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Chemico-Biological Interactions 184 (2010) 484–491

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

rotective effects of pre-germinated brown rice diet on low levels of Pb-inducedearning and memory deficits in developing rat

ong Zhanga, Hongzhi Lua, Su Tiana, Jie Yinb, Qing Chena, Li Maa, Shijie Cuia, Yujie Niua,∗

Department of Occupational Health and Environmental Health, School of Public Health, Hebei Medical University, Zhongshan East Road 361,hijiazhuang 050017, Hebei, People’s Republic of ChinaDepartment of Gynaecology and Obstetrics, The Fourth Hospital of Hebei Medical University, Healthy Road 12, Shijiazhuang 050011, Hebei, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 4 December 2009eceived in revised form 28 January 2010ccepted 28 January 2010vailable online 6 February 2010

eywords:ead (Pb)re-germinated brown riceeveloping nervous systemABAxidative damage

a b s t r a c t

Lead (Pb) is a known neurotoxicant in humans and experimental animals. Numerous studies have pro-vided evidence that humans, especially young children, and animals chronically intoxicated with lowlevels of Pb show learning and memory impairments. Unfortunately, Pb-poisoning cases continue tooccur in many countries. Because the current treatment options are very limited, there is a need for alter-native methods to attenuate Pb toxicity. In this study, the weaning (postnatal day 21, PND21) rats wererandomly divided into five groups: the control group (AIN-93G diet, de-ionized water), the lead acetate(PbAC) group (AIN-93G diet, 2 g/L PbAC in de-ionized water), the lead acetate + WR group (white ricediet, 2 g/L PbAC in de-ionized water; PbAC + WR), the lead acetate + BR group (brown rice diet, 2 g/L PbACin de-ionized water; PbAC + BR) and the lead acetate + PR group (pre-germinated brown rice diet, 2 g/LPbAC in de-ionized water; PbAC + PR). The animals received the different diets until PND60, and then theexperiments were terminated. The protective effects of pre-germinated brown rice (PR) on Pb-inducedlearning and memory impairment in weaning rats were assessed by the Morris water maze and one-trial-

learning passive avoidance test. The anti-oxidative effects of feeding a PR diet to Pb-exposed rats wereevaluated. The levels of reactive oxygen species (ROS) were determined by flow cytometry. The levels of8-hydroxy-2-deoxyguanosine (8-OHdG), �-aminobutyric acid (GABA) and glutamate were determinedby HPLC. Our data showed that feeding a PR diet decreased the accumulation of lead and decreasedPb-induced learning and memory deficits in developing rats. The mechanisms might be related to theanti-oxidative effects and large amount of GABA in PR. Our study provides a regimen to reduce Pb-induced

learn

toxicity, especially future

. Introduction

Pb is an environmental neurotoxicant known to produce detri-ental effects in the nervous system. Numerous studies have

rovided evidence that animals chronically intoxicated with lowevels of Pb show cognitive deficits [1–3]. Epidemiological analysisndicated that children exposed to low levels of Pb are at high risk of

earning and memory impairments [4]. Morphologically, Pb affectsxonal and synaptic elaboration; neurochemically, it replaces cal-ium resulting in altered calcium channel and NMDA receptorunction; and metabolically, it causes oxidative stress and damages

Abbreviations: AP, alkaline phosphatase; BR, brown rice; 2′-dG, 2′-eoxyguanosine; EDTA, ethylenediamine tetraacetic acid; GABA, �-aminobutyriccid; Glu, glutamate; HPLC, high-performance liquid chromatography; NP1, nucle-se P1; PR, pre-germinated brown rice; ROS, reactive oxygen species; WR, whiteice; 8-OHdG, 8-hydroxy-2-deoxyguanosine.∗ Corresponding author. Tel.: +86 311 86265632; fax: +86 311 86265632.

E-mail address: yjniu@hebmu.edu.cn (Y. Niu).

009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.01.043

ing and memory deficits in the developing brain.© 2010 Elsevier Ireland Ltd. All rights reserved.

the developing brain [5,6]. In many countries Pb-poisoning casescontinue to occur, and current treatment options are very limited;thus, there is a need of alternative methods to attenuate Pb toxicity[7,8].

Recently, the neuroprotective potential of many drugs (e.g.,chelators) and regimens to reduce Pb-induced toxicity has beenexplored. However, there are no randomized, clinical trials ofPb-poisoned children providing evidence that treatment withchelators improves clinical outcomes, particularly future cogni-tive development. In large population studies, children who havedietary deficiencies in iron, calcium, vitamin C, or zinc are moresusceptible to injury from environmental sources of lead, whereaspreschool urban children who have higher dietary iron intake havelower blood lead levels [9]. Therefore, treatment regimens have

focused on reducing Pb-induced toxicity especially future learningand memory [10].

