Protective effect of selenium on gentamicin-induced oxidative stress and nephrotoxicity in rats

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Introduction

Gentamicin (GM) is an aminoglycoside antibiotic com-monly used in the treatment of life-threatening Gram-negative bacterial infections (Rao et al., 2006). However, the usefulness of GM is limited by the development of nephrotoxicity. Despite the introduction of less nephro-toxic antibiotics against Gram-negative microorganisms, it is still used clinically because of its high antibacterial efficacy, rapid onset of action, low rate of true resistance, synergy with β-lactam antibiotics, and low cost (Edson and Terrell, 1999). GM nephrotoxicity involves renal free radical generation, reduction in antioxidant defense mechanisms, acute tubular necrosis, and glomerular con-gestion (Martinez-Salgado et al., 2007; Mingeot-Leclercq

and Tulkens, 1999; Geleilete et al., 2002; Abdel-Raheem et al., 2009), resulting in diminished glomerular filtration rate and renal dysfunction. The mechanisms involved in GM-induced cell injury are not clearly understood. However, several studies demonstrated that reactive oxygen species (ROS) may be important in GM-induced nephrotoxicity (Cuzzocrea et al., 2002; Yanagida et al., 2004). ROS directly act on cell components, including lipids, proteins, and DNA, destroying their structure. On the other hand, in vivo and in vitro studies have shown that the scavengers of reactive oxygen metabolites are protective in GM-induced renal failure (Fryer, 1997; Pedraza-Chaverrı et al., 2000). The administration of several compounds with antioxidant activity has been

RESEARCH ARTICLE

Protective effect of selenium on gentamicin-induced oxidative stress and nephrotoxicity in rats

Pavle Randjelovic1, Slavimir Veljkovic1, Nenad Stojiljkovic1, Ljubinka Velickovic2, Dusan Sokolovic3, Milan Stoiljkovic,4 and Ivan Ilic2

Departments of 1Physiology, 2Pathology, 3Biochemistry, and 4Pharmacology and Toxicology, Faculty of Medicine, University of Nis, Nis, Serbia

AbstractGentamicin (GM) is a widely used antibiotic against serious, life-threatening infections, but its usefulness is limited by the development of nephrotoxicity. The present study was designed to determine the protective effect of selenium (Se) in GM-induced nephrotoxicity in rats. Experiments were done on 32 adult Wistar rats divided into four groups of 8 animals each. The GM group received gentamicin (100 mg/kg), whereas the GM+Se group received the same dose of GM and selenium (1 mg/kg) by intraperitoneal (i.p.) injections on a daily basis. Animals in the Se group, serving as a positive control, received only selenium (1 mg/kg) and the control group received saline (1 mL/day), both given i.p. All groups were treated during 8 consecutive days. Quantitative evaluation of GM-induced structural alterations and degree of functional alterations in the kidneys were performed by histopathological and biochemical analyses in order to determine potential beneficial effects of selenium coadministration with GM. GM was observed to cause a severe nephrotoxicity, which was evidenced by an elevation of serum urea and creatinine levels. The significant increases in malondialdehyde levels and protein carbonyl groups indicated that GM-induced tissue injury was mediated through oxidative reactions. On the other hand, simultaneous selenium administration protected kidney tissue against oxidative damage and the nephrotoxic effect caused by GM treatment. Exposure to GM caused necrosis of tubular epithelial cells. Necrosis of tubules was found to be prevented by selenium pretreatment.

The results from our study indicate that selenium supplementation attenuates oxidative-stress–associated renal injury by reducing oxygen free radicals and lipid peroxidation in GM-treated rats.

Keywords: Gentamicin, selenium, nephrotoxicity, MDA, oxidative stress, rat

Address for Correspondence: Pavle Randjelovic, Department of Physiology, Faculty of Medicine, University of Nis, Boulevard Dr. Zorana Djindjica 81, 18000 Nis, Serbia; Fax: +381-18-238–770; E-mail: [email protected]

(Received 15 February 2011; revised 19 March 2011; accepted 02 May 2011)

Drug and Chemical Toxicology, 2012; 35(2): 141–148© 2012 Informa Healthcare USA, Inc.ISSN 0148-0545 print/ISSN 1525-6014 onlineDOI: 10.3109/01480545.2011.589446

Drug and Chemical Toxicology

2012

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15 February 2011

19 March 2011

02 May 2011

0148-0545

1525-6014

© 2012 Informa Healthcare USA, Inc.

