Abstract. – OBJECTIVE: Malathion (MLT) isan organophosphate (OP) pesticide widely usedin agriculture and for domestic purposes forseveral years. Intravenous lipid emulsion (ILE)has been reported to reduce toxicity caused bysome lipid soluble agents. The aim of this studywas to investigate the possible protective effectsof ILE treatment on acute malathion toxicity inovarian tissue of female rats.

MATERIALS AND METHODS: Twenty-oneadult female Wistar rats (weighted 200-250 g)were divided into three groups; control (corn oil,gavage), MLT (one administration of 100 mg/kg/by gavage), 20% ILE (one intravenous adminis-tration of 3 ml/kg) plus the MLT group. Bloodsamples were collected for biochemical tests.The ovaries were removed and fixed forhistopathological and immunohistochemicalanalyses. Malondialdehyde (MDA), superoxidedismutase (SOD), and glutathione peroxidase(GSH-Px) were investigated in ovarian tissues.Histopathological and immunohistochemicalevaluations were performed through scoringovarian tissue damage and bax/caspase-3 im-munoreactivity, respectively.

RESULTS: SOD activity decreased in MLTgroup compared to the control group in tissuesamples (p = 0.012). ILE treatment significantlyincreased SOD activity in MLT+ILE group com-pared to MLT group in tissue samples (p =0.017). MLT treatment increased significantlycaspase-3 and bax immunoreactivity while ILEdecreased bax and caspase-3 immunoreactivity.However, no significant difference was found forMDA levels and GSH-Px activity in both bloodand tissue samples and for histopathological re-sults.

CONCLUSIONS: The present study revealedthat acute oral MLT administration increased ox-idative stress and apoptosis in the rats. ILE

European Review for Medical and Pharmacological Sciences

Protective effect of intravenous lipidemulsion treatment on malathion-inducedovarian toxicity in female rats

A.Z. OZSOY1, A.F. NURSAL2, M.F. KARSLI3, M. UYSAL4, O. ALICI5,I. BUTUN6, U. TAS4, I.B. DELIBAS1

1Department of Obstetrics and Gynecology, School of Medicine, Gaziosmanpasa University, Tokat,Turkey2Department of Medical Genetics, School of Medicine, Giresun University, Giresun, Turkey3Departments of Obstetrics and Gynecology, School of Medicine, Hitit University, Corum, Turkey4Department of Anatomy, School of Medicine, Gaziosmanpasa University, Tokat, Turkey5Department of Patology, School of Medicine, Gaziosmanpasa University, Tokat, Turkey6Department of Biochemistry, School of Medicine, Gaziosmanpasa University, Tokat, Turkey

Corresponding Author: Asker Zeki Ozsoy, MD; e-mail: drzekiozsoy@hotmail.com 2425

treatment partially decreased deleterious effectsof MLT. Further controlled animal studies are re-quired to define the role of ILE in acute OP poi-sonings.

Key Words:Organophosphate, Malathion, Intravenous lipid

emulsion, Ovary.

Introduction

Organophosphate (OP) pesticides are syntheticchemicals widely used since the mid-1940s forpest control1. They inhibit acetylcholinesterase,an enzyme hydrolyzing acetylcholine in choliner-gic synapses and neuromuscular junctions. Thisinhibition causes acetylcholine to accumulateand leads to subsequent activation of cholinergicmuscarinic and nicotinic receptors1.Malathion (S-(1,2-dicarbethoxyethyl) O,O-di-

methyldithiophosphate) (MLT) is one of the mostwidely used OPs in agriculture, industry, and vet-erinary medicine because it has relatively loweracute toxicity compared to other OPs2. Severalpapers reported that it may have neurotoxic ef-fects including various neuromotor, cholinergic,affective and cognitive disorders, when con-sumed at doses producing cholinesterase inhibi-tion3. Owing to its lipophilic nature, MLT inter-acts with the cell membrane and results in distur-bances in phospholipids bilayer structure of in-ternal organs4. Recent studies have shown thatOPs are likely to cause oxidative stress via dis-rupting the oxidant-antioxidant balance in the

2016; 20: 2425-2434

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A.Z. Ozsoy, A.F. Nursal, M.F. Karsli, M. Uysal, O. Alici, I. Butun, U. Tas, I.B. Delibas

Istanbul, Turkey) anesthesia, 12 hours after thelast oral dose administration. The ovaries wereremoved and left ovary was fixed in 10% forma-lin for histological and immunohistochemicalanalysis. The right ovary was removed in all ani-mals for biochemical analysis of MDA level,SOD and GSH-Px activity.

