NeuroToxicology 33 (2012) 280–289

The protective effect of green tea extract on lead induced oxidative and DNAdamage on rat brain

A.A. Khalaf a,1, Walaa A. Moselhy b,*, Marwa I. Abdel-Hamed c,1

a Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Cairo University, Giza, Egyptb Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egyptc Department of Biochemistry, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

A R T I C L E I N F O

Article history:

Received 22 September 2011

Accepted 2 February 2012

Available online 11 February 2012

Keywords:

Albino rats

Lead

Neurotoxicity

Green tea

Oxidative stress

A B S T R A C T

The role of green tea in protection against neurotoxicity induced by lead acetate was investigated in rats.

Five equal groups, each of ten rats were used. The first group was served as control, the second, third, and

fourth groups were given lead acetate, lead acetate and green tea, and green tea only, respectively, for

one month, the fifth group was administered lead acetate for one month followed by green tea for 15

days. Lead acetate was given orally at a dose of 100 mg/kg b. wt, while green tea was given in drinking

water at a concentration of 5 g/L. Lead acetate administration induced loss of body weight and decreased

concentration of reduced glutathione and SOD activity in brain tissues as well as significantly high DNA

fragmentation and pathological changes. Co-administration of green tea with lead acetate significantly

alleviated these adverse effects.

� 2012 Elsevier Inc. All rights reserved.

Contents lists available at SciVerse ScienceDirect

NeuroToxicology

1. Introduction

In recent years, the level of heavy metals, particularly lead hasincreased in air, water and soil in both urban and periurban areas(Gupta, 2007). It is well known that heavy metals induce toxic effectson different systems and apparatuses. Furthermore, because of theirlong half-life, heavy metals also induce accumulation phenomena,which in turn produce an experimental increase of their concentra-tion in blood and tissues. Besides their effects and their implicationin chronic respiratory disease, there is a risk that heavy metalsintoxication may lead to damage of the nervous system (Mameliet al., 2001). Among heavy metals, lead represents the mainenvironmental poison. This pollutant causes well documentedneurological impairment (Soong et al., 1999). Lead is a non essentialtoxic heavy metal widely distributed in the environment andchronic exposure to low levels of lead has been a matter of publichealth concern in many countries (Moreira et al., 2001). A number ofstudies demonstrated that prolonged exposure to lead inducesslower nerve conduction and an alteration of calcium homeostasis(Orrenius et al., 1992). Because neurotransmitter release is alsodependent on calcium transport, lead intoxication induces severeimpairment to the dopaminergic system, acting both directly on

* Corresponding author. Tel.: +20 822327982; fax: +20 822327982.

E-mail addresses: drwalaamoselhy@yahoo.com (W.A. Moselhy),

marwa99@yahoo.com (M.I. Abdel-Hamed).1 Tel.: +20 822327982; fax: +20 822327982.

0161-813X/$ – see front matter � 2012 Elsevier Inc. All rights reserved.

doi:10.1016/j.neuro.2012.02.003

neurotransmitter synthesis and indirectly on calcium transport. As aconsequence, the signal conduction and transmission in neuronalcircuits of the CNS and peripheral nervous system could be seriouslyimpaired (Mameli et al., 2001). Catecholaminergic and serotonergictransmission in brain have been altered by lead (Antonio and Leret,2000). The neurotoxicity associated with lead exposure may be theresult of a series of small perturbations in brain metabolism, wherelead enhances lipid peroxidation through inhibition of superoxidedismutase and other related enzymes (Villeda-Hernandez et al.,2001). Moreover, lead may damage the membrane entirely byinactivation of the essential thiol group membrane proteins (Herak-Kramberger and Sabolic, 2001). One possible molecular mechanisminvolved in lead neurotoxicity is the disruption of the prooxidant/antioxidant balance, which can lead to brain injury via oxidativedamage to critical biomolecules, such as lipids, proteins and DNA(Adonaylo and Oteiza, 1999a). Also lead exposure results in changesin neuronal mitochondria, including swelling and partial loss ofcristae, in addition partial chromatin dissolution and dispersedarrangement of the rough endoplasmic reticulum (Zhang et al.,2009).

The antioxidant capacity of dietary components has been linkedto the prevention of cancer, cardiovascular disease, neurodegen-erative disease, and other degenerative diseases (Duthie et al.,2000; Koo and Cho, 2004). Dietary components, such as fruits,vegetables, spices and extracts of herbs, particularly tea have beenstudied for their antioxidant properties in vitro (Pellegrini et al.,2006). It is known that tea, one of the most popular beveragesconsumed world wide by the man, has received a great deal of

A.A. Khalaf et al. / NeuroToxicology 33 (2012) 280–289 281

attention regarding to its possible contribution in prevention ofcoronary artery and capillary resistance activity as well as tumorprogression and cardiovascular diseases (Dreosti et al., 1997).Green tea polyphenols have been reported to reduce aortic lesionformation and diminished lipid oxidation, indicating an athero-sclerotic property (Cook and Sammn, 1996; Tijburg et al., 1997).Several studies have demonstrated that green tea componentsscavenge reactive oxygen species (Guo et al., 1996) that areformed in vitro by the various systems, resulting in changes incellular biomembranes. Several compounds of green tea havebeen reported to suppress lipid peroxidation in model systemsand in several biological systems, such as mitochondria ormicrosomes (Middleton et al., 2000). One of such potentiallyhealth-promoting beverages is green tea, whose components aremainly catechins and catechin derivatives which have antioxi-dant properties (Guo et al., 1996; Rice-Evans et al., 1996). In thisperspective, green tea (GT) has been in focus, as it is a rich sourceof brain-accessible polyphenols (Abd El Mohsen et al., 2002;Mandel et al., 2006; Youdim et al., 2004). Many of thesecompounds are monomeric catechins, which have been shown toexert antioxidant effects acting directly as radical scavengers ormetal-chelators and indirectly through modulation of transcrip-tion factors or enzymes (Cabrera et al., 2006; Higdon and Frei,2003). Furthermore, favorable effects of GT catechins on brainage. The protective effects of the green tea extract on the centralnervous tissue expression in decreased level of lipid peroxida-tion products were shown (Graham, 1992; Rice-Evans et al.,1996). Hence, the goal of the present study has been toinvestigate the efficacy of green tea, as a source of water-soluble antioxidants on brain of mature rats exposed to anoxidative stress induced by lead.

