Toxicology in Vitro 26 (2012) 823–830

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Toxicology in Vitro

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Cytotoxicity induced by deltamethrin and its metabolites in SH-SY5Y cellscan be differentially prevented by selected antioxidants

Alejandro Romero ⇑, Eva Ramos, Víctor Castellano, María Aranzazu Martínez, Irma Ares,Marta Martínez, María Rosa Martínez-Larrañaga, Arturo AnadónDepartment of Toxicology and Pharmacology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain

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

Article history:Received 21 October 2011Accepted 14 May 2012Available online 23 May 2012

Keywords:CytotoxicityDeltamethrinMetabolitesSH-SY5Y cellsAntioxidants

0887-2333/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.tiv.2012.05.004

Abbreviations: 3-PBA, 3-Phenoybenzoic acid; NO,cysteine; MEL, melatonin; MDA, malondialdehyde;reactive substance; DMSO, dimethylsulfoxide; SDS, soEagles minimum essential medium; FBS, fetal bovineylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide;2,5,7,8-tetramethylchromane-2-carboxylic acid.⇑ Corresponding author. Address: Departamento de

Facultad de Veterinaria, Universidad ComplutenseHierro s/n, 28040 Madrid, Spain. Tel.: +34 913943834

E-mail address: aromero@vet.ucm.es (A. Romero).

Deltamethrin, an a-cyano pyrethroid insecticide, is a relatively potent neurotoxicant. The main delta-methrin metabolism mechanisms are ester cleavage and oxidation at the 20 and 40 position of the termi-nal aromatic ring. Although some aspects of the toxicity properties of deltamethrin have been reported,limited information is available about the metabolites cytotoxic actions. The aims of this study are toexamine in vitro neurotoxicity of deltamethrin and its metabolites 3-phenoxybenzoic acid (3-PBA), 20-OH-deltamethrin, and 40-OH-deltamethrin and to evaluate melatonin (0.1, 1 lM), trolox (0.3, 1 lM)and N-acetylcysteine (500, 1000 lM) protective role in SH-SY5Y cells. MTT and neutral red uptake(NRU) assays were carried out to assess the cytotoxicity of deltamethrin and its metabolites. Of the threemetabolites tested, while 3-PBA (0.01–1000 lM) did not show neurotoxicity, 20-OH- and 40-OH-delta-methrin (10–1000 lM) were more toxic than deltamethrin (10–1000 lM). Levels of both nitric oxide(NO) and lipid peroxides measured as malondialdehyde were significantly increased in deltamethrinand 40-OH-deltamethrin-treated cells. Compared to other antioxidants, 1 lM MEL treatment effectivelyprotected against deltamethrin and 40-OH-deltamethrin-induced lipid peroxidation and amelioratedthe NO adverse effect that might have been caused. These results suggest that oxidative stress observedis one of the major mechanisms of deltamethrin-induced neurotoxicity and it may be attributed in part todeltamethrin disposition and metabolism.

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1. Introduction

Deltamethrin, a type II pyrethroid insecticide, is considered asthe most potent neurotoxic pyrethroid (Ruzo and Casida, 1977;Pham et al., 1984). Most pyrethroid insecticides exist as at leasttwo isomers, unlike deltamethrin is marketed as a single isomer(cis) (Elliott et al., 1974) and it is widely used in veterinary prod-ucts as well as in agricultural formulations. The selective neurotox-icity of deltamethrin is attributed to its effect on voltage sensitivesodium channels (VSSCs) (Shafer et al., 2005) and to its interactionwith the GABA receptor–ionophore complex (Lawrence and Casida,

ll rights reserved.

nitric oxide; NAC, N-acetyl-TBARS, thiobarbituric acid

dium dodecyl sulfate; EMEM,serum; MTT, 3-[4,5 dimeth-TROLOX, [(±)-6-hydroxy-

Toxicología y Farmacología,de Madrid, Avda. Puerta de; fax: +34 913943840.

1983). Although initially it was thought to have a low toxicity, anumber of reports demonstrated deltamethrin toxicity in mamma-lian and nonmammalian laboratory and wildlife animal species(Vais et al., 2001; Das et al., 2001; Bradberry et al., 2005). Delta-methrin is readily absorbed by the oral route (Anadón et al.,1996), therefore, the main sources of exposure to this pesticidemight be contaminated food and water.

