Journal of Surgical Research 175, e17–e23 (2012)doi:10.1016/j.jss.2011.11.002

Protective Effects of Melatonin and S-Methylisothiourea on

Mechlorethamine Induced Nephrotoxicity

Zeki Ilker Kunak, M.D., Ph.D.,* Enis Macit, Ph.D.,† Hakan Yaren, M.D., Ph.D.,* Halil Yaman,‡,1 Erdinc Cakir,‡Ibrahim Aydin, M.D.,‡ Turker Turker, M.D.,§ Yasemin Gulcan Kurt, M.D.,‡ Ayhan Ozcan, M.D.,k

Bulent Uysal, M.D.,{ Salim Isbilir, M.D.,# Emin Ozgur Akgul, M.D.,‡ Tuncer Cayci, M.D.,‡Ahmet Korkmaz, M.D.,{ and Levent Kenar, M.D., Ph.D.*

*Department of CBRN Defense, Gulhane Military Medical Academy, Ankara, Turkey; †Department of Pharmacology, Gulhane MilitaryMedical Academy, Ankara, Turkey; ‡Department of Biochemistry, GulhaneMilitary Medical Academy, Ankara, Turkey; §Department ofEpidemiology, Gulhane Military Medical Academy, Ankara, Turkey; kDepartment of Pathology, Gulhane Military Medical Academy,Ankara, Turkey; {Department of Physiology, Gulhane Military Medical Academy, Ankara, Turkey; and #Department of Biochemistry,

Beytepe Military Hospital, Ankara, Turkey

Originally submitted August 11, 2011; accepted for publication November 1, 2011

Background. In this study, we aimed to investigatethe protective effects of melatonin (MEL) and S-meth-ylisothiourea (SMT) on mechlorethamine (MEC) in-duced nephrotoxicity.Materials and Methods. A total of 36 male Sprague-

Dawley rats were divided into four groups: control,MEC, MECDMEL, and MECDSMT. Three groups re-ceived single dose of MEC (3.5 mg/kg) via transdermalroute. Control animals were given saline only viatransdermal route. MEL (100 mg/kg) was administeredintraperitoneally 30 min after the application of MEC,andafter the samedose ofMELwas given every 12 h fora total of six doses. SMT (50mg/kg)was also given intra-peritoneally 30 min after the application of MEC.Results. The tissue TNF-a, IL-1b, and NOx levels

were found significantly different for all groups (P <0.001). MEC application resulted in severe histopatho-logical changes. Melatonin showedmeaningful protec-tion against kidney damage. But protection by SMTwas weaker. TNF-a and IL-1b levels increased signifi-cantly with MEC application, and MEL and SMT ame-liorated these increases in kidney tissue. MEC alsoelevated NOx levels in kidney tissue.Conclusions. Both inflammation and oxidative

stress may have an important role in the MEC inducednephrotoxicity. MEL and SMT may also have anti-inflammatory properties, as well as anti-oxidantproperties. � 2012 Elsevier Inc. All rights reserved.

1 To whom correspondence and reprint requests should beaddressed at Department of Biochemistry, Gulhane Military MedicalAcademy,Etlik-06018, Ankara, Turkey. E-mail: hyaman@gata.edu.tr.

e17

Key Words: mechlorethamine; melatonin; S-methy-lisothioure; pro-inflammatory cytokines; nephro-toxicity.

INTRODUCTION

Nitrogen mustard (NM) and sulfur mustard (SM) arestrong alkylating cytotoxic chemical warfare agentsand vesicants that cause severe and debilitating dam-age to target organs [1, 2]. Some NM analogous are stillin use as cytostatic drugs, including mechlorethamine,cyclophosphamide, melphalan, chlorambucil, and ifos-famide [3]. Mechlorethamine [bis(2-chloroethyl)me-thylamine, MEC] is a nitrogen mustard that has beendesignated by the military as HN2. MEC is a bifunc-tional alkylating agent containing two reactive chlor-oethyl groups [4]. The primary targets of mustardsare the skin, cornea, and respiratory tissues. Followingan asymptomatic period of variable length, symptomsand signs of injury depend on the target organs andthe level and dose of exposure [5].

Mustard has hydrophobic nature. Therefore, it easilypenetrates and accumulates in the lipid component ofexposed tissues. In case of contact with skin, approxi-mately 20% is absorbed by the skin. In this process, itnot only accumulates in the skin but also distributesto other tissues. While approximately 10% of absorbedmustard is retained in the skin, 90% of mustard passesthrough the systemic circulation and reaches other tis-sues such as brain, kidney, muscle, spleen, liver, and

0022-4804/$36.00� 2012 Elsevier Inc. All rights reserved.