2. Materials and methods

2.1. Experimental animals

Fifty mature male albino rats were obtained from the Breedingunit of the Veterinary Hygiene and Management Department,Faculty of Veterinary Medicine, Cairo University. The animals werehoused in cages at identical conditions of the animal housing. Feedand water were provided ad libitum.

2.2. Chemicals

Lead acetate (C4H6O4Pb�H2O) with molecular weight of 379.33in the form of pure crystals was obtained from Riedel-De Haen AG-Seelze-Hannover Germany.

Green tea. Lipton Green Tea Unilever brand, packed in theUnited Arab Emirates by Unilever Gulf FZE was dissolved in thedrinking water at a concentration of 5 g/L. Tea was preparedfreshly three times per week and stored at 4 8C until use. Thecontent of drinking vessels was renewed every day. Green teaextract contained epigallocatechin gallate (337 mg/l), epigalloca-techin (268 mg/l), epicatechin (90 mg/l), epicatechin gallate(60 mg/l), and coffeic acid (35 mg/l) as determined by HPLCmethod (Maiani et al., 1977).

Biodiagnostic Kits for determination of glutathione reduced(GSH), super oxide dismutase (SOD) and total antioxidant capacitywere obtained from bio-diagnostic company, Egypt, in addition toa Kit of DNA extraction for agarose gel electrophoresis wasperformed using DNeasy Qiagen kit (catmo s 69504).

2.3. Animals and treatment

Fifty experimental mature male rats were randomly assignedinto five equal groups:

(1) Group I: contained 10 male rats without any treatment andserved as a control group.

(2) Group II: received lead acetate by stomach tube in a dose of100 mg/kg b. wt for one month (Zhang et al., 2009).

(3) Group III: received lead acetate as in group II besides green teain drinking water at a concentration of 5 g/L (Skrzydlewskaet al., 2002) for one month.

(4) Group IV: received green tea only in drinking water asdescribed in group III.

(5) Group V (withdrawal group): lead acetate intubation is stoppedafter one month and replaced by green tea in the drinkingwater at a concentration of 5 g/L for 15 days.

2.4. Tissue preparation and methods

After one month, ten rats from each group were taken; bodyweight was done and sacrificed for obtaining brain samples whichweighed and prepared to study biochemical, DNA and histopatho-logical changes in the brain tissue of the treated rats. Ten rats werealso taken from group V after the withdrawal time (15 days) andsacrificed under the anaesthesia to obtain brain tissue.

2.4.1. Brain tissue for biochemical assay

Prior to dissection, brain tissue was perfused with phosphatebuffer saline (50 mM potassium phosphate pH 7.4 containing0.16 mg/ml heparin) to remove any red blood cells and clots. Thetissue was homogenized in 5–10 ml cold buffer (i.e. �50 mMpotassium phosphate which composed of 9.4 ml of 1 M monobasicsolution and 40.6 ml of 1 M dibasic solution and complete to 1 L bydistilled water, pH 5.1 mM EDTA) per gram tissue using tissuehomogenizer and centrifuged at 4000 rpm for 15 min at 4 8C. Thesupernatant was separated for assay and stored on ice. The finalsupernatant was washed and subjected to assay the activity ofreduced glutathione (Beutler et al., 1963), activity of superoxidedismutase (Nishikimi et al., 1972) and concentration of totalantioxidant (Koracevic et al., 2001).

2.4.2. Brain tissue for DNA fragmentation

Brain tissue was lysed in 1 ml buffer (10 mM Tris–HCl, pH 7.4,10 mM EDTA, 0.5% Triton X-100) by the method of Sellins andCohen (1987). The pellets containing total intact DNA (designatedP) and the supernatants containing smaller fragments of DNA(designated S) were treated separately with 0.5 ml of 25%trichloroacetic acid (TCA). Both the sets were left overnight at4 8C and DNA precipitated was collected by centrifugation. Eachsample was treated with 80 ml of 5% TCA followed by heattreatment at 90 8C for 15 min. Freshly prepared 1 ml diphenyl-amine (DPA) reagent was added in each sample, tubes wereallowed to stand overnight at room temperature and OD wasrecorded at 600 nm. Percent DNA fragmentation was calculated as:% DNA fragmentation = [S/(S + P)] � 100.

2.4.2.1. Agarose gel electrophoresis of fragmented DNA. For electro-phoretic analysis of fragmented DNA, the total nuclear DNA wasisolated from tissue according to the method of Kuo et al. (2005).Briefly, tissue were homogenized in 1 ml of lysis buffer [20 mMTris–Cl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100 and 25 mM Na2 pyrophosphate] at 37 8C for 1 h. Toprecipitate out proteins, 0.4 ml of saturated NaCl was added ineach set of cell lysate, tubes were left on ice for 5 min andcentrifuged at 3000 � g for 30 min. DNA was then precipitated byadding two times chilled ethanol (v/v). Samples were frozen at�20 8C overnight. The DNA precipitated was collected bycentrifugation and dissolved in TAE buffer (40 mM Tris-aceta-te + 1 mM EDTA). The DNA samples were prepared in a loading

Table 1Mean values of brain/body weight ratio in exposed rats (mean � S.E.).

Group

C

Group I

Pb

Group II

Pb + GT

Group III

GT

Group IV

Withd

Group V

Brain weight (%) 1.564 � 0.0350 1.506 � 0.0280a 1.516 � 0.0389a 1.556 � 0.0361b 1.543 � 0.0423a,b

Values are means � S.E.; N (number of animals) = 10; LSD (least significant difference) at the 5%level = 0.01650. aSuperscript in the same row differ significantly at P < 0.05 with

control (C). bSuperscript in the same row differ significantly at P < 0.05 with lead group (Pb).

Fig. 1. Mean values of brain/body weight ratio in exposed rats (mean � S.E.).

A.A. Khalaf et al. / NeuroToxicology 33 (2012) 280–289282

solution (0.25% bromophenol blue, 0.25% xylene cyanol FF and 30%glycerol) in the ratio of 1:5. The samples containing10 mg DNAwere loaded in each well of 1% agarose gel containing 0.5 mg/mlethidium bromide. The electrophoresis was carried out in TAEbuffer for 2–3 h. The DNA bands in gel were observed under UVtrans-illuminator and photographed.