Biochemical AnalysisEvidence of oxidative stress was determined

based on ovarian tissue homogenates by measur-ing the levels of malondialdehyde (MDA), glu-tathione peroxidase (GSH-Px) and superoxidedismutase (SOD) activities. All assays were car-ried out at room temperature. The ovarian tissueswere first washed with a cold isotonic saline so-lution. Then, tissues were homogenized using ahomogenizer (IKA Ultra-Turrax t 25 Basic,Staufen, Germany) at 3,000 rpm for 3 min. Ho-mogenates were then filtered and centrifuged.Supernatants obtained were used to determinethe enzymatic activities.Measurement of MDA level: Levels of MDA

were measured as described by Esterbauer et al12.This method is based on the reaction of MDAwith thiobarbituric acid (TBA) at 90-100 °C. Af-ter the MDA and TBA reaction, a pink pigmentwith an absorption maximum at 532 nm is pro-duced. Results were expressed as nanomoles pergram protein (nmol/ g protein).Measurement of GSH-Px activity: GPx activi-

ty was determined according to the method de-scribed by Paglia and Valentine13. In this method,the enzymatic reaction was initiated by the addi-tion of H2O2, and the change in absorbance at340 nm was monitored with a spectrophotometer.Measurement of SOD activity: Total (Cu/Zn

and Mn) SOD activity was determined using themethod described by Sun et al14. This method isbased on the inhibition of nitroblue tetrazolium(NBT) reduction. One unit of SOD is defined asthe amount of enzyme causing 50% inhibition inthe NBT reduction rate. Both GPx and SOD ac-tivities are expressed as units per gram protein(U/ g protein).

Histopathological ExaminationThe ovaries were removed and fixed in 10%

formaldehyde solution at room temperature, thendehydrated through ascending grades of alcohol,cleared in xylene and embedded in paraffinblocks. Ovary tissue sections (4-5 µm in thick-ness) were stained with hematoxylin and eosin(H&E). Histological evaluation was performed

body5. Increases in lipid peroxidation and de-creases in antioxidant defense capacity are im-portant results associated with OP toxicity6. MLThas toxic effects on experimental animals and ex-posed workers. It was observed that MLT is cor-related with the dysfunction of several organ sys-tems; among them are liver7, testis8 and brain9.Intralipid is the brand name of the first safe fat

emulsion developed for human use and was in-troduced in 1962. Intravenous lipid emulsion(ILE) is always used in parenteral nutrition thera-py. ILE is available in 10%, 20% and 30% con-centrations3. ILE has proved to be an effectivetreatment for local anesthetic systemic toxicity inhumans and animals and is promising as a novelantidote for a wide range of other lipophilic drugpoisonings10,11. In this respect, we aimed to deter-mine the acute effects of MLT on the ovarian tis-sue of female rats and to assess whether these ef-fects could be improved by ILE treatment.

Material and Methods

ChemicalsMLT (Malaxon 65 EC®, Astranova Inc.,

Nigde, Turkey) and ILE (20% Clinoleic®, BaxterHealthcare Limited, Norfolk, England) were pur-chased. Other chemicals used in this study weredescribed as needed.