10.3109/01480545.2011.589446

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142 Pavle Randjelovic et al.


successfully used to prevent or ameliorate GM-induced nephrotoxicity (Ali, 2003; Stojiljkovic et al., 2010).

Selenium (Se) is a fundamental trace element that plays an important role in a number of physiological processes, including the elimination of reactive oxygen species (ROS) and the modulation of redox-sensitive enzyme cascades. Humans and animals require selenium for the function of a number of selenium-dependent enzymes, also known as selenoproteins [Se-dependent glutathione peroxi-dase (GPx), thioredoxin reductase, and selenoprotein P] (Rotruck et al., 1973; Kazura and Meshnick, 1984; Callahan et al., 1988; Barceloux, 1999). The GPx enzyme was the first established selenoenzyme that could prevent oxidative damage of cell membranes. GPx not only protects cells against damage by free radicals, but also permits regen-eration of a membrane lipid molecule through reacylation (McPherson, 1994). Selenium was used in the treatment of free radical–associated diseases, such as diabetes (Kahler et al., 1993), aflatoxin Bl-induced lipid peroxidation (LPO) (Shen et al., 1991), lead (Abdollahi et al., 2001), and ben-zene toxicity (Stankovic et al., 2001). The recommended dietary allowance of selenium for both men and women is 55 µg per day for optimum biological performance. According to the National Research Council (Committee on animal nutrition, Subcommittee on selenium, 1983), selenium requirements for most animals fall in the range of 0.05–0.3 mg/kg in the dry diet. Common forms of sele-nium in dietary supplements are selenomethionine and sodium selenite. The latter is frequently used in different experimental approaches (Ates et al., 2008).

Potential therapeutic approaches to protect or reverse renal GM damage would have very important clini-cal consequences in increasing the safety of the drug. Therefore, the present study was designed to determine the protective effect of Se in GM-induced nephrotoxicity in rats.

Methods

Male Wistar albino rats (250–300 g; n = 32) were placed in a temperature- (22 ± 2°C) and humidity-controlled room with a 12-hour light-dark cycle and with free access to food and water. The standard laboratory animal diet used in the study contained less than 0.1 mg of selenium per kilogram. All experimental procedures were conducted in accord with the principles for the care and use of laboratory animals in research and approved by the local ethics committee. All efforts were made to minimize suf-fering and the number of animals used.

Experimental protocolAfter a quarantine period of 7 days, 32 rats were ran-domly divided into four groups, each consisting of 8 animals. Group 1 was used as a control and received 1 mL of saline intraperitoneally (i.p.) per day. Group 2 received only sodium selenite in a single dose of 1 mg/kg i.p. daily. Group 3 received GM (Galenika AD, Belgrade, Serbia) on a daily basis in a single dose of

100 mg/kg by i.p. injection. Group 4 was given sodium selenite (Sigma-Aldrich, St. Louis, Missouri, USA) in a single i.p. dose of 1 mg/kg, along with the same dose of GM as group 3 each day throughout the experiment. All groups were treated over 8 consecutive days. Twenty-four hours after the administration of the last doses of GM and Se, on day 9, rats were anesthetized by an i.p. injection of ketamine (Ketamidor 10%; Richter Pharma AG, Wels, Austria) and sacrificed. Renal cortical tissues were separated into two parts for biochemical analysis and light microscopic examination. Blood samples were also taken by cardiac puncture to assess the serum levels of urea and creatinine.

Biochemical analysisSerum urea and creatinine levels were determined with an automatic biochemical analyzer (A25 Biosystems, Barcelona, Spain). For estimation of oxidative stress, kidney tissue was cut in small pieces and homogenized in ice-cold water by using a homogenizer (RJ 22713–00; IKA® Works de Brasil Ltda Taquara, Rio de Janeiro, Brazil). The homogenates (10% w/v) were centrifuged at 1,500×g at 4°C for 10 minutes.

Determination of proteinsProteins were determined according to Lowry’s method (Lowry et al., 1951), using bovine serum albumin as the standard.

Determination of malondialdehydeThe intensity of LPO in kidney tissue was spectrophoto-metrically measured based on the thiobarbituric (TBA) response products (Ohkawa et al., 1979). Homogenate absorption was measured at 532 nm. The malondial-dehyde (MDA)/LPO end-product concentration was expressed per mg/protein using the molecular extinction coefficient of MDA (1.56 × 10−5 mol cm−1).