The Pb-induced impairment of synaptic plasticity and itseffects on learning and memory deficits have been adequatelystudied. Previous studies suggested that oxidative stress, intracel-

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ular calcium, long-term potentiation, the specific roles of GABA,he metabotropic glutamate receptor, the cholinergic nicotiniceceptor and nitric oxide are related to Pb exposure-induced devel-pmental neurotoxicity including learning and memory defects11]. These toxic mechanisms have led scientists to study therotective qualities of regimens with anti-oxidation and other pro-ective mechanisms on lead toxicity. White rice (WR), the stapleood of Asians, is manufactured by eliminating the fiber-rich branayer from unpolished rice, that is, brown rice (BR). Pre-germinatedrown rice (PR) has been developed by soaking BR in water to

nduce slight germination. After germination, there is the advan-age that PR does not have the hardness of brown rice. Moremportantly, following germination, the levels of several valuableutrients within the grain increase, especially oryzanol and �-minobutyric acid (GABA). The quantity of nutrients contained inR compared to milled rice are 13 times for oryzanol, 10 times forABA, nearly 4 times for dietary fiber, vitamin E, niacin and lysine,nd about 3 times for vitamin B1, B6 and magnesium. Previousesearch has demonstrated that the beneficial effects of abundanthenolic compounds in PR have been attributed mainly to theirnti-oxidant activities [12]. It has recently been reported that PRacilitates spatial learning, and therapeutically, PR may preventlzheimer’s disease (AD) [13]. However, it remains to be clarified

f these nutrients improve brain function, particularly learning andemory abilities in the developing brain after Pb exposure.Therefore, the effects of feeding three rice diets (WR, BR and

R) on learning and memory impairment induced by Pb in wean-ng rats were evaluated in this study. The phenolic compounds inR are known to have anti-oxidant properties, while developmen-al exposure to Pb has been shown to be elevated in AD and isnown to generate reactive oxygen species (ROS) in the aging brain14]. Therefore, we examined whether feeding developing rats withifferent rice diets can counter the oxidative damage induced byb. Our results showed that feeding a PR diet could decrease theccumulation of lead and improve learning and memory deficitsn developing rats after Pb exposure. These mechanisms might beelated to the anti-oxidative effects of phenolic compounds andhe large amount of GABA in PR. This study provides a regimen toeduce Pb-induced toxicity, especially future learning and memoryn developing brain.

. Materials and methods

.1. Animals and foods

Sprague–Dawley rats (75 males and 75 females; 48 ± 6 g) at theeaning age of 21 days (PND21) were procured from the Experi-ental Animal Center of Hebei Medical University (Shijiazhuang,

hina) and housed in polycarbonate cages with filter tops. Ani-als were maintained under standard conditions of temperature

25 ± 2 ◦C), relative humidity (50 ± 2%), and natural light–dark cycleor 1 week to acclimate. All animals were fed a diet and waterd libitum in stainless cages, and they received humane treatmentn compliance with the Principles of Laboratory Animal Care for-

ulated by the National Society for Medical Research and theuide for the Care and Use of Laboratory Animals prepared by

he National Academy of Sciences and published by the Nationalnstitutes of Health (NIH Publication No. 80-23, revised 1978). Thethical regulations have been followed in accordance with nationalnd institutional guidelines for the protection of animal welfare

uring experiments.

All rice and control diets were manufactured as pellets by thexperimental Animal Center of Chinese Academy of Medical Sci-nces (Beijing, China). The control diet (AIN-93G) was composedf cornstarch [39.7% (w/w)], �-cornstarch [13.2% (w/w)], casein

teractions 184 (2010) 484–491 485

[20.0% (w/w)], l-cysteine [0.3% (w/w)], sucrose [10% (w/w)], soy-bean oil [7.0% (w/w)], cellulose powder [5.0% (w/w)], mineral mix[3.5% (w/w)], vitamin mix [1.0% (w/w)], choline bicitrates [0.25%(w/w)] and t-butylhydroquinone [0.0014% (w/w)]. The PR, BR, orWR diets were produced by replacing cornstarch and �-cornstarchwith pre-germinated brown, brown, or white rice, respectively [15].

The weaning rats were randomly divided into 5 groups (30 ratsper treatment group): the control group (AIN-93G diet, de-ionizedwater), the lead acetate (PbAC) group (AIN-93G diet, 2 g/L leadacetate in de-ionized water), the lead acetate + WR group (whiterice diet, 2 g/L PbAC in de-ionized water; PbAC + WR), the leadacetate + BR group (brown rice diet, 2 g/L PbAC in de-ionized water;PbAC + BR) and the lead acetate + PR group (pre-germinated brownrice diet, 2 g/L PbAC in de-ionized water; PbAC + PR).

On PND60, six animals per group were used for Morris watermaze test, and other six animals per group were used for passiveavoidance test. The remaining animals were decapitated after beinganesthetized by ether for further assessing the biochemical param-eters. Six animals per groups were used for oxidative stress markers(GSH-Px, SOD, MDA and 8-OHdG levels), while other six animalsper group were used to determine the levels of lead, ALA, GABAand glutamate. The remaining six animals per group were used todetermine the ROS levels.

After the animals were anesthetized, blood samples were col-lected from femoral artery. Serum was harvested by centrifugationat 700 × g for 10 min. Brains were removed and placed on an ice-cold plate. The hippocampus was excised from the undersurfaceof the corpus callosum for ROS measurement or stored in liquidnitrogen for further analysis.

