Food and Chemical Toxicology 52 (2013) 129–136

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Food and Chemical Toxicology

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Merit of quinacrine in the decrease of ingested sulfite-induced toxic actionin rat brain

Ceren Kencebay a, Narin Derin a,⇑, Ozlem Ozsoy b, Dijle Kipmen-Korgun c, Gamze Tanriover d,Nihal Ozturk a, Goksun Basaranlar a, Piraye Yargicoglu-Akkiraz a, Berna Sozen d, Aysel Agar b

a Akdeniz University, Medical School, Department of Biophysics, Antalya, Turkeyb Akdeniz University, Medical School, Department of Physiology, Antalya, Turkeyc Akdeniz University, Medical School, Department of Biochemistry, Antalya, Turkeyd Akdeniz University, Medical School, Department of Histology and Embryology, Antalya, Turkey

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 September 2012Accepted 8 November 2012Available online 17 November 2012

Keywords:SulfiteSomatosensory evoked potentialssPLA2QuinacrineLipid peroxidation

0278-6915/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.fct.2012.11.015

Abbreviations: ADI, acceptable daily intake; HSO�

calcium dependent phospholipase A2; iPLA2, cytphospholipase A2; DNA, deoxyribonucleic acid; HCpararosaline hydrochloride; PBS, phosphate buffercyanide; K2S2O5, potassium metabisulfite; K2SO3, poglandin H synthase; sPLA2, secretory phospholipase ANaCl, sodium chloride; NaOH, sodium hydroxide; NaNa2SO3, sodium sulfite; SEP, somatosensory evoked poSO3

�2, sulfite; SO3OO, sulfite peroxyl radical; SO3+,

trioxide anion radical; SO2, sulfurdioxide; TBA, tthiobarbituric acid reactive substances; TBS, tris bHealth Organization.⇑ Corresponding author. Address: Akdeniz Univers

ment of Biophysics, 07070 Antalya, Turkey. Tel.: +90 09242 2496903.

E-mail address: narinderin@akdeniz.edu.tr (N. Der

We aimed at investigating the effects of sulfite-induced lipid peroxidation and apoptosis mediated bysecretory phospholipase A2 (sPLA2) on somatosensory evoked potentials (SEP) alterations in rats. Thirtymale albino Wistar rats were randomized into three experimental groups as follows; control (C), sodiummetabisulfite treated (S), sodium metabisulfite + quinacrine treated (SQ). Sodium metabisulfite (100 mg/kg/day) was given by gastric gavage for 5 weeks and 10 mg/kg/day quinacrine was applied as a singledose of intraperitoneal injection for the same period. The latencies of SEP components were significantlyprolonged in the S group and returned to control levels following quinacrine administration. Plasma-S-sulfonate level was increased in S and SQ groups. TBARS levels in the S group were significantly higherthan those detected in controls. Quinacrine significantly decreased brain TBARS levels in the SQ groupcompared with the S group. Quinacrine treatment did not have an effect on the increased sPLA2 levelof the sulfite administered group. Immunohistochemistry showed that sulfite caused an increase in cas-pase-3 and TUNEL positive cells, restored to control levels via quinacrine administration. This studyshowed that sPLA2 might play a role in ingested sulfite-induced SEP alterations, oxidative stress, apop-totic cell death and DNA damage in the brain.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Since 1959, FDA (Food and Drug Administration) has acceptedsulfite compounds as safe and therefore these chemical agentsare used as a food additive agent (Schroeter, 1966). Five sulfitesalts including sodium metabisulfite (Na2S2O5), potassium metabi-

ll rights reserved.

3, bisulfite; cPLA2, cytosolicosolic calcium independentl, hydrochloric acid; PRA,ed saline; KCN, potassiumtassium sulfite; PGH, prosta-2; NaHSO3, sodium bisulfite;2S2O5, sodium metabisulfite;tential; SO4

�, sulfate radical;sulfite radical; SO3

�, sulfurhiobarbituric acid; TBARS,uffered saline; WHO, World

ity, Medical School, Depart-0 242 2496905; fax: +90 090

in).

sulfite (K2S2O5), sodium bisulfite (NaHSO3), potassium sulfite(K2SO3), and sodium sulfite (Na2SO3) are commonly used as antiox-idants in food and pharmaceutical preparations (Gunnison andJacobsen, 1987). Once ingested, sulfite salts react with waterleading to the generation of bisulfite (HSO�3), sulfite (SO3

�2), andsulfurdioxide (SO2) (Gunnison, 1981; Mottley and Mason, 1988).Previous studies have shown that ingested sulfite enters the sys-temic circulation by gastrointestinal absorption and distributedessentially to all body tissues including the brain (Gunnison andJacobsen, 1987; Gunnison and Benton, 1971).