JOURNAL OF SURGICAL RESEARCH: VOL. 175, NO. 1, JUNE 1, 2012e18

bone marrow [6]. Following acute mustard exposure,analyses have show that it accumulates at a high ratein thigh fat, brain, abdominal skin, kidney, and muscletissues [7].

In addition to the alkylating action of mustard, theinjuries of mustard and analogs are mediated by theformation and action of reactive oxygen species (ROS)[8]. Alkylating agents such asMEC can also induce glu-tathione depletion [9]. The depletion of glutathioneleads to accumulation of ROS and reactive nitrogen spe-cies (RNS) within cells, inactivation of sulfhydryl-containing enzymes, loss of calcium homeostasis, lipidperoxidation, cellular membrane breakdown, and ulti-mately cell death [10]. MEC also induces the secretionof pro-inflammatory cytokines, including tumor necro-sis factor-alpha (TNF-a), interleukin (IL)-1, IL-6, IL-8,and IL-15 from differentiated human respiratory epi-thelial cells [11]. On the other hand, inflammation char-acterized by accumulation of granulocytes andmacrophages at sites of injury has a significant role inSM and analogs induced tissue toxicity [12, 13]. In addi-tion, inflammatory cells generate additional ROS thatcontribute to oxidative stress in the SM induced skin in-jury [14].

More evidences suggest that the mechanisms under-lying mustard toxicity include DNA alkylation, gluta-thione depletion, activation of proteases, production offree radicals, and free radical-mediated oxidativestress, inflammation, activation of pro-inflammatorycytokines such as TNF-a, IL-1b, IL-6, IL-8, inducible ni-tric oxide synthase (iNOS) activation and poly(ADP-ribose) polymerase activation [15, 16].

Melatonin (N-acetyl-5-methoxytryptamine, MEL) isthe major product of the pineal gland that has a signifi-cant role in modulation of sleep, sexual behavior, im-mune function, and circadian rhythm. Additionally, itis a potent ROS-scavenger [17–19]. MEL and its metab-olites have also powerful anti-inflammatory propertiesand have proven to be highly effective in a variety of dis-orders linked to oxidative stress and inflammation inexperimental animals [17].

Nitric oxide (NO) is an unstable nitrogen radical thatis generated in different cell types by the concomitantconversion of L-arginine into citrulline through theenzyme NO synthase (NOS). The iNOS induced byendotoxins and cytokines is continuously active and re-leases NO. NO reacts with superoxide anion and ulti-mately form peroxynitrite, a potent reactive oxidant,which leads to direct damage to mitochondrial enzymeandDNA [20]. S-methylisothiourea (SMT) is not a selec-tive iNOS inhibitor and reduces plasma NO levels. Butit is 10- to 20-fold more potent than NG methyl-L-arginine or other iNOS inhibitors known [21].

In this study, we aimed to investigate the protectiveeffects of MEL and SMT on MEC induced nephrotoxi-

city and to determine the levels of TNF-a, IL-1b andnitrite plus nitrate (NOx) in kidney tissues.

MATERIALS AND METHODS

This project was approved by the Experimental Ethics Committeeof Gulhane Military Medical Academy, Ankara, Turkey, and the Na-tional Institute of Health’s Guide for the Care and Use of LaboratoryAnimals was followed.

Animal and Experimental Protocol

A total of 36 male Sprague-Dawley rats (Health sciences Institute,Gulhane Military Medical Academy, Ankara, Turkey), with bodyweights of 220–280 g, were included in this study. They were givenfood and water ad libitum. All animals received humane care in com-pliance with the institution’s guidelines, as outlined in the Guide forthe Care and Use of Laboratory Animals, published by the NationalInstitutes of Health.

They were randomly assigned into four groups containing nine ratseach: control, MEC, MECþMEL, and MECþSMT. All animals exceptthe control groupwere given, via transdermal route, a dose of 3.5mg/kgMEC (dissolved in 500 mL saline). Control animals were given 2 mLsaline only via transdermal route. MEL (100 mg/kg) was adminis-tered intraperitoneally 30min after the application of MEC, and afterthe same dose of MEL was given every 12 h for a total of six doses.SMT (50 mg/kg) was also given intraperitoneally 30 min after the ap-plication of MEC. All surviving animals were killed on d 3 of experi-ment via decapitation, the abdomen was opened, and then kidneyswere harvested for analysis. One kidney was fixed in 4% bufferedformaldehyde for histopathologic evaluation and the other waswashed with saline to remove residual blood, placed in tubes, frozenwith liquid nitrogen, and stored at �80�C.