Animals and TreatmentAdult female Wistar albino rats (weighed 200-

250 g) were used in the study. Before the treat-ment, the animals were held at room temperature(22 ± 1 °C) and at 40-50% humidity. The lightpattern was set to be 12 hour day and 12 hournight. They were left free in terms of eating anddrinking. The rats were kept under observationfor one week and they were physically examineddaily. This work was performed in the experi-mental research unit after obtaining the permis-sion of the local Ethics Committee (2014HADYEK 77).Rats were randomly divided into three groups:

(1) control group (corn oil), (2) MLT treatmentgroup (100 mg/kg), and (3) MLT + 20% ILE 3ml/kg treatment group. All groups consisted ofseven rats. MLT was administrated via gavagefor once. Corn oil (vehicle of MLT) was given inthe same way to rats in the control group. ILEwas intravenously administrated for once.Rats were sacrificed by decapitation under 50

mg/kg i.m. ketamine hydrochloride (Eczacıbası,

under light microscopy by the same pathologist,who was blinded to the study. The sections wereviewed and photographed using a Zeiss Axiolight microscope (Zeiss Axio Lab A1, Jena, Ger-many) and were scored from 0 to 3 according totheir severity, where 0 represents absence of anypathologic finding, and 1, 2, and 3 represent re-spective percentages of involved areas as < 33%,33%-66%, and > 66% of the ovary, respectively,as previously described15. The average scores forfollicular degeneration, vascular congestion, he-morrhage, edema and inflammatory cell infiltra-tion were calculated in each group, and total tis-sue damage score was determined by the sum ofthese scores.

Immunohistochemical AnalysesImmunohistochemical staining was carried out

based on previously described methods16. Inshort, five-micrometer serial sections were col-lected on poly-L-lysine coated slides (Sigma-Aldrich, St. Louis, MO, USA) and incubatedovernight at 56 ºC. Tissue sections were deparaf-finized in xylene and rehydrated in ethanol se-ries. Then, sections were treated in a microwaveoven in 10 mM citrate buffer (pH 6.0) twice for 5minutes each and cooled for 20 min. After wash-ing three times in phosphate buffered saline(PBS), endogenous peroxidase activity wasquenched by 3% hydrogen peroxide in PBS for20 minutes, and washed again three times inPBS. To block non-specific binding sectionswere incubated in a blocking serum (Ultra VBlock, ScyTek Laboratories, West Logan, UT,USA) for 10 minutes. Subsequently, sectionswere kept in a humidified chamber overnight at 4°C using the rabbit polyclonal active-Caspase 3(ab13847, Abcam, UK) and rabbit polyclonal an-ti-Bax (ab7977, Abcam, Germany) primary anti-bodies. Sections were washed with PBS at roomtemperature, and incubated first with biotinylatedpolyvalent antibodies (ScyTek Laboratories) andthen peroxidase-labeled streptavidin (ScyTekLaboratories). Immunohistochemical analysiswas performed using a horseradish peroxidase-labeled streptavidin biotin (SensiTek HRP) kit(ScyTek Laboratories) based on the manufactur-er’s instructions. Bound peroxidase was devel-oped using 3-amino-9-ethylcarbazol (AEC) chro-mogen (ScyTek Laboratories). After being coun-terstained with Mayer’s hematoxylin (ScyTekLaboratories), sections were mounted using Per-mount (Fisher Chemicals, Springfield, NJ, USA)on glass slides. As control, sections were treated

only with normal rabbit serum. The concentra-tions of rabbit serum and the primary antibodyused were the same. All samples for each indi-vidual antibody were subjected to the same pro-tocol. Photomicrographs were taken with a Leicamicroscope (Leica DM2500, Nussloch, Ger-many).

Evaluation of ImmunohistochemicalAnalysesEvaluation of the immunohistochemical label-

ing was performed using H-SCORE analyses aspreviously described16,17. Caspase-3 and Bax im-munoreactivities of the ovarian tissues were se-mi-quantitatively evaluated based on the follow-ing categories: 0 (no staining), 1+ (weak but de-tectable staining), 2+ (moderate or distinct stain-ing), and 3+ (intense staining)18. An H-SCOREwas calculated for each tissue as follows: percent cells stained at each intensity category wascalculated and then was multiplied by theweighted intensity of the staining based on thefollowing formula: H-SCORE = ∑Pi(i+ l); where‘i’ represents the intensity scores, and ‘Pi’ is thecorresponding percentage of the cells. Five ran-domly selected areas were studied under a lightmicroscope on each slide (40× objectives). Twoinvestigators, who were unaware of the type andsource of the tissues, determined the per centcells at each intensity within these areas. The av-erage score of both observers was used.

