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Mutation Research 702 (2010) 1–7

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /gentoxCommuni ty address : www.e lsev ier .com/ locate /mutres

rotective effects of garlic extract and vitamin C against in vivoypermethrin-induced cytogenetic damage in rat bone-marrow

.E. Assayeda,∗, A.A. Khalafb, H.A. Salemc

Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Menoufiya University, Sadat City Branch, Sadat City 32511, EgyptDepartment of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, EgyptDepartment of Pathology, Faculty of Veterinary Medicine, Menoufiya University, Sadat City Branch, Sadat City 32511, Egypt

r t i c l e i n f o

rticle history:eceived 27 July 2009eceived in revised form 12 January 2010ccepted 26 February 2010vailable online 29 July 2010

eywords:ypermethrinaticronucleus

hromosomal aberrationsarlic extractitamin C

a b s t r a c t

The cytogenetic damage inflicted by the synthetic pyrethroid insecticide cypermethrin (CYP) on the bone-marrow of male white rats, as well as possible protective role of two natural elements: garlic extract(GRE, 500 mg/kg) and vitamin C (VTC, 20 mg/kg) against the mutagenic potential of the insecticide wereassessed. CYP was orally intubated in a single treatment (1/2 LD50) or in repeated treatments (1/5 LD50

daily, for 5 successive days), either alone, or concomitantly with repeated oral intubations (5 successivedays) of each individual putative protector, or with their combination (GRE or/and VTC). One hundredand twenty male rats were divided over into five groups of each 24 animals. The groups received nothing,a single dose or repeated treatments with insecticide alone, or associated with putative natural elements,separately or in combinations. Animals were sacrificed at their scheduled times and their femoral bone-marrows were flushed out to be utilized in the micronucleus test and metaphase chromosomal aberrationassay. The results show that CYP administration significantly induced clastogenic effects, as revealed

by the significant increase in the mean frequencies of micronucleated polychromatic erythrocytes andvarious structural chromosomal aberrations in bone-marrow metaphase cells of all groups of treated rats.On the other hand, this investigation clearly revealed the protective role of GRE and VTC, either each aloneor in combination, against the mutagenic potential of cypermethrin: the garlic extract was often moreefficient in its protective action against the insecticide toxicity than vitamin C. while the combinationof both natural elements produced, in most cases, a more pronounced protective effect than when each was administered alone.

. Introduction

The widespread use of pesticides in public health protection andgricultural programs has caused severe environmental pollutionnd health hazards, particularly in developing countries, includ-ng cases of severe acute and chronic human and animal poisonings well as damage to other non-targeted organisms. The introduc-ion of new, more toxic and rapidly disseminating pesticides intohe environment has necessitated accurate identification of theirotential hazards to human and animal health [1]. Every year therere 3 million cases of severe poisoning and 220,000 deaths fromesticides; the majority of these poisonings and 99% of the result-

ng deaths occur in the third world [2]. Cypermethrin, a syntheticyrethroid insecticide has been extensively used in the last twoecades in many of the developing countries, especially in Saudirabia and Egypt, for combating agricultural pests and insects of

∗ Corresponding author. Tel.: +20 2 25190923; fax: +20 482612225.E-mail address: memzat68@yahoo.com (M.E. Assayed).

383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.mrgentox.2010.02.020

© 2010 Elsevier B.V. All rights reserved.

veterinary as well as human concern. The synthetic pyrethroidsinsecticides are widely applied in view of the fact that they haveshown to possess a high insecticidal activity as well as a broadspectrum of high initial toxic action on several types of pest. Thetoxicity of pyrethroid insecticides to mammals has received muchattention in recent years because animals exposed to these insecti-cides showed changes in their physiological activities besides otherpathological features [3–5]. Moreover, the greatly increased agri-cultural use of pesticides besides their vital role in public healthprotection, has introduced serious and novel hazards to humansand their livestock. Studies have shown that prolonged exposureto some of these contaminants causes chronic or persistent neuro-logic syndromes [6,7], immunosuppressive effects [8], malignanttumours [9,10], teratogenic action, abortion and reproductive fail-ure [11].

On the other hand, despite existing knowledge based on studieswith experimental animals it cannot be predicted to what extentchronic exposure of pesticides will affect humans. Free radicals playan important role in toxicity of pesticides and environmental chem-icals. Pesticides may induce oxidative stress leading to generation

2 M.E. Assayed et al. / Mutation R

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f free radicals and alterations in antioxidant status or the oxygenree-radical (OFR)-scavenging enzyme system. Lipid peroxidationas been suggested as one of the molecular mechanisms involved inesticide-induced toxicity [4,12–14]. Because several studies havehown that exposure to pesticides may induce genotoxic effects inccupationally exposed human populations, the evaluation of theenotoxicity of pesticides in use is of immediate concern [15–17].