2.4.3. Brain tissue for histopathological study

Autopsy samples were taken from the brain of rats in differentgroups and fixed in 10% formal saline for 24 h. Washing was donein tap water then serial dilutions of alcohol (methyl, ethyl andabsolute ethyl) were used for dehydration. Specimens were clearedin xylene and embedded in paraffin at 56 8C in hot air oven for 24 h.Paraffin bees wax tissue blocks were prepared for sectioning at4 mm by slidge microtome. The obtained tissue sections werecollected on glass slides, deparffinized and stained by hematoxylin

Table 3Mean values of superoxide dismutase activity (SOD) in brain homogenate (U/g tissue)

Brain region Group

C

Group I

Pb

Group II

Cerebrum (Cr) 611.409 � 14.166 475.952 � 27.615a

Cerebellum (Cl) 574.614 � 17.560 426.111 � 14.262a

Medulla oblongata (Mo) 610.410 � 13.886 541.076 � 13.267a

Values are means � S.E.; N (number of animals) = 10; LSD (least significant difference) at th

control (C). bSuperscript in the same row differ significantly at P < 0.05 with lead group (P

Table 2Mean values of reduced glutathione (GSH) in brain homogenate (mg/g tissue) in expos

Brain region Group

C

Group I

Pb

Group II

Cerebrum (Cr) 7.3324 � 0.0729 5.3304 � 0.0731a

Cerebellum (Cl) 7.0134 � 0.149 4.8794 � 0.0902a

Medulla oblongata (Mo) 6.8952 � 0.2389 4.4234 � 0.1212a

Values are means � S.E.; N (number of animals) = 10; LSD (least significant difference) at t

control (C). bSuperscript in the same row differ significantly at P < 0.05 with lead group (P

and eosin stains according to Banchroft et al. (1996) forhistopathological examination.

2.5. Statistical analysis

Data were expressed as mean � standard error. The data wereanalyzed with one-way analysis of variance (ANOVA).

3. Results

3.1. Brain/body weight ratio

The present data revealed highly significant reduction in brain/body weight ratio in lead exposed rats and lead – green tea group,while in withdrawal group, significant decrease was recorded(Table 1 and Fig. 1).

3.2. Antioxidant enzyme activities

The concentration of glutathione reduced (GSH), activity ofsuperoxide dismutase (SOD) and the concentration of totalantioxidant in brain homogenate of experimental rats wererecorded in Tables 2–4 and Figs. 2–4. It was shown that leadacetate orally administered to male rats induced significantdecrease in the concentration of reduced glutathione, and activityof SOD and concentration of total antioxidant in the three regionsof brain (cerebrum, cerebellum and medulla oblongata) incomparison to the control group, while co-administration of greentea extract with lead acetate improves this inhibition. The sameimprovement was obvious in withdrawal group. Rats treated onlywith green tea extract showed higher activation of the antioxidantenzymes.

in exposed rats (mean � S.E.).

Pb + GT

Group III

GT

Group IV

Withd

Group V

553.739 � 14.034b 657.507 � 14.145b 611.471 � 14.105b

542.376 � 14.018b 611.23 � 13.414b 564.61 � 20.771b

597.691 � 14.202 667.845 � 14.533b 623.306 � 21.748b

e 5%level = 65.01664. aSuperscript in the same row differ significantly at P < 0.05 with

b).

ed rats (mean � S.E.).

Pb + GT

Group III

GT

Group IV

Withd

Group V

6.5924 � 0.1311b 7.8664 � 0.0730b 6.6916 � 0.1704b

6.9456 � 0.1912b 6.5414 � 0.2533b 6.6988 � 0.2407b

6.7659 � 0.1119b 6.9774 � 0.2885b 5.8628 � 0.2480b

he 5%level = 1.2414. aSuperscript in the same row differ significantly at P < 0.05 with

b).

Table 4Mean values of total antioxidant concentration in brain homogenate (mM/g tissue) in exposed rats (mean � S.E.).

Brain region Group

C

Group I

Pb

Group II

Pb + GT

Group III

GT

Group IV

Withd

Group V

Cerebrum (Cr) 0.7802 � 0.0128 0.4754 � 0.0221a 0.686 � 0.01030ab 0.8056 � 0.0061b 0.7492 � 0.0157b

Cerebellum (Cl) 0.774 � 0.0090 0.4718 � 0.0106a 0.6676 � 0.0066ab 0.7772 � 0.0093b 0.7436 � 0.0152b

Medulla oblongata (Mo) 0.7812 � 0.0131 0.5514 � 0.0108a 0.7232 � 0.0078ab 0.8282 � 0.0080b 0.8086 � 0.0057b

Values are means � S.E.; N (number of animals) = 10; LSD (least significant difference) at the 5%level = 0.056990. aSuperscript in the same row differ significantly at P < 0.05 with

control (C). bSuperscript in the same row differ significantly at P < 0.05 with lead group (Pb).

Fig. 2. Mean values of reduced glutathione (GSH) in brain homogenate (mg/g tissue)

in exposed rats (mean � S.E.).

Fig. 4. Mean values of total antioxidant concentration in brain homogenate (mM/g

tissue) in exposed rats (mean � S.E.).

Fig. 5. Mean values of DNA fragmentation in brain regions of experimental lead

treated male rats.

A.A. Khalaf et al. / NeuroToxicology 33 (2012) 280–289 283

3.3. DNA fragmentation assay

Results of DNA fragmentation assay were illustrated in Figs. 5–8and Table 5. Figures revealed that lead induced highly significantpercentages of DNA fragmentation in cerebrum, cerebellum andmedulla oblongata of brain in treated rats. This effect was moremarked in cerebellum than the other regions. Co-administration ofgreen tea extract caused marked inhibition in percentage of DNAfragmentation. The same finding was also shown in withdrawalgroup. In group treated only with green tea extract, the percentageof DNA fragmentation is similar to that of control group or lower.

3.4. Histopathological findings

Histopathological studies of brain samples, showed normalhistopathological structure in control and green tea treated groups,while in lead treated rats, oedema was observed in the

Fig. 3. Mean values of superoxide dismutase activity (SOD) in brain homogenate (U/

g tissue) in exposed rats (mean � S.E.).

hippocampus area and between the cells (Fig. 9) associated withfocal gliosis in the cerebrum (Fig. 10). The cerebellum showedvacuolization (Fig. 11), while the medulla oblongata had neuronaldegeneration and gliosis (Fig. 12). Co-administration of green teaextract with lead acetate ameliorates the histopathologicalchanges and only pericellular and perivascular oedema weredetected in the hippocampus (Fig. 13). In withdrawal group, thechanges were constricted in presence of oedema in the perivas-cular area of the blood capillaries in the cerebrum (Fig. 14).

4. Discussion

Lead is ubiquitously environmental and industrial pollutantthat has been detected in nearly all phases of environment andbiological system. Its persistence in human and animal tissues hasquite often been associated with considerable health risks (Juberget al., 2006).

