INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY

Vol. 26, no. 4, 871-881 (2013)

PROTECTIVE EFFECT OF ALPHA-LIPOIC ACID ON CYPERMETHRIN-INDUCED OXIDATIVE STRESS IN WISTAR RATS F. MIGNINP, C. NASUTP, D. FEDELI l, L. MATTIOLIl, M. COSENZAl, M. ARTIC0 2 and R. GABBIANELLI I

'School ofPharmacy, Experimental Medicine Unit, University ofCamerino, Camerino, Italy; 2Department ofSensory Organs, University ofRome "Sapienza", Rome, Italy Received May 28,2013 -Accepted September 13,2013 Cypermethrin (CY), a class II pyrethroid pesticide, is globally used to control insects in the household and in agriculture. Despite beneficial roles, its uncontrolled and repetitive application leads to unintended effects in non-target organisms. In light of the relevant anti-oxidant properties of alpha-lipoic acid (ALA), in the work described herein we tested the effect of a commercially available ALA formulation on cypermethrin (CY)-induced oxidative stress in Wistar rats. The rats were orally administered with 53.14 mg/kg of ALA and 35.71 mglkg of CY for 60 days. The treatment with CY did not induce changes in either locomotor activities or in body weight. Differences were observed on superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation that were re-established by ALA treatment at similar levels of the placebo group. Furthermore, ALA formulation increased glutathione (GSH) level and glutathione peroxidase (GPx) activity. Because of the widespread use of CY, higher amounts of pesticide residues are present in food, and a diet supplementation with ALA could be an active free radical scavenger protecting against diseases associated with oxidative stress. Synthetic pyrethroid insecticides were introduced into widespread use for the control of insect pests and disease vectors more than three decades ago. In addition to their value in pest-control in agriculture, pyrethroids are at the forefront of efforts to combat malaria and other mosquito-borne diseases and are also common ingredients of household insecticides and domestic animal ectoparasite control products (I). The abundance and variety of pyrethroid use contribute to the risk of exposure and adverse effects in the general population (I). Cypermethrin (CY), a class II pyrethroid pesticide, is globally used to control insects in the household and in agriculture. Despite beneficial roles, its uncontrolled and repetitive application

leads to unintended effects in non-target organisms (2). Several epidemiological and experimental studies have been performed to assess the health risks associated with CY exposure and CY levels in the blood and urine of the people who spray pesticides and other exposed individuals (2). CY has been identified as one of the important constituent pesticides associated with human health risks (3). In mammals, CY can accumulate in body fat, skin, liver, kidneys, adrenal glands, ovaries, lung, blood, and heart (3). However, the main target for CY is the central nervous system. Symptoms of CY toxicity in laboratory animals include pawing, burrowing, salivation, tremors, writhing, and seizures. In humans, high doses of CY result in twitching,

Key words: alpha-lipoic acid. rat, oxidative stress, cypermethrin, lipid peroxidation Mailing address: Prof. Fiorenzo Mignini (MD, PhD) School of Pharmacy, Experimental Medicine Unit, Via Madonna delle Carceri, 9 62032 Camerino (MC), Italy Tel.: +390737403304 Fax: +390737403325 e-mail: [email protected]

0394-6320 (2013)

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Copyright © by BIOLIFE, s.a.s. This publication andlor article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF INTEREST RELEVANT TO THIS ARTICLE.