2.2. Determination of lead levels

To identify the levels of lead exposure of each litter, the leadconcentrations in the sera and hippocampi were measured byatomic absorption spectrophotometry (AAS, TAS-990AFG, BeijingPurkinje General Instrument Co., Ltd., Beijing, China). Hippocampiwere taken out from liquid nitrogen and thawed. About 0.1–0.3 gof each tissue were weighed, digested and analyzed for lead con-tent. Briefly, prior to elemental analysis, the tissues were digestedwith nitric acid (ultrapure grade, Beijing Fine Chemical Ltd., Bei-jing, China) in a polytetrafluoroethylene (PTFE) vessel sealed in asteel vessel overnight. After adding 0.5 mL of H2O2, the mixed solu-tions were completely digested by the microwave digestion system(MDS, Sineo Microwave Chemistry Technology Co., LTD., China).Then, the solutions were heated at 120 ◦C to remove the remainingnitric acid until the solutions were colorless and clear. The remain-ing solutions were diluted to 3 mL with 2% nitric acid. Then, thelead concentration in serum and digested hippocampal sampleswas detected at 283.3 nm.

2.3. Determination of learning and memory

2.3.1. Morris water maze testOn PND60, animals were tested for their spatial memory by the

Morris water maze. The task was adapted from the paradigm origi-nally described in the literature [16]. Briefly, a circular tank (180 cmin diameter × 60 cm in height) was filled with water (26 ± 1 ◦C) to adepth of 40 cm, and the water was made opaque with milk powderto prevent visualization of the platform (10 cm × 10 cm), which wassubmerged 1.5 cm under the water surface. Room lights illuminatedthe pool, and multiple distant cues around the room (window, cab-

inets, and furniture) were kept in the same location throughoutthe experiments. The pool was divided into four quadrants, north-east (NE), northwest (NW), southeast (SE) and southwest (SW).The Plexiglas platform, onto which the rats could escape, was posi-tioned in the middle of quadrant NW. The point of immersion into

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he pool was fixed in the middle of the perimeter of the pool (N, E, Snd W). Rats were trained from PND61 to PND65 during five dailyrials. At the beginning, animals were individually placed into theool facing the wall. If the rat failed to reach the platform in 60 s,

t was gently guided to the platform. Once on the platform, theat was left there for 30 s. Between trials, the rats were toweled,an dried and kept in holding cages for at least 5 min. On PND66,scape latencies from the three quadrants were recorded as theirnal performances.

.3.2. Passive avoidance testAnimals from each group were raised on PND60 to detect

heir learning and memory abilities by one-trial-learning passivevoidance test. A step through the type one-trial-learning passivevoidance test was used as described earlier [17]. A rectangularox (50 cm × 20 cm × 30 cm) consisted of a dark and an illuminatedompartment equipped with a grid floor. The two compartmentsere connected by a little door that allowed the rats pass through.uring passive avoidance conditioning, the illuminated compart-ent was the only light source in the room. On day 1, after

abituation to the dark compartment for 2 min, the rat was placedn the illuminated compartment and allowed to enter the darkompartment. Upon entry, the door was closed, and the animalemained in the dark compartment for 10 s. Thereafter, the ratas placed back in its home cage. Three such training trails were

lso run on day 2, and the latency to enter the dark compartmentas scored. During the third learning trial the rat received a single

crambled electric foot shock of 0.5 mA for 2 s when the rat enteredhe dark compartment. The rat was removed after receiving the foothock and returned to the light compartment by the experimenter.he door was re-opened 10 s later to start the next trial. Trainingontinued in this manner until the rats stayed in the light compart-ent for more than 120 s in a single trial. Twenty-four hours later,

ach animal was placed in the light compartment, and the step-hrough latency was recorded until 300 s had elapsed (retentionrial).

.4. Determination of GABA and glutamate levels

GABA and glutamate levels were quantified by high-erformance liquid chromatography (LC-20AT, Shimadzuorporation, Japan) with a fluorescence detector (RF-10AXL).amples were separated on a Hypersil C18-BDS HPLC column4.6 mm × 250 mm in length and 10 �m beads).

Briefly, the hippocampal samples were taken out from liquiditrogen and homogenized in 2.4 mL 0.04 mol/L of perchloric acid,nd then centrifuged at 10,000 × g for 20 min. The 1 mL supernatantas neutralized with 1 mL of 1.5 mol/L of KHCO3 and then filtered

hrough a 0.22 �m millipore filter. Twenty microliters of the hip-ocampal samples were mixed with 80 �L of methanol. Then, 20 �Lf the mixture was added to 20 �L of OPA-MCE reagent, whichas obtained by mixing 25 mg of OPA (o-phthaldialdehyde, Sigma)ith 0.5 mL of methanol and 50 �L of �-mercaptoethanol (�-MCE,

igma) in 5.0 mL of boric acid buffer (pH 9.6). After 2 min, 20 �L ofhis mixture was injected into an HPLC column. The mobile phaseonsisted of 50 mmol/L of sodium acetate and methanol (55:45,/v) adjusted to pH 6.0. The mobile phase was filtered through0.22 �m millipore filter and ultrasound degassed prior to use.

hromatographic analyses were performed at 25 ± 2 ◦C. The flow

ate was 0.8 mL/min. Amino acids were detected fluorometricallyt an excitation wavelength of 340 nm and an emission wavelengthf 450 nm. Glutamate was eluted with a peak at 2.8 min, followedy GABA at 9.5 min. Standard formulations of GABA and glutamateere purchased from Sigma. All drugs were made up immediatelyrior to the experiment and were applied to the bath.