For this reason, the present work is designed to investigate then vivo cytogenetic effects of the insecticide cypermethrin in the

hite rat as a mammalian model for experimentation, by evalu-tion of its capability to induce chromosome abnormalities in ratone-marrow cells. At the same time, the present work is essen-ially planned for evaluation of the possible protective effects ofwo natural products, viz. garlic extract (Allium sativum) and vita-

in C (l-ascorbic acid) against in vivo cytogenetic effects of thensecticide cypermethrin.

. Materials and methods

.1. The insecticide: cypermethrin®

This insecticide is available as an oily solution containing 250 g/L of the activengredient cypermethrin, which belongs to type-II pyrethroids “cyanopyrethroids”nd has the chemical name: [�-cyano-(3-phenoxyphenyl)methyl(±)-cis/trans--(2,2-dichloro-vinyl)2,2-dimethylcyclo-propanecarboxylate], and molecular for-ula C22H19O3NCl2 (MW 416.3; see Fig. 1). Technical cypermethrin is a mixture of

our isomers and is a yellowish-brown mass of oily to pasty consistency; but above0 ◦C it is a clear yellowish-brown oil. The compound was diluted with distilledater to concentration levels convenient to be used throughout the whole experi-ent, in which 1 ml of diluted working solution contained 25 mg of cypermethrin.

he insecticide was supplied as an emulsifiable concentrate (EC) formulation as wells pure technical grade cypermethrin (92%) by the Department of Mammalian andquatic Organisms Toxicity Research, Central Laboratory of Pesticides, Dokki, Cairo,gypt.

.2. Animals

A total of 120 apparently healthy male white rats (Rattus rattus) of Wistar strainere used throughout the whole work. The animals were obtained from the lab-

ratory animal house of the Modern Veterinary Office, Giza, Egypt. Animals wereept under full hygienic conditions, had free access to fresh water and fresh well-alanced food, and remained under supervision for three weeks before commencinghe experimental work.

.3. Chemicals

Colchicine (fine powder; Sigma–Aldrich, St. Louis, MO, USA) was prepared as amg/ml solution (w/v) in distilled water and was employed as a mitotic inhibitor.etal calf serum (PAA Laboratories, Austria) was used in the micronucleus assay.otassium chloride hypotonic solution 0.56% in distilled water was from Nile Chem-cal Company, Cairo, Egypt. Absolute methyl alcohol (analytical grade) and glacialcetic acid were obtained from Adwic Chemicals Company, Cairo, Egypt. Freshly pre-ared and chilled fixative solution was composed of absolute methanol and glacialcetic acid (3:1, v/v). Diluted Giemsa stain 1:10 in distilled water and Wright stain1.8 g/L in acetone-free methanol) were purchased from BDH Chemicals Co., AT, USA.

.4. Putative natural protectors

.4.1. GarlicAn aqueous extract of whole crude garlic was prepared as follows: freshly peeled

loves of garlic (A. sativum, purchased from an Egyptian market) were sliced intomall pieces and ground in a clean mortar using a mortar pestle to produce a fine

esearch 702 (2010) 1–7

paste. The working solution was then prepared by dissolving 5 g of the paste in100 ml of distilled water, where 1 ml of the extract contains 50 mg of crude gar-lic. Fresh garlic extract was orally administered to rats with the aid of a stomachtube fitted to a graduated syringe, at a dose of 500 mg/kg body weight, daily on 5consecutive days/week [18].

2.4.2. l-Ascorbic acid (vitamin C)A pure form of l-(+) ascorbic acid was supplied as pure crystals (Sigma, AX 1776-

1) by E. Merck Science, a division of EM industries Inc., Darmstadt, West Germany. Afreshly prepared aqueous solution of l-ascorbic acid was orally administered to therats throughout the experiments, either solely or in combination with garlic extract,at a dose of 20 mg/kg bw (i.e. twice the human recommended therapeutic dose of10 mg/kg bw/day), daily on 5 successive days/week [19].

2.5. Cytogenetic investigations

2.5.1. Micronucleus testThe frequency of micronucleated polychromatic erythrocytes [PCE (Mn)] in

femoral bone-marrow preparations were scored and evaluated according to thetechnique described by Schmid [20] with certain modifications in the procedure asrecommended by Hayashi et al. [21].

2.5.2. Chromosomal aberration assayCytogenetic analysis of rat chromosome aberrations (CA) in bone-marrow

metaphase cells was performed according to the technique described earlier [22]with some modifications as recommended by others [23].

2.6. Experimental design

A total of 120 rats were divided over five main groups of 24 animals each. The24 animals in each group were further subdivided into two groups of 12 animalseach: one was used for the micronucleus test (6 rats for single dosing, 6 for repeatedtreatments) while the other subgroup was utilized for the chromosomal aberrationassay (6 rats for single dosing, 6 for repeated treatments), as follows.

