Free Radical Biology & Medicine 43 (2007) 528–534www.elsevier.com/locate/freeradbiomed

Original Contribution

Comparison of the effects of the superoxide dismutase mimeticsEUK-134 and tempol on paraquat-induced nephrotoxicity

Mohamed Samai a, Martyn A. Sharpe b, Paul R. Gard a, Prabal K. Chatterjee a,⁎

a Department of Pharmacology and Therapeutics, University of Brighton, Brighton BN2 4GJ, East Sussex, UKb Biochemistry and Molecular Biology Department, Michigan State University, East Lansing, MI 48824, USA

Received 19 February 2007; accepted 4 May 2007Available online 16 May 2007

Abstract

Paraquat-induced nephrotoxicity involves severe renal cell damage caused by reactive oxygen species (ROS), specifically viaincreasing concentrations of superoxide anions in the kidney. Recently, superoxide dismutase (SOD) mimetics (SODm) have beendeveloped that display safe SOD activities but which also possess additional antioxidant enzyme (e.g., catalase) or ROS-scavengingactivities. The aim of this study was to compare the effects of two such SODm, specifically, EUK-134, a SODm with catalase activity,and tempol, a SODm with ROS-scavenging properties, on paraquat-induced nephrotoxicity of renal NRK-52E cells. Incubation withparaquat (1 mM) for 24 h reduced cell viability and increased necrosis significantly. Paraquat also generated significant quantities ofsuperoxide anions and hydroxyl radicals. Both EUK-134 (10–300 μM) and tempol (0.3–1.0 mM) were able to improve cell viability andreduced paraquat-induced cell death significantly via dismutation or scavenging of superoxide anions and reduced hydroxyl radicalgeneration. The data presented here suggest that SODm such as EUK-134 and tempol, which possess additional catalase and/or ROS-scavenging activities, can significantly reduce renal cell damage caused by paraquat. These effects were evident at concentrations which avoidthe pro-oxidant activities associated with higher concentrations of SOD. Such SODm could therefore prove to be beneficial as therapies forparaquat nephrotoxicity.© 2007 Elsevier Inc. All rights reserved.

Keywords: EUK-134; Kidney; Nephrotoxicity; NRK-52E; Paraquat; Reactive oxygen species; Renal; Superoxide; Superoxide dismutase mimetic; Tempol; Freeradicals

Paraquat (1,1′-dimethyl-4,4′-bipyridium dichloride, alsoknown as methyl viologen) is a widely used broad-spectrumand fast-acting herbicide. However, it is extremely toxic to bothanimals and humans, leading to many thousands of fatalities andhospital admissions due to accidental or intentional pesticidepoisoning, especially in developing countries [1,2]. Paraquatpoisoning causes severe multiple organ failure, with the degreeof poisoning dependent on the route of administration, theamount administered, and the duration of exposure. Paraquat israpidly distributed within the body, with highest concentrationsaccumulating within the kidneys [3], where it produces severenephrotoxicity. Additionally, as paraquat is primarily excretedunchanged via the kidneys, the consequent reduction in renal

⁎ Corresponding author. Fax: +44 1273 642674.E-mail address: pkc1@bton.ac.uk (P.K. Chatterjee).

0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2007.05.014

function also leads to increasing plasma concentrations ofparaquat, which contribute to its toxicity in other, nonrenalorgans, especially the lungs [4,5]. Ultimately, respiratoryfailure, in the presence of paraquat-induced nephrotoxic acuterenal failure, is responsible for most paraquat-associated deaths[6,7]. Therefore, maintaining renal function in patients sufferingfrom paraquat poisoning remains a therapeutically importanttreatment strategy.