teractions 184 (2010) 484–491

2.5. Measurement of oxidative stress state

2.5.1. ROS evaluations in hippocampus cellsThe intracellular ROS was detected using 2′,7′-

dichlorofluorescein diacetate (DCFH-DA) as a probe [18]. DCFH-DAis hydrolyzed by esterase to dichlorofluorescein (DCFH), whichis trapped within the cell. This nonfluorescent molecule is thenoxidized to fluorescent dichlorofluorescein (DCF) by the actionof cellular oxidants. DCFH-DA cannot be appreciably oxidized toa fluorescent state without prior hydrolysis. In order to retainthe activities of esterase and the oxidized action of DCFH by theaction of cellular oxidants within the cell, the acutely isolatedhippocampal cells were used for ROS measurement. Rats weresacrificed under sterile conditions, and the hippocampi wereexcised. The samples were rapidly dissected into small pieces andincubated for 20 min at 37 ◦C in a solution of 2.5 mg/mL trypsin inCa2+- and Mg2+-free Hanks’ balanced salt solution (HBSS) bufferedwith 10 mM HEPES. After letting the tissue fragments settle, thesupernatant was collected and one-tenth volume of fetal bovineserum (FBS) was added to stop the trypsin action. The supernatantwas stored in ice. The remaining tissue fragments were redigestedfor an additional 20 min at 37 ◦C. Then the resulting cell suspen-sion was combined and centrifuged for 5 min at 800 × g afterfiltering with 200 mesh copper grids. The cells were resuspendedin DMEM medium with 10% FBS, 0.5 mM l-glutamine, 100 U/mLpenicillin and 100 U/mL streptomycin. The cells were then dis-tributed to six-well plastic culture plates (Costar, Cambridge,MA) at approximately 1.0 × 106 cells per well in 2 mL of culturemedium. Then, the cells were incubated at a final concentrationof 10 mM DCFH-DA (Bryotime Institute of Biotechnology, China)for 20 min at 37 ◦C in 95% air and 5% CO2. Cells were maintainedin the dark prior to and during the analysis. As soon as the incu-bation was completed, the samples were placed on ice for 5 minto stop the reaction. The formation of the fluorescent-oxidizedderivative of DCF was monitored using a FACS420 flow cytome-ter (Becton Dickinson, USA) at emission wavelength of 525 nmand excitation wavelength of 488 nm. Finally, ROS generationwas quantified by the median fluorescence intensity of 10,000cells.

2.5.2. Quantification of GSH-Px and SOD activities as well as MDAlevels

The activities of anti-oxidase glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and the levels of the lipidperoxidation (LPO) product malonaldehyde (MDA) in sera and hip-pocampi were determined according to the methods describedin the references using commercial kits (Nanjing Jiancheng Bio-eng Inst., China) [19]. To prepare tissue extracts, the hippocampiwere ground with liquid nitrogen in a mortar, and the groundtissues (0.5 g each) were then treated with 4.5 mL of PBS buffer.The mixtures were homogenized using a glass homogenizer for1 min on ice. The homogenates were then filtered and centrifugedusing a refrigerated centrifuge at 4 ◦C. Then, these supernatantswere used to determine the enzymes’ activities using a microplatereader (SynergyTM HT, BioTek Instruments, Inc., USA). The pro-tein levels of samples were measured by the Coomassie BrilliantBlue G-250 method with bovine serum albumin as standard. Theprotein contents were determined according to the manufac-turer’s instructions (Nanjing Jiancheng Bioeng Inst., China). Thedata expressed as units of activity or nanomoles per milligram ofprotein.

2.5.3. Measurement of 8-OHdG and 2′-dGLevels of 8-hydroxy-2-deoxyguanosine (8-OHdG) were quan-

tified according to the previous study described with a minormodification [20]. Briefly, DNA was extracted from hippocampi by

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water maze and positive avoidance test. Fig. 2 shows the perfor-mance in the Morris water maze test. The escape latencies of rats inthe PbAC and PbAC + WR groups were slowly shortened by repeatedtraining, whereas those of the control, PbAC + BR and PbAC + PRgroups were rapidly shortened (Fig. 2A). The final test was per-

Fig. 2. Effects of feeding different diets on Pb-induced learning and memory abili-ties by water maze task. 60-day-old rats took a 5 consecutive-day training and final

R. Zhang et al. / Chemico-Biolog

omogenization in buffer containing 1% sodium dodecyl sulfate,0 mM NaCl, 10 mM EDTA, and a 3-h incubation in 100 �g/mL pro-einase K at 50 ◦C. Homogenates were extracted with Tris–phenolnd trichloromethane. The extracts were mixed with 3 M sodiumcetate and 2 vols of 100% ethanol to precipitate DNA at −20 ◦C. Theamples were washed twice with 70% ethanol, air-dried for 15 minnd dissolved in 100 �L of 10 mM Tris/1 mM EDTA (pH 8.0). Then,NA was digested with 10 unit/�L of nuclease P1 (NP1, Biolong Bio-