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drowsiness, coma, and seizures (2, 3). In addition to neurons, reproductive organs are another toxic target ofCY (2). In previous studies, it was demonstrated that pyrethroids induce enhanced oxidative stress and inflammatory responses in rats treated orally with low doses daily over 2 months. In particular, the treatment induced lipid oxidation, DNA damage, reduction of GSH level on various cell types (i.e. erythrocytes and leukocytes), disruption of antioxidant enzyme activities such as catalase, superoxide dismutase and glutathione peroxidase (4, 5) and increased inflammatory responses (6). Many in vitro studies have shown that dietary antioxidants, such as vitamin C (ascorbic acid), vitamin E (a-tocopherol), ~-carotene, and f1avonoids, act as effective antioxidants in biological systems such as plasma, lipoproteins, and cultured cells (7). In this context, it is remarkable that a positive correlation has been found between dietary supplementation with certain vegetables and plant products and the reduction of toxic effects of various toxicants and environmental contaminants (7). It has already been shown that coenzyme Q10 and vitamin E exert a protective effect against oxidative stress induced by permethrin (a pyrethroid compound) (8). Recently, it was demonstrated that curcumin, a yellow orange dye used as a spice and food-coloring agent in cooking with well known anti-inflammatory and antioxidants properties, can be a potent protective agent against CY-induced biochemical alterations and oxidative damage in rats (9). Alpha-lipoic acid (1,2-dithiolane-3-valeric acid) (ALA), also known as thiotic acid and usually found in small amounts in meats and vegetables, has potent antioxidant properties (I O).ALAexhibitsitsantioxidant activity in both the reduced (dihydrolipoic acid) and oxidized forms (a-lipoic acid). The dihydrolipoic acid (6,8-dimercaptocaprylic acid) can directly regenerate ascorbate due to its low redox potential (-0,32 V), and it is able to regenerate endogenous thiols involved in physiological redox antioxidant systems, such as cysteine and glutathione. The redox couple u-lipoic/dihydrolipoic acid is covalently bound to a lysine residue, forming an essential lipoamide, which functions as a co-enzyme for the E2 subunit of four multi-enzymatic mitochondrial complexes (e.g. pyruvate dehydrogenase) (11). Moreover, the

antioxidant activity of the oxidized form of lipoic acid is based on its scavenger activity against reactive oxygen species, its ability as a metal ion chelator and its capacity to increase glutathione and vitamin C (10). The racemic form of ALA is used as a nutraceutical but in many countries it is a registered pharmaceutical product used for i.v. and oral administration (12, 13). Treatment with lipoic acid reduces oxidative stress in healthy subjects (14) and diabetic patients, also preventing metabolic and neurovascular deficits in several organs and systems (15). It has also shown that ALA induces the expression of cellular antioxidants and phase 2 enzymes including catalase, glutathione reductase, glutathione-S-transferase and nicotinamide adenine dinucleotide phosphate (NADPH) (11). Considering the relevant anti-oxidant properties of ALA, in the work described herein we tested the effect of a commercially available ALA formulation on CY-induced oxidative stress in Wistar rats. MATERIALS AND METHODS Reagents Pure and analytical-grade reagents were used. Technical grade (62.8: 37.2, trans:cis; 96.4% purity) (R,S)a-cyano-3-phenoxybenzyl-( IR,S)-cis.trans-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropane carboxylate, cypermethrin (NRDC 149) was generously donated by Dr. A. Stefanini ofACTIVA, Milan, Italy. Both the formulation containing ALA and the formulation containing only the excipients, as powders, were a gift from Alfa Wassermann S.p.A. (Bologna, Italy). The ALA formulation contained a racemic mixture of RS-(+/-)-alpha-lipoic acid (Giellepi s.p.a, Milan, Italy) (Table I). Animals Twenty-four male Wistar rats (Harlan, Italy), weighing 150--170 g, and about 6 weeks old were used. The animals were housed in plastic (Makrolon) cages (five rats/cage) in a temperature controlled room (21±5°C) and maintained on a laboratory diet with water ad libitum. The light/dark cycle was from 7 p.m. to 7 a.m. Animal use in this study complied with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 20 lOon the protection of animals used for scientific purposes. The experimental protocol was approved by our Institutional Animal Care and Use Committee. Drug preparation and administration A recent study comprising diabetic