2.6.1. Control groupTwenty-four animals (12 for Mn, 12 for CA) were not given cypermethrin nor

exposed to putative protectors. These animals were kept as untreated control.

2.6.2. Group (A)Twenty-four animals were orally intubated with cypermethrin alone at a dose

of 1/2 LD50 as single treatment (6 rats for Mn, 6 for CA) or at 1/5 LD50 as repeatedtreatment (6 rats for Mn, 6 for CA) for 5 consecutive days.

2.6.3. Group (B)Twenty-four animals were orally intubated with whole garlic aqueous extract

at a dose of 500 mg/kg bw, followed by cypermethrin at a dose of 1/2 LD50 as singletreatment (6 rats for Mn, 6 for CA) or 1/5 LD50 as repeated treatment (6 rats for Mn,6 for CA) for 5 consecutive days.

2.6.4. Group (C)Twenty-four animals were orally intubated with a freshly prepared aqueous

solution of l-ascorbic acid at a dose of 20 mg/kg bw, followed by cypermethrin at adose of 1/2 LD50 as single treatment (6 rats for Mn, 6 for CA) or 1/5 LD50 as repeatedtreatment (6 rats for Mn, 6 for CA) for 5 consecutive days.

2.6.5. Group (D)Twenty-four animals were orally intubated with a combination of whole gar-

lic extract and a freshly prepared solution of l-ascorbic acid (500 mg/kg bw and20 mg/kg bw, respectively), followed by cypermethrin at a dose of 1/2 LD50 as singletreatment (6 rats for Mn, 6 for CA) or 1/5 LD50 as repeated treatment (6 rats for Mn,6 for CA) for 5 consecutive days.

2.7. Scheduled time of sacrifice

Rats were sacrificed through cutting of their neck veins at (i) 30 h after the singledose of insecticide (1/2 LD50) or (ii) 30 h after the last of the repeat doses of theinsecticide (1/5 LD50).

2.8. Procedures

2.8.1. Micronucleus testAnimals were sacrificed according to the experimental schedule and both

femurs were dissected out and freed from musculature. The cartilaginous epiphyseswere cut off to facilitate needle insertion. The bone-marrow was flushed out fromboth femurs with 1 ml of fetal calf serum (FCS) and centrifuged at 101 × g for 10 min.The supernatant was discarded and the bone-marrow pellet was re-suspended ina drop of FCS. The evenly spread bone-marrow smear was stained according to theWright stain-Giemsa protocol. Slides were scored at a magnification of 1000× with

M.E. Assayed et al. / Mutation Research 702 (2010) 1–7 3

Table 1Effect of single and repeated oral administration of cypermethrin alone and with natural protectors on the frequency of micronuclei in bone-marrow PCEs of albino rats.

Groups Control Group LSDP < 0.05

No. of Mn/PCE A B C D

Single oral administrationPCE having one Mn 6.83 ± 0.79 71.0 ± 3.32a 46.17 ± 2.70b 51.0 ± 1.95b 11.83 ± 0.60b 6.26PCE having two Mn 1.00 ± 0.37 14.5 ± 0.76a 6.17 ± 0.60b 7.67 ± 0.67b 4.33 ± 0.49b 1.73PCE having more than two Mn 0.33 ± 0.21 10.0 ± 0.58a 2.00 ± 0.26b 2.83 ± 0.40b 1.67 ± 0.33b 1.1Total PCE (Mn) 8.17 ± 1.25 95.5 ± 2.59a 54.33 ± 2.99b 61.5 ± 2.6b 17.83 ± 0.91b 6.49Repeated oral administrationPCE having one Mn 6.67 ± 0.67 77.67 ± 5.23a 41.20 ± 2.72b 49.33 ± 2.47b 13.17 ± 0.60b 8.41PCE having two Mn 1.17 ± 0.31 11.17 ± 0.48a 4.67 ± 0.33b 6.33 ± 0.62b 3.00 ± 0.37b 1.27PCE having more than two Mn 0.67 ± 0.21 8.00 ± 0.58a 1.17 ± 0.17b 1.67 ± 0.33b 0.67 ± 0.21b 1.45Total PCE (Mn) 8.5 ± 0.99 96.83 ± 5.61a 46.83 ± 2.82b 57.33 ± 2.19b 16.83 ± 1.01b 9.62

Group A: cypermethrin administration alone; Group B: cypermethrin + garlic extract; Group C: cypermethrin + l-ascorbic acid; Group D: cypermethrin + garlic extract + l-a ronuc

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scorbic acid; Mn: micronucleus; PCEs: polychromatic erythrocytes; PCE (Mn): mica Significantly different from control group.b Significantly different from group (A).

light microscope. For Mn analysis, 500 polychromatic erythrocytes (PCE) per ani-al were scored. Duplicate slides were examined for each animal to calculate the

CE (Mn) frequencies for each group per thousand cells.