ogical and Technology Co., Ltd., Shanghai, China) and 1 unit/�L oflkaline phosphatase (AP1, Takara Biotechnology CO., LTD., Dalian,hina). The digest was filtered through a 0.22 �m millipore filter,nd then 8-OHdG and 2-deoxyguanosine (2′-dG) levels were mea-ured using high-performance liquid chromatography (LC-20AT,himadzu Corporation, Japan) with a UV-detector (SPD-20A). Stan-ard 8-OHdG and 2′-dG were purchased from Sigma. Samples wereeparated on a Hypersil C18-BDS HPLC column (4.6 mm × 250 mmn length and 10 �m beads). Twenty microliters of DNA digest

as injected into the HPLC column. The mobile phase consisted of0 mmol/L of KH2PO4 and methanol (90:10, v/v) adjusted to pH 5.5.he mobile phase was filtered through 0.22 �m millipore filter andltrasound degassed prior to use. Chromatographic analyses wereerformed at 25 ± 2 ◦C. The flow rate was 1 mL/min. 8-OHdG and′-dG were detected at a wavelength of 260 nm. 2′-dG was elutedith a peak at 8.4 min, followed by 8-OHdG at 12.2 min. Levels of

-OHdG were expressed as 8-oxo-dG/105 dG.

.6. Determination of ı-aminolevulinic acid (ALA) levels in sera

The �-aminolevulinic acid concentration was measured by a col-rimetric method according to the report of Tomokuni and Ogataith a small modification [21]. Briefly, 200 �L of serum was added

o 800 �L of PBS. Then, 1.0 mL of the mixture was added into eachf two 10-mL glass-stoppered tubes, and 1.0 mL of acetate bufferpH 4.6) was added. To one of the tubes, 200 �L of ethyl acetoac-tate was added and mixed with a vibration mixer for about 5 s.thyl acetoacetate was omitted from the other tube, which wasun as a blank. The tubes were placed in a boiling water bath for0 min. After cooling, we added 3.0 mL of ethyl acetate and shookhe tubes 50 times by hand to extract the ALA-pyrrole. After cen-rifuging for 3 min at 1500–2000 × g, we pipetted 2.0 mL of thethyl acetate (upper) layer into another glass tube. Then, we added.0 mL of modified Ehrlich’s reagent and mixed the solution. After0 min, we determined the absorbance of the color solution at53 nm. To calculate the concentration of ALA, we measured thebsorbance of serum with (A) and without ethyl acetoacetate (B),ubtracted the absorbance of B from the absorbance of A, and readhe concentration of ALA from a standard curve prepared fromn aqueous solution of ALA. Standard ALA was purchased fromigma.

.7. Statistical analysis

The figure legends show the means ± SDs. Data were analyzedsing one-way analysis of variance (ANOVA) followed by the postoc Dunnett’s test when F was significant. Differences were con-idered significant when p < 0.05.

. Results

.1. Pb levels after feeding different diets to rats

The Pb levels in sera and hippocampi were elevated after Pbxposure (p < 0.05, Fig. 1). The lead levels in sera and hippocampif rats both in the PbAC + BR and PbAC + PR groups were decreasedignificantly compared with that of the PbAC group (p < 0.05). This

Fig. 1. The levels of Pb in sera and hippocampi of rats on different diets. Data wereexpressed as mean ± SD (n = 6). The Pb levels in sera and hippocampi of rats afterPbAC treatment were significantly increased compared with the control (*p < 0.05),respectively. The Pb levels in sera and hippocampi of rats on BR and PR diet weredecreased significantly compared with that of PbAC group (†p < 0.05), respectively.

indicated that feeding BR and PR diets might prevent Pb accumu-lation to some degree.

3.2. Effects of feeding different diets on learning and memoryabilities

Learning and memory abilities were evaluated by the Morris

test. Data were expressed as mean ± SD escape latency (s) onto a submerged plat-form (n = 6). (A) Latency to find the platform during training sessions. (B) Latencyexamined 24 h after the last training session. The escape latencies of rats in bothPbAC and PbAC + WR groups were increased significantly compared with the con-trol (*p < 0.05). The escape latencies of rat in both PbAC + BR and PbAC + PR groupswere reduced significantly compared with that of the PbAC group (†p < 0.05).

488 R. Zhang et al. / Chemico-Biological Interactions 184 (2010) 484–491

Fig. 3. Effects of feeding different diets on Pb-induced learning and memory abili-ties by one-trial-learning passive avoidance test. Data were expressed as mean ± SDstep-through latency (s) onto a light box (n = 6). The step-through latencies of ratsin PbAC, PbAC + WR and PbAC + BR groups were increased significantly comparedwPt

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ith the control (*p < 0.05), respectively. The step-through latencies of rats in bothbAC + BR and PbAC + PR groups were reduced significantly compared with that ofhe PbAC group (†p < 0.05), respectively.

ormed 24 h after the last training session. The final escape latenciesf rats both in the PbAC and PbAC + WR groups were increased sig-ificantly compared with that of the control group (p < 0.05). Inddition, the final escape latencies of rats both in the PbAC + BRnd PbAC + PR groups were reduced significantly compared withhat of the PbAC group (p < 0.05; Fig. 2B).

Fig. 3 shows the results of performance in the step-throughype passive avoidance test. There were significant increases in thetep-through latencies of rats exposed to lead acetate (in the PbAC,bAC + WR and PbAC + BR groups) in the retention trial comparedith that of the control group (p < 0.05). The step-through latencies

f rats in both the PbAC + BR and PbAC + PR groups were reducedignificantly compared with that of the PbAC group (p < 0.05).