Previous studies of deltamethrin metabolism in rats revealedthat the main mechanisms of metabolism are ester cleavage andoxidation at the 20 and 40 position of the terminal aromatic ring(Ruzo et al., 1978; Rickard and Brodie, 1985; Anand et al., 2006)(Fig. 1). Esterases catalyze hydrolysis of the ester bond to form rel-atively non-toxic acid [metabolite 3-phenoxybenzoic acid (3-PBA)]and alcohol moieties, whereas CYP450s catalyze aromatic hydrox-ylation of deltamethrin, followed by conjugation (Ruzo et al., 1978;Soderlund and Casida, 1977). The parent compound is consideredto be the primary neurotoxicant (Rickard and Brodie, 1985), butthe toxicity of the main oxidative metabolites, 20-OH-deltamethrinand 40-OH-deltamethrin it has not been evaluated so far (Anadónet al., 1996; Dayal et al., 2003; Gray and Rickard, 1982).

Free radicals are produced in cells through normal metabolicprocesses. An increased level of free radicals can lead to the

Fig. 1. Chemical structure of deltamethrin and its main metabolites. Deltamethrin is metabolized by CYP450-mediated oxidation (dotted arrows) and esterase-mediatedhydrolysis (solid arrow).

824 A. Romero et al. / Toxicology in Vitro 26 (2012) 823–830

damage of macromolecules within the cell; it is this damage to lip-ids, proteins, and DNA which can rise to pathological conse-quences. Oxidative stress occurs when the balance of oxidantexceed antioxidant levels present in the cell. During the rapidmetabolism of pyrethroids, free radicals should be generated. Formany pesticides, induction of oxidative stress is one of the mainmechanisms of their toxic action (Banerjee et al., 2001). Recent re-ports have demonstrated the induction of oxidative stress by pyre-throids such as cypermethrin, lambda-cyhalothrin and beta-cyfluthrin (Giray et al., 2001; El-Demerdash, 2007; El-Demerdashet al., 2003; Sadowska-Woda et al., 2010), but there is not dataon the cytotoxic effects of deltamethrin and its oxidativemetabolites.

Human dopaminergic neuroblastoma SH-SY5Y cells possessmany biochemical and functional properties of neurons and havebeen widely used as model of neurons. Cell line SH-SY5Y is a cellu-lar model suitable for exploring the neurotoxic potential of pesti-cides. Dopaminergic cell line SH-SY5Y may be a preferentialtarget of pesticides because of their vulnerability to reactive oxy-gen species-mediated oxidative injury (Bonneh-Barkay et al.,2005). Compared to other neuronal cells, dopaminergic cells aremore sensitive to oxidative injury (Dinis-Oliveira et al., 2006;Romero et al., 2010). The SH-SY5Y neuroblastoma cell line hasbeen widely used in experimental neurological studies, includinganalysis of neuronal differentiation, metabolism, and function re-lated to neurodegenerative and neuroadaptive processes, neuro-toxicity, and neuroprotection (Rios et al., 2003; Skandrani et al.,2006).

Antioxidants are needed to prevent the formation and to revertreactive oxygen and nitrogen species actions, which are generatedin vivo and cause damage to DNA, lipids, proteins, and other bio-molecules (Trushina and McMurray, 2007). They act as free radicalscavengers and slow down not only radical oxidation but also theassociated damage effects in the body. To test a possible protectionagainst oxidative injury in culture cells, the compounds used are

MEL, a potent free radical scavenger and neuroprotective drug(Rodriguez et al., 2004), trolox, a cell-permeative analog of vitaminE, which inhibits reactive oxygen species-induced generation of li-pid peroxyl radicals (Cort et al., 1975) and N-acetyl-cysteine (NAC),a free radical scavenger that acts as a cysteine donor and maintainsor even increases the intracellular levels of glutathione (Li et al.,2007). The objectives of the present study were to characterizethe concentration-dependent cytotoxicity of deltamethrin and itsmetabolites 3-PBA, 20-OH-deltamethrin and 4’-OH-deltamethrin,as well as to determine the different protective roles of the selectedantioxidants, MEL, trolox and NAC on lipid peroxidation in humanneuroblastoma SH-SY5Y cell line.