Sulfite can react with a variety of humoral and cellular compo-nents and can cause toxicity (Gunnison and Palmes, 1976; Hayatsuand Miller, 1972; Rencuzogullari et al., 2001). Hence, sulfite isdetoxified by sulfite oxidase in the mammalian tissues. Sulfite oxi-dase, a molybdenum-containing enzyme located in the intremem-branous space of the mitochondria, oxidizes sulfite to sulfate in atwo-electron oxidation step and protects cells from sulfite toxicity(Cohen and Fridovich, 1971; Feng et al., 2007). If there is deficiencyof sulfite oxidase or exposure to excessive sulfite, the sulfite under-goes one electron oxidation reactions, catalyzed by peroxidases toform sulfur trioxide anion radical (SO3

�) (Mottley and Mason,

130 C. Kencebay et al. / Food and Chemical Toxicology 52 (2013) 129–136

1988). The sulfite radicals can react with oxygen molecules form-ing sulfite peroxyl radical (SO3OO.) and sulfate radical (SO4

�)(Mottley and Mason, 1988). There are in vitro studies suggestingthat sulfite radicals induce DNA injury and (Niknahad and O’Brien,2008; Ozturk et al., 2011; Rencuzogullari et al., 2001) mitochon-drial damage causing excessive reactive oxygen species generation,mediated by lipid peroxidation (Aydin et al., 2005; Derin et al.,2009; Green and Reed, 1998). Reactive oxygen species are pro-duced by a number of cellular oxidative metabolic processes,monoamine oxidases, mitochondrial respiratory chain and PLA2pathway (Adibhatla et al., 2003).

Phospholipase A2 is a family of enzymes that catalyze the cleav-age of fatty acids from sn-2 position of membrane phospholipids torelease free fatty acids (Farooqui and Horrocks, 2006). The PLA2family is classified into three main groups in the brain tissue: cyto-solic calcium dependent PLA2 (cPLA2), cytosolic calcium indepen-dent PLA2 (iPLA2) and secretory PLA2 (sPLA2) (Farooqui et al.,1997). sPLA2 is synthesized in intracellular compartments and se-creted into extracellular space where it binds to cell surface recep-tors, identified N type, in neurons (Matsuzawa et al., 1996). Thehighest activity of sPLA2 is established in hippocampus, medullaoblongata and cerebral cortex (Thwin et al., 2003). sPLA2 causes li-pid peroxidation and apoptosis through its products such as ara-chidonic acid and lysophosphatidylcholine (Toborek et al., 1999).It has been reported that AA produces ROS by regulation of theactivity and the expression of NADPH oxidase and by dysfunctionof mitochondria (Pompeia et al., 2003; Balboa and Balsinde,2006; Farooqui et al. 2007). Arachidonic acid-induced apoptosishas been characterized in various cell models by several assaysthat have shown leakage of cytochrome c from mitochondria,mitochondrial depolarization, phosphatidylserine externalization,caspase activation, poly(ADP)-ribose polymerase cleavage, DNAfragmentation, chromatin condensation, nuclear breakdown andloss of membrane integrity. (Köller et al., 1997; Vento et al.,2000; Garrido et al., 2000; Scorrano et al., 2001). Lysophosphatidyl-choline-induced apoptosis is dependent on activation of Bax andcaspase (Kakisaka et al., 2012). On the other hand, lysophosphati-dylcholine stimulated production of superoxide anion partlythrough membrane-associated NADH-dependent superoxide anionproduction systems (Kugiyama et al., 1999).

Quinacrine, a non selective inhibitor of PLA2, was used to eluci-date the effects of sulfite ingestion on regulation of enzyme proteinlevels. Quinacrine was selected because it is has been shown tocross the blood–brain barrier (Dubin et al., 1982).