Tissue Preparation

The frozen kidney tissues were homogenized in phosphate buffersolution (pH 7.4) by means of a homogenizator (Heidolph Diax 900;Heidolph Elektro GmbH, Kelheim, Germany) on ice cube. Homoge-nates were centrifuged at 14,000 rpm (7530 3 g) in 4�C for 10 min.The supernatants were used for all analysis. The protein content ofkidney homogenates was measured by the method defined by Lowryet al. [22] with bovine serum albumin as the standard.

Biochemical Analyses

Tissue TNF-a, IL-1b andNOx concentrations weremeasured in du-plicate by commercial TNF-a and IL-1b rat ELISA kits (Bender Med-Systems, Vienna, Austria), and NOx colorimetric assay kit (CaymanChemical, Ann Arbor, MI). According to the manufacturer, the sensi-tivities of the assays for TNF-a, IL-1b and NOx were 4.3, 4.4 pg/mL,and 2.5 mmol/L, respectively. TNF-a, IL-1b concentrations were ex-pressed as ng/g protein and NOx concentration was expressed asmol/g protein.

Histopathologic Evaluation

For kidney histopathology analysis, the kidney tissues were pro-cessed for light microscopy. This processing consisted of specimens’fixation in 4% buffered neutral formalin solution for 24 h, embeddingin paraffin wax, slicing sections at 4–5 mm of thickness, and stainingthem with hematoxylin and eosin (H&E). Histopathologic examina-tion was blindly evaluated by a pathologist. All sections of kidneysamples were examined for characteristic histologic changes, includ-ing glomerular, tubular (vacuolization, necrosis, and regeneration),interstitial, and vascular alterations.

III)

Py

(IIversu

sIV

)Py

(III

versu

sIV

)

1.000

0.007

1.000

0.057

0.016

0.030

um).

KUNAK ET AL.: MEL AND SMT IN MEC INDUCED NEPHROTOXICITY e19

Statistical Analysis

All the statistical analyses were performed by using SPSS 13.0 soft-ware (SPSS Inc., Chicago, IL). Distributions were evaluated by usingone-sample Kolmogorov-Smirnov test to determine whether the con-tinuous variables are normally distributed or not. Then, we usedKruskal-Wallis test for the ones that were not normally distributed.For the pair-wise comparisons, we used Bonferroni corrected MannWhitney U test with respect to distribution. We performed a post-hoc power analysis by using Power and Sample Size Calculator ver.3.0 (Vanderbilt University, Nashville, TN). The results were ex-pressed as median (minimum- maximum). A P value < 0.05 was con-sidered statistically significant for overall comparisons.

TABLE

1

BiochemicalResu

ltsin

theallExperim

enta

lGroups

Parameters

Groups

P*

(overall)

Py

(Iversu

sII)

Py

(Iversu

sIII)

Py

(Iversu

sIV

)Py

(IIversu

sCon

trol

(I)

MEC

(II)

MECþM

EL

(III)

MECþS

MT

(IV)

TNF-a

(ng/g

prot)

5.3

(3.6�7

.5)

27.7

(12.2–63.3)

9.5

(3.2–14.3)

24.8

(13.7–31.2)

<0.001

0.009

0.042

0.005

0.013

IL-1b(ng/g

prot)

5.3

(2.6�7

.9)

25.2

(13.4–56.1)

9.3

(2.5–16.7)

21.6

(11.1–42.1)

<0.001

0.009

0.042

0.005

0.013

NOx(m

ol/g

prot)

1.3

(0.6�2

.6)

18.8

(12.9–34.4)

4.7

(2.8–8.1)

1.0

(0.7–4.4)

<0.001

0.009

0.002

1.000

0.009

TNF-a

¼tumor

necrosisfactor-alpha;IL

-1b¼

interleu

kin-1

beta;NOx¼

nitrate/nitrite.Alldata

wereex

pressed

asmed

ian(m

inim

um-m

axim

*Kru

skalWallis

test.

y Bon

ferron

icorrectedMannWhitney

UTest.

