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Chemico-Biological Interactions 182 (2009) 245–252

Contents lists available at ScienceDirect

Chemico-Biological Interactions

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ffect of permethrin insecticide on rat polymorphonuclear neutrophils

osita Gabbianelli a,∗, Maria Letizia Falcionib, Cinzia Nasuti c, Franco Cantalamessac,suke Imadad, Masayasu Inoued

Department of M.C.A. Biology, University of Camerino, Via Gentile III da Varano, I-62032 Camerino (MC), ItalySchool of Advanced Studies “Ageing and Nutrition”, University of Camerino, ItalyDepartment of Experimental Medicine and Public Health, University of Camerino, ItalyDepartment of Biochemistry and Molecular Pathology, Osaka City University Medical School, Japan

r t i c l e i n f o

rticle history:eceived 29 July 2009eceived in revised form0 September 2009ccepted 11 September 2009vailable online 20 September 2009

eywords:olymorphonuclear neutrophilsyperactivationyrethroidlasma membrane fluidityntioxidants

a b s t r a c t

Polymorphonuclear neutrophils are professional phagocytes whose efficacy depends on a multicompo-nent NADPH oxidase for generating superoxide anions and bacterial killing. They can be primed andactivated by different agents that can impair oxidative burst and phagocytosis with opposite effects:reduced capability to destroy bacteria or hyperactivation that induces the generation of large quantitiesof toxic reactive oxygen species, which can damage surrounding tissue and participate in inflammation.

The present study was designed to evaluate the effect of sub-chronic (60 days) permethrin treat-ment (1/10 DL50) on rat polymorphonuclear neutrophils respiratory burst. The results show thatpermethrin treatment increases superoxide anion production (33 times) and the activity of hydrogenperoxide–myeloperoxidase system (67 times). In vitro experiments suggest that this effect can be relatedto permethrin priming and to physico-chemical changes at the plasma membrane level of neutrophils.The antioxidant supplementation with Vitamin E and coenzyme Q10 can protect against the abnormal

rimingrespiratory burst in rat treated with permethrin.

The in vitro studies show that neutrophil apoptosis begins soon after 1 h of incubation with perme-thrin (0.725% of total cells) or its metabolites (3-phenoxybenzyl alcohol, 3-phenoxybenzaldehyde and3-phenoxybenzoic acid 1.36, 2.26 and 1.3 of total cells, respectively) and that the level of apoptotic cellsis very low.

In conclusion, immunotoxicity of permethrin measured in rats could prompt future studies on theconsequences of chronic insecticide exposure.

. Introduction

Polymorphonuclear neutrophils (PMNs) are the most abun-ant type of white blood cells and form an integral part ofhe human immune system. Neutrophils are the major effec-or cells of innate immunity [1–3]. Because of their powerful

icrobicidal equipment, they play a major role in the immunityefence [3]. PMNs are equipped with several antimicrobial sys-ems that allow them to combat a broad spectrum of bacteria,ungi and protozoa. Neutrophils migrate to inflammatory sitesefore monocytes and possess a more elaborate bactericidal poten-

ial compared to monocytes [4]. For this reason PMNs are therst line of defence against pathogens. PMNs have a short lifes-an because after phagocytosis they undergo apoptosis and theyave to be eliminated from the inflammatory site to avoid tissue

∗ Corresponding author. Tel.: +39 0737 403208; fax: +39 0737 403290.E-mail address: rosita.gabbianelli@unicam.it (R. Gabbianelli).

009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2009.09.006

© 2009 Elsevier Ireland Ltd. All rights reserved.

damage. This anti-inflammatory disposal of PMNs is achieved bymacrophages. Therefore apoptosis in neutrophils and phagocyto-sis by macrophages are key steps in the resolution of inflammation[5].

The neutrophil inflammatory response is produced by amulti-step process involving the initial adhesion of circulat-ing neutrophils to activated vascular endothelium, subsequentextravasation and migration of neutrophils towards the inflam-matory site, final elimination of foreign microorganisms byphagocytosis and generation of reactive oxygen species and releaseof microbicidal substances [6]. Exocytosis of granules and secretoryvesicles plays a crucial role in most neutrophil functions startingfrom activation to the destruction of phagocytosed microorgan-isms. Granules contained in neutrophils are an important reservoir

not only of antimicrobial proteins, proteases and componentsof the respiratory burst oxidase, but also of membrane-boundreceptors for endothelial adhesion molecules, extracellular matrixproteins, bacteria products and soluble mediators of inflammation.The mobilization of these granules permits the transformation of

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46 R. Gabbianelli et al. / Chemico-Bio

eutrophils from passive circulating cells to active cells of innatemmunity [4].