Apoptosis, also known as programmed cell death, is a biologicalprocess that plays an important role in the development of the ner-vous system (Sastry and Rao, 2000). The specific morphologicalchanges associated with apoptosis are cell shrinkage, chromatincondensation, internucleosomal DNA fragmentation, and the for-mation of apoptotic bodies (Chandra et al., 2000; Jang et al.,2002). The signaling pathways occurring during apoptosis involvethe activation of cysteine proteases that are part of a large familyof proteins known as caspases (Aggarwal, 2000). There are twotypes of caspases: initiator caspases (caspase 8, 10, 9, 2) and effec-tor caspases (caspase 3, 7, 6). The effector caspases are activated bythe active initiator caspases. In particular, the most widely studiedmember of the caspase family is caspase-3. Caspase-3 is an effectorcaspase, partially or totally responsible for the proteolytic cleavageof several proteins (Cohen, 1997; Ko et al., 2009).

Somatosensory evoked potentials (SEPs) are often used to eval-uate the function of afferent pathways from the sensory receptorsup to the somatosensory cortex (Desmedt and Cheron, 1980; Canuet al., 2003). The components of SEP waves are characterized by thelatency, reflecting the conduction velocity of different stages ofafferent pathway and, the amplitude, corresponding to the post-synaptic response to the quantity of sensory input (Canu et al.,

2003; Herr et al., 2007; Shapiro, 2002). These potentials have be-come an important diagnostic method when evaluating complica-tions caused by neurotoxic agents (Lukács et al., 2007; Herr et al.,2007; Lebrun et al., 2000). Our group previously showed that sulfurdioxide, one of the most common air pollutants, affects SEP laten-cies and amplitudes in rats (Kucukatay et al., 2003). However, theeffect of ingested sulfite-induced SEP alterations has not been re-ported in literature.

This study aimed to investigate whether sPLA2 plays a role insulfite mediated SEP alterations through apoptosis and lipid perox-idation in rats. In this context, thiobarbituric acid reactive sub-stances (TBARS), sPLA2 protein levels, TUNEL and cleavedcaspase-3 levels were determined in the brain. Additionally, plas-ma-S sulfonate levels were also measured as a biomarker of in-gested sulfite accumulation in the plasma.

2. Materials and methods

2.1. Preparation of animals

Healthy male Wistar albino rats, aged three months, weighing 300–350 g wereused in this study. Animals were provided from Akdeniz University Animal CareUnit. All experimental protocols conducted on rats were performed in accordancewith the standards established by the Institutional Animal Care and Use Committeeat Akdeniz University Medical School. Rats were housed in stainless steel cages ingroups of 5 rats per cage and given food and water ad libitum. Animals were main-tained at 12 h light–dark cycles and a constant temperature of 23 ± 1 �C at all times.Rats were divided into three experimental groups consisting of 10 rats each: Group1: control (C); Group 2: rats treated with Na2S2O5 (S); Group 3: rats treated withNa2S2O5 + quinacrine (SQ). Animals in S and SQ groups were given by gastric gavage(100 mg/kg/day) of freshly prepared Na2S2O5 for 5 weeks (Hui et al., 1989; Ozturket al., 2011). Quinacrine, non-spesific PLA2 inhibitor, was applied as a single dose(10 mg/kg/day) of intraperitoneal injection in the SQ group for 5 weeks (Hiroseet al., 2007) while the control group received distilled water by gavage and salineby intraperitoneal injection for the same period.

2.2. SEP recordings

At the end of 5 weeks, rats were deprived of food for 24 h and then SEPs wererecorded. SEPs were recorded with stainless steel subdermal electrodes (Medelec017K024, Medelec Manor Way, Old Woking Surrey, United Kingdom) under etheranesthesia. The active electrode was placed over the left somatosensory area ofthe cerebral cortex (0.4 cm to the right of bregma); the reference was 1.0 cm ante-rior to bregma on the midline. A ground electrode was placed on the animal’s tail(Kanda et al., 1989; Kucukatay et al., 2003).

SEPs were recorded using Biopac MP100 data acquisition equipment (BiopacSystem, Inc.) The electrical stimulus was a square-wave, constant-voltage impulsedelivered at a rate of 1/s transcutaneously to the right posterior tibial nerve atthe ankle. The stimulus duration was 0.5 ms, at an intensity sufficient to the pro-duce a definite twitch of the big toe. Analysis time was set to 150 ms, the samplingrate was 1000 Hz, and the frequency bandwidth of the amplifier was 1–3000 Hz.The gain was selected as 20 lV/div. The body temperature of rats was maintainedbetween 37 �C and 37.5 �C using a heating pad during the SEP recording (Panjwaniet al., 1991). Two hundred responses were averaged. Sweeps contaminated withlarge artifacts were rejected by the computer. To ensure the response reproducibil-ity at least two averages were obtained.

