Toxicity of depleted uranium on isolated rat kidney mitochondria

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Background: Kidney is known as the most sensitive target organ for depleted uranium (DU) toxicity in compar- ison to other organs. Although the oxidative stress ...
Biochimica et Biophysica Acta 1820 (2012) 1940–1950

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Toxicity of depleted uranium on isolated rat kidney mitochondria Fatemeh Shaki a, b, Mir-Jamal Hosseini a, c, Mahmoud Ghazi-Khansari d, Jalal Pourahmad a,⁎ a

Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran Faculty of Pharmacy, Mazandaran University of Medical Sciences, sari, Mazandaran Pharmaceutical Sciences Research Center, Iran c Department of Pharmacology and Toxicology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran d Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 12 July 2012 Received in revised form 15 August 2012 Accepted 17 August 2012 Available online 23 August 2012 Keywords: Depleted uranium Mitochondria Nephrotoxicity Respiratory chain Mitochondrial permeability transition Cytochrome c

a b s t r a c t Background: Kidney is known as the most sensitive target organ for depleted uranium (DU) toxicity in comparison to other organs. Although the oxidative stress and mitochondrial damage induced by DU has been well investigated, the precise mechanism of DU-induced nephrotoxicity has not been thoroughly recognized yet. Methods: Kidney mitochondria were obtained using differential centrifugation from Wistar rats and mitochondrial toxicity endpoints were then determined in both in vivo and in vitro uranyl acetate (UA) exposure cases. Results: Single injection of UA (0, 0.5, 1 and 2 mg/kg, i.p.) caused a significant increase in blood urea nitrogen and creatinine levels. Isolated mitochondria from the UA-treated rat kidney showed a marked elevation in oxidative stress accompanied by mitochondrial membrane potential (MMP) collapse as compared to control group. Incubation of isolated kidney mitochondria with UA (50, 100 and 200 μM) manifested that UA can disrupt the electron transfer chain at complex II and III that leads to induction of reactive oxygen species (ROS) formation, lipid peroxidation, and glutathione oxidation. Disturbances in oxidative phosphorylation were also demonstrated through decreased ATP concentration and ATP/ADP ratio in UA-treated mitochondria. In addition, UA induced a significant damage in mitochondrial outer membrane. Moreover, MMP collapse, mitochondrial swelling and cytochrome c release were observed following the UA treatment in isolated mitochondria. General significance: Both our in vivo and in vitro results showed that UA-induced nephrotoxicity is linked to the impairment of electron transfer chain especially at complex II and III which leads to subsequent oxidative stress. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Uranium (U) is a ubiquitous environmental trace metallo-element which is found in small amounts in food and water supplies as a nonessential inorganic component. Moreover, natural exposure U has also several civilian and military applications that can cause dispersion in the environment [1]. The U which remains after the removal of enriched fraction is known as depleted U(DU), [2,3]. Although DU is less radioactivity compared to natural U but it has the same chemical behavior with natural uranium [4]. The intensive use of DU weapons in the Balkans conflict, Afghanistan war and the second War of Persian Gulf have continued to stimulate the research into the toxicity of DU [5].

Abbreviations: U, Uranium; DU, Depleted uranium; UA, uranyl acetate; ROS, Reactive oxygen species; GSH, reduced glutathione; DCF-DA, 2′,7′-dichlorofluorescein diacetate; TBARs, thiobarbituric acid reactive substances; Cs A, Cyclosporin A; MDA, Malondialdehyde; Rh123, Rhodamine 123; BSA, bovine serum albumin; MPT, mitochondrial permeability transition; MMP, mitochondrial membrane potential; BHT, butylatedhydroxytoluene; BUN, blood urea nitrogen ⁎ Corresponding author at: Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, P.O. Box: 14155‐6153 Iran. Tel.: +98 21 2255 8786; fax: +98 21 8820 9620. E-mail address: [email protected] (J. Pourahmad). 0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2012.08.015

Kidney and bone are the primary reservoirs for U. Kidney is also the most sensitive target organ for U toxicity [6,7]. Uranium is filtered through the glomerulus and subsequently binds to anionic sites in the proximal tubular epithelial brush border in the form of UO +2; it also enters the cell through the endocytosis [8]. So, an injury in the proximal renal tubular epithelium can occurred even with a low level dosing of DU and renal concentrations ≥3 μg uranium/gram of tissue [6]. Recently, Clinical studies suggest a potential for altered proximal tubular function at U concentrations well below the 3 μg U/gram concentration [9]. The most important toxic mechanism that is suggested for DU toxicity is the involvement of oxidative stress and reactive oxygen species (ROS) [10–12]. ROS are known to play a dual role in biological systems since they can be either harmful or beneficial to the living systems [13]. Previous studies have also showed that the oral uranyl acetate (UA) administration increases the TBARS (thiobarbituric acid reactive substances) in kidneys and testis [14]. Other studies have revealed that the chronic uranyl nitrate ingestion results in an increase in the level of free radicals [15] and lipid peroxidation in CNS [16] and rapid oxidation of glutathione, ROS formation, lipid peroxidation and also decreases the mitochondrial membrane potential (MMP) in isolated rat hepatocytes [11]. These may be based upon the DU-related induction of cellular oxidative stress [15]. Mitochondria are the major source of reactive oxygen in most mammalian cell types and it has been estimated that 1–2% of systemically