Fig. 6. The DNA was electrophoresed on TAE agarose gel for 1.5 h at 80 V. The DNA

fragments were visualized by staining with ethidium bromide. Samples are

cerebellum. Lane 1: negative control group; Lane 2: green tea group; Lane 3: lead

group; Lane 4: lead and green tea group; Lane 5: withdrawal group; M: 100 bp DNA

Ladder.

Fig. 7. The DNA was electrophoresed on TAE agarose gel for 1.5 h at 80 V. The DNA

fragments were visualized by staining with ethidium bromide. Samples are

cerebrum. Lane1: negative control group; Lane 2: green tea group; Lane 3: lead

group; Lane 4: lead and green tea group; Lane 5: withdrawal group; M: 100 bp DNA

ladder.

Fig. 8. The DNA was electrophoresed on TAE agarose gel DNA fragments were

visualized by staining with ethidium bromide. Samples are medulla oblongata. Lane

1: negative control group; Lane 2: lead group; Lane 3: green tea group; Lane 4: lead

and green tea group; Lane 5: withdrawal group; M: 100 bp DNA Ladder.

Table 5Mean values of DNA fragmentation in brain regions of experimental lead-treated male

Cerebellum

Group I (c) 24.10 � 0.1265

Group II (Pb) 51.44 � 0.1690a

Group III (Pb + GT) 35.42 � 0.1281b

Group IV (GT) 23.26 � 0.1327b

Group V (withdrawal) 18.12 � 0.1068b

Values are means � S.E.; N (number of animals) = 10; LSD (least significant difference) at the

control (C). bSuperscript in the same column differ significantly at P < 0.05 with lead grou

A.A. Khalaf et al. / NeuroToxicology 33 (2012) 280–289284

4.1. Body and brain weight

Lead poisoning is a potential factor in brain damage, mentalimpairment and several behavioural problems, as well as anaemia,kidney insufficiency, neuromuscular weakness, and coma (Liujiet al., 2002). In the current study, exposure of rats to lead acetatevia stomach intubation at a dose level of 100 mg/kg b. wt for onemonth induced significant reduction in the mean value of brain/body weight ratio. In parallel with our results those mentioned byAdonaylo and Oteiza (1999a) which revealed that rats treated withlead acetate 1 g/L drinking water for 8 weeks, showed significantdecrease in body weight compared to the control group. AlsoAntonio et al. (2003) reported that lead produced a statisticallysignificant decrease in the weight of the pups and their brain atpost natal days 0 and 21 of dams consumed a drinking watercontaining 300 mg/L lead. Co-administration of green tea extractwith lead acetate in treated rats failed to improve the reduction inthe brain/body weight ratio, while in withdrawal group, slightimprovement was observed. The failure of green tea extract toimprove the significant reduction of brain/body weight ratio wasattributed to what mentioned by several anthers. Sayama et al.(2000) demonstrated that green tea has anti-obesity effects infemale mice fed on diets containing 1, 2 and 4% green tea powderfor 16 weeks, after the administration of green tea, the ovaries,kidneys, adrenals, livers, spleens, brains, pituitary and intraperito-neal adipose tissues in mice were weighed, it was found that body

rats.

Cerebrum Medulla oblongata

26.34 � 0.1691 37.13 � 0.1490

40.28 � 0.1855a 49.36 � 0.1327a

30.66 � 0.1400 41.12 � 0.1020

17.66 � 0.1860b 37.48 � 0.1319b

21.48 � 0.1594b 28.34 � 0.1400b

5%level = 12.56975. aSuperscript in the same column differ significantly at P < 0.05 with

p (Pb).

Fig. 9. Brain of rat in group II showing severe oedema in the hippocampus (o).

Fig. 11. Brain of rat in group II showing vacuolization in the cerebellum (v). E�40.

A.A. Khalaf et al. / NeuroToxicology 33 (2012) 280–289 285

weight increase and intraperitoneal adipose tissues were remark-ably suppressed by the administration of diets containing 2 and 4%green tea powder. Also Zheng et al. (2004) studied the anti-obesityof three major components of green tea, catechins, caffeine andtheanine, female ICR mice were fed on diets containing 2% greentea powder and diets containing 0.3% catechins, 0.05% caffeine and0.03% theamine which correspond respectively to their concen-tration in a 2% green tea powder diets, singly and in combinationfor 16 weeks. They revealed that body gain was significantly

Fig. 10. Brain of rat in group II showing focal gliosis in cerebrum (g). H&E�40.

suppressed by green tea powder (GTP) diets from the 4th, 8th and12th week until end of feeding respectively. They attributed theanti-obesity effect of caffeine in GTP due to enhancement of fatmetabolism. Robertson et al. (1978) and Jung et al. (1981) reportedthat caffeine ingestion elevated the metabolic rate and fatoxidation in vivo through lipolysis in fat cells and the release ofcatecholamines. Moreover, caffeine enhanced nor-adrenaline oradrenaline induced lipolysis in fat cells. (Dulloo et al., 1992; Hanet al., 1999). In the same time, rats treated only with green teaextract had brain/body weight ratio similar to that of controlgroup. In this respect, Assuncao et al. (2011) recorded that meanbody weight of rats treated with green tea extract was similaramong all groups and control, while Khan et al. (2007)demonstrated that rats treated with green tea extract resultedin slight loss of body weight compared with control rats. Theyattributed this reduction to the lowering of blood glucose,cholesterol and protein. These observations are in partial

Fig. 12. Brain of rat in group II showing neuronal degeneration and gliosis in the

medulla oblongata. E�40.

Fig. 13. Brain of rat in group III showing pericellular and perivascular edema in the

hippocampus (c). E�40.

A.A. Khalaf et al. / NeuroToxicology 33 (2012) 280–289286

agreement with some previous reports in humans and animals(Imai and Nakachi, 1995).

4.2. Antioxidant enzymes activity

Exposure to environmental contaminants such as lead, involvesmany complex processes which can be evaluated by antioxidantenzyme activity as well as by lipid peroxidation measures (Romeoet al., 2000). The obtained data revealed that lead may inducesignificant inhibition in the activities of superoxide dismutase(SOD) and concentration of total antioxidant and reducedglutathione (GSH), in cerebrum, cerebellum and medulla oblon-gata of affected rats.

Fig. 14. Brain of rat in group V showing perivascular edema in the cerebrum (c).

E�40.