.8.2. Chromosomal aberration assayAnimals were intra-peritoneally injected with colchicine (4 mg/kg bw) 2 h prior

o the scheduled time of sacrifice, in order to accumulate metaphase cells and pro-ide more readily analyzable chromosomes. The rats were sacrificed 30 h after theast dose for the chromosome aberration assay. Both femurs were dissected outnd cleaned of any adhering muscle, then bone-marrow cells were collected fromoth femurs by flushing in KCl (hypotonic solution, 0.56 mg %, incubated at 38 ◦C for5 min). The cell suspension was centrifuged at 101 × g for 10 min, fixed in aceto-ethanol (acetic acid:methanol, 1:3, v/v). Centrifugation and fixation (in the cold)ere repeated five times at 20-min intervals. The material was re-suspended in a

mall volume of fixative, dropped onto chilled slides, flame-dried, and stained theext day in 5% buffered Giemsa (pH 6.8). One hundred good-quality metaphasesontaining 42 chromosomes were examined per animal to score different aberra-ions.

.9. Statistical analysis

Data were expressed as arithmetic mean ± standard error of the mean (SEM).east significance difference test (LSD) was carried out with a significant probabilityevel of p < 0.05 [24]. Analyses of variance (ANOVA) were conducted to assess theignificance of the effects of treatment and time within each group, and to determineignificant differences among groups and periods.

. Results

.1. Induction of micronuclei in rat bone-marrow PCEs

The obtained data in Table 1 show that the tested pesti-ide cypermethrin induced a potential clastogenic effect in theone-marrow of treated rats, following a single administration137.5 mg/kg, 30 h) as evidenced by the significant increase inhe total number of bone-marrow micronucleated polychromaticrythrocytes [PCE (Mn)], and of frequencies of polychromatic ery-hrocytes (PCE) having one micronucleus (Mn), two micronuclei or

ore than two micronuclei, when compared with control rats.On the other hand, the simultaneous administration of cyper-

ethrin with crude garlic extract, with l-ascorbic acid, or with theombination of these two significantly decreased the total num-er of PCE (Mn), and the numbers of PCE having one Mn, twon and more than two Mn, when compared with those observed

fter cypermethrin alone. With the exception of the number ofCE having more than two micronuclei, the protective effect of

he combination of crude garlic extract and l-ascorbic acid wastronger than that of separate crude garlic extract and l-ascorbiccid, respectively. Nevertheless, the differences among the effectsf the three treatments on the number of PCE having more thanwo micronuclei were insignificant.

leated polychromatic erythrocytes; LSD: least significant difference.

Likely, the results displayed in Table 1 show that the totalnumber of bone-marrow PCE (Mn), and the frequencies of PCEhaving one Mn, two Mn and more than two Mn in the group ofrepeated doses of cypermethrin alone (55.1 mg/kg, 5 days) werehigher than those in control rats. It is also obvious that the con-comitant administration of cypermethrin with either crude garlicextract, l-ascorbic acid, or with the combination of these two sig-nificantly decreased the total number of PCE (Mn) and the numbersof PCE having one Mn, two Mn and more than two Mn when com-pared with the corresponding values when cypermethrin was givenalone. Except for the number of PCE having more than two Mn,the protective effect of the combined administration of crude gar-lic extract and l-ascorbic acid was stronger than that of separateadministration of these two agents. The results of the protectiveeffects of the three treatments on the number of PCE having morethan two micronuclei were not statistically different from eachother and from the control.

3.2. Induction of chromosomal aberrations in rat bone-marrow

3.2.1. Frequency of aberrant cellsTable 2 shows that rats given cypermethrin as a single treat-

ment (137.5 mg/kg, 30 h) have the highest number of aberrantcells, comprising cells having one and more than one aberrationin comparison with control rats and the other groups. Conversely,the concomitant individual administration of crude garlic extractor l-ascorbic acid with cypermethrin greatly increased the num-ber of normal cells by significantly decreasing the frequency ofaberrant cells. It is clear that the protective effect of crude garlicextract exceeded that of ascorbic acid. In addition, the number ofaberrant cells in rats that received the combination of crude garlicextract and l-ascorbic acid was statistically similar to that of controlrats.

The results also demonstrate that repeated administration ofcypermethrin (55.1 mg/kg, 5 days) induced a significant increasein the total number of aberrant cells, comprising cells with oneand more than one aberration, in comparison with the control. Incontrast, the effect of separate oral doses of crude garlic extractor l-ascorbic acid, combined with cypermethrin ameliorated theeffects of cypermethrin alone, albeit that the ameliorative effect of

crude garlic extract was stronger than that of l-ascorbic acid. It isworth noting that the number of the various aberrant cells (withone or more than one aberration) in rats that received combinedadministration of crude garlic extract and l-ascorbic acid and thatof control was statistically the same.