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consumed oxygen is converted to reactive oxygen by mitochondria [17]; it should be noted that mitochondria is responsible for the production of 60–80% of H2O2 in cells under the normal condition [18] which can be increased in pathological conditions and through the inhibition of mitochondrial electron transfer chain complexes [19,20]. On the other hand, numerous studies have revealed that mitochondrial-generated ROS facilitates the process of mitochondrial permeability transition (MPT) pores opening, leading to mitochondrial inner membrane potential collapse [21]. In fact, the MMP represents the integrity of mitochondrial membrane and its metabolic activity [22] and can be used as a key indicator of mitochondrial functionality even as a marker of cellular viability [22]. Furthermore, MMP is a crucial factor for the regulation of mitochondrial activity [23] and the MMP collapse is the important stimuli for apoptosis and necrosis [24]. Presently, isolated mitochondria are considered as powerful tools for the precise examination of an external agent that affects the mitochondrial function, with an emphasis on the mechanism of changes in MMP [25]. In accordance with the previous studies demonstrating that DU causes oxidative stress [11,14–16], the significant MMP collapse [11,26] and mitochondrial swelling [27] has lead us to the fact that mitochondria can also be considered as the important target in DU nephrotoxicity. Despite many investigations that have documented the production of ROS in kidney and several extra renal tissues in DU-treated animals, the original mechanisms of DU-induced ROS formation and the toxicity including mitochondrial involvement have not been fully investigated yet. The aim of the current study was to explore the impact of mitochondria in DU-induced nephrotoxicity using isolated rat kidney mitochondria. 2. Materials and methods 2.1. Materials Uranyl acetate was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Uranyl acetate (U238 = 99.74%, U235= 0.26%, U234 = 0.001%), had 1.459E4 Bq/g specific activity based on manufacturer data. 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), antimycin A(AA), rotenone (Rot), myxothiazole (myx), DMSO, D-mannitol, thiobarbutiric acid (TBA), MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide), cyclosporin A (CsA), dithiobis2-nitrobenzoic acid (DTNB), reduced glutathione (GSH), 2′,7′dichlorofluorescein diacetate (DCFH-DA), Malondialdehyde (MDA), Horseradish peroxidase, Tris–HCl, sodium succinate, sulfuric acid, n-butanol, Tetramethoxypropane (TEP), homovanillic acid, horseradish peroxidase, pyruvate, malate, sucrose, KCl, Na2HPO4, MgCl2, MnCl2, potassium phosphate, butylated hydroxytoluene (BHT),Rhodamine 123 (Rh 123), Coomassie blue, Ethylene glycol-bis (2-aminoethylether)N,N,N′,N′-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals were of analytical grade, HPLC grade or the best pharmaceutical grade. 2.2. Animals treatment Male Wistar rats (250–300 g) were housed in an air-conditioned room with controlled temperature of 25 ± 2 °C and maintained on a 12:12 h light cycle with free access to food and water. All experimental procedures were conducted according to the ethical standards and protocols approved by the Committee of Animal Experimentation of Shahid Beheshti University of Medical Sciences, Tehran, Iran. All efforts were made to minimize the number of animals and their suffering. For studies in vivo rats were fasted overnight, then animals were divided into two groups, with six rats in each group. The control group (vehicle) received a single intraperitoneal (i.p.) injection of saline solution (1 ml per 100 g body weight). Uranyl acetate was dissolved in normal saline. Rats were treated with single intraperitoneal

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(i.p.) injections of UA in doses 0.5, 1 and 2 mg/kg body weight. These dosages was selected based on previous studies [28], which is sufficient to induce oxidative stress in kidney without causing death and none died within the duration of experiments. Blood urea nitrogen (BUN) and creatinine, marker of kidney dysfunction, were determined by commercial reagents (obtained from Parsazmoon Co., Iran). The rats were killed by decapitation 24 h after injection. The kidney were immediately removed and placed in ice-cold mitochondria isolation medium (0.225 M D-mannitol, 75 mM sucrose, and 0.2 mM EDTA, pH = 7.4).

2.3. Mitochondrial preparation Mitochondria were prepared from Wistar rat's kidneys using differential centrifugation [29]. Tissues were minced and homogenized with glass handheld homogenizer. The nuclei and broken cell debris were sedimented through centrifuging at 1500 ×g for 10 min at 4 °C and the pellet was discarded. The supernatant was subjected to a further centrifugation at 10,000 ×g for 10 min and the superior layer was carefully discarded. The mitochondrial pellet was washed by gently suspending in the isolation medium and centrifuged again at 10,000 ×g for 10 min. Final mitochondrial pellets were suspended in Tris buffer containing (0.05 M Tris–HCl, 0.25 M sucrose, 20 Mm KCl, 2.0 mM MgCl2, and 1.0 mM Na2HPO4, pH of 7.4) at 4 °C, except for mitochondria used to assess ROS production, MMP and swelling, which were suspended in respiration buffer (0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 μM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate), MMP assay buffer (220 mM sucrose, 68 mM D-mannitol, 10 mM KCl, 5 mM KH2PO4, 2 mM MgCl2, 50 μM EGTA, 5 mM sodium succinate, 10 mM HEPES, 2 μM Rotenone) and swelling buffer (70 mM sucrose, 230 mM mannitol, 3 mM HEPES, 2 mM Tris-phosphate, 5 mM succinate and 1 μM of rotenone). Protein concentrations were determined through the Coomassie blue protein-binding method as explained by Bradford [30]. The isolation of mitochondria was confirmed by the measurement of succinate dehydrogenase [31]. Mitochondria were prepared fresh for each experiment and used within 4 h of isolation and all steps were strictly operated on ice to guarantee the isolation of high-quality mitochondrial preparation. For in vitro experiments, UA was dissolved in distilled water. The concentrations of UA (50, 100, 200 μmol/l) were chosen based on the previous study [11] and mitochondrial fractions were incubated in Tris buffer with different concentrations of UA for 1 h. In experiments where electron transport inhibitors were employed, mitochondria were preincubated with the indicated mitochondrial complex inhibitors (rotenone, antimycin and myxothiazole) for 5 min before UA and substrate were added to incubation mixtures.

2.4. Quantification of mitochondrial ROS level The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 μM) in respiration buffer containing (0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 μM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 μM) to mitochondria and was then incubated for 10 min. Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.

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2.5. Evaluation of complex I or III effect on UA-induced H2O2 formation The role of complex I and III in uranyl acetate-induced H2O2 production was measured spectrophotofluorometrically using homovanillic acid and horseradish peroxidase at 312 nm excitation and 420 nm emissions wave lengths [33]. The incubation medium consisted of KCl (145 mM), Hepes (30 mM), KH2PO4 (5 mM), MgCl2 (3 mM), EGTA (0.1 mM) and 0.1% fatty-acid-free albumin (pH =7.4;T=37 °C). The following solutions are prepared in fore-mentioned incubation medium without albumin: high purity horseradish peroxidase (70 U/ml), homovanillic acid (4 mM), pyruvate/malate (125 mM), and succinate (250 mM) at pH of 7.4. The order of addition was as follows: mitochondria (0.5 mg protein/ml), horseradish peroxidase (6 U/ml), homovanillic acid (0.1 mM) and the substrate (2.5 mM pyruvate/2.5 mM malate or succinate 5 mM+rotenone 2 μM) to start the reaction [34]. The volume of added incubation medium should be around 85% of the total reaction volume (1.5 ml). After 15 min of addition with constant agitation in a temperature-controlled water bath at 37 °C, the reaction stopped transferring the samples to an ice-cold bath, 0.5 ml of glycine-NaOH (0.1 M; pH=12) containing 25 mM of EDTA was added (per each 1.5 ml of reaction volume), and the fluorescence was measured at 312 nm excitation and 420 nm emission [33]. Appropriate blanks were also run to correct for the positive fluorescence of the mitochondria themselves. These blanks have mitochondria and all the reaction components but do not contain substrate. Values are obtained through subtracting the fluorescence of the blanks from the fluorescence of the samples and dividing the results by 15 min of incubation. Antimycin A (2 μM) and rotenone (2 μM) as mitochondrial respiratory inhibitors were also added to the reaction in some groups. 2.6. Complex II activity assay using MTT test The activity of mitochondrial complex II was assayed by measuring the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide). Briefly, 100 μl of mitochondrial suspensions (0.5 mg protein/ml) was incubated with different concentration of UA (0, 50, 100 and 200 μM) at 37 °C for 20 min; then, 0.4% of MTT was added to the medium and incubated at 37 °C for 30 min. The product of formazan crystals were dissolved in 100 μl DMSO and the absorbance at 570 nm was measured with an ELISA reader (Tecan, Rainbow Thermo, Austria)[35]. 2.7. Measurement of lipid peroxidation The content of MDA was determined using the method of Zhang et al. 2008 [36]. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C; 0.25 ml sulfuric acid (0.05 M) was added to 0.2 ml mitochondrial fractions afterwards, with the addition of 0.3 ml 0.2% TBA. All the microtubes were placed in a boiling water bath for 30 min. At the end, the tubes were shifted to an ice-bath and 0.4 ml n-butanol was added to each tube. Then, they were centrifuged at 3500 ×g for 10 min. The amount of MDA formed in each of the samples was assessed through measuring the absorbance of the supernatant at 532 nm with an ELISA reader (Tecan, Rainbow Thermo, Austria). Tetramethoxypropane (TEP) was used as standard and MDA content was expressed as nmol/mg protein [36]. 2.8. Measurement of GSH content GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed

yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as μg/mg protein [37]. 2.9. Determination of the MMP Mitochondrial uptake of the cationic fluorescent dye, rhodamine 123, has been used for the estimation of mitochondrial membrane potential. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate and then 10 μM of rhodamine 123 was added to mitochondrial solution in MMP assay buffer (220 mM sucrose, 68 mM D-mannitol, 10 mM KCl, 5 mM KH2PO4, 2 mM MgCl2, 50 μM EGTA, 5 mM sodium succinate, 10 mM HEPES, 2 μM Rotenone). The fluorescence was monitored using Schimadzou RF-5000U fluorescence spectrophotometer at the excitation and emission wavelength of 490 nm and 535 nm, respectively [38]. 2.10. Determination of mitochondrial swelling Analysis of mitochondrial swelling after the isolated mitochondria (0.5 mg protein/ml) was estimated through changes in light scattering as monitored spectrophotometrically at 540 nm (30 °C) as described [35]. Briefly, isolated mitochondria were suspended in swelling buffer (70 mM sucrose, 230 mM mannitol, 3 mM HEPES, 2 mM tris-phosphate, 5 mM succinate and 1 μM of rotenone) and incubated at 30 °C with 50, 100 and 500 μM of uranyl acetate. The absorbance was measured at 549 nm at 10 min time intervals with an ELISA reader (Tecan, Rainbow Thermo, Austria). A decrease in absorbance indicates an increase in mitochondrial swelling. 2.11. Measurement of cytochrome c oxidase activity and assessment of outer mitochondrial membrane damage Both mitochondrial cytochrome c oxidase activity and outer membrane integrity were evaluated using cytochrome-c oxidase assay kit (Sigma, St. Louis, MO). The colorimetric assay was based on the observation that a decrease in absorbance of ferrocytochrome c at 550 nm was caused by its oxidation to ferricytochrome c by cytochrome c oxidase. Experimental procedures were performed according to the manufacturer's protocol; 20 μg of freshly isolated mitochondrial fraction were used for each reaction, and duplicate reactions were conducted for each assay. For the measurement of total mitochondrial cytochrome-c oxidase activity, the mitochondrial fraction was diluted in the enzyme dilution buffer (10 mM Tris–HCl, pH = 7.0, containing 250 mM sucrose) with 1 mM n-dodecyl β-D-maltoside and incubated on ice for 30 min. The reaction was initiated by adding freshly prepared ferrocytochrome-c substrate solution (0.22 mM) to the sample. The decrease in absorbance at 550 nm is related to the oxidation of ferrocytochrome-c by cytochrome-c oxidase. Cytochrome-c oxidase activities were calculated and normalized for the amount of protein per reaction and results were expressed as units per milligram mitochondrial protein. Mitochondrial outer membrane integrity was assessed through measuring the cytochrome-c oxidase activity of mitochondria in the presence or absence of the detergent, n-dodecyl β-D-maltoside. The mitochondrial outer membrane damage was assayed from the ratio between cytochrome-c oxidase activity with and without detergent. 2.12. Assay of ATP and ATP/ADP ratio The ATP and ATP/ADP ratio level were measured by luciferase enzyme as described by Tafreshi et al. [39]. Bioluminescence intensity was measured using Sirius tube luminometer (Berthold Detection System, Germany).

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2.13. Cytochrome c release assay The concentration of cytochrome c was determined through using the Quantikine Rat/Mouse Cytochrome c Immunoassay kit provided by R & D Systems, Inc. (Minneapolis, Minn.). Briefly, a monoclonal antibody specific for rat/mouse cytochrome c was precoated onto the microplate. Seventy-five microliter of conjugate (containing monoclonal antibody specific for cytochrome c conjugated to horseradish peroxidase) and 50 μl of standard and positive control were added to each well of the microplate. One microgram of protein from each supernatant fraction was added to the sample wells. All of the standards, controls, and samples were added to two wells of the microplate. After 2 h of incubation, the substrate solution (100 μl) was added to each well and incubated for 30 min. After 100 μl of the stop solution was added to each well; the optical density of each well was determined by the aforementioned microplate spectrophotometer set to 450 nm. 2.14. Statistical analysis Results are presented as mean±SD. All statistical analyses were performed using the SPSS software, version 17. Assays were performed in triplicate and the mean was used for statistical analysis. Statistical significance was determined using the one-way ANOVA test, followed by the post-hoc Tukey test. In some experiments, the two-way ANOVA test followed by the post-hoc Bonferroni test was also performed. Statistical significance was set at Pb 0.05. 3. Results 3.1. Effect of UA on serum marker of kidney dysfunction Table 1 show that injection of different doses of UA (i.p.) caused significant increase in the levels of BUN compared with that of control (P b 0.05). A marked increase in creatinine was noted in UA treated group compared with that of control (Table 1) (P b 0.05). 3.2. Effect of in vivo UA administration on mitochondrial dysfunction parameters The rates of ROS production in kidney mitochondria isolated from rats treated with UA (0.5, 1 and 2 mg/kg i.p.) 24 h before being killed, are shown in Table 2. ROS production increased in a dose dependent manner after UA injection. TBARS formation, an indicator of lipid peroxidation was also measured in kidney mitochondria isolated from rats treated with UA (0.5, 1 and 2 mg/kg i.p.). As shown in Table 2, TBARS formation significantly elevated in mitochondria obtained from UA treated rats compared with control rats suggesting of increased lipid peroxidation due to UA treatment (P b 0.05). However, increased ROS production in kidney mitochondria following UA treatment was accompanied by a dose-dependent decrease in mitochondrial GSH content (Table 2). In addition, a dose dependent decrease in rhodamine fluorescence (membrane potential) observed in Table 1 Effect of in vivo administration of uranyl acetate (UA) on blood urea nitrogen (BUN) and serum creatinine in the rat blood. Uranyl acetate