.3. Effects of feeding different diets on GABA and glutamateontent in rat hippocampi

The levels of GABA and glutamate in hippocampi were mea-ured with HPLC (Fig. 4). After Pb treatment, the GABA content wasignificantly decreased compared with the control (*p < 0.05). TheABA levels in hippocampi from rats in the PbAC + BR and PbAC + PR

roups were significantly increased compared with that of the PbACroup (p < 0.05). The glutamate levels in rat hippocampi from thebAC group were significantly increased compared with the controlp < 0.05).

ig. 4. The levels of GABA and glutamate in hippocampi. Data were expressed asean ± SD (n = 6). After Pb treatment, the GABA levels were significantly decreased

ompared with the control (*p < 0.05). The GABA levels in hippocampi of rats inbAC + BR and PbAC + PR groups were significantly increased compared with PbACroup (†p < 0.05), respectively. The glutamate levels in hippocampi of rats in PbACroup were significantly increased compared with the control (*p < 0.05).

of rats in PbAC group were significantly different compared with that of the con-trol (*p < 0.05). The ROS levels of rats with different rice diet (PbAC + WR, PbAC + BRand PbAC + PR) were decreased significantly compared with that of PbAC group(†p < 0.05).

3.4. Effects of feeding different diets on oxidative stress levels

The imbalance between the levels of cellular ROS and activity ofanti-oxidants results in oxidative stress in biological systems. Fig. 5shows that the ROS levels in hippocampal cells of PbAC-treatedrats on the AIN-93G diet were significantly higher compared withthe control, whereas the ROS levels were significantly decreasedin hippocampal cells of rats on the rice diets (PbAC + WR, PbAC + BRand PbAC + PR groups) compared with that of the PbAC-treated ratson the AIN-93G diet (p < 0.05). These results indicated that feedingthe rice diets might prevent ROS generation in the hippocampi ofPb-exposed rats.

ROS could react with lipids to generate MDA, which couldform DNA adducts and cause oxidative cell damage [22]. In thisstudy, we observed significantly increased MDA levels in serafrom rats fed with the normal diet (PbAC) and rice diet groups(PbAC + WR and PbAC + BR) compared with the control (p < 0.05).However, the levels of MDA were not increased significantly inthe PbAC + PR group compared with the control. The MDA levels insera from PbAC + WR-, PbAC + BR- and PbAC + PR-treated animalswere reduced significantly compared with that of the PbAC group(p < 0.05), but no significant changes were seen in the hippocampifrom rats on the different diet groups. These results indicated thatthe rice diet played an important anti-oxidative role on Pb-exposedrats, especially the PR diet.

Studies suggested that one of the important mechanisms asso-ciated with toxic effects of lead is oxidative stress caused bydisruption of the oxidant/anti-oxidant balance in animals includ-ing humans. Reduced levels of glutathione (GSH) and activities ofGSH-Px and SOD in tissues or blood are most commonly used toevaluate lead-induced oxidative damage [22]. In this study, theGSH-Px and SOD activities in sera and hippocampi from PbAC-treated animals fed with different diets were decreased comparedwith those of the control (Table 1). The GSH-Px and SOD activitiesin sera of rats in PbAC treatment groups feeding different rice diets(PbAC + WR, PbAC + BR and PbAC + PR) were significantly increased(p < 0.05) compared with those of PbAC-fed animals. The SOD activ-ities in hippocampi of rats in the PbAC + BR and PbAC + PR groupswere significantly increased compared with that of the PbAC group(p < 0.05).

Another mechanism of free-radical generation and adduct for-mation by Pb may involve ALA, the heme precursor whose levels

are elevated by lead exposure through feedback inhibition of theenzyme ALA dehydratase [23]. In this study, we found that the ALAlevels in sera were significantly different among groups (Fig. 6).After Pb exposure, the ALA levels were increased by 3.8-fold com-pared with the control. ALA can generate free radicals [24] and

R. Zhang et al. / Chemico-Biological Interactions 184 (2010) 484–491 489

Table 1Levels of GSH-Px, SOD and MDA in sera and hippocampi of rats.

Groups GSH-Px SOD MDA

Serum (U/mL) Hippocampus (U/mg pro) Serum (U/mL) Hippocampus (U/mg pro) Serum (nmol/mL) Hippocampus (nmol/mg pro)

Control 280.00 ± 1.49 129.34 ± 20.06 31.82 ± 0.13 8.15 ± 1.06 2.20 ± 0.26 1.63 ± 0.29PbAC 230.54 ± 6.82* 90.28 ± 12.67* 25.71 ± 1.34* 1.08 ± 0.18* 17.36 ± 1.96* 2.18 ± 0.43PbAC + WR 253.52 ± 3.38*† 91.74 ± 13.16* 28.78 ± 0.46*† 1.17 ± 0.26* 13.93 ± 1.67*† 2.07 ± 0.61PbAC + BR 269.51 ± 2.82*† 102.41 ± 14.55* 31.16 ± 0.41*† 6PbAC + PR 277.93 ± 2.03† 108.66 ± 17.03* 29.87 ± 0.76*† 7

Data represented as means ± SD (n = 6). Values marked with an “*” are significantly differ