Determination of protein oxidationCarbonyl group concentration, as the level of oxidative modified proteins, was determined spectrophotometri-cally (Levine et al., 1994) using 2.4 dinitrophenylhydra-zine, a traditional carbonyl reagent. Reactive carbonyl derivatives were calculated using the DPNH molar extinc-tion coefficient at 370 nm (22 × 103 L/mol/cm) and expressed in μmol/g of protein.

Histopathological examinationsHistopathological evaluation was made in kidney tissues. Kidneys were dissected immediately and preserved in 10% buffered formaldehyde for further histopathological examinations. Tissue samples were embedded in paraf-fin, and 5–6-μm sections were cut using a rotary micro-tome and stained with hematoxilin and eosin (H&E). Other paraffin sections were stained with periodic acid-Schiff (PAS) to detect other pathological alterations clearly. A minimum of eight fields for each kidney section was examined and assigned for severity of changes by

Protective effect of selenium on gentamicin nephrotoxicity 143


an observer blinded to the treatments of the animals. All sections were examined with a Leica DMR (Leica Microsystems AG, Wetzlar, Germany) light microscope. To evaluate the level of damages, indexes such as tubu-lar degeneration, tubular necrosis, mononuclear cell infiltration, and hyaline casts were scored numerically. Evaluation criteria were as follows: 0 for no detectable lesion, 1 for mild changes, 2 for moderate changes, and 3 for severe changes.

Statistical analysisResults were expressed as the mean ± standard deviation (SD). Statistical significant difference was determined by one-way analysis of variance, followed by Tukey’s post-hoc test for multiple comparison (Graphpad Prism version 5.03; GraphPad Software, San Diego, California, USA). Probability values (P) less than 0.05 were consid-ered to be statistically significant.

Results

Levels of urea in control and Se groups were 5.60 ± 0.91 and 6.22 ± 1.05 mmol/L, respectively (Table 1). Chronic GM administration significantly elevated urea levels to 18.91 ± 2.86 mmol/L (P < 0.001), and this elevation was prevented by Se pretreatment (8.21 ± 3.78 mmol/L; P < 0.001). Consistent with nephrotoxicity, creatinine values were 48.63 ± 2.34 µmol/L in the control group and 51.25 ± 3.59 µmol/L in the Se group. In the GM group, cre-atinine was increased to 71.71 ± 9.43 µmol/L (P < 0.001), and Se-pretreated rats exhibited similar values to con-trols (57.12 ± 15.45 µmol/L).

Kidney MDA levels were found to be significantly higher in GM-treated rats (7.36 ± 0.44 µmol/mg pro-tein) than those of control animals (6.07 ± 0.54 µmol/mg protein) and the Se group (6.31 ± 1.11 µmol/mg protein) (P < 0.05). Se coadministration with GM decreased these values to 6.17 ± 0.78 µmol/mg protein (P < 0.05) (Figure 1). Carbonyl concentrations were 8.3 ± 2.5 µmol/mg protein in the control group and 8.13 ± 3.18 µmol/mg protein in the Se group. In the GM group, it was increased to 14.98 ± 2.58 µmol/mg protein (P < 0.001), and Se-pretreated rats exhibited similar values to the GM group (16.77 ± 3.12 µmol/mg protein) (Figure 2).

Histopathological changes in kidneys in all groups are summarized in Table 2. For the microscopic

examination of kidney tissues of the control group, rats revealed normal renal glomeruli surrounded by capsule and normal proximal, distal, and convoluted tubules (Figure 3A). Kidneys of animals of the Se group also showed normal histoarchitecture (Figure 3B).

Treatment with GM alone caused a marked proximal tubular necrosis, desquamation, and degeneration in the epithelial cells of the proximal tubules (Figure 3C). Vacuolation, hydrophic degeneration, desquamation, and necrosis were observed in the epithelial cells of the proximal tubules in rats of the GM group. In addition, the tubular lumens were frequently filled with hyaline casts (Figure 3D). Severe inflammatory infiltrate, in the form of mononuclear cells, was observed in the renal sections of this group (Figure 3E).

Mild histopathological lesions were observed in the glomerulus and renal tubules of kidney tissues of rats treated with GM+Se, when compared with the GM-treated group (Figure 3F). Kidneys showed reparative tendencies in the GM+Se-treated group. Cotreatment with selenium slightly decreased tubular necrosis and caused epithelial and nuclear ameliorative changes, compared with the GM-treated group.