RESULTS

Three rats in the MEC group and two rats in theMECþSMT group are died until the end of experiment.The kidney TNF-a, IL-1b, and NOx concentrations areshown in Table 1. The tissue TNF-a IL-1b and NOxlevels were found significantly different for all groups(P < 0.001).

The control group had the lowest kidney TNF-a levels[5.3 (3.6–7.5) ng/g protein], and these concentrationswere significantly different compared with the MEC[27.7 (12.2–63.3) ng/g protein], the MECþMEL [9.5(3.2–14.3) ng/g protein], and the MECþSMT [24.8(13.7–31.2) ng/g protein] groups (P ¼ 0.009, P ¼ 0.042,and P ¼ 0.005, respectively) (Fig. 1). TNF-a levels inthe MEC group were significantly higher than theMECþMEL and the MECþSMT groups, respectively(P ¼ 0.013 and P ¼ 1.000). Additionally, in theMECþMEL group, TNF-a concentrations were signifi-cantly lower than the MECþSMT group (P ¼ 0.007).However, there was no difference between the MECand the MECþSMT groups in terms of TNF-aconcentrations.

The concentrations of IL-1b in the control group [5.3(2.6–7.9) ng/g protein] were significantly different com-pared with theMEC [25.2 (13.4–56.1) ng/g protein], theMECþMEL [9.3 (2.5–16.7) ng/g protein] and theMECþSMT [21.6 (11.1–42.1) ng/g protein] groups(P ¼ 0.009, P ¼ 0.042, and P ¼ 0.005, respectively)(Fig. 2). On the other hand, in the MEC group, IL-1blevels were significantly higher than the MECþMELgroup (P ¼ 0.013). But there was no difference betweenthe MEC and the MECþSMT groups, the MECþMELand the MECþSMT groups in terms of IL-1bconcentrations.

The lowest kidney NOx concentrations [1.3 (0.6–2.6)mol/g protein] were also in the control group. These con-centrations were significantly different than the MEC[18.8 (12.9–34.4) mol/g protein] and the MECþMEL[4.7 (2.8–8.1) mol/g protein] groups (P ¼ 0.009 andP ¼ 0.002, respectively) (Fig. 3). There was no statisti-cal difference between the control and the MECþSMT[1.0 (0.7–4.4) mol/g protein] groups. In the MEC group,

FIG. 3. Kidney NOx levels in all experimental groups.FIG. 1. Kidney TNF-a levels in all experimental groups.

JOURNAL OF SURGICAL RESEARCH: VOL. 175, NO. 1, JUNE 1, 2012e20

NOx levels were significantly higher than in theMECþMEL and the MECþSMT groups (P ¼ 0.009and P ¼ 0.016, respectively). In the MECþSMT group,the kidney NOx concentrations were higher than thecontrol group, but it was not statistically significant.Additionally, tissue NOx levels in the MECþMELgroup were higher compared with the MECþSMTgroup (P ¼ 0.030).

Since this is a first stage animal experiments, localethic committee prohibited use more than nine subjectsfor each group.Many of our power calculations were be-low 0.80 but higher than 0.50. Power calculations aboutMEC versusMECþSMT groups were higher than 0.80.Our least power calculation was between MEC versusMECþSMT groups for TNF-a, IL-1b, and NOx vari-ables (power < 0.02).

All histopathologic changes were scored as describedin Table 2. Histopathologic images are also shown in

FIG. 2. Kidney IL-1b levels in all experimental groups.

Fig. 4. Histopathology of control rat kidney shows nor-mal glomerulus and tubules, blood vessels with con-necting renal parenchyma. MEC treatment resultedin severe kidney toxicity. Histopathologic observationsfollowingMEC treatment in rat kidney showed glomer-ular focal congestion, mild mesangiolysis, mild intersti-tial edema, and severe peritubular congestion.However, glomerular focal congestion, mild mesangiol-ysis, and severe peritubular congestion were observedin theMECþMEL group. There were diffuse segmentalglomerular congestion, focal and mild glomerular me-sangiolysis, mild tubular vacuolar degeneration andnecrosis, severe tubular regeneration, and mild peri-tubular congestion in the MECþSMT group.

DISCUSSION

There are many studies showing the role of inflam-matory mediators and cytokines in the NM and SMinduced skin and lung toxicities. However, the role ofpro-inflammatory cytokines and oxidative stress inMEC induced kidney injury is unknown. In the presentstudy, we characterized kidney toxicity and inflamma-tory and oxidative responses to MEC in a rat model.MEC induced nephrotoxicitymay be associated with in-creased cytokine production and changing in the bal-ance oxidants/antioxidants.