Although the capacity of PMNs to kill bacteria is related to theroduction of powerful oxidants and proteinases, an adverse effectould be measured in host tissues when the PMNs activation is outf control [4,7]. After initial stimulation, PMNs are left in a primedtate, but on encountering a second stimulus, they proceed to fullctivation, releasing reactive oxygen species, granule contents andnflammatory mediators to the surrounding environment. So prim-ng is the effect of an agent that cannot induce complete activationf PMNs because it is present at low concentration compared tohe amount necessary to give rise to a response. Following a sec-nd stimulus by another agent, primed PMNs can respond withstronger ROS production compared to that obtained by a sin-

le agent alone. In this context, the release of proinflammatoryytokines by PMNs can give rise to an abnormal increase in ROSroduction with consequent damage at the tissue level [5,8,9].

Different chemicals can modify the neutrophil response [4,8,10].nsecticides can influence the immune system activity of mammals11–16]. In rats and humans, pyrethroid insecticides are similarly

etabolized by esterases or hydrolases indicating that the ratesre similar between species. This supports the use of rodent mod-ls for studies of pyrethroid toxicity [11,17–19]. The metabolitesre eliminated in urine within 24 h after oral uptake, partiallyn the form of corresponding conjugates (glucoronides, sulfates,tc.) [20–22]. Some authors suggest that pyrethroids toxicity maye related to their metabolites, e.g. 3-phenoxybenzyl alcohol, 3-henoxybenzaldehyde and 3-phenoxybenzoic acid deriving fromermethrin (PERM), a common pyrethroid [11,19,23].

The present study was designed to evaluate the in vivo effectf PERM treatment on rat PMNs respiratory burst. Since PERM cane metabolized by mammals, the in vitro effect due to these com-ounds was also studied.

Furthermore, the influence of antioxidant treatment, using Vita-in E alone or with coenzyme Q10 (Q10), on PMNs respiratory

urst in rats treated with PERM was evaluated. Although Q9 ishe major form of Qn in rodents, we used Q10 rather than Q9ecause amongst the various Q-homologs administered orally toats, intestinal absorption of Q9 is fairly low compared with othern [24].

Additional in vitro experiments were also performed in order tonvestigate the mechanisms related to the change in PMNs respi-atory burst observed following permethrin treatment in rats.

. Materials and methods

.1. Materials

All reagents were of pure and analytical grade. Mono-Polyesolving Medium was obtained from ICN, Biomedicals, Milan,

taly. Phorbol myristate acetate (PMA), luminol, lucigenin,anthine, xanthine–oxidase, myeloperoxidase (MPO), 2,4-initrophenylhydrazine (DNPH), substance P, 3-phenoxybenzyllcohol, 3-phenoxybenzaldehyde and 3-phenoxybenzoic acid wereurchased from Sigma Chemical Co. 1,6-Diphenyl-1,3,5-hexatrieneDPH) and 6-lauroyl-2-dimethylaminonaphtalene (laurdan) wereurchased from Molecular Probes (Eugene, OR, USA). The vibrantpoptosis assay kit was obtained from Molecular Probes. 8-mino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)ione (L-012) was donated by Takeda Chem. Ind. (Osaka, Japan).

oenzyme Q10 was obtained from Nutralina Essentials Co-Q10,armaderbe, Pradamano, Udine Italy. Vitamin E was obtainedrom Roche S.p.A., Milano. Technical grade (75:25, trans:cis; 94%urity) 3-phenoxybenzyl-(1R,S)-cis,trans-3-(2,2-dichlorovinyl)-,2-dimethylcyclopropanecarboxyl-ate (permethrin, PERM)

Interactions 182 (2009) 245–252

(NRDC 143) were generously donated by Dr. A. Stefanini of ACTIVA,Milan, Italy.

2.2. Animals

Male Wistar rats from Charles River (Calco, LC, Italy), weigh-ing 120–130 g and about 5 weeks old were used. The animalswere housed in plastic (Makrolon) cages (five rats/cage) in atemperature-controlled room (21 ± 5 ◦C) and maintained on a lab-oratory diet (pellet 4RF from Mucedula, Settimo Milanese, Italy)with tap water ad libitum. The light/dark cycle was from 7 a.m. to7 p.m. Animal use in this study complied with the Italian Govern-ment’s guidelines for the care and use of laboratory animals (D.L.n. 116 of January 27, 1992).