4 M.E. Assayed et al. / Mutation Research 702 (2010) 1–7

Table 2Effect of single and repeated oral administration of cypermethrin alone and with natural protectors on the frequency of aberrant cells in bone-marrow of albino rats.

Groups Control Group LSDP < 0.05

Aberrant cells A B C D

Single oral administrationCells with one aberr. 6.67 ± 1.20 22.33 ± 0.76a 11.67 ± 1.02b 15.83 ± 1.54b 7.17 ± 0.79b 3.206Cells with more than one aberr. 0.83 ± 0.31 15.5 ± 1.33a 2.17 ± 0.48b 5.33 ± 0.76b 1.17 ± 0.17b 2.15Total aberrant cells 7.17 ± 1.25 37.83 ± 0.95a 13.83 ± 1.35b 21.17 ± 1.17b 8.33 ± 0.88b 3.302Repeated oral administrationCells with one aberr. 5.67 ± 0.88 15.67 ± 1.30a 8.5 ± 1.05b 12.7 ± 1.28b 8.33 ± 0.61b 3.091Cells with more than one aberr. 0.50 ± 0.22 7.33 ± 0.71a 1.00 ± 0.36b 2.33 ± 0.42b 0.67 ± 0.33b 1.292Total aberrant cells 6.17 ± 1.01 23.0 ± 1.2a 9.5 ± 1.3b 15.0 ± 1.1b 9.0 ± 0.86b 3.247

Group A: cypermethrin administration alone; Group B: cypermethrin + garlic extract; Group C: cypermethrin + l-ascorbic acid; Group D: cypermethrin + garlic extract + l-a

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scorbic acid; aberr.: aberration(s); LSD: least significant difference.a Significantly different from control group.b Significantly different from group (A).

.2.2. Frequency of chromatid-type aberrationsTable 3 shows that cypermethrin alone induced a signifi-

ant increase in the numbers of chromatid gaps (tg), chromatidreaks (tb), chromatid deletions (td) and acentric fragments (aF)s chromatid-type structural aberrations in comparison with theontrol. Nonetheless, the separate dosings of crude garlic extractr l-ascorbic acid with cypermethrin led to significant reduction inhese structural aberrations. The ameliorating effect of crude garlicxtract was stronger than that of l-ascorbic acid on (td) chromatid-ype aberration. Furthermore, the alleviating effect of the combinedreatment with crude garlic extract and ascorbic acid resulted in aumber of chromatid-type structural aberrations that was statisti-ally similar to that of the control rats.

It is also evident that repeated administration with cyperme-hrin caused an increase in the frequencies of chromatid gapstg), chromatid breaks (tb), chromatid deletions (td), acentric frag-

ents (aF) and minutes or double minutes (min or Dmin) – thehromatid-type structural aberrations – in comparison with theontrol. But the individual administration of crude garlic extract or-ascorbic acid together with cypermethrin had the opposite effect

o treatment with cypermethrin alone; in addition, the effect ofrude garlic extract in decreasing the number of chromatid gapsmong the chromatid-type aberrations was stronger than that of-ascorbic acid. The difference between the numbers of chromatid-ype aberrations in the control rats and in rats that had received

able 3ffect of single and repeated oral administration of cypermethrin alone and with naturaone-marrow of albino rats.

Groups Control Group

Chromatid-type aberrations A

Single oral administrationChromatid gap (tg) 4.67 ± 0.88 11.83 ± 0.70a

Chromatid break (tb) 0.50 ± 0.34 9.33 ± 0.88a

Chromatid deletion (td) 0.33 ± 0.21 5.8 ± 0.47a

Acentric fragment (aF) 3.17 ± 0.07 13.83 ± 0.87a

Minute and double minute (min and Dmin) 0.83 ± 0.30 3.16 ± 0.60a

Triradial and quadriradial (Tr and Qr) 0.0 ± 0.0 0.5 ± 0.34Repeated oral administrationChromatid gap (tg) 2.67 ± 0.42 7.83 ± 0.60a

Chromatid break (tb) 0.33 ± 0.21 5.00 ± 0.73a

Chromatid deletion (td) 0.33 ± 0.33 3.83 ± 0.40a

Acentric fragment (aF) 2.5 ± 0.42 7.83 ± 0.94a

Minute and double minute (min and Dmin) 1.33 ± 0.49 4.66 ± 0.49a

Triradial and quadriradial (Tr and Qr) 0.0 ± 0.0 0.0 ± 0.0

roup A: cypermethrin administration alone; Group B: cypermethrin + garlic extract; Grscorbic acid; aberr.: aberration(s); LSD: least significant difference.

a Significantly different from control group.b Significantly different from group (A).

the combination of crude garlic extract and ascorbic acid was notstatistically significant.