2. Materials and methods

2.1. Chemicals and reagents

Analytical standards of deltamethrin [(S)-alpha-cyano-3-phen-oxybenzyl-(1R, cis)-2,2-dimethyl-3-(2,2-dibromovinyl)-cyclopro-panecarboxylate] and its metabolites, 20-OH-deltamethrin, 40-OH-deltamethrin and 3-PBA (P98%) were provided by Roussel Uclaf(Romainville, France). The compounds 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), Neutral Red solution,Melatonin (N-acetyl-5-methoxytryptamine) (MEL), N-acetyl-cys-teine (NAC), trolox [(±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid], malondialdehyde tetrabutylammonium salt(MDA) and F-12 Nutrient Mixture were obtained from Sigma (StLouis, MO, USA). Fetal bovine serum (FBS), penicillin, and strepto-mycin were obtained from Invitrogen (Madrid, Spain). The otherchemicals used were of analytical grade.

2.2. Culture of SH-SY5Y cells

Human dopaminergic neuroblastoma SH-SY5Y cells were main-tained in a 1:1 mixture of Nutrient Mixture F-12 and Eagles

A. Romero et al. / Toxicology in Vitro 26 (2012) 823–830 825

minimum essential medium (EMEM) supplemented with 15 non-essential amino acids, 1 mM sodium pyruvate, 10% heat-inacti-vated FBS, 100 units/ml penicillin, and 100 lg/ml streptomycin.Cultures were seeded into flasks containing supplemented med-ium and maintained at 37 �C in a humidified atmosphere of 5%CO2 and 95% air. For assays, SH-SY5Y cells were subcultured in48-well plates at a seeding density of 1 � 105 cells per well. Cellswere treated with the drugs before confluence in F-12/EMEM with1% FBS. A vehicle group containing 0.1% DMSO was employed inparallel for each experiment. All SH-SY5Y cells used in this studywere used at a low passage number (<13).

2.3. MTT assay and cell viability

Cell viability, virtually the mitochondrial activity of living cells,was measured by quantitative colorimetric assay with MTT, as de-scribed previously (Denizot and Lang, 1986). Briefly, 50 ll of theMTT labeling reagent, at a final concentration of 0.5 mg/ml, wasadded to each well at the end of the incubation period and theplate was placed in a humidified incubator at 37 �C with 5% CO2

and 95% air (v/v) for an additional 2 h period. Metabolically activecells convert the yellow MTT tetrazolium compound to a purpleformazan product. Then, the insoluble formazan was dissolvedwith dimethylsulfoxide; colorimetric determination of MTT reduc-tion was measured at 540 nm. Control cells treated with F-12/EMEM were taken as 100% viability.

2.4. Neutral red uptake assay

Neutral red uptake (NRU) assay was performed following theprocedure as described by Borenfreund and Puerner (1984). Briefly,cells were seeded (80,000 cells/well) on 96-well plates, grown for24 h prior to experiments, and then exposed 1 lM of melatoninfor 24 h. After removing the medium, cells were exposed to10 lM of deltamethrin and 4’-OH-deltamethrin for 24 h. Cells werewashed with 1� PBS twice and incubated for 3 h in medium sup-plemented with neutral red (50 lg/ml). Cells were washed with1� PBS followed by the addition of a solution of 1% glacial aceticacid in 50% ethanol and 49% deionized water (neutral red destainsolution). Plates were shaken and the absorbance was measuredat 540 nm in a plate reader (Biochrom ASYS UVM 340, Cambridge,UK).

2.5. Determination of lipid peroxidation

MDA is a breakdown product of the oxidative degradation ofcell membrane lipids and it is generally considered an indicatorof lipid peroxidation. We evaluated lipid peroxidation induced bydeltamethrin (10 lM) and 40-OH-deltamethrin (10 lM) with orwithout MEL (1 lM) for an 8 h incubation period. We selected8 h co-incubation because at this time the maximum MDA levelswithout necrosis were found (data not shown). Intracellular MDAproduction was quantified using a thiobarbituric acid reactive sub-stance (TBARS) assay kit (Cell Biolabs Inc., San Diego, CA). Briefly,1 � 106 cells per well were seeded in a six-well plate, then col-lected in 200 ll of culture medium and sonicated for 3 � 5 s inter-vals at 40 V over ice. SDS Lysis solution (100 ll) was added to thesample solution and the MDA standards in a microcentrifuge tubeand mix thoroughly. Then, 250 ll of TBA reagent were added toeach sample and standard to be tested, and incubate at 95 �C for45–60 min. Each sample and standard (200 ll) were loaded (induplicate) into a clear 96-well plate and the absorbance at532 nm was recorded using a microplate reader (Biochrom ASYSUVM 340, Cambridge, UK). The content of MDA was calculatedfor each sample from a standard curve.