2.3. Treatment

Permethrin was dissolved in corn oil and administered orally(5 ml/kg) (n = 30) for 60 days, at a dose of 150 mg/kg bodyweight/day (1/10 of LD50) [25] by intragastric tube. The animalstreated with PERM were divided into 3 groups: one group wastreated only with PERM (n = 14), the second group was treatedwith PERM plus Vitamin E (n = 8) and the third group was treatedwith PERM + Vitamin E + Q10 (n = 8). Vitamin E (280 mg/kg bodyweight/day) alone or plus Q10 (10 mg/kg/3 ml) was dissolved incorn oil and administered orally.

Rats serving as controls (n = 8) received 5 ml/kg body weightof corn oil orally for 60 days by intragastric tube. The substanceswere administered daily in the rats and the volume administeredwas based on body weight. The animals were observed daily andweighed at regular intervals.

After 60 days of treatment the rats were killed by CO2 asphyxiafor biochemical experiments.

2.4. PMNs separation

Blood was drawn by cardiac puncture from Wistar rats andcollected in vials containing heparin (250 I.U.). PMNs were iso-lated using Mono-Poly Resolving Medium (ICN, Biomedicals, Milan,Italy).

2.5. In vitro and ex vivo PMNs treatment

106 PMNs were suspended in 1 ml of Krebs-Ringer Phosphatesolution (KRP) containing 1 mg/ml of glucose and incubated for60 min at RT with 10 �l of DMSO or PERM (10 �M) or PERM metabo-lites (10 �M) (3-phenoxybenzyl alcohol, 3-phenoxybenzaldehydeand 3-phenoxybenzoic acid) or substance P (10 �M) used as posi-tive control (in vitro treatment). PERM or PERM metabolites weresolubilised in DMSO so that in in vitro experiments, DMSO was usedas control. Substance P was solubilised in distilled water. A con-centration of 10 �M PERM or PERM metabolites was chosen basedon preliminary dose- and time-dependent experiments (data notshown).

PMNs collected from animals treated for 60 days with PERM orPERM + antioxidants are indicated as ex vivo treatment.

Supernatants obtained from PMNs ex vivo (50 �l) were incu-bated in vitro with PMNs for 60 min before activation with 0.3 �M

PMA.

In experiments using undiluted blood plasma ex vivo, 50 �l ofplasma from control, PERM or PERM + Vitamin E + Q10 was incu-bated in vitro with PMNs for 60 min before activation with 0.3 �MPMA.

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.6. Respiratory burst of PMNs

A reaction mixture containing 106 cells ex vivo or in vitro inRP (treated as described above) was prepared. Lucigenin (150 �M)r luminol (100 �M) or L-012 (0.4 mM) was used as luminogenicrobes to amplify the reaction in order to detect superoxide anion26], hydrogen peroxide [27], and hydroxyl radical, respectively28,29]. Chemiluminescence (CL) was measured soon after activa-ion with 0.3 �M PMA in an AutoLumat LB 953. The instrumentemperature was set at 37 ◦C. The CL signal was recorded for 60 min.ata from ex vivo experiments are expressed as counts per minute

cpm), while all the other chemiluminescence results are shown asercentage change in area under the curve (% change in AUC) of thereated sample compared to the control one (DMSO). The % changen AUC vs control was calculated as follows:

change in AUC vs control = treated sample area

× 100/control sample area

.7. Evaluation of PERM hydrogen peroxide interaction

In vitro studies (without cells) were performed in the pres-nce and absence of 2.5 �g/ml MPO, 33 �M H2O2, 1 mM luminolr 0.4 �M L-012 and 10 �M PERM or 10 �l DMSO in PBS. Chemilu-inescence was followed for 60 min and the results are shown as

ounts per minute (cpm).

.8. Degranulation measurements

Degranulation measurement was estimated in the supernatantf leukocytes. 106 leukocytes were incubated for 60 min with0 �M PERM or 10 �l DMSO or phosphate buffer saline (PBS). After

ncubation, 900 �l of supernatant, for each sample, was mixed with.5 mM H2O2 + 1 mM luminol in PBS. Chemiluminescence was fol-

owed for 30 min and results are expressed as AUC.