patients

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with symptomatic distal symmetric polyneuropathy demonstrated that oral treatment with ALA at doses of 600, 1200 and 1800 mg/day for 5 weeks improved the positive sensory symptom (16). However, the lower dose (600 mg/day) provided the optimum risk-to-benefit ratio and was designed as the recommended dose. To determine the ALA dose from humans to rats, the human dose (8.57 mglkg) was multiplied by a conversion factor (6.2), according to the method of body surface area (17). Thus, ALA dosage was 53.14 mg/kg body weight. Since the used ALA formulation contains 58.53% of ALA, the rats received a dose of 90.79 mg/kg body weight. Regarding the placebo formulation, containing only excipients (as reported in Table I), a dose of 37.65 mg/ kg body weight was administered. The ALA formulation was prepared by solubilizing the powder containing the racemic mixture of RS-(+/-)-alpha-lipoic acid and the excipients in saline. The placebo formulation, containing only the excipients present in the ALA formulation, was solubilized in saline. CY was dissolved in com oil (Sigma, Italy) and administered at a dose of 35.71 mg/ kg body weight (1 mL/kg) corresponding to 1/10 of LDso (18). All formulations were freshly prepared each day before treatment. All animals were divided into 4 groups, placebo (n=6), ALA (n=6), CY+placebo (n=6) and CY+ALA (n=6), and administered daily by oral gavage through gastric tubes for 60 days. The placebo group received I mL/kg of com oil plus 2 mL/kg of placebo formulation; the ALA group received I mL/kg of com oil plus 2 mL/kg of ALA formulation; the CY+placebo group was treated with I mL/kg of CY solution plus 2 mL/kg of placebo formulation; the CY+ALA group received I mL/kg of CY solution plus 2 mL/kg of ALA formulation. The volume of the compound administered was adjusted daily based on body weight measured during the dosing period. All groups received the same volume of com oil per body weight. At the end of the treatment, all animals were sacrificed by exposure to CO 2, and their blood was collected for analysis. Measurement oflocomotor activity Automated locomotor activity boxes (MedAssociates, VT 05478) were used to assess the behavioral activity. Each animal was placed in the activity box, and spontaneous locomotor activity parameters were monitored. Activity was automatically recorded for 5 min by interruptions of two orthogonal light beams which were connected to automatic softwares (Activity Monitor, MedAssociates). The behavioral tests were carried out at the end of the treatment period (60 days) before blood collection. The behavioral parameters observed were locomotion (number of ambulatory episodes), rearings (number of rears), and stereotype counts (number of grooming movements).

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Locomotion counts were recorded when the low row of photocells was interrupted, while rearing counts were recorded by taking the higher row of photocells. Blood collection After 60 days of treatment, all the rats were sacrificed by exposure to CO 2, and their blood was collected for analysis. The plasma was separated from erythrocytes by centrifugation at 3000 x g for 5 min. Samples were kept frozen until the following analyses were carried out. Superoxide dismutase determination Superoxide dismutase (SOD) was determined on hemolysated blood according to the method of Misra and Fridovich (19). Briefly, hemolysated blood was normalized to hemoglobin concentration of5.5 mg/mL; to I mL of solution was added I mL of chloroform:ethanol (I :2), centrifuged at 3000 x g and the supernatant constituted of ethanolic extracted was collected. SOD activity was monitored in a reaction mixture containing 0.05 M carbonate buffer, 0.1 M EDTA, pH 10.2, 0.05 M adrenaline and 50 ml of ethanolic extracted. Change in absorbance was recorded spectrophotometrically at 480 nm at 37°C. One unit of enzyme activity is defined as the amount of SOD causing 50% inhibition of adrenaline to adrenochrome reaction. A standard curve was used to report the results as mg SOD/mg hemoglobin. Catalase determination Catalase (CAT) activity was determined using the Luck's method (20). Briefly, the assay mixture consisted of 3 mL 0.066 M phosphate buffer pH 7.0,40 /-11 of 9 M HP2 and 10 /-11 hemolysates normalized to a concentration of hemoglobin of Img/mL. The change in absorbance was measured spectrophotometrically at 240 nm. The enzyme concentration was calculated using a standard curve. The results were expressed as mg CAT/mg hemoglobin Glutathione peroxidase activity Glutathione peroxidase (GPx) activity was measured using the method of Paglia and Valentine (21) on hemolysates normalized to 4 mg/mL hemoglobin concentration. The reaction mixture contained: 0.05 M phosphate buffer, 5 mM EDTA, pH 7.0, 0.125 M NaN 3, 0.0084 M NADPH, 0.15 M glutathione (GSH), 7.6 Units of glutathione reductase (E.C.1.6.4.2,) 0.0022 M hydrogen peroxide and 30 /-11 of normalized samples pretreated with Drabkin's reagent. GPx catalyses the oxidation of GSH by hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidized glutathione is immediately converted to the reduced form with the concomitant oxidation of NADPH to NADP, measured spectrophotometrically at 340 nm. The enzyme