BUN (mg/dl)

Creatinine (mg/dl)

Control 0.5 mg/kg 1 mg/kg 2 mg/kg

28 ± 3.6 39 ± 4⁎ 45 ± 2.6⁎⁎ 58 ± 3⁎⁎⁎

0.4 ± 0.02 0.51 ± 0.03 0.7 ± 0.07⁎⁎ 1.1 ± 0.13⁎⁎⁎

Values are expressed as mean ± SD for six rats in each group. ⁎ Significantly different when compared to the control (*P b 0.05). ⁎⁎ Significantly different when compared to the control (**P b 0.01). ⁎⁎⁎ Significantly different when compared to the control (***P b 0.001).

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Table 2 Effect of in vivo administration of uranyl acetate (UA) on ROS production (fluorescence Intensity), lipid peroxidation (μg/mg protein), glutathione (μg/mg protein) and MMP (fluorescence Intensity) in the rat kidney mitochondrial. Uranyl acetate

ROS production

Lipid peroxidation

Glutathione

MMP

Control 0.5 mg/kg 1 mg/kg 2 mg/kg

25 ± 8 31 ± 7 45.3 ± 6.5* 58 ± 10.5**

6.6 ± 1.3 6.8 ± 1.9 9.4 ± 1.3* 13.07 ± 2**

18.4 ± 2.5 17 ± 2 14.45 ± 1.6* 10.5 ± 1.4**

201.2 ± 29 232 ± 30 240 ± 27 285 ± 25*

Values are expressed as mean ± SD for six rats in each group. * Significantly different when compared to the control (*P b 0.05). ** Significantly different when compared to the control (**P b 0.01).

UA treated rats. At dose of 1 and 2 mg/kg, UA significantly induced MMP collapse compared with that of control rats. In vitro studies were performed to investigate the potential mechanisms underlying the observed increase in ROS production and mitochondrial dysfunction following UA treatment and to relate this effect to UA induced oxidative stress. 3.3. Effect of in vitro UA treatment on mitochondrial ROS production As shown in Fig. 1, different concentration of UA (50, 100 and 200 μM) induced significant H2O2 formation in isolated kidney mitochondria in vitro using flow cytometry assay. UA-induced elevation in H2O2 production was concentration and time dependent manner in comparison with the control mitochondria. However, low concentration of UA (50 μM) did not significantly increase H2O2 the generation until 60 min, whereas the rate of H2O2 formation was significantly increased at 30 and 60 min, following exposure to UA (100 μM) comparing to control mitochondria. A more substantial increase in mitochondrial H2O2 formation was observed in higher concentration (200 μM) at 30 and 60 min (Pb 0.05). To assess the involvement of complex I and III on UA-induced H2O2 production in kidney mitochondria, a fluorescent dye/horseradish peroxidase detecting system was used to employ the peroxidase substrate homovanillic acid (Fig. 2A and B). As demonstrated in Fig. 2A, UA could not produce significant amounts of H2O2 in isolated kidney mitochondria respiring on the NAD-linked substrates malate and pyruvate. Here, the addition of respiratory Complex I inhibitor rotenone produced greater amounts of H2O2 in isolated kidney mitochondria than those of UA alone (Pb 0.05). There was a significant elevation in H2O2 production following the addition of UA treated-isolated kidney mitochondria to the incubation medium containing Complex III-linked substrate succinate (Fig. 2B), which was greater than the one present in complex I substrate (pyruvate/malate). Maximum level of UA induced-H2O2 production was achieved following the addition of Antimycin A (specific inhibitors of complex III) to the medium (Fig. 2B). However, the addition of rotenone (complex I inhibitor) and myxothiazol (semiquinone formation inhibitor) led to a reduction in H2O2 production (P b 0.05) (Fig. 2B). 3.4. Effect of in vitro UA treatment on mitochondrial complex II and IV We also assessed the succinate dehydrogenase activity (complex II) using the MTT test after 1 h of incubation in mitochondria with different concentrations of UA (50, 100 and 200 μM). Fig. 3 shows a significant concentration-dependent decrease in the mitochondrial metabolism of MTT to formazan (Pb 0.05). Cytochrome-c oxidase activity (complex IV), an important enzyme in the mitochondrial respiratory complex, was determined in the isolated kidney mitochondria after the exposure to UA. No change in the activity of this enzyme was observed during the incubation time (Fig. 4).

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A

B

C

D Flourescence Intensity

100 80

5 min 30 min 60 min

**

***

*** *** **

60 40 20

co U ntr A ol U 50µ A 10 M U 0µ A M 20 0µ M

co U ntr A ol U 50µ A 10 M U 0µ A M 20 0µ M

co U ntr A ol U 50µ A 10 M U 0µ A M 20 0µ M

0

Fig. 1. ROS formation in UA-treated mitochondria. ROS formation after the addition of various concentrations of UA (0, 50, 100 and 200 μM) at intervals of A) 5 min after the addition, B) 30 min after the addition and C) 60 min after the addition. D) ROS formation was determined through flow cytometry using DCF-DA as described in Materials and methods. FL1: the fluorescence intensity of DCF. *P b 0.05; **P b 0.01; ***P b 0.001 compared with control mitochondria.

3.5. Effect of in vitro UA treatment on mitochondrial lipid peroxidation

3.6. Effect of in vitro UA treatment on mitochondrial Glutathione

Lipid peroxidation (MDA formation) was assayed in the rat kidney mitochondria that were exposed to different concentration of UA (50, 100 and 200 μM). As shown in Fig. 5, there is a concentrationdependent relation between lipid peroxidation and UA concentration in the isolated kidney mitochondria. The amounts of MDA formation in renal mitochondria were 7.4 ± 1.2, 10.6 ± 1.2 and 16 ± 1.3 μg MDA/mg protein at 50, 100 and 200 μM UA concentration, respectively, whereas that of control group was 5.28 ± 0.7 μg MDA/mg protein. The resulted data shows that the MDA was influenced by UA exposure in vitro.