3.2.3. Frequency of chromosome-type aberrationsData presented in Table 4 show that a single dose of

cypermethrin induced a significant increase in the numbers of chro-mosome gaps (Sg), chromosome breaks (Sb), ring chromosomes(R) and dicentric chromosomes (Dic) – i.e. the main categories ofchromosome-type aberrations – in comparison with the control.In contrast, parallel individual dosing either of crude garlic extractor l-ascorbic acid together with cypermethrin caused a significantreduction in the number of these chromosome-type aberrations incomparison with treatment with cypermethrin only. Moreover, theeffect of crude garlic extract in reducing the frequency of chromo-some gaps was stronger than that of l-ascorbic acid. The number ofchromosome-type structural aberrations in rats that had receivedthe combined treatment of crude garlic extract and l-ascorbic acidwas not statistically significantly different from that in controlrats.

The data also demonstrate that repeated cypermethrin treat-

ment resulted in a significant increase in the number ofchromosome gaps (Sg) and ring chromosomes (R) – i.e. forms ofchromosome-type aberrations – in comparison with that of con-trol. However, concomitant separate administration of crude garlicextract or l-ascorbic acid together with cypermethrin decreased

l putative protectors on the frequency of chromatid-type structural aberrations in

LSDP < 0.05

C D

6.5 ± 0.76b 8.66 ± 0.88b 3.33 ± 0.42b 2.2351.33 ± 0.61b 2.66 ± 0.42b 0.66 ± 0.42b 1.6621.83 ± 0.60b 3.16 ± 0.30b 0.66 ± 0.21b 1.1453.66 ± 0.66b 5.66 ± 0.76b 3.16 ± 0.40b 2.2962.80 ± 0.60 4.66 ± 0.84 2.33 ± 0.55 1.767

0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 –

3.83 ± 0.75b 6.00 ± 0.93b 3.00 ± 0.63b 2.0040.5 ± 0.22b 1.00 ± 0.25b 0.33 ± 0.21b 1.12

0.83 ± 0.30b 1.16 ± 0.03b 1.00 ± 0.24b 0.9474.00 ± 0.57b 5.83 ± 0.01b 4.00 ± 0.57b 2.1691.83 ± 0.30b 3.00 ± 0.55b 1.66 ± 0.33b 1.321

0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 –

oup C: cypermethrin + l-ascorbic acid; Group D: cypermethrin + garlic extract + l-

M.E. Assayed et al. / Mutation Research 702 (2010) 1–7 5

Table 4Effect of single and repeated oral administration of cypermethrin alone and with natural protectors on the frequency of chromosome-type structural aberrations in bone-marrow of albino rats.

Groups Control Group LSDP < 0.05

Chromosome-type aberrations A B C D

Single oral administrationChromosome gap (sg) 0.0 ± 0.0 8.83 ± 0.60a 0.50 ± 0.22b 1.83 ± 0.31b 0.17 ± 0.17b 0.951Chromosome break (sb) 0.0 ± 0.0 5.17 ± 0.60a 0.0 ± 0.0b 0.67 ± 0.33b 0.0 ± 0.0b 0.895Ring Chromosome (R) 0.0 ± 0.0 5.80 ± 0.78a 0.17 ± 0.17b 0.33 ± 0.21b 0.0 ± 0.0b 1.014Dicentric chromosome (Dic) 0.0 ± 0.0 1.00 ± 0.25a 0.0 ± 0.0b 0.0 ± 0.0b 0.0 ± 0.0b 0.434Repeated oral administrationChromosome gap (sg) 0.0 ± 0.0 3.33 ± 0.42a 0.17 ± 0.01b 0.5 ± 0.34b 0.0 ± 0.0b 0.732Chromosome break (sb) 0.0 ± 0.0 1.33 ± 0.21 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 –Ring chromosome (R) 0.0 ± 0.0 2.83 ± 0.3a 0.33 ± 0.21b 0.67 ± 0.33b 0.0 ± 0.0b 0.651Dicentric chromosome (Dic) 0.0 ± 0.0 0.17 ± 0.01 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 –

Group A: cypermethrin administration alone; Group B: cypermethrin + garlic extract; Group C: cypermethrin + l-ascorbic acid; Group D: cypermethrin + garlic extract + l-a

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scorbic acid; aberr.: aberration(s); LSD: least significant difference.a Significantly different from control group.b Significantly different from group (A).

he number of these chromosome-type aberrations in comparisonith cypermethrin alone. In addition, the reducing effect of the

ombination of both these natural protectors on the number of thisype of aberration was more obvious than for each one alone.