Alkylating agents such as SM and NM lead to alkyl-ation of cell compounds, and production and release ofinflammatory mediators and cytokines. The cell re-sponds to this situation as inflammation and tissuedamage [23]. The activated cells in the inflammatoryareas cause release of several toxic agents such as pro-teases and free radicals that lead to destruction of cellsand regional necrosis in the renal papillary after expo-sure to MEC [24].

TABLE

2

MeanScoreofHisto

path

ologic

Changesin

allAnim

alGroups

Group

Glomerularch

anges

Tubularch

anges

Interstitialch

anges

Vascularch

anges

All

scores

Con

ges-

tion

Hem

orrh

age

Necrosis

(mesangiolisis)

Vacu

olar

deg

eneration

Necrosis

Reg

enera-

tion

Edem

aPeritubular

congestion

Hem

or-

rhage

Inflam-

mation

Vasculitis

Fibrinoid

necrosis

Fibrointimal

thicken

ing

Con

trol

00

00

00

00

00

00

00

00

00

MEC

10

12

22

48

14

10

60

00

016

MEC þM

EL

10

12

00

00

04

00

40

00

06

MEC þSMT

30

14

31

26

01

00

10

00

011

0:Normal/nopatholog

icch

anges

1:Focal*

andsegmen

tal

2:Focalandglobal

3:Diffuse**

andsegmen

tal

4:Diffuse

andglobal

(*)Theinvolvem

entrestricted

inless

than50%

ofglomeruli

(**)

Theinvolvem

entrestricted

inmorethan50%

ofglomeruli

0:Normal/nopatholog

icch

anges

1:Focalandmild

2:Focalandsevere

3:Diffuse

andmild

4:Diffuse

andsevere

0:Normal/nopatholog

icch

anges

1:Focalandmild

2:Focalandsevere

3:Diffuse

andmild

4:Diffuse

andsevere

0:Normal/opatholog

icch

anges

1:Minim

al

2:Mild

3:Mod

erate

4:Sev

ere

MEC¼

mechlorethamine;

MEL¼

melatonin;SMT¼

S-m

ethylisothiourea.

KUNAK ET AL.: MEL AND SMT IN MEC INDUCED NEPHROTOXICITY e21

In a mustard induced toxicity, cytotoxic ROS andRNS stimulate synthesis and release of pro-inflammatory cytokines. On the other hand, inflamma-tory responses induce oxidative and nitrosative stressand also decrease cellular antioxidants such as gluta-thione. In the inflammation, the overproduction ofROS and RNS leads to an extreme oxidative stress, cel-lular injury, and apoptosis by various mechanisms, in-cluding peroxidation of membrane lipids, proteinnitration, and DNA damage [25]. In recent studies, re-searchers have reported that mustard gas exposure en-hances intracellular oxidative stress and antioxidantdefense mechanisms. These antioxidant responsesmay induce expression of the pro-inflammatory cyto-kines such as IL-1b and IL-6 [26]. The exposure to mus-tard and analogs results in depletion of cellularantioxidants, and the lung injury induced by theseagents is ameliorated in animal studies by treatmentwith various antioxidants [27, 28].

Production of pro-inflammatory cytokines by macro-phages may also play a key role. Blockade of eitherTNF-a or IL-1b produced by glomerular macrophageswas able to suppress glomerular inflammation ina number of disease models [29]. The present studydemonstrates that MEC caused a significant increasein the levels of kidney tissue TNF-a and IL-1b followingMEC administration to rats and there is a possible roleof inflammation in the MEC induced kidney injury.

