADP+ (for HK and G6PD) by taking the decrease or increase of the absorbance (A340 ), in reaction mixtures at 25 ◦ C for 1-min period. Tissue protein levels were determined by the Lowry procedure, with bovine serum albumin as a standard [10]. Activities were given in milliunits (mU) and units (U) per milligram protein (mg protein−1 ). All samples were assayed in duplicate. 2.3. Data analysis Data are expressed as means±standard error. The one-way ANOVA analysis of variance and post-hoc multiple comparison tests (least significant difference, LSD) were performed on the data of biochemical variables to examine differences among groups. All analyses were made using the SPSS statistical software package. A P-value of <0.05 were considered as statistically significant.
3. Results There were no macroscopic changes in kidney tissues after cisplatin application. As shown in Table 1, HK and G6PH activities were found to be increased significantly in rat treated with cisplatin alone (2.74 ± 0.09 mU mg protein−1 , 3.34 ± 0.15 mU mg protein−1 , respectively) when compared to control animal (1.65 ± 0.18 mU mg protein−1 , 1.50 ± 0.08 mU mg protein−1 , respectively) (P < 0.0001). LDH activity was found to be unchanged in cisplatin group when compared to control group (P > 0.05). However, there was a little decrease in MDH activity of renal tissue of rat receiving cisplatin when compared to control rat (P > 0.05).
Table 1 Enzyme activities of kidney tissues in cisplatin, cisplatin plus erdostein, and control groups in a rat model of cisplatin-induced nephrotoxicity
(I) Control (n = 6) (II) Cisplatin (n = 9) (III) Cisplatin + erdosteine (n = 8)
HK (mU mg protein−1 )
G6PD (mU mg protein−1 )
LDH (U mg protein−1 )
MDH (U mg protein−1 )
1.65 ± 0.18 2.74 ± 0.09 2.67 ± 0.16
1.50 ± 0.08 3.34 ± 0.15 2.56 ± 0.19
0.44 ± 0.01 0.45 ± 0.01 0.42 ± 0.01
2.21 ± 0.03 2.06 ± 0.11 1.85 ± 0.05
0.0001 0.0001 0.001
ns ns ns
ns 0.029 ns
P values (I) and (II) (I)–(III) (II) and (III)
0.0001 0.0001 ns
Results were expressed as mean ± S.E.M. Abbreviations: HK, hexokinase; G6PD, glucose-6-phosphate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; ns, not significant.
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Erdosteine co-administration with cisplatin resulted in a decrease in G6PD (2.56 ± 0.19 mU mg protein−1 ) when compared to cisplatin treated rats (3.34±0.15 mU mg protein−1 ) (P < 0.001). Although there were decreases in HK, MDH, and LDH activities of erdosteine treated rats when compared to cisplatin group, the differences were not statistically significant.
4. Discussion The result of the present study indicated that cisplatin administration at a dose of 7 mg kg−1 BW resulted in a significant increase in HK and G6PD activities in the damaged rat kidney. Hexokinase is responsible for the phosphorylation of glucose into its metabolically active from glucose-6-phosphate. Hexokinase enzyme is on the pathway for the net conversion of adenosine diphosphate into ATP [11]. Glucose-6-phosphate dehydrogenase is the key regulatory enzyme in the pentose phosphate cycle catalyzing the oxidation of glucose-6-phosphate to 6-phosphogluconolactone. The production of reducing equivalents in the form of NADPH in this reaction meets the cellular needs for reductive biosynthesis and maintenance of the cellular redox status [12]. It was shown that the serum activities of glutathione reductase and isocitrate dehydrogenase were significantly increased and much greater changes were found in the kidneys, with increased activity of G6PD [13,14]. In cisplatin-treated rats, although statistically insignificant, MDH activity was found to be decreased in the kidney tissues. Similarly, Bogin et al. [14] found that in kidneys of cisplatin-treated rats, the activities of aspartate and alanine aminotransferase, alkaline phospatase, malic dehydrogenase, sorbitol dehydrogenase and gamma-glutamyltransferase were decreased. On