Statistical AnalysisStatistical analyses were performed using IBM-

SPSS 20 program. Data were presented as mean ±standard deviation (SD). One-way ANOVA testwas used to compare groups in terms of H-scorevalues, and Tukey test was used as a post-hoc test.p < 0.05 was considered statistically significant.

Results

Biochemical ResultsTissue MDA, SOD and GSH-Px values in all

groups were given in Table I. SOD activities inovarian tissue were significantly lower in theMLT group compared with the control group(p = 0.012). ILE treatment significantly in-creased SOD activity in the MLT+ILE groupcompared with the MLT group (p = 0.017).MDA levels in ovarian tissue in MLT group wassignificantly higher than that in the control andMLT+ILE group but the difference was not sig-

2427

Malathion-induced ovarian toxicity

2428

nificant (p = 0.222 and p = 0.300, respectively).The difference among the study groups for GPxactivity in tissue samples was not significant.Tissue GSH-Px and SOD activities decreased

and MDA levels increased in the MLT groupcompared with the control and the MLT+ILEgroup. Although ILE treatment reduced MDAlevel and improved GSH-Px and SOD activity,the differences were not significant.

Histopathological ResultsMicroscopic images of histopathological

changes in all groups were shown in Figure 1.Also, histopathological findings of ovarian tissuesamples were summarized in Table II. When thetissue damage scores were comparatively evalu-ated, it was seen that pathological findings wereminimum in the control group. Tissue damagescores demonstrating vascular congestion, hem-orrhage, edema, inflammatory cell infiltrationand follicular degeneration were slightly, but notsignificantly, higher in the MLT group comparedto the control group (p = 0.310). Co-administra-tion of MLT and ILE significantly decreased allof the pathological finding scores in theMLT+ILE group compared to MLT group, butthis decrease was not significant (p = 0.690).

Immunohistochemical FindingsBax and caspase-3 proteins were immunohis-

tochemically stained and H-score results werepresented in Figure 2. Immunoreactivity was notobserved in negative control staining (Figures 2and 3). H-score analysis revealed that bax andcaspase-3 immunoreactivities significantly in-creased in the MLT group compared to the con-trol and the MLT+ILE group (p < 0.001 and p <0.01, respectively). Also, the bax and caspase-3activities in the MLT+ILE group were signifi-cantly lower compared to the control group (p <0.01) (Figures 2 and 3).

Discussion

Although OPs are commonly used as insecti-cides in agriculture all over the world, their spe-cific effect on the female reproductive system isnot sufficiently clear. In the present study, westudied biochemical, histological and immuno-histochemical alterations caused by acute MLTtoxicity on rat ovarian tissue and ameliorative ef-fects of ILE treatment on these alterations.Acute, subchronic and chronic toxicity studies

indicated that MLT is highly toxic to mammals.

A.Z. Ozsoy, A.F. Nursal, M.F. Karsli, M. Uysal, O. Alici, I. Butun, U. Tas, I.B. Delibas

Control MLT MLT+ILE

MDA (nmol/g) 2.4 ± 0.3 3.4 ± 1.8 2.8 ± 1.1GPx (U/g) 228 ± 31 177 ± 67 225 ± 42SOD (U/mg) 0.8 ± 0.2 0.3 ± 0.1*,# 0.6 ± 0.1

Table I. MDA levels, SOD and GSH-Px activities in tissue samples.

MDA: malondialdehyde; GSH-Px: glutathione peroxidase; SOD: superoxide dismutase. Values are expressed as mean ± SD.*p= 0.012, control vs. MLT #p = 0.017, MLT+ILE vs. MLT.

Figure 1. A, In the control group, ovarian stroma had a normal appearance. B, Ovarian tissues of the MLT group demonstratedpathological changes as indicated by the appearance of diffuse congestion, interstitial edema, and follicular degeneration. C, In theMLT+ILE group, milder congestion and hemorrhage were observed. (HE, ×10 objective).