Table 1. Effects of selenium (1 mg/kg/day) on the levels of serum urea and creatinine of rats treated with gentamicin (100 mg/kg/day).Parameters Control Se GM GM + SeUrea (mmol/L)

5.60 ± 0.91* 6.22 ± 1.05* 18.91 ± 2.86 8.21 ± 3.78*

Creatinine (µmol/L)

48.63 ± 2.34* 51.25 ± 3.59* 71.71 ± 9.43 57.12 ± 15.45**#

Data are presented as mean ± SD.*P < 0.001 versus GM;**#P < 0.05 versus GM.

Figure 1. Effects of selenium on kidney malondialdehyde in rats treated with GM. Values are means ± SD. *P < 0.05 versus control, Se, and GM+Se groups.

Figure 2. Effects of selenium on kidney protein carbonyl content in rats treated with GM. Values are means ± SD. *P < 0.001 versus control and Se groups.



Discussion

The aminoglycoside antibiotic, GM, continues to be an important agent against life-threatening infections. However, nephrotoxicity is a major complication of GM administration. GM-induced nephrotoxicity is an important cause of renal failure (Perazella, 2003). Thus, amelioration of nephrotoxicity would enhance its clinical use. Several approaches involving the use of chemical compounds have been used to reduce GM nephrotoxicity (Cuzzocrea et al., 2002; Parlakpinar et al., 2005). It has been shown that the specificity of GM renal toxicity is related to its preferential accumulation in renal convoluted tubules and lysosomes (Nagai, 2004). Aminogylcoside antibiotics such as GM are not metabo-lized in the live organism, and the injected dose is essentially eliminated by glomerular filtration, whereas a fraction accumulates in the renal proximal tubule cells where the concentration of aminoglycosides is several times higher than in plasma. GM also undergoes partial reabsorption by proximal tubular cells as a consequence of adsorptive endocytosis (Beauchamp et al., 1997; Mingeot-Leclerc and Tulkens, 1999). Acidic phospholip-ids, broadly distributed in plasma membranes in various tissues, were considered to be the binding site of amino-glycosides in the brush-border membrane of proximal tubular cells (Nagai and Takano, 2004; Nagai, 2006). GM-loaded endocytic vacuoles fuse with lysosomes, where the drug accumulates. This accumulation leads to the development of a lysosomal phospholipidosis, characterized by an impairment of phospholipase A1 and sphingomyelinase activities and by phospholipid accumulation within the lysosomal compartment. The overloaded lysosomes continue to swell. In vivo, this may result in the loss of integrity of the lysosomal mem-branes and the release of large amounts of hydrolytic enzymes, phospholipids, and aminoglycosides into the cytosol. Thus, these aminoglycosides can gain access to and injure other organelles, such as mitochondria, and disturb their functional integrity, which rapidly leads to cell death as a consequence of cell apoptosis and necro-sis (Beauchamp et al., 1997; El Mouedden et al., 2000). The concentrated accumulation of aminoglycosides in the proximal tubule cells is associated with nephrotoxic-ity (Abdel-Naim et al., 1999).

As treatment with some antioxidants protects against GM-induced renal injury, we studied here the effect of Se, a potent antioxidant and free radical scavenger, on the renal damage and oxidative injury induced by GM.

There are some experimental data suggesting that nephrotoxic drugs may also change levels of MDA, GPx, creatinine, and urea (Ozbek et al., 2000; Parlakpinar et al., 2003), which are commonly used to monitor the development and extent of renal tubular damage caused by oxidative stress. Results of this study confirmed that GM, at a dose of 100 mg/kg/day, produces nephrotoxic-ity, as evidenced by the reduction in glomerular filtration rate (GFR), which is indicated by an increase in serum creatinine. This impairment in glomerular function was accompanied by an increase in urea level.

Vasoconstriction induced by ROS is involved in the decrease in glomerular filtration rate, and ROS produce cellular injury and necrosis via several mechanisms, including lipoperoxidation and protein modification. The above information may explain why antioxidants, and Se in particular, are able to prevent GM-induced glomeru-lar and tubular dysfunction. In our study, Se was able to ameliorate the increase in creatinine and urea.

Selenium can enhance antioxidant ability by enhanc-ing the activities of antioxidant enzymes and by increas-ing contents of the antioxidants. Xia et al. (2003) reported that Se is crucial in several enzymes with physiological antioxidant properties, including GPx and thioredoxin reductase. GPx scavenges H

2O

2 and lipid hydroperox-

ides, using reducing equivalents from glutathione and protecting membrane lipids and macromolecules from oxidative damage (Watanabe et al., 1997). Therefore, this trace element could be useful as a free radical scavenger compound against stress conditions in several tissues, including the kidney.