2.3. Biochemical investigations

After SEP recordings, heparinized blood was collected from the abdominal aortaof rats under urethane anesthesia and used for the determination of plasma-S-sul-fonate levels. For other biochemical analysis, brains of rats were perfused transcar-dially with heparinized saline, removed immediately and stored at �80 �C. On theother hand, for immunohistochemical studies brains were perfused with by 10%formalin and embedded in paraffin.

2.4. Plasma-S-sulfonate analysis

Plasma-S-sulfonate levels were measured by the method of Gunnison and Pal-mes (Gunnison and Palmes, 1973). One milliliter of plasma was mixed with 0.2 mlof a solution containing 0.027 mM NaOH and 0,125 mM KCN. The mixture wasincubated at 35 ± 1 �C under nitrogen for 1 h. Following incubation, the mixturewas cooled in ice and transferred to a cellulose dialysis bag and dialyzed at 4 �Cagainst 5 ml of dialysate containing 10 mM glycine buffers at pH 10.2 for 4 h. Afterthe dialysis, 200 ll of each reagent given below was added to 1.4 ml of dialysate inthe following order. 0.15 M HCl, sodium tetrachloromercurate solution [0.18 M

C. Kencebay et al. / Food and Chemical Toxicology 52 (2013) 129–136 131

HgCl2 (Merck, Darmstadt, Germany) and 0.43 M NaCl (Sigma, Steinheim, Germany)in distilled water], distilled water, formaldehyde solution [1:200 dilution of 37%formaldehyde (Merck, Germany) in distilled water] and pararosaline hydrochloride(PRA) (SigmaAldrich, Steinheim, Germany). The obtained solution (2.4 ml) wasmixed for 20 min at room temperature and the absorbance of the sulfite-PRA-form-aldehyde reaction product was measured spectrophotometrically at 560 nm (Shi-madzu UV-1601, Kyoto, Japan). The sulfite level in the dialysate was calculatedfrom a standard curve obtained by sulfite standards (27–206 lmol/ml) preparedby dissolving adequate amounts of Na2O5S2 (Merck, Darmstadt, Germany), in dis-tilled water. The results were expressed as lmol/ml.

2.5. TBARS assay

Levels of TBARS were measured by a fluorimetric method described by Was-owicz et al. (1993), using 1,1,3,3-tetraethoxypropane as a standard. Brain tissueswere weighed and homogenized (Bio-Gen Pro-200) in icecold 50 mM potassiumphosphate buffer at pH: 7. Homogenates were centrifuged (10,000g, 15 min, 4 �C)(Sigma 3–18 K centrifuge) and supernatants were used for lipid peroxidation anal-ysis. Supernatants (50 ll) were introduced into a tube containing 29 mmol/l thio-barbituric acid (TBA) in acetic acid (8.75 mol/l), samples were placed in a waterbath and heated for 1 h at 95–100 �C. After the samples were cooled, 25 ll of 5 MHCl was added and the reaction mixture was extracted by agitation for 5 min with3.5 ml n-butanol. After centrifugation, the butanol phase was separated and thefluorescence of the butanol extract was measured in a fluorescence plate reader(Biotek-synergy Mx) using wavelengths of 525 nm for excitation, and 547 nm foremission. The results are given as nmol/g protein.

2.6. sPLA2 Western blot analysis

Brain tissues were resuspended in lysis buffer (Invitrogen, Carlsbad, CA, USA)supplemented with protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO,USA). After sonication, samples were centrifuged at 14, 000 rpm for 20 min at4 �C. Supernatants were collected and stored at �80 �C. Protein concentrations weredetermined for each sample using the BCA protein assay kit according to theinstructions of the manufacturer. Equal amounts of protein were subjected to 15%SDS polyacrylamide gel and transferred onto nitrocellulose. The membranes wereblocked for 1 h with 5% w/v nonfat dry milk in TBS containing 0.1% Tween 20 (Sig-ma–Aldrich, St. Louis, MO, USA) at room temperature. Membranes were incubatedwith a 1:100 dilution of sPLA2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) over-night at 4 �C. After washing with TBS containing 0.1% Tween 20, membranes wereincubated with horseradish peroxidase conjugated donkey anti-goat IgG (SantaCruz Biotechnology, Inc., Santa Cruz, CA) in blocking solution for 2 h at room tem-perature. After three washings with TBS containing 0.1% Tween 20, the immunola-beling was visualized using the chemiluminescence-based SuperSignal CL-HRPSubstrate System (Pierce Chemical Co., Rockford, IL) according to the instructionsof the manufacturer. Membranes were exposed to Hyperfilm (Amersham Biosci-ences, Sweden), which was subsequently analyzed using an Alpha Digi Doc 1000gel documentation unit (Alpha Innotech Corporation, CA, USA).