. Discussion

In the assessment and evaluation of the toxic characteristics ofsubstance, the determination of acute oral toxicity is usually an

nitial step that provides information on health hazards likely torise from a short-term exposure by the oral route [25]. The oraledian lethal dose (oral LD50) of the cypermethrin formulation

ested here was calculated in our previous work to be 275.5 mg/kgw in male Wistar rats [26]. Concerning cytogenetic effects ofypermethrin, our results clearly indicate a significant inductionf micronucleated polychromatic erythrocytes and a significantncrease in the frequency of structural chromosomal aberrations –omprising chromatid-type and chromosome-type aberrations – inhe bone-marrow of rats exposed to both single and repeated dosesf cypermethrin, which is indicative of potential clastogenicity.

The genetic toxicity/mutagenicity studies with syntheticyrethroids have produced controversial results depending on theompound or the assay used. Many previous studies assessing thelastogenicity of these compounds in rodent bone-marrow [27–32]gree with our findings. In contrast, our results disagree with thoseeported by Puig et al. [33] and El-Ashmawy et al. [34], showinghe absence of genotoxic potential of cypermethrin in cultureduman peripheral blood lymphocytes and of allethrin in chromo-omes of rat testicular cells, respectively. Additional factors such ashe different source, different purity, different strains; sub-strainsf animals, doses and treatment schedules, manufacturing processnd the cis:trans ratio of the cypermethrin used must be takennto consideration to explain the inconsistent results with respecto the induction of cytotoxic and genotoxic effects [35]. Unfor-unately, the chemical nature and exact amounts of emulsifiers,tabilizers and solvents, present as additives in the commercial for-ulation of cypermethrin, were not available to us in order to run

arallel controls and rule out any possibility of their contributiono the observed effects. Generally, however, they are often statedo be naturally occurring chemicals, nontoxic to humans, domes-

ic animals and wildlife and of little concern for residual toxicity.nduction of micronuclei and/or chromosomal aberrations by type-I synthetic pyrethroids in rodent bone-marrow cells, as revealed inrevious studies, prompted us, however, to attribute the observedffects solely to the test pyrethroid.

The micronucleus (Mn) is composed of small chromatin frag-ments, which arise from chromosome breaks after clastogenicaction, or whole chromosomes that do not migrate during anaphaseas a result of aneugenic effects. The efficacy of the micronucleustest as an indicator of structural genomic damage has alreadybeen proven and the test has been successfully used as a mea-sure of genotoxic stress, under both laboratory and field conditions[36]. The evaluation of chromosome aberrations is a fully acceptedmethod to reveal genotoxicity, as it is indicative of real geneticeffects [37]. The production of chromosomal aberrations (CA) isa complex cellular process, with mechanisms of chromosomebreakage and re-joining that are not yet completely understood.According to the prevailing theories, structural CA result from: (i)direct DNA breakage, (ii) replication on a damaged DNA template,and (iii) inhibition of DNA synthesis. Under in vivo conditions, thegenotoxicity and in particular the clastogenic potential of an agentis often evaluated by use of the CA assay [23].

The present data of our investigation and those of similar pre-vious studies clearly indicate that cypermethrin possesses thepotential to interact with and to cause alterations in the cellu-lar DNA in mammalian cells in vivo. As previously mentioned, theconsistent correlation of the mutagenic and carcinogenic poten-tial of a variety of chemicals, particularly clastogens, has validatedthe cytogenetic investigation of pyrethroid insecticides, despitethe uncertainties about its molecular mechanisms [38]. Others[39] reported that cypermethrin shows both tumour-initiatingand tumour-promoting potential along with complete carcino-genic activity in mouse skin in long-term in vivo carcinogenicityassays. The authors suggested that the tumour-initiating propertyof cypermethrin observed in their investigation may be attributedto several factors: first, its ability to interact with DNA and dam-age its structure. Such interactions are critical for the initiation ofcells to transform into neo-plastic cells. A second possible mecha-nism is the induction of microsomal enzymes. Covalent binding ofsome pyrethroids to hepatic microsomal proteins has suggested theinvolvement of cytochrome P450-dependent mono-oxygenases inthe metabolism of these pesticides [40].

Our results in Tables 2–4 show that repeated doses of cyperme-thrin seem to have a lower toxicity than a single dose. This outcomecould be explained by the fact that cypermethrin, as a synthetic

pyrethroid, has no tendency to accumulate and concentrate in theanimal body; hence a single large dose (1/2 LD50 = 137.75 mg/kgbw) would be more effective in producing chromosomal damagein sensitive bone-marrow cells, as such dose would overwhelmthe capacity of repair mechanisms within a short time interval

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30 h). Meanwhile, repeated mild doses of the insecticide (1/5D50 = 55.1 mg/kg) may not cause such harm to cells every time theyere administered. Such reduction in clastogenicity is supposed to

e observed because the error-free repair mechanism of the cellperates more efficiently and favorably at low dose rates than atigh dose rates (or a supra-threshold dose). Additionally, in case ofepeated dosing, there is an interval long enough for many of theamaged cells to undergo apoptosis or “programmed cell death”;hese dead cells would be missing from the analysis, which willegatively reflect on the total frequencies of aberrant cells and/orotal aberrations.