.9. Plasma membrane fluidity measurement

The fluorescence measurements were performed using a Hitachi500 spectrofluorometer. The steady-state fluorescence anisotropyr) of DPH was calculated according to Shinitzky and Barenholz [30]quation using as excitation and emission wavelengths of 360 nmnd 430 nm, respectively. To quantify anisotropy, polarized lightas required.

The generalized polarization of laurdan (GP340) was calculatedccording to Parasassi et al. [31]. The excitation wavelength was40 nm while the two emission wavelengths were 440 nm and90 nm.

The samples were normalized after counting the cell numbernd they were treated as described above adding the probes at anal concentration of 10−6 M. Anisotropy and generalized polar-

zation were measured before and after PMA activation. DMSO wassed as control. The measurements were performed three times inriplicate.

.10. Determination of protein oxidation in PMNs

PMNs were stocked at a protein concentration of 1 mg/ml usinghe method of Lowry et al. [32].

10 �l of DMSO or 10 �M PERM (in DMSO) was added to a finalolume of 1 ml of sample and incubated at RT for 1 h. To measurehe protein carbonyl content a modified version of the method ofentz was used [33]. 0.5 ml of each sample ([Pr] = 0.389 mg/ml) wasreated with 0.5 ml of 10 �M DNPH in 2.5 M HCl. Following 1 h of

Interactions 182 (2009) 245–252 247

incubation at RT with continuous shaking, the proteins were pre-cipitated by adding 1 ml of 20% trichloroacetic acid. The sampleswere centrifuged at 3000 × g for 10 min and the protein pellets werewashed three times with ethanol:ethyl acetate (1:1). The pellet wasdissolved in 0.6 ml of 6 M guanidine (in 20 mM phosphate buffer,pH 6.5). Protein carbonyls were read at 370 nm. The absorbancecoefficient is 22,000 M−1 cm−1.

2.11. Measurement of lipid peroxidation in PMNs

The evaluation of PMNs plasma membrane “oxidation index”was used as a relative measurement of conjugated dienes on lipidsextracted according to the Folch method [34]. Dried lipids were re-suspended in ethanol and the absorbance ratio A233/A215 (oxidationindex) was measured on a Carry 1 Varian spectrophotometer at25 ◦C [35].

2.12. Apoptosis

Apoptosis and necrosis were evaluated on PMNs, treated asdescribed earlier (Section 2.5), after 60 min of incubation withPERM or PERM metabolites, using a vibrant apoptosis kit. Cells werewashed with ice cold PBS and the cell concentration was adjustedto 106 cells/ml. Samples were stained by the addition of 5 �l ofannexin V-FITC and 1 �l of 100 �g/ml propidium iodide and incu-bated at RT for 15 min. After this period, the samples were put onice and observed immediately on an Axioskop-2 plus microscope(Carl Zeiss, Germany). Results are shown as percentage of apoptoticand necrotic cells in the sample (% cell). The first was stained withannexin V-FITC and the second with propidium iodide.

2.13. Measurement of plasma superoxide antioxidant capacity

The xanthine and xanthine–oxidase system was used to gener-ate superoxide anion. The chemiluminescence was measured in areaction mixture containing 0.9 U/ml of xanthine–oxidase, 150 �Mlucigenin in 1 ml of KRP. 50 �l of undiluted blood plasma from con-trol, PERM or PERM + Vitamin E + Q10 was added to the system. Thereaction was started by injecting xanthine at a final concentrationof 50 �M and followed for 60 s. The values are expressed as per-centage change in AUC of the sample compared to the control aspreviously described.

2.14. Statistical analysis

Measurements were done on individual rats and the resultswere expressed as mean ±SD.

Statistical analysis was carried out using one-way ANOVA fol-lowed by the Newman–Keuls test. A p value lower than 0.05 wasconsidered statistically significant.

3. Results

3.1. General findings

Rats treated daily during two months with PERM,PERM + Vitamin E, PERM + Vitamin E + Q10, by intragastric tubingshowed no signs of poisoning or gross behavioural abnormalities

throughout the experimental period.

As previous reported [36], data on body weight, in rats treatedwith PERM, PERM + Vitamin E, PERM + Vitamin E + Q10, and control,did not show any changes except for the increase related to the age.