2.6. Nitrite measurement

Changes in NO production were measured indirectly as theaccumulation of nitrites (the end-product of NO metabolism) inthe medium using Griess assay as previously described (Baucheet al., 1998). Neuroblastoma SH-SY5Y cells were incubated withdeltamethrin (10 lM) and 40-OH-deltamethrin (10 lM) for 24 hwith or without MEL (1 lM) pre-incubation for 24 h. Briefly,100 ll of the culture supernatant reacted with 100 ll Griess re-agent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydro-chloride and 2–5% H3PO4) for 10 min at room temperature. Theconcentration of nitrite was measured by spectrophotometry (Bio-chrom ASYS UVM 340, Cambridge, UK) at 540 nm, and the nitriteconcentration was calculated using a standard curve of sodiumnitrite.

2.7. Statistical analysis

Data are represented as means ± standard error of the mean(SEM). Comparisons between experimental and control groupswere performed by one-way ANOVA followed by the Newman–Keuls post hoc test. Statistical difference was accepted whenp 6 0.05. The concentration of deltamethrin or metabolites atwhich 50% of cells in culture die after treatment for a period oftime is termed as inhibitory concentration of viability at 50%(IC50) value compared to untreated controls. IC50 values were cal-culated by concentration–response (Sigmoidal fitting) with Origin-Pro 7.5 software.

3. Results

3.1. Effect of deltamethrin and its metabolites on SH-SY5Y cell viability

We used the MTT test to evaluate cell survival as a function ofmitochondrial viability. Fig. 2 shows the protocol used to evokecytotoxicity. A 24 h incubation period with deltamethrin, 20-OH-and 40-OH-deltamethrin at increasing concentrations (0.01–1000 lM) reduced cell viability in a concentration-dependentmanner compared with vehicle-treated cells (control negative),as shown in Fig. 2(A–C), whereas 3-PBA, did not change cell viabil-ity respect to vehicle-treated cells. Also, there was no noteworthydifference between data of vehicle-treated cells and control cells.At higher doses of treatment (10–1000 lM), deltamethrin and itsmetabolites 20-OH- and 40-OH-deltamethrin induced statisticallysignificant decreases of cell viability, being the metabolites 20-OH- and 40-OH-deltamethrin more potent neurotoxicants thanthe parental compound. In the concentration-dependent curvesshown in Fig. 2(A–C), the IC50 value for deltamethrin was760 lM and for the metabolites 20-OH- and 40-OH-deltamethrin18.6 and 3.85 lM respectively. Therefore, to study deltamethrinand 20-OH- and 40-OH-deltamethrin metabolites induced cytotox-icity, the concentrations used were 10 and 100 lM.

3.2. Effects on SH-SY5Y cell viability of deltamethrin and itsmetabolites after pre-treatment with antioxidants

The antioxidants MEL, NAC and trolox exhibit differences pre-venting decreases in cell viability caused by deltamethrin, 20-OH-and 40-OH-deltamethrin at doses of 10 and 100 lM. After a 24 hpre-incubation period with MEL (0.1 and 1 lM), trolox (0.3 and1 lM) and NAC (500 and 1000 lM), deltamethrin, 20-OH- or40-OH-deltamethrin were added (see protocol in Fig. 3). Only thetrolox treatment did not exert any effect on cytotoxicity inducedby deltamethrin or its metabolites. The effects of MEL and NACwere concentration dependent. The pre-treatment with MEL

Fig. 2. Cytotoxicity of deltamethrin or metabolites after 24 h exposure determined by MTT assay. Experiments were run in parallel as the protocol describes. Figure illustratea concentration–response curve of deltamethrin (A), 20-OH-deltamethrin (B), 40-OH-deltamethrin (C) and 3-PBA (D) on SH-SY5Y cells. Cell viability was measured as MTTreduction (ordinate) and data were normalized as% control (C, white column). Cells treated with DMSO (0.1%) were the negative control (Veh, black column). Data representthe mean ± SEM of five independent experiments in triplicate. ⁄p < 0.05 and ⁄⁄⁄p < 0.001 compared to vehicle.