the other hand, a decreased phosphorylation to oxidation ratio in the mitochondria indicates reduced adenosine triphosphate production. MDH catalyzes the reaction of l-malate and NAD+ to oxalacetate, NADH, and a proton [15]. Previous studies have been shown that cisplatin-induced nephrotoxicity is related to oxidative stress and that lipid peroxidation is one of mechanisms leading to kidney damage [3]. Badary et al. [3] showed that cisplatin induced a significant increase in both renal glutathione and lipid peroxide levels. Glucose-6-phosphate dehydrogenase plays a key role in the hexose monophosphate pathway and through these reactions, generates NADPH, which is required as electron donor in various biosynthetic pathways and for the regeneration of reduced glutathione, which helps protect the cells against oxidative damage [16]. 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 ROS in the pathogenesis of this nephrotoxicity as well as neurotoxicity and ototoxicity [17,18]. Cisplatin induces free radical production causing oxidative renal damage. It has been sug-
289
gested that oxidative stress is an important mechanism of cisplatin-induced toxicity possibly due to depletion of glutathione [19]. Cisplatin has been shown to inhibit the function of respiratory complexes in normal tubular cells [20]. Because mitochondrial electron transport system is an important source of ROS [21], mitochondrial damage could also be a source of ROS induced by cisplatin. On the other hand, cisplatin chemotherapy has been shown to induce a decrease in plasma antioxidant levels, which could involve a failure of the antioxidant defense against free radical damage generated by antitumor drugs [22]. Antioxidants have been shown to be protective in cisplatin nephrotoxicity [16]. 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 [23]. Free radical scavenging effect of erdosteine was found in several previous works [6,24–28]. The active metabolites of erdosteine after metabolization in vivo have two –SH groups. Erdosteine may possibly prevent cisplatin-induced oxidant injury by means of these sulfhydryl-containing metabolites. Previous studies have shown that erdosteine treatment resulted in high antioxidant enzymes and also prevented lipid peroxidation induced by doxorubucin in plasma and erythrocytes of rats [29]. In addition, erdosteine has been shown to significantly attenuate the increase of lipid peroxidation in renal tissue after cisplatin toxicity [7]. Furthermore, it seems to be an important agent for preventing oxidative injury via its MPO reducing activity [30]. Some authors observed that erdosteine has potent free radical scavenging activity against bleomycin-induced lung fibrosis model of rats and that it has a powerful protective effect against bleomycine-induced lung fibrosis in rats, manifested decreasing of hydroxyproline level (a marker of interstitital fibrosis) and histological protection [31]. Based on the similar proposed mechanism for bleomycin and cisplatin (accounting for the pathogenesis of lung fibrosis and nephrotoxicity, respectively), we hypothesized that prophylactic erdosteine administration may prevent cisplatin-induced acute renal failure in rat by its free radical trapping activity. Erdostein caused significant decreases in the activity of G6PD compared to cisplatin group (P < 0.001). Decreases in the activities of HK, LDH and MDH were not significant. The beneficial effects of erdosteine are evidenced by a decrease in kidney G6PD. As a conclusion, co-administration of erdosteine with cisplatin reversed the changes in some metabolic enzyme activities induced by cisplatin.
References [1] Rosenberg B. Fundamental studies with cisplatin. Cancer 1985; 55:2303–16. [2] Kuhlmann MK, Horsch E, Burkhardt G, Wagner M, Kohler H. Reduction of cisplatin toxicity in cultured renal tubular cells by the bioflavonoid quercetin. Arch Toxicol 1998;72:536–40.