248 R. Gabbianelli et al. / Chemico-Biological Interactions 182 (2009) 245–252

Fig. 1. Superoxide anion production (A) and hydrogen peroxide–MPO activity (B) inrRKa

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Fig. 2. Percentage (%) change in the area under the curve (AUC) compared to con-trol samples (DMSO) in PMNs incubated with PERM and its metabolites (A) or withsupernatant from PMNs ex vivo of PERM alone or PERM + Vitamin E + Q10 treatedrats (B). The reaction mixture contained 106 PMNs incubated for 60 min in KRPwith 10 �M of PERM or 3-phenoxybenzyl alcohol or 3-phenoxybenzaldehyde or3-phenoxybenzoic acid or substance P (A) or 50 �l of supernatant from PMNsex vivo (B). PMNs were activated with 0.3 �M PMA in the presence of 150 �M

at PMNs from control, PERM, PERM + Vitamin E and PERM + Vitamin E + Q10 groups.eaction mixture contained 106 PMNs, 150 �M lucigenin or 100 �M luminol inRP. PMNs were activated with 0.3 �M PMA. Chemiluminescence was measureds counts per minute (cpm). The results are indicated as mean values ± SD.

.2. Respiratory burst of PMNs following in vivo treatment

After 60 days of treatment, PMNs from the four rat groupsere isolated and immediately used to evaluate their respiratory

urst. PMNs were activated with PMA in the presence of differentuminogenic probes (lucigenin and luminol) to detect the prod-cts of respiratory burst. When the reaction was measured in theresence of lucigenin, the superoxide anion level was detected. Aseported in Fig. 1A, the superoxide anion production in the groupreated with PERM was significantly increased compared to theontrol group. The superoxide production in the group treated withERM + Vitamin E shows a lower superoxide anion production com-ared to PERM group; however, a greater effect was obtained in theroup treated with PERM + Vitamin E + Q10 where the superoxidenion production was closer to the control one.

When the reaction was monitored in the presence of luminol,he hydrogen peroxide–myeloperoxidase (MPO) activity was mea-ured. Fig. 1B shows the results obtained activating the PMNs withMA in the presence of luminol. As can be observed, a similar out-ome as described above was measured in the four groups.

.3. Respiratory burst of PMNs following in vitro treatment

In order to study the mechanism related to the increasedesponse of PMNs (Fig. 1) following in vivo PERM treatment, somen vitro experiments were performed.

lucigenin. The DMSO group was assumed to be 100% of AUC. The results are indi-cated as mean values ± SD. (A) *p < 0.05 vs DMSO and PERM metabolites; § vs3-phenoxybenzaldehyde. (B) *p < 0.05 vs DMSO and PERM + Vitamin E + Q10.

To investigate if PERM alone or its metabolites can induce prim-ing on PMNs, untreated PMNs were incubated with PERM or itsmetabolites for 60 min and then activated with PMA in the pres-ence of lucigenin. As positive control, substance P, which is knownto induce priming of PMNs was added. The results are reported inFig. 2A. As can be observed, PERM and substance P significantlyincreases the superoxide anion production.

On repeating the above experiments with untreated PMNs andsupernatant obtained from ex vivo treated PMNs, the followingwas observed: when supernatant from PERM group was incu-bated with untreated PMNs, a significant increase in superoxideanion production was measured, whereas with supernatant fromPERM + Vitamin E + Q10 group, no change was observed (Fig. 2B).

On studying the other steps of the respiratory burst usingluminol (Fig. 3A) and L-012 (Fig. 3B) to evaluate hydrogenperoxide–MPO system and hydroxyl radical respectively, differ-ent results were obtained. A decrease in hydrogen peroxide–MPOsystem was measured following the incubation with PERM or itsmetabolites compared to the control (Fig. 3A), while no change wasobserved in hydroxyl radical production (Fig. 3B).

3.4. PERM interaction with hydrogen peroxide

In vitro experiments, without cells, were performed in order toevaluate if PERM interaction with H2O2 or hydrogen peroxide–MPOsystem or hydroxyl radical could influence the chemiluminescenceresponse. When the experiments were performed in the presenceof H2O2 alone, PERM increased significantly the luminol-CL signal

R. Gabbianelli et al. / Chemico-Biological Interactions 182 (2009) 245–252 249

Fig. 3. Percentage (%) change in the area under the curve (AUC) compared to con-trol samples (DMSO) in PMNs incubated with PERM and its metabolites in thepresence of different luminogenic probes luminol (A) and L-012 (B). The reactionmixture contained 106 PMNs incubated for 60 min in KRP with 10 �M of PERM or3-phenoxybenzyl alcohol or 3-phenoxybenzaldehyde or 3-phenoxybenzoic acid orsubstance P. PMNs were activated with 0.3 �M PMA in the presence of 100 �M lumi-nr§