In the present study, GM-induced increase in serum creatinine and urea levels was significantly blocked by Se administration. The protective effect of Se on creatinine and urea concentrations can be attributed to its anti-oxidant properties, as it has been found that ROS may be involved in the impairment of GFR (Pedraza-Chaverri et al., 2000).

These findings correlated well with the histological examination, which revealed tubular necrosis, especially in the renal cortex (Table 2). The kidneys of the control group showed normal histological features (Fig. 3A), but the GM-treated group revealed extensive, marked tubu-lar necrosis. There were leukocytic infiltrations consid-ered, as a prominent response of body tissue facing any injurious effects (Figure 3E). These modifications could be the result of the accumulation of free radicals resulting from an increased LPO in renal tissues of the GM-treated group. Renal lesions were also characterized by vascu-lar congestion as well as tubular obstruction. Similar changes were also reported by Kumar et al. (2000) and Stojiljkovic et al. (2009), who demonstrated structural changes in the renal tissue of GM-treated animals and its reversal by various agents. Glomerular and tubular epithelial changes were considerably mild in the group treated with both GM+Se (Figure 3F). Thus, the improve-ment of the GM-induced histological alterations could be attributed to the antioxidant efficacy of selenium.

Table 2. Degrees of histopathological lesions of kidney sections in rats treated with GM alone or with Se.Histopathological lesions Control Se GM GM + SeTubular necrosis – – ++ –Tubular degeneration – – +++ +Mononuclear cell infiltration – – +++ +Hyaline casts in tubular lumen – – + –Scoring was done as follows: none (–-), mild (+), moderate (++), and severe (+++).



Besides their direct damaging effects on tissues, free radicals seem to trigger the accumulation of leukocytes in the tissue involved and thus cause tissue injury also indirectly through activated neutrophils. It has been shown that activated neutrophils secrete enzymes (e.g., myeloperoxidase, elastase, and proteases) and liberate oxygen radicals (Kettle and Winterbourn, 1997). This finding has been confirmed in our study, as we could see mononuclear cell infiltration in kidneys of GM-treated rats (Figure 3E). In many diseases and acute inflamma-tory disorders, important components of the pathologic process are linked to the neutrophils’ ability to release a complex assortment of agents that can destroy nor-mal cells and dissolve connective tissue (Kettle and Winterbourn, 1997; Reiter et al., 2000). Increasing evi-dence suggests that mesangial cells and neutrophils release chemotactic substances (e.g., interleukin 8), which further promote neutrophil migration to the

kidney, and their activation thus increases glomerular injury (Reiter et al., 2000). These results suggest that neutrophils play an important role in mediating tissue injury with subsequent renal failure (Donnahoo et al., 1999). Oxidative stress and inflammation are inextrica-bly linked as one begins and amplifies the other. In this context, oxidative stress invariably recruits inflamma-tion via activation of NF-κB, which is the general tran-scription factor for various proinflammatory cytokines, chemokines, and adhesion molecules. Production of these mediators promotes leukocyte/macrophage adhe-sion, activation, infiltration, and ROS production. The latter, in turn, trigger oxidative stress. Beside this, release of ROS, reactive chlorine, nitrogen, and other species by activated leukocytes and macrophages in the course of the primary inflammation results in oxidative stress (Stenvinkel et al., 2005; Rodriguez-Iturbe et al., 2005; Anrather et al., 2006).

Figure 3. Photomicrograph of rat kidney section. (A, B) Normal histology of kidney tissue in control and selenium-treated rats (H&E, ×200). (C) Kidney section from rat treated with GM (100 mg/kg) showing marked tubular necrosis (arrows) (H&E, ×200). (D) Sample from GM-treated group featuring hyaline casts in tubular lumens (arrows) (PAS, ×200). (E) Massive mononuclear cell infiltration (arrow) in the cortex of rats in the GM group (PAS, ×200). (F) Section from rat treated with GM (100 mg/kg) plus selenium (1 mg/kg) reveals an almost complete prevention of histopathological alterations (PAS, ×200). (See colour version of this figure online at www.informahealthcare.com/dct)