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[3] Badary OA, Nagi MN, Al-Sawaf HA, Al-Harbi M, Al-Bekairi AM. Effect of l-histidinol on cisplatin nephrotoxicity in the rat. Nephron 1997;77:435–9. [4] Leibbrandt ME, Wolfgang GH, Metz AL, Ozobia AA, Haskins JR. Critical subcellular targets of cisplatin and related platinum analogs in rat proximal tubule cells. Kidney Int 1995;48:761–70. [5] Dechant KL, Noble S. Erdosteine. Drugs 1996;52:875–81. [6] 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. [7] Yildirim Z, Sogut S, Odaci E, Iraz M, Ozyurt H, Kotuk M, et al. Oral erdosteine administration attenuates cisplatin-induced renal tubular damage in rats. Pharmacol Res 2003;47:149–56. [8] Zhang JG, Zhong LF, Zhang M, Xia YX. Protection effects of procaine on oxidative stress and toxicities of renal cortical slices from rats caused by cisplatin in vitro. Arch Toxicol 1992;66:354–8. [9] Boehringer Mannheim. Biochemica information handbook. Boehringer Mannheim GmbH; 1973. [10] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75. [11] Al-Naama MM, Al-Naama LM, Al-Sadoon TA. Glucose-6-phosphate dehydrogenase, hexokinase and pyruvate kinase activities in erythrocytes of neonates and adults in Basrah. Ann Trop Paediatr 1994;14:195–200. [12] Ho HY, Cheng ML, Lu FJ, Chou YH, Stern A, Liang CM, et al. Enhanced oxidative stress and accelerated cellular senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts. Free Radic Biol Med 2000;29:156–69. [13] el Daly ES. Protective effect of cysteine and vitamin E, Crocus sativus and Nigella sativa extracts on cisplatin-induced toxicitiy in rats. J Pharm Belg 1998;53:87–93. [14] Bogin E, Marom M, Levi Y. Changes in serum, liver and kidneys of cisplatin-treated rats; effects of antioxidants. Eur J Clin Chem Clin Biochem 1994;32:843–51. [15] Zauner KP, Conrad M. Enzymatic pattern processing. Naturwissenschaften 2000;87:360–2. [16] Ganczakowski M, Town M, Bowden DK, Vulliamy TJ, Kaneko A, Clegg JB, et al. Multiple glucose-6-phosphate dehydrogenasedeficient variants correlate with malaria endemicity in Vanuatu archipelago (southwestern Pacific). Am J Hum Genet 1995;56:294– 301. [17] Ravi R, Somani SM, Rybak LP. Mechanisms of cisplatin ototoxicity: antioxidant system. Pharmacol Toxicol 1995;76:386–94.
[18] Rybak L, Ravi R, Somani SM. Mechanism of protection by diethyldithiocarbamate against cisplatin ototoxicity: antioxidant system. Fundam Appl Toxicol 1995;26:293–300. [19] Kharbangar A, Khynriam D, Prasad SB. Effect of cisplatin on mitochondrial protein, glutathione, and succinate dehydrogenase in Dalton lymphoma-bearing mice. Cell Biol Toxicol 2000;16:363–73. [20] Kruidering M, van de Water B, de Heer E, Mulder GJ, Nagelkerke JF. Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: mitochondria dysfunction of complex I to IV of the respiratory chain. J Pharmacol Exp Ther 1997;280:638–49. [21] Richter C, Gogvadze V, Laffranchi R, Schlabach R, Schweizer M, Suter M, et al. Oxidants in mitochondria: from physiology to diseases. Biochim Biophys Acta 1995;1271:67–74. [22] Weijl NI, Hopman GD, Wipkink-Bakker A, Lentjes EG, Berger HM, Cleton FJ, et al. Cisplatin combination chemotherapy induces a fall in plasma antioxidants of cancer patients. Ann Oncol 1998;9:1331–7. [23] Baliga R, Ueda N, Walker PD, Shah SV. Oxidant mechanisms in toxic acute renal failure. Drug Metab Rev 1999;31:971–97. [24] Goligorsky MS, Brodsky AV, Noiri E. Nitric oxide in acute ranla failure: NOS versus NOS. Kidney Int 2002;61:855–61. [25] 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. [26] Inglesi M, Nicola M, Fregnan GB, Bradamante S, Pagani G. Synthesis and free radical scavenging properties of the enantiomers of erdosteine. Farmaco 1994;40:703–8. [27] Vagliasindi M, Fregman GB. Erdosteine protection against cigarette smoking-induced functional antiprotease deficiency in human bronchiolo-alveolar structures. Int J Clin Farmacol Ther Toxicol 1989;27:238–41. [28] 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. [29] Fadillioglu E, Erdogan H. Effects of erdosteine treatment against doxorubicin-induced toxicity through erythrocyte and plasma oxidant/antioxidant status in rats. Pharmacol Res 2003;47:317–22. [30] Yagmurca M, Fadillioglu E, Erdogan H, Ucar M, Sogut S, Irmak MK. Erdosteine prevents doxorubicin-induced cardiotoxicity in rats. Pharmacol Res 2003;48:377–82. [31] Sogut S, Ozyurt H, Akyol O, Kart L, Iraz M, Yildirim Z, et al. Protection of erdosteine on bleomycine-induced lung fibrosis in rats. Eur Res J 2002;20(Suppl 38):487.