Although the exact cellular mechanisms involved inparaquat-induced renal dysfunction and associated nephrotoxicacute renal failure remain to be fully established, there is goodevidence that the generation of reactive oxygen species (ROS)such as superoxide anions plays a major part in paraquat-induced nephrotoxicity [8–10]. Current research has thereforefocused on the therapeutic potential of antioxidants againstparaquat-induced toxicity [11], especially those that can remove

529M. Samai et al. / Free Radical Biology & Medicine 43 (2007) 528–534

superoxide anions such as superoxide dismutase (SOD) orreduce their generation due to paraquat. However, due toproblems associated with administering exogenous SOD invivo, many different types of molecules which effectivelymimic SOD activity have been synthesized [12,13]. Such SODmimetics (SODm) include manganese(III) tetrakis (4-benzoicacid) porphyrin and M40401 (a manganese-containing SODm),which have been shown to reduce paraquat-induced lung andbrain injury in mice and rats, respectively [14,15]. Furthermore,increasing SOD concentrations in mammalian cells produce aparadoxical pro-oxidant action by which protection againstoxidative stress is lost [16,17] due to the ability of superoxide toboth initiate and terminate lipid peroxidation [18,19]. Further-more, as SODm rapidly dismutate superoxide into hydrogenperoxide, the possibility that the latter will generate highlydamaging hydroxyl radicals via the Fenton or the Haber–Weissreaction increases significantly, especially if catalase and/orglutathione peroxidase activities or levels are reduced. Addi-tionally, Cu2+ derived from Cu/ZnSOD facilitates oxidativestress in the presence of glutathione [20]. It is thereforeappropriate to examine the protective potential of SODm whichpossess either additional antioxidant enzyme (e.g., catalase) orROS-scavenging activities, which will provide protectionagainst oxidative stress yet allow the consequences of higherSOD concentrations to be avoided.

The aim of this study was to compare the effects of twosuch SODm, specifically, EUK-134 (manganese 3-methoxy N,N1 bis (salicyclidene) ethylenediamine chloride), a SODm withcatalase activity [21,22], and tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), a SODm with ROS-scavengingproperties [23,24], on paraquat-induced nephrotoxicity. Speci-fically, the effects of paraquat on the cellular injury and deathcaused in cultures of a renal proximal epithelial cell linederived from a normal rat kidney (NRK-52E) were examinedand the roles of ROS in the development of paraquatnephrotoxicity confirmed. Second, the protection afforded bytwo SODm with differing profiles against the development ofparaquat-induced cellular injury and death were examined. Thespecific effects of EUK-134 and tempol on the superoxideanion and subsequent hydroxyl radical generation by paraquatwere also investigated.

Materials and methods

Unless otherwise stated, all compounds used in thisstudy were purchased from Sigma–Aldrich Co. Ltd. (Poole,Dorset, UK). EUK-134 was synthesized as described previously[22].

Culture of NRK-52E cells

NRK-52E cells, which maintain characteristics of renalproximal tubular cells in culture [25], were obtained at passage24 from the European Collection of Cell Cultures (Salisbury,Wiltshire, UK). Cells were routinely cultured in 80-cm2 Nuncflasks (Fisher Scientific, Loughborough, Leicestershire, UK)and grown in Dulbecco's modified Eagle's medium (DMEM;

Cambrex Bio Science, Wokingham, Berkshire, UK) containing4.9 g/L glucose and 0.584 g L-glutamine, supplemented with10% (v/v) fetal bovine serum (FBS; Biosera, Ringmer, EastSussex, UK), 1% nonessential amino acid solution, 100 U/mlpenicillin, and 50 μg/ml streptomycin at 37°C in a humidifiedHeraeus incubator supplied with 5% carbon dioxide. Culturemedium was changed every 48 h. NRK-52E cells were sub-cultured at 85–100% confluence using a trypsin (0.1% w/v)/versene (0.02% w/v) mixture into 80-cm2 Nunc culture flasks(Fisher Scientific), for subculture, or onto 6- or 24-well Nuncplates (Fisher Scientific) for in vitro experiments. Cells cul-tured on 6- or 24-well plates were grown in DMEM asdescribed above except for the substitution of 5% (v/v) FBS,which was reduced to 1% (v/v) FBS 24 h before experi-ments. NRK-52E cells were used between passages 28 and46 for experiments.