Our results are in correlation with those mentioned by Moreiraet al. (2001) who revealed significant decrease in the activities ofSOD in the lead-exposed 23-day old pups of rats. Someinvestigators evaluated the activity of antioxidant enzymes inbrain regions instead of whole brain because different regions mayrespond differently to oxidative stress (Sandhir et al., 1994; Shuklaet al., 1988). Also, our findings are in parallel with the results ofWang et al. (2006) who mentioned that Pb-exposed 21 day oldmouse pups showed significant decrease in the activities ofsuperoxide dismutase, glutathione peroxidase (GSH-Px) andglutathione reductase (GSH-Re) in hypothalamus, corpora quad-rigemina and corpus striatum compared with Na-exposed pups.The neurotoxicity of metals was studied by several investigatorsand recorded that one of the major mechanisms behind heavymetal toxicity has been attributed to oxidative stress (Leonardet al., 2004). A growing amount of data provides evidence thatmetal is capable of interacting with nuclear proteins and DNAcausing oxidative deterioration of biological macromolecules.Oxidative stress has been suggested as one possible mechanismfor lead neurotoxicity. Lead-induced oxidative stress damage couldresults from: (1) the inhibition of 5-aminolevulinic acid (ALA)dehydratase leading to accumulation of ALA, a potential endoge-nous source of free radical; (2) direct interaction of lead withbiological membrane, indicating lipid peroxidation; (3) increase ofintracellular calcium, impairing mitochondrial functions; (4)decrease in free radical scavenging enzymes and glutathione(Sandhir et al., 1994; Moreira et al., 2001). Moreover, the harmfuleffects of lead might be related to the protein structure andassembly in cell membrane. Lead may damage the membraneentirely by inactivation of essential thiol groups in membraneproteins (Antonio et al., 2003). On the other hand, neurotoxicityassociated with lead may be the result of a series of smallperturbations in brain metabolism particularly oxidative stress. Ithas been reported that lead enhances lipid peroxidation throughinhibition of superoxide dismutase and other related enzymes(Villeda-Hernandez et al., 2001).

Skrzydlewska et al. (2005) mentioned that a large body ofevidence indicates that the antioxidative abilities decrease and inconsequence oxidative damage of macromolecules such as DNA,protein and lipid occurs more frequently with age and in variousage-related diseases. Therefore, it has been suggested that such anincrease in the oxidative damage, which in turn contributes to theaging process and the degenerative neurons, is the basicpathological change leading to neuro-degenerative diseasesprobably associated with more slight molecular changes in theplasticity of specific neurons and/or synaptic efficacy (Morrisonand Hof, 1997). Our finding revealed that brain is more susceptibleto the toxic effect of lead, as pointed out by Halliwell (2001) whostated that nervous system cells of both human and animals arespecially vulnerable to oxidative damage caused by free radicalsfor a number of reasons. These include high concentration ofreadily oxidizable substrate, in particular membrane lipid poly-unsaturated fatty acid, low level of protective antioxidant enzymes(catalase and glutathione peroxidise) high ratio of membranesurface area to cytoplasmic volume, and extended axonalmorphology prove to peripheral injury.

4.3. Role of green tea

In the present study, green tea co-administered with leadinduced improvement in the activities of SOD and concentration oftotal antioxidant and reduced glutathione in the three regions ofbrain of treated rats. This effect was obvious in withdrawal grouptreated by green tea. In addition, green tea treated group, showedmarked improvement in the antioxidant status similar to that ofcontrol group. Our data are in agreement with Chen et al. (2002)

A.A. Khalaf et al. / NeuroToxicology 33 (2012) 280–289 287

who revealed that tea catechin treatment significantly increasedviability, decreased lipid peroxidation levels and protected cellmembranes fluidity in lead exposed HepG2 cells. Hamed et al.(2010) reported that green tea co-administrated with lead reducedlead contents, increased antioxidant status in blood and braintissue of lead treated rats. The current study was different than thatreported by Hamed et al. (2010) in many points. Firstly wedemonstrated the toxic effect of lead on antioxidant status indifferent regions of brain (cerebrum, cerebellum and medullaoblongata) and not on whole brain homogenate. Also weinvestigate the effect of lead on DNA fragmentation in the threebrain regions previously mentioned, as well as the histopatholog-ical alterations manifested by lead toxicity on brain regions oftreated rats. In addition to know the preventive and curativeefficacy of green tea extract on the undesirable neurotoxicityinduced by lead.

Also, Mehana et al. (2010) demonstrated the protective effect ofgreen tea extract against lead induced liver toxicity in rats.Skrzydlewska et al. (2005) mentioned that green tea has beenimplicated as a regulatory factor for antioxidant enzymes. This ispossible due to its content of polyphenols that are characterized bytheir ability to scavenge free radicals produced during the agingprocess as well as ethanol metabolism. Efficacy of their activity inother tissues such as liver and blood has already been demon-strated (Dobrzynska et al., 2005; Luczaj et al., 2004; Ostrowskaet al., 2004). Tea catechins are strong scavengers againstsuperoxide, hydrogen peroxide, hydroxyl radicals and nitric oxideproduced by various chemicals. They also could chelate withmetals because of catechol structure (Rice-Evans et al., 1996).These characteristics make tea catechins ideal candidates fortreatment of lead toxicity. The most abundant poloyphenols suchas epigallocatechin gallate and epicatechin gallate contained ingreen tea scavenge a wide range of free radicals including the mostactive hydroxyl radical, which may initiate lipid and proteinoxidative modifications. Therefore chemical structure of catechinsis crucial to their antioxidant effect (Guo et al., 1996). Catechin maychelate metal ions especially iron and copper, which in turninhibits the generation of hydroxyl radicals and degeneration oflipid hydroperoxides that cause formation of more reactivealdehydes. Furthermore, the green tea polyphenols have beendemonstrated to inhibit iron-induced oxidation of synaptosomesby scavenging hydroxyl radicals generated in the lecithin/lipooxidase system (Guo et al., 1996). In this respect Skrzydlewskaet al. (2002) revealed that green tea extract representing therichest source of natural polyphenols has in vivo protective effecton the liver, serum and CNS tissue. Green tea increases theactivities of antioxidative enzymes GSH Px and GSSG-R, andcontent of reduced glutathione (GSH) improves total antioxidantstress (TAS) and diminishes the level of lipid peroxidation productsin liver and CNS tissues. Nowadays, tea is considered as a source ofdietary constituents endowed with biological and pharmacologicalactivities with potential benefits to human health. The increasinginterest in the health properties of tea extract and its main catechinpolyphenols have led to a significant rise in scientific investigationfor prevention and therapeutics in several diseases (Mandel et al.,2006; Ostrowska et al., 2004). Tea catechins are strong scavengersagainst superoxide, hydrogen peroxide, hydroxyl radicals andnitric oxide produced by various chemicals. They also could chelatewith metals because of the catechol structure (Rice-Evans et al.,1996). These characteristics make tea catechins ideal candidatesfor treatment of lead toxicity (Chen et al., 2002).