826 A. Romero et al. / Toxicology in Vitro 26 (2012) 823–830

(1 lM but not 0.1 lM) abolished the neurotoxic effect of delta-methrin and its metabolites 20-OH- and 40-OH-deltamethrin (10and 100 lM) on cell viability compared to vehicle group. We alsofound a significant protection in groups previously treated withNAC (1000 lM but not 500 lM) and incubated with deltamethrin(100 lM) (Fig. 3D) and 20-OH-deltamethrin (10 lM) but not with40-OH-deltamethrin (Fig. 3B) respect to vehicle group. These re-sults demonstrated that MEL pre-treatment (1 lM), effectivelyprotected against cell neurotoxicity induced by deltamethrin andits metabolites 20-OH- and 40-OH-deltamethrin.

Because of MTT assay represents the mitochondrial metabo-lism, in order to confirm the observed MEL neuroprotection, theNRU assay, a cell viability test based on the lysosomal capacitywas performed. The pre-treatment with MEL (1 lM) also reducedthe neurotoxic effect of deltamethrin and its metabolite 40-OH-del-tamethrin (10 lM) on cell viability compared to vehicle group(Fig. 4).

3.3. Lipid peroxidation in deltamethrin and 40-OH-deltamethrin-exposed SH-SY5Y cells. Determination of MDA levels

MDA is one of the most important intermediates produced dur-ing lipid peroxidation. Panel (Fig. 5) shows the protocol used toevoke lipid peroxidation, 8 h co-incubation with deltamethrin

(10 lM) or 40-OH-deltamethrin (10 lM) and neuroprotection withMEL (1 lM). Since deltamethrin and its metabolite can be dis-solved in DMSO easily but not in water, the aqueous solution ofthis compound with 0.1% DMSO was examined in the experiment.The results showed that DMSO itself at this concentration did notcause changes in the MDA production respect to vehicle group.Incubation with deltamethrin (Fig. 5A) and 40-OH-deltamethrin(Fig. 5B) for 8 h induced a significant increase in MDA levels (3-foldabove basal and 3.5-fold above basal, respectively) compared tovehicle group. MEL (1 lM) provided a significant decrease ofMDA levels induced by deltamethrin and 4’-OH-deltamethrin.

3.4. Deltamethrin and 40-OH-deltamethrin-stimulated SH-SY5Y cellsproduce NO

A marked increase in NO is detrimental of many pathologicalconditions; furthermore excessive NO can stimulate a neurotoxiccascade. As illustrated in Fig. 6, cells were exposed to deltamethrinand 40-OH-deltamethrin for 24 h. At 10 lM, both deltamethrin and40-OH-deltamethrin increased by 3.5- and by 5-fold NO productionin neuroblastoma SH-SY5Y cells (Fig. 6A and B), respectively. Aftera 24 h pre-incubation period with MEL, NO production was signif-icantly reduced.

Fig. 3. Cytoprotection of Trolox, NAC and MEL after 24 h pre-incubation period against cell death elicited by 10 and 100 lM deltamethrin (A and D), 20-OH-deltamethrin (Band E) and 40-OH-deltamethrin (C and F). The protocol used to evoke cytotoxicity on neuroblastoma cells SH-SY5Y is described in Figure. Data represent the mean ± SEM offive independent experiments in triplicate. ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to vehicle; &p < 0.05; &&p < 0.01 and &&&p < 0.001 compared to deltamethrin or metabolites inthe absence of antioxidants.

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4. Discussion

Central to this study is the finding that deltamethrin and themetabolites 20-OH- and 40-OH-deltamethrin afford cytotoxicity onSH-SY5Y cells in a concentration-dependent manner. According tothe available data the expected neurotoxicity of deltamethrinshould be higher than the metabolites, since the metabolites are ex-pected to be relatively non-toxic. In the present in vitro study, it isdemonstrated that the actions of deltamethrin metabolites, 20-OH-and 40-OH-deltamethrin, may be more cytotoxic than the parentalcompound deltamethrin inducing death of vulnerable neurons.