MLT can adversely affect mammals through oral,dermal and respiratory exposure19. Some aggres-sive tissue damaging factors may impair oxi-dant/antioxidant balance in favor of oxidants in aprocess called oxidative stress. OPs can trigger ox-idative stress, resulting in the production of freeradicals and leading to alterations in antioxidantsor reactive oxygen species (ROS) scavenging en-zymes21. Because of its lipophilic properties, OPsinteracts with the cell membrane and disruptsphospholipids bilayer structure of most internal or-gans4. Thus, OPs may elevate lipid peroxidation(LPO) level through directly interacting with cel-lular membranes and ROS generation. Main toxic-ity of OPs in acute exposure is through its irre-versible binding to enzyme acetylcholinesteraseand preventing its activity, thereby leading to ac-cumulation of acetylcholine which ultimatelyleads to acute muscarinic and nicotinic effects22.There are some recent in vivo studies reporting

OPs-induced oxidative stress in humans and ani-

mals23,24 as well as some in vitro studies in ani-mals25. MDA is an end product of lipid peroxida-tion and has been used to determine oxidativestress level of different tissues. SOD and GSH-Px are the main endogenous antioxidant defenseenzymes. Several studies revealed that MLT in-creased MDA level and decreased SOD andGSH-Px activity26-28. We found that acute MLTexposure significantly decreased SOD activity inovarian tissue compared to control group (p =0.012) (Table I), indicating an impairment in theanti-oxidative system. ILE treatment after MLTexposure restored SOD activity considerably (p= 0.017). However, MDA levels and GSH-Px ac-tivities in ovarian tissue were not significantlydifferent between control and treatment groups(Table I).ILE was introduced in 1961 as a component of

total parenteral nutrition. Then, it became a drugdelivery vehicle and finally an adjunct in the re-suscitation of cardiovascular collapse caused bylocal anesthetics29. Its administration has beenstrongly recommended to patients who devel-oped cardiac arrest due to an overdose of localanesthetics such as bupivacaine30-32. ILE has alsobeen shown to be useful in the treatment of neu-rotoxicity and cardiotoxicity resulting from a va-riety of cardiovascular and psychoactive drugs.Animal studies showed the usefulness of ILE invarious toxicities arising from verapamil33, pro-pranolol34, amiodarone35 and clomipramine36.However, studies describing the effect of ILE onoxidative stress are rare. In one of those rarestudies, oxidative stress parameters in parenteral-ly nourished human preterm infants were not af-fected by ILE37. On the other hand, Basarslan etal3 reported that ILE seriously reduced total oxi-dant status (TOS) in rats with MLT-induced neu-rotoxicity and stated that IFE is a promising safetherapy for acute MLT intoxication.Previous studies38,39 revealed the adverse ef-

fects of OPs on male and female reproductivesystems in rats by affecting the histological func-

2429

Malathion-induced ovarian toxicity

Histopathological findings Control (n = 7) MLT (n = 7) MLT+ILE (n = 7)

Vascular congestion 0.75 1.2 1.1Hemorrhage 0.50 0.8 0.6Edema 0.25 0.5 0.4Inflammatory cell infiltration 0.50 0.7 0.5Follicular degeneration 0 0.5 0.2Tissue damage score 2.0 3.7 2.8

Table II. Distribution of histopathological findings in groups.

Figure 2. H-score values of bax and caspase-3 immunore-activity. *p < 0.001, compared to the control group; ‡p <0.01, compared to the MLT+ILE group; #p < 0.01 comparedto the control group.

2430

tions of the organs. A study designed to investi-gate the effects of Diazinon, an OP, on rat pitu-itary-gonad axis and ovary showed no effect of14-day oral Diazinon application on the meannumber of primary, secondary and graaffian folli-cles. In addition, no difference was found40 be-tween groups with respect to histopathologicalchanges including vascular congestion, hemor-rhage, edema, inflammatory cell infiltration andfollicular degeneration. In another study41 usingmethyl parathion and male rats, on the otherhand, the weight of seminal vesicle and prostatewere found to decrease in a dose-dependent man-ner. A significant reduction in the weight oftestes, epididymis, seminal vesicle and ventralprostate was reported by another study42 in whichMLT was orally administered to rats at 50, 150