The effects of erdosteine on the activities of some metabolic enzymes during cisplatin-induced nephrotoxicity in rats H. Ramazan Yilmaz a,∗ , Mustafa Iraz b , Sadik Sogut c , Huseyin Ozyurt d , Zeki Yildirim e , Omer Akyol f , Serdar Gergerlioglu g a
Department of Medical Biology and Genetics, Faculty of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey b Department of Pharmacology, Faculty of Medicine, Inonu University, Malatya, Turkey c Department of Biochemistry, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey d Department of Biochemistry, Faculty of Medicine, Gaziosmanpasa University, Tokat, Turkey e Department of Pulmonary, Faculty of Medicine, Inonu University, Malatya, Turkey f Department of Biochemistry, Faculty of Medicine, Inonu University, Malatya, Turkey g Department of Physiology, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey Accepted 1 March 2004
Abstract Cisplatin is one of the widely used chemothrapeutic agents. One of the major side effects of the drug is renal toxicity. The aims of the presented study was (1) to investigate the effect of cisplatin on some renal metabolic enzyme activities such as hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) in an experimental model of acute renal failure and (2) to examine the protective role of erdosteine, an expectorant agent which has also antioxidant properties on cisplatin–induced nephrotoxicity and the enzyme activities mentioned above. Female Wistar albino rats were divided into three groups: sham operation group (n = 6), cisplatin group (n = 9), erdostein + cisplatin group (n = 8). All the chemicals used were applied intraperitoneally. Hexokinase, G6PD, LDH, and MDH activities were determined in the kidney supernatant at the end of the surgical procedures. Spectrophotometric methods were used to determine the activities of above-mentioned enzymes in the kidney tissue. Hexokinase and G6PD activities were found to be increased in cisplatin group compared to control group. G6PD activities were found to be decreased in erdosteine + cisplatin group compared to cisplatin group. There were minimal changes in LDH and MDH activities of the two study groups compared with the control group. The results obtained suggested that the glucose metabolizing metabolic pathways of renal tissue were partially affected from cisplatin toxicity and erdosteine have some protective effects on these enzyme activities. © 2004 Elsevier Ltd. All rights reserved. Keywords: Erdosteine; Cisplatin; Hexokinase; Glucose-6-phosphate dehydrogenase; Lactate dehydrogenase; Malate dehydrogenase; Kidney
1. Introduction Cisplatin (cis-dichlorodiammineplatinum(II), CDDP) is widely used in the treatment of solid tumors [1]. Nevertheless, its full clinical utility is limited due to some adverse side effects including renal toxicity [2]. It is known that cisplatin preferentially accumulates in cells of the proximal tubule [3]. Morphological correlates of tubulotoxicity are cellular necrosis, loss of microvilli, alterations in number and size of lysosomes, and mitochondrial vacuolization [2]. Although the production of reactive oxygen species (ROS) by the cisplatin has been implicated in the pathogenesis ∗ Corresponding author. Tel.: +90-246-211-3330; fax: +90-246-237-1165. E-mail address: [email protected] (H.R. Yilmaz).
1043-6618/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2004.03.003
of cisplatin-induced renal injury, the precise mechanisms underlying the disorders remain to be unknown. The free radicals frequently attack lipid component of cell membrane, resulting in lipid peroxidation. Proteins are also subject to free radical-mediated denaturation which may lead to structural or enzymatic deactivation [3], and free radical can also causes mitochondrial dysfunction [4]. Therefore, we hypothesized that the enzymatic deactivation in carbohydrate metabolism as well as mitochondrial dysfunction in the renal cells may contribute to the pathogenesis of cisplatin-mediated renal failure and that free radical scavengers may modify this metabolic pathway by scavenging free radical. Erdosteine (N-(caboxymethylthioacetyl)-homosysteine thiolactone), as a thiol derivate, contains two blocked sulphydryl groups which became free only after hepatic metab-
288
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olization. The reducing potential of these sulphydryl groups accounts for free radical scavenging and antioxidant activity of erdosteine [5,6]. Experimental and clinical studies demonstrate the free radical scavenging properties of erdosteine. In a previous study, authors showed that erdosteine attenuated cisplatin-induced acute renal failure in rats [7]. In the presented study, we further analyzed the damaged renal tissue used in the previous study to investigate the effects of erdosteine on the activities of hexokinase (HK; EC 2.7.1.1), glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49), malate dehydrogenase (MDH; EC 1.1.1.37), and lactate dehydrogenase (LDH; EC 1.1.1.27) in the damaged kidney tissue by cisplatin in rats and possible role of these enzymes in the pathogenesis of cisplatin nephrotoxicity [7].