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concentration was calculated using a standard curve. The results were expressed as ug GPx/mg hemoglobin. Estimation ofglutathione Glutathione (GSH) content was measured in the plasma according to the method previously described by Butler et al. (22). We measured the oxidized GSH by the sulfhydryl reagent 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) on a spectrophotometer at the wavelength of 412 nm with respect to its standard. The GSH concentration was calculated using a standard curve. The results were expressed as mg GSH/ml of plasma. Erythrocyte membrane preparation Plasma membranes were prepared by hypotonic hemolysis from 10 mM Tris pH 7.4 to 2.5 mM Tris pH 7.4 according to Bramley et al. (23). Protein concentration was evaluated using Lowry's method (24). Determination oflipid peroxidation The "oxidation index" in erythrocytes was used as a relative measurement for conjugated dienes on lipids extracted according to the Folch method (25) from plasma membrane erythrocytes normalized to a protein concentration of I mg/mL. Dried lipids were resuspended in ethanol and the absorbance ratio AmiA 215 was measured on a Carry I Yarian spectrophotometer at 25°C (26). Determination ofprotein oxidation A modification of the Lenz's method (27) was used for measurement of protein carbonyls. To summarize, 0.1 mL of 10 mM DNPH in 2.5 M HCI was added to I mL of plasma membrane erythrocyte sample normalized to I mg/mL protein concentration; blank reactions lacked only DNPH. Following I h of incubation with continuous shaking, proteins were precipitated by adding 0.5 mL of 20% TCA and centrifuged at 3000 x g for 10 min. The pellet was washed three times with ethanol:ethyl acetate (I: I) and dissolved in 0.6 mL of 6 M guanidine HCI (pH 6.5). Protein carbonyls were then measured spectrophotometrically at 370 nm using the molar extinction coefficient of22.000 M-I em:'. The results were expressed as llM/mg protein. Nitric oxide detection Nitric oxide (NO) content was measured spectrophotometrically in the plasma of treated and control rats using the kit purchased from Neogen (Neogen Corporation, Lexington KY, USA). The NO concentration was calculated using a standard curve, the results were expressed as concentration 11M of nitric oxide. Cytokines analysis IL-I, IL-I~, IL-2, IL-4, IL-6, IL-IO, IL-12, IL-13,

IFNy, TNF-a, GM-CSF and Rat Rantes levels were determined in triplicate, using commercially available ELISArray Kits Multi-Analyte for Rats (QIAGEN Company, USA) according to the manufacturer's protocol. The plate included a standard curve and known positive and negative controls. Absorbance was read at 450 nm and at 570 nm using a microtiter ELISA reader. Statistics Statistica 9.0 software was used by applying one-way ANaYA to evaluate the effect of procedures on each group of animals. The values were expressed as mean ± SD. If a general effect was determined by ANaYA, post hoc analysis was performed with the Newman-Keuls test with P < 0.05 used as the level of significance.

RESULTS Rats from placebo, ALA, CY+placebo and CY+ALA groups, treated daily for 60 days by intragastric tubing, showed no sign ofgross behavioral abnormalities throughout the experimental period. Data on body weight in all treated and control groups are reported in Fig. I. No difference in body weight was observed among all groups during two months of treatment (p>0.05). In Fig. 2, locomotor activities of rats were tested after 60 days of treatment in an open field task, and performance among groups was comparable; statistical analysis of data did not reveal differences among groups (p>0.05). Fig. 3 shows the GPx, SOD, CAT and GSH concentrations measured in plasma of the four tested group of rats. The GSH level (Fig. 3a) in respect to placebo increased significantly only in ALA treated rats (0. I83±0.00 I vs 0.0067±0.00 I) while no differences were found when rats were exposed to CY and CY+ALA. The treatment with CY induced an increase in GPx concentration in respect to placebo (0.028±0.001 vs 0.009±0.001) but the administration of ALA in CY rats did not report the GPx content to placebo value (0.0 13±0.002 vs 0.009±0.00 I), although it was nearest to the placebo compared to the CY group (0.02±0.002) (Fig. 3b). On the contrary, the CAT activity (Fig. 3c) was significantly decreased in the ALA and CY+placebo groups vs placebo group (l38.6±2.55, 121.3±4.5 vs 151.2±1.8) but the simultaneous treatment with CY and ALA reported the CAT values to levels obtained