2.7. Caspase-3 immunohistochemistry

Formalin-fixed tissue samples were embedded in paraffin and cut into 5 lmsections. 5 lm thick serial sections were collected on poly-L-lysine coated slides(Sigma–Aldrich, St. Louis, MO, USA) and incubated overnight at 56 �C. For cas-pase-3 immunohistochemistry, tissue sections were deparaffinized in xylene andrehydrated in a graded series of ethanol. Sections were then treated in a microwaveoven in 10 mM citrate buffer, pH 6.0, for 5 min twice and left to cool for 20 min.After three washes in phosphate buffered saline (PBS), sections were incubated ina universal blocking reagent (BioGenex, San Ramon, CA, USA) for 7 min at roomtemperature in order to block nonspecific binding. Subsequently, sections wereincubated overnight at 4 �C with anti-rabbit cleaved caspase-3 (Asp 175) (Cell Sig-naling # 9661) antibody (1/250). After several washes in PBS, sections were incu-bated with Alexa 488 donkey anti-rabbit secondary antibody (A21206; 1/400dilution, Invitrogen, Eugene, Oregon, USA) for 1 h and were rinsed with PBS. Thesections were mounted with Vectashield Mounting Medium for fluorescence withDAPI (H-1200; Vector Lab. Inc. Burlingame, CA, USA). Slides were examined usingOlympus BX61 fluorescence microscope. For controls, sections were treated withthe normal rabbit serum depending on the primary antibody used that was dilutedto the same final protein concentration as the primary antibody. All samples for theantibody was exposed to the same protocol and was stained using the same incu-bation periods of staining.

2.8. Apoptosis detection using terminal deoxynucleotidyl transferase-mediated dutpnick and labeling: tunel method

Briefly, paraffin sections of 5-lm thickness from brain tissues were cut and ta-ken onto slides covered with poly-L-lysine. Slides were washed twice in PBS for5 min. Following the incubation of slides with the permeabilisation solution (0.1%Triton X-100 in 0.1% sodium citrate) for 8 min at 4 �C and washing twice with

PBS for 5 min, the labeling reaction was performed using 50 ll TUNEL reagent foreach sample, except negative controls, in which reagent without enzyme was addedand incubated for 1 h at 37 �C. Following PBS washings, slides were mounted withVectashield Mounting Medium for fluorescence with DAPI (H-1200; Vector Lab. Inc.Burlingame, CA, USA). TUNEL was conducted using a Cell Death Detection kit(Roche, Cat No: 11684795910, Mannheim, Germany) and performed according tothe manufacturer’s instructions. Slides were examined using the Olympus BX61fluorescence microscope.

2.9. Determination of protein

Protein concentrations in brain tissues were spectrophotometrically measured(Shimadzu RF-5500, Kyoto, Japan) by a protein assay reagent kit (Pierce, Rockford,IL) via a modified Bradford method (Bradford, 1976). Bovine serum albumin wasused as a standard.

2.10. Statistical analysis

The statistical analyses of the obtained data were performed by SPSS (version18.0) software for windows. The results were expressed as mean standard devia-tion. The latencies and amplitude of SEPs were analyzed via one way analysis of var-iance (ANOVA) and all pairwise multiple comparisons were performed by Tukey’stest. The differences in biochemical parameters were analyzed via Kruskal–Wallis1-way analysis of variance on ranks, and all pairwise multiple comparisons wereperformed by Mann–Whitney U. P values less than 0.05 were considered significant.

3. Results

Characteristic waveforms of SEPs for all groups are presented inFig. 1. In all groups, two positive (P1, P2) and two negative (N1, N2)components were used for the analyses. The mean latencies of eachSEP component recorded from all experimental groups wereshown in Table 1. The latencies of P1, N1, P2 and N2 componentswere significantly prolonged in given sulfite rats compared withthe control group. However, prolonged latencies of SEP compo-nents in sulfite treated rats returned to control levels after quina-crine administration.

Table 2 shows the mean SEP amplitude changes of each groupover the course of the study. No significant difference was ob-served in the recorded amplitudes among the different experimen-tal groups.