The glutathione is a very important antioxidant defense factor against different ROS such as H2O2. GSH can directly react with H2O2 in non-enzymatic reaction and oxidized to GSSG [40]. Consequent to the observation of UA effect on H2O2 generation and lipid peroxidation, it's possible effect on the antioxidant systems was also determined. The GSH concentration in renal mitochondria after 1 h exposure to UA was evaluated to determine the extent of oxidative stress induced by UA. At 50, 100 and 200 μM UA concentrations, GSH levels decreased to 14.34±1.04, 12.47±1.16 and 8.7±1.45 (μg/mg protein) respectively

F. Shaki et al. / Biochimica et Biophysica Acta 1820 (2012) 1940–1950

0.10 0.05 0.00 20 0µ M

50 µM A U

+M A

al /P yr +U M

Fig. 4. Effect of UA on cytochrome c oxidase (complex IV) activity. Cytochrome c oxidase activity was measured using cytochrome c oxidase assay kit as described in Materials and methods. Kidney mitochondria (0.5 mg/ml) were incubated for 1 h with various concentrations of UA (0, 50, 100 and 200 μM). Values represented as mean±SD (n=3). *Pb 0.05; **Pb 0.01; ***Pb 0.001 compared with control mitochondria.

1500

$$

*** 1000

as compared to 17.8 ± 1.4 (μg/mg protein) at control mitochondria (Fig. 6).

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3.7. Effect of in vitro UA treatment on mitochondrial membrane potential

500

yx

ot

A

+M

+R

A

A

U

U

c+

c+

Su

Su

Su

c+

U

Su

A

c+

+A

U

Su

c

A

0

Fig. 2. Influence of respiratory substrates and inhibitors of complex I and III on UA induced ROS formation in isolated kidney mitochondria. The measurements were performed with the homovanilic acid/horseradish peroxidase method, as described in Materials and methods. Panel A: ROS formation was measured in the presence of: pyruvate 2.5 mM + malate 2.5 mM alone (pyr/mal), pyruvate/malate + uranyl acetate 100 μM (pyr/mal + UA), pyruvate/malate + uranyl acetate100μM + Antimycin A 2 μM (pyr/mal + UA + AA), pyruvate/malate + uranyl acetate 100 μM + rotenone 2 μM (pyr/mal + UA + Rot), pyruvate/malate + uranyl acetate 100 μM + myxothiazole 5 μM (pyr/mal + UA + Myx), Values represented as mean ± SD (n = 3). *P b 0.05; **P b 0.01; ***P b 0.001 compared with pyr/mal group. Panel B: ROS formation was measured in the presence of: succinate 5 mM alone (Suc), succinate + uranyl acetate 100 μM (Suc+UA), succinate+uranyl acetate 100 μM+Antimycin A 2 μM (Suc+UA+AA), succinate+uranyl acetate100μM+rotenone 2 μM (Suc+UA+Rot), succinate+uranyl acetate 100 μM+myxothiazole 5 μM (Suc+UA+Myx). Values represented as mean± SD (n=3). *Pb 0.05; **Pb 0.01; ***Pb 0.001 compared with Suc group, $Pb 0.05; $$Pb 0.01; $$$Pb 0.001, compared with Suc+UA group.

MMP is a highly sensitive indicator of the mitochondrial inner membrane condition; therefore, the effect of UA on MMP was measured by Rh123 staining. As shown in Fig. 7, UA concentrations significantly decreased the MMP in a concentration and time-related manner (Pb 0.05) that 50 μM of UA showed the smallest effect on MMP during 60 min of incubation whereas 100 and 200 μM of UA strongly decreased the MMP after 30 min (Fig. 7a). Conversely, the addition of Cs A (1 μM), an inhibitor of MPT pore, and BHT (20 μM), an antioxidant, significantly inhibited the collapse of MMP induced by 100 μM of UA (P b 0.05) (Fig. 7b).

3.8. Effect of in vitro UA treatment on mitochondrial swelling Moreover, we monitored the changes of absorbance at 540 nm (A540) to assay mitochondrial swelling, an indicator of mitochondrial membrane permeability. The addition of UA (50, 100, 200 μM) to mitochondrial suspensions leads to mitochondrial swelling in a concentration and time-dependent manner. Furthermore, Cs A strongly suppressed the decline in A540 of isolated mitochondria exposed to 100 μM of UA (P b 0.05) (Fig. 8).

1.5

1.0

* **

0.5

0.0

MDA(µg/mg proteine)

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Fig. 3. Effect of UA on succinate dehydrogenase (complex II) activity. Succinate dehydrogenase activity was measured using MTT dye as described in Materials and methods. Kidney mitochondria (0.5 mg/ml) were incubated for 1 h with various concentrations of UA (0, 50, 100 and 200 μM). Values represented as mean±SD (n=3). *Pb 0.05; **Pb 0.01; ***Pb 0.001 compared with control mitochondria.

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Fig. 5. Effect of UA on lipid peroxidation in kidney mitochondria. MDA formation was measured using thiobarbituric acid reactive substances assay as described in Materials and methods. Kidney mitochondria (0.5 mg/ml) were incubated for 1 h with various concentrations of UA (0, 50, 100 and 200 μM). Values represented as mean± SD (n = 3). *P b 0.05; **P b 0.01; ***P b 0.001 compared with control mitochondria.

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and 200 μM but did not significantly change in control group at concentration of 50 μM (Pb 0.05) (Fig. 9A). Decline of ATP/ADP ratios in isolated kidney mitochondria was quite significant at UA concentrations of 100 and 200 μM (Pb 0.05) (Fig. 9B). This reduction of mitochondrial ATP content indicates the mitochondrial dysfunction leading to decrease in the ability of oxidative phosphorylation for ATP production. On the other hand, the fall in the ATP content may exacerbate the ROS formation and lipid peroxidation.

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Fig. 6. Effect of UA on mitochondrial GSH content. Kidney mitochondria (0.5 mg/ml) were incubated for 1 h with various concentrations of UA (0, 50, 100 and 200 μM). Values represented as mean ± SD (n = 3). *P b 0.05; **P b 0.01; ***P b 0.001 compared with control mitochondria.