MDA is a physiological compound produced by the peroxidative decomposition of unsaturated lipids as a by-product of arachidonic acid metabolism. ROS, including hydroxyl radical, have been implicated in the etiology of GM-induced nephrotoxicity. It has been dem-onstrated that GM treatment causes enhanced genera-tion of ROS and accelerates the LPO of biomembranes in the renal parenchyma (Baliga et al., 1999; Walker et al., 1999). As a result, tissue levels of MDA, an end-product of LPO, increased, probably due to reduction in GPx activity. The pathological consequence of this process is reflected in the impairment of membrane integrity and membrane-associated function in mitochondria, microsomes, and particularly lysosomes. The destruc-tive action of GM on lysosomal membrane systems has been detected previously. Ngaha and Ogunleye (1983) showed, in vitro, that GM evokes the labilization of rat kidney lysosomes with the marked release of lysosomal acid hydrolases. In their later work, these research-ers confirmed this effect in vivo by measuring urinary excretion of some lysosomal enzymes (Ngaha et al., 1984). Moreover, in both studies, they showed that this GM-induced degradation of the lysosomal membrane could be reduced to a considerable extent by concomi-tant administration of selenium.

In the present study, LPO of lysosomes was observed by the formation of MDA. The high MDA level observed in the group of rats treated only with GM significantly decrease (P < 0.05) in rats receiving GM and selenium at the same time. The lower MDA level in the GM+Se group, which was close to the control value, indicates the selenium attenuation of LPO. This inhibitory effect of selenium might be the result of its ability to interrupt the propagation of the free radical chain reaction already initiated in the membrane lipids of kidney lysosomes and other subcellular organelles. Our observation seems to agree with the proposal of Ngaha and coworkers (Ngaha and Ogunleye, 1983, Ngaha et al., 1984), who have dem-onstrated the lysosomal membrane-stabilizing action of selenium.

In addition to cellular lipids, studies have shown that cellular proteins may also be affected by free radi-cal accumulation. It is generally known that proteins are susceptible to damage by ROS in vitro and in vivo, and oxidative modification of proteins may lead to the structural alteration and functional inactivation of many enzyme proteins (Sitte et al., 2000). Generally oxi-datively modified proteins are degraded more rapidly than native protein by proteolytic systems, which are proposed as a secondary free radical defense system. Hence, the drug-related accumulation of oxidatively modified proteins is the consequence of free radicals and their oxidizing action. Among the various oxida-tive modifications of amino acids in proteins, protein carbonyl formation may be an early marker for protein oxidation (Dursun et al., 2005). This may occur in a vari-ety of physiological and pathological processes, which may be primary or secondary. Primary modifications

occur in metal-catalyzed oxidation, radiation-medi-ated oxidation, and oxidation by ozone or oxides of nitrogen (Stadtman, 1986). Secondary modifications occur when molecules generated by the oxidation of other molecules modify proteins. Oxidative modifica-tion of proteins is also one of the many after effects of oxidative stress (Stadtman, 1986). The highly reactive hydroxyl radical, which is one of the ROS generated in the process leading to oxidative stress, is considered to be responsible for the formation of carbonyl groups in proteins (Oliver, 1987). Protein oxidation can lead to the loss of critical sulfhydryl groups, in addition to a modification of amino acids, leading to the formation of carbonyl and other oxidized moieties (Bainy et al., 1996). This leads to proteolytic degradation, which may affect the structure, function, and integrity of the cell (Carney et al., 1991).

Recent research supports the point that protein car-bonyl derivatives are formed during nephrotoxicity as a consequence of oxidative stress (Orozco-Ibarra et al., 2007; Pari et al., 2007). This finding was confirmed in our study, as protein carbonyl content was significantly increased in rats treated with GM (Figure 2). Similar results were seen in rats where GM and selenium were given together. We have shown that even though selenium has a well-established antioxidant role in living organisms (Sies, 1993), it offers no protection against the oxidative modification of proteins in GM-induced nephrotoxicity.

Conclusions

The results of this study confirm earlier reports that GM-treated rats show accelerated lipid and protein oxi-dation in the renal tissue, as reflected by an increase in MDA and protein carbonyl groups. Pretreatment with Se afforded significant protection against nephrotoxicity induced by GM treatment. The beneficial effect of Se in GM toxicity implies the involvement of free radicals in renal damage. According to our biochemical findings, which were supported by histopathological evidence, the administration of selenium abolished some nephrotoxic effects of GM. These findings indicate that Se supplemen-tation may reduce GM-induced renal injury. We propose that Se acts in the kidney as a potent scavenger of free radicals to prevent the toxic effects of GM, both at the biochemical and histological level.

Declaration of interest

This work was supported by the Ministry of Science and Technological Development, Republic of Serbia (grants 43012, 175092, and 172061).

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