Plasma-S-sulfonate levels (mean ± SD) in all groups are given inFig. 2. When compared to the control (14.9 ± 1.13 lmol/ml) group,plasma-S-sulfonate levels in S (21.5 ± 1.70 lmol/ml) and SQ(20.6 ± 1.36 lmol/ml) groups were significantly increased.

Sulfite treatment increased tissue TBARS levels (0.27 ± 0.05nmol/g protein) in the S group, compared with the control group(0.17 ± 0.01 nmol/g protein). The increase induced by sulfite wascompletely prevented by quinacrine in the SQ group (0.19 ± 0.02nmol/g protein) (Fig. 3).

Fig. 4A and B show Western blot and densitometric analysis ofbrain homogenates, respectively. Western blot analysis showedthat sPLA2 protein was increased in both S and SQ groups com-pared with control (Fig. 4A). The mean densitometric value ofsPLA2 protein was increased in the sulfite treated group comparedwith control (p < 0.05, Fig 4B). In contrast, densitometric value ofsPLA2 did not differ between the S and QS groups.

Immunohistochemistry showed that caspase-3 positive neu-rons were mainly located at somatosensory cortex (Fig. 5). Thoughseveral caspase-3 positive cells were observed in the somatosen-sory cortex in the S group (Fig. 5B and F), caspase-3 immuno-stained cells did not exist in C and SQ group tissues. (Fig. 5A, Eand C, G). No immunoreactivity was observed on the slides whereprimary antibody was replaced with normal rabbit IgG in the braintissues of all groups (Fig. 5D and H).

Nuclear condensation and fragmentation, which are classicalhallmarks of apoptosis, were detected by staining the nuclei withthe Texas red fluorescence TUNEL. No TUNEL-positive cells wereobserved in the C and SQ groups (Fig. 6A, D, G and C, F, I) howeverthere were numerous TUNEL-positive cells in the somatosensorial

Table 1The latencies (ms) of SEP components in the control and experimental groups.

132 C. Kencebay et al. / Food and Chemical Toxicology 52 (2013) 129–136

cortex of S group (Fig. 6 B, E, H). TUNEL analysis data are compat-ible with cleaved caspase-3 immunohistochemical assay results.

Groups P1 (ms) N1 (ms) P2 (ms) N2 (ms)

C (n = 10) 21.9 ± 1.44 41.9 ± 1.91 62.8 ± 3.48 81.09 ± 4.1S (n = 10) 27.7 ± 1.70* 53.6 ± 3.68* 81.1 ± 9.79* 99.7 ± 8.01*

SQ (n = 10) 23.3 ± 2.12# 41.75 ± 3.10# 59.77 ± 2.94# 79.77 ± 3.03#

* p < 0.001, vs. control group.# p < 0.001, vs. sulfite treated group.

Table 2The mean amplitudes of each SEP component were recorded from all groups (lV).control (C), sulfite (S) and sulfite + quinacrine (SQ).

Groups P1N1 (lV) N1P2 (lV) P2N2 (lV)

C (n = 6) 2.62 ± 0.66 3.41 ± 0.43 2.13 ± 0.59S (n = 8) 2.92 ± 0.47 3.69 ± 0.87 2.40 ± 0.37SQ (n = 7) 2.57 ± 0.54 3.09 ± 1.06 2.1 ± 0.5

4. Discussion

The dose of sulfite administered in this study was determined inreference to our previous study (Ozturk et al., 2011) which re-ported that Na2S2O5 leads to increased lipid peroxidation in thebrain tissue when given at a dose of 100 mg/kg/day, an equivalentof 67.3 mg/kg SO2 daily. Although World Health Organization(WHO) has established acceptable daily intake (ADI) level of sul-fites as 0.7 mg/kg/body weight (Nair and Elmore, 2003), Tayloret al. has shown that dose of 180–200 mg/body weight Na2S2O5

can be consumed via food and beverages in a single day (Tayloret al., 1986).

Plasma-S-sulfonate levels are commonly used as a biomarker ofingested sulfite (Gunnison and Palmes, 1973). Previous studieshave reported a correlation between the exposure to sulfite and/or SO2 and plasma-S-sulfonate levels (Gunnison and Jacobsen,1987; Ozturk et al., 2011). In consistency with the literature, ourresearch indicated a statistically significant accumulation of S-sul-fonate in the plasma of sulfite administered rats (Gunnison andPalmes, 1973; Kucukatay et al., 2005; Ozturk et al., 2011).