4.4. The DNA fragmentation assay

The neurotoxic effect of lead is demonstrated by a significantelevation of the DNA fragmentation percentage among

lead-treated group. Such elevation was clear in all tissue samplesif compared to the negative control. The laddering pattern of thefragmented DNA on the agarose gel electrophoresis was obvious inthe lead-treated group. Lead causes encephalopathy, cognitiveimpairment, behavioral disturbances, kidney damage, anaemiaand toxicity to the reproductive system (Lidisky and Schneider,2003). Lead accumulates in different brain regions and high levelsof lipid oxidation were found in the parietal cortex, striatum,hippocampus, thalamus and cerebellum in rats exposed to lead(Villeda-Hernandez et al., 2001). In the present study, thecerebellum samples show marked increase in the DNA fragmen-tation assay if compared to the cerebrum and medulla oblongatasamples. Lead intoxication leads to increased lipid oxidation andalterations in oxidant defenses. Lead interacts with negativelycharged phospholipids in membranes and through the induction ofchanges in membrane physical properties could facilitate thepropagation of lipid oxidation (Adonaylo and Oteiza, 1999b).

4.5. Role of green tea against DNA fragmentation

Compounds such as epigallocatechin from green tea, had beenrecognized for their protective effects against inflammatorydiseases, cancers, cardiovascular and neurodegenerative diseases.These compounds display those beneficial effects through theiranti-oxidative, anti-inflammatory, anti-apoptotic and metal che-lating properties (Rice-Evans and Miller, 1997; Nadiaye et al.,2005). The neuroprotective effect of green tea was documented bymany authors (Dajas et al., 2003; Mandel and Youdim, 2004;Simonyi et al., 2005). In the current study, green tea proved to be aneffective antioxidative and antiapoptotic agent. This is clearthrough the marked decrease in the DNA fragmentation percent-age in all tested brain tissues. Green tea is proved to reducebiomarkers of oxidative DNA damage represented by DNAfragmentation, such that the percentages of fragmented DNAshow marked inclination among the lead and green tea-treatedgroup. These results were obvious in all brain tissue samples. Aprominent decrease in DNA fragmentation was achieved amongthe withdrawal group. These findings proved that green tea couldinhibit an induced DNA damage. Our results were similar to thoseof Hakim et al. (2003) and Tang et al. (2008) who reported thatgreen tea decreased the biomarkers of DNA oxidative damage afterreceiving carcinogenic or oxidative agent.

4.6. Histopathological changes

Microscopical examination of brain sections of control and greentea-treated rats showed normal histopathological structure, while inlead treated rats, brain oedema was observed in the hippocampusarea associated with focal gliosis in the cerebrum. The cerebellumshowed vacuolization, while the medulla oblongata had neuronaldegeneration and gliosis. These findings were similar to thosereported by Amal and Mona (2009) who mentioned that brainsections of rats received 100 mg/L lead acetate in drinking water for3–6 weeks, showed pyknosis of neurons associated with focalgliosis. Also, focal cerebral hemorrhage was evident. Our results arein parallel with the data recorded by Engin (2006), who reported thatrats received lead acetate at a dose of 500 ppm in their drinkingwater for 60 days, showed degeneration in some of neuron cells anddilatation in the lumen of the blood vessels. Pardeep and Bimla(2003), observed that after lead treatment of rats at dose level of50 mg/kg b. wt, and transverse sections of cerebrum, showeddegeneration of neurons. Similar alterations were demonstrated byDilkash et al. (2010) in rats treated with 4% aqueous lead acetateorally for a period of 3 weeks. On gross examination brains fromtreated rats revealed generalized oedema and petechial hemor-rhages. Co-administration of green tea extract with lead acetate

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induced improvement of the histopathological alteration in braincompartment. The same results were revealed in the withdrawalgroup. Histopathological findings were constricted in the presenceof pericellular and perivascular oedema in the hippocampus. Nearlysimilar results were recorded by Amal and Mona (2009) whomentioned that examination of brain of rats treated with lead andantox (mixture of vit. C, vit. A, vit. E and selenium) for 3 or 6 weeksshowed no histopathological changes except pyknosis of someneurons.

5. Conclusion

In the light of results, we can suggest that green tea extract andtheir components are partially efficacious in protection andpreventing disturbances of antioxidant defence system in thebrain of lead treated rats. They may reduce neurodegenerationinduced by lead and promote proper health. These beneficialeffects of green tea can result from inhibition of free radical chainreactions generated during oxidative stress caused by lead andfrom an increase in antioxidant enzyme capacity.

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organizations that couldinappropriately influence or bias the content of the paper.

Acknowledgement

The authors wish to acknowledge Dr. Bakar Ramadan Abdel-Halim, Lecturer of Theriogenology Department, Faculty of Veteri-nary Medicine, Beni-Suef University, Beni-Suef, Egypt for hisreservation of help in preparation of the manuscript.

References

Abd El Mohsen MM, Kuhnle G, Rechner AR, Schroeter H, Rose S, Jenner P, et al. Uptakeand metabolism of epicatechin and its access to the brain after oral ingestion. FreeRadic Biol Med 2002;33:1693–702.

Adonaylo VN, Oteiza PI. Lead intoxication: antioxidant defenses and oxidative damagein rat brain. Toxicology 1999a;135:77–85.

Adonaylo VN, Oteiza PI. Pb2+ promotes lipid oxidation and alterations in membranephysical properties. Toxicology 1999b;132:19–32.

Amal EA, Mona HM. Protective effect of some antioxidants on the brain of adult malealbino rats, Rattus rattus, exposed to heavy metals. Biosci Res 2009;6(1):12–9.

Antonio MT, Leret ML. Study of the neurochemical alterations produced in discretebrain areas by perinatal low-level lead exposure. Life Sci 2000;67:635–42.

Antonio MT, Corredor L, Leret ML. Study of the activity of several brain enzymes likemarkers of the neurotoxicity induced by perinatal exposure to lead/or cadmium.Toxicol Lett 2003;143:331–40.

Assuncao M, Santos M, Carvalho F, Lukoyanov N, Andrade J. Chronic green teaconsumption prevents age-related changes in rat hippocampal formation. Neuro-biology of aging 2011;32(4):707–17.