Investigation of paraquat nephrotoxicity

Confluent cultures of NRK-52E cells on 24-well plates wereincubated for 24 h with increasing concentrations of paraquat(0.003–1 mM) in DMEM. At the end of the incubation period,cellular injury and cell death were determined as describedbelow. From these data, a concentration of paraquat (1 mM) waschosen which produced a submaximal, but significant, cellularinjury (approximately 90% reduction in mitochondrial respira-tion compared to untreated controls) and cell death (approxi-mately 80% of the lactate dehydrogenase (LDH) released bycells treated with Triton X-100).

Investigation of the effects of tempol and EUK-134

To investigate the effects of tempol and EUK-134 oncellular injury and death caused by paraquat, NRK-52E cellswere preincubated (30 min at 37°C) with increasingconcentrations of either EUK-134 (1–300 μM) or tempol(0.3–10 mM). The concentrations of EUK-134 and tempolused in these investigations were based on concentrationspreviously shown to protect primary cultures of rat proximaltubular cells against hydrogen peroxide-mediated oxidativestress [26,27] and are comparable to concentrations which canbe achieved in vivo [26,27]. Paraquat was then added to eachwell as required to produce a final concentration of 1 mM.After 24 h incubation, cellular injury and cell death wereassessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and LDH assays as describedbelow.

Measurement of cellular injury (MTT assay)

Cellular injury or viability was determined as describedpreviously via measurement of the mitochondrial-dependentconversion of MTT into a formazan soluble in dimethylsulfoxide [26–28]. Results were calculated as a percentage ofthe absorbance measured in control (untreated) cells which werenot exposed to paraquat, was were taken as 100% viability andexpressed as cell viability (% untreated controls).

Fig. 1. Effects of paraquat (PQ) on (A) cellular viability/injury and (B) celldeath. NRK-52E cells were incubated with increasing concentrations ofparaquat for 24 h. ★p b 0.05 vs 0 mM paraquat (untreated controls) analyzedusing one-way ANOVA followed by Dunnett's posttest, N = 10.

530 M. Samai et al. / Free Radical Biology & Medicine 43 (2007) 528–534

Measurement of cytotoxicity (LDH assay)

Cell death was determined by measurement of LDH releasedinto the incubation medium due to the loss of membraneintegrity as described previously [26,27,29,30]. Results werecalculated as a percentage of the total LDH released fromcontrol cells (i.e., those not exposed to paraquat) which wereincubated with 1% (w/v) Triton X-100 for 30 min and expressedas LDH release (% TX-100 controls).

Measurement of superoxide anion generation

The generation of superoxide anion was determined using thenitroblue tetrazolium (NBT) assay described previously [31,32].Briefly, NRK-52E cells grown in six-well plates were incubatedfor 24 hwithDMEMcontaining 25 μg/ml NBT in the presence orabsence of 1 mM paraquat. In addition, NRK-52E cells were co-incubated for 24 h with DMEM containing 25 μg/ml NBT and1mMparaquat in the presence of SOD (EC1.15.1.1; 1000U/ml),catalase (EC 1.11.1.6; 1000 U/ml), EUK-134 (300 μM), ortempol (1 mM). The concentrations of SOD and catalase werebased on previous work ranging from 100 to 10,000U/ml [32,33]and those of tempol and EUK-134were based on those providingsignificant protection against paraquat toxicity.

Measurement of hydroxyl radical generation

The production of hydroxyl radicals by NRK-52E cells in theabsence or presence of paraquat was evaluated using thedeoxyribose assay, based on the principle that the pentose sugar2-deoxyribose can be cleaved by hydroxyl radicals to release asubstance that reacts, on heating, with thiobarbituric acid toproduce a pink chromogen [34,35]. Briefly, confluent NRK-52E cells grown in six-well plates were incubated for 4 or 24 hwith DMEM containing 3 mM deoxyribose in the presence ofeither 30 or 1 mM paraquat, respectively. In addition, NRK-52Ecells were co-incubated for the predetermined time periods withDMEM containing 3 mM deoxyribose and 30 or 1 mM paraquatin the presence of SOD (1000 U/ml), catalase (1000 U/ml),EUK-134 (300 μM), or tempol (1 mM). At the end of theincubation period, the cell supernatant was removed andassayed for thiobarbituric acid-reactive substances for MDAas described previously [36]. An aliquot of the cell supernatantwas also used for protein quantification by the Bradford assay[37] and the hydroxyl radical content of samples was expressedas nmol/mg protein.