3.9. Effect of in vitro UA treatment on mitochondrial ATP and ATP/ADP ratio

We also examined mitochondrial outer membrane integrity in the isolated kidney mitochondria 1 h after the exposure to UA (50, 100 and 200 μM). In this assay, cytochrome c oxidase activity was measured in the presence/absence of detergent n-dodecyl β-D-maltoside. This ratio represents the percentage of mitochondrial outer membrane damage. As shown in Fig. 10, mitochondrial outer membrane damage was significantly increased in isolated kidney mitochondria after the exposure to UA in a concentration-dependent manner (P b 0.05). 3.11. Effect of in vitro UA treatment on cytochrome c release

Mitochondrial respiratory processes are required for ATP production in mitochondria, since UA exposure impaired the mitochondrial respiration; therefore, we measured the ATP levels and ATP/ADP ratios in isolated kidney mitochondria following the addition of different concentration of UA. As shown in Fig. 9, mitochondrial ATP levels and ATP/ ADP ratios fell in a concentration dependent manner. Mitochondrial ATP levels were significantly changed by the UA concentrations of 100

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4. Discussion This study supports the role of mitochondria in oxidative injury in kidney resulting from UA exposure. Our data suggests that UA could reach kidney and affect kidney mitochondrial function following in vivo treatment. Results of this study shows single injection of UA in

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Fig. 7. The effect of UA on the mitochondrial membrane potential (MMP) in kidney mitochondria. MMP was measured by rhodamin 123 as described in Materials and methods. A) The effect of UA (0, 50, 100 and 200 μM) on the mitochondrial membrane potential in kidney mitochondria B) The effect of cyclosporine A (1 μM) and BHT (20 μM) on UA induced MMP collapse. Values represented as mean±SD (n=3). *Pb 0.05; **Pb 0.01; ***Pb 0.001 compared with control mitochondria (a) and UA-treated mitochondria (100 μM) (b).

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The results manifest that UA significantly caused the collapse of the mitochondrial membrane potential and disruption of mitochondrial outer membrane integrity. Consequently, it is hypothesized that UA might induce the release of cytochrome c from mitochondria into the cytosolic fraction. As shown in Fig. 11, there were statistically significant differences in the quantity of cytochrome c between the control mitochondria and UA-treated mitochondria (P b 0.05). We also found that the average concentration of expelled cytochrome c from mitochondrial fraction was amplified in a concentration-dependent manner following the treatment with UA, suggesting the capability of cytochrome c release from the mitochondria into the cytosol in intact cells. Significantly, the pretreatment of UA-treated mitochondria with the MPT inhibitor of Cs A and BHT, an antioxidant, inhibited cytochrome c release as compared with UA-treated group (100 μM) (P b 0.01), indicating the role of oxidative stress and MPT pore opening in cytochrome c release.

Time after treatment (min) Fig. 8. The effect of UA on the mitochondrial swelling in kidney mitochondria. Mitochondrial swelling was measured through the determination of absorbance at 540 nm as described in Materials and methods. Values represented as mean±SD (n=3). *Pb 0.05; **P b 0.01; ***P b 0.001 compared with control mitochondria and $P b 0.05; $$P b 0.01; $$$P b 0.01 compared with UA-treated (100 μM) mitochondria.

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Fig. 11. Effect of UA on the cytochrome c release. The amount of expelled cytochrome c from mitochondrial fraction was determined using Rat/Mouse Cytochrome c ELISA kit as described in Materials and methods. Values represented as mean ± SD (n = 3). *P b 0.05; **P b 0.01; ***P b 0.001 compared with control mitochondria and $P b 0.05; $ $P b 0.01; $$$P b 0.01 compared with UA (100 μM) treated mitochondria.

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Fig. 9. Effect of uranyl acetate on mitochondrial ATP level and ATP/ADP ratio. Kidney mitochondria (0.5 mg/ml) were incubated with various concentrations of uranyl acetate (0, 50, 100 and 200 μM) and A) ATP level and B) ATP/ADP ratio were determined using Luciferin/Luciferase Enzyme System as described in Materials and methods. Values represented as mean±SD (n=3). *Pb 0.05; **Pb 0.01; ***Pb 0.001 compared with control mitochondria.

rats leads to deteriorate on of kidney function as evidenced by increase in BUN and serum creatinine (Table 1). These results are consistent with the previous studies on uranium-induced nephrotoxicity in experimental animals which reported a significant increase in the plasma concentration of creatinine and blood urea nitrogen (BUN) after uranium administration [41–47]. Kidney is the major rout of the uranium excretion from the body and uranyl salt binds to anionic sites in the proximal tubular epithelial brush border in the form of UO+2 [47]. So, an injury in the proximal renal tubular epithelium can occurred even with a low 100

Mitochondrial Outer Membrane damage (%)

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**

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Fig. 10. Effect of UA on mitochondrial outer membrane integrity. Kidney mitochondria (0.5 mg/ml) were incubated for 1 h in the presence of different concentration of UA (0, 50, 100 and 200 μM) and mitochondrial outer membrane integrity was measured as described in Materials and methods. Values represented as mean ± SD (n = 3). *P b 0.05; **P b 0.01; ***P b 0.001 compared with control mitochondria.

level dosing of uranium at renal concentrations ≥3 μg uranium/gram of tissue [48]. Recently, clinical studies suggest a potential role for altered proximal tubular function at U concentrations even lower than 3 μg U/gram concentration [9]. On the other hand, several studies showed that regardless of animal strain and route of administration, single doses of a few (>2 mg/kg) are nephrotoxic, as showed by alterations in classical parameters of renal function (BUN, serum creatinine, etc.) during the immediate days after exposure and dose of 0.5 mg/kg i.p. can be considered the toxic threshold for single-dose exposures [28]. Our data shows UA at doses upper than 0.5 mg/kg altered the serum creatinine and BUN. It was reported that disturbances in the oxidative balance might be responsible for uranium mediated nephrotoxicity [28]. The mitochondria are the important source of ROS and also are the powerhouse of cell that provides over 90% of ATP consumed by body. Mitochondria also play an important role in both apoptotic and necrotic cell death. So, mitochondrial dysfunction not only leads to oxidative stress but also results in cell death and finally tissue damage [49]. Several studies demonstrated the central role of mitochondrial dysfunction in nephrotoxicity [50–53]. Our previous study on a hepatocyte model had demonstrated that UA causes an elevation in ROS formation and disrupts the MMP [10,11] while another study in rat kidney proximal cells revealed that UA exposure resulted in ROS production, MMP decrease and apoptosis via mitochondrial pathway [26]. In this study, we assumed that mitochondria might be the most important target of UA in cells and play a crucial role in UA-induced nephrotoxicity. UA-treated rats showed an increased ROS production which was accompanied by increased mitochondrial lipids peroxidation and GSH oxidation that caused an oxidative stress-like condition. On the other hand, rapid MMP collapse and increase in oxidative stress markers observed following the injection of UA (Table 2), further implies that UA-induced mitochondrial damage may be involved in the pathogenesis of nephrotoxicity. Lestaevel et al. injected 144 ± 10 μg DU/kg i.p. to rats and three days after injection found that the amounts of uranium in the kidneys was 2.6 μg of DU/g of tissue [54]. In addition, in vitro data revealed that the exposure of isolated kidney mitochondria to UA-impaired electron transfer chain that leads to the increased ROS formation, lipid peroxidation and GSH oxidation. The impairment of ETC results in reduced ability for ATP synthesis and decline of mitochondrial ATP/ADP ratio. In addition, significant collapse of MMP, mitochondrial swelling and finally disruption of mitochondrial outer membrane integrity occurred. The mitochondria are the important source of ROS since about 1–2% of the total oxygen consumed by mitochondria is converted to O2• - by several components of the mitochondrial respiratory chain [17]. Superoxide anions generated on mitochondria are rapidly converted to H2O2