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Fig. 4. Hydrogen peroxide and hydroxyl radical production in a reaction mixture(without PMNs) containing hydrogen peroxide and luminogenic probes. The reac-tions contained 33 �M H2O2 plus 1 mM luminol (left), 33 �M H2O2, 2.5 �g/ml MPO

indicating that PERM (GP340 = 0.385 ± 0.008) induces a greaterreduction in the fluidity of the outer layer of PMNs compared tothe control (GP340 = 0.239 ± 0.004).

When the same experiment was performed in the presence ofDPH, a significant (p < 0.05) reduction in anisotropy value, related

Fig. 5. Degranulation measurement in supernatant of leukocytes. 106 leukocyteswere incubated for 60 min with 10 �M PERM or 10 �l DMSO or PBS. After incubation,

ol (A) or 0.4 mM L-012 (B). The DMSO group was assumed to be 100% of AUC. Theesults are indicated as mean values ± SD. (A) *p < 0.05 vs 3-phenoxybenzyl alcohol;vs all samples. (B) *p < 0.05 vs all samples.

ompared to the control (Fig. 4, left), whereas when the experimentas monitored in the presence of hydrogen peroxide and MPO, no

hange between DMSO and PERM samples was measured (Fig. 4,entre). In order to detect if PERM can increase hydroxyl radicalevels, experiments in the presence of hydrogen peroxide and L-12 were performed (Fig. 4, right). No change in L-012 CL signalas measured (Fig. 4, right).

.5. Effect of PERM on degranulation

The aim of the in vitro experiments was to elucidate whetherERM was able to induce degranulation.

Leukocytes were incubated with PERM or DMSO or PBS and theelease of MPO was estimated in the supernatant. The results inig. 5 indicate that PERM does not increase degranulation under thexperimental conditions used in this study, since a similar distri-ution was measured in all samples. We also considered threshold

evels to discriminate the cells with increased MPO activity (IMA).he 95th percentile, calculated using the control values as refer-nce, was used as a cut-off point. In this study the cut-off value wasqual to 7.431. Cells with MPO activity under the cut-off do not

how an increase in degranulation, while those above the cut-offalue presented an higher degranulation. The percentages of con-rol (PBS or DMSO) and PERM treated leukocytes having a cut-offver the 95th percentile were 6.52% and 8.69%, respectively.

and 1 mM luminol (centre), 33 �M H2O2 plus 0.4 mM L-012 (right). All samples con-tained 10 �M PERM or 10 �l DMSO in PBS. Chemiluminescence was followed for60 min and the results are shown as counts per minute (cpm). Data are presentedas the mean values of chemiluminescence area ± SD. *p < 0.05 vs control and DMSO.

3.6. Plasma membrane fluidity measurement

Other factors could influence the increased response of PMNsfollowing PERM treatment.

Changes in the physico-chemical properties at the plasma mem-brane level could modify the activity of the proteins presentwithin the membrane. Fluorescent studies to detect any effectdue to PERM incubation were therefore performed. Two fluores-cent probes were employed to evaluate the influence of PERM onPMNs plasma membrane fluidity: laurdan which localizes in thehydrophilic–hydrophobic region of the bilayer and DPH that locatesdeeper within the hydrophobic part of the plasma membrane.

A significant increase (p < 0.05) in GP340 value was observedwhen PMNs were incubated for 1 h with 10 �M PERM(GP340 = 0.262 ± 0.034) vs control (GP340 = 0.165 ± 0.012). Thisincrease was more evident following the activation with PMA

900 �l of supernatant for each sample was mixed with 0.5 mM H2O2 + 1 mM luminolin PBS. Chemiluminescence was followed for 30 min and results are expressed asarea under the curve (AUC) vs the experiment repetition number. The cut-off (95thpercentile) calculated using the control values as reference, measured an AUC valueof 7.431.