Statistical analysis

Results are expressed as means ± SEM.Means were obtainedfrom multiple experiments performed in triplicate. Data wereanalyzed and graphed with commercially available software(Graphpad Prism, version 3.0; Graphpad Software, San Diego,CA, USA). Differences between mean values within the groupswere determined using a Student t test or one-way analysis ofvariance (ANOVA) followed by aDunnett test for comparison ofmultiple means. The level of significance was set at p b 0.05.

Results

Effect of paraquat on cellular viability and LDH release byNRK-52E cells

Incubation of confluent NRK-52E cells with paraquat(0.003–1 mM) for 24 h resulted in a significant reduction incellular viability from 100% for the control (untreated) cells to9% (p b 0.05) (Fig. 1A). Moreover, a significant increase in celldeath from a baseline value of 23% for control (untreated) cellsto 78% was also noted (p b 0.05) for NRK-52E cells incubatedwith increasing concentrations of paraquat (Fig. 1B).

Effects of EUK-134 and tempol on paraquat nephrotoxicity

Once the nephrotoxic actions of paraquat were confirmed,the hypothesis that the SODm EUK-134 and tempol couldprotect NRK-52E cells against paraquat nephrotoxicity wasinvestigated. Co-incubation with paraquat (1 mM) and increas-ing concentrations of EUK-134 (1–300 μM) for 24 h resulted ina significant reduction in paraquat-induced cellular injury anddeath at 100 and 300 μM (p b 0.05) (Figs. 2A and 2B). Higherconcentrations of EUK-134 alone (30–300 μM) produced mo-dest but significant reductions in cellular viability (Fig. 2A);however, this effect was not reflected in cell death measure-ments made at these concentrations (p N 0.05) (Fig. 2B).

A concentration-dependent reduction in the paraquat-mediated cellular injury and death of NRK-52E cells by tempol

Fig. 2. Effects of EUK-134 on (A) reduction of cellular viability and (B)increased cell death caused by paraquat. NRK-52E cells were incubated with1 mM paraquat for 24 h in the presence of increasing concentrations of EUK-134. ★p b 0.05 vs 1 mM PQ+0 μM EUK-134, +p b 0.05 vs 0 μM EUK-134only, analyzed using one-way ANOVA followed by Dunnett's posttest, N = 10.

Fig. 3. Effects of tempol on (A) reduction of cellular viability and (B) increased celldeath caused by paraquat. NRK-52E cells were incubated with 1 mM paraquatfor 24 h in the presence of increasing concentrations of tempol.★p b 0.05 vs 1mMPQ + 0 mM tempol, +p b 0.05 vs 0 mM tempol only, analyzed using one-wayANOVA followed by Dunnett's posttest, N = 10.

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was demonstrated up to a concentration of 1 mM, withreductions produced by 0.1–1 mM tempol observed asstatistically significant (p b 0.05) (Figs. 3A and 3B). However,the protective effect of tempol was lost at higher concentrationsof tempol (3 and 10 mM) and in fact, the highest concentrationof tempol examined (10 mM) produced a modest butnonsignificant increase in the paraquat-mediated cellular injuryand cell death (p N 0.05) (Figs. 3A and 3B). As with EUK-134,higher concentrations of tempol only (1–10 mM) produced amodest yet significant reduction in cellular viability but, incontrast to EUK-134, the highest concentration of tempolincreased cell death significantly after 24 h (p b 0.05) (Figs. 3Aand 3B).