Banchroft JD, Stevens A, Turner DR. Theory and practice of histologic techniques. 4thed. New York/London/San Francisco/Tokyo: Churchil Livingstone; 1996.

Beutler E, Duron O, Kelly MB. Glutathione reagent and method-patent. J Lab Clin Med1963;61:882.

Cabrera C, Artacho R, Gimenez R. Beneficial effects of green tea—a review. J Am CollNutr 2006;25:79–99.

Chen L, Yang X, Jiao H, Zhao B. Tea catechins protect against lead-induced cytotox-icity, lipid peroxidation and membrane fluidity in HepG2 cells. Toxicol Sci2002;69:149–56.

Cook NC, Sammn S. Flavonoids cheme stry, metabolism, cardio-protective effects anddietary sources. J Nutr Biochem 1996;7:66–76.

Dajas F, Rivera F, Blasina F, Arredondo F, Echeverry C, Lafon L, et al. Cell cultureprotection and in vivo neuroprotective capacity of flavonoids. Neurotox Res2003;5:425–32.

Dilkash NA, Husin SM, Khan AA. Effects of lead on the olfactory bulb of the adult albinorats. Curr Neurobiol 2010;1(1):55–7.

Dobrzynska I, Szachowicz-Petelska B, Ostrowska J, Skrzydlewska E, Figaszewski Z.Protective effect of green tea on erythrocyte membrane of different age ratsintoxicated with ethanol. Chem Biol Interact 2005;156:41–53.

Dulloo AG, Seyodoux J, Girardier L. Potentiation of the thermogenic antiobesity effectsof ephedridrine by dietary methylxanthines, adenosine antagonism or phospho-diesterase inhibition. Metabolism 1992;41:1233–41.

Duthie GG, Duthie SJ, Kyle AM. Plant polyphenols in cancer and heart disease:implications as nutritional antioxidants. Nutr Res Rev 2000;13:340–57.

Dreosti IE, Wargovich MJ, Yang CS. Inhibitor of carcinogenesis by tea: the evidence fromexperimental studies. Crit Rev Food Sci Nutr 1997;37:761–70.

Engin D. Ultrastructural effects of lead acetate on brain of rats. Toxicol Ind Health2006;22(10):419–22.

Guo Q, Zhao B, Li M, Shen S, Xin W. Studies on protective mechanisms of fourcomponents of green tea polyphenols against lipid peroxidation in synaptosomes.Biochim Biophys Acta 1996;1304:210–22.

Gupta RC. Veterinary toxicology. Basic and clinical principles. New York: AcademicPress; 2007 pp. 663–725.

Graham HN. Green tea composition, consumption and polyphenols chemistry. PrevMed 1992;21:334–50.

Hakim IA, Harris RB, Brown S, Chow HH, Wiseman S, Agarwal S, et al. Effect of increasetea consumption on oxidative DNA damage among smokers: a randomized con-trolled study. J Nutr 2003;133:3303S–9S.

Halliwell B. Role of free radicals in the neurodegenerative diseases. Therapeuticimplication for antioxidative treatment. Drug Aging 2001;18:685–716.

Hamed EA, Meki MA, Abd El Mottaleb NA. Protective effect of green tea on lead-inducedoxidative damage in rat’s blood and brain tissue homogenates. J Physiol Biochem2010;66:143–51.

Han LK, Takaku T, Li J, Kimura Y, Okuda H. Anti-obesity action of oolong tea. Int J Obes1999;23:98–105.

Herak-Kramberger CM, Sabolic I. The integrity of renal cortical brush border andbasolateral membrane vesicles is damaged in vitro by heavy metals. Toxicology2001;156:139–47.

Higdon JV, Frei B. Tea catechins and polyphenols: health effects, metabolism, andantioxidant functions. Crit Rev Food Sci Nutr 2003;43:89–143.

Imai K, Nakachi K. Cross sectional study of effects of drinking green tea on cardiovas-cular and liver diseases. Br Med J 1995;310:693–6.

Juberg DR, Kleiman CF, Simona CK. Position paper of the American Council on Scienceand Health: lead and human health. Ecotoxicol Environ Saf 2006;38:Error: FPage(s162) is higher than LPage (s180)!.

Jung RT, Shetty PS, Jame WP, Barrand MA, Callingham BA. Caffeine: its effect oncatecolamine and metabolism in lean and obese human. Clin Sci 1981;60:527–35.

Khan SA, Subha MS, Natarajan A, Arivarasu MS. Influence of green tea on enzymes ofcarbohydrate metabolism, antioxidant defense and plasma membrane in rattissues. Basic Nutr Invest 2007;23:687–95.

Koo MW, Cho CH. Pharmacological effects of green tea on the gastrointestinal system.Eur J Pharmacol 2004;500:177–85.

Koracevic D, Koracevic G, Good HF. Methods for measurement of antioxidant activity inhuman fluids. Clin Pathol 2001;54:356–61.

Kuo MW, Lous W, Postlethwait J, Chung BC. Chromosomal organization, evolutionaryrelationship and expression of zebrafish GnRH family members. J Biomed Sci2005;12(4):629–39.

Leonard SS, Harris GK, Shi XL. Metal-induced oxidative stress and signal transduction.Free Rad Biol Med 2004;37:1921–42.

Lidisky TI, Schneider JS. Lead neurotoxicity in children: basic mechanisms and clinicalcorrelates. Brain Rev 2003;126(Pt 1):5–19.

Liuji C, Xianqiang Y, Hongli J. Tea catechins protect against lead induced cytotoxicity,lipid peroxidation, and membrane fluidity in HepG2 cells. Toxicol Sci 2002;69:149–56.

Luczaj W, Waszkiewicz E, Skrzydlewska E, Roszkowska W. Green tea protection againstage-dependant ethanol-induced oxidative stress. J Toxicol Environ Health A2004;67:595–606.

Maiani G, Serafini M, Salicci M, Azzini E, Ferro-Luzzi A. Application of a new highperformance liquid chromatographic method for measuring selected polyphenolsin human plasma. J Chromatogr B 1977;692:311–7.

Mameli O, Caria MA, Solinas A, Tavera C, Tocco M, Flore C, et al. Neurotoxic effect oflead at low concentrations. Brain Res Bull 2001;55(2):269–75.

Mandel S, Youdim MB. Catechin polyphenols: neurodegeneration and neroprotectionin neurodegenerative disease. Free Radic Biol Med 2004;37:304–17.