250 R. Gabbianelli et al. / Chemico-Biological

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ig. 6. Effect of PERM and its metabolites on PMNs apoptosis. 106 PMNs were incu-ated with 10 �M PERM or PERM metabolites or substance P for 60 min beforetaining with annexin V-FITC and propidium iodide. Data are presented as meanalues of % cell positive to stain ± SD. *p < 0.05 vs DMSO

o an increase in fluidity, was observed in the plasma mem-rane of PERM treated PMNs (r = 0.187 ± 0.022) compared to theontrol (r = 0.233 ± 0.016). The change induced by the pesticider = 0.158 ± 0.005) was particularly remarkable subsequent to PMActivation compared to control (r = 0.242 ± 0.021) (p < 0.05).

.7. Determination of protein and lipid oxidation in PMNs

In order to investigate the effect of PERM at the PMNs plasmaembrane level, the oxidative status of proteins and lipids follow-

ng 1 h of incubation with the pesticide was measured. Carbonylroup formation was significantly increased in PERM treatedMNs (12.81 ± 2.08 nmol/mg Prot) compared to the control ones7.24 ± 1.35 nmol/mg Prot).

A similar result was measured for conjugated dienes forma-ion in lipids extracted from membrane of PMNs. The oxidationndex was significantly (p < 0.05) higher in PERM treated PMNs0.442 ± 0.066) than in control ones (0.332 ± 0.012).

.8. Apoptosis

When PMNs were incubated for 60 min with 10 �M PERM orERM metabolites, a significant increase in the % of cells undergoingpoptosis was measured compared to control samples in DMSOFig. 6).

.9. Respiratory burst and antioxidant capacity of plasma

In order to evaluate the positive response (restoration of theespiratory burst vs control value) due to antioxidants in ex vivoxperiments (Fig. 1), some in vitro studies using untreated PMNsncubated with plasma from rats exposed for 60 days to PERM orERM + Vitamin E + Q10 were performed.

Measuring superoxide anion production by PMNs following0 min of incubation with plasma a significant reduction (19%)

n the % change in AUC of groups treated with antioxidants87.6 ± 1.14) vs PERM alone (106.5 ± 9.19) was observed.

To evaluate plasma superoxide antioxidant capacity, plasmaas added to xanthine–xanthine–oxidase system. The capacity to

cavenge superoxide produced by the above reaction was different

or PERM and PERM plus antioxidants treated rats. When the exper-ment was performed with plasma from PERM + Vitamin E + Q10reated rats, an higher superoxide scavenging capacity was mea-ured (75.4 ± 2.99, % change in AUC vs control), compared to PERMlasma (89.6 ± 0.74, % change in AUC vs control) (p < 0.05).

Interactions 182 (2009) 245–252

Because the protein content in the two plasma groups was thesame in all samples, this effect cannot be related to a differentprotein concentration (data not shown).

4. Discussion

PERM is a synthetic pyrethroid (type I pyrethroid), derived frompyrethrins contained in the flower of Chrysanthemum cinerariaefolium. It is a widely used insecticide for wood preservation andto control numerous crops and pests in the public health sec-tor and housing [37,38]. The world-wide use of pyrethroids fordomestic and occupational purposes has caused concern aboutthe risks of long-term exposure [19,22,38]. The general populationis potentially chronically exposed through food consumption andenvironmental contamination [39].

Our previous studies on rats show that monocytes’ respira-tory burst following PERM treatment was reduced [40,41]. Sincethe mechanisms related to inflammatory response are different inmonocytes and in PMNs, in this study we evaluated the effect ofPERM on PMNs respiratory burst.

As is well known, neutrophils respond to infection with a res-piratory burst during which O2 is reduced to superoxide anion(O2

•−) by the NADPH oxidase system [41–43]. Superoxide may bereleased both to the phagosome and to the extracellular compart-ment. The short-lived ROS intermediate O2

•− is potentially hostileto the host since it may exceed certain “antioxidant” levels locallyin the tissue [44]. Moreover, O2

•− generates other reactive oxygenspecies such as hydrogen peroxide (H2O2), hydroxyl radical (

•OH)

and hypochlorous acid (HOCl). These reactions are catalyzed bythe superoxide dismutase (SOD) and the myeloperoxidase (MPO)enzymes, respectively. MPO catalyzes the formation of HOCl fromchlorine and H2O2, a strong microbicidal compound.

All products of respiratory burst can be monitored in vitro usingcompounds such as PMA that can stimulate PMNs similarly to otherin vivo agents.