Effects of EUK-134 and tempol on paraquat-mediated ROSgeneration

Exposure of NRK-52E cells to 1 mM paraquat for 24 hproduced a significant increase in superoxide generation(measured as NBT absorbance) of 52% compared to control(untreated) cells (p b 0.05) (Fig. 4A), with a concomitant 10-fold increase in hydroxyl radical production (p b 0.05) (Fig.4B). As expected, addition of SOD (1000 U/ml) significantlyreduced the paraquat-induced superoxide generation by 40%(p b 0.05), whereas catalase (1000 U/ml) did not have a sig-

nificant effect (Fig. 4A). EUK-134 (300 μM) and tempol(1 mM) also produced a significant reduction in paraquat-mediated superoxide generation, by 45 and 42%, respectively(p b 0.05) (Fig. 4A). In contrast, SOD had no effect uponparaquat-mediated hydroxyl radical generation, whereas cata-lase produced a significant reduction of 68% (p b 0.05) (Fig.4B). EUK-134 and tempol both produced significant reductionsin paraquat-mediated hydroxyl radical production, of 47 and51%, respectively (p b 0.05) (Fig. 4B).

Discussion

The results obtained in this investigation indicate thatparaquat produces a concentration-dependent toxic effect onconfluent renal NRK-52E cells. This nephrotoxic action wassignificantly reduced by both EUK-134 and tempol. However,in contrast to EUK-134, the protection provided by tempolwas lost at higher concentrations and tempol itself was foundto be nephrotoxic at its highest concentration. The role ofsuperoxide generation and subsequent hydroxyl radicalproduction by paraquat was confirmed, as were the SODmproperties of tempol and EUK-134. Furthermore, the abilityof tempol and EUK-134 to reduce hydroxyl radical generationwas demonstrated.

Fig. 4. Effects of SOD, SODm, and catalase on paraquat (PQ)-mediatedgeneration of (A) superoxide anion and (B) hydroxyl radical. NRK-52E cellswere incubated for 24 hwith 1mMparaquat in the presence of SOD (1000U/ml),catalase (CAT; 1000 U/ml), EUK-134 (300 μM), and tempol (1 mM).★pb0.05 vs 0 mM paraquat (untreated cells), ■pb0.05 vs 1 mM PQ only,analyzed using one-way ANOVA followed by Dunnett's posttest, N=8.

532 M. Samai et al. / Free Radical Biology & Medicine 43 (2007) 528–534

In this investigation, tempol, a SODm with additional ROS-scavenging activities [23,24], was able to provide significantprotection against the cellular injury and cell death produced byparaquat at a concentration range of 0.1 to 1 mM via reductionof superoxide anion and hydroxyl radical generation. Tempol isa stable piperidine nitroxide which is a water-soluble analogueof the spin label TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)and an active metabolite of tempone (4-oxo-2,2,6,6-tetramethyl-piperidine-N-oxyl) in vivo [24,26,38]. It has been shown toreduce oxidative stress and ROS-mediated injury both in vivoand in vitro [24] via a variety of mechanisms ranging fromSODm activities to scavenging of ROS to oxidation of Fe2+ toFe3+, thereby reducing the availability of ferric iron to par-ticipate in the Fenton or Haber–Weiss reaction and, conse-quently, inhibiting the generation of hydroxyl radicals fromsuperoxide anions and hydrogen peroxide [23,39,40]. Withinthe kidney, tempol has been shown to reduce renal injury causedby oxidative stress during ischemia–reperfusion and shock[26,41–43] and to protect rat proximal tubules and proximaltubular cells against cellular injury and death caused by hy-drogen peroxide in vitro [26,44].

At higher concentrations, the protection afforded by tempolagainst paraquat-induced nephrotoxicity was reduced and at thehighest concentration (10 mM), tempol even seemed toexacerbate the cell dysfunction or injury caused by paraquat.At this concentration, tempol itself displayed toxicity to renalcells. Such a “bell-shaped” profile of protection is a recognizedfeature of SOD and SODm as increasing SOD concentrations inmammalian cells can produce a paradoxical pro-oxidant actionby which protection against oxidative stress begins to decline[16,17]. Superoxide can both initiate and terminate lipidperoxidation [18] and thus the activities of SOD are limited toa narrow concentration range in which superoxide cytotoxicityis prevented, yet superoxide-dependent termination events areallowed to occur [19]. There is also some evidence that Cu2+