Int. J. Immunopathol. Pharmacol.

Table I. Qualitative and quantitative composition ofALA A Wformulation. Ingredients present in 100 mg ofpowder.

875

INGREDIENT

0/0

Alpha-lipoic acid

58.53

Maltodextins

4.87

Soy lecithin

0.97

Poly vinyl pirrolidone

2.92

Talc

0.97

Silicon dioxide

4.39

Piridoxine HCl (Vit. B6)

0.45

Tiamine HCl (Vit. Bl)

0.33

Riboflavin (Vit. B2)

0.30

Calcium pantothenate (Vit. B5)

1.24

Dibasic calcium phosphate

14.87

Sodium croscaramellose

2.78

Poly vinyl polypirrolidone

1.95

Magnesium stearate

2.92

Sepifilm LP030* (HPMC, MCC, stearic acid)

1.70

Sepisperse dry 5212 Pink/Rose** (HPMC, MCC, E171, EI72)

0.73

in placebo (149.4±4.8 vs 151.2±1.8) CY induces a decrease in the SOD content with respect to placebo (1.5I2±O.069 vs 2.693±0.442), after ALA administration SOD activity was significantly increased vs CY+placebo (2, 131±O.069 vs 1.512±O.069, respectively). The treatment with only ALA did not induce any variation on SOD content in respect to the placebo control (2.34±O.OOI vs 2.693±0.442) (Fig. 3d) In Fig. 4, the lipid peroxidation and the protein oxidation at the plasma membrane erythrocyte level are reported. The CY+placebo group compared to the placebo group showed increased lipid peroxidation (1.033±O.OOI vs O.562±O.0405). The ALA treatment was protective against lipid peroxidation induced by CY as shown by the similar values between the CY+ALA and placebo group (O.523±O.005 and O.562±O.0405, respectively) (Fig. 4a). No significant differences among all groups were instead found in carbonyl formation due to protein oxidation (Fig. 4b). Fig. 5 shows the micromolar concentration of nitric oxide measured in plasma of all groups. The results showed no significant differences in NO content following the different treatments. Only three different cytokines were found in plasma of the tested rat groups, Rat Rantes, TNF-a and GM-CSF (Table 11); no differences among groups (p>O.05) were observed.

100.00

DISCUSSION

TOTAL

(*) Sepifilm LP030: Hydroxy propyl metyl cellulose, Microcrystalline cellulose, Stearic acid. (**) Sepisperse Dry 52 J2 pink/rose: Hydroxy propyl metyl cellulose, Microcrystalline cellulose, E17I, E172 (Iron dioxides).

The aim of the present work was to determine the effect of an ALA formulation, commercially available as nutritional supplement, on CY-induced oxidative stress in Wistar rats. This animal model

Table II. Cytokine concentrations (ug/ml.) measured in plasma ofrats treated with placebo, ALA, CYand CY+ALA. Data are reported as mean values ± standard deviation.

placebo ALA CY+placebo CY+ALA

TNF-a 8.24E-05±2.34E-07 1.26E-04 ±7.26E-07

-

concentration (Jig/mL of plasma) Rat rantes GM-CSF 3.34E-03±8.33E-06 6.39E-05±1.02E-06 3.35E-03±1.32E-05 2.43E-03±1.32E-05 4.56E-05±1.33E-05 1.90E-03±1.05E-05 -

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Int. J. Immunopathol. Pharmacol.

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