Although the biological activity of deltamethrin is certainly de-stroyed by ester hydrolysis, which represents a major detoxifica-tion route (Anand et al., 2006), recent research has raised otherpossibilities for its oxidative metabolites (Anadón et al., 1996; Day-al et al., 1999, 2003). Unfortunately, studies of the toxicity ofmetabolites seem to have been restricted to those produced afterester hydrolysis (Gaughan et al., 1976).

Limited information is available on disposition of pyrethroids(Anadón et al., 1991, 1996, 2006; Godin et al., 2010). Deltamethrinis rapidly absorbed when administered orally or intraperitoneallyand enters the central and peripheral nervous systems (Anadónet al., 1996; Rickard and Brodie, 1985). After oral administrationof deltamethrin in rats, the major metabolite identified in plasmaand nervous tissues was 40-OH-deltamethrin, which is excretedover a period of days (Anadón et al., 1996). Previously, it was de-scribed that CYP450s catalyze aromatic hydroxylation of delta-methrin at various positions, notably the 40 position (Ruzo et al.,1978, 1979).

A common assumption of in vitro studies is that medium con-centration is a predictive marker of tissue concentration. Recent

studies have demonstrated that lipophilic compounds rapidlyaccumulate in cells culture to concentrations much higher thanin the surrounding media, often by two orders of magnitude (Mun-day et al., 2004; Meacham et al., 2005). Concentrations of pyre-throids in the cells, rat cortical neurons, quickly exceed theconcentration of the compound in the medium (Shafer andHughes, 2010). In light of high lipophilicity of the deltamethrin,20-OH-deltamethrin and 40-OH-deltamethrin, they may accumu-late rapidly in SH-SY5Y cells in a concentration-dependent mannerto levels that exceed the concentration in the media much higherthan one or two orders of magnitude. This may explain in partthe observation that accumulation of deltamethrin and its metab-olites afford a cytotoxic effect on human neuroblastoma cells. Inthe present study, the observed cytotoxic concentrations (IC50)of 40-OH-deltamethrin, 3.85 lM, and of deltamethrin, 760 lM,are correlated with the in vivo maximal plasma and nervous tissueconcentrations observed in a previous toxicokinetic study (Anadónet al., 1996).

We evidenced that the cytotoxicity induced by 4’-OH-delta-methrin was 200 times higher than the parental compound. Thepossibility that deltamethrin neurotoxicity may act via metabolitesdeserves to be investigated further, especially given the potentialfor metabolism within the brain (Anadón et al., 1996). Certainly,the metabolites generated from the primary and secondary metab-olism of deltamethrin (Kaneko and Miyamoto, 2001) and the exis-tence of CYP450 activity within the brain (Dayal et al., 2003), couldpoint to the in situ production of 2’-OH and 4’-OH-deltamethrin.

A recent report showed that deltamethrin induced a dose-dependent reduction of cell viability in cultured hippocampal neu-rons (Grosse et al., 2002) and was also found to induce apoptoticcell death in rat brain, suggesting an important role played by

Fig. 4. Cytoprotection of MEL after 24 h pre-incubation period against cell deathelicited by 10 lM deltamethrin (A) and 40-OH-deltamethrin (B) measured byneutral red uptake assay. The protocol used to evoke cytotoxicity on neuroblastomacells SH-SY5Y is described in Figure. Cell viability was measured as neutral reduptake (ordinate) and data were normalized as% control (C, white column). Cellstreated with DMSO (0.1%) were the negative control (Veh, black column). Datarepresent the mean ± SEM of four independent experiments in sextuplicated.⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared to vehicle; &p < 0.05; &&p < 0.01 compared todeltamethrin or metabolite in the absence of melatonin.

Fig. 5. MDA production in SH-SY5Y cells induced by 10 lM deltamethrin (A) or 40-OH-deltamethrin (B) co-incubated with or without MEL (1 lM) for 8 h (as theprotocol describes). Data represent the mean ± SEM of six independent experimentsin triplicate. ⁄⁄⁄p < 0.001 compared to vehicle; &&p < 0.01 and &&&p < 0.001 com-pared deltamethrin or 40-OH-deltamethrin in the absence of MEL.