and 250 mg/kg/body weight/day rates for 60days. Koc et al43 reported that MLT decreased thesize of the ovary in adult Wistar albino rat de-pending on the doses used. In another study,MLT was given as 500 mg/kg body weight (BW)dose to adolescent male mice at the pubertal agefor three days and no histological changes ap-peared in the testis compared to control groups44.MLT has also been reported to reduce spermmotility and viability and to increase the numberof abnormal sperms8,45,46.Here, we observed that MLT caused

histopathological changes in ovarian tissue in-cluding vascular congestion, hemorrhage, edema,inflammatory cell infiltration and follicular de-generation (Figure 1). ILE treatment partially im-proved these problems, but the effect did not

A.Z. Ozsoy, A.F. Nursal, M.F. Karsli, M. Uysal, O. Alici, I. Butun, U. Tas, I.B. Delibas

Figure 3. Evaluation of apoptosis by Bax (immunohistochemical method). A, Bax immunoreactivity in the control group.The green arrows indicate the Bax immunopositive granulose cells in secondary follicle. B, Negative control tissue. C, StrongBax immunoreactivity observed in the MLT group. The green arrows indicate the Bax immunopositive granulose cells in pri-mary follicle and stromal cells. D, In the MLT+ILE group, Bax immunoreactivity was significantly lower in comparison to theMLT group The green arrows indicate the Bax immunopositive granulose cells in primary and secondary follicle, and stromalcells ×40 magnifications).

reach significance level (Table II). This findingcould be due to the duration of exposure to MLTand application doze.Apoptosis is programmed cell death process

and plays an important role in the regulation ofgrowth and development. Apoptosis has beenknown to take place from early developmentalstages to adult stages and is involved in home-ostasis of multicellular organisms, disease devel-opment and different stimuli in different sys-tems47. Apoptosis is regulated by many modula-tors such as ions (e.g. calcium), genes (e.g. c-myc, Bcl-2/Bax, Fas and DR5), proteins (e.g.p53, caspases and IAPs) and even organelles (mi-tochondria and endoplasmic reticulum)47. OPslead to oxidative stress, causing damage to cellu-lar DNA and activation of apoptosis-related p53,caspase 3 and 9 genes. Thus, they have the poten-tial to cause genetic alterations and cellular toxic-ity48. Impairments of several cellular processesassociated with DNA repair and regulation dur-

ing apoptosis are mediated by caspase-3 activa-tion21. OP poisoning has been reported to induceapoptosis via caspase-3 activation49. Four weeksof Diazinon administration was reported to in-crease bax and caspase-2 activity in cardiac tis-sue of rats50. Similarly, chlorpyrifos was conclud-ed to cause apoptosis in rat cortical neuron51. Dif-ferent immunolabeling patterns with caspase-3and -9 were obtained in endometrial epitheliumand stroma specimens from methyl parathion(MPT) treated and untreated rat groups21. In ad-dition, subchronic exposure to dichlorvos was al-so reported to lead to similar differences in en-dometrium tissue52.There were only limited data available con-

cerning histopathological and immunohisto-chemical changes caused by MLT in the ovary.Whether MLT had toxic effects in ovarian tissueremained largely unclear. In the present study,apoptosis was evaluated using bax and caspase-3activity, and a significant difference was found

2431

Malathion-induced ovarian toxicity

Figure 4. Evaluation of apoptosis by caspase-3 (immunohistochemical method). A, Caspase-3 immunoreactivity in the con-trol group. The yellow arrows indicate the Bax immunopositive techa cells in secondary follicle and stromal cells. B, Negativecontrol tissue. C, Strong caspase-3 immunoreactivity observed in the MLT group. The yellow arrows indicate the Bax im-munopositive granulose cells in primary follicle and stromal cells. D, In the MLT+ILE group, caspase-3 immunoreactivity wassignificantly lower in comparison to the MLT group. The yellow arrows indicate the Bax immunopositive techa cells in prima-ry follicle and stromal cells (×40 magnifications).