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by the mitochondrial superoxide dismutase (MnSOD) which is classified as ROS due to its oxidative power [55]. Furthermore, mitochondrial H2O2 production represents 60-80% of the cellular production rate [18]. Following the addition of UA into isolated mitochondria, rapid increase in H2O2 formation was observed, suggesting the probable role of mitochondrial H2O2 in nephrotoxicity associated with UA. Barja showed that mitochondrial site of ROS production is different from organ to organ [56]. It was also shown that Complex I and III are the major sources of ROS production in the respiratory chain [56]. With the aforementioned fact in mind, we decided to figure out the precise site of ROS formation in UA-treated mitochondrial electron transfer chain. To distinguish the involvement of complex I and/or III, We measured the rate of pyruvate/malate (complex I substrate) and succinate (complex III substrate)-supported mitochondrial hydrogen peroxide production in isolated kidney mitochondria which was exposed to different concentrations of UA (Fig. 2A and B). Our data showed that succinate supports the greatest rate of H2O2 production while H2O2 release supported with pyruvate/malate is not significant in comparison with the control group. We observed that succinate-supported H2O2 production in UAexposed isolated kidney mitochondria is fairly inhibited by rotenone, an inhibitor of electron transport at the NADH dehydrogenase region (Fig. 2B); this suggested that reversed electron transfer is involved in succinate-supported H2O2 production. In fact, two pathways contribute to succinate-supported H2O2 production: electron leakage from complex III or reverse electron transport from complex II (succinate dehydrogenase) to complex I [57]. Rotenone inhibited the reverse electron transfer and thus could decrease H2O2 production in succinate dehydrogenase/ubiquinone-cytochrome b region [58]. The slower rate of H2O2 production release with rotenone in succinate, fueled the isolated kidney mitochondria also proposes the role of succinate dehydrogenase/ubiquinone-cytochrome b region and reverse electron transfer in UA-induced H2O2 production in kidney mitochondria. On the other hand, when antimycin A (an inhibitor of electron transport at the ubiquinone-cytochrome b region) plus rotenone were added to incubation medium, no significant change was observed in H2O2 formation. The addition of myxothiazol did not cause a significant reduction in H2O2 production in complex I either but significantly reduced the UA-induced H2O2 production in succinate supported mitochondria. Myxothiazol is an inhibitor of complex III, which prevents the semiquinone formation in this complex. Therefore, the reduction of UA-induced H2O2 production in succinate-supported kidney mitochondria by myxothiazol could confirm the role of complex III in UA nephrotoxicity. We also investigated the effect of UA in the activity of complex II and IV of mitochondrial electron transfer chain. The effect of UA on succinate dehydrogenase activity was determined through the reduction of MTT dye to formazone metabolite. UA significantly reduced the formazone formation suggesting that UA may alter the function of complex II and probably inhibition of this enzyme contributes in UA toxicity. On the other hand, we assayed the activity of complex IV (cytochrome c oxidase). As shown in results, UA did not have considerable impact on cytochrome c oxidase activity. The exposure of isolated kidney mitochondria to UA significantly increased the lipid peroxidation which is demonstrated as the increased TBARS levels. Furthermore, previous studies showed U-induced lipid peroxidation in different tissues such as kidney, testis [59], brain [16]. On the other hand, the oxidation of lipid membrane results in disruption of mitochondrial membrane and consequently the collapse of MMP and cytochrome c release [60]. Glutathione (GSH) is one of the primary non-enzymatic antioxidant systems against hydrogen peroxide and other ROS which constitutes nearly 10–15% of total cellular GSH in the mitochondria [61]. Therefore, the decline of reduced glutathione content in mitochondria could cause severe deficiency in their defense system against oxidative damage, leading to further rise in lipid peroxidation. As our data showed, UA

induced the GSH oxidation and lipid peroxidation in exposed mitochondria that were resulted from the elevation of H2O2 production. Previous studies also reported GSH depletion in UA-exposed cells such as isolated hepatocytes [10,11]. Moreover, GSH is required for the maintenance of reduced form of thiol groups in the mitochondrial membrane proteins [36]. When these thiol groups are oxidized, conformational changes occur in the pore complex that leads to mitochondrial permeability transition (MPT) [62]. Thus, UA-induced ROS production not only leads to lipid peroxidation and GSH oxidation, it could also damage the mitochondrial membrane integrity and open the MPT pores. The opening of the MPT pores and termed permeability transition pores initiates the onset of the MPT that is a determinant key in both necrosis and apoptosis mechanisms [63]. Mitochondrial swelling is a structural change in isolated mitochondria undergoing the permeability transition [64]. Several agents are capable of inducing the permeability transition such as oxidants, heavy metals and sulfhydryl reactive compounds [64]. The opening of the MPT pores by UA is double-confirmed through the mitochondrial swelling determination (Fig. 8) which is subsequent to MMP collapse (Fig. 7a) and in accordance with previous studies that showed the UA-caused significant collapse of MMP in rat isolated hepatocytes treated with UA [11] and Rat Kidney Proximal Cells [26]. Cs A (the MPT inhibitor) significantly inhibited both UA-induced MMP collapse and mitochondrial swelling (Fig. 7B). Besides, the addition of BHT (a typical anti-oxidant) fairly prevented the MMP collapse (Fig. 7B) suggesting that oxidative stress is directly involved in MPT induction. The induction of MPT, not only leads to mitochondrial swelling and outer membrane rupture, but is also associated with the release of apoptogenic factor such as cytochrome c into cytosol [21]. ATP production through oxidative phosphorylation, is the most important mitochondrial function. Furthermore, the ATP level determines the mode of cell death in target cells [65]. In fact, ATP behaves as a switch between apoptosis and necrosis. Execution of apoptosis requires ATP, while depletion of ATP interrupts apoptosis and shifts cells to necrosis [65]. We found that UA exposure decreased the ATP production and ATP/ADP ratio in isolated kidney mitochondria. This might be due to the inhibition of mitochondrial respiratory chain and MPT pore opening. Pereira et al. showed that the inhibition of mitochondrial ETC could lead to a decrease in MMP and ATP production [66]. In fact, when MPT pore opens, the subsequent unlimited proton movement across the inner membrane results in the uncoupling of oxidative phosphorylation [49]. The remaining ATP was immediately consumed for the maintenance of MMP, leading to further reduction of ATP concentration [49]; this results in exacerbation of ROS production. As seen in Fig. 10, UA significantly increased the outer membrane damage. As a result, UA-induced lipid oxidation in kidney mitochondria (as a consequence of respiratory chain impairment and decline of GSH) could promote the mitochondrial membrane damage and finally the loss of mitochondria outer membrane integrity. Mitochondria are essential for cell survival due to their roles as regulators of apoptosis [49]. Programmed cell death may occur when the amount of ROS generated in the mitochondria cannot be neutralized through the radical-scavenging cellular antioxidants. Treatment with specific electron transfer chain inhibitors can cause oxidative stress and ignite the cell cycle signaling [67]. The mechanism of ROS-mediated apoptosis appears to involve the MPT pore opening and release of cytochrome c from mitochondria and their ATP-dependent interaction with cytosolic factors to form apoptosome to activate caspase-9 and caspase-3, respectively [21]. Accordingly, both dissipation of mitochondrial membrane potential and cytochrome c release are the important indicators of cell apoptosis and important endpoints for the determination of mitochondrial dysfunction. As manifested through the results, UA caused significant expulsion of cytochrome c from mitochondria. Moreover, Cs A and BHT pretreatment completely blocked the UA-induced release of cytochrome c from the mitochondria which supports the hypothesis that the apoptosis induction via UA is due to an oxidative stress and depends on the opening of the mitochondrial