2. Materials and methods Animal model of cisplatin-induced renal failure were described in the previous study [7,8]. Briefly, the animals received an intraperitoneal injection of cisplatin (cisplatinum ebewe, 0.5 mg ml−1 ) at a dose of 7 mg kg−1 BW and were sacrificed 5 days after cisplatin injection. Kidneys were obtained for the various biochemical measurements. Erdosteine (Ilsan, Turkey) was dissolved in distilled water with NaHCO3 and administered orally once a day at a dose of 10 mg kg−1 BW via plastic disposable syringes. First dose of erdosteine was given 24 h prior to cisplatin injection and continued until sacrifice. Isotonic saline solution (an equal volume of cisplatin) was injected intraperitoneally into the control rats. NaHCO3 served as control, dissolved with distilled water was given orally (as equal volume of erdosteine) 24 h prior to the saline solution injection, followed by once a day at the same dose until sacrifice. 2.1. Biochemical measurements After kidney tissues were obtained for biochemical analysis, the specimens were washed out from contaminating blood with ice-cold buffered saline. They were weighted and cut very thinly with a clean scalpel blade and then homogenized in 0.15 M ice-cold KCl for 3 min at 16,000 rpm with
a homogenizer (Ultra Turrax Type T-25-B; Labortechnic, Staufen, Germany). The homogenates were then centrifuged for 1 h at 4 ◦ C at 5000 × g. All measurements were done at the supernatant. 2.2. Enzyme activity determinations The activities of HK, G6PD, MDH, and LDH enzymes were determined spectrophotometrically [9], from the oxidation of NADH (for MDH and LDH) or the reduction of NADP+ (for HK and G6PD) by taking the decrease or increase of the absorbance (A340 ), in reaction mixtures at 25 ◦ C for 1-min period. Tissue protein levels were determined by the Lowry procedure, with bovine serum albumin as a standard [10]. Activities were given in milliunits (mU) and units (U) per milligram protein (mg protein−1 ). All samples were assayed in duplicate. 2.3. Data analysis Data are expressed as means±standard error. The one-way ANOVA analysis of variance and post-hoc multiple comparison tests (least significant difference, LSD) were performed on the data of biochemical variables to examine differences among groups. All analyses were made using the SPSS statistical software package. A P-value of <0.05 were considered as statistically significant.
3. Results There were no macroscopic changes in kidney tissues after cisplatin application. As shown in Table 1, HK and G6PH activities were found to be increased significantly in rat treated with cisplatin alone (2.74 ± 0.09 mU mg protein−1 , 3.34 ± 0.15 mU mg protein−1 , respectively) when compared to control animal (1.65 ± 0.18 mU mg protein−1 , 1.50 ± 0.08 mU mg protein−1 , respectively) (P < 0.0001). LDH activity was found to be unchanged in cisplatin group when compared to control group (P > 0.05). However, there was a little decrease in MDH activity of renal tissue of rat receiving cisplatin when compared to control rat (P > 0.05).
Table 1 Enzyme activities of kidney tissues in cisplatin, cisplatin plus erdostein, and control groups in a rat model of cisplatin-induced nephrotoxicity
(I) Control (n = 6) (II) Cisplatin (n = 9) (III) Cisplatin + erdosteine (n = 8)
HK (mU mg protein−1 )
G6PD (mU mg protein−1 )
LDH (U mg protein−1 )
MDH (U mg protein−1 )
1.65 ± 0.18 2.74 ± 0.09 2.67 ± 0.16
1.50 ± 0.08 3.34 ± 0.15 2.56 ± 0.19
0.44 ± 0.01 0.45 ± 0.01 0.42 ± 0.01
2.21 ± 0.03 2.06 ± 0.11 1.85 ± 0.05
0.0001 0.0001 0.001
ns ns ns
ns 0.029 ns
P values (I) and (II) (I)–(III) (II) and (III)
0.0001 0.0001 ns
Results were expressed as mean ± S.E.M. Abbreviations: HK, hexokinase; G6PD, glucose-6-phosphate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; ns, not significant.
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Erdosteine co-administration with cisplatin resulted in a decrease in G6PD (2.56 ± 0.19 mU mg protein−1 ) when compared to cisplatin treated rats (3.34±0.15 mU mg protein−1 ) (P < 0.001). Although there were decreases in HK, MDH, and LDH activities of erdosteine treated rats when compared to cisplatin group, the differences were not statistically significant.