SM induced toxicity is associated with oxidativestress, caused by an imbalance between production ofoxidants and antioxidants in the lung, and this isthought to be a primary event triggering the inflamma-tory cascade and tissue injury [30]. NO readily reactswith superoxide anion forming peroxynitrite. NO andperoxynitrite oxidize and covalently modify membranelipids, thiols, proteins, and DNA, inducing cytotoxicityand perpetuating inflammation. In another study, ithas been demonstrated that the expression of iNOSwas rapidly up-regulated in alveolar macrophages fol-lowing MEC exposure [31]. iNOS is predominantly ex-pressed in inflammatory cells such as macrophages inaffected tissues. The level of iNOS expression is wellcorrelated with the degree of inflammation [32]. Underconditions elevating oxidative stress, the toxicity ofmustards can be greatly enhanced. Stone et al. haveshown that 2-chloroethyl ethyl sulfide, a SM analog,leads to a higher toxicity in cells stimulated by LPS,TNF-a, or IL-1b, which enhance inflammation and oxi-dative stress [33]. Ebselen (a peroxynitrite scavenger)andMEL (a potent anti-oxidant that scavenges both re-active oxygen and nitrogen species) is also classified asa suicidal or terminal antioxidant [34], ameliorate lung-toxicity and oxidative stress induced by NM inrodents, suggesting a potential therapeutic target fortreating mustard gas poisoning [27, 28]. MEL is also

FIG. 4. Representative photographs are showing the histopathologic changes in kidney tissues. (A) Control group: histopathology showsnormal glomeruli and tubuli in rat kidney (H&E,3100). (B) MEC group: glomerular focal congestion and mild mesangiolysis, and mild inter-stitial edema, and severe peritubular congestion (H&E,3200). (C)MECþMEL group: glomerular focal congestion andmildmesangiolysis, andsevere peritubular congestion in the interstitium (H&E,3200). (D) MECþSMT group: moderate to severe tubular vacuolar degeneration andmild necrosis (H&E, 3100).

JOURNAL OF SURGICAL RESEARCH: VOL. 175, NO. 1, JUNE 1, 2012e22

a well-known selective iNOS inhibitor [35]. In an ani-mal study performed Topal et al., rats’ exposure toMEC antioxidant status was evaluated by measuringthe levels of super oxide dismutase, glutathione perox-idase, and MDA. They have shown that MEC adminis-tered animals are exposed to an important oxidantstress. In this study it has been shown that aminogua-nidin, a selective iNOS inhibitor, reduces the toxic ef-fect of acute renal injury by reducing the increasedinduction of iNOS. Additionally, this status suggestsa role of the increased NO production and the inductionof iNOS in the pathogenesis of nephrotoxicity [24].

It has been shown that MEL ameliorates inflamma-tion by blocking transcriptional factors and pro-inflammatory cytokines [36]. MEL has been shown toameliorate inflammation by blocking transcriptionalfactors and TNF-a [37]. These cytokines induce forma-tion of free radicals and promote iNOS activity andtranscriptional factor activation within cells [32].MEL suppresses production and releases of pro-inflammatory cytokines, chemokines, and adhesionmolecules [38]. MEL has been shown to be effective asa protective antioxidant agent against severe free radi-cal mediated toxicity in NM toxicity [27]. Expression ofiNOS in response to cytokine stimulationwas originallyobserved in themacrophage, where it is involved inNO-mediated cell damage and apoptosis [39]. Inhibition ofiNOS by SMT has almost completely abolished thechanges in NOx and proinflammatory cytokines. In

our study, we have found that NOx concentrationswere lower in the MECþSMT group than the MECgroup. These results may indicate the role of NO,iNOS, and oxidative stress in theMEC induced nephro-toxicity. Additionally, the inhibition of iNOS with SMTdecreased kidney NOx levels and improved MEC in-duced kidney damage.

MEC induced oxidative stress can also stimulate in-flammatory responses. In our study, TNF-a and IL-1blevels were significantly decreased in both theMECþMEL and MECþSMT groups compared withthe MEC group. Oxidative stress may accelerate theprogression of renal injury directly by inducing cytotox-icity and indirectly by promoting inflammation. Givingantioxidants such as MEL and SMT reduces inflamma-tion. In addition to its potent antioxidant properties,our results have shown that MEL serves as a potentanti-inflammatory molecule. This situation may showus that there are anti-inflammatory properties ofMEL and SMT as well as their antioxidant properties.

In conclusion, the present study demonstrates thatexposure of rats to MEC induces oxidative stress, in-flammation, and kidney toxicity. In light of thesedata, both inflammation and oxidative stress mayhave an important role in the MEC induced nephrotox-icity. MEL and SMT may also have anti-inflammatoryproperties, as well as antioxidant properties. However,the MEL or SMT treatments may be more effective inthe MEC induced nephrotoxicity, and this treatment

KUNAK ET AL.: MEL AND SMT IN MEC INDUCED NEPHROTOXICITY e23

approachmay be an alternative and effective therapeu-tic strategy for mitigating the toxicity of MEC. Furtherstudies are needed to show these specified effects.

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