In humans, SEP recordings from hand and foot stimulationshave been used extensively to evaluate the corresponding afferentpathways (Desmedt and Cheron, 1980). Furthermore, SEPs are alsoused in experimental toxicology studies in order to investigate theconduction velocity of different stages of afferent pathway and thepostsynaptic response to the quantity of sensory input (Canu et al.,2003; Herr et al., 2007; Shapiro, 2002). Compared with the C group,all SEP components were prolonged in S group. Our finding, reveal-ing that the conduction velocity slows in sulfite treated rats,confirms the previous report (Kucukatay et al., 2003). The mecha-nisms, underlying abnormal SEP changes caused by exposure tosulfite remain unclear. Since sulfite may change the electroconduc-tive properties via excessive oxidative stress, apoptosis and/oractivity of PLA2 enzymes, quinacrine, a non-selective PLA2 inhibi-tor was used in our study. It is observed that prolonged latencies ofSEP components in sulfite treated rats returned to control levelsfollowing quinacrine administration. The relevant data suggest

Fig. 1. Characteristic somatosensorial evoked potentials of the three groups. Two positivand sulfite + quinacrine (SQ).

that PLA2 enzymes might be involved in the mechanisms by whichsulfite alters SEP components.

Brain TBARS level was increased in the S group with respect tothe C group. Likewise, several researches in the literature reportedthe fact that sulfite treatment increases lipid peroxidation (Bakeret al., 2002; Derin et al., 2009; Derin et al., 2006; Kaplan et al.,1975; Ozturk et al., 2011). Mechanisms of sulfite-induced lipid per-oxidation are thought to initially involve SO3

�2 oxidation into asulfite radical (SO3

+) (Baker et al., 2002; Fridovich and Handler,1959). Sulfite radicals can directly react with lipids resulting inthe formation of lipid peroxides. Although there is little informa-tion about the mechanism of sulfite toxicity in neurons, the toxic-ity could be attributed to the formation of sulfur and oxygencentered free radicals (Abedinzadeh, 2001). Our results indicatingthat sulfite exposure caused an increase in lipid peroxidation wasaccompanied by the changes in SEPs. Moreover, Gulati et al. havereported that enhanced lipid peroxidation may cause importantdecrease in myelin specific lipids and result hypomyelination(Gulati et al., 1987). Thus, prolongation of SEP latencies accompa-nied by lipid peroxidation might cause from hypomyelination.

This is the first study to report that PLA2 inhibition decreasesbrain lipid peroxidation, induced by sulfite ingestion. PLA2

e (P1, P2) and two negative (N1, N2) were seen in all groups. Control (C), sulfite (S)

Fig. 2. The mean ± standard deviation (SD) of plasma-S-sulfonate levels.⁄p < 0.05 vs. control group.

Fig. 3. TBARS levels of all groups. Bars represent the group means ± SD. (C: control; S: sulfite and SQ: sulfite and quinacrine) ⁄p < 0.001 vs. control, sulfite + quinacrine group.

Fig. 4. (A) Representative Western blots of sPLA2 and beta actin for control (C), sulfite (S), sulfite and quinacrine (SQ) groups. (B) Densitometric measurements for sPLA2/Betaactin are presented for control (C), sulfite (S), sulfite and quinacrine (SQ) groups. Data were normalized to beta-actin protein. The results represent the mean of at least threeseparate experiments. Values were expressed as mean ± SD.

C. Kencebay et al. / Food and Chemical Toxicology 52 (2013) 129–136 133

mediated lipid peroxidation originates from the release of arachi-donic acid from membrane stores and activation of prostaglandin

H synthase (PGH) (Smythies, 1999). Compatible with our data,Beck-Speier et al. have reported that sulfur/sulfite activates PLA2

Fig. 5. Representative fluorescence photomicrographs of cleaved caspase-3 in somatosensorial cortex of brain (A, B, C, D; magnification is 20�; E, F, G, H: magnification is 40�and inset: grayscale in 20�). Control (A, E); sulfite (B, F); SQ (C, G); negative control (D, H). Cleaved caspase-3 immunostaining was detected in somatosensorial area in sulfite(S) groups (B, F: arrows and also inset). There was not any immunolocalization of caspase-3 in SQ groups. In addition, control group was not expressed caspase-3immunoreaction. Negative-control section. Please note the absence of caspase-3 immunostaining in the negative control staining. Green: cleaved caspase-3 immunoreaction;blue: dapi. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

134 C. Kencebay et al. / Food and Chemical Toxicology 52 (2013) 129–136

enzymes in vitro. Yet, this result does not indicate which PLA2form is involved in the sulfite toxicity (Beck-Speier et al., 2003).Since sPLA2 is the most inflammatory form of PLA2 in the centraland peripheral nervous systems, sPLA2 level is investigated inbrain tissue.