Fig. 6. The levels of �-aminolevulinic acid levels in sera. Data were expressed asmild

hhaitprrtO

4

tei

Fe(Tipwt

ean ± SD (n = 6). After Pb treatment, the �-aminolevulinic acid levels were signif-cantly increased compared with the control (*p < 0.05). The �-aminolevulinic acidevels in sera of rats in PbAC + WR, PbAC + BR and PbAC + PR groups were significantlyecreased compared with PbAC group (†p < 0.05), respectively.

as been shown to cause oxidative damage to DNA in Chineseamster ovary cells in vitro through the formation of 8-OHdGdducts [25,26]. Fig. 7 shows that lead acetate induced a 5.2-foldncrease in the hippocampal 8-OHdG/105 × 2-dG ratio of rats onhe AIN-93G diet (p < 0.05), whereas the levels of the hippocam-al 8-OHdG/105 × 2-dG ratio were 4.4-, 4.5- and 2.1-fold higher inats on the different rice diets (PbAC + WR, PbAC + BR and PbAC + PR,espectively) compared with the control. These results suggestedhat feeding with the rice diets might resist the formation of 8-HdG adducts induced by Pb.

. Discussion

Behavior, which is the net output of sensory, motor, and cogni-ive functions in the nervous system, can be a potentially sensitivendpoint of lead-induced neurotoxicity [27,28]. In previous stud-es, lead exposure impaired the ability to complete behavioral tasks

ig. 7. Comparison of 8-OHdG levels in the hippocampal DNA of rats feeding differ-nt diets. 8-OHdG relative levels were expressed as the ratio of oxidized DNA base8-OHdG) to non-oxidized base (2-dG). Data were expressed as mean ± SD (n = 6).he 8-OHdG levels in hippocampal DNA of rats in PbAC group were significantlyncreased compared with that of the control (*p < 0.05). The 8-OHdG levels in hip-ocampal DNA of rats with different rice diet (PbAC + WR, PbAC + BR and PbAC + PR)ere decreased significantly compared with that of PbAC group (†p < 0.05), respec-

ively.

.02 ± 0.89*† 6.06 ± 0.95*† 1.73 ± 0.11

.95 ± 1.19† 3.07 ± 0.29† 1.72 ± 0.34

ent from control (p < 0.05). Compared with PbAC group, †p < 0.05.

in animals [29] and caused a significant decrease in IQ and otherpsychometric scores in children [30]. Our behavioral tests also con-firmed these results. Compared with the control, the lead-exposedrats showed a significant learning deficit (Fig. 2). Due to currentlyunsuccessful clinical treatments, the regimens of cognitive dys-function have been a focal point of research efforts. Here, learningand memory in lead-exposed, weaning rats on different rice dietswere investigated.

We evaluated spatial learning and memory of animals withthe Morris water maze until maturity (PND66). We found the Pb-exposed rats on different diets took different times to locate theplatform after five days of acquisition training. The final escapelatencies of Pb-exposed rats on AIN-93G and WR diets wereincreased significantly. In addition, the final escape latencies of ratson BR and PR diets were reduced significantly compared with thatof the PbAC group (Fig. 1B), whereas there was no statistical differ-ence in latency of rats on BR and PR diets, compared with that ofthe control group. These results suggested that spatial learning andmemory of lead-exposed rats on the BR and PR diets improved.

The passive avoidance test in a shuttle box is a standard, sen-sitive test of learning and memory in the rats. After lead acetatetreatment, we observed significantly increased step-through laten-cies of Pb-exposed rats on AIN-93G, WR and BR diets in theretention trial compared with that of the control group. The step-through latencies of rats on the BR and PR diets were reducedsignificantly compared with that of the PbAC group. Therefore,feeding BR and PR diets could resist the lead-induced short-term memory deficiency. Increased step-through latencies anddecreased shuttle avoidance responses suggested hyperactivity,decreased exploratory behavior, and impairment in learning andmemory. The impairments in this study may be attributed to theincreased hyperactivity in rats [31]. These alterations in behaviormay also be due to the direct inhibitory effect of lead on neuro-transmitters in the adult brain [32,33].

The studies of biochemical and molecular mechanisms of leadtoxicity suggested that some of the effects of lead may be dueto its interference with calcium-mediated cellular processes, theactivation of protein kinases or the release of neurotransmitters(including GABAergic and glutamatergic neurotransmission) [34].After Pb exposure, synthesis of the inhibitory transmitter GABA isdiminished [35]. PR contains a large amount of GABA, which playsa crucial role in memory processes. In this study, the GABA lev-els in the hippocampi were significantly increased after feedingwith the BR and PR diets. Therefore, the large amount of GABAin PR may regulate the glutamatergic system by enhancing glu-tamate release and/or the sensitivity of NMDA receptors, resultingin memory enhancement [13].