derived from Cu/ZnSOD can facilitate oxidative stress in thepresence of glutathione [20]. Furthermore, as tempol rapidlydismutates superoxide into hydrogen peroxide, this promotesthe generation of hydroxyl radicals via the Fenton or Haber–Weiss reaction, especially if glutathione peroxidase levels oractivity are reduced, as has been reported after exposure toparaquat [45,46]. There is also some evidence that tempol itselfmay act as a free radical or stimulate further production of freeradicals and promote cell death when present in excess of otherintracellular free radicals [47,48]. For example, in the absenceof additional oxidative stress, high doses of tempol can altercellular iron metabolism, resulting in the altered synthesis ofessential iron-dependent proteins, causing an increase in thelow-molecular-weight pool of iron which then becomesavailable to catalyze hydroxyl radical generation [47,48].

EUK-134 is a manganese–salen complex possessing bothsuperoxide dismutase and catalase activities, which can removeboth superoxide and hydrogen peroxide [21,22]. In thisinvestigation, EUK-134 provided significant protection againstthe cellular injury and cell death produced by paraquat at aconcentration range of 10 to 300 μM via reduction ofsuperoxide anion and hydroxyl radical generation. EUK-134,along with a related SOD-catalase mimetic, EUK-8 (manganeseN,N′-bis(salicylidene)ethylenediamine chloride), has beenshown to exert beneficial effects in several in vitro and invivo animal models of human disease which involve oxidativestress ranging from Alzheimer, Parkinson, and motor neurondisease to multiple sclerosis and excitotoxic neuronal injury[49–53]. The EUK compounds have also been shown to affordprotection in animal models of ischemia–reperfusion injury ofthe brain (stroke), heart, and, more recently, kidney [27,54–56].Furthermore EUK-8 has been shown to have a beneficial effectin acute lung injury caused by endotoxic shock in pigs [57] andboth EUK-8 and EUK-134 have reportedly reduced renaldysfunction in rodent models of hemorrhagic and endotoxicshock [58–60].

Although EUK-134 (30–300 μM) alone produced a modestyet significant reduction in cellular viability, this was notaccompanied by a concomitant significant increase in cell death.Thus, at higher concentration, EUK-134 may have causedmitochondrial dysfunction which was not accompanied by celldeath or, more likely, may have interfered with the MTT assaydirectly.

533M. Samai et al. / Free Radical Biology & Medicine 43 (2007) 528–534

This study also demonstrated that paraquat produces asignificant increase in the generation of superoxide anions andhydroxyl radicals in renal NRK-52E cells and confirmed thenotion that ROS generation plays a major part in paraquat-induced damage to the kidneys. As expected, the paraquat-induced increase in the generation of superoxide anions andhydroxyl radicals was significantly reduced by SOD and cata-lase, respectively. Moreover, the SODm, EUK-134 (300 μM)and tempol (1 mM), significantly reduced the paraquat-inducedincreased production of superoxide and hydroxyl radicals. Thus,by reducing the production of ROS, these agents will reduceoxidative stress injury, accounting in part for their cytoprotectiveaction against paraquat-induced nephrotoxicity. This finding isconsistent with several reports in which EUK-134 and tempolhave been documented to be useful against oxidative stressinjury in the kidney [26,27,41,42,54,58].

In conclusion, the cytotoxic effect of paraquat on confluentNRK-52E cells is concentration dependent and is mediated viaoxidative stress injury secondary to increased generation ofROS (superoxide anion and hydroxyl radicals). The SODm,EUK-134 and tempol, can both protect NRK-52E cells againstthe toxic effects of paraquat in part by reducing the generationof ROS. However, the results of this investigation indicate thatEUK-134, a SODm with catalase activity, is more potent andseems to have a safer profile than tempol, a SODm with ROS-scavenging activities. Overall, these results demonstrate thatsuch SODm have the potential to be used for the treatment ofparaquat-induced nephrotoxicity.

Acknowledgments

M.S. is the recipient of a Commonwealth Scholarshipprovided by the Commission Scholarship Commission UK(SLCS-2004-304). P.K.C. acknowledges PABS, University ofBrighton, for additional funding of this research.

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