Considerable emphasis is put on the use of natural dietary con-tituents as a chemoprotective measure to control genetic diseases.one-marrow cytogenetics is a useful short-term technique for elu-idating the mechanism as well as to assess substances for theirlastogenic and anti-clastogenic activity [41]. The data obtained inhis investigation revealed the anti-mutagenic potential of garlicxtract against chromosomal damage induced by cypermethrin.tudies on anti-mutagenic effects of garlic extract against pes-icides in general or against synthetic pyrethroids in particularre not available in the literature. Earlier studies conducted witharlic, however, showed that sulphhydryl compounds and otherrgano-sulphur compounds (such as diallyl sulphide (DAS), dial-yl disulphide (DADS), ajoene, allixin, allyl mercaptans and allyl

ethyl sulphides) are implicated in its anti-mutagenic and anti-arcinogenic effects [42]. Anti-genotoxic agents, especially thoseresent in natural substances, act through different cellular path-ays involving endogenous sequestration of mutagens by various

nzymes. The mechanism for protection of garlic involves scaveng-ng potentially toxic and mutagenic electrophiles and free radicalsnd modification of phase-II enzymes and the phase-I profile,hich enhances detoxification pathways. The allyl group of garlic

onstituents enhances the level of glutathione-S-transferase (GST)hereby accelerating the detoxification of mutagens and carcino-ens [43]. Interference of the thiol moiety of sulphur compoundsn garlic, particularly diallyl sulphide (DAS), with microsomalnzymes that induce inactivation of genotoxic metabolites haseen suggested [44]. Once more, intoxication with cypermethrin

s reported to cause free radical-mediated tissue damage and toeduce total GSH in rats [45–47]. DNA damage caused by reactivexygen species such as hydroxyl radicals, hydrogen peroxides andinglet oxygen has been implicated in mutagenesis, oncogenesisnd ageing [48]. Oxidative lesions in DNA include base modifi-ations, sugar damage, strand breaks and a-basic sites [49,50].xidant-induced DNA damage and mutagenesis are determinants

n the multistage process of cancer; inhibition of these events byhytochemical antioxidants may reduce the risk of the disease51]. Allixin, an important flavonoid in garlic extract, which haseen shown to prevent oxidative modification of LDL [52] andumour promotion [53], inhibits aflatoxin-induced DNA damagend mutagenesis in Salmonella typhimurium in part through theeduction in the DNA-damaging oxidant byproducts that occur dur-ng the induction of P450 enzymes [54]. Garlic extract was furtherroven to enhance the activities of endogenous radical-scavengingnzymes as well. The presence of garlic extract in endothelial cellultures subject to oxidant stress generated increased levels of SOD,atalase, and glutathione peroxidase and suppressed – in a dose-nd time-related fashion – the production of superoxide radicalnd hydrogen peroxide [55]. Additionally, it has become obviousrom the in vivo studies with rodents that some of the chemicalonstituents of garlic can enhance the activity of detoxification

nzymes such as glutathione-S-transferase, glutathione peroxidasend glutathione reductase [56]. It can also induce a number of drug-etabolizing enzymes in liver tissue such as the phase-II enzymesST and the conjugating enzyme, gamma-glutamyltranspeptidase

�GT) [57].

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esearch 702 (2010) 1–7

On the other hand, this study demonstrates that oral admin-istration of l-ascorbic acid (vitamin C) had the ability to reducethe mutagenic effect of cypermethrin, as indicated by the sig-nificant reduction in micronucleated polychromatic erythrocytesand structural chromosomal aberrations. Our results are consistentwith those of Geetanjali et al. [58] who demonstrated that micegiven the organophosphorous insecticide dimethoate and ascorbicacid simultaneously, showed a very low frequency of micronuclei inbone-marrow cells in vivo, which did not differ significantly fromcontrol values in comparison with corresponding values in micegiven the pesticide alone. Our results are also in line with thoserecorded previously [19,59] concerning the anti-clastogenic effectof vitamin C on the metaphase chromosomes of bone-marrow inmice exposed in vivo to the pesticides malathion and rogor, andto endosulphan, phosphamidon or mancozeb, respectively. Highintracellular concentrations of ascorbic acid (vitamin C) reducedthe mutations caused by oxidative stress in human cells in vitroand such reduction is indicative of the role played by vitamin Cin preventing DNA mutagenesis in humans [49]. Recently, someauthors suggested that high doses of ascorbic acid increased thetotal antioxidant status (TAS) in human plasma, and ascorbic acidcould be useful as a free-radical scavenger for paraquat-poisonedpatients [60]. Thus, it is very well possible to assume that vitaminC can provide antioxidant-cellular protection against the oxidativetoxicity of cypermethrin [61].

Conflict of interest

The authors declare that there are no conflicts of interest.

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