2432

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8) UZUN FG, KALENDER S, DURAK D, DEMIR F, KALENDERY. Malathion-induced testicular toxicity in malerats and the protective effect of vitamins C and E.Food Chem Toxicol 2009; 47: 1903-1908.

9) DA SILVA AP, MEOTTI FC, SANTOS AR, FARINA M. Lacta-tional exposure to malathion inhibits brain acetyl-cholinesterase in mice. Neurotoxicology 2006; 27:1101-1105.

10) KAPLAN A, WHELAN M. The use of IV lipid emulsionfor lipophilic drug toxicities. J Am Anim Hosp As-soc 2012; 48: 221-227.

11) FERNANDEZ AL, LEE JA, RAHILLY L, HOVDA L, BRUTLAGAG, ENGEBRETSEN K. The use of intravenous lipidemulsion as an antidote in veterinary toxicology. JVet Emerg Crit Care 2011; 21: 309-320.

12) ESTERBAUER H, CHEESEMAN KH. Determination ofaldehydic lipid peroxidation products: malonalde-hyde and 4-hydroxynonenal. Methods Enzymol1990; 186: 407-421.

13) PAGLIA DE, VALENTINE WN. Studies on the quantita-tive and qualitative characterization of erythrocyteglutathione peroxidase. J Lab Clin Med 1967; 70:158-169.

14) SUN Y, OBERLEY LW, LI Y. A simple method for clini-cal assay of superoxide dismutase. Clin Chem1988; 34: 497-500.

15) YILDIRIM N, YIGITTURK G, SAHINGOZ YILDIRIM AG,AKDEMIR A, LGEN O, YENIEL O, ERGENOGLU M, ERBASO. Octreotide protects ovary against ischemia-reperfusion injury in rats: evaluation of histologicaland biochemical parameters. J Obstet GynaecolRes 2015; 41: 1591-1597.

16) ORTAK H, CAYLI S, TAS U, OCAKLI S, SÖ ÜT E, DEMIR

HD. Expression of p97/VCP and ubiquitin duringpostnatal development of the degenerating ratretina. J Mol Histol 2012; 43:17-25.

17) ACAR N, KORGUN ET, USTUNEL I. Cell cycle inhibitorp57 expression in normal and diabetic rat placen-tas during some stages of pregnancy. HistolHistopathol 2012; 27: 59-68.

18) GÜNEY M, ORAL B, DEMIRIN H, OZGÜNER M, TAKE G,MUNGAN T, ALTUNTAS I. Evaluation of caspase-de-pendent apoptosis during methyl parathion-in-duced endometrial damage in rats: Amelioratingeffect of Vitamins E and C. Environ Toxicol Phar-macol 2007; 23: 221-227.

19) EDWARDS JW, LEE SG, HEATH LM, PISANIELLO DL.Worker exposure and a risk assessment ofmalathion and fenthion used in the control ofMediterranean fruit fly in South Australia. EnvironRes 2007; 103: 38-45.

20) KUNAK CS, KUKULA O, MUTLU E, GENÇ F, PEKER GG,KUYRUKLUYILDIZ U, BINICI O5, ALTUNER D6, ALP HH7.The effect of Etoricoxib on hepatic ıschemia-reperfusion ınjury in rats. Oxid Med Cell Longev2015; 2015:598162.

A.Z. Ozsoy, A.F. Nursal, M.F. Karsli, M. Uysal, O. Alici, I. Butun, U. Tas, I.B. Delibas

between treatment groups. In MLT group, a sig-nificant increase in apoptosis was observed com-pared to control group and MLT+ILE group (p <0.001 and p < 0.01, respectively). Decreasedapoptosis in MLT+ILE group (p < 0.01) is animportant finding.

Conclusions

To our best knowledge, this is the first reportevaluating the effects of MLT on ovarian tissue.The results revealed that the acute MLT exposureinduced ROS production and apoptosis in ovari-an tissue. Although ILE treatment partly im-proved these findings, further studies investigat-ing the effects of ILE on the toxicity of OPs arerequired.

–––––––––––––––––-–––Conflict of InterestThe authors report no conflict of interest. The authors aloneare responsible for the content and writing of the paper

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