4. Discussion The result of the present study indicated that cisplatin administration at a dose of 7 mg kg−1 BW resulted in a significant increase in HK and G6PD activities in the damaged rat kidney. Hexokinase is responsible for the phosphorylation of glucose into its metabolically active from glucose-6-phosphate. Hexokinase enzyme is on the pathway for the net conversion of adenosine diphosphate into ATP [11]. Glucose-6-phosphate dehydrogenase is the key regulatory enzyme in the pentose phosphate cycle catalyzing the oxidation of glucose-6-phosphate to 6-phosphogluconolactone. The production of reducing equivalents in the form of NADPH in this reaction meets the cellular needs for reductive biosynthesis and maintenance of the cellular redox status [12]. It was shown that the serum activities of glutathione reductase and isocitrate dehydrogenase were significantly increased and much greater changes were found in the kidneys, with increased activity of G6PD [13,14]. In cisplatin-treated rats, although statistically insignificant, MDH activity was found to be decreased in the kidney tissues. Similarly, Bogin et al. [14] found that in kidneys of cisplatin-treated rats, the activities of aspartate and alanine aminotransferase, alkaline phospatase, malic dehydrogenase, sorbitol dehydrogenase and gamma-glutamyltransferase were decreased. On the other hand, a decreased phosphorylation to oxidation ratio in the mitochondria indicates reduced adenosine triphosphate production. MDH catalyzes the reaction of l-malate and NAD+ to oxalacetate, NADH, and a proton [15]. Previous studies have been shown that cisplatin-induced nephrotoxicity is related to oxidative stress and that lipid peroxidation is one of mechanisms leading to kidney damage [3]. Badary et al. [3] showed that cisplatin induced a significant increase in both renal glutathione and lipid peroxide levels. Glucose-6-phosphate dehydrogenase plays a key role in the hexose monophosphate pathway and through these reactions, generates NADPH, which is required as electron donor in various biosynthetic pathways and for the regeneration of reduced glutathione, which helps protect the cells against oxidative damage [16]. 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 ROS in the pathogenesis of this nephrotoxicity as well as neurotoxicity and ototoxicity [17,18]. Cisplatin induces free radical production causing oxidative renal damage. It has been sug-
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gested that oxidative stress is an important mechanism of cisplatin-induced toxicity possibly due to depletion of glutathione [19]. Cisplatin has been shown to inhibit the function of respiratory complexes in normal tubular cells [20]. Because mitochondrial electron transport system is an important source of ROS [21], mitochondrial damage could also be a source of ROS induced by cisplatin. On the other hand, cisplatin chemotherapy has been shown to induce a decrease in plasma antioxidant levels, which could involve a failure of the antioxidant defense against free radical damage generated by antitumor drugs [22]. Antioxidants have been shown to be protective in cisplatin nephrotoxicity [16]. 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 [23]. Free radical scavenging effect of erdosteine was found in several previous works [6,24–28]. The active metabolites of erdosteine after metabolization in vivo have two –SH groups. Erdosteine may possibly prevent cisplatin-induced oxidant injury by means of these sulfhydryl-containing metabolites. Previous studies have shown that erdosteine treatment resulted in high antioxidant enzymes and also prevented lipid peroxidation induced by doxorubucin in plasma and erythrocytes of rats [29]. In addition, erdosteine has been shown to significantly attenuate the increase of lipid peroxidation in renal tissue after cisplatin toxicity [7]. Furthermore, it seems to be an important agent for preventing oxidative injury via its MPO reducing activity [30]. Some authors observed that erdosteine has potent free radical scavenging activity against bleomycin-induced lung fibrosis model of rats and that it has a powerful protective effect against bleomycine-induced lung fibrosis in rats, manifested decreasing of hydroxyproline level (a marker of interstitital fibrosis) and histological protection [31]. Based on the similar proposed mechanism for bleomycin and cisplatin (accounting for the pathogenesis of lung fibrosis and nephrotoxicity, respectively), we hypothesized that prophylactic erdosteine administration may prevent cisplatin-induced acute renal failure in rat by its free radical trapping activity. Erdostein caused significant decreases in the activity of G6PD compared to cisplatin group (P < 0.001). Decreases in the activities of HK, LDH and MDH were not significant. The beneficial effects of erdosteine are evidenced by a decrease in kidney G6PD. As a conclusion, co-administration of erdosteine with cisplatin reversed the changes in some metabolic enzyme activities induced by cisplatin.
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