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transition pore. UA in low concentration could induce apoptosis while necrosis is quite likely in higher concentration, (due to severe depletion of ATP level) [68]. 5. Conclusion Based on our findings, UA impaired the electron transfer chain in isolated rat kidney mitochondria at complex II and III leading to increased ROS production. Mitochondrial ROS production contributes to nephrotoxicity of UA, as it leads to the failure of oxidative phosphorylation, ATP cellular declination, mitochondrial membrane potential disruption, mitochondrial swelling, loss of mitochondrial outer membrane integrity and finally the release of cytochrome c from mitochondria. In addition, increased ROS production, lipid peroxidation, GSH depletion, and MMP collapse were also observed in kidney mitochondria following UA injection. These events can justify oxidative damage and nephrotoxicity caused by uranium compounds. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgement The data provided in this article was extracted from the Ph.D. thesis of Dr. Fatemeh Shaki. The thesis was conducted under supervision of Prof. Jalal Pourahmad at Department of Toxicology and Pharmacology, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. This study was supported by a grant from Shahid Beheshti University of Medical Sciences (88/01/94/6533, 2009). References [1] A. Bleise, P.R. Danesi, W. Burkart, Properties, use and health effects of depleted uranium (DU): a general overview, J. Environ. Radioact. 64 (2003) 93–112. [2] WHO, Depleted Uranium: Sources, Exposure and Health Effects - Full Report.In WHO, in: Department of Protection of the Human Environnment, vol. 2001 (WHO/SDE/PHE/01.1). World Health Organization, Geneva, 2001. [3] U.S.EPA, EPA facts about U, in: United States Environment Protection Agency, 2002. [4] Y. Gu´eguen, L. Grandcolas, C. Baudelin, S. Grison, E. Tissandi´e, J.R. Jourdain, F. Paquet, P. Voisin, J. Aigueperse, P. Gourmelon, M. Souidi, Effect of acetaminophen administration to rats chronically exposed to depleted uranium, Toxicology 229 (2007) 62–72. [5] G. Zhu, M. Tan, Y. Li, X. Xiang, H. Hu, S. Zhao, Accumulation and distribution of uranium in rats after implantation with depleted uranium fragments, J. Radiat. Res. 50 (2009) 183–192. [6] D.E. McClain, Depleted uranium: a radiochemical toxicant? Mil. Med. 167 (2002) 125–126. [7] A.P. Gilman, D.C. Villeneuve, V.E. Secours, A.P. Yagminas, B.L. Tracy, J.M. Quinn, V.E. Valli, R.J. Willes, M.A. Moss, Uranyl nitrate: 28-day and 91-day toxicity studies in the Sprague–Dawley rat, Toxicol. Sci. 41 (1998) 117–128. [8] R.W. Leggett, The behavior and chemical toxicity of U in the kidney: a reassessment, Health Phys. 57 (1989) 365–383. [9] K.S. Squibb, R.W. Leggett, M.A. McDiarmid, Prediction of renal concentrations of depleted uranium and radiation dose in Gulf War veterans with embedded shrapnel, Health Phys. 89 (2005) 267–273. [10] J. Pourahmad, F. Shaki, F. Tanbakosazan, R. Ghalandari, H.A. Ettehadi, E. Dahaghin, Protective effects of fungal β-(1→3)-D-glucan against oxidative stress cytotoxicity induced by depleted uranium in isolated rat hepatocytes, Hum. Exp. Toxicol. 30 (2011) 173–181. [11] J. Pourahmad, M. Ghashang, H.A. Ettehadi, R. Ghalandari, A search for cellular and molecular mechanisms involved in depleted uranium(DU) toxicity, Environ. Toxicol. 21 (2006) 349–354. [12] B. Daraie, J. Pourahmad, N. Hamidi-Pour, M.-J. Hosseini, F. Shaki, M. Soleimani, Uranyl acetate induces oxidative stress and mitochondrial membrane potential collapse in the human dermal fibroblast primary cells, IJPR 11 (2012) 495–501. [13] M. Valko, M. Izakovic, M. Mazur, C.J. Rhodes, J. Telser, Role of oxygen radicals in DNA damage and cancer incidence, Mol. Cell. Biochem. 266 (2004) 37–56. [14] V. Linares, M. Bell´es, M.L. Albina, E. Mayayo, D.J. S´anchez, J.L. Domingo, Combined action of U and stress in the rat. II. Effects on male reproduction, Toxicol. Lett. 158 (2005) 186–195. [15] M. Taulan, F. Paquet, C. Maubert, O. Delissen, J. Demaille, M.C. Romey, Renal toxicogenomic response to chronic uranyl nitrate insult in mice, Environ. Health Perspect. 112 (2004) 1628–1635.

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