Mandel S, Amit T, Reznichenko L, Weinreb Oand Youdim MB. Green tea catechins asbrain-permeable, natural iron chelators antioxidants for the treatment of neuro-degenerative disorders. Mol Nutr Food Res 2006;50:229–34.

Mehana EE, Meki MA, Fazili KM. Ameliorated effects of green tea extract on leadinduced liver toxicity in rats. Exp Toxicol Pathol 2010 Oct 12. [Epub ahead of print].

Middleton E, Kandawami C, Theoharides T. The effect of plant flavonoids on mamma-lians cells: implication for inflanimation heart disorse and cancer. Pharmacol Rev2000;52:673–751.

Moreira EG, Rosa GJM, Barros SBM, Vassilieff VS, Vassilieff I. Antioxidant defense in ratbrain regions after developmental lead exposure. Toxicology 2001;169:145–51.

Morrison JH, Hof PR. Life and death of neurons in the aging brain. Science 1997;278:412–9.

Nadiaye M, Chataigneau M, Lobysheva I, Chataigneau T, Schini-KerthV.B.. Red winepolyphenol induced endothelium-dependent NO-mediated relaxation is due to theredox-sensitive P13-kinase/Akt-dependent phosphorylation of indothelial NO-synthetase in the isolated porcine coronary artery. FASEB J 2005;19:455–7.

Nishikimi M, Roa NA, Yogi K. The occurrence of superoxide anion in the reaction ofreduced phenazine methosulfate and molecular oxygen. Biochem Biophys ResCommun 1972;46:849–54.

Orrenius S, Burkitt MJ, Kass GE, Dypbukt JM, Nicoteria P. Calcium ions and oxidative cellinjury. Ann Neurol 1992;32:33–42.

Ostrowska J, Luczaj W, Kasacka I, Rozanski A, Skrzydlewska E. Green tea protectsagainst ethanol induced lipid peroxidation in rat organs. Alcohol 2004;32:25–32.

A.A. Khalaf et al. / NeuroToxicology 33 (2012) 280–289 289

Pardeep S, Bimla N. Lead intoxication, histological and oxidative damage in ratcerebrum and cerebellum. J Trace Elem Exp Med 2003;17(1):45–53.

Pellegrini N, Serafini M, Salvatore S, Del Rio D, Bianchi M, Brighenti F. Totalantioxidant capacity of species, dried fruits, nuts, pulses, cereals and sweetsconsumed in Italy assessed by three different in vitro assays. Mol Nutr Food Res2006;50:1030–8.

Rice-Evans CA, Miller NJ, Paganga G. Structure–antioxidant activity relationship offlavonoids and phenolic acids. Free Radic Biol Med 1996;20:933–56.

Rice-Evans C, Miller N. Measurement of the antioxidant status of dietary constituents,low density lipoproteins and plasma. Prostagladins Leukot Essent Fatty Acids1997;57:499–505.

Robertson D, Frolich JC, Carr RK, Watson JT, Hollifield JW, Shand DG, et al. Effects ofcaffeine on plasma rennin activity, catecholamines and blood pressure. N Engl JMed 1978;298(4):181–6.

Romeo M, Bennani N, Gnassia-Barelli M, Lafaurie M, Girard JP. Cadmium and copperdisplay different responses towards oxidative stress in the kidney of the sea bassDicentraarchus labrax. Aquat Toxicol 2000;48:185–94.

Sandhir R, Julka D, Gillk D. Lipoperoxidative damage on lead exposure in rat brain andits implications on membrane bound enzymes. Pharmacol Toxicol 1994;74:66–71.

Sayama K, Lin S, Zheng G, Oguni I. Effects of green tea on growth, food utilization andlipid metabolism in mice. In Vivo 2000;14(4):481–4.

Sellins A, Cohen S. A flow-cytometric methods to study DNA fragmentation in lym-phocytes. J Immunol Methods 1987;152:171–6.

Shukla GS, Srivastana RS, Chandra SV. Glutathione status and cadmium neurotoxicity:studies in discrete brain regions of growing rats. Fundam Appl Toxicol1988;11:229–35.

Simonyi A, Wang Q, Miller RL, Yusof M, Shelat PB, Sun AY, et al. Polyphenols in cerebralischemia: novel targets for neuroprotection. Mol Neurobiol 2005;31:135–47.

Skrzydlewska E, Ostrowska J, Farbiszewski R, Michalak K. Protective effect of green teaagainst lipid peroxidation in the rat liver, blood serum and the brain. Phytome-dicine 2002;9:232–8.

Skrzydlewska E, Augustyniak A, Michalak K, Farbiszewski R. Green tea supplementa-tion in rats of different ages mitigates ethanol-induced changes in brain antioxi-dant abilities. Alcohol 2005;37:89–98.

Soong WT, Chao KY, Jang CS, Wang JD. Long-term effect of increased lead absorption onintelligence of children. Arch Environ Health 1999;54:297–301.

Tang L, Tang M, Xu L, Luo H, Huang T, Yu J, et al. Modulation of aflatoxin biomarkers inhuman, blood and urine by green tea polyphenols intervention. Carcinogenesis2008;29:411–7.

Tijburg LM, Wiseman SA, Meijer GW, Weststrate JA. Effects of green tea, black tea anddietary lipophylic antioxidant on LDL oxidizability and atherosclerosis in hyperch-olestrolaemic rabbits. Atherosclerosis 1997;135:37–47.

Villeda-Hernandez J, Barroso-Moguel R, Mendez-Armenta M, Nava-Ruiz C, Huerta-Romero R, Riod S. Enhanced brain regional lipid peroxidation in developing ratsexposed to low level lead acetate. Brain Res Bull 2001;55(2):247–51.

Wang J, Wu J, Zhang Z. Oxidative stress in mouse brain exposed to lead. Ann Occup Hyg2006;50(4):405–9.

Youdim KA, Shukitt-Hale B, Joseph JA. Flavonoids and the brain: interactions at theblood–brain barrier and their physiological effects on the central nervous system.Free Radic Biol Med 2004;37:1683–93.

ZhangY.M.. Liu XZ, Lu H, Mei L, Liu ZP. Lipid peroxidation and ultrastructural modifi-cation in brain after perinatal exposure to lead and/or cadmium in rat pups.Biomed Environ Sci 2009;22:423–9.

Zheng G, Sayama K, Ohkubo T, Juneja L, Oguni I. Anti-obesity effects of three majorcomponents of green tea, catechins, caffeine and theanine, in mice. In Vivo2004;18(1):55–62.