Our study shows that production of superoxide anion andhydrogen peroxide–MPO activity was increased in the grouptreated for 60 days with PERM. This effect could be related to var-ious events: one is the priming induced by PERM that could givethis result by itself or by induction of cytokines release. Since invitro experiments show that PMNs incubated with PERM, lead toa superoxide anion production similar to a known priming agent,substance P [9], one can hypothesize that the increased response ofPMNs may be due to priming following PERM treatment. In fact, asimilar superoxide production was measured incubating untreatedPMNs with supernatant from PMNs of rats treated for 60 days withPERM. The priming effect does not seem to be related, under theexperimental conditions used in our study, to PERM metabolitessince these do not increase the PMNs response, as observed in thein vitro study. In fact the production of superoxide anion, hydrogenperoxide–MPO activity and hydroxyl radicals (Figs. 2A, 3A and B)do not change in PMNs incubated with metabolites of PERM.

Metabolites however have an influence on PMNs apoptosis asmeasured in the in vitro experiment. However, we have to considerthat, the percentage of cells that undergo apoptosis following incu-bation with PERM or its metabolites, is small (0.725–2.26% of totalcells, Fig. 6) although statistically significant, so this amount of cellscannot have a significant role in the respiratory burst.

More relevant to respiratory burst could be PERM’s physico-chemical perturbation at the plasma membrane level. PMNsincubated with PERM show changes in fluidity at different levels of

the bilayer. In particular, PERM induces a reduction in fluidity in thehydrophilic–hydrophobic region of the membrane and an increasein the hydrophobic part of the bilayer. These effects are more evi-dent following PMA activation. The alteration in the physical stateof the plasma membrane could influence the NADPH oxidase com-

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R. Gabbianelli et al. / Chemico-Biol

lex activity thus influencing the first step of the respiratory bursts well as superoxide anion production. The changes in the bilayerould be relevantly influenced also by the increased protein andipid oxidation following PERM treatment.

In this context, the antioxidants used in this study during in vivoreatment could explain the restoration of the respiratory burst tolmost control values, measured in the group treated with Vitamin+ Q10 (Fig. 1). The positive effect of antioxidants on PMNs can bebserved also in in vitro experiments where PMNs were incubatedith supernatant from PMNs of rats treated with PERM + Vitamin+ Q10. (Fig. 2B).

Since plasma from rats treated with PERM + Vitamin E + Q10hows an higher superoxide scavenging activity and since PMNsncubated with plasma from the two groups of PERM alone andlus antioxidants give rise to a similar result as in the ex vivo treat-ent, we could assume that the antioxidants supplementation can

ave a protective effect at the plasma membrane level due to theircavenging activity.

In view of the fact that the in vitro study with PERM or itsetabolites did not lead to an increase in hydrogen peroxide–MPO

ystem (Fig. 3A) in contrast to ex vivo results (Fig. 1B), some in vitroxperiments to evaluate the possible interaction between PERMnd hydrogen peroxide–MPO system were performed. The resultsbtained show that the presence of hydrogen peroxide plus PERMncreases the hydrogen peroxide production, while when hydro-en peroxide plus MPO are present, no increase in the signal can beeasured in the samples (Fig. 4). Our finding suggests a pro-oxidant

ffect of PERM that can interact with hydrogen peroxide leading ton increase in this latter compound. In the presence of MPO, theydrogen peroxide is employed for the formation of an interme-iate, compound I, as described by other authors [44–47], so no

ncrease in chemiluminescence can be observed. Moreover, a pos-ible explanation for the difference in the reactivity observed in thexperiments performed in the presence of luminol alone or L-012Fig. 4) could be that the reaction of hydrogen peroxide with Cl-ontaining PERM would have generated hypochlorite that stronglyeacted with luminol but only weakly with L-012.

Although these results are not indicative to explain the differ-nt outcomes of ex vivo and in vitro experiments, they reproducehe normal trend in these reactions. Maybe the experimental con-itions for in vitro experiments (1 h) are too restrictive comparedo the in vivo treatment (60 days), where many other events canontribute to the results obtained.

In conclusion, our study shows that sub-chronic PERM treat-ent increases O2

.− and H2O2–MPO levels during respiratory bursty a priming effect and by perturbation at the plasma membrane

evel. The protective of Vitamin E + Q10 supplementation to limithe abnormal PMNs responses should be considered for tissue pro-ection against the damage due to abnormal neutrophil responses.

Future studies will be performed to investigate if the increasedespiratory burst depends only on PERM or if this effect is mediatedlso by cytokines release.

onflict of interest statement

The authors declare that there are no conflicts of interest.

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