828 A. Romero et al. / Toxicology in Vitro 26 (2012) 823–830

apoptosis in neurotoxicity of deltamethrin (Wu et al., 2003). It hasbeen suggested that deltamethrin causes perturbations in lipid–lipid and lipid–protein interactions, interferes in transport mecha-nisms operating at the membrane level and causes alterations ofmembrane permeability and mitochondrial enzyme activities,effects which would also contribute to the neurotoxic action of del-tamethrin (Braguini et al., 2004). In the present study, three knownantioxidants, MEL, trolox and NAC were compared for their abilityto reduce deltamethrin, 20-OH-deltamethrin and 40-OH-deltameth-rin-induced oxidative damage. Compared to the other antioxi-dants, 1 lM MEL appeared to maintain better the cell viabilityfor doses of 10 and 100 lM deltamethrin and 40-OH-deltamethrin.The lack of protective effect of Trolox and NAC compared to MELmight be due to the fact that the antioxidant capacity of MEL alsoincludes the indirect effect of up-regulating several antioxidativeenzymes and down-regulating pro-oxidant enzymes (Pandi-Peru-mal et al., 2006). Furthermore, some MEL effects are mediated bymembrane receptors MT1 and MT2 (Pandi-Perumal et al., 2006).SH-SY5Y cell line was selected for this study, because it expressesMT1 receptor (Mcmillan et al., 2007). The MEL neuroprotective ef-fect was observed in both MTT and RNU assays indicating that themechanism of protection might involve a stabilization or improve-ment in mitochondrial or lysosomal functions. Our findings sug-gest that MEL should be neuroprotective in vivo and it may have

a useful therapeutic window for deltamethrin toxicity treatment.Further studies are necessary to confirm this hypothesis.

Oxidative stress mediated toxicity has for long been consideredas the mechanism responsible for deltamethrin-induced injury(Yousef et al., 2006; Li et al., 2011). Levels of both NO and lipid per-oxides measured as MDA, which can lead to a loss of membranestructure and function, were found to be significantly increasedin deltamethrin and 40-OH-deltamethrin treated cells (Figs. 5 and6). Similar observations, have been reported in different modelsof neuronal death in vivo (El-Gohary et al., 1999) and in vitro(Wu et al., 2003).

Due its antioxidant or free radical scavenging activity, MEL hasbeen reported to exert neuroprotection against oxidative stress inSH-SY5Y neuroblastoma cells (Romero et al., 2010). Our resultsdemonstrated that 1 lM MEL significantly reversed the effects ofdeltamethrin and 40-OH-deltamethrin on MDA and NO production,and therefore MEL inhibited lipid peroxidation by scavenging morereactive species, which initiates the degradation process.

In conclusion, this study is the first to report that metabolites20-OH-deltamethrin and 40-OH-deltamethrin are more cytotoxicthan the parental compound, deltamethrin. The mechanism ofsuch pathological facts may be prompted by the free radical releaseand the lipid peroxidation that they induce. The use of MEL wasascertained to reduce the harmful effects of deltamethrin and40-OH-deltamethrin in the mentioned toxicity mechanisms. In vitro

Fig. 6. NO production in SH-SY5Y cells pre-incubated 24 h with or without MEL(1 lM) followed by a 24 h treatment period with deltamethrin (10 lM) (A) or 40-OH-deltamethrin (10 lM) (B), as the protocol shows. After the supernatant wascollected, we determined NO production using Griess reagent as described in thematerials and methods. Data represent the mean ± SEM of seven independentexperiments in triplicate. ⁄⁄⁄p < 0.001 compared to control; &p < 0.05 and&&&p < 0.001 compared to deltamethrin or 40-OH-deltamethrin in the absence ofMEL.

A. Romero et al. / Toxicology in Vitro 26 (2012) 823–830 829

cultures of neuroblastoma cells SH-SY5Y have proven to be animportant tool to predict and characterize mechanisms of neuro-toxicity of deltamethrin. The results of this study will be usefulfor making comparisons between in vivo and in vitro studiesregarding effective concentrations of deltamethrin and its metabo-lites 2’-OH-deltamethrin and 4’-OH-deltamethrin. Moreover, ourdata advance the understanding of the deltamethrin oxidativemetabolite toxicity.

Conflicts of interest statement

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the Universidad Complutense deMadrid, Comunidad de Madrid and Ministerio de Educación yCiencia, Projects Refs. GR35/10-A UCM-BSCH, S2009/AGR-1469and Consolider-Ingenio CSD/2007/00063 (FUN-C-FOOD), Madrid,Spain.

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