Lead may activate protein kinase C (PKC) by mimicking cal-cium, and this may result in the production of ROS. Accordingly,

lead increases LPO and the production of ROS in several cell types[34,36]. Accumulating evidence shows that lead causes oxidativestress by inducing the generation of ROS. Excess production of ROSin the brain has been implicated as a common underlying fac-tor for the etiology of lead-induced neurotoxicity [37,38]. Hence,

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ombinational anti-oxidant therapies are warranted. Indirect inivo evidence of oxidative involvement in lead-induced pathotox-city was demonstrated by alleviation of oxidative stress in therythrocytes after treatment with the proven thiol-containing anti-xidants, N-acetyl cysteine, and a succimer in lead-exposed rats39]. Also, ROS-related lead toxicity in rat sperm was prevented byhe supplementation of rat fed with vitamin E and/or vitamin C [40].R and BR contain abundant phenolic compounds with beneficialffects that have been attributed mainly to their anti-oxidant activ-ty [41,42]. In this study, we found a significant decline of ROS levelsn hippocampal cells in rats fed with a PR or BR diet. These recentndings suggest a potential role of PR and BR in the ameliorationf lead toxicity [43].

Lead could also interact with biological membranes, induc-ng LPO [44]. Finally, this metal could decrease the activities ofree-radical scavenging enzymes, such as catalase (CAT), SOD andSH-Px [45]. The latter is mainly attributed to the high affinity ofb for sulfhydryl groups or metal cofactors in these enzymes andolecules. Previous study indicated that the changes of SOD andSH-Px activities in serum and hippocampus could reflect the neu-

onal damage or even neuronal death [46]. And MDA levels in serumould serve as the biomarkers of neuropathy [47]. In this study, thenti-oxidant enzyme activities in sera and hippocampi were mea-ured. We found the GSH-Px and SOD activities in sera of rats inbAC treatment groups that were fed with the different rice dietsere significantly increased. In hippocampi, the SOD activities of

b-exposed rats on BR and PR diets were significantly increasedompared with that of the PbAC group. Our results suggested thateeding with the PR and BR diets could improve the anti-oxidativenzyme activities, which are the major mechanisms for reducingocal levels of ROS such as the superoxide anion radical (O2

•−) andydrogen peroxide (H2O2) [48]. The degree of oxidative stress canlso be evaluated by determining levels of MDA in lead-exposedats on the different diets. A 7.9-fold elevation in serum and a 1.3-old elevation in hippocampal MDA levels indicated the presencef lead-induced LPO. Feeding rice diets following lead exposureeduced the MDA concentrations, and feeding a PR diet reducedhe MDA levels back to normal levels in serum.

Pb-induced oxidative stress damage could result from the inhi-ition of ALAD (d-aminolevulinic acid dehydratase), leading to theccumulation of ALA [49]. ALA in blood might be the best indicatorf the differential lead exposure over baseline levels [50]. In thistudy, we found that feeding PR and BR diets could prevent theccumulation of ALA. These changes in ALA levels in rats on dif-erent diets were due to the changes in Pb concentration in blood.LA is a potential endogenous source of free radicals and has beenhown to cause oxidative damage to DNA in Chinese hamster ovaryells in vitro through the formation of 8-OHdG adducts [51]. Theffect of oxidative damage on DNA in humans from lead toxicitys also supported by a recent study of 7,8-dihydro-9-oxoguaninedducts in lymphocytes collected from persons environmentallyxposed to metals, including lead [52]. In this study, lead acetatenduced a 5.2-fold increase in the hippocampal 8-OHdG/105 × 2-G ratio in rats on the AIN-93G diet. Additionally, the hippocampal-OHdG/105 × 2-dG ratio induced 4.4-, 4.5- and 2.1-fold increases

n rats fed with the different rice diets compared with the control.hese data suggested that feeding with rice diets might prevent theb-induced 8-OHdG adducts. The assumption that oxidative stresss a mechanism that leads to lead neurotoxicity suggests that somenti-oxidants, including vitamins and other minerals as well as theice diet, could ameliorate lead neurotoxicity [43,53,54].

In this study, we found that the protective effects of PR and BRiet on lead-exposed rats were related to their anti-oxidant activ-

ties or to the large amount of GABA present. Moreover, the levelsf Pb in the sera and hippocampi decreased in rats on the PR andR diets, indicating that the BR and PR diets might prevent the Pb

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accumulation to some degree. It is well known that dietary fiber,vitamin E, and vitamin C are abundant in PR. Cellulose supplemen-tation reduced the retention of Pb in rats [55], and dietary fiber frombran can effectively bind Pb ions, preventing toxicity [56]. There-fore, the PR diet might potentially be therapeutic in the treatment ofchronic Pb intoxication. However, the detailed mechanism respon-sible for the falling Pb levels in rats fed with the PR diet requiresadditional investigation.

In conclusion, we have shown that the PR diet improved learn-ing and memory deficits induced by low-level lead in young rats. Inparticular, we concluded that, therapeutically, PR may prevent leadneurotoxicity. However, it is unclear whether the neuronal dam-age induced can be reversed. Several mechanisms may contributeto the apparent positive effects of PR on learning and memory.For instance: (1) the beneficial effects on lead neurotoxicity fromthe abundant phenolic compounds have been mainly attributed totheir associated anti-oxidant activities such as reducing the ROSand MDA levels, as well as the formation of 8-OHdG adducts in thehippocampus and elevating the activities of SOD and GSH-Px; (2)the high levels of GABA in PR might result in memory enhance-ment; and (3) PR diets might prevent Pb accumulation to somedegree. Future studies on chronically low levels of lead exposureand the effect of the PR diet may perhaps be able to answer manyunanswered questions. The PR diet may be a potential therapy inthe treatment of chronic Pb intoxication, primarily during devel-opment.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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