The present study demonstrated that sPLA2 protein level wasincreased in the S and SQ groups compared with control in theWestern blot analysis. Our data imply that sPLA2 might be a medi-ator in sulfite toxicity in the brain. Although, the rats of SQ groupwere treated with quinacrine, sPLA2 level did not change withrespect to S group. Administration of quinacrine did not affect pro-tein levels of the enzyme however it is a well known inhibitor ofPLA2 activity (Koike et al., 1995). Thus the observed beneficial ef-fect of quinacrine on sulfite-induced toxicity is most likely due toinhibition of enzyme activity. sPLA2 mediated membrane lipiddegradation and the consequent lipoxidative metabolism havebeen documented as crucial toxic mechanisms (Farooqui andHorrocks, 2004; Lambeau and Gelb, 2008). Following the activationof sPLA2 arachidonic acid, inducing oxidative stress during its

metabolism and leading to either cell proliferation or apoptosis,is released (Farooqui et al., 1997; Toborek et al., 1999; Ventoet al., 2000).

Caspase-3 is an essential enzyme for normal brain develop-ment. On the other hand, it is the main terminal peptidase playinga critical role in apoptosis (Teschendorf et al., 2008). Additionally,caspase-3 is required for some typical hallmarks of apoptosis suchas, chromatin condensation and DNA fragmentation in all celltypes (Porter and Janicke, 1999). In present study, we showed thatcleaved caspase-3 positive neurons and TUNEL-positive cells weremainly located at somatosensory cortex in the S group. These re-sults are also in agreement with the previous studies (Bai andMeng, 2010; Ercan et al., 2010). Bai and Meng have shown thatboth mRNA levels and numbers of caspase-3, capase-8 and cas-pase-9 in both liver and lung tissues increased after SO2 inhalation(Bai and Meng, 2010). Moreover, Ercan et al. have reported thatintragastric administration of 100 mg/kg sulfite elevated the num-ber of apoptotic cells both in mucosa and submucosa layers of thestomach (Ercan et al., 2010). In consistency with the reports

Fig. 6. Apoptosis were detected by staining the nuclei with the Texas red fluorescent TUNEL. Red is TUNEL reaction (A, B, C), blue is DAPI staining (D, E, F) and also combinedpicture (G, H, I). A, D, G: the somatosensorial cortex of control group. B, E, H: examples of apoptotic cells (arrows) in the somatosensorial cortex of sulfite treated rats, C, F, I:the somatosensorial cortex of SQ group. Magnification is 40�. (For interpretation of the references to color in this figure legend, the reader is referred to the web version ofthis article.)

C. Kencebay et al. / Food and Chemical Toxicology 52 (2013) 129–136 135

implying the neuroprotective effects of PLA2 inhibitors on apopto-sis, our results suggests that sulfite-induced apoptosis is preventedby a non-selective PLA2 inhibitor (quinacrine) (Yagami et al., 2002;Zhao et al., 2002). Hydrolysis of phospholipids by PLA2 generatesarachidonic acid and lysophospholipids that are important mem-bers of the lipid signaling molecules (Zhao et al., 2002). Garridoet al. have reported that exposure of cultured spinal cord neuronsto arachidonic acid can induce apoptosis through the pathwayswhich involve activation of caspase cascade and the release ofcytochrome c from mitochondria into the cytoplasm (Garridoet al., 2001). Disruption of the arachidonic acid pathway byquinacrine via inhibition of PLA2, might lead to prevention of sul-fite-induced apoptosis.

5. Conclusion

This is the first study demonstrating that ingested-sulfite in-duced SEP alterations, increased lipid peroxidation, apoptotic celldeath and DNA damage in the brain are prevented by quinacrinetreatment. Hence, sPLA2 might play a role in sulfite mediated tox-icity in the brain.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by a grant from the ResearchFoundation of Akdeniz University, Turkey (Project number:2009.02.0122.001). This study was carried out as a part of MSc thesisby C. Kencebay presented to Akdeniz University Health SciencesInstitute.

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